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1
Petit, Elise. "Etude des Glutathion Transférases : caractérisation de la classe Kappa et rôle de ces enzymes dans l'hépatotoxicité des Thiopurines." Rennes 1, 2007. http://www.theses.fr/2007REN1B072.
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Les Glutathion Tranférases (GST) constituent un système multienzymatique de détoxication. Elles sont impliquées dans la prévention , le développement des tumeurs et dans la réponse aux anticancéreux. Pendant ma thèse , je me suis intéressée à la GST de classe Kappa. Sa caractérisation nous a permis de mettre en évidence sa présence dans les mitochondries et les peroxysomes. Cette localisation particulière suggère que le GST Kappa pourrait avoir un rôle lié aux fonctions cellulaires de ces deux organites. Les GST étant en outre impliquées dans des phénomènes de résistance à des anticancéreux, je me suis également intéressée à l’hépatotoxicité des thiopurines. Nos résultats montrent que les cellules d’origine humaine sont moins sensibles à un traitement par les thiopurines que les hépatocytes de rat, bien que les composés provoquent une hépatoxicité. En conclusion le GST Kappa est présente dans les mitochondries et les peroxysomes et il pourrait y avoir une relation entre son activité et le métabolisme lipidique. Ce travail a également été l’occasion d’initier pour la première fois une étude sur les htiopurines dans les cellules hépatiques humaines
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Van, Eldik Annamaria Johanna. "Synthesis of glutathione conjugates as selective inhibitors for parasitic glutathione S transferases." Thesis, De Montfort University, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.246521.
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Halfwassen, Kathrin. "Untersuchungen zu Glutathion-sensitiven Farbstoffen in der Meerschweinchen-Retina." Doctoral thesis, Universitätsbibliothek Leipzig, 2012. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-89656.
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Die Glutathionverhältnisse und -verschiebungen zwischen Gliazellen und Ganglienzellen vor und nach oxidativem Stress wurden erstmals im lebenden Zellverband, ex vivo, untersucht. Die Untersuchungen erfolgten an akut isoliertem Retinagewebe vom Meerschweinchen, von welchem Bilder am Laser scanning microscope (LSM) erstellt wurden. Über die Anwendung des in vivo-Fluoreszenzfarbstoffes CellTracker Green wurde dabei dessen Spezifität für Glutathion überprüft und bestätigt.
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Yang, Bo. "Biliary glutathione transport pathways." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0019/MQ52967.pdf.
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Lyon, Robert Patrick. "Enzymology at the dimer interface of cytosolic glutathione S-transferases /." Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/8165.
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Kearns, Pamela Renate. "The role of glutathione and mu class glutathione s-transferases in childhood acute leukaemia." Thesis, University of Newcastle Upon Tyne, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.311134.
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Fläring, Urban. "Glutathione during stress in man /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-799-5/.
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Patzewitz, Eva-Maria. "Glutathione metabolism of Plasmodium falciparum." Thesis, University of Glasgow, 2009. http://theses.gla.ac.uk/913/.
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Apicomplexan parasites of the genus Plasmodium are the causative agent of malaria, one of the most prevalent infectious diseases worldwide. Five different Plasmodium species can cause malaria in humans, leading to a total of approximately 500 million cases each year and of these, P. falciparum causes the most deadly form of the disease and is responsible for more than 1 million deaths annually. A major problem in the global fight against malaria is the widespread resistance of the parasites against the currently available drugs. It is of great importance to identify new drug target as well as to understand the mechanisms that lead to drug resistance in the first instance in order to potentially reverse the resistant phenotypes and to avoid the development of resistance in the future. The tripeptide glutathione (GSH) or γ-glutamylcysteinyl-glycine is the most abundant low molecular weight thiol in most eukaryotic organisms and serves a number of important functions as sulfhydryl-buffer, cofactor for enzymes and for the detoxification of xenobiotics and drugs. GSH is an important component of the antioxidant machinery and because malaria parasites live in an environment rich in iron and oxygen and thus increased oxidative stress, they depend on functional antioxidant systems. The biosynthesis pathway for GSH, consisting of γ-glutamylcysteine synthetase (γGCS) and glutathione synthetase (GS) is present in malaria parasites as well as in their host cells. Previous studies have shown that depletion of GSH has an antimalarial effect, but it remained unclear whether parasites were killed directly or died because their host cell could not survive the depletion of GSH. To address this question, the knockout of both genes encoding the enzymes of the GSH biosynthesis pathway in P. falciparum was attempted. While both gene loci were targeted by control constructs, the knockout of either pfγgcs or pfgs was impossible, indicating both genes are essential for parasite survival in the erythrocytic stages. To analyse the localization of γGCS and GS, GFP-tagged recombinant fusion proteins were expressed in the parasites and showed that GSH biosynthesis is cytosolic. Apart form its other functions GSH has previously been suggested to be involved in resistance to the antimalarial drug chloroquine (CQ). CQ was for a long time the first line antimalarial drug due to its high efficiency, low cost and low toxicity, but is now widely inefficient in the treatment of the disease. CQ resistance is associated with mutations in the CQ resistance transporter (PfCRT), a membrane protein of the digestive vacuole that allows the efflux of the drug form its site of action. However, PfCRT mutations alone cannot explain the full array of phenotypes found in resistant parasites. GSH is able to degrade heme, the target of CQ, in vitro and it has been suggested that elevated GSH levels contribute to CQ resistance. However, analyses of isogenic parasite lines bearing different forms of PfCRT in this study revealed lower GSH levels and higher susceptibility to inhibition of GSH biosynthesis in the CQ resistant lines. These changes did not correlate with changes in the expression of enzymes involved in the de novo biosynthesis or consumption of GSH. However, the cellular accumulation ratio for CQ indicated a decrease of free heme in the resistant parasites. Mutant forms of PfCRT expressed in oocytes of Xenopus laevis were able to transport GSH, while the sensitive wild-type form did not transport the tripeptide. The findings of this study suggest that in parasites bearing mutant PfCRT, GSH is transported into the digestive vacuole where it is able to contribute to resistance by degrading heme, before the tripeptide itself is degraded by peptidases inside the vacuole, consistent with the overall reduction of GSH levels in CQ resistant parasites.
9
Evans, D. C. "Renal processing of glutathione conjugates." Thesis, University of Nottingham, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.383757.
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Di, Ilio C. "Studies on bacterial glutathione transferase." Thesis, Cranfield University, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.333472.
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Praig, Vera Gertraud. "Immobilised glutathione for affinity binding." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620379.
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Knight, Simon Alexander Bowles 1961. "The use of anti-glutathione peroxidase antibodies in the study of selenium-dependent glutathione peroxidase." Thesis, The University of Arizona, 1988. http://hdl.handle.net/10150/276906.
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Liver glutathione peroxidase activity is affected by changes in selenium (Se) status. To investigate the effect of Se status on GSH-Px protein we prepared antibodies against rat liver GSH-Px and used them in an ELISA. The immunoreactivity of the anti-GSH-Px antibodies against GSH-Px was both tissue and species specific. When rats were depleted of Se, liver GSH-Px activity decreased exponentially to zero with a half-life of 2.8 d. Liver GSH-Px protein also decreased exponentially, but not to zero, with a longer half-life of 5.2 d. Dietary repletion of Se-deficient rats with 0.5 mg Se/kg diet increased GSH-Px protein and activity after 1 d. After 14 d of repletion the levels of GSH-Px protein and activity had plateaued at the levels present in Se-adequate rats. When Se-deficient rats were injected with 15 or 60 ug Se, only rats injected with 60 ug Se and killed 24 h later showed an increase in GSH-Px protein and activity. These results suggest that when Se is limiting, GSH-Px protein and GSH-Px activity are coordinately regulated by the available Se, but in Se-adequacy homeostatic processes control the level of GSH-Px.
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Saudrais, Élodie. "Mécanismes de neuroprotection liés au glutathion dans la barrière sang - liquide céphalorachidien choroïdienne au cours du développement périnatal." Thesis, Lyon, 2019. http://www.theses.fr/2019LYSE1026/document.
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Plus de 50 % des handicaps neurodéveloppementaux sont dus à une exposition périnatale à des stress toxiques ou oxydants. Comprendre comment le cerveau est protégé au cours du développement périnatal et pourquoi ses mécanismes de défense sont dépassés lorsque l’enfant est soumis à un stress important est donc crucial. La barrière sang – liquide céphalorachidien (LCR), localisée au niveau des plexus choroïdes, présente une capacité de détoxification élevée et pourrait donc avoir un rôle prépondérant dans la protection du cerveau au stade périnatal. Nous avons étudié la capacité de plusieurs enzymes choroïdiennes à protéger l'environnement liquidien cérébral pendant la période postnatale chez le rat, et évalué si leurs activités pouvaient être induites par la voie du nuclear factor erythroid-2-related factor 2 (Nrf2). Le facteur Nrf2 peut en effet moduler l’expression de différents gènes codant pour des enzymes de détoxification. Nous avons montré que les glutathion transférases (Gst) et les glutathion peroxydases (Gpx), intervenant respectivement dans l’inactivation des molécules toxiques et dans la régulation du stress oxydant, présentaient des activités choroïdiennes élevées pendant la période postnatale, et avons caractérisé fonctionnellement leur capacités de neuroprotection. Le traitement des ratons avec du diméthylfumarate (DMF), inducteur de la voie Nrf2, induit la migration nucléaire de Nrf2, augmente l’activité choroïdienne Gst, et réduit de 40 % le passage cérébral de toxiques substrats des Gst. Ces données montrent la capacité neuroprotectrice précoce des plexus choroïdes, et indique qu’elle peut être induite pharmacologiquementMore than 50 % of intellectual or sensory-motor deficits in children are due to perinatal exposure to oxidative stress or toxicants. Understanding brain protection mechanisms during development is crucial to design therapeutic strategies to address these disabilitating disorders. The choroid plexuses, forming an interface between the blood and the cerebrospinal fluid (CSF), have a high detoxifying capacity, suggesting their involvement in neuroprotection. The nuclear factor erythroid-2-related factor 2 (Nrf2) pathway can modulate the expression of several genes encoding for antioxidant proteins and detoxifying enzymes. We studied the ability of several choroidal enzyme families to protect the brain fluid environment during the postnatal period in rat and explored whether this protection can be enhanced by Nrf2 pathway. We focused on glutathione transferases (Gsts), which conjugate toxic compounds to glutathione, and glutathione peroxidases (Gpxs), which detoxify reactive oxygen species. Gst and Gpx specific activities were high during the postnatal period in choroid plexuses compared to the cerebral cortex, and their neuroprotective functions were efficient. The Nrf2 factor is expressed in choroid plexuses during the perinatal period. Treatment of rat pups with Nrf2 activator dimethylfumarate induced Nrf2 nuclear translocation and increased Gst activities in choroid plexus tissues. The dimethylfumarate treatment resulted in a large decrease of the blood-to-CSF permeability of a prototypical Gst substrate. These data substantiate the early neuroprotective functions of choroid plexuses, which can be enhanced upon treatment with clinically used pharmacological compounds
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Corrigall, Anne Vint. "Human glutathione S-transferases : characterization, tissue distribution and kinetic studies." Doctoral thesis, University of Cape Town, 1988. http://hdl.handle.net/11427/27205.
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In this study the purification of human basic and near-neutral liver, and human basic and acidic lung glutathione S-transferases (GSH S-T) was undertaken. Purification of the basic and near-neutral GSH S-T was achieved using a combination of affinity chromatography, chromatofocusing and immunoaffinity chromatography. Affinity and ion exchange chromatography were employed in the purification of the basic and acidic lung forms. The purified proteins had similar physicochemical characteristics to the GSH S-T purified by others. The binding of 1-chloro-2,4-dinitrobenzene (CDNB) to the 3 classes of human GSH S-T, viz. basic, near-neutral and acidic and the effects of such binding, if any, were examined. Human acidic lung GSH S-T is irreversibly inactivated by CDNB in the absence of the co-substrate glutathione (GSH). The time-dependent inactivation is pseudo-first order and demonstrates saturation kinetics, suggesting that inactivation occurs from an EI complex. GSH protects the enzyme against CDNB inactivation. In contrast, the basic and near-neutral GSH S-T are not significantly inactivated by CDNB. Incubation with [¹⁴C]-CDNB indicated covalent binding to all 3 classes of GSH S-T. When the basic and acidic GSH S-T were incubated with [¹⁴C]-CDNB and GSH, cleaved with cyanogen bromide, and chromatographed by HPLC, a single peptide fraction was found to be labelled in both classes. Incubation in the absence of GSH yielded 1 and 2 additional labelled peptide fractions for the basic and acidic transferases, respectively. These results suggest that while CDNB arylates all 3 classes of human GSH S-T, only the acidic GSH S-T possesses a specific GSH-sensitive CDNB binding site, which when occupied leads to time-dependent inactivation of the enzyme. The tissue distribution and localization of the 3 classes of human GSH S-T in normal and tumour tissue was examined. Antibodies to representatives of the 3 classes were raised in rabbits, and radial immunodiffusion employed to quantitate their concentrations in the cytosol of 18 organs from 9 individuals. The data provide the first direct, quantitative evidence for the inter-individual and inter-organ variation suggested by earlier workers. The absence of the near-neutral GSH S-T in 5 of the 9 individuals studied confirms an earlier suggestion of a "null" allele for this transferase. Basic and acidic GSH S-T (apart from in a single liver), were always present. Near-neutral GSH S-T, when present, were found in all tissues examined. The marked inter-organ and inter-individual variation observed in this study may explain individual and organ susceptibility to drugs, toxins and carcinogens. The immunohistochemical localization of the 3 classes of GSH S-T reveals important differences in their localization, and may provide insight into their functions in various organs and tissues.
15
Nieslanik, Brenda Sue. "A structure-function analysis of the C-terminus in glutathione S-transferase A1-1 /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/8163.
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Rogers, Lynette K. "Mechanisms of nuclear localization of glutathione reductase, subnuclear colocalization with thioredoxin, and genetic analysis of a chemically induced glutathione reductase knockout." Connect to this title online, 2004. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1091194762.
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Thesis (Ph. D.)--Ohio State University, 2004.Title from first page of PDF file. Document formatted into pages; contains xiv, 133 p.; also includes graphics (some col.). Includes bibliographical references (p. 124-133).
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Jorgensen, Karen Virginia. "EFFECTS OF GLUTATHIONE AND GLUTATHIONE-S - TRANSFERASE ON AFLATOXIN B(,1) MUTAGENESIS IN THE AMES TEST." Thesis, The University of Arizona, 1985. http://hdl.handle.net/10150/275310.
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Goold, Richard David. "The glutathione S-transferases : kinetics, binding and inhibition." Doctoral thesis, University of Cape Town, 1989. http://hdl.handle.net/11427/27175.
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The glutathione S-transferases are a group of enzymes which catalyse the conjugation of reduced glutathione with a variety of electrophilic molecules, and they are therefore thought to play a major role in drug biotransformation and the detoxification of xenobiotics. The cytosolic GSH S-transferase isoenzymes of rat, man and mouse have been assigned to three groups, Alpha, Mu and Pi, based on N-terrninal amino acid sequences, substrate specificities, immunological cross-reactivity and sensitivities to inhibitors. The kinetic mechanism of the GSH S-transferases is controversial, due to the observation of non-Michaelian (non-hyperbolic) substrate-rate saturation curves. The most detailed investigations of the steady-state kinetics of glutathione S-transferase have been performed with isoenzyme 3-3 (class Mu) and the substrate 1,2-dichloro-4-nitrobenzene (DCNB). Explanations for the apparently anomalous non-hyperbolic kinetics have included subunit cooperativity, steady-state mechanisms of differing degrees of complexity and the superimposition of either product inhibition or enzyme memory on these mechanisms. This study has confirmed the biphasic kinetics for isoenzyme 3-3 with DCNB and shown non-hyperbolic kinetics for this isoenzyme with 1-chloro-2,4-dinitrobenzene (CDNB) and for isoenzyme 3-4 with DCNB and CDNB. It is proposed that the basic steady-state random sequential Bi Bi mechanism is the simplest mechanism sufficient to explain the non-hyperbolic kinetics of GSH S-transferases 3-3 and 3-4 under initial rate conditions. Neither more complex steady-state mechanisms nor the superimposition of product inhibition or enzyme memory on the simplest steady-state mechanism are necessary.
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Njålsson, Runa Viđarr. "Molecular aspects of glutathione synthetase deficiency /." Stockholm, 2004. http://diss.kib.ki.se/2003/91-7349-808-4.
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Dixon, David Peter. "Glutathione transferases in maize (Zea mays)." Thesis, Durham University, 1998. http://etheses.dur.ac.uk/4788/.
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The glutathione transferases (GSTs) of maize have been the most studied GSTs in plants, however much is still not known about these enzymes. In the course of the current study six GST subunits (Zm GSTs I, II and III, which have been reported previously, and Zm GSTs V, VI and VII, which have not been previously reported) have been identified in the dimers Zm GST I-I, I-II, I-III, V-V, V-VI and V-VII. Maize GSTs are known to be important in herbicide detoxification and the purified maize enzymes were each found to have differing activities toward a number of herbicides, and also a range of other potential GST substrates. Additionally, Zm GST I II and Zm GST V-V possessed glutathione peroxidase activity. The developmental regulation and chemical inducibility of maize GSTs were studied in maize seedlings using western blotting, with different subunits showing markedly different responses. Zm GST I was constitutively present in all plant parts and unaffected by chemical treatment, Zm GST II was only detected in young roots but was induced in roots and shoots by many different chemical treatments, and Zm GST V was present at low levels throughout maize plants, with levels enhanced greatly by treatment with the safener dichlormid but not by other chemicals tested. cDNA clones corresponding to Zm GST subunits I, III, V, VI and VII were isolated by library screening using antibody or DNA probes. The cDNA sequences for Zm GST subunits V, VI and VH were different from those of previously cloned type I (theta class) maize GSTs and were most similar to the auxin-regulated GST family (type III or tau class GSTs) previously only identified in dicotyledonous species. The cloned GSTs were expressed as recombinant proteins in E. coli, allowing further characterisation, including detailed kinetic analysis for recombinant Zm GST I-I and Zm GST V-V.
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Dalton, K. G. "Glutathione transferase isoenzymes in rat hepatocarcinogenesis." Thesis, University of Bradford, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.379829.
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Troughton, P. R. "Isoenzymes of rat glutathione S-transferase." Thesis, University of Bradford, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.371503.
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Dodd, Charlotte Claire. "The role of glutathione in hepatoprotection." Thesis, University of Liverpool, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.250328.
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Fry, V. A. H. "Microglial glutathione and glutamate : regulation mechanisms." Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/18703/.
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Microglia, the immune cells of the central nervous system (CNS), are important in the protection of the CNS, but may be implicated in the pathogenesis of neuroinflammatory disease. Upon activation, microglia produce reactive oxygen and nitrogen species; intracellular antioxidants are therefore likely to be important in their self-defence. Here, it was confirmed that cultured microglia contain high levels of glutathione, the predominant intracellular antioxidant in mammalian cells. The activation of microglia with lipopolysaccharide (LPS) or LPS + interferon-gamma was shown to affect their glutathione levels. GSH levels in primary microglia and those of the BV-2 cell line increased upon activation, whilst levels in N9 microglial cells decreased. Microglial glutathione synthesis is dependent upon cystine uptake via the xc- transporter, which exchanges cystine and glutamate. Glutamate is an excitatory neurotransmitter whose extracellular concentration is tightly regulated by excitatory amino acid transporters, as high levels cause toxicity to neurones and other CNS cell types through overstimulation of glutamate receptors or by causing reversal of xc- transporters. Following exposure to LPS, increased extracellular glutamate and increased levels of messenger ribonucleic acid (mRNA) for xCT, the specific subunit of xc-, were observed in BV-2 and primary microglial cells, suggesting upregulated GSH synthesis. An activation-induced decrease in N9 GSH levels suggests that this cell line is more susceptible to oxidative damage, and may be less able to upregulate GSH synthesis. Albumin, to which microglia may be exposed following blood-brain barrier damage, increased iNOS expression, glutamate release, xCT mRNA levels and intracellular levels of GSH and ATP in BV-2 and primary microglia. Primary and BV-2 microglial conditioned medium contained low levels of GSH, suggesting that microglia may release GSH. Modulation of microglial metabotropic glutamate receptors (mGluRs) may alter microglial activation and neurotoxicity. Here, stimulation of the neuroprotective mGluR5 and group III mGluRs caused a decline in GSH levels in BV-2 and N9 microglia, respectively. In contrast mGluR1 stimulation may increase BV-2 GSH levels. The work presented in this thesis therefore extends current knowledge regarding microglial GSH and its regulation, and contributes to the understanding of microglial neurotoxicity and neuroprotection.
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Waller, Alan Richard. "Glutathione conjugation in non-human primates." Thesis, University of Surrey, 1985. http://epubs.surrey.ac.uk/848541/.
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The scientific literature indicates that the biotransformation of xenobiotics in man is more similar to that observed in the non-human primate than to other laboratory animal species. The main objective of the current investigation was to compare the ability of 3 species of non-human primate, the rhesus monkey (Macaca mulatta), the cynomolgus monkey (Macaca fascicularis) and the baboon (Papio species), to conjugate xenobiotics with glutathione since such information is lacking in the literature. The metabolism and pharmacokinetics of ethacrynic acid, a diuretic known to be extensively metabolised by glutathione conjugation in several other animal species, was studied in the non-human primates. The results indicate that the overall capacity for glutathione conjugation in the non-human primate is large, and demonstrate that various organs and tissues can simultaneously contribute to the detoxication and elimination of various xenobiotics by this pathway. In common with other laboratory animal species, the non-human primate excreted the major proportion of radioactivity, following administration of [14]C-ethacrynic acid, in the bile. The radioactivity in the bile was mainly associated with metabolites which had resulted from the initial conjugation of ethacrynic acid with glutathione, and the subsequent metabolism of this conjugate via the mercapturic acid pathway. There were notable species differences, between the non-human primates and other laboratory animals, in the proportions of the various metabolites which were excreted in the bile. Although the concentrations of glutathione in the various tissues of the non-human primate were similar to those reported for other mammals, species differences occurred in the rates of glutathione conjugation and the subsequent metabolism of the glutathione conjugate. A significant finding concerned the extent to which various aB-unsaturated xenobiotics were conjugated with glutathione. There were marked species differences between the non-human primate and the rat in this respect. The rhesus monkey, cynomolgus monkey and the baboon are similar as regards ethacrynic acid metabolism, tissue glutathione concentrations, tissue glutathione S-transferase activities, and the specific activities of the glutathione S-transferase isoenzymes. The rhesus monkey more closely resembles man in these respects, but both other species of non-human primate would appear to be suitable for use in metabolism and toxicological studies of compounds likely to be conjugated with glutathione.
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Hayes, Peter C. "Glutathione S-transferases in the pancreas." Thesis, University of Edinburgh, 1993. http://hdl.handle.net/1842/19832.
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Glutathione S-transferase (GST) is an important xenobiotic metabolising enzyme which has been extensively studied in the liver. In the first part of this study immunohistochemistry was used to identify the presence and histological localisation of different GST isoenzymes in various gastrointestinal tract tissues in the human in health and disease. GSTP was found throughout the gastrointestinal and biliary tract whilst the position and quantity of other isoenzymes varied locally. Increased expression of GSTP was observed in cholangiocarcinoma and colonic adenocarcinoma, but not hepatocellular carcinoma. In the pancreas GSTP was present in ductal and centroacinar cells, whilst GSTA was present in acinar cells. GSTM was universally present in the cells of islets of Langerhan, not demonstrating genetic polymorphism. In both chronic pancreatitis and pancreatic carcinoma increased expression of GSTP was demonstrated. Using affinity chromatography and high performance liquid chromatography GSTA, P and M were purified from human pancreatic tissue. A novel GST isoenzyme, which ran on SDS/PAGE, similar to GSTP, was identified, purified and confirmed by Western blot analysis to be a GSTA. Feeding rats exclusively on raw soya flour resulted in pancreatic hypertrophy and eventually carcinoma. Serial measurements of GST activity showed only a minor reduction with short term feeding which returned to normal with chronic administration contrary to what has been proposed (Ross & Barrowman, 1987). No selective change in GST isoenzymes was identified. A dominant cytoplasmic protein, shown both enzymatically and by Western blot analysis to be α-amylase fell dramatically with short term administration recovering only marginally with chronic administration.
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Tamaki, Hisanori. "Studies on Yeast Glutathione S-Transferase." Kyoto University, 1991. http://hdl.handle.net/2433/168776.
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本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものであるKyoto University (京都大学)
0048
新制・課程博士
農学博士
甲第4688号
農博第649号
新制||農||585(附属図書館)
学位論文||H3||N2261(農学部図書室)
UT51-91-C106
京都大学大学院農学研究科食品工学専攻
(主査)教授 栃倉 辰六郎, 教授 木村 光, 教授 佐々木 隆造
学位規則第5条第1項該当
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Rawlinson, Rosemary Julia. "Glutathione and the cytosolic heme pool." Thesis, King's College London (University of London), 2018. https://kclpure.kcl.ac.uk/portal/en/theses/glutathione-and-the-cytosolic-heme-pool(e78204fa-e583-41eb-ab9c-1a759b9abf33).html.
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Recently glutathione (GSH) has been proposed as a key component of the cytosolic iron pool, possessing a buffering role for cytosolic iron(II), protecting it from autoxidation. However the chemical nature of the cytosolic heme pool is unknown. We have investigated whether GSH binds heme iron. If so, the resulting complex would be expected to have increased stability and solubility in aqueous solutions, compared to the extremely hydrophobic heme molecule, thereby reducing its ability to partition into membranes. An interaction between glutathione and hematin was established with the affinity constant (Ka) of glutathione for hematin determined by absorption spectroscopy to be 5x104 M-1. Using standard bioassays the influence of GSH on heme oxidase activity and the partitioning of hematin into lipid bilayers was assessed. GSH was found to stabilise hematin in the presence of H2O2 and was found to have a profound effect on the partitioning of hematin into lipid bilayers, reducing partitioning into prepared liposomes by < 70%. The presence of hematin ligated to GSH within the lysate of mammalian cells was established using synthesised [59Fe]hematin, Caco-2 cells and size exclusion HPLC. These results suggest that GSH could be the predominant ligand for the cytosolic heme pool. The effect of glutathione on absorption and catabolism of hematin in Caco-2 cells showed an initial decrease in hematin uptake and a decrease in heme oxygenase 1 expression. Hematin when ligated to GSH, in the presence of ascorbic acid and O2, was found to be rapidly degraded and whilst GSH decreases hematin partitioning into erythrocyte plasma membranes, the effect was not as dramatic as was observed in liposomes. These results did not provide further support for glutathione serving as the predominant ligand for the organic iron pool and led to the conclusion that heme is chaperoned (not by glutathione) and encapsulated within endosomes in the cytosol. It is proposed that GSH and ascorbic acid function cooperatively to rapidly ligate and degrade any heme which escapes from the endosome system into the cytosol, hence preventing ferroptosis.
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Dixit, Vaishali S. "Inactivation of glutathione s transferase zeta by dichloroacetic acid." [Gainesville, Fla.] : University of Florida, 2005. http://purl.fcla.edu/fcla/etd/UFE0010021.
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Thesis (Ph. D.)--University of Florida, 2005.Typescript. Title from title page of source document. Document formatted into pages; contains 98 pages. Includes Vita. Includes bibliographical references.
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Yilmaz, Can. "Glutathione S-transferase Activity And Glutathione Levels In Drought Stressed Pinus Brutia Ten. Trees Growing In Ankara." Master's thesis, METU, 2006. http://etd.lib.metu.edu.tr/upload/12607732/index.pdf.
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Turkish red pine is coastal tree and is a drought resistant pine that withstands more aridity and poor soils than most other timber species growing in the same climatic conditions. In Turkey, this species grows in southern and western Anatolia and is also found in the Marmara region.
Drought results in a water deficit in plant tissues, which, in turn, can lead to an imbalance in the redox poise of plant cells, and thus inducing oxidative stress in plants.
Resistance to conditions associated with oxidative-stress must, in part, rely on endogenous antioxidative defense mechanisms required to maintain cellular homeostasis. Glutathione is one of the major endogenous antioxidants in plants known to play an important role in plant defense mechanisms. Glutathione S-transferase (GST, EC 2.5.1.18) is a GSH dependent detoxifying enzyme in plants, which catalyzes the conjugation of GSH.
In this study, we investigated the changes in cytosolic glutathione S-transferase enzyme activity using CDNB as substrate and total thiol amount in Pinus brutia Ten., related to the drought stress during four months, June to September. The osmotic pressure in the needles was also determined as an indirect measure of drought condition. Together with the increase in the temperature values from June to July, GST enzyme activity increased from 15,78 ±1,36 µ
moles min-1 mg protein-1 to 22,91 ±
1,99 µ
moles min-1 mg protein-1 which was statistically significant. However in August, GST activity had fallen to 16,54 ±
1,61 µ
moles/min/mg protein, which may be because of a local rainfall at the beginning of the August in the sampling area. In September, GST activity significantly increased with respect to June, in accordance with high temperatures. The total thiol amount was not changed significantly during the sampling period. Although there were statistically significant changes in osmotic pressure in the needdles collected during the same sampling period, it did not exactly correlated to the changes in GST activity.
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Bryant, David. "Glutathione conjugation of herbicides and fungicides in plants and fungi : functional characterization of glutathione transferases from phytopathogens." Thesis, Durham University, 2004. http://etheses.dur.ac.uk/3117/.
Full textAbstract:
The aim of this research was to determine the relative importance of glutathione S-transferase (GST) mediated herbicide and fungicide detoxification in wheat plants and to functionally characterise GSTs cloned from phytopathogenic fungi. Wheat seedlings treated with and without safeners were used for in vivo and in vitro pesticide metabolism studies with the herbicide fenoxaprop-P-ethyl and the fungicide fluquinconazole, which are both known to undergo glutathione conjugation. Fenoxaprop-P-ethyl underwent rapid detoxification by this route and the rates of metabolism were enhanced in safener treated wheat. In contrast, fluquinconazole was poorly metabolised with only a small proportion detoxified by glutathlonylation irrespective of whether or not the plants were safener treated. Subsequent studies confirmed that whereas fenoxaprop was a substrate for wheat GSTs, fluquinconazole was not. Similarly, when a diverse range of systemic and contact fungicides was tested, none were found to undergo conjugation mediated by wheat GSTs, even though several compounds underwent conjugation with glutathione in alkaline conditions. Metabolite profiling by HPLC-MS established that the alternative thiol present in wheat, hydroxymethyl glutathione, was used in addition to glutathione in fenoxaprop detoxification. The S-conjugates were then further metabolised by peptidases and glucosyl- and malonyl transferases to yield polar derivatives such as the cysteinyl-malonyl-glucosyl derivative. This rapid processing was in contrast to that determined in herbicide-susceptible and herbicide-resistant black-grass, where the glutathione derivative was the major metabolite of fenoxaprop. Using an informatics approach, GSTs in IVIagnaporthe griseae and Phytoptithora infestans were identified cloned and then expressed in E. coli, with the pure enzymes assayed for glutathione-dependent activities. MgGSTX1 of Magnaporthe griseae was found to be a member of a novel class of GSTs (termed xi class) and was shown to function as a thioltransferase, with a specific activity of 50.4 ± 3.4 nKat mg (^-1)protein. Unusually, the catalytic mechanism of thiol transfer was insensitive to the alkylating agent iodoacetamide, indicating that free cysteines were not involved in catalysis. MgGSTX1 did not conjugate any of the experimental xenobiotics tested with GSH. The theta class GST P/GSTT1 cloned from P. infestans exhibited glutathione peroxidase activity and readily detoxified fungitoxic oxylipins produced by potato as antimicrobial defence by potato through the action of lipoxygenases. Using anti-sera raised against the purified recombinant protein, immunoblotting experiments revealed expression of P/GSTT1 in the in vitro cultured fungus as well as during colonisation of potato. P/GSTT1 did not demonstrate GSH conjugating toward any of the xenobiotic substrates tested including herbicides and fungicides. Based on the sequences of GSTs identified in this study an extension of the existing classification system is suggested to include the GSTs of fungal phytopathogens
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Abdu, Habibu U. "Investigating the role of glutathione and glutathione biosynthetic genes in the adaptation of Anopheles gambiae to insecticides." Thesis, Abertay University, 2015. https://rke.abertay.ac.uk/en/studentTheses/d30eb4df-64c0-43fe-ba6d-0fe778ff6b80.
Full textAbstract:
Malaria remains a serious public health challenge in the tropical world, with 584,000 deaths globally in 2013, of which 90% occurred in Africa, and mostly in pregnant women and children under the age of five. Anopheles gambiae (An. gambiae) is the principal malaria vector in Africa, where vector control measures involve the use of insecticides in the forms of long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS). The development of insecticides resistance mitigates these approaches. Glutathione (GSH) is widely distributed among all living organisms, and is associated with detoxification pathways, especially the Glutathione S-transferases (GSTs). Its direct involvement and relevance in insecticide resistance in An. gambiae has not been determined. Thus, this work examines the contribution of GSH, its biosynthetic genes (GCLM, GCLC) and their possible transcriptional regulator Nrf2 in insecticide resistance in An. gambiae sampled from agricultural setting (areas of intensive agriculture) and residential setting (domestic area). Bioinformatics analysis, W.H.O. adult susceptibility bioassays and molecular techniques were employed to investigate. Total RNA was first isolated from the adults An. gambiae mosquitoes raised from agricultural and residential field-caught larvae which had been either challenged or unchallenged with insecticides. Semi-quantitative RT-PCR using gel image densitometry was used to determine the expression levels of GCLM, GCLC genes and Nrf2. Bioinformatics’ results established the presence of putative AGAP010259 (AhR) and AGAP005300 (Nf2e1) transcription factor binding sites in An. gambiae GCLC and GCLM promoters in silico. An. gambiae s.l. studied here were highly resistant to DDT and permethrin but less resistant to bendiocarb. Both knockdown resistance (kdr) mutation variants L1014S and L1014F that confers resistance to pyrethroid insecticides were identified in both An. coluzzii and An. arabiensis sampled from northern Nigeria. The L1014F was much associated with An. coluzzii. A significant positive correlation (P=0.04) between the frequency of the L1014F point mutation and resistance to DDT and permethrin was observed. However, a weak or non-significant correlation (P=0.772) between the frequency of the L1014S point mutation and resistance was also found. L1014S and L1014F mutations co-occurred in both agricultural and residential settings with high frequencies. However, the frequencies of the two mutations were greater in the agricultural settings than in the residential settings. The levels of total, reduced and oxidized GSH were significantly higher in mosquitoes from agricultural sites than those from residential sites. Increased oxidized GSH levels appears to correlate with higher DDT resistance. The expression levels of GCLM, GCLC and Nrf2 were also significantly up-regulated in adults An. gambiae raised from agricultural and residential field-caught larvae when challenged with insecticide. However, there was higher constitutive expression of GCLM, GCLC and Nrf2 in mosquitoes from agricultural setting. The increased expression levels of these genes and also GSH levels in this population suggest their roles in the response and adaptation of An. gambiae to insecticide challenges. There exists the feasibility of using GSH status in An. gambiae to monitor adaptation and resistance to insecticides.
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Dolan, Catherine. "Regulation of mouse hepatic glutathione S-transferases." Thesis, University of Edinburgh, 1991. http://hdl.handle.net/1842/23855.
Full textAbstract:
The glutathione S-transferases (GST) are a multi-gene family of dimeric proteins which catalyse the conjugation of glutathione to a wide range of electrophilic compounds. Three classes of mouse cytosolic GST have been isolated, alpha, mu and pi, comprising Ya-, Yb- and Yf-type subunits respectively. A marked sexual dimorphism in mouse hepatic GST has been observed. The YfYf GST is the most abundant form in the male, constituting approximately 70% of total hepatic GST content. By contrast, the Yf subunit represents only a minor form in the livers of female mice. The hormonal controls which regulate the expression of the YfYf GST in mouse liver have been investigated. Testosterone, the major male sex hormone, is found to regulate the levels of Yf in mouse liver. Castration of the male leads to a decline in the levels of Yf to that observed in females. Replacement therapy with testosterone partially restores the levels of Yf. Testosterone treatment induces expression of this subunit in the female. Growth hormone secretion from the pituitary gland differs markedly between the sexes. Androgens act to produce the male pattern of growth hormone secretion which regulates the sex-specific expression of numerous hepatic proteins. Male 'little mice', specifically defective in the production of growth hormone, exhibit a feminine pattern of GST expression, despite having normal levels of testosterone. Testosterone treatment has no effect on the expression of YfYf in little mice. In contrast, growth hormone replacement therapy, administered to simulate the male-specific pattern causes an increase in the expression of the Yf subunit. These findings strongly suggest that testosterone regulates the hepatic expression of the Yf subunit indirectly through the male-specific pattern of growth hormone secretion. The effects of the xenobiotics, phenobarbital, dexamethasone and 1,4-Bis[2-(3,5-dichloropyridyloxy)]-benzene (TCBOP) on mouse hepatic GST content have been investigated in two strains of mice, C57BL/6 and DBA/2. All three compounds were found to induce hepatic GST in both strains and sexes, predominately affecting expression of members of the mu class. TCBOP was the most potent inducer. Hypophysectomy did not significantly affect induction of GST by these compounds.
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Ristoff, Ellinor. "Inborn errors in the metabolism of glutathione /." Stockholm, 2002. http://diss.kib.ki.se/2002/91-7349-392-9/.
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Ren, Pei. "Glutathione reductase, redox homeostasis, and mitochondrial dysfunction." Thesis, Kent State University, 2014. http://pqdtopen.proquest.com/#viewpdf?dispub=1555282.
Full textAbstract:
Overproduction of reactive oxygen species and increasing oxidative stress have emerged as the major factors leading to mitochondrial dysfunction during myocardial ischemia and reperfusion (I/R) injury. Under normal physiology conditions, glutathione (GSH) concentration is high in the mitochondrial redox pool However, GSH is oxidized to GSSG (oxidized glutathione) upon the exposure of oxidative stress, such as the overproduction of ·O 2– and ·O2 – derived oxidants during I/R injury. Decreased GSH/GSSG ratio was found to enhance protein S-glutathiolation of complex I, which serves as a redox marker for mitochondrial dysfunction in vivo. Excess GSSG normally is recycled to GSH by mitochondrial glutathione reductase (GR2 isoform) in maintaining the basal level of complex I S-glutathiolation and redox homeostasis in mitochondria. Available evidence has indicated that protein S-glutathiolation in vitro can be induced by GSSG through the reaction of protein thiol-disulfide exchange. Thus we hypothesize that GR2 deficiency will impair mitochondrial function and subsequently heart function via enhancing S-glutathiolation of complex I. Furthermore, there is little understanding toward the role of GR2 in mitochondrial dysfunction, and the mechanism of GR2-mediated S-glutathiolation of Complex I. We first performed the study in the heart of a post-ischemic rat model to evaluate the alteration in GR2 activity after myocardial infarction. Rats received intraperitoneal administration of 3-bischoloroethyl-1-nitrosourea(BCNU), a GR2 inhibitor. We observed systolic dysfunction, decreased respiratory control ratio, and increased ·O2– generation under the condition of state 3 respiration after BCNU treatment. We also found decreased GSH/GSSG ratio, increased redox activity and increased Complex I S-glutathiolation. These data suggest that mitochondrial GR deficiency and mitochondrial oxidative stress and associated redox modification are the molecular mechanisms of BCNU induced systolic dysfunction. The above studies were further evaluated using the animal model of cardiac-specific SOD2 transgenic mice. The overexpression of SOD2 reversed the BCNU-induced GR2 inhibition and mitochondrial impairment.
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Al-Timari, A. A. A. K. "Binding determinants for some glutathione-dependent enzymes." Thesis, University of Essex, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.354003.
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Lewis, Alexander David. "Glutathione-dependent enzyme expression in drug resistance." Thesis, University of Edinburgh, 1988. http://hdl.handle.net/1842/19050.
Full textAbstract:
Glutathione and glutathione-dependent enzymes play a central role in the protection of cells from cytotoxic chemicals. In particular, reduced glutathione (GSH) and glutathione S- transferases (GST's) have been studied in relation to intrinsic and acquired resistance of tumours to cytotoxic drugs. There are however, other glutathione-dependent enzymes which may also be involved in drug resistance: these include glutathione perox-idase (GPX) and the enzymes, fglutamyltranspeptides (fGT), fglutamyl cysteinyl synthetase ('yGCS) and glutathione reductase (GRD). The latter enzymes are involved in the maintenance of reduced GSH levels within cells. GSH and the above glutathione-dependent enzymes have been studied in a series of drug resistance models, a) drug resistant tumour cell lines generated in vitro and in vivo; b) chemotherapeutic agent induced resistance in normal bone marrow cells; c) in oxygen resistant lung cells and d) in preneoplastic foci, in order to evaluate whether GSH and associated glutathione-dependent enzymes form part of an adaptive response involved in protection against the environment. Several models of drug resistance involving cell lines in culture were taken for study. A CHO cell line resistant to chlorambucil, an ovarian cell line generated in vivo, resistant to cis-Platinum and chlorambucil, and a sarcoma cell line resistant to adriamycin. In all these cell lines GST, GSH and 7GT were significantly elevated. In the latter two cell lines selenium dependent GPX was also induced. In the CHO line the elevated GST activity was explained by a 40 fold induction of the alpha class Yc GST subunit and a 2 fold elevation in the Ya subunit. In mouse bone marrow cells following the administration in vivo of a low 'primary dose' of cyclophosphamide, transient increase in alpha Ya and particularly mu Yb GST subunits were found. These increases have been associated with a subsequent protection against a higher lethal dose of the same agent. The changes observed in GST isozyme composition were confined to the granulocyte population. Differences in selenium-dependent GPX, GSH and fGT were also found in cells resistant to high oxygen, with only marginal changes in GST sub-unit profiles. In preneoplastic foci, significant elevations in pi class Yf GST and also alpha Ya and mu Yb were detected. Selenium- dependent GPX was decreased and TfGCS and GRD elevated in this model. These studies indicate therefore: 1) GST levels in certain cells appear to be directly related to resistance to cytotoxic chemicals; 2) Changes in GST expression associated with preneoplasia and in acquired drug resistance are not confined to one sub group of GST enzymes; 3) Changes in GST expression are often paralleled by changes in GSH and other glutathione- dependent enzymes; 4) The most consistent phenotypic changes observed in the drug resistant models studied were in GSH and fGT levels; 5) There seemed to be an inverse relationship in the regulation of selenium and non-selenium-dependent GPX activity in different drug resistant models. GSH and glutathione-dependent enzyme changes in acquired drug resistance, appear to be due to an adaptive response which may be of central importance in the resistance of tumours to chemotherapeutic agents.
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Meikle, Ian. "Glutathione S-transferases in the adrenal cortex." Thesis, University of Edinburgh, 1992. http://hdl.handle.net/1842/19139.
Full textAbstract:
Data available prior to this thesis had shown that, of all bovine organs examined, the adrenal cortex exhibited the second highest level of glutathione S-transferase (GST) expression behind the liver. This finding, along with increasing evidence implicating the importance of GST in endogenous detoxification processes, formed the basis for a further extensive investigation of the GST isoenzymes expressed by the adrenal cortex. Investigation of the GST isoenzymes expressed by a number of different bovine organs using affinity chromatography on S-hexylglutathione-Sepharose 6B (S-hexG-Ag) revealed a marked organ-specific distribution of these enzymes. Bovine adrenal cortex, in particular, expressed isoenzymes from each GST class, as determined by immunoblotting experiments. GST activity determinations of these enzyme pools using a number of model substrates revealed the bovine enzymes to possess a specificity distinct to that of rat and human GST. Isoelectric focusing of the bovine adrenal cortex isoenzymes showed them to possess pl values similar to those found in other species. The affinity-purified mu- and pi-glass isoenzymes were resolved using anion-exchange chromatography, followed by reverse-phase hplc. Using this approach, at least 3 mu-class GST subunits and 1 pi-class GST subunit were identified. Ion-exchange chromatography failed to resolve the affinity-purified alpha-class GSTs, and reverse-phase hplc analysis resolved 2 polypeptides, designated Ya1 and Ya3 respectively. Analysis of the protein that failed to bind to the S-hexG-Ag column revealed that about 35% of GST activity remained in this fraction. Application of this material to glutathione-Sepharose 6B (GSH-Ag) resulted in the purification of an abundant alpha-class GST (1.3% total cytosolic protein). This GST was found to exhibit marked peroxidase and Δ5-ketosteroid isomerase activities, in addition to high activity with 4-hydroxynonenal. SDS/PAGE analysis revealed 2 distinct polypeptides of Mr 25900 and 26500, the former being equivalent to the Ya3 subunit purified on S-hexG-Ag, and the latter named Ya2. Ion-exchange chromatography of the GSH-Ag purified alpha-class GST isoenzyme pool resulted in a complex picture, suggesting there to be at least 3 distinct subunits in this pool.
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Ahmad, Laziana. "Characterisation of glutathione transferase from Arabidopsis thaliana." Thesis, University of York, 2017. http://etheses.whiterose.ac.uk/17477/.
Full textAbstract:
Glutathione transferases (GSTs) are soluble enzymes with activity towards a wide range of xenobiotic and endogenous compounds. The Arabidopsis genome encoded 54 GSTs, which have been classified into eight classes including Tau and Phi. Members of these Tau and Phi clades are strongly upregulated in response to abiotic stresses such as xenobiotics and biotic stresses including pathogen attack. The most characterised activity of GSTs is the transfer of glutathione to an electrophilic centre to form a polar glutathionylated conjugate. However, increasing number of research demonstrated a non-catalytic activity plants GSTs, especially in the transportation of flavonoids from the cytosol to the vacuole. Despite the wealth of investigations into GSTs, and probably as a result of overlapping substrate specificities, the endogenous roles for the vast majority of these plant GSTs remains unknown. Here, the binding interaction of camalexin, indole-3-aldehyde, quercetrin and quercetin to GSTF2 in a non-catalytic fashion was observed in three different sites; two identical sites of L1 and one L2 from the X-ray crystallography data. Mutagenesis of the active residues, Q73L, H77A, Y97A and R154A were performed and using isothermal calorimetry (ITC) techniques, lower binding affinities were observed for all mutants towards all ligands except for Y97A and Q73L which showed higher binding affinities with indole-3-aldehyde. This unexpected finding was likely due to the conformational change of the mutant compared to the wild type, as observed in the structure of mutant Y97A. On elucidating the catalytic activity of GSTs, the structure of GSTU25 in complex with disulphide glutathione was obtained. The GSTU25 has been recently identified to catalyse the denitration of TNT to form 2-glutathionyl- 4,6-dinitrotoluene, a potentially more amenable product for subsequent degradation. This structure complex provides insights of GSTU25 folding upon substrate binding. The involvement of GSTU25 in the detoxification of TNT was further analysed using CRISPR/Cas9 technology. Subclades of GSTU25, including GSTU24, GSTU21, GSTU19 were knockout with the aim to remove overlapping substrate specificities and to finally reveal TNT-specific phenotypes. Only gstU25 Cas9 segregated Arabidopsis were obtained from the experiment that could potentially be optimised in the future study.
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REN, PEI. "Glutathione Reductase, Redox Homeostasis, and Mitochondrial Dysfunction." Kent State University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=kent1366643205.
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Dinescu, Adriana. "Modeling wild type and mutant glutathione synthetase." Thesis, University of North Texas, 2004. https://digital.library.unt.edu/ark:/67531/metadc5556/.
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Glutathione syntethase (GS) is an enzyme that belongs to the ATP-grasp superfamily and catalyzes the second step in the biosynthesis of glutathione. GS has been purified and sequenced from a variety of biological sources; still, its exact mechanism is not fully understood. Four highly conserved residues were identified in the binding site of human GS. Additionally, the G-loop residues that close the active site during catalysis were found to be conserved. Since these residues are important for catalysis, their function was studied computationally by site-directed mutagenesis. Starting from the reported crystal structure of human GS, different conformations for the wild type and mutants were obtained using molecular dynamics technique. The key interactions between residues and ligands were detected and found to be essential for enzyme activity.
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Hill, Alison Elspeth. "Regulation of glutathione S-transferases during stress." Thesis, University of Edinburgh, 1994. http://hdl.handle.net/1842/19846.
Full textAbstract:
Elevation of glutathione S-transferases (GST) in tumour cells can be responsible for resistance to a variety of chemotherapeutic agents. It has been hypothesised that GSTs may be induced as part of a stress response, similar to that of the prokaryotic adaptive response. To further this investigation I studied the induction of GSTs in a variety of permanent and transient stress models. A chlorambucil resistant CHO cell line which was known to express increased levels of Alpha-class GST was studied to determine the nature of the increase in protein. Northern and Southern blot analysis revealed a 4-8 fold amplification in the DNA encoding the Alpha-class GSTs with an accompanying increase in mRNA levels. Elevated levels of an Alpha-class GST were noted in oxygen resistant CHO cells. Transient exposure to 98% oxygen also induced the same Alpha-class GST. A heat shocked lung tumour cell line as well as heat selected sublines showed some changes in the levels of Pi- and Mu-class GSTs. A novel putative Mu-class GST subunit has been identified in the nucleus of heat shocked cells. The nature of the GST level variations at the RNA and DNA levels were studied. These studies do not suggest co-ordinate regulation of the GSTs as part of a general stress response. It does not exclude the possibility of GST π and perhaps the nuclear Mu-class GST are induced as part of a more limited response either to heat or in certain tissues. Inconsistencies in the data from the preliminary induction experiments led to the investigation of the effect of growth conditions on GST levels. Unexpectedly isoenzymes from three classes of GST were found to be elevated by increased confluence and a low frequency of feeding. This response was found to be mediated through the culture media.
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La, Pira Lucia. "Post-translational mechanisms regulating Glutathione Peroxidase 4." Doctoral thesis, Università degli studi di Padova, 2016. http://hdl.handle.net/11577/3424680.
Full textAbstract:
Glutathione peroxidase 4 (GPx4) is a selenocysteine-containing homolog of the vertebrate glutathione peroxidase family, peculiarly reducing membrane hydroperoxide by glutathione (GSH). GPx4 was first purified in 1982 as a ‘peroxidation inhibiting protein’, and is emerging to date, together with its substrate GSH, as a major determinant in regulating cell balance between proliferation and death. Recently, it emerged that GPx4 inactivation or GSH depletion in cells causes death by a novel subroutine relying on lipid peroxidation, named ferroptosis. Consistently, reverse genetics studies indicate that all the tissues where GPx4 is in so far silenced undergo degeneration. Understanding therefore GPx4 turnover is a crucial issue.
Major aim of this work was therefore clarifying whether GPx4 could be a Nedd4 substrate, and thus proteasomal degradation could have a role in GPx4 turnover.
This hypothesis was suggested by the presence of a conserved LPXY motif observed in silico at the C-terminal end of GPx4, a canonic position typical of many substrates of the WW domains-containing E3 ligases of the Nedd4 family.
In Nedd4.1/Nedd4.2 and GPx4 co-transfected cells, the Nedd4 isoforms are co-immunoprecipitated by GPx4, while mutation at the conserved C-terminal LPXY motif on GPx4, yields a decreased signal of the Nedd4 band, suggesting that the binding between GPx4 and the two Nedd4 isoforms relies indeed on the LPXY motif. Ubiquitylation is observed in co-transfected cells immunoprecipitated as above, but the ubiquitylation pattern is not affected when an inactive mutant of the catalytic Cys of Nedd4.1 or Nedd4.2 is co-transfected with GPx4. Purified rat GPx4 is not ubiquitylated in an in vitro assay containing home made recombinant Nedd4.1, E1, E2, ATP and ubiquitin (Ub). Furthermore, overexpressed Nedd4.1 fails to accelerate endogenous GPx4 clearance following a ferroptotic signal, such as GSH depletion or GPx4 modification by the ferroptosis-inducing compound 1S,3R RSL3. In the GSH depletion model, however, GPx4 protein and activity decreases in the cytosol, while, surprisingly, it appears in the pellet, containing the membrane fraction, in a non-ubiquitylated form. Dissociation constant measurement by SPR analysis on model liposomes interacting with purified GPx4 indicates a decrease in the absence of GSH, while a combined docking and molecular dynamic approach shows that GPx4 binds to the polar head of membrane phospholipid by its basic zone adjacent to the catalytic site. Furthermore, upon oxidation, GSH releases the enzyme in the cytosol by interacting with some residues of the basic zone involved in phospholipid binding. Thus our work, although showing a LPXY motif-dependent binding between both the Nedd4 isoforms and GPx4, fails to validate the hypothesis that GPx4 is ubiquitylated by these ligases, which appear indeed not involved in GPx4 turnover. Yet, the observation that GPx4 translocates to membrane under GSH depletion opens interesting perspectives in the understanding of the mechanism underlying the action of GPx4 on biological membranes.La Glutatione Perossidasi 4 (GPx4) è un omologo dei vertebrati delle glutation perossidasi. La selenocisteina nel sito catalitico è l’amminoacido redox-attivo, implicato nella riduzione degli idroperossidi di membrana a spese del glutatione (GSH). La GPx4 fu per la prima volta purificata nel 1982, come ‘proteina inibente la perossidazione’ e ad oggi sta emergendo, insieme al suo substrato GSH, come un elemento determinante nel regolare l’equilibrio tra proliferazione e morte cellulare. Infatti risultati recenti indicano che l’inattivazione della GPx4 o la deplezione di GSH causa morte cellulare attraverso un nuovo meccanismo programmato basato sulla perossidazione lipidica, chiamato ferroptosi. Studi di genetica inversa indicano che tutti i tessuti dove la GPx4 è silenziata, subiscono degenerazione. La comprensione, perciò, dei meccanismi che sottendono il ricambio (turn-over) della GPx4 appaiono cruciali. Il principale obiettivo del lavoro è stato quelle chiarire se la GPx4 potrebbe essere un substrato di Nedd4, e se la degradazione proteosomale potrebbe avere un ruolo nel ricambio del selenoenzima. Questa ipotesi è stata suggerita dalla presenza di un motivo strettamente conservato LPXY, osservato in silico all’estremità C-terminale della GPx4. Questo motivo infatti è noto per essere riconosciuto dai domini WW contenuto nelle E3-ligasi della famiglia delle Nedd4 ed è presente in molte proteine che sono substrato di Nedd4. Nelle cellule co-trasfettate con Nedd4.1/Nedd4.2 e GPx4, l’isoforma di Nedd4 co-immunoprecipita con la GPx4, mentre la mutazione del motivo C-terminale LPXY della GPx4, provoca un decremento del segnale della banda di Nedd4, suggerendo che il legame tra la GPx4 e le due isoforme di Nedd4 dipenda dal motivo LPXY. Si osserva anche ubiquitinazione nelle cellule co-trasfettate e immunoprecipitate come sopra, ma il ‘pattern’ di ubiquitinazione non è influenzato se un mutante inattivo della cisteina catalitica di Nedd4.1 o Nedd4.2 è co-trasfettato con la GPx4. Inoltre la GPx4 di ratto purificata non è ubiquitinata in un saggio di ubiquitinazione in vitro contenente la Nedd4.1, gli enzimi E1 e E2, ATP e ubiquitina (Ub). Inoltre, la sovra espressione di Nedd4.1 non induce un’accelerazione del ricambio della perossidasi, sia quando le cellule non sono trattate che a seguito di uno stimolo ferroptotico, come la deplezione di GSH o l’alchilazione di GPx4 tramite 1S,3R-RSL3. Nel modello di deplezione del GSH, comunque, la proteina GPx4 e la sua attività subiscono una riduzione nel citoplasma, mentre sorprendentemente, la proteina appare nel ‘pellet’, che contiene la frazione delle membrane in forma non ubiquitinata. La costante di dissociazione dell’interazione di GPx4 e di liposomi contenenti tetraoleilcardiolipina (TOCL), misurato con la tecnica Surface Plasmon Resonance (SPR) diminuisce in assenza di GSH. Inoltre un approccio combinato di docking e dinamica molecolare indica che la GPx4 si lega alle teste polari dei fosfolipidi di membrana attraverso un’ area basica adiacente al sito catalitico. Inoltre, dopo ossidazione della GPx4, il GSH rilascia la perossidasi nel citoplasma mediante interazione con alcuni residui della area basica coinvolta nel legame con i fosfolipidi. In conclusione il nostro lavoro dimostra un legame tra entrambe le isoforme di Nedd4 e la GPx4 che dipende dal motivo LPXY, ma non valida l’ipotesi iniziale che la GPx4 possa essere ubiquitinata da queste ligasi, che quindi non sembrerebbero essere coinvolte nel ricambio della proteina GPx4. Tuttavia, l’osservazione che la GPx4 trasloca nelle membrane in condizioni di deplezione di GSH apre interessanti prospettive nella comprensione dei meccanismi che sottendono l’interazione della GPx4 con le membrane biologiche.
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Slavík, Jan. "Elektrochemické a matematické studium interakcí selenu s biologicky aktivními thioly." Master's thesis, Vysoké učení technické v Brně. Fakulta elektrotechniky a komunikačních technologií, 2012. http://www.nusl.cz/ntk/nusl-219501.
Full textAbstract:
Proteins with thiol groups interact with metal ions in the human body. They maintain their homeostasis, participate in cell signaling, protect the cell against the effects of toxic metals and detoxify them. This work is focused on proteins with thiol groups glutathione and metallothionein and their effects on selenium. The method of study is electrochemical.
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Bangash, Sajid Ali Khan [Verfasser]. "Subcellular glutathione homeostasis and characterisation of glutathione transport across the plasma membrane in Arabidopsis thaliana / Sajid Ali Khan Bangash." Bonn : Universitäts- und Landesbibliothek Bonn, 2017. http://d-nb.info/1174670940/34.
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Lu, Weiya Douglas. "Photophysical consequences from interactions of glutathione S-transferases with the photodynamic sensitizer hypericin /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/8638.
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Ingle, Brandall L. "The Mechanisms of Human Glutathione Synthetase and Related Non-Enyzmatic Catalysis." Thesis, University of North Texas, 2015. https://digital.library.unt.edu/ark:/67531/metadc801927/.
Full textAbstract:
Human glutathione synthetase (hGS) is a homodimeric enzymes that catalyzes the second step in the biological synthesis of glutathione, a critical cellular antioxidant. The enzyme exhibits negative cooperativity towards the γ-glutamylcysteine (γ-GC) substrate. In this type of allosteric regulation, the binding of γ-GC at one active site significantly reduces substrate affinity at a second active site over 40 Å away. The presented work explores protein-protein interactions, substrate binding, and allosteric communication through investigation of three regions of hGS: the dimer interface, the S-loop, and the E-loop. Strong electrostatic interactions across the dimer interface of hGS maintain the appropriate tertiary and quaternary enzymatic structure needed for activity. The S-loop and E-loop of hGS form walls of the active site near γ-GC, with some residues serving to bind and position the negatively cooperative substrate. These strong interactions in the active site serve as a trigger for allosteric communication, which then passes through hydrophobic interactions at the interface. A comprehensive computational and experimental approach relates hGS structure with activity and regulation. ATP-grasp enzymes, including hGS, utilize ATP in the nucleophilic attack of a carboxylic acid in a reaction thought to proceed through the formation of an acylphosphate intermediate. Small metal cations are known to chelate the terminal phosphates of actives site ATP, yet the role of these atoms remains unclear. In the presented work, a computational metal substitution study establishes the role these divalent cations in the catalysis of peptide bonds. The simple model is used to determine the impact of metal cations on the thermodynamics and kinetics, an important stepping stone in understanding the importance of metal cations in larger biological systems.
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Daher, Boutaina. "Induction de la mort ferroptotique dans les cellules de cancer pancréatique : rôle essentiel de la cystéine et du transporteur xCT." Electronic Thesis or Diss., Université Côte d'Azur, 2020. http://www.theses.fr/2020COAZ6031.
Full textAbstract:
Les cellules cancéreuses possèdent un profil de prolifération et un métabolisme très accru nécessitant de sérieuses adaptations. Les tumeurs ont par conséquent, un besoin d’import de nutriments élevé mais aussi des défenses antioxydantes solides afin de combattre la toxicité des espèces réactives de l’oxygène (ROS). Les acides aminés, éléments clé de la structure et fonctionnalité des protéines, sont l’un des trois macronutriments essentiels pour la croissance tumorale. La cystéine est un acide aminé présentant une double fonction au sein de la cellule car en plus de son rôle protéogénique, la cystéine joue un rôle majeur dans les fonctions antioxydantes. C’est en effet le facteur limitant dans la biosynthèse de l’acteur clé du maintient du potentiel redox intracellulaire : le glutathion. Le potentiel redox de ce tripeptide (glutamate, cystéine et glycine) est notamment utilisé par de multiples enzymes dont les peroxydases afin de détoxifier les produits des ROS et parmi une large famille de peroxydases, la glutathion peroxydase 4 (GPx4) assure la réduction des lipides peroxydés. Le maintien d’un niveau de cystéine intracellulaire se fait par un ensemble de transporteurs incomplètement caractérisés dont le principal est le transporteur de la forme oxydée cystine: xCT (échangeur glutamate/cystine 1 :1). Ce transporteur fait parti d’un ‘trio’ minimal de transporteurs surexprimés dans la majorité des cancers (LAT1, ASCT2/1 et xCT). Une perturbation de l’homéostasie intracellulaire de cystéine et de glutathion entraine un stress oxydatif provoquant une accumulation des lipides peroxydés et conduit à une mort non-apoptotique récemment décrite : ‘ferroptose’ rappelant le rôle clé de la biologie du fer dans ce mécanisme. Dans la présente thèse, nous nous sommes initialement intéressé à l’étude de l’impact de l’invalidation génétique (CRISPR-Cas9) du transporteur xCT sur différentes lignées d’adénocarcinome canalaire pancréatique (PDAC). Les cellules xCT-KO obtenues présentent un stress nutritif avec une activation de la voie de stress nutritionnelle GCN2/ATF4 ainsi qu’une inhibition de la synthèse protéique générale par une inhibition de mTORC1. De plus, ces cellules entrent en ferroptose lors de l’absence d’apport en source de cystéine notamment par l’ajout de N-Acétylcystéine (NAC) dans le milieu. En effet, la déplétion en glutathion dans ces cellules induit une accumulation notable de lipides peroxydés altérant ainsi l’intégrité membranaire des cellules. Par conséquent, l’axe xCT/Cystéine/Glutathion/GPx4 joue ainsi un rôle crucial dans la mort par ferroptose. Par la suite, dans la deuxième partie de cette thèse, nous nous sommes intéressés au rôle joué par le glutathion dans la survenu de la ferroptose, indépendamment de la cystéine. Pour cela nous avons invalidé la sous unité catalytique du gène responsable de sa biosynthèse, la γ-Glutamate Cystéine Ligase (GCLC). De manière intéressante, nos résultats démontrent que la mort cellulaire qui survient dans les cellules dépourvues en glutathion, mais ayant gardé un pool intracellulaire en cystéine inchangé, ne présentent pas le même profil de mort cellulaire que les cellules xCT-KO entrant en ferroptose et non pas en apoptose. En conclusion, les études menées durant cette thèse démontrent: 1) que l’axe xCT/Cysteine/Glutathion/GPx4 joue un rôle crucial dans la mort par ferroptose 2) que la suppression de la biosynthèse du glutathion sans impact sur l’homéostasie de la cystéine déclenche une mort cellulaire progressive par apoptose. Ces résultats mettent ainsi en évidence le rôle de la cystéine comme co-substrat pour la GPx4 en l’absence de glutathionWhen compared with normal, non-transformed counterparts, cancer cells exhibit a modified proliferation profile, supported by intensified metabolic activity. Consequently, this leads to greater requirements of the cancer cells for nutrients, but also for antioxidant defence that can counteract increased production of reactive oxygen species (ROS) as a necessary by-product of the metabolism. Cysteine is an amino acid with a dual function within the cell. Indeed, in addition to its proteogenic role like all the other 21 amino acids, cysteine plays an important role in the antioxidant functions of the cell. Namely, cysteine is a rate-limiting amino acid for synthesis of the major redox molecule of the cell: the glutathione (GSH). GSH serves as a reducing equivalent for many antioxidant enzymes, including large family of peroxidases (glutathione peroxidases, GPx), such as GPx4 that ensures the reduction of lipid peroxides in the membrane compartment of the cell. The maintenance of an intracellular cysteine level is done by a set of incompletely characterized transporters, among which the best known is xCT – the transporter of the oxidized form of cysteine (cystine). This transporter is part of a “minimal set” of transporters overexpressed in the majority of cancers, together with LAT1 and ASCT1/2. A disbalance in the intracellular cysteine and glutathione homeostasis leads to disturbed cellular redox status, an accumulation of lipid peroxides, and finall to a recently described non-apoptotic type of cell death named ferroptosis. In this thesis, we initially investigated the impact of a genetic invalidation (CRISPR-Cas9) of the xCT transporter on different pancreatic ductal adenocarcinoma (PDAC) cell lines. The resulting xCT-KO cells exhibit nutrient stress with an activation of the GCN2/ATF4 pathway and an inhibition of general protein synthesis via mTORC1 suppresion. In addition, these cells undergo ferroptosis unless alternative source of cysteine (N-acetylcysteine, NAC) is added. Indeed, the depletion of glutathione in these cells induces a notable accumulation of lipid peroxides, thus compromising the membrane integrity of cells. Therefore, according to the data, the xCT/cysteine/GSH/GPx4 axis seems crucial for ferroptotic cell death in PDAC cells. Subsequently, in the second part of this thesis, we look at the role played by glutathione in the occurrence of ferroptosis, independently of intracellular cysteine. For this we invalidated the catalytic subunit of the gene responsible for its biosynthesis, γ-Glutamate Cysteine Ligase (GCLC). These GCLC-KO cells also undergo cell death; however, interestingly, our results showed that the cells lacking glutathione but not cysteine die in a ferroptosis-independent manner (it could not be prevented by classical ferroptosis inhibitors) and are partially saved by apoptosis inhibitors. In conclusion, the studies carried out during this thesis demonstrated that: 1) the xCT/cysteine/GSH/GPx4 axis plays a crutial role in ferroptosis of PDAC cells, and 2) the suppression of GSH biosynthesis, without affecting the cysteine intracellular pool, results in progressive cell death by apoptosis. These results thus highlight the complexity of cysteine role in the occurrence of cell death by ferroptosis independently of glutathione biosynthesis/availability
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Han, Yi. "Functional analysis of glutathione and autophagy in response to oxidative stress." Thesis, Paris 11, 2012. http://www.theses.fr/2012PA112392/document.
Full textAbstract:
Le H2O2 est reconnu comme un signal dans l’activation des mécanismes de défense en réponse à divers stress, et son accumulation est donc régulée étroitement par le système antioxydant des plantes. Puisque la signalisation par le H2O2 peut être transmise par des processus thiol-dépendants, le statut du glutathion pourrait jouer un rôle important. Le rôle de ce composé en tant que molécule antioxydante est bien établi; cependant, son importance en tant que signal reste à élucider. Afin d’étudier cette question, ce travail a utilisé un mutant, cat2, ayant un défaut dans son métabolisme du H2O2 peroxysomal qui engendre, d’une manière conditionnelle, une oxydation et une accumulation du glutathion. Les modifications du glutathion dans cat2 sont accompagnées par l’activation à la fois de réponses dépendantes de l’acide salicylique (SA) ainsi que l’expression de gènes associés à l’acide jasmonique (JA). L’activation des deux voies phytohormonales par le stress oxydant intracellulaire est largement empêchée en bloquant génétiquement l’accumulation du glutathion dans un double mutant, cat2 cad2, qui porte une mutation additionnelle dans la voie de synthèse du glutathion. Les phénotypes contrastants de cat2 cad2 et cat2 gr1, dans lequel la perte de l’activité GR1 aggrave le stress oxydant, suggèrent que des processus glutathion-dépendants relient le H2O2 et l’activation des réponses de pathogenèse SA-dépendantes par un effet qui est additionnel aux fonctions antioxydantes du glutathion. Des comparaisons directes de cat2 cad2 et cat2 npr1 indiquent que les effets de bloquer l’accumulation du glutathion sur l’induction des voies SA et JA chez cat2 ne sont pas causés par une déficience dans la fonction de la NPR1. L’autophagie a été impliquée dans des processus comme la sénescence, et interagirait à la fois avec le stress oxydant et avec la signalisation par le SA. Afin d’explorer des relations entre autophagie et stress oxydant, des mutants atg ont été sélectionnés et croisés avec le cat2. Des analyses phénotypiques ont révélé que l’étendue de lésions SA-dépendantes observée chez cat2 cultivé en jours longs est similaire chez trois double mutants cat2 atg, alors que l’augmentation de la disponibilité en H2O2 peroxysomal liée à la mutation cat2 retarde la sénescence précoce observée chez les mutants atg. Dans son ensemble, le travail suggère que (1) des nouvelles fonctions glutathion-dépendantes sont importantes pour relier la disponibilité en H2O2 intracellulaire et activation des voies de signalisation SA et JA, et (2) que le H2O2 produit par la photorespiration pourrait jouer un rôle antagoniste dans les phénotypes de sénescence précoce observée chez les mutants atgH2O2 is a recognized signal in activation of defence mechanisms in response to various stresses, and its accumulation is thus tightly controlled by plant antioxidant systems. Because H2O2 signals may be transmitted by thiol-dependent processes, glutathione status could play an important role. While the antioxidant role of this compound is long established, the importance of glutathione in signaling remains unclear. To study this question, this work exploited a stress mimic mutant, cat2, which has a defect in metabolism of peroxisomal H2O2 that conditionally leads to oxidation and accumulation of glutathione. In cat2, changes in glutathione are accompanied by activation of both salicylic acid (SA)-dependent responses and jasmonic acid (JA)-associated genes in a time-dependent manner. This up-regulation of both phytohormone signaling pathway by intracellular oxidative stress can be largely prevented by genetically blocking glutathione accumulation in a double mutant, cat2 cad2, that additionally carries a mutation in the pathway of glutathione synthesis. Contrasting phenotypes between cat2 cad2 and cat2 gr1, in which loss of GR1 activity exacerbates oxidative stress, suggest that glutathione-dependent processes couple H2O2 to activation of SA-dependent pathogenesis responses through an effect that is additional to glutathione antioxidant functions. Direct comparison of cat2 cad2 and cat2 npr1 double mutants suggests that the effects of blocking glutathione accumulation on cat2-triggered up-regulation of both SA and JA pathways are not mediated by defective NPR1 function. Autophagy has been implicated in processes such as senescence, and may interact with oxidative stress and SA signaling. To explore relationships between autophagy and oxidative stress, selected atg mutants were crossed with cat2. Phenotypic analysis revealed that SA-dependent lesion spread observed in cat2 grown in long days is similar in three cat2 atg double mutants, whereas increased peroxisomal H2O2 availability in cat2 delays an oxidative stress related-senescence triggered by atg in short days. Overall, the work suggests that (1) novel glutathione-dependent functions are important to couple intracellular H2O2 availability to the activation of both SA and JA signaling pathways and (2) H2O2 produced through photorespiration may play an antagonistic role in the early senescence phenotype observed in atg mutants
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Roland, Aurélie. "Influence des phénomènes d'oxydation lors de l'élaboration des moûts sur la qualité aromatique des vins de Melon B. et de Sauvignon Blanc en Val de Loire." Thesis, Montpellier, SupAgro, 2010. http://www.theses.fr/2010NSAM0016/document.
Full textAbstract:
Afin de caractériser les moûts de Melon B. et de Sauvignon Blanc en composition et de modéliser leur profil d'oxydation, diverses méthodologies analytiques quantitatives ont été développées et validées. La quantification par dilution isotopique des précurseurs de thiols a nécessité la synthèse de molécules marquées qui ont également servies de traceurs lors d'études de filiation dans des milieux complexes. Ainsi, au cours de la fermentation alcoolique, nous avons pu démontrer que le S-3-(hexan-1-ol)-glutathion (G3MH) et la S-4-(4-méthylpentan-2-one)-glutathion (G4MMP) sont métabolisés par la levure et libèrent le 3-mercaptohexan-1-ol (3MH) et la 4-méthyl-4-mercaptopentan-2-one (4MMP) avec des rendements de conversion molaires proches de 4,4 % et 0,3 % respectivement. La modélisation des phénomènes d'oxydation à l'échelle laboratoire a permis par ailleurs de vérifier, comme le laissaient supposer leurs structures chimiques, que les précurseurs de thiols ne sont pas dégradés par les mécanismes oxydatifs affectant les moûts. Au contraire, une formation de G3MH concomitante avec le pic de production du Grape Reaction Product (GRP) est observée. La validation de ces observations à l'échelle industrielle a été conduite par comparaison des pressurages traditionnel et inerté. L' élaboration d'un moût de Melon B. sous gaz inerte n'est pas favorable à la production de G3MH d'origine pré-fermentaire, et a pour conséquence une diminution des teneurs en 3MH dans les vins correspondants, sans pour autant, déprécier les qualités organoleptiques des vins jeunes. Pour le Sauvignon Blanc, le potentiel aromatique de type thiol n'est pas affecté par le type de pressurage, mais une diminution très significative en 3MH dans les vins issus des cuvées obtenues sous gaz inerte est observée. La voie du (E)-2-hexènal qui est certainement impliquée, pourrait expliquer cette perte aromatique. Ainsi, de manière globale, dans nos conditions expérimentales, une oxydation ménagée et raisonnée des moûts de Melon B., et dans une moindre mesure de Sauvignon Blanc, est favorable à la qualité aromatique des vins du Val de LoireIn order to characterize Melon B. and Sauvignon Blanc musts in composition and to study their oxidation profiles, several analytical methodologies have been developed and validated. The quantification of thiols precursors by Stable Isotope Dilution Assay required the synthesis of labeled molecules, which have been used either as analytical standards or as tracers for relationship studies in complex matrices. Thus, we established that, during the alcoholic fermentation, the S-3-(hexan-1-ol)-glutathione (G3MH) and the S-4-(4-méthylpentan-2-one)-glutathione (G4MMP) are metabolized by the yeast to release the 3-mercaptohexan-1-ol (3MH) and the 4-méthyl-4-mercaptopentan-2-one (4MMP) with molar conversion yields close to 4.4 % and 0.3 % respectively. Oxidation mechanisms study at laboratory scale demonstrated that aromatic potential was not affected by oxidative reactions, as expected in regard to their chemical structures. On the contrary, the G3M H was produced in the same time as the Grape Reaction Product peak (GRP). The validation of these observations at industrial scale was conducted by comparing traditional and inerted pressing systems. The elaboration of a Melon B. must under inert gas was not in favor of a G3MH pre-fermentary production, which induced a decrease of 3MH concentration in wine without affecting the organoleptic qualities of young wines. For Sauvignon Blanc must, the aromatic potential was not affected by the kind of pressing systems but a significant decrease in 3MH was observed in the wines obtained with juices from the beginning of pressing. The E-(2)-hexenal pathway could certainly explain such aromatic losses. Thus, under our experimental conditions, a mild and controlled oxidation of Melon B. must and, in a certain extend of Sauvignon Blanc must, is in favor of the aromatic quality of wines from Loire Valley