Glutathione Redox Homeostasis Research Articles

Age-Related Shift in Cardiac and Metabolic Phenotyping Linked to Inflammatory Cytokines and Antioxidant Status in Mice.

Age-related alterations in cardiac function, metabolic, inflammatory and antioxidant profiles are associated with an increased risk of cardiovascular mortality and morbidity. Here, we examined cardiac and metabolic phenotypes in relation to inflammatory status and antioxidant capacity in young, middle-aged and old mice. Real-time reverse transcription-polymerase chain reactions were performed on myocardium and immunoassays on plasma. Left ventricular (LV) structure and function were assessed by echocardiography using high-frequency ultrasound. Middle-aged mice exhibited an altered metabolic profile and antioxidant capacity compared to young mice, whereas myocardial expression of inflammatory factors (TNFα, IL1β, IL6 and IL10) remained unchanged. In contrast, old mice exhibited increased expression of inflammatory cytokines and plasma levels of resistin compared to young and middle-aged mice (p < 0.05). The pro-inflammatory signature of aged hearts was associated with alterations in glutathione redox homeostasis and elevated contents of 4-hydroxynonenal (4-HNE), a marker of lipid peroxidation and oxidative stress. Furthermore, echocardiographic parameters of LV systolic and diastolic functions were significantly altered in old mice compared to young mice. Taken together, these findings suggest age-related shifts in cardiac phenotype encompass the spectrum of metabo-inflammatory abnormalities and altered redox homeostasis.

Excessive N-acetylcysteine exaggerates glutathione redox homeostasis and apoptosis during acetaminophen exposure in Huh-7 human hepatoma cells

Acetaminophen (APAP) hepatotoxicity is one of the biggest drawbacks of this relatively safe and widely used drug. In addition to its hepatotoxicity, APAP also cause comparable levels of toxicity on human hepatoma cells. Here we show activation of the intrinsic caspase-9/3 pathway of apoptosis followed by gasdermin E (GSDME) cleavage and subsequent ballooning in APAP (10 mM, 72 h)-treated Huh-7 human hepatocarcinoma cells. N-acetylcysteine (NAC), an antioxidant currently used as an antidote for APAP overdose, does not alleviate APAP toxicity in Huh-7 cells; NAC overdose (10 mM) rather aggravates APAP toxicity. NAC overdose not only aggravates cell death, but also decreases the cellular GSH/GSSG ratio, an indicator of redox homeostasis of glutathione. These results show for the first time that APAP-induced apoptosis in hepatoma cells is followed by secondary necrosis via the caspase-3/GSDME pathway. NAC overdose (10 mM) not only worsens the glutathione redox status, but also accelerates this pathway.

Transcriptomic analysis reveals antibacterial mechanism of flavonoids from Sedum aizoon L. against Pseudomonas fragi

Flavonoids in Sedum aizoon L. (FSAL) have been used traditional medicine due to its antibacterial properties and anti-inflammatory. In this study, the molecular mechanisms involved in the action of FSAL on Pseudomonas fragi ( P. fragi ) were investigated for the first time using the transcriptome. The results showed that FSAL treatment (1.0 MIC) disrupted the intracellular sulfate assimilation pathway and led to disturbances in glutathione redox homeostasis. A total of 298 genes were found to be responsive to FSAL, including 233 up-regulated and 65 down-regulated. The highlighted changes were those related to sulfur metabolism, ABC transporters, aminobenzoate degradation and benzoate degradation. In summary, the intervention of FSAL resulted in the disturbance of cellular metabolic processes and redox homeostasis, which inhibited P. fragi . The results revealed the mechanism of P. fragi inhibition under FSAL treatment, which could be helpful for the application of FSAL in food industry and food safety. • Transcriptome analysis of P. fragi in response to FSAL. • Sulfur metabolism is an important pathway to inhibit the growth of the P. fragi. • GSH is a key limiting factor in the sulfate assimilation pathway.

The critical role of glutathione redox homeostasis towards oxidation in ermanin-induced melanogenesis

Vitiligo is a depigmented disease featured as diagnosis simplicity and cure difficulty. Its occurrence and development are associated with a variety of factors, including oxidative stress, heredity and immunity, etc. Existing drugs for the treatment of vitiligo are to reduce the death of melanocytes and induce pigment accumulation as the main treatment strategy. Ermanin, a member of the flavonoids, is extracted from bee glue which is wildly used to treat vitiligo in traditional Chinese medicine. Therefore, this article discusses the relationship between melanogenesis and glutathione redox homeostasis by ermanin via biochemical and free radical approaches in vivo and in vitro. In this study, we found that ermanin effectively increased the melanin content at the in vivo model (zebrafish). Moreover, the melanin levels at the in vitro models (B16F10 cells and primary melanocytes) were also increased significantly accompanied with a shift of glutathione redox homeostasis towards oxidation.Ermanin also significantly enhanced the activity of tyrosinase. Meanwhile, ermanin increased the expression levels of TYR, TRP-1, and DCT genes, while ROS accumulation and glutathione depletion mediated the accumulation of pigments caused by ermanin, which increased the production of pigments and regulated the expression mRNA levels of TYR and DCT genes. From the perspective of pigment production regulation pathways, western blot showed that the pigment accumulation caused by ermanin was closely related to the CREB-MITF pathways, it activated CREB, TYR, TRP-1, and DCT proteins. The use of CREB specific inhibitor 666-15 and MITF inhibitor ML329 confirmed that the pigment accumulation caused by ermanin was positively correlated with CREB and MITF proteins. Our findings revealed the potential mechanisms by which ermanin promoted the production of melanin through activated CREB-MITF signaling pathway and glutathione redox homeostasis towards oxidation function as a signal are beneficial to melanin production and will help develop novel therapeutic approaches for vitiligo.

Moringa oleifera Leaf Extract Upregulates Nrf2/HO-1 Expression and Ameliorates Redox Status in C2C12 Skeletal Muscle Cells.

Moringa oleifera is a multi-purpose herbal plant with numerous health benefits. In skeletal muscle cells, Moringa oleifera leaf extract (MOLE) acts by increasing the oxidative metabolism through the SIRT1-PPARα pathway. SIRT1, besides being a critical energy sensor, is involved in the activation related to redox homeostasis of transcription factors such as the nuclear factor erythroid 2-related factor (Nrf2). The aim of the present study was to evaluate in vitro the capacity of MOLE to influence the redox status in C2C12 myotubes through the modulation of the total antioxidant capacity (TAC), glutathione levels, Nrf2 and its target gene heme oxygenase-1 (HO-1) expression, as well as enzyme activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and transferase (GST). Moreover, the impact of MOLE supplementation on lipid peroxidation and oxidative damage (i.e., TBARS and protein carbonyls) was evaluated. Our results highlight for the first time that MOLE increased not only Nrf2 and HO-1 protein levels in a dose-dependent manner, but also improved glutathione redox homeostasis and the enzyme activities of CAT, SOD, GPx and GST. Therefore, it is intriguing to speculate that MOLE supplementation could represent a valuable nutrition for the health of skeletal muscles.

The requirement for pyruvate dehydrogenase in leukemogenesis depends on cell lineage

Cancer cells are metabolically similar to their corresponding normal tissues. Differences between cancers and normal tissues may reflect reprogramming during transformation or maintenance of the metabolism of the specific normal cell type that originated the cancer. Here, we compare glucose metabolism in hematopoiesis and leukemia. Thymus Tcell progenitors were glucose avid and oxidized more glucose in the tricarboxylic acid cycle through pyruvate dehydrogenase (PDH) as compared with other hematopoietic cells. PDH deletion decreased double-positive Tcell progenitor cells but had no effect on hematopoietic stem cells, myeloid progenitors, or other hematopoietic cells. PDH deletion blocked the development of Pten-deficient Tcell leukemia, but not the development of a Pten-deficient myeloid neoplasm. Therefore, the requirement for PDH in leukemia reflected the metabolism of the normal cell of origin independently of the driver genetic lesion. PDH was required to prevent pyruvate accumulation and maintain glutathione levels and redox homeostasis.

Alterations in glutathione redox homeostasis among adolescents with obesity and anemia

The reduced (GSH)-to-oxidized (GSSG) glutathione ratio represents a dynamic balance between oxidants and antioxidants. However, redox status in adolescents with obesity and anemia has not been investigated. This study investigated the association of erythrocyte GSH redox status (GSH, GSH:GSSG ratio, and glutathione peroxidase [GPx] activity) with anemia and adiposity in adolescents. This case–control study nested in a cross-sectional study enrolled 524 adolescents (268 boys; 256 girls). The prevalence of anemia in overweight and obesity (OWOB) was 5.2% in boys and 11.7% in girls. The GSH:GSSG ratio and GPx activity were significantly higher in girls than in boys (p < 0.001), in anemic than in non-anemic subjects (p < 0.001), and in OWOB than in normal-weight subjects (p < 0.001). Similarly, significantly higher GSH: GSSG level (p < 0.001) and GPx activity (p < 0.001) were found in subjects with 90th percentile waist circumference than in those with < 90th percentile. GPx and GSH:GSSG were positively associated with anemia after adjusting for age, sex, and body mass index (adjusted odds ratio, adjOR [95% confidence interval, CI] 2.18 [1.44–3.29]) or tertiles (adjOR [95% CI], T3 = 2.49 [1.03–6.01]). A similar association was noted for GSH and GPx. A compensatory increased redox defense mechanism exists in anemia and obesity among adolescents without metabolic disturbances.

Glutathione as a mediator of cartilage oxidative stress resistance and resilience during aging and osteoarthritis

ABSTRACTPurpose: An underlying cause of osteoarthritis (OA) is the inability of chondrocytes to maintain homeostasis in response to changing stress conditions. The purpose of this article was to review and experimentally evaluate oxidative stress resistance and resilience concepts in cartilage using glutathione redox homeostasis as an example. This framework may help identify novel approaches for promoting chondrocyte homeostasis during aging and obesity.Materials and Methods: Changes in glutathione content and redox ratio were evaluated in three models of chondrocyte stress: (1) age- and tissue-specific changes in joint tissues of 10 and 30-month old F344BN rats, including ex vivo patella culture experiments to evaluate N-acetylcysteine dependent resistance to interleukin-1beta; (2) effect of different durations and patterns of cyclic compressive loading in bovine cartilage on glutathione stress resistance and resilience pathways; (3) time-dependent changes in GSH:GSSG in primary chondrocytes from wild-type and Sirt3 deficient mice challenged with the pro-oxidant menadione.Results: Glutathione was more abundant in cartilage than meniscus or infrapatellar fat pad, although cartilage was also more susceptible to age-related glutathione oxidation. Glutathione redox homeostasis was sensitive to the duration of compressive loading such that load-induced oxidation required unloaded periods to recover and increase total antioxidant capacity. Exposure to a pro-oxidant stress enhanced stress resistance by increasing glutathione content and GSH:GSSG ratio, especially in Sirt3 deficient cells. However, the rate of recovery, a marker of resilience, was delayed without Sirt3.Conclusions: OA-related models of cartilage stress reveal multiple mechanisms by which glutathione provides oxidative stress resistance and resilience.

Arabidopsis glutathione reductase 2 is indispensable in plastids, while mitochondrial glutathione is safeguarded by additional reduction and transport systems.

A highly negative glutathione redox potential (EGSH ) is maintained in the cytosol, plastids and mitochondria of plant cells to support fundamental processes, including antioxidant defence, redox regulation and iron-sulfur cluster biogenesis. Out of two glutathione reductase (GR) proteins in Arabidopsis, GR2 is predicted to be dual-targeted to plastids and mitochondria, but its differential roles in these organelles remain unclear. We dissected the role of GR2 in organelle glutathione redox homeostasis and plant development using a combination of genetic complementation and stacked mutants, biochemical activity studies, immunogold labelling and in vivo biosensing. Our data demonstrate that GR2 is dual-targeted to plastids and mitochondria, but embryo lethality of gr2 null mutants is caused specifically in plastids. Whereas lack of mitochondrial GR2 leads to a partially oxidised glutathione pool in the matrix, the ATP-binding cassette (ABC) transporter ATM3 and the mitochondrial thioredoxin system provide functional backup and maintain plant viability. We identify GR2 as essential in the plastid stroma, where it counters GSSG accumulation and developmental arrest. By contrast a functional triad of GR2, ATM3 and the thioredoxin system in the mitochondria provides resilience to excessive glutathione oxidation.

Loss of glutathione redox homeostasis impairs proteostasis by inhibiting autophagy-dependent protein degradation.

In the presence of aggregation-prone proteins, the cytosol and endoplasmic reticulum (ER) undergo a dramatic shift in their respective redox status, with the cytosol becoming more oxidized and the ER more reducing. However, whether and how changes in the cellular redox status may affect protein aggregation is unknown. Here, we show that C. elegans loss-of-function mutants for the glutathione reductase gsr-1 gene enhance the deleterious phenotypes of heterologous human, as well as endogenous worm aggregation-prone proteins. These effects are phenocopied by the GSH-depleting agent diethyl maleate. Additionally, gsr-1 mutants abolish the nuclear translocation of HLH-30/TFEB transcription factor, a key inducer of autophagy, and strongly impair the degradation of the autophagy substrate p62/SQST-1::GFP, revealing glutathione reductase may have a role in the clearance of protein aggregates by autophagy. Blocking autophagy in gsr-1 worms expressing aggregation-prone proteins results in strong synthetic developmental phenotypes and lethality, supporting the physiological importance of glutathione reductase in the regulation of misfolded protein clearance. Furthermore, impairing redox homeostasis in both yeast and mammalian cells induces toxicity phenotypes associated with protein aggregation. Together, our data reveal that glutathione redox homeostasis may be central to proteostasis maintenance through autophagy regulation.

Glutathione: subcellular distribution and membrane transport 1.

Glutathione (γ-l-glutamyl-l-cysteinylglycine) is a small tripeptide found at millimolar concentrations in nearly all eukaryotes as well as many prokaryotic cells. Glutathione synthesis is restricted to the cytosol in animals and fungi and to the cytosol and plastids in plants. Nonetheless, glutathione is found in virtually all subcellular compartments. This implies that transporters must exist that facilitate glutathione transport into and out of the various subcellular compartments. Glutathione may also be exported and imported across the plasma membrane in many cells. However, in most cases, the molecular identity of these transporters remains unclear. Whilst glutathione transport is essential for the supply and replenishment of subcellular glutathione pools, recent evidence supports a more active role for glutathione transport in the regulation of subcellular glutathione redox homeostasis. However, our knowledge of glutathione redox homeostasis at the level of specific subcellular compartments remains remarkably limited and the role of glutathione transport remains largely unclear. In this review, we discuss how new tools and techniques have begun to yield insights into subcellular glutathione distribution and glutathione redox homeostasis. In particular, we discuss the known and putative glutathione transporters and examine their contribution to the regulation of subcellular glutathione redox homeostasis.

Changes in Glutathione Redox Potential Are Linked to Aβ42-Induced Neurotoxicity

Glutathione is the major low-molecular weight thiol ofeukaryotic cells. It is central to one of the two major NADPH-dependent reducing systems and is likely to play a role in combating oxidative stress, a process suggested to play a key role in Alzheimer's disease (AD). However, the nature and relevance of redox changes in the onset and progression of AD are still uncertain. Here, we combine genetically encoded redox sensors with our Drosophila models of amyloid-beta (Aβ) aggregation. We find that changes in glutathione redox potential (EGSH) closely correlate with disease onset and progression. We observe this redox imbalance specifically in neurons, but not in glia cells. EGSH changes and Aβ42 deposition are also accompanied by increased JNK stress signaling. Furthermore, pharmacologic and genetic manipulation of glutathione synthesis modulates Aβ42-mediated neurotoxicity, suggesting a causal relationship between disturbed glutathione redox homeostasis and early AD pathology.

Glutathione redox cycle enzymes as potential targets for heme-mediated oxidation under hemolysis: in silico analysis

Glutathione (g-glutamylcysteinylglycine) redox homeostasis in human erythrocytes is dependent on the activities of glutathione peroxidase (GPX1, EC 1.11.1.9), glutathione reductase (GSR, EC 1.8.1.7), glutaredoxin 1 (GRX1) and NADPH-generating enzymes of pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (PGD, EC 1.1.1.44). Free heme accumulation under hemolysis can affect proteins activity thereby in silico analysis of glutathione redox cycle enzymes structure was performed in order to reveal putative heme-binding sites and oxidizable cysteine residues. Protein annotations were taken from UniProt. Heme docking was performed by PatchDock with clustering RMSD 1,5 Å using PDB structures of proteins and heme. Cysteines oxidation potential was estimated by Cy-Preds. Heme binding GSR monomers (1DNC, 3DJJ, 3DK9, 2GRT) and dimers (3SQP, 2GH5) was predicted through His81 close to interchain disulfide bond and through Cys59 near FAD and GSSG binding sites. Heme-binding areas in GPX1 (2F8A) and GPX3 (2R37) also were revealed in the interchain region and in active centre (His80). GLRX1 (4RQR) was predicted to bind heme almost exclusively near the N-end in spite of accessibility of all cysteines including CPYC motif in active centre. G6PD monomer (2BH9, 5UKW) revealed heme-docking areas in NADP+ binding region and a-helix 437–447 located in dimer 2BHL at the interchain surface. Heme docking to PGD (4GWG, 4GWK) was in substrate binding region near His187. So enzymes active centres and chain interaction regions were revealed in the most of heme docking variants. From one (in PGD) to three (GSR) cysteines susceptible to oxidation were found in each protein including cysteines that were predicted to bind heme. Heme-mediated oxidative effect on glutathione redox cycle enzymes in erythrocytes and blood plasma could be an important mechanism of hemolysis progression under stress and pathologies.

Glutathione, Glutaredoxin And S-Glutathionylation In Lung Disease

Chronic lung diseases, like idiopathic pulmonary fibrosis (IPF) and COPD are accompanied by marked changes in redox homeostasis, that affect glutathione. The covalent attachment of glutathione to reactive protein cysteines, a process known as S-glutathionylation (PSSG), has become recognized as a mechanism whereby changes in glutathione redox homeostasis may contribute to chronic diseases. Our laboratory has developed approaches to detect PSSG in intact tissues and demonstrated strong increases in PSSG in lungs from patients with IPF, notably in epithelial cells in areas of active disease progression. Our laboratory has furthermore demonstrated that Fas (CD95) is a critical target for S-glutathionylation, that S-glutathionylation of Fas is a prominent apoptotic signal in lung epithelial cells, and that this contributes to the development of fibrosis. The biochemical processes that control S-glutathionylation will be discussed, and their impact for progression of pulmonary fibrosis presented. Lastly, opportunities to harness S-glutathionylation chemistry as a potential treatment modality for pulmonary fibrosis also will be covered.

Cytochrome c peroxidase regulates intracellular reactive oxygen species and methylglyoxal via enzyme activities of erythroascorbate peroxidase and glutathione-related enzymes in Candida albicans.

D-erythroascorbate peroxidase (EAPX1) deficiency causes glutathione deprivation, leading to the accumulation of methylglyoxal and reactive oxygen species (ROS), and especially, induction of cytochrome c peroxidase (Ccp1) in Candida albicans. Nevertheless, reciprocal effects between changes in Ccp1 activity and the antioxidative D-erythroascorbic acid- and glutathione-dependent redox status, which reflects methylglyoxal biosynthesis altering pathophysiology are unclear in eukaryotes. To elucidate the effect of CCP1 expression on EAPX1 and glutathione reductase (Glr1) activity-mediated D-erythroascorbic acid biosynthesis and redox homeostasis, the CCP1 gene was disrupted and overexpressed. First, we demonstrated both glutathione-independent and-dependent metabolite contents and their corresponding gene transcripts and enzyme activities (i.e., Ccp1, catalase-peroxidase [KatG], superoxide dismutase [Sod], Eapx1, and Glr1) in CCP1 mutants. Second, methylglyoxal-oxidizing alcohol dehydrogenase (Adh1) and methylglyoxal-reducing oxidoreductase activity on glycolytic methylglyoxal and pyruvate production and NAD(P)H content were determined in these mutants. Contrary to our expectation, CCP1 disruption (42.19±3.22nmolO2h-1mgwetcell-1) failed to affect cell respiration compared to the wild-type strain (41.62±7.11nmolO2h-1mgwetcell-1) under cyanide treatment, and in contrast to hydrogen peroxide (H2O2) treatment (21.74±1.03nmol O2h-1mgwetcell-1). Additionally, Ccp1 predominantly detoxified H2O2 rather than negligible scavenging activities towards methylglyoxal and other oxidants. CCP1 deficiency stimulated Sod and Adh1 activity but downregulated Glr1, Eapx1, catalase, and peroxidase activity while enhancing KatG, EAPX1, and GLR1 transcription by decreasing glutathione and D-erythroascorbic acid and increasing pyruvate. Noticeably, the ROS-accumulating CCP1-deficient mutant maintained steady-state levels of methylglyoxal, which was revealed to be regulated by methylglyoxal-oxidizing and -reducing activity with drastic changes in NAD(P)H. We confirmed and clarified our results by showing that CCP1/EAPX1 double disruptants underwent severe growth defects due to the D-erythroascorbic acid and glutathione depletion because of pyruvate overaccumulation. These observations were made in both budding and hyphal-growing CCP1 mutants. The revealed metabolic network involving Ccp1 and other redox regulators affected ROS and methylglyoxal through D-erythroascorbic acid and glutathione-dependent metabolites, thereby influencing dimorphism. This is the first report of the Ccp1-mediated D-erythroascorbic acid and glutathione biosynthesis accompanying methylglyoxal scavengers for full fungal virulence.

Mitochondrial Glutathione: Regulation and Functions.

Mitochondrial glutathione fulfills crucial roles in a number of processes, including iron-sulfur cluster biosynthesis and peroxide detoxification. Recent Advances: Genetically encoded fluorescent probes for the glutathione redox potential (EGSH) have permitted extensive new insights into the regulation of mitochondrial glutathione redox homeostasis. These probes have revealed that the glutathione pools of the mitochondrial matrix and intermembrane space (IMS) are highly reduced, similar to the cytosolic glutathione pool. The glutathione pool of the IMS is in equilibrium with the cytosolic glutathione pool due to the presence of porins that allow free passage of reduced glutathione (GSH) and oxidized glutathione (GSSG) across the outer mitochondrial membrane. In contrast, limited transport of glutathione across the inner mitochondrial membrane ensures that the matrix glutathione pool is kinetically isolated from the cytosol and IMS. In contrast to the situation in the cytosol, there appears to be extensive crosstalk between the mitochondrial glutathione and thioredoxin systems. Further, both systems appear to be intimately involved in the removal of reactive oxygen species, particularly hydrogen peroxide (H2O2), produced in mitochondria. However, a detailed understanding of these interactions remains elusive. We postulate that the application of genetically encoded sensors for glutathione in combination with novel H2O2 probes and conventional biochemical redox state assays will lead to fundamental new insights into mitochondrial redox regulation and reinvigorate research into the physiological relevance of mitochondrial redox changes. Antioxid. Redox Signal. 27, 1162-1177.

Regulative roles of glutathione reductase and four glutaredoxins in glutathione redox, antioxidant activity, and iron homeostasis of Beauveria bassiana.

Multiple glutaredoxins (Grx) and glutathione reductase (Glr) are vital for the thiol-disulfide redox system in budding yeast but generally unexplored in filamentous fungi. Here we characterized the Beauveria bassiana redox system comprising dithiol Grx1, monothiol Grx2-4, Grx-like Grx5, and Glr orthologue. Each grx or glr deletion was compensated by increased transcripts of some other grx genes in normal cultures. Particularly, grx3 compensated the absence of grx1, grx2, grx5, or glr under oxidative stress while its absence was compensated only by undeletable grx4 under normal conditions but by most of other undeleted grx and glr genes in response to menadione. Consequently, the redox state was disturbed in Δglr more than in Δgrx3 but not in Δgrx1/2/5. Superoxide dismutases were more active in normal Δgrx1-3 cultures but less in Δgrx5 or Δglr response to menadione. Total catalase activity increased differentially in all the mutant cultures stressed with or without H2O2 while total peroxidase activity decreased more in the normal or H2O2-stressed culture of Δglr than of Δgrx3. Among the mutants, Δgrx3 showed slightly increased sensitivity to menadione or H2O2; Δglr exhibited greater sensitivity to thiol-oxidizing diamide than thiol-reducing 1-chloro-2,4-dinitrobenzene as well as increased sensitivity to the two oxidants. Intriguingly, all the mutants grew slower in a Fe(3+)-inclusive medium perhaps due to elevated transcripts of two Fe(3+) transporter genes. More or fewer phenotypes linked with biocontrol potential were altered in four deletion mutants excluding Δgrx5. All the changes were restored by targeted gene complementation. Overall, Grx3 played more critical role than other Grx homologues in the Glr-dependent redox system of the fungal entomopathogen.

Mitochondrial respiration and redox coupling in articular chondrocytes.

IntroductionChondrocytes rely primarily on glycolysis to meet cellular energy needs, but recent studies implicate impaired mitochondrial function in osteoarthritis (OA) pathogenesis. Our objectives were to investigate the ability of chondrocytes to upregulate mitochondrial respiration when challenged with a nutrient stress and determine the effect on mediators of chondrocyte oxidative homeostasis.MethodsPrimary bovine chondrocytes were isolated and cultured in alginate beads. Mitochondrial respiration was stimulated by culturing cells with galactose-supplemented media for a period of 1 or 5 days. Metabolic flexibility was assessed by measuring metabolite and enzymatic biomarkers of glycolytic and mitochondrial metabolism. Oxidative homeostasis was assessed by measuring (1) cellular glutathione content and redox homeostasis, (2) rates of nitric oxide and superoxide production, and (3) the abundance and activity of cellular anti-oxidant proteins, especially the mitochondrial isoform of superoxide dismutase (SOD2). The regulatory role of hypoxia-inducible factor 2α (HIF-2α) in mediating the metabolic and redox responses was evaluated by chemical stabilization with cobalt chloride (CoCl2).ResultsAfter 5 days of galactose culture, lactate production and lactate dehydrogenase activity were reduced by 92% (P <0.0001) and 28% (P = 0.051), respectively. Conversely, basal oxygen consumption increased 35% (P = 0.042) without increasing mitochondrial content. Glutathione redox homeostasis was unaffected by galactose culture. However, the production of nitric oxide and superoxide and the expression and activity of SOD2 were significantly reduced after 5 days in galactose culture. Nuclear protein expression and gene expression of HIF-2α, a transcription factor for SOD2, were significantly downregulated (more than twofold; P <0.05) with galactose culture. CoCl2-mediated stabilization of HIF-2α during the initial galactose response phase attenuated the reduction in SOD2 (P = 0.028) and increased cell death (P = 0.003).ConclusionsChondrocyte metabolic flexibility promotes cell survival during a nutrient stress by upregulating mitochondrial respiration and reducing the rate of reactive nitrogen and oxygen species production. These changes are coupled to a substantial reduction in the expression and activity of the mitochondrial anti-oxidant SOD2 and its pro-catabolic transcription factor HIF-2α, suggesting that an improved understanding of physiologic triggers of chondrocyte metabolic flexibility may provide new insight into the etiology of OA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0566-9) contains supplementary material, which is available to authorized users.

Antioxidant plasticity and thermal sensitivity in four types of Symbiodinium sp.

Warmer than average summer sea surface temperature is one of the main drivers for coral bleaching, which describes the loss of endosymbiotic dinoflagellates (genus: Symbiodinium) in reef-building corals. Past research has established that oxidative stress in the symbiont plays an important part in the bleaching cascade. Corals hosting different genotypes of Symbiodinium may have varying thermal bleaching thresholds, but changes in the symbiont's antioxidant system that may accompany these differences have received less attention. This study shows that constitutive activity and up-regulation of different parts of the antioxidant network under thermal stress differs between four Symbiodinium types in culture and that thermal susceptibility can be linked to glutathione redox homeostasis. In Symbiodinium B1, C1 and E, declining maximum quantum yield of PSII (Fv /Fm ) and death at 33°C were generally associated with elevated superoxide dismutase (SOD) activity and a more oxidized glutathione pool. Symbiodinium F1 exhibited no decline in Fv /Fm or growth, but showed proportionally larger increases in ascorbate peroxidase (APX) activity and glutathione content (GSx), while maintaining GSx in a reduced state. Depressed growth in Symbiodinium B1 at a sublethal temperature of 29°C was associated with transiently increased APX activity and glutathione pool size, and an overall increase in glutathione reductase (GR) activity. The collapse of GR activity at 33°C, together with increased SOD, APX and glutathione S-transferase activity, contributed to a strong oxidation of the glutathione pool with subsequent death. Integrating responses of multiple components of the antioxidant network highlights the importance of antioxidant plasticity in explaining type-specific temperature responses in Symbiodinium.

Effects of copper overload in P19 neurons: impairment of glutathione redox homeostasis and crosstalk between caspase and calpain protease systems in ROS-induced apoptosis.

Copper, a transition metal with essential biological functions, exerts neurotoxic effects when present in excess. The aim of the present study was to better elucidate cellular and molecular mechanisms of CuSO4 toxicity in differentiated P19 neurons. Exposure to 0.5mM CuSO4 for 24h provoked moderate decrease in viability, accompanied with barely increased generation of reactive oxygen species (ROS) and caspase-3/7 activity. Glutathione (GSH) and ATP contents were depleted, lactate dehydrogenase inactivated, and glyceraldehyde-3-phosphate dehydrogenase overexpressed. In severely damaged neurons exposed to only two times higher concentration, classical caspase-dependent apoptosis was triggered as evidenced by marked caspase-3/7 activation and chromatin condensation. Multifold increase in ROS, together with very pronounced ATP and GSH loss, strongly suggests impairment of redox homeostasis. At higher copper concentration protease calpains were also activated, and neuronal injury was prevented in the presence of calpain inhibitor leupeptin through the mechanism that affects caspase activation. MK-801 and nifedipine, inhibitors of calcium entry, and H-89 and UO126, inhibitors of PKA and ERK signaling respectively, exacerbated neuronal death only in severely damaged neurons, while ROS-scavenger quercetin and calcium chelator BAPTA attenuated toxicity only at lower concentration. In a dose-dependent manner copper also provoked transcriptional changes of genes involved in intracellular signaling and induction of apoptosis (p53, c-fos, Bcl-2 and Bax). The obtained results emphasize differences in triggered neuronal-death processes in a very narrow range of concentrations and give further insight into the molecular mechanisms of copper toxicity with the potential to improve current therapeutic approaches in curing copper-related neurodegenerative diseases.