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Study of interaction of antimicrobial peptides with cells in culture
Kroupová, Hilda ; Stiborová, Marie (advisor) ; Votruba, Ivan (referee)
In English The thesis deals with research of novel antimicrobial peptides (AMP) Halictines (HAL-1, GMWSKILGHLIR-NH2 a HAL-2, GKWMSLLKHILK-NH2) and their structural analogs isolated from the venom of the wild bee Halictus sexcinctus. The structure and antimicrobial activity of these peptides had been described earlier [1]. The goal of this diploma thesis is to find peptide which is strongly toxic only for cancer cells and nontoxic for normal cells. Using of the fluorescent marked peptides we aimed to acquire the information about mechanism of action of the studied peptides on the cells. Using the MTT test (determination of valuation IC50), the toxicity of HAL-1 and HAL-2 and their analogs against 2 normal cell lines (Human umbilical vein endothelial cells, HUVEC, and normal rat intestinal cells, IEC) and against 2 cancer cell lines (cancer cells of suppository uterine, HeLa-S3 and cancer cells of human colorectal carcinoma, CRC SW 480) was determined. First we tested antimicrobial peptides with antimicrobial activity and low hemolytic activity. For verification the toxicity of less active analogs was also determined. We found out that the HeLa-S3 cells are the most sensitive to these peptides. The most toxic peptides (HAL-1/9, HAL-1/18, HAL-2/2) kill 50% of cells in the concentration 2,5 - 10 µM. To obtain...
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The study of activation and detoxication metabolism of the anticancer drug ellipticine by the cytochrome P450 system in vitro and in vivo…
Kotrbová, Věra ; Stiborová, Marie (advisor) ; Hansíková, Hana (referee) ; Souček, Pavel (referee)
Gen. Physiol. Biophys. (2006), 25, 245-261 245 Oxidation Pattern of the Anticancer Drug Ellipticine by Hepatic Microsomes - Similarity Between Human and Rat Systems M. Stiborová1 , L. Bořek-Dohalská1 , D. Aimová1 , V. Kotrbová1 , K. Kukačková1 , K. Janouchová1 , M. Rupertová1 , H. Ryšlavá1 , J. Hudeček1 and E. Frei2 1 Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic 2 Division of Molecular Toxicology, German Cancer Research Center, Heidelberg, Germany Abstract. Ellipticine is an antineoplastic agent, whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of DNA adducts mediated by cytochrome P450 (CYP). We investigated the ability of CYP enzymes in rat, rabbit and human hepatic microsomes to oxidize ellip- ticine and evaluated suitable animal models mimicking its oxidation in humans. Ellipticine is oxidized by microsomes of all species to 7-hydroxy-, 9-hydroxy-, 12- hydroxy-, 13-hydroxyellipticine and ellipticine N2 -oxide. However, only rat micro- somes generated the pattern of ellipticine metabolites reproducing that formed by human microsomes. While rabbit microsomes favored the production of ellipticine N2 -oxide, human and rat microsomes predominantly formed 13-hydroxyellipticine. The species difference in...
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The role of glycoconjugates in cell signalling - CEACAM1 and Fc ε RI
Dudková, Lenka ; Šmíd, František (advisor) ; Stiborová, Marie (referee) ; Bílková, Zuzana (referee)
Carcinoembryonic cell adhesion molecule 1 (CEACAM1) is human membrane glycoprotein. The purpose of the first part of this thesis was to isolate CEACAM1 glycoprotein from bile and characterise its purity and recovery that have not been described before. Affinity chromatography of CEACAM1 on hydrazide-activated cellulose with immobilized monoclonal anti-CEA F34-187 antibodies is described. The method is based on the oxidation of the immunoglobulin carbohydrate component located on the Fc fragment of antibody by periodate and oriented bounding to hydrazide-activated matrix. Crude protein fraction from bile was applied onto the affinity column and CEACAM1 was eluted with 6 M guanidine-HCl. A single immunopositive 85 kDa band of CEACAM1 was detected on the western blot with anti-CEA antibody. Probably due to the high glycosylation of CEACAM1 any common method of protein staining was not applicable except staining with lectin.
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