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Conformational states of the opiod receptors
Provazníková, Adéla ; Hudeček, Jiří (advisor) ; Entlicher, Gustav (referee)
This Thesis elucidates the relationships between structure and function of opioid receptors. The mechanism of opioid receptor function could be better understood on the basis of the recent knowledge of their three-dimensional structure. In the first part of the Thesis, methods for membrane protein crystallization and X-ray structural analysis are reviewed with a special focus on in meso crystallization. Next, the three opioid receptor subtype crystal structures are described and analysed, starting with their general architecture and proceeding to the detailed description including the binding pocket for opioid ligands with the help of visualization with the PyMol software. In its third part, this Thesis re-examines the structure-function correlations predicted on the basis of site-directed mutagenesis (Décaillot F.M. et al, Nature Structural Biology 10, 629, 2003) in the light of now available crystal structure. This analysis is pointing to the potentially important contacts of aspartate D128, suggesting that ligand binding and receptor activation might involve changes in its interaction with tyrosine Y308.
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Effects of valproic acid and its combinations with cytostatic agents on tumor cells in vitro
Hinďoš Hřebačková, Jana ; Dyr, Jan (advisor) ; Vávrová, Jiřina (referee) ; Entlicher, Gustav (referee)
Cancer is one of the most challenging problems the modern medicine is facing today. An increasing incidence and a great variability of tumor cells are the main reasons those drive the research to develop better diagnostics and therapeutic protocols. Histone deacetylase inhibitors, a group of epigenetic chemotherapeutics, are able to improve the performance of currently used anticancer agents. Vaplroic acid that is commonly used as antiepileptic drug exhibits a remarkable anticancer activity by itself as well as it is capable of therapy potentiation based on other therapeutic agents. Its effect to inhibit growth of tumor cells and induce apoptotic cell death seems to be even greater under hypoxic conditions (<1% O2). This study is focused on effect of valproic acid on neuroblastoma cell lines in vitro under normoxic and hypoxic conditions. We observed significantly greater efficacy of valproic acid in hypoxia compared to normoxia. The mechanism of induction of apoptotic cell death is based on disruption of the balance between pro- and antiapoptoic proteins. Intrinsic apoptotic pathway is probably initiated by the action of 19 kDa variant of proapoptotic protein Bax on mitochondrial membrane. Moreover, we examined the efficiency of a combined treatment of neuroblastoma cells with valproic acid and...
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Theoretical study of enzymes related to carcinogenesis: DNA polymerase β and cytochromes P450
Jeřábek, Petr ; Martínek, Václav (advisor) ; Entlicher, Gustav (referee) ; Ettrich, Rüdiger (referee)
Present doctoral thesis contributed to understanding of mechanistic principles of two enzymes participating in the process of carcinogenesis; DNA polymerase (pol ) and cytochromes P450 (CYP). Pol is part of the DNA base-excision repair mechanism (BER). The primary role of pol in, the BER mechanism, is inserting a new nucleotide into a DNA strand according to Watson-Crick base pairing rules. Pol plays an important role in the process of carcinogenesis, approximately 30 % of human tumors express pol mutants. The ability of pol to discriminate between "right" and "wrong" nucleotide during the insertion process is called fidelity. We employed computational methods to elucidate molecular basis of the fidelity of pol . First, the relative free energy calculation method LRA was employed to compare differences in free energies between the "right" and "wrong" nucleotide during its insertion into DNA. The results indicated a better stabilization of transition-state of the nucleophilic substitution catalyzed by pol in the case of the "right" versus "wrong" nucleotide. This difference resulted in an 80-fold contribution to its fidelity. Further, computational methods FEP and LIE were used to examine how mutations effect fidelity of pol . Results were than correlated with experimental data...
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The study of fluorescent products in the mitochondria after an attack by free radicals
Ivica, Joško ; Wilhelm, Jiří (advisor) ; Entlicher, Gustav (referee) ; Drahota, Zdeněk (referee)
Lipofuscin-like pigments are products of reactions involving free radical attack onto molecules with nucleophilic groups. They can be for med, for example, in the reactions between lipid peroxidation decomposition products, such as aldehydes, and amino-group containing compounds, e.g. phospholipids, peptides. Owing to their intrinsic fluorescent properties LFP can be easily measured. LFP are relatively stable and therefore have been successfully used as robust markers of oxidative damage. We undertook the metabolomic studies, where fluorescent LFP were first analysed spectrofluorimetrically by using tridimensional and differential fluorescence spectral arrays. After that, certain LFP were analysed by means of high performance liquid chromatography, in order to resolve the mixture of compounds into distinct fractions. For this purpose we used LFP prepared after isolated heart itochondria had been exposed in vitro to oxidative stress initiated by various triggers. LFP were also analysed during early development in rat brain, which is accompanied by transient increase in oxygen concentration, and in erythrocytes from patients with Alzheimer's disease. We developed HPLC methods for qualitative analysis of LFP of different origin. This analysis unfolded that LFP indeed consist of many chromatographically...
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Biochemical changes accompanying the aging of red wine
Vacková, Tereza ; Hudeček, Jiří (advisor) ; Entlicher, Gustav (referee)
Flavonoids are plant secondary metabolites, which belong to several groups varying in their chemical structure. Anthocyanins and tannins are important flavonoid components of wine that are responsible for its color, taste and other sensory properties. The concentration of anthocyanins in wine is affected by grape variety, processing technology, and climatic conditions. In this Thesis, we studied the changes in color and in related chemical composition, using three non-commercial samples of red wine: Svatovavřinecké (year 2010 and 2012), and home-made wine (prepared without addition of SO2). These changes in color were determined using standard colorimetric method (CIELab) and also a simplified two-parametric spectrophotometric method (tint/color density). The content of anthocyanins was followed using analytical RP-HPLC method. In paralel, simplified oenologic methods for estimation of phenolic compounds were used. Generally the wine samples changed color to darker tint. Chemically, this was caused by polymerisation reactions between anthocyanins and phenolic compounds. This led to the formation of stable pigments characterised by a higher absorption maximum at longetr wavelength, hence a darker tint. Key words: anthocyanins, color, red wine, phenolic compounds, malvidin-3-glucosid, polymeric reactions,...
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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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Preparation of chicken antibodies against phosphoserine residues of phosphoproteins
Benýšek, Jakub ; Tichá, Marie (advisor) ; Entlicher, Gustav (referee)
New type of immunogen for the preparation of chicken antibodies specific for O- phosphorylated amino acid residues of phosphoproteins was prepared: the α-casein phosphopeptide mixture was coupled to maleinylated derivative of bovine serum albumin. Affinity chromatography on immobilized Fe(III) ions (Fe(III)-IDA-Sepharose) was used for the separation of the α-casein phosphopeptide fraction from the α-casein peptide mixture obtained by the proteolytic digestion using trypsin. The presence of coupled phosphopeptide was shown by means of MALDI-TOF MS analysis. After chicken immunisaion with the prepared immunogen the immunoglobulin fraction was isolated from egg yolks that was further purified using affinity chromatography on α-casein-Sepharose and bovine serum albumin immobilized on Sepharose (BSA-Sepharose). The specificity of obtaine immunoglobulin fractions was tested by means of ELISA tests. The obtained results showed, that the prepared chicken antibodies and their fractions obtained by affinity chromatography on α-casein-Sepharose do not recognize the O-fosforyl-L-serine residues of phosphoproteins (α-casein, phosvitin, ovalbumin). Using the prepared different antigens it has been shown, that prepared chicken antibodies interact with maleinylated chain coupled to carrier proteins. .
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Eukaryotic and prokaryotic nitric oxide synthase - structure-function studies
Mikula, Ivan ; Martásek, Pavel (advisor) ; Entlicher, Gustav (referee) ; Král, Vladimír (referee)
Nitric oxide (NO) is an important signaling molecule in organisms. It plays a role in wide spectrum of physiological and pathophysiological processes, including vasodilatation, neurotransmission and host defense. The gaseous molecule of NO is produced by oxidative reaction catalyzed by proteins from the family of nitric oxide synthases (NOSs). Three NOS isoforms were identified in mammals, endothelial (eNOS), neuronal (nNOS) and inducible or immunologic (iNOS). Some bacteria harbor genes coding for proteins homologous to the mammalian NOS oxygenase domain and showing NO-producing activity in vitro. NO generated by pathologic organisms such as B. anthracis and S. aureus is supposed to play a critical role in the pathophysiological processes during the infection. Comparative study of bacterial NOS-like proteins and mammalian NOSs confirmed their principal similarity, but also revealed differences in the interactions of distinct bacterial proteins and mammalian NOS isoforms with different analogs of substrate L-arginine and various ligands. On the basis of the kinetics measurement of NO-rebinding a second NO-binding site in the active center of NOS was predicted. Further, the regulation of NO dynamic and release from the protein by the active site Hbonding network connecting the heme, the substrate and BH4...
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Metabolism of carcinogenic o-nitroanisol and its metabolite o-nitrophenol and two environmental pollutants 2-nitrobenzanthrone and 3-nitrobenzanthrone
Svobodová, Martina ; Stiborová, Marie (advisor) ; Entlicher, Gustav (referee) ; Souček, Pavel (referee)
CHARLES UNIVERSITY IN PRAGUE FACULTY OF SCIENCE DEPARTMENT OF BIOCHEMISTRY Metabolism of carcinogenic o-nitroanisole, its metabolite o-nitrophenol and environmental pollutants 2-nitrobenzanthrone and 3-nitrobenzanthrone Summary of PhD Thesis RNDr. Martina Svobodová Supervisor: Prof. RNDr. Marie Stiborová, DrSc. Prague 2010 RNDr. Martina Svobodová Introduction -1- INTRODUCTION 2-Nitroanisole 2-Nitroanisole (2-methoxynitrobenzene, 2-NA, figure 1) is an important industrial pollutant and a strong carcinogen for rodents causing neoplastic transformation in the urinary bladder and, to a lesser extent, in the spleen, liver and kidney [19, 30, 31] . 2-NA is also a toxic compound, causing anemia. 2-NA is used primarily as a precursor in the synthesis of o-anisidine (2-methoxyaniline), which is an intermediate in the production of many azo dyes. This compound is used in pharmaceutical industry as an intermediate in the synthesis of some medicaments [30, 31] . In spite of potent rodent carcinogenicity of 2-NA, this chemical is weakly mutagenic in the Ames test with the Salmonella typhimurium. This carcinogen also exhibits a low activity in cytogenetic tests. It induces a slight increase in chromosomal aberration and in sister chromatid exchanges, but only at high concentrations [31] . 2-nitroanisole may be...
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