A Caenorhabditis elegans mutant lacking functional nicotinamide nucleotide transhydrogenase displays increased sensitivity to oxidative stress

Proton-translocating mitochondrial nicotinamide nucleotide transhydrogenase (NNT) was investigated regarding its physiological role in Caenorhabditis elegans. NNT catalyzes the reduction of NADP(+) by NADH driven by the electrochemical proton gradient, Deltap, and is thus a potentially important source of mitochondrial NADPH. Mitochondrial detoxification of reactive oxygen species (ROS) by glutathione-dependent peroxidases depends on NADPH for regeneration of reduced glutathione. Transhydrogenase may therefore be directly involved in the defense against oxidative stress. nnt-1 deletion mutants of C. elegans, nnt-1(sv34), were isolated and shown to grow essentially as wild type under normal laboratory conditions, but with a strongly lowered GSH/GSSG ratio. Under conditions of oxidative stress, caused by the superoxide-generating agent methyl viologen, growth of worms lacking nnt-1 activity was severely impaired. A similar result was obtained by using RNAi. Reintroducing nnt-1 in the nnt-1(sv34) knockout mutant led to a partial rescue of growth under oxidative stress conditions. These results provide evidence for the first time that nnt-1 is important in the defense against mitochondrial oxidative stress.     

Autophagy is required for tolerance of drought and salt stress in plants

Autophagy is a protein degradation process in which cells recycle cytoplasmic contents when subjected to environmental stress conditions or during certain stages of development. Upon the induction of autophagy, a double membrane autophagosome forms around cytoplasmic components and delivers them to the vacuole or lysosome for degradation. In plants, autophagy has been shown previously to be induced during abiotic stresses including nutrient starvation and oxidative stress. In this paper, we demonstrate the induction of autophagy in high salt and osmotic stress conditions, concomitant with the upregulation of expression of an Arabidopsis thaliana autophagy-related gene AtATG18a. Autophagy-defective RNAi-AtATG18a plants are more sensitive to salt and drought conditions than wild-type plants, demonstrating a role for autophagy in the response to these stresses. NADPH oxidase inhibitors block autophagy induction upon nutrient starvation and salt stress, but not during osmotic stress, indicating that autophagy can be activated by NADPH oxidase-dependent or -independent pathways. Together our results indicate that diverse environmental stresses can induce autophagy and that autophagy is regulated by distinct signaling pathways in different conditions.     

NADPH recycling systems in oxidative stressed pea nodules: a key role for the NADP<Superscript>+</Superscript>-dependent isocitrate dehydrogenase

The symbiosis between legumes and rhizobia is characterised by the formation of dinitrogen-fixing root nodules. In natural conditions, nitrogen fixation is strongly impaired by abiotic stresses which generate over-production of reactive oxygen species. Since one of the nodule main antioxidant systems is the ascorbate–glutathione cycle, NADPH recycling that is involved in glutathione reduction is of great relevance under stress conditions. NADPH is mainly produced by glucose 6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) from the oxidative pentose phosphate pathway, and also by NADP⁺-dependent isocitrate dehydrogenase (ICDH; EC 1.1.1.42). In this work, 10 μM paraquat (PQ) was applied to pea roots in order to determine the in vivo relationship between oxidative stress and the activity of the NADPH-generating enzymes in nodules. Whereas G6PDH and 6PGDH activities remained unchanged, a remarkable induction of ICDH gene expression and a dramatic increase of the ICDH activity was observed during the PQ treatment. These results support that ICDH has a key role in NADPH recycling under oxidative stress conditions in pea root nodules.     

Oxidized NADH Oxidase Inhibits Activity of an ATP/NAD Kinase from a Thermophilic Archaeon

NADH oxidases (NOXs) are important enzymes in detoxifying oxidative stress and regenerating oxidized pyridine nucleotides. In the present study, a NOX from Thermococcus kodakarensis KOD1 (NOXtk) was recombinantly expressed in Escherichia coli and purified to homogeneity. NOXtk displayed NADH oxidase activity that was inhibited by oxidization. Under physiological conditions, unoxidized and oxidized NOXtk formed dimers and hexamers, respectively. Mutating the single cysteine residue Cys45 to alanine (NOXtkC45A) decreased NADH oxidase activity without affecting dimerization or hexamerization, suggesting that oligomerization does not occur through disulfide bond formation. Pull-down assay results indicated that an ATP/NAD kinase from T. kodakarensis KOD1 (ANKtk) binds to NOXtk. Use of several assays revealed that ANKtk can only bind to oxidized hexameric NOXtk, through which it inhibits ANKtk activity. Because ANKtk converts NADH to NADPH (an important factor in oxidative stress protection), a model based on in vitro result was proposed in which NOXtk hexamerization under oxic conditions inhibits both NOXtk and ANKtk activities, thereby sensitizing cells to oxidative stress-induced death.     

Reactive oxygen species mediate Na+-induced SOS1 mRNA stability in Arabidopsis

Salt Overly Sensitive 1 (SOS1), a plasma membrane Na⁺/H⁺ antiporter in Arabidopsis, is a salt tolerance determinant crucial for the maintenance of ion homeostasis in saline stress conditions. SOS1 mRNA is unstable at normal growth conditions, but its stability is substantially increased under salt stress and other ionic and dehydration stresses. In addition, H₂O₂ treatment increases the stability of SOS1 mRNA. SOS1 mRNA is inherently unstable and rapidly degraded with a half-life of approximately 10 min. Rapid decay of SOS1 mRNA requires new protein synthesis. Stress-induced SOS1 mRNA stability is mediated by reactive oxygen species (ROS). NADPH oxidase is also involved in the upregulation of SOS1 mRNA stability, presumably through the control of extracellular ROS production. The cis -element required for SOS1 mRNA instability resides in the 500-bp region within the 2.2 kb at the 3' end of the SOS1 mRNA. Furthermore, mutations in the SOS1 gene render sos1 mutants more tolerant to paraquat, a non-selective herbicide causing oxidative stress, indicating that SOS1 plays negative roles in tolerance of oxidative stress. A hypothetical model for the signaling pathway involving SOS1-mediated pH changes, NADPH oxidase activation, apoplastic ROS production and downstream signaling transduction is proposed, and the biological significance of ROS-mediated induction of SOS1 mRNA stability is discussed.     

Impact of small RNA RaoN on nitrosative-oxidative stress resistance and virulence of Salmonella enterica serovar Typhimurium

RaoN is a Salmonella-specific small RNA that is encoded in the cspH-envE intergenic region on Salmonella pathogenicity island-11. We previously reported that RaoN is induced under conditions of acid and oxidative stress combined with nutrient limitation, contributing to the intramacrophage growth of Salmonella enterica serovar Typhimurium. However, the role of RaoN in nitrosative stress response and virulence has not yet been elucidated. Here we show that the raoN mutant strain has increased susceptibility to nitrosative stress by using a nitric oxide generating acidified nitrite. Extending previous research on the role of RaoN in oxidative stress resistance, we found that NADPH oxidase inhibition restores the growth of the raoN mutant in LPS-treated J774A.1 macrophages. Flow cytometry analysis further revealed that the inactivation of raoN leads to an increase in the intracellular level of reactive oxygen species (ROS) in Salmonella-infected macrophages, suggesting that RaoN is involved in the inhibition of NADPH oxidase-mediated ROS production by mechanisms not yet resolved. Moreover, we evaluated the effect of raoN mutation on the virulence in murine systemic infection and determined that the raoN mutant is less virulent than the wild-type strain following oral inoculation. In conclusion, small regulatory RNA RaoN controls nitrosative-oxidative stress resistance and is required for virulence of Salmonella in mice.     

Reactive oxygen species mediate Na+-induced SOS1 mRNA stability in Arabidopsis.

Salt Overly Sensitive 1 (SOS1), a plasma membrane Na+/H+ antiporter in Arabidopsis, is a salt tolerance determinant crucial for the maintenance of ion homeostasis in saline stress conditions. SOS1 mRNA is unstable at normal growth conditions, but its stability is substantially increased under salt stress and other ionic and dehydration stresses. In addition, H2O2 treatment increases the stability of SOS1 mRNA. SOS1 mRNA is inherently unstable and rapidly degraded with a half-life of approximately 10 min. Rapid decay of SOS1 mRNA requires new protein synthesis. Stress-induced SOS1 mRNA stability is mediated by reactive oxygen species (ROS). NADPH oxidase is also involved in the upregulation of SOS1 mRNA stability, presumably through the control of extracellular ROS production. The cis-element required for SOS1 mRNA instability resides in the 500-bp region within the 2.2 kb at the 3' end of the SOS1 mRNA. Furthermore, mutations in the SOS1 gene render sos1 mutants more tolerant to paraquat, a non-selective herbicide causing oxidative stress, indicating that SOS1 plays negative roles in tolerance of oxidative stress. A hypothetical model for the signaling pathway involving SOS1-mediated pH changes, NADPH oxidase activation, apoplastic ROS production and downstream signaling transduction is proposed, and the biological significance of ROS-mediated induction of SOS1 mRNA stability is discussed.     

Enzymatic and physiological study of d-xylose metabolism by Candida shehatae

Summary Candida shehatae carbon metabolic pathways were correlated with fermentative activity under different growth conditions. Reduced nicotine adenine dinucleotide (NADPH) is the coenzyme preferred for xylose reductase by C. shehatae under in vitro anoxic cell culture conditions. To prevent a redox imbalance derived from intracellular accumulation of NADH in the second enzymatic step of xylose metabolism, the operation of phosphoketolase via in addition the classic pentose phosphate pathway essential for NADH dissimilation is suggested. Variation in cultivation conditions showed a different NADH/NADPH ratio coupled to xylose reductase activity. The existence of two xylose reductases is discussed. Like ethanol, xylitol accumulates only under oxygen-limited or anaerobic conditions. Xylitol accumulaiton under unaerobic conditions was higher when using respiring cells than respirofermentative cells. This fact suggests that cells pregrown under oxygen limitation are better adapted to starting alcoholic fermentation than cells previously grown under aerobic conditions.Offprint requests to: M. T. Amaral-Collaço     

<Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>, a model system for functional validation of abiotic stress responsive genes

Stress tolerance is a multigenic character and there are many stress responsive genes, which are stress specific. Although many of these have been cloned, their functional significance remains fragmentary. Hence it is important to identify the relevant stress genes involved in altering the metabolism for adaptation. Overexpression is one of the several approaches and Chlamydomonas is a suitable system to study the functional relevance of stress genes. Stress responses can only be assessed on prior exposure to sublethal induction stress. In this study the acclimation response of Chlamydomonas was assessed for different abiotic stresses using physiological screens like chlorophyll stability, membrane damage, cell viability, accumulation of free radicals, survival and recovery growth. We demonstrate that Chlamydomonas responds to diverse stresses and is a potential system to study the relevance of stress genes. The relevance of choline oxidase A (codA), a key enzyme in glycinebetaine biosynthesis, was examined by developing transformants expressing codA gene from Arthrobacter globiformis. Southern positive transformants showed enhanced accumulation of glycinebetaine. The transformants also showed enhanced growth under salinity, high light coupled with methylviologen-induced oxidative stress, high temperature and cold stress. However the transgenics were not tolerant to PEG-mediated simulated osmotic stress, LiCl, menadione and UV stress. Increased cell survival and decreased chlorophyll degradation in transformants under acclimated conditions further confirmed the relevance of codA in imparting stress tolerance. Our results indicated that the relevance of stress responsive genes can be efficiently validated for diverse abiotic stresses using Chlamydomonas system. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. R. Hema and M. Senthil-Kumar contributed equally.     

Rac1 Is a Possible Link Between Obesity and Oxidative Stress in Chinese Overweight Adolescents

Enhanced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in the monocytes occurred in metabolic syndrome, hypertension, diabetes and obese patients in adults. However, whether NADPH oxidase is involved in the oxidative stress of overweight adolescents without comorbidities is still unclear. This study aimed to identify whether and how NADPH oxidase plays a crucial role in overweight adolescents. The study was performed in 93 overweight adolescents and 31 normal weight controls. Moreover, 87 overweight adolescents were enrolled in weight‐loss program. Demographics characteristics, anthropometrics, composition and clinical characteristics were analyzed. Oxidative stress indexes including the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in plasma and the expression of NADPH oxidase in the monocytes were examined. Overweight adolescents showed a higher oxidative stress state, as indicated by decreased SOD activity and elevated MDA level (P < 0.01). Furthermore, increased NADPH oxidase activity in the monocytes was accompanied by Rac1 upregulation. A significant positive bivariate correlation was found between Rac1 expression and MDA (r = 0.289). There also was a significant positive bivariate correlation between Rac1 expression and obesity‐related indexes including BMI (r = 0.227) and percentage of trunk fat (r = 0.233). Data from weight‐loss program reinforced the results. Partial correlation analysis indicated that obesity‐induced oxidative stress and Rac1 expression is a consequence of aberrant glucose‐lipid metabolism in overweight adolescents. In conclusion, we provided novel data showing that NADPH oxidase in the monocytes was highly activated by enhancing Rac1 expression in Chinese overweight adolescents and Rac1 may act as a link between obesity and oxidative stress in overweight adolescents.     

Control of the NADPH supply and GSH recycling for oxidative stress management in hepatoma and liver mitochondria

To unveil what controls mitochondrial ROS detoxification, the NADPH supply and GSH/GSSG recycling for oxidative stress management were analyzed in cancer and non-cancer mitochondria. Therefore, proteomic and kinetomic analyses were carried out of the mitochondrial (i) NADPH producing and (ii) GSH/GSSG recycling enzymes associated to oxidative stress management. The protein contents of the eight enzymes analyzed were similar or even higher in AS-30D rat hepatoma mitochondria (HepM) than in rat liver (RLM) and rat heart (RHM) mitochondria, suggesting that the NADPH/GSH/ROS pathway was fully functional in cancer mitochondria.The Vmax values of IDH-2 were much greater than those of GDH, TH and ME, suggesting that IDH-2 is the predominant NADPH producer in the three mitochondrial types; in fact, the GDH reverse reaction was favored. The Vmax values of GR and GPx were lower in HepM than in RLM, suggesting that the oxidative stress management is compromised in cancer mitochondria. The Km values of IDH-2, GR and GPx were all similar among the different mitochondrial types.Kinetic modeling revealed that the oxidative stress management was mainly controlled by GR, GPx and IDH. Modeling and experimentation also revealed that, due to their higher IDH-2 activity and lower GPx activity presumably by acetylation, HepM (i) showed higher steady-state NADPH levels; (ii) required greater peroxide concentrations to achieve reliable steady-state fluxes and metabolite concentration; and (iii) endured higher peroxide concentrations without collapsing their GSH/GSSG ratios. Then, to specifically prompt lower GSH/GSSG ratios under oxidative stress thus compromising cancer mitochondria functioning, GPx should be re-activated.     

Cross talk between mitochondria and NADPH oxidases

Reactive oxygen species (ROS) play an important role in physiological and pathological processes. In recent years, a feed-forward regulation of the ROS sources has been reported. The interactions between the main cellular sources of ROS, such as mitochondria and NADPH oxidases, however, remain obscure. This work summarizes the latest findings on the role of cross talk between mitochondria and NADPH oxidases in pathophysiological processes. Mitochondria have the highest levels of antioxidants in the cell and play an important role in the maintenance of cellular redox status, thereby acting as an ROS and redox sink and limiting NADPH oxidase activity. Mitochondria, however, are not only a target for ROS produced by NADPH oxidase but also a significant source of ROS, which under certain conditions may stimulate NADPH oxidases. This cross talk between mitochondria and NADPH oxidases, therefore, may represent a feed-forward vicious cycle of ROS production, which can be pharmacologically targeted under conditions of oxidative stress. It has been demonstrated that mitochondria-targeted antioxidants break this vicious cycle, inhibiting ROS production by mitochondria and reducing NADPH oxidase activity. This may provide a novel strategy for treatment of many pathological conditions including aging, atherosclerosis, diabetes, hypertension, and degenerative neurological disorders in which mitochondrial oxidative stress seems to play a role. It is conceivable that the use of mitochondria-targeted treatments would be effective in these conditions.     

Sugar metabolism, redox balance and oxidative stress response in the respiratory yeast Kluyveromyces lactis

A lot of studies have been carried out on Saccharomyces cerevisiae, an yeast with a predominant fermentative metabolism under aerobic conditions, which allows exploring the complex response induced by oxidative stress. S. cerevisiae is considered a eukaryote model for these studies. We propose Kluyveromyces lactis as a good alternative model to analyse variants in the oxidative stress response, since the respiratory metabolism in this yeast is predominant under aerobic conditions and it shows other important differences with S. cerevisiae in catabolic repression and carbohydrate utilization. The knowledge of oxidative stress response in K. lactis is still a developing field. In this article, we summarize the state of the art derived from experimental approaches and we provide a global vision on the characteristics of the putative K. lactis components of the oxidative stress response pathway, inferred from their sequence homology with the S. cerevisiae counterparts. Since K. lactis is also a well-established alternative host for industrial production of native enzymes and heterologous proteins, relevant differences in the oxidative stress response pathway and their potential in biotechnological uses of this yeast are also reviewed.     

The dehydrogenase-mediated recycling of NADPH is a key antioxidant system against salt-induced oxidative stress in olive plants

NADPH is an important molecule in the redox balance of the cell. In this paper, using olive tissue cultures as a model of the function of the NADPH-generating dehydrogenases in the mechanism of oxidative stress induced by severe salinity conditions was studied. When olive (Olea europaea) plants were grown with 200 mM NaCl, a 40% reduction in leaf fresh weight was produced. The content of non-enzymatic antioxidants such as ascorbate and glutathione was diminished between 20% to 39%, whereas the H2O2 content was increased threefold. In contrast, the analysis of the activity and protein contents of the main antioxidative enzymes showed a significant increase of catalase, superoxide dismutase and glutathione reductase. Overall, these changes strongly suggests that NaCl induces oxidative stress in olive plants. On the other hand, while the content of glucose-6-phosphate was increased almost eightfold in leaves of plants grown under salt stress, the content of NAD(P)H (reduced and oxided forms) did not show significant variations. Under salt stress conditions, the activity and protein contents of the main NADPH-recycling enzymes, glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (ICDH), malic enzyme (ME) and ferrodoxin-NADP reductase (FNR) showed an enhancement of 30-50%. In leaves of olive plants grown with 200 mM NaCl, analysis of G6PDH by immunocytochemistry and confocal laser scanning microscopy showed a general increase of this protein in epidermis, palisade and spongy mesophyll cells. These results indicate that in olive plants, salinity causes reactive oxygen species (ROS)-mediated oxidative stress, and plants respond to this situation by inducing different antioxidative enzymes, especially the NADPH-producing dehydrogenases in order to recycle NADPH necessary for the protection against oxidative damages. These NADP-dehydrogenases appear to be key antioxidative enzymes in olive plants under salt stress conditions.