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Intracellular pathogens are responsible for much of the world-wide morbidity and mortality due to infectious diseases. To colonize their hosts successfully, pathogens must sense their environment and regulate virulence gene expression appropriately. Accordingly, on entry into mammalian cells, the facultative intracellular bacterial pathogen Listeria monocytogenes remodels its transcriptional program by activating the master virulence regulator PrfA. Here we show that bacterial and host-derived glutathione are required to activate PrfA. In this study a genetic selection led to the identification of a bacterial mutant in glutathione synthase that exhibited reduced virulence gene expression and was attenuated 150-fold in mice. Genome sequencing of suppressor mutants that arose spontaneously in vivo revealed a single nucleotide change in prfA that locks the protein in the active conformation (PrfA*) and completely bypassed the requirement for glutathione during infection. Biochemical and genetic studies support a model in which glutathionedependent PrfA activation is mediated by allosteric binding of glutathione to PrfA. Whereas glutathione and other low-molecular-weight thiols have important roles in redox homeostasis in all forms of life, here we demonstrate that glutathione represents a critical signalling molecule that activates the virulence of an intracellular pathogen., Listeria monocytogenes is a Gram-positive pathogen of animals and humans that cycles between a saprophytic lifestyle and an intracellular pathogen that escapes from a vacuole and grows in the cytosol [...]

Cotton (Gossypium hirsutum L.) seedlings are considered to be sensitive to temperature stress. The exposure of plants to high and low temperatures can result in the production of reactive oxygen species that contribute to diminished plant performance. Plants have mechanisms that metabolize oxidants into less harmful chemicals, and enhancement of antioxidant metabolism has been shown to be beneficial to plants exposed to acute oxidative stresses. Transgenic overexpression of glutathione reductase (GR) in cotton could result in faster seedling emergence at nonoptimal temperatures. Transgenic GR and 5 nontransgenic cotton lines were planted at four dates in the field in Lubbock, TX, and in constant temperature growth boxes. The temperatures in the field were typical for the cotton planting period in the region, and in growth boxes ranged from 15 to 45[degrees]C. Emergence rates were determined for all lines, and the activity of GR and the levels of malondialdehyde in the cotyledons were monitored to verify altered enzyme activity and to detect differences in oxidative damage, respectively. Enhancement of GR activity did not improve emergence or reduce oxidative damage. It is concluded that GR activity did not limit emergence.

BACKGROUND: Exposure to toxic methylmercury (MeHg) through fish consumption is a large problem worldwide, and it has led to governmental recommendations of reduced fish consumption and blacklisting of mercury-contaminated fish. [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2007.06.044 Byline: Namakkal S. Rajasekaran (1), Patrice Connell (2), Elisabeth S. Christians (2)(3), Liang-Jun Yan (2), Ryan P. Taylor (1), Andras Orosz (1), Xiu Q. Zhang (1), Tamara J. Stevenson (1), Ronald M. Peshock (2)(4), Jane A. Leopold (5), William H. Barry (1), Joseph Loscalzo (5), Shannon J. Odelberg (1), Ivor J. Benjamin (1)(2) Keywords: HUMDISEASE; SIGNALING Abstract: The autosomal dominant mutation in the human [alpha]B-crystallin gene inducing a R120G amino acid exchange causes a multisystem, protein aggregation disease including cardiomyopathy. The pathogenesis of cardiomyopathy in this mutant (hR120GCryAB) is poorly understood. Here, we show that transgenic mice overexpressing cardiac-specific hR120GCryAB recapitulate the cardiomyopathy in humans and find that the mice are under reductive stress. The myopathic hearts show an increased recycling of oxidized glutathione (GSSG) to reduced glutathione (GSH), which is due to the augmented expression and enzymatic activities of glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase, and glutathione peroxidase. The intercross of hR120GCryAB cardiomyopathic animals with mice with reduced G6PD levels rescues the progeny from cardiac hypertrophy and protein aggregation. These findings demonstrate that dysregulation of G6PD activity is necessary and sufficient for maladaptive reductive stress and suggest a novel therapeutic target for abrogating R120GCryAB cardiomyopathy and heart failure in humans. Author Affiliation: (1) Center for Cardiovascular Translational Biomedicine, Division of Cardiology, Department of Internal Medicine, University of Utah School of Medicine, 30 North 1900 East, Room 4A100, Salt Lake City, UT 84132, USA (2) Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA (3) Centre for Developmental Biology UMR5547, 118 route de Narbonne, 31062 Toulouse, France (4) Department of Radiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA (5) Cardiovascular Medicine Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 77 Avenue Louis Pasteur Boston, MA 02115, USA Article History: Received 25 January 2007; Revised 26 April 2007; Accepted 22 June 2007 Article Note: (miscellaneous) Published: August 9, 2007

Glutathione protects cells and organisms from oxygen species and peroxides and is indispensable for aerobically living organisms. Moreover, it acts against xenobiotics and drugs by the formation and excretion of glutathione S conjugates. In this study, we show that the yliA, -B, -C, and -D genes of Escherichia coli K-12 encode a glutathione transporter with the ATP-binding cassette. The transporter imports extracellular glutathione into the cytoplasm in an ATP-dependent manner. This transporter, along with [gamma]-glutamyltranspeptidase, has an important role in E. coli growth with glutathione as a sole sulfur source.

A 68 kDa fibronectin-binding protein (Fbp68) from Clostridium difficile displaying significant homology to several established or putative Fbps from other bacteria was identified. The one-copy gene is highly conserved in C. difficile isolates. Fbp68 was expressed in Escherichia coli in fusion with glutathione S-transferase; the fusion protein and the native Fbp68 were purified. Immunoblot analysis and cell fractionation experiments revealed that Fbp68 is present on the surface of the bacteria. Far-immuno dot-blotting demonstrated that Fbp68 was capable of fixing fibronectin. Indirect immunofluorescence and ELISA were employed to demonstrate that C. difficile could bind both soluble and immobilized fibronectin. With competitive adherence inhibition assays it was shown that antibodies raised against Fbp68 partially inhibited attachment of C. difficile to fibronectin and Vero cells. Furthermore, Vero cells could fix purified membrane-immobilized Fbp68. Thus Fbp68 appears to be one of the several adhesins identified to date in C. difficile.

The biochemical characteristics of the gst-like Rhizobium leguminosarum gene belonging to the glutathione S-transferase (GST) gene family were analyzed in Escherchia coli. Analysis of Rhizobium leguminasarum gene overexpressed in Escherichia coli indicated the presence of a 21990 Da polypeptide with a 203 amino acid sequence that was encoded by the GST gene. Furthermore, the GST gene exhibited an antigenic domain that was different from mammalian GST genes.

The bioactivity of endothelium-derived nitric oxide (NO) is an important component of vascular homeostasis that is sensitive to intracellular redox status. Because glutathione (GSH) is a major determinant of intracellular redox state, we sought to define its role in modulating endothelial NO bioactivity. In porcine aortic endothelial cells (PAECs), we depleted intracellular GSH ([is greater than] 70%) using 1) buthionine-(S,R)-sulfoximine (BSO), which inhibits GSH synthesis; 2) diamide, which oxidizes thiols; or 3) 1-chloro-2,4-dinitrobenzene (CDNB), which putatively depletes GSH through glutathione S-transferase activity. Cellular GSH depletion with BSO had no effect on endothelial NO bioactivity measured as A-23187-induced cGMP accumulation. In contrast, oxidation of intracellular thiols with diamide inhibited both A-23187-induced cGMP accumulation and the cGMP response to exogenous NO. Diamide treatment of either PAECs, PAEC membrane fractions, or purified endothelial nitric oxide synthase (eNOS) resulted in significant inhibition (~75%) of eNOS catalytic activity measured as L-[[sup.3]H]arginine-to-L-[[sup.3]H]citrulline conversion. This effect appeared related to oxidation of eNOS thiols as it was completely reversed by dithiothreitol. Glutathione depletion with CDNB inhibited A-23187-stimulated cGMP accumulation but not the cGMP response to exogenous NO. Rather, CDNB treatment impaired eNOS catalytic activity in intact PAECs, and this effect was reversed by excess NADPH in isolated purified eNOS assays. Consistent with these results, we found spectral evidence that CDNB reacts with NADPH and renders it inactive as a cofactor for either eNOS or glutathione reductase. Thus thiol-modulating agents exert pleiotropic effects on endothelial NO bioactivity, and these data may help to resolve a number of conflicting previous studies linking GSH status with endothelial cell NO bioactivity. thiols; nitric oxide; antioxidants; endothelium; nitric oxide synthase; glutathione

Thioredoxin (Trx) and glutathione (GSH) systems are considered to be two major redox systems in animal cells. They are reduced by NADPH via Trx reductase (TR) or oxidized GSH (GSSG) reductase and further supply electrons for deoxyribonucleotide synthesis, antioxidant defense, and redox regulation of signal transduction, transcription, cell growth, and apoptosis. We cloned and characterized a pyridine nucleotide disulfide oxidoreductase, Trx and GSSG reductase (TGR), that exhibits specificity for both redox systems. This enzyme contains a selenocysteine residue encoded by the TGA codon. TGR can reduce Trx, GSSG, and a GSH-linked disulfide in in vitro assays. This unusual substrate specificity is achieved by an evolutionary conserved fusion of the TR and glutaredoxin domains. These observations, together with the biochemical probing and molecular modeling of the TGR structure, suggest a mechanism whereby the C-terminal selenotetrapeptide serves a role of a protein-linked GSSG and shuttles electrons from the disulfide center within the TR domain to either the glutaredoxin domain or Trx.

Induction of phase 2 enzymes and elevations of glutathione are major and sufficient strategies for protecting mammals and their cells against the toxic and carcinogenic effects of electrophiles and reactive forms of oxygen. Inducers belong to nine chemical classes and have few common properties except for their ability to modify sulfhydryl groups by oxidation, reduction, or alkylation. Much evidence suggests that the cellular 'sensor' molecule that recognizes the inducers and signals the enhanced transcription of phase 2 genes does so by virtue of unique and highly reactive sulfhydryl functions that recognize and covalently react with the inducers. Benzylidene-alkanones and -cycloalkanones are Michael reaction acceptors whose inducer potency is profoundly increased by the presence of ortho- (but not other) hydroxyl substituent(s) on the aromatic ring(s). This enhancement correlates with more rapid reactivity of the ortho-hydroxylated derivatives with model sulfhyclryl compounds. Proton NMR spectroscopy provides no evidence for increased electrophilicity of the/3-vinyl carbons (the presumed site of nucleophilic attack) on the hydroxylated inducers. Surprisingly, these ortho-hydroxyl groups display a propensity for extensive intermolecular hydrogen bond formation, which may raise the reactivity and facilitate addition of mercaptans, thereby raising inducer potencies.

In Arabidopsis thaliana, trichome cells are specialized unicellular structures with uncertain functions. Based on earlier observations that one of the genes involved in cysteine biosynthesis (Atcys-3A) is highly expressed in trichomes, we have extended our studies in trichome cells to determine their capacity for glutathione (GSH) biosynthesis. First, we have analyzed by in situ hybridization the tissue-specific expression of the genes Atcys-3A and sat5, which encode O-acetylserine(thio)lyase (OASTL) and serine acetyltransferase (SAT), respectively, as well as gshl and gsh2, which encode [Gamma]-glutamylcysteine synthetase and glutathione synthetase, respectively. The four genes are highly expressed in leaf trichomes of Arabidopsis, and their mRNA accumulate to high levels. Second, we have directly measured cytoplasmic GSH concentration in intact cells by laser-scanning microscopy after labeling with monochlorobimane as a GSH-specific probe. From these measurements, cytosolic GSH concentrations of 238 [+ or -] 25, 80 [+ or -] 2, and 144 [+ or -] 19 [micro]M were estimated for trichome, basement, and epidermal cells, respectively. Taking into account the volume of the cells measured using stereological techniques, the trichomes have a total GSH content more than 300-fold higher than the basement and epidermal cells. Third, after NaCI treatment, GSH biosynthesis is markedly decreased in trichomes. Atcys-3A, sat5, gsh 1, and gsh2 mRNA levels show a decrease in transcript abundance, and [GSH][sup.cyt] is reduced to 47 [+ or -] 5 [micro]M. These results suggest the important physiological significance of trichome cells related to GSH biosynthesis and their possible role as a sink during detoxification processes. cysteine | glutathione | two-photon laser-scanning microscopy | O-acetylserine(thiol)lyase | sulfate assimilation

The porcine glutathione-peroxidase-5 (GPX5) gene, which may play a role in the interaction of sperm and egg, and thus be a candidate for boar fertility, has been mapped to pig chromosome 7. Topics discussed include the source and description of primers, primer sequences, method of detection, sequencing and polymorphisms, inheritance pattern, allele frequencies, and chromosomal location.