biographies and abstracts - Fish Bites
biographies and abstracts - Fish Bites
biographies and abstracts - Fish Bites
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
Welcome!<br />
We were absolutely delighted that the IST awarded us the privilege<br />
of arranging <strong>and</strong> hosting the 15 th World Congress on Animal, Plant<br />
<strong>and</strong> Microbial Toxins, <strong>and</strong> we are very pleased to welcome you to<br />
Glasgow.<br />
We think that we have an exciting <strong>and</strong> fascinating programme<br />
covering all aspects of toxinology, <strong>and</strong> we have a great mix of<br />
invited speakers <strong>and</strong> other participants. We hope that you will enjoy<br />
the next few days, meeting old friends <strong>and</strong> making new ones, <strong>and</strong><br />
re-discovering the fascinating insights that toxins can reveal to us.<br />
Our grateful thanks go to our premier sponsors <strong>and</strong> co-sponsors for<br />
the outst<strong>and</strong>ing support they have given us.<br />
We have been very lucky to be helped in our organisational tasks<br />
by Angelfish. Together, we will try to make your participation in the<br />
Congress as enjoyable <strong>and</strong> rewarding as possible.<br />
Best wishes,<br />
Alan Harvey <strong>and</strong> Edward Rowan<br />
Congress Organising Committee<br />
Department of Physiology <strong>and</strong> Pharmacology<br />
University of Strathclyde<br />
27 Taylor Street<br />
Glasgow G4 0NR, UK<br />
Contents<br />
Scientific programme in outline page 3<br />
Social Programme page 5<br />
Scientific timetable day-by-day<br />
Abstracts<br />
Monday page 7<br />
Tuesday page 9<br />
Wednesday page 11<br />
Thursday page 12<br />
Friday page 15<br />
Plenary lectures<br />
Symposia<br />
W P Stemmer page 19<br />
J E Rothman page 21<br />
Redi Award page 23<br />
J W Daly page 25<br />
M J Browstein page 27<br />
Toxins <strong>and</strong> drug discovery page 29<br />
Lipids <strong>and</strong> toxins page 35<br />
From anecdotes to antidotes page 41<br />
Ion channel toxins page 53<br />
Toxins <strong>and</strong> hemostasis page 59<br />
Venomics page 70<br />
Oral communications<br />
Posters<br />
Monday page 88<br />
Tuesday page 112<br />
Thursday page 130<br />
Friday page 146<br />
Monday page 170<br />
Tuesday page 226<br />
Thursday page 281<br />
Friday page 331
Scientific programme in outline<br />
The programme is composed of plenary lectures, symposia, oral<br />
communications, <strong>and</strong> poster presentations.<br />
The plenary lectures <strong>and</strong> symposia will be held in the John Anderson<br />
Building, lecture theatre K3.25.<br />
The oral communications will be in three parallel sessions in lecture<br />
theatres K3.14, K3.17 <strong>and</strong> K3.25 in the John Anderson Building.<br />
The poster sessions <strong>and</strong> trade exhibition will be in the Colville Building<br />
(adjacent to the John Anderson Building), rooms 5.11 <strong>and</strong> 5.12.<br />
Conference Office: this will be opposite the entry to the main lecture theatre<br />
K3.25 in the John Anderson Building. Registration will be open from 8.00am on<br />
Monday 24 July, <strong>and</strong> the Conference Office will be open throughout each day of<br />
the Congress.<br />
3
Time Monday 24th Tuesday 25th Wednesday 26th Thursday 27th Friday 28th<br />
8.30-9.00 Registration <strong>and</strong> congress opening Registration Registration Registration Registration<br />
9.00-11.15 Toxins & Drug Discovery symposium Lipids & Toxins From Anecdotes to Ion channel symposium: “Venomics” symposium:<br />
symposium<br />
Antidotes<br />
Sponsored by the<br />
Sponsored by CEA<br />
symposium<br />
International Society for<br />
Neurochemistry <strong>and</strong> the<br />
Physiological Society<br />
Saclay<br />
11.15-<br />
11.45<br />
coffee coffee coffee coffee coffee<br />
11.45- Elsevier-Toxicon Plenary lecture-<br />
Plenary lecture- Redi Award lecture - Plenary lecture- John W. Plenary lecture- Michael<br />
12.45 Sponsored by Elsevier Life Sciences - James E. Rothman to be announced Daly “Alkaloids as probes Brownstein "What can<br />
Willem Pim Stemmer “The advantages of “Membrane fusion in<br />
for ionic channels”<br />
genomics teach you<br />
disulfide-rich microproteins as scaffolds<br />
for the construction of antibody-mimetic<br />
pharmaceuticals”<br />
the cell”<br />
about toxins?"<br />
12.45-1.45 Lunch Lunch Lunch Lunch Lunch<br />
1.45-2.45 Posters Posters Conference outing<br />
to Burrell Collection<br />
<strong>and</strong> Pollok Park<br />
(from 2.00 to 5.00)<br />
Posters Posters<br />
2.45-3.00 Posters <strong>and</strong> coffee Posters <strong>and</strong> coffee Posters <strong>and</strong> coffee Posters <strong>and</strong> coffee<br />
3.00-5.40 Free oral communications (3 sessions in Free oral<br />
Toxins <strong>and</strong> hemostasis Free oral<br />
parallel)<br />
communications (3<br />
symposium<br />
communications (3<br />
sessions in parallel)<br />
with parallel poster<br />
<strong>and</strong> oral sessions<br />
sessions in parallel)<br />
5.00-6.00 IST business meeting<br />
ISTH Exogenous<br />
Hemostatic Factors<br />
subgroup business meeting<br />
End of Congress<br />
Evening Civic reception at City Chambers An evening of play at Free Celebration dinner <strong>and</strong><br />
event<br />
the Glasgow Science<br />
ceilidh, Bute Hall, Glasgow<br />
Centre<br />
University
Social Programme<br />
Sunday 23rd July<br />
Pre-Congress Registration & Welcome, Barony Hall - University of Strathclyde<br />
(corner of Castle Street <strong>and</strong> Rottenrow - a 2 min walk from campus accommodation)<br />
Pre-Congress Registration Opening - 4pm - 8pm<br />
Originally a church, the Barony Hall was the competition winning design of J.J. Burnet <strong>and</strong> JA<br />
Campbell in 1886. This red s<strong>and</strong>stone building replaced a Gothic church by John Robertson <strong>and</strong> James<br />
Adam (1793-1800) <strong>and</strong> now serves as the main ceremonial hall of the University of Strathclyde.<br />
The pre-congress registration will open at 4pm in the Barony Hall on Sunday 23rd July. This will allow<br />
you the opportunity to register your arrival <strong>and</strong> collect your Congress information. There will be a<br />
relaxed pre-congress welcome drinks reception with light refreshments also taking place in the Barony<br />
Hall which will offer you the perfect opportunity to meet up with new <strong>and</strong> old friends <strong>and</strong> the hosts of<br />
the IST 15th World Congress on Animal, Plant <strong>and</strong> Microbial Toxins.<br />
Monday 24th July, 7pm - 8.30pm<br />
Civic Welcome Reception - Glasgow City Chambers<br />
In the very heart of Glasgow (<strong>and</strong> a few minutes walk from the Congress location) st<strong>and</strong>s one of the<br />
city’s most important <strong>and</strong> prestigious buildings - the City Chambers.<br />
A gr<strong>and</strong> <strong>and</strong> imposing edifice overlooking George Square, the City Chambers is an impressive symbol<br />
of Glasgow’s political strength <strong>and</strong> historical wealth. Completed in 1888, the City Chambers has for<br />
over a hundred years been the headquarters of successive councils serving the City of Glasgow.<br />
The Lord Provost will welcome you to the city with a Civic Welcome Reception providing you with an<br />
opportunity to network with fellow delegates following the first day of the Congress whilst enjoying a<br />
relaxing drink in beautiful <strong>and</strong> elegant surroundings.<br />
Tuesday 25th July, 7.30pm - 9.30pm<br />
An Evening of Play, Glasgow Science Centre<br />
Glasgow Science Centre is Scotl<strong>and</strong>’s flagship Millennium Project. Housed in three stunning buildings<br />
at Pacific Quay on the River Clyde, GSC brings science <strong>and</strong> technology to life through hundreds of<br />
interactive exhibits in the Science Mall <strong>and</strong> the unique experiences of the GSC IMAX® Theatre <strong>and</strong><br />
the Glasgow Tower. For more information on the venue, see www.glasgowsciencecentre.org<br />
Enjoy a relaxed evening with your fellow delegates at Glasgow’s Science Centre. Here you can enjoy<br />
the drinks reception whilst amusing yourselves with the Science Centre's collection of attractions which<br />
we are sure will illuminate, intrigue, inspire <strong>and</strong> fire your imagination!<br />
Transport to <strong>and</strong> from the Science Centre will be provided.<br />
Wednesday 26th July, 2pm- 5pm<br />
Congress Social Afternoon, Burrell Collection<br />
The City of Glasgow owns one of the richest art collections in Europe, displayed in 13 museums across<br />
the city. The award winning Burrell Collection is set in Pollok Country Park <strong>and</strong> more than 8,000 art<br />
objects amassed in a lifetime by the Glasgow shipping magnate Sir William Burrell are in the museum<br />
(http://www.glasgowmuseums.com/venue/index). The light <strong>and</strong> airy building, its woodl<strong>and</strong> setting <strong>and</strong><br />
the amazing breadth of Sir William Burrell's collection make a visit to The Burrell Collection an<br />
experience not to be missed. The park <strong>and</strong> woodl<strong>and</strong> also offer plenty of opportunity for enjoying a<br />
walk around the estate, <strong>and</strong> to see some prize Highl<strong>and</strong> cattle.<br />
After the morning congress <strong>and</strong> lunch, you will be taken by coach to spend the afternoon at the Burrell<br />
Collection. Pollok House is also located within the grounds of Pollok Estate, but please note that entry<br />
to Pollock House is not included in your booking. If you wish to visit the House you are free to do so<br />
but there will be a charge payable on admission to the venue of approximately £8 per adult (free<br />
admission to the gardens, shop <strong>and</strong> restaurant). The house is a 10 minute walk from the Burrell <strong>and</strong><br />
there is a shuttle service provided by the estate between the two venues. Pollok Estate has been the<br />
home of the Maxwell family since the mid-13th century. The current house is an impressive 18th<br />
century mansion, filled with wonderful collections of Spanish art, antique furniture, silverware <strong>and</strong><br />
ceramics, <strong>and</strong> an impressive library. See http://www.glasgowmuseums.com/venue/index<br />
Transport to <strong>and</strong> from the Burrell Collection will be provided. A packed lunch will be available before<br />
departure from the Congress venue.<br />
Thursday 27th July, 7pm - midnight<br />
Congress Celebration Dinner & Ceilidh, Bute Hall - University of Glasgow<br />
The sheer scale of the University of Glasgow’s most famous venue is, quite simply, breathtaking. This<br />
gr<strong>and</strong> Victorian hall will be the setting for a fun <strong>and</strong> relaxed Scottish Gala Dinner to mark the closing<br />
of the Congress <strong>and</strong> to celebrate the 40 years of IST Congresses. This is the perfect opportunity to<br />
experience some true Scottish hospitality whilst allowing us all to bid farewell to our old <strong>and</strong> new<br />
friends. You will be treated to a fabulous Scottish themed menu <strong>and</strong> will be entertained by a fun <strong>and</strong><br />
energetic Ceilidh b<strong>and</strong> who will have you up dancing the night away…so don’t forget your dancing<br />
shoes.<br />
Transport to <strong>and</strong> from the venue will be provided.<br />
6
Scientific timetable day-by-day<br />
Monday 24 July<br />
Symposium: Toxins <strong>and</strong> Drug Discovery (K3.25)<br />
9.00-9.45: George Miljanich (CEO, AIRMID LLC, California)<br />
PRIALT (ziconotide intrathecal infusion): a conopeptide for treating<br />
severe chronic pain<br />
9.45-10.30: Michael Hanley (Vice President of Discovery Research,<br />
Amylin Pharmaceuticals, California)<br />
Physiological fluids of venomous animals: novel resources for drug<br />
discovery<br />
10.30-11.15: George Ch<strong>and</strong>y (University California Irvine)<br />
From marine toxins to therapy for autoimmune diseases<br />
The Elsevier-Toxicon Lecture (K3.25):<br />
11.45am-12.45pm<br />
Willem Pim Stemmer (CEO <strong>and</strong> Founder of Amunix, Inc, California)<br />
The advantages of disulfide-rich microproteins as scaffolds for the<br />
construction of antibody-mimetic pharmaceuticals<br />
Poster sessions (Colville 5.11/5.12)<br />
1.45-3.00 pm<br />
Nerve, muscle <strong>and</strong> myotoxicity<br />
Ion channels<br />
7
Monday 24th<br />
July<br />
Track 1: Acetylcholine Receptors<br />
Oral Presentations K3.14<br />
3.00-3.20 Servent, D. : MT7 muscarinic toxin as tool to study<br />
direct allosteric interactions on M1 muscarinic<br />
receptor<br />
3.20-3.40 Tsetlin, V.I.:. α-Conotoxins And Their Targets:<br />
From Photolabeling To The Three-Dimensional<br />
Structures<br />
3.40-4.00 Wildeboer, K.M.: Interaction of Erythrina <strong>and</strong><br />
Phelline Alkaloids with the Neuronal α4β2 Nicotinic<br />
Acetylcholine Receptor (nAChR)<br />
4.00-4.20 Kem, W.R.: Binding of 3-(Benzylidene)-<br />
Anabaseines to Mammalian Nicotinic Acetylcholine<br />
Receptors <strong>and</strong> Molluscan ACh Binding Proteins<br />
4.20-4.40 Adams, D.J.: The Structural Characterisation <strong>and</strong><br />
Receptor Specificity of α-Conotoxin Vc1.1<br />
4.40-5.00 Molgó, J.: Gymnodimine-A targets muscular <strong>and</strong><br />
neuronal nicotinic acetylcholine receptors with high<br />
affinity<br />
5.00-5.20 Daly, N.L.: The unique cysteine spacing of BuIA<br />
destabilizes the globular fold commonly found in αconotoxins<br />
5.20-5.40 Marchot, P.: Structural analysis of AChBP<br />
complexes with nicotinic agonists <strong>and</strong> antagonists<br />
Track 2: Cardiovascular Themes <strong>and</strong><br />
Phospholipases<br />
K3.17<br />
Gopalakrishnakone, P.: Designing Peptide Drugs<br />
from Python Serum: Dual Inhibitors of sPLA2 <strong>and</strong><br />
MMP-1 as Therapeutic Option for Treatment of<br />
Inflammation<br />
Wen-guey Wu : Dynamin dependent <strong>and</strong><br />
independent endocytic pathway of cobra<br />
cardiotoxins: role of distinct cholesterol <strong>and</strong><br />
heparan sulfate domain for toxin selection<br />
Yamazaki, Y.: Snake venom VEGF-F is a most<br />
potent inducer of vascular permeability<br />
Tokunaga, Y.: C-terminal peptide of snake venom<br />
VEGF-F specifically blocks VEGF-A165 activity by<br />
binding to heparin-like molecules<br />
Flight, S.M.: A factor Xa-like enzyme from<br />
Pseudonaja textilis venom is a powerful<br />
procoagulant that reduces blood loss in a topical<br />
anti-bleeding model.<br />
Perchuc, A.M.: Heparin-neutralizing properties of<br />
two novel Lys49 PLA2 variants from the Bothrops<br />
moojeni venom.<br />
Faure, G. FXa-binding studies of the anticoagulant<br />
phospholipases A2 from Viperidae <strong>and</strong> Crotalidae<br />
venom<br />
Guarnieri, M.C. BE-I-PLA2, a novel acidic<br />
phospholipase A2 from Bothrops erythromelas<br />
venom: purification, cloning <strong>and</strong> characterization as<br />
potent anti-platelet <strong>and</strong> inductor of PGI release by<br />
endothelial cells<br />
8<br />
Track 3: Structural <strong>and</strong> Biochemical Aspects<br />
K3.25<br />
Winter, K.L. : Stability studies on the venom of the<br />
major Australian box jellyfish (Chironex fleckeri).<br />
Kintner A.: Variation in venom between two<br />
Australian box jellyfish: prey-specific adaptations<br />
Ward, R. J.: Structural Determinants of Lys49<br />
Phospholipase A2 Activity Probed by Scanning<br />
Alanine Mutagenesis<br />
Souza, G.H.M.F.: Screening of Bothrops snake<br />
venoms using bidimensional capillary liquid<br />
chromatography coupled to t<strong>and</strong>em nanoelectrospray<br />
quadrupole time-of-flight mass<br />
spectrometry: identification of novel peptides<br />
Gillet, D.: Role of histidines <strong>and</strong> the N-terminal<br />
helices in the function of the diphtheria toxin T<br />
domain<br />
Bieber, A.L.: Detection <strong>and</strong> Quantitation of<br />
Detrimental Levels of Staphylococcal Enterotoxin B<br />
by Mass Spectral Immunoassay<br />
Tulayakul, P.: Chemical modification of mycotoxin<br />
metabolism by green tea extract <strong>and</strong> coumarin in<br />
piglets<br />
Stöcklin, R.: Novel proteolytic enzymes from<br />
pathogenic fungi <strong>and</strong> their use for biotechnology<br />
<strong>and</strong> health
Scientific timetable day-by-day<br />
Tuesday 25 July<br />
Symposium on Lipids <strong>and</strong> Toxins (K3.25)<br />
9.00-9.45: Edward A. Dennis (Distinguished Professor, University of<br />
California San Diego)<br />
LIPID MAPS <strong>and</strong> eicosanoid lipidomics<br />
9.45-10.30: Cesare Montecucco (Professor of General Pathology,<br />
University of Padova, Italy)<br />
How snake <strong>and</strong> bacterial presynaptic enzyme toxins block nerve<br />
terminals<br />
10.30-11.15: Uroš Petrovic (Department of Biochemistry & Molecular<br />
Biology, Jozef Stefan Institute, Slovenia<br />
Genome-wide analysis of the molecular mechanism of action of<br />
ammodytoxin, a neurotoxic sPLA2, in yeast cells<br />
Plenary lecture (K3.25)<br />
11.45am-12.45pm<br />
James E. Rothman (Director of the Columbia Genome Center, Professor<br />
of Chemical Biology, Columbia University, New York)<br />
Membrane fusion in the cell<br />
Poster sessions (Colville 5.11/5.12)<br />
1.45-3.00 pm<br />
Venomics, transcriptomics, cloning<br />
Toxin discovery, purification, biochemistry<br />
9
Tuesday 25th<br />
July<br />
Track 1: Clinical Envenoming<br />
Oral Presentations K3.14<br />
3.00-3.20 Thomas, L.: Post mortem examination of a fatal<br />
case of Bothrops lanceolatus envenoming<br />
3.20-3.40 Kuch, U.: Kraits with 17 dorsal scale rows (Bungarus<br />
sindanus complex): unrecognised causes of severe<br />
neurotoxic envenoming in South Asia<br />
3.40-4.00 Faiz, M.A.: Severe neurotoxic <strong>and</strong> myotoxic<br />
envenoming by the Greater Black Krait (Bungarus<br />
niger) in Chittagong Division, Bangladesh<br />
4.00-4.20 Ariaratnam, A.: Distinctive epidemiology <strong>and</strong> clinical<br />
features of common krait (Bungarus caeruleus) bite<br />
in Sri Lanka<br />
4.20-4.40 Warrell, D.A.: First authenticated cases of lifethreatening<br />
envenoming by the hump-nosed pit viper<br />
(Hypnale hypnale) in India, <strong>and</strong> Severe systemic<br />
envenoming by hump-nosed vipers (Hypnale<br />
hypnale) in Sri Lanka<br />
4.40-5.00 Haddad Jr., V.: Injuries caused by venomous<br />
animals occurred in domestic <strong>and</strong> commercial<br />
Aquariums: A study of 24 cases<br />
5.00-5.40 IST business meeting – K3.25. All IST members are<br />
urged to attend<br />
Track 2: Model Test Systems<br />
K3.17<br />
Sevcik, C.: A dynamic interpretation of the<br />
pharmacokinetic volume of distribution <strong>and</strong> its<br />
relation to the pharmacokinetics of F(ab')2 <strong>and</strong> other<br />
drugs<br />
D´Suze, G.: Effect of Leukocyte Inhibitors<br />
Benzydamine <strong>and</strong> Cyclophosphamide on Lung Injury<br />
Caused by Tityus discrepans Scorpion Venom<br />
Ratanabanangkoon, K.: Pig as an experimental<br />
model for the study of local tissue necrosis caused<br />
by snake venoms<br />
Carlini, C.R.: Ureases display biological effects<br />
independent of enzymatic activity. Is there a<br />
connection to diseases caused by urease-producing<br />
bacteria?<br />
Arthur, P.K.: The Use Of Indigenous Knowledge in<br />
Rural Development: A Case Study In The Use Of<br />
Herbal Medicine In The Treatment of Diseases In<br />
The Northern Region Of Ghana<br />
IST business meeting –K3.25. All IST members are<br />
urged to attend<br />
10<br />
Track 3: Toxins, receptors <strong>and</strong> ion channels<br />
K3.25<br />
Nicholson, G.M.: Pharmacophore mapping of the katracotoxins:<br />
selective insect potassium channel<br />
blockers that reveal a novel insecticide target<br />
Cuypers, E. : Jellyfish <strong>and</strong> other Cnidarians cause<br />
pain by affecting TRPV1 channels<br />
Marí, F.: Novel Conotoxin Frameworks<br />
King, G,F. : A dual-target, self-synergizing peptide<br />
toxin from spider venom<br />
Petit, K.E. : Fulvol Acetate, a novel activator of<br />
transient Low-Voltage Activated (LVA) Ca 2+ current<br />
Kauferstein, S. : A novel family of conotoxins target<br />
to nACh-receptors<br />
IST business meeting – K3.25. All IST members are<br />
urged to attend
Scientific timetable day-by-day<br />
Wednesday 26 July<br />
Symposium: From anecdotes to antidotes (K3.25)<br />
9.00-9.30: José María Gutiérrez (Subdirector, Instituto Clodomiro Picado,<br />
Costa Rica)<br />
Novel possibilities for the treatment of local effects in snakebite<br />
envenomation<br />
9.30-10.00: Isaac Asuzu (Professor, Department of Veterinary Physiology<br />
<strong>and</strong> Pharmacology, University of Nigeria, Nsukka)<br />
Myths <strong>and</strong> realities of plant-based remedies for snake bite<br />
“An integrated approach to the development, optimization <strong>and</strong><br />
evaluation of a polyvalent antivenom for Sub-Saharan Africa”<br />
10.00-10.20: Jean-Philippe Chippaux<br />
Mangement of snake bite in Africa: how to stop the vicious cycle?<br />
10.20-10.35: Alej<strong>and</strong>ro Alagón<br />
Development of a polyvalent F(ab’) antivenom for Sub-Sahara African<br />
snakes<br />
10.35-10.45: Roberto P. Stock<br />
The spectrum of protection: serological cross-reactivity between<br />
venoms of African vipers <strong>and</strong> elapids<br />
10.45-100.05: Achille Massougbodji<br />
Clinical trial of a polyvalent antivenom specific for African snakes<br />
Redi Award lecture (K3.25)<br />
11.45am-12.45<br />
The winner of the Redi Award will be announced at the Congress <strong>and</strong><br />
will present the Redi Award lecture<br />
11<br />
Scientific timetable day-by-day<br />
Thursday 27 July<br />
ISN Symposium: Ion channel toxins: tools to explore ion<br />
channel structure, function <strong>and</strong> physiology (K3.25)<br />
A symposium sponsored by The International Society for<br />
Neurochemistry, with support from The Physiological Society<br />
9.00-9.45: Brian Robertson (Professor of Neurobiology, University of<br />
Leeds)<br />
Potassium channel toxins - a trail from cortex to channel subtype<br />
9.45-10.30: Frederic Meunier (Laboratory of Molecular Dynamics of<br />
Synaptic Function, University of Queensl<strong>and</strong>, Australia)<br />
Glycerotoxin, a new tool to dissect synaptic vesicle recycling<br />
10.30-11.15: Michael Gurevitz (Professor, Department of Plant Sciences,<br />
Tel Aviv University, Israel)<br />
Structural commonality versus functional specificity in scorpion toxins<br />
that affect voltage-gated sodium channels<br />
Plenary lecture (K3.25)<br />
11.45am-12.45pm<br />
John W. Daly (NIH Scientist Emeritus, NIH, Maryl<strong>and</strong>)<br />
Alkaloids as probes for ionic channels<br />
Poster sessions (Colville 5.11/5.12)<br />
1.45-3.00 pm<br />
Marine toxins<br />
Pain <strong>and</strong> CNS<br />
Inflammation <strong>and</strong> cytokines<br />
Cytotoxicity <strong>and</strong> antimicrobial<br />
12
Symposium: Toxins <strong>and</strong> hemostasis (K3.25)<br />
Sponsored by Pentapharm, Latoxan <strong>and</strong> Venom Supplies<br />
3.00-3.30: Kenneth J. Clemetson (Professor of Biochemistry, Theodor<br />
Kocher Institute)<br />
Snake venom proteins affecting platelets<br />
3.30-4.00: Aura Kamiguti (University of Liverpool)<br />
Platelet interaction with multidomain soluble snake venom<br />
metalloproteinases<br />
4.00-4.30: Russolina Zingali (Associate Professor, Universidade Federal<br />
do Rio de Janeiro)<br />
Structural <strong>and</strong> pharmacological studies of bothrojaracin, a<br />
(pro)thrombin inhibitor<br />
4.30-5.00: Frank S Markl<strong>and</strong> (Professor of Biochemistry <strong>and</strong> Molecular<br />
Biology, University of Southern California)<br />
Contortrostatin <strong>and</strong> its anti-cancer action<br />
5.00-5.30: Manjunatha Kini (Professor, National University of Singapore)<br />
Origin, evolution <strong>and</strong> recruitment of venom prothrombin activators in<br />
Australian elapids<br />
5.30-6.00: meeting of the ISTH SSC Registry of Exogenous Hemostatic<br />
Factors<br />
13
Thursday<br />
27th July<br />
Track 1: Toxins <strong>and</strong> Na<br />
Oral Presentations<br />
+ channels<br />
K3.14<br />
3.00-3.20 Ilan N.: Scorpion Beta-Toxins Interact with their<br />
Na-channel Receptor Via a Novel 'Ratchet<br />
Mechanism'<br />
3.20-3.40 Beress, L.: A new toxin from the sea anemone<br />
Condylactis gigantea with effect on sodium<br />
channel inactivation<br />
3.40-4.00 Gordon, D.: Allosteric interactions between<br />
scorpion toxin receptor sites on voltage-gated Na<br />
channels imply a novel role for weakly active<br />
components in arthropod venom<br />
4.00-4.20 Jalali, A.: OD1, α-like toxin isolated from the<br />
Iranian yellow scorpion Odonthobuthus doriae<br />
venom<br />
4.20-4.40 Cohen L.: Direct Evidence that Receptor Site-4<br />
of Sodium Channel Gating Modifiers is not<br />
Dipped in the Phospholipid Bilayer of Neuronal<br />
Membranes<br />
4.40-5.00 Norton, R.S.: Structure <strong>and</strong> alanine scan of a<br />
spider toxin that affects the activation of<br />
mammalian voltage-gated Na + channels<br />
5.00-5.20 Karbat I.: X-ray structure <strong>and</strong> point mutagenesis<br />
of the scorpion depressant toxin LqhIT2 reveals<br />
key determinants crucial for activity <strong>and</strong> antiinsect<br />
selectivity<br />
5.20-5.40<br />
Martin-Eauclaire, M.-F. : New β-type toxins<br />
from the Androctonus scorpion venoms?<br />
Track 2: Cytotoxins, <strong>and</strong> therapeutics<br />
K3.17<br />
Cahan, R.: Cyt2Ba of Bacillus thuringiensis<br />
subsp. israelensis: activation by putative<br />
endogenous protease<br />
Turk, T. : New type of cytolysin <strong>and</strong> an unusual<br />
phospholipase A2 like protein isolated from the<br />
Northern Pacific sea anemone Urticina<br />
crassicornis<br />
Vassilevski, A.A.: Latarcins – a group of spider<br />
venom cytolytic peptides with high structural<br />
diversity<br />
Anderluh, G.: Mechanism of sphingomyelin<br />
specificity of actinoporins, pore-forming toxins<br />
from sea anemones<br />
Mbugua, P. M.: In vitro evaluation of<br />
antibacterial activity of crude aqueous extract of<br />
Azadirachta indica (Neem) leaves<br />
Saha, A. : Non-protein/non-peptide compounds<br />
from cobra venom having therapeutic potential<br />
Gawade, S. P.: Pharmaco-photodynamics of<br />
snake venoms: Perspectives to access drugs for<br />
the future<br />
EL-Latif, S.A.A.: Yeast culture Saccharomyces<br />
cerevisiae as feed additive in Poultry<br />
14<br />
Please note the Toxins <strong>and</strong> Hemostasis<br />
symposium will also run in K3.25 at the same<br />
time as these other Oral Sessions
Scientific timetable day-by-day<br />
Friday 28 July<br />
Symposium: Venomics (K3.25)<br />
Sponsored by CEA<br />
9.00-9.10: André Ménez (DIEP, CEA Saclay, France, Head of Dept of<br />
Protein Study & Engineering of the Life Sciences Division at the French<br />
Atomic Energy Commission)<br />
Introducing the Venomics Project<br />
9.10-9.15: Reto Stöcklin (Owner <strong>and</strong> Head of Atheris Laboratories)<br />
Use of mass spectroscopy in venomics<br />
9.15-9.20: Dietrich Mebs<br />
Venom gl<strong>and</strong>s in old snakes<br />
9.20-9.40: Bryan Greig Fry (Deputy director of the Australian Venom<br />
Research Unit at the University of Melbourne)<br />
Evolution of the venom system in squamate reptiles<br />
9.40-10.00: Baldomero Olivera (Distinguished Prof of Biology at the<br />
University of Utah, Associate Prof of Biochemistry at the University of<br />
the Philippines)<br />
Exogenomics <strong>and</strong> post-translational modification of Conus peptides<br />
10.00-10.20: Motonori Ohno (Professor of Biochemistry at Sojo<br />
University)<br />
Venomics of protobothrops flavoviridis venom gl<strong>and</strong> phospholipase A2<br />
isozymes<br />
10.20-10.40: Eugene Grishin (Professor <strong>and</strong> Deputy director of the<br />
Shemyakin-Ovchinnikov Institute of Bio-organic Chemistry at the<br />
Russian Academy of Sciences)<br />
Wide variety <strong>and</strong> multiplicity of spider toxins: structural aspects<br />
10.40-11.00: Manjunatha Kini (Professor, National University of<br />
Singapore)<br />
Protein scaffolds in snake venoms<br />
Dr Richard Lewis (Co-founder of the Xenome, joint appointment as<br />
Xenome's CSO & Principal investigator (molecular pharmacology) at the<br />
Institute for Molecular Bioscience (IMB) at the University of Queensl<strong>and</strong><br />
Venomics to drugs: the Xen2174 story<br />
Plenary lecture (K3.25)<br />
11.45am-12.45pm<br />
Michael Brownstein (Director of Functional Genomics, J. Craig Venter<br />
Institute, Maryl<strong>and</strong>)<br />
What can genomics teach you about toxins?<br />
Poster sessions (Colville 5.11/5.12)<br />
1.45-3.00 pm<br />
Clinical aspects<br />
Cardiovascular<br />
Toxins from novel sources<br />
Plant toxins<br />
16
Friday 28th<br />
July<br />
Track 1: Clinical aspects – Antivenoms <strong>and</strong><br />
treatments; toxins <strong>and</strong> analgesic discovery<br />
Oral Presentations K3.14<br />
3.00-3.20 Wagstaff, S.C.: Bioinformatics <strong>and</strong> multi-epitope<br />
DNA immunisation to design rational snake<br />
antivenom<br />
3.20-3.40 Laing, G.D. : Pre-clinical assessment of equine<br />
<strong>and</strong> ovine antivenoms raised against saw-scaled<br />
viper (Echis ocellatus) venom for use in Nigeria<br />
3.40-4.00 Williams, D. J.: What price, salvation? Antivenom<br />
costs <strong>and</strong> availability in Papua New Guinea 1992-<br />
2004<br />
4.00-4.20 Winkel K.D.: Developing a toxinology toolkit for an<br />
under-developed<br />
4.20-4.40 Jensen S.D.: Teaching clinical toxinology in the<br />
developing world: Setting an example in Papua<br />
New Guinea<br />
4.40-5.00 Cury, Y.: Analgesic action of Crotalphine, a novel<br />
opioid receptor agonist from the venom of the<br />
South American rattlesnake Crotalus durissus<br />
terrificus (CdtV)<br />
5.00-5.20 Chacur, M.: Analgesic effect induced by Naja<br />
kaouthia snake venom <strong>and</strong> α-cobratoxin isolated<br />
from this venom<br />
5.20-5.40 Zaharenko, A.J.: BcIV, a new paralyzing peptide<br />
from the venom of the sea anemone Bunodosoma<br />
caissarum. A comparison with the Na + channel<br />
toxin BcIII<br />
Track 2: Venomics, genomics, proteomics,<br />
transcriptomics<br />
K3.17<br />
Sher, D.: Beyond prey capture – a comparative<br />
bioinformatic approach to cnidarian allomones<br />
Moran, Y.: When Positive Selection of Neurotoxin<br />
Genes is Missing - the Riddle of the Sea Anemone<br />
Nematostella vectensis<br />
Escoubas, P.: Venom l<strong>and</strong>scapes: Towards the<br />
discovery of novel pharmacological tools via the<br />
combined use of LC-MALDI-TOF <strong>and</strong> MALDI-<br />
TOF/TOF with cDNA analysis<br />
Calvete, J.J.: Snake venomics<br />
St Pierre, L.: Identification <strong>and</strong> characterisation of<br />
toxin-specific transcripts from the venom gl<strong>and</strong>s of<br />
Australian elapid snakes<br />
Earl, S.T.: Proteomic analysis of Australian snake<br />
venoms for the discovery <strong>and</strong> development of new<br />
human therapeutics<br />
Harrison, R.A.: Molecular characterization of<br />
hyaluronidase-encoding genes from venom gl<strong>and</strong><br />
cDNA libraries of Echis, Bitis <strong>and</strong> Cerastes viper<br />
species<br />
Juárez, P.: Miniminization of protein structure <strong>and</strong><br />
gene organization along the evolution of the short<br />
disintegrin ocellatusin from a dimeric disintegrin<br />
precursor<br />
Track 3: Structural aspects of toxins<br />
K3.25<br />
Ménez, A. : The same small three-fingered fold for<br />
snake toxins <strong>and</strong> human uPAR<br />
Araki, S.: 3D-structure of VAP1, a high-molecular<br />
snake venom metalloprotease, reveals<br />
metalloproteinase/disintegrin/cysteine-rich<br />
architecture of SVMPs <strong>and</strong> ADAMs<br />
Kahn R. : Unique Molecular Formations in Crystals<br />
of Scorpion Toxins that Affect Voltage-Gated Na-<br />
Channels<br />
Ducancel, F.: The increasing molecular diversity of<br />
sarafotoxins<br />
Matsunaga, Y.: Molecular diversity of snake<br />
venom VEGFs<br />
Obayashi, S.: Isolation <strong>and</strong> characterization of a<br />
VEGF-like protein from the venom of Bitis arietans<br />
Fujisawa D.: VEGF receptor-binding potential is a<br />
common property for myotoxic phospholipase A2<br />
Utkin, Y.N.: Low Abundant Proteins in Cobra<br />
Venom
Plenary lectures <strong>and</strong><br />
symposia<br />
Speakers’ <strong>biographies</strong><br />
<strong>and</strong> <strong>abstracts</strong><br />
All plenary lectures <strong>and</strong> symposia will be<br />
held in lecture theatre K3.25 in the John<br />
Anderson Building<br />
Premier sponsors:<br />
18<br />
Willem ‘Pim’ Stemmer, Ph.D. is CEO <strong>and</strong> Founder of microPROTEINS,<br />
Inc, a biotechnology company located in Menlo Park, CA, focused on creating therapeutics that<br />
combine the best properties of small molecules (size, stability, non-immunogenicity) with the best<br />
properties of proteins (affinity, specificity, safety, design <strong>and</strong> manufacturing process,<br />
hydrophilicity, halflife). The company’s microprotein products are derived from small proteins with<br />
high disulfide density. The 3-6kD size, temperature/protease stability, hydrophilicity <strong>and</strong> E. coli<br />
expression properties of microproteins make them ideally suited for therapeutics. Products are<br />
broadly applicable across a variety of therapeutic areas <strong>and</strong> are designed to have sub-nanomolar<br />
activity, long halflife, low immunogenicity, long shelflife, low cost of goods, low royalty stack <strong>and</strong><br />
high margin, creating an important advantage over antibodies or antibody fragments.<br />
Pim previously founded Avidia Inc. in Mountain View, California. Pim invented the company’s<br />
technology in 2001 <strong>and</strong> <strong>and</strong> served as its chief scientific officer for four years. Avidia's proteins,<br />
called ‘Avimers’, bind targets multivalently with multiple domains to inhibit or activate a specific<br />
biologic function. Avidia was founded in July 2003 as a spin-out from Maxygen to focus on the<br />
commercial development of this novel protein technology.<br />
In 1997, Pim co-founded Maxygen (Nasdaq: MAXY) with Drs. Alej<strong>and</strong>ro Zaffaroni, Isaac Stein<br />
<strong>and</strong> Russell Howard to commercialize DNA Shuffling <strong>and</strong> he served as VP R&D until 2003. In<br />
1993, Pim invented Maxygen’s core technology, called DNA Shuffling or Molecular Breeding.<br />
Pim is the inventor on over 70 issued patents <strong>and</strong> over 100 pending applications. He has<br />
authored over 60 research publications <strong>and</strong> has given over 200 invited scientific lectures in many<br />
different fields. He received the David Perlman Award <strong>and</strong> the Doisy Award. He was instrumental<br />
in obtaining $25M in DARPA grants for Maxygen.<br />
From 1987 to 1992, Pim was a research scientist at Hybritech, Inc, working on antibody fragment<br />
engineering in E. coli <strong>and</strong> mammalian cells, focused on applications for cancer therapy. In 1992,<br />
he was awarded ‘scientist of the year’. Pim obtained his Ph. D. from the University of Wisconsin-<br />
Madison in 1985 on bacterial virulence mechanisms.<br />
Silverman J, Lu Q, Bakker A, To W, Duguay A, Alba BM, Smith R, Rivas A, Li P, Le H, Whitehorn<br />
E, Moore KW, Swimmer C, Perlroth V, Vogt M, Kolkman J, Stemmer WP (2005) Multivalent<br />
avimer proteins evolved by exon shuffling of a family of human receptor domains. Nat Biotechnol.<br />
23: 1556-61.<br />
Wright A, Semyonov A, Dawes G, Crameri A, Lyons R, Stemmer WP, Apt D, Punnonen J. (2005)<br />
Diverse plasmid DNA vectors by directed molecular evolution of cytomegalovirus promoters.Hum<br />
Gene Ther. 16: 881-92.<br />
Leong SR, Chang JC, Ong R, Dawes G, Stemmer WP, Punnonen J. (2003) Optimized expression<br />
<strong>and</strong> specific activity of IL-12 by directed molecular evolution. Proc Natl Acad Sci USA 100: 1163-8.<br />
Leong SR, Chang JC, Ong R, Dawes G, Stemmer WP, Punnonen J. (2003) Optimized expression<br />
<strong>and</strong> specific activity of IL-12 by directed molecular evolution. Proc Natl Acad Sci USA 100: 1163-8.<br />
19
Monday 24 July: 11.45 am-12.45 pm<br />
Lecture theatre K3.25<br />
The Elsevier-Toxicon Lecture<br />
20<br />
Sponsored by<br />
The advantages of disulfide-rich microproteins as scaffolds for the<br />
construction of antibody-mimetic pharmaceuticals<br />
Willem ‘Pim’ Stemmer<br />
Amunix Inc., Menlo Park, CA 94025, USA<br />
pstemmer@amunix.com<br />
Disulfide-rich microprotein scaffolds offer important advantages for the creation<br />
of antibody-like therapeutics. Scaffolds of special interest are those of natural<br />
injectable microproteins (ie leeches, snakes, snails, spiders, scorpions,<br />
anemones) <strong>and</strong> microproteins that are natural orally available (ie KalataB1).<br />
Whereas the folding of typical protein domains is mediated by a large<br />
hydrophobic core, in most families of microproteins this is replaced by a few<br />
disulfides. As a consequence, protein domains based on disulfide scaffolds are<br />
much smaller, less hydrophobic <strong>and</strong> allow a higher level of amino acid<br />
substitution, which allows these proteins to be evolved for tight binding to novel<br />
therapeutic targets, for use as antibody replacements. This reduction in size <strong>and</strong><br />
in hydrophobic amino acids (esp. aliphatic) greatly decreases the predicted<br />
MHC-binding affinity <strong>and</strong> the expected level of immunogenicity of these proteins.<br />
Drugs based on such scaffolds can be designed to have exceptional stability to<br />
heat <strong>and</strong> proteases <strong>and</strong> can be formulated to very high concentrations. These<br />
properties may allow products to be delivered orally or by home injection,<br />
creating an important advantage over antibodies.<br />
Dr James Rothman is currently Director of the Columbia Genome Center <strong>and</strong><br />
Professor of Chemical Biology at The College of Physicians <strong>and</strong> Surgeons at<br />
Columbia University, New York. Dr Rothman has made fundamental<br />
contributions to the underst<strong>and</strong>ing of protein transport between membranebound<br />
compartments of cells. He has developed assays that have allowed the<br />
function of the Golgi apparatus to be dissected at the biochemical level <strong>and</strong><br />
has purified several proteins that catalyze vesicular formation <strong>and</strong> fusion with<br />
target membranes. He was awarded the Albert Lasker Award for Basic<br />
Medical Research in 2002 for his work on membrane vesicles. Dr Rothman was also awarded the<br />
Dr H.P. Heineken Prize for Biochemistry <strong>and</strong> Biophysics in 2000 for clarifying the mechanism of<br />
intracellular membrane fusion.<br />
He made the historic discovery that the cell contains very small membrane-enveloped vesicles<br />
that carry a large variety of proteins between different compartments in the cytoplasm. This<br />
delivery process, which involves vesicle flow <strong>and</strong> membrane fusion, is vital for the growth <strong>and</strong><br />
division of every cell. How this process comes about was a great mystery, <strong>and</strong> one of the great<br />
unsolved questions of biochemistry <strong>and</strong> cell biology. James Rothman discovered the molecular<br />
principles of intracellular membrane fusion <strong>and</strong> demonstrated that the specificity of fusion was<br />
dictated by the pairing of SNARE proteins between membranes. This historic discovery provided<br />
a single unified principle for underst<strong>and</strong>ing important physiological processes, including the<br />
release of insulin into the blood, communication between nerve cells in the brain <strong>and</strong> the entry of<br />
viruses like HIV (the AIDS virus) to infect cells. Defects in the control of these pathways are<br />
important in diabetes <strong>and</strong> most likely also in certain cancers. Currently a major effort is under way<br />
to develop a new generation of drugs to control AIDS by blocking the membrane fusion process.<br />
James Rothman was born in Haverhill, Massachusetts, USA. He has a Ph.D. in Biological<br />
Chemistry (Harvard Medical School), <strong>and</strong> he worked at the Sloan-Kettering Institute in New<br />
York from 1991 until 2003 when he moved to Columbia University College of Physicians <strong>and</strong><br />
Surgeons to establish a new Center for Chemical Biology. Amongst his other honours, Dr<br />
Rothman has received the Gairdner Foundation International Award (1996), the King Faisal<br />
International Prize in Science (1996) <strong>and</strong> the Lounsbery Award of the National Academy of<br />
Sciences (1997).<br />
Cosson P, Ravazzola M, Varlamov O, Sollner TH, Di Liberto M, Volchuk A, Rothman JE, Orci<br />
L. (2005) Dynamic transport of SNARE proteins in the Golgi apparatus. Proc Natl Acad Sci<br />
USA 102:14647-52.<br />
Fix M, Melia TJ, Jaiswal JK, Rappoport JZ, You D, Sollner TH, Rothman JE, Simon SM. (2004)<br />
Imaging single membrane fusion events mediated by SNARE proteins. Proc Natl Acad Sci<br />
USA 101:7311-6.<br />
Varlamov O, Volchuk A, Rahimian V, Doege CA, Paumet F, Eng WS, Arango N, Parlati F,<br />
Ravazzola M, Orci L, Sollner TH, Rothman JE. (2004) i-SNAREs: inhibitory SNAREs that finetune<br />
the specificity of membrane fusion. J Cell Biol. 164:79-88.<br />
Hu C, Ahmed M, Melia TJ, Sollner TH, Mayer T, Rothman JE. (2003) Fusion of cells by flipped<br />
SNAREs. Science 300:1745-9.<br />
21
Tuesday 25 July: 11.45 am-12.45 pm<br />
Lecture theatre K3.25<br />
James E. Rothman (Director of the Columbia Genome Center, Professor of<br />
Chemical Biology, Columbia University, New York)<br />
Membrane fusion in the cell<br />
A vast array of physiological processes – passing of information between nerves in the<br />
brain, control of blood sugar by insulin, fertilization of the egg, to name just a few –<br />
require the fusion of membranes. Intracellular membrane fusion results when the<br />
cytoplasmic domains of matching SNARE proteins link-up between membranes to form<br />
a helical bundle termed a SNAREpin. Since only designated SNAREs can link up, fusion<br />
is highly specific. All nucleated cells – whether from yeast, animals, plants, or man –<br />
have families of related SNARE proteins that fall into two overall categories: v-SNAREs<br />
that primarily mark transport vesicles <strong>and</strong> t-SNAREs that primarily mark target<br />
membranes; matching pairs are distributed among membranes in a pattern that reflects<br />
the pattern of membrane flow among them. Proteolytic neurotoxins, such as botulinum<br />
toxins, specifically target SNARE proteins mediating exocytosis. Additional machinery<br />
exists to regulate the activity of SNARE proteins, allowing local or global control of the<br />
rate <strong>and</strong> location of vesicle flow in the cell.<br />
In particular, neuronal synapses influx of calcium ions stimulates the release of<br />
neurotransmitter. However the mechanism by which synaptic vesicle fusion is coupled to<br />
calcium has been unclear, despite the identification of both the core fusion machinery<br />
(SNAREs) <strong>and</strong> the principal calcium sensor (synaptotagmin). We have recently<br />
established what may represent a basic principle of the coupling mechanism: a reversible<br />
clamping protein (complexin) that can freeze the SNAREpin, an assembled fusioncompetent<br />
intermediate en route to fusion. When calcium binds to the calcium sensor<br />
synaptotagmin, the clamp would then be released. SNARE proteins, <strong>and</strong> key regulators<br />
like synaptotagmin <strong>and</strong> complexin, can be ectopically expressed on the cell surface. Cells<br />
expressing such “flipped” synaptic SNAREs fuse constitutively, but when we coexpressed<br />
complexin fusion was blocked. Adding back calcium triggered fusion from<br />
this intermediate in the presence of synaptotagmin.<br />
22<br />
Redi Award lecture<br />
The International Society on Toxinology confers the Redi Award every three<br />
years in recognition of distinguished work in the field of toxinology. The recipient<br />
of the 2006 Redi Award will be announced at the Congress <strong>and</strong> will deliver a<br />
plenary lecture.<br />
Previous recipients:<br />
Findlay E. Russell 1967<br />
Paul Boquet 1970<br />
Andre de Vries 1974<br />
Chen-Yuan Lee 1976<br />
Hugh Alistair Reid 1979<br />
Nobuo Tamiya 1982<br />
Philip Rosenberg 1982<br />
Sherman A. Minton 1985<br />
Paul A. Christensen 1988<br />
Ernst Habermann 1991<br />
Elazar Kochva 1994<br />
Evert Karlsson 1997<br />
Alan L. Harvey 2000<br />
André Ménez 2000<br />
Baldomero M. Olivera 2003<br />
23
Wednesday 26 July: 11.45 am-12.45 pm<br />
Lecture theatre K3.25<br />
Redi Award lecture - to be announced at the Congress<br />
Sponsored by International Society on Toxinology<br />
24<br />
Dr. John Daly was born <strong>and</strong> raised in Portl<strong>and</strong>, Oregon. He received in<br />
1954 a bachelors degree in Biochemistry <strong>and</strong> in 1955 a masters degree in Organic Chemistry at<br />
Oregon State College <strong>and</strong> then a Ph.D. in Organic Chemistry at Stanford University in 1958.<br />
After a two year postdoctorate in the Laboratory of Chemistry at NIH, he became a permanent<br />
member of the staff in 1960, a section chief in 1969 <strong>and</strong> the founding chief of the Laboratory of<br />
Bioorganic Chemistry in 1981. He became a NIH Scientist Emeritus in January 2003. During his<br />
five decades at NIH, his research has spanned many disciplines, being involved in isolation,<br />
structure elucidation <strong>and</strong> synthesis of novel natural products <strong>and</strong> receptor agonists/ antagonists<br />
<strong>and</strong> in the elucidation of their mechanism of action with a focus on receptors, ion channels, <strong>and</strong><br />
second messenger formation in the nervous system. High points in his career include the<br />
discovery of the NIH Shift, the introduction of a prelabeling technique for investigation of cyclic<br />
AMP generation in intact cells, the introduction of a variety of selective agonists <strong>and</strong> antagonists<br />
as probes <strong>and</strong> radiolig<strong>and</strong>s for adenosine receptors, the delineation of biological targets for<br />
caffeine <strong>and</strong> other xanthines in the central nervous system, the discovery of the direct activation<br />
of adenylyl cyclase by forskolin, the discovery of the activation of phosphoinositide breakdown by<br />
maitotoxin, <strong>and</strong> the discovery that loperamide is a unique modulator of a capacitative calcium<br />
influx pathway <strong>and</strong> that N-substituted dihydropyridines are selective blockers of such channels.<br />
Over four decades, he has been involved both in field collection <strong>and</strong> as a chemist <strong>and</strong><br />
pharmacologist in the discovery <strong>and</strong> structure elucidation of a wide range of over 800 alkaloids of<br />
twenty some structural classes in amphibian skin <strong>and</strong> elucidation of the basis for their biological<br />
activity. Such compounds include batrachotoxins, histrionicotoxins, pumiliotoxins, <strong>and</strong><br />
epibatidine. Certain of these alkaloids are now widely used as research tools; batrachotoxin as a<br />
selective activator of sodium channels, histrionicotoxins as blockers of nicotinic channels,<br />
pumiliotoxins as sodium channel agents with biomedical potential as cardiotonic agents <strong>and</strong><br />
epibatidine as nicotinic agonist with potent analgetic activity. Analogues of epibatidine are the<br />
subject of clinical trials for a treatment of chronic pain of neuropathies, cancer <strong>and</strong> arthritis. Many<br />
of the alkaloids discovered by Dr. Daly have been the target of synthetic research in laboratories<br />
world-wide. Dr. Daly’s research presently focuses on the structure <strong>and</strong> biological activity of<br />
further novel alkaloids found in amphibian skin <strong>and</strong> on the dietary origin of such alkaloids. Dr.<br />
Daly has been the recipient of other awards in recognition of his achievements. He was elected<br />
to the National Academy of Sciences, USA in 1997. Besides his abiding interest in field work <strong>and</strong><br />
research, Dr. Daly is an avid fisherman.<br />
1. Seamon, K.B., <strong>and</strong> Daly, J.W.: Forskolin: A unique diterpene activator of cyclic AMP-generating systems. J. Cyclic<br />
Nucleotide Res. 7: 201-224, 1982.<br />
2. Badio, B., <strong>and</strong> Daly, J.W. Epibatidine, a potent analgetic <strong>and</strong> nicotinic agonist. Mol Pharmacol. 45: 563-569, 1994.<br />
3. Daly, J.W., Kaneko, T., Wilham, J., Garraffo, H.M., Sp<strong>and</strong>e, T.F., A. Espinosa <strong>and</strong> Donnelly, M.A. Bioactive alkaloids<br />
of frog skin: Combinatorial bioprospecting reveals that pumiliotoxins have an arthropod source. Proc. Natl. Acad. Sci.<br />
U.S.A. 99: 13996-14001, 2002.<br />
4. Daly, J.W. Ernest Guenther Award in Chemistry of Natural Products. Amphibian skin: A remarkable source of<br />
biologically active arthropod alkaloids. J. Med. Chem. 46: 445-452, 2003.<br />
5. Fitch, R.W., Garraffo, H.M., Sp<strong>and</strong>e, T.F., Yeh, H.J.C. <strong>and</strong> Daly, J.W. Bioassay-guided isolation of epiquinamide, a<br />
novel quinolizidine alkaloid <strong>and</strong> nicotinic agonist from an Ecuadoran poison frog, Epipedobates tricolor. J. Nat. Prod. 66:<br />
1345-1350, 2003.<br />
6. Dumbacher, J.P., Wako, A., Derrickson, S.R., Samuelson, A., Sp<strong>and</strong>e, T.F., <strong>and</strong> Daly, J.W. Melyrid beetles<br />
(Choresine): A putative source for the batrachotoxin alkaloids found in poison-dart frogs <strong>and</strong> toxic passerine birds. Proc.<br />
Natl. Acad. Sci. U.S.A. 101: 15857-15860, 2004.<br />
7. Daly, J.W., Sp<strong>and</strong>e, T.F., <strong>and</strong> Garraffo, H.M. Alkaloids from amphibian skin: A tabulation of over eight-hundred<br />
compounds. J. Nat. Prod. 68: 1556-1575, 2005.<br />
25
Thursday 27 July: 11.45 am-12.45 pm<br />
Lecture theatre K3.25<br />
Alkaloids as Probes for Ionic Channels:<br />
John William Daly.<br />
Laboratory of Bioorganic Chemistry, NIDDK, NIH, Bethesda, MD, USA<br />
Alkaloids represent an impressive source of probes for the study of ion<br />
channels in nerve, muscle <strong>and</strong> other tissues. Although most alkaloids have been<br />
derived from plant sources, over 800 alkaloids have been identified from amphibian<br />
skin 1 . These include 1) the sam<strong>and</strong>arines that block sodium channels, 2) the<br />
batrachotoxins that activate sodium channels, 3) the pumiliotoxins, allopumiliotoxins<br />
<strong>and</strong> homopumiliotoxins that are positive modulators of sodium channels <strong>and</strong> thereby<br />
myotonic <strong>and</strong> cardiotonic agents, 4) the histrionicotoxins <strong>and</strong> decahydroquinolines,<br />
izidines, tricyclics, pyrrolidines, piperidines, spiropyrrolizidine oximes <strong>and</strong><br />
pseudophrynamines that are noncompetitive blockers of nicotinic receptor-channels,<br />
5) the epibatidines that are potent agonists at nicotinic receptors <strong>and</strong> thereby powerful<br />
analgetics. From the plant kingdom many alkaloids have provided ion channel<br />
probes 2,3 . These include 1) nicotine, cytisine, anatoxin, ferruginine, anabaseine <strong>and</strong><br />
lobeline as nicotinic agonists, 2) tubocurarine, erythryodine, methyllyaconitine <strong>and</strong><br />
ibogaine as blockers <strong>and</strong> galanthamine as a positive modulator of nicotinic receptor<br />
channels, 3) veratridine <strong>and</strong> aconitine as activators of sodium channels <strong>and</strong> quinine<br />
<strong>and</strong> cocaine as blockers, 4) strychine as an antagonist of glycine receptors, 5)<br />
bicuculline as an antagonist of GABA receptors, 6) ryanodine <strong>and</strong> caffeine as<br />
modulators of calcium-activated calcium channels of the endoplasmic <strong>and</strong><br />
sarcoplasmic reticulum. Natural products continue to be an incredibly rich source of<br />
bioactive compounds with major impact on biomedical research.<br />
1) Daly, J.W., Sp<strong>and</strong>e, T.F. <strong>and</strong> Garraffo, H.M. J. Nat. Prod. 68: 1556-1575, 2005<br />
2) Daly, J.W. Cell. Mol. Neurobiol. 25: 513-552, 2005.<br />
3) Wink, M. Studies in Natural Product Chemistry. 21:3-121, 2000.<br />
o Alkaloids<br />
o Ion Channels<br />
o Nicotinic receptors<br />
o Epibatidine<br />
26<br />
Dr Brownstein earned his A.B. from the Columbia College <strong>and</strong> his M.D.<br />
<strong>and</strong> Ph.D. degrees at the University of Chicago. After completing an internship at the Boston<br />
Children’s Hospital, he went to the NIH in 1972 as a Pharmacology Research Associate to work<br />
with Julie Axelrod. Initially he studied the pineal gl<strong>and</strong>, but he began to develop more sensitive<br />
techniques for measuring neurotransmitters <strong>and</strong> their biosynthetic enzymes. He met Miklos<br />
Palkovits, who had just devised a novel brain microdissection method, in 1973, <strong>and</strong> together they<br />
mapped many “classical” transmitters <strong>and</strong> neuropeptides in the central nervous system. Dr.<br />
Brownstein was the first to point out that neurons make <strong>and</strong> likely release more than one<br />
chemical messenger, <strong>and</strong> the first to show that “hypothalamic hormones” must have much<br />
broader roles in the brain <strong>and</strong> periphery.<br />
He <strong>and</strong> Dr. Harold Gainer used pulse-chase studies in vivo to show that vasopressin <strong>and</strong><br />
oxytocin are synthesized as parts of larger precursor proteins, that these precursors are<br />
processed by proteases in vesicles while they are being transported from the cell body to its<br />
axon terminals, <strong>and</strong> that precursor synthesis <strong>and</strong> processing are both regulated by dem<strong>and</strong>.<br />
Dr. Brownstein <strong>and</strong> Hiroto Okayama developed robust protocols for making cDNA libraries <strong>and</strong><br />
expressing the inserts in mammalian cells. Dr. Brownstein’s goal at the time was to use such<br />
methods for expression cloning of vasopressin <strong>and</strong> oxytocin receptors—the next essential step<br />
in learning about the biology of these peptide hormones. He <strong>and</strong> his coworkers ultimately<br />
achieved these goals, <strong>and</strong> they cloned a number of additional important cDNAs. Some of<br />
these have proven especially important to members of the mental health community: e.g.,<br />
cDNAs that encode the serotonin transporter (the target of SSRIs), the dopamine transporter<br />
(the target of cocaine), the cannabinoid receptor (the target of a novel <strong>and</strong> very promising drug<br />
for treating obesity <strong>and</strong> addictive behavior), <strong>and</strong> the vasopressin V1b receptor (an attractive<br />
target for development of antidepressant compounds).<br />
One of the vasopressin receptors that Dr. Brownstein’s group cloned was the V2 (kidney)<br />
subtype. He understood that mutations in this receptor could very well be responsible for Xlinked<br />
nephrogenic diabetes insipidus, <strong>and</strong> rapidly demonstrated that this was indeed the<br />
case. This was his reintroduction to human genetics, <strong>and</strong> he decided to learn more about<br />
genetic analyses by working in the NHGRI. He was among the first people there to do highthroughput,<br />
fluorescence-based genotyping, <strong>and</strong> he developed a method for modifying the<br />
primers used to study microsatellite markers (“Pig tailing”) that was subsequently licensed by<br />
Applied Biosystems <strong>and</strong> applied to all of the primer pairs that they manufactured. To date tens<br />
of millions of genotypes have been done with such markers.<br />
In his final years at the NIH, Dr. Brownstein’s focus was complex traits genetics <strong>and</strong><br />
genomics. He <strong>and</strong> the members of his team defined causative mutations in a number of<br />
genetic diseases, ranging from prostate cancer to cataracts, <strong>and</strong> continued to develop<br />
important methods, notably ones for labeling probes for microarray studies.<br />
In 2005, Dr. Brownstein retired from the civil service, <strong>and</strong> accepted a position at the J. Craig<br />
Venter Institute, Rockville, MD, where he is now director of functional genomics. He has also<br />
served on numerous editorial boards, scientific advisory boards, <strong>and</strong> review committees, <strong>and</strong><br />
has received a number of awards, most recently, an honorary Doctorate from the University of<br />
Lund in Sweden.<br />
27
Friday 28 July: 11.45 am-12.45 pm<br />
Lecture theatre K3.25<br />
Michael J. Brownstein, MD, PhD<br />
Director of Functional Genomics, J. Craig Venter Institute, Rockville,<br />
Maryl<strong>and</strong> 20850, USA<br />
What can genomics teach you about toxins?<br />
There are still many partially answered or unanswered questions about the constituents of<br />
venoms <strong>and</strong> the specialized tissues that make, store, <strong>and</strong> deliver them. These include the<br />
following: How are the genes that encode toxins organized? How many such genes are<br />
there? How are the diverse toxins distributed among these genes? Are all of toxinencoding<br />
sequences expressed or is there a large reservoir of unused sequences to draw<br />
on? Are toxin genes distributed across the entire genome or located at one or more<br />
specific loci? In the future, we hope to learn more about the complexity of certain toxin<br />
families--e.g., the conopeptides—<strong>and</strong> whether mechanisms resembling those responsible<br />
for antibody diversity (including recombination <strong>and</strong> hypermutability) could act during the<br />
life of an organism to increase its toxin repertoire. To answer these questions, we will<br />
need to characterize the genes, mRNAs, <strong>and</strong> protein products of one or more venomous<br />
species, <strong>and</strong> subsequently to look at transcripts <strong>and</strong> peptides in several organs of multiple<br />
individuals. Until recently, such work was time consuming, labor intensive, <strong>and</strong><br />
enormously expensive. New methods are reducing costs, increasing speed, <strong>and</strong><br />
improving the quality of the results obtained. I will review these methods for you <strong>and</strong> tell<br />
you about advances that are likely to occur in the next few years.<br />
Some recent publications<br />
AHN, JI, et al. Comprehensive transcriptome analysis of differentiation of embryonic stem cells into midbrain <strong>and</strong> hindbrain neurons.<br />
DEV. BIOL. 265: 491-501, 2004.<br />
MUTSUGA, N. et al Selective gene expression in magnocellular neurons in rat supraoptic nucleus. J. NEUROSCIENCE 24: 7174-<br />
85, 2004.<br />
ARROZTOA, JA et al. Identification of genes expressed in primary primordial oocytes. HUMAN REPRODUCTION 20: 476-483,<br />
2005. Epub 2004 Dec 2.<br />
MUTSUGA, N. et al Regulation of gene expression in magnocellular neurons in rat supraoptic nucleus during sustained<br />
hypoosmolality. ENDOCRINOLOGY 146: 1254-1267, 2005. Epub 2004 Dec 9.<br />
XIANG, CC, et al. Using DSP, a reversible cross-linker, to fix tissue sections for immunostaining, microdissection <strong>and</strong> expression<br />
profiling. NUCLEIC ACIDS RESEARCH 32: e185, 2004.<br />
KUO, J, et al. Evaluation of vector-primed cDNA library production from microgram quantities of total RNA. NUCLEIC ACIDS<br />
RESEARCH 32: e183, 2004.<br />
ALTUVIA, Y, LANDGRAF, P., LITHWICK, G. ELFANT, N. PFEFFER, S. ARAVIN, A., BROWNSTEIN, M. J. TUSCHL, T.,<br />
AND MARGALIT, H. Clustering <strong>and</strong> conservation patterns of human microRNAs. NUCLEIC ACIDS RESEARCH 33: 2697-2706,<br />
2005.<br />
SEWER, A., PAUL, N. LANDGRAF, P. ARAVIN, A. PFEFFER, S. BROWNSTEIN, M.J., TUSCHL, T., VAN NIMWEGEN, E.,<br />
AND ZAVOLAN, M. Identification of clustered microRNAs using an ab initio prediction method. BMC BIOINFORMATICS. 6:<br />
267, 2005.<br />
28<br />
George Miljanich has been developing venom peptide therapeutics, ion channel therapeutics,<br />
<strong>and</strong> analgesics for more than 20 years. He is author or co-author of over 50 scientific<br />
publications, <strong>and</strong> is co-inventor on 15 issued U.S. patents <strong>and</strong> numerous corresponding non-U.S.<br />
patents. Dr. Miljanich is currently Chief Executive Officer of the U.S. biotechnology company,<br />
AIRMID LLC, where he leads the effort to discover, develop, <strong>and</strong> commercialize highly potent <strong>and</strong><br />
selective potassium channel blockers - including venom peptide toxin <strong>and</strong> plant toxin derivatives -<br />
to treat a variety of autoimmune disorders. Prior to joining AIRMID, he was Senior Medical<br />
Director at Elan Pharmaceuticals, where he played a key role in progressing the novel analgesic,<br />
PRIALT ® , through the final stages of development, toward FDA <strong>and</strong> EMEA approval, <strong>and</strong> to the<br />
analgesia market.<br />
Dr. Miljanich received his BS degree in Chemistry from the University of California, Berkeley, <strong>and</strong><br />
his PhD in Chemistry from U.C. Santa Cruz. He then conducted post-doctoral research at U.C.<br />
San Francisco, studying presynaptic function. As a faculty member at the University of Southern<br />
California, he continued these investigations, including studying the biochemistry <strong>and</strong><br />
pharmacology of calcium channels, thus becoming acquainted with the research applications of<br />
calcium channel-blocking toxins from cone snails. Dr. Miljanich joined the Neurex Corporation to<br />
initiate the translation of these research tools into pharmaceuticals. He contributed to the<br />
development of the conopeptide, ziconotide (PRIALT ® ), through preclinical studies to Phase III<br />
clinical trials, while also leading several drug discovery projects targeted at a variety of<br />
presynaptic proteins. With the acquisition of Neurex by Elan, Dr. Miljanich became head of<br />
analgesia research at Elan <strong>and</strong> oversaw several research projects aimed at developing novel<br />
therapeutics for pain.<br />
In addition to heading AIRMID, he is an advisor to several companies in the pharmaceutical<br />
industry. Dr Miljanich may be contacted at gmiljanich@comcast.com.<br />
Some relevant publications:<br />
Miljanich, G.P., Ziconotide: Neuronal Calcium Blocker for Treating Severe Chronic Pain. Current<br />
Medicinal Chemistry 11, 3129-3140 (2004)<br />
Newcomb R. <strong>and</strong> Miljanich G. “Neurotoxins of Cone Snail Venoms.” In: H<strong>and</strong>book of<br />
Neurotoxicology, 2002, ed., Massaro, EJ. (Humana Press, Totowa, New Jersey) pp. 617-651<br />
Newcomb, R., et al., Diversity of neuronal R-type calcium currents revealed by a selective peptide<br />
antagonist for the class E calcium channel from the venom of the tarantula, Hysterocrates gigas.<br />
Biochemistry, 37, 15353-15362 (1998)<br />
See also: “A Toxin Against Pain” by Gary Stix in Scientific American, April 2005 pp. 88-93.<br />
29
Symposia<br />
Monday 24 July: 9.00 am- 11.15 am<br />
Lecture theatre K3.25<br />
Symposium: Toxins <strong>and</strong> drug discovery<br />
9.00-9.45: George Miljanich (CEO, AIRMID LLC, California):<br />
PRIALT® (ziconotide intrathecal infusion): A Conopeptide for Treating Severe<br />
Chronic Pain<br />
Airmid LLC, 600 Castle Hill Road, Redwood City CA USA 94061, gmiljanich@comcast.net<br />
The analgesic, PRIALT ® (ziconotide), is the first conopeptide to receive approval for<br />
marketing in the US <strong>and</strong> EU. Ziconotide is a non-opioid treatment for severe chronic pain<br />
in patients warranting intrathecal (intraspinal) therapy <strong>and</strong> who are intolerant or refractory<br />
to other treatment, such as adjunctive therapy, systemic analgesics, or intrathecal opiates.<br />
Ziconotide is the synthetic equivalent of omega-conotoxin MVIIA originally found in<br />
Conus magus venom <strong>and</strong> is the first in a new class of therapeutics: N-type calcium channel<br />
blockers. Approval was based in part on: 1) safety data from ~1250 pain patients <strong>and</strong> 2)<br />
analgesic efficacy demonstrated in a placebo-controlled, double-blind study of 220 patients<br />
suffering severe chronic pain. This study showed ziconotide is analgesic in patients who<br />
failed to obtain adequate pain relief from oral <strong>and</strong>/or intrathecal opiate therapy, as well as<br />
in patients taking oral opiates concomitantly with ziconotide. Ziconotide does not elicit<br />
respiratory depression, analgesic tolerance, or withdrawal syndrome upon discontinuation,<br />
nor does it pose a risk of drug dependency. Adverse effects attributed to ziconotide<br />
(incidence >10%; % affected ziconotide patients minus % affected placebo patients)<br />
include dizziness, ataxia, abnormal gait, confusion, memory impairment, nausea, <strong>and</strong><br />
fatigue. Psychiatric symptoms may also occur with ziconotide treatment. Ziconotide’s<br />
clinical profile is consistent with its unique mechanism of action as determined by<br />
extensive non-clinical studies which show ziconotide diminishes pain behaviors by<br />
blocking N-channels on sensory nerves, thereby inhibiting transmission of noxious signals<br />
to the brain. Ziconotide’s non-clinical pharmacology <strong>and</strong> therapeutic profile demonstrate<br />
that 1) N-channels play a key role in pain perception, 2) N-channels are a valid target for<br />
analgesic therapy, <strong>and</strong> 3) ziconotide is a non-opioid alternative for treating severe chronic<br />
pain.<br />
Reference: Ziconotide: neuronal calcium channel blocker for treating severe chronic pain.<br />
Miljanich GP, Curr Med Chem. 23, 29-40 (2004).<br />
o PRIALT<br />
o ziconotide<br />
o conopeptide<br />
o calcium channel blocker<br />
30<br />
Dr. Michael Hanley is Vice President of Discovery Research at Amylin Pharmaceuticals, which<br />
launched two first-in-class diabetes therapeutics in 2005. Prior to joining Amylin, Dr. Hanley held<br />
tenured faculty positions at the Department of Biochemistry,Imperial College, London, the<br />
Medical Research Council Laboratories, Cambridge, <strong>and</strong> the University of California at Davis,<br />
where he was Professor of Biological Chemistry. He has over 160 papers covering fields from<br />
signal transduction to molecular neurobiology to peptidomics, <strong>and</strong> been an editor or advisor to<br />
over a dozen journals. Dr. Hanley has served on advisory or review panels for the National<br />
Institutes of Health, the Medical Research Council <strong>and</strong> Wellcome Trust of Great Britain, <strong>and</strong> for<br />
the governments of Australia, Singapore, New Zeal<strong>and</strong>, Hong Kong, Denmark <strong>and</strong> Japan. From<br />
1997 to 2003, Dr. Hanley was a senior consultant for healthcare investors in the venture capital<br />
<strong>and</strong> banking communities <strong>and</strong> for biotechnology companies such as Cell Therapeutics,<br />
Zymogenetics, Elan Pharmaceuticals, <strong>and</strong> Chiron Corporation. Dr. Hanley has also set-up <strong>and</strong><br />
directed research programs in privately-held start-ups, such as Chemocentryx, PsychoGenics,<br />
<strong>and</strong> most recently Harvard-based Resolvyx Pharmaceuticals. He received his B.S. in<br />
Biochemistry <strong>and</strong> his Ph.D. in Molecular Biology from the University of California, Berkeley.<br />
31
9.45-10.30: Physiological fluids of venomous animals: novel resources for drug<br />
discovery<br />
Hanley, M.R.<br />
Amylin Pharmaceuticals, Inc, 9360 Towne Centre Drive, San Diego CA, 92121 USA,<br />
michael.hanley@amylin.com<br />
Venom gl<strong>and</strong>s constitute precedented resources of chemically diverse natural products<br />
with therapeutic potential..However, venomous <strong>and</strong> poisonous animals have also coevolved<br />
specialisation of other secretory gl<strong>and</strong>s to provide novel variants of neural <strong>and</strong><br />
circulating.factors. Thus, looking beyond the constituents of venom itself, venomous<br />
organisms provide rich opportunities for discovery from physiological fluids; particularly<br />
saliva <strong>and</strong> blood.<br />
The point is ideally illustrated by glucagon-like peptide-1 (GLP-1). In mammals, GLP-1 is<br />
an incretin - broadly defined as a glucose-dependent insulinotropic hormone - which<br />
regulates blood glucose in a self-limiting manner. Incretins are therefore highly attractive<br />
targets for drugs to treat Type II diabetes. As an example of species-specific adaptation, the<br />
gila monster (Heloderma suspectum) expresses a both a GLP-1 as well as a second GLP-1<br />
variant, exendin-4 (Ex-4). Ex-4 is produced <strong>and</strong> secreted by the salivary gl<strong>and</strong>s, as well as<br />
other tissues. Exendin-4 (“exenatide”) turns out to have a superior pharmaceutical<br />
chemistry compared to natural GLP-1orthologues or synthetic analogues.<br />
The peptide has progressed from concept to clinic to commercialisation., <strong>and</strong> thus<br />
provides many lessons on drug development of naturally-occurring peptides; including<br />
solutions for immunogenicity, optimal solubility, low aggregation potential, patientfriendly<br />
drug delivery, beneficial pharmacokinetics, enhanced chemical stability, <strong>and</strong><br />
reverse engineering of favourable drug properties. These lessons will be discussed in terms<br />
of their general applicability to peptide therapeutics;<br />
Going forward, we are extending the approach of finding pharmaceutically-enhanced<br />
natural variants of our pipeline, using comparative genomic <strong>and</strong> experimental peptide<br />
discovery platforms. The results have been combined to build proprietary computational<br />
tools <strong>and</strong> libraries of novel peptide <strong>and</strong> protein drug c<strong>and</strong>idates.<br />
o GLP-1<br />
o Salivary gl<strong>and</strong><br />
o Diabetes<br />
o Drug development<br />
32<br />
Professor George Ch<strong>and</strong>y completed his MBBS at the Christian Medical College, Vellore, India,<br />
<strong>and</strong> his PhD in Immunology at the University of Birmingham, UK. He joined the University of<br />
California Irvine in 1983 as a postdoctoral researcher <strong>and</strong> is currently Professor of Physiology<br />
<strong>and</strong> Biophysics at that university.<br />
Dr. Ch<strong>and</strong>y’s group focuses on potassium channels – structure, function, functional role <strong>and</strong><br />
therapeutic targets. Dr. Ch<strong>and</strong>y proposed the nomenclature for potassium channels that is widely<br />
accepted internationally. Dr. Ch<strong>and</strong>y <strong>and</strong> his colleagues were the first to discover potassium<br />
channels in immune cells in the early 1980s, <strong>and</strong> they cloned the genes encoding these<br />
potassium channels in the 1990s. Recent studies by Dr. Ch<strong>and</strong>y’s group have identified the Kv1.3<br />
potassium channel as a potentially important therapeutic target for diverse autoimmune diseases.<br />
They have developed a highly selective <strong>and</strong> potent inhibitor of the Kv1.3 channel by modifying the<br />
ShK peptide derived from the Cuban sea anemone, Stichodactyla helianthus. Kv1.3 blockers<br />
ameliorate disease in animal models of multiple sclerosis, rheumatoid arthritis <strong>and</strong> type-1<br />
diabetes. Studies are underway to develop Kv1.3 blockers for human autoimmune diseases.<br />
Dr Ch<strong>and</strong>y has published more than 100 research papers <strong>and</strong> he is a named inventor on 17<br />
patents or patent applications.<br />
Wulff, H., Beeton, C., Ch<strong>and</strong>y, K. G. (2003) Potassium channels as therapeutic targets for autoimmune<br />
disorders. Current Opinions in Drug Discovery <strong>and</strong> Development 6, 640.<br />
Shakkottai , V.G, Chou, C-h, Oddo, S, Sailer, C.A., Knaus, H-G, Gutman, G.A., Barish, M.E, LaFerla, F.M.,<br />
Ch<strong>and</strong>y, K.G. (2004). Enhanced neuronal excitability in the absence of neurodegeneration induces<br />
cerebellar ataxia. J. Clinical Investigation 113, 582.<br />
Villalobos C, Shakkottai VG, Ch<strong>and</strong>y KG, Michelhaugh, S. K., Andrade R. (2004) SKCa channels mediate the<br />
medium but not the slow calcium-activated afterhyperpolarization in cortical neurons. J.<br />
Neuroscience 24, 3537.<br />
Ch<strong>and</strong>y, K.G., Wulff, H., Beeton, C., Pennington, M., Gutman, G. A., Cahalan, M. D. (2004). Potassium<br />
channels as targets for specific immunomodulation. Trends in Pharmacological Sciences 25, 280.<br />
Kolski-Andreaco, A. A., Tomita, H., Shakkottai, V.G., Gutman, G. A., Cahalan, M. D., Gargus, J. J., Ch<strong>and</strong>y,<br />
K. G. (2004) SK3-1C: a dominant-negative suppressor of SKCa <strong>and</strong> IKCa channels. J. Biol. Chem.<br />
279, 6893.<br />
Vennekamp, J., Wulff, H., Beeton, C., Calabresi, P. A., Grissmer, S., Hansel, W., Ch<strong>and</strong>y, K. G. (2004)<br />
Kv1.3 Blocking 5-Phenylalkoxypsoralens: A new Class of Immunomodulators Molecular<br />
Pharmacology 65, 1364.<br />
Wulff, H., Knaus, H-G, Pennington, M., Ch<strong>and</strong>y, K. G. (2004) K + channel expression during B-cell<br />
differentiation: implications for immunomodulation <strong>and</strong> autoimmune disorders. J. Immunol. 173,<br />
776.<br />
Beeton, C., Pennington, M. W., Singh, S., Nugent, D., Crossley, G., Khaytin, I., Calabresi, P. A., Ch<strong>and</strong>y, K.<br />
G. (2005) Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for<br />
therapy of autoimmune diseases. Molecular Pharmacology 67, 1369.<br />
Beeton, C., Ch<strong>and</strong>y, K. G. (2005) Potassium channels, memory cells <strong>and</strong> multiple sclerosis. Neuroscientist<br />
11, 550.<br />
Rus, H, Pardo CA, Hu L, Darrah E, Cudrici C, Niculescu T, Niculescu F, Mullen KM,, Allie R, Guo L, Wulff H,<br />
Beeton C, Judge SIV, Kerr DA, Knaus H-G, Ch<strong>and</strong>y KG, <strong>and</strong> Calabresi PA (2005). The voltagegated<br />
potassium channel Kv1.3 is highly expressed on inflammatory infiltrates in multiple sclerosis<br />
brain. Proc. Natl. Acad. Sci. USA 102, 11094.<br />
33
10.30-11.15: From marine toxins to therapy for autoimmune diseases<br />
K. George Ch<strong>and</strong>y, Christine Beeton, Michael Pennington, Heike Wulff, Alex<strong>and</strong>ra<br />
Grino, GA Gutman<br />
University of California Irvine <strong>and</strong> Bachem Bioscience Inc.<br />
T-cell mediated autoimmune diseases afflict millions of people globally.<br />
Disease-modifying immunotherapies have improved the management of these diseases,<br />
but each of these therapies is known to induce specific side effects. Consequently, there is<br />
an unmet medical need for novel immunomodulators with different mechanisms of action<br />
<strong>and</strong>/or adverse-effect profiles from existing drugs. We show that the disease-associated<br />
autoreactive T cells from patients with multiple sclerosis, type-1 diabetes mellitus or<br />
rheumatoid arthritis are effector memory T lymphocytes, <strong>and</strong> therapies that selectively<br />
suppress these cells without affecting other lymphoid subsets would have immense value.<br />
Kv1.3, one of 76 human K + -channel genes, regulates membrane potential <strong>and</strong> calcium<br />
signaling in human effector memory T cells. We have developed a peptide analog of the<br />
ShK toxin from the sea anemone Stichodactyla helianthus called ShK(L5)-amide, which<br />
blocks Kv1.3 at low picomolar concentrations <strong>and</strong> exhibits greater than 100-fold<br />
selectivity for Kv1.3 over other channels. ShK(L5)-amide suppresses calcium signaling,<br />
cytokine production <strong>and</strong> proliferation of autoantigen-specific effector memory T cells at<br />
picomolar concentrations while sparing other classes of T cells.<br />
ShK(L5)-amide administered as single daily subcutaneous injections (100 μg /kg /day)<br />
ameliorated pristane-induced arthritis in rats, a model for rheumatoid arthritis, <strong>and</strong><br />
prevented <strong>and</strong> treated experimental autoimmune encephalomyelitis in rats, a model for<br />
multiple sclerosis. Repeated dosing with ShK(L5)-amide in rats has not revealed systemic<br />
toxicity, <strong>and</strong> rhesus monkeys tolerated the peptide administered as a single i.v. injection.<br />
Further development of Kv1.3 blockers for autoimmune disease-therapy appears<br />
warranted.<br />
34<br />
Dr Edward A. Dennis: Distinguished Professor of Chemistry <strong>and</strong> Biochemistry<br />
<strong>and</strong> Pharmacology, School of Medicine, University of California, San Diego<br />
(UCSD), La Jolla, California, U.S.A.<br />
B.A. Yale University, 1963; M.A. Harvard University, 1965; Ph.D. Harvard<br />
University, 1968; Postdoctoral Fellow, Harvard Medical School, 1967-1969;<br />
Appointed to UCSD faculty, 1970-; Guggenheim Fellow, 1983-1984; Amer. Soc.<br />
Biochemistry <strong>and</strong> Molecular Biology Avanti Award, 2000; Editor-in-Chief, Journal<br />
of Lipid Research, 2003-; Director, LIPID MAPS Initiative, 2003-<br />
Dr Dennis’s research laboratory is focused on underst<strong>and</strong>ing the regulation of lipid second<br />
messengers <strong>and</strong> signal transduction processes <strong>and</strong> especially the role of various phospholipases<br />
in their generation. Special attention is paid to the cytosolic, secreted, <strong>and</strong> membrane-bound<br />
phospholipase A2s (PLA2) responsible for the control of arachidonic acid derived prostagl<strong>and</strong>in<br />
<strong>and</strong> leukotriene biosynthesis in macrophage cells. The goal is to elucidate the regulatory<br />
mechanisms of phospholipase A2s in vitro, ex vivo, <strong>and</strong> in vivo.<br />
In studies on the regulation <strong>and</strong> detailed mechanism of action of Group IA PLA2 from cobra<br />
venom at membrane <strong>and</strong> other lipid-water interfaces, Dr Dennis has developed in vitro systems<br />
for studying their detailed mechanism of action. PLA2s represent the smallest (molecular weight<br />
13,000) <strong>and</strong> perhaps the simplest enzymes of complex lipid metabolism known <strong>and</strong> are ideally<br />
suited for mechanistic studies. PLA2s have several kinds of phospholipid binding sites including<br />
activator sites, interfacial sites, <strong>and</strong> catalytic sites. It is important to define the precise role of the<br />
amino acid residues involved in these interactions <strong>and</strong> the laboratory is now carrying out HD/MS<br />
deuterium exchange studies using mass spectrometric analysis of surface interactions on the<br />
cobra venom enzyme to define the activator <strong>and</strong> other interfacial sites.<br />
Dr Dennis is involved in design <strong>and</strong> synthesis of chemical inhibitors of phospholipase A2. Many<br />
different inhibitor classes have been developed. He is also studying synthetic oxidized<br />
phospholipids <strong>and</strong> their role in LDL scavenger receptor uptake, autoantibody formation, <strong>and</strong><br />
apoptosis <strong>and</strong> their connection to atherosclerosis. Dr. Dennis is also the Director of the LIPID<br />
MAPS lipidomics initiative which aims to identify <strong>and</strong> quantify all of the lipid molecular species in<br />
the macrophage <strong>and</strong> their changes induced by activation of phospholipase A2.<br />
Six, D. A., <strong>and</strong> Dennis, E.A., Essential Ca 2+ -Independent Role of the Group IVA Cytosolic Phospholipase A2<br />
C2 Domain for Interfacial Activity, J. Biol. Chem., 278, 23842 (2003).<br />
Phillips, R., Six, D. A., Dennis, E. A., <strong>and</strong> Ghosh, P., Protection from Cytotoxicity of ExoU-expressing<br />
Pseudomonas Aeruginosa by Phospholipase A2 Inhibitors, J. Biol. Chem., 278 41326-41332 (2003).<br />
Boegeman, S. C., Deems, R. A., <strong>and</strong> Dennis, E. A., Phospholipid Binding <strong>and</strong> the Activation of Group IA<br />
Secreted Phospholipase A2, Biochemistry, 43, 3907-3916 (2004).<br />
Kokotos,G., Six,D.A., Loukas,V., Smith,T., Constantinou-Kokotou,V., Hadjipavlou-Litina,D., Kotsovolou,S.,<br />
Chiou,A., Beltzner,C.C., <strong>and</strong> Dennis,E.A., Inhibition of Group IVA Cytosolic Phospholipase A2 by Novel 2-<br />
Oxoamides in vitro, in Cells, <strong>and</strong> in vivo., J.Med.Chem, 47, 3615-3628 (2004).<br />
Killermann Lucas,K., Svensson,C.I., Hua,X.Y., Yaksh,T.L., <strong>and</strong> Dennis,E.A., Spinal Phospholipase A2 in<br />
Inflammatory Hyperalgesia: Role of Group IVA cPLA2, British Journal of Pharmacology , 144, 940-952<br />
(2005).<br />
Fahy,E., Subramaniam,S., Brown,H.A., Glass,C.K., Merrill,A.H., Murphy,R.C., Raetz,C.R.H., Russell,D.W.,<br />
Seyama,Y., Shaw,W., Shimizu,T., Spener,F., Van Meer,G., VanNieuwenhze,M., White,S., Witztum,J.L., <strong>and</strong><br />
Dennis,E.A., A Comprehensive Classification System for Lipids, Journal of Lipid Research, 46, 839-861<br />
(2005).<br />
35
Tuesday 25 July: 9.00 am- 11.15 am<br />
Lecture theatre K3.25<br />
Symposium: Lipids <strong>and</strong> toxins<br />
9.00-9.45: LIPID MAPS <strong>and</strong> Eicosanoid Lipidomics<br />
Edward A. Dennis<br />
Department of Chemistry <strong>and</strong> Biochemistry <strong>and</strong> Department of Pharmacology, School of<br />
Medicine, University of California at San Diego, La Jolla, CA 92093-0601, U.S.A.<br />
The metabolic pathways involving lipids are complex <strong>and</strong> intertwined. Developing an<br />
integrated metabolomic system capable of characterizing the global changes in lipid<br />
metabolites (“lipidomics”) is a daunting task but one that is important to undertake in<br />
light of the significant returns produced by the global approaches of genomics <strong>and</strong><br />
proteomics. Our consortium has developed a Lipid Metabolites And Pathways Strategy,<br />
termed LIPID MAPS, that applies a global integrated approach to the study of lipidomics.<br />
The specific aim of LIPID MAPS is to develop the requisite technology <strong>and</strong> conduct an<br />
integrated research program that will establish lipidomics as a fully functioning research<br />
field. We are employing a rigorously maintained set of common biological, biochemical,<br />
<strong>and</strong> analytical technologies in each of the consortium laboratories, <strong>and</strong> have developed<br />
an extensive informatics infrastructure. After introducing LIPID MAPS<br />
[http://www.lipidmaps.org] <strong>and</strong> its new classification system [J. Lipid Res.,46, 839-861<br />
(2005)], its application <strong>and</strong> use in lipid <strong>and</strong> protein databases will be summarized. Then<br />
new LC/MS results from the LIPID MAPS Fatty Acid/Eicosanoid Core that establish<br />
techniques for the detection <strong>and</strong> quanitation of various eicosanoid molecules generated<br />
by macrophages in response to stimuli such as LPS <strong>and</strong> a defined species Kdo2-Lipid A<br />
[J. Lipid Res., 47, 1097-1111 (2006)] will be reported. This includes the identification of<br />
the major <strong>and</strong> minor eicosanoid products <strong>and</strong> the quantification of the increases <strong>and</strong><br />
decreases of metabolites as a function of time during cell stimulation. Approaches to the<br />
identification of unknown eicosanoids will also be explored. Correlations of lipid<br />
metabolite changes across lipid categories will be reported. U54 GM069338<br />
36<br />
Cesare Montecucco graduated in Chemistry (1971) <strong>and</strong> Biology (1975) cum laude from the<br />
University of Padova, where he is currently Professor of General Pathology <strong>and</strong> Vice-Director of<br />
the Scuola Galileiana. He has carried out research in the University of Cambridge <strong>and</strong> Utrecht,<br />
the Pasteur Institute of Paris <strong>and</strong> the EMBL of Heidelberg. He studies the molecular <strong>and</strong> cellular<br />
mechanisms underlying the pathogenesis of diseases caused by pathogenic bacteria: anthrax,<br />
botulism, tetanus, <strong>and</strong> pylori gastrointestinal pathologies associated with Helicobacter, as well as<br />
studies on presynaptically acting snake toxins. Moreover, he collaborates actively with Chiron-<br />
Vaccines of Siena on the development of an anti-H. pylori vaccine.<br />
His major scientific achievements are: a) the discovery of the metalloproteolytic activity of the<br />
clostridial neurotoxins responsible for tetanus <strong>and</strong> botulism specific for VAMP/synaptobrevin,<br />
SNAP-25 <strong>and</strong> syntaxin (SNARE proteins), providing the key demonstration of the role of the<br />
SNARE proteins in exocytosis; b) co-discovered the activity <strong>and</strong> substrate of the anthrax lethal<br />
factor <strong>and</strong> that this toxin has an immunosuppressive activity <strong>and</strong> of the immunosuppressive<br />
synergism of the anthrax edema <strong>and</strong> lethal toxins; c) discovery of the mechanism of action of the<br />
vacuolating VacA cytotoxin <strong>and</strong> of the neutrophil-activating protein (HP-NAP) of H. pylori, the<br />
bacterium which colonizes the stomach of the majority of the human populations <strong>and</strong> is<br />
associated with severe pathologies including active chronic gastritis, peptic ulcers <strong>and</strong> stomach<br />
cancers; <strong>and</strong> d) provided compelling evidence that presynaptic snake PLA2 neurotoxins act on<br />
the plasma membrane by producing lysophosphatydilcholine <strong>and</strong> fatty acid which promote<br />
exocytosis via hemifusion intermediates. In addition, he has provided theoretical contributions on<br />
the mode of binding of clostridial neurotoxins, on the mechanism of membrane translocation of<br />
bacterial protein toxins <strong>and</strong> on the assembly of the SNARE apparatus.<br />
He has published more than two hundred articles in international scientific journals as well as two<br />
books. Prof. Montecucco received the 1993 prize of Harvard Medical School, the 1998 prize of<br />
the Italian Consortium for the Biotechnologies, the 2000 prize of the Deutsche Gesellschat fur<br />
Hygiene und Microbiology, the 2003 prize of the Masi Foundation for the Venetian Civilization<br />
<strong>and</strong> the 2004 Feltrinelli Prize for Medicine. He has served or is serving in several Editorial Boards<br />
of scientific journals <strong>and</strong> in the Scientific Councils of major research institutions. He is member of<br />
EMBO, Academia Europaea, Leopoldina Academy <strong>and</strong> Istituto Veneto di Scienze Lettere ed Arti.<br />
Rigoni, M et al. Equivalent effects of snake PLA2 neurotoxins <strong>and</strong> lysophospholipid-fatty acid<br />
mixtures. Science (2005) 310, 1678-1680<br />
Tonello et al. Pharmacology: Screening inhibitors of anthrax lethal factor. Nature (2002) 418,<br />
386.<br />
Montecucco, C. & Rappuoli, R. Living dangerously: how Helicobacter pylori survives in the human<br />
stomach. Nature Rev. Cell Biol. (2001) 2, 457-466.<br />
Schiavo G et al. Identification of the nerve-terminal targets of botulinum neurotoxins serotypes A,<br />
D <strong>and</strong> E. J. Biol. Chem. (1993) 268, 23784-23787.<br />
Schiavo G et al. Tetanus <strong>and</strong> botulinum B neurotoxins block neurotransmitter release by<br />
proteolytic cleavage of synaptobrevin. Nature (1992), 359, 832-835.<br />
37
9.45-10.30: How snake <strong>and</strong> bacterial presynaptic enzyme toxins block nerve<br />
terminals<br />
Cesare MONTECUCCO<br />
Department of Biomedical Sciences, University of Padova, Italy<br />
e.mail: cesare.montecucco@unipd.it<br />
Botulinum neurotoxins (BoNTs) <strong>and</strong> snake presynaptic phospholipase A2 neurotoxins<br />
(SPANs) are very potent toxins which paralyze the neuromuscular junction (NMJ),<br />
without killing the nerve, which fully recovers its function. BoNTs bind to the<br />
presynaptic membrane <strong>and</strong> are internalized inside synaptic vesicles whereform they enter<br />
the cytosol. Here, they display a very specific proteolytic activity versus any of the three<br />
SNARE proteins which form the core of the neuroexocytosis apparatus. Consequently,<br />
the process of synaptic vesicles fusion with the nerve membrane is prevented, no<br />
neurotransmitter is released <strong>and</strong> the synapse is blocked. SPANs bind to the presynaptic<br />
membrane <strong>and</strong> act on the cell surface by hydrolysing phospholipids with production of<br />
fatty acids (FA) <strong>and</strong> lysophospholipids (LPL). FA partition between the two leaflets of<br />
the membrane bilayer, whilst LPL remains on the external leaflet. This membrane<br />
configuration promotes the transition from an hemifuion membrane intermediate to<br />
membrane pore formation with release of neurotransmitter. At the same time, for the<br />
same reason, vesicle endocytosis is inhibited. This SPAN action leads to synaptic vesicle<br />
depletion <strong>and</strong> nerve terminals are paralysed. Thus, the two groups of neurotoxins have an<br />
opposite effects on the process of neurotransmitter release, yet achieve the same goal of<br />
paralysing nerve terminals causing animal death by respiratory failure. The elucitdation<br />
of their mechanism of action provides a strong evidence that SNARE proteins are<br />
essential for exocytosis <strong>and</strong> that the formation of hemifusion membrane intermediates is a<br />
key steps in membrane fusion during exocytosis (1).<br />
(1) Rossetto O, Morbiato L, Caccin P, Rigoni M, Montecucco C. Presynaptic enzymatic<br />
neurotoxins. J Neurochem. (2006) 97:1534-45.<br />
38<br />
The research of Dr Petrovič is focused on the molecular mechanisms <strong>and</strong> physiological role of<br />
deacylating phospholipases in yeast Saccharomyces cerevisiae. Genome-wide approaches, such<br />
as DNA microarrays <strong>and</strong> synthetic genetic array analysis, are being used to identify the molecular<br />
targets of activity of both exogenous <strong>and</strong> endogenous enzymes. Ammodytoxin, a neurotoxic<br />
secretory phospholipase A2 from the long-nosed viper venom, has been used as a model to<br />
underst<strong>and</strong> the mechanism of activity of neurotoxic sPLA2s in his work. One of the aims of his<br />
research is to develop yeast as a robust model system to analyze on a genome-wide scale the<br />
molecular mechanisms of intracellularly acting toxic proteins <strong>and</strong> peptides. Dr Petrovič is also<br />
involved in development of bioinformatics tools for analysis of functional genomics data.<br />
Selected publications:<br />
Petrovič U, Šribar J, Matis M, Anderluh G, Peter-Katalinič J, Križaj I, Gubenšek F. (2005)<br />
Ammodytoxin, a secretory phospholipase A2, inhibits G2 cell cycle arrest in yeast S. cerevisiae.<br />
Biochem J. 391:383-388.<br />
Petrovič U, Šribar J, Pariš A, Rupnik M, Kržan M, Vardjan N, Gubenšek F, Zorec R, Križaj I.<br />
(2004) Ammodytoxin, a neurotoxic secreted phospholipase A2, can act in the cytosol of the nerve<br />
cell. Biochem Biophys Res Commun 324:981-985.<br />
Curk T, Demšar J, Xu Q, Leban G, Petrovič U, Bratko I, Shaulsky G <strong>and</strong> Zupan B. (2004)<br />
Microarray data mining with visual programming. Bioinformatics 21:396-398.<br />
39
10.30-11.15: Genome-wide analysis of the molecular mechanism of action of<br />
ammodytoxin, a neurotoxic sPLA2, in yeast cells<br />
Petrovič, U.* 1 , Mattiazzi, M. 1 , Cvetek, J. 1 , Jenko, Z. 1 , Logonder, U. 1 , Bavdek, A. 2 , Kovačič, L. 1 ,<br />
Pungerčar, J. 1 , Rowan, E.G. 3 , Anderluh, G. 2 <strong>and</strong> Križaj, I. 1<br />
1 Jožef Stefan Institute, Jamova 39, Ljubljana, Slovenia, * uros.petrovic@ijs.si<br />
2 University of Ljubljana, Dept. of Biology, Večna pot 111, Ljubljana, Slovenia<br />
3 University of Strathclyde, SIBS, 27 Taylor Street, Glasgow, Scotl<strong>and</strong>, UK<br />
Neurotoxic secretory phospholipases A2 (sPLA2s) are enzymes of substantial medical <strong>and</strong><br />
potential pharmacological importance. However, reliable treatment of intoxication caused<br />
by them, <strong>and</strong> their biotechnological exploitation are both hindered by lack of<br />
underst<strong>and</strong>ing of their mechanism of action. We have been using a genetically tractable<br />
model organism, yeast Saccharomyces cerevisiae, to obtain a global insight into the<br />
mechanism of action of ammodytoxin, the neurotoxic sPLA2 from the venom of the longnosed<br />
viper. This approach is useful in generating hypotheses for its mechanism of action<br />
in mammalian systems, which can then be tested in relevant cellular models. We<br />
demonstrated that intracellular, active ammodytoxin inhibits endocytosis in yeast cells. By<br />
expressing ammodytoxin in its active form in the cells of all ~4700 viable yeast single<br />
gene deletion mutant strains we identified the proteins with endocytosis-related function<br />
that are specifically affected by ammodytoxin. Subsequent experiments suggest that in the<br />
yeast cells ammodytoxin binds to 14-3-3 proteins <strong>and</strong> is thus recruited onto clathrin-coated<br />
membrane invaginations, where it presumably inhibits the scission step in the generation of<br />
endocytotic vesicles. In another example, the combination of the yeast mutant approach<br />
with transcriptome analysis revealed the possible involvement of the yeast homologue of<br />
the AMP-dependent kinase in ammodytoxin-mediated cellular events. Subsequent<br />
experiments on neuromuscular preparations showed that, by pharmacologically modifying<br />
the AMP-dependent kinase activity, it is possible to modulate the level of motor neurontriggered<br />
muscle contraction. In the case of ammodytoxin, the yeast model system thus<br />
proved to be effective in identifying potential molecular targets of a vertebrate toxin.<br />
This work has been funded partially by the Slovenian Research Agency (grant J1-6507)<br />
<strong>and</strong> by the NATO Security Through Science Programme (grant 980899).<br />
o phospholipase A2<br />
o neurotoxicity<br />
o Saccharomyces cerevisiae<br />
o functional genomics<br />
40<br />
José María Gutiérrez has a BS in Microbiology from<br />
the University of Costa Rica (1977) <strong>and</strong> PhD in Physiological Sciences from Oklahoma State<br />
University (USA). Since 1977, he has been working at Instituto Clodomiro Picado, University of<br />
Costa Rica, with teaching responsibilities at the School of Microbiology, University of Costa<br />
Rica. From 1988 to 1996, he was appointed Director of this Institute. From 1999 to 2005, he was<br />
Head of the Research Division of this Institute, <strong>and</strong> currently he is Subdirector of the<br />
Institute. From 1986 to 1988, he was Director of the Graduate Program in Microbiology.<br />
University of Costa Rica.<br />
Dr Gutiérrez was awarded the National Award in Science <strong>and</strong> Technology in Costa Rica (1980),<br />
the TWAS-CONICIT award for young scientists of Costa Rica in the field of Biological Sciences<br />
(1990), <strong>and</strong> the Sven Brohult Award, given by the International Foundation for Science (1997).<br />
His fields of interest have been mostly (a) the study of the biochemical characteristics <strong>and</strong><br />
mechanism of action of snake venom toxins involved in the local pathology (hemorrhage <strong>and</strong><br />
myonecrosis) of envenomation, particularly those of Bothrops sp venoms, (2) the study of the<br />
ability of antivenoms to neutralize the most relevant snake venoms in Latin America, <strong>and</strong> (3) the<br />
study of novel technological ways to improve the quality of antivenoms for the treatment of<br />
snakebite envenomations. As a result of these research efforts, he has authored or co-authored<br />
250 papers in scientific journals.<br />
Mora, R., Valverde, B., Díaz, C., Lomonte, B. & Gutiérrez, J.M. (2005) A Lys49 phospholipase A2<br />
homologue from Bothrops asper snake venom induces proliferation, apoptosis <strong>and</strong> necrosis in a<br />
lymphoblastoid cell line. Toxicon 45: 651-660.<br />
Teixeira, C.F.P., Chaves, F., Zamunér, S.R., Fern<strong>and</strong>es, C.M., Zuliani, J.P., Cruz-Hofling, M.A., Fern<strong>and</strong>es,<br />
I. & Gutiérrez, J.M. (2005) Effects of neutrophil depletion in the local pathological alterations <strong>and</strong> muscle<br />
regeneration in mice injected with Bothrops jararaca snake venom. Int. J. Exp. Pathol. 86: 107-115.<br />
Gutiérrez, J.M., Rojas, E., Quesada, L., León, G., Núñez, J., Laing, G.D., Sasa, M., Renjifo, J.M., Nasidi, A.,<br />
Warrell, D.A., Theakston, R.D.G. & Rojas, G. (2005) Pan-African polyspecific antivenom produced by<br />
caprylic acid purification of horse IgG: an alternative to the antivenom crisis in Africa. Transactions of the<br />
Royal Society of Tropical Medicine <strong>and</strong> Hygiene 99, 468-475.<br />
Gutiérrez, J.M., Rucavado, A., Escalante, T. & Díaz, C. (2005) Hemorrhage induced by snake venom<br />
metalloproteinases: biochemical <strong>and</strong> biophysical mechanisms involved in microvessel damage. Toxicon 45,<br />
997-1011.<br />
Flores-Díaz, M., Alape-Girón, A., Clark, G., Catimel, B., Hirabayashi, Y., Nice, E., Gutiérrez, J.M., Titball, R.<br />
& Thelestam, M. (2005) A cellular deficiency of gangliosides causes hypersensitivity to Clostridium<br />
perfringens phospholipase C. Journal of Biological Chemistry 280, 26680-26689.<br />
Murakami, M.T., Arruda, E.Z., Melo, P.A., Martinez, A.B., Calil-Elias, S., Tomaz, M.A., Lomonte, B.,<br />
Gutiérrez, J.M. & Arni, R.K. (2005) Inhibition of myotoxic activity of Bothrops asper myotoxin II by the antitrypanosomal<br />
drug suramin. Journal of Molecular Biology 350, 416-426.<br />
Rucavado, A., Soto, M., Escalante, T., Loría, G.D., Arni, R.K. & Gutiérrez, J.M. (2005) Thrombocytopenia<br />
<strong>and</strong> platelet hypoaggregation induced by Bothrops asper snake venom. Toxins involved <strong>and</strong> their<br />
contribution to metalloproteinase-induced pulmonary hemorrhage. Thrombosis & Haemostasis 94, 123-131.<br />
Zuliani, J.P., Gutiérrez, J.M., Casais e Silva, L.L., Sampaio, S.C., Lomonte, B. & Teixeira, C.F.P. (2005)<br />
Activation of cellular functions in macrophages by venom secretory Asp-49 <strong>and</strong> Lys-49 phospholipases A2.<br />
Toxicon 46, 523-532.<br />
41
Wednesday 26 July: 9.00 am- 11.15 am<br />
Lecture theatre K3.25<br />
Symposium: From anecdotes to antidotes<br />
9.00-9.30: Novel possibilities for the treatment of local effects in snakebite<br />
envenomation<br />
Gutiérrez, J.M.<br />
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica,<br />
jgutierr@icp.ucr.ac.cr<br />
The development of severe local pathological alterations, i.e. hemorrhage, edema,<br />
blistering, myonecrosis <strong>and</strong> dermonecrosis, constitutes one of the most serious<br />
consequences of snakebites, which often lead to permanent disability. The pathogenesis of<br />
these complex alterations is predominantly associated with the direct action of myotoxic<br />
phospholipases A2, hemorrhagic metalloproteinases <strong>and</strong> hyaluronidases. In addition, a<br />
prominent inflammatory reaction develops in the affected tissues, which may further<br />
contribute to these pathological events. Despite the therapeutic success of antivenoms in<br />
reducing mortality <strong>and</strong> neutralizing systemically-acting toxins, they are poorly effective in<br />
the prevention of local tissue damage. Recent developments in our underst<strong>and</strong>ing of the<br />
biochemistry of locally-acting toxins, the pathogenesis of local effects, <strong>and</strong> the<br />
pharmacokinetic-pharmacodynamic relationships of antivenoms have opened a window of<br />
opportunity for the development of novel therapeutic strategies to confront this aspect of<br />
snakebite-induced pathology. Some of these possibilities are: (1) The use of phospholipase<br />
A2, metalloproteinase <strong>and</strong> hyaluronidase inhibitors, many of which have been developed to<br />
inhibit endogenous enzymes having homology with snake venom enzymes <strong>and</strong> being<br />
currently used in the clinical setting. It is likely that a rapid application of these inhibitors<br />
at the site of venom injection may block tissue-damaging enzymes in situ. (2) The<br />
modulation of local inflammatory events, guided by an adequate underst<strong>and</strong>ing of the role<br />
of inflammatory mediators in the pathogenesis of tissue damage, which may halt the<br />
deleterious effects of inflammation while maintaining the reparative <strong>and</strong> regenerative roles<br />
of the tissue response to venom-induced damage. (3) The engineering of antibodies <strong>and</strong><br />
antibody fragments of different molecular characteristics that have the ability to rapidly<br />
reach tissue compartments. Future developments will undoubtedly contribute to the<br />
improvement of the therapy of this complex aspect of snakebite envenomation.<br />
o Snakebite envenomation<br />
o Local effects treatment<br />
o Antivenoms<br />
o Enzyme inhibitors<br />
42<br />
Isaac Asuzu obtained his DVM <strong>and</strong> PhD degrees from the University of Nigeria Nsukka. He was<br />
previously Head of Department of Veterinary Physiology <strong>and</strong> Pharmacology <strong>and</strong> Dean of the<br />
Faculty of Veterinary Medicine. He is currently a full Professor of Veterinary Pharmacology in the<br />
Department of Veterinary Physiology <strong>and</strong> Pharmacology, University of Nigeria <strong>and</strong> a Fellow of<br />
Veterinary Surgeons Nigeria (FCVSN).<br />
Isaac Asuzu has published several scientific papers on the pharmacology <strong>and</strong> toxicology of<br />
medicinal plant extracts. He is presently working on plants that have anti-snake venom <strong>and</strong> antidiabetic<br />
activities. He interacts with local herbalists in the course of collecting plant materials for<br />
his studies. Professor Asuzu has visited many laboratories overseas as Visiting Scientist,<br />
including the University of Strathclyde, Glasgow <strong>and</strong> the University of Trieste in Italy. He is a<br />
recipient of the Commonwealth Academic Staff Fellowship <strong>and</strong> the Wellcome Trust Fellowship.<br />
Selected Publications<br />
1. Asuzu, I.U., Gray, A.I. <strong>and</strong> Waterman, P.G. (1999) The anthelmintic activity of D-3- Omethylchiroinositol<br />
isolated from Piliostigma thonningii stem bark extract. Fitoterapia. 70, 77.<br />
2. Uchendu, C.N., Kamalu, T.N. <strong>and</strong> Asuzu, I.U. (2000) A preliminary evaluation of<br />
the antifertility activity of a triterpenoid glycoside (DSS) from Dalbergia saxatilis in female Wistar<br />
rats. Pharmacological Research. 41. 521-525.<br />
3. Asuzu, I.U. <strong>and</strong> Harvey, A.L. (2003) The antisnake venom activities of Parkia biglobosa<br />
(Mimosaceae) stem extract. Toxicon . 42, 763-768.<br />
4. Anaga, A.O., Njoku, C.J., Ekejiuba, E.S., Esiaka, M.N. <strong>and</strong> Asuzu, I.U. (2004) Investigations of<br />
the methanolic leaf extract of Costus afer. Ker for pharmacological activities in vitro <strong>and</strong> in vivo.<br />
Phytomedicine 11, 242-248.<br />
5. Asuzu, I.U. (2005) A review on Croton penduliflorus seed extract. Recent Progress in<br />
Medicinal Plants. Stadium Press LLC, Texas USA. Pp 32.<br />
43
9.30-10.00: MYTHS AND REALITIES OF PLANT-BASED REMEDY FOR<br />
SNAKEBITE<br />
Isaac U. Asuzu, DVM, MSc., PhD, FCVSN.<br />
Department of Veterinary Physiology & Pharmacology,<br />
University of Nigeria, Nsukka<br />
Enugu State, Nigeria.<br />
Plant based-remedies have been used since centuries ago for the treatment of snakebites,<br />
particularly in the rural areas of Africa, South America <strong>and</strong> Asia. The broad geographic<br />
distribution of poisonous snakes in general, presumably accounts for at least some of the<br />
high diversity of plants used as antidotes. Classical pharmacological investigations of<br />
plant-based remedies tend to narrow down to a single plant, whereas a typical plant-based<br />
remedy as instituted by a traditional healer involves a group of plant parts, which are<br />
usually more than three. Among the group, one may contribute the main active<br />
ingredient, which may block the mechanism of toxicity – the one usually investigated by<br />
the pharmacologist, while the other members may contribute compounds which have<br />
palliative effects on the toxic situation. Examples include the relief of pain (analgesic),<br />
prevention of inflammation (anti-inflammatory), reduction of venom absorption from site<br />
of envenomation (chelating effect) <strong>and</strong> prevention of bacterial contamination<br />
(antibacterial effect) to mention a few. It is therefore not enough to dismiss a plant-based<br />
remedy as a myth whereas the full compliment of the various contributors to its overall<br />
activity is not investigated. Although many claims of successful treatment of snakebites<br />
with plant-based remedies are anecdotal, abundant evidence is available to prove the<br />
reality of plant-based remedies from laboratory experiments <strong>and</strong> chemical<br />
characterisation of the active constituents of the plants. Generally, these plants contain<br />
bioactive compounds in the form of alkaloids, tannins, steroids, glycosides, phenols <strong>and</strong><br />
volatile oils. These are secondary metabolites that in addition to specific anti-snake<br />
venom activity exhibit other ancillary but complimentary actions. Most of these plants<br />
are considered ‘multifunctional’ in the sense that more than one biochemical or<br />
pharmacological property has been demonstrated for them. Due to the variety of active<br />
compounds present in any one plant, coupled with their complementary actions, plantbased<br />
remedies are efficacious for the treatment of snakebites.<br />
44<br />
Dr. Chippaux was born in France in 1954 <strong>and</strong> received his M.D.<br />
degree in 1980 at the University of Marseille with a thesis on the epidemiology of<br />
snakebite in Ivory Coast, <strong>and</strong> his Ph.D. in Public Health at the Université de Paris VI on<br />
the control of dracunculiasis (Guinea Worm) in 1991. He has resided in Western <strong>and</strong><br />
Central African countries for most of his life, carrying out research in public health issues<br />
concerning the surveillance, prevention <strong>and</strong> control of endemic conditions prevalent in<br />
developing countries such as malaria, schistosomiasis, dracunculiasis, loasis, meningitis<br />
<strong>and</strong> envenomations. He is the author of over a hundred articles published in international<br />
peer-reviewed journals, of numerous chapters in books, <strong>and</strong> of eight books dealing,<br />
respectively, with snakes of the Ivory Coast, the snakes of French Guyana, the control of<br />
dracunculiasis <strong>and</strong> schistosomiasis in Western Africa, the snakes of Sub-Saharan Africa<br />
<strong>and</strong> snake venoms <strong>and</strong> envenomations. As a researcher of the Institut de Recherche pour<br />
le Développement (IRD, formerly ORSTOM) he has done research in epidemiology <strong>and</strong><br />
public health as a resident in Ivory Coast, Benin, Cameroon, Niger, Senegal, French<br />
Guyana, <strong>and</strong> has developed collaborations with numerous groups in other African<br />
countries (such as Guinea-Conakry, Burkina Faso, Republic of Congo <strong>and</strong> Democratic<br />
Republic of Congo, among others). He is now working in La Paz on the implementation<br />
of measures for the control of the American trypanosomiasis in endemic regions of<br />
Bolivia.<br />
Chippaux JP, Vieillefosse S, Sall O, Mafouta R, Diallo A. Appraisal of snakebite incidence in Senegal, West<br />
Africa. Bull Soc Pathol Exot. 2005; 98: 277-82.<br />
Chippaux JP. Ophidian envenomations <strong>and</strong> emergencies in Sub-Saharan Africa. Bull Soc Pathol Exot.<br />
2005;98: 263-8.<br />
Herrera M, Leon G, Segura A, Meneses F, Lomonte B, Chippaux JP, Gutierrez JM. Factors associated with<br />
adverse reactions induced by caprylic acid-fractionated whole IgG preparations: comparison<br />
between horse, sheep <strong>and</strong> camel IgGs. Toxicon. 2005; 46: 775-81.<br />
Chippaux JP, Rage-Andrieux V, Le Mener-Delore V, Charrondiere M, Sagot P, Lang J. Epidemiology of<br />
snake envenomations in northern Cameroon. Bull Soc Pathol Exot. 2002; 95: 184-7.<br />
Chippaux JP, Lang J, Amadi-Eddine S, Fagot P, Le Mener V. Short report: treatment of snake<br />
envenomations by a new polyvalent antivenom composed of highly purified F(ab)2: results of a<br />
clinical trial in northern Cameroon. Am J Trop Med Hyg. 1999; 61: 1017-1018.<br />
Chippaux JP, Amadi-Eddine S, Fagot P. Diagnosis <strong>and</strong> monitoring of hemorrhage due to viper<br />
envenomation in the African savanna. Bull Soc Pathol Exot. 1999; 92: 109-13.<br />
Chippaux JP, Lang J, Eddine SA, Fagot P, Rage V, Peyrieux JC, Le Mener V. Clinical safety of a polyvalent<br />
F(ab')2 equine antivenom in 223 African snake envenomations: a field trial in Cameroon. VAO<br />
(Venin Afrique de l'Ouest) Investigators. Trans R Soc Trop Med Hyg. 1998; 92: 657-62.<br />
Chippaux JP. Snakebites: appraisal of the global situation. Bull World Health Org. 1998; 76: 515-24.<br />
Chippaux JP. The development <strong>and</strong> use of immunotherapy in Africa. Toxicon. 1998; 36: 1503-6.<br />
Chippaux JP, Goyffon M. Venoms, antivenoms <strong>and</strong> immunotherapy. Toxicon. 1998; 36: 823-46.<br />
Chippaux JP Les serpents d’Afrique Occidentale et Centrale. IRD éd., Paris, coll. « Faune et flore<br />
tropicales » 2001.<br />
Chippaux J.-P.- Venins de serpent et envenimations. IRD éd., Paris, coll. « Didactiques », 2002.<br />
Chippaux J.-P.- Pratique des essais cliniques en Afrique. IRD éd., Paris, coll. « Didactiques », 2004.<br />
45
The following presentations represent a report on “An integrated approach to the<br />
development, optimization <strong>and</strong> evaluation of a polyvalent antivenom for Sub-Saharan<br />
Africa”<br />
10.00-10.20: Management of snake bite in Africa: how to stop the vicious cycle?<br />
Chippaux, J.-P.<br />
Institute de Recherche pour le Développement (IRD), La Paz, Bolivia. chippaux@ird.fr<br />
In Africa, snake bite remains a public health problem that can be summarily described by<br />
two types of data: i) the high number of annual bites (more than 1 million leading to<br />
20,000 deaths), <strong>and</strong> ii) the failure of snake bite management (less than 1% of all<br />
envenomations are treated with antivenom). Efforts made during the last years to improve<br />
the quality of antivenom proved to be ineffective to solve this problem. At-risk populations<br />
are mostly rural, mainly males from 15 to 50 years old. Human activities <strong>and</strong> snake<br />
behaviour largely explain man-snake contacts <strong>and</strong>, as a consequence, the high snake bite<br />
incidence in most African rural areas, both in savanna <strong>and</strong> forest. Time of arrival to a<br />
health care facility has a considerable impact on morbidity <strong>and</strong> mortality. Delayed<br />
admission is mainly due to care-seeking behaviour, the scattered distribution of health<br />
centers <strong>and</strong> the unavailability of both medical staff <strong>and</strong> drugs, especially antivenom. The<br />
antivenom supply is irregular because of its high cost, inadequate organisation of<br />
distribution, <strong>and</strong> poor training of medical workers. The less antivenom is used, the more its<br />
price increases <strong>and</strong> high costs lead to further reduction in use. Several pragmatic solutions<br />
would be proposed: a) to target <strong>and</strong> evaluate needs by enhancing epidemiological studies,<br />
b) to reduce the antivenom cost by developing antivenom production in pharmaceutical<br />
industries of emergent countries, c) to improve the marketing of drugs, d) to make<br />
treatment protocols more explicit <strong>and</strong> simple, e) to train health workers <strong>and</strong> f) to sensitize<br />
the populations to the existence of an effective treatment. Financial <strong>and</strong> logistical resources<br />
should be obtained from governments, local communities, private companies <strong>and</strong> patients<br />
themselves.<br />
o Africa<br />
o Snake bite<br />
o Epidemiology<br />
o Antivenom<br />
46<br />
Dr. Alagón was born in Mexico in 1954, obtained his M.D. degree <strong>and</strong><br />
a Ph.D. in Biochemistry at the Universidad Nacional Autónoma de México (UNAM) <strong>and</strong> did<br />
postdoctoral research at Rockefeller University, specializing in the immunology <strong>and</strong> biochemistry<br />
of venoms. He is currently laboratory head at the Instituto de Biotecnología of the UNAM in<br />
Cuernavaca, where he has been active in the molecular biology of Entamoeba histolytica as well<br />
as basic <strong>and</strong> applied toxinology research. He has been particularly interested in the development<br />
of diagnostic systems <strong>and</strong> new antivenoms against elapid <strong>and</strong> viperine snakes, spiders <strong>and</strong><br />
scorpions, <strong>and</strong> the application of novel technologies to the production, evaluation <strong>and</strong> quality<br />
control of therapeutic antibodies in general. He was the discoverer of a powerful thrombolytic<br />
agent, the plasminogen activator from the saliva of the common vampire bat Desmodus rotundus,<br />
which is being clinically tested worldwide (phase III) for the treatment of thromboembolic brain<br />
stroke. He is the author numerous scientific articles in international peer-reviewed journals.<br />
Almagro JC, Martinez L, Smith SL, Alagon A, Estevez J, Paniagua J. Analysis of the horse V(H) repertoire<br />
<strong>and</strong> comparison with the human IGHV germline genes, <strong>and</strong> sheep, cattle <strong>and</strong> pig V(H) sequences.<br />
Mol Immunol. 2005 Dec 6.<br />
Vazquez H, Chavez-Haro A, Garcia-Ubbelohde W, Mancilla-Nava R, Paniagua-Solis J, Alagon A, Sevcik C.<br />
Pharmacokinetics of a F(ab')2 scorpion antivenom in healthy human volunteers. Toxicon. 2005;46:<br />
797-805.<br />
Chippaux JP, Stock RP, Alagon A. Report of the 2nd International Conference on Envenomations in Africa<br />
(Deuxieme Colloque International sur les Envenomations en Afrique). Toxicon. 2005; 46: 115-8.<br />
Sanchez R, Saralegui A, Olivos-Garcia A, Scapolla C, Damonte G, Sanchez-Lopez R, Alagon A, Stock RP.<br />
Entamoeba histolytica: intracellular distribution of the sec61alpha subunit of the secretory pathway<br />
<strong>and</strong> down-regulation by antisense peptide nucleic acids. Exp Parasitol. 2005; 109: 241-51.<br />
de Roodt AR, Estevez-Ramirez J, Paniagua-Solis JF, Litwin S, Carvajal-Saucedo A, Dolab JA, Robles-Ortiz<br />
LE, Alagon A. Toxicity of venoms from snakes of medical importance in Mexico. Gac Med Mex.<br />
2005; 141: 13-21.<br />
Ramos-Cerrillo B, Olvera A, Odell GV, Zamudio F, Paniagua-Solis J, Alagon A, Stock RP. Genetic <strong>and</strong><br />
enzymatic characterization of sphingomyelinase D isoforms from the North American fiddleback<br />
spiders Loxosceles boneti <strong>and</strong> Loxosceles reclusa. Toxicon. 2004; 44: 507-14.<br />
de Roodt AR, Paniagua-Solis JF, Dolab JA, Estevez-Ramirez J, Ramos-Cerrillo B, Litwin S, Dokmetjian JC,<br />
Alagon A. Effectiveness of two common antivenoms for North, Central, <strong>and</strong> South American<br />
Micrurus envenomations. J Toxicol Clin Toxicol. 2004; 42: 171-8.<br />
D'Suze G, Moncada S, Gonzalez C, Sevcik C, Aguilar V, Alagon A. Relationship between plasmatic levels of<br />
various cytokines, tumour necrosis factor, enzymes, glucose <strong>and</strong> venom concentration following<br />
Tityus scorpion sting. Toxicon. 2003; 41: 367-75.<br />
Stock RP, Olvera A, Sanchez R, Saralegui A, Scarfi S, Sanchez-Lopez R, Ramos MA, Boffa LC, Benatti U,<br />
Alagon A. Inhibition of gene expression in Entamoeba histolytica with antisense peptide nucleic<br />
acid oligomers. Nat Biotechnol. 2001; 19: 231-4.<br />
Noeske-Jungblut C, Haendler B, Donner P, Alagon A, Possani L, Schleuning WD. Triabin, a highly potent<br />
exosite inhibitor of thrombin. J Biol Chem. 1995; 270: 28629-34.<br />
Noeske-Jungblut C, Kratzschmar J, Haendler B, Alagon A, Possani L, Verhallen P, Donner P, Schleuning<br />
WD. An inhibitor of collagen-induced platelet aggregation from the saliva of Triatoma pallidipennis.<br />
J Biol Chem. 1994; 269: 5050-3.<br />
Schleuning WD, Alagon A, Boidol W, Bringmann P, Petri T, Kratzschmar J, Haendler B, Langer G, Baldus B,<br />
Witt W. Plasminogen activators from the saliva of Desmodus rotundus (common vampire bat):<br />
unique fibrin specificity. Ann N Y Acad Sci. 1992; 667: 395-403.<br />
Cevallos MA, Navarro-Duque C, Varela-Julia M, Alagon AC. Molecular mass determination <strong>and</strong> assay of<br />
venom hyaluronidases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Toxicon.<br />
1992; 30: 925-30.<br />
47
10.20-10.35: Development of a polyvalent F(ab’)2 antivenom for sub-Sahara African<br />
snakes.<br />
Roberto P. Stock 1 , Judith Estévez 2 , Penélope Magaña 2 , Rita Mancilla 2 , Jorge-Paniagua-<br />
Solís 3 , Jean-Philippe Chippaux 4 <strong>and</strong> Alej<strong>and</strong>ro Alagón 1, *.<br />
(1)<br />
Instituto de Biotecnología-UNAM. Av. Universidad 2001, Cuernavaca, Morelos 62210, Mexico.<br />
(*) alagon@ibt.unam.mx<br />
(2)<br />
Instituto Bioclon, S.A. de C. V., México, DF, 14050, Mexico.<br />
(3)<br />
Dirección de Investigación. Laboratorios Silanes S.A. de C.V. México, DF, 83100, Mexico.<br />
(4)<br />
Institut de Recherche pour le Développement (IRD), La Paz, Bolivia.<br />
This work represents our efforts to help to ameliorate the current shortage in antivenom<br />
production <strong>and</strong> supply in sub-Saharan Africa <strong>and</strong> is our response to a call made during the<br />
WHO workshop on the st<strong>and</strong>ardization <strong>and</strong> control of antivenoms held at the NIBSC,<br />
Potters Bar, Engl<strong>and</strong>, 7–9 February 2001. We have prepared a polyvalent antivenom for<br />
snakes of sub-Saharan African. The final product is composed of purified F(ab’)2 (Fabtherapeutic)<br />
obtained after pepsin digestion of immunoglobulins from hyperimmunized<br />
horses (US Pat. 6,709,655 B2). The African Fab-therapeutic complies the same strict high<br />
st<strong>and</strong>ards as for the more than 300,000 vials of antivenom employed per year in Mexico<br />
with extremely low incidence (less than 1 in 50,000 cases) of severe secondary reactions.<br />
For this purpose we immunized groups of five horses each with either a viperid or a elapid<br />
venom mix. The immunogen viperid venom mix consisted of venoms from Bitis arietans,<br />
B. gabonica, Echis ocellatus, E. pyramidum <strong>and</strong> E. leucogaster while the elapid mix<br />
contained venom from the species Naja nigricollis, N. haje, N. pallida, N. melanoleuca,<br />
Dendroaspis viridis <strong>and</strong> D. polylepis. The antibody horse response against venoms was<br />
very high, with neutralization potencies higher than the 250 DL50 per venom species set as<br />
our lower limit per vial. The only exception was D. viridis in which the neutralization<br />
potency was not as high as with the other venoms, a problem we are working on at this<br />
time <strong>and</strong> that will be solved in the next lots of antivenom. We have already produced<br />
around 5,000 vials of antivenom <strong>and</strong> we expect to produce 40,000 more within this year.<br />
Acknowledgements. We wish to thank Y. Doljansky of Latoxan (Valence, France) for<br />
valuable advice.<br />
o African snakes<br />
o Antivenom<br />
o Neutralization potency<br />
o Composition<br />
48<br />
Dr. Stock was born in Uruguay in 1963. He obtained a degree in<br />
Biochemistry at Albright College, Pennsylvania, USA, a M.Sc. degree in Immnunology in the<br />
Hebrew University of Jerusalem, Israel, <strong>and</strong> a Ph.D. in Chemical Sciences in the University of<br />
Granada, Spain. He is laboratory head at the Instituto de Biotecnología of the UNAM in Mexico.<br />
He has done molecular <strong>and</strong> cellular biology research on P. falciparum, T. cruzi <strong>and</strong> E. histolytica,<br />
<strong>and</strong> is currently active on the antigenic characterization of venoms of African snakes of medical<br />
importance (notably of the genera Bitis, Dendroaspis, Echis <strong>and</strong> Naja) as well as on the<br />
enzymatic <strong>and</strong> genetic characterization of the venoms of the spiders of the genera Loxosceles<br />
<strong>and</strong> Latrodectus in order to apply techniques of molecular genetics to the production <strong>and</strong><br />
evaluation of antivenoms, such as the utilization of recombinant venom components for the<br />
production of antivenoms.<br />
Sánchez, R., Saralegui, A., Olivos-García, A., Scapolla, C., Damonte, G., Sanchez-Lopez, R., Alagón, A.<br />
<strong>and</strong> Stock, R.P. (2005) “Entamoeba histolytica: Intracellular distribution of the Sec61 subunit of<br />
the secretory pathway <strong>and</strong> down-regulation by antisense peptide nucleic acids.” Exp. Parasitol.<br />
109: 241-251.<br />
Chippaux, J-P., Stock, R.P. <strong>and</strong> Alagón, A. (2005) Report of the 2 nd International Conference on<br />
Envenomations in Africa (Deuxième Colloque International sur les Envenimations en Afrique).<br />
Toxicon 46: 115–118.<br />
Martínez, C., Paredes, R., Stock, R.P., Saralegui, A., Andreu, Cabezon, C., M., Ehrlich, R. <strong>and</strong> Galanti, N.<br />
(2005) “Cellular organization <strong>and</strong> appearance of differentiated structures in developing stages of<br />
the parasitic platyhelminth Echinococcus granulosus.” J. Cell. Biochem. 94: 327–335.<br />
Ramos-Cerrillo, B., Olvera, A., Odell, G.V., Zamudio, F., Paniagua-Solís, J., Alagón, A. <strong>and</strong> Stock, R.P.<br />
(2004) “Genetic <strong>and</strong> enzymatic characterization of Sphingomyelinase D isoforms from the North<br />
American fiddleback spiders Loxosceles boneti <strong>and</strong> Loxosceles reclusa.” Toxicon 44: 507-514.<br />
Scarfì, S., Giovine, M., Pintus, R., Millo, E., Clavarino, E., Pozzolini, M., Sturla, L., Stock, R.P., Benatti, U.<br />
<strong>and</strong> Damonte, G. (2003) “Selective inhibition of inducible cyclooxygenase-2 expression by<br />
antisense Peptide Nucleic Acids in intact murine macrophages.” Biotechnol. Appl. Biochem. 38: 61-<br />
69.<br />
Cerecedo D., Stock, R.P., González, S., Reyes, E., Mondragón, R. (2002) “Spatial distribution <strong>and</strong> structural<br />
correlation of actin, myosin <strong>and</strong> tubulin during platelet adhesion through confocal microscopy.”<br />
Haematologica 87:1165-1176.<br />
Stock, R.P., Olvera, A., Sánchez, R., Saralegui, A., Scarfì, S., Sanchez-Lopez, R., Ramos, M.A., Boffa, L.C.,<br />
Benatti, U. <strong>and</strong> Alagón, A. (2001) “Inhibition of gene expression in Entamoeba histolytica with<br />
antisense peptide nucleic acid (PNA) oligomers.” Nature Biotechnol. 19: 231-234.<br />
Ramos, M.A., Mercado, G.C., Salgado, L.M., Sánchez-López, R., Stock, R.P., Lizardi, P.M. <strong>and</strong> Alagón, A.<br />
(1997) “Entamoeba histolytica contains a gene encoding a homologue to the 54 KDa subunit of the<br />
signal recognition particle.” Mol. Biochem. Parasitol. 88: 225-235.<br />
49
10.35-10.45: The spectrum of protection: serological cross-reactivity between<br />
venoms of African vipers <strong>and</strong> elapids.<br />
Stock, R.P. 1,* , Estévez, J. 2 , Penélope Magaña, P. 2 , de Roodt, A. 1,5 , Jorge-Paniagua-Solís 3 ,<br />
J., Chippaux, J.-P. 4 <strong>and</strong> Alej<strong>and</strong>ro Alagón 1 .<br />
(1)<br />
Instituto de Biotecnología-UNAM. Av. Universidad 2001, Cuernavaca, Morelos 62210, Mexico.<br />
(*) rstock@ibt.unam.mx<br />
(2)<br />
Instituto Bioclon, S.A. de C. V., México, DF, 14050, Mexico.<br />
(3)<br />
Dirección de Investigación. Laboratorios Silanes S.A. de C.V. México, DF, 83100, Mexico.<br />
(4)<br />
Institut de Recherche pour le Développement (IRD), La Paz, Bolivia.<br />
(5)<br />
National Institute for the Production of Biologicals, Administración Nacional de<br />
Laboratorios e Instituto de Salud “Dr. Carlos G. Malbran”, Buenos Aires, Argentina.<br />
The spectrum of protection of an antivenom for snake bite can be significantly increased<br />
by taking into account the antigenic relationships between species the same genus, thus<br />
enabling a foreknowledge of the paraspecific neutralization potential of a given mono- or<br />
polyvalent antivenom. In this study we report a systematic comparison of paraspecific<br />
recognition of venoms of species belonging to the major genera of snakes of medical<br />
relevance in Africa in vitro by paraspecific titer determination (by ELISA) <strong>and</strong> in vivo by<br />
neutralization of lethality (as ED50) in mice. The genera studied included species of Bitis,<br />
Echis, Naja <strong>and</strong> Dendroaspis in vitro using experimental monovalent antisera raised in<br />
rabbits. Antigenic groupings, as measured by the degree of cross reactivity to the<br />
monospecific antisera, can be established within each genus. In vivo, a polyvalent<br />
antivenom (Antiophidien Panafricain ® ) raised against several species belonging to these<br />
genera significantly neutralizes lethality of venoms of species of the same genera not<br />
included in the original immunization mixture. In situations such as those prevalent in<br />
most of Africa, where antivenom distribution (where available) is difficult <strong>and</strong> the<br />
epidemiology of snake bite complex <strong>and</strong> varied, even within countries, the assessment of<br />
the full spectrum of protection of any given antivenom may considerably simplify<br />
distribution - by promoting regionalization schemes of the greatest possible amplitude<br />
(ideally one antivenom for the vast majority of cases in all of Africa)- <strong>and</strong> therapeutic<br />
protocols (ideally simplified for treatment of generalized viper <strong>and</strong> elapid syndromes).<br />
Also, minimization of the number of venoms used for generation of antivenoms of the<br />
highest protective potency <strong>and</strong> widest protective spectrum may have an impact on the cost<br />
of production <strong>and</strong> comercialization, <strong>and</strong> therefore on the implementation of national<br />
strategies for snake bite treatment in often severely under-funded African public health<br />
institutions.<br />
o African snakes<br />
o In vitro cross-reactivity<br />
o Antivenom<br />
o In vivo protection<br />
50<br />
Dr. Massougbodji was born in Abomey in 1949. He studied<br />
medicine in Dakar (Senegal) <strong>and</strong> Montpellier (France) where he specialized in tropical medicine,<br />
parasitology <strong>and</strong> public health. He resides in the Republic of Benin, where he is currently head of<br />
the Unit of Parasitology, Mycology <strong>and</strong> Ecology at the Faculté des Sciences de la Santé in<br />
Cotonou. He has worked on the epidemiology, treatment <strong>and</strong> control of many endemic infectious<br />
conditions throughout rural <strong>and</strong> urban Benin, such as malaria, schistosomiasis, dracunculiasis,<br />
filariasis <strong>and</strong> also on clinical <strong>and</strong> epidemiological aspects of envenomation by snakebite, which in<br />
some regions of Benin accounts for very high morbidity <strong>and</strong> mortality rates. He is the author of 75<br />
scientific publications in national <strong>and</strong> international scientific <strong>and</strong> medical journals <strong>and</strong> a leading<br />
figure in many programs <strong>and</strong> courses bearing on the ethics, development, practice <strong>and</strong> evaluation<br />
of medicine <strong>and</strong> medical programs in Africa.<br />
Lymphatic filariasis in a hyperendemic region : a ten years, follow-up panel survey. Myung K.,<br />
Massougbodji A., Ekoue S., Atchade P., Kiki-Fagla V. Klion A.D. Am. J. Trop. Med. Hyg. 1998; 59:<br />
222-226.<br />
Molecular characterisation of Plasmodium reichenowi apical membrane antigen-1 (AMA1), comparison with<br />
P; falciparum AMA1 <strong>and</strong> antibodiy-mediated inhibition of red cell invasion. Kocken C.H.M., Narum<br />
D.L., Massougbodji A., Ayivi B., Dubbeld M. A., Van Der Wel A., Conway D.J., Sanni A. And<br />
Thomas A.W. Molecular <strong>and</strong> Biochemical Parasitology 2000; 109: 147 – 156.<br />
Le risque de paludisme transfusionnel à Cotonou, Bénin. Garba A., Kinde-Gazard D., Makoutode M., Boyer<br />
N., Ernould J.C., Chippaux J.P. Massougbodji A. Cahiers santé 2000; 10 : 389-92.<br />
Meningococcal immunisation in Ghana. De Chabalier F., Chippaux J.P., Massougbodji A. Lancet. 2000;<br />
355: 2252 .<br />
A r<strong>and</strong>omized, double-blind study on the efficacy <strong>and</strong> safety of a practical three-day regimen with artesunate<br />
<strong>and</strong> mefloquine for the treatment of uncomplicated Plasmodium falciparum malaria in Africa.<br />
Massougbodji A., Kone M., Kinde-Gazard D., Same-Ekobo A., Cambon N. <strong>and</strong> Muller A. Trans. R.<br />
Soc. Trop. Med. Hyg., 2002; 96: 655 - 659.<br />
Limits <strong>and</strong> weaknesses of intermittent treatment in malaria prevention. Chippaux JP, Le Hesran Jy, Cot M,<br />
Massougbodji A. Bull Soc Pathol Exot. 2003;96: 75-6.<br />
Geoclimatology <strong>and</strong> severity of snake bite envenomations in Benin. Massougbodji M, Chobli M, Assouto P,<br />
Lokossou T, Sanoussi H, Sossou A, Massougbodji A. Bull Soc Pathol Exot. 2002; 95: 175-7.<br />
Epidemiological data on snake bite cases reported in Benin from 1994 to 2000. Fayomi B, Massougbodji A,<br />
Chobli M. Bull Soc Pathol Exot. 2002;95: 178-80.<br />
Round table <strong>and</strong> synthesis of the meeting. Chippaux JP, Goyffon M, Benguedda AC, El Ayeb M, Griguer F,<br />
Massougbodji A, Mion G. Bull Soc Pathol Exot. 2002; 95: 217-2199<br />
Comités d’éthique en Afrique de l’Ouest et Afrique Centrale : des prémices concrets pour un partenariat de<br />
la recherche clinique. Effa P., Massougbodji A., Ntoumi F., Derme A., Ndemanga-Kamoune J.,<br />
Nguembo J., Impouma B., Akue J-P., Ehouman A., Dieye A., Kilama W., Vicari M.,Crawley F P.,<br />
Hirsch F et Debois H. Les Cahiers du C.C.N.E., n037, 2003,45 -49.<br />
Round table of November 20th, 2004: recommendations for improving the management of envenomations.<br />
Chippaux JP, Massougbodji A, Goyffon M. Bull Soc Pathol Exot. 2005; 98: 316-9.<br />
51
10.45-11.05: Clinical trial of a polyvalent antivenom specific for African snakes<br />
Massougbodji, A.<br />
Faculté des Sciences de la Santé (FSS), Cotonou, Benin. massougbodjiachille@yahoo.fr<br />
A clinical trial of Antiophidien Panafricain ® was carried out in 11 health centers or<br />
hospitals of Benin between July 2005 <strong>and</strong> June 2006. At the end of April 2006, 317 snake<br />
bites had been registered, of which 234 were included. The other 83 were not treated by<br />
Antiophidien Panafricain ® because i) they did not show envenomation, ii) they refused to<br />
be included, or iii) the antivenom was not available. The average age was 23 with a<br />
majority of men (163 men versus 71 women) <strong>and</strong> adults (134 adults versus 100 children<br />
under 16). The average delay of treatment was approximately 8 hours, with a wide range of<br />
time (30 minutes to 12 days). A biological (TCTS) <strong>and</strong>/or clinical hemorrhagic syndrome<br />
was observed in 153 patients out of the 218 among those in which the coagulation status<br />
was assessed (70%). The average number of doses administered was 3.6 vials of 10 mL.<br />
Twelve patients presented benign adverse effects (5%). Late tolerance was studied in 46<br />
patients (20%) <strong>and</strong> no serum sickness was observed. The average duration of<br />
hospitalization was 3.4 days. There are slight differences in recruitment characteristics<br />
between the centers but no significant differences in results between them. Five deaths<br />
were observed. Three of these deaths were due to late arrival at health centers with already<br />
severe clinical complications (cerebral hemorrhage, neurotoxic coma). Another one<br />
followed an envenomation by Atractaspis whose venom is not neutralized by Antiophidien<br />
Panafricain ® . The last one also arrived with hemorrhagic complications, <strong>and</strong> died during<br />
transfer to another center. Lethality was 2.1% of the envenomations, to be compared to a<br />
lethality of 8% in the group not included for various reasons (83 people) <strong>and</strong>, within this<br />
group, to the 12.5% of envenomated patients not treated by Antiophidien Panafricain ® .<br />
Moreover, historical hospital lethality reaches 24.4% in the same geographical area,<br />
according the available literature <strong>and</strong> previous surveys.<br />
o Clinical trial<br />
o Antivenom<br />
o Snake bite<br />
o West Africa<br />
52<br />
Brian Robertson: BSc (Hons) Aberdeen University, 1982; PhD, John Curtin School of Medical<br />
Research, Australian National University 1986; Postdoctoral Fellow, S<strong>and</strong>oz Institute 1986-1988;<br />
Principal Scientist, Wyeth Research UK 1988- 1994; Governors’ Lecturer, Biochemistry, Imperial<br />
College London 1995- 2002; Professor of Neuroscience, University of Strathclyde 2002-2004;<br />
Eberhard Buhl Chair of Neurobiology, University of Leeds (2004 - ); Wellcome Trust<br />
Neurosciences <strong>and</strong> Mental Health Committee; Scientific Advisory Board, Biofocus PLC; MRC<br />
Brain Sciences Panel; Reviewing <strong>and</strong> Topical Reviews Senior Editor, Journal of Physiology.<br />
Research programme: Underst<strong>and</strong>ing the role of neuronal ion channels<br />
The challenge of underst<strong>and</strong>ing brain <strong>and</strong> neuronal function in health <strong>and</strong> disease requires an<br />
interdisciplinary attack at multiple levels, from genomic information through molecular, cellular<br />
<strong>and</strong> systems approaches. My background is in physiology with my present research focussing<br />
mainly on the characterization of voltage-gated potassium channels. Potassium channels are<br />
crucial regulators of the excitability of nerve cells, <strong>and</strong> they have consequently become an<br />
important target for academic <strong>and</strong> drug research. Indeed, I first worked on these in the<br />
pharmaceutical industry. My interests <strong>and</strong> experience ranges from obscure biophysical<br />
measurements of channel function up to complex neuronal networks in brain slices <strong>and</strong> isolated,<br />
intact, cerebellum. Throughout my career, I’ve come back again <strong>and</strong> again to using toxins as<br />
selective tools to dissect out roles of specific ion channels. Our lab employs a wide range of<br />
techniques <strong>and</strong> has developed some novel preparations. At the most basic level, we employ site<br />
directed mutagenesis techniques to unravel which parts of cloned potassium channel subunits<br />
contribute to their opening <strong>and</strong> their distinctive pharmacology. Furthermore, we examine ‘native’<br />
voltage gated potassium currents in nerve cells, trying to underst<strong>and</strong> the molecular identity of<br />
these using antibody labelling <strong>and</strong> physiological <strong>and</strong> pharmacological ‘fingerprinting’ <strong>and</strong><br />
transgenic animals.<br />
Another major interest in our lab is the study of changes in sensory neurons following nerve<br />
injury. Neuropathic pain is a huge health problem, <strong>and</strong> unfortunately, despite intensive study, we<br />
still know very little about the fundamental mechanisms involved. Using animal <strong>and</strong> organotypic<br />
culture models, we are investigating excitability changes often associated with anatomical<br />
remodelling, including sprouting of axonal terminals around primary afferent somata in dorsal root<br />
ganglia.<br />
elected recent publications:<br />
JM MILLAR, L BARRATT, AP SOUTHAN, KM PAGE, REW FYFFE, B ROBERTSON, & A MATHIE (2000) A functional<br />
role for the two-pore domain potassium channel TASK-1 in cerebellar granule neurons. Proceedings of the National<br />
Academy of Sciences (USA) 97, 3614-3618.<br />
T BUSHELL, C CLARKE, A MATHIE & B ROBERTSON (2002) Pharmacological characterisation of a non-inactivating<br />
outward current in mouse cerebellar Purkinje neurones. British Journal of Pharmacology 135, 705-712.<br />
NP MORRIS, REW FYFFE & B ROBERTSON (2004) Characterisation of the hyperpolarization-activated current (Ih) in the<br />
medial septum/diagonal b<strong>and</strong> complex in the mouse. Brain Research, 1006, 74-86.<br />
SYM YEUNG, D THOMPSON, Z WANG, D FEDIDA, B ROBERTSON (2005) Modulation of Kv3 subfamily potassium<br />
currents by the sea anemone toxin BDS: Significance for CNS <strong>and</strong> biophysical studies. Journal of Neuroscience, 25,<br />
8735-8745<br />
53
Thursday 27 July: 9.00 am- 11.15 am<br />
Lecture theatre K3.25<br />
ISN Symposium: Ion channel toxins: tools to explore ion channel<br />
structure, function <strong>and</strong> physiology<br />
Sponsored by the International Society for Neurochemistry<br />
<strong>and</strong> supported by the Physiological Society<br />
9.00- 9.45: Potassium channel toxins - a trail from cortex to channel subtype<br />
Brian Robertson<br />
(Eberhard Buhl Professor of Neurobiology, Neuroscience Research Group, Institute of Membrane <strong>and</strong><br />
Systems Biology, University of Leeds, LEEDS LS2 9JT)<br />
Voltage-gated potassium channels are ubiquitously expressed in neurones <strong>and</strong> are crucial regulators of<br />
their excitability, finely tuning firing rates, action potential width, dendritic integration <strong>and</strong> transmitter<br />
release. After many years of working with individual cloned potassium (Kv) channels in expression<br />
systems such as oocytes <strong>and</strong> cell lines, we decided to apply the results of such physiological <strong>and</strong><br />
pharmacological ‘fingerprinting’ to central neurones, using electrophysiological methods in brain<br />
slices. We chose to study the basket cell–Purkinje cell connection in the mouse cerebellum. The<br />
cerebellum has a relatively simple architecture <strong>and</strong> it is straightforward to identify most target cells.<br />
Several groups showed with immunocytochemical techniques that the basket–Purkinje cell synapse<br />
was particularly well endowed with Kv1 subunit channels, of which three (Kv1.1, 1.2 <strong>and</strong> 1.6) are<br />
sensitive to dendrotoxins derived from mamba venom. (It is also of interest to note that basket cells<br />
synapse around the cell soma <strong>and</strong> axon initial segment of Purkinje cells, <strong>and</strong> are therefore ideally<br />
placed to influence the final output of the cerebellar cortex.) Therefore, studying the potassium<br />
channels at this connection would offer us a good chance to record the behaviour <strong>and</strong> determine the<br />
roles of Kv1 channels in their native environment. We found that about half the voltage activated K +<br />
current present in the synaptic terminal was due to Kv1 subfamily channels, since dendrotoxins could<br />
only inhibit half the total conductance. A number of different lines of evidence (physiological,<br />
immunocytochemical <strong>and</strong> pharmacological) led us to the conclusion that the remaining current was<br />
probably from the Kv3 subfamily of K + channels. Around that time, it was suggested by Michel<br />
Lazdunski <strong>and</strong> colleagues that the toxin BDS, from sea anemone, was a selective blocker of Kv3.4<br />
subunits <strong>and</strong> since we had antibody labelling for this protein in the basket cell terminals we were<br />
surprised to see no effect of this toxin in our slice patch clamp experiments. In the process of making<br />
sure the toxin did block Kv3.4 subunits in mammalian cell lines, we also discovered that it inhibited<br />
Kv3.2 <strong>and</strong> Kv3.1 subunits equally well. However, BDS did not behave like a classical open channel<br />
blocker, but instead slowed current activation, shifted voltage dependence to the right <strong>and</strong> blocked<br />
Kv3 channels only from the outside <strong>and</strong> could inhibit both open <strong>and</strong> closed channels. I will describe<br />
how we began unravel the sites of action of BDS on Kv3 channels <strong>and</strong> determine how it acts on Kv3<br />
channels.<br />
KEY WORDS: Dendrotoxin; BDS; potassium channel; neurone; cerebellum<br />
54<br />
Dr Meunier’s research focuses on underst<strong>and</strong>ing, at the molecular level, the dynamics events<br />
mediating neuronal/hormonal secretions <strong>and</strong> synaptic plasticity (how neurons survive, connect<br />
<strong>and</strong> communicate). To study these fundamental physiological processes, two main strategies are<br />
used: first, taking advantage of the exquisite selectivity of neurotoxins to selectively dissect these<br />
mechanisms, <strong>and</strong> second, examining the role played by members of certain classes of lipid in<br />
modulating various steps of neuronal secretions. The hope is to establish novel avenues in our<br />
underst<strong>and</strong>ing of neuronal communications that will lead to novel therapeutic strategies to tackle<br />
neuronal diseases.<br />
Selected Publications:<br />
Rickman, C., Meunier, F. A., Binz, T. <strong>and</strong> Davletov, B. A. (2004). High affinity interaction of<br />
syntaxin <strong>and</strong> SNAP-25 on the plasma membrane is abolished by botulinum toxin E. J. Biol. Chem.<br />
279:644-51.<br />
Foran, P.G., Davletov, B., <strong>and</strong> Meunier F. A. (2003). Getting muscles moving again after<br />
botulinum toxin: novel therapeutic challenges. Trends in Molecular Medicine 9: 9291-9299.<br />
Meunier, F.A., Feng, Z.P., Molgo, J., Zamponi, G. <strong>and</strong> Schiavo G. (2002). Glycerotoxin from<br />
Glycera convoluta stimulates neurosecretion by targeting N-type Ca 2+ channels Cav2.2. EMBO J.<br />
21: 6733-6743.<br />
Osborne, S.L., Meunier, F. A. <strong>and</strong> Schiavo G. (2001). Phosphoinositides as Key Regulators of<br />
Synaptic Function. Neuron 32: 9-12.<br />
55
9.45-10.30: Glycerotoxin, a new tool to dissect synaptic vesicle recycling<br />
Frederic A. Meunier 1 , Jordi Molgo 2 , Giampietro Schiavo 3<br />
1 Molecular Dynamics of Synaptic Function Laboratory, The School of Biomedical<br />
Sciences, The University of Queensl<strong>and</strong>, St. Lucia, 4072 Queensl<strong>and</strong>, Australia;<br />
2 Laboratoire de Neurobiologie Cellulaire et Moléculaire, Unité Propre de Recherche<br />
9040, Centre National de la Recherche Scientifique, 1 avenue de la Terrasse, 91198 Gifsur-Yvette,<br />
France; 3 Molecular Neuropathobiology Laboratory, Cancer Research UK<br />
London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields,<br />
London WC2A 3PX, UK<br />
Multi-quantal release of neurotransmitter is driven by voltage-gated calcium channels,<br />
including Cav2.2 (N-type), which tightly couple action potentials to neurotransmitter<br />
release. Following stimulation of neurotransmitter release, the number of synaptic<br />
vesicles at the presynaptic nerve terminal is maintained via the vesicle recycling process.<br />
We have recently discovered that glycerotoxin (GLTx), a 320 kDa neurotoxin purified<br />
from the venom of the polychaete annelids Glycera convoluta, selectively activates<br />
Cav2.2 channels <strong>and</strong> promotes a large increase in neurotransmitter release in Cav2.2expressing<br />
neurons from the peripheral <strong>and</strong> central nervous system (Meunier et al., 2002;<br />
Schenning et al., 2006).<br />
Surprisingly, we found that GLTx stimulates spontaneous release of ACh at very high<br />
rate (20-100 quanta/s) for more than 10 hours at the amphibian neuromuscular junction.<br />
An estimate of the number of vesicle undergoing exocytosis during that period of time<br />
largely exceeds the number of vesicles present in motor nerve terminals.<br />
Ultrastructural examination of GLTx-treated nerve terminals revealed no significant<br />
changes in the number of synaptic vesicles. Using styryl dyes we found that GLTx upregulates<br />
synaptic vesicle recycling, an effect blocked by Cav2.2 selective inhibitor ωconotoxin<br />
MVIIA. After photoconverting the styryl dye, electron microscopy<br />
examination revealed that most recycled vesicles emanate from large endosomal<br />
structures. Our initial analysis shows that the majority of recycling vesicles were<br />
undistinguishable in size <strong>and</strong> shape from small synaptic vesicles.<br />
Our experimental data suggest that synaptic vesicle recycling is selectively enhanced<br />
through the specific activation of Cav2.2. GLTx therefore represents a new tool with<br />
potential to further dissect the role of Cav2.2 channels in controlling the coupling between<br />
exocytosis <strong>and</strong> endocytosis in presynaptic terminals.<br />
Meunier et al., EMBO J (2002) 21(24):6733-43.<br />
Schenning et al., J. Neurochem. (2006) Epub.<br />
56<br />
Michael Gurevitz was born in 1946 in Germany <strong>and</strong> received his BS, MSc<br />
<strong>and</strong> PhD degrees at the Hebrew University of Jerusalem. He spent six years (1981-1986) in the<br />
United States before he joined Tel Aviv University, where he serves now as a Professor at The<br />
Department of Plant Sciences. His interests divide between toxinology <strong>and</strong> plant molecular<br />
biology. In the early 90s he has established a successful expression system for scorpion toxins<br />
affecting voltage-gated sodium channels, which has been used extensively to study structureactivity<br />
relationship <strong>and</strong> the mechanism by which these toxins interact with their target channels.<br />
The Gurevitz laboratory focuses in their toxinology research on molecular mechanisms underlying<br />
the mode of action <strong>and</strong> specific recognition of insect <strong>and</strong> mammalian Na + channels by a variety of<br />
pharmacologically distinct scorpion toxins. This study is motivated by the need to develop new<br />
approaches to the design of environmentally-safe, biodegradable insecticides <strong>and</strong> selective drugs<br />
in medicine. The experimental approach combines methods of various disciplines (molecular<br />
genetics, neuropharmacology, electrophysiology, structural biology) <strong>and</strong> involves extensive<br />
collaboration with several research groups in Israel <strong>and</strong> abroad.<br />
Cohen L, Karbat I, Gilles N, Froy O, Corzo G, Angelovici R, Gordon D & Gurevitz M (2004)<br />
Dissection of the functional surface of an anti-insect excitatory toxin illuminates a putative ‘hot<br />
spot’ common to all scorpion α-toxins affecting Na + channels. J Biol Chem 279:8206-8211.<br />
Karbat I, Cohen L, Gilles N, Gordon D & Gurevitz M (2004) Conversion of a scorpion toxin<br />
agonist into an antagonist highlights an acidic residue involved in voltage sensor trapping during<br />
activation of neuronal Na + channels. FASEB J 18:683-689.<br />
Karbat I, Frolow F, Froy O, Gilles N, Cohen L, Turkov M, Gordon D & Gurevitz M (2004)<br />
Molecular basis of the high insecticidal potency of scorpion α-toxins. J Biol Chem 279:31679-<br />
31686.<br />
Tan J, Liu Z, Wang R, Huang Z, Chen AC, Gurevitz M & Dong K (2005) Identification of amino<br />
acid residues critical for pyrethroid binding to insect sodium channels. Mol Pharmacol 67:513-<br />
522.<br />
Corzo G, Escoubas E, Villegas E, Karbat I, Gordon D, Gurevitz M, Nakajima T & Gilles N (2005)<br />
A spider toxin that induces a typical effect of scorpion α-toxins but competes with β-toxins on<br />
binding to insect sodium channels. Biochemistry 44:1542-1549.<br />
Cohen L, Karbat I, Gilles N, Ilan N, Gordon D & Gurevitz M (2005) Common features in the<br />
functional surface of scorpion β-toxins <strong>and</strong> elements that confer specificity for insect <strong>and</strong><br />
mammalian voltage-gated Na-channels. J Biol Chem 280:5045-5053.<br />
Strugatsky D, Zilberberg N, Ilan N, Turkov M, Cohen L, Stankiewicz M, Pelhate M, Gilles N,<br />
Gordon D & Gurevitz M (2005) Expression of a gene family encoding a novel scorpion<br />
depressant toxin illuminates the key role of asparagine-58 in activity on insect neuronal sodium<br />
channels. Biochemistry 44:9179-9187.<br />
57
10.30-11.15 : Structural commonality versus functional specificity in scorpion toxins<br />
that affect voltage-gated sodium channels<br />
Izhar Karbat 1 , Lior Cohen 1 , Nitza Ilan 1 , Roy Kahn 1 , Michael Turkov 1 , Maya Gur 1 ,<br />
Nicolas Gilles 2 , Dalia Gordon 1 & Michael Gurevitz 1<br />
1 Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv<br />
University, Ramat Aviv 69978, Israel; 2 CEA Saclay, Département d’Ingéniérie des<br />
Protéines, Gif-sur Yvette, France<br />
The functional surface of scorpion toxins representing all major pharmacological<br />
groups (anti-mammalian α, α-anti-insect, α-like, anti-mammalian β, anti-insect<br />
depressant <strong>and</strong> excitatory) was elucidated by mutagenesis followed by toxicity,<br />
binding, electrophysiological, <strong>and</strong> structural studies. Despite the vast differences in<br />
their effects on voltage-gated sodium channels (Navs), in all toxins the loop preceding<br />
the α-helix motif <strong>and</strong> the C-tail are important for function, which may result from their<br />
common ancestry <strong>and</strong> structural resemblance. Molecular dissection of alpha-toxins<br />
revealed two bioactive domains: a conserved Core-domain, formed by residues of the<br />
molecule core, <strong>and</strong> a variable NC-domain, formed by the five-residue-turn <strong>and</strong> the Ctail,<br />
<strong>and</strong> shown to be associated with toxin selectivity. This was verified by exchange of<br />
active sites between two distinct toxins leading to inversion of selectivity toward insect<br />
<strong>and</strong> mammalian brain channels. Analysis of the electrophysiological effects of alphatoxin<br />
mutants <strong>and</strong> chimeras on the rat brain channel Nav1.2a has suggested a two-step<br />
binding, first of the Core-domain that associates with a solvent accessible channel<br />
region, followed by interaction of the NC-domain with another less accessible channel<br />
region. In β-toxins, mutagenesis of the anti-insect excitatory <strong>and</strong> depressant toxins, BjxtrIT<br />
<strong>and</strong> LqhIT2, <strong>and</strong> the anti-mammalian β-toxin, Css4, highlighted a common<br />
‘pharmacophore’ whereas their C-tail region was associated with toxin specificity. A<br />
conserved glutamate (E15) in Css4 was shown to be involved in ‘trapping’ of the<br />
channel voltage sensor. Substitution E15R in Bj-xtrIT <strong>and</strong> Css4 abolished activity with<br />
no change in binding affinity. The Bj-xtrIT-E15R mutant has become an efficient<br />
antagonist of the unmodified toxin. These studies contribute to clarification of the<br />
mechanism by which scorpion toxins interact with Navs, <strong>and</strong> may pave the way for<br />
design of insecticides <strong>and</strong> drugs with high specificity to Nav subtypes.<br />
58<br />
Kenneth Clemetson<br />
Born 1943 in Newcastle-upon Tyne, Engl<strong>and</strong><br />
Christ’s College/Dept. of Chemistry, Univ. of Cambridge PhD, 1968 in organic<br />
chemistry (affinity-labelling with galactosides).<br />
Post-Docs at the University of Alberta, Edmonton, Canada 1968-1970 <strong>and</strong> the<br />
University of California, Santa Barbara 1970-1972 on carbohydrate chemistry<br />
(antibiotic synthesis) <strong>and</strong> then its applications in biochemistry <strong>and</strong> microbiology.<br />
Theodor Kocher Institute since December 1972 with Ernst Lüscher <strong>and</strong> as<br />
Professor of Biochemistry since 1990. Initially, studies on techniques for the<br />
isolation <strong>and</strong> characterisation of cell receptors <strong>and</strong> then with platelets from 1974.<br />
Prizes/Honours:<br />
1990 Mogens-Ellerman Prize of the Swiss Society of Haematology<br />
1991- date President, Berne Biochemical Society<br />
1993-1997 President, European Thrombosis Research Organization<br />
1998-2004 Member of Council, International Society on Thrombosis <strong>and</strong><br />
Haemostasis<br />
2000 Owren Lecturer, University of Oslo, Norway<br />
2002 Fellow Royal Society of Chemistry<br />
2003 Distinguished Career Award, International Society on Thrombosis <strong>and</strong><br />
Haemostasis<br />
Highlights of research:<br />
Isolation <strong>and</strong> characterisation of platelet receptors. Platelet receptors in bleeding<br />
disorders. 2D separations of carbohydrate surface labelled platelets (early<br />
proteomics!). First identification of several platelet receptor components. Cloning<br />
GPVI. Platelet receptor signalling. Crystal structure of GPIb. COAT platelets/role<br />
of serotonin. Snake venom proteins <strong>and</strong> platelet receptors.<br />
59
Thursday 27 July: 3.00 pm- 5.30 pm<br />
Lecture theatre K3.25<br />
Symposium: Toxins <strong>and</strong> hemostasis<br />
Incorporating a meeting of the ISTH SSC Registry of Exogenous Hemostatic Factors<br />
Supported by Latoxan, Pentapharm <strong>and</strong> Venom Supplies Pty Ltd<br />
3.00-3.30: Snake venom proteins affecting platelets<br />
Kenneth J. Clemetson <strong>and</strong> Jeannine M. Clemetson<br />
Theodor Kocher Institute, University of Berne, Freiestrasse 1, CH-3012, Berne,<br />
Switzerl<strong>and</strong><br />
Snake venoms are complex mixtures of biologically active proteins <strong>and</strong> peptides. These<br />
components have been classified into various families, such as serine proteases,<br />
metalloproteinases, C-type lectins, disintegrins <strong>and</strong> phospholipases. Many of them affect<br />
haemostasis by activating or inhibiting coagulant factors or platelets, or by affecting<br />
endothelial or smooth muscle cells. Venom proteins can affect platelet function by<br />
binding to or proteolysing platelet receptors to prevent or, more often induce, platelet<br />
activation or degrade vWF or cause it to bind to GPIb <strong>and</strong> activate platelets. Thus, there<br />
are disintegrins in nearly all venoms that block the integrin IIb 3 on platelets <strong>and</strong> a<br />
variety of others have been identified that affect other integrins, such as 2 1, present<br />
on platelets <strong>and</strong> other vascular cells. C-type lectin-like proteins are a major group, either<br />
inhibiting platelet function by blocking receptors such as GPIb <strong>and</strong> 2 1, or, when<br />
multimeric, by clustering receptors such as GPVI <strong>and</strong> GPIb to activate platelets <strong>and</strong> thus<br />
remove them more efficiently. Metalloproteases are also constituents of major classes<br />
binding to receptors <strong>and</strong> either blocking function directly or by targeted cleavage.<br />
Phospholipases A2 are another major class blocking or activating platelet function <strong>and</strong>,<br />
since many are not enzymatically active, must function via specific receptors.<br />
Other venom components act by cleaving protease-activated receptors or modulating<br />
released ADP <strong>and</strong> thromboxane A2 formation. Some venom enzymes cleave key<br />
basement membrane components <strong>and</strong> directly affect platelet interactions with capillary<br />
blood vessels to cause haemorrhaging. L-amino acid oxidases activate or, in some cases,<br />
inhibit platelets via H2O2 production.<br />
o C-type lectins<br />
o Disintegrins<br />
o L-amino acid oxidases<br />
o Metalloproteinases<br />
o Phospholipases<br />
o serine proteases<br />
60<br />
Aura Kamiguti:<br />
BSc degree in 1970 (Pharmacy <strong>and</strong> Biochemistry), University of Sao Paulo, Brazil,<br />
MSc in 1988 (Pharmacology), University of Sao Paulo, Brazil <strong>and</strong>, PhD in 1995 (Haematology), University<br />
of Liverpool, U.K.<br />
Retired Lecturer (since October 2005), Dept. of Haematology, University of Liverpool. Member<br />
of the International Society of Thrombosis <strong>and</strong> Haemostasis (ISTH); of the Subcommittee on<br />
Exogenous Products Active on Haemostasis for the International Society of Thrombosis <strong>and</strong><br />
Haemostasis; of the International Society of Toxinology, <strong>and</strong> of the Brazilian Society of<br />
Toxinology. Major interest in haemostastic defects due to exogenous substances, in particular<br />
snake venom metalloproteinases with platelets, <strong>and</strong> in cell signalling in platelets <strong>and</strong> in malignant<br />
B cells. Intense collaboration with different groups with similar aims. Participation in several<br />
scientific events as invited speaker by organisers of meetings on behalf of above societies.<br />
Kamiguti AS, Serr<strong>and</strong>er L, Lin K, Harris RJ, Cawley JC, Allsup DJ, Slupsky JR, Krause KH <strong>and</strong><br />
Zuzel M (2005) Expression <strong>and</strong> activity of NOX5 in the circulating malignant B cells of hairy-cell<br />
leukaemia. J. Immunol. 175, 8424-8430.<br />
Laing GD, Compton SJ, Ramach<strong>and</strong>ran R, Fuller GL, Wilkinson MC, Wagstaff SC, Watson SP,<br />
Kamiguti AS, Theakston RDG <strong>and</strong> Senis YA (2005) Characterization of a novel protein from<br />
Proatheris superciliaris venom: proatherocytin, a 34-kDa platelet receptor PAR1 agonist. Toxicon<br />
46, 490-499.<br />
Allsup DJ, Kamiguti AS, Lin K, Sherrington PD, Matrai Z, Slupsky JR, Cawley JC <strong>and</strong> Zuzel M<br />
(2005) B-cell receptor translocation to lipid rafts <strong>and</strong> associated signaling differ between<br />
prognostically important subgroups of chronic lymphocytic leukaemia. Cancer Res. 65, 7328-<br />
7337.<br />
Howes JM, Kamiguti AS, Theakston RDG, Wilkinson MC <strong>and</strong> Laing GD (2005) Effects of three<br />
novel metalloproteinases from the venom of the West African saw-scaled viper, Echis ocellatus<br />
on blood coagulation <strong>and</strong> platelets. Biochim. Biophys. Acta 1724, 194-202.<br />
Kamiguti AS (2005) Platelets as targets of snake venom metalloproteinases. Toxicon 45, 1041-<br />
1049.<br />
61
3.30-4.00: Platelet interaction with multidomain soluble snake venom<br />
metalloproteinases<br />
Aura S. Kamiguti<br />
Department of Haematology, University of Liverpool<br />
Platelets play a central role in haemostasis during vascular injury. Adhesion of these cells<br />
to the exposed sub-endothelium, essential to stop haemorrhage, is initiated by the<br />
engagement of platelet surface glycoproteins to collagen fibres <strong>and</strong> collagen-bound von<br />
Willebr<strong>and</strong> factor (vWF). Following systemic envenoming by viperid or crotalid sankes,<br />
powerful enzymes such as snake venom metalloproteinases (SVMPs) contribute to<br />
haemorrhage. This is mainly attributed to their proteolytic activity on the extracellular<br />
matrix surrounding blood vessels. In addition, SVMPs are capable of inhibiting platelet<br />
responses to collagen. These secreted enzymes are multidomain proteins<br />
(metalloproteinase, disintegrin-like <strong>and</strong> cysteine-rich domains). Proteins containing both<br />
the disintegrin-like <strong>and</strong> cysteine-rich domains have been either purified or expressed <strong>and</strong><br />
have been demonstrated to have similar inhibitory effects on the platelet/collagen<br />
interaction. This observation has prompted investigations on the identification of the<br />
site(s) in both domains responsible for such effects. Both domains in fact contain relevant<br />
sequences that have the ability to inhibit platelet interaction with collagen.<br />
62<br />
Russolini Zingali<br />
1986 B.S. São Paulo University (Pharmacist)<br />
1989 M.Sc. Federal University from São Paulo (Molecular Biology)<br />
1993 Ph.D. Federal University from Rio de Janeiro (Biological Chemistry)<br />
Professional Experience / Present Position: Associate Professor, Institute of Medical<br />
Biochemistry, since January 1995, Universidade Federal do Rio de Janeiro, Rio de Janeiro.<br />
Research Interest: Snake venoms components acting on hemostasis<br />
My laboratory has been mainly involved in the characterization of anti-hemostatic compounds,<br />
having in mind the search for peptides <strong>and</strong> proteins which can be used as prototypes <strong>and</strong> models<br />
for the development of antithrombotic drugs.<br />
The major objective of the laboratory is to characterize in detail, in vitro as well as in vivo, the<br />
mechanism of action of snake venoms components that have a potential anti-hemostatic action.<br />
Lately, my interest has also turned to the suitable proteomics techiniques for the analysis of<br />
complex biological fluids such as venoms <strong>and</strong> also their effects on different cells <strong>and</strong> tissues.<br />
1. Castro KN, Carvalho AL, Almeida AP, Oliveira DB, Borba HR, Costa SS, Zingali RB. 2003<br />
Preliminary in vitro studies on the Marsypianthes chamaedrys (boia-caa) extracts at<br />
fibrinoclotting induced by snake venoms. Toxicon. Jun;41(7):929-32.<br />
2. Castro HC,. Lemos MGJ, Bon C, Zingali RB (2003). Comparative evaluation of immunological<br />
<strong>and</strong> structural similarities of snake venom C-type lectin proteins” Toxicon 41: 525-528.<br />
3. Moreira MF Coelho HSL, Zingali RB, Oliveira PL, Masuda, H (2003) Nitrophorins during life<br />
cycle of the blood-sucking bug Rhodnius prolixus. Insect Biochemistry <strong>and</strong> Molecular<br />
Biology. 33(1):23-8<br />
4. Monteiro R Q., Foguel D, Castro HC., <strong>and</strong> Zingali R B.(2003) “Subunit dissociation, unfolding,<br />
<strong>and</strong> inactivation of bothrojaracin, a C-type lectin-like protein from snake venom”<br />
Biochemistry. 42(2):509-15.<br />
5. Coelho AL, De Freitas MS, Mariano-Oliveira A, Rapozo DC, Pinto LF, Niewiarowski S, Zingali<br />
RB, Marcinkiewicz C, Barja-Fidalgo C. RGD- <strong>and</strong> MLD-disintegrins, jarastatin <strong>and</strong> EC3,<br />
activate integrin-mediated signaling modulating the human neutrophils chemotaxis,<br />
apoptosis <strong>and</strong> IL-8 gene expression.Exp Cell Res. 2004 Jan 15;292(2):371-84.<br />
6 .Guimarães-Gomes V (M), Oliveira-Carvalho AL, Junqueira-de-Azevedo ILM; Dutra*, DLS.,<br />
Pujol-Luz M. (IC),. Castro H. C, Lee Ho P e. Zingali RB (2004) Cloning, characterization<br />
<strong>and</strong> structural analysis of a C-type lectin from Bothrops insularis (BiL) venom. Arch.<br />
Biophys <strong>and</strong> Biochem.<br />
7. Lima LM; Zingali RB; Foguel D; Monteiro RQ (2004) New Insights On Conformational And<br />
Functional Stability Of Human [[Alpha]]-Thrombin Probed By High Hydrostatic Pressure<br />
Eur. J. Biochem.<br />
8. Wermelinger LS, Dutra DL, Oliveira-Carvalho AL, Soares MR, Bloch C Jr, Zingali RB. (2005)<br />
Fast analysis of low molecular mass compounds present in snake venom: identification of<br />
ten new pyroglutamate-containing peptides. Rapid Commun Mass<br />
Spectrom.;19(12):1703-8.<br />
9. Soares MR, Oliveira-Carvalho AL, Wermelinger LS, Zingali RB, Ho PL, Junqueira-de-Azevedo<br />
ID, Diniz MR. (2005). Identification of novel bradykinin-potentiating peptides <strong>and</strong> C-type<br />
natriuretic peptide from Lachesis muta venom. Toxicon.;46(1):31-38.<br />
63
4.00-4.30: Structural <strong>and</strong> pharmacological studies of bothrojaracin, a<br />
(pro)thrombin inhibitor<br />
Zingali, R. B. 1 , Castro, H.C. 2 , Monteiro, RQ 1 , Assafim, M. 1 , Bon C. 3<br />
1<br />
Instituto de Bioquímica Médica, CCS, UFRJ; Rio de Janeiro Brazil<br />
2<br />
Departamento de Biologia Molecular e Celular. UFF. Rio de Janeiro Brazil;<br />
3<br />
Département : Régulations, Développement et Diversité moléculaire Muséum national d'Histoire<br />
naturelle (MNHN) Paris -France<br />
Bothrojaracin (BJC), a 27 kDa C-type lectin-like protein from Bothrops jararaca<br />
venom it has only 11 of the 13 amino-acid residues that are important for the Carbohydrate<br />
Recognition Domain (CRD), thus it does not bind to carbohydrates. Bothrojaracin-like<br />
molecules were detected in other snake venoms from Brazil. Structural analysis, by<br />
molecular modeling, <strong>and</strong> specially the electrostatic potential map of bothroinsularin<br />
purified from B. insularis venoms, which is very similar to BJC (α= 94 <strong>and</strong> β= 98),<br />
revealed significant differences that may be involved in the biological activity. BJC is a<br />
selective <strong>and</strong> potent thrombin inhibitor (KD = 0.6 nM) which interacts with both thrombin<br />
anion binding exosites (I <strong>and</strong> II) but not with the enzymes catalytic site. BJC also binds<br />
with high affinity (KD = 75 nM) to proexosite I. This ability would confer to BJC a new<br />
mechanism of action for an antithrombotic drug. We further analyzed the in vivo<br />
antithrombotic effect of BJC on a venous thrombosis model in rats that combines stasis<br />
<strong>and</strong> hypercoagulability. It was observed that intravenous administration of 3 mg/kg of<br />
thromboplastin combined with stasis caused 100% of thrombus incidence weighting 9.1 ±<br />
2.0 mg. In contrast, co-administration of 1 mg/kg of BJC decreased thrombus weight by<br />
95% (to 0.5 ± 0.1 mg). This effect was maintained at least for 48 hrs after drug<br />
administration. Accordingly, ex vivo aPTT was enhanced by 1.7-fold for BJC doses of 1<br />
mg/kg. In addition, we observed that this BJC doses caused significant hemorrhagic effect<br />
as compared to control animals. Using another animal model where the thromboembolism<br />
is induced by thrombin we observed that 80% of animal group died. When BJC (1mg/kg)<br />
is administered, 60 min prior to thromboembolism induction, it protects 100% of the mice<br />
group from dead. Western blotting assays using ex vivo rats or mice plasma showed an<br />
interaction between BJC <strong>and</strong> prothrombin along 120 min or 12 h, respectively. Altogether,<br />
our data show that BJC is a potent antithrombotic agent that could further help the<br />
development of new dual mechanistic drugs directed to prothrombin <strong>and</strong> thrombin<br />
inhibition. Financial Support: FAPERJ, IFS, CNPq, <strong>and</strong> FINEP.<br />
o Bothrojaracin<br />
o Bothrops jararaca venom<br />
o Anti-thrombotic<br />
o Thrombin inhibitor<br />
64<br />
Francis S. Markl<strong>and</strong>, Jr., PhD, is Associate Dean for Scientific Affairs <strong>and</strong> Professor of<br />
Biochemistry <strong>and</strong> Molecular Biology at the University of Southern California (USC) Keck School<br />
of Medicine. Dr. Markl<strong>and</strong> received his Ph.D. from Johns Hopkins University in 1964 in the<br />
laboratory of Drs. Albert Lehninger <strong>and</strong> Charles Wadkins. He then did a two year NIH post<br />
doctoral fellowship in protein chemistry with Dr. Emil Smith in the Biochemistry Department,<br />
University of California, Los Angeles, School of Medicine, where he was appointed assistant<br />
professor in 1966. He received an NIH Career Development Award from 1968-1973 while at<br />
UCLA. In 1974 he moved to the USC School of Medicine as Associate Professor in the<br />
Biochemistry Department <strong>and</strong> was appointed a member of the USC Comprehensive Cancer<br />
Center. In 1983 he was promoted to Professor of Biochemistry. He served as Acting Chair of the<br />
Biochemistry Department from 1996-1998 <strong>and</strong> as Vice Chair from 1988-1992. In 2004 he was<br />
appointed Associate Dean for Scientific Affairs at the USC Keck School of Medicine. He served<br />
as a member of the NIH Biochemical Endocrinology Study Section from 1996-2000 <strong>and</strong> served<br />
on several study sections during 2002-2003 to evaluate NCI grants in the small business<br />
program. Dr. Markl<strong>and</strong> is an internationally recognized expert on proteins from snake venoms<br />
<strong>and</strong> their potential clinical application. One of the proteins he originally purified from southern<br />
copperhead, called fibrolase, is presently in clinical trials, in a slightly altered forma <strong>and</strong> as a<br />
recombinant protein called alfimeprase, as therapy for peripheral arterial occlusive disease. He is<br />
presently working with nanospheres (lioposomes) as a delivery modality for a novel antiangiogenic<br />
agent that his laboratory purified from southern copperhead venom. Recently his<br />
laboratory perfected a technique, using as highly engineered bacterial expression system, to<br />
produce a recombinant version of the anti-angiogenic protein, which we now call vicrostatin.<br />
65
4.30-5.00: Contortrostatin <strong>and</strong> its anti-cancer action<br />
Markl<strong>and</strong>, F.S. 1* , Swenson, S. 1 , Minea, R. 1 , Costa, F.K. 1 , Fujii, G. 2 , Ernst, W. 2 ,<br />
Wang, Q. 1 , Pinski, J. 1<br />
1<br />
University of Southern California, 1303 North Mission Road, Los Angeles, California, USA<br />
*markl<strong>and</strong>@usc.edu<br />
2<br />
Molecular Express, Inc.,13310 South Figueroa Street, Los Angeles, California, USA<br />
Introduction: We provide an update on a homodimeric disintegrin, contortrostatin (CN),<br />
from southern copperhead venom. Recently we developed an expression system to prepare<br />
a recombinant version of CN <strong>and</strong> show that, like the natural protein, it has potent antitumor/anti-angiogenic<br />
activity in vivo. Interestingly, the recombinant protein, which we<br />
call vicrostatin (VN), is a monomer. Here we describe the anti-cancer activity of CN in<br />
prostate cancer (PC) <strong>and</strong> VN in breast cancer (BC) mouse models. Methods: CN was<br />
purified from venom by a four step HPLC procedure. VN was prepared using a highly<br />
engineered E. coli expression system. We used xenograft animal models of human PC<br />
(PC-3) <strong>and</strong> BC (MDA-MB-435); cells were implanted subcutaneously into the flanks of<br />
nude mice (PC-3) or into the mammary fat pads (MDA-MB-435). Therapy was initiated<br />
using an Alzet osmotic pump for CN in PC (315 μg/week) <strong>and</strong> liposomal delivery for VN<br />
in BC (LVN, intravenous twice weekly, 210 μg/week). Results: Using combination<br />
therapy in the PC-3 model, there is a significant enhancement of therapeutic efficacy with<br />
the combination of CN <strong>and</strong> docetaxel. Tumor volumes observed after four weeks of<br />
treatment: control, 378 mm 3 ; CN alone, 195 mm 3 ; docetaxel, 259 mm 3 , <strong>and</strong> the<br />
combination, 95 mm 3 . The impressive effect observed with the combination indicates that<br />
these two drugs complement each other very effectively <strong>and</strong> may offer a viable clinical<br />
option for therapy. For studies with VN in the MDA-MB-435 tumor, LVN was<br />
administered with no visible toxicity; 7-week treatment resulted in significant inhibition of<br />
tumor growth (80%). These results indicate that VN functions as effectively as CN in BC.<br />
For analysis of angiogenesis, BC tumor slices from treated <strong>and</strong> untreated mice were<br />
prepared for immunohistochemistry <strong>and</strong> incubated with monoclonal antibody to CD31 to<br />
detect angiogenic vessels. There is a 92% reduction of angiogenesis (p
5.00-5.30: Origin, evolution <strong>and</strong> recruitment of venom prothrombin activators in<br />
Australian Elapids<br />
Kini RM<br />
*<br />
Department of Biological Sciences, Faculty of Science, National University of<br />
Singapore, Singapore 117543 <strong>and</strong><br />
† Department of Biochemistry, VCU Medical Center, Virginia Commonwealth University,<br />
Richmond, Virginia 23298-0614, USA<br />
The parallel prothrombin activator (PA) system of Australian elapids provides an<br />
excellent opportunity to study proteins with similarity in structure <strong>and</strong> enzymatic<br />
properties, but differences in their physiological roles. Their venom contains prothrombin<br />
activators, which act as a toxin <strong>and</strong> induce microclots leading to cyanosis <strong>and</strong> death of the<br />
prey, whereas their plasma contains blood coagulation factors that activate prothrombin<br />
during injury <strong>and</strong> prevent excessive blood loss. These two PA are structurally similar, but<br />
with distinct physiological roles because of their highly tissue-specific expression. We<br />
have studied the protein <strong>and</strong> gene structure of group C <strong>and</strong> group D PAs. Group C PAs<br />
resemble factor Xa-Va complex, while group D PAs resemble factor Xa. The plasma<br />
coagulation factors are structurally similar to the venom PAs. Interestingly, we found an<br />
evolutionary intermediate between the venom <strong>and</strong> plasma prothrombin activator in the<br />
liver of P. textilis (PFX2) which is expressed ~56,000 times lower amounts compared to<br />
its functional plasma FX (PFX1). Recently, we determined the complete gene structure of<br />
FX <strong>and</strong> trocarin D from Tropidechis carinatus including their promoter regions. Both<br />
these plasma <strong>and</strong> venom prothrombin activator has similar gene structure. Both genes<br />
have eight exons with identical exon-intron boundaries. All the introns of trocarin D gene<br />
are nearly identical to that of FX gene, but for the two deletions (255 bp <strong>and</strong> 1,406 bp)<br />
<strong>and</strong> three insertions (214 bp, 1975 bp <strong>and</strong> 2174 bp) in the first intron. One of these<br />
insertions has a potential scaffold/matrix attached region. In addition, trocarin D<br />
promoter has a 264-bp insertion, which carries core promoter sequences <strong>and</strong> cis-elements<br />
that are known to induce high level of expression. This insertion may be responsible for<br />
the gene recruitment <strong>and</strong> may act as a switch for venom gl<strong>and</strong>-specific expression. We<br />
named this segment as VERSE (Venom Recruitment/Switch Element). Our studies<br />
provide the first molecular evidence for the origin <strong>and</strong> recruitment of venom PA gene<br />
from liver PA gene by gene duplication followed by changes in the cis-elements in the<br />
promoter <strong>and</strong> first intron leading towards functional diversification.<br />
68<br />
Professor André Ménez is the head of the Department of Protein Study <strong>and</strong><br />
Engineering of the Life Sciences Division at the French Atomic Energy<br />
Commission. He is Professor at the Institute of Technical <strong>and</strong> Nuclear Sciences.<br />
He has mostly studied proteins at the molecular level, including several toxins.<br />
He has received national <strong>and</strong> international awards, including the Redi Award<br />
from the International Society on Toxinology (2000). He was recently elected<br />
President of the IST. He has published more than 260 scientific papers <strong>and</strong><br />
written the book ‘The Subtle Beast. Snakes : from Myth to Medicine’ (Taylor &<br />
Francis).<br />
69
Friday 28 July: 9.00 am- 11.15 am<br />
Lecture theatre K3.25<br />
Symposium: Venomics<br />
9.00-9.10: Venomics: a project of the IST<br />
Ménez, A. 1 , Stöcklin, R. 2 <strong>and</strong> Mebs, D. 3<br />
70<br />
Sponsored by<br />
1<br />
Département d’Ingénierie et d’Etudes des Protéines, CEA/Saclay, Bât. 152, 91191 Gifsur-Yvette<br />
cedex, France<br />
2<br />
Atheris Laboratories, Case Postale 314, CH-1233 Bernex, Geneva, Switzerl<strong>and</strong><br />
3<br />
Zentrum der Rechtsmedizin, University of Frankfurt, Kennedyallee 104, D-60596<br />
Frankfurt, Germany<br />
Venomous animals possess potent factories, the venom gl<strong>and</strong>s, which produce a diversity<br />
of compounds tailored to act efficiently on key physiological systems of vertebrates<br />
<strong>and</strong>/or invertebrates. The Venomics project consists in studying the genetics,<br />
transcriptomics <strong>and</strong> proteomics of this evolutionary tripartite combination: animal,<br />
venomous system <strong>and</strong> toxins. The project is anticipated to clarify various essential<br />
biological aspects of this harmonious triad, <strong>and</strong> especially their associated evolutionary<br />
processes. It is also expected to have important practical consequences, including the<br />
discovery of new drug c<strong>and</strong>idates <strong>and</strong> the development of novel protective strategies<br />
against envenomation.<br />
- genomics<br />
- transcriptomics<br />
- proteomics<br />
Reto Stöcklin specialises in protein chemistry, mass spectrometry, proteomics<br />
<strong>and</strong> biocomputing. He co-founded the Swiss Proteomics Society <strong>and</strong> is a<br />
member of its Executive Committee. He is the owner <strong>and</strong> head of Atheris<br />
Laboratories, a company he founded in Geneva in 1995, which focuses primarily<br />
on research <strong>and</strong> development in life sciences. More recently, he established<br />
FunZyme BioTechnologies S.A., a company aimed at exploiting enzymes of<br />
fungal origins.<br />
71
9.10-9.15: Use of Mass Spectrometry in Venomics<br />
Reto Stöcklin<br />
Atheris Laboratories, case postale 314, CH-1233 Bernex-Geneva, Switzerl<strong>and</strong>,<br />
*reto.stocklin@atheris.ch - http://www.atheris.com<br />
Due to their complexity <strong>and</strong> diversity, animal venoms represent an extensive source of<br />
bioactive compounds for the development of drugs <strong>and</strong> therapeutic agents with two recent<br />
approvals (Prialt <strong>and</strong> Exenatide). Conventional approaches (extraction, fractionation <strong>and</strong><br />
screening) for their characterization often require large quantities of biological material<br />
<strong>and</strong> efficient biological assays. Current mass spectrometry (MS) techniques through a<br />
variety of approaches now give access to a wealth of information in a short working time<br />
frame with minute sample amount. Currently, MS-based peptide drug discovery may rely<br />
on computer-assisted strategies going backwards from structural information to functional<br />
activity. MALDI-TOF MS requires minimal sample preparation <strong>and</strong> can be used to rapidly<br />
perform molecular mass fingerprints of venoms at a high sensitivity to identifiy peptide<br />
families. Further characterization is achieved by de novo MS/MS peptide sequencing. This<br />
approach offers rapid access to primary peptide structures. The importance of<br />
complementary genomic <strong>and</strong> transcriptomic information will be highlighted.<br />
Favreau P., Menin L., Michalet S., Perret F., Cheneval O., Stöcklin M., Bulet P., Stöcklin<br />
R., 2006. Mass spectrometry strategies for venom mapping <strong>and</strong> peptide sequencing from<br />
crude venoms: case applications with single arthropod specimen. Toxicon, 47(6):676-87.<br />
Ménez A., Stöcklin R., Mebs, D., 2006. 'Venomics' or: The venomous systems genome<br />
project. Editorial. Toxicon, 47(3):255-259.<br />
o Venomics<br />
o Mass spectrometry<br />
o Proteomics<br />
72<br />
Prof. Dietrich Mebs<br />
Zentrum der Rechtsmedizin, Johann Wolfgang Goethe University Frankfurt,<br />
Kennedyallee 104, D-60596 Frankfurt, Germany.<br />
E-mail: mebs@em.uni-frankfurt.de<br />
Prof. Dietrich Mebs, born in Frankfurt, Germany, 1942, studied biology <strong>and</strong> biochemistry<br />
at the University of Frankfurt. He spent a half year for studies on the biochemistry of<br />
snake venoms at the Instituto Butantan in São Paulo, Brazil, <strong>and</strong> obtained his PhD in<br />
1968 from the University of Frankfurt. After graduating, Prof. Mebs joined the Center of<br />
Forensic Medicine, where he spent his entire career. As a postdoc researcher he stayed<br />
for a half year at the Institute of Protein Research, University of Osaka, Japan, in<br />
1970/71, where he elucidated the primary structure of alpha-bungarotoxin. After his<br />
habilitation in Forensic Sciences in 1979, he was promoted to Professor in 1985. Beside<br />
his forensic work in toxicology <strong>and</strong> DNA-typing his research interest is devoted to all<br />
aspects of toxinology, e.g. venoms <strong>and</strong> poisons of plant <strong>and</strong> animal origin, their<br />
chemistry <strong>and</strong> mode of action. Prof. Mebs has published more than 250 scientific articles<br />
<strong>and</strong> four books, among them "Venomous <strong>and</strong> Poisonous Animals" which first appeared<br />
in German ("Gifttiere") 1992. He teaches forensic medicine, toxicology <strong>and</strong> toxinology at<br />
the university as well as overseas (Philippines, Australia, South-Africa). Since 1982, he is<br />
Secretary-Treasurer of the International Society on Toxinology.<br />
73
9.15-9.20: Venom gl<strong>and</strong>s in old snakes<br />
Mebs, D.<br />
Zentrum der Rechtsmedizin, University of Frankfurt, Kennedyallee 104, D-60596 Frankfurt, Germany<br />
The study of delivery systems, particularly of venom producing gl<strong>and</strong>ular tissues, is part of<br />
the Venomics Project. Snakes developed a highly efficient venom apparatus, but fossil<br />
records supporting its gradual refinement during evolution are poor. The recent discovery<br />
of fossil snake fangs in the Oppenheim/Nierstein quarry (Germany) dating back to the<br />
Lower Miocene (about 23 million years before present) revealed a surprising similarity <strong>and</strong><br />
virtual identity with the fangs of modern elapid <strong>and</strong> viperid snakes (1). This implies that<br />
these snakes had venom gl<strong>and</strong>s much like their modern counterparts <strong>and</strong> must have used a<br />
similar potent mixture of proteins <strong>and</strong> peptides to quickly subdue their prey.<br />
Reference:<br />
1. Kuch, U., Müller, J., Mödden, C., Mebs, D., 2006, Naturwissenschaften 93, 84-87<br />
o Snake evolution<br />
o venom gl<strong>and</strong>s<br />
o snake fangs<br />
74<br />
Dr. Bryan Grieg Fry has a Ph.D. in Biochemistry from the Centre for Drug Design &<br />
Development, Institute for Molecular Biosciences <strong>and</strong> the Department of Biochemistry, University<br />
of Queensl<strong>and</strong>. He is currently the Deputy Director of the Australian Venom Research Unit at the<br />
University of Melbourne.<br />
Fry, BG (2005) “From genome to ‘venome’: Molecular origin <strong>and</strong> evolution of the snake venom proteome<br />
inferred from phylogenetic analysis of toxin sequences <strong>and</strong> related body proteins.” Genome<br />
Research 15:403-420.<br />
Fry BG, Lumsden N, Wüster W, Wickramaratna J, Hodgson WC, Kini RM. (2003b) “Isolation of a neurotoxin<br />
(alpha-colubritoxin) from a ‘non-venomous’ colubrid: evidence for early origin of venom in snakes.<br />
Journal of Molecular Evolution 57(4):446-452.<br />
Fry BG, Vidal N, Norman JA, Vonk FJ, Scheib H, Ramjan R, Kuruppu S, Fung K, Hedges SB, Richardson<br />
MK, Hodgson WC, Ignjatovic V, Summerhayes R <strong>and</strong> Kochva E (2006) “Early evolution of the venom<br />
system in lizards <strong>and</strong> snakes” Nature 439(7076):509-632; Advance online publication November 17,<br />
2005 doi:10.1038/nature04328.<br />
Fry BG, Wickramaratana J, Lemme S, Beuve A, Garbers D, Hodgson WC, Alewood P (2005) “Novel<br />
natriuretic peptides from the venom of the inl<strong>and</strong> taipan (Oxyuranus microlepidotus): Isolation,<br />
chemical <strong>and</strong> biological characterization” Biochemical <strong>and</strong> Biophysical Research Communications<br />
327:1011-1015.<br />
Fry BG, Wickramaratna JC, Hodgson WC, Alewood PF, Kini RM, Ho H, Wuster W. (2002) "Electrospray<br />
liquid chromatography/mass spectrometry fingerprinting of Acanthophis (death adder) venoms:<br />
taxonomic <strong>and</strong> toxinological implications" Rapid Communications in Mass Spectrometry 16:600-608.<br />
Fry BG, Wüster W (2004) “Assembling an arsenal: Origin <strong>and</strong> evolution of the snake venom proteome<br />
inferred from phylogenetic analysis of toxin sequences”. Molecular Biology <strong>and</strong> Evolution 21(5): 870-<br />
883.<br />
Fry BG, Wuster W, Kini RM., Brusic V, Khan A, Venkataraman D, Rooney AP. (2003a) “Molecular evolution<br />
of elapid snake venom three finger toxins” Journal of Molecular Evolution 57(1):110-129.<br />
Fry BG, Wüster W, Ramjan SFR, Jackson T, Martelli P, Kini RM. (2003c) “LC/MS (liquid chromatography,<br />
mass spectrometry) analysis of Colubroidea snake venoms: evolutionary <strong>and</strong> toxinological<br />
implications.” Rapid Communications in Mass Spectrometry 17: 2047-2062.<br />
Li M, Fry BG, Kini RM (2005) “Putting the brakes on snake venom evolution: the unique molecular<br />
evolutionary patterns of Aipysurus eydouxii (Marbled sea snake) phospholipase A2 toxins.” Molecular<br />
Biology <strong>and</strong> Evolution 22(4):934-941.<br />
Li M, Fry BG, Kini RM (2005) “Eggs only diet: the shift in preferred prey by the Marbled sea snake<br />
(Aipysurus eydouxii) resulting in a loss of postsynaptic neurotoxicity.” Journal of Molecular Evolution<br />
60(1):81-9.<br />
Wüster W, Dumbrell AJ, Hay C, Pook CE, Williams DJ, Fry BG (2004) “Snakes across the Strait: Trans-<br />
Torresian Phylogeographic Relationships in Three Genera of Australasian Snakes (Serpentes:<br />
Elapidae: Acanthophis, Oxyuranus <strong>and</strong> Pseudechis)” Molecular Phylogenetics <strong>and</strong> Evolution 34: 1-<br />
14.<br />
75
9.20-9.40: Evolution of the venom system in squamate reptiles<br />
Bryan G. Fry<br />
Australian Venom Research Unit, University of Melbourne, Parkville, Vic 3010 Australia<br />
bgf@unimelb.edu.au<br />
The origin, evolution <strong>and</strong> secreted bioactive properties of the reptile venom system was<br />
investigated through:<br />
• Phylogenetic reconstruction of the evolutionary relationships of secreted proteins<br />
in the dental gl<strong>and</strong>s of major squamate lineages.<br />
• Analysis of mechanisms of evolution in multigene families.<br />
• Macroecological investigation of the relationships between secreted protein types<br />
<strong>and</strong> features of squamate ecology, behaviour <strong>and</strong> venom gl<strong>and</strong> structure.<br />
• Bioactivity studies of representative purified proteins.<br />
A major outcome was the determination that the venomous function in squamates had a<br />
single, early origin; that Anguimorpha, Iguania <strong>and</strong> Serpentes share a common venomous<br />
ancestor. Bioactivity testing of the venom components from lizards previously<br />
considered ‘non-venomous’ demonstrated potent actions upon a myriad of physiological<br />
systems. This research reveals a vast treasure trove of novel biomolecules for use in drug<br />
design <strong>and</strong> development while also opening up new avenues of evolutionary studies.<br />
76<br />
After his Bachelor’s degree in chemistry from the University of the Philippines,<br />
Baldomero “Toto” Olivera obtained a PhD in biophysical chemistry from<br />
Caltech <strong>and</strong> subsequently undertook postdoctoral research at Stanford<br />
University. His early work was on DNA ligase <strong>and</strong> DNA replication, <strong>and</strong> he<br />
published a series of papers in Proceedings of the National Academy of<br />
Sciences, Journal of Biological Chemistry <strong>and</strong> Nature. After returning to the<br />
University of the Philippines as an Associate Professor of Biochemistry, Dr<br />
Olivera began what has been his continued <strong>and</strong> continuing interest in biologically<br />
active molecules from the venoms of marine cone snails.<br />
From 1970, Dr Olivera has been associated with the University of Utah, where he<br />
is currently Distinguished Professor of Biology. Throughout that time <strong>and</strong> in<br />
association with his longst<strong>and</strong>ing collaborator, Dr Lourdes Cruz, Dr Olivera has<br />
greatly developed our underst<strong>and</strong>ing of the chemistry, biology <strong>and</strong> pharmacology<br />
of Conus peptides <strong>and</strong> toxins. He has been involved in the discovery <strong>and</strong><br />
classification of many important types of bioactive molecules, including αconotoxin,<br />
ω-conotoxin, μ-conotoxin, κ-conotoxin, δ-conotoxin, ψ-conotoxin, <strong>and</strong><br />
conantokin peptides. Many of these are in use every day as pharmacological<br />
tools, <strong>and</strong> some have provided leads for novel therapeutic agents.<br />
Professor Olivera has produced over 230 publications <strong>and</strong> has received many<br />
awards, including the Redi Award from the International Society on Toxinology<br />
(2003).<br />
77
9.40-10.00: EXOGENOMICS AND POST-TRANSLATIONAL MODIFICATION<br />
OF CONUS PEPTIDES<br />
B. M. Olivera<br />
Dept. of Biology, University of Utah, Salt Lake City, Utah 84112, USA<br />
Most Conus venom peptides are encoded by a small number of gene superfamilies that<br />
are initially translated as prepropeptide precursors which are then posttranslationally<br />
modified. In some Conus peptide gene families, there is extensive <strong>and</strong> diverse posttranslational<br />
modification of the mature peptide toxins. A hallmark of these gene<br />
superfamilies is that they are rapidly diversifying; a biological rationale for this rapid<br />
diversification will be presented. Conus peptide gene superfamilies may represent the<br />
first examples of a distinctive part of the genome of all animals, which we propose to<br />
refer to as the exogenome.<br />
78<br />
Professor Ohno has been Professor of Biochemistry at Sojo University,<br />
Kumamoto, Japan since 1997. Prior to that, he has been Professor at Kyushu<br />
University <strong>and</strong> a Visiting Associate <strong>and</strong> Visiting Scientist at the National Institute<br />
of Health, USA. He has undergraduate <strong>and</strong> postgraduate degrees in chemistry<br />
from Kyushu University.<br />
79
10.00-10.20: Venomics of Protobothrops flavoviridis Venom-gl<strong>and</strong> Phospholipase A2<br />
Isozymes<br />
Chijiwa,T. 1 , Ikeda, N. 1 , Kariu, T. 2 , Ogawa, T. 3 , Oda-Ueda, N. 2 , Hattori, S. 4 , Ohno, M. 1<br />
1 Sojo University, Faculty of Bioscience <strong>and</strong> Biotechnology, Kumamoto 860-0082, Japan<br />
2 Sojo University, Faculty of Pharmaceutical Science, Kumamoto 860-0082, Japan<br />
3 Tohoku University, Department of Life Science, Sendai 981-8555, Japan<br />
4 University of Tokyo, Institute of Medical Science, Oshima-gun, Kagoshima 894-1531, Japan<br />
Introduction: Pairwise comparison of five Protobothrops flavoviridis venom-gl<strong>and</strong><br />
phospholipase A2 (PLA2) isozyme genes showed that the numbers of nucleotide<br />
substitution per nonsynonymous site are larger than or close to the numbers of nucleotide<br />
substitution per synonymous site, indicating that the genes have evolved in an accelerated<br />
manner. Such accelerated evolution is now thought to be universal for the genes coding for<br />
venom proteins <strong>and</strong> to have occurred for acquisition of particular physiological activities.<br />
The cause of accelerated evolution must be hidden in the genomic sequences encoding<br />
venom isozymes. In the present study a part of the genomic sequences encoding P.<br />
flavoviridis venom-gl<strong>and</strong> PLA2 isozymes was analyzed for their venomics.<br />
Here the term “venomics” is used in a narrow sense. This “venomics” includes<br />
elucidation of the mechanism of unique evolutionary phenomena <strong>and</strong> the function of<br />
venomous isozymes in addition to their genomics, transcriptomics <strong>and</strong> proteomics.<br />
Results <strong>and</strong> Discussion: P. flavoviridis genome contains 16-32 PLA2 isozyma genes (dot<br />
blot analysis) <strong>and</strong> it was suggested that they form a cluster on one microchromosome<br />
(FISH analysis). Shearing of P. flavoviridis liver DNA gave a 40 kbp fragment named<br />
NER1. Its 3’ terminal half (about 20 kbp long) contained the genes encoding edemainducing<br />
PLA-B, neurotoxic PLA-N <strong>and</strong> necrotic BPII. Five inverted repeat (IR)<br />
sequences (60 bp) <strong>and</strong> partial fragments of three retrotransposon-like elements were found.<br />
Reverse transcriptase encoded by retrotransposon might have played a role for gene<br />
duplication in ancient times. The fragment called NER2 (9 kbp) contained the gene coding<br />
for highly lipolytic <strong>and</strong> necrotic PLA2 <strong>and</strong> an inactive gene. Three IR sequences (110 bp)<br />
which are different from those in NER1 were also found in <strong>and</strong> between these genes.<br />
Expressed sequence tag (EST) analysis for P. flavoviridis venom gl<strong>and</strong> gave 466 cDNA<br />
clones. Most (42%) of the clones coded for PLA2 isozymes <strong>and</strong> 35% for nontoxic proteins.<br />
More time is required for getting more data.<br />
Key Words: Protobothrops flavoviridis, Phospholipase A2 isozymes, Accelerated<br />
evolution, Venomics.<br />
80<br />
Prof. Eugene V. Grishin is currently a Professor <strong>and</strong> Deputy Director of the<br />
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry (IBCh) at the Russian<br />
Academy of Sciences. He graduated in 1969 from the Chemical<br />
Department at the Moscow State University. He received his Ph.D. in<br />
Chemistry (1973) <strong>and</strong> D.Sc. in Chemistry (1985) from the IBCh. He began<br />
his scientific career in 1969 as a junior scientist at the IBCh in Moscow. In 1987,<br />
he was appointed Head of the Laboratory for Neuroreceptors <strong>and</strong><br />
Neuroregulators at the IBCh <strong>and</strong> became Deputy Director of the IBCh in<br />
1988. Since 1988, he has been a Professor of Bioorganic Chemistry at the<br />
Biological Department of Moscow State University. In 1997, he was elected to<br />
become part of the body of the Russian Academy of Sciences. His current<br />
scientific interests include toxinology, protein chemistry, neurochemistry, <strong>and</strong><br />
molecular neurobiology in general.<br />
81
10.20-10.40: Wide variety <strong>and</strong> multiplicity of spider toxins: structural aspects<br />
Grishin, E.V, Kozlov, S.A.<br />
Shemyakin-Ovchinnikov Institute, Russian Academy of Sciences, Miklukho-Maklaya, 16/10, Moscow<br />
V437, 117997, Russia, grev@ibch.ru<br />
Spider toxins can be divided into four major groups: polyamine toxins, high molecular<br />
mass toxins, cystine knot <strong>and</strong> non-cystine knot polypeptide toxins. Commonly, spider<br />
venom contains one or two groups of toxins. The majority of known toxins are small<br />
polypeptides with molecular weight 4-7 kDa cross-linked by 3-5 disulfide bonds. Such<br />
toxins are the most investigated <strong>and</strong> the corresponding structural <strong>and</strong> genomic information<br />
is also available. Generally, individual venom contains about ten structural families of<br />
polypeptide toxins, the number of homologues in each family ranges from 1 up to several<br />
dozen. For this reason, spider venom represents a peculiar naturally edited combinatorial<br />
library of polypeptide molecules. Sequence analysis of the available spider toxins resulted<br />
in formulation of common patterns present in these molecules. The principal structural<br />
motif (PSM) <strong>and</strong> the extra structural motif (ESM) describe the position of cysteine residues<br />
in the polypeptide molecule. One can propose five main structural classes of spider toxins.<br />
1 – toxins with PSM only, 2 – toxins with PSM <strong>and</strong> ESM, 3 – toxins with ESM only, 4 –<br />
toxins with the lack of PSM <strong>and</strong> ESM, 5 – cysteine-free toxins. Analysis of spider venom<br />
gl<strong>and</strong> EST database allowed us to elucidate the structural features typical of toxin<br />
precursor organization <strong>and</strong> the process of toxin maturation.<br />
o spider toxin<br />
o structural motif<br />
o toxin precursor<br />
82<br />
R.M. Kini<br />
Present Appointment: Professor, National University of Singapore; Affiliate Associate<br />
Professor, Medical College of Virginia, USA; Adjunct Research Scientist, DMRI, Singapore<br />
Research Areas: Structure-function relationships <strong>and</strong> mechanism of<br />
protein toxins; Protein-protein interactions; Protein design & engineering; Thrombosis <strong>and</strong><br />
hemostasis.<br />
Academic/Professional Qualifications:<br />
BSc (1977); MSc (1979); PhD (1983), University of Mysore. Mysore, India<br />
Awards/Honours (Post-PhD):<br />
• Japanese Government Ministry of Education Monbusho Fellowship (1985-86)<br />
• John B. Foote Award from Medical College of Virginia (1991-92)<br />
• French Academic Exchange Fellowship to visit France (1996)<br />
Career History:<br />
• Scientific Officer, National Institute of Immunology, New Delhi, INDIA (1984-85)<br />
• Postdoctoral Fellow, Kyushu University, Fukuoka, JAPAN (1985-86)<br />
• Lecturer in Biochemistry, University of Mysore, Mysore, INDIA (1986)<br />
• Postdoctoral Fellow (1986-92); Research Associate (1992-94) Medical College of Virginia, Virginia<br />
Commonwealth University, Richmond, U.S.A.<br />
• Research Fellow (1994-95); Senior Scientist (1996-2000); Associate Professor (2000-2004);<br />
Professor (2005-present) Department of Biological Sciences, National University of Singapore,<br />
Singapore<br />
Entrepreneurship:<br />
• Co-founder of ProScience, Inc., Richmond, Virginia, USA (1993-2003)<br />
• Founder of ProTherapeutics Private Limited, Singapore (2005-present)<br />
Professional/Consulting Activities:<br />
• Consultant to General Medical Industries, Virginia, USA (1998-Present)<br />
• Consultant to Perkin Elmer, Singapore (1999-Present)<br />
Publications:<br />
• 28 Patents; 108 Research papers; 18 Reviews <strong>and</strong> 8 Book chapters<br />
• Edited a monograph “Venom Phospholipase A2 Enzymes; Structure, Function <strong>and</strong> Mechanism”<br />
John Wiley, 1997<br />
• Guest editor, Proceedings of “International Conference on Exogenous Factors Affecting<br />
Thrombosis <strong>and</strong> Haemostasis”, Haemostasis Vol. 31, pp. 117-311, S. Karger, 2002<br />
• Guest editor, “Exogenous Factors Affecting Thrombosis <strong>and</strong> Hemostasis” in Current Drug<br />
Targets – Cardiovascular <strong>and</strong> Hematologic Disorders. Bentham Publishers, 2004<br />
83
10.40-11.00: Protein scaffolds in snake venoms<br />
Kini RM<br />
*<br />
Department of Biological Sciences, Faculty of Science, National University of<br />
Singapore, Singapore 117543 <strong>and</strong><br />
† Department of Biochemistry, VCU Medical Center, Virginia Commonwealth University,<br />
Richmond, Virginia 23298-0614, USA<br />
Snake venoms are complex mixtures of pharmacologically active peptides <strong>and</strong> proteins.<br />
These protein toxins belong to a small number of superfamilies of proteins. The members<br />
in a single family show remarkable similarities in their primary, secondary <strong>and</strong> tertiary<br />
structures. At times, however, they differ from each other in their biological targeting<br />
<strong>and</strong> hence their pharmacological effects. Thus, each family of protein toxins has a<br />
similar molecular scaffold but exhibit multiple functions. Thus structure-function<br />
relationships <strong>and</strong> the mechanisms of action of snake venom proteins are intriguing <strong>and</strong><br />
pose exciting challenges to scientists. So far, we have understood structure-function<br />
relationships of only a small number of toxins in the some of these families. An<br />
overview of this theme of molecular molds with multiple missions will be illustrated by<br />
the structure-function relationships of families of snake venom toxins.<br />
84<br />
Dr Richard Lewis is a co-founder of Xenome <strong>and</strong> holds joint appointments as<br />
Xenome's CSO <strong>and</strong> Principle Investigator (Molecular Pharmacology) at the<br />
Institute for Molecular Bioscience (IMB), University of Queensl<strong>and</strong>. He has over<br />
20 years experience in the field of marine toxin pharmacology, particularly ion<br />
channel <strong>and</strong> transporter modulation. He has managed several commercial<br />
venom research programs at IMB, <strong>and</strong> spearheaded the development of new<br />
classes of peptide therapeutic c<strong>and</strong>idates. He currently heads a NHMRC<br />
Program Grant focused on dissecting pain pathways using conotoxins. Richard<br />
has published extensively in major peer reviewed international journals.<br />
85
11.00-11.20: Venomics to drugs: the Xen2174 story<br />
Richard Lewis 1,2<br />
1 Institute for Molecular Bioscience, University of Queensl<strong>and</strong>, Brisbane, QLD,<br />
Australia, <strong>and</strong> 2 Xenome Ltd, Brisbane, QLD, Australia.<br />
Venom peptides are increasingly being explored as a valuable source of novel<br />
bioactive molecules. Cone snail venom contains some 50,000 small bioactive<br />
peptides that target numerous specific ion channels <strong>and</strong> receptors. A number of<br />
classes of conotoxins have emerged with therapeutic potential. Recent studies<br />
indicate that several of these conopeptides may be useful in the treatment of<br />
neuropathic <strong>and</strong> inflammatory pain states. Xenome Ltd was founded to mine the<br />
diversity <strong>and</strong> complexity of Australian venoms, especially the conopeptides, for<br />
novel peptide therapeutics. In this presentation, the discovery, pharmacological<br />
characterization, analgaesic action <strong>and</strong> clinical development of χ-conopeptides<br />
are described. Through inhibition of the norepinephrine transporter (NET), χconopeptides<br />
elevate the levels of NE leading to the amplification of inhibitory<br />
pathways <strong>and</strong> the reduction in the strength of pain signals reaching the brain.<br />
Xen2174, an unnatural χ-conopeptides analogue with improved potency,<br />
selectivity, stability <strong>and</strong> ease of synthesis, was found to be well tolerated<br />
intravenously in a Phase I safety trial. Xen2174 is currently being evaluated<br />
intrathecally in a Phase I/IIa safety trial in cancer patients suffering severe<br />
otherwise unmanageable pain.<br />
86<br />
87
Oral communications<br />
Abstracts<br />
All oral communications will be in lecture<br />
theatres K3.14, K3.17 <strong>and</strong> K3.25 in the<br />
John Anderson Building<br />
Premier sponsors:<br />
88<br />
Monday Track 1, K3.14 3.00-3.20pm<br />
MT7 muscarinic toxin as tool to study direct allosteric interactions on M1<br />
muscarinic receptor<br />
Servent, D.* , Fruchart-Gaillard, C., Mourier, G., Marquer, C. , Ménez,<br />
A.<br />
CEA, Département d’Ingénierie et d’Etudes des Protéines, 91191 Gifsur-Yvette,<br />
France, * denis.servent@cea.fr<br />
There is now clear evidence showing that muscarinic receptors, as<br />
many other GPCRs, are susceptible to allosteric modulation. The effects<br />
of allosteric lig<strong>and</strong>s are consistent with a ternary complex model in<br />
which the orthosteric <strong>and</strong> allosteric agents bind simultaneously to the<br />
receptor <strong>and</strong> modify each other’s affinities. Until now, all<br />
pharmacological characterizations of allosteric agents interacting with<br />
M1 muscarinic receptor were derived from their indirect effects on the<br />
binding of orthosteric lig<strong>and</strong> such as NMS. Thus, the lack of<br />
radiolabeled allosteric selective lig<strong>and</strong>s prevents a direct investigation<br />
of the interaction of allosteric agents with their specific binding sites.<br />
For instance, pharmacological studies of the MT7-hM1 interaction have<br />
been always done indirectly <strong>and</strong> the mode of action of this allosteric<br />
toxin is not yet fully understood.<br />
We’ll present recent results obtained by equilibrium <strong>and</strong> kinetic<br />
experiments of the interaction of a monoiodinated MT7 toxin with hM1<br />
receptor in its free <strong>and</strong> NMS-occupied states. Thus, the negative<br />
cooperativity between MT7 <strong>and</strong> NMS has been evaluated directly <strong>and</strong><br />
the effect of various orthosteric <strong>and</strong> allosteric agents on the [125I]-MT7<br />
binding was measured <strong>and</strong> sheds new light on the mode of interaction<br />
of these lig<strong>and</strong>s.<br />
Our results suggest that MT7 toxin interacts with hM1 receptor at a<br />
specific allosteric site which may partially overlap those previously<br />
identified for “classical” or “atypical” allosteric agents <strong>and</strong> highlight the<br />
potential of this new allosteric tracer in studying allosterism at<br />
muscarinic receptors.<br />
- muscarinic toxin<br />
- muscarinic acetylcholine receptors<br />
- allosteric interaction<br />
89
Monday Track 1, K3.14 3.20-3.40pm<br />
α-Conotoxins And Their Targets: From Photolabeling To The Three-Dimensional<br />
Structures<br />
Tsetlin, V.I. 1* , Kasheverov, I. E 1 , Cohen, J.B 2 ., Smit, A.B 3 ., Sixma T.K 4 .<br />
1<br />
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya<br />
street 16/10, Moscow, Russia, *vits@ibch.ru.<br />
2<br />
Dept. Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston MA 02115, USA<br />
3<br />
Department of Molecular <strong>and</strong> Cellular Neurobiology, Vrijie Universiteit, De Bolelaan 1085, 1081 HV<br />
Amsterdam, The Netherl<strong>and</strong>s<br />
4<br />
Netherl<strong>and</strong>s Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherl<strong>and</strong>s<br />
Introduction. α-Conotoxins (αCt), short neurotoxic peptides from the Conus snails,<br />
distinguish various nicotinic acetylcholine receptors (nAChRs). The X-ray structure of the<br />
acetylcholine-binding protein (AChBP) is an excellent model for lig<strong>and</strong>-binding domains<br />
of nAChRs. Our purpose was to elucidate the spatial structures of the αCt complexes with<br />
the AChBP <strong>and</strong> with the Torpedo californica AChR.<br />
Methods. The αCt PnIA[A10L, D14K] has been obtained by solid-phase peptide synthesis<br />
<strong>and</strong> shown to interact with neuronal α7 nAChR <strong>and</strong> AChBPs from A.californica <strong>and</strong><br />
L.stagnalis. G1(Bpa12), a photoactivatable analog of αCt G1 bearing a<br />
benzoylphenylalanine (Bpa) residues in position 12, has been synthesized. UV-irradiation<br />
of the Torpedo californica AChR complex with radioiodinated G1(Bpa12) resulted in<br />
specific labeling of the α, γ <strong>and</strong> δ subunits.<br />
Results. X-ray structure (2.4Å) has been established for the complex of PnI[A10L, D14K]<br />
<strong>and</strong> A.californica AChBP [1]. The toxin retains its conformation but induces<br />
conformational changes in AChBP, the most pronounced in the C-loop. For G1 (Bpa12),<br />
photolabeling sites in the Torpedo nAChR were mapped to the fragment starting at<br />
αSer173 <strong>and</strong> including loop C; in γ-subunit, crosslinks are in the fragments including<br />
segments F <strong>and</strong> D.<br />
Discussion. The X-ray structure of the AChBP complex sheds light on the interaction of<br />
αCt with distinct nAChRs. It was used to build models of α7 nAChR-αCt complexes <strong>and</strong> to<br />
rationalize the crosslinking results suggesting the two possible orientations of the Bpa side<br />
chain in the complex.<br />
References. 1. Celie P.H.N., Kasheverov I.E., Mordvintsev D.Y., Hogg R.C., van Nierop<br />
P., van Elk R., van Rossum-Finkert S.E., Zhmak M.N., Bertr<strong>and</strong> D., Tsetlin V., Sixma<br />
T.K., Smit A.B. Nature Str.Mol.Biol.12, 582-588 (2005)<br />
2. Kasheverov I.E., Chiara D.C., Zhmak M.N., Maslennikov I.V., Pashkov V.S., Arseniev<br />
A.S., Utkin Yu.N., Cohen J.B., Tsetlin V.I. FEBS J. 273, 1373-1388(2006)<br />
o α-conotoxins<br />
o acetylcholine-binding protein<br />
o photolabeling<br />
o nicotinic acetylcholine receptor<br />
90<br />
Monday Track 1, K3.14 3.40-4.00pm<br />
Interaction of Erythrina <strong>and</strong> Phelline Alkaloids with the Neuronal α4β2 Nicotinic<br />
Acetylcholine Receptor (nAChR)<br />
Wildeboer, K.M 1* , Soti, F. 1 , Langlois, N. 2 , Kem, W.R. 1<br />
1 University of Florida, COM, 1600 SW Archer Road, Gainesville, Florida, USA, kwildebo@ufl.edu<br />
2 Institut de Chimie des Substances Naturelles, CNRS, 91198 Gif-sur-Yvette Cedex, Paris, France<br />
Neuronal nAChRs have been implicated in neurodegenerative diseases <strong>and</strong> substance<br />
abuse. The high affinity nAChR subtype α4β2 participates in the dopamine-releasing <strong>and</strong><br />
cognition-enhancing effects of nicotine. Involvement of this <strong>and</strong> other β2-subunit<br />
containing receptors in addiction has led these receptors to become targets for the design of<br />
new smoking cessation drugs. The development of selective α4β2 antagonists would<br />
permit a more rigorous testing of the involvement of these receptors in various neuronal<br />
processes <strong>and</strong> nicotine addiction. Seeds (coral beans) from the plant genus Erythrina<br />
contain alkaloids that are nAChR antagonists. These compounds share a common scaffold<br />
consisting of four fused rings. Early studies determined that Erythrina alkaloids produce<br />
curare-like blockade of neuromuscular <strong>and</strong> ganglionic transmission. A semi-synthetic<br />
compound, dihydro-β-erythroidine (DHβE), has been widely used to investigate the<br />
neuronal α4β2 nAChR. One aromatic compound, erysodine, was shown to be more potent<br />
than DHβE on rat α4β2 receptors. Our goal was to assess structure-activity relationships<br />
of these alkaloids for the α4β2 nAChR using radiolig<strong>and</strong> binding on rat <strong>and</strong> human<br />
receptors <strong>and</strong> functional (Flexstation) methods on human α4β2. Two groups of Erythrina<br />
alkaloids were studied: the D-ring lactones (β-erythroidines) <strong>and</strong> the D-ring aromatic<br />
compounds. A related group of homoerythrina alkaloids from the genus Phelline were<br />
also tested. The aromatic Erythrina compounds were the most active. Erysovine had the<br />
highest affinity <strong>and</strong> potency for human α4β2 nAChRs. Opening the D-ring of βerythroidine<br />
did not result in loss of activity; therefore major determinants for binding<br />
must occur within the other three rings. DHβE was more active than the diene or<br />
tetrahydro-forms of β-erythroidine. The Phelline alkaloid, O-methylisophellibiline<br />
(identical structure to DHβE except it contains a seven-membered C-ring), bound 11-fold<br />
less tightly than DHβE. Overall, various modifications of the D-ring still allow high<br />
affinity binding to the α4β2 nAChR <strong>and</strong> inhibition of the receptor response.<br />
o Nicotinic acetylcholine receptor<br />
o Nicotinic receptor antagonist<br />
o Erythrina alkaloid<br />
o Nicotine addiction<br />
91
Monday Track 1, K3.14 4.00-4.20pm<br />
Binding of 3-(Benzylidene)-Anabaseines to Mammalian Nicotinic Acetylcholine<br />
Receptors <strong>and</strong> Molluscan ACh Binding Proteins<br />
Kem, W.R 1* , LeFrancois, S 1 , Talley, T 2 , Prokai, L 1 , Taylor, P 2 , MacDougall, K 1 , Gallo, R 1 , Soti, F 1<br />
1 Department of Pharmacology <strong>and</strong> Therapeutics, University of Florida, 1600 SW Archer Road,<br />
Gainesville, FL, USA, *kem@pharmacology.ufl.edu<br />
2 Department of Pharmacology,University of California San Diego, 9500 Gilman Drive, La Jolla, CA, USA<br />
The marine toxin anabaseine is converted into a selective partial agonist for α7<br />
nicotinic acetylcholine receptors (nAChRs) by addition of a benzylidene or<br />
cinnamylidene moiety at the 3-position of its tetrahydropyridyl ring. One benzylideneanabaseine<br />
(BA), DMXBA [3-(2,4-dimethoxybenzylidene)-anabaseine, also called GTS-<br />
21)], is in clinical trials for treatment of cognition deficits in schizophrenia <strong>and</strong><br />
Alzheimer’s disease. Substituents on the benzylidene moiety influence compound<br />
ionization, lipophilicity <strong>and</strong> steric properties <strong>and</strong> influence nAChR binding <strong>and</strong><br />
activation. We measured the nAChR binding affinities of >40 benzylidene-anabaseine<br />
derivatives to infer a quantitative structure activity relationship (QSAR) for each of the<br />
two major mammalian brain receptor subtypes, α4β2 <strong>and</strong> α7. To sample a relatively<br />
large chemical space, BAs containing substituents representing a wide range of electrondonating,<br />
lipophilicity <strong>and</strong> steric bulk properties were synthesized <strong>and</strong> tested. Using a<br />
QSAR program, several parameters (the number of electronic charges on heteroatoms,<br />
calculated dipoles <strong>and</strong> experimentally derived pKa’s of the compounds) were utilized to<br />
calculate QSAR equations representing the best fit of the binding data for α7 <strong>and</strong> α4β2<br />
nAChRs. There was good agreement between the predicted <strong>and</strong> the experimental<br />
compound affinities when only three descriptors were used. Substituent influence on BA<br />
binding to three molluscan AChBPs, with some notable exceptions, was generally found<br />
to be similar to binding to that of rat brain α7 receptors. A notable exception was the<br />
relatively low binding affinity of anabaseine <strong>and</strong> its analog PTHP for the AChBPs.<br />
QSAR <strong>and</strong> related data for these compounds will be useful in predicting binding of<br />
virtual compounds prior to their synthesis <strong>and</strong> pharmacological characterization.<br />
(Supported by NIH grants to both labs).<br />
o Nicotinic acetylcholine receptor<br />
o Acetylcholine binding protein<br />
o Anabaseine<br />
o GTS-21<br />
92<br />
Monday Track 1, K3.14 4.20-4.40pm<br />
The Structural Characterisation <strong>and</strong> Receptor Specificity of α-Conotoxin Vc1.1<br />
Adams, D.J 1* , Clark, R.J 2 , Fischer, H 1 , Nevin, S.T 1 , Craik, D.J. 2<br />
1 School of Biomedical Sciences <strong>and</strong> 2 Institute for Molecular Bioscience, The University of Queensl<strong>and</strong>,<br />
Brisbane, Queensl<strong>and</strong>, 4072, Australia, *dadams@uq.edu.au<br />
The α-conotoxin Vc1.1 was first discovered using a PCR screen of cDNAs from the<br />
venom ducts of Conus victoriae (1). The cysteine spacing within the sequence of Vc1.1<br />
suggests that it is a member of the 4/7 subclass of α-conotoxins. α-Conotoxin Vc1.1 is a<br />
small disulfide bonded peptide currently in development as a treatment for neuropathic<br />
pain (2). In the present study, the structure <strong>and</strong> activity of Vc1.1 <strong>and</strong> two derivatives, vc1a<br />
<strong>and</strong> [P6O]-Vc1.1, which contain the post-translationally modified residues hydroxyproline<br />
<strong>and</strong> γ-carboxyglutamate were examined using NMR spectroscopy <strong>and</strong> electrophysiological<br />
techniques. Vc1.1 inhibited reversibly nicotine-evoked membrane currents in isolated<br />
bovine chromaffin cells in a concentration dependant manner. Vc1.1 also inhibited the<br />
ACh-evoked membrane currents of recombinant nAChR’s expressed in Xenopus oocytes<br />
preferentially targeting peripheral nAChR subtypes over central subtypes. Specifically,<br />
Vc1.1 is selective for α3-containing nAChR subtypes, α3β2 <strong>and</strong> α3β4 having IC50 values<br />
of 7.3 <strong>and</strong> 4.2 μM, respectively, whereas the central <strong>and</strong> muscle nicotinic subtypes<br />
α4β2, α4β4, α7 <strong>and</strong> α1β1γδ, all exhibited IC50 values > 30 μM. Lowering the external pH<br />
from 7.4 to 6.0, increased the potency of Vc1.1 for α3β4 suggesting that a protonated<br />
histidine may be important for the receptor interaction. Application of 10-30 μM vc1a or<br />
[P6O]-Vc1.1 failed to inhibit ACh-evoked currents mediated by all nAChR subunit<br />
combinations expressed in oocytes. The 3D structure of Vc1.1 comprises a small α-helix<br />
spanning residues P6 to D11 <strong>and</strong> is braced by the I-II, III-IV disulfide connectivity seen in<br />
other α-conotoxins. Comparison of the structure of Vc1.1 with other α-conotoxins, taken<br />
together with nAChR selectivity data, suggests that the conserved proline at position 6 is<br />
important for binding while a number of residues in the C-terminal portion of the peptide<br />
contribute towards the selectivity. This structure should open new opportunities for further<br />
development of Vc1.1 or analogues as analgesic agents.<br />
1. S<strong>and</strong>all et al. (2003) Biochemistry, 42, 6904-6911.<br />
2. Satkunanathan et al. (2005) Brain Research, 1059,149-158.<br />
o nicotinic receptor<br />
o α-conotoxin<br />
o synthetic peptide<br />
o NMR structure<br />
93
Monday Track 1, K3.14 4.40-5.00pm<br />
Gymnodimine-A targets muscular <strong>and</strong> neuronal nicotinic acetylcholine receptors<br />
with high affinity<br />
Kharrat, R. 1,2 , Servent, D. 3 , Girard, E. 1 , Ouanounou, G. 1 , Molgó, J 1 *<br />
1 CNRS, Institut de Neurobiologie Alfred Fessard – FRC2118, Laboratoire de Neurobiologie<br />
Cellulaire et Moléculaire – UPR9040, Gif sur Yvette, France, *Jordi.Molgo@nbcm.cnrs-gif.fr<br />
2 Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, Tunisie.<br />
3 CEA/Saclay, Département d’Ingénierie et d’Etudes des Protéines, Gif sur YVette, France.<br />
Gymnodimines (GYM) are bioactive phycotoxins, isolated from contaminated shellfish<br />
<strong>and</strong> produced by the dinoflagellate Karenia selliformis. GYM exhibit unusual structural<br />
features, including a spirocyclic imine ring system <strong>and</strong> a trisubstituted tetrahydrofuran<br />
embedded within a 16-membered macrocycle. GYM are designated as "fast-acting toxins"<br />
due to the rapid onset of neurological symptoms <strong>and</strong> rapid death following intraperitoneal<br />
or intracerebroventricular injection to mice. To get information on the toxic potential <strong>and</strong><br />
the molecular target of GYM, we have studied the mechanism of action of highly purified<br />
gymnodimine-A (GYM-A) isolated from contaminated clams harvested in Tunisian coasts.<br />
For this, we used mouse bioassays, isolated frog <strong>and</strong> mouse neuromuscular preparations,<br />
cultured Xenopus laevis skeletal myocytes, cultured cells expressing different muscular<br />
<strong>and</strong> neuronal nicotinic acetylcholine (ACh) receptors (nAChRs) subtypes <strong>and</strong> conventional<br />
electrophysiological <strong>and</strong> binding-competition assays. The results show that GYM-A<br />
produced a concentration- <strong>and</strong> time-dependent blockade of twitch responses evoked by<br />
phrenic-nerve stimulation in mouse hemidiaphragm preparations. As GYM-A did not<br />
block twitch responses elicited by direct muscle stimulation, the GYM-A activity cannot<br />
be attributed to a direct inhibitory effect on muscle contractility, but to the blockade of<br />
postsynaptic nAChRs of the neuromuscular junction. This was confirmed by recording<br />
subthreshold endplate potentials <strong>and</strong> by the complete blockade of miniature endplate<br />
potentials in neuromuscular preparations treated with GYM-A, <strong>and</strong> by patch-clamp<br />
recordings showing that nicotinic currents evoked by constant <strong>and</strong> brief iontophoretical<br />
ACh pulses applied to the surface of skeletal myocytes were markedly reduced.<br />
Furthermore, competition-binding assays revealed that GYM-A is a powerful lig<strong>and</strong><br />
interacting with subnanomolar affinities not only with muscular, but also with<br />
homopentameric (α7) <strong>and</strong> heteropentameric (α4β2) neuronal nAChRs. In conclusion, our<br />
data show for the first time that GYM-A broadly targets nAChRs, <strong>and</strong> explain the basis of<br />
its neurotoxicity to various animal species.<br />
o gymnodimines<br />
o marine toxin<br />
o muscular nicotinic acetylcholine receptors<br />
o neuronal nicotinic acetylcholine receptors<br />
94<br />
Monday Track 1, K3.14 5.00-5.20pm<br />
The unique cysteine spacing of BuIA destabilizes the globular fold commonly<br />
found in α-conotoxins<br />
Daly, N.L 1 , Jin, A 1 , Br<strong>and</strong>stätter, H 1 , Nevin, S.T 2 , Adams, D.J 2 , Alewood, P.F 1 , Craik, D.J. 1<br />
1 Institute for Molecular Bioscience, University of Queensl<strong>and</strong>, Brisbane, Australia, n.daly@imb.uq.edu.au<br />
2 School of Biomedical Sciences, University of Queensl<strong>and</strong>, Brisbane, Australia<br />
α-Conotoxins isolated from the venom of cone snails have exciting therapeutic potential<br />
based on their high selectivity <strong>and</strong> affinity at nicotinic acetylcholine receptors (nAChRs).<br />
BuIA is a recently discovered neuronal α-conotoxin derived from a gene sequence with a<br />
unique intercysteine loop spacing. It is the only example in the α-conotoxins with four<br />
residues in the second loop, <strong>and</strong> makes an unusual 4/4 loop spacing framework. In the<br />
current study we have used an orthogonal cysteine-protection strategy to synthesise the<br />
globular (CysI-CysIII, CysII-CysIV) <strong>and</strong> ribbon (CysI-CysIV, CysII-CysIII) disulfide<br />
isomers of BuIA, <strong>and</strong> examined their structures <strong>and</strong> activity. Interestingly, the globular<br />
form, which is assumed to be the native connectivity as this is found in all α-conotoxins<br />
discovered to date, is active at the α6 nAChRs but displays multiple conformations in<br />
solution. In contrast, the ribbon connectivity isomer forms a well-defined single<br />
conformation in solution, but does not maintain activity at the nAChR. It appears the<br />
unique spacing of BuIA has a significant influence on the structure <strong>and</strong> destabilizes the<br />
globular form commonly found in α-conotoxins. Furthermore, a single well-defined<br />
solution conformation is not crucial for activity at the nAChR, <strong>and</strong> conformational<br />
flexibility may indeed facilitate binding.<br />
o conotoxin<br />
o structure<br />
o NMR<br />
o Disulfide connectivity<br />
95
Monday Track 1, K3.14 5.20-5.40pm<br />
Structural analysis of AChBP complexes with nicotinic agonists <strong>and</strong> antagonists<br />
Scott B. HANSEN 1,2 , Gerlind SULZENBACHER 3 , Todd T. TALLEY 1 , Tom HUXFORD 2 , Palmer TAYLOR 1 ,<br />
Yves BOURNE 3 , & Pascale MARCHOT 4*<br />
1 Departments of Pharmacology <strong>and</strong> 2 Chemistry <strong>and</strong> Biochemistry, UCSD, La Jolla, CA, USA.<br />
3 Architecture et Fonction des Macromolécules Biologiques, CNRS/Univ, Marseille, France.<br />
4 Ingénierie des Protéines, CNRS/Univ, IFR Jean Roche, Université de la Méditerranée, Faculté de<br />
Médecine Secteur Nord, Marseille, France, * marchot.p@jean-roche.univ-mrs.fr.<br />
Acetylcholine binding proteins (AChBP) are soluble homopentameric surrogates of the<br />
extracellular lig<strong>and</strong> binding domain of the nicotinic acetylcholine receptor (nAChR). To<br />
study the conformational changes in the nAChR lig<strong>and</strong> binding domain that occur upon<br />
lig<strong>and</strong> binding <strong>and</strong> those that allosterically trigger channel gating upon agonist binding, we<br />
have solved crystal structures of Lymnaea stagnalis <strong>and</strong> Aplysia californica AChBPs in<br />
various complexes with peptidic <strong>and</strong> alkaloid antagonists <strong>and</strong> with alkaloid agonists,<br />
bound at the subunit interfaces [1,2]. Compared with the AChBP apo conformation,<br />
opening of loops C <strong>and</strong> F, that border the lig<strong>and</strong> binding pocket on each face of the<br />
interface, is associated with binding of the peptidic antagonists, α-cobratoxin <strong>and</strong> αconotoxin<br />
ImI, while no major conformational change occurs upon binding of the alkaloid<br />
antagonist, methyllycaconitine. In contrast, loop closure is associated with binding of the<br />
alkaloid agonists, lobeline <strong>and</strong> epibatidine, trapped within the binding pocket. The<br />
structures also reveal extended <strong>and</strong> non-overlapping interaction surfaces for the αconotoxin<br />
<strong>and</strong> methyllycaconitine antagonists, outside the binding loci for the agonists.<br />
This comprehensive set of structures suggests that substantial fluctuations in the AChBP<br />
conformation preexist lig<strong>and</strong> binding <strong>and</strong> shows that binding of antagonists <strong>and</strong> agonists<br />
distinctively locks the selected open or closed conformations. Hence it reflects a dynamic<br />
template for delineating further the conformational changes of the nAChR lig<strong>and</strong> binding<br />
domain elicited by lig<strong>and</strong> binding, of which loop closure may be an essential feature linked<br />
to channel opening.<br />
1. Bourne Y, Talley TT, Hansen SB, Taylor P, Marchot P (2005) Crystal structure of a Cbtx-AChBP complex<br />
reveals essential interactions between snake α-neurotoxins <strong>and</strong> nicotinic receptors. EMBO J 24, 1512–<br />
1522.<br />
2. Hansen SB, Sulzenbacher G, Huxford T, Marchot P, Taylor P, Bourne Y (2005) Structures of Aplysia<br />
AChBP complexes with nicotinic agonists <strong>and</strong> antagonists reveal distinctive binding interfaces <strong>and</strong><br />
conformations. EMBO J 24, 3635–3646.<br />
o Nicotinic agonist <strong>and</strong> antagonist<br />
o AChBP<br />
o High affinity complex<br />
o Crystal structure<br />
96<br />
Monday Track 2, K3.17 3.00-3.20pm<br />
Designing Peptide Drugs from Python Serum: Dual Inhibitors of sPLA2 <strong>and</strong> MMP-<br />
1 as Therapeutic Option for Treatment of Inflammation<br />
Gopalakrishnakone, P 1* , Thwin, M.M 1 , Sato, K. 2<br />
1 Venom & Toxin Research Programme, Department of Anatomy, Yong Loo Lin School<br />
of Medicine, National University of Singapore, Singapore117597, *antgopal@nus.edu.sg<br />
2 Fukuoka Women's University, Fukuoka 813-8529, Japan<br />
The objective of this study is to confirm the anti-inflammatory effect of the potent <strong>and</strong><br />
selective secretory phospholipase A2 (sPLA2-IIA) inhibitor, derived from the primary<br />
structure of an endogenous protein termed “Phospholipase Inhibitor from Python (PIP)”.<br />
Tg197 mice (CBAxC57BL/6) carrying human TNF genes that develop polyarthritis were<br />
used. Clinical <strong>and</strong> histopathological scores along with the morphological evaluations<br />
indicate that the articular cartilage <strong>and</strong> the synovium of the peptide-treated Tg197 mice<br />
reverted to that observed in control wild-type mice. In the rat (Sprague-Dawley) incisional<br />
hernia model, the peptide significantly (Student’s-t test; P < 0.05) reduced the extent of<br />
postsurgical peritoneal adhesions in the peritoneal tissues of rats at 7th postoperative day.<br />
In organotypic cultures, kainate-induced neuronal cell death was significantly reduced in<br />
the peptide-treated hippocampal neurons than that of the untreated cells. Among the<br />
peptide mutants examined, the 18-residue linear peptide displayed potent inhibition against<br />
the activity of human recombinant synovial sPLA2 <strong>and</strong> human recombinant matrix<br />
metalloproteinase-1 (MMP-1). It showed remarkable clinical improvement of arthritis in<br />
transgenic mice, <strong>and</strong> markedly suppressed secreted MMP-1 activity from IL-1 β -<br />
stimulated rabbit synovial fibroblasts. mRNA <strong>and</strong> protein expressions of MMP-1, MMP-2,<br />
MMP-9 <strong>and</strong> sPLA2 were found to be significantly decreased after treatment of the IL-1β -<br />
stimulated cultured rabbit synovial fibroblasts. The peptide acts against the gelatinases<br />
mainly through downregulation of the expression of genes, while its action against MMP-1<br />
is through suppression of gene expression combined with enzyme inhibition. This<br />
optimized peptide analogue presents strong in vitro evidence for control <strong>and</strong> modulation of<br />
enzyme activity <strong>and</strong>/or transcription of both MMP <strong>and</strong> sPLA2, <strong>and</strong> provides new<br />
therapeutic options in the treatment of arthritis <strong>and</strong> cancer.<br />
o Secretory phospholipase A2 inhibitor<br />
o Matrix metalloproteinase-1 inhibitor<br />
o Tg197 mice<br />
o Antiinflammatory peptide<br />
97
Monday Track 2, K3.17 3.20-3.40pm<br />
Dynamin dependent <strong>and</strong> independent endocytic pathway of cobra cardiotoxins: role<br />
of distinct cholesterol <strong>and</strong> heparan sulfate domain for toxin selection<br />
Lee, S.C 1, # , Wang, C.H. 1, # , Tsai C.M. 1 , Lin J.L. 1 , Liu Y.A. 1 <strong>and</strong> Wen-guey Wu 1,2,*<br />
# These authors contributed equally to this work.<br />
1 Institute of Bioinformatics <strong>and</strong> Structural Biology, National Tsing Hua University<br />
2 National Synchrotron Radiation Research Center, Hsinchu, Taiwan *wgwu@nsrrc.org.tw<br />
Cobra produces a series of three-fingered β-sheet cardiotoxins homologues with distinct<br />
glycosaminoglycan, glycosphingolipid <strong>and</strong> integrin binding ability that cause systolic heart<br />
arrest <strong>and</strong> severe tissue inflammation with retarded wound healing process. In addition to<br />
membrane pore forming activities responsible for its cytotoxicity, cardiotoxins can also be<br />
uptaken via multiple internalization pathways to target other intracellular organelle <strong>and</strong><br />
induce necrotic <strong>and</strong>/or apoptosis cell death depending on the studied cells <strong>and</strong> cardiotoxin<br />
homologues. Although gylcosphingolipid lipid such as sulfatide has been suggested to<br />
facilitate such a process on the major cobra cardiotoxin from Naja atra, i. e., CTX A3, it is<br />
not clear how other cardiotoxins are internalized <strong>and</strong> whether there are other membrane<br />
factors involved in the process. We show in this study that other cardiotoxin homologues<br />
such as CTX A2 <strong>and</strong> CTX A4 are internalized via a dynamin dependent endocytic<br />
pathway, in sharp contrast to the dynamin independent internalization of CTX A3. The two<br />
endocytic pathways are both cholesterol sensitive, but sterol appears to modulate the<br />
process in an opposite manner. Specifically, while depletion of cholesterol inhibits the<br />
internalization of CTX A2 <strong>and</strong> A4, it enhances that of CTX A3. Since CTX A2 <strong>and</strong> A4<br />
also exhibit differential sensitivity toward heparinase I <strong>and</strong> III treated cell in terms of cell<br />
retention <strong>and</strong> cell internalization, our results suggest that distinct cholesterol <strong>and</strong> heparin<br />
sulfate domains on cell surface may allow the exploration of highly homologous<br />
cardiotoxins, sometimes with single site mutation, for the selection of not only cell types<br />
but also action mechanism.<br />
Cardiotoxins<br />
Dynamin<br />
Internalization<br />
heparin<br />
98<br />
Monday Track 2, K3.17 3.40-4.00pm<br />
Snake venom VEGF-F is a most potent inducer of vascular permeability<br />
Yamazaki, Y. 1 , Imamura, T. 2 , Morita, T. 1 *<br />
1 Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo, Japan, *tmorita@my-pharm.ac.jp<br />
2 Kumamoto University, 1-1-1 Honjo, Kumamoto, Japan<br />
– Introduction – Vascular endothelial growth factor (VEGF165) is an endothelial cell<br />
mitogen <strong>and</strong> also functions as a potent vascular permeability factor. We previously isolated<br />
<strong>and</strong> characterized VEGF homologous proteins designated VEGF-Fs that specifically<br />
recognize KDR (VEGFR-2), from two viper venoms (1). VEGF-Fs show a strong<br />
endothelial cell proliferative effect in vitro <strong>and</strong> a potent hypotensive effect in vivo when<br />
compared with human VEGF165 (1). Crystal structural analysis of VEGF-Fs has revealed<br />
substantial variation from other VEGF subtypes with respect to three structural features: an<br />
amino acid insertion in receptor-binding loop 3, a negatively charged surface potential<br />
rather than the positively charged potential seen in VEGF165, <strong>and</strong> an additional salt bridge<br />
formation that is not observed in VEGF165 (2). In this study, VEGF-F was quantitatively<br />
evaluated in a Miles assay for its ability to induce vascular permeability.<br />
– Results <strong>and</strong> Discussion – Both vammin, a VEGF-F from Vipera a. ammodytes venom,<br />
<strong>and</strong> VEGF165 similarly stimulated dye leakage when administered at low concentration (up<br />
to 4 × 10 -9 M), however, vammin was a more potent inducer of vascular permeability than<br />
VEGF165 at higher concentration (>1.2 × 10 -8 M). To clarify the mechanism of the potent<br />
effect of VEGF-F, we next evaluated the effects of histamine <strong>and</strong> bradykinin antagonists,<br />
which are well-known inducers of vascular permeability. Neither histamine nor bradykinin<br />
antagonist blocked the dye leakage stimulated by vammin <strong>and</strong> VEGF165. Our results<br />
indicate that VEGF-F exhibits potent activity even including the induction of vascular<br />
permeability <strong>and</strong> this action may be mediated through a pathway similar to that of<br />
VEGF165. We conclude that snake venom VEGF-F is the most potent inducer of vascular<br />
permeability among the previously discovered molecules <strong>and</strong> assists in the penetration of<br />
the toxin.<br />
–References–<br />
1. Yamazaki Y, Takani K, Atoda H, Morita T (2003) J. Biol. Chem. 278, 51985-51988.<br />
2. Suto K, Yamazaki Y, Morita T, Mizuno H (2005) J. Biol. Chem. 280, 2126-2131.<br />
o Snake venom<br />
o Vascular endothelial growth factor (VEGF)<br />
o Vascular permeability<br />
99
Monday Track 2, K3.17 4.00-4.20pm<br />
C-terminal peptide of snake venom VEGF-F specifically blocks VEGF-A165<br />
activity by binding to heparin-like molecules<br />
Tokunaga, Y 1 , Yamazaki, Y 1 , <strong>and</strong> Morita, T 1*<br />
1 Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo, Japan, *tmorita@my-pharm.ac.jp<br />
[Background] Heparan sulfate/heparin-like molecules are known to modulate the<br />
interaction between vascular endothelial growth factor (VEGF-A165) <strong>and</strong> its receptor,<br />
KDR (VEGFR-2). A C-terminal heparin-binding region deficient form of VEGF-A165,<br />
composed of 110 residues, significantly reduced mitogenic potency compared to the<br />
intact form. We previously found two novel heparin-binding VEGFs from two distinct<br />
viper venoms. 1) Snake venom VEGFs, designated VEGF-F, selectively recognize KDR,<br />
<strong>and</strong> exhibit a more potent mitogenic activity compared to VEGF-A165. VEGF-Fs<br />
generally lack the C-terminal heparin-binding region (16~17 amino acid residues) when<br />
compared with VEGF-A165 (55 amino acid residues), despite the heparin-binding<br />
potential of VEGF-Fs. We herein report the identification of the heparin-binding region<br />
of VEGF-F <strong>and</strong> VEGF-blocking activity of the corresponding peptide.<br />
[Results <strong>and</strong> Discussion] A synthetic peptide representing the C-terminal region of<br />
VEGF-F was prepared. The synthetic peptide <strong>and</strong> intact VEGF-F were tested for<br />
heparin-binding ability using heparin affinity chromatography. The affinities of VEGF-F<br />
<strong>and</strong> the synthetic peptide were nearly equivalent as determined by the NaCl<br />
concentrations required for elution (0.33 <strong>and</strong> 0.36 M, respectively), indicating that the Cterminal<br />
region mediates the heparin-binding ability of VEGF-F. The synthetic peptide<br />
completely blocked both VEGF-F <strong>and</strong> VEGF-A165-induced endothelial cell proliferation<br />
<strong>and</strong> hypotension, despite having only a slight inhibitory effect on proliferation induced<br />
by basic fibroblast growth factor (bFGF), which is a well-characterized heparindependent<br />
growth factor. We conclude that the C-terminal region of VEGF-F<br />
specifically inhibits the biological activity of VEGF-A165 by binding to VEGF165bondable<br />
heparin structure. 2)<br />
1) Yamazaki, Y., Takani, K., Atoda, H., <strong>and</strong> Morita, T. (2003) J. Biol. Chem. 278,<br />
51985-51988, 2) Yamazaki, Y., Tokunaga, Y., Takani, K., <strong>and</strong> Morita, T. (2005)<br />
Biochemistry 44, 8858-8864<br />
o VEGF<br />
o Snake venom VEGF<br />
o heparin<br />
o synthetic peptide<br />
100<br />
Monday Track 2, K3.17 4.20-4.40pm<br />
A Factor Xa-like enzyme from Pseudonaja textilis venom is a powerful procoagulant<br />
that reduces blood loss in a topical anti-bleeding model.<br />
Flight, S.M 1* , Warner, R.L 2 , Lavin, M.F 3 , de Jersey J 4 , Masci, P. P 1<br />
1<br />
University of Queensl<strong>and</strong>, School of Medicine, Princess Alex<strong>and</strong>ra Hospital, Brisbane, Australia<br />
*sflight@soms.uq.edu.au<br />
2.<br />
University of Michigan, Department of Pathology, University of Michigan Medical School, Ann Arbor,<br />
USA.<br />
3<br />
Queensl<strong>and</strong> Institute of Medical Research, Royal Brisbane Hospital, Brisbane, Australia.<br />
4<br />
University of Queensl<strong>and</strong>, School of Molecular <strong>and</strong> Microbial Sciences, Brisbane, Australia<br />
Introduction: The prothrombin activating complex in the venom of the Australian brown<br />
snake Pseudonaja textilis contains a nonenzymatic factor Va-like component <strong>and</strong> a factor<br />
Xa-like enzyme. In vitro <strong>and</strong> in vivo analyses were performed to characterise the factor Xa<br />
component <strong>and</strong> assess its efficacy as a topical anti-bleeding agent. Methods: The snake<br />
factor Xa component was purified using affinity <strong>and</strong> size exclusion chromatography. The<br />
clotting times were measured using a range of concentrations of snake factor Xa, human<br />
factor Xa <strong>and</strong> thrombin in citrated plasma with <strong>and</strong> without added calcium. The same<br />
enzymes were added to citrated whole blood <strong>and</strong> coagulation analysed using<br />
thromboelastography. A rat dermal incision model was used to assess the blood lost from a<br />
wound in the presence of 100, 250 <strong>and</strong> 1000 mcg/mL of the snake factor Xa compound<br />
<strong>and</strong> 1000 U/mL of thrombin (n=10 animals/treatment). Results: Snake factor Xa was<br />
purified by column chromatography. The purified enzyme clotted citrated plasma in the<br />
absence of added calcium at concentrations as low as 10 nM whereas no clotting was<br />
observed using human factor Xa at concentrations up to 700 nM. In this assay, prior<br />
recalcification of the plasma led to a further 2-4-fold decrease in clotting time on addition<br />
of 10 nM snake factor Xa. Similar results were obtained using thromboelastography with<br />
citrated whole blood. The snake factor Xa reduced blood loss in the dermal incision model<br />
from 44.3 ± 34.3 mcL blood lost (saline control) to 2.8 ± 2.5 mcL blood lost with 250<br />
mcg/mL of enzyme (p
Monday Track 2, K3.17 4.40-5.00pm<br />
Heparin-neutralizing properties of two novel Lys49 PLA2 variants from the<br />
Bothrops moojeni venom.<br />
Anna Maria Perchuc 1* , Laure Menin 2 , Philippe Favreau 2 , Beatrice Bühler 1 , Philippe Bulet 2 , Reto<br />
Schöni 1 <strong>and</strong> Reto Stöcklin 2<br />
1 Pentapharm Ltd., Engelgasse 109, P.O. Box, CH-4002 Basel, Switzerl<strong>and</strong>,<br />
* annamaria.perchuc@pentapharm.com<br />
2 Atheris Laboratories, Case Postale 314, CH-1233 Bernex – Geneva, Switzerl<strong>and</strong><br />
Introduction: In the present work proteomics <strong>and</strong> peptidomics approaches have been used<br />
in combination with newly developed bioassays screening in order to identify new<br />
compounds in the venom of Bothrops moojeni snake.<br />
The most interesting compounds are thought to be used as active pharmaceutical (APIs) or<br />
active diagnostic (ADIs) ingredients mainly in the field of haemostasis <strong>and</strong> fibrinolysis.<br />
Methods: Among the peptides <strong>and</strong> proteins already characterized in B. moojeni venom in<br />
the framework of our project, two phospholipases A2 (PLA2) have been purified <strong>and</strong> fully<br />
sequenced by ESI-MS/MS techniques. Their inhibitory action towards heparin has been<br />
found by the means of specific assays designed in order to analyze the anticoagulant<br />
properties of heparins in plasma <strong>and</strong> the neutralization thereof.<br />
Results: ESI-MS/MS analysis revealed novel proteins, distinct from the three PLA2<br />
(MjTX-I <strong>and</strong> MjTX-II <strong>and</strong> MOO-1) of B. moojeni already described in the literature. Both<br />
of them belong to the enzymatically non-active Lys49 variants of PLA2. They consist of<br />
122 amino acids <strong>and</strong> share a characteristic sequence in the C-terminal region composed of<br />
clusters of basic amino acids known from the literature to interact with heparin.<br />
The novel PLA2 variants we have isolated <strong>and</strong> characterized as well as their synthetic Cterminal<br />
fragments interact in vitro with unfractionated (UFH) <strong>and</strong> low molecular weight<br />
(LMWH) heparins, neutralizing their anticoagulant activity. The synthetic peptides are also<br />
able to inhibit the anticoagulant effects of LMWH to a higher extent than protamine<br />
hydrochloride, the known heparin antidote, used in its therapeutically recommended<br />
concentration.<br />
Discussion/Conclusion: Although it is well known that different PLA2s from snake venoms<br />
influence the blood coagulation system <strong>and</strong> some of them interact with heparin, the use of<br />
those substances or their fragments to antagonize the anticoagulant effect of heparin in vivo<br />
or in vitro has never been proposed. Their possible use in diagnostic <strong>and</strong>/or pharmaceutical<br />
applications is currently under investigation.<br />
o Bothrops moojeni<br />
o Lys49 PLA2 variant<br />
o Heparin inhibition<br />
102<br />
Monday Track 2, K3.17 5.00-5.20pm<br />
FXa-binding studies of the anticoagulant phospholipases A2 from Viperidae <strong>and</strong><br />
Crotalidae venom<br />
FAURE, G 1* ., GOWDA 2 , V. T., BON 3 , C & R.C. MAROUN 4<br />
1 Unité d’Immunologie Structurale, Institut Pasteur, 25 rue du Dr. Roux, Paris, France * fgrazyna@pasteur.fr<br />
2 University of Mysore, Department of Studies in Biochemistry, Manasagangothri, India<br />
3 MNHN, Lab. de Biochimie des Substances Naturelles, 63 rue Buffon, Paris, France<br />
4 Unité de Bioinformatique Structurale, Institut Pasteur, 25 rue du Dr. Roux, Paris, France<br />
The snake venom group IIA secreted phospholipases A2 (sPLA2s) from Viperidae <strong>and</strong><br />
Crotalidae families are multifunctional proteins that exhibit a wide range of toxic <strong>and</strong><br />
pharmacological effects. These sPLA2s catalyse the hydrolysis of the ester bond of<br />
phospholipids <strong>and</strong> also selectively interact with various protein targets. These proteinprotein<br />
interactions, rather than protein-phospholipid interactions, play an important role<br />
in determining the specific function of PLA2s.<br />
In particular, the sPLA2s that interact with presynaptic receptors [1,2] block the release<br />
of acetylcholine <strong>and</strong> the same neurotoxic enzymes interfere with blood coagulation<br />
exhibiting a strong anticoagulant function [3, 4].<br />
Using Surface Plasmon Resonance (SPR) <strong>and</strong> an in vitro biological test of the<br />
inhibition of prothrombinase activity, we identified several Viperidae <strong>and</strong> Crotalidae<br />
sPLA2s that inhibit blood coagulation through direct binding to human blood coagulation<br />
factor Xa (FXa). These anticoagulant sPLA2s that inhibit coagulation by a non-enzymatic<br />
pathway represent a novel family of anticoagulant agents, useful in identifying the sites of<br />
interaction of anticoagulants at the level of specific amino acid residues. Molecular<br />
electrostatic potentials calculated at the solvent-accessible surface of obtained 3D models<br />
<strong>and</strong> available crystal structures of these FXa-binding molecules show a correlation with<br />
their anticoagulant activity. Analysis of the complexes obtained through docking<br />
simulations allows us to map four sPLA2 binding regions at the interface. We compare<br />
these regions to those mapped previously by site-directed mutagenesis <strong>and</strong> SPR studies<br />
for human group IIA sPLA2 [4] <strong>and</strong> for AtxA [3]. The structural information that we<br />
obtain about the binding of sPLA2s to FXa will be useful in the 3D structure-based design<br />
of therapeutic agents.<br />
[1] Krizaj, I., Faure, G., Gubensek, F. & Bon, C. (1997) Biochemistry, 36, 2779<br />
[2] Faure, G, Copic, A., Le Porrier, S, Gubensek, F., Bon, C & Krizaj, I. (2003) Toxicon, 40, 509<br />
[3] Prijatelj, P., Charnay, M., Ivanovski, G., Jenko, Z., Pungarcar, J. Krizaj, I. & Faure, G. (2006) Biochimie,<br />
88, 69<br />
[4] Mounier C. M., Luchetta, P., Lecut, C., Koduri, R. S., Faure, G., Lambeau, G., et al., (2000) Eur. J. Bioch<br />
em. 267, 4960<br />
anticoagulant phospholipase A2,<br />
human factor Xa<br />
surface plasmon resonance<br />
docking simulations<br />
103
Monday Track 2, K3.17 5.20-5.40pm<br />
BE-I-PLA2, a novel acidic phospholipase A2 from Bothrops erythromelas venom:<br />
purification, cloning <strong>and</strong> characterization as potent anti-platelet <strong>and</strong> inductor of PGI<br />
release by endothelial cells<br />
Modesto, J.C.A 1,3 , Spencer, P.J 2 , Fritzen, M 1 , Oliva, M.L.V 3 , Silva, MB1 4 , Chudzinski-Tavassi, A.M 1 ,<br />
Guarnieri, M.C 4<br />
1 Instituto Butantan, São Paulo SP, Brasil<br />
2 Instituto de Pesquisas Energéticas e Nucleares, São Paulo SP, Brasil<br />
3 Universidade Federal de São Paulo, São Paulo SP, Brasil<br />
4 Universidade Federal de Pernambuco, Recife PE, Brasil.<br />
A novel acidic Asp49 phospholipase A2 was isolated from Bothrops erythromelas (jararaca<br />
malha-de-cascavel) snake venom by four chromatographic steps. BE-I-PLA2 present a<br />
molecular weight of 13649.57 Da as estimated by mass spectrometry. N-teminal <strong>and</strong> four<br />
internal peptides were sequenced, covering around one third of the complete toxin<br />
sequence. The complete BE-I-PLA2 cDNA was cloned from a B. erythromelas venomgl<strong>and</strong><br />
cDNA library. The cDNA sequence possesses 753 bp <strong>and</strong> encodes a protein with<br />
significant sequence similarity to many other PLA2s from snake venoms. When tested in<br />
platelet rich plasma, the enzyme showed a potent inhibitory effect on aggregation induced<br />
by arachidonic acid <strong>and</strong> collagen, but not ADP. On the other h<strong>and</strong>, BE-I-PLA2 did not<br />
modify aggregation in washed platelet. Furthermore, no action of BE-I-PLA2 on the<br />
principal platelets receptors was observed. Chemical modification with p-bromophenacyl<br />
bromide abolished the enzymatic activity of BE-I-PLA2, but its anti-platelet activity was<br />
only partially inhibited. In HUVEC, BE-I-PLA2 was neither apoptotic nor proliferative but<br />
stimulated endothelial cells to release PGI2, suggesting an increase of its potential antiplatelet<br />
activity in vivo. Further studies are required in order to determine the exact<br />
mechanism of action of BE-I-PLA2 in the inhibition of platelet aggregationAbstract text<br />
here:<br />
o Phospholipase<br />
o Anti-platelet<br />
o Snake Venom<br />
o Cloning<br />
104<br />
Monday Track 3, K3.25 3.00-3.20pm<br />
Stability studies on the venom of the major Australian box jellyfish (Chironex<br />
fleckeri).<br />
1 2 3 1*<br />
Winter, K.L., Isbister, G.K., Seymour, J.E., Hodgson, W.C.<br />
1<br />
Monash Venom Group, Dept. Pharmacology, Monash University, Victoria, Australia, 3800. *<br />
kelly.winter@med.monash.edu.au<br />
2<br />
Tropical Toxicology Unit, Menzies School of Health Research, Northern Territory, Australia.<br />
3<br />
Dept. Tropical Biology, James Cook University, Queensl<strong>and</strong>, Australia, 4878<br />
Introduction: It has been suggested that jellyfish venoms are difficult to work with <strong>and</strong><br />
that they are sensitive to pH <strong>and</strong> chemicals, thermolabile, <strong>and</strong> have a tendency to<br />
aggregate, disaggregate <strong>and</strong> stick to the surface of equipment (Ramasamy et al., 2003).<br />
We have previously characterised the pharmacological activity of a number of jellyfish<br />
venoms. The current study aimed to examine the working parameters of the venom of the<br />
Australian box jellyfish Chironex fleckeri, to aid fractionation using HPLC analysis.<br />
Methods: Anaesthetised (pentobarbitone sodium, 100 mg/kg, i.p.) rats were used to<br />
examine the cardiovascular effects of the venom. Venom was made up fresh each day <strong>and</strong><br />
subjected to a number of different environments. A pH range of 5-9 <strong>and</strong> a temperature<br />
range of 4-30 ºC were examined. In addition, the effect of freeze drying <strong>and</strong> reconstituting<br />
the venom was investigated.<br />
Results: Venom (50 μg/kg, i.v.) produced a transient hypertensive response followed by<br />
cardiovascular collapse. This effect was not significantly effected by preparation of the<br />
venom at pH’s of 5, 7 <strong>and</strong> 9 (p>0.05, n=3; one way ANOVA). Similarly the venom (50<br />
μg/kg) did not display loss of activity when exposed to temperatures of 4 ºC, 20 ºC or 30<br />
ºC for 1.5 hr. However, the cardiovascular activity of the venom was abolished by boiling<br />
the venom (p
Monday Track 3, K3.25 3.20-3.40pm<br />
Variation in venom between two Australian box jellyfish: prey-specific<br />
adaptations<br />
Kintner A. 1* , Seymour J. 2 , Carrette T. 3<br />
1 Monash Venom Group, Monash University, Melbourne Vic Australia, *anna.kintner@med.monash.edu.au<br />
2 Tropical Australian Stinger Research Unit, James Cook University, Cairns Qld Australia<br />
3 Tropical Australian Stinger Research Unit, James Cook University, Cairns Qld Australia<br />
Introduction. This study examines the venoms of two species of closely-related box<br />
jellyfish: Chironex fleckeri, which has caused 70+ deaths in Australia, <strong>and</strong> Chiropsalmus<br />
sp., which has never caused serious injury in Australian waters. The two share a similar<br />
nematocyst complement <strong>and</strong> prey of small shrimps during their juvenile stages. However,<br />
as C. fleckeri matures, its proportion of venom-delivering nematocysts increases, allowing<br />
it to inject 15 times more venom than Chiropsalmus sp, <strong>and</strong> simultaneously, its prey focus<br />
shifts to include fish 1 . This study investigates adaptations of the two species’ venoms, by<br />
examining their cardiac effects in prey model animals (e.g. crustaceans <strong>and</strong> fish) <strong>and</strong> by<br />
comparing their protein constructs. This will elucidate reasons for difference in sting<br />
lethality <strong>and</strong> facilitate further investigation into clinical significance of C. fleckeri stings.<br />
Methods. Venoms from both species were injected into crustacean <strong>and</strong> fish prey models<br />
(Cherax quadricarinatus, <strong>and</strong> Lepidozygus tapeikosoma respectively) <strong>and</strong> their effects <strong>and</strong><br />
dose-dependency assessed via Doppler ultrasound cardiac monitoring. Examination of the<br />
venom fractions was undertaken using SDS-PAGE analysis.<br />
Results. C. fleckeri <strong>and</strong> Chiropsalmus sp. venoms were roughly equivalent in a crustacean<br />
model, but widely disparate in a vertebrate model, with C. fleckeri venom being more<br />
efficient. C. fleckeri venom was also more complex, containing roughly twice as many<br />
components as Chiropsalmus sp. venom.<br />
Discussion/Conclusion. Our results demonstrate that Chiropsalmus sp. would be<br />
incapable of preying on fish or causing significant envenoming in humans. Interestingly,<br />
both venoms contain protein fractions of similar size <strong>and</strong> charge, suggesting that the two<br />
may share invertebrate-specific toxins, with C. fleckeri venom containing additional<br />
vertebrate-specific toxins. We suggest future avenues of investigation to confirm this.<br />
References. Carrette T., Alderslade P., <strong>and</strong> Seymour J., 2002. Nematocyst ratio <strong>and</strong> prey<br />
in two Australian cubomedusans, Chironex fleckeri <strong>and</strong> Chiropsalmus sp. Toxicon 40 (11),<br />
1547-1551.<br />
o venom ecology<br />
o box jellyfish<br />
o nematocyst<br />
o cubozoan<br />
106<br />
Monday Track 3, K3.25 3.40-4.00pm<br />
Structural Determinants of Lys49 Phospholipase A2 Activity Probed by Scanning<br />
Alanine Mutagenesis.<br />
Chioato, L, Aragão, E. A, Lopes, T. F, Ruller, R, Ward, R. J*<br />
Universidade de São Paulo, FFCLRP, Av. B<strong>and</strong>eirantes 3900, Ribeirão Preto-SP, Brazil.<br />
rjward@ffclrp.usp.br<br />
Lysine 49 Phospholipases A2 (Lys49-PLA2) are naturally occurring class II<br />
phospholipases, in which the active site Asp49 is replaced by a lysine. Although the<br />
Lys49-PLA2s do not hydrolyze phospholipid substrates, they show a wide range of<br />
pharmacological effects, are bactericidal, <strong>and</strong> damage artificial membranes by a Ca 2+ -<br />
independent mechanism. As part of our ongoing efforts to underst<strong>and</strong> the<br />
structure/function relationships of PLA2, we have undertaken a scanning alanine<br />
mutagenesis study of the bothropstoxin-I, a Lys49-PLA2 isolated from the venom of the<br />
snake Bothrops jararacussu.<br />
All cationic residues together with all residues between positions 112-131 were substituted<br />
by alanine, <strong>and</strong> aromatic residues in the C terminal loop region (residues 116-127) were<br />
substituted by tryptophan. After expression in E. coli BL21 <strong>and</strong> purification by cation<br />
exchange chromatography, the native-like secondary structure of 33 mutants was<br />
confirmed by circular dichroism spectroscopy. The effects of mutagenesis were evaluated<br />
on the bactericidal activity against E. coli K12, the membrane damaging activity (release<br />
of liposome entrapped marker), <strong>and</strong> the in vivo myotoxic effect. For each functional assay,<br />
the positions of mutants that altered the activity were mapped on the calculated protein<br />
surface in the three dimensional structure.<br />
Comparison of the structural determinants of all activities revealed that Ca 2+ -independent<br />
membrane damage against artificial liposome membranes correlated well with the<br />
bactericidal activity against E. coli, suggesting that a common mechanism underlies the<br />
bactericidal <strong>and</strong> Ca 2+ -independent membrane damaging activities. In contrast, the<br />
structural determinants of the myotoxic activity were restricted to the C-terminal loop<br />
region, suggesting that the myotoxic effect is not due to Ca 2+ -independent membrane<br />
damage. These results show that although the structural determinants of the bactericidal<br />
<strong>and</strong> myotoxic activities are partly overlapping, the underlying mechanisms of action<br />
against the two membranes are different.<br />
o Bactericidal<br />
o Myotoxic<br />
o Membrane damage<br />
107
Monday Track 3, K3.25 4.00-4.20pm<br />
Screening of Bothrops snake venoms using bidimensional capillary liquid<br />
chromatography coupled to t<strong>and</strong>em nano-electrospray quadrupole time-of-flight<br />
mass spectrometry: identification of novel peptides<br />
Souza, G.H.M.F. 1,2,* , Ifa, D.R. 2,3 , Eberlin, M.N. 2 , Hyslop, S. 1<br />
1<br />
Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas<br />
(UNICAMP), CP 6111, 13083-970, Campinas, SP, Brazil. *desouz@fcm.unicamp.br<br />
2<br />
Laboratório Thomson de Espectrometria de Massas, Instituto de Química, Universidade Estadual de<br />
Campinas (UNICAMP), 13083-970, Campinas, SP, Brazil.<br />
3<br />
Aston Laboratory for Mass Spectrometry, Department of Chemistry, Purdue University, Oval Drive,<br />
West Lafayette, IN, USA.<br />
Introduction: Bothrops snake venoms are a mixture of proteins <strong>and</strong> peptides, with the<br />
latter including bradykinin-potentiating peptides (BPPs) <strong>and</strong> C-type natriuretic peptides<br />
(CNPs) [1,2]. However, the occurrence of peptides other than BPPs <strong>and</strong> CNPs has not been<br />
extensively investigated. In this work, we used mass spectrometry to analyze the peptide<br />
content of a low molecular mass fraction obtained by ultrafiltration of several Bothrops (B.<br />
alternatus, B. erythromelas, B. insularis, B. jararaca, B. jararacussu, B. leucurus, B.<br />
moojeni) venoms. Methods: Lyophilized or dessicated Bothrops venoms were dissolved in<br />
0.1% trifluoroacetic acid containing 50% acetonitrile <strong>and</strong> ultrafiltered by centrifugation<br />
through a 5 kDa cut-off membrane (Millipore). The resulting low molecular mass fraction<br />
was analyzed by quadrupole/time-of-flight mass spectrometry (Qtof) using capillary<br />
liquid-chromatography <strong>and</strong> nano-electrospray ionization. Results: Eighty-five peptides<br />
with molecular masses of 400-1900 kDa were identified <strong>and</strong> sequenced by 2D-capillary<br />
liquid chromatography coupled to t<strong>and</strong>em mass spectrometry using nano-electrospray<br />
ionization quadrupole time-of-flight mass spectrometry (nanoESI-MS/MS), although no<br />
single venom contained all of these peptides. Various peptides were identified as BPPs or<br />
putative BPPs. However, a larger number did not show the characteristic BPP structure.<br />
BLAST searches of the latter peptides revealed homologies (based on 80% similarity) with<br />
various vertebrate proteins, as well as with a few snake venom proteins, particularly Lamino<br />
acid oxidase, myotoxic phopholipases A2 <strong>and</strong> (hemorrhagic) metalloproteinases.<br />
Conclusion: Bothrops venoms contain a variety of peptides other than BPPs <strong>and</strong> CNPs.<br />
Although the functions of these peptides are still unknown, the finding that several of them<br />
are identical to internal regions of larger venom proteins (enzymes) suggests that these<br />
peptides may have important as yet unidentified functions.<br />
References: 1. Murayama, N. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 1189-1193. 2.<br />
Wermelinger, L.S. et al. (2005) Rapid Commun. Mass. Spectrom. 19, 1703-1708.<br />
Financial support: CAPES, CNPq, FAPESP<br />
o Bothrops<br />
o Mass spectrometry<br />
o Peptides<br />
o Venom<br />
108<br />
Monday Track 3, K3.25 4.20-4.40pm<br />
Role of histidines <strong>and</strong> the N-terminal helices in the function of the diphtheria<br />
toxin T domain<br />
Perier, A. 1 , Chassaing, A. 1 , Montagner, C. 2 , Pichard, S. 1 , Vernier, G. 2 , Ménez, A. 1 ,<br />
Forge, V. 2 , Chenal, A. 3 <strong>and</strong> Gillet, D. 1*<br />
1 Département d’Ingénierie et d’Etudes des Protéines, CEA-Saclay, 91191 Gif sur Yvette<br />
cedex, Franc. *daniel.gillet@cea.fr<br />
2 Biophysique Moléculaire et Cellulaire, UMR 5090, Département Réponse et Dynamique<br />
Cellulaires, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble cedex 9, France<br />
3 Département de Biochimie Structurale et Chimie, Unité Postulante Biochimie des<br />
Interactions Macromoléculaires, Institut Pasteur, 25 rue du Dr Roux, 75014 Paris, France<br />
During intoxication of a cell, the translocation (T) domain of the diphtheria toxin<br />
helps the passage of the catalytic domain across the membrane of the endosome<br />
into the cytoplasm. The interaction of the T domain with the membrane of the<br />
endosome has been related to the formation of a molten globule state in solution at<br />
acidic pH. We have found that protonation of histidines was required for the<br />
formation of this molten globule state <strong>and</strong> probed the role of each histidine by site<br />
directed mutagenesis. We have also investigated the behavior of the N-terminal<br />
region of the T domain during the successive steps of its interaction with<br />
membranes using fluorescence <strong>and</strong> trypsin digestion. The change of the<br />
environment of this region was monitored using mutant W281F carrying a single<br />
native Trp at position 206 at the tip of helix TH1. The role of each helix of the Nterminus<br />
of the T domain, TH1, TH2, TH3 <strong>and</strong> TH4 was studied using synthetic<br />
peptides. The N-terminal region of the T domain was not involved in the binding of<br />
the domain to the membrane, which occurred between pH 7 <strong>and</strong> pH 6 mainly<br />
through hydrophobic effects. At that stage of the interaction, the N-terminal region<br />
remained strongly solvated. Further acidification eliminated repulsive electrostatic<br />
interactions between this region <strong>and</strong> the membrane, allowing its penetration into the<br />
membrane by attractive electrostatic interactions involving TH1 <strong>and</strong> TH4 <strong>and</strong><br />
hydrophobic effects involving TH3.<br />
The data give information on the physicochemical properties of the T domain<br />
required for its penetration into the membrane <strong>and</strong> on the successive steps involved<br />
during this process.<br />
- diphtheria toxin<br />
- translocation<br />
- molten globule<br />
- membrane interaction<br />
- amphiphilic helices<br />
109
Monday Track 3, K3.25 4.40-5.00pm<br />
Detection <strong>and</strong> Quantitation of Detrimental Levels of Staphylococcal Enterotoxin B<br />
by Mass Spectral Immunoassay<br />
Bieber, A.L., Niederkofler, E.E., Tubbs, K.A., Kiernan, U.A., Nedelkov, D.<br />
<strong>and</strong> Nelson, R.W.<br />
Intrinsic Bioprobes, Inc., 625 S. Smith Road, Suite 22, Tempe, AZ, USA 85281*abieber@intrinsicbio.com<br />
Introduction: The need to develop early detection devices for natural toxins is evident from<br />
their potential use as agents for terrorist attacks. Low detection limits are a requirement for<br />
detection <strong>and</strong> quantitation of these agents from complex biological <strong>and</strong> environmental<br />
systems. An aspect not addressed by current detection methods is the presence of variant<br />
forms of toxins. The application of mass spectral immunoassay (MSIA) to directly detect<br />
Staphylococcal Enterotoxin B (SEB) <strong>and</strong> associated variants is described.<br />
Methods: Highly sensitive MSIA technology was applied to detect biologically relevant<br />
levels of SEB. Pico-molar concentrations of SEB were extracted from milk, buffer <strong>and</strong><br />
water through the use of affinity pipette tips (small porous micro-columns fitted inside 200<br />
microliter pipet tips) derivatized with anti-SEB polyclonal antibody. Repeated passage of<br />
sample through the affinity pipette selectively extracted the targeted analytes from large<br />
volumes of solution for subsequent mass spectrometric analysis. Analyses of multiple<br />
samples were accomplished by use of a robotic pipetting workstation.<br />
Results: The application of MSIA for the detection of SEB indicated a lower detection<br />
limit of 0.5 ng/mL from as little as 1mL of sample. Accurate identification of SEB <strong>and</strong><br />
SEB variants, which may be produced through chemical <strong>and</strong>/or biological modifications,<br />
was accomplished. High content MS data pertaining to the target analyte <strong>and</strong> variants was<br />
obtained in the absence of other proteins that are present in crude samples. SEB<br />
quantitation by MSIA was achieved using a chemically modified form of SEB as an<br />
internal reference. Furthermore, a robotic pipetting workstation was used to process<br />
multiple samples in parallel. The three test solutions, present in all wells of separate 96<br />
position microtiter plates, were subjected to MSIA. SEB was subsequently identified from<br />
each of the 288 samples, thereby validating the reproducibility of MSIA. Overall, the<br />
results indicate that the application of MSIA for early detection of biological pathogens,<br />
such as SEB, has great potential for rapidly <strong>and</strong> accurately identifying the presence of<br />
known pathogenic agents <strong>and</strong> their variants in crude biological <strong>and</strong> environmental samples.<br />
o immunoassay<br />
o enterotoxin B<br />
o mass spectrometry<br />
o quantitation<br />
110<br />
Monday Track 3, K3.25 5.00-5.20pm<br />
Chemical modification of mycotoxin metabolism by green tea extract <strong>and</strong><br />
coumarin in piglets.<br />
Tulayakul, P. 1 , Dong, K.S. 1 , Li, J.Y. 2 , Kumagai, S. 1*<br />
1<br />
Laboratory of Veterinary Public Health, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo,<br />
Japan,*askuma@mail.ecc.u-tokyo.ac.jp.<br />
2.<br />
Research Unit for Animal Life Sciences, Animal Resource Science Center, University of Tokyo, Ibaraki-<br />
Iwama, Japan.<br />
Pig is known to be a sensitive species to aflatoxin B1 toxicity. We have found that the<br />
CYP450 activity to form AFB1-epoxide is relatively high, while the GST activity to<br />
detoxify AFB1 is low, in piglets compared with other animal species (1). The purpose of<br />
this study is to clarify the effects of feeding piglets with green tea extract (Salphenol) <strong>and</strong><br />
coumarin on AFB1 metabolism, which is a determinant of the adverse effect of the toxin.<br />
Method: Piglets were fed with the diet containing 0.2% w/w green tea extract or 0.5% w/w<br />
coumarin for 3 weeks, <strong>and</strong> then analysed for aflatoxin B1 metabolism in their liver <strong>and</strong><br />
intestinal tissues in vitro.<br />
Results: The liver microsomes activity to bind AFB1 to DNA was reduced by coumarin,<br />
but not by green tea extract. The intestinal microsomes activity was not changed by either<br />
compound. The GST activity toward AFB1-epoxide was not changed in the liver, but that<br />
was increased in the intestine, by both the compounds. The GST activity toward CDNB<br />
was increased by both the compounds in the liver <strong>and</strong> intestine. The formation of free<br />
AFQ1 <strong>and</strong> AFM1 by liver microsomes was reduced by coumarin treatment, while the<br />
formation of aflatoxicol <strong>and</strong> AFB2a was increased in the liver <strong>and</strong> intestine by coumarin<br />
treatment, respectively. On the other h<strong>and</strong>, AFQ1 <strong>and</strong> aflatoxicol formation was increased<br />
in the intestine of piglets treated with green tea extract.<br />
Discussion/Conclusion: Coumarin increased the intestinal GST activity toward AFB1, <strong>and</strong><br />
reduced the liver P450 activity to form AFB1-DNA adduct. Also green tea extracts<br />
increased the intestinal GST activity toward AFB1, but exerted no effects on P450<br />
activities to form AFB1-epoxide. These results indicate that green tea may be an effective<br />
c<strong>and</strong>idate for protecting piglets from toxicity of AFB1, although its activity is lower than<br />
coumarin.<br />
References: 1. Tulayakul et al. (2005) Toxicon. 46, 204-209.<br />
o green tea<br />
o piglets<br />
o AFB1<br />
o metabolism<br />
111
Monday Track 3, K3.25 5.20-5.40pm<br />
Novel proteolytic enzymes from pathogenic fungi <strong>and</strong> their use for biotechnology<br />
<strong>and</strong> health<br />
Stöcklin, R. 1* , Grouzmann, E. 2 , Monod, M. 3 , Michalet, S. 1 <strong>and</strong> Gaertner, H. 1<br />
1<br />
Atheris Laboratories & FunZyme Biotechnologies S.A., case postale 314, CH-1233 Bernex-Geneva,<br />
Switzerl<strong>and</strong>, *reto.stocklin@atheris.ch - http://www.atheris.com<br />
2<br />
Division de Pharmacologie et Toxicologie Cliniques Centre Hospitalier Universitaire Vaudois, CH-1011<br />
Lausanne, Switzerl<strong>and</strong><br />
3<br />
Service de Dermatologie, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerl<strong>and</strong><br />
We have isolated <strong>and</strong> characterized a set of potent proteolytic enzymes secreted by<br />
pathogenic dermatophyte fungi. One of our aminopeptidases is able to completely degrade<br />
any peptide from its N-terminal extremity like a zipper, but it is stopped when it reaches a<br />
Yaa-Pro sequence. A complementary enzyme is capable to exclusively remove the Yaa-<br />
Pro sequence. The use of these proteases - either alone or in combination - has many<br />
promising industrial applications ranging from highly specific <strong>and</strong> controlled modification<br />
of protein’s extremities to complete degradation of large resistant proteins.<br />
On the one h<strong>and</strong>, our enzymes can be used to specifically remove affinity tags, thus<br />
facilitating the bio-processing of recombinant protein production <strong>and</strong> chemical peptide<br />
synthesis. On the other h<strong>and</strong>, our enzymes are able in vitro to fully degrade proteins down<br />
to dipeptides <strong>and</strong> single amino acids, which has never been reported before. This<br />
unprecedented efficacy of our enzymes has convinced us of their possible use for oral food<br />
implementation in gastrointestinal disorders due to resistance or intolerance to proteins.<br />
Celiac disease is a pathology characterized by intolerance to gluten (a major protein of<br />
wheat, rye <strong>and</strong> barley) that is not properly digested by intestinal tract of affected people.<br />
This results in the accumulation of gliadins, toxic peptides (fragments of gluten) that are<br />
resistant to degradation in the gastrointestinal tract <strong>and</strong> that contain several T-cell<br />
stimulatory epitopes. The accumulation of gliadins causes an immune reaction leading to<br />
the destruction of the intestine. Despite its prevalence of ~1:100, there is no drug on the<br />
market, <strong>and</strong> the only solution is a strictly gluten-free diet. We intend to develop a first drug<br />
for Celiac disease based on an oral administration of our enzymes to fully degrade gliadins.<br />
Monod, M., Lechenne, B., Jousson, O., Gr<strong>and</strong>, D., Zaugg, C., Stöcklin, R. <strong>and</strong> Grouzmann,<br />
E. (2005). Microbiology, 151:145-55.<br />
o Fungal enzymes<br />
o Bio-processing<br />
o Drug discovery<br />
o Celiac disease<br />
112<br />
Tuesday Track 1, K3.14 3.00-3.20pm<br />
Post mortem examination of a fatal case of Bothrops lanceolatus envenoming<br />
Thomas, L. 1* , Malbranque, S. 1 , Piercecchi- Marti, M.D. 2 , Coursier, D. 1 , Barbey, D. 1 , Bucher, B. 1 ,<br />
Ridarch, A 1 , Smadja, D. 1<br />
1 Hôpital Universitaire, 97200 Fort-de-France, Martinique, * laurent.thomas@chu-fortdefrance.fr<br />
2 Laboratoire de Médecine légale, Faculté de Médecine Timone, Marseille, France<br />
We describe the first documented case of autopsy proven multiple systemic infarctions<br />
following snake bite by Bothrops lanceolatus.<br />
Case report: a 74-yo healthy man was bitten on the left elbow. He did not seek immediate<br />
medical attention. Two days later he fell in a coma <strong>and</strong> was hospitalised. On admission he<br />
presented with typical fang marks <strong>and</strong> swelling of the bitten limb, altered sensorium,<br />
aphasia, <strong>and</strong> tetraplegia. Breathing, arterial pressure, urine output <strong>and</strong> coagulation were<br />
normal. Platelet count was 95,000/mm 3 . Specific BothroFav antivenom therapy was<br />
initiated. MRI demonstrated multiple cerebral infarcts. Myocardial infarction was<br />
confirmed by characteristic electrocardiographic changes <strong>and</strong> elevated serum troponin.<br />
Coronarography was normal. No subsequent neurological improvement was demonstrated.<br />
Six days after the bite he developed atrial fibrillation <strong>and</strong> left ventricular failure.<br />
Echocardiography showed rupture of the mitral valve. Treatment was ineffective. The<br />
patient died 10 days after the bite. Necropsy findings: Incision of the heart revealed a<br />
thickening of the wall of the left ventricle, a posterior infarction combined with a rupture<br />
of the posterior leaflet of the mitral valve <strong>and</strong> no vegetation, <strong>and</strong> a fibrinous pericarditis.<br />
On slicing of the brain hemispheres <strong>and</strong> cerebellum, multiple small symmetrical infarcts<br />
<strong>and</strong> liquefaction necrosis were noticed. Opening of the abdominal cavity revealed a<br />
localised infarct of an ileal loop. No thrombosis of large vessels could be seen.<br />
Histopathology: Microscopic examination demonstrated widespread ischemic lesions,<br />
typically ranging from a few days old to recent in the heart (left ventricle) <strong>and</strong> brain. On<br />
examination of the small vessels, fibrinous clots were uncovered on dissected endothelium,<br />
leading to complete occlusion of arterioles <strong>and</strong> capillaries. No inflammatory change was<br />
seen. The organs involved included the brain, heart, ileum, lungs, <strong>and</strong> kidneys to a lesser<br />
extent. Conclusion: The pathological findings suggest a disseminated thrombotic<br />
microangiopathy process of the endothelium, without vasculitis, consistent with a local<br />
activation phenomenon. Further research is now m<strong>and</strong>ated in the field of toxinology.<br />
o Bothrops<br />
o Snake bites<br />
o Infarction<br />
o Thrombotic microangiopathy<br />
113
Tuesday Track 1, K3.14 3.20-3.40pm<br />
Kraits with 17 dorsal scale rows (Bungarus sindanus complex): unrecognised<br />
causes of severe neurotoxic envenoming in South Asia<br />
Kuch, U. 1,2* , Faiz, M.A. 3 , Pillai, L. 4 , Ahasan, H.A.M.N. 5 , Captain, A. 6 , Khaire, A. 7 , Harris, J.B. 2 ,<br />
Theakston, R.D.G. 8 , Mebs, D. 1 , Warrell, D.A. 8,9<br />
1 Zentrum der Rechtsmedizin, Klinikum der Johann Wolfgang Goethe-Universität, Kennedyallee 104, 60596<br />
Frankfurt am Main, Germany, *U.Kuch@em.uni-frankfurt.de 2 School of Neurology, Neurobiology <strong>and</strong><br />
Psychiatry, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK<br />
3 Dhaka Medical College <strong>and</strong> Hospital, Dhaka 1000, Bangladesh 4 Critical Care Department, Lokmanya Hospital,<br />
Chinchwad, Pune 411033, Maharashtra, India 5 Department of Medicine, Khulna Medical College, 109 South<br />
Central Road, Khulna 9100, Bangladesh 6 3/1 Boat Club Road, Pune 411001, Maharashtra, India<br />
7 PCMC Zoo, Sambhaji Nagar, Chinchwad, Pune 411019, Maharashtra, India 8 Liverpool School of<br />
Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK 9 University of Oxford, Nuffield Department<br />
of Clinical Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK<br />
Introduction: Kraits bites in South Asia are generally attributed to Common Kraits<br />
(Bungarus caeruleus). These, however, share most of their range with superficially very<br />
similar kraits of the B. sindanus complex such as the Sind Krait (B. s. sindanus) <strong>and</strong> Wall's<br />
Krait (B. s. walli). We aimed to compare the venoms of B. sindanus <strong>and</strong> B. caeruleus <strong>and</strong><br />
their clinical effects in human snake bite victims.<br />
Methods: Analysis of case histories <strong>and</strong> literature review; morphological identification of<br />
responsible snakes; mtDNA sequence analysis; liquid chromatography/electrospray massspectrometry.<br />
Results: Molecular <strong>and</strong> mass spectrometry analyses supported the separation of B.<br />
caeruleus <strong>and</strong> B. sindanus. They are reliably distinguished by the number of dorsal scale<br />
rows (15 vs. 17–19, respectively) <strong>and</strong> differ in behaviour <strong>and</strong> ecology. Among 4 proven<br />
<strong>and</strong> 1 suspected cases of B. s. walli envenoming in India (Pune District, Maharashtra, <strong>and</strong><br />
Indore, Madhya Pradesh), 4 died, including 2 bitten by the same krait. In Bangladesh, 4 out<br />
of 6 black-<strong>and</strong>-white b<strong>and</strong>ed kraits causing bites in Dhaka <strong>and</strong> Khulna Divisions were B. s.<br />
walli. Three B. s. walli bites in India occurred inside temporary huts on construction sites;<br />
a farmer in Bangladesh was bitten by a B. s. walli entangled in his fishing net. Two<br />
patients with detailed clinical histories developed paralytic symptoms within 2.5–3 hr after<br />
the bite, fasciculations, <strong>and</strong> generalised flaccid paralysis; one showed evidence of<br />
generalised myocardial damage. There was no response to anticholinesterase or polyvalent<br />
antivenom. Recovery followed prolonged ventilation.<br />
Conclusion: More clinical studies of bites by accurately identified species are needed in<br />
South Asia. Without such evidence, it must not be assumed that the clinical syndrome of<br />
krait envenoming in this region is caused by B. caeruleus alone. Unusual clinical features<br />
such as fasciculations <strong>and</strong> myocardial damage warrant further investigation of the venoms<br />
of members of the B. sindanus complex <strong>and</strong> consideration of their inclusion in antivenom<br />
production.<br />
o Bangladesh<br />
o India<br />
o Bungarus sindanus sindanus<br />
o Bungarus sindanus walli<br />
114<br />
Tuesday Track 1, K3.14 3.40-4.00pm<br />
Severe neurotoxic <strong>and</strong> myotoxic envenoming by the Greater Black Krait (Bungarus<br />
niger) in Chittagong Division, Bangladesh<br />
Faiz, M.A. 1 , Rahman M.R. 1 , Ahsan, M.F. 2 , Kuch, U. 3,4 , Harris, J.B. 4 , Theakston, R.D.G. 5 ,<br />
Warrell,D.A. 5,6 *<br />
1 Dhaka Medical College <strong>and</strong> Hospital, Dhaka 1000, Bangladesh 2 Department of Zoology, University of<br />
Chittagong, Chittagong 4331, Bangladesh 3 Zentrum der Rechtsmedizin, Klinikum der Johann Wolfgang<br />
Goethe-Universität, Kennedyallee 104, 60596 Frankfurt am Main, Germany 4 School of Neurology,<br />
Neurobiology <strong>and</strong> Psychiatry, The Medical School, University of Newcastle upon Tyne, Newcastle upon<br />
Tyne NE2 4HH, UK 5 Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK<br />
6 University of Oxford, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford OX3<br />
9DU, UK, *david.warrell@ndm.ox.ac.uk<br />
Introduction: the venomous fauna of Bangladesh is poorly characterised. We aimed to<br />
discover which species were responsible for bites <strong>and</strong> to define clinical syndromes of<br />
envenoming.<br />
Methods: a prospective clinical study was established at Chittagong Medical College<br />
using enzyme immunoassay (EIA) <strong>and</strong> morphological <strong>and</strong> molecular studies of snakes<br />
brought with bite victims for species diagnosis.<br />
Results: among 18 venomous snakes responsible for bites, 4 proved to be Greater Black<br />
Kraits (Bungarus niger). A 40-year-old labourer was bitten while cutting wood. Paralytic<br />
symptoms began 4-5 hr later <strong>and</strong> on admission, 9.5 hr post- bite, he was unconscious with<br />
flaccid paralysis. Generalised rhabdomyolysis evolved <strong>and</strong> he died 47 hr post-bite. A 35year-old<br />
man, bitten while asleep, died within a few hours. A 12-year-old boy, also bitten<br />
while asleep developed progressive paralysis 11 hr post-bite but recovered after 22 hr of<br />
mechanical ventilation. An 18-year-old man, bitten while fishing, became paralysed 5 hr<br />
later but did not need mechanical ventilation.<br />
Bungarus caeruleus has not been found in Chittagong District but with EIA, sera of 22<br />
snake bite cases, including 2 of the B. niger cases, reacted weakly with B. caeruleus<br />
diagnostic antiserum. This antiserum reacted only very weakly with Nepalese B. niger<br />
venom but strongly with B. caeruleus venom.<br />
Discussion: before this study, B. niger was known only from Nepal, Bhutan <strong>and</strong> NE India.<br />
This is the first report of envenoming by this species anywhere <strong>and</strong> the first report of<br />
generalised rhabdomyolysis caused by envenoming by any Asian or African elapid snake.<br />
Our preliminary data suggest that B. niger may be wide-ranging in Bangladesh <strong>and</strong> an<br />
important cause of snake bites. Its venom should be considered when antivenoms are<br />
designed for this region. The striking finding of rhabdomyolysis should prompt further<br />
investigation of venom properties.<br />
o Bangladesh<br />
o Bungarus niger<br />
o neurotoxicity<br />
o rhabdomyolysis<br />
115
Tuesday Track 1, K3.14 4.00-4.20pm<br />
Distinctive epidemiology <strong>and</strong> clinical features of common krait (Bungarus<br />
caeruleus) bite in Sri Lanka<br />
Ariaratnam, A. 1 , Sheriff, M.H.R. 1 , Theakston, R.D.G. 2 , Warrell, D.A. 2,3<br />
1Faculty of Medicine, University of Colombo, Kynsey Road, Colombo 08, Sri Lanka 2 Liverpool School;<br />
of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK 3 University of Oxford, Nuffield<br />
Department of Clinical Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK.<br />
*david.warrell@ndm.ox.ac.uk<br />
Introduction:10% of snakes brought by snake bite victims during a prospective study at<br />
10 Sri Lankan provincial hospitals were identified as common kraits (Bungarus<br />
caeruleus). However, 75% of snake bite victims did not bring the snake. This study<br />
aimed to define the clinical syndrome of krait bite to aid case identification.<br />
Methods: we analysed proformas completed for all patients bitten by identified snakes.<br />
Results:<br />
Feature 88 cases of proven B.<br />
caeruleus envenoming<br />
674 bites by other<br />
venomous species<br />
Sex ratio (M:F) 1.6:1 2.9:1<br />
Bitten while sleeping on<br />
ground<br />
100% 1%<br />
Bitten 2300-0500 hr 100% 49%<br />
Bitten indoors 100% 3%<br />
Not bitten on lower limb 77% 18%<br />
Local envenoming 30% (mild swelling only) 93%<br />
Respiratory paralysis 64% 2%<br />
Case fatality 6% 3%<br />
91% of krait bite victims complained of abdominal pain<br />
Discussion: this highly distinctive syndrome of Bungarus envenoming has also been<br />
reported from India <strong>and</strong> Thail<strong>and</strong>. Although 60% of krait bite cases were admitted to<br />
hospital within 4 hours of the bite, the case fatality was double that of envenoming by<br />
other species, emphasising the need for improved first aid, transport to hospital <strong>and</strong><br />
respiratory support. Prevention depends on encouraging people not to sleep on the floor<br />
of their dwellings.<br />
o Common krait<br />
o Bungarus caeruleus<br />
o Sri Lanka<br />
o Clinical syndrome<br />
116<br />
Tuesday Track 1, K3.14 4.20-4.40pm<br />
First authenticated cases of life-threatening envenoming by the hump-nosed pit<br />
viper (Hypnale hypnale) in India.<br />
Joseph, J. K 1 , Simpson, I. D 1 , Menon, N. C. S 1 , Jose, M. P 1 , Kulkarni, K. J 1 , Raghavendra, G. B 1 ,<br />
Warrell D. A 2* .<br />
1<br />
Little Flower Hospital <strong>and</strong> Research Centre, Snakebite Research Unit, P.O. Box 23, Angamaly 683572,<br />
Kerala, India<br />
2<br />
University of Oxford, Headington, Oxford, OX3 9DU, UK, *david.warrell@ndm.ox.ac.uk<br />
Introduction: in Kerala, south-western India, Russell’s viper (Daboia russelii) “annali”<br />
(Malayalam) is the commonest proven or suspected cause of snake bite, causing<br />
coagulopathy, bleeding, shock, “permeability syndrome” <strong>and</strong> acute renal failure. However,<br />
some patients thought to have been envenomed by hump-nosed pit vipers (Hypnale<br />
hypnale) “churutta” <strong>and</strong> saw-scaled vipers (Echis carinatus) “chenat<strong>and</strong>en” also develop<br />
coagulopathy, bleeding <strong>and</strong> acute renal failure. Their haemostatic abnormalities fail to<br />
respond to Indian polyvalent antivenoms which covers only “the big four” venomous<br />
snakes of India (cobra, krait, Russell’s <strong>and</strong> saw-scaled vipers).<br />
Methods: a clinical study based in Little Flower Hospital, Angamaly <strong>and</strong> Jubilee Mission<br />
Hospital, Thrissur involved the strict identification the species of snake responsible for<br />
cases of life-threatening envenoming.<br />
Results: five patients developed systemic envenoming after bites by H. hypnale, proved by<br />
identification of the snakes responsible. Two snakes had been misidentified as E. carinatus<br />
while three were unidentified. Symptoms of local envenoming were pain, swelling,<br />
haemorrhagic blistering, bruising <strong>and</strong> regional lymphadenopathy. Systemic symptoms<br />
included headache, nausea, vomiting <strong>and</strong> abdominal <strong>and</strong> chest pain. There was some<br />
evidence of haemostatic dysfunction in all cases (coagulopathy, fibrinolysis,<br />
thrombocytopenia or spontaneous systemic haemorrhage) <strong>and</strong> of microangiopathic<br />
haemolysis in two. Two patients were haemodialysed for acute renal failure one of whom<br />
developed pulmonary oedema requiring mechanical ventilation.<br />
Discussion: in India, H. hypnale had not previously been regarded as a cause of frequent or<br />
potentially-dangerous envenoming. It now appears to be second only to Russell’s viper as<br />
a cause of serious envenoming in parts of SW India. Its medical importance has been<br />
overlooked throughout its geographical range (southern Kerala to Radhanagari,<br />
Maharashtra) because of confusion with other small snakes. An effective antivenom is<br />
urgently needed in South India <strong>and</strong> in Sri Lanka where this species is also a common cause<br />
of bites.<br />
o Hypnale hypnale<br />
o haemostatic disturbances<br />
o acute renal failure<br />
o untreatable<br />
117
Tuesday Track 1, K3.14 4.20-4.40pm (as part of preceding presentation)<br />
Severe systemic envenoming by hump-nosed vipers (Hypnale hypnale) in Sri Lanka<br />
Ariaratnam, A. 1 , Sheriff, M.H.R. 1 , de Silva, A. 2 , Theakston, R.D.G. 3 , Warrell, D.A. 3,4<br />
1 Faculty of Medicine, University of Colombo, Kynsey Road, Colomob 08, Sri Lanka<br />
2 Rajarata University, Sri Lanka<br />
3 Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK<br />
4 University of Oxford, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford OX3<br />
9DU, UK *david.warrell@ndm.ox.ac.uk<br />
Introduction: in Sri Lanka, the hump-nosed pit viper (H. hypnale) is one of the<br />
commonest causes of snake bite. This small species is distributed throughout the isl<strong>and</strong>. It<br />
is considered by some to be capable of causing only painful local swelling, there have been<br />
reports of severe systemic envenoming <strong>and</strong> fatalities since the 1970s. This study aimed to<br />
discover the true pattern of envenoming attributable to this species.<br />
Methods: in 10 hospitals in Colombo, Panadura, Watthupitiwela, Chillaw, Matale,<br />
Pollonnaruwa <strong>and</strong> Anuradhapura patients who brought the H. hypnale responsible for their<br />
bites were studied prospectively.<br />
Results: among the 302 hump-nosed vipers responsible for causing bites, 301 H, hypnale<br />
<strong>and</strong> 1 H. nepa were identified. Most of the victims were males (4:1) bitten while walking<br />
in the dark (between 1800 <strong>and</strong> 2400 hr) close to their homes. 80% of bites were on the<br />
lower limbs. 90% developed local envenoming (pain, swelling, bruising, blistering)<br />
complicated by local necrosis that required amputation of digits in a few cases. 40% had<br />
incoagulable blood with spontaneous systemic bleeding <strong>and</strong> 10% went on to develop acute<br />
renal failure. No neurological signs were observed in contrast to victims of Russell’s viper<br />
(Daboia russelii). 28% were treated with non-specific Indian polyspecific antivenom<br />
without effect. There were 3 deaths from complications of acute renal failure <strong>and</strong> 2 delayed<br />
deaths in patients with bilateral renal cortical necrosis causing chronic renal failure.<br />
Discussion: this first study of a large group of proven H. hypnale bites revealed a<br />
surprisingly high incidence of haemostatic abnormalities which are potentially lifethreatening.<br />
Acute <strong>and</strong> chronic renal failure proved fatal in 2% of our patients. Only Indian<br />
polyvalent antivenom is available in Sri Lanka. It has proved ineffective both clinically <strong>and</strong><br />
in laboratory assays <strong>and</strong> is dangerous, causing anaphylactoid reactions in 53% of patients.<br />
A specific H. hypnale antivenom is urgently needed.<br />
o Hump-nosed pit viper<br />
o Hypnale hypnale<br />
o Haemostatic disturbances<br />
o Renal failure<br />
118<br />
Tuesday Track 1, K3.14 4.40-5.00pm<br />
INJURIES CAUSED BY VENOMOUS ANIMALS OCURRED IN DOMESTIC<br />
AND COMMERCIAL AQUARIUMS: A STUDY OF 24 CASES.<br />
1,2<br />
Haddad Jr, V .<br />
1<br />
Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, Caixa Postal 557, 18618-000, São Paulo<br />
State, Brazil, haddadjr@fmb.unesp.br<br />
2<br />
Vital Brazil Hospital, Butantan Institute, Avenida Vital Brazil, 1500, Butantan, São Paulo State, Brazil.<br />
INTRODUCTION/OBJECTIVES - Among the recreation activities that more grew in the<br />
last years, the aquarism wins every day new followers in the entire world. Impelled by<br />
beautiful fish, plants <strong>and</strong> decoration objects, the habit involves some problems, as<br />
envenoming after contact with venomous animals. This communication reports the clinical<br />
aspects <strong>and</strong> the animals most frequently involved with envenoming <strong>and</strong> the therapeutic <strong>and</strong><br />
preventive measures for control of the problem. The other objectives are to alert for the<br />
possibility of accidents <strong>and</strong> to investigate the level of information of the proprietors about<br />
the animals present in the aquarium. METHODS - A prospective study was accomplished<br />
for three years being looked for the detection of injuries for venomous animals in<br />
aquariums. These data served as base for epidemiologic, clinical <strong>and</strong> therapeutic studies on<br />
the problem. RESULTS/DISCUSSION - in near 600 injuries by venomous aquatic<br />
animals, fish <strong>and</strong> sea urchins in aquariums caused 28 or near 4,5% of the total 1 . The<br />
animals associated to the injuries were marine stingrays (4), freshwater stingrays (2),<br />
freshwater catfish (2), s<strong>and</strong> moray eel (1), sea urchins (3) <strong>and</strong> lionfish (12). The owners of<br />
aquariums most of the time do not have information about the risks, what intervenes with<br />
the prevention measures that would have to be taken when dealing with the animals. The<br />
use of thick rubber gloves is recommended when it is necessary the introduction of the<br />
h<strong>and</strong>s in an aquarium (domestic or not). The use of nets is also recommended when to be<br />
manipulated dangerous species of animals. The envenoming are not common, but they can<br />
occur if the necessary cares will not be taken <strong>and</strong> a good knowledge of the animal kept in<br />
the aquarium does not exist. The centers of studies on toxicology <strong>and</strong> toxinology should be<br />
alert for the problem.<br />
(1). Haddad Jr V (2000). Atlas of Brazilian dangerous aquatic animals: a medical guide of diagnosis <strong>and</strong> treatment. São<br />
Paulo: Editora Roca, 145 pp.<br />
o venomous aquatic animals<br />
o aquarium<br />
o venomous fishes<br />
o sea urchins<br />
119
Tuesday Track 2, K3.17 3.00-3.20pm<br />
A dynamic interpretation of the pharmacokinetic volume of distribution <strong>and</strong> its relation to the<br />
pharmacokinetics of F(ab')2 <strong>and</strong> other drugs.<br />
C. Sevcik 1* , V. Salazar 2 , P. Díaz 1 , G. D'Suze 1 .<br />
1 Laboratory of Cellular Neuropharmacology, 2 Histology Service from the Centro de Biofísica y<br />
Bioquímica Instituto Venezolano de Investigaciones Científicas (IVIC), Apartado 21827,<br />
Caracas 1020A, Venezuela. * csevcik@ivic.ve<br />
Introduction: We use fluorescent microscopy <strong>and</strong> numerical methods to study the<br />
pharmacokinetics of fluorescin isothiocyanate labeled F(ab’)2 [FITC-F(ab’)2] in<br />
mice.<br />
Methods: We developed the equation V d t = D A<br />
120<br />
z<br />
AUC ∞[∑ i= 1<br />
−<br />
Ci τi e<br />
t<br />
τ i]<br />
¿[∑<br />
z<br />
i= 1<br />
−<br />
C i e<br />
t<br />
τ i] to<br />
describe the dependency of the volume of distribution with time. Where DA is the<br />
amount of drug available in de body after an i.v. bolus, AUC∞ is the<br />
pharmacokinetic total area under the concentration curve, Ci are the<br />
concentrations at t=0 for each of the z components of a multiexponential time<br />
course of the plasmatic concentration of drug, <strong>and</strong> τi are the time constant for each<br />
of these components. This equation is less restricted that one previously proposed<br />
by Nazi (1976).<br />
Results: Fluorescent microscopy show that the rapid initial decay in plasmatic<br />
F(ab')2 concentration (Sevcik et al., 2004; Vázquez et al., 2005) may be related to<br />
uptake of F(ab')2 by vascular endothelium which, in combination with accumulation<br />
in the vascular wall connective tissue may produce an intermediate plateau in<br />
F(ab')2 Vd(t) time course a <strong>and</strong> to produce a slow time course of F(ab')2 Vd(t) which<br />
requires up to 10 h to reach its final steady value.<br />
Discussion: The half time of decay of plasmatic concentration of F(ab’)2 <strong>and</strong> other<br />
drugs has little use to estimate how a drug distributes through the body to exert its<br />
action, <strong>and</strong> in some instances predicts that intermediate plateaus in the time<br />
course of Vd(t) exist. We also used data from the literature, as well as the equation<br />
above to show that the kinetic considerations discussed ahead may also apply to<br />
o Antivenoms<br />
o Pharmacokinetics<br />
o Distribution<br />
o<br />
F(ab’)2<br />
Tuesday Track 2, K3.17 3.20-3.40pm<br />
Effect of Leukocyte Inhibitors Benzydamine <strong>and</strong> Cyclophosphamide on Lung<br />
Injury Caused by Tityus discrepans Scorpion Venom<br />
D´Suze, G 1* , Salazar, V 2 , Díaz, P 1 , Sevcik, C 1 .<br />
1 Celullar Neuropharmacology Lab. 2 Light Microscopy Service. IVIC, Biophysics <strong>and</strong> Biochemistry<br />
Centre, Apartado 21827, Caracas 1020-A, Venezuela, *gdsuze@ivic.ve<br />
Introduction: Severe scorpion envenoming produces a systemic inflammatory response<br />
syndrome causing pulmonary distress. Previously we suggested a close relationship among<br />
leukocyte activation, fibrin alveolar deposition <strong>and</strong> lung injury. This study was aimed to<br />
investigate the effect of a nonsteroidal anti-inflammatory drug <strong>and</strong> an immunosuppressive<br />
alkylating agent on lung injury caused by T. discrepans venom.<br />
Methods: Male IVIC strain mice (~40 g) were previously treated with benzydamine (BZ,<br />
20 mg/kg), saline or cyclophosphamide (CP, 100 mg/kg). Severe experimental envenoming<br />
was induced by sc venom (56 μg/mouse) injection. Animals were sacrificed after 5 h by<br />
cervical dislocation. Lungs were fixed by infusion through the trachea <strong>and</strong> immersion in<br />
4% w/v formaldehyde. The Carstairs' method was used for fibrin staining. Stereological<br />
<strong>and</strong> morphometric study was carried out using ImageJ determining the fractional area<br />
occupied by nuclei (FAN), fibrin (FAF), parenchyma (FAP) <strong>and</strong> alveolar space (FAA).<br />
Results: FAN was increased by 210% of the control in venom alone, by 56% of the<br />
control in BZ <strong>and</strong> by a statistically insignificant 9% in CP. FAF was increased by 188% of<br />
the control in venom alone, by 25% of the control in BZ <strong>and</strong> by 42% in CP. FAP was<br />
increased by 75% of the control in venom alone, by 36% of the control in BZ <strong>and</strong> by a<br />
statistically insignificant 5% in CP. FAA was decreased by 19% of the control in venom<br />
alone, by 13% of the control in BZ <strong>and</strong> by a statistically insignificant 0.3% in CP.<br />
Discussion: Both leukocyte inhibitors antagonised venom effects on all parameters studied.<br />
Cyclophosphamide was more potent as inhibitor that benzydamine, with the exception of<br />
their effects on FAF which was more inhibited by BZ. These findings support our previous<br />
proposal that leukocytes play a major role in the production of lung injury by T. discrepans<br />
venom. (Financed in part by Lab. Silanes/Inst. Bioclón <strong>and</strong> FONACIT grant S1-<br />
2001000908)<br />
o Cyclophosphamide<br />
o Benzydamine<br />
o Tityus<br />
o Lung<br />
121
Tuesday Track 2, K3.17 3.40-4.00pm<br />
Pig as an experimental model for the study of local tissue necrosis caused by snake<br />
venoms<br />
Imkhieo, S. 1 , Nakthong, C. 2 , Kespichayawattana, W. 1 , Sirimujalin, R. 2 , <strong>and</strong> Ratanabanangkoon, K. 1*<br />
1 Laboratory of Immunology, Chulabhorn Research Institute, Bangkok, Thail<strong>and</strong>,*sckrt@mahidol.ac.th<br />
2 Faculty of Veterinary Medicine, Mahidol University, Salaya, Nakorn Pathom, Thail<strong>and</strong><br />
Introduction: Snake venoms can cause serious toxicity locally or systemically or both.<br />
While systemic poisoning can be treated successfully with specific antivenoms, local tissue<br />
necrosis (LTN) which in some cases may be severe <strong>and</strong> require skin graft/amputation,<br />
remains to be tackled. Experiments on LTN have been carried out mostly in rodents. Pig,<br />
which unlike rodents, has skin structure similar to that of human, was studied here. The<br />
aim was to find manifestations induced by snake venoms that can be precisely quantitated.<br />
Furthermore, the experiments should be short so as to minimize suffering, while<br />
maximizing the number of data points per animal.<br />
Materials <strong>and</strong> Methods: Pigs weighing 20±2 kg <strong>and</strong> fresh C.rhodostoma venom (CRV)<br />
were used. Plasma cortisol <strong>and</strong> IL-6 were quantitated using commercial immunoassay kits.<br />
Creatine kinase was assayed spectrophotometrically.<br />
Results <strong>and</strong> Discussion: I.d. injection of CRV on the lateral sides of pig induced<br />
induration, the area of which peaked at about 60 min <strong>and</strong> was dose-dependent.<br />
Furthermore, dissection of these injection sites after 3-4 hours exposed subcutaneous<br />
hemorrhage; whose area was also dose dependent. A total of 24 data points (injection sites)<br />
could be obtained per pig. By implanting a catheter into the jugular vein, blood could be<br />
drawn gently <strong>and</strong> serially. After i.m. injection of CRV, plasma creatine kinase <strong>and</strong> cortisol<br />
could be determined, these markers peaked at 20 hrs <strong>and</strong> 2 hrs, respectively. Plasma IL-6<br />
could not be detected during 72 hours after the venom injection. Finally, histological study<br />
could be made by serial punch biopsy to study the pathological changes at various skin<br />
layers during the first few minutes of venom injection. Such study is not possible in<br />
human, especially with hemotoxic venoms which could lead to severe hemorrhage. This<br />
animal model should be useful to the study of LTN <strong>and</strong> its treatment modality.<br />
This study was supported by a grant from Thail<strong>and</strong> Research Fund.<br />
o Snake venom<br />
o Local tissue necrosis<br />
o Animal model<br />
o pig<br />
122<br />
Tuesday Track 2, K3.17 4.00-4.20pm<br />
Ureases display biological effects independent of enzymatic activity. Is there a<br />
connection to diseases caused by urease-producing bacteria ?<br />
Olivera-Severo, D 1 , Wassermann, G.E 1 , Carlini, C.R 1,2<br />
1 Graduate Program in Cellular <strong>and</strong> Molecular Biology, Center of Biotecnology, Universidade Federal<br />
do Rio Gr<strong>and</strong>e do Sul, Porto Alegre, Av. Bento Gonçalves, 9500, CEP 91.501-970, Porto Alegre, Brazil,<br />
2 Department of Biophysics, Universidade Federal do Rio Gr<strong>and</strong>e do Sul, Porto Alegre, Av. Bento<br />
Gonçalves, 9500, CEP 91.501-970, Porto Alegre, Brazil. ccarlini@ufrgs.br<br />
Introduction: Ureases are highly homologous enzymes from plants, fungi <strong>and</strong> bacteria that<br />
hydrolyze urea into ammonia <strong>and</strong> carbon dioxide. Fungal <strong>and</strong> plant ureases are homooligomers<br />
of 90-kDa subunits, while bacterial enzymes are multimers of two or three<br />
subunit complexes. We showed that jack bean urease <strong>and</strong> its isoform canatoxin induce<br />
exocytosis in a number of systems, releasing histamine from mast cells, insulin from<br />
pancreatic cells, neurotransmitters from brain synaptosomes <strong>and</strong> activating platelet<br />
aggregation (ED50 0.015 mg/mL). In vivo canatoxin induces rat paw edema <strong>and</strong> neutrophil<br />
chemotaxis. These effects are independent of ureolytic activity <strong>and</strong> require activation of<br />
eicosanoid metabolism <strong>and</strong> calcium channels. Here we investigated if bacterial ureases<br />
induce platelet aggregation or have pro-inflammatory activity.<br />
Methods: purified Bacillus pasteurii urease <strong>and</strong> purified recombinant Helicobacter pylori<br />
urease were tested for platelet-aggregating activity <strong>and</strong> in the rat paw edema model.<br />
Inhibitors of eicosanoid metabolism or blockers of platelet receptors were used to<br />
investigate signaling transduction.<br />
Results: B. pasteurii <strong>and</strong> H. pylori ureases induced platelet aggregation (ED50 0.4 <strong>and</strong> 0.1<br />
mg protein/mL, respectively) recruiting the same signal transduction pathways previously<br />
described for canatoxin. We also observed that 50 micrograms of purified H. pylori urease<br />
induced maximal pro-inflammatory activity in the rat paw edema model.<br />
Discussion: H. pylori causes gastric ulcers <strong>and</strong> cancer by a mechanism that is not fully<br />
understood. H. pylori urease, accounting for ~10% of bacterial protein, is important for<br />
gastric colonization as it neutralizes the acidic medium permitting survival in the stomach.<br />
Our findings that H. pylori urease has biological properties not related to its enzymatic<br />
activity could be relevant to elucidate the role of this protein in the pathogenesis of the<br />
gastrointestinal disease caused by this bacterium.<br />
o urease<br />
o Helicobacter pylori<br />
o Platelet aggregation<br />
o eicosanoids<br />
123
Tuesday Track 2, K3.17 4.20-4.40pm<br />
The Use Of Indigenous Knowledge in Rural Development: A Case Study In The<br />
Use Of Herbal Medicine In The Treatment of Diseases In The Northern Region Of<br />
Ghana.<br />
Arthur,P.K, Opoku, K. F<br />
University of Cape Coast, School of Agriculture, PMB, Cape Coast, Ghana,*patrickarthur84@yahoo.co.uk<br />
In Africa, herbal medicine is often used as primary treatment for various diseases.<br />
Generally, traditional medicines are not well researched <strong>and</strong> are poorly regulated. This<br />
paper examines the use of herbal medicines in the treatment of diseases in northern Ghana.<br />
It discusses the role of indigenous knowledge in rural development. It outlines the<br />
development in interest in herbal medicine <strong>and</strong> the rising benefit that may accrue from<br />
indigenous health resources. Field studies were conducted to investigate the medicinal<br />
plants, through identification, collection <strong>and</strong> domestication of these plants in Northern<br />
Region of Ghana. Questionnaire, personal interview <strong>and</strong> review <strong>and</strong> review of available<br />
records showed that herbal medicines were used in the treatment of diseases in both human<br />
beings <strong>and</strong> animals. The researched centered on pharmacology, toxicology <strong>and</strong> the<br />
pharmacokinetics of these herbal medicines. This paper reviews the relation between<br />
indigenous medicines <strong>and</strong> pharmacology, encompassing human health <strong>and</strong> indigenous<br />
medicine; vertinary medicine <strong>and</strong> animal health; maternal <strong>and</strong> child health; <strong>and</strong> sexual<br />
health <strong>and</strong> disease. Although no clinical trials of efficacy exist on the use of herbal<br />
medicines, efforts should be made by mainstream health professionals to provide validated<br />
information to traditional healers <strong>and</strong> patients on the judicious use of herbal remedies.<br />
References:<br />
Ayo Walberg, health, vol. 10, No. 2, 123-147<br />
Mensah-Dapaah, K, traditional healing, Ghana Journal of Science,1968, 21, 16-21<br />
o Pharmacology<br />
o Pharmacokinetics<br />
o Toxicology<br />
o Indigenous<br />
124<br />
Tuesday Track 3, K3.25 3.00-3.20pm<br />
Pharmacophore mapping of the κ-atracotoxins: selective insect potassium channel<br />
blockers that reveal a novel insecticide target<br />
Gunning, S.J. 1 , Maggio, F. 2 , Valenzuela, S. 1 Broady, K.W. 1 , King, G.F. 2 , Nicholson,<br />
G.M. 1,*<br />
1 Department of Medical & Molecular Biosciences, University of Technology, Sydney, Broadway, NSW,<br />
2007 Australia, *Graham.Nicholson@uts.edu.au<br />
2 Department of Molecular, Microbial <strong>and</strong> Structural Biology, University of Connecticut Health Center,<br />
263 Farmington Ave., Farmington CT 06032-3305, USA<br />
The κ-atracotoxins (κ-ACTXs) are a family of insect-selective peptide neurotoxins<br />
containing 36-37 residues, first isolated from the venom of the Blue Mountains funnel-web<br />
spider, Hadronyche versuta. The amino acid sequence <strong>and</strong> 3D structure of κ-ACTX<br />
provided few clues as to its likely molecular target. In order to characterise the site of<br />
action <strong>and</strong> phylogenetic specificity of these toxins, whole-cell patch-clamp<br />
electrophysiology was employed using isolated DUM neurons from the American<br />
cockroach (Periplaneta americana). The prototypic family member κ-ACTX-Hv1c had no<br />
effect on the gating or kinetics of INa or ICa at concentrations up to 1 μM. However, at the<br />
same concentration, it reduced outward Kv channel currents. Subsequent experiments using<br />
insect DUM neurons indicated that inhibition of the macroscopic IK was due to a block of<br />
KCa channels, with an IC50 of 3.1 ± 0.1 nM, <strong>and</strong> not ‘A-type’ or delayed-rectifier Kv<br />
channels. Insect selectivity was confirmed by a lack of activity on rat dorsal root ganglion<br />
neuron global IK as well as IK(Ca) at doses up to 1 µM. κ-ACTX-Hv1c is a selective insect<br />
KCa channel pore-blocker, not a gating modifier, as inhibition of insect IK(Ca) occurred in<br />
the absence of any voltage-dependent actions on channel activation, <strong>and</strong> the block could be<br />
reduced 36-fold by increasing [K + ]i. The insect target was further validated by κ-ACTX-<br />
Hv1c induced inhibition of IK(Ca) from cloned cockroach KCa (pSlo) channels expressed in<br />
HEK293 cells. Additional experiments using alanine mutants confirmed that the<br />
pharmacophore of κ-ACTX-Hv1c comprises Arg 8 , Pro 9 , Val 29 , Tyr 31 as well as IIe 2 <strong>and</strong> the<br />
vicinal disulfide (Cys 13 -Cys 14 ), previously identified by toxicity tests in crickets.<br />
Interestingly, an R8K mutant was 155-fold less active than the native toxin, whereas the<br />
Lys residue in the functional diad of vertebrate Kv channel toxins normally inserts into the<br />
channel pore. Thus, κ-ACTX-Hv1c appears to bind to an insect-specific site in the channel<br />
vestibule <strong>and</strong> occlude the pore without binding to conserved pore residues found in<br />
vertebrate <strong>and</strong> insect channels. This study therefore identifies insect KCa channels as a<br />
novel insecticide target.<br />
o spider toxin<br />
o Kv channels<br />
o insecticide<br />
o electrophysiology<br />
125
Tuesday Track 3, K3.25 3.20-3.40pm<br />
Jellyfish <strong>and</strong> other Cnidarians cause pain by affecting TRPV1 channels.<br />
Cuypers, E. 1 , Yanagihara, A. 2 , Karlsson, E. 3 & Tytgat, J. 1*<br />
1<br />
University of Leuven, Laboratory of Toxicology, O&N2, Herestraat 49, P.O. Box 922, 3000 Leuven,<br />
Belgium, * jan.tytgat@pharm.kuleuven.be<br />
2<br />
University of Hawaii at Manoa, Békésy Laboratory of Neurobiology, 1993 East West Road, Honolulu,<br />
HI, 96822, USA<br />
3<br />
Lindsbergsgatan 11A, Uppsala, Sweden<br />
Jellyfish <strong>and</strong> other cnidarian envenomations are characterised by a burning-pain<br />
sensation, the precise underlying mechanisms of which are unclear. Activation of<br />
TRPV1, a non-selective cation channel that is highly expressed in nociceptive<br />
neurons, leads to an inward flow of monovalent <strong>and</strong> bivalent cations, resulting in<br />
depolarisation of the cell <strong>and</strong> generation of burning pain.<br />
Here, we show in vitro (heterologous expression system with Xenopus laevis<br />
oocytes) <strong>and</strong> in vivo (rat studies) evidence for TRPV1 activation in cnidarian<br />
envenomations. Cnidaria venoms, unlike those from other non-cnidarian species,<br />
interfered with the desensitization of TRPV1 channels in an allosteric manner but<br />
did not change the voltage dependent open probability. Furthermore, the cnidarian<br />
venom from Cyanea capillata induced a nociceptive reactivity, comparable to<br />
capsaicin, in laboratory rats, <strong>and</strong> the selective TRPV1 antagonist, BCTC, was able<br />
to reduce both capsaicin- <strong>and</strong> venom-induced nociceptive behaviours, suggestive<br />
of some unique anti-nociceptive properties of VR1 antagonists.<br />
These findings, which to our knowledge are the first to explain at least part of the<br />
symptomology of cnidarian envenomations, provide insights into the design of<br />
more effective treatments for this global public health problem.<br />
1. Caterina et al. (1997) Nature 389, 816-24.<br />
2. Bailey, P. M. et al. (2003) Med J Aust 178, 34-7.<br />
3. Nilius, B. et al. (2005) J Physiol 567, 35-44.<br />
4. Swanson, D. M. et al. (2005) J Med Chem 48, 1857-72.<br />
o Cnidarians<br />
o TRPV1<br />
o Pain<br />
o Desensitization<br />
126<br />
Tuesday Track 3, K3.25 3.40-4.00pm<br />
Novel Conotoxin Frameworks<br />
Möller, C. 1 , Rahmankhah, S. 1 , Urdaneta, A.M. 1 , Castillo, C. 2 , Martinez, J.C. 2 , Hern<strong>and</strong>ez, D. 2 , Moran, O. 3 ,<br />
Fields G.B. 1 <strong>and</strong> Marí, F. 1 *<br />
1<br />
Department of Chemistry & Biochemistry <strong>and</strong> Center of Excellence in Biomedical & Marine<br />
Biotechnology, Florida Atlantic University, Boca Raton, Fl 33431, USA. mari@fau.edu<br />
2<br />
Center of Biosciences <strong>and</strong> Molecular Biology. Instituto de Estudios Avanzados (IDEA). Sartenejas,<br />
Venezuela.<br />
3<br />
Instituto di Biofisica. Consiglio Nazionale delle Ricerche, Genova, Italy.<br />
Cone snails are marine venomous predatory molluscs that excel at producing constrained<br />
peptide toxins (Conotoxins). Conotoxins have been recognized as great<br />
neuropharmacological agents including the development of Prialt TM , a powerful<br />
painkiller that has been approved by the FDA in the U.S., which is the first drug of<br />
marine origin to receive such status. From the venomics of cones snail species from the<br />
Americas, we have characterized several novel conotoxin frameworks, with the<br />
following Cys/Loop arrangements: a 4-Cys/3-loop (Framework 14 = F14-conotoxins), a<br />
6-Cys/4-loop (F15-conotoxins) that features a vicinal disulphide bond, 4-Cys/2-loop<br />
(F16-conotoxins) with a “reverse alpha” Cys arrangement, 6-Cys/3-loop (F17conotoxins),<br />
6-Cys/2-loop (F18-conotoxins) <strong>and</strong> 6-Cys/6-loop (F19-conotoxins). All of<br />
these frameworks are likely to define new conotoxin gene superfamilies <strong>and</strong> represent<br />
unique peptidic scaffolds not seen before in any organism (except for F14).<br />
These scaffolds exp<strong>and</strong> the structural diversity of the known conopeptide library<br />
enhancing the neuropharmacological reach of conotoxins. Three F14-conotoxins have<br />
been purified from Conus floridanus floridensis (flf14a, flf14b <strong>and</strong> flf14c) <strong>and</strong> one from<br />
Conus villepinii (vil14a). Their disulfide bonding pattern (C1-C4; C2-C3) was<br />
determined by limited proteolysis <strong>and</strong> analysis by MS. The nanoNMR spectra (< 100<br />
nanomoles) of the native peptides showed a very well defined structure in solution, <strong>and</strong><br />
in agreement with their CD spectra, all four conotoxins revealed a Helix-Loop-Helix as<br />
their main 3D fold. These novel Conus peptides structurally resemble the κ-hefutoxins<br />
found in scorpions. vil14a has a conserved amino acid dyad defining for the binding to<br />
the voltage-gated potassium channels. Patch-clamp measurements in HEK cells<br />
transfected with Kv1.3, indicate that vil14a blocks this potassium channels as predicted<br />
by our structural analysis. The details on the isolation, structural <strong>and</strong> functional<br />
characterization of the F14-19 conotoxins frameworks will be discussed.<br />
o Cone snails<br />
o Venom<br />
o Conotoxins<br />
o Potassium channels<br />
127
Tuesday Track 3, K3.25 4.00-4.20pm<br />
A dual-target, self-synergizing peptide toxin from spider venom<br />
Sollod, B,L. 1 , Gunning, S. 2 , Wen, S. 2 , Nicholson, G.M. 2 , <strong>and</strong> King, G,F. 1,*<br />
1<br />
Department of Molecular, Microbial <strong>and</strong> Structural Biology, University of Connecticut Health Center,<br />
263 Farmington Ave., Farmington CT 06032-3305, USA *glenn@psel.uchc.edu<br />
2<br />
Department of Medical & Molecular Biosciences, University of Technology, Sydney, Broadway NSW<br />
2007, Australia<br />
Excluding insects, which are their primary prey, spiders are the most successful terrestrial<br />
invertebrates. Whereas most invertebrates produce an array of neuropeptides for internal<br />
regulation of various physiological <strong>and</strong> behavioural processes, spiders synthesize in their<br />
venom gl<strong>and</strong>s a combinatorial library of neuropeptides that are designed to kill or paralyze<br />
envenomated prey 1 . These neurotoxins are initially produced as prepropeptide precursors<br />
that are posttranslationally processed to yield the mature toxins. The complete venom is<br />
often remarkably complex—recent peptidomic analyses reveal that a single spider venom<br />
can comprise more than 1000 different peptide toxins 2 .<br />
We have been exploring the potential of spider venoms to contribute to the development of<br />
insecticides with novel modes of action. We demonstrated previously that it is possible to<br />
isolate spider toxins that potently block specific subtypes of insect ion channels while<br />
being devoid of activity against vertebrate orthologs of these channels 3 . More recently, via<br />
analyses of venom-gl<strong>and</strong> cDNA libraries, we isolated a novel family of insecticidal peptide<br />
toxins that appear to be ubiquitously expressed in the venom of Australian funnel-web<br />
spiders (genera Atrax <strong>and</strong> Hadronyche). These toxins are remarkable for their ability to<br />
potently block both voltage-gated calcium channels <strong>and</strong> calcium-activated potassium<br />
channels in insect neurons. These toxins are the most potent insecticidal peptide toxins<br />
isolated thus far from spider venoms, <strong>and</strong> their potency appears to result from a selfsynergistic<br />
effect on the two targeted ion channels. In essence, this dual-target toxin<br />
corresponds to a toxin cabal encoded within a single polypeptide chain. The implications<br />
of using a dual-target approach for the development of novel insecticides will be discussed.<br />
1. Sollod, B.L., Wilson, D., Zhaxybayeva, O., Gogarten, J.P., Drinkwater, R., <strong>and</strong> King,<br />
G.F. (2005) Peptides 26, 131–139.<br />
2. Escoubas, P, Sollod, B.L., <strong>and</strong> King, G.F. (2006) Toxicon 47, 650–663.<br />
3. Tedford, H.W., Gilles, N., Ménez, A., Doering, C.J., Zamponi, G.W., <strong>and</strong> King, G.F.<br />
(2004) J. Biol. Chem. 279, 44133–44140.<br />
o spider toxin<br />
o ion channel<br />
o self-synergism<br />
o insecticide<br />
128<br />
Tuesday Track 3, K3.25 4.20-4.40pm<br />
Fulvol Acetate, a novel activator of transient Low-Voltage Activated (LVA) Ca 2+<br />
current<br />
Petit, K.E. 1* , Grolleau, F. 2 , Lapied, B. 3 , Hamon, A. 3 , Biard, J.F. 1<br />
1<br />
Université de Nantes, Nantes Atlantique Universités, SMAB, EA 2160, Faculté de Pharmacie, 1 rue G.<br />
Veil – BP 53508, Nantes, F-44000 France * karina.petit@univ-nantes.fr<br />
2<br />
Laboratoire Biologie Neurovasculaire Intégrée, UMR CNRS 6214 / Inserm 771, UFR Sciences<br />
Médicales, Rue Haute de Reculée, F-49045 Angers cedex 01 France<br />
3<br />
Université d'Angers, RCIM, EA 2647, Faculté des Sciences, 2 boulevard Lavoisier, Angers cedex, F-<br />
49045 France<br />
Fulvol Acetate is a new norsesquiterpene isolated from the soft coral Rhytisma fulvum. The<br />
unique <strong>and</strong> novel effect on neuronal transient Low-Voltage Activated (LVA) calcium<br />
current led us to deposit a patent. 1<br />
Electrophysiological studies were conducted on isolated short term cultured Dorsal<br />
Unpaired Median (DUM) neurons of the cockroach (Periplaneta americana) using the<br />
whole cell patch-clamp technique. Parallel double intracellular microelectrode<br />
investigations were also performed on three α-subunits of the T-type LVA calcium<br />
channels (Cav 3.1, 3.2 <strong>and</strong> 3.3) expressed in Xenopus oocytes.<br />
In current-clamp, external application of Fulvol Acetate on DUM neurons induced an<br />
increase of the action potential discharge frequency together with a potentialisation of the<br />
posthyperpolarisation. This seemed to indicate the participation of calcium currents. This<br />
was confirmed with additional voltage-clamp experiments indicating that transient LVA<br />
calcium current was specifically increased (60 %) in DUM neurons. No effect of Fulvol<br />
Acetate was observed on both sodium <strong>and</strong> High Voltage-Activated (HVA) calcium<br />
currents. Similar results were observed when Fulvol Acetate was intracellularly applied<br />
into DUM neuron cell body through the patch pipette. By contrast, external application of<br />
Fulvol Acetate onto oocytes expressing the different α-subunits of the T-type LVA<br />
calcium channels did not show any effects.<br />
These results led us to suggest that Fulvol Acetate could act indirectly on the LVA calcium<br />
channel via, among other possibilities an accessory protein. Based on these findings, the<br />
specific increase of the LVA calcium current amplitude produced by Fulvol Acetate makes<br />
this compound the first activator of such channels never reported before.<br />
1 Petit et al. (2003) Patent PCT/FR04/03030.<br />
2 Lacinova et al. (2000) Neuropharmacology, 39, 1254-1266.<br />
3 Yaksh (2006) The Journal of Pain, 7(1), S13-S30.<br />
o Marine natural substance<br />
o Activator of tLVA<br />
o Research tool<br />
o Drug<br />
129
Tuesday Track 3, K3.25 4.40-5.00pm<br />
A novel family of conotoxins target to nACh-Receptors<br />
Kauferstein, S. 1 , Nicke, A. 2 , Kendel, Y. 1 ., Zamudio, F.Z. 3 , Stöcklin, R. 4 , Mebs, D. 1<br />
1University of Frankfurt, Kennedyallee 104, D-60596 Frankfurt am Main, Germany,<br />
kauferstein@em.uni-frankfurt.de<br />
2 Max-Planck-Institute for Brain Research, D-60528 Frankfurt am Main, Germany<br />
3 Instituto de Biotecnologia-UNAM, Avenida Universidad, 2001, Cuernavaca 62210, Mexico<br />
4 Atheris Laboratories, CH-1233 Bernex-Geneva, Switzerl<strong>and</strong><br />
Introduction: Over the last 50 million years, cone snails developed a highly efficient<br />
venomous system.Using their complex venom apparatus they inject a mixture of peptides,<br />
which almost instantly paralyzes the prey. These peptides are consisting mainly of 11 to 35<br />
amino acid residues with a variable number of disulfide bridges, exhibiting a great<br />
variability in their amino acid sequence. The venom of cone snails is one of the most<br />
complex peptide mixtures acting on a wide range of target tissues in the nervous system,<br />
e.g. ion channels <strong>and</strong> receptors. The high affinity of these toxins to these targets made<br />
them indispensable tools in neurochemistry <strong>and</strong> neurophysiology.<br />
Methods: Crude venom samples from Conus capitaneus were tested on nAChRs expressed<br />
in oocytes using the two-electrode voltage-clamp method. Fractionation of the samples<br />
was performed by HPLC followed by liquid chromatography-electrospray-mass<br />
spectrometry analysis. N-terminal amino acid sequence analysis was performed by<br />
automated Edman degradation.<br />
Results: From the venom of Conus capitaneus a protein was isolated which represents a<br />
new family of conotoxins acting at nicotinic AChRs. It has a remarkably high molecular<br />
weight of 11 kDa <strong>and</strong>, as SDS-PAGE revealed the toxin is a dimer. Its N-terminal<br />
sequence exhibits no similarity to any known proteins. Analysis of the subtype selectivity<br />
of the purified toxin on nAChRs revealed subnanomolar potency on α7 <strong>and</strong> nanomolar<br />
potency on� α3β2 nAChRs subtypes with IC50 values of 300 pM <strong>and</strong> 3 nM respectively.<br />
The IC50 value for the �α4β2 subtype was 30 nM, making it the most potent conotoxin<br />
acting on this AchR-subtype known so far.<br />
Discussion/Conclusion: We suggest that some cone snail species, besides families of small<br />
peptides, developed an alternative class of large conopeptides to target the nAChR.<br />
o Conotoxins<br />
o nAChR<br />
o Cone snails<br />
o Voltage clamp<br />
130<br />
Thursday Track 1, K3.14 3.00-3.20pm<br />
Scorpion Beta-Toxins Interact with their Na-channel Receptor Via a Novel 'Ratchet<br />
Mechanism'<br />
Ilan N 1* , Karbat I 1 , Cohen L 1 , Catterall WA 2 , Gordon D 1 , Gurevitz M 1<br />
1<br />
Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Israel<br />
*nitzai@post.tau.ac.il<br />
2<br />
Department of Pharmacology, University of Washington, Seattle, WA 98195-7280, USA<br />
Gating modifiers constitute a large group of polypeptide toxins that interact with the<br />
voltage-sensing module of ion channels. Among them, scorpion beta-toxins induce a<br />
negative shift in the voltage dependence of sodium channel activation following a priming<br />
depolarization. To explain their effect, a ‘voltage sensor trapping’ model has been<br />
proposed, in which the voltage sensor of domain-II (DIIS4) is trapped in an outward,<br />
activated position by a pre-bound beta-toxin upon membrane depolarization (1). Using the<br />
scorpion excitatory beta-toxin, Bj-xtrIT, we demonstrate uncoupling of toxin activity from<br />
its binding affinity by a single substitution E15R, thereby obtaining an efficient antagonist<br />
of the native toxin (2). Kinetic analysis of the effect of the anti-mammalian scorpion betatoxin<br />
Css4 on the rat brain Na-channel, Nav1.2a, revealed a biphasic onset of toxin effect<br />
with a voltage-dependent fast time constant of
Thursday Track 1, K3.14 3.20-3.40pm<br />
A new toxin from the sea anemone Condylactis gigantea with effect on sodium<br />
channel inactivation<br />
Béress, L. 1, 2 , Ständker, L. 1,6 , John, H. 1 , Forssmann, W.G. 1 , Pérez-Castells, J. 6 , López-Méndez, B. 6 ,<br />
Jiménez-Barbero, J. 6 , Giménez-Gallego, G. 6 , Garateix, A. 3 , Aneiros, A. 3† , Christ, T. 4 , Salceda, E. 5 , Soto,<br />
E. 5 , Ravens, U. 4*<br />
1 IPF PharmaCeuticals GmbH, 30625 Hannover, Germany 2 Institut für Experimentelle Toxikologie,<br />
Universitäts-Klinikum, 24105 Kiel, Germany 3 Centre of Marine Bioproducts (CEBIMAR), Loma y 37,<br />
Vedado, Ciudad Habana, Cuba 4* Institut für Pharmakologie und Toxikologie, Medizinischen Fakultät der<br />
TU Dresden, Fetscherstraße 74, 01307 Dresden, Germany, * ravens@rcs.urz.tu-dresden.de<br />
5 Institute of Physiology, University Autonomous of Puebla, 14 Sur 6301, CP 72570, Puebla,México. 6 Centro<br />
de Investigaciones Biologicas (CIB/CSIC), C. Ramiro de Maeztu, 9, 28040 Madrid, Spain<br />
Our aim was to isolate <strong>and</strong> characterize a peptide toxin representing the main crab<br />
paralyzing activity in the sea anemone Condylactis gigantea. Methods: Applying ethanolic<br />
acetic acid extraction of the sea anemone Condylactis gigantea <strong>and</strong> a multistep<br />
chromatographic procedure employing cation exchange, size exclusion, <strong>and</strong> reversed phase<br />
chromatography, a peptide toxin was isolated. The toxic activity was monitored by its crab<br />
paralyzing effects <strong>and</strong> biophysical <strong>and</strong> structural features of the purified peptide were<br />
characterized by mass spectrometry, sequence <strong>and</strong> NMR analysis. Its electrophysiological<br />
properties were analysed on rat dorsal root ganglion neurons <strong>and</strong> on myocardial cells of<br />
guinea pigs. A new peptide toxin exhibiting a MW of 5043 Da (av.) <strong>and</strong> comprising 47<br />
amino acid residues was isolated <strong>and</strong> exhibited a strong paralytic activity on crustacea<br />
(LD50 approx. 1 μg/kg). Complete sequence analysis of the toxic peptide revealed the<br />
isolation of a new member of type I sea anemone sodium channel toxins. The toxin termed<br />
CgNa contained the typical pattern of six cysteine residues. From 11 kg of wet starting<br />
material approximately 1 g of the peptide toxin was isolated. Using whole-cell patch clamp<br />
technique (n = 60) under current clamp condition CgNa increased action potential duration<br />
of rat dorsal root ganglion neuronns. It prolonged the cardiac action potential duration <strong>and</strong><br />
enhanced contractile force albeit at 100-fold higher concentrations than the Anemonia<br />
sulcata toxin ATXII. Homonuclear NMR experiments have been used to determine the 3D<br />
structure of the peptide. The structure consists of four beta str<strong>and</strong>s <strong>and</strong> a long nonstructured<br />
loop between residues 7-19. Root mean squared deviation for the best 20<br />
structures (CYANA) is better than 1 Å. The electrophysiological effects of CgNa may be<br />
due to slowing down of the TTX-S sodium current inactivation, without modifying the<br />
activation process. The action on sodium channel inactivation <strong>and</strong> on cardiac excitation<br />
contraction coupling resemble previous results with peptides obtained from sea anemones,<br />
probably by binding to receptor site 3 of the mammalian sodium channel. We suggest that<br />
CgNa may represent the main peptide toxin of this sea anemone species.<br />
o peptide toxin<br />
o sodium channel inactivation<br />
o sea anemone<br />
o 3D structure<br />
132<br />
Thursday Track 1, K3.14 3.40-4.00pm<br />
Allosteric interactions between scorpion toxin receptor sites on voltage-gated Na channels<br />
imply a novel role for weakly active components in arthropod venom<br />
Cohen, L 1 , Lipstein, N 1 , Gordon, D 1 .<br />
1 Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat<br />
Aviv, Tel Aviv 69978, Israel; dgordon@post.tau.ac.il<br />
Scorpion beta <strong>and</strong> alpha-toxins modify the activation <strong>and</strong> inactivation of voltage-gated<br />
sodium channels. Although the two types of toxin bind at two distinct receptor sites on<br />
the same sodium channel, they exhibit synergic effects when co-injected into insects<br />
(1). To clarify the basis of this synergism we examined the mutual effects of alpha <strong>and</strong><br />
beta toxin representatives in radio-lig<strong>and</strong> binding assays. We found positive allosteric<br />
interactions between receptor site-4 of the excitatory Bj-xtrIT <strong>and</strong> the depressant<br />
LqhIT2 beta toxins <strong>and</strong> receptor site-3 of the alpha toxin LqhαIT, on locust neuronal<br />
membranes. Unexpectedly, a non-toxic mutant Bj-xtrIT-E15R (2), which binds with<br />
high affinity to receptor site-4, was able to enhance LqhαIT binding <strong>and</strong> toxicity<br />
similarly to the unmodified Bj-xtrIT. This result indicates that mere binding of a nontoxic<br />
lig<strong>and</strong> to receptor site-4 ('silent binding') induces a conformational change that<br />
does not alter channel gating, but influences toxin binding at receptor site-3 leading to<br />
enhanced toxicity. This finding suggests a new functional role for weakly toxic<br />
polypeptides in that they enhance the effect of other active neurotoxins in the arthropod<br />
venom. Such 'silent binding' may have also valuable implications in attempts to<br />
improve drug efficacy by combining potent drugs with non-active allosteric enhancers.<br />
References: (1) Herrmann R, Moskowitz H, Zlotkin E, Hammock B (1995) Positive<br />
cooperativity among insecticidial scorpion neurotoxins. Toxicon 33:1099-1102<br />
(2) Karbat I, Cohen L, Gilles N, Gordon D, Gurevitz M (2004) Conversion of a<br />
scorpion toxin agonist into an antagonist highlights an acidic residue involved in<br />
voltage sensor trapping during activation of neuronal Na channels. FASEB J 18:683-89<br />
o Scorpion toxin<br />
o sodium channel<br />
o alpha-like-toxin<br />
o receptor recognition<br />
133
Thursday Track 1, K3.14 4.00-4.20pm<br />
OD1, α-like toxin isolated from the Iranian yellow scorpion Odonthobuthus doriae<br />
venom<br />
Jalali, A 1 *, Bosmans, F 2 , Amininasab, M 3 , Clynen, E 4 , Cuypers, E 2 , Zaremirakabadi,<br />
A 5 , Sarbolouki, M.N 3 , Schoofs, L 4 , Vatanpour, H 6 , Tytgat, J 2 **<br />
1<br />
Dept. of Pharmacology <strong>and</strong> Toxicology, Jundishapour Univ. of Med. Sci., Ahwaz, Iran,<br />
*Amir.Jalali@ajums.ac.ir<br />
2<br />
Lab. of Toxicology, Univ. of Leuven, Leuven, Belgium, ** Jan.Tytgat@pharm.kuleuven.ac.be<br />
3<br />
IBB Institute, Tehran Univ. of Med. Sci., Tehran, Iran.<br />
4<br />
Lab. for Developmental Physiology <strong>and</strong> Mol. Biology, Univ. of Leuven, Belgium.<br />
5<br />
Razi Institute of Vaccine <strong>and</strong> Serum, Karaj, Iran.<br />
6<br />
Dept. of Pharmacology <strong>and</strong> Toxicology, Shaheed Beheshti Univ. of Med. Sci., Tehran, Iran.<br />
Within the most dangerous scorpions in Iran, the Odonthobuthus doriae sting can cause<br />
fatal envenoming with unknown mechanism of action. With view to this <strong>and</strong> similarity of<br />
clinical effects to toxins that affect Na + channels, this investigation was conducted to<br />
access to the bioactive substances in the scarcely studied Iranian scorpion venom.<br />
In this study, we isolated <strong>and</strong> pharmacologically characterized the α-like toxin from the<br />
Odonthobuthus doriae venom, termed OD1 <strong>and</strong> its primary sequence was determined:<br />
GVRDAYIADDKNCVYTCASNGYCNTECTKNGAESGYCQWIGRYGNACWCIKLPDEVPIRIPGKCR<br />
Using the two-electrode voltage clamp technique, the pharmacological effects of OD1<br />
were studied on three cloned voltage-gated Na + channels expressed in Xenopus laevis<br />
oocytes (Na(v)1.2/beta1, Na(v)1.5/beta1, para/tipE). The inactivation process of the<br />
insect channel, para/tipE, was severely hampered by 200nM of OD1 (EC50= 80 ±14nM)<br />
while Na(v)1.2/beta1 still was not affected at concentrations up to 5microM.<br />
Na(v)1.5/beta1 was influenced at micromolar concentrations.<br />
Our results show that OD1 can inhibit the inactivation process of both mammalian (Nav1.5)<br />
<strong>and</strong> insect VGSCs (para). However, OD1 does posses a distinct preference for para.<br />
134<br />
Thursday Track 1, K3.14 4.20-4.40pm<br />
Direct Evidence that Receptor Site-4 of Sodium Channel Gating Modifiers is not<br />
Dipped in the Phospholipid Bilayer of Neuronal Membranes<br />
Cohen L 1* , Gilles N 2 , Karbat I 1 , Ilan N 1 , Gordon D 1 , Gurevitz M 1<br />
1<br />
Department of Plant Sciences, George S Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv<br />
69978, Tel-Aviv, Israel *liorcoh@post.tau.ac.il<br />
2<br />
CEA, Department d'Ingenierie et d'Etudes des Proteines, CE Saclay F-91191 Gif Sur Yvette Cedex,<br />
France<br />
In a recent note to Nature R. MacKinnon has raised the possibility that potassium channel<br />
gating modifiers are able to partition in the phospholipid bilayer of neuronal membranes<br />
<strong>and</strong> by increasing their partial concentration adjacent to their receptor affect channel<br />
function with apparent high affinity (1). This suggestion was adopted by Smith et al. (2),<br />
who analyzed the partitioning of sodium channel modifiers in liposomes. They found that<br />
certain modifiers were able to partition in these artificial membranes <strong>and</strong> on this basis<br />
have extrapolated that scorpion beta-toxins interact with their channel receptor in a<br />
similar mechanism as that proposed by MacKinnon. Since this hypothesis has actually<br />
raised a new conception, we examined it in binding assays using a number of<br />
pharmacologically distinct scorpion beta-toxins <strong>and</strong> insect <strong>and</strong> mammalian neuronal<br />
membrane preparations, as well as by analyzing the rate by which the toxin effect on<br />
gating of Drosophila DmNav1 <strong>and</strong> rat brain rNav1.2a develops. We show that in general<br />
scorpion beta-toxins do not partition in neuronal membranes <strong>and</strong> that in the case where a<br />
depressant beta-toxin partitions in insect neuronal membranes, this partitioning is<br />
unrelated to its interaction with the receptor site <strong>and</strong> the effect on the gating properties of<br />
the sodium channel. These results negate the hypothesis that the high affinity of betatoxins<br />
for sodium channels is gained by their ability to partition in the phospholipid<br />
bilayer, <strong>and</strong> clearly indicate that the receptor site for scorpion beta-toxins is accessible to<br />
the extra-cellular solvent.<br />
References<br />
1. Lee SY & MacKinnon R (2004) Nature 430, 232-235<br />
2. Smith JJ, Alphy S, Seibert AL & Blumenthal KM (2005) J Biol Chem 280, 11127-<br />
11133<br />
3. Cohen L, Gilles N, Karbat I, Ilan N, Gordon D & Gurevitz M (2006) J Biol Chem In<br />
press<br />
o Scorpion beta-toxins<br />
o Phospholipid bilayer<br />
o Voltage-gated sodium channels<br />
o Partition<br />
135
Thursday Track 1, K3.14 4.40-5.00pm<br />
Structure <strong>and</strong> alanine scan of a spider toxin that affects the activation of<br />
mammalian voltage-gated Na + channels<br />
Norton, R.S. 1 , Corzo, G. 2 , Sabo, J.K. 1 , Bosmans, F. 3 , Villegas, E. 4 , Tytgat, J. 3<br />
1<br />
Walter <strong>and</strong> Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville 3050, Australia.<br />
ray.norton@wehi.edu.au<br />
2<br />
Institute of Biotechnology-UNAM, Av. Universidad 2001, Cuernavaca, Morelos, 62210, Mexico<br />
3<br />
Laboratory of Toxicology, University of Leuven, Campus Gasthuisberg, O&N2, PO Box 922, 3000 Leuven,<br />
Belgium<br />
4<br />
Centro de Investigacion en Biotecnologia UAEM, Av. Universidad 2001, Cuernavaca, Morelos, 62210, Mexico<br />
Magi 5, from the hexathelid spider Macrothele gigas, is a 29-residue polypeptide stabilized<br />
by three disulfide bridges. It binds specifically to receptor site 4 on mammalian voltagegated<br />
sodium channels, <strong>and</strong> competes with classical scorpion β-toxins, such as Css IV from<br />
Centruroides suffusus suffusus. As a consequence, Magi 5 shifts the activation voltage of<br />
the mammalian Nav1.2 channel to more hyperpolarized voltages, whereas the insect<br />
channel, para, is not affected.<br />
To gain insight into toxin-channel interactions, Magi 5 <strong>and</strong> 23 analogues were produced by<br />
peptide synthesis. The three-dimensional structure of Magi 5 in aqueous solution was<br />
determined <strong>and</strong> its voltage-gated sodium channel binding surfaces were mapped onto this<br />
structure using data from electrophysiological measurements on a series of Ala-substituted<br />
analogues. The structure clearly resembles the inhibitor cystine knot (ICK) structural<br />
motif, although the triple-str<strong>and</strong>ed β-sheet typically found in that motif is partly distorted<br />
in Magi 5.<br />
The interactive surface of Magi 5 towards voltage-gated sodium channels resembles in<br />
some respects the Janus-faced atracotoxins, with functionally important charged residues<br />
on one side of the toxin <strong>and</strong> hydrophobic residues on the other. Magi 5 also resembles the<br />
scorpion β-toxin Css IV, which displays distinct non-polar <strong>and</strong> charged surfaces that are<br />
critical for voltage-gated sodium channel binding <strong>and</strong> has a key Glu involved in voltage<br />
sensor trapping (1). It is likely that these two distinct classes of toxin, with different amino<br />
acid sequences <strong>and</strong> different structures, utilize similar groups of residues on their surface to<br />
achieve the common end of modifying voltage-gated sodium channel function. Magi 5 thus<br />
represents a valuable new probe of receptor site 4 on mammalian VGSC.<br />
1. Cohen, L., Karbat, I., Gilles, N., Ilan, N., Benveniste, M., Gordon, D., <strong>and</strong> Gurevitz, M.<br />
(2005) J Biol Chem 280, 5045-5053<br />
o polypeptide<br />
o sodium channel<br />
o structure<br />
o binding surface<br />
136<br />
Thursday Track 1, K3.14 5.00-5.20pm<br />
X-ray structure <strong>and</strong> point mutagenesis of the scorpion depressant toxin LqhIT2<br />
reveals key determinants crucial for activity <strong>and</strong> anti-insect selectivity<br />
Izhar Karbat 1 , Michael Turkov 1 , Lior Cohen 1 , Roy Kahn 1 , Dalia Gordon 1 , Michael Gurevitz 1* <strong>and</strong> Felix<br />
Frolow 2<br />
Departments of 1 Plant Sciences <strong>and</strong> 2 Molecular Microbiology & Biotechnology, George S. Wise Faculty of Life<br />
Sciences, Tel-Aviv University, Tel-Aviv , Israel, *karbat@post.tau.ac.il<br />
Scorpion depressant β-toxins are 61 amino acid long polypeptides that show preference<br />
for insect voltage gated sodium channels (Navs) <strong>and</strong> modulate their activation process by<br />
shifting the voltage dependence of activation to more negative membrane potentials.<br />
Although a number of depressant toxins were identified <strong>and</strong> characterized only a few were<br />
cloned 1 <strong>and</strong> just little is known about residues important for their activity 2 . In addition,<br />
data pertaining to the three-dimensional structure of these toxins are currently lacking.<br />
Here we report about the crystal structure of LqhIT2, a depressant toxin from the venom<br />
of Leiurus quinquestriatus hebreus, refined at 1.2Å resolution. Comparison of LqhIT2<br />
structure to those of scorpion β-toxins highly active on mammals reveals a similar overall<br />
structure composed of two lobes, the Core-lobe <strong>and</strong> the NC-lobe. Mutagenesis of LqhIT2<br />
illuminates a continuous bioactive surface encompassing a groove formed between the<br />
Core- <strong>and</strong> NC- lobes (N-groove), a conserved pharmacophore 3 (Glu24, Tyr26), <strong>and</strong> Trp53<br />
<strong>and</strong> Asn58 of the C-tail. The fine surface topology of the N-groove region <strong>and</strong> its overall<br />
hydrophobicity are critical for toxin activity <strong>and</strong> anti-insect selectivity. Yet, NMR studies<br />
of similar β-toxins suggest that this region is highly flexible in solution. Combined with<br />
previous studies, we offer a model in which the initial docking of the toxin on the channel<br />
involves interactions between the structurally rigid pharmacophore <strong>and</strong> C-tail to a<br />
stationary channel region, followed by 'voltage-sensor trapping' achieved through a<br />
conformational change in the N-groove, which is triggered by channel activation.<br />
References<br />
1. Possani LD, Becerril B, Delepierre M & Tytgat J (1999) Eur J Biochem 264:287-300<br />
2. Strugatsky D, Zilberberg N, Ilan N, Turkov M, Cohen L, Stankiewicz M, Pelhate M, Gilles N,<br />
Gordon D & Gurevitz M (2005) Biochemistry 44:9179-91873<br />
3. Cohen L, Karbat I, Gilles N, Ilan N, Gordon D & Gurevitz M (2005) J Biol Chem 280:5045-<br />
5053<br />
o Scorpion depressant toxin<br />
o X-ray structureBioactive surface<br />
o Voltage-gated sodium channels<br />
o Bioactive surface<br />
137
Thursday Track 1, K3.14 5.20-5.40pm<br />
New β-type toxins from the Androctonus scorpion venoms ?<br />
Martin-Eauclaire, M-F 1 *, Céard, B 1 , Bosmans, F 2 , Rosso, J-P 1 , Tytgat, J 2 <strong>and</strong> Bougis,<br />
P. E. 1<br />
1 CNRS FRE 2738, Faculté de Médecine secteur Nord, Institut Jean Roche, Université de la Méditerranée, Bd<br />
Pierre Dramard, 13916, Marseille, Cedex 20, France.mail : eauclaire.mf@jean-roche.univ-mrs.fr<br />
2 Laboratory of Toxicology, University of Leuven, E. Van Evenstraat 4, 3000 Leuven, Belgium.<br />
Since our main objective was the discovery of new selective insect voltage-gated<br />
Na+ channels (NavCh) modulators, we have analyzed our collection of uncharacterized<br />
protein fractions from the venom of the North African scorpion Androctonus australis. In<br />
this venom, more than 90 % of the lethality for mice <strong>and</strong> insects was caused by “longchain”<br />
toxins, which modify the gating properties of NavCh (e.g. the α-toxins AaH I, II, III<br />
<strong>and</strong> IV specifically active on mammals <strong>and</strong> the “excitatory” insect-specific toxin AaH IT).<br />
From the venom of the scorpion Androctonus australis, we have isolated a new<br />
bioactive polypeptide termed AaBTX-L1. When tested on the insect NavCh (para) of the<br />
fruit fly, this toxin was able to induce a clear shift in activation (V1/2) resulting in the<br />
opening of the channel at more negative membrane potentials. Furthermore, AaBTX-L1<br />
was totally devoid of toxicity when injected into mice intracerebroventricularly <strong>and</strong> did not<br />
compete with radiolabeled voltage-gated K + <strong>and</strong> Na + channel toxins in binding<br />
experiments on rat brain synaptosomes. Using its N-terminal amino acid sequence to<br />
design degenerate primers, several clones were amplified by PCR from the Androctonus<br />
australis venom gl<strong>and</strong> cDNA library. As a consequence, seven full oligonucleotide<br />
sequences encoding “long-chain” polypeptides with only three disulfide bridges have been<br />
cloned for the first time <strong>and</strong> are described here. Remarkably, they share a high similarity<br />
with the anti-insect toxin Birtoxin from Parabuthus transvaalicus.<br />
Classically, scorpion β-toxins produce a hyperpolarizing shift of the voltage dependence of<br />
activation in NavCh. The unambiguous electrophysiological effect observed on the insect<br />
NavCh of Drosophila by AaBTX-L1 is fully consistent with this β-type activity.<br />
Previously, a weak β-type activity, associated with another type of anti-insect toxin (AaH<br />
IT4) both active on insects <strong>and</strong> mammals, was identified in the Androctonus australis<br />
venom using competition against the β-toxin 125 I-Css II bound on its receptor site 4 on rat<br />
brain synaptosomes. With the results of our study, the new structural group of scorpion<br />
toxins related to Birtoxin has increased significantly.<br />
o scorpion toxins<br />
o insects<br />
o cDNA<br />
o voltage-gated sodium channels<br />
138<br />
Thursday Track 2, K3.17 3.00-3.20pm<br />
Cyt2Ba of Bacillus thuringiensis subsp. israelensis: activation by putative<br />
endogenous protease<br />
Cahan, R. 1* , Nisnevitch, M. 1 , Cohen, S. 1,2 , Ben-Dov, E. 2 , Zaritsky, A. 2 <strong>and</strong> Sofer, Y. 2<br />
1* College of Judea <strong>and</strong> Samaria, Ariel 44837, Israel. rivkac@yosh.ac.il<br />
2 Ben-Gurion University of the Negev,POB 653, Beer-Sheva 84105, Israel .<br />
Introduction: Bacillus thuringiensis subsp. israelensis (Bti) produces parasporal<br />
crystalline proteins, which possess larvicidal <strong>and</strong> cytolytic activities. The crystal is<br />
composed of four main proteins, Cry4Aa, Cry4Ba, Cry11Aa <strong>and</strong> Cyt1Aa. The genes for<br />
these <strong>and</strong> other Cry <strong>and</strong> Cyt proteins are located on the large plasmid (128 kb) pBtoxis.<br />
Analysis of the plasmid sequence revealed besides cyt1Aa, an additional gene cyt2Ba<br />
encoding another Cyt protein. The aim of this research was to purify the Cyt2Ba <strong>and</strong> to<br />
identify its activity.<br />
Methods: The gene cyt2Ba of (Bti) was cloned for expression, together with p20, in an<br />
acrystalliferous strain IPS78/11. This strain was grown 4 days, centrifuged, <strong>and</strong> the<br />
sediment, including crystals, spores <strong>and</strong> cell debris, was divided into two portions for<br />
further purification. The first portion was only solubilized. The second portion was<br />
partially purified by biphasic separation <strong>and</strong> the crystals obtained were solubilized <strong>and</strong><br />
activated by exogenous proteases.<br />
Results: The supernatant of the sediment that was only solubilized consisted of a<br />
predominant protein with molecular mass of 22 kDa which was designated Cyt2Ba-E<br />
for endogenous bacterial proteases that seem to digest the intact Cyt2Ba. The Cyt2Ba-E<br />
had a relatively high haemolytic activity. Mass spectrometric analysis revealed that the<br />
N-terminus of Cyt2Ba-E, having Threonine-35 as the first residue. The protein b<strong>and</strong> of<br />
Cyt2Ba-E included another protein that was discovered by Blast analysis as a metallo<br />
protease camelysin. The supernatant fraction of the solubilized sediment that was<br />
partially purified by biphasic separation contained a protein, with molecular mass of<br />
about 24 kDa. Edman degradation analysis revealed that the N-terminal sequence is<br />
MHLNN the same as the original Cyt2Ba thereby designated as Cyt2Ba-N (N-for<br />
native). The haemolytic activity of Cyt2Ba-N was negligible however soluble Cyt2Ba-<br />
N that was incubated with exogenous proteases yielded a major b<strong>and</strong> of 22 kDa with the<br />
same size <strong>and</strong> haemolytic activity as Cyt2Ba-E.<br />
Discussion: Cyt2Ba crystals solubilization in the presence of endogenous proteases,<br />
most likely by a camelysin, enabled quick <strong>and</strong> simple procedure to obtain rather pure<br />
<strong>and</strong> active Cyt2Ba toxin.<br />
o Bacillus thuringiensis subsp. israelensis,<br />
o Cyt2Ba<br />
o camelysin<br />
139
Thursday Track 2, K3.17 3.20-3.40pm<br />
New type of cytolysin <strong>and</strong> an unusual phospholipase A2 like protein isolated from<br />
the Northern Pacific sea anemone Urticina crassicornis<br />
Razpotnik, A. 1 , Križaj I. 2 , Šribar, J. 2 , Maček P. 1 , <strong>and</strong> Turk, T. 1*<br />
1 University of Ljubljana, Department of Biology, 1000-Ljubljana, Slovenia *tom.turk@bf.uni-lj.si<br />
2 Jožef Stefan Institute, Deapartment of Biochemistry, 1000-Ljubljana, Slovenia<br />
Sea anemones generally possess two types of toxins: neurotoxins <strong>and</strong> cytolysins. The<br />
former are relative short polypeptides acting on voltage gated sodium or potassium<br />
channels. The later are (i) actinoporins which disrupt biological membranes by forming<br />
discrete oligomeric pores or very rarely (ii) phospholipase A like enzymes which<br />
hydrolyze membrane phospholipids. As observed previously, Northern Pacific sea<br />
anemone Urticina crassicornis possesses polypeptides with potassium channel blocking<br />
activity <strong>and</strong> proteins having cytolytic activity.<br />
From freeze-dried specimens we have isolated a novel type of cytolytic protein which is<br />
clearly different from those of actinoporin family. It is a 28 kDa protein which is<br />
inhibited by sphingomyelin, but can only disrupt lipid vesicles or lipid monolayers<br />
composed of sphingomyelin/cholesterol mixtures. Such lipid composition is<br />
characteristic for so called lipid rafts, therefore new cytolysin may be used as a new<br />
tool for localization of these important cell membrane domains. We report a partial<br />
amino acid sequence <strong>and</strong> some characteristics of this novel cytolysin.<br />
Fresh extracts milked from tentacles <strong>and</strong> gastric fluid of the same sea anemone were used<br />
for isolation of a novel phospholipase A2 type protein which has some unique structural<br />
features not previously known within this protein family. cDNA deduced amino acid<br />
sequence reveals a large degree of homology to β-bungarotoxin or notexin, a well known<br />
examples of snake neurotoxic phospholipases. However, new protein has a unique<br />
structural feature at the position 25 where absolutely conserved Cys residue is replaced by<br />
an Asp residue. New phospholipase like protein is also about 10 amino acid residues<br />
shorter than its snake neurotoxic relatives. New protein clearly show concentration<br />
dependent phospholipase enzymatic activity using pyr-PG as a substrate, which is,<br />
however, only about 2.5% of that obtained with ammoditoxin C. We also report on some<br />
other characteristics of this unique phospholipase A2 like protein.<br />
o Sea anemone<br />
o cytolysin<br />
o phospholipase A2<br />
o new type<br />
140<br />
Thursday Track 2, K3.17 3.40-4.00pm<br />
Latarcins – a group of spider venom cytolytic peptides with high structural<br />
diversity<br />
Vassilevski, A.A. 1* , Kozlov, S.A. 1 , Feofanov, A.V. 1 , Surovoy, A.Y. 1 , Karpunin, D.V. 1 , Grishin, E.V. 1<br />
1 Shemyakin <strong>and</strong> Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul.<br />
Miklukho-Maklaya, 16/10, 117997 Moscow, Russia, *avas@ibch.ru<br />
A combination of chromatography, mass spectrometry, <strong>and</strong> EST database analysis was<br />
used to identify antimicrobial <strong>and</strong> cytolytic peptides in the venom of the spider Lachesana<br />
tarabaevi. In total, sequences of 11 new short linear cationic <strong>and</strong> amphipathic peptides<br />
named latarcins were retrieved <strong>and</strong> split into 7 groups based on similarity; latarcins showed<br />
negligible level of homology to other known polypeptides. 7 of the peptides from 5 groups<br />
were isolated from the venom. These peptides were chemically synthesized <strong>and</strong> were<br />
found to lyse different cell types – bacteria, yeasts, <strong>and</strong> erythrocytes – at micromolar<br />
concentrations. Under physiological membrane potential, all latarcins caused scaled<br />
membrane destabilization when tested in planar lipid bilayers. In contact with target<br />
membranes, the peptides were suggested to adopt amphipathic α-helical structure, as<br />
indicated by CD spectroscopy. The “carpet” model was proposed to account for latarcins’<br />
mode of action. Thus, the spider was found to produce a cocktail of structurally diverse but<br />
functionally similar venom peptides. Such biomolecular diversity seems to be a common<br />
evolutionary trend for venomous animals <strong>and</strong> venom compounds – neurotoxins <strong>and</strong><br />
cytolytic peptides.<br />
In addition, latarcin precursor protein sequences were retrieved from the venom gl<strong>and</strong> EST<br />
database. The precursors showed a considerable level of variation <strong>and</strong> were split into 3<br />
groups: simple precursors with a conventional prepropeptide structure, binary precursors<br />
with 2 mature chains arranged sequentially into a typical modular block organization, <strong>and</strong><br />
complex precursors of modular structure suggested to be cleaved into mature chains of 2<br />
different types. In each case, a signal peptide <strong>and</strong> an acidic pro-sequence were identified<br />
<strong>and</strong> the biosynthetic pathway was suggested to involve targeted proteolysis at a specific<br />
processing site, known as the Processing Quadruplet Motif (PQM).<br />
o spider venom<br />
o cytolytic peptide<br />
o antimicrobial peptide<br />
o precursor protein<br />
141
Thursday Track 2, K3.17 4.00-4.20pm<br />
Mechanism of sphingomyelin specificity of actinoporins, pore-forming toxins from<br />
sea anemones<br />
Anderluh, G. 1* , Bakrač, B. 1 , Podlesek, Z. 1 , Maček, P. 1 , Separovic, F. 2 , Norton, R.S. 3 , Lakey, J.H. 4<br />
1<br />
University of Ljubljana, Department of Biology, Večna pot 111, 1000 Ljubljana, Slovenia,<br />
*gregor.<strong>and</strong>erluh@bf.uni-lj.si<br />
2<br />
School of Chemistry, University of Melbourne, 3010, Australia<br />
3<br />
Walter <strong>and</strong> Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville 3050, Australia<br />
4<br />
University of Newcastle upon Tyne, Framlington Place, Newcastle, Engl<strong>and</strong>, UK<br />
Pathogens <strong>and</strong> protein toxins use lipid domains or ‘rafts’ to invade cells by binding to the<br />
lipid components sphingomyelin or cholesterol. Possible molecular mechanisms that<br />
enable association of proteins with such domains are becoming an important issue in cell<br />
biology <strong>and</strong> medicine. Surprisingly, there are very few examples of protein-sphingomyelin<br />
interactions defined at a molecular level. Here we provide some clues on how<br />
sphingomyelin is recognised by equinatoxin, the most studied member of the actinoporins,<br />
a conserved family of sea anemone pore-forming toxins. As shown by lipid dot-blots <strong>and</strong><br />
surface plasmon resonance experiments, equinatoxin directly binds sphingomyelin but not<br />
phosphatidylcholine even though it presents the same choline headgroup. The toxin<br />
obviously recognizes the chemical structure of sphingomyelin below the lipid headgroup.<br />
The two closest residues that could fulfil this role are Trp112 <strong>and</strong> Tyr113. Just recently we<br />
were able to introduce 19 F label on tryptophans <strong>and</strong> confirm with NMR that Trp112 is<br />
involved in sphingomyelin recognition (1). Mutations of Trp112 to Phe or Leu, two amino<br />
acids that are found at equivalent positions in some homologues of equinatoxin, <strong>and</strong><br />
mutation of Tyr113 to Phe, slightly decrease calcein release <strong>and</strong> binding to liposomes.<br />
Mutant W112A is almost inactive <strong>and</strong> does not bind sphingomyelin in a dot-blot assay <strong>and</strong><br />
sphingomyelin analogues as measured by SPR. Mutation of Tyr113 to Ala causes complete<br />
loss of permeabilising activity <strong>and</strong> there is no binding to liposomes.<br />
Trp112 stabilizes the bound form of the toxin in the lipid-water interface <strong>and</strong> could interact<br />
with sphingomyelin in the region below the headgroup. On the other h<strong>and</strong>, Tyr113 is close<br />
enough to form a hydrogen bond with the amide nitrogen of sphingomyelin <strong>and</strong> could in<br />
this way discriminate between the two lipids.<br />
1. Anderluh, G., Razpotnik, A., Podlesek, Z., Maček, P., Separovic, F. <strong>and</strong> Norton, R.S.<br />
(2005) J Mol Biol 347, 27-39.<br />
o cytolysin<br />
o actinoporins<br />
o equinatoxin<br />
o sphingomyelin specificity<br />
142<br />
Thursday Track 2, K3.17 4.20-4.40pm<br />
In vitro evaluation of antibacterial activity of crude aqueous extract of Azadirachta<br />
indica (Neem) leaves.<br />
Kihara, M. W 1 , Mbugua, P. M 1* , Kioy, P. G 1 , Makumi, J. N 2 , Ngeranwa, J. M. 2<br />
1 University of Nairobi, Nairobi, Kenya, *paulmmbugua@yahoo.com<br />
2 Kenyatta University, Nairobi, Kenya.<br />
Azadirachta indica (Neem tree) is widely grown in Kenya <strong>and</strong> from it numerous types of<br />
herbal products are currently being manufactured <strong>and</strong> sold to the public. Every part of the<br />
plant is used as raw material. Aqueous freeze dried leave extract was used to determine the<br />
spectrum of activity <strong>and</strong> potency by disk diffusion, minimum inhibition concentration<br />
(MIC), minimum bactericidal concentration (MBC) <strong>and</strong> time-kill methods. Out 108<br />
bacterial isolates, 60.2% (+-10.1) were sensitive at varying degree (7-11.5 mm diameter)<br />
by disc diffusion method at 5% confidence interval. Of 32 gram positive isolates tested,<br />
71.8% (+-9.3) were sensitive while 28.2% were resistant. Of 76 gram negative organism<br />
tested 55.2% (+-10.3) were sensitive at 5% confidence interval. MIC <strong>and</strong> MBC values by<br />
microtitre plate method for (Staphyloccocus aureas, Escherichia coli, Salmonella typhi,<br />
Pseudomonas eurogenosa <strong>and</strong> Streptoccocus pyogenes) selected organisms ranged from<br />
1.5-6.0mg/ml <strong>and</strong> 2-13mg/ml respectively. For comparison, MIC <strong>and</strong> MBC values<br />
obtained for chloramphenical on the same organisms ranged from 0.0625-1.2 mg/ml <strong>and</strong><br />
0.106-4.8 mg/ml respectively. Both the extract <strong>and</strong> chloramphenical showed poor<br />
bactericidal properties by tube method. Both could not eliminate a population of 10 6 of<br />
organism on incubation for 24hrs even at concentration two times the MBC. They<br />
however considerably suppressed growth of the organisms. The neem leave aqueous<br />
extract exhibited broad spectrum antibacterial activity. The results explain its popularity in<br />
controlling bacterial infections traditionally but also indicate necessity to perform<br />
susceptibility tests for all isolates as same species showed different responses with<br />
different isolates. Neem extracts would be very useful in multiple infections. The<br />
organisms that responded best included,;Staphyloccocus, Escherichia, Sallmonella <strong>and</strong><br />
Streptoccocus species. Shigella, Neisseria <strong>and</strong> Haemophilus species showed least<br />
response. A methicilline resistant strain of Staphyloccocus aureas was resistant while an<br />
Escherichia coli isolates which had proved resistant to a battery of antibacterial agents was<br />
suppressed.<br />
o Azadirachta indica<br />
o In vitro<br />
o Antibacterial<br />
o Leave<br />
143
Thursday Track 2, K3.17 4.40-5.00pm<br />
Non-protein/non-peptide compounds from cobra venom having therapeutic<br />
potential<br />
Saha, A. 1* , Gomes, A.<br />
1 Lab. Of Toxinology & Experimental Pharmacodynamics, Department of Physiology, University of<br />
Calcutta, Kolkata, India, *archita_s1@rediffmail.com<br />
Introduction: The present communication reports the purification <strong>and</strong> characterization of<br />
three non-protein/non-peptide compounds from King Cobra (Ophiophagus hannah) venom<br />
(KCV) <strong>and</strong> Monocled Cobra (Naja kaouthia) venom (MCV) <strong>and</strong> evaluating their<br />
therapeutic potential (antiarrhythmic, antipyretic <strong>and</strong> anticonvulsant, antineoplastic).<br />
Methods: Swiss albino male mice, Charles Foster male rats, guinea pigs. Human myeloid<br />
leukemic cell lines (U937,K562). TLC, column chromatography, IEC, gel filtration, HPLC<br />
- for purification. Structural characterization by UV, IR, NMR, Mass. Biological studies.<br />
Results: KC-MMT1 – saturated aliphatic acid isolated from KCV, showed anti-arrhythmic<br />
property established through in vitro studies on guinea pig heart/ auricle preparations <strong>and</strong><br />
in vivo ECG recordings <strong>and</strong> cardiac marker enzyme assay on aconitine induced arrhythmic<br />
rats <strong>and</strong> mice. KC-MMT2 – unsaturated aliphatic acid isolated from KCV, showed CNS<br />
activity (lowered body temperature of normothermic <strong>and</strong> hyperthermic mice <strong>and</strong><br />
potentiated sleeping time) <strong>and</strong> anticonvulsant activity. NK-ANF1 – polyhydroxylated nonaromatic<br />
compound isolated from MCV had antineoplastic potential (cell growth inhibition<br />
<strong>and</strong> apoptogenic observed by fluorescence, confocal <strong>and</strong> scan electron microscopy, FACS)<br />
on cell lines. (Statistics – Mean ± SEM, Student’s t-test, p
Thursday Track 2, K3.17 5.20-5.40pm<br />
Yeast culture Saccharomyces cerevisiae as feed additive in poultry<br />
S. A. Abd EL-Latif<br />
Department of Animal Production, Faculty of Agriculture, Minia University, Minia, Egypt<br />
E-mail:- shaker7112001@yahoo.com<br />
The chickens are stressed by various factors such as overcrowding, vaccination,<br />
chilling, <strong>and</strong>/or overheating. Under such circumstances feed additives like yeast culture<br />
have great significance for enhancing feed quality, productivity <strong>and</strong> health. Yeast culture<br />
consists of natural live yeast cells grown <strong>and</strong> fermented in the media like molasses <strong>and</strong><br />
grain mashes in the form of spores at low temperature. It includes both media <strong>and</strong> yeast<br />
conditioned for immediate enzymatic action upon entering the digestive tract. It is neither<br />
an antibiotic nor chemotherapeutic agent <strong>and</strong> is distinctly different from baker's yeast.<br />
Yeast culture is added as a digestive aid to increase the nutrient availability <strong>and</strong> they will<br />
act by producing certain digestive enzymes.<br />
Addition of yeast culture Saccharomyces cerevisiae in drinking water resulted in<br />
the reduction of number of days required to reach the market weight, improving the<br />
livability <strong>and</strong> feed conversion in broilers. Dietary inclusion of Saccharomyces cerevisiae (<br />
1 kg per ton ) of feed in layer resulted in improved egg production, feed conversion <strong>and</strong><br />
egg shell quality. The broiler breeder diet with 0.5kg/ton of live yeast culture (Yeasacc) at<br />
20 weeks of age, <strong>and</strong> increased it to 1 kg/ton at 47 weeks of age resulting in no consistent<br />
effect on female reproductive characteristics, rate of lay, egg specific gravity <strong>and</strong> egg<br />
weight.<br />
This review article discuss the effect of yeast culture Saccharomyces cerevisiae as<br />
feed additives in poultry diets on production <strong>and</strong> some metabolic functions.<br />
o yeast culture<br />
o poultry<br />
o production<br />
o metabolic functions<br />
146<br />
Friday Track 1, K3.14 3.00-3.20pm<br />
Bioinformatics <strong>and</strong> multi-epitope DNA immunisation to design rational snake<br />
antivenom<br />
Wagstaff, S.C.*, Laing, G.D., Theakston, R.D.G., Papaspyridis, C., Harrison, R.A.<br />
Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool,<br />
L3 5QA, UK *simonw@liv.ac.uk<br />
The rational design of toxin-specific snake bite immunotherapy is a realistic prospective if<br />
recombinant immunogens that generate toxin-specific antibody responses could be<br />
developed as alternatives to conventional, whole venom, immunisation protocols currently<br />
used to generate antivenoms. In this paper, we describe the use of bioinformatics <strong>and</strong><br />
venom gl<strong>and</strong> expressed sequence tag (vgEST) to design novel immunogens that elicit<br />
cross-generic <strong>and</strong> toxin-neutralising antibody responses.<br />
Using a bioinformatic approach that combines algorithms that identify surface exposed,<br />
antigenic <strong>and</strong> highly represented epitopes within EST databases, we identified seven<br />
epitopes conserved throughout the highly divergent snake venom metalloproteinases<br />
(SVMP) family from Echis ocellatus.<br />
To confirm our bioinformatic predictions, we raised antisera to each of these epitopes <strong>and</strong><br />
confirmed their immunological conservation in the venoms of several disparate species<br />
distributed throughout African, Asia <strong>and</strong> America. Furthermore, we designed <strong>and</strong><br />
constructed either a single DNA or recombinant protein immunogen, containing all seven<br />
epitopes as a string, which generated antibody responses to epitopes contained within a<br />
wide range of SVMPs that inhibited haemorrhage induced by the venoms of<br />
geographically distinct vipers, E. ocellatus <strong>and</strong> Cerastes cerastes cerastes.<br />
Extending the scope of this work by selecting epitopes from multiple toxin groups<br />
conserved throughout other medically important viper <strong>and</strong> elapid species, offers new<br />
opportunities to design antivenoms which are toxin-specific, have improved clinical<br />
efficacy <strong>and</strong> may be engineered to provide geographical polyspecificity to regions of high<br />
incidence of snake bite.<br />
o EST<br />
o Antivenom<br />
o Metalloproteinase<br />
o Bioinformatics<br />
147
Friday Track 1, K3.14 3.20-3.40pm<br />
Pre-clinical assessment of equine <strong>and</strong> ovine antivenoms raised against saw-scaled<br />
viper (Echis ocellatus) venom for use in Nigeria<br />
Laing, G.D. 1 *, Warrell, D.A. 2 <strong>and</strong> R.D.G. Theakston 1 .<br />
1<br />
Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool,<br />
L3 5QA, UK. *gavin.laing@liv.ac.uk<br />
2<br />
Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 9DU, UK<br />
The Echis genus is responsible for more snakebite deaths worldwide than any other<br />
venomous snake. The saw-scaled viper (Echis ocellatus) is the most abundant snake in<br />
savannah Nigeria <strong>and</strong> a major health concern. At some times of the year victims can<br />
occupy 74% of regional hospital beds. The severe shortage of antivenoms for Africa has<br />
prompted an international collaboration to produce antivenoms as a means of resolving the<br />
crisis in the short-term. In order to select c<strong>and</strong>idate antivenoms for a planned clinical trial<br />
in late 2006, we have assessed the potency of several antivenoms derived from ovine or<br />
equine IgG <strong>and</strong> F(ab’)2 fragments using WHO-approved tests.<br />
Four antivenoms were developed using venoms extracted from wild-caught Nigerian<br />
(Kaltungo) specimens of E. ocellatus, Bitis arietans <strong>and</strong> Naja nigricollis maintained in the<br />
Liverpool School of Tropical Medicine. The antivenoms tested were an equine caprylic<br />
acid derived IgG (Instituto Clodomiro Picado, Costa Rica), an equine pepsin digested<br />
F(ab / )2 (Vacsera, Egypt) (both trispecific antivenoms raised against E. ocellatus, B.<br />
arietans <strong>and</strong> N. nigricollis venoms), an ovine caprylic acid derived IgG <strong>and</strong> an ovine<br />
pepsin digested F(ab / )2 product (both MicroPharm, UK) (both monospecific antivenoms<br />
raised against E. ocellatus venom). Tests for estimating the neutralising effects of these<br />
antivenoms against the lethal effects of E. ocellatus venom were carried out.<br />
All four antivenoms showed neutralising potency against the immunising venoms <strong>and</strong><br />
results were expressed as the number of LD50 neutralising units/ampoule of antivenom.<br />
Comparisons with established antivenoms are made <strong>and</strong> the selection of c<strong>and</strong>idate<br />
antivenoms for the forthcoming clinical trial is discussed.<br />
The results demonstrate that the commitment of international antivenom manufacturers<br />
to address the shortfall of antivenom supply for Africa has resulted in the production of<br />
potent <strong>and</strong> efficacious therapies.<br />
o Echis<br />
o antivenom<br />
o potency<br />
o effective dose<br />
148<br />
Friday Track 1, K3.14 3.40-4.00pm<br />
What price, salvation? Antivenom costs <strong>and</strong> availability in Papua New Guinea<br />
1992-2004<br />
Williams, D. J. 1*<br />
1 Australian Venom Research Unit, University of Melbourne, Parkville, Vic, 3010. Australia<br />
* d.williams4@pgrad.unimelb.edu.au<br />
Introduction: Papua New Guinea has some of the highest incidence rates for snakebite in<br />
the world, yet for almost two decades antivenoms have become increasingly expensive,<br />
<strong>and</strong> increasingly scarce. A study was conducted to examine antivenom costs <strong>and</strong><br />
availability <strong>and</strong> to investigate supply <strong>and</strong> distribution anomalies.<br />
Methods: Analysis of purchasing, inventory <strong>and</strong> distribution records.<br />
Results: The PNG government purchased 5,571 vials of antivenom between May 1992 <strong>and</strong><br />
April 2005 at a total cost of more than K11.1 million, yet in yearly terms the availability of<br />
antivenom has decreased by 40.2% since 1986. The cost of a single patient treatment with<br />
antivenom has increased by 884% since 1987, <strong>and</strong> the average treatment cost is now<br />
K3,060. Rising costs have been largely driven by international economics, but reliance on<br />
expensive polyvalent antivenom which increased from 49.2% (1992-1995) to 82.0%<br />
(1997-2000) added K511 to the costs of single patient treatment. This increased average<br />
annual expenditure by K266,742; sufficient to have funded the purchase of an additional<br />
150 vials of taipan antivenom <strong>and</strong> 50 additional vials of death adder antivenom. In 2003<br />
while total national pharmaceutical <strong>and</strong> medical supplies purchases totalled K14.7 million;<br />
more than K3.4 million (24.7%) was spent on antivenoms. Despite large spending<br />
antivenom supplies were poorly managed <strong>and</strong> badly distributed. From January 1998 to<br />
December 2004 2,040 vials of antivenom were recorded as being distributed from Area<br />
Medical Store (POM), but the dispositions of another 416 vials of various antivenoms<br />
worth over K1.2 million were not recorded. Only 47 health facilities throughout PNG<br />
received antivenom in this period; Port Moresby General Hospital received 717 (35.1%)<br />
vials of which 616 (85.9%) were CSL polyvalent.<br />
Conclusions: The purchasing, warehousing <strong>and</strong> distribution of antivenom requires urgent<br />
review with an emphasis on inventory security <strong>and</strong> purchase/delivery cross-matching. An<br />
alternative system linked to snakebite research <strong>and</strong> surveillance efforts has now been<br />
proposed.<br />
o antivenoms<br />
o costs<br />
o accountability<br />
o availability<br />
149
Friday Track 1, K3.14 4.00-4.20pm<br />
Developing a toxinology toolkit for an under-developed world<br />
Williams D.J. 1* , Jensen S.D. 1,2 , Matainaho T. 2 , Wüster W. 3 , Nimorakiotakis B. 1 , Winkel K.D. 1<br />
1 Australian Venom Research Unit, University of Melbourne, Parkville, Vic, 3010. Australia<br />
2 School of Medicine & Health Sciences, University of Papua New Guinea, Boroko, Papua New Guinea<br />
3 School of Biological Sciences, University of Wales, Bangor, Wales, UK<br />
* d.williams4@pgrad.unimelb.edu.au<br />
Introduction: The global burden of snakebite is greatest in the world’s poorest tropical<br />
countries. We have been working in this arena to develop a new approach to snakebite<br />
management <strong>and</strong> associated resource use aimed at improving both local systems <strong>and</strong><br />
infrastructure <strong>and</strong> the individual prognoses of envenomed patients.<br />
Methods: As part of a major study of snakebite in Papua New Guinea we are combining<br />
the use of zoogeographical, ecological, epidemiological, clinical, economic, <strong>and</strong> GIS<br />
studies with public <strong>and</strong> medical education to both resolve unanswered questions, <strong>and</strong> to<br />
develop local capacity for managing snakebite <strong>and</strong> improving clinical outcomes. Specific<br />
approaches are being developed for (1) epidemiological surveillance <strong>and</strong> reporting, (2)<br />
mapping <strong>and</strong> GIS analysis, (3) efficient pursuit of clinical investigations, (4) resolution of<br />
taxonomic questions, (5) improved resource management <strong>and</strong> distribution, (6) medical <strong>and</strong><br />
community education, <strong>and</strong> (7) improving clinical outcomes.<br />
Results: In Papua New Guinea our approach has lead to the establishment of a new plan<br />
for addressing the snakebite burden. A new National Snakebite & Antivenom Unit will use<br />
epidemiological, GIS <strong>and</strong> zoogeographic data in combination with m<strong>and</strong>atory reporting of<br />
snakebite cases by health service providers, locally initiated clinical studies, <strong>and</strong> the phased<br />
introduction of EIA-based venom immunotype identification to better manage expensive<br />
antivenom supplies; ensuring needs-based distribution, stock accountability <strong>and</strong> cost<br />
minimization. Coordinated programmes of public education <strong>and</strong> nationwide training of<br />
health workers <strong>and</strong> medical professionals have also been implemented.<br />
Discussion: Although clinical <strong>and</strong> epidemiological studies add significantly to our overall<br />
knowledge, they rarely produce lasting practical benefits for the countries in which they<br />
take place. Our approach combines this essential quest for knowledge with the creation of<br />
a functional legacy: improved local skills, resources <strong>and</strong> systems which can sustainably<br />
reduce morbidity, mortality <strong>and</strong> financial costs. We believe that offering this approach to<br />
others in the so-called ‘under-developed’ world may enable them to reap similar rewards.<br />
o snakebite<br />
o public health<br />
o resource management<br />
o clinical outcomes<br />
150<br />
Friday Track 1, K3.14 4.20-4.40pm<br />
Teaching clinical toxinology in the developing world: Setting an example in Papua<br />
New Guinea<br />
Williams D.J, 1* Jensen S.D, 1,2 Nimorakiotakis B, 1 Winkel K.D. 1<br />
1<br />
Australian Venom Research Unit, University of Melbourne, Parkville, Vic, 3010. Australia<br />
2<br />
School of Medicine & Health Sciences, University of Papua New Guinea, Boroko, Papua New Guinea<br />
* d.williams4@pgrad.unimelb.edu.au<br />
Envenomation is an important cause of morbidity <strong>and</strong> mortality in many areas of the<br />
developing world, <strong>and</strong> the prognosis is often influenced by the knowledge <strong>and</strong> skills of<br />
local health workers. In Papua New Guinea the incidence of snakebite is one of the highest<br />
in the world, <strong>and</strong> in the face of spiralling antivenom costs <strong>and</strong> reduced availability, health<br />
workers <strong>and</strong> doctors are often faced with difficult clinical decisions. We have developed<br />
<strong>and</strong> successfully implemented a st<strong>and</strong>-alone training programme for Papua New Guinea’s<br />
medical <strong>and</strong> allied health community that encompasses a broad curriculum in applied<br />
clinical toxinology.<br />
This training course, the first of its kind to ever be offered in a developing tropical country<br />
anywhere in the world, provides instruction in a broad range of subjects including first aid<br />
management of patient’s in remote areas; patient assessment <strong>and</strong> diagnosis; specific<br />
evidence-based treatment protocols; syndromic management of neurotoxicity,<br />
coagulopathy <strong>and</strong> other venom-induced conditions; antivenom use <strong>and</strong> administration;<br />
recognition <strong>and</strong> treatment of adverse antivenom reactions; advanced airway protection <strong>and</strong><br />
respiratory support; <strong>and</strong> non-antivenom treatment strategies for remote rural health centres.<br />
An inaugural course, taught in September 2004 was attended by 62 participants from 8<br />
provinces; a second course conducted in September 2005 had an intake of 105 participants.<br />
To meet dem<strong>and</strong> the course will be run twice in 2006. A comprehensive h<strong>and</strong>book was<br />
written to accompany the course in 2005, <strong>and</strong> corporate funding has enabled this book to<br />
be produced for free distribution to health workers throughout Papua New Guinea. The<br />
course offers financial assistance to participants from under-resourced health centres who<br />
can demonstrate need. The success of the course will hopefully be measured in future years<br />
not just by improvements in the clinical outcomes of PNG’s snakebite victims, but also<br />
though application of this model to address clinical toxinology training needs elsewhere in<br />
the developing world.<br />
o Clinical toxinology<br />
o Training<br />
o Education<br />
o Envenomation<br />
151
Friday Track 1, K3.14 4.40-5.00pm<br />
Analgesic action of Crotalphine, a novel opioid receptor agonist from the venom of<br />
the South American rattlesnake Crotalus durissus terrificus (CdtV)<br />
Cury, Y. 1* , Konno, K. 2 , Picolo, G. 1 , Brigatte, P. 1 , Gutierrez, V. 1 , Zambelli, V. 1<br />
Laboratory of 1 Pathophysiology <strong>and</strong> 2 Center of Applied Toxinology (CAT), Butantan Institute, Av. Vital<br />
Brazil, 1500. 05503-900, Sao Paulo, Brazil, * yarac@attglobal.net<br />
Introduction: CdtV induces antinociception mediated by opioid receptors <strong>and</strong> activation<br />
of peripheral L-arginine-NO-cGMP pathway <strong>and</strong> opening of ATP-sensitive K+ channels.<br />
Recently, a synthetic 14 aminoacids peptide, named Crotalphine, was obtained based on<br />
the sequence of the natural analgesic factor isolated from crude venom. The aim of the<br />
present work was to evaluate the analgesic effect of Crotalphine in acute (prostagl<strong>and</strong>in E2induced<br />
hyperalgesia) <strong>and</strong> chronic (neuropathic or cancer) pain models <strong>and</strong> to determine<br />
the mechanisms involved in this effect.<br />
Methods: The analgesic effect of CNF was evaluated in male Wistar rats (160-180 g)<br />
Acute hyperalgesia was determined 3 hours after PGE2 injection (100 ng/paw). Cancer<br />
pain was determined 5 days after intraplantar injection of Walker 256 carcinoma cells<br />
(1x10 6 ). Neuropathic pain was evaluated 14 days after chronic constriction of sciatic nerve.<br />
For characterization of chronic pain, hyperalgesia, allodynia <strong>and</strong> spontaneous pain were<br />
measured. The rat paw pressure test <strong>and</strong> von Frey test were used for evaluation of<br />
hyperalgesia <strong>and</strong> allodynia, respectively. Spontaneous pain was evaluated by the<br />
determination of the duration of lifting <strong>and</strong> licking of paws.<br />
Results: Crotalphine (1 to 6 µg/kg) administered by oral route, induced antinociception in<br />
all models evaluated. This effect was a long lasting effect, since it persisted for 2 (chronic<br />
pain) to 5 (acute pain) days after Crotalphine administration. Antinociception was not<br />
modified by CTOP (20 μg/paw). On the other h<strong>and</strong>, nor-BNI (50 μg/paw) blocked the<br />
analgesic effect observed on PGE2-induced hyperalgesia, whereas ICI 174,864 (10 μ<br />
g/paw) <strong>and</strong> nor-BNI inhibited the effect of Crotalphine on neuropathic <strong>and</strong> cancer pain.<br />
Conclusion: These data suggest that Crotalphine induces a long-lasting analgesic effect on<br />
acute <strong>and</strong> chronic pain mediated by activation of peripheral κ <strong>and</strong> δ opioid receptors.<br />
Financial support: CAT/CEPID/FAPESP, COINFAR Pesquisa e Desenvolvimento<br />
o Crotalphine<br />
o Crotalus durissus terrificus venom<br />
o analgesia<br />
o peripheral opioid receptors<br />
152<br />
Friday Track 1, K3.14 5.00-5.20pm<br />
Analgesic effect induced by Naja kaouthia snake venom <strong>and</strong> α-cobratoxin isolated<br />
from this venom<br />
1Marucia Chacur, 1 Vanessa Zambelli, 1 Vanessa Gutierrez, 2 Peter Mirtschin, 1 Yara Cury, Frank Madaras 3 ,<br />
Paul Masci 4 , <strong>and</strong> Simone Flight 4<br />
1 Laboratory of Pathophysiology, Butantan Institute, Av. Vital Brazil, 1500, 05503-900, SP, Brazil, 2 Venom<br />
Supplies Pty Ltd, Tanunda South Australia 5352, Australia, 3 Venom Science Pty Ltd, Tanunda South<br />
Australia 5352 <strong>and</strong> 4 Southern Clinical School, University of Queensl<strong>and</strong>, Brisbane, Queensl<strong>and</strong>, Australia<br />
4102<br />
Naja kaouthia is responsible for most cases of snakebite mortality <strong>and</strong> morbidity in<br />
Thail<strong>and</strong> <strong>and</strong> is a major cause of envenoming in a number of other Asian countries.<br />
Important toxic components of N. kaouthia are the postsynaptic neurotoxins. These toxins<br />
block nerve transmission by binding specifically to the nicotinic acetylcholine receptor.<br />
Despite neurotoxicity, some reports exist on the analgesic activity displayed by venoms<br />
from the Asiatic Naja genus. There is no specific data in the literature however, about the<br />
possible analgesic effect of the venom from Naja kaouthia. The aim of the present study is<br />
to investigate, in male Wistar rats, the possible antinociceptive effect induced by Naja<br />
kaouthia (Nk) venom <strong>and</strong> by the postsynaptic neurotoxin, α-cobratoxin (α-cobra) isolated<br />
from this venom <strong>and</strong> to characterize the peripheral mechanisms involved in this activity.<br />
Methods: The antinociceptive action of Nk venom <strong>and</strong> α-cobra was evaluated in the<br />
prostagl<strong>and</strong>in E2 (PGE2)-induced hyperalgesia. The rat paw pressure test was used to<br />
evaluate pain threshold. In this test, a force (in g) was applied the hind paw, before <strong>and</strong> 3 h<br />
after the intraplantar (i.pl.) injection of PGE2. The force (in g) needed to induce the<br />
withdrawal of the paw represents the pain threshold.<br />
Results: The i.pl. injection of PGE2 (100 ng/paw) reduced the pain threshold of the rats,<br />
causing mechanical hyperalgesia. Nk (40 µg) or α-cobra (20 µg), administered p.o.<br />
immediately before PGE2, significantly inhibited hyperalgesia. Naloxone (1μg/paw), a<br />
nonspecific opioid receptor antagonist, injected i.pl. simultaneously with PGE2, abolished<br />
the antinociceptive effect on the venom <strong>and</strong> α-cobratoxin. Nor-BNI (50 µg/paw), an<br />
antagonist of kappa opioid receptors, partially antagonized the antinociceptive effect of αcobra,<br />
whereas ICI 174,864 (10 µg/paw) an antagonist of delta opioid receptors abolished<br />
this effect. On the other h<strong>and</strong>, CTOP (20 µg/paw) an antagonist of mu opioid receptors did<br />
not modify the α-cobra antinociceptive effect.<br />
Conclusions: These data suggest that Naja kaouthia venom <strong>and</strong> α-cobratoxin are able to<br />
induce antinociceptive effect mediated by peripheral opioid receptors. Kappa <strong>and</strong> delta, but<br />
not mu opioid receptors, are involved in antinociception induced by α-cobratoxin.<br />
153
Friday Track 1, K3.14 5.20-5.40pm<br />
BcIV, a new paralyzing peptide from the venom of the sea anemone Bunodosoma<br />
caissarum. A comparison with the Na + channel toxin BcIII<br />
Zaharenko, A.J. 1,2* , Oliveira, J.S. 1 , Ferreira-Jr., W.A. 1,2 , Konno, K. 2 , Shida, C.S. 3 , Richardson, M. 4 , Lúcio,<br />
A.D. 5 , Beirão,P.S.L. 5 , Freitas, J.C. 1,2<br />
1 Universidade de São Paulo, Depto. de Fisiologia, IBUSP, São Paulo,BRAZIL-<br />
*a.j.zaharenko@ig.com.br 2 Center for Applied Toxinology, CAT-CEPID, Instituto Butantan, São Paulo,<br />
BRAZIL 3 Centro Interdisciplinar de Investigação Bioquímica, Universidade de Mogi das Cruzes, Mogi<br />
das Cruzes, BRAZIL. 4 Fundação Nacional Ezequiel Dias- FUNED, Belo Horizonte, BRAZIL. 5<br />
Universidade Federal de Minas Gerais, Depto. de Bioquímica e Imunologia, Belo Horizonte, BRAZIL<br />
Sea anemones produce a wide variety of biologically active compounds, such as the<br />
proteinaceous neurotoxins <strong>and</strong> cytolysins. Herein we report a new peptide, purified to<br />
homogeneity from the neurotoxic fraction of B. caissarum venom, by using gel filtration<br />
followed by rp-HPLC, naming it as BcIV. BcIV is a 41 amino acid peptide (molecular<br />
mass of 4669 Da) possessing 6 cysteines covalently linked by three disulfide bonds. This<br />
toxin has 45 <strong>and</strong> 48% of identity when compared to APETx1 <strong>and</strong> APETx2 from<br />
Anthopleura elegantissima, respectively, <strong>and</strong> 42% of identity with AmII <strong>and</strong> BDS-I <strong>and</strong> -II<br />
obtained from Antheopsis maculata <strong>and</strong> Anemonia sulcata, respectively. This neurotoxin<br />
presents only a weak paralysing action (minimal Lethal Dose close to 2000μg/kg) in<br />
swimming crabs Callinectes danae. This appears to be a different effect to that caused by<br />
the type 1 sea anemone toxin BcIII that is lethal to the same animals at lower doses (LD50 =<br />
219 μg/kg). Circular dichroism spectra of BcIII <strong>and</strong> BcIV show a high content of β-str<strong>and</strong><br />
secondary structure in both peptides, very similar to type 1 sodium channel toxins from<br />
various sea anemones, <strong>and</strong> to APETx1 <strong>and</strong> APETx2 from A. elegantissima, a HERG<br />
channel modulator <strong>and</strong> an ASIC3 inhibitor, respectively. Interestingly, BcIII <strong>and</strong> BcIV<br />
have similar effects on the action potential of the crab leg nerves, suggesting the same<br />
target in this tissue. As BcIII was previously reported as a Na + channel effector <strong>and</strong> BcIV is<br />
inactive over Na + currents of mammalian GH3 cells, we propose a species-specific action<br />
for this new molecule. A molecular model of BcIV was constructed using the structure of<br />
the APETx1 as template <strong>and</strong> putative key residues are discussed.<br />
o Bunodosoma caissarum<br />
o MS/MS Spectrometry<br />
o Sea Anemone<br />
o Molecular Modelling<br />
154<br />
Friday Track 2, K3.17 3.00-3.20pm<br />
Beyond prey capture – a comparative bioinformatic approach to cnidarian<br />
allomones<br />
Sher, D.*, Knebel, A., Bsor, T., Nesher, N., Tal, T., Morgenstern, D., Cohen, E., <strong>Fish</strong>man, Y. <strong>and</strong> Zlotkin,<br />
E.<br />
Department of Cell <strong>and</strong> Animal Biology, Silberman Institute of Life Sciences, Hebrew University,<br />
Jerusalem 91904, Israel. * dsher@pob.huji.ac.il<br />
Cnidarians such as hydrae <strong>and</strong> sea anemones are sessile, predatory, soft bodied animals<br />
which depend on a complex array of offensive <strong>and</strong> defensive allomones for prey capture<br />
<strong>and</strong> survival. These allomones are distributed throughout the entire organism both in<br />
specialized stinging cells (nematocytes) <strong>and</strong> in the body tissues (1). Cnidarians are entering<br />
the genomic era with the sequencing of the genome of the sea anemone Nematostella<br />
vectensis <strong>and</strong> a large EST database from Hydra magnipapillata, allowing for the first time<br />
to study cnidarians <strong>and</strong> their allomonal systems as a whole. This should facilitate the<br />
discovery of novel toxins, trace the evolution of cnidarian allomones, <strong>and</strong> help underst<strong>and</strong><br />
how the various allomones interact to paralyze the prey or fulfil other biological roles.<br />
To gain a global view of toxins in Hydra <strong>and</strong> Nematostella, bioinformatic methods were<br />
used to search the genome <strong>and</strong> EST databases for orthologs of toxins from cnidarians <strong>and</strong><br />
other organisms, <strong>and</strong> to determine the likelihood that detected sequences have the expected<br />
biological effect (2). Several toxin types, including venom phopholipase A2s (PLA2),<br />
were detected in both the hydra <strong>and</strong> the anemone. However, marked differences exist<br />
between the two organisms in the toxin genes detected. For example, the “classic” short<br />
chain neurotoxins, which are found in many anemones including Nematostella <strong>and</strong> are<br />
expected to be a major cause of prey paralysis, are absent in Hydra. Many other putative<br />
toxins were detected in one or both of these organisms, including several not previously<br />
detected in cnidarians. These include pore-forming toxins, elapid-like PLA2s, CRISP<br />
proteins, Prokineticin-like polypeptides <strong>and</strong> toxic Deoxyribonucleases.<br />
Our results illustrate the usefulness of a genomic approach to study toxins, revealing a<br />
high level of complexity in cnidarian allomonal systems. We suggest the ancestral<br />
cnidarians utilized neurotoxic PLA2s to paralyze prey, similar to modern day hydrae,<br />
while Anthozoans have additionally evolved short-chain neurotoxins. Finally, we raise the<br />
possibility that similar proteins may fulfil endogenous <strong>and</strong> allomonal roles in cnidaria.<br />
1) Zhang, M., et al. (2003) Biochemistry 42, 8939-44 2)Sher, D., et al. (2005) Toxicon 45, 865-79<br />
o Cnidaria<br />
o Genome<br />
o phospholipase<br />
o neurotoxin<br />
155
Friday Track 2, K3.17 3.20-3.40pm<br />
When Positive Selection of Neurotoxin Genes is Missing - the Riddle of the Sea<br />
Anemone Nematostella vectensis<br />
Moran Y*, Gurevitz M<br />
Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv,<br />
Israel, *moranyeh@post.tau.ac.il<br />
Rapid evolution driven by positive Darwinian Selection appears in toxins of vipers,<br />
scorpions, <strong>and</strong> marine snails. Although the vast phylogenetic distances between these<br />
animals suggest that this phenomenon is common, the recent release of the genome of<br />
Nematostella vectensis (Starlet anemone) as a collection of contigs portrays another<br />
extreme. Besides ShKT domains (resemble potassium channel blockers) embedded in<br />
various genes, only one gene family encoding for Na-channel neurotoxins has been found,<br />
<strong>and</strong> the putative mature product of ten family members is identical. Whereas the existence<br />
of a single toxin encoded by multiple genes may be explained by the unique ecology of N.<br />
vectensis, the complete absence of substitutions including synonymous ones is surprising<br />
<strong>and</strong> suggests either that these genes have been duplicated recently, or that their total<br />
conservation was advantageous. A retro-element identified downstream to one of the genes<br />
offers a possible mechanism of enhanced toxin gene duplication. This assumption still<br />
awaits further verification as soon as the various contigs will be assigned within larger<br />
genomic fragments.<br />
References<br />
1. Sullivan JC, Ryan JF, Watson JA, Webb J, Mullikin JC, Rokhsar D & Finnerty JR<br />
(2006) Nucleic Acids Res 34, D495-499<br />
o Sea anemone toxins<br />
o Genomics<br />
o Positive selection<br />
o Retro elements<br />
156<br />
Friday Track 2, K3.17 3.40-4.00pm<br />
Venom l<strong>and</strong>scapes: Towards the discovery of novel pharmacological tools via the<br />
combined use of LC-MALDI-TOF <strong>and</strong> MALDI-TOF/TOF with cDNA analysis<br />
Escoubas, P. 1* , Sollod, B. 2 , Nicholson, G.M. 3 , Wilson, D.T.R. 4 , Vinh, J. 5 , King, G.F 2 ,<br />
1<br />
Institut de Pharmacologie Moléculaire et Cellulaire – CNRS, 660 Route des Lucioles, 06560 Valbonne,<br />
France, *escoubas@ipmc.cnrs.fr<br />
2<br />
Department of Molecular, Microbial <strong>and</strong> Structural Biology, University of Connecticut Health Center,<br />
263 Farmington Ave., Farmington CT 06032-3305, USA<br />
3<br />
Department of Medical <strong>and</strong> Molecular Biosciences, University of Technology, Sydney, Broadway NSW<br />
2007, Australia<br />
4<br />
Xenome Inc., 50 Meiers Rd, Indooroopilly, Brisbane, QLD 4068, Australia<br />
5<br />
ESPCI Laboratoire de biologie CNRS UMR7637 10 rue Vauquelin 75005 Paris France<br />
The extent <strong>and</strong> diversity of peptide toxins that are expressed in the venom gl<strong>and</strong>s of cone<br />
snails, scorpions, <strong>and</strong> spiders results in a diverse array of leads for drug <strong>and</strong> insecticide<br />
discovery. But this diversity also represents a daunting analytical challenge. With the goal<br />
of streamlining the discovery process we have explored the complexity of Australian<br />
funnel-web spider venoms via the combined use of MALDI-TOF <strong>and</strong> MALDI-TOF/TOF<br />
MS coupled with chromatographic separation (LC-MALDI) <strong>and</strong> the analysis of venomgl<strong>and</strong><br />
cDNA libraries. Venom collected from male <strong>and</strong> female spiders of several species of<br />
Australian funnel-web spiders (Atrax <strong>and</strong> Hadronyche spp.) was fractionated by reversedphase<br />
HPLC. Fractions were analyzed by MALDI-TOF MS <strong>and</strong> data plotted as intensity<br />
(z) vs. m/z (x) vs. fraction number (y) in 3D contour graphs termed “venom l<strong>and</strong>scapes”.<br />
The results show that these spider venoms are far more complex than previously realized<br />
<strong>and</strong> contain many hundreds of peptides that follow a bimodal distribution, with about 75%<br />
of the peptides having a mass of 3,000–5,000 Da. Analysis of the venom peptidome of the<br />
spider H. versuta revealed 1018 peptides with mass less than 9 kDa.<br />
Toxin families were identified by matching the experimentally observed masses with those<br />
predicted from peptide sequences derived from analysis of venom-gl<strong>and</strong> cDNA libraries.<br />
The combination of measured parameters with prediction of molecular weights, HPLC<br />
retention times <strong>and</strong> data from the literature permits the interpretation of the LC-MALDI<br />
analysis. Groups of peptides of selected pharmacology (eg. Cav, Kv or Nav channel<br />
blockers) can be distinguished as discrete “peaks” with a specific signature on the 3D plot.<br />
Analysis of 11 venoms <strong>and</strong> ca. 400 fractions demonstrates common features of the funnelweb<br />
spider venoms, leading to the possibility of generating entire combinatorial<br />
pharmacological libraries for each toxin class. Additional validation of the method was<br />
achived using MALDI-TOF/TOF analysis of A. robustus female venom <strong>and</strong> permitted<br />
determination of the number of disulfide bridges. We propose that this approach may have<br />
predictive value for the discovery of novel pharmacological tools in complex venoms.<br />
o Spider toxins<br />
o Mass spectrometry analysis<br />
o cDNA analysis<br />
o MALDI-TOF<br />
157
Friday Track 2, K3.17 4.00-4.20pm<br />
Snake venomics<br />
Calvete, J.J. 1,* , Juárez, P. 1 , Sanz, L. 1<br />
1<br />
Instituto de Biomedicina de Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain.<br />
*jcalvete@ibv.csic.es<br />
Venoms of Viperidae <strong>and</strong> Crotalidae snakes (vipers <strong>and</strong> rattlesnakes) contain a number of<br />
different proteins that interfere with the coagulation cascade, the normal haemostatic<br />
system <strong>and</strong> tissue repair. Consequently, the envenomenations by these snakes generally<br />
result in persistent bleeding. Snake bite is still a serious threat in both developed <strong>and</strong><br />
underdeveloped countries. The world-wide mortality caused by snake envenomation is<br />
estimated at 50.000 deaths annually. The only effective treatment for systemic<br />
envenomation is the intravenous administration of an antivenom. However, polyclonal<br />
antisera include numerous antibodies with specificities not confined to the toxic target<br />
molecules. Hence, knowledge of the toxin composition of venoms could be devised to<br />
design immunization protocols with toxin-specific antibodies with greater specificity <strong>and</strong><br />
effectiveness that conventional systems. Despite its potential value, little is known about<br />
the venom protein composition of most vipers. The protein composition of the crude<br />
venoms of a number of genera from Viperinae (Macrovipera, Cerastes, Echis, Bitis) <strong>and</strong><br />
Crotalinae (Sistrurus, Crotalus, Agkistrodon) were analyzed by RP-HPLC, N-terminal<br />
sequence analysis, MALDI-TOF MS, <strong>and</strong> in-gel tryptic digestion followed by peptide<br />
mass fingerprinting <strong>and</strong> CID-MS/MS of selected peptide ions in a quadrupole-linear ion<br />
trap instrument. As expected from the rapid amino acid sequence divergence of venom<br />
proteins by accelerated evolution, with a few exceptions, the product ion spectra did not<br />
match to any known protein using the MASCOT search program. The CID-MS/MS spectra<br />
were therefore manually interpreted <strong>and</strong> the deduced peptide ion sequences submitted to<br />
BLAST sequence similarity searches. This approach allowed us to assign unambiguously<br />
all of the isolated venom fractions to protein families present in the non-redundant<br />
databases. Our proteomic approach complement transcriptomic studies by showing the<br />
relative abundance of the proteins that are actually secreted into the venoms. Our results<br />
show that the venom proteomes are composed of proteins belonging to only a few protein<br />
families. However, each venom showed distinct degree of protein composition complexity.<br />
o Venom proteome<br />
o Mass spectrometry<br />
o N-terminal sequencing<br />
o Disulfide bonds<br />
158<br />
Friday Track 2, K3.17 4.20-4.40pm<br />
Identification <strong>and</strong> characterisation of toxin-specific transcripts from the venom<br />
gl<strong>and</strong>s of Australian elapid snakes.<br />
St Pierre, L 1,2* , Masci, P.P 2 , Earl, S.T 1,2 , Flight, S 2 , de Jersey, J 3 <strong>and</strong> Lavin, M.F. 1,2<br />
1<br />
Queensl<strong>and</strong> Institute of Medical Research. PO Box Royal Brisbane Hospital, Brisbane, QLD, 4029,<br />
Australia. *liamS@qimr.edu.au<br />
2<br />
Faculty of Health Sciences, The University of Queensl<strong>and</strong>, Brisbane, QLD, 4067, Australia.<br />
3<br />
Faculty of Biological <strong>and</strong> Chemical Sciences, The University of Queensl<strong>and</strong>, Brisbane, QLD, 4067,<br />
Australia.<br />
The venom of Australian snakes contain a complex mixture of polypeptides that are<br />
typically small in size, disulfide rich, <strong>and</strong> highly stable, potent <strong>and</strong> specific in their<br />
mechanism of action, <strong>and</strong> hence are the ideal targets for the identification of novel<br />
therapeutic c<strong>and</strong>idates. Australian elapid venoms are amongst the most toxic in the world<br />
with numerous significant clinical outcomes, however they remain largely understudied at<br />
the molecular level. To gauge an underst<strong>and</strong>ing of the total complement of toxins within<br />
the venom (increasingly referred to as the “Venome”) of Australian snakes, we employed a<br />
transcriptomic analysis of the venom gl<strong>and</strong>, complemented by recombinant protein<br />
expression to further characterise specific toxin function.<br />
A comparative analysis of multiple toxin families was performed from ten medically<br />
significant Australian snakes: the inl<strong>and</strong> <strong>and</strong> coastal taipan, common brown, red-bellied<br />
black, mulga, rough-scaled, Stephen’s b<strong>and</strong>ed, small-eyed, black whip <strong>and</strong> tiger snakes.<br />
Toxin transcripts were initially identified from a coastal taipan venom gl<strong>and</strong> cDNA<br />
microarray with subsequent isolation <strong>and</strong> comparative analysis of isoforms from all ten<br />
snakes. These included the sequences of toxins such as factor X- <strong>and</strong> factor V-like<br />
prothrombin activators, phospholipase A2 isoforms, long <strong>and</strong> short chain neurotoxins,<br />
cysteine rich secretory proteins, venom natriuretic peptides, L-amino acid oxidases, along<br />
with number of proteins involved in regular cellular maintenance. The activity of unique<br />
toxin sequences were then characterised by recombinant protein expression. Examples<br />
include natriuretic peptides, which were shown to demonstrate a dose dependent inhibition<br />
of ACE, a factor X-like protein from the coastal taipan capable of cleaving prothrombinlike<br />
substrates <strong>and</strong> novel neurotoxins which also demonstrated potent inhibitory effects<br />
upon neuronal receptors. This study represents the most comprehensive survey to date of<br />
the genes responsible for the envenomation process in Australian snakes <strong>and</strong> sheds much<br />
light on the evolutionary <strong>and</strong> functional relationships between these species.<br />
o Australian elapid snake<br />
o Venom gl<strong>and</strong><br />
o Transcriptomics<br />
o Recombinant protein expression<br />
159
Friday Track 2, K3.17 4.40-5.00pm<br />
Proteomic analysis of Australian snake venoms for the discovery <strong>and</strong> development<br />
of new human therapeutics #<br />
Earl, S.T 1,2* , Birrell, G.W 1 , St Pierre, L.D 1 , Wallis, T.P 1,3 , Gorman, J.J 1,3 , Masci, P.P 4 , de Jersey, J 5 <strong>and</strong><br />
Lavin, M.F 1,2 .<br />
1<br />
Queensl<strong>and</strong> Institute of Medical Research PO Box Royal Brisbane Hospital, Brisbane 4029, Australia ,<br />
*Stephen.Earl@qimr.edu.au<br />
2<br />
The School of Medicine, Central Clinical Division, The University of Queensl<strong>and</strong>, Brisbane, Australia<br />
3<br />
The Institute for Molecular Biosciences, The University of Queensl<strong>and</strong>, Brisbane, Australia<br />
4<br />
The School of Medicine, Southern Clinical Division, Princess Alex<strong>and</strong>ra Hospital, The University of<br />
Queensl<strong>and</strong>, Brisbane, Australia<br />
5<br />
The School of Molecular <strong>and</strong> Microbial Sciences, The University of Queensl<strong>and</strong>, Brisbane, Australia<br />
Australian elapid species possess the most toxic snake venoms in the world. However, in<br />
comparison to Asian <strong>and</strong> American snake species, much less is known about overall<br />
venom composition. Australian snake venoms are lethal protein cocktails that interfere<br />
with several mammalian physiological processes, including the blood coagulation <strong>and</strong><br />
nervous systems. In many cases, these venom proteins have a stable disulfide-rich<br />
structure, are very specific in their site of action <strong>and</strong> act with high efficacy. For these<br />
reasons, we hypothesise that Australian elapid venoms are a potential source of new<br />
human therapeutics. The aim of this study was to systematically examine the venoms of 20<br />
Australian elapid snakes using proteomic methods such as 2-dimensional gel<br />
electrophoresis (2-DE) with mass spectrometry (MS) to identify all venom components<br />
<strong>and</strong> select novel venom proteins with potential for development as new human<br />
therapeutics. When separated by 2-DE, each venom showed approximately 100-200<br />
discrete protein spots, varying in molecular weight from 7 to over 100 kDa with pIs from 3<br />
to 10. Using MS, the majority of venom protein spots have been identified. These include<br />
previously characterised venom proteins such as phospholipase A2 enzymes, neurotoxins<br />
<strong>and</strong> prothrombin-activating proteins, along with proteins not previously reported to be<br />
components of Australian elapid venoms such as acetylcholinesterase <strong>and</strong> nerve growth<br />
factor. A number of novel venom proteins have also been identified <strong>and</strong> selected as<br />
potential therapeutic c<strong>and</strong>idates. In addition, we have used several enzymes, protein stains<br />
<strong>and</strong> specific antisera to examine different post-translational modifications amongst<br />
Australian elapid venom proteins. This work represents the most comprehensive analysis<br />
<strong>and</strong> identification of Australian elapid venom proteins yet undertaken.<br />
#<br />
This project was supported by our commercial partner QRxPharma <strong>and</strong> the Australian<br />
Research Council.<br />
o Snake venom<br />
o Australian elapid<br />
o proteomics<br />
o drug discovery<br />
160<br />
Friday Track 2, K3.17 5.00-5.20pm<br />
Molecular characterization of hyaluronidase-encoding genes from venom gl<strong>and</strong><br />
cDNA libraries of Echis, Bitis <strong>and</strong> Cerastes viper species<br />
Harrison, R.A,* Ibison, F., Wilbraham, D. Wagstaff, S.C.<br />
Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool,<br />
UK *R.Harrison@liverpool.ac.uk<br />
The killing of prey species by snakes is most efficiently achieved by the rapid<br />
dissemination of tissue-embedded venom. Hyaluronidase, the “venom spreading factor”<br />
has long been associated with tissue distribution of venom <strong>and</strong> several reports have<br />
demonstrated that immunological or biochemical inhibition of hyaluronidase reduces<br />
venom-induced pathology. Venom hyaluronidases have been isolated in native form but<br />
their extensive characterisation has been restricted by the lack of any protein or gene<br />
sequence data – the objective of this study.<br />
We r<strong>and</strong>omly sequenced 1000 Echis ocellatus (Nigeria) venom gl<strong>and</strong> cDNAs (ESTs)<br />
<strong>and</strong> this transcriptome was annotated (using annot8r_blast2GO) with gene ontology (GO)terms<br />
that functionally classifies EST sequences into proteins with, for example, sugar<br />
binding or growth factor activities. Examination of each EST in the “protein, lipid <strong>and</strong><br />
carbohydrate metabolism” categories revealed a single sequence matching the GOannotation<br />
of hyalurononglucosaminadase-activity (GO:000415). Because this singleton<br />
cluster (Eo00242) was clearly a partial transcript lacking 5’ sequence we designed PCR<br />
primers that amplified the full-length sequence with extensive 5’ <strong>and</strong> 3’ UTR sequence.<br />
Since the latter sequences are typically more conserved across species than protein-coding<br />
sequences, we utilised a series of primers complimentary to the 5’ <strong>and</strong> 3’ UTR of the E.<br />
ocellatus sequence to amplify analogous cDNAs from venom gl<strong>and</strong> cDNA libraries of E.<br />
pyramidum leakeyi (Kenya), Cerastes cerastes cerastes (Egypt) <strong>and</strong> Bitis arietans<br />
(Nigeria).<br />
We will describe the extraordinarily high degree of sequence similarity between the E.<br />
ocellatus, E.p. leakeyi, C.c. cerastes <strong>and</strong> B. arietans hyaluronidase genes <strong>and</strong> illustrate<br />
that, although they are very different from the bee venom <strong>and</strong> four mammalian classes of<br />
hyaluronidase, they retain several specific residues <strong>and</strong> the overall architecture that<br />
characterise this class of carbohdrate metabolising hydrolases.<br />
o Hyaluronidase<br />
o EST database<br />
o E. ocellatus<br />
o B. arietans<br />
161
Friday Track 2, K3.17 5.20-5.40pm<br />
Miniminization of protein structure <strong>and</strong> gene organization along the evolution of<br />
the short disintegrin ocellatusin from a dimeric disintegrin precursor<br />
Juárez, P. 1* , Wagstaff, S.C. 2 , Sanz, L. 1 , Harrison, R.A. 2 , Calvete, J.J. 1<br />
1<br />
Instituto de Biomedicina de Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain.<br />
*pjuarez@ibv.csic.es<br />
2<br />
Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool<br />
L3 5QA, Engl<strong>and</strong>, UK<br />
Venom represents a key innovation in ophidian evolution that allowed advanced snakes to<br />
transition from a mechanical (constriction) to a chemical (venom) means of subduing <strong>and</strong><br />
digesting prey larger than themselves. Venom toxins likely evolved from endogenous<br />
proteins with normal physiological functions that were recruited into the venom proteome<br />
before the radiation of the advanced snakes at the base of the Colubroid radiation. Venoms<br />
of Viperidae <strong>and</strong> Crotalidae snakes contain proteins that interfere with the coagulation<br />
cascade, the normal haemostatic system <strong>and</strong> tissue repair. Among them, disintegrins<br />
represent a family of integrin receptor antagonists that are released in viper venoms by<br />
proteolytic processing of PII snake venom metalloproteinase (SVMP) precursors or<br />
synthesized from short-coding mRNAs. The disintegrin family has been divided into five<br />
groups according to their polypeptide length <strong>and</strong> number of disulfide bonds, <strong>and</strong> the<br />
current view is that the structural diversity of disintegrins has been achieved through the<br />
selective loss of disulfide bonds. To underst<strong>and</strong> the genomic basis of the accelerated<br />
evolution of disintegrins, <strong>and</strong> the molecular mechanism underlying their structural<br />
diversification, we have undertaken the analysis of cDNAs encoding disintegrins from a<br />
venom gl<strong>and</strong> library of Echis ocellatus (Eo) <strong>and</strong> the genomic organization of the Eo<br />
disintegrin genes. The cDNA sequences coding for (RGD/KGD/WGD/MLD-containing)<br />
dimeric disintegrins lack the metalloproteinase domain <strong>and</strong> belong thus to the short-coding<br />
class. Analysis of two distinct messengers coding for the short disintegrin ocellatusin,<br />
obtained by PCR (Eo 10c-10) <strong>and</strong> from ESTs (Eo-00006), strongly argues for a common<br />
ancestry of short- <strong>and</strong> dimeric distintegrin subunits. Two nucleotide mutations (Cys->Tyr<br />
<strong>and</strong> Ser->Cys) are hypothesized to represent key events in the dimeric->short disintegrin<br />
transition. On the other h<strong>and</strong>, comparison of the exon-intron organization of genes for<br />
medium-sized, dimeric, <strong>and</strong> short disintegrins revealed that the evolutionary pathway<br />
leading to the diversification of disintegrins (medium->dimeric->short) also involved the<br />
miniminization of the gene organization by the sucessive removal of introns.<br />
o Echis ocellatus venom<br />
o Disintegrin evolution<br />
o Ocellatusin cDNA<br />
162<br />
Friday Track 3, K3.25 3.00-3.20pm<br />
The same small three-fingered fold for snake toxins <strong>and</strong> human uPAR.<br />
Llinas, P. 1 , Le Du, M.H. 1 , Gardsvoll, H. 2 , Dano, K. 2 , Ploug,M. 2 , Gilquin, B. 1 , Stura,<br />
E.A. 1 , Ménez, A. 1* .<br />
1 Département d’Ingénierie et d’Etudes des Protéines, Bât 142, CEA, Saclay, 91191,<br />
Gif-sur-Yvette, France. *<strong>and</strong>re.menez@cea.fr<br />
2 Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark.<br />
Human urokinase-type plasminogen activator receptor (uPAR; CD87) is a glycolipidanchored<br />
modular protein made of a single chain polypeptide (283 amino acids)<br />
organized into three extracellular domains. Each domain comprises about 90 residues<br />
with four to five disulphide bonds. The primary function of uPAR is to bind uPA with<br />
high affinity <strong>and</strong> hence to focalize the cellular conversion of plasminogen to plasmin.<br />
uPAR is involved in the pathology of human cancers. To underst<strong>and</strong> the structural<br />
properties of human uPAR, we have solved the crystal structure of a soluble form of<br />
the receptor at 2.7 Å, in complex with a peptide that has a high affinity for uPAR <strong>and</strong><br />
inhibits its capacity to bind uPA. uPAR is composed of three consecutive domains that<br />
are organized in an almost circular manner, <strong>and</strong> which generate both a large external<br />
surface <strong>and</strong> a deep internal cavity where the peptide binds in a helical conformation.<br />
Each domain adopts a typical three-fingered structure, the first two superimposing well<br />
with the structure of buc<strong>and</strong>in <strong>and</strong> the third one with that of CD59. Therefore, the<br />
building blocks of uPAR are folded like the multifunctional three-fingered snake<br />
toxins. This work further demonstrates that the three-fingered fold is a highly<br />
divergent protein structure.<br />
Llinas, P., Le Du, M-H., Gårdsvoll, H. Danø, K., Ploug, M., Gilquin, B., Stura E. A.<br />
<strong>and</strong> Ménez, A. 2005, EMBO J., 24, 1655-1663<br />
ubiquitous toxin folds<br />
163
Friday Track 3, K3.25 3.20-3.40pm<br />
3D-structure of VAP1, a high-molecular snake venom metalloprotease, reveals<br />
metalloproteinase/disintegrin/cysteine-rich architecture of SVMPs <strong>and</strong> ADAMs<br />
Takeda, S 1, 2 , Igarashi, T 1 , Mori, H 1 , Araki, S 3<br />
1 National Cardiovascular Center Research Institute, Fujishiro-dai, Suita, Japan<br />
2 Riken Harima Institute at SPring-8,1-1-1 Kouto, Mikazuki, Sayo, Japan<br />
3 Nagoya University, MBL, Toba, Japan, *saraki@bio.nagoya-u.ac.jp<br />
Introduction: High-molecular snake venom metalloproteases (SVMPs) <strong>and</strong> ADAMs<br />
possess metalloproteinase/disintegrin/cysteine-rich (MDC) domains in common. However,<br />
the physiological functions of the multidomains remain unclear. One of the reasons is the<br />
lack of information of three-dimensional (3D) structures of the multidomain components.<br />
To clarify the architecture, we used vascular apoptosis-inducing protein 1 (VAP1), which<br />
is a dimer-type high-molecular SVMP <strong>and</strong> stable to make crystals for X-ray analysis.<br />
Methods: VAP1 was isolated from crude western diamondback rattlesnake Crotalus atrox<br />
venom (Sigma-Aldrich, USA) <strong>and</strong> subjected to sitting- or hanging-drop vapor diffusion<br />
crystallization. X-ray diffraction data were collected at SPring-8 beamlines.<br />
Results: 3D structures of VAP1 are determined from the crystals at 2.5-Å resolution. The<br />
disintegrin (D)-domain protrudes from the metalloprotease (M)-domain opposing the<br />
catalytic site <strong>and</strong> constituting a C-shaped arm with cores of Ca2+ ions. The disintegrinloop<br />
is packed by the cysteine-rich (C)-domain <strong>and</strong> inaccessible for protein-binding.<br />
Instead, the hyper-variable region (HVR) in the C-domain, which has a novel fold<br />
stabilized by the strictly conserved disulfide bridges, constitutes a potential protein-protein<br />
adhesive interface. The HVR is located at the distal end of the arm <strong>and</strong> faces toward the<br />
catalytic site. The amino acid sequence alignment indicates that the architecture is common<br />
to most of SVMPs <strong>and</strong> ADAMs.<br />
Discussion/Conclusion: Here for the first time, we reveal the 3D architecture of MDC<br />
domains of SVMPs <strong>and</strong> ADAMs. The unique C-shaped structure of MDC implies<br />
interplay between the proteolytic <strong>and</strong> adhesive domains. The HVR may be important to<br />
search physiological targets of SVMPs <strong>and</strong> ADAMs <strong>and</strong> provide insights into the future<br />
design of drugs with higher specificity.<br />
o snake venom<br />
o metalloproteinase/disintegrin<br />
o 3D-structure<br />
o ADAM<br />
164<br />
Friday Track 3, K3.25 3.40-4.00pm<br />
Unique Molecular Formations in Crystals of Scorpion Toxins that Affect Voltage-Gated<br />
Na-Channels<br />
Kahn R 1 *, Karbat I 1 , Gurevitz M 1 , Frolow F 2<br />
1<br />
Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Israel<br />
*nitzai@post.tau.ac.il<br />
2<br />
Department of Molecular Microbiology <strong>and</strong> Biotechnology, George S. Wise Faculty of Life Sciences, Tel-Aviv<br />
University, Tel-Aviv, Israel<br />
Long-chain scorpion toxins bind <strong>and</strong> modulate the function of voltage-gated sodium<br />
channels (Navs) in excitable tissues. The toxins are approximately 61-76 aa residue long<br />
globular polypeptides reticulated by four disulfide bonds <strong>and</strong> very similar in structure.<br />
They divide into two classes, alpha <strong>and</strong> beta, according to their mode of action. Each class<br />
is subdivided to distinct pharmacological groups according to their ability to displace one<br />
another from the receptor site of channels of various origin. We cloned <strong>and</strong> produced in<br />
recombinant form representative toxins of all pharmacological groups <strong>and</strong> analyzed them<br />
by mutagenesis <strong>and</strong> X-ray crystallography. Determination of the structure of the alphatoxin<br />
Lqh-alpha-IT at 1.1Å resolution revealed cubic space group with complicated<br />
packing arrangements in the crystal. Close inspection of these arrangements revealed a 24mer<br />
shell-like 'particle' of a 432 symmetry. With the objective to create novel 'molecular<br />
formation' we crystallized <strong>and</strong> analyzed several other toxins. Determination of the structure<br />
of a triple mutant of Lqh-alpha-IT, LqhαIT 8D-9D-10V , which was devoid of activity, at 1.6Å<br />
resolution exhibited a dodecameric particle formed from a dimer in the asymmetric unit by<br />
interplay of I4122 symmetry. We further solved the structure of a toxin chimera,<br />
Lqh2 LqhαIT(face) , composed of the bioactive surface of Lqh-alpha-IT on the scaffold of Lqh2,<br />
at 1.2Å resolution <strong>and</strong> found novel formations of I422 symmetry. We than analyzed the<br />
crystal structure of the beta-toxin Bj-xtrIT (solved at a resolution of 2.1Å). This crystal<br />
reveals a mono-molecular filament-like pattern, where the crystallographic translational<br />
repeat is of eight molecules, 191.4Å long, <strong>and</strong> is due to interplay of I41 symmetry<br />
operators. Close inspection of these arrangements reveals interplay among symmetry<br />
elements, molecular size, shape <strong>and</strong> bioactive surface that provide unique formations of<br />
potential nano-technological importance.<br />
o Scorpion toxins<br />
o Crystal structure<br />
o Packing<br />
o Molecular formations<br />
165
Friday Track 3, K3.25 4.00-4.20pm<br />
The increasing molecular diversity of sarafotoxins<br />
Ducancel 1 F.*, Quinton 3 L., Menin 4 L., Hayashi 2 , M.A.F., Lamthanh 1 , H., Chamot-<br />
Rooke 3 J., Stöcklin 4 R.<br />
1<br />
Department of Protein Engineering (DIEP), Bâtiment 152, CEA de Saclay, 91191<br />
Gif sur Yvette Cedex (France), *Tel: +33 169088154, Fax: +33 169089071, e-mail:<br />
frederic.ducancel@cea.fr<br />
2<br />
Biochemical <strong>and</strong> Biophysical Laboratory, Instituto Butantan, SP 05503-900, Sao<br />
Paulo, Brazil.<br />
3<br />
Laboratoire des Mécanismes Réactionnels, UMR 7651 CNRS, Ecole Polytechnique,<br />
F-91128, Palaiseau, France. 4 Atheris Laboratories, case postale 314, CH-1233<br />
Bernex, Geneva, Switzerl<strong>and</strong>.<br />
Abstract.<br />
Snake venom sarafotoxins (SRTXs) <strong>and</strong> mammalian endothelins (ETs)<br />
comprise structurally <strong>and</strong> functionally related potent vasoconstrictor<br />
isopeptides that act on the vascular system via identical receptors. This<br />
similarity is remarkable, since SRTXs are highly toxic components<br />
isolated from the venoms of snakes of the genus Atractaspis in the<br />
Atractaspididae family, while ETs are endogenous mammalian<br />
hormones. Since the first functional <strong>and</strong> structural description of<br />
SRTXs in 1988, the full extent of their natural diversity has become<br />
increasingly apparent, <strong>and</strong> this has led to the characterization of new<br />
families of sarafotoxins peptides. Based on a combination of<br />
conventional biochemical approaches <strong>and</strong> the latest molecular biology<br />
<strong>and</strong> mass spectrometry techniques, the more recent panel of SRTX<br />
isopeptides isolated from various snake species within the<br />
Atractaspididae family will be presented. Also, the similarities <strong>and</strong><br />
differences that exist between sarafotoxins <strong>and</strong> endothelins in terms of<br />
their metabolism, genetic origin, structure <strong>and</strong> functional sites will be<br />
discussed.<br />
166<br />
Friday Track 3, K3.25 4.20-4.40pm<br />
Molecular diversity of snake venom VEGFs<br />
Matsunaga, Y. 1 , Yamazaki, Y. 1 , Morita, T. 1*<br />
1 Meiji Pharmaceutical Univ., 2-522-1 Noshio, Kiyose-city, Tokyo, Japan, *tmorita@my-pharm.ac.jp<br />
[Background] Vascular endothelial growth factor (VEGF-A) plays a critical role in the<br />
embryonic vasculogenesis <strong>and</strong> in the pathological angiogenesis observed in several<br />
endothelium proliferative diseases, including cancer. Recently, we isolated <strong>and</strong><br />
characterized novel VEGFs (vammin <strong>and</strong> VR-1) from the venoms of snakes, Vipera<br />
ammodytes ammodytes <strong>and</strong> Daboia russelli russelli (Vipera r. russelli, also known as<br />
Russell’s viper) 1 . Compared with human VEGF-A, the snake venom derived VEGFs show<br />
stronger proliferative affects in vitro <strong>and</strong> more potent hypotensive effects in vivo 1 .<br />
Furthermore, the snake venom VEGFs specifically recognize KDR (VEGF receptor 2), but<br />
not other VEGF receptors 1 . The crystal structures of snake venom VEGFs reveal similar<br />
but significantly different architectures from the known structures of other human VEGFs:<br />
insertion of an amino acid resulted in a different structure for receptor-binding loop 3, <strong>and</strong><br />
a difference in surface potential of the molecule 2 . Thus, these recent findings regarding<br />
snake venom VEGFs demonstrate unique <strong>and</strong> distinct biological activities <strong>and</strong> also<br />
substantially different structural features from other known VEGF subtypes. To identify<br />
novel VEGFs derived from several snake venoms, we cloned cDNAs encoding VEGF-like<br />
proteins using the venom gl<strong>and</strong> cDNAs from several venomous snakes.<br />
[Result <strong>and</strong> Discussion] We first performed PCR using snake venom VEGF specific<br />
primers. In addition to the Vipera species, specific amplified PCR products were observed<br />
when the venom gl<strong>and</strong>s cDNAs from Echis, Bitis, Agkistrodon <strong>and</strong> Crotalus species were<br />
used as templates. Encoding proteins exhibited high identity to known snake venom<br />
derived VEGFs, but showed significant variation in their receptor-binding loop 3 <strong>and</strong> Cterminal<br />
heparin-binding regions. These data suggest that snake venom contains other<br />
novel VEGFs possessing unknown, conceivably interesting, properties.<br />
1. Yamazaki, Y., Takani, K., Atoda, H., Morita, T. (2003) JBC 278, 51985-51988<br />
2. Suto, K., Yamazaki, Y., Morita, T., Mizuno, H. (2005) JBC 280, 2126-2131<br />
o vascular endothelial growth factor<br />
o snake venom<br />
o cDNA cloning<br />
o molecular diversity<br />
167
Friday Track 3, K3.25 4.40-5.00pm<br />
Isolation <strong>and</strong> characterization of a VEGF-like protein from the venom of Bitis<br />
arietans<br />
Obayashi S 1 , Matsunaga Y 1 , Yamazaki Y 1 , Morita T 1*<br />
1 Department of Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-<br />
8588, Japan * tmorita@my-pharm.ac.jp<br />
Background: Vascular endothelial growth factor-A (VEGF-A165) exerts multiple effects<br />
upon binding to VEGFR-1 (Flt-1), VEGFR-2 (KDR), <strong>and</strong> neuropilin-1. We recently<br />
identified <strong>and</strong> characterized two novel snake venom VEGFs (vammin <strong>and</strong> VR-1) which<br />
possess unique properties. These VEGFs, designated as VEGF-Fs, are highly specific<br />
lig<strong>and</strong>s for KDR 1) . In this study, we presumed that snake venom-derived VEGFs may<br />
exhibit the same molecular diversity observed in other snake venom proteins, such as Ctype<br />
lectin-like proteins 2) <strong>and</strong> phospholipase A2. In fact, other snake venom VEGFs<br />
(TfsvVEGF <strong>and</strong> Pm-VEGF) demonstrating distinct receptor selectivity relative to vammin<br />
<strong>and</strong> VR-1 have recently been identified in the venom of both Trimeresurus <strong>and</strong><br />
Protobothrops species 3, 4) . Subsequently, we screened several snake venoms using antivammin<br />
antiserum. As a result, we detected an immunoreactive protein in the venom of<br />
Bitis arietans. Results <strong>and</strong> Discussion: The immunoreactive protein was purified by three<br />
steps column chromatographies using an anti-vammin antiserum as a detection regent. The<br />
purified protein, named barietin, was obtained with a yield of 0.08 % in relation to total<br />
venom protein. Sequence analysis revealed that barietin exhibits significant homology with<br />
the VEGF family; however barietin generally lacks most of the C-terminal sequence, when<br />
compared with other heparin-binding VEGFs including vammin 5) . Heparin-binding affinity<br />
of barietin was measured using analytical heparin affinity chromatography. Barietin could<br />
bind heparin with high affinity as well as human VEGF-A165. These results indicate that<br />
barietin has distinct molecular properties from the previously described VEGFs such as<br />
snake venom-derived VEGF (VEGF-F) <strong>and</strong> mammalian VEGF-A165.<br />
1) Yamazaki Y, Takani K, Atoda H, <strong>and</strong> Morita T (2003) JBC 278, 51985-51988<br />
2) Morita T (2005) Toxicon 45, 1099-1114<br />
3) Takahashi H, et al. (2004) JBC 279, 46304-46314<br />
4) Chen YL, Tsai IH, Hong TM, <strong>and</strong> Tsai SH (2005) Thromb. Haemost. 93, 331-338<br />
5) Yamazaki Y, Tokunaga Y, Takani K, <strong>and</strong> Morita T (2005) Biochemistry 44, 8858-8864<br />
o vascular endothelial growth factor<br />
o snake venom<br />
o heparin<br />
168<br />
Friday Track 3, K3.25 5.00-5.20pm<br />
VEGF receptor-binding potential is a common property for myotoxic<br />
phospholipase A2.<br />
Fujisawa D., Matsunaga Y., Yamazaki Y. <strong>and</strong> Morita T.*<br />
Department of Biochemistry, Meiji Pharmaceutical University, 2-522-1, Noshio, Kiyose, Tokyo, Japan, *<br />
tmorita@my-pharm.ac.jp<br />
Introduction: Vascular endothelial growth factor (VEGF165) plays a central role in<br />
angiogenesis through binding to the VEGF receptor-2 (KDR). We previously found a<br />
novel VEGF subtype designated VEGF-F, which selectively binds to KDR, from two viper<br />
venoms 1) . Recently, we identified a VEGF receptor-binding protein, abbreviated as KDRbp,<br />
from the venom of the eastern cottonmouth (Agkistrodon piscivorus piscivorus), which<br />
binds to the extracellular domain of KDR with 10 -8 M affinity 2) . Sequence analysis<br />
revealed that the KDR-bp molecule is identical to an inactive phospholipase A2 (PLA2)<br />
homologue, Lys49PLA2. Lys49PLA2 has been shown to exhibit myotoxicity; however, the<br />
molecular mechanism remains unknown. The aim of this study is to examine the<br />
relationship between KDR-binding potential <strong>and</strong> myotoxicity through the analysis of<br />
KDR-binding potential of several PLA2 <strong>and</strong> PLA2 homologues derived from the venom of<br />
five snake species.<br />
Results <strong>and</strong> Discussion: PLA2 <strong>and</strong> its homologues were purified by gel filtration, ionexchange<br />
chromatography <strong>and</strong> HPLC. Isolated PLA2s were identified by N-terminal<br />
sequencing <strong>and</strong> MALDI-TOF MS. KDR-binding potential was evaluated using the Biacore<br />
system. As a result, all myotoxic PLA2 homologues were bound to the extracellular<br />
domain of KDR, while no specific interaction was observed in other PLA2 homologues<br />
such as neurotoxic or anticoagulant PLA2s; showing strong correlation between KDRbinding<br />
ability <strong>and</strong> myotoxicity. In addition to the inactive PLA2 homologues,<br />
Asp49PLA2, an active PLA2 from the venom of A. p. piscivorus possessing myotoxicity,<br />
also displayed KDR-binding potential. These results indicate that KDR-binding potential is<br />
independent of PLA2 enzymatic activity. Sequence comparisons suggest that the Nterminal<br />
α-helix region, β-wing region <strong>and</strong> C-terminal loop region may be involved in<br />
KDR-binding.<br />
1). Yamazaki Y., Takani K., Atoda H. <strong>and</strong> Morita T. (2003) JBC, 278, 51985-51988.<br />
2). Yamazaki Y., Matsunaga Y., Nakano Y. <strong>and</strong> Morita T. (2005) JBC, 280, 29989-29992.<br />
o Snake venom<br />
o Vascular endothelial growth factor (VEGF)<br />
o VEGF receptor-2 (KDR)<br />
o Myotoxic phospholipase A2<br />
169
Friday Track 3, K3.25 5.20-5.40pm<br />
Low Abundant Proteins in Cobra Venom<br />
Utkin, Y.N., Osipov, A.V., Makarova, Ya.V.<br />
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, ul. Miklukho-Maklaya 16/10, Moscow, Russia,<br />
utkin@ibch.ru<br />
Cobra venoms are complex mixtures of different components, mostly of polypeptide<br />
nature. The main components of cobra venom are cytotoxins, phospholipases A2 <strong>and</strong><br />
neurotoxins. The properties <strong>and</strong> biological effects of these proteins are very well studied.<br />
At the same time cobra venoms contain less abundant <strong>and</strong> not so well studied proteins of<br />
different structural types. The aim of this work is to identify, isolate <strong>and</strong> investigate low<br />
abundant proteins in cobra venom. Our approach is based on the use of different kinds of<br />
liquid chromatography (gel-filtration, ion-exchange <strong>and</strong> reversed phase) for separation of<br />
the venom in combination with MALDI mass-spectroscopy for identification of new<br />
proteins. Such a proteomic approach has allowed us to identify practically all the known<br />
proteins in cobra Naja kaouthia venom as well as to find several new ones.<br />
We have found the first representative of glycosylated three-fingered toxins – cytotoxin 3<br />
containing a carbohydrate residue on Asn29. This modification results in substantial<br />
decrease in cytotoxicity. A series of so called “weak” or non-conventional toxins was<br />
identified in N. kaouthia venom. The in vivo effects for one of them (WTX) were studied,<br />
<strong>and</strong> data obtained suggest the action of WTX on several biological targets. A series of<br />
toxins highly homologous to muscarinic toxins from mamba venom was found in cobra<br />
venom as well. Several novel proteins belonging to the family of cysteine-rich secretory<br />
proteins (CRISP) were isolated from the venoms of N. kaouthia <strong>and</strong> N. haje cobra. Four<br />
CRISP variants were identified in N. kaouthia venom <strong>and</strong> three proteins in N. haje venom.<br />
Our results suggest that each cobra venom contains a pool of different CRISPs. In addition<br />
to acute toxicity determination, the activity of new toxins was tested on rat<br />
pheochromocytoma cell line PC12. Depending on the protein studied we have observed<br />
different effects ranging from cytotoxicity (for cytotoxins) to neurite outgrowth inducing<br />
activity (for nerve growth factor <strong>and</strong> phospholipase A2).<br />
Thus, using the proteomic approach we have identified <strong>and</strong> characterized several new low<br />
abundant proteins in cobra venom.<br />
o Cobra venom<br />
o New proteins<br />
o isolation<br />
o properties<br />
170<br />
Monday 24 July: poster session<br />
(Colville Building 5.11/5.12)<br />
Nerve, muscle, <strong>and</strong> myotoxicity<br />
1. Ability of suramin to antagonize myotoxic <strong>and</strong> phospholipase activities of bee<br />
venom. El Kik, C.Z., Fern<strong>and</strong>es, F.F.A., Sanches, F.V.,Arruda, E.Z., Melo, P.A.<br />
2. Antimyotoxic effects of suramin <strong>and</strong> polyethylene glycol (PEG) against<br />
Agkistrodon contortrix laticinctus crude venom <strong>and</strong> its myotoxin. Tomaz, M. A. 1 ,<br />
Serafim, A.D., Williams, K.,Murakami, M.T., Arni, R.K., Ownby, C.L.; Melo, P.A.<br />
3. Ability of a synthetic coumestan named LQB93 to antagonize some Bothrops<br />
snake venoms activity. Melo, P. A., Pinheiro, D. A.; Fern<strong>and</strong>es, F. F. A.; Tomaz, M.<br />
A.;Moraes, R. A. M. 1 ; Ricardo, H. D 1 . da Silva, A. J. M 2 . ; Costa, P. R. R 2 .<br />
4. Dexamethasone shows in vivo but not in vitro antimyotoxic effect against Bothrops<br />
jararacussu snake venom. Patrão-Neto, F.C. 1 ; Tomaz, M.A. 2 ; Calil-Elias, S. 2,3 , Melo,<br />
P.A.<br />
5. Improvement of mouse skeletal muscle regeneration by heparin after lesion<br />
induced by Bothrops jararacussu venom. Calil-Elias, S., Tomaz, M. A., Costa, M.L.,<br />
Mermelstein, C.L., Martinez;A. M. B., Melo, P.A.<br />
6. Mouse extensor digitorum longus <strong>and</strong> soleus have a different sensitivity to some<br />
snake venoms myotoxicity. Tomaz, M. A 1 ., Calil-Elias, S 1,3 .,Martinez, A.M.B 2 ., Melo,<br />
P.A 1 .<br />
7. αδ-Bungarotoxin, a new reversible long-chain neurotoxin from Malayan Krait<br />
(Bungarus c<strong>and</strong>idus) venom with high toxicity <strong>and</strong> site-selective binding at the<br />
muscle nicotinic acetylcholine receptor. Kuch, U. 1* , Molles, B.E. 2 , Bergman, T. 3 ,<br />
Cederlund, E. 3 , Alvelius, G. 3 , Jörnvall, H. 3 , Chanhome, L. 4 , Omori-Satoh, T. 4 , Chen,<br />
Y.M. 5 , Samejima, Y. 6 , Warrell, D.A. 7 , Mebs, D. 1<br />
8. Novel recombinant expression system for myonecrotic Lys49 phospholipase A2<br />
<strong>and</strong> its mutants identifying the functional residues for necrosis. Tomohisa Ogawa 1, * ,<br />
Minae Seto 1 , Koji Muramoto 1 , Motonori Ohno 2<br />
9. Non-cholinergic contractile activity of Dispholidus typus (Boomslang snake) in the<br />
rat isolated vas deferens. Lumsden, N. G 1 , Hodgson, W. C *2 , Ventura, S 3 , Lewis, R. J 1<br />
10. Equivalent effects of snake PLA2 neurotoxins <strong>and</strong> lysophospholipid–fatty acid<br />
mixtures. Rigoni, M 1 Caccin, P 1 , Gschmeissner, S 2 , Koster, G 3 , Postle, A. D 3<br />
,Rossetto, O 1 , Schiavo, G 2 <strong>and</strong> Montecucco, C 1<br />
171
11. EFFECT OF COLUBER RAVERGIERI VENOM ON SYNAPTIC PASSAGE.<br />
Kazakov I., Abubakirova M.E.<br />
12. Effect of Duvernoy's gl<strong>and</strong> secretion of snake Coluber ravergieri on frog<br />
nerve-muscle preparation. Akhmedov K.J., Makhkamov B.T., R.S.Reimbaeva.,<br />
M.E.Abubakirova., I.K.Kazakov<br />
13. Myotoxic activity from the venoms of six species of Micrurus. Néstor Lago 1 ;<br />
Adolfo R. de Roodt 2 ; Claudio Mirabelli 1 ; Rodrigo D. Laskowicz 2 ; Judith Estévez 3 ;<br />
Víctor M. Manzaneli 2 ; Jorge Paniagua 4 ; Eduardo Scarlatto 5 .<br />
14. Development of an in vivo model for the study of snake venom myotoxicity.<br />
Andrew J. Hart 1 , Geoffrey K. Isbister 1,2 , Sharmaine Ramasamy 1 , Wayne C.<br />
Hodgson 1<br />
15. Inotropic effects of Tityus cambridgei scorpion venom on skeletal muscle.<br />
Borja-Oliveira C.R. 1,2 , Rodrigues-Simioni L. 2 , Spisni A. 1,3*<br />
16. Neutralization of the neurotoxicity of Micrurus altirostris (Uruguayan coral<br />
snake) venom by coral snake antivenom. Abreu, V.A. 1 , Leite, G.B. 1 , Borja-Oliveira,<br />
C.R. 1 , Hyslop, S. 1 , Furtado, M.F.D. 2 , Rodrigues-Simioni, L. 1*<br />
17. Peptide inhibitors of sPLA2 do not prevent the neuromuscular blocking actions<br />
of β-bungarotoxin in vitro. Harvey, A.L. 1 , Young, L.C. 1 , Gopalakrishnakone, P. 2 ,<br />
Thwin, M.M 2<br />
18. Gambierol markedly enhances evoked quantal acetylcholine release from motor<br />
nerve terminals at vertebrate skeletal neuromuscular junctions. Girard, E 1* , Benoit,<br />
E 1 , Sasaki, M 2 , Fuwa, H 2 , Cagide, E 3 , Louzao, M.C 3 , Botana, L.M 3 , Molgó, J 1<br />
19. Neutralization of neuromuscular activity of bothropstoxin-I (BthTX-I) by a<br />
methanolic fraction from Casearia sylvestris Sw. Francischinelli, M. C 1 , Gerenutti, M 1 ,<br />
Silva, M. G 1 , Andréo-Filho, N 1 , Oliveira, S. J 1 , Leite, G. B 3 , Dal Belo, C. A 3 , Cintra, A.<br />
C. O 4 , Cruz-Höfling, M. A 2 , Rodrigues-Simioni, L 3 , Oshima-Franco, Y 1,3*<br />
20. In vivo study of lethal effects of ostreolysin, a cytolysin from the edible oyster<br />
mushroom in rodents. Žužek, M.C 1 , Maček, P 2 , Sepčić, K 2 , Cestnik,V 1 , Frangež, R 1* .<br />
21. Effects of Bothrops marajoensis venom on the mouse <strong>and</strong> chick nerve-muscle<br />
preparations. Cavalcante, W.L.G 1 , Hern<strong>and</strong>ez-Oliveira, S 1 , Leite, G.B 1 , Ponce-Soto,<br />
L.A 2 , Marangoni, S 2 , Rodrigues-Simioni, L. 1*<br />
22. Purification <strong>and</strong> N- terminal sequence of two presynaptic neurotoxic PLA2,<br />
neuwieditoxin-I (NeuTX-I) <strong>and</strong> neuwieditoxin-II (NeuTX-II), from Bothrops<br />
neuwiedi pauloensis (jararaca-pintada) snake venom. Borja-Oliveira C.R. 1 , Kassab<br />
172<br />
B.H. 1 , Soares A.M. 2 , Toyama M.H. 1 , Giglio J.R. 3 , Marangoni S. 1 , Re L. 4 , Rodrigues-<br />
Simioni L. 1*<br />
23. The twitch potentiation induced by crotamine homologs from Crotalus durissus<br />
ruruima <strong>and</strong> C. d. terrificus venoms. Hern<strong>and</strong>ez-Oliveira S.S., Silva S.O., Borja-<br />
Oliveira C.R., Hyslop S., Marangoni S., Rodrigues-Simioni L.*<br />
24. Purification <strong>and</strong> Pharmacological Characterization of BtII-2 a Novel Presynaptic PLA2<br />
Isolated From Bothrops alternatus Venom. Ponce-Soto, L.A 1 ; Barros, JC 2 ; Hernadez-Oliveira, S 2 ,<br />
Novello, JC 1 , Dal Belo, CA 2 ; Marangoni, S 1 .; Rodrigues-Simioni, L 2* .<br />
25. Antiophidian Activity of Galactia glaucescens Ethanolic Extract (GGE) Against<br />
Crotalic <strong>and</strong> Bothropic Venoms. Colares, AV 4 ; Oshima-Franco, Y 1 ; Corrado, AP 2 ;<br />
Ticli, FK 3 ; Sampaio, SV 3 ; Cintra, ACO 3 ; Rodrigues-Simioni, L 1 .;dos Santos, MG 4 ; Dal<br />
Belo, CA 1,4,5* .<br />
26. Neutralization of snake venoms phospholipase A2 activities in mice<br />
neuromuscular preparation by aqueous extract of Casearia sylvestris<br />
(Flacourtiaceae). Campos, T.C 1 , Cavalcante W.L.G 1 , Pai-Silva M.D 1 , Pereira P.S. 2 ,<br />
Soares A.M 3 , Gallacci M 1* .<br />
Ion channels<br />
27. Expression <strong>and</strong> Mutagenesis of the Sea Anemone Toxin Av2 Reveals Key Amino<br />
Acid Residues Important for Activity on Voltage-Gated Sodium Channels. Moran<br />
Y*, Cohen L, Kahn R, Karbat I, Gordon D, Gurevitz M<br />
28. Characterization of Voltage-dependent Calcium Channel Blocker Peptides<br />
Isolated from Tarantula Venom. Ono, S., Kimura T., Kubo T.<br />
29. Recombinant expression of a four disulfide-bridged scorpion toxin antagonist of<br />
voltage-gated sodium channels. Estrada, G., García, B.I., Ortiz, E.,Riano, L., Becerril,<br />
B., Possani, L.D., Corzo, G.<br />
30. Structure <strong>and</strong> function of huwentoxin- , a novel spider toxin targeting both<br />
serine proteinase <strong>and</strong> potassium channels. Chunhua Yuan , Kuan Peng, Jianbo Diao<br />
<strong>and</strong> Songping Liang<br />
31. A novel family of conotoxins target the nACh-Receptors. Kauferstein, S. 1 , Nicke,<br />
A. 2 , Kendel, Y. 1 ., Possani, L.D. 3 , Stöcklin, R. 4 , Mebs, D. 1<br />
32. Isolation <strong>and</strong> characterisation of a potent novel tarantula toxin targeting the<br />
acid sensing ion channel ASIC1a. Rash LD 1 , Adams DJ 2 <strong>and</strong> Alewood PF 1 .<br />
173
33. Modulation of voltage-gated Na + <strong>and</strong> K + channels by pumiliotoxin 251D: a “joint<br />
venture” alkaloid from arthropods <strong>and</strong> amphibians. V<strong>and</strong>endriessche, T. 1 , Maertens,<br />
C. 1 , Abdel-Mottaleb, Y. 1 , Cuypers, E. 1 , Nubbemeyer, U. 2 , Mebs, D. 3 & Tytgat, J. 1*<br />
34. Structure-function relationships of Magi 4, a spider neurotoxin targeting insect<br />
<strong>and</strong> mammalian voltage-gated sodium channels. Little, M.J. 1 , Yamaji, N. 2 , Villegas,<br />
E. 3 , Corzo, G. 4 , Nicholson, G.M. 1*<br />
35. The natural anatoxin Amm VIII from Androctonus mauretanicus induces<br />
neutralizing antibodies against the most potent “Old-World” alpha-toxins. Martin-<br />
Eauclaire, M-F, Alami, M, Rosso, J-P. <strong>and</strong> Bougis, P.E*.<br />
36. Scorpion β-toxins: surveying the species-selectivity of five ‘classics’.<br />
Bosmans, F. 1 , Martin-Eauclaire, M.F. 2 , Tytgat, J. 1*<br />
37. Potent modulation of the voltage-gated sodium channel Nav1.7 by OD1, a toxin<br />
from the scorpion Odonthobuthus doriae. Maertens, C. 1* , Cuypers, E. 1 , Amininasab,<br />
M. 2 , Jalali, A. 3 , Vatanpour, H. 4 , Tytgat, J. 1<br />
38. Electrophysiological Characterization of Marine Snail Conus californicus<br />
Venom in Potassium Ion Channels. Juárez-Moreno, K. O. 1 , Prior-Mier y Teran A 1 ,<br />
García-Valdés, J. 2 , Aguilar M 3 , Morales E 1 , Waumann D 4 <strong>and</strong> Licea-Navarro, A. 1*<br />
39. GAMBIEROL-INDUCED CYTOSOLIC CALCIUM INCREASE IN HUMAN<br />
NEUROBLASTOMA CELLS. Cagide, E 1 , Louzao, M.C 1* , Vieytes, M.R 2 , Sasaki, M 3 ,<br />
Fuwa, H 3 , Yasumoto, T 4 , Botana, L.M. 1<br />
40. New modulator of P-type Ca 2+ -channels from Lycosa sp. venom. Pluzhnikov, K. 1 ,<br />
Korolkova, Y. 1* , Vassilevski, A. 1 , Nikolsky, A. 1 , Fisyunov, A. 2 , Tsintsadze, V. 2 ,<br />
Krishtal, O. 2 , Grishin, E. 1<br />
41. Evidence for a novel epitope for hERG channel blocking activity. Yousra<br />
Abdel-Mottaleb 1 , Praveen Kumar 1 , Brigitte Céard 2 , Pierre E. Bougis 2 , Marie-<br />
France Martin-Eauclaire 2 , Jan Tytgat 1*<br />
42. The first potassium channel toxin from the venom of the Iranian scorpion<br />
Odonthobuthus doriae. Yousra Abdel-Mottaleb 1 , Amir Jalali 2 , Elke Clynen 3 , Frank<br />
Bosmans 1 , Liliane Schoofs 3 , Hossein Vatanpour 2 , Jan Tytgat 1*<br />
43. Novel insect-selective neurotoxins from the venom of the tarantula Eucratoscelus<br />
longiceps target insect Kv channels. Escoubas, P. 1* , Lee, M. 2 , Ross, G. 2 , Lazdunski,<br />
M. 1 Nicholson, G.M. 2<br />
44. Short insecticidal toxins from the Asian scorpions Buthus martensi <strong>and</strong><br />
Mesobuthus tamulus modulate insect neuronal voltage-dependent <strong>and</strong> calcium-<br />
174<br />
activated chloride channels. Grolleau F 1 ., Stankiewicz M. 3 , Herrmann R. 4 , Moskowitz<br />
H. 4 , Rajendra W. 4 , Hammock B.D. 4 , Romi-Lebrun R. 5 , Wu F.Q. 6 , Nakajima T. 5 Escoubas<br />
P. 2,5*<br />
45. Phlotoxin 1, a toxin from tarantula venom, is a potent modulator of Nav1.7<br />
sodium channels <strong>and</strong> a potential analgesic. Escoubas P. 1* , Bosmans F. 2 , Cuypers E. 2 ,<br />
Diochot S. 1 , Mebs D. 3 , Craik D. 4 , Hill J. 4 , Maertens C. 2 , Nakajima T. 5 , Lazdunski M. 1 ,<br />
Tytgat J. 2<br />
46. ON SOME BIOPHYSICAL TRAITS OF COLUBRIDAE SNAKE VENOMS.<br />
Kazakov I., Abubakirova M.E., Reimbaeva R.S.<br />
47. THE EFFECT OF BOOPHILUS CALCARATUS VENOM ON BILAYER<br />
LIPID AND MITOCHONDRIAL MEMBRANES. Kazakov I., Rakhimova Sh.H.,<br />
Abubakirova M.E., Azimov D.A.<br />
48. THE EFFECT OF HELMINTH EXTRACT ON THE BILAYER LIPID AND<br />
MITHOCHONDRIAL MEMBRANES. I.Kazakov, M.E.Abubakirova, A.E.Kuchboev,<br />
D.A.Azimov<br />
49. ω-ACTX-Ar1a: a novel insect-selective voltage-gated calcium channel blocker<br />
from the venom of the Sydney funnel-web spider. Chong, Y 1* , Wen, S 1 , Hayes, J.L 1 ,<br />
Hodgson, W.C. 2 , Hains, P 3 , Broady, K.W 1 , King, G.F 4 , Nicholson, G.M 1<br />
50. The Effects of the Sodium Channel Blocker Lidocaine on Cardiovascular <strong>and</strong><br />
Respiratory Actions of the Yellow Scorpion Leiurus Quinquestriatus in Rabbits. Al-<br />
Shanawani, A. R * , Fatani, A.J.<br />
51. Postsynaptic neuromuscular activity of Cerastes cerastes cerastes crude snake<br />
venom. Soliman M. M 1 , Abu-Sinna G 1 , Abd-Elbaset A 2 , Harvey A.L 3 <strong>and</strong> Rowan,E.G 3<br />
52: The bioactive surface of the scorpion depressant toxin LqhIT2. Michael Turkov,<br />
Izhar Karbat, Lior Cohen, Roy Kahn, Dalia Gordon <strong>and</strong> Michael Gurevitz<br />
175
Poster 1: Ability of suramin to antagonize myotoxic <strong>and</strong> phospholipase activities of<br />
bee venom venom<br />
El Kik, C.Z. 1 , Fern<strong>and</strong>es, F.F.A. 1 , Sanches, F.V 1 .,Arruda, E.Z. 1 , Melo, P.A. 1<br />
1<br />
Departameto de Farmacologia Básica e Clínica, Universidade Federal do Rio de Janeiro-UFRJ 21941-<br />
590 Rio de Janeiro, RJ, Brazil.<br />
We evaluate the ability of suramin, a polyanion, to antagonize the phospholipase (PLA2)<br />
<strong>and</strong> myotoxicity activities of Apis mellifera venom. Myotoxicity in vitro was assed by<br />
perfusion of isolated mouse extensor digitorius longus (EDL) muscle exposed to the<br />
venom (25 μg/ml) <strong>and</strong> measured the rate of CK release. EDL muscle had the rate of CK<br />
release increased from 0.4 ± 0.1 (n=4) to 12.64 ± 2.3 U.g -1 .h -1 (n=4) after 60 min of venom<br />
exposition. When we added suramin to the bath solution (10 μM) we observed a significant<br />
reduction to 1.86 ± 0.6 U.g -1 .h -1 (n=4). The bee venom PLA2 activity, evaluated by the<br />
modified Marinetti’s method * , showed a concentration-dependent activity that was also<br />
inhibited by suramin (1-30 μM). Adult male mice weighting 20-25g were separated in 5<br />
groups . Control group received i.m. injection of physiological saline solution <strong>and</strong> treated<br />
groups received i.m. of venom(0.5 mg/Kg) alone or venom plus suramin(30 mg/kg)<br />
preincubated (30 min before),or by i.p. route 15 min. before or after the venom injection.<br />
Two hours after i.m. venom injection, with the anesthetized mice, the blood was collected<br />
in the orbital plexus, centrifuged <strong>and</strong> plasma CK activity was measured. The i.m. injection<br />
of bee venom induced an increase of plasma CK activity from 228.60 ±14.00 U/L to<br />
1158.0±124.0 U/L which was inhibited circa of 50% by suramin in the pre incubated<br />
protocol. At this same dose, pre or post-treatment did not protected against the myotoxic<br />
effect. Suramin effect seems to be due to the interaction of its charges with the polycations<br />
present in the bee venom.<br />
References: Marinetti, GV (1965) Biochem. Biophys. Acta, 98, 554-565.<br />
Supported by CNPq, FAPERJ, CAPES, PRONEX<br />
o Bee venom<br />
o Phospholipase activity<br />
o Myotoxicity<br />
o Suramin<br />
176<br />
Poster 2: Antimyotoxic effects of suramin <strong>and</strong> polyethylene glycol (PEG) against<br />
Agkistrodon contortrix laticinctus crude venom <strong>and</strong> its myotoxin<br />
Tomaz, M. A. 1 , Serafim, A.D. 1 , Williams, K 3 .,Murakami, M.T. 2 , Arni, R.K. 2 , Ownby, C.L. 4 ; Melo, P.A. 1<br />
1 Departamento de Farmacologia Básica e Clínica ICB, CCS, Universidade Federal do Rio de Janeiro,<br />
21941-590, Rio de, Janeiro, RJ, Brazil. pamelo@farmaco.ufrj.br.<br />
2 Departament of Physics IBILCE/UNESP, São José do Rio Preto, SP, Brazil<br />
3 Minority International Research Program, University of Maryl<strong>and</strong> at Baltimore, Baltimore, MD, USA<br />
4 Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, USA<br />
We assessed the effects of suramin <strong>and</strong> polyethylene glycol (PEG400) against the<br />
myotoxicity of Agkistrodon contortrix laticinctus (ACL) crude venom <strong>and</strong> its myotoxin on<br />
mouse isolated extensor digitorum longus (EDL) <strong>and</strong> neuromuscular diaphragm<br />
preparation. Isolated EDL were exposed to ACL crude venom <strong>and</strong> myotoxin (25 µg/mL)<br />
alone or together with suramin (10 <strong>and</strong> 30 µM) <strong>and</strong> PEG (10 <strong>and</strong> 30 mM). The rate of<br />
creatine kinase (CK) release from the muscles was measured, indicating tissue damage.<br />
Basal CK release from EDL was 0,43±0,06 U.g -1 .h -1 , <strong>and</strong> after 60 minutes of exposure to<br />
the myotoxin <strong>and</strong> treatments it increased to 13,76±3,76 (ACL myotoxin), 5,90±1,19 <strong>and</strong><br />
2,10±0,86 (myotoxin plus suramin 10 <strong>and</strong> 30 µM, respectively), <strong>and</strong> 8,87±3,63 <strong>and</strong><br />
5,80±2,04 (myotoxin plus PEG 10 <strong>and</strong> 30 mM, respectively). ACL crude venom (60 min)<br />
also induced an increase in the rate of CK release up to 32,07±5,43 U.g -1 .h -1 , which was<br />
significantly inhibited by PEG 100 mM (6,47±3,14 U.g -1 .h -1 ). Mouse neuromuscular<br />
diaphragm preparation was exposed to ACL crude venom <strong>and</strong> venom plus suramin. The<br />
preparation was directly (muscle) <strong>and</strong> indirectly (nerve) stimulated, <strong>and</strong> twitch amplitude<br />
was recorded. The snake venom (12,5 <strong>and</strong> 25 µg/mL) decreased twitch amplitude down to<br />
25% of the control in approximately 30 to 40 min in both protocols. Suramin prevented in<br />
a concentration-dependent fashion the venom’s effect (20, 25 <strong>and</strong> 50 μM caused a 5, 40<br />
<strong>and</strong> 100% inhibition on the depressing effect of ACL crude venom, respectively). Data<br />
show that suramin <strong>and</strong> PEG are able to prevent the myotoxic activities of ACL crude<br />
venom <strong>and</strong> its myotoxin in mouse isolated muscle <strong>and</strong> suramin prevents the neuromuscular<br />
effects of ACL crude venom.<br />
Research supported by CNPq, CAPES, FAPERJ, PRONEX, FAPESP, Fundação<br />
Universitária José Bonifácio (FUJB, UFRJ),<strong>and</strong> Fogarty Minority International Research<br />
Training Program.<br />
o Agkistrodom contortrix laticinctus venom<br />
o Myotoxicity <strong>and</strong> Suramin<br />
o Myotoxicity <strong>and</strong> polyethylene glycol<br />
177
Poster 3: Ability of a synthetic coumestan named LQB93 to antagonize some<br />
Bothrops snake venoms activity<br />
Melo, P. A. 1 Pinheiro, D. A 1 .; Fern<strong>and</strong>es, F. F. A. 1 ; Tomaz, M. A 1 .;Moraes, R. A. M. 1 ;<br />
Ricardo, H. D 1 . da Silva, A. J. M 2 . ; Costa, P. R. R 2 .<br />
1<br />
Departamento de Farmacologia Básica e Clínica, ICB<br />
2 Núcleo de Pesquisas de Produtos Naturais, NPPN, CCS; Universidade Federal do Rio de Janeiro-UFRJ<br />
21941-590, Rio de Janeiro RJ, Brazil<br />
In this work we studied the anti-ophidic effect of synthetic coumestan LQB93 against<br />
some activities of the venoms of Bothrops jararacussu <strong>and</strong> Bothrops jararaca in adult<br />
mice. The anti-myotoxic activity of the coumestan was evaluated by in vitro <strong>and</strong> in vivo<br />
tests. This substance, (30 μM), reduced 100% of the increase of creatine kinase (CK)<br />
release rate of isolated mouse extensor digitorum longus muscle induced by B.<br />
jararacussu (25 μg/ml) in vitro (IC50=0.0291 μM). In vivo experiments, B. jararacussu (1<br />
μg/g) venom was pre-incubated with LBQ93 (0.1-30 μg/g), during 30 min, for later<br />
injection in mice paw <strong>and</strong> evaluation of coumestan antimyotoxic <strong>and</strong> anti-edematogenic<br />
effects. The myotoxicity was evaluate by the increase of plasma CK activity <strong>and</strong> the paw<br />
edema was measured using the sliding caliper. The coumestan antimyotoxic <strong>and</strong> antiedematogenic<br />
effects were similar in both protocols <strong>and</strong> this effect was dose-dependent<br />
with ID50 of 0.1728 μg/g <strong>and</strong> 0.1434 μg/g, respectively. The hemorrhage, induced by the<br />
intradermal injection of B. jararaca (1 µg/g) venom in the mice skin, was evaluated<br />
measuring the optic density of the hemorrhagic area. LQB93 (10 µg/g), pre-incubated with<br />
venom, abolished the hemorrhagic effect. The pro-coagulating activity of B. jararaca (10<br />
µg) was analyzed by Lee-White modified method, <strong>and</strong> showed complete inhibition by<br />
LQB93, in the proportion of 30 µg coumestan per microgram of venom. This synthetic<br />
coumestan has the skill to inhibit the effects of two important Bothrops sp venoms<br />
reproducing some of the properties of wedelolactone a natural compound isolated from the<br />
plant Eclipta prostrata which is active against snake venoms.<br />
o Snake venoms<br />
o Antivenoms<br />
o Natural & Synthetic coumestan<br />
o Wedelolactone<br />
178<br />
Poster 4: Dexamethasone shows in vivo but not in vitro antimyotoxic effect against<br />
Bothrops jararacussu snake venon.<br />
Patrão-Neto, F.C. 1 ; Tomaz, M.A. 2 ; Calil-Elias, S. 2,3 , Melo, P.A. 2<br />
1<br />
Faculdade de Medicina de Campos, Campos dos Goytacazes, RJ - Brazil<br />
2<br />
Departamento de Farmacologia Básica e Clínica, Universidade Federal do Rio de Janeiro-UFRJ 21941-<br />
590 Rio de Janeiro RJ. Brazil<br />
3<br />
Departamento de Farmácia e Administração Farmacêutica, Faculdade de Farmácia, Universidade<br />
Federal Fluminense, Niterói, RJ, Brazil<br />
We studied the antivenom effect of Dexamethasone (DEXA) alone or its association with a<br />
crude extract of an antiophidic plant named * Eclipta prostrata (EP), in vitro <strong>and</strong> in vivo<br />
protrocols. On the in vivo experiments, a sham group of adult Swiss mice (25.0 ± 3.0 g)<br />
received 100 μL of saline solution (PSS), in a perimuscular injection close to extensor<br />
digitorum longus (EDL). Control group received B. jararacussu venom (1.0 mg/kg) alone.<br />
Treated groups received venom plus DEXA (1.0 mg/kg), a mixture of venom preincubated<br />
with EP (50 mg/kg), or a mixture of both agents. After 3 days we measured the<br />
creatine kinase (CK) muscular content <strong>and</strong> performed a histological analysis. For in vitro<br />
experiments, we analysed the rate of CK release from isolated mouse EDL muscles which<br />
were bathed in PSS <strong>and</strong> exposed to B. jararacussu venom (25 μg/ml) alone, the venom<br />
plus DEXA (25 μg/ml), or it plus EP (25-100 μg/ml) that was added to the bath. Venom<br />
injection reduced the EDL CK content (U/g) from 908.9±21.6 n=3 to 223.9±33.7 n=3.<br />
Venom plus DEXA limited this reduction to 566.2±73.8 n=3, while EP crude extract to<br />
712.3±44.4 n=3. The venom plus the mixture of DEXA+EP to 850.4±48.6 n=3, (U/g).<br />
These in vivo observations were confirmed by light microscopy examination. After 90<br />
min. of EDL in vitro exposure to B. jararacussu venom an increase of the rate of CK<br />
release from 1.06±0.09 to 37.5±3.5 U.g-1.h-1 n=7 was observed. Venom plus DEXA alone<br />
(39.7±0.4 n=3) did not reduce the venom effect, while EP at different concentrations, 25<br />
(16.5±1.7 U.g-1.h-1 n=3) or 100 μg/ml (2.4±0.8 U.g-1.h-1 n=3) inhibited the venom<br />
effect. These data confirm our previously observations that EP has an antimyotoxic effect<br />
<strong>and</strong> show that DEXA has in vivo ability to protect the muscle from B. jararacussu<br />
myotoxicity, but not in vitro.<br />
Melo, P.A. et al., (1994)Toxicon, 32, 595-603<br />
Supported by: FAPERJ; CAPES; CNPq; FUJB-UFRJ; PRONEX<br />
o Mouse EDL<br />
o Bothrops jararacussu venom<br />
o Myotoxicity,<br />
o Dexamethasone & Eclipta prostrata<br />
179
Poster 5: Improvement of mouse skeletal muscle regeneration by heparin after<br />
lesion induced by Bothrops jararacussu venom<br />
Calil-Elias, S., Tomaz, M. A., Costa, M.L., Mermelstein, C.L., Martinez;A. M. B.,<br />
Melo, P.A. pamelo@farmaco.ufrj.br<br />
1<br />
Departamento de Farmacologia Básica e Clínica, Universidade Federal do Rio de<br />
Janeiro, Rio de Janeiro, RJ. Brazil<br />
2<br />
Departamento de Histologia e Embriologia, Instituto de Ciências Biomédicas,<br />
Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ. Brazil<br />
3<br />
Departamento de Farmácia e Administração Farmacêutica, Faculdade de Farmácia,<br />
Universidade Federal Fluminense, Niterói, RJ, Brazil<br />
We examined the effect of treatment with heparin <strong>and</strong> polyvalent antivenom on the<br />
regeneration of mouse Extensor digitorum longus (EDL) muscle after damage by Bothrops<br />
jararacussu venom. The mice received i.v. treatment, with either low molecular weight<br />
heparin (LMWH), regular heparin (H) or polyvalent antivenom (PAV) at 15 minutes <strong>and</strong> 4<br />
hours after the venom injection. Twenty-one days later, the muscles were dissected <strong>and</strong><br />
processed for electron microscopy or for electrophoresis. The muscle total Creatine<br />
Kinase(CK) content was also evaluated 3 <strong>and</strong> 21 days after injury. The regenerated muscle<br />
without treatment showed a complete disorganization of myofibrils. The treatments with<br />
heparins induced a good regeneration, with well-organized myofibrils <strong>and</strong> recovery of the<br />
muscle total CK content was observed with LMWH. The electrophoresis showed a<br />
decrease of muscle myosin heavy chains in the muscle that received only the venom.<br />
However the treated muscles showed the protein b<strong>and</strong>s similar to the control muscle. It is<br />
concluded that heparins improves muscle regeneration.<br />
Calil-Elias, et al., 2002, Braz J Med Biol Res. 35:1233-1235 .<br />
Calil-Elias et al., 2002, Histol Histopathol. 17:463-70<br />
o Bothrops jararacussu venom<br />
o Myotoxicity<br />
o Muscle regeneration<br />
o Heparin<br />
180<br />
Poster 6: Mouse extensor digitorum longus <strong>and</strong> soleus have a different sensitivity to<br />
some snake venoms myotoxicity<br />
Tomaz, M. A 1 ., Calil-Elias, S 1,3 .,Martinez, A.M.B 2 ., Melo, P.A 1 .<br />
1<br />
Departamento de Farmacologia Básica e Clínica, Universidade Federal do Rio de Janeiro-UFRJ 21941-<br />
590 Rio de Janeiro RJ. Brazil<br />
2<br />
Departamento de Histologia e Embriologia, Instituto de Ciências Biomédicas, Universidade Federal do<br />
Rio de Janeiro, Rio de Janeiro, RJ. Brazil<br />
3<br />
Departamento de Farmácia e Administração Farmacêutica, Faculdade de Farmácia, Universidade<br />
Federal Fluminense, Niterói, RJ, Brazil<br />
Mouse extensor digitorum longus (EDL) <strong>and</strong> soleus (SOL), present distinct properties:<br />
EDL is a fast-twitch, white muscle with predominantly glycolotic fibers, while SOL is a<br />
slow-twitch, red muscle with predominantly oxidative fibers. These muscles were exposed<br />
to an in vitro preparation, to the venoms of Bothrops jararacussu, Agkistrodon contortrix<br />
laticinctus, Crotalus viridis viridis, Crotalus durissus terrificus (25 μg/ml).EDL<br />
presented higher increase of the rate of creatine kinase (CK) release than SOL. The basal<br />
CK release to the bathing solution was 0,43 ± 0,06 U.g -1 .h -1 for EDL <strong>and</strong> 0,29 ± 0,06 U.g -<br />
1 .h -1 for SOL. After 60 minutes of exposure to B. jararacussu venom, EDL <strong>and</strong> SOL<br />
presented CK release rates of, respectively, 13,2 ± 1,5 <strong>and</strong> 2,9 ± 0,7 U.g -1 .h -1 (p
Poster 7: αδ-Bungarotoxin, a new reversible long-chain neurotoxin from Malayan<br />
Krait (Bungarus c<strong>and</strong>idus) venom with high toxicity <strong>and</strong> site-selective binding at<br />
the muscle nicotinic acetylcholine receptor<br />
Kuch, U. 1* , Molles, B.E. 2 , Bergman, T. 3 , Cederlund, E. 3 , Alvelius, G. 3 , Jörnvall, H. 3 , Chanhome, L. 4 ,<br />
Omori-Satoh, T. 4 , Chen, Y.M. 5 , Samejima, Y. 6 , Warrell, D.A. 7 , Mebs, D. 1<br />
1 Zentrum der Rechtsmedizin, Klinikum der Johann Wolfgang Goethe-Universität, Kennedyallee 104, 60596<br />
Frankfurt am Main, Germany, *U.Kuch@em.uni-frankfurt.de 2 Unité de Récepteurs et Cognition, Département de<br />
Neuroscience, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris Cedex 15, France 3 Protein Analysis Center,<br />
Department of Medical Biochemistry <strong>and</strong> Biophysics, Karolinska Institutet, Scheeles vag 2, 17177 Stockholm,<br />
Sweden 4 Queen Saovabha Memorial Institute, The Thai Red Cross Society, Rama IV Road, Bangkok 10330,<br />
Thail<strong>and</strong> 5 Department of Pharmacology, College of Medicine, National Taiwan University, 1 Jen Ai Road, 1 st<br />
Section, Taipei, Taiwan 6 Institute of Medicinal Chemistry, Hoshi University, Ebara 2-4-41, Shinagawa-ku, Tokyo<br />
142-8501, Japan 7 University of Oxford, Nuffield Department of Clinical Medicine, John Radcliffe Hospital,<br />
Oxford OX3 9DU, UK<br />
Introduction: Studying the binding of selective peptide toxins to the two subunit<br />
interfaces (α-ε/γ <strong>and</strong> α-δ) serving as lig<strong>and</strong> binding sites of the nicotinic acetylcholine<br />
receptor (nAChR) has offered special opportunities to distinguish features on their<br />
surfaces, <strong>and</strong> their lig<strong>and</strong> specificity characteristics. We report a new snake neurotoxin, αδbungarotoxin<br />
(αδ-BgTx), that combines binding site selectivity <strong>and</strong> reversible binding with<br />
great structural similarity to the classic receptor probe α-bungarotoxin (α-BgTx).<br />
Methods: Isolation of αδ-BgTx from the venom of Malayan Kraits (Bungarus c<strong>and</strong>idus)<br />
from southern Thail<strong>and</strong> by gel-filtration-, cation-exchange-, <strong>and</strong> reversed-phase-HPLC;<br />
mass determination by SDS-PAGE <strong>and</strong> electrospray-ionization mass-spectrometry;<br />
complete amino acid sequence determination by Edman degradation; cloning <strong>and</strong><br />
nucleotide sequence analysis of the toxin-coding region of αδ-BgTx genes; LD50<br />
determination in mice; functional studies in the isolated chick biventer-cervicis nervemuscle<br />
preparation <strong>and</strong> by competition binding with 125 I-α-BgTx using mouse muscle<br />
nAChRs expressed in HEK293 cells.<br />
Results: The 73-amino acid, 8031 Da polypeptide chain of αδ-BgTx differs in only ten<br />
substitutions <strong>and</strong> one deletion from the Ala 31 variant of α-BgTx. Two additional isoforms<br />
of αδ-BgTx were identified by genomic DNA analysis. The toxicity of αδ-BgTx (LD50<br />
0.17–0.28 μg/g mouse, i.p. injection) is essentially as high as that of α-BgTx. αδ-BgTx<br />
completely abolishes the response to acetylcholine in the isolated chick biventer-cervicis<br />
nerve-muscle preparation, but in contrast to the block induced by α-BgTx, acetylcholine<br />
response is fully reversible. In addition, αδ-BgTx distinctly prefers the α-δ over the α-ε<br />
binding site interface of the mouse nAChR, whereas α-BgTx shows no such selectivity.<br />
Discussion: αδ-BgTx is the major postsynaptic toxin of Malayan Kraits from southern<br />
Thail<strong>and</strong>. Ancillary anticholinesterase treatment has proved beneficial in a victim of B.<br />
c<strong>and</strong>idus envenoming (Br Med J 1983; 286:678–80). A test dose is recommended but the<br />
most lethal toxins in the venom of these snakes are presynaptically-acting β-bungarotoxins.<br />
o Bungarus c<strong>and</strong>idus<br />
o neurotoxin<br />
o nicotinic acetylcholine receptor<br />
o Thail<strong>and</strong><br />
182<br />
Poster 8: Novel recombinant expression system for myonecrotic Lys49<br />
phospholipase A2 <strong>and</strong> its mutants identifying the functional residues for necrosis<br />
Tomohisa Ogawa 1, * , Minae Seto 1 , Koji Muramoto 1 , Motonori Ohno 2<br />
1 Graduate School of Life Sciences, Tohoku University, Sendai 981-8555 Japan,<br />
*ogawa@biochem.tohoku.ac.jp<br />
2 Department of Applied Life Science, Sojo University, Kumamoto 860-0082, Japan.<br />
Recombinant expression systems by E. coli are the most efficient widely used system for<br />
recombinant proteins production. With the advent of the post-genomic era, the necessity of<br />
the useful systems in E. coli are increased for analyzing functions of a growing number of<br />
genes from different organisms including venomous animals. However, many of these<br />
genes, especially venomous protein genes, severely interfere with the survival of E. coli<br />
cells. They lead to bacteria death or cause significant defects in bacteria growth or the<br />
expression efficiency. Fortunately, even if the bacteria expressed the recombinant proteins<br />
they are inactive form such as inclusion bodies.<br />
In the present study, we have developed the novel recombinant expression system using<br />
conger eel galectin, congerin, as a tag <strong>and</strong> chaperon protein in E.coli, <strong>and</strong> applied for the<br />
functional expression of myotoxic Lys49 phospholipase A2 (PLA2) from Protobothrops<br />
flavoviridis (Pf) <strong>and</strong> its mutants. Fused recombinant protein of Pf Lys49PLA2 <strong>and</strong><br />
congerin has been successfully expressed in soluble fraction with correct disulfide bond<br />
formation <strong>and</strong> folding. After removal digestion of tag protein by restricted protease, fully<br />
active recombinant Lys49PLA2 was obtained.<br />
Mutants of Pf Lys49PLA2, of which lysine residues at positions at 114, 118 <strong>and</strong> 119 in<br />
the C terminal region were substituted into alanine, were also prepared. These mutations<br />
decreased their necrotic activities, indicating that the C terminal basic lysine residues of Pf<br />
Lys49PLA2 are responsible for their necrotic activity.<br />
o myonecrosis<br />
o Phospholipase A2<br />
o Protobothrops flavoviridis<br />
o recombinant protein<br />
183
Poster 9: Non-cholinergic contractile activity of Dispholidus typus (Boomslang<br />
snake) in the rat isolated vas deferens<br />
Lumsden, N. G 1 , Hodgson, W. C *2 , Ventura, S 3 , Lewis, R. J 1<br />
1 Institute for Molecular Bioscience, The University of Queensl<strong>and</strong>, Brisbane, Queensl<strong>and</strong> 4072, Australia.<br />
2 Department of Pharmacology, Monash University, Clayton, Victoria 3800., Australia,<br />
* wayne.hodgson@med.monash.edu.au<br />
3 Department of Pharmaceutical Biology <strong>and</strong> Pharmacology, Victorian College of Pharmacy, Monash<br />
University, Parkville, Victoria 3052, Australia.<br />
Dispholidus typus venom has been reported to exhibit contractile activity in the rat<br />
isolated duodenum # . This effect was abolished by the muscarinic receptor antagonist<br />
atropine indicating cholinomimetic activity. The current study further examines the<br />
pharmacological activity of D. typus venom upon the rat (male Wistar 150-250 g) isolated<br />
vas deferens setup in 5 ml organ baths containing Krebs solution (NaCl, 118.4 mM; KCl,<br />
4.7 mM; MgSO4, 1.2 mM; KHPO4, 1.2 mM; CaCl2, 2.5 mM; NaHCO3, 25 mM <strong>and</strong><br />
glucose, 11.1 mM). Venom (0.01-50 µg/ml) produced concentration-dependant contractile<br />
activity in epididymal segments. The responses were transient (i.e. not sustained) with a<br />
slow onset, typically reaching peak amplitude within 10 min. As responses to venom (0.8<br />
μg/ml) were at least 3-fold greater in the epididymal segment than in the prostatic segment,<br />
further experimentation was carried out using the former preparation. Suramin (0.3 mM),<br />
but not atropine (1-10 μM) or prazosin (10 μM), significantly attenuated the contractile<br />
activity of the venom (0.8 μg/ml). However, P2X receptor desensitisation by α, β-mATP<br />
(10 μM) was without effect on venom responses (0.8 μg/ml) indicating that the blockade<br />
produced by suramin was unlikely to be due to antagonism of P2X receptors.<br />
Unremarkable anticholinesterase activity was noted since responses to the cholinesterase<br />
substrate, acetylcholine (1 mM), were not significantly augmented in the presence of<br />
venom (0.8 μg/ml). Nifedipine (1 μM) <strong>and</strong> the South African Institute of Medical Research<br />
(SAIMR) Boomslang antivenom (40 μl/ml) significantly attenuated responses to venom<br />
(0.8 μg/ml). These results suggest activation of L-type calcium channels by D. typus<br />
venom that is not mediated by purinergic, adrenergic or cholinergic receptors in the rat<br />
isolated vas deferens epididymal segment <strong>and</strong> therefore reveal additional smooth muscle<br />
activity to that previously reported.<br />
#<br />
Boomslang, smooth muscle, contraction, rat vas deferens<br />
184<br />
Poster 10: Equivalent effects of snake PLA2 neurotoxins <strong>and</strong> lysophospholipid–fatty<br />
acid mixtures<br />
Rigoni, M 1 Caccin, P 1 , Gschmeissner, S 2 , Koster, G 3 , Postle, A. D 3 ,Rossetto, O 1 , Schiavo, G 2 <strong>and</strong><br />
Montecucco, C 1<br />
1<br />
Department of Biomedical Sciences <strong>and</strong> Consiglio Nazionale Ricerche Institute of Neuroscience,<br />
University of Padova, Italy<br />
2<br />
Cancer Research UK, London Research Institute, London, UK<br />
3<br />
School of Medicine, University of Southampton, UK<br />
Corresponding author: cesare.montecucco@unipd.it<br />
Snake presynaptic phospholipase A2 neurotoxins (SPANs) paralyze the neuromuscular<br />
junction (NMJ): upon intoxication, the NMJ enlarges <strong>and</strong> has a reduced content of synaptic<br />
vesicles (1). Exposure of primary neuronal cultures to nanomolar concentrations of four<br />
different SPANs (beta-bungarotoxin, taipoxin, notexin <strong>and</strong> textilotoxin) induces a dosedependent<br />
formation of discrete bulges at various sites of neuronal projections. Neuronal<br />
bulging is paralleled by the redistribution of vesicle markers to the bulges <strong>and</strong> by the<br />
exposure of the lumenal domain of synaptotagmin I on the cell surface. Concomitantly,<br />
these neurotoxins induce glutamate release from cultured neurons <strong>and</strong> loss of FM 1-43 dye<br />
(2,3). These findings indicate that the observed phenotype results at least partially from the<br />
fusion of synaptic vesicles with the plasma membrane not balanced by an adequate<br />
membrane retrieval <strong>and</strong> closely resemble those occurring at neuromuscular junctions of<br />
intoxicated animals.<br />
The involvement of the enzymatic activity of these toxins in the NMJ blockade is still<br />
debated: indeed, there is only a partial correlation between PLA2 activity <strong>and</strong> neurotoxicity<br />
among the different SPANs <strong>and</strong> no overlap of surface residues required for neurotoxicity<br />
with those essential for PLA2 activity (4). We addressed this problem by comparing the<br />
effects of SPANs on the mouse NMJ hemidiaphragm preparation <strong>and</strong> on neurons in culture<br />
with those of their hydrolysis products: lysophospholipids (LysoPLs) <strong>and</strong> fatty acids<br />
(FAs). We found that an equimolar mixture of LysoPLs <strong>and</strong> FAs closely mimics all of the<br />
biological effects of SPANs (5). These results draw attention to the possible role of local<br />
lipid changes in synaptic vesicle release <strong>and</strong> provide new tools for the study of exocytosis.<br />
1. R.M. Kini, Ed., Venom Phospholipase A2 Enzymes (Wiley, Chichester, UK, 1997).<br />
2. M. Rigoni et al., J. Cell Sci. 15, 3561 (2004).<br />
3. D. Bonanomi et al., Mol. Pharmacol. 67, 1901 (2005).<br />
4. C.C. Yang, in Venom Phospholipase A2 Enzymes, R. M. Kini, Ed. (Wiley, Chichester, UK,1997), pp.<br />
185-204.<br />
5. M. Rigoni et al., Science 310, 1678 (2005).<br />
o neurotrasmission<br />
o PLA2 activity<br />
o snake neurotoxins<br />
o lysophospholipids <strong>and</strong> fatty acids<br />
185
Poster 11: EFFECT OF COLUBER RAVERGIERI VENOM ON SYNAPTIC<br />
PASSAGE<br />
Kazakov I., Abubakirova M.E.<br />
Institute of Zoology of Uzbek Academy of Sciences<br />
A. Niyazov Street 1, Tashkent 700095, Uzbekistan<br />
In the experiments on nerve-muscular synapses of frogs we established that the<br />
extract of Duvernoy , s gl<strong>and</strong> (venom) of Coluber ravergieri at the concentration of 100<br />
µg/ml caused a decrease in the amplitude of miniature endplate potential (MEPP). The<br />
amount of 185 µg/ml caused a complete inhibition of MEPP. It was established that the<br />
venom of Coluber ravergieri did not produce a significant effect on the miniature<br />
potential MP of muscle tissues, the frequency of MEPP <strong>and</strong> the quantum composition of<br />
endplate potential (EPP). These data suggest that the venom of Coluber ravergieri acts at<br />
the level of post-synaptic membrane.<br />
To confirm this, we studied the response to the microsuperfusion. In the norm, the<br />
superfusion of acetylcholine (100 mM) in the synapse caused a postsynaptic response at<br />
the 5mB amplitude. The addition of 185 µg/ml of venom into a solution washing the<br />
preparation caused a gradual drop in the amplitude of responses inflicted by<br />
microsuperfusion of acetylcholine, which completely disappeared within 20 min. The<br />
washing of the preparation for 30 min with a normal Ringer solution leads to a partial<br />
restoration of acetylcholine responses.<br />
Thus, data obtained suggest that the venom of Coluber ravergieri contains a<br />
component, which, like neurotoxins of cobra, interacting with choline receptors of<br />
postsynaptic membranes blocks them, which results in the violation of synaptic passage.<br />
186<br />
Poster 12: Effect of Duvernoy's gl<strong>and</strong> secretion of snake Coluber ravergieri on<br />
frog nerve-muscle preparation.<br />
Akhmedov K.J., Makhkamov B.T., R.S.Reimbaeva., M.E.Abubakirova., I.K.Kazakov<br />
Uzbek Academy of Science, Uzbekistan<br />
kdakhm@yahoo.com<br />
The effect of Duvernoy’s gl<strong>and</strong> secretion Coluber ravergieri Menet on<br />
neuromuscular transmission has been examined by means of twitch tension using<br />
the frog nerve-muscle preparations. Freshly dried crystal of Duvernoy's gl<strong>and</strong><br />
secretion 10 micrograms/ml, <strong>and</strong> more during 2-4 min reduced amplitude of<br />
electrically induced twitch response followed by irreversible blockade. The secret<br />
of gl<strong>and</strong>s has dose <strong>and</strong>-time dependant activity, 2 micrograms/ml blocked twitch<br />
tension in 10-15min, after 1h potassium-evoked contractures <strong>and</strong> direct stimulation<br />
also have been blocked irreversibly. During experiment we do not observed<br />
contraction or destruction of nerve-muscle preparation fibers. Following<br />
fractionation on a Sephacryl S-100 gives five peaks, but we lost potent blockage<br />
activity of Duvernoy’s gl<strong>and</strong> secretion. Only second peak with 20-30 kDa<br />
molecular weight has weak <strong>and</strong> slow blockage activity. By paralyzing nerve-muscle<br />
preparation the secret of Duvernoy’s gl<strong>and</strong> blocks along from nerve to muscle all<br />
transmission.<br />
187
Poster 13: Myotoxic activity from the venoms of six species of Micrurus.<br />
Néstor Lago 1 ; Adolfo R. de Roodt 2 ; Claudio Mirabelli 1 ; Rodrigo D. Laskowicz 2 ; Judith Estévez 3 ;<br />
Víctor M. Manzaneli 2 ; Jorge Paniagua 4 ; Eduardo Scarlatto 5 .<br />
1 GEMA-Biotech, Bs. As., Argentina.<br />
2 Instituto Nacional de Producción de Biológicos – A.N.L.I.S. “Dr. Carlos G. Malbrán”, Min. De Salud y<br />
Ambiente, Av. Vélez Sarsfield 563, CP 1281, Bs.As., Argentina.<br />
3 Instituto Bioclon, Mexico DF, Mexico.<br />
4 Laboratorios Silanes, Mexico DF, Mexico.<br />
5 Servicio de Toxicología, Hospital de Clínicas “José de San Martín”, Facultad de Medicina U.B.A.<br />
The lethal, hemolytic, phospholipasic <strong>and</strong> miotoxic activities of the venoms from M.<br />
altirrostris, M. mesopotamicus, M. pyrrhocryptus (the three species from Argentina), M.<br />
surinamensis (Colombia), M. nigrocinctus (Costa Rica) <strong>and</strong> M. fulvius (United States)<br />
were studied. The direct <strong>and</strong> indirect hemolytic activity was studied in a liquid system<br />
containing red blood cells with (indirect activity) or without (direct activity) egg yolk. The<br />
phospholipase activity was studied by radial hydrolysis of egg yolk in agarose plates. The<br />
miotoxicity was studied by the intramuscular injection of the venom in Whistar rats (250<br />
g). The levels of creatin quinase (CK) were recorded 2, 6 <strong>and</strong> 24 h after injection. Samples<br />
of muscles <strong>and</strong> kidneys were taken at 24 h post inoculation for pathological studies. None<br />
of the venoms showed direct hemolytic activity in the conditions of the study. The venoms<br />
of M. nigrocinctus <strong>and</strong> M. fulvius showed the highest indirect hemolytic activity, <strong>and</strong> the<br />
lower activity was detected in M. mesopotamicus <strong>and</strong> M. surinamensis venoms. All<br />
venoms showed high phospholipase activity, with the exception of the venom of M.<br />
surinamensis, which only showed hydrolysis at high concentrations. All the venoms<br />
caused elevation of the CK levels, being the highest level detected 6 hours after venom<br />
inoculation <strong>and</strong> descending by 24 h, but maintaining the level above that of the controls (p<br />
< 0.05); M. nigrocinctus, M. fulvius <strong>and</strong> M. pyrrhocryptus venoms showed the highest<br />
activity, whereas M. surinamensis <strong>and</strong> M. altirrostris showed the lowest activity.<br />
Histological analysis of muscles showed severe myofiber necrosis in rats injected with M.<br />
fulvius <strong>and</strong> M. nigrocintus venoms, <strong>and</strong> an important myonecrosis in those injected with<br />
M. pyrrhocryptus venom; the lesions caused by the other venoms were of minor<br />
importance or absent. The kidneys showed extensive tubular necrosis with protein deposits<br />
in rats injected with M. fulvius <strong>and</strong> M. nigrocinctus venoms. The other venoms produced<br />
isolated mild renal lesions, inclusive those with higher lethal doses. The envenomation by<br />
M. fulvius <strong>and</strong> M. nigrocinctus venoms caused macroscopic myoglobinuria in CF-1 mice<br />
(18-20 g).<br />
o Micrurus<br />
o Venom<br />
o Myotoxicity<br />
o nephrotoxicity<br />
188<br />
Poster 14: Development of an in vivo model for the study of snake venom<br />
myotoxicity<br />
Andrew J. Hart 1 , Geoffrey K. Isbister 1,2 , Sharmaine Ramasamy 1 , Wayne C. Hodgson 1<br />
1 Monash Venom Group, Department of Pharmacology, Monash University, Clayton, Australia,<br />
2 Topical Toxinology Unit, Menzies School of Health Research, Darwin, Northern Territory, Australia<br />
Introduction: Myotoxicity is a serious clinical manifestation of snake envenoming <strong>and</strong><br />
venoms with myotoxic activity occur in snakes from most parts of the world. Currently,<br />
there is no st<strong>and</strong>ard in vivo technique to investigate myotoxicity <strong>and</strong> its outcomes.<br />
Development of such a model will lead to further underst<strong>and</strong>ing of the pathophysiology of<br />
myotoxic envenoming <strong>and</strong> allow assessment of therapy. This study aimed to develop a<br />
suitable model. The whole venom <strong>and</strong> an isolated myotoxin (i.e. mulgatoxin) from the<br />
Australian Mulga snake (Pseudechis australis) were used as the test compounds due to well<br />
documented myotoxic activity (Chen et al., 1994; Ponraj & Gopalakrishnakone, 1995;<br />
Ramasamy et al., 2004).<br />
Method: Rats were anaesthetised with pentobarbitone sodium (60-100 mg/kg, i.p.). Blood<br />
pressure was monitored via the carotid artery, <strong>and</strong> blood samples (0.5 ml) taken via the<br />
jugular vein <strong>and</strong> collected in MiniCollect LH/Gel separation tubes. A blood sample was<br />
taken prior to venom/toxin administration <strong>and</strong> then every 2 h for a period of 8 h. after<br />
venom/toxin administration (i.e. a total of 5 samples per rat). Samples were tested for<br />
serum electrolytes (i.e. sodium, potassium, chloride <strong>and</strong> bicarbonate levels) <strong>and</strong> creatine<br />
kinase (CK) concentration.<br />
Results: P. australis venom (20 µg/kg, i.v., n=6) or mulgatoxin (30 µg/kg, i.v., n=4) both<br />
produced increases in CK concentrations, indicative of myotoxicity, which were<br />
significantly different from control (n=4) after 8 hours (one-way ANOVA, P
Poster 15: Inotropic effects of Tityus cambridgei scorpion venom on skeletal muscle<br />
Borja-Oliveira C.R. 1,2 , Rodrigues-Simioni L. 2 , Spisni A. 1,3*<br />
1<br />
Center for Structural Molecular Biology, Brazilian Synchrotron Light Laboratory-LNLS, Campinas-SP,<br />
Brazil,<br />
2<br />
Department of Pharmacology, State University of Campinas (UNICAMP), Cidade Universitária Zeferino<br />
Vaz, Campinas, SP, Brazil<br />
3<br />
Department of Experimental Medicine, University of Parma, Italy (permanent address),<br />
*alberto.spisni@unipr.it.<br />
Toxins that block voltage-dependent K + channels, such as TsTX-K-alpha (1), <strong>and</strong> those<br />
that modify Na + channel gating, such as crotamine (2) have inotropic effects in skeletal<br />
muscle. In order to screen the presence of some inotropic activity in crude venoms <strong>and</strong> to<br />
investigate the role of voltage-dependent Na + <strong>and</strong> K + channels in inotropism, we studied<br />
the neuromuscular effects of T. cambridgei <strong>and</strong> T. serrulatus venom, using isolated mouse<br />
phrenic nerve-diaphragm <strong>and</strong> chick biventer cervicis. A significantly different<br />
strengthening of indirect elicited twitches in phrenic nerve-diaphragm of 90 ± 8 % <strong>and</strong><br />
254.5 ± 7.6 % is induced by T. cambridgei venom, 5 μg/ml, <strong>and</strong> T. serrulatus venom, 0.5<br />
μg/ml, respectively. In the case of T. serrulatus venom this effect is prevented by the Na +<br />
channel blocker tetrodotoxin (TTX), 30 nM, <strong>and</strong> attenuated by the K + channel openers<br />
cromakalim (CRO), 200 µM <strong>and</strong> diclofenac (DIC), 5 μg/ml, while for T. cambridgei<br />
venom the effect is inhibited only by TTX <strong>and</strong> CRO. From the studied venoms, only T.<br />
cambridgei venom, 10 μg/ml, potentiated the direct evoked twitches of curarized<br />
preparations (d-tubocurarine 7.3 µM): this effect being prevented by TTX. When tested on<br />
biventer cervicis, T. serrulatus venom, 1 μg/ml, potentiated (26.0 ± 8.4 %), both the<br />
indirectly elicited twitches <strong>and</strong> the response to acetylcholine <strong>and</strong> KCl, after 90 min, while<br />
T. cambridgei venom, 10 μg/ml, partially inhibited the response to KCl. Overall, these<br />
results suggest that neuromuscular effects are associated to modifications of TTX-sensitive<br />
Na + channel gating, although blocking of K + channel may be involved as well.<br />
Noteworthy, while T. serrulatus venom seems to improve muscle strength by interacting<br />
mainly with nerve ion channels, T. cambridgei venom appears to act directly on skeletal<br />
muscle ion channels thus suggesting it contains active factors potentially relevant for<br />
clinical applications. Partially support by CNPq.<br />
1. van Lunteren E, Moyer M. (1999). J. Appl. Phisiol, 86, 1009-16.<br />
2. Toyama MH, Marangoni S, Novello JC, Leite GB, Prado-Franceschi J, Cruz-Hofling<br />
MA, Rodrigues-Simioni L (2003). Toxicon, 41, 493-500.<br />
o Phrenic nerve-diaphragm preparation<br />
o Chick biventer cervicis preparation<br />
o Neuromuscular transmission<br />
o Skeletal muscle force<br />
190<br />
Poster 16: Neutralization of the neurotoxicity of Micrurus altirostris (Uruguayan<br />
coral snake) venom by coral snake antivenom<br />
Abreu, V.A. 1 , Leite, G.B. 1 , Borja-Oliveira, C.R. 1 , Hyslop, S. 1 , Furtado, M.F.D. 2 , Rodrigues-Simioni, L. 1*<br />
1<br />
Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas<br />
(UNICAMP), Campinas, SP, Brazil, *simioni@unicamp.br<br />
2<br />
Laboratório de Herpetologia, Instituto Butantan, Avenida Vital Brazil, 1500, São Paulo, SP, Brazil.<br />
Coral snake (Micrurus) venoms have a high lethality mediated by pre- <strong>and</strong> postsynaptic<br />
neurotoxins that can be neutralized by antivenom. However, the lethality of Micrurus<br />
altirostris venom is not neutralized by commercial antivenom (Instituto Butantan) raised<br />
against M. corallinus <strong>and</strong> M. frontalis venoms [4]. In this work, we examined the<br />
neuromuscular activity of M. altirostris venom in vitro <strong>and</strong> assessed the ability of<br />
antivenom to neutralize the neurotoxicity in vitro <strong>and</strong> in vivo. The neurotoxicity of M.<br />
altirostris venom (0.1-10 μg/ml) <strong>and</strong> its neutralization by antivenom were assayed using<br />
indirectly stimulated chick biventer cervicis [1] <strong>and</strong> mouse phrenic nerve-diaphragm [2]<br />
preparations. Neutralization in vivo was tested in chicks (LD50 0.042 mg/kg) <strong>and</strong> mice<br />
(LD50 0.255 mg/kg [4]) injected i.m. <strong>and</strong> i.p., respectively, with venom (5 LD50):<br />
antivenom mixtures (n=6 animals/group). The venom:antivenom ratios used in all assays<br />
were 1:1, 1:2.5, 1:5, 1:10 <strong>and</strong> 1:20, assuming that 1 ml of antivenom neutralizes 1.5 mg of<br />
Micrurus spp. venom (antivenom package insert). M. altirostris venom produced total<br />
neuromuscular blockade in both nerve-muscle preparations at all concentrations tested.<br />
The blockade was not reversed by washing or by neostigmine. In chick preparations, the<br />
venom totally blocked the contracture to exogenous ACh without affecting the response to<br />
KCl. Antivenom failed to neutralize the neuromuscular blockade in the proportion<br />
recommended by the manufacturer. Only the venom:antivenom ratio of 1:20 provided total<br />
neutralization of the neuromuscular blockade in vitro <strong>and</strong> protected chicks <strong>and</strong> mice<br />
against 5 LD50 of venom. M. altirostris venom is highly neurotoxic <strong>and</strong> irreversibly blocks<br />
neuromuscular transmission. Commercial antivenom showed low efficacy in neutralizing<br />
this neurotoxicity. Hence, there may be a need for more specific antivenoms to neutralize<br />
the activities of Brazilian coral snake species other than M. corallinus <strong>and</strong> M. frontalis [3].<br />
1. Bülbring E (1946) Br. J. Pharmacol. 1, 38-61. 2. Ginsborg BL, Warriner J (1960) Br. J.<br />
Pharmacol. 15, 410-411. 3. Higashi HG et al. (1995) Braz. J. Med. Biol. Res. 28, 767-771.<br />
4. Moraes FV et al. (2003) Toxicon 41, 71-79. Supported by: CNPq.<br />
o chick biventer cervicis<br />
o lethality<br />
o mouse phrenic nerve-diaphragm<br />
o neuromuscular junction<br />
191
Poster 17: Peptide inhibitors of sPLA2 do not prevent the neuromuscular blocking<br />
actions of β-bungarotoxin in vitro<br />
Harvey, A.L. 1 , Young, L.C. 1 , Gopalakrishnakone, P. 2 , Thwin, M.M 2<br />
1 Department of Physiology <strong>and</strong> Pharmacology, University of Strathclyde, Glasgow G4 0NR, UK, <strong>and</strong><br />
2 Venom & Toxin Research Programme, Department of Anatomy, Yong Loo Lin School<br />
of Medicine, National University of Singapore, Singapore117597<br />
The objective of this study was to determine whether phospholipase A2 (sPLA2-IIA)<br />
inhibitory peptides, derived from the primary structure of an endogenous protein termed<br />
“Phospholipase Inhibitor from Python (PIP)” could prevent the neuromuscular paralysis<br />
induced by β-bungarotoxin. Experiments were conducted on the chick biventer cervicis<br />
isolated nerve-muscle preparation. Two types of experiments were performed: 1. peptides<br />
were premixed with β-bungarotoxin before adding to the preparations, <strong>and</strong> 2. peptides<br />
were tested in experiments where they were added to the preparations 30 min before the<br />
toxin.<br />
In premixing experiments, a mixture containing 5 μM of each peptide with 25 nM βbungarotoxin<br />
was made <strong>and</strong> kept at room temperature for 30 min before adding to<br />
indirectly stimulated chick biventer cervicis preparations. Eight experimental preparations<br />
were run simultaneously with four time-matched preparations that received toxin only.<br />
There were no significant differences in time to block in any of the experimental sets<br />
compared with controls with toxin.<br />
In preparations that had been incubated with peptides before adding toxin, there was no<br />
effect of any peptide on the blocking action of β-bungarotoxin.<br />
Contractures to exogenously added acetylcholine were greater in the presence of some of<br />
the peptides. This could be explained by an anticholinesterase effect, <strong>and</strong> this was<br />
confirmed by biochemical assays.<br />
o secretory phospholipase A2 inhibitor<br />
o β-bungarotoxin<br />
o neuromuscular<br />
o acetylcholinesterase<br />
192<br />
Poster 18: Gambierol markedly enhances evoked quantal acetylcholine release<br />
from motor nerve terminals at vertebrate skeletal neuromuscular junctions<br />
Girard, E 1* , Benoit, E 1 , Sasaki, M 2 , Fuwa, H 2 , Cagide, E 3 , Louzao, M.C 3 , Botana, L.M 3 , Molgó, J 1<br />
1<br />
CNRS, Institut de Neurobiologie Alfred Fessard – FRC2118, Laboratoire de Neurobiologie<br />
Cellulaire et Moléculaire – UPR9040, Gif sur Yvette, France, *girard@nbcm.cnrs-gif.fr<br />
2<br />
Graduate School of Life Sciences, Tohoku University, 1-1 Tsutsumidoori-Amamiya, Aoba-ku, Sendai,<br />
Japan<br />
3 Departamento de Farmacologia, Facultad de Veterianaria. Universidad de Santiago de Compostela.<br />
Campus de Lugo, Lugo, Spain<br />
Gambierol is a marine polyether toxin isolated with ciguatoxin congeners from cultures of<br />
the toxic dinoflagellate Gambierdiscus toxicus. To date, two total syntheses of gambierol<br />
have been accomplished. Gambierol has shown toxicity against mice with symptoms<br />
resembling those of the ciguatoxins (CTXs), inferring the possibility that gambierol is<br />
involved in ciguatera, a worldwide-spread food poisoning caused by the consumption of<br />
fish contaminated with toxins produced by G. toxicus. Our previous observations suggest<br />
that CTXs induce neurological disturbances by acting at low concentrations on voltagegated<br />
Na + channels, <strong>and</strong> at higher concentrations on voltage-dependent K + channels in<br />
excitable tissues. The aim of the present study was to explore the effects of gambierol on<br />
isolated frog cutaneous pectoris <strong>and</strong> mouse phrenic-hemidiaphragm nerve-muscle<br />
preparations, using conventional electrophysiological techniques. Gambierol in the<br />
nanomolar range greatly increased the quantal content of endplate potentials (EPPs) in<br />
isolated neuromuscular preparations equilibrated in a low-Ca 2+ -high Mg 2+ medium. In<br />
preparations in which gambierol produced a 120-fold increase in evoked quantal<br />
acetylcholine (ACh) release, no change was detected in the muscle membrane potential.<br />
Spontaneous quantal ACh release recorded as miniature endplate potential frequency<br />
remained unaffected by sub-micromolar concentrations of gambierol. Gambierol (100 nM-<br />
1 μM) evoked asynchronous nerve action potentials that elicited EPPs which in turn<br />
triggered muscle action potentials <strong>and</strong> muscle contractions. In the light of the data<br />
obtained, gambierol is suggested to block K + channels in motor nerve terminals allowing a<br />
greater phasic Ca 2+ influx into the endings. This greater Ca 2+ influx during the presynaptic<br />
action potential is responsible for the increase in evoked quantal ACh release. In<br />
conclusion, our data show for the first time that gambierol is a potent modulator of evoked<br />
ACh release <strong>and</strong> that its actions at the neuromuscular junction differ from those previously<br />
reported with the CTXs. Gambierol appears to be about 1,000 times more active than 3,4diaminopyridine,<br />
a well known blocker of K + channels in motor nerve terminals.<br />
o Gambierol<br />
o acetylcholine release<br />
o nerve terminals<br />
o ciguatoxins<br />
193
Poster 19: Neutralization of neuromuscular activity of bothropstoxin-I (BthTX-I)<br />
by a methanolic fraction from Casearia sylvestris Sw.<br />
Francischinelli, M. C 1 , Gerenutti, M 1 , Silva, M. G 1 , Andréo-Filho, N 1 , Oliveira, S. J 1 , Leite, G. B 3 , Dal<br />
Belo, C. A 3 , Cintra, A. C. O 4 , Cruz-Höfling, M. A 2 , Rodrigues-Simioni, L 3 , Oshima-Franco, Y 1,3*<br />
1 University of Sorocaba, Sorocaba, SP, Brazil, *yofranco@terra.com.br<br />
2 Dept .of Histology <strong>and</strong> Embryology, IB, <strong>and</strong> 3 Dept. Pharmacology, Faculty of Medical Sciences, State<br />
University of Campinas, (UNICAMP), SP, Brazil,<br />
4 Faculty of Medicine, University of São Paulo, Ribeirão Preto, SP, Brazil.<br />
Introduction: Bothropstoxin-I (BthTX-I), a PLA2 myotoxin from Bothrops jararacussu<br />
snake venom, shows neurotoxic activity in vitro, that is prevented by a hydroalcoholic<br />
extract of Casearia sylvestris Sw. (CS). Since antivenom therapy is efficient against the<br />
systemic, but not the local actions of B. jararacussu venom, we examined the ability of a<br />
methanolic fraction (MF) of CS to neutralize the myotoxicity of BthTX-I in mouse phrenic<br />
nerve-diaphragm preparations (PND).<br />
Methods: A methanolic fraction of CS leaves was obtained by the Soxhlet procedure <strong>and</strong><br />
its constituents were visualized by thin layer chromatography. Myographic <strong>and</strong> histological<br />
analyses were done in PND incubated with BthTX-I (40 μg/mL), a mixture of BthTX-I +<br />
MF (0.2 mg/mL), MF alone (0.2 mg/mL) or Tyrode solution (control) for 120 min.<br />
Student’s t-test was used for statistical analyses with p
Poster 21: Effects of Bothrops marajoensis venom on the mouse <strong>and</strong> chick nervemuscle<br />
preparations<br />
Cavalcante, W.L.G 1 , Hern<strong>and</strong>ez-Oliveira, S 1 , Leite, G.B 1 , Ponce-Soto, L.A 2 , Marangoni, S 2 , Rodrigues-<br />
Simioni, L. 1*<br />
1<br />
University of Campinas, UNICAMP, Department of Pharmacology, 13084-971,Campinas, Brazil,<br />
*simioni@unicamp.br<br />
2<br />
University of Campinas, UNICAMP, Department of Biology, 13083-970, Campinas, Brazil<br />
Introduction: The venoms of some Bothrops species produce neuromuscular blockade in<br />
avian <strong>and</strong> mammalian nerve-muscle preparations in vitro. In this study, we investigated the<br />
neuromuscular activities (neurotoxicity <strong>and</strong> myotoxicity) of Bothrops marajoensis venom<br />
(Bmj) in mouse phrenic nerve-diaphragm muscle (PNDp) <strong>and</strong> chick biventer cervicis<br />
muscle preparations (BCp).<br />
Methods: The preparations were mounted in a 5 ml organ bath (PNDp - Tyrod solution;<br />
BCp - Krebs solution), aerated (95% O2 <strong>and</strong> 5% CO2) at 37 ºC for indirectly stimulation.<br />
Myotoxicity was assessed by light microscopic analysis. Data (mean ± S.E.M.; n=4-6)<br />
were analyzed by ANOVA (p
Poster 23: The twitch potentiation induced by crotamine homologs from Crotalus<br />
durissus ruruima <strong>and</strong> C. d. terrificus venoms<br />
Hern<strong>and</strong>ez-Oliveira S.S., Silva S.O., Borja-Oliveira C.R., Hyslop S., Marangoni S., Rodrigues-Simioni<br />
L.*<br />
State University of Campinas (UNICAMP), Campinas, SP, Brazil, *simioni@unicamp.br<br />
Several subspecies of the tropical rattlesnake, Crotalus durissus, occur in Brazil. In this<br />
work, we compared the neuromuscular activities of Crotalus durissus terrificus, C. d.<br />
ruruima, C. d. cascavella, <strong>and</strong> C. d. collilineatus venoms <strong>and</strong> their respective crotoxin<br />
(CrTX) homologs, besides crotamine (CrTM) homologs from C. d. terrificus <strong>and</strong> C. d.<br />
ruruima (crotamine-positive venoms), in indirectly stimulated mouse phrenic nervediaphragm<br />
<strong>and</strong> chick biventer cervicis preparations. CrTX <strong>and</strong> CrTM were isolated from<br />
C. d. ruruima venom by chromatography on Sephadex G-75 followed by reverse-phase<br />
HPLC. All of the venoms <strong>and</strong> their crotoxin homologs (10 μg/ml each) caused complete<br />
neuromuscular blockade within 120 min in both preparations. However, only C. d.<br />
terrificus <strong>and</strong> C. d. ruruima venoms (10 μg/ml each) produced an initial increase in twitchtension<br />
(187 ± 40% <strong>and</strong> 167 ± 29%, respectively; mean+S.E.M., n=7 <strong>and</strong> 6, respectively)<br />
in mouse preparations. Crotamine alone (10 μg/ml) from C. d. terrificus <strong>and</strong> C. d. ruruima<br />
venoms caused an initial facilitation (167 ± 38%, n=8, <strong>and</strong> 163± 23%, n=11, respectively,<br />
in mouse preparations, <strong>and</strong> 37 ± 12% <strong>and</strong> 21 ± 7%, respectively, in chick preparations, n=5<br />
each). The facilitation were not significantly different when crotoxin was added with<br />
crotamine. In mouse <strong>and</strong> chick preparations, crotoxin from the four subspecies caused<br />
complete <strong>and</strong> irreversible neuromuscular blockade within 60-70 min <strong>and</strong> 15-30 min,<br />
respectively. None of the crude venoms or crotoxin <strong>and</strong> crotamine homologs inhibited<br />
ACh- or KCl-induced contractures in chick preparations. Since C. d. terrificus <strong>and</strong> C. d.<br />
ruruima are the only crotamine-positive venoms within the studied venoms, we suggest<br />
that the pronounced <strong>and</strong> initial facilitatory effect of these venoms is induced by crotamine.<br />
In addition, these results show that crotoxin homologs from studied venoms <strong>and</strong> crotamine<br />
homologs from C. d. terrificus <strong>and</strong> C. d. ruruima have very similar neuromuscular effects.<br />
We conclude that the twitch potentiation followed by neuromusculau blockade caused by<br />
the crude venoms studied, was due the presence of crotamine.<br />
Supported by CNPq.<br />
o Crotalus durissus cascavella<br />
o Crotalus durissus collilineatus<br />
o Crotoxin<br />
o Nerve-muscle preparations<br />
198<br />
Poster 24: Purification <strong>and</strong> Pharmacological Characterization of BtII-2 a Novel Presynaptic<br />
PLA2 Isolated From Bothrops alternatus Venom<br />
Ponce-Soto, L.A 1 ; Barros, JC 2 ; Hernadez-Oliveira, S 2 , Novello, JC 1 , Dal Belo, CA 2 ; Marangoni, S 1 .; Rodrigues-Simioni,<br />
L 2* .<br />
1<br />
State University of Campinas, UNICAMP, Department of Biology, 13083-970, Campinas-SP, Brazil.<br />
2<br />
State University of Campinas, UNICAMP, Department of Pharmacology, 13084-971, Campinas-SP,<br />
Brazil.*simioni@unicamp.br<br />
Introduction: BtII-2 is a novel presynaptic PLA2 (K49) isolated from Bothrops alternatus<br />
snake venom.<br />
Methods: Toxin was purified using reverse phase HPLC <strong>and</strong> its molecular mass was<br />
estimated by SDS-PAGE <strong>and</strong> MALDI-TOF mass spectrometry. BtII-2 was assayed at<br />
mouse hemidiaphragm preparations (PND) <strong>and</strong> at chick biventer cervicis preparations<br />
(CBC) mounted as described by Cogo et al. (1993). Quantal content of end-plate potentials<br />
(QC) was determined according to Dal Belo et al. (2005) using mouse hemidiaphragm<br />
preparations.<br />
Results: BtII-2 has a molecular mass of 13898.7121 Da. The first eight residues of the N-terminal<br />
sequence were SLFELGKMILQETGKNPAKS YGAYYCYC… sharing high homo-<br />
logy with other Lys49 PLA2. The high content of Lys, Tyr, Gly, Pro <strong>and</strong> 14 half-Cys resi-<br />
dues indicated to be a basic PLA2. At (CBC) BtII-2 (0.1-20 µg/ml) induced an irreversible<br />
<strong>and</strong> concentration time-dependent blockade of neurotransmission (n=6, p
Poster 25: Antiophidian Activity of Galactia glaucescens Ethanolic Extract (GGE)<br />
Against Crotalic <strong>and</strong> Bothropic Venoms<br />
Colares, AV 4 ; Oshima-Franco, Y 1 ; Corrado, AP 2 ; Ticli, FK 3 ; Sampaio, SV 3 ; Cintra, ACO 3 ; Rodrigues-<br />
Simioni, L 1 .;dos Santos, MG 4 ; Dal Belo, CA 1,4,5* .<br />
1 State University of Campinas, UNICAMP, Department of Pharmacology, 13084-971, Campinas-SP,<br />
Brazil.<br />
2 University of São Paulo, USP, Department of Pharmacology, Avenida B<strong>and</strong>eirantes 3900, 14049-900,<br />
Ribeirão Preto-SP, Brazil.<br />
3 University of São Paulo, USP, Department of Clinical <strong>and</strong> Bromatological Analysis, Avenida do Café s/n,<br />
14040-903, Ribeirão Preto-SP, Brazil.<br />
4 Federal University of Tocantins, UFT, PGCIAMB, Avenida NS 15, ALCNO 14, 77020-120, Palmas-TO,<br />
Brazil. *chariston@uft.edu.br<br />
5 Instituto de Ensino Superior de Porto Nacional, IESPEN, Rua Antônio Ayres Primo 2071, 77500-000,<br />
Porto Nacional, Tocantins, Brazil.<br />
Introduction: A crude GGE was tested on neurotoxicity, oedema <strong>and</strong> hemorrhagic<br />
activities induced by crotalic <strong>and</strong> bothropic venoms in mouse preparations.<br />
Methods: All protocols were performed using male Swiss white mice (18-22g). To the<br />
neurotoxicity assays mice were anaesthetized (chloral hydrate 300 mg/kg, i.p.) <strong>and</strong><br />
sacrificed by exsanguination. Phrenic nerve-hemidiaphragm preparations (PND) were<br />
mounted as described by Bülbring (1946) for rats. Oedema inhibition studies were<br />
performed after i.d. injection in the right foot pad (Soares et al., 2000a) by incubating a<br />
PLA2 Bothropstoxin I (BthTX I) from Bothrops jararacussu venom with GGE. Control<br />
groups received an injection of PBS (50µl), pH7.2, or GGE alone. The progression of<br />
oedema was evaluated with a low-pressure pachymeter (Mitutoyo, Japan) at various time<br />
intervals. Hemorrhagic activity was measure according Kondo et al. (1960).<br />
Results: GGE (10 µg/25ml, n=5) inhibited by 50% oedema formation induced by BthTX I<br />
(1µg/25ml, p
Poster 27: Expression <strong>and</strong> Mutagenesis of the Sea Anemone Toxin Av2 Reveals<br />
Key Amino Acid Residues Important for Activity on Voltage-Gated Sodium<br />
Channels<br />
Moran Y*, Cohen L, Kahn R, Karbat I, Gordon D, Gurevitz M<br />
Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv,<br />
Israel, *moranyeh@post.tau.ac.il<br />
Type I sea anemone toxins are highly potent modulators of voltage-gated Na-channels<br />
(Navs) <strong>and</strong> compete with the structurally dissimilar scorpion alpha-toxins on binding to<br />
receptor site-3. While these features provide two structurally different probes for studying<br />
this receptor site <strong>and</strong> channel fast inactivation, the bioactive surface of sea anemone toxins<br />
has not been fully resolved. We established an efficient expression system for Av2 (known<br />
as ATX II), a highly insecticidal sea anemone toxin from Anemonia viridis (named<br />
previously A. sulcata), <strong>and</strong> mutagenized it throughout. Each toxin mutant was analyzed in<br />
toxicity <strong>and</strong> binding assays, as well as by circular dichroism spectroscopy to discern<br />
effects derived from structural perturbation from those related to bioactivity. Six residues<br />
were found to constitute the anti-insect bioactive surface of Av2 (Val2, Leu5, Asn16,<br />
Leu18, Ile41). Further analysis of nine Av2 mutants on the human heart channel Nav1.5<br />
expressed in Xenopus oocytes indicated that the bioactive surfaces toward insects <strong>and</strong><br />
mammals practically coincide, but differ from the bioactive surface of a structurally similar<br />
sea anemone toxin, Anthopleurin B, from Anthopleura xanthogrammica (1). Hence, not<br />
only that our results demonstrate clear differences in the bioactive surfaces of Av2 <strong>and</strong><br />
scorpion alpha-toxins, they also indicate that despite the general conservation in structure<br />
<strong>and</strong> importance of the Arg14 loop <strong>and</strong> its flanking residues Gly10 <strong>and</strong> Gly20 for function,<br />
the surface of interaction between different sea anemone toxins <strong>and</strong> Navs varies (2).<br />
References<br />
1. Seibert AL, Liu J, Hanck DA & Blumenthal KM (2004) Biochemistry 43, 7082-7089<br />
2. Moran Y, Cohen L, Kahn R, Karbat I, Gordon D & Gurevitz M (2006) Biochemistry, In<br />
press.<br />
o Sea anemone toxin<br />
o Voltage-gated sodium channels<br />
o Molecular dissection<br />
o High insecticidal activity<br />
202<br />
Poster 28: Characterization of Voltage-dependent Calcium Channel Blocker<br />
Peptides Isolated from Tarantula Venom<br />
Ono, S., Kimura T., Kubo T.*<br />
National Institute of Advanced Industrial Science <strong>and</strong> Technology (AIST), 1-1-1 Higashi, Tsukuba,<br />
Ibaraki 305-8566, Japan, *tai.kubo@aist.go.jp<br />
Introduction<br />
Voltage-dependent ion channels control transmembrane ion flux via electrical excitability.<br />
Some of the neurological <strong>and</strong> cardiac diseases, such as epilepsy, arrhythmia <strong>and</strong><br />
hypertension, are caused by dysfunction of ion channels. Toxins that recognize ion<br />
channel subtypes are versatile tools for channel studies, <strong>and</strong> thus contribute to drug<br />
discovery. Spider venoms contain arthropod-specific neurotoxins <strong>and</strong> are expected to be a<br />
rich source of ion channel blockers. In this study, we screened the tarantula venom to<br />
identify calcium channel blocker peptides that specifically recognize a variety of the<br />
channel subfamilies.<br />
Methods<br />
Crude venom was collected from the venom gl<strong>and</strong>s of tarantula Grammostola spatulata.<br />
The supernatant was separated on a C18 reversed-phase liquid chromatography. Voltagedependent<br />
calcium channels, Cav1.2, Cav2.1, Cav2.2, Cav2.3 <strong>and</strong> Cav3.1, were expressed in<br />
Xenopus oocytes <strong>and</strong> current modulations by the separated fractions were measured in twoelectrode<br />
voltage clump configuration. Purified active peptides were sequenced by Edman<br />
degradation after alkylation.<br />
Results<br />
Three peptides were identified that inhibit different types of calcium channel. One of the<br />
peptides (35 amino acids) suppressed the Cav2.1 (P/Q-type) current. The sequence is<br />
identical with that of omega-GsTx SIA except valine residue in the C-terminus. The<br />
second peptide (known as GsAFII) with 30 amino acid residues blocked L-type Ca 2+<br />
channel (Cav1.2). We also isolated a novel peptide, which specifically targets the T-type<br />
Ca 2+ channel (Cav3.1). The peptide has sequence homology with sodium channel blocker<br />
peptides. These spider venom peptides so far identified have different structural features<br />
with different cysteine motifs, as characterized as the Inhibitory Cystine Knot structure.<br />
o Voltage-dependent calcium channel<br />
o Spider venom<br />
o Channel blocker<br />
o Peptide<br />
203
Poster 29: Recombinant expression of a four disulfide-bridged scorpion toxin<br />
antagonist of voltage-gated sodium channels†<br />
Estrada, G., García, B.I., Ortiz, E.,Riano, L., Becerril, B., Possani, L.D., Corzo, G.<br />
Institute of Biotechnology-UNAM, Av. Universidad 2001, Cuernavaca, Morelos, 62210, Mexico.<br />
*corzo@ibt.unam.mx<br />
Css2 is a neurotoxin that affects voltage-gated sodium channels. Css2 is the major toxic<br />
component of the venom of the scorpion Centruroides suffusus suffusus (1). The gene of<br />
Css2 was cloned into the expression vector, pQE-30 containing a 6His-tag <strong>and</strong> an Fxa<br />
enzymatic cleavage site. The pQE-30 vector was transformed into E. coli BL21 cells, <strong>and</strong><br />
the expression was induced with isopropyl thiogalactoside (IPTG). The level of expression<br />
was 24 mg/L of culture medium, <strong>and</strong> the His tagged recombinant toxin (hrCss2) was found<br />
only in inclusion bodies with no evidence of in the soluble fraction. The recombinant Css2<br />
was extracted from inclusion bodies using a 0.2 M Tris buffer pH 8.0 solution, containing<br />
6 M Gnd-HCl. The hrCss2 peptide was purified by affinity <strong>and</strong> hydrophobic interaction<br />
chromatography. The reverse-phase HPLC profile, <strong>and</strong> the single molecular mass of<br />
9,392.2 Da in several protein fractions indicated that the expressed hrCss2 had multiple<br />
disulfide bridge arrangements. All hrCss2 structural forms were reduced using 0.2 M Tris<br />
buffer pH 8.0 containing 50 mM DTT to obtain a single protein fraction with a molecular<br />
mass of 9,400.2. An in vitro folding process generate a single hrCss2 form with the<br />
expected molecular mass of 9,392.2 Da. After an enzymatic cleavage with Fxa, the<br />
recombinant Css2 (rCss2) was obtained. The molecular mass of rCss2 was 7,538.6 Da as<br />
expected. Because of the natural C-terminus amidation of Css2, they both Css2 <strong>and</strong> rCss2<br />
differ each other in 1 Da. Nevertheless, both Css2 <strong>and</strong> rCss2 had similar LD50s in mice<br />
when injected intracranially. Also, the recombinant Css2 was recognized by antibodies<br />
raise against Css2.<br />
1. Martin, M..F. et al. (1987) J Biol Chem. 262:4452-4459.<br />
†This work was supported in part by a grant from Laboratorios Silanes S.A. de C.V. <strong>and</strong><br />
Institute Bioclón S.A. de C.V., <strong>and</strong> by a grant from the Dirección General de Asuntos del<br />
Personal Académico (DGAPA-UNAM) IN226006.<br />
o Antimicrobial<br />
o Pore-forming<br />
o Scorpion<br />
o Recombinant<br />
204<br />
Poster 30: Structure <strong>and</strong> function of huwentoxin- , a novel spider toxin<br />
targeting both serine proteinase <strong>and</strong> potassium channels<br />
Chunhua Yuan , Kuan Peng, Jianbo Diao <strong>and</strong> Songping Liang<br />
College of Life Sciences, Hunan Normal University,Changsha, Hunan 410081, China<br />
A novel peptide, denoted Huwentoxin- (HWTX- ), which inhibits serine proteinase<br />
such as trypsin <strong>and</strong> chymotrypsin <strong>and</strong> also behaves as a blocker of voltage-sensitive K +<br />
channels, has been isolated from the venom of Chinese bird spider Ornithoctonus huwena<br />
Wang. HWTX- is a 55 amino acid peptide cross-linked with three disulfide bridges.<br />
The full-length cDNA of HWTX- was isolated by the method of rapid amplification of<br />
cDNA ends. Its dissociation constant for trypsin is about 1×10 -12 M measured by a<br />
BIAcore binding assay system. Whole-cell configuration patch clamp recording indicated<br />
that HWTX- showed no effects on voltage-gated sodium channels or calcium channels<br />
in dorsal root ganglion neurons, whereas it significantly inhibited outward delay-rectified<br />
potassium currents in rat dorsal root ganglion neurons. This was confirmed by testing its<br />
effect on Kv1.1, Kv1.2 <strong>and</strong> Kv1.3 channels expressed in Xenopus oocytes. The solution<br />
structure of HWTX-XI was obtained by heteronuclear multidimensional NMR<br />
experiments. The structure of HWTX-XI adopts the same structural motif as that of<br />
BPTI,a trypsin inhibitor from bovine, <strong>and</strong> α-DTX, a potassium channel inhibitor from<br />
snake. The 3D structure of HWTX-XI shows some distinct differences compared with<br />
that of BPTI <strong>and</strong> α-DTX, which might be related to the dual inhibitory activity of the<br />
toxin.<br />
205
Poster 31: A novel family of conotoxins target to nACh-Receptors**<br />
Kauferstein, S. 1 , Nicke, A. 2 , Kendel, Y. 1 ., Zamudio, F.Z. 3 , Stöcklin, R. 4 , Mebs, D. 1<br />
1University of Frankfurt, Kennedyallee 104, D-60596 Frankfurt am Main, Germany,<br />
kauferstein@em.uni-frankfurt.de<br />
2 Max-Planck-Institute for Brain Research, D-60528 Frankfurt am Main, Germany<br />
3 Instituto de Biotecnologia-UNAM, Avenida Universidad, 2001, Cuernavaca 62210, Mexico<br />
4 Atheris Laboratories, CH-1233 Bernex-Geneva, Switzerl<strong>and</strong><br />
Introduction: Over the last 50 million years, cone snails developed a highly efficient<br />
venomous system.Using their complex venom apparatus they inject a mixture of peptides,<br />
which almost instantly paralyzes the prey. These peptides are consisting mainly of 11 to 35<br />
amino acid residues with a variable number of disulfide bridges, exhibiting a great<br />
variability in their amino acid sequence. The venom of cone snails is one of the most<br />
complex peptide mixtures acting on a wide range of target tissues in the nervous system,<br />
e.g. ion channels <strong>and</strong> receptors. The high affinity of these toxins to these targets made<br />
them indispensable tools in neurochemistry <strong>and</strong> neurophysiology.<br />
Methods: Crude venom samples from Conus capitaneus were tested on nAChRs expressed<br />
in oocytes using the two-electrode voltage-clamp method. Fractionation of the samples<br />
was performed by HPLC followed by liquid chromatography-electrospray-mass<br />
spectrometry analysis. N-terminal amino acid sequence analysis was performed by<br />
automated Edman degradation.<br />
Results: From the venom of Conus capitaneus a protein was isolated which represents a<br />
new family of conotoxins acting at nicotinic AChRs. It has a remarkably high molecular<br />
weight of 11 kDa <strong>and</strong>, as SDS-PAGE revealed the toxin is a dimer. Its N-terminal<br />
sequence exhibits no similarity to any known proteins. Analysis of the subtype selectivity<br />
of the purified toxin on nAChRs revealed subnanomolar potency on α7 <strong>and</strong> nanomolar<br />
potency on� α3β2 nAChRs subtypes with IC50 values of 300 pM <strong>and</strong> 3 nM respectively.<br />
The IC50 value for the �α4β2 subtype was 30 nM, making it the most potent conotoxin<br />
acting on this AchR-subtype known so far.<br />
Discussion/Conclusion: We suggest that some cone snail species, besides families of small<br />
peptides, developed an alternative class of large conopeptides to target the nAChR.<br />
o Conotoxins<br />
o nAChR<br />
o Cone snails<br />
o Voltage clamp<br />
**this presentation will be given as an oral communication on Tuesday at 4.40pm<br />
in K3.25<br />
206<br />
Poster 32: Isolation <strong>and</strong> characterisation of a potent novel tarantula toxin<br />
targeting the acid sensing ion channel ASIC1a.<br />
Rash LD 1 , Adams DJ 2 <strong>and</strong> Alewood PF 1 .<br />
1<br />
Institute for Molecular Bioscience, University of Queensl<strong>and</strong>, St Lucia, Queensl<strong>and</strong> 4072<br />
l.rash@imb.uk.edu.au<br />
2<br />
School of Biomedical Sciences, University of Queensl<strong>and</strong>, St Lucia, Queensl<strong>and</strong> 4072.<br />
Changes in local tissue pH can occur in many physiological <strong>and</strong> pathophysiological<br />
circumstances, most notably in ischaemia <strong>and</strong> inflammation. Acid sensing ion channels<br />
(ASICs) are a recently discovered group of non voltage-gated sodium channels belonging<br />
to the epithelial / degenerin Na channel family (ENaC/DEG). These channels are gated by<br />
protons <strong>and</strong> widely distributed throughout both the central <strong>and</strong> peripheral nervous systems.<br />
There are 4 ASIC genes encoding 6 proteins (1a, 1b, 2a, 2b, 3 <strong>and</strong> 4) with ASIC1a, 1b, 2a<br />
<strong>and</strong> 3 being able to form functional tetrameric channels on their own (1). ASIC1a has been<br />
shown to be involved in central processes such as memory, learning <strong>and</strong> synaptic plasticity<br />
(2), neuronal excitotoxicity such as in stroke (3). Due to their ability to sense pH change<br />
they are also implicated in pain transduction. At present there are very few specific<br />
pharmacological tools with which to study the structure, function <strong>and</strong> role of these<br />
channels. Two toxins have been reported to date. The tarantula toxin PcTx1 selectively<br />
blocks ASIC1a channels (4) while the sea anemone peptide APETX2 blocks ASIC3 <strong>and</strong><br />
several ASIC3 containing heteromers (5) however both of these toxins are readily<br />
reversible. Here we report the isolation <strong>and</strong> characterisation of a novel tarantula toxin<br />
which causes a more slowly reversible inhibition of ASIC1a channels expressed in<br />
Xenopus oocytes. CcrTx1 is a peptide isolated from the venom of the African spider<br />
Citharischius crawshayi. CcrTx1 inhibits ASIC1a with an IC50 of ~5 nM <strong>and</strong> most<br />
interestingly the inhibition at 100nM is stable for > 30 mins. Due to its stable inhibition of<br />
the channel, CcrTx1 should prove to be an invaluable tool for studies of the structure <strong>and</strong><br />
function of this interesting channel family.<br />
1. Waldmann R (1999). Ann NY Acad Sci. 868, 67-76.<br />
2. Wemmie J. (2002). Neuron 34, 463-77.<br />
3. Xiong Z. (2004). Cell 118, 687-98.<br />
4. Escoubas P. (2000). J Biol Chem. 275<br />
5. Diochot S. (2004). EMBO J. 23, 1516-25.<br />
o Acid sensing ion channels<br />
o Tarantula peptide<br />
o Channel blocker<br />
207
Poster 33: Modulation of voltage-gated Na + <strong>and</strong> K + channels by pumiliotoxin<br />
251D: a “joint venture” alkaloid from arthropods <strong>and</strong> amphibians<br />
V<strong>and</strong>endriessche, T. 1 , Maertens, C. 1 , Abdel-Mottaleb, Y. 1 , Cuypers, E. 1 , Nubbemeyer, U. 2 , Mebs, D. 3<br />
& Tytgat, J. 1*<br />
1 Laboratory of Toxicology, University of Leuven, O&N2, Herestraat 49, P.O. Box 922, 3000 Leuven,<br />
Belgium, * jan.tytgat@pharm.kuleuven.be<br />
2 Institute of Organic Chemistry, University of Mainz, Duesbergweg 10-14, 55128 Mainz, Germany<br />
3 Zentrum der Rechtsmedizin, University of Frankfurt, Kennedyvallee 104, 60596 Frankfurt, Germany<br />
Several amphibians defend themselves by accumulating lipophilic alkaloids from dietary<br />
alkaloid containing arthropods. Among these alkaloids, pumiliotoxins represent a major,<br />
widespread group found in virtually all anurans. Although pumiliotoxins are known as<br />
positive modulators of voltage-gated sodium channels (VGSCs), some of them, like<br />
pumiliotoxin 251D (PTX 251D), do not share this characteristic. However, mice <strong>and</strong> insect<br />
studies showed that this alkaloid is highly toxic, but the basis for the in vivo effects of PTX<br />
251D remains unexplained [1, 2].<br />
In the present work we searched for the possible target of PTX 251D. The synthetic toxin<br />
was tested on four VGSCs (mammalian Nav1.2/β1, Nav1.4/β1, Nav1.5/β1 <strong>and</strong> insect<br />
para/tipE) <strong>and</strong> five voltage-gated potassium channels (VGPCs) (mammalian Kv1.1, Kv1.2,<br />
Kv1.3, Kv11.1 (hERG) <strong>and</strong> insect Shaker IR) in a heterologous expression system (Xenopus<br />
laevis oocytes), by means of the two-electrode voltage clamp technique.<br />
PTX 251D not only inhibited the Na + -influx through all three mammalian VGSCs but also<br />
affected the activation <strong>and</strong> steady-state inactivation while, in the insect ortholog, the<br />
inactivation process was dramatically affected. Interestingly, PTX 251D inhibited the K + -<br />
efflux through all five VGPCs <strong>and</strong> slowed down the deactivation of the mammalian<br />
VGPCs. Kv1.3 is the most sensitive channel, with an IC50 value 10,8 ± 0,5 µM, as<br />
compared to VGSCs <strong>and</strong> the other VGPCs tested. To the best of our knowledge this is the<br />
first report of a pumiliotoxin affecting VGPCs, helping to underst<strong>and</strong> the in vivo effects<br />
observed in mice.<br />
1. Daly, J.W. et al. (2003) Proc. Natl. Acad. Sci. U.S.A 100, 11092-11097.<br />
2. Bargar, T.M. et al. (1995) Journal of Agricultural <strong>and</strong> Food Chemistry 43, 1044-1051.<br />
o Pumiliotoxin 251D<br />
o Voltage-gated sodium channels<br />
o Voltage-gated potassium channels<br />
o Two-electrode voltage clamp technique<br />
208<br />
Poster 34: Structure-function relationships of Magi 4, a spider neurotoxin<br />
targeting insect <strong>and</strong> mammalian voltage-gated sodium channels<br />
Little, M.J. 1 , Yamaji, N. 2 , Villegas, E. 3 , Corzo, G. 4 , Nicholson, G.M. 1*<br />
1<br />
Department of Medical & Molecular Biosciences, University of Technology, Sydney, Broadway NSW<br />
2007, Australia, *Graham.Nicholson@uts.edu.au<br />
2<br />
Division of Spectroscopic <strong>and</strong> Structural Research, Suntory Institute for Bioorganic Research,<br />
Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan<br />
3<br />
Centro de Investigacion en Biotecnologia UAEM, Av. Universidad 2001, Cuernavaca, Morelos, 62210,<br />
Mexico.<br />
4<br />
Institute of Biotechnology-UNAM, Av. Universidad 2001 Cuernavaca, Morelos, 62210 Mexico.<br />
Magi 4 is a peptide neurotoxin isolated from the venom of the Japanese funnel-web spider<br />
Macrothele gigas. This toxin is composed of 43 amino acids with a molecular mass of<br />
5150 Da <strong>and</strong> shows between 50 <strong>and</strong> 60% homology with the δ-ACTX-1 family of toxins<br />
from Australian funnel-web spiders. Following determination of the NMR solution<br />
structure of Magi 4 we found that it has a similar cysteine spacing <strong>and</strong> inhibitory cystine<br />
knot motif to the δ-ACTX-1 toxins. Furthermore, several basic (K4, R5, K9, K34), acidic<br />
(E10) <strong>and</strong> hydrophobic (W7, Y22) residues occupy a similar surface position to those<br />
believed to be important in the pharmacophore of δ-ACTX-1 toxins. Given this structural<br />
homology it is likely that Magi 4 targets Nav channels similarly to δ-ACTX-1 toxins.<br />
Accordingly, we have employed the whole-cell patch-clamp technique to characterise the<br />
actions of Magi 4 on INa in both neonatal rat dorsal root ganglion (DRG) neurons <strong>and</strong><br />
cockroach dorsal unpaired medial (DUM) neurons. Similarly to the δ-ACTXs, Magi 4<br />
caused a concentration-dependent slowing of tetrodotoxin (TTX)-sensitive INa inactivation<br />
in DRGs, with an EC50 of 45.7 ± 2.7 nM, <strong>and</strong> no effect on TTX-resistant INa. Similarly,<br />
Magi 4 also caused a 10 mV depolarising shift in the voltage-dependence of TTX-sensitive<br />
Nav channel activation. However, unlike δ-ACTX-1s, the slowing of inactivation was<br />
almost entirely reversible on washout in toxin-free solution (τoff = 99.2 sec at 300 nM).<br />
Also, Magi 4 failed to inhibit peak INa amplitude. In insect DUM neurons Magi 4, like δ-<br />
ACTXs, also slows Nav channel inactivation again accompanied by a depolarising shift in<br />
the voltage-dependence of activation. Importantly, while Magi 4 is toxic by i.c.v. injection<br />
into mice, it failed to displace 125 I-Lqh2 binding to neurotoxin receptor site 3 on Nav<br />
channels in rat brain synaptosomes, a hallmark of the actions of δ-ACTX-1 toxins <strong>and</strong><br />
scorpion α-toxins. Thus, while Magi 4 clearly modulates the gating of TTX-sensitive Nav<br />
channels in DRGs it does not target Nav1.1 or 1.2 channels, the predominant Nav channel<br />
subtypes in brain. Therefore the actions of Magi 4 are similar to scorpion α-like toxins that<br />
show CNS toxicity via an action on the Nav1.6 channel subtype in brain.<br />
o Magi 4<br />
o Voltage-gated sodium channels<br />
o NMR structure<br />
o Electrophysiology<br />
209
Poster 35: The natural anatoxin Amm VIII from Androctonus mauretanicus induces<br />
neutralizing antibodies against the most potent “Old-World” alpha-toxins<br />
Martin-Eauclaire, M-F, Alami, M, Rosso, J-P. <strong>and</strong> Bougis, P.E*.<br />
CNRS FRE 2738, IFR Jean-Roche, Université de la Méditerranée, Faculté de Médecine Secteur Nord,<br />
Bd Pierre Dramard, 13916, MARSEILLE, Cedex 20, France.*bougis.p@jean-roche.univ-mrs.fr<br />
Scorpion envenomations constitute a real public health problem in Maghreb, where<br />
epidemiological data indicate about 100 000 cases of scorpion stings per year. Fatality<br />
rates among children can reach 7%. The most lethal scorpions in North Africa are the<br />
Buthidae Androctonus mauretanicus (Amm) in Morocco, Androctonus australis (AaH) in<br />
Algeria <strong>and</strong> Tunisia <strong>and</strong> Leiurus quinquestriatus (Lqq) in Sudan.<br />
Up to 90 % of the lethality for mammals is induced by the binding of the alpha-toxins<br />
(about 65 amino acids residues long, four disulphide bridges) to site 3 of the voltagesensitive<br />
sodium channels (Nav) of nerve <strong>and</strong> muscle cells. Antivenom immunotherapy<br />
still remains the most useful specific treatment for scorpion envenomation. However, in<br />
spite of a broadly similar 3D-structure, the scorpion toxins display large sequence<br />
variations <strong>and</strong> are classified into several structural groups according to their sequence<br />
homologies. A consequence of this structural polymorphism is that no immunological<br />
cross-reactivity is observed between the toxins belonging to different groups when specific<br />
antisera are used, even if the toxins are found in the same venom. Thus, this wide antigenic<br />
polymorphism remains a persistent barrier to the preparation of an efficient <strong>and</strong> safe<br />
universal antiserum against “Old-World” scorpion alpha-toxins.<br />
In this study, we have used Amm VIII, a natural anatoxin from the scorpion Androctonus<br />
mauretanicus, to elicit specific polyclonal antibodies into rabbit. Using liquid-phase<br />
radioimmunoassay, we have studied its selectivity <strong>and</strong> its neutralizing activity both in vitro<br />
<strong>and</strong> in vivo for the most lethal scorpion alpha-toxins described, in particular the alpha-toxin<br />
of reference AaH II. We have shown that the anti-Amm VIII serum prevents the<br />
association of 125 I-AaH II with its receptor <strong>and</strong> is able to remove 125 I-AaH II already<br />
bound to its site (the half-life of the complex 125 I-AaH II-receptor site was 12 min in the<br />
absence of anti-Amm VIII serum but decreased to only 2 min in the presence of anti-Amm<br />
VIII serum). In vivo, the serum also has a protective effect in mice: 42 LD50 of AaH II by<br />
ml are neutralized, measured by subcutaneous injection.<br />
o scorpion<br />
o alpha-toxins<br />
o voltage-sensitive sodium channel<br />
o immunotherapy<br />
210<br />
Poster 36: Scorpion β-toxins: surveying the species-selectivity of five ‘classics’<br />
Bosmans, F. 1 , Martin-Eauclaire, M.F. 2 , Tytgat, J. 1*<br />
1<br />
Laboratory of Toxicology, University of Leuven, O&N 2, P.O. Box 922, Herestraat 49, 3000 Leuven, Belgium,*<br />
jan.tytgat@pharm.kuleuven.be<br />
2<br />
CNRS FRE 2738, Ingénierie des Protéines, Faculté de Médecine secteur Nord, Institut Jean Roche, Université de<br />
la Méditerranée, Bd Pierre Dramard, 13916, Marseille, Cedex 20, France<br />
In general, scorpion β-toxins have been well examined. However, little in-depth studies<br />
have been devoted to species selectivity <strong>and</strong> affinity comparisons on the different voltagegated<br />
Na + channels since they have become available as cloned channels that can be<br />
studied in heterologous expression systems. As a result, their classification is rather<br />
historical <strong>and</strong> dates from early in vivo experiments on mice <strong>and</strong> cockroach <strong>and</strong> fly larvae.<br />
In this study, we aimed to provide an updated overview of species selectivity <strong>and</strong> affinity<br />
of scorpion β-toxins towards voltage-gated Na + channels. As pharmacological tools, we<br />
used the classic β-toxins AaHIT, Css II, Css IV, Css VI <strong>and</strong> Ts VII <strong>and</strong> tested them on the<br />
neuronal vertebrate voltage-gated Na + channel, Nav1.2. For comparison, its invertebrate<br />
counterpart, the insect channel, para, was also tested.<br />
Nav1.2 <strong>and</strong> the insect ortholog, para, were heterologously expressed in Xenopus laevis<br />
oocytes <strong>and</strong> measured with the two-electrode voltage-clamp technique. We supplemented<br />
this data with several binding displacement studies on rat brain synaptosomes.<br />
As a result, we propose a general classification <strong>and</strong> a novel nomenclature of scorpion βtoxins<br />
based on pharmacological function.<br />
o scorpion<br />
o β-toxin<br />
o voltage-gated Na + channel<br />
o species-selectivity<br />
211
Poster 37: Potent modulation of the voltage-gated sodium channel Nav1.7 by OD1,<br />
a toxin from the scorpion Odonthobuthus doriae<br />
Maertens, C. 1* , Cuypers, E. 1 , Amininasab, M. 2 , Jalali, A. 3 , Vatanpour, H. 4 , Tytgat, J. 1<br />
1 Laboratory of Toxicology, University of Leuven, Leuven, Belgium<br />
*Chantal.Maertens@pharm.kuleuven.be<br />
2 Dept. of Cell <strong>and</strong> Molecular Biology, Faculty of Science, University of Tehran, Tehran, Iran<br />
3 Dept. of Toxicology <strong>and</strong> Pharmacology, Jundishapour University of Medical Sciences, Ahvaz, Iran<br />
4 Dept. of Toxicology <strong>and</strong> Pharmacology, Shaheed Beheshti University of Medical Science, Tehran, Iran<br />
Voltage-gated sodium channels are essential for the propagation of action potentials in<br />
nociceptive neurons. Nav1.7 is found in peripheral sensory <strong>and</strong> sympathetic neurons <strong>and</strong><br />
involved in acute <strong>and</strong> inflammatory pain. Nav1.8 <strong>and</strong> Nav1.3 are major players in<br />
nociception <strong>and</strong> neuropathic pain, respectively.<br />
In our effort to identify isoform-specific <strong>and</strong> high-affinity lig<strong>and</strong>s for these channels, we<br />
investigated the effects of OD1, a scorpion toxin isolated from the venom of the scorpion<br />
Odonthobuthus doriae. Nav1.3, Nav1.7 <strong>and</strong> Nav1.8 channels were coexpressed with β1subunits<br />
in Xenopus oocytes. Na + currents were recorded with the two-electrode voltageclamp<br />
technique.<br />
OD1 modulates Nav1.7 at low nanomolar concentrations: (1) Fast inactivation is impaired,<br />
with an EC50 value of 4.5 nM. (2) OD1 dramatically increases the peak current at all<br />
voltages. (3) OD1 induces a substantial persistent current. Nav1.8 was not affected by<br />
concentrations up to 2 micromolar, whereas Nav1.3 was only sensitive to concentrations<br />
higher than 100 nM. OD1 impairs the inactivation process of Nav1.3 with an EC50 value of<br />
1127 nM. Finally, the effects of OD1 were compared with a classical α-toxin, AahII from<br />
Androctonus australis hector <strong>and</strong> a classical α-like toxin, BmK M1 from Buthus martensii<br />
Karsch. At a concentration of 50 nM, both toxins affected Nav1.7. Nav1.3 was sensitive to<br />
AahII, but not to BmK M1, whereas Nav1.8 was not affected by both toxins.<br />
In conclusion, the present study shows that the scorpion toxin OD1 is a potent modulator<br />
of Nav1.7, with a unique selectivity pattern.<br />
o Scorpion toxin<br />
o Voltage-gated sodium channel<br />
o Nav1.7<br />
o Pain<br />
212<br />
Poster 38: Electrophysiological Characterization of Marine Snail Conus californicus<br />
Venom in Potassium Ion Channels<br />
Juárez-Moreno, K. O. 1 , Prior-Mier y Teran A 1 , García-Valdés, J. 2 , Aguilar M 3 , Morales E 1 , Waumann D 4<br />
<strong>and</strong> Licea-Navarro, A. 1*<br />
1<br />
Molecular Immunology <strong>and</strong> Biotoxins Laboratory Centro de Investigación Científica y de Educación<br />
Superior de Ensenada, Baja California, Mexico, *alicea@cicese.mx<br />
2.<br />
Bioelectrochemical Laboratory. Chemistry Faculty Universidad Nacional Autónoma de México, Mexico<br />
City.<br />
3.<br />
Neurobiology Institute, Universidad Nacional Autónoma de México, Queretaro City.<br />
4.<br />
Sciences School. Universidad Autonoma de Baja California<br />
The marine snail Conus californicus has a venom apparatus used to kill its prey; the venom<br />
contain an unique set of 50 to 200 different peptides called conotoxins 1 . Those have a<br />
highly targeting selectivity of voltage-gated <strong>and</strong> lig<strong>and</strong>-gated ion channels, acting as<br />
blockers of voltage-gated Ca 2+ , Na + <strong>and</strong> K + channels 2 . Due to the high selectivity <strong>and</strong><br />
affinity of these toxins, several of them are in various stages of clinical development for<br />
treatment of human diseases 2 . Despite of its variability, only few conotoxins acting on K +<br />
channels have been identified <strong>and</strong> characterized, <strong>and</strong> were commonly called k-conotoxins.<br />
Potassium channels are a big family of proteins greatly distributed among all the<br />
kingdoms, <strong>and</strong> have been implicated in relevant neurodegenerative pathologies such as<br />
Parkinson’s <strong>and</strong> Alzheimer’s diseases <strong>and</strong> chronic pain.<br />
In this study we purified different fractions of the venom of C. californicus by RP-HPLC<br />
according to they retention time. We tested the effect of those fractions in heterologous<br />
expressed K + channels as Shaker IR <strong>and</strong> Hslo channel in Xenopus laevis oocytes using the<br />
Two Electrode Voltage Clamp technique.<br />
Up to date, our results indicated that in the venom of C. californicus there are three<br />
fractions corresponding to minutes 5, 28 <strong>and</strong> 44 of elution time, which caused a significant<br />
decrease of the current when were tested on the Shaker IR channel in 40%, 55% <strong>and</strong> 44%<br />
respectively. We already have a partial amino acid sequence of the fraction corresponding<br />
to the fraction of the 28 minute of elution time.<br />
In the case of the Hslo channel, there are two fractions corresponding to the minutes 20-25<br />
<strong>and</strong> 45-50 of elution time, which diminished the current of the channel in 20% <strong>and</strong> 12%<br />
respectively, we tried to test the individual minutes but was unable because of its low<br />
concentration. Also, we purpose that the venom of the marine snail C. californicus had a<br />
different activity according with the season in which the organisms were collected.<br />
1.Olivera, B. M. (1999) J. Comp. Physiol A. 185: 353-359.<br />
2 Terlau, H. & Olivera, B. M. (2003) Physiol. Review. 84: 41-68.<br />
o Conus californicus<br />
o κ-conotoxins<br />
o Potassium Ion Channels<br />
o Two Electrode Voltage Clamp<br />
213
Poster 39: GAMBIEROL-INDUCED CYTOSOLIC CALCIUM INCREASE IN<br />
HUMAN NEUROBLASTOMA CELLS<br />
Cagide, E 1 , Louzao, M.C 1* , Vieytes, M.R 2 , Sasaki, M 3 , Fuwa, H 3 , Yasumoto, T 4 , Botana, L.M. 1<br />
1 2<br />
Departamento de Farmacologia, Departamento de Fisiologia. Facultad de Veterianaria. Universidad<br />
de Santiago de Compostela. Campus de Lugo, Lugo, Spain. *ffmclooj@lugo.usc.es<br />
3<br />
Graduate School of Life Sciences, Tohoku University, 1-1 Tsutsumidoori-Amamiya, Aoba-ku, Sendai,<br />
Japan.<br />
4<br />
Japan Food Research Laboratories, Tama Laboratory, Nagayama, Tama, Tokyo, Japan.<br />
Gambierol is a polycyclic ether toxin isolated from Gambierdiscus toxicus, the same<br />
marine dinoflagellate causative of Ciguatera <strong>Fish</strong> Poisoning. High concentrations of this<br />
toxin activate sodium channels in human neuroblastoma cells. As a consequence gambierol<br />
induces a cytosolic calcium increment. We deeply investigate mechanisms responsible of<br />
the elevation of intracellular calcium in neuroblastoma cells by using the dye fura-2. We<br />
observed that preincubation of cells with nifedipine <strong>and</strong> mibefradil or KB-R7943<br />
significantly reduced gambierol-induced cytosolic calcium increment. Interestingly, when<br />
all inhibitors were added together this calcium influx was completely blocked. Those<br />
results indicate that gambierol stimulate Ca 2+ entry through voltage-gated calcium channels<br />
<strong>and</strong> the Na + /Ca 2+ exchanger working in reverse mode.<br />
o Gambierol<br />
o neuroblastoma<br />
o voltage-gated calcium channels<br />
o Na + /Ca 2+<br />
214<br />
Poster 40: New modulator of P-type Ca 2+ -channels from Lycosa sp. venom.<br />
Pluzhnikov, K. 1 , Korolkova, Y. 1* , Vassilevski, A. 1 , Nikolsky, A. 1 , Fisyunov, A. 2 , Tsintsadze, V. 2 ,<br />
Krishtal, O. 2 , Grishin, E. 1<br />
1 Shemyakin <strong>and</strong> Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,<br />
Miklukho-Maklaya, 16/10, 117997 Moscow, Russia,* july@ibch.ru<br />
2 Bogomoletz Institute of Physiology, 4 Bogomoletz Street, Kyiv 01024, Ukraine<br />
The potent modulator of P-type Ca 2+ channels, named Lsp-1, has been recently isolated<br />
from the venom of the spider Lycosa sp. (Fisyunov, A., Pluzhnikov, K., Molyavka, A.,<br />
Grishin, E., Lozovaya, N., Krishtal, O., 2005, Toxicology 207, 129-136). Lsp-1 action on<br />
P-current in mammalian central neurons (prominent deceleration of the activation kinetics<br />
<strong>and</strong> partial decrease of the peak amplitude) notably differs from the effects of other wellknown<br />
Ca 2+ -toxins. The amino acid sequence of Lsp-1 was determined using a<br />
combination of Edman degradation, mass spectrometry <strong>and</strong> specific proteolytic<br />
fragmentation techniques. The peptide consists of 47 amino acid residues including 8<br />
cysteines involved in 4 intramolecular disulfide bridges, <strong>and</strong> presumably forms the ICK<br />
fold. Lsp-1 displays little homology to other known spider toxins affecting Ca 2+ -channels,<br />
but possesses a marked cluster of positively charged residues (-RKEKK-) in the C-terminal<br />
part of the molecule which may play a role in the action specificity.<br />
For comprehensive structure-function investigation, recombinant Lsp-1 was produced. The<br />
gene of Lsp-1 was constructed from synthetic oligonucleotides, <strong>and</strong> expressed as a soluble<br />
fusion protein with thioredoxin in E. coli Origami B cells. The fusion protein was purified<br />
by metal affinity chromatography, <strong>and</strong> cleaved with human enteropeptidase catalytic<br />
subunit. Lsp-1 was recovered by reverse-phase HPLC. The recombinant Lsp-1 was shown<br />
to be indistinguishable from the native peptide when tested on P-currents in rat cerebellar<br />
Purkinje cells.<br />
As voltage-dependent calcium channels are potential targets for therapeutics directed<br />
against migraine, intractable pain <strong>and</strong> some forms of epilepsy, Lsp-1 is attractive from both<br />
scientific <strong>and</strong> clinical perspectives.<br />
o spider toxin<br />
o calcium channel modulator<br />
o functional expression<br />
215
Poster 41: Evidence for a novel epitope for hERG channel blocking activity<br />
Yousra Abdel-Mottaleb 1 , Praveen Kumar 1 , Brigitte Céard 2 , Pierre E. Bougis 2 , Marie-France Martin-<br />
Eauclaire 2 , Jan Tytgat 1*<br />
1Laboratory of Toxicology, University of Leuven, Onderwijs <strong>and</strong> Navorsing 2, Herestraat 49, Postbus<br />
922, 3000 Leuven, Belgium, * jan.tytgat@pharm.kuleuven.be<br />
2CNRS UMR 6560, Ingénierie des proteines, Faculté de Médecine secteur Nord, institut Jean Roche,<br />
Université de la Méditerranée, Bd Pierre Dramard, 13916, Marseille, cedex 20, France<br />
To date, a total of 28 hERG blockers are known; 27 of which are grouped into γ-KTXs [1]<br />
<strong>and</strong> only one, BmTX3, belongs to subfamily α-KTX 15. The hERG blocking activity in<br />
BmTX3 has been assigned to 2 positively charged residues R18 <strong>and</strong> K19 on the α-helix<br />
side of the peptide [2].<br />
To further investigate the interaction between the α-KTX family <strong>and</strong> hERG channels, a<br />
natural mutant of BmTX3 which is AmmTX3 (91% sequence homology with BmTX3) [3],<br />
was tested on cloned hERG channels in Xenopus laevis oocytes, using the 2 electrode<br />
voltage clamp technique.<br />
Complementary testing was carried out on other members of subfamily α-KTX 15 (Aa1,<br />
Discrepin) as well as some selected toxins belonging to the α-KTX family <strong>and</strong> possessing<br />
the 2 positively charged residues R18 or K18 <strong>and</strong> K 19 on the α-helix side of the peptide<br />
(BmTx2, Tsκ <strong>and</strong> HsTx1).<br />
The effects obtained were then compared to those from a control group of toxins lacking<br />
the 2 positively charged residues <strong>and</strong> belonging to the same α-KTX subfamilies as the test<br />
group (PBTx3, TsTXKα <strong>and</strong> Spinotoxin).<br />
Our results indicate that the presence of 2 positively charged residues R18 (or K18) <strong>and</strong><br />
K19 on the α-helix side of the peptide, confers a hERG channel blocking activity to these<br />
α-KTXs.<br />
1. Korolkova,Y.V. et al., 2004, J. Mol. Recognit., 17, 209-217.<br />
2. Huys, I. et al., 2003, Biochem. J., 378, 745-752.<br />
3. Vacher, H. et al., 2002, Eur. J. Biochem., 269, 6037-6041.<br />
o hERG channels<br />
o α-KTX<br />
o voltage clamp technique<br />
216<br />
Poster 42: The first potassium channel toxin from the venom of the Iranian<br />
scorpion Odonthobuthus doriae<br />
Yousra Abdel-Mottaleb 1 , Amir Jalali 2 , Elke Clynen 3 , Frank Bosmans 1 , Liliane Schoofs 3 , Hossein<br />
Vatanpour 2 , Jan Tytgat 1*<br />
1Laboratory of Toxicology, University of Leuven, Onderwijs <strong>and</strong> Navorsing 2, Herestraat 49, Postbus<br />
922, 3000 Leuven, Belgium, * jan.tytgat@pharm.kuleuven.be<br />
2Department of Toxicology <strong>and</strong> Pharmacology, Shaheed Beheshti University of Medical science,<br />
Tehran, Iran<br />
3Laboratory for Developmental Physiology <strong>and</strong> Molecular biology, University of Leuven,<br />
Naamsestraat 59, 3000 Leuven, Belgium<br />
The first member of K + channels toxins from the venom of the Iranian scorpion<br />
Odonthobuthus doriae (OD16) was purified, sequenced <strong>and</strong> its pharmacological profile<br />
determined.<br />
OD16 is composed of 29 amino acids <strong>and</strong> has the six conserved cysteines. The toxin’s<br />
molecular weight <strong>and</strong> pI value are respectively 3188.5 <strong>and</strong> 4.95.<br />
Name α-KTX Sequence I%<br />
PO1 8.1 VSCEDCPEHCSTQKAQAKCDNDKCVCEPI 90<br />
BmPO1 8.2 ATCEDCPEHCATQNARAKCDNDKCVCEPK 88.5<br />
LpII 8.3 VSCEDCPDHCSTQKARAKCDNDKCVCEPI 92<br />
LpIII 8.4 VSCEDCPDHCSTQKARAKCDNDKCVCEPK 92<br />
OD16 8.5 VSCEDCPEHCSTQKARAKCDNDKCVCESV 100<br />
Based on multiple sequence alignments <strong>and</strong> homology modelling of the toxin structure,<br />
OD16 belongs to subfamily α-KTX 8, therefore, it was classified as α-KTX 8.5 <strong>and</strong> it was<br />
shown to adopt a cysteine-stabilized α/β scaffold. It is interesting to note that the toxin<br />
bears a basic residue (K) before the 3 rd cysteine, which is a property typical for the dyad<br />
present in many α-KTX’s, however, it was found that the aromatic counterpart is not<br />
present, raising our interest in the toxin’s bioactivity. The pharmacological effects of<br />
OD16 were studied on six different cloned K + channels (vertebrate Kv1.1-Kv1.5 <strong>and</strong> the<br />
insect ortholog Shaker IR) expressed in Xenopus laevis oocytes using the two-electrode<br />
voltage clamp technique. The toxin induced full current inhibition of Kv1.2 channels<br />
(EC50=182.6±2.6 nM) but did not affect any of the other channels. Therefore, among the<br />
channels tested, OD16 is selective to Kv1.2 <strong>and</strong> might serve as a good c<strong>and</strong>idate for<br />
structure-function <strong>and</strong> channel assembly studies as well as the identification of the<br />
physiological role of Kv1.2 in multiple sclerosis.<br />
o OD16<br />
o Odonthobuthus doriae<br />
o α-KTX 8.5<br />
o Scorpion<br />
217
Poster 43: Novel insect-selective neurotoxins from the venom of the tarantula<br />
Eucratoscelus longiceps target insect Kv channels<br />
Escoubas, P. 1* , Lee, M. 2 , Ross, G. 2 , Lazdunski, M. 1 Nicholson, G.M. 2<br />
1<br />
Institut de Pharmacologie Moléculaire et Cellulaire – CNRS, 660 Route des Lucioles, 06560 Valbonne,<br />
France, *escoubas@ipmc.cnrs.fr<br />
2<br />
Department of Medical <strong>and</strong> Molecular Biosciences, University of Technology, Sydney, Broadway NSW<br />
2007, Australia<br />
Arthropod pests are vectors for the transmission of many diseases <strong>and</strong> destroy an<br />
estimated 20–30% of the world’s food supply. Currently available insecticides are<br />
severely limited by toxicity <strong>and</strong>/or insect resistance <strong>and</strong> in many cases are undergoing<br />
use cancellation. Thus there is an urgent need to develop safer <strong>and</strong> more selective<br />
insecticides <strong>and</strong> to identify new insecticide targets. Spider venoms have been proven to<br />
be a rich source of insecticidal compounds with unique properties, that target a wide<br />
array of insect neuronal receptor subtypes. With the goal of discovering novel<br />
insecticidal toxins from spider venoms, we have assayed an array of New <strong>and</strong> Old World<br />
tarantula venoms for insecticidal activity by intrathoracic injection into juvenile crickets<br />
(Gryllus bimaculatus) <strong>and</strong> selected the most potent venoms for further investigation. The<br />
most potent of these was the venom from the stoutleg baboon spider Eucratoscelus<br />
longiceps. Bioassay-guided fractionation of the venom using reversed-phase <strong>and</strong> cationexchange<br />
HPLC, yielded a family of three novel insecticidal toxins each with 29 residues<br />
<strong>and</strong> three disulfide bonds. Both ElTx1 <strong>and</strong> ElTx2 were identified to have insect<br />
neurotoxicity with LD50 values in crickets of 331 <strong>and</strong> 1033 pmol/g, respectively, but<br />
without overt signs of mammalian toxicity. ElTx3, with five non-conserved amino acid<br />
substitutions, also showed neurotoxicity in mice following icv injection. All three<br />
peptides display high homology to short ICK (inhibitory cystine knot) toxins from other<br />
tarantula venoms especially the phrixotoxins <strong>and</strong> both GsAFI <strong>and</strong> II. These toxins have<br />
been reported to target Kv4 channels, or possibly mechanosensitive cation channels. The<br />
target of ElTx1 was investigated by whole-cell patch-clamp analysis of DUM neurons<br />
from the American cockroach (Periplaneta americana). At 160 nM, ElTx1 blocked 55 ±<br />
14% (n = 3) of the global Kv channel current with no shift in the voltage-dependence of<br />
activation. Further experiments are currently determining the specific Kv channel subtype<br />
involved nevertheless this finding indicates that ElTx1 blocks invertebrate ion channels<br />
not previously targeted by conventional insecticides <strong>and</strong> validates Kv channels as novel<br />
insecticide targets.<br />
o Spider toxins<br />
o Kv channels<br />
o insecticide<br />
o patch-clamp electrophysiology<br />
218<br />
Poster 44: Short insecticidal toxins from the Asian scorpions Buthus<br />
martensi <strong>and</strong> Mesobuthus tamulus modulate insect neuronal voltagedependent<br />
<strong>and</strong> calcium-activated chloride channels<br />
Grolleau F 1 ., Stankiewicz M. 3 , Herrmann R. 4 , Moskowitz H. 4 , Rajendra W. 4 ,<br />
Hammock B.D. 4 , Romi-Lebrun R. 5 , Wu F.Q. 6 , Nakajima T. 5 Escoubas P. 2,5*<br />
1 Laboratoire Biologie Neurovasculaire Intégrée, UMR CNRS 6214 / Inserm 771, UFR Sciences Medicales<br />
Rue Haute de Reculée 49045 Angers cedex 01, France 2 Institut de Pharmacologie Moléculaire et<br />
Cellulaire – CNRS, 660 Route des Lucioles, 06560 Valbonne, France, *escoubas@ipmc.cnrs.fr 3<br />
Laboratory of Biophysics, Institute of General <strong>and</strong> Molecular Biology, N. Copernicus University, Torun,<br />
Pol<strong>and</strong><br />
4 Department of Entomology <strong>and</strong> Cancer Research Center UCDMC, University of California Davis, Davis<br />
CA 95616-8584, USA 5 Suntory Institute for Bioorganic Research. Mishima-Gun, Shimamoto-Cho,<br />
Wakayamadai, Osaka 618-8503 Japan 6 Sichuan Province Institute for Antibiotics. Sanbanqiao 9,<br />
Chengdu, China<br />
Five novel short insecticidal toxins have been isolated from the venoms of the<br />
Asian scorpions Buthus martensi <strong>and</strong> Mesobuthus tamulus, following a bioassay-guided<br />
procedure based on their insecticidal activity. Their full sequences show a high degree of<br />
similarity with previously described scorpion “short insectotoxins”, with 35-38 amino<br />
acids <strong>and</strong> molecular masses ranging from 3735 to 4019 Da. Insecticidal assays against<br />
three insect species show paralytic activity in the 1-10 ng/mg of insect range, with three of<br />
the toxins inactive against crickets. This level of activity, 10 to 100 times lower than the<br />
activity of the scorpion toxins acting on sodium channels, as well as differences in the<br />
paralysis symptoms suggest a different molecular target. We also report the unusual<br />
electrophysiological effects of toxins Bm20-IV <strong>and</strong> Mt-V6 isolated respectively from B.<br />
martensi <strong>and</strong> M. tamulus. Applied on cockroach neurosecretory dorsal unpaired median<br />
(DUM) neurons, the two peptides (2.5 µM) reduced the amplitude of the<br />
hyperpolarization-activated calcium sensitive chloride current (IClCa) by more than 40%,<br />
without any effect on reversal potential or voltage dependence. The inhibitory effects of<br />
Bm20-IV <strong>and</strong> Mt-V6 were mimicked by the specific chloride channel blocker DIDS (0.5<br />
mM) as well as commercial chlorotoxin (0.6 µM) from Leiurus scorpion venom, which is<br />
presumed to target chloride channels. In current-clamp experiments, Bm20-IV <strong>and</strong> Mt-V6<br />
induced hyperpolarization of the membrane potential associated with an increase of the<br />
input membrane resistance. DUM neuron sodium <strong>and</strong> potassium currents were insensitive<br />
to the toxins. Structure-activity relationships are discussed as these peptides might have<br />
great potential for 1°) ion channel mapping studies <strong>and</strong> 2°) as chloride channel inhibitors<br />
similar to chlorotoxin, which is currently used in clinical trials for the treatment of glioma.<br />
o Scorpion toxins<br />
o Chloride channels<br />
219
Poster 45: Phlotoxin 1, a toxin from tarantula venom, is a potent<br />
modulator of Nav1.7 sodium channels <strong>and</strong> a potential analgesic<br />
Escoubas P. 1* , Bosmans F. 2 , Cuypers E. 2 , Diochot S. 1 , Mebs D. 3 , Craik D. 4 , Hill J. 4 ,<br />
Maertens C. 2 , Nakajima T. 5 , Lazdunski M. 1 , Tytgat J. 2<br />
1<br />
Institut de Pharmacologie Moléculaire et Cellulaire – CNRS, Valbonne, France,<br />
*escoubas@ipmc.cnrs.fr<br />
2<br />
Laboratory of Toxicology, University of Leuven, Leuven, Belgium<br />
3<br />
Zentrum der Rechtsmedizin, University of Frankfurt, Frankfurt, Germany<br />
4<br />
Institute for Molecular Bioscience, University of Queensl<strong>and</strong>, Brisbane, Australia<br />
5<br />
Suntory Institute for Bioorganic Research, Mishima-Gun, Shimamoto-Cho, Osaka, Japan<br />
Voltage-gated sodium (Nav) channels play an essential role in the biophysical properties of<br />
nociceptive neurons. Several Nav subtypes are key components of nociceptive<br />
neurotransmission <strong>and</strong> are potential targets for the development of novel analgesic<br />
strategies. Importantly, the Nav1.7 channel is found in peripheral primary sensory, <strong>and</strong><br />
sympathetic, neurons <strong>and</strong> is involved in acute <strong>and</strong> inflammatory pain. We report here the<br />
discovery, structural characterization <strong>and</strong> pharmacological properties of phlotoxin 1<br />
(PhlTx1), a novel 34-residue peptide toxin from the venom of a tarantula of the genus<br />
Phlogiellus, which selectively inhibits Nav1.7.<br />
PhlTx1 was isolated as the major component of the venom <strong>and</strong> its sequence was<br />
determined <strong>and</strong> confirmed by solid-phase peptide synthesis. Homology modelling revealed<br />
that it belongs to the inhibitory cystine knot (ICK) family of spider toxins <strong>and</strong> mass<br />
spectrometry indicates three disulfide bridges. PhlTx1 shows limited homology with other<br />
Nav toxins from Chinese tarantulas (Hainantoxins, Huwentoxins). PhlTx1 almost<br />
completely blocked (~90%) Nav1.7 channel currents at a concentration of 1.2 µM <strong>and</strong> the<br />
dose-response relationship on heterologously expressed channels revealed an IC50 value of<br />
260 nM. The voltage-dependence of steady-state Nav1.7 channel activation <strong>and</strong><br />
inactivation were not affected, suggesting that Phltx1 does not act as a gating-modifier<br />
toxin but rather blocks or impedes ion flux through the channel pore. PhlTx1 was almost<br />
exclusively selective for the Nav1.7 channel subtype, failing to modulate other Nav<br />
channels at concentrations up to 2 μM<br />
In a model of inflammatory pain (mouse paw injection of formalin), intrathecal injection of<br />
PhlTtx1 resulted in a significantly reduced response to the pain stimulus in both the acute<br />
<strong>and</strong> inflammatory phases, strongly suggesting that PhlTx1 produces an analgesic effect<br />
through inhibition of the Nav1.7 channel in dorsal root ganglion neurones. Phlotoxin 1 may<br />
therefore represent a lead for the development of novel analgesic agents.<br />
o Spider toxins<br />
o Sodium channels<br />
o Pain<br />
o Analgesia<br />
220<br />
Poster 46: ON SOME BIOPHYSICAL TRAITS OF COLUBRIDAE SNAKE<br />
VENOMS<br />
Kazakov I., Abubakirova M.E., Reimbaeva R.S.<br />
Institute of Zoology of Uzbek Academy of Sciences<br />
A. Niyazov Street 1, Tashkent 700095, Uzbekistan<br />
Venoms of Colubridae snakes (Coluber ravergieri, C. rodorochochis, C. tyria<br />
Linneus, Elaphe dione Pallas, Natrix tesselata) were studied. The doses 0.10-0.40 µg/ml<br />
caused unstable channels for univalent ions, while large concentrations (100-200 µg/ml)<br />
decreased the instability of membranes. Labilization of membranes <strong>and</strong> their quick<br />
destruction were connected with the fact that snake venoms show the phospholipase <strong>and</strong><br />
protease activities.<br />
In some respect, of special interest was the venom of the Psammophis Lineolatum<br />
Br<strong>and</strong>t of the family Colubridae. Our experiments showed the venom of Psammophis<br />
Lineolatum Br<strong>and</strong>t caused changes in the BLM conductance <strong>and</strong> forms channels with<br />
conductance for univalent <strong>and</strong> divalent cations. 140 ±10 pS, 96±4,2 pS in mediums<br />
containing 100 mM KCl, 100 mM CaCl2, 5mM tris HCl, pH=7,5).<br />
Thus, data obtained suggest that the venoms of Colubridae snakes contain<br />
components able to from unstable <strong>and</strong> discrete single channels.<br />
221
Poster 47: THE EFFECT OF BOOPHILUS CALCARATUS VENOM ON<br />
BILAYER LIPID AND MITOCHONDRIAL MEMBRANES<br />
Kazakov I.., Rakhimova Sh.H., Abubakirova M.E., Azimov D.A.<br />
Institute of Zoology of Uzbek Academy of Sciences<br />
A. Niyazov Street 1, Tashkent 700095, Uzbekistan<br />
LD50 of crude venom isolated from salivary gl<strong>and</strong>s of mite Boophilus calcaratus is<br />
100µg/kg. It shows phospholipase, protease <strong>and</strong> hemorrhagic activities.<br />
In low concentrations (below 170 µg/ml of protein), this venom causes stimulation<br />
of mitochondrial respiration at states 2 <strong>and</strong> 4, but produces no effect on the oxygen<br />
consumption at state 3, which is accompanied with the drop of respiratory control (RC)<br />
<strong>and</strong> ADP/O. The dose of this venom at 250 µg/ml almost completely inhibits ATP<br />
synthesizing functions of mitochondria, this effect growing at the absence of EDTA in<br />
the medium.<br />
Channels with the conductance of 120±10 pS (medium: 100 mM KCl <strong>and</strong> 5 mM<br />
tris-HCl, pH=7,5) are formed on bilayer lipid membranes under the effect of the crude<br />
venom.<br />
Thus, the venom of mite Boophilus calcaratus contains components able to form<br />
single ion channels in bilayer lipid membranes. It disturbs membrane-metabolic<br />
processes in mitochondria: at low doses it significantly increases the free oxidation not<br />
coupled with the ATP synthesis, while high concentrations inhibit the rate of<br />
phosphorylating respiration.<br />
222<br />
Poster 48: THE EFFECT OF HELMINTH EXTRACT ON THE BILAYER LIPID<br />
AND MITHOCHONDRIAL MEMBRANES<br />
I. Kazakov, M.E.Abubakirova, A.E.Kuchboev, D.A.Azimov<br />
Institute of Zoology, Uzbek Academy of Science, 700095 Tashkent, Uzbekistan<br />
The addition of 30 µg/ml of the five helminth species Echinococcus granulosus,<br />
Moniezia expanza, Fasciola hepatica, Ascaridia galli <strong>and</strong> Ascaris suum to one of the<br />
sections of the experimental cell is shown to cause a spasmodic increase in the membrane<br />
conductance. The further increase in the content of the extract was accompanied by a<br />
decrease in the lifetime of the membrane, i.e. it underwent destabilization <strong>and</strong><br />
destruction. At a level of isolated mitochondria the increase of respiration rate at<br />
metabolic state 4 <strong>and</strong> some decrease of it in state 3 were recorded. The data obtained<br />
show that the extract of helminth membrane-active components form ion channels <strong>and</strong><br />
modify functions of mitochondrial membranes. The presence of the receptor-enzymatic<br />
complexes is suggested.<br />
223
Poster 49: ω-ACTX-Ar1a: a novel insect-selective voltage-gated calcium channel<br />
blocker from the venom of the Sydney funnel-web spider<br />
Chong, Y 1* , Wen, S 1 , Hayes, J.L 1 , Hodgson, W.C. 2 , Hains, P 3 , Broady, K.W 1 , King, G.F 4 , Nicholson,<br />
G.M 1<br />
1<br />
Neurotoxin Research Group, Department of Medical & Molecular Biosciences, University of<br />
Technology, Sydney, Broadway NSW 2007 Australia, * youmie.chong@uts.edu.au<br />
2<br />
Monash Venom Group, Department of Pharmacology, Monash University, Clayton, Victoria 3800,<br />
Australia<br />
3<br />
Save Sight Institute, Sydney Eye Hospital, Macquarie Street, Sydney NSW 2001, Australia<br />
4<br />
Department of Molecular, Microbial <strong>and</strong> Structural Biology, University of Connecticut Health Center,<br />
263 Farmington Ave., Farmington CT 06032-3305, USA<br />
Insect-selective toxins have the potential to ameliorate the growing problem of<br />
agrochemical resistance in pest insects. Here we report the isolation <strong>and</strong> characterisation of<br />
a novel insect-selective toxin, ω-ACTX-Ar1a, from the venom of the female Sydney<br />
funnel-web spider, Atrax robustus (Hexathelidae: Atracinae). Crude venom was purified<br />
using C18 reverse-phase HPLC, <strong>and</strong> a fraction containing a 4003 Da peptide neurotoxin<br />
was identified by toxicity bioassay using intrathoracic injections into House crickets<br />
(Acheta domesticus). Acute toxicity tests revealed a depressant lethal phenotype with an<br />
LD50 of 250 ± 28 pmol/g, <strong>and</strong> a KD50 (median knockdown dose) of 149 ±16 pmol/g at 48<br />
hours. Using isolated chick biventer cervicis nerve-muscle <strong>and</strong> mouse vas deferens<br />
preparations, the purified toxin was confirmed to lack overt vertebrate toxicity at<br />
concentrations up to 1 μM. Edman degradation revealed a 37-residue toxin with six<br />
cysteines <strong>and</strong> high sequence homology with insect voltage-gated calcium (Cav) channel<br />
blockers from the ω-ACTX-1 family. Importantly, there was conservation of the cysteine<br />
spacing <strong>and</strong> pharmacophore of ω-ACTX-Hv1a from Hadronyche versuta venom.<br />
Accordingly, whole-cell patch-clamp analysis was undertaken using dorsal unpaired<br />
median (DUM) neurons from the terminal abdominal ganglia of the cockroach Periplaneta<br />
americana to investigate the effect of ω-ACTX-Ar1a on insect Cav channels. A<br />
concentration-dependent voltage-independent block of IBa currents through Cav channels<br />
was observed at all membrane potentials, with no shift in the threshold of Cav activation.<br />
The IC50 values on insect Cav channels were 853 nM <strong>and</strong> 766 nM for ω-ACTX-Ar1a <strong>and</strong><br />
ω-ACTX-Hv1a, respectively. There was no block of DUM neuron macroscopic Kv channel<br />
currents. However ω-ACTX-Ar1a did produce a modest block of peak INa amplitude, as<br />
has been previously observed with other Cav channel toxins such as ω-agatoxin IVA. In<br />
conclusion, ω-ACTX-Ar1a is an insect Cav pore blocker that both blocks low- <strong>and</strong> highvoltage<br />
activated insect Cav channels <strong>and</strong> further validates the ω-ACTX-1 pharmacophore.<br />
o Sydney funnel-web spider<br />
o electrophysiology<br />
o voltage-gated calcium channel<br />
o insect-selective toxin<br />
224<br />
Poster 50: The Effects of the Sodium Channel Blocker Lidocaine on Cardiovascular<br />
<strong>and</strong> Respiratory Actions of the Yellow Scorpion Leiurus Quinquestriatus in Rabbits<br />
Al-Shanawani, A. R 1* , Fatani, A.J. 2<br />
King Saud University, Department of Pharmacology, College of Pharmacy, AlMalaz Street, Riyadh, Saudi<br />
Arabia, ash77_00@hotmail.com<br />
.<br />
Introduction: Scorpion venom generally produce similar effects by acting mainly on Na +<br />
channels, causing an exaggerated release of neurotransmitters <strong>and</strong> mediators leading to<br />
pathological changes in several major systems in the body, mainly respiratory <strong>and</strong><br />
cardiovascular systems. This study was performed to determine whether the Na + channel<br />
blocker, lidocaine, would be useful in preventing cardiovascular <strong>and</strong> respiratory actions of<br />
the venom of the common yellow scorpion Leiurus quinquestriatus quinquestriatus (LQQ).<br />
Methods: Arterial blood pressure (BP), respiration <strong>and</strong> ECG were recorded in urethaneanaesthetized<br />
New Zeal<strong>and</strong> white male rabbits according to method of Ismail et al. (1972).<br />
Animals were injected with LQQ (0.5 mg kg -1 , i.v.) followed by lidocaine (1 mg kg -1 , i.v.<br />
bolus plus i.v. infusion of 50 μg kg -1 min -1 at a rate of 4.5 ml hr -1 ) at 5, 15 or 30 min after<br />
venom. Arterial BP, heart rate, respiration <strong>and</strong> ECG changes were recorded throughout the<br />
time limit of the experiment (300 min). Kruskal-Wallis non-parametric analysis of variance<br />
<strong>and</strong> Dunn’s Multiple comparisons post test were used in cardiovascular <strong>and</strong> respiratory<br />
experiments. Survival of the rabbits was then analyzed utilizing Covariance Wilcoxon<br />
Analysis. Results were expressed as mean ± S.E.M of 5 animals, with values of P≤ 0.05<br />
considered significant.<br />
Results: Lidocaine was capable of significantly (P
Poster 51: Postsynaptic neuromuscular activity of Cerastes cerastes cerastes crude<br />
snake venom.<br />
Soliman M. M 1 , Abu-Sinna G 1 , Abd-Elbaset A 2 , Harvey A.L 3 <strong>and</strong> Rowan,E.G 3<br />
1 Department of Zoology, Faculty of Science, Ain Shams University, Cairo, Egypt,<br />
*mohamed.soliman@strath.ac.uk<br />
2 Medical Research Center, Faculty of Medicine, Ain Shams University, Cairo, Egypt<br />
3 University of Strathclyde, SIBS, 27 Taylor Street, Glasgow, Scotl<strong>and</strong>, UK<br />
Introduction: Cerastes cerastes cerastes is an Egyptian viper that belongs to the family of<br />
Viperidae <strong>and</strong> the genus Cerastes. Many biochemical <strong>and</strong> pharmacological studies have<br />
been carried out on this venom, however, little is known of the effect of the venom on<br />
neuromuscular transmission. Hence, the present study was undertaken to test the<br />
neuromuscular activity of Cerastes venom.<br />
Methods: Mouse phrenic nerve hemidiaphragm preparation <strong>and</strong> chick biventer cervicis<br />
(CBC) nerve muscle preparation were mounted in 10 ml organ baths in physiological salt<br />
solution. Motor nerves were stimulated at the desired frequency at a voltage greater than<br />
that required to produce a maximal twitch. On CBC preparations submaximal<br />
concentrations of acetylcholine, carbachol <strong>and</strong> KCl were added to the bath prior to <strong>and</strong> at<br />
the end of the experiment. Data is expressed as means ± sem.<br />
Results: Cerastes venom dose dependently inhibited the contractile responses of indirectly<br />
stimulated (nerve stimulation) mouse hemidiaphragm preparations . Venom (10µg/ml)<br />
reduced twitches to around 63.6 ± 2.1 % of control in one hour, while with 20µg/ml of<br />
venom twitches were reduced further to 77±1.6% of control. CBC nerve muscle<br />
preparations were less sensitive to the venom than the hemidiaphragm preparation,<br />
10µg/ml, <strong>and</strong> 20µg/ml had no effect on twitches. However, 40µg/ml of venom reduced<br />
twitches to about 37.1 ± 12 of control. The venom (40µg/ml) also reduced contractile<br />
response to exogenous acetylcholine (10 -3 M) to about 4.7±2.1%, to exogenous carbachol<br />
(2x10 -6 M) to about 5.1±2.5% <strong>and</strong> also to exogenous KCl (60mM) to about 21.7±2.6%. It is<br />
concluded that the Cerastes venom has neurotoxic activity <strong>and</strong> act primarily<br />
postsynaptically to depress muscle contractility.<br />
Ref.<br />
Wayne C Hodgson <strong>and</strong> Janith C Wikramaratna (2002): In vitro neuromuscular activity of<br />
snake venoms. Clin Exp Pharmacol Physiol, 29(9):807-14<br />
• Neurotoxicity<br />
• Snake venom<br />
• Phrenic nerve<br />
• Chick biventer<br />
226<br />
Tuesday 25 July: poster session (Colville<br />
Building 5.11/5.12)<br />
Venomics, transcriptomics, cloning<br />
1. Inter-specific comparison of the toxin encoding transcriptomes of medically<br />
important African vipers, E. ocellatus, C. cerastes <strong>and</strong> B. arietans. Wagstaff, S.C.*,<br />
<strong>and</strong> Harrison, R.A.<br />
2. An O-conotoxin-like Cys framework <strong>and</strong> hydroxyproline in peptides of the<br />
venom of the turrid Polystira albida from the Bay of Campeche, México. Aguilar,<br />
M.B.*, Chan de la Rosa, R.A., Falcón, A., Heimer de la Cotera, E.P.<br />
3. Gene Expression Profiling <strong>and</strong> Fingerprinting of Human Genome Following<br />
Exposure to Natural Toxins <strong>and</strong> Nerve Agents – TOXINOGENOMICS.<br />
Gopalakrishnakone, P *, Pachiappan, A.<br />
4. Molecular cloning of dipeptidyl-peptidase IV (CD26) from Bothrops alternatus<br />
(urutu) snake venom gl<strong>and</strong> cDNA. Cardoso, K.C. 1 , da Silva, J.M. 2,3 , Souza,<br />
G.H.M.F. 1 , Menossi, M.T. 2,3 , Hyslop, S. 1<br />
5. Phylogeny of Protobothrops Snakes Inhabiting the Southwestern Isl<strong>and</strong>s of<br />
Japan. Chijiwa, T 1 *, Tomino, H 1 , So, S 1 , Oda-Ueda, N 2 , Hattori, S 3 , Ohno, M.<br />
6. The main toxins of the Colubridae snake Philodryas olfersii revealed from a<br />
Duvernoy´s (venom) gl<strong>and</strong> transcriptome. Ching, A.T.C. 1* , Rocha, M.M.T. 2 , Paes-<br />
Leme, A. F. 3 , Pimenta, D. C. 3 , Furtado, M. F. D. 2 , Serrano, S. M. T. 3 ; Ho, P. L. 1 ,<br />
Junqueira-de-Azevedo, I.L.M. 1<br />
7. A comparison of the venom from left <strong>and</strong> right fangs of three Australian<br />
elapid’s (Pseudechis australis, Psuedonaja textiles <strong>and</strong> Notechis scutatus). Winter,<br />
K.L., 1* Mirtschin, P.J., 2 Hodgson, W.C. 1<br />
8. Gene Structures of Two Functionally Diverse Prothrombin Activators, Trocarin<br />
D <strong>and</strong> Coagulation Factor X in Tropidechis carinatus Snake: Gene Duplication<br />
<strong>and</strong> Recruitment of Factor X Gene to the Venom Gl<strong>and</strong>. Reza, MA 1 , Swarup, S 1 ,<br />
1, 2<br />
Kini, RM<br />
9. Modulation of transcription activities between group IA <strong>and</strong> IB phospholipase<br />
A2 genes in sea snake Laticauda semifasciata. Hayashi, Y,, Sagitani, A., Fujimi, T. J.,<br />
Kanzawa, N., Tsuchiya, T., Tamiya, T.*<br />
227
10. Maturation signatures in the sequences of natural toxins. Kozlov S., Grishin E.<br />
11. Proteomic analysis of the venom from the Mexican scorpion Centruroides<br />
limpidus limpidus. Gurrola, G.B., Batista, C.V.F., Zamudio, F.Z. <strong>and</strong> Possani, L.D.<br />
12. Venom Proteomes of Closely-related Sistrurus Rattlesnakes with Divergent<br />
Diets. Sanz, L. 1 , Gibbs, H.L. 2 , Mackessy, S.P. 3 , Calvete, J.J. 1,*<br />
13. Molecular cloning of a non-venom-secreted PII disintegrin-like transcript, BA-<br />
5A, from a Bitis arietans cDNA library reveals a pathway for the evolution of the<br />
long-chain disintegrin bitistatin from a PIII disintegrin-like precursor.<br />
Juárez, P. 1* , Wagstaff, S.C. 2 , Oliver, J. 2 , Sanz, L. 1 , Harrison, R.A. 2 , Calvete, J.J. 1<br />
14. Spider venom proteome mining <strong>and</strong> de novo toxin sequencing by LC-MALDI-<br />
TOF <strong>and</strong> FT-ICR mass spectrometry. Escoubas P. 1* , Quinton, L. 2 , Hu Renjie 2 , Wen<br />
S. 3 , Chamot-Rooke J. 2 , Nicholson G.M. 3<br />
15. Gene <strong>and</strong> primary structure of an insect-specific peptide from the venom of<br />
the theraposid Brachypelma smithi. Corzo, G., Diego, E., Clement, H., Estrada, G.,<br />
Odell, G., Batista, C., Possani, L.D., Alagón, A.<br />
16. TRANSCRIPTOME ANALYSIS OF EXPRESSED SEQUENCE TAGS<br />
FROM Thalassophryne nattereri FISH VENOM GLANDS. Magalhães G.S 1,2 ,<br />
Junqueira de Azevedo I.L.M 3 , Lopes-Ferreira M 1 , Lorenzini D.M 4 , Ho P.L 3 , Mourada-Silva<br />
A.M<br />
17. CLONING AND CHARACTERIZATION OF A C-TYPE LECTIN FROM<br />
Thalassophryne nattereri FISH VENOM GLAND. Lopes-Ferreira M 1 , Magalhães<br />
G.S 1,2 , Junqueira de Azevedo I.L.M 3 , Elífio S.L 4 , Valente R.H 5,6 , Fox, J .W 6 , Ho P.L 3 ,<br />
Moura-da-Silva A.M 1* .<br />
18. Biochemical evidence that crotasin is a fully active gene in tissues of the South<br />
American rattlesnake Crotalus durissus terrificus Collares, M. de A. 1 ; Silva, A. R. P.<br />
da 1 ; Rádis-Baptista, G. 2 ; Grego, K. F. 1 ; Oguiura, N. 1*<br />
19. The Gene Family of Crotamine <strong>and</strong> Variations of its Content in Venoms of the<br />
South American Rattlesnake Crotalus durissus. Collares, M. de A. 1 ; Corrêa, P. G. 1 ;<br />
Suzuki, H. 2 ; Furtado, M. F. D. 1 ; Oguiura, N. 1*<br />
20. A profile of typical Viperidae toxins in the Lachesis muta venom gl<strong>and</strong>s<br />
transcriptome. Junqueira-de-Azevedo, I.L.M. 1* , Ching, A.T.C. 1 , Faria, F. 2 ,<br />
Nishiyama Jr, M.Y. 3 , Ho, P.L. 1 , Diniz, M.R.V. 4<br />
21. Atypical Viperidae cDNAs from Lachesis muta venom gl<strong>and</strong>s <strong>and</strong> their<br />
implication in snake toxin repertoire evolution. Junqueira-de-Azevedo, I.L.M. 1* ,<br />
Ching, A.T.C. 1 , Carvalho, E. 1 , Ho, P.L. 1 , Diniz, M.R.V. 2<br />
228<br />
22. The Bitis gabonica gabonica venom proteome: venomics vs. transcriptomics.<br />
Calvete, J.J. 1,* , Marcinkiewicz, C. 2 , Sanz, L. 1<br />
23. Loss of introns along the evolutionary diversification pathway of snake venom<br />
disintegrins evidenced by sequence analysis of genomic DNA from Macrovipera<br />
lebetina transmediterranea. Bazaa, A. 1 , Juárez, P. 2 , Marrakchi, N. 1 , Bel Lasfer, Z. 1 , El<br />
Ayeb, M. 1 , Calvete, J.J. 2,* , Sanz, L.<br />
24. Current state of Bothrops insularis venom proteome. A joint effort of the Rio<br />
de Janeiro Proteomic Network. Perales, J 1,5 ., Neves Ferreira, A.G.C 1,5 ., Valente,<br />
R.H 1,5 ., Chapeaurouge, D.A 1,5 ., Trugilho, M.R.O 1,5 ., Rocha, S.L.G 1,5 ., Junqueira,<br />
M 1,4,5 ., Leon, I.R 1,5 ., Ho, P.L 2 ., Junqueira de Azevedo, I.L.M 2 ., Dutra, D.L.S 3,5 .,<br />
Oliveira-Carvalho, A.L 3,5 ., Wermelinger, L.S 3,5 , Zingali, R.B 3,5 . <strong>and</strong> Domont, G.B 4 .<br />
25. Bothrops jararaca Venom Proteome. Valente, R.H. 1,4 ; Coelho, F.E.M.R.F. 1,4 ;<br />
Neves-Ferreira, A.G.C. 1,4 ; Leon, I.R. 1,4 ; Cidade, D.A. 2 , Albano, R.M. 2 , Domont,<br />
G.B. 3,4 <strong>and</strong> Perales, J. 1,4<br />
Toxin discovery, purification, biochemistry<br />
26. Post-translational amino acid epimerization in an excitatory conotoxin.<br />
Structures of the L- <strong>and</strong> D-forms. Norton, R.S. 1 , Wei, D., 1 Yang, X., 1 Babon, J.J., 1<br />
Buczek, O., 2 Olivera, B.M., 2 Bulaj, G., 2,3<br />
27. Biochemical characterization of venom from the South American colubrid<br />
Philodryas patagoniensis. Carreiro da Costa, R.S. 1 , Ferrari, E.F. 1 , Leonardo, S.D. 1 ,<br />
Prudêncio, L. 1 , Souza, G.H.M.F. 1 , Hyslop, S. 2 , Cogo, J.C. 1,*<br />
28. Miwaprin, a whey acidic protein-like in coral snake (Micrurus frontalis)<br />
venom. Moreira, K. G 1,2* , Silva, L. P 2 , Bloch Jr, C 2 .<br />
29. Purification <strong>and</strong> partial characterization of a deoxyribonuclease II from<br />
Bothrops alternatus (urutu) venom. Nascimento, J.M. 1,2,* , Collares-Buzato, C.B. 3 ,<br />
Hyslop, S. 1<br />
30. LC-MS Analysis of All PSP Toxins Using Anion Exchange <strong>and</strong> Reverse Phase<br />
Columns Connected in Series. Sachio Nishio 1* ,Takefumi Sagara 1 , Shigeto<br />
Taniyama 2 , Tamiko Hashimoto 1 , Naoyoshi Nishibori 1 <strong>and</strong> Manabu Asakawa 2<br />
31. <strong>Fish</strong>ing Toxins out of Snake Venoms Using the Antitoxin DM43 as a Tool for<br />
the Analysis of Subproteomes. Rocha, S.L.G 1 ., Neves-Ferreira, A.G.C 1 .,<br />
Chapeaurouge, A 1 ., Valente, RH 1 ., Trugilho, M.R.O 1 ., Léon, I.R 1 ., Domont, G.B 2 . <strong>and</strong><br />
Perales, J 1* .<br />
229
32. Novel peptides in Philodryas olfersii <strong>and</strong> Philodryas patagoniensis venoms<br />
detected by bidimensional liquid chromatography coupled to t<strong>and</strong>em nanoelectrospray<br />
mass spectrometry <strong>and</strong> de novo mass spectrum analysis. Souza,<br />
G.H.M.F. 1,2,* , Cogo, J.C. 3 , Eberlin, M.N. 2 , Hyslop, S. 1<br />
33. Development of a novel fluorometric assay for the evaluation of BoNT/A<br />
protease activity using genetically engineering a fluorogenic substrate. Ban, B. 1<br />
Yang, G.H. 1 1, 2<br />
, <strong>and</strong> Jung, H. H.<br />
34. PURIFICATION AND PARTIAL ENZYMATIC CHARACTERIZATION<br />
OF THE HYALURONIDASE FROM Crotalus durissus terrificus SNAKE<br />
VENOM. Cordeiro, A. T. 1 ; Tolini, M.M. 1 ; Bertazzi, D. T. 1 ; Giglio, J. R. 2 ; Arantes, E.<br />
C. 1 *<br />
35. Mass spectrometry strategies for venom mapping <strong>and</strong> peptide sequencing from<br />
crude venom of a single Bombus specimen. Menin L., Favreau P., Michalet S., Perret<br />
F., Cheneval O., Stöcklin M., Bulet P. <strong>and</strong> Stöcklin R.<br />
36. A New Family of Peptides Discovered in Snake Venoms from the Atheris genus<br />
by Mass Spectrometry Techniques. Favreau, P. 1 , Cheneval, O. 1 , Menin L. 1 ,<br />
Michalet, S. 1 , Principaud, F. 2 , Thai, R. 3 , Ménez, A. 3 , Bulet, P. 1 <strong>and</strong> Stöcklin, R. 1 *<br />
37. Quantitative high-throughput LC-MS detection of cyanotoxins in aquatic<br />
tissue samples. Lähde, M.-R. , Meriluoto, J.A.O.<br />
38. Improved EC method for the determination of cyanide in feed. M. Baltussen, D.<br />
Luykx, A. Koot, R. Frankhuizen <strong>and</strong> S. van Ruth<br />
39. The sulfo-SBED derivative of ammodytoxin, a photoprobe for studying the<br />
molecular basis of phospholipase A2 β-neurotoxicity. Kovačič, L. * , Šribar, J., Križaj,<br />
I.<br />
40. Purification <strong>and</strong> characterisation of two apoptosis-inducing L-amino acid<br />
oxidases from the venoms of selected Australian elapids. Bateman, E.H, 1* Venning,<br />
M.G, 2 Mirtschin, P.J 2,3 <strong>and</strong> Woods, A.E 2<br />
41. Discovery of a novel conopeptide-AmIXA: Prediction of functional<br />
pharmacophore(s) <strong>and</strong> mechanism of action. Saminathan, R 1 , Jois, S.D.S 2 , Sato,<br />
K 3 , Gopalakrishnakone, P 1* .<br />
42. Kunitz-type serine protease inhibitors from snake venoms. Trabi, M. 1,2* , Flight,<br />
S. 3 , Masci, P.P. 3 , de Jersey, J. 2 , Lavin, M.F. 1<br />
43. Identification of the active-site residues of the fungal virulence factor,<br />
phospholipase B. Tsukamoto, K 1* , Inoue, Y 1 , Tsuruga, A 1 <strong>and</strong> Obata, Y. 2<br />
230<br />
44. A Comparative Study of the Enzymatic <strong>and</strong> Physiological Activities of Two<br />
Viper Venom L-Amino Acid Oxidases. Samel, M. 1 , Tõnismägi, K. 1 , Trummal, K. 1 ,<br />
Rönnholm, G. 2 , Siigur, J. 1 , Kalkkinen, N. 2 , Siigur, E. 1 *<br />
45. Heterodimeric Disintegrin VLT-2 from Vipera lebetina Snake Venom. Siigur,<br />
J.*, Vija, H., Aaspõllu, A., Samel, M., Tõnismägi. K., Trummal, K., Pikver, R., Subbi,<br />
J., Siigur, E.<br />
46. Discovery <strong>and</strong> pharmacology of Conopressin-T, a novel Vasopressin-like<br />
peptide from Conus Tulipa. Daniel Croker 1 , Sebastien Dutertre 2,3 , Paul Alewood 2 ,<br />
Richard Lewis 2<br />
47. Isolation <strong>and</strong> characterisation of Conomap-Vt, a D-amino acid containing<br />
excitatory peptide from the venom of a vermivorous cone snail. Sébastien<br />
Dutertre 1 , Natalie Lumsden, Paul F. Alewood <strong>and</strong> Richard J. Lewis<br />
48. Tityus perijanensis (Scorpiones, Buthidae) from Zulia State, western<br />
Venezuela: Geographic Distribution <strong>and</strong> Venom Molecular <strong>and</strong><br />
Immunological Characterization. Borges, A. 1* , Blumer-Lairet, V. 1 , Poliwoda, S. 1 ,<br />
Alfonzo, M.J. 1 , Rojas-Runjaic, F. 2 , De Sousa, L. 3<br />
49. Error-prone DNA polymerase k from Protobothrops flavoviridis, member of<br />
the DinB superfamily. Oda-Ueda, N 1 ., Kariu, T 1 ., Fukuhara, A 2 ., Takazaki, S 1 ,<br />
Chijiwa, T 2 ., Ohno, M 2 .<br />
50. Post-translational modifications of venom components in Conus victoriae.<br />
Townsend, A 1, 2 , Scanlon, D 2 , O’Donnell, P 2 , Inserra, M 1 , Bingham, J-P 3 ,<br />
Satkunanathan, N 1 , Khalil Z 1, 4 , Purcell, A 1, 2 1, 2*<br />
, Livett, B.G.<br />
231
Poster 1: Inter-specific comparison of the toxin encoding transcriptomes of<br />
medically important African vipers, E. ocellatus, C. cerastes <strong>and</strong> B. arietans.<br />
Wagstaff, S.C.*, <strong>and</strong> Harrison, R.A.<br />
Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool,<br />
L3 5QA, UK *simonw@liv.ac.uk<br />
Generating expressed sequence tags (EST) from medically important venomous snakes<br />
provides an unbiased global panorama of venom gl<strong>and</strong> transcriptional activity, <strong>and</strong> by<br />
inference, insights into the composition of the venom proteome. Here, we describe the<br />
multi-isoform toxin encoding profiles of three identically constructed venom gl<strong>and</strong> EST<br />
libraries from medically important <strong>and</strong> geographically distinct vipers, E. ocellatus<br />
(Nigeria), C. cerastes cerastes (Egypt) <strong>and</strong> B. arietans (Nigeria).<br />
To assist interpretation of these typically complex, multi-toxin, multi isoform<br />
transcriptomes we employed a variety of bioinformatic methodologies including BLAST,<br />
st<strong>and</strong>ard gene ontology (GO) annotation <strong>and</strong> motif searches in order to highlight<br />
potentially important differences between isoforms.<br />
In addition to identifying several transcripts that may play unique roles in the venomous<br />
strategy of individual species, inter-specific comparison of these transcriptomes also<br />
revealed clear differences in toxins encoding profiles, most notably variations in the<br />
diversity <strong>and</strong> pseudo-expression levels of serine proteinases relative to snake venom<br />
metalloproteinases. This analysis also revealed several motifs within catalytic or integrin<br />
adhesive motifs of several venom toxins that may have important functional implications.<br />
The significance of these findings is discussed.<br />
o EST<br />
o Transcriptome<br />
o Venom<br />
o Bioinformatics<br />
232<br />
Poster 2: An O-conotoxin-like Cys framework <strong>and</strong> hydroxyproline in peptides of<br />
the venom of the turrid Polystira albida from the Bay of Campeche, México<br />
Aguilar, M.B.*, Chan de la Rosa, R.A., Falcón, A., Heimer de la Cotera, E.P.<br />
National Autonomous University of Mexico, Campus UNAM-UAQ Juriquilla, Juriquilla, Qro., México,<br />
*maguilar@servidor.unam.mx<br />
Introduction: Superfamily Conoidea (families Conidae, Terebridae <strong>and</strong> Turridae) includes<br />
predatory, venomous snails. Conotoxins are short, disulfide-rich peptides that often contain<br />
post-translational modifications <strong>and</strong> that target ion channels, receptors, <strong>and</strong> transporters.<br />
Conotoxins are classified into gene superfamilies <strong>and</strong> pharmacological families according<br />
to their signal peptides <strong>and</strong> molecular targets, respectively, <strong>and</strong> have provided molecular<br />
tools, diagnostic agents, pharmaceuticals, <strong>and</strong> drug leads. The venom of the terebrid<br />
Terebra subulata contains some "augertoxins" similar to the O- <strong>and</strong> I-conotoxins, with no<br />
post-translational modifications other than disulfides, whereas those of the turrids<br />
Gemmula periscelida <strong>and</strong> Polystira albida include long, methionine-rich "turritoxins".<br />
Our objective was to find conotoxin-like peptides in the venom of the turrid P. albida.<br />
Methods: The venom ducts of 10 specimens of Polystira albida were dissected <strong>and</strong> used<br />
to prepare a crude extract, which was fractionated by RP-HPLC (C18). Selected peptides<br />
were further purified (C18, C8) <strong>and</strong> then subjected to automated Edman degradation.<br />
Results: Three peptides were purified from three medium-sized peaks in the crude extract.<br />
The partial amino acid sequences obtained are (C, putative Cys; O, hydroxy-Pro):<br />
Pa1: YGDSVTSDECQTISCYEAHVGOVCQOGEYD...; Pa2: ECTPVHFSCTCTGLE...;<br />
Pa3: NVCDGDACPDGVCCSGCTTDFNVAQRKDTCFYPQ... .<br />
Discussion/Conclusion: The three turritoxins characterized in this work seem to contain<br />
multiple Cys residues, as do the conotoxins. Pa3 has an O-conotoxin-like framework <strong>and</strong> is<br />
more similar to ω-conotoxins SVIA, GVIIA, <strong>and</strong> GVIIB than to augertoxins s6a <strong>and</strong> s7a.<br />
Recently, mRNAs encoding O-, P-, <strong>and</strong> I-conotoxin-like peptides have been found in the<br />
turrid Lophiotoma olangoensis Olivera 2002 (1). Within Conoidea, Pa1 is the first non-<br />
Conus peptide shown to contain hydroxy-Pro. This finding implies that hydroxylation of<br />
Pro occurred in the venom of the common ancestor of families Conidae <strong>and</strong> Turridae.<br />
References: 1. Watkins, M. et al. (2006) J. Mol. Evol., 62, 247-256.<br />
Supported by PAPIIT-UNAM (IN206701 <strong>and</strong> IX211904) <strong>and</strong> CONACYT (41477-Q).<br />
Key Words: type your keywords here (no more than four words)<br />
o Turridae<br />
o Polystira albida<br />
o O-conotoxin-like<br />
o Hydroxyproline<br />
233
Poster 3: Gene Expression Profiling <strong>and</strong> Fingerprinting of Human Genome<br />
Following Exposure to Natural Toxins <strong>and</strong> Nerve Agents – TOXINOGENOMICS<br />
Gopalakrishnakone, P *, Pachiappan, A.<br />
Venom <strong>and</strong> Toxin Research Programme, Department of Anatomy, Yong Loo Lin School of Medicine,<br />
National University of Singapore, Singapore 119260, *antgopal@nus.edu.sg<br />
Conventional methods such as northern blotting <strong>and</strong> RNAse protection assays generally do<br />
not provide sufficient throughput to exploit the genomic data. High-density oligonucleotide<br />
gene profiling studies hold promise for biomarker identification <strong>and</strong> for delineation of<br />
global alterations in molecular expression after exposure of cells <strong>and</strong> tissues to toxins. In<br />
the present study, individual functions of predicted ORF’s within the genome <strong>and</strong> the<br />
relationship between genes at the expression level are investigated through the use of<br />
genome sequence data accumulated after exposure of human glial cells to natural toxins<br />
<strong>and</strong> nerve agents. We have used six toxins of three different classes (Neurotoxins, Nerveagents,<br />
Hepatotoxins). Following exposure of human brain cell lines to neurotoxins –<br />
c<strong>and</strong>oxin (1), tetrodotoxin (2) <strong>and</strong> domoic acid, <strong>and</strong> to nerve-agents - soman, <strong>and</strong> sarin,<br />
gene expression profiling was done using Affymetrix (HG-U133A) human GeneChips. For<br />
comparison, human liver cell lines were exposed to hepatotoxin – aflatoxin B, with rigid<br />
filtering st<strong>and</strong>ards used to fish-out relevant genes of interest. With the aid of GeneSpring<br />
(PCA) analysis, 257 genes whose expression was significantly (p < 0.01) altered by at least<br />
2.5-fold, were selected <strong>and</strong> clustered by genesis software. The emerging data from this<br />
study indicate a significantly different global expression pattern seen with the toxinexposed<br />
human brain cell lines as compared to unexposed cells. Such data, when used as a<br />
fingerprint to characterize toxins, will provide possible insights into the mechanism/s of<br />
actions of these toxins at molecular level.<br />
o High density oligonucleotide gene profiling<br />
o Tetrodotoxin<br />
o Sarin<br />
o Human glial cells<br />
234<br />
Poster 4: Molecular cloning of dipeptidyl-peptidase IV (CD26) from Bothrops<br />
alternatus (urutu) snake venom gl<strong>and</strong> cDNA<br />
Cardoso, K.C. 1 , da Silva, J.M. 2,3 , Souza, G.H.M.F. 1 , Menossi, M.T. 2,3 , Hyslop, S. 1<br />
1 Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas<br />
(UNICAMP), CP 6111, 13083-970, Campinas, SP, Brazil *kiara@fcm.unicamp.br;<br />
hyslop@fcm.unicamp.br<br />
2 Departamento de Genética e Evolução, Instituto de Biologia <strong>and</strong> 4 Centro de Biologia Molecular e<br />
Genética (CBMEG), Universidade Estadual de Campinas (UNICAMP), CP 6109, 13083-970, Campinas,<br />
SP, Brazil<br />
Introduction: Snake venoms contain a variety of peptidases, including dipeptidylpeptidase<br />
IV (DPP IV) or CD26 that may contribute to envenoming [1-3]. DPP IV,<br />
which cleaves after proline residues in neuropeptide Y, peptide YY, growth hormonereleasing<br />
hormone, glucagon-like peptides 1 <strong>and</strong> 2, <strong>and</strong> substance P, may have a role in<br />
venom-induced hypotension [1]. In this work, we isolated <strong>and</strong> sequenced a cDNA<br />
encoding DPP IV from a cDNA library prepared from poly (A) + RNA of the venom<br />
gl<strong>and</strong> of the snake Bothrops alternatus. Methods: Total RNA was extracted from the<br />
venom gl<strong>and</strong>s three days after venom milking. A cDNA library was prepared using<br />
st<strong>and</strong>ard procedures <strong>and</strong> the DPP IV gene was cloned from this library. R<strong>and</strong>om positive<br />
clones were sequenced using a Perkin-Elmer ABI Prism Model 310 sequencer. Results:<br />
An open reading frame (ORF) sequence of 2286 base pairs encoded a mature, 762-amino<br />
acid protein with a calculated molecular mass of 86.2 kDa <strong>and</strong> a theoretical pI of 6.11.<br />
Multiple sequence alignment using Clustal X <strong>and</strong> phylogenetic analysis showed that this<br />
protein grouped with other DPP IV from various vertebrate species <strong>and</strong> was essentially<br />
identical to DPP IV from Gloydius blomhoffi brevicaudus (NCBI no.<br />
AB158224/158225). Computer modeling showed that the protein contained numerous αhelix<br />
<strong>and</strong> β-sheet regions, with the N-terminal region being highly conserved, as in other<br />
DPP IV. The active site contained Serine630, which is characteristic of this group of<br />
enzymes. Conclusion: DPP IV from B. alternatus venom is a high molecular mass<br />
protein that shares similarities with other venom <strong>and</strong> non-venom DPP IV. The successful<br />
expression of this protein should provide material to allow study of its biological<br />
activity. This is the first characterization of a DPP IV from Bothrops snake venoms.<br />
References: 1. Aird SD (2002) Toxicon 40, 335-393; 2. Anderson L, Bussler B, Martins<br />
H, Dufton M (1998) Toxicon 36, 719-728; 3. Gasparello-Clemente E, Silveira PF (2002)<br />
Toxicon 40, 1617-1626.<br />
Financial support: CAPES, CNPq, FAPESP.<br />
o Bothrops alternatus<br />
o Peptidases<br />
o Venom gl<strong>and</strong> library<br />
o Dipeptidyl-peptidase IV<br />
235
Poster 5: Phylogeny of Protobothrops Snakes Inhabiting the Southwestern Isl<strong>and</strong>s<br />
of Japan<br />
Chijiwa, T 1 *, Tomino, H 1 , So, S 1 , Oda-Ueda, N 2 , Hattori, S 3 , Ohno, M. 1<br />
1<br />
Sojo University, Faculty of Biological <strong>and</strong> Bioscience, 4-22-1 Ikeda, Kumamoto, Japan, *chijiwa@life.sojou.ac.jp<br />
2<br />
Sojo University, Faculty of Pharmacological, 4-22-1 Ikeda, Kumamoto, Japan<br />
3<br />
University of Tokyo, Institute of Medical Science, Tean-Sude802 Setouchi-chou, Ohshima-gun, Kagoshima,<br />
Japan<br />
Introduction: Three species of Protobothrops snakes inhabit the south western isl<strong>and</strong>s of<br />
Japan: P. flavoviridis (Habu snake) in Amami-Oshima, Tokunoshima, Okinawa <strong>and</strong><br />
Kumejima isl<strong>and</strong>s, P. tokarensis (Tokara Habu) in the Tokara Isl<strong>and</strong>s (Takarajima <strong>and</strong><br />
Kodakarajima isl<strong>and</strong>s) <strong>and</strong> P. elegans (Sakaishima Habu) in the Sakaishima Isl<strong>and</strong>s<br />
(Ishigaki <strong>and</strong> Iriomote isl<strong>and</strong>s). The classification of these snakes had been made in the<br />
conventional way, mainly based on their morphological features. However, the recent<br />
comparative chromatographic <strong>and</strong> biochemical studies showed that the constituents of the<br />
venom-gl<strong>and</strong> isozymes of these snakes have diversified depending on their environments.<br />
This may provide a new clue to consider the relationship between geological history of the<br />
isl<strong>and</strong>s <strong>and</strong> evolution of the snakes. In this work, to ascertain the phylogenetic relationship<br />
of these snakes, the D-loop region of mitochondrial DNA of each snake was sequenced<br />
<strong>and</strong> the phylogenetic tree was constructed.<br />
Methods: The mitochondrial DNAs were extracted from livers of P. flavoviridis snakes in<br />
Amami-Oshima, Tokunoshima, Okinawa, Kumejima isl<strong>and</strong>, of P. tokarensis in<br />
Kodakarajima isl<strong>and</strong> <strong>and</strong> of P. elegans in Ishigaki isl<strong>and</strong>. The D-loop region of each<br />
mitochondrial DNA was amplified with specific primers <strong>and</strong> subcloned into the plasmid<br />
vector <strong>and</strong> sequenced. The nucleotide sequences were aligned <strong>and</strong> their relative evolutional<br />
distances were estimated by the neighbour-joining method to construct the phylogenetic<br />
tree.<br />
Results: The topology of the phylogenetic tree showed that Protobothrops genus in the<br />
southwestern isl<strong>and</strong>s of Japan could be classified into three major groups; the Satsunan<br />
Isl<strong>and</strong>s’ group (Kodakarajima, Amami-Oshima <strong>and</strong> Tokunoshima isl<strong>and</strong>s), the Ryukyu<br />
Isl<strong>and</strong>s’ (Okinawa <strong>and</strong> Kumejima isl<strong>and</strong>s) <strong>and</strong> the Sakishima Isl<strong>and</strong>s’ (Ishigaki isl<strong>and</strong>).<br />
Discussion/Conclusion In contrast to their morphological variation, the phylogeny of<br />
Protobothrops genus snakes in the southwestern isl<strong>and</strong>s of Japan is well in accordance<br />
with the geological history of the isl<strong>and</strong> where they lived.<br />
o Protobothrops genus<br />
o Mitochondria D-loop<br />
o Phylogeny<br />
o Evolution<br />
236<br />
Poster 6: The main toxins of the Colubridae snake Philodryas olfersii revealed from<br />
a Duvernoy´s (venom) gl<strong>and</strong> transcriptome<br />
Ching, A.T.C. 1* , Rocha, M.M.T. 2 , Paes-Leme, A. F. 3 , Pimenta, D. C. 3 , Furtado, M. F. D. 2 , Serrano, S.<br />
M. T. 3 ; Ho, P. L. 1 , Junqueira-de-Azevedo, I.L.M. 1<br />
1<br />
Centro de Biotecnologia, Instituto Butantan, Av. Vital Brasil, 1500 - São Paulo, Brazil- 05503-900<br />
*atung@butantan.gov.br<br />
2<br />
Laboratório de Herpetologia, Instituto Butantan, São Paulo, Brazil<br />
3<br />
Laboratório Especial de Toxinologia Aplicada, CAT-CEPID, Instituto Butantan, São Paulo, Brazil<br />
Introduction: The interest about venoms of Colubridae, a family of snakes relatively<br />
neglected in the venom studies, is increasing due to its importance in human envenoming<br />
accidents. Philodryas olfersii, a representative species of this group <strong>and</strong> the one involved<br />
in most accidents in Brazil, has a Duvernoy's gl<strong>and</strong> that produces a secretion exhibiting<br />
high hemorrhagic activity.<br />
Methods: We constructed a cDNA library from P. olfersii Duvernoy’s gl<strong>and</strong> for the<br />
generation of an Expressed Sequence Tags database (dbEST) aiming to characterize its<br />
transcriptome. The ESTs were clustered, searched against public databases <strong>and</strong> catalogued.<br />
Results: We obtained 2194 clones with an average of 415.7 bp that were grouped in 172<br />
clusters with two or more sequences <strong>and</strong> 1113 singlets. Thirty percent of all transcripts of<br />
P. olfersii Duvernoy's gl<strong>and</strong> corresponded to toxin sequences, indicating the presence of<br />
most of the main toxin classes found in the Viperidae family. The P-III class of<br />
metalloproteases is the predominant (53% of toxin ESTs), but also serine proteases, C-type<br />
lectins, Cystein Rich Secretory Proteins (CRISPs), <strong>and</strong> a C-type natriuretic peptides<br />
(CNPs) were found. The corresponding proteins were detected in a 2-D gel of the venom<br />
<strong>and</strong> identified by mass spectrometry, showing a low venom complexity when compared to<br />
other snake families. Phylogenetic analysis of the CNP precursor showed it as a linker<br />
between the multifunctional precursors found in Viperidae <strong>and</strong> Elapidae snakes. We<br />
suggest that this precursor constitutes a monophyletic group derived from the vertebrate<br />
CNPs. Other sequences were investigated <strong>and</strong> suggested as possible toxin c<strong>and</strong>idates.<br />
Conclusion: The P. olfesii dbEST is the first effort to massively identify cDNA sequences<br />
from a Colubridae species. It matches the venom composition, provides an overview of a<br />
Colubridae venom <strong>and</strong> supports the important role of metalloproteases in the hemorrhagic<br />
effect elicited during P. olfersii envenoming.<br />
Support: FAPESP<br />
o Colubridae<br />
o transcriptome<br />
o Duvernoy's gl<strong>and</strong><br />
o C-type natrituretic peptide<br />
237
Poster 7: A comparison of the venom from left <strong>and</strong> right fangs of three Australian<br />
elapid’s (Pseudechis australis, Psuedonaja textiles <strong>and</strong> Notechis scutatus).<br />
Winter, K.L., 1* Mirtschin, P.J., 2 Hodgson, W.C. 1<br />
1<br />
Monash Venom Group, Dept. Pharmacology, Monash University, Victoria, Australia, 3800. *<br />
kelly.winter@med.monash.edu.au<br />
2<br />
Venom Supplies Pty. Ltd, Tanunda, South Australia, Australia, 5352.<br />
Introduction: Australia is home to many of the world’s most venomous snakes. Previous<br />
studies have shown differences in venom components within the same species from<br />
different geographical locations. Similarly an examination of specimens with different<br />
body <strong>and</strong> head sizes has shown that these are related to differences in venom yields<br />
(Mirtschin et al., 2002). This study examined venom yields <strong>and</strong> activity from the left <strong>and</strong><br />
right fangs of individual specimens of Pseudechis australis (Mulga snake), Psuedonaja<br />
textilis (eastern brown snake) <strong>and</strong> Notechis scutatus (common tiger snake).<br />
Methods: Venom from the left <strong>and</strong> right fangs of individual specimens was milked.<br />
Reverse phase (rp)HPLC was used to compare elution profiles of the venoms. The chick<br />
biventer cervicis nerve-muscle preparation (0.1 Hz, 0.2 ms, supramaximal V) was used to<br />
examine the venoms for differences in neurotoxic activity.<br />
Results: There was a significant difference in the amount of venom produced by the left<br />
<strong>and</strong> right fangs of individual snakes. However, rpHPLC showed no major differences in<br />
the composition of the venoms from each of the species (i.e. no major peaks present or<br />
missing from either the left or right fang). No difference in neurotoxic activity, as<br />
determined by the time taken to produce 90% inhibition of indirect twitches (i.e. t90), was<br />
identified between left <strong>and</strong> right fangs from the three species examined (see Table).<br />
Discussion/Conclusions: Although the venom yields between the left <strong>and</strong> right fangs<br />
Species / fang side t90 (min), n=4<br />
P. australis left 111 ± 5<br />
P. australis right 116 ± 5<br />
N. scutatus left 83 ± 13<br />
N. scutatus right 76 ± 16<br />
P. textilis left 33 ± 6<br />
P. textilis right 29 ± 4<br />
o snake<br />
o venom<br />
o neurotoxic<br />
showed variations in the amount of venom<br />
produced there appears to be no major difference<br />
in the composition of the venom <strong>and</strong> the<br />
neurotoxic activity. The reasons for the<br />
differences in yields between the left <strong>and</strong> right<br />
fangs are not yet apparent.<br />
Reference: Mirtschin et al., (2002) Toxicon, 40,<br />
1581-1592.<br />
238<br />
Poster 8: Gene Structures of Two Functionally Diverse Prothrombin Activators,<br />
Trocarin D <strong>and</strong> Coagulation Factor X in Tropidechis carinatus Snake: Gene<br />
Duplication <strong>and</strong> Recruitment of Factor X Gene to the Venom Gl<strong>and</strong><br />
Reza, MA 1 , Swarup, S 1 , Kini, RM 1, 2<br />
1 National University of Singapore, Singapore<br />
2 Virginia Commonwealth University, Richmond, Virginia<br />
The occurrence of structurally <strong>and</strong> functionally similar proteins in specialized organs with<br />
highly diverse physiological roles within a single organism is a rare phenomenon. Origin<br />
<strong>and</strong> evolution of such specialized organs <strong>and</strong> their functions are of great interest. Recently,<br />
we showed that several Australian elapid snakes have two similar prothrombin activating<br />
systems. One exists in plasma <strong>and</strong> plays a crucial role in hemostasis, whereas the other<br />
exists in the venom gl<strong>and</strong> <strong>and</strong> acts as toxin Here we present the complete gene structure of<br />
these two divergent prothrombin activators from Tropidechis carinatus. Both of the genes<br />
have 8 exons <strong>and</strong> all the exon-intron boundaries are almost at the same position. Introns of<br />
these two genes show high identity (>85%), indicating a recent gene duplication event.<br />
Real time PCR results indicate that these two genes are expressed in a highly tissuespecific<br />
manner. Interestingly, the promoter of trocarin D has an insertion of 264 bp (-29 to<br />
-293). This small insertion carries core promoter sequences <strong>and</strong> cis-elements that are<br />
known to induce high level of expression. We speculate that this insert might be<br />
responsible for the gene recruitment <strong>and</strong> acts as a switch for venom gl<strong>and</strong>-specific<br />
expression <strong>and</strong> we named this segment as VERSE (Venom Recruitment/Switch Element).<br />
This is the first molecular evidence demonstrating the recruitment of a duplicated gene<br />
expressed in the liver for expression in a highly specialized organ (venom gl<strong>and</strong>s) by a<br />
simple insertion.<br />
o Prothrombin activation<br />
o Factor X<br />
o Haemostasis<br />
o Snake venom<br />
239
Poster 9: Modulation of transcription activities between group IA <strong>and</strong> IB<br />
phospholipase A2 genes in sea snake Laticauda semifasciata<br />
Hayashi, Y,, Sagitani, A., Fujimi, T. J., Kanzawa, N., Tsuchiya, T., Tamiya, T.*<br />
Sophia University 7-1, Kioi-cho,Chiyoda-ku, Tokyo 102-8554, Japan,* t_tamiya@sophia.ac.jp<br />
Phospholipase A2 (PLA2) genes expressed in the venom gl<strong>and</strong>s of the sea snake Laticauda<br />
semifasciata, were investigated. Both kinds of mRNAs, encoding group IA (without a<br />
pancreatic loop) <strong>and</strong> group IB (with a pancreatic loop), were detected in the venom gl<strong>and</strong>s<br />
by Northern blot hybridization analysis <strong>and</strong> RT-PCR. Quantitative PCR analysis revealed<br />
that the amount of group IA mRNA was about 100-300 times greater than that of group IB<br />
mRNA. Recently, we determined the abundance ratio of group IA to IB PLA2 genes (2:1)<br />
in the genome of L. semifasciata. The difference between the amount of transcribed<br />
mRNAs encoding group IA <strong>and</strong> IB PLA2s did not agree with the number of gene copies.<br />
Nucleotide sequences of group IA <strong>and</strong> IB PLA2 genes in the upstream regions were<br />
determined. The sequences from the transcription initiation site to -33 <strong>and</strong> -445 to -633 bp<br />
in the group IA PLA2 genes <strong>and</strong> from there to -220 bp in the group IB PLA2 genes were<br />
highly homologous (ecIAIB consensus sequences). Electrophoresis mobility shift assay<br />
<strong>and</strong> DNase I footprint assay were carried to verify transcription factor binding sites in the<br />
ecIAIB conserved region. All the binding sites detected were already registered in the<br />
Transcription Factor Database. In the group IA gene upstream sequence, the ecIAIB<br />
consensus sequence was interrupted by a sequence specific to group IA PLA2 genes. A<br />
CCAAT box (-91 to -87) in the group IB gene was shifted more than 400 bp upstream in<br />
the group IA gene by the inserted sequence. Thus, the sequence dramatically changed the<br />
structure of the promoter region between the group IA <strong>and</strong> IB genes of L. semifasciata.<br />
Luciferase reporter gene assays were carried out to examine the importance of the group<br />
IA specific sequence for transcription. The transcription activity of a chimera mutant<br />
(which had the group IA specific sequence inserted in the appropriate position of the group<br />
IB gene) showed the same level of group IA PLA2 genes upstream. The IA-specific<br />
sequence modulated the expression levels of the group IA <strong>and</strong> IB genes in the venom<br />
gl<strong>and</strong>s.<br />
o Snake toxin gene<br />
o Phospholipase A2 genes<br />
o Promoter region<br />
240<br />
Poster 10: Maturation signatures in the sequences of natural toxins<br />
Kozlov S., Grishin E.<br />
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-<br />
Maklaya, 16/10, 117997 Moscow, Russia<br />
Recent data collected for animal toxins reveal an important role of maturation processes in<br />
active molecules formation. Every novel precursor sequence deduced from gene should be<br />
analyzed to define mature polypeptide chain, before synthesis <strong>and</strong> activity measurements<br />
are carried out. In many cases, multiple sequence alignment with known homologues<br />
produces faithful results, but the exact primary structure can be determined precisely by<br />
finding specific maturation motifs. We analyze the available sequence data of animal toxin<br />
precursors obtained from several species of Conus snails, sea anemones, spiders,<br />
scorpions, bees, ants, <strong>and</strong> amphibians. The analysis reveals specific motifs for directed<br />
enzymatic cleavage simultaneously present in toxin precursor sequences immediately<br />
before <strong>and</strong> after the mature chain. One of the motifs is well defined for specific maturation<br />
of polypeptide molecules in various animals, whereas other motifs are found only in<br />
venomous species. In case of spider <strong>and</strong> frog cytotoxins, we describe a new “paired” motif<br />
typical for precursors that are processed into more than two mature active compounds. The<br />
information from the primary structure of precursor proteins allowed us to produce<br />
assumable maturation maps that are quite species-specific. Differences were found not<br />
only in precursor protein organization, but also in the set of specific enzymes thought to be<br />
involved in toxin processing events.<br />
o primary structure motif<br />
o precursor maturation<br />
o toxins processing<br />
241
Poster 11: Proteomic analysis of the venom from the Mexican scorpion Centruroides<br />
limpidus limpidus.<br />
Gurrola, G.B., Batista, C.V.F., Zamudio, F.Z. <strong>and</strong> Possani, L.D*.<br />
Department of Molecular Medicine <strong>and</strong> Bioprocesses. Institute of Biotechnology –<br />
National Autonomous University of Mexico *possani@ibt.unam.mx<br />
Scorpion venoms are complex mixtures of peptides that display a variety of different<br />
biological activities. These venoms have been studied in the light of their pharmacological<br />
targets <strong>and</strong> many of their constituents were described to affect the function of a variety of<br />
ion channels of excitable <strong>and</strong> non excitable membranes. This communication describes the<br />
separation of approximately 90 different components by high performance liquid<br />
chromatography (HPLC) of the venom from the scorpion Centruroides limpidus limpidus .<br />
Mass spectrometry analysis of the soluble venom shed light on the molecular composition<br />
of the venom <strong>and</strong> facilitated the search for novel pharmacologically active components. At<br />
least 318 distinct molecular weight components were identified. The complete amino acid<br />
sequence of 3 peptides was determined <strong>and</strong> the partial sequence of 10 additional<br />
components was also identified. The molecular masses of peptides eluting at 30 to 40 min<br />
elution time were from 6.5 to 8.0 kDa. Most of them correspond to long chain toxins<br />
specific for voltage-gated Na + channels. The components eluting at 20 to 30 min elution<br />
time have molecular weights from 2.5 to 5.0 kDa, among which are most of the short-chain<br />
peptides that block K + -channels. At the higher molecular weight range an enzyme with<br />
hyaluronidase activity was found, with molecular mass of 44.396 kDa. This enzyme was<br />
shown to be glycosilated <strong>and</strong> highly active using hyaluran as substrate. Separation by<br />
HPLC in the presence of trifluoacetic acid <strong>and</strong> acetonitrile resulted in a series of isoforms<br />
with the same molecular masses, but different elution times. The amino acid sequence of<br />
the N-terminal region of the enzyme was determined.<br />
Acknowledgements: Supported in part by DGAPA-UNAM IN-206003 to LDP <strong>and</strong> GBG;<br />
CONACyT grant number 47879-Q to CVFB <strong>and</strong> grant from Instituto Bioclon S.A. de C.V.<br />
(Mexico)<br />
o Scorpion toxin<br />
o Mass spectrometry<br />
o Centruroides limpidus limpidus<br />
o Hyaluronidase<br />
242<br />
Poster 12: Venom Proteomes of Closely-related Sistrurus Rattlesnakes with<br />
Divergent Diets<br />
Sanz, L. 1 , Gibbs, H.L. 2 , Mackessy, S.P. 3 , Calvete, J.J. 1,*<br />
1<br />
Instituto de Biomedicina de Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain.<br />
*Libia.Sanz@ibv.csic.es<br />
2<br />
Department of Evolution, Ecology <strong>and</strong> Organismal Biology, Ohio State University, 300 Aronoff<br />
Laboratory, 318 W. 12th Ave., Columbus, OH 43210-1293, USA<br />
3<br />
School of Biological Sciences, University of Northern Colorado, 501 20th St., CB 92, Greeley, CO<br />
80639-0017, USA<br />
Venoms represent the critical innovation that allowed advanced snakes to transition from a<br />
mechanical (constriction) to a chemical (venom) means of subduing <strong>and</strong> digesting prey<br />
larger than themselves, <strong>and</strong> as such, venom proteins have multiple functions including<br />
immobilizing, paralyzing, killing <strong>and</strong> digesting prey. Given the central role that diet has<br />
played in the adaptive radiation of snakes 14 , venom thus represents a key adaptation that<br />
has played an important role in the diversification of these animals.The protein<br />
composition of the venoms of the three subspecies of Sistrurus catenatus (S. c. catenatus,<br />
tergeminus, <strong>and</strong> edwardsii) <strong>and</strong> a basal species, Sistrurus miliarius barbouri, were<br />
analyzed by RP-HPLC, N-terminal sequencing, MALDI-TOF peptide mass fingerprinting<br />
<strong>and</strong> CID-MS/MS. The venoms of the four Sistrurus taxa contain proteins from 11 families.<br />
The protein family profile <strong>and</strong> the relative abundance of each protein group in the different<br />
venoms are not conserved. Myotoxins <strong>and</strong> 2-chain PLA2s were present only in S.c.<br />
catenatus <strong>and</strong> S.c. tergeminus, whereas C-type BPP <strong>and</strong> Kunitz-type inhibitors were<br />
exclusively found in S.c. edwardsii <strong>and</strong> Sistrurus miliarius barbouri. Among major protein<br />
families, taxa were most similar in their metalloproteases (protein similarity coefficient<br />
value: 34%) while most divergent in PLA2s (12%), with values for disintegrins <strong>and</strong> serine<br />
proteases lying between these extremes (25% <strong>and</strong> 20% respectively). The patterns of<br />
venom diversity points to either a gain in complexity in S. catenatus taxa or a loss of<br />
venom diversity occurring early on in the evolution of the group involving the lineage<br />
connecting S. milarius to the other taxa. The high degree of differentiation in the venom<br />
proteome among recently-evolved congeneric taxa emphasizes the uniqueness of the<br />
venom composition of even closely related species which have different diets.<br />
Comparative proteomic analysis of Sistrurus venoms provides a comprehensive catalogue<br />
of secreted proteins, which may contribute to a deeper underst<strong>and</strong>ing of the biology <strong>and</strong><br />
ecology of these North American snakes, <strong>and</strong> may also serve as a starting point for<br />
studying structure-function correlations of individual toxins.<br />
o Sistrurus<br />
o Snake venomics<br />
o Mass spectrometry<br />
o Venom protein families<br />
243
Poster 13: Molecular cloning of a non-venom-secreted PII disintegrin-like<br />
transcript, BA-5A, from a Bitis arietans cDNA library reveals a pathway for the<br />
evolution of the long-chain disintegrin bitistatin from a PIII disintegrin-like<br />
precursor<br />
Juárez, P. 1* , Wagstaff, S.C. 2 , Oliver, J. 2 , Sanz, L. 1 , Harrison, R.A. 2 , Calvete, J.J. 1<br />
1<br />
Instituto de Biomedicina de Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain.<br />
*pjuarez@ibv.csic.es<br />
2<br />
Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool<br />
L3 5QA, Engl<strong>and</strong>, UK<br />
Disintegrins represent a family of integrin receptor antagonists that are released in viper<br />
venoms by proteolytic processing of PII snake venom metalloproteinase (SVMP)<br />
precursors or synthesized from short-coding mRNAs. Functional diversification between<br />
disintegrins is mainly due to amino acid substitutions within the active loop, whereas<br />
structural diversification was driven through a disulfide bond engineering mechanism<br />
involving the selective loss of pairs of cysteine residues engaged in the formation of<br />
disulfide bonds. The evolutionary pressure acting to promote high levels of variation in<br />
venom proteins may be part of a predator-prey arms race that allow the snake to adapt to a<br />
variety of different prey, each of which are most efficiently subdued with a different<br />
venom formulation. However, the molecular details of the mechanisms leading to snake<br />
venom toxin diversification remains unclear.We report the cloning <strong>and</strong> sequence analysis<br />
of BA-5A from Bitis arietans, a novel <strong>and</strong> unique ECD-disintegrin-like domain.It contains<br />
the 16 cysteine residues that are conserved in all PIII disintegrin-like domains but lacks the<br />
cysteine-rich domain. These features suggest that BA-5A represents an intermediate in the<br />
evolutionary pathway of the long disintegrin bitistatin, <strong>and</strong> that removal of the cysteinerich<br />
domain <strong>and</strong> loss of the PIII-specific disulfide bond were separate events along the<br />
structural diversification pathway of disintegrins, the former predating the latter. BA-5A<br />
could not be detected in the venom proteome of Bitis arietans using proteomic techniques.<br />
The occurrence of this very low abundance (
Poster 15: Gene <strong>and</strong> primary structure of an insect-specific peptide from the<br />
venom of the theraposid Brachypelma smithi†<br />
Corzo, G., Diego, E., Clement, H., Estrada, G., Odell, G., Batista, C., Possani, L.D., Alagón, A.<br />
Institute of Biotechnology-UNAM, Av. Universidad 2001, Cuernavaca, Morelos, 62210, Mexico.<br />
*corzo@ibt.unam.mx<br />
The soluble venom of the theraposid spider Brachypelma smithi (B. smithi) was screened<br />
for insecticidal toxins based on toxicity to house crickets. This venom was fractionated<br />
using reverse-phase <strong>and</strong> cation exchange chromatographies. An insecticidal peptide,<br />
named Bs1 was obtained. It contains 41 amino acids cross-linked by three disulfide<br />
bridges. The amino acid sequence of Bs1 is highly similar to the insecticidal peptides from<br />
the hexathelid spider Macrothele gigas (Japan) (1), <strong>and</strong> the theraposid spider<br />
Ornithoctonus huwena (China) (2) indicating their close phylogenetic relationship. The<br />
gene of Bs1 was cloned from a cDNA library, <strong>and</strong> genes related to Bs1 showed the<br />
presence of a wide range of similar toxins with slight variations in primary structure.<br />
Proteins codified by this family of genes contain signal peptides, which seem to be excised<br />
at a region rich in acidic <strong>and</strong> basic residues (3). Moreover, their C-terminals are amidated,<br />
where the residue that dictates such protein modification is just glycine. The exact target of<br />
this toxin is not known. Thus insecticidal peptides represent interesting new tools for the<br />
discovery of new cell receptors in insects. Generation of knowledge on the molecular basis<br />
of insect toxin specificity has a motivating potential, since they represent a possible<br />
application, via recombinant baculovirus in crop protection.<br />
1. Corzo, G. et al. (2003) FEBS Lett. 547:43-50.<br />
2. Yi, T.F et al. (2002) Zool. Res. 23:463-466.<br />
3. Satake, H. et al. (2004) Toxicon 44:149-156.<br />
†This work was supported in part by a grant from the Dirección General de Asuntos del<br />
Personal Académico (DGAPA-UNAM) IN226006.<br />
o Insect-specific<br />
o Genes<br />
o Spider<br />
o Peptide<br />
246<br />
Poster 16: TRANSCRIPTOME ANALYSIS OF EXPRESSED SEQUENCE TAGS<br />
FROM Thalassophryne nattereri FISH VENOM GLANDS<br />
Magalhães G.S 1,2 , Junqueira de Azevedo I.L.M 3 , Lopes-Ferreira M 1 , Lorenzini D.M 4 , Ho P.L 3 , Mourada-Silva<br />
A.M 1* .<br />
1 2 3<br />
Laboratório de Imunopatologia, Laboratório de Imunogenética <strong>and</strong> Centro de Biotecnologia, Instituto<br />
Butantan, São Paulo, Brazil; * anamoura@butantan.gov.br<br />
4<br />
Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade São Paulo, Brazil<br />
Thalassophryne nattereri (niquim) is a venomous fish found in northern <strong>and</strong> northeastern<br />
Brazilian coast. T. nattereri fish induces hundreds of human accidents characterized by<br />
excruciating local pain, edema, <strong>and</strong> necrosis that may lead to permanent disabilities. In<br />
experimental models, T. nattereri venom toxins showed to be highly active causing edema<br />
<strong>and</strong> nociception that has been correlated to human symptoms <strong>and</strong> dependent on venom<br />
kininogenase activity. Other activities such as myotoxicity; impairment of blood flow;<br />
platelet lysis <strong>and</strong> cytotoxicity on endothelial cells were also observed. In an attempt to<br />
characterize the primary structure of T. nattereri venom toxins, a list of transcripts within<br />
the venom gl<strong>and</strong> was accomplished using the expressed sequence tag (EST) strategy. The<br />
analysis of 775 ESTs from the venom gl<strong>and</strong> allowed us to isolate the cDNAs sequence of a<br />
family of kininogenases never before described in the databases. These sequences were<br />
named natterin 1, 2, 3, 4 <strong>and</strong> P. Except natterin 4, all the others were found in the whole<br />
venom. Furthermore, we found others putative toxins sequences also unknown to the<br />
databases, as well as interesting findings such as sequences matching with cocaine- <strong>and</strong><br />
amphetamine-regulated transcript (CART), which has been shown to be involved in food<br />
intake regulation (1.3%), retrotransposon-like sequences (1.0%), which might be related to<br />
the occurrence <strong>and</strong> diversity of many paralogous forms of toxins in this venom gl<strong>and</strong> <strong>and</strong>,<br />
a considerable number of sequences (32%) did not show any similarities in the databases,<br />
which indicates that a great number of new toxins/proteins are still to be discovered.<br />
o Thalassophrine nattereri<br />
o ESTs<br />
o natterin<br />
o toxin<br />
247
Poster 17: CLONING AND CHARACTERIZATION OF A C-TYPE LECTIN<br />
FROM Thalassophryne nattereri FISH VENOM GLAND<br />
Lopes-Ferreira M 1 , Magalhães G.S 1,2 , Junqueira de Azevedo I.L.M 3 , Elífio S.L 4 , Valente R.H 5,6 , Fox, J<br />
.W 6 , Ho P.L 3 , Moura-da-Silva A.M 1* .<br />
1 Laboratório de Imunopatologia, 2 Laboratório de Imunogenética <strong>and</strong> 3 Centro de Biotecnologia, Instituto<br />
Butantan, São Paulo, Brazil; * anamoura@butantan.gov.br 4 PUC,<br />
Curitiba, Brazil; 5 Fiocruz, Rio de Janeiro, Brazil; 6 University of Virginia, Charlottesville, USA.<br />
Using a venom gl<strong>and</strong> cDNA library from Thalassophryne nattereri fish, a C-type lectin<br />
transcript was cloned <strong>and</strong> characterized. Its 984 pb cDNA contains a 363 bp open reading<br />
frame from which a 121-amino-acid was deduced. The deduced amino acid sequence was<br />
found to contain a carbohydrate recognition domain (CRD) homologous with those of<br />
some known C-type animal lectins, although showing low general homology with those<br />
lectins. This novel C-type lectin was purified from T. nattereri venom whose N-terminal<br />
showed to match with the deduced amino acid sequence. This protein, named nattectin,<br />
revealed a single b<strong>and</strong> with an apparent molecular weight of 15 kDa under reduced <strong>and</strong><br />
unreduced conditions on SDS-PAGE gel. Analysis of the purified protein by MALD-TOF<br />
revealed a mass of ~15,087 indicating that this protein is composed of a single monomer,<br />
unlike C-type lectins from snake venoms that are composed exclusively of homodimers or<br />
homooligomers. Comparison of nattectin with a well characterized rat mannose-binding<br />
protein showed that it is a galactose-binding lectin that also contains conserved residues for<br />
Ca 2+ binding. Hemaglutination analysis showed that this lectin is Ca 2+ dependent. To our<br />
knowledge, this is the first characterization of a lectin from a venomous fish <strong>and</strong> this<br />
finding may contribute to elucidate the functions of other venom C-type lectins.<br />
o C-type lectins<br />
o Thalassophryne nattereri<br />
o cDNA library<br />
o toxin<br />
248<br />
Poster 18: Biochemical evidence that crotasin is a fully active gene in tissues of the<br />
South American rattlesnake Crotalus durissus terrificus<br />
Collares, M. de A. 1 ; Silva, A. R. P. da 1 ; Rádis-Baptista, G. 2 ; Grego, K. F. 1 ; Oguiura, N. 1*<br />
1 Laboratório de Herpetologia – Instituto Butantan, Av. Doutor Vital Brasil 1500 – CEP 05503-900 São<br />
Paulo, SP, Brazil, *nancyoguiura@butantan.gov.br.<br />
2 Dept. de Bioquímica e Laboratório de Imunopatologia Keizo Asami- Universidade Federal de Pernambuco<br />
(UFPE), Av. Professor Moraes Rego S/N – CEP 53670-910 Recife, PE, Brazil<br />
Crotasin, Cts-p2, is a crotamine-related gene of Crotalus durissus terrificus, a South<br />
American rattlesnake. Both crotamine <strong>and</strong> crotasin genes have three exons interrupted by<br />
two introns, one long <strong>and</strong> the other short. In the Cts-p2 gene, the longer intron showed an<br />
insertion of approximately 750 bp, <strong>and</strong> the exon 2 presented most of the non-synonymous<br />
substitutions. In fact, the overall gene organization shared by crotasin <strong>and</strong> crotamine is<br />
indicative of gene duplication <strong>and</strong> independent molecular evolution. A high level of<br />
expression of Cts-p2 transcripts was detected by reversed transcription coupled to<br />
polymerase chain reaction (RT-PCR) in pancreas, heart, liver, brain, <strong>and</strong> kidneys.<br />
Interestingly, <strong>and</strong> unlike crotamine, crotasin transcripts were scarcely detected in venom<br />
gl<strong>and</strong>s 1 . The biological function of crotasin is still unknown. The predicted amino acid<br />
sequence of crotasin shares a primary structure similarity with Gallus gallus beta-defensin<br />
2 (GI49037274), which belongs to the diverse class of antimicrobial peptide, <strong>and</strong> with the<br />
recently identified peptide POGL2-CLP from the lizard Pogona barbata 2 . This fact<br />
motivated us to investigate the biological <strong>and</strong>/or pharmacological activities of crotasin.<br />
Hence, we evaluated the crotasin expression in snake tissues by Immunoblot, <strong>and</strong> prepared<br />
recombinant crotasin fused with thioredoxin. A customized antibody was prepared with the<br />
synthetic peptide (cys-pro-ser-gly-thr-thr-ser-ile-gly-gln-gln-asp) conjugated with KLH<br />
carrier by rabbit immunization. Heart, liver, <strong>and</strong> pancreas proteins were then extracted,<br />
resolved by SDS-PAGE (15%T/2.6%C) <strong>and</strong> transferred to a nitrocellulose membrane<br />
using a semi-dry apparatus. To reveal the pattern of peptide expression in snake tissues, we<br />
used anti-crotasin as the primary antibody <strong>and</strong> anti-rabbit IgG conjugated with peroxidase<br />
as the secondary one. The immune complexes were viewed with DAB/CoCl2. As expected,<br />
crotasin peptide was detected in pancreas, liver <strong>and</strong> heart protein extracts, indicating that<br />
crotasin is a fully active gene in rattlesnakes, <strong>and</strong> that it may be involved in the innate<br />
defense mechanism of snake organs. Supported by FAPESP, FUNDAP. 1 Rádis-Baptista et<br />
al., 2004, Toxicon 43:751-759. 2 Fry et al., 2006, Nature 439: 584-588.<br />
o rattlesnake<br />
o crotasin<br />
o crotamine<br />
o antimicrobial peptide<br />
249
Poster 19: The Gene Family of Crotamine <strong>and</strong> Variations of its Content in Venoms<br />
of the South American Rattlesnake Crotalus durissus<br />
Collares, M. de A. 1 ; Corrêa, P. G. 1 ; Suzuki, H. 2 ; Furtado, M. F. D. 1 ; Oguiura, N. 1*<br />
1 Laboratório de Herpetologia <strong>and</strong> 2 Laboratório de Biologia Celular – Instituto Butantan, Av. Doutor Vital<br />
Brasil, 1500 – CEP 05503-900 São Paulo, SP, Brazil<br />
*nancyoguiura@butantan.gov.br<br />
Crotalus durissus is a rattlesnake that occurs from southern Mexico to the Argentinean<br />
pampas. Crotamine, which was first isolated from the venom of C. d. terrificus, belongs to<br />
a group of closely related small basic polypeptide myotoxins commonly found in Crotalus<br />
venoms <strong>and</strong> its content, averaging 17% (w/w) of the crude venom, varies according to the<br />
snake’s geographical location. The crotamine-positive rattlesnake has its crotamine gene<br />
with 1.8 kbp organized into three exons separated by a long phase-1 (900 bp) <strong>and</strong> a short<br />
phase-2 (140 bp) intron. The amount of crotamine in the venoms of C. durissus from the<br />
states of Paraná, São Paulo, Mato Grosso do Sul, Goiás, Bahia, <strong>and</strong> Maranhão were<br />
determined. The crotamine was quantified by ELISA <strong>and</strong> the protein content determined<br />
by the Lowry method. To analyze the crotamine genes, we amplified the sequence of part<br />
of exon 2, intron 2 <strong>and</strong> exon 3 by PCR using 5crot <strong>and</strong> 3UTRas primers. This fragment<br />
was cloned, <strong>and</strong> seven clones of each snake were sequenced. The quantity varied from<br />
0.006 to 0.5 µg of crotamine per µg of protein in the venom. Based on these results, the<br />
venoms were classified into three categories: crotamine-negative, less than 10% <strong>and</strong> more<br />
than 10% of crotamine content, which was plotted on a map. We found a large number of<br />
crotamine-plus venoms in the state of Paraná, including venom with a crotamine<br />
concentration exceeding 10%. We analyzed crotamine gene sequences of specimens from<br />
Paraná, São Paulo, Mato Grosso do Sul, Goiás <strong>and</strong> which showed an identical sequence to<br />
that described by Rádis-Baptista et al. (2003) 1 . The changes found in sequences were point<br />
mutations in introns <strong>and</strong> in an untranslated region, amino acid changes, <strong>and</strong> complete<br />
deletion of intron 2. These results indicated that crotamine is a gene family, since the same<br />
rattlesnake may have up to four different sequences. The rattlesnakes with the highest<br />
amount of crotamine showed no alteration in their crotamine genes.<br />
Supported by FAPESP <strong>and</strong> FUNDAP.<br />
1 Rádis-Baptista et al., 2003, Toxicon 42: 747-752.<br />
o Rattlesnake venom<br />
o Crotamine<br />
o Gene family<br />
o Crotalus durissus<br />
250<br />
Poster 20: A profile of typical Viperidae toxins in the Lachesis muta venom gl<strong>and</strong>s<br />
transcriptome<br />
Junqueira-de-Azevedo, I.L.M. 1* , Ching, A.T.C. 1 , Faria, F. 2 , Nishiyama Jr, M.Y. 3 , Ho, P.L. 1 , Diniz,<br />
M.R.V. 4<br />
1<br />
Centro de Biotecnologia, Instituto Butantan - Av. Vital Brasil, 1500 - São Paulo, Brazil- 05503-900<br />
*ijuncaze@butantan.gov.br<br />
2<br />
Laboratório de Bioquímica e Biofísica, Instituto Butantan - São Paulo, Brazil - 05503-900<br />
3<br />
Instituto de Química, Universidade de São Paulo – São Paulo, Brazil<br />
4<br />
Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias - Belo Horizonte, Brazil.<br />
Introduction: Snake families possess hundreds of toxin isoforms from a few structural<br />
types spread within each family but often absent in other. To describe the repertoire of<br />
common molecules <strong>and</strong> to reveal the possible new toxins from a Viperidae we carried on a<br />
transcriptomic analysis of the venom gl<strong>and</strong>s of L. muta, the world largest Viperidae snake.<br />
Methods: A cDNA library was constructed from venom gl<strong>and</strong> mRNA, validated <strong>and</strong> over<br />
2500 r<strong>and</strong>om clones were sequenced through an Expressed Sequence Tags (ESTs)<br />
approach. The ESTs were clustered <strong>and</strong> searched against public databases.<br />
Results: The analysis of 2095 ESTs clustered in 1162 contigs yielded many new<br />
representative molecules of nine toxin classes commonly present in Viperidae <strong>and</strong> their<br />
expression levels, being mainly BPPs (bradykinin potentiating peptide precursor), metallo-<br />
<strong>and</strong> serine proteases, C-type lectins. The BPPs are highly expressed (72% of toxins) <strong>and</strong><br />
revealed an unusual peptide. Surprisingly, we found mRNAs matching two toxin classes<br />
exclusively described in Elapidae or Colubridae snakes (detailed in a separated abstract).<br />
Besides the sequences matching venom molecules, other transcripts could be suggested as<br />
new toxin c<strong>and</strong>idates. These include non-toxin proteins whose activities match known<br />
features of envenoming (e.g., 5´ nucleotidases <strong>and</strong> ADAMs) <strong>and</strong> toxin inhibitors never<br />
observed in venom or venom gl<strong>and</strong>s. The profile of non-toxin transcripts categories also<br />
shows the specialization of venom gl<strong>and</strong>s in synthesizing toxins, with special care to<br />
disulfide bond assembly, being the PDI (proteins disulfide isomerase) the major non-toxin<br />
transcript.<br />
Conclusion: We defined a catalog of transcripts from L. muta, allowing the identification<br />
of the most common classes of toxins present in Viperidae venoms, which parallels the<br />
complex physiological effects evoked by the venom, <strong>and</strong> some new putative toxin<br />
molecules, providing also a comprehensive resource of reptilian expressed sequences.<br />
Supported by FAPESP.<br />
o Transcriptome<br />
o Expressed Sequence Tags<br />
o cDNA<br />
o Viperidae<br />
251
Poster 21: Atypical Viperidae cDNAs from Lachesis muta venom gl<strong>and</strong>s <strong>and</strong> their<br />
implication in snake toxin repertoire evolution<br />
Junqueira-de-Azevedo, I.L.M. 1* , Ching, A.T.C. 1 , Carvalho, E. 1 , Ho, P.L. 1 , Diniz, M.R.V. 2<br />
1<br />
Centro de Biotecnologia, Instituto Butantan - Av. Vital Brasil, 1500 - São Paulo, Brazil- 05503-900<br />
*ijuncaze@butantan.gov.br<br />
2<br />
Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias - Belo Horizonte, Brazil.<br />
Introduction: Efforts to describe toxins from the two major families of venomous snakes<br />
(Viperidae <strong>and</strong> Elapidae) usually reveal proteins belonging to few structural types,<br />
particular of each family. To investigate possible unusual toxins in a Viperidae venom, we<br />
generated a database transcriptomic sequences from Lachesis muta snake <strong>and</strong> carried on a<br />
search for atypical molecules.<br />
Methods: A cDNA library was constructed from venom gl<strong>and</strong> mRNA, validated <strong>and</strong> over<br />
2500 r<strong>and</strong>om clones were sequenced through an Expressed Sequence Tags (ESTs)<br />
approach. The ESTs were clustered <strong>and</strong> cluster sequences were searched against public<br />
databases. Atypical molecules were further full-length re-sequenced <strong>and</strong> phylogeneticaly<br />
analyzed through Bayesian models.<br />
Results: Besides nine classes of typical toxins found on the transcriptome (presented in a<br />
separated abstract), atypical sequences never observed in the hundreds of Viperidae snakes<br />
studied so far were shown to be highly expressed: (i) a diverging C-type lectin that is<br />
related to Viperidae toxins but appears to be independently originated; (ii) an ohanin-like,<br />
that would be the third member of the recently described class of Elapidae toxins, related<br />
to human butyrophilin <strong>and</strong> B30.2 proteins; <strong>and</strong> (iii) a 3FTx-like, a new diverging member<br />
of the widely studied three-finger family of proteins, which includes major Elapidae<br />
neurotoxins, CD59 antigen <strong>and</strong> a growing number of human proteins.<br />
Discussion: The presence of these common <strong>and</strong> uncommon molecules suggests that the<br />
repertoire of toxins could be more conserved between snake families than it was used to be<br />
considered <strong>and</strong> their features indicate a dynamic process of venom evolution through<br />
molecular mechanisms, such as multiple recruitments of important scaffolds <strong>and</strong> domain<br />
exchange between paralogs, always keeping a minimalist nature in most toxin structures in<br />
opposition to their non-toxin counterparts.<br />
Supported by FAPESP.<br />
o Transcriptome<br />
o Molecular Evolution<br />
o Three finger toxins<br />
o Ohanin<br />
252<br />
Poster 22: The Bitis gabonica gabonica venom proteome: venomics vs.<br />
transcriptomics<br />
Calvete, J.J. 1,* , Marcinkiewicz, C. 2 , Sanz, L. 1<br />
1<br />
Instituto de Biomedicina de Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain.<br />
*jcalvete@ibv.csic.es<br />
2 Biotechnology Center, Temple University College of Science <strong>and</strong> Technology, 1900 N. 12th Street,<br />
Philadelphia, PA 19122-6078, USA<br />
Snakes of the family Viperidae (vipers <strong>and</strong> pitvipers) produce a complex mixture of a large<br />
number of distinct proteins. However, venom proteins belong to only a few major protein<br />
families, including enzymes (serine proteinases, Zn 2+ -metalloproteases, L-amino acid<br />
oxidase, group II PLA2) <strong>and</strong> proteins without enzymatic activity (disintegrins, C-type<br />
lectins, natriuretic peptides, myotoxins, CRISP toxins, nerve <strong>and</strong> vascular endothelium<br />
growth factors, cystatin <strong>and</strong> Kunitz-type protease inhibitors). In additon to underst<strong>and</strong>ing<br />
how venoms evolve, characterization of the protein content of snake venoms also has a<br />
number of potential benefits for basic research, clinical diagnosis, development of new<br />
research tools <strong>and</strong> drugs of potential clinical use, <strong>and</strong> for antivenom production strategies.<br />
Despite its potential value, little is known about the venom protein composition of most<br />
vipers. Recent reports surveyed gene transcriptional activity (transcriptome) of the snake<br />
venom gl<strong>and</strong>s of Bothrops insularis, Bothrops jararacussu, Bitis gabonica, <strong>and</strong><br />
Agkistrodon acutus by generation of expressed sequence tags (ESTs) or construction of a<br />
cDNA library followed by sequencing of the clones. These works have provided catalogs<br />
of full-length venom gl<strong>and</strong> mRNAs. Our proteomic approach complements these studies<br />
by showing the relative abundance of the proteins that are actually secreted into the<br />
venoms.We have analyzed the protein composition of the crude venom of Bitis gabonica<br />
by RP-HPLC, N-terminal sequence analysis, MALDI-TOF MS, <strong>and</strong> in-gel tryptic<br />
digestion followed by CID-MS/MS. This approach allowed us to assign unambiguously all<br />
of the isolated venom fractions to protein families present in the non-redundant databases.<br />
Our proteomic approach complements transcriptomic studies by showing the relative<br />
abundance of the proteins that are actually secreted into the venoms. On the other h<strong>and</strong>,<br />
comparison of the proteomic versus the transcriptomic data showed significant differences<br />
for a number of protein families, indicating lack of correlation between the transcriptional<br />
<strong>and</strong> the translational activity of the venom gl<strong>and</strong>. The use of protein-chemical techniques<br />
allowed us also to determine the subunit composition of expressed venom proteins.<br />
o Venom proteome<br />
o Mass spectrometry<br />
o N-terminal sequencing<br />
o Bitis gabonica gabonica<br />
253
Poster 23: Loss of introns along the evolutionary diversification pathway of snake<br />
venom disintegrins evidenced by sequence analysis of genomic DNA from<br />
Macrovipera lebetina transmediterranea<br />
Bazaa, A. 1 , Juárez, P. 2 , Marrakchi, N. 1 , Bel Lasfer, Z. 1 , El Ayeb, M. 1 , Calvete, J.J. 2,* , Sanz, L. 2<br />
1<br />
Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, B.P. 74, 1002 Tunis-Belvédère, Tunisia.<br />
2<br />
Instituto de Biomedicina de Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain.<br />
*jcalvete@ibv.csic.es<br />
Venom toxins likely evolved from endogenous proteins with normal physiological<br />
functions that were recruited into the venom proteome. Notably, most venom toxins are<br />
extensively cross-linked by disulfide bonds <strong>and</strong> have flourished into functionally diverse,<br />
toxin multigene families that exhibit interfamily, intergenus, interspecies, <strong>and</strong> intraspecific<br />
variability. The existence in the same venom of a functionally diverse isoforms of the same<br />
protein family reflects accelerated Darwinian evolution. The evolutionary pressure acting<br />
to promote high levels of variation in venom proteins may be part of a predator-prey arms<br />
race that allow the snake to adapt to a variety of different prey, each of which are most<br />
efficiently subdued with a different venom formulation. Disintegrins, a broad family of<br />
small, cysteine-rich polypeptides isolated from venoms of vipers <strong>and</strong> rattlesnakes, are<br />
released in viper venoms <strong>and</strong> selectively block the function of cell surface adhesive<br />
receptors of the integrin family. Analysis of cDNAs from Macrovipera lebetina<br />
transmediterranea <strong>and</strong> Echis ocellatus venom gl<strong>and</strong> libraries encoding disintegrins<br />
strongly argued for a common ancestry of the messengers of short disintegrins <strong>and</strong> those<br />
for precursors of dimeric disintegrin chains. We now report the sequence analysis of<br />
disintegrin-coding genes from Macrovipera lebetina transmediterranea. Genomic DNAs<br />
for dimeric disintegrin subunits Ml_G1 <strong>and</strong> Ml_G2 contain single 1 kb-introns exhibiting<br />
the 5´-GTAAG (donor)/3´-AG (acceptor) consensus signature of group I self-splicing<br />
introns. On the other h<strong>and</strong>, the short RTS-disintegrin Ml_G3 is transcribed from an<br />
intronless gene, indicating that the evolutionary pathway leading to the emergence of short<br />
disintegrins involved the removal of all intronic sequences. The insertion position of the<br />
Ml_G1 <strong>and</strong> Ml_G2 intron is conserved in the genes for vertebrate ADAM´s disintegrinlike<br />
domains <strong>and</strong> within the gene for the medium-size snake disintegrins halystatin 2 <strong>and</strong> 3.<br />
However, a comparative analysis of currently available disintegrin(-like) genes outline the<br />
view that a minimization of both the gene organization <strong>and</strong> the protein structure underlie<br />
the evolution of the snake venom disintegrin family.<br />
o Macrovipera lebetina transmediterranea<br />
o Evolution of disintegrins<br />
o Disintegrin gene structure<br />
o Genomic DNA sequencing<br />
254<br />
Poster 24: Current state of Bothrops insularis venom proteome. A joint effort of the<br />
Rio de Janeiro Proteomic Network.<br />
Perales, J 1,5 ., Neves Ferreira, A.G.C 1,5 ., Valente, R.H 1,5 ., Chapeaurouge, D.A 1,5 ., Trugilho, M.R.O 1,5 .,<br />
Rocha, S.L.G 1,5 ., Junqueira, M 1,4,5 ., Leon, I.R 1,5 ., Ho, P.L 2 ., Junqueira de Azevedo, I.L.M 2 ., Dutra, D.L.S 3,5 .,<br />
Oliveira-Carvalho, A.L 3,5 ., Wermelinger, L.S 3,5 , Zingali, R.B 3,5 . <strong>and</strong> Domont, G.B 4 .<br />
1<br />
Physiology <strong>and</strong> Pharmacodynamics Department, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ,<br />
Brazil<br />
2<br />
Instituto Butantan, São Paulo, SP, Brazil<br />
3<br />
Medical Biochemistry Institute, UFRJ, Rio de Janeiro, RJ, Brazil<br />
4<br />
Biochemistry Department, Instituto de Química, UFRJ, Rio de Janeiro, RJ, Brazil<br />
5<br />
Rio de Janeiro Proteomic Network<br />
Bothrops insularis is an endemic snake found in Queimada Gr<strong>and</strong>e isl<strong>and</strong>, Brazilian coast,<br />
that feeds on birds <strong>and</strong> some invertebrates available in its habitat. It is thought that<br />
evolutionary pressure could have resulted in an adaptation of its venom components<br />
providing divergent toxins to better capture the preys. Dr Paulo L Ho <strong>and</strong> colleagues<br />
(Instituto Butantan, SP, Brazil) are performing a survey of gene expression <strong>and</strong> diversity in<br />
B. insularis venom gl<strong>and</strong>s <strong>and</strong> through the generation of ESTs putting out a wealthy<br />
genomic data. Thus, B. insularis venom seems to be a good model system in order to study<br />
toxin expression. The strategy for the elucidation of the proteome used 2D-PAGE.<br />
Samples were dissolved in 8-9.5M Urea, 2%(w/v) CHAPS, 20mM Dithiotreitol,<br />
0.002%(w/v) Bromophenol blue <strong>and</strong> 4-7 IPG buffer or 3-10 Biolyte. The first dimension<br />
was performed on 4-7 IPG strips <strong>and</strong> the second dimension on a 15%T SDS-PAGE under<br />
reducing conditions. From the reference gel [1.8 mg, dry weight, venom by in gel<br />
rehydration with 0.5% (v/v) 4-7 IPG buffer] major spots were used to generate tryptic<br />
hydrolysates for peptide mapping by MALDI-TOF MS <strong>and</strong>/or sequencing by LC-ESI-ION<br />
TRAP MS <strong>and</strong> MALDI-TOF-TOF MS/MS. Searches in Gene/ ESTs data banks allowed<br />
identification <strong>and</strong> a possible function assignment to 67 proteins scattered all over our 2D<br />
reference, distributed between 12-70 kDa <strong>and</strong> 4-7 pI. They include metalloproteinases,<br />
serineproteinases, phospholipases A2, platelet agglutinating inhibiting protein, lectins,<br />
platelet glycoprotein Ib-binding protein alpha <strong>and</strong> beta subunits, vascular endothelial<br />
growth factor, convulxin beta, anticoagulant protein A <strong>and</strong> metalloproteinase degradation<br />
products. They also showed that the horizontal streaking observed in our reference gel is<br />
not due to artifacts or bad sample preparation but rather to a large number of protein<br />
isoforms <strong>and</strong>/or glycosylated molecules, which make snake venom 2D analysis not so<br />
straightforward as expected due to the ease of sample preparation.<br />
Supported by: FAPESP, FAPERJ <strong>and</strong> CNPq<br />
o B. insularis<br />
o Venom<br />
o Proteome<br />
o 2D-PAGE<br />
255
Poster: 25: Bothrops jararaca Venom Proteome.<br />
Valente, R.H. 1,4 ; Coelho, F.E.M.R.F. 1,4 ; Neves-Ferreira, A.G.C. 1,4 ; Leon, I.R. 1,4 ; Cidade, D.A. 2 , Albano,<br />
R.M. 2 , Domont, G.B. 3,4 <strong>and</strong> Perales, J. 1,4<br />
1 Physiology <strong>and</strong> Pharmacodynamics Department, Instituto Oswaldo Cruz, FIOCRUZ, RJ, Brazil<br />
2 Biochemistry Department, UERJ, RJ, Brazil<br />
3 Biochemistry Department, Instituto de Química, UFRJ, RJ, Brazil<br />
4 Rio de Janeiro Proteomic Network<br />
Mortality <strong>and</strong> morbidity data indicate that snake bite envenomation remains a public health<br />
problem in Brazil. Approximately 85 percent of reported envenomations are caused by<br />
bothropic species <strong>and</strong> most of these specifically by Bothrops jararaca. The study of its<br />
venom proteome could shed some light on mechanisms of action by unraveling previously<br />
unknown proteic components (toxins, enzymes, biologically active peptides etc.) that could<br />
not be detected by traditional protein chemistry. As an aid to protein identification we have<br />
generated expressed sequence tags (ESTs) from a venomous gl<strong>and</strong> cDNA library <strong>and</strong> made<br />
the data available as an EST sequence bank. 2D-PAGE <strong>and</strong> mass spectrometry analyses of<br />
tryptic hydrolysates of gel spots were used to separate <strong>and</strong> identify proteins. Venom<br />
separation performed using 3-10 non-linear <strong>and</strong> 4-7 linear pH IPG strips (IEF, first<br />
dimension) <strong>and</strong> 15%T (SDS-PAGE, second) yielded by Coomassie blue staining, 337 <strong>and</strong><br />
521 spots, respectively. Identified proteins represent toxins known to be present in this<br />
snake venom. B. jararaca venom was also submitted to size-exclusion chromatography<br />
<strong>and</strong> three pools were analyzed by 4-7 linear pH range 2D-PAGE in order to examine<br />
venom subproteomes. Results indicate enrichment in different regions of the gel when<br />
compared to whole venom fractionation. Detection of low molecular weight components in<br />
the high molecular weight size exclusion fraction indicates their possible interaction in the<br />
native state <strong>and</strong>/or artifactual data due to abnormal behavior (eg. interaction with column<br />
resin) during chromatography. The use of proteomic techniques like 2D-PAGE <strong>and</strong> mass<br />
spectrometry can be a very productive approach to snake venom studies allowing better<br />
underst<strong>and</strong>ing of venom complexity <strong>and</strong> toxic properties leading to more effective<br />
antivenom therapy in the near future.<br />
Supported by FAPERJ, FIOCRUZ <strong>and</strong> CNPq<br />
o Bothrops jararaca<br />
o Venom<br />
o Proteome<br />
o 2D-PAGE<br />
256<br />
Poster 26: Post-translational amino acid epimerization in an excitatory conotoxin.<br />
Structures of the L- <strong>and</strong> D-forms.<br />
Norton, R.S. 1 , Wei, D., 1 Yang, X., 1 Babon, J.J., 1 Buczek, O., 2 Olivera, B.M., 2 Bulaj, G., 2,3<br />
1<br />
Walter <strong>and</strong> Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville 3050, Australia.<br />
ray.norton@wehi.edu.au<br />
2<br />
Department of Biology, University of Utah, Salt Lake City, Utah, 84112, USA<br />
3<br />
Cognetix, Inc., 421 Wakara Way, Suite 201, Salt Lake City, Utah, 84108, USA<br />
The 46-residue conotoxin r11a, from the fish-hunting species Conus radiatus, is a member<br />
of the recently characterized I-superfamily. Conotoxins belonging to this group exhibit<br />
excitotoxic activity: several members were shown to block activity of K + channels. R11a<br />
has four disulfide cross-links. Phe44, the third residue from the C terminus, is posttranslationally<br />
modified to a D- configuration. The naturally occurring D-Phe form of r11a<br />
is significantly more active as an excitotoxin than the L-Phe analogue both in vitro <strong>and</strong> in<br />
vivo (1).<br />
In this study we have determined the solution structures by using NMR spectroscopy of<br />
both forms of this toxin. The connectivities of the eight half-cystines were determined<br />
from preliminary structure calculations <strong>and</strong> confirmed chemically to be 5-19, 12-22, 18-27,<br />
<strong>and</strong> 21-38. This pattern suggests that r11a has an ICK motif structure with one additional<br />
disulfide (21-38). Indeed, it is the same pattern seen in the funnel-web spider toxin,<br />
robustoxin, ie.1-4/2-6/3-7/5-8 (2).<br />
Apart from the first few residues, the structure of r11a is well defined up to around residue<br />
35, after which the molecule becomes increasingly disordered. The ordered region does<br />
indeed adopt an ICK motif structure. The C-terminal region, where Phe44 resides, is<br />
highly disordered <strong>and</strong> has chemical shifts consistent with a lack of structure. Comparison<br />
of the synthetic D-Phe44 <strong>and</strong> L-Phe44 forms of the molecule indicates that this<br />
isomerization has very little effect on the structured region. However, the effect of this<br />
isomerization on activity implies that that this region makes contact with the relevant<br />
receptor <strong>and</strong> presumably becomes more ordered as a result.<br />
1. Buczek, O., Yoshikami, D., Bulaj, G., Jimenez, E.C., <strong>and</strong> Olivera, B.M. (2005)<br />
J. Biol. Chem. 280, 4247–4253.<br />
2. Pallaghy, P.K., Alewood, D., Alewood, P.F., <strong>and</strong> Norton, R.S. (1997) FEBS Lett. 419,<br />
191-196.<br />
o Conotoxin<br />
o Post-translational modification<br />
o Structure<br />
o Disulfide connectivities<br />
257
Poster 27: Biochemical characterization of venom from the South American<br />
colubrid Philodryas patagoniensis<br />
Carreiro da Costa, R.S. 1 , Ferrari, E.F. 1 , Leonardo, S.D. 1 , Prudêncio, L. 1 , Souza, G.H.M.F. 1 , Hyslop, S. 2 ,<br />
Cogo, J.C. 1,*<br />
1 Laboratório de Fisiologia, Instituto de Pesquisa e Desenvolvimento (IPD) e Serpentário do Centro de<br />
Estudos da Natureza, Universidade do Vale do Paraíba (UNIVAP), Av. Shishima Hifumi, 2911,<br />
Urbanova, 12244-000, São José dos Campos –SP, Brazil. * jccogo@univap.br<br />
2 Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de<br />
Campinas (UNICAMP), 13083-970, Campinas, SP, Brazil<br />
Introduction: The venoms of the South American colubrids Philodryas olfersii <strong>and</strong><br />
Philodryas patagoniensis cause edema, hemorrhage, necrosis, alterations in blood<br />
coagulation <strong>and</strong> neuromuscular blockade in vitro. In this work, we examined some of the<br />
biochemical activities of P. patagoniensis venom. Methods: Venom was obtained from<br />
adult specimens of P. patagoniensis maintained in the serpentarium at UNIVAP <strong>and</strong><br />
lyophilized. The esterase, phospholipase A2 (PLA2) <strong>and</strong> proteolytic activities were<br />
assayed. SDS-PAGE was used to determine the electrophoretic profile <strong>and</strong> venom was<br />
fractionated by reverse-phase chromatography (RPC) on a Source 15 column<br />
(Pharmacia). The neuromuscular activity was tested in chick biventer cervicis muscle<br />
preparations, <strong>and</strong> creatine kinase (CK) activity was measured using a commercial kit.<br />
Results: The esterase, PLA2 <strong>and</strong> proteolytic activities were 7.0+0.1 U/mg, 17+0.5 ΔA420<br />
nm/mg <strong>and</strong> 162.3+1.3 U/mg, respectively. For comparison, the corresponding values for<br />
venom of the pitviper Bothrops jararaca were 49.0+4.5, 124+26.3 <strong>and</strong> 47+0.9,<br />
respectively (mean+SEM, n=4 each). SDS-PAGE showed that the venom contained<br />
proteins with molecular masses of
Poster 29: Purification <strong>and</strong> partial characterization of a deoxyribonuclease II from<br />
Bothrops alternatus (urutu) venom<br />
Nascimento, J.M. 1,2,* , Collares-Buzato, C.B. 3 , Hyslop, S. 1<br />
1<br />
Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas<br />
(UNICAMP), CP 6111, 13083-970, Campinas, SP, Brazil. *jminardi@unicamp.br; hyslop@fcm.unicamp.br<br />
2<br />
Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), CP 6109,<br />
13083-970, Campinas, SP, Brazil<br />
3<br />
Departamento de Histologia e Embriologia, Instituto de Biologia, Universidade Estadual de Campinas<br />
(UNICAMP), CP 6109, 13083-970, Campinas, SP, Brazil<br />
Introduction: Snake venoms contain a variety of enzymes that degrade nucleic acids <strong>and</strong><br />
their constituents, including deoxyribonuclease, 5´-nucleotidase, phosphatases,<br />
phosphodiesterase <strong>and</strong> ribonuclease. In mammals, acidic DNase II has been implicated in<br />
DNA fragmentation during apoptosis. In this work, we describe the purification <strong>and</strong><br />
characterization of DNase II from Bothrops alternatus snake venom. Methods:<br />
Deoxyribonuclease II was purified from B. alternatus venom using a combination of ionexchange<br />
(SP-Sepharose, heparin-Sepharose), gel filtration, affinity ConA-Sepharose <strong>and</strong><br />
reverse-phase chromatographies. All fractions were screened for enzymatic activity<br />
(increase in A260 nm) using salmon testes DNA (75 μg/ml) in 0.15 M acetate buffer, pH<br />
4.7. The cross-reactivity with commercial equine antivenom was assessed by<br />
immunoblotting <strong>and</strong> by ELISA, the latter using affinity-purified IgG from commercial<br />
antibothropic antivenom (Instituto Butantan) followed by detection with a rabbit anti-horse<br />
IgG-peroxidase conjugate. Results: DNase II was purified with a specific activity of<br />
1.9x10 3 units/mg compared to 36.1 units/mg for venom (purification factor = 51.2) <strong>and</strong> a<br />
protein yield of 1.75%. SDS-PAGE showed a single b<strong>and</strong> with a molecular mass of 24.6<br />
kDa that was unaffected by dithiothreitol or β-mercaptoethanol. Immunoblotting also gave<br />
a single protein b<strong>and</strong> with the same molecular mass <strong>and</strong>, together with ELISA, indicated<br />
that this protein was immunogenic since it interacted with antivenom <strong>and</strong> purified IgG.<br />
DNase II cleaved double-str<strong>and</strong>ed DNA, denatured DNA, circular DNA from the plasmids<br />
pGEM <strong>and</strong> pBR 322, <strong>and</strong> nucleosomal DNA isolated from rat spleen cells; there was no<br />
degradation of RNA. The enzyme was active in the pH range of 4.5-5.5, with an optimum<br />
of pH 4.7; activity was lost at >50 o C. Enzymatic activity was inhibited by<br />
aurintricarboxylic acid (25 µM), iodoacetamide (1 mM), <strong>and</strong> Zn 2+ (10 mM). Conclusion:<br />
Bothrops alternatus venom contains a DNase II that shares several characteristics with<br />
mammalian acidic DNases. This enzyme could contribute to DNA degradation <strong>and</strong><br />
apoptosis following envenomation. Financial support: CNPq, FAPESP<br />
o Bothrops alternatus<br />
o Deoxyribonuclease II<br />
o Purification<br />
o Characterization<br />
260<br />
Poster 30: LC-MS Analysis of All PSP Toxins Using Anion Exchange <strong>and</strong> Reverse<br />
Phase Columns Connected in Series<br />
Sachio Nishio 1* ,Takefumi Sagara 1 , Shigeto Taniyama 2 , Tamiko Hashimoto 1 , Naoyoshi Nishibori 1 <strong>and</strong><br />
Manabu Asakawa 2<br />
1<br />
Faculty of Junior College, Shikoku University, 123-1, Ebisuno, Furukawa, Ohjin-cho, Tokushima,<br />
Japan, * sachio-nishio@shikoku-u.ac.jp<br />
2<br />
Graduate School of Biosphere Science, Hiroshima University, 1-4-4, Kagamiyama, Higashi-Hiroshima,<br />
Hiroshima, Japan<br />
All PSP toxins, such as disufonated protogonyautoxins (PXs, C toxins), monosufonated<br />
gonyautoxins (GTXs) <strong>and</strong> basic saxitoxins (STXs), were measured with newly developed<br />
two-column chromatography. A Hitachi Gel 3013N column (Hitachi, 50 mm x 2.7 mm<br />
i.d.) <strong>and</strong> a Deverosil RP-AQUEOUS-AR column (Nomura Chem., 250mm x 3 mm i.d.)<br />
were connected in series. Both a mobile phase A, 0.1% heptafluorobutylic acid /10mM<br />
ammoniumacetate, <strong>and</strong> a mobile phase B, 0.1% pentafluoropropionic acid /10mM<br />
ammoniumacetate /4% acetonitorile, were adopted under a gradient system at a flow rate<br />
of 0.25 mL/minutes. PXs <strong>and</strong> GTXs were eluted from the columns with the mobile phase<br />
A. The 100% A was changed at 20 minutes to 100% B. The former column was<br />
maintained at 50˚C throughout the run. And the latter one was at 20˚C until 30 minutes for<br />
the both detection of PXs <strong>and</strong> GTXs , <strong>and</strong> then the column was heated to 40˚C to perform<br />
the dissociation of STXs with the mobile phase B. The PSP toxins eluted from the twocolumn<br />
were identified from the results of mass chromatograms <strong>and</strong> mass spectra on<br />
measurement of their LC-MS. The mass spectrometry, HITACHI M-8000 LC-MS, was<br />
successfully measured to detect the PSP components eluted from the two-column. It was<br />
equipped with sonic spray ionization interface (SSI). The mass numbers of desulfonated<br />
<strong>and</strong> protonated ions of gonyautoxin5 (GTX5) <strong>and</strong> protonated saxitoxin (STX) were<br />
calculated as m/z 300. Those of GTX6, GTX3, GTX2, PX1 (C1), PX2 (C2), nSTX <strong>and</strong><br />
hySTX were m/z 316, GTX1, GTX4, hynSTX <strong>and</strong> PX3(C3) were m/z 332, dcGTX2,<br />
dcGTX3 <strong>and</strong> dcnSTX were m/z 273, <strong>and</strong> dcSTX was m/z 257, respectively.<br />
A new method of two-column HPLC made it possible to determine all PSP components<br />
in one chromatographic run of 60 minutes. A good separation <strong>and</strong> an unvaried retention<br />
time for each PSP toxins were clarified by repeat determinations. In addition, a fine<br />
correlation between the LC-SSI-MS <strong>and</strong> LC with post-column oxidation <strong>and</strong> fluorescence<br />
detection (LC-ox-FLD) were performed in this study.<br />
o All PSP toxins<br />
o LC-MS<br />
o LC-ox-FLD<br />
261
Poster 31: <strong>Fish</strong>ing Toxins out of Snake Venoms Using the Antitoxin DM43 as a Tool<br />
for the Analysis of Subproteomes<br />
Rocha, S.L.G 1 ., Neves-Ferreira, A.G.C 1 ., Chapeaurouge, A 1 ., Valente, RH 1 ., Trugilho, M.R.O 1 ., Léon,<br />
I.R 1 ., Domont, G.B 2 . <strong>and</strong> Perales, J 1* .<br />
1Departamento de Fisiologia e Farmacodinâmica, Instituto Oswaldo Cruz, Rio de Janeiro, Brasil<br />
jperales@ioc.fiocruz.br <strong>and</strong> 2 Departamento de Química de Proteínas, Instituto de Química, Universidade<br />
Federal do Rio de Janeiro, Rio de Janeiro, Brasil.<br />
Snake venoms are complex mixtures of proteins <strong>and</strong> peptides with different biological<br />
activities, many of them very toxic. Several animals, including the opossum Didelphis<br />
marsupialis, are resistant to snake venoms due to the presence of neutralizing factors in<br />
their blood. An antihaemorrhagic protein named DM43 was isolated from opossum serum.<br />
It inhibits snake venom metalloproteinases through non covalent complex formation with<br />
these enzymes. In this study, we have used DM43 <strong>and</strong> proteomic techniques to explore<br />
snake venom subproteomes. Several venoms were chromatographed through an affinity<br />
column containing immobilized DM43. Bound fractions were analyzed either by SDS-<br />
PAGE <strong>and</strong>/or 2D-PAGE, followed by identification by MALDI-TOF/TOF MS. Following<br />
this methodology, we could classify venoms from Bothrops alternatus, B. asper, B. atrox,<br />
B. insularis, B. jararaca, B. jararacussu, B. moojeni, B. neuwiedi, Crotalus adamanteus,<br />
C. atrox, C. durissus terrificus, Lachesis muta muta <strong>and</strong> Naja naja atra according to their<br />
relative content of metalloproteinases (PI, PIII <strong>and</strong>/or their fragments). Venom fractions<br />
not bound to DM43 column were equally analyzed <strong>and</strong> were composed basically of serine<br />
proteinases, C-type lectins, L-amino acid oxidases, nerve growth factor, metalloproteinases<br />
<strong>and</strong>/or some unidentified spots. Studied venoms presented important proteic variability,<br />
with frequent detection of multiple forms of the same protein <strong>and</strong> several members of the<br />
same protein family. DM43 affinity chromatography associated with proteomic techniques<br />
showed to be useful tools to separate <strong>and</strong> identify proteins from snake venoms.<br />
Financial Support: FAPERJ, CNPq <strong>and</strong> Fiocruz.<br />
o Snake Venoms<br />
o Proteome - Subproteomes<br />
o Antitoxin DM43<br />
o Didelphis marsupialis<br />
262<br />
Poster 32: Novel peptides in Philodryas olfersii <strong>and</strong> Philodryas patagoniensis<br />
venoms detected by bidimensional liquid chromatography coupled to t<strong>and</strong>em<br />
nano-electrospray mass spectrometry <strong>and</strong> de novo mass spectrum analysis<br />
Souza, G.H.M.F. 1,2,* , Cogo, J.C. 3 , Eberlin, M.N. 2 , Hyslop, S. 1<br />
1 Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas<br />
(UNICAMP), CP 6111, 13083-970, Campinas, SP, Brazil. *desouz@fcm.unicamp.br<br />
2 Laboratório Thomson de Espectometria de Massas, Instituto de Química, Universidade Estadual de<br />
Campinas (UNICAMP), 13083-970, Campinas, SP, Brazil.<br />
3 Laboratório de Fisiologia e Farmacodinâmica, Instituto de Pesquisa e Desenvolvimento (IPD) <strong>and</strong><br />
Serpentário do Centro de Estudos da Natureza (CEN), Universidade do Vale do Paraíba<br />
(UNIVAP), São José dos Campos, SP, Brazil.<br />
Introduction: Colubrid venoms contain a variety of enzymes <strong>and</strong> toxins, but their content<br />
of low molecular mass peptides remains poorly studied. In this work, we used mass<br />
spectrometry to examine the peptide content of venoms from the South American<br />
colubrids Philodryas olfersii <strong>and</strong> Philodryas patagoniensis.<br />
Methods: Venom was obtained from adult specimens of P. olfersii <strong>and</strong> P. patagoniensis<br />
maintained at UNIVAP. Lyophilized venom was dissolved in 0.1% trifluoroacetic acid<br />
containing 50% acetonitrile <strong>and</strong> filtered by centrifugation through a 5 kDa cut-off<br />
membrane (Millipore). The resulting low molecular mass fraction was analyzed by<br />
quadrupole/time-of-flight mass spectrometry (Qtof) using capillary liquid-chromatography<br />
<strong>and</strong> nano-electrospray ionization.<br />
Results: Qtof-MS revealed 21 peptides in P. olfersii venom <strong>and</strong> 51 peptides in P.<br />
patagoniensis, with molecular masses of 500-2500 Da. Complementary online CapLCnano-ESI-MS<br />
confirmed the presence of these 72 peptides. Various peptides showed posttranslational<br />
modifications such as an amidated C-terminus <strong>and</strong> pyroglutamic acid at the<br />
N-terminus. The peptides that gave the most intense ion signals were EAGTPLSV (754.27<br />
Da) in P. olfersii <strong>and</strong> NEPPLWVPG (1007.44 Da) in P. patagoniensis. BLAST searches<br />
for homologies to the peptides failed to identify any corresponding venom peptides or<br />
proteins, but revealed considerable homology (at least 80% similarity) with other<br />
vertebrate (chicken, chimpanzee, duck, frog, ox, human, mouse) proteins.<br />
Conclusion: The venoms of P. olfersii <strong>and</strong> P. patagoniensis contain numerous novel<br />
peptides, the biological functions of which remain to be determined. The lack of homology<br />
with other snake venom proteins (principally crotalid, elapid, <strong>and</strong> viperid) indicates either<br />
that there are indeed no venom proteins homologous with these peptides or that our current<br />
knowledge of snake venom gl<strong>and</strong> composition is far from complete. Screening of these<br />
peptides using bioassays in vitro <strong>and</strong> in vivo could lead to the identification of compounds<br />
with potential therapeutic applications. Financial support: CAPES, CNPq, FAPESP.<br />
o Mass spectrometry<br />
o Peptides<br />
o Venom<br />
263
Poster 33: Development of a novel fluorometric assay for the evaluation of BoNT/A<br />
protease activity using genetically engineering a fluorogenic substrate<br />
Ban, B. 1 Yang, G.H. 1 1, 2<br />
, <strong>and</strong> Jung, H. H.<br />
Microbial Toxin Research Institute, Medy-Tox Inc. 1 100, Kalsan ri, Tangjeong myeon, Asan si, Chungnam,<br />
336-708, South Korea<br />
Division of Applied Molecular Biology, Sun Moon University 2 100, Kalsan ri, Tangjeong myeon, Asan si,<br />
Chungnam, 336-708, South Korea<br />
Botulinum neurotoxins (BoNTs), the most toxic biological substances known to human, cause<br />
botulism by entering neurons <strong>and</strong> cleaving SNARE proteins that mediate neurotransmitter release;<br />
disruption of exocytosis results in paralysis <strong>and</strong> death. The lack of high-throughput screening (HTS) assay<br />
has limited the screening of new antidotes against botulism, <strong>and</strong> also rapid determination of exposure to<br />
BoNT is an important public health goal. In this study, a novel fluorometric assay was developed <strong>and</strong><br />
successfully applied for the assessment of protease activity of BoNT/A. Target truncated protein (substrate)<br />
for BoNT/A was engineered to be recombinant fusion protein where a green fluorescent protein is linked to<br />
cellulose binding domain via the substrate protein that is cleavable by BoNT/A. The cleavage of fusion<br />
protein by BoNT/A results in emission of fluorescence light that is easily detected <strong>and</strong> quantitated by<br />
fluorometric assay. Even though the concentration around 1ng/ml of BoNT/A produced significant<br />
fluorescence signals; the routine toxin concentration was 10 ng/ml or higher. In total, it could be concluded<br />
that this assay is a convenient, reliable, <strong>and</strong> less labor intensive alternative for the detecting protease<br />
activity of BoNT/A in vitro, <strong>and</strong> even suitable for screening large numbers of compounds for potential<br />
anti-BoNT/A drugs.<br />
Botulinum neurotoxins (BoNTs), High-throughput screening (HTS), Enhanced Green Fluorescence<br />
Protein (EGFP), Cellulose Binding Domain (CBD)<br />
264<br />
Poster 34: PURIFICATION AND PARTIAL ENZYMATIC<br />
CHARACTERIZATION OF THE HYALURONIDASE FROM Crotalus durissus<br />
terrificus SNAKE VENOM<br />
Cordeiro, A. T. 1 ; Tolini, M.M. 1 ; Bertazzi, D. T. 1 ; Giglio, J. R. 2 ; Arantes, E. C. 1 *<br />
1University<br />
of São Paulo, FCFRP, Dept. Física e Química, Av. do Café s/n, Ribeirão Preto, SP, Brazil.<br />
2<br />
University of São Paulo, FMRP,Depto. Bioquímica e Imunologia, Av. B<strong>and</strong>eirantes, Ribeirão Preto, SP,<br />
Brazil. * ecabraga@fcfrp.usp.br<br />
Introduction: Hyaluronidases are enzymes that catalyze the breakdown of hyaluronan, a<br />
high molecular weight polysaccharide found in the extracellular matrix. They are produced<br />
by a variety of bacteria <strong>and</strong> are also found in leech, snake <strong>and</strong> insect venoms, as well as in<br />
malignant tissues. Hyaluronidases have been employed therapeutically to increase the<br />
speed of absorption of fluids, to promote resorption of extravasated blood <strong>and</strong> to increase<br />
the effectiveness of local anesthetics. Hyaluronidases in venoms are spreading factors<br />
because they facilitate toxin diffusion into the tissues of the prey, thus contributing to<br />
systemic envenomation. The aim of this study was the isolation <strong>and</strong> partial enzymatic<br />
characterization of the hyaluronidase from Crotalus durissus terrificus snake venom<br />
(CdtV).<br />
Methods: The purification procedure involves basically an ion-exchange chromatography<br />
on CM-Sepharose FF, which was equilibrated <strong>and</strong> eluted with 0.05 M, pH 5.5 sodium<br />
acetate buffer up to 50 mL effluent, when a linear concentration gradient was started from<br />
0 to 1M NaCl in the same buffer. The active fraction III was filtered on Sepharose G75<br />
column at pH 5.5. In order to find the optimal conditions for hyaluronidase activity, CdtV<br />
<strong>and</strong> hyaluronic acid were incubated for 15 min at different pHs (2 to 10) <strong>and</strong> temperatures (0<br />
to 70°C) <strong>and</strong> the enzymatic activity was determined according to Pukrittayakamee et al.<br />
(Toxicon, 26, 639, 1988).<br />
Results: The optima pH <strong>and</strong> temperature for maximum activity of the CdtV were 5.5 <strong>and</strong><br />
40 o C, respectively. The specific activity of the isolated enzyme (~80 kDa) was 48,000<br />
turbidity reducing units (TRU)/mg against 182 TRU/mg for the soluble venom,<br />
representing a 263 to 264 fold purification range<br />
Conclusion: Despite being relatively simple, this purification procedure was able to afford<br />
a highly purified <strong>and</strong> active hyaluronidase from C.d. terrificus venom.<br />
Financial Support: CNPq <strong>and</strong> FAPESP.<br />
o Hyaluronidase<br />
o Snake venom<br />
o Crotalus durissus terrificus<br />
265
Poster 35: Mass spectrometry strategies for venom mapping <strong>and</strong> peptide sequencing<br />
from crude venom of a single Bombus specimen<br />
Menin L., Favreau P., Michalet S., Perret F., Cheneval O., Stöcklin M., Bulet P. <strong>and</strong> Stöcklin R.<br />
Atheris Laboratories, Case Postale 314, CH 1233 Bernex - Geneva, Switzerl<strong>and</strong>.<br />
admin@atheris.ch<br />
Venoms are a complex set of bioactive molecules including peptides <strong>and</strong> proteins that<br />
display a wide array of functions. Due to their complexity <strong>and</strong> diversity, animal venoms<br />
represent an extensive source of bioactive compounds for the development of drugs <strong>and</strong><br />
therapeutic agents. Conventional approaches for their characterization often require large<br />
quantities of biological material <strong>and</strong> efficient biological assays. Current mass spectrometry<br />
(MS) techniques through a variety of approaches now give access to a wealth of<br />
information in a short working time frame with minute sample amount. Currently, MSbased<br />
peptide <strong>and</strong> protein discovery may rely on strategies going from structure<br />
information to the functional activity. MALDI-TOF MS is a soft ionization method that<br />
requires minimal sample preparation <strong>and</strong> can be used to rapidly perform molecular mass<br />
fingerprints of venoms at a high sensitivity. Such molecular mass peptide profiles allow<br />
the quick identification of known toxins as well as of the identification of new compounds.<br />
Nevertheless, further characterization requires the use of de novo MS/MS peptide<br />
sequencing. This approach of peptide sequencing offers rapid access to total or partial<br />
primary peptide structures. A specific instrumentation is required for such studies such as<br />
ion traps, triple quadrupole, quadrupole time-of-flight, quadrupole ion trap or MALDI-<br />
TOF-TOF. We will exemplify an approach with the molecular mass fingerprint analyses<br />
<strong>and</strong> structural characterizations of components of the venom collected from a single<br />
bumblebee Bombus lapidarius specimen. Among the numerous components detected in the<br />
Bombus venom, three major peptides were fully characterized on a working day basis by<br />
de novo sequencing using ESI-QqTOF <strong>and</strong> MALDI-LIFT-TOF-TOF mass spectrometers.<br />
These peptides belong to the mast cell degranulating peptides named bombolitin <strong>and</strong><br />
previously characterized from a Megabombus species (1).<br />
References: 1. Argiolas <strong>and</strong> Pisano (1985) J. Biol. Chem. 260, 1437-1444.<br />
o Venom mass fingerprint<br />
o MS/MS de novo sequencing<br />
o Toxins<br />
o Bombus venom<br />
266<br />
Poster 36: A New Family of Peptides Discovered in Snake Venoms from the<br />
Atheris genus by Mass Spectrometry Techniques<br />
Favreau, P. 1 , Cheneval, O. 1 , Menin L. 1 , Michalet, S. 1 , Principaud, F. 2 , Thai, R. 3 , Ménez, A. 3 , Bulet, P. 1<br />
<strong>and</strong> Stöcklin, R. 1 *<br />
1 Atheris Laboratories, case postale 314, CH-1233 Bernex-Geneva, Switzerl<strong>and</strong><br />
2 Latoxan, 20 rue Léon Blum, F-26000 Valence, France<br />
3 CEA/Saclay, Département d'Ingénierie et d'Etudes des Protéines, F-91191 Gif sur Yvette, France<br />
The discovery of novel molecules of potential biomedical interest can be initiated from a<br />
structurally driven strategy based on mass spectrometry (MS) techniques. The use of MSbased<br />
strategies is greatly cost-effective regarding the amount of material needed to obtain<br />
structural information from complex mixtures 1 .<br />
In order to find new components, the venoms of numerous snakes were investigated using<br />
matrix-assisted laser desorption ionisation time-of-flight <strong>and</strong> electrospray ionisation<br />
t<strong>and</strong>em mass spectrometry. Molecular mass fingerprints were obtained <strong>and</strong> this allowed<br />
the identification <strong>and</strong> characterization of an original set of peptides with molecular masses<br />
ranging from 2 to 3 kDa in the snake venoms of the Atheris genus.<br />
Following de novo sequencing by t<strong>and</strong>em mass spectrometry, numerous peptides were<br />
found to exhibit characteristic structural features consisting of contiguous poly-His <strong>and</strong><br />
poly-Gly segments. Total amino acid sequence assignments were completed by Edman<br />
sequencing <strong>and</strong> these compounds were named pHpG peptides because of their original<br />
structural organisation. Using a similar strategy, we have detected pHpG peptides in<br />
venoms from other Viperidae but also from some Crotalidae, indicating that the occurrence<br />
of the pHpG peptide family is not restricted to the Atheris genus.<br />
Bioinformatic investigations did not reveal any relevant homology in protein databases.<br />
However, a deeper investigation revealed interesting sequence similarities with several<br />
bradykinin potentiating peptide (BPP) <strong>and</strong> C-type natriuretic peptide (CNP) precursors,<br />
indicating that pHpG peptides may be coded by the BPP/CNP precursors. As no clear cut<br />
similarity was observed between pHpG <strong>and</strong> other already identified peptides, the exact<br />
function of this new set of peptides remains to be elucidated.<br />
References: Favreau P., Menin L., Michalet S., Perret F., Cheneval O., Stöcklin M., Bulet<br />
P., Stöcklin R. (2006) Mass spectrometry strategies for venom mapping <strong>and</strong> peptide<br />
sequencing from crude venoms: case applications with single arthropod specimen.<br />
Toxicon, 47, 676-687.<br />
o snake venom<br />
o mass spectrometry<br />
o poly-His poly-Gly peptide<br />
o BPP/CNP<br />
267
Poster 37: Quantitative high-throughput LC-MS detection of cyanotoxins in<br />
aquatic tissue samples<br />
Lähde, M.-R. , Meriluoto, J.A.O.<br />
Åbo Akademi University, Department of Biochemistry <strong>and</strong> Pharmacy, Biocity, Turku/Åbo, Finl<strong>and</strong>,<br />
mlahde@abo.fi<br />
Introduction: Microcystins <strong>and</strong> nodularins (MC/NOD) are hepatotoxic bioaccumulating<br />
peptides produced by cyanobacteria. High-throughput (HTP) detection methods for<br />
cyanotoxins are needed in e.g. hygienic <strong>and</strong> ecotoxicological investigations. The aim of<br />
this study was to test an HTP LC-ESI-MS system for quantitative detection of MC/NOD in<br />
aquatic tissue samples.<br />
Methods: LC-MS analysis on two triple-quadrupole instruments: a Micromass Quattro II<br />
coaxial electrospray, <strong>and</strong> a Quattro Micro Z-spray electrospray. The stationary phase was a<br />
Purospher STAR RP-18e 30 mm x 4 mm <strong>and</strong> the gradient mobile phase consisted of 0.1%<br />
formic acid <strong>and</strong> ACN (injection cycle 4.2 min). Crusian carp livers <strong>and</strong> blue mussels were<br />
spiked with NOD-R <strong>and</strong> extracted with aqueous MeOH. The chromatographic<br />
performance <strong>and</strong> signal responses in SIR <strong>and</strong> MRM modes were evaluated in extended<br />
sample series. Two sample pre-treatment methods were assessed: the SPE procedure<br />
involved extraction on a C18 solid phase extraction cartridge, <strong>and</strong> the RAW procedure<br />
merely filtration.<br />
Results: Chromatographic performance could be retained throughout the extended sample<br />
series. The SPE-treated tissue samples analysed with Quattro Micro gave steady MS<br />
signals for NOD-R throughout the analysis whereas the Quattro II signal response<br />
fluctuated considerably, possibly due to sample cone fouling <strong>and</strong> uncontrolled signal<br />
enhancement <strong>and</strong> suppression. The RAW pre-treatment was inadequate for HTP analysis.<br />
Discussion: An earlier evaluation (1) showed a similar HTP method to be suitable for >300<br />
cyanobacterial extract samples on a coaxial probe LC-ESI-MS. The sample clean-up<br />
procedures reported in (2) are being evaluated as a part of the methodology development.<br />
The current study shows promising results for the LC-MS part, but the bottleneck for HTP<br />
analysis is the extraction <strong>and</strong> SPE of tissue samples.<br />
References: 1 Meriluoto J. et al. (2004) Chromatographia, 59, 291-298.<br />
2 Karlsson K.M. et al. (2005) Environ. Toxicol. 20, 381-389.<br />
o High-throughput analysis<br />
o LC-ESI-MS<br />
o Tissue samples<br />
o Nodularin<br />
268<br />
Poster 38: Improved EC method for the determination of cyanide in feed.<br />
M. Baltussen, D. Luykx, A. Koot, R. Frankhuizen <strong>and</strong> S. van Ruth<br />
RIKILT Institute of Food Safety, P.O. Box 230 NL-6700 AE Wageningen, The Netherl<strong>and</strong>s<br />
Introduction: Cyanide is highly toxic <strong>and</strong> therefore it is of paramount importance that the<br />
method for determination of cyanide in feed is reliable, sensitive <strong>and</strong> robust. Until now, the<br />
reference method as described in First Commission Directive 71/250/EEC was used in the<br />
EC to determine cyanide in feed. However, this method proved to be not sensitive enough<br />
for levels of 10 mg cyanide/kg feed <strong>and</strong> it could lead to false positive results. CEN has<br />
asked RIKILT to develop a new reference method under a new m<strong>and</strong>ate of the EC.<br />
Methods: Several steps in the EC protocol were altered to optimize the extraction of<br />
cyanoglycosides from feed, the formation <strong>and</strong> extraction of hydrogen cyanide <strong>and</strong> the<br />
determination of cyanide. Flaxseed containing high levels of cyanoglycosides <strong>and</strong> blank<br />
feed spiked with a cyanoglycoside, were used for testing the alterations.<br />
Results: The extraction of cyanoglycosides from feed was improved by using an acid<br />
treatment. The formation of hydrogen cyanide was optimized by using pure enzyme<br />
instead of an almond suspension. In the EC protocol, a titrimetric method was described to<br />
determine cyanide. In this study, cyanide was derivatized with a fluorogenic label <strong>and</strong><br />
determined by HPLC with fluorescence detection (Sano et al. 1992). Levels well below 10<br />
mg cyanide/kg feed could be detected <strong>and</strong> no interferences with other compounds were<br />
seen in the chromatogram. Moreover, the cyanide yield increased (from 210 mg/kg to 245<br />
mg/kg in flaxseed) <strong>and</strong> recovery of the spikes increased (from ~60% to ~90%).<br />
Discussion/Conclusion: The new method for cyanide determination in feed including<br />
HPLC analysis developed by RIKILT for CEN proved to be more sensitive, selective <strong>and</strong><br />
resulted in higher yields of cyanide.<br />
Reference: Sano A., Takimoto N. <strong>and</strong> Takitani S., J of Chrom., 1992, vol. 582, pp 131-135<br />
o cyanide<br />
o feed<br />
o HPLC<br />
o CEN<br />
269
Poster 39: The sulfo-SBED derivative of ammodytoxin, a photoprobe for studying<br />
the molecular basis of phospholipase A2 β-neurotoxicity<br />
Kovačič, L. * , Šribar, J., Križaj, I.<br />
Department of Biochemistry <strong>and</strong> Molecular Biology, Jožef Stefan Institute, Jamova 39, SI-1000<br />
Ljubljana, Slovenia, *lidija.kovacic@ijs.si<br />
The neurotoxicity of ammodytoxin, a secreted phospholipase A2 from long-nosed viper<br />
venom, has been proposed to be mediated by certain binding proteins. Interactions of<br />
ammodytoxin with the M-type receptor of secreted phospholipases A2 (1), calmodulin (2),<br />
14-3-3 proteins (3), protein disulfide isomerase (4) <strong>and</strong> a still unidentified mitochondrial<br />
membrane protein R25 (5) have been partially characterized. For further identification of<br />
ammodytoxin-binding proteins, we have explored the use of a biotin-containing crosslinking<br />
reagent sulfo-SBED (6). This trifunctional cross-linker possesses one aminereactive<br />
<strong>and</strong> one photo-reactive site <strong>and</strong>, additionally, allows affinity-based concentration<br />
of cross-linker containing species. Sulfo-SBED was coupled to ammodytoxin C whose<br />
specific binding was demonstrated by biotin-tagging of the known ammodytoxin targets,<br />
calmodulin, 14-3-3 proteins, protein disulfide isomerase <strong>and</strong> R25, but not other proteins.<br />
Using this tool, two additional proteins having affinity for ammodytoxin, of 45 <strong>and</strong> 46<br />
kDa, were identified in the mitochondrial-synaptosomal fraction of porcine cerebral cortex,<br />
that were not detected with previously used methods. The methodology of preparing a<br />
photoreactive phospholipase A2 derivative <strong>and</strong> labeling the respective binding proteins is<br />
generally applicable. In this way, not only the molecular basis of ß-neurotoxicity but other<br />
aspects of the (patho)physiological action of phospholipases A2 can also be studied.<br />
References:<br />
1. N.Vardjan et al. (2001), Biochem. Biophys. Res. Commun. 289, 143-149<br />
2. J. Šribar et al. (2001), J. Biol. Chem. 276, 12493-12496<br />
3. J. Šribar et al. (2003) Biochem. Biophys. Res. Commun. 302, 691-696<br />
4. J. Šribar et al. (2005) Biochem. Biophys. Res. Commun. 329, 733-737<br />
5. J. Šribar et al. (2003a), FEBS Lett. 553, 309-314<br />
6. T. Larsson et al. (2000), FEBS Lett. 469, 155-158<br />
o phospholipase A2,<br />
o neurotoxicity,<br />
o affinity-labeling <strong>and</strong><br />
o binding proteins.<br />
270<br />
Poster 40: Purification <strong>and</strong> characterisation of two apoptosis-inducing L-amino<br />
acid oxidases from the venoms of selected Australian elapids<br />
Bateman, E.H, 1* Venning, M.G, 2 Mirtschin, P.J 2,3 <strong>and</strong> Woods, A.E 2<br />
1 University of Otago, Dunedin Public Hospital, Great King St, Dunedin, New Zeal<strong>and</strong><br />
*emma.bateman@stonebow.otago.ac.nz<br />
2 University of South Australia, Frome Road, Adelaide, Australia.<br />
3 Venom Supplies, Tanunda, South Australia.<br />
Introduction - A previous study 1 demonstrated that crude venoms from Australian elapids<br />
Austrelaps superbus <strong>and</strong> Hoplocephalus stephensii elicited an apoptotic response in<br />
tumour-associated microvascular endothelial cells (TAMECs) which was similar to<br />
apoptotic effects elicited by a panel of exotic viperid snakes. The focus of the current study<br />
was to purify, identify <strong>and</strong> characterise the apoptotic agent(s) within these venoms.<br />
Methods – Crude, lyophilised venoms from A.superbus <strong>and</strong> H.stephensii were fractionated<br />
via size exclusion chromatography <strong>and</strong> fractions were screened at various time/dose<br />
combinations on TAMEC cultures to identify the fraction(s) responsible for apoptotic<br />
effects. These fractions were then pooled <strong>and</strong> characterised by native <strong>and</strong> denaturing<br />
polyacrylamide gel electrophoresis (PAGE), biochemical assays <strong>and</strong> peptide sequencing.<br />
Results – Both A.superbus <strong>and</strong> H.stephensii venoms demonstrated significant apoptotic<br />
activity in fractions of approximately 140-160kDa. Pooling of active fractions <strong>and</strong><br />
subsequent native PAGE showed pure, single b<strong>and</strong>s at 158kDa (A.superbus apoptotic<br />
fractions = AS1a) <strong>and</strong> 141kDa (H.stephensii apoptotic fractions = HS1a); denaturing<br />
PAGE demonstrated heterodimerism for AS1a <strong>and</strong> homodimerism for HS1a. Both AS1a<br />
<strong>and</strong> HS1a demonstrated specific catabolism of L-leucine, were inhibited by catalase <strong>and</strong><br />
stained positive under periodic acid-Schiff staining. Peptide sequencing then identified the<br />
apoptotic agents as L-amino acid oxidases (LAOs). Conclusions – This is the first study to<br />
report the isolation <strong>and</strong> identification of LAOs from Australian elapid venoms. The agents<br />
share similar capabilities <strong>and</strong> attributes with several LAOs from snake species exotic to<br />
Australia, including the capacity to elicit an apoptotic response in endothelial cells. 2,3<br />
Further studies to fully sequence AS-LAO <strong>and</strong> HS-LAO <strong>and</strong> characterise their biochemical<br />
properties are required, as well as further investigation to elucidate the exact mechanisms<br />
of LAO apoptotic activity.<br />
1 Bateman et al. 2002. 14th World Congress on Animal, Plant & Microbial Toxins, Abstract<br />
22501, page 75. 2 Torii et al., 1997. Journal of Biological Chemistry, v272(14), pp9539-<br />
9542.<br />
3 Suhr <strong>and</strong> Kim, 1999. Journal of Biochemistry, v125, pp305-309.<br />
o Apoptosis<br />
o Australian elapid venoms<br />
o L-amino acid oxidase<br />
o Tumour-associated endothelial cells<br />
271
Poster 41: Discovery of a novel conopeptide-AmIXA: Prediction of functional<br />
pharmacophore(s) <strong>and</strong> mechanism of action<br />
Saminathan, R 1 , Jois, S.D.S 2 , Sato, K 3 , Gopalakrishnakone, P 1* .<br />
1<br />
Venom <strong>and</strong> Toxin Research Programme, Department of Anatomy, Yong Loo Lin School of Medicine,<br />
National University of Singapore, 4 Medical Drive, Singapore-117597, *antgopal@nus.edu.sg<br />
2<br />
Department of Pharmacy, Faculty of Science, National University of Singapore, 4 Science Drive,<br />
Singapore-117543<br />
3<br />
Fukuoka Women's University, Kasumigaoka, Higashi-ku, Fukuoka, 813-8529, Japan<br />
Drugs <strong>and</strong> drugable c<strong>and</strong>idates obtained from the venoms of marine cone snails are<br />
appreciated clinically to treat conditions from chronic pain to epilepsy. The venom element<br />
is usually gene encoded, conformationally constrained <strong>and</strong> disulfide bond rich shortpeptides<br />
(
Poster 43: Identification of the active-site residues of the fungal virulence factor,<br />
phospholipase B<br />
Tsukamoto, K 1* , Inoue, Y 1 , Tsuruga, A 1 <strong>and</strong> Obata, Y. 2<br />
1<br />
Showa Pharmaceutical University, 3-3165 Higashi Tamagawagakuen, Machida, Tokyo, Japan,<br />
*kikuot@ac.shoyaku.ac.jp<br />
2<br />
Aichi Medical University, Nagakute, Aichi, Japan<br />
Fungal phospholipase B (PLB) has been known to be a virulence factor in fungal infection<br />
such as Cryptococcus neoformans <strong>and</strong> C<strong>and</strong>ida albicans. The enzyme catalyzes the<br />
hydrolysis both of 1- <strong>and</strong> 2-acyl ester bonds of glycerophospholipid, <strong>and</strong> also exhibits<br />
lysophospholipase <strong>and</strong> transacylase activities. To investigate the structure-function<br />
relationship of fungal PLBs, we first cloned the plb1 gene of Saccharomyces cerevisiae<br />
W303-1A as a representative one. According to the sequence similarity between fungal<br />
PLBs <strong>and</strong> mammalian cytosolic phospholipase A2, we predicted the three-dimensional<br />
structure of the PLB1 by the homology modelling method. In the structural model,<br />
conserved residues among fungal PLBs converged on the cleft of the enzyme. On the<br />
basis of the structural characteristics of PLB1, we introduced site-directed mutagenesis to<br />
the conserved residues, <strong>and</strong> evaluate the effect of mutaions on the enzyme activity using<br />
the plb1 knocked-out S. cerevisiae strain as a host. Experimental results showed that<br />
conserved Ser147 <strong>and</strong> Asp403 are critical residues in catalysis, <strong>and</strong> conserved Arg109<br />
must contribute to the substrate hydrolysis through electrostatic interaction. These suggest<br />
that PLB1 belongs to the serine super family. For purification <strong>and</strong> the accurate functional<br />
analysis of PLB1, we established the baculoviral expression system of PLB1. The system<br />
expressed the PLB1 activity more 25-fold efficient than the reconstructed system using the<br />
plb1 knocked-out S. cerevisiae strain. The PLB1 protein was homogeneously purified<br />
from the culture medium of the baculoviral PLB1 expression clone. Effect of the PLB1 on<br />
mammalian cells would be discussed.<br />
o Phospholipase b<br />
o Invasion factor<br />
o cPLA2<br />
o homology modelling<br />
274<br />
Poster 44: A Comparative Study of the Enzymatic <strong>and</strong> Physiological Activities of<br />
Two Viper Venom L-Amino Acid Oxidases<br />
Samel, M. 1 , Tõnismägi, K. 1 , Trummal, K. 1 , Rönnholm, G. 2 , Siigur, J. 1 , Kalkkinen, N. 2 , Siigur, E. 1 *<br />
1<br />
National Institute of Chemical Physics <strong>and</strong> Biophysics, Akadeemia tee 23, Tallinn, Estonia,<br />
*enesi@kbfi.ee<br />
2<br />
Institute of Biotechnology, FIN-00014 University of Helsinki, Finl<strong>and</strong><br />
L-Amino acid oxidases (LAAO) are flavoenzymes able to stereospecific catalysis of the<br />
oxidative deamination of L-amino acids to alpha-keto acids, hydrogen peroxide <strong>and</strong><br />
ammonia. Snake venom is the richest source of the enzyme. Before 1990s the studies were<br />
mainly focused on the enzymatic properties of LAAOs but in the last 10-15 years the main<br />
interest is connected with effects of LAAO on various cells: platelets, vascular endothelial<br />
cells, different cancer cell lines. In this study the LAAOs from Vipera lebetina <strong>and</strong> Vipera<br />
berus berus venoms are characterized.<br />
The LAAOs have been isolated by size exclusion, ion exchange <strong>and</strong> affinity<br />
chromatography with protein yields of 1.8% (V. berus berus) <strong>and</strong> 2.5% (V. lebetina). Both<br />
enzymes are homodimeric FAD-containing glycoproteins with monomeric molecular<br />
masses of 57.7 (V. berus berus) <strong>and</strong> 60.9 (V.lebetina) kDa. They have identical N-terminal<br />
sequences <strong>and</strong> are characterized by tryptic peptides sharing high homology with other<br />
snake venom LAAOs. The most specific substrates for both enzymes are hydrophobic<br />
amino acids L-Leu, L-Met, L-Trp or L-Phe. V. b. berus LAAO inhibited ADP-induced<br />
platelet aggregation in the range of 0.03-0.10 microM, V. lebetina LAAO inhibited ADP-<br />
or collagen-induced aggregation dose-dependently in the range of 1.0-2.5 microM. In case<br />
of the ADP-induction the inhibition was more pronounced on the second wave of<br />
aggregation. V. b. berus LAAO induced apoptosis in the HeLa <strong>and</strong> K562 cells, V. lebetina<br />
LAAO inhibited growth of B. subtilis <strong>and</strong> E. coli. All physiological effects were abolished<br />
by catalase pointing to the role of hydrogen peroxide liberated in the oxidation process.<br />
Acknowledgement. The study has been financially supported by Estonian Science<br />
Foundation Grant 5938.<br />
o L-Amino acid oxidase<br />
o Vipera lebetina<br />
o Vipera berus berus<br />
o Snake venom<br />
275
Poster 45: Heterodimeric Disintegrin VLT-2 from Vipera lebetina Snake Venom<br />
Siigur, J.*, Vija, H., Aaspõllu, A., Samel, M., Tõnismägi. K., Trummal, K., Pikver, R., Subbi, J., Siigur,<br />
E.<br />
National Institute of Chemical Physics <strong>and</strong> Biophysics, Akadeemia tee 23,Tallinn 12618, Estonia, *siigur@kbfi.ee<br />
Vipera lebetina venoms of different subspecies contain several monomeric <strong>and</strong> dimeric<br />
disintegrins: obtustatin, VLO-4, VLO-5 from Vipera lebetina obtusa venom (Calvete et al<br />
2003, 2005); lebein (Gasmi et al 2001); lebein-1, lebein-2 (Eble et al 2003) <strong>and</strong> lebestatin<br />
(Sanz et al 2006) from V. lebetina mediterranea (Tunesian snake) venom. We purified a<br />
heterodimeric disintegrin VLT-2 from Vipera lebetina turanica venom obtained from<br />
Uzbekistan, using size exclusion chromatography on Sephadex G-100sf. <strong>and</strong> HPLC on<br />
C18 column. The VLT-2 chains were separated using SDS-PAGE; molecular masses of<br />
tryptic peptides from both chains <strong>and</strong> of native heterodimeric VLT-2 were detected by<br />
MALDI-TOF mass-spectrometry. Using cDNA library of the venom gl<strong>and</strong> of a single<br />
V.lebetina snake the VLT-2 coding cDNAs were cloned <strong>and</strong> sequenced. VLT-2 chains are<br />
synthesized from different genes. One chain, containing VGD sequence in disintegrin loop,<br />
is synthesized with PII type fibrinolytic metalloproteinase Le-3 (Lebetase), which is<br />
cleaved posttranslationally. The other chain, containing MLD sequence in disintegrin loop,<br />
is synthesized from the gene without metalloproteinase domain. The translated MLD<br />
containing sequence is longer than the protein counterpart purified from the venom. Most<br />
probably the C-terminus is processed proteolytically by venom proteinases. VLT-2<br />
primary structure is very similar to that of VLO-5: Le-3 disintegrin-like sequence is<br />
identical to VLO-5 A- chain, MLD containing sequence has two differences in the<br />
sequence compared with VLO-5 B-chain. VLT-2 dose-dependently inhibited collagen-<br />
induced platelet aggregation with IC50= 240 nM <strong>and</strong> ADP-induced aggregation with IC50=<br />
480 nM. The nucleotide sequences are registered in GenBank under Accession numbers<br />
X97894 <strong>and</strong> DQ295886.<br />
References: Gasmi et al (2001) BBA 1547,51-66; Calvete et al (2003) Biochem. J.<br />
372,725-734; Eble et al (2003) J.Biol. Chem. 278, 26488-26496; Calvete et al (2005)<br />
Toxicon 45,1063-1074; Sanz et al (2006) Biochem. J. 395, 385-392.<br />
The work was supported by Estonian Science Foundation Grant No. 5554.<br />
o Vipera lebetina<br />
o Heterodimeric disintegrin<br />
o Snake venom<br />
o Inhibition of platelet aggregation<br />
276<br />
Poster 46: Discovery <strong>and</strong> pharmacology of Conopressin-T, a novel Vasopressin-like<br />
peptide from Conus Tulipa.<br />
Daniel Croker 1 , Sebastien Dutertre 2,3 , Paul Alewood 2 , Richard Lewis 2<br />
1<br />
Xenome Ltd, Indooroopilly, Australia<br />
2<br />
Institute for Molecular Bioscience, The University of Queensl<strong>and</strong>, 4072 Qld, Australia.<br />
r.lewis@imb.uq.edu.au<br />
3<br />
Present address: Department of Neurochemistry, Max-Planck-Institute for Brain Research,<br />
Deutschordenstrasse 46, 60528 Frankfurt, Germany. dutertre@mpih-frankfurt.mpg.de<br />
To date, only two conopressins were isolated from cone snail venom. Conopressin-S was<br />
isolated from C. striatus venom, while conopressin-G was isolated from C. geographus<br />
venom. Here we report the discovery <strong>and</strong> pharmacology of a novel conopressin,<br />
conopressin-T (Con-T), which was isolated from the venom of C. Tulipa. Following<br />
isolation, Con-T showed a novel sequence that differed at two highly conserved residues of<br />
this peptide family. Synthesis of Con-T <strong>and</strong> its analogue [L7P]-Con-T (reversion to a<br />
conserved residue), were tested for there activity at the vasopressin/oxytocin receptor<br />
family. These peptides were assessed, for their biological activity by radiolig<strong>and</strong> binding<br />
assay <strong>and</strong> also by functional assay. The binding data showed that Con-T has modest<br />
activity at the oxytocin receptor, but is selective for the vasopressin V1A receptor. In<br />
addition, the analogue L7P-Con-T was 7- to 20-fold more active than Con-T at the V1A,<br />
<strong>and</strong> conserved the V1A selectivity. This preliminary SAR study could help in the design of<br />
novel vasopressin subtype selective drugs. Hence we have discovered a new novel<br />
Conopressin that has an unusual sequence compared to other peptides in this family <strong>and</strong><br />
displays an interesting selectivity profile at its target receptors.<br />
o Cone snail<br />
o Conopressin<br />
o Vasopressin<br />
o Pharmacology<br />
277
Poster 47: Isolation <strong>and</strong> characterisation of Conomap-Vt, a D-amino acid<br />
containing excitatory peptide from the venom of a vermivorous cone snail<br />
Sébastien Dutertre 1 , Natalie Lumsden, Paul F. Alewood <strong>and</strong> Richard J. Lewis<br />
Institute for Molecular Bioscience, The University of Queensl<strong>and</strong>, 4072 Qld, Australia.<br />
r.lewis@imb.uq.edu.au<br />
1 Present address: Department of Neurochemistry, Max-Planck-Institute for Brain Research,<br />
Deutschordenstrasse 46, 60528 Frankfurt, Germany. dutertre@mpih-frankfurt.mpg.de<br />
Cone snail venom is a rich source of bioactives, in particular small disulfide rich peptides<br />
that disrupt synaptic transmission. Here, we report the discovery of Conomap-Vt, an<br />
unusual linear tetradecapeptide isolated from Conus vitulinus venom. The sequence<br />
displays no homology to known conopeptides, but displays significant homology to<br />
peptides of the MATP (myoactive tetradecapeptide) family, which are important<br />
endogenous neuromodulators in molluscs, annelids <strong>and</strong> insects. Conomap-Vt showed<br />
potent excitatory activity in several snail isolated tissue preparations. Similar to ACh,<br />
repeated doses of conomap-Vt were tachyphylactic. Since nicotinic <strong>and</strong> muscarinic<br />
antagonists failed to block its effect <strong>and</strong> conomap-Vt desensitised tissue remained<br />
responsive to ACh, it appears that conomap-Vt contractions were non-cholinergic in<br />
origin. Finally, biochemical studies revealed that conomap-Vt is the first member of the<br />
MATP family with a D-amino acid. Interestingly, the isomerization of L-Phe to D-Phe<br />
enhanced biological activity, suggesting that this post-translational modified conopeptide<br />
may have evolved for prey capture.<br />
o Cone snail<br />
o D-amino acid<br />
o Conopeptide<br />
o isomerization<br />
278<br />
Poster 48: Tityus perijanensis (Scorpiones, Buthidae) from Zulia State, western<br />
Venezuela: Geographic Distribution <strong>and</strong> Venom Molecular <strong>and</strong><br />
Immunological Characterization<br />
Borges, A. 1* , Blumer-Lairet, V. 1 , Poliwoda, S. 1 , Alfonzo, M.J. 1 , Rojas-Runjaic, F. 2 , De Sousa, L. 3<br />
1 Instituto de Medicina Experimental, Universidad Central de Venezuela, Caracas *borgesa@ucv.ve<br />
2 Museo de Historia Natural La Salle, Caracas, Venezuela<br />
3 Centro de Investigaciones en Ciencias de la Salud, Universidad de Oriente, Puerto La Cruz<br />
Tityus perijanensis is a buthid scorpion endemic to the Perijá range, in the border between<br />
Colombia <strong>and</strong> Venezuela, which has caused severe accidents with central neurological<br />
sequelae. Since only scarce toxinological information is available on this species, an<br />
investigation of its toxicology, distribution <strong>and</strong> venom peptide composition was<br />
undertaken. Originally described from Ayajpaina (alto Río Negro), southern Zulia State,<br />
collection at various sites allowed extension of its geographical range to 250 km north of<br />
the type locality. Specimens throughout the extended distribution range shared the same<br />
cytochrome oxidase I subunit nucleotide sequence. Estimation of T. perijanensis venom<br />
toxicity was performed through LD50 assays by i.v. injections in mice. In vivo<br />
neutralizations tests were also carried out to evaluate the efficacy of the anti-T. discrepans<br />
(a northcentral Venezuelan species) antivenom to protect against envenoming by T.<br />
perijanensis. Lethality tests showed that T. perijanensis venom (LD50 = 0.9 mg/kg) is 2.7fold<br />
more toxic than T. discrepans. The venom was poorly neutralized by the anti-T.<br />
discrepans antivenom. Chromatographic fractionation of T. perijanensis venom through<br />
HPLC indicated that protein composition was different from other Venezuelan Tityus<br />
venoms of medical importance. Analysis of the T. perijanensis toxin repertoire was studied<br />
through amplification by RT-PCR of venom gl<strong>and</strong> mRNA using a combination of a<br />
degenerate primer (binding at the region encoding the toxin signal peptide) <strong>and</strong> a modified<br />
oligo-d(T) primer. Sequences of putative T. perijanensis sodium channel-active toxins<br />
encoded by isolated mRNAs differed from T. zulianus (a toxic Andean species) <strong>and</strong> T.<br />
discrepans equivalent peptides, suggesting the existence of regional differences in scorpion<br />
toxin antigenic reactivity within Venezuela (Supported by Fonacit Grant S1-2001000674<br />
to A.B.).<br />
Key Words: Neurotoxins, Scorpion, Tityus perijanensis, Venezuela<br />
279
Poster 49: Error-prone DNA polymerase k from Protobothrops flavoviridis,<br />
member of the DinB superfamily.<br />
Oda-Ueda, N 1 ., Kariu, T 1 ., Fukuhara, A 2 ., Takazaki, S 1 , Chijiwa, T 2 ., Ohno, M 2 .<br />
1 2<br />
Dept. Phamaceutical Sciences, Dept. Life Science, Sojo University, Kumamoto Japan,<br />
*naoko@life.sojo-u.ac.jp<br />
1. Introduction: We found that venom isozymes with a variety of physiological activities<br />
from Protobothrops (formerly known as Trimeresurus) flavoviridis have evolved in an<br />
accelerated manner <strong>and</strong> thought that mostlikely the diversity has been produced by<br />
accumulation of point mutations in their genes. To underst<strong>and</strong> the molecular mechanism<br />
underlying such mutation in P. flavoviridis, we have cloned a gene homologous to<br />
Echerichia coli dinB gene which encodes DNA polymerase IV known as error-prone DNA<br />
polymerase κ.<br />
2. Methods: Total RNA from P. flavoviridis oocyte was used as template for first str<strong>and</strong><br />
cDNA synthesis by using SMART cDNA Library construction kit. Degenerate primers<br />
were designed based on the conserved sequences of the polymerases in Xenopus laevis<br />
(frog) <strong>and</strong> Gallus gallus (chicken). Multiple rounds of 5’ <strong>and</strong> 3’ rapid amplification of<br />
cDNA ends were employed to extend the putative P. flavoviridis genes using BD SMART<br />
RACE amplification kit. Fibroblast cells from P. flavoviridis embryo were prepared for<br />
chromosomal mapping with fluorescence in situ hybridization (FISH). Multiple alignment<br />
was made with the CLUSTAL X program.<br />
3. Results <strong>and</strong> Discussion: A 2412-bp cDNA of P. flavoviridis DinB contained a ORF of<br />
2503 bp that encodes a protein of 833 amino acids. The amino acid sequence consisted of<br />
N-terminal nucleotidyl transferase domain, two t<strong>and</strong>em HhH domains implicated in DNA<br />
binding, nuclear localization signal like domain <strong>and</strong> proliferating cell nuclear antigen<br />
domain, as all being conserved in DinB superfamily, <strong>and</strong> shares significant identity (~84%)<br />
with G. gallus enzyme. In a phylogenetic tree of DinB superfamily, P. flavoviridis DinB<br />
constitutes a branch together with G. gallus enzyme out of the enzymes from other<br />
eukaryotes such as H. sapiens or mouse. Chromosomal FISH showed that P. flavoviridis<br />
DinB gene is localized in chromosome 6 (macrochromosome) different from the case of G.<br />
gallus DinB which is localized in microchromosome.<br />
o P. flavoviridis<br />
o Error-prone DNA polymerase<br />
280<br />
Poster 50: Post-translational modifications of venom components in Conus<br />
victoriae.<br />
1, 2 2 2 1 3 1 1, 4<br />
Townsend, A , Scanlon, D , O’Donnell, P , Inserra, M , Bingham, J-P , Satkunanathan, N , Khalil Z ,<br />
Purcell, A 1, 2 1, 2*<br />
, Livett, B.G.<br />
1<br />
University of Melbourne, Biochemistry <strong>and</strong> Molecular Biology, Parkville, Victoria, AUSTRALIA,<br />
*b.livett@unimelb.edu.au<br />
2<br />
Bio21 Institute, 30 Flemington Rd.,Parkville,Victoria, AUSTRALIA<br />
3<br />
Clarkson University, 8 Clarkson Ave.,Potsdam,New York, USA<br />
4<br />
University of Sharjah, College of Pharmacy,UAE.<br />
Introduction: Chemical analysis of Conus venoms has revealed a variety of biologically<br />
active small <strong>and</strong> intermediate size peptides rich in post-translational modifications (PTMs).<br />
Known PTMs include hydroxylation of proline, gamma-carboxylation of glutamate,<br />
bromination of tryptophan, sulfation of tyrosine, <strong>and</strong> glycosylation. A range of PTM<br />
patterns have been observed, including differential/preferential hydroxylation <strong>and</strong><br />
hyperhydroxylation (1). The functional role(s) of PTMs are not known but they could<br />
contribute to specificity of action, while stabilizing conopeptide structure <strong>and</strong>/or providing<br />
resistance to proteolysis. We have studied PTM-conotoxins in C. victoriae venom (2).<br />
Methods: Solid phase FMOC synthesis of conotoxin Vc1.1 sequence (deduced from the<br />
venom duct cDNA) <strong>and</strong> of an analogue (Vc1.1-ptm) where Pro 6 was replaced by hydroxy-<br />
Pro <strong>and</strong> Glu 14 by gammacarboxy-Glu. Preparative/analytical RP-HPLC was performed on<br />
an Agilent 1200 HPLC System. Crude venom was analysed with Agilent in-line<br />
chromatography for ESI TOF MS with sequence determination using an Agilent HPLC<br />
chip for ESI ion-trap MS/ MS. Antagonism of neuronal nicotinic receptors was assayed in<br />
vitro using monolayer cultures of bovine adrenal chromaffin cells. In vivo tests for<br />
hyperalgesia <strong>and</strong> allodynia were performed in adult female Sprague Dawley rats.<br />
Results: HPLC purification of the air-oxidized Vc1.1 (Mr 1807) revealed one major peak<br />
whereas the Vc1.1-ptm (Mr 1823) revealed three major peaks, representing two disulfide<br />
isomers (globular <strong>and</strong> ribbon) <strong>and</strong> a dimer (Mr 3643). Unlike Vc1.1, the Vc1.1-ptm did not<br />
inhibit the neuronal nicotinic receptor response <strong>and</strong> was not an analgesic. However, it<br />
retained the ability to accelerate functional nerve recovery of injured nerves.<br />
Discussion/Conclusion: These results on C. victoriae indicate that dimeric <strong>and</strong> higher<br />
order forms of conotoxins exist within the venom (3) <strong>and</strong> may have distinct biological<br />
activities.<br />
References: 1.Buczek, O. et al (2005) Cell Mol Life Sci.62:3067-3079; 2.Jakubowski,<br />
J.A., et al (2004) J. Mass Spectrom 39: 548-557; 3.Loughnan, M. et al (2006) JBC. [in<br />
press].<br />
Acknowledgement: Supported by DEST Australia, Innovation Grant #CG03 0017 to BGL<br />
o conotoxin<br />
o posttranslational modification<br />
o nicotinic receptor<br />
o analgesics<br />
281
Thursday 27 July: poster session<br />
(Colville Building 5.11/5.12)<br />
Marine toxins<br />
1. Poly-APS induced Ca 2+ entry in ECV cells. Maružin, M. 1 *, Hawlina, S. 2 , Bunc,<br />
M. 2 , Šuput, D. 1<br />
.<br />
2. Occurrence of tetrodotoxin in two new gastropods collected from west southern<br />
Taiwan. P.-A. Hwang a,b , Yung-Hsiang Tsai c , Shin-Jung Lin d , <strong>and</strong> Deng-Fwu<br />
Hwang b,*<br />
3. Occurrence of Tetrodotoxin <strong>and</strong> Paralytic Shellfish Poisons of a Gastropod in<br />
Southern Taiwan. Hsiao-Chin Jen 1 , Shin-Jung Lin 2 , I-Chiu Liao 1 , Osamu<br />
Arakawa 3 ,Shin-Yuan Lin 1 <strong>and</strong> Deng-Fwu Hwang 1*<br />
4. Influence of the sample toxic profile on the suitability of an HPLC method for<br />
Official PSP Control. Ben-Gigirey, B. 1* , Rodríguez-Velasco, M.L 1 , Botana, L.M. 1,2<br />
5. Evidence for the involvement of nitric oxide pathway in ciguatoxin-induced<br />
swelling of amphibian red blood cells. Sauviat, M.-P 1 , Boydron - Le Garrec, R 1, 2 ,<br />
Masson, J.-B 1 , Lewis, R.L 3 , Vernoux, J.-P 4 , Molgó, J 5 , Laurent, D 2 , Benoit, E 5 *<br />
6. Severe seafood poisoning in French Polynesia: a retrospective analysis of<br />
severity criteria in 129 hospital medical files. Gatti, C. 1* , Oehler, E. 2 , Valence, A. 2 ,<br />
Tamarii, D. 2 <strong>and</strong> Legr<strong>and</strong>, A.-M.<br />
7. Paralytic Toxicity in a Ribbon Worm Cephalothrix species (Nemertean)<br />
Adherent to Cultured Oysters in Hiroshima Bay, Hiroshima Prefecture, Japan.<br />
Manabu, Asakawa 1* , Shuhei Matsuda 1 , Shintaro Tsuruda 1 , Hiroshi Kajihara 2 ,<br />
Shigeto Taniyama 1<br />
8. Transfer profile <strong>and</strong> immnopotentiating effect of tetrodotoxin administered<br />
intraperitoneally into non-toxic cultured pufferfish Takifugu rubripes. Honda, S 1* ,<br />
Murakami, Y 1 , Ichibu, T 1 , Takatani, T 2 , Tachibana, K 2 , Arakawa, O 2 , Noguchi, T. 3<br />
9. Toxicity of edible dried fish in the Philippines. Shigeto Taniyama 1 , Takefumi<br />
Sagara 2 , Ryoichi Kuroki 1 , Satoshi Takamoto 3 , Shintaro Tsuruda 3 , Gloria Gomez<br />
Delan 4 , Sachio Nishio 2 , Manabu Asakawa 1*<br />
Pain <strong>and</strong> CNS<br />
10. ANTINOCICEPTIVE AND ANTIHYPERNOCICEPTIVE ACTIVITY OF<br />
THE METHANOLIC EXTRACT OF Phyllomedusa rohdei. Marinho, E.A.V. 1,3 ;<br />
282<br />
Zaharenko, A.J. 2 ; Paraventi, C 1 ; Jimenez, R.S. 1 ; Molska, G.R. 1 ; Paula-Freire, L.I.G. 1 ;<br />
Zanoni, C.I.S. 4 ; Almeida, E. 3 ; Frussa-Filho, R. 3 ; Parada, C.A. 4 ; Ferreira, S.H. 4 ;<br />
Malpezzi-Marinho, E.L.A 1<br />
11. ACTIVITY OF EXTRACTS FROM Phyllomedusa rohdei (AMPHIBIA,<br />
ANURA) SKIN ON THE MICE BEHAVIOUR IN THE OPEN FIELD AND<br />
ROTA ROD. Silva, R.W. 1 ; Zaharenko, A.J. 2 ; Paula-Freire, L.I.G. 1 ; Almeida, E. 3 ;<br />
Frussa-Filho, R. 3 ; Malpezzi-Marinho, E.L.A 1 , Marinho, E.A.V. 1,3<br />
12. κ AND μ OPIOID RECEPTORS ARE INVOLVED IN THE<br />
ANTINOCICEPTIVE ACTIVITY OF METHANOL EXTRACT OF<br />
Phyllomedusa rohdei ON THE HOT PLATE MODEL. Zanoni, C.I.S. 4 ; Molska,<br />
G.R. 1 ; Zaharenko, A.J. 2 ; Paraventi, C 1 ; Jimenez, R.S. 1 ; Paula-Freire, L.I.G. 1 ; Parada,<br />
C.A. 4 ; Ferreira, S.H. 4 ; Malpezzi-Marinho, E.L.A 1 ; Marinho, E.A.V. 1,3<br />
13. Xen2174: A novel NET inhibitor that enhances α2-adrenoceptor inhibition of<br />
spinal pain pathways. Croker, D.E 1 , Palant, E 1 , Nielsen C 2 , Drinkwater, R 1 , Patterson,<br />
M 3 , McCumber, D 3 , Yaksh, T 3 , Wilson, D 1* , Smith, M 2 <strong>and</strong> Lewis, R.J. 1<br />
14. Crotoxin inhibits neuropathic pain <strong>and</strong> the development of neuromas. Cury,<br />
Y. 1* , Nogueira-Neto, F. 1,2 , Amorim, R.L. 2 , Brigatte, P. 1 , Picolo, G. 1 , Ferreira Jr., W.A.<br />
1 , Nicoletti, J.L.M. 2<br />
15. Phospholipases A2 (PLA2) Isolated From Brazilian Coral Snake (Micrurus<br />
lemniscatus) Induced Neuronal Injury: In Vivo And In Vitro Studies. Carvalho,<br />
N.D 1 ., Oliveira, D.A. 1 , Casais e Silva, L.L. 3 ,Lebrun, I. 2 , Afeche, S.C 1 , S<strong>and</strong>oval,<br />
M.R.L. 1*<br />
Inflammation <strong>and</strong> cytokines<br />
16. ENHANCEMENT OF TNF-α AND IL-1 PRODUCTION IN ALVEOLAR<br />
MACROPHAGES BY C-FIBER DEGENERATION. DeSouza, IA *1 Camargo,<br />
EA 1 ; Franco-Penteado, CF 1 ; Antunes, E 1<br />
17. PRELIMINARY STUDIES OF STRUCTURAL AND BIOLOGICAL<br />
ACTIVITY OF GALATROX, A β-GALACTOSIDE-BINDING LECTIN FROM<br />
THE VENOM OF Bothrops atrox SNAKE. Mendonça-Franqueiro, E.P. 1 ; Callejon,<br />
D.R. 1 ; Paiva, H.H. 1 , Rosa, J.C. 2 ; Alves, R.M. 1 ; Franco, J.J. 1 ; Dias-Baruffi, M. 1 ;<br />
Sampaio, S.V. 1<br />
18. ASSESSMENT OF THE INHIBITION INDUCED BY Bufo paracnemis<br />
POISON AND ITS FRACTIONS ON COMPLEMENT SYSTEM AND<br />
LEUKOCYTE RECRUITMENT. Marongio, A. F. Q. 1 , Bertazzi, D. T. 1 , Arantes,<br />
E. C*<br />
283
19. Do cytokines <strong>and</strong> nitric oxide play a role in the actions of Leiurus<br />
quinquestriatus scorpion venom in animals? Abdoon, N. A 1 *, Elsayed Ali, A 2 ,<br />
Fatani, A. J. 3<br />
20. EVALUATION OF THE ACUTE LUNG INJURY IN A MURINE MODEL<br />
INDUCED BY THE SCORPAENA PLUMIERI FISH VENOM. Boletini-Santos<br />
D 1 , Haddad Jr V. 2* , Figueiredo S.G. 3 , Lopes-Ferreira M. 1 , Lima C.<br />
21. INFLAMMATORY MEDIATORS INVOLVED IN PHOSPHOLIPASE A2<br />
(PLA2)-INDUCED ACUTE PANCREATITIS. Camargo, E.A. *1 ; Ferreira, T. 1 ;<br />
L<strong>and</strong>ucci E.C.T. 1 ; Antunes, E.<br />
22. Effect of eugenol <strong>and</strong> local anesthetic agents on tongue <strong>and</strong> skin edema<br />
induced by Dieffenbachia picta Schott in mice. Dip, E.C. 1 ; Pereira, N.A. 1 ; Gaban,<br />
G.A. 1 ; Fonseca, T. 1 ; El Kik, C.Z. 1 ; Tomaz, M.A 1 ; Calil-Elias, S. 1 ; Martinez, A.M.B.<br />
2 ; Melo, P.A. 1 .<br />
23. Importance of jararhagin disintegrin-like <strong>and</strong> cysteine rich domains in the<br />
early events of local inflammatory response. Clissa, P.B. 1 ; Lopes-Ferreira, M. 1 ;<br />
Della-Casa, M.S. 1 ; Farsky, S.H.P. 2 ; Moura-da-Silva, A.M. 1<br />
24. Short-time analysis of pulmonary mechanics on acute lung injury induced by<br />
Crotalus durissus terrificus venom. Nonaka, P. N 1 , Peres, A. C. P 1 , Ferrari, E. F,<br />
Carreiro da Costa, R. S 1 , Silva, C. A. M 2 , Ribeiro, W, R, Cogo, J.C 1 , Oliveira, L.V.F. 1<br />
25: Effect of Crotalus durissus terrificus snake venom <strong>and</strong> crotoxin on cytokine<br />
secretion by macrophage. Cury, Y. * , Sampaio, SC., Brigatte, P, Curi, R., Sousa-e-<br />
Silva, MCC.<br />
Cytotoxicity <strong>and</strong> antimicrobial<br />
26. Hydralysins – novel proteins from cnidaria revealing a sequence signature<br />
with a role in pore formation common to various beta pore forming toxins. Sher,<br />
D.* 1 , <strong>Fish</strong>man, Y. 1 , Zhang, M. 1 , Lebendiker, M 1 , Mancheño, J.M. 2 <strong>and</strong> Zlotkin, E. 1<br />
27. Caissarolysin I (Bcs I), a new hemolytic toxin from the Brazilian sea anemone<br />
Bunodosoma caissarum: Purification <strong>and</strong> biological characterization. Zaharenko,<br />
A.J. 1,2* , Oliveira, J.S. 1,3 , Freitas, J.C. 1 , Konno, K. 2 , Andrade, S.A. 3 , Portaro, F.C.V. 2 ,<br />
Richardson, M. 4 , Sant’Anna, O.A. 3 , Tambourgi, D.V. 3<br />
28. Beta-Lactamase in Escherichia coli. Siham S. Shaokat*, Hamoudi A.<br />
Hameed, Israa H.J.<br />
284<br />
29. Membrane Interactions of the Pore-forming Protein Equinatoxin II:<br />
19 F NMR Studies of the role of Trp <strong>and</strong> Tyr. Norton, R.S. 1 , Bakrač, B. 2 , Anderluh.<br />
G. 2 , Podlesek, Z. 2 , Razpotnik, A. 2 , Separovic, F. 3<br />
30. Toxicity of Gambierdiscus sp. <strong>and</strong> Ostreopsis sp. collected from the coasts of<br />
western Japan. Takefumi Sagara 1* , Shigeto Taniyama 2 , Osamu Arakawa 3 , Tamiko<br />
Hashimoto 1 , Naoyoshi Nishibori 1 , Manabu Asakawa 2 <strong>and</strong> Sachio Nishio 1<br />
31. Syphaxin 1.5, a novel antimicrobial peptide isolated from the skin secretions of<br />
the anuran Leptodactylus syphax. Dourado, F. S 1,2* , Moreira, K. G 2 , Br<strong>and</strong>, G. D 2 ,<br />
Melo, J. T 2 , Leite, J, R. S. A 2 , Silva, L. P 2 , Bloch Jr, C 2 , Schwartz, E. F. 1<br />
32. Cytoskeletal rearrangement <strong>and</strong> cell death induced by Bothrops alternatus<br />
snake venom in cultured Madin-Darby canine kidney cells. Nascimento, J.M. 1,2 ,<br />
Franchi, G. 3 , Novill, A. 3 , Collares-Buzato, C.B. 4 , Hyslop, S. 1,*<br />
33. Influence of salinity on gliotoxin excretion by marine <strong>and</strong> terrestrial<br />
Aspergillus fumigatus strains. Kerzaon, I.*, Grovel, O., Le Pape, P., Robiou du Pont,<br />
T., Pouchus, Y.F.<br />
34. Effect of the Disintegrin Eristostatin on Melanoma Cell Signal Transduction.<br />
Paquette-Straub, C.A, DiRosato, S.C, McLane, M.A.<br />
35. Ostreolysin, a mushroom pore-forming toxin specifically binding to<br />
sphingomyelin/cholesterol-rich membrane domains. Rebolj, K. 1 , Maček, P. 1 <strong>and</strong><br />
Sepčić, K. 1*<br />
36. Time- <strong>and</strong> concentration-dependent cytotoxicity of ricin in human lung<br />
epithelial cells. Ramasamy, S. 1* <strong>and</strong> Proll, D. 1<br />
37. Cytotoxic Effect of Primulaceae on Normal Fibroblasts <strong>and</strong> Cancer Cell Lines.<br />
Young, L.C 1* , Abbott, G. M 1 , Harvey, A.L. 1<br />
38. A crystalline nonprotein compound (BM-ANF) from toad (Bufo melanostictus,<br />
Schneider) skin extract having antiproliferative <strong>and</strong> apoptogenic activity. Giri,<br />
B 1* , Mishra, R 1 , Dasgupta, S.C 1 , Gomes, A. 1<br />
39. A new protein toxin (drCT1) from Dabuia russellii venom having<br />
antiproliferative <strong>and</strong> apoptogenic activity. Dasgupta, S.C 1* , Saha, A., Giri, B.,<br />
Roychoudhury, S., Gomes, A.<br />
40. ANTIMICROBIAL ACTIVITY OF EXTRACTS FROM Phyllomedusa rohdei<br />
(AMPHIBIA, ANURA) SKIN. Gomez, J.G.C. 1 ; Zaharenko, A.J. 2 ; Rocha, L.M. 1 ;<br />
Wuo, V.G. 1 ; Marinho, E.A.V. 1,3 ; Malpezzi-Marinho, E.L.A 1<br />
285
41. EFFECT OF NOVEL PECTENOTOXINS ON ACTIN CYTOSKELETON.<br />
Louzao, M.C. 1* , Ares, I.R. 1 , Cagide, E. 1 , Vieytes, M.R. 2 , Miles, C.O. 3 , Yasumoto, T. 4<br />
<strong>and</strong> Botana, L.M. 1<br />
42. A Cardiotoxin (NK31) from Indian Monocled Cobra (Naja kaouthia) shows<br />
activity against human chronic myelogenic cell-line (K562). Debnath, A, Gomes,<br />
A*, Vedasiromoni, J.R.<br />
43. Antileukemic activity of Indian Black Scorpion (Heterometrus bengalensis)<br />
venom against human leukemic U937 <strong>and</strong> K562 cells. Gomes, A * , Das Gupta, S ,<br />
Vedasiromoni, J.R.<br />
44. Haemolytic protein from pedicellaria of violet sea urchin Sphaerechinus<br />
granularis (Toxopneustidae). Turk, T 1 <strong>and</strong> Kem, W. R.<br />
45: An alpha-conotoxin from Conus victoriae is effective at alleviating neuropathic<br />
pain in an animal model of diabetic neuropathy. Satkunanathan, N 1 , Khodr, B 1 ,<br />
Georgiou, G 1 , McIntosh, D 2 , Belyea, C 2 , Khalil Z 1, 3 , Livett, B.G. 1*<br />
286<br />
Poster 1: Poly-APS induced Ca 2+ entry in ECV cells<br />
Maružin, M. 1 *, Hawlina, S. 2 , Bunc, M. 2 , Šuput, D. 1<br />
.<br />
1<br />
University of Ljubljana, Medical faculty, Institute of Pathophysiology, Zaloška 2, Ljubljana, Slovenia;<br />
*mitja.maruzin@mf.uni-lj.si<br />
2<br />
Clinical centre in Ljubljana, Zaloška 2, Ljubljana, Slovenia<br />
Introduction: Of the many distinct chemical weapons produced by sessile marine<br />
sponges, a number of them are novel alkylpyridinium salts (APS) that have interesting<br />
biological properties that may be exploited. Polymeric APS (poly-APS) are isolated from<br />
the aqueous extract of the marine sponge Reniera sarai. Diverse biological activities have<br />
been identified for different 1,3-APS oligomer preparations. These include cytotoxicity,<br />
neurotoxicity <strong>and</strong> inhibition of action potentials, stimulation of transmitter release,<br />
inhibition of K + -conductances <strong>and</strong> anticholinesterase activity. Some preparations of these<br />
toxins that contain cocktails of APS have been found to produce hemolysis. These actions<br />
may be attributed to the formation of lesions or pores in cell membranes, an effect that can<br />
also be produced in artificial lipid bilayers. We tried to explain in-vivo vascular effects by<br />
cytotoxic action of poly-APS on endothelial cells.<br />
Methods: We used fura-2 Ca 2+ imaging <strong>and</strong> confocal microscopy to compare the<br />
cytotoxic action of poly-APS on ECV-304 cell culture with the action of equinatoxin II<br />
(EqT II), which produced irreversible cell swelling <strong>and</strong> cytolysis.<br />
Results: Addition of poly-APS (0,5-5µg/ml) to the culture medium was followed by a<br />
dose-dependent rise in intracellular Ca 2+ . At concentrations used there were some<br />
sporadical changes in volume observed but no cytolysis.<br />
Discussion: Till recently, there were no reports of the toxic effects of these novel<br />
substances in vivo. Bunc M. et al.(1) evaluated the role of AChE inhibitory activity as well<br />
as the role of other effects of poly-APS observed in vitro by monitoring their effects on<br />
cardiovascular <strong>and</strong> respiratory activity in vivo. At higher doses the possible AChE<br />
inhibitory effects were not observed as they were probably masked by other, faster <strong>and</strong><br />
more pronounced lethal effects of the toxin, such as the initiation of blood coagulation,<br />
platelet aggregation <strong>and</strong> cytotoxity. An increase of intracellular [Ca 2+ ] could also be<br />
considered as a possible mechanism of trombocyte activation.<br />
References: 1. Bunc, M. et al. (2002). Toxicon 40 (7): 843-849.<br />
o Alkylpyridinium salt<br />
o Endothelial cell<br />
o Intracellular calcium<br />
o Cytotoxicity<br />
287
Poster 2: Occurrence of tetrodotoxin in two new gastropods collected from west<br />
southern Taiwan<br />
P.-A. Hwang a,b , Yung-Hsiang Tsai c , Shin-Jung Lin d , <strong>and</strong> Deng-Fwu Hwang b,*<br />
a Division of Food Technolgy, <strong>Fish</strong>eries Research Institute, Keelung, Taiwan<br />
b Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan<br />
c Department of Food Sanitation, Tajen Institute of Technology, Pingtung, Taiwan<br />
d<br />
Department of Food Science <strong>and</strong> Technology, Ching-Kuo Institute of Management <strong>and</strong><br />
Health, Keelung, Taiwan<br />
Abstract<br />
The gastropod Harpa articularis (8 specimens) <strong>and</strong> Hindsia sinensis (6 specimens) were,<br />
respectively, collected from west southern Taiwan were toxic. The average toxicities in<br />
digestive gl<strong>and</strong> <strong>and</strong> muscle were 14±3 (mean±S.D.) <strong>and</strong> 19±5 mouse unit per gram<br />
(MU/g) for H. articularis, <strong>and</strong> 31±10 <strong>and</strong> 23±7 MU/g for H. sinensis, respectively. But<br />
the other twenty-one specimens of both species collected from eastern Taiwan were not<br />
toxic. The toxin of both toxic species was extracted with methanol, defatted with<br />
dichloromethane, concentrated, purified through Bio-Gel P-2 column <strong>and</strong> eluted with<br />
0.03 M acetic acid. The high performance liquid chromatography <strong>and</strong> liquid<br />
chromatography/mass spectrometry analyses demonstrated that the toxin of both toxic<br />
species was composed of tetrodotoxin (TTX) <strong>and</strong> anhydro-TTX.<br />
288<br />
Poster 3: Occurrence of Tetrodotoxin <strong>and</strong> Paralytic Shellfish Poisons of a<br />
Gastropod in Southern Taiwan<br />
Hsiao-Chin Jen 1 , Shin-Jung Lin 2 , I-Chiu Liao 1 , Osamu Arakawa 3 ,<br />
Shin-Yuan Lin 1 <strong>and</strong> Deng-Fwu Hwang 1*<br />
1<br />
Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan,<br />
R.O.C.<br />
2<br />
Department of Food Science <strong>and</strong> Technology, Ching Kuo Institute of Management <strong>and</strong><br />
Health, Keelung, Taiwan, R.O.C.<br />
3<br />
Faculty of <strong>Fish</strong>eries Nagasaki University, Bunkyo-machi 1-14, Nagasaki, 852-8521,<br />
Japan.<br />
The toxicity of gastropod Nassarius papillosus implicated to a food paralytic poisoning<br />
incident in Liuchiu Isl<strong>and</strong>, Taiwan in October, 2005 was reported. The symptoms of a<br />
victim (67 years old) were featured by general paresthesia, paralysis of phalanges <strong>and</strong><br />
extremities, paralysis, coma <strong>and</strong> aphasia. The remained specimens of shell were assayed<br />
for toxicity. The range of specimen toxicity was 63 to 474 mouse units per specimen<br />
(MU/ specimen) TTX for N. papillosus. The average toxicity of the digestive gl<strong>and</strong> <strong>and</strong><br />
other portions was 296 ± 120 (mean ± S.D.) <strong>and</strong> 382 ± 156 MU in N. papillosus. The<br />
toxin was partially purified from the acidic methanol extract of the gastropod by using a<br />
C18 solid-phase extraction column <strong>and</strong> then the eluate was filtered through a 3,000 MW<br />
cut-off ultrafree microcentrifuge filter. The toxin purified from gastropods was analyzed<br />
by HPLC <strong>and</strong> LC/MS showed that the toxin contained TTX <strong>and</strong> related compound anh-<br />
TTX (about 90%), whereas along with minor GTX2,3 <strong>and</strong> neo-STX (about 10%).<br />
289
Poster 4: Influence of the sample toxic profile on the suitability of an HPLC<br />
method for Official PSP Control<br />
Ben-Gigirey, B. 1* , Rodríguez-Velasco, M.L 1 , Botana, L.M. 1,2<br />
1 CommunityReference Laboratory of Marine Biotoxins (CRLMB), Estación Marítima S/N, Muelle de<br />
Trasatlánticos, Vigo, Spain, *bbeng@msc.es<br />
2 Departamento de Farmacología, Facultad de Veterinaria, Campus Universitario, Lugo, Spain<br />
An HPLC-FLD method, involving pre-chromatographic oxidation of the PSP toxins with<br />
hydrogen peroxide <strong>and</strong> periodate, has been AOAC validated through a collaborative trial<br />
<strong>and</strong> adopted as AOAC Official Method. This method could be a c<strong>and</strong>idate for replacing the<br />
mouse bioassay (MBA) for the Official Control of PSP toxins at European level, once<br />
accepted by the legislation. An interlaboratory exercise has been organized by the CRLMB<br />
to evaluate its “fitness for purpose” for the Official Control of PSP toxins in the E.U.<br />
laboratories. Eighteen participant laboratories analyzed six bivalve mollusc samples (two<br />
of them blind duplicates) with several PSP toxic profiles.<br />
The method used for this interlaboratory exercise was the Lawrence HPLC method as<br />
appears in AOAC Official Method 2005.06 (First Action). A detailed protocol with<br />
instructions for the application of the method for this exercise was facilitated to<br />
participants.<br />
Samples assigned values were calculated by consensus from participants valid results after<br />
removing outliers (Cochran & Grubbs / Dixon (α=0.05) tests). St<strong>and</strong>ard deviations for<br />
proficiency assessment were derived from the precision data obtained at the collaborative<br />
trial. For each sample z-scores were calculated for valid data.<br />
The HPLC validated method is only applicable for Official PSP Control for certain<br />
samples. This depends on sample PSP toxic profile. Results obtained for a sample with a<br />
toxic profile dominated by GTX6 <strong>and</strong> suspected to contain also C1,2 & C3,4 were not<br />
satisfactory. Only GTX5 <strong>and</strong> dc-STX could be quantified <strong>and</strong> the results achieved (total<br />
toxicity) were much lower (1/8) than those obtained by MBA. The lack of several PSP<br />
st<strong>and</strong>ards, the fact that the method is not validated for all the PSP toxins, <strong>and</strong> several<br />
drawbacks found in its application are a h<strong>and</strong>icap to implement it for Official PSP Control.<br />
o PSP toxins<br />
o Interlaboratory exercise<br />
o HPLC method<br />
290<br />
Poster 5: Evidence for the involvement of nitric oxide pathway in ciguatoxininduced<br />
swelling of amphibian red blood cells<br />
Sauviat, M.-P 1 , Boydron - Le Garrec, R 1, 2 , Masson, J.-B 1 , Lewis, R.L 3 , Vernoux, J.-P 4 , Molgó, J 5 ,<br />
Laurent, D 2 , Benoit, E 5 *<br />
1 Laboratoire d'Optique et Biosciences, INSERM U696, UMR CNRS 7645, X / ENSTA, Ecole<br />
Polytechnique, Palaiseau, France<br />
2 Laboratoire de Pharmacochimie des Substances Naturelles et Pharmacophores Redox, UMR 152,<br />
IRD-Université Paul Sabatier, Centre IRD de Nouméa, Nouméa, New Caledonia<br />
3 School of Biomedical Sciences, The University of Queensl<strong>and</strong>, Brisbane, Australia<br />
4 Microbiologie Alimentaire, USC INRA, Université de Caen Basse-Norm<strong>and</strong>ie, Caen, France<br />
5 CNRS, Institut de Neurobiologie Alfred Fessard – FRC2118, Laboratoire de Neurobiologie<br />
Cellulaire et Moléculaire – UPR9040, Gif sur Yvette, France, *benoit@nbcm.cnrs-gif.fr<br />
The cyclic polyethers ciguatoxins (CTXs), produced by toxic dinoflagellates, are<br />
responsible for ciguatera, a human ichthyosarcotoxism acquired by eating contaminated<br />
species of fish. Although these toxins are well known to activate voltage-gated Na +<br />
channels, they were recently reported to produce a swelling of frog erythrocytes that was<br />
dependent on the activation of voltage-gated L-type Ca 2+ channels. In order to get further<br />
information on the mode of action of CTXs, we have studied the cellular mechanisms<br />
involved in the effects of the Pacific (P-CTX-1) <strong>and</strong> Caribbean (C-CTX-1) ciguatoxins on<br />
the rheological behaviour of frog red blood cells (RBCs). For these purpose, the length,<br />
width <strong>and</strong> surface of the elliptic shape of RBCs, obtained from the blood evicted from the<br />
frog (Rana esculenta) heart, were measured with a graduated eyepiece, <strong>and</strong> intracellular<br />
Ca 2+ changes were determined by microspectrofluorometry using the Ca 2+ -sensitive<br />
fluorescent dye fura-2/AM. Comparisons between values were done using paired Student’s<br />
t-test <strong>and</strong> considering that a P value of less than 0.05 was significant. The results show that<br />
nanomolar concentrations of P-CTX-1 <strong>and</strong> C-CTX-1 induced an increase in intracellular<br />
Ca 2+ that was sensitive to the L-type Ca 2+ channels' inhibitors Cd 2+ <strong>and</strong> verapamil, <strong>and</strong> to<br />
the nitric oxide (NO) synthase (NOS) blocker NG-methyl-L-arginine (L-NMA), as well as<br />
a cell swelling that was prevented <strong>and</strong>/or reversed by L-NMA, by blocking Ca 2+ -dependent<br />
K + channels with apamin or Sr 2+ , <strong>and</strong> by inhibiting soluble guanylate cyclase (sGC) with<br />
H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one. In addition, cytochalasin D, a potent<br />
inhibitor of actin filament <strong>and</strong> contractile microfilaments, mimicked the CTXs' effects but<br />
did not prevent the CTXs-induced L-NMA-sensitive extra increase in RBCs' surface. We<br />
conclude that the molecular mechanisms underlying CTXs-induced RBCs' swelling<br />
involve the NO pathway by activating first inducible NOS <strong>and</strong> then sGC which modulates<br />
intracellular cGMP <strong>and</strong> regulates L-type Ca 2+ channels. In turn, the resulting increase in<br />
intracellular Ca 2+ is likely to disrupt the actin cytoskeleton, causing thus a water influx, <strong>and</strong><br />
to activate the SK2 isoform of Ca 2+ -dependent K + channels.<br />
o Ciguatoxins<br />
o Frog erythrocytes<br />
o Cell swelling<br />
o Nitric oxide pathway<br />
291
Poster 6: Severe seafood poisoning in French Polynesia: a retrospective analysis of<br />
severity criteria in 129 hospital medical files<br />
Gatti, C. 1* , Oehler, E. 2 , Valence, A. 2 , Tamarii, D. 2 <strong>and</strong> Legr<strong>and</strong>, A.-M. 1<br />
1 Institut Louis Malardé, Papeete, Tahiti, French Polynesia, *cgatti@ilm.pf<br />
2 Centre Hospitalier de Polynésie française, Papeete, Tahiti.<br />
Seafood poisonings (SFP) are commonly encountered in tropical areas, especially<br />
in Pacific isl<strong>and</strong>s where fish is an important source of proteins. The diseases appear usually<br />
benign, nevertheless a significant number of severe cases require hospital care. Herein is<br />
presented a retrospective study of 129 medical files concerning SFP registered at the<br />
central hospital of Tahiti between 1999 <strong>and</strong> 2005. During that period we registered in all<br />
four different SFP: tetrodo-intoxication, carcha-intoxication, lyngbya-intoxication <strong>and</strong><br />
ciguatera poisoning respectively due to the consumption of some species of<br />
tetraodon/diodon family, shark, sea turtle or reef fish. Micro-organisms responsible for<br />
such intoxication types, respectively induced by tetrodotoxins, carchatoxins, lyngbyatoxins<br />
or ciguatoxins, have been identified for some of them as bacteria, cyanobacteria or<br />
dinoflagellates. Most of the described cases (96%) concerned the ichtyosarcotoxism called<br />
ciguatera. In those files, cardiovascular symptoms were the primarily criteria of severity<br />
with bradycardia <strong>and</strong> hypotension observed in respectively 74 % <strong>and</strong> 42 % of the cases.<br />
Neurological manifestations (such as cerebellar syndrome, language troubles, diplopia, or<br />
polyradiculoneuritis), trouble <strong>and</strong>/or loss of conscience <strong>and</strong> dyspnoea, intervene as<br />
secondary cause of severity. Besides, our attention focused on the body temperature<br />
reported becoming under 36.5°C in 48 upon 80 documented files. This observation, which<br />
had never been raised yet in human, may be related to possible central effects of the<br />
ingested toxin. Indeed, hypothalamus is known to be implicated in thermoregulation <strong>and</strong><br />
hypothermia has been reported as a central effect in experimentally acute intoxication of<br />
mice with ciguatoxins. The last remark concerns two extremely severe cases of ciguatera<br />
fish poisoning in which physicians had suspected a Guillain-Barré Syndrome (GBS), an<br />
autoimmune disease associated with nerve demyelinization <strong>and</strong> characterised by an<br />
ascendant reversible paralysis. It is precipitate to formulate any correlation between the<br />
intoxication <strong>and</strong> the appearance of GBS but it is interesting to underline that in both<br />
pathologies, morphological disturbances of nerves fibres have been reported. Could<br />
ciguatoxins be possible inductors of GBS?<br />
o ciguatera<br />
o sea food poisoning<br />
o severe symptoms<br />
o hospitalisation<br />
292<br />
Poster 7: Paralytic Toxicity in a Ribbon Worm Cephalothrix species (Nemertean)<br />
Adherent to Cultured Oysters in Hiroshima Bay, Hiroshima Prefecture, Japan<br />
Manabu, Asakawa 1* , Shuhei Matsuda 1 , Shintaro Tsuruda 1 , Hiroshi Kajihara 2 , Shigeto Taniyama 1<br />
1 Graduate School of Biosphere Science <strong>and</strong> Technology, Hiroshima University, 1-4-4, Kagamiyama<br />
Higashi-Hiroshima, Hiroshima, 739-8528 Japan,* asakawa@hiroshima-u.ac.jp<br />
2 Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan<br />
Specimens of ribbon worm Cephalothrix sp. were collected in Hiroshima Bay between<br />
November <strong>and</strong> May from 1998 to 2005 almost every two weeks during the harvest time of<br />
cultured oysters. A total of 764 specimens were collected, <strong>and</strong> assayed for toxicity. All the<br />
specimens assayed were found to be toxic throughout the season covered, <strong>and</strong> toxicity<br />
scores ranged from 169 MU/g to 25,590 MU/g (as tetrodotoxin). The ratio of strongly<br />
toxic ( 1,000 MU/g) to total specimens was 80%. On the other h<strong>and</strong>, the frequency of the<br />
specimens possessing toxicity scores more than 2,000 MU/g to total specimens was the<br />
high score of 48%. A relationship between size <strong>and</strong> toxicity of this ribbon worm was not<br />
obvious. The highest toxicity detected was 25,590 MU/g in a specimen collected in Jun.<br />
25(1999). The total toxicity of this one was roughly calculated to be 5,631 MU, the value<br />
which is approximately equivalent to half of the minimum lethal dose of TTX in human,<br />
which is reported to be 10,000 MU. In this connection, this lethal potency was about 51 or<br />
47 times larger than the highest score so far recorded for two species of ribbon worm,<br />
Lineus fuscoviridis <strong>and</strong> Tubulanus punctatus inhabiting the surface of rocks or soft mud on<br />
the seashore (Miyazawa et al., 1986, 1988). It has been reported that TTX <strong>and</strong> related<br />
substances are present in highly toxic species of the ribbon worm C. linearis (Ali et al.,<br />
1990). No paralytic toxicity was detected in the shucked meat of the oysters fouled with<br />
the ribbon worms. As the culturing of edible bivalves such as oysters <strong>and</strong> scallops is a<br />
flourishing industry in Japan, surveillance for the distribution of toxic ribbon worms in<br />
other areas besides Hiroshima Bay is urgently needed. Some seasonal variation of lethal<br />
potency of this ribbon worm could be recognized. Specimens collected in October 28,<br />
2004 to January 20, 2005 showed mean toxicities of 1,855-4,076 MU/g. But on February<br />
22, 2005, their toxicities had sharply decreased down to a mean of 1,578 MU/g. At the<br />
beginning of December, their toxicities increased to a mean of 4,510 MU/g.<br />
References: 1. Ali A.E. et al. (1990). Toxicon. 28, 1083-1093. 2. Miyazawa, K. et al.<br />
(1986). Toxicon. 24, 645-650. 3. Miyazawa, K. et al. (1988). Toxicon. 26, 867-874.<br />
o Ribbon worm<br />
o Oyster<br />
o Tetrodotoxin<br />
o Hiroshima Bay<br />
293
Poster 8: Transfer profile <strong>and</strong> immnopotentiating effect of tetrodotoxin<br />
administered intraperitoneally into non-toxic cultured pufferfish Takifugu<br />
rubripes<br />
Honda, S 1* , Murakami, Y 1 , Ichibu, T 1 , Takatani, T 2 , Tachibana, K 2 , Arakawa, O 2 , Noguchi, T. 3<br />
1<br />
Graduate School of Science <strong>and</strong> Technology, Nagasaki University, 1-14, Bunkyo-machi, Nagasaki 852-<br />
8521, Japan, *number3000jp@yahoo.co.jp<br />
2<br />
Faculty of <strong>Fish</strong>eries, Nagasaki University, 1-14, Bunkyo-machi, Nagasaki 852-8521, Japan<br />
3<br />
Faculty of Healthcare, Tokyo Health Care University, 3-11-3, Setagaya, Setagaya-ku, Tokyo 154-8568,<br />
Japan<br />
Introduction: As a part of studies to clarify the accumulation mechanism <strong>and</strong> biological<br />
functions of tetrodotoxin (TTX) in pufferfish, two types of TTX preparations, crude<br />
(cTTX) <strong>and</strong> purified (pTTX), were administered intraperitoneally into the pufferfish<br />
Takifugu rubripes, <strong>and</strong> their transfer profiles <strong>and</strong> activating effect for splenocyte<br />
proliferation reactions (SPRs) were investigated.<br />
Methods: Three groups of non-toxic cultured T. rubripes specimens (body weight 144±16<br />
g; 30 individuals in each group) were administered intraperitonieally with saline, cTTX<br />
(crude extract from toxic tissues of the wild puffer T. vermicularis), <strong>and</strong> pTTX (95%<br />
purity), respectively, <strong>and</strong> reared in aerated tanks (90 l) up to 168 hrs. The test fish of each<br />
group were taken up from the tank 1, 4, 8, 12, 24, 72 <strong>and</strong> 168 hrs after the toxin<br />
administration, <strong>and</strong> determined for the toxicity of each tissue by the mouse bioassay. In<br />
addition, their SPRs were also examined at 72 <strong>and</strong> 168 hrs.<br />
Results: Transfer profiles of cTTX <strong>and</strong> pTTX in puffer body after intraperitoneal<br />
administration were somewhat different from each other. pTTX moved to the liver <strong>and</strong><br />
skin more rapidly than cTTX, but at the end of rearing period (168 hrs after<br />
administration), it remained only in the skin in a small quantity (2.4-3.0 MU/g), whereas<br />
cTTX was retained mainly in the liver in a relatively large quantity (11-19 MU/g). On the<br />
other h<strong>and</strong>, the test fish of pTTX-administered group exhibited slightly higher SPRs than<br />
those of the other groups at 72 hrs, while the test fish of cTTX-administered group showed<br />
significantly higher SPRs than those of the other groups at 168 hrs. The temporal changes<br />
in SPRs appeared to coincide with the transfer profiles of TTX.<br />
Discussion/Conclusion: From the results it can be inferred that an unknown substance(s)<br />
coexisting with TTX in cTTX, possibly a TTX-binding protain (1), might be involved in<br />
the longer retention of TTX in puffer liver. It was also suggested that TTX could exhibit an<br />
immnopotentiating effect on T. rubripes by acting on the spleen, a main organ coordinating<br />
immunity in fish.<br />
References: 1. Matsui et al. (2000) Toxicon, 38, 463-468.<br />
o Tetrodotoxin (TTX)<br />
o Pufferfish<br />
o Takifugu rubripes<br />
o Splenocyte proliferation reaction<br />
294<br />
Poster 9: Toxicity of edible dried fish in the Philippines<br />
Shigeto Taniyama 1 , Takefumi Sagara 2 , Ryoichi Kuroki 1 , Satoshi Takamoto 3 , Shintaro Tsuruda 3 , Gloria<br />
Gomez Delan 4 , Sachio Nishio 2 , Manabu Asakawa 1*<br />
1 Graduate School of Biosphere Science <strong>and</strong> Technology, Hiroshima University, 1-4-4, Kagamiyama<br />
Higashi-Hiroshima, Hiroshima 739-8528 Japan, *asakawa@hiroshima-u.ac.jp<br />
2 Department of Science of Living, Shikoku University, Junior College, 123-1 Furukawa, Ohjin-cho, Tokushima<br />
771-1192, Japan<br />
3 Faculty of Applied Biological Science, Hiroshima University, 1-4-4, Kagamiyama Higashi-Hiroshima,<br />
Hiroshima 739-8528 Japan<br />
4 College of <strong>Fish</strong>eries Technology, Cebu State College Science <strong>and</strong> Technology, Carmen, Cebu Campus,<br />
Philippines<br />
In the Philippines, many kind of dried fish are consumed as popular marine product.<br />
During the studies on the utilization of fish as source of protein, ciguateric toxicities were<br />
detected in some dried fish sold in the fish market. In this study, attempts were made to<br />
clear some characteristics of the toxin contained in them. In July <strong>and</strong> October, 2005, 46<br />
specimens of dried fish from mainly Scarus niger, S. ghobban, Bolbometopon bicolor <strong>and</strong><br />
Cheilinus chlorourus, were collected from the local market in Negros Is. <strong>and</strong> Cebu Is. At<br />
first, meat of 13 specimens was homogenized with acetone respectively. The extract was<br />
treated with diethyl ether <strong>and</strong> partitioned between a 90% methanol <strong>and</strong> an n-hexane.<br />
Ciguateric toxicities of ninety % of methanol fractions suspended in 1% Tween60 saline<br />
solution were examined by mouse bioassay to determine ciguatoxins (CTXs). On the other<br />
h<strong>and</strong>, by using of ciguatera fish poison test kit (Cigua-Check), CTX is in all specimens or<br />
not. Eight fat-soluble extracts were found be not only toxic (0.025-0.05 MU/g), but also<br />
positive in Cigua-Check kit. Thirty-two specimens were too small to clarify their toxicities<br />
by mouse bioassay. But they showed positive or weakly positive by the kit. Moreover, the<br />
meat of 12 specimens, 3 of which were combined, was extracted with 75% ethanol (pH<br />
3.5), defatted with diethyl ether. The extracts of an aqueous layer were submitted to<br />
biochemical test. All of water-soluble extract were toxic, <strong>and</strong> their toxicity ranged from 0.5<br />
to 2.0 MU/g. The symptoms derived from administration (i.p.) to mice were convulsion,<br />
wobbling gait, jumping, drowsiness <strong>and</strong> collapse. They died within 24 hrs. Further, in<br />
hemolysis test, their toxin showed delayed hemolytic activity with mouse or human<br />
erythrocytes at sample concentration of 0.05-0.2 MU/ml with incubation time of 4 hrs at<br />
37°C. From these data, it is suggested that most of dried fish were contaminated with<br />
CTXs in the Philippines, <strong>and</strong> at least 5 specimens were contained CTX <strong>and</strong> another<br />
toxin(s) based on the lethal potency <strong>and</strong> the delayed haemolytic activity. However, studies<br />
along these lines are now in progress.<br />
o Dried fish<br />
o Ciguatoxins<br />
o The Philippines<br />
295
Poster 10: ANTINOCICEPTIVE AND ANTIHYPERNOCICEPTIVE ACTIVITY<br />
OF THE METHANOLIC EXTRACT OF Phyllomedusa rohdei<br />
Marinho, E.A.V. 1,3 ; Zaharenko, A.J. 2 ; Paraventi, C 1 ; Jimenez, R.S. 1 ; Molska, G.R. 1 ; Paula-Freire,<br />
L.I.G. 1 ; Zanoni, C.I.S. 4 ; Almeida, E. 3 ; Frussa-Filho, R. 3 ; Parada, C.A. 4 ; Ferreira, S.H. 4 ; Malpezzi-<br />
Marinho, E.L.A 1<br />
1- Braz Cubas University, Health Science Area, Mogi das Cruzes, SP, Brazil, edumarinho@hotmail.com<br />
2- University of São Paulo, Biosciences Institute, Dept. of Physiology, São Paulo, SP, Brazil,<br />
a.j.zaharenko@ig.com.br<br />
3- Federal University of São Paulo, Dept. of Pharmacology, São Paulo, SP, Brazil<br />
4- University of São Paulo, Ribeirão Preto, SP, Brazil<br />
Amphibians of the genus Phyllomedusa produce <strong>and</strong> secrete peptides <strong>and</strong> alkaloids in the<br />
skin. P. bicolor is used in rituals <strong>and</strong> their secretion is applied on skin burning, promoting<br />
vomit, salivation <strong>and</strong> arterial pressure increase. Analysis of the secretion indicated opioid<br />
compounds. The aim of this study was evaluate the antinociceptive activity of the P.<br />
rohdei skin extract (EPr). Skin from 5 animals was removed, maintained 1 month in<br />
methanol, which was filtered <strong>and</strong> freeze-dried. To the writhing test male mice were used<br />
(25-35g) in groups (n=6), treated by ip injection, 20min before ip injection of 0,4mL of<br />
acetic acid 0,6%, observed <strong>and</strong> the contortions computed during 5min. The mean±SEM of<br />
the groups treated with EPr 0,3 (27±3), 1 (12±3) <strong>and</strong> 3mg/kg (3±2) reduced when<br />
compared to the C (34±3). To the inhibition of hypernociception, male rats (200g) were<br />
used, <strong>and</strong> the EPr (60μL) was applied ip 2h40’ after the sc injection of PGE2 (100ng-25μL)<br />
in the back right paw <strong>and</strong> was evaluated by the increasing pressure on the paw (electronic<br />
Von Frey) during 90min (each 30min) since the injection of the EPr <strong>and</strong> computed as the<br />
variation in the pressure (Δg) before <strong>and</strong> after the injection of the extract. The doses of 1<br />
<strong>and</strong> 3mg/kg reduced significantly the Δ of nociception (between 2.6 <strong>and</strong> 5g until the min<br />
60) when compared to the PGE2 group (~9.2g until the min 60). The Rota-rod test<br />
evaluated the depressive effect of EPr. Male mice (35-45g) were selected (plus that 1min<br />
in the equipment), in groups (n=6) <strong>and</strong> in the test day, pre-exposed 30min before <strong>and</strong><br />
10min after the treatments, <strong>and</strong> in the times 0, 30, 60 <strong>and</strong> 90min were evaluated the<br />
latencies (L) in the apparatus. In C group the mean L was ~60s, diazepam (2mg/kg)<br />
reduced the L (26±11s) <strong>and</strong> EPr 0,3; 1 <strong>and</strong> 3mg/kg did not cause alterations (L ~60s). The<br />
results indicated that the EPr has antinociceptive <strong>and</strong> antihypernociceptive action in these<br />
doses <strong>and</strong> models, did not change the motor control, with good performance in the Rotarod.<br />
o Antinociception<br />
o Antihypernociception<br />
o Phyllomedusa rohdei<br />
o Amphibians<br />
296<br />
Poster 11: ACTIVITY OF EXTRACTS FROM Phyllomedusa rohdei (AMPHIBIA,<br />
ANURA) SKIN ON THE MICE BEHAVIOUR IN THE OPEN FIELD AND<br />
ROTA ROD<br />
Silva, R.W. 1 ; Zaharenko, A.J. 2 ; Paula-Freire, L.I.G. 1 ; Almeida, E. 3 ; Frussa-Filho, R. 3 ; Malpezzi-<br />
Marinho, E.L.A 1 , Marinho, E.A.V. 1,3<br />
5- Braz Cubas University, Health Science Area, Mogi das Cruzes, SP, Brazil, edumarinho@hotmail.com<br />
6- University of São Paulo, Biosciences Institute, Dept. of Physiology, São Paulo, SP, Brazil,<br />
a.j.zaharenko@ig.com.br<br />
7- Federal University of São Paulo, Dept. of Pharmacology, São Paulo, SP, Brazil<br />
Phyllomedusa produces in the skin amines <strong>and</strong> active peptides <strong>and</strong> is used in rituals of<br />
Amazon tribes. The aim was evaluate the action of extracts of P. rohdei on the male mice<br />
(25-30g) behaviour in the open field. It was computed the mean±SEM (n=10) of<br />
peripheral, central <strong>and</strong> total locomotion (squares), grooming (s), rearing, immobilization<br />
(s) <strong>and</strong> movement (s) after treatment with the total extract (TEPr 0.3; 1; 3mg/kg), the polar<br />
fraction (PFPr 0.03; 0.1; 0.3mg/kg) <strong>and</strong> the apolar fraction (AFPr 0.3; 1; 3mg/kg). The<br />
TEPr reduced the total locomotion from 92±7 to the control (C) to 70±9 (0.3mg/kg), 52±9<br />
(1mg/kg) <strong>and</strong> 27±4 (3mg/kg). With the PFPr the total locomotion was 59±9 (0.03mg/kg),<br />
71±10 (0.1mg/kg) <strong>and</strong> 39±10 (0.3mg/kg). With the AFPr the total locomotion was 91±7<br />
(0.3mg/kg), 92±8 (1mg/kg) <strong>and</strong> 83±7 (3mg/kg). The TEPr reduced the rearing from 32±3<br />
(C) to 27±4 (0.3mg/kg), 10±3 (1mg/kg) <strong>and</strong> 3±1 (3mg/kg). With PFPr the rearings were<br />
18±3 (0.03mg/kg), 16±4 (0.1 mg/kg) <strong>and</strong> 6±2 (0.3mg/kg) <strong>and</strong> with the AFPr 29±4<br />
(0.3mg/kg), 31±3 (1mg/kg) <strong>and</strong> 27±4 (3mg/kg). The immobilization increased; in the C<br />
was 6±3s <strong>and</strong> with the TEPr 41±18s (0.3mg/kg), 79±18s (1mg/kg) <strong>and</strong> 162±12s (3mg/kg).<br />
With the PFPr 38±12s (0.03mg/kg), 56±11s (0.1mg/kg) <strong>and</strong> 131±17s (0.3mg/kg). With the<br />
AFPr 4±2s (0.3mg/kg), 2±1s (1mg/kg) <strong>and</strong> 12±7s (3mg/kg). The grooming time of the C<br />
was 17±3s; with the TEPr 12±4s (0.3mg/kg), 7±2s (1mg/kg) <strong>and</strong> 1±1s (3mg/kg), with the<br />
PFPr 11±3s (0.03mg/kg), 1±0.5s (0.1mg/kg) <strong>and</strong> 1±0.5s (0.3mg/kg) <strong>and</strong> with the AFPr<br />
11±2s (0.3mg/kg), 15±3s (1mg/kg) <strong>and</strong> 14 ± 3s (3 mg/kg). The results indicate that the<br />
TEPr <strong>and</strong> the PFPr change the behaviour in the open field. The Rota-rod test indicates that<br />
the TEPr do not affect the motor function of the animals (56±4s 0.3mg/kg; 59±1s 1mg/kg;<br />
51±5s 3mg/kg <strong>and</strong> 57±3s C), different to the diazepam (2mg/kg 26±11s). This suggests<br />
that the alterations may be caused either by the peptides (opioids <strong>and</strong> adenorreguline) or<br />
opioid receptor antagonists from the skin of P. rohdei.<br />
o Phyllomedusa rohdei<br />
o Open field<br />
o Behavior<br />
o opioids<br />
297
Poster 12: κ AND μ OPIOID RECEPTORS ARE INVOLVED IN THE<br />
ANTINOCICEPTIVE ACTIVITY OF METHANOL EXTRACT OF<br />
Phyllomedusa rohdei ON THE HOT PLATE MODEL<br />
Zanoni, C.I.S. 4 ; Molska, G.R. 1 ; Zaharenko, A.J. 2 ; Paraventi, C 1 ; Jimenez, R.S. 1 ; Paula-Freire, L.I.G. 1 ;<br />
Parada, C.A. 4 ; Ferreira, S.H. 4 ; Malpezzi-Marinho, E.L.A 1 ; Marinho, E.A.V. 1,3<br />
8- Braz Cubas University, Health Science Area, Mogi das Cruzes, SP, Brazil, edumarinho@hotmail.com<br />
9- University of São Paulo, Biosciences Institute, Dept. of Physiology, São Paulo, SP, Brazil,<br />
a.j.zaharenko@ig.com.br<br />
10- Federal University of São Paulo, Dept. of Pharmacology, São Paulo, SP, Brazil<br />
11- University of São Paulo, Ribeirão Preto, SP, Brazil<br />
Amphibians of the genus Phyllomedusa produce <strong>and</strong> secrete peptides <strong>and</strong> alkaloids in the<br />
skin. P. bicolor is used in rituals <strong>and</strong> their secretion is applied on skin burning, promoting<br />
vomit, salivation <strong>and</strong> AP increase. Analysis of the skin secretion indicated opioid<br />
compounds. The aim of this study was to evaluate the antinociceptive activity of the P<br />
rohdei skin extract (EPr). Skin from 5 animals was removed, maintained 1 month in<br />
methanol, which was filtered <strong>and</strong> freeze-dried. Male mice were employed (25-30g), under<br />
light/dark cycle of 12h <strong>and</strong> water <strong>and</strong> food ad libitum. A curve (groups with n=5) of the<br />
EPr during the time (paw licking <strong>and</strong> jumping) of the mice on hot plate (55±1 o C) (0 to<br />
240min each 30min) was obtained. The EPr (1 <strong>and</strong> 3mg/kg) inhibited the response of the<br />
animals (10-20s) compared to the control (C) (2-8s).<br />
So, the dose of 1mg/kg was defined to test selective <strong>and</strong> non-selective antagonists to opioid<br />
receptors (systemic subcutaneously). Each group (n=5) receives an antagonist (except C)<br />
<strong>and</strong> 40min after the EPr. After 20min, each animal was placed on the hot plate computing<br />
the reaction time. The C group shows a time of 2 to 8s, the treated with EPr 10 to 18s until<br />
180min. In the groups pre-treated with naloxone (1mg/kg) <strong>and</strong> EPr the reaction time was<br />
similar to the C, indicating the presence of opioid receptors. With the selective antagonists<br />
NorBNI (κ) e Ciprodime (μ) the response to the EPr was similar to C indicating the<br />
participation of these receptors. Animals pre-treated with Naltrindol (δ) did not show<br />
differences to the EPr indicating the no participation of these receptors. When naloxone<br />
was applied in the paw (1μg) it was verified that the EPr has peripheral action, with<br />
significant reduction during the time. We suggest the participation of opioid compounds,<br />
or opioid receptor antagonists, in the EPr mediating the antinociceptive effects.<br />
o Phyllomedusa rohdei<br />
o Hot plate<br />
o Opioids receptors<br />
o Antinociception<br />
298<br />
Poster: 13: Xen2174: A novel NET inhibitor that enhances a2-adrenoceptor<br />
inhibition of spinal pain pathways<br />
Croker, D.E 1 , Palant, E 1 , Nielsen C 2 , Drinkwater, R 1 , Patterson, M 3 , McCumber, D 3 , Yaksh, T 3 , Wilson,<br />
D 1* , Smith, M 2 <strong>and</strong> Lewis, R.J. 1<br />
1Xenome<br />
Ltd, 120 Meiers Rd, Indooroopilly, Australia, *david.wilson@xenome.com<br />
2<br />
School of Pharmacy, The University of Queensl<strong>and</strong>, St Lucia, Brisbane, Australia<br />
3<br />
Department of Anaesthesiology, Research Clinical Teaching Facility, University of California-San<br />
Diego (UCSD), San Diego, CA, USA<br />
Xenome Ltd was founded to mine the therapeutic potential of Australian venoms. Venom<br />
duct DNA has been used to guide the production of synthetic venom peptide libraries that<br />
have already delivered safe <strong>and</strong> effective peptide therapeutics. The discovery platform has<br />
demonstrated its effectiveness across an increasing number of drug targets including<br />
validated members of the GPCR, kinase, phosphatase, protease, <strong>and</strong> ion channel target<br />
families. Xenome’s first product, Xen2174 a novel norepinephrine transporter inhibitor,<br />
has been progressed to Phase I/IIa trials for the treatment of severe chronic pain. Xen2174<br />
acts non-competitively to enhance the levels of noradrenaline that accumulate after release<br />
from the nerve endings of descending inhibitory pain pathways. These elevated levels of<br />
noradrenaline produce greater activation of alpha2-adrenoceptors (α2-AR) found at the<br />
nerve terminals of ascending pain pathways. Consistent with this mechanism of action, the<br />
effects of a bolus IT dose of Xen2174 is fully abolished when co-administered with a<br />
saturating dose of the α2-AR blocker yohimbine. The SAR <strong>and</strong> development of this new<br />
class of therapeutic will be briefly outlined.<br />
o Xenome<br />
o Xen2174<br />
o NET<br />
o pain<br />
299
Poster 14: Crotoxin inhibits neuropathic pain <strong>and</strong> the development of neuromas<br />
Cury, Y. 1* , Nogueira-Neto, F. 1,2 , Amorim, R.L. 2 , Brigatte, P. 1 , Picolo, G. 1 , Ferreira Jr., W.A. 1 , Nicoletti,<br />
J.L.M. 2<br />
1<br />
Laboratory of Pathophysiology, Butantan Institute, Av. Vital Brazil, 1500. 05503-900, Sao Paulo, Brazil,<br />
*<br />
yarac@attglobal.net<br />
2<br />
Faculty of Veterinary Medicine <strong>and</strong> Zootechny, UNESP, Distrito Rubiao Junior S/N, 18618-000, Botucatu,<br />
Sao Paulo, Brazil<br />
Introduction: Crotalus durissus terrificus snake venom(CdtV) induces analgesia mediated<br />
by opioid receptors. The factor responsible for the analgesic action of the venom, named<br />
crotalphine, was recently isolated <strong>and</strong> characterized. However, recent data have indicated<br />
that crotoxin (CTX), the main neurotoxic component of CdtV, exerts antinociceptive effect<br />
in an experimental model of cancer pain. The aim of the present study is to characterize<br />
further the analgesic action of CTX, evaluating the effect of the toxin on neuropathic pain<br />
<strong>and</strong> determining the mechanisms involved in this effect.<br />
Methods: For induction of neuropathic pain, the sciatic nerve of male Wistar rats was<br />
exposed unilaterally by careful dissection, transected in two locations at the mid-thigh<br />
level <strong>and</strong> 0.5 cm of the nerve was removed. Pain-related behavior <strong>and</strong> development of<br />
neuromas were analyzed over a 64-day period after surgery. The rat paw pressure test was<br />
used for hyperalgesia evaluation. The presence of neuromas was determined by<br />
histological analysis of the nerves.<br />
Results: Hyperalgesia was detected 2 h after surgery <strong>and</strong> persisted for 64 days. Few<br />
animals developed neuromas until day 7, however 80 % of the rats presented neuromas on<br />
day 64. Neuromas often occur at the proximal stump of the transected nerve. CTX (0.01<br />
mM) applied to the proximal <strong>and</strong> distal nerve stumps, immediately after nerve transection,<br />
blocked hyperalgesia. The analgesic effect was observed 2 h after CTX treatment <strong>and</strong><br />
persisted for 64 days. CTX-induced analgesia was blocked or significantly inhibited by i.p.<br />
administration of atropine (10 mg/kg) <strong>and</strong> yohimbine (2 mg/kg), respectively.<br />
Methysergide (5 mg/kg), atenolol (1 mg/kg), <strong>and</strong> naloxone (1mg/kg) did not interfere with<br />
this analgesic activity. Histological analysis showed that CTX delays <strong>and</strong>/or significantly<br />
inhibits the development of neuromas.<br />
Conclusions: The results indicate that CTX induces a long-lasting analgesic effect on<br />
neuropathic pain <strong>and</strong> inhibits the development of the neuropathy. The analgesic effect is<br />
mediated by muscarinic receptors <strong>and</strong> α-adrenoceptors.<br />
o Crotoxin<br />
o Neuropathic pain<br />
o Neuromas<br />
o Muscarinic receptors<br />
300<br />
Poster 15: Phospholipases A2 (PLA2) Isolated From Brazilian Coral Snake<br />
(Micrurus lemniscatus) Induced Neuronal Injury: In Vivo And In Vitro Studies.<br />
Carvalho,N.D 1 ., Oliveira, D.A 1 ., Casais e Silva, L.L. 3 ,Lebrun, I. 2 , Afeche, S.C 1 , S<strong>and</strong>oval, M.R.L. 1*<br />
1Laboratory of Pharmacology <strong>and</strong> 2 Laboratory of Biochemistry <strong>and</strong> Biophysics,Butantan Institute, av. Dr.<br />
Vital Brasil, 1500, São Paulo, SP, 05503 900. Brazil. 1* mrls<strong>and</strong>o@butantan.gov.br<br />
3 Laboratory of Animais Peçonhentos, Federal University of Bahia, Brazil<br />
Neurotoxicity from Micrurus venoms from Americas has been poorly studied. The<br />
aim of this work is to investigate behavioural, EEG <strong>and</strong> histological effects of two PLA2s,<br />
Ml-8 <strong>and</strong> ML-9, isolated from the venom of Micrurus lemniscatus. We also analysis the<br />
neurotoxicity induced by these toxins in cultured hippocampal neurons. Method: Cannula<br />
<strong>and</strong> electrodes were implanted in the CA1 hippocampus (Hpc) 4 days prior the Ml-8, Ml-9<br />
or phosphate buffer injection into the Hpc. EEG <strong>and</strong> behaviours were recorded during 8<br />
hours. Seven days after, the brains were processed for histological analyses. In vitro<br />
studies: Neurons were dissociated from hippocampi of E18-E19 Wistar rat embryos, after<br />
treatment with trypsin 0,25% <strong>and</strong> deoxyribonuclease (0,15mg/ml) neurons were cultured in<br />
B27-supplemented Neurobasal Medium (GIBCO). Cultures were kept at 37 0 C in a<br />
humidified incubator in 5% CO2. Cells were plated at a density of 200 neurons/mm 2 . After<br />
incubation of cultures neurons (at day7) with of Ml-8 or Ml-9 (0.1 – 1000 ng/ml) toxins,<br />
for 12 or 24 h assessment of neuronal injury was made by using ethidium bromide<br />
(Mercille & Massie (1994) B. Cytotechnoligy, 15: 117). It was counted 200 neurons in 10<br />
fields in each coverslip. PLA2 activity was previously determined (date not yet published).<br />
Results: Ml-8 (2.1μg/μl), Ml-9 (2.4 ug/ul) induced circling behavior, jumping, intense<br />
scratching, forelimb clonus <strong>and</strong> the EEG record showed spikes <strong>and</strong> epileptiform<br />
discharges. Lesions on CA1 region of the Hpc were also observed. Cultured neurons<br />
survival was significantly decreased when compared with the control group (ANOVA <strong>and</strong><br />
Dunnett test): control (25±5,9); Ml-8 0,1 (20,2±4,7); 1,0 (18,1±3,6), 10,0 (4,8±3,3), 100,0<br />
(1,1±1,8) or 1000,0 (1,8±3,1) ng/ml ; Ml-9 0,1 (26,4±5,1)1,0 (17,2±13,8); 10,0 (17,3±6,6);<br />
100,0 (7,8±2,2); 1000,0 (2,4±2,1) ng/ml. Conclusion: Ml-8 <strong>and</strong> Ml-9 caused neuronal<br />
death by apoptosis <strong>and</strong> necrosis in neuronal culture. In vivo studies showed behavioral <strong>and</strong><br />
EEG seizures <strong>and</strong> hpc injury. The toxicity might be associated with the binding of these<br />
toxins with specific brain site (Lambeau et al, J. Biol. Chem. 1989, 264, 11503). These<br />
PLA2 can be a useful tool for exploring the death- signaling pathways of neurotoxicity.<br />
o Micrurus venom<br />
o Neurotoxic PLA2<br />
o Neuronal injury<br />
o Seizure<br />
301
Poster 16: ENHANCEMENT OF TNF-α AND IL-1 PRODUCTION IN<br />
ALVEOLAR MACROPHAGES BY C-FIBER DEGENERATION<br />
DeSouza, IA*Camargo, EA; Franco-Penteado, CF; Antunes, E.<br />
Department of Pharmacology, Faculty of Medical Sciences, UNICAMP; Campinas, São<br />
Paulo, Brazil; *ivanidesouza@fcm.unicamp.br<br />
Objectives: Permanent loss of C-fibers in the rat lungs lead to an exacerbation of<br />
neutrophil influx in response to ovalbumin <strong>and</strong> staphylotoxins (SEA <strong>and</strong> SEB). In an<br />
attempt to further underst<strong>and</strong> the communication of C-fibers <strong>and</strong> pulmonary cells, we have<br />
evaluated the in vitro TNF-α <strong>and</strong> IL-1 production by alveolar macrophages stimulated with<br />
SEA <strong>and</strong> SE. Alveolar macrophages were collected from control or capsaicin-treated<br />
animals.<br />
Methods <strong>and</strong> Results: Alveolar macrophages (AM) were isolated from brochoalveolar<br />
lavage of control <strong>and</strong> male Wistar rats (250-300g) treated with capsaicin as neonates. AM<br />
were ressuspended in culture medium <strong>and</strong> transferred to tissue culture wells. They were<br />
allowed to adhere for 2 h (37 o C, 5% CO2) <strong>and</strong> then incubated for 4 h in the presence of<br />
either SEA or SEB (3 μg/ml each). The supernatant was colleted <strong>and</strong> used to determinate<br />
TNF-α <strong>and</strong> IL-1 concentrations by ELISA. Incubation of AM with either SEB or SEA<br />
increased by 30% <strong>and</strong> 42% the TNF-α production above basal levels, respectively<br />
(P
Poster 18: ASSESSMENT OF THE INHIBITION INDUCED BY Bufo paracnemis<br />
POISON AND ITS FRACTIONS ON COMPLEMENT SYSTEM AND<br />
LEUKOCYTE RECRUITMENT<br />
Marongio, A. F. Q. 1 , Bertazzi, D. T. 1 , Arantes, E. C* 1<br />
1<br />
University of São Paulo, FCFRP, Av. do Café s/n, 14040-903, Ribeirão Preto, SP,<br />
Brazil. *ecabraga@fcfrp.usp.br<br />
Previous study showed that Bufo paracnemis poison (BpP) affects the lytic activity<br />
of the complement system (CS) (Experientia, 41: 940, 1985). Complement activation<br />
occurs through classical (CP), lectin (LP) <strong>and</strong> alternative pathways (AP), leading to a<br />
cascade of component interactions <strong>and</strong> generation of products with biological activities<br />
such as anaphylaxis, chemotaxis, opsonization, immune complex solubilization,<br />
participation in the immune response, etc. The aim of the present study was to investigate<br />
the effect of BpP <strong>and</strong> its fractions on the CS <strong>and</strong> on leukocyte recruitment.<br />
Methods <strong>and</strong> Results: The fractionation procedure of BpP involves basically a cationicexchange<br />
chromatography on CM-Celulose followed by a anionic-exchange<br />
chromatography of the active fraction on DEAE-celulose column. Four fractions, named<br />
D1 to D4, were obtained. The effects of BpP <strong>and</strong> its fractions on CP/LP pathways were<br />
evaluated using in vitro haemolytic assays. In vitro chemotaxis assays were performed<br />
using the Boyden chamber model. Poison <strong>and</strong> the active fractions (C1, D3 <strong>and</strong> D4)<br />
induced a reduction in hemolytic activity of the CP/LP complement pathways. Samples<br />
containing 225 μg <strong>and</strong> 375μg of BpP reduced 37,8 ± 0.1 % <strong>and</strong> 50 ± 0.3 % the complement<br />
activity, respectively. Fractions C1, D3 <strong>and</strong> D4 induced reduction of 93 ± 0.8 %, 86 ± 8.6<br />
% <strong>and</strong> 62 ± 0.2 %, respectively. Incubation of normal human serum with BpP reduced<br />
neutrophil chemotaxis when compared to that observed with control serum (BpP: 10 ± 1.4<br />
μm <strong>and</strong> control: 60 ± 1.2 μm).<br />
Our results showed that BpP inhibits the complement system <strong>and</strong> reduces serum<br />
chemotactic activity. Therefore, it may have an important potential as biological tools for<br />
studies of processes involving the complement system.<br />
Financial Support: CAPES <strong>and</strong> FAPESP.<br />
o Bufo paracnemis<br />
o complement system<br />
o neutrophil chemotaxis<br />
o toad poison<br />
304<br />
Poster 19: Do cytokines <strong>and</strong> nitric oxide play a role in the actions of Leiurus<br />
quinquestriatus scorpion venom in animals?<br />
Abdoon, N. A 1 *, Elsayed Ali, A 2 , Fatani, A. J. 3<br />
1 National antivenom <strong>and</strong> vaccine production center, King Fahd National Guard hospital,<br />
Riyadh,KSA,*Nozabdoon@hotmail.com<br />
2 Dept. of Pharmacology, College of Pharmacy, Khartoom University, Khartoom,Sudan<br />
3 Dept. of Pharmacology College of pharmacy, King Saud University,Riyadh,KSA<br />
Introduction: The actions of scorpion neurotoxins are complex <strong>and</strong> may be traced to<br />
activation of different ion channels with subsequent release of various modulators<br />
including inflammatory mediators. This would lead to various pathological manifestations<br />
<strong>and</strong> even death. The purpose of this project was to study these pathological changes <strong>and</strong><br />
examine the ability of inhibitors of different steps in the inflammatory process, to<br />
ameliorate the detrimental actions <strong>and</strong> prolong survival of animals injected with Leiurus<br />
quinqestriatus (LQQ) scorpion venom.<br />
Methods: LQQ venom (0.4 mg kg -1 , s.c.) was injected into conscious male New Zeal<strong>and</strong><br />
rabbits. Changes in blood pressure (BP) were measured up to 12hours via central ear artery<br />
using blood pressure transducer <strong>and</strong> physiograph. The BP was correlated with selected<br />
serum biochemical <strong>and</strong> hematological parameters including nitric oxide <strong>and</strong> cytokines (IL-<br />
8 & TNF). In addition, montelukast (10 <strong>and</strong> 20 mg kg –1, po), indomethacin (10 <strong>and</strong> 20 mg<br />
kg –1 ,iv) <strong>and</strong> hydrocortisone (5 <strong>and</strong>10 mg kg –1 ,iv), were administered to mice prior to LQQ<br />
venom (0.25 <strong>and</strong> 0.3mg kg –1, sc), <strong>and</strong> survival recorded after 24hr.<br />
Results: LQQ caused a triphasic effect on BP: initial reduction, prolonged increase <strong>and</strong><br />
gradual terminal hypotension <strong>and</strong> bradycardia, followed by death. Increases in serum nitric<br />
oxide, IL-8 & TNF in addition to leucocytosis were evident in the terminal hypotension.<br />
All drugs used significantly prolonged survival time <strong>and</strong> %survival in mice, with<br />
montelukast having greater protective abilities.<br />
Conclusions: Anti-inflammatory drugs protect against lethal effects of LQQ venom.<br />
especially those occurring during the late terminal hypotensive phase indicating that early<br />
treatment of envenomed victims is essential. (1,2,3,4)<br />
References: 1. Ismail, M. 1995, Toxicon, 33, 825-858. 2. Barraviera, B. 1997, Toxicon, 35,<br />
13-14. 3. Fukuhara, Y.M.D, Reis, M.L, Dellabibera-Joviliano, R, Cunha,Q.C, Donadi, E.<br />
A. 2003, Toxicon, 41, 49-55. 4. Meki,A.A,Mohamed,Z.M.MoheyElDeen,H.M.2003,<br />
o Leiurus quinqestriatus<br />
o Nitric oxide<br />
o cytokines<br />
305
Poster 20: EVALUATION OF THE ACUTE LUNG INJURY IN A MURINE<br />
MODEL INDUCED BY THE SCORPAENA PLUMIERI FISH VENOM<br />
Boletini-Santos D 1 , Haddad Jr V. 2 *, Figueiredo S.G. 3 , Lopes-Ferreira M. 1 , Lima C. 1 .<br />
1Laboratório<br />
de Imunopatologia do Instituto Butantan, São Paulo, Brazil.<br />
2<br />
Departamento de Dermatologia, Faculdade de Medicina, UNESP, Botucatu. ZIP 18618-<br />
000, * haddadjr@fmb.unesp.br.<br />
3<br />
Departamento de Ciências Fisiológicas, UFES, Brasil.<br />
INTRODUCTION: The Brazilian Scorpaena plumieri fish provoked human accidents<br />
causing severe injuries, characterized by local <strong>and</strong> systemic effects. The aim of this work<br />
was to study the effect of venom on target organs distant from the inoculation site.<br />
METHODS: BALB/c mice injected i.p. with 10, 30, <strong>and</strong> 100 μg of venom were killed after<br />
2, 6, 24, <strong>and</strong> 48 h <strong>and</strong> the tracheas were cannulated for collection of bronchoalveolar<br />
lavage fluid (BAL). The pellet <strong>and</strong> supernatants from BAL were used for cell counts <strong>and</strong><br />
determination of protein <strong>and</strong> NO levels, respectively. The homogenates of lung tissue were<br />
used for cytokines analysis <strong>and</strong> zymography. RESULTS: When 10 or 30 μg venom was<br />
injected a large number of leukocytes, mainly macrophages, was observed in BAL<br />
collected 6 h after. However with 100 μg venom, an earlier lung inflammation (2 h),<br />
predominantly of macrophages, was observed. All doses of venom induced a significant<br />
increase in protein level in BAL at 24 <strong>and</strong> 48 h, but only 100 μg venom induced a rapid<br />
<strong>and</strong> sustained extravasation of protein. Only IL-6 was detected in lung at 2 <strong>and</strong> 24 h. IL-<br />
1β, TNF-α, MCP-1 or NO were not detected in BAL in any circumstance. Gelatinolytic<br />
activity was detected in lungs 2 <strong>and</strong> 24 h after injection of 100 μg venom <strong>and</strong> by SDS-<br />
PAGE showed correlation with a 100 kDa b<strong>and</strong>. Immunohistochemical staining for S.<br />
plumieri venom showed the presence of the venom in alveolar epithelial cells <strong>and</strong> around<br />
the vessels <strong>and</strong> abundant distribution throughout of the lung. CONCLUSION: The results<br />
showed by the first time that S.plumieri venom induces acute lung injury by protein<br />
passage from the vascular to the alveolar space <strong>and</strong> infiltration of macrophages, secretion<br />
of cytokines <strong>and</strong> proteinases with gelatinolytic activity. Supported by Fapesp.<br />
Haddad Jr V, Martins IA, Makyama HM. Injuries caused by scorpionfishes (Scorpaena plumieri Bloch, 1789<br />
<strong>and</strong> Scorpaena brasiliensis Cuvier, 1829) in the Southwestern Atlantic Ocean (Brazilian coast):<br />
epidemiologic, clinic <strong>and</strong> therapeutic aspects of 23 stings in humans. Toxicon 42: 79-83, 2003.<br />
o Scorpaena<br />
o Scorpaenidae<br />
o human injuries for fish<br />
o venomous fish<br />
306<br />
Poster 21: INFLAMMATORY MEDIATORS INVOLVED IN PHOSPHOLIPASE<br />
A2 (PLA2)-INDUCED ACUTE PANCREATITIS<br />
Camargo, E.A.* 1 ; Ferreira, T. 1 ; L<strong>and</strong>ucci E.C.T. 1 ; Antunes, E 1 .<br />
1 Department of Pharmacology, Faculty of Medical Sciences, UNICAMP; Campinas, São<br />
Paulo, Brazil; *eapcamargo@fcm.unicamp.br<br />
Objectives: PLA2s induce acute pancreatitis when injected into the pancreaticobilliary<br />
duct of rats (Camargo et al., 2005). We have investigated the inflammatory mediators<br />
involved in PLA2-induced acute pancreatitis. Methods: Acute pancreatitis was induced by<br />
the injection of PLA2 from Naja mocambique mocambique venom (PLA2-Nmm; 300<br />
μg/kg) into the common bile duct of male Wistar rats (220-260g, n=5-8). Four h later, the<br />
pancreatic plasma protein extravasation (PPE), pancreatic <strong>and</strong> lung myeloperoxidase<br />
(MPO), serum amylase (sAM) <strong>and</strong> TNF-α (sTNF-α) were measured. Results: The PLA2-<br />
Nmm induced a marked increase in PPE (65%), pancreatic MPO (309%), lung MPO<br />
(34%), sAM (155%) <strong>and</strong> sTNF-α (260%). Pretreatment with the B2 receptor antagonist<br />
Icatibant (100 nmol/kg) reduced significantly the PPE, pancreatic MPO, lung MPO, sAM<br />
<strong>and</strong> sTNF-α. The neurokinin-1 (NK1) receptor antagonist SR140333 (120 nmol/kg)<br />
reduced the PPE, pancreatic MPO <strong>and</strong> s-TNF-α, but did not affect the lung MPO <strong>and</strong><br />
sAM. The PAF receptor antagonist PCA4248 (5 mg/kg) decreased significantly the lung<br />
MPO <strong>and</strong> sTNF-α, without causing any change in the other parameters. The non-selective<br />
NO inhibition with L-NAME (20 mg/kg) reduced significantly the PPE <strong>and</strong> sTNF-α, but<br />
rather caused an increase in the pancreatic MPO. The lung MPO <strong>and</strong> sAM were not<br />
significantly affected by L-NAME. Aminoguanidine (inducible NO synthase inhibitor, 50<br />
mg/kg) treatment had no effect in any parameter evaluetad. Indomethacin (cyclooxygenase<br />
inhibitor, 5mg/kg) decreased significantly the lung MPO <strong>and</strong> sTNF-α, without causing any<br />
other changes. The treatment with celecoxib (cyclooxygenase -2 selective inhibitor, 20<br />
mg/kg) decreased the PPE without affecting other parameters. Conclusions: Acute<br />
pancreatitis induced by exogenous PLA2 involves the activation of B2 <strong>and</strong> NK1 receptors,<br />
endothelial NO synthase-derived NO <strong>and</strong> cyclooxygenase -2 metabolites in the pancreatic<br />
tissue. The remote lung injury seems to involve cyclooxygenase -1 metabolites <strong>and</strong> the<br />
activation of B2 <strong>and</strong> PAF receptors. Reference: Camargo et al., Toxicon, 2005, 46, 921-6.<br />
Financial Support: Fundação de Apoio à Pesquisa do Estado de São Paulo<br />
o Acute pancreatitis<br />
o Inflammatory mediators<br />
o Phospholipases A2<br />
307
Poster 22: Effect of eugenol <strong>and</strong> local anesthetic agents on tongue <strong>and</strong> skin edema<br />
induced by Dieffenbachia picta Schott in mice<br />
Dip, E.C. 1 ; Pereira, N.A. 1 ; Gaban, G.A. 1 ; Fonseca, T. 1 ; El Kik, C.Z. 1 ; Tomaz, M.A 1 ;<br />
Calil-Elias, S. 1 ; Martinez, A.M.B. 2 ; Melo, P.A. 1 .<br />
1<br />
Departamento de Farmacologia Básica e Clínica ICB, CCS Universidade Federal do Rio de Janeiro,<br />
21941-590, Rio de, Janeiro, RJ, Brazil. pamelo@farmaco.ufrj.br.<br />
2<br />
Departamento de Histologia e Embriologia, ICB, Universidade Federal do Rio de Janeiro, Rio de Janeiro,<br />
RJ, Brazil .<br />
Dieffenbachia picta Schott is a tropical poisonous an ornamental plant which induce<br />
angioedema, glottis obstruction, respiratory compromise <strong>and</strong> death in mammals. These<br />
manifestations can be partially antagonized by eugenol <strong>and</strong> some anti-inflammatory drugs.<br />
We examined the tongue <strong>and</strong> skin, edema induced by D. picta juice <strong>and</strong> the effect of<br />
denervation or the local anesthetic agent, benzocaine (20%) lidocaine (10%) <strong>and</strong> eugenol<br />
(0.1- 0.3%). Tongue edema was induced by topical application of 100 μL of D. picta juice<br />
<strong>and</strong> it was evaluated with a digital tachymeter during 2 hours. For the skin edema study,<br />
mice received i.v. injection of Evan’s blue dye (2.5% solution; 25 mg/kg) 60 min. before<br />
the intradermal injection of 100 μL of D. picta juice. Skin edema were evaluated by<br />
measuring the dyed plasma extravasation <strong>and</strong> accumulation in the skin in a<br />
spectrophometer. The amount of dye in the dried skin were evaluated The tongue edema<br />
reached the maximum at 60 min. after topical application of 100 μL of D. picta juice in the<br />
control mice, <strong>and</strong> it was completely inhibited by topical application of benzocaine <strong>and</strong><br />
partially inhibited by lidocaine (circa of 30%) or eugenol(circa of 31.6%). Preincubation of<br />
D. picta juice with lidocaine, 2.5<strong>and</strong> 10% decrease the skin plasma extravasation from<br />
42,93% ± 12.333% to 99.68 ± 9,03 respectively (n=8 for each group) while eugenol 50<br />
mg/kg (n=10) reduced in 31.96% ±2.30%. Our results showed that local anesthetic agents<br />
can decrease the acute inflammatory response induced by D. picta juice in mice.<br />
Dip et al., (2004) Toxicon 43:729-735<br />
o Dieffenbachia picta,<br />
o tongue edema <strong>and</strong> inflammation,<br />
o local anesthetic<br />
308<br />
Poster 23: Importance of jararhagin disintegrin-like <strong>and</strong> cysteine rich domains in<br />
the early events of local inflammatory response.<br />
Clissa, P.B. 1 ; Lopes-Ferreira, M. 1 ; Della-Casa, M.S. 1 ; Farsky, S.H.P. 2 ; Moura-da-Silva, A.M. 1<br />
1. Immunopathology Laboratory – Butantan Institute - Sao Paulo – Brazil<br />
2. Department of Clinical <strong>and</strong> Toxicological Analyses, School of Pharmaceutical Sciences, University of<br />
Sao Paulo – Brazil<br />
Jararhagin is a multidomain SVMP from Bothrops jararaca venom comprising catalytic,<br />
disintegrin-like <strong>and</strong> cysteine-rich domains, which cause a local reaction manifested by<br />
hemorrhage, edema, cytokine release <strong>and</strong> inflammatory cell recruitment. In this study, the<br />
importance of disintegrin-like/cysteine-rich domains of jararhagin was addressed by<br />
analyzing the effects of jararhagin-C, which lacks the catalytic domain, in induction of<br />
leukocyte rolling <strong>and</strong> release of pro-inflammatory cytokines. Jararhagin-C was isolated<br />
from B. jararaca venom conserving the same ability of complete jararhagin molecule in<br />
inhibiting collagen-induced platelet-aggregation. Treatment of trans-illuminated cremaster<br />
muscle in vivo with jararhagin-C increased number of rolling leukocytes (approximately<br />
250%) in post-capilary venules in all periods analyzed, without interfering with<br />
microvasculature haemodinamic, like vessel diameter, the eritrocyte speed or the blood<br />
flow rate. The release of pro-inflammatory cytokines TNF-α, IL-1β <strong>and</strong> IL-6 was<br />
significantly enhanced in the local of jararhagin-C injection, showing the maximum levels<br />
in periods between 2 <strong>and</strong> 4 hours after treatment. Besides the action of jararhagin-C, the<br />
presence of the inactivated catalytic domain in o-phenanthrolin-treated jararhagin was<br />
related to a higher increase in the number of rolling leukocytes. Moreover, the levels of IL-<br />
6 <strong>and</strong> IL-1β induced by catalytically active jararhagin were higher than those induced by<br />
jararhagin-C. In conclusion, our findings suggest that the disintegrin-like/cysteine-rich<br />
domains of jararhagin are sufficient to locally activate the early events of an acute<br />
inflammatory response as leukocyte rolling <strong>and</strong> pro-inflammatory cytokine release <strong>and</strong> this<br />
action may add to the effect of catalysis, which enhances the primary cell activation.<br />
Supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP)<br />
o Jararhagin<br />
o Jararhagin-C<br />
o Disintegrin-like <strong>and</strong> cysteine-rich domains<br />
o Inflammation<br />
309
Poster 24: Short-time analysis of pulmonary mechanics on acute lung injury<br />
induced by Crotalus durissus terrificus venom<br />
Nonaka, P. N 1 , Peres, A. C. P 1 , Ferrari, E. F, Carreiro da Costa, R. S 1 , Silva, C. A. M 2 , Ribeiro, W, R,<br />
Cogo, J.C 1 , Oliveira, L.V.F. 1<br />
1<br />
Universidade do Vale do Paraíba-UNIVAP, IP&D, 2911 Shishima Hifumi Av, São José dos Campos-<br />
SP, Brazil, paulanaomi@gmail.com<br />
2<br />
Universidade de Brasília-UNB, Laboratório de Fisiologia Respiratória, Brasília-DF, Brazil.<br />
There are very few reports related to specific inquiries on the repercussions of the Crotalus<br />
durissus snakes venom in the respiratory system, therefore, the behavior of the mechanical<br />
properties of the respiratory system, the characterization of the pulmonary structures <strong>and</strong><br />
the quantification of the generated inflammatory process are less known. The aim of this<br />
study is to test if this venom could induce pulmonary mechanics alterations at a short-time.<br />
Eighteen Swiss mice, were analysed three hours after intramuscular injection of saline<br />
(control group) or Crotalus durissus terrificus crude venom (0,6µg.g -1 ). The sub-lethal<br />
dose used in venom group, was determined by the LD50 previously found. The mechanical<br />
parameters were obtained by End Inspiratory Occlusion Method. The statistical analysis<br />
was carried through the Kolmogorov-Smirnov test for normality <strong>and</strong> the independent t-test<br />
in a significance level of 5%. Static elastance (p
Poster 26: Hydralysins – novel proteins from cnidaria revealing a sequence<br />
signature with a role in pore formation common to various beta pore forming toxins<br />
Sher, D.* 1 , <strong>Fish</strong>man, Y. 1 , Zhang, M. 1 , Lebendiker, M 1 , Mancheño, J.M. 2 <strong>and</strong> Zlotkin, E. 1<br />
1<br />
Department of Cell <strong>and</strong> Animal Biology, Silberman Institute of Life Sciences, Hebrew University,<br />
Jerusalem 91904, Israel. * dsher@pob.huji.ac.il<br />
2<br />
Instituto de Quimica-Fisica Rocasolano, CSIC, Departamento de Cristalografia, Serrano 119, E-28006<br />
Madrid, Spain<br />
Beta Pore-forming toxins (βPFTs) are a diverse family of water soluble proteins able to<br />
interact with <strong>and</strong> enter into biological membranes to create transmembrane pores. They<br />
are found in animals, plants <strong>and</strong> fungi, <strong>and</strong> are involved in the pathogenicity of many<br />
bacteria. While the stages of pore formation by different βPFTs are similar, they differ<br />
widely in their primary amino acid sequences. Thus, it has been difficult to generalize the<br />
information gained from model βPFTs on the relationship between structure <strong>and</strong> function<br />
<strong>and</strong> apply it to other members of this protein family.<br />
We have previously shown that the green hydra Chlorohydra viridissima produces nonnematocystic<br />
paralytic <strong>and</strong> cytolytic toxins, named hydralysins (Hlns) (1). Here we show<br />
that Hlns are a family of βPFTs, which are distinct from other cnidarian toxins but similar<br />
in activity <strong>and</strong> structure to bacterial <strong>and</strong> fungal toxins (2). Functional characterization of<br />
soluble Hln monomers using UV circular dichroism (UV-CD) <strong>and</strong> computational analysis<br />
reveals that they are rich in beta-structure. Hlns bind erythrocyte membranes <strong>and</strong> form<br />
discrete pores with an internal diameter of ~1.2nm. The cytolytic effect of Hlns is cell-type<br />
selective, suggesting a specific receptor. Multiple sequence alignment reveals that Hlns<br />
share a set of conserved sequence motifs with known βPFTs such as aerolysin, epsilon<br />
toxin, alpha toxin <strong>and</strong> LSL, <strong>and</strong> that these sequence motifs are found in <strong>and</strong> around the<br />
toxins’ pore-forming domain. The importance of this “sequence signature” is revealed by<br />
the cloning, expression <strong>and</strong> mutagenesis of three Hln isoforms which strongly differ in<br />
their hemolytic <strong>and</strong> paralytic activities.<br />
The sequence signature common to Hlns <strong>and</strong> other βPFTs reveals previously undetected<br />
conserved residues, <strong>and</strong> provides a platform for comparative structure-function analysis of<br />
βPFTs. Proteins containing this sequence signature are widely distributed, suggesting<br />
novel biological roles for βPFT-like proteins in nature.<br />
1. Zhang, M., et al. (2003) Biochemistry 42, 8939-8944<br />
2. Sher, D., et al. (2005) J Biol Chem 280, 22847-22855<br />
o Pore-forming toxins<br />
o sequence<br />
o mutagenesis<br />
o structure-function<br />
312<br />
Poster 27: Caissarolysin I (Bcs I), a new hemolytic toxin from the Brazilian sea<br />
anemone Bunodosoma caissarum: Purification <strong>and</strong> biological characterization.<br />
Zaharenko, A.J. 1,2* , Oliveira, J.S. 1,3 , Freitas, J.C. 1 , Konno, K. 2 , Andrade, S.A. 3 , Portaro, F.C.V. 2 ,<br />
Richardson, M. 4 , Sant’Anna, O.A. 3 , Tambourgi, D.V. 3<br />
1<br />
Universidade de São Paulo, Depto. de Fisiologia, Instituto de Biociências, São Paulo, BRAZIL.<br />
*a.j.zaharenko@ig.com.br.<br />
2<br />
Center for Applied Toxinology, CAT-CEPID, Instituto Butantan, São Paulo, BRAZIL.<br />
3<br />
Laboratório de Imunoquímica, Instituto Butantan, São Paulo, BRAZIL.<br />
4<br />
Fundação Nacional Ezequiel Dias- FUNED, Belo Horizonte, BRAZIL.<br />
Two cationic proteins, C1 <strong>and</strong> C3, were purified to homogeneity from the hemolytic<br />
fraction of the venom of Bunodosoma caissarum sea anemone. The purification processes<br />
employed gel filtration followed by ion exchange chromatography (FPLC), being the<br />
purity <strong>and</strong> molecular weight confirmed by SDS-PAGE <strong>and</strong> mass spectrometry. Protein C1<br />
represented the second major peak of the hemolytic fraction <strong>and</strong> was previously believed<br />
to be a cytolysin belonging to a new class of hemolysins. The C1 protein has a molecular<br />
weight of 15495 Da <strong>and</strong> was assayed for hemolysis, PLA2 activity <strong>and</strong> acute toxicity in<br />
crabs <strong>and</strong> mice, showing no activity in these assays. It has an amino terminal with no<br />
similarity to all known hemolysins <strong>and</strong>, therefore, should not be considered a toxin, being<br />
its function completely unknown. The protein C3 (MW19757), that also lacks PLA2<br />
activity, was recognized by antiserum against Eqt II <strong>and</strong> presented high hemolytic activity<br />
to human erythrocytes (ED50 of 0.270 μg/ml), being named Caissarolysin I (Bcs I). Its<br />
activity was inhibited by pre-incubation with sphingomyelin (SM) <strong>and</strong> also when in<br />
presence of erythrocytes pre-treated with the SMase P2, a phospholypase D from the<br />
brown spider Loxosceles intermedia, indicating that SM is the main target of Bcs I.<br />
Caissarolysin I is the first hemolysin purified from a sea anemone belonging to the genus<br />
Bunodosoma <strong>and</strong> belongs to the Actinoporin family of sea anemone hemolysins.<br />
All this data was published in the paper Oliveira et al., (2006).<br />
References: Oliveira, J.S., Zaharenko, A.J., Freitas, J.C., Konno, K., Andrade, S.A.,<br />
Portaro, F.C.V., Richardson, M., Sant’Anna, O.A. <strong>and</strong> Tambourgi, D.V. (2006) Biochimica<br />
et Biophysica Acta-General Subjects, 1760(3), 453-561.<br />
Financial Support: FAPESP, CNPq.<br />
o Hemolysin<br />
o Sea anemone<br />
o Bunodosoma caissarum<br />
o sphingomyelin<br />
313
Poster 28: Beta-Lactamase in Escherichia coli<br />
Siham S. Shaokat* Hamoudi A. Hameed 1<br />
ٍٍٍ<br />
Israa H.J. 2<br />
*Prof. Of Pharmaceutical Microbiology/ Food & Drugs Sector/Nidal Street/Baghdad/Iraq/ email:<br />
albiatyss84@yahoo.com M: 07901867274<br />
1<br />
Prof. Of Chemistry. / Ministry of Industry & Minerals<br />
Chief Food & Drugs Sector/hamodiabas@yahoo.com/M:07901918147<br />
Introduction: The aims of this study was to evaluate the Production of transferable betalactamases<br />
in 58 strains of E.coli isolated from urinary tract infections collected from two<br />
hospitals in Iraq .<br />
Methods: identification of E.coli by the Api systems.<br />
The antimicrobial susceptibility test& the minimum inhibitory concentrations were<br />
determined, by Kirby-Bauer method. The antibiotic resistance rates were: Amoxycillin<br />
(100%), Carbenicillin (79.3%), Cefalothin (37.93%), Amoxycillin\ potassium Clavulanate<br />
(20.7%), Cefotaxime (17.25%), Ceftrixone (8.62%), Ceftazidime (0%);<br />
Trimethoprim100%, Tetracycline (77.6%), Rifampicin (67.24%), Chloramphenicol<br />
(48.28%), &Nalidixic acid (32.8%). Transfer tests were done <strong>and</strong> cell free β- lactamases<br />
were prepared <strong>and</strong> detected by macro-iodometric method.<br />
Results: The most frequent mechanism of resistance was due to Production of transferable<br />
beta-lactamases in 36 strains, Production of cephalosporinases 12 strains <strong>and</strong> combination<br />
of these two mechanisms 10 strains.<br />
UTIs due to resistant strains of E.coli may be successfully treated with β- lactam<br />
antibiotics, <strong>and</strong> the activity was in the following order: Ceftazidime, Ceftrixone,<br />
Cefotaxime, &Amoxycillin\ Clavulanate.<br />
Conclusions<br />
UTIs due to resistant strains of E.coli may be successfully treated with β- lactam <strong>and</strong> the<br />
activity was in the following order: Ceftazidime, Ceftrixone, Cefotaxime, &Amoxycillin\<br />
Clavulanate.<br />
Mushtaq, -S; Woodford,-N; Potz,-N; Livermore,-D-M.Detection of CTX-M-15 extendedspectrum<br />
beta-lactamase in the United Kingdom.<br />
J-Antimicrob-Chemother. 2003 Sep; 52(3): 528-9<br />
o Beta-lactamase<br />
o E. coli<br />
o Beta-lactam<br />
314<br />
Poster 29: Membrane Interactions of the Pore-forming Protein Equinatoxin II:<br />
19 F NMR Studies of the role of Trp <strong>and</strong> Tyr<br />
Norton, R.S. 1 , Bakrač, B. 2 , Anderluh. G. 2 , Podlesek, Z. 2 , Razpotnik, A. 2 , Separovic, F. 3<br />
1<br />
Walter <strong>and</strong> Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville 3050, Australia.<br />
ray.norton@wehi.edu.au<br />
2<br />
Department of Biology, Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia<br />
3<br />
School of Chemistry, University of Melbourne, 3010, Australia<br />
Sea anemones produce a family of 18-20 kDa proteins, the actinoporins, which lyse cells<br />
by forming pores in cell membranes. Sphingomyelin (SM) plays an important role in their<br />
lytic activity, with membranes lacking this lipid being largely refractory to these toxins. As<br />
a means of characterising membrane binding by the actinoporin equinatoxin II (EqTII), we<br />
have used 19 F NMR to probe the environment of Trp <strong>and</strong> Tyr residues in the presence of<br />
micelles <strong>and</strong> bicelles. Trp <strong>and</strong> Tyr were chosen as previous data from mutational studies<br />
<strong>and</strong> truncated analogues had identified the N-terminal helix of EqTII <strong>and</strong> the surface<br />
aromatic cluster including Trp 112 <strong>and</strong> 116 <strong>and</strong> Tyr 113, 133, 137 <strong>and</strong> 138 as being<br />
important for membrane interactions. In two separate analogues, the five Trp residues were<br />
replaced with 5-fluoro-Trp <strong>and</strong> assigned by site-directed mutagenesis <strong>and</strong> the 11 Tyr by 3fluoro-Tyr.<br />
Changes in the Trp 19 F resonances upon EqTII binding to phospholipid (PC)<br />
micelles or bicelles, <strong>and</strong> subsequently upon addition of SM, allowed a model to be<br />
constructed of how EqTII interacts initially with membranes (1). However, in the model<br />
membranes studied here, interaction with SM was not sufficient to trigger dissociation of<br />
the N-terminal helix from the beta-s<strong>and</strong>wich, which forms the bulk of the protein; this<br />
dissociation is considered to be essential for pore formation (2). Further characterization of<br />
the membrane interactions of EqTII is underway using F-Tyr labelled EqTII.<br />
Our results illustrate the value of 19 F NMR in studies of the interactions of proteins with<br />
membranes, with good quality spectra attainable in relatively short acquisition times <strong>and</strong><br />
chemical shifts that are exquisitely sensitive to the local environment. Moreover, the 19 Flabelled<br />
proteins are useful in solid-state NMR experiments.<br />
1. Anderluh, G., Razpotnik, A., Podlesek, Z., Maček, P., Separovic, F. <strong>and</strong> Norton, R.S.<br />
(2005) J Mol Biol 347, 27-39.<br />
2. Hong Q, Gutiérrez-Aguirre I, Barlič A, Malovrh P, Kristan K, Podlesek Z, Maček P,<br />
Turk D, González-Mañas JM, Lakey JH, Anderluh G. (2002) J Biol Chem 277, 41916-24.<br />
o cytolysin<br />
o protein<br />
o membrane binding<br />
o fluorine<br />
315
Poster 30: Toxicity of Gambierdiscus sp. <strong>and</strong> Ostreopsis sp. collected from the<br />
coasts of western Japan<br />
Takefumi Sagara 1* , Shigeto Taniyama 2 , Osamu Arakawa 3 , Tamiko Hashimoto 1 , Naoyoshi Nishibori 1 ,<br />
Manabu Asakawa 2 <strong>and</strong> Sachio Nishio 1<br />
1<br />
Faculty of Junior College, Shikoku University, 123-1, Ebisuno, Furukawa, Ohjin-cho, Tokushima,<br />
Japan, *takefumi-sagara@shikoku-u.ac.jp<br />
2<br />
Graduate School of Biosphere Science, Hiroshima University, 1-4-4, Kagamiyama, Higashi-Hiroshima,<br />
Hiroshima, Japan<br />
3<br />
Faculty of <strong>Fish</strong>eries, Nagasaki University, 1-14, Bunkyo-machi, Nagasaki, Japan<br />
Recently, ciguatoxin (CTX) <strong>and</strong> palytoxin (PTX) food poisoning incidents due to the<br />
ingestion of toxic marine fish have occurred in the western parts of Japan. As previously<br />
reported, CTXs are produced by the benthic dinoflagellate Gambierdiscus toxicus<br />
distributed in the Gambier Isl<strong>and</strong>s of French Polynesia (1). The benthic dinoflagellate<br />
Ostreopsis sp. has been known to produce analogue of PTX (2), <strong>and</strong> might be the origin of<br />
PTX in toxic parrotfish, Scarus ovifrons, inhabiting Japan (3). Between 2004 <strong>and</strong> 2006,<br />
benthic dinoflagellates were collected from algal samples in four stationary points,<br />
Miyazaki, Nagasaki, Tokushima <strong>and</strong> Kochi Prefectures, those are located in the North<br />
Temperate Zone, <strong>and</strong> are the reported areas of CTX <strong>and</strong> PTX poisoning in Japan.<br />
Gambierdiscus sp. was attached to algal surface in Tokushima <strong>and</strong> Kochi. In May, 2005,<br />
especially, a maximum of 212 cells of Gambierdiscus sp. was observed to adhere per one<br />
gram of seaweed from Kochi <strong>and</strong> they were positive in the rapid detection test kit, Cigua-<br />
Check, for CTX. The clone cell was cultured in an ESM medium for 30 days, the cell<br />
pellets (about 10 6 cells) were extracted <strong>and</strong> examined using biochemical analyses. The<br />
crude extract was found to be positive at a sample concentration in Cigua-Check. On the<br />
other h<strong>and</strong>, Ostreopsis sp. was distributed in all stationary points, <strong>and</strong> each clone isolated<br />
from them was cultured according to the previous conditions. All crude extracts from<br />
cultured clones of Ostreopsis sp. had detected a lethal potency (0.5 x 10 -4 - 1.0 x 10 -4<br />
MU/cell) in the mouse test for PTXs. Further, their toxins showed delayed haemolytic<br />
activity with mouse <strong>and</strong> human erythrocytes <strong>and</strong> the activity with latter erythrocytes was<br />
inhibited by g-strophanthin as well as PTX. Thus, it is suggested that some species of<br />
marine fish may be toxified due to toxic benthic dinoflagellate Gambierdiscus sp. <strong>and</strong><br />
Ostreopsis sp. in Japan.<br />
1. Yasumoto et al. (1977). Bull. Jpn. Soc. Sci. <strong>Fish</strong>., 43, 1021-1026.<br />
2. Usami et al. (1995). J. Am. Chem. Soc., 177, 5389-5390.<br />
3. Taniyama et al. (2003). Toxicon, 42, 29-33.<br />
o Ciguatoxin<br />
o Palytoxin<br />
o Gambierdiscus sp.<br />
o Ostreopsis sp.<br />
316<br />
Poster 31: Syphaxin 1.5, a novel antimicrobial peptide isolated from the skin<br />
secretions of the anuran Leptodactylus syphax.<br />
Dourado, F. S 1,2* , Moreira, K. G 2 , Br<strong>and</strong>, G. D 2 , Melo, J. T 2 , Leite, J, R. S. A 2 , Silva, L. P 2 , Bloch Jr, C 2 ,<br />
Schwartz, E. F. 1<br />
1<br />
Universidade de Brasília – Laboratório de Toxinologia, Brasília-DF Brazil, *fsdourado@unb.br<br />
2<br />
EMBRAPA - Recursos Genéticos e Biotecnologia – Laboratório de Espectrometria de Massa, Brasília-<br />
DF, Brazil<br />
Introduction: Antimicrobial peptides (AMPs) are considered part of the innate immune<br />
system of the majority of living organisms. Mostly of them are cationic, amphiphilic, <strong>and</strong><br />
have molecular mass smaller than 10 kDa. This work describes a new AMP active against<br />
Staphylococcus aureus <strong>and</strong> Escherichia coli without cytotoxic activity to human blood cells.<br />
Methods: The skin secretion of two specimens of Leptodactylus syphax (Brasília, DF, Brazil)<br />
was extracted by mild electrical stimulation, dried, resuspended with TFA 0.1%, <strong>and</strong> filtered<br />
(0.22 µm). The filtered material was submitted to RP-HPLC (218TP510 <strong>and</strong> 218TP54 Vydac<br />
columns) using a linear gradient of TFA 0.1% (v/v) <strong>and</strong> acetonitrile/TFA 0.1% (v/v).<br />
Syphaxin 1.5 (SPX 1.5) was de novo sequenced by MALDI-TOF/TOF (Bruker Ultraflex II).<br />
The activity of SPX 1.5 against S. aureus <strong>and</strong> E. coli was tested by the broth microdilution<br />
assay <strong>and</strong> the minimum inhibitory concentration (MICs) obtained was compared with MICs<br />
obtained from commercial antibiotics. The cytotoxic activity was tested on human blood cells<br />
<strong>and</strong> performed in Cell-Dyn 3500 SC/SL flow cytometry automated hematology analyzer.<br />
Results: The purified peptide (SPX 1.5) had the experimental mass of [M+H] + = 1577.83 Da<br />
<strong>and</strong> the sequence GVLDILKGAAKDLAGH. SPX 1.5 inhibited both S. aureus <strong>and</strong> E. coli<br />
ATCC strains, with MICs of 40.5 <strong>and</strong> 10.1 μM, respectively. SPX 1.5 did not exhibit<br />
significant lytic activity on human blood cells.<br />
Conclusion: SPX 1.5 was only the fifth AMP isolated from the skin secretions of<br />
Leptodactylus genus frogs (1, 2 <strong>and</strong> 3). A ClustalW cladogram<br />
(http://www.ebi.ac.uk/clustalw/) of the five peptides described to Leptodactylus genus<br />
positioned SPX 1.5 in the same cluster as the peptides from L. pentadactylus group, as<br />
expected by phylogenetic relationships of these anurans.<br />
References: 1. Nascimento et al. (2004) Protein J. 23:501-8. 2. Rollins-Smith et al. (2005)<br />
Regulatory Pept. 124:173-8. 3. King et al. (2005) Comp. Biochem. Physiol. C: 141:393-7.<br />
Financial Support: FUB, CAPES <strong>and</strong> FINATEC.<br />
o Antimicrobial Peptides<br />
o Leptodactylus syphax<br />
o Skin secretion<br />
o Syphaxin 1.5<br />
317
Poster 32: Cytoskeletal rearrangement <strong>and</strong> cell death induced by Bothrops<br />
alternatus snake venom in cultured Madin-Darby canine kidney cells<br />
Nascimento, J.M. 1,2 , Franchi, G. 3 , Novill, A. 3 , Collares-Buzato, C.B. 4 , Hyslop, S. 1,*<br />
1<br />
Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas<br />
(UNICAMP), CP 6111, 13083-970, Campinas, SP, Brazil *hyslop@fcm.unicamp.br<br />
2<br />
Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), CP<br />
6109, 13083-970, Campinas, SP, Brazil<br />
3<br />
Centro Integrado de Pesquisas Oncológicas Infantis (CIPOI), Faculdade de Ciências Médicas, Universidade<br />
Estadual de Campinas (UNICAMP), CP 6111, 13083-970, Campinas, SP, Brazil<br />
4 Departamento de Histologia e Embriologia, Instituto de Biologia, Universidade Estadual de Campinas<br />
(UNICAMP), CP 6109, 13083-970, Campinas, SP, Brazil<br />
Introduction: Bothrops snake venoms cause renal damage that can lead to renal failure,<br />
the principal cause of death in humans bitten by these snakes. In this work, we<br />
investigated the cytoskeletal rearrangement <strong>and</strong> cytotoxicity caused by Bothrops<br />
alternatus venom in cultured Madin-Darby canine kidney (MDCK) cells, a renal<br />
epithelial cell line. Methods: MDCK cells were incubated with 10 or 100 μg of<br />
venom/ml <strong>and</strong> cytotoxicity was assayed based on neutral red uptake <strong>and</strong> changes in the<br />
transepithelial electrical resistance (RT). MDCK morphology was assessed by the<br />
Feulgen reaction, toluidine blue staining <strong>and</strong> scanning electron microscopy (SEM).<br />
Staining with rhodamine-conjugated phalloidin was used to detect F-actin. Cell death<br />
was quantified by flow cytometry using annexinV-FITC <strong>and</strong> propidium iodide. Results:<br />
Venom (10 or 100 μg/ml) significantly (p
Poster 34: Effect of the Disintegrin Eristostatin on Melanoma Cell Signal<br />
Transduction.<br />
Paquette-Straub, C.A, DiRosato, S.C, McLane, M.A*.<br />
University of Delaware, 305 Willard Hall Educ. Bldg., Newark, DE, USA, *mclane@udel.edu<br />
Metastasis is a major obstacle to treatment of melanoma. Previous researchers have<br />
shown that the venom-derived disintegrin eristostatin inhibits both human <strong>and</strong> murine<br />
melanoma lung or liver colonization in hematogenous mouse metastasis models. The<br />
mechanism for this inhibition is not yet known. Recent studies have shown that a possible<br />
mode of action for this inhibition may be through inhibiting cell motility. In order to better<br />
underst<strong>and</strong> the intracellular signalling, RNA was isolated from five melanoma cell lines<br />
(C8161, M24met, MV3, WM164 <strong>and</strong> 1205Lu) after treatment with or without eristostatin<br />
(3000nM). The RNA was used to create a cRNA probe for hybridization to<br />
oligonucleotide microarrays. After hybridization, the microarrays were developed using<br />
enhanced chemiluminescence. The results were photographed <strong>and</strong> imported into analysis<br />
software supplied by the manufacturer of the microarrays. A comparison of treated <strong>and</strong><br />
untreated cells showed a change in expression of several genes, including MMP2, MMP9,<br />
AKT1, PTEN <strong>and</strong> several integrins. Interestingly, the changes were not consistent across<br />
all five cell lines, increasing in some, while decreasing in others. Concurrently, cells that<br />
had been treated with or without eristostatin for varying time points were lysed <strong>and</strong><br />
analysed for changes in protein tyrosine phosphorylation. The change in phosphorylation<br />
was different for all five cell lines. Proteins at molecular weights of 100-120 kDa, 60-65<br />
kDa, <strong>and</strong> 40-50 kDa all had a cyclic change in phosphorylation across the time course.<br />
These molecular weights may correspond to proteins such as FGFR, Grb10, the Src family<br />
of proteins, PTEN <strong>and</strong> Nck. The involvement of these proteins would suggest eristostatin<br />
is affecting focal adhesion formation (1). Further studies are now being performed using<br />
Real Time PCR <strong>and</strong> confocal microscopy to further elucidate the mode of action of<br />
eristostatin.<br />
1. Staiano, N. et al. 1997. European Journal of Cell Biology 73, 298-305.<br />
o Eristostatin<br />
o Disintegrin<br />
o Cancer<br />
o Signal Transduction<br />
320<br />
Poster 35: Ostreolysin, a mushroom pore-forming toxin specifically binding to<br />
sphingomyelin/cholesterol-rich membrane domains<br />
Rebolj, K. 1 , Maček, P. 1 <strong>and</strong> Sepčić, K. 1*<br />
1 University of Ljubljana, Biotechnical faculty, Department of biology, Večna pot 111, Ljubljana,<br />
Slovenia, *kristina.sepcic@bf.uni-lj.si<br />
Introduction: Ostreolysin (Oly) 1 is an acidic 15-kDa cytolytic protein from the edible<br />
mushroom Pleurotus ostreatus. It is haemolytic, cytolytic, <strong>and</strong> toxic to mice with a LD50 of<br />
1170 μg/kg. Its lytic activity is a consequence of specific interaction with cholesterol-<br />
enriched membrane domains that exist in the liquid ordered (lo) phase (lipid rafts). In this<br />
work, we have investigated the determinants of the lipid structure dictating lipid raft<br />
formation, Oly membrane binding <strong>and</strong> pore-forming activity.<br />
Methods: Surface plasmon resonance was applied to study the binding of Oly on lipid<br />
mono- <strong>and</strong> bilayers with different composition, <strong>and</strong> measurements of calcein release to<br />
determine its permeabilization activity.<br />
Results: The activity of Oly is the highest on sphingomyelin (SM)/Cholesterol (Chol)<br />
membranes. The activity decreases if Chol is replaced with other steroids, or if SM is<br />
replaced by fully saturated phosphatidylcholine (PC). Introduction of less saturated PCs<br />
decreases the activity even further. Activity is also abolished by lysophospholipids.<br />
Discussion/Conclusions: The observed preferential permeabilization <strong>and</strong> binding of Oly to<br />
SM/Chol-containing membranes are highly cooperative with respect to Chol concentration,<br />
reflecting the importance of membrane lateral distribution <strong>and</strong> accessibility of Chol<br />
molecules for protein binding. The inhibitory effects of unsaturated PCs <strong>and</strong><br />
lysophospholipids on Oly activity are very likely a consequence of lo domain disruption.<br />
Our results indicate that fluorescently labelled Oly mutants deprived of toxic activity could<br />
be useful markers in studies of Chol-enriched lipid rafts.<br />
References: 1 Sepčić K, Berne S, Rebolj K, Batista U, Plemenitaš A, Šentjurc M, Maček P (2004) FEBS Lett. 575(1-<br />
3): 81-85.<br />
o pore-forming toxin<br />
o ostreolysin<br />
o lipid rafts<br />
o liquid ordered domains<br />
321
Poster 36: Time- <strong>and</strong> concentration-dependent cytotoxicity of ricin in human lung<br />
epithelial cells<br />
Ramasamy, S. 1* <strong>and</strong> Proll, D. 1<br />
1 DSTO, Human Protection <strong>and</strong> Performance Division, <strong>Fish</strong>ermans Bend, Victoria, Australia, 3207.<br />
* sharmaine.ramasamy@dsto.defence.gov.au<br />
Ricin, a potent ribosome-inactivating hetero-dimer protein toxin (66kDa) produced in the<br />
seeds of the castor oil plant (Ricinus communis), is a Category B Agent on the Centres for<br />
Disease Control (CDC) Select Agent List. Accordingly, there is a need to develop<br />
prophylactic <strong>and</strong> therapeutic countermeasures for ricin intoxication. This is the first<br />
reported study to investigate the time- <strong>and</strong> dose-dependent cytotoxic effects of ricin in<br />
human lung small airway epithelial (SAE) cells. Ricin (1-100pM) produced a time- <strong>and</strong><br />
dose-dependent decrease in SAE cell survival after exposure to the toxin. Ricin (1pM)<br />
reduced cell survival to 75.5 ± 7.3% of the control, compared with only 12.2 ± 5.3% <strong>and</strong><br />
12.1 ± 11.3% of cells surviving 24 hour exposure to higher concentrations of ricin (10 <strong>and</strong><br />
100pM; n=4; p100 60 57 56 20<br />
Primula veris root <strong>and</strong> leaf 11 9 10 14 4.1<br />
Primula vulgaris leaf xx xx xx xx 95<br />
Primula vulgaris flower xx xx xx xx xx<br />
xx-no cytotoxic effect detected<br />
Discussion/Conclusion: The cyclamen <strong>and</strong> P. veris extracts showed cytotoxic effects<br />
against both normal fibroblasts <strong>and</strong> cancer cell lines. Primula vulgaris leaf extract was<br />
cytotoxic against a leukaemia cell line (Jurkat) <strong>and</strong> not normal fibroblasts.<br />
References: 1. Tokalov SV, Kind B, Wollenweber E, Gutzeit GO. (2004) J Agric Food<br />
Chem, 52, 239-245.<br />
o Primulaceae<br />
o cell<br />
o cytotoxic<br />
o extract<br />
323
Poster 38: A crystalline nonprotein compound (BM-ANF) from toad (Bufo<br />
melanostictus, Schneider) skin extract having antiproliferative <strong>and</strong> apoptogenic<br />
activity**<br />
Giri, B 1* , Mishra, R 1 , Dasgupta, S.C 1 , Gomes, A. 1<br />
1 Laboratory of Toxinology & Experimental Pharmacodynamics, Department of Physiology, University<br />
of Calcutta, 92, APC Road, Calcutta 700009, India, *biplab_giri@rediffmail.com<br />
Introduction: Present study is an effort to isolate <strong>and</strong> characterise an antiproliferative <strong>and</strong><br />
apoptogenic compound from the methanolic extract of Indian toad (Bufo melanostictus)<br />
skin <strong>and</strong> to establish its biological activity.<br />
Methods: Dried methanolic extract of toad skin was subjected to alumina gel column<br />
chromatography <strong>and</strong> HPLC. Desired active fraction was crystallised <strong>and</strong> solubility, melting<br />
temperature <strong>and</strong> spectral analysis (UV, IR, TOF-MS, etc.) were done. Biological testing of<br />
pure compound (BM-ANF) was done by U937 & K562 cell growth inhibition, MTT assay,<br />
PCNA expression, Annexin-V binding study, Fluorescent microscopy <strong>and</strong> SEM.<br />
Results: BM-ANF was eluted in chloroform : methanol (60 : 40) <strong>and</strong> further purified<br />
through HPLC. It was crystallised <strong>and</strong> partially characterised (MT > 325°C, UVλmax<br />
229.2 nm, MW 782.52 Da). IC50 dose of BM-ANF in U937 & K562 cells were 14 μg/100<br />
μl <strong>and</strong> 10 μg/100 μl respectively. BM-ANF (IC 50 doses) produced time dependent (24,<br />
48, 72 hrs.) significant inhibition of U937 & K562 cell growth (One way Anova,<br />
P
Poster 40: ANTIMICROBIAL ACTIVITY OF EXTRACTS FROM Phyllomedusa<br />
rohdei (AMPHIBIA, ANURA) SKIN<br />
Gomez, J.G.C. 1 ; Zaharenko, A.J. 2 ; Rocha, L.M. 1 ; Wuo, V.G. 1 ; Marinho, E.A.V. 1,3 ; Malpezzi-Marinho,<br />
E.L.A 1<br />
12- Braz Cubas University, Health Science Area, Mogi das Cruzes, SP, Brazil, gomez@brazcubas.br<br />
13- University of São Paulo, Biosciences Institute, Dept. of Physiology, São Paulo, SP, Brazil,<br />
a.j.zaharenko@ig.com.br<br />
14- Federal University of São Paulo, Dept. of Pharmacology, São Paulo, SP, Brazil<br />
The production of peptides showing antimicrobial activity in skin granular gl<strong>and</strong>s of<br />
Phyllomedusa has been described. This work aimed to evaluate a skin methanolic extract<br />
from P. rohdei regarding its antimicrobial activity. The methanol skin extract was<br />
fractioned using methylene chloride <strong>and</strong> water to obtain a polar <strong>and</strong> non polar fractions<br />
respectively. No antimicrobial activity was detected in the methanol extract as well as in<br />
the polar fraction. The non polar fraction showed antimicrobial activity against Gram<br />
negative <strong>and</strong> Gram positive bacteria as well as fungi. A minimal inhibitory concentration<br />
(MIC) of 0.5 mg/mL was verified for the Gram negative bacteria Escherichia coli ATCC<br />
11229 <strong>and</strong> Pseudomonas aeruginosa ATCC 15442. The MIC value was 0.25 mg/mL for<br />
the yeast C<strong>and</strong>ida albicans ATCC 10233. The higher antimicrobial activity was detected<br />
against the Gram positive bacteria Staphylococcus aureus ATCC 6538 with MIC value<br />
reaching 0.06 mg/mL. To evaluate the antimicrobial activity against S. aureus ATCC<br />
6538, it was cultivated in nutrient broth. The non polar fraction of methanol extract of P.<br />
rohdei skin at the concentration of 0.5 mg/mL reduced 90% of the growth capacity of S.<br />
aureus (one log unity). These results are compatible with a microbiostatic activity in the<br />
extract of P. rohdei skin.<br />
o Phyllomedusa rohdei<br />
o Antimicrobial activity<br />
o Microbiostatic<br />
326<br />
Poster 41: EFFECT OF NOVEL PECTENOTOXINS ON ACTIN<br />
CYTOSKELETON<br />
Louzao, M.C. 1* , Ares, I.R. 1 , Cagide, E. 1 , Vieytes, M.R. 2 , Miles, C.O. 3 , Yasumoto, T. 4 <strong>and</strong> Botana, L.M. 1<br />
(1) Departamento de Farmacología, * ffmclooj@lugo.usc.es (2) Departamento de Fisiología Animal.<br />
Facultad de Veterinaria. Campus de Lugo. Universidad de Santiago de Compostela. 27002 Lugo. Spain<br />
(3) National Veterinary Institute, PB 8156 Dep., N-0033 Oslo, Norway; AgResearch Ltd, Ruakura<br />
Research Centre, Private Bag 3123, Hamilton, New Zeal<strong>and</strong><br />
(4) Japan Food Research Laboratories, Tama, Tokyo 206-0025, Japan<br />
In recent years new congeners belonging to the marine pectenotoxins family have been<br />
detected. Pectenotoxin-11 has been recently isolated from toxic phytoplankton <strong>and</strong><br />
shellfish in New Zeal<strong>and</strong>. Pectenotoxin-2 seco acid, another novel compound of this group,<br />
is generated by hydrolysis of pectenotoxin-2 within of the bivalve’s tissues. So far, there is<br />
no data available about their mechanism of action. We investigated the biological activity<br />
of pectenotoxin-11 <strong>and</strong> pectenotoxin-2 seco acid on actin cytoskeleton <strong>and</strong> morphology of<br />
neuroblastoma cells. In addition, their effects were compared with those found with<br />
pectenotoxin-2 in the same conditions. Fluorescent phalloidin was utilized for detecting<br />
quantitative changes in actin level through a laser scanning cytometer. Likewise, confocal<br />
microscopy allowed to visualize the organization of filamentous actin as well as changes in<br />
morphology. The results showed that pectenotoxin-11 triggered an important<br />
depolymerizing effect on actin cytoskeleton as well as modifications in shape of<br />
neuroblastoma cells. Interestingly, these effects were very similar to those observed with<br />
pectenotoxin-2. Otherwise, pectenotoxin-2 seco acid induced no alteration in any of the<br />
parameters studied. This has suggested that actin cytoskeleton is a common target for<br />
pectenotoxin-11 <strong>and</strong> pectenotoxin-2, but not for its derivative pectenotoxin-2 seco acid.<br />
o Pectenotoxins<br />
o neuroblastoma<br />
o actin cytoskeleton<br />
o cellular morphology<br />
327
Poster 42: A Cardiotoxin (NK31) from Indian Monocled Cobra (Naja kaouthia)<br />
shows activity against human chronic myelogenic cell-line (K562)<br />
Debnath, A, Gomes, A*, Vedasiromoni, J.R.<br />
Drug Development Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur-700032,<br />
West Bengal, INDIA. *gomes_aparna@yahoo.com<br />
Introduction: In our previous studies, it was found that Indian monocled cobra (Naja<br />
kaouthia) venom (NKV) was active on carcinoma, sarcoma <strong>and</strong> leukemia models. We have<br />
tried to isolate, identify <strong>and</strong> characterize the factor(s) responsible for the anti-neoplastic<br />
effect of NKV.<br />
Methods: IEC using CM cellulose, HPLC, TOF-Mass spectrometry, Amino acid<br />
sequencing, Cell-culture, Trypan blue exclusion method, MTT reduction assay, Confocal,<br />
SEM, FACS (AnnexinV-FITC/PI), Cell-cycle analysis. Statistical analysis by student’s ‘t’<br />
test with p
Poster 44: Haemolytic protein from pedicellaria of violet sea urchin Sphaerechinus<br />
granularis (Toxopneustidae)<br />
Turk, T 1 <strong>and</strong> Kem, W. R 2<br />
1<br />
University of Ljubljana, Department of Biology, 1000-Ljubljana, Slovenia *tom.turk@bf.uni-lj.si<br />
2<br />
University of Florida, Collegel of Medicine, Department of Pharmacology <strong>and</strong> Therapeutics,<br />
Gainesville, Fla, USA<br />
In terms of toxins sea urchins are poorly characterized group of marine animals although<br />
some of the species, mainly those belonging to the family Toxopneustide, can cause<br />
serious medical conditions in humans.<br />
An extract from pedicellaria of live violet sea urchin Sphaerechinus granularis, the only<br />
Mediterranean member of family Toxopneustide, was toxic to crabs. Isolation of toxic<br />
proteins by means of different chromatographic techniques revealed several haemolytic<br />
protein peaks. One of the proteins was purified to homogeneity. It is a 28 kDa protein with<br />
an Ip of 6.5. The protein is lytic to red blood cells but does not cause their agglutination.<br />
Obtained protein appears to be thermostable. The hemagglutinating activity reported for<br />
several proteins isolated from sea urchins was generally absent from our samples. In terms<br />
of molecular weight <strong>and</strong> its activity new protein resembles a haemolytic protein isolated<br />
from coelomic fluid of Tripneustes gratilla, another member of Toxopneustidae family.<br />
However, in our case the haemolytic activity was Ca 2+ independent <strong>and</strong> without lectin<br />
characteristics typical for T. gratilla lectin.<br />
o sea urchin<br />
o pedicellaria<br />
o haemolytic activity<br />
o crab toxicity<br />
330<br />
Poster 45: An alpha-conotoxin from Conus victoriae is effective at alleviating<br />
neuropathic pain in an animal model of diabetic neuropathy.<br />
Satkunanathan, N 1 , Khodr, B 1 , Georgiou, G 1 , McIntosh, D 2 , Belyea, C 2 , Khalil Z 1, 3 , Livett, B.G. 1*<br />
1 University of Melbourne, Biochemistry <strong>and</strong> Molecular Biology <strong>and</strong> Bio21 Institute, 30 Flemington<br />
Rd.,Parkville,Victoria, AUSTRALIA, *b.livett@unimelb.edu.au<br />
2 Metabolic Pharmaceuticals Pty Ltd, St. Kilda Rd., Melbourne,Victoria, AUSTRALIA<br />
3 University of Sharjah, College of Pharmacy,UAE.<br />
Introduction: The present study was performed to evaluate the antiallodynic effects of αconotoxin<br />
Vc1.1, in rats with diabetic neuropathy. With an increasing older demographic,<br />
diabetic neuropathy is an increasing problem in the community. Oxidative stress has been<br />
implicated in the pathogenesis of diabetic neuropathy, the most common complication of<br />
diabetes mellitus affecting more than 50% of diabetic patients (1). α-conotoxin Vc1.1<br />
(ACV1) is a synthetic peptide of 16 amino acids, whose amino acid sequence was deduced<br />
from cDNA created from the venom duct of the Australian cone snail Conus victoriae (2).<br />
ACV1 is a competitive antagonist of the neuronal nicotinic acetylcholine receptor<br />
(nAChR) in animals <strong>and</strong> humans. Previous studies in our laboratory (3) have shown that<br />
ACV1 is a potent analgesic in several rat models of human neuropathic pain.<br />
Methods: The streptozotocin-induced diabetic rat model of peripheral neuropathy was<br />
used. Mechanical allodynia was assessed using von Frey filaments. Treatment with<br />
ACV1 started after the development of allodynia (6 weeks after induction of diabetes) <strong>and</strong><br />
continued for 4 weeks. ACV1 was administered via subcutaneous bolus injection at the<br />
back of the neck at doses of 3μg/kg, 30μg/kg <strong>and</strong> 300μg/kg. Sciatic nerve biopsies <strong>and</strong><br />
plasma were collected one week after cessation of treatment to assess the oxidative stress.<br />
In addition, the effect of ACV1 treatment on levels of lipid hydroperoxides in injured<br />
sciatic nerve <strong>and</strong> on the levels of nitrotyrosine in plasma proteins was investigated.<br />
Results: An antiallodynic effect was observed at 30μg/kg <strong>and</strong> 300μg/kg, within 1 hr postdosing.<br />
ACV1 attenuated allodynia for up to a week after cessation of treatment. ACV1<br />
also reduced oxidative stress markers such as lipid hydroperoxides <strong>and</strong> nitrotyrosine.<br />
Discussion/Conclusion: The results indicate that ACV1 has an antiallodynic effect <strong>and</strong><br />
that one possible mechanism could be related to reduction in oxidative stress associated<br />
with diabetic peripheral neuropathy.<br />
References: 1. Sayyed, S.G. et al (2006) Life Sci.(in press); 2. S<strong>and</strong>all, D.W. et al (2003)<br />
Biochemistry 42: 6904-6911; 3. Satkunanathan, N. et al (2005) Brain Res. 1059: 149-158;.<br />
Satkunanathan, N. et al (2005) Brain Res. 1059 : 149-158 ; 3. Sayyed, S.G. et al (2006)<br />
Life Sci. in press.<br />
o conotoxin<br />
o analgesic<br />
o diabetic neuropathy<br />
o nicotinic acetylcholine receptor<br />
331
Friday 28 July: poster session<br />
(Colville Building 5.11/5.12)<br />
Clinical aspects<br />
1. Venomous Bite <strong>and</strong> Sting Hospitalisations in Australia 2000-2002. Winkel<br />
K.D.* 1 Kreisfeld, R 2 , Harrison, J.E. 2<br />
2. Death from brown snake (Genus: Pseudonaja) envenomation in Australia: is<br />
antivenom to blame? Winkel K.D. 1 , Lowe R. 1 , McGain F. 1 , Nimorakiotakis, B. 1 ,<br />
Williams D.J. 1<br />
3. Efficacy <strong>and</strong> Safety of an F(ab’)2 Antivenom for Scorpion Envenomation.<br />
Boyer, L.V. 1 , Alagon A. 2 , Mallie J. 1<br />
4. Neurotoxicity in Children Hospitalized due to North American Scorpion<br />
Envenomation. Boyer, L.V. 1 , Mallie J 1 , Chase, P.B. 1 , Theodorou, A. 1 , Schmier, C. 1<br />
5. A new venomous scorpion responsible of human deaths in Argentine: Tityus<br />
confluens. Adolfo R. de Roodt 1 ; Néstor Lago 2 ; Judith Estevez 3 ; Estela Neder 4 ; Alicia<br />
Montero 4 ; Oscar D. Salomón 1 ; Jorge F. Paniagua 5 ; Raúl López 6 ; Valeria Vega 7 .<br />
6. Toxicity of five scorpion venoms from the Old World, <strong>and</strong> its immunological<br />
reactivity with American scorpion antivenoms. Adolfo Rafael de Roodt 1 ; Néstor R.<br />
Lago 2 ; Judith Estevez 3 ; Eduardo Scarlatto 4 ; Rodrigo D. Laskowicz 2 ; Jorge F.<br />
Paniagua 5 ; Hernán García 6 ; Victor M. Manzanelli 2 , Alej<strong>and</strong>ro Alagon 7 .<br />
7. Neutralization of the haemorrhagic activity of Echis ocellatus (saw-scaled viper)<br />
venom <strong>and</strong> a venom metalloproteinase using synthetic peptide inhibitors <strong>and</strong><br />
chelators. Howes, J-M, Theakston, R.D.G., Laing, G.D.*<br />
8. The potential advantages of camelid IgG for the development of antivenoms.<br />
Harrison, R.A 1 *, Cook, D 1 , Hasson, S.S 2 , Harmsen, M 3 , Laing, G.D 1 , Conrath, K 4 ,<br />
Theakston, R.D.G 1 .<br />
9. The Antifungal Activity of Punica Granatum L Peels powder <strong>and</strong> extracts.<br />
Siham S. Shaokat* <strong>and</strong> Hamoudi A. Hameed 1<br />
ٍٍٍ<br />
10. Epidemiology <strong>and</strong> management of snakebites in Burkina Faso. Sondo Blaise 1, 2 ,<br />
Kou<strong>and</strong>a Séni 2 , Some Noya 2<br />
332<br />
11. UNCOMMON ENVENOMING PROVOKED BY ARTHROPODS: TOXINS<br />
FOR KNOWING. Haddad Jr, V.<br />
12. North <strong>and</strong> South American Loxosceles spiders: Development of a polyvalent<br />
antivenom with recombinant Sphingomyelinase D as antigens. Olvera, A 1* , Ramos-<br />
Cerrillo, B 1 , Paniagua-Solís, J 2 , Stock, R.P 1 <strong>and</strong> Alagón, A 1 .<br />
13. EPIDEMIOLOGY OF SNAKEBITES BASED ON HOSPITAL SURVEY IN<br />
CHITWAN AND NAWALPARASI DISTRICTS, NEPAL. P<strong>and</strong>ey, Deb Prasad<br />
14. Analysis of clinical outcomes <strong>and</strong> cost-benefits associated with the use of CSL<br />
Snake Venom Detection Kits in the management of snakebite at Port Moresby<br />
General Hospital, Papua New Guinea. Williams D.J, 1* Jensen S.D, 1,2 Winkel K.D. 1<br />
15. Epidemiology of snakebite in Guinea. Baldé M. C., Barry A. O., Tamboura B. A.<br />
16. RENAL AND BLOOD VESSEL ALTERATIONS INDUCED BY GAMMA<br />
TOXIN OF Tityus serrulatus VENOM. Monteiro, H.S.A. 1 *; Alves, R.S. 1 ; Martins,<br />
R.D. 1 ; Amora, D.N. 1 ; Barbosa, P.S.F. 1 ; Sousa, D.F. 1 ; Alves, C.D. 1 ; Toyama, M.H. 2 ;<br />
Menezes, D.B. 3 ; Martins, A.M.C. 4 ; Fonteles, M.C. 1 ; Monteiro, F. C. D. 1<br />
17. Biological <strong>and</strong> Clinical Evaluations of Antivenom Immunotherapy. Bon, C. 1* ,<br />
Audebert, F. 1 , Calderon, E. 2 , Choumet, V. 1 , ElAyeb, M. 3 , Krifi, M. 3 , Possani, L. 2 ,<br />
Rivière, G. 1 , Sorkine, M.<br />
Cardiovascular<br />
18. Amelioration of the cardiovascular effects of the yellow scorpion Leiurus<br />
quinquestriatus envenomation in rats by the red grape seeds proanthocyanidins.<br />
El-Alfy, A 1 ., Ahmed, A 1 ., Kader, F 1 ., Fatani, A 1 .<br />
19. Can the antioxidant, ginkgo biloba extract, <strong>and</strong> the protease inhibitor,<br />
aprotinin, protect rats against Leiurus quinquestriatus scorpion venom-induced<br />
cardio-vascular effects? Fatani, AJ, Yaqub, HI, Ibrahim, M, Al-Zuhair, H, Qadir, F,<br />
Al-Qutub, M.<br />
20. Alterations to the expression <strong>and</strong> distribution of adhesion molecules in rat<br />
renal glomeruli after Bothrops moojeni envenoming. *Cruz-Höfling, M. A.,<br />
Collares-Buzato, C. B.<br />
21. Snake Venom Metalloproteinases (SVMPs) binds to collagen <strong>and</strong> α2β1<br />
integrin by two different motifs. Tanjoni,I. 1 , Butera, D. 1 , Della-Casa, M.S. 1 ,<br />
Fern<strong>and</strong>es, I. 1 , Clissa, P.B. 1 , Evangelista, K.S. 2 , Eble, J. 2 , Moura-da-Silva, A.M. 1*<br />
333
22. Distribution <strong>and</strong> effects of ε-toxin-GFP in mouse brain <strong>and</strong> kidneys. Juan<br />
Blasi 1* , Alex Soler 1 , Jonatan Dorca 1 , Josep Saura 2 , Josep Maria Tussell 2 , Maryse<br />
Gibert 3 , Michel Poppof 3 , Joan Serratosa 2 <strong>and</strong> Mireia Martín-Satué 1 .<br />
23. Evaluation of potential ricin inhibitors on pulmonary <strong>and</strong> digestive epithelial<br />
cells. Emilie Gobbo 1 , Roman Lopez 2 , Goulven Merer 2 , Bernard Rousseau 2 , André<br />
Ménez 1 , Bruno Beaumelle 3 , Daniel Gillet 1* <strong>and</strong> Julien Barbier 1<br />
24. Antagonism of the cardiotoxic effect of Bothrops jararacussu by Suramin.<br />
Sifuentes, N. D. 1 , El-Kik, C. Z. 2 , Ricardo, H.D. 2 ,Schwartz, E.N.F. 1 , Melo, P.A. 2<br />
25. EFFECT OF CROTOXIN ON MICROCIRCULATION AND<br />
CIRCULATING LYMPHOCYTES. Zambelli, VO 1 ; Sampaio, SC 1 , Britto, LRG 2 ,<br />
Zychar,B 1 ; Gonçalves, LRC 1 ; Cury, Y 1 .<br />
26. EFFECTS OF THE SEA ANEMONE Bunodosoma caissarum VENOM ON<br />
RENAL PERFUSION AND MESENTERIC BED VESSELS. Monteiro, H.S.A 1 *.;<br />
Martins, R. D. 1 ; Alves, R. S. 1 ; Amora, D. N. 1 ; Silva Neto, A.G. 1 ; Maia, D.G. 1 ; Toyama,<br />
M. H. 2 ; Martins, A M.C. 3 ; Barbosa, P.S.F. 1 ; Fonteles, M. C. 1 , Monteiro, F. C. D. 1<br />
27. Tityus serrulatus Hypotensive Peptides: a novel class of hypotensive agents.<br />
Verano-Braga, T. 1,2* , Silva, D.M.R. 4 , Santos, C.F.F. 4 , Bemquerer, M.P. 2,3 , Santos,<br />
R.A.S. 4 , Bougis, P.E. 5 , Martin-Eauclaire, M.F. 5 , De Lima, M.E. 1,2 <strong>and</strong> Pimenta,<br />
A.M.C. 1,2<br />
28. Snake Venom Components as Pharmacological Tools <strong>and</strong> Molecular Models in<br />
Thrombosis <strong>and</strong> Haemostasis. Bon, C., Arocas V., Braud S., Francischetti I., Leduc<br />
M., Maroun R., Saliou B., Wisner A., Zhang Y., Zingali R..<br />
29. Venom Peptides from Calloselasma rhodostoma --- New Twist in Vasoactive<br />
Peptides? Higuchi, S 1,2* , Khow, O 3 , Suntrarachun, S 3 , Noiphrom, J 3 , Miyamoto, S 1 ,<br />
Kawai, A 1 , Sitprija,V 3 , Sato,T. 3<br />
30. Potent dromedary antibody fragments for using in immunotherapy against<br />
scorpion envenomation. Bouhaouala-Zahar, B. 1 *, Conrath, K. 2 , Ben abderrazek R. 1 ,<br />
Hmila I. 1 , Benlasfar Z. 1 , Hammadi M. 3 , Khorchani T. 3 , Muyldermans S. 2 . & El Ayeb,<br />
M. 1<br />
31. Purification <strong>and</strong> characterization of ancistron–Bu, a novel fibrinogen-clotting<br />
serine protease from Agkistrodon blomhoffii ussuriensis venom. Karbovskiy V.<br />
L 1,2* , Savchuk A.N 2 , Volkov G.L. 2<br />
32. “Blomus-B” – a novel non-enzymatic platelet aggregation inhibitor purified<br />
from Agkistrodon blomhoffii ussuriensis venom. Karbovskiy V. L 1,2* , Savchuk A.N.<br />
334<br />
Toxins from novel sources<br />
33. Advances in the studies in the field of animal venoms <strong>and</strong> their toxins in Brazil<br />
from 1900 until today. Borja-Oliveira CR <strong>and</strong> Rodrigues-Simioni L*.<br />
34. Geographical <strong>and</strong> Toxinological Characterization of Tityus quirogae<br />
(Scorpiones, Buthidae), a Species of Medical Importance from northeastern<br />
Venezuela. De Sousa, L. 1* , Borges, A. 2 , Manzanilla, J. 3 , Gregoriani, T. 1 , Romero, L.<br />
1 , Parrilla-Alvarez, P. 4 , Quiroga, M. 4<br />
35. Modulation of the content of naturally released scorpion venom by a,<br />
presumably, rapid holocrinic secretion in the venom gl<strong>and</strong>. Morgenstern D 1* ,<br />
Matthews G 1,2 , Sher D 1 , Zlotkin E 1 .<br />
36. A study on “Black Widow” spiders from Argentina. Adolfo Rafael de Roodt 1 ;<br />
Rodrigo D. Laskowicz 1 ; Victor M. Manzanelli 1 ; Judith Estevez 2 .<br />
37. Sex-linked variation of Thalassophryne maculosa fish venom. Sosa-Rosales J.I. 1 ,<br />
Pereira C. 1 , Bruni F.M. 2 , Pareja-dos-Santos A. 2 , Boletini-dos-Santos D. 2 , Ramos A.D. 2 ,<br />
Kadura A.M. 2 , Conceição K. 3 , Pimenta D.C. 3 , Lima C. 2 , Lopes-Ferreira M. 2<br />
38. Comparative Studies Between Secretory Cells Of Sting Apparatus And Venoms<br />
Obtained from Freshwater (Potamotrygon falkneri) And Marine (Dasyatis guttata)<br />
Stingrays. Barbaro, K.C. 1 *, Lira, M.S. 1 , Malta, M.B. 1 , Soares, S.L. 1 , Santoro, M.L 2 ,<br />
Pedroso, C.M. 3 , Antoniazzi, M.M. 3 , Jared, C. 3 , Cardoso, J.L.C. 4 , Garrone Neto, D. 5 ,<br />
Haddad Jr, V.<br />
Plant toxins<br />
39. Componnt Characteristics <strong>and</strong> Food Safety of Star Fruit. Jou-Fang Deng 1,2 , Yu-<br />
Lan Hung 3 , Ming-Ling Wu 1 , Yen-Hung Yeh 4 <strong>and</strong> Deng-Fwu Hwang 3,*<br />
40. ALLELOPATHIC EFFECTS OF CROP PLANTS ON OTHER CROP<br />
PLANTS. OMID ALIZADEH<br />
41. THE EFFECT OF SAFFLOWER (Carthamus tinctorius) ON<br />
REPRODUCTIVE PHYSIOLOGY OF THE MALE MICE. Mehrdad Modaresi<br />
<strong>and</strong> Fateme Matboo Sobhani<br />
335
Poster 1: Venomous Bite <strong>and</strong> Sting Hospitalisations in Australia 2000-2002<br />
Winkel K.D.* 1 Kreisfeld, R 2 , Harrison, J.E. 2<br />
1 Australian Venom Research Unit, Department of Pharmacology, University of Melbourne, Parkville,<br />
Vic, 3010. Australia *kdw@unimelb.edu.au<br />
2 National Injury Surveillance Unit, Flinders University, GPO Box 2100, Adelaide, 5042 Australia.<br />
Introduction: Little systematic national data has been available to assess the current burden<br />
of venomous bite <strong>and</strong> sting injury in Australia. National, systematically coded, hospital<br />
separation data therefore provides a new method of monitoring overall venom-related<br />
injury.<br />
Methods: Cases that met the following criteria were selected for analysis: Australian<br />
hospital separations occurring from July 1 st , 2000 to June 30 th , 2002. This period was<br />
chosen because it centres on the date of the 2001 Census, which enables certain analyses<br />
by location that cannot be done so well for period more distant from a census.<br />
Results: In the period 2000/01–2001/02, 7,407 people were admitted to hospital as the<br />
result of bites <strong>and</strong> stings by a venomous animal or plant. Spiders, bees, <strong>and</strong> snakes <strong>and</strong><br />
lizards were the most common sources of bites <strong>and</strong> stings. There were some marked<br />
differences between males <strong>and</strong> females. For the categories snakes <strong>and</strong> lizards, wasps, bees<br />
<strong>and</strong> marine animals <strong>and</strong> plants, cases involving females were less than half as frequent as<br />
those involving males. All categories of bites <strong>and</strong> stings resulted in a similar mean length<br />
of stay in hospital [1.3 days]. Rates of bites <strong>and</strong> stings were fairly similar for children <strong>and</strong><br />
adults in the middle years but began to decline from the age of 60. Rates for bites <strong>and</strong><br />
stings as a whole increased relative to the remoteness of the location in which they<br />
occurred. There were notable differences between latitudes, with most venomous injuries<br />
increasing in frequency as one progressed further north The all-Australia rate for these<br />
bites <strong>and</strong> stings as a whole was 19.1 per 100,000 population.<br />
Conclusions: This report represents significant progress in our underst<strong>and</strong>ing of the<br />
venomous bite <strong>and</strong> sting injury burden in Australia. The excess of rural <strong>and</strong> remote area<br />
admissions reinforces the need for specific strategies to recognise <strong>and</strong> manage venomrelated<br />
injuries in these areas.<br />
o Australia<br />
o Hospitalisations<br />
o Injury<br />
o Venomous bites <strong>and</strong> stings<br />
336<br />
Poster 2: Death from brown snake (Genus: Pseudonaja) envenomation in<br />
Australia: is antivenom to blame?<br />
Winkel K.D. 1 , Lowe R. 1 , McGain F. 1 , Nimorakiotakis, B. 1 , Williams D.J. 1<br />
1<br />
Australian Venom Research Unit, University of Melbourne, Parkville, Vic, 3010. Australia<br />
* kdw@unimelb.edu.au<br />
Introduction: Recent investigators have questioned the efficacy of Australian brown snake<br />
antivenom manufactured by CSL Limited 1 . We reviewed definite brown snake related<br />
fatalities to ascertain the circumstances of death <strong>and</strong> the possible contribution of antivenom<br />
to the outcome.<br />
Methods: Retrospective review of definite brown snake-related fatalities using data from<br />
hospital records, autopsy <strong>and</strong> coronial findings held by the Australian Venom Research<br />
Unit (the Australian Bite & Sting Fatality Database).<br />
Results: 24 definite cases were identified between January 1980 <strong>and</strong> December 2004 .<br />
Patients ranged in age from 1-75 yrs, with a mean adult age of 50.2 years <strong>and</strong> with a<br />
male:female ratio of 3:1. There were only two paediatric deaths (unwitnessed snakebite<br />
that went unrecognised prehospital). Patients >50 yrs were over-represented (57% of<br />
cases). Almost half the deaths (47%) occurred in Queensl<strong>and</strong>. The identification of the<br />
immunotype or species was made on the basis of Snake Venom Detection Kit (CSL<br />
Limited) in 88% of cases (mostly bite site swab), <strong>and</strong> on the basis of physical identification<br />
of the snake (11%). Antivenom was administered to 58% of patients but most (95%) had a<br />
prior, early loss of consciousness from which they never recovered (either pre-hospital or<br />
on presentation <strong>and</strong>/or upon intra-hospital release of first aid). Time to death was between<br />
20 minutes to three weeks. All but one patient sustained bites to either upper or lower limb<br />
extremities. Death was attributable to pre-antivenom events in 95% of cases: most cases<br />
lacked early, adequate pressure-immobilisation first-aid but rapid release of b<strong>and</strong>ages,<br />
including tourniquet, was also associated with intra-hospital collapse in some cases.<br />
Conclusion: This study reveals that death after brown snake envenomation typically<br />
involves older Australians <strong>and</strong> is precipitated by critical events that occur prior to the<br />
administration of antivenom.<br />
References: 1. Judge et al (2006) Toxicology & Applied Pharmacology, 213: 117-125.<br />
o Envenomation<br />
o Fatality<br />
o Genus: Pseudonaja<br />
o CSL brown snake antivenom<br />
337
Poster 3: Efficacy <strong>and</strong> Safety of an F(ab’)2 Antivenom for Scorpion Envenomation<br />
Boyer, L.V. 1 , Alagon A. 2 , Mallie J. 1<br />
1 University of Arizona Health Sciences Center, Tucson, Arizona, USA, boyer@pharmacy.arizona.edu<br />
2 Instituto de Tecnologîa, Universidad Nacional Autonoma de Mexico, Cuernavaca, MX<br />
Introduction: Neurotoxicity <strong>and</strong> ventilatory compromise from North American Buthid<br />
scorpion sting may be life-threatening in children. Management of the syndrome with<br />
sedation <strong>and</strong> intensive supportive care is associated with sedative dosing in excess of that<br />
commonly accepted as safe, <strong>and</strong> adverse events are common.<br />
Methods: An equine F(ab’)2 antivenom commercially available in Mexico was<br />
administered to children presenting in the US <strong>and</strong> Mexico with clinically important signs<br />
of scorpion envenomation. Three closely related protocols were used in a multicenter study<br />
adapted to varied local needs. Patient demographics, time of sting, nature <strong>and</strong> duration of<br />
syndrome, <strong>and</strong> adverse events were recorded. Serum venom levels were determined by<br />
specific ELISA. Descriptive statistics were derived <strong>and</strong> clinically interpreted.<br />
Results: Forty-one children <strong>and</strong> 3 adults were enrolled. The study population was 51.2%<br />
male, with average age of children 4.0 years <strong>and</strong> weight 17.3 kg. Time from sting to study<br />
drug infusion averaged 114 minutes. Overall, clinically important signs were present in<br />
100% at baseline, 62% at 1 hour, 12.2% at 2 hours, <strong>and</strong> 0% at 4 hours after infusion.<br />
Venom levels declined by an order of magnitude, within one hour of antivenom infusion.<br />
Adverse events, none attributed to the study drug, affected 9.8% of patients.<br />
Discussion/Conclusion: Compared with historical controls, patients who received<br />
antivenom recovered faster, with fewer adverse events. Venom levels declined concurrent<br />
with syndrome resolution, suggesting a surrogate marker of efficacy for future studies.<br />
F(ab’)2 antivenom appears to provoke fewer hypersensitivity reactions than does wholeimmunoglobulin<br />
antivenom in a similar population (1).<br />
References: 1. LoVecchio F et al. (1999) Incidence of immediate <strong>and</strong> delayed<br />
hypersensitivity to Centruroides antivenom, Ann Emerg Med 34:5; 615-619.<br />
o scorpion<br />
o antivenom<br />
o clinical trial<br />
o neurotoxicity<br />
338<br />
Poster 4: Neurotoxicity in Children Hospitalized due to North American Scorpion<br />
Envenomation<br />
Boyer, L.V. 1 , Mallie J 1 , Chase, P.B. 1 , Theodorou, A. 1 , Schmier, C. 1<br />
1 University of Arizona Health Sciences Center, Tucson, Arizona, USA, boyer@pharmacy.arizona.edu<br />
Introduction: Envenomation by North American Buthid scorpions may involve profound<br />
neurotoxicity <strong>and</strong> ventilatory compromise. Formal clinical trials of antivenom under way<br />
in the U.S. required establishment of a control group for comparison to treated patients.<br />
Methods: Patient records at hospitals where antivenom was not historically used were<br />
formally reviewed by a physician/nurse team. Patient demographics, time of sting, duration<br />
of syndrome, dose of adjunctive sedative, blood count <strong>and</strong> seru chemistries, <strong>and</strong> adverse<br />
events were recorded. Descriptive statistics were derived <strong>and</strong> clinically interpreted.<br />
Results: Ninety-seven evaluable records were identified <strong>and</strong> reviewed. The study<br />
population was 53.6% male, with average age 3.9 years <strong>and</strong> weight 18.3 kg. Time from<br />
sting to last documented clinically important sign of envenomation averaged 764 minutes<br />
(SD 370, range 180-2160). The most consistently documented signs of toxicity were<br />
thrashing limbs (in 94.8%) <strong>and</strong> abnormal eye movements (74.2%). On average, midazolam<br />
sedation continued until 858 minutes after the sting (SD 311, range 200-2025). Total dose<br />
averaged 6.56 mg/kg. Adverse events affected 39.2% of patients.<br />
Discussion/Conclusion: Without specific antivenom, children with serious neurotoxicity<br />
remain critically ill for an average of 12.7, <strong>and</strong> up to 36 hours, following scorpion sting.<br />
Treatment is frequently complicated by adverse events. Benzodiazepine dosing is<br />
substantial, averaging 20 times the maximum dose generally used for conscious sedation.<br />
These findings are consistent with <strong>and</strong> exp<strong>and</strong> upon the findings of others (1,2).<br />
References: 1. Curry SC et al. (1983-1984) Envenomation by the Scorpion Centruroides<br />
sculpturatus, J Tox Clin Tox 21 (4&5); 417-449. 2. Gibley R et al. (1999) Continuous<br />
intravenous midazolam infusion for Centruroides exilicauda scorpion envenomation, Ann<br />
Emerg Med 34:5; 620-625.<br />
o scorpion<br />
o envenomation<br />
o neurotoxicity<br />
o natural history<br />
339
Poster 5: A new venomous scorpion responsible of human deaths in Argentine:<br />
Tityus confluens.<br />
Adolfo R. de Roodt 1 ; Néstor Lago 2 ; Judith Estevez 3 ; Estela Neder 4 ; Alicia Montero 4 ; Oscar D. Salomón 1 ;<br />
Jorge F. Paniagua 5 ; Raúl López 6 ; Valeria Vega 7 .<br />
1 Administración Nacional de Laboratórios e Institutos de Salud “Dr. Carlos G. Malbrán”, Ministerio de<br />
Salud y Ambiente. Av. Velez Sarsfield 563, CP 1281, Bs.As., Argentina.<br />
2 GEMA-Biotech, Bs.As., Argentina.<br />
3 InsitutoBioclon, México DF, Mexico.<br />
4 Universidad Nacional de Jujuy, Jujuy, Argentina.<br />
6 Ministerio de Salud de Catamarca, Catamarca, Argentina.<br />
7 Universidad Nacional de Tucumán, Tucumán, Argentina.<br />
In Argentine the principal scorpion responsible for severe envenomation in humans is T.<br />
trivittatus from the North of Patagonian region to the whole Northern of the country, with<br />
a lethality of 3‰. None of the other Buthiidae species have been confirmed as cause of<br />
death. In year 2003, in the province of Jujuy (in the North) two children died after scorpion<br />
bite. The search of scorpions in their homes gave as a result the finding of several<br />
specimens of T. confluens <strong>and</strong> some other scorpions but not T. trivittatus. In year 2005 <strong>and</strong><br />
2006, in the provinces of Catamarca <strong>and</strong> Tucumán in the Northwest of Argentina, three<br />
deaths of children were registered with the typical signs of the scorpion sting. The only<br />
scorpion found was T. confluens. We studied the toxicity of telson homogenates of T.<br />
confluens from the province of Jujuy <strong>and</strong> Catamarca, <strong>and</strong> found that the LD50, in NIH<br />
mice, was 143 µg [118-174 µg] for those from Jujuy <strong>and</strong> 380 µg [387-390 µg] for those<br />
from Catamarca). These values are in the order of that measured for T. trivittatus<br />
venomous gl<strong>and</strong>s homogenate, from regions were human deaths have been recorded<br />
recorded. The injection of the venom showed in all the animals generalized congestion <strong>and</strong><br />
hepatic lesions consistent in diffuse citoplasmatic vacuolization <strong>and</strong> sinusoidal congestion.<br />
Pancreatic damage was observed in 40% of the injected animals. Lungs showed congestion<br />
<strong>and</strong> foci of hemorrhage <strong>and</strong> mild edema. Most of the hearths (80%) showed myocardical<br />
eosinophylic injury of the fibers. The protein extracted from venomous gl<strong>and</strong>s <strong>and</strong> the<br />
electrophoretic profiles were in the order <strong>and</strong> similar to those observed for T. trivittatus<br />
from different regions of Argentina. The venomous gl<strong>and</strong> homogenates showed high<br />
reactivity with Anti-T. trivittatus (Anti-Escorpión from INPB, Argentine), anti-T.<br />
serrulatus (Antiaracnídico <strong>and</strong> Antiescorpión from Butantan Institute, Brasil) <strong>and</strong><br />
reactivity against Anti-Centruroides sp. (Alacramyn, Bioclon. Mexico) antivenoms. The<br />
sting of this scorpion must be considered of risk in the same degree of the stings of T.<br />
trivittatus or T. bahiensis.<br />
o Scorpion<br />
o Venom<br />
o Toxicity<br />
o Immunology<br />
340<br />
Poster 6: Toxicity of five scorpion venoms from the Old World, <strong>and</strong> its<br />
immunological reactivity with American scorpion antivenoms.<br />
Adolfo Rafael de Roodt 1 ; Néstor R. Lago 2 ; Judith Estevez 3 ; Eduardo Scarlatto 4 ; Rodrigo D. Laskowicz<br />
2 ; Jorge F. Paniagua 5 ; Hernán García 6 ; Victor M. Manzanelli 2 , Alej<strong>and</strong>ro Alagon 7 .<br />
1 Instituto Nacional de Producción de Biológicos – A.N.L.I.S. “Dr. Carlos G. Malbrán”, Min. de Salud y<br />
Ambiente, Av. Vélez Sarsfield 563, CP 1281, Bs.As., Argentina.<br />
2 GEMA-Biotech, Bs. As., Argentina.<br />
3 Instituto Bioclon, Mexico DF, Mexico.<br />
4 Servicio de Toxicología, Hospital “José de San Martín”, Fac. de Medicina U.B.A., Bs.As., Argentina.<br />
5 Laboratorios Silanes, Mexico DF, Mexico.<br />
6 Servicio de Patología, Hospital Italiano de Buenos Aires, Buenos Aires, Argentina.<br />
7 Instituto de Biotecnología de la Universidad Autónoma de México, Cuernavaca, Morelos, Mexico.<br />
The toxic activity of the venom from Buthus occitanus (Bo), Androctonus australis (Aa),<br />
Leiurus quinquestriatus (Lq), Parabuthus granulatus (Pg) <strong>and</strong> Parabuthus transvaalicus<br />
(Pt) was studied in NIH mice. The immunochemical reactivity against four antivenoms<br />
(AV) against T. trivittatus (Suero antiescorpión, INPB, Argentina, A), T. serrulatus (Soro<br />
antiaracnídico <strong>and</strong> Soro Antiescorpiónico, Butantan Institute, Brasil) <strong>and</strong> Centruroides sp.<br />
(Bioclon, Mexico, B) antivenoms was studied. The i.p. LD50s found were of 132 µg (Pt), 47<br />
µg (Pg), 14 µg (Bo), 8 µg (Aa) <strong>and</strong> 11µg (Lq). The Aa venom showed the highest potency<br />
(p < 0.05) closely followed by Bo <strong>and</strong> Lq venoms. The venom from South African scorpions<br />
showed the lower potency (p < 0.05), being that of Pt the one with the lowest lethal<br />
potency. The histopathological study of lungs showed congestion, edema <strong>and</strong> intraalveolar<br />
hemorrhage. The most important findings in hearths were eosinophilic infiltration of the<br />
muscle fibers <strong>and</strong> severe generalized injury. These findings were observed in all the animals<br />
studied (n=5 by venom). The immunochemical reactivity was poor as observed by double<br />
immunodifussion, but all antivenoms gave diffuse precipitation b<strong>and</strong>s with all venoms<br />
tested; the highest reactivity was observed with the venom of Lq. Neutralization<br />
experiments showed different degrees of protection conferred by the INPB <strong>and</strong> Alacramyn<br />
heterologous antivenoms (challenge dose 3 LD50 i.p.). The ED50s of the AVs against Bo<br />
venom were 184 µl <strong>and</strong> 214 µl for B <strong>and</strong> A, respectively, against Aa venom were 233 µl for<br />
B <strong>and</strong> > 250 µl for A, <strong>and</strong> against Lq venom were 440 µl for B <strong>and</strong> over 500 µl for A. The<br />
neutralization against Parabuthus venoms was poor for both antivenoms. The venom of P.<br />
transvaalicus was very poorly neutralized by anyone of the three antivenoms, although the<br />
neutralization by the South American antivenoms was slightly better (ED50 over 500µl).<br />
The venom of P. granulatus was neutralized with ED50s values of 110 μl <strong>and</strong> 165 µl for A<br />
<strong>and</strong> B, respectively. We conclude that the AVs from North or South America can neutralize<br />
at high doses the toxicity of venoms of scorpions from Africa.<br />
o Scorpion<br />
o Venom<br />
o Toxicity<br />
o Antivenom<br />
341
Poster 7: Neutralization of the haemorrhagic activity of Echis ocellatus (saw-scaled<br />
viper) venom <strong>and</strong> a venom metalloproteinase using synthetic peptide inhibitors <strong>and</strong><br />
chelators<br />
Howes, J-M, Theakston, R.D.G., Laing, G.D.*<br />
Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3<br />
5QA, UK. *gavin.laing@liv.ac.uk<br />
Envenoming by the West African saw-scaled viper, Echis ocellatus, resembles that<br />
of most vipers, in that it results in local blistering, necrosis <strong>and</strong> sometimes life-threatening<br />
systemic haemorrhage. While effective against systemic envenoming, current antivenoms<br />
have little or no effect against local tissue damage. The major mediators of local venom<br />
pathology are the zinc-dependant snake venom metalloproteinases (SVMPs). The<br />
extensive structural <strong>and</strong> functional similarity between SVMPs <strong>and</strong> the mammalian matrix<br />
metalloproteinases (MMPs) suggests that substrate/inhibitor interactions between these<br />
subfamilies are likely to be equally similar.<br />
In this study, four MMP inhibitors (Marimastat, AG-3340, CGS-270 23A <strong>and</strong> Bay-<br />
12 9566) <strong>and</strong> three metal ion chelators (EDTA, TPEN <strong>and</strong> BAPTA) were evaluated for<br />
their ability to inhibit the haemorrhagic activities of medically-important viperine venoms<br />
<strong>and</strong> isolated SVMPs. While both marimastat <strong>and</strong> CGS-270 23A inhibited local<br />
haemorrhage following injection of the isolated PIII SVMP EoVMP2, neither was<br />
effective against the haemorrhagic activity of whole E. ocellatus venom. AG-3340<br />
successfully inhibited the haemorrhagic activities of both EoVMP2 <strong>and</strong> E. ocellatus<br />
venom, but Bay-12 9566 was ineffective against haemorrhage induced by the whole<br />
venom. We suggest that an inhibitor mixture may be more effective against local venom<br />
pathology than individual compounds. The validation of this theory would be a logical<br />
progression of this study, <strong>and</strong> would ensure that a close structural counterpart for each<br />
SVMP is represented, proving a more effective broad-range treatment for local<br />
envenoming.<br />
o Echis<br />
o Snake venom metalloproteinase<br />
o Inhibitor<br />
o Haemorrhage<br />
342<br />
Poster 8: The potential advantages of camelid IgG for the development of<br />
antivenoms<br />
Harrison, R.A 1 *, Cook, D 1 , Hasson, S.S 2 , Harmsen, M 3 , Laing, G.D 1 , Conrath, K 4 , Theakston, R.D.G 1 .<br />
1<br />
Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool,<br />
UK<br />
*R.Harrison@liverpool.ac.uk<br />
2<br />
Medical Research centre, Faculty of Medical Sciences, University of Science <strong>and</strong> Technology, PO Box 13064, The<br />
Republic of Yemen.<br />
3<br />
Institute for Animal Science <strong>and</strong> Health of Wageningen University <strong>and</strong> Research Centre, 8219 PH lelystad, The<br />
Netherl<strong>and</strong>s.<br />
4<br />
Laboratory of cellular <strong>and</strong> Molecular Immunology, Dept of Molecular <strong>and</strong> Cellular Interactions, Vlaams<br />
Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, B-1050 Brussels, Belgium.<br />
Envenoming by snakes results in severe systemic <strong>and</strong> local pathology. Intravenous<br />
administration of antivenom, prepared from IgG of venom immunised horses or sheep, is<br />
the only effective treatment of systemic envenoming. Conventional antivenoms,<br />
formulated as intact IgG, papain-cleaved (Fab) or pepsin-cleaved F(ab / )2 fragments, are<br />
however ineffective against the local venom effects because of their inability to penetrate<br />
the blood/tissue barrier. We have embarked on a new research program to examine (i)<br />
whether the unusually small (15 kDa) antigen-binding fragment of camelid heavy chain<br />
IgG (VHH) can be exploited to neutralise the local effects of envenoming <strong>and</strong> (ii) whether a<br />
novel antivenom to treat both the systemic <strong>and</strong> local effects of envenoming can be<br />
formulated by combining anti-snake venom VHH <strong>and</strong> conventional F(ab / )2 <strong>and</strong> (iii) to<br />
determine whether the comparatively low immunogenicity <strong>and</strong> complement activation of<br />
camelid IgG [Herrera et al, 2005] indicates that camelid antivenom may have a lower risk<br />
of adverse effects than equine or ovine IgG antivenoms.<br />
The objectives of this preliminary study were to determine whether (i) camels (Camelus<br />
dromedarius) <strong>and</strong> llamas (Lama glama) immunised with venom raised antibodies of the<br />
appropriate titre <strong>and</strong> venom-neutralising efficacy <strong>and</strong> (ii) there were immunological or<br />
therapeutic reasons to select one camelid species over the other for future development of a<br />
camelid snake antivenom.<br />
We demonstrate that camels <strong>and</strong> llamas respond to immunisation with Echis ocellatus<br />
venom with high antibody titres <strong>and</strong> broad antigen specificity. These encouraging<br />
immunological results were matched by the successful elimination of venom-induced<br />
haemorrhage by IgG from the venom-immunised camels <strong>and</strong> llamas.<br />
Herrera, M., León, G., Segura, A., Meneses, F., Lomonte, B., Chippaux J.P., Gutiérrez, J-M. Factors<br />
associated with adverse reactions induced by caprylic acid-fractionated whole IgG preparations: comparison<br />
between horse, sheep <strong>and</strong> camel IgGs. Toxicon 46,(7):775-781.<br />
o Antivenom<br />
o Camelid<br />
o Echis ocellatus<br />
343
Poster 9: The Antifungal Activity of Punica Granatum L Peels powder <strong>and</strong> extracts.<br />
Siham S. Shaokat* Hamoudi A. Hameed 1<br />
ٍٍٍ<br />
*Prof. Of Pharmaceutical Microbiology/ Food & Drugs Sector/Nidal Street/Baghdad/Iraq/ email:<br />
albiatyss84@yahoo.com M: 07901867274<br />
1<br />
Prof. Of Chemistry. / Ministry of Industry & Minerals<br />
Chief Food & Drugs Sector/hamodiabas@yahoo.com/M:07901918147<br />
Introduction<br />
The aims of this study are to investigate anti-fungal properties of water <strong>and</strong><br />
ethanol, extracts & powder of Punica granatum L.Peels for treatment of several skin<br />
infections <strong>and</strong> inflammatory disorders .<br />
Method<br />
Sixty five patients (2-47)years old, suffered from, infected skin, fissures, rushes, boils,<br />
itching. Eight of them (12.24%) were infected with (Tinea pedis ) ( athlete foot), 6 patients<br />
(9.23%) suffered from erythematous, were infected with(Tinea cruris) ( jock itch) , 9<br />
patients (13.9%) were infected with (C<strong>and</strong>ida albicans), 2 patients(3.1%), with circular<br />
patches indicated (Tinea corporis ) ( ring worm) , 26 patients (40%) with fungus inside <strong>and</strong><br />
on surface of hair (Tinea capitis) &14 (21.53%) Cases showed infected nails thickened or<br />
crumbling (Tinea unguium).<br />
Results<br />
The anti-fungal activity of the extracts was evaluated by treating the cases above during<br />
two months by water hot extract <strong>and</strong> ethanol extract of the punica granatum peels as well<br />
as the dried powder on athlete foot <strong>and</strong> other infections.<br />
Conclusions<br />
These results suggest the Pomegranate Peels extract which contains gallotanic acid as a<br />
promising anti-fungal agent.<br />
References:<br />
-Robert A Neurath, Nathan S, Yun-Yao Li,<strong>and</strong> AsimK Debnath. Punica granatum juice provi<br />
V-1 entry inhibitor <strong>and</strong> c<strong>and</strong>idate topical microbicide. BMC Infectious diseases<br />
2004,4:41 do i: 10.1186/1471-2334<br />
o Antifungal agents<br />
o Plant extracts<br />
344<br />
Poster 10: Epidemiology <strong>and</strong> management of snakebites in Burkina Faso<br />
Sondo Blaise 1, 2 , Kou<strong>and</strong>a Séni 2 , Some Noya 2<br />
1. Faculté de médecine, Université de Ouagadougou<br />
2. IRSS, Ouagadougou, Burkina Faso<br />
Snakebites remain an important public health problem in Burkina Faso. For the past 15<br />
years, the average annual incidence of snakebite reported by the health care system of<br />
Burkina Faso was approx. 70 cases per 100,000 inhabitants, representing 0.25% of the<br />
total consultations in primary care medical facilities. The incidence increased, from 60<br />
cases per 100,000 inhabitants 15 years ago to 90 by 2005. A probable reason for this<br />
progression is that the use of public health care is increasing rather than that the incidence<br />
of snakebite is increasing. Hospital consultations following snakebites also increased<br />
during this period, from an average of less than 2,000 envenomations per year to more<br />
than 3,600. However, envenomations represented more than 5% of total hospitalizations<br />
in 1990 whereas they correspond to 2% in 2005. Hospital mortality rate resulting from<br />
envenomations still remains very high: 3% of total deaths occurring in hospitals but 8%<br />
of patients treated for snakebite.<br />
It is also known that the data from the health care system underestimate the incidence <strong>and</strong><br />
mortality resulting from snakebites by 60-80% because the majority of victims are treated<br />
first, <strong>and</strong> often exclusively, by traditional healers.<br />
The high cost of the antivenin currently available in Burkina Faso (nearly 100 US$) may<br />
explain this failure. It is predicted that an effective <strong>and</strong> cheaper antivenin would be better<br />
able to compete with the traditional treatments generally used, more as a result of their<br />
reduced cost than because of belief in their effectiveness.<br />
Epidemiology<br />
Snakebite<br />
Antivenin<br />
Burkina Faso<br />
345
Poster 11: UNCOMMON ENVENOMING PROVOKED BY ARTHROPODS:<br />
TOXINS FOR KNOWING<br />
Haddad Jr, V 1,2 .<br />
1<br />
Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, Caixa Postal 557, 18618-000, São Paulo<br />
State, Brazil, haddadjr@fmb.unesp.br<br />
2<br />
Vital Brazil Hospital, Butantan Institute, Avenida Vital Brazil, 1500, Butantan, São Paulo State, Brazil.<br />
INTRODUCTION/OBJECTIVES: The arthropods constitute the most numerous group of<br />
animals of the Nature. Several of them present toxins as mechanisms of attack <strong>and</strong><br />
defense, being able to cause injuries in human beings. There is clinical interest <strong>and</strong> an<br />
immense pharmacological potential in these toxins, practically unexplored. MATERIAL<br />
AND METHODS: The accidents had been selected in the casuistry of the author, in<br />
accordance with the rarity of the occurrence <strong>and</strong> the potential interest for toxinologists.<br />
RESULTS/DISCUSSION: the clinical aspects of envenoming for Millepede or Diplopoda<br />
are presented. These animals can eject irritating fluids that cause brown pigmentation <strong>and</strong><br />
occasionally severe inflammation <strong>and</strong> blisters in the skin. The stink bugs (Pentatomidae)<br />
have gl<strong>and</strong>s that produce a mixture of hidrocarbonates that function as a repellent of<br />
predators <strong>and</strong> paralysis in prey. In humans, the secretion causes inflammatory skin lesions.<br />
The effects of the secretion are described in the first observation of lesions caused by<br />
Pentatomidae in humans. Finally, an accident caused for a toc<strong>and</strong>ira ant is presented<br />
(Paraponera clavata). The toc<strong>and</strong>ira is a giant ant which sting provokes violent pain <strong>and</strong><br />
systemic manifestations in the victim. The intention of this presentation is to demonstrate<br />
uncommon injuries caused by venomous <strong>and</strong> poisonous arthropods <strong>and</strong> to offer new paths<br />
for interested on the involved toxins, poorly studied until today.<br />
1. HADDAD Jr, V, CARDOSO JLC; MORAES RHP. Skin lesions caused by stink bugs<br />
(Pentatomidae): first report of dermatological injuries in humans. Wild. <strong>and</strong> Env. Med.<br />
13(1): 48-50, 2002. 2. HADDAD Jr V, CARDOSO JLC, MORAIS RHP. Description of an<br />
injury in a human caused by a false toc<strong>and</strong>ira (Dinoponera gigantea, PERTY, 1883) with a<br />
revision on folkloric, pharmacological <strong>and</strong> clinical aspects of the giant ants of he genera<br />
Paraponera e Dinoponera (sub-family Ponerinae). Rev. Instit. Med. Trop. de São Paulo,<br />
47 (4): 235-238, 2005.<br />
o venomous animals<br />
o arthopods<br />
o toxins<br />
o stink bugs<br />
346<br />
Poster 12: North <strong>and</strong> South American Loxosceles spiders: Development of a<br />
polyvalent<br />
antivenom with recombinant Sphingomyelinase D as antigens.<br />
Olvera, A 1* , Ramos-Cerrillo, B 1 , Paniagua-Solís, J 2 , Stock, R.P 1 <strong>and</strong> Alagón, A 1 .<br />
1<br />
Instituto de Biotecnología-UNAM. Av. Universidad 2001, Chamilpa, Cuernavaca, Mor. México.<br />
* aolvera@ibt.unam.mx<br />
2<br />
Laboratorios Silanes S.A. de C.V.831000, Mexico City, Mexico.<br />
The bite of Loxosceles spiders can induce dermonecrotic lesions <strong>and</strong> systemic syndromes<br />
such as kidney failure <strong>and</strong> hemolysis. Sphingomyelinase D (SMD) is the most abundant<br />
protein component of Loxosceles venom. It has a molecular mass of approximately 33<br />
kDa <strong>and</strong> catalyzes the cleavage of sphingomyelin, releasing 1-phosphoceramide <strong>and</strong><br />
choline.<br />
In recent studies it has been shown that a sphingomyelinase D activity is responsible for<br />
the pathological effects of the Loxosceles bite (Ramos-Cerrillo, et al.). The aim of the<br />
present study was to generate recombinant SMD antigens for the production of a<br />
neutralizing antivenom. Here we report the cloning of cDNA coding for SMD from L.<br />
reclusa, L. laeta (Perú) <strong>and</strong> L. boneti, into bacterial E. coli expression systems as well as<br />
optimization of expression conditions so as to obtain soluble <strong>and</strong> active recombinant<br />
enzymes. The recombinant mature SMDs, tagged with a histidine tail at the N- or Ctermini,<br />
were compared in terms of toxicity <strong>and</strong> enzymatic activity, <strong>and</strong> were used as<br />
immunogens for the production of monovalent antisera in rabbits <strong>and</strong> F(ab’)2<br />
preparations in animals used for commercial antivenom production (horses). We<br />
performed studies on in vitro antigenic cross-reactivity of the recombinant enzymes <strong>and</strong><br />
inhibition of enzymatic activity by antibodies generated against the tagged proteins. We<br />
also present <strong>and</strong> discuss the results of studies on the specific <strong>and</strong> para-specific in vivo<br />
protective potential of the rabbit <strong>and</strong> equine antibody preparations against the<br />
recombinant proteins themselves <strong>and</strong> natural venom preparations. Our conclusions<br />
support the feasibility of using recombinant SMDs for production <strong>and</strong> evaluation of<br />
polyvalent anti-Loxosceles antivenoms, <strong>and</strong> we offer data on the potential of paraspecific<br />
neutralization in the context of the antigenic groupings <strong>and</strong> the molecular phylogeny of<br />
those active SMDs for which amino acid sequence information is available.<br />
Ramos-Cerrillo B., et al. 2004. Toxicon (44) 507-514.<br />
o Loxosceles<br />
o Sphingomyelinase D<br />
o Recombinant antigens<br />
o Polyvalent antivenom<br />
347
Poster 13: EPIDEMIOLOGY OF SNAKEBITES BASED ON HOSPITAL<br />
SURVEY IN CHITWAN AND NAWALPARASI DISTRICTS, NEPAL.<br />
P<strong>and</strong>ey, Deb Prasad; Part-time Lecturer, Birendra Multiple Campus, Tribhuvan<br />
University, Bharatpur, Chitwan; President, Association for Nature Conservation <strong>and</strong><br />
Social Upliftment, Nepal<br />
OBJECTIVE: To characterize <strong>and</strong> compare the epidemiology of snakebite in Chitwan<br />
<strong>and</strong> Nawalparasi Districts. METHODS: Two hospitals, one from each district, selected<br />
deliberately were visited for 7 days/month commencing from 15 Sept. 2004 to 15 Sept.<br />
2005 <strong>and</strong> abstracted information using predesigned <strong>and</strong> pretested data collection sheet<br />
from the hospital record file. RESULTS: Total snakebite cases recorded from 17 months<br />
hospital-record files were 860, of which, 23.72% were recorded from Kali G<strong>and</strong>aki<br />
Hospital, Nawalparasi <strong>and</strong> 76.28% from Bharatpur Hospital, Chitwan; 59.07% were from<br />
Nawalparasi <strong>and</strong> 40.93% were from Chitwan. Of the total cases recorded during 15 April<br />
2004 to 15 April 2005(12 months), the maximum cases (33.78%) were found in 15 June<br />
to 15 July (Ashad) <strong>and</strong> null in the next four months starting from 15 Nov. 2004 to 15<br />
March 2005; the maximum (68.89%) were in summer season with no cases in winter. Of<br />
the totality, 49.30% patients were males <strong>and</strong> 50.70% were females; the maximum (i.e.<br />
22.56%) were between 20 – 30 yrs.; the maximum (39.07%) snakebite occurred in<br />
Bramin caste; 104 (12.093 %) were venomous cases with 25% hospital case fatality rate.<br />
Of the total fatality, 26.92% was recorded in the age group of 0-10 yrs. The fatality rate<br />
was found to be gradually less in the older people. 31.84 anti-snake venom vials per<br />
patient were used. DISCUSSION: Seasonal <strong>and</strong> monthly incidence of sankebite<br />
demonstrated a distinct pattern closely related to rainfall, temperature <strong>and</strong> abundance of<br />
the foods of snakes which was comparable to the findings of H<strong>and</strong>sdak et al. 1998,<br />
P<strong>and</strong>ey 2005. The sex-, age- & Caste-wise incidence of snakebite victims throw light on<br />
the vulnerable section of the population which was similar to the findings of P<strong>and</strong>ey,<br />
2005. CONCLUSION: The largest numbers of snakebite cases were recorded from<br />
Nawalparasi. Maximum snakebite cases in Bharatur Hospital reflect the facility of antisnake<br />
venom vials in free of cost <strong>and</strong> intensive care of patients. The maximum snakebites<br />
in Ashad <strong>and</strong> in summer season reflect optimum environment to snakes in terai, greater<br />
agricultural <strong>and</strong> other outdoor <strong>and</strong> indoor activities of people <strong>and</strong> no snakebite cases in<br />
winter reflect hibernation of snakes in winter. Children below the 10 yrs were in great<br />
risk of death because of greater amount of toxin/kg body weight.<br />
Key words- snakebite, toxin, epidemiology, fatality, incidence,<br />
348<br />
Poster 14: Analysis of clinical outcomes <strong>and</strong> cost-benefits associated with the use<br />
of CSL Snake Venom Detection Kits in the management of snakebite at Port<br />
Moresby General Hospital, Papua New Guinea<br />
Williams D.J, 1* Jensen S.D, 1,2 Winkel K.D. 1<br />
1<br />
Australian Venom Research Unit, University of Melbourne, Parkville, Vic, 3010. Australia<br />
2<br />
School of Medicine & Health Sciences, University of Papua New Guinea, Boroko, Papua New Guinea<br />
* d.williams4@pgrad.unimelb.edu.au<br />
Introduction: A commercial enzyme immunoassay (CSL Snake Venom Detection Kit) has<br />
been in common use in Australia for over 25 years, but has not been adopted in nearby<br />
Papua New Guinea, despite the two countries sharing several species with the same venom<br />
immunotypes. Ahead of the introduction of CSL SVDKs in PNG, a cost-benefit analysis<br />
was conducted to assess potential clinical <strong>and</strong> financial benefits.<br />
Methods: Patients recruited to a prospective study of snakebite at Port Moresby General<br />
Hospital who had positive signs of envenoming had either bite site swabs or urine samples<br />
taken for testing using the CSL SVDK as per manufacturer instructions. All patients in<br />
which a venom immunotype was detected were promptly treated with a single vial of<br />
appropriate antivenom after premedication with subcutaneous adrenaline (0.25 mg 1:1000<br />
adults; 0.05 mg/kg 1:10000 children). The subsequent outcomes were monitored <strong>and</strong> costs<br />
of hospitalisation calculated from internal hospital budget documents. Clinical outcomes<br />
<strong>and</strong> costs were compared to a representative cohort of patients treated previously without<br />
CSL SVDK testing.<br />
Results: 37 envenomed patients were tested. 33 patients positive for the taipan venom<br />
immunotype received monovalent taipan antivenom; 1 patient positive for brown snake<br />
immunotype received polyvalent antivenom; <strong>and</strong> 2 of 3 patients negative for any of the 5<br />
test immunotypes also received polyvalent antivenom, the third patient was managed with<br />
anticholinesterase therapy. 18 patients were kept in the Emergency Dept for 24 hours after<br />
antivenom, <strong>and</strong> discharged home. 10 required intubation <strong>and</strong> assisted ventilation in ICU,<br />
<strong>and</strong> 9 patients were admitted to the High Dependency Unit for further observation. 2<br />
patients died. Detailed analysis of antivenom <strong>and</strong> hospitalisation cost benefits is presented.<br />
Discussion: Compared to recent historical outcomes, the use of CSL SVDKs significantly<br />
improved clinical outcomes, <strong>and</strong> reduced treatment costs. Phased introduction of broader<br />
use of these diagnostic tools has now been recommended to Health administrators.<br />
o CSL Snake Venom Detection Kit<br />
o Envenomation<br />
o Health economics<br />
o Clinical management<br />
349
Poster 15: Epidemiology of snakebite in Guinea<br />
Baldé M. C., Barry A. O., Tamboura B. A.<br />
Institut Pasteur de Guinée, B.P. 146, Kindia, Guinée<br />
The Institut Pasteur de Guinée carried out epidemiological studies for several years based<br />
on data available at medical facilities <strong>and</strong> on household surveys. Comparing these two<br />
sources of information, the incidence of snakebite, mortality, the use of medical facilities,<br />
hospital mortality <strong>and</strong> the incidence of bites by the various venomous species in the<br />
country are evaluated.<br />
3,047 households (23,442 inhabitants) were investigated in the area of Kindia (Low<br />
Guinea, forest-savanna limits). Surveys in health centers serving this population were<br />
used to quantify annual incidence of snakebite. An annual rate of 375 bites, of which 19.2<br />
were fatal, per 100,000 inhabitants was observed. However, it was observed that 80% of<br />
the surveyed victims used only traditional medicine.<br />
A survey of 990 households (12,850 people) in the area of Kankan (Northern Guinea,<br />
savanna) showed that the annual incidence of bites was 1,758 per 100,000 inhabitants<br />
with an annual mortality rate of 108 per 100,000 inhabitants. These dramatic results need<br />
to be confirmed. 88% of the victims initially consulted a traditional healer, <strong>and</strong> only 20%<br />
of them went to the hospital after treatment by the traditional healer failed.<br />
Notifications by health centres showed high mortality rate in hospitals ranging from 7 to<br />
22% (average approx. 15% over ten years). This high mortality rate resulted from the late<br />
arrival of patients at the hospital, often with severe complications. The average time of<br />
arrival was more than 150 hours after snakebite. Other possible reasons are the lack or<br />
scarcity of antivenins resulting in insufficient treatment. Finally, the frequency of bites by<br />
the various venomous species (15% by Bitis, 21% by Naja, 12% by Dendroaspis) may<br />
also explain this high rate of mortality.<br />
Epidemiology<br />
Snakebite<br />
Mortality<br />
Guinea<br />
350<br />
Poster 16: RENAL AND BLOOD VESSEL ALTERATIONS INDUCED BY<br />
GAMMA TOXIN OF Tityus serrulatus VENOM.<br />
Monteiro, H.S.A. 1 *; Alves, R.S. 1 ; Martins, R.D. 1 ; Amora, D.N. 1 ; Barbosa, P.S.F. 1 ;<br />
Sousa, D.F. 1 ; Alves, C.D. 1 ; Toyama, M.H. 2 ; Menezes, D.B. 3 ; Martins, A.M.C. 4 ; Fonteles,<br />
M.C. 1 ; Monteiro, F. C. D. 1<br />
1. Departamento de Fisiologia e Farmacologia, Coronel Nunes de Melo street, 1127/ Universidade<br />
Federal do Ceará/ Brazil *serrazul@truenet-ce.com.br<br />
2. Departamento de Bioquímica, Praça Infante Dom Henrique s/nº -São Vicente/SP; Universidade<br />
Estadual Paulista Júlio de Mesquita Filho/ Brazil<br />
3. Departamento de Patologia e Medicina Legal, Coronel Nunes de Melo street, 1127/ Universidade<br />
Federal do Ceará/ Brazil<br />
4. Departamento de Análises Clínicas e Toxicológicas, Capitão Francisco Pedro street, 1210/<br />
Universidade Federal do Ceará/ Brazil<br />
About 8000 cases of scorpion envenomation are reported yearly in Brazil according to<br />
the Ministry of Health. Most cases are due to the sting of Tityus serrulatus, the most<br />
dangerous scorpion in Brazil 1 . The aim was to test the effects of gamma toxin (gT) of T.<br />
serrulatus venom in perfused isolated rat kidney <strong>and</strong> mesenteric blood vessels. Wistar<br />
rats kidneys were perfused with Krebs-Henseleit solution containing 6g% bovine<br />
albumin 2 . The effects of gT at 3µg/mL (n=6) concentration were studied on perfusion<br />
pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration<br />
rate (GFR), sodium (%TNa), potassium (%TK) <strong>and</strong> chloride (%TCl) tubular transport.<br />
Mesenteric bed 3 was perfused with Krebs solution <strong>and</strong> effects of gT (3µg/mL/min; n=6)<br />
were examined <strong>and</strong> compared to the infusion of the perfuse solution. Effects of gT were<br />
compared to external control (C). Data were analyzed by Student’s t-test (*p
Poster 17 : Biological <strong>and</strong> Clinical Evaluations of Antivenom Immunotherapy<br />
Bon, C. 1* , Audebert, F. 1 , Calderon, E. 2 , Choumet, V. 1 , ElAyeb, M. 3 , Krifi, M. 3 , Possani, L. 2 ,<br />
Rivière, G. 1 , Sorkine, M. 1<br />
1 Unité des Venins, Institut Pasteur, Paris, France, cbon@mnhn.fr ; 2 Laboratoire des Venins et Toxines,<br />
Institut Pasteur, Tunis, Tunisie ; 3 UNAM Instituto de Biotecnologia, Cuernavaca, Mexico<br />
Antivenom immunotherapy is the unique specific treatment of envenomations.<br />
Although widely used <strong>and</strong> medically accepted, it is still empirically administered. Its<br />
improvement requires accurate criteria to assess when this treatment has to be used or not.<br />
It also requires accurate guidelines to improve the efficacy <strong>and</strong> the safety of this treatment.<br />
ELISA tests were set up to follow the venom toxins in patients’ blood during<br />
envenomations <strong>and</strong> pharmacokinetics were followed in experimentally envenomed rabbits,<br />
in the absence of, or after antivenom therapy. In a first step, ELISAs were used in parallel<br />
with clinical grading of viper <strong>and</strong> scorpion envenomations. In both cases, a good<br />
correlation was observed between the venom levels in the blood <strong>and</strong> the clinical<br />
symptoms. In a second step, we examined the venom kinetics, in the absence of, <strong>and</strong> after<br />
antivenom administration. Investigations were carried out in the case of envenomations by<br />
viper (V. aspis) in France <strong>and</strong> by scorpion (A. a. garzonii) in Tunisia <strong>and</strong> (C. l. limpidus) in<br />
Mexico. After intramuscular injection of viper venom, the resorption of the venom follows<br />
a complex process: it is fast during the first 24 hr then it occurs at a slower rate over the<br />
subsequent 72 hr, resulting in a long half-life of elimination (36 hr). On the other h<strong>and</strong>, the<br />
absorption of scorpion toxins is very fast <strong>and</strong> complete <strong>and</strong> its half-life of elimination is<br />
short (2 hr). The effect of antivenom therapy was further tested. It appeared that the<br />
detoxification process could be explained by a redistribution of the venom from the<br />
extravascular compartment to the vascular one, where it is sequestered by the antibodies.<br />
Intravenous injection was shown to be the most effective route for antivenom<br />
administration. The relative efficacy of Fab'2 <strong>and</strong> Fab was tested also <strong>and</strong>, in case of viper<br />
envenomations, it was shown that Fab'2 are more efficient than Fab because of more<br />
appropriate pharmacokinetic parameters.<br />
These experimental studies provide an experimental model to optimize the antivenom<br />
therapy. However, questions remain to be solved to improve further this treatment:<br />
pharmacodynamic of toxins, their local versus systemic actions, their elimination route.<br />
o Envenomation<br />
o Antivenom<br />
o Toxicokinetics<br />
o Venom neutralization<br />
352<br />
Poster 18: Amelioration of the cardiovascular effects of the yellow scorpion<br />
Leiurus quinquestriatus envenomation in rats by the red grape seeds<br />
proanthocyanidins<br />
El-Alfy, A 1 ., Ahmed, A 1 ., Kader, F 1 ., Fatani, A 1 .<br />
1<br />
Pharmacology Department, Faculty of Pharmacy, King Saud University, P.O. Box 22452, Riyadh 11495,<br />
Saudi Arabi (amfatani@yahoo.com).<br />
Introduction: Cardiovascular effects induced by Leiurus quinsuestriatus (LQQ) venom<br />
plays a key role in the morbidity <strong>and</strong> lethality of scorpion envenoming. It has been<br />
recently suggested that free radical generation may contribute to the damaging effect of<br />
scorpion venoms on the myocardium. Hence the objectives of this study were to determine<br />
the involvement of oxidative stress in LQQ scorpion envenomation <strong>and</strong> the possible<br />
protective actions of the potent antioxidants red grape seeds proanthocyanidins (GSP).<br />
Methods: Lethality studies were conducted in mice. Animals were treated with 100 or 200<br />
mgkg -1 day -1 GSP (p.o.) for 10 days prior to injection of 250 or 350 ugkg -1 scorpion venom<br />
(s.c.). Blood pressure, via the carotid artery following tracheostomy was measured in<br />
anaesthetized rats. Animals were treated with GSP (100 or 200 mgkg -1 day -1 p.o.) or saline<br />
for 10 days followed by venom injection (350 μgkg -1 , s.c.) <strong>and</strong> blood pressure (MABP),<br />
heart rate (HR), <strong>and</strong> ECG changes were recorded periodically up to death.<br />
Results: Results revealed significant protection of GSP against LQQ venom lethality.<br />
Additionally, pretreatment with GSP significantly (p
Poster 19: Can the antioxidant, ginkgo biloba extract, <strong>and</strong> the protease inhibitor,<br />
aprotinin, protect rats against Leiurus quinquestriatus scorpion venom-induced<br />
cardio-vascular effects?<br />
Fatani, AJ, Yaqub, HI, Ibrahim, M, Al-Zuhair, H, Qadir, F, Al-Qutub, M.<br />
Dept of Pharmacology, College of Pharmacy, King Saud University, P.O Box 616, Riyadh, 11421, Saudi<br />
Arabia, amfatani@ksu.edu.sa<br />
Introduction: Scorpion toxins act on ionic channels causing dysfunction in several systems,<br />
with death attributed to cardiovascular (CV) damage 1 . Oxidative stress <strong>and</strong> proteases are<br />
implicated in several diseases, including those of the CV system 2,3 . This study was<br />
undertaken to assess if antioxidants <strong>and</strong> protease inhibitors could protect animals against<br />
the detrimental cardiovascular manifestations caused by the venom of a common scorpion<br />
Leiurus quinquestriatus (LQQ) <strong>and</strong> if they would prolong survival.<br />
Methods: Male Swiss albino mice were injected with LQQ venom (250 μg kg -1 , s.c n=10)<br />
alone or after Gin (150 mg kg -1 , p.o, 3 weeks before venom) <strong>and</strong>/or Apr (46000 KIU kg -1 ,<br />
i.p, 30 min before venom). Control groups were injected with saline or treatments alone<br />
Percent surviving after 24 hr plus time of death were recorded <strong>and</strong> Covariance Wilcoxon<br />
Survival analysis utilized. In addition, blood pressure (BP), heart rate (HR) <strong>and</strong> lung<br />
edema index were measured in anaesthetized male wistar rats (n=5) pretreated with Gin<br />
<strong>and</strong>/or Apr prior to LQQ venom injection (doses as above) <strong>and</strong> Kruskal Wallis nonparametric<br />
ANOVA was utilized for analysis.<br />
Results: Pretreatment with Apr showed greater efficacy than Gin in prolonging the survival<br />
(P
Poster 21: Snake Venom Metalloproteinases (SVMPs) binds to collagen <strong>and</strong> α2β1<br />
integrin by two different motifs<br />
Tanjoni,I. 1 , Butera, D. 1 , Della-Casa, M.S. 1 , Fern<strong>and</strong>es, I. 1 , Clissa, P.B. 1 , Evangelista, K.S. 2 , Eble, J. 2 ,<br />
Moura-da-Silva, A.M. 1*<br />
1 *<br />
Laboratório de Imunopatologia, Instituto Butantan, Brazil, anamoura@butantan.gov.br<br />
2 Department of Physiological Chemistry, University of Münster, Germany<br />
Several integrin inhibitors that interfere with platelet functions have been isolated from<br />
snake venoms. Jararhagin, a P-III snake venom metalloproteinase, from Bothrops jararaca<br />
venom presents disintegrin activity as a collagen receptor α2β1 antagonist. To study the<br />
structure/ function relationships of jararhagin, our group has developed seven monoclonal<br />
antibodies (MAJar 1-7) <strong>and</strong> mapped their respective epitopes. In this study, the<br />
neutralizing activity of MAJars of jararhagin potential in inhibiting collagen-induced<br />
platelet-aggregation was evaluated. Pre-incubation of jararhagin with MAJar 3, which<br />
recognized the C-terminal portion of the disintegrin domain, blocked its ability to inhibit<br />
collagen-induced platelet-aggregation. MAJars 1 <strong>and</strong> 3 efficiently neutralized jararhagin<br />
binding to collagen in solid phase assays. However, the binding of jararhagin to the α2β1<br />
integrin was not reduced by its pre-incubation with MAJars either in dotblots of<br />
mammalian transfected cells with α2 integrin subunit (α2k562), nor by solid phase assays<br />
using microtiter plates coated with recombinant soluble human α2β1 integrin, suggesting<br />
that the epitopes recognized by the MAJars are not involved in the binding of jararhagin to<br />
α2β1 integrin. These data indicate the existence of two independent motifs for jararhagin<br />
binding to collagen or α2β1 integrin. The collagen-binding motif, which is predicted to be<br />
located in the C-terminal portion of the disintegrin-like domain, is sufficient for the<br />
expression of jararhagin activity related to the inhibition of the platelet function.<br />
Supported by FAPESP <strong>and</strong> CNPq.<br />
snake venom<br />
metalloproteinase<br />
platelets<br />
disintegrin<br />
356<br />
Poster 22: Distribution <strong>and</strong> effects of ε-toxin-GFP in mouse brain <strong>and</strong> kidneys<br />
Juan Blasi 1* , Alex Soler 1 , Jonatan Dorca 1 , Josep Saura 2 , Josep Maria Tussell 2 , Maryse Gibert 3 , Michel<br />
Poppof 3 , Joan Serratosa 2 <strong>and</strong> Mireia Martín-Satué 1 .<br />
1 Departament de Patologia i Terapèutica Experimental. Universitat de Barcelona-IDIBELL. Feixa Llarga<br />
s/n, 08907 L’Hospitalet Spain. * blasi@ub.edu<br />
2 Departament de Farmacologia i Toxicologia. IIBB-CSIC, IDIBAPS. Rosselló, 161, 6ª - 08036 Barcelona<br />
3 CNR Anaérobies, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France.<br />
ε- toxin, from Clostridium perfringens (B <strong>and</strong> D strains), is a protein of about 32 kDa that<br />
causes a very severe <strong>and</strong> often fatal form of enterotoxaemia in sheep, goats, <strong>and</strong><br />
occasionally calves <strong>and</strong> other animals. In mice, intravenous injection of the toxin produces<br />
its accumulation in several defined organs, but mainly in kidneys <strong>and</strong> central nervous<br />
system. This accumulation correlates with the toxic effect at cellular level, producing<br />
selective death of epithelial cells from the distal tubule of the nephron <strong>and</strong> cerebral edema<br />
with occasionally neuronal death. In this study ε-toxin-GFP was intravenous injected into<br />
mice in order to monitor the “in vivo” distribution of this protein in the brain <strong>and</strong> kidneys.<br />
The toxin was detected in kidney tubules, both in the apical or basolateral region of<br />
proximal <strong>and</strong> distal tubules respectively. When similar amounts of GFP were injected, only<br />
the apical region of proximal tubules was labeled suggesting that a non-specific binding<br />
previously found was due to the toxin accumulation in proximal renal tubules. After 5-10<br />
minutes of the i.v. injection, ε-toxin-GFP was found lining vascular endothelium of brain<br />
microvasculature. This binding was no visible at longer times after injection, suggesting<br />
that the toxin could cross the endothelia (<strong>and</strong> blood brain barrier) <strong>and</strong> gain access to the<br />
nervous tissue. Consequently, the possible effect of the toxin on glial cells <strong>and</strong> isolated<br />
nerve terminals (synaptosomes) was studied. The permeabilizing effect of the toxin on<br />
vascular endothelia was also studied by co-injection of Alexa688 coupled bovine serum<br />
albumin (Alexa688-BSA). All together, the results show the “in vivo” differential<br />
distribution of ε-toxin-GFP after i.v. injection <strong>and</strong> the use of the fusion protein (ε-toxin-<br />
GFP) as a valuable tool for the study of the intoxication pathway.<br />
Supported by a grant from MEC (BFU2005-02202) <strong>and</strong> ISCIII (PI040778 <strong>and</strong> PI050658).<br />
JD is a recipient of a predoctoral fellowship from IDIBELL.<br />
o Epsilon toxin<br />
o Clostridial toxins<br />
o enterotoxaemia<br />
o GFP fusion protein<br />
357
Poster 23: Evaluation of potential ricin inhibitors on pulmonary <strong>and</strong> digestive<br />
epithelial cells<br />
Emilie Gobbo 1 , Roman Lopez 2 , Goulven Merer 2 , Bernard Rousseau 2 , André Ménez 1 ,<br />
Bruno Beaumelle 3 , Daniel Gillet 1* <strong>and</strong> Julien Barbier 1<br />
1<br />
Département d’Ingénierie et d’Etudes des Protéines, *daniel.gillet@cea.fr<br />
2<br />
Service de Marquage Moléculaire et de Chimie Bioorganique, CEA-Saclay, 91191 Gif<br />
sur Yvette, France,<br />
3 UMR 5539 CNRS, Université Montpellier II, 34095 Montpellier, France<br />
We have studied the inhibitory power of eleven drug molecules against ricin using a<br />
cellular assay. These drugs were chosen because their use in humans is authorized for<br />
other diseases <strong>and</strong> data in the literature suggest possible anti-ricin activity for several of<br />
those. Dexamethasone <strong>and</strong> di-fluoromethylornithine were described for delaying slightly<br />
the death of intoxicated mice. Antiviral nucleotidic analogues (AZT, ddC) or pteroic acid<br />
were shown to inhibit toxicity on cells or to bind weakly to the catalytic site of the toxin<br />
in vitro with millimolar dissociation constants. Finally, other antiviral nucleotide<br />
analogues (d4T, 3TC, ddI, ribavirine) or drugs with related structures (methotrexate <strong>and</strong><br />
diazepam) were also tested.<br />
The assay was performed on pulmonary (A549) <strong>and</strong> digestive (Caco2) epithelial cells <strong>and</strong><br />
measured the inhibition of protein synthesis by incorporation of [ 3 H]-Leucine. Cells were<br />
intoxicated with varying concentrations of ricin in the presence of the highest possible<br />
concentration of inhibitors which was non toxic <strong>and</strong> soluble (from 3x10 -4 to 2x10 -3 M<br />
depending on the drug).<br />
No inhibition was observed with any of the drugs. Only millimolar concentrations of<br />
lactose (<strong>and</strong> to a lesser extent galactose), already known to inhibit binding of the toxin to<br />
cell surface glycoproteins shown some protective effect. However, these concentrations<br />
are incompatible with human treatment.<br />
- ricin<br />
- inhibitor<br />
- cellular assay<br />
358<br />
Poster 24: Antagonism of the cardiotoxic effect of Bothrops jararacussu by Suramin<br />
Sifuentes, N. D. 1 , El-Kik, C. Z. 2 , Ricardo, H.D. 2 ,Schwartz, E.N.F. 1 , Melo, P.A. 2<br />
1<br />
Laboratório de Toxinologia, Departamento de Ciências Fisiológicas, Universidade de Brasilia, Brasilia,<br />
DF, Brazil;<br />
2<br />
Departamento de Farmacologia Básica e Clínica, ICB, CCS, UFRJ. 21941-590 - Rio de Janeiro - RJ,<br />
Brazil pamelo@farmaco.ufrj.br.<br />
Bothrops snakes are responsible for a great number of accidents in Brazil within this<br />
genus, the species whose venom causes the greatest percentage of tissue necrosis following<br />
accidents is B. jararacussu. We have investigated many alternatives treatments for these<br />
accidents, <strong>and</strong> one frequently studied in our group is the effect of polyanion substances.<br />
Within these compounds, suramin have shown to be capable of inhibiting great part of<br />
Bothrops snake venoms effects. Until now the citotoxicity activity B. jararacussu venom<br />
on the heart has never been investigated. The objective of this work was to show, for the<br />
first time, the citotoxicity of B. jararacussu crude venom on mammal heart, <strong>and</strong> its<br />
inhibition by suramin. The in vitro Langendorff (modified) isolated rat heart preparation<br />
was used, perfused with a physiological saline solution plus B. jararacussu venom (2.5–10<br />
mcg/mL), pre-incubated or not with suramin( 1-100 micromolar) <strong>and</strong>/or antibothropic<br />
antivenom(2.0 microliters/ml), analyzing patterns of EKG waves, cardiac tension, CK<br />
release, histology <strong>and</strong> coronary pression; <strong>and</strong> in vivo anesthetized rats analyzing arterial<br />
blood pressure <strong>and</strong> EKG, with the same treatments. B. jararacussu venom(0.1- 1mcg/kg),<br />
reduced cardiac tension <strong>and</strong> arterial pressure leading to collapse <strong>and</strong> induced many<br />
alterations on EKG waves, proving to be cardiotoxic, <strong>and</strong> dose or concentration dependent.<br />
These activities probably occurred due to lesions on the cardiac cells, shown by histology<br />
<strong>and</strong> increase on the CK release level. When suramin was pre-incubated with the venom, it<br />
inhibited the cardiac collapse, in a dose or concentration dependent way. When associated<br />
with the antivenom, suramin totally inhibited the cardiotoxic activity, keeping parameters<br />
close to control. Thus, in this study it was shown that B. jararacussu venom is cardiotoxic,<br />
<strong>and</strong> that suramin was able to inhibit this activity dose dependently.<br />
Arruda et al. Braz J Med Biol Res. 2002 35:723-7266<br />
Murakami et al., 2005 J Mol Biol 350:416-426<br />
o Bothrops jararacussu venom<br />
o Cardiotoxicity<br />
o Suramin<br />
359
Poster 25: EFFECT OF CROTOXIN ON MICROCIRCULATION AND<br />
CIRCULATING LYMPHOCYTES<br />
Zambelli, VO 1 ; Sampaio, SC 1 , Britto, LRG 2 , Zychar,B 1 ; Gonçalves, LRC 1 ; Cury, Y 1 .<br />
1 2<br />
Lab. of Pathophisiology; Butantan Institute, SP; Dep. of Physiology <strong>and</strong> Biophysics Biomedical Sciences<br />
Institute- USP, SP.<br />
Crotoxin (CTX), the main neurotoxin component of the South American rattlesnake<br />
Crotalus durissus terrificus, is composed of a non-toxic <strong>and</strong> non- enzymatic polypeptide<br />
named crotapotin <strong>and</strong> of a weakly toxic fosfolipase A2 (PLA2). CTX <strong>and</strong> PLA2 reduce the<br />
number of lymphocytes in blood <strong>and</strong> lymph. The aim of this study was to investigate the<br />
effect of CTX <strong>and</strong> PLA2 on lymphoid tissue (spleen <strong>and</strong> mesenteric lymph node),<br />
evaluating the expression of T <strong>and</strong> B-lymphocytes, as well as the dynamics of cellular<br />
events occurring in microcirculation. Moreover, mesenteric lymph nodes <strong>and</strong> spleens of<br />
male Wistar rats were removed for analyses 2h after subcutaneous (s.c.) injection of<br />
CTX (18µg/animal), PLA2 (10.4µg/animal) or saline (300µL). Paraffin-embedded tissue<br />
sections were stained with hematoxylin-eosin for histopathological assessments. The<br />
expression of T <strong>and</strong> B lymphocyte was determined by immunohistochemistry.<br />
Microcirculation was analysed by intravital microscopy of the internal spermatic fascia.<br />
Rolling, adherence <strong>and</strong> migration phenomena in post-capillary venules were evaluated.<br />
CTX <strong>and</strong> PLA2 promoted, in mesenteric lymph nodes, follicular hyperplasia with<br />
increase of germinal center. Hyperplasia of spleen white pulp was also detected. In<br />
addiction, toxin treatment increases T <strong>and</strong> B lymphocyte immunostain. Microcirculation<br />
analyses showed that CTX increases, adhered leukocyte (12.2 CTX vs. 3.25 saline), <strong>and</strong><br />
their migration (11 CTX vs. 2.25 saline) at 1h. A decrease in the rolling (5.75 CTX vs.<br />
20.25 saline) <strong>and</strong> an increase in migration of lymphocyte (10.75 CTX vs. 1 saline) were<br />
observed 2h after treatment. These results indicate that toxins mobilize leukocytes to<br />
extravascular tissue, promote morphological alterations <strong>and</strong> an increase of lymphocytes<br />
in lymphatic tissue.<br />
Supported by: CAPES, CNPq<br />
360<br />
Poster 26: EFFECTS OF THE SEA ANEMONE Bunodosoma caissarum VENOM<br />
ON RENAL PERFUSION AND MESENTERIC BED VESSELS.<br />
Monteiro, H.S.A 1 *.; Martins, R. D. 1 ; Alves, R. S. 1 ; Amora, D. N. 1 ; Silva Neto, A.G. 1 ; Maia, D.G. 1 ;<br />
Toyama, M. H. 2 ; Martins, A M.C. 3 ; Barbosa, P.S.F. 1 ; Fonteles, M. C. 1 , Monteiro, F. C. D. 1<br />
5. Departamento de Fisiologia e Farmacologia, Coronel Nunes de Melo street, 1127/ Universidade<br />
Federal do Ceará/ Brazil *serrazul@truenet-ce.com.br<br />
6. Departamento de Bioquímica, Praça Infante Dom Henrique s/nº -São Vicente/SP; Universidade<br />
Estadual Paulista Júlio de Mesquita Filho/ Brazil<br />
7. Departamento de Análises Clínicas e Toxicológicas, Capitão Francisco Pedro street, 1210/<br />
Universidade Federal do Ceará/ Brazil<br />
Sea anemones contain a variety of interesting biological active compounds including<br />
some potent toxins. For this reason many investigators have focused attention on the<br />
biological activities of protein molecules from various sea anemones species, as<br />
Bunodosoma caissarum, which is endemic in Brazilian Southern coast 1 . The aim of the<br />
present work was to study the potential functional alterations produced by the<br />
Bunodosoma caissarum venom (BcV) on isolated rat kidney <strong>and</strong> its effects on vascular<br />
reactivity on isolated perfused rat arteriolar mesenteric bed. Isolated kidneys from Wistar<br />
rats weighing 250 to 300g were perfused with Krebs-Henseleit solution containing 6g%<br />
bovine serum albumin 2 . BcV (10μg/mL/min; n=6) was added to the system 30 min after<br />
the beginning of each experiment. Mesenteric bed 3 was perfused with Krebs solution<br />
kept warm at 37°C by a constant flow (4mL/min) <strong>and</strong> variation of perfusion pressure was<br />
measured throughout 80 min after the equilibration period. Data were analyzed by<br />
Student’s t-test with the level of significance of p
Poster 27: Tityus serrulatus Hypotensive Peptides: a novel class of hypotensive<br />
agents<br />
Verano-Braga, T. 1,2* , Silva, D.M.R. 4 , Santos, C.F.F. 4 , Bemquerer, M.P. 2,3 , Santos, R.A.S. 4 , Bougis,<br />
P.E. 5 , Martin-Eauclaire, M.F. 5 , De Lima, M.E. 1,2 <strong>and</strong> Pimenta, A.M.C. 1,2<br />
1 Núcleo de Biomoléculas, 2 Laboratório de Venenos e Toxinas Animais <strong>and</strong> 3 Laboratório de Enzimologia e Físico-<br />
Química de Proteínas, Depto. de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade<br />
Federal de Minas Gerais, Av.Antônio Carlos 6627, Belo Horizonte, Brazil, *thiagovb@icb.ufmg.br<br />
4 Laboratório de Hipertensão, Depto. de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade<br />
Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, Brazil.<br />
5 CNRS-FRE 2738, Ingénierie des Protéines, Université de la Méditerranée, UMR6560, IFR Jean Roche, 15, Bd<br />
Pierre Dramard, 13916 Marseille, France<br />
Facilities in using micro-scale analytical techniques, such as mass spectrometry <strong>and</strong><br />
proteomics, have led to a novel approach to prospect bioactive molecules in animal<br />
venoms. By this approach, we aimed to prospect new structural families of scorpion toxins<br />
<strong>and</strong> were able to find a new structural family of peptides in the venom of the Brazilian<br />
yellow scorpion Tityus serrulatus, named TsHptP (Tityus serrulatus Hypotensive<br />
Peptides(1)). Structurally, these are r<strong>and</strong>om-coiled linear peptides, ranging from 2,5 to 3,0<br />
kDa <strong>and</strong> have a similar bradykinin-potentiating peptide (BPP) amino acid signature.<br />
TsHpt-I (2,7 kDa), a member of this peptide family, was able to potentiate the hypotensive<br />
effects of bradykinin (BK) in normotensive rats. To optimize the pharmacokinetics <strong>and</strong> the<br />
stability of TsHpt-I, few synthetic analogs with lower mass ranges were constructed using<br />
peptide synthesis <strong>and</strong> the TsHpt-I as a template. These analogs held the BK-potentiating<br />
effect. A relevant hypotensive action independent on BK was observed to all analogs,<br />
indicating that they are themselves hypotensive agents. We used two hypertensive rats<br />
strains (SHR <strong>and</strong> TGR) to study this hypotensive effect <strong>and</strong> it has been shown that these<br />
analogs induces a strong <strong>and</strong> long-lasting hypotensive effect. To evaluate this action, we<br />
examined the vasorelaxation effect of aortic rings derived from male Wistar rats. The<br />
analogs were able to induce ± 20% of vasorelaxation (10 -7 M). One analog was orally<br />
administrated in SHR rats being able to reduce the blood pressure, indicating that it is<br />
stable <strong>and</strong> can be absorbed in the gastrointestinal tract. Although these peptides have a<br />
similar amino acid sequence with the classical BPPs (2), differences in the primary<br />
structure are crucial to the new pharmacological effects observed in these peptides.<br />
1. De Lima, M.E., Diniz, C.R., Santos, R.A.S., Bougis, P.E., Martin-Eauclaire, M.F.,<br />
Pimenta, A.M.C. Patent Application 2002 PI 0202157-9, 2003 PCT/BR03/00073,<br />
2006 USPTO 20060014928.<br />
2. Ianzer, D.; Konno, K.; Marques-Porto, R.; Portaro, F.C.V; Stöcklin, R.; Camargo,<br />
A.C.M.; Pimenta, D.C. (2004) Peptides, 25, 1085-92.<br />
o Bradykinin-Potentiating Peptide<br />
o Tityus serrulatus<br />
o Hypotensive Peptide<br />
o Hypertension<br />
362<br />
Poster 28: Snake Venom Components as Pharmacological Tools <strong>and</strong> Molecular<br />
Models in Thrombosis <strong>and</strong> Haemostasis<br />
Bon, C., Arocas V., Braud S., Francischetti I., Leduc M., Maroun R., Saliou B., Wisner A.,<br />
Zhang Y., Zingali R..<br />
Unité des Venins, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, France<br />
Numerous components in snake venoms affect haemostatic mechanisms. Several of<br />
them have been employed to develop diagnostic kits, while others are useful<br />
pharmacological tools <strong>and</strong>/or molecular models, in the development antithrombotic <strong>and</strong><br />
thrombolytic agents.<br />
We have shown that the C-type lectin convulxin, from C. d. terrificus venom, activates<br />
blood platelets by binding specifically with a high affinity to GPVI, the main collagen<br />
receptor. Hence convulxin, the two subunits of which have been cloned, sequenced <strong>and</strong><br />
expressed as a recombinant fusion protein with Bungarus venom acetylcholinestrase,<br />
appears to be a precious tool to study GPVI functions.<br />
The C-type lectin bothrojaracin, discovered in B. jararaca venom, is a specific <strong>and</strong><br />
potent thrombin inhibitor that interacts with thrombin exosites I <strong>and</strong> II but does not block<br />
its catalytic site. Having cloned <strong>and</strong> sequenced the cDNAs encoding the subunits of<br />
bothrojaracin, we used site-directed mutagenesis in a structure-function studies to design<br />
new antithrombotic agents.<br />
We identified a specific plasminogen activator, TSV-PA, in the venom of T. stejnegeri.<br />
At variance with t-PA, TSV-PA is insensitive to the physiological inhibitor PAI-1. We<br />
cloned the cDNA encoding TSV-PA <strong>and</strong> expressed it in E. coli. We also established its 3D<br />
structure by crystallography, showing that TSV-PA resembles the catalytic domain of t-<br />
PA. However, subtle differences have been proved, by site-directed mutagenesis, to play a<br />
critical role in the insensitivity of TSV-PA to physiological inhibitors.<br />
Strongly anticoagulant snake venom PLA2s selectively inhibit the prothrombinase<br />
complex. Their action is independent of their enzymatic activity <strong>and</strong> of phospholipids. We<br />
proved that the strongly anticoagulant PLA2s bind selectively with a high affinity to factor<br />
X, preventing its activation by factor Va. Interestingly, human PLA2 that is liberated by<br />
platelets during their activation behaves as the strongly anticoagulant PLA2s from snake<br />
venom, exerting a negative control to the pro-coagulant action of activated platelets.<br />
o Thrombosis & Haemostasis<br />
o Molecular biology<br />
o Expression & Site directed mutagenesis<br />
o Structure Function<br />
363
Poster 29: Venom Peptides from Calloselasma rhodostoma --- New Twist in<br />
Vasoactive Peptides?<br />
Higuchi, S 1,2* , Khow, O 3 , Suntrarachun, S 3 , Noiphrom, J 3 , Miyamoto, S 1 , Kawai, A 1 , Sitprija,V 3 ,<br />
Sato,T. 3<br />
1<br />
Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto 860-0082, Japan,<br />
*higuchis@ph.sojo-u.ac.jp<br />
2<br />
School of Pharmaceutical Sciences, Showa University, Tokyo 142-8555, Japan<br />
3<br />
Queen Saovabha Memorial Institute, The Thai Red Cross Society, Bangkok 10330, Thail<strong>and</strong><br />
Findings that crotaline snakes (e.g. B. jararaca) have genes coding for the precursor<br />
polypeptide bearing angiotensin converting enzyme inhibitors (ACEIs, also known as<br />
bradykinin potentiating peptides (BPPs)) <strong>and</strong> a C-type natriuretic peptide (CNP) led us to<br />
investigate the venom gl<strong>and</strong> cDNA library of C. rhodostoma. The precursor polypeptide<br />
comprised 333 amino acid residues <strong>and</strong> had the same structure as those from other species:<br />
at the N-terminus, t<strong>and</strong>em repeats of sequence for ACEI-like peptide <strong>and</strong> a C-type<br />
natriuretic peptide (CNP) sequence at the C-terminus. Some of ACEI-like peptide<br />
sequences had specific features of ACE inhibitor, i.e. glutamine residue in the amino<br />
terminus which is transformed into pyroglutamic acid (Pyr) in the mature form <strong>and</strong> Pro-Pro<br />
motif in the carboxyl terminus. The CNP sequence was highly similar to other venom<br />
CNPs, having a 17-membered ring structure through forming a disulfide bond.<br />
In order to know the peptides mentioned above are actually expressed <strong>and</strong> milked in the<br />
venom of C. rhodostoma, the low molecular weight components were collected with the<br />
use of Sephadex G100 gel chromatography <strong>and</strong> then applied onto a C18-reversed phase<br />
HPLC for further fractionation. Mass spectrometric analysis was conducted for each HPLC<br />
fractions. The ions at m/z 1281.7 <strong>and</strong> 1116.6 are assignable to ACE inhibitors with 12 <strong>and</strong><br />
10 amino acid residues in the precursor sequence, respectively. A distinct ion at m/z 596.3<br />
was subjected to MS/MS analysis <strong>and</strong> a pentapeptide with the sequence PyrGRPR<br />
emerged as a result. Intriguingly, this peptide appears to arise from the amino-terminal part<br />
common to four t<strong>and</strong>emly aligned ACEI-like sequences. On the contrary, any of the<br />
putative ACEI-like peptides were not detected in the mass spectrometric measurements.<br />
Although the function of this peptide is unknown, the C-terminal arginine <strong>and</strong> the<br />
penultimate proline suggests a similarity to blomhotin from A. blomhoffi (1) <strong>and</strong> a novel<br />
peptide found in C. d. collilineatus (2).<br />
1. Yanoshita, R. et al. (1999) Toxicon, 37, 1761-1770.<br />
2. Higuchi, S. et al. (2006) Comp. Biochem. Physiol. C, in press.<br />
o Calloselasma rhodostoma<br />
o ACE inhibitor<br />
o Mass spectrometric analysis<br />
o a pentapeptide PyrGRPR<br />
364<br />
Poster 30: Potent dromedary antibody fragments for using in immunotherapy<br />
against scorpion envenomation<br />
Bouhaouala-Zahar, B. 1 *, Conrath, K. 2 , Ben abderrazek R. 1 , Hmila I. 1 , Benlasfar Z. 1 , Hammadi M. 3 ,<br />
Khorchani T. 3 , Muyldermans S. 2 . & El Ayeb, M. 1<br />
1-Laboratoire LVT-Institut Pasteur de Tunis. 13 Place Pasteur BP74, 1002 Tunis-TUNISIA*<br />
balkiss.bouhaouala@pasteur.rns.tn<br />
2- Laboratory of Cellular <strong>and</strong> Molecular Immunology, Pleinlaan 2, 1050 Brussel, BELGIUM.<br />
3-Institut des regions arides de Medenine -TUNISIA<br />
The importance <strong>and</strong> great promise of specific antibodies against toxins is still<br />
understudied. The few studies that exist concern poly- <strong>and</strong> monoclonal antibodies,<br />
produced against these toxins as a means of specific treating envenoming <strong>and</strong> seem to<br />
favour this approach. They correspond to purified heterologous IgGs or F(ab)'2 fractions<br />
prepared from hyperimmune horse sera. However, such treatments have several<br />
limitations. Their preparation involving protease digestion of the Fc part of the antibody is<br />
tedious.<br />
Herein we report results of genetic engineering approach, leading to smaller recombinant<br />
antigen binding fragments, such as camel single-domain antibody fragment VHH, <strong>and</strong> their<br />
of significant relevance for the improvement of serotherapy applications.<br />
Thus, we previously demonstrated that the toxic fraction AahG50 elicited in dromedaries a<br />
high venom neutralising antibody response. Furthermore, the camel Heavy-Chain Ab<br />
subclasses recognize <strong>and</strong> neutralize efficiently the toxic peptides of the venom.<br />
Recently we cloned an ‘immune’ VHH library from the cDNA of the peripheral blood<br />
lymphocytes of a dromedary immunized with AahG50 toxic fraction of scorpion venom.<br />
From this immune VHH library we identified four individual clones that encoded a VHH<br />
which recognises specifically the AahII toxin. The affinity of the recombinant VHH to<br />
interact with the toxin was measured <strong>and</strong> in progress to be compared to mouse<br />
monoclonals <strong>and</strong> other classical antibodies. In addition, preliminary neutralising activity<br />
tests of these binders were initiated <strong>and</strong> were positively evaluated in relation to other antitoxin<br />
antibody fragments.<br />
Meddeb-Mouelhi et al., 2003 Toxicon, 42, 785-791.<br />
Bouhaouala-Zahar et al. (in progress to be published)<br />
This work was supported in part by an IFS grant<br />
o Scorpion toxin<br />
o VHH fragment<br />
o serotherapy<br />
o neutralizing activity<br />
365
Poster 31: Purification <strong>and</strong> characterization of ancistron–Bu, a novel fibrinogenclotting<br />
serine protease from Agkistrodon blomhoffii ussuriensis venom<br />
Karbovskiy V. L 1,2* , Savchuk A.N 2 , Volkov G.L. 2<br />
1 National Taras Shevchenko University of Kiev, Biotechnology department, 2 Glushkova Street, Kiev,<br />
Ukraine, *Vital-kiev@ukr.net<br />
2 Palladin Institute of Biochemistry NAS of Ukraine, FEBS, 9 Leontovicha Street, Kiev, Ukraine,<br />
A novel fibrinogen-clotting enzyme (named ancistron–Bu) has been homogeneity<br />
purified from the Agkistrodon blomhoffii ussuriensis venom using affinity chromatography<br />
on Blue Sepharose FF <strong>and</strong> Sephadex G 25 gel filtration. Ancistron–Bu, obtained by this<br />
way, proved as homogeneous <strong>and</strong> show molecular weight approximately 13,5 <strong>and</strong> 27 kDa<br />
in reducing <strong>and</strong> non-reducing conditions of sodium dodecyl sulphate polyacrylamide gel<br />
electrophoresis respectively. The protein purity was about 99%. Its yield was 8,4% of total<br />
protein <strong>and</strong> purity increasing was 20,5 fold. This enzyme showed specific fibrinogenclotting<br />
activity equivalent to 46,4 international thrombin units/mg. Unlike alphathrombin,<br />
ancistron–Bu split off fibrinopeptide A without releasing fibrinopeptide B from<br />
fibrinogen. The optimal pH range for the clotting activity of ancistron–Bu was 7,4-8,0. The<br />
hydrolytic activity of ancistron–Bu is strongly inhibited by diisopropyl fluorophosphate (it<br />
is obvious that this enzyme is serine protease), but heparin–antithrombin-III complex has a<br />
small effect on ancistron–Bu catalytic properties. This enzyme did not activate factor XIII<br />
<strong>and</strong> had not fibrinolytic <strong>and</strong> caseinolytic activities.<br />
Thrombin-specific substrate (D-Phe-Pip-Arg-p-NA) <strong>and</strong> protein C substrate<br />
(pyroGlu-Pro-Arg-p-NA) were most susceptible to hydrolysis by ancistron–Bu. In that time<br />
this enzyme did not showed any detectable amidolytic activity on the tested chromogenic<br />
substrates for plasmin <strong>and</strong> plasma factor Xa. Ancistron–Bu did not induce aggregation of<br />
washed normal platelets by itself, but caused plateletes aggregation in the presence of<br />
exogenous fibrinogen in a manner entirely different from that of thrombin.<br />
Ancistron time (like reptilase time) is a simple alternative to the thrombin time for<br />
rapid fibrinogen assay in samples containing heparin <strong>and</strong> is particularly useful in the assay<br />
of antithrombin-III where plasma can be prepared free of fibrinogen. The presence of fibrin<br />
degradation products, hypofibrinogenaemia <strong>and</strong> defects in fibrin polymerisation will<br />
prolong the ancistron time.<br />
o Snake venom<br />
o Thrombin-like enzymes<br />
o Serine protease<br />
366<br />
Poster 32: “Blomus-B” – a novel non-enzymatic platelet aggregation inhibitor<br />
purified from Agkistrodon blomhoffii ussuriensis venom.<br />
Karbovskiy V. L 1,2* , Savchuk A.N 2<br />
1 National Taras Shevchenko University of Kiev, Biotechnology department, 2 Glushkova Street, Kiev,<br />
Ukraine, *Vital-kiev@ukr.net<br />
2 Palladin Institute of Biochemistry NAS of Ukraine, FEBS, 9 Leontovicha Street, Kiev, Ukraine,<br />
Platelet aggregation is mediated by the interaction of membrane αIIbβ3 receptor with<br />
fibrinogen <strong>and</strong> can be inhibited by many factors. The largest <strong>and</strong> the most well studied<br />
group of thease ihibitors are desintegrins – non-enzymatic, smal molecular weight proteins,<br />
isolated from snake venoms, which specificly bound with αIIbβ3 integrin receptor.<br />
A novel platelet aggregation inhibitor (named “Blomus-B”) has been homogeneity<br />
purified from the Agkistrodon blomhoffii ussuriensis venom using affinity <strong>and</strong> ionexchange<br />
chromatography. “Blomus-B”, obtained by this way, proved as homogeneous<br />
single-chain protein with molecular weight, approximately 13,2 kDa estimated by SDS-<br />
PAGE <strong>and</strong> analytical size exclusion chromatography. The protein purity was about 99%.<br />
Its yield was nearly 4% of total venom proteins.<br />
This protein did not showe any detectable amidolytic activity on the tested<br />
chromogenic substrates for plasmin, thrombin <strong>and</strong> protein C <strong>and</strong> hadn’t fibrinolytic,<br />
caseinolytic <strong>and</strong> phospholipase activities. In that time “Blomus-B” strongly supressed<br />
platelet aggregation induced by ADP <strong>and</strong> adrenaline with IC50 230nM <strong>and</strong> 120nM<br />
respectively.<br />
Flow-cytometric analysis of washed platelets, preincubated with 250 nM “Blomus-<br />
B” for 10 min, showed the changes of platelets shape. This fact can be evidence of<br />
activation proceedings that originate after iteraction between platelet inhibitor <strong>and</strong> its<br />
receptor on cell membrane.<br />
Obtained resalts make possible to draw a conclusion that platelet aggregation<br />
inhibitor isolated from Agkistrodon blomhoffii ussuriensis venom is a novel low molecular<br />
weight desintegrin <strong>and</strong> it can be used as hightspecific <strong>and</strong> effective antiplatelet agent.<br />
o Snake venom<br />
o Platelets<br />
o Platelet aggregation inhibitor<br />
o Disintegrins<br />
367