biographies and abstracts - Fish Bites

Welcome! 

We were absolutely delighted that the IST awarded us the privilege 

of arranging and hosting the 15 th World Congress on Animal, Plant 

and Microbial Toxins, and we are very pleased to welcome you to 

Glasgow. 

We think that we have an exciting and fascinating programme 

covering all aspects of toxinology, and we have a great mix of 

invited speakers and other participants. We hope that you will enjoy 

the next few days, meeting old friends and making new ones, and 

re-discovering the fascinating insights that toxins can reveal to us. 

Our grateful thanks go to our premier sponsors and co-sponsors for 

the outstanding support they have given us. 

We have been very lucky to be helped in our organisational tasks 

by Angelfish. Together, we will try to make your participation in the 

Congress as enjoyable and rewarding as possible. 

Best wishes, 

Alan Harvey and Edward Rowan 

Congress Organising Committee 

Department of Physiology and Pharmacology 

University of Strathclyde 

27 Taylor Street 

Glasgow G4 0NR, UK 

Contents 

Scientific programme in outline page 3 

Social Programme page 5 

Scientific timetable day-by-day 

Abstracts 

Monday page 7 

Tuesday page 9 

Wednesday page 11 

Thursday page 12 

Friday page 15 

Plenary lectures 

Symposia 

W P Stemmer page 19 

J E Rothman page 21 

Redi Award page 23 

J W Daly page 25 

M J Browstein page 27 

Toxins and drug discovery page 29 

Lipids and toxins page 35 

From anecdotes to antidotes page 41 

Ion channel toxins page 53 

Toxins and hemostasis page 59 

Venomics page 70 

Oral communications 

Posters 

Monday page 88 



Friday page 146 

Monday page 170 



Friday page 331

Scientific programme in outline 

The programme is composed of plenary lectures, symposia, oral 

communications, and poster presentations. 

The plenary lectures and symposia will be held in the John Anderson 

Building, lecture theatre K3.25. 

The oral communications will be in three parallel sessions in lecture 

theatres K3.14, K3.17 and K3.25 in the John Anderson Building. 

The poster sessions and trade exhibition will be in the Colville Building 

(adjacent to the John Anderson Building), rooms 5.11 and 5.12. 

Conference Office: this will be opposite the entry to the main lecture theatre 

K3.25 in the John Anderson Building. Registration will be open from 8.00am on 

Monday 24 July, and the Conference Office will be open throughout each day of 

the Congress. 

3

Time Monday 24th Tuesday 25th Wednesday 26th Thursday 27th Friday 28th 

8.30-9.00 Registration and congress opening Registration Registration Registration Registration 

9.00-11.15 Toxins & Drug Discovery symposium Lipids & Toxins From Anecdotes to Ion channel symposium: “Venomics” symposium: 

symposium 

Antidotes 

Sponsored by the 

Sponsored by CEA 

symposium 

International Society for 

Neurochemistry and the 

Physiological Society 

Saclay 

11.15- 

11.45 

coffee coffee coffee coffee coffee 

11.45- Elsevier-Toxicon Plenary lecture- 

Plenary lecture- Redi Award lecture - Plenary lecture- John W. Plenary lecture- Michael 

12.45 Sponsored by Elsevier Life Sciences - James E. Rothman to be announced Daly “Alkaloids as probes Brownstein "What can 

Willem Pim Stemmer “The advantages of “Membrane fusion in 

for ionic channels” 

genomics teach you 

disulfide-rich microproteins as scaffolds 

for the construction of antibody-mimetic 

pharmaceuticals” 

the cell” 

about toxins?" 

12.45-1.45 Lunch Lunch Lunch Lunch Lunch 

1.45-2.45 Posters Posters Conference outing 

to Burrell Collection 

and Pollok Park 

(from 2.00 to 5.00) 

Posters Posters 

2.45-3.00 Posters and coffee Posters and coffee Posters and coffee Posters and coffee 

3.00-5.40 Free oral communications (3 sessions in Free oral 

Toxins and hemostasis Free oral 

parallel) 

communications (3 

symposium 

communications (3 

sessions in parallel) 

with parallel poster 

and oral sessions 

sessions in parallel) 

5.00-6.00 IST business meeting 

ISTH Exogenous 

Hemostatic Factors 

subgroup business meeting 

End of Congress 

Evening Civic reception at City Chambers An evening of play at Free Celebration dinner and 

event 

the Glasgow Science 

ceilidh, Bute Hall, Glasgow 

Centre 

University

Social Programme 

Sunday 23rd July 

Pre-Congress Registration & Welcome, Barony Hall - University of Strathclyde 

(corner of Castle Street and Rottenrow - a 2 min walk from campus accommodation) 

Pre-Congress Registration Opening - 4pm - 8pm 

Originally a church, the Barony Hall was the competition winning design of J.J. Burnet and JA 

Campbell in 1886. This red sandstone building replaced a Gothic church by John Robertson and James 

Adam (1793-1800) and now serves as the main ceremonial hall of the University of Strathclyde. 

The pre-congress registration will open at 4pm in the Barony Hall on Sunday 23rd July. This will allow 

you the opportunity to register your arrival and collect your Congress information. There will be a 

relaxed pre-congress welcome drinks reception with light refreshments also taking place in the Barony 

Hall which will offer you the perfect opportunity to meet up with new and old friends and the hosts of 

the IST 15th World Congress on Animal, Plant and Microbial Toxins. 

Monday 24th July, 7pm - 8.30pm 

Civic Welcome Reception - Glasgow City Chambers 

In the very heart of Glasgow (and a few minutes walk from the Congress location) stands one of the 

city’s most important and prestigious buildings - the City Chambers. 

A grand and imposing edifice overlooking George Square, the City Chambers is an impressive symbol 

of Glasgow’s political strength and historical wealth. Completed in 1888, the City Chambers has for 

over a hundred years been the headquarters of successive councils serving the City of Glasgow. 

The Lord Provost will welcome you to the city with a Civic Welcome Reception providing you with an 

opportunity to network with fellow delegates following the first day of the Congress whilst enjoying a 

relaxing drink in beautiful and elegant surroundings. 

Tuesday 25th July, 7.30pm - 9.30pm 

An Evening of Play, Glasgow Science Centre 

Glasgow Science Centre is Scotland’s flagship Millennium Project. Housed in three stunning buildings 

at Pacific Quay on the River Clyde, GSC brings science and technology to life through hundreds of 

interactive exhibits in the Science Mall and the unique experiences of the GSC IMAX® Theatre and 

the Glasgow Tower. For more information on the venue, see www.glasgowsciencecentre.org 

Enjoy a relaxed evening with your fellow delegates at Glasgow’s Science Centre. Here you can enjoy 

the drinks reception whilst amusing yourselves with the Science Centre's collection of attractions which 

we are sure will illuminate, intrigue, inspire and fire your imagination! 

Transport to and from the Science Centre will be provided. 

Wednesday 26th July, 2pm- 5pm 

Congress Social Afternoon, Burrell Collection 

The City of Glasgow owns one of the richest art collections in Europe, displayed in 13 museums across 

the city. The award winning Burrell Collection is set in Pollok Country Park and more than 8,000 art 

objects amassed in a lifetime by the Glasgow shipping magnate Sir William Burrell are in the museum 

(http://www.glasgowmuseums.com/venue/index). The light and airy building, its woodland setting and 

the amazing breadth of Sir William Burrell's collection make a visit to The Burrell Collection an 

experience not to be missed. The park and woodland also offer plenty of opportunity for enjoying a 

walk around the estate, and to see some prize Highland cattle. 

After the morning congress and lunch, you will be taken by coach to spend the afternoon at the Burrell 

Collection. Pollok House is also located within the grounds of Pollok Estate, but please note that entry 

to Pollock House is not included in your booking. If you wish to visit the House you are free to do so 

but there will be a charge payable on admission to the venue of approximately £8 per adult (free 

admission to the gardens, shop and restaurant). The house is a 10 minute walk from the Burrell and 

there is a shuttle service provided by the estate between the two venues. Pollok Estate has been the 

home of the Maxwell family since the mid-13th century. The current house is an impressive 18th 

century mansion, filled with wonderful collections of Spanish art, antique furniture, silverware and 

ceramics, and an impressive library. See http://www.glasgowmuseums.com/venue/index 

Transport to and from the Burrell Collection will be provided. A packed lunch will be available before 

departure from the Congress venue. 

Thursday 27th July, 7pm - midnight 

Congress Celebration Dinner & Ceilidh, Bute Hall - University of Glasgow 

The sheer scale of the University of Glasgow’s most famous venue is, quite simply, breathtaking. This 

grand Victorian hall will be the setting for a fun and relaxed Scottish Gala Dinner to mark the closing 

of the Congress and to celebrate the 40 years of IST Congresses. This is the perfect opportunity to 

experience some true Scottish hospitality whilst allowing us all to bid farewell to our old and new 

friends. You will be treated to a fabulous Scottish themed menu and will be entertained by a fun and 

energetic Ceilidh band who will have you up dancing the night away…so don’t forget your dancing 

shoes. 

Transport to and from the venue will be provided. 

6


Monday 24 July 

Symposium: Toxins and Drug Discovery (K3.25) 

9.00-9.45: George Miljanich (CEO, AIRMID LLC, California) 

PRIALT (ziconotide intrathecal infusion): a conopeptide for treating 

severe chronic pain 

9.45-10.30: Michael Hanley (Vice President of Discovery Research, 

Amylin Pharmaceuticals, California) 

Physiological fluids of venomous animals: novel resources for drug 

discovery 

10.30-11.15: George Chandy (University California Irvine) 

From marine toxins to therapy for autoimmune diseases 

The Elsevier-Toxicon Lecture (K3.25): 

11.45am-12.45pm 

Willem Pim Stemmer (CEO and Founder of Amunix, Inc, California) 

The advantages of disulfide-rich microproteins as scaffolds for the 

construction of antibody-mimetic pharmaceuticals 

Poster sessions (Colville 5.11/5.12) 

1.45-3.00 pm 

Nerve, muscle and myotoxicity 

Ion channels 

7

Monday 24th 

July 

Track 1: Acetylcholine Receptors 

Oral Presentations K3.14 

3.00-3.20 Servent, D. : MT7 muscarinic toxin as tool to study 

direct allosteric interactions on M1 muscarinic 

receptor 

3.20-3.40 Tsetlin, V.I.:. α-Conotoxins And Their Targets: 

From Photolabeling To The Three-Dimensional 

Structures 

3.40-4.00 Wildeboer, K.M.: Interaction of Erythrina and 

Phelline Alkaloids with the Neuronal α4β2 Nicotinic 

Acetylcholine Receptor (nAChR) 

4.00-4.20 Kem, W.R.: Binding of 3-(Benzylidene)- 

Anabaseines to Mammalian Nicotinic Acetylcholine 

Receptors and Molluscan ACh Binding Proteins 

4.20-4.40 Adams, D.J.: The Structural Characterisation and 

Receptor Specificity of α-Conotoxin Vc1.1 

4.40-5.00 Molgó, J.: Gymnodimine-A targets muscular and 

neuronal nicotinic acetylcholine receptors with high 

affinity 

5.00-5.20 Daly, N.L.: The unique cysteine spacing of BuIA 

destabilizes the globular fold commonly found in αconotoxins 

5.20-5.40 Marchot, P.: Structural analysis of AChBP 

complexes with nicotinic agonists and antagonists 

Track 2: Cardiovascular Themes and 

Phospholipases 

K3.17 

Gopalakrishnakone, P.: Designing Peptide Drugs 

from Python Serum: Dual Inhibitors of sPLA2 and 

MMP-1 as Therapeutic Option for Treatment of 

Inflammation 

Wen-guey Wu : Dynamin dependent and 

independent endocytic pathway of cobra 

cardiotoxins: role of distinct cholesterol and 

heparan sulfate domain for toxin selection 

Yamazaki, Y.: Snake venom VEGF-F is a most 

potent inducer of vascular permeability 

Tokunaga, Y.: C-terminal peptide of snake venom 

VEGF-F specifically blocks VEGF-A165 activity by 

binding to heparin-like molecules 

Flight, S.M.: A factor Xa-like enzyme from 

Pseudonaja textilis venom is a powerful 

procoagulant that reduces blood loss in a topical 

anti-bleeding model. 

Perchuc, A.M.: Heparin-neutralizing properties of 

two novel Lys49 PLA2 variants from the Bothrops 

moojeni venom. 

Faure, G. FXa-binding studies of the anticoagulant 

phospholipases A2 from Viperidae and Crotalidae 

venom 

Guarnieri, M.C. BE-I-PLA2, a novel acidic 

phospholipase A2 from Bothrops erythromelas 

venom: purification, cloning and characterization as 

potent anti-platelet and inductor of PGI release by 

endothelial cells 

8 

Track 3: Structural and Biochemical Aspects 

K3.25 

Winter, K.L. : Stability studies on the venom of the 

major Australian box jellyfish (Chironex fleckeri). 

Kintner A.: Variation in venom between two 

Australian box jellyfish: prey-specific adaptations 

Ward, R. J.: Structural Determinants of Lys49 

Phospholipase A2 Activity Probed by Scanning 

Alanine Mutagenesis 

Souza, G.H.M.F.: Screening of Bothrops snake 

venoms using bidimensional capillary liquid 

chromatography coupled to tandem nanoelectrospray 

quadrupole time-of-flight mass 

spectrometry: identification of novel peptides 

Gillet, D.: Role of histidines and the N-terminal 

helices in the function of the diphtheria toxin T 

domain 

Bieber, A.L.: Detection and Quantitation of 

Detrimental Levels of Staphylococcal Enterotoxin B 

by Mass Spectral Immunoassay 

Tulayakul, P.: Chemical modification of mycotoxin 

metabolism by green tea extract and coumarin in 

piglets 

Stöcklin, R.: Novel proteolytic enzymes from 

pathogenic fungi and their use for biotechnology 

and health


Tuesday 25 July 

Symposium on Lipids and Toxins (K3.25) 

9.00-9.45: Edward A. Dennis (Distinguished Professor, University of 

California San Diego) 

LIPID MAPS and eicosanoid lipidomics 

9.45-10.30: Cesare Montecucco (Professor of General Pathology, 

University of Padova, Italy) 

How snake and bacterial presynaptic enzyme toxins block nerve 

terminals 

10.30-11.15: Uroš Petrovic (Department of Biochemistry & Molecular 

Biology, Jozef Stefan Institute, Slovenia 

Genome-wide analysis of the molecular mechanism of action of 

ammodytoxin, a neurotoxic sPLA2, in yeast cells 

Plenary lecture (K3.25) 

11.45am-12.45pm 

James E. Rothman (Director of the Columbia Genome Center, Professor 

of Chemical Biology, Columbia University, New York) 

Membrane fusion in the cell 


1.45-3.00 pm 

Venomics, transcriptomics, cloning 

Toxin discovery, purification, biochemistry 

9

Tuesday 25th 

July 

Track 1: Clinical Envenoming 


3.00-3.20 Thomas, L.: Post mortem examination of a fatal 

case of Bothrops lanceolatus envenoming 

3.20-3.40 Kuch, U.: Kraits with 17 dorsal scale rows (Bungarus 

sindanus complex): unrecognised causes of severe 

neurotoxic envenoming in South Asia 

3.40-4.00 Faiz, M.A.: Severe neurotoxic and myotoxic 

envenoming by the Greater Black Krait (Bungarus 

niger) in Chittagong Division, Bangladesh 

4.00-4.20 Ariaratnam, A.: Distinctive epidemiology and clinical 

features of common krait (Bungarus caeruleus) bite 

in Sri Lanka 

4.20-4.40 Warrell, D.A.: First authenticated cases of lifethreatening 

envenoming by the hump-nosed pit viper 

(Hypnale hypnale) in India, and Severe systemic 

envenoming by hump-nosed vipers (Hypnale 

hypnale) in Sri Lanka 

4.40-5.00 Haddad Jr., V.: Injuries caused by venomous 

animals occurred in domestic and commercial 

Aquariums: A study of 24 cases 

5.00-5.40 IST business meeting – K3.25. All IST members are 

urged to attend 

Track 2: Model Test Systems 

K3.17 

Sevcik, C.: A dynamic interpretation of the 

pharmacokinetic volume of distribution and its 

relation to the pharmacokinetics of F(ab')2 and other 

drugs 

D´Suze, G.: Effect of Leukocyte Inhibitors 

Benzydamine and Cyclophosphamide on Lung Injury 

Caused by Tityus discrepans Scorpion Venom 

Ratanabanangkoon, K.: Pig as an experimental 

model for the study of local tissue necrosis caused 

by snake venoms 

Carlini, C.R.: Ureases display biological effects 

independent of enzymatic activity. Is there a 

connection to diseases caused by urease-producing 

bacteria? 

Arthur, P.K.: The Use Of Indigenous Knowledge in 

Rural Development: A Case Study In The Use Of 

Herbal Medicine In The Treatment of Diseases In 

The Northern Region Of Ghana 

IST business meeting –K3.25. All IST members are 

urged to attend 

10 

Track 3: Toxins, receptors and ion channels 

K3.25 

Nicholson, G.M.: Pharmacophore mapping of the katracotoxins: 

selective insect potassium channel 

blockers that reveal a novel insecticide target 

Cuypers, E. : Jellyfish and other Cnidarians cause 

pain by affecting TRPV1 channels 

Marí, F.: Novel Conotoxin Frameworks 

King, G,F. : A dual-target, self-synergizing peptide 

toxin from spider venom 

Petit, K.E. : Fulvol Acetate, a novel activator of 

transient Low-Voltage Activated (LVA) Ca 2+ current 

Kauferstein, S. : A novel family of conotoxins target 

to nACh-receptors 

IST business meeting – K3.25. All IST members are 

urged to attend


Wednesday 26 July 

Symposium: From anecdotes to antidotes (K3.25) 

9.00-9.30: José María Gutiérrez (Subdirector, Instituto Clodomiro Picado, 

Costa Rica) 

Novel possibilities for the treatment of local effects in snakebite 

envenomation 

9.30-10.00: Isaac Asuzu (Professor, Department of Veterinary Physiology 

and Pharmacology, University of Nigeria, Nsukka) 

Myths and realities of plant-based remedies for snake bite 

“An integrated approach to the development, optimization and 

evaluation of a polyvalent antivenom for Sub-Saharan Africa” 

10.00-10.20: Jean-Philippe Chippaux 

Mangement of snake bite in Africa: how to stop the vicious cycle? 

10.20-10.35: Alejandro Alagón 

Development of a polyvalent F(ab’) antivenom for Sub-Sahara African 

snakes 

10.35-10.45: Roberto P. Stock 

The spectrum of protection: serological cross-reactivity between 

venoms of African vipers and elapids 

10.45-100.05: Achille Massougbodji 

Clinical trial of a polyvalent antivenom specific for African snakes 

Redi Award lecture (K3.25) 

11.45am-12.45 

The winner of the Redi Award will be announced at the Congress and 

will present the Redi Award lecture 

11 


Thursday 27 July 

ISN Symposium: Ion channel toxins: tools to explore ion 

channel structure, function and physiology (K3.25) 

A symposium sponsored by The International Society for 

Neurochemistry, with support from The Physiological Society 

9.00-9.45: Brian Robertson (Professor of Neurobiology, University of 

Leeds) 

Potassium channel toxins - a trail from cortex to channel subtype 

9.45-10.30: Frederic Meunier (Laboratory of Molecular Dynamics of 

Synaptic Function, University of Queensland, Australia) 

Glycerotoxin, a new tool to dissect synaptic vesicle recycling 

10.30-11.15: Michael Gurevitz (Professor, Department of Plant Sciences, 

Tel Aviv University, Israel) 

Structural commonality versus functional specificity in scorpion toxins 

that affect voltage-gated sodium channels 


11.45am-12.45pm 

John W. Daly (NIH Scientist Emeritus, NIH, Maryland) 

Alkaloids as probes for ionic channels 


1.45-3.00 pm 

Marine toxins 

Pain and CNS 

Inflammation and cytokines 

Cytotoxicity and antimicrobial 

12

Symposium: Toxins and hemostasis (K3.25) 

Sponsored by Pentapharm, Latoxan and Venom Supplies 

3.00-3.30: Kenneth J. Clemetson (Professor of Biochemistry, Theodor 

Kocher Institute) 

Snake venom proteins affecting platelets 

3.30-4.00: Aura Kamiguti (University of Liverpool) 

Platelet interaction with multidomain soluble snake venom 

metalloproteinases 

4.00-4.30: Russolina Zingali (Associate Professor, Universidade Federal 

do Rio de Janeiro) 

Structural and pharmacological studies of bothrojaracin, a 

(pro)thrombin inhibitor 

4.30-5.00: Frank S Markland (Professor of Biochemistry and Molecular 

Biology, University of Southern California) 

Contortrostatin and its anti-cancer action 

5.00-5.30: Manjunatha Kini (Professor, National University of Singapore) 

Origin, evolution and recruitment of venom prothrombin activators in 

Australian elapids 

5.30-6.00: meeting of the ISTH SSC Registry of Exogenous Hemostatic 

Factors 

13

Thursday 

27th July 

Track 1: Toxins and Na 

Oral Presentations 

+ channels 

K3.14 

3.00-3.20 Ilan N.: Scorpion Beta-Toxins Interact with their 

Na-channel Receptor Via a Novel 'Ratchet 

Mechanism' 

3.20-3.40 Beress, L.: A new toxin from the sea anemone 

Condylactis gigantea with effect on sodium 

channel inactivation 

3.40-4.00 Gordon, D.: Allosteric interactions between 

scorpion toxin receptor sites on voltage-gated Na 

channels imply a novel role for weakly active 

components in arthropod venom 

4.00-4.20 Jalali, A.: OD1, α-like toxin isolated from the 

Iranian yellow scorpion Odonthobuthus doriae 

venom 

4.20-4.40 Cohen L.: Direct Evidence that Receptor Site-4 

of Sodium Channel Gating Modifiers is not 

Dipped in the Phospholipid Bilayer of Neuronal 

Membranes 

4.40-5.00 Norton, R.S.: Structure and alanine scan of a 

spider toxin that affects the activation of 

mammalian voltage-gated Na + channels 

5.00-5.20 Karbat I.: X-ray structure and point mutagenesis 

of the scorpion depressant toxin LqhIT2 reveals 

key determinants crucial for activity and antiinsect 

selectivity 

5.20-5.40 

Martin-Eauclaire, M.-F. : New β-type toxins 

from the Androctonus scorpion venoms? 

Track 2: Cytotoxins, and therapeutics 

K3.17 

Cahan, R.: Cyt2Ba of Bacillus thuringiensis 

subsp. israelensis: activation by putative 

endogenous protease 

Turk, T. : New type of cytolysin and an unusual 

phospholipase A2 like protein isolated from the 

Northern Pacific sea anemone Urticina 

crassicornis 

Vassilevski, A.A.: Latarcins – a group of spider 

venom cytolytic peptides with high structural 

diversity 

Anderluh, G.: Mechanism of sphingomyelin 

specificity of actinoporins, pore-forming toxins 

from sea anemones 

Mbugua, P. M.: In vitro evaluation of 

antibacterial activity of crude aqueous extract of 

Azadirachta indica (Neem) leaves 

Saha, A. : Non-protein/non-peptide compounds 

from cobra venom having therapeutic potential 

Gawade, S. P.: Pharmaco-photodynamics of 

snake venoms: Perspectives to access drugs for 

the future 

EL-Latif, S.A.A.: Yeast culture Saccharomyces 

cerevisiae as feed additive in Poultry 

14 

Please note the Toxins and Hemostasis 

symposium will also run in K3.25 at the same 

time as these other Oral Sessions


Friday 28 July 

Symposium: Venomics (K3.25) 

Sponsored by CEA 

9.00-9.10: André Ménez (DIEP, CEA Saclay, France, Head of Dept of 

Protein Study & Engineering of the Life Sciences Division at the French 

Atomic Energy Commission) 

Introducing the Venomics Project 

9.10-9.15: Reto Stöcklin (Owner and Head of Atheris Laboratories) 

Use of mass spectroscopy in venomics 

9.15-9.20: Dietrich Mebs 

Venom glands in old snakes 

9.20-9.40: Bryan Greig Fry (Deputy director of the Australian Venom 

Research Unit at the University of Melbourne) 

Evolution of the venom system in squamate reptiles 

9.40-10.00: Baldomero Olivera (Distinguished Prof of Biology at the 

University of Utah, Associate Prof of Biochemistry at the University of 

the Philippines) 

Exogenomics and post-translational modification of Conus peptides 

10.00-10.20: Motonori Ohno (Professor of Biochemistry at Sojo 

University) 

Venomics of protobothrops flavoviridis venom gland phospholipase A2 

isozymes 

10.20-10.40: Eugene Grishin (Professor and Deputy director of the 

Shemyakin-Ovchinnikov Institute of Bio-organic Chemistry at the 

Russian Academy of Sciences) 

Wide variety and multiplicity of spider toxins: structural aspects 

10.40-11.00: Manjunatha Kini (Professor, National University of 

Singapore) 

Protein scaffolds in snake venoms 

Dr Richard Lewis (Co-founder of the Xenome, joint appointment as 

Xenome's CSO & Principal investigator (molecular pharmacology) at the 

Institute for Molecular Bioscience (IMB) at the University of Queensland 

Venomics to drugs: the Xen2174 story 


11.45am-12.45pm 

Michael Brownstein (Director of Functional Genomics, J. Craig Venter 

Institute, Maryland) 

What can genomics teach you about toxins? 


1.45-3.00 pm 

Clinical aspects 

Cardiovascular 

Toxins from novel sources 

Plant toxins 

16

Friday 28th 

July 

Track 1: Clinical aspects – Antivenoms and 

treatments; toxins and analgesic discovery 


3.00-3.20 Wagstaff, S.C.: Bioinformatics and multi-epitope 

DNA immunisation to design rational snake 

antivenom 

3.20-3.40 Laing, G.D. : Pre-clinical assessment of equine 

and ovine antivenoms raised against saw-scaled 

viper (Echis ocellatus) venom for use in Nigeria 

3.40-4.00 Williams, D. J.: What price, salvation? Antivenom 

costs and availability in Papua New Guinea 1992- 

2004 

4.00-4.20 Winkel K.D.: Developing a toxinology toolkit for an 

under-developed 

4.20-4.40 Jensen S.D.: Teaching clinical toxinology in the 

developing world: Setting an example in Papua 

New Guinea 

4.40-5.00 Cury, Y.: Analgesic action of Crotalphine, a novel 

opioid receptor agonist from the venom of the 

South American rattlesnake Crotalus durissus 

terrificus (CdtV) 

5.00-5.20 Chacur, M.: Analgesic effect induced by Naja 

kaouthia snake venom and α-cobratoxin isolated 

from this venom 

5.20-5.40 Zaharenko, A.J.: BcIV, a new paralyzing peptide 

from the venom of the sea anemone Bunodosoma 

caissarum. A comparison with the Na + channel 

toxin BcIII 

Track 2: Venomics, genomics, proteomics, 

transcriptomics 

K3.17 

Sher, D.: Beyond prey capture – a comparative 

bioinformatic approach to cnidarian allomones 

Moran, Y.: When Positive Selection of Neurotoxin 

Genes is Missing - the Riddle of the Sea Anemone 

Nematostella vectensis 

Escoubas, P.: Venom landscapes: Towards the 

discovery of novel pharmacological tools via the 

combined use of LC-MALDI-TOF and MALDI- 

TOF/TOF with cDNA analysis 

Calvete, J.J.: Snake venomics 

St Pierre, L.: Identification and characterisation of 

toxin-specific transcripts from the venom glands of 

Australian elapid snakes 

Earl, S.T.: Proteomic analysis of Australian snake 

venoms for the discovery and development of new 

human therapeutics 

Harrison, R.A.: Molecular characterization of 

hyaluronidase-encoding genes from venom gland 

cDNA libraries of Echis, Bitis and Cerastes viper 

species 

Juárez, P.: Miniminization of protein structure and 

gene organization along the evolution of the short 

disintegrin ocellatusin from a dimeric disintegrin 

precursor 

Track 3: Structural aspects of toxins 

K3.25 

Ménez, A. : The same small three-fingered fold for 

snake toxins and human uPAR 

Araki, S.: 3D-structure of VAP1, a high-molecular 

snake venom metalloprotease, reveals 

metalloproteinase/disintegrin/cysteine-rich 

architecture of SVMPs and ADAMs 

Kahn R. : Unique Molecular Formations in Crystals 

of Scorpion Toxins that Affect Voltage-Gated Na- 

Channels 

Ducancel, F.: The increasing molecular diversity of 

sarafotoxins 

Matsunaga, Y.: Molecular diversity of snake 

venom VEGFs 

Obayashi, S.: Isolation and characterization of a 

VEGF-like protein from the venom of Bitis arietans 

Fujisawa D.: VEGF receptor-binding potential is a 

common property for myotoxic phospholipase A2 

Utkin, Y.N.: Low Abundant Proteins in Cobra 

Venom

Plenary lectures and 

symposia 

Speakers’ biographies 

and abstracts 

All plenary lectures and symposia will be 

held in lecture theatre K3.25 in the John 

Anderson Building 

Premier sponsors: 

18 

Willem ‘Pim’ Stemmer, Ph.D. is CEO and Founder of microPROTEINS, 

Inc, a biotechnology company located in Menlo Park, CA, focused on creating therapeutics that 

combine the best properties of small molecules (size, stability, non-immunogenicity) with the best 

properties of proteins (affinity, specificity, safety, design and manufacturing process, 

hydrophilicity, halflife). The company’s microprotein products are derived from small proteins with 

high disulfide density. The 3-6kD size, temperature/protease stability, hydrophilicity and E. coli 

expression properties of microproteins make them ideally suited for therapeutics. Products are 

broadly applicable across a variety of therapeutic areas and are designed to have sub-nanomolar 

activity, long halflife, low immunogenicity, long shelflife, low cost of goods, low royalty stack and 

high margin, creating an important advantage over antibodies or antibody fragments. 

Pim previously founded Avidia Inc. in Mountain View, California. Pim invented the company’s 

technology in 2001 and and served as its chief scientific officer for four years. Avidia's proteins, 

called ‘Avimers’, bind targets multivalently with multiple domains to inhibit or activate a specific 

biologic function. Avidia was founded in July 2003 as a spin-out from Maxygen to focus on the 

commercial development of this novel protein technology. 

In 1997, Pim co-founded Maxygen (Nasdaq: MAXY) with Drs. Alejandro Zaffaroni, Isaac Stein 

and Russell Howard to commercialize DNA Shuffling and he served as VP R&D until 2003. In 

1993, Pim invented Maxygen’s core technology, called DNA Shuffling or Molecular Breeding. 

Pim is the inventor on over 70 issued patents and over 100 pending applications. He has 

authored over 60 research publications and has given over 200 invited scientific lectures in many 

different fields. He received the David Perlman Award and the Doisy Award. He was instrumental 

in obtaining $25M in DARPA grants for Maxygen. 

From 1987 to 1992, Pim was a research scientist at Hybritech, Inc, working on antibody fragment 

engineering in E. coli and mammalian cells, focused on applications for cancer therapy. In 1992, 

he was awarded ‘scientist of the year’. Pim obtained his Ph. D. from the University of Wisconsin- 

Madison in 1985 on bacterial virulence mechanisms. 

Silverman J, Lu Q, Bakker A, To W, Duguay A, Alba BM, Smith R, Rivas A, Li P, Le H, Whitehorn 

E, Moore KW, Swimmer C, Perlroth V, Vogt M, Kolkman J, Stemmer WP (2005) Multivalent 

avimer proteins evolved by exon shuffling of a family of human receptor domains. Nat Biotechnol. 

23: 1556-61. 

Wright A, Semyonov A, Dawes G, Crameri A, Lyons R, Stemmer WP, Apt D, Punnonen J. (2005) 

Diverse plasmid DNA vectors by directed molecular evolution of cytomegalovirus promoters.Hum 

Gene Ther. 16: 881-92. 

Leong SR, Chang JC, Ong R, Dawes G, Stemmer WP, Punnonen J. (2003) Optimized expression 

and specific activity of IL-12 by directed molecular evolution. Proc Natl Acad Sci USA 100: 1163-8. 

Leong SR, Chang JC, Ong R, Dawes G, Stemmer WP, Punnonen J. (2003) Optimized expression 

and specific activity of IL-12 by directed molecular evolution. Proc Natl Acad Sci USA 100: 1163-8. 

19

Monday 24 July: 11.45 am-12.45 pm 

Lecture theatre K3.25 

The Elsevier-Toxicon Lecture 

20 

Sponsored by 

The advantages of disulfide-rich microproteins as scaffolds for the 

construction of antibody-mimetic pharmaceuticals 

Willem ‘Pim’ Stemmer 

Amunix Inc., Menlo Park, CA 94025, USA 

pstemmer@amunix.com 

Disulfide-rich microprotein scaffolds offer important advantages for the creation 

of antibody-like therapeutics. Scaffolds of special interest are those of natural 

injectable microproteins (ie leeches, snakes, snails, spiders, scorpions, 

anemones) and microproteins that are natural orally available (ie KalataB1). 

Whereas the folding of typical protein domains is mediated by a large 

hydrophobic core, in most families of microproteins this is replaced by a few 

disulfides. As a consequence, protein domains based on disulfide scaffolds are 

much smaller, less hydrophobic and allow a higher level of amino acid 

substitution, which allows these proteins to be evolved for tight binding to novel 

therapeutic targets, for use as antibody replacements. This reduction in size and 

in hydrophobic amino acids (esp. aliphatic) greatly decreases the predicted 

MHC-binding affinity and the expected level of immunogenicity of these proteins. 

Drugs based on such scaffolds can be designed to have exceptional stability to 

heat and proteases and can be formulated to very high concentrations. These 

properties may allow products to be delivered orally or by home injection, 

creating an important advantage over antibodies. 

Dr James Rothman is currently Director of the Columbia Genome Center and 

Professor of Chemical Biology at The College of Physicians and Surgeons at 

Columbia University, New York. Dr Rothman has made fundamental 

contributions to the understanding of protein transport between membranebound 

compartments of cells. He has developed assays that have allowed the 

function of the Golgi apparatus to be dissected at the biochemical level and 

has purified several proteins that catalyze vesicular formation and fusion with 

target membranes. He was awarded the Albert Lasker Award for Basic 

Medical Research in 2002 for his work on membrane vesicles. Dr Rothman was also awarded the 

Dr H.P. Heineken Prize for Biochemistry and Biophysics in 2000 for clarifying the mechanism of 

intracellular membrane fusion. 

He made the historic discovery that the cell contains very small membrane-enveloped vesicles 

that carry a large variety of proteins between different compartments in the cytoplasm. This 

delivery process, which involves vesicle flow and membrane fusion, is vital for the growth and 

division of every cell. How this process comes about was a great mystery, and one of the great 

unsolved questions of biochemistry and cell biology. James Rothman discovered the molecular 

principles of intracellular membrane fusion and demonstrated that the specificity of fusion was 

dictated by the pairing of SNARE proteins between membranes. This historic discovery provided 

a single unified principle for understanding important physiological processes, including the 

release of insulin into the blood, communication between nerve cells in the brain and the entry of 

viruses like HIV (the AIDS virus) to infect cells. Defects in the control of these pathways are 

important in diabetes and most likely also in certain cancers. Currently a major effort is under way 

to develop a new generation of drugs to control AIDS by blocking the membrane fusion process. 

James Rothman was born in Haverhill, Massachusetts, USA. He has a Ph.D. in Biological 

Chemistry (Harvard Medical School), and he worked at the Sloan-Kettering Institute in New 

York from 1991 until 2003 when he moved to Columbia University College of Physicians and 

Surgeons to establish a new Center for Chemical Biology. Amongst his other honours, Dr 

Rothman has received the Gairdner Foundation International Award (1996), the King Faisal 

International Prize in Science (1996) and the Lounsbery Award of the National Academy of 

Sciences (1997). 

Cosson P, Ravazzola M, Varlamov O, Sollner TH, Di Liberto M, Volchuk A, Rothman JE, Orci 

L. (2005) Dynamic transport of SNARE proteins in the Golgi apparatus. Proc Natl Acad Sci 

USA 102:14647-52. 

Fix M, Melia TJ, Jaiswal JK, Rappoport JZ, You D, Sollner TH, Rothman JE, Simon SM. (2004) 

Imaging single membrane fusion events mediated by SNARE proteins. Proc Natl Acad Sci 

USA 101:7311-6. 

Varlamov O, Volchuk A, Rahimian V, Doege CA, Paumet F, Eng WS, Arango N, Parlati F, 

Ravazzola M, Orci L, Sollner TH, Rothman JE. (2004) i-SNAREs: inhibitory SNAREs that finetune 

the specificity of membrane fusion. J Cell Biol. 164:79-88. 

Hu C, Ahmed M, Melia TJ, Sollner TH, Mayer T, Rothman JE. (2003) Fusion of cells by flipped 

SNAREs. Science 300:1745-9. 

21

Tuesday 25 July: 11.45 am-12.45 pm 


James E. Rothman (Director of the Columbia Genome Center, Professor of 

Chemical Biology, Columbia University, New York) 

Membrane fusion in the cell 

A vast array of physiological processes – passing of information between nerves in the 

brain, control of blood sugar by insulin, fertilization of the egg, to name just a few – 

require the fusion of membranes. Intracellular membrane fusion results when the 

cytoplasmic domains of matching SNARE proteins link-up between membranes to form 

a helical bundle termed a SNAREpin. Since only designated SNAREs can link up, fusion 

is highly specific. All nucleated cells – whether from yeast, animals, plants, or man – 

have families of related SNARE proteins that fall into two overall categories: v-SNAREs 

that primarily mark transport vesicles and t-SNAREs that primarily mark target 

membranes; matching pairs are distributed among membranes in a pattern that reflects 

the pattern of membrane flow among them. Proteolytic neurotoxins, such as botulinum 

toxins, specifically target SNARE proteins mediating exocytosis. Additional machinery 

exists to regulate the activity of SNARE proteins, allowing local or global control of the 

rate and location of vesicle flow in the cell. 

In particular, neuronal synapses influx of calcium ions stimulates the release of 

neurotransmitter. However the mechanism by which synaptic vesicle fusion is coupled to 

calcium has been unclear, despite the identification of both the core fusion machinery 

(SNAREs) and the principal calcium sensor (synaptotagmin). We have recently 

established what may represent a basic principle of the coupling mechanism: a reversible 

clamping protein (complexin) that can freeze the SNAREpin, an assembled fusioncompetent 

intermediate en route to fusion. When calcium binds to the calcium sensor 

synaptotagmin, the clamp would then be released. SNARE proteins, and key regulators 

like synaptotagmin and complexin, can be ectopically expressed on the cell surface. Cells 

expressing such “flipped” synaptic SNAREs fuse constitutively, but when we coexpressed 

complexin fusion was blocked. Adding back calcium triggered fusion from 

this intermediate in the presence of synaptotagmin. 

22 

Redi Award lecture 

The International Society on Toxinology confers the Redi Award every three 

years in recognition of distinguished work in the field of toxinology. The recipient 

of the 2006 Redi Award will be announced at the Congress and will deliver a 

plenary lecture. 

Previous recipients: 

Findlay E. Russell 1967 

Paul Boquet 1970 

Andre de Vries 1974 

Chen-Yuan Lee 1976 

Hugh Alistair Reid 1979 

Nobuo Tamiya 1982 

Philip Rosenberg 1982 

Sherman A. Minton 1985 

Paul A. Christensen 1988 

Ernst Habermann 1991 

Elazar Kochva 1994 

Evert Karlsson 1997 

Alan L. Harvey 2000 

André Ménez 2000 

Baldomero M. Olivera 2003 

23

Wednesday 26 July: 11.45 am-12.45 pm 


Redi Award lecture - to be announced at the Congress 

Sponsored by International Society on Toxinology 

24 

Dr. John Daly was born and raised in Portland, Oregon. He received in 

1954 a bachelors degree in Biochemistry and in 1955 a masters degree in Organic Chemistry at 

Oregon State College and then a Ph.D. in Organic Chemistry at Stanford University in 1958. 

After a two year postdoctorate in the Laboratory of Chemistry at NIH, he became a permanent 

member of the staff in 1960, a section chief in 1969 and the founding chief of the Laboratory of 

Bioorganic Chemistry in 1981. He became a NIH Scientist Emeritus in January 2003. During his 

five decades at NIH, his research has spanned many disciplines, being involved in isolation, 

structure elucidation and synthesis of novel natural products and receptor agonists/ antagonists 

and in the elucidation of their mechanism of action with a focus on receptors, ion channels, and 

second messenger formation in the nervous system. High points in his career include the 

discovery of the NIH Shift, the introduction of a prelabeling technique for investigation of cyclic 

AMP generation in intact cells, the introduction of a variety of selective agonists and antagonists 

as probes and radioligands for adenosine receptors, the delineation of biological targets for 

caffeine and other xanthines in the central nervous system, the discovery of the direct activation 

of adenylyl cyclase by forskolin, the discovery of the activation of phosphoinositide breakdown by 

maitotoxin, and the discovery that loperamide is a unique modulator of a capacitative calcium 

influx pathway and that N-substituted dihydropyridines are selective blockers of such channels. 

Over four decades, he has been involved both in field collection and as a chemist and 

pharmacologist in the discovery and structure elucidation of a wide range of over 800 alkaloids of 

twenty some structural classes in amphibian skin and elucidation of the basis for their biological 

activity. Such compounds include batrachotoxins, histrionicotoxins, pumiliotoxins, and 

epibatidine. Certain of these alkaloids are now widely used as research tools; batrachotoxin as a 

selective activator of sodium channels, histrionicotoxins as blockers of nicotinic channels, 

pumiliotoxins as sodium channel agents with biomedical potential as cardiotonic agents and 

epibatidine as nicotinic agonist with potent analgetic activity. Analogues of epibatidine are the 

subject of clinical trials for a treatment of chronic pain of neuropathies, cancer and arthritis. Many 

of the alkaloids discovered by Dr. Daly have been the target of synthetic research in laboratories 

world-wide. Dr. Daly’s research presently focuses on the structure and biological activity of 

further novel alkaloids found in amphibian skin and on the dietary origin of such alkaloids. Dr. 

Daly has been the recipient of other awards in recognition of his achievements. He was elected 

to the National Academy of Sciences, USA in 1997. Besides his abiding interest in field work and 

research, Dr. Daly is an avid fisherman. 

1. Seamon, K.B., and Daly, J.W.: Forskolin: A unique diterpene activator of cyclic AMP-generating systems. J. Cyclic 

Nucleotide Res. 7: 201-224, 1982. 

2. Badio, B., and Daly, J.W. Epibatidine, a potent analgetic and nicotinic agonist. Mol Pharmacol. 45: 563-569, 1994. 

3. Daly, J.W., Kaneko, T., Wilham, J., Garraffo, H.M., Spande, T.F., A. Espinosa and Donnelly, M.A. Bioactive alkaloids 

of frog skin: Combinatorial bioprospecting reveals that pumiliotoxins have an arthropod source. Proc. Natl. Acad. Sci. 

U.S.A. 99: 13996-14001, 2002. 

4. Daly, J.W. Ernest Guenther Award in Chemistry of Natural Products. Amphibian skin: A remarkable source of 

biologically active arthropod alkaloids. J. Med. Chem. 46: 445-452, 2003. 

5. Fitch, R.W., Garraffo, H.M., Spande, T.F., Yeh, H.J.C. and Daly, J.W. Bioassay-guided isolation of epiquinamide, a 

novel quinolizidine alkaloid and nicotinic agonist from an Ecuadoran poison frog, Epipedobates tricolor. J. Nat. Prod. 66: 

1345-1350, 2003. 

6. Dumbacher, J.P., Wako, A., Derrickson, S.R., Samuelson, A., Spande, T.F., and Daly, J.W. Melyrid beetles 

(Choresine): A putative source for the batrachotoxin alkaloids found in poison-dart frogs and toxic passerine birds. Proc. 

Natl. Acad. Sci. U.S.A. 101: 15857-15860, 2004. 

7. Daly, J.W., Spande, T.F., and Garraffo, H.M. Alkaloids from amphibian skin: A tabulation of over eight-hundred 

compounds. J. Nat. Prod. 68: 1556-1575, 2005. 

25

Thursday 27 July: 11.45 am-12.45 pm 


Alkaloids as Probes for Ionic Channels: 

John William Daly. 

Laboratory of Bioorganic Chemistry, NIDDK, NIH, Bethesda, MD, USA 

Alkaloids represent an impressive source of probes for the study of ion 

channels in nerve, muscle and other tissues. Although most alkaloids have been 

derived from plant sources, over 800 alkaloids have been identified from amphibian 

skin 1 . These include 1) the samandarines that block sodium channels, 2) the 

batrachotoxins that activate sodium channels, 3) the pumiliotoxins, allopumiliotoxins 

and homopumiliotoxins that are positive modulators of sodium channels and thereby 

myotonic and cardiotonic agents, 4) the histrionicotoxins and decahydroquinolines, 

izidines, tricyclics, pyrrolidines, piperidines, spiropyrrolizidine oximes and 

pseudophrynamines that are noncompetitive blockers of nicotinic receptor-channels, 

5) the epibatidines that are potent agonists at nicotinic receptors and thereby powerful 

analgetics. From the plant kingdom many alkaloids have provided ion channel 

probes 2,3 . These include 1) nicotine, cytisine, anatoxin, ferruginine, anabaseine and 

lobeline as nicotinic agonists, 2) tubocurarine, erythryodine, methyllyaconitine and 

ibogaine as blockers and galanthamine as a positive modulator of nicotinic receptor 

channels, 3) veratridine and aconitine as activators of sodium channels and quinine 

and cocaine as blockers, 4) strychine as an antagonist of glycine receptors, 5) 

bicuculline as an antagonist of GABA receptors, 6) ryanodine and caffeine as 

modulators of calcium-activated calcium channels of the endoplasmic and 

sarcoplasmic reticulum. Natural products continue to be an incredibly rich source of 

bioactive compounds with major impact on biomedical research. 

1) Daly, J.W., Spande, T.F. and Garraffo, H.M. J. Nat. Prod. 68: 1556-1575, 2005 

2) Daly, J.W. Cell. Mol. Neurobiol. 25: 513-552, 2005. 

3) Wink, M. Studies in Natural Product Chemistry. 21:3-121, 2000. 

o Alkaloids 

o Ion Channels 

o Nicotinic receptors 

o Epibatidine 

26 

Dr Brownstein earned his A.B. from the Columbia College and his M.D. 

and Ph.D. degrees at the University of Chicago. After completing an internship at the Boston 

Children’s Hospital, he went to the NIH in 1972 as a Pharmacology Research Associate to work 

with Julie Axelrod. Initially he studied the pineal gland, but he began to develop more sensitive 

techniques for measuring neurotransmitters and their biosynthetic enzymes. He met Miklos 

Palkovits, who had just devised a novel brain microdissection method, in 1973, and together they 

mapped many “classical” transmitters and neuropeptides in the central nervous system. Dr. 

Brownstein was the first to point out that neurons make and likely release more than one 

chemical messenger, and the first to show that “hypothalamic hormones” must have much 

broader roles in the brain and periphery. 

He and Dr. Harold Gainer used pulse-chase studies in vivo to show that vasopressin and 

oxytocin are synthesized as parts of larger precursor proteins, that these precursors are 

processed by proteases in vesicles while they are being transported from the cell body to its 

axon terminals, and that precursor synthesis and processing are both regulated by demand. 

Dr. Brownstein and Hiroto Okayama developed robust protocols for making cDNA libraries and 

expressing the inserts in mammalian cells. Dr. Brownstein’s goal at the time was to use such 

methods for expression cloning of vasopressin and oxytocin receptors—the next essential step 

in learning about the biology of these peptide hormones. He and his coworkers ultimately 

achieved these goals, and they cloned a number of additional important cDNAs. Some of 

these have proven especially important to members of the mental health community: e.g., 

cDNAs that encode the serotonin transporter (the target of SSRIs), the dopamine transporter 

(the target of cocaine), the cannabinoid receptor (the target of a novel and very promising drug 

for treating obesity and addictive behavior), and the vasopressin V1b receptor (an attractive 

target for development of antidepressant compounds). 

One of the vasopressin receptors that Dr. Brownstein’s group cloned was the V2 (kidney) 

subtype. He understood that mutations in this receptor could very well be responsible for Xlinked 

nephrogenic diabetes insipidus, and rapidly demonstrated that this was indeed the 

case. This was his reintroduction to human genetics, and he decided to learn more about 

genetic analyses by working in the NHGRI. He was among the first people there to do highthroughput, 

fluorescence-based genotyping, and he developed a method for modifying the 

primers used to study microsatellite markers (“Pig tailing”) that was subsequently licensed by 

Applied Biosystems and applied to all of the primer pairs that they manufactured. To date tens 

of millions of genotypes have been done with such markers. 

In his final years at the NIH, Dr. Brownstein’s focus was complex traits genetics and 

genomics. He and the members of his team defined causative mutations in a number of 

genetic diseases, ranging from prostate cancer to cataracts, and continued to develop 

important methods, notably ones for labeling probes for microarray studies. 

In 2005, Dr. Brownstein retired from the civil service, and accepted a position at the J. Craig 

Venter Institute, Rockville, MD, where he is now director of functional genomics. He has also 

served on numerous editorial boards, scientific advisory boards, and review committees, and 

has received a number of awards, most recently, an honorary Doctorate from the University of 

Lund in Sweden. 

27

Friday 28 July: 11.45 am-12.45 pm 


Michael J. Brownstein, MD, PhD 

Director of Functional Genomics, J. Craig Venter Institute, Rockville, 

Maryland 20850, USA 

What can genomics teach you about toxins? 

There are still many partially answered or unanswered questions about the constituents of 

venoms and the specialized tissues that make, store, and deliver them. These include the 

following: How are the genes that encode toxins organized? How many such genes are 

there? How are the diverse toxins distributed among these genes? Are all of toxinencoding 

sequences expressed or is there a large reservoir of unused sequences to draw 

on? Are toxin genes distributed across the entire genome or located at one or more 

specific loci? In the future, we hope to learn more about the complexity of certain toxin 

families--e.g., the conopeptides—and whether mechanisms resembling those responsible 

for antibody diversity (including recombination and hypermutability) could act during the 

life of an organism to increase its toxin repertoire. To answer these questions, we will 

need to characterize the genes, mRNAs, and protein products of one or more venomous 

species, and subsequently to look at transcripts and peptides in several organs of multiple 

individuals. Until recently, such work was time consuming, labor intensive, and 

enormously expensive. New methods are reducing costs, increasing speed, and 

improving the quality of the results obtained. I will review these methods for you and tell 

you about advances that are likely to occur in the next few years. 

Some recent publications 

AHN, JI, et al. Comprehensive transcriptome analysis of differentiation of embryonic stem cells into midbrain and hindbrain neurons. 

DEV. BIOL. 265: 491-501, 2004. 

MUTSUGA, N. et al Selective gene expression in magnocellular neurons in rat supraoptic nucleus. J. NEUROSCIENCE 24: 7174- 

85, 2004. 

ARROZTOA, JA et al. Identification of genes expressed in primary primordial oocytes. HUMAN REPRODUCTION 20: 476-483, 

2005. Epub 2004 Dec 2. 

MUTSUGA, N. et al Regulation of gene expression in magnocellular neurons in rat supraoptic nucleus during sustained 

hypoosmolality. ENDOCRINOLOGY 146: 1254-1267, 2005. Epub 2004 Dec 9. 

XIANG, CC, et al. Using DSP, a reversible cross-linker, to fix tissue sections for immunostaining, microdissection and expression 

profiling. NUCLEIC ACIDS RESEARCH 32: e185, 2004. 

KUO, J, et al. Evaluation of vector-primed cDNA library production from microgram quantities of total RNA. NUCLEIC ACIDS 

RESEARCH 32: e183, 2004. 

ALTUVIA, Y, LANDGRAF, P., LITHWICK, G. ELFANT, N. PFEFFER, S. ARAVIN, A., BROWNSTEIN, M. J. TUSCHL, T., 

AND MARGALIT, H. Clustering and conservation patterns of human microRNAs. NUCLEIC ACIDS RESEARCH 33: 2697-2706, 

2005. 

SEWER, A., PAUL, N. LANDGRAF, P. ARAVIN, A. PFEFFER, S. BROWNSTEIN, M.J., TUSCHL, T., VAN NIMWEGEN, E., 

AND ZAVOLAN, M. Identification of clustered microRNAs using an ab initio prediction method. BMC BIOINFORMATICS. 6: 

267, 2005. 

28 

George Miljanich has been developing venom peptide therapeutics, ion channel therapeutics, 

and analgesics for more than 20 years. He is author or co-author of over 50 scientific 

publications, and is co-inventor on 15 issued U.S. patents and numerous corresponding non-U.S. 

patents. Dr. Miljanich is currently Chief Executive Officer of the U.S. biotechnology company, 

AIRMID LLC, where he leads the effort to discover, develop, and commercialize highly potent and 

selective potassium channel blockers - including venom peptide toxin and plant toxin derivatives - 

to treat a variety of autoimmune disorders. Prior to joining AIRMID, he was Senior Medical 

Director at Elan Pharmaceuticals, where he played a key role in progressing the novel analgesic, 

PRIALT ® , through the final stages of development, toward FDA and EMEA approval, and to the 

analgesia market. 

Dr. Miljanich received his BS degree in Chemistry from the University of California, Berkeley, and 

his PhD in Chemistry from U.C. Santa Cruz. He then conducted post-doctoral research at U.C. 

San Francisco, studying presynaptic function. As a faculty member at the University of Southern 

California, he continued these investigations, including studying the biochemistry and 

pharmacology of calcium channels, thus becoming acquainted with the research applications of 

calcium channel-blocking toxins from cone snails. Dr. Miljanich joined the Neurex Corporation to 

initiate the translation of these research tools into pharmaceuticals. He contributed to the 

development of the conopeptide, ziconotide (PRIALT ® ), through preclinical studies to Phase III 

clinical trials, while also leading several drug discovery projects targeted at a variety of 

presynaptic proteins. With the acquisition of Neurex by Elan, Dr. Miljanich became head of 

analgesia research at Elan and oversaw several research projects aimed at developing novel 

therapeutics for pain. 

In addition to heading AIRMID, he is an advisor to several companies in the pharmaceutical 

industry. Dr Miljanich may be contacted at gmiljanich@comcast.com. 

Some relevant publications: 

Miljanich, G.P., Ziconotide: Neuronal Calcium Blocker for Treating Severe Chronic Pain. Current 

Medicinal Chemistry 11, 3129-3140 (2004) 

Newcomb R. and Miljanich G. “Neurotoxins of Cone Snail Venoms.” In: Handbook of 

Neurotoxicology, 2002, ed., Massaro, EJ. (Humana Press, Totowa, New Jersey) pp. 617-651 

Newcomb, R., et al., Diversity of neuronal R-type calcium currents revealed by a selective peptide 

antagonist for the class E calcium channel from the venom of the tarantula, Hysterocrates gigas. 

Biochemistry, 37, 15353-15362 (1998) 

See also: “A Toxin Against Pain” by Gary Stix in Scientific American, April 2005 pp. 88-93. 

29

Symposia 

Monday 24 July: 9.00 am- 11.15 am 


Symposium: Toxins and drug discovery 

9.00-9.45: George Miljanich (CEO, AIRMID LLC, California): 

PRIALT® (ziconotide intrathecal infusion): A Conopeptide for Treating Severe 

Chronic Pain 

Airmid LLC, 600 Castle Hill Road, Redwood City CA USA 94061, gmiljanich@comcast.net 

The analgesic, PRIALT ® (ziconotide), is the first conopeptide to receive approval for 

marketing in the US and EU. Ziconotide is a non-opioid treatment for severe chronic pain 

in patients warranting intrathecal (intraspinal) therapy and who are intolerant or refractory 

to other treatment, such as adjunctive therapy, systemic analgesics, or intrathecal opiates. 

Ziconotide is the synthetic equivalent of omega-conotoxin MVIIA originally found in 

Conus magus venom and is the first in a new class of therapeutics: N-type calcium channel 

blockers. Approval was based in part on: 1) safety data from ~1250 pain patients and 2) 

analgesic efficacy demonstrated in a placebo-controlled, double-blind study of 220 patients 

suffering severe chronic pain. This study showed ziconotide is analgesic in patients who 

failed to obtain adequate pain relief from oral and/or intrathecal opiate therapy, as well as 

in patients taking oral opiates concomitantly with ziconotide. Ziconotide does not elicit 

respiratory depression, analgesic tolerance, or withdrawal syndrome upon discontinuation, 

nor does it pose a risk of drug dependency. Adverse effects attributed to ziconotide 

(incidence >10%; % affected ziconotide patients minus % affected placebo patients) 

include dizziness, ataxia, abnormal gait, confusion, memory impairment, nausea, and 

fatigue. Psychiatric symptoms may also occur with ziconotide treatment. Ziconotide’s 

clinical profile is consistent with its unique mechanism of action as determined by 

extensive non-clinical studies which show ziconotide diminishes pain behaviors by 

blocking N-channels on sensory nerves, thereby inhibiting transmission of noxious signals 

to the brain. Ziconotide’s non-clinical pharmacology and therapeutic profile demonstrate 

that 1) N-channels play a key role in pain perception, 2) N-channels are a valid target for 

analgesic therapy, and 3) ziconotide is a non-opioid alternative for treating severe chronic 

pain. 

Reference: Ziconotide: neuronal calcium channel blocker for treating severe chronic pain. 

Miljanich GP, Curr Med Chem. 23, 29-40 (2004). 

o PRIALT 

o ziconotide 

o conopeptide 

o calcium channel blocker 

30 

Dr. Michael Hanley is Vice President of Discovery Research at Amylin Pharmaceuticals, which 

launched two first-in-class diabetes therapeutics in 2005. Prior to joining Amylin, Dr. Hanley held 

tenured faculty positions at the Department of Biochemistry,Imperial College, London, the 

Medical Research Council Laboratories, Cambridge, and the University of California at Davis, 

where he was Professor of Biological Chemistry. He has over 160 papers covering fields from 

signal transduction to molecular neurobiology to peptidomics, and been an editor or advisor to 

over a dozen journals. Dr. Hanley has served on advisory or review panels for the National 

Institutes of Health, the Medical Research Council and Wellcome Trust of Great Britain, and for 

the governments of Australia, Singapore, New Zealand, Hong Kong, Denmark and Japan. From 

1997 to 2003, Dr. Hanley was a senior consultant for healthcare investors in the venture capital 

and banking communities and for biotechnology companies such as Cell Therapeutics, 

Zymogenetics, Elan Pharmaceuticals, and Chiron Corporation. Dr. Hanley has also set-up and 

directed research programs in privately-held start-ups, such as Chemocentryx, PsychoGenics, 

and most recently Harvard-based Resolvyx Pharmaceuticals. He received his B.S. in 

Biochemistry and his Ph.D. in Molecular Biology from the University of California, Berkeley. 

31

9.45-10.30: Physiological fluids of venomous animals: novel resources for drug 

discovery 

Hanley, M.R. 

Amylin Pharmaceuticals, Inc, 9360 Towne Centre Drive, San Diego CA, 92121 USA, 

michael.hanley@amylin.com 

Venom glands constitute precedented resources of chemically diverse natural products 

with therapeutic potential..However, venomous and poisonous animals have also coevolved 

specialisation of other secretory glands to provide novel variants of neural and 

circulating.factors. Thus, looking beyond the constituents of venom itself, venomous 

organisms provide rich opportunities for discovery from physiological fluids; particularly 

saliva and blood. 

The point is ideally illustrated by glucagon-like peptide-1 (GLP-1). In mammals, GLP-1 is 

an incretin - broadly defined as a glucose-dependent insulinotropic hormone - which 

regulates blood glucose in a self-limiting manner. Incretins are therefore highly attractive 

targets for drugs to treat Type II diabetes. As an example of species-specific adaptation, the 

gila monster (Heloderma suspectum) expresses a both a GLP-1 as well as a second GLP-1 

variant, exendin-4 (Ex-4). Ex-4 is produced and secreted by the salivary glands, as well as 

other tissues. Exendin-4 (“exenatide”) turns out to have a superior pharmaceutical 

chemistry compared to natural GLP-1orthologues or synthetic analogues. 

The peptide has progressed from concept to clinic to commercialisation., and thus 

provides many lessons on drug development of naturally-occurring peptides; including 

solutions for immunogenicity, optimal solubility, low aggregation potential, patientfriendly 

drug delivery, beneficial pharmacokinetics, enhanced chemical stability, and 

reverse engineering of favourable drug properties. These lessons will be discussed in terms 

of their general applicability to peptide therapeutics; 

Going forward, we are extending the approach of finding pharmaceutically-enhanced 

natural variants of our pipeline, using comparative genomic and experimental peptide 

discovery platforms. The results have been combined to build proprietary computational 

tools and libraries of novel peptide and protein drug candidates. 

o GLP-1 

o Salivary gland 

o Diabetes 

o Drug development 

32 

Professor George Chandy completed his MBBS at the Christian Medical College, Vellore, India, 

and his PhD in Immunology at the University of Birmingham, UK. He joined the University of 

California Irvine in 1983 as a postdoctoral researcher and is currently Professor of Physiology 

and Biophysics at that university. 

Dr. Chandy’s group focuses on potassium channels – structure, function, functional role and 

therapeutic targets. Dr. Chandy proposed the nomenclature for potassium channels that is widely 

accepted internationally. Dr. Chandy and his colleagues were the first to discover potassium 

channels in immune cells in the early 1980s, and they cloned the genes encoding these 

potassium channels in the 1990s. Recent studies by Dr. Chandy’s group have identified the Kv1.3 

potassium channel as a potentially important therapeutic target for diverse autoimmune diseases. 

They have developed a highly selective and potent inhibitor of the Kv1.3 channel by modifying the 

ShK peptide derived from the Cuban sea anemone, Stichodactyla helianthus. Kv1.3 blockers 

ameliorate disease in animal models of multiple sclerosis, rheumatoid arthritis and type-1 

diabetes. Studies are underway to develop Kv1.3 blockers for human autoimmune diseases. 

Dr Chandy has published more than 100 research papers and he is a named inventor on 17 

patents or patent applications. 

Wulff, H., Beeton, C., Chandy, K. G. (2003) Potassium channels as therapeutic targets for autoimmune 

disorders. Current Opinions in Drug Discovery and Development 6, 640. 

Shakkottai , V.G, Chou, C-h, Oddo, S, Sailer, C.A., Knaus, H-G, Gutman, G.A., Barish, M.E, LaFerla, F.M., 

Chandy, K.G. (2004). Enhanced neuronal excitability in the absence of neurodegeneration induces 

cerebellar ataxia. J. Clinical Investigation 113, 582. 

Villalobos C, Shakkottai VG, Chandy KG, Michelhaugh, S. K., Andrade R. (2004) SKCa channels mediate the 

medium but not the slow calcium-activated afterhyperpolarization in cortical neurons. J. 

Neuroscience 24, 3537. 

Chandy, K.G., Wulff, H., Beeton, C., Pennington, M., Gutman, G. A., Cahalan, M. D. (2004). Potassium 

channels as targets for specific immunomodulation. Trends in Pharmacological Sciences 25, 280. 

Kolski-Andreaco, A. A., Tomita, H., Shakkottai, V.G., Gutman, G. A., Cahalan, M. D., Gargus, J. J., Chandy, 

K. G. (2004) SK3-1C: a dominant-negative suppressor of SKCa and IKCa channels. J. Biol. Chem. 

279, 6893. 

Vennekamp, J., Wulff, H., Beeton, C., Calabresi, P. A., Grissmer, S., Hansel, W., Chandy, K. G. (2004) 

Kv1.3 Blocking 5-Phenylalkoxypsoralens: A new Class of Immunomodulators Molecular 

Pharmacology 65, 1364. 

Wulff, H., Knaus, H-G, Pennington, M., Chandy, K. G. (2004) K + channel expression during B-cell 

differentiation: implications for immunomodulation and autoimmune disorders. J. Immunol. 173, 

776. 

Beeton, C., Pennington, M. W., Singh, S., Nugent, D., Crossley, G., Khaytin, I., Calabresi, P. A., Chandy, K. 

G. (2005) Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for 

therapy of autoimmune diseases. Molecular Pharmacology 67, 1369. 

Beeton, C., Chandy, K. G. (2005) Potassium channels, memory cells and multiple sclerosis. Neuroscientist 

11, 550. 

Rus, H, Pardo CA, Hu L, Darrah E, Cudrici C, Niculescu T, Niculescu F, Mullen KM,, Allie R, Guo L, Wulff H, 

Beeton C, Judge SIV, Kerr DA, Knaus H-G, Chandy KG, and Calabresi PA (2005). The voltagegated 

potassium channel Kv1.3 is highly expressed on inflammatory infiltrates in multiple sclerosis 

brain. Proc. Natl. Acad. Sci. USA 102, 11094. 

33

10.30-11.15: From marine toxins to therapy for autoimmune diseases 

K. George Chandy, Christine Beeton, Michael Pennington, Heike Wulff, Alexandra 

Grino, GA Gutman 

University of California Irvine and Bachem Bioscience Inc. 

T-cell mediated autoimmune diseases afflict millions of people globally. 

Disease-modifying immunotherapies have improved the management of these diseases, 

but each of these therapies is known to induce specific side effects. Consequently, there is 

an unmet medical need for novel immunomodulators with different mechanisms of action 

and/or adverse-effect profiles from existing drugs. We show that the disease-associated 

autoreactive T cells from patients with multiple sclerosis, type-1 diabetes mellitus or 

rheumatoid arthritis are effector memory T lymphocytes, and therapies that selectively 

suppress these cells without affecting other lymphoid subsets would have immense value. 

Kv1.3, one of 76 human K + -channel genes, regulates membrane potential and calcium 

signaling in human effector memory T cells. We have developed a peptide analog of the 

ShK toxin from the sea anemone Stichodactyla helianthus called ShK(L5)-amide, which 

blocks Kv1.3 at low picomolar concentrations and exhibits greater than 100-fold 

selectivity for Kv1.3 over other channels. ShK(L5)-amide suppresses calcium signaling, 

cytokine production and proliferation of autoantigen-specific effector memory T cells at 

picomolar concentrations while sparing other classes of T cells. 

ShK(L5)-amide administered as single daily subcutaneous injections (100 μg /kg /day) 

ameliorated pristane-induced arthritis in rats, a model for rheumatoid arthritis, and 

prevented and treated experimental autoimmune encephalomyelitis in rats, a model for 

multiple sclerosis. Repeated dosing with ShK(L5)-amide in rats has not revealed systemic 

toxicity, and rhesus monkeys tolerated the peptide administered as a single i.v. injection. 

Further development of Kv1.3 blockers for autoimmune disease-therapy appears 

warranted. 

34 

Dr Edward A. Dennis: Distinguished Professor of Chemistry and Biochemistry 

and Pharmacology, School of Medicine, University of California, San Diego 

(UCSD), La Jolla, California, U.S.A. 

B.A. Yale University, 1963; M.A. Harvard University, 1965; Ph.D. Harvard 

University, 1968; Postdoctoral Fellow, Harvard Medical School, 1967-1969; 

Appointed to UCSD faculty, 1970-; Guggenheim Fellow, 1983-1984; Amer. Soc. 

Biochemistry and Molecular Biology Avanti Award, 2000; Editor-in-Chief, Journal 

of Lipid Research, 2003-; Director, LIPID MAPS Initiative, 2003- 

Dr Dennis’s research laboratory is focused on understanding the regulation of lipid second 

messengers and signal transduction processes and especially the role of various phospholipases 

in their generation. Special attention is paid to the cytosolic, secreted, and membrane-bound 

phospholipase A2s (PLA2) responsible for the control of arachidonic acid derived prostaglandin 

and leukotriene biosynthesis in macrophage cells. The goal is to elucidate the regulatory 

mechanisms of phospholipase A2s in vitro, ex vivo, and in vivo. 

In studies on the regulation and detailed mechanism of action of Group IA PLA2 from cobra 

venom at membrane and other lipid-water interfaces, Dr Dennis has developed in vitro systems 

for studying their detailed mechanism of action. PLA2s represent the smallest (molecular weight 

13,000) and perhaps the simplest enzymes of complex lipid metabolism known and are ideally 

suited for mechanistic studies. PLA2s have several kinds of phospholipid binding sites including 

activator sites, interfacial sites, and catalytic sites. It is important to define the precise role of the 

amino acid residues involved in these interactions and the laboratory is now carrying out HD/MS 

deuterium exchange studies using mass spectrometric analysis of surface interactions on the 

cobra venom enzyme to define the activator and other interfacial sites. 

Dr Dennis is involved in design and synthesis of chemical inhibitors of phospholipase A2. Many 

different inhibitor classes have been developed. He is also studying synthetic oxidized 

phospholipids and their role in LDL scavenger receptor uptake, autoantibody formation, and 

apoptosis and their connection to atherosclerosis. Dr. Dennis is also the Director of the LIPID 

MAPS lipidomics initiative which aims to identify and quantify all of the lipid molecular species in 

the macrophage and their changes induced by activation of phospholipase A2. 

Six, D. A., and Dennis, E.A., Essential Ca 2+ -Independent Role of the Group IVA Cytosolic Phospholipase A2 

C2 Domain for Interfacial Activity, J. Biol. Chem., 278, 23842 (2003). 

Phillips, R., Six, D. A., Dennis, E. A., and Ghosh, P., Protection from Cytotoxicity of ExoU-expressing 

Pseudomonas Aeruginosa by Phospholipase A2 Inhibitors, J. Biol. Chem., 278 41326-41332 (2003). 

Boegeman, S. C., Deems, R. A., and Dennis, E. A., Phospholipid Binding and the Activation of Group IA 

Secreted Phospholipase A2, Biochemistry, 43, 3907-3916 (2004). 

Kokotos,G., Six,D.A., Loukas,V., Smith,T., Constantinou-Kokotou,V., Hadjipavlou-Litina,D., Kotsovolou,S., 

Chiou,A., Beltzner,C.C., and Dennis,E.A., Inhibition of Group IVA Cytosolic Phospholipase A2 by Novel 2- 

Oxoamides in vitro, in Cells, and in vivo., J.Med.Chem, 47, 3615-3628 (2004). 

Killermann Lucas,K., Svensson,C.I., Hua,X.Y., Yaksh,T.L., and Dennis,E.A., Spinal Phospholipase A2 in 

Inflammatory Hyperalgesia: Role of Group IVA cPLA2, British Journal of Pharmacology , 144, 940-952 

(2005). 

Fahy,E., Subramaniam,S., Brown,H.A., Glass,C.K., Merrill,A.H., Murphy,R.C., Raetz,C.R.H., Russell,D.W., 

Seyama,Y., Shaw,W., Shimizu,T., Spener,F., Van Meer,G., VanNieuwenhze,M., White,S., Witztum,J.L., and 

Dennis,E.A., A Comprehensive Classification System for Lipids, Journal of Lipid Research, 46, 839-861 

(2005). 

35

Tuesday 25 July: 9.00 am- 11.15 am 


Symposium: Lipids and toxins 

9.00-9.45: LIPID MAPS and Eicosanoid Lipidomics 

Edward A. Dennis 

Department of Chemistry and Biochemistry and Department of Pharmacology, School of 

Medicine, University of California at San Diego, La Jolla, CA 92093-0601, U.S.A. 

The metabolic pathways involving lipids are complex and intertwined. Developing an 

integrated metabolomic system capable of characterizing the global changes in lipid 

metabolites (“lipidomics”) is a daunting task but one that is important to undertake in 

light of the significant returns produced by the global approaches of genomics and 

proteomics. Our consortium has developed a Lipid Metabolites And Pathways Strategy, 

termed LIPID MAPS, that applies a global integrated approach to the study of lipidomics. 

The specific aim of LIPID MAPS is to develop the requisite technology and conduct an 

integrated research program that will establish lipidomics as a fully functioning research 

field. We are employing a rigorously maintained set of common biological, biochemical, 

and analytical technologies in each of the consortium laboratories, and have developed 

an extensive informatics infrastructure. After introducing LIPID MAPS 

[http://www.lipidmaps.org] and its new classification system [J. Lipid Res.,46, 839-861 

(2005)], its application and use in lipid and protein databases will be summarized. Then 

new LC/MS results from the LIPID MAPS Fatty Acid/Eicosanoid Core that establish 

techniques for the detection and quanitation of various eicosanoid molecules generated 

by macrophages in response to stimuli such as LPS and a defined species Kdo2-Lipid A 

[J. Lipid Res., 47, 1097-1111 (2006)] will be reported. This includes the identification of 

the major and minor eicosanoid products and the quantification of the increases and 

decreases of metabolites as a function of time during cell stimulation. Approaches to the 

identification of unknown eicosanoids will also be explored. Correlations of lipid 

metabolite changes across lipid categories will be reported. U54 GM069338 

36 

Cesare Montecucco graduated in Chemistry (1971) and Biology (1975) cum laude from the 

University of Padova, where he is currently Professor of General Pathology and Vice-Director of 

the Scuola Galileiana. He has carried out research in the University of Cambridge and Utrecht, 

the Pasteur Institute of Paris and the EMBL of Heidelberg. He studies the molecular and cellular 

mechanisms underlying the pathogenesis of diseases caused by pathogenic bacteria: anthrax, 

botulism, tetanus, and pylori gastrointestinal pathologies associated with Helicobacter, as well as 

studies on presynaptically acting snake toxins. Moreover, he collaborates actively with Chiron- 

Vaccines of Siena on the development of an anti-H. pylori vaccine. 

His major scientific achievements are: a) the discovery of the metalloproteolytic activity of the 

clostridial neurotoxins responsible for tetanus and botulism specific for VAMP/synaptobrevin, 

SNAP-25 and syntaxin (SNARE proteins), providing the key demonstration of the role of the 

SNARE proteins in exocytosis; b) co-discovered the activity and substrate of the anthrax lethal 

factor and that this toxin has an immunosuppressive activity and of the immunosuppressive 

synergism of the anthrax edema and lethal toxins; c) discovery of the mechanism of action of the 

vacuolating VacA cytotoxin and of the neutrophil-activating protein (HP-NAP) of H. pylori, the 

bacterium which colonizes the stomach of the majority of the human populations and is 

associated with severe pathologies including active chronic gastritis, peptic ulcers and stomach 

cancers; and d) provided compelling evidence that presynaptic snake PLA2 neurotoxins act on 

the plasma membrane by producing lysophosphatydilcholine and fatty acid which promote 

exocytosis via hemifusion intermediates. In addition, he has provided theoretical contributions on 

the mode of binding of clostridial neurotoxins, on the mechanism of membrane translocation of 

bacterial protein toxins and on the assembly of the SNARE apparatus. 

He has published more than two hundred articles in international scientific journals as well as two 

books. Prof. Montecucco received the 1993 prize of Harvard Medical School, the 1998 prize of 

the Italian Consortium for the Biotechnologies, the 2000 prize of the Deutsche Gesellschat fur 

Hygiene und Microbiology, the 2003 prize of the Masi Foundation for the Venetian Civilization 

and the 2004 Feltrinelli Prize for Medicine. He has served or is serving in several Editorial Boards 

of scientific journals and in the Scientific Councils of major research institutions. He is member of 

EMBO, Academia Europaea, Leopoldina Academy and Istituto Veneto di Scienze Lettere ed Arti. 

Rigoni, M et al. Equivalent effects of snake PLA2 neurotoxins and lysophospholipid-fatty acid 

mixtures. Science (2005) 310, 1678-1680 

Tonello et al. Pharmacology: Screening inhibitors of anthrax lethal factor. Nature (2002) 418, 

386. 

Montecucco, C. & Rappuoli, R. Living dangerously: how Helicobacter pylori survives in the human 

stomach. Nature Rev. Cell Biol. (2001) 2, 457-466. 

Schiavo G et al. Identification of the nerve-terminal targets of botulinum neurotoxins serotypes A, 

D and E. J. Biol. Chem. (1993) 268, 23784-23787. 

Schiavo G et al. Tetanus and botulinum B neurotoxins block neurotransmitter release by 

proteolytic cleavage of synaptobrevin. Nature (1992), 359, 832-835. 

37

9.45-10.30: How snake and bacterial presynaptic enzyme toxins block nerve 

terminals 

Cesare MONTECUCCO 

Department of Biomedical Sciences, University of Padova, Italy 

e.mail: cesare.montecucco@unipd.it 

Botulinum neurotoxins (BoNTs) and snake presynaptic phospholipase A2 neurotoxins 

(SPANs) are very potent toxins which paralyze the neuromuscular junction (NMJ), 

without killing the nerve, which fully recovers its function. BoNTs bind to the 

presynaptic membrane and are internalized inside synaptic vesicles whereform they enter 

the cytosol. Here, they display a very specific proteolytic activity versus any of the three 

SNARE proteins which form the core of the neuroexocytosis apparatus. Consequently, 

the process of synaptic vesicles fusion with the nerve membrane is prevented, no 

neurotransmitter is released and the synapse is blocked. SPANs bind to the presynaptic 

membrane and act on the cell surface by hydrolysing phospholipids with production of 

fatty acids (FA) and lysophospholipids (LPL). FA partition between the two leaflets of 

the membrane bilayer, whilst LPL remains on the external leaflet. This membrane 

configuration promotes the transition from an hemifuion membrane intermediate to 

membrane pore formation with release of neurotransmitter. At the same time, for the 

same reason, vesicle endocytosis is inhibited. This SPAN action leads to synaptic vesicle 

depletion and nerve terminals are paralysed. Thus, the two groups of neurotoxins have an 

opposite effects on the process of neurotransmitter release, yet achieve the same goal of 

paralysing nerve terminals causing animal death by respiratory failure. The elucitdation 

of their mechanism of action provides a strong evidence that SNARE proteins are 

essential for exocytosis and that the formation of hemifusion membrane intermediates is a 

key steps in membrane fusion during exocytosis (1). 

(1) Rossetto O, Morbiato L, Caccin P, Rigoni M, Montecucco C. Presynaptic enzymatic 

neurotoxins. J Neurochem. (2006) 97:1534-45. 

38 

The research of Dr Petrovič is focused on the molecular mechanisms and physiological role of 

deacylating phospholipases in yeast Saccharomyces cerevisiae. Genome-wide approaches, such 

as DNA microarrays and synthetic genetic array analysis, are being used to identify the molecular 

targets of activity of both exogenous and endogenous enzymes. Ammodytoxin, a neurotoxic 

secretory phospholipase A2 from the long-nosed viper venom, has been used as a model to 

understand the mechanism of activity of neurotoxic sPLA2s in his work. One of the aims of his 

research is to develop yeast as a robust model system to analyze on a genome-wide scale the 

molecular mechanisms of intracellularly acting toxic proteins and peptides. Dr Petrovič is also 

involved in development of bioinformatics tools for analysis of functional genomics data. 

Selected publications: 

Petrovič U, Šribar J, Matis M, Anderluh G, Peter-Katalinič J, Križaj I, Gubenšek F. (2005) 

Ammodytoxin, a secretory phospholipase A2, inhibits G2 cell cycle arrest in yeast S. cerevisiae. 

Biochem J. 391:383-388. 

Petrovič U, Šribar J, Pariš A, Rupnik M, Kržan M, Vardjan N, Gubenšek F, Zorec R, Križaj I. 

(2004) Ammodytoxin, a neurotoxic secreted phospholipase A2, can act in the cytosol of the nerve 

cell. Biochem Biophys Res Commun 324:981-985. 

Curk T, Demšar J, Xu Q, Leban G, Petrovič U, Bratko I, Shaulsky G and Zupan B. (2004) 

Microarray data mining with visual programming. Bioinformatics 21:396-398. 

39

10.30-11.15: Genome-wide analysis of the molecular mechanism of action of 

ammodytoxin, a neurotoxic sPLA2, in yeast cells 

Petrovič, U.* 1 , Mattiazzi, M. 1 , Cvetek, J. 1 , Jenko, Z. 1 , Logonder, U. 1 , Bavdek, A. 2 , Kovačič, L. 1 , 

Pungerčar, J. 1 , Rowan, E.G. 3 , Anderluh, G. 2 and Križaj, I. 1 

1 Jožef Stefan Institute, Jamova 39, Ljubljana, Slovenia, * uros.petrovic@ijs.si 

2 University of Ljubljana, Dept. of Biology, Večna pot 111, Ljubljana, Slovenia 

3 University of Strathclyde, SIBS, 27 Taylor Street, Glasgow, Scotland, UK 

Neurotoxic secretory phospholipases A2 (sPLA2s) are enzymes of substantial medical and 

potential pharmacological importance. However, reliable treatment of intoxication caused 

by them, and their biotechnological exploitation are both hindered by lack of 

understanding of their mechanism of action. We have been using a genetically tractable 

model organism, yeast Saccharomyces cerevisiae, to obtain a global insight into the 

mechanism of action of ammodytoxin, the neurotoxic sPLA2 from the venom of the longnosed 

viper. This approach is useful in generating hypotheses for its mechanism of action 

in mammalian systems, which can then be tested in relevant cellular models. We 

demonstrated that intracellular, active ammodytoxin inhibits endocytosis in yeast cells. By 

expressing ammodytoxin in its active form in the cells of all ~4700 viable yeast single 

gene deletion mutant strains we identified the proteins with endocytosis-related function 

that are specifically affected by ammodytoxin. Subsequent experiments suggest that in the 

yeast cells ammodytoxin binds to 14-3-3 proteins and is thus recruited onto clathrin-coated 

membrane invaginations, where it presumably inhibits the scission step in the generation of 

endocytotic vesicles. In another example, the combination of the yeast mutant approach 

with transcriptome analysis revealed the possible involvement of the yeast homologue of 

the AMP-dependent kinase in ammodytoxin-mediated cellular events. Subsequent 

experiments on neuromuscular preparations showed that, by pharmacologically modifying 

the AMP-dependent kinase activity, it is possible to modulate the level of motor neurontriggered 

muscle contraction. In the case of ammodytoxin, the yeast model system thus 

proved to be effective in identifying potential molecular targets of a vertebrate toxin. 

This work has been funded partially by the Slovenian Research Agency (grant J1-6507) 

and by the NATO Security Through Science Programme (grant 980899). 

o phospholipase A2 

o neurotoxicity 

o Saccharomyces cerevisiae 

o functional genomics 

40 

José María Gutiérrez has a BS in Microbiology from 

the University of Costa Rica (1977) and PhD in Physiological Sciences from Oklahoma State 

University (USA). Since 1977, he has been working at Instituto Clodomiro Picado, University of 

Costa Rica, with teaching responsibilities at the School of Microbiology, University of Costa 

Rica. From 1988 to 1996, he was appointed Director of this Institute. From 1999 to 2005, he was 

Head of the Research Division of this Institute, and currently he is Subdirector of the 

Institute. From 1986 to 1988, he was Director of the Graduate Program in Microbiology. 

University of Costa Rica. 

Dr Gutiérrez was awarded the National Award in Science and Technology in Costa Rica (1980), 

the TWAS-CONICIT award for young scientists of Costa Rica in the field of Biological Sciences 

(1990), and the Sven Brohult Award, given by the International Foundation for Science (1997). 

His fields of interest have been mostly (a) the study of the biochemical characteristics and 

mechanism of action of snake venom toxins involved in the local pathology (hemorrhage and 

myonecrosis) of envenomation, particularly those of Bothrops sp venoms, (2) the study of the 

ability of antivenoms to neutralize the most relevant snake venoms in Latin America, and (3) the 

study of novel technological ways to improve the quality of antivenoms for the treatment of 

snakebite envenomations. As a result of these research efforts, he has authored or co-authored 

250 papers in scientific journals. 

Mora, R., Valverde, B., Díaz, C., Lomonte, B. & Gutiérrez, J.M. (2005) A Lys49 phospholipase A2 

homologue from Bothrops asper snake venom induces proliferation, apoptosis and necrosis in a 

lymphoblastoid cell line. Toxicon 45: 651-660. 

Teixeira, C.F.P., Chaves, F., Zamunér, S.R., Fernandes, C.M., Zuliani, J.P., Cruz-Hofling, M.A., Fernandes, 

I. & Gutiérrez, J.M. (2005) Effects of neutrophil depletion in the local pathological alterations and muscle 

regeneration in mice injected with Bothrops jararaca snake venom. Int. J. Exp. Pathol. 86: 107-115. 

Gutiérrez, J.M., Rojas, E., Quesada, L., León, G., Núñez, J., Laing, G.D., Sasa, M., Renjifo, J.M., Nasidi, A., 

Warrell, D.A., Theakston, R.D.G. & Rojas, G. (2005) Pan-African polyspecific antivenom produced by 

caprylic acid purification of horse IgG: an alternative to the antivenom crisis in Africa. Transactions of the 

Royal Society of Tropical Medicine and Hygiene 99, 468-475. 

Gutiérrez, J.M., Rucavado, A., Escalante, T. & Díaz, C. (2005) Hemorrhage induced by snake venom 

metalloproteinases: biochemical and biophysical mechanisms involved in microvessel damage. Toxicon 45, 

997-1011. 

Flores-Díaz, M., Alape-Girón, A., Clark, G., Catimel, B., Hirabayashi, Y., Nice, E., Gutiérrez, J.M., Titball, R. 

& Thelestam, M. (2005) A cellular deficiency of gangliosides causes hypersensitivity to Clostridium 

perfringens phospholipase C. Journal of Biological Chemistry 280, 26680-26689. 

Murakami, M.T., Arruda, E.Z., Melo, P.A., Martinez, A.B., Calil-Elias, S., Tomaz, M.A., Lomonte, B., 

Gutiérrez, J.M. & Arni, R.K. (2005) Inhibition of myotoxic activity of Bothrops asper myotoxin II by the antitrypanosomal 

drug suramin. Journal of Molecular Biology 350, 416-426. 

Rucavado, A., Soto, M., Escalante, T., Loría, G.D., Arni, R.K. & Gutiérrez, J.M. (2005) Thrombocytopenia 

and platelet hypoaggregation induced by Bothrops asper snake venom. Toxins involved and their 

contribution to metalloproteinase-induced pulmonary hemorrhage. Thrombosis & Haemostasis 94, 123-131. 

Zuliani, J.P., Gutiérrez, J.M., Casais e Silva, L.L., Sampaio, S.C., Lomonte, B. & Teixeira, C.F.P. (2005) 

Activation of cellular functions in macrophages by venom secretory Asp-49 and Lys-49 phospholipases A2. 

Toxicon 46, 523-532. 

41

Wednesday 26 July: 9.00 am- 11.15 am 


Symposium: From anecdotes to antidotes 

9.00-9.30: Novel possibilities for the treatment of local effects in snakebite 

envenomation 

Gutiérrez, J.M. 

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica, 

jgutierr@icp.ucr.ac.cr 

The development of severe local pathological alterations, i.e. hemorrhage, edema, 

blistering, myonecrosis and dermonecrosis, constitutes one of the most serious 

consequences of snakebites, which often lead to permanent disability. The pathogenesis of 

these complex alterations is predominantly associated with the direct action of myotoxic 

phospholipases A2, hemorrhagic metalloproteinases and hyaluronidases. In addition, a 

prominent inflammatory reaction develops in the affected tissues, which may further 

contribute to these pathological events. Despite the therapeutic success of antivenoms in 

reducing mortality and neutralizing systemically-acting toxins, they are poorly effective in 

the prevention of local tissue damage. Recent developments in our understanding of the 

biochemistry of locally-acting toxins, the pathogenesis of local effects, and the 

pharmacokinetic-pharmacodynamic relationships of antivenoms have opened a window of 

opportunity for the development of novel therapeutic strategies to confront this aspect of 

snakebite-induced pathology. Some of these possibilities are: (1) The use of phospholipase 

A2, metalloproteinase and hyaluronidase inhibitors, many of which have been developed to 

inhibit endogenous enzymes having homology with snake venom enzymes and being 

currently used in the clinical setting. It is likely that a rapid application of these inhibitors 

at the site of venom injection may block tissue-damaging enzymes in situ. (2) The 

modulation of local inflammatory events, guided by an adequate understanding of the role 

of inflammatory mediators in the pathogenesis of tissue damage, which may halt the 

deleterious effects of inflammation while maintaining the reparative and regenerative roles 

of the tissue response to venom-induced damage. (3) The engineering of antibodies and 

antibody fragments of different molecular characteristics that have the ability to rapidly 

reach tissue compartments. Future developments will undoubtedly contribute to the 

improvement of the therapy of this complex aspect of snakebite envenomation. 

o Snakebite envenomation 

o Local effects treatment 

o Antivenoms 

o Enzyme inhibitors 

42 

Isaac Asuzu obtained his DVM and PhD degrees from the University of Nigeria Nsukka. He was 

previously Head of Department of Veterinary Physiology and Pharmacology and Dean of the 

Faculty of Veterinary Medicine. He is currently a full Professor of Veterinary Pharmacology in the 

Department of Veterinary Physiology and Pharmacology, University of Nigeria and a Fellow of 

Veterinary Surgeons Nigeria (FCVSN). 

Isaac Asuzu has published several scientific papers on the pharmacology and toxicology of 

medicinal plant extracts. He is presently working on plants that have anti-snake venom and antidiabetic 

activities. He interacts with local herbalists in the course of collecting plant materials for 

his studies. Professor Asuzu has visited many laboratories overseas as Visiting Scientist, 

including the University of Strathclyde, Glasgow and the University of Trieste in Italy. He is a 

recipient of the Commonwealth Academic Staff Fellowship and the Wellcome Trust Fellowship. 

Selected Publications 

1. Asuzu, I.U., Gray, A.I. and Waterman, P.G. (1999) The anthelmintic activity of D-3- Omethylchiroinositol 

isolated from Piliostigma thonningii stem bark extract. Fitoterapia. 70, 77. 

2. Uchendu, C.N., Kamalu, T.N. and Asuzu, I.U. (2000) A preliminary evaluation of 

the antifertility activity of a triterpenoid glycoside (DSS) from Dalbergia saxatilis in female Wistar 

rats. Pharmacological Research. 41. 521-525. 

3. Asuzu, I.U. and Harvey, A.L. (2003) The antisnake venom activities of Parkia biglobosa 

(Mimosaceae) stem extract. Toxicon . 42, 763-768. 

4. Anaga, A.O., Njoku, C.J., Ekejiuba, E.S., Esiaka, M.N. and Asuzu, I.U. (2004) Investigations of 

the methanolic leaf extract of Costus afer. Ker for pharmacological activities in vitro and in vivo. 

Phytomedicine 11, 242-248. 

5. Asuzu, I.U. (2005) A review on Croton penduliflorus seed extract. Recent Progress in 

Medicinal Plants. Stadium Press LLC, Texas USA. Pp 32. 

43

9.30-10.00: MYTHS AND REALITIES OF PLANT-BASED REMEDY FOR 

SNAKEBITE 

Isaac U. Asuzu, DVM, MSc., PhD, FCVSN. 

Department of Veterinary Physiology & Pharmacology, 

University of Nigeria, Nsukka 

Enugu State, Nigeria. 

Plant based-remedies have been used since centuries ago for the treatment of snakebites, 

particularly in the rural areas of Africa, South America and Asia. The broad geographic 

distribution of poisonous snakes in general, presumably accounts for at least some of the 

high diversity of plants used as antidotes. Classical pharmacological investigations of 

plant-based remedies tend to narrow down to a single plant, whereas a typical plant-based 

remedy as instituted by a traditional healer involves a group of plant parts, which are 

usually more than three. Among the group, one may contribute the main active 

ingredient, which may block the mechanism of toxicity – the one usually investigated by 

the pharmacologist, while the other members may contribute compounds which have 

palliative effects on the toxic situation. Examples include the relief of pain (analgesic), 

prevention of inflammation (anti-inflammatory), reduction of venom absorption from site 

of envenomation (chelating effect) and prevention of bacterial contamination 

(antibacterial effect) to mention a few. It is therefore not enough to dismiss a plant-based 

remedy as a myth whereas the full compliment of the various contributors to its overall 

activity is not investigated. Although many claims of successful treatment of snakebites 

with plant-based remedies are anecdotal, abundant evidence is available to prove the 

reality of plant-based remedies from laboratory experiments and chemical 

characterisation of the active constituents of the plants. Generally, these plants contain 

bioactive compounds in the form of alkaloids, tannins, steroids, glycosides, phenols and 

volatile oils. These are secondary metabolites that in addition to specific anti-snake 

venom activity exhibit other ancillary but complimentary actions. Most of these plants 

are considered ‘multifunctional’ in the sense that more than one biochemical or 

pharmacological property has been demonstrated for them. Due to the variety of active 

compounds present in any one plant, coupled with their complementary actions, plantbased 

remedies are efficacious for the treatment of snakebites. 

44 

Dr. Chippaux was born in France in 1954 and received his M.D. 

degree in 1980 at the University of Marseille with a thesis on the epidemiology of 

snakebite in Ivory Coast, and his Ph.D. in Public Health at the Université de Paris VI on 

the control of dracunculiasis (Guinea Worm) in 1991. He has resided in Western and 

Central African countries for most of his life, carrying out research in public health issues 

concerning the surveillance, prevention and control of endemic conditions prevalent in 

developing countries such as malaria, schistosomiasis, dracunculiasis, loasis, meningitis 

and envenomations. He is the author of over a hundred articles published in international 

peer-reviewed journals, of numerous chapters in books, and of eight books dealing, 

respectively, with snakes of the Ivory Coast, the snakes of French Guyana, the control of 

dracunculiasis and schistosomiasis in Western Africa, the snakes of Sub-Saharan Africa 

and snake venoms and envenomations. As a researcher of the Institut de Recherche pour 

le Développement (IRD, formerly ORSTOM) he has done research in epidemiology and 

public health as a resident in Ivory Coast, Benin, Cameroon, Niger, Senegal, French 

Guyana, and has developed collaborations with numerous groups in other African 

countries (such as Guinea-Conakry, Burkina Faso, Republic of Congo and Democratic 

Republic of Congo, among others). He is now working in La Paz on the implementation 

of measures for the control of the American trypanosomiasis in endemic regions of 

Bolivia. 

Chippaux JP, Vieillefosse S, Sall O, Mafouta R, Diallo A. Appraisal of snakebite incidence in Senegal, West 

Africa. Bull Soc Pathol Exot. 2005; 98: 277-82. 

Chippaux JP. Ophidian envenomations and emergencies in Sub-Saharan Africa. Bull Soc Pathol Exot. 

2005;98: 263-8. 

Herrera M, Leon G, Segura A, Meneses F, Lomonte B, Chippaux JP, Gutierrez JM. Factors associated with 

adverse reactions induced by caprylic acid-fractionated whole IgG preparations: comparison 

between horse, sheep and camel IgGs. Toxicon. 2005; 46: 775-81. 

Chippaux JP, Rage-Andrieux V, Le Mener-Delore V, Charrondiere M, Sagot P, Lang J. Epidemiology of 

snake envenomations in northern Cameroon. Bull Soc Pathol Exot. 2002; 95: 184-7. 

Chippaux JP, Lang J, Amadi-Eddine S, Fagot P, Le Mener V. Short report: treatment of snake 

envenomations by a new polyvalent antivenom composed of highly purified F(ab)2: results of a 

clinical trial in northern Cameroon. Am J Trop Med Hyg. 1999; 61: 1017-1018. 

Chippaux JP, Amadi-Eddine S, Fagot P. Diagnosis and monitoring of hemorrhage due to viper 

envenomation in the African savanna. Bull Soc Pathol Exot. 1999; 92: 109-13. 

Chippaux JP, Lang J, Eddine SA, Fagot P, Rage V, Peyrieux JC, Le Mener V. Clinical safety of a polyvalent 

F(ab')2 equine antivenom in 223 African snake envenomations: a field trial in Cameroon. VAO 

(Venin Afrique de l'Ouest) Investigators. Trans R Soc Trop Med Hyg. 1998; 92: 657-62. 

Chippaux JP. Snakebites: appraisal of the global situation. Bull World Health Org. 1998; 76: 515-24. 

Chippaux JP. The development and use of immunotherapy in Africa. Toxicon. 1998; 36: 1503-6. 

Chippaux JP, Goyffon M. Venoms, antivenoms and immunotherapy. Toxicon. 1998; 36: 823-46. 

Chippaux JP Les serpents d’Afrique Occidentale et Centrale. IRD éd., Paris, coll. « Faune et flore 

tropicales » 2001. 

Chippaux J.-P.- Venins de serpent et envenimations. IRD éd., Paris, coll. « Didactiques », 2002. 

Chippaux J.-P.- Pratique des essais cliniques en Afrique. IRD éd., Paris, coll. « Didactiques », 2004. 

45

The following presentations represent a report on “An integrated approach to the 

development, optimization and evaluation of a polyvalent antivenom for Sub-Saharan 

Africa” 

10.00-10.20: Management of snake bite in Africa: how to stop the vicious cycle? 

Chippaux, J.-P. 

Institute de Recherche pour le Développement (IRD), La Paz, Bolivia. chippaux@ird.fr 

In Africa, snake bite remains a public health problem that can be summarily described by 

two types of data: i) the high number of annual bites (more than 1 million leading to 

20,000 deaths), and ii) the failure of snake bite management (less than 1% of all 

envenomations are treated with antivenom). Efforts made during the last years to improve 

the quality of antivenom proved to be ineffective to solve this problem. At-risk populations 

are mostly rural, mainly males from 15 to 50 years old. Human activities and snake 

behaviour largely explain man-snake contacts and, as a consequence, the high snake bite 

incidence in most African rural areas, both in savanna and forest. Time of arrival to a 

health care facility has a considerable impact on morbidity and mortality. Delayed 

admission is mainly due to care-seeking behaviour, the scattered distribution of health 

centers and the unavailability of both medical staff and drugs, especially antivenom. The 

antivenom supply is irregular because of its high cost, inadequate organisation of 

distribution, and poor training of medical workers. The less antivenom is used, the more its 

price increases and high costs lead to further reduction in use. Several pragmatic solutions 

would be proposed: a) to target and evaluate needs by enhancing epidemiological studies, 

b) to reduce the antivenom cost by developing antivenom production in pharmaceutical 

industries of emergent countries, c) to improve the marketing of drugs, d) to make 

treatment protocols more explicit and simple, e) to train health workers and f) to sensitize 

the populations to the existence of an effective treatment. Financial and logistical resources 

should be obtained from governments, local communities, private companies and patients 

themselves. 

o Africa 

o Snake bite 

o Epidemiology 

o Antivenom 

46 

Dr. Alagón was born in Mexico in 1954, obtained his M.D. degree and 

a Ph.D. in Biochemistry at the Universidad Nacional Autónoma de México (UNAM) and did 

postdoctoral research at Rockefeller University, specializing in the immunology and biochemistry 

of venoms. He is currently laboratory head at the Instituto de Biotecnología of the UNAM in 

Cuernavaca, where he has been active in the molecular biology of Entamoeba histolytica as well 

as basic and applied toxinology research. He has been particularly interested in the development 

of diagnostic systems and new antivenoms against elapid and viperine snakes, spiders and 

scorpions, and the application of novel technologies to the production, evaluation and quality 

control of therapeutic antibodies in general. He was the discoverer of a powerful thrombolytic 

agent, the plasminogen activator from the saliva of the common vampire bat Desmodus rotundus, 

which is being clinically tested worldwide (phase III) for the treatment of thromboembolic brain 

stroke. He is the author numerous scientific articles in international peer-reviewed journals. 

Almagro JC, Martinez L, Smith SL, Alagon A, Estevez J, Paniagua J. Analysis of the horse V(H) repertoire 

and comparison with the human IGHV germline genes, and sheep, cattle and pig V(H) sequences. 

Mol Immunol. 2005 Dec 6. 

Vazquez H, Chavez-Haro A, Garcia-Ubbelohde W, Mancilla-Nava R, Paniagua-Solis J, Alagon A, Sevcik C. 

Pharmacokinetics of a F(ab')2 scorpion antivenom in healthy human volunteers. Toxicon. 2005;46: 

797-805. 

Chippaux JP, Stock RP, Alagon A. Report of the 2nd International Conference on Envenomations in Africa 

(Deuxieme Colloque International sur les Envenomations en Afrique). Toxicon. 2005; 46: 115-8. 

Sanchez R, Saralegui A, Olivos-Garcia A, Scapolla C, Damonte G, Sanchez-Lopez R, Alagon A, Stock RP. 

Entamoeba histolytica: intracellular distribution of the sec61alpha subunit of the secretory pathway 

and down-regulation by antisense peptide nucleic acids. Exp Parasitol. 2005; 109: 241-51. 

de Roodt AR, Estevez-Ramirez J, Paniagua-Solis JF, Litwin S, Carvajal-Saucedo A, Dolab JA, Robles-Ortiz 

LE, Alagon A. Toxicity of venoms from snakes of medical importance in Mexico. Gac Med Mex. 

2005; 141: 13-21. 

Ramos-Cerrillo B, Olvera A, Odell GV, Zamudio F, Paniagua-Solis J, Alagon A, Stock RP. Genetic and 

enzymatic characterization of sphingomyelinase D isoforms from the North American fiddleback 

spiders Loxosceles boneti and Loxosceles reclusa. Toxicon. 2004; 44: 507-14. 

de Roodt AR, Paniagua-Solis JF, Dolab JA, Estevez-Ramirez J, Ramos-Cerrillo B, Litwin S, Dokmetjian JC, 

Alagon A. Effectiveness of two common antivenoms for North, Central, and South American 

Micrurus envenomations. J Toxicol Clin Toxicol. 2004; 42: 171-8. 

D'Suze G, Moncada S, Gonzalez C, Sevcik C, Aguilar V, Alagon A. Relationship between plasmatic levels of 

various cytokines, tumour necrosis factor, enzymes, glucose and venom concentration following 

Tityus scorpion sting. Toxicon. 2003; 41: 367-75. 

Stock RP, Olvera A, Sanchez R, Saralegui A, Scarfi S, Sanchez-Lopez R, Ramos MA, Boffa LC, Benatti U, 

Alagon A. Inhibition of gene expression in Entamoeba histolytica with antisense peptide nucleic 

acid oligomers. Nat Biotechnol. 2001; 19: 231-4. 

Noeske-Jungblut C, Haendler B, Donner P, Alagon A, Possani L, Schleuning WD. Triabin, a highly potent 

exosite inhibitor of thrombin. J Biol Chem. 1995; 270: 28629-34. 

Noeske-Jungblut C, Kratzschmar J, Haendler B, Alagon A, Possani L, Verhallen P, Donner P, Schleuning 

WD. An inhibitor of collagen-induced platelet aggregation from the saliva of Triatoma pallidipennis. 

J Biol Chem. 1994; 269: 5050-3. 

Schleuning WD, Alagon A, Boidol W, Bringmann P, Petri T, Kratzschmar J, Haendler B, Langer G, Baldus B, 

Witt W. Plasminogen activators from the saliva of Desmodus rotundus (common vampire bat): 

unique fibrin specificity. Ann N Y Acad Sci. 1992; 667: 395-403. 

Cevallos MA, Navarro-Duque C, Varela-Julia M, Alagon AC. Molecular mass determination and assay of 

venom hyaluronidases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Toxicon. 

1992; 30: 925-30. 

47

10.20-10.35: Development of a polyvalent F(ab’)2 antivenom for sub-Sahara African 

snakes. 

Roberto P. Stock 1 , Judith Estévez 2 , Penélope Magaña 2 , Rita Mancilla 2 , Jorge-Paniagua- 

Solís 3 , Jean-Philippe Chippaux 4 and Alejandro Alagón 1, *. 

(1) 

Instituto de Biotecnología-UNAM. Av. Universidad 2001, Cuernavaca, Morelos 62210, Mexico. 

(*) alagon@ibt.unam.mx 

(2) 

Instituto Bioclon, S.A. de C. V., México, DF, 14050, Mexico. 

(3) 

Dirección de Investigación. Laboratorios Silanes S.A. de C.V. México, DF, 83100, Mexico. 

(4) 

Institut de Recherche pour le Développement (IRD), La Paz, Bolivia. 

This work represents our efforts to help to ameliorate the current shortage in antivenom 

production and supply in sub-Saharan Africa and is our response to a call made during the 

WHO workshop on the standardization and control of antivenoms held at the NIBSC, 

Potters Bar, England, 7–9 February 2001. We have prepared a polyvalent antivenom for 

snakes of sub-Saharan African. The final product is composed of purified F(ab’)2 (Fabtherapeutic) 

obtained after pepsin digestion of immunoglobulins from hyperimmunized 

horses (US Pat. 6,709,655 B2). The African Fab-therapeutic complies the same strict high 

standards as for the more than 300,000 vials of antivenom employed per year in Mexico 

with extremely low incidence (less than 1 in 50,000 cases) of severe secondary reactions. 

For this purpose we immunized groups of five horses each with either a viperid or a elapid 

venom mix. The immunogen viperid venom mix consisted of venoms from Bitis arietans, 

B. gabonica, Echis ocellatus, E. pyramidum and E. leucogaster while the elapid mix 

contained venom from the species Naja nigricollis, N. haje, N. pallida, N. melanoleuca, 

Dendroaspis viridis and D. polylepis. The antibody horse response against venoms was 

very high, with neutralization potencies higher than the 250 DL50 per venom species set as 

our lower limit per vial. The only exception was D. viridis in which the neutralization 

potency was not as high as with the other venoms, a problem we are working on at this 

time and that will be solved in the next lots of antivenom. We have already produced 

around 5,000 vials of antivenom and we expect to produce 40,000 more within this year. 

Acknowledgements. We wish to thank Y. Doljansky of Latoxan (Valence, France) for 

valuable advice. 

o African snakes 

o Antivenom 

o Neutralization potency 

o Composition 

48 

Dr. Stock was born in Uruguay in 1963. He obtained a degree in 

Biochemistry at Albright College, Pennsylvania, USA, a M.Sc. degree in Immnunology in the 

Hebrew University of Jerusalem, Israel, and a Ph.D. in Chemical Sciences in the University of 

Granada, Spain. He is laboratory head at the Instituto de Biotecnología of the UNAM in Mexico. 

He has done molecular and cellular biology research on P. falciparum, T. cruzi and E. histolytica, 

and is currently active on the antigenic characterization of venoms of African snakes of medical 

importance (notably of the genera Bitis, Dendroaspis, Echis and Naja) as well as on the 

enzymatic and genetic characterization of the venoms of the spiders of the genera Loxosceles 

and Latrodectus in order to apply techniques of molecular genetics to the production and 

evaluation of antivenoms, such as the utilization of recombinant venom components for the 

production of antivenoms. 

Sánchez, R., Saralegui, A., Olivos-García, A., Scapolla, C., Damonte, G., Sanchez-Lopez, R., Alagón, A. 

and Stock, R.P. (2005) “Entamoeba histolytica: Intracellular distribution of the Sec61 subunit of 

the secretory pathway and down-regulation by antisense peptide nucleic acids.” Exp. Parasitol. 

109: 241-251. 

Chippaux, J-P., Stock, R.P. and Alagón, A. (2005) Report of the 2 nd International Conference on 

Envenomations in Africa (Deuxième Colloque International sur les Envenimations en Afrique). 

Toxicon 46: 115–118. 

Martínez, C., Paredes, R., Stock, R.P., Saralegui, A., Andreu, Cabezon, C., M., Ehrlich, R. and Galanti, N. 

(2005) “Cellular organization and appearance of differentiated structures in developing stages of 

the parasitic platyhelminth Echinococcus granulosus.” J. Cell. Biochem. 94: 327–335. 

Ramos-Cerrillo, B., Olvera, A., Odell, G.V., Zamudio, F., Paniagua-Solís, J., Alagón, A. and Stock, R.P. 

(2004) “Genetic and enzymatic characterization of Sphingomyelinase D isoforms from the North 

American fiddleback spiders Loxosceles boneti and Loxosceles reclusa.” Toxicon 44: 507-514. 

Scarfì, S., Giovine, M., Pintus, R., Millo, E., Clavarino, E., Pozzolini, M., Sturla, L., Stock, R.P., Benatti, U. 

and Damonte, G. (2003) “Selective inhibition of inducible cyclooxygenase-2 expression by 

antisense Peptide Nucleic Acids in intact murine macrophages.” Biotechnol. Appl. Biochem. 38: 61- 

69. 

Cerecedo D., Stock, R.P., González, S., Reyes, E., Mondragón, R. (2002) “Spatial distribution and structural 

correlation of actin, myosin and tubulin during platelet adhesion through confocal microscopy.” 

Haematologica 87:1165-1176. 

Stock, R.P., Olvera, A., Sánchez, R., Saralegui, A., Scarfì, S., Sanchez-Lopez, R., Ramos, M.A., Boffa, L.C., 

Benatti, U. and Alagón, A. (2001) “Inhibition of gene expression in Entamoeba histolytica with 

antisense peptide nucleic acid (PNA) oligomers.” Nature Biotechnol. 19: 231-234. 

Ramos, M.A., Mercado, G.C., Salgado, L.M., Sánchez-López, R., Stock, R.P., Lizardi, P.M. and Alagón, A. 

(1997) “Entamoeba histolytica contains a gene encoding a homologue to the 54 KDa subunit of the 

signal recognition particle.” Mol. Biochem. Parasitol. 88: 225-235. 

49

10.35-10.45: The spectrum of protection: serological cross-reactivity between 

venoms of African vipers and elapids. 

Stock, R.P. 1,* , Estévez, J. 2 , Penélope Magaña, P. 2 , de Roodt, A. 1,5 , Jorge-Paniagua-Solís 3 , 

J., Chippaux, J.-P. 4 and Alejandro Alagón 1 . 

(1) 

Instituto de Biotecnología-UNAM. Av. Universidad 2001, Cuernavaca, Morelos 62210, Mexico. 

(*) rstock@ibt.unam.mx 

(2) 

Instituto Bioclon, S.A. de C. V., México, DF, 14050, Mexico. 

(3) 

Dirección de Investigación. Laboratorios Silanes S.A. de C.V. México, DF, 83100, Mexico. 

(4) 

Institut de Recherche pour le Développement (IRD), La Paz, Bolivia. 

(5) 

National Institute for the Production of Biologicals, Administración Nacional de 

Laboratorios e Instituto de Salud “Dr. Carlos G. Malbran”, Buenos Aires, Argentina. 

The spectrum of protection of an antivenom for snake bite can be significantly increased 

by taking into account the antigenic relationships between species the same genus, thus 

enabling a foreknowledge of the paraspecific neutralization potential of a given mono- or 

polyvalent antivenom. In this study we report a systematic comparison of paraspecific 

recognition of venoms of species belonging to the major genera of snakes of medical 

relevance in Africa in vitro by paraspecific titer determination (by ELISA) and in vivo by 

neutralization of lethality (as ED50) in mice. The genera studied included species of Bitis, 

Echis, Naja and Dendroaspis in vitro using experimental monovalent antisera raised in 

rabbits. Antigenic groupings, as measured by the degree of cross reactivity to the 

monospecific antisera, can be established within each genus. In vivo, a polyvalent 

antivenom (Antiophidien Panafricain ® ) raised against several species belonging to these 

genera significantly neutralizes lethality of venoms of species of the same genera not 

included in the original immunization mixture. In situations such as those prevalent in 

most of Africa, where antivenom distribution (where available) is difficult and the 

epidemiology of snake bite complex and varied, even within countries, the assessment of 

the full spectrum of protection of any given antivenom may considerably simplify 

distribution - by promoting regionalization schemes of the greatest possible amplitude 

(ideally one antivenom for the vast majority of cases in all of Africa)- and therapeutic 

protocols (ideally simplified for treatment of generalized viper and elapid syndromes). 

Also, minimization of the number of venoms used for generation of antivenoms of the 

highest protective potency and widest protective spectrum may have an impact on the cost 

of production and comercialization, and therefore on the implementation of national 

strategies for snake bite treatment in often severely under-funded African public health 

institutions. 

o African snakes 

o In vitro cross-reactivity 

o Antivenom 

o In vivo protection 

50 

Dr. Massougbodji was born in Abomey in 1949. He studied 

medicine in Dakar (Senegal) and Montpellier (France) where he specialized in tropical medicine, 

parasitology and public health. He resides in the Republic of Benin, where he is currently head of 

the Unit of Parasitology, Mycology and Ecology at the Faculté des Sciences de la Santé in 

Cotonou. He has worked on the epidemiology, treatment and control of many endemic infectious 

conditions throughout rural and urban Benin, such as malaria, schistosomiasis, dracunculiasis, 

filariasis and also on clinical and epidemiological aspects of envenomation by snakebite, which in 

some regions of Benin accounts for very high morbidity and mortality rates. He is the author of 75 

scientific publications in national and international scientific and medical journals and a leading 

figure in many programs and courses bearing on the ethics, development, practice and evaluation 

of medicine and medical programs in Africa. 

Lymphatic filariasis in a hyperendemic region : a ten years, follow-up panel survey. Myung K., 

Massougbodji A., Ekoue S., Atchade P., Kiki-Fagla V. Klion A.D. Am. J. Trop. Med. Hyg. 1998; 59: 

222-226. 

Molecular characterisation of Plasmodium reichenowi apical membrane antigen-1 (AMA1), comparison with 

P; falciparum AMA1 and antibodiy-mediated inhibition of red cell invasion. Kocken C.H.M., Narum 

D.L., Massougbodji A., Ayivi B., Dubbeld M. A., Van Der Wel A., Conway D.J., Sanni A. And 

Thomas A.W. Molecular and Biochemical Parasitology 2000; 109: 147 – 156. 

Le risque de paludisme transfusionnel à Cotonou, Bénin. Garba A., Kinde-Gazard D., Makoutode M., Boyer 

N., Ernould J.C., Chippaux J.P. Massougbodji A. Cahiers santé 2000; 10 : 389-92. 

Meningococcal immunisation in Ghana. De Chabalier F., Chippaux J.P., Massougbodji A. Lancet. 2000; 

355: 2252 . 

A randomized, double-blind study on the efficacy and safety of a practical three-day regimen with artesunate 

and mefloquine for the treatment of uncomplicated Plasmodium falciparum malaria in Africa. 

Massougbodji A., Kone M., Kinde-Gazard D., Same-Ekobo A., Cambon N. and Muller A. Trans. R. 

Soc. Trop. Med. Hyg., 2002; 96: 655 - 659. 

Limits and weaknesses of intermittent treatment in malaria prevention. Chippaux JP, Le Hesran Jy, Cot M, 

Massougbodji A. Bull Soc Pathol Exot. 2003;96: 75-6. 

Geoclimatology and severity of snake bite envenomations in Benin. Massougbodji M, Chobli M, Assouto P, 

Lokossou T, Sanoussi H, Sossou A, Massougbodji A. Bull Soc Pathol Exot. 2002; 95: 175-7. 

Epidemiological data on snake bite cases reported in Benin from 1994 to 2000. Fayomi B, Massougbodji A, 

Chobli M. Bull Soc Pathol Exot. 2002;95: 178-80. 

Round table and synthesis of the meeting. Chippaux JP, Goyffon M, Benguedda AC, El Ayeb M, Griguer F, 

Massougbodji A, Mion G. Bull Soc Pathol Exot. 2002; 95: 217-2199 

Comités d’éthique en Afrique de l’Ouest et Afrique Centrale : des prémices concrets pour un partenariat de 

la recherche clinique. Effa P., Massougbodji A., Ntoumi F., Derme A., Ndemanga-Kamoune J., 

Nguembo J., Impouma B., Akue J-P., Ehouman A., Dieye A., Kilama W., Vicari M.,Crawley F P., 

Hirsch F et Debois H. Les Cahiers du C.C.N.E., n037, 2003,45 -49. 

Round table of November 20th, 2004: recommendations for improving the management of envenomations. 

Chippaux JP, Massougbodji A, Goyffon M. Bull Soc Pathol Exot. 2005; 98: 316-9. 

51

10.45-11.05: Clinical trial of a polyvalent antivenom specific for African snakes 

Massougbodji, A. 

Faculté des Sciences de la Santé (FSS), Cotonou, Benin. massougbodjiachille@yahoo.fr 

A clinical trial of Antiophidien Panafricain ® was carried out in 11 health centers or 

hospitals of Benin between July 2005 and June 2006. At the end of April 2006, 317 snake 

bites had been registered, of which 234 were included. The other 83 were not treated by 

Antiophidien Panafricain ® because i) they did not show envenomation, ii) they refused to 

be included, or iii) the antivenom was not available. The average age was 23 with a 

majority of men (163 men versus 71 women) and adults (134 adults versus 100 children 

under 16). The average delay of treatment was approximately 8 hours, with a wide range of 

time (30 minutes to 12 days). A biological (TCTS) and/or clinical hemorrhagic syndrome 

was observed in 153 patients out of the 218 among those in which the coagulation status 

was assessed (70%). The average number of doses administered was 3.6 vials of 10 mL. 

Twelve patients presented benign adverse effects (5%). Late tolerance was studied in 46 

patients (20%) and no serum sickness was observed. The average duration of 

hospitalization was 3.4 days. There are slight differences in recruitment characteristics 

between the centers but no significant differences in results between them. Five deaths 

were observed. Three of these deaths were due to late arrival at health centers with already 

severe clinical complications (cerebral hemorrhage, neurotoxic coma). Another one 

followed an envenomation by Atractaspis whose venom is not neutralized by Antiophidien 

Panafricain ® . The last one also arrived with hemorrhagic complications, and died during 

transfer to another center. Lethality was 2.1% of the envenomations, to be compared to a 

lethality of 8% in the group not included for various reasons (83 people) and, within this 

group, to the 12.5% of envenomated patients not treated by Antiophidien Panafricain ® . 

Moreover, historical hospital lethality reaches 24.4% in the same geographical area, 

according the available literature and previous surveys. 

o Clinical trial 

o Antivenom 

o Snake bite 

o West Africa 

52 

Brian Robertson: BSc (Hons) Aberdeen University, 1982; PhD, John Curtin School of Medical 

Research, Australian National University 1986; Postdoctoral Fellow, Sandoz Institute 1986-1988; 

Principal Scientist, Wyeth Research UK 1988- 1994; Governors’ Lecturer, Biochemistry, Imperial 

College London 1995- 2002; Professor of Neuroscience, University of Strathclyde 2002-2004; 

Eberhard Buhl Chair of Neurobiology, University of Leeds (2004 - ); Wellcome Trust 

Neurosciences and Mental Health Committee; Scientific Advisory Board, Biofocus PLC; MRC 

Brain Sciences Panel; Reviewing and Topical Reviews Senior Editor, Journal of Physiology. 

Research programme: Understanding the role of neuronal ion channels 

The challenge of understanding brain and neuronal function in health and disease requires an 

interdisciplinary attack at multiple levels, from genomic information through molecular, cellular 

and systems approaches. My background is in physiology with my present research focussing 

mainly on the characterization of voltage-gated potassium channels. Potassium channels are 

crucial regulators of the excitability of nerve cells, and they have consequently become an 

important target for academic and drug research. Indeed, I first worked on these in the 

pharmaceutical industry. My interests and experience ranges from obscure biophysical 

measurements of channel function up to complex neuronal networks in brain slices and isolated, 

intact, cerebellum. Throughout my career, I’ve come back again and again to using toxins as 

selective tools to dissect out roles of specific ion channels. Our lab employs a wide range of 

techniques and has developed some novel preparations. At the most basic level, we employ site 

directed mutagenesis techniques to unravel which parts of cloned potassium channel subunits 

contribute to their opening and their distinctive pharmacology. Furthermore, we examine ‘native’ 

voltage gated potassium currents in nerve cells, trying to understand the molecular identity of 

these using antibody labelling and physiological and pharmacological ‘fingerprinting’ and 

transgenic animals. 

Another major interest in our lab is the study of changes in sensory neurons following nerve 

injury. Neuropathic pain is a huge health problem, and unfortunately, despite intensive study, we 

still know very little about the fundamental mechanisms involved. Using animal and organotypic 

culture models, we are investigating excitability changes often associated with anatomical 

remodelling, including sprouting of axonal terminals around primary afferent somata in dorsal root 

ganglia. 

elected recent publications: 

JM MILLAR, L BARRATT, AP SOUTHAN, KM PAGE, REW FYFFE, B ROBERTSON, & A MATHIE (2000) A functional 

role for the two-pore domain potassium channel TASK-1 in cerebellar granule neurons. Proceedings of the National 

Academy of Sciences (USA) 97, 3614-3618. 

T BUSHELL, C CLARKE, A MATHIE & B ROBERTSON (2002) Pharmacological characterisation of a non-inactivating 

outward current in mouse cerebellar Purkinje neurones. British Journal of Pharmacology 135, 705-712. 

NP MORRIS, REW FYFFE & B ROBERTSON (2004) Characterisation of the hyperpolarization-activated current (Ih) in the 

medial septum/diagonal band complex in the mouse. Brain Research, 1006, 74-86. 

SYM YEUNG, D THOMPSON, Z WANG, D FEDIDA, B ROBERTSON (2005) Modulation of Kv3 subfamily potassium 

currents by the sea anemone toxin BDS: Significance for CNS and biophysical studies. Journal of Neuroscience, 25, 

8735-8745 

53

Thursday 27 July: 9.00 am- 11.15 am 


ISN Symposium: Ion channel toxins: tools to explore ion channel 

structure, function and physiology 

Sponsored by the International Society for Neurochemistry 

and supported by the Physiological Society 

9.00- 9.45: Potassium channel toxins - a trail from cortex to channel subtype 

Brian Robertson 

(Eberhard Buhl Professor of Neurobiology, Neuroscience Research Group, Institute of Membrane and 

Systems Biology, University of Leeds, LEEDS LS2 9JT) 

Voltage-gated potassium channels are ubiquitously expressed in neurones and are crucial regulators of 

their excitability, finely tuning firing rates, action potential width, dendritic integration and transmitter 

release. After many years of working with individual cloned potassium (Kv) channels in expression 

systems such as oocytes and cell lines, we decided to apply the results of such physiological and 

pharmacological ‘fingerprinting’ to central neurones, using electrophysiological methods in brain 

slices. We chose to study the basket cell–Purkinje cell connection in the mouse cerebellum. The 

cerebellum has a relatively simple architecture and it is straightforward to identify most target cells. 

Several groups showed with immunocytochemical techniques that the basket–Purkinje cell synapse 

was particularly well endowed with Kv1 subunit channels, of which three (Kv1.1, 1.2 and 1.6) are 

sensitive to dendrotoxins derived from mamba venom. (It is also of interest to note that basket cells 

synapse around the cell soma and axon initial segment of Purkinje cells, and are therefore ideally 

placed to influence the final output of the cerebellar cortex.) Therefore, studying the potassium 

channels at this connection would offer us a good chance to record the behaviour and determine the 

roles of Kv1 channels in their native environment. We found that about half the voltage activated K + 

current present in the synaptic terminal was due to Kv1 subfamily channels, since dendrotoxins could 

only inhibit half the total conductance. A number of different lines of evidence (physiological, 

immunocytochemical and pharmacological) led us to the conclusion that the remaining current was 

probably from the Kv3 subfamily of K + channels. Around that time, it was suggested by Michel 

Lazdunski and colleagues that the toxin BDS, from sea anemone, was a selective blocker of Kv3.4 

subunits and since we had antibody labelling for this protein in the basket cell terminals we were 

surprised to see no effect of this toxin in our slice patch clamp experiments. In the process of making 

sure the toxin did block Kv3.4 subunits in mammalian cell lines, we also discovered that it inhibited 

Kv3.2 and Kv3.1 subunits equally well. However, BDS did not behave like a classical open channel 

blocker, but instead slowed current activation, shifted voltage dependence to the right and blocked 

Kv3 channels only from the outside and could inhibit both open and closed channels. I will describe 

how we began unravel the sites of action of BDS on Kv3 channels and determine how it acts on Kv3 

channels. 

KEY WORDS: Dendrotoxin; BDS; potassium channel; neurone; cerebellum 

54 

Dr Meunier’s research focuses on understanding, at the molecular level, the dynamics events 

mediating neuronal/hormonal secretions and synaptic plasticity (how neurons survive, connect 

and communicate). To study these fundamental physiological processes, two main strategies are 

used: first, taking advantage of the exquisite selectivity of neurotoxins to selectively dissect these 

mechanisms, and second, examining the role played by members of certain classes of lipid in 

modulating various steps of neuronal secretions. The hope is to establish novel avenues in our 

understanding of neuronal communications that will lead to novel therapeutic strategies to tackle 

neuronal diseases. 

Selected Publications: 

Rickman, C., Meunier, F. A., Binz, T. and Davletov, B. A. (2004). High affinity interaction of 

syntaxin and SNAP-25 on the plasma membrane is abolished by botulinum toxin E. J. Biol. Chem. 

279:644-51. 

Foran, P.G., Davletov, B., and Meunier F. A. (2003). Getting muscles moving again after 

botulinum toxin: novel therapeutic challenges. Trends in Molecular Medicine 9: 9291-9299. 

Meunier, F.A., Feng, Z.P., Molgo, J., Zamponi, G. and Schiavo G. (2002). Glycerotoxin from 

Glycera convoluta stimulates neurosecretion by targeting N-type Ca 2+ channels Cav2.2. EMBO J. 

21: 6733-6743. 

Osborne, S.L., Meunier, F. A. and Schiavo G. (2001). Phosphoinositides as Key Regulators of 

Synaptic Function. Neuron 32: 9-12. 

55

9.45-10.30: Glycerotoxin, a new tool to dissect synaptic vesicle recycling 

Frederic A. Meunier 1 , Jordi Molgo 2 , Giampietro Schiavo 3 

1 Molecular Dynamics of Synaptic Function Laboratory, The School of Biomedical 

Sciences, The University of Queensland, St. Lucia, 4072 Queensland, Australia; 

2 Laboratoire de Neurobiologie Cellulaire et Moléculaire, Unité Propre de Recherche 

9040, Centre National de la Recherche Scientifique, 1 avenue de la Terrasse, 91198 Gifsur-Yvette, 

France; 3 Molecular Neuropathobiology Laboratory, Cancer Research UK 

London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, 

London WC2A 3PX, UK 

Multi-quantal release of neurotransmitter is driven by voltage-gated calcium channels, 

including Cav2.2 (N-type), which tightly couple action potentials to neurotransmitter 

release. Following stimulation of neurotransmitter release, the number of synaptic 

vesicles at the presynaptic nerve terminal is maintained via the vesicle recycling process. 

We have recently discovered that glycerotoxin (GLTx), a 320 kDa neurotoxin purified 

from the venom of the polychaete annelids Glycera convoluta, selectively activates 

Cav2.2 channels and promotes a large increase in neurotransmitter release in Cav2.2expressing 

neurons from the peripheral and central nervous system (Meunier et al., 2002; 

Schenning et al., 2006). 

Surprisingly, we found that GLTx stimulates spontaneous release of ACh at very high 

rate (20-100 quanta/s) for more than 10 hours at the amphibian neuromuscular junction. 

An estimate of the number of vesicle undergoing exocytosis during that period of time 

largely exceeds the number of vesicles present in motor nerve terminals. 

Ultrastructural examination of GLTx-treated nerve terminals revealed no significant 

changes in the number of synaptic vesicles. Using styryl dyes we found that GLTx upregulates 

synaptic vesicle recycling, an effect blocked by Cav2.2 selective inhibitor ωconotoxin 

MVIIA. After photoconverting the styryl dye, electron microscopy 

examination revealed that most recycled vesicles emanate from large endosomal 

structures. Our initial analysis shows that the majority of recycling vesicles were 

undistinguishable in size and shape from small synaptic vesicles. 

Our experimental data suggest that synaptic vesicle recycling is selectively enhanced 

through the specific activation of Cav2.2. GLTx therefore represents a new tool with 

potential to further dissect the role of Cav2.2 channels in controlling the coupling between 

exocytosis and endocytosis in presynaptic terminals. 

Meunier et al., EMBO J (2002) 21(24):6733-43. 

Schenning et al., J. Neurochem. (2006) Epub. 

56 

Michael Gurevitz was born in 1946 in Germany and received his BS, MSc 

and PhD degrees at the Hebrew University of Jerusalem. He spent six years (1981-1986) in the 

United States before he joined Tel Aviv University, where he serves now as a Professor at The 

Department of Plant Sciences. His interests divide between toxinology and plant molecular 

biology. In the early 90s he has established a successful expression system for scorpion toxins 

affecting voltage-gated sodium channels, which has been used extensively to study structureactivity 

relationship and the mechanism by which these toxins interact with their target channels. 

The Gurevitz laboratory focuses in their toxinology research on molecular mechanisms underlying 

the mode of action and specific recognition of insect and mammalian Na + channels by a variety of 

pharmacologically distinct scorpion toxins. This study is motivated by the need to develop new 

approaches to the design of environmentally-safe, biodegradable insecticides and selective drugs 

in medicine. The experimental approach combines methods of various disciplines (molecular 

genetics, neuropharmacology, electrophysiology, structural biology) and involves extensive 

collaboration with several research groups in Israel and abroad. 

Cohen L, Karbat I, Gilles N, Froy O, Corzo G, Angelovici R, Gordon D & Gurevitz M (2004) 

Dissection of the functional surface of an anti-insect excitatory toxin illuminates a putative ‘hot 

spot’ common to all scorpion α-toxins affecting Na + channels. J Biol Chem 279:8206-8211. 

Karbat I, Cohen L, Gilles N, Gordon D & Gurevitz M (2004) Conversion of a scorpion toxin 

agonist into an antagonist highlights an acidic residue involved in voltage sensor trapping during 

activation of neuronal Na + channels. FASEB J 18:683-689. 

Karbat I, Frolow F, Froy O, Gilles N, Cohen L, Turkov M, Gordon D & Gurevitz M (2004) 

Molecular basis of the high insecticidal potency of scorpion α-toxins. J Biol Chem 279:31679- 

31686. 

Tan J, Liu Z, Wang R, Huang Z, Chen AC, Gurevitz M & Dong K (2005) Identification of amino 

acid residues critical for pyrethroid binding to insect sodium channels. Mol Pharmacol 67:513- 

522. 

Corzo G, Escoubas E, Villegas E, Karbat I, Gordon D, Gurevitz M, Nakajima T & Gilles N (2005) 

A spider toxin that induces a typical effect of scorpion α-toxins but competes with β-toxins on 

binding to insect sodium channels. Biochemistry 44:1542-1549. 

Cohen L, Karbat I, Gilles N, Ilan N, Gordon D & Gurevitz M (2005) Common features in the 

functional surface of scorpion β-toxins and elements that confer specificity for insect and 

mammalian voltage-gated Na-channels. J Biol Chem 280:5045-5053. 

Strugatsky D, Zilberberg N, Ilan N, Turkov M, Cohen L, Stankiewicz M, Pelhate M, Gilles N, 

Gordon D & Gurevitz M (2005) Expression of a gene family encoding a novel scorpion 

depressant toxin illuminates the key role of asparagine-58 in activity on insect neuronal sodium 

channels. Biochemistry 44:9179-9187. 

57

10.30-11.15 : Structural commonality versus functional specificity in scorpion toxins 

that affect voltage-gated sodium channels 

Izhar Karbat 1 , Lior Cohen 1 , Nitza Ilan 1 , Roy Kahn 1 , Michael Turkov 1 , Maya Gur 1 , 

Nicolas Gilles 2 , Dalia Gordon 1 & Michael Gurevitz 1 

1 Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv 

University, Ramat Aviv 69978, Israel; 2 CEA Saclay, Département d’Ingéniérie des 

Protéines, Gif-sur Yvette, France 

The functional surface of scorpion toxins representing all major pharmacological 

groups (anti-mammalian α, α-anti-insect, α-like, anti-mammalian β, anti-insect 

depressant and excitatory) was elucidated by mutagenesis followed by toxicity, 

binding, electrophysiological, and structural studies. Despite the vast differences in 

their effects on voltage-gated sodium channels (Navs), in all toxins the loop preceding 

the α-helix motif and the C-tail are important for function, which may result from their 

common ancestry and structural resemblance. Molecular dissection of alpha-toxins 

revealed two bioactive domains: a conserved Core-domain, formed by residues of the 

molecule core, and a variable NC-domain, formed by the five-residue-turn and the Ctail, 

and shown to be associated with toxin selectivity. This was verified by exchange of 

active sites between two distinct toxins leading to inversion of selectivity toward insect 

and mammalian brain channels. Analysis of the electrophysiological effects of alphatoxin 

mutants and chimeras on the rat brain channel Nav1.2a has suggested a two-step 

binding, first of the Core-domain that associates with a solvent accessible channel 

region, followed by interaction of the NC-domain with another less accessible channel 

region. In β-toxins, mutagenesis of the anti-insect excitatory and depressant toxins, BjxtrIT 

and LqhIT2, and the anti-mammalian β-toxin, Css4, highlighted a common 

‘pharmacophore’ whereas their C-tail region was associated with toxin specificity. A 

conserved glutamate (E15) in Css4 was shown to be involved in ‘trapping’ of the 

channel voltage sensor. Substitution E15R in Bj-xtrIT and Css4 abolished activity with 

no change in binding affinity. The Bj-xtrIT-E15R mutant has become an efficient 

antagonist of the unmodified toxin. These studies contribute to clarification of the 

mechanism by which scorpion toxins interact with Navs, and may pave the way for 

design of insecticides and drugs with high specificity to Nav subtypes. 

58 

Kenneth Clemetson 

Born 1943 in Newcastle-upon Tyne, England 

Christ’s College/Dept. of Chemistry, Univ. of Cambridge PhD, 1968 in organic 

chemistry (affinity-labelling with galactosides). 

Post-Docs at the University of Alberta, Edmonton, Canada 1968-1970 and the 

University of California, Santa Barbara 1970-1972 on carbohydrate chemistry 

(antibiotic synthesis) and then its applications in biochemistry and microbiology. 

Theodor Kocher Institute since December 1972 with Ernst Lüscher and as 

Professor of Biochemistry since 1990. Initially, studies on techniques for the 

isolation and characterisation of cell receptors and then with platelets from 1974. 

Prizes/Honours: 

1990 Mogens-Ellerman Prize of the Swiss Society of Haematology 

1991- date President, Berne Biochemical Society 

1993-1997 President, European Thrombosis Research Organization 

1998-2004 Member of Council, International Society on Thrombosis and 

Haemostasis 

2000 Owren Lecturer, University of Oslo, Norway 

2002 Fellow Royal Society of Chemistry 

2003 Distinguished Career Award, International Society on Thrombosis and 

Haemostasis 

Highlights of research: 

Isolation and characterisation of platelet receptors. Platelet receptors in bleeding 

disorders. 2D separations of carbohydrate surface labelled platelets (early 

proteomics!). First identification of several platelet receptor components. Cloning 

GPVI. Platelet receptor signalling. Crystal structure of GPIb. COAT platelets/role 

of serotonin. Snake venom proteins and platelet receptors. 

59

Thursday 27 July: 3.00 pm- 5.30 pm 


Symposium: Toxins and hemostasis 

Incorporating a meeting of the ISTH SSC Registry of Exogenous Hemostatic Factors 

Supported by Latoxan, Pentapharm and Venom Supplies Pty Ltd 

3.00-3.30: Snake venom proteins affecting platelets 

Kenneth J. Clemetson and Jeannine M. Clemetson 

Theodor Kocher Institute, University of Berne, Freiestrasse 1, CH-3012, Berne, 

Switzerland 

Snake venoms are complex mixtures of biologically active proteins and peptides. These 

components have been classified into various families, such as serine proteases, 

metalloproteinases, C-type lectins, disintegrins and phospholipases. Many of them affect 

haemostasis by activating or inhibiting coagulant factors or platelets, or by affecting 

endothelial or smooth muscle cells. Venom proteins can affect platelet function by 

binding to or proteolysing platelet receptors to prevent or, more often induce, platelet 

activation or degrade vWF or cause it to bind to GPIb and activate platelets. Thus, there 

are disintegrins in nearly all venoms that block the integrin IIb 3 on platelets and a 

variety of others have been identified that affect other integrins, such as 2 1, present 

on platelets and other vascular cells. C-type lectin-like proteins are a major group, either 

inhibiting platelet function by blocking receptors such as GPIb and 2 1, or, when 

multimeric, by clustering receptors such as GPVI and GPIb to activate platelets and thus 

remove them more efficiently. Metalloproteases are also constituents of major classes 

binding to receptors and either blocking function directly or by targeted cleavage. 

Phospholipases A2 are another major class blocking or activating platelet function and, 

since many are not enzymatically active, must function via specific receptors. 

Other venom components act by cleaving protease-activated receptors or modulating 

released ADP and thromboxane A2 formation. Some venom enzymes cleave key 

basement membrane components and directly affect platelet interactions with capillary 

blood vessels to cause haemorrhaging. L-amino acid oxidases activate or, in some cases, 

inhibit platelets via H2O2 production. 

o C-type lectins 

o Disintegrins 

o L-amino acid oxidases 

o Metalloproteinases 

o Phospholipases 

o serine proteases 

60 

Aura Kamiguti: 

BSc degree in 1970 (Pharmacy and Biochemistry), University of Sao Paulo, Brazil, 

MSc in 1988 (Pharmacology), University of Sao Paulo, Brazil and, PhD in 1995 (Haematology), University 

of Liverpool, U.K. 

Retired Lecturer (since October 2005), Dept. of Haematology, University of Liverpool. Member 

of the International Society of Thrombosis and Haemostasis (ISTH); of the Subcommittee on 

Exogenous Products Active on Haemostasis for the International Society of Thrombosis and 

Haemostasis; of the International Society of Toxinology, and of the Brazilian Society of 

Toxinology. Major interest in haemostastic defects due to exogenous substances, in particular 

snake venom metalloproteinases with platelets, and in cell signalling in platelets and in malignant 

B cells. Intense collaboration with different groups with similar aims. Participation in several 

scientific events as invited speaker by organisers of meetings on behalf of above societies. 

Kamiguti AS, Serrander L, Lin K, Harris RJ, Cawley JC, Allsup DJ, Slupsky JR, Krause KH and 

Zuzel M (2005) Expression and activity of NOX5 in the circulating malignant B cells of hairy-cell 

leukaemia. J. Immunol. 175, 8424-8430. 

Laing GD, Compton SJ, Ramachandran R, Fuller GL, Wilkinson MC, Wagstaff SC, Watson SP, 

Kamiguti AS, Theakston RDG and Senis YA (2005) Characterization of a novel protein from 

Proatheris superciliaris venom: proatherocytin, a 34-kDa platelet receptor PAR1 agonist. Toxicon 

46, 490-499. 

Allsup DJ, Kamiguti AS, Lin K, Sherrington PD, Matrai Z, Slupsky JR, Cawley JC and Zuzel M 

(2005) B-cell receptor translocation to lipid rafts and associated signaling differ between 

prognostically important subgroups of chronic lymphocytic leukaemia. Cancer Res. 65, 7328- 

7337. 

Howes JM, Kamiguti AS, Theakston RDG, Wilkinson MC and Laing GD (2005) Effects of three 

novel metalloproteinases from the venom of the West African saw-scaled viper, Echis ocellatus 

on blood coagulation and platelets. Biochim. Biophys. Acta 1724, 194-202. 

Kamiguti AS (2005) Platelets as targets of snake venom metalloproteinases. Toxicon 45, 1041- 

1049. 

61

3.30-4.00: Platelet interaction with multidomain soluble snake venom 

metalloproteinases 

Aura S. Kamiguti 

Department of Haematology, University of Liverpool 

Platelets play a central role in haemostasis during vascular injury. Adhesion of these cells 

to the exposed sub-endothelium, essential to stop haemorrhage, is initiated by the 

engagement of platelet surface glycoproteins to collagen fibres and collagen-bound von 

Willebrand factor (vWF). Following systemic envenoming by viperid or crotalid sankes, 

powerful enzymes such as snake venom metalloproteinases (SVMPs) contribute to 

haemorrhage. This is mainly attributed to their proteolytic activity on the extracellular 

matrix surrounding blood vessels. In addition, SVMPs are capable of inhibiting platelet 

responses to collagen. These secreted enzymes are multidomain proteins 

(metalloproteinase, disintegrin-like and cysteine-rich domains). Proteins containing both 

the disintegrin-like and cysteine-rich domains have been either purified or expressed and 

have been demonstrated to have similar inhibitory effects on the platelet/collagen 

interaction. This observation has prompted investigations on the identification of the 

site(s) in both domains responsible for such effects. Both domains in fact contain relevant 

sequences that have the ability to inhibit platelet interaction with collagen. 

62 

Russolini Zingali 

1986 B.S. São Paulo University (Pharmacist) 

1989 M.Sc. Federal University from São Paulo (Molecular Biology) 

1993 Ph.D. Federal University from Rio de Janeiro (Biological Chemistry) 

Professional Experience / Present Position: Associate Professor, Institute of Medical 

Biochemistry, since January 1995, Universidade Federal do Rio de Janeiro, Rio de Janeiro. 

Research Interest: Snake venoms components acting on hemostasis 

My laboratory has been mainly involved in the characterization of anti-hemostatic compounds, 

having in mind the search for peptides and proteins which can be used as prototypes and models 

for the development of antithrombotic drugs. 

The major objective of the laboratory is to characterize in detail, in vitro as well as in vivo, the 

mechanism of action of snake venoms components that have a potential anti-hemostatic action. 

Lately, my interest has also turned to the suitable proteomics techiniques for the analysis of 

complex biological fluids such as venoms and also their effects on different cells and tissues. 

1. Castro KN, Carvalho AL, Almeida AP, Oliveira DB, Borba HR, Costa SS, Zingali RB. 2003 

Preliminary in vitro studies on the Marsypianthes chamaedrys (boia-caa) extracts at 

fibrinoclotting induced by snake venoms. Toxicon. Jun;41(7):929-32. 

2. Castro HC,. Lemos MGJ, Bon C, Zingali RB (2003). Comparative evaluation of immunological 

and structural similarities of snake venom C-type lectin proteins” Toxicon 41: 525-528. 

3. Moreira MF Coelho HSL, Zingali RB, Oliveira PL, Masuda, H (2003) Nitrophorins during life 

cycle of the blood-sucking bug Rhodnius prolixus. Insect Biochemistry and Molecular 

Biology. 33(1):23-8 

4. Monteiro R Q., Foguel D, Castro HC., and Zingali R B.(2003) “Subunit dissociation, unfolding, 

and inactivation of bothrojaracin, a C-type lectin-like protein from snake venom” 

Biochemistry. 42(2):509-15. 

5. Coelho AL, De Freitas MS, Mariano-Oliveira A, Rapozo DC, Pinto LF, Niewiarowski S, Zingali 

RB, Marcinkiewicz C, Barja-Fidalgo C. RGD- and MLD-disintegrins, jarastatin and EC3, 

activate integrin-mediated signaling modulating the human neutrophils chemotaxis, 

apoptosis and IL-8 gene expression.Exp Cell Res. 2004 Jan 15;292(2):371-84. 

6 .Guimarães-Gomes V (M), Oliveira-Carvalho AL, Junqueira-de-Azevedo ILM; Dutra*, DLS., 

Pujol-Luz M. (IC),. Castro H. C, Lee Ho P e. Zingali RB (2004) Cloning, characterization 

and structural analysis of a C-type lectin from Bothrops insularis (BiL) venom. Arch. 

Biophys and Biochem. 

7. Lima LM; Zingali RB; Foguel D; Monteiro RQ (2004) New Insights On Conformational And 

Functional Stability Of Human [[Alpha]]-Thrombin Probed By High Hydrostatic Pressure 

Eur. J. Biochem. 

8. Wermelinger LS, Dutra DL, Oliveira-Carvalho AL, Soares MR, Bloch C Jr, Zingali RB. (2005) 

Fast analysis of low molecular mass compounds present in snake venom: identification of 

ten new pyroglutamate-containing peptides. Rapid Commun Mass 

Spectrom.;19(12):1703-8. 

9. Soares MR, Oliveira-Carvalho AL, Wermelinger LS, Zingali RB, Ho PL, Junqueira-de-Azevedo 

ID, Diniz MR. (2005). Identification of novel bradykinin-potentiating peptides and C-type 

natriuretic peptide from Lachesis muta venom. Toxicon.;46(1):31-38. 

63

4.00-4.30: Structural and pharmacological studies of bothrojaracin, a 

(pro)thrombin inhibitor 

Zingali, R. B. 1 , Castro, H.C. 2 , Monteiro, RQ 1 , Assafim, M. 1 , Bon C. 3 

1 

Instituto de Bioquímica Médica, CCS, UFRJ; Rio de Janeiro Brazil 

2 

Departamento de Biologia Molecular e Celular. UFF. Rio de Janeiro Brazil; 

3 

Département : Régulations, Développement et Diversité moléculaire Muséum national d'Histoire 

naturelle (MNHN) Paris -France 

Bothrojaracin (BJC), a 27 kDa C-type lectin-like protein from Bothrops jararaca 

venom it has only 11 of the 13 amino-acid residues that are important for the Carbohydrate 

Recognition Domain (CRD), thus it does not bind to carbohydrates. Bothrojaracin-like 

molecules were detected in other snake venoms from Brazil. Structural analysis, by 

molecular modeling, and specially the electrostatic potential map of bothroinsularin 

purified from B. insularis venoms, which is very similar to BJC (α= 94 and β= 98), 

revealed significant differences that may be involved in the biological activity. BJC is a 

selective and potent thrombin inhibitor (KD = 0.6 nM) which interacts with both thrombin 

anion binding exosites (I and II) but not with the enzymes catalytic site. BJC also binds 

with high affinity (KD = 75 nM) to proexosite I. This ability would confer to BJC a new 

mechanism of action for an antithrombotic drug. We further analyzed the in vivo 

antithrombotic effect of BJC on a venous thrombosis model in rats that combines stasis 

and hypercoagulability. It was observed that intravenous administration of 3 mg/kg of 

thromboplastin combined with stasis caused 100% of thrombus incidence weighting 9.1 ± 

2.0 mg. In contrast, co-administration of 1 mg/kg of BJC decreased thrombus weight by 

95% (to 0.5 ± 0.1 mg). This effect was maintained at least for 48 hrs after drug 

administration. Accordingly, ex vivo aPTT was enhanced by 1.7-fold for BJC doses of 1 

mg/kg. In addition, we observed that this BJC doses caused significant hemorrhagic effect 

as compared to control animals. Using another animal model where the thromboembolism 

is induced by thrombin we observed that 80% of animal group died. When BJC (1mg/kg) 

is administered, 60 min prior to thromboembolism induction, it protects 100% of the mice 

group from dead. Western blotting assays using ex vivo rats or mice plasma showed an 

interaction between BJC and prothrombin along 120 min or 12 h, respectively. Altogether, 

our data show that BJC is a potent antithrombotic agent that could further help the 

development of new dual mechanistic drugs directed to prothrombin and thrombin 

inhibition. Financial Support: FAPERJ, IFS, CNPq, and FINEP. 

o Bothrojaracin 

o Bothrops jararaca venom 

o Anti-thrombotic 

o Thrombin inhibitor 

64 

Francis S. Markland, Jr., PhD, is Associate Dean for Scientific Affairs and Professor of 

Biochemistry and Molecular Biology at the University of Southern California (USC) Keck School 

of Medicine. Dr. Markland received his Ph.D. from Johns Hopkins University in 1964 in the 

laboratory of Drs. Albert Lehninger and Charles Wadkins. He then did a two year NIH post 

doctoral fellowship in protein chemistry with Dr. Emil Smith in the Biochemistry Department, 

University of California, Los Angeles, School of Medicine, where he was appointed assistant 

professor in 1966. He received an NIH Career Development Award from 1968-1973 while at 

UCLA. In 1974 he moved to the USC School of Medicine as Associate Professor in the 

Biochemistry Department and was appointed a member of the USC Comprehensive Cancer 

Center. In 1983 he was promoted to Professor of Biochemistry. He served as Acting Chair of the 

Biochemistry Department from 1996-1998 and as Vice Chair from 1988-1992. In 2004 he was 

appointed Associate Dean for Scientific Affairs at the USC Keck School of Medicine. He served 

as a member of the NIH Biochemical Endocrinology Study Section from 1996-2000 and served 

on several study sections during 2002-2003 to evaluate NCI grants in the small business 

program. Dr. Markland is an internationally recognized expert on proteins from snake venoms 

and their potential clinical application. One of the proteins he originally purified from southern 

copperhead, called fibrolase, is presently in clinical trials, in a slightly altered forma and as a 

recombinant protein called alfimeprase, as therapy for peripheral arterial occlusive disease. He is 

presently working with nanospheres (lioposomes) as a delivery modality for a novel antiangiogenic 

agent that his laboratory purified from southern copperhead venom. Recently his 

laboratory perfected a technique, using as highly engineered bacterial expression system, to 

produce a recombinant version of the anti-angiogenic protein, which we now call vicrostatin. 

65

4.30-5.00: Contortrostatin and its anti-cancer action 

Markland, F.S. 1* , Swenson, S. 1 , Minea, R. 1 , Costa, F.K. 1 , Fujii, G. 2 , Ernst, W. 2 , 

Wang, Q. 1 , Pinski, J. 1 

1 

University of Southern California, 1303 North Mission Road, Los Angeles, California, USA 

*markland@usc.edu 

2 

Molecular Express, Inc.,13310 South Figueroa Street, Los Angeles, California, USA 

Introduction: We provide an update on a homodimeric disintegrin, contortrostatin (CN), 

from southern copperhead venom. Recently we developed an expression system to prepare 

a recombinant version of CN and show that, like the natural protein, it has potent antitumor/anti-angiogenic 

activity in vivo. Interestingly, the recombinant protein, which we 

call vicrostatin (VN), is a monomer. Here we describe the anti-cancer activity of CN in 

prostate cancer (PC) and VN in breast cancer (BC) mouse models. Methods: CN was 

purified from venom by a four step HPLC procedure. VN was prepared using a highly 

engineered E. coli expression system. We used xenograft animal models of human PC 

(PC-3) and BC (MDA-MB-435); cells were implanted subcutaneously into the flanks of 

nude mice (PC-3) or into the mammary fat pads (MDA-MB-435). Therapy was initiated 

using an Alzet osmotic pump for CN in PC (315 μg/week) and liposomal delivery for VN 

in BC (LVN, intravenous twice weekly, 210 μg/week). Results: Using combination 

therapy in the PC-3 model, there is a significant enhancement of therapeutic efficacy with 

the combination of CN and docetaxel. Tumor volumes observed after four weeks of 

treatment: control, 378 mm 3 ; CN alone, 195 mm 3 ; docetaxel, 259 mm 3 , and the 

combination, 95 mm 3 . The impressive effect observed with the combination indicates that 

these two drugs complement each other very effectively and may offer a viable clinical 

option for therapy. For studies with VN in the MDA-MB-435 tumor, LVN was 

administered with no visible toxicity; 7-week treatment resulted in significant inhibition of 

tumor growth (80%). These results indicate that VN functions as effectively as CN in BC. 

For analysis of angiogenesis, BC tumor slices from treated and untreated mice were 

prepared for immunohistochemistry and incubated with monoclonal antibody to CD31 to 

detect angiogenic vessels. There is a 92% reduction of angiogenesis (p

5.00-5.30: Origin, evolution and recruitment of venom prothrombin activators in 

Australian Elapids 

Kini RM 

* 

Department of Biological Sciences, Faculty of Science, National University of 

Singapore, Singapore 117543 and 

† Department of Biochemistry, VCU Medical Center, Virginia Commonwealth University, 

Richmond, Virginia 23298-0614, USA 

The parallel prothrombin activator (PA) system of Australian elapids provides an 

excellent opportunity to study proteins with similarity in structure and enzymatic 

properties, but differences in their physiological roles. Their venom contains prothrombin 

activators, which act as a toxin and induce microclots leading to cyanosis and death of the 

prey, whereas their plasma contains blood coagulation factors that activate prothrombin 

during injury and prevent excessive blood loss. These two PA are structurally similar, but 

with distinct physiological roles because of their highly tissue-specific expression. We 

have studied the protein and gene structure of group C and group D PAs. Group C PAs 

resemble factor Xa-Va complex, while group D PAs resemble factor Xa. The plasma 

coagulation factors are structurally similar to the venom PAs. Interestingly, we found an 

evolutionary intermediate between the venom and plasma prothrombin activator in the 

liver of P. textilis (PFX2) which is expressed ~56,000 times lower amounts compared to 

its functional plasma FX (PFX1). Recently, we determined the complete gene structure of 

FX and trocarin D from Tropidechis carinatus including their promoter regions. Both 

these plasma and venom prothrombin activator has similar gene structure. Both genes 

have eight exons with identical exon-intron boundaries. All the introns of trocarin D gene 

are nearly identical to that of FX gene, but for the two deletions (255 bp and 1,406 bp) 

and three insertions (214 bp, 1975 bp and 2174 bp) in the first intron. One of these 

insertions has a potential scaffold/matrix attached region. In addition, trocarin D 

promoter has a 264-bp insertion, which carries core promoter sequences and cis-elements 

that are known to induce high level of expression. This insertion may be responsible for 

the gene recruitment and may act as a switch for venom gland-specific expression. We 

named this segment as VERSE (Venom Recruitment/Switch Element). Our studies 

provide the first molecular evidence for the origin and recruitment of venom PA gene 

from liver PA gene by gene duplication followed by changes in the cis-elements in the 

promoter and first intron leading towards functional diversification. 

68 

Professor André Ménez is the head of the Department of Protein Study and 

Engineering of the Life Sciences Division at the French Atomic Energy 

Commission. He is Professor at the Institute of Technical and Nuclear Sciences. 

He has mostly studied proteins at the molecular level, including several toxins. 

He has received national and international awards, including the Redi Award 

from the International Society on Toxinology (2000). He was recently elected 

President of the IST. He has published more than 260 scientific papers and 

written the book ‘The Subtle Beast. Snakes : from Myth to Medicine’ (Taylor & 

Francis). 

69

Friday 28 July: 9.00 am- 11.15 am 


Symposium: Venomics 

9.00-9.10: Venomics: a project of the IST 

Ménez, A. 1 , Stöcklin, R. 2 and Mebs, D. 3 

70 

Sponsored by 

1 

Département d’Ingénierie et d’Etudes des Protéines, CEA/Saclay, Bât. 152, 91191 Gifsur-Yvette 

cedex, France 

2 

Atheris Laboratories, Case Postale 314, CH-1233 Bernex, Geneva, Switzerland 

3 

Zentrum der Rechtsmedizin, University of Frankfurt, Kennedyallee 104, D-60596 

Frankfurt, Germany 

Venomous animals possess potent factories, the venom glands, which produce a diversity 

of compounds tailored to act efficiently on key physiological systems of vertebrates 

and/or invertebrates. The Venomics project consists in studying the genetics, 

transcriptomics and proteomics of this evolutionary tripartite combination: animal, 

venomous system and toxins. The project is anticipated to clarify various essential 

biological aspects of this harmonious triad, and especially their associated evolutionary 

processes. It is also expected to have important practical consequences, including the 

discovery of new drug candidates and the development of novel protective strategies 

against envenomation. 

- genomics 

- transcriptomics 

- proteomics 

Reto Stöcklin specialises in protein chemistry, mass spectrometry, proteomics 

and biocomputing. He co-founded the Swiss Proteomics Society and is a 

member of its Executive Committee. He is the owner and head of Atheris 

Laboratories, a company he founded in Geneva in 1995, which focuses primarily 

on research and development in life sciences. More recently, he established 

FunZyme BioTechnologies S.A., a company aimed at exploiting enzymes of 

fungal origins. 

71

9.10-9.15: Use of Mass Spectrometry in Venomics 

Reto Stöcklin 

Atheris Laboratories, case postale 314, CH-1233 Bernex-Geneva, Switzerland, 

*reto.stocklin@atheris.ch - http://www.atheris.com 

Due to their complexity and diversity, animal venoms represent an extensive source of 

bioactive compounds for the development of drugs and therapeutic agents with two recent 

approvals (Prialt and Exenatide). Conventional approaches (extraction, fractionation and 

screening) for their characterization often require large quantities of biological material 

and efficient biological assays. Current mass spectrometry (MS) techniques through a 

variety of approaches now give access to a wealth of information in a short working time 

frame with minute sample amount. Currently, MS-based peptide drug discovery may rely 

on computer-assisted strategies going backwards from structural information to functional 

activity. MALDI-TOF MS requires minimal sample preparation and can be used to rapidly 

perform molecular mass fingerprints of venoms at a high sensitivity to identifiy peptide 

families. Further characterization is achieved by de novo MS/MS peptide sequencing. This 

approach offers rapid access to primary peptide structures. The importance of 

complementary genomic and transcriptomic information will be highlighted. 

Favreau P., Menin L., Michalet S., Perret F., Cheneval O., Stöcklin M., Bulet P., Stöcklin 

R., 2006. Mass spectrometry strategies for venom mapping and peptide sequencing from 

crude venoms: case applications with single arthropod specimen. Toxicon, 47(6):676-87. 

Ménez A., Stöcklin R., Mebs, D., 2006. 'Venomics' or: The venomous systems genome 

project. Editorial. Toxicon, 47(3):255-259. 

o Venomics 

o Mass spectrometry 

o Proteomics 

72 

Prof. Dietrich Mebs 

Zentrum der Rechtsmedizin, Johann Wolfgang Goethe University Frankfurt, 

Kennedyallee 104, D-60596 Frankfurt, Germany. 

E-mail: mebs@em.uni-frankfurt.de 

Prof. Dietrich Mebs, born in Frankfurt, Germany, 1942, studied biology and biochemistry 

at the University of Frankfurt. He spent a half year for studies on the biochemistry of 

snake venoms at the Instituto Butantan in São Paulo, Brazil, and obtained his PhD in 

1968 from the University of Frankfurt. After graduating, Prof. Mebs joined the Center of 

Forensic Medicine, where he spent his entire career. As a postdoc researcher he stayed 

for a half year at the Institute of Protein Research, University of Osaka, Japan, in 

1970/71, where he elucidated the primary structure of alpha-bungarotoxin. After his 

habilitation in Forensic Sciences in 1979, he was promoted to Professor in 1985. Beside 

his forensic work in toxicology and DNA-typing his research interest is devoted to all 

aspects of toxinology, e.g. venoms and poisons of plant and animal origin, their 

chemistry and mode of action. Prof. Mebs has published more than 250 scientific articles 

and four books, among them "Venomous and Poisonous Animals" which first appeared 

in German ("Gifttiere") 1992. He teaches forensic medicine, toxicology and toxinology at 

the university as well as overseas (Philippines, Australia, South-Africa). Since 1982, he is 

Secretary-Treasurer of the International Society on Toxinology. 

73

9.15-9.20: Venom glands in old snakes 

Mebs, D. 

Zentrum der Rechtsmedizin, University of Frankfurt, Kennedyallee 104, D-60596 Frankfurt, Germany 

The study of delivery systems, particularly of venom producing glandular tissues, is part of 

the Venomics Project. Snakes developed a highly efficient venom apparatus, but fossil 

records supporting its gradual refinement during evolution are poor. The recent discovery 

of fossil snake fangs in the Oppenheim/Nierstein quarry (Germany) dating back to the 

Lower Miocene (about 23 million years before present) revealed a surprising similarity and 

virtual identity with the fangs of modern elapid and viperid snakes (1). This implies that 

these snakes had venom glands much like their modern counterparts and must have used a 

similar potent mixture of proteins and peptides to quickly subdue their prey. 

Reference: 

1. Kuch, U., Müller, J., Mödden, C., Mebs, D., 2006, Naturwissenschaften 93, 84-87 

o Snake evolution 

o venom glands 

o snake fangs 

74 

Dr. Bryan Grieg Fry has a Ph.D. in Biochemistry from the Centre for Drug Design & 

Development, Institute for Molecular Biosciences and the Department of Biochemistry, University 

of Queensland. He is currently the Deputy Director of the Australian Venom Research Unit at the 

University of Melbourne. 

Fry, BG (2005) “From genome to ‘venome’: Molecular origin and evolution of the snake venom proteome 

inferred from phylogenetic analysis of toxin sequences and related body proteins.” Genome 

Research 15:403-420. 

Fry BG, Lumsden N, Wüster W, Wickramaratna J, Hodgson WC, Kini RM. (2003b) “Isolation of a neurotoxin 

(alpha-colubritoxin) from a ‘non-venomous’ colubrid: evidence for early origin of venom in snakes. 

Journal of Molecular Evolution 57(4):446-452. 

Fry BG, Vidal N, Norman JA, Vonk FJ, Scheib H, Ramjan R, Kuruppu S, Fung K, Hedges SB, Richardson 

MK, Hodgson WC, Ignjatovic V, Summerhayes R and Kochva E (2006) “Early evolution of the venom 

system in lizards and snakes” Nature 439(7076):509-632; Advance online publication November 17, 

2005 doi:10.1038/nature04328. 

Fry BG, Wickramaratana J, Lemme S, Beuve A, Garbers D, Hodgson WC, Alewood P (2005) “Novel 

natriuretic peptides from the venom of the inland taipan (Oxyuranus microlepidotus): Isolation, 

chemical and biological characterization” Biochemical and Biophysical Research Communications 

327:1011-1015. 

Fry BG, Wickramaratna JC, Hodgson WC, Alewood PF, Kini RM, Ho H, Wuster W. (2002) "Electrospray 

liquid chromatography/mass spectrometry fingerprinting of Acanthophis (death adder) venoms: 

taxonomic and toxinological implications" Rapid Communications in Mass Spectrometry 16:600-608. 

Fry BG, Wüster W (2004) “Assembling an arsenal: Origin and evolution of the snake venom proteome 

inferred from phylogenetic analysis of toxin sequences”. Molecular Biology and Evolution 21(5): 870- 

883. 

Fry BG, Wuster W, Kini RM., Brusic V, Khan A, Venkataraman D, Rooney AP. (2003a) “Molecular evolution 

of elapid snake venom three finger toxins” Journal of Molecular Evolution 57(1):110-129. 

Fry BG, Wüster W, Ramjan SFR, Jackson T, Martelli P, Kini RM. (2003c) “LC/MS (liquid chromatography, 

mass spectrometry) analysis of Colubroidea snake venoms: evolutionary and toxinological 

implications.” Rapid Communications in Mass Spectrometry 17: 2047-2062. 

Li M, Fry BG, Kini RM (2005) “Putting the brakes on snake venom evolution: the unique molecular 

evolutionary patterns of Aipysurus eydouxii (Marbled sea snake) phospholipase A2 toxins.” Molecular 

Biology and Evolution 22(4):934-941. 

Li M, Fry BG, Kini RM (2005) “Eggs only diet: the shift in preferred prey by the Marbled sea snake 

(Aipysurus eydouxii) resulting in a loss of postsynaptic neurotoxicity.” Journal of Molecular Evolution 

60(1):81-9. 

Wüster W, Dumbrell AJ, Hay C, Pook CE, Williams DJ, Fry BG (2004) “Snakes across the Strait: Trans- 

Torresian Phylogeographic Relationships in Three Genera of Australasian Snakes (Serpentes: 

Elapidae: Acanthophis, Oxyuranus and Pseudechis)” Molecular Phylogenetics and Evolution 34: 1- 

14. 

75

9.20-9.40: Evolution of the venom system in squamate reptiles 

Bryan G. Fry 

Australian Venom Research Unit, University of Melbourne, Parkville, Vic 3010 Australia 

bgf@unimelb.edu.au 

The origin, evolution and secreted bioactive properties of the reptile venom system was 

investigated through: 

• Phylogenetic reconstruction of the evolutionary relationships of secreted proteins 

in the dental glands of major squamate lineages. 

• Analysis of mechanisms of evolution in multigene families. 

• Macroecological investigation of the relationships between secreted protein types 

and features of squamate ecology, behaviour and venom gland structure. 

• Bioactivity studies of representative purified proteins. 

A major outcome was the determination that the venomous function in squamates had a 

single, early origin; that Anguimorpha, Iguania and Serpentes share a common venomous 

ancestor. Bioactivity testing of the venom components from lizards previously 

considered ‘non-venomous’ demonstrated potent actions upon a myriad of physiological 

systems. This research reveals a vast treasure trove of novel biomolecules for use in drug 

design and development while also opening up new avenues of evolutionary studies. 

76 

After his Bachelor’s degree in chemistry from the University of the Philippines, 

Baldomero “Toto” Olivera obtained a PhD in biophysical chemistry from 

Caltech and subsequently undertook postdoctoral research at Stanford 

University. His early work was on DNA ligase and DNA replication, and he 

published a series of papers in Proceedings of the National Academy of 

Sciences, Journal of Biological Chemistry and Nature. After returning to the 

University of the Philippines as an Associate Professor of Biochemistry, Dr 

Olivera began what has been his continued and continuing interest in biologically 

active molecules from the venoms of marine cone snails. 

From 1970, Dr Olivera has been associated with the University of Utah, where he 

is currently Distinguished Professor of Biology. Throughout that time and in 

association with his longstanding collaborator, Dr Lourdes Cruz, Dr Olivera has 

greatly developed our understanding of the chemistry, biology and pharmacology 

of Conus peptides and toxins. He has been involved in the discovery and 

classification of many important types of bioactive molecules, including αconotoxin, 

ω-conotoxin, μ-conotoxin, κ-conotoxin, δ-conotoxin, ψ-conotoxin, and 

conantokin peptides. Many of these are in use every day as pharmacological 

tools, and some have provided leads for novel therapeutic agents. 

Professor Olivera has produced over 230 publications and has received many 

awards, including the Redi Award from the International Society on Toxinology 

(2003). 

77

9.40-10.00: EXOGENOMICS AND POST-TRANSLATIONAL MODIFICATION 

OF CONUS PEPTIDES 

B. M. Olivera 

Dept. of Biology, University of Utah, Salt Lake City, Utah 84112, USA 

Most Conus venom peptides are encoded by a small number of gene superfamilies that 

are initially translated as prepropeptide precursors which are then posttranslationally 

modified. In some Conus peptide gene families, there is extensive and diverse posttranslational 

modification of the mature peptide toxins. A hallmark of these gene 

superfamilies is that they are rapidly diversifying; a biological rationale for this rapid 

diversification will be presented. Conus peptide gene superfamilies may represent the 

first examples of a distinctive part of the genome of all animals, which we propose to 

refer to as the exogenome. 

78 

Professor Ohno has been Professor of Biochemistry at Sojo University, 

Kumamoto, Japan since 1997. Prior to that, he has been Professor at Kyushu 

University and a Visiting Associate and Visiting Scientist at the National Institute 

of Health, USA. He has undergraduate and postgraduate degrees in chemistry 

from Kyushu University. 

79

10.00-10.20: Venomics of Protobothrops flavoviridis Venom-gland Phospholipase A2 

Isozymes 

Chijiwa,T. 1 , Ikeda, N. 1 , Kariu, T. 2 , Ogawa, T. 3 , Oda-Ueda, N. 2 , Hattori, S. 4 , Ohno, M. 1 

1 Sojo University, Faculty of Bioscience and Biotechnology, Kumamoto 860-0082, Japan 

2 Sojo University, Faculty of Pharmaceutical Science, Kumamoto 860-0082, Japan 

3 Tohoku University, Department of Life Science, Sendai 981-8555, Japan 

4 University of Tokyo, Institute of Medical Science, Oshima-gun, Kagoshima 894-1531, Japan 

Introduction: Pairwise comparison of five Protobothrops flavoviridis venom-gland 

phospholipase A2 (PLA2) isozyme genes showed that the numbers of nucleotide 

substitution per nonsynonymous site are larger than or close to the numbers of nucleotide 

substitution per synonymous site, indicating that the genes have evolved in an accelerated 

manner. Such accelerated evolution is now thought to be universal for the genes coding for 

venom proteins and to have occurred for acquisition of particular physiological activities. 

The cause of accelerated evolution must be hidden in the genomic sequences encoding 

venom isozymes. In the present study a part of the genomic sequences encoding P. 

flavoviridis venom-gland PLA2 isozymes was analyzed for their venomics. 

Here the term “venomics” is used in a narrow sense. This “venomics” includes 

elucidation of the mechanism of unique evolutionary phenomena and the function of 

venomous isozymes in addition to their genomics, transcriptomics and proteomics. 

Results and Discussion: P. flavoviridis genome contains 16-32 PLA2 isozyma genes (dot 

blot analysis) and it was suggested that they form a cluster on one microchromosome 

(FISH analysis). Shearing of P. flavoviridis liver DNA gave a 40 kbp fragment named 

NER1. Its 3’ terminal half (about 20 kbp long) contained the genes encoding edemainducing 

PLA-B, neurotoxic PLA-N and necrotic BPII. Five inverted repeat (IR) 

sequences (60 bp) and partial fragments of three retrotransposon-like elements were found. 

Reverse transcriptase encoded by retrotransposon might have played a role for gene 

duplication in ancient times. The fragment called NER2 (9 kbp) contained the gene coding 

for highly lipolytic and necrotic PLA2 and an inactive gene. Three IR sequences (110 bp) 

which are different from those in NER1 were also found in and between these genes. 

Expressed sequence tag (EST) analysis for P. flavoviridis venom gland gave 466 cDNA 

clones. Most (42%) of the clones coded for PLA2 isozymes and 35% for nontoxic proteins. 

More time is required for getting more data. 

Key Words: Protobothrops flavoviridis, Phospholipase A2 isozymes, Accelerated 

evolution, Venomics. 

80 

Prof. Eugene V. Grishin is currently a Professor and Deputy Director of the 

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry (IBCh) at the Russian 

Academy of Sciences. He graduated in 1969 from the Chemical 

Department at the Moscow State University. He received his Ph.D. in 

Chemistry (1973) and D.Sc. in Chemistry (1985) from the IBCh. He began 

his scientific career in 1969 as a junior scientist at the IBCh in Moscow. In 1987, 

he was appointed Head of the Laboratory for Neuroreceptors and 

Neuroregulators at the IBCh and became Deputy Director of the IBCh in 

1988. Since 1988, he has been a Professor of Bioorganic Chemistry at the 

Biological Department of Moscow State University. In 1997, he was elected to 

become part of the body of the Russian Academy of Sciences. His current 

scientific interests include toxinology, protein chemistry, neurochemistry, and 

molecular neurobiology in general. 

81

10.20-10.40: Wide variety and multiplicity of spider toxins: structural aspects 

Grishin, E.V, Kozlov, S.A. 

Shemyakin-Ovchinnikov Institute, Russian Academy of Sciences, Miklukho-Maklaya, 16/10, Moscow 

V437, 117997, Russia, grev@ibch.ru 

Spider toxins can be divided into four major groups: polyamine toxins, high molecular 

mass toxins, cystine knot and non-cystine knot polypeptide toxins. Commonly, spider 

venom contains one or two groups of toxins. The majority of known toxins are small 

polypeptides with molecular weight 4-7 kDa cross-linked by 3-5 disulfide bonds. Such 

toxins are the most investigated and the corresponding structural and genomic information 

is also available. Generally, individual venom contains about ten structural families of 

polypeptide toxins, the number of homologues in each family ranges from 1 up to several 

dozen. For this reason, spider venom represents a peculiar naturally edited combinatorial 

library of polypeptide molecules. Sequence analysis of the available spider toxins resulted 

in formulation of common patterns present in these molecules. The principal structural 

motif (PSM) and the extra structural motif (ESM) describe the position of cysteine residues 

in the polypeptide molecule. One can propose five main structural classes of spider toxins. 

1 – toxins with PSM only, 2 – toxins with PSM and ESM, 3 – toxins with ESM only, 4 – 

toxins with the lack of PSM and ESM, 5 – cysteine-free toxins. Analysis of spider venom 

gland EST database allowed us to elucidate the structural features typical of toxin 

precursor organization and the process of toxin maturation. 

o spider toxin 

o structural motif 

o toxin precursor 

82 

R.M. Kini 

Present Appointment: Professor, National University of Singapore; Affiliate Associate 

Professor, Medical College of Virginia, USA; Adjunct Research Scientist, DMRI, Singapore 

Research Areas: Structure-function relationships and mechanism of 

protein toxins; Protein-protein interactions; Protein design & engineering; Thrombosis and 

hemostasis. 

Academic/Professional Qualifications: 

BSc (1977); MSc (1979); PhD (1983), University of Mysore. Mysore, India 

Awards/Honours (Post-PhD): 

• Japanese Government Ministry of Education Monbusho Fellowship (1985-86) 

• John B. Foote Award from Medical College of Virginia (1991-92) 

• French Academic Exchange Fellowship to visit France (1996) 

Career History: 

• Scientific Officer, National Institute of Immunology, New Delhi, INDIA (1984-85) 

• Postdoctoral Fellow, Kyushu University, Fukuoka, JAPAN (1985-86) 

• Lecturer in Biochemistry, University of Mysore, Mysore, INDIA (1986) 

• Postdoctoral Fellow (1986-92); Research Associate (1992-94) Medical College of Virginia, Virginia 

Commonwealth University, Richmond, U.S.A. 

• Research Fellow (1994-95); Senior Scientist (1996-2000); Associate Professor (2000-2004); 

Professor (2005-present) Department of Biological Sciences, National University of Singapore, 

Singapore 

Entrepreneurship: 

• Co-founder of ProScience, Inc., Richmond, Virginia, USA (1993-2003) 

• Founder of ProTherapeutics Private Limited, Singapore (2005-present) 

Professional/Consulting Activities: 

• Consultant to General Medical Industries, Virginia, USA (1998-Present) 

• Consultant to Perkin Elmer, Singapore (1999-Present) 

Publications: 

• 28 Patents; 108 Research papers; 18 Reviews and 8 Book chapters 

• Edited a monograph “Venom Phospholipase A2 Enzymes; Structure, Function and Mechanism” 

John Wiley, 1997 

• Guest editor, Proceedings of “International Conference on Exogenous Factors Affecting 

Thrombosis and Haemostasis”, Haemostasis Vol. 31, pp. 117-311, S. Karger, 2002 

• Guest editor, “Exogenous Factors Affecting Thrombosis and Hemostasis” in Current Drug 

Targets – Cardiovascular and Hematologic Disorders. Bentham Publishers, 2004 

83

10.40-11.00: Protein scaffolds in snake venoms 

Kini RM 

* 

Department of Biological Sciences, Faculty of Science, National University of 

Singapore, Singapore 117543 and 

† Department of Biochemistry, VCU Medical Center, Virginia Commonwealth University, 

Richmond, Virginia 23298-0614, USA 

Snake venoms are complex mixtures of pharmacologically active peptides and proteins. 

These protein toxins belong to a small number of superfamilies of proteins. The members 

in a single family show remarkable similarities in their primary, secondary and tertiary 

structures. At times, however, they differ from each other in their biological targeting 

and hence their pharmacological effects. Thus, each family of protein toxins has a 

similar molecular scaffold but exhibit multiple functions. Thus structure-function 

relationships and the mechanisms of action of snake venom proteins are intriguing and 

pose exciting challenges to scientists. So far, we have understood structure-function 

relationships of only a small number of toxins in the some of these families. An 

overview of this theme of molecular molds with multiple missions will be illustrated by 

the structure-function relationships of families of snake venom toxins. 

84 

Dr Richard Lewis is a co-founder of Xenome and holds joint appointments as 

Xenome's CSO and Principle Investigator (Molecular Pharmacology) at the 

Institute for Molecular Bioscience (IMB), University of Queensland. He has over 

20 years experience in the field of marine toxin pharmacology, particularly ion 

channel and transporter modulation. He has managed several commercial 

venom research programs at IMB, and spearheaded the development of new 

classes of peptide therapeutic candidates. He currently heads a NHMRC 

Program Grant focused on dissecting pain pathways using conotoxins. Richard 

has published extensively in major peer reviewed international journals. 

85

11.00-11.20: Venomics to drugs: the Xen2174 story 

Richard Lewis 1,2 

1 Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD, 

Australia, and 2 Xenome Ltd, Brisbane, QLD, Australia. 

Venom peptides are increasingly being explored as a valuable source of novel 

bioactive molecules. Cone snail venom contains some 50,000 small bioactive 

peptides that target numerous specific ion channels and receptors. A number of 

classes of conotoxins have emerged with therapeutic potential. Recent studies 

indicate that several of these conopeptides may be useful in the treatment of 

neuropathic and inflammatory pain states. Xenome Ltd was founded to mine the 

diversity and complexity of Australian venoms, especially the conopeptides, for 

novel peptide therapeutics. In this presentation, the discovery, pharmacological 

characterization, analgaesic action and clinical development of χ-conopeptides 

are described. Through inhibition of the norepinephrine transporter (NET), χconopeptides 

elevate the levels of NE leading to the amplification of inhibitory 

pathways and the reduction in the strength of pain signals reaching the brain. 

Xen2174, an unnatural χ-conopeptides analogue with improved potency, 

selectivity, stability and ease of synthesis, was found to be well tolerated 

intravenously in a Phase I safety trial. Xen2174 is currently being evaluated 

intrathecally in a Phase I/IIa safety trial in cancer patients suffering severe 

otherwise unmanageable pain. 

86 

87

Oral communications 

Abstracts 

All oral communications will be in lecture 

theatres K3.14, K3.17 and K3.25 in the 

John Anderson Building 

Premier sponsors: 

88 

Monday Track 1, K3.14 3.00-3.20pm 

MT7 muscarinic toxin as tool to study direct allosteric interactions on M1 

muscarinic receptor 

Servent, D.* , Fruchart-Gaillard, C., Mourier, G., Marquer, C. , Ménez, 

A. 

CEA, Département d’Ingénierie et d’Etudes des Protéines, 91191 Gifsur-Yvette, 

France, * denis.servent@cea.fr 

There is now clear evidence showing that muscarinic receptors, as 

many other GPCRs, are susceptible to allosteric modulation. The effects 

of allosteric ligands are consistent with a ternary complex model in 

which the orthosteric and allosteric agents bind simultaneously to the 

receptor and modify each other’s affinities. Until now, all 

pharmacological characterizations of allosteric agents interacting with 

M1 muscarinic receptor were derived from their indirect effects on the 

binding of orthosteric ligand such as NMS. Thus, the lack of 

radiolabeled allosteric selective ligands prevents a direct investigation 

of the interaction of allosteric agents with their specific binding sites. 

For instance, pharmacological studies of the MT7-hM1 interaction have 

been always done indirectly and the mode of action of this allosteric 

toxin is not yet fully understood. 

We’ll present recent results obtained by equilibrium and kinetic 

experiments of the interaction of a monoiodinated MT7 toxin with hM1 

receptor in its free and NMS-occupied states. Thus, the negative 

cooperativity between MT7 and NMS has been evaluated directly and 

the effect of various orthosteric and allosteric agents on the [125I]-MT7 

binding was measured and sheds new light on the mode of interaction 

of these ligands. 

Our results suggest that MT7 toxin interacts with hM1 receptor at a 

specific allosteric site which may partially overlap those previously 

identified for “classical” or “atypical” allosteric agents and highlight the 

potential of this new allosteric tracer in studying allosterism at 

muscarinic receptors. 

- muscarinic toxin 

- muscarinic acetylcholine receptors 

- allosteric interaction 

89


α-Conotoxins And Their Targets: From Photolabeling To The Three-Dimensional 

Structures 

Tsetlin, V.I. 1* , Kasheverov, I. E 1 , Cohen, J.B 2 ., Smit, A.B 3 ., Sixma T.K 4 . 

1 

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 

street 16/10, Moscow, Russia, *vits@ibch.ru. 

2 

Dept. Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston MA 02115, USA 

3 

Department of Molecular and Cellular Neurobiology, Vrijie Universiteit, De Bolelaan 1085, 1081 HV 

Amsterdam, The Netherlands 

4 

Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands 

Introduction. α-Conotoxins (αCt), short neurotoxic peptides from the Conus snails, 

distinguish various nicotinic acetylcholine receptors (nAChRs). The X-ray structure of the 

acetylcholine-binding protein (AChBP) is an excellent model for ligand-binding domains 

of nAChRs. Our purpose was to elucidate the spatial structures of the αCt complexes with 

the AChBP and with the Torpedo californica AChR. 

Methods. The αCt PnIA[A10L, D14K] has been obtained by solid-phase peptide synthesis 

and shown to interact with neuronal α7 nAChR and AChBPs from A.californica and 

L.stagnalis. G1(Bpa12), a photoactivatable analog of αCt G1 bearing a 

benzoylphenylalanine (Bpa) residues in position 12, has been synthesized. UV-irradiation 

of the Torpedo californica AChR complex with radioiodinated G1(Bpa12) resulted in 

specific labeling of the α, γ and δ subunits. 

Results. X-ray structure (2.4Å) has been established for the complex of PnI[A10L, D14K] 

and A.californica AChBP [1]. The toxin retains its conformation but induces 

conformational changes in AChBP, the most pronounced in the C-loop. For G1 (Bpa12), 

photolabeling sites in the Torpedo nAChR were mapped to the fragment starting at 

αSer173 and including loop C; in γ-subunit, crosslinks are in the fragments including 

segments F and D. 

Discussion. The X-ray structure of the AChBP complex sheds light on the interaction of 

αCt with distinct nAChRs. It was used to build models of α7 nAChR-αCt complexes and to 

rationalize the crosslinking results suggesting the two possible orientations of the Bpa side 

chain in the complex. 

References. 1. Celie P.H.N., Kasheverov I.E., Mordvintsev D.Y., Hogg R.C., van Nierop 

P., van Elk R., van Rossum-Finkert S.E., Zhmak M.N., Bertrand D., Tsetlin V., Sixma 

T.K., Smit A.B. Nature Str.Mol.Biol.12, 582-588 (2005) 

2. Kasheverov I.E., Chiara D.C., Zhmak M.N., Maslennikov I.V., Pashkov V.S., Arseniev 

A.S., Utkin Yu.N., Cohen J.B., Tsetlin V.I. FEBS J. 273, 1373-1388(2006) 

o α-conotoxins 

o acetylcholine-binding protein 

o photolabeling 

o nicotinic acetylcholine receptor 

90 


Interaction of Erythrina and Phelline Alkaloids with the Neuronal α4β2 Nicotinic 

Acetylcholine Receptor (nAChR) 

Wildeboer, K.M 1* , Soti, F. 1 , Langlois, N. 2 , Kem, W.R. 1 

1 University of Florida, COM, 1600 SW Archer Road, Gainesville, Florida, USA, kwildebo@ufl.edu 

2 Institut de Chimie des Substances Naturelles, CNRS, 91198 Gif-sur-Yvette Cedex, Paris, France 

Neuronal nAChRs have been implicated in neurodegenerative diseases and substance 

abuse. The high affinity nAChR subtype α4β2 participates in the dopamine-releasing and 

cognition-enhancing effects of nicotine. Involvement of this and other β2-subunit 

containing receptors in addiction has led these receptors to become targets for the design of 

new smoking cessation drugs. The development of selective α4β2 antagonists would 

permit a more rigorous testing of the involvement of these receptors in various neuronal 

processes and nicotine addiction. Seeds (coral beans) from the plant genus Erythrina 

contain alkaloids that are nAChR antagonists. These compounds share a common scaffold 

consisting of four fused rings. Early studies determined that Erythrina alkaloids produce 

curare-like blockade of neuromuscular and ganglionic transmission. A semi-synthetic 

compound, dihydro-β-erythroidine (DHβE), has been widely used to investigate the 

neuronal α4β2 nAChR. One aromatic compound, erysodine, was shown to be more potent 

than DHβE on rat α4β2 receptors. Our goal was to assess structure-activity relationships 

of these alkaloids for the α4β2 nAChR using radioligand binding on rat and human 

receptors and functional (Flexstation) methods on human α4β2. Two groups of Erythrina 

alkaloids were studied: the D-ring lactones (β-erythroidines) and the D-ring aromatic 

compounds. A related group of homoerythrina alkaloids from the genus Phelline were 

also tested. The aromatic Erythrina compounds were the most active. Erysovine had the 

highest affinity and potency for human α4β2 nAChRs. Opening the D-ring of βerythroidine 

did not result in loss of activity; therefore major determinants for binding 

must occur within the other three rings. DHβE was more active than the diene or 

tetrahydro-forms of β-erythroidine. The Phelline alkaloid, O-methylisophellibiline 

(identical structure to DHβE except it contains a seven-membered C-ring), bound 11-fold 

less tightly than DHβE. Overall, various modifications of the D-ring still allow high 

affinity binding to the α4β2 nAChR and inhibition of the receptor response. 

o Nicotinic acetylcholine receptor 

o Nicotinic receptor antagonist 

o Erythrina alkaloid 

o Nicotine addiction 

91


Binding of 3-(Benzylidene)-Anabaseines to Mammalian Nicotinic Acetylcholine 

Receptors and Molluscan ACh Binding Proteins 

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 

1 Department of Pharmacology and Therapeutics, University of Florida, 1600 SW Archer Road, 

Gainesville, FL, USA, *kem@pharmacology.ufl.edu 

2 Department of Pharmacology,University of California San Diego, 9500 Gilman Drive, La Jolla, CA, USA 

The marine toxin anabaseine is converted into a selective partial agonist for α7 

nicotinic acetylcholine receptors (nAChRs) by addition of a benzylidene or 

cinnamylidene moiety at the 3-position of its tetrahydropyridyl ring. One benzylideneanabaseine 

(BA), DMXBA [3-(2,4-dimethoxybenzylidene)-anabaseine, also called GTS- 

21)], is in clinical trials for treatment of cognition deficits in schizophrenia and 

Alzheimer’s disease. Substituents on the benzylidene moiety influence compound 

ionization, lipophilicity and steric properties and influence nAChR binding and 

activation. We measured the nAChR binding affinities of >40 benzylidene-anabaseine 

derivatives to infer a quantitative structure activity relationship (QSAR) for each of the 

two major mammalian brain receptor subtypes, α4β2 and α7. To sample a relatively 

large chemical space, BAs containing substituents representing a wide range of electrondonating, 

lipophilicity and steric bulk properties were synthesized and tested. Using a 

QSAR program, several parameters (the number of electronic charges on heteroatoms, 

calculated dipoles and experimentally derived pKa’s of the compounds) were utilized to 

calculate QSAR equations representing the best fit of the binding data for α7 and α4β2 

nAChRs. There was good agreement between the predicted and the experimental 

compound affinities when only three descriptors were used. Substituent influence on BA 

binding to three molluscan AChBPs, with some notable exceptions, was generally found 

to be similar to binding to that of rat brain α7 receptors. A notable exception was the 

relatively low binding affinity of anabaseine and its analog PTHP for the AChBPs. 

QSAR and related data for these compounds will be useful in predicting binding of 

virtual compounds prior to their synthesis and pharmacological characterization. 

(Supported by NIH grants to both labs). 

o Nicotinic acetylcholine receptor 

o Acetylcholine binding protein 

o Anabaseine 

o GTS-21 

92 


The Structural Characterisation and Receptor Specificity of α-Conotoxin Vc1.1 

Adams, D.J 1* , Clark, R.J 2 , Fischer, H 1 , Nevin, S.T 1 , Craik, D.J. 2 

1 School of Biomedical Sciences and 2 Institute for Molecular Bioscience, The University of Queensland, 

Brisbane, Queensland, 4072, Australia, *dadams@uq.edu.au 

The α-conotoxin Vc1.1 was first discovered using a PCR screen of cDNAs from the 

venom ducts of Conus victoriae (1). The cysteine spacing within the sequence of Vc1.1 

suggests that it is a member of the 4/7 subclass of α-conotoxins. α-Conotoxin Vc1.1 is a 

small disulfide bonded peptide currently in development as a treatment for neuropathic 

pain (2). In the present study, the structure and activity of Vc1.1 and two derivatives, vc1a 

and [P6O]-Vc1.1, which contain the post-translationally modified residues hydroxyproline 

and γ-carboxyglutamate were examined using NMR spectroscopy and electrophysiological 

techniques. Vc1.1 inhibited reversibly nicotine-evoked membrane currents in isolated 

bovine chromaffin cells in a concentration dependant manner. Vc1.1 also inhibited the 

ACh-evoked membrane currents of recombinant nAChR’s expressed in Xenopus oocytes 

preferentially targeting peripheral nAChR subtypes over central subtypes. Specifically, 

Vc1.1 is selective for α3-containing nAChR subtypes, α3β2 and α3β4 having IC50 values 

of 7.3 and 4.2 μM, respectively, whereas the central and muscle nicotinic subtypes 

α4β2, α4β4, α7 and α1β1γδ, all exhibited IC50 values > 30 μM. Lowering the external pH 

from 7.4 to 6.0, increased the potency of Vc1.1 for α3β4 suggesting that a protonated 

histidine may be important for the receptor interaction. Application of 10-30 μM vc1a or 

[P6O]-Vc1.1 failed to inhibit ACh-evoked currents mediated by all nAChR subunit 

combinations expressed in oocytes. The 3D structure of Vc1.1 comprises a small α-helix 

spanning residues P6 to D11 and is braced by the I-II, III-IV disulfide connectivity seen in 

other α-conotoxins. Comparison of the structure of Vc1.1 with other α-conotoxins, taken 

together with nAChR selectivity data, suggests that the conserved proline at position 6 is 

important for binding while a number of residues in the C-terminal portion of the peptide 

contribute towards the selectivity. This structure should open new opportunities for further 

development of Vc1.1 or analogues as analgesic agents. 

1. Sandall et al. (2003) Biochemistry, 42, 6904-6911. 

2. Satkunanathan et al. (2005) Brain Research, 1059,149-158. 

o nicotinic receptor 

o α-conotoxin 

o synthetic peptide 

o NMR structure 

93


Gymnodimine-A targets muscular and neuronal nicotinic acetylcholine receptors 

with high affinity 

Kharrat, R. 1,2 , Servent, D. 3 , Girard, E. 1 , Ouanounou, G. 1 , Molgó, J 1 * 

1 CNRS, Institut de Neurobiologie Alfred Fessard – FRC2118, Laboratoire de Neurobiologie 

Cellulaire et Moléculaire – UPR9040, Gif sur Yvette, France, *Jordi.Molgo@nbcm.cnrs-gif.fr 

2 Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, Tunisie. 

3 CEA/Saclay, Département d’Ingénierie et d’Etudes des Protéines, Gif sur YVette, France. 

Gymnodimines (GYM) are bioactive phycotoxins, isolated from contaminated shellfish 

and produced by the dinoflagellate Karenia selliformis. GYM exhibit unusual structural 

features, including a spirocyclic imine ring system and a trisubstituted tetrahydrofuran 

embedded within a 16-membered macrocycle. GYM are designated as "fast-acting toxins" 

due to the rapid onset of neurological symptoms and rapid death following intraperitoneal 

or intracerebroventricular injection to mice. To get information on the toxic potential and 

the molecular target of GYM, we have studied the mechanism of action of highly purified 

gymnodimine-A (GYM-A) isolated from contaminated clams harvested in Tunisian coasts. 

For this, we used mouse bioassays, isolated frog and mouse neuromuscular preparations, 

cultured Xenopus laevis skeletal myocytes, cultured cells expressing different muscular 

and neuronal nicotinic acetylcholine (ACh) receptors (nAChRs) subtypes and conventional 

electrophysiological and binding-competition assays. The results show that GYM-A 

produced a concentration- and time-dependent blockade of twitch responses evoked by 

phrenic-nerve stimulation in mouse hemidiaphragm preparations. As GYM-A did not 

block twitch responses elicited by direct muscle stimulation, the GYM-A activity cannot 

be attributed to a direct inhibitory effect on muscle contractility, but to the blockade of 

postsynaptic nAChRs of the neuromuscular junction. This was confirmed by recording 

subthreshold endplate potentials and by the complete blockade of miniature endplate 

potentials in neuromuscular preparations treated with GYM-A, and by patch-clamp 

recordings showing that nicotinic currents evoked by constant and brief iontophoretical 

ACh pulses applied to the surface of skeletal myocytes were markedly reduced. 

Furthermore, competition-binding assays revealed that GYM-A is a powerful ligand 

interacting with subnanomolar affinities not only with muscular, but also with 

homopentameric (α7) and heteropentameric (α4β2) neuronal nAChRs. In conclusion, our 

data show for the first time that GYM-A broadly targets nAChRs, and explain the basis of 

its neurotoxicity to various animal species. 

o gymnodimines 

o marine toxin 

o muscular nicotinic acetylcholine receptors 

o neuronal nicotinic acetylcholine receptors 

94 


The unique cysteine spacing of BuIA destabilizes the globular fold commonly 

found in α-conotoxins 

Daly, N.L 1 , Jin, A 1 , Brandstätter, H 1 , Nevin, S.T 2 , Adams, D.J 2 , Alewood, P.F 1 , Craik, D.J. 1 

1 Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia, n.daly@imb.uq.edu.au 

2 School of Biomedical Sciences, University of Queensland, Brisbane, Australia 

α-Conotoxins isolated from the venom of cone snails have exciting therapeutic potential 

based on their high selectivity and affinity at nicotinic acetylcholine receptors (nAChRs). 

BuIA is a recently discovered neuronal α-conotoxin derived from a gene sequence with a 

unique intercysteine loop spacing. It is the only example in the α-conotoxins with four 

residues in the second loop, and makes an unusual 4/4 loop spacing framework. In the 

current study we have used an orthogonal cysteine-protection strategy to synthesise the 

globular (CysI-CysIII, CysII-CysIV) and ribbon (CysI-CysIV, CysII-CysIII) disulfide 

isomers of BuIA, and examined their structures and activity. Interestingly, the globular 

form, which is assumed to be the native connectivity as this is found in all α-conotoxins 

discovered to date, is active at the α6 nAChRs but displays multiple conformations in 

solution. In contrast, the ribbon connectivity isomer forms a well-defined single 

conformation in solution, but does not maintain activity at the nAChR. It appears the 

unique spacing of BuIA has a significant influence on the structure and destabilizes the 

globular form commonly found in α-conotoxins. Furthermore, a single well-defined 

solution conformation is not crucial for activity at the nAChR, and conformational 

flexibility may indeed facilitate binding. 

o conotoxin 

o structure 

o NMR 

o Disulfide connectivity 

95


Structural analysis of AChBP complexes with nicotinic agonists and antagonists 

Scott B. HANSEN 1,2 , Gerlind SULZENBACHER 3 , Todd T. TALLEY 1 , Tom HUXFORD 2 , Palmer TAYLOR 1 , 

Yves BOURNE 3 , & Pascale MARCHOT 4* 

1 Departments of Pharmacology and 2 Chemistry and Biochemistry, UCSD, La Jolla, CA, USA. 

3 Architecture et Fonction des Macromolécules Biologiques, CNRS/Univ, Marseille, France. 

4 Ingénierie des Protéines, CNRS/Univ, IFR Jean Roche, Université de la Méditerranée, Faculté de 

Médecine Secteur Nord, Marseille, France, * marchot.p@jean-roche.univ-mrs.fr. 

Acetylcholine binding proteins (AChBP) are soluble homopentameric surrogates of the 

extracellular ligand binding domain of the nicotinic acetylcholine receptor (nAChR). To 

study the conformational changes in the nAChR ligand binding domain that occur upon 

ligand binding and those that allosterically trigger channel gating upon agonist binding, we 

have solved crystal structures of Lymnaea stagnalis and Aplysia californica AChBPs in 

various complexes with peptidic and alkaloid antagonists and with alkaloid agonists, 

bound at the subunit interfaces [1,2]. Compared with the AChBP apo conformation, 

opening of loops C and F, that border the ligand binding pocket on each face of the 

interface, is associated with binding of the peptidic antagonists, α-cobratoxin and αconotoxin 

ImI, while no major conformational change occurs upon binding of the alkaloid 

antagonist, methyllycaconitine. In contrast, loop closure is associated with binding of the 

alkaloid agonists, lobeline and epibatidine, trapped within the binding pocket. The 

structures also reveal extended and non-overlapping interaction surfaces for the αconotoxin 

and methyllycaconitine antagonists, outside the binding loci for the agonists. 

This comprehensive set of structures suggests that substantial fluctuations in the AChBP 

conformation preexist ligand binding and shows that binding of antagonists and agonists 

distinctively locks the selected open or closed conformations. Hence it reflects a dynamic 

template for delineating further the conformational changes of the nAChR ligand binding 

domain elicited by ligand binding, of which loop closure may be an essential feature linked 

to channel opening. 

1. Bourne Y, Talley TT, Hansen SB, Taylor P, Marchot P (2005) Crystal structure of a Cbtx-AChBP complex 

reveals essential interactions between snake α-neurotoxins and nicotinic receptors. EMBO J 24, 1512– 

1522. 

2. Hansen SB, Sulzenbacher G, Huxford T, Marchot P, Taylor P, Bourne Y (2005) Structures of Aplysia 

AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and 

conformations. EMBO J 24, 3635–3646. 

o Nicotinic agonist and antagonist 

o AChBP 

o High affinity complex 

o Crystal structure 

96 


Designing Peptide Drugs from Python Serum: Dual Inhibitors of sPLA2 and MMP- 

1 as Therapeutic Option for Treatment of Inflammation 

Gopalakrishnakone, P 1* , Thwin, M.M 1 , Sato, K. 2 

1 Venom & Toxin Research Programme, Department of Anatomy, Yong Loo Lin School 

of Medicine, National University of Singapore, Singapore117597, *antgopal@nus.edu.sg 

2 Fukuoka Women's University, Fukuoka 813-8529, Japan 

The objective of this study is to confirm the anti-inflammatory effect of the potent and 

selective secretory phospholipase A2 (sPLA2-IIA) inhibitor, derived from the primary 

structure of an endogenous protein termed “Phospholipase Inhibitor from Python (PIP)”. 

Tg197 mice (CBAxC57BL/6) carrying human TNF genes that develop polyarthritis were 

used. Clinical and histopathological scores along with the morphological evaluations 

indicate that the articular cartilage and the synovium of the peptide-treated Tg197 mice 

reverted to that observed in control wild-type mice. In the rat (Sprague-Dawley) incisional 

hernia model, the peptide significantly (Student’s-t test; P < 0.05) reduced the extent of 

postsurgical peritoneal adhesions in the peritoneal tissues of rats at 7th postoperative day. 

In organotypic cultures, kainate-induced neuronal cell death was significantly reduced in 

the peptide-treated hippocampal neurons than that of the untreated cells. Among the 

peptide mutants examined, the 18-residue linear peptide displayed potent inhibition against 

the activity of human recombinant synovial sPLA2 and human recombinant matrix 

metalloproteinase-1 (MMP-1). It showed remarkable clinical improvement of arthritis in 

transgenic mice, and markedly suppressed secreted MMP-1 activity from IL-1 β - 

stimulated rabbit synovial fibroblasts. mRNA and protein expressions of MMP-1, MMP-2, 

MMP-9 and sPLA2 were found to be significantly decreased after treatment of the IL-1β - 

stimulated cultured rabbit synovial fibroblasts. The peptide acts against the gelatinases 

mainly through downregulation of the expression of genes, while its action against MMP-1 

is through suppression of gene expression combined with enzyme inhibition. This 

optimized peptide analogue presents strong in vitro evidence for control and modulation of 

enzyme activity and/or transcription of both MMP and sPLA2, and provides new 

therapeutic options in the treatment of arthritis and cancer. 

o Secretory phospholipase A2 inhibitor 

o Matrix metalloproteinase-1 inhibitor 

o Tg197 mice 

o Antiinflammatory peptide 

97


Dynamin dependent and independent endocytic pathway of cobra cardiotoxins: role 

of distinct cholesterol and heparan sulfate domain for toxin selection 

Lee, S.C 1, # , Wang, C.H. 1, # , Tsai C.M. 1 , Lin J.L. 1 , Liu Y.A. 1 and Wen-guey Wu 1,2,* 

# These authors contributed equally to this work. 

1 Institute of Bioinformatics and Structural Biology, National Tsing Hua University 

2 National Synchrotron Radiation Research Center, Hsinchu, Taiwan *wgwu@nsrrc.org.tw 

Cobra produces a series of three-fingered β-sheet cardiotoxins homologues with distinct 

glycosaminoglycan, glycosphingolipid and integrin binding ability that cause systolic heart 

arrest and severe tissue inflammation with retarded wound healing process. In addition to 

membrane pore forming activities responsible for its cytotoxicity, cardiotoxins can also be 

uptaken via multiple internalization pathways to target other intracellular organelle and 

induce necrotic and/or apoptosis cell death depending on the studied cells and cardiotoxin 

homologues. Although gylcosphingolipid lipid such as sulfatide has been suggested to 

facilitate such a process on the major cobra cardiotoxin from Naja atra, i. e., CTX A3, it is 

not clear how other cardiotoxins are internalized and whether there are other membrane 

factors involved in the process. We show in this study that other cardiotoxin homologues 

such as CTX A2 and CTX A4 are internalized via a dynamin dependent endocytic 

pathway, in sharp contrast to the dynamin independent internalization of CTX A3. The two 

endocytic pathways are both cholesterol sensitive, but sterol appears to modulate the 

process in an opposite manner. Specifically, while depletion of cholesterol inhibits the 

internalization of CTX A2 and A4, it enhances that of CTX A3. Since CTX A2 and A4 

also exhibit differential sensitivity toward heparinase I and III treated cell in terms of cell 

retention and cell internalization, our results suggest that distinct cholesterol and heparin 

sulfate domains on cell surface may allow the exploration of highly homologous 

cardiotoxins, sometimes with single site mutation, for the selection of not only cell types 

but also action mechanism. 

Cardiotoxins 

Dynamin 

Internalization 

heparin 

98 


Snake venom VEGF-F is a most potent inducer of vascular permeability 

Yamazaki, Y. 1 , Imamura, T. 2 , Morita, T. 1 * 

1 Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo, Japan, *tmorita@my-pharm.ac.jp 

2 Kumamoto University, 1-1-1 Honjo, Kumamoto, Japan 

– Introduction – Vascular endothelial growth factor (VEGF165) is an endothelial cell 

mitogen and also functions as a potent vascular permeability factor. We previously isolated 

and characterized VEGF homologous proteins designated VEGF-Fs that specifically 

recognize KDR (VEGFR-2), from two viper venoms (1). VEGF-Fs show a strong 

endothelial cell proliferative effect in vitro and a potent hypotensive effect in vivo when 

compared with human VEGF165 (1). Crystal structural analysis of VEGF-Fs has revealed 

substantial variation from other VEGF subtypes with respect to three structural features: an 

amino acid insertion in receptor-binding loop 3, a negatively charged surface potential 

rather than the positively charged potential seen in VEGF165, and an additional salt bridge 

formation that is not observed in VEGF165 (2). In this study, VEGF-F was quantitatively 

evaluated in a Miles assay for its ability to induce vascular permeability. 

– Results and Discussion – Both vammin, a VEGF-F from Vipera a. ammodytes venom, 

and VEGF165 similarly stimulated dye leakage when administered at low concentration (up 

to 4 × 10 -9 M), however, vammin was a more potent inducer of vascular permeability than 

VEGF165 at higher concentration (>1.2 × 10 -8 M). To clarify the mechanism of the potent 

effect of VEGF-F, we next evaluated the effects of histamine and bradykinin antagonists, 

which are well-known inducers of vascular permeability. Neither histamine nor bradykinin 

antagonist blocked the dye leakage stimulated by vammin and VEGF165. Our results 

indicate that VEGF-F exhibits potent activity even including the induction of vascular 

permeability and this action may be mediated through a pathway similar to that of 

VEGF165. We conclude that snake venom VEGF-F is the most potent inducer of vascular 

permeability among the previously discovered molecules and assists in the penetration of 

the toxin. 

–References– 

1. Yamazaki Y, Takani K, Atoda H, Morita T (2003) J. Biol. Chem. 278, 51985-51988. 

2. Suto K, Yamazaki Y, Morita T, Mizuno H (2005) J. Biol. Chem. 280, 2126-2131. 

o Snake venom 

o Vascular endothelial growth factor (VEGF) 

o Vascular permeability 

99


C-terminal peptide of snake venom VEGF-F specifically blocks VEGF-A165 

activity by binding to heparin-like molecules 

Tokunaga, Y 1 , Yamazaki, Y 1 , and Morita, T 1* 

1 Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo, Japan, *tmorita@my-pharm.ac.jp 

[Background] Heparan sulfate/heparin-like molecules are known to modulate the 

interaction between vascular endothelial growth factor (VEGF-A165) and its receptor, 

KDR (VEGFR-2). A C-terminal heparin-binding region deficient form of VEGF-A165, 

composed of 110 residues, significantly reduced mitogenic potency compared to the 

intact form. We previously found two novel heparin-binding VEGFs from two distinct 

viper venoms. 1) Snake venom VEGFs, designated VEGF-F, selectively recognize KDR, 

and exhibit a more potent mitogenic activity compared to VEGF-A165. VEGF-Fs 

generally lack the C-terminal heparin-binding region (16~17 amino acid residues) when 

compared with VEGF-A165 (55 amino acid residues), despite the heparin-binding 

potential of VEGF-Fs. We herein report the identification of the heparin-binding region 

of VEGF-F and VEGF-blocking activity of the corresponding peptide. 

[Results and Discussion] A synthetic peptide representing the C-terminal region of 

VEGF-F was prepared. The synthetic peptide and intact VEGF-F were tested for 

heparin-binding ability using heparin affinity chromatography. The affinities of VEGF-F 

and the synthetic peptide were nearly equivalent as determined by the NaCl 

concentrations required for elution (0.33 and 0.36 M, respectively), indicating that the Cterminal 

region mediates the heparin-binding ability of VEGF-F. The synthetic peptide 

completely blocked both VEGF-F and VEGF-A165-induced endothelial cell proliferation 

and hypotension, despite having only a slight inhibitory effect on proliferation induced 

by basic fibroblast growth factor (bFGF), which is a well-characterized heparindependent 

growth factor. We conclude that the C-terminal region of VEGF-F 

specifically inhibits the biological activity of VEGF-A165 by binding to VEGF165bondable 

heparin structure. 2) 

1) Yamazaki, Y., Takani, K., Atoda, H., and Morita, T. (2003) J. Biol. Chem. 278, 

51985-51988, 2) Yamazaki, Y., Tokunaga, Y., Takani, K., and Morita, T. (2005) 

Biochemistry 44, 8858-8864 

o VEGF 

o Snake venom VEGF 

o heparin 

o synthetic peptide 

100 


A Factor Xa-like enzyme from Pseudonaja textilis venom is a powerful procoagulant 

that reduces blood loss in a topical anti-bleeding model. 

Flight, S.M 1* , Warner, R.L 2 , Lavin, M.F 3 , de Jersey J 4 , Masci, P. P 1 

1 

University of Queensland, School of Medicine, Princess Alexandra Hospital, Brisbane, Australia 

*sflight@soms.uq.edu.au 

2. 

University of Michigan, Department of Pathology, University of Michigan Medical School, Ann Arbor, 

USA. 

3 

Queensland Institute of Medical Research, Royal Brisbane Hospital, Brisbane, Australia. 

4 

University of Queensland, School of Molecular and Microbial Sciences, Brisbane, Australia 

Introduction: The prothrombin activating complex in the venom of the Australian brown 

snake Pseudonaja textilis contains a nonenzymatic factor Va-like component and a factor 

Xa-like enzyme. In vitro and in vivo analyses were performed to characterise the factor Xa 

component and assess its efficacy as a topical anti-bleeding agent. Methods: The snake 

factor Xa component was purified using affinity and size exclusion chromatography. The 

clotting times were measured using a range of concentrations of snake factor Xa, human 

factor Xa and thrombin in citrated plasma with and without added calcium. The same 

enzymes were added to citrated whole blood and coagulation analysed using 

thromboelastography. A rat dermal incision model was used to assess the blood lost from a 

wound in the presence of 100, 250 and 1000 mcg/mL of the snake factor Xa compound 

and 1000 U/mL of thrombin (n=10 animals/treatment). Results: Snake factor Xa was 

purified by column chromatography. The purified enzyme clotted citrated plasma in the 

absence of added calcium at concentrations as low as 10 nM whereas no clotting was 

observed using human factor Xa at concentrations up to 700 nM. In this assay, prior 

recalcification of the plasma led to a further 2-4-fold decrease in clotting time on addition 

of 10 nM snake factor Xa. Similar results were obtained using thromboelastography with 

citrated whole blood. The snake factor Xa reduced blood loss in the dermal incision model 

from 44.3 ± 34.3 mcL blood lost (saline control) to 2.8 ± 2.5 mcL blood lost with 250 

mcg/mL of enzyme (p


Heparin-neutralizing properties of two novel Lys49 PLA2 variants from the 

Bothrops moojeni venom. 

Anna Maria Perchuc 1* , Laure Menin 2 , Philippe Favreau 2 , Beatrice Bühler 1 , Philippe Bulet 2 , Reto 

Schöni 1 and Reto Stöcklin 2 

1 Pentapharm Ltd., Engelgasse 109, P.O. Box, CH-4002 Basel, Switzerland, 

* annamaria.perchuc@pentapharm.com 

2 Atheris Laboratories, Case Postale 314, CH-1233 Bernex – Geneva, Switzerland 

Introduction: In the present work proteomics and peptidomics approaches have been used 

in combination with newly developed bioassays screening in order to identify new 

compounds in the venom of Bothrops moojeni snake. 

The most interesting compounds are thought to be used as active pharmaceutical (APIs) or 

active diagnostic (ADIs) ingredients mainly in the field of haemostasis and fibrinolysis. 

Methods: Among the peptides and proteins already characterized in B. moojeni venom in 

the framework of our project, two phospholipases A2 (PLA2) have been purified and fully 

sequenced by ESI-MS/MS techniques. Their inhibitory action towards heparin has been 

found by the means of specific assays designed in order to analyze the anticoagulant 

properties of heparins in plasma and the neutralization thereof. 

Results: ESI-MS/MS analysis revealed novel proteins, distinct from the three PLA2 

(MjTX-I and MjTX-II and MOO-1) of B. moojeni already described in the literature. Both 

of them belong to the enzymatically non-active Lys49 variants of PLA2. They consist of 

122 amino acids and share a characteristic sequence in the C-terminal region composed of 

clusters of basic amino acids known from the literature to interact with heparin. 

The novel PLA2 variants we have isolated and characterized as well as their synthetic Cterminal 

fragments interact in vitro with unfractionated (UFH) and low molecular weight 

(LMWH) heparins, neutralizing their anticoagulant activity. The synthetic peptides are also 

able to inhibit the anticoagulant effects of LMWH to a higher extent than protamine 

hydrochloride, the known heparin antidote, used in its therapeutically recommended 

concentration. 

Discussion/Conclusion: Although it is well known that different PLA2s from snake venoms 

influence the blood coagulation system and some of them interact with heparin, the use of 

those substances or their fragments to antagonize the anticoagulant effect of heparin in vivo 

or in vitro has never been proposed. Their possible use in diagnostic and/or pharmaceutical 

applications is currently under investigation. 

o Bothrops moojeni 

o Lys49 PLA2 variant 

o Heparin inhibition 

102 


FXa-binding studies of the anticoagulant phospholipases A2 from Viperidae and 

Crotalidae venom 

FAURE, G 1* ., GOWDA 2 , V. T., BON 3 , C & R.C. MAROUN 4 

1 Unité d’Immunologie Structurale, Institut Pasteur, 25 rue du Dr. Roux, Paris, France * fgrazyna@pasteur.fr 

2 University of Mysore, Department of Studies in Biochemistry, Manasagangothri, India 

3 MNHN, Lab. de Biochimie des Substances Naturelles, 63 rue Buffon, Paris, France 

4 Unité de Bioinformatique Structurale, Institut Pasteur, 25 rue du Dr. Roux, Paris, France 

The snake venom group IIA secreted phospholipases A2 (sPLA2s) from Viperidae and 

Crotalidae families are multifunctional proteins that exhibit a wide range of toxic and 

pharmacological effects. These sPLA2s catalyse the hydrolysis of the ester bond of 

phospholipids and also selectively interact with various protein targets. These proteinprotein 

interactions, rather than protein-phospholipid interactions, play an important role 

in determining the specific function of PLA2s. 

In particular, the sPLA2s that interact with presynaptic receptors [1,2] block the release 

of acetylcholine and the same neurotoxic enzymes interfere with blood coagulation 

exhibiting a strong anticoagulant function [3, 4]. 

Using Surface Plasmon Resonance (SPR) and an in vitro biological test of the 

inhibition of prothrombinase activity, we identified several Viperidae and Crotalidae 

sPLA2s that inhibit blood coagulation through direct binding to human blood coagulation 

factor Xa (FXa). These anticoagulant sPLA2s that inhibit coagulation by a non-enzymatic 

pathway represent a novel family of anticoagulant agents, useful in identifying the sites of 

interaction of anticoagulants at the level of specific amino acid residues. Molecular 

electrostatic potentials calculated at the solvent-accessible surface of obtained 3D models 

and available crystal structures of these FXa-binding molecules show a correlation with 

their anticoagulant activity. Analysis of the complexes obtained through docking 

simulations allows us to map four sPLA2 binding regions at the interface. We compare 

these regions to those mapped previously by site-directed mutagenesis and SPR studies 

for human group IIA sPLA2 [4] and for AtxA [3]. The structural information that we 

obtain about the binding of sPLA2s to FXa will be useful in the 3D structure-based design 

of therapeutic agents. 

[1] Krizaj, I., Faure, G., Gubensek, F. & Bon, C. (1997) Biochemistry, 36, 2779 

[2] Faure, G, Copic, A., Le Porrier, S, Gubensek, F., Bon, C & Krizaj, I. (2003) Toxicon, 40, 509 

[3] Prijatelj, P., Charnay, M., Ivanovski, G., Jenko, Z., Pungarcar, J. Krizaj, I. & Faure, G. (2006) Biochimie, 

88, 69 

[4] Mounier C. M., Luchetta, P., Lecut, C., Koduri, R. S., Faure, G., Lambeau, G., et al., (2000) Eur. J. Bioch 

em. 267, 4960 

anticoagulant phospholipase A2, 

human factor Xa 

surface plasmon resonance 

docking simulations 

103


BE-I-PLA2, a novel acidic phospholipase A2 from Bothrops erythromelas venom: 

purification, cloning and characterization as potent anti-platelet and inductor of PGI 

release by endothelial cells 

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 , 

Guarnieri, M.C 4 

1 Instituto Butantan, São Paulo SP, Brasil 

2 Instituto de Pesquisas Energéticas e Nucleares, São Paulo SP, Brasil 

3 Universidade Federal de São Paulo, São Paulo SP, Brasil 

4 Universidade Federal de Pernambuco, Recife PE, Brasil. 

A novel acidic Asp49 phospholipase A2 was isolated from Bothrops erythromelas (jararaca 

malha-de-cascavel) snake venom by four chromatographic steps. BE-I-PLA2 present a 

molecular weight of 13649.57 Da as estimated by mass spectrometry. N-teminal and four 

internal peptides were sequenced, covering around one third of the complete toxin 

sequence. The complete BE-I-PLA2 cDNA was cloned from a B. erythromelas venomgland 

cDNA library. The cDNA sequence possesses 753 bp and encodes a protein with 

significant sequence similarity to many other PLA2s from snake venoms. When tested in 

platelet rich plasma, the enzyme showed a potent inhibitory effect on aggregation induced 

by arachidonic acid and collagen, but not ADP. On the other hand, BE-I-PLA2 did not 

modify aggregation in washed platelet. Furthermore, no action of BE-I-PLA2 on the 

principal platelets receptors was observed. Chemical modification with p-bromophenacyl 

bromide abolished the enzymatic activity of BE-I-PLA2, but its anti-platelet activity was 

only partially inhibited. In HUVEC, BE-I-PLA2 was neither apoptotic nor proliferative but 

stimulated endothelial cells to release PGI2, suggesting an increase of its potential antiplatelet 

activity in vivo. Further studies are required in order to determine the exact 

mechanism of action of BE-I-PLA2 in the inhibition of platelet aggregationAbstract text 

here: 

o Phospholipase 

o Anti-platelet 

o Snake Venom 

o Cloning 

104 


Stability studies on the venom of the major Australian box jellyfish (Chironex 

fleckeri). 

1 2 3 1* 

Winter, K.L., Isbister, G.K., Seymour, J.E., Hodgson, W.C. 

1 

Monash Venom Group, Dept. Pharmacology, Monash University, Victoria, Australia, 3800. * 

kelly.winter@med.monash.edu.au 

2 

Tropical Toxicology Unit, Menzies School of Health Research, Northern Territory, Australia. 

3 

Dept. Tropical Biology, James Cook University, Queensland, Australia, 4878 

Introduction: It has been suggested that jellyfish venoms are difficult to work with and 

that they are sensitive to pH and chemicals, thermolabile, and have a tendency to 

aggregate, disaggregate and stick to the surface of equipment (Ramasamy et al., 2003). 

We have previously characterised the pharmacological activity of a number of jellyfish 

venoms. The current study aimed to examine the working parameters of the venom of the 

Australian box jellyfish Chironex fleckeri, to aid fractionation using HPLC analysis. 

Methods: Anaesthetised (pentobarbitone sodium, 100 mg/kg, i.p.) rats were used to 

examine the cardiovascular effects of the venom. Venom was made up fresh each day and 

subjected to a number of different environments. A pH range of 5-9 and a temperature 

range of 4-30 ºC were examined. In addition, the effect of freeze drying and reconstituting 

the venom was investigated. 

Results: Venom (50 μg/kg, i.v.) produced a transient hypertensive response followed by 

cardiovascular collapse. This effect was not significantly effected by preparation of the 

venom at pH’s of 5, 7 and 9 (p>0.05, n=3; one way ANOVA). Similarly the venom (50 

μg/kg) did not display loss of activity when exposed to temperatures of 4 ºC, 20 ºC or 30 

ºC for 1.5 hr. However, the cardiovascular activity of the venom was abolished by boiling 

the venom (p


Variation in venom between two Australian box jellyfish: prey-specific 

adaptations 

Kintner A. 1* , Seymour J. 2 , Carrette T. 3 

1 Monash Venom Group, Monash University, Melbourne Vic Australia, *anna.kintner@med.monash.edu.au 

2 Tropical Australian Stinger Research Unit, James Cook University, Cairns Qld Australia 

3 Tropical Australian Stinger Research Unit, James Cook University, Cairns Qld Australia 

Introduction. This study examines the venoms of two species of closely-related box 

jellyfish: Chironex fleckeri, which has caused 70+ deaths in Australia, and Chiropsalmus 

sp., which has never caused serious injury in Australian waters. The two share a similar 

nematocyst complement and prey of small shrimps during their juvenile stages. However, 

as C. fleckeri matures, its proportion of venom-delivering nematocysts increases, allowing 

it to inject 15 times more venom than Chiropsalmus sp, and simultaneously, its prey focus 

shifts to include fish 1 . This study investigates adaptations of the two species’ venoms, by 

examining their cardiac effects in prey model animals (e.g. crustaceans and fish) and by 

comparing their protein constructs. This will elucidate reasons for difference in sting 

lethality and facilitate further investigation into clinical significance of C. fleckeri stings. 

Methods. Venoms from both species were injected into crustacean and fish prey models 

(Cherax quadricarinatus, and Lepidozygus tapeikosoma respectively) and their effects and 

dose-dependency assessed via Doppler ultrasound cardiac monitoring. Examination of the 

venom fractions was undertaken using SDS-PAGE analysis. 

Results. C. fleckeri and Chiropsalmus sp. venoms were roughly equivalent in a crustacean 

model, but widely disparate in a vertebrate model, with C. fleckeri venom being more 

efficient. C. fleckeri venom was also more complex, containing roughly twice as many 

components as Chiropsalmus sp. venom. 

Discussion/Conclusion. Our results demonstrate that Chiropsalmus sp. would be 

incapable of preying on fish or causing significant envenoming in humans. Interestingly, 

both venoms contain protein fractions of similar size and charge, suggesting that the two 

may share invertebrate-specific toxins, with C. fleckeri venom containing additional 

vertebrate-specific toxins. We suggest future avenues of investigation to confirm this. 

References. Carrette T., Alderslade P., and Seymour J., 2002. Nematocyst ratio and prey 

in two Australian cubomedusans, Chironex fleckeri and Chiropsalmus sp. Toxicon 40 (11), 

1547-1551. 

o venom ecology 

o box jellyfish 

o nematocyst 

o cubozoan 

106 


Structural Determinants of Lys49 Phospholipase A2 Activity Probed by Scanning 

Alanine Mutagenesis. 

Chioato, L, Aragão, E. A, Lopes, T. F, Ruller, R, Ward, R. J* 

Universidade de São Paulo, FFCLRP, Av. Bandeirantes 3900, Ribeirão Preto-SP, Brazil. 

rjward@ffclrp.usp.br 

Lysine 49 Phospholipases A2 (Lys49-PLA2) are naturally occurring class II 

phospholipases, in which the active site Asp49 is replaced by a lysine. Although the 

Lys49-PLA2s do not hydrolyze phospholipid substrates, they show a wide range of 

pharmacological effects, are bactericidal, and damage artificial membranes by a Ca 2+ - 

independent mechanism. As part of our ongoing efforts to understand the 

structure/function relationships of PLA2, we have undertaken a scanning alanine 

mutagenesis study of the bothropstoxin-I, a Lys49-PLA2 isolated from the venom of the 

snake Bothrops jararacussu. 

All cationic residues together with all residues between positions 112-131 were substituted 

by alanine, and aromatic residues in the C terminal loop region (residues 116-127) were 

substituted by tryptophan. After expression in E. coli BL21 and purification by cation 

exchange chromatography, the native-like secondary structure of 33 mutants was 

confirmed by circular dichroism spectroscopy. The effects of mutagenesis were evaluated 

on the bactericidal activity against E. coli K12, the membrane damaging activity (release 

of liposome entrapped marker), and the in vivo myotoxic effect. For each functional assay, 

the positions of mutants that altered the activity were mapped on the calculated protein 

surface in the three dimensional structure. 

Comparison of the structural determinants of all activities revealed that Ca 2+ -independent 

membrane damage against artificial liposome membranes correlated well with the 

bactericidal activity against E. coli, suggesting that a common mechanism underlies the 

bactericidal and Ca 2+ -independent membrane damaging activities. In contrast, the 

structural determinants of the myotoxic activity were restricted to the C-terminal loop 

region, suggesting that the myotoxic effect is not due to Ca 2+ -independent membrane 

damage. These results show that although the structural determinants of the bactericidal 

and myotoxic activities are partly overlapping, the underlying mechanisms of action 

against the two membranes are different. 

o Bactericidal 

o Myotoxic 

o Membrane damage 

107


Screening of Bothrops snake venoms using bidimensional capillary liquid 

chromatography coupled to tandem nano-electrospray quadrupole time-of-flight 

mass spectrometry: identification of novel peptides 

Souza, G.H.M.F. 1,2,* , Ifa, D.R. 2,3 , Eberlin, M.N. 2 , Hyslop, S. 1 

1 

Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas 

(UNICAMP), CP 6111, 13083-970, Campinas, SP, Brazil. *desouz@fcm.unicamp.br 

2 

Laboratório Thomson de Espectrometria de Massas, Instituto de Química, Universidade Estadual de 

Campinas (UNICAMP), 13083-970, Campinas, SP, Brazil. 

3 

Aston Laboratory for Mass Spectrometry, Department of Chemistry, Purdue University, Oval Drive, 

West Lafayette, IN, USA. 

Introduction: Bothrops snake venoms are a mixture of proteins and peptides, with the 

latter including bradykinin-potentiating peptides (BPPs) and C-type natriuretic peptides 

(CNPs) [1,2]. However, the occurrence of peptides other than BPPs and CNPs has not been 

extensively investigated. In this work, we used mass spectrometry to analyze the peptide 

content of a low molecular mass fraction obtained by ultrafiltration of several Bothrops (B. 

alternatus, B. erythromelas, B. insularis, B. jararaca, B. jararacussu, B. leucurus, B. 

moojeni) venoms. Methods: Lyophilized or dessicated Bothrops venoms were dissolved in 

0.1% trifluoroacetic acid containing 50% acetonitrile and ultrafiltered by centrifugation 

through a 5 kDa cut-off membrane (Millipore). The resulting low molecular mass fraction 

was analyzed by quadrupole/time-of-flight mass spectrometry (Qtof) using capillary 

liquid-chromatography and nano-electrospray ionization. Results: Eighty-five peptides 

with molecular masses of 400-1900 kDa were identified and sequenced by 2D-capillary 

liquid chromatography coupled to tandem mass spectrometry using nano-electrospray 

ionization quadrupole time-of-flight mass spectrometry (nanoESI-MS/MS), although no 

single venom contained all of these peptides. Various peptides were identified as BPPs or 

putative BPPs. However, a larger number did not show the characteristic BPP structure. 

BLAST searches of the latter peptides revealed homologies (based on 80% similarity) with 

various vertebrate proteins, as well as with a few snake venom proteins, particularly Lamino 

acid oxidase, myotoxic phopholipases A2 and (hemorrhagic) metalloproteinases. 

Conclusion: Bothrops venoms contain a variety of peptides other than BPPs and CNPs. 

Although the functions of these peptides are still unknown, the finding that several of them 

are identical to internal regions of larger venom proteins (enzymes) suggests that these 

peptides may have important as yet unidentified functions. 

References: 1. Murayama, N. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 1189-1193. 2. 

Wermelinger, L.S. et al. (2005) Rapid Commun. Mass. Spectrom. 19, 1703-1708. 

Financial support: CAPES, CNPq, FAPESP 

o Bothrops 


o Peptides 

o Venom 

108 


Role of histidines and the N-terminal helices in the function of the diphtheria 

toxin T domain 

Perier, A. 1 , Chassaing, A. 1 , Montagner, C. 2 , Pichard, S. 1 , Vernier, G. 2 , Ménez, A. 1 , 

Forge, V. 2 , Chenal, A. 3 and Gillet, D. 1* 

1 Département d’Ingénierie et d’Etudes des Protéines, CEA-Saclay, 91191 Gif sur Yvette 

cedex, Franc. *daniel.gillet@cea.fr 

2 Biophysique Moléculaire et Cellulaire, UMR 5090, Département Réponse et Dynamique 

Cellulaires, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble cedex 9, France 

3 Département de Biochimie Structurale et Chimie, Unité Postulante Biochimie des 

Interactions Macromoléculaires, Institut Pasteur, 25 rue du Dr Roux, 75014 Paris, France 

During intoxication of a cell, the translocation (T) domain of the diphtheria toxin 

helps the passage of the catalytic domain across the membrane of the endosome 

into the cytoplasm. The interaction of the T domain with the membrane of the 

endosome has been related to the formation of a molten globule state in solution at 

acidic pH. We have found that protonation of histidines was required for the 

formation of this molten globule state and probed the role of each histidine by site 

directed mutagenesis. We have also investigated the behavior of the N-terminal 

region of the T domain during the successive steps of its interaction with 

membranes using fluorescence and trypsin digestion. The change of the 

environment of this region was monitored using mutant W281F carrying a single 

native Trp at position 206 at the tip of helix TH1. The role of each helix of the Nterminus 

of the T domain, TH1, TH2, TH3 and TH4 was studied using synthetic 

peptides. The N-terminal region of the T domain was not involved in the binding of 

the domain to the membrane, which occurred between pH 7 and pH 6 mainly 

through hydrophobic effects. At that stage of the interaction, the N-terminal region 

remained strongly solvated. Further acidification eliminated repulsive electrostatic 

interactions between this region and the membrane, allowing its penetration into the 

membrane by attractive electrostatic interactions involving TH1 and TH4 and 

hydrophobic effects involving TH3. 

The data give information on the physicochemical properties of the T domain 

required for its penetration into the membrane and on the successive steps involved 

during this process. 

- diphtheria toxin 

- translocation 

- molten globule 

- membrane interaction 

- amphiphilic helices 

109


Detection and Quantitation of Detrimental Levels of Staphylococcal Enterotoxin B 

by Mass Spectral Immunoassay 

Bieber, A.L., Niederkofler, E.E., Tubbs, K.A., Kiernan, U.A., Nedelkov, D. 

and Nelson, R.W. 

Intrinsic Bioprobes, Inc., 625 S. Smith Road, Suite 22, Tempe, AZ, USA 85281*abieber@intrinsicbio.com 

Introduction: The need to develop early detection devices for natural toxins is evident from 

their potential use as agents for terrorist attacks. Low detection limits are a requirement for 

detection and quantitation of these agents from complex biological and environmental 

systems. An aspect not addressed by current detection methods is the presence of variant 

forms of toxins. The application of mass spectral immunoassay (MSIA) to directly detect 

Staphylococcal Enterotoxin B (SEB) and associated variants is described. 

Methods: Highly sensitive MSIA technology was applied to detect biologically relevant 

levels of SEB. Pico-molar concentrations of SEB were extracted from milk, buffer and 

water through the use of affinity pipette tips (small porous micro-columns fitted inside 200 

microliter pipet tips) derivatized with anti-SEB polyclonal antibody. Repeated passage of 

sample through the affinity pipette selectively extracted the targeted analytes from large 

volumes of solution for subsequent mass spectrometric analysis. Analyses of multiple 

samples were accomplished by use of a robotic pipetting workstation. 

Results: The application of MSIA for the detection of SEB indicated a lower detection 

limit of 0.5 ng/mL from as little as 1mL of sample. Accurate identification of SEB and 

SEB variants, which may be produced through chemical and/or biological modifications, 

was accomplished. High content MS data pertaining to the target analyte and variants was 

obtained in the absence of other proteins that are present in crude samples. SEB 

quantitation by MSIA was achieved using a chemically modified form of SEB as an 

internal reference. Furthermore, a robotic pipetting workstation was used to process 

multiple samples in parallel. The three test solutions, present in all wells of separate 96 

position microtiter plates, were subjected to MSIA. SEB was subsequently identified from 

each of the 288 samples, thereby validating the reproducibility of MSIA. Overall, the 

results indicate that the application of MSIA for early detection of biological pathogens, 

such as SEB, has great potential for rapidly and accurately identifying the presence of 

known pathogenic agents and their variants in crude biological and environmental samples. 

o immunoassay 

o enterotoxin B 

o mass spectrometry 

o quantitation 

110 


Chemical modification of mycotoxin metabolism by green tea extract and 

coumarin in piglets. 

Tulayakul, P. 1 , Dong, K.S. 1 , Li, J.Y. 2 , Kumagai, S. 1* 

1 

Laboratory of Veterinary Public Health, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 

Japan,*askuma@mail.ecc.u-tokyo.ac.jp. 

2. 

Research Unit for Animal Life Sciences, Animal Resource Science Center, University of Tokyo, Ibaraki- 

Iwama, Japan. 

Pig is known to be a sensitive species to aflatoxin B1 toxicity. We have found that the 

CYP450 activity to form AFB1-epoxide is relatively high, while the GST activity to 

detoxify AFB1 is low, in piglets compared with other animal species (1). The purpose of 

this study is to clarify the effects of feeding piglets with green tea extract (Salphenol) and 

coumarin on AFB1 metabolism, which is a determinant of the adverse effect of the toxin. 

Method: Piglets were fed with the diet containing 0.2% w/w green tea extract or 0.5% w/w 

coumarin for 3 weeks, and then analysed for aflatoxin B1 metabolism in their liver and 

intestinal tissues in vitro. 

Results: The liver microsomes activity to bind AFB1 to DNA was reduced by coumarin, 

but not by green tea extract. The intestinal microsomes activity was not changed by either 

compound. The GST activity toward AFB1-epoxide was not changed in the liver, but that 

was increased in the intestine, by both the compounds. The GST activity toward CDNB 

was increased by both the compounds in the liver and intestine. The formation of free 

AFQ1 and AFM1 by liver microsomes was reduced by coumarin treatment, while the 

formation of aflatoxicol and AFB2a was increased in the liver and intestine by coumarin 

treatment, respectively. On the other hand, AFQ1 and aflatoxicol formation was increased 

in the intestine of piglets treated with green tea extract. 

Discussion/Conclusion: Coumarin increased the intestinal GST activity toward AFB1, and 

reduced the liver P450 activity to form AFB1-DNA adduct. Also green tea extracts 

increased the intestinal GST activity toward AFB1, but exerted no effects on P450 

activities to form AFB1-epoxide. These results indicate that green tea may be an effective 

candidate for protecting piglets from toxicity of AFB1, although its activity is lower than 

coumarin. 

References: 1. Tulayakul et al. (2005) Toxicon. 46, 204-209. 

o green tea 

o piglets 

o AFB1 

o metabolism 

111


Novel proteolytic enzymes from pathogenic fungi and their use for biotechnology 

and health 

Stöcklin, R. 1* , Grouzmann, E. 2 , Monod, M. 3 , Michalet, S. 1 and Gaertner, H. 1 

1 

Atheris Laboratories & FunZyme Biotechnologies S.A., case postale 314, CH-1233 Bernex-Geneva, 

Switzerland, *reto.stocklin@atheris.ch - http://www.atheris.com 

2 

Division de Pharmacologie et Toxicologie Cliniques Centre Hospitalier Universitaire Vaudois, CH-1011 

Lausanne, Switzerland 

3 

Service de Dermatologie, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland 

We have isolated and characterized a set of potent proteolytic enzymes secreted by 

pathogenic dermatophyte fungi. One of our aminopeptidases is able to completely degrade 

any peptide from its N-terminal extremity like a zipper, but it is stopped when it reaches a 

Yaa-Pro sequence. A complementary enzyme is capable to exclusively remove the Yaa- 

Pro sequence. The use of these proteases - either alone or in combination - has many 

promising industrial applications ranging from highly specific and controlled modification 

of protein’s extremities to complete degradation of large resistant proteins. 

On the one hand, our enzymes can be used to specifically remove affinity tags, thus 

facilitating the bio-processing of recombinant protein production and chemical peptide 

synthesis. On the other hand, our enzymes are able in vitro to fully degrade proteins down 

to dipeptides and single amino acids, which has never been reported before. This 

unprecedented efficacy of our enzymes has convinced us of their possible use for oral food 

implementation in gastrointestinal disorders due to resistance or intolerance to proteins. 

Celiac disease is a pathology characterized by intolerance to gluten (a major protein of 

wheat, rye and barley) that is not properly digested by intestinal tract of affected people. 

This results in the accumulation of gliadins, toxic peptides (fragments of gluten) that are 

resistant to degradation in the gastrointestinal tract and that contain several T-cell 

stimulatory epitopes. The accumulation of gliadins causes an immune reaction leading to 

the destruction of the intestine. Despite its prevalence of ~1:100, there is no drug on the 

market, and the only solution is a strictly gluten-free diet. We intend to develop a first drug 

for Celiac disease based on an oral administration of our enzymes to fully degrade gliadins. 

Monod, M., Lechenne, B., Jousson, O., Grand, D., Zaugg, C., Stöcklin, R. and Grouzmann, 

E. (2005). Microbiology, 151:145-55. 

o Fungal enzymes 

o Bio-processing 

o Drug discovery 

o Celiac disease 

112 

Tuesday Track 1, K3.14 3.00-3.20pm 

Post mortem examination of a fatal case of Bothrops lanceolatus envenoming 

Thomas, L. 1* , Malbranque, S. 1 , Piercecchi- Marti, M.D. 2 , Coursier, D. 1 , Barbey, D. 1 , Bucher, B. 1 , 

Ridarch, A 1 , Smadja, D. 1 

1 Hôpital Universitaire, 97200 Fort-de-France, Martinique, * laurent.thomas@chu-fortdefrance.fr 

2 Laboratoire de Médecine légale, Faculté de Médecine Timone, Marseille, France 

We describe the first documented case of autopsy proven multiple systemic infarctions 

following snake bite by Bothrops lanceolatus. 

Case report: a 74-yo healthy man was bitten on the left elbow. He did not seek immediate 

medical attention. Two days later he fell in a coma and was hospitalised. On admission he 

presented with typical fang marks and swelling of the bitten limb, altered sensorium, 

aphasia, and tetraplegia. Breathing, arterial pressure, urine output and coagulation were 

normal. Platelet count was 95,000/mm 3 . Specific BothroFav antivenom therapy was 

initiated. MRI demonstrated multiple cerebral infarcts. Myocardial infarction was 

confirmed by characteristic electrocardiographic changes and elevated serum troponin. 

Coronarography was normal. No subsequent neurological improvement was demonstrated. 

Six days after the bite he developed atrial fibrillation and left ventricular failure. 

Echocardiography showed rupture of the mitral valve. Treatment was ineffective. The 

patient died 10 days after the bite. Necropsy findings: Incision of the heart revealed a 

thickening of the wall of the left ventricle, a posterior infarction combined with a rupture 

of the posterior leaflet of the mitral valve and no vegetation, and a fibrinous pericarditis. 

On slicing of the brain hemispheres and cerebellum, multiple small symmetrical infarcts 

and liquefaction necrosis were noticed. Opening of the abdominal cavity revealed a 

localised infarct of an ileal loop. No thrombosis of large vessels could be seen. 

Histopathology: Microscopic examination demonstrated widespread ischemic lesions, 

typically ranging from a few days old to recent in the heart (left ventricle) and brain. On 

examination of the small vessels, fibrinous clots were uncovered on dissected endothelium, 

leading to complete occlusion of arterioles and capillaries. No inflammatory change was 

seen. The organs involved included the brain, heart, ileum, lungs, and kidneys to a lesser 

extent. Conclusion: The pathological findings suggest a disseminated thrombotic 

microangiopathy process of the endothelium, without vasculitis, consistent with a local 

activation phenomenon. Further research is now mandated in the field of toxinology. 

o Bothrops 

o Snake bites 

o Infarction 

o Thrombotic microangiopathy 

113


Kraits with 17 dorsal scale rows (Bungarus sindanus complex): unrecognised 

causes of severe neurotoxic envenoming in South Asia 

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 , 

Theakston, R.D.G. 8 , Mebs, D. 1 , Warrell, D.A. 8,9 

1 Zentrum der Rechtsmedizin, Klinikum der Johann Wolfgang Goethe-Universität, Kennedyallee 104, 60596 

Frankfurt am Main, Germany, *U.Kuch@em.uni-frankfurt.de 2 School of Neurology, Neurobiology and 

Psychiatry, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK 

3 Dhaka Medical College and Hospital, Dhaka 1000, Bangladesh 4 Critical Care Department, Lokmanya Hospital, 

Chinchwad, Pune 411033, Maharashtra, India 5 Department of Medicine, Khulna Medical College, 109 South 

Central Road, Khulna 9100, Bangladesh 6 3/1 Boat Club Road, Pune 411001, Maharashtra, India 

7 PCMC Zoo, Sambhaji Nagar, Chinchwad, Pune 411019, Maharashtra, India 8 Liverpool School of 

Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK 9 University of Oxford, Nuffield Department 

of Clinical Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK 

Introduction: Kraits bites in South Asia are generally attributed to Common Kraits 

(Bungarus caeruleus). These, however, share most of their range with superficially very 

similar kraits of the B. sindanus complex such as the Sind Krait (B. s. sindanus) and Wall's 

Krait (B. s. walli). We aimed to compare the venoms of B. sindanus and B. caeruleus and 

their clinical effects in human snake bite victims. 

Methods: Analysis of case histories and literature review; morphological identification of 

responsible snakes; mtDNA sequence analysis; liquid chromatography/electrospray massspectrometry. 

Results: Molecular and mass spectrometry analyses supported the separation of B. 

caeruleus and B. sindanus. They are reliably distinguished by the number of dorsal scale 

rows (15 vs. 17–19, respectively) and differ in behaviour and ecology. Among 4 proven 

and 1 suspected cases of B. s. walli envenoming in India (Pune District, Maharashtra, and 

Indore, Madhya Pradesh), 4 died, including 2 bitten by the same krait. In Bangladesh, 4 out 

of 6 black-and-white banded kraits causing bites in Dhaka and Khulna Divisions were B. s. 

walli. Three B. s. walli bites in India occurred inside temporary huts on construction sites; 

a farmer in Bangladesh was bitten by a B. s. walli entangled in his fishing net. Two 

patients with detailed clinical histories developed paralytic symptoms within 2.5–3 hr after 

the bite, fasciculations, and generalised flaccid paralysis; one showed evidence of 

generalised myocardial damage. There was no response to anticholinesterase or polyvalent 

antivenom. Recovery followed prolonged ventilation. 

Conclusion: More clinical studies of bites by accurately identified species are needed in 

South Asia. Without such evidence, it must not be assumed that the clinical syndrome of 

krait envenoming in this region is caused by B. caeruleus alone. Unusual clinical features 

such as fasciculations and myocardial damage warrant further investigation of the venoms 

of members of the B. sindanus complex and consideration of their inclusion in antivenom 

production. 

o Bangladesh 

o India 

o Bungarus sindanus sindanus 

o Bungarus sindanus walli 

114 


Severe neurotoxic and myotoxic envenoming by the Greater Black Krait (Bungarus 

niger) in Chittagong Division, Bangladesh 

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 , 

Warrell,D.A. 5,6 * 

1 Dhaka Medical College and Hospital, Dhaka 1000, Bangladesh 2 Department of Zoology, University of 

Chittagong, Chittagong 4331, Bangladesh 3 Zentrum der Rechtsmedizin, Klinikum der Johann Wolfgang 

Goethe-Universität, Kennedyallee 104, 60596 Frankfurt am Main, Germany 4 School of Neurology, 

Neurobiology and Psychiatry, The Medical School, University of Newcastle upon Tyne, Newcastle upon 

Tyne NE2 4HH, UK 5 Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK 

6 University of Oxford, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford OX3 

9DU, UK, *david.warrell@ndm.ox.ac.uk 

Introduction: the venomous fauna of Bangladesh is poorly characterised. We aimed to 

discover which species were responsible for bites and to define clinical syndromes of 

envenoming. 

Methods: a prospective clinical study was established at Chittagong Medical College 

using enzyme immunoassay (EIA) and morphological and molecular studies of snakes 

brought with bite victims for species diagnosis. 

Results: among 18 venomous snakes responsible for bites, 4 proved to be Greater Black 

Kraits (Bungarus niger). A 40-year-old labourer was bitten while cutting wood. Paralytic 

symptoms began 4-5 hr later and on admission, 9.5 hr post- bite, he was unconscious with 

flaccid paralysis. Generalised rhabdomyolysis evolved and he died 47 hr post-bite. A 35year-old 

man, bitten while asleep, died within a few hours. A 12-year-old boy, also bitten 

while asleep developed progressive paralysis 11 hr post-bite but recovered after 22 hr of 

mechanical ventilation. An 18-year-old man, bitten while fishing, became paralysed 5 hr 

later but did not need mechanical ventilation. 

Bungarus caeruleus has not been found in Chittagong District but with EIA, sera of 22 

snake bite cases, including 2 of the B. niger cases, reacted weakly with B. caeruleus 

diagnostic antiserum. This antiserum reacted only very weakly with Nepalese B. niger 

venom but strongly with B. caeruleus venom. 

Discussion: before this study, B. niger was known only from Nepal, Bhutan and NE India. 

This is the first report of envenoming by this species anywhere and the first report of 

generalised rhabdomyolysis caused by envenoming by any Asian or African elapid snake. 

Our preliminary data suggest that B. niger may be wide-ranging in Bangladesh and an 

important cause of snake bites. Its venom should be considered when antivenoms are 

designed for this region. The striking finding of rhabdomyolysis should prompt further 

investigation of venom properties. 

o Bangladesh 

o Bungarus niger 


o rhabdomyolysis 

115


Distinctive epidemiology and clinical features of common krait (Bungarus 

caeruleus) bite in Sri Lanka 

Ariaratnam, A. 1 , Sheriff, M.H.R. 1 , Theakston, R.D.G. 2 , Warrell, D.A. 2,3 

1Faculty of Medicine, University of Colombo, Kynsey Road, Colombo 08, Sri Lanka 2 Liverpool School; 

of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK 3 University of Oxford, Nuffield 

Department of Clinical Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK. 

*david.warrell@ndm.ox.ac.uk 

Introduction:10% of snakes brought by snake bite victims during a prospective study at 

10 Sri Lankan provincial hospitals were identified as common kraits (Bungarus 

caeruleus). However, 75% of snake bite victims did not bring the snake. This study 

aimed to define the clinical syndrome of krait bite to aid case identification. 

Methods: we analysed proformas completed for all patients bitten by identified snakes. 

Results: 

Feature 88 cases of proven B. 

caeruleus envenoming 

674 bites by other 

venomous species 

Sex ratio (M:F) 1.6:1 2.9:1 

Bitten while sleeping on 

ground 

100% 1% 

Bitten 2300-0500 hr 100% 49% 

Bitten indoors 100% 3% 

Not bitten on lower limb 77% 18% 

Local envenoming 30% (mild swelling only) 93% 

Respiratory paralysis 64% 2% 

Case fatality 6% 3% 

91% of krait bite victims complained of abdominal pain 

Discussion: this highly distinctive syndrome of Bungarus envenoming has also been 

reported from India and Thailand. Although 60% of krait bite cases were admitted to 

hospital within 4 hours of the bite, the case fatality was double that of envenoming by 

other species, emphasising the need for improved first aid, transport to hospital and 

respiratory support. Prevention depends on encouraging people not to sleep on the floor 

of their dwellings. 

o Common krait 

o Bungarus caeruleus 

o Sri Lanka 

o Clinical syndrome 

116 


First authenticated cases of life-threatening envenoming by the hump-nosed pit 

viper (Hypnale hypnale) in India. 

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 , 

Warrell D. A 2* . 

1 

Little Flower Hospital and Research Centre, Snakebite Research Unit, P.O. Box 23, Angamaly 683572, 

Kerala, India 

2 

University of Oxford, Headington, Oxford, OX3 9DU, UK, *david.warrell@ndm.ox.ac.uk 

Introduction: in Kerala, south-western India, Russell’s viper (Daboia russelii) “annali” 

(Malayalam) is the commonest proven or suspected cause of snake bite, causing 

coagulopathy, bleeding, shock, “permeability syndrome” and acute renal failure. However, 

some patients thought to have been envenomed by hump-nosed pit vipers (Hypnale 

hypnale) “churutta” and saw-scaled vipers (Echis carinatus) “chenatanden” also develop 

coagulopathy, bleeding and acute renal failure. Their haemostatic abnormalities fail to 

respond to Indian polyvalent antivenoms which covers only “the big four” venomous 

snakes of India (cobra, krait, Russell’s and saw-scaled vipers). 

Methods: a clinical study based in Little Flower Hospital, Angamaly and Jubilee Mission 

Hospital, Thrissur involved the strict identification the species of snake responsible for 

cases of life-threatening envenoming. 

Results: five patients developed systemic envenoming after bites by H. hypnale, proved by 

identification of the snakes responsible. Two snakes had been misidentified as E. carinatus 

while three were unidentified. Symptoms of local envenoming were pain, swelling, 

haemorrhagic blistering, bruising and regional lymphadenopathy. Systemic symptoms 

included headache, nausea, vomiting and abdominal and chest pain. There was some 

evidence of haemostatic dysfunction in all cases (coagulopathy, fibrinolysis, 

thrombocytopenia or spontaneous systemic haemorrhage) and of microangiopathic 

haemolysis in two. Two patients were haemodialysed for acute renal failure one of whom 

developed pulmonary oedema requiring mechanical ventilation. 

Discussion: in India, H. hypnale had not previously been regarded as a cause of frequent or 

potentially-dangerous envenoming. It now appears to be second only to Russell’s viper as 

a cause of serious envenoming in parts of SW India. Its medical importance has been 

overlooked throughout its geographical range (southern Kerala to Radhanagari, 

Maharashtra) because of confusion with other small snakes. An effective antivenom is 

urgently needed in South India and in Sri Lanka where this species is also a common cause 

of bites. 

o Hypnale hypnale 

o haemostatic disturbances 

o acute renal failure 

o untreatable 

117

Tuesday Track 1, K3.14 4.20-4.40pm (as part of preceding presentation) 

Severe systemic envenoming by hump-nosed vipers (Hypnale hypnale) in Sri Lanka 

Ariaratnam, A. 1 , Sheriff, M.H.R. 1 , de Silva, A. 2 , Theakston, R.D.G. 3 , Warrell, D.A. 3,4 

1 Faculty of Medicine, University of Colombo, Kynsey Road, Colomob 08, Sri Lanka 

2 Rajarata University, Sri Lanka 

3 Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK 

4 University of Oxford, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford OX3 

9DU, UK *david.warrell@ndm.ox.ac.uk 

Introduction: in Sri Lanka, the hump-nosed pit viper (H. hypnale) is one of the 

commonest causes of snake bite. This small species is distributed throughout the island. It 

is considered by some to be capable of causing only painful local swelling, there have been 

reports of severe systemic envenoming and fatalities since the 1970s. This study aimed to 

discover the true pattern of envenoming attributable to this species. 

Methods: in 10 hospitals in Colombo, Panadura, Watthupitiwela, Chillaw, Matale, 

Pollonnaruwa and Anuradhapura patients who brought the H. hypnale responsible for their 

bites were studied prospectively. 

Results: among the 302 hump-nosed vipers responsible for causing bites, 301 H, hypnale 

and 1 H. nepa were identified. Most of the victims were males (4:1) bitten while walking 

in the dark (between 1800 and 2400 hr) close to their homes. 80% of bites were on the 

lower limbs. 90% developed local envenoming (pain, swelling, bruising, blistering) 

complicated by local necrosis that required amputation of digits in a few cases. 40% had 

incoagulable blood with spontaneous systemic bleeding and 10% went on to develop acute 

renal failure. No neurological signs were observed in contrast to victims of Russell’s viper 

(Daboia russelii). 28% were treated with non-specific Indian polyspecific antivenom 

without effect. There were 3 deaths from complications of acute renal failure and 2 delayed 

deaths in patients with bilateral renal cortical necrosis causing chronic renal failure. 

Discussion: this first study of a large group of proven H. hypnale bites revealed a 

surprisingly high incidence of haemostatic abnormalities which are potentially lifethreatening. 

Acute and chronic renal failure proved fatal in 2% of our patients. Only Indian 

polyvalent antivenom is available in Sri Lanka. It has proved ineffective both clinically and 

in laboratory assays and is dangerous, causing anaphylactoid reactions in 53% of patients. 

A specific H. hypnale antivenom is urgently needed. 

o Hump-nosed pit viper 

o Hypnale hypnale 

o Haemostatic disturbances 

o Renal failure 

118 


INJURIES CAUSED BY VENOMOUS ANIMALS OCURRED IN DOMESTIC 

AND COMMERCIAL AQUARIUMS: A STUDY OF 24 CASES. 

1,2 

Haddad Jr, V . 

1 

Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, Caixa Postal 557, 18618-000, São Paulo 

State, Brazil, haddadjr@fmb.unesp.br 

2 

Vital Brazil Hospital, Butantan Institute, Avenida Vital Brazil, 1500, Butantan, São Paulo State, Brazil. 

INTRODUCTION/OBJECTIVES - Among the recreation activities that more grew in the 

last years, the aquarism wins every day new followers in the entire world. Impelled by 

beautiful fish, plants and decoration objects, the habit involves some problems, as 

envenoming after contact with venomous animals. This communication reports the clinical 

aspects and the animals most frequently involved with envenoming and the therapeutic and 

preventive measures for control of the problem. The other objectives are to alert for the 

possibility of accidents and to investigate the level of information of the proprietors about 

the animals present in the aquarium. METHODS - A prospective study was accomplished 

for three years being looked for the detection of injuries for venomous animals in 

aquariums. These data served as base for epidemiologic, clinical and therapeutic studies on 

the problem. RESULTS/DISCUSSION - in near 600 injuries by venomous aquatic 

animals, fish and sea urchins in aquariums caused 28 or near 4,5% of the total 1 . The 

animals associated to the injuries were marine stingrays (4), freshwater stingrays (2), 

freshwater catfish (2), sand moray eel (1), sea urchins (3) and lionfish (12). The owners of 

aquariums most of the time do not have information about the risks, what intervenes with 

the prevention measures that would have to be taken when dealing with the animals. The 

use of thick rubber gloves is recommended when it is necessary the introduction of the 

hands in an aquarium (domestic or not). The use of nets is also recommended when to be 

manipulated dangerous species of animals. The envenoming are not common, but they can 

occur if the necessary cares will not be taken and a good knowledge of the animal kept in 

the aquarium does not exist. The centers of studies on toxicology and toxinology should be 

alert for the problem. 

(1). Haddad Jr V (2000). Atlas of Brazilian dangerous aquatic animals: a medical guide of diagnosis and treatment. São 

Paulo: Editora Roca, 145 pp. 

o venomous aquatic animals 

o aquarium 

o venomous fishes 

o sea urchins 

119


A dynamic interpretation of the pharmacokinetic volume of distribution and its relation to the 

pharmacokinetics of F(ab')2 and other drugs. 

C. Sevcik 1* , V. Salazar 2 , P. Díaz 1 , G. D'Suze 1 . 

1 Laboratory of Cellular Neuropharmacology, 2 Histology Service from the Centro de Biofísica y 

Bioquímica Instituto Venezolano de Investigaciones Científicas (IVIC), Apartado 21827, 

Caracas 1020A, Venezuela. * csevcik@ivic.ve 

Introduction: We use fluorescent microscopy and numerical methods to study the 

pharmacokinetics of fluorescin isothiocyanate labeled F(ab’)2 [FITC-F(ab’)2] in 

mice. 

Methods: We developed the equation V d t = D A 

120 

z 

AUC ∞[∑ i= 1 

− 

Ci τi e 

t 

τ i] 

¿[∑ 

z 

i= 1 

− 

C i e 

t 

τ i] to 

describe the dependency of the volume of distribution with time. Where DA is the 

amount of drug available in de body after an i.v. bolus, AUC∞ is the 

pharmacokinetic total area under the concentration curve, Ci are the 

concentrations at t=0 for each of the z components of a multiexponential time 

course of the plasmatic concentration of drug, and τi are the time constant for each 

of these components. This equation is less restricted that one previously proposed 

by Nazi (1976). 

Results: Fluorescent microscopy show that the rapid initial decay in plasmatic 

F(ab')2 concentration (Sevcik et al., 2004; Vázquez et al., 2005) may be related to 

uptake of F(ab')2 by vascular endothelium which, in combination with accumulation 

in the vascular wall connective tissue may produce an intermediate plateau in 

F(ab')2 Vd(t) time course a and to produce a slow time course of F(ab')2 Vd(t) which 

requires up to 10 h to reach its final steady value. 

Discussion: The half time of decay of plasmatic concentration of F(ab’)2 and other 

drugs has little use to estimate how a drug distributes through the body to exert its 

action, and in some instances predicts that intermediate plateaus in the time 

course of Vd(t) exist. We also used data from the literature, as well as the equation 

above to show that the kinetic considerations discussed ahead may also apply to 

o Antivenoms 

o Pharmacokinetics 

o Distribution 

o 

F(ab’)2 


Effect of Leukocyte Inhibitors Benzydamine and Cyclophosphamide on Lung 

Injury Caused by Tityus discrepans Scorpion Venom 

D´Suze, G 1* , Salazar, V 2 , Díaz, P 1 , Sevcik, C 1 . 

1 Celullar Neuropharmacology Lab. 2 Light Microscopy Service. IVIC, Biophysics and Biochemistry 

Centre, Apartado 21827, Caracas 1020-A, Venezuela, *gdsuze@ivic.ve 

Introduction: Severe scorpion envenoming produces a systemic inflammatory response 

syndrome causing pulmonary distress. Previously we suggested a close relationship among 

leukocyte activation, fibrin alveolar deposition and lung injury. This study was aimed to 

investigate the effect of a nonsteroidal anti-inflammatory drug and an immunosuppressive 

alkylating agent on lung injury caused by T. discrepans venom. 

Methods: Male IVIC strain mice (~40 g) were previously treated with benzydamine (BZ, 

20 mg/kg), saline or cyclophosphamide (CP, 100 mg/kg). Severe experimental envenoming 

was induced by sc venom (56 μg/mouse) injection. Animals were sacrificed after 5 h by 

cervical dislocation. Lungs were fixed by infusion through the trachea and immersion in 

4% w/v formaldehyde. The Carstairs' method was used for fibrin staining. Stereological 

and morphometric study was carried out using ImageJ determining the fractional area 

occupied by nuclei (FAN), fibrin (FAF), parenchyma (FAP) and alveolar space (FAA). 

Results: FAN was increased by 210% of the control in venom alone, by 56% of the 

control in BZ and by a statistically insignificant 9% in CP. FAF was increased by 188% of 

the control in venom alone, by 25% of the control in BZ and by 42% in CP. FAP was 

increased by 75% of the control in venom alone, by 36% of the control in BZ and by a 

statistically insignificant 5% in CP. FAA was decreased by 19% of the control in venom 

alone, by 13% of the control in BZ and by a statistically insignificant 0.3% in CP. 

Discussion: Both leukocyte inhibitors antagonised venom effects on all parameters studied. 

Cyclophosphamide was more potent as inhibitor that benzydamine, with the exception of 

their effects on FAF which was more inhibited by BZ. These findings support our previous 

proposal that leukocytes play a major role in the production of lung injury by T. discrepans 

venom. (Financed in part by Lab. Silanes/Inst. Bioclón and FONACIT grant S1- 

2001000908) 

o Cyclophosphamide 

o Benzydamine 

o Tityus 

o Lung 

121


Pig as an experimental model for the study of local tissue necrosis caused by snake 

venoms 

Imkhieo, S. 1 , Nakthong, C. 2 , Kespichayawattana, W. 1 , Sirimujalin, R. 2 , and Ratanabanangkoon, K. 1* 

1 Laboratory of Immunology, Chulabhorn Research Institute, Bangkok, Thailand,*sckrt@mahidol.ac.th 

2 Faculty of Veterinary Medicine, Mahidol University, Salaya, Nakorn Pathom, Thailand 

Introduction: Snake venoms can cause serious toxicity locally or systemically or both. 

While systemic poisoning can be treated successfully with specific antivenoms, local tissue 

necrosis (LTN) which in some cases may be severe and require skin graft/amputation, 

remains to be tackled. Experiments on LTN have been carried out mostly in rodents. Pig, 

which unlike rodents, has skin structure similar to that of human, was studied here. The 

aim was to find manifestations induced by snake venoms that can be precisely quantitated. 

Furthermore, the experiments should be short so as to minimize suffering, while 

maximizing the number of data points per animal. 

Materials and Methods: Pigs weighing 20±2 kg and fresh C.rhodostoma venom (CRV) 

were used. Plasma cortisol and IL-6 were quantitated using commercial immunoassay kits. 

Creatine kinase was assayed spectrophotometrically. 

Results and Discussion: I.d. injection of CRV on the lateral sides of pig induced 

induration, the area of which peaked at about 60 min and was dose-dependent. 

Furthermore, dissection of these injection sites after 3-4 hours exposed subcutaneous 

hemorrhage; whose area was also dose dependent. A total of 24 data points (injection sites) 

could be obtained per pig. By implanting a catheter into the jugular vein, blood could be 

drawn gently and serially. After i.m. injection of CRV, plasma creatine kinase and cortisol 

could be determined, these markers peaked at 20 hrs and 2 hrs, respectively. Plasma IL-6 

could not be detected during 72 hours after the venom injection. Finally, histological study 

could be made by serial punch biopsy to study the pathological changes at various skin 

layers during the first few minutes of venom injection. Such study is not possible in 

human, especially with hemotoxic venoms which could lead to severe hemorrhage. This 

animal model should be useful to the study of LTN and its treatment modality. 

This study was supported by a grant from Thailand Research Fund. 

o Snake venom 

o Local tissue necrosis 

o Animal model 

o pig 

122 


Ureases display biological effects independent of enzymatic activity. Is there a 

connection to diseases caused by urease-producing bacteria ? 

Olivera-Severo, D 1 , Wassermann, G.E 1 , Carlini, C.R 1,2 

1 Graduate Program in Cellular and Molecular Biology, Center of Biotecnology, Universidade Federal 

do Rio Grande do Sul, Porto Alegre, Av. Bento Gonçalves, 9500, CEP 91.501-970, Porto Alegre, Brazil, 

2 Department of Biophysics, Universidade Federal do Rio Grande do Sul, Porto Alegre, Av. Bento 

Gonçalves, 9500, CEP 91.501-970, Porto Alegre, Brazil. ccarlini@ufrgs.br 

Introduction: Ureases are highly homologous enzymes from plants, fungi and bacteria that 

hydrolyze urea into ammonia and carbon dioxide. Fungal and plant ureases are homooligomers 

of 90-kDa subunits, while bacterial enzymes are multimers of two or three 

subunit complexes. We showed that jack bean urease and its isoform canatoxin induce 

exocytosis in a number of systems, releasing histamine from mast cells, insulin from 

pancreatic cells, neurotransmitters from brain synaptosomes and activating platelet 

aggregation (ED50 0.015 mg/mL). In vivo canatoxin induces rat paw edema and neutrophil 

chemotaxis. These effects are independent of ureolytic activity and require activation of 

eicosanoid metabolism and calcium channels. Here we investigated if bacterial ureases 

induce platelet aggregation or have pro-inflammatory activity. 

Methods: purified Bacillus pasteurii urease and purified recombinant Helicobacter pylori 

urease were tested for platelet-aggregating activity and in the rat paw edema model. 

Inhibitors of eicosanoid metabolism or blockers of platelet receptors were used to 

investigate signaling transduction. 

Results: B. pasteurii and H. pylori ureases induced platelet aggregation (ED50 0.4 and 0.1 

mg protein/mL, respectively) recruiting the same signal transduction pathways previously 

described for canatoxin. We also observed that 50 micrograms of purified H. pylori urease 

induced maximal pro-inflammatory activity in the rat paw edema model. 

Discussion: H. pylori causes gastric ulcers and cancer by a mechanism that is not fully 

understood. H. pylori urease, accounting for ~10% of bacterial protein, is important for 

gastric colonization as it neutralizes the acidic medium permitting survival in the stomach. 

Our findings that H. pylori urease has biological properties not related to its enzymatic 

activity could be relevant to elucidate the role of this protein in the pathogenesis of the 

gastrointestinal disease caused by this bacterium. 

o urease 

o Helicobacter pylori 

o Platelet aggregation 

o eicosanoids 

123


The Use Of Indigenous Knowledge in Rural Development: A Case Study In The 

Use Of Herbal Medicine In The Treatment of Diseases In The Northern Region Of 

Ghana. 

Arthur,P.K, Opoku, K. F 

University of Cape Coast, School of Agriculture, PMB, Cape Coast, Ghana,*patrickarthur84@yahoo.co.uk 

In Africa, herbal medicine is often used as primary treatment for various diseases. 

Generally, traditional medicines are not well researched and are poorly regulated. This 

paper examines the use of herbal medicines in the treatment of diseases in northern Ghana. 

It discusses the role of indigenous knowledge in rural development. It outlines the 

development in interest in herbal medicine and the rising benefit that may accrue from 

indigenous health resources. Field studies were conducted to investigate the medicinal 

plants, through identification, collection and domestication of these plants in Northern 

Region of Ghana. Questionnaire, personal interview and review and review of available 

records showed that herbal medicines were used in the treatment of diseases in both human 

beings and animals. The researched centered on pharmacology, toxicology and the 

pharmacokinetics of these herbal medicines. This paper reviews the relation between 

indigenous medicines and pharmacology, encompassing human health and indigenous 

medicine; vertinary medicine and animal health; maternal and child health; and sexual 

health and disease. Although no clinical trials of efficacy exist on the use of herbal 

medicines, efforts should be made by mainstream health professionals to provide validated 

information to traditional healers and patients on the judicious use of herbal remedies. 

References: 

Ayo Walberg, health, vol. 10, No. 2, 123-147 

Mensah-Dapaah, K, traditional healing, Ghana Journal of Science,1968, 21, 16-21 

o Pharmacology 

o Pharmacokinetics 

o Toxicology 

o Indigenous 

124 


Pharmacophore mapping of the κ-atracotoxins: selective insect potassium channel 

blockers that reveal a novel insecticide target 

Gunning, S.J. 1 , Maggio, F. 2 , Valenzuela, S. 1 Broady, K.W. 1 , King, G.F. 2 , Nicholson, 

G.M. 1,* 

1 Department of Medical & Molecular Biosciences, University of Technology, Sydney, Broadway, NSW, 

2007 Australia, *Graham.Nicholson@uts.edu.au 

2 Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, 

263 Farmington Ave., Farmington CT 06032-3305, USA 

The κ-atracotoxins (κ-ACTXs) are a family of insect-selective peptide neurotoxins 

containing 36-37 residues, first isolated from the venom of the Blue Mountains funnel-web 

spider, Hadronyche versuta. The amino acid sequence and 3D structure of κ-ACTX 

provided few clues as to its likely molecular target. In order to characterise the site of 

action and phylogenetic specificity of these toxins, whole-cell patch-clamp 

electrophysiology was employed using isolated DUM neurons from the American 

cockroach (Periplaneta americana). The prototypic family member κ-ACTX-Hv1c had no 

effect on the gating or kinetics of INa or ICa at concentrations up to 1 μM. However, at the 

same concentration, it reduced outward Kv channel currents. Subsequent experiments using 

insect DUM neurons indicated that inhibition of the macroscopic IK was due to a block of 

KCa channels, with an IC50 of 3.1 ± 0.1 nM, and not ‘A-type’ or delayed-rectifier Kv 

channels. Insect selectivity was confirmed by a lack of activity on rat dorsal root ganglion 

neuron global IK as well as IK(Ca) at doses up to 1 µM. κ-ACTX-Hv1c is a selective insect 

KCa channel pore-blocker, not a gating modifier, as inhibition of insect IK(Ca) occurred in 

the absence of any voltage-dependent actions on channel activation, and the block could be 

reduced 36-fold by increasing [K + ]i. The insect target was further validated by κ-ACTX- 

Hv1c induced inhibition of IK(Ca) from cloned cockroach KCa (pSlo) channels expressed in 

HEK293 cells. Additional experiments using alanine mutants confirmed that the 

pharmacophore of κ-ACTX-Hv1c comprises Arg 8 , Pro 9 , Val 29 , Tyr 31 as well as IIe 2 and the 

vicinal disulfide (Cys 13 -Cys 14 ), previously identified by toxicity tests in crickets. 

Interestingly, an R8K mutant was 155-fold less active than the native toxin, whereas the 

Lys residue in the functional diad of vertebrate Kv channel toxins normally inserts into the 

channel pore. Thus, κ-ACTX-Hv1c appears to bind to an insect-specific site in the channel 

vestibule and occlude the pore without binding to conserved pore residues found in 

vertebrate and insect channels. This study therefore identifies insect KCa channels as a 

novel insecticide target. 


o Kv channels 

o insecticide 

o electrophysiology 

125


Jellyfish and other Cnidarians cause pain by affecting TRPV1 channels. 

Cuypers, E. 1 , Yanagihara, A. 2 , Karlsson, E. 3 & Tytgat, J. 1* 

1 

University of Leuven, Laboratory of Toxicology, O&N2, Herestraat 49, P.O. Box 922, 3000 Leuven, 

Belgium, * jan.tytgat@pharm.kuleuven.be 

2 

University of Hawaii at Manoa, Békésy Laboratory of Neurobiology, 1993 East West Road, Honolulu, 

HI, 96822, USA 

3 

Lindsbergsgatan 11A, Uppsala, Sweden 

Jellyfish and other cnidarian envenomations are characterised by a burning-pain 

sensation, the precise underlying mechanisms of which are unclear. Activation of 

TRPV1, a non-selective cation channel that is highly expressed in nociceptive 

neurons, leads to an inward flow of monovalent and bivalent cations, resulting in 

depolarisation of the cell and generation of burning pain. 

Here, we show in vitro (heterologous expression system with Xenopus laevis 

oocytes) and in vivo (rat studies) evidence for TRPV1 activation in cnidarian 

envenomations. Cnidaria venoms, unlike those from other non-cnidarian species, 

interfered with the desensitization of TRPV1 channels in an allosteric manner but 

did not change the voltage dependent open probability. Furthermore, the cnidarian 

venom from Cyanea capillata induced a nociceptive reactivity, comparable to 

capsaicin, in laboratory rats, and the selective TRPV1 antagonist, BCTC, was able 

to reduce both capsaicin- and venom-induced nociceptive behaviours, suggestive 

of some unique anti-nociceptive properties of VR1 antagonists. 

These findings, which to our knowledge are the first to explain at least part of the 

symptomology of cnidarian envenomations, provide insights into the design of 

more effective treatments for this global public health problem. 

1. Caterina et al. (1997) Nature 389, 816-24. 

2. Bailey, P. M. et al. (2003) Med J Aust 178, 34-7. 

3. Nilius, B. et al. (2005) J Physiol 567, 35-44. 

4. Swanson, D. M. et al. (2005) J Med Chem 48, 1857-72. 

o Cnidarians 

o TRPV1 

o Pain 

o Desensitization 

126 


Novel Conotoxin Frameworks 

Möller, C. 1 , Rahmankhah, S. 1 , Urdaneta, A.M. 1 , Castillo, C. 2 , Martinez, J.C. 2 , Hernandez, D. 2 , Moran, O. 3 , 

Fields G.B. 1 and Marí, F. 1 * 

1 

Department of Chemistry & Biochemistry and Center of Excellence in Biomedical & Marine 

Biotechnology, Florida Atlantic University, Boca Raton, Fl 33431, USA. mari@fau.edu 

2 

Center of Biosciences and Molecular Biology. Instituto de Estudios Avanzados (IDEA). Sartenejas, 

Venezuela. 

3 

Instituto di Biofisica. Consiglio Nazionale delle Ricerche, Genova, Italy. 

Cone snails are marine venomous predatory molluscs that excel at producing constrained 

peptide toxins (Conotoxins). Conotoxins have been recognized as great 

neuropharmacological agents including the development of Prialt TM , a powerful 

painkiller that has been approved by the FDA in the U.S., which is the first drug of 

marine origin to receive such status. From the venomics of cones snail species from the 

Americas, we have characterized several novel conotoxin frameworks, with the 

following Cys/Loop arrangements: a 4-Cys/3-loop (Framework 14 = F14-conotoxins), a 

6-Cys/4-loop (F15-conotoxins) that features a vicinal disulphide bond, 4-Cys/2-loop 

(F16-conotoxins) with a “reverse alpha” Cys arrangement, 6-Cys/3-loop (F17conotoxins), 

6-Cys/2-loop (F18-conotoxins) and 6-Cys/6-loop (F19-conotoxins). All of 

these frameworks are likely to define new conotoxin gene superfamilies and represent 

unique peptidic scaffolds not seen before in any organism (except for F14). 

These scaffolds expand the structural diversity of the known conopeptide library 

enhancing the neuropharmacological reach of conotoxins. Three F14-conotoxins have 

been purified from Conus floridanus floridensis (flf14a, flf14b and flf14c) and one from 

Conus villepinii (vil14a). Their disulfide bonding pattern (C1-C4; C2-C3) was 

determined by limited proteolysis and analysis by MS. The nanoNMR spectra (< 100 

nanomoles) of the native peptides showed a very well defined structure in solution, and 

in agreement with their CD spectra, all four conotoxins revealed a Helix-Loop-Helix as 

their main 3D fold. These novel Conus peptides structurally resemble the κ-hefutoxins 

found in scorpions. vil14a has a conserved amino acid dyad defining for the binding to 

the voltage-gated potassium channels. Patch-clamp measurements in HEK cells 

transfected with Kv1.3, indicate that vil14a blocks this potassium channels as predicted 

by our structural analysis. The details on the isolation, structural and functional 

characterization of the F14-19 conotoxins frameworks will be discussed. 

o Cone snails 

o Venom 

o Conotoxins 

o Potassium channels 

127


A dual-target, self-synergizing peptide toxin from spider venom 

Sollod, B,L. 1 , Gunning, S. 2 , Wen, S. 2 , Nicholson, G.M. 2 , and King, G,F. 1,* 

1 

Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, 

263 Farmington Ave., Farmington CT 06032-3305, USA *glenn@psel.uchc.edu 

2 

Department of Medical & Molecular Biosciences, University of Technology, Sydney, Broadway NSW 

2007, Australia 

Excluding insects, which are their primary prey, spiders are the most successful terrestrial 

invertebrates. Whereas most invertebrates produce an array of neuropeptides for internal 

regulation of various physiological and behavioural processes, spiders synthesize in their 

venom glands a combinatorial library of neuropeptides that are designed to kill or paralyze 

envenomated prey 1 . These neurotoxins are initially produced as prepropeptide precursors 

that are posttranslationally processed to yield the mature toxins. The complete venom is 

often remarkably complex—recent peptidomic analyses reveal that a single spider venom 

can comprise more than 1000 different peptide toxins 2 . 

We have been exploring the potential of spider venoms to contribute to the development of 

insecticides with novel modes of action. We demonstrated previously that it is possible to 

isolate spider toxins that potently block specific subtypes of insect ion channels while 

being devoid of activity against vertebrate orthologs of these channels 3 . More recently, via 

analyses of venom-gland cDNA libraries, we isolated a novel family of insecticidal peptide 

toxins that appear to be ubiquitously expressed in the venom of Australian funnel-web 

spiders (genera Atrax and Hadronyche). These toxins are remarkable for their ability to 

potently block both voltage-gated calcium channels and calcium-activated potassium 

channels in insect neurons. These toxins are the most potent insecticidal peptide toxins 

isolated thus far from spider venoms, and their potency appears to result from a selfsynergistic 

effect on the two targeted ion channels. In essence, this dual-target toxin 

corresponds to a toxin cabal encoded within a single polypeptide chain. The implications 

of using a dual-target approach for the development of novel insecticides will be discussed. 

1. Sollod, B.L., Wilson, D., Zhaxybayeva, O., Gogarten, J.P., Drinkwater, R., and King, 

G.F. (2005) Peptides 26, 131–139. 

2. Escoubas, P, Sollod, B.L., and King, G.F. (2006) Toxicon 47, 650–663. 

3. Tedford, H.W., Gilles, N., Ménez, A., Doering, C.J., Zamponi, G.W., and King, G.F. 

(2004) J. Biol. Chem. 279, 44133–44140. 


o ion channel 

o self-synergism 

o insecticide 

128 


Fulvol Acetate, a novel activator of transient Low-Voltage Activated (LVA) Ca 2+ 

current 

Petit, K.E. 1* , Grolleau, F. 2 , Lapied, B. 3 , Hamon, A. 3 , Biard, J.F. 1 

1 

Université de Nantes, Nantes Atlantique Universités, SMAB, EA 2160, Faculté de Pharmacie, 1 rue G. 

Veil – BP 53508, Nantes, F-44000 France * karina.petit@univ-nantes.fr 

2 

Laboratoire Biologie Neurovasculaire Intégrée, UMR CNRS 6214 / Inserm 771, UFR Sciences 

Médicales, Rue Haute de Reculée, F-49045 Angers cedex 01 France 

3 

Université d'Angers, RCIM, EA 2647, Faculté des Sciences, 2 boulevard Lavoisier, Angers cedex, F- 

49045 France 

Fulvol Acetate is a new norsesquiterpene isolated from the soft coral Rhytisma fulvum. The 

unique and novel effect on neuronal transient Low-Voltage Activated (LVA) calcium 

current led us to deposit a patent. 1 

Electrophysiological studies were conducted on isolated short term cultured Dorsal 

Unpaired Median (DUM) neurons of the cockroach (Periplaneta americana) using the 

whole cell patch-clamp technique. Parallel double intracellular microelectrode 

investigations were also performed on three α-subunits of the T-type LVA calcium 

channels (Cav 3.1, 3.2 and 3.3) expressed in Xenopus oocytes. 

In current-clamp, external application of Fulvol Acetate on DUM neurons induced an 

increase of the action potential discharge frequency together with a potentialisation of the 

posthyperpolarisation. This seemed to indicate the participation of calcium currents. This 

was confirmed with additional voltage-clamp experiments indicating that transient LVA 

calcium current was specifically increased (60 %) in DUM neurons. No effect of Fulvol 

Acetate was observed on both sodium and High Voltage-Activated (HVA) calcium 

currents. Similar results were observed when Fulvol Acetate was intracellularly applied 

into DUM neuron cell body through the patch pipette. By contrast, external application of 

Fulvol Acetate onto oocytes expressing the different α-subunits of the T-type LVA 

calcium channels did not show any effects. 

These results led us to suggest that Fulvol Acetate could act indirectly on the LVA calcium 

channel via, among other possibilities an accessory protein. Based on these findings, the 

specific increase of the LVA calcium current amplitude produced by Fulvol Acetate makes 

this compound the first activator of such channels never reported before. 

1 Petit et al. (2003) Patent PCT/FR04/03030. 

2 Lacinova et al. (2000) Neuropharmacology, 39, 1254-1266. 

3 Yaksh (2006) The Journal of Pain, 7(1), S13-S30. 

o Marine natural substance 

o Activator of tLVA 

o Research tool 

o Drug 

129


A novel family of conotoxins target to nACh-Receptors 

Kauferstein, S. 1 , Nicke, A. 2 , Kendel, Y. 1 ., Zamudio, F.Z. 3 , Stöcklin, R. 4 , Mebs, D. 1 

1University of Frankfurt, Kennedyallee 104, D-60596 Frankfurt am Main, Germany, 

kauferstein@em.uni-frankfurt.de 

2 Max-Planck-Institute for Brain Research, D-60528 Frankfurt am Main, Germany 

3 Instituto de Biotecnologia-UNAM, Avenida Universidad, 2001, Cuernavaca 62210, Mexico 

4 Atheris Laboratories, CH-1233 Bernex-Geneva, Switzerland 

Introduction: Over the last 50 million years, cone snails developed a highly efficient 

venomous system.Using their complex venom apparatus they inject a mixture of peptides, 

which almost instantly paralyzes the prey. These peptides are consisting mainly of 11 to 35 

amino acid residues with a variable number of disulfide bridges, exhibiting a great 

variability in their amino acid sequence. The venom of cone snails is one of the most 

complex peptide mixtures acting on a wide range of target tissues in the nervous system, 

e.g. ion channels and receptors. The high affinity of these toxins to these targets made 

them indispensable tools in neurochemistry and neurophysiology. 

Methods: Crude venom samples from Conus capitaneus were tested on nAChRs expressed 

in oocytes using the two-electrode voltage-clamp method. Fractionation of the samples 

was performed by HPLC followed by liquid chromatography-electrospray-mass 

spectrometry analysis. N-terminal amino acid sequence analysis was performed by 

automated Edman degradation. 

Results: From the venom of Conus capitaneus a protein was isolated which represents a 

new family of conotoxins acting at nicotinic AChRs. It has a remarkably high molecular 

weight of 11 kDa and, as SDS-PAGE revealed the toxin is a dimer. Its N-terminal 

sequence exhibits no similarity to any known proteins. Analysis of the subtype selectivity 

of the purified toxin on nAChRs revealed subnanomolar potency on α7 and nanomolar 

potency on� α3β2 nAChRs subtypes with IC50 values of 300 pM and 3 nM respectively. 

The IC50 value for the �α4β2 subtype was 30 nM, making it the most potent conotoxin 

acting on this AchR-subtype known so far. 

Discussion/Conclusion: We suggest that some cone snail species, besides families of small 

peptides, developed an alternative class of large conopeptides to target the nAChR. 

o Conotoxins 

o nAChR 

o Cone snails 

o Voltage clamp 

130 

Thursday Track 1, K3.14 3.00-3.20pm 

Scorpion Beta-Toxins Interact with their Na-channel Receptor Via a Novel 'Ratchet 

Mechanism' 

Ilan N 1* , Karbat I 1 , Cohen L 1 , Catterall WA 2 , Gordon D 1 , Gurevitz M 1 

1 

Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Israel 

*nitzai@post.tau.ac.il 

2 

Department of Pharmacology, University of Washington, Seattle, WA 98195-7280, USA 

Gating modifiers constitute a large group of polypeptide toxins that interact with the 

voltage-sensing module of ion channels. Among them, scorpion beta-toxins induce a 

negative shift in the voltage dependence of sodium channel activation following a priming 

depolarization. To explain their effect, a ‘voltage sensor trapping’ model has been 

proposed, in which the voltage sensor of domain-II (DIIS4) is trapped in an outward, 

activated position by a pre-bound beta-toxin upon membrane depolarization (1). Using the 

scorpion excitatory beta-toxin, Bj-xtrIT, we demonstrate uncoupling of toxin activity from 

its binding affinity by a single substitution E15R, thereby obtaining an efficient antagonist 

of the native toxin (2). Kinetic analysis of the effect of the anti-mammalian scorpion betatoxin 

Css4 on the rat brain Na-channel, Nav1.2a, revealed a biphasic onset of toxin effect 

with a voltage-dependent fast time constant of


A new toxin from the sea anemone Condylactis gigantea with effect on sodium 

channel inactivation 

Béress, L. 1, 2 , Ständker, L. 1,6 , John, H. 1 , Forssmann, W.G. 1 , Pérez-Castells, J. 6 , López-Méndez, B. 6 , 

Jiménez-Barbero, J. 6 , Giménez-Gallego, G. 6 , Garateix, A. 3 , Aneiros, A. 3† , Christ, T. 4 , Salceda, E. 5 , Soto, 

E. 5 , Ravens, U. 4* 

1 IPF PharmaCeuticals GmbH, 30625 Hannover, Germany 2 Institut für Experimentelle Toxikologie, 

Universitäts-Klinikum, 24105 Kiel, Germany 3 Centre of Marine Bioproducts (CEBIMAR), Loma y 37, 

Vedado, Ciudad Habana, Cuba 4* Institut für Pharmakologie und Toxikologie, Medizinischen Fakultät der 

TU Dresden, Fetscherstraße 74, 01307 Dresden, Germany, * ravens@rcs.urz.tu-dresden.de 

5 Institute of Physiology, University Autonomous of Puebla, 14 Sur 6301, CP 72570, Puebla,México. 6 Centro 

de Investigaciones Biologicas (CIB/CSIC), C. Ramiro de Maeztu, 9, 28040 Madrid, Spain 

Our aim was to isolate and characterize a peptide toxin representing the main crab 

paralyzing activity in the sea anemone Condylactis gigantea. Methods: Applying ethanolic 

acetic acid extraction of the sea anemone Condylactis gigantea and a multistep 

chromatographic procedure employing cation exchange, size exclusion, and reversed phase 

chromatography, a peptide toxin was isolated. The toxic activity was monitored by its crab 

paralyzing effects and biophysical and structural features of the purified peptide were 

characterized by mass spectrometry, sequence and NMR analysis. Its electrophysiological 

properties were analysed on rat dorsal root ganglion neurons and on myocardial cells of 

guinea pigs. A new peptide toxin exhibiting a MW of 5043 Da (av.) and comprising 47 

amino acid residues was isolated and exhibited a strong paralytic activity on crustacea 

(LD50 approx. 1 μg/kg). Complete sequence analysis of the toxic peptide revealed the 

isolation of a new member of type I sea anemone sodium channel toxins. The toxin termed 

CgNa contained the typical pattern of six cysteine residues. From 11 kg of wet starting 

material approximately 1 g of the peptide toxin was isolated. Using whole-cell patch clamp 

technique (n = 60) under current clamp condition CgNa increased action potential duration 

of rat dorsal root ganglion neuronns. It prolonged the cardiac action potential duration and 

enhanced contractile force albeit at 100-fold higher concentrations than the Anemonia 

sulcata toxin ATXII. Homonuclear NMR experiments have been used to determine the 3D 

structure of the peptide. The structure consists of four beta strands and a long nonstructured 

loop between residues 7-19. Root mean squared deviation for the best 20 

structures (CYANA) is better than 1 Å. The electrophysiological effects of CgNa may be 

due to slowing down of the TTX-S sodium current inactivation, without modifying the 

activation process. The action on sodium channel inactivation and on cardiac excitation 

contraction coupling resemble previous results with peptides obtained from sea anemones, 

probably by binding to receptor site 3 of the mammalian sodium channel. We suggest that 

CgNa may represent the main peptide toxin of this sea anemone species. 

o peptide toxin 

o sodium channel inactivation 

o sea anemone 

o 3D structure 

132 


Allosteric interactions between scorpion toxin receptor sites on voltage-gated Na channels 

imply a novel role for weakly active components in arthropod venom 

Cohen, L 1 , Lipstein, N 1 , Gordon, D 1 . 

1 Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat 

Aviv, Tel Aviv 69978, Israel; dgordon@post.tau.ac.il 

Scorpion beta and alpha-toxins modify the activation and inactivation of voltage-gated 

sodium channels. Although the two types of toxin bind at two distinct receptor sites on 

the same sodium channel, they exhibit synergic effects when co-injected into insects 

(1). To clarify the basis of this synergism we examined the mutual effects of alpha and 

beta toxin representatives in radio-ligand binding assays. We found positive allosteric 

interactions between receptor site-4 of the excitatory Bj-xtrIT and the depressant 

LqhIT2 beta toxins and receptor site-3 of the alpha toxin LqhαIT, on locust neuronal 

membranes. Unexpectedly, a non-toxic mutant Bj-xtrIT-E15R (2), which binds with 

high affinity to receptor site-4, was able to enhance LqhαIT binding and toxicity 

similarly to the unmodified Bj-xtrIT. This result indicates that mere binding of a nontoxic 

ligand to receptor site-4 ('silent binding') induces a conformational change that 

does not alter channel gating, but influences toxin binding at receptor site-3 leading to 

enhanced toxicity. This finding suggests a new functional role for weakly toxic 

polypeptides in that they enhance the effect of other active neurotoxins in the arthropod 

venom. Such 'silent binding' may have also valuable implications in attempts to 

improve drug efficacy by combining potent drugs with non-active allosteric enhancers. 

References: (1) Herrmann R, Moskowitz H, Zlotkin E, Hammock B (1995) Positive 

cooperativity among insecticidial scorpion neurotoxins. Toxicon 33:1099-1102 

(2) Karbat I, Cohen L, Gilles N, Gordon D, Gurevitz M (2004) Conversion of a 

scorpion toxin agonist into an antagonist highlights an acidic residue involved in 

voltage sensor trapping during activation of neuronal Na channels. FASEB J 18:683-89 

o Scorpion toxin 

o sodium channel 

o alpha-like-toxin 

o receptor recognition 

133


OD1, α-like toxin isolated from the Iranian yellow scorpion Odonthobuthus doriae 

venom 

Jalali, A 1 *, Bosmans, F 2 , Amininasab, M 3 , Clynen, E 4 , Cuypers, E 2 , Zaremirakabadi, 

A 5 , Sarbolouki, M.N 3 , Schoofs, L 4 , Vatanpour, H 6 , Tytgat, J 2 ** 

1 

Dept. of Pharmacology and Toxicology, Jundishapour Univ. of Med. Sci., Ahwaz, Iran, 

*Amir.Jalali@ajums.ac.ir 

2 

Lab. of Toxicology, Univ. of Leuven, Leuven, Belgium, ** Jan.Tytgat@pharm.kuleuven.ac.be 

3 

IBB Institute, Tehran Univ. of Med. Sci., Tehran, Iran. 

4 

Lab. for Developmental Physiology and Mol. Biology, Univ. of Leuven, Belgium. 

5 

Razi Institute of Vaccine and Serum, Karaj, Iran. 

6 

Dept. of Pharmacology and Toxicology, Shaheed Beheshti Univ. of Med. Sci., Tehran, Iran. 

Within the most dangerous scorpions in Iran, the Odonthobuthus doriae sting can cause 

fatal envenoming with unknown mechanism of action. With view to this and similarity of 

clinical effects to toxins that affect Na + channels, this investigation was conducted to 

access to the bioactive substances in the scarcely studied Iranian scorpion venom. 

In this study, we isolated and pharmacologically characterized the α-like toxin from the 

Odonthobuthus doriae venom, termed OD1 and its primary sequence was determined: 

GVRDAYIADDKNCVYTCASNGYCNTECTKNGAESGYCQWIGRYGNACWCIKLPDEVPIRIPGKCR 

Using the two-electrode voltage clamp technique, the pharmacological effects of OD1 

were studied on three cloned voltage-gated Na + channels expressed in Xenopus laevis 

oocytes (Na(v)1.2/beta1, Na(v)1.5/beta1, para/tipE). The inactivation process of the 

insect channel, para/tipE, was severely hampered by 200nM of OD1 (EC50= 80 ±14nM) 

while Na(v)1.2/beta1 still was not affected at concentrations up to 5microM. 

Na(v)1.5/beta1 was influenced at micromolar concentrations. 

Our results show that OD1 can inhibit the inactivation process of both mammalian (Nav1.5) 

and insect VGSCs (para). However, OD1 does posses a distinct preference for para. 

134 


Direct Evidence that Receptor Site-4 of Sodium Channel Gating Modifiers is not 

Dipped in the Phospholipid Bilayer of Neuronal Membranes 

Cohen L 1* , Gilles N 2 , Karbat I 1 , Ilan N 1 , Gordon D 1 , Gurevitz M 1 

1 

Department of Plant Sciences, George S Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv 

69978, Tel-Aviv, Israel *liorcoh@post.tau.ac.il 

2 

CEA, Department d'Ingenierie et d'Etudes des Proteines, CE Saclay F-91191 Gif Sur Yvette Cedex, 

France 

In a recent note to Nature R. MacKinnon has raised the possibility that potassium channel 

gating modifiers are able to partition in the phospholipid bilayer of neuronal membranes 

and by increasing their partial concentration adjacent to their receptor affect channel 

function with apparent high affinity (1). This suggestion was adopted by Smith et al. (2), 

who analyzed the partitioning of sodium channel modifiers in liposomes. They found that 

certain modifiers were able to partition in these artificial membranes and on this basis 

have extrapolated that scorpion beta-toxins interact with their channel receptor in a 

similar mechanism as that proposed by MacKinnon. Since this hypothesis has actually 

raised a new conception, we examined it in binding assays using a number of 

pharmacologically distinct scorpion beta-toxins and insect and mammalian neuronal 

membrane preparations, as well as by analyzing the rate by which the toxin effect on 

gating of Drosophila DmNav1 and rat brain rNav1.2a develops. We show that in general 

scorpion beta-toxins do not partition in neuronal membranes and that in the case where a 

depressant beta-toxin partitions in insect neuronal membranes, this partitioning is 

unrelated to its interaction with the receptor site and the effect on the gating properties of 

the sodium channel. These results negate the hypothesis that the high affinity of betatoxins 

for sodium channels is gained by their ability to partition in the phospholipid 

bilayer, and clearly indicate that the receptor site for scorpion beta-toxins is accessible to 

the extra-cellular solvent. 

References 

1. Lee SY & MacKinnon R (2004) Nature 430, 232-235 

2. Smith JJ, Alphy S, Seibert AL & Blumenthal KM (2005) J Biol Chem 280, 11127- 

11133 

3. Cohen L, Gilles N, Karbat I, Ilan N, Gordon D & Gurevitz M (2006) J Biol Chem In 

press 

o Scorpion beta-toxins 

o Phospholipid bilayer 

o Voltage-gated sodium channels 

o Partition 

135


Structure and alanine scan of a spider toxin that affects the activation of 

mammalian voltage-gated Na + channels 

Norton, R.S. 1 , Corzo, G. 2 , Sabo, J.K. 1 , Bosmans, F. 3 , Villegas, E. 4 , Tytgat, J. 3 

1 

Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville 3050, Australia. 

ray.norton@wehi.edu.au 

2 

Institute of Biotechnology-UNAM, Av. Universidad 2001, Cuernavaca, Morelos, 62210, Mexico 

3 

Laboratory of Toxicology, University of Leuven, Campus Gasthuisberg, O&N2, PO Box 922, 3000 Leuven, 

Belgium 

4 

Centro de Investigacion en Biotecnologia UAEM, Av. Universidad 2001, Cuernavaca, Morelos, 62210, Mexico 

Magi 5, from the hexathelid spider Macrothele gigas, is a 29-residue polypeptide stabilized 

by three disulfide bridges. It binds specifically to receptor site 4 on mammalian voltagegated 

sodium channels, and competes with classical scorpion β-toxins, such as Css IV from 

Centruroides suffusus suffusus. As a consequence, Magi 5 shifts the activation voltage of 

the mammalian Nav1.2 channel to more hyperpolarized voltages, whereas the insect 

channel, para, is not affected. 

To gain insight into toxin-channel interactions, Magi 5 and 23 analogues were produced by 

peptide synthesis. The three-dimensional structure of Magi 5 in aqueous solution was 

determined and its voltage-gated sodium channel binding surfaces were mapped onto this 

structure using data from electrophysiological measurements on a series of Ala-substituted 

analogues. The structure clearly resembles the inhibitor cystine knot (ICK) structural 

motif, although the triple-stranded β-sheet typically found in that motif is partly distorted 

in Magi 5. 

The interactive surface of Magi 5 towards voltage-gated sodium channels resembles in 

some respects the Janus-faced atracotoxins, with functionally important charged residues 

on one side of the toxin and hydrophobic residues on the other. Magi 5 also resembles the 

scorpion β-toxin Css IV, which displays distinct non-polar and charged surfaces that are 

critical for voltage-gated sodium channel binding and has a key Glu involved in voltage 

sensor trapping (1). It is likely that these two distinct classes of toxin, with different amino 

acid sequences and different structures, utilize similar groups of residues on their surface to 

achieve the common end of modifying voltage-gated sodium channel function. Magi 5 thus 

represents a valuable new probe of receptor site 4 on mammalian VGSC. 

1. Cohen, L., Karbat, I., Gilles, N., Ilan, N., Benveniste, M., Gordon, D., and Gurevitz, M. 

(2005) J Biol Chem 280, 5045-5053 

o polypeptide 

o sodium channel 

o structure 

o binding surface 

136 


X-ray structure and point mutagenesis of the scorpion depressant toxin LqhIT2 

reveals key determinants crucial for activity and anti-insect selectivity 

Izhar Karbat 1 , Michael Turkov 1 , Lior Cohen 1 , Roy Kahn 1 , Dalia Gordon 1 , Michael Gurevitz 1* and Felix 

Frolow 2 

Departments of 1 Plant Sciences and 2 Molecular Microbiology & Biotechnology, George S. Wise Faculty of Life 

Sciences, Tel-Aviv University, Tel-Aviv , Israel, *karbat@post.tau.ac.il 

Scorpion depressant β-toxins are 61 amino acid long polypeptides that show preference 

for insect voltage gated sodium channels (Navs) and modulate their activation process by 

shifting the voltage dependence of activation to more negative membrane potentials. 

Although a number of depressant toxins were identified and characterized only a few were 

cloned 1 and just little is known about residues important for their activity 2 . In addition, 

data pertaining to the three-dimensional structure of these toxins are currently lacking. 

Here we report about the crystal structure of LqhIT2, a depressant toxin from the venom 

of Leiurus quinquestriatus hebreus, refined at 1.2Å resolution. Comparison of LqhIT2 

structure to those of scorpion β-toxins highly active on mammals reveals a similar overall 

structure composed of two lobes, the Core-lobe and the NC-lobe. Mutagenesis of LqhIT2 

illuminates a continuous bioactive surface encompassing a groove formed between the 

Core- and NC- lobes (N-groove), a conserved pharmacophore 3 (Glu24, Tyr26), and Trp53 

and Asn58 of the C-tail. The fine surface topology of the N-groove region and its overall 

hydrophobicity are critical for toxin activity and anti-insect selectivity. Yet, NMR studies 

of similar β-toxins suggest that this region is highly flexible in solution. Combined with 

previous studies, we offer a model in which the initial docking of the toxin on the channel 

involves interactions between the structurally rigid pharmacophore and C-tail to a 

stationary channel region, followed by 'voltage-sensor trapping' achieved through a 

conformational change in the N-groove, which is triggered by channel activation. 

References 

1. Possani LD, Becerril B, Delepierre M & Tytgat J (1999) Eur J Biochem 264:287-300 

2. Strugatsky D, Zilberberg N, Ilan N, Turkov M, Cohen L, Stankiewicz M, Pelhate M, Gilles N, 

Gordon D & Gurevitz M (2005) Biochemistry 44:9179-91873 

3. Cohen L, Karbat I, Gilles N, Ilan N, Gordon D & Gurevitz M (2005) J Biol Chem 280:5045- 

5053 

o Scorpion depressant toxin 

o X-ray structureBioactive surface 


o Bioactive surface 

137


New β-type toxins from the Androctonus scorpion venoms ? 

Martin-Eauclaire, M-F 1 *, Céard, B 1 , Bosmans, F 2 , Rosso, J-P 1 , Tytgat, J 2 and Bougis, 

P. E. 1 

1 CNRS FRE 2738, Faculté de Médecine secteur Nord, Institut Jean Roche, Université de la Méditerranée, Bd 

Pierre Dramard, 13916, Marseille, Cedex 20, France.mail : eauclaire.mf@jean-roche.univ-mrs.fr 

2 Laboratory of Toxicology, University of Leuven, E. Van Evenstraat 4, 3000 Leuven, Belgium. 

Since our main objective was the discovery of new selective insect voltage-gated 

Na+ channels (NavCh) modulators, we have analyzed our collection of uncharacterized 

protein fractions from the venom of the North African scorpion Androctonus australis. In 

this venom, more than 90 % of the lethality for mice and insects was caused by “longchain” 

toxins, which modify the gating properties of NavCh (e.g. the α-toxins AaH I, II, III 

and IV specifically active on mammals and the “excitatory” insect-specific toxin AaH IT). 

From the venom of the scorpion Androctonus australis, we have isolated a new 

bioactive polypeptide termed AaBTX-L1. When tested on the insect NavCh (para) of the 

fruit fly, this toxin was able to induce a clear shift in activation (V1/2) resulting in the 

opening of the channel at more negative membrane potentials. Furthermore, AaBTX-L1 

was totally devoid of toxicity when injected into mice intracerebroventricularly and did not 

compete with radiolabeled voltage-gated K + and Na + channel toxins in binding 

experiments on rat brain synaptosomes. Using its N-terminal amino acid sequence to 

design degenerate primers, several clones were amplified by PCR from the Androctonus 

australis venom gland cDNA library. As a consequence, seven full oligonucleotide 

sequences encoding “long-chain” polypeptides with only three disulfide bridges have been 

cloned for the first time and are described here. Remarkably, they share a high similarity 

with the anti-insect toxin Birtoxin from Parabuthus transvaalicus. 

Classically, scorpion β-toxins produce a hyperpolarizing shift of the voltage dependence of 

activation in NavCh. The unambiguous electrophysiological effect observed on the insect 

NavCh of Drosophila by AaBTX-L1 is fully consistent with this β-type activity. 

Previously, a weak β-type activity, associated with another type of anti-insect toxin (AaH 

IT4) both active on insects and mammals, was identified in the Androctonus australis 

venom using competition against the β-toxin 125 I-Css II bound on its receptor site 4 on rat 

brain synaptosomes. With the results of our study, the new structural group of scorpion 

toxins related to Birtoxin has increased significantly. 

o scorpion toxins 

o insects 

o cDNA 

o voltage-gated sodium channels 

138 


Cyt2Ba of Bacillus thuringiensis subsp. israelensis: activation by putative 

endogenous protease 

Cahan, R. 1* , Nisnevitch, M. 1 , Cohen, S. 1,2 , Ben-Dov, E. 2 , Zaritsky, A. 2 and Sofer, Y. 2 

1* College of Judea and Samaria, Ariel 44837, Israel. rivkac@yosh.ac.il 

2 Ben-Gurion University of the Negev,POB 653, Beer-Sheva 84105, Israel . 

Introduction: Bacillus thuringiensis subsp. israelensis (Bti) produces parasporal 

crystalline proteins, which possess larvicidal and cytolytic activities. The crystal is 

composed of four main proteins, Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa. The genes for 

these and other Cry and Cyt proteins are located on the large plasmid (128 kb) pBtoxis. 

Analysis of the plasmid sequence revealed besides cyt1Aa, an additional gene cyt2Ba 

encoding another Cyt protein. The aim of this research was to purify the Cyt2Ba and to 

identify its activity. 

Methods: The gene cyt2Ba of (Bti) was cloned for expression, together with p20, in an 

acrystalliferous strain IPS78/11. This strain was grown 4 days, centrifuged, and the 

sediment, including crystals, spores and cell debris, was divided into two portions for 

further purification. The first portion was only solubilized. The second portion was 

partially purified by biphasic separation and the crystals obtained were solubilized and 

activated by exogenous proteases. 

Results: The supernatant of the sediment that was only solubilized consisted of a 

predominant protein with molecular mass of 22 kDa which was designated Cyt2Ba-E 

for endogenous bacterial proteases that seem to digest the intact Cyt2Ba. The Cyt2Ba-E 

had a relatively high haemolytic activity. Mass spectrometric analysis revealed that the 

N-terminus of Cyt2Ba-E, having Threonine-35 as the first residue. The protein band of 

Cyt2Ba-E included another protein that was discovered by Blast analysis as a metallo 

protease camelysin. The supernatant fraction of the solubilized sediment that was 

partially purified by biphasic separation contained a protein, with molecular mass of 

about 24 kDa. Edman degradation analysis revealed that the N-terminal sequence is 

MHLNN the same as the original Cyt2Ba thereby designated as Cyt2Ba-N (N-for 

native). The haemolytic activity of Cyt2Ba-N was negligible however soluble Cyt2Ba- 

N that was incubated with exogenous proteases yielded a major band of 22 kDa with the 

same size and haemolytic activity as Cyt2Ba-E. 

Discussion: Cyt2Ba crystals solubilization in the presence of endogenous proteases, 

most likely by a camelysin, enabled quick and simple procedure to obtain rather pure 

and active Cyt2Ba toxin. 

o Bacillus thuringiensis subsp. israelensis, 

o Cyt2Ba 

o camelysin 

139


New type of cytolysin and an unusual phospholipase A2 like protein isolated from 

the Northern Pacific sea anemone Urticina crassicornis 

Razpotnik, A. 1 , Križaj I. 2 , Šribar, J. 2 , Maček P. 1 , and Turk, T. 1* 

1 University of Ljubljana, Department of Biology, 1000-Ljubljana, Slovenia *tom.turk@bf.uni-lj.si 

2 Jožef Stefan Institute, Deapartment of Biochemistry, 1000-Ljubljana, Slovenia 

Sea anemones generally possess two types of toxins: neurotoxins and cytolysins. The 

former are relative short polypeptides acting on voltage gated sodium or potassium 

channels. The later are (i) actinoporins which disrupt biological membranes by forming 

discrete oligomeric pores or very rarely (ii) phospholipase A like enzymes which 

hydrolyze membrane phospholipids. As observed previously, Northern Pacific sea 

anemone Urticina crassicornis possesses polypeptides with potassium channel blocking 

activity and proteins having cytolytic activity. 

From freeze-dried specimens we have isolated a novel type of cytolytic protein which is 

clearly different from those of actinoporin family. It is a 28 kDa protein which is 

inhibited by sphingomyelin, but can only disrupt lipid vesicles or lipid monolayers 

composed of sphingomyelin/cholesterol mixtures. Such lipid composition is 

characteristic for so called lipid rafts, therefore new cytolysin may be used as a new 

tool for localization of these important cell membrane domains. We report a partial 

amino acid sequence and some characteristics of this novel cytolysin. 

Fresh extracts milked from tentacles and gastric fluid of the same sea anemone were used 

for isolation of a novel phospholipase A2 type protein which has some unique structural 

features not previously known within this protein family. cDNA deduced amino acid 

sequence reveals a large degree of homology to β-bungarotoxin or notexin, a well known 

examples of snake neurotoxic phospholipases. However, new protein has a unique 

structural feature at the position 25 where absolutely conserved Cys residue is replaced by 

an Asp residue. New phospholipase like protein is also about 10 amino acid residues 

shorter than its snake neurotoxic relatives. New protein clearly show concentration 

dependent phospholipase enzymatic activity using pyr-PG as a substrate, which is, 

however, only about 2.5% of that obtained with ammoditoxin C. We also report on some 

other characteristics of this unique phospholipase A2 like protein. 

o Sea anemone 

o cytolysin 

o phospholipase A2 

o new type 

140 


Latarcins – a group of spider venom cytolytic peptides with high structural 

diversity 

Vassilevski, A.A. 1* , Kozlov, S.A. 1 , Feofanov, A.V. 1 , Surovoy, A.Y. 1 , Karpunin, D.V. 1 , Grishin, E.V. 1 

1 Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. 

Miklukho-Maklaya, 16/10, 117997 Moscow, Russia, *avas@ibch.ru 

A combination of chromatography, mass spectrometry, and EST database analysis was 

used to identify antimicrobial and cytolytic peptides in the venom of the spider Lachesana 

tarabaevi. In total, sequences of 11 new short linear cationic and amphipathic peptides 

named latarcins were retrieved and split into 7 groups based on similarity; latarcins showed 

negligible level of homology to other known polypeptides. 7 of the peptides from 5 groups 

were isolated from the venom. These peptides were chemically synthesized and were 

found to lyse different cell types – bacteria, yeasts, and erythrocytes – at micromolar 

concentrations. Under physiological membrane potential, all latarcins caused scaled 

membrane destabilization when tested in planar lipid bilayers. In contact with target 

membranes, the peptides were suggested to adopt amphipathic α-helical structure, as 

indicated by CD spectroscopy. The “carpet” model was proposed to account for latarcins’ 

mode of action. Thus, the spider was found to produce a cocktail of structurally diverse but 

functionally similar venom peptides. Such biomolecular diversity seems to be a common 

evolutionary trend for venomous animals and venom compounds – neurotoxins and 

cytolytic peptides. 

In addition, latarcin precursor protein sequences were retrieved from the venom gland EST 

database. The precursors showed a considerable level of variation and were split into 3 

groups: simple precursors with a conventional prepropeptide structure, binary precursors 

with 2 mature chains arranged sequentially into a typical modular block organization, and 

complex precursors of modular structure suggested to be cleaved into mature chains of 2 

different types. In each case, a signal peptide and an acidic pro-sequence were identified 

and the biosynthetic pathway was suggested to involve targeted proteolysis at a specific 

processing site, known as the Processing Quadruplet Motif (PQM). 

o spider venom 

o cytolytic peptide 

o antimicrobial peptide 

o precursor protein 

141


Mechanism of sphingomyelin specificity of actinoporins, pore-forming toxins from 

sea anemones 

Anderluh, G. 1* , Bakrač, B. 1 , Podlesek, Z. 1 , Maček, P. 1 , Separovic, F. 2 , Norton, R.S. 3 , Lakey, J.H. 4 

1 

University of Ljubljana, Department of Biology, Večna pot 111, 1000 Ljubljana, Slovenia, 

*gregor.anderluh@bf.uni-lj.si 

2 

School of Chemistry, University of Melbourne, 3010, Australia 

3 

Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville 3050, Australia 

4 

University of Newcastle upon Tyne, Framlington Place, Newcastle, England, UK 

Pathogens and protein toxins use lipid domains or ‘rafts’ to invade cells by binding to the 

lipid components sphingomyelin or cholesterol. Possible molecular mechanisms that 

enable association of proteins with such domains are becoming an important issue in cell 

biology and medicine. Surprisingly, there are very few examples of protein-sphingomyelin 

interactions defined at a molecular level. Here we provide some clues on how 

sphingomyelin is recognised by equinatoxin, the most studied member of the actinoporins, 

a conserved family of sea anemone pore-forming toxins. As shown by lipid dot-blots and 

surface plasmon resonance experiments, equinatoxin directly binds sphingomyelin but not 

phosphatidylcholine even though it presents the same choline headgroup. The toxin 

obviously recognizes the chemical structure of sphingomyelin below the lipid headgroup. 

The two closest residues that could fulfil this role are Trp112 and Tyr113. Just recently we 

were able to introduce 19 F label on tryptophans and confirm with NMR that Trp112 is 

involved in sphingomyelin recognition (1). Mutations of Trp112 to Phe or Leu, two amino 

acids that are found at equivalent positions in some homologues of equinatoxin, and 

mutation of Tyr113 to Phe, slightly decrease calcein release and binding to liposomes. 

Mutant W112A is almost inactive and does not bind sphingomyelin in a dot-blot assay and 

sphingomyelin analogues as measured by SPR. Mutation of Tyr113 to Ala causes complete 

loss of permeabilising activity and there is no binding to liposomes. 

Trp112 stabilizes the bound form of the toxin in the lipid-water interface and could interact 

with sphingomyelin in the region below the headgroup. On the other hand, Tyr113 is close 

enough to form a hydrogen bond with the amide nitrogen of sphingomyelin and could in 

this way discriminate between the two lipids. 

1. Anderluh, G., Razpotnik, A., Podlesek, Z., Maček, P., Separovic, F. and Norton, R.S. 

(2005) J Mol Biol 347, 27-39. 

o cytolysin 

o actinoporins 

o equinatoxin 

o sphingomyelin specificity 

142 


In vitro evaluation of antibacterial activity of crude aqueous extract of Azadirachta 

indica (Neem) leaves. 

Kihara, M. W 1 , Mbugua, P. M 1* , Kioy, P. G 1 , Makumi, J. N 2 , Ngeranwa, J. M. 2 

1 University of Nairobi, Nairobi, Kenya, *paulmmbugua@yahoo.com 

2 Kenyatta University, Nairobi, Kenya. 

Azadirachta indica (Neem tree) is widely grown in Kenya and from it numerous types of 

herbal products are currently being manufactured and sold to the public. Every part of the 

plant is used as raw material. Aqueous freeze dried leave extract was used to determine the 

spectrum of activity and potency by disk diffusion, minimum inhibition concentration 

(MIC), minimum bactericidal concentration (MBC) and time-kill methods. Out 108 

bacterial isolates, 60.2% (+-10.1) were sensitive at varying degree (7-11.5 mm diameter) 

by disc diffusion method at 5% confidence interval. Of 32 gram positive isolates tested, 

71.8% (+-9.3) were sensitive while 28.2% were resistant. Of 76 gram negative organism 

tested 55.2% (+-10.3) were sensitive at 5% confidence interval. MIC and MBC values by 

microtitre plate method for (Staphyloccocus aureas, Escherichia coli, Salmonella typhi, 

Pseudomonas eurogenosa and Streptoccocus pyogenes) selected organisms ranged from 

1.5-6.0mg/ml and 2-13mg/ml respectively. For comparison, MIC and MBC values 

obtained for chloramphenical on the same organisms ranged from 0.0625-1.2 mg/ml and 

0.106-4.8 mg/ml respectively. Both the extract and chloramphenical showed poor 

bactericidal properties by tube method. Both could not eliminate a population of 10 6 of 

organism on incubation for 24hrs even at concentration two times the MBC. They 

however considerably suppressed growth of the organisms. The neem leave aqueous 

extract exhibited broad spectrum antibacterial activity. The results explain its popularity in 

controlling bacterial infections traditionally but also indicate necessity to perform 

susceptibility tests for all isolates as same species showed different responses with 

different isolates. Neem extracts would be very useful in multiple infections. The 

organisms that responded best included,;Staphyloccocus, Escherichia, Sallmonella and 

Streptoccocus species. Shigella, Neisseria and Haemophilus species showed least 

response. A methicilline resistant strain of Staphyloccocus aureas was resistant while an 

Escherichia coli isolates which had proved resistant to a battery of antibacterial agents was 

suppressed. 

o Azadirachta indica 

o In vitro 

o Antibacterial 

o Leave 

143


Non-protein/non-peptide compounds from cobra venom having therapeutic 

potential 

Saha, A. 1* , Gomes, A. 

1 Lab. Of Toxinology & Experimental Pharmacodynamics, Department of Physiology, University of 

Calcutta, Kolkata, India, *archita_s1@rediffmail.com 

Introduction: The present communication reports the purification and characterization of 

three non-protein/non-peptide compounds from King Cobra (Ophiophagus hannah) venom 

(KCV) and Monocled Cobra (Naja kaouthia) venom (MCV) and evaluating their 

therapeutic potential (antiarrhythmic, antipyretic and anticonvulsant, antineoplastic). 

Methods: Swiss albino male mice, Charles Foster male rats, guinea pigs. Human myeloid 

leukemic cell lines (U937,K562). TLC, column chromatography, IEC, gel filtration, HPLC 

- for purification. Structural characterization by UV, IR, NMR, Mass. Biological studies. 

Results: KC-MMT1 – saturated aliphatic acid isolated from KCV, showed anti-arrhythmic 

property established through in vitro studies on guinea pig heart/ auricle preparations and 

in vivo ECG recordings and cardiac marker enzyme assay on aconitine induced arrhythmic 

rats and mice. KC-MMT2 – unsaturated aliphatic acid isolated from KCV, showed CNS 

activity (lowered body temperature of normothermic and hyperthermic mice and 

potentiated sleeping time) and anticonvulsant activity. NK-ANF1 – polyhydroxylated nonaromatic 

compound isolated from MCV had antineoplastic potential (cell growth inhibition 

and apoptogenic observed by fluorescence, confocal and scan electron microscopy, FACS) 

on cell lines. (Statistics – Mean ± SEM, Student’s t-test, p


Yeast culture Saccharomyces cerevisiae as feed additive in poultry 

S. A. Abd EL-Latif 

Department of Animal Production, Faculty of Agriculture, Minia University, Minia, Egypt 

E-mail:- shaker7112001@yahoo.com 

The chickens are stressed by various factors such as overcrowding, vaccination, 

chilling, and/or overheating. Under such circumstances feed additives like yeast culture 

have great significance for enhancing feed quality, productivity and health. Yeast culture 

consists of natural live yeast cells grown and fermented in the media like molasses and 

grain mashes in the form of spores at low temperature. It includes both media and yeast 

conditioned for immediate enzymatic action upon entering the digestive tract. It is neither 

an antibiotic nor chemotherapeutic agent and is distinctly different from baker's yeast. 

Yeast culture is added as a digestive aid to increase the nutrient availability and they will 

act by producing certain digestive enzymes. 

Addition of yeast culture Saccharomyces cerevisiae in drinking water resulted in 

the reduction of number of days required to reach the market weight, improving the 

livability and feed conversion in broilers. Dietary inclusion of Saccharomyces cerevisiae ( 

1 kg per ton ) of feed in layer resulted in improved egg production, feed conversion and 

egg shell quality. The broiler breeder diet with 0.5kg/ton of live yeast culture (Yeasacc) at 

20 weeks of age, and increased it to 1 kg/ton at 47 weeks of age resulting in no consistent 

effect on female reproductive characteristics, rate of lay, egg specific gravity and egg 

weight. 

This review article discuss the effect of yeast culture Saccharomyces cerevisiae as 

feed additives in poultry diets on production and some metabolic functions. 

o yeast culture 

o poultry 

o production 

o metabolic functions 

146 

Friday Track 1, K3.14 3.00-3.20pm 

Bioinformatics and multi-epitope DNA immunisation to design rational snake 

antivenom 

Wagstaff, S.C.*, Laing, G.D., Theakston, R.D.G., Papaspyridis, C., Harrison, R.A. 

Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, 

L3 5QA, UK *simonw@liv.ac.uk 

The rational design of toxin-specific snake bite immunotherapy is a realistic prospective if 

recombinant immunogens that generate toxin-specific antibody responses could be 

developed as alternatives to conventional, whole venom, immunisation protocols currently 

used to generate antivenoms. In this paper, we describe the use of bioinformatics and 

venom gland expressed sequence tag (vgEST) to design novel immunogens that elicit 

cross-generic and toxin-neutralising antibody responses. 

Using a bioinformatic approach that combines algorithms that identify surface exposed, 

antigenic and highly represented epitopes within EST databases, we identified seven 

epitopes conserved throughout the highly divergent snake venom metalloproteinases 

(SVMP) family from Echis ocellatus. 

To confirm our bioinformatic predictions, we raised antisera to each of these epitopes and 

confirmed their immunological conservation in the venoms of several disparate species 

distributed throughout African, Asia and America. Furthermore, we designed and 

constructed either a single DNA or recombinant protein immunogen, containing all seven 

epitopes as a string, which generated antibody responses to epitopes contained within a 

wide range of SVMPs that inhibited haemorrhage induced by the venoms of 

geographically distinct vipers, E. ocellatus and Cerastes cerastes cerastes. 

Extending the scope of this work by selecting epitopes from multiple toxin groups 

conserved throughout other medically important viper and elapid species, offers new 

opportunities to design antivenoms which are toxin-specific, have improved clinical 

efficacy and may be engineered to provide geographical polyspecificity to regions of high 

incidence of snake bite. 

o EST 

o Antivenom 

o Metalloproteinase 

o Bioinformatics 

147


Pre-clinical assessment of equine and ovine antivenoms raised against saw-scaled 

viper (Echis ocellatus) venom for use in Nigeria 

Laing, G.D. 1 *, Warrell, D.A. 2 and R.D.G. Theakston 1 . 

1 


L3 5QA, UK. *gavin.laing@liv.ac.uk 

2 

Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 9DU, UK 

The Echis genus is responsible for more snakebite deaths worldwide than any other 

venomous snake. The saw-scaled viper (Echis ocellatus) is the most abundant snake in 

savannah Nigeria and a major health concern. At some times of the year victims can 

occupy 74% of regional hospital beds. The severe shortage of antivenoms for Africa has 

prompted an international collaboration to produce antivenoms as a means of resolving the 

crisis in the short-term. In order to select candidate antivenoms for a planned clinical trial 

in late 2006, we have assessed the potency of several antivenoms derived from ovine or 

equine IgG and F(ab’)2 fragments using WHO-approved tests. 

Four antivenoms were developed using venoms extracted from wild-caught Nigerian 

(Kaltungo) specimens of E. ocellatus, Bitis arietans and Naja nigricollis maintained in the 

Liverpool School of Tropical Medicine. The antivenoms tested were an equine caprylic 

acid derived IgG (Instituto Clodomiro Picado, Costa Rica), an equine pepsin digested 

F(ab / )2 (Vacsera, Egypt) (both trispecific antivenoms raised against E. ocellatus, B. 

arietans and N. nigricollis venoms), an ovine caprylic acid derived IgG and an ovine 

pepsin digested F(ab / )2 product (both MicroPharm, UK) (both monospecific antivenoms 

raised against E. ocellatus venom). Tests for estimating the neutralising effects of these 

antivenoms against the lethal effects of E. ocellatus venom were carried out. 

All four antivenoms showed neutralising potency against the immunising venoms and 

results were expressed as the number of LD50 neutralising units/ampoule of antivenom. 

Comparisons with established antivenoms are made and the selection of candidate 

antivenoms for the forthcoming clinical trial is discussed. 

The results demonstrate that the commitment of international antivenom manufacturers 

to address the shortfall of antivenom supply for Africa has resulted in the production of 

potent and efficacious therapies. 

o Echis 

o antivenom 

o potency 

o effective dose 

148 


What price, salvation? Antivenom costs and availability in Papua New Guinea 

1992-2004 

Williams, D. J. 1* 

1 Australian Venom Research Unit, University of Melbourne, Parkville, Vic, 3010. Australia 

* d.williams4@pgrad.unimelb.edu.au 

Introduction: Papua New Guinea has some of the highest incidence rates for snakebite in 

the world, yet for almost two decades antivenoms have become increasingly expensive, 

and increasingly scarce. A study was conducted to examine antivenom costs and 

availability and to investigate supply and distribution anomalies. 

Methods: Analysis of purchasing, inventory and distribution records. 

Results: The PNG government purchased 5,571 vials of antivenom between May 1992 and 

April 2005 at a total cost of more than K11.1 million, yet in yearly terms the availability of 

antivenom has decreased by 40.2% since 1986. The cost of a single patient treatment with 

antivenom has increased by 884% since 1987, and the average treatment cost is now 

K3,060. Rising costs have been largely driven by international economics, but reliance on 

expensive polyvalent antivenom which increased from 49.2% (1992-1995) to 82.0% 

(1997-2000) added K511 to the costs of single patient treatment. This increased average 

annual expenditure by K266,742; sufficient to have funded the purchase of an additional 

150 vials of taipan antivenom and 50 additional vials of death adder antivenom. In 2003 

while total national pharmaceutical and medical supplies purchases totalled K14.7 million; 

more than K3.4 million (24.7%) was spent on antivenoms. Despite large spending 

antivenom supplies were poorly managed and badly distributed. From January 1998 to 

December 2004 2,040 vials of antivenom were recorded as being distributed from Area 

Medical Store (POM), but the dispositions of another 416 vials of various antivenoms 

worth over K1.2 million were not recorded. Only 47 health facilities throughout PNG 

received antivenom in this period; Port Moresby General Hospital received 717 (35.1%) 

vials of which 616 (85.9%) were CSL polyvalent. 

Conclusions: The purchasing, warehousing and distribution of antivenom requires urgent 

review with an emphasis on inventory security and purchase/delivery cross-matching. An 

alternative system linked to snakebite research and surveillance efforts has now been 

proposed. 

o antivenoms 

o costs 

o accountability 

o availability 

149


Developing a toxinology toolkit for an under-developed world 

Williams D.J. 1* , Jensen S.D. 1,2 , Matainaho T. 2 , Wüster W. 3 , Nimorakiotakis B. 1 , Winkel K.D. 1 

1 Australian Venom Research Unit, University of Melbourne, Parkville, Vic, 3010. Australia 

2 School of Medicine & Health Sciences, University of Papua New Guinea, Boroko, Papua New Guinea 

3 School of Biological Sciences, University of Wales, Bangor, Wales, UK 


Introduction: The global burden of snakebite is greatest in the world’s poorest tropical 

countries. We have been working in this arena to develop a new approach to snakebite 

management and associated resource use aimed at improving both local systems and 

infrastructure and the individual prognoses of envenomed patients. 

Methods: As part of a major study of snakebite in Papua New Guinea we are combining 

the use of zoogeographical, ecological, epidemiological, clinical, economic, and GIS 

studies with public and medical education to both resolve unanswered questions, and to 

develop local capacity for managing snakebite and improving clinical outcomes. Specific 

approaches are being developed for (1) epidemiological surveillance and reporting, (2) 

mapping and GIS analysis, (3) efficient pursuit of clinical investigations, (4) resolution of 

taxonomic questions, (5) improved resource management and distribution, (6) medical and 

community education, and (7) improving clinical outcomes. 

Results: In Papua New Guinea our approach has lead to the establishment of a new plan 

for addressing the snakebite burden. A new National Snakebite & Antivenom Unit will use 

epidemiological, GIS and zoogeographic data in combination with mandatory reporting of 

snakebite cases by health service providers, locally initiated clinical studies, and the phased 

introduction of EIA-based venom immunotype identification to better manage expensive 

antivenom supplies; ensuring needs-based distribution, stock accountability and cost 

minimization. Coordinated programmes of public education and nationwide training of 

health workers and medical professionals have also been implemented. 

Discussion: Although clinical and epidemiological studies add significantly to our overall 

knowledge, they rarely produce lasting practical benefits for the countries in which they 

take place. Our approach combines this essential quest for knowledge with the creation of 

a functional legacy: improved local skills, resources and systems which can sustainably 

reduce morbidity, mortality and financial costs. We believe that offering this approach to 

others in the so-called ‘under-developed’ world may enable them to reap similar rewards. 

o snakebite 

o public health 

o resource management 

o clinical outcomes 

150 


Teaching clinical toxinology in the developing world: Setting an example in Papua 

New Guinea 

Williams D.J, 1* Jensen S.D, 1,2 Nimorakiotakis B, 1 Winkel K.D. 1 

1 

Australian Venom Research Unit, University of Melbourne, Parkville, Vic, 3010. Australia 

2 

School of Medicine & Health Sciences, University of Papua New Guinea, Boroko, Papua New Guinea 


Envenomation is an important cause of morbidity and mortality in many areas of the 

developing world, and the prognosis is often influenced by the knowledge and skills of 

local health workers. In Papua New Guinea the incidence of snakebite is one of the highest 

in the world, and in the face of spiralling antivenom costs and reduced availability, health 

workers and doctors are often faced with difficult clinical decisions. We have developed 

and successfully implemented a stand-alone training programme for Papua New Guinea’s 

medical and allied health community that encompasses a broad curriculum in applied 

clinical toxinology. 

This training course, the first of its kind to ever be offered in a developing tropical country 

anywhere in the world, provides instruction in a broad range of subjects including first aid 

management of patient’s in remote areas; patient assessment and diagnosis; specific 

evidence-based treatment protocols; syndromic management of neurotoxicity, 

coagulopathy and other venom-induced conditions; antivenom use and administration; 

recognition and treatment of adverse antivenom reactions; advanced airway protection and 

respiratory support; and non-antivenom treatment strategies for remote rural health centres. 

An inaugural course, taught in September 2004 was attended by 62 participants from 8 

provinces; a second course conducted in September 2005 had an intake of 105 participants. 

To meet demand the course will be run twice in 2006. A comprehensive handbook was 

written to accompany the course in 2005, and corporate funding has enabled this book to 

be produced for free distribution to health workers throughout Papua New Guinea. The 

course offers financial assistance to participants from under-resourced health centres who 

can demonstrate need. The success of the course will hopefully be measured in future years 

not just by improvements in the clinical outcomes of PNG’s snakebite victims, but also 

though application of this model to address clinical toxinology training needs elsewhere in 

the developing world. 

o Clinical toxinology 

o Training 

o Education 

o Envenomation 

151


Analgesic action of Crotalphine, a novel opioid receptor agonist from the venom of 

the South American rattlesnake Crotalus durissus terrificus (CdtV) 

Cury, Y. 1* , Konno, K. 2 , Picolo, G. 1 , Brigatte, P. 1 , Gutierrez, V. 1 , Zambelli, V. 1 

Laboratory of 1 Pathophysiology and 2 Center of Applied Toxinology (CAT), Butantan Institute, Av. Vital 

Brazil, 1500. 05503-900, Sao Paulo, Brazil, * yarac@attglobal.net 

Introduction: CdtV induces antinociception mediated by opioid receptors and activation 

of peripheral L-arginine-NO-cGMP pathway and opening of ATP-sensitive K+ channels. 

Recently, a synthetic 14 aminoacids peptide, named Crotalphine, was obtained based on 

the sequence of the natural analgesic factor isolated from crude venom. The aim of the 

present work was to evaluate the analgesic effect of Crotalphine in acute (prostaglandin E2induced 

hyperalgesia) and chronic (neuropathic or cancer) pain models and to determine 

the mechanisms involved in this effect. 

Methods: The analgesic effect of CNF was evaluated in male Wistar rats (160-180 g) 

Acute hyperalgesia was determined 3 hours after PGE2 injection (100 ng/paw). Cancer 

pain was determined 5 days after intraplantar injection of Walker 256 carcinoma cells 

(1x10 6 ). Neuropathic pain was evaluated 14 days after chronic constriction of sciatic nerve. 

For characterization of chronic pain, hyperalgesia, allodynia and spontaneous pain were 

measured. The rat paw pressure test and von Frey test were used for evaluation of 

hyperalgesia and allodynia, respectively. Spontaneous pain was evaluated by the 

determination of the duration of lifting and licking of paws. 

Results: Crotalphine (1 to 6 µg/kg) administered by oral route, induced antinociception in 

all models evaluated. This effect was a long lasting effect, since it persisted for 2 (chronic 

pain) to 5 (acute pain) days after Crotalphine administration. Antinociception was not 

modified by CTOP (20 μg/paw). On the other hand, nor-BNI (50 μg/paw) blocked the 

analgesic effect observed on PGE2-induced hyperalgesia, whereas ICI 174,864 (10 μ 

g/paw) and nor-BNI inhibited the effect of Crotalphine on neuropathic and cancer pain. 

Conclusion: These data suggest that Crotalphine induces a long-lasting analgesic effect on 

acute and chronic pain mediated by activation of peripheral κ and δ opioid receptors. 

Financial support: CAT/CEPID/FAPESP, COINFAR Pesquisa e Desenvolvimento 

o Crotalphine 

o Crotalus durissus terrificus venom 

o analgesia 

o peripheral opioid receptors 

152 


Analgesic effect induced by Naja kaouthia snake venom and α-cobratoxin isolated 

from this venom 

1Marucia Chacur, 1 Vanessa Zambelli, 1 Vanessa Gutierrez, 2 Peter Mirtschin, 1 Yara Cury, Frank Madaras 3 , 

Paul Masci 4 , and Simone Flight 4 

1 Laboratory of Pathophysiology, Butantan Institute, Av. Vital Brazil, 1500, 05503-900, SP, Brazil, 2 Venom 

Supplies Pty Ltd, Tanunda South Australia 5352, Australia, 3 Venom Science Pty Ltd, Tanunda South 

Australia 5352 and 4 Southern Clinical School, University of Queensland, Brisbane, Queensland, Australia 

4102 

Naja kaouthia is responsible for most cases of snakebite mortality and morbidity in 

Thailand and is a major cause of envenoming in a number of other Asian countries. 

Important toxic components of N. kaouthia are the postsynaptic neurotoxins. These toxins 

block nerve transmission by binding specifically to the nicotinic acetylcholine receptor. 

Despite neurotoxicity, some reports exist on the analgesic activity displayed by venoms 

from the Asiatic Naja genus. There is no specific data in the literature however, about the 

possible analgesic effect of the venom from Naja kaouthia. The aim of the present study is 

to investigate, in male Wistar rats, the possible antinociceptive effect induced by Naja 

kaouthia (Nk) venom and by the postsynaptic neurotoxin, α-cobratoxin (α-cobra) isolated 

from this venom and to characterize the peripheral mechanisms involved in this activity. 

Methods: The antinociceptive action of Nk venom and α-cobra was evaluated in the 

prostaglandin E2 (PGE2)-induced hyperalgesia. The rat paw pressure test was used to 

evaluate pain threshold. In this test, a force (in g) was applied the hind paw, before and 3 h 

after the intraplantar (i.pl.) injection of PGE2. The force (in g) needed to induce the 

withdrawal of the paw represents the pain threshold. 

Results: The i.pl. injection of PGE2 (100 ng/paw) reduced the pain threshold of the rats, 

causing mechanical hyperalgesia. Nk (40 µg) or α-cobra (20 µg), administered p.o. 

immediately before PGE2, significantly inhibited hyperalgesia. Naloxone (1μg/paw), a 

nonspecific opioid receptor antagonist, injected i.pl. simultaneously with PGE2, abolished 

the antinociceptive effect on the venom and α-cobratoxin. Nor-BNI (50 µg/paw), an 

antagonist of kappa opioid receptors, partially antagonized the antinociceptive effect of αcobra, 

whereas ICI 174,864 (10 µg/paw) an antagonist of delta opioid receptors abolished 

this effect. On the other hand, CTOP (20 µg/paw) an antagonist of mu opioid receptors did 

not modify the α-cobra antinociceptive effect. 

Conclusions: These data suggest that Naja kaouthia venom and α-cobratoxin are able to 

induce antinociceptive effect mediated by peripheral opioid receptors. Kappa and delta, but 

not mu opioid receptors, are involved in antinociception induced by α-cobratoxin. 

153


BcIV, a new paralyzing peptide from the venom of the sea anemone Bunodosoma 

caissarum. A comparison with the Na + channel toxin BcIII 

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, 

A.D. 5 , Beirão,P.S.L. 5 , Freitas, J.C. 1,2 

1 Universidade de São Paulo, Depto. de Fisiologia, IBUSP, São Paulo,BRAZIL- 

*a.j.zaharenko@ig.com.br 2 Center for Applied Toxinology, CAT-CEPID, Instituto Butantan, São Paulo, 

BRAZIL 3 Centro Interdisciplinar de Investigação Bioquímica, Universidade de Mogi das Cruzes, Mogi 

das Cruzes, BRAZIL. 4 Fundação Nacional Ezequiel Dias- FUNED, Belo Horizonte, BRAZIL. 5 

Universidade Federal de Minas Gerais, Depto. de Bioquímica e Imunologia, Belo Horizonte, BRAZIL 

Sea anemones produce a wide variety of biologically active compounds, such as the 

proteinaceous neurotoxins and cytolysins. Herein we report a new peptide, purified to 

homogeneity from the neurotoxic fraction of B. caissarum venom, by using gel filtration 

followed by rp-HPLC, naming it as BcIV. BcIV is a 41 amino acid peptide (molecular 

mass of 4669 Da) possessing 6 cysteines covalently linked by three disulfide bonds. This 

toxin has 45 and 48% of identity when compared to APETx1 and APETx2 from 

Anthopleura elegantissima, respectively, and 42% of identity with AmII and BDS-I and -II 

obtained from Antheopsis maculata and Anemonia sulcata, respectively. This neurotoxin 

presents only a weak paralysing action (minimal Lethal Dose close to 2000μg/kg) in 

swimming crabs Callinectes danae. This appears to be a different effect to that caused by 

the type 1 sea anemone toxin BcIII that is lethal to the same animals at lower doses (LD50 = 

219 μg/kg). Circular dichroism spectra of BcIII and BcIV show a high content of β-strand 

secondary structure in both peptides, very similar to type 1 sodium channel toxins from 

various sea anemones, and to APETx1 and APETx2 from A. elegantissima, a HERG 

channel modulator and an ASIC3 inhibitor, respectively. Interestingly, BcIII and BcIV 

have similar effects on the action potential of the crab leg nerves, suggesting the same 

target in this tissue. As BcIII was previously reported as a Na + channel effector and BcIV is 

inactive over Na + currents of mammalian GH3 cells, we propose a species-specific action 

for this new molecule. A molecular model of BcIV was constructed using the structure of 

the APETx1 as template and putative key residues are discussed. 

o Bunodosoma caissarum 

o MS/MS Spectrometry 

o Sea Anemone 

o Molecular Modelling 

154 


Beyond prey capture – a comparative bioinformatic approach to cnidarian 

allomones 

Sher, D.*, Knebel, A., Bsor, T., Nesher, N., Tal, T., Morgenstern, D., Cohen, E., Fishman, Y. and Zlotkin, 

E. 

Department of Cell and Animal Biology, Silberman Institute of Life Sciences, Hebrew University, 

Jerusalem 91904, Israel. * dsher@pob.huji.ac.il 

Cnidarians such as hydrae and sea anemones are sessile, predatory, soft bodied animals 

which depend on a complex array of offensive and defensive allomones for prey capture 

and survival. These allomones are distributed throughout the entire organism both in 

specialized stinging cells (nematocytes) and in the body tissues (1). Cnidarians are entering 

the genomic era with the sequencing of the genome of the sea anemone Nematostella 

vectensis and a large EST database from Hydra magnipapillata, allowing for the first time 

to study cnidarians and their allomonal systems as a whole. This should facilitate the 

discovery of novel toxins, trace the evolution of cnidarian allomones, and help understand 

how the various allomones interact to paralyze the prey or fulfil other biological roles. 

To gain a global view of toxins in Hydra and Nematostella, bioinformatic methods were 

used to search the genome and EST databases for orthologs of toxins from cnidarians and 

other organisms, and to determine the likelihood that detected sequences have the expected 

biological effect (2). Several toxin types, including venom phopholipase A2s (PLA2), 

were detected in both the hydra and the anemone. However, marked differences exist 

between the two organisms in the toxin genes detected. For example, the “classic” short 

chain neurotoxins, which are found in many anemones including Nematostella and are 

expected to be a major cause of prey paralysis, are absent in Hydra. Many other putative 

toxins were detected in one or both of these organisms, including several not previously 

detected in cnidarians. These include pore-forming toxins, elapid-like PLA2s, CRISP 

proteins, Prokineticin-like polypeptides and toxic Deoxyribonucleases. 

Our results illustrate the usefulness of a genomic approach to study toxins, revealing a 

high level of complexity in cnidarian allomonal systems. We suggest the ancestral 

cnidarians utilized neurotoxic PLA2s to paralyze prey, similar to modern day hydrae, 

while Anthozoans have additionally evolved short-chain neurotoxins. Finally, we raise the 

possibility that similar proteins may fulfil endogenous and allomonal roles in cnidaria. 

1) Zhang, M., et al. (2003) Biochemistry 42, 8939-44 2)Sher, D., et al. (2005) Toxicon 45, 865-79 

o Cnidaria 

o Genome 

o phospholipase 

o neurotoxin 

155


When Positive Selection of Neurotoxin Genes is Missing - the Riddle of the Sea 

Anemone Nematostella vectensis 

Moran Y*, Gurevitz M 

Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, 

Israel, *moranyeh@post.tau.ac.il 

Rapid evolution driven by positive Darwinian Selection appears in toxins of vipers, 

scorpions, and marine snails. Although the vast phylogenetic distances between these 

animals suggest that this phenomenon is common, the recent release of the genome of 

Nematostella vectensis (Starlet anemone) as a collection of contigs portrays another 

extreme. Besides ShKT domains (resemble potassium channel blockers) embedded in 

various genes, only one gene family encoding for Na-channel neurotoxins has been found, 

and the putative mature product of ten family members is identical. Whereas the existence 

of a single toxin encoded by multiple genes may be explained by the unique ecology of N. 

vectensis, the complete absence of substitutions including synonymous ones is surprising 

and suggests either that these genes have been duplicated recently, or that their total 

conservation was advantageous. A retro-element identified downstream to one of the genes 

offers a possible mechanism of enhanced toxin gene duplication. This assumption still 

awaits further verification as soon as the various contigs will be assigned within larger 

genomic fragments. 

References 

1. Sullivan JC, Ryan JF, Watson JA, Webb J, Mullikin JC, Rokhsar D & Finnerty JR 

(2006) Nucleic Acids Res 34, D495-499 

o Sea anemone toxins 

o Genomics 

o Positive selection 

o Retro elements 

156 


Venom landscapes: Towards the discovery of novel pharmacological tools via the 

combined use of LC-MALDI-TOF and MALDI-TOF/TOF with cDNA analysis 

Escoubas, P. 1* , Sollod, B. 2 , Nicholson, G.M. 3 , Wilson, D.T.R. 4 , Vinh, J. 5 , King, G.F 2 , 

1 

Institut de Pharmacologie Moléculaire et Cellulaire – CNRS, 660 Route des Lucioles, 06560 Valbonne, 

France, *escoubas@ipmc.cnrs.fr 

2 



3 

Department of Medical and Molecular Biosciences, University of Technology, Sydney, Broadway NSW 


4 

Xenome Inc., 50 Meiers Rd, Indooroopilly, Brisbane, QLD 4068, Australia 

5 

ESPCI Laboratoire de biologie CNRS UMR7637 10 rue Vauquelin 75005 Paris France 

The extent and diversity of peptide toxins that are expressed in the venom glands of cone 

snails, scorpions, and spiders results in a diverse array of leads for drug and insecticide 

discovery. But this diversity also represents a daunting analytical challenge. With the goal 

of streamlining the discovery process we have explored the complexity of Australian 

funnel-web spider venoms via the combined use of MALDI-TOF and MALDI-TOF/TOF 

MS coupled with chromatographic separation (LC-MALDI) and the analysis of venomgland 

cDNA libraries. Venom collected from male and female spiders of several species of 

Australian funnel-web spiders (Atrax and Hadronyche spp.) was fractionated by reversedphase 

HPLC. Fractions were analyzed by MALDI-TOF MS and data plotted as intensity 

(z) vs. m/z (x) vs. fraction number (y) in 3D contour graphs termed “venom landscapes”. 

The results show that these spider venoms are far more complex than previously realized 

and contain many hundreds of peptides that follow a bimodal distribution, with about 75% 

of the peptides having a mass of 3,000–5,000 Da. Analysis of the venom peptidome of the 

spider H. versuta revealed 1018 peptides with mass less than 9 kDa. 

Toxin families were identified by matching the experimentally observed masses with those 

predicted from peptide sequences derived from analysis of venom-gland cDNA libraries. 

The combination of measured parameters with prediction of molecular weights, HPLC 

retention times and data from the literature permits the interpretation of the LC-MALDI 

analysis. Groups of peptides of selected pharmacology (eg. Cav, Kv or Nav channel 

blockers) can be distinguished as discrete “peaks” with a specific signature on the 3D plot. 

Analysis of 11 venoms and ca. 400 fractions demonstrates common features of the funnelweb 

spider venoms, leading to the possibility of generating entire combinatorial 

pharmacological libraries for each toxin class. Additional validation of the method was 

achived using MALDI-TOF/TOF analysis of A. robustus female venom and permitted 

determination of the number of disulfide bridges. We propose that this approach may have 

predictive value for the discovery of novel pharmacological tools in complex venoms. 

o Spider toxins 

o Mass spectrometry analysis 

o cDNA analysis 

o MALDI-TOF 

157


Snake venomics 

Calvete, J.J. 1,* , Juárez, P. 1 , Sanz, L. 1 

1 

Instituto de Biomedicina de Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain. 

*jcalvete@ibv.csic.es 

Venoms of Viperidae and Crotalidae snakes (vipers and rattlesnakes) contain a number of 

different proteins that interfere with the coagulation cascade, the normal haemostatic 

system and tissue repair. Consequently, the envenomenations by these snakes generally 

result in persistent bleeding. Snake bite is still a serious threat in both developed and 

underdeveloped countries. The world-wide mortality caused by snake envenomation is 

estimated at 50.000 deaths annually. The only effective treatment for systemic 

envenomation is the intravenous administration of an antivenom. However, polyclonal 

antisera include numerous antibodies with specificities not confined to the toxic target 

molecules. Hence, knowledge of the toxin composition of venoms could be devised to 

design immunization protocols with toxin-specific antibodies with greater specificity and 

effectiveness that conventional systems. Despite its potential value, little is known about 

the venom protein composition of most vipers. The protein composition of the crude 

venoms of a number of genera from Viperinae (Macrovipera, Cerastes, Echis, Bitis) and 

Crotalinae (Sistrurus, Crotalus, Agkistrodon) were analyzed by RP-HPLC, N-terminal 

sequence analysis, MALDI-TOF MS, and in-gel tryptic digestion followed by peptide 

mass fingerprinting and CID-MS/MS of selected peptide ions in a quadrupole-linear ion 

trap instrument. As expected from the rapid amino acid sequence divergence of venom 

proteins by accelerated evolution, with a few exceptions, the product ion spectra did not 

match to any known protein using the MASCOT search program. The CID-MS/MS spectra 

were therefore manually interpreted and the deduced peptide ion sequences submitted to 

BLAST sequence similarity searches. This approach allowed us to assign unambiguously 

all of the isolated venom fractions to protein families present in the non-redundant 

databases. Our proteomic approach complement transcriptomic studies by showing the 

relative abundance of the proteins that are actually secreted into the venoms. Our results 

show that the venom proteomes are composed of proteins belonging to only a few protein 

families. However, each venom showed distinct degree of protein composition complexity. 

o Venom proteome 


o N-terminal sequencing 

o Disulfide bonds 

158 


Identification and characterisation of toxin-specific transcripts from the venom 

glands of Australian elapid snakes. 

St Pierre, L 1,2* , Masci, P.P 2 , Earl, S.T 1,2 , Flight, S 2 , de Jersey, J 3 and Lavin, M.F. 1,2 

1 

Queensland Institute of Medical Research. PO Box Royal Brisbane Hospital, Brisbane, QLD, 4029, 

Australia. *liamS@qimr.edu.au 

2 

Faculty of Health Sciences, The University of Queensland, Brisbane, QLD, 4067, Australia. 

3 

Faculty of Biological and Chemical Sciences, The University of Queensland, Brisbane, QLD, 4067, 

Australia. 

The venom of Australian snakes contain a complex mixture of polypeptides that are 

typically small in size, disulfide rich, and highly stable, potent and specific in their 

mechanism of action, and hence are the ideal targets for the identification of novel 

therapeutic candidates. Australian elapid venoms are amongst the most toxic in the world 

with numerous significant clinical outcomes, however they remain largely understudied at 

the molecular level. To gauge an understanding of the total complement of toxins within 

the venom (increasingly referred to as the “Venome”) of Australian snakes, we employed a 

transcriptomic analysis of the venom gland, complemented by recombinant protein 

expression to further characterise specific toxin function. 

A comparative analysis of multiple toxin families was performed from ten medically 

significant Australian snakes: the inland and coastal taipan, common brown, red-bellied 

black, mulga, rough-scaled, Stephen’s banded, small-eyed, black whip and tiger snakes. 

Toxin transcripts were initially identified from a coastal taipan venom gland cDNA 

microarray with subsequent isolation and comparative analysis of isoforms from all ten 

snakes. These included the sequences of toxins such as factor X- and factor V-like 

prothrombin activators, phospholipase A2 isoforms, long and short chain neurotoxins, 

cysteine rich secretory proteins, venom natriuretic peptides, L-amino acid oxidases, along 

with number of proteins involved in regular cellular maintenance. The activity of unique 

toxin sequences were then characterised by recombinant protein expression. Examples 

include natriuretic peptides, which were shown to demonstrate a dose dependent inhibition 

of ACE, a factor X-like protein from the coastal taipan capable of cleaving prothrombinlike 

substrates and novel neurotoxins which also demonstrated potent inhibitory effects 

upon neuronal receptors. This study represents the most comprehensive survey to date of 

the genes responsible for the envenomation process in Australian snakes and sheds much 

light on the evolutionary and functional relationships between these species. 

o Australian elapid snake 

o Venom gland 

o Transcriptomics 

o Recombinant protein expression 

159


Proteomic analysis of Australian snake venoms for the discovery and development 

of new human therapeutics # 

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 and 

Lavin, M.F 1,2 . 

1 

Queensland Institute of Medical Research PO Box Royal Brisbane Hospital, Brisbane 4029, Australia , 

*Stephen.Earl@qimr.edu.au 

2 

The School of Medicine, Central Clinical Division, The University of Queensland, Brisbane, Australia 

3 

The Institute for Molecular Biosciences, The University of Queensland, Brisbane, Australia 

4 

The School of Medicine, Southern Clinical Division, Princess Alexandra Hospital, The University of 

Queensland, Brisbane, Australia 

5 

The School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Australia 

Australian elapid species possess the most toxic snake venoms in the world. However, in 

comparison to Asian and American snake species, much less is known about overall 

venom composition. Australian snake venoms are lethal protein cocktails that interfere 

with several mammalian physiological processes, including the blood coagulation and 

nervous systems. In many cases, these venom proteins have a stable disulfide-rich 

structure, are very specific in their site of action and act with high efficacy. For these 

reasons, we hypothesise that Australian elapid venoms are a potential source of new 

human therapeutics. The aim of this study was to systematically examine the venoms of 20 

Australian elapid snakes using proteomic methods such as 2-dimensional gel 

electrophoresis (2-DE) with mass spectrometry (MS) to identify all venom components 

and select novel venom proteins with potential for development as new human 

therapeutics. When separated by 2-DE, each venom showed approximately 100-200 

discrete protein spots, varying in molecular weight from 7 to over 100 kDa with pIs from 3 

to 10. Using MS, the majority of venom protein spots have been identified. These include 

previously characterised venom proteins such as phospholipase A2 enzymes, neurotoxins 

and prothrombin-activating proteins, along with proteins not previously reported to be 

components of Australian elapid venoms such as acetylcholinesterase and nerve growth 

factor. A number of novel venom proteins have also been identified and selected as 

potential therapeutic candidates. In addition, we have used several enzymes, protein stains 

and specific antisera to examine different post-translational modifications amongst 

Australian elapid venom proteins. This work represents the most comprehensive analysis 

and identification of Australian elapid venom proteins yet undertaken. 

# 

This project was supported by our commercial partner QRxPharma and the Australian 

Research Council. 

o Snake venom 

o Australian elapid 

o proteomics 

o drug discovery 

160 


Molecular characterization of hyaluronidase-encoding genes from venom gland 

cDNA libraries of Echis, Bitis and Cerastes viper species 

Harrison, R.A,* Ibison, F., Wilbraham, D. Wagstaff, S.C. 


UK *R.Harrison@liverpool.ac.uk 

The killing of prey species by snakes is most efficiently achieved by the rapid 

dissemination of tissue-embedded venom. Hyaluronidase, the “venom spreading factor” 

has long been associated with tissue distribution of venom and several reports have 

demonstrated that immunological or biochemical inhibition of hyaluronidase reduces 

venom-induced pathology. Venom hyaluronidases have been isolated in native form but 

their extensive characterisation has been restricted by the lack of any protein or gene 

sequence data – the objective of this study. 

We randomly sequenced 1000 Echis ocellatus (Nigeria) venom gland cDNAs (ESTs) 

and this transcriptome was annotated (using annot8r_blast2GO) with gene ontology (GO)terms 

that functionally classifies EST sequences into proteins with, for example, sugar 

binding or growth factor activities. Examination of each EST in the “protein, lipid and 

carbohydrate metabolism” categories revealed a single sequence matching the GOannotation 

of hyalurononglucosaminadase-activity (GO:000415). Because this singleton 

cluster (Eo00242) was clearly a partial transcript lacking 5’ sequence we designed PCR 

primers that amplified the full-length sequence with extensive 5’ and 3’ UTR sequence. 

Since the latter sequences are typically more conserved across species than protein-coding 

sequences, we utilised a series of primers complimentary to the 5’ and 3’ UTR of the E. 

ocellatus sequence to amplify analogous cDNAs from venom gland cDNA libraries of E. 

pyramidum leakeyi (Kenya), Cerastes cerastes cerastes (Egypt) and Bitis arietans 

(Nigeria). 

We will describe the extraordinarily high degree of sequence similarity between the E. 

ocellatus, E.p. leakeyi, C.c. cerastes and B. arietans hyaluronidase genes and illustrate 

that, although they are very different from the bee venom and four mammalian classes of 

hyaluronidase, they retain several specific residues and the overall architecture that 

characterise this class of carbohdrate metabolising hydrolases. 

o Hyaluronidase 

o EST database 

o E. ocellatus 

o B. arietans 

161


Miniminization of protein structure and gene organization along the evolution of 

the short disintegrin ocellatusin from a dimeric disintegrin precursor 

Juárez, P. 1* , Wagstaff, S.C. 2 , Sanz, L. 1 , Harrison, R.A. 2 , Calvete, J.J. 1 

1 


*pjuarez@ibv.csic.es 

2 

Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool 

L3 5QA, England, UK 

Venom represents a key innovation in ophidian evolution that allowed advanced snakes to 

transition from a mechanical (constriction) to a chemical (venom) means of subduing and 

digesting prey larger than themselves. Venom toxins likely evolved from endogenous 

proteins with normal physiological functions that were recruited into the venom proteome 

before the radiation of the advanced snakes at the base of the Colubroid radiation. Venoms 

of Viperidae and Crotalidae snakes contain proteins that interfere with the coagulation 

cascade, the normal haemostatic system and tissue repair. Among them, disintegrins 

represent a family of integrin receptor antagonists that are released in viper venoms by 

proteolytic processing of PII snake venom metalloproteinase (SVMP) precursors or 

synthesized from short-coding mRNAs. The disintegrin family has been divided into five 

groups according to their polypeptide length and number of disulfide bonds, and the 

current view is that the structural diversity of disintegrins has been achieved through the 

selective loss of disulfide bonds. To understand the genomic basis of the accelerated 

evolution of disintegrins, and the molecular mechanism underlying their structural 

diversification, we have undertaken the analysis of cDNAs encoding disintegrins from a 

venom gland library of Echis ocellatus (Eo) and the genomic organization of the Eo 

disintegrin genes. The cDNA sequences coding for (RGD/KGD/WGD/MLD-containing) 

dimeric disintegrins lack the metalloproteinase domain and belong thus to the short-coding 

class. Analysis of two distinct messengers coding for the short disintegrin ocellatusin, 

obtained by PCR (Eo 10c-10) and from ESTs (Eo-00006), strongly argues for a common 

ancestry of short- and dimeric distintegrin subunits. Two nucleotide mutations (Cys->Tyr 

and Ser->Cys) are hypothesized to represent key events in the dimeric->short disintegrin 

transition. On the other hand, comparison of the exon-intron organization of genes for 

medium-sized, dimeric, and short disintegrins revealed that the evolutionary pathway 

leading to the diversification of disintegrins (medium->dimeric->short) also involved the 

miniminization of the gene organization by the sucessive removal of introns. 

o Echis ocellatus venom 

o Disintegrin evolution 

o Ocellatusin cDNA 

162 


The same small three-fingered fold for snake toxins and human uPAR. 

Llinas, P. 1 , Le Du, M.H. 1 , Gardsvoll, H. 2 , Dano, K. 2 , Ploug,M. 2 , Gilquin, B. 1 , Stura, 

E.A. 1 , Ménez, A. 1* . 

1 Département d’Ingénierie et d’Etudes des Protéines, Bât 142, CEA, Saclay, 91191, 

Gif-sur-Yvette, France. *andre.menez@cea.fr 

2 Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark. 

Human urokinase-type plasminogen activator receptor (uPAR; CD87) is a glycolipidanchored 

modular protein made of a single chain polypeptide (283 amino acids) 

organized into three extracellular domains. Each domain comprises about 90 residues 

with four to five disulphide bonds. The primary function of uPAR is to bind uPA with 

high affinity and hence to focalize the cellular conversion of plasminogen to plasmin. 

uPAR is involved in the pathology of human cancers. To understand the structural 

properties of human uPAR, we have solved the crystal structure of a soluble form of 

the receptor at 2.7 Å, in complex with a peptide that has a high affinity for uPAR and 

inhibits its capacity to bind uPA. uPAR is composed of three consecutive domains that 

are organized in an almost circular manner, and which generate both a large external 

surface and a deep internal cavity where the peptide binds in a helical conformation. 

Each domain adopts a typical three-fingered structure, the first two superimposing well 

with the structure of bucandin and the third one with that of CD59. Therefore, the 

building blocks of uPAR are folded like the multifunctional three-fingered snake 

toxins. This work further demonstrates that the three-fingered fold is a highly 

divergent protein structure. 

Llinas, P., Le Du, M-H., Gårdsvoll, H. Danø, K., Ploug, M., Gilquin, B., Stura E. A. 

and Ménez, A. 2005, EMBO J., 24, 1655-1663 

ubiquitous toxin folds 

163


3D-structure of VAP1, a high-molecular snake venom metalloprotease, reveals 

metalloproteinase/disintegrin/cysteine-rich architecture of SVMPs and ADAMs 

Takeda, S 1, 2 , Igarashi, T 1 , Mori, H 1 , Araki, S 3 

1 National Cardiovascular Center Research Institute, Fujishiro-dai, Suita, Japan 

2 Riken Harima Institute at SPring-8,1-1-1 Kouto, Mikazuki, Sayo, Japan 

3 Nagoya University, MBL, Toba, Japan, *saraki@bio.nagoya-u.ac.jp 

Introduction: High-molecular snake venom metalloproteases (SVMPs) and ADAMs 

possess metalloproteinase/disintegrin/cysteine-rich (MDC) domains in common. However, 

the physiological functions of the multidomains remain unclear. One of the reasons is the 

lack of information of three-dimensional (3D) structures of the multidomain components. 

To clarify the architecture, we used vascular apoptosis-inducing protein 1 (VAP1), which 

is a dimer-type high-molecular SVMP and stable to make crystals for X-ray analysis. 

Methods: VAP1 was isolated from crude western diamondback rattlesnake Crotalus atrox 

venom (Sigma-Aldrich, USA) and subjected to sitting- or hanging-drop vapor diffusion 

crystallization. X-ray diffraction data were collected at SPring-8 beamlines. 

Results: 3D structures of VAP1 are determined from the crystals at 2.5-Å resolution. The 

disintegrin (D)-domain protrudes from the metalloprotease (M)-domain opposing the 

catalytic site and constituting a C-shaped arm with cores of Ca2+ ions. The disintegrinloop 

is packed by the cysteine-rich (C)-domain and inaccessible for protein-binding. 

Instead, the hyper-variable region (HVR) in the C-domain, which has a novel fold 

stabilized by the strictly conserved disulfide bridges, constitutes a potential protein-protein 

adhesive interface. The HVR is located at the distal end of the arm and faces toward the 

catalytic site. The amino acid sequence alignment indicates that the architecture is common 

to most of SVMPs and ADAMs. 

Discussion/Conclusion: Here for the first time, we reveal the 3D architecture of MDC 

domains of SVMPs and ADAMs. The unique C-shaped structure of MDC implies 

interplay between the proteolytic and adhesive domains. The HVR may be important to 

search physiological targets of SVMPs and ADAMs and provide insights into the future 

design of drugs with higher specificity. 

o snake venom 

o metalloproteinase/disintegrin 

o 3D-structure 

o ADAM 

164 


Unique Molecular Formations in Crystals of Scorpion Toxins that Affect Voltage-Gated 

Na-Channels 

Kahn R 1 *, Karbat I 1 , Gurevitz M 1 , Frolow F 2 

1 

Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Israel 

*nitzai@post.tau.ac.il 

2 

Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel-Aviv 

University, Tel-Aviv, Israel 

Long-chain scorpion toxins bind and modulate the function of voltage-gated sodium 

channels (Navs) in excitable tissues. The toxins are approximately 61-76 aa residue long 

globular polypeptides reticulated by four disulfide bonds and very similar in structure. 

They divide into two classes, alpha and beta, according to their mode of action. Each class 

is subdivided to distinct pharmacological groups according to their ability to displace one 

another from the receptor site of channels of various origin. We cloned and produced in 

recombinant form representative toxins of all pharmacological groups and analyzed them 

by mutagenesis and X-ray crystallography. Determination of the structure of the alphatoxin 

Lqh-alpha-IT at 1.1Å resolution revealed cubic space group with complicated 

packing arrangements in the crystal. Close inspection of these arrangements revealed a 24mer 

shell-like 'particle' of a 432 symmetry. With the objective to create novel 'molecular 

formation' we crystallized and analyzed several other toxins. Determination of the structure 

of a triple mutant of Lqh-alpha-IT, LqhαIT 8D-9D-10V , which was devoid of activity, at 1.6Å 

resolution exhibited a dodecameric particle formed from a dimer in the asymmetric unit by 

interplay of I4122 symmetry. We further solved the structure of a toxin chimera, 

Lqh2 LqhαIT(face) , composed of the bioactive surface of Lqh-alpha-IT on the scaffold of Lqh2, 

at 1.2Å resolution and found novel formations of I422 symmetry. We than analyzed the 

crystal structure of the beta-toxin Bj-xtrIT (solved at a resolution of 2.1Å). This crystal 

reveals a mono-molecular filament-like pattern, where the crystallographic translational 

repeat is of eight molecules, 191.4Å long, and is due to interplay of I41 symmetry 

operators. Close inspection of these arrangements reveals interplay among symmetry 

elements, molecular size, shape and bioactive surface that provide unique formations of 

potential nano-technological importance. 

o Scorpion toxins 

o Crystal structure 

o Packing 

o Molecular formations 

165


The increasing molecular diversity of sarafotoxins 

Ducancel 1 F.*, Quinton 3 L., Menin 4 L., Hayashi 2 , M.A.F., Lamthanh 1 , H., Chamot- 

Rooke 3 J., Stöcklin 4 R. 

1 

Department of Protein Engineering (DIEP), Bâtiment 152, CEA de Saclay, 91191 

Gif sur Yvette Cedex (France), *Tel: +33 169088154, Fax: +33 169089071, e-mail: 

frederic.ducancel@cea.fr 

2 

Biochemical and Biophysical Laboratory, Instituto Butantan, SP 05503-900, Sao 

Paulo, Brazil. 

3 

Laboratoire des Mécanismes Réactionnels, UMR 7651 CNRS, Ecole Polytechnique, 

F-91128, Palaiseau, France. 4 Atheris Laboratories, case postale 314, CH-1233 

Bernex, Geneva, Switzerland. 

Abstract. 

Snake venom sarafotoxins (SRTXs) and mammalian endothelins (ETs) 

comprise structurally and functionally related potent vasoconstrictor 

isopeptides that act on the vascular system via identical receptors. This 

similarity is remarkable, since SRTXs are highly toxic components 

isolated from the venoms of snakes of the genus Atractaspis in the 

Atractaspididae family, while ETs are endogenous mammalian 

hormones. Since the first functional and structural description of 

SRTXs in 1988, the full extent of their natural diversity has become 

increasingly apparent, and this has led to the characterization of new 

families of sarafotoxins peptides. Based on a combination of 

conventional biochemical approaches and the latest molecular biology 

and mass spectrometry techniques, the more recent panel of SRTX 

isopeptides isolated from various snake species within the 

Atractaspididae family will be presented. Also, the similarities and 

differences that exist between sarafotoxins and endothelins in terms of 

their metabolism, genetic origin, structure and functional sites will be 

discussed. 

166 


Molecular diversity of snake venom VEGFs 

Matsunaga, Y. 1 , Yamazaki, Y. 1 , Morita, T. 1* 

1 Meiji Pharmaceutical Univ., 2-522-1 Noshio, Kiyose-city, Tokyo, Japan, *tmorita@my-pharm.ac.jp 

[Background] Vascular endothelial growth factor (VEGF-A) plays a critical role in the 

embryonic vasculogenesis and in the pathological angiogenesis observed in several 

endothelium proliferative diseases, including cancer. Recently, we isolated and 

characterized novel VEGFs (vammin and VR-1) from the venoms of snakes, Vipera 

ammodytes ammodytes and Daboia russelli russelli (Vipera r. russelli, also known as 

Russell’s viper) 1 . Compared with human VEGF-A, the snake venom derived VEGFs show 

stronger proliferative affects in vitro and more potent hypotensive effects in vivo 1 . 

Furthermore, the snake venom VEGFs specifically recognize KDR (VEGF receptor 2), but 

not other VEGF receptors 1 . The crystal structures of snake venom VEGFs reveal similar 

but significantly different architectures from the known structures of other human VEGFs: 

insertion of an amino acid resulted in a different structure for receptor-binding loop 3, and 

a difference in surface potential of the molecule 2 . Thus, these recent findings regarding 

snake venom VEGFs demonstrate unique and distinct biological activities and also 

substantially different structural features from other known VEGF subtypes. To identify 

novel VEGFs derived from several snake venoms, we cloned cDNAs encoding VEGF-like 

proteins using the venom gland cDNAs from several venomous snakes. 

[Result and Discussion] We first performed PCR using snake venom VEGF specific 

primers. In addition to the Vipera species, specific amplified PCR products were observed 

when the venom glands cDNAs from Echis, Bitis, Agkistrodon and Crotalus species were 

used as templates. Encoding proteins exhibited high identity to known snake venom 

derived VEGFs, but showed significant variation in their receptor-binding loop 3 and Cterminal 

heparin-binding regions. These data suggest that snake venom contains other 

novel VEGFs possessing unknown, conceivably interesting, properties. 

1. Yamazaki, Y., Takani, K., Atoda, H., Morita, T. (2003) JBC 278, 51985-51988 

2. Suto, K., Yamazaki, Y., Morita, T., Mizuno, H. (2005) JBC 280, 2126-2131 

o vascular endothelial growth factor 

o snake venom 

o cDNA cloning 

o molecular diversity 

167


Isolation and characterization of a VEGF-like protein from the venom of Bitis 

arietans 

Obayashi S 1 , Matsunaga Y 1 , Yamazaki Y 1 , Morita T 1* 

1 Department of Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204- 

8588, Japan * tmorita@my-pharm.ac.jp 

Background: Vascular endothelial growth factor-A (VEGF-A165) exerts multiple effects 

upon binding to VEGFR-1 (Flt-1), VEGFR-2 (KDR), and neuropilin-1. We recently 

identified and characterized two novel snake venom VEGFs (vammin and VR-1) which 

possess unique properties. These VEGFs, designated as VEGF-Fs, are highly specific 

ligands for KDR 1) . In this study, we presumed that snake venom-derived VEGFs may 

exhibit the same molecular diversity observed in other snake venom proteins, such as Ctype 

lectin-like proteins 2) and phospholipase A2. In fact, other snake venom VEGFs 

(TfsvVEGF and Pm-VEGF) demonstrating distinct receptor selectivity relative to vammin 

and VR-1 have recently been identified in the venom of both Trimeresurus and 

Protobothrops species 3, 4) . Subsequently, we screened several snake venoms using antivammin 

antiserum. As a result, we detected an immunoreactive protein in the venom of 

Bitis arietans. Results and Discussion: The immunoreactive protein was purified by three 

steps column chromatographies using an anti-vammin antiserum as a detection regent. The 

purified protein, named barietin, was obtained with a yield of 0.08 % in relation to total 

venom protein. Sequence analysis revealed that barietin exhibits significant homology with 

the VEGF family; however barietin generally lacks most of the C-terminal sequence, when 

compared with other heparin-binding VEGFs including vammin 5) . Heparin-binding affinity 

of barietin was measured using analytical heparin affinity chromatography. Barietin could 

bind heparin with high affinity as well as human VEGF-A165. These results indicate that 

barietin has distinct molecular properties from the previously described VEGFs such as 

snake venom-derived VEGF (VEGF-F) and mammalian VEGF-A165. 

1) Yamazaki Y, Takani K, Atoda H, and Morita T (2003) JBC 278, 51985-51988 

2) Morita T (2005) Toxicon 45, 1099-1114 

3) Takahashi H, et al. (2004) JBC 279, 46304-46314 

4) Chen YL, Tsai IH, Hong TM, and Tsai SH (2005) Thromb. Haemost. 93, 331-338 

5) Yamazaki Y, Tokunaga Y, Takani K, and Morita T (2005) Biochemistry 44, 8858-8864 

o vascular endothelial growth factor 

o snake venom 

o heparin 

168 


VEGF receptor-binding potential is a common property for myotoxic 

phospholipase A2. 

Fujisawa D., Matsunaga Y., Yamazaki Y. and Morita T.* 

Department of Biochemistry, Meiji Pharmaceutical University, 2-522-1, Noshio, Kiyose, Tokyo, Japan, * 

tmorita@my-pharm.ac.jp 

Introduction: Vascular endothelial growth factor (VEGF165) plays a central role in 

angiogenesis through binding to the VEGF receptor-2 (KDR). We previously found a 

novel VEGF subtype designated VEGF-F, which selectively binds to KDR, from two viper 

venoms 1) . Recently, we identified a VEGF receptor-binding protein, abbreviated as KDRbp, 

from the venom of the eastern cottonmouth (Agkistrodon piscivorus piscivorus), which 

binds to the extracellular domain of KDR with 10 -8 M affinity 2) . Sequence analysis 

revealed that the KDR-bp molecule is identical to an inactive phospholipase A2 (PLA2) 

homologue, Lys49PLA2. Lys49PLA2 has been shown to exhibit myotoxicity; however, the 

molecular mechanism remains unknown. The aim of this study is to examine the 

relationship between KDR-binding potential and myotoxicity through the analysis of 

KDR-binding potential of several PLA2 and PLA2 homologues derived from the venom of 

five snake species. 

Results and Discussion: PLA2 and its homologues were purified by gel filtration, ionexchange 

chromatography and HPLC. Isolated PLA2s were identified by N-terminal 

sequencing and MALDI-TOF MS. KDR-binding potential was evaluated using the Biacore 

system. As a result, all myotoxic PLA2 homologues were bound to the extracellular 

domain of KDR, while no specific interaction was observed in other PLA2 homologues 

such as neurotoxic or anticoagulant PLA2s; showing strong correlation between KDRbinding 

ability and myotoxicity. In addition to the inactive PLA2 homologues, 

Asp49PLA2, an active PLA2 from the venom of A. p. piscivorus possessing myotoxicity, 

also displayed KDR-binding potential. These results indicate that KDR-binding potential is 

independent of PLA2 enzymatic activity. Sequence comparisons suggest that the Nterminal 

α-helix region, β-wing region and C-terminal loop region may be involved in 

KDR-binding. 

1). Yamazaki Y., Takani K., Atoda H. and Morita T. (2003) JBC, 278, 51985-51988. 

2). Yamazaki Y., Matsunaga Y., Nakano Y. and Morita T. (2005) JBC, 280, 29989-29992. 

o Snake venom 

o Vascular endothelial growth factor (VEGF) 

o VEGF receptor-2 (KDR) 

o Myotoxic phospholipase A2 

169


Low Abundant Proteins in Cobra Venom 

Utkin, Y.N., Osipov, A.V., Makarova, Ya.V. 

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, ul. Miklukho-Maklaya 16/10, Moscow, Russia, 

utkin@ibch.ru 

Cobra venoms are complex mixtures of different components, mostly of polypeptide 

nature. The main components of cobra venom are cytotoxins, phospholipases A2 and 

neurotoxins. The properties and biological effects of these proteins are very well studied. 

At the same time cobra venoms contain less abundant and not so well studied proteins of 

different structural types. The aim of this work is to identify, isolate and investigate low 

abundant proteins in cobra venom. Our approach is based on the use of different kinds of 

liquid chromatography (gel-filtration, ion-exchange and reversed phase) for separation of 

the venom in combination with MALDI mass-spectroscopy for identification of new 

proteins. Such a proteomic approach has allowed us to identify practically all the known 

proteins in cobra Naja kaouthia venom as well as to find several new ones. 

We have found the first representative of glycosylated three-fingered toxins – cytotoxin 3 

containing a carbohydrate residue on Asn29. This modification results in substantial 

decrease in cytotoxicity. A series of so called “weak” or non-conventional toxins was 

identified in N. kaouthia venom. The in vivo effects for one of them (WTX) were studied, 

and data obtained suggest the action of WTX on several biological targets. A series of 

toxins highly homologous to muscarinic toxins from mamba venom was found in cobra 

venom as well. Several novel proteins belonging to the family of cysteine-rich secretory 

proteins (CRISP) were isolated from the venoms of N. kaouthia and N. haje cobra. Four 

CRISP variants were identified in N. kaouthia venom and three proteins in N. haje venom. 

Our results suggest that each cobra venom contains a pool of different CRISPs. In addition 

to acute toxicity determination, the activity of new toxins was tested on rat 

pheochromocytoma cell line PC12. Depending on the protein studied we have observed 

different effects ranging from cytotoxicity (for cytotoxins) to neurite outgrowth inducing 

activity (for nerve growth factor and phospholipase A2). 

Thus, using the proteomic approach we have identified and characterized several new low 

abundant proteins in cobra venom. 

o Cobra venom 

o New proteins 

o isolation 

o properties 

170 

Monday 24 July: poster session 

(Colville Building 5.11/5.12) 

Nerve, muscle, and myotoxicity 

1. Ability of suramin to antagonize myotoxic and phospholipase activities of bee 

venom. El Kik, C.Z., Fernandes, F.F.A., Sanches, F.V.,Arruda, E.Z., Melo, P.A. 

2. Antimyotoxic effects of suramin and polyethylene glycol (PEG) against 

Agkistrodon contortrix laticinctus crude venom and its myotoxin. Tomaz, M. A. 1 , 

Serafim, A.D., Williams, K.,Murakami, M.T., Arni, R.K., Ownby, C.L.; Melo, P.A. 

3. Ability of a synthetic coumestan named LQB93 to antagonize some Bothrops 

snake venoms activity. Melo, P. A., Pinheiro, D. A.; Fernandes, F. F. A.; Tomaz, M. 

A.;Moraes, R. A. M. 1 ; Ricardo, H. D 1 . da Silva, A. J. M 2 . ; Costa, P. R. R 2 . 

4. Dexamethasone shows in vivo but not in vitro antimyotoxic effect against Bothrops 

jararacussu snake venom. Patrão-Neto, F.C. 1 ; Tomaz, M.A. 2 ; Calil-Elias, S. 2,3 , Melo, 

P.A. 

5. Improvement of mouse skeletal muscle regeneration by heparin after lesion 

induced by Bothrops jararacussu venom. Calil-Elias, S., Tomaz, M. A., Costa, M.L., 

Mermelstein, C.L., Martinez;A. M. B., Melo, P.A. 

6. Mouse extensor digitorum longus and soleus have a different sensitivity to some 

snake venoms myotoxicity. Tomaz, M. A 1 ., Calil-Elias, S 1,3 .,Martinez, A.M.B 2 ., Melo, 

P.A 1 . 

7. αδ-Bungarotoxin, a new reversible long-chain neurotoxin from Malayan Krait 

(Bungarus candidus) venom with high toxicity and site-selective binding at the 

muscle nicotinic acetylcholine receptor. Kuch, U. 1* , Molles, B.E. 2 , Bergman, T. 3 , 

Cederlund, E. 3 , Alvelius, G. 3 , Jörnvall, H. 3 , Chanhome, L. 4 , Omori-Satoh, T. 4 , Chen, 

Y.M. 5 , Samejima, Y. 6 , Warrell, D.A. 7 , Mebs, D. 1 

8. Novel recombinant expression system for myonecrotic Lys49 phospholipase A2 

and its mutants identifying the functional residues for necrosis. Tomohisa Ogawa 1, * , 

Minae Seto 1 , Koji Muramoto 1 , Motonori Ohno 2 

9. Non-cholinergic contractile activity of Dispholidus typus (Boomslang snake) in the 

rat isolated vas deferens. Lumsden, N. G 1 , Hodgson, W. C *2 , Ventura, S 3 , Lewis, R. J 1 

10. Equivalent effects of snake PLA2 neurotoxins and lysophospholipid–fatty acid 

mixtures. Rigoni, M 1 Caccin, P 1 , Gschmeissner, S 2 , Koster, G 3 , Postle, A. D 3 

,Rossetto, O 1 , Schiavo, G 2 and Montecucco, C 1 

171

11. EFFECT OF COLUBER RAVERGIERI VENOM ON SYNAPTIC PASSAGE. 

Kazakov I., Abubakirova M.E. 

12. Effect of Duvernoy's gland secretion of snake Coluber ravergieri on frog 

nerve-muscle preparation. Akhmedov K.J., Makhkamov B.T., R.S.Reimbaeva., 

M.E.Abubakirova., I.K.Kazakov 

13. Myotoxic activity from the venoms of six species of Micrurus. Néstor Lago 1 ; 

Adolfo R. de Roodt 2 ; Claudio Mirabelli 1 ; Rodrigo D. Laskowicz 2 ; Judith Estévez 3 ; 

Víctor M. Manzaneli 2 ; Jorge Paniagua 4 ; Eduardo Scarlatto 5 . 

14. Development of an in vivo model for the study of snake venom myotoxicity. 

Andrew J. Hart 1 , Geoffrey K. Isbister 1,2 , Sharmaine Ramasamy 1 , Wayne C. 

Hodgson 1 

15. Inotropic effects of Tityus cambridgei scorpion venom on skeletal muscle. 

Borja-Oliveira C.R. 1,2 , Rodrigues-Simioni L. 2 , Spisni A. 1,3* 

16. Neutralization of the neurotoxicity of Micrurus altirostris (Uruguayan coral 

snake) venom by coral snake antivenom. 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* 

17. Peptide inhibitors of sPLA2 do not prevent the neuromuscular blocking actions 

of β-bungarotoxin in vitro. Harvey, A.L. 1 , Young, L.C. 1 , Gopalakrishnakone, P. 2 , 

Thwin, M.M 2 

18. Gambierol markedly enhances evoked quantal acetylcholine release from motor 

nerve terminals at vertebrate skeletal neuromuscular junctions. 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 

19. Neutralization of neuromuscular activity of bothropstoxin-I (BthTX-I) by a 

methanolic fraction from Casearia sylvestris Sw. Francischinelli, M. C 1 , Gerenutti, M 1 , 

Silva, M. G 1 , Andréo-Filho, N 1 , Oliveira, S. J 1 , Leite, G. B 3 , Dal Belo, C. A 3 , Cintra, A. 

C. O 4 , Cruz-Höfling, M. A 2 , Rodrigues-Simioni, L 3 , Oshima-Franco, Y 1,3* 

20. In vivo study of lethal effects of ostreolysin, a cytolysin from the edible oyster 

mushroom in rodents. Žužek, M.C 1 , Maček, P 2 , Sepčić, K 2 , Cestnik,V 1 , Frangež, R 1* . 

21. Effects of Bothrops marajoensis venom on the mouse and chick nerve-muscle 

preparations. Cavalcante, W.L.G 1 , Hernandez-Oliveira, S 1 , Leite, G.B 1 , Ponce-Soto, 

L.A 2 , Marangoni, S 2 , Rodrigues-Simioni, L. 1* 

22. Purification and N- terminal sequence of two presynaptic neurotoxic PLA2, 

neuwieditoxin-I (NeuTX-I) and neuwieditoxin-II (NeuTX-II), from Bothrops 

neuwiedi pauloensis (jararaca-pintada) snake venom. Borja-Oliveira C.R. 1 , Kassab 

172 

B.H. 1 , Soares A.M. 2 , Toyama M.H. 1 , Giglio J.R. 3 , Marangoni S. 1 , Re L. 4 , Rodrigues- 

Simioni L. 1* 

23. The twitch potentiation induced by crotamine homologs from Crotalus durissus 

ruruima and C. d. terrificus venoms. Hernandez-Oliveira S.S., Silva S.O., Borja- 

Oliveira C.R., Hyslop S., Marangoni S., Rodrigues-Simioni L.* 

24. Purification and Pharmacological Characterization of BtII-2 a Novel Presynaptic PLA2 

Isolated From Bothrops alternatus Venom. Ponce-Soto, L.A 1 ; Barros, JC 2 ; Hernadez-Oliveira, S 2 , 

Novello, JC 1 , Dal Belo, CA 2 ; Marangoni, S 1 .; Rodrigues-Simioni, L 2* . 

25. Antiophidian Activity of Galactia glaucescens Ethanolic Extract (GGE) Against 

Crotalic and Bothropic Venoms. Colares, AV 4 ; Oshima-Franco, Y 1 ; Corrado, AP 2 ; 

Ticli, FK 3 ; Sampaio, SV 3 ; Cintra, ACO 3 ; Rodrigues-Simioni, L 1 .;dos Santos, MG 4 ; Dal 

Belo, CA 1,4,5* . 

26. Neutralization of snake venoms phospholipase A2 activities in mice 

neuromuscular preparation by aqueous extract of Casearia sylvestris 

(Flacourtiaceae). Campos, T.C 1 , Cavalcante W.L.G 1 , Pai-Silva M.D 1 , Pereira P.S. 2 , 

Soares A.M 3 , Gallacci M 1* . 

Ion channels 

27. Expression and Mutagenesis of the Sea Anemone Toxin Av2 Reveals Key Amino 

Acid Residues Important for Activity on Voltage-Gated Sodium Channels. Moran 

Y*, Cohen L, Kahn R, Karbat I, Gordon D, Gurevitz M 

28. Characterization of Voltage-dependent Calcium Channel Blocker Peptides 

Isolated from Tarantula Venom. Ono, S., Kimura T., Kubo T. 

29. Recombinant expression of a four disulfide-bridged scorpion toxin antagonist of 

voltage-gated sodium channels. Estrada, G., García, B.I., Ortiz, E.,Riano, L., Becerril, 

B., Possani, L.D., Corzo, G. 

30. Structure and function of huwentoxin- , a novel spider toxin targeting both 

serine proteinase and potassium channels. Chunhua Yuan , Kuan Peng, Jianbo Diao 

and Songping Liang 

31. A novel family of conotoxins target the nACh-Receptors. Kauferstein, S. 1 , Nicke, 

A. 2 , Kendel, Y. 1 ., Possani, L.D. 3 , Stöcklin, R. 4 , Mebs, D. 1 

32. Isolation and characterisation of a potent novel tarantula toxin targeting the 

acid sensing ion channel ASIC1a. Rash LD 1 , Adams DJ 2 and Alewood PF 1 . 

173

33. Modulation of voltage-gated Na + and K + channels by pumiliotoxin 251D: a “joint 

venture” alkaloid from arthropods and amphibians. Vandendriessche, T. 1 , Maertens, 

C. 1 , Abdel-Mottaleb, Y. 1 , Cuypers, E. 1 , Nubbemeyer, U. 2 , Mebs, D. 3 & Tytgat, J. 1* 

34. Structure-function relationships of Magi 4, a spider neurotoxin targeting insect 

and mammalian voltage-gated sodium channels. Little, M.J. 1 , Yamaji, N. 2 , Villegas, 

E. 3 , Corzo, G. 4 , Nicholson, G.M. 1* 

35. The natural anatoxin Amm VIII from Androctonus mauretanicus induces 

neutralizing antibodies against the most potent “Old-World” alpha-toxins. Martin- 

Eauclaire, M-F, Alami, M, Rosso, J-P. and Bougis, P.E*. 

36. Scorpion β-toxins: surveying the species-selectivity of five ‘classics’. 

Bosmans, F. 1 , Martin-Eauclaire, M.F. 2 , Tytgat, J. 1* 

37. Potent modulation of the voltage-gated sodium channel Nav1.7 by OD1, a toxin 

from the scorpion Odonthobuthus doriae. Maertens, C. 1* , Cuypers, E. 1 , Amininasab, 

M. 2 , Jalali, A. 3 , Vatanpour, H. 4 , Tytgat, J. 1 

38. Electrophysiological Characterization of Marine Snail Conus californicus 

Venom in Potassium Ion Channels. Juárez-Moreno, K. O. 1 , Prior-Mier y Teran A 1 , 

García-Valdés, J. 2 , Aguilar M 3 , Morales E 1 , Waumann D 4 and Licea-Navarro, A. 1* 

39. GAMBIEROL-INDUCED CYTOSOLIC CALCIUM INCREASE IN HUMAN 

NEUROBLASTOMA CELLS. Cagide, E 1 , Louzao, M.C 1* , Vieytes, M.R 2 , Sasaki, M 3 , 

Fuwa, H 3 , Yasumoto, T 4 , Botana, L.M. 1 

40. New modulator of P-type Ca 2+ -channels from Lycosa sp. venom. Pluzhnikov, K. 1 , 

Korolkova, Y. 1* , Vassilevski, A. 1 , Nikolsky, A. 1 , Fisyunov, A. 2 , Tsintsadze, V. 2 , 

Krishtal, O. 2 , Grishin, E. 1 

41. Evidence for a novel epitope for hERG channel blocking activity. Yousra 

Abdel-Mottaleb 1 , Praveen Kumar 1 , Brigitte Céard 2 , Pierre E. Bougis 2 , Marie- 

France Martin-Eauclaire 2 , Jan Tytgat 1* 

42. The first potassium channel toxin from the venom of the Iranian scorpion 

Odonthobuthus doriae. Yousra Abdel-Mottaleb 1 , Amir Jalali 2 , Elke Clynen 3 , Frank 

Bosmans 1 , Liliane Schoofs 3 , Hossein Vatanpour 2 , Jan Tytgat 1* 

43. Novel insect-selective neurotoxins from the venom of the tarantula Eucratoscelus 

longiceps target insect Kv channels. Escoubas, P. 1* , Lee, M. 2 , Ross, G. 2 , Lazdunski, 

M. 1 Nicholson, G.M. 2 

44. Short insecticidal toxins from the Asian scorpions Buthus martensi and 

Mesobuthus tamulus modulate insect neuronal voltage-dependent and calcium- 

174 

activated chloride channels. Grolleau F 1 ., Stankiewicz M. 3 , Herrmann R. 4 , Moskowitz 

H. 4 , Rajendra W. 4 , Hammock B.D. 4 , Romi-Lebrun R. 5 , Wu F.Q. 6 , Nakajima T. 5 Escoubas 

P. 2,5* 

45. Phlotoxin 1, a toxin from tarantula venom, is a potent modulator of Nav1.7 

sodium channels and a potential analgesic. Escoubas P. 1* , Bosmans F. 2 , Cuypers E. 2 , 

Diochot S. 1 , Mebs D. 3 , Craik D. 4 , Hill J. 4 , Maertens C. 2 , Nakajima T. 5 , Lazdunski M. 1 , 

Tytgat J. 2 

46. ON SOME BIOPHYSICAL TRAITS OF COLUBRIDAE SNAKE VENOMS. 

Kazakov I., Abubakirova M.E., Reimbaeva R.S. 

47. THE EFFECT OF BOOPHILUS CALCARATUS VENOM ON BILAYER 

LIPID AND MITOCHONDRIAL MEMBRANES. Kazakov I., Rakhimova Sh.H., 

Abubakirova M.E., Azimov D.A. 

48. THE EFFECT OF HELMINTH EXTRACT ON THE BILAYER LIPID AND 

MITHOCHONDRIAL MEMBRANES. I.Kazakov, M.E.Abubakirova, A.E.Kuchboev, 

D.A.Azimov 

49. ω-ACTX-Ar1a: a novel insect-selective voltage-gated calcium channel blocker 

from the venom of the Sydney funnel-web spider. 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, G.M 1 

50. The Effects of the Sodium Channel Blocker Lidocaine on Cardiovascular and 

Respiratory Actions of the Yellow Scorpion Leiurus Quinquestriatus in Rabbits. Al- 

Shanawani, A. R * , Fatani, A.J. 

51. Postsynaptic neuromuscular activity of Cerastes cerastes cerastes crude snake 

venom. Soliman M. M 1 , Abu-Sinna G 1 , Abd-Elbaset A 2 , Harvey A.L 3 and Rowan,E.G 3 

52: The bioactive surface of the scorpion depressant toxin LqhIT2. Michael Turkov, 

Izhar Karbat, Lior Cohen, Roy Kahn, Dalia Gordon and Michael Gurevitz 

175

Poster 1: Ability of suramin to antagonize myotoxic and phospholipase activities of 

bee venom venom 

El Kik, C.Z. 1 , Fernandes, F.F.A. 1 , Sanches, F.V 1 .,Arruda, E.Z. 1 , Melo, P.A. 1 

1 

Departameto de Farmacologia Básica e Clínica, Universidade Federal do Rio de Janeiro-UFRJ 21941- 

590 Rio de Janeiro, RJ, Brazil. 

We evaluate the ability of suramin, a polyanion, to antagonize the phospholipase (PLA2) 

and myotoxicity activities of Apis mellifera venom. Myotoxicity in vitro was assed by 

perfusion of isolated mouse extensor digitorius longus (EDL) muscle exposed to the 

venom (25 μg/ml) and measured the rate of CK release. EDL muscle had the rate of CK 

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 

exposition. When we added suramin to the bath solution (10 μM) we observed a significant 

reduction to 1.86 ± 0.6 U.g -1 .h -1 (n=4). The bee venom PLA2 activity, evaluated by the 

modified Marinetti’s method * , showed a concentration-dependent activity that was also 

inhibited by suramin (1-30 μM). Adult male mice weighting 20-25g were separated in 5 

groups . Control group received i.m. injection of physiological saline solution and treated 

groups received i.m. of venom(0.5 mg/Kg) alone or venom plus suramin(30 mg/kg) 

preincubated (30 min before),or by i.p. route 15 min. before or after the venom injection. 

Two hours after i.m. venom injection, with the anesthetized mice, the blood was collected 

in the orbital plexus, centrifuged and plasma CK activity was measured. The i.m. injection 

of bee venom induced an increase of plasma CK activity from 228.60 ±14.00 U/L to 

1158.0±124.0 U/L which was inhibited circa of 50% by suramin in the pre incubated 

protocol. At this same dose, pre or post-treatment did not protected against the myotoxic 

effect. Suramin effect seems to be due to the interaction of its charges with the polycations 

present in the bee venom. 

References: Marinetti, GV (1965) Biochem. Biophys. Acta, 98, 554-565. 

Supported by CNPq, FAPERJ, CAPES, PRONEX 

o Bee venom 

o Phospholipase activity 

o Myotoxicity 

o Suramin 

176 

Poster 2: Antimyotoxic effects of suramin and polyethylene glycol (PEG) against 

Agkistrodon contortrix laticinctus crude venom and its myotoxin 

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 

1 Departamento de Farmacologia Básica e Clínica ICB, CCS, Universidade Federal do Rio de Janeiro, 

21941-590, Rio de, Janeiro, RJ, Brazil. pamelo@farmaco.ufrj.br. 

2 Departament of Physics IBILCE/UNESP, São José do Rio Preto, SP, Brazil 

3 Minority International Research Program, University of Maryland at Baltimore, Baltimore, MD, USA 

4 Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, USA 

We assessed the effects of suramin and polyethylene glycol (PEG400) against the 

myotoxicity of Agkistrodon contortrix laticinctus (ACL) crude venom and its myotoxin on 

mouse isolated extensor digitorum longus (EDL) and neuromuscular diaphragm 

preparation. Isolated EDL were exposed to ACL crude venom and myotoxin (25 µg/mL) 

alone or together with suramin (10 and 30 µM) and PEG (10 and 30 mM). The rate of 

creatine kinase (CK) release from the muscles was measured, indicating tissue damage. 

Basal CK release from EDL was 0,43±0,06 U.g -1 .h -1 , and after 60 minutes of exposure to 

the myotoxin and treatments it increased to 13,76±3,76 (ACL myotoxin), 5,90±1,19 and 

2,10±0,86 (myotoxin plus suramin 10 and 30 µM, respectively), and 8,87±3,63 and 

5,80±2,04 (myotoxin plus PEG 10 and 30 mM, respectively). ACL crude venom (60 min) 

also induced an increase in the rate of CK release up to 32,07±5,43 U.g -1 .h -1 , which was 

significantly inhibited by PEG 100 mM (6,47±3,14 U.g -1 .h -1 ). Mouse neuromuscular 

diaphragm preparation was exposed to ACL crude venom and venom plus suramin. The 

preparation was directly (muscle) and indirectly (nerve) stimulated, and twitch amplitude 

was recorded. The snake venom (12,5 and 25 µg/mL) decreased twitch amplitude down to 

25% of the control in approximately 30 to 40 min in both protocols. Suramin prevented in 

a concentration-dependent fashion the venom’s effect (20, 25 and 50 μM caused a 5, 40 

and 100% inhibition on the depressing effect of ACL crude venom, respectively). Data 

show that suramin and PEG are able to prevent the myotoxic activities of ACL crude 

venom and its myotoxin in mouse isolated muscle and suramin prevents the neuromuscular 

effects of ACL crude venom. 

Research supported by CNPq, CAPES, FAPERJ, PRONEX, FAPESP, Fundação 

Universitária José Bonifácio (FUJB, UFRJ),and Fogarty Minority International Research 

Training Program. 

o Agkistrodom contortrix laticinctus venom 

o Myotoxicity and Suramin 

o Myotoxicity and polyethylene glycol 

177

Poster 3: Ability of a synthetic coumestan named LQB93 to antagonize some 

Bothrops snake venoms activity 

Melo, P. A. 1 Pinheiro, D. A 1 .; Fernandes, F. F. A. 1 ; Tomaz, M. A 1 .;Moraes, R. A. M. 1 ; 

Ricardo, H. D 1 . da Silva, A. J. M 2 . ; Costa, P. R. R 2 . 

1 

Departamento de Farmacologia Básica e Clínica, ICB 

2 Núcleo de Pesquisas de Produtos Naturais, NPPN, CCS; Universidade Federal do Rio de Janeiro-UFRJ 

21941-590, Rio de Janeiro RJ, Brazil 

In this work we studied the anti-ophidic effect of synthetic coumestan LQB93 against 

some activities of the venoms of Bothrops jararacussu and Bothrops jararaca in adult 

mice. The anti-myotoxic activity of the coumestan was evaluated by in vitro and in vivo 

tests. This substance, (30 μM), reduced 100% of the increase of creatine kinase (CK) 

release rate of isolated mouse extensor digitorum longus muscle induced by B. 

jararacussu (25 μg/ml) in vitro (IC50=0.0291 μM). In vivo experiments, B. jararacussu (1 

μg/g) venom was pre-incubated with LBQ93 (0.1-30 μg/g), during 30 min, for later 

injection in mice paw and evaluation of coumestan antimyotoxic and anti-edematogenic 

effects. The myotoxicity was evaluate by the increase of plasma CK activity and the paw 

edema was measured using the sliding caliper. The coumestan antimyotoxic and antiedematogenic 

effects were similar in both protocols and this effect was dose-dependent 

with ID50 of 0.1728 μg/g and 0.1434 μg/g, respectively. The hemorrhage, induced by the 

intradermal injection of B. jararaca (1 µg/g) venom in the mice skin, was evaluated 

measuring the optic density of the hemorrhagic area. LQB93 (10 µg/g), pre-incubated with 

venom, abolished the hemorrhagic effect. The pro-coagulating activity of B. jararaca (10 

µg) was analyzed by Lee-White modified method, and showed complete inhibition by 

LQB93, in the proportion of 30 µg coumestan per microgram of venom. This synthetic 

coumestan has the skill to inhibit the effects of two important Bothrops sp venoms 

reproducing some of the properties of wedelolactone a natural compound isolated from the 

plant Eclipta prostrata which is active against snake venoms. 

o Snake venoms 

o Antivenoms 

o Natural & Synthetic coumestan 

o Wedelolactone 

178 

Poster 4: Dexamethasone shows in vivo but not in vitro antimyotoxic effect against 

Bothrops jararacussu snake venon. 

Patrão-Neto, F.C. 1 ; Tomaz, M.A. 2 ; Calil-Elias, S. 2,3 , Melo, P.A. 2 

1 

Faculdade de Medicina de Campos, Campos dos Goytacazes, RJ - Brazil 

2 

Departamento de Farmacologia Básica e Clínica, Universidade Federal do Rio de Janeiro-UFRJ 21941- 

590 Rio de Janeiro RJ. Brazil 

3 

Departamento de Farmácia e Administração Farmacêutica, Faculdade de Farmácia, Universidade 

Federal Fluminense, Niterói, RJ, Brazil 

We studied the antivenom effect of Dexamethasone (DEXA) alone or its association with a 

crude extract of an antiophidic plant named * Eclipta prostrata (EP), in vitro and in vivo 

protrocols. On the in vivo experiments, a sham group of adult Swiss mice (25.0 ± 3.0 g) 

received 100 μL of saline solution (PSS), in a perimuscular injection close to extensor 

digitorum longus (EDL). Control group received B. jararacussu venom (1.0 mg/kg) alone. 

Treated groups received venom plus DEXA (1.0 mg/kg), a mixture of venom preincubated 

with EP (50 mg/kg), or a mixture of both agents. After 3 days we measured the 

creatine kinase (CK) muscular content and performed a histological analysis. For in vitro 

experiments, we analysed the rate of CK release from isolated mouse EDL muscles which 

were bathed in PSS and exposed to B. jararacussu venom (25 μg/ml) alone, the venom 

plus DEXA (25 μg/ml), or it plus EP (25-100 μg/ml) that was added to the bath. Venom 

injection reduced the EDL CK content (U/g) from 908.9±21.6 n=3 to 223.9±33.7 n=3. 

Venom plus DEXA limited this reduction to 566.2±73.8 n=3, while EP crude extract to 

712.3±44.4 n=3. The venom plus the mixture of DEXA+EP to 850.4±48.6 n=3, (U/g). 

These in vivo observations were confirmed by light microscopy examination. After 90 

min. of EDL in vitro exposure to B. jararacussu venom an increase of the rate of CK 

release from 1.06±0.09 to 37.5±3.5 U.g-1.h-1 n=7 was observed. Venom plus DEXA alone 

(39.7±0.4 n=3) did not reduce the venom effect, while EP at different concentrations, 25 

(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 

effect. These data confirm our previously observations that EP has an antimyotoxic effect 

and show that DEXA has in vivo ability to protect the muscle from B. jararacussu 

myotoxicity, but not in vitro. 

Melo, P.A. et al., (1994)Toxicon, 32, 595-603 

Supported by: FAPERJ; CAPES; CNPq; FUJB-UFRJ; PRONEX 

o Mouse EDL 

o Bothrops jararacussu venom 

o Myotoxicity, 

o Dexamethasone & Eclipta prostrata 

179

Poster 5: Improvement of mouse skeletal muscle regeneration by heparin after 

lesion induced by Bothrops jararacussu venom 

Calil-Elias, S., Tomaz, M. A., Costa, M.L., Mermelstein, C.L., Martinez;A. M. B., 

Melo, P.A. pamelo@farmaco.ufrj.br 

1 

Departamento de Farmacologia Básica e Clínica, Universidade Federal do Rio de 

Janeiro, Rio de Janeiro, RJ. Brazil 

2 

Departamento de Histologia e Embriologia, Instituto de Ciências Biomédicas, 

Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ. Brazil 

3 

Departamento de Farmácia e Administração Farmacêutica, Faculdade de Farmácia, 

Universidade Federal Fluminense, Niterói, RJ, Brazil 

We examined the effect of treatment with heparin and polyvalent antivenom on the 

regeneration of mouse Extensor digitorum longus (EDL) muscle after damage by Bothrops 

jararacussu venom. The mice received i.v. treatment, with either low molecular weight 

heparin (LMWH), regular heparin (H) or polyvalent antivenom (PAV) at 15 minutes and 4 

hours after the venom injection. Twenty-one days later, the muscles were dissected and 

processed for electron microscopy or for electrophoresis. The muscle total Creatine 

Kinase(CK) content was also evaluated 3 and 21 days after injury. The regenerated muscle 

without treatment showed a complete disorganization of myofibrils. The treatments with 

heparins induced a good regeneration, with well-organized myofibrils and recovery of the 

muscle total CK content was observed with LMWH. The electrophoresis showed a 

decrease of muscle myosin heavy chains in the muscle that received only the venom. 

However the treated muscles showed the protein bands similar to the control muscle. It is 

concluded that heparins improves muscle regeneration. 

Calil-Elias, et al., 2002, Braz J Med Biol Res. 35:1233-1235 . 

Calil-Elias et al., 2002, Histol Histopathol. 17:463-70 


o Myotoxicity 

o Muscle regeneration 

o Heparin 

180 

Poster 6: Mouse extensor digitorum longus and soleus have a different sensitivity to 

some snake venoms myotoxicity 

Tomaz, M. A 1 ., Calil-Elias, S 1,3 .,Martinez, A.M.B 2 ., Melo, P.A 1 . 

1 

Departamento de Farmacologia Básica e Clínica, Universidade Federal do Rio de Janeiro-UFRJ 21941- 

590 Rio de Janeiro RJ. Brazil 

2 

Departamento de Histologia e Embriologia, Instituto de Ciências Biomédicas, Universidade Federal do 

Rio de Janeiro, Rio de Janeiro, RJ. Brazil 

3 

Departamento de Farmácia e Administração Farmacêutica, Faculdade de Farmácia, Universidade 

Federal Fluminense, Niterói, RJ, Brazil 

Mouse extensor digitorum longus (EDL) and soleus (SOL), present distinct properties: 

EDL is a fast-twitch, white muscle with predominantly glycolotic fibers, while SOL is a 

slow-twitch, red muscle with predominantly oxidative fibers. These muscles were exposed 

to an in vitro preparation, to the venoms of Bothrops jararacussu, Agkistrodon contortrix 

laticinctus, Crotalus viridis viridis, Crotalus durissus terrificus (25 μg/ml).EDL 

presented higher increase of the rate of creatine kinase (CK) release than SOL. The basal 

CK release to the bathing solution was 0,43 ± 0,06 U.g -1 .h -1 for EDL and 0,29 ± 0,06 U.g - 

1 .h -1 for SOL. After 60 minutes of exposure to B. jararacussu venom, EDL and SOL 

presented CK release rates of, respectively, 13,2 ± 1,5 and 2,9 ± 0,7 U.g -1 .h -1 (p

Poster 7: αδ-Bungarotoxin, a new reversible long-chain neurotoxin from Malayan 

Krait (Bungarus candidus) venom with high toxicity and site-selective binding at 

the muscle nicotinic acetylcholine receptor 

Kuch, U. 1* , Molles, B.E. 2 , Bergman, T. 3 , Cederlund, E. 3 , Alvelius, G. 3 , Jörnvall, H. 3 , Chanhome, L. 4 , 

Omori-Satoh, T. 4 , Chen, Y.M. 5 , Samejima, Y. 6 , Warrell, D.A. 7 , Mebs, D. 1 

1 Zentrum der Rechtsmedizin, Klinikum der Johann Wolfgang Goethe-Universität, Kennedyallee 104, 60596 

Frankfurt am Main, Germany, *U.Kuch@em.uni-frankfurt.de 2 Unité de Récepteurs et Cognition, Département de 

Neuroscience, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris Cedex 15, France 3 Protein Analysis Center, 

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles vag 2, 17177 Stockholm, 

Sweden 4 Queen Saovabha Memorial Institute, The Thai Red Cross Society, Rama IV Road, Bangkok 10330, 

Thailand 5 Department of Pharmacology, College of Medicine, National Taiwan University, 1 Jen Ai Road, 1 st 

Section, Taipei, Taiwan 6 Institute of Medicinal Chemistry, Hoshi University, Ebara 2-4-41, Shinagawa-ku, Tokyo 

142-8501, Japan 7 University of Oxford, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, 

Oxford OX3 9DU, UK 

Introduction: Studying the binding of selective peptide toxins to the two subunit 

interfaces (α-ε/γ and α-δ) serving as ligand binding sites of the nicotinic acetylcholine 

receptor (nAChR) has offered special opportunities to distinguish features on their 

surfaces, and their ligand specificity characteristics. We report a new snake neurotoxin, αδbungarotoxin 

(αδ-BgTx), that combines binding site selectivity and reversible binding with 

great structural similarity to the classic receptor probe α-bungarotoxin (α-BgTx). 

Methods: Isolation of αδ-BgTx from the venom of Malayan Kraits (Bungarus candidus) 

from southern Thailand by gel-filtration-, cation-exchange-, and reversed-phase-HPLC; 

mass determination by SDS-PAGE and electrospray-ionization mass-spectrometry; 

complete amino acid sequence determination by Edman degradation; cloning and 

nucleotide sequence analysis of the toxin-coding region of αδ-BgTx genes; LD50 

determination in mice; functional studies in the isolated chick biventer-cervicis nervemuscle 

preparation and by competition binding with 125 I-α-BgTx using mouse muscle 

nAChRs expressed in HEK293 cells. 

Results: The 73-amino acid, 8031 Da polypeptide chain of αδ-BgTx differs in only ten 

substitutions and one deletion from the Ala 31 variant of α-BgTx. Two additional isoforms 

of αδ-BgTx were identified by genomic DNA analysis. The toxicity of αδ-BgTx (LD50 

0.17–0.28 μg/g mouse, i.p. injection) is essentially as high as that of α-BgTx. αδ-BgTx 

completely abolishes the response to acetylcholine in the isolated chick biventer-cervicis 

nerve-muscle preparation, but in contrast to the block induced by α-BgTx, acetylcholine 

response is fully reversible. In addition, αδ-BgTx distinctly prefers the α-δ over the α-ε 

binding site interface of the mouse nAChR, whereas α-BgTx shows no such selectivity. 

Discussion: αδ-BgTx is the major postsynaptic toxin of Malayan Kraits from southern 

Thailand. Ancillary anticholinesterase treatment has proved beneficial in a victim of B. 

candidus envenoming (Br Med J 1983; 286:678–80). A test dose is recommended but the 

most lethal toxins in the venom of these snakes are presynaptically-acting β-bungarotoxins. 

o Bungarus candidus 

o neurotoxin 


o Thailand 

182 

Poster 8: Novel recombinant expression system for myonecrotic Lys49 

phospholipase A2 and its mutants identifying the functional residues for necrosis 

Tomohisa Ogawa 1, * , Minae Seto 1 , Koji Muramoto 1 , Motonori Ohno 2 

1 Graduate School of Life Sciences, Tohoku University, Sendai 981-8555 Japan, 

*ogawa@biochem.tohoku.ac.jp 

2 Department of Applied Life Science, Sojo University, Kumamoto 860-0082, Japan. 

Recombinant expression systems by E. coli are the most efficient widely used system for 

recombinant proteins production. With the advent of the post-genomic era, the necessity of 

the useful systems in E. coli are increased for analyzing functions of a growing number of 

genes from different organisms including venomous animals. However, many of these 

genes, especially venomous protein genes, severely interfere with the survival of E. coli 

cells. They lead to bacteria death or cause significant defects in bacteria growth or the 

expression efficiency. Fortunately, even if the bacteria expressed the recombinant proteins 

they are inactive form such as inclusion bodies. 

In the present study, we have developed the novel recombinant expression system using 

conger eel galectin, congerin, as a tag and chaperon protein in E.coli, and applied for the 

functional expression of myotoxic Lys49 phospholipase A2 (PLA2) from Protobothrops 

flavoviridis (Pf) and its mutants. Fused recombinant protein of Pf Lys49PLA2 and 

congerin has been successfully expressed in soluble fraction with correct disulfide bond 

formation and folding. After removal digestion of tag protein by restricted protease, fully 

active recombinant Lys49PLA2 was obtained. 

Mutants of Pf Lys49PLA2, of which lysine residues at positions at 114, 118 and 119 in 

the C terminal region were substituted into alanine, were also prepared. These mutations 

decreased their necrotic activities, indicating that the C terminal basic lysine residues of Pf 

Lys49PLA2 are responsible for their necrotic activity. 

o myonecrosis 

o Phospholipase A2 

o Protobothrops flavoviridis 

o recombinant protein 

183

Poster 9: Non-cholinergic contractile activity of Dispholidus typus (Boomslang 

snake) in the rat isolated vas deferens 

Lumsden, N. G 1 , Hodgson, W. C *2 , Ventura, S 3 , Lewis, R. J 1 

1 Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia. 

2 Department of Pharmacology, Monash University, Clayton, Victoria 3800., Australia, 

* wayne.hodgson@med.monash.edu.au 

3 Department of Pharmaceutical Biology and Pharmacology, Victorian College of Pharmacy, Monash 

University, Parkville, Victoria 3052, Australia. 

Dispholidus typus venom has been reported to exhibit contractile activity in the rat 

isolated duodenum # . This effect was abolished by the muscarinic receptor antagonist 

atropine indicating cholinomimetic activity. The current study further examines the 

pharmacological activity of D. typus venom upon the rat (male Wistar 150-250 g) isolated 

vas deferens setup in 5 ml organ baths containing Krebs solution (NaCl, 118.4 mM; KCl, 

4.7 mM; MgSO4, 1.2 mM; KHPO4, 1.2 mM; CaCl2, 2.5 mM; NaHCO3, 25 mM and 

glucose, 11.1 mM). Venom (0.01-50 µg/ml) produced concentration-dependant contractile 

activity in epididymal segments. The responses were transient (i.e. not sustained) with a 

slow onset, typically reaching peak amplitude within 10 min. As responses to venom (0.8 

μg/ml) were at least 3-fold greater in the epididymal segment than in the prostatic segment, 

further experimentation was carried out using the former preparation. Suramin (0.3 mM), 

but not atropine (1-10 μM) or prazosin (10 μM), significantly attenuated the contractile 

activity of the venom (0.8 μg/ml). However, P2X receptor desensitisation by α, β-mATP 

(10 μM) was without effect on venom responses (0.8 μg/ml) indicating that the blockade 

produced by suramin was unlikely to be due to antagonism of P2X receptors. 

Unremarkable anticholinesterase activity was noted since responses to the cholinesterase 

substrate, acetylcholine (1 mM), were not significantly augmented in the presence of 

venom (0.8 μg/ml). Nifedipine (1 μM) and the South African Institute of Medical Research 

(SAIMR) Boomslang antivenom (40 μl/ml) significantly attenuated responses to venom 

(0.8 μg/ml). These results suggest activation of L-type calcium channels by D. typus 

venom that is not mediated by purinergic, adrenergic or cholinergic receptors in the rat 

isolated vas deferens epididymal segment and therefore reveal additional smooth muscle 

activity to that previously reported. 

# 

Boomslang, smooth muscle, contraction, rat vas deferens 

184 

Poster 10: Equivalent effects of snake PLA2 neurotoxins and lysophospholipid–fatty 

acid mixtures 

Rigoni, M 1 Caccin, P 1 , Gschmeissner, S 2 , Koster, G 3 , Postle, A. D 3 ,Rossetto, O 1 , Schiavo, G 2 and 

Montecucco, C 1 

1 

Department of Biomedical Sciences and Consiglio Nazionale Ricerche Institute of Neuroscience, 

University of Padova, Italy 

2 

Cancer Research UK, London Research Institute, London, UK 

3 

School of Medicine, University of Southampton, UK 

Corresponding author: cesare.montecucco@unipd.it 

Snake presynaptic phospholipase A2 neurotoxins (SPANs) paralyze the neuromuscular 

junction (NMJ): upon intoxication, the NMJ enlarges and has a reduced content of synaptic 

vesicles (1). Exposure of primary neuronal cultures to nanomolar concentrations of four 

different SPANs (beta-bungarotoxin, taipoxin, notexin and textilotoxin) induces a dosedependent 

formation of discrete bulges at various sites of neuronal projections. Neuronal 

bulging is paralleled by the redistribution of vesicle markers to the bulges and by the 

exposure of the lumenal domain of synaptotagmin I on the cell surface. Concomitantly, 

these neurotoxins induce glutamate release from cultured neurons and loss of FM 1-43 dye 

(2,3). These findings indicate that the observed phenotype results at least partially from the 

fusion of synaptic vesicles with the plasma membrane not balanced by an adequate 

membrane retrieval and closely resemble those occurring at neuromuscular junctions of 

intoxicated animals. 

The involvement of the enzymatic activity of these toxins in the NMJ blockade is still 

debated: indeed, there is only a partial correlation between PLA2 activity and neurotoxicity 

among the different SPANs and no overlap of surface residues required for neurotoxicity 

with those essential for PLA2 activity (4). We addressed this problem by comparing the 

effects of SPANs on the mouse NMJ hemidiaphragm preparation and on neurons in culture 

with those of their hydrolysis products: lysophospholipids (LysoPLs) and fatty acids 

(FAs). We found that an equimolar mixture of LysoPLs and FAs closely mimics all of the 

biological effects of SPANs (5). These results draw attention to the possible role of local 

lipid changes in synaptic vesicle release and provide new tools for the study of exocytosis. 

1. R.M. Kini, Ed., Venom Phospholipase A2 Enzymes (Wiley, Chichester, UK, 1997). 

2. M. Rigoni et al., J. Cell Sci. 15, 3561 (2004). 

3. D. Bonanomi et al., Mol. Pharmacol. 67, 1901 (2005). 

4. C.C. Yang, in Venom Phospholipase A2 Enzymes, R. M. Kini, Ed. (Wiley, Chichester, UK,1997), pp. 

185-204. 

5. M. Rigoni et al., Science 310, 1678 (2005). 

o neurotrasmission 

o PLA2 activity 

o snake neurotoxins 

o lysophospholipids and fatty acids 

185

Poster 11: EFFECT OF COLUBER RAVERGIERI VENOM ON SYNAPTIC 

PASSAGE 

Kazakov I., Abubakirova M.E. 

Institute of Zoology of Uzbek Academy of Sciences 

A. Niyazov Street 1, Tashkent 700095, Uzbekistan 

In the experiments on nerve-muscular synapses of frogs we established that the 

extract of Duvernoy , s gland (venom) of Coluber ravergieri at the concentration of 100 

µg/ml caused a decrease in the amplitude of miniature endplate potential (MEPP). The 

amount of 185 µg/ml caused a complete inhibition of MEPP. It was established that the 

venom of Coluber ravergieri did not produce a significant effect on the miniature 

potential MP of muscle tissues, the frequency of MEPP and the quantum composition of 

endplate potential (EPP). These data suggest that the venom of Coluber ravergieri acts at 

the level of post-synaptic membrane. 

To confirm this, we studied the response to the microsuperfusion. In the norm, the 

superfusion of acetylcholine (100 mM) in the synapse caused a postsynaptic response at 

the 5mB amplitude. The addition of 185 µg/ml of venom into a solution washing the 

preparation caused a gradual drop in the amplitude of responses inflicted by 

microsuperfusion of acetylcholine, which completely disappeared within 20 min. The 

washing of the preparation for 30 min with a normal Ringer solution leads to a partial 

restoration of acetylcholine responses. 

Thus, data obtained suggest that the venom of Coluber ravergieri contains a 

component, which, like neurotoxins of cobra, interacting with choline receptors of 

postsynaptic membranes blocks them, which results in the violation of synaptic passage. 

186 

Poster 12: Effect of Duvernoy's gland secretion of snake Coluber ravergieri on 

frog nerve-muscle preparation. 

Akhmedov K.J., Makhkamov B.T., R.S.Reimbaeva., M.E.Abubakirova., I.K.Kazakov 

Uzbek Academy of Science, Uzbekistan 

kdakhm@yahoo.com 

The effect of Duvernoy’s gland secretion Coluber ravergieri Menet on 

neuromuscular transmission has been examined by means of twitch tension using 

the frog nerve-muscle preparations. Freshly dried crystal of Duvernoy's gland 

secretion 10 micrograms/ml, and more during 2-4 min reduced amplitude of 

electrically induced twitch response followed by irreversible blockade. The secret 

of glands has dose and-time dependant activity, 2 micrograms/ml blocked twitch 

tension in 10-15min, after 1h potassium-evoked contractures and direct stimulation 

also have been blocked irreversibly. During experiment we do not observed 

contraction or destruction of nerve-muscle preparation fibers. Following 

fractionation on a Sephacryl S-100 gives five peaks, but we lost potent blockage 

activity of Duvernoy’s gland secretion. Only second peak with 20-30 kDa 

molecular weight has weak and slow blockage activity. By paralyzing nerve-muscle 

preparation the secret of Duvernoy’s gland blocks along from nerve to muscle all 

transmission. 

187

Poster 13: Myotoxic activity from the venoms of six species of Micrurus. 

Néstor Lago 1 ; Adolfo R. de Roodt 2 ; Claudio Mirabelli 1 ; Rodrigo D. Laskowicz 2 ; Judith Estévez 3 ; 

Víctor M. Manzaneli 2 ; Jorge Paniagua 4 ; Eduardo Scarlatto 5 . 

1 GEMA-Biotech, Bs. As., Argentina. 

2 Instituto Nacional de Producción de Biológicos – A.N.L.I.S. “Dr. Carlos G. Malbrán”, Min. De Salud y 

Ambiente, Av. Vélez Sarsfield 563, CP 1281, Bs.As., Argentina. 

3 Instituto Bioclon, Mexico DF, Mexico. 

4 Laboratorios Silanes, Mexico DF, Mexico. 

5 Servicio de Toxicología, Hospital de Clínicas “José de San Martín”, Facultad de Medicina U.B.A. 

The lethal, hemolytic, phospholipasic and miotoxic activities of the venoms from M. 

altirrostris, M. mesopotamicus, M. pyrrhocryptus (the three species from Argentina), M. 

surinamensis (Colombia), M. nigrocinctus (Costa Rica) and M. fulvius (United States) 

were studied. The direct and indirect hemolytic activity was studied in a liquid system 

containing red blood cells with (indirect activity) or without (direct activity) egg yolk. The 

phospholipase activity was studied by radial hydrolysis of egg yolk in agarose plates. The 

miotoxicity was studied by the intramuscular injection of the venom in Whistar rats (250 

g). The levels of creatin quinase (CK) were recorded 2, 6 and 24 h after injection. Samples 

of muscles and kidneys were taken at 24 h post inoculation for pathological studies. None 

of the venoms showed direct hemolytic activity in the conditions of the study. The venoms 

of M. nigrocinctus and M. fulvius showed the highest indirect hemolytic activity, and the 

lower activity was detected in M. mesopotamicus and M. surinamensis venoms. All 

venoms showed high phospholipase activity, with the exception of the venom of M. 

surinamensis, which only showed hydrolysis at high concentrations. All the venoms 

caused elevation of the CK levels, being the highest level detected 6 hours after venom 

inoculation and descending by 24 h, but maintaining the level above that of the controls (p 

< 0.05); M. nigrocinctus, M. fulvius and M. pyrrhocryptus venoms showed the highest 

activity, whereas M. surinamensis and M. altirrostris showed the lowest activity. 

Histological analysis of muscles showed severe myofiber necrosis in rats injected with M. 

fulvius and M. nigrocintus venoms, and an important myonecrosis in those injected with 

M. pyrrhocryptus venom; the lesions caused by the other venoms were of minor 

importance or absent. The kidneys showed extensive tubular necrosis with protein deposits 

in rats injected with M. fulvius and M. nigrocinctus venoms. The other venoms produced 

isolated mild renal lesions, inclusive those with higher lethal doses. The envenomation by 

M. fulvius and M. nigrocinctus venoms caused macroscopic myoglobinuria in CF-1 mice 

(18-20 g). 

o Micrurus 

o Venom 

o Myotoxicity 

o nephrotoxicity 

188 

Poster 14: Development of an in vivo model for the study of snake venom 

myotoxicity 

Andrew J. Hart 1 , Geoffrey K. Isbister 1,2 , Sharmaine Ramasamy 1 , Wayne C. Hodgson 1 

1 Monash Venom Group, Department of Pharmacology, Monash University, Clayton, Australia, 

2 Topical Toxinology Unit, Menzies School of Health Research, Darwin, Northern Territory, Australia 

Introduction: Myotoxicity is a serious clinical manifestation of snake envenoming and 

venoms with myotoxic activity occur in snakes from most parts of the world. Currently, 

there is no standard in vivo technique to investigate myotoxicity and its outcomes. 

Development of such a model will lead to further understanding of the pathophysiology of 

myotoxic envenoming and allow assessment of therapy. This study aimed to develop a 

suitable model. The whole venom and an isolated myotoxin (i.e. mulgatoxin) from the 

Australian Mulga snake (Pseudechis australis) were used as the test compounds due to well 

documented myotoxic activity (Chen et al., 1994; Ponraj & Gopalakrishnakone, 1995; 

Ramasamy et al., 2004). 

Method: Rats were anaesthetised with pentobarbitone sodium (60-100 mg/kg, i.p.). Blood 

pressure was monitored via the carotid artery, and blood samples (0.5 ml) taken via the 

jugular vein and collected in MiniCollect LH/Gel separation tubes. A blood sample was 

taken prior to venom/toxin administration and then every 2 h for a period of 8 h. after 

venom/toxin administration (i.e. a total of 5 samples per rat). Samples were tested for 

serum electrolytes (i.e. sodium, potassium, chloride and bicarbonate levels) and creatine 

kinase (CK) concentration. 

Results: P. australis venom (20 µg/kg, i.v., n=6) or mulgatoxin (30 µg/kg, i.v., n=4) both 

produced increases in CK concentrations, indicative of myotoxicity, which were 

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 

Borja-Oliveira C.R. 1,2 , Rodrigues-Simioni L. 2 , Spisni A. 1,3* 

1 

Center for Structural Molecular Biology, Brazilian Synchrotron Light Laboratory-LNLS, Campinas-SP, 

Brazil, 

2 

Department of Pharmacology, State University of Campinas (UNICAMP), Cidade Universitária Zeferino 

Vaz, Campinas, SP, Brazil 

3 

Department of Experimental Medicine, University of Parma, Italy (permanent address), 

*alberto.spisni@unipr.it. 

Toxins that block voltage-dependent K + channels, such as TsTX-K-alpha (1), and those 

that modify Na + channel gating, such as crotamine (2) have inotropic effects in skeletal 

muscle. In order to screen the presence of some inotropic activity in crude venoms and to 

investigate the role of voltage-dependent Na + and K + channels in inotropism, we studied 

the neuromuscular effects of T. cambridgei and T. serrulatus venom, using isolated mouse 

phrenic nerve-diaphragm and chick biventer cervicis. A significantly different 

strengthening of indirect elicited twitches in phrenic nerve-diaphragm of 90 ± 8 % and 

254.5 ± 7.6 % is induced by T. cambridgei venom, 5 μg/ml, and T. serrulatus venom, 0.5 

μg/ml, respectively. In the case of T. serrulatus venom this effect is prevented by the Na + 

channel blocker tetrodotoxin (TTX), 30 nM, and attenuated by the K + channel openers 

cromakalim (CRO), 200 µM and diclofenac (DIC), 5 μg/ml, while for T. cambridgei 

venom the effect is inhibited only by TTX and CRO. From the studied venoms, only T. 

cambridgei venom, 10 μg/ml, potentiated the direct evoked twitches of curarized 

preparations (d-tubocurarine 7.3 µM): this effect being prevented by TTX. When tested on 

biventer cervicis, T. serrulatus venom, 1 μg/ml, potentiated (26.0 ± 8.4 %), both the 

indirectly elicited twitches and the response to acetylcholine and KCl, after 90 min, while 

T. cambridgei venom, 10 μg/ml, partially inhibited the response to KCl. Overall, these 

results suggest that neuromuscular effects are associated to modifications of TTX-sensitive 

Na + channel gating, although blocking of K + channel may be involved as well. 

Noteworthy, while T. serrulatus venom seems to improve muscle strength by interacting 

mainly with nerve ion channels, T. cambridgei venom appears to act directly on skeletal 

muscle ion channels thus suggesting it contains active factors potentially relevant for 

clinical applications. Partially support by CNPq. 

1. van Lunteren E, Moyer M. (1999). J. Appl. Phisiol, 86, 1009-16. 

2. Toyama MH, Marangoni S, Novello JC, Leite GB, Prado-Franceschi J, Cruz-Hofling 

MA, Rodrigues-Simioni L (2003). Toxicon, 41, 493-500. 

o Phrenic nerve-diaphragm preparation 

o Chick biventer cervicis preparation 

o Neuromuscular transmission 

o Skeletal muscle force 

190 

Poster 16: Neutralization of the neurotoxicity of Micrurus altirostris (Uruguayan 

coral snake) venom by coral snake antivenom 

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* 

1 


(UNICAMP), Campinas, SP, Brazil, *simioni@unicamp.br 

2 

Laboratório de Herpetologia, Instituto Butantan, Avenida Vital Brazil, 1500, São Paulo, SP, Brazil. 

Coral snake (Micrurus) venoms have a high lethality mediated by pre- and postsynaptic 

neurotoxins that can be neutralized by antivenom. However, the lethality of Micrurus 

altirostris venom is not neutralized by commercial antivenom (Instituto Butantan) raised 

against M. corallinus and M. frontalis venoms [4]. In this work, we examined the 

neuromuscular activity of M. altirostris venom in vitro and assessed the ability of 

antivenom to neutralize the neurotoxicity in vitro and in vivo. The neurotoxicity of M. 

altirostris venom (0.1-10 μg/ml) and its neutralization by antivenom were assayed using 

indirectly stimulated chick biventer cervicis [1] and mouse phrenic nerve-diaphragm [2] 

preparations. Neutralization in vivo was tested in chicks (LD50 0.042 mg/kg) and mice 

(LD50 0.255 mg/kg [4]) injected i.m. and i.p., respectively, with venom (5 LD50): 

antivenom mixtures (n=6 animals/group). The venom:antivenom ratios used in all assays 

were 1:1, 1:2.5, 1:5, 1:10 and 1:20, assuming that 1 ml of antivenom neutralizes 1.5 mg of 

Micrurus spp. venom (antivenom package insert). M. altirostris venom produced total 

neuromuscular blockade in both nerve-muscle preparations at all concentrations tested. 

The blockade was not reversed by washing or by neostigmine. In chick preparations, the 

venom totally blocked the contracture to exogenous ACh without affecting the response to 

KCl. Antivenom failed to neutralize the neuromuscular blockade in the proportion 

recommended by the manufacturer. Only the venom:antivenom ratio of 1:20 provided total 

neutralization of the neuromuscular blockade in vitro and protected chicks and mice 

against 5 LD50 of venom. M. altirostris venom is highly neurotoxic and irreversibly blocks 

neuromuscular transmission. Commercial antivenom showed low efficacy in neutralizing 

this neurotoxicity. Hence, there may be a need for more specific antivenoms to neutralize 

the activities of Brazilian coral snake species other than M. corallinus and M. frontalis [3]. 

1. Bülbring E (1946) Br. J. Pharmacol. 1, 38-61. 2. Ginsborg BL, Warriner J (1960) Br. J. 

Pharmacol. 15, 410-411. 3. Higashi HG et al. (1995) Braz. J. Med. Biol. Res. 28, 767-771. 

4. Moraes FV et al. (2003) Toxicon 41, 71-79. Supported by: CNPq. 

o chick biventer cervicis 

o lethality 

o mouse phrenic nerve-diaphragm 

o neuromuscular junction 

191

Poster 17: Peptide inhibitors of sPLA2 do not prevent the neuromuscular blocking 

actions of β-bungarotoxin in vitro 

Harvey, A.L. 1 , Young, L.C. 1 , Gopalakrishnakone, P. 2 , Thwin, M.M 2 

1 Department of Physiology and Pharmacology, University of Strathclyde, Glasgow G4 0NR, UK, and 

2 Venom & Toxin Research Programme, Department of Anatomy, Yong Loo Lin School 

of Medicine, National University of Singapore, Singapore117597 

The objective of this study was to determine whether phospholipase A2 (sPLA2-IIA) 

inhibitory peptides, derived from the primary structure of an endogenous protein termed 

“Phospholipase Inhibitor from Python (PIP)” could prevent the neuromuscular paralysis 

induced by β-bungarotoxin. Experiments were conducted on the chick biventer cervicis 

isolated nerve-muscle preparation. Two types of experiments were performed: 1. peptides 

were premixed with β-bungarotoxin before adding to the preparations, and 2. peptides 

were tested in experiments where they were added to the preparations 30 min before the 

toxin. 

In premixing experiments, a mixture containing 5 μM of each peptide with 25 nM βbungarotoxin 

was made and kept at room temperature for 30 min before adding to 

indirectly stimulated chick biventer cervicis preparations. Eight experimental preparations 

were run simultaneously with four time-matched preparations that received toxin only. 

There were no significant differences in time to block in any of the experimental sets 

compared with controls with toxin. 

In preparations that had been incubated with peptides before adding toxin, there was no 

effect of any peptide on the blocking action of β-bungarotoxin. 

Contractures to exogenously added acetylcholine were greater in the presence of some of 

the peptides. This could be explained by an anticholinesterase effect, and this was 

confirmed by biochemical assays. 

o secretory phospholipase A2 inhibitor 

o β-bungarotoxin 

o neuromuscular 

o acetylcholinesterase 

192 

Poster 18: Gambierol markedly enhances evoked quantal acetylcholine release 

from motor nerve terminals at vertebrate skeletal neuromuscular junctions 

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 

1 

CNRS, Institut de Neurobiologie Alfred Fessard – FRC2118, Laboratoire de Neurobiologie 

Cellulaire et Moléculaire – UPR9040, Gif sur Yvette, France, *girard@nbcm.cnrs-gif.fr 

2 

Graduate School of Life Sciences, Tohoku University, 1-1 Tsutsumidoori-Amamiya, Aoba-ku, Sendai, 

Japan 

3 Departamento de Farmacologia, Facultad de Veterianaria. Universidad de Santiago de Compostela. 

Campus de Lugo, Lugo, Spain 

Gambierol is a marine polyether toxin isolated with ciguatoxin congeners from cultures of 

the toxic dinoflagellate Gambierdiscus toxicus. To date, two total syntheses of gambierol 

have been accomplished. Gambierol has shown toxicity against mice with symptoms 

resembling those of the ciguatoxins (CTXs), inferring the possibility that gambierol is 

involved in ciguatera, a worldwide-spread food poisoning caused by the consumption of 

fish contaminated with toxins produced by G. toxicus. Our previous observations suggest 

that CTXs induce neurological disturbances by acting at low concentrations on voltagegated 

Na + channels, and at higher concentrations on voltage-dependent K + channels in 

excitable tissues. The aim of the present study was to explore the effects of gambierol on 

isolated frog cutaneous pectoris and mouse phrenic-hemidiaphragm nerve-muscle 

preparations, using conventional electrophysiological techniques. Gambierol in the 

nanomolar range greatly increased the quantal content of endplate potentials (EPPs) in 

isolated neuromuscular preparations equilibrated in a low-Ca 2+ -high Mg 2+ medium. In 

preparations in which gambierol produced a 120-fold increase in evoked quantal 

acetylcholine (ACh) release, no change was detected in the muscle membrane potential. 

Spontaneous quantal ACh release recorded as miniature endplate potential frequency 

remained unaffected by sub-micromolar concentrations of gambierol. Gambierol (100 nM- 

1 μM) evoked asynchronous nerve action potentials that elicited EPPs which in turn 

triggered muscle action potentials and muscle contractions. In the light of the data 

obtained, gambierol is suggested to block K + channels in motor nerve terminals allowing a 

greater phasic Ca 2+ influx into the endings. This greater Ca 2+ influx during the presynaptic 

action potential is responsible for the increase in evoked quantal ACh release. In 

conclusion, our data show for the first time that gambierol is a potent modulator of evoked 

ACh release and that its actions at the neuromuscular junction differ from those previously 

reported with the CTXs. Gambierol appears to be about 1,000 times more active than 3,4diaminopyridine, 

a well known blocker of K + channels in motor nerve terminals. 

o Gambierol 

o acetylcholine release 

o nerve terminals 

o ciguatoxins 

193

Poster 19: Neutralization of neuromuscular activity of bothropstoxin-I (BthTX-I) 

by a methanolic fraction from Casearia sylvestris Sw. 

Francischinelli, M. C 1 , Gerenutti, M 1 , Silva, M. G 1 , Andréo-Filho, N 1 , Oliveira, S. J 1 , Leite, G. B 3 , Dal 

Belo, C. A 3 , Cintra, A. C. O 4 , Cruz-Höfling, M. A 2 , Rodrigues-Simioni, L 3 , Oshima-Franco, Y 1,3* 

1 University of Sorocaba, Sorocaba, SP, Brazil, *yofranco@terra.com.br 

2 Dept .of Histology and Embryology, IB, and 3 Dept. Pharmacology, Faculty of Medical Sciences, State 

University of Campinas, (UNICAMP), SP, Brazil, 

4 Faculty of Medicine, University of São Paulo, Ribeirão Preto, SP, Brazil. 

Introduction: Bothropstoxin-I (BthTX-I), a PLA2 myotoxin from Bothrops jararacussu 

snake venom, shows neurotoxic activity in vitro, that is prevented by a hydroalcoholic 

extract of Casearia sylvestris Sw. (CS). Since antivenom therapy is efficient against the 

systemic, but not the local actions of B. jararacussu venom, we examined the ability of a 

methanolic fraction (MF) of CS to neutralize the myotoxicity of BthTX-I in mouse phrenic 

nerve-diaphragm preparations (PND). 

Methods: A methanolic fraction of CS leaves was obtained by the Soxhlet procedure and 

its constituents were visualized by thin layer chromatography. Myographic and histological 

analyses were done in PND incubated with BthTX-I (40 μg/mL), a mixture of BthTX-I + 

MF (0.2 mg/mL), MF alone (0.2 mg/mL) or Tyrode solution (control) for 120 min. 

Student’s t-test was used for statistical analyses with p

Poster 21: Effects of Bothrops marajoensis venom on the mouse and chick nervemuscle 

preparations 

Cavalcante, W.L.G 1 , Hernandez-Oliveira, S 1 , Leite, G.B 1 , Ponce-Soto, L.A 2 , Marangoni, S 2 , Rodrigues- 

Simioni, L. 1* 

1 

University of Campinas, UNICAMP, Department of Pharmacology, 13084-971,Campinas, Brazil, 

*simioni@unicamp.br 

2 

University of Campinas, UNICAMP, Department of Biology, 13083-970, Campinas, Brazil 

Introduction: The venoms of some Bothrops species produce neuromuscular blockade in 

avian and mammalian nerve-muscle preparations in vitro. In this study, we investigated the 

neuromuscular activities (neurotoxicity and myotoxicity) of Bothrops marajoensis venom 

(Bmj) in mouse phrenic nerve-diaphragm muscle (PNDp) and chick biventer cervicis 

muscle preparations (BCp). 

Methods: The preparations were mounted in a 5 ml organ bath (PNDp - Tyrod solution; 

BCp - Krebs solution), aerated (95% O2 and 5% CO2) at 37 ºC for indirectly stimulation. 

Myotoxicity was assessed by light microscopic analysis. Data (mean ± S.E.M.; n=4-6) 

were analyzed by ANOVA (p

Poster 23: The twitch potentiation induced by crotamine homologs from Crotalus 

durissus ruruima and C. d. terrificus venoms 

Hernandez-Oliveira S.S., Silva S.O., Borja-Oliveira C.R., Hyslop S., Marangoni S., Rodrigues-Simioni 

L.* 

State University of Campinas (UNICAMP), Campinas, SP, Brazil, *simioni@unicamp.br 

Several subspecies of the tropical rattlesnake, Crotalus durissus, occur in Brazil. In this 

work, we compared the neuromuscular activities of Crotalus durissus terrificus, C. d. 

ruruima, C. d. cascavella, and C. d. collilineatus venoms and their respective crotoxin 

(CrTX) homologs, besides crotamine (CrTM) homologs from C. d. terrificus and C. d. 

ruruima (crotamine-positive venoms), in indirectly stimulated mouse phrenic nervediaphragm 

and chick biventer cervicis preparations. CrTX and CrTM were isolated from 

C. d. ruruima venom by chromatography on Sephadex G-75 followed by reverse-phase 

HPLC. All of the venoms and their crotoxin homologs (10 μg/ml each) caused complete 

neuromuscular blockade within 120 min in both preparations. However, only C. d. 

terrificus and C. d. ruruima venoms (10 μg/ml each) produced an initial increase in twitchtension 

(187 ± 40% and 167 ± 29%, respectively; mean+S.E.M., n=7 and 6, respectively) 

in mouse preparations. Crotamine alone (10 μg/ml) from C. d. terrificus and C. d. ruruima 

venoms caused an initial facilitation (167 ± 38%, n=8, and 163± 23%, n=11, respectively, 

in mouse preparations, and 37 ± 12% and 21 ± 7%, respectively, in chick preparations, n=5 

each). The facilitation were not significantly different when crotoxin was added with 

crotamine. In mouse and chick preparations, crotoxin from the four subspecies caused 

complete and irreversible neuromuscular blockade within 60-70 min and 15-30 min, 

respectively. None of the crude venoms or crotoxin and crotamine homologs inhibited 

ACh- or KCl-induced contractures in chick preparations. Since C. d. terrificus and C. d. 

ruruima are the only crotamine-positive venoms within the studied venoms, we suggest 

that the pronounced and initial facilitatory effect of these venoms is induced by crotamine. 

In addition, these results show that crotoxin homologs from studied venoms and crotamine 

homologs from C. d. terrificus and C. d. ruruima have very similar neuromuscular effects. 

We conclude that the twitch potentiation followed by neuromusculau blockade caused by 

the crude venoms studied, was due the presence of crotamine. 

Supported by CNPq. 

o Crotalus durissus cascavella 

o Crotalus durissus collilineatus 

o Crotoxin 

o Nerve-muscle preparations 

198 

Poster 24: Purification and Pharmacological Characterization of BtII-2 a Novel Presynaptic 

PLA2 Isolated From Bothrops alternatus Venom 

Ponce-Soto, L.A 1 ; Barros, JC 2 ; Hernadez-Oliveira, S 2 , Novello, JC 1 , Dal Belo, CA 2 ; Marangoni, S 1 .; Rodrigues-Simioni, 

L 2* . 

1 

State University of Campinas, UNICAMP, Department of Biology, 13083-970, Campinas-SP, Brazil. 

2 

State University of Campinas, UNICAMP, Department of Pharmacology, 13084-971, Campinas-SP, 

Brazil.*simioni@unicamp.br 

Introduction: BtII-2 is a novel presynaptic PLA2 (K49) isolated from Bothrops alternatus 

snake venom. 

Methods: Toxin was purified using reverse phase HPLC and its molecular mass was 

estimated by SDS-PAGE and MALDI-TOF mass spectrometry. BtII-2 was assayed at 

mouse hemidiaphragm preparations (PND) and at chick biventer cervicis preparations 

(CBC) mounted as described by Cogo et al. (1993). Quantal content of end-plate potentials 

(QC) was determined according to Dal Belo et al. (2005) using mouse hemidiaphragm 

preparations. 

Results: BtII-2 has a molecular mass of 13898.7121 Da. The first eight residues of the N-terminal 

sequence were SLFELGKMILQETGKNPAKS YGAYYCYC… sharing high homo- 

logy with other Lys49 PLA2. The high content of Lys, Tyr, Gly, Pro and 14 half-Cys resi- 

dues indicated to be a basic PLA2. At (CBC) BtII-2 (0.1-20 µg/ml) induced an irreversible 

and concentration time-dependent blockade of neurotransmission (n=6, p

Poster 25: Antiophidian Activity of Galactia glaucescens Ethanolic Extract (GGE) 

Against Crotalic and Bothropic Venoms 

Colares, AV 4 ; Oshima-Franco, Y 1 ; Corrado, AP 2 ; Ticli, FK 3 ; Sampaio, SV 3 ; Cintra, ACO 3 ; Rodrigues- 

Simioni, L 1 .;dos Santos, MG 4 ; Dal Belo, CA 1,4,5* . 

1 State University of Campinas, UNICAMP, Department of Pharmacology, 13084-971, Campinas-SP, 

Brazil. 

2 University of São Paulo, USP, Department of Pharmacology, Avenida Bandeirantes 3900, 14049-900, 

Ribeirão Preto-SP, Brazil. 

3 University of São Paulo, USP, Department of Clinical and Bromatological Analysis, Avenida do Café s/n, 

14040-903, Ribeirão Preto-SP, Brazil. 

4 Federal University of Tocantins, UFT, PGCIAMB, Avenida NS 15, ALCNO 14, 77020-120, Palmas-TO, 

Brazil. *chariston@uft.edu.br 

5 Instituto de Ensino Superior de Porto Nacional, IESPEN, Rua Antônio Ayres Primo 2071, 77500-000, 

Porto Nacional, Tocantins, Brazil. 

Introduction: A crude GGE was tested on neurotoxicity, oedema and hemorrhagic 

activities induced by crotalic and bothropic venoms in mouse preparations. 

Methods: All protocols were performed using male Swiss white mice (18-22g). To the 

neurotoxicity assays mice were anaesthetized (chloral hydrate 300 mg/kg, i.p.) and 

sacrificed by exsanguination. Phrenic nerve-hemidiaphragm preparations (PND) were 

mounted as described by Bülbring (1946) for rats. Oedema inhibition studies were 

performed after i.d. injection in the right foot pad (Soares et al., 2000a) by incubating a 

PLA2 Bothropstoxin I (BthTX I) from Bothrops jararacussu venom with GGE. Control 

groups received an injection of PBS (50µl), pH7.2, or GGE alone. The progression of 

oedema was evaluated with a low-pressure pachymeter (Mitutoyo, Japan) at various time 

intervals. Hemorrhagic activity was measure according Kondo et al. (1960). 

Results: GGE (10 µg/25ml, n=5) inhibited by 50% oedema formation induced by BthTX I 

(1µg/25ml, p

Poster 27: Expression and Mutagenesis of the Sea Anemone Toxin Av2 Reveals 

Key Amino Acid Residues Important for Activity on Voltage-Gated Sodium 

Channels 

Moran Y*, Cohen L, Kahn R, Karbat I, Gordon D, Gurevitz M 

Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, 

Israel, *moranyeh@post.tau.ac.il 

Type I sea anemone toxins are highly potent modulators of voltage-gated Na-channels 

(Navs) and compete with the structurally dissimilar scorpion alpha-toxins on binding to 

receptor site-3. While these features provide two structurally different probes for studying 

this receptor site and channel fast inactivation, the bioactive surface of sea anemone toxins 

has not been fully resolved. We established an efficient expression system for Av2 (known 

as ATX II), a highly insecticidal sea anemone toxin from Anemonia viridis (named 

previously A. sulcata), and mutagenized it throughout. Each toxin mutant was analyzed in 

toxicity and binding assays, as well as by circular dichroism spectroscopy to discern 

effects derived from structural perturbation from those related to bioactivity. Six residues 

were found to constitute the anti-insect bioactive surface of Av2 (Val2, Leu5, Asn16, 

Leu18, Ile41). Further analysis of nine Av2 mutants on the human heart channel Nav1.5 

expressed in Xenopus oocytes indicated that the bioactive surfaces toward insects and 

mammals practically coincide, but differ from the bioactive surface of a structurally similar 

sea anemone toxin, Anthopleurin B, from Anthopleura xanthogrammica (1). Hence, not 

only that our results demonstrate clear differences in the bioactive surfaces of Av2 and 

scorpion alpha-toxins, they also indicate that despite the general conservation in structure 

and importance of the Arg14 loop and its flanking residues Gly10 and Gly20 for function, 

the surface of interaction between different sea anemone toxins and Navs varies (2). 

References 

1. Seibert AL, Liu J, Hanck DA & Blumenthal KM (2004) Biochemistry 43, 7082-7089 

2. Moran Y, Cohen L, Kahn R, Karbat I, Gordon D & Gurevitz M (2006) Biochemistry, In 

press. 

o Sea anemone toxin 


o Molecular dissection 

o High insecticidal activity 

202 

Poster 28: Characterization of Voltage-dependent Calcium Channel Blocker 

Peptides Isolated from Tarantula Venom 

Ono, S., Kimura T., Kubo T.* 

National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, 

Ibaraki 305-8566, Japan, *tai.kubo@aist.go.jp 

Introduction 

Voltage-dependent ion channels control transmembrane ion flux via electrical excitability. 

Some of the neurological and cardiac diseases, such as epilepsy, arrhythmia and 

hypertension, are caused by dysfunction of ion channels. Toxins that recognize ion 

channel subtypes are versatile tools for channel studies, and thus contribute to drug 

discovery. Spider venoms contain arthropod-specific neurotoxins and are expected to be a 

rich source of ion channel blockers. In this study, we screened the tarantula venom to 

identify calcium channel blocker peptides that specifically recognize a variety of the 

channel subfamilies. 

Methods 

Crude venom was collected from the venom glands of tarantula Grammostola spatulata. 

The supernatant was separated on a C18 reversed-phase liquid chromatography. Voltagedependent 

calcium channels, Cav1.2, Cav2.1, Cav2.2, Cav2.3 and Cav3.1, were expressed in 

Xenopus oocytes and current modulations by the separated fractions were measured in twoelectrode 

voltage clump configuration. Purified active peptides were sequenced by Edman 

degradation after alkylation. 

Results 

Three peptides were identified that inhibit different types of calcium channel. One of the 

peptides (35 amino acids) suppressed the Cav2.1 (P/Q-type) current. The sequence is 

identical with that of omega-GsTx SIA except valine residue in the C-terminus. The 

second peptide (known as GsAFII) with 30 amino acid residues blocked L-type Ca 2+ 

channel (Cav1.2). We also isolated a novel peptide, which specifically targets the T-type 

Ca 2+ channel (Cav3.1). The peptide has sequence homology with sodium channel blocker 

peptides. These spider venom peptides so far identified have different structural features 

with different cysteine motifs, as characterized as the Inhibitory Cystine Knot structure. 

o Voltage-dependent calcium channel 

o Spider venom 

o Channel blocker 

o Peptide 

203

Poster 29: Recombinant expression of a four disulfide-bridged scorpion toxin 

antagonist of voltage-gated sodium channels† 

Estrada, G., García, B.I., Ortiz, E.,Riano, L., Becerril, B., Possani, L.D., Corzo, G. 

Institute of Biotechnology-UNAM, Av. Universidad 2001, Cuernavaca, Morelos, 62210, Mexico. 

*corzo@ibt.unam.mx 

Css2 is a neurotoxin that affects voltage-gated sodium channels. Css2 is the major toxic 

component of the venom of the scorpion Centruroides suffusus suffusus (1). The gene of 

Css2 was cloned into the expression vector, pQE-30 containing a 6His-tag and an Fxa 

enzymatic cleavage site. The pQE-30 vector was transformed into E. coli BL21 cells, and 

the expression was induced with isopropyl thiogalactoside (IPTG). The level of expression 

was 24 mg/L of culture medium, and the His tagged recombinant toxin (hrCss2) was found 

only in inclusion bodies with no evidence of in the soluble fraction. The recombinant Css2 

was extracted from inclusion bodies using a 0.2 M Tris buffer pH 8.0 solution, containing 

6 M Gnd-HCl. The hrCss2 peptide was purified by affinity and hydrophobic interaction 

chromatography. The reverse-phase HPLC profile, and the single molecular mass of 

9,392.2 Da in several protein fractions indicated that the expressed hrCss2 had multiple 

disulfide bridge arrangements. All hrCss2 structural forms were reduced using 0.2 M Tris 

buffer pH 8.0 containing 50 mM DTT to obtain a single protein fraction with a molecular 

mass of 9,400.2. An in vitro folding process generate a single hrCss2 form with the 

expected molecular mass of 9,392.2 Da. After an enzymatic cleavage with Fxa, the 

recombinant Css2 (rCss2) was obtained. The molecular mass of rCss2 was 7,538.6 Da as 

expected. Because of the natural C-terminus amidation of Css2, they both Css2 and rCss2 

differ each other in 1 Da. Nevertheless, both Css2 and rCss2 had similar LD50s in mice 

when injected intracranially. Also, the recombinant Css2 was recognized by antibodies 

raise against Css2. 

1. Martin, M..F. et al. (1987) J Biol Chem. 262:4452-4459. 

†This work was supported in part by a grant from Laboratorios Silanes S.A. de C.V. and 

Institute Bioclón S.A. de C.V., and by a grant from the Dirección General de Asuntos del 

Personal Académico (DGAPA-UNAM) IN226006. 

o Antimicrobial 

o Pore-forming 

o Scorpion 

o Recombinant 

204 

Poster 30: Structure and function of huwentoxin- , a novel spider toxin 

targeting both serine proteinase and potassium channels 

Chunhua Yuan , Kuan Peng, Jianbo Diao and Songping Liang 

College of Life Sciences, Hunan Normal University,Changsha, Hunan 410081, China 

A novel peptide, denoted Huwentoxin- (HWTX- ), which inhibits serine proteinase 

such as trypsin and chymotrypsin and also behaves as a blocker of voltage-sensitive K + 

channels, has been isolated from the venom of Chinese bird spider Ornithoctonus huwena 

Wang. HWTX- is a 55 amino acid peptide cross-linked with three disulfide bridges. 

The full-length cDNA of HWTX- was isolated by the method of rapid amplification of 

cDNA ends. Its dissociation constant for trypsin is about 1×10 -12 M measured by a 

BIAcore binding assay system. Whole-cell configuration patch clamp recording indicated 

that HWTX- showed no effects on voltage-gated sodium channels or calcium channels 

in dorsal root ganglion neurons, whereas it significantly inhibited outward delay-rectified 

potassium currents in rat dorsal root ganglion neurons. This was confirmed by testing its 

effect on Kv1.1, Kv1.2 and Kv1.3 channels expressed in Xenopus oocytes. The solution 

structure of HWTX-XI was obtained by heteronuclear multidimensional NMR 

experiments. The structure of HWTX-XI adopts the same structural motif as that of 

BPTI,a trypsin inhibitor from bovine, and α-DTX, a potassium channel inhibitor from 

snake. The 3D structure of HWTX-XI shows some distinct differences compared with 

that of BPTI and α-DTX, which might be related to the dual inhibitory activity of the 

toxin. 

205

Poster 31: A novel family of conotoxins target to nACh-Receptors** 

Kauferstein, S. 1 , Nicke, A. 2 , Kendel, Y. 1 ., Zamudio, F.Z. 3 , Stöcklin, R. 4 , Mebs, D. 1 

1University of Frankfurt, Kennedyallee 104, D-60596 Frankfurt am Main, Germany, 

kauferstein@em.uni-frankfurt.de 

2 Max-Planck-Institute for Brain Research, D-60528 Frankfurt am Main, Germany 

3 Instituto de Biotecnologia-UNAM, Avenida Universidad, 2001, Cuernavaca 62210, Mexico 

4 Atheris Laboratories, CH-1233 Bernex-Geneva, Switzerland 

Introduction: Over the last 50 million years, cone snails developed a highly efficient 

venomous system.Using their complex venom apparatus they inject a mixture of peptides, 

which almost instantly paralyzes the prey. These peptides are consisting mainly of 11 to 35 

amino acid residues with a variable number of disulfide bridges, exhibiting a great 

variability in their amino acid sequence. The venom of cone snails is one of the most 

complex peptide mixtures acting on a wide range of target tissues in the nervous system, 

e.g. ion channels and receptors. The high affinity of these toxins to these targets made 

them indispensable tools in neurochemistry and neurophysiology. 

Methods: Crude venom samples from Conus capitaneus were tested on nAChRs expressed 

in oocytes using the two-electrode voltage-clamp method. Fractionation of the samples 

was performed by HPLC followed by liquid chromatography-electrospray-mass 

spectrometry analysis. N-terminal amino acid sequence analysis was performed by 

automated Edman degradation. 

Results: From the venom of Conus capitaneus a protein was isolated which represents a 

new family of conotoxins acting at nicotinic AChRs. It has a remarkably high molecular 

weight of 11 kDa and, as SDS-PAGE revealed the toxin is a dimer. Its N-terminal 

sequence exhibits no similarity to any known proteins. Analysis of the subtype selectivity 

of the purified toxin on nAChRs revealed subnanomolar potency on α7 and nanomolar 

potency on� α3β2 nAChRs subtypes with IC50 values of 300 pM and 3 nM respectively. 

The IC50 value for the �α4β2 subtype was 30 nM, making it the most potent conotoxin 

acting on this AchR-subtype known so far. 

Discussion/Conclusion: We suggest that some cone snail species, besides families of small 

peptides, developed an alternative class of large conopeptides to target the nAChR. 

o Conotoxins 

o nAChR 

o Cone snails 

o Voltage clamp 

**this presentation will be given as an oral communication on Tuesday at 4.40pm 

in K3.25 

206 

Poster 32: Isolation and characterisation of a potent novel tarantula toxin 

targeting the acid sensing ion channel ASIC1a. 

Rash LD 1 , Adams DJ 2 and Alewood PF 1 . 

1 

Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland 4072 

l.rash@imb.uk.edu.au 

2 

School of Biomedical Sciences, University of Queensland, St Lucia, Queensland 4072. 

Changes in local tissue pH can occur in many physiological and pathophysiological 

circumstances, most notably in ischaemia and inflammation. Acid sensing ion channels 

(ASICs) are a recently discovered group of non voltage-gated sodium channels belonging 

to the epithelial / degenerin Na channel family (ENaC/DEG). These channels are gated by 

protons and widely distributed throughout both the central and peripheral nervous systems. 

There are 4 ASIC genes encoding 6 proteins (1a, 1b, 2a, 2b, 3 and 4) with ASIC1a, 1b, 2a 

and 3 being able to form functional tetrameric channels on their own (1). ASIC1a has been 

shown to be involved in central processes such as memory, learning and synaptic plasticity 

(2), neuronal excitotoxicity such as in stroke (3). Due to their ability to sense pH change 

they are also implicated in pain transduction. At present there are very few specific 

pharmacological tools with which to study the structure, function and role of these 

channels. Two toxins have been reported to date. The tarantula toxin PcTx1 selectively 

blocks ASIC1a channels (4) while the sea anemone peptide APETX2 blocks ASIC3 and 

several ASIC3 containing heteromers (5) however both of these toxins are readily 

reversible. Here we report the isolation and characterisation of a novel tarantula toxin 

which causes a more slowly reversible inhibition of ASIC1a channels expressed in 

Xenopus oocytes. CcrTx1 is a peptide isolated from the venom of the African spider 

Citharischius crawshayi. CcrTx1 inhibits ASIC1a with an IC50 of ~5 nM and most 

interestingly the inhibition at 100nM is stable for > 30 mins. Due to its stable inhibition of 

the channel, CcrTx1 should prove to be an invaluable tool for studies of the structure and 

function of this interesting channel family. 

1. Waldmann R (1999). Ann NY Acad Sci. 868, 67-76. 

2. Wemmie J. (2002). Neuron 34, 463-77. 

3. Xiong Z. (2004). Cell 118, 687-98. 

4. Escoubas P. (2000). J Biol Chem. 275 

5. Diochot S. (2004). EMBO J. 23, 1516-25. 

o Acid sensing ion channels 

o Tarantula peptide 

o Channel blocker 

207

Poster 33: Modulation of voltage-gated Na + and K + channels by pumiliotoxin 

251D: a “joint venture” alkaloid from arthropods and amphibians 

Vandendriessche, T. 1 , Maertens, C. 1 , Abdel-Mottaleb, Y. 1 , Cuypers, E. 1 , Nubbemeyer, U. 2 , Mebs, D. 3 

& Tytgat, J. 1* 

1 Laboratory of Toxicology, University of Leuven, O&N2, Herestraat 49, P.O. Box 922, 3000 Leuven, 

Belgium, * jan.tytgat@pharm.kuleuven.be 

2 Institute of Organic Chemistry, University of Mainz, Duesbergweg 10-14, 55128 Mainz, Germany 

3 Zentrum der Rechtsmedizin, University of Frankfurt, Kennedyvallee 104, 60596 Frankfurt, Germany 

Several amphibians defend themselves by accumulating lipophilic alkaloids from dietary 

alkaloid containing arthropods. Among these alkaloids, pumiliotoxins represent a major, 

widespread group found in virtually all anurans. Although pumiliotoxins are known as 

positive modulators of voltage-gated sodium channels (VGSCs), some of them, like 

pumiliotoxin 251D (PTX 251D), do not share this characteristic. However, mice and insect 

studies showed that this alkaloid is highly toxic, but the basis for the in vivo effects of PTX 

251D remains unexplained [1, 2]. 

In the present work we searched for the possible target of PTX 251D. The synthetic toxin 

was tested on four VGSCs (mammalian Nav1.2/β1, Nav1.4/β1, Nav1.5/β1 and insect 

para/tipE) and five voltage-gated potassium channels (VGPCs) (mammalian Kv1.1, Kv1.2, 

Kv1.3, Kv11.1 (hERG) and insect Shaker IR) in a heterologous expression system (Xenopus 

laevis oocytes), by means of the two-electrode voltage clamp technique. 

PTX 251D not only inhibited the Na + -influx through all three mammalian VGSCs but also 

affected the activation and steady-state inactivation while, in the insect ortholog, the 

inactivation process was dramatically affected. Interestingly, PTX 251D inhibited the K + - 

efflux through all five VGPCs and slowed down the deactivation of the mammalian 

VGPCs. Kv1.3 is the most sensitive channel, with an IC50 value 10,8 ± 0,5 µM, as 

compared to VGSCs and the other VGPCs tested. To the best of our knowledge this is the 

first report of a pumiliotoxin affecting VGPCs, helping to understand the in vivo effects 

observed in mice. 

1. Daly, J.W. et al. (2003) Proc. Natl. Acad. Sci. U.S.A 100, 11092-11097. 

2. Bargar, T.M. et al. (1995) Journal of Agricultural and Food Chemistry 43, 1044-1051. 

o Pumiliotoxin 251D 


o Voltage-gated potassium channels 

o Two-electrode voltage clamp technique 

208 

Poster 34: Structure-function relationships of Magi 4, a spider neurotoxin 

targeting insect and mammalian voltage-gated sodium channels 

Little, M.J. 1 , Yamaji, N. 2 , Villegas, E. 3 , Corzo, G. 4 , Nicholson, G.M. 1* 

1 

Department of Medical & Molecular Biosciences, University of Technology, Sydney, Broadway NSW 

2007, Australia, *Graham.Nicholson@uts.edu.au 

2 

Division of Spectroscopic and Structural Research, Suntory Institute for Bioorganic Research, 

Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan 

3 

Centro de Investigacion en Biotecnologia UAEM, Av. Universidad 2001, Cuernavaca, Morelos, 62210, 

Mexico. 

4 

Institute of Biotechnology-UNAM, Av. Universidad 2001 Cuernavaca, Morelos, 62210 Mexico. 

Magi 4 is a peptide neurotoxin isolated from the venom of the Japanese funnel-web spider 

Macrothele gigas. This toxin is composed of 43 amino acids with a molecular mass of 

5150 Da and shows between 50 and 60% homology with the δ-ACTX-1 family of toxins 

from Australian funnel-web spiders. Following determination of the NMR solution 

structure of Magi 4 we found that it has a similar cysteine spacing and inhibitory cystine 

knot motif to the δ-ACTX-1 toxins. Furthermore, several basic (K4, R5, K9, K34), acidic 

(E10) and hydrophobic (W7, Y22) residues occupy a similar surface position to those 

believed to be important in the pharmacophore of δ-ACTX-1 toxins. Given this structural 

homology it is likely that Magi 4 targets Nav channels similarly to δ-ACTX-1 toxins. 

Accordingly, we have employed the whole-cell patch-clamp technique to characterise the 

actions of Magi 4 on INa in both neonatal rat dorsal root ganglion (DRG) neurons and 

cockroach dorsal unpaired medial (DUM) neurons. Similarly to the δ-ACTXs, Magi 4 

caused a concentration-dependent slowing of tetrodotoxin (TTX)-sensitive INa inactivation 

in DRGs, with an EC50 of 45.7 ± 2.7 nM, and no effect on TTX-resistant INa. Similarly, 

Magi 4 also caused a 10 mV depolarising shift in the voltage-dependence of TTX-sensitive 

Nav channel activation. However, unlike δ-ACTX-1s, the slowing of inactivation was 

almost entirely reversible on washout in toxin-free solution (τoff = 99.2 sec at 300 nM). 

Also, Magi 4 failed to inhibit peak INa amplitude. In insect DUM neurons Magi 4, like δ- 

ACTXs, also slows Nav channel inactivation again accompanied by a depolarising shift in 

the voltage-dependence of activation. Importantly, while Magi 4 is toxic by i.c.v. injection 

into mice, it failed to displace 125 I-Lqh2 binding to neurotoxin receptor site 3 on Nav 

channels in rat brain synaptosomes, a hallmark of the actions of δ-ACTX-1 toxins and 

scorpion α-toxins. Thus, while Magi 4 clearly modulates the gating of TTX-sensitive Nav 

channels in DRGs it does not target Nav1.1 or 1.2 channels, the predominant Nav channel 

subtypes in brain. Therefore the actions of Magi 4 are similar to scorpion α-like toxins that 

show CNS toxicity via an action on the Nav1.6 channel subtype in brain. 

o Magi 4 


o NMR structure 

o Electrophysiology 

209

Poster 35: The natural anatoxin Amm VIII from Androctonus mauretanicus induces 

neutralizing antibodies against the most potent “Old-World” alpha-toxins 

Martin-Eauclaire, M-F, Alami, M, Rosso, J-P. and Bougis, P.E*. 

CNRS FRE 2738, IFR Jean-Roche, Université de la Méditerranée, Faculté de Médecine Secteur Nord, 

Bd Pierre Dramard, 13916, MARSEILLE, Cedex 20, France.*bougis.p@jean-roche.univ-mrs.fr 

Scorpion envenomations constitute a real public health problem in Maghreb, where 

epidemiological data indicate about 100 000 cases of scorpion stings per year. Fatality 

rates among children can reach 7%. The most lethal scorpions in North Africa are the 

Buthidae Androctonus mauretanicus (Amm) in Morocco, Androctonus australis (AaH) in 

Algeria and Tunisia and Leiurus quinquestriatus (Lqq) in Sudan. 

Up to 90 % of the lethality for mammals is induced by the binding of the alpha-toxins 

(about 65 amino acids residues long, four disulphide bridges) to site 3 of the voltagesensitive 

sodium channels (Nav) of nerve and muscle cells. Antivenom immunotherapy 

still remains the most useful specific treatment for scorpion envenomation. However, in 

spite of a broadly similar 3D-structure, the scorpion toxins display large sequence 

variations and are classified into several structural groups according to their sequence 

homologies. A consequence of this structural polymorphism is that no immunological 

cross-reactivity is observed between the toxins belonging to different groups when specific 

antisera are used, even if the toxins are found in the same venom. Thus, this wide antigenic 

polymorphism remains a persistent barrier to the preparation of an efficient and safe 

universal antiserum against “Old-World” scorpion alpha-toxins. 

In this study, we have used Amm VIII, a natural anatoxin from the scorpion Androctonus 

mauretanicus, to elicit specific polyclonal antibodies into rabbit. Using liquid-phase 

radioimmunoassay, we have studied its selectivity and its neutralizing activity both in vitro 

and in vivo for the most lethal scorpion alpha-toxins described, in particular the alpha-toxin 

of reference AaH II. We have shown that the anti-Amm VIII serum prevents the 

association of 125 I-AaH II with its receptor and is able to remove 125 I-AaH II already 

bound to its site (the half-life of the complex 125 I-AaH II-receptor site was 12 min in the 

absence of anti-Amm VIII serum but decreased to only 2 min in the presence of anti-Amm 

VIII serum). In vivo, the serum also has a protective effect in mice: 42 LD50 of AaH II by 

ml are neutralized, measured by subcutaneous injection. 

o scorpion 

o alpha-toxins 

o voltage-sensitive sodium channel 

o immunotherapy 

210 

Poster 36: Scorpion β-toxins: surveying the species-selectivity of five ‘classics’ 

Bosmans, F. 1 , Martin-Eauclaire, M.F. 2 , Tytgat, J. 1* 

1 

Laboratory of Toxicology, University of Leuven, O&N 2, P.O. Box 922, Herestraat 49, 3000 Leuven, Belgium,* 

jan.tytgat@pharm.kuleuven.be 

2 

CNRS FRE 2738, Ingénierie des Protéines, Faculté de Médecine secteur Nord, Institut Jean Roche, Université de 

la Méditerranée, Bd Pierre Dramard, 13916, Marseille, Cedex 20, France 

In general, scorpion β-toxins have been well examined. However, little in-depth studies 

have been devoted to species selectivity and affinity comparisons on the different voltagegated 

Na + channels since they have become available as cloned channels that can be 

studied in heterologous expression systems. As a result, their classification is rather 

historical and dates from early in vivo experiments on mice and cockroach and fly larvae. 

In this study, we aimed to provide an updated overview of species selectivity and affinity 

of scorpion β-toxins towards voltage-gated Na + channels. As pharmacological tools, we 

used the classic β-toxins AaHIT, Css II, Css IV, Css VI and Ts VII and tested them on the 

neuronal vertebrate voltage-gated Na + channel, Nav1.2. For comparison, its invertebrate 

counterpart, the insect channel, para, was also tested. 

Nav1.2 and the insect ortholog, para, were heterologously expressed in Xenopus laevis 

oocytes and measured with the two-electrode voltage-clamp technique. We supplemented 

this data with several binding displacement studies on rat brain synaptosomes. 

As a result, we propose a general classification and a novel nomenclature of scorpion βtoxins 

based on pharmacological function. 

o scorpion 

o β-toxin 

o voltage-gated Na + channel 

o species-selectivity 

211

Poster 37: Potent modulation of the voltage-gated sodium channel Nav1.7 by OD1, 

a toxin from the scorpion Odonthobuthus doriae 

Maertens, C. 1* , Cuypers, E. 1 , Amininasab, M. 2 , Jalali, A. 3 , Vatanpour, H. 4 , Tytgat, J. 1 

1 Laboratory of Toxicology, University of Leuven, Leuven, Belgium 

*Chantal.Maertens@pharm.kuleuven.be 

2 Dept. of Cell and Molecular Biology, Faculty of Science, University of Tehran, Tehran, Iran 

3 Dept. of Toxicology and Pharmacology, Jundishapour University of Medical Sciences, Ahvaz, Iran 

4 Dept. of Toxicology and Pharmacology, Shaheed Beheshti University of Medical Science, Tehran, Iran 

Voltage-gated sodium channels are essential for the propagation of action potentials in 

nociceptive neurons. Nav1.7 is found in peripheral sensory and sympathetic neurons and 

involved in acute and inflammatory pain. Nav1.8 and Nav1.3 are major players in 

nociception and neuropathic pain, respectively. 

In our effort to identify isoform-specific and high-affinity ligands for these channels, we 

investigated the effects of OD1, a scorpion toxin isolated from the venom of the scorpion 

Odonthobuthus doriae. Nav1.3, Nav1.7 and Nav1.8 channels were coexpressed with β1subunits 

in Xenopus oocytes. Na + currents were recorded with the two-electrode voltageclamp 

technique. 

OD1 modulates Nav1.7 at low nanomolar concentrations: (1) Fast inactivation is impaired, 

with an EC50 value of 4.5 nM. (2) OD1 dramatically increases the peak current at all 

voltages. (3) OD1 induces a substantial persistent current. Nav1.8 was not affected by 

concentrations up to 2 micromolar, whereas Nav1.3 was only sensitive to concentrations 

higher than 100 nM. OD1 impairs the inactivation process of Nav1.3 with an EC50 value of 

1127 nM. Finally, the effects of OD1 were compared with a classical α-toxin, AahII from 

Androctonus australis hector and a classical α-like toxin, BmK M1 from Buthus martensii 

Karsch. At a concentration of 50 nM, both toxins affected Nav1.7. Nav1.3 was sensitive to 

AahII, but not to BmK M1, whereas Nav1.8 was not affected by both toxins. 

In conclusion, the present study shows that the scorpion toxin OD1 is a potent modulator 

of Nav1.7, with a unique selectivity pattern. 


o Voltage-gated sodium channel 

o Nav1.7 

o Pain 

212 

Poster 38: Electrophysiological Characterization of Marine Snail Conus californicus 

Venom in Potassium Ion Channels 

Juárez-Moreno, K. O. 1 , Prior-Mier y Teran A 1 , García-Valdés, J. 2 , Aguilar M 3 , Morales E 1 , Waumann D 4 

and Licea-Navarro, A. 1* 

1 

Molecular Immunology and Biotoxins Laboratory Centro de Investigación Científica y de Educación 

Superior de Ensenada, Baja California, Mexico, *alicea@cicese.mx 

2. 

Bioelectrochemical Laboratory. Chemistry Faculty Universidad Nacional Autónoma de México, Mexico 

City. 

3. 

Neurobiology Institute, Universidad Nacional Autónoma de México, Queretaro City. 

4. 

Sciences School. Universidad Autonoma de Baja California 

The marine snail Conus californicus has a venom apparatus used to kill its prey; the venom 

contain an unique set of 50 to 200 different peptides called conotoxins 1 . Those have a 

highly targeting selectivity of voltage-gated and ligand-gated ion channels, acting as 

blockers of voltage-gated Ca 2+ , Na + and K + channels 2 . Due to the high selectivity and 

affinity of these toxins, several of them are in various stages of clinical development for 

treatment of human diseases 2 . Despite of its variability, only few conotoxins acting on K + 

channels have been identified and characterized, and were commonly called k-conotoxins. 

Potassium channels are a big family of proteins greatly distributed among all the 

kingdoms, and have been implicated in relevant neurodegenerative pathologies such as 

Parkinson’s and Alzheimer’s diseases and chronic pain. 

In this study we purified different fractions of the venom of C. californicus by RP-HPLC 

according to they retention time. We tested the effect of those fractions in heterologous 

expressed K + channels as Shaker IR and Hslo channel in Xenopus laevis oocytes using the 

Two Electrode Voltage Clamp technique. 

Up to date, our results indicated that in the venom of C. californicus there are three 

fractions corresponding to minutes 5, 28 and 44 of elution time, which caused a significant 

decrease of the current when were tested on the Shaker IR channel in 40%, 55% and 44% 

respectively. We already have a partial amino acid sequence of the fraction corresponding 

to the fraction of the 28 minute of elution time. 

In the case of the Hslo channel, there are two fractions corresponding to the minutes 20-25 

and 45-50 of elution time, which diminished the current of the channel in 20% and 12% 

respectively, we tried to test the individual minutes but was unable because of its low 

concentration. Also, we purpose that the venom of the marine snail C. californicus had a 

different activity according with the season in which the organisms were collected. 

1.Olivera, B. M. (1999) J. Comp. Physiol A. 185: 353-359. 

2 Terlau, H. & Olivera, B. M. (2003) Physiol. Review. 84: 41-68. 

o Conus californicus 

o κ-conotoxins 

o Potassium Ion Channels 

o Two Electrode Voltage Clamp 

213

Poster 39: GAMBIEROL-INDUCED CYTOSOLIC CALCIUM INCREASE IN 

HUMAN NEUROBLASTOMA CELLS 

Cagide, E 1 , Louzao, M.C 1* , Vieytes, M.R 2 , Sasaki, M 3 , Fuwa, H 3 , Yasumoto, T 4 , Botana, L.M. 1 

1 2 

Departamento de Farmacologia, Departamento de Fisiologia. Facultad de Veterianaria. Universidad 

de Santiago de Compostela. Campus de Lugo, Lugo, Spain. *ffmclooj@lugo.usc.es 

3 

Graduate School of Life Sciences, Tohoku University, 1-1 Tsutsumidoori-Amamiya, Aoba-ku, Sendai, 

Japan. 

4 

Japan Food Research Laboratories, Tama Laboratory, Nagayama, Tama, Tokyo, Japan. 

Gambierol is a polycyclic ether toxin isolated from Gambierdiscus toxicus, the same 

marine dinoflagellate causative of Ciguatera Fish Poisoning. High concentrations of this 

toxin activate sodium channels in human neuroblastoma cells. As a consequence gambierol 

induces a cytosolic calcium increment. We deeply investigate mechanisms responsible of 

the elevation of intracellular calcium in neuroblastoma cells by using the dye fura-2. We 

observed that preincubation of cells with nifedipine and mibefradil or KB-R7943 

significantly reduced gambierol-induced cytosolic calcium increment. Interestingly, when 

all inhibitors were added together this calcium influx was completely blocked. Those 

results indicate that gambierol stimulate Ca 2+ entry through voltage-gated calcium channels 

and the Na + /Ca 2+ exchanger working in reverse mode. 

o Gambierol 

o neuroblastoma 

o voltage-gated calcium channels 

o Na + /Ca 2+ 

214 

Poster 40: New modulator of P-type Ca 2+ -channels from Lycosa sp. venom. 

Pluzhnikov, K. 1 , Korolkova, Y. 1* , Vassilevski, A. 1 , Nikolsky, A. 1 , Fisyunov, A. 2 , Tsintsadze, V. 2 , 

Krishtal, O. 2 , Grishin, E. 1 

1 Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 

Miklukho-Maklaya, 16/10, 117997 Moscow, Russia,* july@ibch.ru 

2 Bogomoletz Institute of Physiology, 4 Bogomoletz Street, Kyiv 01024, Ukraine 

The potent modulator of P-type Ca 2+ channels, named Lsp-1, has been recently isolated 

from the venom of the spider Lycosa sp. (Fisyunov, A., Pluzhnikov, K., Molyavka, A., 

Grishin, E., Lozovaya, N., Krishtal, O., 2005, Toxicology 207, 129-136). Lsp-1 action on 

P-current in mammalian central neurons (prominent deceleration of the activation kinetics 

and partial decrease of the peak amplitude) notably differs from the effects of other wellknown 

Ca 2+ -toxins. The amino acid sequence of Lsp-1 was determined using a 

combination of Edman degradation, mass spectrometry and specific proteolytic 

fragmentation techniques. The peptide consists of 47 amino acid residues including 8 

cysteines involved in 4 intramolecular disulfide bridges, and presumably forms the ICK 

fold. Lsp-1 displays little homology to other known spider toxins affecting Ca 2+ -channels, 

but possesses a marked cluster of positively charged residues (-RKEKK-) in the C-terminal 

part of the molecule which may play a role in the action specificity. 

For comprehensive structure-function investigation, recombinant Lsp-1 was produced. The 

gene of Lsp-1 was constructed from synthetic oligonucleotides, and expressed as a soluble 

fusion protein with thioredoxin in E. coli Origami B cells. The fusion protein was purified 

by metal affinity chromatography, and cleaved with human enteropeptidase catalytic 

subunit. Lsp-1 was recovered by reverse-phase HPLC. The recombinant Lsp-1 was shown 

to be indistinguishable from the native peptide when tested on P-currents in rat cerebellar 

Purkinje cells. 

As voltage-dependent calcium channels are potential targets for therapeutics directed 

against migraine, intractable pain and some forms of epilepsy, Lsp-1 is attractive from both 

scientific and clinical perspectives. 


o calcium channel modulator 

o functional expression 

215

Poster 41: Evidence for a novel epitope for hERG channel blocking activity 

Yousra Abdel-Mottaleb 1 , Praveen Kumar 1 , Brigitte Céard 2 , Pierre E. Bougis 2 , Marie-France Martin- 

Eauclaire 2 , Jan Tytgat 1* 

1Laboratory of Toxicology, University of Leuven, Onderwijs and Navorsing 2, Herestraat 49, Postbus 

922, 3000 Leuven, Belgium, * jan.tytgat@pharm.kuleuven.be 

2CNRS UMR 6560, Ingénierie des proteines, Faculté de Médecine secteur Nord, institut Jean Roche, 

Université de la Méditerranée, Bd Pierre Dramard, 13916, Marseille, cedex 20, France 

To date, a total of 28 hERG blockers are known; 27 of which are grouped into γ-KTXs [1] 

and only one, BmTX3, belongs to subfamily α-KTX 15. The hERG blocking activity in 

BmTX3 has been assigned to 2 positively charged residues R18 and K19 on the α-helix 

side of the peptide [2]. 

To further investigate the interaction between the α-KTX family and hERG channels, a 

natural mutant of BmTX3 which is AmmTX3 (91% sequence homology with BmTX3) [3], 

was tested on cloned hERG channels in Xenopus laevis oocytes, using the 2 electrode 

voltage clamp technique. 

Complementary testing was carried out on other members of subfamily α-KTX 15 (Aa1, 

Discrepin) as well as some selected toxins belonging to the α-KTX family and possessing 

the 2 positively charged residues R18 or K18 and K 19 on the α-helix side of the peptide 

(BmTx2, Tsκ and HsTx1). 

The effects obtained were then compared to those from a control group of toxins lacking 

the 2 positively charged residues and belonging to the same α-KTX subfamilies as the test 

group (PBTx3, TsTXKα and Spinotoxin). 

Our results indicate that the presence of 2 positively charged residues R18 (or K18) and 

K19 on the α-helix side of the peptide, confers a hERG channel blocking activity to these 

α-KTXs. 

1. Korolkova,Y.V. et al., 2004, J. Mol. Recognit., 17, 209-217. 

2. Huys, I. et al., 2003, Biochem. J., 378, 745-752. 

3. Vacher, H. et al., 2002, Eur. J. Biochem., 269, 6037-6041. 

o hERG channels 

o α-KTX 

o voltage clamp technique 

216 

Poster 42: The first potassium channel toxin from the venom of the Iranian 

scorpion Odonthobuthus doriae 

Yousra Abdel-Mottaleb 1 , Amir Jalali 2 , Elke Clynen 3 , Frank Bosmans 1 , Liliane Schoofs 3 , Hossein 

Vatanpour 2 , Jan Tytgat 1* 

1Laboratory of Toxicology, University of Leuven, Onderwijs and Navorsing 2, Herestraat 49, Postbus 

922, 3000 Leuven, Belgium, * jan.tytgat@pharm.kuleuven.be 

2Department of Toxicology and Pharmacology, Shaheed Beheshti University of Medical science, 

Tehran, Iran 

3Laboratory for Developmental Physiology and Molecular biology, University of Leuven, 

Naamsestraat 59, 3000 Leuven, Belgium 

The first member of K + channels toxins from the venom of the Iranian scorpion 

Odonthobuthus doriae (OD16) was purified, sequenced and its pharmacological profile 

determined. 

OD16 is composed of 29 amino acids and has the six conserved cysteines. The toxin’s 

molecular weight and pI value are respectively 3188.5 and 4.95. 

Name α-KTX Sequence I% 

PO1 8.1 VSCEDCPEHCSTQKAQAKCDNDKCVCEPI 90 

BmPO1 8.2 ATCEDCPEHCATQNARAKCDNDKCVCEPK 88.5 

LpII 8.3 VSCEDCPDHCSTQKARAKCDNDKCVCEPI 92 

LpIII 8.4 VSCEDCPDHCSTQKARAKCDNDKCVCEPK 92 

OD16 8.5 VSCEDCPEHCSTQKARAKCDNDKCVCESV 100 

Based on multiple sequence alignments and homology modelling of the toxin structure, 

OD16 belongs to subfamily α-KTX 8, therefore, it was classified as α-KTX 8.5 and it was 

shown to adopt a cysteine-stabilized α/β scaffold. It is interesting to note that the toxin 

bears a basic residue (K) before the 3 rd cysteine, which is a property typical for the dyad 

present in many α-KTX’s, however, it was found that the aromatic counterpart is not 

present, raising our interest in the toxin’s bioactivity. The pharmacological effects of 

OD16 were studied on six different cloned K + channels (vertebrate Kv1.1-Kv1.5 and the 

insect ortholog Shaker IR) expressed in Xenopus laevis oocytes using the two-electrode 

voltage clamp technique. The toxin induced full current inhibition of Kv1.2 channels 

(EC50=182.6±2.6 nM) but did not affect any of the other channels. Therefore, among the 

channels tested, OD16 is selective to Kv1.2 and might serve as a good candidate for 

structure-function and channel assembly studies as well as the identification of the 

physiological role of Kv1.2 in multiple sclerosis. 

o OD16 

o Odonthobuthus doriae 

o α-KTX 8.5 

o Scorpion 

217

Poster 43: Novel insect-selective neurotoxins from the venom of the tarantula 

Eucratoscelus longiceps target insect Kv channels 

Escoubas, P. 1* , Lee, M. 2 , Ross, G. 2 , Lazdunski, M. 1 Nicholson, G.M. 2 

1 

Institut de Pharmacologie Moléculaire et Cellulaire – CNRS, 660 Route des Lucioles, 06560 Valbonne, 

France, *escoubas@ipmc.cnrs.fr 

2 

Department of Medical and Molecular Biosciences, University of Technology, Sydney, Broadway NSW 


Arthropod pests are vectors for the transmission of many diseases and destroy an 

estimated 20–30% of the world’s food supply. Currently available insecticides are 

severely limited by toxicity and/or insect resistance and in many cases are undergoing 

use cancellation. Thus there is an urgent need to develop safer and more selective 

insecticides and to identify new insecticide targets. Spider venoms have been proven to 

be a rich source of insecticidal compounds with unique properties, that target a wide 

array of insect neuronal receptor subtypes. With the goal of discovering novel 

insecticidal toxins from spider venoms, we have assayed an array of New and Old World 

tarantula venoms for insecticidal activity by intrathoracic injection into juvenile crickets 

(Gryllus bimaculatus) and selected the most potent venoms for further investigation. The 

most potent of these was the venom from the stoutleg baboon spider Eucratoscelus 

longiceps. Bioassay-guided fractionation of the venom using reversed-phase and cationexchange 

HPLC, yielded a family of three novel insecticidal toxins each with 29 residues 

and three disulfide bonds. Both ElTx1 and ElTx2 were identified to have insect 

neurotoxicity with LD50 values in crickets of 331 and 1033 pmol/g, respectively, but 

without overt signs of mammalian toxicity. ElTx3, with five non-conserved amino acid 

substitutions, also showed neurotoxicity in mice following icv injection. All three 

peptides display high homology to short ICK (inhibitory cystine knot) toxins from other 

tarantula venoms especially the phrixotoxins and both GsAFI and II. These toxins have 

been reported to target Kv4 channels, or possibly mechanosensitive cation channels. The 

target of ElTx1 was investigated by whole-cell patch-clamp analysis of DUM neurons 

from the American cockroach (Periplaneta americana). At 160 nM, ElTx1 blocked 55 ± 

14% (n = 3) of the global Kv channel current with no shift in the voltage-dependence of 

activation. Further experiments are currently determining the specific Kv channel subtype 

involved nevertheless this finding indicates that ElTx1 blocks invertebrate ion channels 

not previously targeted by conventional insecticides and validates Kv channels as novel 

insecticide targets. 


o Kv channels 

o insecticide 

o patch-clamp electrophysiology 

218 

Poster 44: Short insecticidal toxins from the Asian scorpions Buthus 

martensi and Mesobuthus tamulus modulate insect neuronal voltagedependent 

and calcium-activated chloride channels 

Grolleau F 1 ., Stankiewicz M. 3 , Herrmann R. 4 , Moskowitz H. 4 , Rajendra W. 4 , 

Hammock B.D. 4 , Romi-Lebrun R. 5 , Wu F.Q. 6 , Nakajima T. 5 Escoubas P. 2,5* 

1 Laboratoire Biologie Neurovasculaire Intégrée, UMR CNRS 6214 / Inserm 771, UFR Sciences Medicales 

Rue Haute de Reculée 49045 Angers cedex 01, France 2 Institut de Pharmacologie Moléculaire et 

Cellulaire – CNRS, 660 Route des Lucioles, 06560 Valbonne, France, *escoubas@ipmc.cnrs.fr 3 

Laboratory of Biophysics, Institute of General and Molecular Biology, N. Copernicus University, Torun, 

Poland 

4 Department of Entomology and Cancer Research Center UCDMC, University of California Davis, Davis 

CA 95616-8584, USA 5 Suntory Institute for Bioorganic Research. Mishima-Gun, Shimamoto-Cho, 

Wakayamadai, Osaka 618-8503 Japan 6 Sichuan Province Institute for Antibiotics. Sanbanqiao 9, 

Chengdu, China 

Five novel short insecticidal toxins have been isolated from the venoms of the 

Asian scorpions Buthus martensi and Mesobuthus tamulus, following a bioassay-guided 

procedure based on their insecticidal activity. Their full sequences show a high degree of 

similarity with previously described scorpion “short insectotoxins”, with 35-38 amino 

acids and molecular masses ranging from 3735 to 4019 Da. Insecticidal assays against 

three insect species show paralytic activity in the 1-10 ng/mg of insect range, with three of 

the toxins inactive against crickets. This level of activity, 10 to 100 times lower than the 

activity of the scorpion toxins acting on sodium channels, as well as differences in the 

paralysis symptoms suggest a different molecular target. We also report the unusual 

electrophysiological effects of toxins Bm20-IV and Mt-V6 isolated respectively from B. 

martensi and M. tamulus. Applied on cockroach neurosecretory dorsal unpaired median 

(DUM) neurons, the two peptides (2.5 µM) reduced the amplitude of the 

hyperpolarization-activated calcium sensitive chloride current (IClCa) by more than 40%, 

without any effect on reversal potential or voltage dependence. The inhibitory effects of 

Bm20-IV and Mt-V6 were mimicked by the specific chloride channel blocker DIDS (0.5 

mM) as well as commercial chlorotoxin (0.6 µM) from Leiurus scorpion venom, which is 

presumed to target chloride channels. In current-clamp experiments, Bm20-IV and Mt-V6 

induced hyperpolarization of the membrane potential associated with an increase of the 

input membrane resistance. DUM neuron sodium and potassium currents were insensitive 

to the toxins. Structure-activity relationships are discussed as these peptides might have 

great potential for 1°) ion channel mapping studies and 2°) as chloride channel inhibitors 

similar to chlorotoxin, which is currently used in clinical trials for the treatment of glioma. 

o Scorpion toxins 

o Chloride channels 

219

Poster 45: Phlotoxin 1, a toxin from tarantula venom, is a potent 

modulator of Nav1.7 sodium channels and a potential analgesic 

Escoubas P. 1* , Bosmans F. 2 , Cuypers E. 2 , Diochot S. 1 , Mebs D. 3 , Craik D. 4 , Hill J. 4 , 

Maertens C. 2 , Nakajima T. 5 , Lazdunski M. 1 , Tytgat J. 2 

1 

Institut de Pharmacologie Moléculaire et Cellulaire – CNRS, Valbonne, France, 

*escoubas@ipmc.cnrs.fr 

2 

Laboratory of Toxicology, University of Leuven, Leuven, Belgium 

3 

Zentrum der Rechtsmedizin, University of Frankfurt, Frankfurt, Germany 

4 

Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia 

5 

Suntory Institute for Bioorganic Research, Mishima-Gun, Shimamoto-Cho, Osaka, Japan 

Voltage-gated sodium (Nav) channels play an essential role in the biophysical properties of 

nociceptive neurons. Several Nav subtypes are key components of nociceptive 

neurotransmission and are potential targets for the development of novel analgesic 

strategies. Importantly, the Nav1.7 channel is found in peripheral primary sensory, and 

sympathetic, neurons and is involved in acute and inflammatory pain. We report here the 

discovery, structural characterization and pharmacological properties of phlotoxin 1 

(PhlTx1), a novel 34-residue peptide toxin from the venom of a tarantula of the genus 

Phlogiellus, which selectively inhibits Nav1.7. 

PhlTx1 was isolated as the major component of the venom and its sequence was 

determined and confirmed by solid-phase peptide synthesis. Homology modelling revealed 

that it belongs to the inhibitory cystine knot (ICK) family of spider toxins and mass 

spectrometry indicates three disulfide bridges. PhlTx1 shows limited homology with other 

Nav toxins from Chinese tarantulas (Hainantoxins, Huwentoxins). PhlTx1 almost 

completely blocked (~90%) Nav1.7 channel currents at a concentration of 1.2 µM and the 

dose-response relationship on heterologously expressed channels revealed an IC50 value of 

260 nM. The voltage-dependence of steady-state Nav1.7 channel activation and 

inactivation were not affected, suggesting that Phltx1 does not act as a gating-modifier 

toxin but rather blocks or impedes ion flux through the channel pore. PhlTx1 was almost 

exclusively selective for the Nav1.7 channel subtype, failing to modulate other Nav 

channels at concentrations up to 2 μM 

In a model of inflammatory pain (mouse paw injection of formalin), intrathecal injection of 

PhlTtx1 resulted in a significantly reduced response to the pain stimulus in both the acute 

and inflammatory phases, strongly suggesting that PhlTx1 produces an analgesic effect 

through inhibition of the Nav1.7 channel in dorsal root ganglion neurones. Phlotoxin 1 may 

therefore represent a lead for the development of novel analgesic agents. 


o Sodium channels 

o Pain 

o Analgesia 

220 

Poster 46: ON SOME BIOPHYSICAL TRAITS OF COLUBRIDAE SNAKE 

VENOMS 

Kazakov I., Abubakirova M.E., Reimbaeva R.S. 



Venoms of Colubridae snakes (Coluber ravergieri, C. rodorochochis, C. tyria 

Linneus, Elaphe dione Pallas, Natrix tesselata) were studied. The doses 0.10-0.40 µg/ml 

caused unstable channels for univalent ions, while large concentrations (100-200 µg/ml) 

decreased the instability of membranes. Labilization of membranes and their quick 

destruction were connected with the fact that snake venoms show the phospholipase and 

protease activities. 

In some respect, of special interest was the venom of the Psammophis Lineolatum 

Brandt of the family Colubridae. Our experiments showed the venom of Psammophis 

Lineolatum Brandt caused changes in the BLM conductance and forms channels with 

conductance for univalent and divalent cations. 140 ±10 pS, 96±4,2 pS in mediums 

containing 100 mM KCl, 100 mM CaCl2, 5mM tris HCl, pH=7,5). 

Thus, data obtained suggest that the venoms of Colubridae snakes contain 

components able to from unstable and discrete single channels. 

221

Poster 47: THE EFFECT OF BOOPHILUS CALCARATUS VENOM ON 

BILAYER LIPID AND MITOCHONDRIAL MEMBRANES 

Kazakov I.., Rakhimova Sh.H., Abubakirova M.E., Azimov D.A. 



LD50 of crude venom isolated from salivary glands of mite Boophilus calcaratus is 

100µg/kg. It shows phospholipase, protease and hemorrhagic activities. 

In low concentrations (below 170 µg/ml of protein), this venom causes stimulation 

of mitochondrial respiration at states 2 and 4, but produces no effect on the oxygen 

consumption at state 3, which is accompanied with the drop of respiratory control (RC) 

and ADP/O. The dose of this venom at 250 µg/ml almost completely inhibits ATP 

synthesizing functions of mitochondria, this effect growing at the absence of EDTA in 

the medium. 

Channels with the conductance of 120±10 pS (medium: 100 mM KCl and 5 mM 

tris-HCl, pH=7,5) are formed on bilayer lipid membranes under the effect of the crude 

venom. 

Thus, the venom of mite Boophilus calcaratus contains components able to form 

single ion channels in bilayer lipid membranes. It disturbs membrane-metabolic 

processes in mitochondria: at low doses it significantly increases the free oxidation not 

coupled with the ATP synthesis, while high concentrations inhibit the rate of 

phosphorylating respiration. 

222 

Poster 48: THE EFFECT OF HELMINTH EXTRACT ON THE BILAYER LIPID 

AND MITHOCHONDRIAL MEMBRANES 

I. Kazakov, M.E.Abubakirova, A.E.Kuchboev, D.A.Azimov 

Institute of Zoology, Uzbek Academy of Science, 700095 Tashkent, Uzbekistan 

The addition of 30 µg/ml of the five helminth species Echinococcus granulosus, 

Moniezia expanza, Fasciola hepatica, Ascaridia galli and Ascaris suum to one of the 

sections of the experimental cell is shown to cause a spasmodic increase in the membrane 

conductance. The further increase in the content of the extract was accompanied by a 

decrease in the lifetime of the membrane, i.e. it underwent destabilization and 

destruction. At a level of isolated mitochondria the increase of respiration rate at 

metabolic state 4 and some decrease of it in state 3 were recorded. The data obtained 

show that the extract of helminth membrane-active components form ion channels and 

modify functions of mitochondrial membranes. The presence of the receptor-enzymatic 

complexes is suggested. 

223

Poster 49: ω-ACTX-Ar1a: a novel insect-selective voltage-gated calcium channel 

blocker from the venom of the Sydney funnel-web spider 

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, 

G.M 1 

1 

Neurotoxin Research Group, Department of Medical & Molecular Biosciences, University of 

Technology, Sydney, Broadway NSW 2007 Australia, * youmie.chong@uts.edu.au 

2 

Monash Venom Group, Department of Pharmacology, Monash University, Clayton, Victoria 3800, 

Australia 

3 

Save Sight Institute, Sydney Eye Hospital, Macquarie Street, Sydney NSW 2001, Australia 

4 



Insect-selective toxins have the potential to ameliorate the growing problem of 

agrochemical resistance in pest insects. Here we report the isolation and characterisation of 

a novel insect-selective toxin, ω-ACTX-Ar1a, from the venom of the female Sydney 

funnel-web spider, Atrax robustus (Hexathelidae: Atracinae). Crude venom was purified 

using C18 reverse-phase HPLC, and a fraction containing a 4003 Da peptide neurotoxin 

was identified by toxicity bioassay using intrathoracic injections into House crickets 

(Acheta domesticus). Acute toxicity tests revealed a depressant lethal phenotype with an 

LD50 of 250 ± 28 pmol/g, and a KD50 (median knockdown dose) of 149 ±16 pmol/g at 48 

hours. Using isolated chick biventer cervicis nerve-muscle and mouse vas deferens 

preparations, the purified toxin was confirmed to lack overt vertebrate toxicity at 

concentrations up to 1 μM. Edman degradation revealed a 37-residue toxin with six 

cysteines and high sequence homology with insect voltage-gated calcium (Cav) channel 

blockers from the ω-ACTX-1 family. Importantly, there was conservation of the cysteine 

spacing and pharmacophore of ω-ACTX-Hv1a from Hadronyche versuta venom. 

Accordingly, whole-cell patch-clamp analysis was undertaken using dorsal unpaired 

median (DUM) neurons from the terminal abdominal ganglia of the cockroach Periplaneta 

americana to investigate the effect of ω-ACTX-Ar1a on insect Cav channels. A 

concentration-dependent voltage-independent block of IBa currents through Cav channels 

was observed at all membrane potentials, with no shift in the threshold of Cav activation. 

The IC50 values on insect Cav channels were 853 nM and 766 nM for ω-ACTX-Ar1a and 

ω-ACTX-Hv1a, respectively. There was no block of DUM neuron macroscopic Kv channel 

currents. However ω-ACTX-Ar1a did produce a modest block of peak INa amplitude, as 

has been previously observed with other Cav channel toxins such as ω-agatoxin IVA. In 

conclusion, ω-ACTX-Ar1a is an insect Cav pore blocker that both blocks low- and highvoltage 

activated insect Cav channels and further validates the ω-ACTX-1 pharmacophore. 

o Sydney funnel-web spider 

o electrophysiology 

o voltage-gated calcium channel 

o insect-selective toxin 

224 

Poster 50: The Effects of the Sodium Channel Blocker Lidocaine on Cardiovascular 

and Respiratory Actions of the Yellow Scorpion Leiurus Quinquestriatus in Rabbits 

Al-Shanawani, A. R 1* , Fatani, A.J. 2 

King Saud University, Department of Pharmacology, College of Pharmacy, AlMalaz Street, Riyadh, Saudi 

Arabia, ash77_00@hotmail.com 

. 

Introduction: Scorpion venom generally produce similar effects by acting mainly on Na + 

channels, causing an exaggerated release of neurotransmitters and mediators leading to 

pathological changes in several major systems in the body, mainly respiratory and 

cardiovascular systems. This study was performed to determine whether the Na + channel 

blocker, lidocaine, would be useful in preventing cardiovascular and respiratory actions of 

the venom of the common yellow scorpion Leiurus quinquestriatus quinquestriatus (LQQ). 

Methods: Arterial blood pressure (BP), respiration and ECG were recorded in urethaneanaesthetized 

New Zealand white male rabbits according to method of Ismail et al. (1972). 

Animals were injected with LQQ (0.5 mg kg -1 , i.v.) followed by lidocaine (1 mg kg -1 , i.v. 

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 

venom. Arterial BP, heart rate, respiration and ECG changes were recorded throughout the 

time limit of the experiment (300 min). Kruskal-Wallis non-parametric analysis of variance 

and Dunn’s Multiple comparisons post test were used in cardiovascular and respiratory 

experiments. Survival of the rabbits was then analyzed utilizing Covariance Wilcoxon 

Analysis. Results were expressed as mean ± S.E.M of 5 animals, with values of P≤ 0.05 

considered significant. 

Results: Lidocaine was capable of significantly (P

Poster 51: Postsynaptic neuromuscular activity of Cerastes cerastes cerastes crude 

snake venom. 

Soliman M. M 1 , Abu-Sinna G 1 , Abd-Elbaset A 2 , Harvey A.L 3 and Rowan,E.G 3 

1 Department of Zoology, Faculty of Science, Ain Shams University, Cairo, Egypt, 

*mohamed.soliman@strath.ac.uk 

2 Medical Research Center, Faculty of Medicine, Ain Shams University, Cairo, Egypt 

3 University of Strathclyde, SIBS, 27 Taylor Street, Glasgow, Scotland, UK 

Introduction: Cerastes cerastes cerastes is an Egyptian viper that belongs to the family of 

Viperidae and the genus Cerastes. Many biochemical and pharmacological studies have 

been carried out on this venom, however, little is known of the effect of the venom on 

neuromuscular transmission. Hence, the present study was undertaken to test the 

neuromuscular activity of Cerastes venom. 

Methods: Mouse phrenic nerve hemidiaphragm preparation and chick biventer cervicis 

(CBC) nerve muscle preparation were mounted in 10 ml organ baths in physiological salt 

solution. Motor nerves were stimulated at the desired frequency at a voltage greater than 

that required to produce a maximal twitch. On CBC preparations submaximal 

concentrations of acetylcholine, carbachol and KCl were added to the bath prior to and at 

the end of the experiment. Data is expressed as means ± sem. 

Results: Cerastes venom dose dependently inhibited the contractile responses of indirectly 

stimulated (nerve stimulation) mouse hemidiaphragm preparations . Venom (10µg/ml) 

reduced twitches to around 63.6 ± 2.1 % of control in one hour, while with 20µg/ml of 

venom twitches were reduced further to 77±1.6% of control. CBC nerve muscle 

preparations were less sensitive to the venom than the hemidiaphragm preparation, 

10µg/ml, and 20µg/ml had no effect on twitches. However, 40µg/ml of venom reduced 

twitches to about 37.1 ± 12 of control. The venom (40µg/ml) also reduced contractile 

response to exogenous acetylcholine (10 -3 M) to about 4.7±2.1%, to exogenous carbachol 

(2x10 -6 M) to about 5.1±2.5% and also to exogenous KCl (60mM) to about 21.7±2.6%. It is 

concluded that the Cerastes venom has neurotoxic activity and act primarily 

postsynaptically to depress muscle contractility. 

Ref. 

Wayne C Hodgson and Janith C Wikramaratna (2002): In vitro neuromuscular activity of 

snake venoms. Clin Exp Pharmacol Physiol, 29(9):807-14 

• Neurotoxicity 

• Snake venom 

• Phrenic nerve 

• Chick biventer 

226 

Tuesday 25 July: poster session (Colville 

Building 5.11/5.12) 

Venomics, transcriptomics, cloning 

1. Inter-specific comparison of the toxin encoding transcriptomes of medically 

important African vipers, E. ocellatus, C. cerastes and B. arietans. Wagstaff, S.C.*, 

and Harrison, R.A. 

2. An O-conotoxin-like Cys framework and hydroxyproline in peptides of the 

venom of the turrid Polystira albida from the Bay of Campeche, México. Aguilar, 

M.B.*, Chan de la Rosa, R.A., Falcón, A., Heimer de la Cotera, E.P. 

3. Gene Expression Profiling and Fingerprinting of Human Genome Following 

Exposure to Natural Toxins and Nerve Agents – TOXINOGENOMICS. 

Gopalakrishnakone, P *, Pachiappan, A. 

4. Molecular cloning of dipeptidyl-peptidase IV (CD26) from Bothrops alternatus 

(urutu) snake venom gland cDNA. Cardoso, K.C. 1 , da Silva, J.M. 2,3 , Souza, 

G.H.M.F. 1 , Menossi, M.T. 2,3 , Hyslop, S. 1 

5. Phylogeny of Protobothrops Snakes Inhabiting the Southwestern Islands of 

Japan. Chijiwa, T 1 *, Tomino, H 1 , So, S 1 , Oda-Ueda, N 2 , Hattori, S 3 , Ohno, M. 

6. The main toxins of the Colubridae snake Philodryas olfersii revealed from a 

Duvernoy´s (venom) gland transcriptome. 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. M. T. 3 ; Ho, P. L. 1 , 

Junqueira-de-Azevedo, I.L.M. 1 

7. A comparison of the venom from left and right fangs of three Australian 

elapid’s (Pseudechis australis, Psuedonaja textiles and Notechis scutatus). Winter, 

K.L., 1* Mirtschin, P.J., 2 Hodgson, W.C. 1 

8. Gene Structures of Two Functionally Diverse Prothrombin Activators, Trocarin 

D and Coagulation Factor X in Tropidechis carinatus Snake: Gene Duplication 

and Recruitment of Factor X Gene to the Venom Gland. Reza, MA 1 , Swarup, S 1 , 

1, 2 

Kini, RM 

9. Modulation of transcription activities between group IA and IB phospholipase 

A2 genes in sea snake Laticauda semifasciata. Hayashi, Y,, Sagitani, A., Fujimi, T. J., 

Kanzawa, N., Tsuchiya, T., Tamiya, T.* 

227

10. Maturation signatures in the sequences of natural toxins. Kozlov S., Grishin E. 

11. Proteomic analysis of the venom from the Mexican scorpion Centruroides 

limpidus limpidus. Gurrola, G.B., Batista, C.V.F., Zamudio, F.Z. and Possani, L.D. 

12. Venom Proteomes of Closely-related Sistrurus Rattlesnakes with Divergent 

Diets. Sanz, L. 1 , Gibbs, H.L. 2 , Mackessy, S.P. 3 , Calvete, J.J. 1,* 

13. Molecular cloning of a non-venom-secreted PII disintegrin-like transcript, BA- 

5A, from a Bitis arietans cDNA library reveals a pathway for the evolution of the 

long-chain disintegrin bitistatin from a PIII disintegrin-like precursor. 

Juárez, P. 1* , Wagstaff, S.C. 2 , Oliver, J. 2 , Sanz, L. 1 , Harrison, R.A. 2 , Calvete, J.J. 1 

14. Spider venom proteome mining and de novo toxin sequencing by LC-MALDI- 

TOF and FT-ICR mass spectrometry. Escoubas P. 1* , Quinton, L. 2 , Hu Renjie 2 , Wen 

S. 3 , Chamot-Rooke J. 2 , Nicholson G.M. 3 

15. Gene and primary structure of an insect-specific peptide from the venom of 

the theraposid Brachypelma smithi. Corzo, G., Diego, E., Clement, H., Estrada, G., 

Odell, G., Batista, C., Possani, L.D., Alagón, A. 

16. TRANSCRIPTOME ANALYSIS OF EXPRESSED SEQUENCE TAGS 

FROM Thalassophryne nattereri FISH VENOM GLANDS. 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 

A.M 

17. CLONING AND CHARACTERIZATION OF A C-TYPE LECTIN FROM 

Thalassophryne nattereri FISH VENOM GLAND. 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 .W 6 , Ho P.L 3 , 

Moura-da-Silva A.M 1* . 

18. Biochemical evidence that crotasin is a fully active gene in tissues of the South 

American rattlesnake Crotalus durissus terrificus Collares, M. de A. 1 ; Silva, A. R. P. 

da 1 ; Rádis-Baptista, G. 2 ; Grego, K. F. 1 ; Oguiura, N. 1* 

19. The Gene Family of Crotamine and Variations of its Content in Venoms of the 

South American Rattlesnake Crotalus durissus. Collares, M. de A. 1 ; Corrêa, P. G. 1 ; 

Suzuki, H. 2 ; Furtado, M. F. D. 1 ; Oguiura, N. 1* 

20. A profile of typical Viperidae toxins in the Lachesis muta venom glands 

transcriptome. 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, M.R.V. 4 

21. Atypical Viperidae cDNAs from Lachesis muta venom glands and their 

implication in snake toxin repertoire evolution. Junqueira-de-Azevedo, I.L.M. 1* , 

Ching, A.T.C. 1 , Carvalho, E. 1 , Ho, P.L. 1 , Diniz, M.R.V. 2 

228 

22. The Bitis gabonica gabonica venom proteome: venomics vs. transcriptomics. 

Calvete, J.J. 1,* , Marcinkiewicz, C. 2 , Sanz, L. 1 

23. Loss of introns along the evolutionary diversification pathway of snake venom 

disintegrins evidenced by sequence analysis of genomic DNA from Macrovipera 

lebetina transmediterranea. Bazaa, A. 1 , Juárez, P. 2 , Marrakchi, N. 1 , Bel Lasfer, Z. 1 , El 

Ayeb, M. 1 , Calvete, J.J. 2,* , Sanz, L. 

24. Current state of Bothrops insularis venom proteome. A joint effort of the Rio 

de Janeiro Proteomic Network. 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 ., 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 ., 

Oliveira-Carvalho, A.L 3,5 ., Wermelinger, L.S 3,5 , Zingali, R.B 3,5 . and Domont, G.B 4 . 

25. Bothrops jararaca Venom Proteome. 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, R.M. 2 , Domont, 

G.B. 3,4 and Perales, J. 1,4 

Toxin discovery, purification, biochemistry 

26. Post-translational amino acid epimerization in an excitatory conotoxin. 

Structures of the L- and D-forms. 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 

27. Biochemical characterization of venom from the South American colubrid 

Philodryas patagoniensis. 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 , Cogo, J.C. 1,* 

28. Miwaprin, a whey acidic protein-like in coral snake (Micrurus frontalis) 

venom. Moreira, K. G 1,2* , Silva, L. P 2 , Bloch Jr, C 2 . 

29. Purification and partial characterization of a deoxyribonuclease II from 

Bothrops alternatus (urutu) venom. Nascimento, J.M. 1,2,* , Collares-Buzato, C.B. 3 , 

Hyslop, S. 1 

30. LC-MS Analysis of All PSP Toxins Using Anion Exchange and Reverse Phase 

Columns Connected in Series. Sachio Nishio 1* ,Takefumi Sagara 1 , Shigeto 

Taniyama 2 , Tamiko Hashimoto 1 , Naoyoshi Nishibori 1 and Manabu Asakawa 2 

31. Fishing Toxins out of Snake Venoms Using the Antitoxin DM43 as a Tool for 

the Analysis of Subproteomes. Rocha, S.L.G 1 ., Neves-Ferreira, A.G.C 1 ., 

Chapeaurouge, A 1 ., Valente, RH 1 ., Trugilho, M.R.O 1 ., Léon, I.R 1 ., Domont, G.B 2 . and 

Perales, J 1* . 

229

32. Novel peptides in Philodryas olfersii and Philodryas patagoniensis venoms 

detected by bidimensional liquid chromatography coupled to tandem nanoelectrospray 

mass spectrometry and de novo mass spectrum analysis. Souza, 

G.H.M.F. 1,2,* , Cogo, J.C. 3 , Eberlin, M.N. 2 , Hyslop, S. 1 

33. Development of a novel fluorometric assay for the evaluation of BoNT/A 

protease activity using genetically engineering a fluorogenic substrate. Ban, B. 1 

Yang, G.H. 1 1, 2 

, and Jung, H. H. 

34. PURIFICATION AND PARTIAL ENZYMATIC CHARACTERIZATION 

OF THE HYALURONIDASE FROM Crotalus durissus terrificus SNAKE 

VENOM. Cordeiro, A. T. 1 ; Tolini, M.M. 1 ; Bertazzi, D. T. 1 ; Giglio, J. R. 2 ; Arantes, E. 

C. 1 * 

35. Mass spectrometry strategies for venom mapping and peptide sequencing from 

crude venom of a single Bombus specimen. Menin L., Favreau P., Michalet S., Perret 

F., Cheneval O., Stöcklin M., Bulet P. and Stöcklin R. 

36. A New Family of Peptides Discovered in Snake Venoms from the Atheris genus 

by Mass Spectrometry Techniques. Favreau, P. 1 , Cheneval, O. 1 , Menin L. 1 , 

Michalet, S. 1 , Principaud, F. 2 , Thai, R. 3 , Ménez, A. 3 , Bulet, P. 1 and Stöcklin, R. 1 * 

37. Quantitative high-throughput LC-MS detection of cyanotoxins in aquatic 

tissue samples. Lähde, M.-R. , Meriluoto, J.A.O. 

38. Improved EC method for the determination of cyanide in feed. M. Baltussen, D. 

Luykx, A. Koot, R. Frankhuizen and S. van Ruth 

39. The sulfo-SBED derivative of ammodytoxin, a photoprobe for studying the 

molecular basis of phospholipase A2 β-neurotoxicity. Kovačič, L. * , Šribar, J., Križaj, 

I. 

40. Purification and characterisation of two apoptosis-inducing L-amino acid 

oxidases from the venoms of selected Australian elapids. Bateman, E.H, 1* Venning, 

M.G, 2 Mirtschin, P.J 2,3 and Woods, A.E 2 

41. Discovery of a novel conopeptide-AmIXA: Prediction of functional 

pharmacophore(s) and mechanism of action. Saminathan, R 1 , Jois, S.D.S 2 , Sato, 

K 3 , Gopalakrishnakone, P 1* . 

42. Kunitz-type serine protease inhibitors from snake venoms. Trabi, M. 1,2* , Flight, 

S. 3 , Masci, P.P. 3 , de Jersey, J. 2 , Lavin, M.F. 1 

43. Identification of the active-site residues of the fungal virulence factor, 

phospholipase B. Tsukamoto, K 1* , Inoue, Y 1 , Tsuruga, A 1 and Obata, Y. 2 

230 

44. A Comparative Study of the Enzymatic and Physiological Activities of Two 

Viper Venom L-Amino Acid Oxidases. Samel, M. 1 , Tõnismägi, K. 1 , Trummal, K. 1 , 

Rönnholm, G. 2 , Siigur, J. 1 , Kalkkinen, N. 2 , Siigur, E. 1 * 

45. Heterodimeric Disintegrin VLT-2 from Vipera lebetina Snake Venom. Siigur, 

J.*, Vija, H., Aaspõllu, A., Samel, M., Tõnismägi. K., Trummal, K., Pikver, R., Subbi, 

J., Siigur, E. 

46. Discovery and pharmacology of Conopressin-T, a novel Vasopressin-like 

peptide from Conus Tulipa. Daniel Croker 1 , Sebastien Dutertre 2,3 , Paul Alewood 2 , 

Richard Lewis 2 

47. Isolation and characterisation of Conomap-Vt, a D-amino acid containing 

excitatory peptide from the venom of a vermivorous cone snail. Sébastien 

Dutertre 1 , Natalie Lumsden, Paul F. Alewood and Richard J. Lewis 

48. Tityus perijanensis (Scorpiones, Buthidae) from Zulia State, western 

Venezuela: Geographic Distribution and Venom Molecular and 

Immunological Characterization. Borges, A. 1* , Blumer-Lairet, V. 1 , Poliwoda, S. 1 , 

Alfonzo, M.J. 1 , Rojas-Runjaic, F. 2 , De Sousa, L. 3 

49. Error-prone DNA polymerase k from Protobothrops flavoviridis, member of 

the DinB superfamily. Oda-Ueda, N 1 ., Kariu, T 1 ., Fukuhara, A 2 ., Takazaki, S 1 , 

Chijiwa, T 2 ., Ohno, M 2 . 

50. Post-translational modifications of venom components in Conus victoriae. 

Townsend, A 1, 2 , Scanlon, D 2 , O’Donnell, P 2 , Inserra, M 1 , Bingham, J-P 3 , 

Satkunanathan, N 1 , Khalil Z 1, 4 , Purcell, A 1, 2 1, 2* 

, Livett, B.G. 

231

Poster 1: Inter-specific comparison of the toxin encoding transcriptomes of 

medically important African vipers, E. ocellatus, C. cerastes and B. arietans. 

Wagstaff, S.C.*, and Harrison, R.A. 


L3 5QA, UK *simonw@liv.ac.uk 

Generating expressed sequence tags (EST) from medically important venomous snakes 

provides an unbiased global panorama of venom gland transcriptional activity, and by 

inference, insights into the composition of the venom proteome. Here, we describe the 

multi-isoform toxin encoding profiles of three identically constructed venom gland EST 

libraries from medically important and geographically distinct vipers, E. ocellatus 

(Nigeria), C. cerastes cerastes (Egypt) and B. arietans (Nigeria). 

To assist interpretation of these typically complex, multi-toxin, multi isoform 

transcriptomes we employed a variety of bioinformatic methodologies including BLAST, 

standard gene ontology (GO) annotation and motif searches in order to highlight 

potentially important differences between isoforms. 

In addition to identifying several transcripts that may play unique roles in the venomous 

strategy of individual species, inter-specific comparison of these transcriptomes also 

revealed clear differences in toxins encoding profiles, most notably variations in the 

diversity and pseudo-expression levels of serine proteinases relative to snake venom 

metalloproteinases. This analysis also revealed several motifs within catalytic or integrin 

adhesive motifs of several venom toxins that may have important functional implications. 

The significance of these findings is discussed. 

o EST 

o Transcriptome 

o Venom 

o Bioinformatics 

232 

Poster 2: An O-conotoxin-like Cys framework and hydroxyproline in peptides of 

the venom of the turrid Polystira albida from the Bay of Campeche, México 

Aguilar, M.B.*, Chan de la Rosa, R.A., Falcón, A., Heimer de la Cotera, E.P. 

National Autonomous University of Mexico, Campus UNAM-UAQ Juriquilla, Juriquilla, Qro., México, 

*maguilar@servidor.unam.mx 

Introduction: Superfamily Conoidea (families Conidae, Terebridae and Turridae) includes 

predatory, venomous snails. Conotoxins are short, disulfide-rich peptides that often contain 

post-translational modifications and that target ion channels, receptors, and transporters. 

Conotoxins are classified into gene superfamilies and pharmacological families according 

to their signal peptides and molecular targets, respectively, and have provided molecular 

tools, diagnostic agents, pharmaceuticals, and drug leads. The venom of the terebrid 

Terebra subulata contains some "augertoxins" similar to the O- and I-conotoxins, with no 

post-translational modifications other than disulfides, whereas those of the turrids 

Gemmula periscelida and Polystira albida include long, methionine-rich "turritoxins". 

Our objective was to find conotoxin-like peptides in the venom of the turrid P. albida. 

Methods: The venom ducts of 10 specimens of Polystira albida were dissected and used 

to prepare a crude extract, which was fractionated by RP-HPLC (C18). Selected peptides 

were further purified (C18, C8) and then subjected to automated Edman degradation. 

Results: Three peptides were purified from three medium-sized peaks in the crude extract. 

The partial amino acid sequences obtained are (C, putative Cys; O, hydroxy-Pro): 

Pa1: YGDSVTSDECQTISCYEAHVGOVCQOGEYD...; Pa2: ECTPVHFSCTCTGLE...; 

Pa3: NVCDGDACPDGVCCSGCTTDFNVAQRKDTCFYPQ... . 

Discussion/Conclusion: The three turritoxins characterized in this work seem to contain 

multiple Cys residues, as do the conotoxins. Pa3 has an O-conotoxin-like framework and is 

more similar to ω-conotoxins SVIA, GVIIA, and GVIIB than to augertoxins s6a and s7a. 

Recently, mRNAs encoding O-, P-, and I-conotoxin-like peptides have been found in the 

turrid Lophiotoma olangoensis Olivera 2002 (1). Within Conoidea, Pa1 is the first non- 

Conus peptide shown to contain hydroxy-Pro. This finding implies that hydroxylation of 

Pro occurred in the venom of the common ancestor of families Conidae and Turridae. 

References: 1. Watkins, M. et al. (2006) J. Mol. Evol., 62, 247-256. 

Supported by PAPIIT-UNAM (IN206701 and IX211904) and CONACYT (41477-Q). 

Key Words: type your keywords here (no more than four words) 

o Turridae 

o Polystira albida 

o O-conotoxin-like 

o Hydroxyproline 

233

Poster 3: Gene Expression Profiling and Fingerprinting of Human Genome 

Following Exposure to Natural Toxins and Nerve Agents – TOXINOGENOMICS 

Gopalakrishnakone, P *, Pachiappan, A. 

Venom and Toxin Research Programme, Department of Anatomy, Yong Loo Lin School of Medicine, 

National University of Singapore, Singapore 119260, *antgopal@nus.edu.sg 

Conventional methods such as northern blotting and RNAse protection assays generally do 

not provide sufficient throughput to exploit the genomic data. High-density oligonucleotide 

gene profiling studies hold promise for biomarker identification and for delineation of 

global alterations in molecular expression after exposure of cells and tissues to toxins. In 

the present study, individual functions of predicted ORF’s within the genome and the 

relationship between genes at the expression level are investigated through the use of 

genome sequence data accumulated after exposure of human glial cells to natural toxins 

and nerve agents. We have used six toxins of three different classes (Neurotoxins, Nerveagents, 

Hepatotoxins). Following exposure of human brain cell lines to neurotoxins – 

candoxin (1), tetrodotoxin (2) and domoic acid, and to nerve-agents - soman, and sarin, 

gene expression profiling was done using Affymetrix (HG-U133A) human GeneChips. For 

comparison, human liver cell lines were exposed to hepatotoxin – aflatoxin B, with rigid 

filtering standards used to fish-out relevant genes of interest. With the aid of GeneSpring 

(PCA) analysis, 257 genes whose expression was significantly (p < 0.01) altered by at least 

2.5-fold, were selected and clustered by genesis software. The emerging data from this 

study indicate a significantly different global expression pattern seen with the toxinexposed 

human brain cell lines as compared to unexposed cells. Such data, when used as a 

fingerprint to characterize toxins, will provide possible insights into the mechanism/s of 

actions of these toxins at molecular level. 

o High density oligonucleotide gene profiling 

o Tetrodotoxin 

o Sarin 

o Human glial cells 

234 

Poster 4: Molecular cloning of dipeptidyl-peptidase IV (CD26) from Bothrops 

alternatus (urutu) snake venom gland cDNA 

Cardoso, K.C. 1 , da Silva, J.M. 2,3 , Souza, G.H.M.F. 1 , Menossi, M.T. 2,3 , Hyslop, S. 1 

1 Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas 

(UNICAMP), CP 6111, 13083-970, Campinas, SP, Brazil *kiara@fcm.unicamp.br; 

hyslop@fcm.unicamp.br 

2 Departamento de Genética e Evolução, Instituto de Biologia and 4 Centro de Biologia Molecular e 

Genética (CBMEG), Universidade Estadual de Campinas (UNICAMP), CP 6109, 13083-970, Campinas, 

SP, Brazil 

Introduction: Snake venoms contain a variety of peptidases, including dipeptidylpeptidase 

IV (DPP IV) or CD26 that may contribute to envenoming [1-3]. DPP IV, 

which cleaves after proline residues in neuropeptide Y, peptide YY, growth hormonereleasing 

hormone, glucagon-like peptides 1 and 2, and substance P, may have a role in 

venom-induced hypotension [1]. In this work, we isolated and sequenced a cDNA 

encoding DPP IV from a cDNA library prepared from poly (A) + RNA of the venom 

gland of the snake Bothrops alternatus. Methods: Total RNA was extracted from the 

venom glands three days after venom milking. A cDNA library was prepared using 

standard procedures and the DPP IV gene was cloned from this library. Random positive 

clones were sequenced using a Perkin-Elmer ABI Prism Model 310 sequencer. Results: 

An open reading frame (ORF) sequence of 2286 base pairs encoded a mature, 762-amino 

acid protein with a calculated molecular mass of 86.2 kDa and a theoretical pI of 6.11. 

Multiple sequence alignment using Clustal X and phylogenetic analysis showed that this 

protein grouped with other DPP IV from various vertebrate species and was essentially 

identical to DPP IV from Gloydius blomhoffi brevicaudus (NCBI no. 

AB158224/158225). Computer modeling showed that the protein contained numerous αhelix 

and β-sheet regions, with the N-terminal region being highly conserved, as in other 

DPP IV. The active site contained Serine630, which is characteristic of this group of 

enzymes. Conclusion: DPP IV from B. alternatus venom is a high molecular mass 

protein that shares similarities with other venom and non-venom DPP IV. The successful 

expression of this protein should provide material to allow study of its biological 

activity. This is the first characterization of a DPP IV from Bothrops snake venoms. 

References: 1. Aird SD (2002) Toxicon 40, 335-393; 2. Anderson L, Bussler B, Martins 

H, Dufton M (1998) Toxicon 36, 719-728; 3. Gasparello-Clemente E, Silveira PF (2002) 

Toxicon 40, 1617-1626. 

Financial support: CAPES, CNPq, FAPESP. 

o Bothrops alternatus 

o Peptidases 

o Venom gland library 

o Dipeptidyl-peptidase IV 

235

Poster 5: Phylogeny of Protobothrops Snakes Inhabiting the Southwestern Islands 

of Japan 

Chijiwa, T 1 *, Tomino, H 1 , So, S 1 , Oda-Ueda, N 2 , Hattori, S 3 , Ohno, M. 1 

1 

Sojo University, Faculty of Biological and Bioscience, 4-22-1 Ikeda, Kumamoto, Japan, *chijiwa@life.sojou.ac.jp 

2 

Sojo University, Faculty of Pharmacological, 4-22-1 Ikeda, Kumamoto, Japan 

3 

University of Tokyo, Institute of Medical Science, Tean-Sude802 Setouchi-chou, Ohshima-gun, Kagoshima, 

Japan 

Introduction: Three species of Protobothrops snakes inhabit the south western islands of 

Japan: P. flavoviridis (Habu snake) in Amami-Oshima, Tokunoshima, Okinawa and 

Kumejima islands, P. tokarensis (Tokara Habu) in the Tokara Islands (Takarajima and 

Kodakarajima islands) and P. elegans (Sakaishima Habu) in the Sakaishima Islands 

(Ishigaki and Iriomote islands). The classification of these snakes had been made in the 

conventional way, mainly based on their morphological features. However, the recent 

comparative chromatographic and biochemical studies showed that the constituents of the 

venom-gland isozymes of these snakes have diversified depending on their environments. 

This may provide a new clue to consider the relationship between geological history of the 

islands and evolution of the snakes. In this work, to ascertain the phylogenetic relationship 

of these snakes, the D-loop region of mitochondrial DNA of each snake was sequenced 

and the phylogenetic tree was constructed. 

Methods: The mitochondrial DNAs were extracted from livers of P. flavoviridis snakes in 

Amami-Oshima, Tokunoshima, Okinawa, Kumejima island, of P. tokarensis in 

Kodakarajima island and of P. elegans in Ishigaki island. The D-loop region of each 

mitochondrial DNA was amplified with specific primers and subcloned into the plasmid 

vector and sequenced. The nucleotide sequences were aligned and their relative evolutional 

distances were estimated by the neighbour-joining method to construct the phylogenetic 

tree. 

Results: The topology of the phylogenetic tree showed that Protobothrops genus in the 

southwestern islands of Japan could be classified into three major groups; the Satsunan 

Islands’ group (Kodakarajima, Amami-Oshima and Tokunoshima islands), the Ryukyu 

Islands’ (Okinawa and Kumejima islands) and the Sakishima Islands’ (Ishigaki island). 

Discussion/Conclusion In contrast to their morphological variation, the phylogeny of 

Protobothrops genus snakes in the southwestern islands of Japan is well in accordance 

with the geological history of the island where they lived. 

o Protobothrops genus 

o Mitochondria D-loop 

o Phylogeny 

o Evolution 

236 

Poster 6: The main toxins of the Colubridae snake Philodryas olfersii revealed from 

a Duvernoy´s (venom) gland transcriptome 

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. 

M. T. 3 ; Ho, P. L. 1 , Junqueira-de-Azevedo, I.L.M. 1 

1 

Centro de Biotecnologia, Instituto Butantan, Av. Vital Brasil, 1500 - São Paulo, Brazil- 05503-900 

*atung@butantan.gov.br 

2 

Laboratório de Herpetologia, Instituto Butantan, São Paulo, Brazil 

3 

Laboratório Especial de Toxinologia Aplicada, CAT-CEPID, Instituto Butantan, São Paulo, Brazil 

Introduction: The interest about venoms of Colubridae, a family of snakes relatively 

neglected in the venom studies, is increasing due to its importance in human envenoming 

accidents. Philodryas olfersii, a representative species of this group and the one involved 

in most accidents in Brazil, has a Duvernoy's gland that produces a secretion exhibiting 

high hemorrhagic activity. 

Methods: We constructed a cDNA library from P. olfersii Duvernoy’s gland for the 

generation of an Expressed Sequence Tags database (dbEST) aiming to characterize its 

transcriptome. The ESTs were clustered, searched against public databases and catalogued. 

Results: We obtained 2194 clones with an average of 415.7 bp that were grouped in 172 

clusters with two or more sequences and 1113 singlets. Thirty percent of all transcripts of 

P. olfersii Duvernoy's gland corresponded to toxin sequences, indicating the presence of 

most of the main toxin classes found in the Viperidae family. The P-III class of 

metalloproteases is the predominant (53% of toxin ESTs), but also serine proteases, C-type 

lectins, Cystein Rich Secretory Proteins (CRISPs), and a C-type natriuretic peptides 

(CNPs) were found. The corresponding proteins were detected in a 2-D gel of the venom 

and identified by mass spectrometry, showing a low venom complexity when compared to 

other snake families. Phylogenetic analysis of the CNP precursor showed it as a linker 

between the multifunctional precursors found in Viperidae and Elapidae snakes. We 

suggest that this precursor constitutes a monophyletic group derived from the vertebrate 

CNPs. Other sequences were investigated and suggested as possible toxin candidates. 

Conclusion: The P. olfesii dbEST is the first effort to massively identify cDNA sequences 

from a Colubridae species. It matches the venom composition, provides an overview of a 

Colubridae venom and supports the important role of metalloproteases in the hemorrhagic 

effect elicited during P. olfersii envenoming. 

Support: FAPESP 

o Colubridae 

o transcriptome 

o Duvernoy's gland 

o C-type natrituretic peptide 

237

Poster 7: A comparison of the venom from left and right fangs of three Australian 

elapid’s (Pseudechis australis, Psuedonaja textiles and Notechis scutatus). 

Winter, K.L., 1* Mirtschin, P.J., 2 Hodgson, W.C. 1 

1 

Monash Venom Group, Dept. Pharmacology, Monash University, Victoria, Australia, 3800. * 

kelly.winter@med.monash.edu.au 

2 

Venom Supplies Pty. Ltd, Tanunda, South Australia, Australia, 5352. 

Introduction: Australia is home to many of the world’s most venomous snakes. Previous 

studies have shown differences in venom components within the same species from 

different geographical locations. Similarly an examination of specimens with different 

body and head sizes has shown that these are related to differences in venom yields 

(Mirtschin et al., 2002). This study examined venom yields and activity from the left and 

right fangs of individual specimens of Pseudechis australis (Mulga snake), Psuedonaja 

textilis (eastern brown snake) and Notechis scutatus (common tiger snake). 

Methods: Venom from the left and right fangs of individual specimens was milked. 

Reverse phase (rp)HPLC was used to compare elution profiles of the venoms. The chick 

biventer cervicis nerve-muscle preparation (0.1 Hz, 0.2 ms, supramaximal V) was used to 

examine the venoms for differences in neurotoxic activity. 

Results: There was a significant difference in the amount of venom produced by the left 

and right fangs of individual snakes. However, rpHPLC showed no major differences in 

the composition of the venoms from each of the species (i.e. no major peaks present or 

missing from either the left or right fang). No difference in neurotoxic activity, as 

determined by the time taken to produce 90% inhibition of indirect twitches (i.e. t90), was 

identified between left and right fangs from the three species examined (see Table). 

Discussion/Conclusions: Although the venom yields between the left and right fangs 

Species / fang side t90 (min), n=4 

P. australis left 111 ± 5 

P. australis right 116 ± 5 

N. scutatus left 83 ± 13 

N. scutatus right 76 ± 16 

P. textilis left 33 ± 6 

P. textilis right 29 ± 4 

o snake 

o venom 

o neurotoxic 

showed variations in the amount of venom 

produced there appears to be no major difference 

in the composition of the venom and the 

neurotoxic activity. The reasons for the 

differences in yields between the left and right 

fangs are not yet apparent. 

Reference: Mirtschin et al., (2002) Toxicon, 40, 

1581-1592. 

238 

Poster 8: Gene Structures of Two Functionally Diverse Prothrombin Activators, 

Trocarin D and Coagulation Factor X in Tropidechis carinatus Snake: Gene 

Duplication and Recruitment of Factor X Gene to the Venom Gland 

Reza, MA 1 , Swarup, S 1 , Kini, RM 1, 2 

1 National University of Singapore, Singapore 

2 Virginia Commonwealth University, Richmond, Virginia 

The occurrence of structurally and functionally similar proteins in specialized organs with 

highly diverse physiological roles within a single organism is a rare phenomenon. Origin 

and evolution of such specialized organs and their functions are of great interest. Recently, 

we showed that several Australian elapid snakes have two similar prothrombin activating 

systems. One exists in plasma and plays a crucial role in hemostasis, whereas the other 

exists in the venom gland and acts as toxin Here we present the complete gene structure of 

these two divergent prothrombin activators from Tropidechis carinatus. Both of the genes 

have 8 exons and all the exon-intron boundaries are almost at the same position. Introns of 

these two genes show high identity (>85%), indicating a recent gene duplication event. 

Real time PCR results indicate that these two genes are expressed in a highly tissuespecific 

manner. Interestingly, the promoter of trocarin D has an insertion of 264 bp (-29 to 

-293). This small insertion carries core promoter sequences and cis-elements that are 

known to induce high level of expression. We speculate that this insert might be 

responsible for the gene recruitment and acts as a switch for venom gland-specific 

expression and we named this segment as VERSE (Venom Recruitment/Switch Element). 

This is the first molecular evidence demonstrating the recruitment of a duplicated gene 

expressed in the liver for expression in a highly specialized organ (venom glands) by a 

simple insertion. 

o Prothrombin activation 

o Factor X 

o Haemostasis 

o Snake venom 

239

Poster 9: Modulation of transcription activities between group IA and IB 

phospholipase A2 genes in sea snake Laticauda semifasciata 

Hayashi, Y,, Sagitani, A., Fujimi, T. J., Kanzawa, N., Tsuchiya, T., Tamiya, T.* 

Sophia University 7-1, Kioi-cho,Chiyoda-ku, Tokyo 102-8554, Japan,* t_tamiya@sophia.ac.jp 

Phospholipase A2 (PLA2) genes expressed in the venom glands of the sea snake Laticauda 

semifasciata, were investigated. Both kinds of mRNAs, encoding group IA (without a 

pancreatic loop) and group IB (with a pancreatic loop), were detected in the venom glands 

by Northern blot hybridization analysis and RT-PCR. Quantitative PCR analysis revealed 

that the amount of group IA mRNA was about 100-300 times greater than that of group IB 

mRNA. Recently, we determined the abundance ratio of group IA to IB PLA2 genes (2:1) 

in the genome of L. semifasciata. The difference between the amount of transcribed 

mRNAs encoding group IA and IB PLA2s did not agree with the number of gene copies. 

Nucleotide sequences of group IA and IB PLA2 genes in the upstream regions were 

determined. The sequences from the transcription initiation site to -33 and -445 to -633 bp 

in the group IA PLA2 genes and from there to -220 bp in the group IB PLA2 genes were 

highly homologous (ecIAIB consensus sequences). Electrophoresis mobility shift assay 

and DNase I footprint assay were carried to verify transcription factor binding sites in the 

ecIAIB conserved region. All the binding sites detected were already registered in the 

Transcription Factor Database. In the group IA gene upstream sequence, the ecIAIB 

consensus sequence was interrupted by a sequence specific to group IA PLA2 genes. A 

CCAAT box (-91 to -87) in the group IB gene was shifted more than 400 bp upstream in 

the group IA gene by the inserted sequence. Thus, the sequence dramatically changed the 

structure of the promoter region between the group IA and IB genes of L. semifasciata. 

Luciferase reporter gene assays were carried out to examine the importance of the group 

IA specific sequence for transcription. The transcription activity of a chimera mutant 

(which had the group IA specific sequence inserted in the appropriate position of the group 

IB gene) showed the same level of group IA PLA2 genes upstream. The IA-specific 

sequence modulated the expression levels of the group IA and IB genes in the venom 

glands. 

o Snake toxin gene 

o Phospholipase A2 genes 

o Promoter region 

240 

Poster 10: Maturation signatures in the sequences of natural toxins 

Kozlov S., Grishin E. 

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho- 

Maklaya, 16/10, 117997 Moscow, Russia 

Recent data collected for animal toxins reveal an important role of maturation processes in 

active molecules formation. Every novel precursor sequence deduced from gene should be 

analyzed to define mature polypeptide chain, before synthesis and activity measurements 

are carried out. In many cases, multiple sequence alignment with known homologues 

produces faithful results, but the exact primary structure can be determined precisely by 

finding specific maturation motifs. We analyze the available sequence data of animal toxin 

precursors obtained from several species of Conus snails, sea anemones, spiders, 

scorpions, bees, ants, and amphibians. The analysis reveals specific motifs for directed 

enzymatic cleavage simultaneously present in toxin precursor sequences immediately 

before and after the mature chain. One of the motifs is well defined for specific maturation 

of polypeptide molecules in various animals, whereas other motifs are found only in 

venomous species. In case of spider and frog cytotoxins, we describe a new “paired” motif 

typical for precursors that are processed into more than two mature active compounds. The 

information from the primary structure of precursor proteins allowed us to produce 

assumable maturation maps that are quite species-specific. Differences were found not 

only in precursor protein organization, but also in the set of specific enzymes thought to be 

involved in toxin processing events. 

o primary structure motif 

o precursor maturation 

o toxins processing 

241

Poster 11: Proteomic analysis of the venom from the Mexican scorpion Centruroides 

limpidus limpidus. 

Gurrola, G.B., Batista, C.V.F., Zamudio, F.Z. and Possani, L.D*. 

Department of Molecular Medicine and Bioprocesses. Institute of Biotechnology – 

National Autonomous University of Mexico *possani@ibt.unam.mx 

Scorpion venoms are complex mixtures of peptides that display a variety of different 

biological activities. These venoms have been studied in the light of their pharmacological 

targets and many of their constituents were described to affect the function of a variety of 

ion channels of excitable and non excitable membranes. This communication describes the 

separation of approximately 90 different components by high performance liquid 

chromatography (HPLC) of the venom from the scorpion Centruroides limpidus limpidus . 

Mass spectrometry analysis of the soluble venom shed light on the molecular composition 

of the venom and facilitated the search for novel pharmacologically active components. At 

least 318 distinct molecular weight components were identified. The complete amino acid 

sequence of 3 peptides was determined and the partial sequence of 10 additional 

components was also identified. The molecular masses of peptides eluting at 30 to 40 min 

elution time were from 6.5 to 8.0 kDa. Most of them correspond to long chain toxins 

specific for voltage-gated Na + channels. The components eluting at 20 to 30 min elution 

time have molecular weights from 2.5 to 5.0 kDa, among which are most of the short-chain 

peptides that block K + -channels. At the higher molecular weight range an enzyme with 

hyaluronidase activity was found, with molecular mass of 44.396 kDa. This enzyme was 

shown to be glycosilated and highly active using hyaluran as substrate. Separation by 

HPLC in the presence of trifluoacetic acid and acetonitrile resulted in a series of isoforms 

with the same molecular masses, but different elution times. The amino acid sequence of 

the N-terminal region of the enzyme was determined. 

Acknowledgements: Supported in part by DGAPA-UNAM IN-206003 to LDP and GBG; 

CONACyT grant number 47879-Q to CVFB and grant from Instituto Bioclon S.A. de C.V. 

(Mexico) 



o Centruroides limpidus limpidus 


242 

Poster 12: Venom Proteomes of Closely-related Sistrurus Rattlesnakes with 

Divergent Diets 

Sanz, L. 1 , Gibbs, H.L. 2 , Mackessy, S.P. 3 , Calvete, J.J. 1,* 

1 


*Libia.Sanz@ibv.csic.es 

2 

Department of Evolution, Ecology and Organismal Biology, Ohio State University, 300 Aronoff 

Laboratory, 318 W. 12th Ave., Columbus, OH 43210-1293, USA 

3 

School of Biological Sciences, University of Northern Colorado, 501 20th St., CB 92, Greeley, CO 

80639-0017, USA 

Venoms represent the critical innovation that allowed advanced snakes to transition from a 

mechanical (constriction) to a chemical (venom) means of subduing and digesting prey 

larger than themselves, and as such, venom proteins have multiple functions including 

immobilizing, paralyzing, killing and digesting prey. Given the central role that diet has 

played in the adaptive radiation of snakes 14 , venom thus represents a key adaptation that 

has played an important role in the diversification of these animals.The protein 

composition of the venoms of the three subspecies of Sistrurus catenatus (S. c. catenatus, 

tergeminus, and edwardsii) and a basal species, Sistrurus miliarius barbouri, were 

analyzed by RP-HPLC, N-terminal sequencing, MALDI-TOF peptide mass fingerprinting 

and CID-MS/MS. The venoms of the four Sistrurus taxa contain proteins from 11 families. 

The protein family profile and the relative abundance of each protein group in the different 

venoms are not conserved. Myotoxins and 2-chain PLA2s were present only in S.c. 

catenatus and S.c. tergeminus, whereas C-type BPP and Kunitz-type inhibitors were 

exclusively found in S.c. edwardsii and Sistrurus miliarius barbouri. Among major protein 

families, taxa were most similar in their metalloproteases (protein similarity coefficient 

value: 34%) while most divergent in PLA2s (12%), with values for disintegrins and serine 

proteases lying between these extremes (25% and 20% respectively). The patterns of 

venom diversity points to either a gain in complexity in S. catenatus taxa or a loss of 

venom diversity occurring early on in the evolution of the group involving the lineage 

connecting S. milarius to the other taxa. The high degree of differentiation in the venom 

proteome among recently-evolved congeneric taxa emphasizes the uniqueness of the 

venom composition of even closely related species which have different diets. 

Comparative proteomic analysis of Sistrurus venoms provides a comprehensive catalogue 

of secreted proteins, which may contribute to a deeper understanding of the biology and 

ecology of these North American snakes, and may also serve as a starting point for 

studying structure-function correlations of individual toxins. 

o Sistrurus 

o Snake venomics 


o Venom protein families 

243

Poster 13: Molecular cloning of a non-venom-secreted PII disintegrin-like 

transcript, BA-5A, from a Bitis arietans cDNA library reveals a pathway for the 

evolution of the long-chain disintegrin bitistatin from a PIII disintegrin-like 

precursor 

Juárez, P. 1* , Wagstaff, S.C. 2 , Oliver, J. 2 , Sanz, L. 1 , Harrison, R.A. 2 , Calvete, J.J. 1 

1 


*pjuarez@ibv.csic.es 

2 

Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool 

L3 5QA, England, UK 

Disintegrins represent a family of integrin receptor antagonists that are released in viper 

venoms by proteolytic processing of PII snake venom metalloproteinase (SVMP) 

precursors or synthesized from short-coding mRNAs. Functional diversification between 

disintegrins is mainly due to amino acid substitutions within the active loop, whereas 

structural diversification was driven through a disulfide bond engineering mechanism 

involving the selective loss of pairs of cysteine residues engaged in the formation of 

disulfide bonds. The evolutionary pressure acting to promote high levels of variation in 

venom proteins may be part of a predator-prey arms race that allow the snake to adapt to a 

variety of different prey, each of which are most efficiently subdued with a different 

venom formulation. However, the molecular details of the mechanisms leading to snake 

venom toxin diversification remains unclear.We report the cloning and sequence analysis 

of BA-5A from Bitis arietans, a novel and unique ECD-disintegrin-like domain.It contains 

the 16 cysteine residues that are conserved in all PIII disintegrin-like domains but lacks the 

cysteine-rich domain. These features suggest that BA-5A represents an intermediate in the 

evolutionary pathway of the long disintegrin bitistatin, and that removal of the cysteinerich 

domain and loss of the PIII-specific disulfide bond were separate events along the 

structural diversification pathway of disintegrins, the former predating the latter. BA-5A 

could not be detected in the venom proteome of Bitis arietans using proteomic techniques. 

The occurrence of this very low abundance (

Poster 15: Gene and primary structure of an insect-specific peptide from the 

venom of the theraposid Brachypelma smithi† 

Corzo, G., Diego, E., Clement, H., Estrada, G., Odell, G., Batista, C., Possani, L.D., Alagón, A. 

Institute of Biotechnology-UNAM, Av. Universidad 2001, Cuernavaca, Morelos, 62210, Mexico. 

*corzo@ibt.unam.mx 

The soluble venom of the theraposid spider Brachypelma smithi (B. smithi) was screened 

for insecticidal toxins based on toxicity to house crickets. This venom was fractionated 

using reverse-phase and cation exchange chromatographies. An insecticidal peptide, 

named Bs1 was obtained. It contains 41 amino acids cross-linked by three disulfide 

bridges. The amino acid sequence of Bs1 is highly similar to the insecticidal peptides from 

the hexathelid spider Macrothele gigas (Japan) (1), and the theraposid spider 

Ornithoctonus huwena (China) (2) indicating their close phylogenetic relationship. The 

gene of Bs1 was cloned from a cDNA library, and genes related to Bs1 showed the 

presence of a wide range of similar toxins with slight variations in primary structure. 

Proteins codified by this family of genes contain signal peptides, which seem to be excised 

at a region rich in acidic and basic residues (3). Moreover, their C-terminals are amidated, 

where the residue that dictates such protein modification is just glycine. The exact target of 

this toxin is not known. Thus insecticidal peptides represent interesting new tools for the 

discovery of new cell receptors in insects. Generation of knowledge on the molecular basis 

of insect toxin specificity has a motivating potential, since they represent a possible 

application, via recombinant baculovirus in crop protection. 

1. Corzo, G. et al. (2003) FEBS Lett. 547:43-50. 

2. Yi, T.F et al. (2002) Zool. Res. 23:463-466. 

3. Satake, H. et al. (2004) Toxicon 44:149-156. 

†This work was supported in part by a grant from the Dirección General de Asuntos del 

Personal Académico (DGAPA-UNAM) IN226006. 

o Insect-specific 

o Genes 

o Spider 

o Peptide 

246 

Poster 16: TRANSCRIPTOME ANALYSIS OF EXPRESSED SEQUENCE TAGS 

FROM Thalassophryne nattereri FISH VENOM GLANDS 

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 

A.M 1* . 

1 2 3 

Laboratório de Imunopatologia, Laboratório de Imunogenética and Centro de Biotecnologia, Instituto 

Butantan, São Paulo, Brazil; * anamoura@butantan.gov.br 

4 

Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade São Paulo, Brazil 

Thalassophryne nattereri (niquim) is a venomous fish found in northern and northeastern 

Brazilian coast. T. nattereri fish induces hundreds of human accidents characterized by 

excruciating local pain, edema, and necrosis that may lead to permanent disabilities. In 

experimental models, T. nattereri venom toxins showed to be highly active causing edema 

and nociception that has been correlated to human symptoms and dependent on venom 

kininogenase activity. Other activities such as myotoxicity; impairment of blood flow; 

platelet lysis and cytotoxicity on endothelial cells were also observed. In an attempt to 

characterize the primary structure of T. nattereri venom toxins, a list of transcripts within 

the venom gland was accomplished using the expressed sequence tag (EST) strategy. The 

analysis of 775 ESTs from the venom gland allowed us to isolate the cDNAs sequence of a 

family of kininogenases never before described in the databases. These sequences were 

named natterin 1, 2, 3, 4 and P. Except natterin 4, all the others were found in the whole 

venom. Furthermore, we found others putative toxins sequences also unknown to the 

databases, as well as interesting findings such as sequences matching with cocaine- and 

amphetamine-regulated transcript (CART), which has been shown to be involved in food 

intake regulation (1.3%), retrotransposon-like sequences (1.0%), which might be related to 

the occurrence and diversity of many paralogous forms of toxins in this venom gland and, 

a considerable number of sequences (32%) did not show any similarities in the databases, 

which indicates that a great number of new toxins/proteins are still to be discovered. 

o Thalassophrine nattereri 

o ESTs 

o natterin 

o toxin 

247

Poster 17: CLONING AND CHARACTERIZATION OF A C-TYPE LECTIN 

FROM Thalassophryne nattereri FISH VENOM GLAND 

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 

.W 6 , Ho P.L 3 , Moura-da-Silva A.M 1* . 

1 Laboratório de Imunopatologia, 2 Laboratório de Imunogenética and 3 Centro de Biotecnologia, Instituto 

Butantan, São Paulo, Brazil; * anamoura@butantan.gov.br 4 PUC, 

Curitiba, Brazil; 5 Fiocruz, Rio de Janeiro, Brazil; 6 University of Virginia, Charlottesville, USA. 

Using a venom gland cDNA library from Thalassophryne nattereri fish, a C-type lectin 

transcript was cloned and characterized. Its 984 pb cDNA contains a 363 bp open reading 

frame from which a 121-amino-acid was deduced. The deduced amino acid sequence was 

found to contain a carbohydrate recognition domain (CRD) homologous with those of 

some known C-type animal lectins, although showing low general homology with those 

lectins. This novel C-type lectin was purified from T. nattereri venom whose N-terminal 

showed to match with the deduced amino acid sequence. This protein, named nattectin, 

revealed a single band with an apparent molecular weight of 15 kDa under reduced and 

unreduced conditions on SDS-PAGE gel. Analysis of the purified protein by MALD-TOF 

revealed a mass of ~15,087 indicating that this protein is composed of a single monomer, 

unlike C-type lectins from snake venoms that are composed exclusively of homodimers or 

homooligomers. Comparison of nattectin with a well characterized rat mannose-binding 

protein showed that it is a galactose-binding lectin that also contains conserved residues for 

Ca 2+ binding. Hemaglutination analysis showed that this lectin is Ca 2+ dependent. To our 

knowledge, this is the first characterization of a lectin from a venomous fish and this 

finding may contribute to elucidate the functions of other venom C-type lectins. 

o C-type lectins 

o Thalassophryne nattereri 

o cDNA library 

o toxin 

248 

Poster 18: Biochemical evidence that crotasin is a fully active gene in tissues of the 

South American rattlesnake Crotalus durissus terrificus 

Collares, M. de A. 1 ; Silva, A. R. P. da 1 ; Rádis-Baptista, G. 2 ; Grego, K. F. 1 ; Oguiura, N. 1* 

1 Laboratório de Herpetologia – Instituto Butantan, Av. Doutor Vital Brasil 1500 – CEP 05503-900 São 

Paulo, SP, Brazil, *nancyoguiura@butantan.gov.br. 

2 Dept. de Bioquímica e Laboratório de Imunopatologia Keizo Asami- Universidade Federal de Pernambuco 

(UFPE), Av. Professor Moraes Rego S/N – CEP 53670-910 Recife, PE, Brazil 

Crotasin, Cts-p2, is a crotamine-related gene of Crotalus durissus terrificus, a South 

American rattlesnake. Both crotamine and crotasin genes have three exons interrupted by 

two introns, one long and the other short. In the Cts-p2 gene, the longer intron showed an 

insertion of approximately 750 bp, and the exon 2 presented most of the non-synonymous 

substitutions. In fact, the overall gene organization shared by crotasin and crotamine is 

indicative of gene duplication and independent molecular evolution. A high level of 

expression of Cts-p2 transcripts was detected by reversed transcription coupled to 

polymerase chain reaction (RT-PCR) in pancreas, heart, liver, brain, and kidneys. 

Interestingly, and unlike crotamine, crotasin transcripts were scarcely detected in venom 

glands 1 . The biological function of crotasin is still unknown. The predicted amino acid 

sequence of crotasin shares a primary structure similarity with Gallus gallus beta-defensin 

2 (GI49037274), which belongs to the diverse class of antimicrobial peptide, and with the 

recently identified peptide POGL2-CLP from the lizard Pogona barbata 2 . This fact 

motivated us to investigate the biological and/or pharmacological activities of crotasin. 

Hence, we evaluated the crotasin expression in snake tissues by Immunoblot, and prepared 

recombinant crotasin fused with thioredoxin. A customized antibody was prepared with the 

synthetic peptide (cys-pro-ser-gly-thr-thr-ser-ile-gly-gln-gln-asp) conjugated with KLH 

carrier by rabbit immunization. Heart, liver, and pancreas proteins were then extracted, 

resolved by SDS-PAGE (15%T/2.6%C) and transferred to a nitrocellulose membrane 

using a semi-dry apparatus. To reveal the pattern of peptide expression in snake tissues, we 

used anti-crotasin as the primary antibody and anti-rabbit IgG conjugated with peroxidase 

as the secondary one. The immune complexes were viewed with DAB/CoCl2. As expected, 

crotasin peptide was detected in pancreas, liver and heart protein extracts, indicating that 

crotasin is a fully active gene in rattlesnakes, and that it may be involved in the innate 

defense mechanism of snake organs. Supported by FAPESP, FUNDAP. 1 Rádis-Baptista et 

al., 2004, Toxicon 43:751-759. 2 Fry et al., 2006, Nature 439: 584-588. 

o rattlesnake 

o crotasin 

o crotamine 

o antimicrobial peptide 

249

Poster 19: The Gene Family of Crotamine and Variations of its Content in Venoms 

of the South American Rattlesnake Crotalus durissus 

Collares, M. de A. 1 ; Corrêa, P. G. 1 ; Suzuki, H. 2 ; Furtado, M. F. D. 1 ; Oguiura, N. 1* 

1 Laboratório de Herpetologia and 2 Laboratório de Biologia Celular – Instituto Butantan, Av. Doutor Vital 

Brasil, 1500 – CEP 05503-900 São Paulo, SP, Brazil 

*nancyoguiura@butantan.gov.br 

Crotalus durissus is a rattlesnake that occurs from southern Mexico to the Argentinean 

pampas. Crotamine, which was first isolated from the venom of C. d. terrificus, belongs to 

a group of closely related small basic polypeptide myotoxins commonly found in Crotalus 

venoms and its content, averaging 17% (w/w) of the crude venom, varies according to the 

snake’s geographical location. The crotamine-positive rattlesnake has its crotamine gene 

with 1.8 kbp organized into three exons separated by a long phase-1 (900 bp) and a short 

phase-2 (140 bp) intron. The amount of crotamine in the venoms of C. durissus from the 

states of Paraná, São Paulo, Mato Grosso do Sul, Goiás, Bahia, and Maranhão were 

determined. The crotamine was quantified by ELISA and the protein content determined 

by the Lowry method. To analyze the crotamine genes, we amplified the sequence of part 

of exon 2, intron 2 and exon 3 by PCR using 5crot and 3UTRas primers. This fragment 

was cloned, and seven clones of each snake were sequenced. The quantity varied from 

0.006 to 0.5 µg of crotamine per µg of protein in the venom. Based on these results, the 

venoms were classified into three categories: crotamine-negative, less than 10% and more 

than 10% of crotamine content, which was plotted on a map. We found a large number of 

crotamine-plus venoms in the state of Paraná, including venom with a crotamine 

concentration exceeding 10%. We analyzed crotamine gene sequences of specimens from 

Paraná, São Paulo, Mato Grosso do Sul, Goiás and which showed an identical sequence to 

that described by Rádis-Baptista et al. (2003) 1 . The changes found in sequences were point 

mutations in introns and in an untranslated region, amino acid changes, and complete 

deletion of intron 2. These results indicated that crotamine is a gene family, since the same 

rattlesnake may have up to four different sequences. The rattlesnakes with the highest 

amount of crotamine showed no alteration in their crotamine genes. 

Supported by FAPESP and FUNDAP. 

1 Rádis-Baptista et al., 2003, Toxicon 42: 747-752. 

o Rattlesnake venom 

o Crotamine 

o Gene family 

o Crotalus durissus 

250 

Poster 20: A profile of typical Viperidae toxins in the Lachesis muta venom glands 

transcriptome 

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, 

M.R.V. 4 

1 

Centro de Biotecnologia, Instituto Butantan - Av. Vital Brasil, 1500 - São Paulo, Brazil- 05503-900 

*ijuncaze@butantan.gov.br 

2 

Laboratório de Bioquímica e Biofísica, Instituto Butantan - São Paulo, Brazil - 05503-900 

3 

Instituto de Química, Universidade de São Paulo – São Paulo, Brazil 

4 

Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias - Belo Horizonte, Brazil. 

Introduction: Snake families possess hundreds of toxin isoforms from a few structural 

types spread within each family but often absent in other. To describe the repertoire of 

common molecules and to reveal the possible new toxins from a Viperidae we carried on a 

transcriptomic analysis of the venom glands of L. muta, the world largest Viperidae snake. 

Methods: A cDNA library was constructed from venom gland mRNA, validated and over 

2500 random clones were sequenced through an Expressed Sequence Tags (ESTs) 

approach. The ESTs were clustered and searched against public databases. 

Results: The analysis of 2095 ESTs clustered in 1162 contigs yielded many new 

representative molecules of nine toxin classes commonly present in Viperidae and their 

expression levels, being mainly BPPs (bradykinin potentiating peptide precursor), metallo- 

and serine proteases, C-type lectins. The BPPs are highly expressed (72% of toxins) and 

revealed an unusual peptide. Surprisingly, we found mRNAs matching two toxin classes 

exclusively described in Elapidae or Colubridae snakes (detailed in a separated abstract). 

Besides the sequences matching venom molecules, other transcripts could be suggested as 

new toxin candidates. These include non-toxin proteins whose activities match known 

features of envenoming (e.g., 5´ nucleotidases and ADAMs) and toxin inhibitors never 

observed in venom or venom glands. The profile of non-toxin transcripts categories also 

shows the specialization of venom glands in synthesizing toxins, with special care to 

disulfide bond assembly, being the PDI (proteins disulfide isomerase) the major non-toxin 

transcript. 

Conclusion: We defined a catalog of transcripts from L. muta, allowing the identification 

of the most common classes of toxins present in Viperidae venoms, which parallels the 

complex physiological effects evoked by the venom, and some new putative toxin 

molecules, providing also a comprehensive resource of reptilian expressed sequences. 

Supported by FAPESP. 


o Expressed Sequence Tags 

o cDNA 

o Viperidae 

251

Poster 21: Atypical Viperidae cDNAs from Lachesis muta venom glands and their 

implication in snake toxin repertoire evolution 

Junqueira-de-Azevedo, I.L.M. 1* , Ching, A.T.C. 1 , Carvalho, E. 1 , Ho, P.L. 1 , Diniz, M.R.V. 2 

1 

Centro de Biotecnologia, Instituto Butantan - Av. Vital Brasil, 1500 - São Paulo, Brazil- 05503-900 

*ijuncaze@butantan.gov.br 

2 

Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias - Belo Horizonte, Brazil. 

Introduction: Efforts to describe toxins from the two major families of venomous snakes 

(Viperidae and Elapidae) usually reveal proteins belonging to few structural types, 

particular of each family. To investigate possible unusual toxins in a Viperidae venom, we 

generated a database transcriptomic sequences from Lachesis muta snake and carried on a 

search for atypical molecules. 

Methods: A cDNA library was constructed from venom gland mRNA, validated and over 

2500 random clones were sequenced through an Expressed Sequence Tags (ESTs) 

approach. The ESTs were clustered and cluster sequences were searched against public 

databases. Atypical molecules were further full-length re-sequenced and phylogeneticaly 

analyzed through Bayesian models. 

Results: Besides nine classes of typical toxins found on the transcriptome (presented in a 

separated abstract), atypical sequences never observed in the hundreds of Viperidae snakes 

studied so far were shown to be highly expressed: (i) a diverging C-type lectin that is 

related to Viperidae toxins but appears to be independently originated; (ii) an ohanin-like, 

that would be the third member of the recently described class of Elapidae toxins, related 

to human butyrophilin and B30.2 proteins; and (iii) a 3FTx-like, a new diverging member 

of the widely studied three-finger family of proteins, which includes major Elapidae 

neurotoxins, CD59 antigen and a growing number of human proteins. 

Discussion: The presence of these common and uncommon molecules suggests that the 

repertoire of toxins could be more conserved between snake families than it was used to be 

considered and their features indicate a dynamic process of venom evolution through 

molecular mechanisms, such as multiple recruitments of important scaffolds and domain 

exchange between paralogs, always keeping a minimalist nature in most toxin structures in 

opposition to their non-toxin counterparts. 

Supported by FAPESP. 


o Molecular Evolution 

o Three finger toxins 

o Ohanin 

252 

Poster 22: The Bitis gabonica gabonica venom proteome: venomics vs. 

transcriptomics 

Calvete, J.J. 1,* , Marcinkiewicz, C. 2 , Sanz, L. 1 

1 



2 Biotechnology Center, Temple University College of Science and Technology, 1900 N. 12th Street, 

Philadelphia, PA 19122-6078, USA 

Snakes of the family Viperidae (vipers and pitvipers) produce a complex mixture of a large 

number of distinct proteins. However, venom proteins belong to only a few major protein 

families, including enzymes (serine proteinases, Zn 2+ -metalloproteases, L-amino acid 

oxidase, group II PLA2) and proteins without enzymatic activity (disintegrins, C-type 

lectins, natriuretic peptides, myotoxins, CRISP toxins, nerve and vascular endothelium 

growth factors, cystatin and Kunitz-type protease inhibitors). In additon to understanding 

how venoms evolve, characterization of the protein content of snake venoms also has a 

number of potential benefits for basic research, clinical diagnosis, development of new 

research tools and drugs of potential clinical use, and for antivenom production strategies. 

Despite its potential value, little is known about the venom protein composition of most 

vipers. Recent reports surveyed gene transcriptional activity (transcriptome) of the snake 

venom glands of Bothrops insularis, Bothrops jararacussu, Bitis gabonica, and 

Agkistrodon acutus by generation of expressed sequence tags (ESTs) or construction of a 

cDNA library followed by sequencing of the clones. These works have provided catalogs 

of full-length venom gland mRNAs. Our proteomic approach complements these studies 

by showing the relative abundance of the proteins that are actually secreted into the 

venoms.We have analyzed the protein composition of the crude venom of Bitis gabonica 

by RP-HPLC, N-terminal sequence analysis, MALDI-TOF MS, and in-gel tryptic 

digestion followed by CID-MS/MS. This approach allowed us to assign unambiguously all 

of the isolated venom fractions to protein families present in the non-redundant databases. 

Our proteomic approach complements transcriptomic studies by showing the relative 

abundance of the proteins that are actually secreted into the venoms. On the other hand, 

comparison of the proteomic versus the transcriptomic data showed significant differences 

for a number of protein families, indicating lack of correlation between the transcriptional 

and the translational activity of the venom gland. The use of protein-chemical techniques 

allowed us also to determine the subunit composition of expressed venom proteins. 

o Venom proteome 


o N-terminal sequencing 

o Bitis gabonica gabonica 

253

Poster 23: Loss of introns along the evolutionary diversification pathway of snake 

venom disintegrins evidenced by sequence analysis of genomic DNA from 

Macrovipera lebetina transmediterranea 

Bazaa, A. 1 , Juárez, P. 2 , Marrakchi, N. 1 , Bel Lasfer, Z. 1 , El Ayeb, M. 1 , Calvete, J.J. 2,* , Sanz, L. 2 

1 

Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, B.P. 74, 1002 Tunis-Belvédère, Tunisia. 

2 



Venom toxins likely evolved from endogenous proteins with normal physiological 

functions that were recruited into the venom proteome. Notably, most venom toxins are 

extensively cross-linked by disulfide bonds and have flourished into functionally diverse, 

toxin multigene families that exhibit interfamily, intergenus, interspecies, and intraspecific 

variability. The existence in the same venom of a functionally diverse isoforms of the same 

protein family reflects accelerated Darwinian evolution. The evolutionary pressure acting 

to promote high levels of variation in venom proteins may be part of a predator-prey arms 

race that allow the snake to adapt to a variety of different prey, each of which are most 

efficiently subdued with a different venom formulation. Disintegrins, a broad family of 

small, cysteine-rich polypeptides isolated from venoms of vipers and rattlesnakes, are 

released in viper venoms and selectively block the function of cell surface adhesive 

receptors of the integrin family. Analysis of cDNAs from Macrovipera lebetina 

transmediterranea and Echis ocellatus venom gland libraries encoding disintegrins 

strongly argued for a common ancestry of the messengers of short disintegrins and those 

for precursors of dimeric disintegrin chains. We now report the sequence analysis of 

disintegrin-coding genes from Macrovipera lebetina transmediterranea. Genomic DNAs 

for dimeric disintegrin subunits Ml_G1 and Ml_G2 contain single 1 kb-introns exhibiting 

the 5´-GTAAG (donor)/3´-AG (acceptor) consensus signature of group I self-splicing 

introns. On the other hand, the short RTS-disintegrin Ml_G3 is transcribed from an 

intronless gene, indicating that the evolutionary pathway leading to the emergence of short 

disintegrins involved the removal of all intronic sequences. The insertion position of the 

Ml_G1 and Ml_G2 intron is conserved in the genes for vertebrate ADAM´s disintegrinlike 

domains and within the gene for the medium-size snake disintegrins halystatin 2 and 3. 

However, a comparative analysis of currently available disintegrin(-like) genes outline the 

view that a minimization of both the gene organization and the protein structure underlie 

the evolution of the snake venom disintegrin family. 

o Macrovipera lebetina transmediterranea 

o Evolution of disintegrins 

o Disintegrin gene structure 

o Genomic DNA sequencing 

254 

Poster 24: Current state of Bothrops insularis venom proteome. A joint effort of the 

Rio de Janeiro Proteomic Network. 

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 ., 

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 ., 

Oliveira-Carvalho, A.L 3,5 ., Wermelinger, L.S 3,5 , Zingali, R.B 3,5 . and Domont, G.B 4 . 

1 

Physiology and Pharmacodynamics Department, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, 

Brazil 

2 

Instituto Butantan, São Paulo, SP, Brazil 

3 

Medical Biochemistry Institute, UFRJ, Rio de Janeiro, RJ, Brazil 

4 

Biochemistry Department, Instituto de Química, UFRJ, Rio de Janeiro, RJ, Brazil 

5 

Rio de Janeiro Proteomic Network 

Bothrops insularis is an endemic snake found in Queimada Grande island, Brazilian coast, 

that feeds on birds and some invertebrates available in its habitat. It is thought that 

evolutionary pressure could have resulted in an adaptation of its venom components 

providing divergent toxins to better capture the preys. Dr Paulo L Ho and colleagues 

(Instituto Butantan, SP, Brazil) are performing a survey of gene expression and diversity in 

B. insularis venom glands and through the generation of ESTs putting out a wealthy 

genomic data. Thus, B. insularis venom seems to be a good model system in order to study 

toxin expression. The strategy for the elucidation of the proteome used 2D-PAGE. 

Samples were dissolved in 8-9.5M Urea, 2%(w/v) CHAPS, 20mM Dithiotreitol, 

0.002%(w/v) Bromophenol blue and 4-7 IPG buffer or 3-10 Biolyte. The first dimension 

was performed on 4-7 IPG strips and the second dimension on a 15%T SDS-PAGE under 

reducing conditions. From the reference gel [1.8 mg, dry weight, venom by in gel 

rehydration with 0.5% (v/v) 4-7 IPG buffer] major spots were used to generate tryptic 

hydrolysates for peptide mapping by MALDI-TOF MS and/or sequencing by LC-ESI-ION 

TRAP MS and MALDI-TOF-TOF MS/MS. Searches in Gene/ ESTs data banks allowed 

identification and a possible function assignment to 67 proteins scattered all over our 2D 

reference, distributed between 12-70 kDa and 4-7 pI. They include metalloproteinases, 

serineproteinases, phospholipases A2, platelet agglutinating inhibiting protein, lectins, 

platelet glycoprotein Ib-binding protein alpha and beta subunits, vascular endothelial 

growth factor, convulxin beta, anticoagulant protein A and metalloproteinase degradation 

products. They also showed that the horizontal streaking observed in our reference gel is 

not due to artifacts or bad sample preparation but rather to a large number of protein 

isoforms and/or glycosylated molecules, which make snake venom 2D analysis not so 

straightforward as expected due to the ease of sample preparation. 

Supported by: FAPESP, FAPERJ and CNPq 

o B. insularis 

o Venom 

o Proteome 

o 2D-PAGE 

255

Poster: 25: Bothrops jararaca Venom Proteome. 

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, 

R.M. 2 , Domont, G.B. 3,4 and Perales, J. 1,4 

1 Physiology and Pharmacodynamics Department, Instituto Oswaldo Cruz, FIOCRUZ, RJ, Brazil 

2 Biochemistry Department, UERJ, RJ, Brazil 

3 Biochemistry Department, Instituto de Química, UFRJ, RJ, Brazil 

4 Rio de Janeiro Proteomic Network 

Mortality and morbidity data indicate that snake bite envenomation remains a public health 

problem in Brazil. Approximately 85 percent of reported envenomations are caused by 

bothropic species and most of these specifically by Bothrops jararaca. The study of its 

venom proteome could shed some light on mechanisms of action by unraveling previously 

unknown proteic components (toxins, enzymes, biologically active peptides etc.) that could 

not be detected by traditional protein chemistry. As an aid to protein identification we have 

generated expressed sequence tags (ESTs) from a venomous gland cDNA library and made 

the data available as an EST sequence bank. 2D-PAGE and mass spectrometry analyses of 

tryptic hydrolysates of gel spots were used to separate and identify proteins. Venom 

separation performed using 3-10 non-linear and 4-7 linear pH IPG strips (IEF, first 

dimension) and 15%T (SDS-PAGE, second) yielded by Coomassie blue staining, 337 and 

521 spots, respectively. Identified proteins represent toxins known to be present in this 

snake venom. B. jararaca venom was also submitted to size-exclusion chromatography 

and three pools were analyzed by 4-7 linear pH range 2D-PAGE in order to examine 

venom subproteomes. Results indicate enrichment in different regions of the gel when 

compared to whole venom fractionation. Detection of low molecular weight components in 

the high molecular weight size exclusion fraction indicates their possible interaction in the 

native state and/or artifactual data due to abnormal behavior (eg. interaction with column 

resin) during chromatography. The use of proteomic techniques like 2D-PAGE and mass 

spectrometry can be a very productive approach to snake venom studies allowing better 

understanding of venom complexity and toxic properties leading to more effective 

antivenom therapy in the near future. 

Supported by FAPERJ, FIOCRUZ and CNPq 

o Bothrops jararaca 

o Venom 

o Proteome 

o 2D-PAGE 

256 

Poster 26: Post-translational amino acid epimerization in an excitatory conotoxin. 

Structures of the L- and D-forms. 

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 

1 



2 

Department of Biology, University of Utah, Salt Lake City, Utah, 84112, USA 

3 

Cognetix, Inc., 421 Wakara Way, Suite 201, Salt Lake City, Utah, 84108, USA 

The 46-residue conotoxin r11a, from the fish-hunting species Conus radiatus, is a member 

of the recently characterized I-superfamily. Conotoxins belonging to this group exhibit 

excitotoxic activity: several members were shown to block activity of K + channels. R11a 

has four disulfide cross-links. Phe44, the third residue from the C terminus, is posttranslationally 

modified to a D- configuration. The naturally occurring D-Phe form of r11a 

is significantly more active as an excitotoxin than the L-Phe analogue both in vitro and in 

vivo (1). 

In this study we have determined the solution structures by using NMR spectroscopy of 

both forms of this toxin. The connectivities of the eight half-cystines were determined 

from preliminary structure calculations and confirmed chemically to be 5-19, 12-22, 18-27, 

and 21-38. This pattern suggests that r11a has an ICK motif structure with one additional 

disulfide (21-38). Indeed, it is the same pattern seen in the funnel-web spider toxin, 

robustoxin, ie.1-4/2-6/3-7/5-8 (2). 

Apart from the first few residues, the structure of r11a is well defined up to around residue 

35, after which the molecule becomes increasingly disordered. The ordered region does 

indeed adopt an ICK motif structure. The C-terminal region, where Phe44 resides, is 

highly disordered and has chemical shifts consistent with a lack of structure. Comparison 

of the synthetic D-Phe44 and L-Phe44 forms of the molecule indicates that this 

isomerization has very little effect on the structured region. However, the effect of this 

isomerization on activity implies that that this region makes contact with the relevant 

receptor and presumably becomes more ordered as a result. 

1. Buczek, O., Yoshikami, D., Bulaj, G., Jimenez, E.C., and Olivera, B.M. (2005) 

J. Biol. Chem. 280, 4247–4253. 

2. Pallaghy, P.K., Alewood, D., Alewood, P.F., and Norton, R.S. (1997) FEBS Lett. 419, 

191-196. 

o Conotoxin 

o Post-translational modification 

o Structure 

o Disulfide connectivities 

257

Poster 27: Biochemical characterization of venom from the South American 

colubrid Philodryas patagoniensis 

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 , 

Cogo, J.C. 1,* 

1 Laboratório de Fisiologia, Instituto de Pesquisa e Desenvolvimento (IPD) e Serpentário do Centro de 

Estudos da Natureza, Universidade do Vale do Paraíba (UNIVAP), Av. Shishima Hifumi, 2911, 

Urbanova, 12244-000, São José dos Campos –SP, Brazil. * jccogo@univap.br 

2 Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de 

Campinas (UNICAMP), 13083-970, Campinas, SP, Brazil 

Introduction: The venoms of the South American colubrids Philodryas olfersii and 

Philodryas patagoniensis cause edema, hemorrhage, necrosis, alterations in blood 

coagulation and neuromuscular blockade in vitro. In this work, we examined some of the 

biochemical activities of P. patagoniensis venom. Methods: Venom was obtained from 

adult specimens of P. patagoniensis maintained in the serpentarium at UNIVAP and 

lyophilized. The esterase, phospholipase A2 (PLA2) and proteolytic activities were 

assayed. SDS-PAGE was used to determine the electrophoretic profile and venom was 

fractionated by reverse-phase chromatography (RPC) on a Source 15 column 

(Pharmacia). The neuromuscular activity was tested in chick biventer cervicis muscle 

preparations, and creatine kinase (CK) activity was measured using a commercial kit. 

Results: The esterase, PLA2 and proteolytic activities were 7.0+0.1 U/mg, 17+0.5 ΔA420 

nm/mg and 162.3+1.3 U/mg, respectively. For comparison, the corresponding values for 

venom of the pitviper Bothrops jararaca were 49.0+4.5, 124+26.3 and 47+0.9, 

respectively (mean+SEM, n=4 each). SDS-PAGE showed that the venom contained 

proteins with molecular masses of

Poster 29: Purification and partial characterization of a deoxyribonuclease II from 

Bothrops alternatus (urutu) venom 

Nascimento, J.M. 1,2,* , Collares-Buzato, C.B. 3 , Hyslop, S. 1 

1 


(UNICAMP), CP 6111, 13083-970, Campinas, SP, Brazil. *jminardi@unicamp.br; hyslop@fcm.unicamp.br 

2 

Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), CP 6109, 

13083-970, Campinas, SP, Brazil 

3 

Departamento de Histologia e Embriologia, Instituto de Biologia, Universidade Estadual de Campinas 

(UNICAMP), CP 6109, 13083-970, Campinas, SP, Brazil 

Introduction: Snake venoms contain a variety of enzymes that degrade nucleic acids and 

their constituents, including deoxyribonuclease, 5´-nucleotidase, phosphatases, 

phosphodiesterase and ribonuclease. In mammals, acidic DNase II has been implicated in 

DNA fragmentation during apoptosis. In this work, we describe the purification and 

characterization of DNase II from Bothrops alternatus snake venom. Methods: 

Deoxyribonuclease II was purified from B. alternatus venom using a combination of ionexchange 

(SP-Sepharose, heparin-Sepharose), gel filtration, affinity ConA-Sepharose and 

reverse-phase chromatographies. All fractions were screened for enzymatic activity 

(increase in A260 nm) using salmon testes DNA (75 μg/ml) in 0.15 M acetate buffer, pH 

4.7. The cross-reactivity with commercial equine antivenom was assessed by 

immunoblotting and by ELISA, the latter using affinity-purified IgG from commercial 

antibothropic antivenom (Instituto Butantan) followed by detection with a rabbit anti-horse 

IgG-peroxidase conjugate. Results: DNase II was purified with a specific activity of 

1.9x10 3 units/mg compared to 36.1 units/mg for venom (purification factor = 51.2) and a 

protein yield of 1.75%. SDS-PAGE showed a single band with a molecular mass of 24.6 

kDa that was unaffected by dithiothreitol or β-mercaptoethanol. Immunoblotting also gave 

a single protein band with the same molecular mass and, together with ELISA, indicated 

that this protein was immunogenic since it interacted with antivenom and purified IgG. 

DNase II cleaved double-stranded DNA, denatured DNA, circular DNA from the plasmids 

pGEM and pBR 322, and nucleosomal DNA isolated from rat spleen cells; there was no 

degradation of RNA. The enzyme was active in the pH range of 4.5-5.5, with an optimum 

of pH 4.7; activity was lost at >50 o C. Enzymatic activity was inhibited by 

aurintricarboxylic acid (25 µM), iodoacetamide (1 mM), and Zn 2+ (10 mM). Conclusion: 

Bothrops alternatus venom contains a DNase II that shares several characteristics with 

mammalian acidic DNases. This enzyme could contribute to DNA degradation and 

apoptosis following envenomation. Financial support: CNPq, FAPESP 

o Bothrops alternatus 

o Deoxyribonuclease II 

o Purification 

o Characterization 

260 

Poster 30: LC-MS Analysis of All PSP Toxins Using Anion Exchange and Reverse 

Phase Columns Connected in Series 

Sachio Nishio 1* ,Takefumi Sagara 1 , Shigeto Taniyama 2 , Tamiko Hashimoto 1 , Naoyoshi Nishibori 1 and 

Manabu Asakawa 2 

1 

Faculty of Junior College, Shikoku University, 123-1, Ebisuno, Furukawa, Ohjin-cho, Tokushima, 

Japan, * sachio-nishio@shikoku-u.ac.jp 

2 

Graduate School of Biosphere Science, Hiroshima University, 1-4-4, Kagamiyama, Higashi-Hiroshima, 

Hiroshima, Japan 

All PSP toxins, such as disufonated protogonyautoxins (PXs, C toxins), monosufonated 

gonyautoxins (GTXs) and basic saxitoxins (STXs), were measured with newly developed 

two-column chromatography. A Hitachi Gel 3013N column (Hitachi, 50 mm x 2.7 mm 

i.d.) and a Deverosil RP-AQUEOUS-AR column (Nomura Chem., 250mm x 3 mm i.d.) 

were connected in series. Both a mobile phase A, 0.1% heptafluorobutylic acid /10mM 

ammoniumacetate, and a mobile phase B, 0.1% pentafluoropropionic acid /10mM 

ammoniumacetate /4% acetonitorile, were adopted under a gradient system at a flow rate 

of 0.25 mL/minutes. PXs and GTXs were eluted from the columns with the mobile phase 

A. The 100% A was changed at 20 minutes to 100% B. The former column was 

maintained at 50˚C throughout the run. And the latter one was at 20˚C until 30 minutes for 

the both detection of PXs and GTXs , and then the column was heated to 40˚C to perform 

the dissociation of STXs with the mobile phase B. The PSP toxins eluted from the twocolumn 

were identified from the results of mass chromatograms and mass spectra on 

measurement of their LC-MS. The mass spectrometry, HITACHI M-8000 LC-MS, was 

successfully measured to detect the PSP components eluted from the two-column. It was 

equipped with sonic spray ionization interface (SSI). The mass numbers of desulfonated 

and protonated ions of gonyautoxin5 (GTX5) and protonated saxitoxin (STX) were 

calculated as m/z 300. Those of GTX6, GTX3, GTX2, PX1 (C1), PX2 (C2), nSTX and 

hySTX were m/z 316, GTX1, GTX4, hynSTX and PX3(C3) were m/z 332, dcGTX2, 

dcGTX3 and dcnSTX were m/z 273, and dcSTX was m/z 257, respectively. 

A new method of two-column HPLC made it possible to determine all PSP components 

in one chromatographic run of 60 minutes. A good separation and an unvaried retention 

time for each PSP toxins were clarified by repeat determinations. In addition, a fine 

correlation between the LC-SSI-MS and LC with post-column oxidation and fluorescence 

detection (LC-ox-FLD) were performed in this study. 

o All PSP toxins 

o LC-MS 

o LC-ox-FLD 

261

Poster 31: Fishing Toxins out of Snake Venoms Using the Antitoxin DM43 as a Tool 

for the Analysis of Subproteomes 

Rocha, S.L.G 1 ., Neves-Ferreira, A.G.C 1 ., Chapeaurouge, A 1 ., Valente, RH 1 ., Trugilho, M.R.O 1 ., Léon, 

I.R 1 ., Domont, G.B 2 . and Perales, J 1* . 

1Departamento de Fisiologia e Farmacodinâmica, Instituto Oswaldo Cruz, Rio de Janeiro, Brasil 

jperales@ioc.fiocruz.br and 2 Departamento de Química de Proteínas, Instituto de Química, Universidade 

Federal do Rio de Janeiro, Rio de Janeiro, Brasil. 

Snake venoms are complex mixtures of proteins and peptides with different biological 

activities, many of them very toxic. Several animals, including the opossum Didelphis 

marsupialis, are resistant to snake venoms due to the presence of neutralizing factors in 

their blood. An antihaemorrhagic protein named DM43 was isolated from opossum serum. 

It inhibits snake venom metalloproteinases through non covalent complex formation with 

these enzymes. In this study, we have used DM43 and proteomic techniques to explore 

snake venom subproteomes. Several venoms were chromatographed through an affinity 

column containing immobilized DM43. Bound fractions were analyzed either by SDS- 

PAGE and/or 2D-PAGE, followed by identification by MALDI-TOF/TOF MS. Following 

this methodology, we could classify venoms from Bothrops alternatus, B. asper, B. atrox, 

B. insularis, B. jararaca, B. jararacussu, B. moojeni, B. neuwiedi, Crotalus adamanteus, 

C. atrox, C. durissus terrificus, Lachesis muta muta and Naja naja atra according to their 

relative content of metalloproteinases (PI, PIII and/or their fragments). Venom fractions 

not bound to DM43 column were equally analyzed and were composed basically of serine 

proteinases, C-type lectins, L-amino acid oxidases, nerve growth factor, metalloproteinases 

and/or some unidentified spots. Studied venoms presented important proteic variability, 

with frequent detection of multiple forms of the same protein and several members of the 

same protein family. DM43 affinity chromatography associated with proteomic techniques 

showed to be useful tools to separate and identify proteins from snake venoms. 

Financial Support: FAPERJ, CNPq and Fiocruz. 

o Snake Venoms 

o Proteome - Subproteomes 

o Antitoxin DM43 

o Didelphis marsupialis 

262 

Poster 32: Novel peptides in Philodryas olfersii and Philodryas patagoniensis 

venoms detected by bidimensional liquid chromatography coupled to tandem 

nano-electrospray mass spectrometry and de novo mass spectrum analysis 

Souza, G.H.M.F. 1,2,* , Cogo, J.C. 3 , Eberlin, M.N. 2 , Hyslop, S. 1 

1 Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas 

(UNICAMP), CP 6111, 13083-970, Campinas, SP, Brazil. *desouz@fcm.unicamp.br 

2 Laboratório Thomson de Espectometria de Massas, Instituto de Química, Universidade Estadual de 

Campinas (UNICAMP), 13083-970, Campinas, SP, Brazil. 

3 Laboratório de Fisiologia e Farmacodinâmica, Instituto de Pesquisa e Desenvolvimento (IPD) and 

Serpentário do Centro de Estudos da Natureza (CEN), Universidade do Vale do Paraíba 

(UNIVAP), São José dos Campos, SP, Brazil. 

Introduction: Colubrid venoms contain a variety of enzymes and toxins, but their content 

of low molecular mass peptides remains poorly studied. In this work, we used mass 

spectrometry to examine the peptide content of venoms from the South American 

colubrids Philodryas olfersii and Philodryas patagoniensis. 

Methods: Venom was obtained from adult specimens of P. olfersii and P. patagoniensis 

maintained at UNIVAP. Lyophilized venom was dissolved in 0.1% trifluoroacetic acid 

containing 50% acetonitrile and filtered by centrifugation through a 5 kDa cut-off 

membrane (Millipore). The resulting low molecular mass fraction was analyzed by 

quadrupole/time-of-flight mass spectrometry (Qtof) using capillary liquid-chromatography 

and nano-electrospray ionization. 

Results: Qtof-MS revealed 21 peptides in P. olfersii venom and 51 peptides in P. 

patagoniensis, with molecular masses of 500-2500 Da. Complementary online CapLCnano-ESI-MS 

confirmed the presence of these 72 peptides. Various peptides showed posttranslational 

modifications such as an amidated C-terminus and pyroglutamic acid at the 

N-terminus. The peptides that gave the most intense ion signals were EAGTPLSV (754.27 

Da) in P. olfersii and NEPPLWVPG (1007.44 Da) in P. patagoniensis. BLAST searches 

for homologies to the peptides failed to identify any corresponding venom peptides or 

proteins, but revealed considerable homology (at least 80% similarity) with other 

vertebrate (chicken, chimpanzee, duck, frog, ox, human, mouse) proteins. 

Conclusion: The venoms of P. olfersii and P. patagoniensis contain numerous novel 

peptides, the biological functions of which remain to be determined. The lack of homology 

with other snake venom proteins (principally crotalid, elapid, and viperid) indicates either 

that there are indeed no venom proteins homologous with these peptides or that our current 

knowledge of snake venom gland composition is far from complete. Screening of these 

peptides using bioassays in vitro and in vivo could lead to the identification of compounds 

with potential therapeutic applications. Financial support: CAPES, CNPq, FAPESP. 


o Peptides 

o Venom 

263

Poster 33: Development of a novel fluorometric assay for the evaluation of BoNT/A 

protease activity using genetically engineering a fluorogenic substrate 

Ban, B. 1 Yang, G.H. 1 1, 2 

, and Jung, H. H. 

Microbial Toxin Research Institute, Medy-Tox Inc. 1 100, Kalsan ri, Tangjeong myeon, Asan si, Chungnam, 

336-708, South Korea 

Division of Applied Molecular Biology, Sun Moon University 2 100, Kalsan ri, Tangjeong myeon, Asan si, 

Chungnam, 336-708, South Korea 

Botulinum neurotoxins (BoNTs), the most toxic biological substances known to human, cause 

botulism by entering neurons and cleaving SNARE proteins that mediate neurotransmitter release; 

disruption of exocytosis results in paralysis and death. The lack of high-throughput screening (HTS) assay 

has limited the screening of new antidotes against botulism, and also rapid determination of exposure to 

BoNT is an important public health goal. In this study, a novel fluorometric assay was developed and 

successfully applied for the assessment of protease activity of BoNT/A. Target truncated protein (substrate) 

for BoNT/A was engineered to be recombinant fusion protein where a green fluorescent protein is linked to 

cellulose binding domain via the substrate protein that is cleavable by BoNT/A. The cleavage of fusion 

protein by BoNT/A results in emission of fluorescence light that is easily detected and quantitated by 

fluorometric assay. Even though the concentration around 1ng/ml of BoNT/A produced significant 

fluorescence signals; the routine toxin concentration was 10 ng/ml or higher. In total, it could be concluded 

that this assay is a convenient, reliable, and less labor intensive alternative for the detecting protease 

activity of BoNT/A in vitro, and even suitable for screening large numbers of compounds for potential 

anti-BoNT/A drugs. 

Botulinum neurotoxins (BoNTs), High-throughput screening (HTS), Enhanced Green Fluorescence 

Protein (EGFP), Cellulose Binding Domain (CBD) 

264 

Poster 34: PURIFICATION AND PARTIAL ENZYMATIC 

CHARACTERIZATION OF THE HYALURONIDASE FROM Crotalus durissus 

terrificus SNAKE VENOM 

Cordeiro, A. T. 1 ; Tolini, M.M. 1 ; Bertazzi, D. T. 1 ; Giglio, J. R. 2 ; Arantes, E. C. 1 * 

1University 

of São Paulo, FCFRP, Dept. Física e Química, Av. do Café s/n, Ribeirão Preto, SP, Brazil. 

2 

University of São Paulo, FMRP,Depto. Bioquímica e Imunologia, Av. Bandeirantes, Ribeirão Preto, SP, 

Brazil. * ecabraga@fcfrp.usp.br 

Introduction: Hyaluronidases are enzymes that catalyze the breakdown of hyaluronan, a 

high molecular weight polysaccharide found in the extracellular matrix. They are produced 

by a variety of bacteria and are also found in leech, snake and insect venoms, as well as in 

malignant tissues. Hyaluronidases have been employed therapeutically to increase the 

speed of absorption of fluids, to promote resorption of extravasated blood and to increase 

the effectiveness of local anesthetics. Hyaluronidases in venoms are spreading factors 

because they facilitate toxin diffusion into the tissues of the prey, thus contributing to 

systemic envenomation. The aim of this study was the isolation and partial enzymatic 

characterization of the hyaluronidase from Crotalus durissus terrificus snake venom 

(CdtV). 

Methods: The purification procedure involves basically an ion-exchange chromatography 

on CM-Sepharose FF, which was equilibrated and eluted with 0.05 M, pH 5.5 sodium 

acetate buffer up to 50 mL effluent, when a linear concentration gradient was started from 

0 to 1M NaCl in the same buffer. The active fraction III was filtered on Sepharose G75 

column at pH 5.5. In order to find the optimal conditions for hyaluronidase activity, CdtV 

and hyaluronic acid were incubated for 15 min at different pHs (2 to 10) and temperatures (0 

to 70°C) and the enzymatic activity was determined according to Pukrittayakamee et al. 

(Toxicon, 26, 639, 1988). 

Results: The optima pH and temperature for maximum activity of the CdtV were 5.5 and 

40 o C, respectively. The specific activity of the isolated enzyme (~80 kDa) was 48,000 

turbidity reducing units (TRU)/mg against 182 TRU/mg for the soluble venom, 

representing a 263 to 264 fold purification range 

Conclusion: Despite being relatively simple, this purification procedure was able to afford 

a highly purified and active hyaluronidase from C.d. terrificus venom. 

Financial Support: CNPq and FAPESP. 


o Snake venom 

o Crotalus durissus terrificus 

265

Poster 35: Mass spectrometry strategies for venom mapping and peptide sequencing 

from crude venom of a single Bombus specimen 

Menin L., Favreau P., Michalet S., Perret F., Cheneval O., Stöcklin M., Bulet P. and Stöcklin R. 

Atheris Laboratories, Case Postale 314, CH 1233 Bernex - Geneva, Switzerland. 

admin@atheris.ch 

Venoms are a complex set of bioactive molecules including peptides and proteins that 

display a wide array of functions. Due to their complexity and diversity, animal venoms 

represent an extensive source of bioactive compounds for the development of drugs and 

therapeutic agents. Conventional approaches for their characterization often require large 

quantities of biological material and efficient biological assays. Current mass spectrometry 

(MS) techniques through a variety of approaches now give access to a wealth of 

information in a short working time frame with minute sample amount. Currently, MSbased 

peptide and protein discovery may rely on strategies going from structure 

information to the functional activity. MALDI-TOF MS is a soft ionization method that 

requires minimal sample preparation and can be used to rapidly perform molecular mass 

fingerprints of venoms at a high sensitivity. Such molecular mass peptide profiles allow 

the quick identification of known toxins as well as of the identification of new compounds. 

Nevertheless, further characterization requires the use of de novo MS/MS peptide 

sequencing. This approach of peptide sequencing offers rapid access to total or partial 

primary peptide structures. A specific instrumentation is required for such studies such as 

ion traps, triple quadrupole, quadrupole time-of-flight, quadrupole ion trap or MALDI- 

TOF-TOF. We will exemplify an approach with the molecular mass fingerprint analyses 

and structural characterizations of components of the venom collected from a single 

bumblebee Bombus lapidarius specimen. Among the numerous components detected in the 

Bombus venom, three major peptides were fully characterized on a working day basis by 

de novo sequencing using ESI-QqTOF and MALDI-LIFT-TOF-TOF mass spectrometers. 

These peptides belong to the mast cell degranulating peptides named bombolitin and 

previously characterized from a Megabombus species (1). 

References: 1. Argiolas and Pisano (1985) J. Biol. Chem. 260, 1437-1444. 

o Venom mass fingerprint 

o MS/MS de novo sequencing 

o Toxins 

o Bombus venom 

266 

Poster 36: A New Family of Peptides Discovered in Snake Venoms from the 

Atheris genus by Mass Spectrometry Techniques 

Favreau, P. 1 , Cheneval, O. 1 , Menin L. 1 , Michalet, S. 1 , Principaud, F. 2 , Thai, R. 3 , Ménez, A. 3 , Bulet, P. 1 

and Stöcklin, R. 1 * 

1 Atheris Laboratories, case postale 314, CH-1233 Bernex-Geneva, Switzerland 

2 Latoxan, 20 rue Léon Blum, F-26000 Valence, France 

3 CEA/Saclay, Département d'Ingénierie et d'Etudes des Protéines, F-91191 Gif sur Yvette, France 

The discovery of novel molecules of potential biomedical interest can be initiated from a 

structurally driven strategy based on mass spectrometry (MS) techniques. The use of MSbased 

strategies is greatly cost-effective regarding the amount of material needed to obtain 

structural information from complex mixtures 1 . 

In order to find new components, the venoms of numerous snakes were investigated using 

matrix-assisted laser desorption ionisation time-of-flight and electrospray ionisation 

tandem mass spectrometry. Molecular mass fingerprints were obtained and this allowed 

the identification and characterization of an original set of peptides with molecular masses 

ranging from 2 to 3 kDa in the snake venoms of the Atheris genus. 

Following de novo sequencing by tandem mass spectrometry, numerous peptides were 

found to exhibit characteristic structural features consisting of contiguous poly-His and 

poly-Gly segments. Total amino acid sequence assignments were completed by Edman 

sequencing and these compounds were named pHpG peptides because of their original 

structural organisation. Using a similar strategy, we have detected pHpG peptides in 

venoms from other Viperidae but also from some Crotalidae, indicating that the occurrence 

of the pHpG peptide family is not restricted to the Atheris genus. 

Bioinformatic investigations did not reveal any relevant homology in protein databases. 

However, a deeper investigation revealed interesting sequence similarities with several 

bradykinin potentiating peptide (BPP) and C-type natriuretic peptide (CNP) precursors, 

indicating that pHpG peptides may be coded by the BPP/CNP precursors. As no clear cut 

similarity was observed between pHpG and other already identified peptides, the exact 

function of this new set of peptides remains to be elucidated. 

References: Favreau P., Menin L., Michalet S., Perret F., Cheneval O., Stöcklin M., Bulet 

P., Stöcklin R. (2006) Mass spectrometry strategies for venom mapping and peptide 

sequencing from crude venoms: case applications with single arthropod specimen. 

Toxicon, 47, 676-687. 

o snake venom 

o mass spectrometry 

o poly-His poly-Gly peptide 

o BPP/CNP 

267

Poster 37: Quantitative high-throughput LC-MS detection of cyanotoxins in 

aquatic tissue samples 

Lähde, M.-R. , Meriluoto, J.A.O. 

Åbo Akademi University, Department of Biochemistry and Pharmacy, Biocity, Turku/Åbo, Finland, 

mlahde@abo.fi 

Introduction: Microcystins and nodularins (MC/NOD) are hepatotoxic bioaccumulating 

peptides produced by cyanobacteria. High-throughput (HTP) detection methods for 

cyanotoxins are needed in e.g. hygienic and ecotoxicological investigations. The aim of 

this study was to test an HTP LC-ESI-MS system for quantitative detection of MC/NOD in 

aquatic tissue samples. 

Methods: LC-MS analysis on two triple-quadrupole instruments: a Micromass Quattro II 

coaxial electrospray, and a Quattro Micro Z-spray electrospray. The stationary phase was a 

Purospher STAR RP-18e 30 mm x 4 mm and the gradient mobile phase consisted of 0.1% 

formic acid and ACN (injection cycle 4.2 min). Crusian carp livers and blue mussels were 

spiked with NOD-R and extracted with aqueous MeOH. The chromatographic 

performance and signal responses in SIR and MRM modes were evaluated in extended 

sample series. Two sample pre-treatment methods were assessed: the SPE procedure 

involved extraction on a C18 solid phase extraction cartridge, and the RAW procedure 

merely filtration. 

Results: Chromatographic performance could be retained throughout the extended sample 

series. The SPE-treated tissue samples analysed with Quattro Micro gave steady MS 

signals for NOD-R throughout the analysis whereas the Quattro II signal response 

fluctuated considerably, possibly due to sample cone fouling and uncontrolled signal 

enhancement and suppression. The RAW pre-treatment was inadequate for HTP analysis. 

Discussion: An earlier evaluation (1) showed a similar HTP method to be suitable for >300 

cyanobacterial extract samples on a coaxial probe LC-ESI-MS. The sample clean-up 

procedures reported in (2) are being evaluated as a part of the methodology development. 

The current study shows promising results for the LC-MS part, but the bottleneck for HTP 

analysis is the extraction and SPE of tissue samples. 

References: 1 Meriluoto J. et al. (2004) Chromatographia, 59, 291-298. 

2 Karlsson K.M. et al. (2005) Environ. Toxicol. 20, 381-389. 

o High-throughput analysis 

o LC-ESI-MS 

o Tissue samples 

o Nodularin 

268 

Poster 38: Improved EC method for the determination of cyanide in feed. 

M. Baltussen, D. Luykx, A. Koot, R. Frankhuizen and S. van Ruth 

RIKILT Institute of Food Safety, P.O. Box 230 NL-6700 AE Wageningen, The Netherlands 

Introduction: Cyanide is highly toxic and therefore it is of paramount importance that the 

method for determination of cyanide in feed is reliable, sensitive and robust. Until now, the 

reference method as described in First Commission Directive 71/250/EEC was used in the 

EC to determine cyanide in feed. However, this method proved to be not sensitive enough 

for levels of 10 mg cyanide/kg feed and it could lead to false positive results. CEN has 

asked RIKILT to develop a new reference method under a new mandate of the EC. 

Methods: Several steps in the EC protocol were altered to optimize the extraction of 

cyanoglycosides from feed, the formation and extraction of hydrogen cyanide and the 

determination of cyanide. Flaxseed containing high levels of cyanoglycosides and blank 

feed spiked with a cyanoglycoside, were used for testing the alterations. 

Results: The extraction of cyanoglycosides from feed was improved by using an acid 

treatment. The formation of hydrogen cyanide was optimized by using pure enzyme 

instead of an almond suspension. In the EC protocol, a titrimetric method was described to 

determine cyanide. In this study, cyanide was derivatized with a fluorogenic label and 

determined by HPLC with fluorescence detection (Sano et al. 1992). Levels well below 10 

mg cyanide/kg feed could be detected and no interferences with other compounds were 

seen in the chromatogram. Moreover, the cyanide yield increased (from 210 mg/kg to 245 

mg/kg in flaxseed) and recovery of the spikes increased (from ~60% to ~90%). 

Discussion/Conclusion: The new method for cyanide determination in feed including 

HPLC analysis developed by RIKILT for CEN proved to be more sensitive, selective and 

resulted in higher yields of cyanide. 

Reference: Sano A., Takimoto N. and Takitani S., J of Chrom., 1992, vol. 582, pp 131-135 

o cyanide 

o feed 

o HPLC 

o CEN 

269

Poster 39: The sulfo-SBED derivative of ammodytoxin, a photoprobe for studying 

the molecular basis of phospholipase A2 β-neurotoxicity 

Kovačič, L. * , Šribar, J., Križaj, I. 

Department of Biochemistry and Molecular Biology, Jožef Stefan Institute, Jamova 39, SI-1000 

Ljubljana, Slovenia, *lidija.kovacic@ijs.si 

The neurotoxicity of ammodytoxin, a secreted phospholipase A2 from long-nosed viper 

venom, has been proposed to be mediated by certain binding proteins. Interactions of 

ammodytoxin with the M-type receptor of secreted phospholipases A2 (1), calmodulin (2), 

14-3-3 proteins (3), protein disulfide isomerase (4) and a still unidentified mitochondrial 

membrane protein R25 (5) have been partially characterized. For further identification of 

ammodytoxin-binding proteins, we have explored the use of a biotin-containing crosslinking 

reagent sulfo-SBED (6). This trifunctional cross-linker possesses one aminereactive 

and one photo-reactive site and, additionally, allows affinity-based concentration 

of cross-linker containing species. Sulfo-SBED was coupled to ammodytoxin C whose 

specific binding was demonstrated by biotin-tagging of the known ammodytoxin targets, 

calmodulin, 14-3-3 proteins, protein disulfide isomerase and R25, but not other proteins. 

Using this tool, two additional proteins having affinity for ammodytoxin, of 45 and 46 

kDa, were identified in the mitochondrial-synaptosomal fraction of porcine cerebral cortex, 

that were not detected with previously used methods. The methodology of preparing a 

photoreactive phospholipase A2 derivative and labeling the respective binding proteins is 

generally applicable. In this way, not only the molecular basis of ß-neurotoxicity but other 

aspects of the (patho)physiological action of phospholipases A2 can also be studied. 

References: 

1. N.Vardjan et al. (2001), Biochem. Biophys. Res. Commun. 289, 143-149 

2. J. Šribar et al. (2001), J. Biol. Chem. 276, 12493-12496 

3. J. Šribar et al. (2003) Biochem. Biophys. Res. Commun. 302, 691-696 

4. J. Šribar et al. (2005) Biochem. Biophys. Res. Commun. 329, 733-737 

5. J. Šribar et al. (2003a), FEBS Lett. 553, 309-314 

6. T. Larsson et al. (2000), FEBS Lett. 469, 155-158 

o phospholipase A2, 

o neurotoxicity, 

o affinity-labeling and 

o binding proteins. 

270 

Poster 40: Purification and characterisation of two apoptosis-inducing L-amino 

acid oxidases from the venoms of selected Australian elapids 

Bateman, E.H, 1* Venning, M.G, 2 Mirtschin, P.J 2,3 and Woods, A.E 2 

1 University of Otago, Dunedin Public Hospital, Great King St, Dunedin, New Zealand 

*emma.bateman@stonebow.otago.ac.nz 

2 University of South Australia, Frome Road, Adelaide, Australia. 

3 Venom Supplies, Tanunda, South Australia. 

Introduction - A previous study 1 demonstrated that crude venoms from Australian elapids 

Austrelaps superbus and Hoplocephalus stephensii elicited an apoptotic response in 

tumour-associated microvascular endothelial cells (TAMECs) which was similar to 

apoptotic effects elicited by a panel of exotic viperid snakes. The focus of the current study 

was to purify, identify and characterise the apoptotic agent(s) within these venoms. 

Methods – Crude, lyophilised venoms from A.superbus and H.stephensii were fractionated 

via size exclusion chromatography and fractions were screened at various time/dose 

combinations on TAMEC cultures to identify the fraction(s) responsible for apoptotic 

effects. These fractions were then pooled and characterised by native and denaturing 

polyacrylamide gel electrophoresis (PAGE), biochemical assays and peptide sequencing. 

Results – Both A.superbus and H.stephensii venoms demonstrated significant apoptotic 

activity in fractions of approximately 140-160kDa. Pooling of active fractions and 

subsequent native PAGE showed pure, single bands at 158kDa (A.superbus apoptotic 

fractions = AS1a) and 141kDa (H.stephensii apoptotic fractions = HS1a); denaturing 

PAGE demonstrated heterodimerism for AS1a and homodimerism for HS1a. Both AS1a 

and HS1a demonstrated specific catabolism of L-leucine, were inhibited by catalase and 

stained positive under periodic acid-Schiff staining. Peptide sequencing then identified the 

apoptotic agents as L-amino acid oxidases (LAOs). Conclusions – This is the first study to 

report the isolation and identification of LAOs from Australian elapid venoms. The agents 

share similar capabilities and attributes with several LAOs from snake species exotic to 

Australia, including the capacity to elicit an apoptotic response in endothelial cells. 2,3 

Further studies to fully sequence AS-LAO and HS-LAO and characterise their biochemical 

properties are required, as well as further investigation to elucidate the exact mechanisms 

of LAO apoptotic activity. 

1 Bateman et al. 2002. 14th World Congress on Animal, Plant & Microbial Toxins, Abstract 

22501, page 75. 2 Torii et al., 1997. Journal of Biological Chemistry, v272(14), pp9539- 

9542. 

3 Suhr and Kim, 1999. Journal of Biochemistry, v125, pp305-309. 

o Apoptosis 

o Australian elapid venoms 

o L-amino acid oxidase 

o Tumour-associated endothelial cells 

271

Poster 41: Discovery of a novel conopeptide-AmIXA: Prediction of functional 

pharmacophore(s) and mechanism of action 

Saminathan, R 1 , Jois, S.D.S 2 , Sato, K 3 , Gopalakrishnakone, P 1* . 

1 

Venom and Toxin Research Programme, Department of Anatomy, Yong Loo Lin School of Medicine, 

National University of Singapore, 4 Medical Drive, Singapore-117597, *antgopal@nus.edu.sg 

2 

Department of Pharmacy, Faculty of Science, National University of Singapore, 4 Science Drive, 

Singapore-117543 

3 

Fukuoka Women's University, Kasumigaoka, Higashi-ku, Fukuoka, 813-8529, Japan 

Drugs and drugable candidates obtained from the venoms of marine cone snails are 

appreciated clinically to treat conditions from chronic pain to epilepsy. The venom element 

is usually gene encoded, conformationally constrained and disulfide bond rich shortpeptides 

(

Poster 43: Identification of the active-site residues of the fungal virulence factor, 

phospholipase B 

Tsukamoto, K 1* , Inoue, Y 1 , Tsuruga, A 1 and Obata, Y. 2 

1 

Showa Pharmaceutical University, 3-3165 Higashi Tamagawagakuen, Machida, Tokyo, Japan, 

*kikuot@ac.shoyaku.ac.jp 

2 

Aichi Medical University, Nagakute, Aichi, Japan 

Fungal phospholipase B (PLB) has been known to be a virulence factor in fungal infection 

such as Cryptococcus neoformans and Candida albicans. The enzyme catalyzes the 

hydrolysis both of 1- and 2-acyl ester bonds of glycerophospholipid, and also exhibits 

lysophospholipase and transacylase activities. To investigate the structure-function 

relationship of fungal PLBs, we first cloned the plb1 gene of Saccharomyces cerevisiae 

W303-1A as a representative one. According to the sequence similarity between fungal 

PLBs and mammalian cytosolic phospholipase A2, we predicted the three-dimensional 

structure of the PLB1 by the homology modelling method. In the structural model, 

conserved residues among fungal PLBs converged on the cleft of the enzyme. On the 

basis of the structural characteristics of PLB1, we introduced site-directed mutagenesis to 

the conserved residues, and evaluate the effect of mutaions on the enzyme activity using 

the plb1 knocked-out S. cerevisiae strain as a host. Experimental results showed that 

conserved Ser147 and Asp403 are critical residues in catalysis, and conserved Arg109 

must contribute to the substrate hydrolysis through electrostatic interaction. These suggest 

that PLB1 belongs to the serine super family. For purification and the accurate functional 

analysis of PLB1, we established the baculoviral expression system of PLB1. The system 

expressed the PLB1 activity more 25-fold efficient than the reconstructed system using the 

plb1 knocked-out S. cerevisiae strain. The PLB1 protein was homogeneously purified 

from the culture medium of the baculoviral PLB1 expression clone. Effect of the PLB1 on 

mammalian cells would be discussed. 

o Phospholipase b 

o Invasion factor 

o cPLA2 

o homology modelling 

274 

Poster 44: A Comparative Study of the Enzymatic and Physiological Activities of 

Two Viper Venom L-Amino Acid Oxidases 

Samel, M. 1 , Tõnismägi, K. 1 , Trummal, K. 1 , Rönnholm, G. 2 , Siigur, J. 1 , Kalkkinen, N. 2 , Siigur, E. 1 * 

1 

National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, Tallinn, Estonia, 

*enesi@kbfi.ee 

2 

Institute of Biotechnology, FIN-00014 University of Helsinki, Finland 

L-Amino acid oxidases (LAAO) are flavoenzymes able to stereospecific catalysis of the 

oxidative deamination of L-amino acids to alpha-keto acids, hydrogen peroxide and 

ammonia. Snake venom is the richest source of the enzyme. Before 1990s the studies were 

mainly focused on the enzymatic properties of LAAOs but in the last 10-15 years the main 

interest is connected with effects of LAAO on various cells: platelets, vascular endothelial 

cells, different cancer cell lines. In this study the LAAOs from Vipera lebetina and Vipera 

berus berus venoms are characterized. 

The LAAOs have been isolated by size exclusion, ion exchange and affinity 

chromatography with protein yields of 1.8% (V. berus berus) and 2.5% (V. lebetina). Both 

enzymes are homodimeric FAD-containing glycoproteins with monomeric molecular 

masses of 57.7 (V. berus berus) and 60.9 (V.lebetina) kDa. They have identical N-terminal 

sequences and are characterized by tryptic peptides sharing high homology with other 

snake venom LAAOs. The most specific substrates for both enzymes are hydrophobic 

amino acids L-Leu, L-Met, L-Trp or L-Phe. V. b. berus LAAO inhibited ADP-induced 

platelet aggregation in the range of 0.03-0.10 microM, V. lebetina LAAO inhibited ADP- 

or collagen-induced aggregation dose-dependently in the range of 1.0-2.5 microM. In case 

of the ADP-induction the inhibition was more pronounced on the second wave of 

aggregation. V. b. berus LAAO induced apoptosis in the HeLa and K562 cells, V. lebetina 

LAAO inhibited growth of B. subtilis and E. coli. All physiological effects were abolished 

by catalase pointing to the role of hydrogen peroxide liberated in the oxidation process. 

Acknowledgement. The study has been financially supported by Estonian Science 

Foundation Grant 5938. 

o L-Amino acid oxidase 

o Vipera lebetina 

o Vipera berus berus 

o Snake venom 

275

Poster 45: Heterodimeric Disintegrin VLT-2 from Vipera lebetina Snake Venom 

Siigur, J.*, Vija, H., Aaspõllu, A., Samel, M., Tõnismägi. K., Trummal, K., Pikver, R., Subbi, J., Siigur, 

E. 

National Institute of Chemical Physics and Biophysics, Akadeemia tee 23,Tallinn 12618, Estonia, *siigur@kbfi.ee 

Vipera lebetina venoms of different subspecies contain several monomeric and dimeric 

disintegrins: obtustatin, VLO-4, VLO-5 from Vipera lebetina obtusa venom (Calvete et al 

2003, 2005); lebein (Gasmi et al 2001); lebein-1, lebein-2 (Eble et al 2003) and lebestatin 

(Sanz et al 2006) from V. lebetina mediterranea (Tunesian snake) venom. We purified a 

heterodimeric disintegrin VLT-2 from Vipera lebetina turanica venom obtained from 

Uzbekistan, using size exclusion chromatography on Sephadex G-100sf. and HPLC on 

C18 column. The VLT-2 chains were separated using SDS-PAGE; molecular masses of 

tryptic peptides from both chains and of native heterodimeric VLT-2 were detected by 

MALDI-TOF mass-spectrometry. Using cDNA library of the venom gland of a single 

V.lebetina snake the VLT-2 coding cDNAs were cloned and sequenced. VLT-2 chains are 

synthesized from different genes. One chain, containing VGD sequence in disintegrin loop, 

is synthesized with PII type fibrinolytic metalloproteinase Le-3 (Lebetase), which is 

cleaved posttranslationally. The other chain, containing MLD sequence in disintegrin loop, 

is synthesized from the gene without metalloproteinase domain. The translated MLD 

containing sequence is longer than the protein counterpart purified from the venom. Most 

probably the C-terminus is processed proteolytically by venom proteinases. VLT-2 

primary structure is very similar to that of VLO-5: Le-3 disintegrin-like sequence is 

identical to VLO-5 A- chain, MLD containing sequence has two differences in the 

sequence compared with VLO-5 B-chain. VLT-2 dose-dependently inhibited collagen- 

induced platelet aggregation with IC50= 240 nM and ADP-induced aggregation with IC50= 

480 nM. The nucleotide sequences are registered in GenBank under Accession numbers 

X97894 and DQ295886. 

References: Gasmi et al (2001) BBA 1547,51-66; Calvete et al (2003) Biochem. J. 

372,725-734; Eble et al (2003) J.Biol. Chem. 278, 26488-26496; Calvete et al (2005) 

Toxicon 45,1063-1074; Sanz et al (2006) Biochem. J. 395, 385-392. 

The work was supported by Estonian Science Foundation Grant No. 5554. 

o Vipera lebetina 

o Heterodimeric disintegrin 

o Snake venom 

o Inhibition of platelet aggregation 

276 

Poster 46: Discovery and pharmacology of Conopressin-T, a novel Vasopressin-like 

peptide from Conus Tulipa. 

Daniel Croker 1 , Sebastien Dutertre 2,3 , Paul Alewood 2 , Richard Lewis 2 

1 

Xenome Ltd, Indooroopilly, Australia 

2 

Institute for Molecular Bioscience, The University of Queensland, 4072 Qld, Australia. 

r.lewis@imb.uq.edu.au 

3 

Present address: Department of Neurochemistry, Max-Planck-Institute for Brain Research, 

Deutschordenstrasse 46, 60528 Frankfurt, Germany. dutertre@mpih-frankfurt.mpg.de 

To date, only two conopressins were isolated from cone snail venom. Conopressin-S was 

isolated from C. striatus venom, while conopressin-G was isolated from C. geographus 

venom. Here we report the discovery and pharmacology of a novel conopressin, 

conopressin-T (Con-T), which was isolated from the venom of C. Tulipa. Following 

isolation, Con-T showed a novel sequence that differed at two highly conserved residues of 

this peptide family. Synthesis of Con-T and its analogue [L7P]-Con-T (reversion to a 

conserved residue), were tested for there activity at the vasopressin/oxytocin receptor 

family. These peptides were assessed, for their biological activity by radioligand binding 

assay and also by functional assay. The binding data showed that Con-T has modest 

activity at the oxytocin receptor, but is selective for the vasopressin V1A receptor. In 

addition, the analogue L7P-Con-T was 7- to 20-fold more active than Con-T at the V1A, 

and conserved the V1A selectivity. This preliminary SAR study could help in the design of 

novel vasopressin subtype selective drugs. Hence we have discovered a new novel 

Conopressin that has an unusual sequence compared to other peptides in this family and 

displays an interesting selectivity profile at its target receptors. 

o Cone snail 

o Conopressin 

o Vasopressin 

o Pharmacology 

277

Poster 47: Isolation and characterisation of Conomap-Vt, a D-amino acid 

containing excitatory peptide from the venom of a vermivorous cone snail 

Sébastien Dutertre 1 , Natalie Lumsden, Paul F. Alewood and Richard J. Lewis 

Institute for Molecular Bioscience, The University of Queensland, 4072 Qld, Australia. 

r.lewis@imb.uq.edu.au 

1 Present address: Department of Neurochemistry, Max-Planck-Institute for Brain Research, 

Deutschordenstrasse 46, 60528 Frankfurt, Germany. dutertre@mpih-frankfurt.mpg.de 

Cone snail venom is a rich source of bioactives, in particular small disulfide rich peptides 

that disrupt synaptic transmission. Here, we report the discovery of Conomap-Vt, an 

unusual linear tetradecapeptide isolated from Conus vitulinus venom. The sequence 

displays no homology to known conopeptides, but displays significant homology to 

peptides of the MATP (myoactive tetradecapeptide) family, which are important 

endogenous neuromodulators in molluscs, annelids and insects. Conomap-Vt showed 

potent excitatory activity in several snail isolated tissue preparations. Similar to ACh, 

repeated doses of conomap-Vt were tachyphylactic. Since nicotinic and muscarinic 

antagonists failed to block its effect and conomap-Vt desensitised tissue remained 

responsive to ACh, it appears that conomap-Vt contractions were non-cholinergic in 

origin. Finally, biochemical studies revealed that conomap-Vt is the first member of the 

MATP family with a D-amino acid. Interestingly, the isomerization of L-Phe to D-Phe 

enhanced biological activity, suggesting that this post-translational modified conopeptide 

may have evolved for prey capture. 

o Cone snail 

o D-amino acid 

o Conopeptide 

o isomerization 

278 

Poster 48: Tityus perijanensis (Scorpiones, Buthidae) from Zulia State, western 

Venezuela: Geographic Distribution and Venom Molecular and 

Immunological Characterization 

Borges, A. 1* , Blumer-Lairet, V. 1 , Poliwoda, S. 1 , Alfonzo, M.J. 1 , Rojas-Runjaic, F. 2 , De Sousa, L. 3 

1 Instituto de Medicina Experimental, Universidad Central de Venezuela, Caracas *borgesa@ucv.ve 

2 Museo de Historia Natural La Salle, Caracas, Venezuela 

3 Centro de Investigaciones en Ciencias de la Salud, Universidad de Oriente, Puerto La Cruz 

Tityus perijanensis is a buthid scorpion endemic to the Perijá range, in the border between 

Colombia and Venezuela, which has caused severe accidents with central neurological 

sequelae. Since only scarce toxinological information is available on this species, an 

investigation of its toxicology, distribution and venom peptide composition was 

undertaken. Originally described from Ayajpaina (alto Río Negro), southern Zulia State, 

collection at various sites allowed extension of its geographical range to 250 km north of 

the type locality. Specimens throughout the extended distribution range shared the same 

cytochrome oxidase I subunit nucleotide sequence. Estimation of T. perijanensis venom 

toxicity was performed through LD50 assays by i.v. injections in mice. In vivo 

neutralizations tests were also carried out to evaluate the efficacy of the anti-T. discrepans 

(a northcentral Venezuelan species) antivenom to protect against envenoming by T. 

perijanensis. Lethality tests showed that T. perijanensis venom (LD50 = 0.9 mg/kg) is 2.7fold 

more toxic than T. discrepans. The venom was poorly neutralized by the anti-T. 

discrepans antivenom. Chromatographic fractionation of T. perijanensis venom through 

HPLC indicated that protein composition was different from other Venezuelan Tityus 

venoms of medical importance. Analysis of the T. perijanensis toxin repertoire was studied 

through amplification by RT-PCR of venom gland mRNA using a combination of a 

degenerate primer (binding at the region encoding the toxin signal peptide) and a modified 

oligo-d(T) primer. Sequences of putative T. perijanensis sodium channel-active toxins 

encoded by isolated mRNAs differed from T. zulianus (a toxic Andean species) and T. 

discrepans equivalent peptides, suggesting the existence of regional differences in scorpion 

toxin antigenic reactivity within Venezuela (Supported by Fonacit Grant S1-2001000674 

to A.B.). 

Key Words: Neurotoxins, Scorpion, Tityus perijanensis, Venezuela 

279

Poster 49: Error-prone DNA polymerase k from Protobothrops flavoviridis, 

member of the DinB superfamily. 

Oda-Ueda, N 1 ., Kariu, T 1 ., Fukuhara, A 2 ., Takazaki, S 1 , Chijiwa, T 2 ., Ohno, M 2 . 

1 2 

Dept. Phamaceutical Sciences, Dept. Life Science, Sojo University, Kumamoto Japan, 

*naoko@life.sojo-u.ac.jp 

1. Introduction: We found that venom isozymes with a variety of physiological activities 

from Protobothrops (formerly known as Trimeresurus) flavoviridis have evolved in an 

accelerated manner and thought that mostlikely the diversity has been produced by 

accumulation of point mutations in their genes. To understand the molecular mechanism 

underlying such mutation in P. flavoviridis, we have cloned a gene homologous to 

Echerichia coli dinB gene which encodes DNA polymerase IV known as error-prone DNA 

polymerase κ. 

2. Methods: Total RNA from P. flavoviridis oocyte was used as template for first strand 

cDNA synthesis by using SMART cDNA Library construction kit. Degenerate primers 

were designed based on the conserved sequences of the polymerases in Xenopus laevis 

(frog) and Gallus gallus (chicken). Multiple rounds of 5’ and 3’ rapid amplification of 

cDNA ends were employed to extend the putative P. flavoviridis genes using BD SMART 

RACE amplification kit. Fibroblast cells from P. flavoviridis embryo were prepared for 

chromosomal mapping with fluorescence in situ hybridization (FISH). Multiple alignment 

was made with the CLUSTAL X program. 

3. Results and Discussion: A 2412-bp cDNA of P. flavoviridis DinB contained a ORF of 

2503 bp that encodes a protein of 833 amino acids. The amino acid sequence consisted of 

N-terminal nucleotidyl transferase domain, two tandem HhH domains implicated in DNA 

binding, nuclear localization signal like domain and proliferating cell nuclear antigen 

domain, as all being conserved in DinB superfamily, and shares significant identity (~84%) 

with G. gallus enzyme. In a phylogenetic tree of DinB superfamily, P. flavoviridis DinB 

constitutes a branch together with G. gallus enzyme out of the enzymes from other 

eukaryotes such as H. sapiens or mouse. Chromosomal FISH showed that P. flavoviridis 

DinB gene is localized in chromosome 6 (macrochromosome) different from the case of G. 

gallus DinB which is localized in microchromosome. 

o P. flavoviridis 

o Error-prone DNA polymerase 

280 

Poster 50: Post-translational modifications of venom components in Conus 

victoriae. 

1, 2 2 2 1 3 1 1, 4 

Townsend, A , Scanlon, D , O’Donnell, P , Inserra, M , Bingham, J-P , Satkunanathan, N , Khalil Z , 

Purcell, A 1, 2 1, 2* 

, Livett, B.G. 

1 

University of Melbourne, Biochemistry and Molecular Biology, Parkville, Victoria, AUSTRALIA, 

*b.livett@unimelb.edu.au 

2 

Bio21 Institute, 30 Flemington Rd.,Parkville,Victoria, AUSTRALIA 

3 

Clarkson University, 8 Clarkson Ave.,Potsdam,New York, USA 

4 

University of Sharjah, College of Pharmacy,UAE. 

Introduction: Chemical analysis of Conus venoms has revealed a variety of biologically 

active small and intermediate size peptides rich in post-translational modifications (PTMs). 

Known PTMs include hydroxylation of proline, gamma-carboxylation of glutamate, 

bromination of tryptophan, sulfation of tyrosine, and glycosylation. A range of PTM 

patterns have been observed, including differential/preferential hydroxylation and 

hyperhydroxylation (1). The functional role(s) of PTMs are not known but they could 

contribute to specificity of action, while stabilizing conopeptide structure and/or providing 

resistance to proteolysis. We have studied PTM-conotoxins in C. victoriae venom (2). 

Methods: Solid phase FMOC synthesis of conotoxin Vc1.1 sequence (deduced from the 

venom duct cDNA) and of an analogue (Vc1.1-ptm) where Pro 6 was replaced by hydroxy- 

Pro and Glu 14 by gammacarboxy-Glu. Preparative/analytical RP-HPLC was performed on 

an Agilent 1200 HPLC System. Crude venom was analysed with Agilent in-line 

chromatography for ESI TOF MS with sequence determination using an Agilent HPLC 

chip for ESI ion-trap MS/ MS. Antagonism of neuronal nicotinic receptors was assayed in 

vitro using monolayer cultures of bovine adrenal chromaffin cells. In vivo tests for 

hyperalgesia and allodynia were performed in adult female Sprague Dawley rats. 

Results: HPLC purification of the air-oxidized Vc1.1 (Mr 1807) revealed one major peak 

whereas the Vc1.1-ptm (Mr 1823) revealed three major peaks, representing two disulfide 

isomers (globular and ribbon) and a dimer (Mr 3643). Unlike Vc1.1, the Vc1.1-ptm did not 

inhibit the neuronal nicotinic receptor response and was not an analgesic. However, it 

retained the ability to accelerate functional nerve recovery of injured nerves. 

Discussion/Conclusion: These results on C. victoriae indicate that dimeric and higher 

order forms of conotoxins exist within the venom (3) and may have distinct biological 

activities. 

References: 1.Buczek, O. et al (2005) Cell Mol Life Sci.62:3067-3079; 2.Jakubowski, 

J.A., et al (2004) J. Mass Spectrom 39: 548-557; 3.Loughnan, M. et al (2006) JBC. [in 

press]. 

Acknowledgement: Supported by DEST Australia, Innovation Grant #CG03 0017 to BGL 

o conotoxin 

o posttranslational modification 

o nicotinic receptor 

o analgesics 

281

Thursday 27 July: poster session 


Marine toxins 

1. Poly-APS induced Ca 2+ entry in ECV cells. Maružin, M. 1 *, Hawlina, S. 2 , Bunc, 

M. 2 , Šuput, D. 1 

. 

2. Occurrence of tetrodotoxin in two new gastropods collected from west southern 

Taiwan. P.-A. Hwang a,b , Yung-Hsiang Tsai c , Shin-Jung Lin d , and Deng-Fwu 

Hwang b,* 

3. Occurrence of Tetrodotoxin and Paralytic Shellfish Poisons of a Gastropod in 

Southern Taiwan. Hsiao-Chin Jen 1 , Shin-Jung Lin 2 , I-Chiu Liao 1 , Osamu 

Arakawa 3 ,Shin-Yuan Lin 1 and Deng-Fwu Hwang 1* 

4. Influence of the sample toxic profile on the suitability of an HPLC method for 

Official PSP Control. Ben-Gigirey, B. 1* , Rodríguez-Velasco, M.L 1 , Botana, L.M. 1,2 

5. Evidence for the involvement of nitric oxide pathway in ciguatoxin-induced 

swelling of amphibian red blood cells. 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 , Laurent, D 2 , Benoit, E 5 * 

6. Severe seafood poisoning in French Polynesia: a retrospective analysis of 

severity criteria in 129 hospital medical files. Gatti, C. 1* , Oehler, E. 2 , Valence, A. 2 , 

Tamarii, D. 2 and Legrand, A.-M. 

7. Paralytic Toxicity in a Ribbon Worm Cephalothrix species (Nemertean) 

Adherent to Cultured Oysters in Hiroshima Bay, Hiroshima Prefecture, Japan. 

Manabu, Asakawa 1* , Shuhei Matsuda 1 , Shintaro Tsuruda 1 , Hiroshi Kajihara 2 , 

Shigeto Taniyama 1 

8. Transfer profile and immnopotentiating effect of tetrodotoxin administered 

intraperitoneally into non-toxic cultured pufferfish Takifugu rubripes. Honda, S 1* , 

Murakami, Y 1 , Ichibu, T 1 , Takatani, T 2 , Tachibana, K 2 , Arakawa, O 2 , Noguchi, T. 3 

9. Toxicity of edible dried fish in the Philippines. Shigeto Taniyama 1 , Takefumi 

Sagara 2 , Ryoichi Kuroki 1 , Satoshi Takamoto 3 , Shintaro Tsuruda 3 , Gloria Gomez 

Delan 4 , Sachio Nishio 2 , Manabu Asakawa 1* 

Pain and CNS 

10. ANTINOCICEPTIVE AND ANTIHYPERNOCICEPTIVE ACTIVITY OF 

THE METHANOLIC EXTRACT OF Phyllomedusa rohdei. Marinho, E.A.V. 1,3 ; 

282 

Zaharenko, A.J. 2 ; Paraventi, C 1 ; Jimenez, R.S. 1 ; Molska, G.R. 1 ; Paula-Freire, 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-Marinho, E.L.A 1 

11. ACTIVITY OF EXTRACTS FROM Phyllomedusa rohdei (AMPHIBIA, 

ANURA) SKIN ON THE MICE BEHAVIOUR IN THE OPEN FIELD AND 

ROTA ROD. Silva, R.W. 1 ; Zaharenko, A.J. 2 ; Paula-Freire, L.I.G. 1 ; Almeida, E. 3 ; 

Frussa-Filho, R. 3 ; Malpezzi-Marinho, E.L.A 1 , Marinho, E.A.V. 1,3 

12. κ AND μ OPIOID RECEPTORS ARE INVOLVED IN THE 

ANTINOCICEPTIVE ACTIVITY OF METHANOL EXTRACT OF 

Phyllomedusa rohdei ON THE HOT PLATE MODEL. 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 ; Parada, 

C.A. 4 ; Ferreira, S.H. 4 ; Malpezzi-Marinho, E.L.A 1 ; Marinho, E.A.V. 1,3 

13. Xen2174: A novel NET inhibitor that enhances α2-adrenoceptor inhibition of 

spinal pain pathways. Croker, D.E 1 , Palant, E 1 , Nielsen C 2 , Drinkwater, R 1 , Patterson, 

M 3 , McCumber, D 3 , Yaksh, T 3 , Wilson, D 1* , Smith, M 2 and Lewis, R.J. 1 

14. Crotoxin inhibits neuropathic pain and the development of neuromas. Cury, 

Y. 1* , Nogueira-Neto, F. 1,2 , Amorim, R.L. 2 , Brigatte, P. 1 , Picolo, G. 1 , Ferreira Jr., W.A. 

1 , Nicoletti, J.L.M. 2 

15. Phospholipases A2 (PLA2) Isolated From Brazilian Coral Snake (Micrurus 

lemniscatus) Induced Neuronal Injury: In Vivo And In Vitro Studies. Carvalho, 

N.D 1 ., Oliveira, D.A. 1 , Casais e Silva, L.L. 3 ,Lebrun, I. 2 , Afeche, S.C 1 , Sandoval, 

M.R.L. 1* 

Inflammation and cytokines 

16. ENHANCEMENT OF TNF-α AND IL-1 PRODUCTION IN ALVEOLAR 

MACROPHAGES BY C-FIBER DEGENERATION. DeSouza, IA *1 Camargo, 

EA 1 ; Franco-Penteado, CF 1 ; Antunes, E 1 

17. PRELIMINARY STUDIES OF STRUCTURAL AND BIOLOGICAL 

ACTIVITY OF GALATROX, A β-GALACTOSIDE-BINDING LECTIN FROM 

THE VENOM OF Bothrops atrox SNAKE. Mendonça-Franqueiro, E.P. 1 ; Callejon, 

D.R. 1 ; Paiva, H.H. 1 , Rosa, J.C. 2 ; Alves, R.M. 1 ; Franco, J.J. 1 ; Dias-Baruffi, M. 1 ; 

Sampaio, S.V. 1 

18. ASSESSMENT OF THE INHIBITION INDUCED BY Bufo paracnemis 

POISON AND ITS FRACTIONS ON COMPLEMENT SYSTEM AND 

LEUKOCYTE RECRUITMENT. Marongio, A. F. Q. 1 , Bertazzi, D. T. 1 , Arantes, 

E. C* 

283

19. Do cytokines and nitric oxide play a role in the actions of Leiurus 

quinquestriatus scorpion venom in animals? Abdoon, N. A 1 *, Elsayed Ali, A 2 , 

Fatani, A. J. 3 

20. EVALUATION OF THE ACUTE LUNG INJURY IN A MURINE MODEL 

INDUCED BY THE SCORPAENA PLUMIERI FISH VENOM. Boletini-Santos 

D 1 , Haddad Jr V. 2* , Figueiredo S.G. 3 , Lopes-Ferreira M. 1 , Lima C. 

21. INFLAMMATORY MEDIATORS INVOLVED IN PHOSPHOLIPASE A2 

(PLA2)-INDUCED ACUTE PANCREATITIS. Camargo, E.A. *1 ; Ferreira, T. 1 ; 

Landucci E.C.T. 1 ; Antunes, E. 

22. Effect of eugenol and local anesthetic agents on tongue and skin edema 

induced by Dieffenbachia picta Schott in mice. Dip, E.C. 1 ; Pereira, N.A. 1 ; Gaban, 

G.A. 1 ; Fonseca, T. 1 ; El Kik, C.Z. 1 ; Tomaz, M.A 1 ; Calil-Elias, S. 1 ; Martinez, A.M.B. 

2 ; Melo, P.A. 1 . 

23. Importance of jararhagin disintegrin-like and cysteine rich domains in the 

early events of local inflammatory response. Clissa, P.B. 1 ; Lopes-Ferreira, M. 1 ; 

Della-Casa, M.S. 1 ; Farsky, S.H.P. 2 ; Moura-da-Silva, A.M. 1 

24. Short-time analysis of pulmonary mechanics on acute lung injury induced by 

Crotalus durissus terrificus venom. 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, Cogo, J.C 1 , Oliveira, L.V.F. 1 

25: Effect of Crotalus durissus terrificus snake venom and crotoxin on cytokine 

secretion by macrophage. Cury, Y. * , Sampaio, SC., Brigatte, P, Curi, R., Sousa-e- 

Silva, MCC. 

Cytotoxicity and antimicrobial 

26. Hydralysins – novel proteins from cnidaria revealing a sequence signature 

with a role in pore formation common to various beta pore forming toxins. Sher, 

D.* 1 , Fishman, Y. 1 , Zhang, M. 1 , Lebendiker, M 1 , Mancheño, J.M. 2 and Zlotkin, E. 1 

27. Caissarolysin I (Bcs I), a new hemolytic toxin from the Brazilian sea anemone 

Bunodosoma caissarum: Purification and biological characterization. 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 , 

Richardson, M. 4 , Sant’Anna, O.A. 3 , Tambourgi, D.V. 3 

28. Beta-Lactamase in Escherichia coli. Siham S. Shaokat*, Hamoudi A. 

Hameed, Israa H.J. 

284 

29. Membrane Interactions of the Pore-forming Protein Equinatoxin II: 

19 F NMR Studies of the role of Trp and Tyr. Norton, R.S. 1 , Bakrač, B. 2 , Anderluh. 

G. 2 , Podlesek, Z. 2 , Razpotnik, A. 2 , Separovic, F. 3 

30. Toxicity of Gambierdiscus sp. and Ostreopsis sp. collected from the coasts of 

western Japan. Takefumi Sagara 1* , Shigeto Taniyama 2 , Osamu Arakawa 3 , Tamiko 

Hashimoto 1 , Naoyoshi Nishibori 1 , Manabu Asakawa 2 and Sachio Nishio 1 

31. Syphaxin 1.5, a novel antimicrobial peptide isolated from the skin secretions of 

the anuran Leptodactylus syphax. Dourado, F. S 1,2* , Moreira, K. G 2 , Brand, G. D 2 , 

Melo, J. T 2 , Leite, J, R. S. A 2 , Silva, L. P 2 , Bloch Jr, C 2 , Schwartz, E. F. 1 

32. Cytoskeletal rearrangement and cell death induced by Bothrops alternatus 

snake venom in cultured Madin-Darby canine kidney cells. Nascimento, J.M. 1,2 , 

Franchi, G. 3 , Novill, A. 3 , Collares-Buzato, C.B. 4 , Hyslop, S. 1,* 

33. Influence of salinity on gliotoxin excretion by marine and terrestrial 

Aspergillus fumigatus strains. Kerzaon, I.*, Grovel, O., Le Pape, P., Robiou du Pont, 

T., Pouchus, Y.F. 

34. Effect of the Disintegrin Eristostatin on Melanoma Cell Signal Transduction. 

Paquette-Straub, C.A, DiRosato, S.C, McLane, M.A. 

35. Ostreolysin, a mushroom pore-forming toxin specifically binding to 

sphingomyelin/cholesterol-rich membrane domains. Rebolj, K. 1 , Maček, P. 1 and 

Sepčić, K. 1* 

36. Time- and concentration-dependent cytotoxicity of ricin in human lung 

epithelial cells. Ramasamy, S. 1* and Proll, D. 1 

37. Cytotoxic Effect of Primulaceae on Normal Fibroblasts and Cancer Cell Lines. 

Young, L.C 1* , Abbott, G. M 1 , Harvey, A.L. 1 

38. A crystalline nonprotein compound (BM-ANF) from toad (Bufo melanostictus, 

Schneider) skin extract having antiproliferative and apoptogenic activity. Giri, 

B 1* , Mishra, R 1 , Dasgupta, S.C 1 , Gomes, A. 1 

39. A new protein toxin (drCT1) from Dabuia russellii venom having 

antiproliferative and apoptogenic activity. Dasgupta, S.C 1* , Saha, A., Giri, B., 

Roychoudhury, S., Gomes, A. 

40. ANTIMICROBIAL ACTIVITY OF EXTRACTS FROM Phyllomedusa rohdei 

(AMPHIBIA, ANURA) SKIN. 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, E.L.A 1 

285

41. EFFECT OF NOVEL PECTENOTOXINS ON ACTIN CYTOSKELETON. 

Louzao, M.C. 1* , Ares, I.R. 1 , Cagide, E. 1 , Vieytes, M.R. 2 , Miles, C.O. 3 , Yasumoto, T. 4 

and Botana, L.M. 1 

42. A Cardiotoxin (NK31) from Indian Monocled Cobra (Naja kaouthia) shows 

activity against human chronic myelogenic cell-line (K562). Debnath, A, Gomes, 

A*, Vedasiromoni, J.R. 

43. Antileukemic activity of Indian Black Scorpion (Heterometrus bengalensis) 

venom against human leukemic U937 and K562 cells. Gomes, A * , Das Gupta, S , 

Vedasiromoni, J.R. 

44. Haemolytic protein from pedicellaria of violet sea urchin Sphaerechinus 

granularis (Toxopneustidae). Turk, T 1 and Kem, W. R. 

45: An alpha-conotoxin from Conus victoriae is effective at alleviating neuropathic 

pain in an animal model of diabetic neuropathy. Satkunanathan, N 1 , Khodr, B 1 , 

Georgiou, G 1 , McIntosh, D 2 , Belyea, C 2 , Khalil Z 1, 3 , Livett, B.G. 1* 

286 

Poster 1: Poly-APS induced Ca 2+ entry in ECV cells 

Maružin, M. 1 *, Hawlina, S. 2 , Bunc, M. 2 , Šuput, D. 1 

. 

1 

University of Ljubljana, Medical faculty, Institute of Pathophysiology, Zaloška 2, Ljubljana, Slovenia; 

*mitja.maruzin@mf.uni-lj.si 

2 

Clinical centre in Ljubljana, Zaloška 2, Ljubljana, Slovenia 

Introduction: Of the many distinct chemical weapons produced by sessile marine 

sponges, a number of them are novel alkylpyridinium salts (APS) that have interesting 

biological properties that may be exploited. Polymeric APS (poly-APS) are isolated from 

the aqueous extract of the marine sponge Reniera sarai. Diverse biological activities have 

been identified for different 1,3-APS oligomer preparations. These include cytotoxicity, 

neurotoxicity and inhibition of action potentials, stimulation of transmitter release, 

inhibition of K + -conductances and anticholinesterase activity. Some preparations of these 

toxins that contain cocktails of APS have been found to produce hemolysis. These actions 

may be attributed to the formation of lesions or pores in cell membranes, an effect that can 

also be produced in artificial lipid bilayers. We tried to explain in-vivo vascular effects by 

cytotoxic action of poly-APS on endothelial cells. 

Methods: We used fura-2 Ca 2+ imaging and confocal microscopy to compare the 

cytotoxic action of poly-APS on ECV-304 cell culture with the action of equinatoxin II 

(EqT II), which produced irreversible cell swelling and cytolysis. 

Results: Addition of poly-APS (0,5-5µg/ml) to the culture medium was followed by a 

dose-dependent rise in intracellular Ca 2+ . At concentrations used there were some 

sporadical changes in volume observed but no cytolysis. 

Discussion: Till recently, there were no reports of the toxic effects of these novel 

substances in vivo. Bunc M. et al.(1) evaluated the role of AChE inhibitory activity as well 

as the role of other effects of poly-APS observed in vitro by monitoring their effects on 

cardiovascular and respiratory activity in vivo. At higher doses the possible AChE 

inhibitory effects were not observed as they were probably masked by other, faster and 

more pronounced lethal effects of the toxin, such as the initiation of blood coagulation, 

platelet aggregation and cytotoxity. An increase of intracellular [Ca 2+ ] could also be 

considered as a possible mechanism of trombocyte activation. 

References: 1. Bunc, M. et al. (2002). Toxicon 40 (7): 843-849. 

o Alkylpyridinium salt 

o Endothelial cell 

o Intracellular calcium 

o Cytotoxicity 

287

Poster 2: Occurrence of tetrodotoxin in two new gastropods collected from west 

southern Taiwan 

P.-A. Hwang a,b , Yung-Hsiang Tsai c , Shin-Jung Lin d , and Deng-Fwu Hwang b,* 

a Division of Food Technolgy, Fisheries Research Institute, Keelung, Taiwan 

b Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan 

c Department of Food Sanitation, Tajen Institute of Technology, Pingtung, Taiwan 

d 

Department of Food Science and Technology, Ching-Kuo Institute of Management and 

Health, Keelung, Taiwan 

Abstract 

The gastropod Harpa articularis (8 specimens) and Hindsia sinensis (6 specimens) were, 

respectively, collected from west southern Taiwan were toxic. The average toxicities in 

digestive gland and muscle were 14±3 (mean±S.D.) and 19±5 mouse unit per gram 

(MU/g) for H. articularis, and 31±10 and 23±7 MU/g for H. sinensis, respectively. But 

the other twenty-one specimens of both species collected from eastern Taiwan were not 

toxic. The toxin of both toxic species was extracted with methanol, defatted with 

dichloromethane, concentrated, purified through Bio-Gel P-2 column and eluted with 

0.03 M acetic acid. The high performance liquid chromatography and liquid 

chromatography/mass spectrometry analyses demonstrated that the toxin of both toxic 

species was composed of tetrodotoxin (TTX) and anhydro-TTX. 

288 

Poster 3: Occurrence of Tetrodotoxin and Paralytic Shellfish Poisons of a 

Gastropod in Southern Taiwan 

Hsiao-Chin Jen 1 , Shin-Jung Lin 2 , I-Chiu Liao 1 , Osamu Arakawa 3 , 

Shin-Yuan Lin 1 and Deng-Fwu Hwang 1* 

1 

Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan, 

R.O.C. 

2 

Department of Food Science and Technology, Ching Kuo Institute of Management and 

Health, Keelung, Taiwan, R.O.C. 

3 

Faculty of Fisheries Nagasaki University, Bunkyo-machi 1-14, Nagasaki, 852-8521, 

Japan. 

The toxicity of gastropod Nassarius papillosus implicated to a food paralytic poisoning 

incident in Liuchiu Island, Taiwan in October, 2005 was reported. The symptoms of a 

victim (67 years old) were featured by general paresthesia, paralysis of phalanges and 

extremities, paralysis, coma and aphasia. The remained specimens of shell were assayed 

for toxicity. The range of specimen toxicity was 63 to 474 mouse units per specimen 

(MU/ specimen) TTX for N. papillosus. The average toxicity of the digestive gland and 

other portions was 296 ± 120 (mean ± S.D.) and 382 ± 156 MU in N. papillosus. The 

toxin was partially purified from the acidic methanol extract of the gastropod by using a 

C18 solid-phase extraction column and then the eluate was filtered through a 3,000 MW 

cut-off ultrafree microcentrifuge filter. The toxin purified from gastropods was analyzed 

by HPLC and LC/MS showed that the toxin contained TTX and related compound anh- 

TTX (about 90%), whereas along with minor GTX2,3 and neo-STX (about 10%). 

289

Poster 4: Influence of the sample toxic profile on the suitability of an HPLC 

method for Official PSP Control 

Ben-Gigirey, B. 1* , Rodríguez-Velasco, M.L 1 , Botana, L.M. 1,2 

1 CommunityReference Laboratory of Marine Biotoxins (CRLMB), Estación Marítima S/N, Muelle de 

Trasatlánticos, Vigo, Spain, *bbeng@msc.es 

2 Departamento de Farmacología, Facultad de Veterinaria, Campus Universitario, Lugo, Spain 

An HPLC-FLD method, involving pre-chromatographic oxidation of the PSP toxins with 

hydrogen peroxide and periodate, has been AOAC validated through a collaborative trial 

and adopted as AOAC Official Method. This method could be a candidate for replacing the 

mouse bioassay (MBA) for the Official Control of PSP toxins at European level, once 

accepted by the legislation. An interlaboratory exercise has been organized by the CRLMB 

to evaluate its “fitness for purpose” for the Official Control of PSP toxins in the E.U. 

laboratories. Eighteen participant laboratories analyzed six bivalve mollusc samples (two 

of them blind duplicates) with several PSP toxic profiles. 

The method used for this interlaboratory exercise was the Lawrence HPLC method as 

appears in AOAC Official Method 2005.06 (First Action). A detailed protocol with 

instructions for the application of the method for this exercise was facilitated to 

participants. 

Samples assigned values were calculated by consensus from participants valid results after 

removing outliers (Cochran & Grubbs / Dixon (α=0.05) tests). Standard deviations for 

proficiency assessment were derived from the precision data obtained at the collaborative 

trial. For each sample z-scores were calculated for valid data. 

The HPLC validated method is only applicable for Official PSP Control for certain 

samples. This depends on sample PSP toxic profile. Results obtained for a sample with a 

toxic profile dominated by GTX6 and suspected to contain also C1,2 & C3,4 were not 

satisfactory. Only GTX5 and dc-STX could be quantified and the results achieved (total 

toxicity) were much lower (1/8) than those obtained by MBA. The lack of several PSP 

standards, the fact that the method is not validated for all the PSP toxins, and several 

drawbacks found in its application are a handicap to implement it for Official PSP Control. 

o PSP toxins 

o Interlaboratory exercise 

o HPLC method 

290 

Poster 5: Evidence for the involvement of nitric oxide pathway in ciguatoxininduced 

swelling of amphibian red blood cells 

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 , 

Laurent, D 2 , Benoit, E 5 * 

1 Laboratoire d'Optique et Biosciences, INSERM U696, UMR CNRS 7645, X / ENSTA, Ecole 

Polytechnique, Palaiseau, France 

2 Laboratoire de Pharmacochimie des Substances Naturelles et Pharmacophores Redox, UMR 152, 

IRD-Université Paul Sabatier, Centre IRD de Nouméa, Nouméa, New Caledonia 

3 School of Biomedical Sciences, The University of Queensland, Brisbane, Australia 

4 Microbiologie Alimentaire, USC INRA, Université de Caen Basse-Normandie, Caen, France 

5 CNRS, Institut de Neurobiologie Alfred Fessard – FRC2118, Laboratoire de Neurobiologie 

Cellulaire et Moléculaire – UPR9040, Gif sur Yvette, France, *benoit@nbcm.cnrs-gif.fr 

The cyclic polyethers ciguatoxins (CTXs), produced by toxic dinoflagellates, are 

responsible for ciguatera, a human ichthyosarcotoxism acquired by eating contaminated 

species of fish. Although these toxins are well known to activate voltage-gated Na + 

channels, they were recently reported to produce a swelling of frog erythrocytes that was 

dependent on the activation of voltage-gated L-type Ca 2+ channels. In order to get further 

information on the mode of action of CTXs, we have studied the cellular mechanisms 

involved in the effects of the Pacific (P-CTX-1) and Caribbean (C-CTX-1) ciguatoxins on 

the rheological behaviour of frog red blood cells (RBCs). For these purpose, the length, 

width and surface of the elliptic shape of RBCs, obtained from the blood evicted from the 

frog (Rana esculenta) heart, were measured with a graduated eyepiece, and intracellular 

Ca 2+ changes were determined by microspectrofluorometry using the Ca 2+ -sensitive 

fluorescent dye fura-2/AM. Comparisons between values were done using paired Student’s 

t-test and considering that a P value of less than 0.05 was significant. The results show that 

nanomolar concentrations of P-CTX-1 and C-CTX-1 induced an increase in intracellular 

Ca 2+ that was sensitive to the L-type Ca 2+ channels' inhibitors Cd 2+ and verapamil, and to 

the nitric oxide (NO) synthase (NOS) blocker NG-methyl-L-arginine (L-NMA), as well as 

a cell swelling that was prevented and/or reversed by L-NMA, by blocking Ca 2+ -dependent 

K + channels with apamin or Sr 2+ , and by inhibiting soluble guanylate cyclase (sGC) with 

H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one. In addition, cytochalasin D, a potent 

inhibitor of actin filament and contractile microfilaments, mimicked the CTXs' effects but 

did not prevent the CTXs-induced L-NMA-sensitive extra increase in RBCs' surface. We 

conclude that the molecular mechanisms underlying CTXs-induced RBCs' swelling 

involve the NO pathway by activating first inducible NOS and then sGC which modulates 

intracellular cGMP and regulates L-type Ca 2+ channels. In turn, the resulting increase in 

intracellular Ca 2+ is likely to disrupt the actin cytoskeleton, causing thus a water influx, and 

to activate the SK2 isoform of Ca 2+ -dependent K + channels. 

o Ciguatoxins 

o Frog erythrocytes 

o Cell swelling 

o Nitric oxide pathway 

291

Poster 6: Severe seafood poisoning in French Polynesia: a retrospective analysis of 

severity criteria in 129 hospital medical files 

Gatti, C. 1* , Oehler, E. 2 , Valence, A. 2 , Tamarii, D. 2 and Legrand, A.-M. 1 

1 Institut Louis Malardé, Papeete, Tahiti, French Polynesia, *cgatti@ilm.pf 

2 Centre Hospitalier de Polynésie française, Papeete, Tahiti. 

Seafood poisonings (SFP) are commonly encountered in tropical areas, especially 

in Pacific islands where fish is an important source of proteins. The diseases appear usually 

benign, nevertheless a significant number of severe cases require hospital care. Herein is 

presented a retrospective study of 129 medical files concerning SFP registered at the 

central hospital of Tahiti between 1999 and 2005. During that period we registered in all 

four different SFP: tetrodo-intoxication, carcha-intoxication, lyngbya-intoxication and 

ciguatera poisoning respectively due to the consumption of some species of 

tetraodon/diodon family, shark, sea turtle or reef fish. Micro-organisms responsible for 

such intoxication types, respectively induced by tetrodotoxins, carchatoxins, lyngbyatoxins 

or ciguatoxins, have been identified for some of them as bacteria, cyanobacteria or 

dinoflagellates. Most of the described cases (96%) concerned the ichtyosarcotoxism called 

ciguatera. In those files, cardiovascular symptoms were the primarily criteria of severity 

with bradycardia and hypotension observed in respectively 74 % and 42 % of the cases. 

Neurological manifestations (such as cerebellar syndrome, language troubles, diplopia, or 

polyradiculoneuritis), trouble and/or loss of conscience and dyspnoea, intervene as 

secondary cause of severity. Besides, our attention focused on the body temperature 

reported becoming under 36.5°C in 48 upon 80 documented files. This observation, which 

had never been raised yet in human, may be related to possible central effects of the 

ingested toxin. Indeed, hypothalamus is known to be implicated in thermoregulation and 

hypothermia has been reported as a central effect in experimentally acute intoxication of 

mice with ciguatoxins. The last remark concerns two extremely severe cases of ciguatera 

fish poisoning in which physicians had suspected a Guillain-Barré Syndrome (GBS), an 

autoimmune disease associated with nerve demyelinization and characterised by an 

ascendant reversible paralysis. It is precipitate to formulate any correlation between the 

intoxication and the appearance of GBS but it is interesting to underline that in both 

pathologies, morphological disturbances of nerves fibres have been reported. Could 

ciguatoxins be possible inductors of GBS? 

o ciguatera 

o sea food poisoning 

o severe symptoms 

o hospitalisation 

292 

Poster 7: Paralytic Toxicity in a Ribbon Worm Cephalothrix species (Nemertean) 

Adherent to Cultured Oysters in Hiroshima Bay, Hiroshima Prefecture, Japan 

Manabu, Asakawa 1* , Shuhei Matsuda 1 , Shintaro Tsuruda 1 , Hiroshi Kajihara 2 , Shigeto Taniyama 1 

1 Graduate School of Biosphere Science and Technology, Hiroshima University, 1-4-4, Kagamiyama 

Higashi-Hiroshima, Hiroshima, 739-8528 Japan,* asakawa@hiroshima-u.ac.jp 

2 Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan 

Specimens of ribbon worm Cephalothrix sp. were collected in Hiroshima Bay between 

November and May from 1998 to 2005 almost every two weeks during the harvest time of 

cultured oysters. A total of 764 specimens were collected, and assayed for toxicity. All the 

specimens assayed were found to be toxic throughout the season covered, and toxicity 

scores ranged from 169 MU/g to 25,590 MU/g (as tetrodotoxin). The ratio of strongly 

toxic ( 1,000 MU/g) to total specimens was 80%. On the other hand, the frequency of the 

specimens possessing toxicity scores more than 2,000 MU/g to total specimens was the 

high score of 48%. A relationship between size and toxicity of this ribbon worm was not 

obvious. The highest toxicity detected was 25,590 MU/g in a specimen collected in Jun. 

25(1999). The total toxicity of this one was roughly calculated to be 5,631 MU, the value 

which is approximately equivalent to half of the minimum lethal dose of TTX in human, 

which is reported to be 10,000 MU. In this connection, this lethal potency was about 51 or 

47 times larger than the highest score so far recorded for two species of ribbon worm, 

Lineus fuscoviridis and Tubulanus punctatus inhabiting the surface of rocks or soft mud on 

the seashore (Miyazawa et al., 1986, 1988). It has been reported that TTX and related 

substances are present in highly toxic species of the ribbon worm C. linearis (Ali et al., 

1990). No paralytic toxicity was detected in the shucked meat of the oysters fouled with 

the ribbon worms. As the culturing of edible bivalves such as oysters and scallops is a 

flourishing industry in Japan, surveillance for the distribution of toxic ribbon worms in 

other areas besides Hiroshima Bay is urgently needed. Some seasonal variation of lethal 

potency of this ribbon worm could be recognized. Specimens collected in October 28, 

2004 to January 20, 2005 showed mean toxicities of 1,855-4,076 MU/g. But on February 

22, 2005, their toxicities had sharply decreased down to a mean of 1,578 MU/g. At the 

beginning of December, their toxicities increased to a mean of 4,510 MU/g. 

References: 1. Ali A.E. et al. (1990). Toxicon. 28, 1083-1093. 2. Miyazawa, K. et al. 

(1986). Toxicon. 24, 645-650. 3. Miyazawa, K. et al. (1988). Toxicon. 26, 867-874. 

o Ribbon worm 

o Oyster 

o Tetrodotoxin 

o Hiroshima Bay 

293

Poster 8: Transfer profile and immnopotentiating effect of tetrodotoxin 

administered intraperitoneally into non-toxic cultured pufferfish Takifugu 

rubripes 

Honda, S 1* , Murakami, Y 1 , Ichibu, T 1 , Takatani, T 2 , Tachibana, K 2 , Arakawa, O 2 , Noguchi, T. 3 

1 

Graduate School of Science and Technology, Nagasaki University, 1-14, Bunkyo-machi, Nagasaki 852- 

8521, Japan, *number3000jp@yahoo.co.jp 

2 

Faculty of Fisheries, Nagasaki University, 1-14, Bunkyo-machi, Nagasaki 852-8521, Japan 

3 

Faculty of Healthcare, Tokyo Health Care University, 3-11-3, Setagaya, Setagaya-ku, Tokyo 154-8568, 

Japan 

Introduction: As a part of studies to clarify the accumulation mechanism and biological 

functions of tetrodotoxin (TTX) in pufferfish, two types of TTX preparations, crude 

(cTTX) and purified (pTTX), were administered intraperitoneally into the pufferfish 

Takifugu rubripes, and their transfer profiles and activating effect for splenocyte 

proliferation reactions (SPRs) were investigated. 

Methods: Three groups of non-toxic cultured T. rubripes specimens (body weight 144±16 

g; 30 individuals in each group) were administered intraperitonieally with saline, cTTX 

(crude extract from toxic tissues of the wild puffer T. vermicularis), and pTTX (95% 

purity), respectively, and reared in aerated tanks (90 l) up to 168 hrs. The test fish of each 

group were taken up from the tank 1, 4, 8, 12, 24, 72 and 168 hrs after the toxin 

administration, and determined for the toxicity of each tissue by the mouse bioassay. In 

addition, their SPRs were also examined at 72 and 168 hrs. 

Results: Transfer profiles of cTTX and pTTX in puffer body after intraperitoneal 

administration were somewhat different from each other. pTTX moved to the liver and 

skin more rapidly than cTTX, but at the end of rearing period (168 hrs after 

administration), it remained only in the skin in a small quantity (2.4-3.0 MU/g), whereas 

cTTX was retained mainly in the liver in a relatively large quantity (11-19 MU/g). On the 

other hand, the test fish of pTTX-administered group exhibited slightly higher SPRs than 

those of the other groups at 72 hrs, while the test fish of cTTX-administered group showed 

significantly higher SPRs than those of the other groups at 168 hrs. The temporal changes 

in SPRs appeared to coincide with the transfer profiles of TTX. 

Discussion/Conclusion: From the results it can be inferred that an unknown substance(s) 

coexisting with TTX in cTTX, possibly a TTX-binding protain (1), might be involved in 

the longer retention of TTX in puffer liver. It was also suggested that TTX could exhibit an 

immnopotentiating effect on T. rubripes by acting on the spleen, a main organ coordinating 

immunity in fish. 

References: 1. Matsui et al. (2000) Toxicon, 38, 463-468. 

o Tetrodotoxin (TTX) 

o Pufferfish 

o Takifugu rubripes 

o Splenocyte proliferation reaction 

294 

Poster 9: Toxicity of edible dried fish in the Philippines 

Shigeto Taniyama 1 , Takefumi Sagara 2 , Ryoichi Kuroki 1 , Satoshi Takamoto 3 , Shintaro Tsuruda 3 , Gloria 

Gomez Delan 4 , Sachio Nishio 2 , Manabu Asakawa 1* 

1 Graduate School of Biosphere Science and Technology, Hiroshima University, 1-4-4, Kagamiyama 

Higashi-Hiroshima, Hiroshima 739-8528 Japan, *asakawa@hiroshima-u.ac.jp 

2 Department of Science of Living, Shikoku University, Junior College, 123-1 Furukawa, Ohjin-cho, Tokushima 

771-1192, Japan 

3 Faculty of Applied Biological Science, Hiroshima University, 1-4-4, Kagamiyama Higashi-Hiroshima, 

Hiroshima 739-8528 Japan 

4 College of Fisheries Technology, Cebu State College Science and Technology, Carmen, Cebu Campus, 

Philippines 

In the Philippines, many kind of dried fish are consumed as popular marine product. 

During the studies on the utilization of fish as source of protein, ciguateric toxicities were 

detected in some dried fish sold in the fish market. In this study, attempts were made to 

clear some characteristics of the toxin contained in them. In July and October, 2005, 46 

specimens of dried fish from mainly Scarus niger, S. ghobban, Bolbometopon bicolor and 

Cheilinus chlorourus, were collected from the local market in Negros Is. and Cebu Is. At 

first, meat of 13 specimens was homogenized with acetone respectively. The extract was 

treated with diethyl ether and partitioned between a 90% methanol and an n-hexane. 

Ciguateric toxicities of ninety % of methanol fractions suspended in 1% Tween60 saline 

solution were examined by mouse bioassay to determine ciguatoxins (CTXs). On the other 

hand, by using of ciguatera fish poison test kit (Cigua-Check), CTX is in all specimens or 

not. Eight fat-soluble extracts were found be not only toxic (0.025-0.05 MU/g), but also 

positive in Cigua-Check kit. Thirty-two specimens were too small to clarify their toxicities 

by mouse bioassay. But they showed positive or weakly positive by the kit. Moreover, the 

meat of 12 specimens, 3 of which were combined, was extracted with 75% ethanol (pH 

3.5), defatted with diethyl ether. The extracts of an aqueous layer were submitted to 

biochemical test. All of water-soluble extract were toxic, and their toxicity ranged from 0.5 

to 2.0 MU/g. The symptoms derived from administration (i.p.) to mice were convulsion, 

wobbling gait, jumping, drowsiness and collapse. They died within 24 hrs. Further, in 

hemolysis test, their toxin showed delayed hemolytic activity with mouse or human 

erythrocytes at sample concentration of 0.05-0.2 MU/ml with incubation time of 4 hrs at 

37°C. From these data, it is suggested that most of dried fish were contaminated with 

CTXs in the Philippines, and at least 5 specimens were contained CTX and another 

toxin(s) based on the lethal potency and the delayed haemolytic activity. However, studies 

along these lines are now in progress. 

o Dried fish 

o Ciguatoxins 

o The Philippines 

295

Poster 10: ANTINOCICEPTIVE AND ANTIHYPERNOCICEPTIVE ACTIVITY 

OF THE METHANOLIC EXTRACT OF Phyllomedusa rohdei 

Marinho, E.A.V. 1,3 ; Zaharenko, A.J. 2 ; Paraventi, C 1 ; Jimenez, R.S. 1 ; Molska, G.R. 1 ; Paula-Freire, 

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- 

Marinho, E.L.A 1 

1- Braz Cubas University, Health Science Area, Mogi das Cruzes, SP, Brazil, edumarinho@hotmail.com 

2- University of São Paulo, Biosciences Institute, Dept. of Physiology, São Paulo, SP, Brazil, 

a.j.zaharenko@ig.com.br 

3- Federal University of São Paulo, Dept. of Pharmacology, São Paulo, SP, Brazil 

4- University of São Paulo, Ribeirão Preto, SP, Brazil 

Amphibians of the genus Phyllomedusa produce and secrete peptides and alkaloids in the 

skin. P. bicolor is used in rituals and their secretion is applied on skin burning, promoting 

vomit, salivation and arterial pressure increase. Analysis of the secretion indicated opioid 

compounds. The aim of this study was evaluate the antinociceptive activity of the P. 

rohdei skin extract (EPr). Skin from 5 animals was removed, maintained 1 month in 

methanol, which was filtered and freeze-dried. To the writhing test male mice were used 

(25-35g) in groups (n=6), treated by ip injection, 20min before ip injection of 0,4mL of 

acetic acid 0,6%, observed and the contortions computed during 5min. The mean±SEM of 

the groups treated with EPr 0,3 (27±3), 1 (12±3) and 3mg/kg (3±2) reduced when 

compared to the C (34±3). To the inhibition of hypernociception, male rats (200g) were 

used, and the EPr (60μL) was applied ip 2h40’ after the sc injection of PGE2 (100ng-25μL) 

in the back right paw and was evaluated by the increasing pressure on the paw (electronic 

Von Frey) during 90min (each 30min) since the injection of the EPr and computed as the 

variation in the pressure (Δg) before and after the injection of the extract. The doses of 1 

and 3mg/kg reduced significantly the Δ of nociception (between 2.6 and 5g until the min 

60) when compared to the PGE2 group (~9.2g until the min 60). The Rota-rod test 

evaluated the depressive effect of EPr. Male mice (35-45g) were selected (plus that 1min 

in the equipment), in groups (n=6) and in the test day, pre-exposed 30min before and 

10min after the treatments, and in the times 0, 30, 60 and 90min were evaluated the 

latencies (L) in the apparatus. In C group the mean L was ~60s, diazepam (2mg/kg) 

reduced the L (26±11s) and EPr 0,3; 1 and 3mg/kg did not cause alterations (L ~60s). The 

results indicated that the EPr has antinociceptive and antihypernociceptive action in these 

doses and models, did not change the motor control, with good performance in the Rotarod. 

o Antinociception 

o Antihypernociception 

o Phyllomedusa rohdei 

o Amphibians 

296 

Poster 11: ACTIVITY OF EXTRACTS FROM Phyllomedusa rohdei (AMPHIBIA, 

ANURA) SKIN ON THE MICE BEHAVIOUR IN THE OPEN FIELD AND 

ROTA ROD 

Silva, R.W. 1 ; Zaharenko, A.J. 2 ; Paula-Freire, L.I.G. 1 ; Almeida, E. 3 ; Frussa-Filho, R. 3 ; Malpezzi- 

Marinho, E.L.A 1 , Marinho, E.A.V. 1,3 





Phyllomedusa produces in the skin amines and active peptides and is used in rituals of 

Amazon tribes. The aim was evaluate the action of extracts of P. rohdei on the male mice 

(25-30g) behaviour in the open field. It was computed the mean±SEM (n=10) of 

peripheral, central and total locomotion (squares), grooming (s), rearing, immobilization 

(s) and movement (s) after treatment with the total extract (TEPr 0.3; 1; 3mg/kg), the polar 

fraction (PFPr 0.03; 0.1; 0.3mg/kg) and the apolar fraction (AFPr 0.3; 1; 3mg/kg). The 

TEPr reduced the total locomotion from 92±7 to the control (C) to 70±9 (0.3mg/kg), 52±9 

(1mg/kg) and 27±4 (3mg/kg). With the PFPr the total locomotion was 59±9 (0.03mg/kg), 

71±10 (0.1mg/kg) and 39±10 (0.3mg/kg). With the AFPr the total locomotion was 91±7 

(0.3mg/kg), 92±8 (1mg/kg) and 83±7 (3mg/kg). The TEPr reduced the rearing from 32±3 

(C) to 27±4 (0.3mg/kg), 10±3 (1mg/kg) and 3±1 (3mg/kg). With PFPr the rearings were 

18±3 (0.03mg/kg), 16±4 (0.1 mg/kg) and 6±2 (0.3mg/kg) and with the AFPr 29±4 

(0.3mg/kg), 31±3 (1mg/kg) and 27±4 (3mg/kg). The immobilization increased; in the C 

was 6±3s and with the TEPr 41±18s (0.3mg/kg), 79±18s (1mg/kg) and 162±12s (3mg/kg). 

With the PFPr 38±12s (0.03mg/kg), 56±11s (0.1mg/kg) and 131±17s (0.3mg/kg). With the 

AFPr 4±2s (0.3mg/kg), 2±1s (1mg/kg) and 12±7s (3mg/kg). The grooming time of the C 

was 17±3s; with the TEPr 12±4s (0.3mg/kg), 7±2s (1mg/kg) and 1±1s (3mg/kg), with the 

PFPr 11±3s (0.03mg/kg), 1±0.5s (0.1mg/kg) and 1±0.5s (0.3mg/kg) and with the AFPr 

11±2s (0.3mg/kg), 15±3s (1mg/kg) and 14 ± 3s (3 mg/kg). The results indicate that the 

TEPr and the PFPr change the behaviour in the open field. The Rota-rod test indicates that 

the TEPr do not affect the motor function of the animals (56±4s 0.3mg/kg; 59±1s 1mg/kg; 

51±5s 3mg/kg and 57±3s C), different to the diazepam (2mg/kg 26±11s). This suggests 

that the alterations may be caused either by the peptides (opioids and adenorreguline) or 

opioid receptor antagonists from the skin of P. rohdei. 


o Open field 

o Behavior 

o opioids 

297

Poster 12: κ AND μ OPIOID RECEPTORS ARE INVOLVED IN THE 

ANTINOCICEPTIVE ACTIVITY OF METHANOL EXTRACT OF 

Phyllomedusa rohdei ON THE HOT PLATE MODEL 

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 ; 

Parada, C.A. 4 ; Ferreira, S.H. 4 ; Malpezzi-Marinho, E.L.A 1 ; Marinho, E.A.V. 1,3 





11- University of São Paulo, Ribeirão Preto, SP, Brazil 

Amphibians of the genus Phyllomedusa produce and secrete peptides and alkaloids in the 

skin. P. bicolor is used in rituals and their secretion is applied on skin burning, promoting 

vomit, salivation and AP increase. Analysis of the skin secretion indicated opioid 

compounds. The aim of this study was to evaluate the antinociceptive activity of the P 

rohdei skin extract (EPr). Skin from 5 animals was removed, maintained 1 month in 

methanol, which was filtered and freeze-dried. Male mice were employed (25-30g), under 

light/dark cycle of 12h and water and food ad libitum. A curve (groups with n=5) of the 

EPr during the time (paw licking and jumping) of the mice on hot plate (55±1 o C) (0 to 

240min each 30min) was obtained. The EPr (1 and 3mg/kg) inhibited the response of the 

animals (10-20s) compared to the control (C) (2-8s). 

So, the dose of 1mg/kg was defined to test selective and non-selective antagonists to opioid 

receptors (systemic subcutaneously). Each group (n=5) receives an antagonist (except C) 

and 40min after the EPr. After 20min, each animal was placed on the hot plate computing 

the reaction time. The C group shows a time of 2 to 8s, the treated with EPr 10 to 18s until 

180min. In the groups pre-treated with naloxone (1mg/kg) and EPr the reaction time was 

similar to the C, indicating the presence of opioid receptors. With the selective antagonists 

NorBNI (κ) e Ciprodime (μ) the response to the EPr was similar to C indicating the 

participation of these receptors. Animals pre-treated with Naltrindol (δ) did not show 

differences to the EPr indicating the no participation of these receptors. When naloxone 

was applied in the paw (1μg) it was verified that the EPr has peripheral action, with 

significant reduction during the time. We suggest the participation of opioid compounds, 

or opioid receptor antagonists, in the EPr mediating the antinociceptive effects. 


o Hot plate 

o Opioids receptors 

o Antinociception 

298 

Poster: 13: Xen2174: A novel NET inhibitor that enhances a2-adrenoceptor 

inhibition of spinal pain pathways 

Croker, D.E 1 , Palant, E 1 , Nielsen C 2 , Drinkwater, R 1 , Patterson, M 3 , McCumber, D 3 , Yaksh, T 3 , Wilson, 

D 1* , Smith, M 2 and Lewis, R.J. 1 

1Xenome 

Ltd, 120 Meiers Rd, Indooroopilly, Australia, *david.wilson@xenome.com 

2 

School of Pharmacy, The University of Queensland, St Lucia, Brisbane, Australia 

3 

Department of Anaesthesiology, Research Clinical Teaching Facility, University of California-San 

Diego (UCSD), San Diego, CA, USA 

Xenome Ltd was founded to mine the therapeutic potential of Australian venoms. Venom 

duct DNA has been used to guide the production of synthetic venom peptide libraries that 

have already delivered safe and effective peptide therapeutics. The discovery platform has 

demonstrated its effectiveness across an increasing number of drug targets including 

validated members of the GPCR, kinase, phosphatase, protease, and ion channel target 

families. Xenome’s first product, Xen2174 a novel norepinephrine transporter inhibitor, 

has been progressed to Phase I/IIa trials for the treatment of severe chronic pain. Xen2174 

acts non-competitively to enhance the levels of noradrenaline that accumulate after release 

from the nerve endings of descending inhibitory pain pathways. These elevated levels of 

noradrenaline produce greater activation of alpha2-adrenoceptors (α2-AR) found at the 

nerve terminals of ascending pain pathways. Consistent with this mechanism of action, the 

effects of a bolus IT dose of Xen2174 is fully abolished when co-administered with a 

saturating dose of the α2-AR blocker yohimbine. The SAR and development of this new 

class of therapeutic will be briefly outlined. 

o Xenome 

o Xen2174 

o NET 

o pain 

299

Poster 14: Crotoxin inhibits neuropathic pain and the development of neuromas 

Cury, Y. 1* , Nogueira-Neto, F. 1,2 , Amorim, R.L. 2 , Brigatte, P. 1 , Picolo, G. 1 , Ferreira Jr., W.A. 1 , Nicoletti, 

J.L.M. 2 

1 

Laboratory of Pathophysiology, Butantan Institute, Av. Vital Brazil, 1500. 05503-900, Sao Paulo, Brazil, 

* 

yarac@attglobal.net 

2 

Faculty of Veterinary Medicine and Zootechny, UNESP, Distrito Rubiao Junior S/N, 18618-000, Botucatu, 

Sao Paulo, Brazil 

Introduction: Crotalus durissus terrificus snake venom(CdtV) induces analgesia mediated 

by opioid receptors. The factor responsible for the analgesic action of the venom, named 

crotalphine, was recently isolated and characterized. However, recent data have indicated 

that crotoxin (CTX), the main neurotoxic component of CdtV, exerts antinociceptive effect 

in an experimental model of cancer pain. The aim of the present study is to characterize 

further the analgesic action of CTX, evaluating the effect of the toxin on neuropathic pain 

and determining the mechanisms involved in this effect. 

Methods: For induction of neuropathic pain, the sciatic nerve of male Wistar rats was 

exposed unilaterally by careful dissection, transected in two locations at the mid-thigh 

level and 0.5 cm of the nerve was removed. Pain-related behavior and development of 

neuromas were analyzed over a 64-day period after surgery. The rat paw pressure test was 

used for hyperalgesia evaluation. The presence of neuromas was determined by 

histological analysis of the nerves. 

Results: Hyperalgesia was detected 2 h after surgery and persisted for 64 days. Few 

animals developed neuromas until day 7, however 80 % of the rats presented neuromas on 

day 64. Neuromas often occur at the proximal stump of the transected nerve. CTX (0.01 

mM) applied to the proximal and distal nerve stumps, immediately after nerve transection, 

blocked hyperalgesia. The analgesic effect was observed 2 h after CTX treatment and 

persisted for 64 days. CTX-induced analgesia was blocked or significantly inhibited by i.p. 

administration of atropine (10 mg/kg) and yohimbine (2 mg/kg), respectively. 

Methysergide (5 mg/kg), atenolol (1 mg/kg), and naloxone (1mg/kg) did not interfere with 

this analgesic activity. Histological analysis showed that CTX delays and/or significantly 

inhibits the development of neuromas. 

Conclusions: The results indicate that CTX induces a long-lasting analgesic effect on 

neuropathic pain and inhibits the development of the neuropathy. The analgesic effect is 

mediated by muscarinic receptors and α-adrenoceptors. 

o Crotoxin 

o Neuropathic pain 

o Neuromas 

o Muscarinic receptors 

300 

Poster 15: Phospholipases A2 (PLA2) Isolated From Brazilian Coral Snake 

(Micrurus lemniscatus) Induced Neuronal Injury: In Vivo And In Vitro Studies. 

Carvalho,N.D 1 ., Oliveira, D.A 1 ., Casais e Silva, L.L. 3 ,Lebrun, I. 2 , Afeche, S.C 1 , Sandoval, M.R.L. 1* 

1Laboratory of Pharmacology and 2 Laboratory of Biochemistry and Biophysics,Butantan Institute, av. Dr. 

Vital Brasil, 1500, São Paulo, SP, 05503 900. Brazil. 1* mrlsando@butantan.gov.br 

3 Laboratory of Animais Peçonhentos, Federal University of Bahia, Brazil 

Neurotoxicity from Micrurus venoms from Americas has been poorly studied. The 

aim of this work is to investigate behavioural, EEG and histological effects of two PLA2s, 

Ml-8 and ML-9, isolated from the venom of Micrurus lemniscatus. We also analysis the 

neurotoxicity induced by these toxins in cultured hippocampal neurons. Method: Cannula 

and electrodes were implanted in the CA1 hippocampus (Hpc) 4 days prior the Ml-8, Ml-9 

or phosphate buffer injection into the Hpc. EEG and behaviours were recorded during 8 

hours. Seven days after, the brains were processed for histological analyses. In vitro 

studies: Neurons were dissociated from hippocampi of E18-E19 Wistar rat embryos, after 

treatment with trypsin 0,25% and deoxyribonuclease (0,15mg/ml) neurons were cultured in 

B27-supplemented Neurobasal Medium (GIBCO). Cultures were kept at 37 0 C in a 

humidified incubator in 5% CO2. Cells were plated at a density of 200 neurons/mm 2 . After 

incubation of cultures neurons (at day7) with of Ml-8 or Ml-9 (0.1 – 1000 ng/ml) toxins, 

for 12 or 24 h assessment of neuronal injury was made by using ethidium bromide 

(Mercille & Massie (1994) B. Cytotechnoligy, 15: 117). It was counted 200 neurons in 10 

fields in each coverslip. PLA2 activity was previously determined (date not yet published). 

Results: Ml-8 (2.1μg/μl), Ml-9 (2.4 ug/ul) induced circling behavior, jumping, intense 

scratching, forelimb clonus and the EEG record showed spikes and epileptiform 

discharges. Lesions on CA1 region of the Hpc were also observed. Cultured neurons 

survival was significantly decreased when compared with the control group (ANOVA and 

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 

(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); 

100,0 (7,8±2,2); 1000,0 (2,4±2,1) ng/ml. Conclusion: Ml-8 and Ml-9 caused neuronal 

death by apoptosis and necrosis in neuronal culture. In vivo studies showed behavioral and 

EEG seizures and hpc injury. The toxicity might be associated with the binding of these 

toxins with specific brain site (Lambeau et al, J. Biol. Chem. 1989, 264, 11503). These 

PLA2 can be a useful tool for exploring the death- signaling pathways of neurotoxicity. 

o Micrurus venom 

o Neurotoxic PLA2 

o Neuronal injury 

o Seizure 

301

Poster 16: ENHANCEMENT OF TNF-α AND IL-1 PRODUCTION IN 

ALVEOLAR MACROPHAGES BY C-FIBER DEGENERATION 

DeSouza, IA*Camargo, EA; Franco-Penteado, CF; Antunes, E. 

Department of Pharmacology, Faculty of Medical Sciences, UNICAMP; Campinas, São 

Paulo, Brazil; *ivanidesouza@fcm.unicamp.br 

Objectives: Permanent loss of C-fibers in the rat lungs lead to an exacerbation of 

neutrophil influx in response to ovalbumin and staphylotoxins (SEA and SEB). In an 

attempt to further understand the communication of C-fibers and pulmonary cells, we have 

evaluated the in vitro TNF-α and IL-1 production by alveolar macrophages stimulated with 

SEA and SE. Alveolar macrophages were collected from control or capsaicin-treated 

animals. 

Methods and Results: Alveolar macrophages (AM) were isolated from brochoalveolar 

lavage of control and male Wistar rats (250-300g) treated with capsaicin as neonates. AM 

were ressuspended in culture medium and transferred to tissue culture wells. They were 

allowed to adhere for 2 h (37 o C, 5% CO2) and then incubated for 4 h in the presence of 

either SEA or SEB (3 μg/ml each). The supernatant was colleted and used to determinate 

TNF-α and IL-1 concentrations by ELISA. Incubation of AM with either SEB or SEA 

increased by 30% and 42% the TNF-α production above basal levels, respectively 

(P

Poster 18: ASSESSMENT OF THE INHIBITION INDUCED BY Bufo paracnemis 

POISON AND ITS FRACTIONS ON COMPLEMENT SYSTEM AND 

LEUKOCYTE RECRUITMENT 

Marongio, A. F. Q. 1 , Bertazzi, D. T. 1 , Arantes, E. C* 1 

1 

University of São Paulo, FCFRP, Av. do Café s/n, 14040-903, Ribeirão Preto, SP, 

Brazil. *ecabraga@fcfrp.usp.br 

Previous study showed that Bufo paracnemis poison (BpP) affects the lytic activity 

of the complement system (CS) (Experientia, 41: 940, 1985). Complement activation 

occurs through classical (CP), lectin (LP) and alternative pathways (AP), leading to a 

cascade of component interactions and generation of products with biological activities 

such as anaphylaxis, chemotaxis, opsonization, immune complex solubilization, 

participation in the immune response, etc. The aim of the present study was to investigate 

the effect of BpP and its fractions on the CS and on leukocyte recruitment. 

Methods and Results: The fractionation procedure of BpP involves basically a cationicexchange 

chromatography on CM-Celulose followed by a anionic-exchange 

chromatography of the active fraction on DEAE-celulose column. Four fractions, named 

D1 to D4, were obtained. The effects of BpP and its fractions on CP/LP pathways were 

evaluated using in vitro haemolytic assays. In vitro chemotaxis assays were performed 

using the Boyden chamber model. Poison and the active fractions (C1, D3 and D4) 

induced a reduction in hemolytic activity of the CP/LP complement pathways. Samples 

containing 225 μg and 375μg of BpP reduced 37,8 ± 0.1 % and 50 ± 0.3 % the complement 

activity, respectively. Fractions C1, D3 and D4 induced reduction of 93 ± 0.8 %, 86 ± 8.6 

% and 62 ± 0.2 %, respectively. Incubation of normal human serum with BpP reduced 

neutrophil chemotaxis when compared to that observed with control serum (BpP: 10 ± 1.4 

μm and control: 60 ± 1.2 μm). 

Our results showed that BpP inhibits the complement system and reduces serum 

chemotactic activity. Therefore, it may have an important potential as biological tools for 

studies of processes involving the complement system. 

Financial Support: CAPES and FAPESP. 

o Bufo paracnemis 

o complement system 

o neutrophil chemotaxis 

o toad poison 

304 

Poster 19: Do cytokines and nitric oxide play a role in the actions of Leiurus 

quinquestriatus scorpion venom in animals? 

Abdoon, N. A 1 *, Elsayed Ali, A 2 , Fatani, A. J. 3 

1 National antivenom and vaccine production center, King Fahd National Guard hospital, 

Riyadh,KSA,*Nozabdoon@hotmail.com 

2 Dept. of Pharmacology, College of Pharmacy, Khartoom University, Khartoom,Sudan 

3 Dept. of Pharmacology College of pharmacy, King Saud University,Riyadh,KSA 

Introduction: The actions of scorpion neurotoxins are complex and may be traced to 

activation of different ion channels with subsequent release of various modulators 

including inflammatory mediators. This would lead to various pathological manifestations 

and even death. The purpose of this project was to study these pathological changes and 

examine the ability of inhibitors of different steps in the inflammatory process, to 

ameliorate the detrimental actions and prolong survival of animals injected with Leiurus 

quinqestriatus (LQQ) scorpion venom. 

Methods: LQQ venom (0.4 mg kg -1 , s.c.) was injected into conscious male New Zealand 

rabbits. Changes in blood pressure (BP) were measured up to 12hours via central ear artery 

using blood pressure transducer and physiograph. The BP was correlated with selected 

serum biochemical and hematological parameters including nitric oxide and cytokines (IL- 

8 & TNF). In addition, montelukast (10 and 20 mg kg –1, po), indomethacin (10 and 20 mg 

kg –1 ,iv) and hydrocortisone (5 and10 mg kg –1 ,iv), were administered to mice prior to LQQ 

venom (0.25 and 0.3mg kg –1, sc), and survival recorded after 24hr. 

Results: LQQ caused a triphasic effect on BP: initial reduction, prolonged increase and 

gradual terminal hypotension and bradycardia, followed by death. Increases in serum nitric 

oxide, IL-8 & TNF in addition to leucocytosis were evident in the terminal hypotension. 

All drugs used significantly prolonged survival time and %survival in mice, with 

montelukast having greater protective abilities. 

Conclusions: Anti-inflammatory drugs protect against lethal effects of LQQ venom. 

especially those occurring during the late terminal hypotensive phase indicating that early 

treatment of envenomed victims is essential. (1,2,3,4) 

References: 1. Ismail, M. 1995, Toxicon, 33, 825-858. 2. Barraviera, B. 1997, Toxicon, 35, 

13-14. 3. Fukuhara, Y.M.D, Reis, M.L, Dellabibera-Joviliano, R, Cunha,Q.C, Donadi, E. 

A. 2003, Toxicon, 41, 49-55. 4. Meki,A.A,Mohamed,Z.M.MoheyElDeen,H.M.2003, 

o Leiurus quinqestriatus 

o Nitric oxide 

o cytokines 

305

Poster 20: EVALUATION OF THE ACUTE LUNG INJURY IN A MURINE 

MODEL INDUCED BY THE SCORPAENA PLUMIERI FISH VENOM 

Boletini-Santos D 1 , Haddad Jr V. 2 *, Figueiredo S.G. 3 , Lopes-Ferreira M. 1 , Lima C. 1 . 

1Laboratório 

de Imunopatologia do Instituto Butantan, São Paulo, Brazil. 

2 

Departamento de Dermatologia, Faculdade de Medicina, UNESP, Botucatu. ZIP 18618- 

000, * haddadjr@fmb.unesp.br. 

3 

Departamento de Ciências Fisiológicas, UFES, Brasil. 

INTRODUCTION: The Brazilian Scorpaena plumieri fish provoked human accidents 

causing severe injuries, characterized by local and systemic effects. The aim of this work 

was to study the effect of venom on target organs distant from the inoculation site. 

METHODS: BALB/c mice injected i.p. with 10, 30, and 100 μg of venom were killed after 

2, 6, 24, and 48 h and the tracheas were cannulated for collection of bronchoalveolar 

lavage fluid (BAL). The pellet and supernatants from BAL were used for cell counts and 

determination of protein and NO levels, respectively. The homogenates of lung tissue were 

used for cytokines analysis and zymography. RESULTS: When 10 or 30 μg venom was 

injected a large number of leukocytes, mainly macrophages, was observed in BAL 

collected 6 h after. However with 100 μg venom, an earlier lung inflammation (2 h), 

predominantly of macrophages, was observed. All doses of venom induced a significant 

increase in protein level in BAL at 24 and 48 h, but only 100 μg venom induced a rapid 

and sustained extravasation of protein. Only IL-6 was detected in lung at 2 and 24 h. IL- 

1β, TNF-α, MCP-1 or NO were not detected in BAL in any circumstance. Gelatinolytic 

activity was detected in lungs 2 and 24 h after injection of 100 μg venom and by SDS- 

PAGE showed correlation with a 100 kDa band. Immunohistochemical staining for S. 

plumieri venom showed the presence of the venom in alveolar epithelial cells and around 

the vessels and abundant distribution throughout of the lung. CONCLUSION: The results 

showed by the first time that S.plumieri venom induces acute lung injury by protein 

passage from the vascular to the alveolar space and infiltration of macrophages, secretion 

of cytokines and proteinases with gelatinolytic activity. Supported by Fapesp. 

Haddad Jr V, Martins IA, Makyama HM. Injuries caused by scorpionfishes (Scorpaena plumieri Bloch, 1789 

and Scorpaena brasiliensis Cuvier, 1829) in the Southwestern Atlantic Ocean (Brazilian coast): 

epidemiologic, clinic and therapeutic aspects of 23 stings in humans. Toxicon 42: 79-83, 2003. 

o Scorpaena 

o Scorpaenidae 

o human injuries for fish 

o venomous fish 

306 

Poster 21: INFLAMMATORY MEDIATORS INVOLVED IN PHOSPHOLIPASE 

A2 (PLA2)-INDUCED ACUTE PANCREATITIS 

Camargo, E.A.* 1 ; Ferreira, T. 1 ; Landucci E.C.T. 1 ; Antunes, E 1 . 

1 Department of Pharmacology, Faculty of Medical Sciences, UNICAMP; Campinas, São 

Paulo, Brazil; *eapcamargo@fcm.unicamp.br 

Objectives: PLA2s induce acute pancreatitis when injected into the pancreaticobilliary 

duct of rats (Camargo et al., 2005). We have investigated the inflammatory mediators 

involved in PLA2-induced acute pancreatitis. Methods: Acute pancreatitis was induced by 

the injection of PLA2 from Naja mocambique mocambique venom (PLA2-Nmm; 300 

μg/kg) into the common bile duct of male Wistar rats (220-260g, n=5-8). Four h later, the 

pancreatic plasma protein extravasation (PPE), pancreatic and lung myeloperoxidase 

(MPO), serum amylase (sAM) and TNF-α (sTNF-α) were measured. Results: The PLA2- 

Nmm induced a marked increase in PPE (65%), pancreatic MPO (309%), lung MPO 

(34%), sAM (155%) and sTNF-α (260%). Pretreatment with the B2 receptor antagonist 

Icatibant (100 nmol/kg) reduced significantly the PPE, pancreatic MPO, lung MPO, sAM 

and sTNF-α. The neurokinin-1 (NK1) receptor antagonist SR140333 (120 nmol/kg) 

reduced the PPE, pancreatic MPO and s-TNF-α, but did not affect the lung MPO and 

sAM. The PAF receptor antagonist PCA4248 (5 mg/kg) decreased significantly the lung 

MPO and sTNF-α, without causing any change in the other parameters. The non-selective 

NO inhibition with L-NAME (20 mg/kg) reduced significantly the PPE and sTNF-α, but 

rather caused an increase in the pancreatic MPO. The lung MPO and sAM were not 

significantly affected by L-NAME. Aminoguanidine (inducible NO synthase inhibitor, 50 

mg/kg) treatment had no effect in any parameter evaluetad. Indomethacin (cyclooxygenase 

inhibitor, 5mg/kg) decreased significantly the lung MPO and sTNF-α, without causing any 

other changes. The treatment with celecoxib (cyclooxygenase -2 selective inhibitor, 20 

mg/kg) decreased the PPE without affecting other parameters. Conclusions: Acute 

pancreatitis induced by exogenous PLA2 involves the activation of B2 and NK1 receptors, 

endothelial NO synthase-derived NO and cyclooxygenase -2 metabolites in the pancreatic 

tissue. The remote lung injury seems to involve cyclooxygenase -1 metabolites and the 

activation of B2 and PAF receptors. Reference: Camargo et al., Toxicon, 2005, 46, 921-6. 

Financial Support: Fundação de Apoio à Pesquisa do Estado de São Paulo 

o Acute pancreatitis 

o Inflammatory mediators 

o Phospholipases A2 

307

Poster 22: Effect of eugenol and local anesthetic agents on tongue and skin edema 

induced by Dieffenbachia picta Schott in mice 

Dip, E.C. 1 ; Pereira, N.A. 1 ; Gaban, G.A. 1 ; Fonseca, T. 1 ; El Kik, C.Z. 1 ; Tomaz, M.A 1 ; 

Calil-Elias, S. 1 ; Martinez, A.M.B. 2 ; Melo, P.A. 1 . 

1 

Departamento de Farmacologia Básica e Clínica ICB, CCS Universidade Federal do Rio de Janeiro, 

21941-590, Rio de, Janeiro, RJ, Brazil. pamelo@farmaco.ufrj.br. 

2 

Departamento de Histologia e Embriologia, ICB, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 

RJ, Brazil . 

Dieffenbachia picta Schott is a tropical poisonous an ornamental plant which induce 

angioedema, glottis obstruction, respiratory compromise and death in mammals. These 

manifestations can be partially antagonized by eugenol and some anti-inflammatory drugs. 

We examined the tongue and skin, edema induced by D. picta juice and the effect of 

denervation or the local anesthetic agent, benzocaine (20%) lidocaine (10%) and eugenol 

(0.1- 0.3%). Tongue edema was induced by topical application of 100 μL of D. picta juice 

and it was evaluated with a digital tachymeter during 2 hours. For the skin edema study, 

mice received i.v. injection of Evan’s blue dye (2.5% solution; 25 mg/kg) 60 min. before 

the intradermal injection of 100 μL of D. picta juice. Skin edema were evaluated by 

measuring the dyed plasma extravasation and accumulation in the skin in a 

spectrophometer. The amount of dye in the dried skin were evaluated The tongue edema 

reached the maximum at 60 min. after topical application of 100 μL of D. picta juice in the 

control mice, and it was completely inhibited by topical application of benzocaine and 

partially inhibited by lidocaine (circa of 30%) or eugenol(circa of 31.6%). Preincubation of 

D. picta juice with lidocaine, 2.5and 10% decrease the skin plasma extravasation from 

42,93% ± 12.333% to 99.68 ± 9,03 respectively (n=8 for each group) while eugenol 50 

mg/kg (n=10) reduced in 31.96% ±2.30%. Our results showed that local anesthetic agents 

can decrease the acute inflammatory response induced by D. picta juice in mice. 

Dip et al., (2004) Toxicon 43:729-735 

o Dieffenbachia picta, 

o tongue edema and inflammation, 

o local anesthetic 

308 

Poster 23: Importance of jararhagin disintegrin-like and cysteine rich domains in 

the early events of local inflammatory response. 

Clissa, P.B. 1 ; Lopes-Ferreira, M. 1 ; Della-Casa, M.S. 1 ; Farsky, S.H.P. 2 ; Moura-da-Silva, A.M. 1 

1. Immunopathology Laboratory – Butantan Institute - Sao Paulo – Brazil 

2. Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of 

Sao Paulo – Brazil 

Jararhagin is a multidomain SVMP from Bothrops jararaca venom comprising catalytic, 

disintegrin-like and cysteine-rich domains, which cause a local reaction manifested by 

hemorrhage, edema, cytokine release and inflammatory cell recruitment. In this study, the 

importance of disintegrin-like/cysteine-rich domains of jararhagin was addressed by 

analyzing the effects of jararhagin-C, which lacks the catalytic domain, in induction of 

leukocyte rolling and release of pro-inflammatory cytokines. Jararhagin-C was isolated 

from B. jararaca venom conserving the same ability of complete jararhagin molecule in 

inhibiting collagen-induced platelet-aggregation. Treatment of trans-illuminated cremaster 

muscle in vivo with jararhagin-C increased number of rolling leukocytes (approximately 

250%) in post-capilary venules in all periods analyzed, without interfering with 

microvasculature haemodinamic, like vessel diameter, the eritrocyte speed or the blood 

flow rate. The release of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 was 

significantly enhanced in the local of jararhagin-C injection, showing the maximum levels 

in periods between 2 and 4 hours after treatment. Besides the action of jararhagin-C, the 

presence of the inactivated catalytic domain in o-phenanthrolin-treated jararhagin was 

related to a higher increase in the number of rolling leukocytes. Moreover, the levels of IL- 

6 and IL-1β induced by catalytically active jararhagin were higher than those induced by 

jararhagin-C. In conclusion, our findings suggest that the disintegrin-like/cysteine-rich 

domains of jararhagin are sufficient to locally activate the early events of an acute 

inflammatory response as leukocyte rolling and pro-inflammatory cytokine release and this 

action may add to the effect of catalysis, which enhances the primary cell activation. 

Supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) 

o Jararhagin 

o Jararhagin-C 

o Disintegrin-like and cysteine-rich domains 

o Inflammation 

309

Poster 24: Short-time analysis of pulmonary mechanics on acute lung injury 

induced by Crotalus durissus terrificus venom 

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, 

Cogo, J.C 1 , Oliveira, L.V.F. 1 

1 

Universidade do Vale do Paraíba-UNIVAP, IP&D, 2911 Shishima Hifumi Av, São José dos Campos- 

SP, Brazil, paulanaomi@gmail.com 

2 

Universidade de Brasília-UNB, Laboratório de Fisiologia Respiratória, Brasília-DF, Brazil. 

There are very few reports related to specific inquiries on the repercussions of the Crotalus 

durissus snakes venom in the respiratory system, therefore, the behavior of the mechanical 

properties of the respiratory system, the characterization of the pulmonary structures and 

the quantification of the generated inflammatory process are less known. The aim of this 

study is to test if this venom could induce pulmonary mechanics alterations at a short-time. 

Eighteen Swiss mice, were analysed three hours after intramuscular injection of saline 

(control group) or Crotalus durissus terrificus crude venom (0,6µg.g -1 ). The sub-lethal 

dose used in venom group, was determined by the LD50 previously found. The mechanical 

parameters were obtained by End Inspiratory Occlusion Method. The statistical analysis 

was carried through the Kolmogorov-Smirnov test for normality and the independent t-test 

in a significance level of 5%. Static elastance (p

Poster 26: Hydralysins – novel proteins from cnidaria revealing a sequence 

signature with a role in pore formation common to various beta pore forming toxins 

Sher, D.* 1 , Fishman, Y. 1 , Zhang, M. 1 , Lebendiker, M 1 , Mancheño, J.M. 2 and Zlotkin, E. 1 

1 

Department of Cell and Animal Biology, Silberman Institute of Life Sciences, Hebrew University, 

Jerusalem 91904, Israel. * dsher@pob.huji.ac.il 

2 

Instituto de Quimica-Fisica Rocasolano, CSIC, Departamento de Cristalografia, Serrano 119, E-28006 

Madrid, Spain 

Beta Pore-forming toxins (βPFTs) are a diverse family of water soluble proteins able to 

interact with and enter into biological membranes to create transmembrane pores. They 

are found in animals, plants and fungi, and are involved in the pathogenicity of many 

bacteria. While the stages of pore formation by different βPFTs are similar, they differ 

widely in their primary amino acid sequences. Thus, it has been difficult to generalize the 

information gained from model βPFTs on the relationship between structure and function 

and apply it to other members of this protein family. 

We have previously shown that the green hydra Chlorohydra viridissima produces nonnematocystic 

paralytic and cytolytic toxins, named hydralysins (Hlns) (1). Here we show 

that Hlns are a family of βPFTs, which are distinct from other cnidarian toxins but similar 

in activity and structure to bacterial and fungal toxins (2). Functional characterization of 

soluble Hln monomers using UV circular dichroism (UV-CD) and computational analysis 

reveals that they are rich in beta-structure. Hlns bind erythrocyte membranes and form 

discrete pores with an internal diameter of ~1.2nm. The cytolytic effect of Hlns is cell-type 

selective, suggesting a specific receptor. Multiple sequence alignment reveals that Hlns 

share a set of conserved sequence motifs with known βPFTs such as aerolysin, epsilon 

toxin, alpha toxin and LSL, and that these sequence motifs are found in and around the 

toxins’ pore-forming domain. The importance of this “sequence signature” is revealed by 

the cloning, expression and mutagenesis of three Hln isoforms which strongly differ in 

their hemolytic and paralytic activities. 

The sequence signature common to Hlns and other βPFTs reveals previously undetected 

conserved residues, and provides a platform for comparative structure-function analysis of 

βPFTs. Proteins containing this sequence signature are widely distributed, suggesting 

novel biological roles for βPFT-like proteins in nature. 

1. Zhang, M., et al. (2003) Biochemistry 42, 8939-8944 

2. Sher, D., et al. (2005) J Biol Chem 280, 22847-22855 

o Pore-forming toxins 

o sequence 

o mutagenesis 

o structure-function 

312 

Poster 27: Caissarolysin I (Bcs I), a new hemolytic toxin from the Brazilian sea 

anemone Bunodosoma caissarum: Purification and biological characterization. 

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 , 

Richardson, M. 4 , Sant’Anna, O.A. 3 , Tambourgi, D.V. 3 

1 

Universidade de São Paulo, Depto. de Fisiologia, Instituto de Biociências, São Paulo, BRAZIL. 

*a.j.zaharenko@ig.com.br. 

2 

Center for Applied Toxinology, CAT-CEPID, Instituto Butantan, São Paulo, BRAZIL. 

3 

Laboratório de Imunoquímica, Instituto Butantan, São Paulo, BRAZIL. 

4 

Fundação Nacional Ezequiel Dias- FUNED, Belo Horizonte, BRAZIL. 

Two cationic proteins, C1 and C3, were purified to homogeneity from the hemolytic 

fraction of the venom of Bunodosoma caissarum sea anemone. The purification processes 

employed gel filtration followed by ion exchange chromatography (FPLC), being the 

purity and molecular weight confirmed by SDS-PAGE and mass spectrometry. Protein C1 

represented the second major peak of the hemolytic fraction and was previously believed 

to be a cytolysin belonging to a new class of hemolysins. The C1 protein has a molecular 

weight of 15495 Da and was assayed for hemolysis, PLA2 activity and acute toxicity in 

crabs and mice, showing no activity in these assays. It has an amino terminal with no 

similarity to all known hemolysins and, therefore, should not be considered a toxin, being 

its function completely unknown. The protein C3 (MW19757), that also lacks PLA2 

activity, was recognized by antiserum against Eqt II and presented high hemolytic activity 

to human erythrocytes (ED50 of 0.270 μg/ml), being named Caissarolysin I (Bcs I). Its 

activity was inhibited by pre-incubation with sphingomyelin (SM) and also when in 

presence of erythrocytes pre-treated with the SMase P2, a phospholypase D from the 

brown spider Loxosceles intermedia, indicating that SM is the main target of Bcs I. 

Caissarolysin I is the first hemolysin purified from a sea anemone belonging to the genus 

Bunodosoma and belongs to the Actinoporin family of sea anemone hemolysins. 

All this data was published in the paper Oliveira et al., (2006). 

References: Oliveira, J.S., Zaharenko, A.J., Freitas, J.C., Konno, K., Andrade, S.A., 

Portaro, F.C.V., Richardson, M., Sant’Anna, O.A. and Tambourgi, D.V. (2006) Biochimica 

et Biophysica Acta-General Subjects, 1760(3), 453-561. 

Financial Support: FAPESP, CNPq. 

o Hemolysin 

o Sea anemone 

o Bunodosoma caissarum 

o sphingomyelin 

313

Poster 28: Beta-Lactamase in Escherichia coli 

Siham S. Shaokat* Hamoudi A. Hameed 1 

ٍٍٍ 

Israa H.J. 2 

*Prof. Of Pharmaceutical Microbiology/ Food & Drugs Sector/Nidal Street/Baghdad/Iraq/ email: 

albiatyss84@yahoo.com M: 07901867274 

1 

Prof. Of Chemistry. / Ministry of Industry & Minerals 

Chief Food & Drugs Sector/hamodiabas@yahoo.com/M:07901918147 

Introduction: The aims of this study was to evaluate the Production of transferable betalactamases 

in 58 strains of E.coli isolated from urinary tract infections collected from two 

hospitals in Iraq . 

Methods: identification of E.coli by the Api systems. 

The antimicrobial susceptibility test& the minimum inhibitory concentrations were 

determined, by Kirby-Bauer method. The antibiotic resistance rates were: Amoxycillin 

(100%), Carbenicillin (79.3%), Cefalothin (37.93%), Amoxycillin\ potassium Clavulanate 

(20.7%), Cefotaxime (17.25%), Ceftrixone (8.62%), Ceftazidime (0%); 

Trimethoprim100%, Tetracycline (77.6%), Rifampicin (67.24%), Chloramphenicol 

(48.28%), &Nalidixic acid (32.8%). Transfer tests were done and cell free β- lactamases 

were prepared and detected by macro-iodometric method. 

Results: The most frequent mechanism of resistance was due to Production of transferable 

beta-lactamases in 36 strains, Production of cephalosporinases 12 strains and combination 

of these two mechanisms 10 strains. 

UTIs due to resistant strains of E.coli may be successfully treated with β- lactam 

antibiotics, and the activity was in the following order: Ceftazidime, Ceftrixone, 

Cefotaxime, &Amoxycillin\ Clavulanate. 

Conclusions 

UTIs due to resistant strains of E.coli may be successfully treated with β- lactam and the 

activity was in the following order: Ceftazidime, Ceftrixone, Cefotaxime, &Amoxycillin\ 

Clavulanate. 

Mushtaq, -S; Woodford,-N; Potz,-N; Livermore,-D-M.Detection of CTX-M-15 extendedspectrum 

beta-lactamase in the United Kingdom. 

J-Antimicrob-Chemother. 2003 Sep; 52(3): 528-9 

o Beta-lactamase 

o E. coli 

o Beta-lactam 

314 

Poster 29: Membrane Interactions of the Pore-forming Protein Equinatoxin II: 

19 F NMR Studies of the role of Trp and Tyr 

Norton, R.S. 1 , Bakrač, B. 2 , Anderluh. G. 2 , Podlesek, Z. 2 , Razpotnik, A. 2 , Separovic, F. 3 

1 



2 

Department of Biology, Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia 

3 

School of Chemistry, University of Melbourne, 3010, Australia 

Sea anemones produce a family of 18-20 kDa proteins, the actinoporins, which lyse cells 

by forming pores in cell membranes. Sphingomyelin (SM) plays an important role in their 

lytic activity, with membranes lacking this lipid being largely refractory to these toxins. As 

a means of characterising membrane binding by the actinoporin equinatoxin II (EqTII), we 

have used 19 F NMR to probe the environment of Trp and Tyr residues in the presence of 

micelles and bicelles. Trp and Tyr were chosen as previous data from mutational studies 

and truncated analogues had identified the N-terminal helix of EqTII and the surface 

aromatic cluster including Trp 112 and 116 and Tyr 113, 133, 137 and 138 as being 

important for membrane interactions. In two separate analogues, the five Trp residues were 

replaced with 5-fluoro-Trp and assigned by site-directed mutagenesis and the 11 Tyr by 3fluoro-Tyr. 

Changes in the Trp 19 F resonances upon EqTII binding to phospholipid (PC) 

micelles or bicelles, and subsequently upon addition of SM, allowed a model to be 

constructed of how EqTII interacts initially with membranes (1). However, in the model 

membranes studied here, interaction with SM was not sufficient to trigger dissociation of 

the N-terminal helix from the beta-sandwich, which forms the bulk of the protein; this 

dissociation is considered to be essential for pore formation (2). Further characterization of 

the membrane interactions of EqTII is underway using F-Tyr labelled EqTII. 

Our results illustrate the value of 19 F NMR in studies of the interactions of proteins with 

membranes, with good quality spectra attainable in relatively short acquisition times and 

chemical shifts that are exquisitely sensitive to the local environment. Moreover, the 19 Flabelled 

proteins are useful in solid-state NMR experiments. 

1. Anderluh, G., Razpotnik, A., Podlesek, Z., Maček, P., Separovic, F. and Norton, R.S. 

(2005) J Mol Biol 347, 27-39. 

2. Hong Q, Gutiérrez-Aguirre I, Barlič A, Malovrh P, Kristan K, Podlesek Z, Maček P, 

Turk D, González-Mañas JM, Lakey JH, Anderluh G. (2002) J Biol Chem 277, 41916-24. 

o cytolysin 

o protein 

o membrane binding 

o fluorine 

315

Poster 30: Toxicity of Gambierdiscus sp. and Ostreopsis sp. collected from the 

coasts of western Japan 

Takefumi Sagara 1* , Shigeto Taniyama 2 , Osamu Arakawa 3 , Tamiko Hashimoto 1 , Naoyoshi Nishibori 1 , 

Manabu Asakawa 2 and Sachio Nishio 1 

1 

Faculty of Junior College, Shikoku University, 123-1, Ebisuno, Furukawa, Ohjin-cho, Tokushima, 

Japan, *takefumi-sagara@shikoku-u.ac.jp 

2 

Graduate School of Biosphere Science, Hiroshima University, 1-4-4, Kagamiyama, Higashi-Hiroshima, 

Hiroshima, Japan 

3 

Faculty of Fisheries, Nagasaki University, 1-14, Bunkyo-machi, Nagasaki, Japan 

Recently, ciguatoxin (CTX) and palytoxin (PTX) food poisoning incidents due to the 

ingestion of toxic marine fish have occurred in the western parts of Japan. As previously 

reported, CTXs are produced by the benthic dinoflagellate Gambierdiscus toxicus 

distributed in the Gambier Islands of French Polynesia (1). The benthic dinoflagellate 

Ostreopsis sp. has been known to produce analogue of PTX (2), and might be the origin of 

PTX in toxic parrotfish, Scarus ovifrons, inhabiting Japan (3). Between 2004 and 2006, 

benthic dinoflagellates were collected from algal samples in four stationary points, 

Miyazaki, Nagasaki, Tokushima and Kochi Prefectures, those are located in the North 

Temperate Zone, and are the reported areas of CTX and PTX poisoning in Japan. 

Gambierdiscus sp. was attached to algal surface in Tokushima and Kochi. In May, 2005, 

especially, a maximum of 212 cells of Gambierdiscus sp. was observed to adhere per one 

gram of seaweed from Kochi and they were positive in the rapid detection test kit, Cigua- 

Check, for CTX. The clone cell was cultured in an ESM medium for 30 days, the cell 

pellets (about 10 6 cells) were extracted and examined using biochemical analyses. The 

crude extract was found to be positive at a sample concentration in Cigua-Check. On the 

other hand, Ostreopsis sp. was distributed in all stationary points, and each clone isolated 

from them was cultured according to the previous conditions. All crude extracts from 

cultured clones of Ostreopsis sp. had detected a lethal potency (0.5 x 10 -4 - 1.0 x 10 -4 

MU/cell) in the mouse test for PTXs. Further, their toxins showed delayed haemolytic 

activity with mouse and human erythrocytes and the activity with latter erythrocytes was 

inhibited by g-strophanthin as well as PTX. Thus, it is suggested that some species of 

marine fish may be toxified due to toxic benthic dinoflagellate Gambierdiscus sp. and 

Ostreopsis sp. in Japan. 

1. Yasumoto et al. (1977). Bull. Jpn. Soc. Sci. Fish., 43, 1021-1026. 

2. Usami et al. (1995). J. Am. Chem. Soc., 177, 5389-5390. 

3. Taniyama et al. (2003). Toxicon, 42, 29-33. 

o Ciguatoxin 

o Palytoxin 

o Gambierdiscus sp. 

o Ostreopsis sp. 

316 

Poster 31: Syphaxin 1.5, a novel antimicrobial peptide isolated from the skin 

secretions of the anuran Leptodactylus syphax. 

Dourado, F. S 1,2* , Moreira, K. G 2 , Brand, G. D 2 , Melo, J. T 2 , Leite, J, R. S. A 2 , Silva, L. P 2 , Bloch Jr, C 2 , 

Schwartz, E. F. 1 

1 

Universidade de Brasília – Laboratório de Toxinologia, Brasília-DF Brazil, *fsdourado@unb.br 

2 

EMBRAPA - Recursos Genéticos e Biotecnologia – Laboratório de Espectrometria de Massa, Brasília- 

DF, Brazil 

Introduction: Antimicrobial peptides (AMPs) are considered part of the innate immune 

system of the majority of living organisms. Mostly of them are cationic, amphiphilic, and 

have molecular mass smaller than 10 kDa. This work describes a new AMP active against 

Staphylococcus aureus and Escherichia coli without cytotoxic activity to human blood cells. 

Methods: The skin secretion of two specimens of Leptodactylus syphax (Brasília, DF, Brazil) 

was extracted by mild electrical stimulation, dried, resuspended with TFA 0.1%, and filtered 

(0.22 µm). The filtered material was submitted to RP-HPLC (218TP510 and 218TP54 Vydac 

columns) using a linear gradient of TFA 0.1% (v/v) and acetonitrile/TFA 0.1% (v/v). 

Syphaxin 1.5 (SPX 1.5) was de novo sequenced by MALDI-TOF/TOF (Bruker Ultraflex II). 

The activity of SPX 1.5 against S. aureus and E. coli was tested by the broth microdilution 

assay and the minimum inhibitory concentration (MICs) obtained was compared with MICs 

obtained from commercial antibiotics. The cytotoxic activity was tested on human blood cells 

and performed in Cell-Dyn 3500 SC/SL flow cytometry automated hematology analyzer. 

Results: The purified peptide (SPX 1.5) had the experimental mass of [M+H] + = 1577.83 Da 

and the sequence GVLDILKGAAKDLAGH. SPX 1.5 inhibited both S. aureus and E. coli 

ATCC strains, with MICs of 40.5 and 10.1 μM, respectively. SPX 1.5 did not exhibit 

significant lytic activity on human blood cells. 

Conclusion: SPX 1.5 was only the fifth AMP isolated from the skin secretions of 

Leptodactylus genus frogs (1, 2 and 3). A ClustalW cladogram 

(http://www.ebi.ac.uk/clustalw/) of the five peptides described to Leptodactylus genus 

positioned SPX 1.5 in the same cluster as the peptides from L. pentadactylus group, as 

expected by phylogenetic relationships of these anurans. 

References: 1. Nascimento et al. (2004) Protein J. 23:501-8. 2. Rollins-Smith et al. (2005) 

Regulatory Pept. 124:173-8. 3. King et al. (2005) Comp. Biochem. Physiol. C: 141:393-7. 

Financial Support: FUB, CAPES and FINATEC. 

o Antimicrobial Peptides 

o Leptodactylus syphax 

o Skin secretion 

o Syphaxin 1.5 

317

Poster 32: Cytoskeletal rearrangement and cell death induced by Bothrops 

alternatus snake venom in cultured Madin-Darby canine kidney cells 

Nascimento, J.M. 1,2 , Franchi, G. 3 , Novill, A. 3 , Collares-Buzato, C.B. 4 , Hyslop, S. 1,* 

1 


(UNICAMP), CP 6111, 13083-970, Campinas, SP, Brazil *hyslop@fcm.unicamp.br 

2 

Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), CP 

6109, 13083-970, Campinas, SP, Brazil 

3 

Centro Integrado de Pesquisas Oncológicas Infantis (CIPOI), Faculdade de Ciências Médicas, Universidade 

Estadual de Campinas (UNICAMP), CP 6111, 13083-970, Campinas, SP, Brazil 

4 Departamento de Histologia e Embriologia, Instituto de Biologia, Universidade Estadual de Campinas 

(UNICAMP), CP 6109, 13083-970, Campinas, SP, Brazil 

Introduction: Bothrops snake venoms cause renal damage that can lead to renal failure, 

the principal cause of death in humans bitten by these snakes. In this work, we 

investigated the cytoskeletal rearrangement and cytotoxicity caused by Bothrops 

alternatus venom in cultured Madin-Darby canine kidney (MDCK) cells, a renal 

epithelial cell line. Methods: MDCK cells were incubated with 10 or 100 μg of 

venom/ml and cytotoxicity was assayed based on neutral red uptake and changes in the 

transepithelial electrical resistance (RT). MDCK morphology was assessed by the 

Feulgen reaction, toluidine blue staining and scanning electron microscopy (SEM). 

Staining with rhodamine-conjugated phalloidin was used to detect F-actin. Cell death 

was quantified by flow cytometry using annexinV-FITC and propidium iodide. Results: 

Venom (10 or 100 μg/ml) significantly (p

Poster 34: Effect of the Disintegrin Eristostatin on Melanoma Cell Signal 

Transduction. 

Paquette-Straub, C.A, DiRosato, S.C, McLane, M.A*. 

University of Delaware, 305 Willard Hall Educ. Bldg., Newark, DE, USA, *mclane@udel.edu 

Metastasis is a major obstacle to treatment of melanoma. Previous researchers have 

shown that the venom-derived disintegrin eristostatin inhibits both human and murine 

melanoma lung or liver colonization in hematogenous mouse metastasis models. The 

mechanism for this inhibition is not yet known. Recent studies have shown that a possible 

mode of action for this inhibition may be through inhibiting cell motility. In order to better 

understand the intracellular signalling, RNA was isolated from five melanoma cell lines 

(C8161, M24met, MV3, WM164 and 1205Lu) after treatment with or without eristostatin 

(3000nM). The RNA was used to create a cRNA probe for hybridization to 

oligonucleotide microarrays. After hybridization, the microarrays were developed using 

enhanced chemiluminescence. The results were photographed and imported into analysis 

software supplied by the manufacturer of the microarrays. A comparison of treated and 

untreated cells showed a change in expression of several genes, including MMP2, MMP9, 

AKT1, PTEN and several integrins. Interestingly, the changes were not consistent across 

all five cell lines, increasing in some, while decreasing in others. Concurrently, cells that 

had been treated with or without eristostatin for varying time points were lysed and 

analysed for changes in protein tyrosine phosphorylation. The change in phosphorylation 

was different for all five cell lines. Proteins at molecular weights of 100-120 kDa, 60-65 

kDa, and 40-50 kDa all had a cyclic change in phosphorylation across the time course. 

These molecular weights may correspond to proteins such as FGFR, Grb10, the Src family 

of proteins, PTEN and Nck. The involvement of these proteins would suggest eristostatin 

is affecting focal adhesion formation (1). Further studies are now being performed using 

Real Time PCR and confocal microscopy to further elucidate the mode of action of 

eristostatin. 

1. Staiano, N. et al. 1997. European Journal of Cell Biology 73, 298-305. 

o Eristostatin 

o Disintegrin 

o Cancer 

o Signal Transduction 

320 

Poster 35: Ostreolysin, a mushroom pore-forming toxin specifically binding to 

sphingomyelin/cholesterol-rich membrane domains 

Rebolj, K. 1 , Maček, P. 1 and Sepčić, K. 1* 

1 University of Ljubljana, Biotechnical faculty, Department of biology, Večna pot 111, Ljubljana, 

Slovenia, *kristina.sepcic@bf.uni-lj.si 

Introduction: Ostreolysin (Oly) 1 is an acidic 15-kDa cytolytic protein from the edible 

mushroom Pleurotus ostreatus. It is haemolytic, cytolytic, and toxic to mice with a LD50 of 

1170 μg/kg. Its lytic activity is a consequence of specific interaction with cholesterol- 

enriched membrane domains that exist in the liquid ordered (lo) phase (lipid rafts). In this 

work, we have investigated the determinants of the lipid structure dictating lipid raft 

formation, Oly membrane binding and pore-forming activity. 

Methods: Surface plasmon resonance was applied to study the binding of Oly on lipid 

mono- and bilayers with different composition, and measurements of calcein release to 

determine its permeabilization activity. 

Results: The activity of Oly is the highest on sphingomyelin (SM)/Cholesterol (Chol) 

membranes. The activity decreases if Chol is replaced with other steroids, or if SM is 

replaced by fully saturated phosphatidylcholine (PC). Introduction of less saturated PCs 

decreases the activity even further. Activity is also abolished by lysophospholipids. 

Discussion/Conclusions: The observed preferential permeabilization and binding of Oly to 

SM/Chol-containing membranes are highly cooperative with respect to Chol concentration, 

reflecting the importance of membrane lateral distribution and accessibility of Chol 

molecules for protein binding. The inhibitory effects of unsaturated PCs and 

lysophospholipids on Oly activity are very likely a consequence of lo domain disruption. 

Our results indicate that fluorescently labelled Oly mutants deprived of toxic activity could 

be useful markers in studies of Chol-enriched lipid rafts. 

References: 1 Sepčić K, Berne S, Rebolj K, Batista U, Plemenitaš A, Šentjurc M, Maček P (2004) FEBS Lett. 575(1- 

3): 81-85. 

o pore-forming toxin 

o ostreolysin 

o lipid rafts 

o liquid ordered domains 

321

Poster 36: Time- and concentration-dependent cytotoxicity of ricin in human lung 

epithelial cells 

Ramasamy, S. 1* and Proll, D. 1 

1 DSTO, Human Protection and Performance Division, Fishermans Bend, Victoria, Australia, 3207. 

* sharmaine.ramasamy@dsto.defence.gov.au 

Ricin, a potent ribosome-inactivating hetero-dimer protein toxin (66kDa) produced in the 

seeds of the castor oil plant (Ricinus communis), is a Category B Agent on the Centres for 

Disease Control (CDC) Select Agent List. Accordingly, there is a need to develop 

prophylactic and therapeutic countermeasures for ricin intoxication. This is the first 

reported study to investigate the time- and dose-dependent cytotoxic effects of ricin in 

human lung small airway epithelial (SAE) cells. Ricin (1-100pM) produced a time- and 

dose-dependent decrease in SAE cell survival after exposure to the toxin. Ricin (1pM) 

reduced cell survival to 75.5 ± 7.3% of the control, compared with only 12.2 ± 5.3% and 

12.1 ± 11.3% of cells surviving 24 hour exposure to higher concentrations of ricin (10 and 

100pM; n=4; p100 60 57 56 20 

Primula veris root and leaf 11 9 10 14 4.1 

Primula vulgaris leaf xx xx xx xx 95 

Primula vulgaris flower xx xx xx xx xx 

xx-no cytotoxic effect detected 

Discussion/Conclusion: The cyclamen and P. veris extracts showed cytotoxic effects 

against both normal fibroblasts and cancer cell lines. Primula vulgaris leaf extract was 

cytotoxic against a leukaemia cell line (Jurkat) and not normal fibroblasts. 

References: 1. Tokalov SV, Kind B, Wollenweber E, Gutzeit GO. (2004) J Agric Food 

Chem, 52, 239-245. 

o Primulaceae 

o cell 

o cytotoxic 

o extract 

323

Poster 38: A crystalline nonprotein compound (BM-ANF) from toad (Bufo 

melanostictus, Schneider) skin extract having antiproliferative and apoptogenic 

activity** 

Giri, B 1* , Mishra, R 1 , Dasgupta, S.C 1 , Gomes, A. 1 

1 Laboratory of Toxinology & Experimental Pharmacodynamics, Department of Physiology, University 

of Calcutta, 92, APC Road, Calcutta 700009, India, *biplab_giri@rediffmail.com 

Introduction: Present study is an effort to isolate and characterise an antiproliferative and 

apoptogenic compound from the methanolic extract of Indian toad (Bufo melanostictus) 

skin and to establish its biological activity. 

Methods: Dried methanolic extract of toad skin was subjected to alumina gel column 

chromatography and HPLC. Desired active fraction was crystallised and solubility, melting 

temperature and spectral analysis (UV, IR, TOF-MS, etc.) were done. Biological testing of 

pure compound (BM-ANF) was done by U937 & K562 cell growth inhibition, MTT assay, 

PCNA expression, Annexin-V binding study, Fluorescent microscopy and SEM. 

Results: BM-ANF was eluted in chloroform : methanol (60 : 40) and further purified 

through HPLC. It was crystallised and partially characterised (MT > 325°C, UVλmax 

229.2 nm, MW 782.52 Da). IC50 dose of BM-ANF in U937 & K562 cells were 14 μg/100 

μl and 10 μg/100 μl respectively. BM-ANF (IC 50 doses) produced time dependent (24, 

48, 72 hrs.) significant inhibition of U937 & K562 cell growth (One way Anova, 

P

Poster 40: ANTIMICROBIAL ACTIVITY OF EXTRACTS FROM Phyllomedusa 

rohdei (AMPHIBIA, ANURA) SKIN 

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, 

E.L.A 1 

12- Braz Cubas University, Health Science Area, Mogi das Cruzes, SP, Brazil, gomez@brazcubas.br 




The production of peptides showing antimicrobial activity in skin granular glands of 

Phyllomedusa has been described. This work aimed to evaluate a skin methanolic extract 

from P. rohdei regarding its antimicrobial activity. The methanol skin extract was 

fractioned using methylene chloride and water to obtain a polar and non polar fractions 

respectively. No antimicrobial activity was detected in the methanol extract as well as in 

the polar fraction. The non polar fraction showed antimicrobial activity against Gram 

negative and Gram positive bacteria as well as fungi. A minimal inhibitory concentration 

(MIC) of 0.5 mg/mL was verified for the Gram negative bacteria Escherichia coli ATCC 

11229 and Pseudomonas aeruginosa ATCC 15442. The MIC value was 0.25 mg/mL for 

the yeast Candida albicans ATCC 10233. The higher antimicrobial activity was detected 

against the Gram positive bacteria Staphylococcus aureus ATCC 6538 with MIC value 

reaching 0.06 mg/mL. To evaluate the antimicrobial activity against S. aureus ATCC 

6538, it was cultivated in nutrient broth. The non polar fraction of methanol extract of P. 

rohdei skin at the concentration of 0.5 mg/mL reduced 90% of the growth capacity of S. 

aureus (one log unity). These results are compatible with a microbiostatic activity in the 

extract of P. rohdei skin. 


o Antimicrobial activity 

o Microbiostatic 

326 

Poster 41: EFFECT OF NOVEL PECTENOTOXINS ON ACTIN 

CYTOSKELETON 

Louzao, M.C. 1* , Ares, I.R. 1 , Cagide, E. 1 , Vieytes, M.R. 2 , Miles, C.O. 3 , Yasumoto, T. 4 and Botana, L.M. 1 

(1) Departamento de Farmacología, * ffmclooj@lugo.usc.es (2) Departamento de Fisiología Animal. 

Facultad de Veterinaria. Campus de Lugo. Universidad de Santiago de Compostela. 27002 Lugo. Spain 

(3) National Veterinary Institute, PB 8156 Dep., N-0033 Oslo, Norway; AgResearch Ltd, Ruakura 

Research Centre, Private Bag 3123, Hamilton, New Zealand 

(4) Japan Food Research Laboratories, Tama, Tokyo 206-0025, Japan 

In recent years new congeners belonging to the marine pectenotoxins family have been 

detected. Pectenotoxin-11 has been recently isolated from toxic phytoplankton and 

shellfish in New Zealand. Pectenotoxin-2 seco acid, another novel compound of this group, 

is generated by hydrolysis of pectenotoxin-2 within of the bivalve’s tissues. So far, there is 

no data available about their mechanism of action. We investigated the biological activity 

of pectenotoxin-11 and pectenotoxin-2 seco acid on actin cytoskeleton and morphology of 

neuroblastoma cells. In addition, their effects were compared with those found with 

pectenotoxin-2 in the same conditions. Fluorescent phalloidin was utilized for detecting 

quantitative changes in actin level through a laser scanning cytometer. Likewise, confocal 

microscopy allowed to visualize the organization of filamentous actin as well as changes in 

morphology. The results showed that pectenotoxin-11 triggered an important 

depolymerizing effect on actin cytoskeleton as well as modifications in shape of 

neuroblastoma cells. Interestingly, these effects were very similar to those observed with 

pectenotoxin-2. Otherwise, pectenotoxin-2 seco acid induced no alteration in any of the 

parameters studied. This has suggested that actin cytoskeleton is a common target for 

pectenotoxin-11 and pectenotoxin-2, but not for its derivative pectenotoxin-2 seco acid. 

o Pectenotoxins 

o neuroblastoma 

o actin cytoskeleton 

o cellular morphology 

327

Poster 42: A Cardiotoxin (NK31) from Indian Monocled Cobra (Naja kaouthia) 

shows activity against human chronic myelogenic cell-line (K562) 

Debnath, A, Gomes, A*, Vedasiromoni, J.R. 

Drug Development Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur-700032, 

West Bengal, INDIA. *gomes_aparna@yahoo.com 

Introduction: In our previous studies, it was found that Indian monocled cobra (Naja 

kaouthia) venom (NKV) was active on carcinoma, sarcoma and leukemia models. We have 

tried to isolate, identify and characterize the factor(s) responsible for the anti-neoplastic 

effect of NKV. 

Methods: IEC using CM cellulose, HPLC, TOF-Mass spectrometry, Amino acid 

sequencing, Cell-culture, Trypan blue exclusion method, MTT reduction assay, Confocal, 

SEM, FACS (AnnexinV-FITC/PI), Cell-cycle analysis. Statistical analysis by student’s ‘t’ 

test with p

Poster 44: Haemolytic protein from pedicellaria of violet sea urchin Sphaerechinus 

granularis (Toxopneustidae) 

Turk, T 1 and Kem, W. R 2 

1 

University of Ljubljana, Department of Biology, 1000-Ljubljana, Slovenia *tom.turk@bf.uni-lj.si 

2 

University of Florida, Collegel of Medicine, Department of Pharmacology and Therapeutics, 

Gainesville, Fla, USA 

In terms of toxins sea urchins are poorly characterized group of marine animals although 

some of the species, mainly those belonging to the family Toxopneustide, can cause 

serious medical conditions in humans. 

An extract from pedicellaria of live violet sea urchin Sphaerechinus granularis, the only 

Mediterranean member of family Toxopneustide, was toxic to crabs. Isolation of toxic 

proteins by means of different chromatographic techniques revealed several haemolytic 

protein peaks. One of the proteins was purified to homogeneity. It is a 28 kDa protein with 

an Ip of 6.5. The protein is lytic to red blood cells but does not cause their agglutination. 

Obtained protein appears to be thermostable. The hemagglutinating activity reported for 

several proteins isolated from sea urchins was generally absent from our samples. In terms 

of molecular weight and its activity new protein resembles a haemolytic protein isolated 

from coelomic fluid of Tripneustes gratilla, another member of Toxopneustidae family. 

However, in our case the haemolytic activity was Ca 2+ independent and without lectin 

characteristics typical for T. gratilla lectin. 

o sea urchin 

o pedicellaria 

o haemolytic activity 

o crab toxicity 

330 

Poster 45: An alpha-conotoxin from Conus victoriae is effective at alleviating 

neuropathic pain in an animal model of diabetic neuropathy. 

Satkunanathan, N 1 , Khodr, B 1 , Georgiou, G 1 , McIntosh, D 2 , Belyea, C 2 , Khalil Z 1, 3 , Livett, B.G. 1* 

1 University of Melbourne, Biochemistry and Molecular Biology and Bio21 Institute, 30 Flemington 

Rd.,Parkville,Victoria, AUSTRALIA, *b.livett@unimelb.edu.au 

2 Metabolic Pharmaceuticals Pty Ltd, St. Kilda Rd., Melbourne,Victoria, AUSTRALIA 

3 University of Sharjah, College of Pharmacy,UAE. 

Introduction: The present study was performed to evaluate the antiallodynic effects of αconotoxin 

Vc1.1, in rats with diabetic neuropathy. With an increasing older demographic, 

diabetic neuropathy is an increasing problem in the community. Oxidative stress has been 

implicated in the pathogenesis of diabetic neuropathy, the most common complication of 

diabetes mellitus affecting more than 50% of diabetic patients (1). α-conotoxin Vc1.1 

(ACV1) is a synthetic peptide of 16 amino acids, whose amino acid sequence was deduced 

from cDNA created from the venom duct of the Australian cone snail Conus victoriae (2). 

ACV1 is a competitive antagonist of the neuronal nicotinic acetylcholine receptor 

(nAChR) in animals and humans. Previous studies in our laboratory (3) have shown that 

ACV1 is a potent analgesic in several rat models of human neuropathic pain. 

Methods: The streptozotocin-induced diabetic rat model of peripheral neuropathy was 

used. Mechanical allodynia was assessed using von Frey filaments. Treatment with 

ACV1 started after the development of allodynia (6 weeks after induction of diabetes) and 

continued for 4 weeks. ACV1 was administered via subcutaneous bolus injection at the 

back of the neck at doses of 3μg/kg, 30μg/kg and 300μg/kg. Sciatic nerve biopsies and 

plasma were collected one week after cessation of treatment to assess the oxidative stress. 

In addition, the effect of ACV1 treatment on levels of lipid hydroperoxides in injured 

sciatic nerve and on the levels of nitrotyrosine in plasma proteins was investigated. 

Results: An antiallodynic effect was observed at 30μg/kg and 300μg/kg, within 1 hr postdosing. 

ACV1 attenuated allodynia for up to a week after cessation of treatment. ACV1 

also reduced oxidative stress markers such as lipid hydroperoxides and nitrotyrosine. 

Discussion/Conclusion: The results indicate that ACV1 has an antiallodynic effect and 

that one possible mechanism could be related to reduction in oxidative stress associated 

with diabetic peripheral neuropathy. 

References: 1. Sayyed, S.G. et al (2006) Life Sci.(in press); 2. Sandall, D.W. et al (2003) 

Biochemistry 42: 6904-6911; 3. Satkunanathan, N. et al (2005) Brain Res. 1059: 149-158;. 

Satkunanathan, N. et al (2005) Brain Res. 1059 : 149-158 ; 3. Sayyed, S.G. et al (2006) 

Life Sci. in press. 

o conotoxin 

o analgesic 

o diabetic neuropathy 


331

Friday 28 July: poster session 


Clinical aspects 

1. Venomous Bite and Sting Hospitalisations in Australia 2000-2002. Winkel 

K.D.* 1 Kreisfeld, R 2 , Harrison, J.E. 2 

2. Death from brown snake (Genus: Pseudonaja) envenomation in Australia: is 

antivenom to blame? Winkel K.D. 1 , Lowe R. 1 , McGain F. 1 , Nimorakiotakis, B. 1 , 

Williams D.J. 1 

3. Efficacy and Safety of an F(ab’)2 Antivenom for Scorpion Envenomation. 

Boyer, L.V. 1 , Alagon A. 2 , Mallie J. 1 

4. Neurotoxicity in Children Hospitalized due to North American Scorpion 

Envenomation. Boyer, L.V. 1 , Mallie J 1 , Chase, P.B. 1 , Theodorou, A. 1 , Schmier, C. 1 

5. A new venomous scorpion responsible of human deaths in Argentine: Tityus 

confluens. Adolfo R. de Roodt 1 ; Néstor Lago 2 ; Judith Estevez 3 ; Estela Neder 4 ; Alicia 

Montero 4 ; Oscar D. Salomón 1 ; Jorge F. Paniagua 5 ; Raúl López 6 ; Valeria Vega 7 . 

6. Toxicity of five scorpion venoms from the Old World, and its immunological 

reactivity with American scorpion antivenoms. Adolfo Rafael de Roodt 1 ; Néstor R. 

Lago 2 ; Judith Estevez 3 ; Eduardo Scarlatto 4 ; Rodrigo D. Laskowicz 2 ; Jorge F. 

Paniagua 5 ; Hernán García 6 ; Victor M. Manzanelli 2 , Alejandro Alagon 7 . 

7. Neutralization of the haemorrhagic activity of Echis ocellatus (saw-scaled viper) 

venom and a venom metalloproteinase using synthetic peptide inhibitors and 

chelators. Howes, J-M, Theakston, R.D.G., Laing, G.D.* 

8. The potential advantages of camelid IgG for the development of antivenoms. 

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 . 

9. The Antifungal Activity of Punica Granatum L Peels powder and extracts. 

Siham S. Shaokat* and Hamoudi A. Hameed 1 

ٍٍٍ 

10. Epidemiology and management of snakebites in Burkina Faso. Sondo Blaise 1, 2 , 

Kouanda Séni 2 , Some Noya 2 

332 

11. UNCOMMON ENVENOMING PROVOKED BY ARTHROPODS: TOXINS 

FOR KNOWING. Haddad Jr, V. 

12. North and South American Loxosceles spiders: Development of a polyvalent 

antivenom with recombinant Sphingomyelinase D as antigens. Olvera, A 1* , Ramos- 

Cerrillo, B 1 , Paniagua-Solís, J 2 , Stock, R.P 1 and Alagón, A 1 . 

13. EPIDEMIOLOGY OF SNAKEBITES BASED ON HOSPITAL SURVEY IN 

CHITWAN AND NAWALPARASI DISTRICTS, NEPAL. Pandey, Deb Prasad 

14. Analysis of clinical outcomes and cost-benefits associated with the use of CSL 

Snake Venom Detection Kits in the management of snakebite at Port Moresby 

General Hospital, Papua New Guinea. Williams D.J, 1* Jensen S.D, 1,2 Winkel K.D. 1 

15. Epidemiology of snakebite in Guinea. Baldé M. C., Barry A. O., Tamboura B. A. 

16. RENAL AND BLOOD VESSEL ALTERATIONS INDUCED BY GAMMA 

TOXIN OF Tityus serrulatus VENOM. Monteiro, H.S.A. 1 *; Alves, R.S. 1 ; Martins, 

R.D. 1 ; Amora, D.N. 1 ; Barbosa, P.S.F. 1 ; Sousa, D.F. 1 ; Alves, C.D. 1 ; Toyama, M.H. 2 ; 

Menezes, D.B. 3 ; Martins, A.M.C. 4 ; Fonteles, M.C. 1 ; Monteiro, F. C. D. 1 

17. Biological and Clinical Evaluations of Antivenom Immunotherapy. Bon, C. 1* , 

Audebert, F. 1 , Calderon, E. 2 , Choumet, V. 1 , ElAyeb, M. 3 , Krifi, M. 3 , Possani, L. 2 , 

Rivière, G. 1 , Sorkine, M. 

Cardiovascular 

18. Amelioration of the cardiovascular effects of the yellow scorpion Leiurus 

quinquestriatus envenomation in rats by the red grape seeds proanthocyanidins. 

El-Alfy, A 1 ., Ahmed, A 1 ., Kader, F 1 ., Fatani, A 1 . 

19. Can the antioxidant, ginkgo biloba extract, and the protease inhibitor, 

aprotinin, protect rats against Leiurus quinquestriatus scorpion venom-induced 

cardio-vascular effects? Fatani, AJ, Yaqub, HI, Ibrahim, M, Al-Zuhair, H, Qadir, F, 

Al-Qutub, M. 

20. Alterations to the expression and distribution of adhesion molecules in rat 

renal glomeruli after Bothrops moojeni envenoming. *Cruz-Höfling, M. A., 

Collares-Buzato, C. B. 

21. Snake Venom Metalloproteinases (SVMPs) binds to collagen and α2β1 

integrin by two different motifs. Tanjoni,I. 1 , Butera, D. 1 , Della-Casa, M.S. 1 , 

Fernandes, I. 1 , Clissa, P.B. 1 , Evangelista, K.S. 2 , Eble, J. 2 , Moura-da-Silva, A.M. 1* 

333

22. Distribution and effects of ε-toxin-GFP in mouse brain and kidneys. Juan 

Blasi 1* , Alex Soler 1 , Jonatan Dorca 1 , Josep Saura 2 , Josep Maria Tussell 2 , Maryse 

Gibert 3 , Michel Poppof 3 , Joan Serratosa 2 and Mireia Martín-Satué 1 . 

23. Evaluation of potential ricin inhibitors on pulmonary and digestive epithelial 

cells. Emilie Gobbo 1 , Roman Lopez 2 , Goulven Merer 2 , Bernard Rousseau 2 , André 

Ménez 1 , Bruno Beaumelle 3 , Daniel Gillet 1* and Julien Barbier 1 

24. Antagonism of the cardiotoxic effect of Bothrops jararacussu by Suramin. 

Sifuentes, N. D. 1 , El-Kik, C. Z. 2 , Ricardo, H.D. 2 ,Schwartz, E.N.F. 1 , Melo, P.A. 2 

25. EFFECT OF CROTOXIN ON MICROCIRCULATION AND 

CIRCULATING LYMPHOCYTES. Zambelli, VO 1 ; Sampaio, SC 1 , Britto, LRG 2 , 

Zychar,B 1 ; Gonçalves, LRC 1 ; Cury, Y 1 . 

26. EFFECTS OF THE SEA ANEMONE Bunodosoma caissarum VENOM ON 

RENAL PERFUSION AND MESENTERIC BED VESSELS. 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 ; Toyama, 

M. H. 2 ; Martins, A M.C. 3 ; Barbosa, P.S.F. 1 ; Fonteles, M. C. 1 , Monteiro, F. C. D. 1 

27. Tityus serrulatus Hypotensive Peptides: a novel class of hypotensive agents. 

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, P.E. 5 , Martin-Eauclaire, M.F. 5 , De Lima, M.E. 1,2 and Pimenta, 

A.M.C. 1,2 

28. Snake Venom Components as Pharmacological Tools and Molecular Models in 

Thrombosis and Haemostasis. Bon, C., Arocas V., Braud S., Francischetti I., Leduc 

M., Maroun R., Saliou B., Wisner A., Zhang Y., Zingali R.. 

29. Venom Peptides from Calloselasma rhodostoma --- New Twist in Vasoactive 

Peptides? Higuchi, S 1,2* , Khow, O 3 , Suntrarachun, S 3 , Noiphrom, J 3 , Miyamoto, S 1 , 

Kawai, A 1 , Sitprija,V 3 , Sato,T. 3 

30. Potent dromedary antibody fragments for using in immunotherapy against 

scorpion envenomation. Bouhaouala-Zahar, B. 1 *, Conrath, K. 2 , Ben abderrazek R. 1 , 

Hmila I. 1 , Benlasfar Z. 1 , Hammadi M. 3 , Khorchani T. 3 , Muyldermans S. 2 . & El Ayeb, 

M. 1 

31. Purification and characterization of ancistron–Bu, a novel fibrinogen-clotting 

serine protease from Agkistrodon blomhoffii ussuriensis venom. Karbovskiy V. 

L 1,2* , Savchuk A.N 2 , Volkov G.L. 2 

32. “Blomus-B” – a novel non-enzymatic platelet aggregation inhibitor purified 

from Agkistrodon blomhoffii ussuriensis venom. Karbovskiy V. L 1,2* , Savchuk A.N. 

334 

Toxins from novel sources 

33. Advances in the studies in the field of animal venoms and their toxins in Brazil 

from 1900 until today. Borja-Oliveira CR and Rodrigues-Simioni L*. 

34. Geographical and Toxinological Characterization of Tityus quirogae 

(Scorpiones, Buthidae), a Species of Medical Importance from northeastern 

Venezuela. De Sousa, L. 1* , Borges, A. 2 , Manzanilla, J. 3 , Gregoriani, T. 1 , Romero, L. 

1 , Parrilla-Alvarez, P. 4 , Quiroga, M. 4 

35. Modulation of the content of naturally released scorpion venom by a, 

presumably, rapid holocrinic secretion in the venom gland. Morgenstern D 1* , 

Matthews G 1,2 , Sher D 1 , Zlotkin E 1 . 

36. A study on “Black Widow” spiders from Argentina. Adolfo Rafael de Roodt 1 ; 

Rodrigo D. Laskowicz 1 ; Victor M. Manzanelli 1 ; Judith Estevez 2 . 

37. Sex-linked variation of Thalassophryne maculosa fish venom. Sosa-Rosales J.I. 1 , 

Pereira C. 1 , Bruni F.M. 2 , Pareja-dos-Santos A. 2 , Boletini-dos-Santos D. 2 , Ramos A.D. 2 , 

Kadura A.M. 2 , Conceição K. 3 , Pimenta D.C. 3 , Lima C. 2 , Lopes-Ferreira M. 2 

38. Comparative Studies Between Secretory Cells Of Sting Apparatus And Venoms 

Obtained from Freshwater (Potamotrygon falkneri) And Marine (Dasyatis guttata) 

Stingrays. Barbaro, K.C. 1 *, Lira, M.S. 1 , Malta, M.B. 1 , Soares, S.L. 1 , Santoro, M.L 2 , 

Pedroso, C.M. 3 , Antoniazzi, M.M. 3 , Jared, C. 3 , Cardoso, J.L.C. 4 , Garrone Neto, D. 5 , 

Haddad Jr, V. 

Plant toxins 

39. Componnt Characteristics and Food Safety of Star Fruit. Jou-Fang Deng 1,2 , Yu- 

Lan Hung 3 , Ming-Ling Wu 1 , Yen-Hung Yeh 4 and Deng-Fwu Hwang 3,* 

40. ALLELOPATHIC EFFECTS OF CROP PLANTS ON OTHER CROP 

PLANTS. OMID ALIZADEH 

41. THE EFFECT OF SAFFLOWER (Carthamus tinctorius) ON 

REPRODUCTIVE PHYSIOLOGY OF THE MALE MICE. Mehrdad Modaresi 

and Fateme Matboo Sobhani 

335

Poster 1: Venomous Bite and Sting Hospitalisations in Australia 2000-2002 

Winkel K.D.* 1 Kreisfeld, R 2 , Harrison, J.E. 2 

1 Australian Venom Research Unit, Department of Pharmacology, University of Melbourne, Parkville, 

Vic, 3010. Australia *kdw@unimelb.edu.au 

2 National Injury Surveillance Unit, Flinders University, GPO Box 2100, Adelaide, 5042 Australia. 

Introduction: Little systematic national data has been available to assess the current burden 

of venomous bite and sting injury in Australia. National, systematically coded, hospital 

separation data therefore provides a new method of monitoring overall venom-related 

injury. 

Methods: Cases that met the following criteria were selected for analysis: Australian 

hospital separations occurring from July 1 st , 2000 to June 30 th , 2002. This period was 

chosen because it centres on the date of the 2001 Census, which enables certain analyses 

by location that cannot be done so well for period more distant from a census. 

Results: In the period 2000/01–2001/02, 7,407 people were admitted to hospital as the 

result of bites and stings by a venomous animal or plant. Spiders, bees, and snakes and 

lizards were the most common sources of bites and stings. There were some marked 

differences between males and females. For the categories snakes and lizards, wasps, bees 

and marine animals and plants, cases involving females were less than half as frequent as 

those involving males. All categories of bites and stings resulted in a similar mean length 

of stay in hospital [1.3 days]. Rates of bites and stings were fairly similar for children and 

adults in the middle years but began to decline from the age of 60. Rates for bites and 

stings as a whole increased relative to the remoteness of the location in which they 

occurred. There were notable differences between latitudes, with most venomous injuries 

increasing in frequency as one progressed further north The all-Australia rate for these 

bites and stings as a whole was 19.1 per 100,000 population. 

Conclusions: This report represents significant progress in our understanding of the 

venomous bite and sting injury burden in Australia. The excess of rural and remote area 

admissions reinforces the need for specific strategies to recognise and manage venomrelated 

injuries in these areas. 

o Australia 

o Hospitalisations 

o Injury 

o Venomous bites and stings 

336 

Poster 2: Death from brown snake (Genus: Pseudonaja) envenomation in 

Australia: is antivenom to blame? 

Winkel K.D. 1 , Lowe R. 1 , McGain F. 1 , Nimorakiotakis, B. 1 , Williams D.J. 1 

1 


* kdw@unimelb.edu.au 

Introduction: Recent investigators have questioned the efficacy of Australian brown snake 

antivenom manufactured by CSL Limited 1 . We reviewed definite brown snake related 

fatalities to ascertain the circumstances of death and the possible contribution of antivenom 

to the outcome. 

Methods: Retrospective review of definite brown snake-related fatalities using data from 

hospital records, autopsy and coronial findings held by the Australian Venom Research 

Unit (the Australian Bite & Sting Fatality Database). 

Results: 24 definite cases were identified between January 1980 and December 2004 . 

Patients ranged in age from 1-75 yrs, with a mean adult age of 50.2 years and with a 

male:female ratio of 3:1. There were only two paediatric deaths (unwitnessed snakebite 

that went unrecognised prehospital). Patients >50 yrs were over-represented (57% of 

cases). Almost half the deaths (47%) occurred in Queensland. The identification of the 

immunotype or species was made on the basis of Snake Venom Detection Kit (CSL 

Limited) in 88% of cases (mostly bite site swab), and on the basis of physical identification 

of the snake (11%). Antivenom was administered to 58% of patients but most (95%) had a 

prior, early loss of consciousness from which they never recovered (either pre-hospital or 

on presentation and/or upon intra-hospital release of first aid). Time to death was between 

20 minutes to three weeks. All but one patient sustained bites to either upper or lower limb 

extremities. Death was attributable to pre-antivenom events in 95% of cases: most cases 

lacked early, adequate pressure-immobilisation first-aid but rapid release of bandages, 

including tourniquet, was also associated with intra-hospital collapse in some cases. 

Conclusion: This study reveals that death after brown snake envenomation typically 

involves older Australians and is precipitated by critical events that occur prior to the 

administration of antivenom. 

References: 1. Judge et al (2006) Toxicology & Applied Pharmacology, 213: 117-125. 


o Fatality 

o Genus: Pseudonaja 

o CSL brown snake antivenom 

337

Poster 3: Efficacy and Safety of an F(ab’)2 Antivenom for Scorpion Envenomation 

Boyer, L.V. 1 , Alagon A. 2 , Mallie J. 1 

1 University of Arizona Health Sciences Center, Tucson, Arizona, USA, boyer@pharmacy.arizona.edu 

2 Instituto de Tecnologîa, Universidad Nacional Autonoma de Mexico, Cuernavaca, MX 

Introduction: Neurotoxicity and ventilatory compromise from North American Buthid 

scorpion sting may be life-threatening in children. Management of the syndrome with 

sedation and intensive supportive care is associated with sedative dosing in excess of that 

commonly accepted as safe, and adverse events are common. 

Methods: An equine F(ab’)2 antivenom commercially available in Mexico was 

administered to children presenting in the US and Mexico with clinically important signs 

of scorpion envenomation. Three closely related protocols were used in a multicenter study 

adapted to varied local needs. Patient demographics, time of sting, nature and duration of 

syndrome, and adverse events were recorded. Serum venom levels were determined by 

specific ELISA. Descriptive statistics were derived and clinically interpreted. 

Results: Forty-one children and 3 adults were enrolled. The study population was 51.2% 

male, with average age of children 4.0 years and weight 17.3 kg. Time from sting to study 

drug infusion averaged 114 minutes. Overall, clinically important signs were present in 

100% at baseline, 62% at 1 hour, 12.2% at 2 hours, and 0% at 4 hours after infusion. 

Venom levels declined by an order of magnitude, within one hour of antivenom infusion. 

Adverse events, none attributed to the study drug, affected 9.8% of patients. 

Discussion/Conclusion: Compared with historical controls, patients who received 

antivenom recovered faster, with fewer adverse events. Venom levels declined concurrent 

with syndrome resolution, suggesting a surrogate marker of efficacy for future studies. 

F(ab’)2 antivenom appears to provoke fewer hypersensitivity reactions than does wholeimmunoglobulin 

antivenom in a similar population (1). 

References: 1. LoVecchio F et al. (1999) Incidence of immediate and delayed 

hypersensitivity to Centruroides antivenom, Ann Emerg Med 34:5; 615-619. 

o scorpion 

o antivenom 

o clinical trial 


338 

Poster 4: Neurotoxicity in Children Hospitalized due to North American Scorpion 

Envenomation 

Boyer, L.V. 1 , Mallie J 1 , Chase, P.B. 1 , Theodorou, A. 1 , Schmier, C. 1 

1 University of Arizona Health Sciences Center, Tucson, Arizona, USA, boyer@pharmacy.arizona.edu 

Introduction: Envenomation by North American Buthid scorpions may involve profound 

neurotoxicity and ventilatory compromise. Formal clinical trials of antivenom under way 

in the U.S. required establishment of a control group for comparison to treated patients. 

Methods: Patient records at hospitals where antivenom was not historically used were 

formally reviewed by a physician/nurse team. Patient demographics, time of sting, duration 

of syndrome, dose of adjunctive sedative, blood count and seru chemistries, and adverse 

events were recorded. Descriptive statistics were derived and clinically interpreted. 

Results: Ninety-seven evaluable records were identified and reviewed. The study 

population was 53.6% male, with average age 3.9 years and weight 18.3 kg. Time from 

sting to last documented clinically important sign of envenomation averaged 764 minutes 

(SD 370, range 180-2160). The most consistently documented signs of toxicity were 

thrashing limbs (in 94.8%) and abnormal eye movements (74.2%). On average, midazolam 

sedation continued until 858 minutes after the sting (SD 311, range 200-2025). Total dose 

averaged 6.56 mg/kg. Adverse events affected 39.2% of patients. 

Discussion/Conclusion: Without specific antivenom, children with serious neurotoxicity 

remain critically ill for an average of 12.7, and up to 36 hours, following scorpion sting. 

Treatment is frequently complicated by adverse events. Benzodiazepine dosing is 

substantial, averaging 20 times the maximum dose generally used for conscious sedation. 

These findings are consistent with and expand upon the findings of others (1,2). 

References: 1. Curry SC et al. (1983-1984) Envenomation by the Scorpion Centruroides 

sculpturatus, J Tox Clin Tox 21 (4&5); 417-449. 2. Gibley R et al. (1999) Continuous 

intravenous midazolam infusion for Centruroides exilicauda scorpion envenomation, Ann 

Emerg Med 34:5; 620-625. 

o scorpion 

o envenomation 


o natural history 

339

Poster 5: A new venomous scorpion responsible of human deaths in Argentine: 

Tityus confluens. 

Adolfo R. de Roodt 1 ; Néstor Lago 2 ; Judith Estevez 3 ; Estela Neder 4 ; Alicia Montero 4 ; Oscar D. Salomón 1 ; 

Jorge F. Paniagua 5 ; Raúl López 6 ; Valeria Vega 7 . 

1 Administración Nacional de Laboratórios e Institutos de Salud “Dr. Carlos G. Malbrán”, Ministerio de 

Salud y Ambiente. Av. Velez Sarsfield 563, CP 1281, Bs.As., Argentina. 

2 GEMA-Biotech, Bs.As., Argentina. 

3 InsitutoBioclon, México DF, Mexico. 

4 Universidad Nacional de Jujuy, Jujuy, Argentina. 

6 Ministerio de Salud de Catamarca, Catamarca, Argentina. 

7 Universidad Nacional de Tucumán, Tucumán, Argentina. 

In Argentine the principal scorpion responsible for severe envenomation in humans is T. 

trivittatus from the North of Patagonian region to the whole Northern of the country, with 

a lethality of 3‰. None of the other Buthiidae species have been confirmed as cause of 

death. In year 2003, in the province of Jujuy (in the North) two children died after scorpion 

bite. The search of scorpions in their homes gave as a result the finding of several 

specimens of T. confluens and some other scorpions but not T. trivittatus. In year 2005 and 

2006, in the provinces of Catamarca and Tucumán in the Northwest of Argentina, three 

deaths of children were registered with the typical signs of the scorpion sting. The only 

scorpion found was T. confluens. We studied the toxicity of telson homogenates of T. 

confluens from the province of Jujuy and Catamarca, and found that the LD50, in NIH 

mice, was 143 µg [118-174 µg] for those from Jujuy and 380 µg [387-390 µg] for those 

from Catamarca). These values are in the order of that measured for T. trivittatus 

venomous glands homogenate, from regions were human deaths have been recorded 

recorded. The injection of the venom showed in all the animals generalized congestion and 

hepatic lesions consistent in diffuse citoplasmatic vacuolization and sinusoidal congestion. 

Pancreatic damage was observed in 40% of the injected animals. Lungs showed congestion 

and foci of hemorrhage and mild edema. Most of the hearths (80%) showed myocardical 

eosinophylic injury of the fibers. The protein extracted from venomous glands and the 

electrophoretic profiles were in the order and similar to those observed for T. trivittatus 

from different regions of Argentina. The venomous gland homogenates showed high 

reactivity with Anti-T. trivittatus (Anti-Escorpión from INPB, Argentine), anti-T. 

serrulatus (Antiaracnídico and Antiescorpión from Butantan Institute, Brasil) and 

reactivity against Anti-Centruroides sp. (Alacramyn, Bioclon. Mexico) antivenoms. The 

sting of this scorpion must be considered of risk in the same degree of the stings of T. 

trivittatus or T. bahiensis. 

o Scorpion 

o Venom 

o Toxicity 

o Immunology 

340 

Poster 6: Toxicity of five scorpion venoms from the Old World, and its 

immunological reactivity with American scorpion antivenoms. 

Adolfo Rafael de Roodt 1 ; Néstor R. Lago 2 ; Judith Estevez 3 ; Eduardo Scarlatto 4 ; Rodrigo D. Laskowicz 

2 ; Jorge F. Paniagua 5 ; Hernán García 6 ; Victor M. Manzanelli 2 , Alejandro Alagon 7 . 

1 Instituto Nacional de Producción de Biológicos – A.N.L.I.S. “Dr. Carlos G. Malbrán”, Min. de Salud y 

Ambiente, Av. Vélez Sarsfield 563, CP 1281, Bs.As., Argentina. 

2 GEMA-Biotech, Bs. As., Argentina. 

3 Instituto Bioclon, Mexico DF, Mexico. 

4 Servicio de Toxicología, Hospital “José de San Martín”, Fac. de Medicina U.B.A., Bs.As., Argentina. 

5 Laboratorios Silanes, Mexico DF, Mexico. 

6 Servicio de Patología, Hospital Italiano de Buenos Aires, Buenos Aires, Argentina. 

7 Instituto de Biotecnología de la Universidad Autónoma de México, Cuernavaca, Morelos, Mexico. 

The toxic activity of the venom from Buthus occitanus (Bo), Androctonus australis (Aa), 

Leiurus quinquestriatus (Lq), Parabuthus granulatus (Pg) and Parabuthus transvaalicus 

(Pt) was studied in NIH mice. The immunochemical reactivity against four antivenoms 

(AV) against T. trivittatus (Suero antiescorpión, INPB, Argentina, A), T. serrulatus (Soro 

antiaracnídico and Soro Antiescorpiónico, Butantan Institute, Brasil) and Centruroides sp. 

(Bioclon, Mexico, B) antivenoms was studied. The i.p. LD50s found were of 132 µg (Pt), 47 

µg (Pg), 14 µg (Bo), 8 µg (Aa) and 11µg (Lq). The Aa venom showed the highest potency 

(p < 0.05) closely followed by Bo and Lq venoms. The venom from South African scorpions 

showed the lower potency (p < 0.05), being that of Pt the one with the lowest lethal 

potency. The histopathological study of lungs showed congestion, edema and intraalveolar 

hemorrhage. The most important findings in hearths were eosinophilic infiltration of the 

muscle fibers and severe generalized injury. These findings were observed in all the animals 

studied (n=5 by venom). The immunochemical reactivity was poor as observed by double 

immunodifussion, but all antivenoms gave diffuse precipitation bands with all venoms 

tested; the highest reactivity was observed with the venom of Lq. Neutralization 

experiments showed different degrees of protection conferred by the INPB and Alacramyn 

heterologous antivenoms (challenge dose 3 LD50 i.p.). The ED50s of the AVs against Bo 

venom were 184 µl and 214 µl for B and A, respectively, against Aa venom were 233 µl for 

B and > 250 µl for A, and against Lq venom were 440 µl for B and over 500 µl for A. The 

neutralization against Parabuthus venoms was poor for both antivenoms. The venom of P. 

transvaalicus was very poorly neutralized by anyone of the three antivenoms, although the 

neutralization by the South American antivenoms was slightly better (ED50 over 500µl). 

The venom of P. granulatus was neutralized with ED50s values of 110 μl and 165 µl for A 

and B, respectively. We conclude that the AVs from North or South America can neutralize 

at high doses the toxicity of venoms of scorpions from Africa. 

o Scorpion 

o Venom 

o Toxicity 

o Antivenom 

341

Poster 7: Neutralization of the haemorrhagic activity of Echis ocellatus (saw-scaled 

viper) venom and a venom metalloproteinase using synthetic peptide inhibitors and 

chelators 

Howes, J-M, Theakston, R.D.G., Laing, G.D.* 

Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 

5QA, UK. *gavin.laing@liv.ac.uk 

Envenoming by the West African saw-scaled viper, Echis ocellatus, resembles that 

of most vipers, in that it results in local blistering, necrosis and sometimes life-threatening 

systemic haemorrhage. While effective against systemic envenoming, current antivenoms 

have little or no effect against local tissue damage. The major mediators of local venom 

pathology are the zinc-dependant snake venom metalloproteinases (SVMPs). The 

extensive structural and functional similarity between SVMPs and the mammalian matrix 

metalloproteinases (MMPs) suggests that substrate/inhibitor interactions between these 

subfamilies are likely to be equally similar. 

In this study, four MMP inhibitors (Marimastat, AG-3340, CGS-270 23A and Bay- 

12 9566) and three metal ion chelators (EDTA, TPEN and BAPTA) were evaluated for 

their ability to inhibit the haemorrhagic activities of medically-important viperine venoms 

and isolated SVMPs. While both marimastat and CGS-270 23A inhibited local 

haemorrhage following injection of the isolated PIII SVMP EoVMP2, neither was 

effective against the haemorrhagic activity of whole E. ocellatus venom. AG-3340 

successfully inhibited the haemorrhagic activities of both EoVMP2 and E. ocellatus 

venom, but Bay-12 9566 was ineffective against haemorrhage induced by the whole 

venom. We suggest that an inhibitor mixture may be more effective against local venom 

pathology than individual compounds. The validation of this theory would be a logical 

progression of this study, and would ensure that a close structural counterpart for each 

SVMP is represented, proving a more effective broad-range treatment for local 

envenoming. 

o Echis 

o Snake venom metalloproteinase 

o Inhibitor 

o Haemorrhage 

342 

Poster 8: The potential advantages of camelid IgG for the development of 

antivenoms 

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 . 

1 


UK 

*R.Harrison@liverpool.ac.uk 

2 

Medical Research centre, Faculty of Medical Sciences, University of Science and Technology, PO Box 13064, The 

Republic of Yemen. 

3 

Institute for Animal Science and Health of Wageningen University and Research Centre, 8219 PH lelystad, The 

Netherlands. 

4 

Laboratory of cellular and Molecular Immunology, Dept of Molecular and Cellular Interactions, Vlaams 

Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, B-1050 Brussels, Belgium. 

Envenoming by snakes results in severe systemic and local pathology. Intravenous 

administration of antivenom, prepared from IgG of venom immunised horses or sheep, is 

the only effective treatment of systemic envenoming. Conventional antivenoms, 

formulated as intact IgG, papain-cleaved (Fab) or pepsin-cleaved F(ab / )2 fragments, are 

however ineffective against the local venom effects because of their inability to penetrate 

the blood/tissue barrier. We have embarked on a new research program to examine (i) 

whether the unusually small (15 kDa) antigen-binding fragment of camelid heavy chain 

IgG (VHH) can be exploited to neutralise the local effects of envenoming and (ii) whether a 

novel antivenom to treat both the systemic and local effects of envenoming can be 

formulated by combining anti-snake venom VHH and conventional F(ab / )2 and (iii) to 

determine whether the comparatively low immunogenicity and complement activation of 

camelid IgG [Herrera et al, 2005] indicates that camelid antivenom may have a lower risk 

of adverse effects than equine or ovine IgG antivenoms. 

The objectives of this preliminary study were to determine whether (i) camels (Camelus 

dromedarius) and llamas (Lama glama) immunised with venom raised antibodies of the 

appropriate titre and venom-neutralising efficacy and (ii) there were immunological or 

therapeutic reasons to select one camelid species over the other for future development of a 

camelid snake antivenom. 

We demonstrate that camels and llamas respond to immunisation with Echis ocellatus 

venom with high antibody titres and broad antigen specificity. These encouraging 

immunological results were matched by the successful elimination of venom-induced 

haemorrhage by IgG from the venom-immunised camels and llamas. 

Herrera, M., León, G., Segura, A., Meneses, F., Lomonte, B., Chippaux J.P., Gutiérrez, J-M. Factors 

associated with adverse reactions induced by caprylic acid-fractionated whole IgG preparations: comparison 

between horse, sheep and camel IgGs. Toxicon 46,(7):775-781. 

o Antivenom 

o Camelid 

o Echis ocellatus 

343

Poster 9: The Antifungal Activity of Punica Granatum L Peels powder and extracts. 

Siham S. Shaokat* Hamoudi A. Hameed 1 

ٍٍٍ 

*Prof. Of Pharmaceutical Microbiology/ Food & Drugs Sector/Nidal Street/Baghdad/Iraq/ email: 

albiatyss84@yahoo.com M: 07901867274 

1 

Prof. Of Chemistry. / Ministry of Industry & Minerals 

Chief Food & Drugs Sector/hamodiabas@yahoo.com/M:07901918147 

Introduction 

The aims of this study are to investigate anti-fungal properties of water and 

ethanol, extracts & powder of Punica granatum L.Peels for treatment of several skin 

infections and inflammatory disorders . 

Method 

Sixty five patients (2-47)years old, suffered from, infected skin, fissures, rushes, boils, 

itching. Eight of them (12.24%) were infected with (Tinea pedis ) ( athlete foot), 6 patients 

(9.23%) suffered from erythematous, were infected with(Tinea cruris) ( jock itch) , 9 

patients (13.9%) were infected with (Candida albicans), 2 patients(3.1%), with circular 

patches indicated (Tinea corporis ) ( ring worm) , 26 patients (40%) with fungus inside and 

on surface of hair (Tinea capitis) &14 (21.53%) Cases showed infected nails thickened or 

crumbling (Tinea unguium). 

Results 

The anti-fungal activity of the extracts was evaluated by treating the cases above during 

two months by water hot extract and ethanol extract of the punica granatum peels as well 

as the dried powder on athlete foot and other infections. 

Conclusions 

These results suggest the Pomegranate Peels extract which contains gallotanic acid as a 

promising anti-fungal agent. 

References: 

-Robert A Neurath, Nathan S, Yun-Yao Li,and AsimK Debnath. Punica granatum juice provi 

V-1 entry inhibitor and candidate topical microbicide. BMC Infectious diseases 

2004,4:41 do i: 10.1186/1471-2334 

o Antifungal agents 

o Plant extracts 

344 

Poster 10: Epidemiology and management of snakebites in Burkina Faso 

Sondo Blaise 1, 2 , Kouanda Séni 2 , Some Noya 2 

1. Faculté de médecine, Université de Ouagadougou 

2. IRSS, Ouagadougou, Burkina Faso 

Snakebites remain an important public health problem in Burkina Faso. For the past 15 

years, the average annual incidence of snakebite reported by the health care system of 

Burkina Faso was approx. 70 cases per 100,000 inhabitants, representing 0.25% of the 

total consultations in primary care medical facilities. The incidence increased, from 60 

cases per 100,000 inhabitants 15 years ago to 90 by 2005. A probable reason for this 

progression is that the use of public health care is increasing rather than that the incidence 

of snakebite is increasing. Hospital consultations following snakebites also increased 

during this period, from an average of less than 2,000 envenomations per year to more 

than 3,600. However, envenomations represented more than 5% of total hospitalizations 

in 1990 whereas they correspond to 2% in 2005. Hospital mortality rate resulting from 

envenomations still remains very high: 3% of total deaths occurring in hospitals but 8% 

of patients treated for snakebite. 

It is also known that the data from the health care system underestimate the incidence and 

mortality resulting from snakebites by 60-80% because the majority of victims are treated 

first, and often exclusively, by traditional healers. 

The high cost of the antivenin currently available in Burkina Faso (nearly 100 US$) may 

explain this failure. It is predicted that an effective and cheaper antivenin would be better 

able to compete with the traditional treatments generally used, more as a result of their 

reduced cost than because of belief in their effectiveness. 

Epidemiology 

Snakebite 

Antivenin 

Burkina Faso 

345

Poster 11: UNCOMMON ENVENOMING PROVOKED BY ARTHROPODS: 

TOXINS FOR KNOWING 

Haddad Jr, V 1,2 . 

1 

Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, Caixa Postal 557, 18618-000, São Paulo 

State, Brazil, haddadjr@fmb.unesp.br 

2 

Vital Brazil Hospital, Butantan Institute, Avenida Vital Brazil, 1500, Butantan, São Paulo State, Brazil. 

INTRODUCTION/OBJECTIVES: The arthropods constitute the most numerous group of 

animals of the Nature. Several of them present toxins as mechanisms of attack and 

defense, being able to cause injuries in human beings. There is clinical interest and an 

immense pharmacological potential in these toxins, practically unexplored. MATERIAL 

AND METHODS: The accidents had been selected in the casuistry of the author, in 

accordance with the rarity of the occurrence and the potential interest for toxinologists. 

RESULTS/DISCUSSION: the clinical aspects of envenoming for Millepede or Diplopoda 

are presented. These animals can eject irritating fluids that cause brown pigmentation and 

occasionally severe inflammation and blisters in the skin. The stink bugs (Pentatomidae) 

have glands that produce a mixture of hidrocarbonates that function as a repellent of 

predators and paralysis in prey. In humans, the secretion causes inflammatory skin lesions. 

The effects of the secretion are described in the first observation of lesions caused by 

Pentatomidae in humans. Finally, an accident caused for a tocandira ant is presented 

(Paraponera clavata). The tocandira is a giant ant which sting provokes violent pain and 

systemic manifestations in the victim. The intention of this presentation is to demonstrate 

uncommon injuries caused by venomous and poisonous arthropods and to offer new paths 

for interested on the involved toxins, poorly studied until today. 

1. HADDAD Jr, V, CARDOSO JLC; MORAES RHP. Skin lesions caused by stink bugs 

(Pentatomidae): first report of dermatological injuries in humans. Wild. and Env. Med. 

13(1): 48-50, 2002. 2. HADDAD Jr V, CARDOSO JLC, MORAIS RHP. Description of an 

injury in a human caused by a false tocandira (Dinoponera gigantea, PERTY, 1883) with a 

revision on folkloric, pharmacological and clinical aspects of the giant ants of he genera 

Paraponera e Dinoponera (sub-family Ponerinae). Rev. Instit. Med. Trop. de São Paulo, 

47 (4): 235-238, 2005. 

o venomous animals 

o arthopods 

o toxins 

o stink bugs 

346 

Poster 12: North and South American Loxosceles spiders: Development of a 

polyvalent 

antivenom with recombinant Sphingomyelinase D as antigens. 

Olvera, A 1* , Ramos-Cerrillo, B 1 , Paniagua-Solís, J 2 , Stock, R.P 1 and Alagón, A 1 . 

1 

Instituto de Biotecnología-UNAM. Av. Universidad 2001, Chamilpa, Cuernavaca, Mor. México. 

* aolvera@ibt.unam.mx 

2 

Laboratorios Silanes S.A. de C.V.831000, Mexico City, Mexico. 

The bite of Loxosceles spiders can induce dermonecrotic lesions and systemic syndromes 

such as kidney failure and hemolysis. Sphingomyelinase D (SMD) is the most abundant 

protein component of Loxosceles venom. It has a molecular mass of approximately 33 

kDa and catalyzes the cleavage of sphingomyelin, releasing 1-phosphoceramide and 

choline. 

In recent studies it has been shown that a sphingomyelinase D activity is responsible for 

the pathological effects of the Loxosceles bite (Ramos-Cerrillo, et al.). The aim of the 

present study was to generate recombinant SMD antigens for the production of a 

neutralizing antivenom. Here we report the cloning of cDNA coding for SMD from L. 

reclusa, L. laeta (Perú) and L. boneti, into bacterial E. coli expression systems as well as 

optimization of expression conditions so as to obtain soluble and active recombinant 

enzymes. The recombinant mature SMDs, tagged with a histidine tail at the N- or Ctermini, 

were compared in terms of toxicity and enzymatic activity, and were used as 

immunogens for the production of monovalent antisera in rabbits and F(ab’)2 

preparations in animals used for commercial antivenom production (horses). We 

performed studies on in vitro antigenic cross-reactivity of the recombinant enzymes and 

inhibition of enzymatic activity by antibodies generated against the tagged proteins. We 

also present and discuss the results of studies on the specific and para-specific in vivo 

protective potential of the rabbit and equine antibody preparations against the 

recombinant proteins themselves and natural venom preparations. Our conclusions 

support the feasibility of using recombinant SMDs for production and evaluation of 

polyvalent anti-Loxosceles antivenoms, and we offer data on the potential of paraspecific 

neutralization in the context of the antigenic groupings and the molecular phylogeny of 

those active SMDs for which amino acid sequence information is available. 

Ramos-Cerrillo B., et al. 2004. Toxicon (44) 507-514. 

o Loxosceles 

o Sphingomyelinase D 

o Recombinant antigens 

o Polyvalent antivenom 

347

Poster 13: EPIDEMIOLOGY OF SNAKEBITES BASED ON HOSPITAL 

SURVEY IN CHITWAN AND NAWALPARASI DISTRICTS, NEPAL. 

Pandey, Deb Prasad; Part-time Lecturer, Birendra Multiple Campus, Tribhuvan 

University, Bharatpur, Chitwan; President, Association for Nature Conservation and 

Social Upliftment, Nepal 

OBJECTIVE: To characterize and compare the epidemiology of snakebite in Chitwan 

and Nawalparasi Districts. METHODS: Two hospitals, one from each district, selected 

deliberately were visited for 7 days/month commencing from 15 Sept. 2004 to 15 Sept. 

2005 and abstracted information using predesigned and pretested data collection sheet 

from the hospital record file. RESULTS: Total snakebite cases recorded from 17 months 

hospital-record files were 860, of which, 23.72% were recorded from Kali Gandaki 

Hospital, Nawalparasi and 76.28% from Bharatpur Hospital, Chitwan; 59.07% were from 

Nawalparasi and 40.93% were from Chitwan. Of the total cases recorded during 15 April 

2004 to 15 April 2005(12 months), the maximum cases (33.78%) were found in 15 June 

to 15 July (Ashad) and null in the next four months starting from 15 Nov. 2004 to 15 

March 2005; the maximum (68.89%) were in summer season with no cases in winter. Of 

the totality, 49.30% patients were males and 50.70% were females; the maximum (i.e. 

22.56%) were between 20 – 30 yrs.; the maximum (39.07%) snakebite occurred in 

Bramin caste; 104 (12.093 %) were venomous cases with 25% hospital case fatality rate. 

Of the total fatality, 26.92% was recorded in the age group of 0-10 yrs. The fatality rate 

was found to be gradually less in the older people. 31.84 anti-snake venom vials per 

patient were used. DISCUSSION: Seasonal and monthly incidence of sankebite 

demonstrated a distinct pattern closely related to rainfall, temperature and abundance of 

the foods of snakes which was comparable to the findings of Handsdak et al. 1998, 

Pandey 2005. The sex-, age- & Caste-wise incidence of snakebite victims throw light on 

the vulnerable section of the population which was similar to the findings of Pandey, 

2005. CONCLUSION: The largest numbers of snakebite cases were recorded from 

Nawalparasi. Maximum snakebite cases in Bharatur Hospital reflect the facility of antisnake 

venom vials in free of cost and intensive care of patients. The maximum snakebites 

in Ashad and in summer season reflect optimum environment to snakes in terai, greater 

agricultural and other outdoor and indoor activities of people and no snakebite cases in 

winter reflect hibernation of snakes in winter. Children below the 10 yrs were in great 

risk of death because of greater amount of toxin/kg body weight. 

Key words- snakebite, toxin, epidemiology, fatality, incidence, 

348 

Poster 14: Analysis of clinical outcomes and cost-benefits associated with the use 

of CSL Snake Venom Detection Kits in the management of snakebite at Port 

Moresby General Hospital, Papua New Guinea 

Williams D.J, 1* Jensen S.D, 1,2 Winkel K.D. 1 

1 


2 

School of Medicine & Health Sciences, University of Papua New Guinea, Boroko, Papua New Guinea 


Introduction: A commercial enzyme immunoassay (CSL Snake Venom Detection Kit) has 

been in common use in Australia for over 25 years, but has not been adopted in nearby 

Papua New Guinea, despite the two countries sharing several species with the same venom 

immunotypes. Ahead of the introduction of CSL SVDKs in PNG, a cost-benefit analysis 

was conducted to assess potential clinical and financial benefits. 

Methods: Patients recruited to a prospective study of snakebite at Port Moresby General 

Hospital who had positive signs of envenoming had either bite site swabs or urine samples 

taken for testing using the CSL SVDK as per manufacturer instructions. All patients in 

which a venom immunotype was detected were promptly treated with a single vial of 

appropriate antivenom after premedication with subcutaneous adrenaline (0.25 mg 1:1000 

adults; 0.05 mg/kg 1:10000 children). The subsequent outcomes were monitored and costs 

of hospitalisation calculated from internal hospital budget documents. Clinical outcomes 

and costs were compared to a representative cohort of patients treated previously without 

CSL SVDK testing. 

Results: 37 envenomed patients were tested. 33 patients positive for the taipan venom 

immunotype received monovalent taipan antivenom; 1 patient positive for brown snake 

immunotype received polyvalent antivenom; and 2 of 3 patients negative for any of the 5 

test immunotypes also received polyvalent antivenom, the third patient was managed with 

anticholinesterase therapy. 18 patients were kept in the Emergency Dept for 24 hours after 

antivenom, and discharged home. 10 required intubation and assisted ventilation in ICU, 

and 9 patients were admitted to the High Dependency Unit for further observation. 2 

patients died. Detailed analysis of antivenom and hospitalisation cost benefits is presented. 

Discussion: Compared to recent historical outcomes, the use of CSL SVDKs significantly 

improved clinical outcomes, and reduced treatment costs. Phased introduction of broader 

use of these diagnostic tools has now been recommended to Health administrators. 

o CSL Snake Venom Detection Kit 


o Health economics 

o Clinical management 

349

Poster 15: Epidemiology of snakebite in Guinea 

Baldé M. C., Barry A. O., Tamboura B. A. 

Institut Pasteur de Guinée, B.P. 146, Kindia, Guinée 

The Institut Pasteur de Guinée carried out epidemiological studies for several years based 

on data available at medical facilities and on household surveys. Comparing these two 

sources of information, the incidence of snakebite, mortality, the use of medical facilities, 

hospital mortality and the incidence of bites by the various venomous species in the 

country are evaluated. 

3,047 households (23,442 inhabitants) were investigated in the area of Kindia (Low 

Guinea, forest-savanna limits). Surveys in health centers serving this population were 

used to quantify annual incidence of snakebite. An annual rate of 375 bites, of which 19.2 

were fatal, per 100,000 inhabitants was observed. However, it was observed that 80% of 

the surveyed victims used only traditional medicine. 

A survey of 990 households (12,850 people) in the area of Kankan (Northern Guinea, 

savanna) showed that the annual incidence of bites was 1,758 per 100,000 inhabitants 

with an annual mortality rate of 108 per 100,000 inhabitants. These dramatic results need 

to be confirmed. 88% of the victims initially consulted a traditional healer, and only 20% 

of them went to the hospital after treatment by the traditional healer failed. 

Notifications by health centres showed high mortality rate in hospitals ranging from 7 to 

22% (average approx. 15% over ten years). This high mortality rate resulted from the late 

arrival of patients at the hospital, often with severe complications. The average time of 

arrival was more than 150 hours after snakebite. Other possible reasons are the lack or 

scarcity of antivenins resulting in insufficient treatment. Finally, the frequency of bites by 

the various venomous species (15% by Bitis, 21% by Naja, 12% by Dendroaspis) may 

also explain this high rate of mortality. 

Epidemiology 

Snakebite 

Mortality 

Guinea 

350 

Poster 16: RENAL AND BLOOD VESSEL ALTERATIONS INDUCED BY 

GAMMA TOXIN OF Tityus serrulatus VENOM. 

Monteiro, H.S.A. 1 *; Alves, R.S. 1 ; Martins, R.D. 1 ; Amora, D.N. 1 ; Barbosa, P.S.F. 1 ; 

Sousa, D.F. 1 ; Alves, C.D. 1 ; Toyama, M.H. 2 ; Menezes, D.B. 3 ; Martins, A.M.C. 4 ; Fonteles, 

M.C. 1 ; Monteiro, F. C. D. 1 

1. Departamento de Fisiologia e Farmacologia, Coronel Nunes de Melo street, 1127/ Universidade 

Federal do Ceará/ Brazil *serrazul@truenet-ce.com.br 

2. Departamento de Bioquímica, Praça Infante Dom Henrique s/nº -São Vicente/SP; Universidade 

Estadual Paulista Júlio de Mesquita Filho/ Brazil 

3. Departamento de Patologia e Medicina Legal, Coronel Nunes de Melo street, 1127/ Universidade 

Federal do Ceará/ Brazil 

4. Departamento de Análises Clínicas e Toxicológicas, Capitão Francisco Pedro street, 1210/ 

Universidade Federal do Ceará/ Brazil 

About 8000 cases of scorpion envenomation are reported yearly in Brazil according to 

the Ministry of Health. Most cases are due to the sting of Tityus serrulatus, the most 

dangerous scorpion in Brazil 1 . The aim was to test the effects of gamma toxin (gT) of T. 

serrulatus venom in perfused isolated rat kidney and mesenteric blood vessels. Wistar 

rats kidneys were perfused with Krebs-Henseleit solution containing 6g% bovine 

albumin 2 . The effects of gT at 3µg/mL (n=6) concentration were studied on perfusion 

pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration 

rate (GFR), sodium (%TNa), potassium (%TK) and chloride (%TCl) tubular transport. 

Mesenteric bed 3 was perfused with Krebs solution and effects of gT (3µg/mL/min; n=6) 

were examined and compared to the infusion of the perfuse solution. Effects of gT were 

compared to external control (C). Data were analyzed by Student’s t-test (*p

Poster 17 : Biological and Clinical Evaluations of Antivenom Immunotherapy 

Bon, C. 1* , Audebert, F. 1 , Calderon, E. 2 , Choumet, V. 1 , ElAyeb, M. 3 , Krifi, M. 3 , Possani, L. 2 , 

Rivière, G. 1 , Sorkine, M. 1 

1 Unité des Venins, Institut Pasteur, Paris, France, cbon@mnhn.fr ; 2 Laboratoire des Venins et Toxines, 

Institut Pasteur, Tunis, Tunisie ; 3 UNAM Instituto de Biotecnologia, Cuernavaca, Mexico 

Antivenom immunotherapy is the unique specific treatment of envenomations. 

Although widely used and medically accepted, it is still empirically administered. Its 

improvement requires accurate criteria to assess when this treatment has to be used or not. 

It also requires accurate guidelines to improve the efficacy and the safety of this treatment. 

ELISA tests were set up to follow the venom toxins in patients’ blood during 

envenomations and pharmacokinetics were followed in experimentally envenomed rabbits, 

in the absence of, or after antivenom therapy. In a first step, ELISAs were used in parallel 

with clinical grading of viper and scorpion envenomations. In both cases, a good 

correlation was observed between the venom levels in the blood and the clinical 

symptoms. In a second step, we examined the venom kinetics, in the absence of, and after 

antivenom administration. Investigations were carried out in the case of envenomations by 

viper (V. aspis) in France and by scorpion (A. a. garzonii) in Tunisia and (C. l. limpidus) in 

Mexico. After intramuscular injection of viper venom, the resorption of the venom follows 

a complex process: it is fast during the first 24 hr then it occurs at a slower rate over the 

subsequent 72 hr, resulting in a long half-life of elimination (36 hr). On the other hand, the 

absorption of scorpion toxins is very fast and complete and its half-life of elimination is 

short (2 hr). The effect of antivenom therapy was further tested. It appeared that the 

detoxification process could be explained by a redistribution of the venom from the 

extravascular compartment to the vascular one, where it is sequestered by the antibodies. 

Intravenous injection was shown to be the most effective route for antivenom 

administration. The relative efficacy of Fab'2 and Fab was tested also and, in case of viper 

envenomations, it was shown that Fab'2 are more efficient than Fab because of more 

appropriate pharmacokinetic parameters. 

These experimental studies provide an experimental model to optimize the antivenom 

therapy. However, questions remain to be solved to improve further this treatment: 

pharmacodynamic of toxins, their local versus systemic actions, their elimination route. 


o Antivenom 

o Toxicokinetics 

o Venom neutralization 

352 

Poster 18: Amelioration of the cardiovascular effects of the yellow scorpion 

Leiurus quinquestriatus envenomation in rats by the red grape seeds 

proanthocyanidins 

El-Alfy, A 1 ., Ahmed, A 1 ., Kader, F 1 ., Fatani, A 1 . 

1 

Pharmacology Department, Faculty of Pharmacy, King Saud University, P.O. Box 22452, Riyadh 11495, 

Saudi Arabi (amfatani@yahoo.com). 

Introduction: Cardiovascular effects induced by Leiurus quinsuestriatus (LQQ) venom 

plays a key role in the morbidity and lethality of scorpion envenoming. It has been 

recently suggested that free radical generation may contribute to the damaging effect of 

scorpion venoms on the myocardium. Hence the objectives of this study were to determine 

the involvement of oxidative stress in LQQ scorpion envenomation and the possible 

protective actions of the potent antioxidants red grape seeds proanthocyanidins (GSP). 

Methods: Lethality studies were conducted in mice. Animals were treated with 100 or 200 

mgkg -1 day -1 GSP (p.o.) for 10 days prior to injection of 250 or 350 ugkg -1 scorpion venom 

(s.c.). Blood pressure, via the carotid artery following tracheostomy was measured in 

anaesthetized rats. Animals were treated with GSP (100 or 200 mgkg -1 day -1 p.o.) or saline 

for 10 days followed by venom injection (350 μgkg -1 , s.c.) and blood pressure (MABP), 

heart rate (HR), and ECG changes were recorded periodically up to death. 

Results: Results revealed significant protection of GSP against LQQ venom lethality. 

Additionally, pretreatment with GSP significantly (p

Poster 19: Can the antioxidant, ginkgo biloba extract, and the protease inhibitor, 

aprotinin, protect rats against Leiurus quinquestriatus scorpion venom-induced 

cardio-vascular effects? 

Fatani, AJ, Yaqub, HI, Ibrahim, M, Al-Zuhair, H, Qadir, F, Al-Qutub, M. 

Dept of Pharmacology, College of Pharmacy, King Saud University, P.O Box 616, Riyadh, 11421, Saudi 

Arabia, amfatani@ksu.edu.sa 

Introduction: Scorpion toxins act on ionic channels causing dysfunction in several systems, 

with death attributed to cardiovascular (CV) damage 1 . Oxidative stress and proteases are 

implicated in several diseases, including those of the CV system 2,3 . This study was 

undertaken to assess if antioxidants and protease inhibitors could protect animals against 

the detrimental cardiovascular manifestations caused by the venom of a common scorpion 

Leiurus quinquestriatus (LQQ) and if they would prolong survival. 

Methods: Male Swiss albino mice were injected with LQQ venom (250 μg kg -1 , s.c n=10) 

alone or after Gin (150 mg kg -1 , p.o, 3 weeks before venom) and/or Apr (46000 KIU kg -1 , 

i.p, 30 min before venom). Control groups were injected with saline or treatments alone 

Percent surviving after 24 hr plus time of death were recorded and Covariance Wilcoxon 

Survival analysis utilized. In addition, blood pressure (BP), heart rate (HR) and lung 

edema index were measured in anaesthetized male wistar rats (n=5) pretreated with Gin 

and/or Apr prior to LQQ venom injection (doses as above) and Kruskal Wallis nonparametric 

ANOVA was utilized for analysis. 

Results: Pretreatment with Apr showed greater efficacy than Gin in prolonging the survival 

(P

Poster 21: Snake Venom Metalloproteinases (SVMPs) binds to collagen and α2β1 

integrin by two different motifs 

Tanjoni,I. 1 , Butera, D. 1 , Della-Casa, M.S. 1 , Fernandes, I. 1 , Clissa, P.B. 1 , Evangelista, K.S. 2 , Eble, J. 2 , 

Moura-da-Silva, A.M. 1* 

1 * 

Laboratório de Imunopatologia, Instituto Butantan, Brazil, anamoura@butantan.gov.br 

2 Department of Physiological Chemistry, University of Münster, Germany 

Several integrin inhibitors that interfere with platelet functions have been isolated from 

snake venoms. Jararhagin, a P-III snake venom metalloproteinase, from Bothrops jararaca 

venom presents disintegrin activity as a collagen receptor α2β1 antagonist. To study the 

structure/ function relationships of jararhagin, our group has developed seven monoclonal 

antibodies (MAJar 1-7) and mapped their respective epitopes. In this study, the 

neutralizing activity of MAJars of jararhagin potential in inhibiting collagen-induced 

platelet-aggregation was evaluated. Pre-incubation of jararhagin with MAJar 3, which 

recognized the C-terminal portion of the disintegrin domain, blocked its ability to inhibit 

collagen-induced platelet-aggregation. MAJars 1 and 3 efficiently neutralized jararhagin 

binding to collagen in solid phase assays. However, the binding of jararhagin to the α2β1 

integrin was not reduced by its pre-incubation with MAJars either in dotblots of 

mammalian transfected cells with α2 integrin subunit (α2k562), nor by solid phase assays 

using microtiter plates coated with recombinant soluble human α2β1 integrin, suggesting 

that the epitopes recognized by the MAJars are not involved in the binding of jararhagin to 

α2β1 integrin. These data indicate the existence of two independent motifs for jararhagin 

binding to collagen or α2β1 integrin. The collagen-binding motif, which is predicted to be 

located in the C-terminal portion of the disintegrin-like domain, is sufficient for the 

expression of jararhagin activity related to the inhibition of the platelet function. 

Supported by FAPESP and CNPq. 

snake venom 

metalloproteinase 

platelets 

disintegrin 

356 

Poster 22: Distribution and effects of ε-toxin-GFP in mouse brain and kidneys 

Juan Blasi 1* , Alex Soler 1 , Jonatan Dorca 1 , Josep Saura 2 , Josep Maria Tussell 2 , Maryse Gibert 3 , Michel 

Poppof 3 , Joan Serratosa 2 and Mireia Martín-Satué 1 . 

1 Departament de Patologia i Terapèutica Experimental. Universitat de Barcelona-IDIBELL. Feixa Llarga 

s/n, 08907 L’Hospitalet Spain. * blasi@ub.edu 

2 Departament de Farmacologia i Toxicologia. IIBB-CSIC, IDIBAPS. Rosselló, 161, 6ª - 08036 Barcelona 

3 CNR Anaérobies, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France. 

ε- toxin, from Clostridium perfringens (B and D strains), is a protein of about 32 kDa that 

causes a very severe and often fatal form of enterotoxaemia in sheep, goats, and 

occasionally calves and other animals. In mice, intravenous injection of the toxin produces 

its accumulation in several defined organs, but mainly in kidneys and central nervous 

system. This accumulation correlates with the toxic effect at cellular level, producing 

selective death of epithelial cells from the distal tubule of the nephron and cerebral edema 

with occasionally neuronal death. In this study ε-toxin-GFP was intravenous injected into 

mice in order to monitor the “in vivo” distribution of this protein in the brain and kidneys. 

The toxin was detected in kidney tubules, both in the apical or basolateral region of 

proximal and distal tubules respectively. When similar amounts of GFP were injected, only 

the apical region of proximal tubules was labeled suggesting that a non-specific binding 

previously found was due to the toxin accumulation in proximal renal tubules. After 5-10 

minutes of the i.v. injection, ε-toxin-GFP was found lining vascular endothelium of brain 

microvasculature. This binding was no visible at longer times after injection, suggesting 

that the toxin could cross the endothelia (and blood brain barrier) and gain access to the 

nervous tissue. Consequently, the possible effect of the toxin on glial cells and isolated 

nerve terminals (synaptosomes) was studied. The permeabilizing effect of the toxin on 

vascular endothelia was also studied by co-injection of Alexa688 coupled bovine serum 

albumin (Alexa688-BSA). All together, the results show the “in vivo” differential 

distribution of ε-toxin-GFP after i.v. injection and the use of the fusion protein (ε-toxin- 

GFP) as a valuable tool for the study of the intoxication pathway. 

Supported by a grant from MEC (BFU2005-02202) and ISCIII (PI040778 and PI050658). 

JD is a recipient of a predoctoral fellowship from IDIBELL. 

o Epsilon toxin 

o Clostridial toxins 

o enterotoxaemia 

o GFP fusion protein 

357

Poster 23: Evaluation of potential ricin inhibitors on pulmonary and digestive 

epithelial cells 

Emilie Gobbo 1 , Roman Lopez 2 , Goulven Merer 2 , Bernard Rousseau 2 , André Ménez 1 , 

Bruno Beaumelle 3 , Daniel Gillet 1* and Julien Barbier 1 

1 

Département d’Ingénierie et d’Etudes des Protéines, *daniel.gillet@cea.fr 

2 

Service de Marquage Moléculaire et de Chimie Bioorganique, CEA-Saclay, 91191 Gif 

sur Yvette, France, 

3 UMR 5539 CNRS, Université Montpellier II, 34095 Montpellier, France 

We have studied the inhibitory power of eleven drug molecules against ricin using a 

cellular assay. These drugs were chosen because their use in humans is authorized for 

other diseases and data in the literature suggest possible anti-ricin activity for several of 

those. Dexamethasone and di-fluoromethylornithine were described for delaying slightly 

the death of intoxicated mice. Antiviral nucleotidic analogues (AZT, ddC) or pteroic acid 

were shown to inhibit toxicity on cells or to bind weakly to the catalytic site of the toxin 

in vitro with millimolar dissociation constants. Finally, other antiviral nucleotide 

analogues (d4T, 3TC, ddI, ribavirine) or drugs with related structures (methotrexate and 

diazepam) were also tested. 

The assay was performed on pulmonary (A549) and digestive (Caco2) epithelial cells and 

measured the inhibition of protein synthesis by incorporation of [ 3 H]-Leucine. Cells were 

intoxicated with varying concentrations of ricin in the presence of the highest possible 

concentration of inhibitors which was non toxic and soluble (from 3x10 -4 to 2x10 -3 M 

depending on the drug). 

No inhibition was observed with any of the drugs. Only millimolar concentrations of 

lactose (and to a lesser extent galactose), already known to inhibit binding of the toxin to 

cell surface glycoproteins shown some protective effect. However, these concentrations 

are incompatible with human treatment. 

- ricin 

- inhibitor 

- cellular assay 

358 

Poster 24: Antagonism of the cardiotoxic effect of Bothrops jararacussu by Suramin 

Sifuentes, N. D. 1 , El-Kik, C. Z. 2 , Ricardo, H.D. 2 ,Schwartz, E.N.F. 1 , Melo, P.A. 2 

1 

Laboratório de Toxinologia, Departamento de Ciências Fisiológicas, Universidade de Brasilia, Brasilia, 

DF, Brazil; 

2 

Departamento de Farmacologia Básica e Clínica, ICB, CCS, UFRJ. 21941-590 - Rio de Janeiro - RJ, 

Brazil pamelo@farmaco.ufrj.br. 

Bothrops snakes are responsible for a great number of accidents in Brazil within this 

genus, the species whose venom causes the greatest percentage of tissue necrosis following 

accidents is B. jararacussu. We have investigated many alternatives treatments for these 

accidents, and one frequently studied in our group is the effect of polyanion substances. 

Within these compounds, suramin have shown to be capable of inhibiting great part of 

Bothrops snake venoms effects. Until now the citotoxicity activity B. jararacussu venom 

on the heart has never been investigated. The objective of this work was to show, for the 

first time, the citotoxicity of B. jararacussu crude venom on mammal heart, and its 

inhibition by suramin. The in vitro Langendorff (modified) isolated rat heart preparation 

was used, perfused with a physiological saline solution plus B. jararacussu venom (2.5–10 

mcg/mL), pre-incubated or not with suramin( 1-100 micromolar) and/or antibothropic 

antivenom(2.0 microliters/ml), analyzing patterns of EKG waves, cardiac tension, CK 

release, histology and coronary pression; and in vivo anesthetized rats analyzing arterial 

blood pressure and EKG, with the same treatments. B. jararacussu venom(0.1- 1mcg/kg), 

reduced cardiac tension and arterial pressure leading to collapse and induced many 

alterations on EKG waves, proving to be cardiotoxic, and dose or concentration dependent. 

These activities probably occurred due to lesions on the cardiac cells, shown by histology 

and increase on the CK release level. When suramin was pre-incubated with the venom, it 

inhibited the cardiac collapse, in a dose or concentration dependent way. When associated 

with the antivenom, suramin totally inhibited the cardiotoxic activity, keeping parameters 

close to control. Thus, in this study it was shown that B. jararacussu venom is cardiotoxic, 

and that suramin was able to inhibit this activity dose dependently. 

Arruda et al. Braz J Med Biol Res. 2002 35:723-7266 

Murakami et al., 2005 J Mol Biol 350:416-426 


o Cardiotoxicity 

o Suramin 

359

Poster 25: EFFECT OF CROTOXIN ON MICROCIRCULATION AND 

CIRCULATING LYMPHOCYTES 

Zambelli, VO 1 ; Sampaio, SC 1 , Britto, LRG 2 , Zychar,B 1 ; Gonçalves, LRC 1 ; Cury, Y 1 . 

1 2 

Lab. of Pathophisiology; Butantan Institute, SP; Dep. of Physiology and Biophysics Biomedical Sciences 

Institute- USP, SP. 

Crotoxin (CTX), the main neurotoxin component of the South American rattlesnake 

Crotalus durissus terrificus, is composed of a non-toxic and non- enzymatic polypeptide 

named crotapotin and of a weakly toxic fosfolipase A2 (PLA2). CTX and PLA2 reduce the 

number of lymphocytes in blood and lymph. The aim of this study was to investigate the 

effect of CTX and PLA2 on lymphoid tissue (spleen and mesenteric lymph node), 

evaluating the expression of T and B-lymphocytes, as well as the dynamics of cellular 

events occurring in microcirculation. Moreover, mesenteric lymph nodes and spleens of 

male Wistar rats were removed for analyses 2h after subcutaneous (s.c.) injection of 

CTX (18µg/animal), PLA2 (10.4µg/animal) or saline (300µL). Paraffin-embedded tissue 

sections were stained with hematoxylin-eosin for histopathological assessments. The 

expression of T and B lymphocyte was determined by immunohistochemistry. 

Microcirculation was analysed by intravital microscopy of the internal spermatic fascia. 

Rolling, adherence and migration phenomena in post-capillary venules were evaluated. 

CTX and PLA2 promoted, in mesenteric lymph nodes, follicular hyperplasia with 

increase of germinal center. Hyperplasia of spleen white pulp was also detected. In 

addiction, toxin treatment increases T and B lymphocyte immunostain. Microcirculation 

analyses showed that CTX increases, adhered leukocyte (12.2 CTX vs. 3.25 saline), and 

their migration (11 CTX vs. 2.25 saline) at 1h. A decrease in the rolling (5.75 CTX vs. 

20.25 saline) and an increase in migration of lymphocyte (10.75 CTX vs. 1 saline) were 

observed 2h after treatment. These results indicate that toxins mobilize leukocytes to 

extravascular tissue, promote morphological alterations and an increase of lymphocytes 

in lymphatic tissue. 

Supported by: CAPES, CNPq 

360 

Poster 26: EFFECTS OF THE SEA ANEMONE Bunodosoma caissarum VENOM 

ON RENAL PERFUSION AND MESENTERIC BED VESSELS. 

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 ; 

Toyama, M. H. 2 ; Martins, A M.C. 3 ; Barbosa, P.S.F. 1 ; Fonteles, M. C. 1 , Monteiro, F. C. D. 1 

5. Departamento de Fisiologia e Farmacologia, Coronel Nunes de Melo street, 1127/ Universidade 

Federal do Ceará/ Brazil *serrazul@truenet-ce.com.br 

6. Departamento de Bioquímica, Praça Infante Dom Henrique s/nº -São Vicente/SP; Universidade 

Estadual Paulista Júlio de Mesquita Filho/ Brazil 

7. Departamento de Análises Clínicas e Toxicológicas, Capitão Francisco Pedro street, 1210/ 

Universidade Federal do Ceará/ Brazil 

Sea anemones contain a variety of interesting biological active compounds including 

some potent toxins. For this reason many investigators have focused attention on the 

biological activities of protein molecules from various sea anemones species, as 

Bunodosoma caissarum, which is endemic in Brazilian Southern coast 1 . The aim of the 

present work was to study the potential functional alterations produced by the 

Bunodosoma caissarum venom (BcV) on isolated rat kidney and its effects on vascular 

reactivity on isolated perfused rat arteriolar mesenteric bed. Isolated kidneys from Wistar 

rats weighing 250 to 300g were perfused with Krebs-Henseleit solution containing 6g% 

bovine serum albumin 2 . BcV (10μg/mL/min; n=6) was added to the system 30 min after 

the beginning of each experiment. Mesenteric bed 3 was perfused with Krebs solution 

kept warm at 37°C by a constant flow (4mL/min) and variation of perfusion pressure was 

measured throughout 80 min after the equilibration period. Data were analyzed by 

Student’s t-test with the level of significance of p

Poster 27: Tityus serrulatus Hypotensive Peptides: a novel class of hypotensive 

agents 

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, 

P.E. 5 , Martin-Eauclaire, M.F. 5 , De Lima, M.E. 1,2 and Pimenta, A.M.C. 1,2 

1 Núcleo de Biomoléculas, 2 Laboratório de Venenos e Toxinas Animais and 3 Laboratório de Enzimologia e Físico- 

Química de Proteínas, Depto. de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade 

Federal de Minas Gerais, Av.Antônio Carlos 6627, Belo Horizonte, Brazil, *thiagovb@icb.ufmg.br 

4 Laboratório de Hipertensão, Depto. de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade 

Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, Brazil. 

5 CNRS-FRE 2738, Ingénierie des Protéines, Université de la Méditerranée, UMR6560, IFR Jean Roche, 15, Bd 

Pierre Dramard, 13916 Marseille, France 

Facilities in using micro-scale analytical techniques, such as mass spectrometry and 

proteomics, have led to a novel approach to prospect bioactive molecules in animal 

venoms. By this approach, we aimed to prospect new structural families of scorpion toxins 

and were able to find a new structural family of peptides in the venom of the Brazilian 

yellow scorpion Tityus serrulatus, named TsHptP (Tityus serrulatus Hypotensive 

Peptides(1)). Structurally, these are random-coiled linear peptides, ranging from 2,5 to 3,0 

kDa and have a similar bradykinin-potentiating peptide (BPP) amino acid signature. 

TsHpt-I (2,7 kDa), a member of this peptide family, was able to potentiate the hypotensive 

effects of bradykinin (BK) in normotensive rats. To optimize the pharmacokinetics and the 

stability of TsHpt-I, few synthetic analogs with lower mass ranges were constructed using 

peptide synthesis and the TsHpt-I as a template. These analogs held the BK-potentiating 

effect. A relevant hypotensive action independent on BK was observed to all analogs, 

indicating that they are themselves hypotensive agents. We used two hypertensive rats 

strains (SHR and TGR) to study this hypotensive effect and it has been shown that these 

analogs induces a strong and long-lasting hypotensive effect. To evaluate this action, we 

examined the vasorelaxation effect of aortic rings derived from male Wistar rats. The 

analogs were able to induce ± 20% of vasorelaxation (10 -7 M). One analog was orally 

administrated in SHR rats being able to reduce the blood pressure, indicating that it is 

stable and can be absorbed in the gastrointestinal tract. Although these peptides have a 

similar amino acid sequence with the classical BPPs (2), differences in the primary 

structure are crucial to the new pharmacological effects observed in these peptides. 

1. De Lima, M.E., Diniz, C.R., Santos, R.A.S., Bougis, P.E., Martin-Eauclaire, M.F., 

Pimenta, A.M.C. Patent Application 2002 PI 0202157-9, 2003 PCT/BR03/00073, 

2006 USPTO 20060014928. 

2. Ianzer, D.; Konno, K.; Marques-Porto, R.; Portaro, F.C.V; Stöcklin, R.; Camargo, 

A.C.M.; Pimenta, D.C. (2004) Peptides, 25, 1085-92. 

o Bradykinin-Potentiating Peptide 

o Tityus serrulatus 

o Hypotensive Peptide 

o Hypertension 

362 

Poster 28: Snake Venom Components as Pharmacological Tools and Molecular 

Models in Thrombosis and Haemostasis 

Bon, C., Arocas V., Braud S., Francischetti I., Leduc M., Maroun R., Saliou B., Wisner A., 

Zhang Y., Zingali R.. 

Unité des Venins, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, France 

Numerous components in snake venoms affect haemostatic mechanisms. Several of 

them have been employed to develop diagnostic kits, while others are useful 

pharmacological tools and/or molecular models, in the development antithrombotic and 

thrombolytic agents. 

We have shown that the C-type lectin convulxin, from C. d. terrificus venom, activates 

blood platelets by binding specifically with a high affinity to GPVI, the main collagen 

receptor. Hence convulxin, the two subunits of which have been cloned, sequenced and 

expressed as a recombinant fusion protein with Bungarus venom acetylcholinestrase, 

appears to be a precious tool to study GPVI functions. 

The C-type lectin bothrojaracin, discovered in B. jararaca venom, is a specific and 

potent thrombin inhibitor that interacts with thrombin exosites I and II but does not block 

its catalytic site. Having cloned and sequenced the cDNAs encoding the subunits of 

bothrojaracin, we used site-directed mutagenesis in a structure-function studies to design 

new antithrombotic agents. 

We identified a specific plasminogen activator, TSV-PA, in the venom of T. stejnegeri. 

At variance with t-PA, TSV-PA is insensitive to the physiological inhibitor PAI-1. We 

cloned the cDNA encoding TSV-PA and expressed it in E. coli. We also established its 3D 

structure by crystallography, showing that TSV-PA resembles the catalytic domain of t- 

PA. However, subtle differences have been proved, by site-directed mutagenesis, to play a 

critical role in the insensitivity of TSV-PA to physiological inhibitors. 

Strongly anticoagulant snake venom PLA2s selectively inhibit the prothrombinase 

complex. Their action is independent of their enzymatic activity and of phospholipids. We 

proved that the strongly anticoagulant PLA2s bind selectively with a high affinity to factor 

X, preventing its activation by factor Va. Interestingly, human PLA2 that is liberated by 

platelets during their activation behaves as the strongly anticoagulant PLA2s from snake 

venom, exerting a negative control to the pro-coagulant action of activated platelets. 

o Thrombosis & Haemostasis 

o Molecular biology 

o Expression & Site directed mutagenesis 

o Structure Function 

363

Poster 29: Venom Peptides from Calloselasma rhodostoma --- New Twist in 

Vasoactive Peptides? 

Higuchi, S 1,2* , Khow, O 3 , Suntrarachun, S 3 , Noiphrom, J 3 , Miyamoto, S 1 , Kawai, A 1 , Sitprija,V 3 , 

Sato,T. 3 

1 

Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto 860-0082, Japan, 

*higuchis@ph.sojo-u.ac.jp 

2 

School of Pharmaceutical Sciences, Showa University, Tokyo 142-8555, Japan 

3 

Queen Saovabha Memorial Institute, The Thai Red Cross Society, Bangkok 10330, Thailand 

Findings that crotaline snakes (e.g. B. jararaca) have genes coding for the precursor 

polypeptide bearing angiotensin converting enzyme inhibitors (ACEIs, also known as 

bradykinin potentiating peptides (BPPs)) and a C-type natriuretic peptide (CNP) led us to 

investigate the venom gland cDNA library of C. rhodostoma. The precursor polypeptide 

comprised 333 amino acid residues and had the same structure as those from other species: 

at the N-terminus, tandem repeats of sequence for ACEI-like peptide and a C-type 

natriuretic peptide (CNP) sequence at the C-terminus. Some of ACEI-like peptide 

sequences had specific features of ACE inhibitor, i.e. glutamine residue in the amino 

terminus which is transformed into pyroglutamic acid (Pyr) in the mature form and Pro-Pro 

motif in the carboxyl terminus. The CNP sequence was highly similar to other venom 

CNPs, having a 17-membered ring structure through forming a disulfide bond. 

In order to know the peptides mentioned above are actually expressed and milked in the 

venom of C. rhodostoma, the low molecular weight components were collected with the 

use of Sephadex G100 gel chromatography and then applied onto a C18-reversed phase 

HPLC for further fractionation. Mass spectrometric analysis was conducted for each HPLC 

fractions. The ions at m/z 1281.7 and 1116.6 are assignable to ACE inhibitors with 12 and 

10 amino acid residues in the precursor sequence, respectively. A distinct ion at m/z 596.3 

was subjected to MS/MS analysis and a pentapeptide with the sequence PyrGRPR 

emerged as a result. Intriguingly, this peptide appears to arise from the amino-terminal part 

common to four tandemly aligned ACEI-like sequences. On the contrary, any of the 

putative ACEI-like peptides were not detected in the mass spectrometric measurements. 

Although the function of this peptide is unknown, the C-terminal arginine and the 

penultimate proline suggests a similarity to blomhotin from A. blomhoffi (1) and a novel 

peptide found in C. d. collilineatus (2). 

1. Yanoshita, R. et al. (1999) Toxicon, 37, 1761-1770. 

2. Higuchi, S. et al. (2006) Comp. Biochem. Physiol. C, in press. 

o Calloselasma rhodostoma 

o ACE inhibitor 

o Mass spectrometric analysis 

o a pentapeptide PyrGRPR 

364 

Poster 30: Potent dromedary antibody fragments for using in immunotherapy 

against scorpion envenomation 

Bouhaouala-Zahar, B. 1 *, Conrath, K. 2 , Ben abderrazek R. 1 , Hmila I. 1 , Benlasfar Z. 1 , Hammadi M. 3 , 

Khorchani T. 3 , Muyldermans S. 2 . & El Ayeb, M. 1 

1-Laboratoire LVT-Institut Pasteur de Tunis. 13 Place Pasteur BP74, 1002 Tunis-TUNISIA* 

balkiss.bouhaouala@pasteur.rns.tn 

2- Laboratory of Cellular and Molecular Immunology, Pleinlaan 2, 1050 Brussel, BELGIUM. 

3-Institut des regions arides de Medenine -TUNISIA 

The importance and great promise of specific antibodies against toxins is still 

understudied. The few studies that exist concern poly- and monoclonal antibodies, 

produced against these toxins as a means of specific treating envenoming and seem to 

favour this approach. They correspond to purified heterologous IgGs or F(ab)'2 fractions 

prepared from hyperimmune horse sera. However, such treatments have several 

limitations. Their preparation involving protease digestion of the Fc part of the antibody is 

tedious. 

Herein we report results of genetic engineering approach, leading to smaller recombinant 

antigen binding fragments, such as camel single-domain antibody fragment VHH, and their 

of significant relevance for the improvement of serotherapy applications. 

Thus, we previously demonstrated that the toxic fraction AahG50 elicited in dromedaries a 

high venom neutralising antibody response. Furthermore, the camel Heavy-Chain Ab 

subclasses recognize and neutralize efficiently the toxic peptides of the venom. 

Recently we cloned an ‘immune’ VHH library from the cDNA of the peripheral blood 

lymphocytes of a dromedary immunized with AahG50 toxic fraction of scorpion venom. 

From this immune VHH library we identified four individual clones that encoded a VHH 

which recognises specifically the AahII toxin. The affinity of the recombinant VHH to 

interact with the toxin was measured and in progress to be compared to mouse 

monoclonals and other classical antibodies. In addition, preliminary neutralising activity 

tests of these binders were initiated and were positively evaluated in relation to other antitoxin 

antibody fragments. 

Meddeb-Mouelhi et al., 2003 Toxicon, 42, 785-791. 

Bouhaouala-Zahar et al. (in progress to be published) 

This work was supported in part by an IFS grant 


o VHH fragment 

o serotherapy 

o neutralizing activity 

365

Poster 31: Purification and characterization of ancistron–Bu, a novel fibrinogenclotting 

serine protease from Agkistrodon blomhoffii ussuriensis venom 

Karbovskiy V. L 1,2* , Savchuk A.N 2 , Volkov G.L. 2 

1 National Taras Shevchenko University of Kiev, Biotechnology department, 2 Glushkova Street, Kiev, 

Ukraine, *Vital-kiev@ukr.net 

2 Palladin Institute of Biochemistry NAS of Ukraine, FEBS, 9 Leontovicha Street, Kiev, Ukraine, 

A novel fibrinogen-clotting enzyme (named ancistron–Bu) has been homogeneity 

purified from the Agkistrodon blomhoffii ussuriensis venom using affinity chromatography 

on Blue Sepharose FF and Sephadex G 25 gel filtration. Ancistron–Bu, obtained by this 

way, proved as homogeneous and show molecular weight approximately 13,5 and 27 kDa 

in reducing and non-reducing conditions of sodium dodecyl sulphate polyacrylamide gel 

electrophoresis respectively. The protein purity was about 99%. Its yield was 8,4% of total 

protein and purity increasing was 20,5 fold. This enzyme showed specific fibrinogenclotting 

activity equivalent to 46,4 international thrombin units/mg. Unlike alphathrombin, 

ancistron–Bu split off fibrinopeptide A without releasing fibrinopeptide B from 

fibrinogen. The optimal pH range for the clotting activity of ancistron–Bu was 7,4-8,0. The 

hydrolytic activity of ancistron–Bu is strongly inhibited by diisopropyl fluorophosphate (it 

is obvious that this enzyme is serine protease), but heparin–antithrombin-III complex has a 

small effect on ancistron–Bu catalytic properties. This enzyme did not activate factor XIII 

and had not fibrinolytic and caseinolytic activities. 

Thrombin-specific substrate (D-Phe-Pip-Arg-p-NA) and protein C substrate 

(pyroGlu-Pro-Arg-p-NA) were most susceptible to hydrolysis by ancistron–Bu. In that time 

this enzyme did not showed any detectable amidolytic activity on the tested chromogenic 

substrates for plasmin and plasma factor Xa. Ancistron–Bu did not induce aggregation of 

washed normal platelets by itself, but caused plateletes aggregation in the presence of 

exogenous fibrinogen in a manner entirely different from that of thrombin. 

Ancistron time (like reptilase time) is a simple alternative to the thrombin time for 

rapid fibrinogen assay in samples containing heparin and is particularly useful in the assay 

of antithrombin-III where plasma can be prepared free of fibrinogen. The presence of fibrin 

degradation products, hypofibrinogenaemia and defects in fibrin polymerisation will 

prolong the ancistron time. 

o Snake venom 

o Thrombin-like enzymes 

o Serine protease 

366 

Poster 32: “Blomus-B” – a novel non-enzymatic platelet aggregation inhibitor 

purified from Agkistrodon blomhoffii ussuriensis venom. 

Karbovskiy V. L 1,2* , Savchuk A.N 2 

1 National Taras Shevchenko University of Kiev, Biotechnology department, 2 Glushkova Street, Kiev, 

Ukraine, *Vital-kiev@ukr.net 

2 Palladin Institute of Biochemistry NAS of Ukraine, FEBS, 9 Leontovicha Street, Kiev, Ukraine, 

Platelet aggregation is mediated by the interaction of membrane αIIbβ3 receptor with 

fibrinogen and can be inhibited by many factors. The largest and the most well studied 

group of thease ihibitors are desintegrins – non-enzymatic, smal molecular weight proteins, 

isolated from snake venoms, which specificly bound with αIIbβ3 integrin receptor. 

A novel platelet aggregation inhibitor (named “Blomus-B”) has been homogeneity 

purified from the Agkistrodon blomhoffii ussuriensis venom using affinity and ionexchange 

chromatography. “Blomus-B”, obtained by this way, proved as homogeneous 

single-chain protein with molecular weight, approximately 13,2 kDa estimated by SDS- 

PAGE and analytical size exclusion chromatography. The protein purity was about 99%. 

Its yield was nearly 4% of total venom proteins. 

This protein did not showe any detectable amidolytic activity on the tested 

chromogenic substrates for plasmin, thrombin and protein C and hadn’t fibrinolytic, 

caseinolytic and phospholipase activities. In that time “Blomus-B” strongly supressed 

platelet aggregation induced by ADP and adrenaline with IC50 230nM and 120nM 

respectively. 

Flow-cytometric analysis of washed platelets, preincubated with 250 nM “Blomus- 

B” for 10 min, showed the changes of platelets shape. This fact can be evidence of 

activation proceedings that originate after iteraction between platelet inhibitor and its 

receptor on cell membrane. 

Obtained resalts make possible to draw a conclusion that platelet aggregation 

inhibitor isolated from Agkistrodon blomhoffii ussuriensis venom is a novel low molecular 

weight desintegrin and it can be used as hightspecific and effective antiplatelet agent. 

o Snake venom 

o Platelets 

o Platelet aggregation inhibitor 

o Disintegrins 

367

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