Glutathione and cysteine enhance porcine preimplantation embryo development in vitro after intracytoplasmic sperm injection

Because intracytoplasmic sperm injection (ICSI) had been introduced to animal science, not only reproductive biology of domestic animals, but also medicine to treat infertility has been developed. This assisted reproductive technology is beneficial for generating transgenic animals, especially pigs, because polyspermy is the greatest hurdle in porcine IVF when researchers make highly qualified preimplantation embryos. However, ICSI-derived embryos expressed high level of reactive oxygen species (ROS), which are known to cause serious dysfunction during preimplantation development. The objective of this study was to investigate the developmental competence, ROS level, and apoptosis index when glutathione (GSH) or cysteine was supplemented into the in vitro culture medium for ICSI-derived porcine embryos. First, we evaluated the effect of different concentrations of GSH or cysteine on developmental ability of porcine ICSI-derived embryos. The cleavage rate (79.6%) and the blastocyst formation rate (20.9%) were significantly improved in culture medium supplemented with 1 mmol/L GSH compared with other concentrations or no supplementation. Also, 1.71 mmol/L cysteine showed a significantly higher proportion of cleavage (80.7%) and blastocyst formation (22.5%) than other cysteine-supplemented groups. Next, we confirmed that intracellular ROS level was significantly reduced in the group of blastocysts cultured with GSH or cysteine after ICSI compared with the no supplementation group. Finally, we found that terminal uridine nick-end labeling index, fragmentation, and total apoptosis were significantly decreased and the total cell number was significantly increased in blastocysts when ICSI-derived embryos were cultured with supplementation of 1.71 mmol/L cysteine or 1 mmol/L GSH. Taken together, these results strongly indicate that GSH or cysteine can improve the developmental competence of porcine ICSI-derived embryos by reducing intracellular ROS level and the apoptosis index.     

Immunomodulation of macrophages by methylglyoxal conjugated with chitosan nanoparticles against Sarcoma-180 tumor in mice

Methylglyoxal (MG), the potent anticancer agent has been conjugated to a nontoxic, biocompatible polymer, chitosan, to protect it from in vivo enzymatic degradation. This polymeric complex, ‘Nano-MG’ shows remarkable antitumor property and elicits macrophage-mediated immunity in tumor bearing mice on intravenous (0.4 mg/kg body wt/day) treatment more efficiently than MG (20 mg/kg body wt/day). These activated macrophages appear more in numbers in the peritoneum and produce more superoxide and nitrite. Moreover, immunomodulatory cytokines and surface receptors of these macrophages like iNOS, IFN-γ, TNF-α, IL-1β, IL-6, M-CSF, TLR-4 and TLR-9 also exhibit marked up-regulation in Sarcoma-180 tumor bearing mice after Nano-MG treatment compared to untreated tumor bearing counterpart. Hence, Nano-MG acts as an immunostimulant in tumor bearing mice to combat cancer at conspicuously lower dose, probably due to its longer circulation time in blood.     

综述: 细胞氧化还原调控与衰老

利用酵母、线虫、果蝇、小鼠等模式生物进行的研究表明,细胞的衰老过程与氧化还原紧密相关.伴随衰老,细胞内GSSG水平升高,GSH、NADPH等水平降低,而氧化还原状态变化将直接影响蛋白质的功能,特别是氧化还原敏感的含巯基蛋白质的功能,从而影响细胞信号转导和细胞命运.氧化还原失衡可能是衰老发生的重要因素.本综述将从氧化还原平衡与衰老、氧化还原调控与信号转导及衰老、氧化损伤与衰老等方面阐述细胞氧化还原调控与衰老研究的最新进展,提出并探讨氧化还原平衡的维持、氧化还原平衡的系统调控及氧化还原调控的个体化等延缓衰老及健康衰老的新策略.     

Sleep deprivation under sustained hypoxia protects against oxidative stress

We previously showed that total sleep deprivation increased antioxidant responses in several rat brain regions. We also reported that chronic hypoxia enhanced antioxidant responses and increased oxidative stress in rat cerebellum and pons, relative to normoxic conditions. In the current study, we examined the interaction between these two parameters (sleep and hypoxia). We exposed rats to total sleep deprivation under sustained hypoxia (SDSH) and compared changes in antioxidant responses and oxidative stress markers in the neocortex, hippocampus, brainstem, and cerebellum to those in control animals left undisturbed under either sustained hypoxia (UCSH) or normoxia (UCN). We measured changes in total nitrite levels as an indicator of nitric oxide (NO) production, superoxide dismutase (SOD) activity and total glutathione (GSHt) levels as markers of antioxidant responses, and levels of thiobarbituric acid-reactive substances (TBARS) and protein carbonyls as signs of lipid and protein oxidation products, respectively. We found that acute (6h) SDSH increased NO production in the hippocampus and increased GSHt levels in the neocortex, brainstem, and cerebellum while decreasing hippocampal lipid oxidation. Additionally, we observed increased hexokinase activity in the neocortex of SDSH rats compared to UCSH rats, suggesting that elevated glucose metabolism may be one potential source of the enhanced free radicals produced in this brain region. We conclude that short-term insomnia under hypoxia may serve as an adaptive response to prevent oxidative stress.     

Characterizing the effect of nitrosative stress in Saccharomyces cerevisiae

Nitrosative stress has various pathophysiological implications. We here present a detailed characterization on the effect of nitrosative stress in Saccharomyces cerevisiae wild-type (Y190) and its isogenic flavohemoglobin mutant (Δyhb1) strain grown in presence of non fermentable carbon source. On addition of sub-toxic dose of nitrosating agent both the strains showed microbiostatic effect. Cellular respiration was found to be significantly affected in both the strains in presence sodium nitroprusside. Although there was no alteration in mitochondrial permeability potential changes and reactive oxygen species production in both the strains but the cellular redox status is differentially regulated in Δyhb1 strain both in cytosol and in mitochondria indicating cellular glutathione is the major player in absence of flavohemoglobin. We also found important role(s) of various redox active enzymes like glutathione reductase and catalase in protection against nitrosative stress. This is the first report of its kind where the effect of nitrosative stress has been evaluated in S. cerevisiae cytosol as well as in mitochondria under respiratory proficient conditions.     

Ecdysone induction of MsrA protects against oxidative stress in Drosophila

The methionine sulfoxide reductases MsrA and MsrB reduce Met(O) to Met in epimer-specific fashion. In Drosophila, the major ecdysone induced protein is MsrA, which is regulated by the EcR-USP complex. We tested Kc cells for induction of MsrA, MsrB, EcR, and CAT by ecdysone and found that MsrA and the EcR were induced by ecdysone, but MsrB and CAT were not. When we tested for resistance to 20mM H2O2 toxicity, viability of Kc cells was reduced 3-fold. Pretreatment with 0.2 microM ecdysone for 48 h prior to exposure to H2O2, increased viability to 77% of controls. The EcR-deficient L57-3-11 knockout line was not responsive to ecdysone, and H2O2 resistance of both control and ecdysone-treated L57-3-11 cells was similar to that of the ecdysone-untreated Kc cells. These results show that hormonal regulation of MsrA is implicated in conferring protection against oxidative stress in the Drosophila model.     

Unravelling how plants benefit from ROS and NO reactions,while resisting oxidative stress

Background and Aims Reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as nitric oxide (NO), play crucial roles in the signal transduction pathways that regulate plant growth, development and defence responses, providing a nexus of reduction/oxidation (redox) control that impacts on nearly every aspect of plant biology. Here we summarize current knowledge and concepts that lay the foundations of a new vision for ROS/RNS functions – particularly through signalling hubs – for the next decade.Scope Plants have mastered the art of redox control using ROS and RNS as secondary messengers to regulate a diverse range of protein functions through redox-based, post-translational modifications that act as regulators of molecular master-switches. Much current focus concerns the impact of this regulation on local and systemic signalling pathways, as well as understanding how such reactive molecules can be effectively used in the control of plant growth and stress responses.Conclusions The spectre of oxidative stress still overshadows much of our current philosophy and understanding of ROS and RNS functions. While many questions remain to be addressed – for example regarding inter-organellar regulation and communication, the control of hypoxia and how ROS/RNS signalling is used in plant cells, not only to trigger acclimation responses but also to create molecular memories of stress – it is clear that ROS and RNS function as vital signals of living cells.     

Enhanced resistance to peroxide stress in Escherichia coli grown outside their niche temperatures

Extended exposure of Escherichia coli to temperatures above and below their growth optimum led to significant changes in oxidant production and antioxidant defense. At 20 °C an increase in the intracellular H₂O₂ concentration and oxidized glutathione (GSSG) level was observed against a background of low levels of reduced glutathione (GSH) and decreased catalase and glutathione reductase (GOR) activities. The intracellular H₂O₂ and GSSG concentrations had minimal values at 30 and 37 °C, but rose again at 42 °C, suggesting that oxidative processes were intensified at high temperatures. An increase in temperature from 20 to 42 °C led to an elevation in the oxygen respiration rate and superoxide production; a 5-fold increase in the intracellular GSH concentration and in the GSH:GSSG ratio occurred simultaneously. Catalase HPI and GOR activities were elevated 4.4- and 1.5-fold, respectively. Prolonged exposure to sublethal temperatures facilitated an adaptation to subsequent oxidative stress produced by the addition of H₂O₂.     

Chitin oligosaccharides inhibit oxidative stress in live cells

The aim of this research is to identify the cellular antioxidant effects of chitin oligosaccharides (NA-COS; M_w 229.21–593.12 Da) produced by acidic hydrolysis of crab chitin. The inhibitory effect of NA-COS on myeloperoxidase (MPO) activity in human myeloid cells (HL-60) and oxidation of DNA and protein in mouse macrophages (Raw 264.7) were identified. Furthermore, their direct radical scavenging effect by 2′,7′-dichlorofluorescein (DCF) intensity and intracellular glutathione (GSH) level were significantly increased in a time dependent manner, respectively. These results suggest that NA-COS act as a potent antioxidant in live cells.     

Increased susceptibility of MER5 (peroxiredoxin III) knockout mice to LPS-induced oxidative stress

MER5 (also called peroxiredoxin III, PrxIII) is a member of peroxiredoxin family that has antioxidant activity. The present study was performed to investigate its in vivo function using MER5 knockout mice. MER5 knockout mice were born in normal frequency and could grow to maturity, but we found that intracellular ROS levels are significantly higher in the macrophages of the knockout mice. We examined roles of MER5 function for the oxidative stress responses by intratracheal inoculation of lipopolysaccharide (LPS) to the mice. Lung inflammation such as inflammatory cell infiltration and airway wall thickening was more severely detected in the knockout mice. At the same time, oxidative damage on DNA and proteins was more strongly detected in lung tissues of the knockout mice, including 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation and protein carbonylation. The degrees of lung inflammation and oxidative damage were positively related with LPS doses. Our results indicate that MER5 knockout mice accumulated higher intracellular ROS levels, which cause LPS-induced lung injury more severely, and thus, suggested that MER5 acts as an important scavenger of reactive oxygen species (ROS) under oxidative stress.     

Cytochrome bd oxidase and bacterial tolerance to oxidative and nitrosative stress

Cytochrome bd is a prokaryotic respiratory quinol:O₂ oxidoreductase, phylogenetically unrelated to the extensively studied heme–copper oxidases (HCOs). The enzyme contributes to energy conservation by generating a proton motive force, though working with a lower energetic efficiency as compared to HCOs. Relevant to patho-physiology, members of the bd-family were shown to promote virulence in some pathogenic bacteria, which makes these enzymes of interest also as potential drug targets. Beyond its role in cell bioenergetics, cytochrome bd accomplishes several additional physiological functions, being apparently implicated in the response of the bacterial cell to a number of stress conditions. Compelling experimental evidence suggests that the enzyme enhances bacterial tolerance to oxidative and nitrosative stress conditions, owing to its unusually high nitric oxide (NO) dissociation rate and a notable catalase activity; the latter has been recently documented in one of the two bd-type oxidases of Escherichia coli. Current knowledge on cytochrome bd and its reactivity with O₂, NO and H₂O₂ is summarized in this review in the light of the hypothesis that the preferential (over HCOs) expression of cytochrome bd in pathogenic bacteria may represent a strategy to evade the host immune attack based on production of NO and reactive oxygen species (ROS). This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

活性氮、活性氧及植物激素在种子休眠解除中的作用及相互关系研究进展

休眠是植物种子对环境变化的适应机制,其机理至今未完全清楚阐明。前期对种子休眠机制的研究主要集中在激素调节上,近期的研究结果表明,一氧化氮（nitric oxide,NO）参与打破种子的休眠,并与其所引起的种子中活性氧的变化有关。本文简要综述活性氮（reactive nitrogen species,RNS）、活性氧（reactive oxygen species,R0s）和植物激素在种子休眠解除中的作用及相互关系研究进展。     

Insulin resistance and diabetes in hyperthyroidism: a possible role for oxygen and nitrogen reactive species

In addition to insulin, glycemic control involves thyroid hormones. However, an excess of thyroid hormone can disturb the blood glucose equilibrium, leading to alterations of carbohydrate metabolism and, eventually, diabetes. Indeed, experimental and clinical hyperthyroidism is often accompanied by abnormal glucose tolerance. A common characteristic of hyperthyroidism and type 2 diabetes is the altered mitochondrial efficiency caused by the enhanced production of reactive oxygen and nitrogen species. It is known that an excess of thyroid hormone leads to increased oxidant production and mitochondrial oxidative damage. It can be hypothesised that these species represent the link between hyperthyroidism and development of insulin resistance and diabetes, even though direct evidence of this relationship is lacking. In this review, we examine the literature concerning the effects of insulin and thyroid hormones on glucose metabolism and discuss alterations of glucose metabolism in hyperthyroid conditions and the cellular and molecular mechanisms that may underline them.     

Proteome-wide profiling of carbonylated proteins and carbonylation sites in HeLa cells under mild oxidative stress conditions

A number of oxidative protein modifications have been well characterized during the past decade. Presumably, reversible oxidative posttranslational modifications (PTMs) play a significant role in redox signaling pathways, whereas irreversible modifications including reactive protein carbonyl groups are harmful, as their levels are typically increased during aging and in certain diseases. Despite compelling evidence linking protein carbonylation to numerous disorders, the underlying molecular mechanisms at the proteome remain to be identified. Recent advancements in analysis of PTMs by mass spectrometry provided new insights into the mechanisms of protein carbonylation, such as protein susceptibility and exact modification sites, but only for a limited number of proteins. Here we report the first proteome-wide study of carbonylated proteins including modification sites in HeLa cells for mild oxidative stress conditions. The analysis relied on our recent strategy utilizing mass spectrometry-based enrichment of carbonylated peptides after DNPH derivatization. Thus a total of 210 carbonylated proteins containing 643 carbonylation sites were consistently identified in three replicates. Most carbonylation sites (284, 44.2%) resulted from oxidation of lysine residues (aminoadipic semialdehyde). Additionally, 121 arginine (18.8%), 121 threonine (18.8%), and 117 proline residues (18.2%) were oxidized to reactive carbonyls. The sequence motifs were significantly enriched for lysine and arginine residues near carbonylation sites (±10 residues). Gene Ontology analysis revealed that 80% of the carbonylated proteins originated from organelles, 50% enrichment of which was demonstrated for the nucleus. Moreover, functional interactions between carbonylated proteins of kinetochore/spindle machinery and centrosome organization were significantly enriched. One-third of the 210 carbonylated proteins identified here are regulated during apoptosis.     

LL202 ameliorates colitis against oxidative stress of macrophage by activation of the Nrf2/HO-1 pathway

LL202, a newly synthesized flavonoid derivative, has been confirmed to inhibit the mitogen-activated protein kinase pathway and activation protein-1 activation in monocytes; however, the anti-inflammatory mechanism has not been clearly studied. Uncontrolled overproduction of reactive oxygen species (ROS) has involved in oxidative damage of inflammatory bowel disease. In this study, we investigated that LL202 reduced lipopolysaccharide (LPS)-induced ROS production and malondialdehyde levels and increased superoxide dismutase, glutathione, and total antioxidant capacity in RAW264.7 cells. Mechanically, LL202 could upregulate heme oxygenase-1 (HO-1) via promoting nuclear translocation of nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) to regulate LPS-induced oxidative stress in macrophages. In vivo, we validated the role of LL202 in dextran sulfate sodium- and TNBS-induced colitis models, respectively. The results showed that LL202 decreased the proinflammatory cytokine expression and regulated colonic oxidative stress by activating the Nrf2/HO-1 pathway. In conclusion, our study showed that LL202 exerts an anti-inflammatory effect by enhancing the antioxidant capacity of the Nrf2/HO-1 pathway to macrophages.     

Inonotus obliquus protects against oxidative stress-induced apoptosis and premature senescence

In this study, we investigated the cytoprotective effects of Inonotus obliquus against oxidative stress-induced apoptosis and premature senescence. Pretreatment with I. obliquus scavenged intracellular ROS and prevented lipid peroxidation in hydrogen peroxide-treated human fibroblasts. As a result, I. obliquus exerted protective effects against hydrogen peroxide-induced apoptosis and premature senescence in human fibroblasts. In addition, I. obliquus suppressed UV-induced morphologic skin changes, such as skin thickening and wrinkle formation, in hairless mice in vivo and increased collagen synthesis through inhibition of MMP-1 and MMP-9 activities in hydrogen peroxide-treated human fibroblasts. Taken together, these results demonstrate that I. obliquus can prevent the aging process by attenuating oxidative stress in a model of stress-induced premature senescence.     

The complex interplay of iron metabolism,reactive oxygen species,and reactive nitrogen species: Insights into the potential of various iron therapies to induce oxidative and nitrosative stress

Production of minute concentrations of superoxide (O₂⁻) and nitrogen monoxide (nitric oxide, NO) plays important roles in several aspects of cellular signaling and metabolic regulation. However, in an inflammatory environment, the concentrations of these radicals can drastically increase and the antioxidant defenses may become overwhelmed. Thus, biological damage may occur owing to redox imbalance—a condition called oxidative and/or nitrosative stress. A complex interplay exists between iron metabolism, O₂⁻, hydrogen peroxide (H₂O₂), and NO. Iron is involved in both the formation and the scavenging of these species. Iron deficiency (anemia) (ID(A)) is associated with oxidative stress, but its role in the induction of nitrosative stress is largely unclear. Moreover, oral as well as intravenous (iv) iron preparations used for the treatment of ID(A) may also induce oxidative and/or nitrosative stress. Oral administration of ferrous salts may lead to high transferrin saturation levels and, thus, formation of non-transferrin-bound iron, a potentially toxic form of iron with a propensity to induce oxidative stress. One of the factors that determine the likelihood of oxidative and nitrosative stress induced upon administration of an iv iron complex is the amount of labile (or weakly-bound) iron present in the complex. Stable dextran-based iron complexes used for iv therapy, although they contain only negligible amounts of labile iron, can induce oxidative and/or nitrosative stress through so far unknown mechanisms. In this review, after summarizing the main features of iron metabolism and its complex interplay with O₂⁻, H₂O₂, NO, and other more reactive compounds derived from these species, the potential of various iron therapies to induce oxidative and nitrosative stress is discussed and possible underlying mechanisms are proposed. Understanding the mechanisms, by which various iron formulations may induce oxidative and nitrosative stress, will help us develop better tolerated and more efficient therapies for various dysfunctions of iron metabolism.     

Paraquat-induced oxidative stress response during amphibian early embryonic development

In addition to the endogenous production of reactive oxygen species (ROS) as a result of normal development, amphibian external development often forces embryos to deal with oxidative stress-producing agents present in the environment. Embryos should therefore develop protective systems to reduce ROS toxicity and achieve successful development. The present work was aimed to characterize the effects produced by the widespread-used ROS-generator pesticide Paraquat during early embryonic development in the toad Chaunus arenarum, as well as to get insights into the defense response elicited by amphibian embryos. The approach consisted in generating a sharp and brief oxidative stress condition early during embryonic development to stimulate the cellular mechanisms involved in ROS-antioxidant response. Results revealed that Paraquat-treatment reduced the ability of embryos to develop normally, leading to arrests of development and severe malformations such as tail abnormalities, abdominal edema, reduced head development and curved dorsal structures. Although Paraquat effects were morphologically evident from gastrula stage on, alterations such as chromatin condensation were observed even at blastula stage by histological examinations. Regarding detoxifying enzymes, a significant induction of Mn-superoxide dismutase activity was detected at stages beyond gastrula in embryos surviving Paraquat treatment, suggesting a major role of this enzyme in the antioxidant response during early embryonic development.