XXII. BIOCHEMICKÝ ZJAZD - Jesseniova lekárska fakulta
XXII. BIOCHEMICKÝ ZJAZD - Jesseniova lekárska fakulta
XXII. BIOCHEMICKÝ ZJAZD - Jesseniova lekárska fakulta
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Slovenská spoločnosť<br />
pre biochémiu a molekulárnu biológiu, člen IUBMB a FEBS<br />
Česká společnost<br />
pro biochemii a molekulární biológii, člen IUBMB a FEBS<br />
Ústav lekárskej biochémie<br />
<strong>XXII</strong>. <strong>BIOCHEMICKÝ</strong> <strong>ZJAZD</strong><br />
8. – 12. septembra 2010<br />
<strong>Jesseniova</strong> <strong>lekárska</strong> <strong>fakulta</strong>, Martin
Proceedings<br />
from <strong>XXII</strong>. Biochemický zjazd<br />
held in Martin 8. – 12. september 2010<br />
Chairman:<br />
Vicechairman:<br />
Members:<br />
Honorary advisory board<br />
J. Turňa<br />
V. Pačes<br />
A. Hrnčiar, D. Mištuna, D. Dobrota, J. Pastorek<br />
Program Committee<br />
J. Lehotský I. Barák A. Breier A. Drgová<br />
Z. Ďuračková P. Griač P. Kaplán J. Kormanec<br />
O. Križanová Ľ. Lacinová J. Korduláková M. Mareková<br />
K. Mikušová P. Račay Z. Sulová S. Stuchlík<br />
Ľ. Škultéty J. Turňa Ľ. Varečka<br />
Organizing Committee<br />
D. Dobrota E. Babušíková M. Bittšanský A. Drgová<br />
A. Evinová J. Hatok M. Chomová J. Jurečeková<br />
P. Kaplán S. Kuka R. Kirschnerová M. Kovalská<br />
J. Lehotský L. Letková T. Matáková P. Račay<br />
M. K. Sivoňová A. Štefaníková Z. Tatarková<br />
Edited by: Eva Babušíková, Jozef Hatok, Ján Lehotský<br />
ISBN<br />
© Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin,<br />
Department of Medical Biochemistry<br />
2<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
<strong>XXII</strong>. Biochemický zjazd is supported by:<br />
MAIN SPONSORS:<br />
BioTech<br />
KRD<br />
K-trade<br />
Lambda Life<br />
Merck<br />
Shimadzu<br />
Sigma-Aldrich<br />
Trigon<br />
SPONSORS:<br />
Biomedica<br />
Bio-Rad<br />
E-Colli<br />
Ecomed<br />
Eppendorf<br />
Fermentas<br />
Mettler-Toledo<br />
MGP<br />
Millipore<br />
Randox<br />
Scintila<br />
THANK YOU.<br />
3<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
TOPICS:<br />
I. BIOCHEMISTRY AND MOLECULAR BIOLOGY OF NERVOUS SYSTEM<br />
II.<br />
III.<br />
IV.<br />
BIOTECHNOLOGY<br />
BIOINFORMATICS<br />
GENOMICS<br />
V. CELL REGULATIONS AND SIGNAL TRANSDUCTION<br />
VI.<br />
GLYCOMICS<br />
VII. MEMBRANE BIOCHEMISTRY AND BIOENERGETICS<br />
VIII. NEW METHODOLOGIC PROCEDURES<br />
IX.<br />
PATHOBIOCHEMISTRY AND TRANSLATIONAL MEDICINE<br />
X. PROTEOMICS AND ENZYMOLOGY<br />
XI.<br />
REACTIVE SPECIES IN BIOMEDICINE<br />
XII. TEACHING OF BIOCHEMISTRY AND MOLECULAR BIOLOGY<br />
XIII. XENOBIOCHEMISTRY<br />
4<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Wednesday, 8 September 2010<br />
14:00 - 18:00 Registration<br />
CC<br />
18:00 - 23:00<br />
WELCOME RECEPTION<br />
CC<br />
Thursday, 9 September 2010<br />
8:15 - 8:45 OPENING CEREMONY<br />
CC<br />
8:45 - 9:00 WINNER CEREMONY CC<br />
9:00 - 9:45<br />
PLENARY LECTURE I.<br />
CC<br />
9:45 - 10:15<br />
COFFEE BREAK<br />
10:15 - 11:55<br />
11:55 - 12:15<br />
12:15 - 12:40<br />
12:40 - 13:45<br />
13:45 - 14:10<br />
14:10 - 15:25<br />
15:25 - 15:45<br />
R<br />
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g<br />
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Cell regulation and signal transduction<br />
CC<br />
Beckman lecture – cell biologyCC<br />
Sigma lecture<br />
Commertial lecture<br />
CC<br />
CC<br />
Reactive species in biomedicine<br />
LUNCH & POSTER VIEWING V, VI, VII, XI<br />
Membrane biochemistry and bioenergetics I.<br />
CC<br />
PLENARY LECTURE II.<br />
COFFEE BREAK<br />
Glycomics<br />
A<br />
A<br />
15:45 - 16:35<br />
16:35 - 17:00<br />
CC<br />
Membrane biochemistry and bioenergetics I.<br />
CC<br />
Glycomics<br />
A<br />
17:00 - 19:00<br />
19:00 - 22:00<br />
Museum<br />
Slovak chamber theatre<br />
Aquapark Turčianské Teplice<br />
Friday, 10 September 2010<br />
8:30 - 9:15 PLENARY LECTURE III.<br />
CC<br />
9:15 - 9:35<br />
9:35 - 11:15<br />
11:15 - 11:40<br />
11:40 - 12:00<br />
12:00 - 13:00<br />
13:00 - 13:45<br />
13:45 - 14:10<br />
R<br />
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Pathobiochemistry and translational<br />
medicine I.<br />
LUNCH<br />
COFFEE BREAK<br />
CC<br />
POSTER VIEWING II, III, IV, IX, XIII<br />
PLENARY LECTURE IV.<br />
Applied molecular biology<br />
Beckman lecture - Genomics A<br />
A<br />
KRD workshop A<br />
14:10 - 15:10<br />
15:10 - 15:30<br />
Xenobiochemistry<br />
Membrane biochemistry and bioenergetics II.<br />
CC<br />
A<br />
COFFEE BREAK<br />
15:30 - 16:50<br />
CC<br />
Xenobiochemistry<br />
CC<br />
Membrane biochemistry and bioenergetics II.<br />
A<br />
17:00 - 18:00<br />
18:00 - 19:00<br />
19:00 - 20:00<br />
Concert: Cantica Collegium Musicum<br />
5<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Saturday, 11 September 2010<br />
9:15 - 10:15<br />
10:15 - 10:35<br />
10:35 - 11:35<br />
11:35 - 12:00<br />
12:00 - 13:00<br />
13:00 - 14:00<br />
14:00 - 14:55<br />
14:55 - 15:15<br />
R<br />
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n<br />
CC<br />
Proteomics and enzymology<br />
Proteomics and enzymology<br />
CC<br />
Beckman lecture - proteomics CC<br />
Pathobiochemistry and translational<br />
medicine II.<br />
CC<br />
COFFEE BREAK<br />
CC<br />
LUNCH<br />
POSTER VIEWING I, VIII, X, XII<br />
COFFEE BREAK<br />
Teaching of biochemistry and molecular biology<br />
A<br />
Teaching of biochemistry and<br />
molecular biology<br />
Biochemistry and molecular biology<br />
of nervous system A<br />
A<br />
15:15 - 15:55<br />
15:55 - 16:20<br />
Pathobiochemistry and translational<br />
medicine II.<br />
CC<br />
Biochemistry and molecular biology<br />
of nervous system<br />
A<br />
16:20 - 16:35<br />
16:35 - 18:00<br />
18:00 - 24:00<br />
Closing ceremony<br />
Farewell party<br />
CC<br />
Sunday, 12 September 2010<br />
8:00 - 12:00<br />
Orava Castle<br />
Šútovo waterfall<br />
13:00<br />
Departure<br />
6<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details: Wednesday<br />
PROGRAM IN DETAILS<br />
WEDNESDAY, 8 SEPTEMBER 2010<br />
Jessenius Faculty of Medicine – Convention Centre<br />
14.00-18.00 REGISTRATION<br />
18.00-22.00 WELCOME RECEPTION<br />
7<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details:Thursday<br />
THURSDAY, 9 SEPTEMBER 2010<br />
Jessenius Faculty of Medicine – Convention Centre<br />
8.00-17.00 REGISTRATION<br />
8.15-08.45 OPENING CEREMONY<br />
8.45-9.00 WINNER´S CEREMONY<br />
9.00-9.45 PLENARY LECTURE I.<br />
Chairs:<br />
Ján Turňa, Dušan Dobrota<br />
09.45-10.15 COFFEE BREAK<br />
Mathias Sprinzl: ELECTROCHEMICAL DETECTION OF NUCLEIC<br />
ACIDS ON BIOSENSORS<br />
10.15-12.40 CELL REGULATIONS AND SIGNAL TRANSDUCTION<br />
Chairs:<br />
Ján Kormanec, Imrich Barák<br />
10.15-10.35 Ján Kormanec, Renáta Nováková, Ľubica Fecková, Peter Kutaš,<br />
Alena Reháková: REGULATION OF AURICIN BIOSYNTHESIS IN<br />
STREPTOMYCES AUREFACIENS CCM 3239<br />
10.35-10.55 Imrich Barák, Katarína Muchová, Naďa Pavlendová, Ján<br />
Jamroškovič: LIPID HELICES FORMATION IN BACILLUS SUBTILIS<br />
CELL MEMBRANE<br />
10.55-11.15 Veronika Benson, Valeria Grobárová, Katarína Hulíková, Jan<br />
Svoboda, Daniel Rozbeský, Daniel Kavan, Alan Kádek, Karel<br />
Křenek, Anna Fišerová, Vladimír Křen, Karel Bezouška: HIGH<br />
AFFINITY CARBOHYDRATE AND NON-CARBOHYDRATE LIGANDS<br />
FOR LECTIN-TYPE ACTIVATION RECEPTORS OF NATURAL KILLER<br />
CELLS REGULATE EFFECTOR FUNCTION THROUGH PI3K<br />
PATHWAY, AND GENERATE PERMANENT IMMUNE PROTECTION<br />
AGAINST MELANOMAS<br />
11.15-11.35 Radim Černý, Elerin Kärner, Christian Unger, Mikael Wendel:<br />
OSTERIX OVER-EXPRESSION IN HUMAN EMBRYONIC STEM CELLS<br />
AND ITS EFFECT ON CELL DIFFERENCIATION<br />
8<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details:Thursday<br />
11.35-11.55 Štefan Zorad, Daniela Ježová, Lucia Gajdošechová, Miroslava<br />
Eckertová, Katarína Kršková: THE ROLE OF ANGIOTENSIN II AND<br />
OXYTOCIN IN REGULATION OF ADIPOCYTE CELL SIZE<br />
LECTURE - BECKMAN CZE: CELL BIOLOGY<br />
11.55-12.15 Jana Morová, Radim Osička, Jiří Mašín, Peter Šebo: RTX<br />
CYTOTOXINS RECOGNIZE β2 INTEGRIN RECEPTORS THROUGH N-<br />
LINKED OLIGOSACHARIDES<br />
LECTURE - SIGMA ALDRICH CZE<br />
12.15-12.30 Lucie Piterková, Jana Kučerová, Karel Indrák, Vladimír Divoký:<br />
INTRONIC LINE-1 INSERTION IN β–GLOBIN GENE CAUSE β–<br />
TALASEMIA DUE TO ABERRANT SPLICING, NONSENSE-MEDIATED<br />
DECAY AND DECREASED RATE OF β–GLOBIN L1 ALLELE<br />
TRANSCRIPTION<br />
LECTURE - SHIMADZU<br />
12.30-12.40 Roman Oros: ADVANCED TECHNOLOGY FOR METABOLIC<br />
INVESTIGATIONS<br />
Jessenius Faculty of Medicine – Lecture Hall A<br />
10.15-12.40 REACTIVE SPECIES IN BIOMEDICINE<br />
Chairs:<br />
Zdenka Ďuračková, Peter Kaplán<br />
10.15-10.50 Karl-Heinz Wagner, Oliver Neubauer: HOW CAN OXIDATIVE<br />
STRESS AND DNA STABILITY BE INFLUENCED BY DIET AND<br />
PHYSICAL ACTIVITY<br />
10.50-11.15 Oľga Pecháňová, Andrej Barta, Stanislava Vranková, Jana<br />
Parohová, Mária Kovácsová: THE CROSS-TALK OF NITRIC OXIDE<br />
AND NUCLEAR FACTOR KAPPA B IN EXPERIMENTAL<br />
HYPERTENSION<br />
11.15-11.40 Jan Borovanský, Adéla Lipšová and Jiří Vachtenheim: FREE<br />
RADICAL SITUATION IN PIGMENT CELLS<br />
11.40-11.55 Jan Pláteník, Juraj Gáll, Jan Škrha, Jr., Richard Buchal, Eva<br />
Sedláčková, Karina Verébová: INDUCTION OF MITOCHONDRIAL<br />
PERMEABILITY TRANSITION BY MICROMOLAR IRON<br />
9<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details:Thursday<br />
11.55-12.10 Zuzana Tatarková, Eva Babušíková, Stanislav Kuka, Ján Lehotský,<br />
Peter Račay, Dušan Dobrota, Peter Kaplán: OXIDATIVE STRESS<br />
AND HEART AGING<br />
12.10-12.25 Eva Babušíková, Miloš Jeseňák, Peter Bánovčin, Dušan Dobrota:<br />
BRONCHIAL ASTHMA AND EFFECT OF OXIDATIVE STRESS ON ITS<br />
DEVELOPMENT<br />
12.25-12.40 Jana Muchová, Zuzana Nagyová, Iveta Ondrejovičová, Zdeňka<br />
Ďuračková: OXIDATIVE RISK IN ATHEROSCLEROSIS<br />
12.30-13.30 LUNCH<br />
13.00-13.45 POSTER VIEWING, Section V, VI, VII, XI (Gymnasium Hall, Malá<br />
Hora 4)<br />
Jessenius Faculty of Medicine – Convention Centre<br />
13.45-14.10 PLENARY LECTURE II: WINNER OF „DROBNICOV MEMORIÁL“<br />
Chair:<br />
Albert Breier<br />
Mária Balážová, Peter Griač: IDENTIFICATION OF<br />
PHOSPHATIDYLGLYCEROL SPECIFIC PHOSPHOLIPASE C IN YEAST<br />
SACCHAROMYCES CEREVISIAE<br />
14.10-16.35 MEMBRANE BIOCHEMISTRY AND BIOENERGETICS I.<br />
Chairs:<br />
Peter Griač, Ľubica Lacinová<br />
14.10-14.50 Anton Horváth, Ingrid Škodová, Anna Gnipová, Alena Zíková,<br />
Vladislava Benkovičová, Zdeněk Verner, Zdeněk Paris, Július<br />
Lukeš: SPECIALITIES OF OXIDATIVE PHOSPHORYLATION OF<br />
TRYPANOSOMATIDS AND EUGLENAS<br />
14.50-15.25 Peter Šmigáň, Monika Vidová, Janette Bobalova, Zuzana<br />
Nováková: ENERGETIC ASPECTS OF A MODIFICATION OF THE<br />
Na + /H + ANTIPORTER ACTIVITY IN A HARMALINE RESISTANT<br />
MUTANT OF METHANOTHERMOBACTER THERMAUTOTROPICUS<br />
15.25-15.45 COFFEE BREAK<br />
10<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details:Thursday<br />
15.45-16.00 Katarína Poloncová, Roman Holič, Peter Griač: IS<br />
PHOSPHATIDYLINOSITOL TRANSFER ACTIVITY ESSENTIAL FOR<br />
THE FUNCTION OF Pdr16p?<br />
16.00-16.15 Andrea Faltinová, Jana Gaburjáková, Ľubica Urbániková, Matúš<br />
Hajduk, Nataša Tomášková, Marián Antalík, Alexandra<br />
Zahradníková: EFFECT OF DOMAIN PEPTIDES OF THE CARDIAC<br />
RYANODINE RECEPTOR ON THE STABILITY OF BILAYER LIPID<br />
MEMBRANES AND ON RYR2 ACTIVITY<br />
16.15-16.35 Iveta Waczulíková, Oľga Uličná, Oľga Vančová, Jarmila<br />
Kucharská, Veronika Ilovská, Libuša Šikurová: ATORVASTIN<br />
CHANGES MEMBRANE LIPID FLUIDITY IN MITOCHONDRIA<br />
ISOLATED FROM VARIOUS TISSUES OF RATS<br />
Jessenius Faculty of Medicine – Lecture Hall A<br />
14.10-17.00 GLYCOMICS<br />
Chairs:<br />
Zdenka Sulová, Katarína Mikušová<br />
14.10-14.35 Igor Tvaroška: MOLECULAR MODELING INSIGHT INTO CATALYTIC<br />
MECHANISMS OF GLYCOSYLTRANSFERASES<br />
14.35-15.00 Marek Baráth, Igor Tvaroška, Ján Hirsch: SYNTHESIS OF GlcNAc-<br />
TS MIMETICS AS A POTENT INHIBITORS OF<br />
GLYCOSYLTRANSFERASES<br />
15.00-15.25 Michaela Wimmerová: LECTINS FROM PATHOGENS: MYSTERY<br />
OF LIFE<br />
15.25-15.45 COFFEE BREAK<br />
15.45-16.10 Vladimír Farkaš: TRANSGLYCOSYLATION - A UNIVERSAL<br />
PRINCIPLE IN TAILORING THE PLANT AND FUNGAL CELL WALLS<br />
16.10-16.35 Mária Vršanská, Katarína Šuchová, Vladimír Puchart, Peter Biely:<br />
DIFFERENCIES BETWEEN TWO ENDOXYLANASES FROM GH5<br />
16.35-17.00 Zuzana Svetlíková, Marcelo E. Guerin, Mary Jackson, Jana<br />
Korduláková, Katarína Mikušová: MYCOBACTERIAL MANNOSYL<br />
TRANSFERASE PimA AS A TARGET FOR THE DEVELOPMENT OF<br />
NEW ANTITUBERCULAR DRUGS<br />
11<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details:Thursday<br />
16.45-21.15 SOCIAL EVENTS<br />
17.30-18.30 Slovak National Museum<br />
16.45-21.15 Aquapark Turčianske Teplice<br />
19.00-21.00 Slovak Chamber Theatre, Martin<br />
12<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details: Friday<br />
FRIDAY, 10 SEPTEMBER 2010<br />
Jessenius Faculty of Medicine – Convention Centre<br />
8.30-9.15 PLENARY LECTURE III.<br />
Chairs:<br />
Ján Lehotský<br />
09.15-09.35 COFFEE BREAK<br />
Peter Biely: MICROBIAL XYLANASES: PROPERTIES AND<br />
APPLICATIONS<br />
9.35-11.15 PATHOBIOCHEMISTRY AND TRANSLATIONAL MEDICINE I.<br />
Chairs:<br />
Peter Račay, Oľga Križanová<br />
09.35-10.00 Juraj Kopáček, Jaromír Pastorek, Silvia Pastoreková: MOLECULAR<br />
MECHANISMS INVOLVED IN RESPONSE TO HYPOXIA<br />
10.00-10.25 Lubomira Lencesova, Marta Sirova, Lucia Csaderova, Marcela<br />
Laukova, Zdena Sulova, Richard Kvetnansky, Olga Krizanova:<br />
CHANGES AND ROLE OF ADRENOCEPTORS IN PC12 CELLS AFTER<br />
PHENYLEPHRINE ADMINISTRATION AND APOPTOSIS INDUCTION<br />
10.25-10.50 Miroslav Barančík, Petra Šimončíková, Monika Ivanová: EFFECTS<br />
OF DOXORUBICIN TREATMENT ON MATRIX<br />
METALLOPROTEINASES IN RATS<br />
10.50-11.15 Attila Ziegelhöffer, Jana Mujkošová, Oľga Uličná, Iveta<br />
Waczulíková, Miroslav Ferko, Norbert Vrbjar, Štefan Polák, Tanya<br />
Ravingerová, Adriana Adameová: FUNCTION AND PROPERTIES<br />
OF HEART AND KIDNEY MITOCHONDRIA (MIT) IN<br />
SPONTANEOUSLY HYPERTENSIVE (HYP) RATS: INFLUENCE OF<br />
CAPTOPRIL AND NIFEDIPINE<br />
13<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details: Friday<br />
Jessenius Faculty of Medicine – Lecture Hall A<br />
9.35-12.00 APPLIED MOLECULAR BIOLOGY<br />
Chairs:<br />
Stanislav Stuchlík, Ján Turňa<br />
09.35-10.00 Peter Májek, Vladimír Špitalský, Gabriel Minárik, Tomáš Szemes:<br />
A METHOD FOR AUTOMATED DETECTION OF HETEROZYGOUS<br />
INSERTION-DELETION MUTATIONS<br />
10.00-10.25 Anna Hrabovska, Véronique Bernard, Eric Krejci: PROTEIN<br />
IMMUNIZATION OF MUTANT MOUSE – AN EFFICIENT WAY TO<br />
GENERATE SELECTIVE AND SENSITIVE ANTIBODIES<br />
10.25-10.50 Michal Kaliňák, Tibor Liptaj: NEW POSSIBILITIES FOR THE STUDY<br />
OF METABOLISM IN SLOVAKIA<br />
10.50-11.15 Martin Benej, Martina Poturnajová: TOXCAT METHOD:<br />
APPLICATION IN MOLECULAR ONCOLOGY<br />
11.15-11.40 Laco Kačáni: FROM BASIC BIOMEDICAL RESEARCH TO<br />
BIOTECHNOLOGY<br />
LECTURE-BECKMAN CZE: GENOMICS<br />
11.40-12.00 Ondřej Mihola, Zdeněk Trachtulec and Jiří Forejt: THE<br />
IDENTIFICATION AND CHARACTERIZATION OF THE FIRST<br />
VERTEBRATE HYBRID STERILITY GENE (HST1/PRDM9)<br />
WORKSHOP<br />
12.00-13.00 KRD Molecular Technologies, s.r.o.<br />
12.00-13.00 LUNCH<br />
13.00-13.45 POSTER VIEWING, Sections II, III, IV,IX, XIII (Gymnasium Hall,<br />
Malá Hora 4)<br />
14<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details: Friday<br />
Jessenius Faculty of Medicine – Convention Centre<br />
13.45-14.10 PLENARY LECTURE IV: WINNER OF „KOŠTÍŘOVA CENA“<br />
Chair:<br />
Václav Pačes<br />
Dana Douděrová, Pavel Martásek: STRUCTURAL-FUNCTIONAL<br />
CORRELATIONS OF HYDROXYMETHYLBILANE SYNTHASE<br />
14.10-16.50 XENOBIOCHEMISTRY<br />
Chairs:<br />
Albert Breier, Ľudovít Varečka<br />
14.10-14.30 Zdena Sulová, Mário Šereš, Miroslav Barančík, Lenka Gibalová,<br />
Branislav Uhrík, Lenka Poleková, Albert Breier: DOES EXISTS ANY<br />
RELATION BETWEEN P-GLYCOPROTEIN MEDIATED MULTIDRUG<br />
RESISTANCE AND INTRACELLULAR CALCIUM HOMEOSTASIS<br />
14.30-14.50 Pavlína Janů, Markéta Thimová, Petra Lovecká, Martina<br />
Macková, Kateřina Demnerová: EVALUATION OF TOXICITY AND<br />
GENOTOXICITY OF ORGANOHALOGEN PESTICIDES<br />
14.50-15.10 Monika Kmeťová Sivoňová, Dušan Dobrota, Tatiana Matáková,<br />
Zuzana Tatarková, Mária Kovalská, Martina Pavlíková, Róbert<br />
Dušenka, Ján Kliment: XENOBIOTIC-METABOLIZING ENZYMES<br />
POLYMORPHISMS AND CANCER RISK<br />
15.10-15.30 COFFEE BREAK<br />
15.30-15.50 Helena Paulíková: ACRIDINES – USEFUL MUTAGENS<br />
15.50-16.10 Helena Mertlíková-Kaiserová, Antonín Holý, Ivan Votruba:<br />
ORIGIN OF ACQUIRED RESISTANCE TO CYTOTOXIC ACYCLIC<br />
NUCLEOSIDE PHOSPHONATES<br />
16.10-16.30 Marie Stiborová, Eva Frei: CYTOCHROME P450- AND<br />
PEROXIDASE-MEDIATED OXIDATION OF ELLIPTICINE DICTATES<br />
ITS ANTI-TUMOR EFFICIENCY<br />
16.30-16.50 Vladimíra Tomečková: SYNTHETIC CYCLIC CHALCONE<br />
ANALOGUES AS NOVEL BIOLOGICALLY ACTIVE DYES<br />
15<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details: Friday<br />
Jessenius Faculty of Medicine – Lecture Hall A<br />
14.10-16.10 MEMBRANE BIOCHEMISTRY AND BIOENERGETICS II.<br />
Chairs:<br />
Peter Griač, Ľubica Lacinová<br />
14.10-14.50 Milan Štengl, František Barták, Roman Sýkora, Jiří Chvojka, Jan<br />
Beneš, Aleš Kroužecký, Ivan Novák, Jitka Švíglerová, Jitka<br />
Kuncová, Martin Matějovič: CARDIAC L-TYPE CALCIUM CURRENT<br />
IN SEPSIS<br />
14.50-15.05 Viera Kominková, Zuzana Tomášková, Ľubica Máleková, Karol<br />
Ondriaš: EFFECT OF ADENINE NUCLEOTIDES AND Mg 2+ IONS ON<br />
MITOCHONDRIAL CHLORIDE CHANNELS<br />
15.05- 15.25 COFFEE BREAK<br />
15.25-15.55 Ľubica Lacinová, Mária Karmažínová: GATING OF THE T-TYPE<br />
CALCIUM CHANNELS<br />
15.55-16.10 Mária Karmažínová, Edward Perez-Reyes, Ľubica Lacinová:<br />
GATING OF THE NEURONAL CA V 3.3 CHANNEL<br />
19.00-20.00 CONCERT: CANTICA COLLEGIUM MUSICUM (EVANGELIC<br />
CHURCH)<br />
20.30-21.30 Biznis meeting: Members of committees Slovak and Czech<br />
biochemical societes<br />
16<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details: Saturday<br />
SATURDAY, 11 SEPTEMBER 2010<br />
Jessenius Faculty of Medicine – Convention Centre<br />
9.15-12.00 PROTEOMICS AND ENZYMOLOGY<br />
Chairs:<br />
Ľudovít Škultéty,Peter Račay<br />
09.15-09.35 Daniela Krajčíková, Denisa Mullerová, Wan Qiang, Per Bullogh,<br />
Jilin Tang, Imrich Barák: ASSEMBLY OF BACILLUS SUBTILIS SPORE<br />
COAT: INVESTIGATION OF PROTEIN-PROTEIN INTERACTIONS<br />
AMONG THE SPORE COAT PROTEINS OF BACILLUS SUBTILIS<br />
09.35-09.55 Katarína Bíliková, Jozef Šimúth: PROTEOMICS OF<br />
MULTIFUNCTIONAL ROYAL JELLY PROTEINS<br />
09.55-10.15 Andrea Antošová, Katarína Šipošová, Martina Koneracká, Vlasta<br />
Závišová, Peter Kopčanský, Zuzana Gažová: INHIBITION OF<br />
INSULIN AMYLOID AGGREGATION WITH ALBUMIN<br />
FUNCTIONALIZED MAGNETIC FLUID<br />
10.15-10.35 COFFEE BREAK<br />
10.35-10.55 H.Mrazek, L. Weignerova, D. Manglova, D. Kavan , , V. Kren, K.<br />
Bezouska: FUNGAL α–N-ACETYLGALACTOSAMINIDASE FROM<br />
ASPERGILLUS NIGER: CLONING AND EXPRESSION IN YEAST<br />
10.55-11.15 Gabriela Flores Ramírez, Zuzana Bílková, Pavol Vadovič, Ľudovít<br />
Škultéty: IDENTIFICATION OF SURFACE PROTEINS OF THE<br />
OBLIGATE INTRACELLULAR BACTERIUM COXIELLA BURNETII<br />
11.15-11.35 Martin Hajduch, Katarína Klubicová, Maksym Danchenko, Ľudovít<br />
Škultéty, Namik Rashydov, Anna Preťová: TWENTY FOUR YEARS<br />
SINCE CHERNOBYL DISASTER: WHAT SEED PROTEIN CAN TELL<br />
US?<br />
LECTURE-BECKMAN CZE: PROTEOMICS<br />
11.35-12.00 Pavel Bouchal, Monika Mudrochová, Eva Budinská, Zbyněk<br />
Bortlíček, Iva Struhárová, Lenka Hernychová, Theodoros<br />
Roumeliotis, Spiros D. Garbis, Roman Hrstka, Petr Müller, Rudolf<br />
Nenutil, Bořivoj Vojtěšek: BIOMARKERS OF LYMPH NODE<br />
METASTASIS IN LOW-GRADE BREAST CANCER: AN INTEGRATED,<br />
PROTEOMICS-BASED APPROACH<br />
17<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details: Saturday<br />
Jessenius Faculty of Medicine – Lecture Hall A<br />
9.15-12.00 TEACHING OF BIOCHEMISTRY AND MOLECULAR BIOLOGY<br />
Chairs:<br />
Anna Drgová, Mária Mareková, Jana Korduláková<br />
09.15-09.35 Ľubomír Tomáška: WHEN MORE IS LESS: A DILEMMA OF<br />
A BIOMEDICAL EDUCATOR<br />
09.35-09.55 Zuzana Kostecká: TEACHING BIOCHEMISTRY AT THE UNIVERSITY<br />
OF VETERINARY MEDICINE AND PHARMACY IN KOŠICE<br />
09.55-10.15 Jiří Hudeček: DO WE TEACH BIOCHEMISTRY IN A LOGICAL WAY?<br />
REMARKS CONCERNING THE CONTENTS AND LEARNING<br />
APPROACH<br />
10.15-10.35 COFFEE BREAK<br />
10.35-10.55 Mária Kožurková, Marián Antalík, Dušan Podhradský: TEACHING<br />
BIOCHEMISTRY AT THE FACULTY OF SCIENCE IN KOŠICE<br />
10.55-11.15 Daniel Rajdl, Jaroslav Racek, Marie Šolcová: E-LEARNING –<br />
FRIEND OF FOE?<br />
11.15-11.35 Zdeňka Ďuračková, Zuzana Országhová: HISTORY AND PRESENT<br />
OF SCIENTIFIC AND PEDAGOGIC CONFERENCES OF TEACHERS<br />
FROM CHEMICAL INSTITUTES AND DEPARTMENTS OF SLOVAK<br />
AND CZECH MEDICAL FACULTIES<br />
11.35-11.55 Mária Mareková, Jana Mašlanková, Peter Urban, Juraj Guzy:<br />
BIOCHEMISTRY IN THE PICTURES – INTERACTIVE BIOCHEMISTRY<br />
12.00-13.00 LUNCH<br />
13.00-14.00 POSTER VIEWING, Sections: I, VIII, X (Gymnasium Hall, Malá<br />
Hora 4)<br />
18<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details: Saturday<br />
Jessenius Faculty of Medicine – Convention Centre<br />
14.00-16.00 PATHOBIOCHEMISTRY AND TRANSLATIONAL MEDICINE II.<br />
Chairs:<br />
Peter Račay, Oľga Križanová<br />
14.00-14.35 Nadežda Lukáčová, Alexandra Dávidová, Ľudmila Capková,<br />
Andrea Kucharíková: SPINAL CORD INJURY: PATHOGENESIS AND<br />
TREATMENT<br />
14.35-14.55 Peter Račay, Jozef Hatok, Mária Chomová, Jana Jurečeková,<br />
Peter Chudý, Juraj Chudej, Andrea Štefániková, Dušan Dobrota:<br />
APOPTOSIS – DOUBLE EDGED SWORD<br />
14.55-15.15 COFFEE BREAK<br />
15.15-15.35 Jozef Hatok, Jana Jurečeková, Peter Chudý, Pavol Hollý, Anton<br />
Dzian, Eduard Huľo, Eva Fabianová, Tatiana Matáková, Peter<br />
Račay: APOPTOSIS IN RELATION TO THE DEVELOPMENT OF<br />
CANCER AND RESISTANCE OF CANCER CELLS TO CYTOSTATICS<br />
15.35-15.55 Lenka Surdeníková, Fei Ru, Marian Kollárik: UTILITY OF SINGLE<br />
CELL RT-PCR (scRT-PCR) FOR THE STUDY OF PRIMARY AFFERENT<br />
NEURONS - PRELIMINARY VALIDATION<br />
Jessenius Faculty of Medicine – Lecture Hall A<br />
14.00-16.00 BIOCHEMISTRY AND MOLECULAR BIOLOGY OF NERVOUS<br />
SYSTEM<br />
Chairs:<br />
Ján Lehotský, Dušan Dobrota<br />
14.00-14.35 Jozef Michalik, Egon Kurča: BIOCHEMICAL MARKERS OF<br />
MULTIPLE SCLEROSIS<br />
14.35-14.55 Eva Babušíková, Dušan Dobrota, Anthony J. Turner, Natalia N.<br />
Nalivaeva: PROCESSING OF AMYLOID PRECURSOR PROTEIN<br />
AFTER IN VIVO INDUCED ISCHEMIA<br />
14.55-15.15 COFFEE BREAK<br />
19<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details: Saturday<br />
15.15-15.35 Dušan Dobrota, Michal Bittšanský: MAGNETIC RESONANCE<br />
SPECTROSCOPY IN DIAGNOSTIC PROTOCOL OF THE BRAIN<br />
DISEASES<br />
15.35-15.55 Michal Bittšanský, Veronika Husárová, Igor Ondrejka, Valéria<br />
Kerná, Pavol Adamík, Huber Poláček, Dušan Dobrota: CHANGES<br />
IN NEURONAL METABOLITES MEASURED BY PROTON MAGNETIC<br />
RESONANCE SPECTROSCOPY IN DEPRESSED PATIENTS DURING<br />
TREATMENT<br />
15:55-16:15 Ján Lehotský, Mária Chomová, Andrea Evinová, Mária Kovalská,<br />
Martina Pavlíková, Zuzana Tatarková, Peter Kaplán, Peter Račay:<br />
INDUCTION OF ISCHEMIC TOLERANCE IN SENSITIVE NEURONS:<br />
COORDINATED ROLE OF MULTIPLE MECHANISMS<br />
16:20-16:35 CLOSING CEREMONY<br />
18.00-22.00 FAREWELL PARTY (Open-air Museum of Slovak Village, Martin)<br />
20<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details: Sunday<br />
SUNDAY, 12 SEPTEMBER 2010<br />
08.00-12.00 GUIDED TOURS<br />
Orava Castle<br />
Šútovo waterfall<br />
13:00 DEPARTURE<br />
21<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
POSTER VIEWING<br />
22<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details:Poster viewing<br />
V.<br />
POSTER VIEWING 1., Section V, VI, VII, XI<br />
Thursday, 9 September 2010,<br />
13.00-13.45<br />
29. Lucia Gajdošechová, Miroslava Eckertová, Katarína Kršková, Štefan Zorad: THE<br />
ROLE OF 14-3-3 PROTEIN IN REGULATION OF GLUCOSE TRANSPORTER GLUT4<br />
TRANSLOCATION TO ADIPOCYTE PLASMA MEMBRANE<br />
30. Dana Grebeňová, Michaela Pluskalová, Zbyněk Hrkal, Kateřina Kuželová:<br />
CHANGES IN COFILIN PHOSPHORYLATION DURING THE APOPTOSIS OF LEUKEMIC<br />
JURL-MK1 CELLS<br />
31. Dagmar Homerová, Bronislava Řežuchová, Henrieta Škovierová, Ján Kormanec:<br />
CHARACTERIZATION OF A GENE ENCODING A SMALL REGULATORY RPOE –<br />
DEPENDENT RNA IN SALMONELLA ENTERICA SEROVAR TYPHIMURIUM<br />
32. Iva Jelínková, Olga Vondálová Blanářová, Jiřina Hofmanová, Petr Sova, Alois<br />
Kozubík, Alena Vaculová: MOLECULAR MECHANISMS INVOLVED IN POTENT<br />
PLATINUM (IV) COMPLEX-MEDIATED COLON CANCER CELL SENSITIZATION TO TRAIL-<br />
INDUCED APOPTOSIS<br />
33. Lenka Kočí, Martina Hýžďalová, Alena Vaculová, Jiřina Hofmanová, Alois Kozubík:<br />
TRAIL-INDUCED APOPTOSIS CAUSES ACTIVATION OF PRO-SURVIVAL PATHWAYS IN<br />
NON-ADHERENTLY GROWING COLON CANCER CELLS<br />
34. Gabriel Kollárovič, Miroslava Kretová, Lucia Lichá, Peter Baráth, Katarína<br />
Luciaková: PREPARATION AND FUNCTIONAL ANALYSIS OF PHOSPHORYLATION<br />
MUTANT FORMS OF THE TRANSCRIPTION FACTOR NFI<br />
35. Soňa Kontseková, Anna Repič, Monika Baráthová, Katarína Polčicová, Jaromír<br />
Pastorek: GENERATION AND CHARACTERIZATION MONOCLONAL ANTIBODIES<br />
AGAINST ENDOSIALIN, THE POTENTIAL MARKER OF TUMOR ANGIOGENESIS<br />
36. Miroslava Kretová, Ľudmila Šabová, Ľudmila Antalová, Katarína Luciaková: THE<br />
ROLE OF NF1 IN P21 GENE EXPRESSION<br />
37. Peter Kutaš, Ľubomíra Fecková, Alena Reháková, Renáta Nováková, Ján Kormanec:<br />
STRICT CONTROL OF AURICIN PRODUCTION IN STREPTOMYCES AUREOFACIES CCM<br />
3239 INVOLVES A FEEDBACK MECHANISM<br />
38. Ingrid Lajdová, Viera Spustová, Adrian Okša, Dušan Chorvát Jr.: ALTERED<br />
CALCIUM SIGNALING IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF CHRONIC<br />
KIDNEY DISEASE PATIENTS<br />
39. Ľubica Ondrušová, Jiří Vachtenheim: EXPRESSION OF MICROPHTHALMIA-<br />
ASSOCIATED TRANSCRIPTION FACTOR CRITICALLY REQUIRES ACTIVE SWI/SNF<br />
CHROMATIN REMODELING COMPLEX<br />
23<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details:Poster viewing<br />
40. Barbora Brodská, Petra Otevřelová, Aleš Holoubek: MCL-1 AS REGULATOR OF<br />
APOPTOSIS IN CML CELL LINE AND PERIPHERAL BLOOD MONONUCLEAR CELLS<br />
41. Jana Plšíková, Ján Kovaľ, Jaromír Mikeš, Mária Kožurková, Peter Fedoročko,<br />
Ladislav Janovec, Ján Ungvarský, Danica Sabolová: NOVEL GUANIDINE DERIVATIVES<br />
AND EVALUATION OF THEIR DNA BINDING AFFINITIES AND POSSIBLE ANTICANCER<br />
EFFECT<br />
42. Michaela Pluskalová, Dana Grebeňová, Zbyněk Hrkal, Kateřina Kuželová: HISTONE<br />
ACETYLATION OF LEUKEMIC JURL-MK1 CELLS WITHIN SAHA TREATMENT<br />
43. Jiřina Procházková, Lenka Umannová, Alois Kozubík, Miroslav Machala, Jan<br />
Vondráček: REGULATION OF PLAKOGLOBIN EXPRESSION, A KEY DESMOSOMAL<br />
CONSTITUENT, BY ARYL HYDROCARBON RECEPTOR AND CAMP SIGNALING<br />
44. Alena Reháková, Renáta Nováková, Ľubomíra Fecková, Peter Kutaš, Ján<br />
Kormanec: CHARAKTERIZATION OF SARP REGULATORY GENE INVOLVED IN POSITIVE<br />
REGULATION OF AN ANGUCYCLINE-LIKE POLYKETIDE ANTIBIOTICS AURICIN GENE<br />
CLUSTER IN STREPTOMYCES AUREOFACIENS CCM 3239<br />
45. Bronislava Řežuchová, Beatrica Ševčíková, Dagmar Homerová, Ján Kormanec: THE<br />
COMPLEX NETWORK REGULATORY CIRCUITS IN THE REGULATION OF SIGMA<br />
FACTORS INVOLVED IN DIFFERENTIATION AND STRESS RESPONSE IN STREPTOMYCES<br />
COELICOLOR A3(2)<br />
VI.<br />
46. Tatiana Kurucová, Helena Kavcová, Kristína Rogozanová, Lucia Messingerová,<br />
Danica Mislovčová, Albert Breier, Zdena Sulová: DIFFERENCES IN INTERACTION OF<br />
LECTINS SPECIFICALLY RECOGNIZING SIALIC ACID RESIDUES WITH SURFACE OF P-GP<br />
NEGATIVE OR POSITIVE L1210 CELLS<br />
47. Zdena Sulová, Peter Ditte, Tatiana Kurucová, Eva Poláková, Kristína Rogozanová<br />
Lucia Škvarková, Ján Sedlák, Jaromír Pastorek, Albert Breier: THE PRESENCE OF P-<br />
GLYCOPROTEIN IN L1210 CELLS DIRECTLY INDUCES DOWN-REGULATION OF CELL<br />
SURFACE SACCHARIDE-TARGETS OF CONCANAVALIN A<br />
VII.<br />
48. Jana Antalíková, Jana Jankovičová, Katarína Michalková, Michal Simon, Ľubica<br />
Horovská: EPITOP OF IVA-520 MONOCLONAL ANTIBODY ON THE BOVINE SPERM<br />
CD46 MOLECULE<br />
49. Cagala M. , Lencesova L., Hudecova S., Csaderova L., Sirova M., Cholujova D.,<br />
Kopacek J., Pastorekova S., Krizanova O.: DIMETHYLOXALLYL GYCINE MODULATES<br />
GENE EXPRESION AND PROTEIN LEVELS OF THE SODIUM CALCIUM EXCHANGER IN<br />
HEK 293 CELL LINE<br />
24<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details:Poster viewing<br />
50. Peter Kohút, Martin Valachovič, Lucia Hronská, Ivan Hapala:<br />
DEHYDROERGOSTEROL ELUCIDATES STEROL UPTAKE PROCESS IN YEAST S. CEREVISIAE<br />
51. Jan Madacki, Katarína Mikušová, Mary Jackson, Jana Korduláková:<br />
MYCOBACTERIAL EPOXIDEHYDROLASE EPHD IS INVOLVED IN FATTY ACID<br />
METABOLISM<br />
52. Boris Lakatoš, Lucia Bialešová, Eva Harnišová: ISOFORMS OF AMP-ACTIVATED<br />
PROTEIN KINASE SUBUNITS IN LYMPHOCYTES AND OBESITY<br />
53. Katarína Michalková, Michal Simon, Jana Antalíková, Ľubica Horovská:<br />
DISTRIBUTION AND BIOCHEMICAL CHARACTERIZATION OF CD52-LIKE MOLECULE IN<br />
BULL EPIDIDYMIS<br />
54. Hana Rauchová, Martina Vokurková, , Tomáš Soukup: IDEBENONE ACTIVATION OF<br />
GLYCEROL-3-PHOSPHATE OXIDATION IN LIVER MITOCHONDRIA FROM CONTROL AND<br />
HYPERTHYROID RATS<br />
55. Oľga Uličná, Oľga Vančová, Jarmila Kucharská, Peter Božek, Iveta Waczulíková,<br />
Libuša Šikurová: EFFECT OF ATORVASTATIN ON BIOENERGETICS OF THE LIVER<br />
MITOCHONDRIA ON A HIGH LIPID DIET<br />
56. Oľga Vančová, Oľga Uličná, Katarína Šebeková, Magdalena Labieniec, Cezary<br />
Watala: EFFECT OF PAMAM G4 DENDRIMER ON LIVER MITOCHONDRIA OXIDATIVE<br />
PHOSPHORYLATION AND LONG-TERM MARKERS OF HYPERGLYCAEMIA IN<br />
EXPERIMENTAL DIABETES<br />
57. Monika Vidová, Zuzana Nováková, Peter Šmigáň: BIOCHEMICAL AND MOLECULAR<br />
ANALYSIS OF NITRATE-RESISTANT MUTANT OF METHANOTHERMOBACTER<br />
THERMAUTOTROPHICUS<br />
XI.<br />
90. Lucia Andrezálová, Zuzana Országhová, Jana Muchová, Zdeňka Ďuračková:<br />
EFFECT OF OMEGA-3 FATTY ACIDS ON PON 1 ARYLESTERASE AND LACTONASE<br />
ACTIVITY IN CHILDREN SUFFERING FROM HYPERCHOLESTEROLEMIA<br />
91. Ima Dovinová, Zuzana Pakanová, Stanislava Vranková, Oľga Pecháňová, Soňa<br />
Čačányiová, František Kristek, Helena Paulíková: ANTIOXIDANT RESPONSE TO<br />
TREATMENT WITH NATURAL COMPOUNDS AND AN NO DONOR IN EXPERIMENTAL<br />
HYPERTENSION<br />
92. Marián Koláček, Jana Muchová, Eva Uhlíková, Viera Kupčová, Ladislav Turecký:<br />
WILSON´S DISEASE AND OXIDATIVE STRESS<br />
93. Stanislav Kuka, Zuzana Tatarková, Peter Račay, Ján Lehotský, Dušan Dobrota,<br />
Peter Kaplán: AGE-RELATED CHANGES IN ACTIVITIES OF MITOCHONDRIAL ELECTRON<br />
TRANSPORT CHAIN COMPLEXES IN THE RAT HEART<br />
25<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details:Poster viewing<br />
94. Daniela Mokrá, Anna Drgová, Rudolf Pullmann st., Andrea Čalkovská: SELECTIVE<br />
PHOSPHODIESTERASE-3 INHIBITOR OLPRINONE ALLEVIATES OXIDATIVE LUNG INJURY<br />
INDUCED BY MECONIUM<br />
95. Zuzana Országhová, Zuzana Paduchová, Ingrid Žitňanová, Cezary Watala, Jana<br />
Muchová, Zdeňka Ďuračková: METYLNICOTINAMIDE AND DNA OXIDATION DAMAGE<br />
IN RATS WITH STREPTOZOTOCINE INDUCED DIABETES MELLITUS<br />
96. Eliška Procházková, Petr Jansa, Lucie Čechová, Ivan Votruba, Helena Mertlíková-<br />
Kaiserová: ANTIOXIDANT CAPACITY OF SELECTED ANALOGS OF NUCLEIC ACID<br />
COMPONENTS: COMPARISON OF CELL-FREE AND CELL-BASED ASSAYS<br />
97. Beáta Veliká, Ivan Kron: EFFECTIVENESS OF PHENOLS AS ANTIOXIDANTS AGAINST<br />
SUPEROXIDE RADICAL<br />
98. Martina Vokurková, Hana Rauchová, Stanislav Pavelka, Tomáš Soukup, Narcis<br />
Tribulová: EFFECT OF N-3 POLYUNSATURATED FATTY ACIDS SUPPLEMENTATION ON<br />
RAT LIVER IN DIFFERENT THYROID STATUS<br />
99. Adéla Zdařilová, Alena Rajnochová Svobodová, Jana Zapletalová, Pavel Štrebl,<br />
Josef Zadražil, Jitka Vostálová: EFFECT OF RENAL TRANSPLANTATION ON OXIDATIVE<br />
STRESS-RELATED BIOMARKERS<br />
POSTER VIEWING 2., Section II, III, IV,IX, XIII<br />
Friday, 10 September 2010,<br />
13.00-13.45<br />
II.<br />
8. Hind Al Alami, Michal Kajsík, Jana Gajdošová, Hana Drahovská, Ján Turňa:<br />
CHARACTERIZATION OF BACTERIOFAGES INFECTING CRONOBACTER STRAINS<br />
9. Andrea Balažová, Víťazoslava Blanáriková, Jindra Valentová, František Bilka, Ivana<br />
Holková: EFFECT OF METHYL JASMONATE ON THE PRODUCTION OF SANGUINARINE<br />
AND ITS PRECURSORS IN OPIUM POPPY SUSPENSION CULTURES<br />
10. František Bilka, Andrea Balažová, Andrea Bilková, Víťazoslava Blanáriková, Ivana<br />
Holková, Marián Vanko: COMPARISON OF SANGUINARINE PRODUCTION OF<br />
PAPAVERACEAE FAMILY PLANTS IN VITRO CULTURES<br />
11. Michaela Kandričáková , Stanislav Stuchlík, Ján Turňa: DESIGN OF AN EXPRESSION<br />
SYSTEM FOR THE PRODUCTION OF RECOMBINANT HUMAN THROMBIN IN<br />
ESCHERICHIA COLI<br />
12. Tatiana Kraková, Jozef Šimúth, Katarína Bíliková: HETEROLOGOUS EXPRESSION<br />
OF ROYAL JELLY APALBUMINS IN E. COLI<br />
26<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details:Poster viewing<br />
13. Mahesh Madyagol, Stanislav Stuchlík, Ján Turňa: EXPRESSION, PURIFICATION AND<br />
FUNCTIONAL CHARACTERIZATION OF TWO FORMS OF AGROBACTERIUM SP. STRAIN<br />
CP4 EPSPS GENE IN ESCHERICHIA COLI FOR HORIZONTAL GENE TRANSFER STUDIES<br />
14. Jozef Parnica, Lukáš Kandráč, Marián Antalík: CONFORMATION TRANSITIONS OF<br />
CYTOCHROME C IN DEEP EUTECTIC SOLVENTS<br />
15. Lucia Pánčiová, Zdenko Levarski, Pavol Utekal, Stanislav Stuchlík, Ján Turňa:<br />
PRODUCTION OF RECOMBINANT PROTEINS USING HIGH CELL DENSITY CULTURES OF<br />
ESCHERICHIA COLI IN BIOREACTOR<br />
16. Michaela Šimšíková, Marián Antalík: SYNTHESIS AND SURFACE MODIFICATION OF<br />
ZINC OXIDE NANOPARTICLES<br />
17. Csaba Tóth, Roland Pálffy, Juraj Gašperík, Stanislav Stuchlík, Ján Turňa: CLONING,<br />
EXPRESSION AND ANTIMICROBIAL ACTIVITY OF THE HUMAN CATHELICIDIN LL-37<br />
18. Ľubomíra Tóthová, Hind Al Alami, Jana Lintnerová, Hana Drahovská, Ján Turňa:<br />
CHARACTERIZATION OF BACTERIOPHAGES INFECTING SALMONELLA ENTERICA<br />
19. Pavol Utekal, Lucia Pánčiová, Stanislav Stuchlík, Ján Turňa: PRODUCTION OF TWO<br />
RECOMBINANT ALCOHOLDEHYDROGENASES SUITABLE FOR BIOTRANSFORMATION<br />
OF C-6 ALDEHYDES INTO CORESPONDING ALCOHOLS<br />
20. Ondřej Vaněk, Petra Celadová, Jan Bláha, Daniel Kavan, Petr Pompach, Karel<br />
Bezouška: EUKARYOTIC EXPRESSION AS AN INDISPENSABLE TOOL FOR PREPARATION<br />
OF NATIVE DIMERIC FORMS OF NK CELL C-TYPE LECTIN-LIKE RECEPTORS<br />
III.<br />
21. Matej Stano, Ľuboš Kľučár: phiGENOME - A WEB-BASED GENOME BROWSER<br />
INTENDED FOR DISPLAY OF PHAGE GENOMES<br />
IV.<br />
22. Jarmila Farkašovská, Andrej Godány: SITE-SPECIFIC INTEGRATION OF<br />
BACTERIOPHAGE µ1/6 INTO THE STREPTOMYCES AUREOFACIENS CHROMOSOME<br />
23. Jana Gajdošová, Natália Kamodyová, Kristína Benedikovičová, Hana Drahovská,<br />
Eva Kaclíková, Ján Turňa: STUDY OF THERMOTOLERANCE ISLAND IN CRONOBACTER<br />
SPP.<br />
24. Lucia Letková, Tatiana Matáková, Erika Halašová, Anna Drgová, Dušan Dobrota:<br />
DNA POLYMORPHISMS OF SELECTED REPAIR GENES AND RISK OF LUNG CANCER<br />
25. Eva Lincová/Slabáková, Zuzana Pernicová, Eva Slavíčková, Alois Kozubík, Karel<br />
Souček: EXPRESSION OF TRANSCRIPTION FACTORS AND microRNAs IN TGF-Β1-<br />
INDUCED EMT OF BENIGN PROSTATE EPITHELIAL CELLS<br />
27<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details:Poster viewing<br />
26. Silvia Mahmood, Lucia Letková, Tatiana Matáková: THE ASSOCIATION BETWEEN<br />
EGF G/A POLYMORPHISM AND COLORECTAL DEVELOPMENT<br />
27. Milena Matejovičová, Michaela Králíková, Jitka Melounová, Martina Vodová, Jana<br />
Žáková, Igor Crha: SPERM DNA INTEGRITY ASSESMENT USING DIFFERENT COMET<br />
ASSAY PROTOCOLS<br />
28. Marika Matoušová, Ivan Votruba, Miroslav Otmar, Helena Mertlíková-Kaiserová:<br />
COMPARATIVE STUDY OF HYPOMETHYLATING ACTIVITIES OF 5-AZACYTIDINE<br />
CONGENERS<br />
IX.<br />
60. Soňa Bálentová, Eva Hajtmanová, Yvetta Mellová, Ivana Kinclová, Marián<br />
Adamkov: EFFECT OF FRACTIONATED DOSES OF GAMA RAYS ON THE ROSTRAL<br />
MIGRATORY STREAM OF ADULTS RATS<br />
61. Ľudmila Capková, Alexandra Dávidová, Andrea Kucháriková, Nadežda Lukáčová:<br />
IS RESPIRATORY PATHWAY ACTING THROUGH NO-SGC?<br />
62. Monika Dvořáková, Jana Muchová, Branislav Trebatický, Ján Breza, Zdeňka<br />
Ďuračková: THE EFFECT OF NATURAL POLYPHENOLS ON ADIPONECTINE LEVEL IN<br />
PATIENTS SUFFERING FROM ERECTILE DYSFUNCTION<br />
63. Jana Jurečeková, Jozef Hatok, Andrea Štefániková, Dušan Dobrota, Peter Račay:<br />
STUDY OF ANTIAPOPTOTIC PROTEINS RESPONSIBLE FOR DEVELOPMENT OF DRUG<br />
RESISTANCE IN ACUTE LEUKEMIA<br />
64. Vlastimil Kulda, Martin Pešta, Ondřej Topolčan, Lukáš Řehoř, Martin Svatoň,<br />
Václav Liška, Václav Babuška, Luboš Holubec, Radim Černý: PROGNOSTIC<br />
SIGNIFICANCE OF MIR-21 AND MIR-143 EXPRESSION IN TISSUE SAMPLES OF<br />
COLORECTAL CARCINOMA AND COLORECTAL LIVER METASTASES<br />
65. Erika Moravčíková, Evžen Křepela, Jan Prochádzka, Jan Čermák, Kamila Benková:<br />
THE FUNCTIONALITY OF APOPTOSOME APPARATUS AND THE EXPRESSION OF ITS<br />
REGULATORS IN NON-SMALL CELL LUNG CARCINOMA<br />
66. Iveta Ondrejovičová, Jana Muchová, Zuzana Paduchová, Zuzana Nagyová, Zdeňka<br />
Ďuračková: EFFECT OF OMEGA-3 PUFA ON LIPID PROFILE AND OXIDATIVE STRESS IN<br />
HYPERCHOLESTEROLEMIC CHILDREN<br />
67. Blanka Stibůrková, Makoto Hosoyamada, Kimiyoshi Ichida, Ivan Šebesta:<br />
ANALYSIS OF URATE TRANSPORTERS SLC22A12 AND SLC2A9 IN PATIENTS WITH<br />
RENAL HYPOURICEMIA IN CZECH POPULATION<br />
68. Andrea Štefániková, Jozef Hatok, Jana Jurečeková, Ivana Plameňová, Dušan<br />
Dobrota, Peter Račay: STUDY OF THE EFFECT OF HISTONE DEACETYLASE INHIBITOR<br />
ON THE SENSITIVITY OF LEUKAEMIC CELLS TO THE CYTOSTATICS<br />
28<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details:Poster viewing<br />
69. Ladislav Vaško, Janka Vašková: CONTENT OF FATTY ACIDS IN FOOD AND HEALTH<br />
STATUS<br />
70. Janka Vašková, Ladislav Vaško: Effect of humic acids in vivo<br />
XIII.<br />
100. Hana Bártíková, Jana Firbasová, Ivan Vokřál, Lenka Skálová, Jiří Lamka, Vladimír<br />
Kubíček, Barbora Szotáková: BIOTRANSFORMATION OF SELECTED ANTHELMINTICS IN<br />
RAT TAPEWORM HYMENOLEPIS DIMINUTA<br />
101. Iva Boušová, Zuzana Průchová, Lucie Trnková, Jaroslav Dršata: INHIBITORY<br />
EFFECT OF NATURAL FLAVONOIDS ON EQUINE LIVER GLUTATHIONE S-TRANSFERASE<br />
102. Lenka Gibalová, Ján Sedlák, Alena Reháková, Martina Labudová, Zdena Sulová,<br />
Albert Breier: MULTIDRUG RESISTANT P-GLYCOPROTEIN POSITIVE CELLS ARE ALSO<br />
CROSS-RESISTANT TO CISPLATIN<br />
103. Veronika Hanušová, Lenka Vildová, Věra Králová, Ladislava Schröterová, Lenka<br />
Trilecová, Alena Pakostová, Lenka Skálová: THE EFFECTIVENESS OF ORACIN IN<br />
ENHANCING THE CYTOTOXICITY OF DOXORUBICIN THROUGH THE INHIBITION OF<br />
DOXORUBICIN DEACTIVATION IN BREAST CANCER CELL LINE MCF-7<br />
104. Věra Kotrbová, Barbora Mrázová, Eva Frei, Marie Stiborová: CYTOCHROME B 5<br />
POTENTIATES ACTIVITIES OF CYTOCHROMES P450 1A1 AND 1A2 TO OXIDIZE<br />
ANTICANCER DRUG ELLIPTICINE TO PHARMACOLOGICALLY EFFICIENT METABOLITES<br />
105. Věra Králová, Emil Rudolf: SELENITE-INDUCED CELL DEATH IN HUMAN COLON<br />
CANCER CELLS<br />
106. Jitka Křížková, Kamila Burdová, Petr Hodek, Marie Stiborová: EFFECTS OF<br />
FLAVONOIDS ON CYTOCHROMES P450 AFTER PERORAL SINGLE DOSE<br />
ADMINISTRATION TO MALE RATS<br />
107. Tamara Lasotová, Hana Bártíková, Ivan Vokřál, Barbora Szotáková, Vladimír<br />
Kubíček, Jiří Lamka, Marián Várady, Lenka Skálová: ACTIVITIES OF DRUG-<br />
METABOLIZING AND ANTIOXIDANT ENZYMES IN HEAMONCHUS CONTORTUS STRAINS<br />
RESISTANT OR SENSITIVE TO ANTHELMINTICS<br />
108. Kateřina Levová, Jana Šístková, Eva Frei, Volker M. Arlt, David H. Phillips, Heinz<br />
H. Schmeiser, Marie Stiborová: CYTOCHROMES P450 1A1/2 OXIDIZE CARCINOGENIC<br />
ARISTOLOCHIC ACID I FORMING ITS DETOXICATION METABOLITE AND DECREASING<br />
LEVELS OF AA-DNA ADDUCTS IN VIVO<br />
109. Anna Sobeková, Ľuboslava Lohajová, Peter Javorský: THE EFFECT OF<br />
BENDIOCARB ON ANTIOXIDANT PARAMETERS IN MALE AND FEMALE ORGANS OF<br />
RABBIT<br />
29<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details:Poster viewing<br />
110. Miroslava Štefanišinová, Mária Kožurková, Vladimíra Tomečková, Mária<br />
Mareková: INTERACTION OF PLASMID DNA AND MITOCHONDRIA WITH CYCLIC<br />
CHALCONE ANALOGUES<br />
111. Tatiana Matáková, Erika Halašová, Lucia Letková, Anton Dzian, Dušan Dobrota:<br />
ASSOCIATION POLYMORPHISMS OF GST, HOGG1 AND XRCC1 GENES WITH LUNG<br />
ADENOCARCINOMA<br />
112. Jana Mizerovská, Helena Dračínská, Volker M. Arlt, Heinz H. Schmeiser, Eva Frei,<br />
Marie Stiborová: OXIDATION OF 3-AMINOBENZANTRONE, A HUMAN METABOLITE OF<br />
CARCINOGENIC 3-NITROBENZANTHRONE, BY HUMAN AND RAT CYTOCHROMES P450<br />
113. Michaela Moserová, Miroslav Šulc, Volker M. Arlt, David H. Phillips, Eva Frei,<br />
Marie Stiborová: METABOLIC ACTIVATION OF CARCINOGENIC BENZO[A]PYRENE BY<br />
CYTOCHROME P450 1A1 IS DICTATED BY COMPOSITION OF THE MIXED-FUNCTION-<br />
MONOOXYGENASE SYSTEM<br />
114. Barbora Mrázová, Eva Martínková, Radek Indra, Eva Frei, Marie Stiborová: THE<br />
STUDY ON THE CYTOCHROME B 5 –MEDIATED STIMULATION OF ELLIPTICINE<br />
OXIDATION BY CYTOCHROME P450 3A4 TO ITS PHARMACOLOGICALLY MORE<br />
EFFICIENT METABOLITES<br />
115. Miloslava Netopilová, Libuše Černá, Lucie Škarydová, Vladimír Wsol:<br />
IMMUNODETECTION OF 11Β-HYDROXYSTEROID DEHYDROGENASE DURING<br />
PURIFICATION OF A NEW HUMAN MEMBRANE-BOUND CARBONYL REDUCING<br />
ENZYME<br />
116. Radka Podlipná, Petra Šídlová, Kotyza Jan, Tomáš Vaněk: PHYTOREMEDIATION –<br />
THE PROMISING METHOD FOR THE REMOVAL OF PHARMACEUTICAL RESIDUES FROM<br />
WASTEWATER<br />
117. Jitka Poljaková, Tomáš Eckschlager, Eva Frei, Marie Stiborová: ELLIPTICINE<br />
CYTOTOXICITY TO HUMAN THYROID CANCER CELL LINES<br />
118. Alena Rajnochová Svobodová, Adéla Zdařilová, Dana Kylarová, Bohumil Zálešák,<br />
Jitka Vostálová: QUALITY AND TIME-STABILITY OF HUMAN SKIN EXPLANTS<br />
119. Anna Sobeková, Katarína Holovská, Peter Javorský: OXIDATIVE STRESS IN<br />
TURKEYS CAUSED BY CHRONIC CADMIUM EXPOSURE<br />
120. Martina Svobodová, Markéta Martínková, Helena Dračínská, Marie Stiborová:<br />
SIMILARITY BETWEEN RAT AND HUMAN ENZYMES INVOLVED IN OXIDATION 2-<br />
NITROPHENOL, A METABOLITE OF CARCINOGENIC 2-NITROANISOLE<br />
121. Mário Šereš, Eva Poláková, Oľga Križanová, Zdena Sulová, Albert Breier:<br />
OVEREXPRESSION OF P-GLYCOPROTEIN IN L1210/VCR CELLS IS ASSOCIATED WITH<br />
CHANGES IN SEVERAL ENDOPLASMIC RETICULUM PROTEINS<br />
122. Lenka Umannová, Miroslav Machala, Alois Kozubík, Jan Vondráček:<br />
ENVIRONMENTAL POLLUTANTS AS FACTOR MODULATING THE INFLAMMATORY<br />
RESPONSE AND FUNCTIONS OF LUNG CELLS<br />
30<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details:Poster viewing<br />
123. Zuzana Vantová, Helena Paulíková, Mária Kožurková, Danica Sabolová, Ima<br />
Dovinová, Pavol Kristián, Ján Imrich, Ján Ungvarský, Ladislav Janovec: THE<br />
MECHANISM OF CYTOTOXIC EFFECT OF NOVEL ACRIDINE INTERCALATORS<br />
124. Jiří Vrba, Jitka Ulrichová: RETINOIC ACID-INDUCED DIFFERENTIATION<br />
MODULATES THE APOPTOTIC EFFECT OF SODIUM VALPROATE IN HL-60 CELLS<br />
POSTER VIEWING 3., Section I, VIII, X<br />
Saturday, 10 September 2010,<br />
13.00-14.00<br />
I.<br />
1. Daniel Čierny, Stanislav Celec, Mária Kovalska, Peter Kaplan, Ivan Ondrejka, Egon<br />
Kurča, Ján Lehotsky: LABORATORY BIOMARKERS IN ISCHEMIC STROKE AND<br />
DEPRESSION IN HUMAN PATIENTS<br />
2. Monika Ďurfinová, Marta Brechtlová, Ľubica Procházková, Peter Kukumberg,<br />
Ľubomír Kuračka, Branislav Líška: IS IT POSSIBLE TO IMPROVE DEMYELINATION<br />
DISEASES MONITORING BY DETERMINATION OF SOME ENZYME ACTIVITIES<br />
CHARACTERISTIC FOR THE CENTRAL NERVOUS SYSTEM?<br />
3. Andrea Evinová, Eva Babušíková, Pavol Adamík, Ivan Ondrejka, Egon Kurča, Milan<br />
Grófik, Ján Lehotský: SELECTED GENE POLYMORPHISMS IN ISCHEMIC STROKE AND<br />
DEPRESSED HUMAN PATIENTS FROM CENTRAL SLOVAKIA<br />
4. Mária Chomová, Peter Račay: AN ANALYSIS OF THE IMPACT OF CNS ISCHEMIA ON<br />
MITOCHONDRIAL RESPIRATORY COMPLEXES<br />
5. Mária Kovalská, Martina Pavlíková, Zuzana Tatarková, Peter Kaplán, Dušan<br />
Dobrota, Marián Adamkov, Ján Lehotský: THE ROLE OF MAP-KINASE PATHWAY IN<br />
GLOBAL ISCHEMIA/REPERFUSION INJURY OF RAT BRAIN AFTER INDUCED<br />
HYPERHOMOCYSTEINEMIA<br />
6. Marcela Martončíková, Juraj Blaško, Judita Orendáčová, Kamila Lievajová, Enikő<br />
Račeková: ANATOMICAL DISTRIBUTION OF DYING CELLS WITHIN ADULT RATS<br />
ROSTRAL MIGRATORY STREAM<br />
7. Martina Pavlíková, Mária Kovalská, Monika Sivoňová, Zuzana Tatarková, Ján<br />
Lehotský: SECRETORY PATHWAYS SPCA1-CA 2+ PUMP EXPRESSION AS A PART OF<br />
ISCHEMIC PRECONDITIONING IN RAT FOREBRAIN<br />
VIII.<br />
58. Katarína Mrvová, Anna Hrabovská: DEVELOPMENT OF A DETECTION TOOL TO<br />
FOLLOW THE SPECIFIC ACTIVITY OF BUTYRYLCHOLINESTERASE IN HUMAN PATIENTS<br />
31<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details:Poster viewing<br />
59. Dominika Neuschlová, Anna Hrabovská: OPTIMALIZATION OF ELLMAN´S ASSAY<br />
TO STUDY THE KINETICS OF CHOLINESTERASES<br />
X.<br />
71. Vladimír Pevala, Jacob A. Bauer, Javier García-Nafría, Gabriela Ondrovičová,<br />
Ľuboš Ambro, Elena Blagova, Vladimir M. Levdikov, Anthony J. Wilkinson, Keith S.<br />
Wilson, Eva Kutejová: HEXAMER FORMATION TRIGGERS A SWITCH FROM AN<br />
INACTIVE TO AN ACTIVE CONFORMATION IN HUMAN MITOCHONDRIAL LON<br />
PROTEASE<br />
72. Milo Bystrický, Martina Beláňová, Mary Jackson, Katarína Mikušová, Jana<br />
Korduláková: BIOCHEMICAL CHARACTERIZATION OF RV1459C PROTEIN – PUTATIVE<br />
GT-C GLYCOSYLTRANSFERASE FROM MYCOBACTERIA<br />
73. Ľubomír Borko, Vladena Bauerová-Hlinková, Eva Hostinová, Juraj Gašperík, Jozef<br />
Ševčík: THE STUDY OF RYANODINE RECEPTOR 2 N-TERMINAL REGION RESPONSIBLE<br />
FOR HEART ARRYTHMIAS AND HEART FALIURE<br />
74. Petronela Dianišková, Jana Korduláková, Henrieta Škovierová, Devinder Kaur,<br />
Mary Jackson, Patrick J. Brennan, Katarína Mikušová: THE FUNCTIONAL<br />
CHARACTERIZATION OF THE PUTATIVE MYCOBACTERIAL ABC TRANSPORTER<br />
MSMEG_6366 - MSMEG_6369<br />
75. Veronika Doubnerová, Lucia Miedzińska, Jana Dobrá, Radomíra Vaňková, Helena<br />
Ryšlavá: EFFECT OF DROUGHT ON THE METABOLISM OF TOBACCO PLANTS<br />
(NICOTIANA TABACUM L.)<br />
76. Diana Fedunová, Zuzana Flachbartová, Jaroslava Bágeľová, Zuzana Gažová,<br />
Marián Antalík: THERMAL STABILITY OF CYTOCHROME C AND Α-LACTALBUMIN<br />
COMPLEXES<br />
77. Peter Grones, Zuzana Odnogová, Jozef Grones: REP 34 PROTEIN ENCODE BY<br />
PLASMID PGP2 FROM ACETOBACTER<br />
78. Hana Kiňová Sepová, Andrea Bilková, František Bilka, Lýdia Bezáková:<br />
PRODUCTION OF 3-HYDROXYPROPIONALDEHYD BY THE STRAINS OF LACTOBACILLUS<br />
REUTERI<br />
79. Michaela Koháryová, Marta Kollárová: THIOREDOXIN SYSTEM OF<br />
STREPTOMYCETES<br />
80. Mária Kožurková, Danica Sabolová, Slávka Hamuľáková, Jana Janočková, Jana<br />
Plšíková, Pavol Kristian, Ján Imrich, Ondrej Holas, Miroslav Pohanka, Kamil Kuča:<br />
STUDIES OF NOVEL BIVALENT TACRINE DERIVATIVES TARGETING CHOLINESTERASES<br />
81. Lucia Lichardusová, Jaroslav Kušnír, Mária Mareková: APPLICATION OF<br />
CONCENTRATION FLUORESCENCE MATRICES TO THE DETECTION OF FLUORESCENCE<br />
METABOLITES IN URINE<br />
32<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Program in details:Poster viewing<br />
82. Marián Mazáň, Noelia Blanco, Javier Arroyo, Vladimír Farkaš: CRH<br />
TRANSGLYCOSYLASES CATALYZE INTER-POLYMERIC LINKAGES IN FUNGAL CELL WALL<br />
83. Ľuboš Nižnanský, Svetlana Kryštofová, Ľudovít Varečka: DELETION OF<br />
GLUTAMATE DECARBOXYLASE GENE FROM TRICHODERMA VIRIDE F-534 STRAIN<br />
84. Helena Ryšlavá, Veronika Doubnerová, Robert Valenta, Kateřina Kloudová, Jana<br />
Trefancová, Helena Synková, Noemi Čeřovská: CHARACTERIZATION OF Β-N-<br />
ACETYLHEXOSAMINIDASE IN LEAVES OF TOBACCO PLANTS<br />
85. Danica Sabolová, Lucia Krajňáková, Jana Plšíková, Mária Kožurková: DNA<br />
BINDING STUDY OF 9-OXO-9,10-DIHYDROACRIDINCARBOXYHYDRAZIDES AS<br />
A POTENT TOPOISOMERASE I INHIBITORS<br />
86. Jana Schubertová Aradská, Dušan Blaškovič, Ján Turňa: IN VIVO CROSS-LINKING<br />
FOR IDENTIFICATION OF TELLURITE RESISTANCE-ASSOCIATED PROTEINS<br />
87. Martin Šimkovič, Anita Gdovinová, Zuzka Zemková, Ľudovít Varečka: MULTIPLE<br />
PROTEASES ARE SECRETED BY VEGETATIVE TRICHODERMA VIRIDE MYCELIUM<br />
CULTIVATED WITH PROTEIN INDUCER<br />
88. Katarína Šipošová, Andrea Antošová, Peter Kutschy, Zuzana Daxnerová, Zuzana<br />
Gažová: PHYTOALEXINS REDUCE INSULIN AMYLOID AGGREGATION<br />
89. Barbora Vidová, Michal Chotár, Andrej Godány: THE LYSM DOMAIN IN SURFACE<br />
IMMUNOGENIC PROTEIN (SIP) AND ITS INFLUENCE ON ELICITATION OF IMMUNITY<br />
AGAINST STREPTOCOCCUS AGALACTIAE<br />
33<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
BOOK OF ABSTRACTS<br />
34<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
PLENARY LECTURES<br />
35<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Plenary lectures<br />
IDENTIFICATION OF PHOSPHATIDYLGLYCEROL SPECIFIC PHOSPHOLIPASE C IN YEAST<br />
SACCHAROMYCES CEREVISIAE<br />
Mária Balážová and Peter Griač<br />
Institute of Animal Biochemistry and Genetics SAV, Ivanka pri Dunaji<br />
Phosphatidylglycerol (PG) is a metabolic precursor to the unique anionic<br />
mitochondrial phospholipid, cardiolipin (CL). CL and PG are phospholipids with<br />
important functions in promoting cell growth, anaerobic metabolism, mitochondrial<br />
functions and biogenesis. Considering their important role in eukaryotic cell<br />
physiology, little is known about the mechanisms by which PG membrane<br />
composition is controlled.<br />
Product of the open reading frame YPL206c, Pgc1p, of the yeast Saccharomyces<br />
cerevisiae is homologous to bacterial and mammalian glycerophosphodiester<br />
phosphodiesterases. Deletion of PGC1 causes accumulation of PG, which was evident<br />
especially under the conditions of inositol limitation. To test if the product of PGC1<br />
has an effect on degradation of PG, an in vitro assay was devised. Data obtained from<br />
this assay indicated that in the strain without Pgc1p, production of NBD-diacylglycerol<br />
(NBD-DAG) is significantly decreased compared to the wild type strain. In addition,<br />
NBD-DAG production was highly increased in the strain with overexpression of the<br />
Pgc1p. Two localizations of GST tagged-Pgs1p were observed: mitochondrion and<br />
lipid particles. However, in vitro phospholipase C activity of Pgc1 protein was<br />
detected only using mitochondrial protein extract. Based on these results we suggest<br />
that the product of YPL206c encodes mitochondrial PG specific phospholipase C<br />
(Pgc1p) involved in regulation of PG levels.<br />
Acknowledgement: Work was supported by LPP-0291-09 and VVCE-0064-07 grants.<br />
36<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Plenary lectures<br />
MICROBIAL XYLANASES: PROPERTIES AND APPLICATIONS<br />
Peter Biely<br />
Institute of Chemistry, Center of Glycomics, Slovak Academy of Sciences, Bratislava<br />
Considerable attention of current research is devoted to development of<br />
environmentally friendly processes for utilization of renewable resources. This effort<br />
includes also bioconversion of the major plant hemicellulose, xylan, after cellulose,<br />
the second most abundant polysaccharide in nature. Xylan is a heteropolysaccharide<br />
with a main chain built of β-1,4-liked xylopyranosyl residues. Depending on a plant<br />
source the main chain is decorated with uronic acids, arabinofuranose or esterified<br />
with acetic acid. Decomposition of xylan in nature by microorganisms is a part of the<br />
carbon cycle and involves concerted action of several enzymes. The enzymes<br />
attacking the xylan main chain are the depolymerizing endo-β-1,4-xylanases and<br />
xylose-releasing β-xylosidases. The acetyl groups and carbohydrate substituents of<br />
the main chain and are liberated with so called accessory enzymes. The group led by<br />
the author contributed significantly to current knowledge on the production of<br />
xylanolytic enzymes, mode of their action, substrate structure requirements and<br />
diversity of endoxylanases and xylosidases. Important impact had the discovery of<br />
hemicellulolytic deacetylases and introduction of efficient assays of xylanolytic<br />
enzymes. Partial amino acid sequences of novel accessory enzymes enabled isolation<br />
of the corresponding genes, their expression and the search for homologous<br />
sequences in known microbial genomes. This work resulted in establishment of new<br />
glycoside hydrolase and carbohydrate esterase families (http://www.cazy.org) with<br />
important synthetic and biotechnological potential. Microbial enzymes hydrolyzing<br />
xylan to oligosaccharides and fermentable sugars, and decreasing viscosity of xylan<br />
solutions became important industrial enzymes. They found applications in the pulp<br />
and paper industry, food industry and animal feed.<br />
37<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Plenary lectures<br />
STRUCTURAL-FUNCTIONAL CORRELATIONS OF HYDROXYMETHYLBILANE SYNTHASE<br />
Dana Douděrová and Pavel Martásek<br />
Department of Pediatrics, 1 st Faculty of Medicine, Charles University in Prague<br />
Acute intermittent porphyria (AIP) is an autosomal dominantly inherited disorder,<br />
classified as acute hepatic porphyria. It is characterized by a deficiency of<br />
hydroxymethylbilane synthase (HMBS, EC 4.3.1.8), the third enzyme in heme<br />
biosynthesis. Clinical features include gastrointestinal, neurologic and cardiovascular<br />
symptoms, but the most common clinical presentation is abdominal pain caused by<br />
neurovisceral crises.<br />
The purpose of this study was first to perform molecular analysis of the AIP patients.<br />
In each affected family, this becomes an important tool for individualised medicine,<br />
allowing for careful drug prescription; in addition, it is very important for the<br />
asymptomatic carriers to be warned of precipitating factors, thus avoiding an acute<br />
attack.<br />
The proper DNA diagnostics can be achieved by a combination of a robust and<br />
effective pre-screening method and a confirmatory DNA sequencing step. We<br />
decided to establish a new generation pre-screening method, which will be highly<br />
sensitive and relatively time- and cost-effective. Our method of choice was highresolution<br />
melting (HRM) analysis using the LightScanner instrument.<br />
Another important aspect of this project was to study the molecular heterogeneity of<br />
AIP in relation to the HMBS protein. We aimed at characterisation of the impact of<br />
the HMBS gene mutation on the structure and function of the enzyme, and<br />
demonstration of how this aids the interpretation of clinical, biochemical and genetic<br />
data in establishing an AIP diagnosis. To demonstrate this, we used expression and<br />
characterisation of mutant HMBS enzymes in the prokaryotic system together with<br />
the use of predictive computer-assisted structure-function correlation studies.<br />
38<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Plenary lectures<br />
Mathias Sprinzl: ELECTROCHEMICAL DETECTION OF NUCLEIC ACIDS ON BIOSENSORS<br />
39<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
LECTURES<br />
40<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
PROCESSING OF AMYLOID PRECURSOR PROTEIN AFTER<br />
IN VIVO INDUCED ISCHEMIA<br />
Eva Babušíková 1 , Dušan Dobrota 1 , Anthony J. Turner 2 and Natalia N. Nalivaeva 2<br />
1 Department of Medical Biochemistry, Comenius University in Bratislava, Jessenius<br />
Faculty of Medicine in Martin, Martin, Slovakia, 2 Institute of Molecular and Cellular<br />
Biology, University of Leeds, Leeds, United Kingdom<br />
Ischemia stroke results from a transient or permanent reduction in cerebral blood<br />
flow. In recent years it has been suggested that neurological disorder in elderly<br />
human population as Alzheimer’s disease (AD) is linked to certain brain pathologies,<br />
which promote its development and progression via accumulation of toxic amyloid<br />
peptide (Aβ) deposits in the brain. In the present study we determined the effect of<br />
the global ischemia (the four-vessel occlusion model) on the amount of amyloid<br />
precursor protein (APP) and some amyloid peptide degrading metalloproteinases. We<br />
observed that ischemia result in increased amyloidogenic processing of APP in<br />
hippocampus and cortex as well. Levels of APP increased significantly after ischemia<br />
as well as the amount of sAPPPβ soluble fragment produced by APP cleavage by β-<br />
secretase (BACE). Levels of BACE were significant increase. Amounts of Aβ degrading<br />
enzymes neprilysin and endothelin-converting enzyme decreased significantly after<br />
ischemia. We observed oxidative damage after ischemia. Oxidative modifications of<br />
proteins were demonstrated by significant accumulation of dityrosines and formation<br />
of lysine conjugates with the lipid peroxidation end products. After ischemia levels of<br />
conjugated dienes increased significantly. Concentrations of free sulfhydryl groups<br />
and thiobarbituric acid-reactive substances did not change during ischemic insult. Our<br />
results suggest that global ischemia may lead to amyloid peptide deposits<br />
accumulation and promote Alzheimer’s disease, which in turn might induce protein<br />
and lipid oxidation and reactive oxygen species formation.<br />
41<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
LIPID HELICES FORMATION IN BACILLUS SUBTILIS CELL MEMBRANE<br />
Imrich Barák, Katarína Muchová, Nada Pavlendová and Ján Jamroškovič<br />
Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 845<br />
51 Bratislava, Slovakia<br />
The domains of different lipid composition are present in eukaryotic and prokaryotic<br />
cell membranes. Using membrane binding fluorescent dyes, we demonstrate<br />
previously, the presence of lipid spirals extending along the long-axis of cells of the<br />
rod-shaped bacterium B. subtilis. These data indicate a higher level of membrane lipid<br />
organization than previously observed. Little is known however of the origin of these<br />
helical structures.<br />
Principally, there are at least three main specifically localized molecular structures in<br />
the membrane or close proximity to it what can help to form or influence the<br />
formation of lipid helixes. In our work we have focused on analyzing these lipid<br />
structures in correlation with other above mentioned helical structures in the cell<br />
membrane or its close proximity. We were analyzing lipid domains by using lipid<br />
specific dyes in protoplasted cells, in Mbl, MreB and MreBH mutant strains. We have<br />
used FRAP and FRET experiments to determine dynamics of lipid domains and colocalization<br />
of lipid dyes with GFP fused proteins, respectively.<br />
We have also studied the role of lipid helices in cell division by directing the Min<br />
system to the helices from pole to pole. We inspected cell division when E. coli Minsystem<br />
was introduced into B. subtilis cells. We show that MinD Ec can partially<br />
substitute function of its B. subtilis protein counterpart. Additionally, we observed<br />
dynamic behavior of MinD Ec and MinE in B. subtilis when expressed together. All<br />
these findings indicate that these two Min systems resemble each other more than<br />
was thought previously<br />
42<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
EFFECTS OF DOXORUBICIN TREATMENT ON MATRIX<br />
METALLOPROTEINASES IN RATS<br />
Miroslav Barančík, Petra Šimončíková and Monika Ivanová<br />
Institute for Heart Research CEKVY SAS, Bratislava<br />
The anthracycline doxorubicin (DOX) is an effective chemotherapeutic agent which is<br />
frequently used in the treatment of many types of malignancies. Limitation of its use<br />
is a cardiotoxicity associated with the development of cardiomyopathy and chronic<br />
heart failure. Matrix metalloproteinases (MMPs) are enzymes that play an important<br />
role in degradation and remodeling of extracellular matrix under physiological and<br />
pathological conditions. Especially MMP-2 and MMP-9 are suggested to play an<br />
important role also in pathogenesis of several cardiovascular diseases. The aim of the<br />
study was to investigate the involvement of MMPs in the responses of rats to<br />
prolonged doxorubicin treatment. In the study, male Wistar rats were used. DOX was<br />
administered to rats by intraperitoneal injections of 7 doses in 3-day’s intervals (total<br />
cumulative dose of DOX was 15 mg per kg of body weight). The control animals were<br />
treated with saline. The samples of tissue or plasma were collected 4, 8 and 12 weeks<br />
after application of last dose of DOX. The protein levels were determined by<br />
immunoblot assay and MMPs activities were measured by gelatin zymography.<br />
Determined were also blood pressure, body weight and weight of several organs<br />
(heart, brain, liver, kidney) and the parameters obtained in DOX-treated rats were<br />
compared with parameters of control animals. The investigation of changes<br />
associated with action of DOX revealed that prolonged exposure of rats to DOX led to<br />
changes in MMPs activation in heart tissue. Moreover, the effects of DOX were<br />
connected with time-dependent changes in plasma MMPs activities. Our results<br />
suggest that MMPs are involved in the responses of rat hearts to chronic DOX<br />
treatment.<br />
Acknowledgement: Supported by VEGA SR 2/0205/09, APVV 51-027404<br />
43<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
SYNTHESIS OF GlcNAc-TS MIMETICS AS A POTENT INHIBITORS OF<br />
GLYCOSYLTRANSFERASES<br />
Marek Baráth, Igor Tvaroška and Ján Hirsch<br />
Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, 845 38<br />
Bratislava, Slovakia<br />
Glysosyltransferases are enzymes that catalyst transfer of monosacharidic unit from<br />
an activated sugar phosphate to an acceptor molecule, usually an alcohol. The result<br />
of glycosyl transfer can be a monosaccharide glycoside, an oligosaccharide or<br />
polysaccharide. Many functions have been implicated for protein glycosylation,<br />
including promoting protein folding or stabilizing cell-surface glycoproteins.<br />
Different strategies have been used in order to identify potent inhibitors of<br />
glycosyltransferases. The main goal of this contribution is to synthesized of the<br />
transition state (TS) analogs starting from the donor UDP-GlcNAc. A leading idea of all<br />
these TS analogs is a „ 1-thio“ linker between a mimetic of GlcNAc in TS geometry and<br />
a mimetic of the acceptor bearing the β-D-psicofuranose, β-D-tagatofuranose and<br />
backbones with the key substitution on position C-1 by the phosphate group and on<br />
position C-2 by the thiophenyl group, which has been designed.<br />
Couple of potential inhibitors have been synthesized bearing β-D-psico and β-Dtagato<br />
configuration using a multi step synthesis. The β-D-psico analogs were as well<br />
as utilized for the synthesis of N-acetylated β-D-fructo analogs as their epimers in<br />
three more steps using a Walden inversion at C-3 position.<br />
Acknowledgement: This work was partially supported by the grants: VEGA 2/6129/27,<br />
2/0128/08 and Centre of Excelence- GLYCOMED.<br />
44<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
CHANGES IN NEURONAL METABOLITES MEASURED BY PROTON MAGNETIC<br />
RESONANCE SPECTROSCOPY IN DEPRESSED PATIENTS DURING TREATMENT<br />
Michal Bittšanský, Veronika Husárová, Igor Ondrejka, Valéria Kerná, Pavol Adamík,<br />
Hubert Poláček and Dušan Dobrota<br />
Jessenius Medical Faculty, Comenius University, Martin-Slovakia<br />
Previous works have shown that the symptoms of depressive disorder can be<br />
correlated to the concentrations of proton MR metabolites. In our study, 20<br />
depressive patients were at the admission to the hospital and after the hospital<br />
treatment clinically examined using MADRS scale (The Montgomery-Åsberg<br />
Depression Rating Scale) and using 1 H MR-spectroscopy (1.5 Tesla, single-voxel<br />
spectroscopy) in both hippocampi. We evaluated the absolute and relative signals of<br />
N-acetyl aspartate (NAA), total creatine (Cre), total cholines (Cho), myo-inositol (mI)<br />
measured with short (30 ms) and long (135 ms) time echo using calibrated LCModel.<br />
We observed statistically significant correlations of MRS-observable metabolites to<br />
the clinical MADRS score, and also significant differences between the patient groups<br />
before and after treatment. Some of the changes seem to reflect changes in the<br />
relaxation times of the metabolites.<br />
Acknowledgement: This work was supported by the grant 2007/57-UK-17 of Slovak<br />
Ministry of Health.<br />
45<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
TOXCAT METHOD: APPLICATION IN MOLECULAR ONCOLOGY<br />
Martin Benej 1 and Martina Poturnajová 2<br />
1 Department of Molecular Biology UK Bratislava,<br />
2 Cancer Research Institute SAS Bratislava<br />
Understanding the molecular mechanisms of cancer onset is one of the goals of<br />
contemporary cancer research. Individual approach to each disease is of crucial<br />
importance in this issue. Moreover, not only each disease, but also each patient<br />
carrying causative mutation in his/her genome requires individual approach. Thus,<br />
a need of a whole set of methods for characterization of causative mutations has<br />
arisen. ToxCAT is a method simulating the natural lipid bilayer environment, enabling<br />
the study of transmembrane (TM) domain interactions in vitro. The method is based<br />
on introduction of chimaeric constructs containing a TM domain of interest into<br />
periplasmic region of E.coli MM39 strain. Interactions of the chimerae result in<br />
expression of chloramphenicol acetyltransferase (CAT) reporter gene. The quantity of<br />
CAT expression corresponds with the strength of TM domain association. We<br />
illustrate the ToxCAT method application on the specific model of our interest –<br />
Medullary thyroid carcinoma (MTC). MTC is caused predominantly by single point<br />
mutations in six RET proto-oncogene exons. RET protein, the product of the RET gene<br />
is a tyrosine kinase cell-surface receptor. Mutations in extracellular domain of the<br />
receptor result in dimerization of the RET protein with other mutant RET molecule,<br />
thus enabling ligand-independent permanent activation of the RET receptor kinase.<br />
For this interaction, the strength of transmembrane domain oligomerization of the<br />
two RET molecules is responsible. We focus on the impact of RET TM domain<br />
mutations of Slovak MTC patients on the strength of TM domain oligomerization, to<br />
investigate a possible correlation with the age of onset and aggressiveness of the<br />
disease.<br />
46<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
HIGH AFFINITY CARBOHYDRATE AND NON-CARBOHYDRATE LIGANDS FOR LECTIN-<br />
TYPE ACTIVATION RECEPTORS OF NATURAL KILLER CELLS REGULATE EFFECTOR<br />
FUNCTION THROUGH PI3K PATHWAY, AND GENERATE PERMANENT IMMUNE<br />
PROTECTION AGAINST MELANOMAS<br />
Veronika Benson 1 , Valeria Grobárová, 1 Katarína Hulíková 1 Jan Svoboda 1 , Daniel<br />
Rozbeský 1,2 , Daniel Kavan 1,2 , Alan Kádek 1,2 , Karel Křenek 1 , Anna Fišerová 1 , Vladimír<br />
Křen 1 and Karel Bezouška 1,2<br />
1 Institute of Microbiology v.v.i., Academy of Sciences of Czech Republic and<br />
2 Department of Biochemistry, Faculty of Science, Charles University Prague, Praha,<br />
Czech Republic<br />
Our laboratories are interested in understanding of complex interactions between<br />
activation receptors of natural killer (NK) cells, their target structures at tumor cell<br />
surface, and intracellular activation pathways resulting in the activation of NK cell<br />
effector functions at molecular and cellular level. To identify high afinity ligands, we<br />
produce stable recombinant soluble forms of NK cell receptors such as NKR-P1, CD69,<br />
and NKG2D and use them in binding, inhibition and precipitation studies based on<br />
standard biochemical assays and oligosaccharide arrays. High affinity ligand mimetics<br />
are constructed by attachment of the active compounds to polyamidoamine or calix<br />
arene cores, or by dimerization of the ligand through a defined chemical linker.<br />
GlcNAc-coated polyamidoamine dendrimers induce upregulation of antibody<br />
formation that triggered by their interaction with mNKR-P1C. GlcNAc-coated calyx<br />
arene downregulated the expression of GlcNAc transferases MGAT3 and MGAT 5,<br />
increased the susceptibility of tumor cells to natural killing, and increased the<br />
expression of mNKG2D through the activation of PI3K–ERK but not phospholipase C-<br />
γ-JNK pathway. GlcNAc dimers can provide permanent protection in 70 % of mice<br />
bearing syngeneic B16S melanomas. This is due to activation of NKT lymphocytes,<br />
and subsequent infiltration of tumors by CD8 + cytotoxic lymphocytes. The exceptional<br />
signaling efficiency of GlcNAc dimers is explaned by sequential cooperative<br />
engagement of mNKR-P1A leading to the formation of large signaling complexes of<br />
about 20 MDa containing G proteins, ß-arrestin, phosphorylated dynamin, Src kinase,<br />
Vav, Rac1, Grb2, and Ras. Use of combined ligand mimetics results in engagement of<br />
several target receptors, and efficient activation of NK cell effector functions due to<br />
effective receptor cross-talk.<br />
Supported by grants by Ministry of Education of Czech Republic (MSM_21620808 and<br />
1M0505), by the Institutional Research Concept for the Institute of Microbiology<br />
(AVOZ50200510), by Czech Science Foundation (303/09/0477 and 305/09/H008),<br />
Grant Agency of Academy of Sciences of the Czech Republic nebo (ASCR)<br />
IAA500200620, and by the European Commission (Project Spine 2 Complexes,<br />
contract LSHG-CT-2006-031220).<br />
47<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
PROTEOMICS OF MULTIFUNCTIONAL ROYAL JELLY PROTEINS<br />
Katarína Bíliková and Jozef Šimúth<br />
Department of molecular apidology, Institute of molecular biology,<br />
SAS, Bratislava<br />
Presented study demonstrated how neofunctionalization results from various<br />
posttranslational modifications of maternal proteins of honeybee royal jelly (RJ). We<br />
have purified a minority protein of RJ, named apalbumin2a. Characterization of<br />
apalbumin2a by LC-MALDI TOF/TOF MS revealed it as a homologue of major basic<br />
royal jelly protein apalbumin2, carrying two fully occupied N-glycosylation sites, one<br />
with high-mannose structure, HexNAc2Hex9, and other carrying complex type<br />
antennary structures, HexNAc4Hex3 and HexNAc5Hex4, while the maternal protein,<br />
apalbumin2, contained only high-mannose N-linked glycans. We have found that<br />
apalbumin2a inhibit growth of Paenibacillus larvae, the primary honeybee pathogen<br />
of American foulbrood disease, similarly to RJ peptide royalisin. In spite of a single<br />
gene in honeybee genome for apalbumin2, presence of various forms of the protein,<br />
having different N-terminal sequences could be a result of specific proteolytic<br />
degradation of mature protein, alternative splicing or heterogonous transcription<br />
start sites by „leakage“ of the RNA transcription machinery. Obtained data call<br />
attention for functional plasticity of RJ proteins with potential impact on fundamental<br />
research, namely studies of novel mechanisms of action of antibacterial proteins, as<br />
well as on the field of drug development and therapeutic application of RJ proteins as<br />
antibiotics.<br />
Acknowledgments: This work was supported by Max-Planck Society for Partner Group<br />
of Slovak Academy of Sciences and by 6RP EU-BeeShop No.: 022568.<br />
48<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
FREE RADICAL SITUATION IN PIGMENT CELLS<br />
Jan Borovanský, Adéla Lipšová and Jiří Vachtenheim<br />
Institute of Biochemistry & Experimental Oncology, 1 st Faculty of Medicine, Charles<br />
University, Prague<br />
Biochemical specificity of pigment cells consists in their capacity to synthesize specific<br />
metabolic products – cytoprotective eumelanins and cytotoxic phaeomelanins in the process<br />
of melanogenesis. Melanogenesis represents a potential threat for the pigment cell because<br />
the intermediates belong to cytotoxic species – quinones, semiquinones and the synthesis of<br />
melanins is accompanied by the production of superoxide anions and H 2 O 2 . For that reason<br />
melanogenesis is strictly compartmentalized to melanosomes. The free radical situation is<br />
quite complex because superoxide anions are tyrosinase substrate and melanin polymer<br />
behaves as a pseudosuperoxide dismutase producing H 2 O 2 . In 1991 we demonstrated the<br />
presence of aberrant melanosomes with membrane defects (with subsequent leakage of<br />
cytotoxic species) as a common phenomenon in melanoma cells. Pigment cells are<br />
protected by scavenging mechanisms (a) intramelanosomal binding of cytotoxic species to<br />
proteins which can be illustrated by our finding of protein-bound dopa in melanosomal<br />
proteins; b) conversion of quinones into adducts with cysteine and GSH into cysteinyldopa<br />
which is excreted via urine; c) prevention of diphenol conversion into quinones by COMT. If<br />
the capacity of scavenging mechanisms is overcome, pathological reactions ensue which can<br />
be exploited in melanoma therapy: a) Trojan horse approach = administration of tyrosine<br />
and DOPA analogues that are converted by tyrosinase specifically in pigment cells to<br />
cytotoxic molecules; b) inhibition of scavenging mechanisms = using COMT inhibitors we<br />
were able to inhibit proliferation of melanoma cells in vitro but not in vivo. Tumour<br />
proliferation is often free radical burden for the host. To our surprise the growth of B16 and<br />
S91 melanomas in mice and MeLiM melanoma in minipigs was not accompanied with signs<br />
of free radical damage. For that reason we compared the activities of antioxidant enzymes in<br />
tumour cells between 7 human melanoma cell lines and human osteosarcoma, glioma,<br />
colorectal carcinoma, lung carcinoma and neuroblastoma cell lines. The comparison showed<br />
significantly higher (p=0,004) catalase activity in melanoma lines compared to<br />
nonmelanoma lines, whereas there were no significant differences as for glutathione<br />
peroxidase, SOD and γ-glutamyltransferase. The total antioxidant status (TAS)of melanoma<br />
cells was also significantly higher than in nonmelanoma cells (p=0.004).Correlation between<br />
catalase activity and TAS (R=0.909, p level = 0,00004) confirmed that the defense of<br />
melanoma was based on catalase activity. Taking into account the role of H 2 O 2 in cell<br />
proliferation, angiogenesis, invasion and metastasizing, apoptosis, the manipulation of<br />
catalase can be promising tool in experimental melanoma therapy.<br />
Ackowledgement: Supported by VZ MSM 21620808.<br />
49<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
BIOMARKERS OF LYMPH NODE METASTASIS IN LOW-GRADE BREAST CANCER: AN<br />
INTEGRATED, PROTEOMICS-BASED APPROACH<br />
Pavel Bouchal 1,2 , Monika Mudrochová 1,2 , Eva Budinská 3 , Zbyněk Bortlíček 3 ,<br />
Iva Struhárová 1,2 , Lenka Hernychová 4 , Theodoros Roumeliotis 5 , Spiros D. Garbis 5 ,<br />
Roman Hrstka 1 , Petr Müller 1 , Rudolf Nenutil 1 and Bořivoj Vojtěšek 1<br />
1 Masaryk Memorial Cancer Institute, Brno; 2 Masaryk University, Faculty of Science,<br />
Brno; 3 Masaryk University, Institute of Biostatistics and Analyses, Brno; 4 University of<br />
Defence, Faculty of Military Health Sciences, Hradec Králové, 5 Academy of Athens,<br />
Greece<br />
Our effort has been focused on biomarker discovery in the set of 96 breast cancer<br />
tumors divided into groups according to grade and presence or absence of lymph<br />
node metastases. Three proteomics approaches were involved in complex protein<br />
analysis of tissue lysates: (i) SELDI-TOF MS enabled us to quantify 130 intact protein<br />
peaks. One protein peak correlating with lymph node metastases was detected and<br />
identified. (ii) Selected set of tumors was analyzed using iTRAQ-2DLC-MS/MS<br />
approach. This approach provides a simultaneous identification and quantitation of<br />
more than 600 proteins. Differentially expressed proteins belong to several functional<br />
groups which will be described in the presentation. (iii) Set of selected tumor lysates<br />
was analyzed also using 2-D SDS-PAGE. Additionally, the expression variability of 26<br />
selected gene products was evaluated using qRT-PCR.<br />
Generally, our results indicate the differential expression of (i) cytoskeletal proteins,<br />
(ii) anterior gradient protein family members and (iii) proteins involved in heme<br />
biosynthesis between the two studied groups.<br />
Acknowledgments: This work was supported by Czech Science Foundation (Project No.<br />
304/10/0868), by Czech Ministry of Health (Project No. MZMOU2005) and by Czech<br />
Ministry of Education (MSM0021622413).<br />
50<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
DOES EXISTS ANY RELATION BETWEEN P-GLYCOPROTEIN MEDIATED MULTIDRUG<br />
RESISTANCE AND INTRACELLULAR CALCIUM HOMEOSTASIS<br />
Zdena Sulová 1 , Mário Šereš 1 , Miroslav Barančík 2 , Lenka Gibalová 1 , Branislav Uhrík 1 ,<br />
Lenka Poleková 1 and Albert Breier 1<br />
1 Institute of Molecular Physiology and Genetics, Centre of Excellence of the Slovak<br />
Research and Development Agency „BIOMEMBRANES2008”, Slovakia, 2 Institute for<br />
Heart Research, SAV, Bratislava<br />
Multidrug resistance (MDR) of neoplastic tissue represents real obstacle in effective<br />
chemotherapy of cancer. Several mechanisms of MDR were identified, from which<br />
over-expression and efflux activity of P-glycoprotein (P-gp) – plasma membrane<br />
ATPase (ABCB1 member of ABC transporter family) – represent most common<br />
observed reason of neoplastic diseases chemotherapy malfunction. Process of P-gp<br />
mediated MDR seems to be related to intracellular calcium homeostasis at least<br />
indirectly because: i. substances blocking calcium influx through L-type of calcium<br />
channels like verapamil were often found to antagonize P-gp mediated MDR; ii.<br />
calcium signal abnormalities were observed in cells over-expressing P-gp; iii. cells<br />
with P-gp mediated MDR were often resistant to thapsigargin; iv. several differences<br />
in intracellular calcium localization were observed when P-gp negative and P-gp<br />
positive cells were compared; v. differences in contents of several proteins of<br />
endoplasmic reticulum involved in calcium homeostasis were observed to be<br />
associated with P-gp over-expression. Current study represents an attempt to<br />
summarize knowledge about possible relations between P-gp mediated MRD and<br />
intracellular calcium homeostasis.<br />
Acknowledgments: This work was supported by: APVV-0084-07, VVCE-0064-07,<br />
VEGA-2/0123/10, VEGA-2/0155/09<br />
51<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
OSTERIX OVER-EXPRESSION IN HUMAN EMBRYONIC STEM CELLS AND ITS EFFECT<br />
ON CELL DIFFERENCIATION<br />
Radim Černý 1 , Elerin Kärner 2 , Christian Unger 3 and Mikael Wendel 2<br />
1 Department of Biochemistry, LFUK Plzeň, 2 Center for Oral Biology and 3 Department<br />
of Medicine, Karolinska Institutet, Stockholm, Sweden<br />
Osterix (Osx) is a recently identified zinc finger-containing transcription factor<br />
encoded by the Sp7 gene, which regulates gene expression in committed osteoblastic<br />
precursor cells, acting downstream of Runx2 (Nakashima et al.: Cell 108, 143, 2002).<br />
We have over-expressed Osx after lentiviral transfer of Osx cDNA recombined with<br />
enhanced green fluorescent protein (EGFP) into the genome of human embryonic<br />
stem cells (HESC) line H9. We obtained two HESC subpopulations expressing two<br />
significantly different levels of Osx. Both subpopulations exhibited spontaneous<br />
differentiation and reduced expression of markers characteristic of the pluripotent<br />
phenotype, such as SSEA3, Tra I-60, and Nanog. The high level of Osx expression,<br />
compared to endogenous levels found in primary human osteoblasts, did not<br />
enhance osteogenic differentiation, and did not up-regulate collagen I expression.<br />
Instead, the high Osx levels induced the commitment towards the hematopoieticendothelial<br />
lineage by up-regulating the expression of CD34 and Gata I. However, low<br />
levels of Osx expression up-regulated collagen I, bone sialoprotein and osteocalcin<br />
production. Conversely, forced high level expression of the homeobox transcription<br />
factor HoxB4, a known regulator for early hematopoiesis, promoted osteogenesis in<br />
HESCs, while low levels of HoxB4 lead to hematopoietic gene expression. We<br />
conclude that for an enhanced osteogenesis originating from in vitro cultured HESCs,<br />
the correct levels of ectopic transcription factors need to be established. Our data<br />
also highlight the notion of close relationship between early blood and bone<br />
development.<br />
52<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
HISTORY AND PRESENT OF SCIENTIFIC AND PEDAGOGIC CONFERENCES OF<br />
TEACHERS FROM CHEMICAL INSTITUTES AND DEPARTMENTS OF SLOVAK AND<br />
CZECH MEDICAL FACULTIES<br />
Zdeňka Ďuračková and Zuzana Országhová<br />
Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry,<br />
Medical School, Comenius University, Bratislava, Slovakia;<br />
The regular meetings of Slovak and Czech teachers from institutes and departments<br />
providing education of chemical and biochemical disciplines at the Medical faculties<br />
are organized more than 50 years. The first meeting of teachers assembled by Prof.<br />
A.F. Richter in Prague was already in 1952.<br />
Primarily these meetings were focused only to the problems of education of<br />
chemistry and biochemistry. The way of interviewing and entrance tests, level of<br />
candidates, postgradual study as well as contemporary methods of education (elearning)<br />
are also discussed.<br />
In the last years our conferences are also place for presentation of research activities<br />
of institutes. The discussions at the meetings and reciprocal deal with practical<br />
experiences are the big contribution to the work of teachers, scientists and<br />
doctorands of participated institutes.<br />
From 20 th to 21 st May 2010 pedagogical conference of Slovak and Czech chemical and<br />
biochemical teachers was organized in Modra by Institute of Medical Chemistry,<br />
Biochemistry and Clinical biochemistry, Medical School, Comenius University in<br />
Bratislava. This conference gave the chance to young colleagues to present the<br />
scientific programmes of individual institutes as well as parts of their own research.<br />
53<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
MAGNETIC RESONANCE SPECTROSCOPY IN<br />
DIAGNOSTIC PROTOCOL OF THE BRAIN DISEASES<br />
Dušan Dobrota and Michal Bittšanský<br />
Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava,<br />
Slovak Republic<br />
Magnetic resonance spectroscopy (MRS) allowed study of some biochemical<br />
changes, and metabolic pathway in vitro and in vivo. MRS is a physical technique that<br />
has been used as an analytical method, predominantly by chemists to describe the<br />
structure of molecules in a specific solution. In biological application the method<br />
allowed to study low molecular compounds containing atoms that have magnetic<br />
properties (e.g. 1 H, 31 P, 13 C, 19 F etc.).Proton magnetic resonance spectroscopy ( 1 H<br />
MRS) can measure levels of cerebral metabolites with the low molecular weight such<br />
as N-acetylaspartate (NAA), choline (Cho), creatine (Cre), lactate (Lac) and some<br />
others. 1 H MRS application in vivo in the diagnostic protocol some brain diseases (<br />
brain tumors, epilepsy, neurodegenerative diseases and schizophrenia) was the<br />
study’s focus. In vivo magnetic resonance spectra were obtained from the different<br />
parts of the brain using clinical scanner Siemens Symphony (1,5T) and standard<br />
protocols. 1 H magnetic resonance spectroscopy (MRS) provides valuable information<br />
about the changes in the concentration of above mentioned metabolites and their<br />
ratios, which are typical for each brain diseases. The great advantage of MRS in vivo<br />
study is that we are allowed study of these biochemical events in real time without<br />
any disturbance of the tissue. We may conclude, that in vitro 1 H MRS provides good<br />
information about biochemical differences in various type of human brain<br />
pathologies and could be used in the standard diagnostic protocol.<br />
Acknowledgement: This work was supported by the Ministry of Education Grant 048-<br />
010UK-8/2008.<br />
54<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
EFFECT OF DOMAIN PEPTIDES OF THE CARDIAC RYANODINE RECEPTOR ON THE<br />
STABILITY OF BILAYER LIPID MEMBRANES AND ON RYR2 ACTIVITY<br />
Andrea Faltinová 1 , Jana Gaburjáková 1 , Ľubica Urbániková 2 , Matúš Hajduk 2 , Nataša<br />
Tomášková 3 , Marián Antalík 3 and Alexandra Zahradníková 1<br />
1 Institute of Molecular Physiology and Genetics SAS, Bratislava, Slovakia, 2 Institute of<br />
Molecular Biology SAS, Bratislava, Slovakia,<br />
3 Institute of Experimental Physics SAS, Košice, Slovakia<br />
The cardiac ryanodine receptor (RyR2) contains one N-terminal, one central and two<br />
C-terminal domains where mutations related to the cardiac arrhythmia, CPVT, tend to<br />
be clustered. It is assumed that interaction between the N-terminal and the central<br />
domain plays a role in forming the “domain switch” that regulates the stability of the<br />
resting (closed) state of the RyR2. The aim of our study was to test whether mutationprone<br />
regions of the RyR2 suppress the stability of the closed conformation.<br />
We constructed two peptides, DPcpvtN2 and DPcpvtC, corresponding to the N-<br />
terminal and central part of the RyR2 with the highest occurrence of CPVT mutations.<br />
We examined their effect on the resting activity of the RyR2. DPcpvtC (20 – 30 µM)<br />
moderately increased the RyR2 open probability, in accordance with the hypothesis.<br />
However, before an effect on the RyR2 activity could be observed, DPcpvtN2<br />
interacted with the BLM. In the concentration range of 0.5 – 2.0 µM the peptide<br />
perforated the BLM regardless of the presence of the RyR2. Secondary structure<br />
analysis of DPcpvtN2 using bioinformatics, CD-spectroscopy and mapping on the<br />
known tertiary structure of the IP3R ligand-binding domain that is homological with<br />
the distal part of the N-terminal domain has shown a high incidence of α-helix (45 - 76<br />
%) as well as ascending hydrophobicity gradient in the DPcpvtN2. These properties<br />
might explain the observed effect of DPcpvtN2 on the stability of BLM.<br />
Supported by grants APVV-0139-06, APVV-0441-09, VEGA 02/0190/10 and by the<br />
European Union Contract No. LSHM-CT-2005-018833/EUGeneHeart.<br />
55<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
TRANSGLYCOSYLATION - A UNIVERSAL PRINCIPLE IN TAILORING THE PLANT AND<br />
FUNGAL CELL WALLS<br />
Vladimír Farkaš<br />
Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, Department<br />
of Glycobiology, Dúbravská cesta 9, 84538 Bratislava, Slovakia<br />
Plant and fungal cell walls are composite structures composed of polysaccharides and<br />
protein-polysaccharides mutually cross-linked by non-covalent interactions and<br />
covalent bonds. Individual wall polymers are being synthesized separately, either<br />
intracellularly or at the plasma membrane and exported into the cell wall. The final<br />
stage of cell wall formation involves the formation of cross-links between the<br />
individual polymer molecules, either of the same or of the diverse types. The<br />
enzymes catalyzing the latter type of reactions are transglycosylases. They are either<br />
GPI-anchored to the plasma membrane or embedded in the cell wall. As an example<br />
from the plant kingdom, the enzyme xyloglucan endotransglycosylase (XET) will be<br />
presented. The enzyme catalyzes cleavage of xyloglucan molecules and transferring<br />
the cleaved fragments to other xyloglucan molecules in the plant cell walls.<br />
Transglycosylases operate also in the fungal cell walls. As the examples can serve the<br />
β-1,3-glucan elongases of the Gas family or the chitin endotransglycosylases of the<br />
Crh family from yeast. Biochemical properties of these enzymes heterologously<br />
expressed in Pichia were determined in vitro using specially devised assays. In these<br />
assays, soluble polysaccharide derivatives were used as the glycosyl donors and<br />
diverse fluorescently labeled oligosaccharides as the acceptors. As the measure of<br />
enzyme activity served the amount of the fluorescence incorporated into the<br />
polymer.<br />
56<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
INHIBITION OF INSULIN AMYLOID AGGREGATION WITH ALBUMIN FUNCTIONALIZED<br />
MAGNETIC FLUID<br />
Andrea Antošová 1 , Katarína Šipošová 2 , Martina Koneracká 1 , Vlasta Závišová 1 ,<br />
Peter Kopčanský 1 and Zuzana Gažová 1<br />
1 Department of Biophysics, Department of Magnetism, Institute of Experimental<br />
Physics SAS, Kosice, Slovakia, 2 Department of Biochemistry, Faculty of Science, P. J.<br />
Safarik University, Kosice, Slovakia<br />
Amyloid-related diseases, such as Alzheimer′s disease or diabetes type II, are<br />
associated with self assembly of protein into amyloid aggregates. Recently, there are<br />
only few reports dealing with the effect of nanomaterials on the amyloid aggregation.<br />
We investigated effect of magnetic fluid consists of Fe 3 O 4 nanoparticles sterically<br />
stabilized by sodium oleate with adsorbed BSA (MF-BSA) on amyloid aggregation of<br />
insulin. The ability of MF-BSA to inhibit formation of insulin amyloid aggregates (Iagg)<br />
in vitro was studied by ThT assay and TEM. We have found that MF-BSA is able to<br />
prevent formation of aggregates, the extent of amyloid formation depends on MF-<br />
BSA concentration with extensive 70% inhibiting activity for ratio Iagg:MF-BSA = 1:7.<br />
The obtained results indicate that presence of MF-BSA led to the inhibition of insulin<br />
amyloid aggregation. Our findings make MF-BSA of potential interest as therapeutic<br />
agents against amyloid-related diseases.<br />
Ackowledgement: This work was supported within the projects Nos. 26220220005,<br />
26220120033 and 26220120021 in frame of SF EU, Centre of Excellence of SAS<br />
Nanofluid, VEGA 0079, 0056, 0077 and VVGS PF 13/2010/ Ch.<br />
57<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
TWENTY FOUR YEARS SINCE CHERNOBYL DISASTER:<br />
WHAT SEED PROTEIN CAN TELL US?<br />
Martin Hajduch 1 , Katarína Klubicová 1 , Maksym Danchenko 1, 3 , Ludovit Škultéty 2 ,<br />
Namik Rashydov 3 and Anna Preťová 1<br />
1 Department of Reproduction and Developmental Biology, Institute of Plant Genetics<br />
and Biotechnology, Slovak Academy of Sciences, Nitra, Slovakia<br />
2 Center for Molecular Medicine, BITCET, Institute of Virology, Slovak Academy of<br />
Sciences, Bratislava, Slovakia, 3 Department of Biophysics and Radiobiology, Institute<br />
of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine,<br />
Kyiv, Ukraine<br />
The explosion of one of the four reactors of Chernobyl nuclear power plant (CNPP) on<br />
26 April 1986 caused the worst environmental nuclear disaster in the history. Huge<br />
amounts of radioactivity were released not only to the close surroundings of the<br />
power plant but also to large parts of Europe. Despite the fact that since 1986<br />
radiation levels in the affected environment have declined several hundred folds,<br />
dangerous long-living isotopes such as 137 Cs and 90 Sr remains as main contaminants.<br />
Now, 24 years after the accident, the question how plants in radio-contaminated<br />
Chernobyl were able to adapt is still open, and needs to be fully answered. Plants are<br />
stationary and thus must adapt to extreme conditions in order to survive. The main<br />
objective of our research is to characterize quantitative differences on protein levels<br />
between soybean (Glycine max) and flax (Linum usitatissimum) grown in<br />
contaminated (~5 km from CNPP) and control (~10 km from CNPP) experimental<br />
fields in order to elucidate molecular mechanisms plants used for adaptation. To<br />
acquire complex proteome information seed proteins are quantitatively analyzed<br />
using two-dimensional gel electrophoresis and identified by tandem mass<br />
spectrometry.<br />
58<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
APOPTOSIS IN RELATION TO THE DEVELOPMENT OF CANCER AND RESISTANCE OF<br />
CANCER CELLS TO CYTOSTATICS<br />
Jozef Hatok 1 , Jana Jurečeková 1 , Peter Chudý 2 , Pavol Hollý 2 , Anton Dzian 3 , Eduard<br />
Huľo 3 , Eva Fabianová 1 , Tatiana Matáková 1 and Peter Račay 1<br />
1 Department of Medical Biochemistry, 2 Clinic of Hematology and Transfusiology,<br />
3 Clinic of Surgery – JFM and MFH in Martin, CU in Bratislava, Slovakia<br />
Apoptosis plays an important role in development and homeostasis of the<br />
multicellular organisms and its deregulation may result in many serious diseases,<br />
including cancer. In addition, dysregulation of apoptosis is associated with resistance<br />
of cancer cells to cytostatics. We studied the expression of mRNA of apoptotic<br />
proteins (p53, Bax, Bcl-2 and Bcl-X L ) in samples from cancer patients. In addition, we<br />
focused on their correlation with the results of chemoresistance testing. We<br />
examined 102 samples from patients with haematological malignancies (60) and solid<br />
tumors (42). To determine the levels of mRNA we used the RT-PCR. The in vitro<br />
chemoresistance of leukaemic cells was evaluated by MTT assay. Statistically<br />
significant differences of mRNA expression of all investigated proteins between the<br />
group of leukaemia samples and leukocytes from healthy volunteers were<br />
determined (p
Lectures<br />
SPECIALITIES OF OXIDATIVE PHOSPHORYLATION OF TRYPANOSOMATIDS AND<br />
EUGLENAS<br />
Anton Horváth 1 , Ingrid Škodová 1 , Anna Gnipová 1 , Alena Zíková 2 , Vladislava<br />
Benkovičová 1 , Zdeněk Verner 2 , Zdeněk Paris 2 and Július Lukeš 2<br />
1 Department of Biochemistry FNS UK, Bratislava, Slovak republic, 2 Institute of<br />
Parasitology, Czech Academy of Sciences, České Budějovice, Czech Republic<br />
Trypanosomatids and euglenas are protists that belong to common phylum<br />
Euglenozoa. Despite their relatively close relations they differ quite strongly. Euglenas<br />
usually live in freshwater environments and contain chloroplasts with functional<br />
photosynthetic apparatus. In contrast with that all trypanosomatids are obligatory<br />
parasites and they are the only known eukaryotes with glycolysis separated from<br />
cytosol to special organelles - glycosomes. Euglenas and trypanosomatids are able<br />
suppress and again activate their oxidative phosphorylation depending on changing<br />
living conditions and they both have some alternative pathways to classical<br />
respiratory chain. So they are good models for study individual enzyme of oxidative<br />
phosphorylation and their importance for metabolism in different living conditions. In<br />
our lab we work with 4 different trypanosomatids and with Euglena gracilis and its<br />
mutants that are not able photosynthesis. Here we report about our two lines of our<br />
research:<br />
1. Localization and activity of two different FAD dependent glycerol-3-phosphate<br />
dehydrogenases and functions of several polypeptides associated with cytochrome C<br />
oxidase in Trypanosoma brucei<br />
2. General characterization of enzymes of respiratory chain in Euglena gracilis and<br />
their significance in cells grown on light and dark<br />
60<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
PROTEIN IMMUNIZATION OF MUTANT MOUSE – AN EFFICIENT WAY TO GENERATE<br />
SELECTIVE AND SENSITIVE ANTIBODIES<br />
Anna Hrabovska 1,2 , Véronique Bernard 1 and Eric Krejci 1<br />
1 Centre d’Etude de la Sensori-Motricité, Université Paris Descartes- CNRS- UMR8194,<br />
45 rue des Saints Pères, 75006 Paris, France<br />
2 Dpt. of pharmacology and toxicology, Faculty of Pharmacy, Comenius University,<br />
Odbojarov 10, 832 32 Bratislava<br />
Despite a long history, the successful generation of specific and selective antibodies is<br />
still a task fraught with considerable uncertainty.<br />
We examine a simple, fast, and highly efficient strategy to produce an antibody,<br />
which utilizes immunization of mutant mouse strains with antigens that the host<br />
strains themselves have been genetically targeted to be deficient for. To test this<br />
strategy we choose butyrylcholinesterase, an antigen that has been considered to be<br />
difficult to generate antibody against. Antigens of different origins all provided a<br />
strong immune response, while the characteristic of the resulting antibodies<br />
depended on the preparation for the antigen prior to the immunization.<br />
This method, introduced previously but since neglected until now due probably to<br />
the lack of specific resources, should at this time, based on our data presented here,<br />
be considered a reasonable and reliable choice for antibody production.<br />
Acknowledgement: Work was supported by grants AFM; grant ANR Neuroscience and<br />
APVV grants (SK-FR-0031-09 a SK-CZ-0028-09).<br />
61<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
DO WE TEACH BIOCHEMISTRY IN A LOGICAL WAY? REMARKS CONCERNING THE<br />
CONTENTS AND LEARNING APPROACH<br />
Jiří Hudeček<br />
Department of Biochemistry, Faculty of Science, Charles University<br />
Hlavova 2030/8, 128 40 Praha 2, Czech Republic<br />
I was always puzzled with the fact that among many of the most motivated and gifted<br />
students of "pure" chemical disciplines, there was a sort of depreciatory attitude<br />
towards biochemistry. (Occasionally, I could feel a similar attitude also among<br />
teachers.) At the same time, biochemical problems are often "invading" the field of<br />
the "pure" chemical disciplines, and one can say that at present a considerable<br />
percentage of all research in our chemical Departments has a strong connection with<br />
biochemistry. Certainly, part of the reasons for this situation might be historical, but<br />
there is a tendency for reproduction of these feelings. After conducting some<br />
interviews with students, I understand now that for the more logically thinking<br />
students, biochemistry is often a discipline too "biological" (in the sense "historical"),<br />
just describing things and showing some a posteriori explanations. Additionally, the<br />
traditional approach to teaching (I call it "synthetical") forces students to learn a lot<br />
of facts, for long time seemingly without much logical coherence. They sometimes<br />
have a difficulty to understand the reasons why to learn, say, details of the citrate<br />
cycle. As the overall view of the intermediate metabolism comes only later, they may<br />
lose the enthusiasm long before coming close to get any sense of the beauty of the<br />
discipline. In the present contribution, I would like to discuss several possibilities how<br />
the situation might be improved: (1) using the "analytical" concept (an overall view<br />
later completed with details), (2) stressing the "chemical logic" in certain parts of<br />
biochemistry (structure and properties of biopolymers), (3) strong connection<br />
between the description (results) and experimental background for it, etc.<br />
62<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
EVALUATION OF TOXICITY AND GENOTOXICITY OF ORGANOHALOGEN PESTICIDES<br />
Pavlína Janů 1 , Markéta Thimová 1 , Petra Lovecká 1 ,<br />
Martina Macková 1 and Kateřina Demnerová 1<br />
Department of Biochemistry and Microbiology, Faculty of Food and Biochemical<br />
Technology, ICT Prague, Technická 5, 166 28 Prague, Czech Republic<br />
This work is focused on toxicity and genotoxicity analysis of the worldwide commonly<br />
used benzonitrile herbicides dichlobenil, chloroxynil, bromoxynil and ioxynil and their<br />
metabolites 2,6-dichlorobenzamid, 2,6-dichlorobenzoic acid, 3,5-dichloro-4-<br />
hydroxybenzoic acid, 3,5-dibromo-4-hydroxybenzoic acid, 3,5-diiodo-4-<br />
hydroxybenzoic acid. The toxicity was also determined for restricted organochlorine<br />
pesticides (fungicide HCB, insecticide γ-HCH, 4,4'-DDT and its metabolites 4,4'-DDA<br />
and 4,4'-DDE).<br />
Sea luminescent bacteria Vibrio fischeri, gramnegative bacteria Escherichia coli,<br />
grampositive sporulating bacteria Bacillus subtilis and microorganism isolated from<br />
contaminated soil (Burkholderia glathei) were chosen as the prokaryotic model<br />
systems for investigation of the acute toxicity. The eukaryotic model systems were<br />
represented by the seeds of Lactuca sativa, var. capitata and Avena sativa, by the<br />
„hairy root“ culture of Solanum nigrum and by the human cell line HEK 293T.<br />
Genotoxicity was determinated using the Ames test with bacteria Salmonella<br />
typhimurium His - TA98 and TA100 and the comet assay with the HEK 293T cell line.<br />
Our data point to that toxicity of pesticides is higher than their corresponding<br />
metabolites practically in all used systems. The toxic effect of studied compounds was<br />
similar in the context of used model system. The comet assay confirmed genotoxicity<br />
of pesticides ioxynil, bromoxynil, hexachlorobenzene and DDE, the metabolite of<br />
DDT.<br />
Acknowledgements: The authors thank for the support of grants Tandem FT –<br />
TA4/101, FT-TA5/043 a MSM 6046137305.<br />
63<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
BRONCHIAL ASTHMA AND EFFECT OF OXIDATIVE STRESS ON ITS DEVELOPMENT<br />
Eva Babušíková 1 , Miloš Jeseňák 2 , Peter Bánovčin 2 and Dušan Dobrota 1<br />
1 Department of Medical Biochemistry, 2 Institute of Children and Adolescents,<br />
Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin, Martin,<br />
Slovakia<br />
Bronchial asthma (BA) is associated with increased oxidative stress. Oxidative stress is<br />
increasing due to the shift of the balance between pro-oxidant and antioxidant to<br />
side of pro-oxidant. Bronchial asthma is a complex chronic inflammatory disorder of<br />
the airways and as a complex disease is multifactorial, with many candidate genes<br />
suspected as being important in its development. In our study we analyzed oxidative<br />
damage of proteins and lipids and polymorphism of glutathione-S-transferase (GST:<br />
GSTT1, GSTM1) which is a BA candidate gene due to its role in protection against<br />
oxidative stress. The total content of sulfhydryl groups was decreased significantly (p<br />
< 0.001) in asthmatic patients compared to healthy children. Concentrations of<br />
thiobarbituric acid-reactive substances were increased significantly (p < 0.001) in<br />
asthma patients. The GSTT1 and GSTM1 null genotypes were more frequent (OR 1.63,<br />
respectively OR 1.18) among the asthmatic patients. The null genotype for both<br />
representatives of glutathione-S-transferase family represented higher risk for<br />
development of BA (OR 2.23) than present of one of them. Asthmatic children with<br />
null genotypes had higher oxidative damage as well. These results suggested that<br />
increased oxidative stress may play role in the asthma pathogenesis. Oxidative stress<br />
and changed antioxidant defense are included in the asthma pathology and therefore<br />
elimination of oxidative stress could be potentially an appropriate strategy for<br />
treatment of bronchial asthma.<br />
Acknowledgements: This work was supported by Ministry of Health of the Slovak<br />
Republic 2007/47-UK-12.<br />
64<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
FROM BASIC BIOMEDICAL RESEARCH TO BIOTECHNOLOGY<br />
Laco Kačáni<br />
CEMIT – Center of Excellence in Medicine &IT GmbH, Innsbruck, Austria<br />
Recent business success of the biotech and pharma industry is mainly based on the<br />
commercialization of basic research discoveries done by researchers at public<br />
universities. As a result of these new economic developments, the strict division<br />
between basic and applied research in life sciences has weakened, thereby linking the<br />
research capacities of universities with the commercial capabilities of industry. The<br />
protection of research results in the life sciences by means of intellectual property<br />
rights is a prerequisite for commercial exploitation of research results in<br />
biotechnology. However, increased awareness among academic researchers for the<br />
commercialization of their research is even more important for the transfer of new<br />
biotechnologies to the business sector.<br />
The way, in which academic researchers cooperate and build partnerships with<br />
businesses, as well as the importance of academic research for biotech and pharma<br />
industry, changed dramatically in recent years. Consequently, new models of<br />
technology transfer and collaboration with biotech industry emerged in the last<br />
decade, some stimulated by state interventions and others formed directly between<br />
universities and biotech companies. These models enable a rapid translation of new<br />
knowledge into technologies, reduce the overall costs of R&D and facilitate the<br />
launching of new products or services onto the market place.<br />
In this talk, various models of technology transfer and collaboration with biotech<br />
industry will be analyzed and exemplified from different points of view, using<br />
examples of successful commercialization of academic biomedical research in<br />
biotechnology.<br />
65<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
NEW POSSIBILITIES FOR THE STUDY OF METABOLISM IN SLOVAKIA<br />
Michal Kaliňák 1 and Tibor Liptaj 2<br />
1 Department of Biochemistry and Microbiology, 2 Department of NMR and MS, Faculty<br />
of Chemical and Food Technology Slovak University of Technology,<br />
Radlinského 9, 812 37 Bratislava<br />
NMR solves chemical structures for decades, but only after some laboratories in<br />
Slovakia were equipped with new NMR spectrometers and appropriate software it is<br />
possible to use metabonomic approach to tackle various biological questions. We<br />
have summarized various aspects of sample preparation, measurement setup and<br />
post-acquisition processing needed to be followed to obtain results giving biologically<br />
relevant information. Restrictions and disadvantages of NMR in terms of sensitivity,<br />
signal overlap and sample volume are presented.<br />
The metabonomic<br />
1 H NMR data of germination, growth and conidiation of<br />
filamentous fungus Trichoderma viride are shown as an example of a study in fungal<br />
microbiology. The onset of metabolic activity can be observed during germination.<br />
Different cultivation conditions can be discerned on the basis of multivariate<br />
statistical analysis without the need to identify individual metabolites.<br />
Acknowledgement: This work was supported by Slovak Grant Agency VEGA<br />
1/0462/08 and APVV 0642-07. NMR experimental part of this work was facilitated by<br />
the support of Slovak National Research and Development Program No.<br />
2003SP200280203.<br />
66<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
GATING OF THE NEURONAL CA V 3.3 CHANNEL<br />
Mária Karmažínová 1 , Edward Perez-Reyes 2 and Ľubica Lacinová 1<br />
1 Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences,<br />
Bratislava, Slovakia, 2 Department of Pharmacology, University of Virginia,<br />
Charlottesville, Virginia 22908, USA<br />
Low-voltage activated Ca V 3 Ca 2+ channels have activation threshold about -60 mV.<br />
Kinetics of their activation at membrane voltages just above activation threshold is<br />
much slower that the activation kinetics of other VDCC. It was demonstrated that<br />
intracellular loop connecting repeats I and II of all three Ca V 3 channels contains socalled<br />
“gating brake”. Disruption of this brake yielded channels that activated at even<br />
more hyperpolarized potentials with significantly accelerated kinetics. We have<br />
compared gating of a wild type Ca V 3.3 channel and a mutated ID12 channel, in which<br />
putative gating brake at proximal part of the I-II loop was removed. The whole cell<br />
Ca 2+<br />
current was measured using the HEKA-10 patch clamp amplifier. Holding<br />
potential (HP) in all experiments was -100 mV. Gating currents were measured by 50<br />
ms long depolarizing pulses to membrane potentials between -90 mV and +70 mV.<br />
Voltage dependence of gating current activation was shifted by 18.5 mV towards<br />
more hyperpolarized potentials in ID12 channel. Kinetics of the on-charge activation<br />
was significantly accelerated. Kinetics of the off-charge was not altered. Value of<br />
maximal on-charge normalized in respect to maximal inward current amplitude was<br />
doubled.<br />
We concluded that the putative gating brake in I-II loop hinders not only opening of<br />
the conducting pore but also the activating movement of voltage sensing S4<br />
segments stabilizing the channel in its closed state.<br />
Acknowledgements: Supported by VVCE-0064-07 and VEGA 2/0195/10.<br />
67<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
MOLECULAR MECHANISMS INVOLVED IN RESPONSE TO HYPOXIA<br />
Juraj Kopáček, Jaromír Pastorek and Silvia Pastoreková<br />
Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9,<br />
845 05 Bratislava<br />
Cellular responses to diminished supply of oxygen include growth arrest, apoptosis,<br />
anaerobic glycolysis, angiogenesis etc. The primary response to the lack of oxygen at<br />
the molecular level is the stabilization of α subunit of HIF-1 transcriptional complex, a<br />
key regulator of the genes involved in adaptation to the hypoxic stress. In normoxia,<br />
HIF-1α undergoes hydroxylation that is required for its interaction with the product<br />
of the with type von Hippel-Lindau (VHL) tumor suppressor gene. This interaction<br />
results in fast ubiquitilation and proteasome degradation of HIF-1α. Loss or mutation<br />
in VHL, the main negative regulator of the hypoxic pathway, leads to development of<br />
the hypoxic phenotype also under normoxic conditions. In addition, stabilization of<br />
HIF-1α could be achieved by signal transduction through the pathways regulated by<br />
activated oncogenes that can contribute to or amplify the effects of of HIF-1<br />
transcriptional complex. HIF-1 is a key regulator of a broad range of cellular and<br />
systemic responses to hypoxia and acts in all mammalian cells. HIF-1 activity is<br />
dependent upon the availability of the HIF-1α subunit, which is in turn regulated by<br />
cellular oxygen levels.<br />
This work was supported by the Research & Development Operational Programme<br />
funded by the ERDF „TRANSMED” and by VEGA 2/0194/09.<br />
68<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
REGULATION OF AURICIN BIOSYNTHESIS IN<br />
STREPTOMYCES AUREOFACIENS CCM 3239<br />
Ján Kormanec, Renáta Nováková, Ľubica Fecková, Peter Kutaš and Alena Reháková<br />
Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 845<br />
51 Bratislava, Slovakia<br />
Gram-positive bacteria Streptomycetes are the main producers of bioactive natural<br />
products including many antibiotics. The polyketides, which are synthesized by<br />
multifunctional enzymes called polyketide synthase (PKS), belong to the most<br />
important classes of antibiotics. We previously identified a type II polyketide synthase<br />
(PKS) gene cluster, aur1, in Streptomyces aureofaciens CCM3239 with the highest<br />
similarity to angucycline polyketide subgroup of PKS clusters. Deletion of two critical<br />
biosynthetic genes resulted in a lack of antibiotic that was named auricin. However,<br />
its purification has been hampered by very low yields. This cryptic phenotype has<br />
been recently described for several homologous angucycline gene clusters, indicating<br />
their tight regulation. Sequence analysis of the whole aur1 cluster revealed a huge<br />
number of regulatory genes, including six genes for homologues of TetR family<br />
repressors, five genes for homologues of specific Streptomyces Antibiotic Regulatory<br />
Proteins (SARP family), a single gene for homologue of AraC/XylS-family regulators, a<br />
single genes for homologue of response regulators of bacterial two-component signal<br />
transduction systems, and two genes for gama-butyrolactone autoregulator-receptor<br />
system. Their partial characterization indicated that several of them play a positive or<br />
negative role in auricin production in a cascade scheme.<br />
Acknowledgements: This work was supported by the Slovak Research and<br />
Development Agency under the contract No. APVV-0017-07.<br />
69<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
TEACHING BIOCHEMISTRY AT THE UNIVERSITY OF VETERINARY MEDICINE AND<br />
PHARMACY IN KOŠICE<br />
Zuzana Kostecká<br />
Institute of Biochemistry, Department of Chemistry, Biochemistry and Biophysics,<br />
The University of Veterinary Medicine and Pharmacy in Košice<br />
Implementation of credit system in the study at the University of Veterinary Medicine<br />
and Pharmacy in Košice was coupled with innovation of curricula of all obligatory<br />
study subjects, creating of new compulsory optional and optional subjects including<br />
biochemistry. Nowadays the teachers of biochemistry institute participate in teaching<br />
of obligatory subjects: Biochemistry for the study pogramme General veterinary<br />
medicine (SP GVM) and SP Food hygiene (SP FH), General Biochemistry for SP<br />
Pharmacy, Aplied Biochemistry for SP Safety of food and feed and Biochemistry for SP<br />
GVM in English language; compulsory optional study subjects: Molecular mechanisms<br />
of metabolic and inherent diseases for SP GVM and SP FH, Molecular mechanisms of<br />
metabolic and inherent diseases for SP GVM in English, Clinical biochemistry for SP<br />
GVM and SP FH and Clinical biochemistry for SP GVM in English. The obligatory<br />
subject Clinical and pathological biochemistry for SP Pharmacy is provided by external<br />
teachers (teachers of Faculty of Medicine of Pavol Jozef Šafárik University in Košice).<br />
The aim of this presentation is to inform about changes in teaching biochemistry and<br />
to evaluate the positives and negatives according to our experiences. Lecture shows<br />
the advantages and disadvantages of credit system of study, including the questions<br />
of actual problems in field of biochemistry teaching at our university, e.g.<br />
organisation of teaching, position of study subjects in curriculum, continuity of study<br />
subjects, using of multimedia technology, etc. In conclusion, considering the possible<br />
solutions and seeking answers to questions will be resulted in successful educational<br />
process.<br />
70<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
TEACHING BIOCHEMISTRY AT THE FACULTY OF SCIENCE<br />
IN KOŠICE<br />
Mária Kožurková, Marián Antalík and Dušan Podhradský<br />
Department of Biochemistry, Institute of Chemistry, Faculty of Science, P. J. Šafárik<br />
University, Košice, Slovakia<br />
We have been teaching biochemistry for 20 year. Our department provides an<br />
education of Biochemistry for students of the bachelor and master degree,<br />
according to the credit system of study at Faculty of Science.<br />
The subject Biochemistry was broken up into part I and II. Biochemistry I taught for<br />
2 hour per week (for students of bachelor study) and will give a survey of new<br />
technologies and materials. The lectures are suitable for both scientific and<br />
pedagogically oriented students. Biochemistry II (for students of master study) will<br />
focus of structures and functions of saccharides and lipids, metabolism, regulation<br />
of metabolic pathways, basic metabolic processes and principle of bioenergetic. The<br />
subject is taught for 2 hours per week and is finished by final written test.<br />
Biochemistry III - Modern trends in biochemistry (for students of master study)<br />
provide biochemical view into the modern trends: evolution of energetic<br />
metabolism, protein splicing, protein sequencing, protein - DNA interaction,<br />
problems of DNA replication, cell division, viruses, and apoptosis. Students are<br />
eligible to graduate with a title of Master of Science upon passing of a state<br />
examination in the areas of Biochemistry, Molecular Biology, Biophysical Chemistry,<br />
Bioorganic Chemistry, Clinical Biochemistry and Biotechnology.<br />
The department is also an educational workplace for post-graduate students in the<br />
field of the structure and functions of biomolecules.<br />
Acknowledgement. This work was supported by the Grant Agency VEGA 1/0053/08<br />
and KEGA 3/6301/08.<br />
71<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
ASSEMBLY OF BACILLUS SUBTILIS SPORE COAT: INVESTIGATION OF PROTEIN-<br />
PROTEIN INTERACTIONS AMONG THE SPORE COAT PROTEINS OF<br />
BACILLLUS SUBTILIS<br />
Daniela Krajčíková 1 , Denisa Mullerová 1 , Wan Qiang 2 , Per Bullogh 2 ,<br />
Jilin Tang 3 and Imrich Barák 1<br />
1 Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia;<br />
2 Krebs Institute for Biomolecular Research, Department of Molecular Biology and<br />
Biotechnology, University of Sheffield, United Kingdom;<br />
3 State Key Laboratory of Electroanaytical Chemistry, Changchun Institute of Applied<br />
Chemistry Chinese Academy of Sciences, Changchun, P. R. China<br />
Spores, dormant cell types of Bacillus subtilis, are incased in thick proteinaceous<br />
multilayered shell, called the coat, with significant protective role from the<br />
environment. While providing high level of resistance, the coat allows the spore to<br />
respond to the renewed presence of nutrients and start the cell growth in the process<br />
called germination. The unique properties of the coat are determined by the<br />
architecture of complex spore coat structure. Being formed by more than 70 different<br />
proteins, two main layer of coat are easily distinguished – the lamellar inner coat and<br />
thick striated outer coat. The order of assembly and final destination of the coat<br />
structural components rely mainly on specific protein-protein interactions and on the<br />
action of small group of morphogenetic proteins which guide the deposition of rest of<br />
the coat components onto the spore surface. Since the process of assembly is still<br />
poorly understood, by searching for direct protein-protein interactions we want to<br />
gradually generate the data to obtain the entire picture of whole coat formation<br />
event.<br />
In our studies we implemented yeast two hybrid system and other genetic and<br />
biochemical methods to examine protein interactions among a group of<br />
morphogenetic coat proteins and proteins of coat insoluble fraction. Investigation of<br />
properties of individual recombinant coat proteins showed, that they frequently form<br />
high molecular weight oligomeric structures in vitro. We employed AFM and EM<br />
microscopy to analyze these structures in detail.<br />
72<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
CHANGES AND ROLE OF ADRENOCEPTORS IN PC12 CELLS AFTER PHENYLEPHRINE<br />
ADMINISTRATION AND APOPTOSIS INDUCTION<br />
Lubomira Lencesova, Marta Sirova, Lucia Csaderova, Marcela Laukova, Zdena Sulova,<br />
Richard Kvetnansky, Olga Krizanova:<br />
73<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
GATING OF THE T-TYPE CALCIUM CHANNELS<br />
Ľubica Lacinová and Mária Karmažínová<br />
Institute of Molecular Physiology and Genetics, SAV, Bratislava, Slovak Republic<br />
T-type calcium channels are distinguished by relatively low voltage threshold for an<br />
activation and steep voltage dependence of activation and inactivation kinetics just<br />
above the activation threshold. Further, while macroscopic current kinetics of Ca V 3.1<br />
and Ca V 3.2 channels are virtually identical, kinetics of the Ca V 3.3 channel is almost<br />
one order more slow. Kinetics and voltage dependence of macroscopic inward<br />
calcium current through Ca V 3 channels was described in a detail. In contrast, very<br />
little information is available on gating current of these channels. Therefore we<br />
compared gating currents measured from all three Ca V 3.1, Ca V 3.2 and Ca V 3.3<br />
channels.<br />
Voltage dependencies of macroscopic current activation are similar for all three Ca V 3<br />
channels. While gating kinetics of macroscopic calcium current is virtually identical<br />
for Ca V 3.1 and Ca V 3.2 channels it is about one order slower for the Ca V 3.3 channel.<br />
Voltage dependencies of charge movement differ dramatically from those for<br />
macroscopic current. First, their slope factors are several-fold bigger that slope<br />
factors of macroscopic current activation. Second, activation mid-point for Ca V 3.3<br />
channels on-gating is shifted to more positive membrane potentials by about 20 mV<br />
compare to Ca V 3.1 and Ca V 3.2 channels, whose activation mid-points are similar. The<br />
same is truth for off-gating voltage dependences. Kinetics of both on- and off-gating<br />
is remarkably faster for Ca V 3.1 and Ca V 3.2 channels compare to Ca V 3.3 channels.<br />
Further, more charge is moved per unit of macroscopic current amplitude in Ca V 3.3<br />
channels compare to Ca V 3.1 and Ca V 3.2 channels.<br />
Acknowledgement: Supported by VVCE-0064-07 and VEGA 2/0195/10.<br />
74<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
INDUCTION OF ISCHEMIC TOLERANCE IN SENSITIVE NEURONS: COORDINATED ROLE<br />
OF MULTIPLE MECHANISMS<br />
Ján Lehotský, Mária Chomová, Andrea Evinová, Mária Kovalská, Martina Pavlíková,<br />
Zuzana Tatarková, Peter Kaplán and Peter Račay<br />
Comenius University, Jessenius Faculty of Medicine, Department of Medical<br />
Biochemistry, Martin, Slovakia<br />
Ischemic brain injuries are among the most common and important causes of disability<br />
and death worldwide. Sublethal ischemia, ischemic preconditioning (IPC), triggers<br />
endogenous responses that protect the brain against a subsequent severe ischemic<br />
insult, a phenomenon known as tolerance. These treatments can initiate and amplify the<br />
endogenous adaptive/restorative processes in brain. The aim of this study was to<br />
determine whether altered interplay between intracellular Ca2 + stores resulting in the<br />
apoptotic and unfolded protein response is linked with the neuronal adapation induced<br />
by ischemic preconditioning. We refer here that ischemic/reperfusion injury (IRI) is<br />
manifested by: i) functional mitochondrial ganges, and ii) altered gene expression and<br />
translation of endoplasmic reticular (ER) key UPR proteins. Tissue response to ischemic<br />
preconditioning includes changes in the: i) level of initiation and execution of apoptosis<br />
ii) activation of inhibition of p53 translocation to mitochondria, iii) changes of<br />
endoplasmic reticular (ER) expression of Ca 2+ binding GRP78 and ATF6 proteins, and iv)<br />
effects on Secretory Pathways Calcium Pump (SPCA) gene expression and partial<br />
recovery of depressed SPCA activity. The results suggest that adaptation process<br />
/ischemic tolerance induced by preischemic challenge includes interplay between<br />
intracellular Ca 2+ stores, mitochondria, ER and Golgi apparatus, and have potential to<br />
translate into novel strategies for the treatment of ischemic stroke. In addition, we<br />
present here an overview of pathways which eventually maturates in tolerant phenotype<br />
of neuronal cells in affected brain areas.<br />
Acknowledgements: This work was supported by the VEGA grant No. 49/09, VVCE 64/07,<br />
55 UK-16/2007 and by project “Center of translational medicine” co-financed from EC<br />
sources and European regional development fund.<br />
75<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
SPINAL CORD INJURY: PATHOGENESIS AND TREATMENT<br />
Nadežda Lukáčová, Alexandra Dávidová, Ľudmila Capková and Andrea Kucharíková<br />
Institute of Neurobiology, Slovak Academy of Sciences, Košice<br />
The current clinical approach to spinal cord injury is mainly symptomatic. Therapies<br />
aimed at limiting the evolution of the neurogenerative disorders affecting motor<br />
neurons are still extensively studied both in experimental and clinical setting.<br />
However, currently there is a lack of effective therapies. The aim of this study was to<br />
find out whether neuronal degeneration correlates with up-regulation of nitric oxide<br />
synthase (NOS) forming nitric oxide, and whether these neurons are protected by<br />
parvalbumin (PV), buffering free intracellular calcium. Fluoro-Jade B was used to<br />
detect dying neurons. In addition, the animals after spinal injury were treated with<br />
GABA B receptor agonist Baclofen (from 7th day 3mg/twice daily), than with NNLA, an<br />
inhibitor of neuronal NOS dosed at 20 mg/b.w., and in the third group the animals<br />
were treated with NNLA in combination with Baclofen. 7 and 14 days after spinal cord<br />
injury both the level of nNOS protein and nNOS mRNA level were significantly<br />
increased in segments below the site of injury. We noted strong nNOS upregulation<br />
in motoneurons and in neurons of laminae VII. However, α-motoneurons were not<br />
Fluoro-Jade B positive. While PV-IR was increased in a number of small neurons in rat,<br />
rabbit′s alfa-motoneurons exhibited very strong PV fluorescent staining. These<br />
results indicate the participation of PV in motor control. Treatment with Baclofen and<br />
NNLA decreased the level of nNOS mRNA in spinal cord, but combined use of both<br />
drugs was not effective.<br />
Acknowledgements: Supported by APVV 0314-06 and by VEGA 2/0015/08.<br />
76<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
A METHOD FOR AUTOMATED DETECTION OF HETEROZYGOUS<br />
INSERTION-DELETION MUTATIONS<br />
Peter Májek 1 , Vladimír Špitalský 1 , Gabriel Minárik 2 and Tomáš Szemes 2<br />
1 ADINIS s.r.o., Bratislava<br />
2 Department of Molecular Biology, FNS, Comenius University in Bratislava<br />
Structural and insertion-deletion (indel) variants are of considerable importance,<br />
mostly because of their phenotypic consequences. Indels shorter than 30 bp<br />
currently constitute about 24 % of all disease causing mutations reported in Human<br />
Gene Mutation Database. However, typical tools for analysis of sequence traces with<br />
indels obtained from diploid samples do not have sufficient accuracy and therefore<br />
extensive manual review of such samples is needed.<br />
We present here a new algorithm, implemented in software ADINIS IndelFinder, for<br />
automated detection of indel mutations from diploid sequence traces. The algorithm<br />
identifies 95% of indel mutations selected by a human expert with 5% false positives<br />
rate on a set of 39 sequence traces of exon 11 of KIT gene of subjects diagnosed with<br />
gastrointestinal cancer.<br />
The algorithm is based on a parametric digitalization of the electropherogram signal<br />
to a discrete set of nucleotide peaks followed by three rounds of the Needleman-<br />
Wunsch (NW) algorithm. The parameters of electropherogram digitalization as well<br />
as the scoring matrices used in the NW are optimized by a Monte-Carlo sampling to<br />
automatically find the best performing set of parameters.<br />
77<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
BIOCHEMISTRY IN THE PICTURES - INTERACTIVE BIOCHEMISTRY<br />
Mária Mareková, Jana Mašlanková, Peter Urban and Juraj Guzy<br />
Department of Medical Chemistry, Biochemistry and Clinical Biochemistry, UPJŠ -<br />
Faculty of Medicine, Tr. SNP 1, 040 01 Košice, Slovak Republic<br />
Modern informatics technologies (IT) including internet, essentially change the<br />
classical teaching methods. Traditional book illustrations cannot compete to<br />
electronic assigns, which provides except high-quality image documentation with<br />
zoom support also video recordings, animations or comments. Electronic form<br />
provides quick searching and intermediate updates. Our aim is to prepare interactive<br />
atlas of biochemistry – biochemistry in the pictures, with pictures, schemes, brief<br />
texts and the testing system for student`s own control. This upcoming atlas will be<br />
adjusted also for multimedia support of teaching, f.e. for it`s using as e-learning<br />
component, which leads students to more active form of studying. For the<br />
development of interactive atlas is also necessary cooperation between teachers and<br />
IT professionals. We are searching for help with preparation and servicing of internet<br />
electronical atlas on internet web servers, using administrator’s rights with possibility<br />
to manage the personal user accounts and with creation of copyright protection for<br />
multimedia content.<br />
Acknowledgments: This work was supported by grant project KEGA3/7130/09.<br />
78<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
ORIGIN OF ACQUIRED RESISTANCE TO CYTOTOXIC ACYCLIC<br />
NUCLEOSIDE PHOSPHONATES<br />
Helena Mertlíková-Kaiserová, Antonín Holý and Ivan Votruba<br />
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech<br />
Republic, Gilead Sciences & IOCB Research Center, 166 10 Prague, Czech Republic<br />
Acquired resistance to chemotherapy upon repeated administration of the cytotoxic<br />
drugs remains a serious clinical issue. Disclosing the mechanisms that lead to its<br />
development is necessary for introduction of successful prevention/reversal<br />
strategies. In our laboratory, we have prepared CCRF-CEM leukemic cells resistant to<br />
high concentrations of cytotoxic nucleotide analogs PMEG and PMEDAP. Employing<br />
[8- 3 H] radiolabeled compounds we aimed to describe changes in membrane<br />
transport and/or intracellular metabolism of the compounds. We found that the<br />
uptake of both PMEG and PMEDAP was unaffected in resistant cells. The resistance is<br />
therefore not due to decreased intracellular concentrations of the parent<br />
compounds. However, their metabolites PMEG/PMEDAP phosphate and<br />
PMEG/PMEDAP diphosphate were only present in sensitive but not resistant cells. As<br />
only the latter two can be incorporated into the DNA and act as chain terminators it is<br />
clear that the origin of resistance lies in the phosphorylation step catalyzed by<br />
relevant nucleoside monophosphate kinases - guanylate kinase (GUK) for PMEG and<br />
mitochondrial adenylate kinase (AK2) for PMEDAP. Expression of these proteins was<br />
indeed decreased in resistant cells. It is possible that apart from lower amount of<br />
GUK and AK2 protein, catalytic activity of these enzymes might also be affected. This<br />
will further be explored.<br />
Acknowledgments: Research project of the IOCB #OZ40550506; Project #1M0508 by<br />
the Ministry of Education, Youth and Sports of the Czech Republic.<br />
79<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
THE IDENTIFICATION AND CHARACTERIZATION OF THE FIRST VERTEBRATE HYBRID<br />
STERILITY GENE (HST1/PRDM9)<br />
Ondřej Mihola, Zdeněk Trachtulec and Jiří Forejt<br />
Department of Mouse Molecular Genetics, Institute of Molecular Genetics, Academy of<br />
Sciences of the Czech Republic, Videnska 1083, 142 20 Prague, Czech Republic<br />
The mouse Hybrid sterility 1 (Hst1) gene participates in a breakdown of<br />
spermatogenesis in the F1 offspring of crosses between some laboratory strains<br />
predominantly of Mus m. domesticus origin (e.g., B6 or B10) and certain mice of Mus<br />
m. musculus (sub)species, such as of the PWD strain. Other hybrid males, e.g. (PWD x<br />
C3H), are fertile. The Hst1 gene has been mapped on a high-resolution ((B10 xC3H) x<br />
B10) backcross and by transgenesis. The Hst1 candidate region was narrowed down<br />
to a single gene, PR-domain 9 (Prdm9) or Meisetz, encoding a histone 3 K4<br />
trimethyltransferase. The gene was confirmed as Hst1 by comparing the phenotypes<br />
of its null allele with the phenotypes of the sterile hybrids. To learn about the<br />
mechanisms regulating the germ-cell development through PRDM9, the expression<br />
profile of fertile and sterile hybrid testes differing only in the allele of Prdm9 were<br />
analysed by microarrays and real-time qRT-PCR. Furthemore, the localization of<br />
PRDM9 protein in germ cells was studied by indirect immunofluorescence. Our data<br />
suggest that Prdm9 is one of the master transactivators regulating meiotic epigenetic<br />
events.<br />
80<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
BIOCHEMICAL MARKERS OF MULTIPLE SCLEROSIS<br />
Jozef Michalik and Egon Kurča<br />
Clinic of Neurology, Jessenius Faculty of Medicine and Faculty Hospital in Martin<br />
Multiple sclerosis is an autoimmune, inflammatory, demyelinating disease of the<br />
central nervous system. Several pathophysiological mechanisms such as alteration of<br />
the immune system, disruption of blood-brain barrier, inflammation, oxidative stress<br />
and excitotoxicity, demyelination, axonal, neuronal damage, gliosis, remyelination and<br />
repair, cortical reorganisation are involved in the pathogenesis of the disease.<br />
Because of the heterogenity in clinical presentations and courses, a subtyping of<br />
patients by clinical, neuroradiological, genetical, neuroimmunological, biochemical<br />
parameters is necessary at present.<br />
This paper discusses the potential applicability of some biological markers for the<br />
diagnosis, disease activity, prediction of clinical courses and response to disease<br />
modifying therapies. There are some immunological markers, biomarkers of<br />
neuronal and axonal damage, biomarkers of demyelination, oxidative stress and<br />
excitotoxicity, gliosis (cytokines and their receptors, adhesion molecules, matrix<br />
metalloproteinases, 24S-hydroxycholesterol, antibodies to myelin oligodendrocyte<br />
glycoprotein, axonal antigens, amyloid precursor protein, 14-3–3 protein,<br />
protein, B cell activating factor of the TNF family - BAFF).<br />
MxA<br />
81<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
RTX CYTOTOXINS RECOGNIZE Β 2 INTEGRIN RECEPTORS THROUGH N-LINKED<br />
OLIGOSACCHARIDES<br />
Jana Morová, Radim Osička, Jiří Mašín and Peter Šebo<br />
Institute of Microbiology of the Academy of Sciences of the Czech Republic,<br />
Czech Republic<br />
Bordetella pertussis Adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) is a bifunctional<br />
protein belonging to the RTX (Repeat in ToXin) family of bacterial<br />
cytotoxins. CyaA delivers into target cells an adenylate cyclase domain, which<br />
catalyzes uncontrolled conversion of ATP to cAMP, a key signaling molecule<br />
subverting cell functions. The toxin utilizes as a specific cellular receptor, the<br />
CD11b/CD18 integrin (α M β 2 , Mac-1, or CR3) that is heavily N-glycosylated. Here, we<br />
demonstrate that deglycosylation of cell surface proteins by glycosidases completely<br />
abolished CyaA binding to CD11b-expressing cells. Moreover, cAMP intoxication of<br />
the deglycosylated cells exposed to the toxin was significantly reduced, suggesting a<br />
requirement of CD11b/CD18 glycosylation. Similar results were obtained, when N-<br />
glycosylation of de novo synthesized cellular proteins was inhibited by the antibiotic<br />
tunicamycin. Moreover, binding of CyaA to CD11b-expressing cells was significantly<br />
inhibited in the presence of excess of free saccharides found in building units of the<br />
oligosaccharide complex of the integrin. On the other hand, saccharides not occurring<br />
in integrin oligosaccharide chains were unable to inhibit CyaA binding to CD11b/CD18<br />
to any significant extent, showing that CyaA selectively recognizes the sugar residues<br />
of N-linked oligosaccharides of the integrin. Furthermore, the requirement for<br />
integrin glycosylation could be demonstrated also for binding of another RTX protein,<br />
the leukotoxin of Aggregatibacter actinomycetemcomitans (LtxA), which specifically<br />
binds to target cells via another receptor of the β 2 integrin family, CD11a/CD18.<br />
These results demonstrate that glycosylation of β 2 integrin receptors facilitates CyaA<br />
and LtxA binding to and killing of various target cells and set a new paradigm for<br />
action of RTX cytotoxins.<br />
82<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
FUNGAL ǡ-N-ACETYLGALACTOSAMINIDASE FROM ASPERGILLUS NIGER: CLONING AND<br />
EXPRESSION IN YEAST<br />
H.Mrazek 1,2 , L. Weignerova 2 , D. Manglova 1,2 , D. Kavan 1,2 , V. Kren 2 and K. Bezouska 2<br />
1 Department of Biochemistry, Faculty of Science, Charles University in Prague, Prague, CZECH<br />
REPUBLIC, 2 Institute of Microbiology v.v.i., Academy of Sciences of Czech Republic, Prague,<br />
CZECH REPUBLIC<br />
Alpha-N-acetylgalactosaminidase is an exoglycosidase specific for the hydrolysis of terminal<br />
ǡ-linked N-acetylgalactosamine in various sugar chain. A large screening study of<br />
extracellular ǡ-N-acetylgalactosaminidase activity of a library of filamentous fungi ( 42<br />
strains ), led to the identification of the best constitutive producer Aspergillus niger CCIM<br />
K2.They occur widely in microorganisms, plants and animals, and have considerable<br />
potential in various industrial application. Stability and activity at high temperatures are<br />
important properties of ǡ-N-acetylgalactosaminidases. This enzyme from Aspergillus niger<br />
has certain unique properties, and it was thus interesting to clone it for further structural<br />
investigations. Alpha-N-acetylgalactosaminidase from Aspergillus niger CCIM K2 was partially<br />
sequenced by Edman degradation and MALDI MS. A gene fragment encoding a putative part<br />
of the ǡ-N-acetylgalactosaminidase was amplified using cDNA prepared from Aspergillus<br />
niger CCIM K2. Degenerated PCR primers were designed according to amino acids found in<br />
ǡ-N-acetylgalactosaminidase from Aspergillus niger CCIM K2. The full-length coding<br />
sequence of ǡ-N-acetylgalactosaminidase was cloned into pPICZǡ and the recombinant<br />
protein was expressed in yeast. The cloned DNA consists of 1450 base pair, and the deduced<br />
amino acid sequence ( 480 amino acid residues with molecular mass 54,814kDa ) is almost<br />
identical to that of purified enzymes ( as determined by SDS-PAGE ). Comparison of this<br />
amino acid sequence with the GenBank database revealed significant homology ( 96%<br />
identity ) with sequence of ǡ-galactosidase ( EC3.2.1.22 ) from Aspergillus niger. Analysis of<br />
the identified sequences shows that ǡ-N-acetylgalactosaminidase is composed of three<br />
domains.<br />
The ǡ-N-acetylgalactosaminidase from Aspergillus niger CCIM K2 was expressed in yeast. In<br />
further experiments we would like to express the individual domains of this complex<br />
enzyme, and to evaluate, if there is an active carbohydrate-binding ( lectin ) domain within<br />
this enzyme.<br />
Acknowledgements: This work was supported, by Grant agency of Charles University in<br />
Prague ( 19309 ) and the Institutional Research Concept for the Institute of Microbiology (<br />
AVOZ5020051 ), and grants from the Czech Science Foundations<br />
( 203/05/0172, 204/06/0771, I)<br />
83<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
OXIDATIVE RISK IN ATHEROSCLEROSIS<br />
Jana Muchová 1 , Zuzana Nagyová 2 , Iveta Ondrejovičová 1 and Zdeňka Ďuračková 1<br />
1 Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry, Medical<br />
School, Comenius University, Bratislava, Slovakia;<br />
2 Juvenalia, Paediatric Center, Dunajská Streda, Slovakia;<br />
Atherosclerosis is the most common pathological process that leads to cardiovascular<br />
diseases and is known to be associated with inflammation, oxidative stress and<br />
endothelial dysfunction. In the vasculature reactive oxidant species may oxidatively<br />
modify lipids and proteins with deleterious consequences for vascular function. The<br />
ROS are common by-products of many oxidative biochemical and physiological<br />
processes. They can be released by increased activation of xanthine oxidase, NAD(P)H<br />
oxidase, lipoxygenases, mitochondria, or the uncoupling of nitric oxide synthase in<br />
vascular cells, as well as decreased cellular antioxidant capacity. ROS mediate various<br />
signaling pathways that underlie vascular inflammation in atherogenesis. The<br />
dysfunctional vasculature is characterized by lipid peroxidation and aberrant lipid<br />
deposition, inflammation, immune cells cell activation, platelet activation, thrombus<br />
formation, and disturbed hemodynamic flow. Each of these pathological states is<br />
associated with increasing of free radical species-derived oxidation products and,<br />
thereby, implicates increased oxidant stress in the pathogenesis of vascular diseases.<br />
84<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
ADVANCED TECHNOLOGY FOR METABOLIC INVESTIGATIONS<br />
Roman Oros<br />
Shimadzu Austria HmbH, Korneuburg, Laaer Strasse 7-9, A-2100, Austria<br />
This lecture presents the use of MS n measurment with prediction software tool to<br />
identify the formulas and structures in fields such as impurity analysis of<br />
pharmacological active substances, metabolic profiling and biomarker research.<br />
Discerning the chemical formula or structure of unknowns is a difficult task that can<br />
be partially alleviated by acquiring high mass accuracy data, however, data<br />
interpretation is tedious and time consuming. By using fragmentation spectra<br />
collected from LCMS-IT-TOF( a hybrid ion-trap time-of-flight mass spectrometer)<br />
along with enhanced formula prediction software, samples are rapidly analyzed to<br />
identify chemical formulas and structures.<br />
The composition prediction is based on three main steps:a) composition calculated<br />
from mass, b)using isotopic pattern, c)MS n spectral filtering<br />
The LCMS n data for metabolite structural detection in a metabolomic field will be<br />
presented.<br />
85<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
ACRIDINES – USEFUL MUTAGENS<br />
Helena Paulíková<br />
Department of Biochemistry and Microbiology, Faculty of Chemical and Food<br />
Technology, Slovak Technical University, SK-81237 Bratislava<br />
Acridine based compounds comprise of an important class of DNA-intercalating<br />
anticancer drugs, and are structurally characterised by the presence of a planar<br />
chromophore capable of intercalation into DNA. However acridines are a doubleedged<br />
sword, they are known as substances cause mutation and cancer. Most<br />
notable among their mutagenic effects is the induction of frameshift mutations. In<br />
spite of their mutagenicity, DNA affinity makes them attractive for the design of<br />
antitumor drug targeting DNA. Four new classes of acridine derivatives have been<br />
investigated by our research group and their anticancer potential has been assessed.<br />
Intercalation into DNA was confirmed by a variety of spectroscopic and<br />
electrophoresis techniques and anti-proliferative activity against three tumor cell<br />
lines (L1210, HL-60; A2780) was observed. The mechanism of antitumor activity of<br />
acridines involves intercalator-dependent formation of irreparable DNA double or<br />
single strand breaks arising from the inhibiting of DNA topoisomerases. Although<br />
acridines are also known as mutagens, the data obtained from the literature and our<br />
results showed that not all acridine intercalators are mutagens and it seems that the<br />
presence of electrophilic functional groups is nescessary for mutagenicity.<br />
Acknowledgements: Supported by Slovak Grant Agency, grants No 1/0097/10 and<br />
1/0053/08.<br />
86<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
THE CROSS-TALK OF NITRIC OXIDE AND NUCLEAR FACTOR KAPPA B IN<br />
EXPERIMENTAL HYPERTENSION<br />
Olga Pecháňová, Anrej Barta, Stanislava Vranková,<br />
Jana Parohová and Mária Kovácsová<br />
Institute of Normal and Pathological Physiology and Centre of Excellence for<br />
Cardiovascular Research, Slovak Academy of Sciences, Bratislava, Slovak Republic<br />
Recently we have demonstrated involvement of NF-κB in the upregulation of<br />
endothelial nitric oxide synthase (eNOS) in hypertension induced by N G -nitro-Larginine<br />
methyl ester (L-NAME). Thus, the goal of our study was to analyze an effect<br />
of NF-κB inhibitor, lactacystin, in L-NAME-induced hypertension. Adult 12-week-old<br />
male Wistar rats were subjected to treatment with L-NAME (40 mg/kg/day) for seven<br />
weeks (n=14). Half of the rats received lactacystin together with L-NAME for last<br />
three weeks. Next 16-week-old male Wistar rats received lactacystin only for 3 weeks<br />
(n=7). Blood pressure was measured by tail-cuff plethysmography every week. Total<br />
NOS activity was determined by measuring the formation of L-[ 3 H] citrulline from L-<br />
[ 3 H] arginine. Endothelial NOS and NF-κB (p65) protein expressions were determined<br />
immunohistochemically and by Western blot analysis. Membrane oxidative damage<br />
was analyzed by conjugated diene (CD) level determination. Lactacystin treatment<br />
did not affect the blood pressure (103±6 mmHg), while 7-week-L-NAME treatment<br />
increased blood pressure (141±3 mmHg) by 38% comparing the age-matched<br />
untreated animals (104±5 mmHg). Addition of lactacystin to the L-NAME increased<br />
blood pressure significantly (159±4 mmHg) by 54% comparing the untreated control<br />
group and by 12% comparing the L-NAME group. L-NAME treatment led to increased<br />
NF-κB expression followed by elevation of both eNOS protein expression and total<br />
NOS activity in the aorta, heart and kidney. Addition of lactacystin blocked, however,<br />
elevated eNOS protein expression in all tissues investigated. CD concentration was<br />
increased by lactacystin treatment.<br />
We hypothesized that NF-κB is responsible for upregulation of eNOS protein<br />
expression which may represent one of the counterregulatory mechanisms activated<br />
to compensate decreased NO production and increased blood pressure after longterm<br />
L-NAME treatment.<br />
Acknowledgements: This study was supported by the grants VEGA 2/0178/09 and<br />
APVV-0538-07.<br />
87<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
INTRONIC LINE-1 INSERTION IN THE Β-GLOBIN GENE CAUSES Β-THALASSEMIA DUE<br />
TO ABERRANT SPLICING, NONSENSE-MEDIATED DECAY AND DECREASED RATE OF Β-<br />
GLOBIN L1 ALLELE TRANSCRIPTION<br />
Lucie Piterková 1 , Jana Kučerová 1 , Karel Indrák 1,2 and Vladimír Divoký 1,2<br />
1 Department of Biology, Faculty of Medicine, UP Olomouc<br />
2 Department of Hemato-Oncology, University Hospital Olomouc<br />
ß-thalassemia is a common hereditary hemoglobin disorder characterized by<br />
quantitative reduction of functional ß globin chains. The patients, mother and<br />
daughter of Ukrainian descent exhibited typical laboratory features of ß -thalassemia<br />
trait. Molecular analyses revealed that the full-length (6 kb) retrotransposon L1 was<br />
inserted in the antisense orientation into the intron-2 of the ß-globin gene<br />
(collaboration with Dr. J. Prchal, Salt Lake City, UT). The total level of expression of<br />
the affected ß-globin gene transcript was reduced to 10-15% of the total ß-globin<br />
mRNA and thus leading to β + -thalassemia. Based on recently published data we<br />
hypothesize that RNA production of mutated gene was affected by the combination<br />
of several events. We demonstrated that the observed reduction in steady-state level<br />
of ß-globin mRNA is partially caused by aberrant splicing followed by activation of<br />
nonsense-mediated decay (NMD) pathway, leading to increased degradation of<br />
aberrant ß-globin mRNA variants. Reduction in expression of ß-globin mRNA from ß -<br />
globin L1 allele comes also from altered rate of transcription. We performed PCRbased<br />
nuclear run-on assay and forty minutes of in vitro transcription revealed 30%<br />
decrease in ß-globin L1 allele transcription rate compared to wild-type ß-globin allele.<br />
We also observed the ß-globin L1 3' enhancer sequence was fully methylated.<br />
However, treatment with a demethylating agent did not increase the expression of<br />
the ß-globin transcript of the ß-globin L1 gene. Therefore the methylation of the ß-<br />
globin L1 3' enhancer sequence is only a secondary event probably associated with<br />
enhancer displacement by L1 insertion. The other known mechanisms of intronic L1-<br />
mediated gene disruption as premature polyadenylation and gene breaking were not<br />
detected in our case.<br />
88<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
INDUCTION OF MITOCHONDRIAL PERMEABILITY<br />
TRANSITION BY MICROMOLAR IRON<br />
Jan Pláteník, Juraj Gáll, Jan Škrha, Jr., Richard Buchal,<br />
Eva Sedláčková and Karina Verébová<br />
Institute of Medical Biochemistry, First Faculty of Medicine,<br />
Charles University in Prague<br />
Mitochondrial permeability transition (MPT) plays an important role in necrotic and<br />
apoptotic cell death. MPT is induced by calcium and promoted by oxidative stress. In<br />
vivo the oxidative stress is often catalyzed by iron. In this study we investigated ability<br />
of micromolar iron to induce MPT in isolated mitochondria. According to literary<br />
data, Fe(II) over-loads antioxidant defense that shifts NAD(P)H/NAD(P) to oxidation,<br />
and the loss of reduced NAD(P)H promotes MPT opening. Iron can also be imported<br />
to mitochondrial matrix by calcium uniporter. In this study, isolated rat liver<br />
mitochondria were initially stabilized with EDTA and bovine serum albumin. They<br />
were energized by succinate or malate/pyruvate, and for MPT induction stimulated<br />
by addition of Ca or Fe(II). We measured mitochondrial swelling (light scatter), the<br />
inner membrane potential (fluorescent probe JC-1) and NAD(P)H oxidation<br />
(autofluorescence 340/465 nm). Both Ca and Fe(II) could induce cyclosporin A-<br />
inhibitable depolarization and swelling (MPT). Fe(II) induced MPT only in the<br />
presence of some residual EDTA that formed an iron complex catalyzing rapid<br />
oxidation of NAD(P)H. Effect of iron also required membrane potential and could be<br />
prevented by post-addition of membrane permeant, but not impermeant iron<br />
chelators. Iron was apparently needed only for induction of MPT, while its<br />
propagation continued through calcium release/reuptake. We conclude that both<br />
iron import and NAD(P)H oxidation must occur simultaneously for MPT to occur. This<br />
observation can help to elucidate mechanism of iron toxicity, which may be involved<br />
in pathogenesis of several liver diseases where cytosolic iron sequestration fails, e.g.<br />
hemochromatosis and alcoholic liver disease.<br />
Acknowledgements: Supported by GAUK 43/2006/C/1.LF and MSM0021620807.<br />
89<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
IS PHOSPHATIDYLINOSITOL TRANSFER ACTIVITY ESSENTIAL FOR<br />
THE FUNCTION OF PDR16P?<br />
Katarína Poloncová, Roman Holič and Peter Griač<br />
Institute of Animal Biochemistry and Genetics SAV, Ivanka pri Dunaji<br />
A common feature of phosphatidylinositol transfer proteins (PITPs) is their ability to<br />
transport phosphatidylinositol (PI) between membranes in vitro. The main PITP of the<br />
yeast Saccharomyces cerevisiae is Sec14p. It is an essential protein that participates<br />
in the vesicular transport from the Golgi membranes. Two point mutations in sites<br />
invariably conserved among all Sec14p homologues present in yeast led to the loss of<br />
PI transfer activity of the Sec14p (Phillips et al. 1999, Molecular Cell 4, p. 187).<br />
Pdr16p is one of the Sec14p homologues with 24% homology of the primary amino<br />
acid sequence to Sec14p. Though the exact function of this protein is yet to be<br />
described, it is known that deletion of the PDR16 gene leads to increased sensitivity<br />
to azole antifungals. Importantly, clinical isolates of Candida albicans and Candida<br />
lusitanie resistant to azoles with increased expression of Pdr16p were identified.<br />
To determine whether the PI transfer activity is essential for the Pdr16p function, we<br />
prepared a mutant that is predicted to be deficient in PI transfer activity of the<br />
Pdr16p. Yeast cells with PI transfer deficient Pdr16p as a sole Pdr16p showed<br />
increased sensitivity to azole antifungals similar to the pdr16′ cells. Sterol<br />
composition of this mutant was also changed compared to the wt and resembles<br />
sterol composition of the pdr16′ cells. These data indicate that PI transfer activity of<br />
the Pdr16p has to be present for this protein to fulfill its cellular function.<br />
Acknowledgements: This work was supported by VEGA 2/0077/10 and VVCE-0064-07<br />
grants.<br />
90<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
APOPTOSIS – DOUBLE EDGED SWORD<br />
Peter Račay 1 , Jozef Hatok 1 , Mária Chomová 1 , Jana Jurečeková 1 , Peter Chudý 2 ,<br />
Juraj Chudej 2 , Andrea Štefániková 1 and Dušan Dobrota 1<br />
1 Department of Medical Biochemistry, 2 Clinic of Haematology and Transfusiology , JLF<br />
UK Martin<br />
Apoptosis is an evolutionarily conserved process that is crucial for development and<br />
homeostasis of tissues of multicellular organisms. Its deregulation can elicit<br />
inappropriate cell death associated with different diseases (e. g. neurodegenerative<br />
diseases, diabetes, etc.) or is associated with survival of undifferentiated or<br />
transformed cells and can lead to development of cancer.<br />
Here we present results documenting initiation of apoptosis after global brain<br />
ischemia as well as dysfunction of apoptosis execution associated with acute<br />
myeloblastic leukaemia. We have documented that global brain ischemia induces<br />
transcription independent p53-mediated mitochondrial apoptosis. Execution of<br />
apoptosis was associated with genomic DNA fragmentation and death of pyramidal<br />
neurones in CA1 layer of rat hippocampus. On the other hand, RT-PCR analysis<br />
documented significant increase of mRNA level of apoptotic protein Bax in leukaemic<br />
cells in comparison to normal leukocytes, indicating transcription dependent<br />
initiation of p53-mediated apoptosis. Despite apoptosis initiation, survival of<br />
leukaemic cells is probably associated with inhibition of apoptosis execution<br />
mediated by increased transcription of both bcl-X and bcl-2 genes.<br />
Our results showed that detailed study of mechanism of apoptosis might by useful in<br />
search for new effective treatment of diseases associated with either cell death or<br />
cell survival due to deregulation of apoptosis.<br />
Acknowledgement: This work was supported by grant VVCE 0064-07<br />
91<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
E-LEARNING – FRIEND OF FOE?<br />
Daniel Rajdl, Jaroslav Racek and Marie Šolcová<br />
Institution of Clinical Biochemistry and Hematology, Medical Faculty, Charles<br />
University in Pilsen and Charles University Hospital in Pilsen<br />
E-learning as a way of teaching and learning is still encountering controversial<br />
reactions. Opponents argue that authoring e-learning materials is very complicated<br />
and dedicated to computer specialists. Furthermore, depersonalization, lack of<br />
dialogue among students plus teachers and poor quality and usability of many<br />
published e-learning materials add further strong arguments to opponents. On the<br />
other hand, e-learning fans appreciate its time flexibility, versatility, better control of<br />
outputs and emphasize enhanced possibilities of individual approach even in the<br />
large groups of students.<br />
In the presentation, we will focus on our personal experience with authoring e-<br />
learning materials and tutoring e-learning courses. We will emphasize Learning<br />
Mangement System Moodle and its usage especially in blended-learning (e-learning<br />
support for presence classes) and testing of students (exam tests). Furthermore, basic<br />
examples of Adobe Captivate (interactive presentations with multimedia),<br />
GoogleDocs (on-line office suite) and Adobe Connect Pro (with emphasis on<br />
videoconferencing) utilization in teaching will also be discussed. Moreover, some tips<br />
for technical equipment useful for interactive teaching (voting systems, interactive<br />
whiteboards) will be presented.<br />
92<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
IDENTIFICATION OF SURFACE PROTEINS OF THE OBLIGATE INTRACELLULAR<br />
BACTERIUM COXIELLA BURNETII<br />
Gabriela Flores Ramírez 1 , Zuzana Bílková 2 , Pavol Vadovič 1 and Ľudovít Škultéty 1,3<br />
1 Department of Rickettsiology. Institute of Virology, SAS, 845 05 Bratislava, Slovakia.<br />
2 Department of Biological and Biochemical Sciences, Faculty of Chemical Technology,<br />
University of Pardubice, Pardubice, Czech Republic, 3 Centre of Molecular Medicine,<br />
Slovak Academy of Sciences, Bratislava, Slovakia.<br />
Coxiella burnetti is an obligatory, intracellular bacterium that causes Q fever in<br />
humans. Due to the important role of surface proteins in adhesion, invasion, and<br />
intracellular survival of pathogens in the host, these proteins represent the crucial<br />
bacterial antigens, drug targets or biomarkers for diagnostic purposes. The aim of our<br />
investigation was to extract and identify the surface proteins of C. burnetii in virulent<br />
phase I.<br />
Two different strategies were applied. The first is based on protein extraction by<br />
detergent Triton X-114 followed by separation of the extracted proteins by SDS-PAGE<br />
and tryptic digestion. The second one is the surface shaving procedure employing<br />
magnetic beads with immobilized trypsin. The tryptic peptides were then separated<br />
by nanocapillary liquid chromatography and the surface proteins were identified by<br />
tandem mass spectrometry.<br />
Each of the presented methods has some advantages over those conventional ones<br />
and they allowed us to identify high numbers of proteins predicted to be surface<br />
localized. In addition to those lipoproteins, ribosomal proteins and metabolic<br />
enzymes were identified.<br />
Acknowledgements: This contribution/publication is the result of the project<br />
implementation: „TRANSMED“ supported by the Research & Development<br />
Operational Programme funded by the ERDF.<br />
93<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
XENOBIOTIC-METABOLIZING ENZYMES POLYMORPHISMS AND CANCER RISK<br />
Monika Kmeťová Sivoňová 1 , Dušan Dobrota 1 , Tatiana Matáková 1 , Zuzana<br />
Tatarková 1 , Mária Kovalská 1 , Martina Pavlíková 1 , Róbert Dušenka 2 and Ján Kliment 2<br />
1 Department of Medical Biochemistry, Comenius University, Jessenius Faculty of<br />
Medicine, Martin, 2 Department of Urology, Comenius University, Jessenius Faculty of<br />
Medicine and University Hospital, Martin<br />
Research in the past few years has shown that genetic, socioeconomic and<br />
environmental factors, particularly diet and lifestyle can affect the process of<br />
carcinogenesis. It is assumed that increased exposure to procarcinogens and<br />
carcinogens contained in tobacco smoke, debris, fermented food, polluted water, air<br />
etc., is implicated in multistage carcinogenesis. Xenobiotic-metabolizing enzymes play<br />
a key role in the metabolism of drugs and environmental chemicals and in the<br />
metabolic activation and detoxification of procarcinogens. Phenotyping analyses have<br />
revealed an association between these enzymes activities and the risk of developing<br />
several forms of cancer. The gene polymorphism of xenobiotic-metabolizing enzymes<br />
can cause interindividual differences in the activation/inactivation of anticancer<br />
agents/carcinogens and understanding of the contribution of these enzymes gene<br />
polymorphisms and their interactions with other relevant factors may improve<br />
screening diagnostic assays for cancer.<br />
Acknowledgements: This work was supported by grants MH SR 2007/45-UK-10, MH<br />
SR 2007/57-UK-17, MVTS Bil/ČR/SR/UK/06, AV 4/0013/05 and project “Center of<br />
Translational Medicine” co-financed from EC sources and European Regional<br />
Development Fund.<br />
94<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
ENERGETIC ASPECTS OF A MODIFICATION OF THE NA + /H + ANTIPORTER ACTIVITY IN<br />
A HARMALINE RESISTANT MUTANT OF METHANOTHERMOBACTER<br />
THERMAUTOTROPHICUS<br />
Peter Šmigáň 1 , Monika Vidová 1 , Janette Bobalova 2 and Zuzana Nováková 1<br />
1 Institute of Animal Biochemistry and Genetics, SAS, Ivanka pri Dunaji,<br />
2 Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Brno<br />
One of the most remarkable features of methanogenic archaea is the coexistence of<br />
two primary ion gradients, Δμ~ + H and Δμ~ + Na participating in ATP synthesis. Na + /H +<br />
antiport is uniquely able to balance these electrochemical gradients. However,<br />
physiological functions and protein components responsible for Na + /H + remain<br />
hypothetical. In this study a spontaneous mutant of M. thermautotrophicus resistant<br />
to the Na + /H + antiporter inhibitor harmaline was isolated. The Na + /H + exchanger<br />
activity in the mutant cells was remarkably decreased in comparison with wild -type<br />
cells. ATP synthesis driven by methanogenic electron transport was significantly<br />
enhanced in the mutant cells. To define the protein basis of harmaline resistance, the<br />
composition of membrane-associated proteins was partially characterized and<br />
compared with that of the wild- type strain. The experimental data revealed the<br />
differential expression in this mutant of A flavoprotein and Molybdenum –containing<br />
formylmethanofuran dehydrogenase 1 subunit C which play a direct role in flavin<br />
based electron bifurcation. The overexpression of these proteins might contribute to<br />
harmaline resistance. Taken together the results indicate that harmaline resistance<br />
in this mutant has arisen as a consequence of mutation(s) in an antiporter gene(s) or<br />
a protein(s) that links or influences an antiporter activity.<br />
Acknowledgements: This investigation was supported in part by the Science and<br />
Technology Assistance Agency (Slovak Republic) APVT-51- 024904, APVV-VVCE 0064-<br />
07 and by Research Grants VEGA 2/0015/09 from the Slovak Academy of Sciences and<br />
the Institutional Research Plan AV0Z40310501 of the Institute of Analytical Chemistry<br />
of the ASCR, v.v.i<br />
95<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
CARDIAC L-TYPE CALCIUM CURRENT IN SEPSIS<br />
Milan Štengl 1 , František Barták 1 , Roman Sýkora 2 , Jiří Chvojka 2 , Jan Beneš 3 , Aleš<br />
Kroužecký 2 , Ivan Novák 2 , Jitka Švíglerová 1 , Jitka Kuncová 1 and Martin Matějovič 2<br />
1 Department of Physiology, 2 First Medical Department, 3 Department of<br />
Anesthesiology and Critical Care Medicine, Charles University in Prague, Faculty of<br />
Medicine and Teaching Hospital in Plzen, Plzen, Czech Republic<br />
Myocardial depression is a well recognized manifestation of sepsis and septic shock.<br />
We hypothesize that reduced L-type calcium current (ICaL) with consequent<br />
shortening of cardiac repolarization contributes to the myocardial depression in a<br />
clinically relevant porcine model of hyperdynamic septic shock. In anesthetized,<br />
mechanically ventilated and instrumented pigs (n=22), sepsis was induced by<br />
bacteremia (central venous infusion of live P. aeruginosa) and continued for 22 hours.<br />
Electrocardiogram was recorded before and 22 hours after induction of bacteremia.<br />
In vitro, action potentials were recorded in right ventricular trabeculae. ICaL was<br />
measured in isolated ventricular myocytes. RR, QT and QTc intervals were<br />
significantly shortened by sepsis. Action potential durations (APD) were shortened in<br />
septic preparations. TNF-α did not influence APD. Peak ICaL density was reduced in<br />
myocytes from septic animals (8.3±0.4 pA/pF vs. 11.2±0.6 pA/pF in control). The<br />
voltage dependence of both ICaL activation and inactivation was shifted to more<br />
negative potentials in myocytes from septic animals. Action potential-clamp<br />
experiments revealed that the contribution of ICaL to the septic action potential was<br />
significantly diminished. In cardiac myocytes incubated with TNF-α ICaL was not<br />
further affected. We conclude that in a clinically relevant porcine model,<br />
hyperdynamic septic shock induced reduction of ICaL and shortening of ventricular<br />
repolarization.<br />
Acknowledgement: Supported by the Research Project MSM 0021620819,<br />
Replacement of and support to some vital organs, from the Ministry of Education,<br />
Czech Republic.<br />
96<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
CYTOCHROME P450- AND PEROXIDASE-MEDIATED OXIDATION OF ELLIPTICINE<br />
DICTATES ITS ANTI-TUMOR EFFICIENCY<br />
Marie Stiborová 1 and Eva Frei 2<br />
1 Department of Biochemistry, Faculty of Science, Charles University in Prague,<br />
2 Division of Molecular Toxicology, German Cancer Research Center, Heidelberg,<br />
Germany<br />
An antineoplastic alkaloid ellipticine is a prodrug, whose pharmacological efficiency is<br />
dependent on its cytochrome P450 (CYP)- and/or peroxidase-mediated activation in<br />
target tissues. The CYP-mediated ellipticine metabolites 9-hydroxy- and 7-<br />
hydroxyellipticine and the product of ellipticine oxidation by peroxidases, the<br />
ellipticine dimer, are the detoxication metabolites of this compound. Two carbenium<br />
ions, ellipticine-13-ylium and ellipticine-12-ylium, derived from two activation<br />
ellipticine metabolites, 13-hydroxyellipticine and 12-hydroxyellipticine, generate two<br />
major deoxyguanosine adducts in DNA found in the human breast adenocarcinoma<br />
MCF-7 cells, leukemia HL-60 and CCRF-CEM cells, neuroblastoma IMR-32, UKF-NB-3<br />
and UKF-NB-4 cells and glioblastoma U87MG cells in vitro and in rat breast carcinoma<br />
in vivo. Formation of these covalent DNA adducts by ellipticine is the predominant<br />
mechanism of its cytotoxicity and anti-tumor activity to these cancer cell lines.<br />
Ellipticine is also an inducer of CYP1A, 1B1 and 3A4 enzymes in the cancer cells<br />
and/or in vivo in rats exposed to this compound, thus modulating its own<br />
pharmacological efficiencies. The study forms the basis to further predict the<br />
susceptibility of human cancers to ellipticine and suggests this alkaloid for treatment<br />
in combination with CYP and/or peroxidase gene transfer increasing the anticancer<br />
potential of this prodrug.<br />
Acknowledgements: Supported by GACR (P301/10/0356) and Czech Ministry of<br />
Education (MSM0021620813 and 1M0505).<br />
97<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
UTILITY OF SINGLE CELL RT-PCR (scRT-PCR) FOR THE STUDY OF PRIMARY AFFERENT<br />
NEURONS - PRELIMINARY VALIDATION<br />
Lenka Surdeníková 1 , Fei Ru 2 and Marian Kollárik 1,2#<br />
1 Pathophysiology, Jessenius Medical School, 2 Medicine, Johns Hopkins School of<br />
Medicine, # correspondence: kollarik@jhmi.edu<br />
We addressed the hypothesis that scRT-PCR detection of selected receptors in<br />
primary sensory neurons provides information predictive of their functional response<br />
mediated by these receptors. scRT-PCR was performed on individual mouse or guinea<br />
pig primary sensory neurons. Functional response of the neurons or their putative<br />
peripheral nerve terminals was evaluated by calcium imaging, whole cell patch clamp<br />
or single nerve fiber recordings. scRT-PCR detection of MrgPrA 3 receptor in neurons<br />
correlated with their responsiveness to the MrgPrA 3 selective agonist chloroquine<br />
(1mM) in calcium imaging studies. MrgPrA 3 mRNA was detected in 8 of 9<br />
chloroquine-responsive neurons but not in 11 chloroquine -unresponsive neurons.<br />
Similarly, detection of TRPV1 receptor correlated with the responsiveness to the<br />
TRPV1 agonist capsaicin (1microM) in patch clamp studies (n=9). Detection of the<br />
purinergic receptors P2X 2 /P2X 3 in the nodose nociceptive neurons correlated with<br />
the P2X 2 /P2X 3 current signature in these neurons in patch clamp studies. scRT-PCR<br />
detection of the adenosine A 1 and A 2A receptors, and the TRPA1 receptor in the vagal<br />
nodose nociceptive neurons correlated with the responsiveness of their putative<br />
nerve terminals to the selective adenosine A 1 and A 2A receptor agonists and TRPA1<br />
agonists, respectively, in single fiber recording studies. We conclude that scRT-PCR<br />
detection of all receptors evaluated thus far in our studies in primary sensory neurons<br />
correlated with the functional response mediated by these receptors. Our data<br />
indicate that scRT-PCR on the individual primary sensory neurons provides<br />
information predictive of their functional response and is a suitable complementary<br />
tool for the study of these neurons.<br />
98<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
MYCOBACTERIAL MANNOSYL TRANSFERASE PimA AS A TARGET FOR THE<br />
DEVELOPMENT OF NEW ANTITUBERCULAR DRUGS<br />
Zuzana Svetlíková 1 , Marcelo E. Guerin 2 , Mary Jackson 3 ,<br />
Jana Korduláková 1 and Katarína Mikušová 1<br />
1 Comenius University, Faculty of Natural Sciences, Bratislava, Slovakia;<br />
2 University of the Basque Country, Unit of Biophysics, Bilbao, Spain;<br />
3 Colorado State University, Department of Microbiology, Immunology and Pathology,<br />
Fort Collins, USA<br />
Tuberculosis still remains one of the most serious infectious diseases in the world<br />
with several million new cases each year. At present there are more than 2 billion<br />
people infected with Mycobacterium tuberculosis, the causative agent of<br />
tuberculosis, with a chance of one in ten for the development of the active disease.<br />
Increased prevalence of multidrug-resistant and extensively drug-resistant strains of<br />
M. tuberculosis diminishes the number of effective drugs available for the treatment<br />
of infections they cause. For this reason there is a need for drugs with new mode of<br />
action. The unique mycobacterial cell envelope provides an array of novel drug<br />
targets since its integrity is necessary for bacterial viability. Phosphatidylinositol<br />
mannosides (PIM) are important part of this crucial structure. They appear to be<br />
involved in host-pathogen interactions and serve as a basis for the biosynthesis of<br />
other key molecules - mycobacterial lipopolysaccharides lipoarabinomannan and<br />
lipomannan. Build up of PIM is initiated by the action of mannosyl transferase PimA<br />
catalyzing the transfer of the mannose residue from GDP-mannose to<br />
phosphatidylinositol. Since the reaction is essential for survival of M. tuberculosis,<br />
PimA represents an attractive target for the drug development.<br />
Acknowledgment: This work was supported by European Comission under contract<br />
LSHP-CT-2005-018923"NM4TB“<br />
99<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
OXIDATIVE STRESS AND HEART AGING.<br />
Zuzana Tatarková, Eva Babušíková, Stanislav Kuka, Ján Lehotský, Peter Račay,<br />
Dušan Dobrota and Peter Kaplán<br />
Department of medical biochemistry JLF UK Martin<br />
Aging plays in our life uniquely role and oxidative stress (OS) is considered by many<br />
authors as a major factor in this process. The causes and consequences of aging<br />
involve the interaction of many processes. Mitochondria are the main sources of ROS<br />
(reactive oxygen species) during normal metabolism. ROS production by<br />
mitochondria is important because it underlies oxidative damage in a lot of<br />
pathologies and is characterized by destruction of structural integrity of membrane,<br />
leading to a decrease in membrane fluidity and enzyme activities. Therefore, the<br />
objective of our study was to determine the specific relationship between heart aging<br />
and changes in the level of OS, lipid peroxidation and OS-sensitive enzymes activities.<br />
We used three different age groups (6-, 15- and 26-months old rats) which<br />
represented adult, old and senescence. The activities of all respiratory enzymes<br />
significantly decreased with the highest activity losses (37%) in complex IV. Inhibition<br />
of citric acid cycle enzymes, α-KGDH and SDH in the hearts of senescent rats, is also<br />
due to reaction of ROS with the thiol groups of these proteins. For this reason we<br />
studied total thiol group content, which decreases by 30%. The whole aging process<br />
was associated with elevation in basal levels of lipid peroxidation-end products, MDA<br />
and HNE. Cells are continually challenged by conditions that cause acute or chronic<br />
stress and there are many antioxidant mechanisms that cope with this situation.<br />
Activity of the Mn-SOD was together with cytosolic CuZn-SOD continuously depressed<br />
with age, but mitochondria creates higher level of MnSOD protein, which possibly<br />
could help to another antioxidant enzymes fight against OS. In all this reasons, OS in<br />
mitochondria may drive myocardial aging. But there are still many processes that we<br />
need investigate.<br />
Acknowledgements: This work was supported by grants VEGA 1/0027/08, VEGA<br />
1/0049/09, VVCE-0064-07.<br />
100<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
WHEN MORE IS LESS: A DILEMMA OF A BIOMEDICAL EDUCATOR<br />
Ľubomír Tomáška<br />
Department of Genetics, Comenius University, Bratislava<br />
The exponential growth of data due to a dramatic increase in the number of active<br />
researchers as well as development of advanced and powerful technologies results<br />
in a general, yet difficult question related to science education: How and what to<br />
teach the future generation of biomedical scientists? Whereas 30 years ago the<br />
classical textbooks covered essentially the whole fields by means of their<br />
presentation in a form of highly readable and entertaining recapitulation of major<br />
discoveries, the modern textbooks, due to their factual nature, do not provide the<br />
reader with an opportunity to „participate“ in the exciting experiments. It seems<br />
logical that in 1976 the key experiment in molecular genetics describing the fine<br />
structure of a gene [1] is elaborated on 15 pages [2], while the most recent edition<br />
of the same textbook barely mentions its main message [3]. The changes of<br />
textbooks in both the richness of their content and style should reflect the content<br />
of undergraduate curricula. Or should they, really? Should the student have an<br />
overview of everything considered important by the major textbooks and thus<br />
naturally gain a shallow and superficial knowledge? Or should we employ key, yet<br />
technologically outdated experiments to catalyze creative thinking risking that the<br />
student will not have comprehensive information about the state-of-the-art in the<br />
corresponding discipline? We need to look for means to dissect this dilemma and<br />
think about the changes in the curricula [4] and ultimately in re-definition of goals of<br />
higher biomedical education [5].<br />
[1] Benzer, S. (1955). Proc. Natl. Acad. Sci. USA 41: 344-354.<br />
[2] Watson, J.D. (1976). Molecular biology of the gene. 3 rd Edition, W.A. Benjamin.<br />
[3] Watson, J.D. et al. (2008). Molecular biology of the gene. 6 th Edition, CSHL Press.<br />
[4] http://www.asbmb.org/CareersAndEducation.aspx?id=432<br />
[5] Connelly. T. et al. (2008). A new biology for the 21 st century. The NAS Press.<br />
101<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
EFFECT OF ADENINE NUCLEOTIDES AND Mg 2+ IONS ON MITOCHONDRIAL CHLORIDE<br />
CHANNELS<br />
Viera Kominková, Zuzana Tomášková, Ľubica Máleková and Karol Ondriaš<br />
Institute of molecular physiology and genetics, Slovak Academy of Sciences,<br />
Bratislava, Slovak Republic<br />
Chloride channels are involved in many physiological and pathological processes such<br />
as the apoptosis, stress, reperfusion injury or cardioprotection. The cells in ischemic<br />
tissues are depleted of oxygen and lose their capacity for ATP production. Due to this<br />
energy deprivation, destructive processes are activated which lead to cell injury or<br />
death. Molecular mechanisms of these processes are still not fully understood. The<br />
aim of our work was to study the possible role of mitochondrial chloride channels in<br />
these processes.<br />
We report the effect of adenine nucleotides (ATP, ADP and AMP-PNP) and Mg 2+ on<br />
the activity of mitochondrial chloride channels. Submitochondrial vesicles isolated<br />
from rat heart were incorporated into bilayer lipid membrane and single chloride<br />
channel currents were measured. We found that ATP inhibited chloride channels and<br />
affected both kinetics and current amplitude. This inhibitory effect was not<br />
dependent on phosphorylation of the channel. Furthermore, ADP did not affect the<br />
channel activity but only decreased the current amplitude. When the effect of ATP<br />
was studied in Mg 2+ free solutions, a hyperbolic current-voltage relationship was<br />
observed after addition of ATP. Addition of MgCl 2 (up to 5mmol/L) partially relieved<br />
this effect of ATP.<br />
ATP, within the range of physiological concentrations, inhibits the mitochondrial<br />
chloride channels, which invokes a hypothesis that these channels have a role during<br />
pathological processes connected with ATP depletion.<br />
Acknowledgements: This work was supported by VEGA 2/0150/10 and VVCE-0064-07.<br />
102<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
SYNTHETIC CYCLIC CHALCONE ANALOGUES AS NOVEL BIOLOGICALLY ACTIVE DYES<br />
Vladimíra Tomečková<br />
Department of Medical Chemistry, Biochemistry, Clinical Biochemistry and LABMED<br />
a.s. UPJŠ Košice<br />
This study demonstrates the behaviour of synthetic fluorescent substituted chalcone<br />
(1) and its cyclic chalcone analogues: indanone (2), tetralone (3), benzosuberone (4)<br />
with dimethylamino substituent in para position in the presence of BSA proteins, egg<br />
yolk lecithine vesicules and cells studied by fluorescence spectroscopy. These dyes<br />
with anticancer properties are yellow and have protein, lipid and cell staining<br />
properties. From the spectral results we can assume that all studied compounds<br />
interacted with lipids and proteins by hydrophobic interactions. Tetralone and<br />
benzosuberone showed the best hydrophobic interaction with lipids and proteins. In<br />
the presence of proteins all compounds showed batochromic shift of the spectra and<br />
increase of fluorescence intensity. Biologically the most active dye (4) was the least<br />
fluorescent and the least biologically active dye (1) was the most fluorescent.<br />
Interaction of these unpolar substances with proteins was concentration dependent<br />
and it was caused by hydrophobic bonding. From these measurements we have<br />
calculated the binding constant of these dyes with BSA protein, which was in order 4<br />
> 3 > 2 > 1. The application of these biologically active dyes (1, 2, 3, 4) on breast<br />
cancer cell lines showed that these fluorescent dyes were able to stain all cellular<br />
components (cytoplasm, nucleus, lipid vesicules). The result fluorescence images of<br />
breast cancer cells staining were aqua tyrkys fluorescence studied by fluorescence<br />
microscope. Nucleus we have costained by DAPI dye, specific for DNA staining. Dye<br />
(3) as well as dye (4) destroyed nuclei of breast cancer cells after 3 hours and all<br />
studied dyes have cytotoxic effect on studied breast cancer cells after 24 hours.<br />
103<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
MOLECULAR MODELING INSIGHT INTO CATALYTIC MECHANISMS OF<br />
GLYCOSYLTRANSFERASES<br />
Igor Tvaroška<br />
Institute of Chemistry, Slovak Academy of Sciences, 845 38 Bratislava, Slovakia<br />
N- and O-linked oligosaccharide chains of glycoproteins play a crucial role in a number of<br />
biological processes. These compounds are found throughout biological systems and<br />
have been implicated in molecular recognition events such as bacterial, viral, and cell-cell<br />
adhesion, inflammation, and tumor invasion. Numerous glycosyltransferases, that<br />
catalyze the addition of a specific glycosyl residue from sugar nucleotide to an acceptor<br />
substrate, were validated as prime targets for therapeutic intervention in human<br />
diseases. Effective inhibitors of these enzymes are not yet available and development of<br />
inhibitors for a specific glycosyltransferase is, therefore, of great interest. Though<br />
transition state analogs are valued tools for drug discovery as potent and specific<br />
inhibitors of enzymes, up to date, the ability to generate transition state analogs of<br />
glycosyltransferases has lagged behind. A transition state analog is a stable compound<br />
that structurally resembles the three-dimensional structure and charge distribution of a<br />
substrate(s) portion of the unstable transition state of an enzymatic reaction. Knowledge<br />
of the geometry and charge distribution of transition state provides blueprint for the<br />
design of the transition state analog inhibitors. It is obvious, that design of transition<br />
state analog inhibitors of an enzyme requires knowledge of the mechanism of the<br />
enzymatic reaction and the structure of transition state. Therefore, we have investigated<br />
the catalytic mechanism for inverting and retaining glycosyltransferases employing high<br />
level ab initio, DFT, and hybrid QM/MM calculations [1-6]. These results provided<br />
detailed insight into the mechanism of the monosaccharide transfer catalyzed by<br />
glycosyltransferases and revealed the main structural features of the transition states.<br />
The purpose of this paper is to summarize the structural insights into the catalytic<br />
mechanism of glycosyltransferases inferred from these calculations.<br />
Acknowledgements: This work was supported by the grants from the Slovak Research<br />
and Development Agency No. APVV-0607-07 and the Slovak Grant Agency VEGA No.<br />
2/0128/08.<br />
104<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
ATORVASTATIN CHANGES MEMBRANE LIPID FLUIDITY IN MITOCHONDRIA<br />
ISOLATED FROM VARIOUS TISSUES OF RATS<br />
Iveta Waczulíková 1 , Oľga Uličná 2 , Oľga Vančová 2 , Jarmila Kucharská 2 ,<br />
Veronika Ilovská 1 and Libuša Šikurová 1<br />
1 Faculty of Mathematics, Physics and Informatics, 2 Pharmacobiochemical Lab., 3 rd<br />
Med. Clinic, Faculty of Medicine, Comenius University, Bratislava, Slovakia<br />
Atorvastatin is a potent cholesterol-lowering agent with well-described benefits and<br />
adverse effects manifested in clinical practice. Hypercholesterolemia leads to<br />
alterations in lipid composition and fluidity of blood cell plasma membranes and<br />
atorvastatin was reported to reverse these alterations. Little is known how the<br />
hypercholesterolemic condition and its therapy may influence mitochondrial<br />
membrane properties. We have examined the effect of two selected doses of<br />
atorvastatin on membrane lipid fluidity and function of mitochondria isolated from<br />
liver, heart and skeletal muscles of control and hypercholesterolemic rats. Wistar rats<br />
were on a high cholesterol diet for 8 weeks. Atorvastatin was administered per os<br />
either at a low or high dose (10/80 mg/kg/day, resp.) for 4 weeks. Fluidity was<br />
assessed spectrofluorometrically and functional parameters of mitochondria with a<br />
Clark oxygen electrode.<br />
Hypercholesterolemic condition affected the investigated properties of mitochondria<br />
in a tissue-dependent manner. This is likely the reason of an ambiguous action of<br />
atorvastatin on membrane fluidity. Atorvastatin seems to eliminate certain types of<br />
membrane damage induced by hypercholesterolemia, if it is controlled by an<br />
appropriate dosage. However, on average, it tended to fluidize the membranes and<br />
compromise capacity of mitochondrial respiratory chain as well as the rate of ATP<br />
formation in mitochondria in both, healthy and hypercholesterolemic rats.<br />
Acknowledgement: Supported by the grants: VEGA 1/0328/10 and 1/0293/08.<br />
105<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
DIFFERENCIES BETWEEN TWO ENDOXYLANASES FROM GH5<br />
Mária Vršanská, Katarína Šuchová, Vladimír Puchart and Peter Biely<br />
Department of enzymology of Carbohydrates, Institute of Chemistry, Center for<br />
Glycomics, SAS, Bratislava<br />
Endoxylanase A(XynA) produced by plant pathogen Erwinia chrysanthemi and<br />
endoxylanase IV(XynIV) isolated from Trichoderma reesei RUT C-30 are classified into<br />
glycoside hydrolase family 5 (GH5). A detailed study of both endoxylanases showed on<br />
differencies in their mode of action on variety of plant glucuronoxylans and linear<br />
xylooligosaccharides.The hydrolytic action of XynA was found to be absolutely<br />
dependend on the presence of 4-O-methyl-D-glucuronosyl (MeGlcA) side residues in<br />
both oligosaccharides and polysaccharides. Neutral linear xylooligosaccharides are<br />
resistant towards enzymatic action of XynA. As a rule, the enzyme attacked the second<br />
glycosidic linkage following the MeGlcA branch towards the reducing end. The<br />
productive complex of XynA with glucuronoxylan is formed in such a way that the<br />
MeGlcA residue is bound to the xylopyranosyl residue accommodated at the<br />
hypothetical subsite -2. Depending on the distribution of MeGlcA residues on<br />
glucuronoxylan main chain, XynA generated series of shorter and longer aldouronic acids<br />
in which the MeGlcA is linked exclusively to the second xylopyranosyl residues from the<br />
reducing end. Endoxylanase Trichoderma reesei XynIV also from GH5 do not recognize<br />
uronic acid chains as substrate determinants. XynIV showed lover afinity toward<br />
deacetylated glucuronoxylan than to acetylated beechwood glucuronoxylan or to<br />
rhodymenan, a sea alga β-1,3-β-1,4-xylan. The behaviour on polymeric substrates<br />
suggested that XynIV prefers xylan with irregularities in the main chain such as the<br />
replacement of β-1,4-linkages by β-1,3-linkages as it is in rhodymenan, or substitution of<br />
xylopyranosyl residues as it is in acetylglucuronoxylan. [1- 3 H]-Linear xylooligosaccharides<br />
are exclusively cleaved by XynIV at the first glycosidic linkage from the reducing end, to<br />
give [1- 3 H]-xylose as the only radioactive product. All experimental evidence suggests<br />
that the substrate-binding site of XynIV is composed of three subsites -II, -I and +I with<br />
a serious steric barrier at the position of the hypothetical subsite +II. Our data point to<br />
a great diversity among endoxylanases belonging to the GH5 family.<br />
106<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
HOW CAN OXIDATIVE STRESS AND DNA STABILITY BE INFLUENCED BY DIET AND<br />
PHYSICAL ACTIVITY?<br />
Karl-Heinz Wagner and Oliver Neubauer<br />
Department of Nutritional Sciences, Emerging Field Oxidative Stress and DNA<br />
Stability, University of Vienna, Austria<br />
Oxidative stress-induced DNA damage and insufficient DNA repair may play an<br />
important role in the etiology of cancer, diabetes and arteriosclerosis. Participation in<br />
regular physical activity as well as plant based diet reduces the risk of developing<br />
such and other lifestyle-dependent diseases. Clear evidence for protective effects<br />
was observed for several foods although no correlations between<br />
oxidative/antioxidative parameter and DNA relevant biomarker can be observed.<br />
Interestingly, acute and strenuous exercise may induce oxidative stress via enhanced<br />
formation of reactive oxygen (ROS) and nitrogen species (RNS) leading to oxidatively<br />
modified lipids, proteins and nucleic acids and possibly to lifestyle dependent<br />
diseases. Exercise-induced DNA damage might either be a consequence of oxidative<br />
stress or inflammatory processes or it is causally involved in inflammation and<br />
immunological alterations after strenuous prolonged exercise (e.g. by inducing<br />
lymphocyte apoptosis and lymphocytopenia). Currently, only a few data have<br />
investigated the influence of exercise on DNA stability and damage with conflicting<br />
results, small study groups and the use of different sample matrices or methods and<br />
result units. The presentation will address results of food based studies as well as the<br />
effects of exercise of various intensities and durations on DNA stability, focusing on<br />
human population studies. It will consider the type of exercise conducted (field or<br />
laboratory based) and the intensity performed (i.e. competitive ultra/endurance<br />
exercise or maximal tests until exhaustion). The findings suggest that competitive<br />
ultra-endurance exercise (>4 h) does not induce persistent DNA damage. However,<br />
when considering the effects of endurance exercise (
Lectures<br />
LECTINS FROM PATHOGENS: MYSTERY OF LIFE<br />
Michaela Wimmerová<br />
National Centre for Biomolecular Research & Department of Biochemistry, Faculty of<br />
Science, Masaryk University, Brno, Czech Republic<br />
Protein/carbohydrate interactions play a crucial role in host cell recognition by<br />
pathogens. Many of them have at disposal a wide arsenal of proteins that selectively<br />
recognize host cabohydrate epitops. In addition, while a general protein/saccharide<br />
binding is usually very weak, pathogens have evolved proteins that can bind even<br />
monosaccharides with unusually high affinities.<br />
Contribution will be focused on some examples of lectins from pathogens, which can<br />
mediate recognition and adhesion to host cells so that they could be important<br />
virulence factors. The combination of binding experiments (isothermal titration<br />
microcalorimetry, surface plasmon resonance,...) and X-ray crystallography<br />
approaches is used to decipher the thermodynamical and structural basis for high<br />
affinity binding of these lectins to host carbohydrates. Site-directed mutagenesis in<br />
combination with structural and functional studies is used for understanding<br />
particular amino acids for sugar specificity and preference.<br />
108<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
FUNCTION AND PROPERTIES OF HEART AND KIDNEY MITOCHONDRIA (MIT) IN<br />
SPONTANEOUSLY HYPERTENSIVE (HYP) RATS: INFLUENCE OF<br />
CAPTOPRIL AND NIFEDIPINE<br />
Attila Ziegelhöffer 1 , Jana Mujkošová 1 , Oľga Uličná 2 , Iveta Waczulíková 3 , Miroslav<br />
Ferko 1 , Norbert Vrbjar 1 , Štefan Polák 4 , Tanya Rav Kingerová 1 and Adriana Adameová 5<br />
1 Inst Heart Res SAS, 2 Pharmacobioch Lab III rd Med Clin MF UK, 3 Dept Biomed Physics<br />
FMFI UK, 4 Inst Histol Embryol MF UK, 5Inst Pharmacol Toxicol, PaF UK, Bratislava<br />
There are only scarce data available about HYP-induced changes in function and<br />
properties of heart MIT and multi-organ studies also involving the effect of anti-HYP<br />
treatment with captopril (CAP) or captopril + nifedipine (CAPNI) are missing completely.<br />
In present study we complete this lack by estimating O 2 consumption and oxidative<br />
phosphorylation (Oxygraph Gilson), DNP stimulated Mg-ATPase activity and membrane<br />
fluidity (fluorescence probe DPH) in MIT isolated from heart and kidney of 16 weeks old<br />
HYP rats treated for the next 4 weeks with CAP (80 mg.kg -1 daily) or CAPNI (80 CAP+10<br />
NI mg.kg -1 daily). HYP induced a moderate elevation of MIT ATP production in response<br />
to HYP-induced increase in energy demands (ED) of the heart. By removing the HYP,<br />
both treatments also decreased the ED and normalized the MIT ATP production. In<br />
kidney MIT HYP decreased the ATP production by depressing the values of OPR. Both<br />
treatments, particularly that with CAPNI even deepened this damage.<br />
Acknowledgements: Grants: VEGA 1/0620/10; 2/0173/08..<br />
109<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Lectures<br />
THE ROLE OF ANGIOTENSIN II AND OXYTOCIN IN REGULATION OF ADIPOCYTE CELL SIZE<br />
Štefan Zorad, Daniela Ježová, Lucia Gajdošechová,<br />
Miroslava Eckertová and Katarína Kršková<br />
Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava<br />
It is well recognized that adipose tissue plays a central role in development of cardiovascular<br />
and metabolic disorders. Beside being a depot for fuel storage the adipose tissue is secreting<br />
a number of peptides and proteins collectively named adipokines. The adipokines represent<br />
substances with wide spectra of action such as proinflammantory leptin, and TNFalpha and<br />
insulin senzitizing adiponectin and visfatin. In general hypertrofic adipose tissue with large<br />
adipocytes is producing more proinflammatory and insulin sensitivity decreasing adipokines<br />
(Zorad et al. 2006). Physical training and dietary interventions are often ineffective in<br />
reduction of adipose tissue mass and adipocyte diameter due to leptin resistance induced<br />
apetite disregulation in obesity. Treatment with thiazolidinediones has two phase effect<br />
with formation of new small adipocytes at the beginning followed by hypertrophy of the<br />
adipocytes (MacKelllar et al. 2009). This paper is presenting data from studies using<br />
angiotensin II AT1 receptor blockade or oxytocin treatment to manipulate adipose tissue<br />
morphology in order to increase production of insulin senzitizing adipokines.<br />
Rats were treated with AT1 blocker candesartan (10mg/kg body weight/day) in drinking<br />
water for 18 weeks Oxytocin (3.6 μg/100 g body weight/day) was administered to rats by<br />
osmotic minipumps during 2 weeks. Adipocytes from epididymal fat tissue were prepared by<br />
collagenase digestion. Diameter of the adipocytes was evaluated by using light microscopy<br />
(Pinterova et al. 2001). Serum concetrations of hormones and adipokines were determined<br />
by commercial radioimmunoassay kits. Protein expression in adipose tissue was studied by<br />
using immunoblot and real-time PCR. Chronic AT1 blockade resulted in an decrease of<br />
adipose tissue mass, adipocyte diameter and expression of leptin and TNFalpha without a<br />
change in food intake. On the contrary, adiponectin serum concetration and its expression as<br />
well as PPARγ expression in adipose tissue increased after candesartan treatment. Oxytocin<br />
treatment did not change the adipose tissue mass and food intake of rats. Despite that the<br />
adipocyte diameter significantly decreased. In addition, significant increase in expression of<br />
aP2, PPARγ, GLUT4, leptin, ACE and CD31was noticed in epididymal adipose tissue.<br />
The effects of long-term Candesartan treatment in adipose tissue are in line with other<br />
described positive effects of renin-angiotensin system inhibition on fat tissue remodeling<br />
and insulin sensitivity. Our oxytocin data suggest a new approach to modulate adipose tissue<br />
morphology in order to elevate expression of some markers of insulin sensitivity.We assume<br />
that oxytocin increases both adipogenesis and angiogenesis in adipose tissue.<br />
Acknowledgement: This work was supported by grant VEGA 2/0162/08.<br />
110<br />
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POSTERS<br />
111<br />
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Posters<br />
I. BIOCHEMISTRY AND MOLECULAR BIOLOGY OF NERVOUS SYSTEM<br />
112<br />
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Posters<br />
1.<br />
LABORATORY BIOMARKERS IN ISCHEMIC STROKE AND<br />
DEPRESSION IN HUMAN PATIENTS<br />
Daniel Čierny 1 , Stanislav Celec 1 , Mária Kovalská 1 , Peter Kaplán 1 , Igor Ondrejka 2 ,<br />
Egon Kurča 3 and Ján Lehotský 1<br />
1 Department of Medical Biochemistry, 2 Psychiatric clinic, 2 Neurological clinic,<br />
Jessenius Fac Med, Comenius University, Martin, 03601, Slovakia<br />
Due to the extreme complex pathogenesis of ischemic stroke, prognostic value of<br />
laboratory parameters is still matter of debate. A number of brain biomarkers for the<br />
diagnosis of ischemic stroke have been evaluated for clinical use in the past several<br />
years. Neurobiological basis of depression is not yet clarified and it is not yet clear the<br />
etipathogenesis of poststroke occurred depression. We present here laboratory<br />
examinations of plasma derievd analytes in the group of ischemic stroke patients, in<br />
the group of patients with depression compared with the group of apparently healthy<br />
subjects.<br />
Laboratory results indicate for a time dependent association between ischemic<br />
damage and biochemical parameters in postischemic plasma. It also suggests for their<br />
use as an additional predictors of tissue damage progression.<br />
Supported by: VEGA 0049/09, COST B30, VVCE 0064/07, MZ 2007/55UK-16.<br />
113<br />
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Posters<br />
2.<br />
IS IT POSSIBLE TO IMPROVE DEMYELINATION DISEASES MONITORING BY<br />
DETERMINATION OF SOME ENZYME ACTIVITIES CHARACTERISTIC FOR<br />
THE CENTRAL NERVOUS SYSTEM?<br />
Monika Ďurfinová 1 , Marta Brechtlová 1 , Ľubica Procházková 2 , Peter Kukumberg 2 ,<br />
Ľubomír Kuračka 1 and Branislav Líška 1<br />
1 Department of Chemistry, Biochemistry and Clinical Biochemistry, LF UK Bratislava,<br />
2 2 nd Department of Neurology, LF UK Bratislava<br />
Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous<br />
system (CNS). It is the most common neurological disease characterized by recurrent<br />
relapses and/or progression that are attributable to multifocal inflammation,<br />
demyelination and axonal pathology within the CNS. MS lesions are typically located<br />
in the periventricular white matter of the brain as well as in superficial areas of the<br />
spinal cord. Since MS lesions are not routinely biopsied, the cerebrospinal fluid (CSF)<br />
is used for measurements of various soluble markers. We have started determination<br />
of enzyme activities in the CSF of MS patients, which have some relationship to the<br />
function of the CNS tissue – phosphodiesterase 3 , ,5 , -cAMP and K + -paranitrophenylphosphatase.<br />
These enzyme activities were at first monitored in model experiments<br />
in vitro. We suppose that K + -paranitrophenylphosphatase might have a connection<br />
with the nerve impulse transmission. Interestingly, the part of phosphodiesterase<br />
3 , ,5 , -cAMP activity, which is calcium-calmodulin dependent, was inhibited by the<br />
conditions of activated oxidative stress. However, several questions still remain to be<br />
elucidated, e.g.: if this CNS inflamatory damage could infuence releasing of these<br />
enzymes into CSF and if these enzymes can reflect the intensity of the patological<br />
process.<br />
114<br />
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Posters<br />
3.<br />
SELECTED GENE POLYMORPHISMS IN ISCHEMIC STROKE AND DEPRESSED HUMAN<br />
PATIENTS FROM CENTRAL SLOVAKIA<br />
1 Andrea Evinová, 1 Eva Babušíková, 2 Pavol Adamík, 2 Igor Ondrejka, 3 Egon Kurča,<br />
3 Milan Grófik and 1 Ján Lehotský<br />
Comenius University, Jessenius Faculty of Medicine, Department of Medical<br />
Biochemistry 1 , Psychiatry Clinic 2 , Neurological Clinic 3 , Martin, Slovakia<br />
The neurobiological basis of ischemic stroke and postischemic depression is not yet<br />
clarified. We have focused on several genes which could be connected with the<br />
pathogenesis of ischemic stroke and depression. Angiotensin converting enzyme<br />
(ACE), as a part of the renin-angiotensin system, is important factor in blood pressure<br />
regulation. Incidence of D allele is associated with higher protein level and its<br />
activity. Serotonin-transporter-linked promoter (HTTLPR) is a degenerate<br />
polymorphic region in the gene that codes the serotonin transporter. Its<br />
polymorphism is connected with neuropsychiatric disorders. Likewise, S allele is<br />
associated with the functional reduction as compared to L allele. Glutathione-Stranferases<br />
(GST) detoxify a broad range of xenobiotics and carcinogens. The most<br />
studied polymorphisms are in families of GSTM1, GSTT1 and GSTP1. In our study we<br />
tested the gene polymorphisms in three groups of patients, with: i) ischemic stroke ii)<br />
depression, and iii) healthy controls. In the DD polymorphism of ACE gene, the<br />
depressed pacients exhibit higher frequency of DD genotypes in comparison to<br />
controls. Stroke patients exhibit only non-significantly lower frequency in comparison<br />
to controls. So far, we did not observe any significant differences in allelic frequency<br />
for HTTLPR gene neither for depressed nor ischemic groups in comparison to<br />
controls. Our results suggest that ACE DD genotype is increased in depressed<br />
pacients. Analysis of selected polymorphisms of GSTM1 and GSTT1 genes show<br />
higher frequency of GSTM1 null and GSTT1 wild genotypes.<br />
This work was supported by the VEGA 49/09 and MZ-2007/55-UK-16<br />
115<br />
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Posters<br />
4.<br />
AN ANALYSIS OF THE IMPACT OF CNS ISCHEMIA ON MITOCHONDRIAL<br />
RESPIRATORY COMPLEXES<br />
Mária Chomová and Peter Račay<br />
Institute of Medical Biochemistry, Jessenius Faculty of Medicine in Martin, Comenius<br />
University in Bratislava<br />
Mitochondria are responsible for aerobic respiration and ATP synthesis by oxidative<br />
phosphorylation. In addition to being crucial for energy production and metabolic<br />
pathways, they also play key roles in the signal cell network. Ischemia/reperfusion is<br />
a multifactorial process that leads to disturbance of key mitochondrial functions and<br />
can initiate a cascade of events, which result in tissue damage and finally in the cell<br />
death. So the existence of mitochondrial signal network evokes numerous responses<br />
and effects, which depend on the signal nature, cell environment and the individual<br />
ability of the cell to deal with the signals. In this regard, the goal of the work was to<br />
contribute to understanding the different processes in mitochondria after ischemic<br />
attack, particularly in connection with mitochondrial respiratory complexes I and IV.<br />
The impact of 15 min global brain ischemia followed by 1, 3, 24 and 72 hours<br />
reperfusion on complexes I and IV in cortex and hippocampus was studied by<br />
spectrophotometric determination of their enzymatic activities by using two electron<br />
acceptors, decylubichinone and ferricyanide and two ways of membrane<br />
permeabilisation (sonification vs detergent). The noticed variations were analyzed by<br />
electrophoretic methods SDS PAGE and two- dimensional native BN PAGE/SDS PAGE.<br />
The posttranslation modifications of selected structural subunits of respiratory<br />
complexes were monitored by Western blotting and immunodetection of 3-<br />
nitrotyrosines as markers of oxidative damage to proteins. Add to this, the interest<br />
was focused on processes of the p53-mitochondrial pathway of apoptosis where the<br />
levels of p53 protein as well as pro- and antiapoptotic members of Bcl 2 family were<br />
monitored. A possible involvement of the complex I structural subunit GRIM-19 in<br />
apoptotic processes was also studied.<br />
116<br />
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Posters<br />
5.<br />
THE ROLE OF MAP-KINASE PATHWAY IN GLOBAL ISCHEMIA/REPERFUSION INJURY<br />
OF RAT BRAIN AFTER INDUCED HYPERHOMOCYSTEINEMIA<br />
Mária Kovalská 1 , Martina Pavlíková 1 , Zuzana Tatarková 1 , Peter Kaplán 1 , Dušan<br />
Dobrota 1 , Marian Adamkov 2 and Ján Lehotský 1<br />
1 Department of Medical Biochemistry, 2 Department of Histology and Embryology<br />
Comenius University, Jessenius Fac Med Martin<br />
An altered cross-talk in intracellular signaling pathways is presumed in the<br />
mechanisms in ischemic injury. The MAPK/ERK is a signal transduction route that<br />
couples intracellular responses to cell surface receptors. ERK protein is part of the<br />
cascade leading to survival of neurons after injury. Tolerance induced by<br />
preconditioning (IPC) is tissue adaptation to sub-lethal ischemia.<br />
Hyperhomocysteinemia (hHcy) is one of the risk factor, which could have negative<br />
impact on the onset/progression of ischemia/reperfusion injury (IRI). In these<br />
experiments, we have studied changes in MAPK pathways and related enzymes after<br />
global IRI in forebrain homogenate. In addition, the effects of: i) ischemic<br />
preconditioning (IPC) and ii) hyperhomocysteinemia (hHcy) on IRI-associated<br />
alternations of protein levels of MAPK pathway was determined. Global forebrain<br />
ischemia was induced by 4-vessels occlusion. Rats were preconditioned by 5 min of<br />
sub-lethal ischemia and 2 days later, 15 min of lethal ischemia with reperfusion<br />
period of 1h, 3h, 24h and 72h was induced. hHCy was induced by twice a day<br />
subcutaneous injection of homocysteine (0.45 µmol/g). Immunohistochemical as well<br />
as Western blot analysis identified MAPK/ERK protein in injured areas. The highest<br />
level of the protein was detected in the reperfusion time after IPC. Converse effect<br />
was observed in the reperfusion time after induced hHcy. This suggests that adaptive<br />
mechanisms in the MAPK signal transduction machinery might have a potential role<br />
in tissues response subjected to IRI and in the phenomenon of tolerance.<br />
Acknowledgments: Supported by VEGA 0049/09, VVCE 0064/07, UK-16/2007 and<br />
UK/10/2010.<br />
117<br />
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Posters<br />
6.<br />
ANATOMICAL DISTRIBUTION OF DYING CELLS WITHIN ADULT RATS ROSTRAL<br />
MIGRATORY STREAM<br />
Marcela Martončíková, Juraj Blaško, Judita Orendáčová,<br />
Kamila Lievajová and Enikő Račeková<br />
Institute of Neurobiology, Slovak Academy of Sciences, Košice, Slovak Republic<br />
The rostral migratory stream (RMS) is a pathway along which newborn cells<br />
originated from the subventricular zone migrate toward the olfactory bulb where<br />
they differentiate into interneurons. This migratory pathway consists of three parts in<br />
caudo-rostral direction: the vertical arm, the elbow and the horizontal arm. The<br />
precursor cells are able to proliferate during migration in the RMS. It has been<br />
reported that these cells may also undergo apoptotic cell death. Dying cells has been<br />
observed along entire extent of the RMS. The number and distribution of dying cells<br />
in the RMS varies according to the methodology used. Commonly applied<br />
methodology for identification of apoptotic cells is a terminal dUTP nick-end-labelling<br />
(TUNEL). Another marker for detection of dying cells is a fluorochrome, Flouro Jade-C<br />
(FJ-C), which stains all degenerating neurons, regardless of mechanism of cell death.<br />
Studies dealing with the cell death in the RMS assess the total number of positive<br />
cells mainly in whole extent of the RMS. In this study we focused on the distribution<br />
of FJ-C positive cells in individual anatomical parts of the RMS. We found that the<br />
number of dying cell is the highest in the caudal part of the RMS – in the vertical arm.<br />
The lowest number of dying cells was observed in the middle part of the RMS –in the<br />
elbow. In the rostral part of the RMS- in the horizontal arm the number of these cells<br />
was higher than in the elbow. Differences in the number of dying cells in the<br />
individual parts of the RMS are probably associated with different developmental<br />
origin of these parts.<br />
Acknowledgments: Supported by VEGA grants: 2/0147/09; 2/058/08.<br />
118<br />
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Posters<br />
7.<br />
SECRETORY PATHWAYS SPCA1- CA2+ PUMP EXPRESSION AS PART OF ISCHEMIC<br />
PRECONDITIONING IN RAT FOREBRAIN<br />
Martina Pavlíková, Mária Kovalská, Monika Kmeťová Sivoňová,<br />
Zuzana Tatarková and Ján Lehotský<br />
Department of Medical Biochemistry Jessenius Faculty of Medicine, Comenius<br />
University, Martin, Slovakia<br />
Neural cells of the brain have important secretory functions. They secrete many<br />
neurotransmitters and secretory proteins. The Golgi apparatus, as part of secretory<br />
pathways (SP), is recognized Ca2+ store, which regulates secretion by SPCA-<br />
Ca2+ATPase. In addition, SP are involved in stress sensing and transduction of<br />
apoptotic signals. Ischemic preconditioning (IPC) is an important phenomenon of<br />
adaptation of tissue to sub-lethal short-term ischemia, which results in increased<br />
tolerance of tissue to the lethal ischemia. In this study we have investigated the<br />
changes in SPCA1 expression after global ischemic injury (IRI) in hippocampal and<br />
cortical areas. In addition, the effects of IPC on IRI associated alternations of mRNA<br />
and protein levels of SPCA1 were determined. Rats were preconditioned by 5 min of<br />
sub-lethal ischemia and 2 days later, 15 min of lethal ischemia followed by<br />
reperfusion for 1h, 3h and 24h. RT-PCR and Western blot analysis clearly detected<br />
expression of SPCA gene in injured area after IRI insult. In addition, tissue response<br />
on the level of mRNA was maximal in the reperfusion period in both paradigms. IPC<br />
did not change significantly the expression profile, however magnitude of cell<br />
response was elevated. Protein level of SPCA was highest in the reperfusion time. Our<br />
results showed that IPC affects gene expression and SPCA translation in forebrain<br />
induced by ischemia. This suggests for a potential role of SPCA regulated secretory<br />
processes in adaptation of neuronal circuits as response to preischemic challenge.<br />
Acknowledgement: This work was supported by grants VEGA 0049/09, UK 10/2010,<br />
VVCE 0064/07.<br />
119<br />
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II. BIOTECHNOLOGY<br />
120<br />
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Posters<br />
8.<br />
CHARACTERIZATION OF BACTERIOPHAGES<br />
INFECTING CRONOBACTER STRAINS<br />
Hind Al Alami, Michal Kajsík, Jana Gajdošová, Hana Drahovská, Ján Turňa<br />
Department of Molecular Biology, Faculty of Natural Sciences, Comenius University,<br />
Mlynská dolina 1, 842 15 Bratislava, Slovensko<br />
Cronobacter spp. (previously known as Enterobacter sakazakii) is an opportunistic<br />
pathogen, implicated in food-borne diseases especially in neonates and infants.<br />
Although the microorganism is widely distributed in the environment, dried infant<br />
milk formula has been implicated as the vehicle of transmission in many clinical<br />
manifestations. The current threat of antibiotic-resistant bacteria has renewed the<br />
interest in exploring bacteriophages as biocontrol agents.<br />
The aim of the our study was to isolate set of bacteriophages infecting Cronobacter<br />
strains and to assess their suitability to be used as antimicrobial agents in food<br />
industry. Six bacteriophages infecting Cronobacter were isolated from wastewater<br />
samples of three Bratislava wastewater treatment plants (Petržalka, Vrakuňa and<br />
Devínska Nová Ves) by propagating on indicator strains. We have determined the<br />
host specificity of bacteriophages on the collection of 16 strains. We have observed,<br />
that bacteriophages were able to infect 3-13 strains, three phages has specificity<br />
restricted to C. sakazakii species, the other three bacteriophages posses the broader<br />
host range specificity; they were able to inhibit growth of 11-13 strains of all<br />
Cronobacter species. By restriction mapping we observed different profiles for each<br />
phage.<br />
121<br />
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Posters<br />
9.<br />
EFFECT OF METHYL JASMONATE ON THE PRODUCTION OF SANGUINARINE AND ITS<br />
PRECURSORS IN OPIUM POPPY SUSPENSION CULTURES<br />
Andrea Balažová 1 , Víťazoslava Blanáriková 1 , Jindra Valentová 2 ,<br />
František Bilka 1 and Ivana Holková 1<br />
1 Department of cellular and molecular biology of drugs, FaF UK Bratislava<br />
2 Department of chemical theory of drugs, FaF UK Bratislava<br />
Suspension cultures of opium poppy are not able to produce morphinan alkaloids but<br />
synthesize and store sanguinarine within a single cells. The morphinans and<br />
sanguinarine share common biosynthetical pathway, leading to (S)-reticuline as a<br />
crutial intermediate. The biosynthesis of these alkaloids begins with the conversion of<br />
tyrosine to both dopamine and 4-hydroxyphenylacetaldehyde. The suspension<br />
cultures of opium poppy enhenced their sanguinarine production after treating with<br />
three different concetrationes of methyl jasmonate (10, 100, 1000 μmol/l). The<br />
presence of elicitor in nutrient medium of suspension cultures also led to the changes<br />
in levels of sanguinarine precursors (DOPA, dopamine, tyrosine and tyramine). The<br />
suspension cultures were exposed to elicitror treatment for 2, 4, 6, 8, 10, 24 and 48 h.<br />
The maximum content of sanguinarine was detected after 8 h exposure to elicitor at<br />
concetration 100 μmol/l (5.8-fold increase over the control). The suspension cultures<br />
produced the highest amount of DOPA between 2-6 h after elicitation with all three<br />
concetrationes of methyl jasmonate. The level of dopamine increased gradually in<br />
elicited cultures. The maximal content of dopamine was detected after 8 h exposure<br />
to elicitor at concentration 10 μmol/l (3.24-fold increase). The level of tyrosine<br />
started to increase later than the levels of DOPA and dopamine. The accumulation of<br />
tyrosine was positively affected mainly by methyl jamonate at concetrationes 10 and<br />
100 μmol/l. The levels of tyramine have not been changed significantly in elicited<br />
suspension cultures.<br />
122<br />
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Posters<br />
10.<br />
COMPARISON OF SANGUINARINE PRODUCTION OF PAPAVERACEAE FAMILY<br />
PLANTS IN VITRO CULTURES<br />
František Bilka, Andrea Balažová, Andrea Bilková, Víťazoslava Blanáriková,<br />
Ivana Holková and Marián Vanko<br />
Department of cellular and molecular biology of drugs, Faculty of Pharmacy,<br />
Comenius University in Bratislava<br />
Intact plants of Papaveraceae family are producers of the whole range of<br />
benzylisoquinoline alkaloids, which are used in pharmaceutical industry. In vitro<br />
cultures derived from plants of Papaveraceae do not have the ability to produce so<br />
broad spectrum of alkaloids – active is only biosynthetic pathway leading to<br />
sanguinarine. We derived in vitro cultures of Papaver somniferum, Eschscholtzia<br />
californica, Chelidonium majus and Macleaya cordata. Their sanguinarine production<br />
abilities were tested and compared. Also the effect of elicitation on the sanguinarine<br />
production was studied. The lowest amounts of sanguinarine from all cultures tested<br />
were accumulated in suspension cultures of opium poppy (0.45 – 0.55 µg in 1 g of<br />
fresh weight). Eschscholtzia calicornica, Chelidonium majus and Macleaya cordata<br />
cultures produced similar amounts of sanguinarine (18.0 – 22.7 µg; 20.5 – 26.3 µg;<br />
15.4 – 20.3 µg in 1 g of fresh weight, resp.). In elicitation studies we used biotic<br />
(Botrytis cinerea hydrolysate) and abiotic (CuSO 4 , methyljasmonate) stressors. In all<br />
cultures treated the increase in sanguinarine accumulation was observed. Among the<br />
elicitors used the most effective was B. cinerea hydrolysate. From all cultures tested<br />
the most intensive response was observed in opium poppy cultures (increse to almost<br />
9 µg of sanguinarine in 1 g of fresh weight), although the amount of sanguinarine in<br />
elicited poppy cultures was lower than in non-elicited cultures of the other cultures.<br />
123<br />
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Posters<br />
11.<br />
DESIGN OF AN EXPRESSION SYSTEM FOR THE PRODUCTION OF RECOMBINANT<br />
HUMAN THROMBIN IN ESCHERICHIA COLI<br />
Michaela Kandričáková 1 , Stanislav Stuchlík 1 and Ján Turňa 1,2<br />
1 Comenius University, Faculty of Natural Sciences, Department of Molecular Biology,<br />
2 Department of Molecular Biology SAV Bratislava<br />
Thrombin is a plasmatic protease, which plays a key role in blood coagulation. This<br />
form of active prothrombin is characterised by many biological functions including<br />
procoagulation and anti coagulation effects. The polyfunctionality of thrombin has<br />
received increasing interest in biofarmaceutical and biomedical research. One of the<br />
possible ways to gain recombinant human thrombin is to use expression systems<br />
based on E.coli cells. E.coli offers many adventages over other host organisms and<br />
therefore remains the most commonly used host for the production of heterologous<br />
proteins. Prethrombin-2, an activation intermediate of thrombin, is often expressed<br />
as a precursor of prothrombin. Presumably, the use of this gene assures higher<br />
transcription and translation efficiency compared to prothrombin gene mainly<br />
because of the smaller size of the mRNA transcript. During the preparation of<br />
prethrombin-2 syntetic gene the known sequence will be used with optimized E.coli<br />
codon usage ensuring higher expression yields.<br />
The aim of this work is the design of proper expression system for the production of<br />
recombinant human thrombin in the E.coli host cells. The study is focused on<br />
selection of proper gene sequence with codon usage optimized for E.coli, selection of<br />
suitable expression system and in the next step we would like to optimise<br />
physiological conditions for the foreign protein expression and subsequent verify<br />
expression of recombinant thrombin.<br />
124<br />
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Posters<br />
12.<br />
HETEROLOGOUS EXPRESSION OF ROYAL JELLY APALBUMINS in E. coli.<br />
Tatiana Kraková, Jozef Šimúth and Katarína Bíliková<br />
Department of Molecular Apidology, Institute of Molecular Biology, SAV Bratislava<br />
The honeybee royal jelly (RJ) contains many valuable components and biologically<br />
active proteins and peptides. Major proteins of RJ, designated as apalbumins belong<br />
to a protein family consisting of nine members with M r of 49-87 kDa. Apalbumin1,<br />
apalbumin2 and apalbumin3 account for 90% of the RJ protein content and are 88%<br />
identical in amino acid sequence. Minor RJ proteins are mainly homologues of<br />
apalbumins, antimicrobial peptides and enzymes. RJ proteins display multifunctional<br />
features. For example, apalbumin1, apalbumin2 stimulated tumor necrosis factor<br />
alpha release. We have focused our attention to the molecular characterization of<br />
the apalbumin2 - second most abundant protein of RJ. Apalbumin2 is a basic protein<br />
of 52.7 kDa with eight isoelectric variants in the range of pI 7.5-8.5. For first time we<br />
purified royal jelly and characterized minority homologue of apalbumin2 named as<br />
apalbumin 2a (48.6 kDa) with antibiotics activities against P. larvae, the inducer of<br />
American foulbrood of honeybee colony.<br />
The aim of our study was a biotechnological preparation of apalbumin1 and<br />
apalbumin2 and their homologues by heterologous expreesion in E. coli with the goal<br />
of study their antibiotics and immunostimulatory properties. For these purposes we<br />
cloned appropriate cDNA or their of PCR fragments into pQE30 and pET28b(+)<br />
expression vectors and transformed into E. coli DH5α and/or BL21-CodonPlus(DE3)-<br />
RIL. We present the molecular properties of recombined aplbumins and their<br />
homologues as well.<br />
125<br />
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Posters<br />
13.<br />
EXPRESSION, PURIFICATION AND FUNCTIONAL CHARACTERIZATION OF TWO<br />
FORMS OF AGROBACTERIUM SP. STRAIN CP4 EPSPS GENE IN ESCHERICHIA COLI FOR<br />
HORIZONTAL GENE TRANSFER STUDIES<br />
Mahesh Madyagol, Stanislav Stuchlík and Jan Turňa<br />
Comenius University, Department of Molecular Biology, Faculty of Natural Sciences,<br />
Bratislava, SLOVAKIA<br />
The Agrobacterium sp. strain CP4 aroA gene encodes an enzyme called 5-enolpyruvylshikimate-3-phosphate<br />
(EPSP) synthase. The enzyme is widely involved in<br />
glyphosate tolerant transgenic plants because it is the primary target of the<br />
nonselective herbicide glyphosate. We have cloned this gene and constructed a<br />
system for the high level expression of a recombinant form of this enzyme by<br />
amplifying the aroA gene from the genetically modified maize genomic DNA and the<br />
coding sequence of EPSPS gene was successfully subcloned in a plasmid-Escherichia<br />
coli system. Furthermore, the truncated form of CP4 EPSPS synthase has been<br />
created in order to compare in vitro glyphosate sensitivity between the two forms of<br />
enzymes. The cells containing the plasmid carrying both forms of EPSPS gene<br />
exhibited enhanced tolerance to herbicide glyphosate, compared to the control. The<br />
resulting plasmids produced the EPSP synthase in large quantities which has been<br />
purified to homogeneity and enzyme activity assay has been carried out. The study of<br />
horizontal gene transfer (HGT) is planned in the future work.<br />
126<br />
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Posters<br />
14.<br />
CONFORMATION TRANSITIONS OF CYTOCHROME C IN DEEP EUTECTIC SOLVENTS<br />
Jozef Parnica 1 , Lukáš Kandráč 1 and Marián Antalík 1, 2<br />
1 Department of Biochemistry, Faculty of Science, P.J. Safarik University, Kosice, Slovak<br />
Republic, 2 Institute of Experimental Physics, SAS, Kosice, Slovak Republic<br />
Original deep eutectic mixtures (DES) were formed by mixing two components<br />
together in different molar ratios that had a melting point much lower than any of<br />
the constituents, until homogenous, colorless liquid was formed. Deep eutectic<br />
solvents were also included in the advanced ionic liquids. They have recently<br />
attracted much attention as “green” alternatives to conventional organic solvents in<br />
various fields including biochemistry and separation processes due to their unique<br />
properties such as high thermal stability, negligible vapor pressure and nonflammability.<br />
In our work, we applied DES’s as alternative solvents to determinate the<br />
stability and conformation transitions of a model protein, ferricytochrome c. We used<br />
urea, malonic acid, and sorbitol as hydrogen bond donors by UV-Vis spectroscopy and<br />
circular dichroism spectroscopy in mixtures of substituted quaternary ammonium<br />
salts e.g. hydroxyethyltrimethyl-ammonium (choline) chloride. We have found DES’s<br />
as suitable solvents for characterization new types of conformation states of<br />
ferricytochrome c that demonstrate their advantages and unique properties, and<br />
suggest opportunities for new applications.<br />
Acknowledgement: This work was supported within the projects Nos., 26220120001,<br />
26220220005, 2622022033 in frame of SF EU, Centre of Excellence of SAS Nanofluid<br />
and VEGA 0056, 0038 and 0079.<br />
127<br />
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Posters<br />
15.<br />
PRODUCTION OF RECOMBINANT PROTEINS USING HIGH CELL DENSITY CULTURES<br />
OF ESCHERICHIA COLI IN BIOREACTORS<br />
Lucia Pánčiová, Zdenko Levarski, Pavol Utekal, Stanislav Stuchlík and Ján Turňa<br />
Comenius University Faculty of Natural science, Department of Molecular Biology<br />
Mathematical modeling of processes, information techniques and database<br />
generation have received increasing interest of biotech industry in recent years. For<br />
the generation of predictable mathematical models and databases of fermentation<br />
processes a vast amount of data is required to precisely characterize the growth<br />
dynamics and microorganism metabolism. These data are generated using systematic<br />
approaches. Systems of metabolic engineering are an ideal way for the development<br />
of bacterial strains producing bioproducts or aminoacids. Production of recombinant<br />
proteins using high cell density cultures of Escherichia coli became one of the most<br />
often used methods in biotech industry, mainly because of the high volumetric<br />
productivity associated with this method, but also because of the vast amount of<br />
data available regarding to host genetics and physiology. Application of different<br />
cultivation techniques results in achievement of cell densities higher than 100 g of dry<br />
cell weight per liter of cultivation media. The most common problems during<br />
cultivation of cells to high densities are limited oxygen solubility in cultivation<br />
medium, formation of growth inhibitory by-products, accumulation of acetate and<br />
CO 2 and generation of heat by the cells. The basic parameter of the growth kinetics<br />
observation is the specific growth rate, which can be different for every<br />
microorganism, type of the cultivation media or cultivation technique used. In this<br />
work, we have focused on the construction of recombinant strains of E.coli, suitable<br />
cultivation media selection, definition of crucial cultivation parameters, optimization<br />
of gene expression, monitoring of growth kinetics and finally determination of cell<br />
viability and biomass concentration.<br />
128<br />
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Posters<br />
16.<br />
SYNTHESIS AND SURFACE MODIFICATION OF ZINC OXIDE NANOPARTICLES<br />
Michaela Šimšíková 1 and Marián Antalík 1,2<br />
1 Department of Biochemistry, Faculty of Science, P.J. Safarik University, Kosice, Slovak<br />
Republic; 2 Institute of Experimental Physics, SAS, Kosice, Slovak Republic<br />
Zinc oxide nanostructures are very attractive for their unique properties and<br />
biocompatibility and in consequence of this have great potencial for applications in<br />
biosystems, for example biolabeling, biosensoring, delivery systems and others,<br />
which can be used in genetics, pathology, criminology, safety of food and many other<br />
industries. For these bioapplications are necessary surface modifications, for example<br />
with water soluble thiols like stabilizating agents, which can made to the<br />
nanostructures to better suit their integration with biological systems, leading to such<br />
interesting properties as enhanced aqueous solubility, bio-recognition or applicability<br />
for biological systems..<br />
For synthesis of ZnO nanoparticles in aqueous solution we used three different<br />
concentrations of 11-mercapto-undecanoic acid MUA (0.1; 0.01 and 0.005 M) as<br />
stabilizing agent. The coating of nanoparticles with MUA could allow their solubility in<br />
a variety of organic solvent and the covalent binding through hydroxyl groups present<br />
in its structure.<br />
The PL spectra indicated that the optical properties of ZnO nanoparticles were<br />
changed with the insertion of MUA, and showed significant influence of this surface<br />
modification on intensity.<br />
Acknowledgements: This work was financially supported by the research grants<br />
VEGA No. 0038, 7055, 0056; VVGS PF 12/2010/CH: Slovak Academy of Science in<br />
frame of CEX NANOFLUID, projects ESF 26220120021 and NFP 26220220005.<br />
129<br />
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Posters<br />
17.<br />
CLONING, EXPRESSION AND ANTIMICROBIAL ACTIVITY OF THE HUMAN<br />
CATHELICIDIN LL-37<br />
Csaba Tóth 1 , Roland Pálffy 1 , Juraj Gašperík 2 , Stanislav Stuchlík 1 and Ján Turňa 1<br />
1 Department of molecular biology, Faculty of Natural Sciences, Comenius University,<br />
2 Institute of Molecular Biology, SAS Bratislava<br />
Antimicrobial peptides are short polypeptides involved in the innate immunity system<br />
and they can be found among all classes of life. They demonstrate antimicrobial<br />
activity against Gram-negative and Gram-positive bacteria, viruses, fungal pathogens<br />
and cancerous cells. LL-37 is a multifunctional peptide and the only antimicrobial<br />
peptide from the cathelicidin family found in human. It is mainly expressed in myeloid<br />
cells, where it is located in specific granules, but it was also described in inflamed<br />
skin, testis, wound fluid, lung epithelia, sweat and saliva. Apart from its’ wide<br />
spectrum of bactericidal activity, LL-37 also plays an important role in the regulation<br />
of the inflammatory response, neutralization of LPS and the promotion of wound<br />
healing.<br />
As the therapeutical and commercial importance of these peptides is rising, simple<br />
expression and purification systems will be needed for their large-scale production.<br />
We have designed and prepared a cost-effective and efficient expression system for<br />
the production of LL-37 in fusion with ketosteroid isomerase in E. coli cells. After the<br />
purification and activation of the peptide, we have tested its’ biological activity<br />
against various bacterial pathogens.<br />
130<br />
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Posters<br />
18.<br />
CHARACTERIZATION OF BACTERIOPHAGES INFECTING<br />
SALMONELLA ENTERICA<br />
Ľubomíra Tóthová, Hind Al Alami, Jana Lintnerová, Hana Drahovská and Ján Turňa<br />
Department of Molecular Biology, Faculty of Natural Sciences, Comenius University,<br />
Bratislava, Slovakia<br />
Food-borne infections caused by Salmonella enterica represent a major healthcare<br />
concern worldwide. Bacteriophages are viruses infecting specifically bacteria. With<br />
regard to their characteristics, they can be considered as a potential antibacterial<br />
agents. In these applications bacteriophages are suitable to prevent or reduce<br />
colonization and diseases in livestock, to decontaminate carcasses and other raw<br />
products, to disinfect equipment and contact surfaces, etc.<br />
The aim of our study was to isolate bacteriophages infecting Salmonella, which can<br />
be used for elimination of these pathogenic bacteria in food technology.<br />
Bacteriophages were isolated from wastewater samples of three Bratislava<br />
wastewater treatment plants (Petržalka, Vrakuňa and DevínskaNováVes) by<br />
propagating on indicator strains. Six bacteriophages infecting Salmonella were<br />
isolated and used for further investigation.<br />
We have determined the host specificity of bacteriophages on the collection of 16<br />
strains belonging to different salmonella serotypes. Two different testing methods,<br />
the cross test and the colorimetric microplate test, were used rendering similar<br />
results. We have observed, that bacteriophages were able to infect 4-14 indicator<br />
strains and that all strains were sensitive to at least one bacteriophage. The<br />
chromosomal DNA of bacteriophages was isolated and characterized by restriction<br />
cleavadge. We observed different restriction profiles for each phage.<br />
131<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
19.<br />
PRODUCTION OF TWO RECOMBINANT ALCOHOLDEHYDROGENASES SUITABLE FOR<br />
BIOTRANSFORMATION OF C-6 ALDEHYDES INTO CORESPONDING ALCOHOLS<br />
Pavol Utekal, Lucia Pánčiová, Stanislav Stuchlík and Ján Turňa<br />
Department of Molecular biology Prif UK Bratislava<br />
C-6 aldehydes and alcohols contribute to the fresh green odor in plants and are<br />
widely used in perfumes and in food technology. Important member of this family is<br />
trans-2-hexenol. Carbonyl compounds such as aldehydes and alcohols are reduced by<br />
chemical methods in industry but it is not appropriate for production of compounds<br />
used in food industry. Therefore, in recent decades biocatalysis is used for these<br />
purposes. The enzymes suitable for reduction of aldehydes are oxidoreductases,<br />
which catalyze the reduction of carbonyl groups of aldehydes and alcohols. The most<br />
suitable enzyme from this class is alcoholdehydrogenase (ADH), which is the last<br />
enzyme involved in lipoxygenase pathway in several species of higher plants, which<br />
converts natural trans-2-hexenal to trans-2-hexenol. In the case of redox reactions<br />
catalyzed by oxidoreductases, which often require stoichiometric oxidation or<br />
reduction of costly coenzymes such as NAD(P) and FAD, efficient coenzyme recycling<br />
must be accomplished if a given application is to be practical from an economic<br />
standpoint. Formate dehydrogenase (FDH) is one of the frequently used biocatalysts<br />
for NADH regeneration. This work is focused on the cloning, expression, purification<br />
and measurements of enzyme activity of two enzymes - ADH from Saccharomyces<br />
cerevisiae and FDH from Candida boidinii.<br />
132<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
20.<br />
EUKARYOTIC EXPRESSION AS AN INDISPENSABLE TOOL FOR PREPARATION OF<br />
NATIVE DIMERIC FORMS OF NK CELL C-TYPE LECTIN-LIKE RECEPTORS<br />
Ondřej Vaněk 1 , Petra Celadová 1 , Jan Bláha 1 , Daniel Kavan 2 ,<br />
Petr Pompach 2 and Karel Bezouška 1,2<br />
1 Department of biochemistry PřF UK Praha, 2 Department of immunology and<br />
gnotobiology MBÚ AVČR Praha<br />
Natural killer cells are able to recognize and kill a variety of tumor and infected cells.<br />
The recognition is mediated by wide repertoire of cell surface receptors,<br />
both activation and inhibitory, belonging to two main structural families:<br />
immunoglobulin-like and C-type lectin-like. While ligands for the Ig-like receptors<br />
were shown to be MHC gp I proteins, ligands only for some of the lectin-like<br />
receptors are known up to date. Both families share relatively weak binding<br />
characteristics and in the case of lectin-like receptors, in vitro oligomerization clearly<br />
improves binding through increase in avidity. However, these lectin-like receptors<br />
were described mostly as dimeric in vivo and this minimal oligomer would be also the<br />
best for structural studies. Moreover, there is no crystal structure known of<br />
extracellular domain of any of these receptors in its full-length natural dimeric form;<br />
probably because prokaryotic expression of native dimers is rather difficult. Here we<br />
present successful design, cloning and production of dimeric forms of some mouse,<br />
rat and human NK cell lectin-like receptors belonging to NKRP1 and Clr families. Fulllength<br />
extracellular receptor parts were expressed via transient transfection of<br />
HEK293 cell lines in suspension culture either alone or as a cleavable fusion with Fc<br />
fragment of human IgG1 to promote native dimerization. This fusion strategy<br />
resulted in cleaved pure covalent dimers of e.g. rat Clrb and purely dimeric Fc fusion<br />
of rat NKRP1B proteins and their structural characterization is currently under way.<br />
133<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
III. BIOINFORMATICS<br />
130<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
21.<br />
phiGENOME – A WEB-BASED GENOME BROWSER INTENDED FOR DISPLAY OF<br />
PHAGE GENOMES<br />
Matej Stano and Lubos Klucar<br />
Institute of Molecular Biology, SAS Bratislava<br />
Graphical representations of genomes convey quick insight into organization of<br />
coding sequences and other functional regions within genomes. Applications<br />
intended for generation of graphical representations of genomes are genome<br />
browsers.<br />
We present phiGENOME, a web-based genome browser providing highly intuitive and<br />
interactive maps of phage genomes stored in phiSITE – database of gene regulation in<br />
bacteriopages. It provides dynamic genome maps built on the Adobe Flash platform<br />
as well as an accessory semi-graphics based on precisely formatted HTML tags.<br />
phiGENOME is a integral part of phiSITE web interface, therefore it was optimized for<br />
visualization of phage genomes with emphasized display of cis-regulatory elements.<br />
Graphical content is generated dynamically; required annotation data are retrieved<br />
promptly from the MySQL back-end. The data transfer from the database to the<br />
genome browser is supplied via XML files.<br />
Genome maps acquired from phiGENOME allow graphically subtle and lucid<br />
inspection of phage genomes. Individual genome features are displayed as coloured<br />
boxes; genes: blue, promoters: red, operators: green, terminators: yellow. All actions<br />
in this browser can be comfortably performed with mouse. Mouse drag enables<br />
continuous sliding along genome; mouse scroll zooms in (up to the level of nucleotide<br />
sequence) and out; mouse over a feature displays tool-tip with feature details; mouse<br />
click highlights selected feature and opens options menu for further actions.<br />
phiGENOME is an innovatory application according to the mode and purpose how the<br />
Flash technology was employed. It is freely accessible at phiSITE web interface:<br />
http://www.phisite.org.<br />
131<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
IV. GENOMICS<br />
132<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
22.<br />
SITE SPECIFIC INTEGRATION OF BACTERIOPHAGE µ1/6 INTO THE STREPTOMYCES<br />
AUREOFACIENS CHROMOSOME<br />
Jarmila Farkašovská and Andrej Godány<br />
Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava<br />
Phage integrases are enzymes that mediate unidirectional site-specific recombination<br />
between two recognition sequences, the phage attachment site, attP, and the<br />
bacterial attachment site, attB. Site-specific recombinases can be classified into two<br />
major families based on amino acid sequence homology and catalytic residues, either<br />
tyrosine or serine. The properties of site-specificity and efficiency make phage<br />
integrases excellent tools for the manipulation of DNA.<br />
The phage µ1/6 integrates site-specifically into the chromosome of tetracycline<br />
producing Streptomyces aureofaciens strains. The region of µ1/6 genome involved in<br />
this process have been identified. The deduced 416 amino acid protein encoded by<br />
orf5 displays significant similarity with site-specific recombinases of the tyrosine<br />
family (sometimes referred to as integrase family). The phage attachment site (attP)<br />
was localized downstream of the putative integrase gene. The junctions (attL and<br />
attR sites) of the prophage with the host genome have been determined, allowing<br />
identification of the host attachment site (attB), which has a common 45-nucleotide<br />
core region with attP. In attB this core sequence consists of the 3 ' end of the putative<br />
tRNA (Thr) (ACA) gene. To prove the functionality of the putative integrase gene (orf5),<br />
an integrative vector pCTint was constructed. This integration plasmid, consisting of<br />
the µ1/6 putative integrase gene and the phage µ1/6 attP, and a selectable marker<br />
(thiostrepton resistance gene for streptomycete) inserted stably into the<br />
chromosome of S. aureofaciens and S. lividans. The µ1/6 integrase with a deduced<br />
molecular mass of 47,5 kDa was overproduced in Escherichia coli and further<br />
analyzed.<br />
133<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
23.<br />
STUDY OF THERMOTOLERANCE ISLAND IN CRONOBACTER SPP<br />
Jana Gajdošová 1 , Natália Kamodyová 1 , Kristína Benedikovičová 1 , Hana Drahovská 1 ,<br />
Eva Kaclíková 2 and Ján Turňa 1<br />
1 Department of Molecular Biology, Faculty of Natural Sciences, Comenius University,<br />
Mlynská dolina 1, 842 15 Bratislava, Slovensko<br />
2 Food Research Institute, Priemyselná 4, Bratislava<br />
Cronobacter spp. is an opportunistic pathogen associated with sporadic cases of<br />
infections in infants. Dried infant milk formula has been identified as a potential<br />
source of the microorganism and the mode of transmission. The spread of<br />
Cronobacter in dried foods is promoted by its higher resistance than other<br />
Enterobacteriaceae to environmental stresses, including heating, drying or osmotic<br />
stress.<br />
The aim of our work was to characterize DNA region, which is present in some<br />
Cronobacter strains and which contributes to their elevated survival at 58°C. The<br />
thermotolerance island was sequenced in C. sakazakii LMG 5740, the length of the<br />
region was 19 kbp and 22 orfs with the size 141 – 2850 bp were detected. The<br />
greatest part of the region contained a cluster of conservative genes, most of them<br />
have significant homologies with bacterial proteins involved in some type of stress<br />
response, including heat, oxidation and acid stress. Similar organization of the region<br />
was observed in all thermotolerant strains. By rt-PCR approach we detected high<br />
expression throughout all thermotolerance gene cluster in both stationary and<br />
exponentially grown bacteria. Part of the thermotolerance genomic island covering<br />
orfD-G was cloned to pUC21 under lac promoter and transformed to E. coli host<br />
strain. Recombinant strain showed 80% increased D 58 value comparing with control<br />
strain containing the vector only.<br />
134<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
24.<br />
DNA POLYMORPHISMS OF SELECTED REPAIR GENES AND RISK OF LUNG CANCER<br />
Lucia Letková 1 , Tatiana Matáková 1 , Erika Halašová 2 ,<br />
Anna Drgová 1 and Dušan Dobrota 1<br />
1 Department of Medical Biochemistry; 2 Department of Medical Biology, Comenius<br />
University, Jessenius Faculty of Medicine, Martin, Slovakia<br />
The presence of polymorphisms of repair genes contributes to individual<br />
susceptibility to the development of lung cancer. We investigated the association<br />
between polymorphisms in DNA repair genes hOGG1 (Ser326Cys) and XRCC1<br />
(Arg399Gln), which play an important role in DNA base excision repair. In this casecontrol<br />
study, we recruited 309 lung cancer cases and 339 healthy controls. Genomic<br />
DNA was extracted from peripheral blood by phenol/chloroform extraction. The<br />
genotypes of hOGG1 and XRCC1genes were determined by PCR-RFLP method. The<br />
differences in the distribution of genotypes and alleles between cases and controls<br />
were analysed using x 2 -test. The odds ratio (OR) and 95% confidence intervals (95%<br />
Cl) were adjusted for the risk of developing lung cancer, alone polymorphisms or<br />
their combinations. The presence of polymorphisms of genes hOGG1 and XRCC1 in<br />
women represents elevated risk of lung cancer compared to men. The frequency of<br />
the genotype XRCC1 Gln/Gln (OR=2,32; P=0,19) represented 2-fold higher risk of lung<br />
cancer compared with genotype Arg/Arg in women. Genotypes Arg/Arg+Ser/Cys (OR=<br />
2,45; P=0,19), Arg/Gln+Ser/Ser (OR=2,3; P=0,08), Arg/Gln+Cys/Cys (OR=2,45; P=0,76)<br />
had a 2-fold increase risk of lung cancer compared with Arg/Arg+Ser/Ser genotype.<br />
The frequency of the Gln/Gln+Ser/Ser (OR=7,36; P=0,012) genotypes was statistically<br />
significantly higher compared with Arg/Arg+Ser/Ser genotype. In summary, variants<br />
alleles Cys326 and Gln399 are associated with reduce enzyme activity of genes and<br />
DNA repair capacity. The presence of these alleles leads to increase susceptibility at<br />
risk of developing lung cancer.<br />
135<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
25.<br />
EXPRESSION OF TRANSCRIPTION FACTORS AND microRNAs<br />
IN TGF-ß1–INDUCED EMT OF BENIGN PROSTATE EPITHELIAL CELLS<br />
E. Lincová/Slabáková 1,2 , Zuzana Pernicová 1,2 , Eva Slavíčková 1,2 ,<br />
Alois Kozubík 1,2 and Karel Souček 1<br />
1<br />
Dept. of Cytokinetics, Institute of Biophysics AS CR, Brno, Czech Republic.<br />
2<br />
Dept of Exp. Biology, Masaryk University, Faculty of Science, Brno, CZ.<br />
Epithelial-mesenchymal transition (EMT), in which epithelial cells become motile<br />
mesenchymal cells, is viewed as an essential early step facilitating dissemination of<br />
tumour cells. MicroRNAs (miRNAs) are small non-protein-coding regulators of gene<br />
expression that play an important role in tumorigenesis and, depending on their<br />
targets, can function as tumour suppressors or oncogenes. Members of the miR-200<br />
family, which regulate expression of ZEB transcription factors, have been found<br />
downregulated during EMT, suggesting an important role in inhibition of EMT. The<br />
aim of this study is to analyze the kinetics of expression of selected transcription<br />
factors and miRNAs in the EMT of prostate cells in order to identify key molecules<br />
responsible for tumorigenicity and invasivity of prostate cancer.<br />
In our experimental system, we induced EMT by TGF-ß1 treatment in BPH-1 cells<br />
derived from non-tumorigenic prostate epithelial cell lines. Among the genes<br />
analyzed, SNAI2/Slug was rapidly and strongly upregulated. Changes in expression<br />
levels of ZEB1 and ZEB2 mRNA and miRNA of miR-200 family are observed after<br />
extended periods of TGF-ß1 treatment and correlate with E-cadherin expression and<br />
progression of EMT but may not be responsible for rapid upregulation of EMT-driving<br />
transcription factors.<br />
Acknowledgements: This work was supported by grants No. 310/07/0961 and<br />
303/09/H048 of the Czech Science Foundation, IGA MZD 9956-4/2008, IGA MZD<br />
9600-4/2008, by the AS CR, grants no. AV0Z50040507 and AV0Z50040702 and by<br />
grant no. CZ.1.07/2.3.00/09.0020.<br />
136<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
26.<br />
THE ASSOCIATION BETWEEN EGF 61 G/A POLYMORPHISM AND COLORECTAL<br />
CANCER DEVELOPMENT<br />
Silvia Mahmood, Lucia Letková and Tatiana Matáková<br />
Department of Medical Biochemistry, Jessenius Faculty of Medicine,<br />
Comenius University, Martin, Slovakia<br />
The epidermal growth factor (EGF) gene has been demonstrated to participate in the<br />
pathogenesis or maintenance of several human cancers of epithelial origin. The<br />
purpose of the present case-control study was to investigate the association of the<br />
single-nucleotide polymorphism of EGF + 61 G/A with the susceptibility to colorectal<br />
cancer (CRC) in a population in the north of Slovakia. We have analyzed the genotype<br />
distribution of this polymorphism in 120 patients (65 [54,2%] men and 55 [45,8%]<br />
women, mean age 64,2 years) and 110 healthy subjects, using the polymerase chain<br />
reaction–restriction fragment length polymorphism technique. Differences in allele<br />
and genotype frequencies were evaluated by the Chi-square (χ 2 ) and Fisher exact<br />
tests. Logistic regression models were used to calculate odds ratios of response to<br />
treatment. A multiscale model of cancer growth based on the genetic and molecular<br />
features of the evolution of colorectal cancer has been proposed according to the<br />
previous published works. Our data suggests that EGF +61 G/A polymorphism may<br />
be used as a genetic susceptibility marker for the colorectal carcinoma.<br />
Acknowledgement: This work was supported by project "Creating a new diagnostic<br />
algorithm for selected cancer diseases" co-financed from EC sources and European<br />
Regional Development Fund.<br />
137<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
27.<br />
SPERM DNA INTEGRITY ASSESMENT USING DIFFERENT COMET ASSAY PROTOCOLS<br />
Milena Matejovičová 1 , Michaela Králíková 1 , Jitka Melounová 1 , Martina Vodová 1 ,<br />
Jana Žáková 2 and Igor Crha 2<br />
1 Department of Biochemistry, Medical Faculty MU, Brno, 2 Center for Reproductive<br />
Medicine, Dept. of Obstetrics and Gynecology, Brno<br />
DNA fragmentation in the individual cells can be measured using comet assay. The<br />
method is based on the electrophoresis of nuclear DNA immobilized in agarose gel<br />
that follows after the cell lysis and DNA denaturation. Cells containing damaged DNA<br />
have appearance of the comet: round „head“ representig unfragmented DNA and<br />
elongated „tail“, which intensity and lenght depends on both the extent of DNA<br />
fragmentation and fragment size. Specificity of the comet assay application on sperm<br />
consists in particullar organization of sperm chromatine, which is much more<br />
compact than in somatic cells and it contains a lot of disulfide bonds between DNA<br />
and protamines. For assesing DNA dammage in sperm cells, effective lysis and DNA<br />
denaturation without background damaging effect to DNA are crucial.<br />
A method of modified alkaline/neutral comet assay was used. We studied basal DNA<br />
damage in sperms from subjects undergoing semen analysis in the Center of Assisted<br />
Reproduction (CAR). Semen was liquified for 1 hour at room temperature and 1 ml<br />
aliquotes were frozen in liquid nitrogen. DNA damage level of frozen sperm samples<br />
was compared with the damage in a fresh samples, analyzed on a day of collection.<br />
No differences between fresh and frozen material were found. Next, we compared<br />
different methods of cell lysis. Two different protocol types were applied: 1)<br />
proteinase K digestion during lysis and 2) lithium diodosalicylate (LIS) as a detergent<br />
for cell membrane and chromatine protein core destruction. In both protocols,<br />
dithiothreitol (DTT) was used for disulfide bonds reduction. In the protocol with<br />
proteinase K, we have found higher level of DNA damage when using DTT than<br />
without this agent. The least damaged DNA was observed in assays with LIS and DTT.<br />
138<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
28.<br />
COMPARATIVE STUDY OF HYPOMETHYLATING<br />
ACTIVITIES OF 5-AZACYTIDINE CONGENERS<br />
Marika Matoušová, Ivan Votruba, Miroslav Otmar and Helena Mertlíková-Kaiserová<br />
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech<br />
Republic, Gilead Sciences & IOCB Research Center, 166 10 Prague, Czech Republic<br />
Cancer cells often display aberrant hypermethylation which leads to transcriptional<br />
inactivity of various gene groups (such as tumor suppressor genes) and is therefore<br />
responsible for genomic instability. The use of hypomethylating agents as anticancer<br />
drugs is intended for reactivate methylation-silenced genes by decreasing the overall<br />
methylation level. Since epigenetic therapy is expected to be more specific, less toxic<br />
and more effective than standard chemotherapy, the search for new<br />
hypomethylating agents is a top-priority task.<br />
The goal of this study was to compare hypomethylating activity of a series of 5-<br />
azacytidine congeners vs. decitabine and zebularine, the well-established DNA<br />
methyltransferase inhibitors. Quantitative methylation-specific PCR was employed to<br />
detect the efficiency of individual agents on thrombospondin-1 and cyclin-dependent<br />
kinase inhibitor 2B hypermethylated gene loci. Overall changes in DNA methylation<br />
level were quantified by direct detection of 5-methylcytosines by HPLC using digested<br />
genomic DNA.<br />
2´-Deoxy-5,6-dihydro-5-azacytidine was identified as a promising drug candidate for<br />
epigenetic therapy. It has similar hypomethylating activity as decitabine, the most<br />
effective hypomethylating drug tested, and is less cytotoxic.<br />
Acknowledgments: Research project of the Institute #OZ40550506; Project #1M0508<br />
by the Ministry of Education, Youth and Sports of the Czech Republic.<br />
139<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
V. CELL REGULATIONS AND SIGNAL TRANSDUCTION<br />
140<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
29.<br />
THE ROLE OF 14-3-3 PROTEIN IN REGULATION OF GLUCOSE TRANSPORTER GLUT4<br />
TRANSLOCATION TO ADIPOCYTE PLASMA MEMBRANE<br />
Lucia Gajdošechová, Miroslava Eckertová, Katarína Kršková and Štefan Zorad<br />
Institute of Experimental Endocrinology, SAS, Bratislava, Slovakia<br />
Basic mechanism of insulin action is translocation of GLUT4 transport vesicles (GTV) from<br />
inner cell compartments to the cell surface and their fusion with plasma membrane. Defect<br />
of this mechanism leads to development of insulin resistance and diabetes. Beta isoform of<br />
14-3-3 protein seems to be a key element of complex mechanism of glucose transporter(s)<br />
translocation in adipose tissue. The 14-3-3 protein binds to Thr-phosphorylated AS160, the<br />
substrate of insulin-activated Akt protein kinase, thereby reducing of AS160 GTP-ase activity<br />
and subsequently activating GTV translocation. The main purpose of the present study was<br />
to evaluate GLUT4 and GLUT1 transporters protein content in epididymal adipose tissue<br />
plasma membranes of young obese Zucker rats and correlate it with the amount of 14-3-3<br />
protein in fat tisssue total homogenate. Obese and lean Zucker rats (Ifa Credo/Charles River,<br />
France) at age 3 months were classified as young. Serum glucose was determined by glucose<br />
oxidase method. Serum insulin, leptin and adiponectin were evaluated by radioimmunoassay<br />
(Linco Research, USA). Plasma membranes from epididymal fat tissue were prepared by<br />
differential centrifugation. GLUT4 and GLUT1 proteins in plasma membrane preparation and<br />
14-3-3 protein in total cell homogenate were evaluated by immunoblot. Protein expression<br />
at mRNA level was determined by real-time PCR method.<br />
Young obese Zucker rats displayed significantly increased weight of epididymal adipose<br />
tissue, hyperinsulinemia, hyperleptinemia and mild hyperglycemia. Surprisingly the serum<br />
concentration of adiponectin was significantly elevated in obese animals despite of<br />
decreased mRNA level in white adipose tissue. GLUT1 protein and mRNA were not changed<br />
in obese animals. GLUT4 mRNA expression was significantly reduced but its protein content<br />
in adipose tissue plasma membrane fraction was significantly increased in obese animals.<br />
We noticed more than 6-fold increase in 14-3-3 protein content in cell homogenate from<br />
obese Zucker rats. Young obese Zucker rats represent a model of obesity and insulin<br />
resistance with mild hyperglycemia. Based on unchanged GLUT1 and elevated GLUT4<br />
content in plasma membrane fraction of obese animals we assume normal insulin sensitivity<br />
with regard to glucose transporter translocation in adipose tissue of 3-month-old obese<br />
Zucker rats. Augmented GLUT4 translocation seems to be due to an enormous increase in<br />
14-3-3 protein which might play a crucial role in glucose transporter activation. The<br />
elucidation of 14-3-3 protein regulation, e.g. by adiponectin, awaits for further investigation.<br />
Ackowledgement: This work was supported by grant VEGA 2/0162/08.<br />
141<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
30.<br />
CHANGES IN COFILIN PHOSPHORYLATION DURING THE APOPTOSIS OF LEUKEMIC<br />
JURL-MK1 CELLS<br />
Dana Grebeňová, Michaela Pluskalová, Zbyněk Hrkal and Kateřina Kuželová<br />
Institute of Hematology and Blood Transfusion, Prague, Czech Republic<br />
Cofilin is a key mediator of actin dynamics which is involved in processes requiring<br />
changes in the cytoskeleton structure, e. g. cell migration, cell division and adhesion<br />
to the extracellular matrix. It promotes actin filament severing and depolymerization,<br />
facilitating the breakdown of existing filaments and the enhancement of filament<br />
growth from newly created barbed ends. Cofilin phosphorylation at Ser3 is known to<br />
prevent its activity. We explored the effect of two antileukemic drugs<br />
(suberoylanilide hydroxamic acid and imatinib mesylate) on JURL-MK1 cell adhesivity<br />
to fibronectin and on cofilin activity. Under certain conditions, both compounds were<br />
able to enhance the cellular adhesivity and cofilin phosphorylation (inactivation)<br />
increased as expected. To the contrary, higher drug doses induced the apoptosis<br />
which was accompagnied by a decrease in cellular adhesivity to fibronectin and by F-<br />
actin disassembly. Surprisingly, cofilin phosphorylation at Ser3 markedly increased<br />
even in these conditions. This increase could be at least partly inhibited by the<br />
apoptosis inhibitor Q-VD-OPh, but not by the inhibitor of ROCK, one of the main<br />
regulators of cofilin activity. We speculate that some prominent actin structures have<br />
to be protected from cofilin-mediated destruction in order to assure the cell<br />
disintegration into apoptotic bodies. The phosphorylated (inactive) cofilin was<br />
concentrated in small distinct spots distributed throughout the cytoplasm.<br />
Ackowledgement: This work was supported by the grant No 301/09/1026 from the<br />
Grant Agency of the Czech Republic.<br />
142<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
31.<br />
CHARACTERIZATION OF A GENE ENCODING A SMALL REGULATORY RPOE –<br />
DEPENDENT RNA IN SALMONELLA ENTERICA SEROVAR TYPHIMURIUM<br />
Dagmar Homerová, Bronislava Řežuchová, Henrieta Škovierová and Ján Kormanec<br />
Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava<br />
Gene regulation of bacteria is a complex process in which proteins have been<br />
believed as the only relevant regulators for a long time. In recent years, the family of<br />
small non-coding RNA (sRNA) with the important role especially in post-transcription<br />
control was described. Enterobacterial pathogen Salmonella enterica serovar<br />
Typhimurium (S. Typhimurium) is an attractive model for studying sRNAs, since the<br />
research could clarify many processes affecting the regulation of bacterial<br />
pathogenicity. So far, more than 70 sRNAs with various roles in control of gene<br />
expression have been identified in S. Typhimurium. Among them, small antisense<br />
RNA MicA, activated by sigma factor RpoE, strongly represses the mRNAs of two<br />
porins, OmpA and LamB. To understand the role of MicA we have investigated its<br />
expression and role in virulence of S. Typhimurium. In vitro transcriptional analysis<br />
revealed presence of a single promoter, micAp, with the high similarity with the<br />
consensus RpoE promoter sequence showing clear dependence upon this sigma<br />
factor. Activity of micAp increased towards stationary phase and was induced by<br />
several stresses including heat shock, cold shock, acid stress, oxidative stress,<br />
ethanol, polymixin B, and by degS-specific stress elicited by unfolded C-terminal outer<br />
membrane protein OmpA. Lack of micA elicited an RpoE-dependent envelope stress<br />
response. Although in vitro phenotypic analysis revealed no significant differences<br />
between wild type and micA mutant strains, in vivo studies showed that the micA<br />
mutant is more virulent in the mouse model.<br />
Ackowledgement: This work was supported by the VEGA grant 2/0104/09 from Slovak<br />
Academy of Sciences.<br />
143<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
32.<br />
MOLECULAR MECHANISMS INVOLVED IN POTENT PLATINUM (IV) COMPLEX-<br />
MEDIATED COLON CANCER CELL SENSITIZATION TO TRAIL-INDUCED APOPTOSIS<br />
Iva Jelínková 1,2 , Olga Vondálová Blanářová 1,2 , Jiřina Hofmanová 1,2 , Petr Sova 3 , Alois<br />
Kozubík 1,2 and Vaculová Alena 1,2<br />
1 Department of Cytokinetics, Institute of Biophysics, AS CR, v.v.i., Brno; 2 Faculty of<br />
Science, Masaryk University Brno; 3 R&D, Pliva-Lachema, a.s., Brno, Czech Republic<br />
Platinum (II) complexes such as cisplatin are used in the therapy of many solid<br />
cancers, but their application is limited due to serious side effects and/or cancer cell<br />
resistance. Recently, platinum (IV) adamantylamine ligand-containing complex LA-12<br />
has been introduced and shown as highly effective in many cancer cells including<br />
colon. TRAIL (TNF-related apoptosis inducing ligand), a cytokine that belongs to the<br />
TNF family, is a potent and promising anticancer agent that triggers apoptosis in<br />
many cancer but not in most normal cells. However, some cancer cells are resistant<br />
to its effects. We demonstrated that LA-12 was very effective in potentiation of<br />
TRAIL-induced apoptosis in colon cancer cells. Molecular mechanisms involved in cell<br />
sensitization were investigated, with a particular focus on the death receptors,<br />
caspases, and mitochondria. We demonstrated that LA-12 was responsible for<br />
significant up-regulation of surface and total TRAIL receptor 2 (DR5) protein level.<br />
Combined treatment of colon cancer with both agents resulted in enhanced caspase<br />
activation, and mitochondrial apoptotic pathway engagement. Our results showed<br />
that LA-12 is a very promising agent to be used in combined cancer therapy with<br />
TRAIL, and suggested the intracellular targets for the therapeutic interventions.<br />
Acknowledgements: This work was supported by grants No. 1QS500040507 IGA AS CR<br />
and 301/07/1557 GACR.<br />
144<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
33.<br />
TRAIL-INDUCED APOPTOSIS CAUSES ACTIVATION OF PRO-SURVIVAL PATHWAYS IN<br />
NON-ADHERENTLY GROWING COLON CANCER CELLS<br />
Lenka Kočí, Martina Hýžďalová, Alena Vaculová,<br />
Jiřina Hofmanová and Alois Kozubík<br />
Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech<br />
Republic, v.v.i., Kralovopolska 135, Brno 61265, Czech republic<br />
Resistance of transformed epithelial cells to the deachment-induced apoptosis<br />
(anoikis) promotes cancer cell invasion and metastasis. We studied the effects of<br />
TNF-related apoptosis inducing ligand (TRAIL) on cytokinetic parameters and adhesive<br />
properties of the cell lines derived from human foetal (FHC cells) and human<br />
adenocarcinoma (HT-29 cells) colon tissues in association with anoikis induction.<br />
We detected the significant decrease of TRAIL-induced apoptosis in the nonadherently<br />
growing HT-29 cells in comparison with the adherent cultivation. Based on<br />
this finding we focused our attention to detail mechanisms of the cell survival under<br />
TRAIL treatment. We confirmed our hypothesis of activation of pro-survival<br />
pathways, actually PI3K/Akt and MAPK/ERK, which are connected with focal adhesion<br />
kinase (FAK) phosphorylation. Increased phosphorylation of Akt and ERK kinases and<br />
also enhanced expression of FLIP and Mcl-1 proteins as downstream molecules of<br />
PI3K/Akt pathway were observed during non-adherent cultivation.<br />
Taken together, our data suggested that the decrease of the TRAIL-mediated<br />
apoptosis of colon epithelial cells induced by non-adherent type of cultivation is<br />
connected with activation of pro-survival pathways.<br />
Acknowledgements: This work was supported by grants 305/09/1526 GACR,<br />
303/09/H048 GACR, and 524/07/1178 GACR.<br />
145<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
34.<br />
PREPARATION AND FUNCTIONAL ANALYSIS OF PHOSPHORYLATION MUTANT<br />
FORMS OF THE TRANSCRIPTION FACTOR NFI<br />
Gabriel Kollárovič 1 , Miroslava Kretová 1 , Lucia Lichá 1 ,<br />
Peter Baráth 1 and Katarína Luciaková 1<br />
1 Laboratory of Molecular Biology, Cancer Research Institute, SAS, Bratislava<br />
Nuclear factors I (NFI) are transcription factors and has been implicated in many<br />
cellular processes such as embryonic development, cell differentiation and<br />
transformation processes. In vertebrates, NFI proteins are encoded by four isogenes<br />
and exist in multiple splice variant and can act both as activators and repressors.<br />
Recent data from the functional proteomic studies identify NFIs as a substrate for<br />
ATM/ATR kinase in response to DNA damage. Such genomic instability results in<br />
activation of a protein cascade(s) that leads to the cell cycle arrest. Regarding our<br />
previous data on the role of NFI in growth-regulated gene expression, a logical<br />
question arises whether NFI proteins indeed play a role in the ATM/ATR signaling<br />
pathway.<br />
For this purpose specific mutations in the ATM/ATR phosphorylation sites in the NFI<br />
was prepared and its effects on the expression of target genes was studied. The<br />
serine to alanine point mutations were prepared by PCR. Simultaneously restriction<br />
site was introduced for restriction enzyme SacI for rapid screening of clones bearing<br />
the mutated forms of NFI.<br />
Acknowledgements: This work was supported by VEGA grant No. 2/0074/08<br />
146<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
35.<br />
GENERATION AND CHARACTERIZATION MONOCLONAL ANTIBODIES AGAINST<br />
ENDOSIALIN, THE POTENTIAL MARKER OF TUMOR ANGIOGENESIS<br />
Soňa Kontseková, Anna Repič, Monika Baráthová,<br />
Katarína Polčicová and Jaromír Pastorek<br />
Institute of Virology, SAS, Dúbravská cesta 9, 84505 Bratislava<br />
Endosialin, also known as TEM1, or CD248, was first discovered with the monoclonal<br />
antibody (mAb) Fb5 as an experimental approach to identify new targets for anticancer<br />
strategies. It was described as a transmembrane glycoprotein selectively<br />
expressed on tumor endothelium, but its expression is barely detectable in normal<br />
human tissue. Recent experiments have challenged the endothelial expression of<br />
endosialin and suggested an expression by activated fibroblasts and pericytes.<br />
Subsequent studies have also confirmed that endosialin is upregulated in blood<br />
vessels in wide range of tumors and its expression is restricted to capillaries, which<br />
means that endosialin is probably involved in vascular reorganisation. It has been<br />
functionally implicated in angiogenesis, a process characterized by vascular branching<br />
and sprouting, which is necessary for tumor expansion and progression. In this work,<br />
we generated monoclonal antibodies against endosialin, characterized their isotypes<br />
and binding epitopes. We also analysed the activity of antibodies in a set of<br />
imunological assays and characterized the most stabile antibody VIII-16 with<br />
potential usage in next studies of endosialin function.<br />
Acknowledgements: This work was supported by VEGA 2/0210/09 and TRANSMED.<br />
147<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
36.<br />
THE ROLE OF NFI IN P21 GENE EXPRESSION<br />
Miroslava Kretová, Ľudmila Šabová, Ľudmila Antalová and Katarína Luciaková<br />
Laboratory of Molecular Biology, Cancer Research Institute SAS Bratislava<br />
p21 protein was originally identified as an inhibitor of cell cycle dependent kinases<br />
(CDK). p21 plays an important role in cell cycle arrest at the G1/ S checkpoint in<br />
response to DNA damage. p21 also modulates various processes such as cell growth,<br />
differentiation and apoptosis. p21 gene expression is mainly regulated at<br />
transcription level. The key regulator of p21 gene expression is tumor suppressor<br />
protein p53. However, expression of p21 may be either p53-dependent or p53-<br />
independent, which results in a network of control mechanisms influencing the cell<br />
cycle.<br />
Transcription factor NFI (nuclear factor-I) is a repressor of p21 transription. NFI and<br />
Sp1-binding sites, which play an important role in p21 gene expression, were<br />
identified in the p21 proximal promoter. The transforming growth factor β (TGF-β)<br />
activates p21 gene in G1 phase of the cell cycle.<br />
Molecular mechanism of the stress-induced expression of p21 is not well understood.<br />
TGF-β may activate signaling pathways affecting the target gene expression either<br />
directly by phosphorylation of Smad proteins or indirectly by activation of the MAPK<br />
signaling pathway. The aim of our work is to identify the signaling pathway(s) playing<br />
a role in the regulation of p21 gene in serum starved cells and in TGF-β-treated cells.<br />
Transfection of recombinant constructs bearing mutations and deletion of NFI<br />
binding sites in the p21 promoter was used to measure the level of p21 expression in<br />
HaCaT, HCT-116 and JEG-3 cells during cellular stress (serum starvation, TGF-β).<br />
Acknowledgements: This work was supported by VEGA grant No. 2/0074/08.<br />
148<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
37.<br />
STRICT CONTROL OF AURICIN PRODUCTION IN STREPTOMYCES AUREOFACIENS CCM<br />
3239 INVOLVES A FEEDBACK MECHANISM<br />
Peter Kutaš, Ľubomíra Fecková, Alena Reháková,<br />
Renáta Nováková and Ján Kormanec<br />
Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 845<br />
51 Bratislava, Slovakia<br />
In Streptomyces aureofaciens CCM 3239, we have previously identified a type II<br />
polyketide synthase gene cluster, aur1, responsible for production of the angucyclinelike<br />
antibiotic auricin. We found out, that auricin was produced at very specific stage<br />
and afterwards it was degraded to a non-active metabolite(s). Two regulatory genes,<br />
aur1P and aur1R, whose deduced products share significant similarity with two<br />
different types of bacterial regulatory proteins, were investigated in order to reveal<br />
tight regulation in S. aureofaciens CCM 3239. Expression of the auricin biosynthetic<br />
genes is under control of the pathway-specific positive regulator Aur1P that belongs<br />
to the family of response regulators of bacterial two-component signal transduction<br />
systems. Transcriptional analysis revealed that the activity of the identified aur1Ap<br />
promoter is dramatically decreased in later stages of stationary phase and the<br />
promoter is directly activated by the auricin pathway specific activator Aur1P at the<br />
entry to stationary phase. Aur1P was shown to bind specifically the aur1Ap promoter<br />
and this binding was abolished by the presence of auricin and/or its intermediates. In<br />
addition, the aur1Pp promoter was negatively regulated by the TetR family Aur1R<br />
repressor, and its binding to the promoter was also obolished by the presence of an<br />
auricin intermediate(s). The results indicate specific feed-back mechanism of auricin<br />
production in S. aureofaciens CCM 3239.<br />
Acknowledgements: This work was supported by the Slovak Research and<br />
Development Agency under the contract No. APVV-0017-07.<br />
149<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
38.<br />
ALTERED CALCIUM SIGNALING IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF<br />
CHRONIC KIDNEY DISEASE PATIENTS<br />
Ingrid Lajdová 1 , Viera Spustová 1 , Adrian Oksa 1 and Dušan Chorvát Jr. 2<br />
1 Slovak Medical University, Department of Clinical and Experimental<br />
Pharmacotherapy , 2 International Laser Centre, Department of Biophotonics,<br />
Bratislava<br />
A rise in the intracellular free calcium concentration ([Ca 2+ ] i ) is a key signal in the<br />
initialization of wide range of cellular events. The elevation of [Ca 2+ ] i have been<br />
found in association with chronic kidney disease (CKD). The aim of this study was to<br />
investigate the [Ca 2+ ] i , intracellular calcium reserves, capacitative calcium entry and<br />
function of purinergic P2X 7 receptors in peripheral blood mononuclear cells (PBMCs)<br />
of early-stage CKD patients.<br />
The study involved 22 healthy volunteers and 22 patients in CKD stage 2-3. The<br />
peripheral blood mononuclear cells (PBMCs) were separated by the Ficoll gradient<br />
centrifugation, and free intracellular calcium was measured using the Fluo-3 AM<br />
fluorimetry. The P2X 7 pore function was evaluated by using a fluorescent dye<br />
ethidium bromide.<br />
Our results demonstrate that 1) [Ca 2+ ] i , intracellular calcium stores and the<br />
capacitative calcium entry were significantly increased already in early stages of CKD;<br />
2) purinergic P2X 7 receptors participate in intracellular calcium homeostasis<br />
regulation in CKD.<br />
Acknowledgements: Supported by grants No. APVT-21-033002 and VG SZU 19-90-07<br />
150<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
39.<br />
EXPRESSION OF MICROPHTHALMIA-ASSOCIATED TRANSCRIPTION FACTOR<br />
CRITICALLY REQUIRES ACTIVE SWI/SNF CHROMATIN REMODELING COMPLEX<br />
Ľubica Ondrušová and Jiří Vachtenheim<br />
Laboratories of Molecular and Cell Biology, University Hospital Bulovka, Prague 8,<br />
Czech Republic<br />
Transcription factor MITF (microphthalmia-associated transcription factor) plays a<br />
central role in the expression of melanocyte-specific genes, lineage determination,<br />
and survival of embryonic, adult and malignant melanocytes. Here we show that the<br />
expression of MITF requires the presence of active SWI/SNF chromatin remodeling<br />
complex, which contains either Brg1 or Brm as a catalytic subunit. The SWI/SNF<br />
components were expressed in melanoma cell lines and only one cell line with Brg1<br />
loss was found (SK-MEL-5 cells). In several Brm/Brg1-expressing melanoma cell lines,<br />
knockdown of Brg1 severely compromised MITF expression with a concomitant<br />
downregulation of MITF targets and decreased cell proliferation. Although Brm was<br />
able to substitute for Brg1, sequential knockdown of both Brm and Brg1 in 501mel<br />
resulted in loss of proliferation and viability. Binding of Brg1 or Brm to the promoter<br />
of MITF was confirmed by chromatin immunoprecipitation. Furthermore, microarray<br />
analysis revealed that osteopontin, IGF1 and survivin, the proteins known to be<br />
associated with tumor progression, were reduced in Brg1-depleted 501mel cells,<br />
suggesting that loss of SWI/SNF function negatively affects survival pathways besides<br />
the MITF cascade. Our results demonstrate an essential role of SWI/SNF for MITF<br />
expression in melanoma cells and suggest that a tissue-aimed inactivation of the<br />
SWI/SNF complex might become an effective approach in the therapy of melanoma.<br />
151<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
40.<br />
MCL-1 AS A REGULATOR OF APOPTOSIS IN CML CELL LINE AND PERIPHERAL BLOOD<br />
MONONUCLEAR CELLS<br />
Barbora Brodská, Petra Otevřelová and Aleš Holoubek<br />
Institute of Hematology and Blood Transfusion, U Nemocnice 1, 128 20 Prague 2,<br />
Czech Republic<br />
Mcl-1 is a Bcl-2 family protein which can act as an apical molecule in apoptosis<br />
control, promoting cell survival by interfering at an early stage in a cascade of events<br />
leading to release of cytochrome c from mitochondria. Mcl-1 has a short half life and<br />
is a highly regulated protein. We investigated changes in the expression of this<br />
protein during combined treatment with demethylating agent decitabine (DAC) and<br />
histone deacetylase inhibitor SAHA (Vorinostat). These drugs represent epigenetic<br />
forces regulating gene and protein expression in cells, affecting cell cycle and cell<br />
death. In our experiments, DAC alone causes substantial increase of p21WAF1<br />
expression, higher levels of proteins p53 and Puma were also detected, but the<br />
viability of the cells, as measured by MTT test, decreased only minimally, and we did<br />
not detect any remarkable apoptotic effect. While in CML-T1 cells we observed a<br />
slight decrease in Mcl-1 expression and PARP fragmentation, none of these effects<br />
have been found in lymphocytes. Addition of SAHA simultaneously with DAC<br />
supressed DAC-induced p21WAF1 up-regulation and substantial down-regulation of<br />
Mcl-1 protein expression together with mitochondrial membrane (MM)<br />
depolarization and PARP fragmentation was also detected. The extent of MM<br />
depolarization and the cell viability decrease observed in both CML-T1 cells and<br />
PBMC after DAC+SAHA treatment implies that this combination has a synergistic<br />
effect on apoptosis and Mcl-1 seems to play a crucial role in this process.<br />
Acknowledgement: This work was supported by the grants IGA NS 9637-4 and IGA<br />
MZOUHKT2005 from the MHCR.<br />
152<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
41.<br />
NOVEL GUANIDINE DERIVATIVES AND EVALUATION OF THEIR DNA BINDING<br />
AFFINITIES AND POSSIBLE ANTICANCER EFFECT<br />
Jana Plšíková 1 , Ján Kovaľ 2 , Jaromír Mikeš 2 , Mária Kožurková 1 , Peter Fedoročko 2 ,<br />
Ladislav Janovec 3 , Ján Ungvarský 3 and Danica Sabolová 1<br />
1 Department of Biochemistry UPJŠ Košice,<br />
2 Department of Biology and Ecology UPJŠ Košice,<br />
3 Department of Organic chemistry UPJŠ Košice<br />
Acridine derivatives represent a well known class of multi-faceted anticancer agents<br />
that generally interfere with DNA and inhibit important regulatory enzymes such as<br />
topoisomerases. In order to identify novel anticancer drugs, we evaluated the<br />
mechanism of action of a novel series of 1`,1``-(acridin–3,6–diyl)–3`,3``dialkyldiguanidines<br />
(etyl to hexyl). The affinity of studied compounds with DNA was<br />
investigated by a variety of techniques including UV-VIS, fluorescence, CD<br />
spectroscopy and electrophoresis. Binding constants for the DNA-drug complexes<br />
were determined from spectrofluorimetric titrations, (K = 1,25 – 5,26 ×10 5 M -1 ).<br />
Moreover, these compounds were capable of inhibiting topoisomerase I. Biological<br />
activities of studied derivatives were determined using flow cytometric methods after<br />
24, 48 and 72 h co-incubation with leukemic cancer cell line HL-60. The most<br />
profound effect on different cell parameters was observed after 72 h incubation with<br />
the pentyl and hexyl derivate. We detected a significant increase in caspase-3<br />
activity, increase of percentage of cells with dissipated mitochondrial membrane<br />
potential, cell accumulation in G1 phase of cell cycle and enhanced DNA<br />
fragmentation.<br />
In summary, the studied derivatives are capable of interacting with ctDNA thought<br />
intercalation and inhibiting topoisomerase I. They also display a significant anticancer<br />
effect in HL 60 cells.<br />
Acknowledgement: This work was supported by VEGA 1/0053/08, 1/0097/10 and by<br />
the Slovak Research and Development Agency under contract no. VVCE-0001-07.<br />
153<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
42.<br />
HISTONE ACETYLATION OF LEUKEMIC JURL-MK1 CELLS WITHIN SAHA TREATMENT<br />
Michaela Pluskalová, Dana Grebeňová, Zbyněk Hrkal and Kateřina Kuželová<br />
Ústav hematologie a krevní transfúze, Praha, Česká republika<br />
The basic unit of chromatin, the nucleosome, is formed by about 146 bp of DNA<br />
wrapped around the histone octamer. The histones play an important role in the<br />
arrangement of nucleosomes. N-terminal tails of histones undergo multiple reversible<br />
posttranslational modifications, which have been proposed to form the so-called<br />
´histone code´. This code (epigenetic information) regulates the binding of<br />
transcription complexes and determines gene transcription rate. The best understood<br />
histone modification is the lysine acetylation which occurs as a result of mutually<br />
opposed activities of histone acetyltransferases (HAT) and histone deacetylases<br />
(HDAC).<br />
Suberoylanilide hydroxamic acid (SAHA) is the first member of the group of HDAC<br />
inhibitors which is used for treating patients with cutaneous T-cell lymphoma. While<br />
it is also evaluated in clinical trials for the treatment of other oncological and<br />
hematological diseases, the mechanism of its action is largely unexplained. We found<br />
that the effect of SAHA on the leukemic cell line JURL-MK1 are strongly dosedependent:<br />
an increase in the cellular adhesivity to fibronectin and cell cycle arrest<br />
was observed for subtoxic SAHA concentration while the apoptosis was triggered at<br />
higher doses. Using specific antibodies against the individual acetylation sites, we<br />
identified dose-dependent changes in the acetylation of histones H2A, H2B and H4<br />
induced by SAHA treatment. We also detected changes in the cellular localization of<br />
these histones (transfer from nuclear to cytoplasmic fraction) due to SAHA.<br />
Acknowledgement: This work was supported by grant 301/09/1026 from the Grant<br />
Agency of the Czech Republic.<br />
154<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
43.<br />
REGULATION OF EXPRESSION OF PLAKOGLOBIN, A KEY DESMOSOMAL<br />
CONSTITUENT, BY ARYL HYDROCARBON RECEPTOR AND CAMP SIGNALING<br />
Jiřina Procházková 1,2 , Lenka Umannová 1,2 , Alois Kozubík 1 ,<br />
Miroslav Machala 2 and Jan Vondráček 1,2<br />
1 Department of Cytokinetics, Insitute of Biophysics AS CR, Brno, 2 Department of<br />
toxicology, pharmacology and immunology, Veterinary Research Insitute, Brno<br />
Aryl hydrocarbon receptor (AhR) has been originally described as a transcription<br />
factor mediating the toxic effects of dioxin-like compounds, but its signaling can be<br />
triggered also by endogenous agents, such as second messenger cyclic adenosine<br />
monophosphate (cAMP). Using an in vitro model of liver progenitor cells,<br />
participating both in liver regeneration and in hepatocarcinogenesis, we first<br />
investigated the potency of cAMP and another protein kinase A activator, forskolin,<br />
to activate AhR signaling pathway, as analyzed at the level of: i) AhR nuclear uptake;<br />
ii) AhR protein degradation; and iii) induction of model target gene Cyp1a1. We<br />
further analyzed the potency of cAMP and forskolin, alone or in combination with<br />
dioxin to modulate expression of Jup gene encoding plakoglobin protein, which plays<br />
a key role in establishment of intercellular junctions. Using WB-F344 cells, we found<br />
that cAMP and forskolin may transiently activate AhR signaling. As both compounds<br />
were able to modulate TCDD-induced changes in Cyp1a1 and Jup expression, our<br />
results support the existence of a possible link between cAMP-activated signaling and<br />
AhR in regulation of both structural and signaling functions of desmosomal and<br />
adherent junctions.<br />
Acknowledgement: This work was funded by the Czech Science Foundation, grants<br />
No. 524/09/1337 and 305/09/1526].<br />
155<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
44.<br />
CHARACTERIZATION OF SARP REGULATORY GENE INVOLVED IN POSITIVE<br />
REGULATION OF AN ANGUCYCLINE-LIKE POLYKETIDE ANTIBIOTICS AURICIN GENE<br />
CLUSTER IN STREPTOMYCES AUREOFACIENS CCM 3239<br />
Alena Reháková, Renáta Nováková , Ľubomíra Fecková,<br />
Peter Kutaš and Ján Kormanec<br />
Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 845<br />
51 Bratislava, Slovakia<br />
Streptomyces aureofaciens CCM 3239 is gram-positive soil bacterium undergoing a<br />
complex developmental life cycle. During aerial mycelium growth S. aureofaciens<br />
CCM 3239 produces their secondary metabolites including antibiotic auricin. The<br />
auricin gene cluster was named aur1. Synthesis of auricin is strictly regulated due to<br />
toxicity of auricin and its regulation is under the complex control of several regulatory<br />
genes. The product of one of these regulatory genes (sa9) belongs to the family of<br />
Streptomyces antibiotic regulatory proteins (SARPs). We characterized this gene and<br />
its impact on auricin production. By using the S1-nuclease mapping we identified a<br />
single promoter, sa9p, which expressed in a narrow stage during growth in rich liquid<br />
Bennet medium. The sa9 gene was disrupted by a homologous recombination in S.<br />
aureofaciens CCM3239. The resulting mutant strain had dramatically decreased<br />
auricin production. In addition we confirmed a direct effect of the aur1P regulatory<br />
gene upon sa9 expression. The auricin pathway-specific activator Aur1P directly<br />
interacted with the sa9p promoter, indicating a cascade regulation of auricin<br />
production.<br />
Acknowledgement: This work was supported by the Slovak Research and<br />
Development Agency under the contract No. APVV-0017-07.<br />
156<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
45.<br />
THE COMLEX NETWORK REGULATORY CIRCUITS IN THE REGULATION OF SIGMA<br />
FACTORS INVOLVED IN DIFFERENTIATION AND STRESS RESPONSE IN<br />
STREPTOMYCES COELICOLOR A3(2)<br />
Bronislava Řežuchová, Beatrica Ševčíková, Dagmar Homerová and Ján Kormanec<br />
Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21,<br />
84551 Bratislava, Slovakia<br />
Genome of the gram-positive soil bacterium Streptomyces coelicolor is a twice as<br />
large as the E. coli genome and contains 7 825 genes including 66 genes encoding<br />
sigma factors of RNA polymerase that is the largest number among the so far<br />
examined bacteria. In B. subtilis a general stress response is under the control of a<br />
single sigma factor, SigB. Its gene is located in the operon together with rsbW and<br />
rsbV genes. The product of rsbW is an anti sigma factor of SigB, which serves as serine<br />
kinase that binds and phosphorylates its negative regulator so-called anti-anti sigma<br />
factor RsbV. However, genome sequence analysis in S. coelicolor has shown nine sigB<br />
homologues, as well as putative 48 anti-sigma factors (RsbW homologues) and 18<br />
antagonists of anti-sigma factors (RsbV homologues). Bacterial two hybrid system<br />
was used to investigate and characterize interaction between nine known<br />
homologues SigB and 15 suggested anti-sigma factors, and between these anti-sigma<br />
factors and 9 selected anti-anti sigma factors. This method allows detect even<br />
relatively weak protein-protein interaction. The efficiency of interactions was<br />
quantified by measuring β-galactosidase activities in liquid cultures. The interactions<br />
were confirmed by native PAGE. The results revealed very complex way of regulation,<br />
where several sigma factors interact with different anti-sigma factors that<br />
additionally interact with several subclasses of anti-sigma factor antagonists.<br />
Acknowledgement: This work was supported by the VEGA grant 2/0104/09 from Slovak<br />
Academy of Sciences.<br />
157<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
VI. GLYCOMICS<br />
158<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
46.<br />
DIFFERENCES IN INTERACTION OF LECTINS SPECIFICALLY RECOGNIZING SIALIC ACID<br />
RESIDUES WITH SURFACE OF P-GP NEGATIVE OR POSITIVE L1210 CELLS<br />
Tatiana Kurucová, Helena Kavcová, Kristína Rogozanová, Lucia Messingerova,<br />
Danica Mislovičová 1 , Albert Breier and Zdena Sulová<br />
Institute of Molecular Physiology and Genetics SAS, Bratislava, Slovakia<br />
1 Institute of Chemistry SAS, Bratislava, Slovakia<br />
Multidrug resistance (MDR) of mouse leukemic cell line L1210/VCR (R) obtained by<br />
adaptation of L1210 cells (S) to vincristine (VCR) is accompanied by overexpression of<br />
P-glycoprotein (P-gp). Selection with VCR that confers overexpression of P-gp is also<br />
inducing various kinds of metabolic alteration. We described recently several<br />
differences in cell surface saccharides between R and S cells.<br />
Aim of this study was to resolve in exist differences in interaction of Triticum vulgaris<br />
lectin (WGA), Maackia amurensis lectin (MAA), Sambucus nigra lectin (SNA) with cell<br />
surface of S, R cells and L1210 cells that express P-gp due to transfection of cells with<br />
plasmid containing human gene encoding this protein (T cells). While MAA was found<br />
to be nontoxic for S, R and T cells and SNA is exerting small cell damage effect, WGA<br />
induced concentration depended cytotoxic effect. Agglutination of cells by this lectin<br />
is more massive to cells expressed P-gp. WGA lectin interact more potently with P-gp<br />
positive cells R and T as with P-gp negative S cells. Spectrum of cell membrane<br />
glycoprotein targets of lectins were assessed by lectin blot methods. Our data<br />
indicated that application of lectins enable to study the differences cell surface<br />
saccharides.<br />
Acknowledgements: This work was supported by: APVV-0084-07, VVCE-0064-07,<br />
VEGA-2/0123/10, VEGA-2/0155/09.<br />
159<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
47.<br />
THE PRESENCE OF P-GLYCOPROTEIN IN L1210 CELLS DIRECTLY INDUCES DOWN-<br />
REGULATION OF CELL SURFACE SACCHARIDE-TARGETS OF CONCANAVALIN A<br />
Zdena Sulová 1 , Peter Ditte 2 , Tatiana Kurucová 1 , Eva Poláková 1 , Kristína<br />
Rogozanová 1 Lucia Škvarková 1 , Ján Sedlák 3 , Jaromír Pastorek 2 and Albert Breier 1<br />
1 Institute of Molecular Physiology and Genetics, 2 Institute of Virology, 3 Istitute of<br />
Experimental Oncology, SAS, Bratislava, Slovak Republic<br />
Overexpression of P-glycoprotein (P-gp), a plasma membrane drug transporter (an<br />
ABCB1 member of the ABC transporter family), is the most prevalent cause of<br />
multidrug resistance in cancer tissues. Lectin Concanavalin A (ConA) induces massive<br />
cell death of L1210 leukemia cells (S). Cell sublines of L1210 in which P-gp<br />
overexpression was induced by selection with vincristine (R) or by stable transfection<br />
with a plasmid encoding full-length human Pglycoprotein (T) were less sensitive to<br />
ConA. Both P-glycoprotein-positive cell lines exhibited typical P-glycoproteinmediated<br />
multidrug resistance. Resistance of R and T cells to ConA was associated<br />
with lower binding of ConA as compared to S cells when analyzed by the following<br />
methods: i) SDS PAGE and electrobloting of proteins in the crude membrane fraction<br />
followed by detection with biotinylated ConA and avidin-peroxidase; ii) fluorescent<br />
cytometry or confocal microscopy of the intact cells with surfaces labeled by FITC-<br />
ConA. These data indicated that the presence of P-glycoprotein in L1210 cells<br />
independently on mode of its expression induced down-regulation of cell surface<br />
saccharide-targets of ConA. Therefore, this feature may be considered as a secondary<br />
cellular response on P-glycoprotein expression.<br />
Acknowledgements: This work was supported by: APVV-0084-07, VVCE-0064-07,<br />
VEGA-2/0123/10, VEGA-2/0155/09.<br />
160<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
VII. MEMBRANE BIOCHEMISTRY AND BIOENERGETICS<br />
161<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
48.<br />
EPITOP OF IVA-520 MONOCLONAL ANTIBODY ON THE<br />
BOVINE SPERM CD46 MOLECULE<br />
Jana Antalíková, Jana Jankovičová, Katarína Michalková,<br />
Michal Simon and Ľubica Horovská<br />
Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Ivanka pri<br />
Dunaji, Slovak Republic, Centre of Excellence of Slovak Research and Development<br />
Agency „Biomembranes 2008“.<br />
Membrane cofactor protein (MCP/CD46) serves as a cofactor for the factor I-<br />
mediated cleavage of C3b and C4b complement components deposited on self<br />
tissues and probably plays also a role in reproduction. In Human and also New World<br />
monkeys the unique pattern of sperm CD46, different from other cells and tissues<br />
were detected. The bovine sperm isoform remains unclear. Using the monoclonal<br />
antibody (mAb) IVA-520 against bovine CD46 (obtained after intrasplenic<br />
immunisation of BALB/c mice with intact bull spermatozoa) the expression of CD46<br />
on bovine spermatozoa has been detected. By SDS-PAGE of detergent solubilised<br />
sperm proteins and immunoblotting with mAb IVA - 520 we found out two bands<br />
with molecular weight of 53 and 43 kDa - different from common blood cells and<br />
tissue isoforms (46-52 lower band, 60-68 upper band), resistant to digestion with N-<br />
glycosidase F and disappearing after neuraminidase treatment (not in other cells and<br />
tissues). The treatment of suspension of whole spermatozoa with neuraminidase<br />
decreased the binding ability of mAb IVA-520 on sperm CD46 molecule. We suppose<br />
the posttranslational modification of the molecule are sperm-specific, the part of<br />
bovine CD46 epitope recognised by mAb IVA - 520 is formed probably by the serine,<br />
threonine and-proline rich region (STP) of MCP molecule and the sialic acid probably<br />
takes part in an epitope conformation of mAb IVA-520.<br />
Acknowledgement: This work was supported by grant VEGA-2/0001/09 and in part by<br />
the grant VVCE-0064-07.<br />
162<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
49.<br />
DIMETHYL-OXALYLGYCINE MODULATES GENE EXPRESION AND PROTEIN LEVELS OF<br />
THE SODIUM CALCIUM EXCHANGER IN HEK 293 CELL LINE<br />
Cagala M. 1 , Lencesova L. 1,2 , Hudecova S. 1 , Csaderova L. 2 , Sirova M. 1 ,<br />
Cholujova D. 3 , Kopacek J. 4 , Pastorekova S. 2,4 and Krizanova O. 1,3<br />
1 IMPG, Center of Excellence for Cardiovascular Research, SAS, Vlarska 5, 833 34<br />
Bratislava, Slovak Republic 2 MMC, SAS, Vlarska 3-7, 831 01 Bratislava, Slovak Republic<br />
3 IEO, SAS, Vlarska 7, 833 91, Bratislava, Slovak Republic 4 IV, Center of Excellence for<br />
Cardiovascular Research, SAS, Dubravska cesta 9, 845 05 Bratislava, Slovak Republic<br />
Up to now a little is known about the effect of hypoxia on the NCX1 expression and<br />
function. Therefore we studied, how dimethyl-oxalylglycine (DMOG), an activator and<br />
stabilizator of the HIF-1α could affect expression of the NCX1 in HEK 293 cell line. We<br />
also tried to determine, whether this activation can result in the development of<br />
apoptosis in HEK 293 cells. We have found that DMOG treatment for 3 hours<br />
significantly increased gene expression and also protein levels of the NCX1. This<br />
increase was accompanied with the decrease in intracellular pH. Wash-out of DMOG<br />
did not result in decrease of NCX1 mRNA and protein to original, control levels,<br />
although pH returned to control values. In the promoter region of the NCX1 we did<br />
not find consensus sequence for HRE, but we have found consensus NF-′B sequence<br />
in this region of the NCX1. Using luciferase reporter assay we observed increase in<br />
NCX1 promoter activity after DMOG treatment, which suggests that NF-′B is involved<br />
in DMOG induced upregulation of the NCX1. Moreover, we also showed that this<br />
upregulation might be involved, at least partially, in the induction of the process of<br />
apoptosis. Nevertheless, further experiments are needed to clarify this issue.<br />
Ackowledgement: This work was supported by grants APVV 51-0397-07, VEGA<br />
2/0082/10 and TRANSMED.<br />
163<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
50.<br />
DEHYDROERGOSTEROL ELUCIDATES STEROL UPTAKE PROCESS IN YEAST S.<br />
CEREVISIAE<br />
Peter Kohút 1 , Martin Valachovič 1,2 , Lucia Hronská 1 and Ivan Hapala 1<br />
1 Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences,<br />
Moyzesova 61, 90028 Ivanka pri Dunaji, Slovakia<br />
2<br />
Medical University Vienna, Christian Doppler Laboratory for Infection Biology, Max F.<br />
Perutz Laboratories, Vienna, Austria<br />
Yeast Saccharomyces cerevisiae is a facultative anaerobic organism - it can grow<br />
either in the presence or absence of oxygen. However, during anaerobic growth it is<br />
unable to synthesize unsaturated fatty acids and ergosterol, which have to be<br />
supplied externally. Uptake of external sterol in yeast consists of three steps: 1)<br />
passage through the cell wall, 2) entry into plasma membrane and 3) integration into<br />
metabolism, but molecular mechanisms of these individual steps are largely<br />
unknown. Sterol uptake can be efficiently studied with fluorescent sterol probes.<br />
Dehydroergosterol (DHE) is a naturally fluorescent sterol and structural similarity<br />
between DHE and ergosterol – native yeast sterol – ensures that the data acquired<br />
using DHE as a probe in the study of sterol uptake in yeast reflect metabolic<br />
processes taking place under physiological conditions. In our experiments we used<br />
DHE to analyze molecular details of sterol uptake in the yeast S. cerevisiae,<br />
particularly to reveal the role of Aus1p and Pdr11p proteins in this process. These<br />
proteins are members of the ABC transporter family and they were previously<br />
identified in a wide-scale screen as proteins involved in sterol uptake in S. cerevisiae.<br />
We used mutants in Aus1p and Pdr11p putative sterol transporters to separate the<br />
first two steps in sterol uptake process. Fluorescent properties of DHE enabled us to<br />
distinguish between membrane-incorporated and cell wall-associated sterol. Our<br />
results indicate that Aus1p and Pdr11p are required for entry of sterols into the<br />
plasma membrane and not for internalization of sterols as suggested in some studies.<br />
This work was supported by grants APVT-51-029504 and VVCE-0064-07<br />
164<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
51.<br />
MYCOBACTERIAL EPOXIDEHYDROLASE EPHD IS INVOLVED IN<br />
FATTY ACID METABOLISM<br />
Jan Madacki 1 , Katarína Mikušová 1 , Mary Jackson 2 and Jana Korduláková 1<br />
1 Department of Biochemistry, Faculty of Natural Sciences, Comenius University,<br />
Bratislava, Slovakia; 2 Department of Microbiology, Immunology, and Pathology,<br />
Colorado State University, Fort Collins, CO, USA<br />
The cell envelope of mycobacteria is a crucial determinant of virulence and drug<br />
resistance in mycobacteria. The major components of the mycobacterial envelope<br />
are mycolic acids – very long branched α-alkyl-, β-hydroxy- fatty acids, which play an<br />
important role in the integrity of the mycobacterial cell, as well as in pathogenicity of<br />
mycobacteria. Experimental observations, accumulated during several decades<br />
allowed to draw a general scheme for the mycolic acid biogenesis. However, many<br />
key questions regarding the molecular basis of the biosynthetic routes leading to the<br />
mature mycolates still have to be answered.<br />
We investigated the function of the protein EphD in the non pathogenic strain of<br />
Mycobacterium smegmatis. The recombinant protein was produced in E. coli and<br />
using the generic substrate we showed that EphD displays the epoxide hydrolase<br />
activity in vitro. Phenotypic analysis of M. smegmatis carrying the disrupted copy of<br />
ephD orthologue revealed that this protein is involved in the fatty acid metabolism,<br />
particularly in the biogenesis of mycolic acids.<br />
Acknowledgement: This work was supported by Slovak Grant Agency VEGA (grant<br />
No. 1/0223/08)<br />
165<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
52.<br />
ISOFORMS OF AMP-ACTIVATED PROTEIN KINASE SUBUNITS IN<br />
LYMPHOCYTES AND OBESITY<br />
Boris Lakatoš, Lucia Bialešová and Eva Harnišová<br />
Institute of Biochemistry, Nutrition and Health Protection, Department of<br />
Biochemistry and Microbiology, Faculty of Chemical and Food Technology, Slovak<br />
University of Technology, Radlinského 9, 812 37 - Bratislava, Slovakia<br />
Obesity becomes to be a real problem in developed countries and it seems that its<br />
prevalence increases. One of consequences of obesity is the increasing incidence of<br />
metabolic syndrome - a state of metabolic dysregulation characterized besides<br />
obesity also with resistance to insulin, changes in blood lipids level, early<br />
atherosclerosis, changes in blood tension, which can result to the appearance of<br />
civilization diseases. Although the obesity could be caused by different factors,<br />
studies in last decades pointed out on fundamental mechanisms leading to the<br />
defects in lipid metabolism, which are regulating energy fluxes in animal metabolism.<br />
It is known that 5´-AMP-activated protein kinase (AMPK) a sensor of metabolic status<br />
plays the key regulatory role. Structurally is AMPK heterotrimeric complex consisting<br />
of catalytic α subunit and regulatory β and γ subunits existing in several isoforms (α 1 ,<br />
α 2 , β 1 , β 2 , γ 1 , γ 2 , γ 3 ). AMPK is widely distributed in all body tissues. In this work we<br />
followed expression of AMPK subunits isoforms in human lymphocytes isolated from<br />
blood of undergraduate students of FCHFT SUT. Simultaneously we measured basic<br />
biochemical parameters of lipid, glucose and energy metabolism. The obtained values<br />
were used to create correlation dependence with body mass index (BMI) and physical<br />
activity.<br />
We found expression of genes for α 2 , β 2 and γ 3 subunits in all blood samples<br />
regardless of gender, BMI or physical activity of probands. Sequential analysis of<br />
obtained PCR products for α 2 subunit of AMPK did not show any polymorphisms.<br />
Biochemical parameters showed rather weak, if any, correlation with BMI.<br />
Acknowledgements: This work was supported by the grant VEGA, nr. 1/0589/08.<br />
166<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
53.<br />
DISTRIBUTION AND BIOCHEMICAL CHARACTERIZATION OF CD52-LIKE MOLECULE IN<br />
BULL EPIDIDYMIS<br />
Katarína Michalková, Michal Simon, Jana Antalíková and Ľubica Horovská<br />
Institute of Animal Biochemistry and Genetics SAS Ivanka pri Dunaji<br />
The aim of this study was to analyze the distribution and biochemical properties of<br />
the antigen in bull genital tract using monoclonal antibody IVA-543 produced in IABG<br />
SAS. CD52 is a GPI linked protein with a molecular weight of about 25 to 29 kDa, it is<br />
expressed on all lymphocytes, and in male genital tract. Except humans, CD52 has<br />
been described in mice, rats, chimps and dogs. Characteristic common to all species is<br />
very similar nature of expression in cells of the epididymal epithelium, the protein in<br />
the genital tract is produced post-testicular and constitutes the main surface<br />
glycopeptide of the sperm from the corpus and cauda epididymis. Our results<br />
revealed that CD52-like molecule in the reproduction system of bull is produced by<br />
epididymal epithelial cells and secreted into the lumen. Immunohistochemical<br />
analysis confirmed that the antigen is synthesized in the epididymis downstream, in<br />
the largest quantities it can be detected in the cauda epididymal tissue. The similar<br />
results we obtained by western blot analysis of the cauda epididymal fluid. FACS<br />
analysis and the indirect immunofluorescence of sperm from of epididymis confirmed<br />
these results, in the proximal part of epididymis CD52-like molecule is present on<br />
4.73 % of sperm and in the distal part it is present on up to 99.45 % of the sperm.<br />
Bovine antigen expression in the distal part of the epididymis points out that the bull<br />
sperm acquire CD52-like molecule on their surface during their transit through the<br />
epididymis, especially in the distal part of this organ. Western blot analysis showed<br />
that the molecular weight of this bull epididymal antigen derived from tissue, fluid or<br />
from sperm is ranging from 22 to 25 kDa.<br />
Acknowledgments: This work was supported by grants VEGA-2/6023/27 and APVV-<br />
VVCE-0064-07.<br />
167<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
54.<br />
IDEBENONE ACTIVATION OF GLYCEROL-3-PHOSPHATE OXIDATION IN LIVER<br />
MITOCHONDRIA FROM CONTROL AND HYPERTHYROID RATS<br />
Hana Rauchová 1,2 , Martina Vokurková 1,2 and Tomáš Soukup 1<br />
1 Institute of Physiology, Academy of Sciences of the Czech Republic,<br />
2 Centre for Cardiovascular Research, Prague, Czech Republic<br />
A synthetically prepared analog of coenzyme Q (CoQ) with lower hydrophobicity,<br />
idebenone (IDE; hydroxydecyl-ubiquinone), was found to be very effective in animal<br />
experiments and human replacement therapy when the synthesis of CoQ was<br />
decreased. In our previous studies, we found that mitochondrial FAD-linked glycerol-<br />
3-phosphate dehydrogenase (GPDH; EC 1.1.99.5) from brown adipose tissue is<br />
activated by IDE. However, in most mammalian tissues expression of GPDH is highly<br />
depressed and enzyme activity is very low. Thyroid hormones are known to have a<br />
marked influence on GPDH activity; they especially induce GPDH activity in liver. The<br />
aim of our study was to test to what extent IDE can activate glycerol phosphate (GP)<br />
oxidation measured as oxygen uptake and GP cytochrome c oxidoreductase activity in<br />
mitochondria from control and hyperthyroid rat liver. We found the significant<br />
increase of GP-dependent oxygen uptake as well as the activation of enzyme activity.<br />
Our results indicate that IDE may be used for the activation of the GP shuttle<br />
catalyzing the interconversion between dihydroxyaceton phosphate and GP (formed<br />
by GPDH together with its cytosolic NADH-dependent counterpart) through which<br />
NADH from cytosol can be oxidized by the mitochondrial respiratory chain to<br />
contribute to the maintenance of the high rate of glycolysis. It might be important in<br />
the case when mitochondrial Complex I is impaired and energy production must be<br />
maintained.<br />
Acknowledgement: This work was supported by the GACR (303/09/0570) and<br />
Ministry of Education, Youth and Sport (1M6798582302 and AV0Z 50110509).<br />
168<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
55.<br />
EFFECT OF ATORVASTATIN ON BIOENERGETICS OF THE LIVER MITOCHONDRIA ON A<br />
HIGH-LIPID DIET<br />
Oľga Uličná 1 , Oľga Vančová 1 , Jarmila Kucharská 1 , Peter Božek 2 ,<br />
Iveta Waczulíková 3 and Libuša Šikurová 3<br />
1 Pharmacobiochemical laboratory 3rd Dept of Int Medicine Med Fac UK Bratislava,<br />
2 Dept of Clin Biochem and Hematol St Michal Hospital Bratislava, 3 Dept of Nuclear<br />
Physic and Biophysic Math Phys Inf Fac UK Bratislava<br />
Statins impair hepatocellular cholesterol production by inhibiting the synthesis of<br />
mevalonate. Mevalonate is a precursor not only of cholesterol but also of<br />
ubiquinone, which is an important carrier of the mitochondrial respiratory chain.<br />
The aim of the study was to evaluate the oxidative phosphorylation in liver<br />
mitochondria in rats on a high-lipid diet. We investigated the toxicity of atorvastatin<br />
on the liver mitochondria. Male Wistar rats (b.w. 210-270g) were divided into four<br />
groups: 1. control group on the Larsen´s diet, 2. hypercholesterolemic (HCh) on a<br />
high-lipid diet (Larsen´s diet containing 4% of cholesterol and 10% of a saturated fat)<br />
3. and 4. HCh treated with atorvastatin at the dose of 10 and 80 mg.kg -1 , respectively.<br />
After 8 weeks, total cholesterol (tChol) and triacylglycerols (TAG) were determined in<br />
the plasma and liver tissues. Liver mitochondria were isolated by differential<br />
centrifugation. Parameters of oxidative phosphorylation were measured on an<br />
oxygraph Gilson 5/6H. We found an increased content of TAG and tChol in the plasma<br />
and liver tissues of HCh group. Atorvastatin at the high dose lowered tChol and TAG<br />
in the plasma and liver tissues. Parameters of oxidative phosphorylation were<br />
significantly impaired in the liver mitochondria of HCh rats. The high dose of<br />
atorvastatin worsened bioenergetics of the liver mitochondria of HCh rats, but the<br />
low dose had no effect. Our results suggest that administration of low doses of<br />
atorvastatin does not exert a negative effect on liver under the high-fat dietary<br />
conditions.<br />
Acknowledgements: Supported by the grants VEGA 1/0328/10 and 1/0293/08.<br />
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56.<br />
EFFECT OF PAMAM G4 DENDRIMER ON LIVER MITOCHONDRIA OXIDATIVE<br />
PHOSPHORYLATION AND METABOLIC CONTROL IN EXPERIMENTAL DIABETES<br />
Oľga Vančová 1 , Oľga Uličná 1 , Katarína Šebeková 2 ,<br />
Magdalena Labieniec 3 and Cezary Watala 3<br />
1 Pharmacobiochemical laboratory 3rd Dept of Int Medicine LFUK Bratislava,<br />
2 Department of Preventive and Clinical Medicine SZU Bratislava, 3 Department of<br />
Haemostasis and Haemostatic Dis Med Univ of Lodz, Poland<br />
Prolonged exposure to hyperglycaemia causes non-enzymatic glycation of proteins<br />
and can lead to production of reactive oxygen species.<br />
Polyamidoamine dendrimer PAMAM G4, a strong nucleophilic molecule with 64 free<br />
primary amino groups at its surface appears as an effective scavenger of excessive<br />
glucose in diabetes.<br />
The aim was to study the ability of PAMAM G4 to lower the concentration of plasma<br />
glucose, to suppress long-term parameters of hyperglycaemia and to improve<br />
oxidative phosphorylation in the liver mitochondria.<br />
Experimental diabetes mellitus was evoked by an injection of streptozotocin in male<br />
Wistar rats. After 7 days half the rats were administered PAMAM 64 i.p. daily. After 8<br />
weeks the concentration of glucose, the end-products of advanced glycation and the<br />
end-products of advanced protein oxidation in plasma and glycated haemoglobin in<br />
blood were determined. Liver tissue was used for mitochondria isolation and<br />
parameters of oxidative phosphorylation were measured using volt-amper method<br />
with a Clark oxygen electrode.<br />
Our results, for the first time in vivo experiment, show that PAMAM G4, regardless of<br />
its known cytotoxicity, significantly reduced hyperglycaemia and all the measured<br />
long-term markers of hyperglycaemia in diabetic rats. This positive effect is not<br />
sufficient to restore the impaired mitochondrial function in experimental diabetes.<br />
Acknowledgement: Supported by grant VEGA 1/0328/10.<br />
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Posters<br />
57.<br />
BIOCHEMICAL AND MOLECULAR ANALYSIS OF NITRATE-RESISTANT MUTANT OF<br />
Methanothermobacter thermautotrophicus<br />
Monika Vidová, Zuzana Nováková and Peter Šmigáň<br />
Institute of Animal Biochemistry and Genetics SAV Bratislava<br />
In spite of many studies over the past decade, the processes of energy conservation<br />
in methanoarchaea have not yet been satisfactorily elucidated. To contribute to the<br />
solution of this complex problem, we started with a systematic genetic approach to<br />
the problem of energy conservation in methanoarchaea. This work presents<br />
a microbiological, biochemical and molecular analysis of a spontaneous mutant of<br />
Methanothermobacter thermautotrophicus resistant to nitrate. Nitrate inhibits the A 1<br />
cytoplasmatic domain of A 1 A 0 -ATP synthase.<br />
Nitrate inhibits methanogenesis in the wild-type cells in the presence of 30 mM<br />
nitrate; however, the nitrate-resistant mutant exhibited two times higher<br />
methanogenesis, even in the presence of 70 mM nitrate. While nitrate profoundly<br />
inhibited ATP synthesis driven by methanogenic electron transport in the wild type,<br />
only a slight inhibition was observed in the mutant strain. These results suggested a<br />
modification in the ATP-synthesizing system of the mutant strain. The sequence of<br />
the complete A 1 A 0 -ATP synthase operon ( MTH952 – MTH961 ) in the wild-type and<br />
mutant strains was determined and compared. Two mutations leading to amino acid<br />
substitutions in two A 1 A 0 -ATP synthase subunits were identified – Ala 337 Val in subunit<br />
A and Ala 292 Ser in subunit B. Moreover, this study revealed the differential expression<br />
of several proteins that may contribute to nitrate resistance. The results imply that<br />
changes of nitrate sensitivities of nitrate-resistant mutant is due to mutational<br />
substitutions in the A 1 A 0 -ATP synthase operon.<br />
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VIII. NEW METHODOLOGIC PROCEDURES<br />
172<br />
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58.<br />
DEVELOPMENT OF A DETECTION TOOL TO FOLLOW THE SPECIFIC ACTIVITY OF<br />
BUTYRYLCHOLINESTERASE IN HUMAN PATIENTS<br />
Katarína Mrvová and Anna Hrabovska<br />
Dpt. of pharmacology and toxicology, Faculty of Pharmacy, Comenius University,<br />
Odbojárov 10, 832 32 Bratislava<br />
Many hypotheses have been proposed in afford to explain a butyrylcholinesterase<br />
(BChE) inter-individual variability in humans, e.g., different expression levels; different<br />
catalytic properties. Yet, it has been impossible to study this topic due to the lack of<br />
an efficient detection tool. However, we have recently generated a selective and<br />
specific antibody against human BChE which allows us to address this problem. The<br />
aim of the project was to develop an ELISA assay for detection of BChE activity and<br />
use this tool to study the inter-individual BChE activity variation in humans.<br />
Human plasma was prepared from the capillary blood of 86 healthy human volunteers<br />
(age 20 – 23; BMI = 17,6-28,4). The Ellman’s assay was used at the conditions<br />
determined in our laboratory (see the abstract of D. Neuschlova). ELISA has been used<br />
to determine specific activity. All samples were tested in triplicates.<br />
In the first step we determined the conditions for the ELISA assay. The lowest<br />
saturating dilution of the primary antibody and the experimental serum dilution were<br />
determined from the saturation curves. In the second step, human sera were tested<br />
for the total BChE activity (Ellman’s assay) and for the specific BChE activity in excess<br />
plasma (ELISA) and compared. Our results suggest that an inter-individual BChE<br />
activity in humans is a caused by both, higher expression level and different catalytic<br />
properties.<br />
Acknowledgement: The project was supported by APVV grant SK-CZ-0028-09.<br />
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59.<br />
OPTIMALIZATION OF ELLMAN’S ASSAY TO STUDY<br />
THE KINETICS OF CHOLINESTERASES<br />
Dominika Neuschlová and Anna Hrabovska<br />
Dpt. of pharmacology and toxicology, Faculty of Pharmacy, Comenius University,<br />
Odbojárov 10, 832 32 Bratislava<br />
Ellman’s assay (EA) has been widely used in experimental research and clinical<br />
practice. The limitations of this method are however a high background in biological<br />
samples, instability of the dissolved substrate (thiocholine) and its sensitivity to the<br />
light exposure. The aim of this project was to determine the favorable conditions for<br />
EA in order to lower the background and the reagent instability and thus allow<br />
detecting even very low activities of cholinesterases.<br />
Human butyrylcholinesterase was chosen to study the conditions of EA.<br />
Butyrylthiocholine iodide was used as a substrate. Phosphate, HEPES and Ringer<br />
buffers were used at pH values 7,0; 7,5; 8,0; and 8,5. Stability of the substrate<br />
dissolved in each buffer was tested over the time. The full spectrum was followed in<br />
each buffer. Velocity of the color product production was followed as a function of<br />
time (v/t curve) and substrate concentration (v/s).<br />
The substrate was the most stable in the presence of HEPES buffer. The charts of full<br />
spectrums and the velocity dependences suggested the same kinetics of<br />
butyrylcholinesterase-catalyzed reaction of butyrylthiocholine in both phosphate and<br />
HEPES buffers.<br />
Based on our results we can conclude that Ellman’s assay performed in HEPES buffer,<br />
in contrary to phosphate buffer, is more suitable for measuring of cholinesterase<br />
activity. This is due to the lower background (raising from substrate instability) and<br />
unaffected kinetics.<br />
Acknowledgement: The project was supported by APVV grant SK-CZ-0028-09.<br />
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IX. PATHOBIOCHEMISTRY AND TRANSLATION MEDICINE<br />
175<br />
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Posters<br />
60.<br />
EFFECT OF FRACTIONATED DOSES OF GAMMA RAYS ON THE ROSTRAL MIGRATORY<br />
STREAM OF ADULT RATS<br />
Soňa Bálentová 1 , Eva Hajtmanová 2 , Yvetta Mellová 3 ,<br />
Ivana Kinclová 2 and Marián Adamkov 1<br />
1 Institute of Histology and Embryology, Jessenius Faculty of Medicine in Martin, CU,<br />
Martin, Slovakia, 2 Department of Radiotherapy and Oncology, Martin Faculty<br />
Hospital, Martin, Slovakia, 3 Institute of Anatomy, Jessenius Faculty of Medicine in<br />
Martin, CU, Martin, Slovakia<br />
Ionizing radiation commonly used in the radiotherapy of brain tumours can cause<br />
adverse side effects to surrounding normal brain tissue. The adult mammalian<br />
subventricular zone (SVZ) of the brain lateral ventricles (LV) and their subsequent<br />
lateral ventricular extension, the rostral migratory stream (RMS), is one of the few<br />
areas, which retains the ability to generate new neurons and glial cells throughout<br />
life. Take into account the fact, that ionizing radiation is one of the strongest<br />
exogenous factors affecting cell proliferation, the aim of the present study was to<br />
investigate the occurence of radiation-induced alterations of apoptosis in the<br />
forebrain's RMS using animal model of radiosurgery. Adult male Wistar rats were<br />
investigated 7, 14 or 21 days after whole-body irradiation with fractionated doses of<br />
gamma rays (the total dose of 3 Gy). Radiation-induced apoptotic cell death was<br />
determined by in situ labeling of DNA nick ends (TUNEL) and light microscopy<br />
evaluation of TUNEL-positive cells. However, the data from quantitative analysis of<br />
the numbers of apoptotic cells are still under evaluation, our preliminary results<br />
showed, that ionizing radiation clearly affect this neurogenic region.<br />
Acknowledgement: This work was supported by AV4/2026/08 Grant.<br />
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61.<br />
IS RESPIRATORY PATHWAY ACTING THROUGH NO-sGC?<br />
Ľudmila Capková, Alexandra Dávidová,Andrea Kucháriková and Nadežda Lukáčová<br />
Institute of Neurobiology, Slovak academy of Sciences, Košice<br />
Most effects of the nitric oxide (NO) are mediated by stimulation of soluble guanylyl<br />
cyclase (sGC) and subsequent increase in cyclic guanosine monophophate (cGMP)<br />
formation. NO/sGC/cGMP signalization was identified in neuronal pathways of the<br />
brain, but little is known about this signaling pathway in the spinal cord. The aim of<br />
our study was to find out, whether bulbospinal respiratory pathway is acting through<br />
NO-sGC signalization. This pathway begins in medulla and project to the phrenic<br />
motoneurons controlling diaphragm activity. The distribution of the neuronal nitric<br />
oxide synthase (nNOS), β1 subunit of soluble guanylyl cyclase (β1sGC) and<br />
synaptophysin (SYN) was explored in control animals and after C2-C3 spinal cord<br />
hemisection in upper part of the respiratory pathway. Retrograde tracer Fluorogold<br />
(FG) was used for identification of respiratory neurons in medulla. Two days after<br />
injection of FG into the phrenic nucleus (PN) at C4 level revealed amount of FG<br />
fluorescent neurons in the ventral respiratory group (VRG) mostly on ipsilateral side.<br />
We showed intense punctate nNOS and SYN-positive terminals of respiratory neurons<br />
in neuropile of PN in control animals and strong depletion of these terminals on<br />
contralateral side and almost entire depletion of nNOS and SYN-positive terminals on<br />
ipsilateral side of lesioned animals. Eight days after the hemisection we have revealed<br />
lightly ß1sGC fluorescent motoneurons of PN and around them a few nNOS<br />
fluorescent boutons on contralateral side. Almost no sign of ß1sGC immunopositivity<br />
could be seen ipsilaterally to the hemisection. These results together suggest that<br />
bulbospinal respiratory pathway is acting through NO-sGC.<br />
Ackowledgement: The experimental work was supported by VEGA Grant 2/0015/08.<br />
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Posters<br />
62.<br />
THE EFFECT OF NATURAL POLYPHENOLS ON ADIPONECTINE LEVEL IN PATIENTS<br />
SUFFERING FROM ERECTILE DYSFUNCTION<br />
Monika Dvořáková 1 , Jana Muchová 1 , Branislav Trebatický 2 ,<br />
Ján Breza 2 and Zdeňka Duračková 1<br />
1 Department of Chemistry, Biochemistry and Clinical Biochemistry, Medical Faculty,<br />
Bratislava, Slovakia, 2 Department of Urology, Faculty of Medicine, Comenius University,<br />
Bratislava, Slovakia<br />
As patients suffering from erectile dysfunction (ED) are considered patients with temporary<br />
or permanent impotence. In the pathophysiology of ED psychological as well as organic<br />
factors (or both together) can take a part. The main role in the erection process plays a<br />
relaxation of smooth muscles inside the arterial system and cavernous tissue. It is controlled<br />
by spinal reflex and can be initialized by visual, osmatic or imaginary stimulation. The most<br />
important blood vessels relaxant is NO. By effect of free radicals, NO becomes more<br />
inactivated, what leads to lowered tissue relaxation caused by change in cGMP/cAMP ratio.<br />
Pycnogenol ® (Pyc) as a mixture of natural polyphenols and polyphenolic extract from gingko<br />
biloba leafs (EGb761) stimulate a constitutive NO synthase, induce vasodilatation and<br />
improve the microcirculation in the blood. Adiponectin is a protein hormone that modulates<br />
glucose regulation and fatty acid catabolism. Levels of the hormone are inversely correlated<br />
with body fat percentage. Levels of adiponectin are reduced in diabetics compared to nondiabetics.<br />
Weight reduction significantly increases circulating levels. Hypoadiponectinemia is<br />
an independent risk factor for developing of e.g. metabolic syndrome, cardiovascular disease<br />
and diabetes mellitus, which are often found as disorders associated to ED. To our double<br />
blind, randomized, placebo controlled study 52 patients suffering from ED were included.<br />
Patients were investigated before, one and three months after supplements administration<br />
and after termination of drugs supplementation. In addition, ED group of patients was split<br />
to diabetes and nondiabets groups to compare adiponectin levels. No change after Pyc or<br />
Egb 761 administration has been found in comparison to placebo group. Similarly, there has<br />
not been found a difference in ED patients with diabetes and ED patients without diabetes<br />
mellitus. ED patients have decreased levels of adiponectine compared to reference values.<br />
But what is the reason of this decrease is hard to say because of the complexity of the<br />
disease.<br />
Ackowledgement: This study was supported by VEGA grants of Ministry of Education of the<br />
Slovak Republic, and Mind and Health, civil association.<br />
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63.<br />
STUDY OF ANTIAPOPTOTIC PROTEINS RESPONSIBLE FOR DEVELOPMENT OF DRUG<br />
RESISTANCE IN ACUTE LEUKEMIA<br />
Jana Jurečeková, Jozef Hatok, Andrea Štefániková, Dušan Dobrota and Peter Račay<br />
Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin,<br />
Department of Medical Biochemistry<br />
Deregulation of apoptosis disrupts the complex and delicate balance between cell<br />
proliferation, survival and cell death and plays a major role in the development of<br />
diseases such as cancer, and particularly acute leukemia. Malignant cells that have<br />
alterations in proteins involed in cell death signaling are very frequently resistant to<br />
chemotherapy and are difficult to treat with chemotherapeutic agents that primarily<br />
act by inducing apoptosis. Structural analysis of antiapoptotic proteins together with<br />
studies of their biochemical mechanisms have outlines strategies for generation of<br />
drugs, resulting in numerous novel chemical entities with mechanism-based activity.<br />
In presented study, the influence of ABT-737, the synthetic inhibitor of antiapoptotic<br />
proteins Bcl-2 and Bcl-xL, on induction of apoptosis and viability of leukemic cell lines<br />
HL-60 and K-562 was tested. Higher sensitivity of HL-60 (EC 50 = 4,5 µM) probably<br />
correlates with mildly lower expression of Mcl-1, which overexpression may be<br />
responsible for resistance to ABT-737. Fragmentation of DNA was observed already<br />
after 3 hours of cultivation with ABT-737, so we confirmed that ABT-737 acts via<br />
induction of apoptosis. ABT-737 was also found to enhance the effects of<br />
chemotherapy in HL-60.<br />
Understanding of the core components of the apoptotic machinery at the molecular<br />
and structural levels may lead to creating a new era in cancer therapy where the<br />
intrinsic and acquired resistance of malignant cells to apoptosis can be<br />
pharmacologically reversed, reinstating natural pathways of cell suicide.<br />
Acknowledgements: This work was supported by the Grant UK/38/2010.<br />
179<br />
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64.<br />
PROGNOSTIC SIGNIFICANCE OF miR-21 AND miR-143 EXPRESSION IN TISSUE<br />
SAMPLES OF COLORECTAL CARCINOMA AND COLORECTAL LIVER METASTASES<br />
Vlastimil Kulda 1 , Martin Pešta 2 , Ondřej Topolčan 2 , Lukáš Řehoř 1 , Martin Svatoň 1 ,<br />
Václav Liška 3 , Václav Babuška 1 , Luboš Holubec 2 and Radim Černý 1<br />
1 Department of biochemistry LF UK Plzeň, 2 Department of internal medicine II LF UK<br />
Plzeň, 3 Department of surgery LF UK Plzeň<br />
Some of the microRNAs, which are the endogenously expressed regulatory small<br />
noncoding RNA molecules, have an altered expression in colorectal cancer. The aim<br />
of our study was to assess the relationship between miR-21 and miR-143 expression<br />
and prognostic/clinicopathological features of colorectal carcinoma (CRC) and<br />
colorectal liver metastases (CLM) as well. The estimation was performed in 46 paired<br />
(tumor and control) tissue samples of CRC. Further we studied 30 tissue samples of<br />
CLM. MiR-21 and miR-143 expressions were quantified using the qRT-PCR method.<br />
Relation of miR-21 and miR-143 expression to DFI (disease free interval) (Wilcoxon;<br />
p=0.0026 and p=0.0191, respectively) was recorded. There was shorter DFI in<br />
patients with a higher expression of miR-21 and surprisingly also in patients with a<br />
higher expression of miR-143, which is a putative tumor suppressor. There was a<br />
higher expression of miR-21 and lower expression of miR-143 in CRC tissue in<br />
comparison with adjacent normal colon tissue (p
Posters<br />
65.<br />
THE FUNCTIONALITY OF APOPTOSOME APPARATUS AND THE EXPRESSION OF ITS<br />
REGULATORS IN NON-SMALL CELL LUNG CARCINOMA<br />
Erika Moravčíková 1 , Evžen Křepela 1 , Jan Procházka 1 ,<br />
Jan Čermák 1 and Kamila Benková 2<br />
1 Department of Pneumology and Thoracic Surgery, 2 Department of Pathology,<br />
University Hospital Bulovka, Prague, Czech Republic<br />
Deficient signaling in the apoptosome pathway may contribute to tumorigenesis and<br />
neoplastic progression of malignant tumors as well as to their chemo- and<br />
radioresistance. In the present study, we investigated the functionality of the<br />
apoptosome apparatus (AA), the expression of mRNAs encoding the activatable and<br />
inactivatable Apaf1 protein variants, and the expression of AA regulators, including<br />
nucling/UACA, APIP, and procaspase-2 (PC-2) in human non-small cell lung carcinoma<br />
(NSCLC) cells and tissues. First, the enzymatic analysis showed that in 2 of 6 examined<br />
NSCLC cell lines and in 18 of 59 NSCLC tissues (from surgically treated patients) the<br />
cytochrome-c (cyt-c) plus dATP could trigger a significant increase in cytosolic<br />
caspase-3-like activity. Furthermore, the endogenous as well as the (cyt-c + dATP)-<br />
induced caspase-3-like activities were higher in NSCLC tissues as compared to<br />
matched lungs. Both in the tumors and the lungs, the expression of mRNAs encoding<br />
the activatable Apaf1-XL and -LC variants was higher than the expression of mRNAs<br />
encoding the inactivatable Apaf1-LN and -S variants. Interestingly, the expression of<br />
both nucling/UACA and APIP was downregulated in NSCLC tissues as compared to the<br />
lungs, but it did not correlate with the AA activation in NSCLC cell lines and tissues<br />
and lungs. The expression of PC-2 mRNA in the tumors was higher as compared to the<br />
lungs, but there was no correlation between PC-2 mRNA and the endogenous<br />
caspase-3-like activity in NSCLC tissues. The results of this study indicate that the<br />
expression of nucling/UACA, APIP and PC-2 did not importantly affect the<br />
functionality of AA in NSCLC.<br />
Acknowledgments: Supported by research projects (MZO 00064211 and NS/9715-3)<br />
from Ministry of Health, Czech Republic.<br />
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66.<br />
EFFECT OF OMEGA-3 PUFA ON LIPID PROFILE AND OXIDATIVE STRESS IN<br />
HYPERCHOLESTEROLEMIC CHILDREN<br />
Iveta Ondrejovičová 1 , Jana Muchová 1 , Zuzana Paduchová 1 ,<br />
Zuzana Nagyová 2 and Zdeňka Ďuračková 1<br />
1 Institute of medical chemistry, biochemistry and clinical biochemistry, Medical Faculty,<br />
Comenius University, Bratislava, Slovak Republic<br />
2 Juvenalia, s.r.o., Pediatric center, Dunajská Streda, Slovak Republic<br />
Hypercholesterolemia is defined as an impairment of lipid metabolism. It is characterized by<br />
an increased level of cholesterol above 6.2 mmol/L in adults and above 4.2 mmol/L in<br />
children. Several studies have proved that every fourth child in Slovakia older than 11 – 12<br />
years has increased cholesterol levels. The aim of our clinical study was to monitor changes<br />
in lipid profile in 35 children with a mild hypercholesterolemia (average age 16 ± 3.34 years),<br />
after daily supplementation with a nutritional product containing omega-3 polyunsaturated<br />
fatty acids (PUFA) (1000 mg EPA/DHA) and phytosterol esters (1300 mg) during 16 weeks<br />
period. Oxidative stress also participates in pathogenesis of hypercholesterolemia and<br />
therefore we have monitored the effect of this product on markers of oxidative damage to<br />
lipids (lipid hydroperoxides, 8-isoprostanes) and the total antioxidative status. We collected<br />
samples before and after 8 and 16 weeks of administration of the supplement.<br />
Serum/plasma was prepared by a standard procedure and was stored at -20 ºC. The total<br />
cholesterol (TCH), LDL-cholesterol (LDL), HDL-cholesterol (HDL), triacylglycerols (TAG), C-<br />
reactive protein, lipoprotein-A, glucose, uric acid, as well as parameters of oxidative stress<br />
were analyzed in serum/plasma samples. After 16 weeks of supplement administration we<br />
have found out that levels of TCH (5.24–4.86 mmol/L, p
Posters<br />
67.<br />
ANALYSIS OF URATE TRANSPORTERS SLC22A12 and SLC2A9 IN PATIENTS WITH<br />
RENAL HYPOURICEMIA IN CZECH POPULATION<br />
Blanka Stibůrková 1 , Makoto Hosoyamada 3 , Kimiyoshi Ichida 4 and Ivan Šebesta 1,2<br />
1 Charles University in Prague, First Faculty of Medicine, Institute of Inherited<br />
Metabolic Disorders and 2 Institute of Clinical Biochemistry and Laboratory Medicine;<br />
3 Division of Pharmacotherapeutics, Faculty of Pharmacy, Keio University, Tokyo;<br />
4 Department of Pathophysiology, Tokyo University of Pharmacy and Life Sciences,<br />
Japan<br />
Renal hypouricemia is a heterogeneous inherited disorder characterised by impaired<br />
tubular uric acid transport, reabsorption insufficiency and/or acceleration of<br />
secretion (OMIM #220150) with severe complications such as acute renal failure and<br />
nephrolithiasis. The most causative genes are SLC22A12 and SLC2A9. We have<br />
selected 14 patients from 10 families for analysis of the SLC22A12 and SLC2A9 from<br />
the group of 569 hypouricemic cases. Sequence analysis of SLC22A12 revealed three<br />
transitions G366R, T467M, R477H and one deletion A416_L418del in seven<br />
heterozygotes, three compound hetero-zygotes and four homozygotes. Sequence<br />
analysis of SLC2A9 revealed three unpublished transitions G216R, D281H, N333S, one<br />
insertion with frame shifting change p.[I118HfsX27] and four published sequence<br />
variants G25R, V282I, R294H and P350L in two heterozygotes, five compound<br />
heterozygotes and two homozygotes. The function and immunohistochemistry<br />
analysis in Xenopus laevis oocytes including subcellular localization, colocalization and<br />
processing dynamics and transport of proteins are in process.<br />
Our finding supports the prediction that intact function of both SLC22A12 and SLC2A9<br />
transporters is necessary for normal urate reabsorption. Further detailed studies<br />
could clarify genotype/phenotype relations in hypo/hyper-uricemia and gout and in<br />
conditions related to hyperuricemia as well.<br />
Acknowledgement: Supported by grant MSM0021620806 Czech Republic.<br />
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Posters<br />
68.<br />
STUDY OF THE EFFECT OF HISTONE DEACETYLASE INHIBITOR ON THE SENSITIVITY<br />
OF LEUKAEMIC CELLS TO THE CYTOSTATICS<br />
Andrea Štefániková 1 , Jozef Hatok 1 , Jana Jurečeková 1 , Ivana Plameňová 2 ,<br />
Dušan Dobrota 1 and Peter Račay 1<br />
1 Department of Medical Biochemistry, 2 Clinic of Haematology and Transfusiology,<br />
JLF UK Martin<br />
One of the possibilities how to overcome a chemoresistance of tumor cells is<br />
a combination of classic cytostatics with substances possessing a potential to block<br />
proliferation or induce apoptosis of these cells. Histone deacetylases are a group of<br />
enzymes that catalyze removing acetic acid residue from histones. This epigenetic<br />
process leads to condensation of chromatin and suppresion of transcription. The<br />
influence of histone deacetylases inhibitors is intensively studied nowadays,<br />
especially because of their connection with antiproliferative and antineoplastic effect.<br />
There has long been known an inhibitor of histone deacetylases class I - sodium<br />
butyrate. It is a short chain fatty acid that has effects at the molecular, cellular, and<br />
tissue level. By performing of in vitro MTT test, we have revealed polyresistance of<br />
leukaemic blasts isolated from blood of AML patient to almost all cytostatics tested.<br />
Addition of sodium butyrate in concentration of 2mM led to significantly increased<br />
chemosensitivity of blasts. In search for molecular mechanism of butyrate action we<br />
have performed analysis of leukaemic cell line HL60 treated with sodium butyrate.<br />
Our results open possibility that addition of butyrate increases chemosensitivity of<br />
blasts by alteration of expression of proteins involved in apoptosis initiation.<br />
Acknowledgement: This work was supported by grant UK/223/2010<br />
Štefaniková.<br />
to Andrea<br />
184<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
69.<br />
CONTENT OF FATTY ACIDS IN FOOD AND HEALTH STATUS<br />
Ladislav Vaško and Janka Vašková<br />
Department of Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty of<br />
Medicine, Pavol Jozef Šafárik University in Košice, 040 66 Košice<br />
Change in blood and tissue fatty acid composition join and trigger quite a few of<br />
pathological variations. Even incorrect lipid nutrition could be negatively affecting the<br />
health status. Know edge of fatty acid content and ratio is important to ensure<br />
suitable lipid nutrition depending on organism physiological needs to prevent<br />
morbidity. In case of congenital afflictions determination of fatty acid content<br />
elaborates diagnostics and predestinates diet treatment with defined fatty acids.<br />
Demand of cognizance the food fatty acid composition, either their influence on<br />
health state or prevention infers possible solution for right lipid nutrition including<br />
direct n-6 and n-3 polyunsaturated fatty acids ratio. In feeding trial on laying hens the<br />
addition of flax oil in first treatment group and fish oil in the second treatment group<br />
led to outstanding increase of polyunsaturated fatty acid content. It concerned,<br />
especially, n-3 acid content in blood, eggs and although in fat tissue of laying hens.<br />
Results attained n-6: n-3 ratio making possible for consumers to improve existing<br />
adverse n-6: n-3 ratio by eating such improved white meat and eggs.<br />
Acknowledgement: Study was supported by Slovak grant agency for Science VEGA<br />
1/0799/09.<br />
185<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
70.<br />
EFFECT OF HUMIC ACIDS IN VIVO<br />
Janka Vašková and Ladislav Vaško<br />
Department of Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty of<br />
Medicine, Pavol Jozef Šafárik University in Košice, 040 66 Košice<br />
Humic acids were described as prospective compounds utilized to ensure sufficient<br />
quantity of food for human population at a high economic profitability of agriculture<br />
production and moreover protection of the environment. In feeding trial on broiler<br />
chickens the effects of humic acids as feeding additives on production parameters<br />
and activities of antioxidant enzymes in blood were tested. 14 800 chickens from<br />
treatment group were compared to 142000 untouched control chickens. Treatment<br />
group showed significant decrease in mortality (0.95%) in comparison to control<br />
(3.5%), better of feed conversion (13%) and faster growth. Comparison of the results<br />
from blood sampling on days 14 and 35 of treatment period showed enhanced<br />
cooperation of antioxidant enzymes as glutathione peroxidase, glutathione<br />
reductase, superoxiddismutase leading to overall lower levels of substrate<br />
peroxidation. Addition of humic acids to feed in treatment group in comparison to<br />
control showed better protection against radical generation.<br />
Acknowledgement: Study was supported by Slovak grant agency for Science VEGA<br />
1/0799/09.<br />
186<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
X. PROTEOMICS AND ENZYMOLOGY<br />
187<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
71.<br />
HEXAMER FORMATION TRIGGERS A SWITCH FROM AN INACTIVE TO AN ACTIVE<br />
CONFORMATION IN HUMAN MITOCHONDRIAL LON PROTEASE<br />
Vladimír Pevala 1 , Jacob A. Bauer 1 , Javier García-Nafría 2 , Gabriela Ondrovičová 1 ,<br />
Ľuboš Ambro 1 , Elena Blagova 2 , Vladimir M. Levdikov 2 , Anthony J. Wilkinson 2 , Keith<br />
S.Wilson 2 and Eva Kutejová 1<br />
1 Institute of Molecular Biology, Department of Biochemistry, SAS, Dúbravská cesta 21,<br />
845 51 Bratislava, Slovakia,<br />
2 Structural Biology Laboratory, Department of Chemistry, University of York, York<br />
YO10 5YW, UK<br />
Although Lon is one of the least complicated ATP-dependent proteases, a structure of<br />
the full-length protein still has not been determined. At present, structures have<br />
been solved of a fragment of the N-terminus, the small α-domain and the proteolytic<br />
domain. We determined the crystal structure of the human mitochondrial Lon<br />
protease. Although the overall structure is very similar to the EcLon one, the<br />
conformation around the active site more closely resembled that seen in the<br />
Methanococcus jannaschii Lon structure. A detailed analysis of these three structures<br />
led us to propose a mechanism by which hexamer formation is coupled to a<br />
conformational transition at the active site, which converts the inactive conformation<br />
seen in the hLon structure to one resembling that seen in the EcLon one. To better<br />
understand the roles of the proteolytic domain in the overall functions of human Lon<br />
protease, we designed several point mutations in this domain based on the known<br />
Lon protease crystal structures. We then tested their influence on protease,<br />
peptidase, and ATPase activity as well as on oligomer formation and stability.<br />
Acknowledgement: This work was supported by VEGA 2/0141/08, APVV-0024-07 and<br />
by European Commission funding SPINE2–COMPLEXES project LSHG–CT–2006–<br />
031220. We thank the staff at the ESRF (beamline ID14-4) for provision of synchrotron<br />
facilities, and Johan Turkenberg and Sam Hart for assistance with data collection.<br />
188<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
72.<br />
BIOCHEMICAL CHARACTERIZATION OF RV1459C PROTEIN PUTATIVE GT-C<br />
GLYCOSYLTRANSFERASE FROM MYCOBACTERIA<br />
Milo Bystrický 1 , Martina Beláňová 1 , Mary Jackson 2 ,<br />
Katarína Mikušová 1 and Jana Korduláková 1<br />
1 Department of Biochemistry, Faculty of Natural Sciences, Comenius University,<br />
Bratislava, Slovakia; 2 Department of Microbiology, Immunology, and Pathology,<br />
Colorado State University, Fort Collins, CO, USA<br />
Mycobacterium tuberculosis and related species of the order Actinomycetales carry<br />
a significant number of glycosyltransferases of the GT-C class. This group of<br />
glycosyltransferases comprises integral membrane proteins with dependency on<br />
polyprenyl-phosphate-linked sugar donors. No protein structure of the GT-C<br />
superfamily has yet been solved. In mycobacteria GT-C glycosyltransferases are<br />
believed to be involved in the later biosynthetic steps of key polysaccharides in the<br />
mycobacterial cell envelope. We focused on the functional characterization of the<br />
mycobacterial protein Rv1459c, putative GT-C glycosyltransferase encoded by the<br />
gene located in the cluster of the genes rv1459c-rv1456c. Homologues of the genes<br />
rv1459c - rv1456c are present in all mycobacterial species sequenced so far. Similar<br />
gene clusters were identified also in corynebacteria and nocardia - organisms with<br />
the cell wall structures very similar to that of mycobacteria. The glycosyltransferase<br />
Rv1459c was produced in E. coli in the form of soluble protein fused with the<br />
maltose binding protein. Heterologous production of the soluble Rv1459c allowed us<br />
to isolate this protein for crystallography purposes, as well as for the investigation of<br />
the interactions between Rv1459c and the subunits of the ABC transporter<br />
Rv1458c/Rv1457c/Rv1456c.<br />
Acknowledgement: This work was supported by Slovak Research and Development<br />
Agency (grant No. APVV-0499-07)<br />
189<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
73.<br />
THE STUDY OF RYANODINE RECEPTOR 2 N-TERMINAL REGION RESPONSIBLE FOR<br />
HEART ARRYTHMIAS AND HEART FAILURE<br />
Ľubomír Borko 1,2 , Vladena Bauerová-Hlinková 2 , Eva Hostinová 2 ,<br />
Juraj Gašperík 2 and Jozef Ševčík 2<br />
1 Department of Molecular Biology, PRIF UK, Mlynská dolina, 842 15 Bratislava<br />
2 Institute of Molecular Biology, SAV, Dúbravská cesta 21, 845 51 Bratislava<br />
Ryanodine receptor (RyR) is a homotetramer composed of four subunits with a<br />
molecular weight of ~560 kDa. In humans there are three isoforms of RyR: RyR1,<br />
RyR2 and RyR3. RyR1 (expressed mostly in sceletal muscle) and RyR2 (in myocardium)<br />
are suggested to be of a vital importance. RyRs are localized in the membrane of<br />
sarcoplasmatic reticulum. The role of RyR is to transport Ca 2+ from sarcopasmatic<br />
reticulum to the myoplasm. The main RyR2 gating regulation mechanism is<br />
considered to be the domain-domain interaction between the N – terminal (aa ~ 1-<br />
600) and central region (aa ~ 2100-2500). A hypothesis was proposed that mutations<br />
in these regions cause regulation failure and thus lead to nonspecific Ca 2+ release<br />
which results in several heart diseases. Ryanodine receptor 2, has a domain structure,<br />
so we decided to prepare DNA fragments coding for residues 384-606, 391-606, 409-<br />
606, 1-606 and 1-655. These fragments were cloned into appropriate pET expression<br />
vectors for heterologous expression in E. coli strains. Recombinant polypeptides RyR2<br />
409-606, NusA_409-606, Trx_409-606, Trx_391-606, Trx_384-606, 1-606 and 1-655<br />
were obtained in high expression levels. Because of high rate of aggregation and<br />
instability of these recombinant proteins it was necessary to use appropriate<br />
detergents, pH values, buffers and salts. Isolation and purification of recombinant<br />
polypeptides was optimized to obtain soluble monomeric products. To study stability<br />
and folding we prepared a number of N-terminal mutations (1-606 fragment), which<br />
are connected to heart arrhythmias and failures. Our work is oriented to study<br />
domain-domain and domain-ligand interactions and structure determination of<br />
interacting regions by X-ray crystallography.<br />
190<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
74.<br />
THE FUNCTIONAL CHARACTERIZATION OF THE PUTATIVE MYCOBACTERIAL ABC<br />
TRANSPORTER MSMEG_6366 - MSMEG_6369<br />
Petronela Dianišková 1 , Jana Korduláková 1 , Henrieta Škovierová 1 , Devinder Kaur 2 ,<br />
Mary Jackson 2 , Patrick J. Brennan 2 and Katarína Mikušová 1<br />
1 Department of Biochemistry, Faculty of Natural Sciences, Comenius University,<br />
Bratislava, Slovakia; 2 Mycobacterial Research Laboratories, Department of<br />
Microbiology, Immunology and Pathology, Colorado State University, Fort Collins,<br />
Colorado, USA<br />
The mycobacterial cell envelope differs substantially from the cell walls of other<br />
bacteria. This unique structure accounts for its unusually low permeability and<br />
resistance towards common antibiotics and thus enables mycobacteria to survive and<br />
multiply within the host. The main covalently linked structural element of<br />
mycobacterial cell wall consists of three entities – peptidoglycan, heteropolymeric<br />
arabinogalactan and highly hydrophobic mycolic acids. During the last years a number<br />
of enzymes involved in the biosynthesis of these components have been identified. In<br />
spite of this fact, a great challenge is to decipher the mechanism of the assembly of<br />
this complex structure including the transport of the cell wall intermediates across<br />
the plasma membrane. In our effort to identify the transport proteins that could be<br />
involved in this process we focused on a putative ABC transporter Rv3781-Rv3783<br />
from M. tuberculosis H37Rv. Rv3781 and Rv3783 genes are located around glfT1, the<br />
gene encoding galactosyl transferase responsible for the initiation of the galactan<br />
biosynthesis. Here we demonstrate that orthologs of all three genes are cotranscribed<br />
in M. smegmatis mc 2 155 and we have also confirmed that recombinant<br />
Rv3781 ortholog (MSMEG_6366) and GlfT1 (MSMEG_6367) from M. smegmatis<br />
mc 2 155 interact with each other suggesting that they form a complex that could be<br />
involved in the biosynthesis of the cell wall in mycobacteria.<br />
Ackowledgement: This work was supported by the Slovak Research and Development<br />
Agency under the contract No. RPEU-0012-06, by European Comission under contract<br />
LSHP-CT-2005-018923"NM4TB“, and by the grant AIDS-FIRCA TW 006487 from NIH,<br />
NIAID.<br />
191<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
75.<br />
EFFECT OF DROUGHT ON THE METABOLISM OF TOBACCO PLANTS<br />
(NICOTIANA TABACUM L.)<br />
Veronika Doubnerová 1 , Lucia Miedzińska 1 , Jana Dobrá 2 ,<br />
Radomíra Vaňková 2 and Helena Ryšlavá 1<br />
1 Department of Biochemistry, Faculty of Natural Science, Charles University in Prague,<br />
2 Institute of Experimental Botany AS CR<br />
Drought is one of the most significant types of abiotic stress worldwide, due to<br />
almost a third of the Earth surface is arid or semi-arid area. In this study the changes<br />
in enzyme activities of NADP-malic enzyme (EC 1.1.1.40; NADP-ME),<br />
phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPC) and pyruvate, phosphate<br />
dikinase (EC 2.7.9.1; PPDK) in tobacco plants (Nicotiana tabacum L., cv. W38) after<br />
drought were investigated. Enzyme activities in tobacco leaves were significantly<br />
increased during 11 days of stress, PEPC 2-fold, PPDK 3,3-fold and NADP-ME 4-fold<br />
compared to control plants. The regulation of NADP-ME and PEPC activities were<br />
studied on transcriptional level by the real-time PCR method and on translational<br />
level-immunochemically. The amount of NADP-ME protein and mRNA for chloroplast<br />
NADP-ME isoform was increased, but mRNA for cytosolic isoform was not affected.<br />
The amount of PEPC protein was unchanged; amount of mRNA for PEPC was little<br />
decreased. Also regulation of PEPC activity was studied, because this enzyme is<br />
regulated at many levels, especially by phosphorylation. We suppose that the<br />
decreased activity after alkaline phosphatase treatment, activation by D-glucose-6-<br />
phosphate and increased ratio of activity at pH 7.1 and 8.1 means enhanced<br />
phosphorylation level of PEPC molecule in stressed plants.<br />
Acknowledgements: This work was supported by the grants of Ministry of Education<br />
of the Czech Republic (grants MSM0021620808 and 1M0505).<br />
192<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
76.<br />
THERMAL STABILITY OF CYTOCHROME C AND α-LACTALBUMIN COMPLEXES<br />
Diana Fedunová 1 , Zuzana Flachbartová 2 , Jaroslava Bágeľová 1 ,<br />
Zuzana Gažová 1 and Marián Antalík 1,2<br />
1 Department of Biophysics, Institute of Experimental Physics SAS, Kosice, Slovakia<br />
2 Institute of Chemical Sciences, Faculty of Science, P. J. Safarik University, Kosice,<br />
Slovakia<br />
Effective activation of apoptosis is one of the important tools for tumor cell<br />
treatment. Cytochrome c (cyt c) plays relevant role during early phase of apoptosis<br />
after release from mitochondria in vivo. A new approach is oriented to the study of<br />
the ability of cyt c to induce apoptosis by its transport from extracellular location. The<br />
active transport of cyt c to the cells requires complexation with compounds<br />
supporting this process. We have studied interactions of cyt c with α-lactalbumin (α-<br />
LA) in order to find optimal conditions for their complexation necessary for using<br />
these complexes in induction of programmed cell death. We have found that α-LA<br />
has only negligible effect on cyt c heme pocket within wide pH interval. The complex<br />
formation is accompanied by turbidity increase even at low protein concentrations.<br />
Thermal stability of the complex depends on protein concentration ratio and absolute<br />
concentration value. The properties of studied complexes depend strongly on ionic<br />
strength.<br />
Acknowledgements: This work was supported within the projects Nos. 26220120021,<br />
26220120001, 26220220005, 2622022033 in frame of SF EU, Centre of Excellence of<br />
SAS Nanofluid and VEGA 0056, 0038 and 0079.<br />
193<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
77.<br />
Rep 34 PROTEIN ENCODE BY PLASMID pGP2 FROM Acetobacter<br />
Peter Grones, Zuzana Odnogová and Jozef Grones<br />
Comenium University, Department of Molecular Biology, Bratislava<br />
The Acetobacter estunensis Rep 34 protein participates in the replication of bacterial<br />
small cryptic plasmid pGP2. The Rep 34 protein (213 aa, 23.65 kDa) from Acetobacter<br />
plasmid pGP2, was cloned to the expression vector, that ensure fusion with a His-tag<br />
sequence (Rep 34 His-tagged), over-expressed in Escherichia coli and purified by metalaffinity<br />
chromatography to yield a highly purified and active protein. On this purified<br />
protein number different activities and motifs were detected. DNA band-shift assays<br />
showed that the Rep 34 His-tagged protein bound to the regulation region for<br />
replication on the linear double-stranded DNA. In the protein was determined<br />
phosphatase activity, ATPase activity and protein is possible to unwind double strand<br />
DNA. After fusion with GFP protein the accruement of protein expression with<br />
confocal microscopy was proven.<br />
194<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
78.<br />
PRODUCTION OF 3-HYDROXYPROPIONALDEHYD BY THE STRAINS OF<br />
LACTOBACILLUS REUTERI<br />
Hana Kiňová Sepová, Andrea Bilková, František Bilka and Lýdia Bezáková<br />
Department of Cell and Molecular Biology of Drugs FaF UK Bratislava<br />
Eight bacterial strains were isolated from stomach mucus of breast fed lamb (C, D,<br />
and E) and kid (KO4b, KO4m, KO5, KG1, KG4). Four of these strains were identified by<br />
sequencing of 16S rDNA as Lactobacillus reuteri (E, KO4b, KO4m, and KO5) and tested<br />
for production of antimicrobial substance 3-hydroxypropionaldehyd (3-HPA). For this<br />
purpose PCR primers targeting gene of large subunit of glycerol dehydratase (GD),<br />
key enzyme involved in 3-HPA synthesis, were designed. Presence of this genefragment<br />
was detected in the case of all four strains and the reference strain L.<br />
reuteri ATCC 55730. Sequencing of PCR product proved that it is fragment of gene of<br />
large subunit of GD. The ability to produce 3-HPA was determined<br />
spectrophotometrically, positive reaction was noticed in the case of L. reuteri E and<br />
reference strain. Quantative analysis of 3-HPA production by L. reuteri E and L. reuteri<br />
ATCC 55730 in aerobic and anaerobic conditions was determined<br />
spectrophotometrically by tryptophan method. The amount of 3-HPA produced<br />
aerobically was in both strains higher than those produced anaerobically. In the case<br />
of L. reuteri E it was aerobically 1.60 mmol/L, anaerobically 0.58 mmol/L and in the<br />
case of L. reuteri ATCC 55730 it was aerobically 184.51 mmol/L and anaerobically 5.71<br />
mmol/L.<br />
Strain L. reuteri E produced lower amounts of 3-HPA than probiotic strain L. reuteri<br />
ATCC 55730. However this does not exclude it from the group of potential probiotics;<br />
it has other important attributes that allow L. reuteri E to be a good probiotic strain<br />
for veterinary use.<br />
195<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
79.<br />
THIOREDOXIN SYSTEM IN STREPTOMYCES<br />
Michaela Koháryová and Marta Kollárová<br />
Department of Biochemistry, Faculty of Natural Sciences, Comenius University,<br />
Mlynská dolina CH-1, 842 15 Bratislava, Slovak Republic<br />
Thiol-disulphide bond balance is generally under control of Trx system and/or<br />
GSH/GSHR system in bacteria. Both systems are the main regulators of redox<br />
homeostasis, which can modulate biological activity of many proteins and also<br />
participate in protection against oxidative stress. S. coelicolor A3(2) (like other<br />
Streptomycetes) is a suitable model organism for studying Trx system, because lacks<br />
GSH/Grx system (Newton et al., 1996).<br />
In the most prokaryotic and eukaryotic organisms are multiple functions<br />
accomplished by single thioredoxin. The complete genome sequence of S. coelicolor<br />
A3(2) (Bentley et al., 2000) revealed several possible thioredoxin genes: three genes<br />
for thioredoxin, two for putative thioredoxins, one for thioredoxin reductase and two<br />
for putative thioredoxin. Their high sequence similarity can predicted similar<br />
physiological functions. All genes encode proteins with putative oxidoreductase<br />
activities, however their biological roles are not determined yet. It seems, that S.<br />
coelicolor has a very complex redox system, what can be responsible for multicellular<br />
development of Streptomyces. We prepared E. coli strains with overproduced<br />
thioredoxins (A, A2, A3) and also thioredoxin reductase TrxB. We purified the<br />
proteins and performed their functional analysis. After first screening we obtained<br />
TrxB protein crystals.<br />
Acknowledgements: This work was supported by project "BIOMAKRO1<br />
ITMS:26240120003", "BIOMAKRO2 ITMS:26240120027", thanks to operating<br />
program Research and progression financed by Europe fund of regional development<br />
and also thanks to VEGA grant 1/03/09.<br />
196<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
80.<br />
STUDIES OF NOVEL BIVALENT TACRINE DERIVATIVES TARGETING<br />
ACETYLCHOLINESTERASES<br />
Mária Kožurková 1 , Danica Sabolová 1 , Slávka Hamuľaková 1 , Jana Janočková 1 , Jana<br />
Plšíková 1 , Pavol Kristian 1 , Ján Imrich 1 , Ladislav Janovec 1 , Ondrej Holas 2 ,<br />
Miroslav Pohanka 2 and Kamil Kuča 2<br />
1 Institute of Chemistry, Faculty of Sciences, P.J. Šafárik University, Košice, SK<br />
2 Faculty of Military Health Sciences, University of Defence, Hradec Králové, CZ<br />
Derivatives of acridine/tacrine are effective drugs against Alzheimer disease (AD)<br />
which is associated with the progressive memory loss and other cognitive<br />
impairments. The primary approach to treating AD is delaying of symptoms of disease<br />
and increasing the acetylcholinesterases levels in the brain by using<br />
acetylcholinesterase inhibitors. The cholinesterase inhibitors have been identified<br />
according to their binding mode.<br />
Eleven compounds of acridine/tacrine derivatives were synthesized and their<br />
properties have been studied by UV-Vis, fluorescence spectrophotometry and circular<br />
dichroism.<br />
The most promising inhibitor of acetylcholine from the newly prepared compounds<br />
were derivative N-{2-[4-(acridine-9-yl) piperazino]etyl}-N-(1,2,3,4-tetrahydroacridine-<br />
9-yl)amine (IC 50 = 0.003 ± 0.0006 µM) and N-(1,2,3,4-tetra-hydroacridine-9-yl)-N´-[2-<br />
(1,2,3,4-tetrahydroacridine-9-ylamino)butyl]thiourea (IC 50 = 0.002 ± 0.0004 µM).<br />
These compounds were two-fold stronger inhibitors compared to standards (tacrine<br />
and 7-MEOTA).<br />
Acknowledgement: This work was supported by the Scientific Grant Agency VEGA<br />
1/0053/08 and 1/0097/10 (Slovak Republic) and Ministry of Defence–<br />
OVUOFVZ200805 (Czech Republic).<br />
197<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
81.<br />
APPLICATION OF CONCENTRATION FLUORESCENCE MATRICES TO THE DETECTION<br />
OF FLUORESCENCE METABOLITES IN URINE<br />
Lucia Lichardusová, Jaroslav Kušnír and Mária Mareková<br />
Institute of Chemistry, Biochemistry, Medical Biochemistry and LABMED a.s., UPJŠ<br />
Košice<br />
Urine contains a variety of organic and inorganic chemicals, including a number of<br />
natural fluorophores, most of which are tryptophan metabolites. The alteration in the<br />
autofluorescence of urine could result from both physiological and pathological<br />
changes.<br />
The synchronous fluorescence spectrum (SFS) is considered to be the characteristic<br />
“fingerprint“, because it is unique for a given mixture and it characterizes it<br />
graphically as one unit. By introducing concentration parameter, the SFS form<br />
concentration fluorescence matrices which provide the information of the precise<br />
mixture ratios of the urine samples.<br />
In this work we present application of concentration fluorescence matrices to the<br />
detection of urine fluorophores, particularly tryptophan and tyrosine metabolites.<br />
The SFS include autofluorescence of individual fluorophores and their interaction.<br />
Every alternation of one fluorophor disturbs balance of fluorophores interaction and<br />
this is shown by alternation graphic record. The CSMF spectra were formed by the<br />
SFS at various Δλ. As the urine is a complex multi-component system, the shape and<br />
intensity of the spectra obtained under different Δλ changed distinctly and contained<br />
rich and varied information.<br />
Acknowledgement: This study was supported by VEGA 1/0402/10.<br />
198<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
82.<br />
CRH TRANSGLYCOSYLASES CATALYZE INTER POLYMERIC LINKAGES IN<br />
FUNGAL CELL WALL<br />
Marián Mazáň 1 , Noelia Blanco 2 , Javier Arroyo 2 and Vladimír Farkaš 1<br />
1<br />
Institute of Chemistry - Center for Glycomics, Slovak Academy of Sciences,<br />
Dúbravská cesta 9, 84538 Bratislava, Slovakia<br />
2<br />
Departamento de Microbiología II, Facultad de Farmacia,<br />
Universidad Complutense de Madrid, 28040 Madrid, Spain<br />
The cell wall of Saccharomyces cerevisiae is an elastic structure that provides osmotic<br />
and physical protection and determines the shape of the cell. Crh1 and Crh2 are GPIlinked<br />
cell wall proteins required for the formation of linkages between chitin and β-<br />
1,3-glucose branches of β-1,6-glucan in the cell wall of budding yeast. According to<br />
the CAZY database, they belong to the glycoside hydrolase family 16.<br />
We developed a fluorescent in vitro assay system for determination of the<br />
transglycosylating activity of Crh proteins. His-tagged Crh1p and Crh2p of S. cerevisiae<br />
were heterologously expressed in Pichia pastoris, purified on Ni-column and their<br />
basic biochemical characteristics were investigated.<br />
The following properties of Crh proteins were determined: pH and temperature<br />
optima, donor and acceptor substrate specificity, effectors, thermal stability and<br />
mode of action.<br />
Acknowledgement: This work was supported by grant no. 2/0011/09 from the Slovak<br />
Grant Agency for Science VEGA.<br />
199<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
83.<br />
DELETION OF GLUTAMATE DECARBOXYLASE GENE FROM TRICHODERMA VIRIDE F-<br />
534 STRAIN<br />
Ľuboš Nižnanský, Svetlana Kryštofova and Ľudovít Varečka<br />
Department of biochemistry and microbiology, Faculty chemical and food technology<br />
Enzyme glutamate decarboxylase (GAD) is present in eukaryotic and prokaryotic<br />
organisms, where is playing role in different processes. GAD catalyses the conversion<br />
of L-glutamate to a non-coded amino acid 4-aminobutyrate (GABA). In yeasts, GAD is<br />
important for oxidative stress response to acid environment. In this study, we used<br />
electroporation to transfer the gad gene disruption cassette with hygromycin<br />
resistance into T. viride conidia and obtained 21 transformants. Deletion of gad gene<br />
was proved by GAD activity measurements and Southern analysis in two<br />
transformants (mutants 10 and 21). Mutants lacking GAD have delayed conidiation.<br />
Submerged mycelia formed pellet-shaped mycelia during submerged cultivation<br />
unlike of wild strain. Microscopic analysis of Δgad10 and Δgad21 mutants revealed<br />
the increase in forming chlamydospores and thinning of hyphae. In submerged<br />
cultivation with sucrose as carbon source, growth rates of these mutants were lower<br />
than that of wild-type strain, while the growth on GABA as a carbon source showed<br />
the opposite pattern. Growth of Δgad10 was more rapid than that of the wild-type<br />
strain using GABA as carbon source but L-glutamate had an opposite effect.<br />
Respiratory quotients of these mutants were higher than that of wild-type strain.<br />
Thus, the gad gene seems to influence basic bioenergetic processes in this fungus<br />
with the impact on growth and conidiation.<br />
Acknowledgement: This work was supported by grant APVV nr. 0642-07<br />
200<br />
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Posters<br />
84.<br />
CHARACTERIZATION OF β-N-ACETYLHEXOSAMINIDASE IN LEAVES OF<br />
TOBACCO PLANTS<br />
Helena Ryšlavá 1 , Veronika Doubnerová 1 , Robert Valenta 1 , Kateřina Kloudová 1 , Jana<br />
Trefancová 1 , Helena Synková 2 and Noemi Čeřovská 2<br />
1 Department of Biochemistry, Faculty of Natural Science, Charles University in Prague,<br />
2<br />
Institute of Experimental Botany AS CR<br />
Plant glycosidases were characterized in seeds, but there is little information about<br />
these enzymes in leaves. In tobacco plants (Nicotiana tabacum L., cv. Petit Havana,<br />
SR1) the activity of α-glucosidase (EC 3.2.1.20), β-glucosidase (EC 3.2.1.21), α-<br />
galactosidase (EC 3.2.1.22), β-galactosidase (EC 3.2.1.23), α-mannosidase (EC<br />
3.2.1.24) and activity of β-N-acetyl-hexosaminidase (EC 3.2.1.52) were determined.<br />
The highest activity was found for β-N-acetyl-hexosaminidase, this enzyme was able<br />
to hydrolyze both chromogenic substrates p-NP-GlcNAc and p-NP-GalNAc. The<br />
products of the reaction were found to be non-competitive inhibitors of the reactions<br />
catalyzed by β-N-acetyl-hexosaminidase. The hydrolysis of another substrate -<br />
chitobiose was studied by capillary electrophoresis. The size of β-N-acetylhexosaminidase<br />
molecule was estimated at 245 000. Under biotic stress caused by<br />
viral infection enhanced activity of β-N-acetyl-hexosaminidase was found.<br />
Acknowledgements: This work was supported by the grants of Ministry of Education<br />
of the Czech Republic (grants MSM0021620808 and 1M0505).<br />
201<br />
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Posters<br />
85.<br />
DNA BINDING STUDY OF 9-OXO-9,10-DIHYDROACRIDIN-CARBOXYHYDRAZIDES AS<br />
POTENT TOPOISOMERASE I INHIBITORS<br />
Danica Sabolová, Lucia Krajňáková, Jana Plšíková and Mária Kožurková<br />
Department of Biochemistry, Institute of Chemistry, Faculty of Science, P.J. Šafárik<br />
University, Moyzesova 11, SK-04167 Košice<br />
DNA, the basic genetic material, is one of the most enigmatic biomolecules and<br />
efforts to understand the subtleties of its behaviour from a structural and energetic<br />
perspective have not yet been fully rewarded. The pivotal role played by DNA in the<br />
synthesis of proteins as well as its own replication makes it an extremely important<br />
potential target for drugs, especially for anticancer, antibiotic and antiviral action.<br />
Regions of DNA involved in vital processes like origin of replication, promotion of<br />
transcription etc. are of particular interest as targets for such drugs.<br />
In this work, the interactions of acridone derivates 9-oxo-9,10-dihydroacridinecarbohydrazides<br />
with DNA were studied by a variety of spectroscopic techniques<br />
including UV-Vis spectrophotometry, fluorimetric titrations and circular dichroism.<br />
From spectrofluorimetric titrations the binding constants for DNA - drug complexes<br />
were determined (K= 1.2 × 10 5 M -1 – 5.1 × 10 5 M -1 ). The effect of investigated<br />
compounds on the thermal denaturation profiles of calf thymus DNA were also<br />
studied. By electrophoretic methods was determined, that the new drugs were able<br />
to inhibit the topoisomerase I.<br />
Acknowledgements: This work was supported by the Slovak Grant Agency VEGA, No.<br />
1/0053/08 and 1/0097/10.<br />
202<br />
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Posters<br />
86.<br />
IN VIVO CROSS-LINKING FOR IDENTIFICATION OF TELLURITE RESISTANCE-<br />
ASSOCIATED PROTEINS<br />
Jana Schubertová Aradská 1 , Dušan Blaškovič 1 and Ján Turňa 1,2<br />
1 Department of molecular biology PriF UK Bratislava, 2 Department of molecular<br />
biology SAV Bratislava<br />
In spite of the fact that a number of tellurite-resistance determinants have been<br />
characterized, little is known about the mechanism responsible for this resistance.<br />
Determinant encoding resistance against pottasium tellurite was discovered in clinical<br />
isolate of Escherichia coli KL53 on a large conjugative plasmid<br />
pTE53. Analyses<br />
showed that genes terB, terC, terD and terE are essential for conservation of the<br />
resistance. In order to understand this mechanism we are looking for tellurite<br />
resistance associated proteins. For initial investigation of protein-protein interactions<br />
we decided to use in vivo chemical cross-linkings to effectively capture and identify<br />
interacting proteins. Chemical cross-linking stabilizes interactions through covalent<br />
bond formation, allowing the detection of protein-protein interactions in native cells<br />
that are weak and/or transient. The most commonly used cross-linking reagent is<br />
formaldehyde, a water-soluble and cell membrane-permeable molecule, which<br />
provides fast and reversible cross-linking reaction. With this in mind, we decided to<br />
investigate the effects of formaldehyde cross-linking. All four genes were cloned to<br />
pET expression system to produce His-tagged proteins and constucts were<br />
transformed to E. coli BL21(DE3). These strains were subequently co-transformed<br />
with plasmid pLK18 which contains the functional part of the tellurite resistance<br />
operon, encompasing terBCDEF. Strains were cultivated on medium with tellurite to<br />
induce oxidative stress and living cells were treated with formaldehyde. Crosslinked<br />
TerB, TerC, TerD and TerE proteins were purificated by Ni-NTA resin and analyzed by<br />
SDS-PAGE. In the next step we would like to identify associated proteins by MS<br />
analyses.<br />
203<br />
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Posters<br />
87.<br />
MULTIPLE PROTEASES ARE SECRETED BY VEGETATIVE TRICHODERMA VIRIDE<br />
MYCELIUM CULTIVATED WITH PROTEIN INDUCER<br />
Martin Šimkovič, Anita Gdovinová, Zuzka Zemková and Ľudovít Varečka<br />
Department of biochemistry and microbiology, Faculty of chemical and food<br />
technology, Slovak Univerzity of Technology, Radlinského 9, 81237 Bratislava<br />
The cultivation of Trichoderma viride mycelium with purified protein substrate (bovine<br />
serum albumin, ovalbumin) resulted in the secretion of protease activity into the<br />
medium under submerged conditions. The secretion began within 30 h and the<br />
greatest secretion activity was observed after 72 h cultivation. The secretion continued<br />
upon the prolonged cultivation (up to 8 d) with lower secreted proteolytic activity.<br />
Gelatine zymography of the secreted protease revealed high-molecular weight<br />
protease(s) (~200 kDa) with high autolytic activity as the only secretory product. Lowmolecular<br />
weight protease(s) involved in the mycoparasitic activity of Trichoderma spp.<br />
was seen after prolonged cultivation only, as a band with m.w. about 30 kDa.<br />
Expression of known Trichoderma spp. genes encoding secreted proteases Prb1, ProA<br />
showed that only Prb1 was expressed after 3-4 days of cultivation, i.e., after fading out<br />
the early secretion phase. The secretory activity of the earlier phase was impaired by<br />
tunicamycin and brefeldin A and was significantly stimulated by uncoupler. The<br />
existence of biphasic fungal secretory response represents a yet not recognized<br />
process. The structure and the biological role of secreted proteases should be<br />
elucidated in the future.<br />
Acknowledgements: This work was supported by the VEGA Grant Agency project (No.<br />
1/0434/08) and by the Science and Technology Assistance Agency under the contracts<br />
Nos. APVV-0642-07, APVT-20-003904 and VVCE-0064-07.<br />
204<br />
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Posters<br />
88.<br />
PHYTOALEXINS REDUCE INSULIN AMYLOID AGGREGATION<br />
Katarína Šipošová 1 , Andrea Antošová 2 , Peter Kutschy 1 ,<br />
Zuzana Daxnerová 3 and Zuzana Gažová 2<br />
1 Institute of Chemical Sciences, 3Institute of Biology and Ecology, Faculty of Science,<br />
P. J. Šafárik University, Košice, Slovakia, 2 Department of Biophysics, Institute of<br />
Experimental Physics SAS, Košice, Slovakia<br />
Amyloid aggregation, a generic behavior of proteins, is related to incurable human<br />
pathologies (amyloid-related diseases) associated with formation of amyloid deposits<br />
in the body. Insulin amyloid deposits have been observed in patients with diabetes<br />
after repeated subcutaneous insulin injection. The recent data confirm the toxic<br />
effect of aggregates on the cells, however, it was found that reduction of amyloid<br />
aggregates plays important role in prevention as well as therapy of amyloidosis. We<br />
investigated capability of phytoalexin derivatives to affect the insulin amyloid<br />
aggregation. Phytoalexins are low molecular weight secondary metabolites produced<br />
by plants after their exposure to biological or physical stress. By ThT and ANS<br />
fluorescence assays, the efficiency of derivatives to inhibit formation of amyloid<br />
aggregates and depolymerize pre-formed amyloid fibrils was studied. We identified<br />
very effective phytoalexin derivates, benzocamalexin and cyclobrassinin, with<br />
significant inhibiting and depolymerizing activities. For these derivatives, the<br />
determined IC50 and DC50 values are at low micromolar concentrations. Our data<br />
suggest the potential therapeutic use of the most effective phytoalexins in the<br />
reduction of insulin amyloid aggregation.<br />
Acknowledgements: This work was supported within the projects Nos. 26220220005,<br />
26220120033 and 26220120021 in frame of SF EU, Centre of Excellence of SAS<br />
Nanofluid, VEGA 0079, 0056 and VVGS PF 13/2010/Ch.<br />
205<br />
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Posters<br />
89.<br />
THE LYSM DOMAIN IN SURFACE IMMUNOGENIC PROTEIN (SIP) AND ITS INFLUECE<br />
ON ELICITATION OF IMMUNITY AGAINST STREPTOCOCCUS AGALACTIAE<br />
Barbora Vidová, Michal Chotár and Andrej Godány<br />
Institute of Molecular Biology, SAS Bratislava<br />
The microbial pathogens are characterized by adapting their surface proteins in order<br />
to protect themselves against the host immune system; accordingly, there is a<br />
possibility of some variability also within the sip gene. This work was focused on<br />
determining of potential variability within the sip gene in the collection of bovine<br />
isolates of S. agalactiae by multiplex PCR method and characterizing the protein Sip<br />
on its nucleotide level, with the aim to study its probable variability and prepare the<br />
basis for future study of its epitope responsible for immunity elicitation.<br />
Concurrently, we also examined the presence of a peptidoglycan-binding anchor, the<br />
LysM domain motif. Because the identification of both variability and anchor motif is<br />
important for the identification of a potential epitope within the Sip, the immune<br />
responses to whole and partial Sip were also tested in the mice model.<br />
Acknowledgements: This work was supported by the VEGA grant no. 2/0121 of the<br />
Slovak Academy of Sciences.<br />
206<br />
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XI. REACTIVE SPECIES IN BIOMEDICINE<br />
207<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
90.<br />
EFFECT OF OMEGA-3 FATTY ACIDS ON PON 1 ARYLESTERASE AND LACTONASE<br />
ACTIVITY IN CHILDREN SUFFERING FROM HYPERCHOLESTEROLEMIA<br />
Lucia Andrezálová, Zuzana Országhová, Jana Muchová and Zdeňka Ďuračková<br />
Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry,<br />
Faculty of Medicine, Comenius University Bratislava, Slovakia<br />
Hypercholesterolemia is characterized by an increased level of blood cholesterol and<br />
give rise to the development of atherosclerosis. The atherosclerosis process begins<br />
already in childhood and is the main risk factor that contributes to the pathology of<br />
cardiovascular diseases in adulthood.<br />
Paraoxonase 1 (Pon 1), HDL-associated enzyme with antioxidative properties, plays<br />
an important role in the antiatherogenic processes. The aim of this study was to<br />
determine PON 1 arylesterase and lactonase activity in 35 moderately<br />
hypercholesterolemic children (average age 16 ± 3.34) and study the changes in its<br />
activity during the daily administration of nutritional supplement (CW) comprising of<br />
omega-3 fatty acids (1000 mg EPA/DHA) and phytosterol esters (1300 mg) over a 16<br />
week period PON 1 arylesterase and lactonase activities were determined<br />
spectophotometrically towards phenylacetate and dihydrocoumarin in serum<br />
samples. CW supplementation did not significantly affect PON 1 activities. While<br />
arylesterase activity was within the physiological range, lactonase activity was<br />
moderately reduced compared to healthy subjects. PON 1 activities were correlated<br />
with other biochemical parameters as well as with lipid peroxides and homocysteine.<br />
Acknowledgement: Authors wish to thank for partial financial support to Obsidian<br />
Research Limited, Pork Talbot (Great Britain), Mind and Health, civil association and<br />
MVTS and VEGA grants of Ministry of Education SR.<br />
208<br />
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Posters<br />
91.<br />
ANTIOXIDANT RESPONSE TO TREATMENT WITH NATURAL COMPOUNDS AND AN<br />
NO DONOR IN EXPERIMENTAL HYPERTENSION<br />
Ima Dovinová 1 , Zuzana Pakanová 2 , StanislavaVranková 1 , Oľga Pecháňová 1 , Soňa<br />
Čačányiová 1 , František Kristek 1 and Helena Paulíková 2<br />
1 Institute of Normal and Pathological Physiology, SAS, Bratislava;<br />
2 Department of Biochemistry and Microbiology, Faculty of Chemical and Food<br />
Technology STU, Bratislava<br />
SOD enzymes play an important role in cardiovascular tissue by protecting NO against<br />
oxidative inactivation by superoxide. Both NO and superoxide are free radicals with<br />
unpaired electrons and can react with one another. Studies with pharmacologically<br />
inhibited SOD1 and SOD3 showed that NO cannot be released from endothelium<br />
without oxidative degradation, that is, SOD enzymes play an important role in<br />
vasodilatation and in protection of NO in blood vessel walls.<br />
In this study, we observed the effect of flavonoids (Alibernet red wine extract),<br />
melatonin, and an NO-donor, PETN, on antioxidant response of blood vessels and the<br />
heart in animal hypertension models, SHR and SHR-cp.<br />
The experiments show that in young hypertensive rats the Alibernet extract increased<br />
the activity of SOD and NOS in the left ventricle of SHR and the total antioxidant<br />
status in plasma.<br />
In adult hypertensive rats, the NO donor, PETN, increased SOD and GPx activities and<br />
decreased left ventricle damage.<br />
The results indicate that the most remarkable effects of the Alibernet red wine<br />
extract in young hypertensive rats, and of the PETN NO donor in adult hypertensive<br />
rats are related to protective effects of SOD enzymes in the heart.<br />
Ackowledgement: The work was supported by VEGA Grant No 2/0066/08.<br />
209<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
92.<br />
WILSON'S DISEASE AND OXIDATIVE STRESS<br />
Marián Koláček 1 , Jana Muchová 1 , Eva Uhlíková 1 ,<br />
Viera Kupčová 2 and Ladislav Turecký 1<br />
1 Institute of medical chemistry, biochemistry a clinical biochemistry<br />
Faculty of medicine Comenius University, Bratislava<br />
2 3 th internal clinic Faculty of medicine Comenius University<br />
Wilson's disease (hepatolenticular degeneration) is autosomal hereditary recessive<br />
disorder of copper metabolism with disorder of copper excretion into the bile and its<br />
excessive storage in some organs. Disorder is caused by mutation of the gene<br />
encoding specific copper transport protein – ATP7B for ATPase type P. Mutated<br />
ATP7B inhibits copper excretion into the bile, stimulates its direct release into the<br />
blood and its storage in organs and this leads to increased free radicals production.<br />
Decreased production of functional ceruloplasmin increases iron toxicity and thus<br />
oxidative stress becomes further increased.<br />
Our goal was to find out how are selected parameters of oxidative stress affected by<br />
Wilson's disease. The studied group consisted of 17 patients with diagnosed Wilson's<br />
disease (9 women and 8 men). We determined total antioxidant capacity of blood<br />
plasma (TEAC), activity of a superoxide dismutase (SOD) in erythrocytes<br />
and nitrotyrosine – marker of damage of proteins by free radicals derived from<br />
nitrogen.<br />
TEAC, as well as SOD activity in erythrocytes of patients with Wilson's disease were<br />
higher compared to control group (2,51 mmol/l vs. 1,22 mmol/l and 11,7 U/g Hb vs.<br />
9,2 U/g Hb). We saw no significant difference in levels of nitrotyrosine of patients and<br />
controls (54,7 nmol/l vs. 53,2 nmol/l).<br />
The increase of TEAC of blood plasma in patients with Wilson's disease may be a<br />
compensatory response to the decline of ferooxidase activity of ceruloplasmin in<br />
serum as well as a response to increased oxidative stress in liver.<br />
210<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
93.<br />
AGE-RELATED CHANGES IN ACTIVITIES OF MITOCHONDRIAL ELECTRON TRANSPORT<br />
CHAIN COMPLEXES IN THE RAT HEART.<br />
Stanislav Kuka, Zuzana Tatarková, Peter Račay, Ján Lehotský,<br />
Dušan Dobrota and Peter Kaplán<br />
Department of Medical Biochemistry Jessenius Faculty of Medicine, Comenius<br />
University, Martin<br />
Oxidative damage of tissues by reactive oxygen species (ROS) has been implicated to<br />
be a major factor of aging process. Mitochondria are considered to be the main<br />
intracellular sources of ROS as well as the major target for their damaging effects.<br />
In our study we examined age-related changes in activities of electron transport chain<br />
complexes in cardiac mitochondria of adult (6 months), old (15 months) and<br />
senescent (26 months) male Wistar rats. Our results display non-uniform changes in<br />
activities of particular ETC complexes. While old rats showed no significant changes in<br />
activities of complexes I and II compared with adult control rats, in senescent rats the<br />
activities of both complexes were significantly decreased (P < 0.01). On the other<br />
hand, complex III activity was significantly lower already at 15 months (P < 0.05) and<br />
further decreased at age of 26 months (P < 0.01). The most pronounced age-related<br />
changes were observed in complex IV activity. Compared to adult, complex IV activity<br />
decreased by 21% (P < 0.01) at old age and by 37% (P
Posters<br />
94.<br />
SELECTIVE PHOSPHODIESTERASE-3 INHIBITOR OLPRINONE ALLEVIATES OXIDATIVE<br />
LUNG INJURY INDUCED BY MECONIUM<br />
Daniela Mokrá 1 , Anna Drgová 2 , Rudolf Pullmann st. 3 and Andrea Čalkovská 1<br />
1 Department of Physiology, 2 Department of Biochemistry, 3 Department of Clinical<br />
Biochemistry, Jessenius Faculty of Medicine, Comenius University, Martin<br />
In the pathophysiology of neonatal meconium aspiration syndrome, inflammation<br />
and oxidation play a key role. This study evaluated effects of phosphodiesterase<br />
(PDE)-3 inhibitor olprinone on meconium-induced lung injury.<br />
Oxygen-ventilated adult rabbits received intratracheally meconium or saline (Sal,<br />
n=7). Thirty minutes after meconium instillation, animals were treated by olprinone<br />
(0.2 mg/kg i.v., Mec+Olp, n=7) or were left without treatment (Mec, n=8). All animals<br />
were oxygen-ventilated for additional 5 hours. Left lungs were saline-lavaged and<br />
differential WBC in the sediment was estimated. Right lungs were used to determine<br />
lung edema by wet/dry weight ratio and oxidative damage by estimation of thiol<br />
content, conjugated dienes, thiobarbituric acid-reactive substances (TBARS),<br />
dityrosines and lysine-lipid peroxidation products concentrations, cytochrome c<br />
oxidase activity (COX) in mitochondria of lung, and total antioxidant status (TAS) in<br />
lung homogenate. In blood plasma, TBARS and TAS were determined.<br />
Meconium instillation increased lung neutrophils and edema formation and<br />
concentrations of oxidation markers and decreased concentration of TAS. Olprinone<br />
reduced the lung edema, lung neutrophils and several oxidation markers and<br />
increased TAS concentrations.<br />
Selective PDE-3 inhibitor olprinone effectively reduced oxidative lung injury in<br />
meconium-instilled animals.<br />
Acknowledgments: Supported by Project “Center of Excellency in Perinatology” No.<br />
26220120016 co-financed by EC, and by Grant VEGA No. 1/0061/08.<br />
212<br />
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Posters<br />
95.<br />
METYLNICOTINAMIDE AND DNA OXIDATION DAMAGE IN RATS WITH<br />
STREPTOZOTOCINE INDUCED DIABETES MELLITUS<br />
Zuzana Országhová 1 , Zuzana Paduchová 1 , Ingrid Žitňanová 1 , Cezary Watala 2 ,<br />
Jana Muchová 1 and Zdeňka Ďuračková 1<br />
1 Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry,<br />
Medical School, Comenius University, Bratislava, Slovakia;<br />
2 Department of Haemostasis and Haemostatic Disorders,<br />
Medical University of Lodz, Poland<br />
Increased oxidative and glycooxidative stresses contribute to the development and<br />
progression of diabetes-associated complications. Under conditions of DM many<br />
important biomolecules including DNA, lipids or proteins are affected.<br />
N(1)methylnicotin-amide chloride (MNA), which is a derivative of nicotinamide, is an<br />
intensively studied agent possessing remarkable anti-inflammatory as well as<br />
antidiabetic properties. This effect is associated with scavenging of reactive forms of<br />
oxygen, in particular superoxide radical anion and hydroxyl radical. In this study we<br />
have studied the level of oxidative damage to DNA in lymphocytes and liver tissue as<br />
well as markers of glycation (AGEs) and oxidation (carbonyls) damage of proteins in<br />
streptozotocine induced diabetic rats. Administration of MNA in dose 200 mg/kg of<br />
weight during 7 weeks significantly decreased DNA damage in lymphocytes and level<br />
of protein carbonyls. All doses of MNA decreased DNA damage in liver tissue.<br />
Advanced glycation of proteins were not affected with MNA. Our results show<br />
possible beneficial protective role of MNA against oxidative stress and damage of<br />
important biomolecules under the conditions of DM.<br />
Acknowledgement: This study was partially supported by MVTS and VEGA grants.<br />
213<br />
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Posters<br />
96.<br />
ANTIOXIDANT CAPACITY OF SELECTED ANALOGS OF NUCLEIC ACID COMPONENTS:<br />
COMPARISON OF CELL-FREE AND CELL-BASED ASSAYS<br />
Eliška Procházková, Petr Jansa, Lucie Čechová, Ivan Votruba and<br />
Helena Mertlíková-Kaiserová<br />
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech<br />
Republic, Gilead Sciences & IOCB Research Center, 166 10 Prague, Czech Republic<br />
Overproduction of reactive oxygen species (ROS) is a phenomenon accompanying a<br />
number of human diseases as well as drug toxicity. Pharmacological potential of the<br />
analogs of nucleic acid components is of considerable interest and so is their ability to<br />
interfere with ROS production. In this study we used a series of poly-substituted<br />
pyrimidines and purines in order to explore their antioxidant capacity. Their ability of<br />
direct radical scavenging was first determined with use of the trolox equivalent<br />
antioxidant capacity (TEAC) assay. Consequently, the effects of the tested compounds<br />
on Fe 2+ /ascorbate-induced lipid peroxidation in rat liver microsomes were studied.<br />
Finally, ROS cellular levels were traced in compound-pretreated HepG2 cells exposed<br />
to oxidative stress by means of a redox-sensitive fluorescein-based probe CM-<br />
H 2 DCFDA. Although remarkable antioxidant capacity of several compounds was<br />
detected in individual assays, we observed that the results largely depend on the<br />
method used and the data therefore have to be interpeted with caution. We suggest<br />
that these variations could be due to different physico-chemical properties of the<br />
compounds.<br />
Acknowledgement: This work was supported by the Research project of the IOCB<br />
OZ40550506 and the Project #1M0508 by the Ministry of Education, Youth and Sports<br />
of the Czech Republic.<br />
214<br />
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Posters<br />
97.<br />
EFFECTIVENESS OF PHENOLS AS ANTIOXIDANTS AGAINST SUPEROXIDE RADICAL<br />
Beáta Veliká and Ivan Kron<br />
Department of Medical Chemistry, Biochemistry, Clinical Biochemistry and LABMED<br />
a.s, Faculty of Mediciny, PJ Šafarik University in Košice, Slovakia<br />
Phenolic antioxidants are included in the category of free radical terminators. It is<br />
known, that phenols reduce rates of oxidation of organic mater by transferring a<br />
hydrogen atom from OH groups to the chain carrying ROO° radicals (Foti, 2007). It<br />
was proved, that the intramolecular hydrogen bonding has an important effect on<br />
the antioxidant properties of phenols. Various ortho-substituents may function as H-<br />
bond acceptors (Foti, 2007).<br />
For the study of antioxidant properties of selected compounds (resorcinol,<br />
hydroquinone, pyrocatechol and pyrogallol) we used a generator of superoxide<br />
radical (Beauchamp and Fridovich, 1971) with NBT as a detector after 5, 10, 15 and<br />
20 min of UV illumination. These compounds are able to capture and acquit an<br />
electron, what depends on the structure of used compounds. The antioxidant activity<br />
of phenols is related to the number and position of hydroxyl groups on benzene<br />
skeleton. The best antioxidant activity showed dihydroxyderivatives in para- and<br />
ortho- position. On the other hand, prooxidant properties were recorded for<br />
resorcinol and pyrogallol in time and concentration dependance. Our study confirms<br />
the relationship between antioxidant properties and structure.<br />
Acknowledgement: VEGA 1/0624/08.<br />
215<br />
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Posters<br />
98.<br />
EFFECT OF N-3 POLYUNSATURATED FATTY ACIDS SUPPLEMENTATION ON RAT LIVER<br />
IN DIFFERENT THYROID STATUSES<br />
Martina Vokurková 1,2 , Hana Rauchová 1,2 , Stanislav Pavelka 1 ,<br />
Tomáš Soukup 1 and Narcis Tribulová 3<br />
1 Institute of Physiology, Academy of Science of the Czech Republic, 2 Centre for<br />
Cardiovascular Research, Prague, Czech Republic, 3 Institute for Heart Research, Slovak<br />
Academy of Sciences, Bratislava, Slovak Republic<br />
It is known that thyroid hormones influence lipid metabolism and oxidative stress.<br />
Epidemiological studies demonstrate that n-3 polyunsaturated fatty acids (PUFAs)<br />
consumption is associated with a reduced risk of atherosclerosis and hyperlipidemia.<br />
The aim of our study was to test how PUFAs supplementation in different thyroid<br />
statuses of rat can affect lipid metabolism and parameters of oxidative stress in the<br />
liver. The different thyroid statuses of the experimental groups (euthyroid,<br />
hyperthyroid and hypothyroid) were defined by the levels of the thyroid hormones in<br />
the plasma and an activity of liver mitochondrial glycerol-3-phosphate<br />
dehydrogenase. In this study, the hyperthyroid rats had significantly decreased levels<br />
of total plasma cholesterol and LDL-cholesterol than the hypothyroid rats. The levels<br />
of HDL-cholesterol were increased in the hyperthyroid rats in comparison with the<br />
euthyroid rats. The concentrations of liver thiol groups differed according to the<br />
thyroid statuses (they were the highest in the hypothyroid group and the lowest in<br />
the hyperthyroid group). However, no differences in the content of conjugated<br />
dienes were observed among the experimental groups. PUFAs supplementation thus,<br />
in our experimental design, modified neither lipid metabolism nor parameters of the<br />
oxidative stress in the liver.<br />
Acknowledgements: This work was supported by the GACR (303/09/0570) and<br />
Ministry of Education, Youth and Sport (1M6798582302 and AV0Z 50110509).<br />
216<br />
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Posters<br />
99.<br />
EFFECT OF RENAL TRANSPLANTATION ON OXIDATIVE STRESS-RELATED<br />
BIOMARKERS<br />
Adéla Zdařilová 1 , Alena Rajnochová Svobodová 1 , Jana Zapletalová 2 , Pavel Štrebl 3 ,<br />
Josef Zadražil 3 and Jitka Vostálová 1<br />
1 Department of Medical Chemistry and Biochemistry, 2 Department of Medical<br />
Biophysics, Faculty of Medicine and Dentistry, Palacký University, Hněvotínská 3, 775<br />
15 Olomouc; 3 Department of Internal Medicine III University Hospital, I. P. Pavlova 6,<br />
775 20 Olomouc, Czech Republic<br />
Patients after kidney transplantation (KT) have a high prevalence of oxidative stress<br />
(OS) that is associated with the excess of cardiovascular complications observed in<br />
this population. Determination of OS-related parameters together with specific<br />
kidney function markers is used to monitor graft function.<br />
Time-dependent changes in OS-related markers in patients (N = 39; male = 23; female<br />
= 16; mean age = 57 ± 10) before (day 0) and after KT (day 1, 7, 30, 90 and 180) were<br />
evaluated. Total antioxidant capacity (TAC), levels of advanced oxidation protein<br />
products (AOPP), lipid peroxidation as thiobarbituric acid-reactive substances (TBARS)<br />
and reduced glutathione (GSH), activities of glutathione peroxidase (GPX), catalase<br />
(CAT) and superoxide dismutase (SOD) and kidney function markers were measured.<br />
AOPP, TAC and TBARS were significantly decreased whereas GSH was significantly<br />
increased after KT. Antioxidant enzyme activities did not changed after KT during<br />
monitored period. Of specific kidney function markers glomerular filtration was<br />
significantly increased and creatinine level was significantly decreased after KT.<br />
Acknowledgements: This work was supported by grants MSM 6198959216 and MZ CR<br />
(NS/9964-4).<br />
217<br />
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XIII. XENOBIOCHEMISTRY<br />
218<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
100.<br />
BIOTRANSFORMATION OF SELECTED ANTHELMINTICS IN RAT TAPEWORM<br />
HYMENOLEPIS DIMINUTA<br />
Hana Bártíková, Jana Firbasová, Ivan Vokřál, Lenka Skálová, Jiří Lamka,<br />
Vladimír Kubíček and Barbora Szotáková<br />
Faculty of Pharmacy, Charles University, Hradec Králové, Czech Republic<br />
In all organisms, drug metabolising enzymes (DME) serve as an efficient defence<br />
against the potential negative action of xenobiotics. While human and mammalian<br />
DME have been intensively studied for many years, DME of helminth parasites have<br />
been relatively little investigated so far in spite of the fact that helminth´ DME can<br />
preserve the parasite against the anthelmintic drug and could contribute to drugresistance<br />
development. The present project was designed to evaluate DME activities<br />
in rat tapeworm (Hymenolepis diminuta), This tapeworm, infecting mammals and<br />
causing hymenolepiasis, is often used as model species for investigation into Cestodes<br />
as the life cycle of H. diminuta is relatively simple, involves rodents (rats primarily) as<br />
the definitive host and beetles (Tribolium or Tenebrio) as the intermediate host. The<br />
implementation of H. Diminuta breeding was the first task of our project. Rats were<br />
experimentally infected with H. diminuta cysticercoids obtained from T. molitor and<br />
then adult worms were collected from rat small intestine. Activities of oxidation<br />
enzymes, carbonyl-reducing enzymes and conjugation enzymes were tested in<br />
subcellular fractions of H. diminuta homogenate. The results showed the ability of H.<br />
diminuta enzymes to reduce effectively the carbonyl group of xenobiotics, e.g.<br />
anthelmintics flubendazole and mebendazole. Catalase, peroxidases, UDPglucuronosyl<br />
transferase and glutathione-S-transferase activities were detected as<br />
well. These results document the ability of H. diminuta to detoxify different<br />
xenobiotics including certain anthelmintics.<br />
Acknowledgements: This project was supported by the Czech Science Foundation,<br />
grant No. P502/10/0217 and by SVV-2010-261-003.<br />
219<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
101.<br />
INHIBITORY EFFECT OF NATURAL FLAVONOIDS ON EQUINE LIVER GLUTATHIONE S-<br />
TRANSFERASE<br />
Iva Boušová, Zuzana Průchová, Lucie Trnková and Jaroslav Dršata<br />
Department of Biochemical Sciences, Charles University in Prague, Faculty of<br />
Pharmacy, Hradec Králové<br />
Mammalian glutathione S-transferases (GST, EC 2.5.1.18), group of intracellular<br />
enzymes involved in detoxification of xenobiotics, belong to the most abundant<br />
cytosolic proteins. They catalyze conjugation of the thiol group of the glutathione<br />
(GSH) to electrophilic xenobiotics, and thereby defend cells against the mutagenic,<br />
carcinogenic, and toxic effects of these compounds. Several classes of GST isozymes<br />
differing in substrate specificity exist.<br />
Flavonoids are a group of polyphenolic compounds that are widely distributed in<br />
plant kingdom. They are known to possess antioxidant, anti-radical, antiviral,<br />
antibacterial, anti-inflammatory, and vasodilatory activity. Besides positive acting,<br />
flavonoids exert also pro-oxidative effects and are able to inhibit various enzymes<br />
(e.g. cyclooxygenase and glutathione peroxidase). We suppose that dietary flavonoids<br />
may inhibit GST and thus influence detoxification of xenobiotics.<br />
GST was incubated in the presence or absence of flavonoids (0-100 µM). Then its<br />
activity was measured using common spectrophotometric assay with 1-chloro-2,4-<br />
dinitrobenzene adapted to 96-well plates. Inhibitory effect of studied flavonoids was<br />
characterized by IC 50 values. The most pronounced inhibition was found in the case of<br />
epigallocatechin gallate, which is the major polyphenolic constituent in the green tea.<br />
The obtained results indicated that GST can be a potential target for inhibitory effect<br />
of flavonoids.<br />
Acknowledgments: This study was supported by the Czech Science Foundation (GAČR,<br />
grant No. 524/09/P121).<br />
220<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
102.<br />
MULTIDRUG RESISTANT P-GLYCOPROTEIN POSITIVE CELLS ARE ALSO<br />
CROSS-RESISTANT TO CISPLATIN<br />
Lenka Gibalová 1 , Ján Sedlák 2 , Alena Reháková 1 , Martina Labudová 3 ,<br />
Zdena Sulová 1 and Albert Breier 1<br />
1 Institute of Molecular Physiology and Genetics SAS Bratislava, 2 Cancer Research<br />
Institute SAS Bratislava, 3 Institute of Virology SAS Bratislava<br />
P-glycoprotein (P-gp, a drug transporter of the plasma membrane) mediated<br />
multidrug resistance of neoplastic cells represents a real problem in the<br />
chemotherapeutic treatment. Mouse leukemia cells L1210 (S) and two drug resistant<br />
cell lines, in which the overexpression of P-gp was induced by i. selection with<br />
vincristine (R) or by ii. transfection of S cells with human gene for P-gp (T) are our<br />
experimental models. Cisplatin (cisPt) is a substance untransportable by P-gp. We<br />
found that R and T cells are also resistant to cisPt. However, cisPt resistance in R and<br />
T cells could not be reversed by verapamil (known P-gp inhibitor), that excluding<br />
responsibility of P-gp transport activity for this resistance. CisPt induced more<br />
pronounced entry to apoptosis in S cells than in R or T cells. While similar levels of<br />
Bax and Bcl-2 proteins were observed in P-gp negative and positive cells, cisPt<br />
induced a more significant decrease of the Bcl-2 levels in S cells than in R and T cells.<br />
Consistent with this, cisPt induced a larger decrease of the Bcl-2 content in the Bcl-<br />
2/Bax heterooligomer in S cells than in R cells. Moreover, cisPt induced apoptotic<br />
DNA fragmentation was observed in all three cell sublines. All observations indicated<br />
that expression of P-gp in L1210 cells is directly associated with reduction of cisPt<br />
induced apoptosis and consequently with resistance to this drug that is not<br />
connected with P-gp induced cisPt efflux.<br />
Ackowledgement: This work was supported by: VVCE-0064-07, APVV-0084-07 and<br />
VEGA 2/0123/10, 2/0155/09.<br />
221<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
103.<br />
THE EFFECTIVENESS OF ORACIN IN ENHANCING THE CYTOTOXICITY OF<br />
DOXORUBICIN THROUGH THE INHIBITION OF DOXORUBICIN DEACTIVATION IN<br />
BREAST CANCER CELL LINE MCF-7<br />
Veronika Hanušová 1 , Lenka Vildová 1 , Věra Králová 2 , Ladislava Schröterová 2 , Lenka<br />
Trilecová 3 , Alena Pakostová 1 and Lenka Skálová 1<br />
1 Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Hradec<br />
Králové, Czech Republic, 2 Department of Biology, Faculty of Medicine, Charles<br />
University, Hradec Králové, Czech Republic 3 Veterinary Research Institute, Brno,<br />
Czech Republic<br />
Doxorubicin (DOX) has played a key role in the treatment of breast cancer, but the<br />
clinical utility of this agent is strictly limited by his associated toxicities, especially<br />
cardiotoxicity. In present project was studied the possibilities of inhibition of the DOX<br />
reduction in breast cancer cell line MCF-7, one possible strategy to improve DOX<br />
efficacy. DOX is deactivated mainly by the action of carbonyl reductases in MCF-7.<br />
Oracin (ORC, 6-[2-(2-hydroxyethyl)aminoethyl]-5,11-dioxo-5,6-dihydro-11Hindeno[1,2-c]<br />
isoquinoline) significantly inhibited DOX reduction in MCF-7 cytosol.<br />
The cytotoxicity of DOX, ORC and DOX+ORC combinations in MCF-7 cells was assayed<br />
using three different end-point tests of cell viability. In all DOX+ORC combinations,<br />
only non-cytotoxic ORC concentrations were used to avoid the possible additive toxic<br />
effect of ORC. After a 48-hour exposition, DOX together with ORC was able to kill<br />
55 % more cells than DOX alone. All DOX+ORC combinations were more toxic in<br />
rapidly proliferating cells than in slowly proliferating cells. ORC significantly increases<br />
DOX efficacy in MCF-7 cells, probably due to the inhibition of DOX reductases. Results<br />
suggest that concomitant use of ORC and DOX may improve the therapeutic index of<br />
DOX in the managements of breast cancer.<br />
222<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
104.<br />
CYTOCHROME b 5 POTENTIATES ACTIVITIES OF CYTOCHROMES P450 1A1 AND 1A2<br />
TO OXIDIZE ANTICANCER DRUG ELLIPTICINE TO PHARMACOLOGICALLY EFFICIENT<br />
METABOLITES<br />
Věra Kotrbová 1 , Barbora Mrázová 1 , Eva Frei 2 and Marie Stiborová 1<br />
1 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030,<br />
128 40 Prague 2, Czech Republic; 2 Department of Molecular Toxicology, German<br />
Cancer Research Center, 69 120 Heidelberg, Germany<br />
Ellipticine is an alkaloid exhibiting significant antineoplastic activities. Its mode of<br />
action is based mainly on cytochrome P450 (CYP)-mediated formation of covalent DNA<br />
adducts. Many CYP-dependent reactions have been shown to be stimulated by<br />
another microsomal protein, cytochrome b 5 (b 5 ). Five ellipticine metabolites, 9-OH-,<br />
12-OH-, 13-OH-, 7-OH-ellipticine and the ellipticine N 2 -oxide, are generated by<br />
CYP1A1/2. The patterns and amounts of ellipticine metabolites vary significantly when<br />
b 5 is present in the incubations. The formation of detoxication products of its oxidation<br />
(7-OH- and 9-OH-ellipticine) is decreased, while generation of 13-OH- and 12-OHellipticine,<br />
the metabolites responsible for formation of DNA adducts, is increased<br />
considerably. The enhanced generation of ellipticine-DNA adducts in the presence of<br />
b 5 in the system was confirmed by 32 P postlabeling. The treatment of rats with<br />
ellipticine resulted in elevated expression of b 5 . Other proteins with or without heme<br />
in their molecules are without such effects on ellipticine oxidation. Cytochrome b 5<br />
affects also the kinetics of ellipticine oxidation by CYP1A1/2. In the case of the<br />
CYP1A1, the presence of b 5 changes the kinetics of ellipticine oxidation to 12-OH- and<br />
13-OH-ellipticine from hyperbolic to sigmoidal, whereas no cooperativity was<br />
observed with CYP1A2.<br />
Acknowledgements: Supported by GACR (203/09/0812, P301/10/0356), the Czech<br />
Ministry of Education (MSM0021620808, 1M0505) and GAUK (127208).<br />
223<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
105.<br />
SELENITE-INDUCED CELL DEATH IN HUMAN COLON CANCER CELLS<br />
Věra Králová and Emil Rudolf<br />
Department of Medical Biology and Genetics, Faculty of Medicine in Hradec Králové<br />
Selenium compounds have been shown to play role in chemoprevention of tumours.<br />
Sodium selenite was reported to inhibit proliferation and induce different types of<br />
cell death in cancer cells in vitro. In our study we tested the effects of sodium selenite<br />
on cell proliferation and cell death in human colon cancer cell line HCT 116. Cell<br />
proliferation was measured by xCELLigence impedance method. Cell morfology was<br />
followed by time-lapse videomicroscopy. Caspase activity and mitochondrial<br />
membrane potential were determined by flow cytometry. Activation of DNA damage<br />
response was detected using western blotting and immunofluorescence. Sodium<br />
selenite at concentration of 10 µM induced cell death in HCT 116 cells characterised<br />
by cell detachment and rounding, decrease in mitochondrial membrane potential and<br />
activation of caspases. Selenite-induced cell death was prevented with antioxidant<br />
MnTMPyP and enhanced by depletion of reduced glutathion with BSO, which<br />
suggests role of oxidative stress. The process was accompanied by increase in ATM<br />
kinase and histone H2A.X phosphorylation.<br />
Acknowledgements: This work was supported by Charles University Grant Agency<br />
(GAUK) Research Project 129609.<br />
224<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
106.<br />
EFFECTS OF FLAVONOIDS ON CYTOCHROMES P450 AFTER PERORAL SINGLE DOSE<br />
ADMINISTRATION TO MALE RATS<br />
Jitka Křížková, Kamila Burdová, Petr Hodek and Marie Stiborová<br />
Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova<br />
2030, Praha 2, 128 43<br />
In recent years, the consumption and use of dietary supplements containing<br />
concentrated phytochemicals (e.g. flavonoids) increased dramatically. Flavonoids, as<br />
foreign compounds (xenobiotics), have great potential to modulate the activity of<br />
cytochrome P450s (CYPs), xenobiotic-metabolizing enzymes involved in the activation<br />
and detoxification of food and environmental carcinogens. Thus, the aim of this study<br />
was to investigate the effects of selected flavonoids on CYPs in rat liver and small<br />
intestine, as the two main organs responsible for xenobiotic metabolism, after p.o.<br />
single dose (60 mg/kg b.w.) administration to male rats.<br />
The expression and specific activity of CYP1A and CYP2B were determined using<br />
Western blotting technique and specific activity assays with alkyl-resorufin<br />
derivatives. The rats were sacrificed 24h, 48h and 72h after the single dose<br />
treatment.<br />
To evaluate the effects of flavonoids on CYPs along small intestine, the tissue was<br />
dissected into proximal (near pylorus), middle and distal parts. Of the two natural<br />
compounds, isoquercitrin was the most efficient CYP1A1 inducer in the proximal part<br />
of small intestine 72h after the single dose treatment. Obtained data demonstrate<br />
the different effects of selected flavonoids on CYP expression in rat liver and small<br />
intestine in dependence on the time after the administration. Since these<br />
phytochemicals are xenobiotics, and thus they can increase the human risk of cancer<br />
development, their consumption in large quantities should be carefully considered.<br />
Acknowledgements: This work was supported by the Czech Science Foundation (Grant<br />
No. 305/09/H008) and the Grant Agency of Charles University (Grant No. 4909).<br />
225<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
107.<br />
ACTIVITIES OF DRUG-METABOLIZING AND ANTIOXIDANT ENZYMES IN<br />
HAEMONCHUS CONTORTUS STRAINS RESISTANT OR SENSITIVE TO ANTHELMINTICS<br />
Tamara Lasotová 1 , Hana Bártíková 1 , Ivan Vokřál 1 , Barbora Szotáková 1 , Vladimír<br />
Kubíček 1 , Jiří Lamka 1 , Marián Várady 2 and Lenka Skálová 1<br />
1 Faculty of Pharmacy, Charles University, Hradec Králové, Czech Republic<br />
2 Institute of Parasitology, Slovak Academy of Sciences, Košice, Slovakia<br />
Haemonchus contortus is one of the most pathogenic parasites of small ruminants<br />
(e.g., sheep and goat). The treatment of haemonchosis is complicated because of<br />
frequent resistance of H. contortus to common anthelmintics. The aim of this project<br />
was to compare the activities of antioxidant enzymes and biotransformation enzymes<br />
toward selected anthelmintics and model xenobiotics in three different strains of H.<br />
contortus: ISE strain (sensitive to common anthelmintics), BR strain (resistant to<br />
benzimidazole anthelmintics) and WR strain (resistant to all common anthelmintics).<br />
For this purpose, lambs were experimentally infected with H. contortus L3 larvae and<br />
then adults worms were collected from ovine abomasums. The biotransformation of<br />
anthelmintics albnedazole and flubendazole was tested in vitro (in subcellular<br />
fractions of H. contortus homogenate) as well as ex vivo (in living nematodes<br />
cultivated in flasks with medium). In vitro, the activities of several carbonyl-reducing<br />
enzymes, UDP-glucosyltransferase, glutathione-S-transferases, peroxidases, catalase,<br />
and superoxid dismutase toward model substrates were tested. The acquired data<br />
showed significant differences in tested activities between sensitive and resistant H.<br />
contortus strains. The altered activities of certain detoxifying enzymes could protect<br />
some parasites against the toxic effect of the drugs and contribute to drug-resistance<br />
of these parasites.<br />
Acknowledgement: This project was supported by the Czech Science Foundation,<br />
grant No. P502/10/0217.<br />
226<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
108.<br />
CYTOCHROMES P450 1A1/2 OXIDIZE CARCINOGENIC ARISTOLOCHIC ACID I<br />
FORMING ITS DETOXICATION METABOLITE AND DECREASING LEVELS OF AA-DNA<br />
ADDUCTS IN VIVO<br />
Kateřina Levová 1 , Jana Šístková 1 , Eva Frei 2 , Volker M. Arlt 3 , David H. Phillips 3 , Heinz<br />
H. Schmeiser 2 and Marie Stiborová 1<br />
1 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030,<br />
128 40 Prague 2, Czech Republic; 2 German Cancer Research Center, 69 120<br />
Heidelberg, Germany; 3 Institute of Cancer Research, Section of Molecular<br />
Carcinogenesis, Sutton, Surrey, SM2 5NG, United Kingdom<br />
Aristolochic acid (AA) causes the development of aristolochic acid nephropathy<br />
(AAN), the Balkan endemic nephropathy (BEN) and urothelial cancer. One of the<br />
common features of AAN and BEN is that not all individuals exposed to AA suffer<br />
from these diseases. We found that hepatic microsomes of wild-type (WT) mice<br />
oxidize AAI in vitro to detoxication metabolite, AAIa, while those of a HRN line were<br />
without this effect. Levels of AA-DNA adducts in organs of WT mice exposed to AAI<br />
were up to more than 10-fold lower than in those of HRN mice. To define the role of<br />
CYP enzymes in AAI oxidation, we used besides hepatic microsomes of these mouse<br />
models, also those of human and rat. Using correlations between the CYP catalytic<br />
activities provided with the set of human hepatic microsomes from 14 different<br />
human donors and the rates of formation of AAIa metabolite in the same set of<br />
human hepatic microsomes, we found a highly significant correlation coefficient<br />
between the rates of phenacetin O-deethylase, a marker for CYP1A2, and the levels<br />
of AAIa. The results demonstrate a major role of hepatic CYPs in AAI detoxication in<br />
vivo and that of CYP1A1/2 in this reaction in vitro.<br />
Acknowledgements: Supported by GACR (303/09/0472, 305/09/H008) and Ministry of<br />
Education (MSM 0021620808).<br />
227<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
109.<br />
THE EFFECT OF BENDIOCARB ON ANTIOXIDANT PARAMETERS IN MALE AND<br />
FEMALE ORGANS OF RABBIT<br />
Anna Sobeková 1 , Ľuboslava Lohajová 1 and Peter Javorský 2<br />
1 Department of Chemistry, Biochemistry and Biophysics UVLF in Košice<br />
2 Institute of Animal Physiology SAV Košice<br />
The toxicity of bendiocarb is believed to be connected with<br />
the acetylcholine esterase inhibition. However, the toxic effect of pesticides is<br />
cumulated from all their metabolites.<br />
Male and female rabbits were orally administered bendiocarb at a dose<br />
5 mg.kg -1 body weight for 30 days. Administration of bendiocarb resulted in<br />
a significant alterations of antioxidant and detoxifying enzymes activities (SODsuperoxide<br />
dismutase, CAT-catalase, GPx-glutathione peroxidase) in liver and kidney<br />
of male and female rabbits. In the male liver, the activities of SOD and CAT were not<br />
affected by bendiocarb, but changes in SOD isoenzymes pattern were observed in the<br />
experimental groups. The activities of GPx were significantly decreased on the 3rd<br />
and the 10th day of an experiment. Significantly higher levels of TBARS indicate the<br />
oxidative stress of the organ. The activity of GPx does not change in female liver. In<br />
the kidney, the activities of all followed enzymes changed without significant<br />
increasing of TBARS.<br />
The inhibition of antioxidant defence system, increased TBARS values and changes in<br />
SOD isoenzyme pattern showed that the toxic effect of bendiocarb is not only in the<br />
acetylcholine esterase inhibition, but probably also in ROS production. ROS can be<br />
produced by cyclic biotransformation of bendiocarb and its metabolites. Our results<br />
also show the alterations in the level of some antioxidant parameters regarding<br />
variation in the response of male and female animals.<br />
228<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
110.<br />
INTERACTION OF PLASMID DNA AND MITOCHONDRIA WITH CYCLIC CHALCONE<br />
ANALOGUES<br />
Miroslava Štefanišinová 1 , Mária Kožurková 2 , Vladimíra Tomečková 1 and<br />
Mária Mareková 1<br />
1Department of Medical Chemistry, Biochemistry and Clinical Biochemistry, UPJŠ -<br />
Faculty of Medicine, Tr. SNP 1, 040 01 Košice, Slovak Republic,<br />
2Department of Biochemistry, Institute of Chemistry, UPJŠ - Faculty of Science,<br />
Moyzesova 11, 040 66 Košice, Slovak Republic<br />
The binding of small molecules to DNA has been of great interest due to the<br />
understanding of the drug-DNA interaction. Substituted chalcone (1) and its cyclic<br />
chalcone analogues: indanone (2), tetralone (3), benzosuberone (4) with<br />
dimethylamino substituent in 2, 4 position are interesting as drugs with antitumor<br />
activity. The purpose of this study was to investigate the interaction of DNA with new<br />
ligands (1 – 4) using by spectroscopy methods. Ligand - DNA binding affinities and<br />
constants (K) of the DNA-drug complexes were determined by UV-Vis and<br />
fluorescence spectrophotometric titration and CD spectroscopy. Generally, these<br />
compounds bound to DNA and show significant decrease of fluorescence and<br />
bathochromic shift of excitation and emission maxima compared to the spectral<br />
characteristics of the free form of ligands in solution phase. The strong<br />
hypochromism, extensive broadening, red-shifting and increased stability of DNA<br />
double helix were observed when these derivatives where bound to DNA. The<br />
compounds have no circular dichroism spectrum when are free in the solution but<br />
they induced CD spectrum when they are in the complex with DNA. The helical band<br />
at 246 nm corresponding to the DNA unwound extent exhibited decrease for all the<br />
compounds in the same order as that of the binding affinity. This phenomenon could<br />
be due to the stabilization of the right-handed B-form of DNA by intercalation.<br />
Acknowledgments: This work was supported by grant project VEGA 1/0402/10<br />
229<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
111.<br />
ASSOCIATION POLYMORPHISMS OF GST, HOGG1 AND XRCC1 GENES WITH LUNG<br />
ADENOCARCINOMA<br />
Tatiana Matáková 1 , Erika Halašová 2 , LuciaLetková 2<br />
Anton Dzian 3 and Dušan Dobrota 1<br />
1 Institute of Medical Biochemistry, JMF CU in Martin; 2 Institute of Medical Biology,<br />
JMF CU in Martin; 3 Clinic of Surgery, JMF CU and MFH in Martin; Slovak Republic<br />
A case-control study with 130 lung cancer cases and 290 controls was conducted.<br />
DNA was extracted from peripheral blood leukocytes, and the polymorphisms of<br />
GSTM1, GSTT1, GSTP1, XRCC1 and hOGG1 enzymes were determined by PCR-based<br />
methods. In this study we determined the genotype distribution of all these genes,<br />
and their combinations. Association between specific genotypes and the<br />
development of lung cancer were examined using logistic regression analysis to<br />
calculate odds ratios (OR) and 95% CI.<br />
The GSTT1 null genotype (OR=2.2; p=0.004) was associated with an elevated risk. The<br />
statistically significant correlation was found also for the combined genotypes of<br />
GSTT1 null and GSTM1 positive (OR=2.2; p=0.04) and GSTT1 null and GSTP1 Ile/Val or<br />
Val/Val (OR = 2.9; p = 0.009). For hOGG1 and XRCC1 were no difference in the<br />
frequency of genotypes between matched cases and controls. The statistically<br />
significant correlation was found also for the combined genotypes of GSTT1 null and<br />
GSTM1 positive (OR=2.2; p=0.04) and GSTT1 null and GSTP1 Ile/Val or Val/Val<br />
(OR=2.9; p=0.009).<br />
The statistically significant correlation was found for the combined genotypes of<br />
GSTT1 null and hOGG1 Ser/Ser (OR=2.2; p=0.02) and GSTT1 null and XRCC1 Arg/Arg<br />
(OR=2.7; p=0.03).<br />
The genotype of selected enzymes affects the risk for lung adenocarcinoma.<br />
Acknowledgments: This work was supported by Ministry of Health of the Slovak<br />
republic under the project No. 2007/48-UK-13 and project “Center of translational<br />
medicine” cofinanced from EC sources and European Regional Development Fund.<br />
230<br />
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Posters<br />
112.<br />
OXIDATION OF 3-AMINOBENZANTRONE, A HUMAN METABOLITE OF<br />
CARCINOGENIC 3-NITROBENZANTHRONE, BY HUMAN AND<br />
RAT CYTOCHROMES P450<br />
Jana Mizerovská 1 , Helena Dračínská 1 , Volker M. Arlt 2 , Heinz H Schmeiser 3 ,<br />
Eva Frei 3 and Marie Stiborová 1<br />
1 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030,<br />
128 40 Prague 2, Czech Republic, 2 Section of Molecular Carcinogenesis, Institute of<br />
Cancer Research, Brookes Lawley Building, Sutton, Surrey SM2 5NG, United Kingdom,<br />
3 German Cancer Research Center, 69 120 Heidelberg<br />
3-Aminobenzanthrone (3-ABA) is the main reductive metabolite of the carcinogenic<br />
environmental pollutant 3-nitrobenzanthrone (3-NBA). 3-ABA was detected in the<br />
urine samples of salt mining workers exposed to diesel emissions, indicating that<br />
exposure to 3-NBA can be detectable. In addition, both parental 3-NBA and its<br />
metabolite, 3-ABA, were found to induce biotransformation enzymes such as<br />
cytochrome P450 1A1 and NAD(P)H:quinone oxidoreductase (NQO1) in several<br />
tissues of rats i.p. treated with both compounds.<br />
Oxidation of 3-ABA was studied using human and rat recombinant CYP enzymes. The<br />
most potent enzymes oxidizing 3-ABA were identified to be CYP1A1 and CYP3A. Using<br />
these enzymes, the kinetic parameters such as K m and V max were determined. Here<br />
we also investigate the induction potential of 3-NBA to induce NQO1 and CYP1A1 in<br />
rats, after the 3-NBA intratracheal instillation. Expression of NQO1 and CYP1A1<br />
protein and its enzymatic activity were induced by 3-NBA in liver, kidney and lung<br />
tissues of rats treated i.t. with a single dose of 0.2 and 2 mg/kg bw of 3-NBA.<br />
Acknowledgments: Supported by GACR (303/09/0472, 203/09/0812 and<br />
305/09/H008) and the Czech Ministry of Education (MSM 0021620808).<br />
231<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
113.<br />
METABOLIC ACTIVATION OF CARCINOGENIC BENZO[A]PYRENE BY CYTOCHROME<br />
P450 1A1 IS DICTATED BY COMPOSITION OF THE MIXED-FUNCTION-<br />
MONOOXYGENASE SYSTEM<br />
Michaela Moserová 1 , Miroslav Šulc 1 , Volker M. Arlt 3 , David H. Phillips 3 ,<br />
Eva Frei 2 and Marie Stiborová 1<br />
1<br />
Department of Biochemistry, Charles University, Albertov 2030, 128 40 Prague 2; 2<br />
Department of Molecular Toxicology, German Cancer Research Center, 69 120<br />
Heidelberg; 3 Section of Molecular Carcinogenesis, Institute of Cancer Research,<br />
Brookes Lawley Building, Sutton, Surrey SM2 5NG, UK<br />
Carcinogenic benzo(a)pyrene (BaP) was investigated for its potential to generate DNA<br />
adducts and to induce cytochrome P450 (CYP)and NADPH:CYP reductase (POR)<br />
enzymes in livers.<br />
BaP induces expression of these enzymes in livers of experimental models, which<br />
leads to increase in their enzymatic activity. In addition, this compound is capable of<br />
generating DNA adducts, predominantly in livers of studied organisms. The<br />
stimulation effect was attributed to induction of CYP1A1/2 and/or enzymes, which<br />
are responsible for oxidative activation of BaP to the metabolites generating major<br />
DNA adducts in vitro. BaP is oxidized by hepatic microsomes from pretreated and<br />
control animals to six metabolites. BaP is also oxidized with purified CYP1A1<br />
reconstituted with NADPH:CYP reductase generating only five metabolites, but<br />
forming two DNA adducts. Activation of BaP by CYP1A1 is dictated by composition of<br />
the mixed-function-monooxygenase system of the endoplasmic reticulum.<br />
Acknowledgments: Supported by Czech Science Foundation (303/09/0472,<br />
305/09/H008), Grant Agency of Charles University (127208) and Ministry of<br />
Education, Youth and Sports (MSM 0021620808).<br />
232<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
114.<br />
THE STUDY ON CYTOCHROME B 5 –MEDIATED STIMULATION OF ELLIPTICINE<br />
OXIDATION BY CYTOCHROME P450 3A4 TO ITS PHARMACOLOGICALLY MORE<br />
EFFICIENT METABOLITES<br />
Barbora Mrázová 1 , Eva Martínková 1 , Radek Indra 1 , Eva Frei 2 and Marie Stiborová 1<br />
1 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128<br />
43 Prague 2, Czech Republic<br />
2 German Cancer Research Center, 69 120 Heidelberg, Germany<br />
Ellipticine alkaloid exhibiting significant antineoplastic activities. CYP3A4 was found to<br />
be the major enzyme responsible for oxidation of ellipticine to 13-hydroxy- and 12-<br />
hydroxyellipticine, two metabolites generating DNA adducts. Many CYP3A4-<br />
dependent reactions have been shown to be stimulated by another microsomal<br />
protein, cytochrome b 5 (b 5 ). Beside 12-hydroxy- and 13-hydroxyellipticine, other three<br />
ellipticine metabolites are generated by CYP3A4. Human b 5 , purified rabbit hepatic b 5<br />
and apo-b 5 reconstituted with heme were capable of stimulating oxidation of<br />
ellipticine by CYP3A4. The patterns and amounts of ellipticine metabolites vary<br />
significantly when b 5 is present in the incubations. Whereas, the formation of<br />
detoxication product of ellipticine oxidation (9-hydroxyellipticine) is practically not<br />
influenced by b 5 , generation of 13-hydroxy- and 12-hydroxyellipticine is increased<br />
significantly by these b 5 proteins. The enhanced generation of ellipticine-DNA adducts<br />
in the presence of b 5 in the system was confirmed by 32 P postlabeling. Other proteins<br />
with or without heme in their molecules are without such effects on ellipticine<br />
oxidation. The hyperbolic kinetics of ellipticine oxidation by CYP3A4 with or without b 5<br />
indicates no cooperativity. The results indicate that the presence of heme in b 5 seems<br />
to be required for the stimulation of CYP3A4-mediated oxidation of ellipticine.<br />
Acknowledgments: Supported by GACR (P303/10/0356, 203/09/0812), the Czech<br />
Ministry of Education (MSM0021620808, 1M0505) and GAUK (1272080).<br />
233<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
115.<br />
IMMUNODETECTION OF 11ß-HYDROXYSTEROID DEHYDROGENASE DURING<br />
PURIFICATION OF A NEW HUMAN MEMBRANE-BOUND CARBONYL REDUCING<br />
ENZYME<br />
Miloslava Netopilová, Libuše Černá, Lucie Škarydová and Vladimír Wsol<br />
Department of Biochemical Sciences, Faculty of Pharmacy, Charles University,<br />
Hradec Králové , Czech Republic<br />
The 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) is a membrane-bound<br />
microsomal protein that mainly catalyzes the conversion of inactive glucocorticoid<br />
cortisone to active cortisol. However, this enzyme has a broad substrate specificity<br />
and can also metabolize various xenobiotics with carbonyl groups, for example the<br />
cytostatic drug oracin. Oracin is reduced by microsomal enzymes to two enantiomers<br />
of 11-dihydrooracin (DHO). Stereospecificity of the conversion depends on type of<br />
tissues and enzymes. While human liver microsomes reduce oracin to (-)-DHO and<br />
(+)-DHO in a ratio of 60:40, purified human liver 11ßHSD1 change oracin more<br />
specifically to (-)-DHO (76%). Because 11ßHSD1 is only known microsomal enzyme<br />
reducing oracin, it is supposed that the liver microsomes contain at least one other<br />
oracin carbonyl reducing enzyme with stereospecific production of (+)-DHO. The<br />
presence of 11ßHSD1 was determinated by immunodetection using Western blotting.<br />
The blot was incubated with a primary polyclonal rabbit antibody against human<br />
11ßHSD1(1:1000, Abcam) and then with a secondary polyclonal swine antibody antirabbit<br />
IgG coupled to horseradish peroxidase (1:1000, Dako). About 30 fractions were<br />
prepared using separation of human microsomes on Q-Sepharose. The highest<br />
reducing activity was found in the flow through fractions Q2-Q4 and in the fractions<br />
Q10-Q14; 11ßHSD1 was detected only in fraction Q11-Q14. The selected fraction Q12<br />
was separated on the Phenyl-Sepharose column and the highest activity was in the<br />
fractions P10-P12. In these fractions, 11ßHSD1 was not found.<br />
Acknowledgement: This project was supported by the Grant Agency of Charles<br />
University, Grant No. 45508/C/2008.<br />
234<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
116.<br />
PHYTOREMEDIATION – THE PROMISING METHOD FOR THE REMOVAL OF<br />
PHARMACEUTICAL RESIDUES FROM WASTEWATER<br />
Radka Podlipná a , Petra Šídlová b , Kotyza Jan c and Tomáš Vaněk a<br />
a<br />
Laboratory of Plant Biotechnologies, Joint Laboratory of the Institute of Experimental<br />
Botany, Academy of Science of the Czech Republic and<br />
Research Institute of Crop Production, Prague<br />
b Department of Biochemical Sciences, Faculty of Pharmacy UK, Hradec Králové<br />
c Faculty of Environmental Technology, Institute of Chemical Technology, Prague<br />
Recently there is an increasing concern about contamination of the environment with<br />
pharmaceutical residues and their metabolites. These contaminants are excreted into<br />
a hospital or municipal wastewater in considerable amount; in addition many of them<br />
are not completely degraded in sewage treatment plants and remains in water<br />
effluent. Although the environmental concentrations of these compounds are usually<br />
on low level (
Posters<br />
117.<br />
ELLIPTICINE CYTOTOXICITY TO HUMAN THYROID CANCER CELL LINES<br />
Jitka Poljaková 1 , Tomáš Eckschlager 2 , Eva Frei 1 and Marie Stiborová 1<br />
1 Department of Biochemistry, Faculty of Science, Charles University in Prague,<br />
2 Department of Pediatric Hematology and Oncology,<br />
2 nd Medical School, Charles University in Prague<br />
Ellipticines are plant alkaloids with an antineoplastic activity. The mode of action is<br />
based mainly on DNA intercalation, inhibition of topoisomerase II and formation of<br />
covalent DNA adducts mediated by cytochromes P450 (CYP) and peroxidases. Such<br />
ellipticine-DNA-adducts are formed in vitro, in human breast adenocarcinoma MCF-7,<br />
leukemia HL-60, CCRF-CEM, neuroblastoma IMR-32, UKF-NB-3, UKF-NB-4 cell lines<br />
and in vivo in rats exposed to ellipticine. Here, the cytotoxicity of ellipticine to human<br />
thyroid cancer cell lines BHT-101, B-CPAP and 8505-C was investigated. Furthermore,<br />
the effect of hypoxic conditions on the ellipticine toxicity to these cells was also<br />
examined. The toxicity of ellipticine to thyroid cancer cells cultivated under the<br />
standard conditions was higher than that to the cell lines grown under hypoxia (1%<br />
oxygen). Another target of this work was to evaluate the effect of ellipticine on<br />
expression of enzymes activating ellipticine, namely on CYP1A1, 1B1, 3A4,<br />
cytochrome b 5 and peroxidases COX-1 and TPO.<br />
Acknowledgement: Supported by GACR (P301/10/0356) and Czech Ministry of<br />
Education (MSM0021620813).<br />
236<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
118.<br />
QUALITY AND TIME-STABILITY OF HUMAN SKIN EXPLANTS<br />
Alena Rajnochová Svobodová 1 , Adéla Zdařilová 1 , Dana Kylarová 2 ,<br />
Bohumil Zálešák 3 and Jitka Vostálová 1<br />
1 Department of Medical Chemistry and Biochemistry, Palacký University Olomouc,<br />
2 Department of Histology and Embryology, Palacký University Olomouc,<br />
3 Department of Plastic and Aesthetic Surgery, Faculty Hospital, Olomouc<br />
Human skin obtained from plastic surgery has been used for isolation of skin cells<br />
such as keratinocytes, fibroblasts, melanocytes or endothelial cells for several<br />
decades. Single cell cultures, co-cultures or 3D cultures of these cells have been<br />
utilized for dermatological studies including phototoxicity and photoprotection<br />
investigation. However, these very simple systems are very different from human<br />
skin. Therefore cultivation of human skin organ culture (ex vivo skin) has been<br />
developed as a more suitable model system.<br />
The aim of this study was to determine explant usage period (quality and stability<br />
during cultivation). Morphological structure and antioxidant parameters (activity of<br />
superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase<br />
(GST) and catalase (CAT) and level of thiobarbituric acid-reactive substances (TBARS)<br />
and glutathione (GSH) were evaluated. Epidermis did not show any significant<br />
changes in its morphology until 72 h cultivation. In dermis moderate acute<br />
inflammatory reaction was observed (48 - 72 h) but it disappeared after 120 h.<br />
Dermal papillae started to be reduced after 96 h and these changes became more<br />
significant after 120 h cultivation. TBARS level together with GSH amount and activity<br />
of SOD have markedly increased for the cultivation period (0 - 144 h). Activities of<br />
GPX and CAT have grown for 72 - 96 h and then decreased. Changes in GST activity<br />
did not have any trend and varied between 70 - 160 % of control (0 h).<br />
In conclusion human skin explants can be utilized for various dermatological<br />
applications including photoprotection or metabolic and xenobiochemic studies.<br />
Acknowledgement: This work was financially supported by the grant MSM<br />
6198959216.<br />
237<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
119.<br />
OXIDATIVE STRESS IN TURKEYS CAUSED BY CHRONIC CADMIUM EXPOSURE<br />
Anna Sobeková 1 , Katarína Holovská 2 and Peter Javorský 3<br />
1 Department of Chemistry, Biochemistry and Biophysics UVLF in Košice, 2 Department<br />
of Anatomy, Histology and Physiology UVLF in Košice, 3 Institute of Animal Physiology<br />
SAV Košice<br />
Heavy metals can cause oxidative stress by increasing the formation of reactive<br />
oxygen species (ROS), which render antioxidants incapable of defence against<br />
growing amounts of free radicals. The alterations of ROS scavenging enzyme activities<br />
are the consequences of this effect. The effect of single Cd and Cd+Zn on the<br />
antioxidant enzymes (SOD-superoxide dismutase, GPx-glutathione peroxidise, GRglutathione<br />
reductase, GST-glutathione-S-transferase) and the content of<br />
thiobarbituric acid reactive species (TBARS) was evaluated in parenchymal tissues<br />
(liver, kidney) from turkeys.<br />
In the liver, the specific activity of SOD was significantly increased in Cd group.<br />
Mitochondria try to decrease intracellular ROS levels by decreasing to consume of<br />
oxygen via formation of „extremely swollen mitochondria“. Ultrastructural changes<br />
were confirmed by electron microscopy. TBARS values were not changed in Cd group.<br />
Antioxidant defence system were able to protect the hepatocytes against oxidative<br />
damage.<br />
Activity of SOD was significantly decreased in the kidney. Cadmium evoked the<br />
increase of activity of another determined antioxidant enzymes (GSHPx, GR, GST).<br />
Higher TBARS values indicate the oxidative stress in this organ. Zinc coadministration<br />
shows protective effect in both observed tissues.<br />
The alterations in the activities of antioxidant defence system and increased TBARS<br />
values showed that toxic effect of cadmium is not only in the high thiol group affinity,<br />
but also in ROS production.<br />
238<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
120.<br />
SIMILARITY BETWEEN RAT AND HUMAN ENZYMES INVOLVED IN OXIDATION 2-<br />
NITROPHENOL, A METABOLITE OF CARCINOGENIC 2-NITROANISOLE<br />
Martina Svobodová, Markéta Martínková, Helena Dračínská and Marie Stiborová<br />
Department of Biochemistry, Faculty of Science, Charles University in Prague<br />
2-Nitrophenol (2-NP) is the main detoxication metabolite of 2-nitroanisole (2-NA)<br />
that is an important industrial pollutant and a potent carcinogen for rodents. The aim<br />
of this study was to investigate the efficiency of rat and human hepatic cytochromes<br />
P450 (CYPs) to metabolize 2-NP, to determine the metabolites formed during such a<br />
metabolism and to compare the efficiencies of rat CYPs with those of human. 2-NP is<br />
oxidized by both liver microsomes to one metabolite, 2,5-dihydroxynitrobenzene<br />
(2,5-DNB). To define the role of rat CYPs in 2-NP oxidation we investigated the<br />
modulation of this reaction by specific inducers and selective inhibitors of these<br />
enzymes. Using microsomes from Baculovirus transfected insect cells expressing<br />
recombinant rat and human CYP enzymes, we found that rat recombinant CYP2E1,<br />
2C11, 2B1, 1A2, 1A1 and 3A4 were the most effective to oxidize 2-NP. Similarly,<br />
human CYP2E1, followed by CYP2A6, 2C6, 3A4 and 2D6 were the most efficient to<br />
oxidize 2-NP. In addition, kinetics of oxidation of 2-NP by the most efficient enzyme<br />
catalyzing this reaction, CYP2E1, was investigated. The present study shows the<br />
similarity between human and rat hepatic enzymes metabolizing 2-NP to 2,5-DNB<br />
and indicate that rat might serve as a suitable model to mimic the fate of 2-NP in<br />
human.<br />
Acknowledgements: Supported by GACR (303/09/0472) and the Czech Ministry of<br />
Education (MSM 0021620808).<br />
239<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
121.<br />
OVEREXPRESSION OF P-GLYCOPROTEIN IN L1210/VCR CELLS IS ASSOCIATED WITH<br />
CHANGES IN SEVERAL ENDOPLASMIC RETICULUM PROTEINS<br />
Mário Šereš, Eva Poláková, Oľga Križanová, Zdena Sulová and Albert Breier<br />
Institute of Molecular Physiology and Genetics SAS Bratislava<br />
Multidrug resistant cells R, which express membrane drug efflux transporter – P-<br />
glycoprotein (P-gp, a member of ABC transporter family), was found to be also less<br />
sensitive to thapsigargin, inhibitor of sarco(endo)plasmic reticulum calcium pump<br />
(SERCA), in contrast to parental drug-sensitive cells L1210 (S). R cells was obtained<br />
from S cells by selection with vincristine (VCR). We have studied differences among S<br />
and R cells in expression of endoplasmic reticulum proteins involved in the regulation<br />
of calcium homeostasis and calcium-dependent processes. Amounts of mRNA<br />
encoding RyR and IP 3 -receptor channels were found to be at similar in S and R cells.<br />
However, mRNAs encoding IP 3 R1 or 2 were decreased in resistant cells cultivated in<br />
the presence of VCR, while mRNA encoding RyR remained unchanged. The amount of<br />
mRNA encoding SERCA2 was lower in R cells as in S cells. This decrease was more<br />
pronounced when R cells were cultivated in the presence of VCR. Calnexin was<br />
believed to be active in P-gp maturation, but surprisingly, calnexin was found to be<br />
less expressed at the protein level in R as in R cells. We have also studied thapsigargin<br />
effect on expression of some endoplasmic reticulum proteins and P-glycoprotein. P-<br />
gp expression was decreased in presence of thapsigargin.<br />
Thus, S and R cells differ in the expression of endoplasmic reticulum proteins involved<br />
in the control of intracellular calcium homeostasis or calcium-dependent processes.<br />
Acknowledgement: This work was supported by: VVCE-0064-07, APVV-0084-07 and<br />
VEGA 2/0123/10, 2/0155/09.<br />
240<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
122.<br />
ENVIRONMENTAL POLLUTANTS AS FACTORS MODULATING THE INFLAMMATORY<br />
RESPONSE AND FUNCTIONS OF LUNG CELLS<br />
Lenka Umannová 1,2 , Miroslav Machala 2 , Alois Kozubík 1 and Jan Vondráček 1,2<br />
1<br />
Laboratory of Cytokinetics, Institute of Biophysics, ASCR v.v.i., 612 65 Brno,<br />
Czech Republic<br />
2<br />
Veterinary Research Institute v.v.i., 621 00 Brno, Czech Republic<br />
Chronic inflammation plays a central role in pathogenesis of lung cancer or chronic<br />
obstructive pulmonary disease, which belong among leading causes of death. Air<br />
pollution represents a significant risk factor for lung cancer incidence. The polluted<br />
atmosphere contains various matrices with carcinogenic potential including vehicle<br />
exhaust, side-stream tobacco smoke, coal combustion effluents and transient metals.<br />
In the present study, we analyzed mutual deregulation of enzymes involved both in<br />
xenobiotic metabolism and inflammatory responses, caused by environmental<br />
pollutant and important tobacco smoke constituent benzo[a]pyrene (BaP) and TNF-α<br />
in rat lung RLE-6TN alveolar type II cell line.<br />
Our results show that BaP modulates the expression/activity of enzymes induced by<br />
TNF-α that are involved in inflammatory responses such as prostaglandinendoperoxide<br />
synthase 2 (COX-2) or inducible nitric oxide synthase (iNOS), leading to<br />
enhanced release of their products such as prostaglandin E2. Moreover, BaP<br />
increases expression of inflammatory cytokines induced by TNF-α in target cells.<br />
Important role in increased expression may play oxidative stress and activation of p38<br />
mitogen activated protein kinases. These results demonstrate that environmental<br />
pollutants affect signaling pathways related to inflammatory response and may thus<br />
contribute to deregulated inflammation and lung epithelial damage.<br />
Acknowledgement: Supported by the Czech Ministry of Agriculture, grant No. MZe<br />
0002716202.<br />
241<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
123.<br />
THE MECHANISM OF CYTOTOXIC EFFECT OF NOVEL ACRIDINE INTERCALATORS<br />
Zuzana Vantová 1 , Helena Paulíková 1 , Mária Kožurková 1 , Danica Sabolová 1 , Ima<br />
Dovinová 1 , Pavol Kristián 2 , Ján Imrich 2 , Ján Ungvarský 2 and Ladislav Janovec 2<br />
1 Department of Biochemistry and Microbiology, STU Bratislava,<br />
2 Department of Biochemistry, UPJŠ Košice<br />
Acridines are well-known chemotherapeutic agents in cancer therapy. New class of<br />
acridine derivatives were synthesized – 2´,2´´-[(acridin-3,6-diyl)]-3´,3´´-dialkyl-1,3-<br />
diimidazolidin-4-on hydrochloride (AcrDIMs). These compounds bearing<br />
imidazolidinone rings, are capable to interact with DNA, mainly by intercalation and<br />
to inhibit activity of topoisomerase I. Their biological activity to inhibit cell<br />
proliferation of leukaemia cells (HL-60 a L1210 cells) and adherent ovarian cancer cell<br />
line A2780 was confirmed. Despite the lowest affinity to DNA (K = 1.9×10 5 M -1 ), 2´,2´´-<br />
[(acridin-3,6-diyl)]-3´,3´´-dihexyl-1,3-diimidazolidin-4-on was the most active<br />
derivative with an IC 50 of 4.61±2.08 µM on a HL 60 cell line, with IC 50 of 5.52±1.08<br />
µM on a L1210 cell line and with IC 50 of 30,83 ± 2,63 µM on a A-2780 cells after 48h<br />
of incubation.. It has been discovered that the cytotoxicity of AcrDIMs correlates with<br />
their hydrophobicity (uptake into cells was monitored). Although apoptosis of treated<br />
cells has not been confirmed it has been shown that dihexyl-AcrDIM arrested cell<br />
cycle, and HL-60 cells were accumulated in G 1 phase after 48 h incubation. Together,<br />
our results support the hypothesis that AcrDIMs exert cytostatic properties by<br />
interaction with DNA inducing arrest of cell cycle and oxidative stress in cancer cells.<br />
Acknowledgement: This work has been supported by VEGA grant 1/0097/10 and<br />
1/0053/08.<br />
242<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
Posters<br />
124.<br />
RETINOIC ACID-INDUCED DIFFERENTIATION MODULATES THE APOPTOTIC EFFECT<br />
OF SODIUM VALPROATE IN HL-60 CELLS<br />
Jiří Vrba and Jitka Ulrichová<br />
Department of medical chemistry and biochemistry UP Olomouc<br />
Differentiation of myeloid leukemia cells results in resistance to various apoptotic<br />
stimuli. We examined whether human leukemia HL-60 cells differentiating by alltrans<br />
retinoic acid (ATRA) into neutrophil-like phenotype became resistant to the<br />
apoptogenic activity of valproate, an inhibitor of histone deacetylases. In<br />
undifferentiated HL-60 cells, valproate induced the G0/G1 cell cycle arrest and<br />
apoptotic changes including dissipation of the inner mitochondrial membrane<br />
potential, loss of plasma membrane asymmetry and/or integrity, activation of<br />
caspase-9 and caspase-3, and appearance of sub-G1 DNA. Cotreatment with ATRA<br />
enhanced the apoptotic response of undifferentiated cells to valproate. On the other<br />
hand, in HL-60 cells pretreated for 72 h with ATRA, valproate in combination with<br />
ATRA induced lower dissipation of mitochondrial membrane potential and weaker<br />
apoptotic changes in the plasma membrane while the DNA fragmentation was not<br />
attenuated in comparison with undifferentiated cells. We conclude that ATRAinduced<br />
differentiation modulates but does not abrogate the apoptotic response of<br />
HL-60 cells to sodium valproate, and nuclei are preferentially affected during<br />
apoptosis of differentiated cells.<br />
Acknowledgements: This research was supported by the Ministry of Education, Youth<br />
and Sports of the Czech Republic (MSM 6198959216).<br />
243<br />
<strong>XXII</strong>. Biochemický zjazd, Martin
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