Influence of carbon and nitrogen sources on glutathione catabolic enzymes inCandida albicans during dimorphism

The effect of carbon sources, glucose and sucrose, and nitrogen sources such as ammonia, glutamate andl-citrulline on the activities of glutathione metabolic enzymes has been studied. Yeast and mycelial cells were used to identify changes in activity levels of glutathione reductase (GSSGR), glutathione transferase (GST), glutathione peroxidase (GPX) and -glutamyl transpeptidase (GGT). Enzyme activities from cells grown in sucrose media were lower than in glucose media regardless of the enzyme tested, morphological form, or the growth interval. In all enzymes except GST, activity was higher in yeast form than in mycelia, regardless of nitrogen source, with lower activity from 24 to 72 h than at 96 h. In citrulline media, yeast form showed the maximum GST, GGT, and GPX activity. In ammonia-amended media, mycelia showed maximum activity in GGT, whereas in glutamate media, mycelia showed the maximum activity in GST. Also, the type of nitrogen source had no effect on GPX activity in the mycelial form. Finally, changing the nitrogen source showed no significant effect on GSSGR activity, either in the yeast or mycelial form.     

Depleted mucosal antioxidant defences in inflammatory bowel disease

Experimental approaches designed to define the role of reactive oxygen and nitrogen species generated by inflammatory cells in the tissue injury seen in inflammatory bowel disease rarely consider the chemical antioxidant defences against such increased oxidant stress in the mucosa. In this investigation, we have analysed components of the aqueous and lipid phase antioxidant mucosal defences by measuring the total peroxyl radical scavenging capacity and the levels of urate, glutathione, -tocopherol, and ubiquinol-10 in paired noninflamed and inflamed mucosal biopsies from inflammatory bowel disease patients. Compared to paired noninflamed mucosa, decreases were observed in inflamed mucosa for total peroxyl radical scavenging capacity (55%, p = 0.0031), urate [Crohn's disease (CD), 62.2%, p = 0.066; ulcerative colitis (UC), 47.3%, p = 0.031], glutathione (UC, 59%, 7/8 patients, ns), total glutathione (UC 65.2%, 6/8 patients, ns), ubiquinol-10 (CD, 75.7%, p = 0.03; UC, 90.5%, p = 0.005). The mean -tocopherol content was unchanged. These observations support our earlier findings of decreased reduced and total ascorbic acid in inflamed IBD mucosa and demonstrate that the loss of chemical antioxidant defences affects almost all the major components. The decreased antioxidant defences may severely compromise the inflamed mucosa, rendering it more susceptible to oxidative tissue damage, hindering recovery of the mucosa and return of epithelial cell layer integrity. The loss of chemical antioxidant components provides a strong rationale for developing novel antioxidant therapies for the treatment of inflammatory bowel disease.     

Inhibition of lipid peroxidation by a novel compound (CV-2619) in brain mitochondria and mode of action of the inhibition

Lipid peroxidation in rat brain mitochondria was induced by NADH in the presence of ADP and FeCl3. CV-2619 inhibited the lipid peroxidation in a concentration-dependent manner; the concentration giving 50% inhibition (IC50) was 84 microM. In addition, the inhibitory effect of CV-2619 was strongly enhanced by adding substrates of mitochondrial respiration; when succinate, glutamate, or succinate plus glutamate was added, the IC50 of CV-2619 was changed to 1.1, 10, or 0.5 microM, respectively. Metabolites of CV-2619 also inhibited the lipid peroxidation. The inhibitory effect of CV-2619 on mitochondrial lipid peroxidation disappeared when TTFA, an inhibitor of complex II in mitochondrial respiratory chain, was added. The results indicate that in mitochondria CV-2619 is changed to its reduced form which inhibits lipid peroxidation.     

Transformation of leukotriene A4 methyl ester to leukotriene C4 monomethyl ester by cytosolic rat glutathione transferases

Six major basic cytosolic glutathione transferases from rat liver catalyzed the conversion of leukotriene A4 methyl ester to the corresponding leukotriene C4 monomethyl ester. Glutathione transferase 4-4, the most active among these enzymes, had a Vmax of 615 nmol X min-1 X mg protein-1 at 30 degrees C in the presence of 5 mM glutathione. It was followed in efficiency by transferase 3-4 which had a Vmax of 160 nmol X min-1 X mg-1 under the same conditions. Transferases 1-1, 1-2, 2-2 and 3-3 had at least 30 times lower Vmax values than transferase 4-4.     

Dehalogenation of dichloromethane by cell extracts of hyphomicrobium DM2

A facultatively methylotrophic bacterium was isolated from enrichment cultures containing dichloromethane as the sole carbon source. It was identified as a Hyphomicrobium species. The organism grew exponentially in batch cultures with 10 mM dichloromethane at a specific growth rate of 0.07 h^-1. The release of Cl^- from dichloromethane and the disapperance of substrate paralleled growth. Resting dichloromethane-grown cells, in the presence of potassium sulphite as a trapping agent, converted cichloromethane methane quantitatively to formaldehyde. The conversion of dichloromethane to formaldehyde by cell extracts was stricly dependent on glutathione. Other thiols were inactive. Glutathione was not consumed in the course of the reaction. The specific activity of the enzymic dehalogenation of dichloromethane amounted to 3.8 mkat/kg protein in extracts of dichloromethane-grown cells and to less than 0.1 mkat/kg protein in extracts from cells grown on methanol.     

高效液相层析法纯化人胎盘谷胱甘肽硫转移酶

足月分娩的新鲜胎盘组织制成匀浆后,经高速离心、超速离心,谷胱甘肽(GSH)Sepharose 6B亲合层析,Amicon pM-10膜超过滤及高效液相层析,最终经SDS-PAGE鉴定,结果呈现单一亚基区带,其亚基分子量为25000。 根据我们现有高效液相设备条件,用ODS柱代替RadulovicL等报道的特异阴离子柱,用磷酸盐洗脱液代替含谷胱甘肽、二硫苏糖醇及氯化钾的梯度洗脱液,从人胎盘组织成功地制备了谷胱甘肽硫转移酶(GST)纯酶,全过程在15min内完成,保留时间及主峰面积的重复性均较理想,7次实验结果的变异系数为0.2％,最终纯化578.9倍。本研究为各种形式GST的纯化制备提供了一个新的、重复性好、分辨率高及回收理想的简易方法。     

Expression and activity of gamma-glutamyl transpeptidase in the rat epididymis.

Following Northern analysis, GGT mRNA was found predominantly within the caput epididymides and kidney. The size of mRNAs for kidney, caput, corpus, and ductus deferens were 2.2, 2.3, 2.2, and 2.3 kb, respectively, whereas cauda showed a doublet of 2.2 and 2.3 kb. GGT transpeptidation and hydrolytic activity within epididymal luminal fluids collected by micropuncture showed caput = corpus greater than cauda and corpus greater than caput greater than cauda, respectively. Caput luminal GGT transpeptidation activity was significantly inhibited by serine-borate and was optimal at pH 8.0. The calculated Km and Vmax values for hydrolysis of GSH by caput luminal GGT were 0.06 microM and 2.19 nmoles/min/microliters luminal fluid at pH 8.5 compared to 0.49 microM and 0.49 nmoles/min/microliters luminal fluid, respectively, at the physiological pH 6.5 of caput fluid. These studies would suggest that the epididymis can control the activity of luminal GGT by pH. Lower Km (0.12 microM) and higher Vmax (1.13 nmoles/min/microliters luminal fluid) values were also calculated when GSSG was used compared to GSH. Results from Triton X-114 partitioning experiments suggest that luminal GGT probably exists in both membrane bound and nonmembrane bound forms. Western blot analysis of proteins within epididymal luminal fluids revealed both subunits of GGT in all epididymal regions studied. However, two lower molecular bands, approximately 22 kDa and 21 kDa, were also observed in cauda fluid. It is suggested that as GGT is transported along the epididymal duct it undergoes degradation, which accounts for its loss of activity in the distal epididymal regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Whole blood superoxide anion generation and efficiency of some erythrocyte antioxidant systems during recombinant human erythropoietin therapy of uremic anemia

Six chronic uremic patients on regular hemodialysis treatment were given recombinant human erythropoietin (r-huEPO) in a dose of 50 U/kg of body weight intravenously thrice weekly for 14 weeks. Following r-huEPO therapy, unstimulated whole blood superoxide anion ( ₂) generation did not change significantly, while opsonized zymosan-stimulated whole blood ₂ generation increased. At the same time, erythrocyte superoxide dismutase and, in particular, glutathione peroxidase activities were found to be reducing with concomitant lowering of erythrocyte malonydialdehyde (MDA) concentrations and increase in plasma MDA concentrations.     

Photosynthetic apparatus of chilling-sensitive plants IX. The involvement of α-tocopherol in the electron transport chain and the anti-oxidizing system in chloroplasts of tomato leaves

1. The role of tocopherols in tomato chloroplasts from fresh, cold and dark-stored as well as stored and illuminated leaves was studied.2. The cold and dark storage of leaves results in a loss of chloroplast α- and γ-tocopherols of about 30–40% accompanied by an increase in chloroplast δ-tocopherol of about 40%. On illumination of stored leaves, an elevation of α- and γ-tocopherol level to about 110 and 95% of the control, respectively, occurs, whilst δ-tocopherol content is not affected.3. Experiments performed with 2,2-diphenyl-1-picrylhydrazyl-treated chloroplasts show that only about 70% of total α-tocopherol is functionally active in the electron transport of Photosystem II between the diphenyl-carbazide (DPC) donation site and the inhibition site of DBMIB.4. A small amount of α-tocopherol quinone (about 10% of α-tocopherol content) is found in chloroplasts from fresh, fresh and illuminated as well as cold and dark-stored tomato leaves, whereas the illumination of the latter increases the chloroplast α-tocopherol quinone content 3-fold. Moreover, following the illumination of chloroplasts from cold and dark-stored as well as stored and illuminated leaves, the oxidation of exogenous α-tocopherol to α-tocopherol quinone is 2-fold faster then in chloroplasts from fresh leaves.5. The primary product (‘α-tocopheroxide’) formed during the α-tocopherol oxidation by illuminated chloroplasts was identified as 8a-hydroxy-α-tocopheron.6. Exogenous α-tocopherol inhibits the lipid photoperoxidation by about 40–50% in chloroplasts from all three kinds of tomato leaf.7. The results seem to suggest that chloroplast α-tocopherol is involved in both electron transport of PS II and antioxidizing system of chloroplasts.     

Coupling of Dopamine Oxidation (Monoamine Oxidase Activity) to Glutathione Oxidation Via the Generation of Hydrogen Peroxide in Rat Brain Homogenates

Abstract: Homogenates of perfused rat brain generated oxidized glutathione from reduced glutathione during incubation with dopamine or serotonin. This activity was blocked by pargyline. a monoamine oxidase inhibitor, or by catalase, a scavenger of hydrogen peroxide. These results demonstrate formation of hydrogen peroxide by monoamine oxidase and the coupling of the peroxide to glutathione peroxidase activity. Oxidized glutathione was measured fluorometrically via the oxidation of NADPH by glutathione reductase. In the absence of added dopamine or serotonin, a much smaller amount of reduced glutathione was oxidized: this activity was blocked by catalase, but not by pargyline. Therefore, endogenous production of hydrogen peroxide, not linked to monoamine oxidase activity, was present. These results indicate that glutathione peroxidase (linked to hexose monophosphate shunt activity) can function to eliminate hydrogen peroxide generated by monoamine oxidase and other endogenous sources in aminergic neurons.