Středa, 29. října 2008 - Faculty of Medicine - Masarykova univerzita

Československá biologická společnost
a
Lékařská fakulta Univerzity Karlovy v Hradci Králové
pořádají
XIX. BIOLOGICKÉ DNY
Biologický výzkum pro lidské zdraví
Hradec Králové
29. - 31. října 2008
Program a sborník abstrakt
Tribun EU
2008

Obsah
Strana
Úvodní slovo 3
Vědecký a organizační výbor 4
Všeobecné a technické informace 5
Program 6
Seznam plakátových sdělení 11
Souhrny ústních sdělení 14
Souhrny plakátových sdělení 49
Seznam účastníků 84
Sponzoři 97
Poznámky 99
Mapa Hradce Králové 101
2

Vážení
Vá ení
účastníci
ú�astníci
XIX.
XIX.
Biologických
Biologických
dnů,
dn�,
jménem
jménem
organizátorů
organizátor�
vám
vám
děkuji
d�kuji
za váš
za vá
zájem
zájem
zúčastnit
zú�astnit
se tradičního
se tradi�ního
setkání
setkání
českých
�eských
a
a
slovenských
slovenských
biologů.
biolog�.
Jsme
Jsme
rádi,
rádi,
že vás
e vás
můžeme
m� eme
přivítat
p�ivítat
v Hradci
v Hradci
Králové,
Králové,
salónu
salónu
republiky,
republiky,
a
a
věřím,
v��ím,
že se
e
přesvědčíte,
se p�esv�d�íte,
že naše
e na
město
e m�sto
si tento
si tento
titul
titul
zaslouží.
zaslou í.
Biologické
Biologické
dny
dny
mají
mají
mezi
mezi
vědeckými
v�deckými
kongresy
kongresy
mimořádné
mimo�ádné
postavení.
postavení.
Je to
Je
dáno
to dáno
nejenom
nejenom
jejich
jejich
dlouhou
dlouhou
nepřerušenou
nep�eru enou
tradicí
tradicí
ale
ale
především
p�edev ím
jejich
jejich
programovým
programovým
zaměřením
zam��ením
na
na
všechny
v echny
obory
obory
biologie.
biologie.
Udržet
Udr
tento
et tento
jednotící
jednotící
princip
princip
není
není
v dnešní
v dne
době
ní dob�
podporující
podporující
úzkou
úzkou
specializaci
specializaci
a v
a
době
v dob�
velké
velké
nabídky
nabídky
vědeckých
v�deckých
setkání
setkání
vůbec
v�bec
jednoduché.
jednoduché.
Jsme
Jsme
proto
proto
rádi,
rádi,
že
e
se i
se
v
i
tomto
v tomto
roce
roce
našel
na
dostatečný
el dostate�ný
počet
po�et
zájemců,
zájemc�,
kteří
kte�í
si chtějí
si cht�jí
zachovat
zachovat
široký
iroký
přehled
p�ehled
o
o
novinkách
novinkách
v biologii
v biologii
a věřím,
a v��ím,
že XIX.
e XIX.
Biologické
Biologické
dny
dny
tento
tento
účel
ú�el
splní.
splní.
Tradiční
Tradi�ní
součástí
sou�ástí
Biologických
Biologických
dnů
dn�
bude
bude
i Babákova
i Babákova
přednáška,
p�edná ka,
letos
letos
již
ji
jedenáctá.
jedenáctá.
V
V
letošním
leto ním
roce
roce
požádal
po ádal
Hlavní
Hlavní
výbor
výbor
Československé
�eskoslovenské
biologické
biologické
společnosti
spole�nosti
o přednesení
o p�ednesení
této
této
přednášky
p�edná
pana
ky pana
doc.
doc.
Ing.
Ing.
Čestmíra
�estmíra
Altanera,
Altanera,
DrSc.
DrSc.
Děkuji
D�kuji
vedení
vedení
lékařské
léka�ské
fakulty,
fakulty,
že nám
e nám
umožnilo
umo nilo
využít
vyu
pro
ít pro
konání
konání
tohoto
tohoto
setkání
setkání
prostory
prostory
lékařské
léka�ské
fakulty.
fakulty.
Je to
Je
tradiční
to tradi�ní
místo
místo
pro
pro
konání
konání
Biologických
Biologických
dnů,
dn�,
v roce
v roce
1995
1995
se XIII.
se XIII.
Biologické
Biologické
dny
dny
konaly
konaly
ve stejných
ve stejných
prostorách.
prostorách.
Takže
Tak
mnozí
e mnozí
z vás
z vás
budou
budou
moci
moci
posoudit,
posoudit,
jak
jak
se naše
se na
fakulta
e fakulta
za uplynulé
za uplynulé
období
období
změnila.
zm�nila.
Děkuji
D�kuji
také
také
všem
v em
sponzorům,
sponzor�m,
bez
bez
jejichž
jejich
podpory
podpory
by realizace
by realizace
vědeckých
v�deckých
setkání
setkání
byla
byla
velmi
velmi
obtížná.
obtí ná.
Věřím,
V��ím,
že toto
e toto
setkání
setkání
vám
vám
přinese
p�inese
mnoho
mnoho
nových
nových
poznatků,
poznatk�,
mnoho
mnoho
nových
nových
osobních
osobních
setkání
setkání
a také
a také
mnoho
mnoho
zážitků
zá itk�
z našeho
z na eho
města.
m�sta.
Přeji
P�eji
XIX.
XIX.
Biologickým
Biologickým
dnům
dn�m
úspěšným
úsp� ným
průběh!
pr�b�h!
Jménem
Jménem
organizátorů
organizátor�
Prof.
Prof.
MUDr.
MUDr.
RNDr.
RNDr.
Miroslav
Miroslav
Červinka,
�ervinka,
CSc.
CSc.
3

Vědecký a organizační výbor
Vědecký výbor
• Prof. MUDr. RNDr. Miroslav Červinka, CSc.
• Prof. MUDr. Fedor Čiampor, DrSc.
• Prof. MUDr. Roman Janisch, DrSc.
• Prof. RNDr. Juraj Krajčovič, CSc.
• Prof. RNDr. Vojtěch Mornstein, CSc.
• Prof. RNDr. Ivan Raška, DrSc.
• Prof. MUDr. Karel Smetana, DrSc. (jun)
• Prof. MUDr. Augustin Svoboda, CSc.
Organizační výbor
• Prof. MUDr. RNDr. Miroslav Červinka, CSc. - předseda
• MUDr. Jana Kolářová, CSc.
• RNDr. Věra Králová
• Doc. PharmDr. Emil Rudolf, PhD.
• RNDr. Ladislava Schröterová, PhD.
4

Všeobecné a technické informace:
Místo konání • Veškerá jednání budou probíhat v prostorách Lékařské fakulty v
Hradci Králové, přednášky, postery a stánky vystavovatelů budou
umístěny v prostorách budovy teoretických ústavů, Šimkova 870.
• Raut dne 29.10. 2008 bude v prostorách Lékařské knihovny v blízkosti
Velkého náměstí na starém městě (viz přiložená mapka)
• Slavnostní večeře bude uspořádána dne 30.10. 2008 v pensionu Nové
Adalbertinum. Zájemci o tuto večeři se přihlásí při registraci a zároveň
zaplatí částku ve výši 250,- Kč.
Registrace
účastníků
Místo pro registraci účastníků je umístěno před Velkou posluchárnou v
budově teoretických ústavů a bude otevřeno
29.10. 8:30 - 18:00
30.10. 8:30 - 18:00
31.10. 8:00 - 12:00
Jednací jazyk Jednacím jazykem je čeština a slovenština. Pouze sekce "Jádro" 30. 10.
bude probíhat v angličtině.
Ústní sdělení PowerPointové prezentace je třeba odevzdat nejméně 20 minut před
zahájením sekce u registrace (pan Pánek). Délka sdělení (včetně diskuse je
uvedena v programu (standardně 15 minut prezentace a 5 minut diskuse)
Plakátová
sdělení
Panely pro postery budou připraveny ve foaye budovy lékařské fakulty.
Postery budou vyvěšeny po celou dobu konání kongresu. Autoři sdělení
musí být u posterů přítomni v průběhu posterové sekce dne 30.10.
odpoledne.
Nouzová linka V případě jakýchkoli problémů kontaktujte paní Lalákovou
(mobil 776 239 086)
5

P R O G R A M
6

8.30 – 10.00 Registrace
10.00 - 10.30 Slavnostní zahájení
10.30 - 12.10 Sekce 1
Předsedající: P. Vodička
7
Středa, 29. října 2008
10.30 - 10.50 R. J. Sram, B. Binkova, O. Beskid, A. Milcova, P. Rossner,
P. Rossner, Jr., A. Rossnerova, I. Solansky, J. Topinka
Biomarkers of exposure and effect interpretation for human risk
10.50 - 11.10 P. Rossner, Jr., V. Svecova, A. Milcova, Z. Lnenickova,
R. J. Sram
Oxidative stress detection in genetic toxicology
11.10 - 11.30 E. Halašová, T. Matáková, Ľ. Mušák, Ľ. Javorka, M. Halaša,
E. Bukovská
Chromosomal damage and polymorphisms of DNA repair genes
XPD, XPG, XPC hOGG1, XRCC1 and XRCC3 in workers
exposed to chromium.
11.30 - 11.50 M. Rédová, P. Chlapek, T. Loja, K. Zitterbart, M. Hermanová,
J. Štěrba, R. Veselská
Modulation of the antineoplastic effect of retinoids in
neuroblastoma and medulloblastoma cell lines
11.50 - 12.10 J. Topinka, B. Binkova, O. Sevastyanova, Z. Novakova,
R.J. Sram
In vitro genotoxicity of complex mixtures extracted from urban
air particles
12.10 - 14.00 Přestávka na oběd
14.00 – 15.20 Sekce 2
Předsedající: Z. Žižka
14:00 - 14.20 M. Pohanka, O. Pavlis, J. Pikula, F. Treml, Jan Marek and
K. Kuca
Tularemia disease modulation using cholinesterase reactivator
HI-6
14:20 - 14.40 D. Šmajs 1 , J. Šmarda 1 , M. Vrba 2 , A. Ševčíková 2
Bacteriocin types produced by Escherichia coli strains isolated
from human and animal samples

14.40 - 15.00 Vesteg M.
Hypotheses for the origin of eukaryotic cytoskeleton
8
Středa, 29. října 2008
15.00 - 15.20 L. Stejskalová, K. Pospěchová, L. Švecová, M. Bitman, R. Vrzal,
Z. Dvořák, P.Pávek
Evidence of cross-talk between aryl hydrocarbon receptor and
glucocorticoid receptor in placental trophoblast cells
15.20 - 15.50 Přestávka na občerstvení
15.50 – 17.30 Sekce 3
Předsedající: R. Janisch
15.50 - 16.10 T. Matáková , E. Halašová , A. Dzian , L. Javorka , D. Dobrota
Polymorphism of GSTs genes in relation to chromosomal damage
in boot workers.
16.10 - 16.30 R. Štětina, P. Vodička, M. Hánová, J. Varvařovská, L. Pácal,
K. Kaňková
Individual DNA repair capacity: from the genetic background to
phenotypic expression
16.30 - 16.50 S. John, L. Klvačová, M. Červinka, E. Rudolf
Antiproliferative effects of zinc in colon cancer cell lines
16.50 - 17.10 V. Králová, K. Brigulová, M. Červinka, E. Rudolf
Effect of sodium selenite on colorectal cell lines with different
p53 status.
17.10 - 17.30 J. Prochazkova, Z. Fiedler
Danger of ethidium bromide and its´ safe alternatives
18.00 – 18.15 Mimořádné valné shromáždění Československé Biologické
společnosti
18.15 – 19.00 Babákova přednáška
Č. Altaner
Stem cells in oncology
19.30 – 21.30 Raut v prostorách Lékařské knihovny na starém městě
(pro všechny registrované účastníky; cena je zahrnuta do
sjezdového poplatku)

9
Thuersday, 30 October 2008
9.00 - 13.10 International Symposium "The Functional Organization of
the Cell Nucleus"
Chair: A. Svoboda
9.05 - 9.30 J. Bartek, C. Lukas, N. Mailand, S. Becker-Jensen, J. Bartkova
and J. Lukas
DNA damage response: Mechanisms, live-cell imaging and
relevance to cancer
9.30 – 9.45 E. Bártová, G. Galiová, J. Krejčí, A. Harničarová, L. Strašák and
S. Kozubek
Chromatin structure of human embryonic stem cells
9.45 - 10.00 J. Bednar, S.H. Syed, D. Anguelov and S. Dimitrov
Nucleosomes with histone variants and the linker histone
incorporation
10.00 – 10.25 S. Ribeiro, J. C. Gatlin, Y. Dong, A. Joglekar, L. Cameron,
D. F. Hudson, B. F. McEwen, E. D. Salmon, P. Vagnarelli and
W. C .Earnshaw
Condensin regulates the stiffness of vertebrate centromeres
10.25 - 10.50 B. Fahrenkrog, D. K. Shumaker, Y. L., T. Shimi, R. D. Goldman
Novel function for a nuclear pore protein in cell cycle control
10.50 - 11.10 Coffee Break
Chair: I. Raška
11.10 - 11.35 R. Foisner, N. Naetar, I. Gotic, U. Pilat, J. Braun
How do mutations in lamins cause disease?
11.35 – 12.00 N. Wiesel, A. Mattout, K. B. Harush, H. Herrmann, U. Aebi,
O. Medalia and Y. Gruenbaum
Laminopathic mutations interfere with the assembly, localization
and dynamics of nuclear lamins in C. elegans
12.00 - 12.15 K. Michalová
Chromosomal deletions and amplifications in malignant cells
12.15 – 12.40 I. Solovei, A. Mardaryev, M. Fessing, Y. Feodorova, S. Kosem,
A. Scharov, V. Botchkarev, T. Cremer, B. Joffe
Establishment of cell type specific nuclear architecture: two cell
types in native tissues
12.40 - 12.55 M. Varecha, P. Matula, M. Kozubek
New approaches to automated cell image acquisition and analysis
using fluorescence microscopy
12.55 - 13.10 Final discussion

13.10 - 14.30 Přestávka na oběd
14.30 – 15.50 Sekce 6
Předsedající: F. Weyda
10
Thuersday, 30 October 2008
14.30 - 14.50 J. Suchánek Foltán
tRNA genes and the genetic code: Presentation of a research
software tool tRNALab
14.50 - 15.10 I. Slaninová, J. Šinkora, E. Táborská
Quaternary benzo(c)phenanthridine alkaloids – new supravital
DNA probes
15.10 - 15.30 M. Červinka, E. Rudolf
Utilisation of time-lapse living cell imaging in the toxicity
assessment and drug discovery
15.30 – 16.20 Přestávka na občerstvení
16.20 - 18.00 Sekce 7 Diskuse u posterů
Předsedající: E. Rudolf
19.00 – 21.00 Slavnostní večeře Nové Adalbertinum
Zájemci o tuto večeři se přihlásí při registraci a zároveň zaplatí
částku ve výši 250,- Kč.

9.00 - 10.40 Sekce 8
Předsedající: J. Krajčovič
11
Pátek, 31. října 2008
9.00 – 9.40 Z. Opatrný
Plant biology, organic farming, GM crops and human health
9.40 – 10.00 B. Koukalová, M. Fojtová, A. Kovařík
Epigenetic changes in dedifferentiated plant cells
10.00 - 10:20 E. Hlinková , R. Tomášová, G. N. Timošenko, S. Vokál,
E. A. Krasavin
Changes in the gene expression and growth processes of peanut
callus culture irradiated with high energetic nuclei
10.20 - 10.50 Přestávka na občerstvení
10.50 – 12.30 Sekce 9
Předsedající: R. Šrám
10.50 – 11.10 T. Soukup, M. Řezáčová, B.Víšek, J. Suchánek, J.Vávrová ,
A.Tichý, L. Kučerová, J. Mokrý
Radiosenzitivity of human dental pulp stem cells
11.10 – 11.30 T. Loja, P. Chlapek, M. Hermanová, I. Zambo, K. Veselý,
K. Zitterbart, J. Štěrba, R. Veselská
The identification of cells with cancer stem cell phenotype in
pediatric solid tumors
11.30 – 11.50 J. Hatina
Cancer stem cells – a new paradigm in tumour biology
11.50 – 12.10 P. Vodicka, R. Kumar, S. Landi, F. Canzian, A. Naccarati, B.
Pardini, L. Vodickova, V. Polakova, E. Tulupova, M. Hanova,
J. Slyskova, A. Foersti, R. Houlston, I.M.P. Tomlinson,
K. Hemminki
Genetic susceptibility to sporadic colorectal cancer
12.30– 12.40 Zakončení XIX. Biologických dnů

SEZNAM PLAKÁTOVÝCH SDĚLENÍ
12

Seznam plakátových sdělení
(abecedně podle jména prvního autora)
E. Babusikova., M. Jesenak, T. .Matakova, J. Hatok, P. Banovcin, D. Dobrota
Characterisation of asthma bronchiale in pediatric patients
V. Bernard, J. Škorpíková, V. Mornstein
The in vitro effect of ultrasound and cisplatin on A2780 and A2780cis cell lines
K. Brigulová, M. Červinka , J. Tošner
Chemoresistence testing of cells isolated from ovarian tumours, effects of freezing
M. Bušová, R. Opatřilová
Food quality in cancerous disease prevention
J. Fukalová, M. Maninová , V. Filimonenko and P.Hozák
Localization of actin-binding proteins in the cell nucleus
E. Halašová, T. Matáková, Ľ. Mušák, Ľ. Javorka, M. Halaša, E. Bukovská
Polymorphisms of GSTM1, GSTT1 and GSTP1 genes in relation to chromosomal damage in
workers exposed to chromium
A. Harničarová, E. Bártová, S. Kozubek
Structure and function of promyelocytic leukaemia nuclear bodies
J. Hochmann
Exploitation of the telephonic mobile apparatus for photography of education preparations in
biology
J. Hochmann
Influence of estrogen and progesterone receptors (ER and PR) on the survival in case of
breast cancer – and questions of exactness of examination of these markers
J. Ipser, P. Češková
Genotoxic effects of Mylecytan in Drosophila melanogaster
J. Ipser, V. Matoušová, P. Češková
Genotoxic effects of Vincristin in Drosophila melanogaster
M. Korabečná , A. Horinek , A. Panczak , S. Opatrna, J. Wirth, F. Sefrna, P. Calda
Concentrations of total cell-free DNA in plasma as potential marker in clinical medicine
D. Krajčí, C. Pellicciari, M-G. Bottone, V. Lisá, V. Mareš
Cell death and survival in astrocytoma cultures after Cisplatin. An EM and cytochemical
study.
13

Kufner, P.*, Hájková, L.*, Reischig, J. (* equally contributed authors)
Melatonin protects mitochondrial transmembrane potential from the effect of antimycin A in
normal as well as cancer cell lines
J. Kůsová, M. Kašová, H. Tomášková
Chromosomal aberrations as a tool of prevention at workers exposed to mutagens and
carcinogens
J. Kůsová, H. Miturová, M. Kašová, H. Tomášková
Biological and chemical evaluation of Ostravian urban air
V. Mareš, J. Burian , V. Lisá, M. Marek, I. Tomandl
Cell death, stress and regeneration induced by Boron-Neutron-Capture Reaction (BNCR) in
situ and in culture
T. Matáková,, E. Halašová, M. Sivoňová, E. Huľo, Ľ. Javorka, P. Žúbor, D. Dobrota
Polymorphisms of GSTM1, GSTT1 and GSTP1 genes in relation to breast cancer
susceptibility.
L. Mikalová, M. Strouhal, P. Matějková, D. Šmajs
Genome differences in the genus Treponema
R. Molíková, A. Šantavá, M. Godava, O. Šmakal, J. Šantavý, M. Bezdičková, O. David
Current findings on genetic regulation of the uropoetic system in fetus and its share in the
incidence of reflective uropathy
P. Nádvorník, V. Pavel, L. Kučerová, S. Bureš
11 DNA microsatellites for the study of paternity in the meadow pipit (Anthus pratensis)
E. Ondroušková, J. Slováčková, V. Pelková, J. Procházková, K. Souček, P. Beneš,
J. Šmarda
Heavy metals induce phosphorylation of the Bcl-2 protein by Jun N-terminal kinase
O. Pavlis, M. Pohanka, J. Pikula, F. Treml, K. Kuca
Antidotes against nerve agents versus tularemia disease progress
M. Pohanka, O. Pavliš, P. Skládal
Tularemia diagnosis using piezoelectric immunonosensor
M. Pohanka, F. Malir, K. Kuca, T. Roubal
Detection of aflatoxins in capsicum spice using an electrochemical immunosensor
K. Rudolf, E. Rudolf, M. Červinka
The involvement of mitochondrial pathway in etoposide-induced demise of melanoma cells
L. Schröterová, P. Hašková, E. Rudolf, M. Červinka
Natural products in chemoprevention of colon cancer
14

I. Slaninová, J. Slanina, I. Tomalová, L. Březinová, M. Broošová, L. Koubíková, K. Krestová
Lignans from Schisandra chinensis restore the cytotoxic action of doxorubicin in drug
resistant lung cancer cells
R. Štětina, M. Jílková
The induction of inter-strand DNA cross-links with sulphur mustard in normal Chinese
hamster cells AA8 and their DNA repair defective mutants UV5 and UV-20
F.Weyda
High speed digital photography with consumer digital camera Casio Exilim Pro EX-F1:
Examples of applications in biology
Z. Žižka, J. Gabriel
Autofluorescence of fruiting bodies of the wood-rotting fungus Piptoporus betulinus
15

SOUHRNY ÚSTNÍCH SDĚLENÍ
16

Souhrny ústních sdělení
(chronologicky)
Stem cells in oncology (Babak's lecture)
Č. Altaner
Cancer Research Institute, Slovak Academy of Sciences, Bratislava
Involvement of stem cells in tumor induction is stressed by existence of cancer stem cells.
According to cancer stem cell hypothesis only a small population of cells with stem cell
characteristics created by mutation events from normal stem cells has the ability to initiate
tumors. Through asymmetric cell division and subsequent aberrant differentiation of
proliferating cellular progeny, heterogeneous cell types comprise a tumor. Addition mutations
and epigenetic changes lead to polyclonal tumor formation in which minor population of
cancer stem cells is persisting. When such a tumor is treated by cytotoxic chemotherapy or
radiotherapy, proliferating cells are killed, but cancer stem due to their properties remain and
can form tumors and metastases many years after the treatment. It is believed that therapy
directed to cancer stem cells may bring improvement in causative tumor treatment.
In the search for more targeted tumor therapy we study the possibility to use mesenchymal
stem cells derived from human adipose tissue for suicide cancer gene therapy. Some our
published results (Cancer Research 2007, 67, 6304-6313, J Gene Med. 2008 10, 1071-1082)
and new developments in this direction will be discussed. Stem cell based gene cancer
therapy seems to be a promising approach, which may target also the cancer stem cells.
17

Biomarkers of exposure and effect – interpretation for human risk
R. J. Sram, B. Binkova, O. Beskid, A. Milcova, P. Rossner, P. Rossner, Jr.,
A. Rossnerova, I. Solansky, J. Topinka
Institute of Experimental Medicine AS CR, v.v.i., 142 20 Prague, Czech Republic
Background. The capital city of Prague becomes one of the most polluted localities of the
Czech Republic. Therefore, the effect of exposure to carcinogenic polycyclic aromatic
hydrocarbons (c-PAHs) adsorbed onto respirable air particles (PM2.5,

Oxidative stress detection in genetic toxicology
P. Rossner, Jr., V. Svecova, A. Milcova, Z. Lnenickova, R. J. Sram
Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR, v.v.i.,
Vídeňská 1083, 142 20 Prague, Czech Republic
Air pollution is associated with many negative health effects, including increased morbidity
and mortality. Particulate matter is a significant component of polluted air. It consists of a
mixture of various chemicals, among them reactive compounds such as quinones, toxic
metals, and benzene, and carcinogenic polycyclic aromatic hydrocarbons may induce
oxidative stress. Oxidative stress, mediated by reactive oxygen species is a process
characterized by the imbalance between pro-oxidants and antioxidant defense of the
organism. Oxidative stress may result in direct DNA damage, lipid peroxidation, protein
oxidation, mitochondrial damage, or membrane disruption. Since the process is complex,
several oxidative stress markers should be analyzed at a time to get a better understanding of
the reactions taking place within the organism. However, for the efficient application of these
markers in molecular-epidemiological studies where large sample sizes are often analyzed the
availability of high-throughput methods is essential.
8-oxodeoxyguanosine, the most abundant and most often studied product of oxidative DNA
damage, is highly mutagenic and if not repaired its presence results in GC>TA transversions.
If repaired, 8-oxodG is excreted in urine. Measurement of 8-oxodG levels in cells, tissues, or
urine is considered a general biomarker of oxidative stress. F2-isoprostanes, compounds
derived from arachidonic acid via a free radical-catalyzed mechanism, belong among
extensively used markers of lipid peroxidation. While several groups of F2-isoprostanes exist,
most studies concentrated on the biological activity of 15-F2t-isoprostane. Isoprostanes are
initially generated from cell membrane-bound arachidonic acid by free radical attack. They
are cleaved from the sites of their origin by phospholipases and then circulate in plasma and
are excreted in urine. F2-isoprostanes can be detected in biological fluids, such as urine, blood
plasma, bronchoalveolar lavage, or cerebrospinal fluid, as well as in tissues. The main
advantage of urinary measurements of F2-isoprostanes is that the compounds are very stable
and are not formed ex vivo. Protein carbonyl levels are the most frequently used biomarker of
protein oxidation. Their accumulation has been observed with aging and in several human
diseases, including cancer. Protein oxidation may occur on amino acid side chains, or protein
backbones and results in changes of protein structure or inactivation of proteins.
The presentation will concentrate on a description of induction of oxidative damage to
macromolecules and detection methods used in molecular epidemiology. Results obtained in
studies conducted in our laboratory will be presented.
Acknowledgements: Supported by the Czech Ministry of the Environment (VaV-SL/5/160/05
and SP/1b3/50/07) and by the Academy of Sciences of the Czech Republic (AVOZ50390512
and 1QS500390506).
19

Chromosomal damage and polymorphisms of DNA repair genes XPD, XPG, XPC
hOGG1, XRCC1 and XRCC3 in workers exposed to chromium.
E. Halašová 1 , T. Matáková 2 , Ľ. Mušák 1 , Ľ. Javorka 3 , M. Halaša 4 , E. Bukovská 1
1
Institute of Medical Biology, Comenius University in Bratislava, Jessenius Faculty
of Medicine in Martin
2
Institute of Medical Biochemistry, Comenius University in Bratislava, Jessenius Faculty
of Medicine in Martin
4
Department of Childbirth Asistance Faculty of Health Catholic University in Ružomberok
Welders have chronically been exposed to hexavalent chromium with potential consequences
on chromosomal integrity. Our study is focused on the level of chromosomal aberrations with
respect to chromium level in the blood of welders as well as on the tentative modulating role
of polymorphisms in DNA repair genes XPD Lys751Gln, XPG Asn114His, XPC Lys939Gln,
hOGG1 Ser326Cys, XRCC1 Arg399Gln and XRCC3 Thr241Met on chromosomal damage.
The study was performed on 41 welders that have been exposed to chromium for 10.2±1.67
years, and 31 control individuals. Conventional cytogenetic analysis was employed for
detection of CAs.
XPD, XPG, XPC, hOGG1 and XRCC1 polymorphisms were assayed for by Taqman SNP
genotyping assay (”Assay-by –Demand”) using Real-Time allelic discrimination on AB 7500
equipment.
Chromium analysis in the blood was performed using the atomic absorption
spectrophotometer.
Higher frequencies of CAs were detected in exposed individuals than in controls (1.96 %
versus 1.55 %, respectively), but this difference was not significant. In the exposed group the
chromosomal damage consisted predominantly of chromosomal-type of breaks (CSAs;
1.03%), which were approximately two-fold higher as compared to the controls (0.55%). The
frequency of chromatid-type breaks was similar in both exposed and control groups (0.92%
vs. 1.00%).
Higher pooled CAs was detected in individuals with homozygous wild type polymorphisms in
hOGG1 Ser326Cys as compared to those with heterozygous and homozygous variant
genotype (1.83% and 1.57% respectively). After the stratification of the cohort, within control
individuals we observed not quite significantly higher frequency of CA associated with wildtype
Ser allele in hOGG1 Ser326Cys (1.71% and 1.20% respectively; P=0.060).
CAs frequencies were the highest in individuals with wild-type Lys/Lys XPD Lys751Gln
genotype. Whether this tendency reflects the true modulating effect of the particular genotype
remains unclear.
Significantly higher pooled CAs was detected in individuals with homozygous wild type
polymorphisms in XRCC1 Arg399Gln gene as compared to those with heterozygous and
homozygous variant genotype (1.33% 1.80% and 2.14% respectively).
This work was supported by the grants of Ministry of Education of Slovak Republic: VEGA
1/3397/06 and of Ministry of Health of Slovak Republic: MZ SR 2007/48-UK-13.
20

Modulation of the antineoplastic effect of retinoids in neuroblastoma and
medulloblastoma cell lines
M. Rédová 1 , P. Chlapek 1 , T. Loja 1 , K. Zitterbart 2 , M. Hermanová 3,4 , J. Štěrba 2 , R. Veselská 1,2
1
Laboratory of Tumor Biology and Genetics, Institute of Experimental Biology, School of
Science, Masaryk University, Brno, Czech Republic
2
Department of Pediatric Oncology, University Hospital Brno, Czech Republic
3 Institute of Pathology, University Hospital Brno, Czech Republic
4 Institute of Pathologic Anatomy, St. Anne’s University Hospital, Brno, Czech Republic
Induced differentiation of transformed cells into mature phenotypes represents one of the
most promising strategies of recent antitumor therapy. Retinoids belong to the frequently used
inductors of differentiation. In the past few years, the attention has been paid also to the
possibilities of combined induction of differentiation as well as to the modulation of
differentiation effects by other compounds. This therapeutic approach was reported especially
in leukemia treatment. Furthermore, this strategy was also demonstrated as effective in
children with various types of relapsed solid tumors Retinoid-induced differentiation may be
influenced - among others - by inhibitors of enzymes that participate in the intracellular
degradation pathway of retinoids, especially of lipoxygenases (LOX) and cyclooxygenases
(COX). Our study is focused on the detailed examination of the effect of combined
application of the LOX/COX inhibitors with ATRA in established neuroblastoma cell lines
(SK-N-BE(2) cell line and SH-SY5Y cell line) and medulloblastoma cell lines (Daoy cell line
and D341 Med). Caffeic acid (CA) as inhibitor of 5-LOX and celecoxibe (CX) as inhibitor of
COX-2 were used in this study. In this presentation, we summarize our results on changes in
differentiation course / cell morphology and in cell proliferation after treatment with various
combinations of the ATRA and LOX/COX inhibitors.
This study was supported by grants IGA NR9341-3/2007, GACR 204/08/H054, and VZ
MSM 0021622415.
21

In vitro genotoxicity of complex mixtures extracted from urban air particles
J. Topinka, B. Binkova, O. Sevastyanova, Z. Novakova, R. J. Sram
Department of Genetic Ecotoxicology, Institute of Experimental Medicine, AS CR, v.v.i.,
Vıdenska 1083, 142 20 Prague 4, Czech Republic
Investigation of the biological effects of ambient air particulate matter has involved a number
of different approaches, including the studies of particle induced genotoxicity. The latter was
shown to be related to chemical compounds bound onto the particles or to particles
themselves. Some studies suggest that genotoxic effect of the particulate matter is due to
polycyclic aromatic hydrocarbons (PAHs) and their derivatives present in the organic fraction
of PM while other studies indicate that some metals, forming PM can catalyze reactions
resulting in oxidative stress and DNA damage. A wide variety of in vitro systems was
developed in order to study the genotoxicity of chemicals and their mixtures, including
complex mixtures of environmental pollutants adsorbed onto respirable air particles (

Tularemia disease modulation using cholinesterase reactivator HI-6
M. Pohanka 1,2 , O. Pavlis 3 , J. Pikula 4 , F. Treml 5 , Jan Marek 1,2 and K. Kuca 1,2
1
Centre of Advanced Studies, Faculty of Military Health Sciences, University of Defence,
Hradec Kralove, Czech Republic
2
Department of Toxicology, Faculty of Military Health Sciences, University of Defence,
Hradec Kralove, Czech Republic
3
Centre of Biological Defence, Techonin, Czech Republic
4
Department of Veterinary Ecology and Environmental Protection, University of Veterinary
and Pharmaceutical Sciences Brno, Czech Republic
5
Department of Infectious Diseases and Epizootiology, University of Veterinary and
Pharmaceutical Sciences Brno, Czech Republic
Cholinesterase reactivator HI-6 is a drug commonly used to treat individuals exposed to nerve
agents. This experimental work has been engaged with studying modulation effects of HI-6
on infection progress. We used BALB/c mice and the causative agent of tularemia, i.e.,
Francisella tularensis, to induce a model bacterial infection. Cultivation tests confirmed
bacteriostatic effects of HI-6. In vivo experiments revealed intriguing effect differences
resulting from HI-6 administration to mice. While the HI-6 dose of 7 mg pro toto induced no
statistically significant effects on infection progress, the much lower dose of 8 µg of HI-6 pro
toto clearly reduced mortality caused by tularemia infection in experimental mice as
compared to only F. tularensis-infected controls (comparison of survival curves using the
logrank test, chi square = 4.335, df = 1, P = 0.0373) . The chemical structure of HI-6
reactivator and expected roles of its organic groups are also discussed in the present study.
Supported by the Ministry of Defense of the Czech Republic (Grant No. FVZ0000604).
23

Bacteriocin types produced by Escherichia coli strains isolated from human and animal
samples
D. Šmajs 1 , J. Šmarda 1 , M. Vrba 2 , A. Ševčíková 2
1 Department of Biology, Masaryk University, Faculty of Medicine, Brno, Czech Republic
2 Department of Clinical Microbiology, Faculty Hospital Brno, Czech Republic
Bacteriocin production was tested in a set of 649 E. coli strains isolated (i) from
gastrointestinal tract (GIT) of healthy persons (195 strains), (ii) from piglet diarrheal feces
(183 strains), (iii) from human urinary tract infections (195 strains), and (iv) from human
extraintestinal inflammatory samples (78 strains). Altogether, 24 colicin and 7 microcin types
were tested in 356 producer strains. In 332 out of 356 producer strains, at least one bacteriocin
type was identified (93.3%). The incidence of colicinogenic strains in individual groups
ranged from 34.6% to 35.9% with the exception of animal samples, where colicinogenic
strains represented 64.5% of investigated strains. The incidence of microcin producing strains
ranged from 14.8% to 26.4% with the exception of strains isolated from human inflammatory
samples outside GIT. In this group, the incidence of microcin producing strains was
significantly higher (48.7%). E. coli strains of animal origin showed high proportion of strains
co-producing three or more colicin types as well as high incidence of strains co-producing
colicins B and M. In addition, few colicin Ia producing strains only were found among animal
strains. Strains isolated from human urinary tract infections showed high incidence of colicin
E1 and colicin N producing strains, respectively. E. coli strains isolated from human
inflammatory samples outside GIT revealed high incidence of colicin E1 and microcin H47
producing strains, respectively. High incidence of colicin E1 production appears to be an
important characteristic of human pathogenic E. coli strains.
This work was supported by Ministry of Education of the Czech Republic Research Project
MSM0021622415.
24

Hypotheses for the origin of eukaryotic cytoskeleton
M. Vesteg
Institute of Cell Biology, Faculty of Natural Sciences, Comenius University, Mlynská dolina,
842 15 Bratislava, Slovakia
e-mail: vesteg@fns.uniba.sk
Several hypotheses for the origin of eukaryotic cytoskeleton exist. These hypotheses are
based on the hypotheses for the origin of eukaryotes. Many of these suggest that prokaryotes,
their symbiotic associations or chimeras were direct ancestors of eukaryotes. According to
these hypotheses prokaryotic proteins were ancestral to eukaryotic cytoskeletal proteins.
However, these hypotheses have various serious pitfalls. As archaea share with eukaryotes
more molecular characteristics than with bacteria, most of these hypotheses suggest that an
archaeal cell was involved in the origin of eukaryotes either as a host cell or as a symbiotic
precursor of the nucleus. However, archaea have different membrane lipids than eukaryotes
and bacteria. Hypotheses postulating prokaryotic origin of eukaryotes (with an archaeal cell
involved) have to propose selectively costly transition of archaeal membrane lipids to
bacterial through an unstable stage of bacterio-archaeal lipid mixture. In addition, these
hypotheses do not explain the origin of specific eukaryotic proteins and processes which have
no homology or even analogy in prokaryotes. Sexual reproduction specific for eukaryotes has
serious consequences for the evolution of eukaryotes and there is no reason to think that it
evolved in an asexual evolutionary stable prokaryotic organism. Moreover, phylogenetic
analyses of slowly evolving genes such as genes encoding ribosomal RNAs and proteins
reveal that eukaryotes are sisters of archaea and do not place eukaryotes within the diversity
of modern archaea. Thus it is possible that eukaryotes are remnants of pre-cellular world
(before the diversification of three domains of life) dominated by lateral gene transfer
mediated by viruses or fusions and fissions of pre-cells. Many eukaryotic cytoskeletal
proteins thus might be traceable to the pre-cell period before diversification of domains. Even
the mobile elements (e.g. viruses) might have themselves used these proteins for segregation
or packing of their genomes while infecting pre-cells.
25

Evidence of cross-talk between Aryl Hydrocarbon Receptor and Glucocorticoid
Receptor in placental trophoblast cells
L.Stejskalová 1 , K.Pospěchová 1 , L. Švecová 1 , M. Bitman 1 , R. Vrzal 2 , Z. Dvořák 2 , P. Pávek 1,3
1 Department of Pharmacology and Toxicology Charles University in Prague; Czech
Republic, Faculty of Pharmacy in Hradec Králové, Heyrovského 1203, Hradec Králové),
2 Department of Medical Chemistry and Biochemistry, Medical Faculty, Palacky University
in Olomouc
3
corresponding author
Aryl Hydrocarbon Receptor (AhR) and Glucocorticoid nuclear receptor (GR) play crucial role
in regulation of drug metabolizing enzymes and in many essentials physiological processes.
CYP1A1 isoform of cytochrome P450 is important biotransformation enzyme which
metabolizes several drugs widely used in pharmacotherapy. On the other side this enzyme
plays a key role in bioactivation of procarcinogens and proteratogens such as polycyclic
aromatic hydrocarbons (PAHs) to form DNA-adducts.
Expression of CYP1A1 is transcriptionally regulated through ligand-activated Aryl
hydrocarbon receptor. Glucocorticoids are reported to modulate the induction of CYP1A1 via
activated AhR.
The aim of this work was to study the cross-talk between these two nuclear receptors in
transactivation of CYP1A1 in placental JEG3 cell line exposed to the prototype AhR ligand
methylcholanthrene (MC) alone and in combination with glucocorticoid dexamethasone.
The effect of dexamethasone on MC-mediated transactivation was assessed employing gene
reporter assay in transiently transfected cells with two gene reporter plasmids containing
promoter or responsive sequences of CYP1A1 gene and cotransfected with expression
plasmids for GR, AhR and ARNT (AhR nuclear translocator). In addition, nuclear
translocation of AhR/ARNT was monitored using Western blotting.
Our preliminary results show that 24-h treatment with dexamethasone decrease AhRmediated
transactivation of CYP1A1 gene in gene reporter assay with both p1A1-luc and
pXRE-luc plasmids. We show that the phenomenon is likely due to decreased nuclear
translocation of AhR after treatment with dexamethasone. Based on out preliminary data, we
can suggest cross-talk of GR/AhR pathways in gene regulation of CYP1A1 in placental
trophoblast, which is not likely at the level of transcriptional regulation.
ACKNOWLEDGEMENT
This project was supported by the grant number GAČR 303/07/0128 (P.P.).
26

Polymorphism of GSTs genes in relation to chromosomal damage in boot workers
T. Matáková 1 , E. Halašová 2 , A. Dzian 3 , L. Javorka 4 , D. Dobrota 1
1
Department of Medical Biochemistry, Comenius University, Jessenius Faculty of Medicine,
Martin
2
Department of Medical Biology, Comenius University, Jessenius Faculty of Medicine,
Martin
3
Clinic of Surgery, Comenius University, Jessenius Faculty of Medicine, Martin
Employees in the footwear manufacturing industry are routinely exposed to complex mixtures
of solvents used in cleaning and as diluents in glues, primers, and degreasers.
The objective of this study was to determine the genotoxic effects in a group of footwearworkers
occupationally exposed to solvent-based adhesive and solutions containing organic
solvents, mainly toluene. Cytogenetic analysis of peripheral blood lymphocytes was used to
compare 40 shoe workers (14 men and 26 women) and 31 subject selected from general
population not exposed to particular mutagenic or carcinogenic agents (control group).
Frequencies of damaged cells, including breaks and rearrangements were scored for both
groups. Polymorphisms in genes GSTT1, GSTM1, and GSTP1 were used as susceptibility
biomarkers. The polymorphisms of GSTM1, GSTT1 and GSTP1 enzymes were determined
by PCR-based methods.
The exposed group showed a very significant increase of genetic damage (P

Individual DNA repair capacity: from the genetic background to phenotypic expression
R. Štětina 1 , P. Vodička 2 , M. Hánová 2 , J. Varvařovská 3 , L. Pácal 4 , K. Kaňková 4
1
Department of Toxicology, Faculty of Military Health Sciences, Hradec Králové, Czech
Republic
2
Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague
3
Pediatric Department, Charles University Hospital, Plzeň, Czech Republic
4
Department of Pathological Physiology, Medical Faculty, Masaryk University, Brno, Czech
republic
We have studied the DNA repair capacity in peripheral lymphocytes of healthy individuals in
a central European population. The influence of polymorphisms in different DNA repair
genes on the level of single-strand breaks (SSBs) and on DNA repair capacity was analysed
both in normal healthy population and in patients with Diabetes melitus type I and in
individuals occupationally exposed to styrene. The DNA repair capacity was estimated 1) by
measuring the rate of repair of single strand DNA breaks (SSB) in peripheral lymphocytes
induced by γ-irradiation 2) by measuring the rate of incision of DNA of target cells (HeLa)
containing 8-oxoguanine (induced with Ro19-8022 + light) during their incubation with
extracts from lymphocytes of tested persons. The DNA damage was estimated using the
modified comet assay.
We have found significantly decreased γ-iradiation-specific DNA repair (0.45 ± 0.47
SSB/10 9 Da) in the group of healthy individuals with XRCC1 Arg399Gln homozygous
variant genotype and with hOGG1 Ser326Cys homozygous variant genotype (0.37 ± 0.28
SSB/10 9 Da) in comparison to wild-type genotype (0.83 ± 0.79 SSB/10 9 Da, P = 0.008).
APE1 Asn148Glu polymorphisms also caused a significant decrease of γ-irradiation-specific
repair rates (P = 0.008). Results show that XRCC1 Arg399Gln and hOGG1 Ser326Cys
polymorphisms seem to exert the predominant modulating effect on γ-irradiation-specific
DNA repair capacity and the capacity to repair DNA oxidative damage, respectively.
Diabetes mellitus (DM) is known to be accompanied by oxidative stress. We have found that
the capacity to repair the oxidative damage of the DNA was significantly increased in diabetic
children (p

Antiproliferative effects of zinc in colon cancer cell lines
S. John, L. Klvačová, M. Červinka, E. Rudolf
Department of Medical Biology, Charles University in Prague, Faculty of Medicine in Hradec
Králové, Hradec Králové, Czech Republic
Zinc is a bioelement involved in many aspects of cellular physiology. Pertubations in zinc
metabolism due to changes in its external or internal concentrations may seriously affect
cellular growth, proliferation and signaling, ultimately leading to cell cycle arrest and cell
death. Colon cancer is among the leading types of cancers in western populations, and in
particular in the Czech Republic. Several studies have shown the involvement of zinc in
colorectal carcinogenesis. Thus the aim of this study was to investigate a potential
chemopreventive role of externally supplemented zinc on growth and proliferation of colon
cancer cell lines representing different stages of this malignancy: HCT-116, HT-29 and
SW620. The results suggest that that there are differences in sensitivity of colon cancer cells
to zinc and that zinc inhibits cell growth and proliferation possibly via several specific and
nonspecific mechanisms including cytoskeleton and cell cycle checkpoints.
This work was supported by GAUK No. 132808.
29

Effect of sodium selenite on colorectal cell lines with different p53 status
V. Králová, K. Brigulová, M. Červinka, E. Rudolf
Department of Medical Biology, Charles University in Prague, Faculty of Medicine in Hradec
Králové, Hradec Králové, Czech Republic
Epidemiological studies have shown an inverse association between selenium levels and
cancer risk in humans, including colorectal cancer. Sodium selenite is a form of selenium
commonly used in dietary supplements and it has been reported to inhibit proliferation and
induce cell death in several types of cancer cells in vitro. In our study we evaluated effects of
sodium selenite on cell proliferation, cell cycle and cell death in colorectal cell lines HCT 116
(p53 wild type) and HCT 116-p53KO. Cell morphology and proliferation was folowed
directly by time-lapse videomicroscopy, indirectly as metabolic activity (WST-1 assay),
protein content ( Brilliant Blue assay), membrane integrity (Neutral red assay) and DNA
synthesis ( BrDU fluorescent staining). Effects of sodium selenite on cell cycle were
determined by analysis of cell cycle distribution, by combined cyclin A2 / 7-AAD staining
and by western blotting. Cell death was measured as the sub G1 fraction of the cell cycle and
by combined annexin V / propidium iodide staining.
We show that sodium selenite inhibits proliferation and induces cell death in both HCT116
and HCT116-p53KO lines. The damaged selenite-treated cells showed typical morphological
changes with intensive vacuolisation. The process was accompanied by decrease in cyclin D1
concentration, increase in cyclin B1 concentration and increase in the number of cyclin A2
positive cells, which suggests accumulation of cells in the S/G2 phases of the cell cycle. HCT
116-p53KO cells showed significantly greater tendency to S/G2 block than the p53 wild type
HCT 116 cell line. Sodium selenite increased the fraction of annexin V / PI positive cells in
both cell lines. It also increased the sub G1 fraction of the cell cycle and this effect was more
profound in HCT 116 p53 wild type cells. These results suggest that sodium selenite inhibits
proliferation of HCT 116 cell lines in a time- and dose- dependent manner by arresting cells
in S/G2 phase of the cell cycle and by inducing cell death and that this process is influenced
by the p53 status of the cell lines.
This work was supported by Ministry of Education of the Czech Republic Research Project
MSM0021620820.
30

Danger of Ethidium Bromide and its´ Safe Alternatives
J. Prochazkova 2 , Z. Fiedler 1,2
1
Charles University in Prague, Faculty of Medicine in Hradec Kralove, Dept Med Biol
Genet, Hradec Kralove, Czech Republic
2
University Pardubice, Faculty of Chemical Technology, Dept Biol Biochem Sci, Pardubice,
Czech Republic
Ethidium bromide (3,8-Diamino-5-ethyl-6-phenylphenanthridinium bromide) is an intercalating
agent commonly used as a nucleic acid stain in molecular biology laboratories for techniques
such as agarose gel electrophoresis and for the polyacrylamide gel staining. Being a strong
mutagen is involved in the complex waste disposal issues. The mutagen potential of the
ethidium bromide described in the literature is summarized.
Experimentally the amount of the ethidium bromide eluted into the electrophoresis buffer
after the routine separation run was determined (2,8 μg per mL of the electrophoresis buffer).
According to the previous data and to the number of the molecular laboratories, potential risk
for the environment stress in some czech cities (Brno, Ceske Budejovice, Praha, Hradec
Kralove) was estimated.
SYBR Green I, SYBR Green II, Mega Fluor, SYBR Gold, SYBR Safe and Goldview are
considered to be a so called safe replacement for the ethidium bromide and are free from the
complex waste disposal issues mentioned above. Nevertheless, some authors published in
1999 and 2001 the experimental data impeaching the safety of these cyanine dyes. We discuss
the safety of the SYBR Green I in comparison with the ethidium bromide and we do propose
the similar waste disposal for the both dyes.
This work was supported by Ministery of Education (MSMT 0021627502).
31

DNA damage response: Mechanisms, live-cell imaging and relevance to cancer
J. Bartek, C. Lukas, N. Mailand, S. Becker-Jensen, J. Bartkova and J. Lukas
Danish Cancer Society, Copenhagen, Denmark
To protect the genome against genotoxic insults, eukaryotic cells evolved surveillance
pathways called checkpoints to coordinate cell cycle progression with repair of the DNA
lesions, resetting the epigenetic marks on the neighboring chromatin, or induction of cell
death. The lecture will first provide some introductory background information on the
significance and mutual coordination of the mechanisms contributing to genome integrity
maintenance. Next, I will provide examples of our recent work on key checkpoint-associated
molecular pathways operating in their physiological environment – in the nucleus of a living
cell, using real-time imaging and laser technology. These examples will focus on our most
recent discovery of a DNA damage signalling pathway through multiple protein
ubiquitylation steps, a new concept we refer to as ‘The ubiquitylation chain reaction’. The
data will be discussed in a broader, interdisciplinary context, including a brief update on the
current status of our concept of the DNA damage response machinery as an inducible
biological barrier against progression of early stages of human tumours in vivo, and in
response to oncogenes in cell culture models. The lecture will be concluded by a general
outline of the intimate involvement of the DNA damage machinery in human physiology and
the impact of such emerging knowledge for individualized management of human diseases,
particularly cancer.
32

Chromatin structure of human embryonic stem cells
E. Bártová, G. Galiová, J. Krejčí, A. Harničarová, L. Strašák and S. Kozubek
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská
135, CZ-612 65, Brno, Czech Republic.
Human embryonic stem cells are characterized by their capacity for sustained self renewal
and their ability to differentiate into the specific cell types of all three germ layers. These
qualities make hESCs promising therapeutic tools in regenerative medicine. Here, we have
studied chromatin structure, nuclear radial arrangement of selected genetic elements and
histone epigenetic patterns in human embryonic stem cells (hESCs) before and after retinoic
acid-induced differentiation.
The genome and epigenome of hESCs underwent differentiation-specific structural changes,
characterized by the redistribution of centromeric heterochromatin, as evidenced by a
perinucleolar accumulation of the centromeric markers CENP-A and H3K9 trimethylation. In
majority of cases, the nuclear patterns of heterochromatic structures and markers, such as
centromeres, inactive chromosome X, H3K9me3, and HP1 protein, were distinct in
pluripotent hESCs, when compared to differentiated cells. Studying the Oct3/4 gene,
responsible for hESC pluripotency, we observed that one or both alleles of Oct3/4 gene can be
located on greatly extended chromatin loops, outside their respective chromosome territories.
However, global gene-density-related radial compartmentalization of chromosome territories
was already largely established in undifferentiated hES cells. This also holds for nuclear
radial distribution of Oct3/4 gene in pluripotent and differentiated hESCs. However, in
differentiated cells Oct3/4 was positioned on the periphery of related chromosome territory.
The results of our experiments showed distinctions between chromatin arrangement in
pluripotent and differentiated hESCs and point out an important nuclear structures and
chromatin marks which are potentially responsible for hESC pluripotency.
We thank the laboratory of Prof. Douglas Melton (HHMI/Harvard University) for providing
us with hES cell lines. This work was supported by the following grants: AVOZ50040507,
IQS500040508, LC535 and 204/06/0978.
33

Nucleosomes with histone variants and the linker histone incorporation
J. Bednar, S.H. Syed 1 , D. Anguelov 1 and S. Dimitrov 1,2
Institute of Cellular Biology and Pathology, First Faculty of Medicine, Charles University in
Prague, and Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i.,
Prague, Czech Republic
1 Laboratoire Joliot-Curie E.N.S Lyon, Lyon, France
2 Institut Albert Bonniot, INSERM U309, La Tronche, France,
The linker histone is the keystone of the structure of the nucleosomal particle and of the
higher order chromatin organization. It is also the most problematic element in the procedure
of chromatin reconstitution in vitro. Due to its highly positively charged C-terminal domain it
exhibits strong, nonspecific affinity towards the DNA which makes its correct, in vivo-like
association to the nucleosomal core particle (NCP) difficult. Very recent identification of the
NAP-1 protein as a native chaperon of the H1 linker histone eased substantially this
procedure. Using cryo-electron microscopy and DNase I and hydroxyradical footprinting we
have studied the involvement of individual domains of H1 in formation of the characteristic
structure at the nucleosomal DNA entry/exit site – so called stem structure - and a capacity of
the NAP-1 to associate them with the NCP.
Three consecutive NCPs (referred as tri-NCP henceforth), were first reconstituted on the
DNA construct containing three tandem repeats of 601 nucleosomal positioning sequence
separated by 50 bp long spacer. Than either complete histone H1 (226 aa) or its truncated
mutants were associated to the tri-NCPs using the Nap-1 system.
Association of the complete histone H1 led to closing of originally opened structure of tri-
NCPs and to formation of the stem structure indistinguishable from the one observed in native
nucleosomes. Association of the H1 globular domain only (residues 35-120) did not influence
the original opened tri-NCP structure, however, the footprinting experiments proved well its
presence at the dyad of the NCPs. This showed that Nap-1 was able to associate the isolated
globular domain to NCP, however, the globular domain alone was not able to associate the
linker DNA and to close the nculeosomal structure. The situation changed when the mutant
H1D100 (residues 1-127)) was associated. Additional 7 residues at the C-terminal were
enough to accomplish the association of the linker DNA and to close the structure, on the
other hand, the formation of the stem structure was not observed. This was confirmed by the
footprinting where none or weak protection of linker DNA was observed. The association of
the mutant H1D50 (residues 1-177) nearly completely recovered the characteristic
nucleosomal configuration, including the stem formation. Although the Nap-1 is very
efficient in H1 association to conventional NCPs the experiments with tri-NCPs containing
the histone H2A variants H2AL2 and H2A.Bbd showed, that their original relaxed structure
remained unchanged after reaction with Nap-1/H1 complex suggesting that either Nap1 is not
capable to associate H1 to such NCPs or that, although correctly associated, H1 is not capable
to close the structure of variant histone containing nucleosomes.
This work was supported by the Grant Agency of the Czech Republic (Grant #304/05/2168),
the Ministry of Education, Youth and Sports (MSM0021620806 and LC535) and the
Academy of Sciences of the Czech Republic (Grant #AV0Z50110509).
34

Condensin regulates the stiffness of vertebrate centromeres
S. Ribeiro, J. C. Gatlin 1 , Y. Dong 2 , A. Joglekar 1 , L. Cameron 1 , D. F. Hudson 1,3 ,
B. F. McEwen 2 , E. D. Salmon 1 , P. Vagnarelli and W.C.Earnshaw
Wellcome Trust Centre for Cell Biology, University of Edinburgh, Mayfield Road
Edinburgh EH9 3JR, U.K.
1 Department of Biology, University of North Carolina, Chapel Hill, NC 27599, USA
2 Wadsworth Center, New York State Department of Health, Albany, New York 12201, USA
3 Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne 3052, AUS
When chromosomes are aligned and bi-oriented at metaphase, the elastic stretch of
centromeric chromatin opposes pulling forces exerted on sister kinetochores by the mitotic
spindle. This produces tension at kinetochores that is important for maintaining chromosome
alignment, stabilizing kinetochore microtubule (kMT) attachments and controlling the spindle
checkpoint. Here we show that condensin is an important regulator of centromere stiffness
and function. Condensin depletion accomplished by shutoff of a regulated SMC2 cDNA in
DT40 conditional knockout cells decreases the stiffness of centromeric chromatin by 50%
when pulling forces are applied to kinetochores. Complementation experiments reveal that
centromeric stiffness requires the ATPase activity of SMC2. However, condensin is
dispensable for the normal level of compaction (rest length) of centromeres. Compaction of
centromeric heterochromatin probably depends on other factors that control higher-order
chromatin folding. Kinetochores also do not require condensin for their protein composition,
structure or motility. Loss of stiffness caused by condensin-depletion produces abnormal
uncoordinated sister kinetochore movements, leads to an increase in Mad2(+) kinetochores
near the metaphase plate and delays anaphase onset. These results are consistent with models
where condensin coordinates the mechanical properties of pairs of chromatin fibers.
This work was supported by the Wellcome Trust, the Caledonian Research Foundation, the
Darwin Trust of Edinburgh and the NIH of the USA.
35

Novel function for a nuclear pore protein in cell cycle control
B. Fahrenkrog, D. K. Shumaker 1 , Y. L., T. Shimi 1 , R. D. Goldman 1
M.E. Müller Institute for Structural Biology, Biozentrum, University of Basel, Switzerland
1
Feinberg School of Medicine, Department of Cell and Molecular Biology, Northwestern
University, Chicago, IL
Nuclear pore complexes (NPCs) present the exclusive sites for macromolecular trafficking
between the cytoplasm and nucleus of eukaryotic cells. NPCs are composed of about 30
different nucleoporins, one of which is Nup153. Based on its amino acid sequence, Nup153
consists of an N-terminal domain required for its targeting to the NE and NPC; a central zincfinger
domain critical for NE breakdown at the onset of mitosis; and a C-terminal domain
housing multiple phenylalanine-glycine repeat motifs that provide interaction sites for a
number of nuclear transport receptors due to which Nup153 functions in nuclear import and
export. Here we show that the interaction of Nup153 with the spindle assembly checkpoint
(SAC) protein Mad1 is important for the correct timing of cytokinesis. Over-expression of
human Nup153 in HeLa cells leads to the appearance of multinucleated cells and the
formation of multipolar spindles that coincide with supernumerary centrosomes and
unresolved cytokinesis. Knocking down Nup153 by using RNA interference demonstrates a
delay in cytokinesis along with an increased number of midbodies. Moreover, the depletion of
Nup153 coincided with a loss of Mad1 from the NPC and during metaphase Nup153 and
Mad1 appear associated at centromeres. Immunoprecipitation and solution binding assays
revealed that Mad1 binds directly to Nup153. Our data suggests that Nup153 affects timing of
cytokinesis by regulating the localization of Mad1, most likely during the
metaphase/anaphase transition.
36

How do mutations in lamins cause disease?
R. Foisner, N. Naetar, I. Gotic, U. Pilat, J. Braun
Max F. Perutz Laboratories, Medical University Vienna, Dr. Bohr-Gasse 9, A-1090 Vienna,
Austria
Lamins are nuclear intermediate filament proteins in multicellular eukaryotes that form a
scaffolding network at the nuclear envelope. While B-type lamins are ubiquitously expressed,
A-type lamins are expressed at later stages of development and in differentiated cells.
Intriguingly, mutations in the LMNA gene, encoding A-type lamins A and C, have been linked
to human diseases, whose pathologies range from striated muscle defects to premature ageing
syndromes. The molecular pathomechanisms, how mutations in lamins affect their functions
and how these can give rise to the diverse pathologies are poorly understood. One disease
hypothesis proposes that mutations impair lamin structure, assembly and stability, while
another hypothesis postulates lamin-linked defects in chromatin organization and gene
expression control in diseased cells. We recently discovered a novel role of lamins in cell
cycle control of adult stem cells and early progenitors, which may significantly contribute to
the disease phenotypes.
We have studied a lamin A-binding protein, termed lamina-associated polypeptide 2 alpha
(LAP2α), which binds chromatin and translocates a subfraction of lamin A/C from the
nuclear periphery to the nuclear interior. We have shown that the nucleoplasmic LAP2αlamin
A/C complex binds to and regulates the retinoblastoma protein (Rb), a major cell cycle
regulator protein. Knockdown of LAP2α in mice revealed a mild postnatal phenotype in
many regenerating tissues. Proliferating early progenitor cells in skin and intestine as well as
proliferating immature erythroblasts in the hematopoietic system accumulated in the tissues of
LAP2α-deficient mice due to inefficient cell cycle exit, yielding tissue hyperplasia. At the
molecular level, loss of LAP2α in proliferating cells caused reduction of the nucleoplasmic
pool of lamin A/C and an impairment of the Rb pathway, as shown by hyperphosphorylation
of Rb and an upregulation of E2F/Rb target genes. We propose that nucleoplasmic lamin
A/C-LAP2α complexes are required for the tight control of proliferation and differentiation of
progenitor cells in an Rb-dependent manner. Disease causing mutations in lamins may disturb
the balance between proliferation and differentiation, and impair tissue homeostasis in
patients.
This study was supported by the Austrian Science Research Fund (FWF P17871) and the
EURO-Laminopathies research project of the European Commission (Contract LSHM-CT-
2005-018690).
37

Laminopathic mutations interfere with the assembly, localization and dynamics of
nuclear lamins in C. elegans
N. Wiesel, A. Mattout, K. B. Harush 1 , H. Herrmann 2 , U. Aebi 3 , O. Medalia 1 and
Y. Gruenbaum
Department of Genetics, The Institute of Life Sciences, The Hebrew University of Jerusalem,
Givat Ram, 91904 Jerusalem, Israel
1
Department of Life Sciences and The NIBN, The Ben-Gurion University, 84120 Beer-Sheva,
Israel
2
Division of Cell Biology, DKFZ, 69120, Heidelberg, Germany
3
M. E. Mueller Institute, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056
Basel, Switzerland
Lamins are nuclear-specific intermediate filament proteins (IFs) that, together with a complex
set of inner nuclear membrane proteins, form a filamentous meshwork between the inner
nuclear membrane and the peripheral chromatin. Lamins are involved in most nuclear
activities; and mutations in human lamins or their associated proteins cause laminopathic
diseases ranging from muscular dystrophies to accelerated ageing. Previous studies showed
that lamin dimers form filamentous structures through ‘head-to-tail’ interactions, which then
form paracrystalline structures in vitro. C. elegans has a single evolutionarily conserved lamin
protein, termed Ce-lamin. This lamin is unique in its ability to form under specific conditions
10-nm filaments, similar to other IFs. We have used both negative staining and cryo-electron
tomography to determine the structure of 10-nm filaments and paracrystalline fibers and to
find how mutations in residues, which are conserved in human lamin A and lead to
laminopathic diseases, affect lamin filaments assembly. We have further analyzed the effects
of these mutations on lamin organization and dynamics in vivo, by creating mutated gfp::lmn-
1 transgenic strains and by performing FRAP experiments and analyzing phenotypes. Our
results the structure of the lamin filament as well as how mutations affect the filament
assembly both in vitro and in vivo.
38

Chromosomal deletions and amplifications in malignant cells
K. Michalová
Center of Oncocytogenetics, Institute of Clinical Biochemistry and Laboratory Diagnostics,
General Faculty Hospital and 1st Medical Faculty of Charles university
U Nemocnice 2, 12808 Prague 2, Czech Republic
Acquired genetic lesions in the cells of malignant tumors are being increasingly recognized as
relevant markers whose identification improves diagnostic refinement, classification and
prognostic assessment of cancer. They are hallmarks of gene deregulation and genome
instability. Specific reciprocal translocations are the most frequent structural changes and
their mechanism of origin and products of gene fusions or oncogene enhancement are known
well specially in hematologic malignant diseases. Second in incidence rate are chromosomal
deletions which can be found as a single primary aberration or they can be adjacent to
translocation breakpoints. It was proved in hematologic malignant tumors that there are
mainly tumor suppressor genes which are deleted and it is probably primary event in
tumorigenesis. On contrary, amplifications are mostly present in solid tumors, they are
reccurrent chromosomal changes, may-be secondary ones and very often consist of
oncogenes. They are connected with poor prognosis and low response to the treatment. Bothdeletions
and amplifications can be detected on chromosomal level by classical technique
with light microscope or they are cryptic and then molecular methods are necessary for their
identification. Examples of both types of aberrations will be presented and their significance
delineated.
However, despite the ability to map the extent of chromosomal imbalances with unparalleled
precision, there is still more to learn about why chromosomal aberrations do or do not have
consequences. Their role for the origin and progression of the tumor is still in the most cases
the matter of speculations and working hypotheses.
Supported by grants, MSM LC535, MZO 00064165 and NR9481-3/2007.
39

Establishment of cell type specific nuclear architecture: two cell types in native tissues
I. Solovei, A. Mardaryev 1 , M. Fessing 1 , Y. Feodorova, S. Kosem, A. Scharov 2 ,
V. Botchkarev 1,2 , T. Cremer, B. Joffe
Dept of Biology II, University of Munich, Germany
1 Dept of Dermatology, University of Bradford, UK
2 Dept of Dermatology, Boston University, USA
Recent studies emphasized the importance of the gene positions for transcriptional regulation.
A number of mechanisms were described in cultured cells, e.g., segregation of
transcriptionally active and inactive nuclear regions or association of loci with one another or
with other nuclear structures. Chromatin repositioning may result from explicit movements of
loci or depend on mitosis. It is important to learn which combinations of mechanisms function
in various native tissues.
In rod photoreceptor cells all genes localize in a thin outer shell enriched in B1 repeats that
also contains transcription and splicing machinery. An inner shell of non-transcribed
chromatin enriched in L1 repeats surrounds the single central chromocenter (subcentromeric
satellite DNA). A hallmark of this inverted architecture are higher order chromatin loops of
L1-rich chromatin (e.g., 3-6 Mb long) stretched in the radial direction, while contigs of B1enriched
chromatin have predominantly tangential orientation. Chromatin remodelling,
accompanied by chromocenter fusion, occurs in postmitotic cells.
In differentiating mouse keratinocytes transcription of many loci including Epidermal
Differentiation Complex (EDC) increases greatly. A change in chromatin folding downstream
Rsp27 gene (1.9 Mb from EDC) moves EDC from the outer to the inner side of the
chromosome territory and therefore into a zone enriched in actively transcribed tissue-specific
and housekeeping genes and nuclear speckles. This position of EDC is also observed in the
basal and in more differentiated postmitotic suprabasal keratinocytes in older animals.
Remarkably, our data suggest that at the onset of the epidermal stratification the EDC locus
obtains some specific epigenetic marking allowing an early establishment of the cell-type
specific chromatin architecture and then propagating it through mitosis.
The features of nuclear architecture shared by these two cell types are: (1) segregation of
transcriptionally active zones, (2) polarized positioning of active and inactive chromatin in
chromosome territories and (3) specific large-scale chromatin folding inflicted during
differentiation.
40

New approaches to automated cell image acquisition and analysis using fluorescence
microscopy
M. Varecha, P. Matula, M. Kozubek
Centre for Biomedical Image Analysis, Masaryk University, Faculty of Informatics, Brno,
Czech Republic
Fluorescence microscopy has become the leading technology to study structure as well as
dynamics of cellular components. The studies can be performed in three-dimensional (3D)
spatial coordinate system as well as in time and spectral dimensions. For example, in cell
nucleus studies, fluorescence microscopy enables to observe not only 3D chromatin structure
(especially spatial organization of selected genes or chromosomes) but also its dynamics in
space (time-lapse imaging of fluorescence-labeled histones within cell nucleus).
At the Centre for Biomedical Image Analysis of Masaryk University in Brno, we have been
developing special systems for automated cell image acquisition and analysis using
fluorescence microscopy. We have called them high-resolution image cytometers (HRCMs)
in literature. The presentation will focus on the latest developments in HRCM technology.
Besides microscopy hardware and software, also examples of possible applications will be
presented.
This work was supported by the Ministry of Education of the Czech Republic (Projects
LC535, 2B06052 and MSM0021622419).
41

tRNA genes and the genetic code: Presentation of a research software tool tRNALab
J. Suchánek Foltán 1,2
1 Dept. of Nuclear Physics and Biophysics, Comenius University, Bratislava, Slovakia
2 Dept. of Informatics, University of Trenčín, Trenčín, Slovakia
The genetic code describes translational assignments between codons and amino acids.
tRNAs and aminoacyl-tRNA synthetases (aaRSs) are those molecules by means of which
these assignments are established. Any aaRS recognizes its tRNAs according to some of their
nucleotides called identity elements. Let a 1Mut-similarity Sim 1Mut
be the average similarity
between such tRNA genes whose codons differ by one point mutation. We showed that 1) a
global maximum of Sim 1Mut
is reached at the standard genetic code 27 times for 4 sets of
identity elements of tRNA genes of eukaryotic species, while it is so only 5 times for
similarities Sim C&R
between all tRNA genes whose codons lie in the same column or row of
the code. Therefore, point mutations of anticodons were tested by nature to recruit tRNAs
from one isoaccepting group to another. 2) Because plain similarities Sim all between tRNA
genes of species within any of the three domains of life are higher than between tRNA genes
of species belonging to different domains, tRNA genes retained information about early
evolution of cells. 3) We searched the order of tRNAs in which they were most probably
assigned to their codons and amino acids. The beginning Ala, (Val), Pro, Ile, Lys, Arg, Trp,
Met, Asp, Cys, (Ser) of our resulting chronology lies under a plateau on a graph of
univ. ancestors
Sim plotted over this chronology for a set S
1Mut,
S IE of all identity elements of tRNA
IE
genes, whose universal ancestors were separately computed for each codon. This plateau has
remained preserved along the whole line of evolution of the code and is consistent with
observations of Ribas de Pouplana and Schimmel [Aminoacy1-tRNA synthetases: potential
markers of genetic code development. Trends Biochem. Sci., 2001, 26, 591–598] that specific
pairs of aaRSs – one from each of their two classes – can be docked simultaneously onto the
acceptor stem of tRNA and hence an interaction existed between their ancestors using a
reduced code. 4) Sharpness of a local maximum of Sim 1Mut
at the standard code is almost
100% along our chronologies.
This all has already been published in tRNA genes and the genetic code. J. Theor. Biol., 2008,
253, 469-482. All computations were performed with our application foltan tRNALab, which
runs under Windows and whose installation file is available at http://web.t-com.sk/hisym.
What we wrote personally in it is more than 54,000 lines of the code in *.cpp and *.h files.
Our application provides a rich interface displaying many intermediate results with
commentaries as well as many additional functions, which all will be presented on the lecture.
We would also want to foreshadow our further results and research intentions in order to
discuss them later in person.
42

Quaternary benzo(c)phenanthridine alkaloids – new supravital DNA probes
I. Slaninová 1 , J. Šinkora 2 , E. Táborská 3
1
Department of Biology, Faculty of Medicine Masaryk University, Brno,
Czech Republic, ipokorna@med.muni.cz
2
Becton Dickinson Czechia, Czech Republic
3
Department of Biochemistry, Faculty of Medicine Masaryk University, Brno,
Czech Republic
Quaternary benzo(c)phenanthridine alkaloids (QBAs) are naturally occurring compounds
isolated from plants in the Fumariaceae, Papaveraceae, Ranunculaceae and Rutaceae
families. In addition to having a wide range of biological activities, they are also attractive
for their fluorescent properties. Fluorescence staining of nucleic acids, especially DNA, is an
important tool used in biology and clinical diagnostics. There are a number of DNA-binding
fluorochromes available today. However, most of them are restricted to use on fixed cells
(e.g., ethidium bromide, propidium iodide, 7-AAD, TOTO3). The most frequently used
membrane permeant DNA stains are DAPI (4'-6-diamidino-2-phenylindole) and the
bisbenzimide dyes Hoechst (33258; 33342; 34580). Both DAPI and Hoechst emit blue
fluorescence under UV illumination when bound to DNA. Thus, their use is restricted to
multilaser systems. Recently some cell permeable DNA dyes, excitable by argon-ion lasers
(488 nm), have been introduced: LDS-751, DRAQ5 TM .
We have found that QBAs - macarpine (MA), chelirubine (CHR), sanguirubine (SR) and
sanguinarine (SA) stain DNA in living cells. The best DNA staining properties showed MA.
When added to intact cell suspensions, water stock solutions of MA stain nuclei of the cells
upon brief (in seconds) incubation at low final concentration (0.01-0.001
mg/ml).Fluorescence microscopy of MA stained cells described the nuclear architecture,
chromosomes and apoptotic fragments. Moreover MA binds to DNA stochiometrically and
can rapidly represent the cellular DNA content of living cells at a resolution adequate for cell
cycle analysis. MA is excitable using common argon lasers (488 nm) emitting at a range of
575 - 755 nm (i.e. fluorescence detectors FL2-5). Spectral characteristics of MA allow
simultaneous surface immunophenotyping. The fact that MA preferentially stain nucleated
cells, allows it to be used to differentiate nucleated cells from cells without a nucleus in a
mixed cell population (especially blood and bone marrow cells). Results, concerning the
staining of blood cells, demonstrate that MA can be used for flow-cytometric cell
classification on the basis of nucleic acid identification and quantification with the possibility
of parallel immunophenotypisation. MA staining allows differentiation of erythrocytes,
reticulocytes and leukocytes in peripheral blood without the need to detect surface antigens.
Furthermore, MA staining can be used in conjunction with commonly used fluorochromes
that are detectable at the FL1 detector (FITC, GFP) of either standard or 2-laser systems.
Parameters of DNA content analysis of intact viable HL60 cells stained with MA were fully
comparable with those stained with DRAQ5. Like DRAQ5, MA is quick and easy to use.
Signals from RNA-MA fluorescence are low and there is no necessity for RNA digestion of
living cells. Above mentioned characteristics allow multiple applications of MA providing a
significant diagnostic utility.
This work was supported by grants from the Czech Science Foundation (GACR 525/08/0819)
and from the Ministry of Education of the Czech Republic (VZ MSM0021622415 and
LC06077).
43

Utilisation of time-lapse living cell imaging in the toxicity assessment and drug discovery
M. Červinka, E. Rudolf
Dep. of Medical Biology and Genetics, Charles University Faculty of Medicine, Simkova
870, 500 38 Hradec Králové, Czech Republic
e-mail: cervinka@lfhk.cuni.cz
Many biologists have realized that in order to describe properly the complex biological effects
in vitro we need information about the dynamic aspects of the biological response of a treated
cell population as well as information about the heterogeneity in this response within a given
cell population. For this purpose we need non-invasive (non-destructive) methods for
assessment of specific biological functions. For many years, the only method fulfilling these
criteria has been the time-lapse microcinematography or more recently time-lapse video
microscopy, the phase contrast being the main technique for visualisation of living cells in
vitro. Recently the situation has changed dramatically, and new technical possibilities emerge.
We believe that there are very important applications for this approach in the field of in vitro
cytotoxicity assessment as well drug discovery.
New technical innovations exist in several fields:
1) There are new possibilities for simultaneous observation of a cell population in the phase
contrast and multidimensional fluorescence. Very sensitive CCD cameras and new
fluorochromes minimize cell damage due to illumination.
2) The introduction of specific fluorochromes enables us to analyse numerous molecular
changes. The microscopy is no longer a morphological discipline. New microscopic
techniques provide data about the molecular mechanism of toxic action.
3) New technical solutions exist for retaining the stability of all basic optical parameters,
mainly the stability of focus and the elimination of vibrations.
4) New devices ensure good cultivation conditions during observation (maintenance of
temperature, humidity, CO2 concentration).
5) Computer controlled XY movement of the specimen enables simultaneous recording of
several places within a given cultivation chamber. This greatly increases the amount of data
available.
6) Digital image analysis systems enable quantification of obtained picture sequences, sharing
visual information with the scientific community, and easy presentation of results.
In our presentation we will document our experience with several systems (including the
Olympus Cell R and the Nikon BioStation IM) designed for time-lapse observation of living
cells in phase contrast and fluorescence mode.
The Czech Ministry of Education grant MSM 0021620820 supported the work.
44

Plant biology, organic farming, GM crops and human health
Z. Opatrný
Department of Plant Physiology, Faculty of Science, Charles University in Prague
Prague, Czech Republic
Of existing 6.5 bill. of world human population more than 50 mil. people are subjected to
continuous hunger and more than 850 mil. suffer from malnutrition. Both quantity and quality
of plant production – production of feed and food, vitamins and additives, pharmaceutically
important substances – drugs, vaccines etc., represents key tool of the human health
improvement. Moreover, the extent and structure of the world agriculture has been directly or
indirectly affecting earth ecosystems and again, vice versa, human life.
Three main forms of the agriculture have been competing both in philosophical and practical
level in feed/food production area: “conventional”, “ecological/organic” and, more and more
intensively, “biotechnological” domains, The last is based on the application of recombinant
technologies and transgenic plants (i.e. genetically modified organism = GMOs).
The advocates of the organic farming postulate that its main dogmas – i.e plant production
free of “industrial/mineral” nutrients, pesticides – and GMOs, guarantee high food/feed
quality, its healthiness. On the contrary – both knowledge of the contemporary plant biology
and the results of careful laboratory analyses prove, among others, high risk of the pest
impairment of the “organic products”, of which the contamination by various mycotoxins
represents main danger.
Plant GMOs – mostly in the form of insect-resistant or herbicide-tolerant crops (maize,
soybean, rapeseed, cotton, rice and others) – entered massively the world agriculture more
than 10 years ago. One of them, Bt corn resistant toward pyralid moth, has been cultured in
our country since 2005 as well. Now it occupies more than 8 500 ha. It needs no pest
application and it gives 10% higher harvest. Worldwide, GM crops have been grown on
millions of hectars – and they offer not only improved food/feed products, but also numerous
types of technical raw materials, including biofuels. The techniques of molecular farming
enabled the use of plants for the production of variable originally microbial, animal or human
metabolites – hormones, vaccines, recombinant antibodies … Pure, reliable – and free of
natural contaminants, like mycoplasmas, viral components.
The insertion and expression of the foreign – usually very distant – genes is of course to
various extent tolerated by plant itself. Side effects like insertion mutagenesis have been
observed as well. The transgenic procedure can be followed by incorrect gene expression,
gene product maturation – with possible consequences in the allergenicity of the compound or
its undesirable immunogenic effects. We know examples of some blind alleys, but in all cases
determined and explained on the laboratory level, before commercial use. No one convincing
example of the human health harm induced by GMO crops has been registered yet.
Gene transfer techniques allowed, among others, wide use of plant experimental models
exploiting not only whole plants but also transgenic plant cell lines – e.g in the study of
common mechanisms of the cell division and differentiation, cell senescence and programmed
cell death. The behaviour of animal /human genes in plant cell as well as plant ones in animal
or yeast model lines have been confronted. The lecture will illustrate these activities –
including the use of our own results.
This work has been supported by Ministry of Education of the Czech Republic Research
Project MSM0021620858 and by Project LC 06034
45

Epigenetic changes in dedifferentiated plant cells
B. Koukalová, M. Fojtová, A. Kovařík
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Brno
Introduction: Methylation of cytosine residue is a common epigenetic modification of DNA,
related to gene expression. Here we report changes in DNA methylation and gene expression
accompanying plant cell dedifferentiation in two loci of tobacco (Nicotiana tabacum (L))
genome: (i) 35S rDNA, (multicopy units coding for ribosomal 18S, 5.8S and 26S RNAs) and
(ii) transgenic loci containing neomycin phosphotransferase reporter transgene driven by the
35S promoter.
Methods: Piece of a leaf was placed on medium inducing callus growth. Dedifferentiated
callus cells were subsequently cultivated. Cytosine methylation was analyzed in
dedifferentiated (callus) and differentiated (leaf) cells using methylation sensitive restriction
endonucleases followed by Southern–blot hybridization.
Results: rDNA of callus (dedifferentiated cells) was hypomethylated in comparison with that
of leaves (differentiated cells). The changes of rDNA methylation started soon after the
induction of callus growth and occurred in both genic and intergenic regions of rDNA units.
rDNA hypomethylation was associated with an increased transcription (analyzed with run-on
method) and elevated levels of mature rRNAs (northern-blot hybridization).
The induced hypomethylated state of rDNA was stably maintained during prolonged callus
cultivation. However, regenerated plants showed partial remethylation and in progeny plants,
an original pattern of methylation was reestablished.
In another part of tobacco genome, epigenetic stability of two types of transgenic loci in
callus culture was investigated. Transgenic locus containing single T-DNA insertion remained
epigenetically stable during long term cultivation; i. e. without methylation along both
promoter and genic regions, expressing the reporter transgene at high level. On the other
hand, important changes were imposed on the transgenic locus encompassing two copies of
T-DNA cassette arranged as the inverted repeat. In parental plant, T-DNA was methylated in
the reporter transgene coding region and neomycin phosphotransferase gene expression was
posttranscriptionally silenced. Interestingly, in the callus, spreading of methylation to the
promoter occurred leading thus to its inactivation. Consequently, the type of the reporter
transgene silencing was changed from posttranscriptional to transcriptional. The changed
epigenetic pattern was stably propagated in regenerated plants over generations.
Conclusion: The results demonstrated that in dedifferentiated cells important epigenetic
changes are installed, but their character and stability are not uniform and depend on the type
of the region analyzed.
46

Changes in the gene expression and growth processes of peanut callus culture irradiated
with high energetic nuclei
E. Hlinková 1,2 , R. Tomášová 3 , G. N. Timošenko 2 , S. Vokál 2 , E. A. Krasavin 2
1 3
Institute of Cell Biology Comenius Universityof Bratislava, Department of Genetics Faculty
of
Natural Sciences Comenius University Bratislava, Mlynska dolina G1, 842 15 Bratislava,
Slovakia
2
JINR Dubna Moscow reg. Russia fed. , elena.hlinkova@fns.uniba.sk
Cancers diseases are succesfully treated with cytostatic compounds or combined effect with
gamma irradiation. Effects after irradiation with gamma rays alone have not have inhibition
effects on growth processes of tumor cells. We would like to present work where were used
two type high energetic particles on peanut callus cells long-term cultivated in vitro
conditions.
Irradiation with high energetic protons (Ep=1GeV/nucleon, D=1,5,10,50Gy; LET=1keV/µm)
and deuterons (Ed=3,5GeV/nucleon, D=0,1; 0,5; 1; 5; 10; 50Gy; LET=0,3keV/µm) showed
that ionizing irradiation with different coefficient LET differed with their effect on gene
expression and growth proceses. High energetic deuterons had stimulated effects on gene
expression and growth processes of peanut callus culture cells in a small interval of used
doses (Dє(1-5Gy)) while high energetic protons had for all used doses inhibition effect.
Stimulating effect depended on the level of cell synchronization on the cell cycles, dose rate
as well as energy of particles. Doses with stimulation effect affected gene expression and
decreased cell polymorphism. Nuclei were higher compared to untreated control samples.
Inhibition effects were obtained for doses D≥10Gy independently on particles used.
Decreasing of the level of the gene expression has shown changes in the content of proteins
what was projected in the small values of growth indexes. Morphological parameters of
irradiated cells compared to control samples were changed in the growth stationary phase
very weakly. The most expressive changes in gene expression were registered immediately
after irradiation, when many proteins were missing. The most new proteins in the patterns of
irradiated callus samples were synthesized in the end of lag-phase. Effect of heavy ions on the
proteins with peroxidase activity was very strong immediately after irradiation. Their amount
changed as qualitatively so quantitatively. Callus samples irradiated with deuterons, doses
D>1Gy, had fully inhibited proteins with peroxidase activity. The 28 days post radiation
interval was short on reparation activity of these enzymes. Peroxidases with the group 6 and 7
(high molecular peroxidases) were missing. High energetic deuterons effected of PNA protein
expression level. Quantitative content of this protein changed in dependence of post radiation
time and the phase of the growth. The highest content of PNA was registered on end of lagphase
(7-th day after irradiation). In the stationary phase of the growth, amount of PNA was
practically equal zero, for callus samples irradiated with doses D> 1Gy. Dose LD50 was equal
for all types of used particles and achieved value D=50Gy.
Acknowledgement: this work was supported by Grant agency Vega, Ministry of Education
SR- project 1/4360/07 and project JINR Dubna 08/9-1015-96/2008,Russia fed.
47

Radiosenzitivity of human dental pulp stem cells
T. Soukup 1 , M. Řezáčová 2 , B.Víšek 1 , J. Suchánek 3 , J.Vávrová 4 , A.Tichý 4 , L. Kučerová 5 ,
J. Mokrý 1
Dept. of Histology and Embryology, Charles University in Prague, Medical Faculty in
Hradec Králové
2
Dept. of Biochemistry, Charles University in Prague, Medical Faculty in Hradec Králové
3
Dept. of Stomatology, Charles University in Prague, Medical Faculty in Hradec Králové
4
Dept. of Radiobiology, University of Defence, Faculty of Military Health Sciences,
Hradec Králové
5
Dept. of Clinical Genetics, Teaching Hospital in Hradec Králové
The aim of our project was to study the effect of ionizing radiation on proliferation, viability
and cell death of human dental pulp stem cells (DPSCs). Dental pulp was isolated from
permanent third molars and exposed to enzymatic treatment. Cell lines were cultivated in the
media with 2% of FCS supplemented with PDGF and EGF growth factors. After reaching
50% confluency cells were irradiated using 60Co irradiator with dose 1 Gy/minute in the
distance 1 m from the source. We have studied doses 2, 6 and 20 Gy. Proliferation of the cells
was determined using Z2 counter, viability using ViCell XR. Analysis of karyotype was
performed every 5th passage in untreated cells and furthermore we have evaluated changes in
karyotype of DPSCs treated by ionizing radiation. Distribution of cells in cell cycle phases
was analyzed by flow-cytometry after staining of fixed cells by propidium iodide. Moreover
we have used dual staining by 7-AAD and mAb anti cyclin A2.Data were analyzed in
Multicycle or WinMDI software. Apoptosis was detected using Annexin V binding and
propidium iodide staining measured by flow-cytometry and using detection of activated
caspase 3 by flow-cytometry. Our results proved that DPSCs react to high doses of ionizing
radiation (6 and 20 Gy) by cell cycle arrest (G2 phase), changes in karyotype and premature
senescence (detected as beta-galactosidase activity), but not by apoptosis. Viability following
irradiation was not affected.
Supported by the project of the Ministry of Education, Czech Repulic No. MSM 0021620820
and project of the Ministry of Health, Czech Republic NR 9182-3/07.
48

The identification of cells with cancer stem cell phenotype in pediatric solid tumors.
T. Loja 1 , P. Chlapek 1 , M. Hermanová 2,3 , I. Zambo 3 , K. Veselý 3 , K. Zitterbart 4 , J. Štěrba 4 ,
R. Veselská 1,4
1
Laboratory of Tumor Biology and Genetics, Institute of Experimental Biology, School of
Science, Masaryk University, Brno, Czech Republic
2
Institute of Pathology, University Hospital Brno, Czech Republic
3
Institute of Pathologic Anatomy, St. Anne’s University Hospital, Brno, Czech Republic
4
Department of Pediatric Oncology, University Hospital Brno, Czech Republic
Cancer stem cells (CSCs) undoubtedly play a key role in the tumor initiation, progression and
metastasis. CSCs were first described in leukemias, but they have been identified also in some
types of human solid tumors. CSCs from different histogenetic types of human malignancies
may vary in their pattern of specific markers; nevertheless, common phenotype characteristics
of these CSCs categories are known. From this point of view, co-expression of nestin and the
CD133 surface molecule is considered to be a marker for CSCs in neurogenic tumors. In our
study, we describe the presence of Nes+/CD133+ subpopulations in some cell lines derived in
our laboratory from pediatric solid tumors of neurogenic origin (neuroblastoma,
medulloblastoma, glioblastoma multiforme). The most surprising finding is the expression of
CD133 and nestin in osteosarcomas, i.e. in tumors of mesenchymal origin. Results obtained
on four newly derived osteosarcoma cell lines work were confirmed using
immunohistochemistry on tissue sections from the corresponding tumors. Moreover, these
findings were verified by detection of Nes+/CD133+ cells established Saos-2 osteosarcoma
cell line that was used as a control in our experiments. These results represent the first
evidence of nestin expression and Nes+/CD133+ in osteosarcomas and suggest that the
presence of cells with Nes+/CD133+ phenotype is not limited to the neurogenic tumors.
This study was supported by grants IGA NR9125-4/2006, GACR 204/08/H054, and VZ
MSM 0021622415.
49

Cancer stem cells – a new paradigm in tumour biology
J. Hatina
Department of Biology, Charles University in Prague, Faculty of Medicine in Pilsen, Czech
Republic
The concept of cancer stem cells represents a recent key development in tumour biology.
Accordingly, tumours are organised following principles, which are essentially the same as in
any other complex tissue or organ. This means that both for tumours and for normal tissues,
there is a rare population of stem cells, uniquely responsible for cell replenishment, and a
majority of cells gradually progressing along a differentiation pathway to ultimately die off.
Crucial biological characteristics of stem cells are self-renewal, self-protection and ability to
yield progeny committed to differentiation. Several different mechanisms underlie the selfprotection
of stem cells, i.e. their ability to resist environmental insults. These include
constitutive expression of drug efflux pumps, drug inactivating enzymatic activity, enhanced
DNA-repair activity as well as a peculiar mode of DNA-replication known as immortal DNAstrand.
Collectively, these mechanisms serve to protect stem cells and in this way to guarantee
the continual cell replenishment. In a physiological sense, this self-protection ability
contributes to tissue renewal, but in the context of cancer, this same ability lies behind therapy
resistance, disease recurrence and metastatic progression. Eradication of cancer stem cells
thus represents a promising way to novel tumour therapies, which could potentially lead to a
real cancer cure. The hierarchical organisation of cell populations is preserved in tumour cell
lines as well, and we have developed a method of visualisation of cancer stem cells in
carcinoma cells cultured in vitro. Our approach is based upon a use of the GFP – reporter
gene, driven by a promoter, which is activated upon doxorubicin treatment. The cancer stem
cells, by virtue of their constitutive drug efflux activity, pump doxorubicin out and
consequently cannot switch on the GFP expression. We believe that our approach will be
helpful not only in identification of putative stem cells or stem cell – directed reagents, but
also in resolving the dynamics of stem cell population in a living cell culture.
This work was supported by Ministry of Education of the Czech Republic Research Project
MSM 0021620819 Replacement of and support to some vital organs.
50

Genetic susceptibility to sporadic colorectal cancer
P. Vodicka 1 , R. Kumar 2 , S. Landi 3 , F. Canzian 2 , A. Naccarati 1 , B. Pardini 1 , L. Vodickova 1 ,
V. Polakova 1 , E. Tulupova 1 , M. Hanova 1 , J. Slyskova 1 , A. Foersti 2 , R. Houlston 4 ,
I. M. P. Tomlinson 5 , K. Hemminki 2
1 Inst. Exper. Med., Acad. Sci. Czech Rep., Prague, Czech Rep.,
2 German Cancer Res. Center (DKFZ), , Heidelberg, FRG,
3 University of Pisa, Pisa, Italy,
4 Inst. Cancer Research, Sutton, UK,
5 University of Oxford, Oxford, UK
Colorectal cancer (CRC) poses a serious health problem in the Czech Republic, as this
country ranks at the top in the incidence worldwide. While inherited susceptibility is
responsible for ~ 35% of CRC, high-risk germline mutations in APC, MMR genes,
MUTYH/MYH, SMAD4, ALK3, and STK11/LKB1 account for about 6% cases. Recently
formulated hypothesis ascribes the CRC risk to common, low-risk variants. Their search
and identification was the main aim of the present investigation.
Concerning DNA repair polymorphisms, variant genotype in the APE1 Asn148Glu was
associated with an increased risk of CRC, in particular with colon cancer (OR 1.50; 95% CI
1.01–2.22). The G-allele in MMR gene hMSH6 -556G>T was associated with increased
CRC risk (OR 1.29; 95% CI 1.02-1.62), while the A-allele for hMSH6 Ex1-145G>A was
associated with decreased risk (OR 0.76; 95% CI 0.60-0.98). The haplotype analysis for 3
SNPs in the hMSH6 gene showed a differential distribution between cases and controls
(global test, P=0.02). No significant associations for any polymorphisms in cell cycle genes
were found, however, we observed differential distribution of haplotypes based on TP53
polymorphisms (PIN3, Arg72Pro, Int7 +72C>T, and Ex11 -363G>A) between CRC cases
and controls (global test, P

SOUHRNY PLAKÁTOVÝCH
SDĚLENÍ
52

Souhrny plakátových sdělení
(abecedně podle jména prvního autora)
Characterisation of asthma bronchiale in pediatric patients
E. Babusikova 1 , M. Jesenak 2 , T. Matakova 1 , J. Hatok 1 , P. Banovcin 2 , D. Dobrota 1
1
Comenius University in Bratislava, 1Institute of Medical Biochemistry, Jessenius Faculty of
Medicine in Martin, Martin, Slovakia
2
Institute of Children and Adolescents, Jessenius Faculty of Medicine in Martin, Martin,
Slovakia
Asthma bronchiale is a complex chronic inflammatory disorder of the airways, involving
variable airflow obstruction with spontaneous reversibility and increased airway
responsiveness to a variety of stimuli. Its prominent symptoms are cough, wheezing,
dyspnoea and tenderness in the chest. Asthma bronchiale has become serious society wide
health problem regarding increasing prevalence. Asthma is the most frequent chronic disease
in children. Early diagnosis, optimal therapy and regularly examination of patient's status can
contribute to the preceding of consequences of asthma in the later age and improve quality of
life. The basic mechanism of the development of asthma has not been yet defined. The key
role in the pathogenesis of asthma plays chronic inflammatory process. It has been suggested,
that the disturbance of the equilibrium among oxidants and antioxidants could contribute to
the development of oxidative stress also in the inflammation causing various respiratory
diseases including asthma bronchiale. Reactive oxygen species (ROS) produced during
oxidative stress are very reactive and can modify proteins, lipids and nucleotides. Oxidative
damage of biomolecules is probably involved in the pathogenesis of many diseases, including
asthma bronchiale.
The enzymes encode by glutathione-S-transferase (GST) supergene family play a critical role
in the protection of cells From ROS.
We determined markers of oxidative stress – total content of sulfhydryl groups and
thiobarbituric acid-reactive substances (TBARS) by spectrophotometry at patients and control
group. Genotyping the polymorphisms in the GSTP1, GSTM1 and GSTT1 genes was
performed using PCR. Content of SH groups and TBARS increased in pediatric patients.
Levels of markers did not change significantly between control and asthmatic group of
children. Patients with asthma had an same prevalence of the GSTT1 null genotype as the
control group. Asthma patients (62%) had a higher prevalence of the GSTM1 null genotype
than the control group. These results suggest that the GSTT1 and GSTM1 null genotypes
could play important roles in the childhood asthma pathogenesis. Other analysis of markers
and gene polymorphisms are necessary to do in the Slovak population.
This work was supported by Ministry of Health grant 2007/47-UK-12.
53

The in vitro effect of ultrasound and cisplatin on A2780 and A2780cis cell lines
V. Bernard, J. Škorpíková, V. Mornstein
Department of Biophysics, Masaryk Univerzity, Faculty of Medicine, Brno, Czech Republic
The human carcinoma ovarian cells A2780 and A2780cis were exposed to the far field of a
horizontal beam of continuous wave ultrasound at the intensity of 0.5 W/cm 2 and 1 W/cm 2 in
a thermostated 37°C water bath for 10 minutes. During the ultrasound exposure the cells were
put in a polyethylene tube fastened to a rotating holder (3 rpm).
The cell viability was compared in following experimental groups: contr - cells incubated
without the addition of cisplatin and without ultrasound exposure, us – cells incubated after
the ultrasound exposure, cisPt+us – cells incubated after the addition of cisplatin and
subsequent ultrasound exposure, us+cisPt - cells incubated after the ultrasound exposure and
subsequent addition of cisplatin. Calculated volumes of the stock cisplatin solution were
added to cells in 96-well tissue culture plates to achieve the resulting 1µM concentration of
cisplatin in each well. For the group cisPt+us, the calculated volume of the cisplatin stock
solution was added to cells before ultrasound exposure to achieve the resulting 1µM cisplatin
concentration in each well. After incubation (72 hours) the cells were subjected to a standard
MTT test of viability. The absorbance of the blue product in individual wells was measured
using microplate reader EL800 at the wavelength of 570 nm.
The conclusions drawn from the experiment clearly documented, that the viability of the
studied cell lines was affected not only by the cisplatin treatment, but also by ultrasound
exposure or by combination of the both factors. A statistically significant difference was
found between the experimental groups that were exposed to different ultrasound intensities.
The viability of the cells affected by equipotent physical factors have statistically significant
difference in the groups with different experimental design (cisPt+us, us+cisPt). For the
experiment with combined effect of cisplatin and ultrasound exposure in the cisPt+us group
the viability was lower compared to that in the us+cisPt groups. The expected effect of cell
stimulation by ultrasound exposure was also confirmed. A possible explanation is the effect of
ultrasound on cell membrane porosity and permeability for cisplatin during ultrasound
exposure. The presence of cisplatin during the ultrasound exposure appears to be the main
factor affecting the decrease in the viability. Based on the earlier and present experiments we
can expect, that the changes in the cell membranes occurred during ultrasound exposure and
the reparation processes started immediately after the ultrasound exposure. The finding that
the influence of the combined effect of cisplatin and ultrasound on cell viability depends on
the experimental design opens new research perspectives.
54

Chemoresistence testing of cells isolated from ovarian tumours, effects of freezing
K. Brigulová 1 , M. Červinka 1 , J. Tošner 2
1 Charles University in Prague, Faculty of Medicine in Hradec Kralove, Department of
Medical Biology and Genetics,
2 Department of Obstetrics and Gynaecology
Ovarian cancer is responsible for 15 % of all malignities in women. Incidence of this disease
is 20/100 000 women, with 1000 new cases in Czech republic every year. New promising
direction in the treatment is an individualization of chemotherapy. In our depatrment we
tested chemoresistence of ovarian cells to selected cytostatics in vitro. Cells were isolated
from ovarian tumours or ascites of patients undergoing surgery. The tissue samples were
mechanically disintegrated and cells were isolated by a density gradient centrifugation.
Isolated cells were exposed to Cisplatin, Paclitaxel, Carboplatin, Doxorubicin, Gemcitabin,
Topotecan, Etoposid, Navelbin and Docetaxel for 72 hours. Effect of cytostatic treatment on
metabolic activity of tumour cells was evaluated by MTT assay, which is based on reduction
of MTT by mitochondrial enzymes and production of insoluble formazan. Intensity of this
colorimetric reaction was measured by spectrophotometry and EC50 value was determined
for individual cytostatics. We repeated the same test on cells that have been frozen for several
weeks. The aim of this part of our work was to determine if there are any changes in
chemosensitivity connected to cryopreservation.
So far we analysed 77 samples including samples of patients after repeated surgery. 58
samples produced sufficient scores of viable tumour cells and were subjected to testing. We
tested 26 samples after freezing and thawing. Chemoresistence testing of ovarian cancer cells
before and after their freezing afforded us 132 coupled values for comparison. From these pair
values there was a higher sensitivity in 33 cases, lower sensitivity in 31 cases and in 68 cases
of pair worths there was no change in reactivity to cytostatics. The results suggested that
process of freezing and thawing influenced the viability of tumour cells and their reactivity to
cytostatic treatment. Our findings have some practical implication in further clinical
applications of chemoresistence testing.
ACKNOWLEDGMENTS: This work was supported by the Czech Ministry of Health Grant
No. NR/ 8768-3.
55

Food quality in cancerous disease prevention
M.Bušová 1 , R.Opatřilová 2
1
Department of Biochemistry, Chemistry and Biophysics, Faculty of Veterinary Hygiene and
Ecology, University of Veterinary and Pharmaceutical Sciences in Brno
2
Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and
Pharmaceutical Sciences in Brno
Apart from physical and chemical factors, also the quality and composition of food impact
human health greatly. Food is taken in a regular and long-term pattern. Thus, its composition,
especially the presence of extraneous substances, can be one of the causes of cancerous
diseases. Food need not contain high concentrations of carcinogens and thanks to the
combined action of many other further external environment factors over a long period of
time, it can lead to the development of the most serious diseases and the most frequent causes
of dying in the Czech Republic. Due to the increasing number of cancerous diseases of the
gastrointestinal tract, especially of colon-rectal carcinomas, which rank among the foremost
places in death rate caused by this disease, efforts of the widest research public are devoted to
this impacted issue.
Xenobiotics enter the organism from the environment in food through the gastrointestinal
tract. Many of them can act as a direct carcinogen. On the whole, however, the process of
carcinogenesis runs a gradual course. Most of chemical carcinogens are transformed upon
entering the organism by oxidative enzymes onto reactive forms which can lead to the
formation of very dangerous adducts with DNA (deoxyribonucleic acid). The cells are
capable of defending against serious damage done by detoxification mechanisms to DNA.
Great importance in the organism detoxification is ascribed to glutathion (γ-Glu-Cys-Gly),
especially its reduced form. Conjugation with metabolites of xenobiotics represents the main
detoxification route preventing thus the formation of adducts with DNA.
The goal of our project is the study of detoxification mechanisms in freshwater fish, an
important food of animal origin. Fish meat ranks among important food items in human
nutrition with regard to the advantageous contents of amino acids and essential amino acids,
good digestibility, low fat content as compared to beef as well as poultry. It also contains ω-3
unsaturated fatty acids that are valuable for the organism for the prevention of cardiovascular
diseases. Attention needs to be paid also to contaminants, especially in fish originating in
ecologically challenged water. Our laboratory focuses on the study of contents of the reduced
form of glutathion in various species of retail freshwater fish of Czech origin. Thus, we can
acquire an overview of this important food and, at the same time, information on the
environment status and feedstuff quality which is used for feeding retail carp.
This project received support from the research plan No. MSM 6215712402 Veterinary
Aspects of Food Safety and Quality.
56

Localization of actin-binding proteins in the cell nucleus
J. Fukalová, M. Maninová, V. Filimonenko and P. Hozák
Institute of Molecular Genetics, Dept. of Biology of the Cell Nucleus, Academy of Sciences
of the Czech Republic, Prague, Czech Republic
Actin is not only a component of the cytoplasm of eukaryotic cells but is abundant also in the
nucleus. However, the functional significance of occurrence of actin in the nucleus is still
largely unknown, although several important roles of the actin in nuclear processes have been
suggested. Nuclear actin participates in regulation of gene transcription, nucleocytoplasmic
transport, and chromatin and nuclear structure.
Numerous actin-binding proteins (ABPs) regulate the dynamics of actin filaments in the
cytoplasm. The presence of some ABPs in the nucleus have been reported ( -actinin, CapG,
emerin, filamin A, gelsolin, profilin, protein 4.1, spectrin, thymosin 4) but little is known
about their nuclear functions.
We address the questions of nuclear localization and of possible nuclear functions of actinin,
filamin, spectrin and vinculin. Spectrin was first characterized as a cytoskeletal protein
underlying the plasma membrane of the erythrocyte and linking actin filaments to an integral
membrane protein. Recent studies has provided evidence that spectrin is present in the
nucleus of mammalian cells. Nonerythroid spectrin ( SpII) is part of nuclear protein
complex involved in DNA repair. Spectrin IV ( SpIV 5) associates with the nuclear
matrix and PML nuclear bodies and spectrin II interacts with nuclear proteins .
Filamin A was the first actin filament cross-linking protein identified in non-muscle cells.
During last few years filamin was observed in the cell nucleus too. Human filamin A
(hsFLNa) interacts with BRCA2 in the nucleus and the lack of hsFLNa results in increased
cellular sensitivity to DNA damaging agents. C-terminal 100kDa fragment of FLNa
colocalized with androgen receptor to the nucleus where it regulates androgen receptor
activity.
Member of spectrin protein family and focal adhesion and stress fiber-associated proteins, αactinin
1 and 4 were identified as class IIa histon deacetylases-interacting proteins and in
addition a novel splice variant of α-actinin 4 which is predominantly localized in the nucleus
was isolated. Vinculin interacts with numerous other junctional proteins and links integral
membrane proteins to actin filaments.
We performed a study on nuclear localization of these actin-binding proteins by means of
electron microscopy. Immunogold labeling on ultrathin sections of HeLa cells was evaluated
using spatial statistics with previously developed plugins to the Ellipse image processiong
program. We demonstrate that several actin-binding proteins reveal characteristic distribution
within the cell nucleus implying important roles in nuclear functioning. The data on
colocalization of ABPs with the key proteins of important nuclear processes, such as
transcription, replication, splicing and cell cycle regulation will be discussed.
This study was supported by the Grant Agency of the Czech Republic (Reg. No.
204/05H023), by the Ministry of Education, Youth and Sports of the Czech Republic (Reg.
No. 2B06063 and LC545) and by Institutional Grant (Reg. No. AV0Z50520514).
57

Polymorphisms of GSTM1, GSTT1 and GSTP1 genes in relation to chromosomal
damage in workers exposed to chromium
E. Halašová 1 , T. Matáková 2 , Ľ. Mušák 1 , Ľ. Javorka 3 , M. Halaša 4 , E. Bukovská 1
1
Institute of Medical Biology, Comenius University in Bratislava, Jessenius Faculty of
Medicine in Martin
2
Institute of Medical Biochemistry, Comenius University in Bratislava, Jessenius Faculty
of Medicine in Martin
3
Department of Childbirth Assistance, Faculty of Health, Catholic University in Ružomberok
4
Clinic of Transplantant and Vascular Surgery, Comenius University, Jessenius Faculty of
Medicine, Martin
Welders are exposed to chromium and to a lesser extent to polycyclic aromatic hydrocarbons
(PAHs). Hexavalent form produces DNA strand breaks, DNA-DNA and DNA-protein crosslinks
and modifies nucleotides, such as 8-hydroxyguanine. Highly reactive intermediates such
as Cr (V) and Cr (IV) formed due to cellular Cr (VI) reduction, are primarily responsible for
the observed genotoxicity. Cellular reducing agents that may be important for Cr(VI)
reduction includes ascorbate and sulfhydryl compounds such as cysteine and glutathione.
Our study is focused on biomonitoring study in welders by employing chrmomosomal
aberations in peripheral blood lymphocytes as a marker of genotoxic effect. Association with
genetic polymorphisms in genes encoding principal metabolizing enzymes GSTM1, GSTT1
and GSTP1 as biomarkers of individual susceptibility to procarcinogens was assessed as well.
The study was performed on a population of 31 welders and 31 control individuals that were
not exposed to any known carcinogens or mutagen.
CAs were analyzed in peripheral blood lymphocytes in two separate tubes using previously
described method. Structural CAs include chromosomal breaks and exchanges visible in
arrested metaphase-stage cells and they were divided into chromosome-type aberrations
(CSAs) and chromatide-type aberrations (CTAs). The GSTM1 (deletion), GSTP1 (alleles
Ile/Val in codon 105 of exon 5) and GSTT1 (deletion) polymorphisms were analyzed by the
multiplex PCR method21.
There was no significant difference (P>0.05) in the number of aberrant cell between the
exposed 1.93 ± 0.17% (±SE) and control 1.54±0.12% groups. Chromatide type - aberrations
(CTA) were higher in the control group 1.03±0.10% in comparison with exposed one
0.70±0.13 %. The difference was not significant (P>0.05). Biologically more serious
chromosome type -aberrations (CHSA) were significantly higher (P

Structure and function of promyelocytic leukaemia nuclear bodies
A. Harničarová, E. Bártová, S. Kozubek
Department of Molecular Cytology and Cytometry, Institute of Biophysics, Academy of
Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65, Brno, Czech Republic.
Promyelocytic leukemia nuclear bodies (PML NBs) represent one of the structures
responsible for the regulation of transcription, apoptosis, tumor suppression, and antiviral
defence. Mammalian nuclei contain several spherical PML NBs, the number of which can
change depending on cell cycle phase. In the present study we investigated the nuclear
arrangement of promyelocytic leukaemia nuclear bodies (PML NBs) in leukaemia and
multiple myeloma cells treated by cytostatics and induced to differentiation.
A reduction in the number of PML bodies, which led to relocation of PML NBs closer to the
nuclear interior, mostly accompanied differentiation processes. In addition, centrally located
PML NBs were associated with RNAP II positive “transcription factories” and nuclear
speckles. These observations support the importance of PML NBs in transcription and RNA
processing which mostly proceed within the nuclear interior. Conversely, the quantity of PML
NBs was increased after cytostatic treatment, which caused the re-distribution of PML NBs
closer to the nuclear envelope. On the basis of our observations we suggest a basic
relationship between the number of PML NBs and average centre-to-PML distances: we
observed that an increased PML number caused relocation of PML bodies more peripherally
within interphase nuclei.
Furthermore, the proteins involved in the PML compartment, such as c-MYC were closely
associated with PML NBs, which changed during various differentiation pathways.
This work was supported by the Ministry of Education and Sport of the Czech Republic
(LC06027) and by the Academy of Sciences of the Czech Republic, grants no.
AV0Z50040507 and AV0Z50040702 and the Grant Agency of the Czech Republic, grant no.:
204/06/0978.
59

Exploitation of the telephonic mobile apparatus for photography of education
preparations in biology
J. Hochmann
Department of Biological and Medical Sciences,Faculty of Pharmacy, Hradec Králové
It is possible to make the microphotographs using the photo-apparatus that is the component part
of the telephonic mobile apparatus – by the attaching of the objective of this „mobile“ to the ocular
of any microscope. It is also possible to make the video-record by this manner. Nevertheless, this
„video“ is tremulous. The better results can be achieved using the simple wood „fixation” adapter
that is fixed to the ocular of the microscope.
We made the microphotographs – predominantly of the paramecia for presentation on this lecture.
60

Influence of estrogen and progesterone receptors (ER and PR) on the survival in case of
breast cancer – and questions of exactness of examination of these markers
J. Hochmann
Department of Biological and Medical Sciences, Faculty of Pharmacy, Hradec Králové
More than 10 years ago we examined the estrogen and progesterone receptors (ER and PR)
using the radio-receptor analysis in breast tumours – to enable the deciding about hormonal
therapy. We were afraid very of the inaccuracies of this difficult method.
This is why we controlled our results using the comparing of the whole our statistical set of
results with the other similar published results. Predominantly, we concentrated on the graph
of increasing dependence of ER on the age. The other control of exactness of our ER and PR
results was the graph of increasing dependence of survival of patients on the receptors
concentration. We suppose that our results are strong because we found the statistical
significance in these graphs. In comparison with literary data – they were correct – as sun as
the increasing graph of the dependence of PR on ER.
The statistical set with nearly only surgical therapy of primary tumour and its pre-operational
and late metastases was analysed for judging of influence of ER and PR. The patients with the
antiestrogen and classical cytostatic treatment were excluded from evaluation.
Now, our students observe statistically in their diploma or bachelor works the results of
different laboratories – again in case of ER and PR – but using immuno-histochemistry. It is
more modern – but we perform it not in our laboratory. We found often the great interlaboratory
variability. It concerns the comparison of international literary sources – as sun as
the unpublished results of different laboratories in our republic.
We recommend to school workplaces that are similar to our one – to exploit the work of
students by the similar way. It could give the control tool to the clinical physicians – by the
use of which they could improve the laboratory results and thereby also the therapy.
61

Genotoxic effects of Mylecytan in Drosophila melanogaster
J. Ipser, P. Češková
Department of Biology, University of J. J. Purkyně in Ústí nad Labem, Faculty of Science,
Ústí nad Labem, Czech republic
Mylecytan is a cytostatic agent frequently used in the treatment of some haematological
diseases. Embryotoxic and mutagenic effects were found in our experiments with Drosophila
melanogaster. Mylecytan was applied into standard nutrition medium at final concentrations
0.01 – 0.1 g/ml. When flies of the standard stock Oregon R developped in the medium with
Mylecytan at concentrations 0.5 – 0.1 g/ml, average number of offsprings was significantly
lowered (52 – 40% in a comparison with control) whereas the most of them died at the stage
of second instar. Similar results were obtained with mutant stocks ebony (black body), white
(white eyes), vestigial (shortened wings) and cinnabar (cinnabar coloured eyes). The wing
spot test carried out with adult survivals showed a low frequency of somatic mutation induced
at the same range of concentrations: 0.04 – 0.070 mwh (multiple wing hair) or flr (flare)
mutations per wing. The frequency of recessive X-linked lethals detected using Basc method
was also slightly increased (0.004 – 0.008) as compared with control. In 11 cases (0.24%)
from the total number of examined flies morfological malformations of wings (enlarged,
narrow, shortened or deflected), head (absence of eye or rudimentary eye) and abdomen
(surface deformation) were observed.
Both embryotoxic, and mutagenic effects did not show any dependence on the pole of treated
individuals. Mylecytan applied at concentrations lower than 0.5 g/ml appeared ineffective
in examined characters.
Conclusions: Mylecytan revealed embryotoxic and mutagenic effects in Drosophila
melanogaster but only at high concentrations and during longterm exposition.
62

Genotoxic effects of Vincristin in Drosophila melanogaster
J. Ipser, V. Matoušová, P. Češková
Department of Biology, University of J. J. Purkyně in Ústí nad Labem, Faculty of Science,
Ústí nad Labem, Czech republic
Vincristin is a cytostatic agent frequently used in chemotherapy of oncological patiens.
Embryotoxic and mutagenic effects found in the experiments with Drosophila melanogaster
are described. Vincristin was dissolved in 0.9% sodium chloride and applied into standard
Drosophila nutrition medium at final concentrations 0.0005 – 0.05 mg/ml. Flies of the
standard stock Oregon R, single mutant stocks cinnabar (cn; cinnabar coloured eyes), ebony
(e; black body), vestigial (vg; shortened wings), white (w; white eyes) and double mutants
cinnabar - ebony (cncn ee), cinnabar - vestigial (cncn vgvg), ebony - vestigial (ee vgvg),
cinnabar - white (cncn X w X w , resp. cncnX w Y) and ebony - white (eeX w X w , resp. eeX w Y)
developped in the medium with Vicristin. Similarly, fruit flies given for the detection of
induced X-linked lethals and somatic mutations have been developped in the medium with
Vincristin. In all cases embryotoxic effect measured as decrease of average numer of
offsprings was significant at concentrations 0.005 – 0.05 g/ml, and ranged from 82 – 63% as
compared with control. The wing spot test carried out with adult survivals showed a low
frequency of somatic mutations induced at the same range of concentrations: 0,003 – 0,014
mwh (multiple wing hair) or flr (flare) mutations per wing. The frequency of recessive
X-linked lethals detected using Basc method was also slightly increased (0.0005 – 0.008) as
compared with control.
Both embryotoxic, and mutagenic effects did not show any dependence on the pole of treated
individuals. Vincristin applied at concentrations lower than 0.005 mg/ml appeared ineffective
in examined characters.
Conclusions: Vincristin revealed embryotoxic and mutagenic effects in Drosophila
melanogaster but only at high concentrations and during longterm exposition.
63

Concentrations of total cell-free DNA in plasma as potential marker in clinical medicine
M. Korabečná 1 , A. Horinek 2 , A. Panczak 2 , S. Opatrna 1 , J. Wirth 1 , F. Sefrna 1 , P. Calda 2
1 Faculty of Medicine, Charles University, Pilsen, Czech Republic,
2 1st Faculty of Medicine, Charles University, Prague, Czech Republic
Although apoptosis and active release of cell-free DNA (cfDNA) by cells are thought to be
the main source of plasma cfDNA, its dynamics and clearance under various pathological
conditions are yet to be clarified not only with respect to the proper interpretation of changes
in cfDNA levels in potential clinical applications but also with respect to explanation of
biological meaning of this phenomenon.
Real-Time PCR method based on amplification of GADPH gene sequences was employed for
quantification of total cfDNA in plasma. We examined samples from 86 physiological
pregnancies bearing single male fetuses in the third trimester and compared them with
samples from pregnant women with long-term tocolysis (n=15), pre-term delivery (n=13),
preeclampsia (n=21) and gestational diabetes (n=5). In second part of our study, we focused
on patients with renal impairment. We compared the values of plasma cfDNA in healthy
volunteers (n=20) and hemodialysed patients (n=17) at the end of short interdialytic interval
(2 days) and immediately after a hemodialysis procedure. Wilcoxon tests were used for
statistical analysis.
In all examined pregnancy-related disorders, we found significant elevations of plasma
cfDNA concentration in comparison with physiological controls. In hemodialysed patients,
we revealed elevated concentrations of plasma cfDNA in interdialytic interval if compared
with healthy subjects, but the difference did not reached statistical significance neither in
short interdialytic interval nor immediately after a hemodialysis procedure.
Concentrations of total cfDNA in plasma seem to provide an interesting view on the character
of pathological changes in organism. Further studies are needed to understand the
mechanisms of release and clearance of plasma cfDNA, study of different clinical conditions
with different involvement of apoptosis and necrosis in pathogenesis may be helpful in this
context.
Supported by the Ministry of Education of the Czech Republic, grants no. MSM 0021620819
and MSM 0021620808.
64

Cell death and survival in astrocytoma cultures after Cisplatin. An EM and ytochemical
study.
D. Krajčí, C. Pellicciari 1 , M-G. Bottone 1 , V. Lisá 2 , V. Mareš 2,3
Department of Anatomy, Faculty of Medicine, Kuwait University, Kuwait
1 Dipartimento di Biologia Animale, Universita di Pavia, Pavia, Italy
2 Institute of Physiology, Academy of Science (LBNB), Prague
3 Faculty of Science, University of J. E. Purkinje, Usti n. Labem, Czech Republic
Cisplatin treatment of tumors causes a wide spectrum of regressive and adaptation reactions
leading to death or survival of cells. Here, we report an in vitro model demonstrating
canonical and alternative routes of programmed cell death (PCD) and changes in
chemoarchitectonics which may support survival of damaged cells.
Cultures of rat astrocyte-like C6 glioma cells grown on glass coverslips were treated with a
pulse of Cisplatin (90 min, 5-10 mg/ml) and examined by electron microscopy (JEM-
1200EXII , JEOL, 80 kV) using a glass plan parallel Araldite sections of cells. DNA content,
activity of γ-glutamyltranspeptidase (γ-GT) and GFAP were determined by conventional
cytochemistry. The cells were currently sampled at 24 h to 192 h post-treatment intervals (pt.
i).
EM showed that cells started dying at 24 h p-t. i via a canonical apoptosis (PCD type-I)
characterized by perinuclear condensation, cell shrinkage and terminal breakdown in
apoptotic bodies. At 48 h, the cells surviving primary hit of the drug displayed intranuclear
bundles of microtubules and microfilaments without perinuclear condensation of chromatin
and severe nuclear lobulation. By 72 h these cells disappeared from the population via PCD
Type-III. Towards 96 h, the still surviving cells became hypertrophic and their nuclei formed
large irregular lobules and micronuclei. They also developed long smooth cytoplasmic
fibers. In these cells, present until the end of the experiment (192 h), hetero- and
autophagocytosis appeared and was often followed by PCD Type-II. As indicated by DNA
cytometry, the PCD Type I and III were entered by cells from G1 phase while PCD Type
II from G2/M. In cells surviving the first attack of Cisplatin, γ-GT and GFAP were upregulated
and the cells resembled reactive astrocyte-like cells. These cells appeared more
resistant to repeated dose of Cisplatin.
It is concluded that Cisplatin induced 3 alternative routes of PCD beginning with canonical
apoptosis (PCD Type I) and followed by atypical pathways PCD Type II and III which
concerned mainly of cells escaping the primary hit of the drug. Up-regulation of γ-GT and
GFAP can interfere with the therapeutic antitumor effects of Cisplatin via attenuation the drug
induced oxidative stress and withdrawal of cells from cycling to differentiation.
Supported by the Ac. Sci. CR Project AV0Z 50110509 and Kuwait University HSC Shared
Facility Project No. GM 01/01.
65

Melatonin protects mitochondrial transmembrane potential from the effect of
antimycin A in normal as well as cancer cell lines
Kufner, P.*, Hájková, L.*, Reischig, J. (* equally contributed authors)
Institute of Biology, Charles University, Faculty of Medicine in Pilsen, Czech Republic.
Melatonin (N-acetyl-5-metoxytryptamin) has been postulated to have beneficial effects in an
ever growing number of pathological conditions. This hormone predominantly of pineal gland
is well-known for its direct (ROS scavenger), as well as many indirect antioxidative effects,
and is therefore a promising candidate for a therapeutic agent potentially with a minimal level
of inauspicious side effects. Deeper understanding is required before its routine clinical use,
however.
Ischemia-reperfusion injury (IRI), a typical phenomenon in various ischemia-related
pathological conditions including e.g. organ transplantations, is caused by an overproduction
of reactive oxygen species (ROS) in the reperfused tissue. The ROS cause cellular oxidative
stress and tissue damage.
The aim of our efforts has been to design a model for monitoring and treatment of hypoxiarelated
processes. We have established an assay for a study of cellular oxidative stress using
cultured mammalian cells. As a measure of the oxidative state of the cells, mitochondrial
transmembrane potential (∆Ψm) was chosen. The potential was monitored in live cells stained
with the fluorescent probe JC-1, whose transition between the monomeric state and JC-1
aggregates, accompanied by a shift of the emission wavelengths (from λ = 527nm to λ =
590nm), is dependent on the value of the mitochondrial transmembrane potential.
Live adherent cells of various cell lines (cancerous as well as normal, of mammalian origin
including human), grown on the bottom glass of a Bachofer perfusion chamber, were
observed in 3D mode in the Olympus Fluoview1000 laser scanning confocal microscope,
using fluorescent as well as DIC microscopy.
The oxidative cellular stress was induced chemically by adding antimycin A, a specific
inhibitor of complex III of the respiratory chain, correspondingly to the action of nitric oxide.
The effect of antimycin A (depolarization of mitochondrial membrane) was measured
as a ratio of average relative fluorescence intensity in green (IF(G)) and red (IF(R)) channels -
(IF(G)) / (IF(R)).
Detection and quantification of the fluorescent signals were optimized by exciting both the
JC-1 monomers and JC-1 aggregates by 488nm laser, and the emissions were submitted to a
strict spectral separation to eliminate any overlap of the signals in the area between the
emission peaks. Minimum of ten view fields per every sample were scanned and quantified.
This model was thereafter employed to examine the potential cytoprotective effect of
melatonin against the consequences of the oxidative challenge.
Pretreatment of the cells with melatonin before their exposure to antimycin A lead to a drop in
the previously observed antimycin-induced decrease of the mitochondrial transmembrane
potential, thus implicating an antioxidative cytoprotective effect of melatonin in this system.
A statistically significant decrease in the IF(G) / IF(R) ratio was observed. Hence, melatonin
stabilizes the mitochondrial transmembrane potential in the studied cell lines and protects the
cells against oxidative stress. The above appears to be a suitable method for estimating and
comparing the protective effects of antioxidants with a therapeutic potential.
The announcement was supported by Ministry of Education of the Czech Republic Research
Project 0021620819 (6096).
66

Chromosomal aberrations as a tool of prevention at workers exposed to mutagens and
carcinogens
J. Kůsová, M. Kašová, H. Tomášková
Regional Institute of Public Health, Ostrava, Czech Republic
Professional safety of workers’ health is usually controlled using an external dose of a
hazardous compound (using its chemical identification) and legislatively fixed limit.
However, biological effect like genotoxic (especially carcinogenity) has multifactorial
character. Real complex mixtures from the environment include except for measured
contaminants a lot of unknown chemical, physical and biological components. The
interactions among all of them are complicated not only outside the organism, but even more
inside it. Biological identification of resulting genotoxic effect could develop more effective
beneficial intervention into this safety.
The use of cytogenetic assays in the surveillance of population occupationally exposed (it
usually means continuous more factors exposure) to genotoxic compounds originates from the
assumption that chromosomal aberrations (CHA) might be causally involved in the early
stages of carcinogenesis and from the results of epidemiological studies, which confirmed
associations between increased risk of development of cancer and increased level of CHA. In
case of CHA detection in peripheral blood of employees professionally exposed to mutagens,
carcinogens and its mixtures, it is possible to detect real biological (genotoxic) effect in vivo
of a complex mixture from the whole environment (including possible cumulative effect).
In Czech Republic (continuing the practice applied in former Czechoslovakia) some
occupationally exposed subjects have been monitored for level of CHAs. CHA detection via
conventional cytogenetic analysis of peripheral blood lymphocytes (CAPL) was used.
This paper presents results of observed groups of workers during 2005 – 2007 periods from
Moravskoslezsky region.
67

Biological and chemical evaluation of Ostravian urban air
J. Kůsová, H. Miturová, M. Kašová, H. Tomášková
Regional Institute of Public Health, Ostrava, Czech Republic
The agglomeration Ostrava-Karvina is one of the most polluted regions both in the Czech
Republic as well as in Europe. Population of the region (consequently of Ostrava city) is
exposed to enormous polluted air. Air quality is frequently evaluated by comparison of
concentration of contaminants with their legislative limits. However, the real environmental
air is a complex mixture of many contaminants (measured and unknown) acting
simultaneously. Their interactions are complicated, not only on the chemical but even more
on the biological level. Especially concerning biological hazard like genotoxic (DNA
damages) effect it seems to be legitimately to take results of biological tests into account first
and foremost.
Our institute provides monitoring of the ambient air in the region. This study elaborates
results of it at three industrial sites of Ostrava (Přívoz, Mariánské Hory a Bartovice) during
2004 – 2007 years. Chemical analyses of selected carcinogenic and mutagenic substances
(about 60 measurements for every pollutant, every year and every site) were carried out and
concurrently air samples were tested using Ames test (the most widely used test to identify
biological – mutagenic effect of air complex mixture).
Annual concentrations of some measured pollutants at observed sites were compared with
their limits. Associations between biological (mutagenic) effect of the air complex mixture
and levels of pollutants for every site and every year were evaluated by correlation analysis.
Annual values of benzo/a/pyrene concentration exceed the annual limit at all sites (Přívoz,
Mariánské Hory, Bartovice), the values of arsenic at two monitored sites (Mariánské Hory,
Bartovice). The benzene annual means exceeds the limit in one site only (Přívoz) during this
period.
As to correlations between biological (mutagenic) effect and levels of pollutants, various
results were found for individual sites. In general, close correlations were noted between
mutagenic effect and levels of polycyclic aromatic hydrocarbons PAHs (although at the most
polluted site with PAHs - Bartovice the correlating coefficients were not the biggest). Weakly
correlations were found between mutagenic effect and arsenic levels (especially at the site
least polluted by metals - Přívoz) and between mutagenic effect and the presence of volatile
compounds (benzene, trichlorethene – not at the most polluted site Přívoz).
Our study confirms that interactions among components of the complex mixture are
complicated and that resulting biological effect is determined by these interactions more than
by legislative limit only. Although taking into account mutagenic potency of Ostravian air the
major contribution seems to occur due to PAHs action. For protecting the populations of
industrially polluted areas biological evaluation of the air/ environment could develop more
effective beneficial intervention into these associations than chemical analyses by themselves.
68

Cell death, stress and regeneration induced by Boron-Neutron-Capture Reaction
(BNCR) in situ and in culture
V. Mareš 1,2 , J. Burian J 3 , V. Lisá 1 , M. Marek 3 , I. Tomandl 4
1 st
Joint Cancer Cell Biol. Lab. (Ac. Sci. C.R. & 1 Med. Faculty, Charles University), Prague
2
University of J. E. Purkinje, Ústí nad Labem
3
Nuclear Res. Institute Řež, plc
4
Nuclear Physics Inst., Ac. Sci., Řež
BNCR is a promising tool for target therapy of brain tumors. The therapeutic success depends
on concentration of 10 B and thermal neutron dose delivered to tumor target. Here, we
demonstrate BNCR in 2 novel experimental biological models suitable for pilot tuning of
irradiation conditions.
1-week-old rat sucklings, used as an in situ model, were injected either with sodium-
borocaptate (BSH) intracranially (i.c.), or subcutaneously (s.c) or 10 B-phenylalanine-fructose
(BPA-F, both drugs from Katchem, Řež, in dose equivalents 50-150 10 B per g b.w) and were
irradiated 90 min later by epithermal neutron beam for 5 min (LVR-15 reactor Řež, 8xx
10 8 n/cm 2 , 9MW, calculated dose 16.1 cGy/min).
It the brain, massive cell death occurred in the germinative regions of the cerebellum and the
forebrain. The lethal BNCR effect exceeded the beam effect per se (Compound Factor, CF)
2.5 to 5 times. No significant damage was observed there after s.c. injection of BSH even at
subtoxic doses. The critical 10 B content in the tissue target (determined by Prompt γ
Activation) was above 20 ug/g brain. This was achieved only by i.c. administration of BSH or
s.c. application of BPA-F which was found to be better tolerated and easier penetrating into
the brain. Moreover, the latter was found to be stimulated by hyperthermia.
In cell cultures fed with BSH or BPA (an equivalent of 100 ug 10 B per ml media, 30 min
irradiation), the lethal effects determined by the number of floating and shrinked cells
appeared after 48 h and were increasing up to 196 h. Surviving cells became hypertrophic
and, as suggested by the deep decrease of the population density, they were inhibited in
cycling. The BNCR induced cytostatic effect started appearing earlier (24 h) and lasted longer
(196 h). The CFs for lethal and cytostatic effect for both drugs ranged from approx. 2 to 7 at
the peak values. Moreover, while population density in cultures irradiated without 10 B started
increasing since 72 h due to recovery of cell division; this has not occurred after BNCR. As
shown by cytochemistry, the cells surviving BCNR up-regulated γ-glutamyltranspeptidase
(γ-GT).
We showed that (i) BCNR induced by the LVR-15 reactor epithermal beam significantly
exceeds the effects of parasitic γ and fast neutrons in both used models. (ii) The s.c.
administration of BPA, especially when combined with hypethermia, enabled induction of
BNCR in the in situ immature rat brain model. (iii) Up-regulation of γ-GT appears to be a
mechanism interfering with therapeutic effects of BNCT.
Supported byNucl.Res. Inst.Řež, plc Project MSM2672244501 and AV0Z50110509.
69

Polymorphisms of GSTM1, GSTT1 and GSTP1 genes in relation to breast cancer
susceptibility.
T. Matáková 1 , E. Halašová 2 , M. Sivoňová 1 , E. Huľo 3 , Ľ. Javorka 4 , P. Žúbor 5 , D. Dobrota 1
1 Department of Medical Biochemistry, Jessenius Faculty of Medicine, Martin
2 Department of Medical Biology, Jessenius Faculty of Medicine, Martin
3 Clinic of Surgery, Jessenius Faculty of Medicine and MFH, Martin
4 Department of Childbirth Asistance Faculty of Health Catholic University in Ružomberok
5 Clinic of Gynaecology, Jessenius Faculty of Medicine and MFH, Martin
The role of the glutathione S-transferase (GST) enzyme family is to detoxify environmental
toxins and carcinogens and to protect organism from their adverse effects, including cancer.
The genes GSTM1, GSTT1 and GSTP1 code for three GSTs involved in the detoxification of
carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) and benzene. In humans,
GSTM1 is deleted in about 50% of the population, GSTT1 is absent in about 20%, whereas the
GSTP1 gene has a single base polymorphism resulting in an enzyme with reduced activity.
Epidemiological studies indicate that GST polymorphisms increase the level of carcinogeninduced
DNA damage and several studies have found a correlation of polymorphisms in one
of the GST genes and an increased risk for certain cancers.
We examined the role of polymorphisms in genes coding for these three GST enzymes in
breast cancer in Slovak women. The study population consisted of 155 incident breast cancer
cases and 189 healthy population controls. Genotyping analyses were performed by
polymerase chain reaction (PCR) for the presence or absence of the GSTM1 and GSTT1 genes
and for GSTP1 single base polymorphisms by PCR/RFLP, and odds ratios (ORs) and 95%
confidence intervals (CIs) were calculated by unconditional logistic regression adjusting for
know or suspected risk factors for breast cancer.
In the case-control study, associations were null for GSTM1 [OR 1.01, 95% confidence
interval (CI):0.6-1.9], GSTT1 [OR 1.4, 95% confidence interval (CI):0.6-1,5] and GSTP1 [OR
0.8, 95% confidence interval (CI):0.5-1.4]. Single polymorphisms of genes GSTM1, and
GSTP1 did not show an association with breast cancer. Only GSTT1 homozygous null
genotype was associated with increased risk of developing breast cancer (OR 1.4), but it was
not statistically significant (P=0.29).
70

Genome differences in the genus Treponema
L. Mikalová, M. Strouhal, P. Matějková, D. Šmajs 1
1 Department of Biology, Masaryk University, Faculty of Medicine, Brno, Czech Republic
The genus Treponema comprises five noncultivable species and subspecies showing various
degrees of invasiveness and pathogenicity to humans. Genomes of 6 strains including Mexico
A, DAL-1 (Treponema pallidum subsp. pallidum), Gauthier, CDC-2 (T. pallidum subsp.
pertenue), Bosnia A (T. pallidum subsp. endemicum), Fribourg-Blanc (unclassified simian
isolate) were compared to the genome of the syphilis spirochete Treponema pallidum subsp.
pallidum strain Nichols using the whole genome fingerprinting (WGF) technique. The
chromosomal DNA was amplified in 130 overlapping regions (TPI intervals) with a median
length of 9,792 bp using a GeneAmp XL PCR kit (Applied Biosystems). Each PCR product
was digested with BamH I, EcoR I and Hind III restriction endonucleases, respectively. To
precisely assess the possible deletions and insertions in the restriction fragments, additional
digestions were performed to reduce the length of each restriction fragment to ≥ 4 kb. This
was achieved by additional digestion with Acc I, Cla I, EcoR V, Kpn I, Mlu I, Nco I, Nhe I,
Nsi I, Rsr II, Sac I, Sap I, Spe I, Sph I, Xba I and Xho I restriction endonucleases or their
combinations. The resulting restriction profiles were used for identification of heterologous
regions in the chromosomal DNA of analyzed strains. Using WGF, deletions, insertions and
nucleotide polymorphisms were found. The length of deletions and insertions ranged
approximately from 30 to 500 bp. The exact nucleotide length of these sequence changes will
be determined by DNA sequencing. Restriction profiles of several TPI intervals indicate that
genomes of T. pallidum subsp. pallidum, T. pallidum subsp. pertenue and T. pallidum
Fribourg-Blanc are closely related and most of the observed sequence differences are
localized in tpr genes and the vinicity of these genes, suggesting their possible role in the host
range and pathogenicity of T. pallidum subsp. pallidum. The high level of relatedness of the
T. pallidum subsp. pallidum and T. pallidum subsp. pertenue genomes will be used in
identification of T. pallidum subsp. pallidum virulence determinants.
This work was supported by grants 310/07/0321 from the Grant Agency of the Czech
Republic and NR/8967-4/2006 from Ministry of Health of the Czech Republic.
71

Current findings on genetic regulation of the uropoetic system in fetus and its share in
the incidence of reflective uropathy
R. Molíková 1 , A. Šantavá 2 , M. Godava 2 , O. Šmakal 3 , J. Šantavý 2 , M. Bezdičková 1 , O. David 4
1
Dept. of Anatomy, Faculty of Medicine and Dentistry Palacký University Olomouc, Czech
Republic
2
Dept. of Medical Genetics and Fetal Medicine, University Hospital and Faculty of Medicine
and Dentistry Palacký University Olomouc, Czech Republic
3
Dept. of Urology, University Hospital and Faculty of Medicine and Dentistry Palacký
University Olomouc, Czech Republic
Structural abnormalities of the kidneys and the urinary tract are amongst the most frequent
congenital abnormalities; its incidence in the general population is known to be around 1%.
These anomalies may occur in the form of monogenetically inherited disorders with a known
gene defect, as part of the polymalformation syndromes where causal mutation is frequently
discovered, or due to chromosomal numerical and structural aberrations. Another group
comprises of isolated abnormalities of the ureteric bud development, most frequently leading
to vesico-ureteric reflux (VUR) which is manifested by recurrent urinary tract infections.
Despite the available pharmaceutical and surgical treatments, VUR remains the cause of renal
failure in ¼ of infants and in 1/5 of all adult patients. One of the causative theories is a
primary aberration in the renal parenchyma.
Recently, 35 candidate genes that probably have their share in ureteric bud development
defects were identified in mouse studies. The position of the ureteric bud is a determining
factor for normal embryogenesis of the kidneys and the urinary tract. Signal molecules and
transcription factors with expression in the mesonephritic channel and in the metanephritic
mesenchyme, which determine this position, were experimentally discovered. One of the
main ones is the transcription factor PAX2, which is expressed during the development of the
mesonephritic channel, the ureteric bud and the metanephritic mesenchyme, and also the glial
neurotropic factor that is expressed in the mesenchyme along the mesonephritic tract prior to
the formation of the ureteric bud. Normal development of the kidneys and the upper urinary
tract is, in addition to PAX2, also influenced by other transcription factors that regulate the
expression of the glial neurotropic factor in the metanephritic mesenchyme.
The incidence of VUR in monozygotic twins, and familiar incidence and the increased risk of
VUR in relatives support the probable hypothesis of autosomal dominant heritability with
incomplete penetration. The aim of the current study is not only to understand the role of gene
regulation in ureteric bud development but also to detect those genes the mutation of which
leads to VUR, or whether these genes are a predisposing factor in primary aberration of the
renal parenchyma.
72

11 DNA microsatellites for the study of paternity in the meadow pipit (Anthus pratensis)
P. Nádvorník 1 , V. Pavel 2 , L. Kučerová 1 , S. Bureš 2
1 Department of Cell Biology and Genetics, Faculty of Science, Palacký University,
Šlechtitelů 11, 783 71, Olomouc, Czech Republic, petr.nadvornik@upol.cz
2 Department of Zoology and Laboratory of Ornithology, Faculty of Science, Palacký
University, Tř. Svobody 26, 771 46, Olomouc, Czech Republic
The study of extra-pair parentage is performed by the microsatellites analysis. The search for
the new microsatellite loci from a ´new´ species usually requires the cloning and sequencing
of their DNA. An alternative way is a cross-species amplification of their genomic DNA with
primers, which are known in other species usually from the same class.
We collected blood samples of 116 adult individuals (males and females) caught in the
Jeseníky Mountains, Northern Moravia, the Czech republic in three years (1999, 2000 and
2001), in the Krkonoše Mountains in the north of the Czech republic (2001 and 2002), and in
the Tydal province in central Norway (1998 and 2000). The DNA from blood in the storage
buffer was isolated using the phenol – chloroform method. 70 Fhy primer pairs (Leder et. al,
2008) were used for the PCR amplification in 8 randomly chosen individuals of meadow
pipit; these primer pairs were previously used to amplify microsatellite loci in pied flycatcher
(Ficedula hypoleuca) and some of them in collared flycatcher (F. albicollis), bluethroat
(Luscinia svecica) and Siberian jay (Perisoreus infaustus). The DNA fragments were run
through 6% denaturing polyacrylamide gel. The visualization of the DNA fragments was
accomplished using silver treatment. At first we used identical PCR conditions as was
described in the case of the original species. Secondly, we tested higher annealing
temperatures (max. 62 °C) for primer pairs, which showed an unscorable multi-band pattern
and lower annealing temperature (min. 48 °C) for primer pairs, which showed no product in
PCR reaction.
After the PCR amplification we excluded all the microsatellite loci, which produced
ambiguous band patterns – no product, poor amplification, confusing stutter bands or multiple
products. 11 of 70 tested primer pairs were to successfully work in meadow pipit for paternity
studies: Fhy 224 (5 alleles), Fhy230 (4 alleles), Fhy234 (2 alleles), Fhy235 (2 alleles), Fhy303
(5 alleles), Fhy310 (2 alleles, occurrence of null allele), Fhy326 (4 alleles), Fhy336 (6 alleles),
Fhy361 (5 alleles), Fhy405 (7 alleles) and Fhy407 (3 alleles).
Leder, E.H., Karaiskou, N. and Primmer, C.R. (2008): Molecular Ecology Resources (8),
874-880.
This work was supported by grant from the Czech Ministry of Education (MŠM
6198959212).
73

Heavy metals induce phosphorylation of the Bcl-2 protein by Jun N-terminal kinase
E. Ondroušková 1 , J. Slováčková 1,2 , V. Pelková 1,3 , J. Procházková 1,4 , K. Souček 4 , P. Beneš 1 ,
J. Šmarda 1
1
Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlářská 2,
611 37 Brno, Czech Republic.
2
Department of Pathology, University Hospital, Jihlavská 20, 625 00 Brno, Czech Republic.
3
Department of Pediatric Hematology and Oncology, 2nd Medical School, Charles
University, V Úvalu 84, 150 06 Prague, Czech Republic.
4
Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135,
612 65 Brno, Czech Republic.
The Bcl-2 protein is one of the key components of biochemical pathways controling
programmed cell death. Function of this protein can be regulated by posttranslational
modifications. Phosphorylation of Bcl-2 has been considered to be significantly associated
with cell cycle arrest in the G2/M phase of the cell cycle, and with cell death caused by
defects of microtubule dynamics.
In this study, we show that heavy metal-induced stress and cell death correlate with induction
of the Bcl-2 protein phosphorylation in several cell lines. Zinc-induced phosphorylation of
Bcl-2 is mediated by the Jun N-terminal kinase pathway, and it is not linked to cell cycle
arrest at G2/M. Cells of breast carcinoma cell line MDA-MB-231 expressing the wild-type
Bcl-2 protein are more resistant to zinc-induced cell death than those expressing mutant
variant of Bcl-2 that cannot be phosphorylated at amino acid residues Ser70, Ser87, Thr69.
These results suggest that phosphorylation of the Bcl-2 protein is an important part of cellular
response to the stress and that this posttranslational modification is significantly involved in
control of the Bcl-2 protein function.
This work was funded by grant MSM0021622415 of the Ministry of Education, Youth and
Sports of the Czech Republic, grant 301/06/0036 of the Grant Agency of the Czech Republic,
grant IAA501630801 of the Grant Agency of Academy of Sciences of the Czech Republic,
grant 204/07/0834 of the Czech Science Foundation and by the Research Plan
AVOZ50040507 of Academy of Sciences of the Czech Republic.
74

Antidotes against nerve agents versus tularemia disease progress
O. Pavlis 1 , M. Pohanka 2,3 , J. Pikula 4 , F. Treml 5 , K. Kuca 2,3
1 Centre of Biological Defence, Techonin, Czech Republic
2 Centre of Advanced Studies, Faculty of Military Health Sciences, University of Defense,
Hradec Kralove, Czech Republic
3 Department of Toxicology, Faculty of Military Health Sciences, University of Defense,
Hradec Kralove, Czech Republic
4 Department of Veterinary Ecology and Environmental Protection, University of Veterinary
and Pharmaceutical Sciences Brno, Czech Republic
5 Department of Infectious Diseases and Epizootiology, University of Veterinary and
Pharmaceutical Sciences Brno, Czech Republic
Cholinesterase antidotes are the drugs commonly used to treat persons exposed to nerve
agents. This experimental work has been engaged with studying modulation effects of
anticholinergic antidotes on infection progress. We used BALB/c mice and the causative
agent of tularemia, i.e., Francisella tularensis, to induce a model bacterial infection. In vivo
tests revealed interesting effect differences resulting from antidotes administration to mice.
Noticeable decrease of mortality due to tularemia was observed when the dose of antidotes
was increased. The achieved data are only preliminary one. It will repeat on laboratory animal
model again.
This work was supported by the Ministry of Defense of the Czech Republic (Grant No.
FVZ0000604).
75

Tularemia diagnosis using piezoelectric immunonosensor
M. Pohanka 1,2 , O. Pavliš 3 , P. Skládal 4
1
Centre of Advanced Studies, Faculty of Military Health Sciences, University of Defence,
Hradec Kralove, Czech Republic
2
Department of Toxicology, Faculty of Military Health Sciences, University of Defence,
Hradec Kralove, Czech Republic
3
Center of Biological Defense, Těchonín, Czech Republic
4
Department of Biochemistry, Masaryk University, Czech Republic
A piezoelectric immunosensor for indirect diagnosis of tularemic infection in mouse serum
was developed. Francisella tularensis LVS antigen was covalently immobilized on the
sensing surface using cystamine and glutardialdehyde for activation and modification of the
gold electrode. The normal mouse serum (NMS) and serum prepared from mice immunized
by Escherichia coli was used as negative control providing signal of 28 Hz during a 5 min
interaction. The tularemic infectious (immunized) mouse serum (IMS) as sample resulted in
the signal above 75 Hz (5th day after infection). Control sensor contained bovine albumin as
sensing element provided signal below 5 Hz with NMS as well IMS. The effects of dilution
degree and purification of sera were tested. To improve resolution of the method, sample
pretreatment steps as precipitation with ammonium sulphate and immunoglobulin extraction
on CBind TM L and MEP Hyper Cel columns were tested. RSD of measurements was 2.3% for
NMS and 2.4% for IMS respectively. The developed method allows indicating the presence
of anti tularemic antibodies shortly (1-3 days) after infection, one analysis is completed in 10
min.
Supported by the Ministry of Defense of the Czech Republic (Grant No. FVZ0000604).
76

Detection of aflatoxins in capsicum spice using an electrochemical immunosensor
M. Pohanka 1,2 , F. Malir 3,4 , K. Kuca 1,2 , T. Roubal 3,4 ,
1
Centre of Advanced Studies, Faculty of Military Health Sciences, University of Defence,
Hradec Kralove, Czech Republic
2
Department of Toxicology, Faculty of Military Health Sciences, University of Defence,
Hradec Kralove, Czech Republic
3
Institute of Public Health, Department of Xenobiochemistry, National Reference Laboratory
for Biomarkers of Mycotoxins, Hradec Kralove, Czech Republic
4
Pedagogic Faulty, Department of Biology, University Hradec Kralove, Czech Republic
Electrochemical immunosensor based on immobilized aflatoxin B1 (AFB) – albumin
conjugate and polyclonal antibody against AFB1 was performed for a competitive assay of
AFB1 in capsicum spice. Spiked samples were used for construction of calibration curve. The
proved limit of detection was 2.4 ppb. The long term stability of immunosensor was also
estimated. The decrease of immunosensor sensitivity was less than 10% when storaged in a
fridge and approximately 22% for the immunosensor preserved in laboratory temperature for
two weeks. The consequent performance of immunosensors for assay of real capsicum spice
samples with proven aflatoxins presence exerted good correlation with the data from valid
method (high-performance liquid chromatography with fluorescence detector).
This work was supported by the grant of the Ministry of Industry and Trade of the Czech
Republic, Grant No. 2A-1TP1/009 that is gratefully acknowledged.
77

The involvement of mitochondrial pathway in etoposide-induced demise of melanoma
cells
K. Rudolf, E. Rudolf 1 , M. Červinka 1
Department of Rheumatology and Clinical Pharmacology, 2 nd Internal Clinic, Faculty
Teaching Hospital in Hradec Králové, Sokolská 581, 500 05 Hradec Králové, Czech Republic
1 Department of Medical Biology, Charles University in Prague, Faculty of Medicine in
Hradec Králové, Hradec Králové, Czech Republic
Melanoma represents the most dangerous form of skin cancer which is responsible for
majority of skin cancer deaths. Unfortunately, when it progresses into metastatic stage it
becomes highly resistant to existing therapies including cytotoxic chemotherapeutic drugs.
Etoposide is a potent antineoplastic agent whose cytotoxicity stems primarily from the
inhibition of toposiomerase II in target cells in addition to complex interaction pattern with
various proapoptotic pathways. Due to this complexity and proposed lack of cytotoxic
efficiency in malignant melanoma our knowledge about individual molecular targets of
etoposide in this type of malignancy remains cursory. Thus the aim of this study was to study
cytotoxicy and apoptosis induced by etoposide during 72h in human melanoma cell line
Bowes. Etoposide initiated DNA-damage signaling via ATM kinase and activated p53
pathway and caspase-2. In response to treatment with etoposide, mitochondria of melanoma
cells first increased their abundance and activity and only at later treatment intervals
underwent dynamic and functional suppression. Observed mitochondrial perturbation was not
preceded by membrane potential loss but cytochrome c release was observed together with a
rise in caspase-9 and caspase-3 activities. The pharmacological inhibition of relevant induced
targets proved the importance of ATM and caspase-2 in etoposide-mediated cytotoxicity and
apoptosis.
This work was supported by Ministry of Education of the Czech Republic Research Project
MSM0021620820.
78

Lignans from Schisandra chinensis restore the cytotoxic action of doxorubicin in drug
resistant lung cancer cells
I. Slaninová, J. Slanina 1 , I. Tomalová 1 , L. Březinová 1 , M. Broošová, L. Koubíková,
K. Krestová
Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
1 Department of Biochemistry, Faculty of Medicine, Masaryk University, Brno,
Czech Republic
Dibenzo[a,c]cyclooctadiene lignans are natural products originated from Schisandra chinensis
(Schisandraceae), a well-known medicinal plant in traditional Chinese medicine. The fruits
and seeds have been used for centuries as a tonic and antitussive. Many studies have indicated
that the active ingredients are lignans possessing an unusual structure derived from
dibenzo[a,c]cyclooctadiene These lignans have been shown to possess a broad range of
biological effects, including hepatoprotective and antiviral properties. Recently,
dibenzocyclooctadiene lignans have been discussed as compounds that are able to overcome
multidrug resistance.
A panel of nine dibenzo[a,c]cyclooctadiene lignans (schizandrin, gomisin A, gomisin N,
gomisin J, angeloylgomisin H, tigloylgomisin P, deoxyschizandrin, γ-schizandrin and
wuweizisu C), isolated by us from seeds of S. chinensis, was examined for their effect on
multidrug resistance, as well as their anti-proliferative and pro-apoptotic activities. COR-
L23/R, a multidrug-resistant sub-line over-expressing multidrug resistance-associated protein
1 (MRP1) together with its parent cell line COR-L23 (human lung cell carcinoma) and HL60
(human promyelocytic leukemia) cell lines were used. We found that deoxyschizandrin and γschizandrin
at relatively non-toxic concentrations restored the cytotoxic action of
doxorubicin, a MRP1 substrate, to COR-L23/R cells. Moreover, deoxyschizandrin and γschizandrin
showed the greatest ability to enhance the accumulation of doxorubicin in drug
resistant cells. Lignans alone had no effect on the cell cycle, however, in combination with
sub-toxic doses of doxorubicin, cell were arrested in the G2/M phase, which is typical for
doxorubicin at toxic doses. Our results suggest that deoxyschizandrin and γ-schizandrin
potentiate the effect of doxorubicin in doxorubicin resistant lung cancer cells by increasing
the accumulation of doxorubicin inside the cells. The common structural feature of both
active lignans and missing from the less active or inactive lignans is the R-biaryl
configuration and the absence of a hydroxy group at C-8. Unlike the reversion effect, the
cytotoxicity of lignans with either the R-biaryl or S-biaryl configurations was approximately
same.
This work was supported by the Czech Science Foundation (project No. 522/07/0995) and the
Ministry of Education of the Czech Republic (VZ MSM0021622415 and LC06077).
79

Natural products in chemoprevention of colon cancer
L. Schröterová, P. Hašková 1 , E. Rudolf, M. Červinka
Department of Medical Biology, Charles University in Prague, Faculty of Medicine in Hradec
Králové, Hradec Králové, Czech Republic
1
Department of biochemical sciences, Charles University in Prague, Faculty of Pharmacy,
Hradec Kralove, Czech Republic
It is widely accepted fact that diet plays a role in the etiology of certain cancers, particularly
in colon cancer. Incidence of colorectal cancer is increasing in the Czech Republic and the
disease does not respond well to cytostatic treatment. Therefore it is necessary to broaden the
spectrum of chemopreventive and therapeutic possibilities. One attractive source of
potentially useful chemicals comprises a various molecules originating from vegetables and
fruits which are often found in our diet. Phytic acid as well as selenium are prime example.
Phytic acid as well as several selenium compounds have been studied in in vitro models as
potential anti cancer agents. Their anti-cancer effects are associated with inhibition of cell
proliferation and induction of apoptosis.
We studied the effect of seleno-L-methionine (SeMet), Se-methyl-L-selenocysteine (SeCys)
and phytic acid on proliferation, metabolic activity and apoptosis in three colorectal cell lines
with different malignant potential (HT-29, SW 480 and SW 620). Generally, SeCys proved to
be more efficient in inducing apoptosis and inhibiting of cell proliferation in employed
colorectal cells compared to SeMet. Phytic acid potent the anti-cancer effect of tested
selenium compounds. In conclusion, this study demonstrates the ability of chosen selenium
compound and phytic acid alone or in combination to reduce the proliferation rate of model
cell lines and increases the proapoptotic effect.
This work was supported by Grant Agency of the Czech Republic (grant 301/06/P047) and
Ministry of Education of the Czech Republic (grant MSM0021620820).
80

The induction of inter-strand DNA cross-links with sulphur mustard (|SM) in normal
Chinese hamster cells AA8 and their DNA repair defective mutants UV5 and UV-20
R. Štětina, M. Jílková
Department of Toxicology, Faculty of Military Health Sciences, Hradec Králové,
Czech Republic
The mechanism of the cytotoxic effect of SM is not fully clarified. SM is known to form
adducts with DNA bases. It binds predominantly at the N7 position of guanine and N3
position of adenine. It can bind also to two guanines positioned in opposite DNA strands and
so to form interstrand cross-links (ICL) in the DNA molecule. ICL are regarded as cytotoxic
DNA lesions inhibiting the DNA replication. Repair mechanisms by which they are removed
from the DNA have not been elucidated.
The nucleotide excision repair (NER) has been suggested to play a crucial role in the SM –
induced ICL removal. We have studied the induction and the repair of ICL in Chinese
hamster ovary mutant cell lines UV- 5 and UV-20 deficient in the incision step of NER using
the comet assay modified for the estimating the number of ICL. While UV-5 line is defective
in ERCC-4 helicase, UV-20 is unable to incise the DNA damage due to the mutation in the
5´- 3´ endonuclease (ERCC-1).
When we measured the inhibition of colony forming capacity of these cell lines we have
found that the UV-5 mutant cells did not differ from the parental wild type line AA8
concerning the sensitivity to SM (LD50 0.2 µM), while UV-20 cells were then 10 times more
sensitive. One hour- treatment with SM induced the dose – related amount of ICL in DNA of
treated cells.The amount of induced ICL was practically the same both in normal AA8 cells
and SM- sensitive mutant UV-20 cells when treated within the range of concentrations from
0.3 to 5 µM. When comparing the repair of ICL in those two cell lines, we found almost no
ICL repair within 2 hours interval after the SM treatment. However substantial proportion of
ICL was removed from the DNA after 24 hours of incubation. In cells treated up to
concentrations of 1.25 µM the ICL were removed completely at this time interval of repair,
while about 30 % or 50% of ICL were removed in cultures treated with 2.5 or 5 µM of SM
respectively. Surprisingly, the repair rate of ICL removal was the same both in normal AA8
and mutant UV-20 cells, showing that the NER, which is defective in UV-20 cells, probably
is not the key mechanism for the removing of ICL. Inhibitors of the polymerisation step of
NER 10 -4 M 1-β-D-furanosylcytosine and 10 -2 M hydroxyurea added into the culture medium
of SM – treated cells for the 24 h period of repair strongly inhibit the ICL removal. The DNA
replication is probably an important step for the repair of ICL. The data may suggest, that the
recombination DNA repair may be the mechanism responsible for the ICL removal.
Acknowledgement: This work was supported by the grant of Ministery of Defence of the
Czech Republic no: OPUOFVZ200603
81

High speed digital photography with consumer digital camera Casio Exilim Pro EX-F1:
Examples of applications in biology
F. Weyda
Biology Center of the Academy of Sciences of the Czech Republic, Institute of Entomology,
Laboratory of Digital Imaging in Entomology and Faculty of Science, University of South
Bohemia, Branisovska 31, CZ-37005 Ceske Budejovice, Czech Republic
In biology there are various processes characterized by high speed. Mostly it is difficult to
document them with current cameras or camcorders. We need specialized and expensive high
speed cameras. Consumer camcorders work usually at frequency 30-50 frames per second
(fps). Exceptionally, some camcorders use 200 fps for very limited time (like 3 seconds).
Consumer digital camera modell Casio Exilim Pro EX-F1 (appeared in end of March 2008)
use sequentional photography at 60 fps (full resolution 6 MP) or videoclips at 300, 600 or
even 1200 fps. It cover many high speed biological processes. In addition to that high speed
recording it can record also two formats: 1) „full HD“ videoclips (1920x1080 pixels) at 60 fps
and 2) „HD“ videoclips (1280x720 pixels) at 30 fps.
We have studied of various biological problems like feeding behaviour of horse chestnut
leafminer larvae, Cameraria ohridella (Lepidoptera, Gracillariidae) at 1200 fps. We were
able to determine speed of mandible moving during feeding. Further, we have also studied
cleaning behaviour of parasitic wasp Closterocerus trifasciatus (Hymenoptera) parasiting on
chestnut leafminer larvae. Digital camera Casio enable us to record flight of insects and
predator birds, jumping of spring-tails (Collembola) and various insects, stridulation of
grasshoppers (Orthoptera) etc.
Recently we try to connect this modell of digital camera to microscope. Special
optomechanic connection adapter between that particular camera (Casio Exilim Pro EX-F1 is
EVF camera, not DSLR) and microscope still does not exists. We will demonstrate some our
preliminary results.
Casio Exilim Pro EX-F1 is capable to record pictures at UV light (350-400 nm) which is
important ability for biological research. On the opposite, it is difficult to record pictures at
N-IR (near infrared light) with this modell.
We edit videoclips (MOV format, Quick Time) in several video editing programs that allows
us to delete unwanted scenes from video files or other serious manipulations. Does not matter
if they are commercial (like Pinnacle Studio) or freeware programs (Virtual Dub).
Acknowledgements
We would like to thank Lubomír Volter for kind determination of parasitic wasps
(Hymenoptera).
This work was supported by grant No. 2B06005 (MSMT, Czech Republic).
82

Autofluorescence of fruiting bodies of the wood-rotting fungus Piptoporus betulinus
Z. Žižka, J. Gabriel
Institute of Microbiology Academy of Sciences, v.v.i., Praha, Czech Republic
The fruiting bodies of Piptoporus betulinus (Basidiomycetes; Hymenomycetes; Polyporales;
Trametoidae) were collected on birch trunks (Betula sp.) in the Krč forest in 2004-2007. Their
autofluorescence (primary fluorescence) was studied by using a Zeiss Jenalumar fluorescence
microscope at a blue (excitation filters: B 226 g, B228 g, B 422 g) and green (excitation
filters: KP 560 – 2x, G 247, B 424 g) excitation. The images were recorded on a
microphotographic device Zeiss on the negative film Kodak Max 400. The surface of the P.
betulinus fruiting bodies often displayed spherical bodies with strong red autofluorescence at
both blue and green excitation. These belonged to coccal algae whose cells sometimes
divided. Apart from the fluorescing hyphae, dark nonfluorescent ones were found in places
where the surface layer was damaged (fissures due to aging). The fluorescent hyphae
exhibited a relatively weak yellow-green autofluorescence at blue excitation and a weak red
autofluorescence at green excitation. Other fluorescent bodies found there were numerous
granules of different sizes with homogeneous or inhomogeneous contents exhibiting intensive
yellow fluorescence at blue excitation and red autofluorescence at green excitation. The
inside of the fruiting body in the trama showed two types of hyphae (a dimitic system) –
smaller-diameter generative hyphae and skeletal larger-diameter hyphae. Both types
possessed mild yellow-green autofluorescence at blue excitation and a mild red fluorescence
at green excitation, as compared with the strong yellow autofluorescence of the granules.
Hyphal walls usually evinced a stronger autofluorescence than their contents, but opposite
situations were also found. In some parts of the fruiting bodies the granules were much more
numerous and exhibited a higher intensity of fluorescence than the hyphae. On comparing the
autofluorescence of hyphae of the wood-rotting fungus with the primary fluorescence of the
fungus Fomes fomentarius the fluorescence intensity of the former is seen to be much weaker
and can sometimes disappear (viz. the dark hyphae in the surface layers of the pileus). P.
betulinus differed from other fungi also in the autofluorescence of hyphal walls, which in this
fungus was stronger than that of hyphal contents. The large number of yellow-fluorescent
granules found in this fungus was also not found in other fungi, e.g. F. fomentarius, Daedalea
quercina, Fomitopsis pinicola, Macrolepida rhacodes and others.
The work was supported by grant 526/07/0620 and the Research Concept AV 0Z 5020 0510.
83

Abecední seznam účastníků
předem registrovaných účastníků
MSc. Eva Babušíková, PhD.
Ústav lekárskej biochémie
Jesseniova lekárska fakulta, Univerzita Komenského v Bratislave,
Malá Hora 4, 036 01 Martin, Slovensko
e-mail: eva.babusikova@gmail.com
Jiří Bártek, Professor, M.D., Ph.D.
Danish Cancer Society
Institute of Cancer Biology
Strandboulevarden 49, DK-2100 Copenhagen Denmark
e-mail: jb@cancer.dk
RNDr. Eva Bártová, Ph.D.
Biofyzikální ústav AV ČR Brno
Královopolská 1356, 612 65 Brno
e-mail: bartova@ibp.cz
RNDr. Jan Bednár, Ph.D.
Ústav buněčné biologie a patologie
1. lékařská fakulta Univerzita Karlova v Praze
Albertov 4, 128 01 Praha 2
e-mail: jbedn@lf1.cuni.cz
Mgr. Vladan Bernard
Biofyzikální ústav
Lékařská fakulta, Masarykova univerzita Brno
Kamenice 3, 625 00 Brno
e-mail: vbernard@med.muni.cz
Mgr.Michal Bitman,
Katedra farmakologie a toxikologie
Farmaceutická fakulta UK Hradec Králové
Heyrovskeho 1203, 500 05 Hradec Králové
e-mail: michal.bitman@faf.cuni.cz
Mgr. Kateřina Brigulová
Ústav lékařské biologie a genetiky
Lékařská fakulta UK v Hradci Králové
Šimkova 870, 500 38 Hradec Králové
e-mail: brigulovak@lfhk.cuni.cz
Ing. Vítězslav Březina, CSc.
Stomatologické výzkumné centrum
Lékařská fakulta Masarykovy univerzity Brno
Komenského nám. 2, 662 43 Brno
e-mail: brezinavita@gmail.com
84

RNDr. Milena Bušová, CSc.
Ústav biochemie, chemie a biofyziky
Veterinární a farmaceutická univerzita Brno
Palackého 1-3, 612 42 Brno
e-mail: busovam@vfu.cz
Prof. MUDr. RNDr. Miroslav Červinka, CSc.
Ústav lékařské biologie a genetiky
Lékařská fakulta UK v Hradci Králové
Šimkova 870, 500 38 Hradec Králové
e-mail: cervinka@lfhk.cuni.cz
Doc. MUDr. Zuzana Červinková, CSc.
Ústav fyziologie
Lékařská fakulta UK v Hradci Králové
Šimkova 870, 500 38 Hradec Králové
e-mail: wolff@lfhk.cuni.cz
Petra Češková
Katedra biologie
Přírodovědecká fakulta UJEP v Ústí nad Labem
České mládeže 8, 400 96 Ústí nad Labem
e-mail: petra.ceska@worldonline.cz
Mgr. Hana Demová
Ústav obecne biologie a genetiky
3. Lékařská fakulta UK v Praze
Ruská 89, 100 000 Praha 10
e-mail: hana.demova@lf3.cuni.cz
Mgr. Dáša Doležalová
Biologický ústav
Lékařská fakulta Masarykovy univerzity Brno
Kamenice 5, 625 00 Brno
e-mail: dasa.dolezalova@gmail.com
William C. Earnshaw, Professor, Ph.D.
Wellcome Trust Centre for Cell Biology
The University of Edinburgh
Mayfield Road, EH9 3JR Edinburgh , United Kingdom
e-mail: bill.earnshaw@ed.ac.uk
PD Dr. Birthe Fahrenkrog
M.E. Mueller Institute
Biozentrum
University of Basel
Klingelbergstr. 70, CH-4056 Basel, Switzerland
e-mail: birthe.fahrenkrog@unibas.ch
85

Roland Foisner, Professor, Ph.D.
Max F. Perutz Laboratories
Medical University Vienna
Dr. Bohr-Gasse 9,1030 Vienna, Austria
e-mail: roland.foisner@meduniwien.ac.at
MUDr. Zdeněk Fiedler, PhD.
Ústav lékařské biologie a genetiky
Lékařská fakulta UK v Hradci Králové
Šimkova 870, 500 38 Hradec Králové
e-mail: ttragelaf@seznam.cz
Mgr. Jana Fukalová
Oddělení Biologie bunečného jádra
Ústav molekulární genetiky AV ČR
Vídeňská 1083, 142 20 Praha 4
e-mail: hofmannova@img.cas.cz
Yosef Gruenbaum, Professor, Ph.D.
Department of Genetics
The Institute of Life Sciences
The Hebrew University of Jerusalem
91904 Jerusalem, Israel
e-mail: gru@vmshuji.ac.il
RNDr. Erika Halašová, PhD.
Ústav lekárskej biológie
Jesseniova lekárska fakulta, Univerzita Komenského v Bratislave
Malá Hora 4, 036 01 Martin, Slovensko
e-mail: halasova@jfmed.uniba.sk
Mgr. Andrea Harničarová
Biofyzikální ústav AV ČR Brno
Královopolská 1356, 612 65 Brno
e-mail: harand@ibp.cz
Doc. Ing. Jiří Hatina , CSc.
Ústav biologie
Lékařská fakulta UK v Plzni
Karlovarská 48, 301 66 Plzeň
e-mail: jiri.hatina@lfp.cuni.cz
RNDr. Elena Hlinková, CSc.
Ústav bunkovej biológie
Prírodovedecká fakulta , Univerzita Komenského v Bratislave
Mlynská dolina B-1, 842 15 Bratislava
e-mail: hlinkova@fns.uniba.sk
86

MUDr. Jiří Hochmann, CSc.
Katedra biologických a lékařských věd
Farmaceutická fakulta UK Hradec Králové
Heyrovského 1203, 500 05 Hradec Králové
e-mail: Jiri.Hochmann@faf.cuni.cz
PharmDr. Zuzana Holubcová
Biologický ústav
Lékařská fakulta, Masarykova univerzita Brno
Kamenice 5 ,625 00 Brno
e-mail: zholub@med.muni.cz
RNDr. Jan Ipser , CSc.
Katedra biologie
Přírodovědecká fakulta UJEP v Ústí nad Labem
České mládeže 8, 400 96 Ústí nad Labem
e-mail: jan.ipser@ujep.cz
Prof. MUDr. Roman Janisch, DrSc.
Biologický ústav
Lékařská fakulta, Masarykova univerzita Brno
Kamenice 5, budova A7, 625 00 Brno
e-mail: rjanisch@med.muni.cz
Stanislav John
Ústav lékařské biologie a genetiky
Lékařská fakulta UK v Praze
Šimkova 870, 500 38 Hradec Králové
e-mail: stanislav.john@gmail.com
Mgr. Alžběta Kalendová
Ústav molekulární genetiky AV ČR, v.v.i
Vídeňská 1083, 142 20 Praha 4
e-mail: kalendova@img.cas.cz
MUDr. Jana Kolářová, CSc.
Ústav lékařské biologie a genetiky
Lékařská fakulta UK v Praze
Šimkova 870, 500 38 Hradec Králové
e-mail: kolarova@lfhk.cuni.cz
Doc. RNDr. Marie Korabečná, PhD.
Biologický ústav
Lékařská fakulta UK v Plzni
Karlovarská 48, 301 66 Plzeň
e-mail: marie.korabecna@lfp.cuni.cz
87

RNDr. Blažena Koukalová, CSc.
Biofyzikální ústav AV ČR,v.v.i,
Královopolská 135, 612 65 Brno
e-mail: blazena@ibp.cz
MUDr. Dimitrolos Krajčí, CSc.
Department of Anatomy
Faculty of Medicine, Kuwait University, Kuwait
Prof. RNDr. Juraj Krajčovič, CSc.
Ústav bunkovej biológie
Prírodovedecká fakulta , Univerzita Komenského v Bratislave
Mlynská dolina B-1, 842 15 Bratislava
e-mail: krajcovic@fns.uniba.sk
RNDr. Věra Králová
Ústav lékařské biologie a genetiky
Lékařská fakulta UK v Praze
Šimkova 870, 500 38 Hradec Králové
e-mail: kralovav@lfhk.cuni.cz
Mgr. Alena Kučerová
Biologický ústav
Lékařská fakulta UK v Plzni
Karlovarská 48, 301 66 Plzeň
e-mail: alena.kucerova@lfp.cuni.cz
MUDr. Petr Kufner
Biologický ústav
Lékařská fakulta UK v Plzni
Karlovarská 48, 301 66 Plzeň
e-mail: Petr.Kufner@lfp.cuni.cz
Doc. RNDr. Jiří Kunert , DrSc.
Ústav biologie
Lékařská fakulta, Univerzita Palackého Olomouc
Hněvotínská 3, 775 15 Olomouc
e-mail: kunert@tunw.upol.cz
RNDr. Jaromíra Kůsová
Zdravotní ústav se sídlem v Ostravě
Partyzánské nám. 7, 702 00 Ostrava
e-mail: jaromira.kusova@zuova.cz
Tomáš Loja
Ústav experimentální biologie
Masarykova univerzita Brno
Kotlářská 2, 611 37 Brno
e-mail: tloja@sci.muni.cz
88

Doc. MUDr. Vladislav Mareš, DrSc.
Fyziologický ústav AV ČR, v.v.i,
Vídeňská 1083, 142 00 Praha 4
e-mail: maresv@biomed.cas.cz
RNDr. Tatiana Matáková
Ústav lekárskej biochémie
Jesseniova lekárska fakulta, Univerzita Komenského v Bratislave
Malá Hora 4, 036 01 Martin, Slovensko
e-mail: matakova@jfmed.uniba.sk
Prof. Ing. Kyra Michalová, DrSc.
Centrum nádorové cytogenetiky
1. Lékařská fakulta UK v Praze a Všeobecná fakultní nemocnice Praha
U Nemocnice 2, 128 08 Praha 12
e-mail: kyra@vfn.cz
Mgr. Lenka Mikalová
Biologický ústav
Lékařská fakulta, Masarykova univerzita Brno
Kamenice 5, 625 00 Brno
e-mail: lmikal@med.muni.cz
Mgr. Radka Molíková, PhD.
Ústav normální anatomie
Lékařská fakulta, Univerzita Palackého Olomouc
Hněvotínská 3, 775 15 Olomouc
e-mail: molikovaradka@centrum.cz
Prof. RNDr. Vojtěch Mornstein, CSc.
Biofyzikální ústav
Lékařská fakulta, Masarykova univerzita Brno
Kamenice 3, 625 00 Brno
e-mail: vmornst@med.muni.cz
RNDr. Petr Nádvorník , PhD.
Katedra buněčné biologie a genetiky
Přírodovědecká fakulta Univerzita Palackého Olomouc
Šlechtitelů 11, 783 71 Olomouc-Holice
e-mail: petr.nadvornik@upol.cz
Mgr. Eva Ondroušková, PhD.
Ústav experimentální biologie
Přírodovědecká fakulta, Masarykova univerzita Brno
ILBIT pavilon A3, Kamenice 5, 625 00, Brno-Bohunice
e-mail: zahradka@sci.muni.cz
89

Prof. RNDr. Zdeněk Opatrný, CSc.
Přírodovědecká fakulta UK Praha
Viničná 5, 128 44 Praha 2
e-mail: opat@natur.cuni.cz
PharmDr. Petr Pávek, PhD.
Katedra farmakologie a toxikologie
Farmaceutická fakulta UK Hradec Králové
Heyrovskeho 1203, 500 05 Hradec Králové
e-mail: petr.pavek@faf.cuni.cz
Mgr. Oto Pavliš
Centrum biologické ochrany Těchonín
561 66 Těchonín
e-mail: oto.pavlis@email.cz
RNDr. Miroslav Pohanka, PhD.
Centrum pokročilých studií,
Fakulta vojenského zdravotnictví, Univerzita obrany
Třebešská 1575, 500 01 Hradec Králové
e-mail: rau@atlas.cz
Prof. RNDr. Ivan Raška, DrSc.
Ústav buněčné biologie a patologie
1. lékařská fakulta UK v Praze
Albertov 4, Praha 2, 128 01
e-mail: ivan.raska@lf1.cuni.cz
Mgr. Martina Rédová
Ústav experimentální biologie
Masarykova univerzita Brno
Kotlářská 2, 611 37 Brno
e-mail: 63800@mail.muni.cz
RNDr. Pavel Rossner, PhD.
Ústav experimentální medicíny AV ČR,v.v.i.
Vídeňská 1083, 142 20 Praha
e-mail: prossner@biomed.cas.cz
MUDr. PharmDr. Kamil Rudolf
Oddělení revmatologie a klinické farmakologie
II. interní klinika, Fakultní nemocnice Hradec Králové
Sokolská 581, 500 05 Hradec Králové
e-mail: rudolfkamil@seznam.cz
doc. PharmDr. Emil Rudolf, PhD.
Ústav lékařské biologie a genetiky
Lékařská fakulta UK v Hradci Králové
Šimkova 870, 500 38 Hradec Králové
e-mail: rudolf@lfhk.cuni.cz
90

RNDr. Ladislava Schröterová, PhD.
Ústav lékařské biologie a genetiky
Lékařská fakulta UK v Hradci Králové
Šimkova 870, 500 38 Hradec Králové
e-mail: schroteroval@lfhk.cz
Mgr. Jiří Slanina, PhD.
Biochemický ústav
Lékařská fakulta, Masarykova univerzita Brno
Kamenice 5, Budova A16, 625 00 Brno
e-mail: jrslanina@med.muni.cz
MUDr. Iva Slaninová, PhD.
Biologický ústav
Biochemický ústav
Lékařská fakulta, Masarykova univerzita Brno
Kamenice 5, Budova A6, 625 00 Brno
e-mail: pokorna@med.muni.cz
Dr. Irina Solovei
Institute of Human Genetics
Biocenter
Ludwig-Maximilians University of Munich
Grosshadernerstr. 2, 82152 Planegg-Martinsried, Germany
e-mail: irina.solovei@lrz.uni-muenchen.de
MUDr. Tomas Soukup
Ústav histologie a embryologie
Lékařská fakulta UK v Hradci Králové
Šimkova 870, 500 38 Hradec Králové
e-mail: soukupto@lfhk.cuni.cz
Mgr.Lucie Stejskalová
Katedra farmakologie a toxikologie
Farmaceutická fakulta UK Hradec Králové
Heyrovskeho 1203, 500 05 Hradec Králové
e-mail: Lucie.stejskalova@faf.cuni.cz
Mgr. Jaromír Suchánek
Katedra informatiky
Fakulta mechatroniky Trenčianská univerzita A. Dubčeka
Študentská 2, 911 50 Trenčín, Slovensko
e-mail: hisym@mail.t-com.sk
Prof. MUDr. Augustin Svoboda, CSc.
Biologický ústav
Lékařská fakulta, Masarykova univerzita Brno
Kamenice 5, 625 00 Brno
e-mail: asvoboda@med.muni.cz
91

MVDr. Jiří Škaloud, CSc.
Ústav pro státní kontrolu veterinárních biopreparátů a léčiv Brno
Hudcova 56 A, 621 00 Brno
e-mail: skaloud@uskvbl.cz
Doc. RNDr. Jiřina Škorpíková, CSc.
Biofyzikální ústav
Lékařská fakulta, Masarykova univerzita Brno
Kamenice 3, 625 00 Brno
e-mail: jskorpik@med.muni.cz
Doc. MUDr. David Šmajs, PhD.
Biologický ústav
Lékařská fakulta, Masarykova univerzita Brno
Kamenice 5, budova A6, 625 00 Brno
e-mail: dsmajs@med.muni.cz
Prof. MUDr. Jan Šmarda, DrSc.
Biologický ústav
Lékařská fakulta, Masarykova univerzita Brno
Kamenice 5, budova A7, 625 00 Brno
e-mail: jsmarda@med.muni.cz
MUDr. Radim J. Šrám, DrSc.
Ústav experimentální mediciny AV ČR, v.v.i
Vídeňská 1083, 142 20 Praha 4
e-mail: sram@biomed.cas.cz
Doc. RNDr. Rudolf Štětina, CSc.
Katedra Toxikologie
Fakulta vojenského zdravotnictví, Univerzita obrany
Třebešská 1575, 500 01 Hradec Králové
e-mail: stetina@pmfhk.cz
Mgr. Lucie Švecová
Katedra farmakologie a toxikologie
Farmaceutická fakulta UK Hradec Králové
Heyrovskeho 1203, 500 05 Hradec Králové
e-mail: svecoval@faf.cuni.cz
Ing. Jan Topinka, DrSc.
Ústav experimentální medicíny AV ČR, v.v.i
Vídeňská 1083, 142 20 Praha 4
e-mail: jtopinka@biomed.cas.cz
Mgr. Miroslav Vařecha, Ph.D.
Centrum pro analýzu biomedicínského obrazu
Fakulta informatiky, Masarykova univerzita Brno
Botanická 68a, 602 00 Brno
e-mail: mvara@fi.muni.cz
92

Mgr. Matej Vesteg
Ústav bunkovej biologie
Prírodovedecká fakulta , Univerzita Komenského v Bratislave
Mlynská dolina B-1, 842 15 Bratislava
e-mail: vesteg@fns.uniba.sk
MUDr. Pavel Vodička, CSc.
Ústav experimentální medicíny AV ČR, v.v.i
Vídeňská 1083, 142 00 Praha 4
e-mail: pvodicka@biomed.cas.cz
Doc. RNDr. František Weyda, CSc.
Entomologický ústav
Biologické centrum AV ČR,v.v.i.
Branišovská 31, 370 05 České Budějovice
e-mail: weydafk@seznam.cz
RNDr. Zdeněk Žižka, DrSc.
Mikrobiologický ústav AV ČR,v.v.i.
Vídeňská 1083, 142 20 Praha 4
e-mail: zizka@biomed.cas.cz
93

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Generální partner
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Poznámky
Poznámky
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Biologický výzkum pro lidské zdraví
Editor M. Červinka
Vydal a vytiskl Tribun EU s.r.o,
Gorkého 41, 602 00 Brno
www.knihovnicka.cz
ISBN 978-80-7399-545-4
V Tribunu EU vydání první
Brno 2008