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.

No description available.

Part I: In the first study of pyrethroids, twenty-one novel pyrethroid esters bearing strong electron-withdrawing groups (e.g., halomethylketo and nitro groups) in the double bond side chain of the cyclopropane acid moiety have been synthesized and evaluated for insect toxicity. Rather than the usually employed Wittig reaction for these syntheses, the novel pyrethroid acid moieties were prepared by amino acidcatalyzed Knoevenagel condensations under mild conditions. In the second study of pyrethroids, fourteen pyrethroid-like carbonates were synthesized by condensation of a variety of alcohols and the chloroformates of the corresponding known pyrethroid alcohols.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy. / A glutathione S-transferase (Sj26GST) from Schistosoma japonicum, which functions in the parasite's Phase II detoxification pathway, is expressed by the Pharmacia pGEX-2T plasmid and is widely used as a fusion-protein affinity tag. It contains all 217 residues of Sj26GST and an ad titional 9-residue peptide linker with a thrombin cleavage site at its C-terminus. Size-exclusion HPLC (SEC-HPLC) and SDS-PAGE studies indicate that purification of the homodimeric protein under nonreducing conditions results in the reversible for-ration of significant amounts of 160 -kDa and larger aggregates without a loss in catalytic activity. The basis for oxidative aggregation can be ascribed to the high degree of exposure of the four cysteine residues per subunit. The conformational stability of the dimeric protein was studied by urea- and temperature-induced unfolding techniques. Fluorescence-spectroscopy, SEC-HPLC, urea- and temperature-gradient gel electrophoresis, ultraviolet melting, differential scanning micro calorimetry , and enzyme activity were employed to monitor structural and functional changes. The unfolding data indicate the absence of thermodynamically stable intermediates and that the umolding/refolding transition is a two-state process involving folded native dimer and unfolded monomer. The stability of the protein was found to be dependent on its concentration with a ~GO(H20) = 26 ±1.7 kcal/mol. The conformational stability was unchanged in the presence of the leading antischistosomal drug Praziquantel, which bound the protein with a Kd = 9 ±1.8 p,M. The strong relationship observed between the m-v,llue and the size of the protein indicates that the amount of protem. surface exposed to solvent upon unfolding is the major structural de.erminant for the dependence of the protein's free energy of unfolding on urea concentration. 'Ihermograms obtained by differential scanning calorimetry also fitted to a two-state irreversible unfolding transition, both in the presence and absence of Praziquantel, with values of ~Cp = 1779 cal mol-IK-I , ~HcaI = 227 kcal/mol, AHVH ::::::233 kcal/mol (r :::::~:HVHIAlIcal = 1.02) and AS = 354 cal mol''K". The low ~Cp and ~S, when compared with the theoretically determined values, implied that the thermal denaturation of Sj26GST did not result in complete unfolding of the protein, / Andrew Chakane 2018

Lo, Yun Chuen. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 69-70). / Summary --- p.1 / Introduction --- p.2 / Methods --- p.6-15 / Subjects / Sample treatment / Protocol / "Equipment, reagents and assays" / Results --- p.15-58 / Technical aspects / Assay characteristics / Stability / Biological variation / Discussion --- p.58-68 / Acknowledgements --- p.68 / References --- p.69

by Yuen Wai Keung. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 99-112). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / Abstract (Chinese Version) --- p.iv / Abbreviations --- p.vi / Table of Contents --- p.viii / Chapter chapter one --- introduction --- p.1 / Chapter 1.1 --- Glutathione S-transferase --- p.2 / Chapter 1.1.1 --- Introduction --- p.2 / Chapter 1.1.2 --- Classification of mammalian GST --- p.2 / Chapter 1.1.3 --- Classification of insect GST --- p.7 / Chapter 1.1.4 --- Substrate specificity --- p.11 / Chapter 1.2 --- The chironomidae --- p.13 / Chapter 1.2.1 --- Biology and life history of chironomidae --- p.13 / Chapter 1.3 --- Chironomidae larvae --- p.16 / Chapter 1.3.1 --- Bloodworm t --- p.6 / Chapter 1.3.2 --- Sources of chironomidae larvae --- p.17 / Chapter 1.4 --- Aim of research --- p.18 / Chapter chapter two --- materials and methods --- p.20 / Chapter 2.1 --- Screening of GST in different subcellular fractions --- p.21 / Chapter 2.1.1 --- Preparation of mitochondria --- p.21 / Chapter 2.1.2 --- Preparation of microsomes --- p.22 / Chapter 2.1.3 --- Preparation of cytosol --- p.22 / Chapter 2.2 --- Assay for GST activity --- p.23 / Chapter 2.2.1 --- Activity Units --- p.23 / Chapter 2.3 --- Protein assay --- p.23 / Chapter 2.4 --- Preparation of glutathione-affinity column --- p.25 / Chapter 2.5 --- Purification of cytosolic GSTs --- p.26 / Chapter 2.5.1 --- Preparation of cytosol --- p.26 / Chapter 2.5.2 --- Chromatography on Sephadex G25 --- p.26 / Chapter 2.5.3 --- Affinity Chromatography --- p.26 / Chapter 2.5.3.1 --- Specific elution of GSTs --- p.26 / Chapter 2.5.3.2 --- Non-specific elution of GSTs --- p.27 / Chapter 2.5.4 --- Fast Protein Liquid Chromatography with Mono Q --- p.27 / Chapter 2.6 --- Determination of molecular mass --- p.29 / Chapter 2.6.1 --- Subunit molecular mass --- p.29 / Chapter 2.6.2 --- Native molecular mass --- p.31 / Chapter 2.7 --- Isoelectric focusing PAGE --- p.31 / Chapter 2.8 --- Enzyme activities and kinetic studies --- p.34 / Chapter 2.8.1 --- Optimum pH --- p.34 / Chapter 2.8.2 --- Heat inactivation assay --- p.34 / Chapter 2.8.3 --- Km and Vmax --- p.34 / Chapter 2.8.4 --- Substrate specificity --- p.35 / Chapter 2.8.5 --- Glutathione peroxidase activity --- p.38 / Chapter 2.9 --- N-terminal amino acid sequence analysis --- p.39 / Chapter 2.9.1 --- Semidry electroblotting --- p.39 / Chapter 2.9.2 --- Staining of proteins on PVDF membrane --- p.40 / Chapter 2.9.3 --- N-terminal amino acid sequence analysis --- p.40 / Chapter 2.9.4 --- On-membrane deblocking of protein --- p.40 / Chapter 2.9.5 --- BLAST search --- p.41 / Chapter chapter three --- results --- p.42 / Chapter 3.1 --- Screening of GST in different subcellular fractions --- p.43 / Chapter 3.2 --- Purification of cytosolic GSTs by chromatography --- p.45 / Chapter 3.2.1 --- Sephadex G25 column --- p.45 / Chapter 3.2.2 --- GSH affinity column --- p.45 / Chapter 3.2.3 --- Mono-Q column --- p.45 / Chapter 3.3 --- Determination of molecular mass --- p.53 / Chapter 3.3.1 --- Subunit molecular mass --- p.53 / Chapter 3.3.2 --- Native molecular mass --- p.53 / Chapter 3.4 --- Isoelectric point determination --- p.53 / Chapter 3.5 --- Enzymes activities and kinetic studies --- p.57 / Chapter 3.5.1 --- Optimum pH --- p.57 / Chapter 3.5.2 --- Thermostability --- p.57 / Chapter 3.5.3 --- Km and Vmax --- p.57 / Chapter 3.5.4 --- Substrate specificity --- p.76 / Chapter 3.5.5 --- Glutathione peroxidase Activity --- p.76 / Chapter 3.6 --- N-terminal amino acid sequence analysis --- p.83 / Chapter chapter four --- discussion --- p.89 / Chapter 4.1 --- GST in different subcellular fractions --- p.90 / Chapter 4.2 --- Purification of cytosolic GST --- p.91 / Chapter 4.3 --- Physical properties --- p.93 / Chapter 4.3.1 --- Subunit molecular mass --- p.93 / Chapter 4.3.2 --- Native molecular mass --- p.93 / Chapter 4.3.3 --- Isoelectric point --- p.95 / Chapter 4.4 --- Kinetic properties --- p.94 / Chapter 4.4.1 --- Optimum pH --- p.94 / Chapter 4.4.2 --- Thermostability --- p.95 / Chapter 4.4.3 --- Km and Vmax --- p.95 / Chapter 4.4.4 --- Substrate specificity --- p.96 / Chapter 4.4.5 --- Glutathione peroxidase activity --- p.96 / Chapter 4.5 --- N-terminal amino acid sequence data --- p.97 / Chapter 4.6 --- Conclusion --- p.98 / references --- p.99

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.

The circadian clock is an endogenous timekeeper that synchronises biological processes with daily external rhythms such as light and temperature cycles. It provides organisms with a competitive advantage by allowing anticipation of daily events. The circadian clock encompasses a network of transcription-translational feedback loops (TTFLs) that orchestrates rhythmic expression of a large part of the genome. This network is regulated at post-transcriptional and post-translational level. Post-translational regulation of clock proteins is essential to ensure stable rhythms and robust timekeeping. Unlike the genes in the TTFL network, modifiers of clock proteins at post-translational level are conserved across taxa. SUMO, a small ubiquitin-related post-translational modifier, regulates timekeeping in mammals through modification of the clock transcription factor BMAL. In this study, SUMO is shown to contribute to oscillator function in Arabidopsis plants. Methods have been developed to prove that mutant lines defective in SUMO machinery, including SUMO-ligase and -protease mutants, display long circadian rhythms. Additionally, sumoylation on the crucial plant clock transcription factor CCA1 is observed in vivo. A fraction of the protein is sumoylated across the expression window of CCA1, with the phase of peak sumoylation in advance of peak total CCA1. The effect of sumoylation of CCA1 was investigated with respect to localisation, stability and DNA binding affinity of the protein, as these are previously described possible effects of sumoylation. The subcellular location of CCA1-YFP fusions in protoplasts was not altered in mutant lines of the SUMO machinery. In vitro experiments show that sumoylation negatively affects the affinity of CCA1 to its cognate promotor element, suggesting that SUMO could act as a reversible attenuator of CCA1 activity. Furthermore, effects of SUMO machinery mutations appear to be differential across a range of physiologically relevant temperatures, implying that sumoylation could be involved in the response to or buffering against fluctuating ambient temperatures. There is an increasing amount of evidence to suggest that metabolic oscillations are not only driven by transcriptional outputs of the clock, but are to some extent self-sustained and can feed timing information back into the clock. Glutathione was investigated as a possible metabolic feedback signal. Expression of clock gene CCA1 was found to be abolished in a mutant of the rate-limiting enzyme for glutathione synthesis (pad2-1). Surprisingly however, the amount of glutathione was not found to oscillate. Combined, the results discussed in this thesis provide a substantial advance on our understanding of post-translational regulation and the integration of metabolic and environmental information into the plant circadian clock.

Flatworm parasites (platyhelminths) cause serious infection diseases in humans, such as schistosomiasis and hydatid disease, mainly prevalent in developing countries. However, the current repertoire of drug armamentarium used to combat flatworm infections is limited. For instance, praziquantel is the only drug available for mass treatment of Schistosoma infections. In contrast to their hosts, flatworm parasites possess a distinct redox arrangement of redox pathways in which the selenoenzyme thioredoxin glutathione reductase (TGR) controls the overall redox homeostasis. Interference with this enzyme leads to parasite death. Hence, this key redox enzyme seems to be a new promising drug target against flatworm infections. Because most flatworms are difficult to cultivate in the laboratory (e.g. Echinococcus granulosus experimental infection in mice takes about 10 month to develop into cysts), this work was focused on Mesocestoides vogae (syn. corti), a non-human flatworm parasite which is an interesting laboratory model to study other flatworm infections: it is very rare in humans, can be easily manipulated both in vivo and in vitro and grows extremely fast in mice. With the aim to assess TGR inhibitors as possible drugs to treat flatworm infections, the thioredoxin and glutathione pathways of M.vogae were studied. Here, the objectives were to study whether the biochemical pathways that maintain the redox homeostasis in M. vogae conform to the general biochemical scenario proposed for other platyhelminth parasites. Here, it was proven that M. vogae extracts possess both thioredoxin and glutathione reductase activities. The thioredoxin and glutathione reductase activities were partially purified from total extracts by a combination of ammonium sulfate precipitation, anion exchange and hydroxyapatite chromatography. Both activities co-purified in all steps which strongly indicates the existence of TGR rather than a single TR and GR. Furthermore partially purified activities could be inhibited by the organogold compound auranofin, a known TGR inhibitor. Moreover, the glutathione reductase activity displays hysteresis (a peculiar kinetic behavior) at high concentrations of oxidised glutathione, a feature typical of flatworm TGRs, but not of conventional GR. Although M. vogae activities could not be purified to homogeneity, the overall results strongly indicate that this flatworm possesses TGR and lacks conventional GR and TR. Furthermore the thiadiazole WPQ75 and the N-oxide VL16E (a furoxan derivate) were identified as inhibitors of TGR activity of M.vogae at a 10 µM concentration. These inhibitors were able to kill M.vogae larval worms in vitro as well as in experimental infection in mice. Due to the existence of TGR activity in M.vogae, the possibility to inhibit this activity with recently discovered inhibitors of flatworm TGR and the successes achieved by testing these inhibitors both in vitro and in vivo, it is strongly evident that M. vogae would be an excellent model to assess TGR inhibitors in flatworm infections. / Charakterisierung von Thioredoxin- und Glutathionreduktase Aktivitäten von Mesocestoides vogae, einem parasitären Plattwurm der als Labormodell für die Testung von Arzneistoffen verwendet werden kann

Electron transport systems exist in the plasma membranes of all cells. Although not well characterised they play roles in cell growth and proliferation, hormone responses and other cell signalling events, but perhaps their most important role, especially in erythrocytes, is enabling the cell to respond to changes in both intra- and extracellular redox environments. Human erythrocytes possess a transmembrane electron transport capability that mediates the transfer of reducing equivalents from reduced intracellular species to oxidised extracellular species and is concomitant with proton extrusion. In the work for this thesis I showed that erythrocyte membranes contain a transmembrane WST-1 (water soluble tetrazolium-1) reductase activity that uses reducing equivalents from intracellular NADH to reduce extracellular WST-1. The rate of WST-1 reduction was increased by the presence of phenazine methosulfate and, although of low activity, it showed similar properties to a previously reported transmembrane NADH-oxidase activity. 1H NMR experiments showed that WST-1 was reversibly bound to the membrane and/or proteins in the membrane within the timeframe of the NMR experiment, confirming the location of the WST-1 reduction. Preliminary attempts to purify NADH:WST-1 reductase and NADH:ferricyanide reductase activities from the erythrocyte plasma membrane were inconclusive. The protein(s) responsible for the reduction of these oxidants appear to be of low abundance in the plasma membrane and may be part of a larger protein complex. Further work on the isolation of these redox activities is required before the protein(s) involved can be identified with any confidence. The ability of cells to export electrons suggests that an electron import mechanism might also exist to re-establish the cell�s redox-buffering equilibrium under conditions of oxidative stress. This hypothesis was tested in glucose-deprived erythrocytes using reduced glutathione and NADH as extracellular electron donors. It was shown that neither reduced glutathione nor NADH donated reducing equivalents through a transmembrane redox system. Extracellular NADH was, however, able to produce profound changes in starvation metabolism and methaemoglobin reduction rates. The addition of extracellular NADH caused a six-fold increase in the rate of lactate production above that observed in glucose-starved controls, together with a concomitant decrease in pyruvate production. In erythrocytes containing high levels of methaemoglobin, extracellular NADH increased the rate of methaemoglobin reduction in both the presence and absence of glucose. These results were explained by the leakage of lactate dehydrogenase from erythrocytes due to an admittedly low level of haemolysis. This caused the displacement of the intracellular pseudo-equilibrium of the lactate dehydrogenase reaction via transmembrane exchange of lactate, allowing the conversion of extracellular pyruvate to lactate and resulted in an increase in intracellular NADH concentrations. The latter increased the rate of methaemoglobin reduction. In conclusion, the work described in this thesis showed that erythrocyte membranes do not contain mechanisms for importing electrons or reducing equivalents from extracellular reduced glutathione or NADH. Erythrocytes do, however, contain an electron export system which can reduce extracellular oxidants such as WST-1 and the activity of this system depends on an intricate balance between intracellular antioxidants and enzyme activities. There is much still to be learnt about plasma membrane redox systems, little is known, for example, about the protein composition, mechanism of action, and the in vivo conditions under which these systems are most active.