N-acetylcysteine effects on a murine model of chronic critical limb ischemia

During chronic limb ischemia, oxidative damage and inflammation are described. Besides oxidative damage, the decrease of tissue oxygen levels is followed by several adaptive responses. The purpose of this study was to determine whether supplementation with N-acetylcysteine (NAC) is effective in an animal model of chronic limb ischemia. Chronic limb ischemia was induced and animals were treated once a day for 30 consecutive days with NAC (30 mg/kg). After this time clinical scores were recorded and soleus muscle was isolated and lactate levels, oxidative damage and inflammatory parameters were determined. In addition, several mechanisms associated with hypoxia adaptation were measured (vascular endothelial growth factor - VEGF and hypoxia inducible factor - HIF levels, ex vivo oxygen consumption, markers of autophagy/mitophagy, and mitochondrial biogenesis). The adaptation to chronic ischemia in this model included an increase in muscle VEGF and HIF levels, and NAC was able to decrease VEGF, but not HIF levels. In addition, ex vivo oxygen consumption under hypoxia was increased in muscle from ischemic animals, and NAC was able to decrease this parameter. This effect was not mediated by a direct effect of NAC on oxygen consumption. Ischemia was followed by a significant increase in muscle myeloperoxidase activity, as well as interleukin-6 and thiobarbituric acid reactive substances species levels. Supplementation with NAC was able to attenuate inflammatory and oxidative damage parameters, and improve clinical scores. In conclusion, NAC treatment decreases oxidative damage and inflammation, and modulates oxygen consumption under hypoxic conditions in a model of chronic limb ischemia.     

Free radicals and anticancer drug resistance: Oxygen free radicals in the mechanisms of drug cytotoxicity and resistance by certain tumors

Certain anticancer agents form free radical intermediates during enzymatic activation. Recent studies have indicated that free radicals generated from adriamycin and mitomycin C may play a critical role in their toxicity to human tumor cells. Furthermore, it is becoming increasingly apparent that reduced drug activation and or enhanced detoxification of reactive oxygen species may be related to the resistance to these anticancer agents by certain tumor cell lines. The purposes of this review are to summarize the evidence pointing toward the significance of free radicals formation in drug toxicity and to evaluate the role of decreased free radical formation and enhanced free radical scavenging and detoxification in the development of anticancer drug resistance by a spectrum of tumor cell types. Studies failing to support the participation of oxyradicals in the cytotoxicity and resistance of adriamycin are also discussed.     

Purification and characterization of a Cys-Gly hydrolase from the gastropod mollusk,Patella caerulea

A magnesium-dependent cysteinyl-glycine hydrolyzing enzyme from the gastropod mollusk Patella caerulea was purified to electrophoretic homogeneity through a simple and rapid purification protocol. The molecular masses of the native protein and the subunit suggest that the enzyme has a homohexameric structure. Structural data in combination with kinetic parameters determined with Cys-Gly and compared with Leu-Gly as a substrate, indicate that the purified enzyme is a member of the peptidase family M17. The finding that an enzyme of the peptidase family M17 is responsible also in mollusks for the breakdown of Cys-Gly confirms the important role of this peptidase family in the glutathione metabolism.     

Glutathione levels during thermal induction of the yeast-to-mycelial transition in Candida albicans

Glutathione (GSH) levels were directly monitored by reverse phase HPLC during the thermal yeast-to-mycelial induction of Candida albicans. The GSH levels decreased approximately 100-fold within 120 min which corresponded to the time of maximal yeast-to-mold conversion. The yeast to mold conversion was inhibited by 1-p-chlorophenyl-4,4-dimethyl-5-diethylamino-1-penten-3-one (CDDP), a thiol-specific alkylator, which prevented the decline in GSH levels. These results are discussed with respect to the potential involvement of intracellular GSH levels in regulation of the yeast-to-mold dimorphism in Candida albicans.     

H<Subscript>2</Subscript>O<Subscript>2</Subscript> production and antioxidant responses in seeds and early seedlings of two different rice varieties exposed to aluminum

The effect of Al stress on H₂O₂ production of rice (Oryza sativa L.) seedlings and difference in responses of antioxidant enzymes between Al-tolerant variety (Azucena) and Al-sensitive rice one (IR 64) were investigated. Aluminum-induced H₂O₂ production and malondialdehyde (MDA) content were more pronounced for IR 64 than for Azucena. In the presence of 2 mM Al, addition of 10 mM imidazole (inhibitor of NADPH oxidase) and 1 mM azide (inhibitor of peroxidase) significantly decreased H₂O₂ production by 16% and 43% for Azucena, and 21% and 68% for IR 64, respectively. Under Al treatment, the Al-tolerant variety Azucena had significantly higher activities of catalase, ascorbate peroxidase, dehydroascorbate reducase, glutathione peroxidase and glutathione reductase, and higher concentrations of reduced glutathione than the Al-sensitive one IR 64. Treatment with buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly increased H₂O₂ production in both varieties in the presence and absence of Al. In contrast, the treatment with GSH significantly decreased the production of H₂O₂ induced by Al stress. Results suggest that GSH may play an important role in scavenging H₂O₂ caused by Al stress.     

Methods for Analysis of Protein Glutathionylation and their Application to Photosynthetic Organisms

Protein S-glutathionylation, the reversible formation of a mixed-disulfide between glutathione and protein thiols, is involved in protection of protein cysteines from irreversible oxidation, but also in protein redox regulation. Recent studies have implicated S-glutathionylation as a cellular response to oxidative/nitrosative stress, likely playing an important role in signaling. Considering the potential importance of glutathionylation, a number of methods have been developed for identifying proteins undergoing glutathionylation. These methods, ranging from analysis of purified proteins in vitro to large-scale proteomic analyses in vivo, allowed identification of nearly 200 targets in mammals. By contrast, the number of known glutathionylated proteins is more limited in photosynthetic organisms, although they are severely exposed to oxidative stress. The aim of this review is to detail the methods available for identification and analysis of glutathionylated proteins in vivo and in vitro. The advantages and drawbacks of each technique will be discussed as well as their application to photosynthetic organisms. Furthermore, an overview of known glutathionylated proteins in photosynthetic organisms is provided and the physiological importance of this post-translational modification is discussed.     

Glutathione metabolism in yeast Saccharomyces cerevisiae. Evidence that gamma-glutamyltranspeptidase is a vacuolar enzyme

In a first experiment we have shown that S. cerevisiae beta-glutamyltranspeptidase is associated with a particulate fraction obtained by differential centrifugation. We have subsequently shown that this enzyme activity followed accurately the distribution of vacuolar markers. Liberation of vacuoles was carried out by mechanical disruption of spheroplast under isotonic conditions and the vacuoles were purified by centrifugation of Ficoll gradients. Yeast beta-glutamyltranspeptidase could be implicated in the exchanges of amino acids between the cytoplasm and the vacuolar sap.     

变铅青链霉菌广谱胁迫蛋白对氧化压力响应的研究

【目的】广谱胁迫蛋白(USP)是一种古老的蛋白家族,在链霉菌属细菌中其功能研究尚未报道。以变铅青链霉菌USP蛋白为对象对其功能进行解析。【方法】使用序列比对的方法分析同源性及保守结构域。纯化USP蛋白,用圆二色谱分析蛋白与环腺苷酸(cAMP)的结合对usp(SLI_7517)进行基因中断。检测野生型和usp基因缺失株对偶氮二甲酰胺造成的氧化压力的耐受能力。使用qPCR荧光定量分析技术,检测野生型菌株与usp缺失株在氧化环境中谷胱甘肽过氧化物酶及巯基过氧化物酶基因转录量的差异。【结果】同源序列分析表明链霉菌属来源的USP蛋白序列相互之间相似性较高,USP-like结构域高度保守。USP蛋白在体外结合cAMP引起CD谱的变化。usp基因缺失株对偶氮二甲酰胺更耐受,同时菌株中谷胱甘肽过氧化物酶基因转录量上升。【结论】变铅青链霉菌中USP蛋白能够结合cAMP。usp参与菌体应对氧化环境的调控,对谷胱甘肽过氧化物酶基因的转录有阻遏作用。     

Ascorbate uptake and antioxidant function in peritoneal macrophages

Since activated macrophages generate potentially deleterious reactive oxygen species, we studied whether ascorbic acid might function as an antioxidant in these cells. Thioglycollate-elicited murine peritoneal macrophages contained about 3 mM ascorbate that was halved by culture in ascorbate-free medium. However, the cells took up added ascorbate to concentrations of 6-8 mM by a high-affinity sodium-dependent transport mechanism. This likely reflected the activity of the SVCT2 ascorbate transporter, since its message and protein were present in the cells. Activation of the cells by phagocytosis of latex particles depleted intracellular ascorbate, although not below the basal levels present in the cells in culture. Glutathione (GSH) was unaffected by phagocytosis, suggesting that ascorbate was more sensitive to the oxidant stress of phagocytosis than GSH. Phagocytosis induced a modest increase in reactive oxygen species as well as a progressive loss of alpha-tocopherol, both of which were prevented in cells loaded with ascorbate. These results suggest that activated macrophages can use ascorbate to lessen self-generated oxidant stress and spare alpha-tocopherol, which may protect these long-lived cells from necrosis or apoptosis.