sborník příspěvků XIII. Pracovní setkání fyzikálních chemiků a elektrochemiků

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1 XIII. Pracovní setkání fyzikálních chemiků a elektrochemiků 13 th Workshop of Physical Chemists and Electrochemists sborník příspěvků Přírodovědecká fakulta Masarykovy univerzity Agronomická fakulta Mendelovy univerzitě v Brně a Fakulta elektrotechniky a komunikačních technologií VUT v Brně

2 ORGANIZACE POŘÁDAJÍCÍ KONFERENCI Přírodovědecká fakulta MU V Brně Ústav chemie Kotlářská Brno Agronomická fakulta MENDELU v Brně Ústav chemie a biochemie Zemědělská Brno Fakulta elektrotechniky a komunikačních technologií VUT v Brně Ústav mikroelektroniky Technická 3058/ , Brno ORGANIZAČNÍ ZABEZPEČENÍ KONFERENCE Libuše Trnková (Ústav chemie, PřF, MU) René Kizek (Ústav chemie a biochemie, AF, MENDELU) Jaromír Hubálek (Ústav mikroelektroniky, FEKT, VUT v Brně) Publikace neprošla jazykovou kontrolou. Jednotlivé příspěvky jsou publikovány tak, jak byly dodány autory. Za věcnou a odbornou správnost jsou plně odpovědni autoři příspěvků. Podrobné informace včetně sborníku příspěvků jsou k dispozici na internetové adrese ISBN:

3 Pracovní setkání bylo podpořeno výzkumnými projekty:

4 sponzoři pracovního setkání Organizátoři děkují všem letošním sponzorům za podporu, která umožnila pořádat tuto, již tradiční, akci: METROHM, spol. s. r. o., EPPENDORF Czech & Slovakia, spol. s. r. o., CHROMSPEC, spol. s. r. o., ILABO, spol. s. r. o., MANEKO, spol. s. r. o., MEDESA, spol. s. r. o., PRAGOLAB, spol. s. r. o., SIGMA, spol. s. r. o., TRIGON plus, spol. s. r. o., CLONESTAR Peptide Services, spol. s. r. o., Stargen EU s. r. o., Chromservis s.r.o., Česká společnost chemická, pobočka Brno

5 Úvodem Milí přátelé, pro konání letošního XII. Pracovního setkání fyzikálních chemiků a elektrochemiků (12 th Workshop of Physical Chemists and Electrochemists) byly zvoleny dva dny na konci května ( a ). Místem konání jsou konferenční prostory s atriem v nové budově Q Univerzity MENDELU v Brně. Naše konference může být považována za jednu z významných akcí v rámci dvou oslav. Brno letos slaví 190 let od narození významného vědce a opata augustiniánského kláštera na Starém Brně Gregora Johanna Mendela, který je díky objevu tří základních zákonů dědičnosti považován za otce genetiky. Pod hlavičkou Mendel 190 si tento významný svátek připomíná různými akcemi (výstavy, konference, přednášky Mendel lectures) nejen univerzita MENDELU, která nese jeho jméno, ale i MASARYKOVA UNIVERZITA. V Mendelově odkazu obě univerzity učí principy genetiky, její základy a rozvoj, uplatňující se jak v říši rostlin a živočichů, tak i v medicíně. Dalším milníkem pro oslavný charakter našeho setkání je 90 let objevu polarografie, za kterým stojí náš nositel Nobelovy ceny v oblasti fyzikální chemie a elektrochemie Jaroslav Heyrovský. Tak jako G.J. Mendel je považován za otce genetiky, J. Heyrovský je považován za otce elektroanalytických metod. Když objevil polarografii, bylo mu pouhých 32 let, kdy za sebou měl úspěšná studia chemie, matematiky a fyziky na Karlově universitě v Praze, a studia u slavných chemiků Sira W. Ramsaye, W. C. McC. Lewise, a F. G. Donnana v Londýně. Jeho metoda, většinou prezentovaná jako analýza roztoků pomocí elektrolýzy, se stala základem pro rozvoj dynamických elektrochemických metod. Záštitu nad konferencí převzali primátor statutárního města Brna Bc. Romana Onderka, MBA, rektoři a děkani vysokých škol, které se na organizaci XII. Pracovního setkání podílely: rektor Masarykovy univerzity Doc. PhDr. Mikuláš Bek, Ph.D. rektor Mendelovy univerzity v Brně Prof. Ing. Jaroslav Hlušek, CSc., dr. h. c., děkan Přírodovědecké fakulty MU Doc. RNDr. Jaromír Leichmann, Dr., děkan Agronomické fakulty MENDELU Prof. Ing. Ladislav Zeman, CSc., děkanka Fakulty elektrotechniky a komunikačních technologií VUT Prof. Ing. Jarmila Dědková, CSc.. Na účastníky z České a Slovenské republiky čekají dva konferenční dny naplněné blokem plenárních (6) a vyzvaných přednášek (13), přednášek nadaných studentů (18) v Sekce mladých (SM). Enormní nárůst přednášek, především v SM, nás velice potěšil.

6 Aby studenti měli dostatek času pro svoje přednášky a diskusi v jazyce anglickém, rozhodli jsme se založit dva SM paralelní bloky, kde přednášky budou hodnoceny dvěma komisemi. Z každého bloku pak budou odměňováni studenti na prvním, druhém a třetím místě. Všichni se mohou těšit na vědecké diskuse, které budou inicializovány nejen přednáškami, ale i prezentacemi velkého počtu posterů (XX). Hodnocení posterů bude probíhat novým způsobem. Autoři plakátových sdělení budou mít možnost se rozhodnout, zda přihlásit svůj poster do soutěže či nikoliv. Potom soutěžící postery budou hodnoceny určenou komisí. Krátká prezentace autora, popř. autorů se bude vyžadovat 3 minuty, diskuse 2 minuty. Všechna abstrakta ve sborníku jsou v jazyce anglickém, mají rozšířenou podobu a obsahují barevné obrázky či grafy. Abstrakta jsou součástí sborníku s ISBN (sborník příspěvků odpovídá kritériím pro hodnocení VaV v kategorii Článek ve sborníku ). V rámci konference bude vydáno speciální číslo v časopise International Journal of Electrochemical Science s IF (ISSN ). Běžná cena tzv. open access free, která umožňuje volnou dostupnost publikovaného díla, je 500 Euro. Pro účastníky konference bude snížený poplatek na vložné do časopisu 400 Euro. Uzávěrka pro dodání příspěvku je stanovena na Následně proběhnou recenzní řízení. Vydání speciální čísla je plánováno na prosinec Vítáme všechny účastníky XII. Pracovního setkání fyzikálních chemiků a elektrochemiků a přejeme všem úspěšnou prezentaci, která může být spolu s bohatou diskusí velmi užitečným pomocníkem v jejich dalším bádání. Libuše Trnková Organizační a vědecký výbor: doc. RNDr. Libuše Trnková, CSc. doc. Ing. René Kizek, Ph.D. doc. Ing. Jaromír Hubálek, Ph.D. doc. RNDr. Vojtěch Adam, Ph.D. Technické zabezpečení Pracovního setkání: Ing. Jiří Sochor, Ph.D. Mgr. Sylvie Holubová Mgr. Olga Kryštofová Mgr. Michal Horák Kristina Nádeníčková Martina Ryvolová

7 Obsah Electrochemical assay of butyrylcholinesterase in biological samples 13 Detection of the six nucleotides repetitions using character based method 14 The Effect of Aristolochic Acid I on NAD(P)H:Quinone Oxidoreductase Expression in Mice and Rats a Comparative Study 16 Utilization of electrotransfer of proteins (Western blot) for a study of histone acetylation mediated by hypoxia in neuroblastoma cells 19 DNA adduct formation by carcinogenic Aristolochic Acid I is dictated by expression of enzymes catalyzing its activation and detoxication 21 Interaction of the Human Prion protein, metals (Zn, Cu) and metallothionein 24 Bacterial production of PrP C mature full length protein for electrochemical analysis 25 Dicoumarol inhibits rat NAD(P)H:quinone oxidoreductase in vitro and induces its expression in vivo 27 Cytochrome b 5 induction by aryl hydrocarbon receptor ligands results in an increase in benzo[a]pyrene-dna adduct formation 30 Utilization of the electrochemical Method of Western blotting for control of heterologous expression of cytochrome P450 2S1 33 Impedance analysis of synthetic oligodeoxynucleotides at the mercury electrode 36 Liposomes use as model membrane and their characterization 38 Sensitive electrochemical determination of adrenaline in human urine using boron-doped diamond film electrode 39 Purification of tulip bulb cytochrome P-450 into electrophoretical homogeneity 41 Use of boron-doped diamond as a new electrode material for immobilization of DNA 44 Kinetic study of metal complexes of cyclen derivatives 46 The effect of cytochrome b 5 on an electron-transporting system of cytochrome P450 1A1 in oxidation of benzo[a]pyrene 47 Copper coated electrodes for electrochemical determination of carbohydrates 50 DNA molecule from the view of physics 52 GFP-RNA transfection of cells containing defective transmembrane conductance regulator. possible approach for genetic treatment of cystic fibrosis? 54 DNA labelling for electrochemical analysis from ultrasensitive detection to DNA sequences analysis 56 Spectroscopic study of quinolone derivatives 57 The relationship between DNA adduct formation by benzo[a]pyrene and expression of its activation enzyme, cytochrome P450 1A1, in rats 59 Electrochemical preparation of gold nanorods for electrode surface modification 62 Glutathione Modified Gold Nanoelectrodes and Electrochemical Characterization 64 Biophysical study of the interaction of zinc ions with DNA 66 Fully automated system for oligo adenine nucleotide detection 67 In vivo investigation of doxorubicin 69 Modification of QCM gold surface by oligonucleotides 70

8 Monitoring of the surface modified electrodes by nucleic acids and electrochemical measurements on the scanning electrochemical microscope 71 Encapsulation of nucleic acid into the apoferritin structure, detection, analysis 72 Apoferritin encapsulated doxorubicin behavior in heart muscle tissue 73 Matematické vyhodnocení získaných experimentálních dat, modelování 75 Design and manufacturing of microfluidic chip with electrochemical detection 77 Lead ions encapsulated in nanotechnology structures 82 Electrochemical biosensor for detection of three influenza subtypes by nanoparticels label 84 Peptidy, bílkoviny a nukleové kyseliny. Adsorpce a jámy 86 Heterologous expression and purification of rat cytochrome P450 1A1 and NADPH: cytochrome P450 oxidoreductase to electrophoretically homogenous proteins 87 Interaction of cytochromes P450 with bacterial electron donor flavodoxin: a theoretical study 92 Benzofurazane based DNA redox labeling and voltammetry at silver amalgam electrode 94 Design and synthesis of a new sensor for dna mutation detection based on förster resonance energy transfer 96 Conformational analyses of some fluoroquinolone precursors 97 Ftir spectroscopy characterization of bitumen from south moravian lignite 99 Determination of water-soluble fractions from lignite 100 Effects of different ions onto adsorption and 2d condensation of 5-fluorocytosine 102 Fluorescence Behaviour of doxorubicin 103 Carbon fiber microelectrodes as electroanalytical tools 105 Fast and efficient calculations of binding Affinities for C8-Substituted GTP analogues to the FtsZ Protein 107 Improvement of enzyme stability by medium and protein engineering 109 Interaction of doxorubicin with metallothionein 110 Electrochemical study of ellipticine and ODN complex on the surface HMDE, Adsorptive Transfer Method 112 Magnetic particles usable in sequencing labeling reaction purification 114 Preparation of chitosan complexes with silver, copper and zinc nanoparticles 115 Impedance measuring of microbial biofilms and interactions with chitosan complexes 116 Ions of lead in the environment and aquatic system 117 Heavy metals in patients with malignant tumors 119 Charge transfer in DNA/ DNA and RNA/DNA hybrids 121 Kinetic study of excited state proton transfer probe 1-naphthol in hyaluronan-surfactant system 123 Quantum dots as a tool for influenza viral protein isolation and detection 125 Determination of relationship among bacteria based on polar representation 127 Correlation between mercury content in muscle and body weight of carp from non-contaminated ponds 129

9 Methods used in characterization of model phospholipid membranes 131 Electrochemical behavior of mixture of phosphatidylcholine and cholesterol in model phospholipid membranes using Impedance spectroscopy 132 Synthesis and NMR spectral studies of fluorinated anilinoethylene derivatives 133 possible application of ultrasensitive lectin biosensors in diagnostics 134 Device for controlled nanopillars growth using anodic aluminum oxide film as template 136 compressed DNA signal representation for classification of long sequences 138 Self-ordered quantum dots array for optical biomolecule detection 139 Platinum complexes. From DNA modifications to anticancer chemotherapy 141 Development of a bead-based sarcosine immunosensor 142 Shape-optimization of controlled-release systems using finite element method 144 RNA modification of hanging mercury drop electrode 145 Use of electrochemical methods for evaluation of critical health condition 146 Atomic, ionic and bohr radii linked via the golden ratio for the elements in DNA: C, N, O, P and H 148 Electrochemical characterization of novel two-electrode thin film system 150 Direct glucose detection on the Cu 2 O based screen-printed electrodes 152 Leporidae family and linkage to geographical distribution based on nucleic acid sequence processing 154 sequencing reaction with respect to study of the fragments 156 physicochemical analogies between description of adsorption of thiosulphate and agglomeration of silver nanoparticles at toxic concentrations 158 Onkolytické viry-další možnost léčby nádorů 160 Sarcosine isolation by paramagnetic microparticles using microfluidic device 162 Spectroelectrochemistry of flavonolignans and their DNA-binding copper complexes 164 study of chemical composition and structure of different humic acids 166 The study of DNA heptamers with different trinucleotide sequences in a loop by electrochemical and spectral methods 168 Interaction of DNA and doxorubicin on gold nanostructured electrode 170 Utilization of diffusion techniques for study on reactivity of modified humic acids 172 Detection of adenine in oligonucleotides using Coper nanoparticle modified GC electrode 174 Behaviour of humic acids in aqueous solutions 176 Separation of alanine and cysteine by ce-ms coupled by sheathless and electrodeless interface 178 The effect of a methyl group on the oxidation of xanthine on a pencil graphite electrode 180

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13 electrochemical assay of butyrylcholinesterase in biological samples Miroslav Pohanka 1*, Vojtech Adam 2,3, Rene Kizek 2,3 1 Faculty of Military Health Sciences, University of Defence, Trebesska 1575, Hradec Kralove, Czech Republic 2 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, Brno, Czech Republic 3 Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, Brno, Czech Republic Abstract In the body, two cholinesterases can be found: acetylcholinesterase and butyrylcholinesterase (BChE). Comparing to acetylcholinesterase, BChE is an enzyme presented in quite high level in the blood plasma where it participates in detoxification reactions but it can be found in organs as well. The BChE presented in plasma comes from liver parenchyma. Lack of BChE plasmatic activity can be used for biochemical diagnosis as a marker of poisoning with some neurotoxins and/or liver damage. In the present work, we gave our attention to construction of an electrochemical method which can be used for a reliable and fast assay of BChE activity in biological samples including plasma. Standard optical, Ellman s, protocol was used for validation of the new method. Screen printed electrodes were used as a platform and butyrylthiocholine as a substrate for BChE. The methods were firstly performed on purified human BChE and the lowest limit of detection, maximal velocity and Michaelis constant were calculated for the both methods. Limit of detection for the electrochemical protocol was less than kat being better than limit of detection for the standard optical protocol. After optimization, the methods were verified on human plasma samples. We can conclude our experiments by a statement that the electrochemical method is suitable for a routine examination of human plasma. ACKNOWLEDGEMENT The Ministry of Education, Youth and Sports (Czech Republic) is gratefully acknowledged for A long-term organization development plan

14 Detection of the six nucleotides repetitions using character based method Jiri Nedved 1*, Vladimira Kubicova 1, Ivo Provaznik 1,2 1 Department of Biomedical Engineering, Faculty of Electrical Engineering and Communication, Brno University of Technology, Technická 3058/10, Brno, Czech Republic 2 International Clinical Research Center Center of Biomedical Engineering, St. Anne s University Hospital Brno, Pekařská 53, Brno, Czech Republic Abstract This research is aimed to detection of the repetition sequences in human genome by a character based method. The detection of these repetitions can help to explain evolution in one species, if the repetitions are shorter, or genetic disorders, if the copies are longer. This work is focused on the detection of the six nucleotides repetitions. INTRODUCTION Nowadays all detections are based on chemical estimation of the copies, which has definitive resolution, need an amount of the time and need somebody working on this in the lab. The estimation using a computer is better for the detection, but now are all methods slow. After improve their codes, they can compute automatically and find regions of interest. [1, 2] Data AND METHODS Data was found in public database NCBI and were chosen two Bioprojects PRNJA1431 (Assembly: WGSA) and PRNJA20837 or PR- NJA19621 (Assembly: HuRef). Both Bioproject have completed human genome divided into chromosomes. [3] Now is used a character based method comparing chosen pattern with a part of the sequence. If the pattern is same as part of the sequence, a counter for this pattern is incremented. After end of the scanning, the pattern is changed and the scanning runs again. Number of the scans is almost equal the length of the scanning sequence. Next program look for tandem repetitions in the results obtained from first program. The tandem repetitions lie closer to each other. RESULTS and discussion The outputs from this detector are written into structure stored information about each pattern, its count of the copies and their positions in the sequence. The results from the second program are figures of the most interesting tandem repetitions. For example, the Figure 1 is shown the beginning of the second chromosome from the Bioproject with assembly HuRef. It is the sequence of the telomere on complementary strand with pattern CCCTAA. The detector has different properties than Tandem Repeat Finder (TRF, [4]) and Spectral Repeat Finder (SRF, [5]). TRF is bit faster than us detector, but finds only tandem repetitions. The other copies are for TRF hidden. SRF chops long sequences into smaller parts and the results are divided for each shorter sequence against us detector, where the results are completed. The results for one sequence length about 300 kbp are computed after few minutes, but it depends on the number of the copies in this sequence. Summed results for first 300 kbp of the chromosome 2 is shown in the next Chart 1 and was competed in seconds. For second program a pattern with maximum number of the copies is used and the output is shown in the Figure 1. Chart 1: The results of the copy detector for first 300 kbp of the human second chromosome. CONCLUSION The detector presented in this paper is good alternative to TRF and SRF, has many preferences, but still is bit slower than TRF in tandem repeats finding. The output from the detector is completed and saved in structure for future work, as the detection longer patterns and the detection with allow mutations. 14

15 REFERENCES [1] Sunstad, D. P., Simmons, M. J.: Principles of Genetics. Fifth edition (2009), pg [2] Mefford, H.C., Trask, B. J.: The complex structure and dynamic evolution of human subtelomeres. Macmillan Magazines Ltd (2002), vol. 3, pg [3] Genome Project Report: nlm.nih.gov/genome/51 [4] Tandem Repeat Finder: bu.edu/trf/trf.html [5] Spectral Repeat Finder: res.in/raghava/srf/ Figure 1: Human telomere of the second chromosome on complementary strand. 15

16 The Effect of Aristolochic Acid I on NAD(P) H:Quinone Oxidoreductase Expression in Mice and Rats a Comparative Study Frantisek Barta 1, Katerina Levova 1, Eva Frei 2, Heinz H. Schmeiser 3, Volker M. Arlt 4, Marie Stiborova 1* 1 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, Prague 2, Czech Republic 2 Division of Preventive Oncology, National Center for Tumor Diseases, German Cancer Research Center (DKFZ), In Neuenheimer Feld 280, Heidelberg, Germany 3 Research Group Genetic Alterations in Carcinogenesis, German Cancer Research Center (DKFZ), In Neuenheimer Feld 280, Heidelberg, Germany 4 Analytical and Environmental Sciences Division, MRC-HPA Centre for Environment and Health, King s College London, London, United Kingdom Abstract Aristolochic acid causes a specific nephropathy, Aristolochic acid nephropathy, Balkan endemic nephropathy, and urothelial malignancies. Using an electrochemical method of Western blotting suitable for determining protein expression, we investigated expression of NAD(P)H:quinone oxidoreductase (NQO1), the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI), in mice and rats. The effect of AAI on NQO1 protein expression and its enzymatic activity in these experimental models was also examined. INTRODUCTION The herbal drug aristolochic acid (AA) derived from Aristolochia species has been shown to be the cause of so-called Chinese herbs nephropathy (CHN), now termed Aristolochic acid nephropathy (AAN) and is hypothesized to be responsible for Balkan endemic nephropathy (BEN) [1,2]. The plant extract AA is a mixture of structurally related nitrophenanthrene carboxylic acids, the major components being aristolochic acid I (AAI) and aristolochic acid II (AAII) [2]. Exposure to AA was demonstrated by identification of specific AA-DNA adducts in urothelial tissue of AAN and BEN patients [2-4]. The most abundant DNA adduct detected in patients is 7-(deoxyadenosin-N 6 -yl)-aristolactam I (da-aai), which causes characteristic AT TA transversions. Such AT TA mutations have been observed in the TP53 tumor suppressor gene in tumors from AAN and BEN patients [3,4], indicating a probable molecular mechanism associated with AA-induced carcinogenesis [2]. More recently, AA exposure was discovered to contribute to the high incidence of upper urinary tract urothelial carcinoma (UUC) in Taiwan, where medicinal use of Aristolochia plants is widespread [5]; again, the TP53 mutational signature in patients with UUC was predominant among otherwise rare AT TA transversions. AA has been classified as a Group I carcinogen in humans by the International Agency for Research on Cancer. The activation pathway for AAI leading to formation of AAI-DNA adducts is nitroreduction, catalyzed by both cytosolic and microsomal enzymes; in this process NAD(P)H:quinone oxidoreductase (NQO1) is the most efficient cytosolic nitroreductase [6,7]. Here, using an electrochemical method of Western blotting suitable for determining protein expression, we investigated expression of this enzyme in mice and rats. MATERIAL AND METHODS Immunoquantitation of NQO1 expressed in liver, kidney and lung of Wistar rats and C57BL/6 mice was carried out by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [8,9]. The membranes were then exposed to specific polyclonal antibodies against this enzyme and the antigen-antibody complex visualized as described [10]. Human recombinant NQO1 (Sigma) was used to identify the band of NOO1 in murine cytosols. NQO1 activity was measured essentially as described [10]. DNA adducts were analyzed by 32 P-postlabeling as shown previously [3, 6-7,10]. RESULTS and discussion Utilizing an electrochemical method of Western blotting we found that NQO1 protein levels in cytosolic fractions isolated from liver, kidney and lung of mice differed from those expressed in these organs of rats (Fig. 1). In mice, the highest levels of NQO1 protein and NQO1 activity were found in the kidney, followed by lung and liver. On the contrary, the levels of NQO1 protein and enzyme activity were lowest in the rat kidney cytosol, whereas the highest amounts of NQO1 protein and activity were found in cytosols of lung, followed by liver. The NQO1 protein and enzyme activity were induced in 16

17 liver and kidney of AAI-pretreated mice, compared with those of untreated mice. The NQO1 protein and enzyme activity were also induced in rat kidney. Furthermore, the increase in hepatic and renal NQO1 enzyme activity (Fig. 1) was associated with bio-activation of AAI and elevated AAI-DNA adduct levels in ex vivo incubations of cytosolic fractions with DNA and AAI (data not shown). In conclusion, AAI appears to increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential. In addition, our studies and the findings of others [11] that certain NQO1 genotypes appear to be associated with increased risk of urothelial cancer in BEN patients, underscore the clinical importance of NQO1 activity in humans exposed to AAI. Figure 1: NQO1 protein expression (blue columns) and NQO1 enzyme activity (red columns) in cytosols isolated from liver, kidney and lung of either untreated mice or mice pretreated with a single oral dose of 50 mg/kg body weight AAI (A) and in those of either untreated rats or rats pretreated with a single i.p. dose of 20 mg/kg body weight AAI (B). CONCLUSION Utilizing Western blot analysis, NQO1 protein levels were analyzed in liver, kidney and lung of untreated and AAI-pretreated mice and rats. Our results demonstrate that AAI has the potential to induce the cytosolic NQO1 nitroreductase activity in liver and kidney of animal models used in the study. The results of this study also indicate that the electrochemical Western blot method is a suitable tool to evaluate expression of NQO1 enzyme in organisms. ACKNOWLEDGEMENT The work has been supported by GACR (303/09/0472) and Charles University in Prague (UNCE /2012). REFERENCES [1] Debelle FD, Vanherweghem JL, Nortier JL: Kidney International, 74 (2008), [2] Schmeiser HH, Stiborova M, Arlt VM: Current Opinion in Drug Discovery and Development, 12 (2009), [3] Arlt VM, Stiborova M, vom Brocke J, et 17

18 18 al.: Carcinogenesis 28 (2007), [4] Grollman A.P., Shibutani S., Moriya M., et al.: Proceedings of American Chemical Society U.S.A., 104 (2007), [5] Chen CH, Dickman KG, Moriya M, et al.: Proceedings of American Chemical Society U.S.A., 109 (2012), [6] Stiborova M, Frei E, Arlt VM, et al.: Mutation Research, 658 (2008), [7] Stiborová M, Frei E, Schmeiser HH: Kidney International, 73 (2008), [8] Poljaková J, Eckschlager T, Kizek R, et al.: International Journal of Electrochemical Science, 8 (2013), [9] Vranová I, Moserová M, Hodek P, et al.: International Journal of Electrochemical Science, 8 (2013), [10] Levova K, Moserova M, Nebert DW, et al.: Toxicology and Applied Pharmacology, 265 (2012), [11] Toncheva DI: Collegium Antropologicum, 30 (Suppl 1) (2006), 34.

19 Utilization of electrotransfer of proteins (Western blot) for a study of histone acetylation mediated by hypoxia in neuroblastoma cells Jitka Poljakova 1, Tomas Groh 1, Zaneta Omana Gudino 1, Jan Hrabeta 2, Rene Kizek 3, Tomas Eckschlager 2, Marie Stiborova 1* 1 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, Prague 2, Czech Republic 2 Department of Pediatric Hematology and Oncology, 2 nd Medical School, Charles University, V Uvalu, Prague 5, Czech Republic 3 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, Brno, and Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, Brno, Czech Republic Abstract Changes in expression levels of acetylated histones H3 and H4 as well as of hypoxia-inducible factor (HIF-1) in human neuroblastoma cell lines cultivated under standard or hypoxic conditions (1% O 2 ) were investigated. The electrochemical method based on protein-electro-transport (Western blot) was utilized for this study. Hypoxic stress increased levels of acetylated histones H3 and H4 in UKF-NB-3 and UKF-NB-4 neuroblastoma cell lines whereas, almost no changes in acetylation of these histones were found in an SK-N-AS neuroblastoma line. INTRODUCTION Neuroblastoma, a tumor of the peripheral sympathetic nervous system, is the most frequent solid extra cranial tumor in children and is a major cause of death from neoplasia in infancy [1]. Treatment of older children with widely disseminated high-risk neuroblastomas remains one of the greatest challenges for pediatric oncologists. Rapidly growing tumors are supplied by oxygen insufficiently, which refers to development of hypoxic regions. Malignant cells adapt to these conditions through angiogenesis, enhanced glycolysis and decreased mitochondrial respiration via up- or down-regulation of gene expression. Of note, hypoxic conditions influence the efficiencies of several drugs that are active to cause death of neuroblastoma cells such as ellipticine and, to a lower extent, doxorubicin, resulting in lowering their cytotoxicities [2-4]. Considerable function in up-regulation of gene expression during hypoxia possess hypoxia-inducible factor (HIF) [5]. Entire lack of oxygen also decreases the expression of cellular adhesion proteins and DNA repair proteins that lead to genetic instability and metastasis of tumor cells. However, the mechanism by which low levels of oxygen repress gene expression is still unknown. Since acetylation of histones is an important epigenetic mark that might be influenced by hypoxia, changes in expression levels of acetylated histones H3 and H4 and hypoxia-inducible factor (HIF-1) in human neuroblastoma cell lines by hypoxia were investigated. MATERIAL AND METHODS Immunoquantitation of H3 and H4 histones, and HIF-alfa in UKF-NB-3, UKF-NB-4 and SK-N-AS neuroblastoma cell lines was done by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The membranes were then exposed to specific polyclonal antibodies against these proteins and the antigen-antibody complex visualized as described [2,6,7]. RESULTS and discussion Changes in levels of acetylated histones H3 and H4 and those of HIF-1 alpha in human neuroblastoma cell lines cultivated under standard or hypoxic conditions (1% O 2 ) were investigated. Using an electrochemical method of Western blotting, we found that hypoxic stress for 6, 12, 24 or 48 h induces acetylation of these histones. Levels of acetylated histones H3 and H4 were increased by hypoxia, particularly in a UKF-NB-4 neuroblastoma cell line derived from high-risk neuroblastoma, whereas essentially no changes in acetylation of these histones were observed in an SK-N-AS neuroblastoma cell line (Fig. 1). Almost no changes in histone deacetylase activity were produced by hypoxia in neuroblastoma cells (data not shown). Likewise, essentially no differences in HIF-1 alpha expression were observed in tested neuroblastoma cells depending on cultivation conditions (Fig. 1). 19

20 Figure 1: Expression of HIF-1 alfa and acetylated histones H3 and H4 by Western blot in human neuroblastoma cell lines. Actin was used as loading control. CONCLUSION Using the electrochemical method (Western blot), we found that hypoxic stress increased levels of acetylated histones H3 and H4, especially in a UKF-NB-4 neuroblastoma cell line derived from high-risk neuroblastoma. In contrast, almost no changes in acetylation of these histones were found in an SK-N-AS line. Insight into changes in levels of acetylated histones H3 and H4 after hypoxic stress among studied neuroblastoma cell lines might be important for partial explanation of tumor aggressive properties. ACKNOWLEDGEMENT The work has been supported by GACR (P301/10/0356) and Charles University in Prague (635712/2012 and UNCE /2012). REFERENCES [1] Maris JMN: England Journal of Medicine, 362 (2010), [2] Poljaková J, Eckschlager T, Hrabeta J, et al.: Biochemical Pharmacology, 77 (2009), (2009). [3] Stiborová M, Rupertová M, Frei E: Biochimica et Biophysica Acta, 1814 (2011), [4] Kizek R, Adam V, Hrabeta J, et al.: Pharmacology & Therapy, 133 (2012), [5] Denko NC: Nature reviews cancer, 8 (2008), [6] Poljaková J, Eckschlager T, Kizek R, et al.: International Journal of Electrochemical Science, 8 (2013), [7] Vranová I, Moserová M, Hodek P, et al.: International Journal of Electrochemical Science, 8 (2013),

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