Glutathione and its Related Enzymes in the Nile Fish

Glutathione (GSH) and related enzymes, glutathione transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR) form an important phase 2 biotransformation enzymes system. The objective of this study was to compare this enzymes system in three fish species from the river Nile, Oreochromis niloticus, Claris lazera and Cyprinus carpio in order to establish the main differences and to purify and characterize GST from the liver of O. niloticus.The level of GSH and the activity of GST, GPx and GR in the liver, kidney and gills of the three fish species were examined. A simple reproducible procedure for the purification of GST from the liver of O. niloticus to homogeneity, which includes chromatography on DEAE- cellulose followed by affinity chromatography on GSH-sepharose was established. The molecular mass was found to be 25,460 Da by SDS-PAGE. The Michaelis-Meneten constants (K_m) of the enzyme for GSH and CDNB were 0.35 mM and 0.42 mM, respectively. The affinity purified enzyme exhibited maximum pH at pH 8.0 and increasing pH above 8.0 did not affect the observed maximum. The purified enzyme acts readily on CDNB, less readily on some standard transferase substrates (1,2-dichloro-4-nitrobenzene and p-nitrophenethyl bromide) and not at all on others (bromosulphophthalein and p-nitrobenzyl chloride). Bromosulfophthalein, cibacron blue and hematin inhibited CDNB-conjugating activity of the purified enzyme with IC₅₀ 0.079, 3.98 and 0.126 μM, respectively.     

Effects of in vitro exposure to ozone and/or hyperoxia on superoxide dismutase, catalase, glutathione and lipid peroxidation in red blood cells and plasma of rainbow trout, Oncorhynchus mykiss (Walbaum)

In aquaculture, ozone is used as a disinfectant. In its production, extensive amounts of oxygen are formed resulting in hyperoxic conditions in culture units. Both ozone and hyperoxia have the potential to be toxic via pro‐oxidant mechanisms and to activate antioxidant defence systems in cultured species. To eliminate systemic effects, blood of rainbow trout, Oncorhynchus mykiss (Walbaum), was exposed in vitro for 5 min to ozone/hyperoxia or hyperoxia, and changes in antioxidant defences and lipid peroxidation were measured after exposure. Ozone exposure caused severe damage in red blood cells (rbc) detected as increased lipid peroxidation and oxidized glutathione (GSSG) levels in both plasma and rbc. Oxygen exposure alone increased intracellular lipid peroxidation and GSSG levels 10 min after exposure and was not evident in the plasma at any time. Ozone, but not oxygen exposure, decreased reduced glutathione (GSH) levels in plasma, and the changes were negatively correlated with increased lipid peroxidation in rbc, indicating that extracellular GSH has a dynamic role in the protection of rbc from direct oxidation by ozone. Both ozone and hyperoxic conditions increased superoxide dismutase (SOD) activity in rbc 3 and 6 h after exposure. In contrast, catalase activity was only increased 10 min after oxygen exposure, suggesting other catalase activation mechanisms rather than enzyme induction. The recovery of lipid peroxidation and GSSG levels in rbc after hyperoxia, but not ozone exposure, indicated a capacity to defend against hyperoxia‐produced oxidative damage, but an overwhelming of antioxidant defences by ozone in rainbow trout rbc in vitro.     

Oxidation of glutathione during hydroperoxide metabolism in isolated hepatocytes of rainbow trout (Salmo gairdneri)

Freshly isolated rainbow trout hepatocytes were exposed to tert-butyl hydroperoxide (BuOOH), a substrate for glutathione peroxidase. BuOOH at a concentration approximately equimolar (1 mM) with intracellular reduced glutathione (GSH) caused a reversible increase in intracellular glutathione disulphide (GSSG) but did not compromise cell viability or damage membrane lipids. BuOOH at 10 mM caused a large irreversible increase in intracellular GSSG followed by efflux into the medium. Considerable leakage of lactate dehydrogenase and loss of highly unsaturated fatty acids, particularly docosahexaenoic acid also occurred. Dependence of hydroperoxide removal on flux through the hexose monophosphate pathway was suggested by the increased release of ¹⁴CO₂ from [1-¹⁴C] glucose from hepatocytes incubated with BuOOH.     

多菌灵对蚯蚓体内3种解毒酶活性的影响

为明确多菌灵质量浓度与蚯蚓体内乙酰胆碱酯酶（AChE）、谷胱甘肽S-转移酶（GSTs）和羧酸酯酶（CarE）3种解毒酶活性之间的剂量效应关系,为多菌灵的安全使用及对相关动物的毒害研究提供参考,利用赤峰周边地区蔬菜大棚的蚯蚓,进行多菌灵0．75 g／L、1．00 g／L、1．25 g／L、1．50 g／L、1．75 g／L 和2．00 g／L 6个质量浓度对蚯蚓体内3种解毒酶活性的影响试验。结果表明：与对照组（蒸馏水）相比,多菌灵对蚯蚓体内的乙酰胆碱酯酶整体起抑制作用,在多菌灵浓度为1．75 g／L 时酶活性最低,为105．625μmol／L;对谷胱甘肽 S-转移酶整体起诱导促进作用,在多菌灵浓度为1．00 g／L 时酶活性最高,为17．343μmol／L;对羧酸酯酶活性,在多菌灵浓度小于1．25 g／L 时表现为诱导促进作用,大于1．25 g／L 时表现为抑制作用。     

磷脂氢谷胱甘肽过氧化物酶在精子发育中的作用研究进展

磷脂氢谷胱甘肽过氧化物酶(PHGPx)是广泛存在于哺乳动物细胞内唯一能够物接还原生物膜上脂类过氧化物的一类含硒酶.该酶在保护细胞膜完整、维持细胞的正常功能中发挥着重要作用;同时PHGPx还作为精子的结构物质,在精子的发育和成熟过程中发挥着重要作用;精液中PHGPx活性是评价精液品质的重要参数之一.对PHGPx的基因结构、体内分布以及与精子发育与成熟相关的生物学特性和功能等研究进展进行了总结.     

Mapping of genes involved in glutathione, carbohydrate and COR14b cold induced protein accumulation during cold hardening in wheat

Using some of the chromosome substitution lines developed from thecrosses of the donor Cheyenne to Chinese Spring we showed that theaccumulation of water soluble carbohydrates during different stages ofhardening was time dependent. Moreover there was a significantcorrelation between the rate of carbohydrate accumulation and the frosttolerance. The expression and regulation of a wheat gene homologous tothe barley cold regulated cor14b gene was compared in frost sensitiveand frost tolerant wheat genotypes at different temperatures. Studies madewith chromosome substitution lines showed that the threshold inductiontemperature polymorphism of the cor14b wheat homologous genewas controlled by loci located on chromosome 5A of wheat, while cor14b gene was mapped, in Triticum monococcum, onto the longarm of chromosome 2A^m. Our study on the effect of cold hardeningon glutathione (GSH) metabolism showed that chromosome 5A of wheathas an influence on the GSH accumulation and on the ratio of reduced andoxidised glutathione as part of a complex regulatory function during coldhardening. In addition, the level of increase in GSH content duringhardening may indicate the degree of the frost tolerance of wheat.     

Alteration of glutathione S-transferase properties during the development of <Emphasis Type="Italic">Micromelalopha troglodyta</Emphasis> larvae (Lepidoptera: Notodontidae)

Micromelalopha troglodyta (Graeser) is an important pest of poplar in China. Glutathione S-transferases (GSTs) are known to be responsible for adaptation mechanisms of M. troglodyta. The activities and kinetic constants of glutathione S-transferases in M. troglodyta were studied. Significant differences in glutathione S-transferase activity and kinetic characteristics were observed among five instars of M. troglodyta larvae. Furthermore, the inhibition of glutathione S-transferase activity in five instars by 24 inhibitors was conducted. The results show the inhibition of GST activity of different instars by 24 inhibitors was different. For GST activity in the 1st instar, chlorpyrifos, lambda-cyhalothrin, endosulfan, abamectin, fipronil and pyridaben were the best inhibitors tested, and for GST activity in the 2nd instar, tannic acid and quercetin were the most potent inhibitors tested, and for GST activity in the 3rd instar, the inhibitory effects of quercetin, chlorpyrifos and lambda-cyhalothrin were the highest, and for GST activity in the 4th instar, quercetin and lambda-cyhalothrin were the best inhibitors, and the inhibitory effect of phoxim was the highest for GST activity in the 5th instar. Our results show that glutathione S-transferases in different instars are qualitatively different in isozyme composition and thus different in sensitivity to inhibitors.     

半边旗提取物6F对HL-60细胞还原型谷胱甘肽(GSH)水平的影响

目的：观察半边旗提取物6F对HL—60细胞内还原型谷胱甘肽(GSH)水平的影响，从氧化机测探讨其抗肿瘤作用的机哩。方法：用邻苯二醛(OPT)荧光分光度法测定细胞内还原型谷胱甘肽．结果：58至231nmol/L 6F作用细胞20h及231nmol/L 6F作用细胞6至24h，HL—60细胞内GSH含量均明显降低，呈明显的时间剂量效应关系。结论：化合物6F可降低HL—60细胞内还原型谷胱甘肽水平，推测通过细胞内氧化机制杀伤HL—60细胞并诱导其细胞凋亡是化合物6F抗肿瘤作用机测之一．     

土壤农杆菌介导的转GST基因拟南芥的选育

将耐盐相关基因盐地碱蓬谷胱甘肽转移酶基因(Glutathione s—transferase，GST)克隆到表达栽体pROKⅡ中以构建植物表达栽体pGST，直接转化法转化土壤农杆菌，PCR验证后的阳性土壤农杆菌利用花序浸泡法转化拟南芥．转化子通过含有卡那霉素的培养基初步筛选，用PCR方法进一步验证外源基因插入到拟南芥基因组中．通过几代选育得到了稳定遗传的转基因拟南芥纯合品系．     

Stimulation of ROS-scavenging systems in squash (Cucurbita pepo L.) plants by compost supplementation under normal and low temperature conditions

The beneficial effect of compost, the final product of aerobic biodegradation of organic matter, on growth, lipid peroxidation [as malondialdehyde (MDA], hydrogen peroxide (H₂O₂) and superoxide anion (O₂^•−), activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR), as well as reduced ascorbate (ASC) and glutathione (GSH) and their oxidized forms was investigated in squash (Cucurbita pepo L. cv. Eskandarany) plants exposed to normal and low temperature (LT) conditions. LT stress of 8 °C significantly reduced the plant growth of untreated plants, but compost alleviated the adverse effect of stress and significantly increased the fresh and dry weights under normal and stress conditions. LT also induced accumulation of H₂O₂ and O₂^•− and resulted in increased lipid peroxidation, pointing out to cellular oxidative stress. Under compost application, such reactive oxygen species (ROS) and peroxidized lipids were markedly reduced, but SOD, CAT, APX and GR activities, key enzymes of ROS-scavenging systems, were significantly increased. Data also indicated that there were general reductions in total ascorbate and glutathione pool in LT control plants, but compost-treated ones considerably have maintained higher levels of such redox metabolites. Significantly higher ratios of ASC/DHA (dehydroascorbate) and GSH/GSSG (glutathione disulfide) were generally found in compost-treated plants than in untreated-ones. It is evident that compost induced enhancement of LT tolerance was related to up-regulation of enzymatic and non-enzymatic antioxidant systems. Such enhancement would eventually protect plant cells from LT-induced oxidative stress reactions via scavenging ROS.