Modulation of glyceraldehyde-3-phosphate dehydrogenase inAnacystis nidulans by glutathione

Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13; GAPDH) from the cyanobacteriumAnacystis nidulans was activated up to five-fold by reduced glutathione (GSH) in the physiological concentration range (0.1–2 mM GSH). Non-physiological reductants, like dithiothreitol (DTT) and -mercaptoethanol, also activated the enzyme. Oxidized glutathione (GSSG) had no effect on the cyanobacterial GAPDH but treatment with H₂O₂ led to a rapid, reversible deactivation of both untreated and GSH-treated enzyme preparations. GSH reversed the inhibition induced by H₂O₂. An oligomeric form of the enzyme (apparentM _r440,000) was dissociated by GSH into a lower-M _r, more active enzyme form (M _r200,000). The enzyme was shown to obey regular Michaelis-Menten kinetics. The activation of GAPDH by GSH was associated with a decrease inK _m and an increase inV _max values of the enzyme for 3-phosphoglycerate. GSH had virtually no effect on a GAPDH preparation isolated from corn chloroplasts and studied for comparison.Abbreviations GAPDH glyceraldehyde-3-phosphate dehydrogenase - GSH reduced glutathione - GSSG oxidized glutathione - DTT dithiothreitol     

Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography

Glutathione reductase (EC 1.6.4.2) was purified from spinach (Spinacia oleracea L.) leaves by affinity chromatography on ADP-Sepharose. The purified enzyme has a specific activity of 246 enzyme units/mg protein and is homogeneous by the criterion of polyacrylamide gel electrophoresis on native and SDS-gels. The enzyme has a molecular weight of 145,000 and consists of two subunits of similar size. The pH optimum of spinach glutathione reductase is 8.5–9.0, which is related to the function it performs in the chloroplast stroma. It is specific for oxidised glutathione (GSSG) but shows a low activity with NADH as electron donor. The pH optimum for NADH-dependent GSSG reduction is lower than that for NADPH-dependent reduction. The enzyme has a low affinity for reduced glutathione (GSH) and for NADP⁺, but GSH-dependent NADP⁺ reduction is stimulated by addition of dithiothreitol. Spinach glutathione reductase is inhibited on incubation with reagents that react with thiol groups, or with heavymetal ions such as Zn²⁺. GSSG protects the enzyme against inhibition but NADPH does not. Pre-incubation of the enzyme with NADPH decreases its activity, so kinetic studies were performed in which the reaction was initiated by adding NADPH or enzyme. The Km for GSSG was approximately 200 M and that for NADPH was about 3 M. NADP⁺ inhibited the enzyme, assayed in the direction of GSSG reduction, competitively with respect to NADPH and non-competitively with respect to GSSG. In contrast, GSH inhibited non-competitively with respect to both NADPH and GSSG. Illuminated chloroplasts, or chloroplasts kept in the dark, contain equal activities of glutathione reductase. The kinetic properties of the enzyme (listed above) suggest that GSH/GSSG ratios in chloroplasts will be very high under both light and dark conditions. This prediction was confirmed experimentally. GSH or GSSG play no part in the light-induced activation of chloroplast fructose diphosphatase or NADP⁺-glyceraldehyde-3-phosphate dehydrogenase. We suggest that GSH helps to stabilise chloroplast enzymes and may also play a role in removing H₂O₂. Glucose-6-phosphate dehydrogenase activity may be required in chloroplasts in the dark in order to provide NADPH for glutathione reductase.Abbreviations GSH reduced form of the tripeptide glutathione - GSSG oxidised form of glutathione     

Changes in the glutathione level induced by transplasma membrane electron transport in maize (Zea mays L.)

Summary We investigated changes of thiols (GSH, GSSG, and cysteine) induced by transplasma membrane electron transport after addition of artificial electron acceptors and the influence of the thiol level on redox activity. GSH, GSSG, and cysteine content of maize (Zea mays L. cv. Golden Bantam) roots and coleoptile segments was determined by high performance liquid chromatography with a fluorescence detector. GSSG increased after treatment with 0.8 mM diamide, an SH-group oxidizer. GSH level of roots increased after treatment with diamide, while GSH levels of coleoptiles decreased. Incubation of roots with the GSH biosynthesis inhibitor buthionine-D,L-sulfoximine for 6 days lowered the glutathione level up to 80%. However, the GSH/GSSG ratio of maize roots remained constant after treatment with both effectors. The GSH/GSSG ratio and the glutathione level were changed by addition of artificial electron acceptors like hexacyanoferrate (III) or hexabromoiridate (IV), which do not permeate the plasma membrane. Hexacyanoferrate (III) reduction was inhibited up to 25% after the cellular glutathione level was lowered by treatment with diamide or buthionine-D,L-sulfoximine. Proton secretion induced by reduction of the electron acceptors was not affected by both modulators. The change in glutathione level is different for roots and coleoptiles. Our data are discussed with regard to the role of GSH in electron donation for a plasma membrane bound electron transport system.Abbreviations Buthionine-D,L-sulfoximine s-n-butyl-homocysteine sulfoximine - cys cysteine - diamide 1,1-azobis (N,N-dimethyl-formamide) - DTE dithioerythritol - EDTA ethylenediaminetetraacetic acid - GSH reduced glutathione - GSSG oxidizied glutathione, glutathione disulfide - HBI IV hexabromoiridate (IV) (K₂[IrBr₆]) - HCF III hexacyanoferrate (III) (K₃[Fe(CN)₆] - NEM N-ethylmaleimide - PM plasma membrane - Tris Tris(hydroxymethyl)aminomethane     

Interaction of sulfate and glutathione transport in cultured tobacco cells

Photoheterotrophic and heterotrophic suspension cultures of tobacco (Nicotiana tabacum L.) were grown with 1 mM glutathione (reduced; GSH) as sole source of sulfur. Addition of sulfate to both cultures did not alter the rate of exponential growth, but affected the removal of GSH and sulfate in different ways. In photoheterotrophic suspensions, addition of sulfate caused a decline in the net uptake of GSH, whereas sulfate was taken up by the green cells immediately. In heterotrophic suspensions, however, addition of sulfate did not affect the net uptake of GSH and sulfate was only taken up by the cells after the GSH supply in the medium had been exhausted. Apparently, GSH uptake in photoheterotrophic cells is inhibited by sulfate, whereas sulfate uptake is inhibited by GSH in heterotrophic cells. The differences in the effect of GSH on sulfate uptake in photoheterotrophic and heterotrophic tobacco suspensions cannot be attributed to differences in the kinetic properties of sulfate carriers. In short-time transport experiments, both cultures took up sulfate almost entirely by an active-transport system as shown by experiments with metabolic inhibitors; sulfate transport of both cultures obeyed monophasic Michaelis-Menten kinetics with similar app. K_m (photoheterotrophic cells: 16.0±2.0 M; heterotrophic cells: 11.8±1.8 M) and V_max (photoheterotrophic cells: 323±50 nmol·min^-1·g^-1 dry weight; heterotrophic cells: 233±3 nmol·min^-1·g^-1 dry weight). Temperature- and pH-dependence of sulfate transport showed almost identical patterns. However, the cultures exhibited remarkable differences in the inhibition of sulfur influx by GSH in short-time transport experiments. Whereas 1 mM GSH inhibited sulfate transport into heterotrophic tobacco cells completely, sulfate transport into photoheterotrophic cells proceeded at more than two-thirds of its maximum velocity at this GSH concentration. The mode of action of GSH on sulfate transport in chloroplast-free tobacco cell does not appear to be direct: a 14-h exposure to 1 mM GSH was found to be necessary to completely block sulfate transport; a 4-h time of exposure did not affect this process. Consequently, glutathione does not seem to be a product of sulfur metabolism acting on sulfate-carrier entities by negative feedback control. When transferred to the whole plant, the observed differences in sulfate and glutathione influx into green and chloroplast-free cells may be interpreted as a regulatory device to prevent the uptake of excess sulfate by plants.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - DNP dinitrophenol - DW dry weight - FW fresh weight - GSH reduced glutathione     

Comparison of glutathione S-transferases of Zea mays responsible for herbicide detoxification in plants and suspension-cultured cells

The metabolism of the s-triazine herbicide atrazine has been compared in Zea mays seedlings and cell suspension cultures. The rapid detoxification observed in the shoots of whole plants was not seen in the cultured cells. This difference in metabolism could be accounted for by the varying substrate specificities of the isoenzymes of glutathione S-transferase (EC 2.5.1.18) present in the plant and the cells. A single form of the enzyme isolated from leaf tissue conjugated both atrazine and the chloracetanilide herbicide metolachlor. However, the two isoenzymes present in suspension-cultured cells although active against metolachlor, showed no activity toward atrazine. Following purification, the major form of transferase present in the cells was physically similar to the enzyme isolated from leaf (M_r=55000). Both proteins were dimers of subunit M_r=26300, and with isoelectric points in the range pH 4.3-4.9. The minor form of the enzyme present in culture showed a greater specificity for metolachlor than the major species. In addition the overall activity and ratio of the two isoenzymes varied over the culture growth cycle. These findings illustrate the need for characterizing enzymes involved in herbicide detoxification in plant cell cultures.Abbreviations CDNB 1-chloro-2,4-dinitrobenzene - DEAE diethylaminoethyl - GSH glutathione (reduced) - GST glutathione S-transferase - HPLC high-pressure liquid chromatography - M_r molecular weight - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Synthesis and role of glutathione in protection against oxidative stress in yeast

Summary

Glutathione (GSH) is an abundant and ubiquitous low-molecular-mass thiol with proposed roles in many cellular processes including amino acid transport, synthesis of proteins and nucleic acids, modulation of enzyme activity and metabolism of xenobiotics, carcinogens and reactive oxygen species. This review describes recent findings in the lower eukaryote Saccharomyces cerevisiae that are leading to a better understanding of the role of this peptide in eukaryotic cell metabolism. In particular, two gene products involved in maintaining the levels of reduced GSH have been studied; namely, GSH1 encoding γ-glutamylcysteine synthetase, the first step in the biosynthesis of GSH, and glutathione reductase, which recycles glutathione to its reduced form. These studies indicate that GSH is an essential metabolite in yeast, and that it is required for protection against oxidative stress produced by mitochondrial metabolism and exogenous reactive oxygen species. These findings are discussed in the light of analogous observations made in higher eukaryotes.     

Influence of sample preparation on cellular glutathione recovery from adherent cells in culture

During the last decade, the unbound glutathione content of cultured adherent cells has become a very important biological marker for many pharmacological and toxicologicalin vitro studies with regard to the protective role of the tripeptide in its reduced form (GSH). However, the literature does not provide extensive information on the influence of sample preparation on cellular GSH and thiol analyses. Using the fibroblast-like V79 cell line as model, we undertook a comparative study of the efficiency of different procedures reported in the literature with respect to GSH recovery. Depending on the preanalytical step, up to 10-fold discrepancies could be observed in the recovery of intracellular GSH. Different parameters that must be controlled in order to maximize GSH recovery are discussed. The optimal strategy consisted in rapid perchloric acid deproteinization performed directly in the dish, which was extremely valuable for preparing GSH samples from adherent cells, and especially from cells expressing elevated -glutamyl transferase activity.Abbreviations EDTA ethylenediaminetetraacetic acid - GGT -glytamyl transferase (EC 2.3.2.2) - GSH reduced glutathione - HPLC high-performance liquid chromatography - PA perchloric acid - PBS Dulbecco's phosphate-buffered saline     

Induction of pumpkin glutathione S-transferases by different stresses and its possible mechanisms

Induction of pumpkin (Cucurbita maxima Duch.) glutathione S-transferases (GSTs) by different stresses and endogenous trans-2-hexenal content were determined in search of a common signal for GST induction. All of the stresses showed significant induction, As₂O₃ causing the highest induction followed by trans-2-hexenal. The trans-2-hexenal content was highest in trans-2-hexenal-treated seedlings and next-highest in methyl jasmonate-treated seedlings, whereas high temperature- and As₂O₃-treated seedlings had trans-2-hexenal contents lower than that of control seedlings. Induction of GST, lipoxygenase (LOX) and hydroperoxide lyase (HPL) was compared, since trans-2-hexenal and methyl jasmonate are the products of the LOX pathway. All four stresses showed weak LOX induction, high temperature causing the highest induction. However, only methyl jasmonate caused weak HPL induction. Both antioxidants or oxidants induced GST to different degrees. Glutathione contents of reduced glutathione (GSH) or oxidized glutathione (GSSG)-treated seedlings were significantly higher than the content of control seedlings, whereas those treated with other antioxidants or oxidants had contents similar to or less than control seedlings. The GSH:GSSG ratio was lowest in GSSG-treated seedlings and next-lowest in GSH-treated seedlings. The results of this study suggest that pumpkin GSTs are not induced through a common signalling pathway and that redox perturbation plays a role in pumpkin GST induction.     

Use of thiopropyl Sepharose for the synthesis of an adsorbent for the affinity chromatography of glutathione S-transferase

Thiopropyl Sepharose 6B in the 2-thiopyridyl-activated form was used for the reversible immobilisation of reduced glutathione (GSH). The resulting affinity matrix was successfully tested as a sorbent for the partial purification of glutathione S-transferase (GST) from pig kidney. The specific elution of the enzyme was performed with 10 mM GSH in Tris-HCl buffer (pH 7.8), non-specific elution with 20 mM dithiotreitol (DTT) in the same buffer.     

Oxidized glutathione fermentation using Saccharomyces cerevisiae engineered for glutathione metabolism

Glutathione is a valuable tripeptide that is widely used in the pharmaceutical, food, and cosmetic industries. Intracellular glutathione exists in two forms, reduced glutathione (GSH) and oxidized glutathione (GSSG). Most of the glutathione produced by fermentation using yeast is in the GSH form because intracellular GSH concentration is higher than GSSG concentration. However, the stability of GSSG is higher than GSH, which makes GSSG more advantageous for industrial production and storage after extraction. In this study, an oxidized glutathione fermentation method using Saccharomyces cerevisiae was developed by following three metabolic engineering steps. First, over-expression of the glutathione peroxidase 3 (GPX3) gene increased the GSSG content better than over-expression of other identified peroxidase (GPX1 or GPX2) genes. Second, the increase in GSSG brought about by GPX3 over-expression was enhanced by the over-expression of the GSH1/GSH2 genes because of an increase in the total glutathione (GSH + GSSG) content. Finally, after deleting the glutathione reductase (GLR1) gene, the resulting GPX3/GSH1/GSH2 over-expressing ΔGLR1 strain yielded 7.3-fold more GSSG compared with the parental strain without a decrease in cell growth. Furthermore, use of this strain also resulted in an enhancement of up to 1.6-fold of the total glutathione content compared with the GSH1/GSH2 over-expressing strain. These results indicate that the increase in the oxidized glutathione content helps to improve the stability and total productivity of glutathione.     

Reduced and oxidized glutathione in brain and convulsions

Concentration changes of reduced glutathione (GSH) and oxidized glutathione (GSSG) were studied by fluorometric assay witho-phthalaldehyde to clarify the relationship between seizure mechanism and the glutathione redox state. In cerebellum the GSH/GSSG ratio was significantly decreased in the interictal stage of E1 mice (stimulated group), but in ddY mice this ratio was decreased before convulsions induced by pentylenetetrazol and during submaximal ECS. No change was found in the GSH/GSSG ratio of the cerebellum during and after convulsions induced by pentylenetetrazol and maximal ECS. GSH levels in cerebrum in the interictal stage of E1 mice (stimulated group) were lower compared to control E1 mice. In ddY mice submaximal ECS increased GSSG levels in cerebrum so that the GSH/GSSG ratio was decreased.     

The regulation of the biosynthesis of glutathione in leaves of barley (Hordeum vulgare L.)

Between 50 and 65% of the glutathione in barley leaves was present in the chloroplasts depending upon the light regime. However, only 66–76% of the chloroplast glutathione was present in the reduced state (GSH) as opposed to 97–98% of that in the cytoplasm. In shoots treated with the catalase inhibitor aminotriazole and in shoots of the catalase deficient barley mutant RPr 79/4 exposed to air, the glutathione level increased 3-fold in 8 h in the light. The increase was accounted for by a rise in both the chloroplast and cytoplasm level of oxidised glutathione (GSSG), the GSH concentration remained relatively constant in both compartments. Only 2–3% of applied ³⁵SO₄ was metabolised to glutathione by wild-type shoots. In aminotriazole-treated plants this value rose to 17.9% and in the mutant RPr 79/4 exposed to air to 32%.     

The Response of Antioxidant Enzymes in Cellular Organelles in Cucumber (Cucumis sativus L.) Leaves to Methyl Viologen-induced Photo-oxidative Stress

In order to clarify the response of antioxidant systems in various cellular organelles to photo-oxidative stress, the activities of superoxide dismutase (SOD) and enzymes of the ascorbate–glutathione (AsA-GSH) cycle were investigated in chloroplasts, mitochondria and cytosol of cucumber leaves subjected to methyl viologen (MV) treatment. Photo-oxidation by MV resulted in significant reductions in net photosynthetic rate (Pn) and increases in the ratio of the quantum efficiency of photosystem II (PSII), Φ_PSII to that of the quantum efficiency of CO₂ fixation (ΦCO₂), followed by increased activities of SOD, and a general increase of AsA-GSH cycle enzymes in chloroplasts, mitochondria and cytosol. These increases were however, most significant in chloroplasts. There were also significant increases in dehydroascorbate (DHA), reduced glutathione (GSH), and oxidized glutathione (GSSG) except that the content of ascorbate (AsA) in chloroplasts and cytosol was slightly decreased and little effected, respectively. However, GSSG in mitochondria and GSH in cytosol were little influenced by the MV treatment. The activity of ascorbate oxidase (AO) in these organelles was independent of the MV treatment while the activity of l-galactono-1,4- lactone dehydrogenase (GLDH) in mitochondria was slightly inhibited by MV treatment. These results indicate that disturbance of electron transport in chloroplasts by MV influenced the metabolism of whole cell by a crosstalk signaling system and that the AsA-GSH cycle played a primary role in sustaining the levels of AsA.     

Identification and clarification of the role of key active site residues in bacterial glutathione S-transferase zeta/maleylpyruvate isomerase

The maleylpyruvate isomerase NagL from Ralstonia sp. strain U2, which has been structurally characterized previously, catalyzes the isomerization of maleylpyruvate to fumarylpyruvate. It belongs to the class zeta glutathione S-transferases (GSTZs), part of the cytosolic GST family (cGSTs). In this study, site-directed mutagenesis was conducted to probe the functions of 13 putative active site residues. Steady-state kinetic information for mutants in the reduced glutathione (GSH) binding site, suggested that (a) Gln64 and Asp102 interact directly with the glutamyl moiety of glutathione, (b) Gln49 and Gln64 are involved in a potential electron-sharing network that influences the ionization of the GSH thiol. The information also suggests that (c) His38, Asn108 and Arg109 interact with the GSH glycine moiety, (d) His104 has a role in the ionization of the GSH sulfur and the stabilization of the maleyl terminal carboxyl group in the reaction intermediate and (e) Arg110 influences the electron distribution in the active site and therefore the ionization of the GSH thiolate. Kinetic data for mutants altered in the substrate-binding site imply that (a) Arg8 and Arg176 are critical for maleylpyruvate orientation and enolization, and (b) Arg109 (exclusive to NagL) participates in k_cat regulation. Surprisingly, the T11A mutant had a decreased GSH K_m value, whereas little impact on maleylpyruvate kinetics was observed, suggesting that this residue plays an important role in GSH binding. An evolutionary trend in this residue appears to have developed not only in prokaryotic and eukaryotic GSTZs, but also among the wider class of cGSTs.     

Efficient production of glutathione in Saccharomyces cerevisiae via a synthetic isozyme system

Glutathione, a tripeptide consisting of cysteine, glutamic acid, and glycine, has multiple beneficial effects on human health. Previous studies have focused on producing glutathione in Saccharomyces cerevisiae by overexpressing γ-glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2), which are the rate-limiting enzymes involved in the glutathione biosynthetic pathway. However, the production yield and titer of glutathione remain low due to the feedback inhibition on GSH1. To overcome this limitation, a synthetic isozyme system consisting of a novel bifunctional enzyme (GshF) from Gram-positive bacteria possessing both GSH1 and GSH2 activities, in addition to GSH1/GSH2, was introduced into S. cerevisiae, as GshF is insensitive to feedback inhibition. Given the HSP60 chaperonin system mismatch between bacteria and S. cerevisiae, co-expression of Group-I HSP60 chaperonins (GroEL and GroES) from Escherichia coli was required for functional expression of GshF. Among various strains constructed in this study, the SKSC222 strain capable of synthesizing glutathione with the synthetic isozyme system produced 240 mg L^-1 glutathione with glutathione content and yield of 4.3% and 25.6 mg_glutathione/g_glucose, respectively. These values were 6.6-, 4.9-, and 4.3-fold higher than the corresponding values of the wild-type strain. In a glucose-limited fed-batch fermentation, the SKSC222 strain produced 2.0 g L^-1 glutathione in 67 h. Therefore, this study highlights the benefits of the synthetic isozyme system in enhancing the production titer and yield of value-added chemicals by engineered strains of S. cerevisiae.     

High level of reduced glutathione contributes to detoxification of lipid peroxide‐derived reactive carbonyl species in transgenic Arabidopsis overexpressing glutathione reductase under aluminum stress

Lipid peroxide‐derived reactive carbonyl species (RCS), generated downstream of reactive oxygen species (ROS), are critical damage‐inducing species in plant aluminum (Al) toxicity. In mammals, RCS are scavenged primarily by glutathione (reduced form of glutathione, GSH), but in plant Al stress, contribution of GSH to RCS detoxification has not been evaluated. In this study, Arabidopsis plants overexpressing the gene AtGR1 (accession code At3g24170), encoding glutathione reductase (GR), were generated, and their performance under Al stress was examined. These transgenic plants (GR‐OE plants) showed higher GSH levels and GSH/GSSG (oxidized form of GSH) ratio, and an improved Al tolerance as they suffered less inhibition of root growth than wild‐type under Al stress. Exogenous application of 4‐hydroxy‐2‐nonenal, an RCS responsible for Al toxicity in roots, markedly inhibited root growth in wild‐type plants. GR‐OE plants suffered significantly smaller inhibition, indicating that the enhanced GSH level increased the capacity of RCS detoxification. The generation of H₂O₂ due to Al stress in GR‐OE plants was lower by 26% than in wild‐type. Levels of various RCS, such as malondialdehyde, butyraldehyde, phenylacetaldehyde, (E)‐2‐heptenal and n‐octanal, were suppressed by more than 50%. These results indicate that high levels of GSH and GSH/GSSG ratio by GR overexpression contributed to the suppression of not only ROS, but also RCS. Thus, the maintenance of GSH level by overexpressing GR reinforces dual detoxification functions in plants and is an efficient approach to enhance Al tolerance.     

Ageing of tomato seeds involves glutathione oxidation

The effect of seed ageing on the oxidation of reduced glutathione (GSH) and the role of GSH oxidation in ageing-induced deterioration were studied in seeds of tomato ( Lycopersicon esculentum Mill. cv. Lerica, Moneymaker and Cromco). Both long-term storage at 15°C/30% relative humidity (RH) and artificial ageing at 20°C/75% RH, 30°C/45% RH and 60°C/45% RH resulted in a marked loss of GSH and a simultaneous, though not proportional, increase in its oxidized form GSSG. The glutathione thiol-disulfide status shifted towards a highly oxidized form, while the total glutathione pool decreased. The extent of GSH oxidation differed between ageing conditions and was not directly related to the extent of seed deterioration. Thiobarbituric acid-reactive substances did not increase in ageing tomato seeds, suggesting that lipid peroxidation did not take place. Hydration of seeds, either upon imbibition in water or by priming in an osmotic solution, resulted in a rapid decrease in GSSG, a shift of the glutathione redox couple to a mainly reduced status and an increase in the glutathione pool, in both control and aged seeds. The results indicate that, in tomato seeds, (1) seed ageing involves GSH oxidation into GSSG, which is indicative of oxidative stress, (2) ageing does not affect the GSSG reduction capacity upon subsequent imbibition, and (3) the lowered viability of aged seeds cannot directly be ascribed to the decreased GSH pool or To the highly oxidized glutathione redox status.     

Blood glutathione redox status in gestational hypertension

Gestational hypertension during the third trimester reflects an exaggerated maternal inflammatory response to pregnancy. We hypothesized that oxidative stress present even in normal pregnancy becomes uncompensated in hypertensive patients. A glucose-6-phosphate dehydrogenase (G6PD) activity sufficient to meet the increased reductive equivalent need of the cells is indispensable for defense against oxidative stress. The erythrocyte glutathione redox system was studied, where G6PD is the only NADPH source. The glutathione (GSH) redox status was measured both in vivo and after an in vitro oxidative challenge in pregnant women with gestational hypertension (n = 19) vs. normotensive pregnant subjects (n = 18) and controls (n = 20). An erythrocyte GSH depletion with an increase in the oxidized form (GSSG) resulted in an elevated ratio GSSG/GSH (0.305 +/- 0.057; mean +/- SD) in hypertensive pregnant women vs. normotensive pregnant or control subjects (0.154 +/- 0.025; 0.168 +/- 0.073; p <.001). In hypertensive pregnant patients, a "GSH stability" decrease after an in vitro oxidative challenge suggested a reduced GSH recycling capacity resulting from an insufficient NADPH supply. The erythrocyte GSSG/GSH ratio may serve as an early and sensitive parameter of the oxidative imbalance and a relevant target for future clinical trials to control the effects of antioxidant treatment in women at increased risk of the pre-eclampsia syndrome.     

Effects of glutathione on thiol redox systems, chromosomal aberrations, and the ultrastructure of meristematic root cells ofPicea abies (L.) Karst.

Summary Young spruce seedlings (Picea abies [L.] Karst.) grown in hydroponic culture were exposed to three different concentrations (50,100, and 500 M) of reduced glutathione for 24 h. These physiologically relevant concentrations of glutathione had a multiple effect on the investigated tissue. Feeding of glutathione to roots increased the concentrations of thiols (glutathione, cysteine, and -glutamyl-cysteine) in roots, decreased the rate of cell divisions, induced mitotic abnormalities, and affected the cell ultrastructure. Electron micrographs showed effects such as advanced vacuolation, dilated rough-endoplasmic-reticulum cisternae, and separations of the plasma membrane from the cell wall.Abbreviations GSH reduced glutathione - GSSG oxidised glu-tathione - rER rough endoplasmic reticulum Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday