Glutathione Oxidoreductase Research Articles

Influences of Shading on Ascorbic Acid Biosynthesis of Blackcurrant (Ribes nigrum L.)

Cultivation conditions may greatly affect fruit quality, especially in the accumulation of functional metabolites. Blackcurrant fruits (Ribes nigrum L.) have high ascorbic acid (AsA) concentrations. The purpose of the current study was to investigate the influence of different shading treatments (full sunlight, and 40% and 60% sunlight) on the fruits’ maturity, and on the levels of fruit firmness, soluble solid, AsA, and enzyme activity involved in AsA biosynthesis and recycling in two blackcurrant (Ribes nigrum) cultivars, ‘Heifeng’ and ‘Adelinia’. Shading conditions of 40% and 60% sunlight delayed fruit ripening and increased fruit firmness in both ‘Adelinia’ and ‘Heifeng’. Soluble solids in ‘Adelinia’ were markedly reduced by shading compared with ‘Heifeng’. Compared with full sunlight, the AsA content was significantly decreased in the ripe fruits under the 40% and 60% shading treatments. Additionally, the AsA content was decreased during the fruit development process under the 60% shading treatment, which was associated with the reduced activity of the enzymes monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione oxidoreductase (GR), ascorbate peroxidase (APX), and L-galactose dehydrogenase (GalDH) involved in the biosynthesis and recycling pathway of AsA. The correlation analysis results showed that the activity of MDHAR, DHAR, GR, APX, and GalDH was significantly positively correlated with AsA concentrations during the 60% shading treatment in ‘Adelinia’ and ‘Heifeng’ fruits, suggesting that AsA biosynthesis and recycling were affected and the two cultivars have similar mechanisms to deal with shading. Our results not only provide a better understanding of the regulation mechanism of AsA accumulation under shading, but also provide a theoretical basis for taking effective cultivation measures aimed at the improvement of AsA levels in blackcurrant fruits.

Inter-study and time-dependent variability of metabolite abundance in cultured red blood cells

BackgroundCultured human red blood cells (RBCs) provide a powerful ex vivo assay platform to study blood-stage malaria infection and propagation. In recent years, high-resolution metabolomic methods have quantified hundreds of metabolites from parasite-infected RBC cultures under a variety of perturbations. In this context, the corresponding control samples of the uninfected culture systems can also be used to examine the effects of these perturbations on RBC metabolism itself and their dependence on blood donors (inter-study variations).MethodsTime-course datasets from five independent studies were generated and analysed, maintaining uninfected RBCs (uRBC) at 2% haematocrit for 48 h under conditions originally designed for parasite cultures. Using identical experimental protocols, quadruplicate samples were collected at six time points, and global metabolomics were employed on the pellet fraction of the uRBC cultures. In total, ~ 500 metabolites were examined across each dataset to quantify inter-study variability in RBC metabolism, and metabolic network modelling augmented the analyses to characterize the metabolic state and fluxes of the RBCs.ResultsTo minimize inter-study variations unrelated to RBC metabolism, an internal standard metabolite (phosphatidylethanolamine C18:0/20:4) was identified with minimal variation in abundance over time and across all the samples of each dataset to normalize the data. Although the bulk of the normalized data showed a high degree of inter-study consistency, changes and variations in metabolite levels from individual donors were noted. Thus, a total of 24 metabolites were associated with significant variation in the 48-h culture time window, with the largest variations involving metabolites in glycolysis and synthesis of glutathione. Metabolic network analysis was used to identify the production of superoxide radicals in cultured RBCs as countered by the activity of glutathione oxidoreductase and synthesis of reducing equivalents via the pentose phosphate pathway. Peptide degradation occurred at a rate that is comparable with central carbon fluxes, consistent with active degradation of methaemoglobin, processes also commonly associated with storage lesions in RBCs.ConclusionsThe bulk of the data showed high inter-study consistency. The collected data, quantification of an expected abundance variation of RBC metabolites, and characterization of a subset of highly variable metabolites in the RBCs will help in identifying non-specific changes in metabolic abundances that may obscure accurate metabolomic profiling of Plasmodium falciparum and other blood-borne pathogens.

Characterization of genes involved in pear ascorbic acid metabolism and their response to bagging treatment during ‘Yali’ fruit development

In this study, a total of 59 genes involved in ascorbic acid (AsA) metabolism were identified from pear genome with diverse chromosome locations, gene structures, and cis-acting elements. Most were derived from WGD/segmental duplication blocks, and purifying selection was the main driving force for their expansion. Their expression profiles were tissue-specific, and postharvest light treatment would alter their expression profiles. During ‘Yali’ pear development, in association with visual and inner quality change, AsA, dehydroascorbate (DHA) and total AsA (T-AsA) concentrations decreased in association with the downregulated activities of glutathione oxidoreductase (GR), l-galactono-1, 4-lactone dehydrogenase (GalLDH), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and ascorbate oxidase (AO) as well as various expression patterns of 59 genes in their metabolic pathway. Bagging treatment obviously suppressed AsA and T-AsA concentrations in both pericarp and cortex, which accompanied with the downregulated GalLDH, GR, MDHAR, and APX activities and the upregulated AO activity. Furthermore, the expression profiles of several genes in the pericarp and cortex were altered after bagging. In combination of the results from quality, physiological-biochemical and gene expression assay, several genes were proposedly responsible for bagging-induced reduction of AsA and T-AsA concentrations in the pericarp and cortex.

Differential suitability of reactive oxygen species and the role of glutathione in regulating paradoxical behavior in gliomas: A mathematical perspective.

Manipulative strategies of ROS in cancer are often exhibited as changes in the redox and thiol ratio of the cells. Cellular responses to oxidative insults are generated in response to these changes which are triggered due to the rerouting of the metabolic framework to maintain survival under stress. However, mechanisms of these metabolic re-routing are not clearly understood and remained debatable. In the present work, we have designed a context-based dynamic metabolic model to establish that the coordinated functioning of glutathione peroxidase (GTHP), glutathione oxidoreductase (GTHO) and NADPH oxidase (NOX) is crucial in determining cancerous transformation, specifically in gliomas. Further, we propose that the puzzling duality of ROS (represented by changes in h2o2 in the present model) in exhibiting varying cellular fates can be determined by considering simultaneous changes in nadph/nadp+ and gsh/gssg that occur during the reprogramming of metabolic reactions. This will be helpful in determining the pro-apoptotic or anti-apoptotic fate of gliomas and can be useful in designing effective pro-oxidant and/or anti-oxidant therapeutic approaches against gliomas.

Intracellular oxidative damage due to antibiotics on gut bacteria reduced by glutathione oxidoreductase-derived antioxidant molecule GM15.

The human gut consists of > 1000 different bacterial species for the smooth functioning of the gut. In normal conditions, the antioxidant system present in cells minimize the effects of reactive oxygen species. Upon exposure to antibiotics, there is a rise in ROS level which induces oxidative stress to the cells, ultimately killing the cells. Two broad-spectrum antibiotics, streptomycin and gentamicin at a concentration of 50µM and 25µM, were treated with Bacillus subtilis SRMIST201901 (MN726522) and B. cereus SRMIST201902 (MN726923); the treatment reduced the cell counts. Considering the bacterial defense property which relies on the antioxidant mechanism, in this study, we have reported an antioxidant peptide (GM15) derived from glutathione oxidoreductase of spirulina (or called cyanobacteria) Arthrospira platensis (Ap) which reduced the intracellular oxidative stress. Cellular ROS detection was confirmed by fluorescent-associated cell sorting (FACS) using the DCFDA dye. Resazurin dye test also confirmed the activity of peptide on the growth of the Bacillus sp. Based on the results obtained, it was concluded that there was a significant (P < 0.05) reduction in the intracellular oxidative stress on treating with GM15 peptide. Overall, the study indicates the influence of antioxidant peptide on the intracellular oxidative stress, leading to the development of an antioxidant drug from glutathione oxidoreductase of A. platensis against oxidative-related stresses.

Design and characterization of a novel Arthrospira platensis glutathione oxido-reductase-derived antioxidant peptide GM15 and its potent anti-cancer activity via caspase-9 mediated apoptosis in oral cancer cells

Glutathione oxido-reductase (GR) is a primary antioxidant enzyme of most living forms which protects the cells from oxidative metabolism by reducing glutathione (GSH) from its oxidized form (GSSG). Although the antioxidant role of the enzyme is well characterized, the specific role of conserved N' peptide sequence in antioxidant mechanism remains unclear. In this study, we have identified an RNA sequence encoding GR enzyme from spirulina, Arthrospira platensis (Ap) and the changes in its gene expression profile was analysed during H2O2 stress. Results showed that H2O2 (10 mM) stimulated the expression of ApGR throughout the timeline of study (0, 5, 10, 15 and 20 days) with highest expression at 5th day post-exposure which confirmed the antioxidant role of ApGR in spirulina during H2O2 induced oxidative stress. A dithiol containing short antioxidant peptide, 39GGTCVIRGCVPKKLM53 (GM15) from ApGR was predicted and its radicals (superoxide and hydroxyl radical) scavenging potential was confirmed by in vitro cell-free assays. GM15 (12.5 μM) reduced the intracellular generalized oxidative stress level, as measured using DCFDA assay in H2O2 exposed leucocytes without affecting any of the cellular population. Further, the biomedical application of the radical scavenging property of GM15 was validated in oral carcinoma (KB) cells where GM15 exhibited significant cytotoxicity. Also, GM15 exhibited heterogenous effects on intracellular oxidative stress level in KB cells: at lower concentration (6.25 μM), the peptide reduced oxidative stress whereas, at higher concentration (25 μM) it increased the intensity of oxidative stress. GM15 (25 μM) induced caspase-9 mediated apoptosis in KB cells along with membrane disruption and DNA degradation which are confirmed by propidium iodide (PI) internalization and comet assays, respectively. Overall, the study shows that GM15 peptide i) scavenges superoxide, hydroxyl radicals, and influences intracellular oxidative stress, and ii) has anti-cancer effect in oral cancer cells.

An investigation into the possible regulation of the expression of genes by yapA in Talaromyces marneffei using the qRT- PCR method.

The pathogenic dimorphic fungus Talaromyces marneffei is known to cause a fatal systemic mycosis in immunocompromised patients, especially in HIV patients in Southeast Asia. The basic leucine-zipper (bZip) transcription factor gene, yapA, has been identified in T. marneffei. A prior study described that yapA was involved in the oxidative and nitrosative stress response in T. marneffei. Interestingly, an essential role of Saccharomyces cerevisiae Yap1p in the oxidative stress response is the activation of the transcription of its target genes. To identify the target genes of yapA in T. marneffei, the qRT-PCR method were used in this study. Investigation into the expression of genes which are probably regulated by yapA revealed that yapA controlled the expression of cat1 (catalase), cpeA (catalase-peroxidase), sodA (copper, zinc superoxide dismutase), gcs1 (glutamate-cysteine ligase), glr1 (glutathione oxidoreductase), trr1/trr2 (thioredoxin reductase), and trxA (thioredoxin) during stress conditions in all forms of conidium, mycelium, and yeast phase. An exception to this was the expression of cat1 under conditions of oxidative stress in the mould phase with a similar relative expression level in all of the wild-type, mutant and complemented strains. These genes are involved in response against oxidative stress and nitrosative stress in this fungus. The data showed that they could be regulated by the yapA gene during stress conditions. Moreover, the yapA gene is also known to control red pigment production by inhibiting the regulation of the five polyketide synthase (pks) genes, pks3 (polyketide synthase), rp1 (transcription activator), rp2 (β-subunit fatty acid synthase), rp3 (α-subunit fatty acid synthase), and rp4 (oxidoreductase) in the mould phase. In addition, it also regulates transcription in the laccase gene cluster including lac (extracellular dihydrogeodin oxidase/laccase), and multicopper oxidase encoding genes (PMAA_050860, PMAA_072680, PMAA_085520, PMAA_082010, and PMAA_082060) in all stages of the T. marneffei lifecycle (conidia, mould, and yeast phase). This study suggests the importance of the role of the yapA gene in the stress response and virulence of T. marneffei.

Selenite Protection of Tellurite Toxicity Toward Escherichia coli.

In this work the influence of selenite on metal resistance in Escherichia coli was examined. Both synergistic and antagonistic resistance and toxicities were found upon co exposure with selenite. In wild type cells co-exposure to selenite had little effect on arsenic resistance, decreased resistance to cadmium and mercury but led to a dramatically increased resistance to tellurite of 32-fold. Due to the potential importance of thiol chemistry in metal biochemistry, deletion strains in γ-glutamylcysteine synthetase (key step in glutathione biosynthesis, encoded by gshA), thioredoxin (trxA), glutaredoxin (grxA), glutathione oxidoreductase (gor), and the periplasmic glutathione transporter (cydD) were also evaluated for resistance to various metals in the presence of selenite. The protective effect of selenite on tellurite toxicity was seen in several of the mutants and was pronounced in the gshA mutant were resistance to tellurite was increased up to 1000-fold relative to growth in the absence of selenite. Thiol oxidation studies revealed a faster rate of loss of reduced thiol content in the cell with selenite than with tellurite, indicating differential thiol reactivity. Selenite addition resulted in reactive oxygen species (ROS) production equivalent to levels associated with H2O2 addition. Tellurite addition resulted in considerably lower ROS generation while vanadate and chromate treatment did not increase ROS production above that of background. This work shows increased resistance toward most oxyanions in mutants of thiol redox suggesting that metalloid reaction with thiol components such as glutathione actually enhances toxicity of some metalloids.

Mutation of the NADH Oxidase Gene ( nox ) Reveals an Overlap of the Oxygen- and Acid-Mediated Stress Responses in Streptococcus mutans

NADH oxidase (Nox) is a flavin-containing enzyme used by Streptococcus mutans to reduce dissolved oxygen encountered during growth in the oral cavity. In this study, we characterized the role of the NADH oxidase in the oxidative and acid stress responses of S. mutans. A nox-defective mutant strain of S. mutans and its parental strain, the genomic type strain UA159, were exposed to various oxygen concentrations at pH values of 5 and 7 to better understand the adaptive mechanisms used by the organism to withstand environmental pressures. With the loss of nox, the activities of oxygen stress response enzymes such as superoxide dismutase and glutathione oxidoreductase were elevated compared to those in controls, resulting in a greater adaptation to oxygen stress. In contrast, the loss of nox led to a decreased ability to grow in a low-pH environment despite an increased resistance to severe acid challenge. Analysis of the membrane fatty acid composition revealed that for both the nox mutant and UA159 parent strain, growth in an oxygen-rich environment resulted in high proportions of unsaturated membrane fatty acids, independent of external pH. The data indicate that S. mutans membrane fatty acid composition is responsive to oxidative stress, as well as changes in environmental pH, as previously reported (E. M. Fozo and R. G. Quivey, Jr., Appl. Environ. Microbiol. 70:929-936, 2004). The heightened ability of the nox strain to survive acidic and oxidative environmental stress suggests a multifaceted response system that is partially dependent on oxygen metabolites.

Sex Determination in the First-Described Sexual Fungus

The original report of sex in fungi dates 2 centuries ago to the species Syzygites megalocarpus (Mucoromycotina). The organism was subsequently used in 1904 to represent self-fertile homothallic species when the concepts of heterothallism and homothallism were developed for the fungal kingdom. In this study, two putative sex/MAT loci were identified in individual strains of S. megalocarpus, accounting for its homothallic behavior. The strains encode both of the high-mobility-group domain-containing proteins, SexM and SexP, flanked by RNA helicase and glutathione oxidoreductase genes that are found adjacent to the mating-type loci in other Mucoromycotina species. The presence of pseudogenes and the arrangement of genes suggest that the origin of homothallism in this species is from a heterothallic relative, obtained via a chromosomal rearrangement to switch two alleles into two separated loci within a single genetic background. Similar events have given rise to homothallic species from heterothallic species in ascomycete fungi, demonstrating that conserved forces shape the evolution of sex determination and speciation in highly diverged fungi.

In vitro effects of rosmarinic acid on glutathione reductase and glucose 6-phosphate dehydrogenase

Context: Glutathione reductase (GR, NADPH:oxidized glutathione oxidoreductase, E.C 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). GR is a crucial enzyme in the antioxidant system by maintaining reduced glutathione (GSH). Glucose 6-phosphate dehydrogenase (G6PD, glucose 6-phosphate (G6P):NADP+ oxidoreductase, EC 1.1.1.49) is the key regulatory enzyme of the pentose phosphate pathway and maintains NADPH for reductive reactions.Objective: Rosmarinic acid (RA; α-O-caffeoyl-3,4-dihydroxyphenyl lactic acid) is an ester of caffeic acid (CA) and 3,4-dihydroxyphenyllactic acid. It has a number of interesting biological activities. The inhibiting activities of the RA on GR and G6PD are investigated here for the first time.Materials and methods: GR and G6PD were purified from tissues, then the effects of RA are investigated.Results: This study reports that RA, which was isolated from Echium vulgare L. (Boraginaceae), inhibits purified GR and G6PD in a concentration-dependent manner. Kinetic characterizations and inhibition constants are investigated.Discussion and conclusion: Because of their importance in the antioxidative defense system, investigation of the inhibitors of these enzymes is important for drug development.

Heterologous expression, purification and characterisation of the extracellular domain of trypanosome invariant surface glycoprotein ISG75

The invariant surface glycoprotein ISG75 is a transmembrane glycoprotein occurring on the surface of the bloodstream-form Trypanozoon. This study describes the expression and purification of the N-terminal extracellular domain of ISG75, a novel target for development of diagnostic tests for trypanosomosis. To facilitate disulfide formation in the cytoplasm, a 1287-bp cDNA fragment encoding ISG75 from Trypanosoma brucei gambiense was expressed in a thioredoxin reductase, glutathione oxidoreductase double mutant Escherichia coli strain. An accessory plasmid pRIL, providing the argI, ileY, and leuW tRNAs, was necessary for efficient heterologous translation of the ISG75 mRNA. The recombinant double-tagged (streptavidine and histidine) ISG75 was purified by two-step affinity chromatography. Addition of l-glutamic acid and l-arginine in the buffer solutions was crucial to stabilise the protein during purification. The purified soluble protein was characterised by circular dichroism spectroscopy, reverse-phase high pressure liquid chromatography and mass spectrometry. It has an alpha-helical folded conformation, is homogeneous and pure (99%). Furthermore, sera of Trypanosoma brucei-infected animals specifically recognise this recombinant ISG75; and rabbit antiserum raised against the recombinant ISG75 detects all species of the Trypanozoon subgenus in parasite preparations.

Purification and kinetic properties of glutathione reductase from bovine liver

Glutathione reductase (GR, NADPH: oxidized glutathione oxidoreductase, EC 1.6.4.2) catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) using NADPH as reducing cofactor. The aim of the present work was to purify and characterize GR from bovine liver. GR was purified using 2', 5' ADP-Sepharose 4B and DEAE-Sepharose Fast Flow columns. The enzyme has been purified 5456-fold and with a yield of 38.4%. The molecular and catalytic properties of bovine liver GR have been studied. Optimum temperature and pH was found to be 50 degrees C and 7, respectively. The activation energy of the reaction catalyzed by the enzyme was 9.065 kcal/mole. The molecular weight of the enzyme was found to be 55 kDa by SDS-PAGE. Kinetic characterization of bovine liver GR was also investigated, Km(NADPH) 0.063 +/- 0.008 mM and Km(GSSG) 0.154 +/- 0.015 mM were determined. It is accepted that parallel lines observed in these double reciprocal plots obeys Ping Pong mechanism and we have showed this in our steady state study. According to our results of statistical analysis, the Ping Pong mechanism is a suitable model since the loss function is less than the other mechanisms. However, competitive inhibition by a product could be accepted in sequential mechanisms but not in a Ping Pong mechanism. In this study, kinetic data are consistent with a branching reaction mechanism previously proposed for GR from other sources by other studies.

Characterization of the OxyR regulon of Neisseria gonorrhoeae

OxyR regulates the expression of the majority of H(2)O(2) responses in Gram-negative organisms. In a previous study we reported the OxyR-dependent derepression of catalase expression in the human pathogen Neisseria gonorrhoeae. In the present study we used microarray expression profiling of N. gonorrhoeae wild-type strain 1291 and an oxyR mutant strain to define the OxyR regulon. In addition to katA (encoding catalase), only one other locus displayed a greater than two-fold difference in expression in the wild type : oxyR comparison. This locus encodes an operon of two genes, a putative peroxiredoxin/glutaredoxin (Prx) and a putative glutathione oxidoreductase (Gor). Mutant strains were constructed in which each of these genes was inactivated. A previous biochemical study in Neisseria meningitidis had confirmed function of the glutaredoxin/peroxiredoxin. Assay of the wild-type 1291 cell free extract confirmed Gor activity, which was lost in the gor mutant strain. Phenotypic analysis of the prx mutant strain in H(2)O(2) killing assays revealed increased resistance, presumably due to upregulation of alternative defence mechanisms. The oxyR, prx and gor mutant strains were deficient in biofilm formation, and the oxyR and prx strains had decreased survival in cervical epithelial cells, indicating a key role for the OxyR regulon in these processes.

Thioredoxin Fusions Increase Folding of Single Chain Fv Antibodies in the Cytoplasm of Escherichia coli: Evidence that Chaperone Activity is the Prime Effect of Thioredoxin

In this study we investigate the effect of thioredoxin (Trx1) protein fusions in the production, oxidation, and folding of single chain Fv (scFv) antibodies in the cytoplasm of Escherichia coli. We analyze the expression levels, solubility, disulfide-bond formation, and antigen-binding properties of Trx1-scFv fusions in E. coli wild-type cells and isogenic strains carrying mutations in the glutathione oxidoreductase (gor) and/or thioredoxin reductase (trxB) genes. We compare the Trx1-scFv fusions with other reported systems for production of scFv in the cytoplasm of E. coli, including protein fusions to the maltose-binding protein. In addition, we analyze the effect of co-expressing a signal-sequence-less derivative of the periplasmic chaperone and disulfide-bond isomerase DsbC (DeltassDsbC), which has been shown to act as a chaperone for scFvs in the cytoplasm. The results reported here demonstrate that Trx1 fusions produce the highest expression level and induce the correct folding of scFvs even in the absence of DeltassDsbC in the cytoplasm of E. coli trxB gor cells. The disulfide bridges of Trx1-scFv fusions were formed correctly in E. coli trxB gor cells, but not in trxB single mutants. Antigen-binding assays showed that Trx1 has only a minor influence in the affinity of the scFv, indicating that Trx1-scFv fusions can be used without removal of the Trx1 moiety. In addition, we proved that a Trx1"AGPA" variant, having its catalytic cysteine residues mutated to alanine, was fully capable of assisting the folding of the fused scFvs. Taken together, our data indicate that the Trx1 moiety acts largely as an intramolecular protein chaperone, not as a disulfide bond catalyst, inducing the correct folding of scFvs in the cytoplasm of E. coli trxB gor cells.

Effects of Cystine and Hydrogen Peroxide on Glutathione Status and Expression of Antioxidant Genes in Escherichia coli

Cysteine or cystine was earlier shown to multiply enhance the toxic effect of hydrogen peroxide on Escherichia coli cells. In the present work, the treatment of E. coli with H2O2 in the presence of cystine increased fivefold the level of extracellular oxidized glutathione (GSSG(out)) and decreased fivefold the GSH/GSSG(out) ratio (from 16.8 to 3.6). The same treatment of cells with deficiency in glutathione oxidoreductase (GOR) resulted in even more severe oxidation of GSH(out), so that the level of oxidized glutathione exceeded that of reduced glutathione and the GSH/GSSG(out) ratio decreased to 0.4. Addition of cystine to the GOR deficient cells resulted in significant oxidation of extracellular glutathione even in the absence of oxidant and in tenfold increase in intracellular oxidized glutathione along with a decrease in the GSH/GSSG(out) ratio from 282 to 26. However, in the cytoplasm of wild type cells, the level of oxidized glutathione (GSSG(in)) was changed insignificantly and the GSH/GSSG(in) ratio increased by 26% (from 330 to 415). Data on glutathione status and cystine reduction in the E. coli gsh and gor mutants suggested that exogenous cystine at first should be reduced with extracellular GSH outside the cells and then imported into them. The high toxicity of H2O2 in the presence of cystine resulted in disorders of membrane functions and inhibition of the expression of genes including those responsible for neutralization of oxidants and DNA repair.

Production of Functional Single-Chain Fv Antibodies in the Cytoplasm of Escherichia coli

Production of intracellular antibodies in Escherichia coli has been thought unlikely owing to an inability to form stable disulfide bonds in the cytoplasm, a necessary step in the folding of most immunoglobulin (Ig) domains. This work investigates whether E. coli strains carrying mutations in the major intracellular disulfide bond-reduction systems (i.e. the thioredoxin and the glutathione/glutaredoxin pathways) allow the oxidation and folding of single chain variable fragment (scFv) antibodies in the cytoplasm. The effect of the co-expression of disulfide bond chaperones in these cells was also examined. An scFv that recognizes the alternative sigma factor σ 54 was used as a model to investigate disulfide bond formation and the folding of Ig domains in E. coli . The results demonstrate that functional intrabodies, with oxidized disulfide bonds in their Ig domains, are produced efficiently in E. coli cells carrying mutations in the glutathione oxidoreductase ( gor) and the thioredoxin reductase ( trxB) genes and co-expressing a signal-sequence-less derivative of the disulfide-bond isomerase DsbC (ΔssDsbC). We obtained evidence indicating that ΔssDsbC acts as a chaperone promoting the correct folding and oxidation of scFvs.

Role of glutathione on acrolein-induced cytotoxicity and mutagenicity in Escherichia coli

The reduced form of glutathione (GSH) is a well-known antioxidant, while there have been few reports indicating the contribution of glutathione to protection of Escherichia coli cells from the lethal effect of oxidative damage. Here, we report that depletion of glutathione causes hypersensitivity of E. coli to acrolein, the structurally simplest α, β-unsaturated aldehyde derived from lipid peroxide-degradation, and that GSH can chemically react with acrolein in vitro thus reducing its toxicity. We further demonstrated that acrolein inactivates glutathione oxidoreductase followed by depletion of glutathione, probably as a part of the toxic effect of acrolein. These results suggested that GSH contributes to cellular defense against the toxic effects.

Role of rpoS in the regulation of glutathione oxidoreductase ( gor) in Escherichia coli

RpoS (sigma-38) is the major regulator of genes for survival of Escherichia coli in the stationary phase. OxyR is a transcriptional regulator that responds to H 2O 2 induced stress in exponential phase. Once considered to act independently of each other, they are now known to be integrally involved in the expression of several oxidative stress genes. While it is known that in the exponential phase, OxyR is the transcriptional regulator of gor, this study has shown that RpoS regulates gor in the stationary phase. β-Galactosidase activity of a gor::lacZ promoter fusion showed no induction in a oxyR rpoS double mutant. Challenge of a gor mutant to several oxidants showed that the gene product was not functioning as a classic antioxidant.

Occurrence of two isoforms of glutathione reductase in the green alga Chlamydomonas reinhardtii

Two isoforms (isoenzymes) of glutathione reductase (NADPH: oxidized glutathione oxidoreductase, EC 1.6.4.2; GR) were clearly resolved when enzyme preparations partially purified from the unicellular alga Chlamydomonas reinhardtii were subjected to column chromatofocusing in the pH range from 8 to 4. One isoform (GR I) exhibited an almost electroneutral isoelectric point (pI, 6.9–7.1) and the other (GR II) was a very acidic protein (pI, 4.7–4.9). Both GRs are, however, homodimeric flavoproteins with similar molecular masses of approx. 127 kDa. Cross-reaction with an antibody against the cyanobacterial GR allowed determination of their subunit molecular masses by Western blotting after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a value of 66 kDa being estimated in both cases. The two algal GR isoforms showed similar K m values for the oxidized form of glutathione (approx. 50 μM). However, the K m values for NADPH were different, being 7 μM and 28 μM for GR I and GR II, respectively. The two isoforms also differed in their optimum pH. Thus, whereas GR I showed a clear maximum at neutral pH, GR II exhibited a broader optimum around pH 8.5 and was more active in the alkaline range. The relative contribution of the two isoforms to the total activity in enzyme preparations of cells disrupted by two different methods indicates that GR I should be a cytoplasmic isoform and GR II a plastidic isoform. The physiological roles of the GR isoenzymes found in Chlamydomonas are discussed and some of their properties compared with those of GRs isolated from other photosynthetic organisms.