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The influence of stress on NADP-dependent enzymes in higher plants.
Kovaľová, Terézia ; Hýsková, Veronika (advisor) ; Dračínská, Helena (referee)
Biotic stress in the form of viral infection, as well as abiotic salt stress, cause leaves injuries, stomata closure and decreased rate of photosynthesis. These factors lead to the limitation of plant growth and to reduced amount of coenzyme NADPH. However NADPH is an important coenzyme for many metabolic pathways such as synthesis of fatty acids, amino acids and secondary metabolites involved in stress responses. NADPH is also a coenzyme for key enzymes of antioxidant system and for many regulatory enzymes. NADP-dependent enzymes are alternative source of NADPH in plants under stress conditions. In this work, activities of four NADP-dependent enzymes: Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), NADP-isocitrate dehydrogenase (NADP-ICDH, EC 1.1.1.42), NADP-malic enzyme (decarboxylating) (NADP-ME, EC 1.1.1.40) and Shikimate dehydrogenase (SDH, EC 1.1.1.25) were studied. Activities of all these enzymes but SDH increased in leaves of tobacco plants (Nicotiana tabacum L.) infected by PVYNTN , The most sensitive enzymes to viral infection were NADP-ICDH and NADP-ME, whose activity was increased in comparison with control plants 3-fold and 2,4-fold, respectively. Changes in activity of studied enzymes were also determined in plants exposed to viral infection in combination with heat-shock...
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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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Study of mechanism of fibrin network formation
Božíková, Paulína ; Martínková, Markéta (advisor) ; Hýsková, Veronika (referee)
Serine protease thrombin plays an important role in the process of fibrin network formation by converting fibrinogen into fibrin monomer which spontaneously polymerizes to form fibrin network. The aim of this work was to characterize interactions between thrombin and surface adsorbed fibrin(ogen) or fibrin network to which thrombin can bind and initiate grow of the fibrin network. Activity of thrombin bound on fibrinogen or fibrin was determined spectrophotometrically in a relation to cleaved chromogenic substrate. Using the method of surface plasmon resonance fibrin network formation initiated by thrombin bound to fibrinogen or fibrin was observed. These networks were also visualized by atomic force microscopy. Determined value of affinity constant KD for interaction of fibrinogen in solution with a fibrin network prepared on surface is in agreement with previous experiments in which KD was determined from interaction of surface covalently bound fibrinogen with fibrin monomers in solution.
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Study on metabolism of 3-aminobenzanthrone and induction of biotransformation enzymes
Mizerovská, Jana ; Stiborová, Marie (advisor) ; Mareš, Jaroslav (referee) ; Hýsková, Veronika (referee)
CHARLES UNIVERSITY IN PRAGUE FACULTY OF SCIENCE DEPARTMENT OF BIOCHEMISTRY Study on metabolism of 3-aminobenzanthrone and induction of biotransformation enzymes Summary of PhD Thesis RNDr. Jana Mizerovská Supervisor: Prof. RNDr. Marie Stiborová, DrSc. Prague 2010 RNDr. Jana Mizerovská Introduction 1 INTRODUCTION 3-Nitrobenzanthron, precursor of 3-ABA The nitroaromatic 3-nitrobenzanthrone (3-nitro-7H-benz de anthracen-7-one, 3-NBA) occurs in diesel exhaust and in airborne particulate matter(8, 9, 14) . 3-NBA is most likely formed during the atmospheric reaction of benzanthrone with nitrogen oxides, especially in the presence of ozone, or during imperfect burning of diesel. 3-NBA exhibits extremely high mutagenic activity(9, 14) and is also a genotoxic carcinogen causing lung tumors in rats(14) . 3-NBA is also evaluated to be a potential carcinogen for humans(14, 1, 9, 19) . The genotoxicity of 3-NBA was documented by the detection of specific 3-NBA-derived DNA adducts in vitro, in human cell lines and also in vivo in rats and mice(12, 13, 15, 2) . The predominant DNA adducts formed by 3-NBA after its metabolic activation by reduction of the nitro group are 2-(2'-deoxyguanosin-N2 -yl)-3- aminobenzanthrone and N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone(9, 14) and these are most probably responsible for the...
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Study of tyrosin kinase inhibitor vandetanibe bound in apoferritin and liposomes
Jáklová, Kateřina ; Indra, Radek (advisor) ; Hýsková, Veronika (referee)
5 Abstract In this thesis the anticancer drug vandetanib was studied. Vandetanib is a tyrosine kinase inhibitor affecting signalling of vascular endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR) or RET protooncogene (REarranged during Transfection). It is primarily used for the treatment of advanced tumors of the thyroid gland. Unfortunately, the usage of vandetanib in the cancer treatment is significantly limited by its toxicity and cardiotoxicity (one of the adverse effects is connected with long QT interval). One way, how to minimize these side effects, is binding a drug into a suitable transporter. Apoferritin and liposomes were used as a transport nanoparticles in this study. The aim of this thesis was to study the stability of the complex of nanoparticle apoferritin with vandetanib molecules (ApoVan) and to study the effect of pH on the release of inhibitor from the ApoVan form. Experiments have shown that ApoVan complex is relatively stable after its storage at 4 řC and - 20 řC for up to 8 weeks. Unfortunately after monitoring the effect of pH on the release of vandetanib from ApoVan, it was found that vandetanib is gradually released from its ApoVan form into the neutral environment at pH 7,4 as well as into the acidic environment at pH 6,5 and the way ApoVan is...
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Biochemical characterization of tomato plants infected with fungal pathogens
Oušková, Marie ; Hýsková, Veronika (advisor) ; Kubíčková, Božena (referee)
Fungal pathogens including Verticillium longisporum which causes verticillium wilt are among the serious diseases of crops that easily spread worldwide. One of the ways to prevent fungal infection is to use fungal biocontrol agents applied as a seed coating. This control agent, the non-pathogenic oomycete Pythium oligandrum, enters the soil together with the seed and acts symbiotically in the plant's root system. On the one hand, it stimulates the plant's defence mechanism by secreting elicitors, and on the other hand, by providing tryptamine, it stimulates growth and increases the plant's fitness with auxin. In this work we studied the effect of seed treatment of Solanum lycopersicum L. cv. Micro-Tom with three different isolates of the genus Pythium (including the commercially used isolate M1 and two yet unused isolates X42 and X48) on the activities of antioxidant and NADP(H)-dependent enzymes in the leaves of plants infected with fungal pathogen V. longisporum. Two weeks after pathogen inoculation, no significant difference was found in the studied enzymes except for increased glucose-6phosphate dehydrogenase and NADP-malate dehydrogenase (oxaloacetate decarboxylation) activity in plants treated with X42 isolate and increased shikimate dehydrogenase activity and antioxidant capacity in plants...
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Nanoparticle forms of anticancer drugs and the mechanisms influencing their efficiency
Urbanová, Tereza ; Stiborová, Marie (advisor) ; Hýsková, Veronika (referee)
Currently, cancer is one of the major diseases of civilization. The disadvantage of conventional chemotherapy, which began in the 1940s, is its non-specific effect, so the cytostatics are toxic to healthy cells. However, if the cytostatic is inserted into a nanotransporter, it increases its specific efficacy and reduces the negative side effects. One of the possible nanotransporters is protein called apoferritin (a protein component of ferritin, an iron-carrying protein) that contains light and heavy subunits differing in their function in iron uptake. In this bachelor thesis, the ability of apoferritin to encapsulate two cytostatics (ellipticine and doxorubicin), depending on its origin and the proportion of light and heavy apoferritin subunits, was studied.
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The inhibitory effect of estrogenic endocrine disruptors on cytochrome P450 activity
Otáhalová, Barbora ; Dračínská, Helena (advisor) ; Hýsková, Veronika (referee)
Endocrine disruptors are exogenous and endogenous compounds that interfere with the production, signaling and metabolism of natural hormones, thereby disturbing the balance of the endocrine system. Exogenous endocrine disruptors include 17α-ethinylestradiol and endogenous endocrine disruptors include 17β-estradiol. This thesis examinates effects of these endocrine disruptors on the specific activities of rat cytochromes P450 1A1 and 3A1. The enzyme specific activity of CYP1A1 is determined by the marker reaction O-deethylation 7-ethoxyresorufin and the activity of CYP3A1 is determined by the marker reaction 6β-hydroxylation of testosterone. It has been confirmed that both estrogens 17β-estradiol and 17α-ethinylestradiol inhibit activity of CYP1A1 and CYP3A1. The stronger inhibitor of CYP1A1 was 17β-estradiol and of CYP3A1 was 17α-ethinylestradiol. The concentration of estrogens causing 50% inhibition (IC50) of enzymes has been determined, for 17β-estradiol it was 4,6 μM and for 17α-ethinylestradiol 7,9 μM. CYP1A1 is a important enzyme for the biotransformation of carcinogens, it can be said that estrogens can modulate the genotoxicity of CYP1A1 activated carcinogens. The results show that CYP1A1 inhibition increased after pre-incubation of estrogens with NADPH (cofactor of cytochromes P450), that...
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