Synthesis and biological evaluation of potential modulators of malarial glutathione-S-transferase(s)

Glutathione-S-transferase(s) (E.C.2.5.1.18, GSTs) have been investigated in parasitic protozoans with respect to their biochemistry and they have been identified as potential vaccine candidates in protozoan parasites and as a target in the synthesis of new antiparasitic agents. In a search towards the identification of novel biochemical targets for antimalarial drug design, the area of Plasmodium glutathione metabolism provides a number of promising chemotherapeutic targets. GST activity was determined in various subcellular fractions of malarial parasites Plasmodium yoelii and was found to be localized mainly in the cytosolic fraction (specific activity, c. 0.058 ± 0.016 μmol/min/mg protein). Hemin, a known inhibitor of mammalian GST(s), maximally inhibited this enzyme from P. yoelii to nearly 86%. In a search towards synthetic modulators of malarial GST(s), 575 compounds belonging to various chemical classes were screened for their effect on crude GST from P. yoelii and 92 compounds belonging to various chemical classes were studied on recombinant GST from P. falciparum. Among all the compounds screened, 83 compounds inhibited/stimulated the enzyme from P. yoelii/P. falciparum to the extent of 40% or more.     

Age-related changes of antioxidant enzyme activities, glutathione status and lipid peroxidation in rat erythrocytes after heat stress

The aim of this study was to determine whether exposure to heat stress would lead to oxidative stress and whether this effect varied with different exposure periods. We kept 1-, 6- and 12-month-old male Wistar rats at an ambient temperature of either 22 degrees C or 40 degrees C for 3 and 7 days and measured glucose-6-phosphate dehydrogenase (G-6-PD), Cu,Zn-superoxide dismutase (Cu,Zn-SOD), catalase (CAT), selenium-dependent glutathione peroxidase (Se-GSH-Px) and glutathione-S-transferase (GST) activities and levels of thiobarbituric acid-reactive substances (TBARS), reduced glutathione (GSH) and oxidized glutathione (GSSG) in erythrocytes and determined GSH/GSSG ratio, total glutathione and the redox index. G-6-PD and CAT activities were found to be significantly increased in 1- and 6-month-old rats after 3 and 7 days of heat stress, but G-6-PD activities decreased in 12-month-old rats. Cu, Zn-SOD activity decreased in 1-month-old rats after heat stress, whereas it increased in 6- and 12-month-old rats. GST activity increased in all groups. GSH and total GSH levels and GSH/GSSG ratios decreased in 1- and 6-month-old rats but they increased in 12-month-old rats after heat stress. GSSG levels increased in 1- and 6-month-old rats but decreased in 12-month-old rats after heat stress. TBARS levels increased in all groups. Seven days of stress is more effective in altering enzyme activities and levels of GSH, GSSG and TBARS. When the effects of both heat stress and aging were examined together, it was interesting to note that they mostly influenced G-6-PD activity.     

Colocalization and identification of interaction sites between IGFBP-3 and GalNAc-T14

GalNAc-T14 was identified as a novel IGFBP-3 binding partner in previous studies. Here, we furtherly confirmed the interaction between them by confocal microscopy, and identified the binding domain and probable interaction sites of GalNAc-T14 with IGFBP-3. The result of subcellular localization indicated that GalNAc-T14 was distributed in the cytosol, whereas IGFBP-3 existed in the cytosol and nucleolus. Confocal analyses demonstrated that IGFBP-3 and GalNAc-T14 colocalized in the cytosol. The result from yeast two hybrid assay showed that the C terminus of GalNAc-T14 (408-552aa) was essential for the interaction between GalNAc-T14 and IGFBP-3, especially Tyr(408), Pro(409), and Glu(410) of GalNAc-T14 may play key roles in the interaction with IGFBP-3. In conclusion, these studies demonstrated that IGFBP-3 and GalNAc-T14 are colocalized in MCF-7 cells and confirmed the interaction between IGFBP-3 and GalNAc-T14. This interaction may play an important role in the functional regulation of IGFBP-3.     

In vitro evaluation of co-exposure of arsenium and an organic nanomaterial (fullerene, C₆₀) in zebrafish hepatocytes

Taking into account the concept of the "Trojan Horse", where contaminants may have its entry into specific organs potentiated by its association with nanomaterials, the aim of this study was to analyze the joint toxic effects induced by an organic nanomaterial, fullerene (C(60)) with the metalloid arsenic (As(III)). Hepatocytes of zebrafish Danio rerio were exposed to As(III) (2.5 or 100 μM), C(60) or As+C(60) for 4h, not altering cells viability. Intracellular reactive oxygen species concentration was reduced in cells exposed only to the C(60) (1mg/L) and in the treatment of 100 μM As(III)+C(60). Co-exposure with C(60) abolished the peak of the antioxidant glutathione (GSH) registered in cells exposed to the lowest As(III) concentration (2.5 μM). A similar result was observed in terms of lipid damage (TBARS). Total antioxidant capacity was significantly higher at both As(III) concentrations co-exposed to C(60) when compared with the control group. Activity of glutathione-S-transferase omega, a limiting enzyme in the methylation pathway of As(III), was reduced in the 100 μM As(III)+C(60) treatment. Cells co-exposed to C(60) had a significantly higher accumulation of As(III), showing a "Trojan Horse" effect which did not result in higher cell toxicity. Instead, co-exposure of As(III) with C(60) showed to reduce cellular injury.     

Effect of temperature in multiple biomarkers of oxidative stress in coastal shrimp

Various studies in captivity and in the wild have pointed to the effect of season, and temperature in particular, in the levels of the oxidative stress biomarkers currently used for environmental quality assessment. However, knowledge on how temperature affects the oxidative stress response is unavailable for most species. This study investigated the effect of increasing temperature on lipid peroxidation, catalase activity, superoxide dismutase and glutathione-S-transferase in the shrimps, Palaemon elegans and Palaemon serratus. It was concluded that increasing temperatures significantly affect all the biomarkers tested in both species, with the exception of superoxide dismutase in P. serratus which was not affected by temperature. The oxidative stress response was more intense in P. elegans, than in P. serratus, producing higher peaks of all biomarkers at temperatures between 22 °C and 26 °C, followed by low levels at higher temperatures. It was concluded that monitoring of ecosystems using oxidative stress biomarkers should take into account the species and thermal history of the organisms. Sampling should be avoided during heat waves and immediately after heat waves.     

人谷胱甘肽硫转移酶A1在乳酸乳球菌中的表达及活性研究

用RTPCR技术从人肝总RNA中分离扩增了人谷胱甘肽硫转移酶A1基因的cDNA序列,克隆至大肠杆菌表达质粒pET23b,采用蛋白表达筛查法及DNA测序证明该cDNA序列完全正确。重组质粒pET23bhgst转化大肠杆菌BL21(DE3),经IPTG诱导获得高效表达的可溶性hGSTA1产物,其表达量约为大肠杆菌可溶性总蛋白的40%。将hGSTA1cDNA亚克隆至乳酸乳球菌表达载体pMG36e,电穿孔法转化乳酸乳球菌MG1363获得hGSTA1乳酸乳球菌表达株。SDSPAGE及Western杂交分析表明该菌株表达预期大小的hGSTA1融合蛋白,经谷胱甘肽琼脂糖亲和层析纯化获得的hGSTA1蛋白具有较高的谷胱甘肽硫转移酶活性。具hGSTA1酶活性的乳酸乳球菌工程菌可望应用于研制防癌保健乳制品。     

Differential Localization of Glutathione-S-Transferase Yp and Yb Subunits in Oligodendrocytes and Astrocytes of Rat Brain

Glutathione-S-transferase Yb subunits were recently identified in rat brain and localized to astrocytes, ependymal cells lining the ventricles, subventricular zone cells, and tanycytes. Another isoform, Yp (pi family), was detected in rat brain by immunoblotting, and its mRNA was detected by Northern hybridizations. Double immunofluorescence localized Yb and Yp in different glial cells. The strongly Yp-positive cells were identified as oligodendrocytes by virtue of their arrangement in rows in white-matter tracts, colocalization in strongly carbonic anhydrase-positive cells, and association with myelinated tracts in the corpus striatum. Ependymal cells in the choroid plexus and ventricular lining were also strongly Yp positive, whereas Yb was not detected in the choroid plexus. The occurrence of Yp at low levels in astrocytes was indicated after immunostaining by a sensitive peroxidase-antiperoxidase method, which revealed weak staining of those cells in the molecular layer of the cortex. The data suggest that Yb and Yp subunits are primarily localized to astrocytes and oligodendrocytes, respectively, and that both are absent from neurons. The glutathione-S-transferase in oligodendrocytes may participate in the removal of toxins from the vicinity of the myelin sheath. The finding of glutathione-S-transferases in ependymal cells and astrocytes in the brain also suggests that this enzyme could be a first line of defense against toxic substances.     

Glutathione-S-Transferase Activity in the Brain: Species, Sex, Regional, and Age Differences

Abstract: Glutathione- S -transferase activity in the brain of male mammals (rat and mouse) was found to be relatively lower than in that of females. In contrast, the male aves (pigeon, kite, vulture, and crow) exhibited comparatively higher activity of brain glutathione- S -transferase than the corresponding females. Postnatal development of cytosolic glutathione-S-transferase activity in the rat brain was also investigated. The day-7 rats showed a low activity of 48 nmol/min/mg protein that gradually increased 3.2-fold over the age of 28 days. No striking differences in brain enzyme activities were observed between the 35- and 90-day-old rats. Discrete brain regions of immature rats were found to possess considerable but lower quantities of glutathione- S -transferase activity than those of the adults. The activity increased with the onset of development and attained a steady state after 21 days of age.     

A Pi Form of Glutathione-S-Transferase Is a Myelin-and Oligodendrocyte-Associated Enzyme in Mouse Brain

The pi form of glutathione-S-transferase (GST), previously found to be oligodendrocyte associated, has also been found in myelin. In the brains of adult mice, immunocytochemical staining for a pi form of GST was observed in myelinated fibers, as well as oligodendrocytes. In contrast, and as previously found in rats, positive immunostaining for mu forms occurred in astrocytes and not in oligodendrocytes or myelinated fibers. Double immunofluorescence staining strengthened the conclusion that pi was in oligodendrocytes and myelin in mouse brains. The results of enzyme assays performed on brain homogenates and purified myelin indicated that GST specific activities in mouse brain myelin were almost as high (0.8-fold) as those in mouse brain homogenates. Low, but reproducible, GST activities were also observed in myelin purified from rat brains, in which pi had been demonstrated in oligodendrocytes and mu in astrocytes. The pi form was also stained by the immunoblot technique in myelin purified from mouse brain. It was concluded that pi is a myelin associated, as well as oligodendrocyte associated, enzyme in mouse brain, and possibly also in rat brain. This is the first report of localization of GSTs in mouse brain and of GST in myelin.