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Cloning and expression of human carbonyl reductase CR-1
Laštovková, Linda ; Wsól, Vladimír (advisor) ; Šimůnek, Tomáš (referee)
The purification of pOTB7 plasmid containing coding sequence of carbonyl reductase1 (CBR1) in cells of Escherichia coli (E. coli) was done by alkali hydrolysis. The sequence of CBR1 was multiplied by polymerase chain reaction (PCR) and synthesized primers with restriction sites were used. The left primer contained sequence for restrictive endonuclease NdeI and the right primer for restrictive endonuclease XhoI. Validation of the first step was confirmed by size measurement of the synthesized fragment with following restrictive analysis realized by restrictive endonuclease BamHI. Prepared sequence CBR1 was cloned into Topo vector, which was transformed into the competent E. coli cells. Topo vector was purified by alkali hydrolysis after innidation of E. coli cells. The size of cyclic Topo vector without CBR1 and Topo vector with CBR1 coding sequence was verified on agar gel by restrictive endonuclease BamHI. This was the validation of the second step. Subcloning of coding sequence CBR1 from Topo vector to express vector pET15b was the last step.
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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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Purification and characterization of selected human microsomal reductase
Skarka, Adam ; Wsól, Vladimír (advisor) ; Živná, Lucie (referee)
Area of human liver microsomal reductases hasn't been fully explored yet. Purification and characterization of still undescribed enzymes is an important step in finishing of this task. There is separation and purification of this reductases described in my work, with usage of hydrophobic interaction chromatography on low pressure chromatography instrument ÄKTA purifier. There were two columns with different substrates used, Phenyl sepharose (low sub) and Octyl sepharose. Separation conditions were the same in both cases. Results obtained by incubation of fractions and measurement on HPLC showed significant differences between both columns and their substrates. But it isn't possible to determine, which column is better for purification, because big area still remain for adjustment and optimization of purification conditions.
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Bioanalytical methodical approaches for the disposition monitoring of a new potential drug in organism using HPLC-PDA-MS-MS
Císař, Přemysl ; Nobilis, Milan (advisor) ; Chládek, Jaroslav (referee) ; Wsól, Vladimír (referee)
1. Summary The aim of this study consisted in the development and validation of a bioanalytical method for the identification and determination of dimefluron and its metabolites in biomatrices. For the purposes of this study, six expected potential dimefluron metabolites were prepared and identified by NMR and MS. Higher homologue of dimefluron (homodimefluron) was selected and synthesized as an internal standard. The chromatographic columns with various types of the stationary phases were tested. The best results were achieved using a pentafluorophenylpropylsilyl silica gel column rinsed with the mobile phase of acetonitrile- phosphate buffer pH = 3 in a gradient mode. After the validation of the bioanalytical method, this chromatographic system was applied to the disposition study of dimefluron in rats. After an intragastric administration of dimefluron to rats, samples of urine and faeces were collected each 24 hours. The elimination of dimefluron and its phase I and phase II metabolites in urine and faeces was studied. Maximum concentrations of dimefluron derivatives in the excrements were found in the time interval of 24 - 48 hours after the administration. 9-O-Desmethyldimefluron and 3-O-desmethyldimefluron were the principal metabolites found in the rat faeces, while the metabolic products of...
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Metabolic study of sibutramine
Link, Marek ; Wsól, Vladimír (advisor) ; Holčapek, Michal (referee) ; Nobilis, Milan (referee)
Obesity represents a serious problem especially in American and European populations. Pharmacotherapy in combination with a reduced calorie diet is recommended for obese patients as a multi-modal approach to weight loss. Sibutramine hydrochloride monohydrate represents one of the few established and well-proven agents available for treatment of obesity. It is sold as a racemic mixture under the trade-name Meridia, Reductil or Lindaxa. It acts as a monoamine reuptake inhibitor. The weight loss of patients induced by sibutramine is thought to be due to a combination of serotonin- and noradrenaline-mediated mechanisms that increase both satiety and energy expenditure. In organisms, sibutramine is rapidly demethylated to form metabolites M1 and M2. These metabolites contribute largely to the pharmacological effects of sibutramine and the pharmacokinetic characteristics of M1 and M2 were thoroughly studied in human plasma. Although sibutramine is widely used for the treatment of obesity almost ten years, the published information on the further metabolic fate of metabolites M1 and M2 as well as on the elimination of sibutramine from the body is almost exclusively limited to package inserts of the product. To address this issue we determined the routes of elimination of sibutramine in humans via urine. LC-API/MS...
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Human microsomal glutathione s-transferase
Hrnčiarik, Karel ; Wsól, Vladimír (advisor) ; Kvasničková, Eva (referee)
The microsomal glutathione S-transferases (GST, EC 2.5.1.18) constitute a group of phase II detoxification enzymes present in eukaryotic cells and prokaryotic organisms. They play significant roles in defense against carcinogenic, toxic, and pharmacologically active electrophilic compounds. The microsomal glutathione S-transferase is a homotrimeric, membrane-bound enzyme, that catalyzes conjugation of electrophilic compounds with glutathione and reduction of lipid hydroperoxides. Peroxidase activity may be of importance for protection against lipid peroxidation under conditions of oxidative stress. A number of biochemical approaches have been used to study the properties of MGST, including enzymatic activity, sub-cellular distribution, substrate specificity, and proteins structure. MGST is considered a member of the MAPEG (membrane- associated proteins in eicosanoid and glutathione metabolism) superfamily of structurally and phylogeneticaily related enzymes, including MGST 1, MGST 2, MGST3, microsomal Ya-GST, MGST 1 Like 1, 5-lipoxygenase activating protein, and leukotriene C4 synthase. Enzymes in this superfamily are involved in detoxification, protection from oxidative stress, and synthesis of prostaglandin E and cysteinyl leukotrienes. On the basis of understanding funcitons MGST is possible...
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The impact of inflammation on muscarinic receptor subtypes expression - Immunohistochemical examination of lacrimal and salivary glands of rat and human
Székelyová, Anita ; Soukup, Ondřej (referee) ; Wsól, Vladimír (advisor)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Anita Székelyová Supervisor: Prof. Gunnar Tobin, O.D., Ph.D. Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: The impact of inflammation on muscarinic receptor subtypes expression - Immunohistochemical examination of lacrimal and salivary glands of rat and human This project was focused on the characterization of muscarinic receptor subtype expression in lacrimal and salivary glands of the rat. Immunohistochemical methods were used to reveal the presence of the M1, M3 and M5 receptors and the expression of these subtypes was further studied under experimentally induced inflammatory conditions. The expression of the above mentioned muscarinic receptor subtypes was also studied in human labial glands, both healthy and with autoimmune sialadenitis compatible with Sjögren's syndrome. M1, M3 and M5 muscarinic receptors were found in the lacrimal and submandibular glands of the rat using immunoenzyme and immunofluorescent methods. The M5 receptor in the lacrimal gland was also detected on intracellular, possibly nuclear localization. The tissue with induced inflammation didn't show any significant differences in the muscarinic receptor expression. The immunofluorescent labeling of the...
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Production and purification of recombinant human xenobiotic nuclear receptors
Menclová, Alena ; Pávek, Petr (referee) ; Wsól, Vladimír (advisor)
The submitted thesis deals with the production and purification of recombinant human xenobiotic nuclear receptors (NRs). This study was focused on the production of ligand - binding domains (LBDs) of human constitutive androstane receptor (hCAR) and human pregnane X receptor (hPXR). These receptors belong to the subfamily NR1I NRs and serve as key regulators of drugs metabolism. They are also implicated in many human diseases such as inflammatory bowel disease or diabetes and obesity. The cDNA of recombinant proteins, which serves for production of hCAR and hPXR LBDs, was cloned into bacterial expression vector pET-15b (Novagen). The gained constructs were propagated in E. coli cells of DH5 strain. The isolated plasmids were analyzed after colony PCR and restriction analysis for the correct insert on agarose gels. The clones with correct inserts were subsequently transformed into E. coli cells strain BL21 DE3 and recombinant protein was synthesized in expression system, which was regulated by concentration of isopropyl-1-thio--D-galactopyranosid (IPTG) under optimal conditions. Production of hCAR and hPXR LBD was observed in defined concentrations of IPTG, temperatures and cell densities (OD600). The results revealed the following optimal conditions for protein production; for the hCAR LBD it was...
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Gene expression profiling after experimental perinatal asphyxia and the effect of complement-derived anaphylatoxin C3a
Šourková, Hana ; Wsól, Vladimír (advisor) ; Šimůnek, Tomáš (referee)
Background: The complement system is involved in neuroprotection and brain repair after brain damage. To understand the molecular mechanisms of these processes, we performed gene expression profiling using quantitative real-time polymerase chain reaction (qPCR), which is the most accurate modern strategy for gene expression analysis. Project: Our project was directly aimed at expression profiling of selected genes potentially involved in loss and rescue of neural tissue during three weeks after hypoxic-ischemic brain injury, an experimental model of perinatal asphyxia. Recent experiments have shown that over-expression of C3a under the control of the GFAP promoter (C3a/GFAP) reduced hippocampal injury after left common carotid artery ligation in neonatal mice by 50%, compared to wild type mice. Here, we assessed how the local expression of C3a/GFAP transgene affects gene expression profiles. Gene expression was measured on samples from hippocampus ipsilateral and contralateral to the injury and ipsilateral part of cortex, taken at the time of injury, 6 and 24 hours; 3, 7 and 21 days after the injury. Results: Our data showed that the regulation of gene expression after hypoxic-ischemic injury differs in timing and intensity and may also be region dependent. The analysed genes belong to families...
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