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Studies on carbonyl reductases 1 and 3 variants with emphasis on S-nitrosoglutathione as substrate and inactivator
Hartmanová, Tereza ; Wsól, Vladimír (advisor) ; Kvasničková, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Tereza Hartmanová Supervisor: Dr. Claudia Staab, Dr. Hans-Jörg Martin, Prof. Ing. Vladimír Wsól, Ph.D. Title of the diploma thesis: Studies on carbonyl reductases 1 and 3 variants with emphasis on S-nitrosoglutathione as substrate and inactivator Human CBR1 and CBR3 (carbonyl reductases 1 and 3) are monomeric, NADPH-dependent enzymes belonging to the short chain dehydrogenase/reductase superfamily. Although they are highly similar at the amino acid level (72 % identity) the enzymes exhibit considerable differences in substrate specificity. The CBR1 substrate spectrum is well described, including a variety of compounds e.g. the endogenous indol isatin, S-nitrosoglutathione (GSNO), prostaglandins, quinones, and many carbonyl group bearing xenobiotics. In contrast, CBR3 shows a distinct and much narrower range of substrates and its role is still not fully clarified. Nevertheless, the dissimilar substrate spectra strongly indicate that CBR1 and CBR3 play different metabolic roles. In the present study, the catalytic properties of CBR1 towards the latest CBR1 substrate described, GSNO, were investigated. CBR3 was assessed for potential GSNO-reducing activity, but no in vitro activity was...
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The influence of bosutinib, neratinib and ibrutinib inhibition on the activity of selected reductases from AKR and SDR superfamilies
Hudáčová, Lenka ; Wsól, Vladimír (advisor) ; Hofman, Jakub (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Lenka Hudáčová Supervisor: Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: The influence of bosutinib, neratinib and ibrutinib inhibition on the activity of selected reductases from AKR and SDR superfamilies Anthracycline antibiotics (ANTs) are antineoplastic drugs. Daunorubicin (DAUN) is used in the treatment of acute leukaemia. Enzymes from aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies mediate the reduction of DAUN to its C-13 alcohol metabolite daunorubicinol (DAUNOL), which is more cardiotoxic, less antineoplastic and is causing anthracycline resistance. In my diploma thesis, I examined the inhibitory effect of bosutnib, neratinib and imatinib on the activity of enzymes from the SDR and AKR superfamilies. The specific enzyme activity and inhibitory effect were estimated on the base of in vitro enzymatic production of DAUNOL by the ultra-high-performance liquid chromatography (UHPLC) system. MTT assay was used to measure DAUN and ibrutinib cytotoxicity effect on HCT116 cell culture. In vitro enzymatic activities for recombinant enzymes were decreased in order CBR1 > AKR1C3 > AKR1B1 > AKR1A1 > AKR7A2 > AKR1B10 > AKR1C1 > AKR1C2 > AKR1C4 > CBR3. The...
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Production and purification of recombinant human xenobiotic nuclear receptors
Menclová, Alena ; Wsól, Vladimír (advisor) ; Pávek, Petr (referee)
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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Cloning, expression and purification of human AKR1C3
Dudová, Radka ; Wsól, Vladimír (advisor) ; Novotná, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Dudová Radka Supervisor: Prof. Ing. Vladimír Wsól, Ph.D Title of diploma thesis: Cloning, expression and purification of human AKR1C3 The purification of the vector pOTB7 containing the coding sequence ( CDS ) of aldo-keto reductase 1C3 ( AKR1C3 ) in Escherichia coli ( E.coli ) cells was performed by an alkaline lysis method. The coding sequence was then amplified by polymerase chain reaction. Result accuracy of the demonstrated step was shown by verification of the size of the synthesized fragment and by a restrictive analysis with the restriction endonuclease PvuII. In the second step, the coding sequence was ligated into the Topo 2.1 vector which was transformed into competent E. coli cells. The cells were multiplied and Topo 2.1 vector with the inserted code sequence was obtained by the alkaline lysis method. The result was verified by comparing of the size of the Topo 2.1 vector without inserted CDS and the vector Topo 2.1 with the CDS on an agarose gel and the subsequent restrictive analysis with the restriction endonuclease HindIII. The coding sequence was verified by the sequenation. There was also carried out the subcloning of the CDS from the Topo 2.1 vector into the express...
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Cloning, expression and purification of human AKR1B1
Lundová, Tereza ; Wsól, Vladimír (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Tereza Lundová Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Cloning, expression and purification of human AKR1B1 Aldose reductase (EC 1.1.1.21) AKR1B1 is one of the 13 human enzymes of the AKR superfamily. All human AKRs are cytosolic and NADP(H) dependent. AKR1B1 plays an important role in metabolism of endogenous and exogenous substances. The main endogenous substrate is glucose. Its reduction to sorbitol is consistently linked to secondary diabetic complications. From xenobiotics metabolized by AKR1B1, daunorubicin, an anticancer drug from the group of anthracyclines, is reduced to daunorubicinol. This metabolite is less active than parent drug and is the cause of anthracycline related cardiotoxicity. At present, many projects are focused on AKR1B1 as a target enzyme and specific inhibitors of AKR1B1 are looking for. The recombinant protein of AKR1B1 was prepared in E. coli together with the pET expression system. First, cDNA for AKR1B1 in pOTB7 was isolated from E. coli. The coding sequence of AKR1B1 was amplified by a PCR. PCR was performed with Phusion Hot Start II polymerase and pair of forward and reverse primers, which contained NdeI and XhoI restriction...
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Inhibition Study of Human Membrane-bound Carbonyl Reductase
Laštovková, Linda ; Wsól, Vladimír (advisor) ; Kvasničková, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Linda Laštovková Supervisor: prof. Ing. Vladimír Wsól, Ph.D. Title of thesis: Inhibition study of human membrane-bound carbonyl reductase. Carbonyl reductases are an important group of enzymes that participate in metabolism of both endogenous substances also xenobiotics. Potential anticancer drug oracin is one of such xenobiotics that is metabolised by various carbonyl reductases to two enantiomers of dihydrooracin. The new human microsomal carbonyl reductase also takes part in biotransformation of oracin. This enzyme was recently purified on Department of biochemical sciences (Faculty of Pharmacy in Hradec Králové). The aim of this study was to find some inhibitors of the new enzyme and distinguish it in terms of inhibitors from another microsomal carbonyl reductase 11β-hydroxysteroid dehydrogenase 1. Flavonoids are substances produced by plants, they have a different both positive and also negative effect on human organism. One of such effect is inhibition effect on diverse enzymes. Carbonyl reductases also fall in this group. It has been described inhibitory effect of different flavonoids on carbonyl reductases. Inhibition study of the new human micorosomal carbonylreductase was...
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Development and applications of affinity carrier for isolation of human carbonyl-reducing enzymes
Andrýs, Rudolf ; Wsól, Vladimír (advisor) ; Šebela, Marek (referee) ; Šatínský, Dalibor (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Rudolf Andrýs Supervisor: Prof. Ing. Vladimír Wsól, Ph.D Title of dissertation thesis: DEVELOPMENT AND APPLICATION OF AFFINITY CARRIER FOR ISOLATION OF HUMAN CARBONYL-REDUCING ENZYMES For several millennia the human medicine is based on application of small bioactive molecules that are administered in the form of plant extracts or synthetic compounds. However, their use in modern medicine is not possible without a detailed understanding of their biochemical effects and identification of their molecular targets. Chemical proteomics based on the specific recognition between the bioactive molecule and the target molecule is currently the most widely used techniques for identification of molecular targets of small molecules. Compared to conventional biochemical methods (e.g. 2D electrophoresis), chemical proteomics represents particularly sensitive and very selective technique that enable successful identification of biomolecules from complex biological samples that are naturally presented in very small concentrations. Carbonyl- reducing enzymes, which play an important role in physiology due to their involvement in metabolism of various endogenous (e.g. prostaglandins, steroid...
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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 ; Wsól, Vladimír (advisor) ; Soukup, Ondřej (referee)
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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Optimization of purification of human membrane-bound carbonyl reductase.
Andrýs, Rudolf ; Wsól, Vladimír (advisor) ; Bílková, Zuzana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Rudolf Andrýs Supervisor: prof. Ing. Vladimír Wsól, Ph.D Title of diploma thesis: Optimalization of purification of a human membrane-bound carbonyl reductase Carbonyl reductases are enzymes participating in metabolic pathways of various eobiotics and xenobiotics. Of all known enzymes metabolizing xenobiotics only 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) is found in the microsomal membrane. It also contributes to metabolism of prochiral anticancer drug oracin, which main metabolic pathway is a carbonyl reduction on the position 11 leading to two enantiomers of (+) an (-) 11-dihydrooracin (DHO). Based on the discrepancy between microsomes and 11β-HSD1 stereospecifity of oracin reduction exist a hypothesis of participation an unknown microsomal enzyme in oracin metabolism. The aim of this study is to purify a new microsomal carbonyl reducing enzyme contributing in the biotransformation of oracin. Human liver microsomes were solubilised and desalted. The prepared sample was used for the first purification step on Q-Sepharose. Captured flow through fraction Q2 was loaded on Phenyl-sepharose. Captured suitable fraction P11 was used for third purification step by gel filtration. All...
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