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Investigation of sugar modified pyrimidine nucleosides as potential inhibitors of Thymidine (TP) and Uridine (UP) phosphorylasis
Růžičková, Markéta ; Wsól, Vladimír (advisor) ; Boušová, Iva (referee)
1 Abstract of the thesis RŮŽIČKOVÁ M.; INVESTIGATION OF SUGAR MODIFIED PYRIMIDINE NUCLEOSIDES AS POTENTIAL INHIBITORS OF THYMIDINE (TP) AND URIDINE (UP) PHOSPHORYLASE Key words: uridine phosphorylase, thymidine phosphorylase, nucleoside, inhibitor, anticancer chemotherapy, substrate specifity The work deals with the investigation of novel potential inhibitors of recombinant thymidine and uridine phosphorylases from E. coli and Salmonella typhimurium. The main goals of this study are the determination of kinetic parameters for natural substrates (thymidine and uridine) for recombinant TP and UP, assessment of the role of phosphate anion in the enzymatic transformations and search for new inhibitors of TP and UP. Study of UP and TP substrate specifity and activity under the various reaction conditions is desired to obtain better view to their functions and to design novel structures of potencial inhibitors. The determination of the Michaelis constant KM and reaction rate Vmax was performed at the spectrophotometer Varian Cary-300 Bio. The representation of obtained kinetic data we used the Lineweawer-Burk plot. The next point of my work was the determination of the enzymatic activity and their substrate specifity for some pyrimidine analogues by HPLC analysis of reaction mixtures. Tested compounds...
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Effect of compounds with antioxidant properties on glycation of aspartate aminotransferase
Bacílková, Eliška ; Boušová, Iva (advisor) ; Dršata, Jaroslav (referee)
Publishing place: Charles University in Prague, Faculty of Pharmacy in Hradec Králové Author: Eliška Bacílková Title: Effect of compounds with antioxidant properties on glycation of aspartate aminotransferase Supervisor: PharmDr. Iva Boušová, Ph.D. In this diploma thesis, I was concerned with the direct and indirect effects of uric acid, aminoguanidine, and hydroxycitric acid on enzymatic activity of aspartate aminotransferase and on its glycation by methylglyoxal. The catalytic activities were measured by kinetic UV method. Stabilities of aminoguanidine and uric acid, their interactions with the molecule of enzyme and influence on formation of glycation products were observed by absorption spectroscopy. Although aminoguanidine decreased enzymatic activity in higher concentration, it showed positive indirect effects on glycation of aspartate aminotransferase and it also decreased amount of rising glycation products. Hydroxycitric acid did not have negative direct effect on enzyme activity and was partially able to protect enzyme from glycation by methylglyoxal. Uric acid exerted neither negative direct nor positive indirect effects. Aminoguanidine and uric acid were stable during experiment and reacted with molecule of protein, which was expressed by modifications of its UV-VIS spectra in the area...
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Interaction of natural compounds with the molecule of HSA
Nováková, Anežka ; Dršata, Jaroslav (referee) ; Boušová, Iva (advisor)
Author: Anežka Nováková Publishing place: Univerzita Karlova v Praze, Farmaceutická fakulta v Hradci Králové Supervisor: PharmDr. Iva Boušová, Ph.D. Title: Interaction of Natural Compounds with the Molecule of HSA This diploma thesis is concerned with the interaction of chosen natural compounds (apigenin, naringenin, kaempferol) with the molecule of human serum albumin. In initial experiments, I observed the stability of these antioxidants under physiological conditions in time by UV-VIS absorption spectroscopy. Then I observed an influence of different storage temperatures on the stability using the same method. The proper interaction of chosen antioxidants with albumin was studied by UV-VIS absorption spectroscopy and fluorescence spectroscopy. Fluorimetric titration proved that mentioned antioxidants quenched the tryptophan fluorescence in the molecule of human serum albumin. Using the Stern-Volmer analysis I found out that a mechanism of fluorescence quenching was static for all antioxidants in the range of initial concentrations (2,5 - 10 µM) of antioxidants. Calculated binding parameters of antioxidants provided more specific information about their interaction with HSA and revealed the relationship between their chemical structure and affinity to HSA. The presence of 3-OH group with 2,3...
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Modulatory effects of apigenin and luteolin on cytochrome P4501A expression and activity in intestinal cells
Šalovská, Barbora ; Boušová, Iva (referee) ; Skálová, Lenka (advisor)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Title, Name, Surname of candidate: Barbora Šalovská Title, Name, Surname of tutor: Doc. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: Modulation effects of apigenin and luteolin on activity and expression of cytochrome P4501A2 in intestinal cells Flavonoids are a large group of polyphenolic compounds that occur naturally in plant kingdom. They have been shown to posses a variety of biological activities including anti-inflammatory, anti-oxidant and anti-mutagenic effects and play a prominent role in cancer prevention. The aim of this work was research of the effect of flavones apigenin and luteolin on an activity and expression of subfamily 1A of cytochromes P450 in human colon cancer cell line HCT-8. The CYP1A isozymes enzymatic activity was measured by EROD/MROD activity assay; the quantity of CYP1A's protein was set by Western blotting. We found out that apigenin and luteolin significantly decreased CYP1A activity in HCT-8 cells. During synchronous application of model inductor CYP1A β-naftoflavon (β-NF) with apigenin or luteolin (at the concentrations of 5µM and higher), the induction effect of β-NF on CYP1A was reduced. Apigenin and luteolin significantly decreased the CYP1A activity...
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Immunodetection of enzymes catalyzing carbonyl reductions (Western blotting)
Černá, Libuše ; Boušová, Iva (referee) ; Netopilová, Miloslava (advisor)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Libuše Černá Supervisor: PharmDr. Miloslava Netopilová, Ph.D. Title of diploma thesis: Immunodetection of enzymes catalyzing karbonyl reductions (Western blotting) The enzymes which reduce carbonyl group are important for metabolism of eobiotics and xenobiotics. There are recognized three superfamilies of carbonyl- reducing enzymes: aldo-keto reductases (AKR), the medium-chain dehydrogenases/reductases (MDR) and the short-chain dehydrogenases/reductases (SDR). The 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is member of the SDR superfamily. This enzyme is essential for activating steroid hormone cortison to its active form cortisol. Its activity is also vital for metabolism of some other xenobiotics, e.g the anticancer drug oracin. Yet the carbonyl group of oracin is also reduced by another unknown enzyme wich is located in microsomal membrane. For the purification of this unknown enzyme Q Sepharose was used. The aim was to find out which fractions after purification contain 11β-HSD1 by using the imudetection (Western blotting). In Western blotting we used nitrocelulose membrane, the primary rabbit polyclonal antibodies against 11β-HSD1 dilution 1:1000, the secondary polyclonal...
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