Total Glutathione Reductase Research Articles

Physiological, Morphological, and Biochemical Responses of Soybean [Glycine max (L.) Merr.] to Loquat (Eriobotrya japonica Lindl.) Leaf Extract Application on Pb-Contaminated Soil

Lead (Pb) is a non-essential element; however, plants uptake it from soils rich in Pb. Soybean [Glycine max (L.) Merr.] is an important legume crop, and Pb toxicity exerts negative impacts on its growth and yield. This study investigated the role of foliar-applied loquat (Eriobotrya japonica Lindl.) leaf extract in improving the morphological, physiological, and biochemical traits of soybean plants under Pb toxicity. Soybean plants were exposed to four Pb concentrations (0, 200, 400, and 800 µg/L) and supplemented with 0% or 5% loquat leaf aqueous extract (EJLE). Data relating to pigments, proline, total soluble sugars, malondialdehyde (MDA), hydrogen peroxide (H2O2), non-enzymatic antioxidant, i.e., [ascorbic acid (AsA), glutathione (GSH), total phenolic contents (TPC), and total flavonoids content (TFC)] and enzymatic antioxidant, i.e., [superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR)] were recorded. Total chlorophyll contents and carotenoids were significantly decreased by Pb stress, while lycopene and anthocyanin contents were increased. Similarly, proline, total soluble sugars, MDA, H2O2, AsA, GSH, TPC, TFC, SOD, CAT, POD, APX, and GR were increased under Pb stress. Foliar spray of EJLE lowered MDA and H2O2 accumulation and increased the contents of chlorophylls, carotenoids, lycopene, anthocyanins, proline, total soluble sugars, and the antioxidant system. The increase in the activities of antioxidant enzymes lowered the adverse effects of Pb stress in soybean. Similarly, the application of EJLE lowered Pb accumulation in different plant parts compared to those receiving no EJLE. It is concluded that EJLE can improve the Pb tolerance of soybean plants by enhancing morphological, physiological, and biochemical traits. However, the actual mechanisms behind these improvements warrant further investigation.

Comparative analysis of the responses to water stress in eggplant (Solanum melongena) cultivars

Little information is available on the physiological and biochemical responses to water stress in eggplant (Solanum melongena). We evaluated four genetically diverse eggplant varieties (MEL3-MEL6) under control and water stress conditions. Measurements were taken for plant growth, tissue water content, levels of chlorophylls a and b, carotenoids, proline, malondialdehyde, total phenolics, total flavonoids, superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) activities. For most traits, the water stress treatment had a greater contribution than the variety effect to the total sums of squares in an ANOVA analysis, except for total flavonoids, SOD, APX, and GR. The water stress treatment had a strong effect on plant growth and tissue water content. In general, water stress reduced the three photosynthetic pigments, increased proline, malondialdehyde, total phenolics, and total flavonoids, although some varietal differences were observed. Different patterns were also detected in the activities of the four enzymes evaluated, but few differences were observed for individual varieties between the control and water stress treatments. Many significant phenotypic correlations were observed among the traits studied, but only eight environmental correlations were detected. A PCA analysis distinctly separated individuals according to the treatment, and revealed a clearer separation of varieties under water stress than under control conditions, pointing to varietal differences in the responses to stress. Our results suggest that proline could be used as a marker for drought stress tolerance in this species. The information obtained provides new insight on the physiological and biochemical responses of eggplant to drought stress.

Tissue Inflammatory Markers and Oxidative Stress in Swine Subjected to a 40% TBSA Burn Injury

Burn injury is well known to induce a robust inflammatory response and hypermetabolic state. Intravenous (IV) crystalloid fluid resuscitation of burn patients is the foundation of modern burn care to improve tissue perfusion and outcomes. However, under military scenarios of far‐forward burn care or under civilian mass casualty situations, the availability of large volumes of such fluids for IV burn resuscitation will be logistically challenging and definitive burn care may be delayed for days. More recently, enteral resuscitation of burns has received renewed attention and some evidence suggests that it may reduce IV fluid requirements to support urine output. The present study investigated the early response of tissue inflammatory markers and evidence for oxidative stress in swine subjected to a 40% burn and resuscitated with enteral fluids compared to no fluids or a standard of care IV fluid regimen. Anesthetized Yorkshire swine (n=6 per group) were subjected to a 40% full thickness total body surface area (TBSA) burn with brass probes heated to 100°C. They were then randomized to 4 groups: 1) no fluid (NF), 2) 15 ml/kg/d WHO oral rehydration solution (ORS) (15‐ORS), 3) 70 ml/kg ORS (70‐ORS) or 4) 2 ml/kg/% burn IV lactated Ringer's (Modified Brooke (MB) formula‐ a standard of care). After 48 h, animals were euthanized and heart, liver, kidney, lung and small intestine collected and analyzed for tissue cytokine levels and indices of oxidative stress. As expected, urine output was higher in the 70‐ORS and MB groups than the NF group (p<0.01). Although plasma creatinine and BUN peaked 6 h after burn, levels returned to baseline by 48 h in all groups except 15‐ORS and NF. In the kidney, levels of IL‐1β, IL‐6 and TNF‐α tended to be higher in the 70‐ORS and MB groups, whereas IL‐10 levels were similar among groups. In liver, heart and jejunum, only IL‐1β levels tended to be higher in MB than the other groups, with a similar trend in IL‐10 concentrations. With regard to oxidant status in kidney, total antioxidant levels, glutathione reductase (GR) and glutathione peroxidase activities were higher in 70‐ORS than the other groups. Mn‐superoxide dismutase (SOD) activity was higher in both 70‐ORS and MB groups, with no differences in catalase activity or nitric oxide levels. In liver, the lowest levels of total antioxidants, SOD, GR and catalase activities were observed in the NF groups compared to the others. No significant differences in indices of antioxidant status were observed in jejunum, heart or lung among groups. Typically, these variables in 15‐ORS were similar to the NF group in all tissues. The results of this study indicate that enteral fluids may be as effective as a standard of care IV fluid regimen in maintaining antioxidant status in vital organs without major changes in pro‐inflammatory cytokines. As such, this oral resuscitation may prove lifesaving in resource poor environments and reduce potential IVfluid‐related complications until definitive clinical care is available.Support or Funding InformationFunded by US Army MRMCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Gingival Periodontal Disease (PD) Level-Butyric Acid Affects the Systemic Blood and Brain Organ: Insights Into the Systemic Inflammation of Periodontal Disease.

Butyric acid (BA) is produced by periodontopathic bacterial pathogens and contributes to periodontal disease (PD) induction. Moreover, PD has been associated with detrimental effects which subsequently may lead to systemic disease (SD) development affecting certain organs. Surprisingly, the potential systemic manifestations and organ-localized effects of BA have never been elucidated. Here, we simulated BA-based oral infection among young (20-week-old) rats and isolated blood cytosol to determine BA effects on stress network-related signals [total heme, hydrogen peroxide (H2O2), catalase (CAT), glutathione reductase (GR), free fatty acid (FFA), NADP/NADPH], inflammation-associated signals [caspases (CASP12 and CASP1), IL-1β, TNF-α, metallomatrix proteinase-9 (MMP-9), and toll-like receptor-2 (TLR2)], and neurological blood biomarkers [presenilin (PS1 and PS2) and amyloid precursor protein (APP)]. Similarly, we extracted the brain from both control and BA-treated rats, isolated the major regions (hippocampus, pineal gland, hypothalamus, cerebrum, and cerebellum), and, subsequently, measured stress network-related signals [oxidative stress: total heme, NADPH, H2O2, GR, and FFA; ER stress: GADD153, calcium, CASP1, and CASP3] and a brain neurodegenerative biomarker (Tau). In the blood, we found that BA was no longer detectable. Nevertheless, oxidative stress and inflammation were induced. Interestingly, amounts of representative inflammatory signals (CASP12, CASP1, IL-1β, and TNF-α) decreased while MMP-9 levels increased which we believe would suggest that inflammation was MMP-9-modulated and would serve as an alternative inflammatory mechanism. Similarly, TLR2 activity was increased which would insinuate that neurological blood biomarkers (APP, PS1, and PS2) were likewise affected. In the brain, BA was not detected, however, we found that both oxidative and ER stresses were likewise altered in all brain regions. Interestingly, tau protein amounts were significantly affected in the cerebellar and hippocampal regions which coincidentally are the major brain regions affected in several neurological disorders. Taken together, we propose that gingival BA can potentially cause systemic inflammation ascribable to prolonged systemic manifestations in the blood and localized detrimental effects within the brain organ.

Differential expression of disulfide reductase enzymes in a free-living platyhelminth (Dugesia dorotocephala).

A search of the disulfide reductase activities expressed in the adult stage of the free-living platyhelminth Dugesia dorotocephala was carried out. Using GSSG or DTNB as substrates, it was possible to obtain a purified fraction containing both GSSG and DTNB reductase activities. Through the purification procedure, both disulfide reductase activities were obtained in the same chromatographic peak. By mass spectrometry analysis of peptide fragments obtained after tryptic digestion of the purified fraction, the presence of glutathione reductase (GR), thioredoxin-glutathione reductase (TGR), and a putative thioredoxin reductase (TrxR) was detected. Using the gold compound auranofin to selectively inhibit the GSSG reductase activity of TGR, it was found that barely 5% of the total GR activity in the D. dorotocephala extract can be assigned to GR. Such strategy did allow us to determine the kinetic parameters for both GR and TGR. Although It was not possible to discriminate DTNB reductase activity due to TrxR from that of TGR, a chromatofocusing experiment with a D. dorotocephala extract resulted in the obtention of a minor protein fraction enriched in TrxR, strongly suggesting its presence as a functional protein. Thus, unlike its parasitic counterparts, in the free-living platyhelminth lineage the three disulfide reductases are present as functional proteins, albeit TGR is still the major disulfide reductase involved in the reduction of both Trx and GSSG. This fact suggests the development of TGR in parasitic flatworms was not linked to a parasitic mode of life.

Interactive zinc, iron, and copper-induced phytotoxicity in wheat roots.

Growth inhibition and antioxidative response were investigated in wheat roots cultured in 1/4 Hoagland solution containing zinc (Zn, 500μM), iron (Fe, 300μM), and copper (Cu, 300μM) in combination. Different Zn, Fe, and Cu interactions inhibited seedling growth and increased Zn, Fe, and Cu contents in roots and shoots, with the most significant inhibition due to Zn+Fe+Cu treatment. The elevation of malondialdehyde content and the loss of cell viability resulted from the increases of total and apoplastic hydrogen peroxide (H2O2) and hydroxyl radical (·OH) contents in all treated roots. Except for Zn+Fe stress, root superoxide anion (O2•-) level significantly decreased at other combined treatments. The application of 10μM diphenylene iodonium suggested that NADPH oxidase activity was lower in Fe+Cu-treated and Zn+Fe+Cu-treated roots than in other roots. Additionally, all combined treatments inhibited superoxide dismutase (SOD) and peroxidase (POD) but stimulated total glutathione reductase (GR) activity in roots. However, in root apoplast, decreased SOD and ascorbate peroxidase activities as well as increased POD, catalase, and GR activities were caused by different Zn, Fe, and Cu interactions. In conclusion, combined Zn, Fe, and Cu stresses exhibited significant inhibition on root growth, with the strongest effect due to Zn+Fe+Cu. Here, it is also indicated that each antioxidantive enzyme including apoplastic enzymes showed specific responses and that the stimulation of some of them played an important protective mechanism against oxidative damage, when wheat roots were treated with different Zn, Fe, and Cu treatments in combination.

Electroacupuncture alleviates cerebral ischemia and reperfusion injury via modulation of the ERK1/2 signaling pathway.

Electroacupuncture (EA) has anti-oxidative and anti-inflammatory actions, but whether the neuroprotective effect of EA against cerebral ischemia-reperfusion (I/R) injury involves modulation of the extracellular regulated kinase 1/2 (ERK1/2) signaling pathway is unclear. Middle cerebral artery occlusion (MCAO) was performed in Sprague-Dawley rats for 2 hours followed by reperfusion for 24 hours. A 30-minute period of EA stimulation was applied to both Baihui (DU20) and Dazhui (DU14) acupoints in each rat (10 mm EA penetration depth, continuous wave with a frequency of 3 Hz, and a current intensity of 1-3 mA) when reperfusion was initiated. EA significantly reduced infarct volume, alleviated neuronal injury, and improved neurological function in rats with MCAO. Furthermore, high mRNA expression of Bax and low mRNA expression of Bcl-2 induced by MCAO was prevented by EA. EA substantially restored total glutathione reductase (GR), glutathione (GSH) and glutathione peroxidase (GSH-Px) levels. Additionally, Nrf2 and glutamylcysteine synthetase (GCS) expression levels were markedly increased by EA. Interestingly, the neuroprotective effects of EA were attenuated when ERK1/2 activity was blocked by PD98059 (a specific MEK inhibitor). Collectively, our findings indicate that activation of the ERK1/2 signaling pathway contributes to the neuroprotective effects of EA. Our study provides a better understanding of the regulatory mechanisms underlying the therapeutic effectiveness of EA.

Gene knockout of glutathione reductase 3 results in increased sensitivity to salt stress in rice.

Glutathione reductase (GR) is one of important antioxidant enzymes in plants. This enzyme catalyzes the reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) with the accompanying oxidation of NADPH. Previously, we showed that salt-stress-responsive GR3 is a functional protein localized in chloroplasts and mitochondria in rice. To learn more about the role of GR3 in salt-stress tolerance, we investigated the response to 100mM NaCl treatment in wild-type rice (WT); GR3 knockout mutant of rice (gr3); and the functional gr3-complementation line (C1). Rice GR3 was primarily expressed in roots at the seedling stage and ubiquitously expressed in all tissues except the sheath at heading stage. GR3 promoter-GUS was expressed in the vascular cylinder and cortex of root tissues in rice seedlings, vascular tissue of nodes, embryo and aleurone layer of seeds, and young flowers. Under both normal and salt-stress conditions, total GR activity was decreased by 20% in gr3. Oxidative stress, indicated by malondialdehyde content, was greater in gr3 than the WT under salt stress. As compared with the WT, gr3 was sensitive to salt and methyl viologen; it showed inhibited growth, decreased maximal efficiency of photosystem II, decreased GSH and GSSG contents, and the ratio of GSH to GSSG. Conversely, the gr3-complementation line C1 rescued the tolerance to methyl viologen and salinity and recovered the growth and physiological damage caused by salinity. These results reveal that GR3 plays an important role in salt stress tolerance by regulating the GSH redox state in rice.

Differential Responses of Antioxidative System to Soil Water Shortage in Barley (<i>Hordeum vulgare</i> L.) Genotypes

Drought is one of the major factors limiting the yield and quality of crops in the world. The activity of antioxidative system to tolerate the drought stress is significant in plants. In the present study, the activities and isoform profiles of catalase (CАТ), ascorbate peroxidase (APX), glutathione reductase (GR), and superoxide dismutase (SOD) were analyzed in four barley genotypes grown under soil water restriction. Drought stress caused increase in the activities of CАТ and SOD in all studied genotypes, while APX activity decreased. The total GR activity increased substantially in genotypes K 2778 and St.Garabag 7 and decreased in No. 77 local and St.Pallidum 596 genotypes under conditions of severe water stress. No detectable differences were observed in the isoenzyme pattern (the appearance of a new isoenzymes and disappearance of another one) between control plants and those subjected to soil drought. However, intensification of corresponding isoforms in electrophoretic spectra was observed in stressed barley leaves relative to watered ones. The obtained results possibly suggest that antioxidant protection in barley plants under drought conditions could be attributed mainly to SOD and CAT.

Water stress reveals differential antioxidant responses of tolerant and non-tolerant sugarcane genotypes

The biochemical responses of the enzymatic antioxidant system of a drought-tolerant cultivar (IACSP 94-2094) and a commercial cultivar in Brazil (IACSP 95-5000) grown under two levels of soil water restriction (70% and 30% Soil Available Water Content) were investigated. IACSP 94-2094 exhibited one additional active superoxide dismutase (Cu/Zn-SOD VI) isoenzyme in comparison to IACSP 95-5000, possibly contributing to the heightened response of IACSP 94-2094 to the induced stress. The total glutathione reductase (GR) activity increased substantially in IACSP 94-2094 under conditions of severe water stress; however, the appearance of a new GR isoenzyme and the disappearance of another isoenzyme were found not to be related to the stress response because the cultivars from both treatment groups (control and water restrictions) exhibited identical changes. Catalase (CAT) activity seems to have a more direct role in H2O2 detoxification under water stress condition and the shift in isoenzymes in the tolerant cultivar might have contributed to this response, which may be dependent upon the location where the excessive H2O2 is being produced under stress. The improved performance of IACSP 94-2094 under drought stress was associated with a more efficient antioxidant system response, particularly under conditions of mild stress.

Oxidative stress and glutathione response in tissue cultures from persons with major depression

There is evidence that major depressive disorder (MDD) is associated with increased peripheral markers of oxidative stress. To explore oxidation and antioxidant response in MDD, we assayed human dermal fibroblast cultures derived from skin biopsies of age-, race-, and sex-matched individuals in depressed and normal control groups (n = 16 each group), cultured in glucose and galactose conditions, for relative protein carbonylation (a measure of oxidative stress), glutathione reductase (GR) expression, and total glutathione concentration. In control-group fibroblasts, galactose induced a significant increase from the glucose condition in both protein carbonylation and GR. The cells from the MDD group showed total protein carbonylation and GR expression in the glucose condition that was significantly higher than control cells in glucose and equivalent to controls in galactose. There was a small decrease in protein carbonylation in MDD cells from glucose to galactose and no significant change in GR. There was no difference in total glutathione among any of the groups. Increased protein carbonylation and GR expression, cellular responses to oxidative stress induced by galactose in control fibroblasts, are present in fibroblasts derived from MDD patients and are not explainable by reduced GR or total glutathione in the depressed patients. These studies support the role of oxidative stress in the pathophysiology of MDD. Further confirmation of these findings could lead to the development of novel antioxidant approaches for the treatment of depression.

Involvement of riboflavin kinase expression in cellular sensitivity against cisplatin

Flavin adenine dinucleotide (FAD) is an essential coenzyme for glutathione reductase (GR) which catalyzes the reduction of oxidized glutathione to regenerate the reduced form involved in protection against oxidative stress. Riboflavin kinase (RFK) also known as flavokinase is involved in the first step of bioactivation of riboflavin (RF) to form flavin mononucleotide (FMN) which can be subsequently converted to FAD in an ATP-dependent reaction catalyzed by FAD synthetase (FADS). We investigated the involvement of RFK in cisplatin resistance using human prostate cancer PC3 cells. RFK overexpression renders cells resistant not only to cisplatin but also to hydrogen peroxide (H2O2) and diamide. Furthermore, knockdown of RFK expression induced apoptosis. We demonstrated that overexpression of RFK increased the levels of FAD, FMN and total glutathione and the expression of GR and glutathione S-transferase-π (GSTπ). RFK expression is up-regulated in cisplatin-resistant P/CDP6 cells in addition to FAD, total glutathione level, GR and GSTπ. Knockdown of RFK expression also sensitized both PC3 and P/CDP6 cells to cisplatin. Moreover, cellular levels of RFK expression correlate well with Gleason score, known as a good indicator of patient prognosis. The present study suggests that RFK expression is involved not only in cellular protection from oxidative stress but also in malignant progression of prostate cancer.

The protective effects of cerium oxide nanoparticles against hepatic oxidative damage induced by monocrotaline

ObjectiveThe objective of the present study was to determine the ability of cerium oxide (CeO2) nanoparticles to protect against monocrotaline (MCT)-induced hepatotoxicity in a rat model.MethodTwenty male Sprague Dawley rats were arbitrarily assigned to four groups: control (received saline), CeO2 (given 0.0001 nmol/kg intraperitoneally [IP]), MCT (given 10 mg/kg body weight IP as a single dose), and MCT + CeO2 (received CeO2 both before and after MCT). Electron microscopic imaging of the rat livers was carried out, and hepatic total glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPX), glutathione S-transferase (GST), superoxide dismutase (SOD), and catalase (CAT) enzymatic activities were quantified.ResultsResults showed a significant MCT-induced decrease in total hepatic GSH, GPX, GR, and GST normalized to control values with concurrent CeO2 administration. In addition, MCT produced significant increases in hepatic CAT and SOD activities, which also ameliorated with CeO2.ConclusionsThese results indicate that CeO2 acts as a putative novel and effective hepatoprotective agent against MCT-induced hepatotoxicity.

Inherited Glutathione Reductase Deficiency and Plasmodium falciparum Malaria—A Case Study

In Plasmodium falciparum-infected red blood cells (RBCs), the flavoenzyme glutathione reductase (GR) regenerates reduced glutathione, which is essential for antioxidant defense. GR utilizes NADPH produced in the pentose phosphate shunt by glucose-6-phosphate dehydrogenase (G6PD). Thus, conditions affecting host G6PD or GR induce increased sensitivity to oxidants. Hereditary G6PD deficiency is frequent in malaria endemic areas and provides protection against severe malaria. Furthermore, GR deficiency resulting from insufficient saturation of the enzyme with its prosthetic group FAD is common. Based on these naturally occurring phenomena, GR of malaria parasites and their host cells represent attractive antimalarial drug targets. Recently we were given the opportunity to examine invasion, growth, and drug sensitivity of three P. falciparum strains (3D7, K1, and Palo Alto) in the RBCs from three homozygous individuals with total GR deficiency resulting from mutations in the apoprotein. Invasion or growth in the GR-deficient RBCs was not impaired for any of the parasite strains tested. Drug sensitivity to chloroquine, artemisinin, and methylene blue was comparable to parasites grown in GR-sufficient RBCs and sensitivity towards paraquat and sodium nitroprusside was only slightly enhanced. In contrast, membrane deposition of hemichromes as well as the opsonizing complement C3b fragments and phagocytosis were strongly increased in ring-infected RBCs of the GR-deficient individuals compared to ring-infected normal RBCs. Also, in one of the individuals, membrane-bound autologous IgGs were significantly enhanced. Thus, based on our in vitro data, GR deficiency and drug-induced GR inhibition may protect from malaria by inducing enhanced ring stage phagocytosis rather than by impairing parasite growth directly.

The NADPH-dependent thioredoxin system constitutes a functional backup for cytosolic glutathione reductase in Arabidopsis

Tight control of cellular redox homeostasis is essential for protection against oxidative damage and for maintenance of normal metabolism as well as redox signaling events. Under oxidative stress conditions, the tripeptide glutathione can switch from its reduced form (GSH) to oxidized glutathione disulfide (GSSG), and thus, forms an important cellular redox buffer. GSSG is normally reduced to GSH by 2 glutathione reductase (GR) isoforms encoded in the Arabidopsis genome, cytosolic GR1 and GR2 dual-targeted to chloroplasts and mitochondria. Measurements of total GR activity in leaf extracts of wild-type and 2 gr1 deletion mutants revealed that approximately 65% of the total GR activity is attributed to GR1, whereas approximately 35% is contributed by GR2. Despite the lack of a large share in total GR activity, gr1 mutants do not show any informative phenotype, even under stress conditions, and thus, the physiological impact of GR1 remains obscure. To elucidate its role in plants, glutathione-specific redox-sensitive GFP was used to dynamically measure the glutathione redox potential (E(GSH)) in the cytosol. Using this tool, it is shown that E(GSH) in gr1 mutants is significantly shifted toward more oxidizing conditions. Surprisingly, dynamic reduction of GSSG formed during induced oxidative stress in gr1 mutants is still possible, although significantly delayed compared with wild-type plants. We infer that there is functional redundancy in this critical pathway. Integrated biochemical and genetic assays identify the NADPH-dependent thioredoxin system as a backup system for GR1. Deletion of both, NADPH-dependent thioredoxin reductase A and GR1, prevents survival due to a pollen lethal phenotype.

Novel Glutaredoxin Activity of the Yeast Prion Protein Ure2 Reveals a Native-like Dimer within Fibrils

Ure2 is the protein determinant of the Saccharomyces cerevisiae prion [URE3]. Ure2 has structural similarity to glutathione transferases, protects cells against heavy metal and oxidant toxicity in vivo, and shows glutathione-dependent peroxidase activity in vitro. Here we report that Ure2 (which has no cysteine residues) also shows thiol-disulfide oxidoreductase activity similar to that of glutaredoxin enzymes. This demonstrates that disulfide reductase activity can be independent of the classical glutaredoxin CXXC/CXXS motif or indeed an intrinsic catalytic cysteine residue. The kinetics of the glutaredoxin activity of Ure2 showed positive cooperativity for the substrate glutathione in both the soluble native state and in amyloid-like fibrils, indicating native-like dimeric structure within Ure2 fibrils. Characterization of the glutaredoxin activity of Ure2 sheds light on its ability to protect yeast from heavy metal ion and oxidant toxicity and suggests a role in reversible protein glutathionylation signal transduction. Observation of allosteric enzyme behavior within amyloid-like Ure2 fibrils not only provides insight into the molecular structure of the fibrils but also has implications for the mechanism of [URE3] prion formation.

Ontogeny of glutathione and glutathione-related antioxidant enzymes in rat liver

We conducted a comprehensive characterization of the ontogeny of glutathione (GSH) and its related enzymes in rat liver. GSH content and activities of glutathione reductase (GR), cytosolic glutathione-S-transferases (GST), and glutathione peroxidase (GPx) were determined in male rat livers (n=4) at different developmental stages. Our results indicate total hepatic GSH content and GR, GST, and GPx activity were low in the perinatal period. GSH content remained relatively constant throughout postnatal development, but GR, GST, and GPx activity underwent significant increases to attain adult levels by late post-weaning. Whether the ontogenic pattern of GSH and its related enzymes explain, in part, the altered susceptibility of neonates to some toxicants requires further investigation. Our study provides fundamental biological information on the ontogeny of cytosolic antioxidant/detoxification enzymes in a relevant toxicological risk assessment species. Additional studies are needed to fully characterize the ontogeny of other xenobiotic detoxification enzymes to further promote the rat as a developmental toxicology model that can identify toxicokinetic reasons for differences in susceptibility to toxicity between the neonate and adult.

Scavenger Enzyme Activities in Subcellular Fractions of White Clover (Trifolium repens L.) under PEG-induced Water Stress

Scavenger enzyme activities in subcellular fractions under polyethylene glycol (PEG)-induced water stress in white clover (Trifolium repens L.) were studied. Water stress decreased ascorbic acid (AA) content and catalase (CAT) activity and increased the contents of hydrogen peroxide (H2O2), thiobarbituric acid reactive substances (TBARS) (measure of lipid peroxidation), and activities of superoxide dismutase (SOD), its various isozymes, ascorbate peroxidase (APOX), and glutathione reductase (GR) in cellular cytosol, chloroplasts, mitochondria, and peroxisomes of Trifolium repens leaves. In both the PEG-treated plants and the control, chloroplastic fractions showed the highest total SOD, APOX, and GR activities, followed by mitochondrial fractions in the case of total SOD and GR activities, whereas cytosolic fractions had the second greatest APOX activity. However, CAT activity was the highest in peroxisomes, followed by the cytosol, mitochondria, and chloroplasts in decreasing order. Although Mn-SOD activity was highest in mitochondrial fractions, residual activity was also observed in cytosolic fractions. Cu/Zn-SOD and Fe-SOD were observed in all subcellular fractions; however, the activities were the highest in chloroplastic fractions for both isoforms. Total Cu/Zn-SOD activity, the sum of activities observed in all fractions, was higher than other SOD isoforms. These results suggest that cytosolic and chloroplastic APOX, chloroplastic and mitochondrial GR, mitochondrial Mn-SOD, cytosolic and chloroplastic Cu/Zn-SOD, and chloroplastic Fe-SOD are the major scavenger enzymes, whereas cellular CAT may play a minor role in scavenging of O2− and H2O2 produced under PEG-induced water stress in Trifolium repens.

Superoxide dismutase and glutathione reductase overexpression in wheat protoplast: photooxidative stress tolerance and changes in cellular redox state

In previous works, we have established a correlation between antioxidant system response and tolerance to drought, osmotic stress and photooxidative stress of different wheat cultivars with contrasting drought tolerance. In the present work, a protocol to obtain and transform wheat protoplasts was established. Transgenic protoplasts with Manganese Superoxide Dismutase (Mn-SOD) (E.C.: 1.15.1.1) and Glutathione Reductase (GR) (E.C.: 1.6.4.2) overexpression in chloroplasts were obtained, and their responses to photooxidative stress were characterized. Protoplasts with Mn-SOD or GR overexpression, showed different responses and tolerance to photooxidative stress. Protoplasts with Mn-SOD overexpression showed lower levels of oxidative damage, higher level of endogenous hydrogen peroxide and a great induction of total SOD and GR activities during photooxidative treatments. In protoplasts with GR overexpression the oxidative damage provoked by the photooxidative treatment was similar to control protoplasts, the GSH content and GSH/GSH + GSSG ratio were higher than control and Mn-SOD transformed protoplast, and total SOD and GR activities were not induced. Our results suggest that the differential responses and tolerance to photooxidative stress given by Mn-SOD or GR overexpression, also depend on the effects of these enzyme activities over the cellular redox state balance, which modulate the responses to photooxidative stress.

Dose dependence of alpha‐tocopherol protection against in vivo ultraviolet radiation and associated alterations in lens redox balance

Abstract Purpose: To investigate the dose dependence of alpha‐tocopherol protection against in vivo ultraviolet radiation (UVR) induced light scattering and associated alterations in the redox balanced in the albino rat lens. Methods: Five groups of twenty, six‐week‐old female albino Sprague Dawley rats were supplemented with 0, 5, 25, 50, and 100 IU/day alpha‐tocopherol orally depending on group belonging for 4 weeks. Then, each rat was unilaterally exposed in vivo to 8 kJ/m2 UVR‐300 nm for 15 min. At 1 week after exposure to UVR, the rats were sacrificed, the lenses were extracted and homogenized. Lens total reduced (GSH) and oxidized (GSSG) glutathione; glutathione‐ reductase (GR) and oxidase (GPx) were determined colorimetrically by enzyme reactions. Results: Alpha‐tocopherol caused a dose‐dependent protection against UVR induced light scattering with a rate constant (1/e) of 4.8 IU/day. The exposure to UVR caused a depletion of total lens GSH at low alpha‐tocopherol supplementation that recovered towards higher alpha‐tocopherol supplementation. The UVR exposure did not cause any alteration of total lens GSSG at any level of alpha‐tocopherol supplementation. The UVR exposure did not cause any alteration of total lens GR‐ or GPx activity at any level of alpha‐tocopherol supplementation. Conclusions: Alpha‐tocopherol dose dependently protects against UVR induced light scattering. The protection of UVR induced light scattering is associated with a reduction of GSH depletion due to UVR exposure without any effect on total lens GSSG or GR‐ or GPx activity