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Oxidation of ellipticine by human cytochromes P450 expressed in prokaryotic and eukaryotic systems
Vejvodová, Lucie ; Stiborová, Marie (advisor) ; Hýsková, Veronika (referee)
Ellipticine is an alkaloid with antitumor activity, whose mechanism of action is based on intercalation into DNA, inhibition of topoisomerase II and formation of covalent adducts with DNA, after its enzymatic activation by cytochromes P450 and/or peroxidases. Ellipticine is oxidized by cytochromes P450 to form up to five metabolites (7-hydroxy-, 9-hydroxy, 12- hydroxy-, 13-hydroxyellipticine and N2 -oxide ellipticine). 9-Hydroxy- and 7- hydroxyellipticine are considered to be detoxification metabolites, whereas 12-hydroxy-, 13- hydroxyellipticine and N2 -oxide of ellipticine are considered as activation metabolites, which are responsible for formation of covalent DNA adducts. The aim of this thesis was to examine the efficiency of human recombinant cytochromes P450 expressed in eukaryotic (SupersomesTM ) and two prokaryotic expression systems (Bactosomes) in oxidation of ellipticine. Cytochromes P450 expressed in prokaryotic systems differed in the amounts of "coexpressed" NADPH:CYP reductase. The resulting ellipticine metabolites were analyzed by HPLC. The results obtained in this thesis demonstrate that human cytochromes P450 2C9/2D6/2C19 expressed in prokaryotic or eukaryotic systems oxidize ellipticine to form up to four metabolites: 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine and N2 -oxide...
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Oxidation of benzo(a)pyrene by cytochrome P450 1A1 expressed in prokaryotic and eukaryotic systems
Kroftová, Natálie ; Stiborová, Marie (advisor) ; Kubíčková, Božena (referee)
Benzo[a]pyrene (BaP) is a human carcinogen, which is metabolized by a variety of enzyms such as cytochrome P450 (CYP) and epoxide hydrolase. The aim of this work was to study BaP metabolism in vitro by the hepatic microsomal system of rats treated with CYP inducers and by human cytochrome P450 1A1 (CYP1A1) expressed in eukaryotic and prokaryotic systems. An eukaryotic expression system consisted of microsomes isolated from insect cells, whereas a prokaryotic expression system was formed by the membrane fragments of E. coli. In the case of recombinant human CYP1A1, we investigated the influence of cytochrome b5, NADPH:cytochrome P450 reductase (CPR) and epoxide hydrolase in BaP oxidation. Isolation and purification of rabbit hepatic CPR was another aim of this work. BaP metabolites were separated by HPLC. The results found in this work demostrate the fact that hepatic microsomal systems of rats treated with an inducer of CYP1A (Sudan I), an inducer of CYP2B (phenobarbital) and an inducer of CYP3A (PCN) exhibit higher efficiency of BaP oxidation than microsomes of control rats. BaP is oxidized by human CYP1A1 expressed in the eukaryotic system to six metabolites (BaP-9,10-dihydrodiol, BaP metabolite with unknown structure, BaP-7,8-dihydrodiol, BaP-1,6-dion, BaP-3,6-dion, BaP-3-ol), whereas by human...
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Expression and purification of N-terminal fragment of filamentous hemagglutinin from Bordetella Pertussis in E. Coli
Jurnečka, David ; Stiborová, Marie (advisor) ; Kavan, Daniel (referee)
: Whooping cough is highly contagious disease caused by gram-negative bacteria Bordetella pertussis. During infection the bacteria produces many types of toxins and adhesive molecules including a filamentous hemagglutinin(FHA). FHA is 220 kDa surface-exposed and secreted protein, which plays a key role in host-cell interactions. The project aims at construction of heterologous expression system for production of the N-terminal part of B. pertussis FHA (FHA1-862) in E. coli. The expression vector is composed of system of two independent T7/Lac promoters and enables secretion of FHA1-862 into the culture media. Downstream of the first promoter is fhaB gene encoding FHA1-862 and the letter is followed by fhaC gen encoding the FhaC transport protein, which allows translocation of FHA from periplasmic space to extracellular milieu. FHA1-862 was successfully secreted in E. coli strain BL21 carrying plasmid pMM100 (Laclq) at 30 řC and purified by affinity chromatography on Cellufine resin. These results indicate that FHA1-862 protein can be produced in E. coli, however, the system is inefficient and the yield of the protein is very low. (In Czech)
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Synthesis and biochemical characterization of hybrid analogues of human insulin and IGF-2
Povalová, Anna ; Stiborová, Marie (advisor) ; Koberová, Monika (referee)
The ever-increasing occurrence of diabetes mellitus brings about the need for development of new therapeutic agents to provide adequate treatment for patients. An important element in this research area is elucidation how insulin works, mainly in connection with insulin-like growth factors (IGF-1 and IGF-2), which show significant structural homology to each other. In addition, their respective receptors - insulin receptor (IR) and receptor for IGF-1 and IGF-2 (IGF-1R) - exhibit very high similarity. As a result, IGF-1 and IGF-2 can bind to IR and insulin can bind to IGF-1R. Of a particular importance is the high affinity binding of IGF-2 to the isoform A of IR. Unlike insulin, which predominantly mediates glucose entry into cells, IGFs induce growth or mitogenic effects. The finding which structural determinants in insulin and IGFs are responsible for the differences in the activation of their cognate receptors could provide an explanation for different functional responses upon binding of these hormones to different target cells. Understanding of this mechanism could also help in the development of functionally selective analogues of these hormones. The aim of this study was the synthesis and characterization of analogues of human insulin extended at the C terminus of the B chain with the amino...
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Preparation of derivatives of anticancer drug ellipticine and their oxidation by cytochromes P450
Škodová, Petra ; Stiborová, Marie (advisor) ; Smrček, Stanislav (referee)
Ellipticine is a plant alkaloid, which exhibits significant antitumor activity; therefore it was a center of interest since 60's of 20th century. There were a lot of ways published to obtain this anticancer drug. Ellipticine is actually a pro-drug, whose pharmacological efficiencies are dependent on the activation of two groups of enzymes, cytochrome P450 and peroxidases. The resulting effect of these groups of enzymes is either detoxification or activation of ellipticine metabolites. 13-Hydroxyellipticine and 12-hydroxyellipticine are products of activation reactions, which subsequently spontaneously cleave to carbenium ions, which in this form are bound to deoxyguanosine while generating DNA adducts. 13-Hydroxyellipticine may have potentially higher biological efficiencies than ellipticine, because it does not need enzymes for its activation and formation of DNA adducts. The aim of the diploma thesis was to synthesize this ellipticine derivative. 9- Nitroellipticin (another derivative of ellipticine) is an intermediate product of the 13- hydroxyellipticine synthesis. Results of this diploma thesis show that 9-nitroellipticine is oxidized by liver microsomal systems, which contain cytochromes P450, forming at least 4 metabolites. 9-Nitroellipticine shows similar behavior as ellipticine. Another...
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The role of human mitochondrial YME1L protease in the biogenesis of the oxidative phosphorylation system
Tesařová, Jana ; Stiborová, Marie (advisor) ; Pecina, Petr (referee)
Mitochondria are found in virtually all eukaryotic cells where their main function is the production of ATP in the oxidative phosphorylation system (OxPhos). OxPhos is build-up of both nuclear and mitochondrial encoded protein subunits. Due to the potential function threatening defects, the quality of these protein subunits is constantly under tight control by specialized proteins. The recognition and selective removal of defective mitochondrial proteins is carried out by specific mitochondrial ATP-dependent proteases. So far, four such proteolytic complexes active within distinct mitochondrial subcompartments were identified. Both i- and m-AAA protease complexes are found in the inner mitochondrial membrane. Whereas the i-AAA protease is active in the intermembrane space, the homologous m-AAA protease functions on the matrix side of the inner membrane. The aim of the present work was to characterize cellular function of the human orthologue YME1L of the yeast i-AAA protease subunit YME1 using human HEK293 cell model. We found that human YME1L is an integral membrane protein with molecular weight of approx. 600-1100 kDa, exposing the carboxy-terminal domain to intermembrane space. The HEK293 cell line with shRNA silenced expression of YME1L showed accumulation of Ndufb6 and ND1 subunits of complex...
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New analogues of human insulin with covalently stabilized cyclic structures in the C-terminus of the B-chain
Kaplan, Vojtěch ; Stiborová, Marie (advisor) ; Flegel, Martin (referee)
Diabetes mellitus is considered as one of world's most common metabolic diseases. Complicated treatment and increasing number of newly diagnosed patients, suffering from diabetes every year, shows the importance and necessity of research in this area. Some of the major aims of this research are the development of new therapeutically utilized drugs and defining the problems of insulin acting in human body. Insulin is a peptide hormone whose main physiological function is to regulate blood glucose level in organism connected with large impact on whole metabolism. Insulin acts through binding of its monomeric form to the insulin receptor. Upon binding to the receptor molecule of insulin undergoes specific structural changes, which put the hormone into an active state. As of now, the structure of the insulin's active monomeric form is still unknown. By testing binding affinities of many modified insulin analogues there was discovered strong evidence between structural conformation of the C-terminus of the B-chain and binding affinity to the receptor. The most crucial data, necessary for this work, were observed from the structure of highly active insulin analogues that possessed unique B26 turn, recently prepared and described by team of Dr. J. Jiráček, IOCB AS CR. The aim of this work was synthesis of...
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Metabolism carcinogens and drugs by the system of monooxygenases
Moserová, Michaela ; Stiborová, Marie (advisor) ; Entlicher, Gustav (referee) ; Čeřovská, Noemi (referee)
Ellipticine, an alkaloid isolated from Apocynaceae plants, exhibits significant antitumor and HIV activities. Ellipticine is a pro-drug, whose pharmacological and genotoxic effects depend on activation by cytochromes P450 (CYP) and peroxidases (Px) to a reactive species generating DNA adducts. To elucidate contribution of CYPs (and which of them) and Px to ellipticine activation, we used rat and mouse models, mice with deleted gene of NADPH:CYP reductase in the liver, thus absenting this enzyme in the liver (HRNTM ) and a control mouse line (WT), rats treated with ellipticine, and microsomal systems isolated from the liver of mouse lines and from the liver, kidney and lung of rats. The purified enzymes, CYP1A1 and 3A4, reconstituted with NADPH:CYP reductase were also used. The effect of cytochrome b5, a facultative component of the mixed function monooxygenase system, on ellipticine oxidation by CYP1A1 and 3A4 was also investigated. Carcinogenic benzo(a)pyrene (BaP), known to covalently bind to DNA after its activation with CYPs, was investigated for its potential to generate DNA adducts and to induce CYP and NADPH:CYP reductase enzymes in mouse livers. We investigated an influence of each of components of the mixed function oxidases (MFO) system on metabolism of BaP. CYP1A1 is widely accepted to be the...
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Biotransformation of phenols by enzymatic systems of Candida tropicalis yeast and Comamonas testosteroni bacteria
Vilímková, Lenka ; Stiborová, Marie (advisor) ; Entlicher, Gustav (referee) ; Mareš, Jaroslav (referee)
Candida tropicalis yeast and bacteria Comamonas testosteroni have been considered to be able to metabolize phenol and utilize it as the only source of carbon and energy. In our laboratory we investigated the cytoplasmic enzymes responsible for the first and second step of phenol degradation, NADPH-dependent phenol hydroxylase of both C. tropicalis and C. testosteroni and catechol 1,2-dioxygenase of C. tropicalis. The aim of our study was to isolate and partially characterize those enzymes. Phenol hydroxylase purification consisted of preparation of cytosol from C. tropicalis yeast by fraction centrifugation, chromatography and re-chromatography on a column of DEAE Sepharose, fractionation by precipitation of the enzyme with polyethylene glycol 6000 and gel permeation chromatography on a column of Sephacryl S-300. Extracellular phenol hydroxylase of C. testosteroni was purified by fraction precipitation with polyethylene glycol 6000 and by gel permeation chromatography on 4B Sepharose and Sephacryl S-300. Catechol 1,2-dioxygenase was purified using the procedure consisting of: chromatography and re- chromatography on a column of DEAE Sepharose, lyophilization of the enzyme and gel permeation chromatography on a column of Sephadex G-100. The enzyme activity was determined by two methods: use of HPLC...
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Analyzing the complexity of auxin-related processes and their regulation
Simon, Sibu ; Zažímalová, Eva (advisor) ; Stiborová, Marie (referee) ; Pavlíček, Jiří (referee)
Phytohormone auxin plays an important role in various aspects of plant growth and development. The necessary concentration maxima at the region of its action are achieved by auxin metabolism, passive diffusion of auxin molecules across plasma membrane and by the carrier-mediated auxin transport, as well as by modulation of these processes. In our study we used a group of compounds structurally related to major endogenous auxin indole-3-acetic acid, as well as synthetic auxins 2,4-dichlorophenoxy acetic acid (2,4- D) and naphthalene-1-acetic acid (NAA). We aimed to characterize the auxin specificity of developmentally important processes such as carrier-mediated auxin transport, and 'genomic' (transcriptional) and 'non-genomic' (transcriptional) auxin signaling. In addition to the characterization of these compounds we also hoped to get an insight into the complex regulatory mechanism of auxin-related processes and to possibly find a particular compound with distinct behavior towards particular processes. By making use of such compounds and other molecular tools we aimed to analyze the mechanism of 'non-genomic' auxin signaling, to understand the mode of action of FM (Fei Mao) styryl dyes on the dynamics of membrane- localized auxin transporters, and to study the involvement of other phytohormones...
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