Optimizing expression and purification of an ATP-binding gene gsiA from Escherichia coli k-12 by using GFP fusion

The cloning, expression and purification of the glutathione (sulfur) import system ATP-binding protein (gsiA) was carried out. The coding sequence of Escherichia coli gsiA, which encodes the ATP-binding protein of a glutathione importer, was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring green fluorescent protein (GFP) reporter gene. The resulting recombinant plasmid pWaldo-GFP-GsiA was transformed into various E. coli strains, and expression conditions were optimized. The effect of five E. coli expression strains on the production of the recombinant gsiA protein was evaluated. E. coli BL21 (DE3) was found to be the most productive strain for GsiA-GFP fusion-protein expression, most of which was insoluble fraction. However, results from in-gel and Western blot analysis suggested that expression of recombinant GsiA in Rosetta (DE3) provides an efficient source in soluble form. By using GFP as reporter, the most suitable host strain was conveniently obtained, whereby optimizing conditions for overexpression and purification of the proteins for further functional and structural studies, became, not only less laborious, but also time-saving

Rapid Dopaminergic Modulation of the Fish Hypothalamic Transcriptome and Proteome

Background - Dopamine (DA) is a major neurotransmitter playing an important role in the regulation of vertebrate reproduction. We developed a novel method for the comparison of transcriptomic and proteomic data obtained from in vivo experiments designed to study the neuroendocrine actions of DA. // Methods and Findings - Female goldfish were injected (i.p.) with DA agonists (D1-specific; SKF 38393, or D2-specific; LY 171555) and sacrificed after 5 h. Serum LH levels were reduced by 57% and 75% by SKF 38393 and LY 171555, respectively, indicating that the treatments produced physiologically relevant responses in vivo. Bioinformatic strategies and a ray-finned fish database were established for microarray and iTRAQ proteomic analysis of the hypothalamus, revealing a total of 3088 mRNAs and 42 proteins as being differentially regulated by the treatments. Twenty one proteins and mRNAs corresponding to these proteins appeared on both lists. Many of the mRNAs and proteins affected by the treatments were grouped into the Gene Ontology categorizations of protein complex, signal transduction, response to stimulus, and regulation of cellular processes. There was a 57% and 14% directional agreement between the differentially-regulated mRNAs and proteins for SKF 38393 and LY 171555, respectively. // Conclusions - The results demonstrate the applicability of advanced high-throughput genomic and proteomic analyses in an amendable well-studied teleost model species whose genome has yet to be sequenced. We demonstrate that DA rapidly regulates multiple hypothalamic pathways and processes that are also known to be involved in pathologies of the central nervous system

Plasma total antioxidant capacity in hemodialyzed patients and its relationships with other biomarkers of oxidative stress and lipid peroxidation

Patients undergoing long-term hemodialysis (HD) exhibit increased levels of oxidative stress, likely contributing to the increased rate of cardiovascular disease. The present study represents a critical evaluation of some of the most widely used oxidative indicators, as applied to the monitoring of hemodialysis-associated oxidative stress. Total plasma antioxidant capacity was determined by two independent procedures, the total antioxidant status (TAS) and the ferric reducing ability of plasma (FRAP) methods. Plasma lipid peroxidation was assessed by determining the peroxidation products malonaldehyde and 4-hydroxynonenal (MDA-4HNE) as well as lipid hydroperoxides ("Fox-2" and "d-ROMs" methods). Total plasma thiols and plasma alpha-tocopherol were also determined. MDA-4HNE levels were higher in HD patients and decreased following HD, possibly due to passive diffusion across dialysis filters. d-ROMs were also higher in HD patients but exhibited a further increase following the dialysis procedure. Serum alpha-tocopherol did not show any significant differences. Plasma thiols were lower in HD patients and were restored following HD. Plasma total antioxidant capacity determined with either method was unexpectedly higher in HD patients compared to controls, and decreased following HD. These data indicate that, of the biomarkers studied, d-ROMs level is the one more accurately reflecting the oxidative alterations taking place in HD patients, while determination of MDA-4HNE fails to detect oxidation occurring during the HD sessions. In addition, our findings point out that the determination of total antioxidant capacity in HD patients is severely affected by the concomitant fluctuations in plasma urate levels and therefore needs careful interpretation

Gamma-glutamyl transpeptidase-dependent iron reduction and LDL oxidation--a potential mechanism in atherosclerosis.

Abstract: Background:gamma-Glutamyl transpeptidase (gamma-GT) is found in serum and in the plasma membranes of virtually all cell types. Its physiologic role is to initiate the hydrolysis of extracellular glutathione (GSH), a tripeptide in which cysteine lies between alpha-glycine and gamma-glutamate residues. Cysteine and other thiol compounds are known to promote LDL oxidation by reducing Fe(In) to redox active Fe(II); therefore, we sought to determine whether similar reactions can be sustained by GSH and influenced by gamma-GT, Methods: Fe(III) reduction and LDL oxidation were studied by monitoring the formation bathophenanthroline-chelatable Fe(II) and the accumulation of thiobarbituric acid-reactive substances, respectively. Human atheromatous tissues were examined by histochemical techniques for the presence of oxidized LDL and their colocalization with cells expressing gamma-GT activity. Results: A series of experiments showed that the gamma-glutamate residue of GSH affected interactions of the juxtaposed cysteine thiol with iron, precluding Fe(III) reduction and hence LDL oxidation, Both processes increased remarkably after addition of purified gamma-GT, which acts by removing the gamma-glutamate residue. GSH-dependent LDL oxidation was similarly promoted by gamma-GT associated with the plasma membrane of human monoblastoid cells, and this process required iron traces that can be found in advanced or late stage atheromas, Collectively, these findings suggested a possible role for gamma-GT in the cellular processes of LDL oxidation and atherogenesis, Histochemical analyses confirmed that this may be the case, showing that gamma-GT activity is expressed by macrophage-derived foam cells within human atheromas, and that these cells colocalize with oxidized LDL. Conclusions: Biochemical and histochemical correlates indicate that gamma-GT can promote LDL oxidation by hydrolyzing GSH into more potent iron reductants, These findings may provide mechanistic clues to the epidemiologic evidence for a possible correlation between persistent elevation of gamma-GT and the risk of fatal reinfarction in patients with ischemic heart disease

Gamma-glutamyl transpeptidase catalyses the extracellular detoxification of cisplatin in a human cell line derived from the proximal convoluted tubule of the kidney

Nephrotoxicity is a side-effect and the main factor limiting the clinical use of cisplatin. In vivo, the administration of the cysteinecontaining tripeptide glutathione (GSH) has been found to reduce nephrotoxicity, but the biochemical mechanism of this protective action is not fully understood. The present study was designed to gain insights into the mechanism by which GSH prevents cisplatin nephrotoxicity. We also wanted to verify the hypothesis of whether the protective action of GSH is mediated by products of the extracellular breakdown of GSH catalysed by g-glutamyl transpeptidase (GGT), an enzyme that is highly expressed in kidney tubular cells. The study was performed in HK-2 cells, derived from the immortalisation of human kidney proximal tubule cells. We investigated the influence of modulators of GGT activity and/or thiols on the antiproliferative activity of cisplatin and on the intracellular GSH content. We determined the antiproliferative activity of cisplatin, platinum cellular accumulation and DNA platination following precomplexing of the drug with thiols. The antiproliferative effect of cisplatin was minimally affected by the addition of GSH. However, when the antiproliferative assay was performed in the presence of glycyl-glycine (GlyGly), to serve as a transpeptidation acceptor and thus to stimulate GGT-mediated GSH catabolism, cisplatin-induced growth inhibition was largely prevented. This effect was not mediated through an increase of intracellular GSH levels, which were not affected by the GlyGly supplementation. The thiol dipeptide cysteinyl-glycine, i.e. the GSH catabolite generated by GGT activity, showed a higher reactivity against cisplatin in vitro than GSH, as was shown by the more rapid oxidation of its \u2013SH groups. The cisplatin/GSH or cisplatin/cysteinyl-glycine adducts did not display an antiproliferative effect. However, 2 h precomplexing with GSH in the presence of GGT, or directly with the GSH catabolite cysteinyl-glycine, decreased the antiproliferative effect of cisplatin and drug-induced DNA platination to a greater extent than precomplexing with GSH alone. The results of the present study show that, in HK-2 cells, extracellular GSH decreases the antiproliferative effects of cisplatin only upon its hydrolysis by GGT, thereby supporting the hypothesis that the extracellular metabolism of GSH by GGT plays a role in modulating cisplatin nephrotoxicity. A primary role in the protection of HK-2 cells appears to be played by cysteinyl-glycine, the proximal product of the GGT-mediated hydrolysis of GSH, which shows a high reactivity against CDDP resulting in the rapid inactivation of the drug