Purification and Biochemical Characterization of Glutathione S-Transferase from Down Syndrome and Normal Children Erythrocytes: A Comparative Study

ERIC Educational Resources Information Center

Hamed, Ragaa R.; Maharem, Tahany M.; Abdel-Meguid, Nagwa; Sabry, Gilane M.; Abdalla, Abdel-Monem; Guneidy, Rasha A.

2011-01-01

Down syndrome (DS) is the phenotypic manifestation of trisomy 21. Our study was concerned with the characterization and purification of glutathione S-transferase enzyme (GST) from normal and Down syndrome (DS) erythrocytes to illustrate the difference in the role of this enzyme in the cell. Glutathione S-transferase and glutathione (GSH) was…

21 CFR 862.1535 - Ornithine carbamyl transferase test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Ornithine carbamyl transferase test system. 862.1535 Section 862.1535 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

Glutathione S-transferase M1 and glutathione S-transferase T1 genotype in chronic pancreatitis: a meta-analysis.

PubMed

Zhong, Yanjun; Zou, Runmei; Cao, Jie; Peng, Mou

2015-02-01

A meta-analysis to determine the association between chronic pancreatitis and glutathione-S transferase (GST) mu 1 (GSTM1) and theta 1 (GSTT1) deletions. Case-control studies concerning the relationship between chronic pancreatitis and GSTM1 or GSTT1 deletions were identified (up to October 2013). Meta-analyses of the association between GSTM1 and GSTT1 genotype and chronic pancreatitis or alcoholic chronic pancreatitis (ACP) were performed. Seven studies were included in the meta-analysis (650 patients/1382 controls for GSTM1 and 536 patients/1304 controls for GSTT1). There were no significant relationships between GSTM1/GSTT1 and chronic pancreatitis or GSTT1 and ACP. There was a significant association between GSTM1 null genotype and ACP (odds ratio 1.16, 95% confidence intervals 1.03, 1.30). The GSTM1 null genotype was significantly associated with ACP risk. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Pleiotropic Functions of Glutathione S-Transferase P

PubMed Central

Zhang, Jie; Grek, Christina; Ye, Zhi-Wei; Manevich, Yefim; Tew, Kenneth D.; Townsend, Danyelle M.

2016-01-01

Glutathione S-transferase P (GSTP) is one member of the GST superfamily that is prevalently expressed in mammals. Known to possess catalytic activity through deprotonating glutathione allowing formation of thioether bonds with electrophilic substrates, more recent discoveries have broadened our understanding of the biological roles of this protein. In addition to catalytic detoxification, other properties so far ascribed to GSTP include chaperone functions, regulation of nitric oxide pathways, regulation of a variety of kinase signaling pathways, and participation in the forward reaction of protein S-glutathionylation. The expression of GSTP has been linked with cancer and other human pathologies and more recently even with drug addiction. With respect to human health, polymorphic variants of GSTP may determine individual susceptibility to oxidative stress and/or be critical in the design and development of drugs that have used redox pathways as a discovery platform. PMID:24974181

Yeast one-hybrid system used to identify the binding proteins for rat glutathione S-transferase P enhancer I.

PubMed

Liao, Ming-Xiang; Liu, Dong-Yuan; Zuo, Jin; Fang, Fu-De

2002-03-01

To detect the trans-factors specifically binding to the strong enhancer element (GPEI) in the upstream of rat glutathione S-transferase P (GST-P) gene. Yeast one-hybrid system was used to screen rat lung MATCHMAKER cDNA library to identify potential trans-factors that can interact with core sequence of GPEI(cGPEI). Electrophoresis mobility shift assay (EMSA) was used to analyze the binding of transfactors to cGPEI. cDNA fragments coding for the C-terminal part of the transcription factor c-Jun and rat adenine nucleotide translocator (ANT) were isolated. The binding of c-Jun and ANT to GPEI core sequence were confirmed. Rat c-jun transcriptional factor and ANT may interact with cGPEI. They could play an important role in the induced expression of GST-P gene.

Enzymatic Glycosylation by Transferases

NASA Astrophysics Data System (ADS)

Blixt, Ola; Razi, Nahid

Glycosyltransferases are important biological catalysts in cellular systems generating complex cell surface glycans involved in adhesion and signaling processes. Recent advances in glycoscience have increased the demands to access significant amount of glycans representing the glycome. Glycosyltransferases are now playing a key role for in vitro synthesis of oligosaccharides and the bacterial genome are increasingly utilized for cloning and over expression of active transferases in glycosylation reactions. This chapter highlights the recent progress towards preparative synthesis of oligosaccharides representing terminal sequences of glycoproteins and glycolipids using recombinant transferases. Transferases are also being explored in the context of solid-phase synthesis, immobilized on resins and over expression in vivo by engineered bacteria.

Global deletion of glutathione S-Transferase A4 exacerbates developmental nonalcoholic steatohepatitis

USDA-ARS?s Scientific Manuscript database

We established a mouse model of developmental nonalcoholic steatohepatitis (NASH) by feeding a high polyunsaturated fat liquid diet to female glutathione-S-transferase 4-4 (Gsta4-/-)/peroxisome proliferator activated receptor a (Ppara-/-) double knockout 129/SvJ mice for 12 weeks from weaning. We us...

GLUTATHIONE S-TRANSFERASE THETA 1-1-DEPENDENT METABOLISM OF THE DISINFECTION BYPRODUCT BROMODICHLOROMETHANE

EPA Science Inventory

ABSTRACT
Bromodichloromethane (BDCM), a prevalent drinking water disinfection by-product, was previously shown to be mutagenic in Salmonella expressing glutathione S-transferase (GST) theta 1-1 (GST T1-1). In the present study, in vitro experiments were performed to study the...

S-Nitrosation destabilizes glutathione transferase P1-1.

PubMed

Balchin, David; Stoychev, Stoyan H; Dirr, Heini W

2013-12-23

Protein S-nitrosation is a post-translational modification that regulates the function of more than 500 human proteins. Despite its apparent physiological significance, S-nitrosation is poorly understood at a molecular level. Here, we investigated the effect of S-nitrosation on the activity, structure, stability, and dynamics of human glutathione transferase P1-1 (GSTP1-1), an important detoxification enzyme ubiquitous in aerobes. S-Nitrosation at Cys47 and Cys101 reduces the activity of the enzyme by 94%. Circular dichroism spectroscopy, acrylamide quenching, and amide hydrogen-deuterium exchange mass spectrometry experiments indicate that the loss of activity is caused by the introduction of local disorder at the active site of GSTP1-1. Furthermore, the modification destabilizes domain 1 of GSTP1-1 against denaturation, smoothing the unfolding energy landscape of the protein and introducing a refolding defect. In contrast, S-nitrosation at Cys101 alone introduces a refolding defect in domain 1 but compensates by stabilizing the domain kinetically. These data elucidate the physical basis for the regulation of GSTP1-1 by S-nitrosation and provide general insight into the consequences of S-nitrosation on protein stability and dynamics.

Glutathione S-transferase P1 (GSTP1) directly influences platinum drug chemosensitivity in ovarian tumour cell lines.

PubMed

Sawers, L; Ferguson, M J; Ihrig, B R; Young, H C; Chakravarty, P; Wolf, C R; Smith, G

2014-09-09

Chemotherapy response in ovarian cancer patients is frequently compromised by drug resistance, possibly due to altered drug metabolism. Platinum drugs are metabolised by glutathione S-transferase P1 (GSTP1), which is abundantly, but variably expressed in ovarian tumours. We have created novel ovarian tumour cell line models to investigate the extent to which differential GSTP1 expression influences chemosensitivity. Glutathione S-transferase P1 was stably deleted in A2780 and expression significantly reduced in cisplatin-resistant A2780DPP cells using Mission shRNA constructs, and MTT assays used to compare chemosensitivity to chemotherapy drugs used to treat ovarian cancer. Differentially expressed genes in GSTP1 knockdown cells were identified by Illumina HT-12 expression arrays and qRT-PCR analysis, and altered pathways predicted by MetaCore (GeneGo) analysis. Cell cycle changes were assessed by FACS analysis of PI-labelled cells and invasion and migration compared in quantitative Boyden chamber-based assays. Glutathione S-transferase P1 knockdown selectively influenced cisplatin and carboplatin chemosensitivity (2.3- and 4.83-fold change in IC50, respectively). Cell cycle progression was unaffected, but cell invasion and migration was significantly reduced. We identified several novel GSTP1 target genes and candidate platinum chemotherapy response biomarkers. Glutathione S-transferase P1 has an important role in cisplatin and carboplatin metabolism in ovarian cancer cells. Inter-tumour differences in GSTP1 expression may therefore influence response to platinum-based chemotherapy in ovarian cancer patients.

DNA BINDING POTENTIAL OF BROMODICHLOROMETHANE MEDIATED BY GLUTATHIONE S-TRANSFERASE THETA 1-1

EPA Science Inventory

DNA BINDING POTENTIAL OF BROMODICHLOROMETHANE MEDIATED BY GLUTATHIONE S-TRANSFERASE THETA 1-1. R A Pegram1 and M K Ross2. 2Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC; 1Pharmacokinetics Branch, NHEERL, ORD, United States Environmental Protection Ag...

A Tyrosine-Reactive Irreversible Inhibitor for Glutathione S-Transferase Pi (GSTP1)

PubMed Central

Crawford, L. A.; Weerapana, E.

2016-01-01

Glutathione S-Transferase Pi (GSTP1) mediates cellular defense against reactive electrophiles. Here, we report LAS17, a dichlorotriazine-containing compound that irreversibly inhibits GSTP1 and is selective for GSTP1 within cellular proteomes. Mass spectrometry and mutational studies identified Y108 as the site of modification, providing a unique mode of GSTP1 inhibition. PMID:27113843

A tyrosine-reactive irreversible inhibitor for glutathione S-transferase Pi (GSTP1).

PubMed

Crawford, L A; Weerapana, E

2016-05-24

Glutathione S-transferase Pi (GSTP1) mediates cellular defense against reactive electrophiles. Here, we report LAS17, a dichlorotriazine-containing compound that irreversibly inhibits GSTP1 and is selective for GSTP1 within cellular proteomes. Mass spectrometry and mutational studies identified Y108 as the site of modification, providing a unique mode of GSTP1 inhibition.

Identification of Protein-Protein Interactions with Glutathione-S-Transferase (GST) Fusion Proteins.

PubMed

Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

2007-08-01

INTRODUCTIONGlutathione-S-transferase (GST) fusion proteins have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis. This article describes the use of GST fusion proteins as probes for the identification of protein-protein interactions.

Glutathione S-transferase P1 (GSTP1) directly influences platinum drug chemosensitivity in ovarian tumour cell lines

PubMed Central

Sawers, L; Ferguson, M J; Ihrig, B R; Young, H C; Chakravarty, P; Wolf, C R; Smith, G

2014-01-01

Background: Chemotherapy response in ovarian cancer patients is frequently compromised by drug resistance, possibly due to altered drug metabolism. Platinum drugs are metabolised by glutathione S-transferase P1 (GSTP1), which is abundantly, but variably expressed in ovarian tumours. We have created novel ovarian tumour cell line models to investigate the extent to which differential GSTP1 expression influences chemosensitivity. Methods: Glutathione S-transferase P1 was stably deleted in A2780 and expression significantly reduced in cisplatin-resistant A2780DPP cells using Mission shRNA constructs, and MTT assays used to compare chemosensitivity to chemotherapy drugs used to treat ovarian cancer. Differentially expressed genes in GSTP1 knockdown cells were identified by Illumina HT-12 expression arrays and qRT–PCR analysis, and altered pathways predicted by MetaCore (GeneGo) analysis. Cell cycle changes were assessed by FACS analysis of PI-labelled cells and invasion and migration compared in quantitative Boyden chamber-based assays. Results: Glutathione S-transferase P1 knockdown selectively influenced cisplatin and carboplatin chemosensitivity (2.3- and 4.83-fold change in IC50, respectively). Cell cycle progression was unaffected, but cell invasion and migration was significantly reduced. We identified several novel GSTP1 target genes and candidate platinum chemotherapy response biomarkers. Conclusions: Glutathione S-transferase P1 has an important role in cisplatin and carboplatin metabolism in ovarian cancer cells. Inter-tumour differences in GSTP1 expression may therefore influence response to platinum-based chemotherapy in ovarian cancer patients. PMID:25010864

Identification of Glutathione S-Transferase Pi as a Protein Involved in Parkinson Disease Progression

PubMed Central

Shi, Min; Bradner, Joshua; Bammler, Theo K.; Eaton, David L.; Zhang, JianPeng; Ye, ZuCheng; Wilson, Angela M.; Montine, Thomas J.; Pan, Catherine; Zhang, Jing

2009-01-01

Parkinson disease (PD) typically affects the cortical regions during the later stages of disease, with neuronal loss, gliosis, and formation of diffuse cortical Lewy bodies in a significant portion of patients with dementia. To identify novel proteins involved in PD progression, we prepared synaptosomal fractions from the frontal cortices of pathologically verified PD patients at different stages along with age-matched controls. Protein expression profiles were compared using a robust quantitative proteomic technique. Approximately 100 proteins displayed significant differences in their relative abundances between PD patients at various stages and controls; three of these proteins were validated using independent techniques. One of the confirmed proteins, glutathione S-transferase Pi, was further investigated in cellular models of PD, demonstrating that its level was intimately associated with several critical cellular processes that are directly related to neurodegeneration in PD. These results have, for the first time, suggested that the levels of glutathione S-transferase Pi may play an important role in modulating the progression of PD. PMID:19498008

21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

Molecular mimicry between cockroach and helminth glutathione S-transferases promotes cross-reactivity and cross-sensitization

USDA-ARS?s Scientific Manuscript database

The extensive similarities between helminth proteins and allergens are thought to contribute to helminth-driven allergic sensitization. We investigated the molecular and structural similarities between Bla g 5, a major glutathione-S transferase (GST) allergen of cockroaches, and the GST of Wucherer...

Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S-transferase π

PubMed Central

Ralat, Luis A.; Colman, Roberta F.

2003-01-01

Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase π (GST π): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST π with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST π at pH 7.0 and 25°C as assayed using mBBr as substrate, with a lesser effect on the enzyme’s use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST π with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites. PMID:14573868

Bisubstrate Kinetics of Glutathione S-Transferase: A Colorimetric Experiment for the Introductory Biochemistry Laboratory

ERIC Educational Resources Information Center

Stefanidis, Lazaros; Scinto, Krystal V.; Strada, Monica I.; Alper, Benjamin J.

2018-01-01

Most biochemical transformations involve more than one substrate. Bisubstrate enzymes catalyze multiple chemical reactions in living systems and include members of the transferase, oxidoreductase, and ligase enzyme classes. Working knowledge of bisubstrate enzyme kinetic models is thus of clear importance to the practicing biochemist. However,…

The glutathione-S-transferase Mu 1 null genotype modulates ozone-induced airway inflammation in humans*

EPA Science Inventory

Background: The Glutathione-S-Transferase Mu 1 null genotype has been reported to be a risk factor for acute respiratory disease associated with increases in ambient air ozone. Ozone is known to cause an immediate decrease in lung function and increased airway inflammation. Howev...

Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S-transferase pi.

PubMed

Ralat, Luis A; Colman, Roberta F

2003-11-01

Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase pi (GST pi): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST pi with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST pi at pH 7.0 and 25 degrees C as assayed using mBBr as substrate, with a lesser effect on the enzyme's use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST pi with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites.

Involvement of glutathione/glutathione S-transferase antioxidant system in butyrate-inhibited vascular smooth muscle cell proliferation.

PubMed

Ranganna, Kasturi; Mathew, Omana P; Yatsu, Frank M; Yousefipour, Zivar; Hayes, Barbara E; Milton, Shirlette G

2007-11-01

Vascular smooth muscle cell (VSMC) proliferation is an important etiological factor in vascular proliferative diseases such as primary atherosclerosis, hypertension, arterial and in-stent restenosis, and transplant vasculopathy. Our studies established that butyrate, a bacterial fermentation product of dietary fiber and a chromatin modulator, is a potent inhibitor of VSMC proliferation. The cardiovascular health benefits of a high-fiber diet, the principle source of butyrate in the body, have been known for a long time, however, very little is known about the antiatherogenic potential of butyrate. Because oxidative stress plays an important role in the pathogenesis of atherosclerosis, we examined involvement of the glutathione/glutathione S-transferase (GST) antioxidant system in butyrate's inhibition of VSMC proliferation. Treatment of proliferating VSMCs with butyrate leads to the induction of several GSTs. Interestingly, our study also demonstrated the nuclear localization of GST-P1 (GST-7-7), which is considered to be a cytosolic protein; this was demonstrated using immunostaining and was corroborated by western blotting. Also, the butyrate-induced antiproliferative action, and the induction of GST-P1 and its nuclear localization are downregulated when butyrate is withdrawn. Furthermore, assessment of intracellular glutathione levels reveals their augmentation by butyrate. Conversely, butyrate treatment reduces the levels of reactive oxygen species in VSMCs. Collectively, the butyrate-treatment-related increase in glutathione content, the reduction in reactive oxygen species, the upregulation of GST and the nuclear localization of GST-P1 in growth-arrested VSMCs imply that butyrate's antiproliferative action involves modulation of the cellular redox state. Thus, induction of the glutathione/GST antioxidant system appears to have other regulatory role(s) besides detoxification and regulation of the cellular redox state, for example, cell-cycle control and cell

Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S -Transferases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stoddard, Ethan G.; Killinger, Bryan J.; Nair, Reji N.

Glutathione S-transferases (GSTs) comprise a highly diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione to various endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured by colorimetric assays, measurement of the individual contribution from specific isoforms and their contribution to the detoxification of xenobiotics in complex biological samples has not been possible. For this reason, we have developed two activity-based probes that characterize active glutathione transferases in mammalian tissues. The GST active site is comprised of a glutathione binding “G site” and a distinct substrate binding “Hmore » site”. Therefore, we developed (1) a glutathione-based photoaffinity probe (GSH-ABP) to target the “G site”, and (2) a probe designed to mimic a substrate molecule and show “H site” activity (GST-ABP). The GSH-ABP features a photoreactive moiety for UV-induced covalent binding to GSTs and glutathione-binding enzymes. The GST-ABP is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and “G” and “H” site specificity was carried out using a series of competitors in liver homogenates. Herein, we present robust tools for the novel characterization of enzyme- and active site-specific GST activity in mammalian model systems.« less

S-Glutathionylation of Keap1: a new role for glutathione S-transferase pi in neuronal protection.

PubMed

Carvalho, Andreia Neves; Marques, Carla; Guedes, Rita C; Castro-Caldas, Margarida; Rodrigues, Elsa; van Horssen, Jack; Gama, Maria João

2016-05-01

Oxidative stress is a key pathological feature of Parkinson's disease (PD). Glutathione S-transferase pi (GSTP) is a neuroprotective antioxidant enzyme regulated at the transcriptional level by the antioxidant master regulator nuclear factor-erythroid 2-related factor 2 (Nrf2). Here, we show for the first time that upon MPTP-induced oxidative stress, GSTP potentiates S-glutathionylation of Kelch-like ECH-associated protein 1 (Keap1), an endogenous repressor of Nrf2, in vivo. S-glutathionylation of Keap1 leads to Nrf2 activation and subsequently increases expression of GSTP. This positive feedback regulatory loop represents a novel mechanism by which GSTP elicits antioxidant protection in the brain. © 2016 Federation of European Biochemical Societies.

Drug resistance in epithelial ovarian cancer: P-glycoprotein and glutation S-transferase. Can they play an important role in detecting response to platinum-based chemotherapy as a first-line therapy.

PubMed

Simşek, T; Ozbilim, G; Gülkesen, H; Kaya, H; Sargin, F; Karaveli, S

2001-01-01

Drug resistance is important for the treatment of ovarian cancer. P-glycoprotein and glutation S-transferase as resistance markers play an important role in the effectivity of chemotherapeutical agents. The role of P-glycoprotein and glutation S-transferase in the treatment of epithelial ovarian cancer is not well understood. We investigated the relation between P-glycoprotein and glutation S-transferase level for response to platinum-based chemotherapy in epithelial ovarian cancer. We reviewed 30 cases diagnosed as epithelial ovarian cancer and treated with platinum-based chemotherapy in the Department of Obstetrics and Gynecology, Akdeniz University School of Medicine. The material was attained from initial parafin-embeded blocks stained for P-glycoprotein and glutation S-transferase. The cases that were diagnosed and treated before attending our clinic were not enrolled in the study. Mean age was 58.2 (25-70) and mean gravida 4.1 (0-10). Twenty-four patients (80%) were glutation S-transferase positive. Three cases (10%) out of 30 had positive reaction for P-glycoprotein. No difference was revealed regarding chemotherapy response rate among the cases showing glutation S-transferase positivity and P-glycoprotein negativity. Detection of glutation S-transferase and P-glycoprotein levels in epithelial ovarian cancer tissue is not important for response to platinum-based chemotherapy as a first line.

The structure of a zeta class glutathione S-transferase from Arabidopsis thaliana: characterisation of a GST with novel active-site architecture and a putative role in tyrosine catabolism.

PubMed

Thom, R; Dixon, D P; Edwards, R; Cole, D J; Lapthorn, A J

2001-05-18

The cis-trans isomerisation of maleylacetoacetate to fumarylacetoacetate is the penultimate step in the tyrosine/phenylalanine catabolic pathway and has recently been shown to be catalysed by glutathione S-transferase enzymes belonging to the zeta class. Given this primary metabolic role it is unsurprising that zeta class glutathione S-transferases are well conserved over a considerable period of evolution, being found in vertebrates, plants, insects and fungi. The structure of this glutathione S-transferase, cloned from Arabidopsis thaliana, has been solved by single isomorphous replacement with anomalous scattering and refined to a final crystallographic R-factor of 19.6% using data from 25.0 A to 1.65 A. The zeta class enzyme adopts the canonical glutathione S-transferase fold and forms a homodimer with each subunit consisting of 221 residues. In agreement with structures of glutathione S-transferases from the theta and phi classes, a serine residue (Ser17) is present in the active site, at a position that would allow it to stabilise the thiolate anion of glutathione. Site-directed mutagenesis of this residue confirms its importance in catalysis. In addition, the role of a highly conserved cysteine residue (Cys19) present in the active site of the zeta class glutathione S-transferase enzymes is discussed. Copyright 2001 Academic Press.

Echinococcus granulosus: Evidence of a heterodimeric glutathione transferase built up by phylogenetically distant subunits.

PubMed

Arbildi, Paula; La-Rocca, Silvana; Lopez, Veronica; Da-Costa, Natalia; Fernandez, Veronica

2017-01-01

In the cestode parasite Echinococcus granulosus, three phylogenetically distant cytosolic glutathione transferases (GSTs) (EgGST1, 2 and 3) were identified. Interestingly, the C-terminal domains of EgGST3 and EgGST2 but not EgGST1, exhibit all amino acids involved in Sigma-class GST dimerization. Here, we provide evidence indicating that EgGST2 and EgGST3 naturally form a heterodimeric structure (EgGST2-3), and also we report the enzymatic activity of the recombinant heterodimer. EgGST2-3 might display novel properties able to influence the infection establishment. This is the first report of a stable heterodimeric GST built up by phylogenetically distant subunits. Copyright © 2016 Elsevier B.V. All rights reserved.

Polymorphisms of Glutathione S-transferases Omega-1 among ethnic populations in China

PubMed Central

Fu, Songbo; Wu, Jie; Chen, Feng; Sun, Dianjun; Fu, Songbin

2008-01-01

Background Glutathione S-transferases (GSTs) is a genetic factor for many diseases and exhibits great diversities among various populations. We assessed association of the genotypes of Glutathione S-transferases Omega-1 (GSTO1) A140D with ethnicity in China. Results Peripheral blood samples were obtained from 1314 individuals from 14 ethnic groups. Polymorphisms of GSTO1 A140D were measured using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Logistic regression was employed to adjustment for regional factor. The frequency of GSTO1 140A allele was 15.49% in the total 14 ethnic populations. Compared to Han ethnic group, two ethnic populations were more likely to have AA or CA genotype [odds ratio (OR): 1.77, 95% confidence interval (95% CI): 1.05–2.98 for Uygur and OR: 1.78, 95% CI: 1.18–2.69 for Hui]. However, there were no statistically significant differences across 14 ethnic groups when region factor was adjusted. In Han ethnicity, region was significantly associated with AA or CA genotype. Han individuals who resided in North-west of China were more likely to have these genotypes than those in South of China (OR: 1.63, 95% CI: 1.21–2.20). Conclusion The prevalence of the GSTO1 140A varied significantly among different regional populations in China, which showed that geography played a more important role in the population differentiation for this allele than the ethnicity/race. PMID:18400112

Glutathione S-transferase M1 polymorphism and endometriosis susceptibility: a meta-analysis.

PubMed

Li, H; Zhang, Y

2015-02-01

Many studies have investigated the association between glutathione S-transferase M1 (GSTM1) null genotype and the risk of endometriosis. However, the effect of the GSTM1 null genotype on endometriosis is still unclear because of apparent inconsistencies among those studies. A meta-analysis was performed to characterize the relationship more accurately. PubMed, Embase, and Web of Science were searched. To derive a more precise estimation of the relationship, a meta-analysis was performed. We estimated the summary odds ratio (OR) with a 95% confidence interval (95% CI) to assess the association. Up to 24 case-control studies with 2,684 endometriosis cases and 3,119 control cases were included into this meta-analysis. Meta-analysis of the 24 studies showed that GSTM1 null genotype was associated with the risk of endometriosis (random effects OR=1.66, 95% CI 1.23 to 2.24). In the subgroup analysis by ethnicity, increased risks were found for both Caucasians (OR=1.26, 95% CI 1.04-1.51) and Asians (OR=1.28, 95% CI 1.06-1.55). No evidence of publication bias was observed. In conclusion, this meta-analysis suggests that the GSTM1 null genotype increases the overall risk of endometriosis. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

A model to environmental monitoring based on glutathione-S-transferase activity and branchial lesions in catfish

NASA Astrophysics Data System (ADS)

Neta, Raimunda Nonata Fortes Carvalho; Torres, Audalio Rebelo

2017-11-01

In this work, we validate the glutathione-S-transferase and branchial lesions as biomarkers in catfish Sciades herzbergii to obtain a predictive model of the environmental impact effects in a harbor of Brazil. The catfish were sampled from a port known to be contaminated with heavy metals and organic compounds and from a natural reserve in São Marcos Bay, Maranhão. Two biomarkers, hepatic glutathione S-transferase (GST) activity and branchial lesions were analyzed. The values for GST activity were modeled with the occurrence of branchial lesions by fitting a third order polynomial. Results from the mathematical model indicate that GST activity has a strong polynomial relationship with the occurrence of branchial lesions in both the wet and the dry seasons, but only at the polluted port site. Our mathematic model indicates that when the GST ceases to act, serious branchial lesions are observed in the catfish of the contaminated port area.

Homogentisate solanesyl transferase (HST) cDNA’s in maize

USDA-ARS?s Scientific Manuscript database

Maize white seedling 3 (w3) has served as a model albino-seedling mutant since its discovery in 1923. We show that the w3 phenotype is caused by disruptions in homogentisate solanesyl transferase (HST), an enzyme that catalyzes the committed step in plastoquinone-9 (PQ9) biosynthesis. This reaction ...

Glutathione S-transferase M1 (GSTM1) polymorphisms and lung cancer: a literature-based systematic HuGE review and meta-analysis.

PubMed

Carlsten, C; Sagoo, G S; Frodsham, A J; Burke, W; Higgins, J P T

2008-04-01

Multiple genes have been studied for potential associations with lung cancer. The gene most frequently associated with increased risk has been glutathione S-transferase M1 (GSTM1). The glutathione S-transferase enzyme family is known to catalyze detoxification of electrophilic compounds, including carcinogens, therapeutic drugs, environmental toxins, and products of oxidative stress. In this review, the authors summarize the available evidence associating lung cancer with the GSTM1 gene. They describe results from an updated meta-analysis of 98 published genetic association studies investigating the relation between the GSTM1 null variant and lung cancer risk including 19,638 lung cancer cases and 25,266 controls (counting cases and controls in each study only once). All studies considered, the GSTM1 null variant was associated with an increased risk of lung cancer (odds ratio (OR) = 1.22, 95% confidence interval (CI): 1.14, 1.30), but no increase in risk was seen (OR = 1.01, 95% CI: 0.91, 1.12) when only the five largest studies (>500 cases each) were considered. Furthermore, while GSTM1 null status conferred a significantly increased risk of lung cancer to East Asians (OR = 1.38, 95% CI: 1.24, 1.55), such a genotype did not confer increased risk to Caucasians. More data regarding the predictive value of GSTM1 genetic testing are needed before population-based testing may be reasonably considered.

Revealing the role of glutathione S-transferase omega in age-at-onset of Alzheimer and Parkinson diseases.

PubMed

Li, Yi-Ju; Scott, William K; Zhang, Ling; Lin, Ping-I; Oliveira, Sofia A; Skelly, Tara; Doraiswamy, Maurali P; Welsh-Bohmer, Kathleen A; Martin, Eden R; Haines, Jonathan L; Pericak-Vance, Margaret A; Vance, Jeffery M

2006-08-01

We previously reported a linkage region on chromosome 10q for age-at-onset (AAO) of Alzheimer (AD) and Parkinson (PD) diseases. Glutathione S-transferase, omega-1 (GSTO1) and the adjacent gene GSTO2, located in this linkage region, were then reported to associate with AAO of AD and PD. To examine whether GSTO1 and GSTO2 (hereafter referred to as GSTO1h) are responsible for the linkage evidence, we identified 39 families in AD that lead to our previous linkage and association findings. The evidence of linkage and association was markedly diminished after removing these 39 families from the analyses, thus providing support that GSTO1h drives the original linkage results. The maximum average AAO delayed by GSTO1h SNP 7-1 (rs4825, A nucleotide) was 6.8 (+/-4.41) years for AD and 8.6(+/-5.71) for PD, respectively. This is comparable to the magnitude of AAO difference by APOE-4 in these same AD and PD families. These findings suggest the presence of genetic heterogeneity for GSTO1h's effect on AAO, and support GSTO1h's role in modifying AAO in these two disorders.

Neuroantibodies (NAB) in African-American Children: Associations with Gender, Glutathione-S-Transferase (GST)Pi Polymorphisms (SNP) and Heavy Metals

EPA Science Inventory

CONTACT (NAME ONLY): Hassan El-Fawal Abstract Details PRESENTATION TYPE: Platform or Poster CURRENT CATEGORY: Neurodegenerative Disease | Biomarkers | Neurotoxicity, Metals KEYWORDS: Autoantibodies, Glutathione-S-Transferase, DATE/TIME LAST MODIFIED: DATE/TIME SUBMITTED: Abs...

Primary and secondary structural analyses of glutathione S-transferase pi from human placenta.

PubMed

Ahmad, H; Wilson, D E; Fritz, R R; Singh, S V; Medh, R D; Nagle, G T; Awasthi, Y C; Kurosky, A

1990-05-01

The primary structure of glutathione S-transferase (GST) pi from a single human placenta was determined. The structure was established by chemical characterization of tryptic and cyanogen bromide peptides as well as automated sequence analysis of the intact enzyme. The structural analysis indicated that the protein is comprised of 209 amino acid residues and gave no evidence of post-translational modifications. The amino acid sequence differed from that of the deduced amino acid sequence determined by nucleotide sequence analysis of a cDNA clone (Kano, T., Sakai, M., and Muramatsu, M., 1987, Cancer Res. 47, 5626-5630) at position 104 which contained both valine and isoleucine whereas the deduced sequence from nucleotide sequence analysis identified only isoleucine at this position. These results demonstrated that in the one individual placenta studied at least two GST pi genes are coexpressed, probably as a result of allelomorphism. Computer assisted consensus sequence evaluation identified a hydrophobic region in GST pi (residues 155-181) that was predicted to be either a buried transmembrane helical region or a signal sequence region. The significance of this hydrophobic region was interpreted in relation to the mode of action of the enzyme especially in regard to the potential involvement of a histidine in the active site mechanism. A comparison of the chemical similarity of five known human GST complete enzyme structures, one of pi, one of mu, two of alpha, and one microsomal, gave evidence that all five enzymes have evolved by a divergent evolutionary process after gene duplication, with the microsomal enzyme representing the most divergent form.

Differential transcription of cytochrome P450s and glutathione S transferases in DDT-susceptible and resistant Drosophila melanogaster strains in response to DDT and oxidative stress

USDA-ARS?s Scientific Manuscript database

Metabolic DDT resistance in Drosophila melanogaster has previously been associated with constitutive over-transcription of cytochrome P450s. Increased P450 activity has also been associated with increased oxidative stress. In contrast, over-transcription of glutathione S transferases (GSTs) has been...

Glutathione S-Transferase Protein Expression in Different Life Stages of Zebrafish (Danio rerio)

PubMed Central

Tierbach, Alena; Groh, Ksenia J; Schönenberger, René; Schirmer, Kristin

2018-01-01

Abstract Zebrafish is a widely used animal model in biomedical sciences and toxicology. Although evidence for the presence of phases I and II xenobiotic defense mechanisms in zebrafish exists on the transcriptional and enzyme activity level, little is known about the protein expression of xenobiotic metabolizing enzymes. Given the important role of glutathione S-transferases (GSTs) in phase II biotransformation, we analyzed cytosolic GST proteins in zebrafish early life stages and different organs of adult male and female fish, using a targeted proteomics approach. The established multiple reaction monitoring-based assays enable the measurement of the relative abundance of specific GST isoenzymes and GST classes in zebrafish through a combination of proteotypic peptides and peptides shared within the same class. GSTs of the classes alpha, mu, pi and rho are expressed in zebrafish embryo as early as 4 h postfertilization (hpf). The majority of GST enzymes are present at 72 hpf followed by a continuous increase in expression thereafter. In adult zebrafish, GST expression is organ dependent, with most of the GST classes showing the highest expression in the liver. The expression of a wide range of cytosolic GST isoenzymes and classes in zebrafish early life stages and adulthood supports the use of zebrafish as a model organism in chemical-related investigations. PMID:29361160

Combined glutathione S transferase M1/T1 null genotypes is associated with type 2 diabetes mellitus

PubMed Central

POROJAN, MIHAI D.; BALA, CORNELIA; ILIES, ROXANA; CATANA, ANDREEA; POPP, RADU A.; DUMITRASCU, DAN L.

2015-01-01

Background Due to new genetic insights, a considerably large number of genes and polymorphic gene variants are screened and linked with the complex pathogenesis of type 2 diabetes (DM). Our study aimed to investigate the association between the two isoforms of the glutathione S-transferase genes (Glutathione S transferase isoemzyme type M1- GSTM1 and Glutathione S transferase isoemzyme type T1-GSTT1) and the prevalence of DM in the Northern Romanian population. Methods We conducted a cross-sectional, randomized, case-control study evaluating the frequency of GSTM1 and GSTT1 null alleles in patients diagnosed with DM. A total of 106 patients diagnosed with DM and 124 healthy controls were included in the study. GSTM1 and GSTT1 null alleles genotyping was carried out using Multiplex PCR amplification of relevant gene fragments, followed by gel electrophoresis analysis of the resulting amplicons. Results Molecular analysis did not reveal an increased frequency of the null GSTM1 and GSTT1 alleles (mutant genotypes) respectively in the DM group compared to controls (p=0.171, OR=1.444 CI=0.852–2.447; p=0.647, OR=0.854, CI=0.436–1.673). Nevertheless, the combined GSTM1/GSTT1 null genotypes were statistically significantly higher in DM patients compared to control subjects (p=0.0021, OR=0.313, CI=0.149–0.655) Conclusions The main finding of our study is that the combined, double GSTM1/GSTT1 null genotypes are to be considered among the polymorphic genetic risk factors for type 2 DM. PMID:26528065

Epidermal growth factor regulation of glutathione S-transferase gene expression in the rat is mediated by class Pi glutathione S-transferase enhancer I.

PubMed

Matsumoto, M; Imagawa, M; Aoki, Y

2000-07-01

Using chloramphenicol acetyltransferase assays we showed that epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and 3,3',4,4',5-pentachlorobiphenyl (PenCB) induce class Pi glutathione S-transferase (GSTP1) in primary cultured rat liver parenchymal cells. GSTP1 enhancer I (GPEI), which is required for the stimulation of GSTP1 expression by PenCB, also mediates EGF and TGF alpha stimulation of GSTP1 gene expression. However, hepatocyte growth factor and insulin did not stimulate GPEI-mediated gene expression. On the other hand, the antioxidant reagents butylhydroxyanisole and t-butylhydroquinone, stimulated GPEI-mediated gene expression, but the level of GSTP1 mRNA was not elevated. Our observations suggest that EGF and TGF alpha induce GSTP1 by the same signal transduction pathway as PenCB. Since the sequence of GPEI is similar to that of the antioxidant responsive element (ARE), some factors which bind to ARE might play a role in GPEI-mediated gene expression.

Epidermal growth factor regulation of glutathione S-transferase gene expression in the rat is mediated by class Pi glutathione S-transferase enhancer I.

PubMed Central

Matsumoto, M; Imagawa, M; Aoki, Y

2000-01-01

Using chloramphenicol acetyltransferase assays we showed that epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and 3,3',4,4',5-pentachlorobiphenyl (PenCB) induce class Pi glutathione S-transferase (GSTP1) in primary cultured rat liver parenchymal cells. GSTP1 enhancer I (GPEI), which is required for the stimulation of GSTP1 expression by PenCB, also mediates EGF and TGF alpha stimulation of GSTP1 gene expression. However, hepatocyte growth factor and insulin did not stimulate GPEI-mediated gene expression. On the other hand, the antioxidant reagents butylhydroxyanisole and t-butylhydroquinone, stimulated GPEI-mediated gene expression, but the level of GSTP1 mRNA was not elevated. Our observations suggest that EGF and TGF alpha induce GSTP1 by the same signal transduction pathway as PenCB. Since the sequence of GPEI is similar to that of the antioxidant responsive element (ARE), some factors which bind to ARE might play a role in GPEI-mediated gene expression. PMID:10861232

The synthesis of ethacrynic acid thiazole derivatives as glutathione S-transferase pi inhibitors.

PubMed

Li, Ting; Liu, Guyue; Li, Hongcai; Yang, Xinmei; Jing, Yongkui; Zhao, Guisen

2012-04-01

Glutathione S-transferase pi (GSTpi) is a phase II enzyme which protects cells from death and detoxifies chemotherapeutic agents in cancer cells. Ethacrynic acid (EA) is a weak GSTpi inhibitor. Structure modifications were done to improve the ability of EA to inhibit GSTpi activity. Eighteen EA thiazole derivatives were designed and synthesized. Compounds 9a, 9b and 9c with a replacement of carboxyl group of EA by a heterocyclic thiazole exhibited improvement over EA to inhibit GSTpi activity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Hibiscus cannabinus feruloyl-coa:monolignol transferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilkerson, Curtis; Ralph, John; Withers, Saunia

The invention relates to isolated nucleic acids encoding a feruloyl-CoA:monolignol transferase and feruloyl-CoA:monolignol transferase enzymes. The isolated nucleic acids and/or the enzymes enable incorporation of monolignol ferulates into the lignin of plants, where such monolignol ferulates include, for example, p-coumaryl ferulate, coniferyl ferulate, and/or sinapyl ferulate. The invention also includes methods and plants that include nucleic acids encoding a feruloyl-CoA:monolignol transferase enzyme and/or feruloyl-CoA:monolignol transferase enzymes.

A study on effects of glutathione s-transferase from silkworm on CCL4-induced mouse liver injury.

PubMed

Yan, Hui; Gui, Zhongzheng; Wang, Bochu

2011-01-01

To assess the hepatoprotective activity of Glutathione S-transferase(GSTsw), extracted and purified from silkworm, in experimental acute mice liver injury and explore mechanisms. Mice were divided into five groups: control group, carbon tetrachloride (CCl4) group, and three treatment groups that received CCl4 and GSTsw at doses of 0.083 mg•g(-1), 0.0415 mg•g(-1) and 0.0207 mg•g(-1) for 3 days. ALT in serum, GST, SOD and T-AOC in liver tissue homogenate, and changes in liver pathology in the five groups were studied. CCl4 administration led to pathological and biochemical evidence of liver injury as compared to untreated controls. GSTsw administration led to significant protection against CCl4-induced changes in liver pathology. It was also associatedwith significantly lower serum ALT levels, higher GST-SOD and T-AOC level in live tissue homogenate. Thus, GSTsw showed protective activity against CCl4-induced hepatotoxicity in mice.

Novel role for glutathione S-transferase pi. Regulator of protein S-Glutathionylation following oxidative and nitrosative stress.

PubMed

Townsend, Danyelle M; Manevich, Yefim; He, Lin; Hutchens, Steven; Pazoles, Christopher J; Tew, Kenneth D

2009-01-02

Glutathione S-transferase Pi (GSTpi) is a marker protein in many cancers and high levels are linked to drug resistance, even when the selecting drug is not a substrate. S-Glutathionylation of proteins is critical to cellular stress response, but characteristics of the forward reaction are not known. Our results show that GSTpi potentiates S-glutathionylation reactions following oxidative and nitrosative stress in vitro and in vivo. Mutational analysis indicated that the catalytic activity of GST is required. GSTpi is itself redox-regulated. S-Glutathionylation on Cys47 and Cys101 autoregulates GSTpi, breaks ligand binding interactions with c-Jun NH2-terminal kinase (JNK), and causes GSTpi multimer formation, all critical to stress response. Catalysis of S-glutathionylation at low pK cysteines in proteins is a novel property for GSTpi and may be a cause for its abundance in tumors and cells resistant to a range of mechanistically unrelated anticancer drugs.

Construction of fusion vectors of corynebacteria: expression of glutathione-S-transferase fusion protein in Corynebacterium acetoacidophilum ATCC 21476.

PubMed

Srivastava, Preeti; Deb, J K

2002-07-02

A series of fusion vectors containing glutathione-S-transferase (GST) were constructed by inserting GST fusion cassette of Escherichia coli vectors pGEX4T-1, -2 and -3 in corynebacterial vector pBK2. Efficient expression of GST driven by inducible tac promoter of E. coli was observed in Corynebacterium acetoacidophilum. Fusion of enhanced green fluorescent protein (EGFP) and streptokinase genes in this vector resulted in the synthesis of both the fusion proteins. The ability of this recombinant organism to produce several-fold more of the product in the extracellular medium than in the intracellular space would make this system quite attractive as far as the downstream processing of the product is concerned.

Feruloyl-CoA:monolignol transferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilkerson, Curtis; Ralph, John; Withers, Saunia

The invention relates to nucleic acids encoding a feruloyl-CoA:monolignol transferase and the feruloyl-CoA:monolignol transferase enzyme that enables incorporation of monolignol ferulates, for example, including p-coumaryl ferulate, coniferyl ferulate, and sinapyl ferulate, into the lignin of plants.

Glutathione S-transferase mediates an ageing response to mitochondrial dysfunction

PubMed Central

Dancy, Beverley M.; Brockway, Nicole; Ramadasan-Nair, Renjini; Yang, Yoing; Sedensky, Margaret M.; Morgan, Philip G.

2016-01-01

To understand primary mitochondrial disease, we utilized a complex I-deficient Caenorhabditis elegans mutant, gas-1. These animals strongly upregulate the expression of gst-14 (encoding a glutathione S-transferase). Knockdown of gst-14 dramatically extends the lifespan of gas-1 and increases hydroxynonenal (HNE) modified mitochondrial proteins without improving complex I function. We observed no change in reactive oxygen species levels as measured by Mitosox staining, consistent with a potential role of GST-14 in HNE clearance. The upregulation of gst-14 in gas-1 animals is specific to the pharynx. These data suggest that an HNE-mediated response in the pharynx could be beneficial for lifespan extension in the context of complex I dysfunction in C. elegans. Thus, whereas HNE is typically considered damaging, our work is consistent with recent reports of its role in signaling, and that in this case, the signal is pro-longevity in a model of mitochondrial dysfunction. PMID:26704446

21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... of the enzyme galactose-1-phosphate uridyl transferase in erythrocytes (red blood cells... hereditary disease galactosemia (disorder of galactose metabolism) in infants. (b) Classification. Class II. ...

Light-induced carotenogenesis in Myxococcus xanthus: evidence that CarS acts as an anti-repressor of CarA.

PubMed

Whitworth, D E; Hodgson, D A

2001-11-01

In the bacterium Myxococcus xanthus, carotenoids are produced in response to illumination, as a result of expression of the crt carotenoid biosynthesis genes. The majority of crt genes are clustered in the crtEBDC operon, which is repressed in the dark by CarA. Genetic data suggest that, in the light, CarS is synthesized and achieves activation of the crtEBDC operon by removing the repressive action of CarA. As CarS contains no known DNA-binding motif, the relief of CarA-mediated repression was postulated to result from a direct interaction between these two proteins. Use of the yeast two-hybrid system demonstrated direct interaction between CarA and CarS. The two-hybrid system also implied that CarA and, possibly, CarS are capable of homodimerization. Direct evidence for CarS anti-repressor action was provided in vitro. A glutathione S-transferase (GST)-CarA protein fusion was shown to bind specifically to a palindromic operator sequence within the crtEBDC promoter. CarA was prevented from binding to its operator, and prebound CarA was removed by the addition of purified CarS. CarS is therefore an anti-repressor.

Antioxidant role of glutathione S-transferases: 4-Hydroxynonenal, a key molecule in stress-mediated signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singhal, Sharad S., E-mail: ssinghal@coh.org; Singh, Sharda P.; Singhal, Preeti

2015-12-15

4-Hydroxy-2-trans-nonenal (4HNE), one of the major end products of lipid peroxidation (LPO), has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. It has been known that glutathione S-transferases (GSTs) can reduce lipid hydroperoxides through their Se-independent glutathione-peroxidase activity and that these enzymes can also detoxify LPO end-products such as 4HNE. Available evidence from earlier studies together with results of recent studies in our laboratories strongly suggests that LPO products, particularly hydroperoxidesmore » and 4HNE, are involved in the mechanisms of stress-mediated signaling and that it can be modulated by the alpha-class GSTs through the regulation of the intracellular concentrations of 4HNE. We demonstrate that 4HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase (JNK) and caspase-3 activation. Cells exposed to mild, transient heat or oxidative stress acquire the capacity to exclude intracellular 4HNE at a faster rate by inducing GSTA4-4 which conjugates 4HNE to glutathione (GSH), and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4HNE (GS-HNE). The balance between formation and exclusion promotes different cellular processes — higher concentrations of 4HNE promote apoptosis; whereas, lower concentrations promote proliferation. In this article, we provide a brief summary of the cellular effects of 4HNE, followed by a review of its GST-catalyzed detoxification, with an emphasis on the structural attributes that play an important role in the interactions with alpha-class GSTA4-4. Taken together, 4HNE is a key signaling molecule and that GSTs being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article. - Highlights: • GSTs are the major

Role of glutathione S-transferase Pi in cisplatin-induced nephrotoxicity.

PubMed

Townsend, Danyelle M; Tew, Kenneth D; He, Lin; King, Jarrod B; Hanigan, Marie H

2009-02-01

One of the dose-limiting toxicities of cisplatin is nephrotoxicity. Renal toxicity is localized to quiescent proximal tubule cells, where the formation of DNA-adducts cannot account for the dose-limiting toxicity. Our earlier results have shown that a glutathione conjugate of cisplatin is metabolized to a nephrotoxicant via gamma-glutamyl transpeptidase (GGT) and a cysteine S-conjugate beta-lyase. The present study was designed to evaluate the potential role of glutathione S-transferase Pi (GSTP) in the initial steps of the bioactivation of cisplatin. Wild-type mice and mice deficient in both murine GSTP genes (GstP1/P2) were treated with cisplatin. Toxicity in both male and female mice was evaluated 5 days after treatment and renal damage was most severe in wild-type male mice. Wild-type males have approximately 10-fold higher levels of GSTP expression in the liver than females, suggesting that hepatic GSTP in the wild-type males contributed to the formation of the nephrotoxic platinum-glutathione conjugate. In GstP1/P2 null mice the gender difference in toxicity was eliminated. Our data show that GSTP expression is a determinant in cisplatin-induced nephrotoxicity and its levels contribute to sex-dependent differences.

Role of Glutathione S-Transferase Pi in Cisplatin Induced Nephrotoxicity

PubMed Central

Townsend, Danyelle M.; Tew, Kenneth D.; He, Lin; King, Jarrod B.; Hanigan, Marie H.

2009-01-01

SUMMARY One of the dose-limiting toxicities of cisplatin is nephrotoxicity. Renal toxicity is localized to quiescent proximal tubule cells, where the formation of DNA-adducts cannot account for the dose-limiting toxicity. Our earlier results have shown that a glutathione-conjugate of cisplatin is metabolized to a nephrotoxicant via gamma-glutamyltranspeptidase (GGT) and a cysteine S-conjugate beta-lyase. The present study was designed to evaluate the potential role of glutathione-S-transferase Pi (GSTP) in the initial steps of the bioactivation of cisplatin. Wild-type mice and mice deficient in both murine GSTP genes (GstP1/P2) were treated with cisplatin. Toxicity in both male and female mice was evaluated 5 days after treatment and renal damage was most severe in wild-type male mice. Wild-type males have ~10-fold higher levels of GSTP expression in the liver than females, suggesting that hepatic GSTP in the wild-type males contributed to the formation of the nephrotoxic platinum-glutathione conjugate. In GstP1/P2 null mice the gender difference in toxicity was eliminated. Our data show that GSTP expression is a determinant in cisplatin-induced nephrotoxicity and its levels contribute to sex-dependent differences. PMID:18819770

The 30 kDa protein co-purified with chick liver glutathione S-transferases is a carbonyl reductase.

PubMed

Tsai, S P; Wang, L Y; Yeh, H I; Tam, M F

1996-02-08

An unidentified 30 kDa protein was co-purified with chick liver glutathione S-transferases from S-hexylglutathione affinity column. The protein was isolated to apparent homogeneity with chromatofocusing. The molecular mass of the protein was determined to be 30 277 +/- 3 dalton by mass spectrometry. The protein was digested with Achromobacter proteinase I. Amino-acid sequence analyses of the resulting peptides show a high degree of identity with those of human carbonyl reductase. The protein is active with menadione as substrate. Thus, it is identified as chick liver carbonyl reductase.

Aniline exposure associated with up-regulated transcriptional responses of three glutathione S-transferase Delta genes in Drosophila melanogaster.

PubMed

Chan, Wen-Chiao; Chien, Yi-Chih; Chien, Cheng-I

2015-03-01

Complex transcriptional profile of glutathione S-transferase Delta cluster genes occurred in the developmental process of the fruit fly Drosophila melanogaster. The purpose of this project was to quantify the expression levels of Gst Delta class genes altered by aniline exposure and to understand the relationship between aniline dosages and the variation of Gst Delta genes expressed in D. melanogaster. Using RT-PCR expression assays, the expression patterns of the transcript mRNAs of the glutathione S-transferase Delta genes were revealed and their expression levels were measured at eggs, larvae, pupae and adults. The adult stage was selected for further dose-response assays. After analysis, the results indicated that three Gst Delta genes (Gst D2, Gst D5 and Gst D6) were found to show a peak of up-regulated transcriptional response at 6-8h of exposure of aniline. Furthermore, the dose-response relationship of their induction levels within the dose regiments (from 1.2 to 2.0 μl/tube) had been measured. The expression patterns and annotations of these genes were discussed in the context. Copyright © 2015 Elsevier B.V. All rights reserved.

Possible prenatal impact of sertraline on human placental glutathione S-transferase-π.

PubMed

Dalmizrak, O; Kulaksiz-Erkmen, G; Ozer, N

2012-05-01

Sertraline (SER), a tricyclic antidepressant, is considered to belong to the group of selective amine reuptake inhibitors. Its ability to cross the blood-brain barrier and transplacental transport has been reported previously. It is widely distributed in the brain and is bound to human glutathione S-transferase-π (GST-π). If SER is taken during pregnancy, it gets accumulated in the embryo and fetus, and some studies have suggested it may cause congenital malformations, thus the study of the interaction of GST-π with antidepressants is crucial. In this study, the interaction of human placental GST-π with SER in the presence of the natural ligand, reduced glutathione (GSH) and a xenobiotic ligand, 1-chloro-2,4-dinitrobenzene (CDNB) was investigated. The V(m) values obtained at variable [CDNB] and variable [GSH] were 61.3 ± 2.3 and 46.4 ± 1.7 U/mg protein, respectively. The k(cat) and k(cat)/K(m) values for GSH and CDNB were 3.63 × 10(6) s(-1), 2.59 × 10(10) M(-1) s(-1) and 4.79 × 10(6) s(-1), 1.29 × 10(10) M(-1) s(-1), respectively. The half maximal inhibitory concentration value for SER was 4.60 mM. At constant [CDNB] and variable [GSH] the inhibition type was linear mixed-type, with K(s), α, and K(i) values of 0.14 ± 0.02, 2.90 ± 1.64, and 2.18 ± 0.80 mM, respectively. On the other hand, at fixed [GSH] and at variable [CDNB], the inhibition type was competitive, with K(i) value of 0.96 ± 0.10 mM. Thus, these findings weaken the importance of the protective role of GST against toxic electrophiles in vivo in adults, but due to its immature enterohepatic system SER may accumulate in the fetus and cause congenital malformations.

Identification and characterization of the zebrafish glutathione S-transferase Pi-1.

PubMed

Abunnaja, Maryam S; Kurogi, Katsuhisa; Mohammed, Yasir I; Sakakibara, Yoichi; Suiko, Masahito; Hassoun, Ezdihar A; Liu, Ming-Cheh

2017-10-01

Zebrafish has in recent years emerged as a popular vertebrate model for use in pharmacological and toxicological studies. While there have been sporadic studies on the zebrafish glutathione S-transferases (GSTs), the zebrafish GST gene superfamily still awaits to be fully elucidated. We report here the identification of 15 zebrafish cytosolic GST genes in NCBI GenBank database and the expression, purification, and enzymatic characterization of the zebrafish cytosolic GST Pi-1 (GSTP1). The cDNA encoding the zebrafish GSTP1 was cloned from a 3-month-old female zebrafish, expressed in Eschelichia coli host cells, and purified. Purified GSTP1 displayed glutathione-conjugating activity toward 1-chloro-2,4-dinitrobenzene as a representative substrate. The enzymatic characteristics of the zebrafish GSTP1, including pH-dependency, effects of metal cations, and kinetic parameters, were studied. Moreover, the expression of zebrafish GSTP1 at different developmental stages during embryogenesis, throughout larval development, onto maturity was examined. © 2017 Wiley Periodicals, Inc.

Glutathione S-transferases in neonatal liver disease.

PubMed Central

Mathew, J.; Cattan, A. R.; Hall, A. G.; Hines, J. E.; Nelson, R.; Eastham, E.; Burt, A. D.

1992-01-01

AIMS: To investigate the distribution of alpha and pi class glutathione S-transferases (GST) in normal fetal, neonatal, and adult liver; and to examine changes in GST expression in neonatal liver disease. METHODS: alpha and pi class GST were immunolocalised in sections of formalin fixed liver tissue obtained from human fetuses (n = 21), neonates (n = 8), young children (n = 9) and adults (n = 10), and from neonates with extrahepatic biliary atresia (n = 15) and neonatal hepatitis (n = 12). Monospecific rabbit polyclonal antibodies were used with a peroxidase-antiperoxidase method. RESULTS: Expression of pi GST was localised predominantly within biliary epithelial cells of developing and mature bile ducts of all sizes from 16 weeks' gestation until term and in neonatal and adult liver. Coexpression of pi and alpha GST was seen in hepatocytes of developing fetal liver between 16 and 34 weeks' gestation. Although pi GST was seen in occasional hepatocytes up to six months of life, this isoenzyme was not expressed by hepatocytes in adult liver. By contrast, alpha GST continued to be expressed by hepatocytes in adult liver; this isoenzyme was also seen in some epithelial cells of large bile ducts in adult liver. No change was observed in the distribution of alpha GST in either neonatal hepatitis or extrahepatic biliary atresia. However, aberrant expression of pi GST was identified in hepatocytes of all but one case of extrahepatic biliary atresia but in only two cases of neonatal hepatitis. CONCLUSIONS: The phenotypic alterations noted in extrahepatic biliary atresia may result from the effect of cholate stasis. Evaluation of the pattern of pi and alpha GST distribution by immunohistochemical staining may provide valuable information in distinguishing between these two forms of neonatal liver disease. Images PMID:1401176

Down-regulation of Glutathione S-transferase Pi in Asthma Contributes to Enhanced Oxidative Stress

PubMed Central

Schroer, Kathy T.; Gibson, Aaron M.; Sivaprasad, Umasundari; Bass, Stacey A.; Ericksen, Mark B.; Wills-Karp, Marsha; LeCras, Tim; Fitzpatrick, Anne M.; Brown, Lou Ann S.; Stringer, Keith F.; Khurana Hershey, Gurjit K.

2011-01-01

Background Glutathione S-transferase Pi (GSTPi) is the predominant redox regulator in the lung. While evidence implicates an important role for GSTPi in asthma, the mechanism for this has remained elusive. Objectives To determine how GSTPi is regulated in asthma and to elucidate its role in maintaining redox homeostasis. Methods We elucidated the regulation of GSTPi in children with asthma and utilized murine models of asthma to determine the role of GSTPi in redox homeostasis. Measurements and Main Results Our findings demonstrate that GSTPi transcript levels are markedly down-regulated in allergen and IL-13 treated mouse models of asthma via STAT6 dependent and independent pathways. Nuclear factor-erythroid 2 related factor 2 (Nrf2) was also down-regulated in these models. The decrease in GSTPi expression was associated with decreased total GST activity in the lungs of mice. Examination of cystine intermediates uncovered a functional role for GSTPi in regulating Cys oxidation, whereby GSTPi-deficient mice exhibited increased oxidative stress (increase in % cystine) compared with wild-type mice following allergen challenge. GSTPi expression was similarly down-regulated in children with asthma. Conclusions These data collectively suggest that down-regulation of GSTPi following allergen challenge may contribute to the asthma phenotype due to disruption of redox homeostasis and increased oxidative stress. Furthermore, GSTPi may be an important therapeutic target for asthma, and evaluation of GSTPi expression may prove beneficial in identifying individuals who would benefit from therapy targeting this pathway. PMID:21570714

Glutathione S-transferase Pi expression in invasive breast cancer and its relation with the clinical outcome.

PubMed

Franco, R L; Schenka, N G M; Schenka, A A A; Rezende, L F; Gurgel, M S C

2012-01-01

Glutathione S-transferase (GST) is a cytosolic enzymatic system involved in cellular detoxifying process. In vitro studies have shown that the presence of this enzymatic system in breast carcinoma cells can accelerate the elimination of drugs commonly used in chemotherapy, thereby decreasing its efficacy. The aim of the present study was to evaluate the association between GST Pi expression by breast carcinoma cells and disease-free and overall survival. Ninety-five female patients with invasive breast carcinoma submitted to surgical treatment and adjuvant chemotherapy from January, 1995 to June, 1997 and followed until August, 2006 were evaluated. The expression of GST Pi in breast carcinoma cells, determined by immunohistochemistry, was correlated with several clinical and pathological parameters of prognostic significance. There were 36 (37.9%) GST Pi-positive cases. GST Pi immunoexpression was not significantly correlated with patient's age, histological tumor type, clinical stage, hormone receptor status and survival. On the other hand, GST Pi positivity showed a significant correlation with a lower histological grade/C-erb-B2 negative breast carcinoma phenotype. The findings suggest that GST Pi expression does not constitute a satisfactory prognostic factor in breast cancer.

Insights into ligand binding to a glutathione S-transferase from mango: Structure, thermodynamics and kinetics

DOE PAGES

Valenzuela-Chavira, Ignacio; Contreras-Vergara, Carmen A.; Arvizu-Flores, Aldo A.; ...

2017-01-17

We studied a mango glutathione S-transferase (GST) ( Mangifera indica) bound to glutathione (GSH) and S-hexyl glutathione (GSX). This GST Tau class (MiGSTU) had a molecular mass of 25.5 kDa. MiGSTU Michaelis-Menten kinetic constants were determined for their substrates obtaining a K m, V max and k cat for CDNB of 0.792 mM, 80.58 mM min -1 and 68.49 s -1 respectively and 0.693 mM, 105.32 mM min -1 and 89.57 s -1, for reduced GSH respectively. MiGSTU had a micromolar affinity towards GSH (5.2 mM) or GSX (7.8 mM). As a result, the crystal structure of the MiGSTU inmore » apo or bound to GSH or GSX generated a model that explains the thermodynamic signatures of binding and showed the importance of enthalpic-entropic compensation in ligand binding to Tau-class GST enzymes.« less

Insights into ligand binding to a glutathione S-transferase from mango: Structure, thermodynamics and kinetics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Valenzuela-Chavira, Ignacio; Contreras-Vergara, Carmen A.; Arvizu-Flores, Aldo A.

We studied a mango glutathione S-transferase (GST) ( Mangifera indica) bound to glutathione (GSH) and S-hexyl glutathione (GSX). This GST Tau class (MiGSTU) had a molecular mass of 25.5 kDa. MiGSTU Michaelis-Menten kinetic constants were determined for their substrates obtaining a K m, V max and k cat for CDNB of 0.792 mM, 80.58 mM min -1 and 68.49 s -1 respectively and 0.693 mM, 105.32 mM min -1 and 89.57 s -1, for reduced GSH respectively. MiGSTU had a micromolar affinity towards GSH (5.2 mM) or GSX (7.8 mM). As a result, the crystal structure of the MiGSTU inmore » apo or bound to GSH or GSX generated a model that explains the thermodynamic signatures of binding and showed the importance of enthalpic-entropic compensation in ligand binding to Tau-class GST enzymes.« less

Organometallic ruthenium anticancer complexes inhibit human glutathione-S-transferase π.

PubMed

Lin, Yu; Huang, Yongdong; Zheng, Wei; Wang, Fuyi; Habtemariam, Abraha; Luo, Qun; Li, Xianchan; Wu, Kui; Sadler, Peter J; Xiong, Shaoxiang

2013-11-01

The organometallic ruthenium(II) anticancer complexes [(η(6)-arene)Ru(en)Cl](+) (arene = p-cymene (1), biphenyl (2) or 9,10-dihydrophenanthrene (3); en = ethylenediamine), exhibit in vitro and in vivo anticancer activities. In the present work, we show that they inhibit human glutathione-S-transferase π (GSTπ) with IC50 values of 59.4 ± 1.3, 63.2 ± 0.4 and 37.2 ± 1.1 μM, respectively. Mass spectrometry revealed that complex 1 binds to the S-donors of Cys15, Cys48 within the G-site and Cys102 at the interface of the GSTπ dimer, while complex 2 binds to Cys48 and Met92 at the dimer interface and complex 3 to Cys15, Cys48 and Met92. Moreover, the binding of complex 1 to Cys15 and Cys102, complex 2 to Cys48 and complex 3 to Cys15 induces the irreversible oxidation of the coordinated thiolates to sulfenates. Molecular modeling studies indicate that the coordination of the {(arene)Ru(en)}(2+) fragment to Cys48 blocks the hydrophilic G-site sterically, perhaps preventing substrate from proper positioning and accounting for the reduction in enzymatic activity of ruthenated GSTπ. The binding of the ruthenium arene complexes to Cys102 or Met92 disrupts the dimer interface which is an essential structural feature for the proper functioning of GSTπ, perhaps also contributing to the inhibition of GSTπ. © 2013.

Fish hepatic glutathione-S-transferase activity is affected by the cestode parasites Schistocephalus solidus and Ligula intestinalis: evidence from field and laboratory studies.

PubMed

Frank, Sabrina Nadine; Faust, Steffen; Kalbe, Martin; Trubiroha, Achim; Kloas, Werner; Sures, Bernd

2011-06-01

The activity of hepatic glutathione-S-transferase (GST) was analysed in 3 different fish species with respect to fish sex and infection with parasites. In both sexes of laboratory bred three-spined sticklebacks (Gasterosteus aculeatus) experimentally infected with Schistocephalus solidus (Cestoda), a significantly lower GST-activity was found for infected fish compared to control. After field sampling of roach (Rutilus rutilus) from Lake Müggelsee (MS) and the Reservoir Listertalsperre (LTS), the GST-activity showed significantly lower values for males infected with Ligula intestinalis from MS (25%) and for infected females from LTS (55%). L. intestinalis-infected female chub (Leuciscus cephalus) from LTS also appeared to have a lower GST-activity. Thus, it could be shown that the presence of parasites significantly affects GST-activity in different fish species resulting in a decreased GST-activity due to infection. Our results therefore emphasize the need for more integrative approaches in environmental pollution research to clearly identify the possible effects of parasites in an effort to develop biomarkers for evaluating environmental health.

Glutathione S-transferase polymorphisms (GSTM1, GSTT1 and GSTP1) and their susceptibility to renal cell carcinoma: an evidence-based meta-analysis.

PubMed

Yang, Xingliang; Long, Shuyu; Deng, Jianping; Deng, Tianxing; Gong, Zhihua; Hao, Ping

2013-01-01

The association of the three Glutathione S-transferases (GSTs) polymorphisms (GSTM1, GSTT1 and GSTP1) genotypes with their individual susceptibilities to renal cell carcinoma (RCC) has not been well established. We performed a quantitative meta-analysis to assess the possible associations between the GSTM1, GSTT1 and GSTP1 genotypes and their individual susceptibilities to renal cell carcinoma. We systematically searched the PubMed, CNKI and Embase databases to identify the relevant studies. Finally, 11 eligible studies were selected. The pooled odds ratios (ORs) with their 95% confidence intervals (CIs) were used to assess the association between the GSTs polymorphisms and the risk of RCC. Multiple subgroup analyses and quality assessment of the included studies were performed based on the available information. None of the GSTs polymorphisms had a significant association with the RCC risk. Similar results were found in the subgroup analyses, except for the GSTs polymorphisms in the situations described below. The GSTM1 and GSTT1 active genotypes in subjects exposed to pesticides (GSTM1: OR = 3.44; 95% CI, 2.04-5.80; GSTT1: OR = 2.84; 95% CI, 1.75-4.60), most of the GSTs genotypes in Asian populations (GSTT1: OR = 2.39, 95% CI = 1.63-3.51; GSTP1: Dominant model: OR = 1.50, 95% CI = 1.14-1.99; Additive model: OR = 1.39, 95% CI = 1.12-1.73; AG vs. AA: OR = 1.47, 95% CI = 1.10-1.97; GG vs. AA: OR = 1.82, 95% CI = 1.07-3.09) and the dual null genotype of GSTT1-GSTP1 (OR = 2.84, 95% CI = 1.75-4.60) showed positive associations with the RCC risk. Our present study provides evidence that the GSTM1, GSTT1 and GSTP1 polymorphisms are not associated with the development of RCC. However, more case-control studies are needed for further confirmation.

Glutathione S-Transferase P-Mediated Protein S-Glutathionylation of Resident Endoplasmic Reticulum Proteins Influences Sensitivity to Drug-Induced Unfolded Protein Response

PubMed Central

Ye, Zhi-Wei; Zhang, Jie; Ancrum, Tiffany; Manevich, Yefim; Townsend, Danyelle M.

2017-01-01

Abstract Aims: S-glutathionylation of cysteine residues, catalyzed by glutathione S-transferase Pi (GSTP), alters structure/function characteristics of certain targeted proteins. Our goal is to characterize how S-glutathionylation of proteins within the endoplasmic reticulum (ER) impact cell sensitivity to ER-stress inducing drugs. Results: We identify GSTP to be an ER-resident protein where it demonstrates both chaperone and catalytic functions. Redox based proteomic analyses identified a cluster of proteins cooperatively involved in the regulation of ER stress (immunoglobulin heavy chain-binding protein [BiP], protein disulfide isomerase [PDI], calnexin, calreticulin, endoplasmin, sarco/endoplasmic reticulum Ca2+-ATPase [SERCA]) that individually co-immunoprecipitated with GSTP (implying protein complex formation) and were subject to reactive oxygen species (ROS) induced S-glutathionylation. S-glutathionylation of each of these six proteins was attenuated in cells (liver, embryo fibroblasts or bone marrow dendritic) from mice lacking GSTP (Gstp1/p2−/−) compared to wild type (Gstp1/p2+/+). Moreover, Gstp1/p2−/− cells were significantly more sensitive to the cytotoxic effects of the ER-stress inducing drugs, thapsigargin (7-fold) and tunicamycin (2-fold). Innovation: Within the family of GST isozymes, GSTP has been ascribed the broadest range of catalytic and chaperone functions. Now, for the first time, we identify it as an ER resident protein that catalyzes S-glutathionylation of critical ER proteins within this organelle. Of note, this can provide a nexus for linkage of redox based signaling and pathways that regulate the unfolded protein response (UPR). This has novel importance in determining how some drugs kill cancer cells. Conclusions: Contextually, these results provide mechanistic evidence that GSTP can exert redox regulation in the oxidative ER environment and indicate that, within the ER, GSTP influences the cellular consequences of the UPR

Characterization of glutathione transferases involved in the pathogenicity of Alternaria brassicicola.

PubMed

Calmes, Benoit; Morel-Rouhier, Mélanie; Bataillé-Simoneau, Nelly; Gelhaye, Eric; Guillemette, Thomas; Simoneau, Philippe

2015-06-18

Glutathione transferases (GSTs) represent an extended family of multifunctional proteins involved in detoxification processes and tolerance to oxidative stress. We thus anticipated that some GSTs could play an essential role in the protection of fungal necrotrophs against plant-derived toxic metabolites and reactive oxygen species that accumulate at the host-pathogen interface during infection. Mining the genome of the necrotrophic Brassica pathogen Alternaria brassicicola for glutathione transferase revealed 23 sequences, 17 of which could be clustered into the main classes previously defined for fungal GSTs and six were 'orphans'. Five isothiocyanate-inducible GSTs from five different classes were more thoroughly investigated. Analysis of their catalytic properties revealed that two GSTs, belonging to the GSTFuA and GTT1 classes, exhibited GSH transferase activity with isothiocyanates (ITC) and peroxidase activity with cumene hydroperoxide, respectively. Mutant deficient for these two GSTs were however neither more susceptible to ITC nor less aggressive than the wild-type parental strain. By contrast mutants deficient for two other GSTs, belonging to the Ure2pB and GSTO classes, were distinguished by their hyper-susceptibility to ITC and low aggressiveness against Brassica oleracea. In particular AbGSTO1 could participate in cell tolerance to ITC due to its glutathione-dependent thioltransferase activity. The fifth ITC-inducible GST belonged to the MAPEG class and although it was not possible to produce the soluble active form of this protein in a bacterial expression system, the corresponding deficient mutant failed to develop normal symptoms on host plant tissues. Among the five ITC-inducible GSTs analyzed in this study, three were found essential for full aggressiveness of A. brassicicola on host plant. This, to our knowledge is the first evidence that GSTs might be essential virulence factors for fungal necrotrophs.

Pharmacogenetics of azathioprine in inflammatory bowel disease: A role for glutathione-S-transferase?

PubMed Central

Stocco, Gabriele; Pelin, Marco; Franca, Raffaella; De Iudicibus, Sara; Cuzzoni, Eva; Favretto, Diego; Martelossi, Stefano; Ventura, Alessandro; Decorti, Giuliana

2014-01-01

Azathioprine is a purine antimetabolite drug commonly used to treat inflammatory bowel disease (IBD). In vivo it is active after reaction with reduced glutathione (GSH) and conversion to mercaptopurine. Although this reaction may occur spontaneously, the presence of isoforms M and A of the enzyme glutathione-S-transferase (GST) may increase its speed. Indeed, in pediatric patients with IBD, deletion of GST-M1, which determines reduced enzymatic activity, was recently associated with reduced sensitivity to azathioprine and reduced production of azathioprine active metabolites. In addition to increase the activation of azathioprine to mercaptopurine, GSTs may contribute to azathioprine effects even by modulating GSH consumption, oxidative stress and apoptosis. Therefore, genetic polymorphisms in genes for GSTs may be useful to predict response to azathioprine even if more in vitro and clinical validation studies are needed. PMID:24707136

Serum glutathione S-transferase Pi as predictor of the outcome and acute kidney injury in premature newborns.

PubMed

Stojanović, Vesna D; Barišić, Nenad A; Radovanović, Tanja D; Kovač, Nataša B; Djuran, Jelena D; Antić, Amira Peco E; Doronjski, Aleksandra D

2018-07-01

The incidence of acute kidney injury (AKI) among the neonates treated at the Neonatal Intensive Care Unit is high with high mortality rates. Glutathione S-transferase (GST) class Pi plays an important role in the protection of cells from cytotoxic and oncogenic agents. The aim of the study was to examine whether the levels of serum glutathione S-transferase Pi (GST Pi) determined after birth have any predictive value for the outcome and development of AKI in premature neonates. The prospective study included 36 premature neonates. The data about morbidity was gathered for all the neonates included in the study. The blood samples were taken in the first 6 h of life and GST Pi levels were measured. The mean values and standard deviations of GST Pi among the neonates who died and who survived were 1.904 ± 0.4535 vs 1.434 ± 0.444 ng/ml (p = 0.0128). Logistic regression revealed a statistically significant, positive correlation between GST Pi levels and death (p = 0.0180, OR7.5954; CI 1.4148-40.7748).The mean value of GST Pi levels in the neonates with AKI was higher than in neonates without AKI (p = 0.011). The conclusion of our study is that high levels of serum GST Pi in the first 6 h after birth are associated with an increased mortality and development of AKI in prematurely born neonates.

Glutathione S-transferase P protects against cyclophosphamide-induced cardiotoxicity in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Conklin, Daniel J., E-mail: dj.conklin@louisville.edu; Institute of Molecular Cardiology, University of Louisville, Louisville, KY 40292; Haberzettl, Petra

2015-06-01

High-dose chemotherapy regimens using cyclophosphamide (CY) are frequently associated with cardiotoxicity that could lead to myocyte damage and congestive heart failure. However, the mechanisms regulating the cardiotoxic effects of CY remain unclear. Because CY is converted to an unsaturated aldehyde acrolein, a toxic, reactive CY metabolite that induces extensive protein modification and myocardial injury, we examined the role of glutathione S-transferase P (GSTP), an acrolein-metabolizing enzyme, in CY cardiotoxicity in wild-type (WT) and GSTP-null mice. Treatment with CY (100–300 mg/kg) increased plasma levels of creatine kinase-MB isoform (CK·MB) and heart-to-body weight ratio to a significantly greater extent in GSTP-null thanmore » WT mice. In addition to modest yet significant echocardiographic changes following acute CY-treatment, GSTP insufficiency was associated with greater phosphorylation of c-Jun and p38 as well as greater accumulation of albumin and protein–acrolein adducts in the heart. Mass spectrometric analysis revealed likely prominent modification of albumin, kallikrein-1-related peptidase, myoglobin and transgelin-2 by acrolein in the hearts of CY-treated mice. Treatment with acrolein (low dose, 1–5 mg/kg) also led to increased heart-to-body weight ratio and myocardial contractility changes. Acrolein induced similar hypotension in GSTP-null and WT mice. GSTP-null mice also were more susceptible than WT mice to mortality associated with high-dose acrolein (10–20 mg/kg). Collectively, these results suggest that CY cardiotoxicity is regulated, in part, by GSTP, which prevents CY toxicity by detoxifying acrolein. Thus, humans with low cardiac GSTP levels or polymorphic forms of GSTP with low acrolein-metabolizing capacity may be more sensitive to CY toxicity. - Graphical abstract: Cyclophosphamide (CY) treatment results in P450-mediated metabolic formation of phosphoramide mustard and acrolein (3-propenal). Acrolein is either metabolized

Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

PubMed Central

Chee, Chin-Soon; Tan, Irene Kit-Ping; Alias, Zazali

2014-01-01

Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively. PMID:24892084

Gene Polymorphisms of Glutathione S-Transferase T1/M1 in Egyptian Children and Adolescents with Type 1 Diabetes Mellitus.

PubMed

Barseem, Naglaa; Elsamalehy, Mona

2017-06-01

Oxidative stress plays an important role in the pathogenesis of type 1 diabetes mellitus (T1DM). To evaluate the association of glutathione S-transferase mu 1 (GST M1) and glutathione S-transferase theta 1 (GST T1) polymorphisms with development of T1DM and disease-related risk factors. Measurement of fasting glucose, serum creatinine, lipid profile, and glycosylated hemoglobin (HbA1c), as well as evaluation of GST T1 and M1 genetic polymorphisms using polymerase chain reaction were done in 64 diabetic children and 41 controls. The diabetic group had significantly higher fasting glucose, HbA1c, and cholesterol levels. GST T1 null genotype was more frequent in the diabetic than the control group with 4.2-fold increased risk of T1DM (odds ratio=4.2; 95% confidence interval=1.6-11.5; p=0.03). Significant positive associations were found with lipid profile, HbA1c, and duration of illness but not with age, age at onset, and body mass index. Gene polymorphisms of the enzyme GST are associated with development of T1DM and disease-related risk factors.

Glutathione -S-Transferase μ 1 Regulates Vascular Smooth Muscle Cell Proliferation, Migration, and Oxidative Stress

PubMed Central

Yang, Yanqiang; Parsons, Kelly K.; Chi, Liqun; Malakauskas, Sandra M.; Le, Thu H.

2009-01-01

Glutathione S-transferase μ-1, GSTM1, belongs to a superfamily of glutathione-S-transferases that metabolize a broad range of reactive oxygen species (ROS) and xenobiotics. Across species, genetic variants that result in decreased expression of the Gstm1 gene are associated with increased susceptibility for vascular diseases, including atherosclerosis in humans. We previously identified Gstm1 as a positional candidate in our gene mapping study for susceptibility to renal vascular injury characterized by medial hypertrophy and hyperplasia of the renal vessels. To determine the role of Gstm1 in vascular smooth muscle cells (VSMCs), we isolated VSMCs from mouse aortas. We demonstrate that VSMCs from the susceptible C57BL/6 mice have reduced expression of Gstm1 mRNA and its protein product compared to that of the resistant 129 mice. After serum stimulation, C57BL/6 VSMCs proliferate and migrate at a much faster rate than 129 VSMCs. Furthermore, C57BL/6 VSMCs have higher levels of ROS, and exhibit exaggerated p38 MAPK phosphorylation after exposure to H2O2. To establish causality, we show that knockdown of Gstm1 by siRNA results in increased proliferation of VSMCs in a dose dependent manner, as well as in increased ROS levels and VSM cell migration. Moreover, Gstm1 siRNA causes increased p38 MAPK phosphorylation, and attenuates the anti-proliferative effect of TEMPOL. Our data suggest that Gstm1 is a novel regulator of VSMC proliferation and migration through its role in handling ROS. Genetic variants that cause a decremental change in expression of Gstm1 may permit an environment of exaggerated oxidative stress, leading to susceptibility to vascular remodeling and atherosclerosis. PMID:19822795

Inhibition of the recombinant cattle tick Rhipicephalus (Boophilus) annulatus glutathione S-transferase.

PubMed

Guneidy, Rasha A; Shahein, Yasser E; Abouelella, Amira M K; Zaki, Eman R; Hamed, Ragaa R

2014-09-01

Rhipicephalus (Boophilus) annulatus is a bloodsucking ectoparasite that causes severe production losses in the cattle industry. This study aims to evaluate the in vitro effects of tannic acid, hematin (GST inhibitors) and different plant extracts (rich in tannic acid) on the activity of the recombinant glutathione S-transferase enzyme of the Egyptian cattle tick R. annulatus (rRaGST), in order to confirm their ability to inhibit the parasitic essential detoxification enzyme glutathione S-transferase. Extraction with 70% ethanol of Hibiscus cannabinus (kenaf flowers), Punica granatum (red and white pomegranate peel), Musa acuminata (banana peel) (Musaceae), Medicago sativa (alfalfa seeds), Tamarindus indicus (seed) and Cuminum cyminum (cumin seed) were used to assess: (i) inhibitory capacities of rRaGST and (ii) their phenolic and flavonoid contents. Ethanol extraction of red pomegranate peel contained the highest content of phenolic compounds (29.95mg gallic acid/g dry tissue) compared to the other studied plant extracts. The highest inhibition activities of rRaGST were obtained with kenaf and red pomegranate peel (P. granatum) extracts with IC50 values of 0.123 and 0.136mg dry tissue/ml, respectively. Tannic acid was the more effective inhibitor of rRaGST with an IC50 value equal to 4.57μM compared to delphinidine-HCl (IC50=14.9±3.1μM). Gossypol had a weak inhibitory effect (IC50=43.7μM), and caffeic acid had almost no effect on tick GST activity. The IC50 values qualify ethacrynic acid as a potent inhibitor of rRaGST activity (IC50=0.034μM). Cibacron blue and hematin showed a considerable inhibition effect on rRaGST activity, and their IC50 values were 0.13μM and 7.5μM, respectively. The activity of rRaGST was highest for CDNB (30.2μmol/min/mg protein). The enzyme had also a peroxidatic activity (the specific activity equals 26.5μmol/min/mg protein). Both tannic acid and hematin inhibited rRaGST activity non-competitively with respect to GSH and

Insights into ligand binding to a Glutathione S-transferase from mango: structure, thermodynamics and kinetics

PubMed Central

Valenzuela-Chavira, Ignacio; Contreras-Vergara, Carmen A.; Arvizu-Flores, Aldo A.; Serrano-Posada, Hugo; Lopez-Zavala, Alonso A.; García-Orozco, Karina D.; Hernandez-Paredes, Javier; Rudiño-Piñera, Enrique; Stojanoff, Vivian; Sotelo-Mundo, Rogerio R.; Islas-Osuna, Maria A.

2017-01-01

We studied a mango glutathione S-transferase (GST) (Mangifera indica) bound to glutathione (GSH) and S-hexyl glutathione (GSX). This GST Tau class (MiGSTU) had a molecular mass of 25.5 kDa. MiGSTU Michaelis-Menten kinetic constants were determined for their substrates obtaining a Km, Vmax and kcat for CDNB of 0.792 mM, 80.58 mM·min−1 and 68.49 s−1 respectively and 0.693 mM, 105.32 mM·min−1 and 89.57 s−1, for reduced GSH respectively. MiGSTU had a micromolar affinity towards GSH (5.2 μM) or GSX (7.8 μM). The crystal structure of the MiGSTU in apo or bound to GSH or GSX generated a model that explains the thermodynamic signatures of binding and showed the importance of enthalpic-entropic compensation in ligand binding to Tau-class GST enzymes. PMID:28104507

Identification and clarification of the role of key active site residues in bacterial glutathione S-transferase zeta/maleylpyruvate isomerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang, Ti; Li, De-Feng; Zhou, Ning-Yi, E-mail: n.zhou@pentium.whiov.ac.cn

2011-07-08

Highlights: {yields} Application of site-directed mutagenesis to probe the active site residues of glutathione-dependent maleylpyruvate isomerase. {yields} Two conserved residues, Arg8 and Arg176, in zeta class glutathione S-transferases are critical for maleylpyruvate orientation and enolization. {yields} Arg109, found exclusively in NagL, participates in k{sub cat} regulation. {yields} The T11A mutant exhibited a significantly decreased K{sub m} value for glutathione with little impact on maleylpyruvate kinetics. {yields} The Thr11 residue appears to have significance in the evolution of glutathione S-transferase classes. -- Abstract: The maleylpyruvate isomerase NagL from Ralstonia sp. strain U2, which has been structurally characterized previously, catalyzes the isomerizationmore » of maleylpyruvate to fumarylpyruvate. It belongs to the class zeta glutathione S-transferases (GSTZs), part of the cytosolic GST family (cGSTs). In this study, site-directed mutagenesis was conducted to probe the functions of 13 putative active site residues. Steady-state kinetic information for mutants in the reduced glutathione (GSH) binding site, suggested that (a) Gln64 and Asp102 interact directly with the glutamyl moiety of glutathione, (b) Gln49 and Gln64 are involved in a potential electron-sharing network that influences the ionization of the GSH thiol. The information also suggests that (c) His38, Asn108 and Arg109 interact with the GSH glycine moiety, (d) His104 has a role in the ionization of the GSH sulfur and the stabilization of the maleyl terminal carboxyl group in the reaction intermediate and (e) Arg110 influences the electron distribution in the active site and therefore the ionization of the GSH thiolate. Kinetic data for mutants altered in the substrate-binding site imply that (a) Arg8 and Arg176 are critical for maleylpyruvate orientation and enolization, and (b) Arg109 (exclusive to NagL) participates in k{sub cat} regulation. Surprisingly, the T11A mutant

Glutathione-S-transferase profiles in the emerald ash borer, Agrilus planipennis.

PubMed

Rajarapu, Swapna Priya; Mittapalli, Omprakash

2013-05-01

The emerald ash borer, Agrilus planipennis Fairmaire is a recently discovered invasive insect pest of ash, Fraxinus spp. in North America. Glutathione-S-transferases (GST) are a multifunctional superfamily of enzymes which function in conjugating toxic compounds to less toxic and excretable forms. In this study, we report the molecular characterization and expression patterns of different classes of GST genes in different tissues and developmental stages plus their specific activity. Multiple sequence alignment of all six A. planipennis GSTs (ApGST-E1, ApGST-E2, ApGST-E3, ApGST-O1, ApGST-S1 and ApGST-μ1) revealed conserved features of insect GSTs and a phylogenetic analysis grouped the GSTs within the epsilon, sigma, omega and microsomal classes of GSTs. Real time quantitative PCR was used to study field collected samples. In larval tissues high mRNA levels for ApGST-E1, ApGST-E3 and ApGST-O1 were obtained in the midgut and Malpighian tubules. On the other hand, ApGST-E2 and ApGST-S1 showed high mRNA levels in fat body and ApGST-μ1 showed constitutive levels in all the tissues assayed. During development, mRNA levels for ApGST-E2 were observed to be the highest in feeding instars, ApGST-S1 in prepupal instars; while the others showed constitutive patterns in all the developmental stages examined. At the enzyme level, total GST activity was similar in all the tissues and developmental stages assayed. Results obtained suggest that A. planipennis is potentially primed with GST-driven detoxification to metabolize ash allelochemicals. To our knowledge this study represents the first report of GSTs in A. planipennis and also in the family of wood boring beetles. Copyright © 2013 Elsevier Inc. All rights reserved.

Association of manganese superoxide dismutase and glutathione S-transferases genotypes with myocardial infarction in patients with type 2 diabetes mellitus.

PubMed

Kariž, Stojan; Nikolajević Starčević, Jovana; Petrovič, Daniel

2012-10-01

In the present study we investigated the association between genetic polymorphisms with functional effects on redox regulation: Val16Ala of manganese superoxide dismutase (MnSOD), polymorphic deletions of glutathione S-transferases M1 (GSTM1) and T1 (GSTT1) and Ile105Val of glutathione S-transferase P1 (GSTP1) and myocardial infarction (MI) in a group of patients with type 2 diabetes mellitus. The study population consisted of 463 Caucasian subjects with type 2 diabetes mellitus of more than 10 years' duration: 206 patients with MI and 257 patients with no history of coronary artery disease (CAD). Genotypes were determined by polymerase chain reaction (PCR) with restriction fragment length polymorphism (RFLP) and with multiplex PCR. The genotype distributions of tested single nucleotide polymorphisms did not show significant difference between cases and controls. After adjustment for age, gender, smoking, BMI, duration of diabetes and lipid parameters carriers of GSTM1/GSTT1-null haplotype showed an increased risk for MI (OR=3.22, 95% CI 1.37-5.04, p=0.03). The GSTM1/GSTT1 haplotype might be a genetic risk factor for MI in patients with type 2 diabetes mellitus. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Isolation and purification of glutathione S-transferases from Brachionus plicatilis and B. calyciflorus (Rotifera).

PubMed

Bowman, B P; Snell, T W; Cochrane, B J

1990-01-01

1. The enzyme glutathione S-transferase (GST), a critical element in xenobiotic metabolism, was isolated from the marine rotifer Brachionus plicatilis and its freshwater congener B. calyciflorus. 2. In B. plicatilis, GST comprised 4.2% of cytosolic protein and was present as three separate isozymes with mol. wts 30,000, 31,400 and 33,700. Specific activity of crude homogenates was 56 nmol min-1 mg-1 protein, while that of affinity chromatography purified GST was 1850. 3. In B. calyciflorus, GST was present as two isozymes with mol. wts of 26,300 and 28,500, representing 1.0% of cytosolic protein. Crude GST specific activity was 1750 nmol min-1 mg-1 protein and purified was 72,400. 4. Rotifer GSTs are unusual because they are monomers whereas all other animals thus far investigated posses dimeric GSTs.

Glutathione S-Transferase Polymorphisms (GSTM1, GSTT1 and GSTP1) and Their Susceptibility to Renal Cell Carcinoma: An Evidence-Based Meta-Analysis

PubMed Central

Deng, Jianping; Deng, Tianxing; Gong, Zhihua; Hao, Ping

2013-01-01

Background The association of the three Glutathione S-transferases (GSTs) polymorphisms (GSTM1, GSTT1 and GSTP1) genotypes with their individual susceptibilities to renal cell carcinoma (RCC) has not been well established. We performed a quantitative meta-analysis to assess the possible associations between the GSTM1, GSTT1 and GSTP1 genotypes and their individual susceptibilities to renal cell carcinoma. Methods We systematically searched the PubMed, CNKI and Embase databases to identify the relevant studies. Finally, 11 eligible studies were selected. The pooled odds ratios (ORs) with their 95% confidence intervals (CIs) were used to assess the association between the GSTs polymorphisms and the risk of RCC. Multiple subgroup analyses and quality assessment of the included studies were performed based on the available information. Results None of the GSTs polymorphisms had a significant association with the RCC risk. Similar results were found in the subgroup analyses, except for the GSTs polymorphisms in the situations described below. The GSTM1 and GSTT1 active genotypes in subjects exposed to pesticides (GSTM1: OR = 3.44; 95% CI, 2.04–5.80; GSTT1: OR = 2.84; 95% CI, 1.75–4.60), most of the GSTs genotypes in Asian populations (GSTT1: OR = 2.39, 95% CI = 1.63–3.51; GSTP1: Dominant model: OR = 1.50, 95% CI = 1.14–1.99; Additive model: OR = 1.39, 95% CI = 1.12–1.73; AG vs. AA: OR = 1.47, 95% CI = 1.10–1.97; GG vs. AA: OR = 1.82, 95% CI = 1.07–3.09) and the dual null genotype of GSTT1-GSTP1 (OR = 2.84, 95% CI = 1.75–4.60) showed positive associations with the RCC risk. Conclusion Our present study provides evidence that the GSTM1, GSTT1 and GSTP1 polymorphisms are not associated with the development of RCC. However, more case-control studies are needed for further confirmation. PMID:23717494

Genetic polymorphisms in glutathione-S-transferases are associated with anxiety and mood disorders in nicotine dependence

PubMed Central

Pizzo de Castro, Márcia Regina; Ehara Watanabe, Maria Angelica; Losi Guembarovski, Roberta; Odebrecht Vargas, Heber; Vissoci Reiche, Edna Maria; Kaminami Morimoto, Helena; Dodd, Seetal; Berk, Michael

2014-01-01

Background Nicotine dependence is associated with an increased risk of mood and anxiety disorders and suicide. The primary hypothesis of this study was to identify whether the polymorphisms of two glutathione-S-transferase enzymes (GSTM1 and GSTT1 genes) predict an increased risk of mood and anxiety disorders in smokers with nicotine dependence. Materials and methods Smokers were recruited at the Centre of Treatment for Smokers. The instruments were a sociodemographic questionnaire, Fagerström Test for Nicotine Dependence, diagnoses of mood disorder and nicotine dependence according to DSM-IV (SCID-IV), and the Alcohol, Smoking and Substance Involvement Screening Test. Anxiety disorder was assessed based on the treatment report. Laboratory assessment included glutathione-S-transferases M1 (GSTM1) and T1 (GSTT1), which were detected by a multiplex-PCR protocol. Results Compared with individuals who had both GSTM1 and GSTT1 genes, a higher frequency of at least one deletion of the GSTM1 and GSTT1 genes was identified in anxious smokers [odds ratio (OR)=2.21, 95% confidence interval (CI)=1.05–4.65, P=0.034], but there was no association with bipolar and unipolar depression (P=0.943). Compared with nonanxious smokers, anxious smokers had a greater risk for mood disorders (OR=4.67; 95% CI=2.24–9.92, P<0.001), lung disease (OR=6.78, 95% CI=1.95–23.58, P<0.003), and suicide attempts (OR=17.01, 95% CI=2.23–129.91, P<0.006). Conclusion This study suggests that at least one deletion of the GSTM1 and GSTT1 genes represents a risk factor for anxious smokers. These two genes may modify the capacity for the detoxification potential against oxidative stress. PMID:24637631

INDUCTION OF DNA-PROTEIN CROSSLINKS BY THE METABOLISM OF DICHLOROMETHANE IN V79 CELL LINES TRANSFECTED WITH THE MURINE GLUTATHIONE-S-TRANSFERASE THETA 1 GENE

EPA Science Inventory

Dichloromethane (DCM) is considered a probable human carcinogen. Laboratory studies have shown an increased incidence of lung and liver cancer in mice but not in rats or hamsters. Despite the correlation between metabolism of DCM by the glutathione-S-transferase (GST) pathway and...

Glutathione-s-transferase-pi expression in early breast cancer: association with outcome and response to chemotherapy.

PubMed

Arun, Banu K; Granville, Laura A; Yin, Guosheng; Middleton, Lavinia P; Dawood, Shaheenah; Kau, Shu-Wan; Kamal, Arif; Hsu, Limin; Hortobagyi, Gabriel N; Sahin, Aysegul A

2010-06-01

Glutathione-S-transferase-pi (GST-pi) is a detoxification enzyme expressed in breast cancer; however its involvement in chemotherapy sensitivity and prognosis is not well understood. We evaluated the expression of GSTpi and its predictive role of chemotherapy response. Breast tumor samples from 166 patients at stage I/II of the disease were immunostained for GST-pi, and the expression was 96 %. There was a trend toward improved disease-free survival with high GST-pi expression (p =.09). There was a statistically non-significant association between high GST-pi expression and improved outcome with adjuvant chemotherapy (p =.055). Further studies should evaluate the role of GST-pi expression in relation to response to different chemotherapies.

Alterations in Glutathione S-transferase pi expression following exposure to MPP+-induced oxidative stress in blood of Parkinson’s disease patients

PubMed Central

Korff, Ane; Pfeiffer, Brenda; Smeyne, Michelle; Kocak, Mehmet; Pfeiffer, Ronald F.

2012-01-01

The major motor symptoms of Parkinson’s disease do not occur until a majority of the dopaminergic neurons in the midbrain SNpc have already died. For this reason, it is critical to identify biomarkers that will allow for the identification of presymptomatic individuals. In this study, we examine the baseline expression of the antioxidant protein Glutathione S-transferase pi (GSTpi) in blood of PD and environmental and age-matched controls and compare it to GSTpi levels following exposure to 1-methyl-4-phenylpyridinium (MPP+), an agent that has been shown to induce oxidative stress. We find that 4 hours of exposure to MPP+, significant increases in GSTpi levels can be observed in the leukocytes of PD patients. No changes were seen in other blood components. This suggests that GSTpi and potentially other members of this and other anti-oxidant families may be viable biomarkers for PD. PMID:21840241

The role of glutathione S-transferases in the detoxification of some organophosphorus insecticides in larvae and pupae of the yellow mealworm, Tenebrio molitor (Coleoptera: Tenebrionidae).

PubMed

Kostaropoulos, I; Papadopoulos, A I; Metaxakis, A; Boukouvala, E; Papadopoulou-Mourkidou, E

2001-06-01

The correlation between the natural levels of glutathione S-transferase (GST) and the tolerance to the organophosphorus insecticides parathion-methyl and paraoxon-methyl, as well as the interaction of affinity-purified enzyme and the insecticides were investigated in order to collect further information on the role of the glutathione S-transferase system as a mechanism of defence against insecticides in insects. The studies were carried out on the larvae and pupae of the coleopteran Tenebrio molitor L, which exhibit varying natural levels of GST activity. Stage-dependent susceptibility of the insect against insecticides was observed during the first 24 h. However, 48 h after treatment, the KD50 value increased significantly due to the recovery of some individuals. Simultaneous injection of insecticide with compounds which inhibit GST activity in vitro caused an alteration in susceptibility of insects 24 or 48 h post-treatment, depending on stage and insecticide used. Inhibition studies combined with competitive fluorescence spectroscopy revealed that the insecticides probably bind to the active site of the enzyme, thus inhibiting its activity towards 1-chloro-2,4-dinitrobenzene in a competitive manner. High-performance liquid chromatography and gas chromatography revealed that T molitor GST catalyses the conjugation of the insecticides studied to a reduced form of glutathione (GSH). From the above experimental results, it is considered that GST offers a protection against the organophosphorus insecticides studied by active site binding and subsequent conjugation with GSH.

Functional variability of glutathione S-transferases in Basque populations.

PubMed

Iorio, Andrea; Piacentini, Sara; Polimanti, Renato; De Angelis, Flavio; Calderon, Rosario; Fuciarelli, Maria

2014-01-01

Glutathione S-transferases (GSTs) are enzymes involved in Phase II reactions. They play a key role in cellular detoxification. Various studies have shown that genes coding for the GST are highly polymorphic and some of these variants are directly associated with a decrease of enzyme activity making individuals more susceptible to different clinical phenotypes. The aim of this study is to investigate the genetic variability of GST genes among human populations. We have focused our attention on the polymorphic variants of the GSTA1, GSTM1, GSTO1, GSTO2, GSTP1, GSTT1, and GSTT2B genes. These polymorphisms were analyzed in a whole sample of 151 individuals: 112 autochthonous Navarrese Basques, and 39 non-autochthonous Navarrese Basques. DNA extraction from plasma was performed by using the phenol:chloroform:isoamylic alcohol method. Genotyping of the gene polymorphisms was performed by PCR Multiplex and the PCR-RFLP method. We applied correspondence analysis and built frequency-maps to compare the genetic structure in worldwide populations. Our results were compared with data available on the Human Genome Diversity Project (HGDP) and on the 1,000 Genomes Project to obtain information on the functional variability of GSTs in Basques. Our data indicated that Basque communities showed a higher differentiation of certain functional GST variants (i.e., GSTM1-positive/null genotype, GSTP1*I105V, and GSTT2B*1/0) than other European and Mediterranean populations. This might account for epidemiological differences in the predisposition to diseases and drug response among Basques and could be used to design and interpret genetic association studies for this particular population. Copyright © 2014 Wiley Periodicals, Inc.

Development of pyrethroid-like fluorescent substrates for glutathione S-transferase

PubMed Central

Huang, Huazhang; Yao, Hongwei; Liu, Jun-Yan; Samra, Aman I.; Kamita, Shizuo G.; Cornel, Anthony J.; Hammock, Bruce D.

2012-01-01

The availability of highly sensitive substrates is critical for the development of precise and rapid assays for detecting changes in glutathione S-transferase (GST) activity that are associated with GST-mediated metabolism of insecticides. In this study, six pyrethroid-like compounds were synthesized and characterized as substrates for insect and mammalian GSTs. All of the substrates were esters composed of the same alcohol moiety, 7-hydroxy-4-methylcoumarin, and acid moieties that structurally mimic some commonly used pyrethroid insecticides including cypermethrin and cyhalothrin. CpGSTD1, a recombinant Delta class GST from the mosquito Culex pipiens, metabolized our pyrethroid-like substrates with both chemical and geometric (i.e., the cis-isomers were metabolized at 2- to 5-fold higher rates than the corresponding trans-isomers) preference. A GST preparation from mouse liver also metabolized most of our pyrethroid-like substrates with both chemical and geometric preference but at 10- to 170-fold lower rates. CpGSTD1 and mouse GSTs metabolized CDNB, a general GST substrate, at more than 200-fold higher rates than our novel pyrethroid-like substrates. There was a 10-fold difference in the specificity constant (kcat/KM ratio) of CpGSTD1 for CDNB and those of CpGSTD1 for cis-DCVC and cis-TFMCVC suggesting that cis-DCVC and cis-TFMCVC may be useful for the detection of GST-based metabolism of pyrethroids in mosquitoes. PMID:23000005

Amitriptyline may have a supportive role in cancer treatment by inhibiting glutathione S-transferase pi (GST-π) and alpha (GST-α).

PubMed

Kulaksiz-Erkmen, Gulnihal; Dalmizrak, Ozlem; Dincsoy-Tuna, Gamze; Dogan, Arın; Ogus, I Hamdi; Ozer, Nazmi

2013-02-01

A tricyclic anti-depressant, amitriptyline, is a highly prescribed drug for cancer patients for mood elevation but there are limited studies about the interaction of amitriptyline with glutathione S-transferases pi (GST-π) and glutathione S-transferases alpha (GST-α). GST isozymes have been implicated in chemotherapeutic drug resistance. We demonstrated that the concentration dependent inhibition of GST-π and GST-α by amitriptyline followed inverse hyperbolic inhibition curves with IC(50) values of 5.54 and 8.32 mM, respectively. When the varied substrate was GSH, amitriptyline inhibited both isozymes competitively and similar K(i) values were found for GST-π (K(i) = 1.61 ± 0.17 mM) and GST-α (K(i) = 1.45 ± 0.20 mM). On the other hand, when the varied substrate was CDNB, the inhibition types were non-competitive for GST-π (K(i) = 1.98 ± 0.31 mM) and competitive for GST-α (K(i) = 1.57 ± 0.16 mM). Amitriptyline, in addition to its antidepressant effect, might also have a minor supportive role on the effectiveness of the anticancer drugs by decreasing their elimination through inhibiting GST-π and GST-α.

S-Glutathionyl-(chloro)hydroquinone reductases: a novel class of glutathione transferases

PubMed Central

XUN, Luying; BELCHIK, Sara M.; XUN, Randy; HUANG, Yan; ZHOU, Huina; SANCHEZ, Emiliano; KANG, ChulHee; BOARD, Philip G.

2010-01-01

Sphingobium chlorophenolicum completely mineralizes PCP (pentachlorophenol). Two GSTs (glutathione transferases), PcpC and PcpF, are involved in the degradation. PcpC uses GSH to reduce TeCH (tetrachloro-p-hydroquinone) to TriCH (trichloro-p-hydroquinone) and then to DiCH (dichloro-p-hydroquinone) during PCP degradation. However, oxidatively damaged PcpC produces GS-TriCH (S-glutathionyl-TriCH) and GS-DiCH (S-glutathionyl-TriCH) conjugates. PcpF converts the conjugates into TriCH and DiCH, re-entering the degradation pathway. PcpF was further characterized in the present study. It catalysed GSH-dependent reduction of GS-TriCH via a Ping Pong mechanism. First, PcpF reacted with GS-TriCH to release TriCH and formed disulfide bond between its Cys53 residue and the GS moiety. Then, a GSH came in to regenerate PcpF and release GS–SG. A TBLASTN search revealed that PcpF homologues were widely distributed in bacteria, halobacteria (archaea), fungi and plants, and they belonged to ECM4 (extracellular mutant 4) group COG0435 in the conserved domain database. Phylogenetic analysis grouped PcpF and homologues into a distinct group, separated from Omega class GSTs. The two groups shared conserved amino acid residues, for GSH binding, but had different residues for the binding of the second substrate. Several recombinant PcpF homologues and two human Omega class GSTs were produced in Escherichia coli and purified. They had zero or low activities for transferring GSH to standard substrates, but all had reasonable activities for GSH-dependent reduction of disulfide bond (thiol transfer), dehydroascorbate and dimethylarsinate. All the tested PcpF homologues reduced GS-TriCH, but the two Omega class GSTs did not. Thus PcpF homologues were tentatively named S-glutathionyl-(chloro)hydroquinone reductases for catalysing the GSH-dependent reduction of GS-TriCH. PMID:20388120

Glutathione S-transferase-encoding gene as a potential probe for environmental bacterial isolates capable of degrading polycyclic aromatic hydrocarbons.

PubMed Central

Lloyd-Jones, G; Lau, P C

1997-01-01

Homologs of the glutathione S-transferase (GST)-encoding gene were identified in a collection of aromatic hydrocarbon-degrading Sphingomonas spp. isolated from New Zealand, Antarctica, and the United States by using PCR primers designed from the GST-encoding gene of Sphingomonas paucimobilis EPA505. Sequence analysis of PCR fragments generated from these isolates and of the GST gene amplified from DNA extracted from polycyclic aromatic hydrocarbon (PAH)-contaminated soil revealed a high degree of conservation, which may make the GST-encoding gene a potentially useful marker for PAH-degrading bacteria. PMID:9251217

Ferrocene labelings as inhibitors and dual electrochemical sensors of human glutathione S-transferase P1-1.

PubMed

Martos-Maldonado, Manuel C; Quesada-Soriano, Indalecio; García-Maroto, Federico; Vargas-Berenguel, Antonio; García-Fuentes, Luís

2012-12-01

The inhibitory and sensor properties of two ferrocene conjugates, in which the ferrocene and glutathione are linked through a spacer arm of different length and chemical structure, on human Pi glutathione S-transferase, were examined by activity assays, ITC, fluorescence spectroscopy and voltammetry. Such ferrocene conjugates are strong competitive inhibitors of this enzyme with an enhanced binding affinity, the one bearing the longest spacer arm being the most potent inhibitor. Voltammetric measurements showed a strong decrease of the peak current intensity and an increase of the oxidation potential upon binding of ferrocene-glutathione conjugates to GST P1-1 showing that both conjugates can be used as dual electrochemical sensors for GST P1-1. Copyright © 2012 Elsevier Ltd. All rights reserved.

Probing the active site of alpha-class rat liver glutathione S-transferases using affinity labeling by monobromobimane.

PubMed Central

Hu, L.; Borleske, B. L.; Colman, R. F.

1997-01-01

Monobromobimane (mBBr) is a substrate of both mu- and alpha-class rat liver glutathione S-transferases, with Km values of 0.63 microM and 4.9 microM for the mu-class isozymes 3-3 and 4-4, respectively, and 26 microM for the alpha-class isozymes 1-1 and 2-2. In the absence of substrate glutathione, mBBr acts as an affinity label of the 1-1 as well as mu-class isozymes, but not of the alpha-class 2-2 isozyme. Incubation of rat liver isozyme 1-1 with mBBr at pH 7.5 and 25 degrees C results in a time-dependent inactivation of the enzyme but at a slower (threefold) rate than for reactions with the mu-class isozyme 3-3 and 4-4. The rate of inactivation of 1-1 isozyme by mBBr is not decreased but, rather, is slightly enhanced by S-methyl glutathione. In contrast, 17 beta-estradiol-3,17-disulfate (500 microM) gives a 12.5-fold decrease in the observed rate constant of inactivation by 4 mM mBBr. When incubated for 60 min with 4 mM mBBr, the 1-1 isozyme loses 60% of its activity and incorporates 1.7 mol reagent/mol subunit. Peptide analysis after thermolysin digestion indicates that mBBr modification is equally distributed between two cysteine residues at positions 17 and 111. Modification at these two sites is reduced equally in the presence of the added protectant, 17 beta-estradiol-3,17-disulfate, suggesting that Cys 17 and Cys 111 reside within or near the enzyme's steroid binding sites. In contrast to the 1-1 isozyme, the other alpha-class isozyme (2-2) is not inactivated by mBBr at concentrations as high as 15 mM. The different reaction kinetics and modification sites by mBBr suggest that distinct binding site structures are responsible for the characteristic substrate specificities of glutathione S-transferase isozymes. PMID:9007975

Probing the active site of alpha-class rat liver glutathione S-transferases using affinity labeling by monobromobimane.

PubMed

Hu, L; Borleske, B L; Colman, R F

1997-01-01

Monobromobimane (mBBr) is a substrate of both mu- and alpha-class rat liver glutathione S-transferases, with Km values of 0.63 microM and 4.9 microM for the mu-class isozymes 3-3 and 4-4, respectively, and 26 microM for the alpha-class isozymes 1-1 and 2-2. In the absence of substrate glutathione, mBBr acts as an affinity label of the 1-1 as well as mu-class isozymes, but not of the alpha-class 2-2 isozyme. Incubation of rat liver isozyme 1-1 with mBBr at pH 7.5 and 25 degrees C results in a time-dependent inactivation of the enzyme but at a slower (threefold) rate than for reactions with the mu-class isozyme 3-3 and 4-4. The rate of inactivation of 1-1 isozyme by mBBr is not decreased but, rather, is slightly enhanced by S-methyl glutathione. In contrast, 17 beta-estradiol-3,17-disulfate (500 microM) gives a 12.5-fold decrease in the observed rate constant of inactivation by 4 mM mBBr. When incubated for 60 min with 4 mM mBBr, the 1-1 isozyme loses 60% of its activity and incorporates 1.7 mol reagent/mol subunit. Peptide analysis after thermolysin digestion indicates that mBBr modification is equally distributed between two cysteine residues at positions 17 and 111. Modification at these two sites is reduced equally in the presence of the added protectant, 17 beta-estradiol-3,17-disulfate, suggesting that Cys 17 and Cys 111 reside within or near the enzyme's steroid binding sites. In contrast to the 1-1 isozyme, the other alpha-class isozyme (2-2) is not inactivated by mBBr at concentrations as high as 15 mM. The different reaction kinetics and modification sites by mBBr suggest that distinct binding site structures are responsible for the characteristic substrate specificities of glutathione S-transferase isozymes.

Characterization of glutathione-S-transferases in zebrafish (Danio rerio).

PubMed

Glisic, Branka; Mihaljevic, Ivan; Popovic, Marta; Zaja, Roko; Loncar, Jovica; Fent, Karl; Kovacevic, Radmila; Smital, Tvrtko

2015-01-01

Glutathione-S-transferases (GSTs) are one of the key enzymes that mediate phase II of cellular detoxification. The aim of our study was a comprehensive characterization of GSTs in zebrafish (Danio rerio) as an important vertebrate model species frequently used in environmental research. A detailed phylogenetic analysis of GST superfamily revealed 27 zebrafish gst genes. Further insights into the orthology relationships between human and zebrafish GSTs/Gsts were obtained by the conserved synteny analysis. Expression of gst genes in six tissues (liver, kidney, gills, intestine, brain and gonads) of adult male and female zebrafish was determined using qRT-PCR. Functional characterization was performed on 9 cytosolic Gst enzymes after overexpression in E. coli and subsequent protein purification. Enzyme kinetics was measured for GSH and a series of model substrates. Our data revealed ubiquitously high expression of gstp, gstm (except in liver), gstr1, mgst3a and mgst3b, high expression of gsto2 in gills and ovaries, gsta in intestine and testes, gstt1a in liver, and gstz1 in liver, kidney and brain. All zebrafish Gsts catalyzed the conjugation of GSH to model GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and monochlorobimane (MCB), apart from Gsto2 and Gstz1 that catalyzed GSH conjugation to dehydroascorbate (DHA) and dichloroacetic acid (DCA), respectively. Affinity toward CDNB varied from 0.28 mM (Gstp2) to 3.69 mM (Gstm3), while affinity toward MCB was in the range of 5 μM (Gstt1a) to 250 μM (Gstp1). Affinity toward GSH varied from 0.27 mM (Gstz1) to 4.45 mM (Gstt1a). Turnover number for CDNB varied from 5.25s(-1) (Gstt1a) to 112s(-1) (Gstp2). Only Gst Pi enzymes utilized ethacrynic acid (ETA). We suggest that Gstp1, Gstp2, Gstt1a, Gstz1, Gstr1, Mgst3a and Mgst3b have important role in the biotransformation of xenobiotics, while Gst Alpha, Mu, Pi, Zeta and Rho classes are involved in the crucial physiological processes. In summary, this study provides the

Phi Class of Glutathione S-transferase Gene Superfamily Widely Exists in Nonplant Taxonomic Groups.

PubMed

Munyampundu, Jean-Pierre; Xu, You-Ping; Cai, Xin-Zhong

2016-01-01

Glutathione S-transferases (GSTs) constitute a superfamily of enzymes involved in detoxification of noxious compounds and protection against oxidative damage. GST class Phi (GSTF), one of the important classes of plant GSTs, has long been considered as plant specific but was recently found in basidiomycete fungi. However, the range of nonplant taxonomic groups containing GSTFs remains unknown. In this study, the distribution and phylogenetic relationships of nonplant GSTFs were investigated. We identified GSTFs in ascomycete fungi, myxobacteria, and protists Naegleria gruberi and Aureococcus anophagefferens. GSTF occurrence in these bacteria and protists correlated with their genome sizes and habitats. While this link was missing across ascomycetes, the distribution and abundance of GSTFs among ascomycete genomes could be associated with their lifestyles to some extent. Sequence comparison, gene structure, and phylogenetic analyses indicated divergence among nonplant GSTFs, suggesting polyphyletic origins during evolution. Furthermore, in silico prediction of functional partners suggested functional diversification among nonplant GSTFs.

Phi Class of Glutathione S-transferase Gene Superfamily Widely Exists in Nonplant Taxonomic Groups

PubMed Central

Munyampundu, Jean-Pierre; Xu, You-Ping; Cai, Xin-Zhong

2016-01-01

Glutathione S-transferases (GSTs) constitute a superfamily of enzymes involved in detoxification of noxious compounds and protection against oxidative damage. GST class Phi (GSTF), one of the important classes of plant GSTs, has long been considered as plant specific but was recently found in basidiomycete fungi. However, the range of nonplant taxonomic groups containing GSTFs remains unknown. In this study, the distribution and phylogenetic relationships of nonplant GSTFs were investigated. We identified GSTFs in ascomycete fungi, myxobacteria, and protists Naegleria gruberi and Aureococcus anophagefferens. GSTF occurrence in these bacteria and protists correlated with their genome sizes and habitats. While this link was missing across ascomycetes, the distribution and abundance of GSTFs among ascomycete genomes could be associated with their lifestyles to some extent. Sequence comparison, gene structure, and phylogenetic analyses indicated divergence among nonplant GSTFs, suggesting polyphyletic origins during evolution. Furthermore, in silico prediction of functional partners suggested functional diversification among nonplant GSTFs. PMID:26884677

Increased resistance to acetaminophen hepatotoxicity in mice lacking glutathione S-transferase Pi

PubMed Central

Henderson, Colin J.; Wolf, C. Roland; Kitteringham, Neil; Powell, Helen; Otto, Diana; Park, B. Kevin

2000-01-01

Overdose of acetaminophen, a widely used analgesic drug, can result in severe hepatotoxicity and is often fatal. This toxic reaction is associated with metabolic activation by the P450 system to form a quinoneimine metabolite, N-acetyl-p-benzoquinoneimine (NAPQI), which covalently binds to proteins and other macromolecules to cause cellular damage. At low doses, NAPQI is efficiently detoxified, principally by conjugation with glutathione, a reaction catalyzed in part by the glutathione S-transferases (GST), such as GST Pi. To assess the role of GST in acetaminophen hepatotoxicity, we examined acetaminophen metabolism and liver damage in mice nulled for GstP (GstP1/P2(−/−)). Contrary to our expectations, instead of being more sensitive, GstP null mice were highly resistant to the hepatotoxic effects of this compound. No significant differences between wild-type (GstP1/P2(+/+)) mice and GstP1/P2(−/−) nulls in either the rate or route of metabolism, particularly to glutathione conjugates, or in the levels of covalent binding of acetaminophen-reactive metabolites to cellular protein were observed. However, although a similar rapid depletion of hepatic reduced glutathione (GSH) was found in both GstP1/P2(+/+) and GstP1/P2(−/−) mice, GSH levels only recovered in the GstP1/P2(−/−) mice. These data demonstrate that GstP does not contribute in vivo to the formation of glutathione conjugates of acetaminophen but plays a novel and unexpected role in the toxicity of this compound. This study identifies new ways in which GST can modulate cellular sensitivity to toxic effects and suggests that the level of GST Pi may be an important and contributing factor in the sensitivity of patients with acetaminophen-induced hepatotoxicity. PMID:11058152

Limonin Methoxylation Influences Induction of Glutathione S-Transferase and Quinone Reductase

PubMed Central

PEREZ, JOSE LUIS; JAYAPRAKASHA, G. K.; VALDIVIA, VIOLETA; MUNOZ, DIANA; DANDEKAR, DEEPAK V.; AHMAD, HASSAN; PATIL, BHIMANAGOUDA S.

2009-01-01

Previous studies have indicated the chemoprevention potential of citrus limonoids due to the induction of phase II detoxifying enzymes. In the present study, three citrus limonoids were purified and identified from sour orange seeds as limonin, limonin glucoside (LG), deacetylnomilinic acid glucoside (DNAG). In addition, limonin was modified to defuran limonin and limonin 7-methoxime. The structures of these compounds were confirmed by NMR studies. These five compounds were used to investigate the influence of Phase II enzymes in female A/J mice. Our results indicated that the highest induction of Glutathione S-Transferase (GST) activity against 1-chloro-2, 4-dinitrobenzene (CDNB) by DNAG (67%) in lung homogenates followed by limonin-7-methoxime (32%) in treated liver homogenates. Interestingly, the limonin-7-methoxime showed the highest GST activity (270%) in liver against 4-nitroquinoline 1-oxide (4NQO), while the same compound in stomach induced GST by 51% compared to the control. DNAG treated group induced 55% in stomach homogenates. Another Phase II enzyme, quinone reductase (QR), was significantly induced by limonin-7-methoxime by 65 and 32% in liver and lung homogenates, respectively. Defuran limonin, induced QR in lung homogenates by 45%. Our results indicated that modification of the limonin have differential induction of phase II enzymes. These findings are indicative of a possible mechanism for the prevention of cancer by aiding in detoxification of xenobiotics. PMID:19480426

Oxidative stress markers and genetic polymorphisms of glutathione S-transferase T1, M1, and P1 in a subset of children with autism spectrum disorder in Lagos, Nigeria.

PubMed

Oshodi, Y; Ojewunmi, O; Oshodi, T A; Ijarogbe, G T; Ogun, O C; Aina, O F; Lesi, Fea

2017-09-01

The role of oxidative stress has been identified in the development of autism spectrum disorder (ASD), and polymorphisms of glutathione S-transferase have been associated with some diseases linked to oxidative stress. Hence, we evaluated the serum levels of oxidative stress markers and investigated genetic polymorphisms of glutathione S-transferase associated with autism. Forty-two children clinically diagnosed with ASD using the Diagnostic and Statistical Manual for Mental Disorders (DSM-5) criteria and a clinical interview were included in the study. Twenty-three age-matched controls without any known genetic/developmental disorder were also recruited. Oxidative stress markers along with the genetic polymorphisms of glutathione S-transferase were determined. Reduced glutathione in ASD patients was significantly lower than the control (P = 0.008), whereas other oxidative stress markers measured were not significantly different in both the control and case populations. The frequencies of GSTT1 and GSTM1 null genotypes were lower among the controls compared with the cases, however, no association risk was observed. The observed risk of carrying Val/Val genotype among the cases was approximately six times that of the controls. Individuals with ASD showed a significant diminished level of reduced glutathione, however, the distribution of GSTT1, GSTM1, and GSTP1 polymorphisms was not found to be associated with autism in this study population.

SKN-1-independent transcriptional activation of glutathione S-transferase 4 (GST-4) by EGF signaling

PubMed Central

Van de Walle, Pieter; Schoofs, Liliane

2016-01-01

ABSTRACT In C. elegans research, transcriptional activation of glutathione S-transferase 4 (gst-4) is often used as a read-out for SKN-1 activity. While many heed an assumed non-exclusivity of the GFP reporter signal driven by the gst-4 promoter to SKN-1, this is also often ignored. We here show that gst-4 can also be transcriptionally activated by EOR-1, a transcription factor mediating effects of the epidermal growth factor (EGF) pathway. Along with enhancing exogenous oxidative stress tolerance, EOR-1 inde-pendently of SKN-1 increases gst-4 transcription in response to augmented EGF signaling. Our findings caution researchers within the C. elegans community to always rely on sufficient experimental controls when assaying SKN-1 transcriptional activity with a gst-4p::gfp reporter, such as SKN-1 loss-of-function mutants and/or additional target genes next to gst-4. PMID:28090393

Association between glutathione S-transferase M1, P1, and NFKB1 polymorphisms and systemic lupus erythematosus susceptibility: a meta-analysis.

PubMed

Lee, Y H; Song, G G

2016-09-30

This study aimed to determine whether Glutathione S-transferase M1 (GSTM1), P1 (GSTT1), NFKB1 polymorphisms confer susceptibility to systemic lupus erythematosus (SLE). We performed a meta-analysis on the associations between GSTM1 and GSTT1 null genotypes, and NFKB1 -94 ins/delATTG polymorphisms and SLE. In total, seven studies were considered for this meta-analysis, which comprised 2,119 SLE patients and 3,014 healthy controls. Meta-analysis of the GSTM1 null polymorphism in 869 SLE and 1,544 control subjects revealed an association between SLE and the GSTM1 null genotype (OR = 1.321, 95% CI = 1.103-1.583, p = 0.002). Stratification by ethnicity indicated an association between the GSTM1 null genotype and SLE in Asians (OR = 1.334, 95% CI = 1.096-1.623, p = 0.004). However, meta-analysis of the GSTT1 null polymorphism, comprising 717 SLE and 1,008 control subjects, revealed no association between SLE and the GSTT1 null genotype overall (OR = 0.850, 95% CI = 0.687-1.051, p = 0.113) or in an Asian population (OR = 0.794, 95% CI = 0.594-1.061, p = 0.119). Meta-analysis of the NFKB1 -94 ins/delATTG polymorphism, comprising 1,250 SLE and 1,127 control subjects, revealed an association between SLE and the NFKB1 D allele (OR = 1.127, 95% CI = 1.011-1.257, p = 0.031). Ethnicity-specific meta-analysis revealed an association between the NFKB1 D allele and SLE in Asians (OR = 1.155, 95% CI = 1.026-1.300, p = 0.017). This meta-analysis demonstrates that the functional GSTM1 and NFKB1 polymorphisms are associated with the SLE risk in Asians.

Characterization of glutathione S-transferase and its immunodiagnostic potential for detecting Taenia multiceps.

PubMed

Sun, Ying; Wang, Yu; Huang, Xing; Gu, Xiaobing; Lai, Weimin; Peng, Xuerong; Yang, Guangyou

2017-08-15

Taenia multiceps is a widespread zoonotic tapeworm parasite which infects cloven-hoofed animals around the world. Animal infection with Coenurus cerebralis, the coenurus larvae of T. multiceps (Tm), is often fatal, which is a major cause of economic losses in stockbreeding. This study amplified the glutathione S-transferase (GST) gene from the total RNA of C. cerebralis. The resulting protein, Tm-GST, consisted of 201 amino acids, and had a predicted molecular mass of 23.1kDa. Its amino acid sequence shares 77.61% similarity with Echinococcus granulosus GST. Recombinant Tm-GST (rTm-GST) was expressed in Escherichia coli. The protein reacted with serum from goats infected with T. multiceps. Immunofluorescence signals indicated that Tm-GST was largely localized in the parenchymatous area of adult T. multiceps; in addition, it was also apparent in the coenurus. An enzyme-linked immunosorbent assay based on rTm-GST showed specificity of 92.8% (13/14) and sensitivity of 90% (18/20) in detecting anti-GST antibodies in serum from naturally infected animals. This study suggests that Tm-GST has the potential to be used as a diagnostic antigen for Coenurosis. Copyright © 2017. Published by Elsevier B.V.

Erk-Creb pathway suppresses glutathione-S-transferase pi expression under basal and oxidative stress conditions in zebrafish embryos.

PubMed

Hrubik, Jelena; Glisic, Branka; Fa, Svetlana; Pogrmic-Majkic, Kristina; Andric, Nebojsa

2016-01-05

Transcriptional activation of phase II enzymes including glutathione-S-transferase pi class (Gst Pi) is important for redox regulation and defense from xenobiotics. The role of extracellular signal-regulated kinase (Erk) and protein kinase B (Akt) in regulation of Gst Pi expression has been described using adult mammalian cells. Whether these signaling pathways contribute to Gst Pi expression during embryogenesis is unknown. Using zebrafish embryo model, we provide novel evidence that Erk signaling acts as a specific suppressor of gstp1-2 mRNA during early embryogenesis. Addition of Erk inhibitor U0126 enhanced gstp1-2 mRNA expression during transition from blastula to the segmentation stage and from pharyngula until the hatching stage. Basal Erk activity did not affect gstp1-2 expression in tert-butylhydroquinone-exposed embryos. Addition of phorbol 12-myristate 13-acetate increased Erk activity leading to suppression of gstp1-2 mRNA. Activation of cAMP/Creb pathway by forskolin prevented gstp1-2 expression, whereas U0126 suppressed Creb phosphorylation, thus setting up Creb as a proximal transmitter of Erk inhibitory effect. Collectively, these findings suggest that Erk-Creb pathway exerts suppressive effect on gstp1-2 mRNA in a narrow developmental window. This study also provides a novel link between Erk and gstp1-2 expression, setting apart a possible differential regulation of gstp1-2 in adult and embryonic cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Decreased Glutathione S-transferase Level and Neonatal Hyperbilirubinemia Associated with Glucose-6-phosphate Dehydrogenase Deficiency: A Perspective Review.

PubMed

Al-Abdi, Sameer Yaseen

2017-02-01

Classically, genetically decreased bilirubin conjugation and/or hemolysis account for the mechanisms contributing to neonatal hyperbilirubinemia associated with glucose-6-phosphate dehydrogenase (G6PD) deficiency. However, these mechanisms are not involved in most cases of this hyperbilirubinemia. Additional plausible mechanisms for G6PD deficiency-associated hyperbilirubinemia need to be considered. Glutathione S-transferases (GST) activity depends on a steady quantity of reduced form of glutathione (GSH). If GSH is oxidized, it is reduced back by glutathione reductase, which requires the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH). The main source of NADPH is the pentose phosphate pathway, in which G6PD is the first enzyme. Rat kidney GSH, rat liver GST, and human red blood cell GST levels have been found to positively correlate with G6PD levels in their respective tissues. As G6PD is expressed in hepatocytes, it is expected that GST levels would be significantly decreased in hepatocytes of G6PD-deficient neonates. As hepatic GST binds bilirubin and prevents their reflux into circulation, hypothesis that decreased GST levels in hepatocytes is an additional mechanism contributing to G6PD deficiency-associated hyperbilirubinemia seems plausible. Evidence for and against this hypothesis are discussed in this article hoping to stimulate further research on the role of GST in G6PD deficiency-associated hyperbilirubinemia. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

JS-K, a glutathione S-transferase-activated nitric oxide donor with antineoplastic activity in malignant gliomas.

PubMed

Weyerbrock, Astrid; Osterberg, Nadja; Psarras, Nikolaos; Baumer, Brunhilde; Kogias, Evangelos; Werres, Anna; Bette, Stefanie; Saavedra, Joseph E; Keefer, Larry K; Papazoglou, Anna

2012-02-01

Glutathione S-transferases (GSTs) control multidrug resistance and are upregulated in many cancers, including malignant gliomas. The diazeniumdiolate JS-K generates nitric oxide (NO) on enzymatic activation by glutathione and GST, showing promising NO-based anticancer efficacy. To evaluate the role of NO-based antitumor therapy with JS-K in U87 gliomas in vitro and in vivo. U87 glioma cells and primary glioblastoma cell lines were exposed to JS-K and a variety of inhibitors to study cell death by necrosis, apoptosis, and other mechanisms. GST expression was evaluated by immunocytochemistry, polymerase chain reaction, and Western blot, and NO release from JS-K was studied with a NO assay. The growth-inhibitory effect of JS-K was studied in a U87 xenograft model in vivo. Dose-dependent inhibition of cell proliferation was observed in human U87 glioma cells and primary glioblastoma cells in vitro. Cell death was partially induced by caspase-dependent apoptosis, which could be blocked by Z-VAD-FMK and Q-VD-OPH. Inhibition of GST by sulfasalazine, cGMP inhibition by ODQ, and MEK1/2 inhibition by UO126 attenuated the antiproliferative effect of JS-K, suggesting the involvement of various intracellular death signaling pathways. Response to JS-K correlated with mRNA and protein expression of GST and the amount of NO released by the glioma cells. Growth of U87 xenografts was reduced significantly, with immunohistochemical evidence for increased necrosis and apoptosis and reduced proliferation. Our data show for the first time the potent antiproliferative effect of JS-K in gliomas in vitro and in vivo. These findings warrant further investigation of this novel NO-releasing prodrug in gliomas.

Glutathione S-transferase P1 Ile105Val Polymorphism and Male Infertility Risk: An Updated Meta-analysis

PubMed Central

Huang, Xue-Kun; Huang, Yong-Han; Huang, Juan-Hua; Liang, Jing-Yao

2017-01-01

Background: Several studies concerning the association between glutathione S-transferase P1 (GSTP1) Ile105Val polymorphism and male infertility risk have reported controversial findings. The present study was aimed to explore this association using a meta-analysis. Methods: The PubMed, EMBASE, China National Knowledge Infrastructure (CNKI), and Wanfang databases were searched. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to estimate the strength of the association. Results: A total of 3282 cases and 3268 controls in nine case-control studies were included. There was no significant association between GSTP1 Ile105Val polymorphism and male infertility in the overall population, but significant associations were found under the dominant (OR = 1.23, 95% CI = 1.04–1.46, I2 = 32.2%) and heterozygote (OR = 1.29, 95% CI = 1.08–1.53, I2 = 26.8%) models after excluding studies for which the data did not satisfy Hardy-Weinberg equilibrium (HWE). Similarly, subgroup analyses revealed no significant association in Asians or Chinese population although a significant association was apparent among Chinese population in studies with HWE under the heterozygote model (OR = 1.25, 95% CI = 1.03–1.52, I2 = 44.1%). Significant heterogeneity could be observed in some genetic models, but this heterogeneity was not significant when stratified by HWE. No evidence for publication bias was found. Conclusions: The GSTP1 Ile105Val polymorphism might not be associated with male infertility risk, and thus additional well-designed studies with larger sample size are warranted. PMID:28397729

Prognostic significance of the null genotype of glutathione S-transferase-T1 in patients with acute myeloid leukemia: increased early death after chemotherapy.

PubMed

Naoe, T; Tagawa, Y; Kiyoi, H; Kodera, Y; Miyawaki, S; Asou, N; Kuriyama, K; Kusumoto, S; Shimazaki, C; Saito, K; Akiyama, H; Motoji, T; Nishimura, M; Shinagawa, K; Ueda, R; Saito, H; Ohno, R

2002-02-01

We investigated the prognostic significance of genetic polymorphism in glutathione-S transferase mu 1 (GSTM1), glutathione-S transferase theta 1 (GSTT1), NAD(P)H:quinone oxidoreductase (NQO1) and myeloperoxidase (MPO), the products of which are associated with drug metabolism as well as with detoxication, in 193 patients with de novo acute myeloid leukemia (AML) other than M3. Of the patients, 64.2% were either homozygous or heterozygous for GSTT1 (GSTT1(+)), while 35.8% showed homozygous deletions of GSTT1 (GSTT1(-)). The GSTT1(-) group had a worse prognosis than the GSTT1(+) group (P = 0.04), whereas other genotypes did not affect the outcome. Multivariate analysis revealed that GSTT1(-) was an independent prognostic factor for overall survival (relative risk: 1.53; P = 0.026) but not for disease-free survival of 140 patients who achieved complete remission (CR). The rate of early death after the initiation of chemotherapy was higher in the GSTT1(-) group than the GSTT1(+) group (within 45 days after initial chemotherapy, P = 0.073; within 120 days, P = 0.028), whereas CR rates and relapse frequencies were similar. The null genotype of GSTT1 might be associated with increased toxicity after chemotherapy.

Glutathione Transferase as a Potential Marker for Gut Epithelial Injury versus the Protective Role of Breast Milk sIgA in Infants with Rota Virus Gastroenteritis

PubMed Central

Sherif, Lobna S.; Raouf, Randaa K. Abdel; Sayede, Rokaya M. El; Wakkadd, Amany S. El; Shoaib, Ashraf R.; Ali, Hanan M.; Refay, Amira S. El

2015-01-01

BACKGROUND: Secretory immunoglobulin A (SIgA) plays an important protective role in the recognition and clearance of enteric pathogens. AIM: This study was designed to assess if mucosal integrity “measured by secretory IgA (SIgA)” is a protective factor from more epithelial alteration “measured by glutathione transferase” in infants with Rota gastroenteritis and its relation to infants’ feeding pattern. PATIENTS AND METHODS: This study was conducted on 79 infants aged 6 months and less from those diagnosed as having gastroenteritis and admitted to Gastroenteritis Department in Abo El Rish Pediatric Hospital, Cairo University. Plasma glutathione s-transferases and Stool SIgA were measured using ELISA technique. Rota virus detection was done by Reverse transcriptase PCR. RESULTS: SIgA was found to be significantly positive in exclusive breast fed infants, Glutathione transferase was significantly more frequently positive in Rota positive cases than Rota negative cases by Reverse transcriptase PCR. A significant negative correlation between Glutathione transferase and Secretory IgA was found, (p < 0.05). CONCLUSION: Breast feeding should be encouraged and highly recommended in the first two years of life as it provides Secretory IgA to breast fed infants who in turn protect them against epithelial damage caused by Rota viral gastroenteritis. PMID:27275307

Thermal- and urea-induced unfolding processes of glutathione S-transferase by molecular dynamics simulation.

PubMed

Li, Jiahuang; Chen, Yuan; Yang, Jie; Hua, Zichun

2015-05-01

The Schistosoma juponicum 26 kDa glutathione S-transferase (sj26GST) consists of the N-terminal domain (N-domain), containing three alpha-helices (named H1-H3) and four anti-parallel beta-strands (S1-S4), and the C-terminal domain (C-domain), comprising five alpha-helices (named H4-H8). In present work, molecular dynamics simulations and fluorescence spectroscopic were used to gain insights into the unfolding process of sj26GST. The molecular dynamics simulations on sj26GST subunit both in water and in 8 M urea were carried out at 300 K, 400 K and 500 K, respectively. Spectroscopic measurements were employed to monitor structural changes. Molecular dynamics simulations of sj26GST subunit induced by urea and temperature showed that the initial unfolding step of sj26GST both in water and urea occurred on N-domain, involving the disruption of helices H2, H3 and strands S3 and S4, whereas H6 was the last region exposed to solution and was the last helix to unfold. Moreover, simulations analyses combining with fluorescence and circular dichroism spectra indicated that N-domain could not fold independent, suggesting that correct folding of N-domain depended on its interactions with C-domain. We further proposed that the folding of GSTs could begin with the hydrophobic collapse of C-domain whose H4, H5, H6 and H7 could move close to each other and form a hydrophobic core, especially H6 wrapped in the hydrophobic center and beginning spontaneous formation of the helix. S3, S4, H3, and H2 could form in the wake of the interaction between C-domain and N-domain. The paper can offer insights into the molecular mechanism of GSTs unfolding. © 2014 Wiley Periodicals, Inc.

Glutathione Transferase U13 Functions in Pathogen-Triggered Glucosinolate Metabolism.

PubMed

Piślewska-Bednarek, Mariola; Nakano, Ryohei Thomas; Hiruma, Kei; Pastorczyk, Marta; Sanchez-Vallet, Andrea; Singkaravanit-Ogawa, Suthitar; Ciesiołka, Danuta; Takano, Yoshitaka; Molina, Antonio; Schulze-Lefert, Paul; Bednarek, Paweł

2018-01-01

Glutathione (GSH) and indole glucosinolates (IGs) exert key functions in the immune system of the model plant Arabidopsis ( Arabidopsis thaliana ). Appropriate GSH levels are important for execution of both pre- and postinvasive disease resistance mechanisms to invasive pathogens, whereas an intact PENETRATION2 (PEN2)-pathway for IG metabolism is essential for preinvasive resistance in this species. Earlier indirect evidence suggested that the latter pathway involves conjugation of GSH with unstable products of IG metabolism and further processing of the resulting adducts to biologically active molecules. Here we describe the identification of Glutathione- S -Transferase class-tau member 13 (GSTU13) as an indispensable component of the PEN2 immune pathway for IG metabolism. gstu13 mutant plants are defective in the pathogen-triggered biosynthesis of end products of the PEN2 pathway, including 4-O-β-d-glucosyl-indol-3-yl formamide, indole-3-ylmethyl amine, and raphanusamic acid. In line with this metabolic defect, lack of functional GSTU13 results in enhanced disease susceptibility toward several fungal pathogens including Erysiphe pisi , Colletotrichum gloeosporioides , and Plectosphaerella cucumerina Seedlings of gstu13 plants fail also to deposit the (1,3)-β-glucan cell wall polymer, callose, after recognition of the bacterial flg22 epitope. We show that GSTU13 mediates specifically the role of GSH in IG metabolism without noticeable impact on other immune functions of this tripeptide. We postulate that GSTU13 connects GSH with the pathogen-triggered PEN2 pathway for IG metabolism to deliver metabolites that may have numerous functions in the innate immune system of Arabidopsis. © 2018 American Society of Plant Biologists. All Rights Reserved.

Structure-activity relationships of 4-hydroxyalkenals in the conjugation catalysed by mammalian glutathione transferases.

PubMed Central

Danielson, U H; Esterbauer, H; Mannervik, B

1987-01-01

The substrate specificities of 15 cytosolic glutathione transferases from rat, mouse and man have been explored by use of a homologous series of 4-hydroxyalkenals, extending from 4-hydroxypentenal to 4-hydroxypentadecenal. Rat glutathione transferase 8-8 is exceptionally active with the whole range of 4-hydroxyalkenals, from C5 to C15. Rat transferase 1-1, although more than 10-fold less efficient than transferase 8-8, is the second most active transferase with the longest chain length substrates. Other enzyme forms showing high activities with these substrates are rat transferase 4-4 and human transferase mu. The specificity constants, kcat./Km, for the various enzymes have been determined with the 4-hydroxyalkenals. From these constants the incremental Gibbs free energy of binding to the enzyme has been calculated for the homologous substrates. The enzymes responded differently to changes in the length of the hydrocarbon side chain and could be divided into three groups. All glutathione transferases displayed increased binding energy in response to increased hydrophobicity of the substrate. For some of the enzymes, steric limitations of the active site appear to counteract the increase in binding strength afforded by increased chain length of the substrate. Comparison of the activities with 4-hydroxyalkenals and other activated alkenes provides information about the active-site properties of certain glutathione transferases. The results show that the ensemble of glutathione transferases in a given species may serve an important physiological role in the conjugation of the whole range of 4-hydroxyalkenals. In view of its high catalytic efficiency with all the homologues, rat glutathione transferase 8-8 appears to have evolved specifically to serve in the detoxication of these reactive compounds of oxidative metabolism. PMID:3426557

Epigenetic alterations are involved in the overexpression of glutathione S-transferase π-1 in human colorectal cancers.

PubMed

Zhang, Rui; Kang, Kyoung Ah; Piao, Mei Jing; Kim, Ki Cheon; Zheng, Jian; Yao, Cheng Wen; Cha, Ji Won; Maeng, Young Hee; Chang, Weon Young; Moon, Pyong-Gon; Baek, Moon-Chang; Hyun, Jin Won

2014-09-01

Glutathione S-transferase π-1 (GSTP-1) is a member of the glutathione S-transferase enzyme superfamily, which catalyzes the conjugation of electrophiles to glutathione during the process of detoxification. In this study, the epigenetic alterations of GSTP-1 expression in human colorectal cancers and the underlying mechanisms were investigated. In 10 colon cancer patients, proteomic analysis revealed that expression of GSTP-1 protein was higher in tumor tissues than in paired adjacent normal tissues. Likewise, in 7 of 10 colon cancer patients, GSTP-1 protein expression was more than 1.5-fold higher in tumor tissues than in adjacent normal tissues, as determined by western blotting. Immunohistochemical data confirmed that GSTP-1 protein was expressed at higher levels in colon cancer tissues compared to normal mucosa. GSTP-1 enzyme activity was closely correlated with GSTP-1 protein expression in colon cancer patients. Consistent with this, GSTP-1 mRNA, protein and activity levels were higher in the colorectal cancer cell lines Caco-2, HCT-116, HT-29, SNU-407 and SNU-1033 compared to the normal colon cell line FHC. Methylation-specific PCR results indicated that the high levels of GSTP-1 in human colorectal cancer cell lines were likely due to the lower degree of promoter methylation in colon cancer cell lines compared to the normal colon cell line, consistent with findings in colon cancer patients. Moreover, the levels of specific activator-protein complexes and histone marks were higher in human colorectal cancer cells compared to the normal human colon cell line, whereas the repressor protein complexes exhibited the opposite pattern. Furthermore, chromatin immunoprecipitation assays demonstrated that expression levels of the transcription factors AP-1 and SP-1 were correlated with the upregulation of GSTP-1 expression in colorectal cancer cells. Finally, knockdown of GSTP-1 promoted the sensitivity of SNU-407 cells to the anticancer agent 5-fluorouracil. These

Lymphocyte DNA damage and plasma antioxidant status in Korean subclinical hypertensive patients by glutathione S-transferase polymorphism

PubMed Central

Han, Jeong-Hwa; Lee, Hye-Jin; Choi, Hee Jeong; Yun, Kyung Eun

2017-01-01

BACKGROUND/OBJECTIVES Glutathione S-transferase (GST) forms a multigene family of phase II detoxification enzymes which are involved in the detoxification of xenobiotics by conjugating substances with glutathione. The aim of this study is to assess the antioxidative status and the degree of DNA damage in the subclinical hypertensive patients in Korea using glutathione S-transferase polymorphisms. SUBJECTS/METHODS We examined whether DNA damage and antioxidative status show a difference between GSTM1 or GSTT1 genotype in 227 newly diagnosed, untreated (systolic blood pressure (BP) ≥ 130 mmHg or diastolic BP ≥ 85 mmHg) subclinical hypertensive patients and 130 normotensive subjects (systolic BP < 120 mmHg and diastolic BP < 80 mmHg). From the blood of the subjects, the degree of the DNA damage in lymphocyte, the activities of erythrocyte superoxide dismutase, the catalase, and the glutathione peroxidase, the level of glutathione, plasma total radical-trapping antioxidant potential (TRAP), anti-oxidative vitamins, as well as plasma lipid profiles and conjugated diene (CD) were analyzed. RESULTS Of the 227 subjects studied, 68.3% were GSTM1 null genotype and 66.5% were GSTT1 null genotype. GSTM1 null genotype had an increased risk of hypertension (OR: 2.104, CI: 1.38-3.35), but no significant association in GSTT1 null genotype (OR 0.982, CI: 0.62-1.55). No difference in erythrocyte activities of superoxide dismutase, catalase, or glutathione peroxidase, and plasma TRAP, CD, lipid profiles, and GSH levels were observed between GSTM1 or GSTT1 genotype. Plasma levels of α-tocopherol increased significantly in GSTT1 wild genotype (P < 0.05); however, plasma level of β-carotene increased significantly in GSTT1 null genotype (P < 0.01). DNA damage assessed by the Comet assay was significantly higher in GSTM1 null genotype than wild genotype (P < 0.05). CONCLUSIONS These results confirm the association between GSTM1 null genotype and risk of hypertension as they suggest

Curcumin activates human glutathione S-transferase P1 expression through antioxidant response element.

PubMed

Nishinaka, Toru; Ichijo, Yusuke; Ito, Maki; Kimura, Masayoshi; Katsuyama, Masato; Iwata, Kazumi; Miura, Takeshi; Terada, Tomoyuki; Yabe-Nishimura, Chihiro

2007-05-15

Curcumin is a plant-derived diferuloylmethane compound extracted from Curcuma longa, possessing antioxidative and anticarcinogenic properties. Antioxidants and oxidative stress are known to induce the expression of certain classes of detoxification enzymes. Since the upregulation of detoxifying enzymes affects the drug metabolism and cell defense system, it is important to understand the gene regulation by such agents. In this study, we demonstrated that curcumin could induce the expression of human glutathione S-transferase P1 (GSTP1). In HepG2 cells treated with 20muM curcumin, the level of GSTP1 mRNA was significantly increased. In luciferase reporter assays, curcumin augmented the promoter activity of a reporter construct carrying 336bp upstream of the 5'-flanking region of the GSTP1 gene. Mutation analyses revealed that the region including antioxidant response element (ARE), which overlaps AP1 in sequence, was essential to the response to curcumin. While the introduction of a wild-type Nrf2 expression construct augmented the promoter activity of the GSTP1 gene, co-expression of a dominant-negative Nrf2 abolished the responsiveness to curcumin. In addition, curcumin activated the expression of the luciferase gene from a reporter construct carrying multiple ARE consensus sequences but not one with multiple AP1 sites. In a gel mobility shift assay with an oligonucleotide with GSTP1 ARE, an increase in the amount of the binding complex was observed in the nuclear extracts of curcumin-treated HepG2 cells. These results suggested that ARE is the primary sequence for the curcumin-induced transactivation of the GSTP1 gene. The induction of GSTP1 may be one of the mechanisms underlying the multiple actions of curcumin.

Delineation of xenobiotic substrate sites in rat glutathione S-transferase M1-1

PubMed Central

Hearne, Jennifer L.; Colman, Roberta F.

2005-01-01

Glutathione S-transferases catalyze the conjugation of glutathione with endogenous and exogenous xenobiotics. Hu and Colman (1995) proposed that there are two distinct substrate sites in rat GST M1-1, a 1-chloro-2,4-dintrobenzene (CDNB) substrate site located in the vicinity of tyrosine-115, and a monobromobimane (mBBr) substrate site. To determine whether the mBBr substrate site is distinguishable from the CDNB substrate site, we tested S-(hydroxyethyl)bimane, a nonreactive derivative of mBBr, for its ability to compete kinetically with the substrates. We find that S-(hydroxyethyl)bimane is a competitive inhibitor (KI = 0.36 μM) when mBBr is used as substrate, but not when CDNB is used as substrate, demonstrating that these two sites are distinct. Using site-directed mutagenesis, we have localized the mBBr substrate site to an area midway through α-helix 4 (residues 90–114) and have identified residues that are important in the enzymatic reaction. Substitution of alanine at positions along α-helix 4 reveals that mutations at positions 103, 104, and 109 exhibit a greater perturbation of the enzymatic reaction with mBBr than with CDNB as substrate. Various other substitutions at positions 103 and 104 reveal that a hydrophobic residue is necessary at each of these positions to maintain optimal affinity of the enzyme for mBBr and preserve the secondary structure of the enzyme. Substitutions at position 109 indicate that this residue is important in the enzyme’s affinity for mBBr but has a minimal effect on Vmax. These results demonstrate that the promiscuity of rat GST M1-1 is in part due to at least two distinct substrate sites. PMID:16195544

Novosphingobium aromaticivorans uses a Nu-class glutathione S-transferase as a glutathione lyase in breaking the β-aryl ether bond of lignin

PubMed Central

Kontur, Wayne S.; Bingman, Craig A.; Olmsted, Charles N.; Wassarman, Douglas R.; Ulbrich, Arne; Gall, Daniel L.; Smith, Robert W.; Yusko, Larissa M.; Fox, Brian G.; Noguera, Daniel R.; Coon, Joshua J.; Donohue, Timothy J.

2018-01-01

As a major component of plant cell walls, lignin is a potential renewable source of valuable chemicals. Several sphingomonad bacteria have been identified that can break the β-aryl ether bond connecting most phenylpropanoid units of the lignin heteropolymer. Here, we tested three sphingomonads predicted to be capable of breaking the β-aryl ether bond of the dimeric aromatic compound guaiacylglycerol-β-guaiacyl ether (GGE) and found that Novosphingobium aromaticivorans metabolizes GGE at one of the fastest rates thus far reported. After the ether bond of racemic GGE is broken by replacement with a thioether bond involving glutathione, the glutathione moiety must be removed from the resulting two stereoisomers of the phenylpropanoid conjugate β-glutathionyl-γ-hydroxypropiovanillone (GS-HPV). We found that the Nu-class glutathione S-transferase NaGSTNu is the only enzyme needed to remove glutathione from both (R)- and (S)-GS-HPV in N. aromaticivorans. We solved the crystal structure of NaGSTNu and used molecular modeling to propose a mechanism for the glutathione lyase (deglutathionylation) reaction in which an enzyme-stabilized glutathione thiolate attacks the thioether bond of GS-HPV, and the reaction proceeds through an enzyme-stabilized enolate intermediate. Three residues implicated in the proposed mechanism (Thr51, Tyr166, and Tyr224) were found to be critical for the lyase reaction. We also found that Nu-class GSTs from Sphingobium sp. SYK-6 (which can also break the β-aryl ether bond) and Escherichia coli (which cannot break the β-aryl ether bond) can also cleave (R)- and (S)-GS-HPV, suggesting that glutathione lyase activity may be common throughout this widespread but largely uncharacterized class of glutathione S-transferases. PMID:29449375

Identification of Small-Molecule Frequent Hitters of Glutathione S-Transferase-Glutathione Interaction.

PubMed

Brenke, Jara K; Salmina, Elena S; Ringelstetter, Larissa; Dornauer, Scarlett; Kuzikov, Maria; Rothenaigner, Ina; Schorpp, Kenji; Giehler, Fabian; Gopalakrishnan, Jay; Kieser, Arnd; Gul, Sheraz; Tetko, Igor V; Hadian, Kamyar

2016-07-01

In high-throughput screening (HTS) campaigns, the binding of glutathione S-transferase (GST) to glutathione (GSH) is used for detection of GST-tagged proteins in protein-protein interactions or enzyme assays. However, many false-positives, so-called frequent hitters (FH), arise that either prevent GST/GSH interaction or interfere with assay signal generation or detection. To identify GST-FH compounds, we analyzed the data of five independent AlphaScreen-based screening campaigns to classify compounds that inhibit the GST/GSH interaction. We identified 53 compounds affecting GST/GSH binding but not influencing His-tag/Ni(2+)-NTA interaction and general AlphaScreen signals. The structures of these 53 experimentally identified GST-FHs were analyzed in chemoinformatic studies to categorize substructural features that promote interference with GST/GSH binding. Here, we confirmed several existing chemoinformatic filters and more importantly extended them as well as added novel filters that specify compounds with anti-GST/GSH activity. Selected compounds were also tested using different antibody-based GST detection technologies and exhibited no interference clearly demonstrating specificity toward their GST/GSH interaction. Thus, these newly described GST-FH will further contribute to the identification of FH compounds containing promiscuous substructures. The developed filters were uploaded to the OCHEM website (http://ochem.eu) and are publicly accessible for analysis of future HTS results. © 2016 Society for Laboratory Automation and Screening.

Glutathione S-transferase Pi mediates proliferation of androgen-independent prostate cancer cells

PubMed Central

Hokaiwado, Naomi; Takeshita, Fumitaka; Naiki-Ito, Aya; Asamoto, Makoto; Ochiya, Takahiro; Shirai, Tomoyuki

2008-01-01

Prostate cancers generally acquire an androgen-independent growth capacity with progression, resulting in resistance to antiandrogen therapy. Therefore, identification of the genes regulated through this process may be important for understanding the mechanisms of prostate carcinogenesis. We here utilized androgen-dependent/independent transplantable tumors, newly established with the ‘transgenic rat adenocarcinoma in prostate’ (TRAP) model, to analyze their gene expression using microarrays. Among the overexpressed genes in androgen-independent prostate cancers compared with the androgen-dependent tumors, glutathione S-transferase pi (GST-pi) was included. In line with this, human prostate cancer cell lines PC3 and DU145 (androgen independent) had higher expression of GST-pi compared with LNCaP (androgen dependent) as determined by semiquantitative reverse transcription–polymerase chain reaction analysis. To investigate the roles of GST-pi expression in androgen-independent human prostate cancers, GST-pi was knocked down by a small interfering RNA (siRNA), resulting in significant decrease of the proliferation rate in the androgen-independent PC3 cell line. In vivo, administration of GST-pi siRNA–atelocollagen complex decreased GST-pi protein expression, resulting in enhanced numbers of TdT mediated dUTP-biotin nick-end labering (TUNEL)-positive apoptotic cells. These findings suggest that GST-pi might play important roles in proliferation of androgen-independent human prostate cancer cells. PMID:18413363

Purification of Glutathione S-Transferase pi from Erythrocytes and Evaluation of the Inhibitory Effect of Hypericin.

PubMed

Turk, Seyhan; Kulaksiz Erkmen, Gulnihal; Dalmizrak, Ozlem; Ogus, I Hamdi; Ozer, Nazmi

2015-12-01

Hypericin is a photosensitizer compound used in the photodynamic therapy (PDT). PDT is an alternative cancer treatment strategy whose function is dependent on the photosensitizers accumulating selectively in tumor cells and following visible or infra-red light induced activation lead to the apoptosis/necrosis of the tumor cells via the formation of reactive oxygen species. Thus, the cellular redox balance is essential for the efficacy of PDT. Among the protective enzyme systems glutathione S-transferases (GST, E.C.2.5.1.18) function in detoxification, protection against oxidative stress and intracellular transport of molecules. It is known that isoenzymes of GST and especially GST-pi is increased in cancer cells and it plays very important functions in the development of resistance to anticancer drugs. Since photosensitizers are used intravenously, it is important to elucidate the effects of photosensitizers on the erythrocyte enzymes. The aim of the present study was to investigate the impact of hypericin on human erythrocyte GST-pi (heGST-pi). Purification yield of 71% and purification fold of 2550 were achieved by using conventional chromatographic methods. The specific activity of the enzyme is found as 51 U/mg protein. Hypericin inhibited heGST-pi in a dose dependent manner and inhibition was biphasic. Noncompetitive type of inhibition was observed with both substrates, GSH and CDNB. The inhibitory constant (K i ) values obtained from Lineweaver-Burk, Dixon, secondary plots; slope and y-intercept versus 1/S (substrate) and from non-linear regression analysis were in good correlation: K i (GSH) was calculated as 0.19 ± 0.01 μM and K i (CDNB) as 0.26 ± 0.03 μM.

Global Analysis of Apicomplexan Protein S-Acyl Transferases Reveals an Enzyme Essential for Invasion

PubMed Central

Frénal, Karine; Tay, Chwen L; Mueller, Christina; Bushell, Ellen S; Jia, Yonggen; Graindorge, Arnault; Billker, Oliver; Rayner, Julian C; Soldati-Favre, Dominique

2013-01-01

The advent of techniques to study palmitoylation on a whole proteome scale has revealed that it is an important reversible modification that plays a role in regulating multiple biological processes. Palmitoylation can control the affinity of a protein for lipid membranes, which allows it to impact protein trafficking, stability, folding, signalling and interactions. The publication of the palmitome of the schizont stage of Plasmodium falciparum implicated a role for palmitoylation in host cell invasion, protein export and organelle biogenesis. However, nothing is known so far about the repertoire of protein S-acyl transferases (PATs) that catalyse this modification in Apicomplexa. We undertook a comprehensive analysis of the repertoire of Asp-His-His-Cys cysteine-rich domain (DHHC-CRD) PAT family in Toxoplasma gondii and Plasmodium berghei by assessing their localization and essentiality. Unlike functional redundancies reported in other eukaryotes, some apicomplexan-specific DHHCs are essential for parasite growth, and several are targeted to organelles unique to this phylum. Of particular interest is DHHC7, which localizes to rhoptry organelles in all parasites tested, including the major human pathogen P. falciparum. TgDHHC7 interferes with the localization of the rhoptry palmitoylated protein TgARO and affects the apical positioning of the rhoptry organelles. This PAT has a major impact on T. gondii host cell invasion, but not on the parasite’s ability to egress. PMID:23638681

Glutathione-S-transferase P protects against endothelial dysfunction induced by exposure to tobacco smoke.

PubMed

Conklin, Daniel J; Haberzettl, Petra; Prough, Russell A; Bhatnagar, Aruni

2009-05-01

Exposure to tobacco smoke impairs endothelium-dependent arterial dilation. Reactive constituents of cigarette smoke are metabolized and detoxified by glutathione-S-transferases (GSTs). Although polymorphisms in GST genes are associated with the risk of cancer in smokers, the role of these enzymes in regulating the cardiovascular effects of smoking has not been studied. The P isoform of GST (GSTP), which catalyzes the conjugation of electrophilic molecules in cigarette smoke such as acrolein, was expressed in high abundance in the mouse lung and aorta. Exposure to tobacco smoke for 3 days (5 h/day) decreased total plasma protein. These changes were exaggerated in GSTP(-/-) mice. Aortic rings isolated from tobacco smoke-exposed GSTP(-/-) mice showed greater attenuation of ACh-evoked relaxation than those from GSTP(+/+) mice. The lung, plasma, and aorta of mice exposed to tobacco smoke or acrolein (for 5 h) accumulated more acrolein-adducted proteins than those tissues of mice exposed to air, indicating that exposure to tobacco smoke results in the systemic delivery of acrolein. Relative to GSTP(+/+) mice, modification of some proteins by acrolein was increased in the aorta of GSTP(-/-) mice. Aortic rings prepared from GSTP(-/-) mice that inhaled acrolein (1 ppm, 5 h/day for 3 days) or those exposed to acrolein in an organ bath showed diminished ACh-induced arterial relaxation more strongly than GSTP(+/+) mice. Acrolein-induced endothelial dysfunction was prevented by pretreatment of the aorta with N-acetylcysteine. These results indicate that GSTP protects against the endothelial dysfunction induced by tobacco smoke exposure and that this protection may be related to the detoxification of acrolein or other related cigarette smoke constituents.

Spectrofluorimetric assay method for glutathione and glutathione transferase using monobromobimane.

PubMed

Yakubu, S I; Yakasai, I A; Musa, A

2011-06-01

The primary role of glutathione transferase is to defend an organism from toxicities through catalyzing the reaction of glutathione (GSH) with potentially toxic compounds or metabolites to their chemically and biologically inert conjugates. The objective of the study was to develop a simple and sensitive spectrofluorimetric assay method for glutathione transferase using monobromobimane (MBB), a non fluorescent compound with electrophilic site. MBB slowly reacted with glutathione to form fluorescent glutathione conjugate and that the reaction was catalysed by glutathione transferase. Both non-enzymatic and enzymatic reaction products of MBB, in presence of GSH in phosphate buffer (pH 6.5), were measured by following increase of fluorescence at wavelength of 475nm. For validation of the assay method, the kinetic parameters such as the apparent Michaelis-Mente constants and maximum rates of conjugate formation as well as the specific activity of rat hepatic glutathione transferase were determined. The method was found to be sensitive, thus, applied to measure glutathione contents of crude preparation of rat hepatic cytosol fraction.

JS-K, a glutathione S-transferase-activated nitric oxide donor with antineoplastic activity in malignant gliomas

PubMed Central

Weyerbrock, Astrid; Osterberg, Nadja; Psarras, Nikolaos; Baumer, Brunhilde; Kogias, Evangelos; Werres, Anna; Bette, Stefanie; Saavedra, Joseph E.; Keefer, Larry K.; Papazoglou, Anna

2011-01-01

Background Glutathione S-transferases (GSTs) control multidrug-resistance and are upregulated in many cancers including malignant gliomas. The diazeniumdiolate JS-K generates nitric oxide (NO) on enzymatic activation by glutathione and GST, showing promising NO-based anticancer efficacy. Objective To evaluate the role of NO-based antitumor therapy with JS-K in U87 gliomas in vitro and in vivo. Methods U87 glioma cells and primary glioblastoma cell lines were exposed to JS-K and a variety of inhibitors to study cell death by necrosis, apoptosis and other mechanisms. GST-expression was evaluated by immunocytochemistry, PCR and Western blot and NO release from JS-K using a NO assay. The growth-inhibitory effect of JS-K was studied in a U87 xenograft model in vivo. Results Dose-dependent inhibition of cell proliferation was observed in human U87 glioma cells and primary glioblastoma cells in vitro. Cell death was partially induced by caspase-dependent apoptosis which could be blocked by Z-VAD-FMK and Q-VD-OPH. GST-inhibition by sulfasalazine, cGMP inhibition by ODQ and MEK 1/2 inhibition by UO126 attenuated the antiproliferative effect of JS-K, suggesting the involvement of various intracellular death signalling pathways. Response to JS-K correlated with mRNA and protein expression of GST and the amount of NO released by the glioma cells. Growth of U87 xenografts was significantly reduced, with immunohistochemical evidence for increased necrosis, apoptosis and reduced proliferation. Conclusion Our data for the first time show the potent antiproliferative effect of JS-K in gliomas in vitro and in vivo. These findings warrant further investigation of this novel NO-releasing prodrug in gliomas. PMID:21849924

Chlortetracycline detoxification in maize via induction of glutathione S-transferases after antibiotic exposure.

PubMed

Farkas, Michael H; Berry, James O; Aga, Diana S

2007-02-15

Soil contamination with nonmetabolized antibiotics is an emerging environmental concern, especially on agricultural croplands that receive animal manure as fertilizer. In this study, phytotoxicity of chlortetracycline (CTC) antibiotics on pinto beans (Phaseolus vulgaris) and maize (Zea mays) was investigated under controlled conditions. When grown in CTC-treated soil, a significant increase in the activities of the plant stress proteins glutathione S-transferases (GST) and peroxidases (POX) were observed in maize plants, but not in pinto beans. In vitro conjugation reactions demonstrated that the induced GST in maize catalyzed the conjugation of glutathione (GSH) with CTC, producing stable conjugates that were structurally characterized using liquid chromatography/mass spectrometry. The antibiotic-induced GST produced CTC-glutathione conjugate at relative concentrations 2-fold higher than that produced by constitutively expressed GST extracted from untreated maize. On the other hand, GST extracted from pinto beans (both treated and untreated) did not efficiently catalyze glutathione conjugation with CTC. These results suggest that maize is able to detoxify chlortetracycline via the glutathione pathway, whereas pinto beans cannot. This may explain the observed stunted growth of pinto beans after antibiotic treatment. This study demonstrates the importance of plant uptake in determining the fate of antibiotics in soil and their potential phytotoxicity to susceptible plants.

A meta-analysis of association between glutathione S-transferase M1 gene polymorphism and Parkinson's disease susceptibility.

PubMed

Weikang, Chen; Jie, Li; Likang, Lan; Weiwen, Qiu; Liping, Lu

2016-01-01

The aim of this meta-analysis was to evaluate whether there was an association between glutathione S-transferase M1(GSTM1)gene polymorphism and Parkinson's disease (PD) susceptibility by pooling published data. We performed comprehensive electronic database search for articles published between February12,2015 and April30 2016. The published case-control or cohort studies related to GSTM1 gene polymorphism and Parkinson's disease susceptibility were screened, reviewed, and included in this meta-analysis. The correlation between GSTM1 gene polymorphism and PD susceptibility was expressed by odds ratio (OR) and its corresponding 95% confidence interval (95%CI). Publication bias was evaluated by Begg's funnel plot and Egger's line regression test. All analysis was done by stata11.0 software. After searching the PubMed, EMBASE, and CNKI databases, seventeen case-control studies with 3,538 PD and 5,180 controls were included in the final meta-analysis. The data was pooled by a fixed-effect model for lack of statistical heterogeneity across the studies; the results showed GSTM1 null expression can significant increase the susceptibility of PD (OR=1.11, 95% CI:1.01-1.21, P<0.05). Subgroup analysis indicated GSTM1 gene polymorphism was associated with PD susceptibility in the Caucasian ethnic group (OR=1.15, 95% CI:1.05-1.27, P<0.05) but not in the Asian ethnic group (OR=0.89, 95% CI:0.70-1.12, P>0.05). Begg's funnel plot and Egger's line regression test showed no significant publication bias. Based on the present evidence, GSTM1 null expression can significant increase the susceptibility of PD in persons of Caucasian ethnicity.

The effect of chemical carcinogenesis on rat glutathione S-transferase P1 gene transcriptional regulation.

PubMed

Liu, D; Liao, M; Zuo, J; Henner, W D; Fan, F

2001-03-01

To investigate mechanisms of rat glutathione S-transferase P1 gene (rGSTP1) expression regulation during chemical carcinogenesis. we studied enhancer elements located in the region between -2.5 kb to -2.2 kb. The region was upstream from the start site of transcription and was divided into two major fragments, GPEI and GPEII. The GPEII fragment was further divided into two smaller fragments, GPEII- I and GPEII-2. Using a luciferase reporter system, we identified a strong enhancer of GPEI and a weak enhancer of GPEII in HeLa and a rat hepatoma cell line CBRH79 19 cell. The enhancer of GPEII was located within the GPEII-I region. Chemical stimulation by glycidyl methatylate (GMA) and phorbol 12-o-tetradecanoate 13-acetate (TPA) analysis revealed that induction of rGSTP1 expression was mainly through GPEI. Although H2O2 could enhance GPEII enhancer activity, the enhancement is not mediated by the NF-kappaB factor that bound the NF-kappaB site in GPEII. Using electrophoretic mobility shift assays (EMSA) and the UV cross-linking assays, we found that HeLa and CBRH7919 cells had proteins that specifically bound GPEI core sequence and a 64 kDa protein that interacted with GPEII-1. The cells from normal rat liver did not express the binding proteins. Therefore, the trans-acting factors seem to be closely related to GPEI, GPEII enhancer activities and may play an important role in high expression of rGSTPI gene.

A SERS protocol as a potential tool to access 6-mercaptopurine release accelerated by glutathione-S-transferase.

PubMed

Wang, Ying; Sun, Jie; Yang, Qingran; Lu, Wenbo; Li, Yan; Dong, Jian; Qian, Weiping

2015-11-21

The developed method for monitoring GST, an important drug metabolic enzyme, could greatly facilitate researches on relative biological fields. In this work, we have developed a SERS technique to monitor the absorbance behaviour of 6-mercaptopurine (6-MP) and its glutathione-S-transferase (GST)-accelerated glutathione (GSH)-triggered release behaviour on the surface of gold nanoflowers (GNFs), using the GNFs as excellent SERS substrates. The SERS signal was used as an indicator of absorbance or release of 6-MP on the gold surface. We found that GST can accelerate GSH-triggered release behaviour of 6-MP from the gold surface. We speculated that GST catalyzes nucleophilic GSH to competitively bind with the electrophilic substance 6-MP. Experimental results have proved that the presented SERS protocol can be utilized as an effective tool for accessing the release of anticancer drugs.

Purification and properties of the glutathione S-transferases from the anoxia-tolerant turtle, Trachemys scripta elegans.

PubMed

Willmore, William G; Storey, Kenneth B

2005-07-01

Glutathione S-transferases (GSTs) play critical roles in detoxification, response to oxidative stress, regeneration of S-thiolated proteins, and catalysis of reactions in nondetoxification metabolic pathways. Liver GSTs were purified from the anoxia-tolerant turtle, Trachemys scripta elegans. Purification separated a homodimeric (subunit relative molecular mass =34 kDa) and a heterodimeric (subunit relative molecular mass = 32.6 and 36.8 kDa) form of GST. The enzymes were purified 23-69-fold and 156-174-fold for homodimeric and heterodimeric GSTs, respectively. Kinetic data gathered using a variety of substrates and inhibitors suggested that both homodimeric and heterodimeric GSTs were of the alpha class although they showed significant differences in substrate affinities and responses to inhibitors. For example, homodimeric GST showed activity with known alpha class substrates, cumene hydroperoxide and p-nitrobenzylchloride, whereas heterodimeric GST showed no activity with cumene hydroperoxide. The specific activity of liver GSTs with chlorodinitrobenzene (CDNB) as the substrate was reduced by 2.6- and 8.7-fold for homodimeric and heterodimeric GSTs isolated from liver of anoxic turtles as compared with aerobic controls, suggesting an anoxia-responsive stable modification of the protein that may alter its function during natural anaerobiosis.

Expression of glutathione S-transferases in poplar trees (Populus trichocarpa) exposed to 2,4,6-trinitrotoluene (TNT).

PubMed

Brentner, Laura B; Mukherji, Sachiyo T; Merchie, Kate M; Yoon, Jong Moon; Schnoor, Jerald L; Van Aken, Benoit

2008-10-01

Twelve Populus genes were identified from Arabidopsis thaliana sequences previously shown to be induced by exposure to 2,4,6-trinitrotoluene (TNT). Using the resources of the Poplar Genome Project and National Center for Biotechnology Information databases, Populus conserved domains were identified and used to design gene specific primers. RNA extracted from root tissues of TNT-exposed hydroponic poplar plants was used to quantify the expression of genes by reverse-transcriptase real-time polymerase chain reaction. Cyclophilin and 18S ribosomal DNA genes were used as internal standards. Exposure to TNT resulted in a significant increase of gene expression of two glutathione S-transferases (GST), peaking at levels of 25.0 +/- 13.1 and 10 +/- 0.7 fold the expression level of non-exposed plants after 24 h for each of the GST genes, respectively. This paper demonstrates the use of functional genomics information from the model plant species, Arabidopsis, to identify genes which may be important in detoxification of TNT in the model phytoremediation species, Populus trichocarpa.

Tyrosine 8 contributes to catalysis but is not required for activity of rat liver glutathione S-transferase, 1-1.

PubMed Central

Wang, J.; Barycki, J. J.; Colman, R. F.

1996-01-01

Reaction of rat liver glutathione S-transferase, isozyme 1-1, with 4-(fluorosulfonyl)benzoic acid (4-FSB), a xenobiotic substrate analogue, results in a time-dependent inactivation of the enzyme to a final value of 35% of its original activity when assayed at pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The rate of inactivation exhibits a nonlinear dependence on the concentration of 4-FSB from 0.25 mM to 9 mM, characterized by a KI of 0.78 mM and kmax of 0.011 min-1. S-Hexylglutathione or the xenobiotic substrate analogue, 2,4-dinitrophenol, protects against inactivation of the enzyme by 4-FSB, whereas S-methylglutathione has little effect on the reaction. These experiments indicate that reaction occurs within the active site of the enzyme, probably in the binding site of the xenobiotic substrate, close to the glutathione binding site. Incorporation of [3,5-3H]-4-FSB into the enzyme in the absence and presence of S-hexylglutathione suggests that modification of one residue is responsible for the partial loss of enzyme activity. Tyr 8 and Cys 17 are shown to be the reaction targets of 4-FSB, but only Tyr 8 is protected against 4-FSB by S-hexylglutathione. DTT regenerates cysteine from the reaction product of cysteine and 4-FSB, but does not reactivate the enzyme. These results show that modification of Tyr 8 by 4-FSB causes the partial inactivation of the enzyme. The Michaelis constants for various substrates are not changed by the modification of the enzyme. The pH dependence of the enzyme-catalyzed reaction of glutathione with CDNB for the modified enzyme, as compared with the native enzyme, reveals an increase of about 0.9 in the apparent pKa, which has been interpreted as representing the ionization of enzyme-bound glutathione; however, this pKa of about 7.4 for modified enzyme remains far below the pK of 9.1 for the -SH of free glutathione. Previously, it was considered that Tyr 8 was essential for GST catalysis. In contrast, we conclude that

Delineation of xenobiotic substrate sites in rat glutathione S-transferase M1-1.

PubMed

Hearne, Jennifer L; Colman, Roberta F

2005-10-01

Glutathione S-transferases catalyze the conjugation of glutathione with endogenous and exogenous xenobiotics. Hu and Colman (1995) proposed that there are two distinct substrate sites in rat GST M1-1, a 1-chloro-2,4-dintrobenzene (CDNB) substrate site located in the vicinity of tyrosine-115, and a monobromobimane (mBBr) substrate site. To determine whether the mBBr substrate site is distinguishable from the CDNB substrate site, we tested S-(hydroxyethyl)bimane, a nonreactive derivative of mBBr, for its ability to compete kinetically with the substrates. We find that S-(hydroxyethyl)bimane is a competitive inhibitor (K(I) = 0.36 microM) when mBBr is used as substrate, but not when CDNB is used as substrate, demonstrating that these two sites are distinct. Using site-directed mutagenesis, we have localized the mBBr substrate site to an area midway through alpha-helix 4 (residues 90-114) and have identified residues that are important in the enzymatic reaction. Substitution of alanine at positions along alpha-helix 4 reveals that mutations at positions 103, 104, and 109 exhibit a greater perturbation of the enzymatic reaction with mBBr than with CDNB as substrate. Various other substitutions at positions 103 and 104 reveal that a hydrophobic residue is necessary at each of these positions to maintain optimal affinity of the enzyme for mBBr and preserve the secondary structure of the enzyme. Substitutions at position 109 indicate that this residue is important in the enzyme's affinity for mBBr but has a minimal effect on Vmax. These results demonstrate that the promiscuity of rat GST M1-1 is in part due to at least two distinct substrate sites.

Cooperativity and pseudo-cooperativity in the glutathione S-transferase from Plasmodium falciparum.

PubMed

Liebau, Eva; De Maria, Francesca; Burmeister, Cora; Perbandt, Markus; Turella, Paola; Antonini, Giovanni; Federici, Giorgio; Giansanti, Francesco; Stella, Lorenzo; Lo Bello, Mario; Caccuri, Anna Maria; Ricci, Giorgio

2005-07-15

Binding and catalytic properties of glutathione S-transferase from Plasmodium falciparum (PfGST) have been studied by means of fluorescence, steady state and pre-steady state kinetic experiments, and docking simulations. This enzyme displays a peculiar reversible low-high affinity transition, never observed in other GSTs, which involves the G-site and shifts the apparent K(D) for glutathione (GSH) from 200 to 0.18 mM. The transition toward the high affinity conformation is triggered by the simultaneous binding of two GSH molecules to the dimeric enzyme, and it is manifested as an uncorrected homotropic behavior, termed "pseudo-cooperativity." The high affinity enzyme is able to activate GSH, lowering its pK(a) value from 9.0 to 7.0, a behavior similar to that found in all known GSTs. Using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, this enzyme reveals a potential optimized mechanism for the GSH conjugation but a low catalytic efficiency mainly due to a very low affinity for this co-substrate. Conversely, PfGST efficiently binds one molecule of hemin/monomer. The binding is highly cooperative (n(H) = 1.8) and occurs only when GSH is bound to the enzyme. The thiolate of GSH plays a crucial role in the intersubunit communication because no cooperativity is observed when S-methylglutathione replaces GSH. Docking simulations suggest that hemin binds to a pocket leaning into both the G-site and the H-site. The iron is coordinated by the amidic nitrogen of Asn-115, and the two carboxylate groups are in electrostatic interaction with the epsilon-amino group of Lys-15. Kinetic and structural data suggest that PfGST evolved by optimizing its binding property with the parasitotoxic hemin rather than its catalytic efficiency toward toxic electrophilic compounds.

Computational QM/MM Study of the Reaction Mechanism of Human Glutathione S-Transferase A3-3

NASA Astrophysics Data System (ADS)

Calvaresi, Matteo; Stenta, Marco; Altoè, Piero; Bottoni, Andrea; Garavelli, Marco; Spinelli, Domenico

2007-12-01

Human Glutathione S-Transferase A3-3(hGSTA3-3) is the most efficient human steroid double-bond isomerase enzyme. It catalyzes the double bond isomerization of Δ5-androstene-3,17-dione (Δ5-AD) and Δ5-pregnene-3,20-dione (Δ5-PD). The isomerization products are the precursors of the steroid hormones testosterone and progesterone. We have carried out a QM/MM study to elucidate some interesting aspects of the enzyme catalytic mechanism. In particular, we have analyzed either a concerted or a stepwise reaction path. Moreover, we have attempted to rationalize the electrostatic effects on the catalytic activity of the residues surrounding the active site. Specifically, we have performed a "finger print" analysis to determine the electrostatic contribution of each aminoacid residue to the global electrostatic term, thus ranking the effect of the various aminoacids in the course of the reaction. In this way, we have highlighted the most important terms affecting the stabilization-destabilization of the enzyme.

Functional characterisation of ganglioside-induced differentiation-associated protein 1 as a glutathione transferase.

PubMed

Shield, Alison J; Murray, Tracy P; Board, Philip G

2006-09-08

Mutations in the ganglioside-induced differentiation-associated protein 1 (GDAP1) gene have been linked with Charcot-Marie-Tooth (CMT) disease. This protein, and its paralogue GDAP1L1, appear to be structurally related to the cytosolic glutathione S-transferases (GST) including an N-terminal thioredoxin fold domain with conserved active site residues. The specific function, of GDAP1 remains unknown. To further characterise their structure and function we purified recombinant human GDAP1 and GDAP1L1 proteins using bacterial expression and immobilised metal affinity chromatography. Like other cytosolic GSTs, GDAP1 protein has a dimeric structure. Although the full-length proteins were largely insoluble, the deletion of a proposed C-terminal transmembrane domain allowed the preparation of soluble protein. The purified proteins were assayed for glutathione-dependent activity against a library of 'prototypic' GST substrates. No evidence of glutathione-dependent activity or an ability to bind glutathione immobilised on agarose was found.

Molecular and biochemical characterization of tomato farnesyl-protein transferase.

PubMed

Schmitt, D; Callan, K; Gruissem, W

1996-10-01

The prenylation of membrane-associated proteins involved in the regulation of eukaryotic cell growth and signal transduction is critically important for their subcellular localization and biological activity. In contrast to mammalian cells and yeast, however, the function of protein prenylation in plants is not well understood and only a few prenylated proteins have been identified. We partially purified and characterized farnesyl-protein transferase from tomato (Lycopersicon esculentum, LeFTase) to analyze its biochemical and molecular properties. Using Ras- and G gamma-specific peptide substrates and competition assays we showed that tomato protein extracts have both farnesyl-protein transferase and geranylgeranyl-protein transferase 1 activities. Compared with the heterologous synthetic peptide substrates, the plant-specific CaaX sequence of the ANJ1 protein is a less efficient substrate for LeFTase in vitro. LeFTase activity profiles and LeFTase beta-subunit protein (LeFTB) levels differ significantly in various tissues and are regulated during fruit development. Partially purified LeFTase requires Zn2+ and Mg2+ for enzymatic activity and has an apparent molecular mass of 100 kD Immunoprecipitation experiments using anti-alpha LeFTB antibodies confirmed that LeFTB is a component of LeFTase but not of tomato geranylgeranyl-protein transferase 1. Based on their conserved bio-chemical activities, we expect that prenyltransferases are likely integrated with the sterol biosynthesis pathway in the control of plant cell growth.

Detection of Glutathione by Glutathione-S-Transferase-Nanoconjugate Ensemble Electrochemical Device.

PubMed

Barman, Ujjwol; Mukhopadhyay, Gargi; Goswami, Namami; Ghosh, Siddhartha Sankar; Paily, Roy P

2017-06-01

This paper reports a novel electrochemical method for detection of Glutathione (GSH) using Glutathione-S-Transferase (GST) - ZnO composite nanoparticles to investigate the prospects of the method for detection of cancer at an early stage. The purified GST enzyme was bound with ZnO nanoparticles by electrostatic interactions and the nanocomposite was dropcast on a silicon dioxide wafer. The GST functionalized deposited layer was then used as a chemiresistive channel to detect conjugation reaction between GSH and 1-Chloro-2, 4-Dinitrobenzene (CDNB). The zeta potential values of the ZnO nanoparticles and the GST were found to be 13.4 mV and-6.21 mV, respectively. Around 73.8% binding was observed between the enzyme and ZnO nanoparticles. I - V analysis of the chemiresistive channel showed an increase in conductivity of the channel due to conjugation reaction between GSH and CDNB as compared with that of GSH or CDNB alone. I - V characterization of the GST functionalized layer was performed at various concentrations of GSH and a sensitivity and limit of detection of 5.68 nA/ [Formula: see text] and 41.9 nM were obtained, respectively. Thus from I - V analysis of the chemiresistivechannel, the detectionand quantification of GSH could be obtained. The kinetic parameters of both GST and nanoconjugate of ZnO nanoparticles andGSTwere determinedwith respect to its substrates, GSH and CDNB, using Michaelis-Mentenmodel. This novel approach of detection of GSH bymeans of ZnO nanoparticle and GST enzyme composite can be further analyzed for in vitro experiments, which will lead us to a new and efficient way of detecting certain types of cancers at an early stage.

Antioxidant role of glutathione S-transferases: 4-Hydroxynonenal, a key molecule in stress-mediated signaling.

PubMed

Singhal, Sharad S; Singh, Sharda P; Singhal, Preeti; Horne, David; Singhal, Jyotsana; Awasthi, Sanjay

2015-12-15

4-Hydroxy-2-trans-nonenal (4HNE), one of the major end products of lipid peroxidation (LPO), has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. It has been known that glutathione S-transferases (GSTs) can reduce lipid hydroperoxides through their Se-independent glutathione-peroxidase activity and that these enzymes can also detoxify LPO end-products such as 4HNE. Available evidence from earlier studies together with results of recent studies in our laboratories strongly suggests that LPO products, particularly hydroperoxides and 4HNE, are involved in the mechanisms of stress-mediated signaling and that it can be modulated by the alpha-class GSTs through the regulation of the intracellular concentrations of 4HNE. We demonstrate that 4HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase (JNK) and caspase-3 activation. Cells exposed to mild, transient heat or oxidative stress acquire the capacity to exclude intracellular 4HNE at a faster rate by inducing GSTA4-4 which conjugates 4HNE to glutathione (GSH), and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4HNE (GS-HNE). The balance between formation and exclusion promotes different cellular processes - higher concentrations of 4HNE promote apoptosis; whereas, lower concentrations promote proliferation. In this article, we provide a brief summary of the cellular effects of 4HNE, followed by a review of its GST-catalyzed detoxification, with an emphasis on the structural attributes that play an important role in the interactions with alpha-class GSTA4-4. Taken together, 4HNE is a key signaling molecule and that GSTs being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article. Copyright © 2015 Elsevier Inc. All rights

1-3-A Resolution Structure of Human Glutathione S-Transferase With S-Hexyl Glutathione Bound Reveals Possible Extended Ligandin Binding Site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trong, I.Le; Stenkamp, R.E.; Ibarra, C.

2005-08-22

Cytosolic glutathione S-transferases (GSTs) play a critical role in xenobiotic binding and metabolism, as well as in modulation of oxidative stress. Here, the high-resolution X-ray crystal structures of homodimeric human GSTA1-1 in the apo form and in complex with S-hexyl glutathione (two data sets) are reported at 1.8, 1.5, and 1.3A respectively. At this level of resolution, distinct conformations of the alkyl chain of S-hexyl glutathione are observed, reflecting the nonspecific nature of the hydrophobic substrate binding site (H-site). Also, an extensive network of ordered water, including 75 discrete solvent molecules, traverses the open subunit-subunit interface and connects the glutathionemore » binding sites in each subunit. In the highest-resolution structure, three glycerol moieties lie within this network and directly connect the amino termini of the glutathione molecules. A search for ligand binding sites with the docking program Molecular Operating Environment identified the ordered water network binding site, lined mainly with hydrophobic residues, suggesting an extended ligand binding surface for nonsubstrate ligands, the so-called ligandin site. Finally, detailed comparison of the structures reported here with previously published X-ray structures reveal a possible reaction coordinate for ligand-dependent conformational changes in the active site and the C-terminus.« less

Prevalence of glutathione S-transferase gene deletions and their effect on sickle cell patients.

PubMed

Sanjay, Pandey; Mani, Mishra Rahasy; Sweta, Pandey; Vineet, Shah; Kumar, Ahuja Rajesh; Renu, Saxena

2012-01-01

Glutathione S-transferase gene deletions are known detoxification agents and cause oxidative damage. Due to the different pathophysiology of anemia in thalassemia and sickle cell disease, there are significant differences in the pathophysiology of iron overload and iron-related complications in these disorders. The aim of this study was to estimate the frequency of the GSTM1 and GSTT1 genotypes in sickle cell disease patients and their effect on iron status. Forty sickle cell anemia and sixty sickle ß-thalassemia patients and 100 controls were evaluated to determine the frequency of GST gene deletions. Complete blood counts were performed by an automated cell analyzer. Hemoglobin F, hemoglobin A, hemoglobin A2 and hemoglobin S were measured and diagnosis of patients was achieved by high performance liquid chromatography with DNA extraction by the phenol-chloroform method. The GST null genotype was determined using multiplex polymerase chain reaction and serum ferritin was measured using an ELISA kit. Statistical analysis was by EpiInfo and GraphPad statistics software. An increased frequency of the GSTT1 null genotype (p-value = 0.05) was seen in the patients. The mean serum ferritin level was higher in patients with the GST genotypes than in controls; this was statistically significant for all genotypes except GSTM1, however the higher levels of serum ferritin were due to blood transfusions in patients. GST deletions do not play a direct role in iron overload of sickle cell patients.

Fluoxetine-induced toxicity results in human placental glutathione S-transferase-π (GST-π) dysfunction.

PubMed

Dalmizrak, Ozlem; Kulaksiz-Erkmen, Gulnihal; Ozer, Nazmi

2016-10-01

The antidepressant drug fluoxetine (FLU) is considered in the group of selective serotonine re-uptake inhibitors. Its distribution in brain and binding to human brain glutathione S-transferase-π (GST-π) have been shown. FLU can cross blood brain barrier and placenta, accumulate in fetus and may cause congenital malformations. To elucidate the interaction of placental GST-π with FLU. First, concentration-dependent inhibition of human placental GST-π was evaluated by using different FLU concentrations and then 0.3125, 0.625, 1.25, 2.5 and 5 mM FLU concentrations were chosen and tested while keeping GSH concentration constant and 1-chloro-2,4-dinitrobenzene (CDNB) concentration varied and vice versa. The data were evaluated with different kinetic models and Statistica 9.00 for Windows. The Vm, at variable [CDNB] (142 ± 16 U/mg protein) was 3 times higher than the Vm obtained at variable [GSH] (49 ± 4 U/mg protein). On the other hand, the Km for CDNB was ∼10 times higher than the Km for GSH (1.99 ± 0.36 mM versus 0.21 ± 0.06 mM). The IC50 value for FLU was 8.6 mM. Both at constant [CDNB] and variable [GSH] and at constant [GSH] and variable [CDNB] the inhibition types were competitive with the Ki values of 5.62 ± 4.37 and 8.09 ± 1.27 mM, respectively. Although the Ki values obtained for FLU in vitro are high, due to their uneven distribution, long elimination time and inhibitory behavior on detoxification systems, it may cause defects in adults but these effects may be much more severe in fetus and result in congenital malformations.

Glutathione-S-transferases pi, alpha, mu and mdr1 mRNA expression in normal lymphocytes and chronic lymphocytic leukemia.

PubMed

Marie, J P; Simonin, G; Legrand, O; Delmer, A; Faussat, A M; Lewis, A D; Sikic, B I; Zittoun, R

1995-10-01

Chronic B cell lymphoproliferative disorders are frequently sensitive to alkylating agents. To assess the glutathione-S-transferases (GSTs) gene expression in B tumoral lymphocytes, possibly responsible for this sensitivity, we developed a sensitive RT-PCR assay for the three isoenzymes GST pi, GST mu and GST alpha mRNA. Normal B and T lymphocytes from 11 blood donors were separated by magnetic beads and tested with this assay. The GST pi was the most abundant transferase, and was detected in all B and T cell samples. GST mu was undetectable ('null' phenotype) in 6/11 normal donors, either in B or T cells. GST alpha was very stable from donor to donor, and was highly correlated between B and T cells of the same individual (P < 0.0001). There is no correlation between the three isoenzymes, and between each isoenzyme and mdr1 gene expression. Twenty-three B lymphoproliferative disorders (20 B-CLL, 3 CD5- chronic lymphoproliferative syndromes) were tested with the same technique. An average decrease of 57% of the GST pi expression was noted in the mononuclear cells of these patients (P < 0.02), with no differences between the untreated and treated cases. The GST alpha and mdr1 mRNA levels did not differ from normal B lymphocytes, but the proportion of patients with no detectable expression of GST mu is lower than in the control (13%). Interestingly, the low content of GST pi in B-CLL could explain the frequent sensitivity of this disease to alkylating agents.

Glutathione S-transferases and UDP-glycosyltransferases Are Involved in Response to Aluminum Stress in Flax

PubMed Central

Dmitriev, Alexey A.; Krasnov, George S.; Rozhmina, Tatiana A.; Kishlyan, Natalya V.; Zyablitsin, Alexander V.; Sadritdinova, Asiya F.; Snezhkina, Anastasiya V.; Fedorova, Maria S.; Yurkevich, Olga Y.; Muravenko, Olga V.; Bolsheva, Nadezhda L.; Kudryavtseva, Anna V.; Melnikova, Nataliya V.

2016-01-01

About 30% of the world's ice-free land area is occupied by acid soils. In soils with pH below 5, aluminum (Al) releases to the soil solution, and becomes highly toxic for plants. Therefore, breeding of varieties that are resistant to Al is needed. Flax (Linum usitatissimum L.) is grown worldwide for fiber and seed production. Al toxicity in acid soils is a serious problem for flax cultivation. However, very little is known about mechanisms of flax resistance to Al and the genetics of this resistance. In the present work, we sequenced 16 transcriptomes of flax cultivars resistant (Hermes and TMP1919) and sensitive (Lira and Orshanskiy) to Al, which were exposed to control conditions and aluminum treatment for 4, 12, and 24 h. In total, 44.9–63.3 million paired-end 100-nucleotide reads were generated for each sequencing library. Based on the obtained high-throughput sequencing data, genes with differential expression under aluminum exposure were revealed in flax. The majority of the top 50 up-regulated genes were involved in transmembrane transport and transporter activity in both the Al-resistant and Al-sensitive cultivars. However, genes encoding proteins with glutathione transferase and UDP-glycosyltransferase activity were in the top 50 up-regulated genes only in the flax cultivars resistant to aluminum. For qPCR analysis in extended sampling, two UDP-glycosyltransferases (UGTs), and three glutathione S-transferases (GSTs) were selected. The general trend of alterations in the expression of the examined genes was the up-regulation under Al stress, especially after 4 h of Al exposure. Moreover, in the flax cultivars resistant to aluminum, the increase in expression was more pronounced than that in the sensitive cultivars. We speculate that the defense against the Al toxicity via GST antioxidant activity is the probable mechanism of the response of flax plants to aluminum stress. We also suggest that UGTs could be involved in cell wall modification and protection

The Relationship Between Glutathione S-Transferase-P1 and Beta-2 Adrenoreceptor Genotypes with Asthmatic Patients in the Turkish Population.

PubMed

Kaymak, Cetin; Aygun Kocabas, Neslihan; Aydın, Nesrin; Oztuna, Derya; Karakaya, Ali Esat

2016-09-01

Individual differences in the activity of enzymes that metabolize xenobiotics can impact health and disease. Beta-2 adrenoreceptor (ADRB2) is a functional G-coupled protein expressed in the vascular endothelium of lungs, alveolar walls, and the ganglions of cholinergic nerves which induces bronchodilation in response to catecholamines. Glutathione S-Transferase-P1 (GSTP1) is a candidate pi class GST gene, which controls pi class glutathione S-transferase activity. In this study we determined the relationship between the ADRB2 Arg16Gly polymorphism and GSTP1 polymorphisms, involved in bronchodilator response and oxidative stress, respectively, with susceptibility to asthma. In this study, 129 asthmatic patients and 127 healthy control cases were recruited to determine ADRB2 and GSTP1 genotypes by allele-specific polymerase chain reaction and restriction fragment length polymorphism assays, respectively. The ADRB2 genotype frequencies of the patients and control cases were found to be 10.9% (Arg16Arg), 48.8% (Arg16Gly), and 40.3% (Gly16Gly) and 24.4% (Arg16Arg), 36.2% (Arg16Gly), and 39.4% (Gly16Gly), respectively. GSTP1 genotype frequencies of patients and control cases were found to be 55% (Ile105Ile), 43.4% (Ile105Val), and 1.6% (Val105Val) and 75.6% (Ile105Ile), 22% (Ile105Val), and 2.4% (Val105Val), respectively. In the case of the GSTP1 gene, we found statistically significant differences in the genotype frequency of Ile105Val and the allele frequency of Val105 in the asthmatic group compared with the controls. Moreover, we observed a relationship between allele frequencies and clinical phenotypes including atopia nocturnal dyspnea, and steroid dependency in the asthmatic patients. Our results suggest that the GSTP1 Ile105Val polymorphism may be linked to the severeness of airway dysfunction.

Glutathione S-transferase 4 is a putative DIF-binding protein that regulates the size of fruiting bodies in Dictyostelium discoideum.

PubMed

Kuwayama, Hidekazu; Kikuchi, Haruhisa; Oshima, Yoshiteru; Kubohara, Yuzuru

2016-12-01

In the development of the cellular slime mold Dictyostelium discoideum , two chlorinated compounds, the differentiation-inducing factors DIF-1 and DIF-2, play important roles in the regulation of both cell differentiation and chemotactic cell movement. However, the receptors of DIFs and the components of DIF signaling systems have not previously been elucidated. To identify the receptors for DIF-1 and DIF-2, we here performed DIF-conjugated affinity gel chromatography and liquid chromatography-tandem mass spectrometry and identified the glutathione S-transferase GST4 as a major DIF-binding protein. Knockout and overexpression mutants of gst4 ( gst4 - and gst4 OE , respectively) formed fruiting bodies, but the fruiting bodies of gst4 - cells were smaller than those of wild-type Ax2 cells, and those of gst4 OE cells were larger than those of Ax2 cells. Both chemotaxis regulation and in vitro stalk cell formation by DIFs in the gst4 mutants were similar to those of Ax2 cells. These results suggest that GST4 is a DIF-binding protein that regulates the sizes of cell aggregates and fruiting bodies in D. discoideum .

Impact of glutathione S-transferase M1 and T1 on anti-tuberculosis drug-induced hepatotoxicity in Chinese pediatric patients.

PubMed

Liu, Fang; Jiao, An-xia; Wu, Xi-rong; Zhao, Wei; Yin, Qing-qin; Qi, Hui; Jiao, Wei-wei; Xiao, Jing; Sun, Lin; Shen, Chen; Tian, Jian-ling; Shen, Dan; Jacqz-Aigrain, Evelyne; Shen, A-dong

2014-01-01

Anti-tuberculosis drug induced hepatotoxicity (ATDH) is a major adverse drug reaction associated for anti-tuberculosis therapy. The glutathione S-transferases (GST) plays a crucial role in the detoxification of hepatotoxic metabolites of anti-tuberculosis drugs.An association between GSTM1/GSTT1 null mutations and increased risk of ATDH has been demonstrated in adults. Given the ethnic differences and developmental changes, our study aims to investigate the potential impacts of GSTM1/GSTT1 genotypes on the development of ATDH in Han Chinese children treated with anti-tuberculosis therapy. Children receiving anti-tuberculosis therapy with or without evidence of ATDH were considered as the cases or controls, respectively. The GSTM1 and GSTT1 genotyping were performed using the polymerase chain reaction. One hundred sixty-three children (20 cases and 143 controls) with a mean age of 4.7 years (range: 2 months-14.1 years) were included. For the GSTM1, 14 (70.0%) cases and 96 (67.1%) controls had homozygous null mutations. For the GSTT1, 13 (65.0%) cases and 97 (67.8%) controls had homozygous null mutations. Neither the GSTM1, nor the GSTT1 polymorphism was significantly correlated with the occurrence of ATHD. Our results did not support the GSTM1 and GSTT1 polymorphisms as the predictors of ADTH in Chinese Han children treated with anti-tuberculosis drugs. An age-related association between pharmacogenetics and ATHD need to be confirmed in the further study.

Functional interaction of glutathione S-transferase pi and peroxiredoxin 6 in intact cells.

PubMed

Zhou, Suiping; Lien, Yu-Chin; Shuvaeva, Tea; DeBolt, Kristine; Feinstein, Sheldon I; Fisher, Aron B

2013-02-01

Peroxiredoxin 6 (Prdx6) is a 1-Cys member of the peroxiredoxin superfamily that plays an important role in antioxidant defense. Glutathionylation of recombinant Prdx6 mediated by π glutathione S-transferase (GST) is required for reduction of the oxidized Cys and completion of the peroxidatic catalytic cycle in vitro. This study investigated the requirement for πGST in intact cells. Transfection with a plasmid construct expressing πGST into MCF7, a cell line that lacks endogenous πGST, significantly increased phospholipid peroxidase activity as measured in cell lysates and protected intact cells against a peroxidative stress. siRNA knockdown indicated that this increased peroxidase activity was Prdx6 dependent. Interaction between πGST and Prdx6, evaluated by the Duolink Proximity Ligation Assay, was minimal under basal conditions but increased dramatically following treatment of cells with the oxidant, tert-butyl hydroperoxide. Interaction was abolished by mutation of C47, the active site for Prdx6 peroxidase activity. Depletion of cellular GSH by treatment of cells with buthionine sulfoximine had no effect on the interaction of Prdx6 and πGST. These data are consistent with the hypothesis that oxidation of the catalytic cysteine in Prdx6 is required for its interaction with πGST and that the interaction plays an important role in regenerating the peroxidase activity of Prdx6. Copyright © 2012 Elsevier Ltd. All rights reserved.

A Glutathione S-Transferase Catalyzes the Dehalogenation of Inhibitory Metabolites of Polychlorinated Biphenyls

PubMed Central

Fortin, Pascal D.; Horsman, Geoff P.; Yang, Hao M.; Eltis, Lindsay D.

2006-01-01

BphK is a glutathione S-transferase of unclear physiological function that occurs in some bacterial biphenyl catabolic (bph) pathways. We demonstrated that BphK of Burkholderia xenovorans strain LB400 catalyzes the dehalogenation of 3-chloro 2-hydroxy-6-oxo-6-phenyl-2,4-dienoates (HOPDAs), compounds that are produced by the cometabolism of polychlorinated biphenyls (PCBs) by the bph pathway and that inhibit the pathway's hydrolase. A one-column protocol was developed to purify heterologously produced BphK. The purified enzyme had the greatest specificity for 3-Cl HOPDA (kcat/Km, ∼104 M−1 s−1), which it dechlorinated approximately 3 orders of magnitude more efficiently than 4-chlorobenzoate, a previously proposed substrate of BphK. The enzyme also catalyzed the dechlorination of 5-Cl HOPDA and 3,9,11-triCl HOPDA. By contrast, BphK did not detectably transform HOPDA, 4-Cl HOPDA, or chlorinated 2,3-dihydroxybiphenyls. The BphK-catalyzed dehalogenation proceeded via a ternary-complex mechanism and consumed 2 equivalents of glutathione (GSH) (Km for GSH in the presence of 3-Cl HOPDA, ∼0.1 mM). A reaction mechanism consistent with the enzyme's specificity is proposed. The ability of BphK to dehalogenate inhibitory PCB metabolites supports the hypothesis that this enzyme was recruited to facilitate PCB degradation by the bph pathway. PMID:16740949

Glutathione S-transferases as antioxidant defence agents confer pyrethroid resistance in Nilaparvata lugens.

PubMed Central

Vontas, J G; Small, G J; Hemingway, J

2001-01-01

Selection of a laboratory colony of the brown planthopper Nilaparvata lugens with the pyrethroids permethrin and lambda-cyhalothrin increased its resistance to both insecticides. Biochemical analysis and synergistic studies with metabolic inhibitors indicated that elevated glutathione S-transferases (GSTs) with a predominant peroxidase activity conferred resistance to both pyrethroids, whereas esterases conferred part of the resistance to permethrin. Purified esterases hydrolysed permethrin at a slow rate, but incubation of either pyrethroid or their primary metabolites with partially purified GSTs had no effect on the metabolic profile. Although GSTs were sensitive to inhibition by both pyrethroids, they did not serve as binding proteins, as previously hypothesized [Grant and Matsumura (1988) Insect Biochem. 18, 615-622]. We demonstrate that pyrethroids, in addition to their neurotoxic effect, induce oxidative stress and lipid peroxidation in insects. Pyrethroid exposure induced lipid peroxides, protein oxidation and depleted reduced glutathione. Elevated GSTs in the resistant strains attenuated the pyrethroid-induced lipid peroxidation and reduced mortality, whereas their in vivo inhibition eliminated their protective role. We therefore hypothesize that the main role of elevated GSTs in conferring resistance in N. lugens is through protecting tissues from oxidative damage. Our study extends the GSTs' range of efficacy to pyrethroid insecticides and possibly explains the role of elevated GSTs in other pyrethroid-resistant insects. PMID:11415437

Monobromobimane as an affinity label of the xenobiotic binding site of rat glutathione S-transferase 3-3.

PubMed

Hu, L; Colman, R F

1995-09-15

Monobromobimane (mBBr), besides being a substrate in the presence of glutathione, inactivates rat liver glutathione S-transferase 3-3 at pH 7.5 and 25 degrees C as assayed using 1-chloro-2,4-dinitrobenzene (CDNB). The rate of inactivation is enhanced about 5-fold by S-methylglutathione. Substrate analogs bromosulfophthalein and 2,4-dinitrophenol decrease the rate of inactivation at least 20-fold. Upon incubation for 60 min with 0.25 mM mBBr and S-methylglutathione, the enzyme loses 91% of its activity toward CDNB and incorporates 2.14 mol of reagent/mol of subunit, whereas incubation under the same conditions but with added protectant 2,4-dinitrophenol yields an enzyme that is catalytically active and contains only 0.89 mol of reagent/mol of subunit. mBBR-modified enzyme is fluorescent, and fluorescence energy transfer occurs between intrinsic tryptophan and covalently bound bimane in modified enzyme. Both Tyr115 and Cys114 are modified, but Tyr115 is the initial reaction target and its modification correlates with loss of activity toward CDNB. The fact that the activity toward mBBr is retained by the enzyme after modification suggests that rat isozyme 3-3 has two binding sites for mBBr.

Perfluorooctanoic acid induces gene promoter hypermethylation of glutathione-S-transferase Pi in human liver L02 cells.

PubMed

Tian, Meiping; Peng, Siyuan; Martin, Francis L; Zhang, Jie; Liu, Liangpo; Wang, Zhanlin; Dong, Sijun; Shen, Heqing

2012-06-14

Perfluorooctanoic acid (PFOA) is one of the most commonly used perfluorinated compounds. Being a persistent environmental pollutant, it can accumulate in human tissues via various exposure routes. PFOA may interfere in a toxic fashion on the immune system, liver, development, and endocrine systems. In utero human exposure had been associated with cord serum global DNA hypomethylation. In light of this, we investigated possible PFOA-induced DNA methylation alterations in L02 cells in order to shed light into its epigenetic-mediated mechanisms of toxicity in human liver. L02 cells were exposed to 5, 10, 25, 50 or 100 mg/L PFOA for 72h. Global DNA methylation levels were determined by LC/ESI-MS, glutathione-S-transferase Pi (GSTP) gene promoter DNA methylation was investigated by methylation-specific polymerase chain reaction (PCR) with bisulfite sequencing, and consequent mRNA expression levels were measured with quantitative real-time reverse transcriptase PCR. A dose-related increase of GSTP promoter methylation at the transcription factor specificity protein 1 (SP1) binding site was observed. However, PFOA did not significantly influence global DNA methylation; nor did it markedly alter the promoter gene methylation of p16 (cyclin-dependent kinase inhibitor 2A), ERα (estrogen receptor α) or PRB (progesterone receptor B). In addition, PFOA significantly elevated mRNA transcript levels of DNMT3A (which mediates de novo DNA methylation), Acox (lipid metabolism) and p16 (cell apoptosis). Considering the role of GSTP in detoxification, aberrant methylation may be pivotal in PFOA-mediated toxicity response via the inhibition of SP1 binding to GSTP promoter. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Functional Dissection of the Bipartite Active Site of the Class I Coenzyme A (CoA)-Transferase Succinyl-CoA:Acetate CoA-Transferase.

PubMed

Murphy, Jesse R; Mullins, Elwood A; Kappock, T Joseph

2016-01-01

Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. In this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. The ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA.

Functional Dissection of the Bipartite Active Site of the Class I Coenzyme A (CoA)-Transferase Succinyl-CoA:Acetate CoA-Transferase

PubMed Central

Murphy, Jesse R.; Mullins, Elwood A.; Kappock, T. Joseph

2016-01-01

Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. In this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. The ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA. PMID:27242998

Functional dissection of the bipartite active site of the class I coenzyme A (CoA)-transferase succinyl-CoA:acetate CoA-transferase

DOE PAGES

Murphy, Jesse R.; Mullins, Elwood A.; Kappock, T. Joseph

2016-05-23

Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. Here in this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes andmore » orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. Finally, the ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA.« less

A glutathione S-transferase gene associated with antioxidant properties isolated from Apis cerana cerana

NASA Astrophysics Data System (ADS)

Liu, Shuchang; Liu, Feng; Jia, Haihong; Yan, Yan; Wang, Hongfang; Guo, Xingqi; Xu, Baohua

2016-06-01

Glutathione S-transferases (GSTs) are an important family of multifunctional enzymes in aerobic organisms. They play a crucial role in the detoxification of exogenous compounds, especially insecticides, and protection against oxidative stress. Most previous studies of GSTs in insects have largely focused on their role in insecticide resistance. Here, we isolated a theta class GST gene designated AccGSTT1 from Apis cerana cerana and aimed to explore its antioxidant and antibacterial attributes. Analyses of homology and phylogenetic relationships suggested that the predicted amino acid sequence of AccGSTT1 shares a high level of identity with the other hymenopteran GSTs and that it was conserved during evolution. Quantitative real-time PCR showed that AccGSTT1 is most highly expressed in adult stages and that the expression profile of this gene is significantly altered in response to various abiotic stresses. These results were confirmed using western blot analysis. Additionally, a disc diffusion assay showed that a recombinant AccGSTT1 protein may be roughly capable of inhibiting bacterial growth and that it reduces the resistance of Escherichia coli cells to multiple adverse stresses. Taken together, these data indicate that AccGSTT1 may play an important role in antioxidant processes under adverse stress conditions.

Hybrid molecule from O2-(2,4-dinitrophenyl)diazeniumdiolate and oleanolic acid: a glutathione S-transferase π-activated nitric oxide prodrug with selective anti-human hepatocellular carcinoma activity and improved stability.

PubMed

Fu, Junjie; Liu, Ling; Huang, Zhangjian; Lai, Yisheng; Ji, Hui; Peng, Sixun; Tian, Jide; Zhang, Yihua

2013-06-13

A series of hybrids from O(2)-(2,4-dinitrophenyl)diazeniumdiolate and oleanolic acid (OA) were designed, synthesized, and biologically evaluated as novel nitric oxide (NO)-releasing prodrugs that could be activated by glutathione S-transferase π (GSTπ) overexpressed in a number of cancer cells. It was discovered that the most active compound, 21, released high levels of NO selectively in HCC cells but not in the normal cells and exhibited potent antiproliferative activity in vitro as well as remarkable tumor-retarding effects in vivo. Compared with the reported GSTπ-activated prodrugs JS-K and PABA/NO, 21 exhibited remarkably improved stability in the absence of GSTπ. Importantly, the decomposition of 21 occurred in the presence of GSTπ and was much more effective than in glutathione S-transferase α. Additionally, 21 induced apoptosis in HepG2 cells by arresting the cell cycle at the G2/M phase, activating both the mitochondrion-mediated pathway and the MAPK pathway and enhancing the intracellular production of ROS.

Genetic Polymorphisms of Glutathione S-Transferase P1 (GSTP1) and the Incidence of Anti-Tuberculosis Drug-Induced Hepatotoxicity.

PubMed

Wu, Shouquan; Wang, You-Juan; Tang, Xiaoyan; Wang, Yu; Wu, Jingcan; Ji, Guiyi; Zhang, Miaomiao; Chen, Guo; Liu, Qianqian; Sandford, Andrew J; He, Jian-Qing

2016-01-01

Anti-tuberculosis drug-induced hepatotoxicity (ATDH) is one of the most common adverse effects associated with tuberculosis (TB) therapy. Animal studies have demonstrated important roles of glutathione S-transferases in the prevention of chemical-induced hepatotoxicity. The aim of this study was to investigate the relationship between single nucleotide polymorphisms (SNPs) of glutathione S-transferase P1 (GSTP1) and ATDH in TB patients. We used two independent samples for this genetic association study. In the initial prospective study, 322 newly diagnosed TB patients were followed up for three months after initiating anti-TB therapy. In an independent retrospective study, 115 ATDH patients and 116 patients without ATDH were selected to verify the results of the prospective study. Tag-SNPs of GSTP1 were genotyped either with the MassARRAY platform or the improved multiple ligase detection reaction (iMLDR) method. The associations between SNPs and ATDH were analyzed by logistic regression analysis adjusting for confounding factors. Of the 322 patients recruited in the prospective cohort, 35 were excluded during the 3 months of follow-up, and 30 were diagnosed with ATDH and were considered as the ATDH group. The remaining 257 subjects without ATDH were considered as the non-ATDH group. After correction for potential confounding factors, significant differences were found for rs1695 (A>G) under an allelic model (OR = 3.876, 95%CI: 1.258011.905; P = 0.018). In the retrospective study, rs1695 allele A also had a higher risk of ATDH (OR = 2.10, 95%CI: 1.17-3.76; P = 0.012). We only found rs4147581AA genotype under a dominant model was related to ATDH in the prospective study (OR = 2.578, 95%CI: 1.076-6.173; P = 0.034). This is the first study to suggest that GSTP1 genotyping can be an important tool for identifying patients who are susceptible to ATDH. This result should be verified in independent large sample studies and also in other ethnic populations.

GSTM1 null polymorphism at the glutathione S-transferase M1 locus: phenotype and genotype studies in patients with primary biliary cirrhosis.

PubMed Central

Davies, M H; Elias, E; Acharya, S; Cotton, W; Faulder, G C; Fryer, A A; Strange, R C

1993-01-01

Studies were carried out to test the hypothesis that the GSTM1 null phenotype at the mu (mu) class glutathione S-transferase 1 locus is associated with an increased predisposition to primary biliary cirrhosis. Starch gel electrophoresis was used to compare the prevalence of GSTM1 null phenotype 0 in patients with end stage primary biliary cirrhosis and a group of controls without evidence of liver disease. The prevalence of GSTM1 null phenotype in the primary biliary cirrhosis and control groups was similar; 39% and 45% respectively. In the primary biliary cirrhosis group all subjects were of the common GSTM1 0, GSTM1 A, GSTM1 B or GSTM1 A, B phenotypes while in the controls, one subject showed an isoform with an anodal mobility compatible with it being a product of the putative GSTM1*3 allele. As the GSTM1 phenotype might be changed by the disease process, the polymerase chain reaction was used to amplify the exon 4-exon 5 region of GSTM1 and show that in 13 control subjects and 11 patients with primary biliary cirrhosis, GSTM1 positive and negative genotypes were associated with corresponding GSTM1 expressing and non-expressing phenotypes respectively. The control subject with GSTM1 3 phenotype showed a positive genotype. Images Figure 1 Figure 2 PMID:8491405

Nuclear translocation of glutathione S-transferase π is mediated by a non-classical localization signal.

PubMed

Kawakatsu, Miho; Goto, Shinji; Yoshida, Takako; Urata, Yoshishige; Li, Tao-Sheng

2011-08-12

Glutathione S-transferase π (GSTπ), a member of the GST family of multifunctional enzymes, is highly expressed in human placenta and involved in the protection of cellular components against electrophilic compounds or oxidative stress. We have recently found that GSTπ is expressed in the cytoplasm, mitochondria, and nucleus in some cancer cells, and that the nuclear expression of GSTπ appears to correlate with resistance to anti-cancer drugs. Although the mitochondrial targeting signal of GSTπ was previously identified in the amino-terminal region, the mechanism of nuclear translocation remains completely unknown. In this study, we find that the region of GSTπ195-208 is critical for nuclear translocation, which is mediated by a novel and non-classical nuclear localization signal. In addition, using an in vitro transport assay, we demonstrate that the nuclear translocation of GSTπ depends on the cytosolic extract and ATP. Although further experiments are needed to understand in depth the precise mechanism of nuclear translocation of GSTπ, our results may help to establish more efficient anti-cancer therapy, especially with respect to resistance to anti-cancer drugs. Copyright © 2011 Elsevier Inc. All rights reserved.

Differential expression of two glutathione S-transferases identified from the American dog tick, Dermacentor variabilis.

PubMed

Dreher-Lesnick, S M; Mulenga, A; Simser, J A; Azad, A F

2006-08-01

Reciprocal signalling and gene expression play a cardinal role during pathogen-host molecular interactions and are prerequisite to the maintenance of balanced homeostasis. Gene expression repertoire changes during rickettsial infection and glutathione-S-transferases (GSTs) were among the genes found up-regulated in Rickettsia-infected Dermacentor variabilis. GSTs are well known to play an important part in cellular stress responses in the host. We have cloned two full-length GSTs from D. variabilis (DvGST1 and DvGST2). Comparison of these two DvGST molecules with those of other species indicate that DvGST1 is related to the mammalian class theta and insect class delta GSTs, while DvGST2 does not seem to fall in the same family. Northern blotting analyses revealed differential expression patterns, where DvGST1 and DvGST2 transcripts are found in the tick gut, with DvGST2 transcripts also present in the ovaries. Both DvGST transcripts are up-regulated upon tick feeding. Challenge of fed adult ticks with Escherichia coli injection showed decreased transcript amounts compared with ticks injected with phosphate-buffered saline (sham) and naïve ticks.

Glutathione S Transferases Polymorphisms Are Independent Prognostic Factors in Lupus Nephritis Treated with Cyclophosphamide

PubMed Central

Verstuyft, Céline; Costedoat-Chalumeau, Nathalie; Hummel, Aurélie; Le Guern, Véronique; Sacré, Karim; Meyer, Olivier; Daugas, Eric; Goujard, Cécile; Sultan, Audrey; Lobbedez, Thierry; Galicier, Lionel; Pourrat, Jacques; Le Hello, Claire; Godin, Michel; Morello, Rémy; Lambert, Marc; Hachulla, Eric; Vanhille, Philippe; Queffeulou, Guillaume; Potier, Jacky; Dion, Jean-Jacques; Bataille, Pierre; Chauveau, Dominique; Moulis, Guillaume; Farge-Bancel, Dominique; Duhaut, Pierre; Saint-Marcoux, Bernadette; Deroux, Alban; Manuzak, Jennifer; Francès, Camille; Aumaitre, Olivier; Bezanahary, Holy; Becquemont, Laurent; Bienvenu, Boris

2016-01-01

Objective To investigate association between genetic polymorphisms of GST, CYP and renal outcome or occurrence of adverse drug reactions (ADRs) in lupus nephritis (LN) treated with cyclophosphamide (CYC). CYC, as a pro-drug, requires bioactivation through multiple hepatic cytochrome P450s and glutathione S transferases (GST). Methods We carried out a multicentric retrospective study including 70 patients with proliferative LN treated with CYC. Patients were genotyped for polymorphisms of the CYP2B6, CYP2C19, GSTP1, GSTM1 and GSTT1 genes. Complete remission (CR) was defined as proteinuria ≤0.33g/day and serum creatinine ≤124 µmol/l. Partial remission (PR) was defined as proteinuria ≤1.5g/day with a 50% decrease of the baseline proteinuria value and serum creatinine no greater than 25% above baseline. Results Most patients were women (84%) and 77% were Caucasian. The mean age at LN diagnosis was 41 ± 10 years. The frequency of patients carrying the GST null genotype GSTT1-, GSTM1-, and the Ile→105Val GSTP1 genotype were respectively 38%, 60% and 44%. In multivariate analysis, the Ile→105Val GSTP1 genotype was an independent factor of poor renal outcome (achievement of CR or PR) (OR = 5.01 95% CI [1.02–24.51]) and the sole factor that influenced occurrence of ADRs was the GSTM1 null genotype (OR = 3.34 95% CI [1.064–10.58]). No association between polymorphisms of cytochrome P450s gene and efficacy or ADRs was observed. Conclusion This study suggests that GST polymorphisms highly impact renal outcome and occurrence of ADRs related to CYC in LN patients. PMID:27002825

A Tau Class Glutathione-S-Transferase is Involved in Trans-Resveratrol Transport Out of Grapevine Cells

PubMed Central

Martínez-Márquez, Ascensión; Martínez-Esteso, María J.; Vilella-Antón, María T.; Sellés-Marchart, Susana; Morante-Carriel, Jaime A.; Hurtado, Elias; Palazon, Javier; Bru-Martínez, Roque

2017-01-01

Vitis vinifera cell cultures respond to pathogens and elicitors by synthesizing and extracellularly accumulating stilbenoid phytoalexins. Large amounts of trans-resveratrol (t-R) are produced when a cell culture is elicited with methylated cyclodextrins (MBCD), either alone or combined with methyl jasmonate (MeJA). t-R transport to the extracellular medium, which represents the apoplastic space, would place this antifungal defense right in the battlefield to efficiently fight against pathogen attack. Yet despite their physiological relevance, these transport pathways are mostly unknown. A broad hypothesis-free DIGE-based proteomic experiment of a temporal series of elicited grapevine cell cultures was performed to explore the expression profiles of t-R biosynthetic proteins and other co-expressing proteins potentially involved in such a cell response. A correlation between two tau class glutathione-S-transferases (GSTs) with several stilbene synthase and phenylalanine ammonia-lyase isoforms, and with the t-R metabolite itself, was found and further assessed by a qRT-PCR gene expression analysis. The best candidate, GSTU-2, was cloned from the cDNA of the MBCD + MeJA-elicited grapevine cells and used for Agrobacterium-mediated grapevine cell transformation. The non-elicited lines that overexpressed GSTU-2 displayed an extracellular t-R accumulating phenotype, but stabilization of t-R required the addition to culture medium of adsorbent compounds, e.g., PVP or β-cyclodextrin. The wild-type cell cultures accumulated no t-R, not even in the presence of adsorbents. The transient expression of the GSTU-2-GFP fusion proteins in grapevine cells showed localisation in the plasma membrane, and the immunoprecipitation of HA-tagged GSTU-2 revealed its interaction with HIR, a plasma membrane-bound protein. These findings are consistent with a functional role in transport. This is the first report providing several pieces of experimental evidence for the involvement of a

Polymorphisms of glutathione S-transferase Mu 1 (GSTM1), Theta 1 (GSTT1), and Pi 1 (GSTP1) genes and epithelial ovarian cancer risk.

PubMed

Oliveira, Cristiane; Lourenço, Gustavo Jacob; Sagarra, Regina Aparecida Martinho; Derchain, Sophie Françoise Mauricette; Segalla, José Getulio; Lima, Carmen Silvia Passos

2012-01-01

Exposure of ovarian cells to estrogen, which is detoxified by glutathione S-transferases (GSTs), has been associated with epithelial ovarian cancer (EOC) development. We tested in this study whether the GSTM1, GSTT1 and GSTP1 Ile105Val polymorphisms alter the risk of EOC. Genomic DNA from 132 EOC patients and 132 controls was analyzed by polymerase chain reaction and restriction fragment length polymorphism methods. The differences between groups were analyzed by χ ^{2} or Fisher's exact test. The frequencies of GSTP1 Ile/Ile (57.6% versus 45.5%, P=0.03), GSTM1 null plus GSTP1 Ile/Ile (43.5% versus 25.8%; P=0.03) and GSTM1 null plus GSTT1 null plus GSTP1 Ile/Ile (30.3% versus 7.7%; P=0.007) genotypes were higher in patients than in controls. Individuals with the respective genotypes had a 1.80 (95% CI: 1.06-3.06), 2.38 (95% CI: 1.08-5.24) and 11.28 (95%CI: 1.95-65.30)-fold increased risks of EOC than those with the remaining genotypes. Our data present preliminary evidence that GSTM1, GSTT1 and GSTP1 polymorphisms, particularly in combination, constitute important inherited EOC determinants in individuals from Southeastern Brazil.

Glutathion-S-Transferase P1 polymorphisms association with broncopulmonary dysplasia in preterm infants

PubMed Central

Karagianni, P; Rallis, D; Fidani, L; Porpodi, M; Kalinderi, K; Tsakalidis, C; Nikolaidis, N

2013-01-01

Background: Oxidative stress, characterized by the excretion of pre-oxidative and anti-oxidative proteases, has a key role in the pathogenesis of bronchopulmonary dysplasia (BPD). One of the many host anti-oxidant enzymes is glutathione-S-transferase P1 (GSTP1), with three polymorphic alleles having been identified: homozygous ile, heterozygous ile/val and homozygous val isomorph. The aim of this study was to examine the genetic predisposition to BPD in the GSTP1 polymorphisms. Methods: A prospective case-control study was carried out in the 2nd Neonatal Intensive Care Unit of Aristotle University in Thessaloniki, Greece during 2008. The genetic polymorphisms of GSTP1 in 28 preterms <32 weeks gestational age (GA) with BPD compared to 74 controls (33 preterms without BPD and 41 healthy terms) were examined. Results: The homozygous ile isomorph was predominant in all groups (preterms with BPD: 82%, preterms without BPD: 70%, healthy terms: 78%), followed by the heterozygous ile/val (14%, 18% and 20% respectively) and the homozygous val isomorph (4%, 12% and 2% respectively). The homozygous ile isomorph was also identified in the majority of preterms with mild (80%), moderate (100%) and severe (73%) BPD. The GSTP1 genetic distribution did not differ between the groups and GSTP1 polymorphisms were not associated with the severity of BPD. Conclusions: This study could not confirm an association between GSTP1 polymorphisms and the development of BPD or the severity of the disease. PMID:25031518

Glutathion-S-Transferase P1 polymorphisms association with broncopulmonary dysplasia in preterm infants.

PubMed

Karagianni, P; Rallis, D; Fidani, L; Porpodi, M; Kalinderi, K; Tsakalidis, C; Nikolaidis, N

2013-10-01

Oxidative stress, characterized by the excretion of pre-oxidative and anti-oxidative proteases, has a key role in the pathogenesis of bronchopulmonary dysplasia (BPD). One of the many host anti-oxidant enzymes is glutathione-S-transferase P1 (GSTP1), with three polymorphic alleles having been identified: homozygous ile, heterozygous ile/val and homozygous val isomorph. The aim of this study was to examine the genetic predisposition to BPD in the GSTP1 polymorphisms. A prospective case-control study was carried out in the 2nd Neonatal Intensive Care Unit of Aristotle University in Thessaloniki, Greece during 2008. The genetic polymorphisms of GSTP1 in 28 preterms <32 weeks gestational age (GA) with BPD compared to 74 controls (33 preterms without BPD and 41 healthy terms) were examined. The homozygous ile isomorph was predominant in all groups (preterms with BPD: 82%, preterms without BPD: 70%, healthy terms: 78%), followed by the heterozygous ile/val (14%, 18% and 20% respectively) and the homozygous val isomorph (4%, 12% and 2% respectively). The homozygous ile isomorph was also identified in the majority of preterms with mild (80%), moderate (100%) and severe (73%) BPD. The GSTP1 genetic distribution did not differ between the groups and GSTP1 polymorphisms were not associated with the severity of BPD. This study could not confirm an association between GSTP1 polymorphisms and the development of BPD or the severity of the disease.

Fluorescent techniques for discovery and characterization of phosphopantetheinyl transferase inhibitors

PubMed Central

Kosa, Nicolas M.; Foley, Timothy L.; Burkart, Michael D.

2016-01-01

Phosphopantetheinyl transferase (E.C. 2.7.8.-) activates biosynthetic pathways that synthesize both primary and secondary metabolites in bacteria. Inhibitors of these enzymes have the potential to serve as antibiotic compounds that function through a unique mode of action and possess clinical utility. Here we report a direct and continuous assay for this enzyme class based upon monitoring polarization of a fluorescent phosphopantetheine analog as it is transferred from a low molecular weight coenzyme A substrate to higher molecular weight protein acceptor. We demonstrate the utility of this method for the biochemical characterization of phosphopantetheinyl transferase Sfp, a canonical representative from this class. We also establish the portability of this technique to other homologs by adapting the assay to function with the human phosphopantetheinyl transferase, a target for which a microplate detection method does not currently exist. Comparison of these targets provides a basis to predict therapeutic index of inhibitor candidates and offers a valuable characterization of enzyme activity. PMID:24192555

Association between glutathione S-transferase pi genetic polymorphisms and oral cancer risk.

PubMed

Park, J Y; Schantz, S P; Stern, J C; Kaur, T; Lazarus, P

1999-08-01

Polymorphisms in the gene encoding the glutathione S-transferase (GST) pi metabolizing enzyme have previously been associated with susceptibility to various cancers. In this study, the importance of GSTP1 genotypes as determinants of risk for oral cancer was assessed by examining the prevalence of GSTP1 alleles in 157 incident oral cancer cases and 260 non-cancer control individuals frequency-matched by race, sex, and age at diagnosis (+/- 5 years). The GSTP1*A, GSTP1*B, GSTP1*C, and GSTP1*D alleles were elucidated by polymerase chain reaction-restriction fragment length polymorphism analysis of polymorphisms present in codons 105 (isoleucine:valine) and 114 (alanine:valine) of the GSTP1 gene. Increased risk for oral cancer was observed in individuals who were homozygous for any combination of GSTP1 polymorphic alleles (i.e. *B, *C, and/or *D alleles; odds ratio = 2.4, 95% confidence interval = 1.2-4.8). Similar risk was observed in both Caucasians (odds ratio = 2.6, 95% confidence interval = 1.1-6.2) and African-Americans (odds ratio = 2.3, 95% CI = 0.68-7.5). A greater risk was observed in individuals with the GSTP1 (Var/Var) genotype who were exposed to low levels of smoking (i.e. < or = 20 pack-years [py], odds ratio = 3.4, 95% confidence interval = 1.1-11) than among heavier smokers (i.e. > 20 pack-years [py], odds ratio = 1.4, 95% confidence interval = 0.48-4.0). These results suggest that GSTP1 genotype may play a role in risk for oral cancer particularly among lighter smokers.

Glutathione S-transferase M1-null genotype as risk factor for SOS in oxaliplatin-treated patients with metastatic colorectal cancer.

PubMed

Vreuls, C P H; Olde Damink, S W M; Koek, G H; Winstanley, A; Wisse, E; Cloots, R H E; van den Broek, M A J; Dejong, C H C; Bosman, F T; Driessen, A

2013-02-19

Oxaliplatin is used as a neo-adjuvant therapy in hepatic colorectal carcinoma metastasis. This treatment has significant side effects, as oxaliplatin is toxic to the sinusoidal endothelial cells and can induce sinusoidal obstruction syndrome (SOS), which is related to decreased overall survival. Glutathione has an important role in the defence system, catalysed by glutathione S-transferase (GST), including two non-enzyme producing polymorphisms (GSTM1-null and GSTT1-null). We hypothesise that patients with a non-enzyme producing polymorphism have a higher risk of developing toxic injury owing to oxaliplatin. In the nontumour-bearing liver, the presence of SOS was studied histopathologically. The genotype was determined by a semi-nested PCR. Thirty-two of the 55 (58%) patients showed SOS lesions, consisting of 27% mild, 22% moderate and 9% severe lesions. The GSTM1-null genotype was present in 25 of the 55 (46%). Multivariate analysis showed that the GSTM1-null genotype significantly correlated with the presence of (moderate-severe) SOS (P=0.026). The GSTM1-null genotype is an independent risk factor for SOS. This finding allows us, in association with other risk factors, to conceive a potential risk profile predicting whether the patient is at risk of developing SOS, before starting oxaliplatin, and subsequently might result in adjustment of treatment.

Molecular Cloning of Adenosinediphosphoribosyl Transferase.

DTIC Science & Technology

1987-09-08

nature of the blocking group is unknown, except its identity with pyroglutamic acid was ruled out by its insensitivity to pyroglutaminase (not shown...AdenosinediphosphoribOSyl Transferase (ADPRT) is: 1) the complete amino acid sequence of this large protein is best determined -from the DNA !equence of the gene, 2...enzyme (I), determination of its peptide structure (II) and application of synthetic DNA probes (III) derived from amino acid sequences, resulting in the

A 4'-phosphopantetheinyl transferase mediates non-ribosomal peptide synthetase activation in Aspergillus fumigatus.

PubMed

Neville, Claire; Murphy, Alan; Kavanagh, Kevin; Doyle, Sean

2005-04-01

Aspergillus fumigatus is a significant human pathogen. Non-ribosomal peptide (NRP) synthesis is thought to be responsible for a significant proportion of toxin and siderophore production in the organism. Furthermore, it has been shown that 4'-phosphopantetheinylation is required for the activation of key enzymes involved in non-ribosomal peptide synthesis in other species. Here we report the cloning, recombinant expression and functional characterisation of a 4'-phosphopantetheinyl transferase from A. fumigatus and the identification of an atypical NRP synthetase (Afpes1), spanning 14.3 kb. Phylogenetic analysis has shown that the NRP synthetase exhibits greatest identity to NRP synthetases from Metarhizium anisolpiae (PesA) and Alternaria brassicae (AbrePsy1). Northern hybridisation and RT-PCR analysis have confirmed that both genes are expressed in A. fumigatus. A 120 kDa fragment of the A. fumigatus NRP synthetase, containing a putative thiolation domain, was cloned and expressed in the baculovirus expression system. Detection of a 4'-phosphopantetheinylated peptide (SFSAMK) from this protein, by MALDI-TOF mass spectrometric analysis after coincubation of the 4'-phosphopantetheinyl transferase with the recombinant NRP synthetase fragment and acetyl CoA, confirms that it is competent to play a role in NRP synthetase activation in A. fumigatus. The 4'-phosphopantetheinyl transferase also activates, by 4'-phosphopantetheinylation, recombinant alpha-aminoadipate reductase (Lys2p) from Candida albicans, a key enzyme involved in lysine biosynthesis.

Susceptibility to endometrial cancer: influence of allelism at p53, glutathione S-transferase (GSTM1 and GSTT1) and cytochrome P-450 (CYP1A1) loci.

PubMed Central

Esteller, M.; García, A.; Martínez-Palones, J. M.; Xercavins, J.; Reventós, J.

1997-01-01

A case-control study was designed to identify associations between polymorphisms at p53, cytochrome P-450 (CYP1A1) and glutathione-S-transferases and endometrial cancer susceptibility. Among all polymorphisms analysed, an insertional variant in p53 (P53PIN3) and two polymorphisms in the 3'-end and exon 7 of CYP1A1 showed significant association with enhanced endometrial cancer risk. Images Figure 1 Figure 2 PMID:9155064

β(1,3)-Glucanosyl-Transferase Activity Is Essential for Cell Wall Integrity and Viability of Schizosaccharomyces pombe

PubMed Central

de Medina-Redondo, María; Arnáiz-Pita, Yolanda; Clavaud, Cécile; Fontaine, Thierry; del Rey, Francisco; Latgé, Jean Paul; Vázquez de Aldana, Carlos R.

2010-01-01

Background The formation of the cell wall in Schizosaccharomyces pombe requires the coordinated activity of enzymes involved in the biosynthesis and modification of β-glucans. The β(1,3)-glucan synthase complex synthesizes linear β(1,3)-glucans, which remain unorganized until they are cross-linked to other β(1,3)-glucans and other cell wall components. Transferases of the GH72 family play important roles in cell wall assembly and its rearrangement in Saccharomyces cerevisiae and Aspergillus fumigatus. Four genes encoding β(1,3)-glucanosyl-transferases -gas1+, gas2+, gas4+ and gas5+- are present in S. pombe, although their function has not been analyzed. Methodology/Principal Findings Here, we report the characterization of the catalytic activity of gas1p, gas2p and gas5p together with studies directed to understand their function during vegetative growth. From the functional point of view, gas1p is essential for cell integrity and viability during vegetative growth, since gas1Δ mutants can only grow in osmotically supported media, while gas2p and gas5p play a minor role in cell wall construction. From the biochemical point of view, all of them display β(1,3)-glucanosyl-transferase activity, although they differ in their specificity for substrate length, cleavage point and product size. In light of all the above, together with the differences in expression profiles during the life cycle, the S. pombe GH72 proteins may accomplish complementary, non-overlapping functions in fission yeast. Conclusions/Significance We conclude that β(1,3)-glucanosyl-transferase activity is essential for viability in fission yeast, being required to maintain cell integrity during vegetative growth. PMID:21124977

Recombinant human glutathione S-transferases catalyse enzymic isomerization of 13-cis-retinoic acid to all-trans-retinoic acid in vitro.

PubMed Central

Chen, H; Juchau, M R

1998-01-01

The steric conversion of 13-cis-retinoic acid (13-cRA) to all-trans-retinoic acid (t-RA) has been proposed as an activation mechanism for the observed therapeutic and teratogenic activities of 13-cRA. Here we have investigated the catalysis of isomerization of 13-cRA to t-RA by recombinant human glutathione S-transferases (GSTs). Substrate was incubated with GST in 0.1 M sodium phosphate buffer, pH 7.5, at 37 degrees C in total darkness. The t-RA generated was measured quantitatively by HPLC. Under the reaction conditions used, GSTP1-1 was far more effective than human GSTM1-1 or human GSTA1-1 in catalysing the isomerization reaction. The reaction catalysed by GSTP1-1 showed substrate saturation and the Km and Vmax values for the reaction were approx. 7 microM and 650 pmol/min per nmol respectively. The reaction rate increased linearly with increasing enzyme concentration. The reaction was inhibited both by heat treatment and by S-decylglutathione (a potent inhibitor of transferase activity associated with GST). Additions of polyclonal rabbit antiserum for human GSTP1-1 to the reaction resulted in a significant decrease in generation of t-RA (70-80%). In addition, ethacrynic acid, a selective substrate for Pi isoforms of GST, also inhibited the isomerization of 13-cRA to t-RA catalysed by GSTP1-1. Under the same reaction conditions, GSTP1-1 was much less effective in catalysing the steric conversion of 9-cis-retinoic acid to t-RA, indicating that the enzyme was stereospecific for the conversion of 13-cRA to t-RA. These observations suggest that enzymic catalysis was the primary mechanism for the GSTP1-1-dependent conversion of 13-cRA to t-RA. Reactions catalysed by a purified rat hepatic GST Pi isoenzyme proceeded more slowly than reactions catalysed by human GSTP1-1. Comparative studies also showed that there were marked species differences in catalytic activities between various purified mammalian hepatic GST mixtures. PMID:9806904

Recombinant human glutathione S-transferases catalyse enzymic isomerization of 13-cis-retinoic acid to all-trans-retinoic acid in vitro.

PubMed

Chen, H; Juchau, M R

1998-11-15

The steric conversion of 13-cis-retinoic acid (13-cRA) to all-trans-retinoic acid (t-RA) has been proposed as an activation mechanism for the observed therapeutic and teratogenic activities of 13-cRA. Here we have investigated the catalysis of isomerization of 13-cRA to t-RA by recombinant human glutathione S-transferases (GSTs). Substrate was incubated with GST in 0.1 M sodium phosphate buffer, pH 7.5, at 37 degrees C in total darkness. The t-RA generated was measured quantitatively by HPLC. Under the reaction conditions used, GSTP1-1 was far more effective than human GSTM1-1 or human GSTA1-1 in catalysing the isomerization reaction. The reaction catalysed by GSTP1-1 showed substrate saturation and the Km and Vmax values for the reaction were approx. 7 microM and 650 pmol/min per nmol respectively. The reaction rate increased linearly with increasing enzyme concentration. The reaction was inhibited both by heat treatment and by S-decylglutathione (a potent inhibitor of transferase activity associated with GST). Additions of polyclonal rabbit antiserum for human GSTP1-1 to the reaction resulted in a significant decrease in generation of t-RA (70-80%). In addition, ethacrynic acid, a selective substrate for Pi isoforms of GST, also inhibited the isomerization of 13-cRA to t-RA catalysed by GSTP1-1. Under the same reaction conditions, GSTP1-1 was much less effective in catalysing the steric conversion of 9-cis-retinoic acid to t-RA, indicating that the enzyme was stereospecific for the conversion of 13-cRA to t-RA. These observations suggest that enzymic catalysis was the primary mechanism for the GSTP1-1-dependent conversion of 13-cRA to t-RA. Reactions catalysed by a purified rat hepatic GST Pi isoenzyme proceeded more slowly than reactions catalysed by human GSTP1-1. Comparative studies also showed that there were marked species differences in catalytic activities between various purified mammalian hepatic GST mixtures.

Glutathione and glutathione S-transferase levels in patients with liver metastases of colorectal cancer and other hepatic disorders.

PubMed

Baltruskeviciene, Edita; Kazbariene, Birutė; Badaras, Robertas; Bagdonaitė, Loreta; Krikštaponienė, Aurelija; Zdanavičius, Linas; Aleknavicius, Eduardas; Didziapetrienė, Janina

2016-07-01

Glutathione and glutathione S-transferases (GST) are involved in cell defence against reactive oxygen species, which induces oxidative stress and are associated with different chronic diseases. The aim of the present study was to determine the differences in reduced glutathione (GSH) and GST levels in patients with different liver diseases. Overall, 114 patients were enrolled in this study: 58 patients with colorectal cancer (18 without and 40 with liver metastases), 27 with liver steatosis, 29 with alcoholic cirrhosis and a group of 40 healthy volunteers. The levels of GSH and GST in blood serum were evaluated by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's guidelines. Significant differences in GSH and GST levels were observed in most of the groups compared to the healthy volunteers (GSH: 52.72 µg/mL, GST: 0.53 ng/mL): with hepatic steatosis (GSH: 17.04 µg/mL, p < 0.001; GST: 5.89 ng/mL, p < 0.001), alcoholic cirrhosis (GSH: 62.04 µg/mL, p < 0.003; GST: 0.94 ng/mL, p < 0.001) and liver metastases (GSH: 37.84 µg/mL, p < 0.001, GST: 1.25 ng/mL, p=0.747). The different GSH and GST levels in patients with colorectal cancer liver metastases, liver steatosis and alcoholic cirrhosis indicate the differences in antioxidative system damage and its compensatory possibilities and could serve as potential biomarkers for its correction.

The inhibition characteristics of human placental glutathione S-transferase-π by tricyclic antidepressants: amitriptyline and clomipramine.

PubMed

Dalmizrak, Ozlem; Kulaksiz-Erkmen, Gulnihal; Ozer, Nazmi

2011-09-01

Tricyclic antidepressants (TCAs) are the non-selective amine re-uptake inhibitors, well absorbed from small intestine, cross the blood-brain barrier, distributed in the brain, and are bound to glutathione S-transferase-π (GST-π). TCAs can pass through placenta, accumulate in utero baby, and cause congenital malformations. Thus, the study of the interaction of GST-π with antidepressants is crucial. In this study, the interaction of GST-π with amitriptyline and clomipramine was investigated. The K (m) values for glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) were found to be 0.16 ± 0.04 and 3.60 ± 1.67 mM, respectively. The V (m) values were varying according to the fixed substrate; [CDNB] fixed, 53 ± 3 and [GSH] fixed 182 ± 63 U/mg protein. At variable [GSH] and variable [CDNB], the k (cat) values of 7.0 × 10(6) and 1.42 × 10(7) s(-1) and the k (cat)/K (m) values of 4.38 × 10(10) and 3.94 × 10(9 )M(-1 )s(-1) were obtained, respectively. At fixed [CDNB] and variable [GSH], amitriptyline (K (s) = 0.16 ± 0.03 mM; α = 2.08; and K (i) = 1.75 ± 0.37 mM) and clomipramine (K (s) = 0.24 ± 0.05 mM; α = 1.57; and K (i) = 3.90 ± 2.26 mM) showed linear mixed-type inhibition whereas when the varied substrate is CDNB, amitriptyline (K (i) = 4.90 ± 0.68 mM) and clomipramine (K (i) = 3.37 ± 0.39 mM) inhibition were noncompetitive. The inhibition of GST-π by TCAs means the destruction of its protective role against toxic electrophiles. The effect of antidepressants on fetus will be much severe, thus, the antidepressant therapy of pregnant women should be done with caution.

Glutathione S-transferase of brown planthoppers (Nilaparvata lugens) is essential for their adaptation to gramine-containing host plants.

PubMed

Sun, Xiao-Qin; Zhang, Mao-Xin; Yu, Jing-Ya; Jin, Yu; Ling, Bing; Du, Jin-Ping; Li, Gui-Hua; Qin, Qing-Ming; Cai, Qing-Nian

2013-01-01

Plants have evolved complex processes to ward off attacks by insects. In parallel, insects have evolved mechanisms to thwart these plant defenses. To gain insight into mechanisms that mediate this arms race between plants and herbivorous insects, we investigated the interactions between gramine, a toxin synthesized by plants of the family Gramineae, and glutathione S transferase (GST), an enzyme found in insects that is known to detoxify xenobiotics. Here, we demonstrate that rice (Oryza sativa), a hydrophytic plant, also produces gramine and that rice resistance to brown planthoppers (Nilaparvata lugens, BPHs) is highly associated with in planta gramine content. We also show that gramine is a toxicant that causes BPH mortality in vivo and that knockdown of BPH GST gene nlgst1-1 results in increased sensitivity to diets containing gramine. These results suggest that the knockdown of key detoxification genes in sap-sucking insects may provide an avenue for increasing their sensitivity to natural plant-associated defense mechanisms.

Glutathione S-Transferase of Brown Planthoppers (Nilaparvata lugens) Is Essential for Their Adaptation to Gramine-Containing Host Plants

PubMed Central

Yu, Jing-Ya; Jin, Yu; Ling, Bing; Du, Jin-Ping; Li, Gui-Hua; Qin, Qing-Ming; Cai, Qing-Nian

2013-01-01

Plants have evolved complex processes to ward off attacks by insects. In parallel, insects have evolved mechanisms to thwart these plant defenses. To gain insight into mechanisms that mediate this arms race between plants and herbivorous insects, we investigated the interactions between gramine, a toxin synthesized by plants of the family Gramineae, and glutathione S transferase (GST), an enzyme found in insects that is known to detoxify xenobiotics. Here, we demonstrate that rice (Oryza sativa), a hydrophytic plant, also produces gramine and that rice resistance to brown planthoppers (Nilaparvata lugens, BPHs) is highly associated with in planta gramine content. We also show that gramine is a toxicant that causes BPH mortality in vivo and that knockdown of BPH GST gene nlgst1-1 results in increased sensitivity to diets containing gramine. These results suggest that the knockdown of key detoxification genes in sap-sucking insects may provide an avenue for increasing their sensitivity to natural plant-associated defense mechanisms. PMID:23700450

Recognition and Detoxification of the Insecticide DDT by Drosophila melanogaster Glutathione S-Transferase D1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Low, Wai Yee; Feil, Susanne C.; Ng, Hooi Ling

2010-06-14

GSTD1 is one of several insect glutathione S-transferases capable of metabolizing the insecticide DDT. Here we use crystallography and NMR to elucidate the binding of DDT and glutathione to GSTD1. The crystal structure of Drosophila melanogaster GSTD1 has been determined to 1.1 {angstrom} resolution, which reveals that the enzyme adopts the canonical GST fold but with a partially occluded active site caused by the packing of a C-terminal helix against one wall of the binding site for substrates. This helix would need to unwind or be displaced to enable catalysis. When the C-terminal helix is removed from the model ofmore » the crystal structure, DDT can be computationally docked into the active site in an orientation favoring catalysis. Two-dimensional {sup 1}H,{sup 15}N heteronuclear single-quantum coherence NMR experiments of GSTD1 indicate that conformational changes occur upon glutathione and DDT binding and the residues that broaden upon DDT binding support the predicted binding site. We also show that the ancestral GSTD1 is likely to have possessed DDT dehydrochlorinase activity because both GSTD1 from D. melanogaster and its sibling species, Drosophila simulans, have this activity.« less

Glutathione S-transferases act as isomerases in isomerization of 13-cis-retinoic acid to all-trans-retinoic acid in vitro.

PubMed

Chen, H; Juchau, M R

1997-11-01

A discovery that rapid enzymic isomerization of 13-cis-retinoic acid (13-cRA) to all-trans-retinoic acid (t-RA) can be catalysed by purified hepatic glutathione S-transferases (GSTs; EC 2.5.1.18) from rat is now reported. Rates of cis-trans isomerization were determined quantitatively by HPLC. GST-catalysed reactions reached equilibrium rapidly, in marked contrast with uncatalysed or GSH-catalysed isomerizations. The GST-catalysed reaction exhibited substrate saturation kinetics with a Km of approx. 8 microM. The maximal velocity of the reaction and the catalytic efficiency of GSTs were determined. The initial rate of the reaction increased linearly as a function of enzyme concentration. Catalysis by GSTs was independent of the presence of GSH, indicating that GSTs act as GSH-independent isomerases as well as transferases. Incubation with guanidine (7-8 M) or heat-inactivation of GSTs (100 degrees C for 3 min) decreased isomerase activities by approx. 50% and 75% respectively. The same heat treatment did not significantly inhibit isomerization catalysed by GSH and apoferritin, indicating that the observed decrease in isomerase activity by heat inactivation was not primarily due to oxidation of protein thiol groups in the GSTs. The specific activity of GSTs was approx. 23- and 340-fold those of GSH and apoferritin respectively when comparisons were made on the basis of free thiol concentrations, indicating that free thiol in GSTs cannot account for the majority of observed isomerase activities and suggesting that specific conformations of GSTs are important for such activities. Complete inhibition of the reaction by low concentrations of N-ethylmaleimide (10 microM) demonstrated that intact protein thiols are required for the isomerase activities of GSTs.

Effects of genetic polymorphisms of glutathione S-transferase genes (GSTM1, GSTT1, GSTP1) on the risk of diabetic nephropathy: a meta-analysis.

PubMed

Orlewski, Jan; Orlewska, Ewa

2015-01-01

Glutathione S-transferases (GSTs) belong to a family of ubiquitous and multifunctional enzymes that protect the cells against oxidative stress. The aim of the study was to evaluate the association between the polymorphisms of glutathione-S-transferase (GST) genes and diabetic nephropathy (DN). PubMed, EMBASE, and Google Scholar databases were systematically searched to identify relevant studies. The odds ratio (OR) for the association was determined using a fixed or random effects model. Tests for heterogeneity of the results and sensitivity analyses were performed. A total of 9 publications (874 patients in the study group, 966 controls) were included. With the exception of 1 study, GSTT1 and GSTM1 genotypes were not assessed by methods that measure a gene copy number. A significantly increased risk of DN was found for the GSTM1(-) genotype (OR, 1.27; 95% CI, 1.02-1.58) and the combination of GSTT1(-)/GSTM1(-) (OR,2.02; 95% CI, 1.22-3.36). We did not observe a correlation between DN and the GSTT1(-) genotype or the presence of Val alleles. In a subgroup analysis, an association between DN and the GSTM1(-) genotype was significant in Asians but not in Caucasians. Our results indicate that the GSTM1(-) genotype and the combination of GSTT1(-)/GSTM1(-) increase the risk of DN. The combination of the GST polymorphisms rather than individual polymorphismshould be investigated. Genotyping allowing a trimodular determination of the GST copy number variations may better describe an association between the risk of disease and a given genotype.

Caribbean yellow band disease compromises the activity of catalase and glutathione S-transferase in the reef-building coral Orbicella faveolata exposed to anthracene.

PubMed

Montilla, Luis Miguel; Ramos, Ruth; García, Elia; Cróquer, Aldo

2016-05-03

Healthy and diseased corals are threatened by different anthropogenic sources, such as pollution, a problem expected to become more severe in the near future. Despite the fact that coastal pollution and coral diseases might represent a serious threat to coral reef health, there is a paucity of controlled experiments showing whether the response of diseased and healthy corals to xenobiotics differs. In this study, we exposed healthy and Caribbean yellow band disease (CYBD)-affected Orbicella faveolata colonies to 3 sublethal concentrations of anthracene to test if enzymatic responses to this hydrocarbon were compromised in CYBD-affected tissues. For this, a 2-factorial fully orthogonal design was used in a controlled laboratory bioassay, using tissue condition (2 levels: apparently healthy and diseased) and pollutant concentration (4 levels: experimental control, 10, 30 and 100 ppb concentration) as fixed factors. A permutation-based ANOVA (PERMANOVA) was used to test the effects of condition and concentration on the specific activity of 3 enzymatic biomarkers: catalase, glutathione S-transferase, and glutathione peroxidase. We found a significant interaction between the concentration of anthracene and the colony condition for catalase (Pseudo-F = 3.84, df = 3, p < 0.05) and glutathione S-transferase (Pseudo-F = 3.29, df = 3, p < 0.05). Moreover, our results indicated that the enzymatic response to anthracene in CYBD-affected tissues was compromised, as the activity of these enzymes decreased 3- to 4-fold compared to healthy tissues. These results suggest that under a potential scenario of increasing hydrocarbon coastal pollution, colonies of O. faveolata affected with CYBD might become more vulnerable to the deleterious effects of chemical pollution.

Glutathione S-transferase (placental) as a marker of transformation in the human cervix uteri: an immunohistochemical study.

PubMed Central

Randall, B. J.; Angus, B.; Akiba, R.; Hall, A.; Cattan, A. R.; Proctor, S. J.; Jones, R. A.; Horne, C. H.

1990-01-01

Using an indirect immunohistochemical technique on paraffin sections, employing a polyclonal antibody to the acidic (placental) form of glutathione-S-transferase (GST), we have evaluated cytoplasmic and nuclear staining in a series of 67 cervical biopsies including normal non neoplastic tissue, immature squamous metaplasia, all grades of cervical intraepithelial neoplasia (CIN) and invasive carcinomas of keratinising and non-keratinising types. No differences in cytoplasmic staining between the varied lesions studied were seen. However, there were marked differences in nuclear staining. While normal non-neoplastic stratified squamous epithelium showed weak staining of the lower one-third of the epithelium only, in immature squamous metaplasia and in all grades of CIN there was intense nuclear staining in all layers of the epithelium. Invasive carcinomas showed generally less intense nuclear staining than CIN lesions. Endocervical cell nuclei also showed intense nuclear staining. These findings indicate that GST is of limited use as a marker of transformation in the human cervix uteri. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:2223578

Characterization of a lambda-cyhalothrin metabolizing glutathione S-transferase CpGSTd1 from Cydia pomonella (L.).

PubMed

Liu, Jiyuan; Yang, Xueqing; Zhang, Yalin

2014-11-01

In insects, glutathione S-transferases (GSTs) are enzymes involved in detoxification of insecticides. However, few data are available for the codling moth, Cydia pomonella (L.). In this study, we cloned a delta class GST gene CpGSTd1 from C. pomonella. Real-time quantitative PCR shows that CpGSTd1 was up-regulated with aging, and the mRNA level of CpGSTd1 was higher in the fat body and silk glands than in other tissues. The expression level of CpGSTd1 exposure to insecticide suggests that CpGSTd1 is up-regulated after chlorpyrifos-methyl and lambda-cyhalothrin treatments. Both lambda-cyhalothrin and chlorpyrifos-methyl altered GST activity in vivo. The purified CpGSTd1 protein exhibits a high catalytic efficiency with CDNB and was inhibited by lambda-cyhalothrin and chlorpyrifos-methyl in vitro. Metabolism assays indicate that lambda-cyhalothrin was significantly metabolized while chlorpyrifos-methyl was not metabolized by CpGSTd1. Binding free energy analysis suggests that CpGSTd1 binding is tighter with lambda-cyhalothrin than with chlorpyrifos-methyl. Our study suggests that CpGSTd1 plays a key role in the metabolism of insecticides in C. pomonella.

Evidence-based management of otitis media: a 5S model approach.

PubMed

Wasson, J D; Yung, M W

2015-02-01

The 5S model proposes five hierarchical levels (systems, summaries, synopses, syntheses and studies) of pre-appraised evidence to guide evidence-based practice. This review aimed to identify and summarise pre-appraised evidence at the highest available 5S level for the management of different subsets of otitis media: acute otitis media, otitis media with effusion, chronic suppurative otitis media and cholesteatoma in both adults and children. Data sources were pre-appraised evidence resources. Evidence freely available from sources at the highest available level of the 5S model were summarised for this review. System level evidence exists for acute otitis media and otitis media with effusion. Summary level evidence exists for recurrent acute otitis media and medical management of chronic suppurative otitis media. There is an absence of randomised controlled trials to prove the efficacy of surgical management of chronic suppurative otitis media and cholesteatoma. Until randomised controlled trial data are generated, consensus publications on the surgical management of chronic suppurative otitis media and cholesteatoma should be used to guide best practice.

Glutathione S-transferase π complexes with and stimulates Na⁺,K⁺-ATPase.

PubMed

Ochiai, Hideo; Eguchi, Hiroshi; Noguchi, Shunsuke; Hayashi, Yutaro; Nishino, Hideaki; Kawamura, Masaru; Wu, Chau H

2013-01-01

Glutathione S-transferase (GST) was found to complex with the Na⁺,K⁺-ATPase as shown by binding assay using quartz crystal microbalance. The complexation was obstructed by the addition of antiserum to the α-subunit of the Na⁺,K⁺-ATPase, suggesting the specificity of complexation between GST and the Na⁺,K⁺-ATPase. Co-immunoprecipitation experiments, using the anti-α-subunit antiserum to precipitate the GST-Na⁺,K⁺-ATPase complex and then using antibodies specific to an isoform of GST to identify the co-precipitated proteins, revealed that GSTπ was complexed with the Na⁺,K⁺-ATPase. GST stimulated the Na⁺,K⁺-ATPase activity up to 1.4-fold. The level of stimulation exhibited a saturable dose-response relationship with the amount of GST added, although the level of stimulation varied depending on the content of GSTπ in the lots of GST received from supplier. The stimulation was also obtained when recombinant GSTπ was used, confirming the results. When GST was treated with reduced glutathione, GST activity was greatly stimulated, whereas the level of stimulation of the Na⁺,K⁺-ATPase activity was similar to that when untreated GST was added. When GST was treated with H₂O₂, GST activity was greatly diminished while the stimulation of the Na⁺,K⁺-ATPase activity was preserved. The results suggest that GSTπ complexes with the Na⁺,K⁺-ATPase and stimulates the latter independent of its GST activity. Copyright © 2012 John Wiley & Sons, Ltd.

Genetic polymorphism in three glutathione s-transferase genes and breast cancer risk

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woldegiorgis, S.; Ahmed, R.C.; Zhen, Y.

The role of the glutathione S-transferase (GST) enzyme family is to detoxify environmental toxins and carcinogens and to protect organisms from their adverse effects, including cancer. The genes GSTM1, GSTP1, and GSTT1 code for three GSTs involved in the detoxification of carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) and benzene. In humans, GSTM1 is deleted in about 50% of the population, GSTT1 is absent in about 20%, whereas the GSTP1 gene has a single base polymorphism resulting in an enzyme with reduced activity. Epidemiological studies indicate that GST polymorphisms increase the level of carcinogen-induced DNA damage and several studies havemore » found a correlation of polymorphisms in one of the GST genes and an increased risk for certain cancers. We examined the role of polymorphisms in genes coding for these three GST enzymes in breast cancer. A breast tissue collection consisting of specimens of breast cancer patients and non-cancer controls was analyzed by polymerase chain reaction (PCR) for the presence or absence of the GSTM1 and GSTT1 genes and for GSTP1 single base polymorphism by PCR/RFLP. We found that GSTM1 and GSTT1 deletions occurred more frequently in cases than in controls, and GSTP1 polymorphism was more frequent in controls. The effective detoxifier (putative low-risk) genotype (defined as presence of both GSTM1 and GSTT1 genes and GSTP1 wild type) was less frequent in cases than controls (16% vs. 23%, respectively). The poor detoxifier (putative high-risk) genotype was more frequent in cases than controls. However, the sample size of this study was too small to provide conclusive results.« less

Biosynthesis of 2-Hydroxyacid-Containing Polyhydroxyalkanoates by Employing butyryl-CoA Transferases in Metabolically Engineered Escherichia coli.

PubMed

David, Yokimiko; Joo, Jeong Chan; Yang, Jung Eun; Oh, Young Hoon; Lee, Sang Yup; Park, Si Jae

2017-11-01

The authors previously reported the production of polyhydroxyalkanoates (PHAs) containing 2-hydroxyacid monomers by expressing evolved Pseudomonas sp. 6-19 PHA synthase and Clostridium propionicum propionyl-CoA transferase in engineered microorganisms. Here, the authors examined four butyryl-CoA transferases from Roseburia sp., Eubacterium hallii, Faecalibacterium prausnitzii, and Anaerostipes caccae as potential CoA-transferases to support synthesis of polymers having 2HA monomer. In vitro activity analyses of the four butyryl-CoA transferases suggested that each butyryl-CoA transferase has different activities towards 2-hydroxybutyrate (2HB), 3-hydroxybutyrate (3HB), and lactate (LA). When Escherichia coli XL1-Blue expressing Pseudomonas sp. 6-19 PhaC1437 along with one butyryl-CoA transferase is cultured in chemically defined MR medium containing 20 g L -1 of glucose, 2 g L -1 of sodium 3-hydroxybutyrate, and various concentrations of sodium 2-hydroxybutyrate, PHAs consisting of 3HB, 2HB, and LA are produced. The monomer composition of PHAs agreed well with the substrate specificities of butyryl-CoA transferases from E. hallii, F. prausnitzii, and A. caccae, but not Roseburia sp. When E. coli XL1-Blue expressing PhaC1437 and E. hallii butyryl-CoA transferase is cultured in MR medium containing 20 g L -1 of glucose and 2 g L -1 of sodium 2-hydroxybutyrate, P(65.7 mol% 2HB-co-34.3 mol% LA) is produced with the highest PHA content of 30 wt%. Butyryl-CoA transferases also supported the production of P(3HB-co-2HB-co-LA) from glucose as the sole carbon source in E. coli XL1-Blue strains when one of these bct genes is expressed with phaC1437, cimA3.7, leuBCD, panE, and phaAB genes. Butyryl-CoA transferases characterized in this study can be used for engineering of microorganisms that produce PHAs containing novel 2-hydroxyacid monomers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Association of genetic polymorphism of glutathione S-transferase (GSTM1, GSTT1, GSTP1) with bladder cancer susceptibility.

PubMed

Safarinejad, Mohammad Reza; Safarinejad, Saba; Shafiei, Nayyer; Safarinejad, Shiva

2013-10-01

The glutathione-S-transferases (GSTs) comprise a class of enzymes that detoxify carcinogenic compounds by conjugating glutathione to facilitate their removal. Polymorphisms in GSTM1, GSTT1, and GSTP1 genes have been related to risk for bladder cancer. Studies focusing on GSTs gene variants relationship with the risk of bladder cancer have produced conflicting and inconsistent results. We examine the association between genetic polymorphism of glutathione S-transferase P1, GSTM1, GSTT1 genes and development of bladder transitional cell carcinoma (TCC). The study population consisted of 166 histologically confirmed male bladder TCC cases and 332 healthy male controls. Genotyping was done using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method and also investigated combined gene interactions. The GSTP1 Val/Val genotype was significantly associated with bladder cancer (OR = 4.32, 95% CI: 2.64-6.34), whereas the association observed for GSTM1 null (OR = 1.32, 95% CI: 0.82-2.62; P = 0.67) and GSTT1 null genotype (OR = 1.18, 95% CI: 0.79-1.67; P = 0.74) did not reach statistical significance. There was a significant multiple interaction between GSTM1, GSTT1, and GSTP1 genotypes in risk of bladder cancer (P for interaction = 0.02). The risk associated with the concurrent presence of GSTM1 positive and GSTP1 Ile/Val or Val/Val (OR = 3.71, 95% CI: 2.34-5.54) and GSTT1 positive and GSTP1 Ile/Val or Val/Val (OR = 2.66, 95% CI: 1.54-4.72) was statistically significant. Patients carrying GSTP1 Val/Val genotype were at increased risk for developing high-grade (OR = 7.68, 95% CI: 4.73-19.25) and muscle invasive (OR = 10.67, 95% CI: 6.34-21.75) bladder cancer. High risk for bladder TCC also was observed with respect to combined GSTT1 null/GSTP1 Ile/Val or Val/Val (OR = 4.76, 95% CI: 2.68-18.72) and GSTM1 null/GSTT1 null/GSTP1 Ile/Val or Val/Val (OR = 6.42, 95% CI: 4.76-14.72) genotype variant. This study suggests that the GSTP1 polymorphism

Genomic organization of plant aminopropyl transferases.

PubMed

Rodríguez-Kessler, Margarita; Delgado-Sánchez, Pablo; Rodríguez-Kessler, Gabriela Theresia; Moriguchi, Takaya; Jiménez-Bremont, Juan Francisco

2010-07-01

Aminopropyl transferases like spermidine synthase (SPDS; EC 2.5.1.16), spermine synthase and thermospermine synthase (SPMS, tSPMS; EC 2.5.1.22) belong to a class of widely distributed enzymes that use decarboxylated S-adenosylmethionine as an aminopropyl donor and putrescine or spermidine as an amino acceptor to form in that order spermidine, spermine or thermospermine. We describe the analysis of plant genomic sequences encoding SPDS, SPMS, tSPMS and PMT (putrescine N-methyltransferase; EC 2.1.1.53). Genome organization (including exon size, gain and loss, as well as intron number, size, loss, retention, placement and phase, and the presence of transposons) of plant aminopropyl transferase genes were compared between the genomic sequences of SPDS, SPMS and tSPMS from Zea mays, Oryza sativa, Malus x domestica, Populus trichocarpa, Arabidopsis thaliana and Physcomitrella patens. In addition, the genomic organization of plant PMT genes, proposed to be derived from SPDS during the evolution of alkaloid metabolism, is illustrated. Herein, a particular conservation and arrangement of exon and intron sequences between plant SPDS, SPMS and PMT genes that clearly differs with that of ACL5 genes, is shown. The possible acquisition of the plant SPMS exon II and, in particular exon XI in the monocot SPMS genes, is a remarkable feature that allows their differentiation from SPDS genes. In accordance with our in silico analysis, functional complementation experiments of the maize ZmSPMS1 enzyme (previously considered to be SPDS) in yeast demonstrated its spermine synthase activity. Another significant aspect is the conservation of intron sequences among SPDS and PMT paralogs. In addition the existence of microsynteny among some SPDS paralogs, especially in P. trichocarpa and A. thaliana, supports duplication events of plant SPDS genes. Based in our analysis, we hypothesize that SPMS genes appeared with the divergence of vascular plants by a processes of gene duplication and the

Characterization of a Bvg-regulated fatty acid methyl-transferase in Bordetella pertussis.

PubMed

Rivera-Millot, Alex; Lesne, Elodie; Solans, Luis; Coutte, Loic; Bertrand-Michel, Justine; Froguel, Philippe; Dhennin, Véronique; Hot, David; Locht, Camille; Antoine, Rudy; Jacob-Dubuisson, Françoise

2017-01-01

The whooping cough agent Bordetella pertussis controls the expression of its large virulence regulon in a coordinated manner through the two-component signal transduction system BvgAS. In addition to the genes coding for bona fide virulence factors, the Bvg regulon comprises genes of unknown function. In this work, we characterized a new Bvg-activated gene called BP2936. Homologs of BP2936 are found in other pathogenic Bordetellae and in several other species, including plant pathogens and environmental bacteria. We showed that the gene product of BP2936 is a membrane-associated methyl-transferase of free fatty acids. We thus propose to name it FmtB, for fatty acid methyl-transferase of Bordetella. The role of this protein was tested in cellular and animal models of infection, but the loss of BP2936 did not appear to affect host-pathogen interactions in those assays. The high level of conservation of BP2936 among B. pertussis isolates nevertheless argues that it probably plays a role in the life cycle of this pathogen.

Characterization of a Bvg-regulated fatty acid methyl-transferase in Bordetella pertussis

PubMed Central

Rivera-Millot, Alex; Lesne, Elodie; Solans, Luis; Coutte, Loic; Bertrand-Michel, Justine; Froguel, Philippe; Dhennin, Véronique; Hot, David; Locht, Camille; Antoine, Rudy

2017-01-01

The whooping cough agent Bordetella pertussis controls the expression of its large virulence regulon in a coordinated manner through the two-component signal transduction system BvgAS. In addition to the genes coding for bona fide virulence factors, the Bvg regulon comprises genes of unknown function. In this work, we characterized a new Bvg-activated gene called BP2936. Homologs of BP2936 are found in other pathogenic Bordetellae and in several other species, including plant pathogens and environmental bacteria. We showed that the gene product of BP2936 is a membrane-associated methyl-transferase of free fatty acids. We thus propose to name it FmtB, for fatty acid methyl-transferase of Bordetella. The role of this protein was tested in cellular and animal models of infection, but the loss of BP2936 did not appear to affect host-pathogen interactions in those assays. The high level of conservation of BP2936 among B. pertussis isolates nevertheless argues that it probably plays a role in the life cycle of this pathogen. PMID:28493897

Response of glutathione S-transferase Pi (GSTP1) to neoadjuvant therapy in rectal adenocarcinoma.

PubMed

Bedford, M R; Anathhanam, S; Saleh, D; Hickson, A; McGregor, A K; Boyle, K; Burke, D

2012-12-01

The response of rectal adenocarcinoma to neoadjuvant therapy is variable. Accurate prediction of response would enable selective administration of therapy. The enzyme glutathione S-transferase Pi (GSTP1) has been shown to influence response to therapy in some solid tumours. Few data are available for rectal cancer. The GSTP1 levels in rectal adenocarcinoma and adjacent normal mucosa were quantified before and after exposure to neoadjuvant therapy. Venous blood samples and biopsies of normal rectal mucosa and tumour were prospectively obtained from patients with primary rectal cancer. Patients were stratified by exposure to neoadjuvant therapy or surgery alone. GSTP1 was quantitatively measured using an enzyme-linked immunosorbent assay. Ninety-two patients (54 men; median age 68 years) were recruited. The median GSTP1 level was significantly higher in rectal adenocarcinoma than in matched normal mucosa [6.59 μg/mg vs 4.57 μg/mg; P < 0.001]. The median tumour GSTP1 level was significantly lower in the therapy group compared with unmatched samples from the no-therapy group [4.47 μg/mg vs 7.76 μg/mg; P < 0.001]. The GSTP1 level is increased in rectal adenocarcinoma compared with adjacent normal mucosa. It decreases following neoadjuvant therapy. Future studies correlating pre-therapy GSTP1 levels with pathological response would be of interest. © 2012 The Authors. Colorectal Disease © 2012 The Association of Coloproctology of Great Britain and Ireland.

Multiple glutathione S-transferase genes: identification and expression in oriental fruit fly, Bactrocera dorsalis.

PubMed

Hu, Fei; Dou, Wei; Wang, Jing-Jing; Jia, Fu-Xian; Wang, Jin-Jun

2014-02-01

The oriental fruit fly, Bactrocera dorsalis (Hendel), is widely distributed in Asia-Pacific regions, where it is a serious pest of a wide range of tropical and subtropical fruit and vegetable crops. In this study, 17 cDNA encoding glutathione S-transferases (GSTs) in B. dorsalis were sequenced and characterised. Phylogenetic analysis revealed that 16 GSTs belonged to five different cytosolic classes, including four in delta, eight in epsilon, two in omega, one in theta, and one in zeta. The remaining GST (BdGSTu1) was unclassified. RT-qPCR assay showed that the relative expression levels of five GST genes were significantly higher in larval stages than in adulthood. Tissue-specific expression analysis found that BdGSTe3, BdGSTe9 and BdGSTd5 were expressed highly in the midgut, BdGSTe4, BdGSTe6, BdGSTd6 and BdGSTz2 were higher in the fat body, and six GSTs were higher in Malpighian tubules. RT-qPCR confirmed that the expressions of nine GST genes were increased by malathion exposure at various times and doses, while BdGSTe4, BdGSTe9 and BdGSTt1 were increased by β-cypermethrin exposure. The increases in GST gene expression levels after malathion and β-cypermethrin exposure in B. dorsalis might increase the ability of this species to detoxify other insecticides and xenobiotics. © 2013 Society of Chemical Industry.

Glutathione S-transferases act as isomerases in isomerization of 13-cis-retinoic acid to all-trans-retinoic acid in vitro.

PubMed Central

Chen, H; Juchau, M R

1997-01-01

A discovery that rapid enzymic isomerization of 13-cis-retinoic acid (13-cRA) to all-trans-retinoic acid (t-RA) can be catalysed by purified hepatic glutathione S-transferases (GSTs; EC 2.5.1.18) from rat is now reported. Rates of cis-trans isomerization were determined quantitatively by HPLC. GST-catalysed reactions reached equilibrium rapidly, in marked contrast with uncatalysed or GSH-catalysed isomerizations. The GST-catalysed reaction exhibited substrate saturation kinetics with a Km of approx. 8 microM. The maximal velocity of the reaction and the catalytic efficiency of GSTs were determined. The initial rate of the reaction increased linearly as a function of enzyme concentration. Catalysis by GSTs was independent of the presence of GSH, indicating that GSTs act as GSH-independent isomerases as well as transferases. Incubation with guanidine (7-8 M) or heat-inactivation of GSTs (100 degrees C for 3 min) decreased isomerase activities by approx. 50% and 75% respectively. The same heat treatment did not significantly inhibit isomerization catalysed by GSH and apoferritin, indicating that the observed decrease in isomerase activity by heat inactivation was not primarily due to oxidation of protein thiol groups in the GSTs. The specific activity of GSTs was approx. 23- and 340-fold those of GSH and apoferritin respectively when comparisons were made on the basis of free thiol concentrations, indicating that free thiol in GSTs cannot account for the majority of observed isomerase activities and suggesting that specific conformations of GSTs are important for such activities. Complete inhibition of the reaction by low concentrations of N-ethylmaleimide (10 microM) demonstrated that intact protein thiols are required for the isomerase activities of GSTs. PMID:9581548

Down-regulation of glutatione S-transferase α 4 (hGSTA4) in the muscle of thermally injured patients is indicative of susceptibility to bacterial infection

PubMed Central

Apidianakis, Yiorgos; Que, Yok-Ai; Xu, Weihong; Tegos, George P.; Zimniak, Piotr; Hamblin, Michael R.; Tompkins, Ronald G.; Xiao, Wenzhong; Rahme, Laurence G.

2012-01-01

Patients with severe burns are highly susceptible to bacterial infection. While immunosuppression facilitates infection, the contribution of soft tissues to infection beyond providing a portal for bacterial entry remains unclear. We showed previously that glutathione S-transferase S1 (gstS1), an enzyme with conjugating activity against the lipid peroxidation byproduct 4-hydroxynonenal (4HNE), is important for resistance against wound infection in Drosophila muscle. The importance of the mammalian functional counterpart of GstS1 in the context of wounds and infection has not been investigated. Here we demonstrate that the presence of a burn wound dramatically affects expression of both human (hGSTA4) and mouse (mGsta4) 4HNE scavengers. hGSTA4 is down-regulated significantly within 1 wk of thermal burn injury in the muscle and fat tissues of patients from the large-scale collaborative Inflammation and the Host Response to Injury multicentered study. Similarly, mGsta4, the murine GST with the highest catalytic efficiency for 4HNE, is down-regulated to approximately half of normal levels in mouse muscle immediately postburn. Consequently, 4HNE protein adducts are increased 4- to 5-fold in mouse muscle postburn. Using an open wound infection model, we show that deletion of mGsta4 renders mice more susceptible to infection with the prevalent wound pathogen Pseudomonas aeruginosa, while muscle hGSTA4 expression negatively correlates with burn wound infection episodes per patient. Our data suggest that hGSTA4 down-regulation and the concomitant increase in 4HNE adducts in human muscle are indicative of susceptibility to infection in individuals with severely thermal injuries.—Apidianakis, Y., Que, Y.-A., Xu, W., Tegos, G. P., Zimniak, P., Hamblin, M. R., Tompkins, R. G., Xiao, W., Rahme, L. G. Down-regulation of glutatione S-transferase α 4 (hGSTA4) in the muscle of thermally injured patients is indicative of susceptibility to bacterial infection. PMID:22038048

A Simple Colorimetric Assay for Specific Detection of Glutathione-S Transferase Activity Associated with DDT Resistance in Mosquitoes

PubMed Central

Rajatileka, Shavanti; Steven, Andrew; Hemingway, Janet; Ranson, Hilary; Paine, Mark; Vontas, John

2010-01-01

Background Insecticide-based methods represent the most effective means of blocking the transmission of vector borne diseases. However, insecticide resistance poses a serious threat and there is a need for tools, such as diagnostic tests for resistance detection, that will improve the sustainability of control interventions. The development of such tools for metabolism-based resistance in mosquito vectors lags behind those for target site resistance mutations. Methodology/Principal Findings We have developed and validated a simple colorimetric assay for the detection of Epsilon class Glutathione transferases (GST)-based DDT resistance in mosquito species, such as Aedes aegypti, the major vector of dengue and yellow fever worldwide. The colorimetric assay is based on the specific alkyl transferase activity of Epsilon GSTs for the haloalkene substrate iodoethane, which produces a dark blue colour highly correlated with AaGSTE2-2-overexpression in individual mosquitoes. The colour can be measured visually and spectrophotometrically. Conclusions/Significance The novel assay is substantially more sensitive compared to the gold standard CDNB assay and allows the discrimination of moderate resistance phenotypes. We anticipate that it will have direct application in routine vector monitoring as a resistance indicator and possibly an important impact on disease vector control. PMID:20824165

Crystal structure of the 28 kDa glutathione S-transferase from Schistosoma haematobium.

PubMed

Johnson, Kenneth A; Angelucci, Francesco; Bellelli, Andrea; Hervé, Maxime; Fontaine, Josette; Tsernoglou, Demetrious; Capron, André; Trottein, François; Brunori, Maurizio

2003-09-02

Schistomiasis is a debilitating parasitic disease which affects 200 million people, causing life-threatening complications in 10% of the patients. This paper reports the crystal structure of the Schistosoma haematobium 28 kDa glutathione S-transferase, a multifunctional enzyme involved in host-parasite interactions and presently considered as a promising vaccine candidate against schistosomiasis. The structures of the GSH-free enzyme, as well as the partially (approximately 40%) and almost fully (approximately 80%) GSH-saturated enzyme, exhibit a unique feature, absent in previous GST structures, concerning the crucial and invariant Tyr10 side chain which occupies two alternative positions. The canonical conformer, which allows an H-bond to be formed between the side chain hydroxyl group and the activated thiolate of GSH, is somewhat less than 50% occupied. The new conformer, with the phenoxyl ring on the opposite side of the mobile loop connecting strand 1 and helix 1, is stabilized by a polar interaction with the guanidinium group of the conserved Arg21 side chain. The presence of two conformers of Tyr10 may provide a clue about clarifying the multiple catalytic functions of Sh28GST and might prove to be relevant for the design of specific antischistosomal drugs. The K(d) for GSH binding was determined by equilibrium fluorescence titrations to be approximately 3 microM and by stopped-flow rapid mixing experiments to be approximately 9 microM. The relatively tight binding of GSH by Sh28GST explains the residually bound GSH in the crystal and supports a possible role of GSH as a tightly bound cofactor involved in the catalytic mechanism for prostaglandin D(2) synthase activity.

Identification and characterisation of multiple glutathione S-transferase genes from the diamondback moth, Plutella xylostella.

PubMed

Chen, Xi'en; Zhang, Ya-lin

2015-04-01

The diamondback moth (DBM), Plutella xylostella, is one of the most harmful insect pests on crucifer crops worldwide. In this study, 19 cDNAs encoding glutathione S-transferases (GSTs) were identified from the genomic and transcriptomic database for DBM (KONAGAbase) and further characterized. Phylogenetic analysis showed that the 19 GSTs were classified into six different cytosolic classes, including four in delta, six in epsilon, three in omega, two in sigma, one in theta and one in zeta. Two GSTs were unclassified. RT-PCR analysis revealed that most GST genes were expressed in all developmental stages, with higher expression in the larval stages. Six DBM GSTs were expressed at the highest levels in the midgut tissue. Twelve purified recombinant GSTs showed varied enzymatic properties towards 1-chloro-2,4-dinitrobenzene and glutathione, whereas rPxGSTo2, rPxGSTz1 and rPxGSTu2 had no activity. Real-time quantitative PCR revealed that expression levels of the 19 DBM GST genes were varied and changed after exposure to acephate, indoxacarb, beta-cypermethrin and spinosad. PxGSTd3 was significantly overexpressed, while PxGSTe3 and PxGSTs2 were significantly downregulated by all four insecticide exposures. The changes in DBM GST gene expression levels exposed to different insecticides indicate that they may play individual roles in tolerance to insecticides and xenobiotics. © 2014 Society of Chemical Industry.

An homolog of the Frz Phosphoenolpyruvate:carbohydrate phosphoTransferase System of extraintestinal pathogenic Escherichia coli is encoded on a genomic island in specific lineages of Streptococcus agalactiae.

PubMed

Patron, Kévin; Gilot, Philippe; Camiade, Emilie; Mereghetti, Laurent

2015-06-01

We identified a Streptococcus agalactiae metabolic region (fru2) coding for a Phosphoenolpyruvate:carbohydrate phosphoTransferase System (PTS) homologous to the Frz system of extraintestinal pathogenic Escherichia coli strains. The Frz system is involved in environmental sensing and regulation of the expression of adaptation and virulence genes in E. coli. The S. agalactiae fru2 region codes three subunits of a PTS transporter of the fructose-mannitol family, a transcriptional activator of PTSs of the MtlR family, an allulose-6 phosphate-3-epimerase, a transaldolase and a transketolase. We demonstrated that all these genes form an operon. The fru2 operon is present in a 17494-bp genomic island. We analyzed by multilocus sequence typing a population of 492 strains representative of the S. agalactiae population and we showed that the presence of the fru2 operon is linked to the phylogeny of S. agalactiae. The fru2 operon is always present within strains of clonal complexes CC 1, CC 7, CC 10, CC 283 and singletons ST 130 and ST 288, but never found in other CCs and STs. Our results indicate that the fru2 operon was acquired during the evolution of the S. agalactiae species from a common ancestor before the divergence of CC 1, CC 7, CC 10, CC 283, ST 130 and ST 288. As S. agalactiae strains of CC 1 and CC 10 are frequently isolated from adults with invasive disease, we hypothesize that the S. agalactiae Fru2 system senses the environment to allow the bacterium to adapt to new conditions encountered during the infection of adults. Copyright © 2015 Elsevier B.V. All rights reserved.

Cloning and expression of clostridium acetobutylicum ATCC 824 acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase in Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cary, J.W.; Petersen, D.J.; Bennett, G.N.

1990-06-01

Coenzyme A (CoA)-transferase (acetoacetyl-CoA:acetate/butyrate:CoA-transferase (butyrate-acetoacetate CoA-transferase) (EC 2.8.3.9)) of Clostridium acetobutylicum ATCC 824 is an important enzyme in the metabolic shift between the acid-producing and solvent-forming states of this organism. The genes encoding the two subunits of this enzyme have been cloned and subsequent subcloning experiments established the position of the structural genes for CoA-transferase. Complementation of Escherichia coli ato mutants with the recombinant plasmid pCoAT4 (pUC19 carrying a 1.8-kilobase insert of C. acetobutylicum DNA encoding CoA-transferase activity) enabled the transformants to grow on butyrate as a sole carbon source. Despite the ability of CoA-transferase to complement the ato defectmore » in E. coli mutants, Southern blot and Western blot (immunoblot) analyses showed showed that neither the C. acetobutylicum genes encoding CoA-transferase nor the enzyme itself shared any apparent homology with its E. coli counterpart. Polypeptides of M{sub r} of the purified CoA-transferase subunits were observed by Western blot and maxicell analysis of whole-cell extracts of E.coli harboring pCoAT4. The proximity and orientation of the genes suggest that the genes encoding the two subunits of CoA-transferase may form an operon similar to that found in E. coli. In the plasmid, however, transcription appears to be primarily from the lac promoter of the vector.« less

The Nrf2/SKN-1-dependent glutathione S-transferase π homologue GST-1 inhibits dopamine neuron degeneration in a Caenorhabditis elegans model of manganism.

PubMed

Settivari, Raja; VanDuyn, Natalia; LeVora, Jennifer; Nass, Richard

2013-09-01

Exposure to high levels of manganese (Mn) results in a neurological condition termed manganism, which is characterized by oxidative stress, abnormal dopamine (DA) signaling, and cell death. Epidemiological evidence suggests correlations with occupational exposure to Mn and the development of the movement disorder Parkinson's disease (PD), yet the molecular determinants common between the diseases are ill-defined. Glutathione S-transferases (GSTs) of the class pi (GSTπ) are phase II detoxification enzymes that conjugate both endogenous and exogenous compounds to glutathione to reduce cellular oxidative stress, and their decreased expression has recently been implicated in PD progression. In this study we demonstrate that a Caenorhabditis elegans GSTπ homologue, GST-1, inhibits Mn-induced DA neuron degeneration. We show that GST-1 is expressed in DA neurons, Mn induces GST-1 gene and protein expression, and GST-1-mediated neuroprotection is dependent on the PD-associated transcription factor Nrf2/SKN-1, as a reduction in SKN-1 gene expression results in a decrease in GST-1 protein expression and an increase in DA neuronal death. Furthermore, decreases in gene expression of the SKN-1 inhibitor WDR-23 or the GSTπ-binding cell death activator JNK/JNK-1 result in an increase in resistance to the metal. Finally, we show that the Mn-induced DA neuron degeneration is independent of the dopamine transporter DAT, but is largely dependent on the caspases CED-3 and the novel caspase CSP-1. This study identifies a C. elegans Nrf2/SKN-1-dependent GSTπ homologue, cell death effectors of GSTπ-associated xenobiotic-induced pathology, and provides the first in vivo evidence that a phase II detoxification enzyme may modulate DA neuron vulnerability in manganism. Copyright © 2013 Elsevier Inc. All rights reserved.

Identification of a novel gene cluster in the upstream region of the S-layer gene sbpA involved in cell wall metabolism of Lysinibacillus sphaericus CCM 2177 and characterization of the recombinantly produced autolysin and pyruvyl transferase.

PubMed

Pleschberger, Magdalena; Hildner, Florian; Rünzler, Dominik; Gelbmann, Nicola; Mayer, Harald F; Sleytr, Uwe B; Egelseer, Eva M

2013-05-01

The S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177 assembles into a square (p4) lattice structure and recognizes a pyruvylated secondary cell wall polymer (SCWP) as the proper anchoring structure to the rigid cell wall layer. Sequencing of 8,004 bp in the 5'-upstream region of the S-layer gene sbpA led to five ORFs-encoding proteins involved in cell wall metabolism. After cloning and heterologous expression of ORF1 and ORF5 in Escherichia coli, the recombinant autolysin rAbpA and the recombinant pyruvyl transferase rCsaB were isolated, purified, and correct folding was confirmed by circular dichroism. Although rAbpA encoded by ORF1 showed amidase activity, it could attack whole cells of Ly. sphaericus CCM 2177 only after complete extraction of the S-layer lattice. Despite the presence of three S-layer-homology motifs on the N-terminal part, rAbpA did not show detectable affinity to peptidoglycan-containing sacculi, nor to isolated SCWP. As the molecular mass of the autolysin lies above the molecular exclusion limit of the S-layer, AbpA is obviously trapped within the rigid cell wall layer by the isoporous protein lattice. Immunogold-labeling of ultrathin-sectioned whole cells of Ly. sphaericus CCM 2177 with a polyclonal rabbit antiserum raised against rCsaB encoded by ORF5, and cell fractionation experiments demonstrated that the pyruvyl transferase was located in the cytoplasm, but not associated with cell envelope components including the plasma membrane. In enzymatic assays, rCsaB clearly showed pyruvyl transferase activity. By using RT-PCR, specific transcripts for each ORF could be detected. Cotranscription could be confirmed for ORF2 and ORF3.

S-glutathionyl-(chloro)hydroquinone reductases: a new class of glutathione transferases functioning as oxidoreductases

PubMed Central

Belchik, Sara M.; Xun, Luying

2011-01-01

Glutathione transferases (GSTs) are best known for transferring glutathione (GSH) to hydrophobic organic compounds, making the conjugates more soluble. However, the omega-class GSTs of animals and the lambda-class GSTs and dehydroascorbate reductases (DHARs) of plants have little or no activity for GSH transfer. Instead, they catalyze GSH-dependent oxidoreductions. The lambda-class GSTs reduce disulfide bonds, the DHARs reduce the disulfide bonds and dehydroascorbate, and the omega-class GSTs can reduce more substrates, including disulfide bonds, dehydroascorbate, and dimethylarsinate. Glutathionyl-(chloro)hydroquinone reductases (GS-HQRs) are the newest class of GSTs that mainly catalyze oxidoreductions. Besides the activities of the other three classes, GS-HQRs also reduce GS-hydroquinones, including GS-trichloro-p-hydroquinone, GS-dichloro-p-hydroquinone, GS-2-hydroxy-p-hydroquinone, and GS-p-hydroquinone. They are conserved and widely distributed in bacteria, fungi, protozoa, and plants, but not in animals. The four classes are phylogenetically more related to each other than to other GSTs, and they share a Cys-Pro motif at the GSH-binding site. Hydroquinones are metabolic intermediates of certain aromatic compounds. They can be auto-oxidized by O2 to benzoquinones, which spontaneously react with GSH to form GS-hydroquinones via Michael’s addition. GS-HQRs are expected to channel GS-hydroquinones, formed spontaneously or enzymatically, back to hydroquinones. When the released hydroquinones are intermediates of metabolic pathways, GS-HQRs play a maintenance role for the pathways. Further, the common presence of GS-HQRs in plants, green algae, cyanobacteria, and halobacteria suggest a beneficial role in the light-using organisms. PMID:21425927

Structural and Biochemical Analyses Reveal the Mechanism of Glutathione S-Transferase Pi 1 Inhibition by the Anti-cancer Compound Piperlongumine*

PubMed Central

Harshbarger, Wayne; Gondi, Sudershan; Ficarro, Scott B.; Hunter, John; Udayakumar, Durga; Gurbani, Deepak; Singer, William D.; Liu, Yan; Li, Lianbo; Marto, Jarrod A.; Westover, Kenneth D.

2017-01-01

Glutathione S-transferase pi 1 (GSTP1) is frequently overexpressed in cancerous tumors and is a putative target of the plant compound piperlongumine (PL), which contains two reactive olefins and inhibits proliferation in cancer cells but not normal cells. PL exposure of cancer cells results in increased reactive oxygen species and decreased GSH. These data in tandem with other information led to the conclusion that PL inhibits GSTP1, which forms covalent bonds between GSH and various electrophilic compounds, through covalent adduct formation at the C7-C8 olefin of PL, whereas the C2-C3 olefin of PL was postulated to react with GSH. However, direct evidence for this mechanism has been lacking. To investigate, we solved the X-ray crystal structure of GSTP1 bound to PL and GSH at 1.1 Å resolution to rationalize previously reported structure activity relationship studies. Surprisingly, the structure showed that a hydrolysis product of PL (hPL) was conjugated to glutathione at the C7-C8 olefin, and this complex was bound to the active site of GSTP1; no covalent bond formation between hPL and GSTP1 was observed. Mass spectrometry (MS) analysis of the reactions between PL and GSTP1 confirmed that PL does not label GSTP1. Moreover, MS data also indicated that nucleophilic attack on PL at the C2-C3 olefin led to PL hydrolysis. Although hPL inhibits GSTP1 enzymatic activity in vitro, treatment of cells susceptible to PL with hPL did not have significant anti-proliferative effects, suggesting that hPL is not membrane-permeable. Altogether, our data suggest a model wherein PL is a prodrug whose intracellular hydrolysis initiates the formation of the hPL-GSH conjugate, which blocks the active site of and inhibits GSTP1 and thereby cancer cell proliferation. PMID:27872191

Radiosensitivity in HeLa cervical cancer cells overexpressing glutathione S-transferase π 1

PubMed Central

YANG, LIANG; LIU, REN; MA, HONG-BIN; YING, MING-ZHEN; WANG, YA-JIE

2015-01-01

The aims of the present study were to investigate the effect of overexpressed exogenous glutathione S-transferase π 1 (GSTP1) gene on the radiosensitivity of the HeLa human cervical cancer cell line and conduct a preliminarily investigation into the underlying mechanisms of the effect. The full-length sequence of human GSTP1 was obtained by performing a polymerase chain reaction (PCR) using primers based on the GenBank sequence of GSTP1. Subsequently, the gene was cloned into a recombinant eukaryotic expression plasmid, and the resulting construct was confirmed by restriction analysis and DNA sequencing. A HeLa cell line that was stably expressing high levels of GSTP1 was obtained through stable transfection of the constructed plasmids using lipofectamine and screening for G418 resistance, as demonstrated by reverse transcription-PCR. Using the transfected HeLa cells, a colony formation assay was conducted to detect the influence of GSTP1 overexpression on the cell radiosensitivity. Furthermore, flow cytometry was used to investigate the effect of GSTP1 overexpression on cell cycle progression, with the protein expression levels of the cell cycle regulating factor cyclin B1 detected using western blot analysis. Colony formation and G2/M phase arrest in the GSTP1-expressing cells were significantly increased compared with the control group (P<0.01). In addition, the expression of cyclin B1 was significantly reduced in the GSTP1-expressing cells. These results demonstrated that increased expression of GSTP1 inhibits radiosensitivity in HeLa cells. The mechanism underlying this effect may be associated with the ability of the GSTP1 protein to reduce cyclin B1 expression, resulting in significant G2/M phase arrest. PMID:26622693

Radiosensitivity in HeLa cervical cancer cells overexpressing glutathione S-transferase π 1.

PubMed

Yang, Liang; Liu, Ren; Ma, Hong-Bin; Ying, Ming-Zhen; Wang, Ya-Jie

2015-09-01

The aims of the present study were to investigate the effect of overexpressed exogenous glutathione S-transferase π 1 ( GSTP1 ) gene on the radiosensitivity of the HeLa human cervical cancer cell line and conduct a preliminarily investigation into the underlying mechanisms of the effect. The full-length sequence of human GSTP1 was obtained by performing a polymerase chain reaction (PCR) using primers based on the GenBank sequence of GSTP1. Subsequently, the gene was cloned into a recombinant eukaryotic expression plasmid, and the resulting construct was confirmed by restriction analysis and DNA sequencing. A HeLa cell line that was stably expressing high levels of GSTP1 was obtained through stable transfection of the constructed plasmids using lipofectamine and screening for G418 resistance, as demonstrated by reverse transcription-PCR. Using the transfected HeLa cells, a colony formation assay was conducted to detect the influence of GSTP1 overexpression on the cell radiosensitivity. Furthermore, flow cytometry was used to investigate the effect of GSTP1 overexpression on cell cycle progression, with the protein expression levels of the cell cycle regulating factor cyclin B1 detected using western blot analysis. Colony formation and G 2 /M phase arrest in the GSTP1 -expressing cells were significantly increased compared with the control group (P<0.01). In addition, the expression of cyclin B1 was significantly reduced in the GSTP1 -expressing cells. These results demonstrated that increased expression of GSTP1 inhibits radiosensitivity in HeLa cells. The mechanism underlying this effect may be associated with the ability of the GSTP1 protein to reduce cyclin B1 expression, resulting in significant G 2 /M phase arrest.

Glutathione S-transferase pi modulates NF-κB activation and pro-inflammatory responses in lung epithelial cells.

PubMed

Jones, Jane T; Qian, Xi; van der Velden, Jos L J; Chia, Shi Biao; McMillan, David H; Flemer, Stevenson; Hoffman, Sidra M; Lahue, Karolyn G; Schneider, Robert W; Nolin, James D; Anathy, Vikas; van der Vliet, Albert; Townsend, Danyelle M; Tew, Kenneth D; Janssen-Heininger, Yvonne M W

2016-08-01

Nuclear Factor kappa B (NF-κB) is a transcription factor family critical in the activation of pro- inflammatory responses. The NF-κB pathway is regulated by oxidant-induced post-translational modifications. Protein S-glutathionylation, or the conjugation of the antioxidant molecule, glutathione to reactive cysteines inhibits the activity of inhibitory kappa B kinase beta (IKKβ), among other NF-κB proteins. Glutathione S-transferase Pi (GSTP) is an enzyme that has been shown to catalyze protein S-glutathionylation (PSSG) under conditions of oxidative stress. The objective of the present study was to determine whether GSTP regulates NF-κB signaling, S-glutathionylation of IKK, and subsequent pro-inflammatory signaling. We demonstrated that, in unstimulated cells, GSTP associated with the inhibitor of NF-κB, IκBα. However, exposure to LPS resulted in a rapid loss of association between IκBα and GSTP, and instead led to a protracted association between IKKβ and GSTP. LPS exposure also led to increases in the S-glutathionylation of IKKβ. SiRNA-mediated knockdown of GSTP decreased IKKβ-SSG, and enhanced NF-κB nuclear translocation, transcriptional activity, and pro-inflammatory cytokine production in response to lipopolysaccharide (LPS). TLK117, an isotype-selective inhibitor of GSTP, also enhanced LPS-induced NF-κB transcriptional activity and pro-inflammatory cytokine production, suggesting that the catalytic activity of GSTP is important in repressing NF-κB activation. Expression of both wild-type and catalytically-inactive Y7F mutant GSTP significantly attenuated LPS- or IKKβ-induced production of GM-CSF. These studies indicate a complex role for GSTP in modulating NF-κB, which may involve S-glutathionylation of IKK proteins, and interaction with NF-κB family members. Our findings suggest that targeting GSTP is a potential avenue for regulating the activity of this prominent pro-inflammatory and immunomodulatory transcription factor. Copyright �

Glutathione S-transferase gene polymorphisms in celiac disease and their correlation with genomic instability phenotype.

PubMed

Fundia, Ariela F; Weich, Natalia; Crivelli, Adriana; La Motta, Graciela; Larripa, Irene B; Slavutsky, Irma

2014-06-01

Genomic instability and reduced glutathione S-transferase (GST) activity have been identified as potential risk factors for malignant complications in celiac disease (CD). In this study, we assessed the possible influence of GST polymorphisms on genome instability phenotypes in a genetically characterised group of celiac patients from previous studies. The deletion polymorphisms in GSTM1 and GSTT1 genes and the single-nucleotide polymorphism GSTP1 c.313A>G were genotyped using PCR in a set of 20 untreated adult patients with a known genomic instability phenotype and 69 age- and sex-matched healthy individuals. The frequencies of variant genotypes in patients were GSTM1-null (30%), GSTT1-null (5%), GSTP1-AG (60%) and GSTP1-GG (15%), and they showed no differences from controls. No significant differences were found in the genotype distribution based on telomere length. Cases with GSTM1-null genotype (83%) and microsatellite stability were more frequent than those with genomic instability. Moreover, carriers of GSTP1-variant genotype (73%) and stable phenotype were significantly increased compared to unstable patients (27%) (P=0.031). No differences were found according to the clinical-pathological characteristics of celiac cases. No association between GST polymorphic variants and celiac-associated genomic instability was proven in our cohort. Future studies should explore the usefulness of other biomarkers to distinguish celiac patients who are susceptible to cancer development. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Glutathione S-transferase iso-enzymes in perfusate from pumped kidneys are associated with delayed graft function

PubMed Central

Hall, Isaac E.; Bhangoo, Ronik S.; Reese, Peter P.; Doshi, Mona D.; Weng, Francis L.; Hong, Kwangik; Lin, Haiqun; Han, Gang; Hasz, Rick D.; Goldstein, Michael J.; Schröppel, Bernd; Parikh, Chirag R.

2014-01-01

Accurate and reliable assessment tools are needed in transplantation. The objective of this prospective, multicenter study was to determine the associations of the alpha and pi iso-enzymes of glutathione S-transferase (GST), measured from perfusate solution at the start and end (base and post) of kidney allograft machine perfusion, with subsequent delayed graft function (DGF). We also compared GST iso-enzyme perfusate levels from discarded versus transplanted kidneys. A total of 428 kidneys were linked to outcomes as recorded by the United Network of Organ Sharing. DGF, defined as any dialysis in the first week of transplant, occurred in 141 recipients (32%). Alpha and pi-GST levels significantly increased during machine perfusion. The adjusted relative risks (95% confidence interval) of DGF with each log-unit increase in base and post pi-GST were 1.14 (1.0-1.28) and 1.33 (1.02-1.72), respectively. Alpha-GST was not independently associated with DGF. There were no significant differences in GST values between discarded and transplanted kidneys, though renal resistance was significantly higher in discarded kidneys. We found pi-GST at the end of machine perfusion to be independently associated with DGF. Further studies should elucidate the utility of GST for identifying injured kidneys with regard to organ allocation, discard and recipient management decisions. PMID:24612768

Identification of an enhancer element of class Pi glutathione S-transferase gene required for expression by a co-planar polychlorinated biphenyl.

PubMed Central

Matsumoto, M; Imagawa, M; Aoki, Y

1999-01-01

3,3',4,4',5-Pentachlorobiphenyl (PenCB), one of the most toxic co-planar polychlorinated biphenyl congeners, specifically induces class Pi glutathione S-transferase (GSTP1) as well as cytochrome P-450 1A1 in primary cultured rat liver parenchymal cells [Aoki, Matsumoto and Suzuki (1993) FEBS Lett. 333, 114-118]. However, the 5'-flanking sequence of the GSTP1 gene does not contain a xenobiotic responsive element, to which arylhydrocarbon receptor binds. Using a chloramphenicol acetyltransferase assay we demonstrate here that the enhancer termed GSTP1 enhancer I (GPEI) is necessary for the stimulation by PenCB of GSTP1 gene expression in primary cultured rat liver parenchymal cells. GPEI is already known to contain a dyad of PMA responsive element-like elements oriented palindromically. It is suggested that a novel signal transduction pathway activated by PenCB contributes to the stimulation of GSTP1 expression. PMID:10051428

Functional complementation of anthocyanin sequestration in the vacuole by widely divergent glutathione S-transferases.

PubMed Central

Alfenito, M R; Souer, E; Goodman, C D; Buell, R; Mol, J; Koes, R; Walbot, V

1998-01-01

Glutathione S-transferases (GSTs) traditionally have been studied in plants and other organisms for their ability to detoxify chemically diverse herbicides and other toxic organic compounds. Anthocyanins are among the few endogenous substrates of plant GSTs that have been identified. The Bronze2 (Bz2) gene encodes a type III GST and performs the last genetically defined step of the maize anthocyanin pigment pathway. This step is the conjugation of glutathione to cyanidin 3-glucoside (C3G). Glutathionated C3G is transported to the vacuole via a tonoplast Mg-ATP-requiring glutathione pump (GS-X pump). Genetically, the comparable step in the petunia anthocyanin pathway is controlled by the Anthocyanin9 (An9) gene. An9 was cloned by transposon tagging and found to encode a type I plant GST. Bz2 and An9 have evolved independently from distinct types of GSTs, but each is regulated by the conserved transcriptional activators of the anthocyanin pathway. Here, a phylogenetic analysis is presented, with special consideration given to the origin of these genes and their relaxed substrate requirements. In particle bombardment tests, An9 and Bz2 functionally complement both mutants. Among several other GSTs tested, only soybean GmGST26A (previously called GmHsp26A and GH2/4) and maize GSTIII were found to confer vacuolar sequestration of anthocyanin. Previously, these genes had not been associated with the anthocyanin pathway. Requirements for An9 and Bz2 gene function were investigated by sequencing functional and nonfunctional germinal revertants of an9-T3529, bz2::Ds, and bz2::Mu1. PMID:9668133

Characterisation of Dermanyssus gallinae glutathione S-transferases and their potential as acaricide detoxification proteins.

PubMed

Bartley, Kathryn; Wright, Harry W; Bull, Robert S; Huntley, John F; Nisbet, Alasdair J

2015-06-26

Glutathione S-transferases (GSTs) facilitate detoxification of drugs by catalysing the conjugation of the reduced glutathione (GSH) to electrophilic xenobiotic substrates and therefore have a function in multi-drug resistance. As a result, knowledge of GSTs can inform both drug resistance in, and novel interventions for, the control of endo- and ectoparasite species. Acaricide resistance and the need for novel control methods are both pressing needs for Dermanyssus gallinae, a highly economically important haematophagous ectoparasite of poultry. A transcriptomic database representing D. gallinae was examined and 11 contig sequences were identified with GST BlastX identities. The transcripts represented by 3 contigs, designated Deg-GST-1, -2 and -3, were fully sequenced and further characterized by phylogenetic analysis. Recombinant versions of Deg-GST-1, -2 and -3 (rDeg-GST) were enzymically active and acaricide-binding properties of the rDeg-GSTs were established by evaluating the ability of selected acaricides to inhibit the enzymatic activity of rDeg-GSTs. 6 of the identified GSTs belonged to the mu class, followed by 3 kappa, 1 omega and 1 delta class molecules. Deg-GST-1 and -3 clearly partitioned with orthologous mu class GSTs and Deg-GST-2 partitioned with delta class GSTs. Phoxim, permethrin and abamectin significantly inhibited rDeg-GST-1 activity by 56, 35 and 17% respectively. Phoxim also inhibited rDeg-2-GST (14.8%) and rDeg-GST-3 (20.6%) activities. Deg-GSTs may have important roles in the detoxification of pesticides and, with the increased occurrence of acaricide resistance in this species worldwide, Deg-GSTs are attractive targets for novel interventions.

In silico genome-wide identification and characterization of the glutathione S-transferase gene family in Vigna radiata.

PubMed

Vaish, Swati; Awasthi, Praveen; Tiwari, Siddharth; Tiwari, Shailesh Kumar; Gupta, Divya; Basantani, Mahesh Kumar

2018-05-01

Plant glutathione S-transferases (GSTs) are integral to normal plant metabolism and biotic and abiotic stress tolerance. The GST gene family has been characterized in diverse plant species using molecular biology and bioinformatics approaches. In the current study, in silico analysis identified 44 GSTs in Vigna radiata. Of the total 44 GSTs identified, chromosomal locations of 31 GSTs were confirmed. The pI value of GST proteins ranged from 5.10 to 9.40. The predicted molecular weights ranged from 13.12 to 50 kDa. Subcellular localization analysis revealed that all GSTs were predominantly localized in the cytoplasm. The active site amino acids were confirmed to be serine in tau, phi, theta, zeta, and TCHQD; cysteine in lambda, DHAR, and omega; and tyrosine in EF1G. The gene architecture conformed to the two-exon/one-intron and three-exon/two-intron organization in the case of tau and phi classes, respectively. MEME analysis identified 10 significantly conserved motifs with the width of 8-50 amino acids. The motifs identified were either specific to a specific GST class or were shared by multiple GST classes. The results of the current study will be of potential importance in the characterization of the GST gene family in V. radiata, an economically important leguminous crop.

PABA/NO lead optimization: Improved targeting of cytotoxicity to glutathione S-transferase P1-overexpressing cancer cells.

PubMed

Kim, Youseung; Maciag, Anna E; Cao, Zhao; Deschamps, Jeffrey R; Saavedra, Joseph E; Keefer, Larry K; Holland, Ryan J

2015-08-01

PABA/NO [O(2)-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino) diazen-1-ium-1,2-diolate] is a nitric oxide (NO)-releasing arylating agent designed to be selectively activated by reaction with glutathione (GSH) on catalysis by glutathione S-transferase P1 (GSTP1), an enzyme frequently overexpressed in cancer cells. PABA/NO has proven active in several cancer models in vitro and in vivo, but its tendency to be metabolized via a variety of pathways, some that generate inactive metabolites and hydrolysis products, limits its potential as a drug. Here we show that a simple replacement of cyano for nitro at the 4 position to give compound 4b ('p-cyano-PABA/NO') has the dual effect of slowing the undesired side reactions while enhancing the proportion of NO release and arylating activity on catalysis by GSTP1. Compound 4b showed increased resistance to hydrolysis and uncatalyzed reaction with GSH, along with a more favorable product distribution in the presence of GSTP1. It also showed significant proapoptotic activity. The data suggest p-cyano-PABA/NO to be a more promising prodrug than PABA/NO, with better selectivity toward cancer cells. Published by Elsevier Ltd.

Glutathione-S-transferase-oxidative stress relationship in the internal spermatic vein blood of infertile men with varicocele.

PubMed

Mostafa, T; Rashed, L A; Zeidan, A S; Hosni, A

2015-02-01

This study aimed to assess glutathione-S-transferase (GST) enzyme- oxidative stress (OS) relationship in the internal spermatic vein (ISV) of infertile men associated with varicocele (Vx). Ninety five infertile oligoasthenoteratozoospemic (OAT) men associated with Vx were subjected to history taking, clinical examination and semen analysis. During inguinal varicocelectomy, GST, malondialdehyde (MDA) and glutathione peroxidase (GPx) were estimated in the blood samples drawn from ISV and median cubital veins. The mean levels of GST, GPx were significantly decreased and the mean level of GPx was significantly increased in the ISV compared with the peripheral blood. The mean level of GST and GPx in the ISV was significantly decreased, and the mean level of MDA was significantly increased in Vx grade III compared with Vx grade II cases. There was nonsignificant difference in the mean level of GST in the ISV in unilateral Vx cases compared with bilateral Vx cases. There was significant positive correlation of GST with sperm count, sperm motility, GPx and significant negative correlation with sperm abnormal forms, MDA. It is concluded that ISV of infertile men associated with Vx has decreased levels of GST compared with peripheral venous circulation that is correlated with both OS and Vx grade. © 2014 Blackwell Verlag GmbH.

Evidence acquisition and evaluation for a U.S. Government Evidence Summit on Protecting Children Outside Family Care.

PubMed

Higgs, Elizabeth S; Zlidar, Vera M; Balster, Robert L

2012-10-01

Recognizing the need for evidence to inform policies, strategies, and programs to care for vulnerable children, the U.S. Government convened an Evidence Summit on Protecting Children Outside of Family Care on December 12-13, 2011, in Washington, DC, USA. This paper summarizes the background and methods for the acquisition and evaluation of the evidence used to achieve the goals of the Summit. A multistep process was undertaken to identify the appropriate evidence for review. It began by identifying crucial focal questions intended to inform low and middle income governments and the U.S. Government about effective systems for protecting children outside family care. This was followed by a systematic attempt to gather relevant peer reviewed and gray literature that would inform these focal questions. The search processes, methods used for screening and quality reviews are described. In addition, members of the Evidence Review Teams were invited to add relevant papers not identified in the initial literature review to complete the bibliographies. These teams were asked to comply with a specific evaluation framework for recommendations on practice and policy based on both expert opinion and the quality of the data. This was the first U.S. Government Evidence Summit originating in the U.S. Agency for International Development Global Health Bureau and valuable lessons were learned on the identification and assessment of evidence informing complex development challenges. Published by Elsevier Ltd.

Relation between glutathione S-transferase genes (GSTM1, GSTT1, and GSTP1) polymorphisms and clinical manifestations of sickle cell disease in Egyptian patients.

PubMed

Ellithy, Hend N; Yousri, Sherif; Shahin, Gehan H

2015-12-01

Clinical manifestations of sickle cell disease (SCD) result from sickling of Hb S due to oxidation, which is augmented by accumulation of oxygen-free radicals. Deficiencies in normal antioxidant protective mechanism might lead to clinical manifestations of SCD like vaso-occlusive crisis (VOC) and acute chest syndrome (ACS). The glutathione system plays an important role in the removal of endogenous products of peroxidation of lipids, thus protecting cells and tissue against damage from oxidative stress. Impairment of the glutathione system due to genetic polymorphisms of glutathione S-transferase (GST) genes is expected to increase the severity of SCD manifestations. This report describes a case control study aimed at studying the ethnic-dependent variation in the frequency of GST gene polymorphisms among participants selected from the Egyptian population and to find out the association between GST gene polymorphisms and the severity of SCD manifestations. We measured the frequency distribution of the three GSTs gene polymorphisms in 100 Egyptian adult SCD patients and 80 corresponding controls. GSTM1 and GSTT1 genotypes were determined by multiplex polymerase chain reaction (PCR). GSTP1 genotyping was conducted with a PCR-restriction fragment length polymorphism assay. The GSTM1 null genotype was significantly associated with ACS and VOC (P = 0.03 and 0.01, respectively). The GSTT1 null genotype was associated with significantly increased requirement of blood transfusion (P = 0.01). Absence of both GSTM1 and GSTT1 genes was significantly associated with pulmonary hypertension (P = 0.04). The non-wild-type GSTP1 polymorphism was not associated with clinical manifestations of SCD. Some GST gene polymorphisms were significantly associated with the worsening of the clinical manifestations of SCD.

Electrostatic association of glutathione transferase to the nuclear membrane. Evidence of an enzyme defense barrier at the nuclear envelope.

PubMed

Stella, Lorenzo; Pallottini, Valentina; Moreno, Sandra; Leoni, Silvia; De Maria, Francesca; Turella, Paola; Federici, Giorgio; Fabrini, Raffaele; Dawood, Kutayba F; Bello, Mario Lo; Pedersen, Jens Z; Ricci, Giorgio

2007-03-02

The possible nuclear compartmentalization of glutathione S-transferase (GST) isoenzymes has been the subject of contradictory reports. The discovery that the dinitrosyl-diglutathionyl-iron complex binds tightly to Alpha class GSTs in rat hepatocytes and that a significant part of the bound complex is also associated with the nuclear fraction (Pedersen, J. Z., De Maria, F., Turella, P., Federici, G., Mattei, M., Fabrini, R., Dawood, K. F., Massimi, M., Caccuri, A. M., and Ricci, G. (2007) J. Biol. Chem. 282, 6364-6371) prompted us to reconsider the nuclear localization of GSTs in these cells. Surprisingly, we found that a considerable amount of GSTs corresponding to 10% of the cytosolic pool is electrostatically associated with the outer nuclear membrane, and a similar quantity is compartmentalized inside the nucleus. Mainly Alpha class GSTs, in particular GSTA1-1, GSTA2-2, and GSTA3-3, are involved in this double modality of interaction. Confocal microscopy, immunofluorescence experiments, and molecular modeling have been used to detail the electrostatic association in hepatocytes and liposomes. A quantitative analysis of the membrane-bound Alpha GSTs suggests the existence of a multilayer assembly of these enzymes at the outer nuclear envelope that could represent an amazing novelty in cell physiology. The interception of potentially noxious compounds to prevent DNA damage could be the possible physiological role of the perinuclear and intranuclear localization of Alpha GSTs.

Evaluation of the in vitro inhibitory impact of hypericin on placental glutathione S-transferase pi.

PubMed

Dalmizrak, Ozlem; Kulaksiz-Erkmen, Gulnihal; Ozer, Nazmi

2012-10-01

St John's Wort (SJW) extracts are herbal products which are available without prescription in most countries and widely used in the treatment of mild to moderate depression. Since it is a herbal product and available without prescription, use of SJW is common among pregnant and/or lactating woman. The principal of the study was to clarify the effects of hypericin, one of the components of SJW, on glutathione S-transferase-pi (GST-pi) purified from human placenta. The K (m) values of GST-pi were 0.21 ± 0.03 mM for glutathione (GSH) and 2.29 ± 0.54 mM for 1-chloro-2,4-dinitrobenzene (CDNB). At fixed [GSH], the V (m) value calculated was about 3 times higher than the conditions in which [CDNB] was fixed; 201 ± 30 U/mg protein versus 74 ± 3 U/mg protein. At constant substrate concentrations (1 mM), an average IC (50) value of 0.70 ± 0.02 μM was obtained. Hypericin inhibited GST-pi competitively with respect to both substrates. When GSH was the varied substrate a K (i) value of 0.31 ± 0.05 μM was found; when CDNB was the varied substrate, a K (i) value of 0.85 ± 0.02 μM was obtained. On the basis of these data considering transplacental transfer of hypericin and immature hepatic clearance of the baby, using this herbal product may cause abnormalites due to the inhibition of one of the most important placental detoxification enzymes, GST-pi.

Alterations in glutathione S-transferase pi expression following exposure to MPP+ -induced oxidative stress in the blood of Parkinson's disease patients.

PubMed

Korff, Ane; Pfeiffer, Brenda; Smeyne, Michelle; Kocak, Mehmet; Pfeiffer, Ronald F; Smeyne, Richard Jay

2011-12-01

The major motor symptoms of Parkinson's disease do not occur until a majority of the dopaminergic neurons in the midbrain SNpc have already died. For this reason, it is critical to identify biomarkers that will allow for the identification of presymptomatic individuals. In this study, we examine the baseline expression of the anti-oxidant protein glutathione S-transferase pi (GSTpi) in the blood of PD patients and environmentally- and age-matched controls and compare it to GSTpi levels following exposure to 1-methyl-4-phenylpyridinium (MPP(+)), an agent that has been shown to induce oxidative stress. We find that after 4 h of exposure to MPP(+), significant increases in GSTpi levels can be observed in the leukocytes of PD patients. No changes were seen in other blood components. This suggests that GSTpi and potentially other members of this and other anti-oxidant families may be viable biomarkers for PD. Copyright © 2011 Elsevier Ltd. All rights reserved.

Variable Levels of Glutathione S-Transferases Are Responsible for the Differential Tolerance to Metolachlor between Maize (Zea mays) Shoots and Roots.

PubMed

Li, Dongzhi; Xu, Li; Pang, Sen; Liu, Zhiqian; Wang, Kai; Wang, Chengju

2017-01-11

Glutathione S-transferases (GSTs) play important roles in herbicide tolerance. However, studies on GST function in herbicide tolerance among plant tissues are still lacking. To explore the mechanism of metolachlor tolerance difference between maize shoots and roots, the effects of metolachlor on growth, GST activity, and the expression of the entire GST gene family were investigated. It was found that this differential tolerance to metolachlor was correlated with contrasting GST activity between the two tissues and can be eliminated by a GST inhibitor. An in vitro metolachlor-glutathione conjugation assay confirmed that the transformation of metolachlor is 2-fold faster in roots than in shoots. The expression analysis of the GST gene family revealed that most GST genes are expressed much higher in roots than shoots, both in control and in metolachlor-treated plants. Taken together, higher level expression of most GST genes, leading to higher GST activity and faster herbicide transformation, appears to be responsible for the higher tolerance to metolachlor of maize roots than shoots.

Transformed yeast (Schizosaccharomyces pombe) overexpressing rice Tau class glutathione S-transferase (OsGSTU30 and OsGSTU41) shows enhanced resistance to hexavalent chromium.

PubMed

Tripathi, Ankita; Indoliya, Yuvraj; Tiwari, Madhu; Tiwari, Poonam; Srivastava, Dipali; Verma, Pankaj kumar; Verma, Shikha; Gautam, Neelam; Chakrabarty, Debasis

2014-08-01

Extensive use of hexavalent chromium [Cr(VI)] in leather tanning, stainless-steel production, wood preservatives and electroplating industries has resulted in widespread environmental pollution and poses a serious threat to human health. A plant's response to Cr(VI) stress results in growth inhibition and toxicity leading to changes in components of antioxidant systems. In a previous study, we observed that a large number of glutathione S-transferase (GST) genes were up-regulated under Cr(VI) stress in rice. In this study, two rice root-specific Tau class GST genes (OsGSTU30 and OsGSTU41) were introduced into yeast (Schizosaccharomyces pombe). Transformed yeast cells overexpressing OsGSTU30 and OsGSTU41 had normal growth, but had much higher levels of GST activities and showed enhanced resistance to Cr(VI) as compared to control cells (transformed with empty vector). Also, a higher accumulation of chromium was found in the transformed yeast cells as compared to the control cells. Manipulation of glutathione biosynthesis by exogenous application of buthionine sulfoximine abolishes the protective effect of OsGSTs against Cr(VI) stress. These results suggest that Tau class OsGSTs play a significant role in detoxification of Cr(VI), probably by chelating and sequestrating glutathione-Cr(VI) complexes into vacuoles.

Madumycin II inhibits peptide bond formation by forcing the peptidyl transferase center into an inactive state

DOE Office of Scientific and Technical Information (OSTI.GOV)

Osterman, Ilya A.; Khabibullina, Nelli F.; Komarova, Ekaterina S.

The emergence of multi-drug resistant bacteria is limiting the effectiveness of commonly used antibiotics, which spurs a renewed interest in revisiting older and poorly studied drugs. Streptogramins A is a class of protein synthesis inhibitors that target the peptidyl transferase center (PTC) on the large subunit of the ribosome. In this work, we have revealed the mode of action of the PTC inhibitor madumycin II, an alanine-containing streptogramin A antibiotic, in the context of a functional 70S ribosome containing tRNA substrates. Madumycin II inhibits the ribosome prior to the first cycle of peptide bond formation. It allows binding of themore » tRNAs to the ribosomal A and P sites, but prevents correct positioning of their CCA-ends into the PTC thus making peptide bond formation impossible. We also revealed a previously unseen drug-induced rearrangement of nucleotides U2506 and U2585 of the 23S rRNA resulting in the formation of the U2506•G2583 wobble pair that was attributed to a catalytically inactive state of the PTC. The structural and biochemical data reported here expand our knowledge on the fundamental mechanisms by which peptidyl transferase inhibitors modulate the catalytic activity of the ribosome.« less

Differences in glutathione S-transferase pi expression in transgenic mice with symptoms of neurodegeneration.

PubMed

Kaźmierczak, Beata; Kuźma-Kozakiewicz, Magdalena; Usarek, Ewa; Barańczyk-Kuźma, Anna

2011-01-01

Glutathione S-transferase pi (GST pi) is an enzyme involved in cell protection against toxic electrophiles and products of oxidative stress. GST pi expression was studied in transgenic mice hybrids (B6-C3H) with symptoms of neurodegeneration harboring SOD1G93A (SOD1/+), Dync1h1 (Cra1/+) and double (Cra1/SOD1) mutations, at presymptomatic and symptomatic stages (age 70, 140, 365 days) using RT-PCR and Western blotting. The main changes in GST pi expression were observed in mice with the SODG93A mutation. In SOD1/+ and Cra1/SOD1 transgenics, with the exception of cerebellum, the changes in GST pi-mRNA accompanied those in GST pi protein. In brain cortex of both groups the expression was unchanged at the presymptomatic (age 70 days) but was lower at the symptomatic stage (age 140 days) and at both stages in hippocampus and spinal cord of SOD1/+ but not of Cra1/SOD1 mice compared to age-matched wild-type controls. In cerebellum of the presymptomatic and the symptomatic SOD1/+ mice and presymptomatic Cra1/SOD1 mice, the GST pi-mRNA was drastically elevated but the protein level remained unchanged. In Cra1/+ transgenics there were no changes in GST pi expression in any CNS region both on the mRNA and on the protein level. It can be concluded that the SOD1G93A but not the Dync1h1 mutation significantly decreases detoxification efficiency of GST pi in CNS, however the Dync1h1 mutation reduces the effects caused by the SOD1G93A mutation. Despite similarities in neurological symptoms, the differences in GST pi expression between SOD1/+ and Cra1/+ transgenics indicate a distinct pathogenic entity of these two conditions.

Atypical localization of the oligodendrocytic isoform (PI) of glutathione-S-transferase in astrocytes during cuprizone intoxication.

PubMed

Cammer, W; Zhang, H

1993-10-01

Immunocytochemical staining for the Pi and Mu isoforms of glutathione-S-transferase was used to investigate changes in the glial cells in the mouse forebrain. During early development in mouse forebrains the localizations of carbonic anhydrase, Pi and Mu were similar to the respective cellular localizations that had been observed in neonatal rat brain. That is, Pi was found in oligodendrocyte precursors, Mu in astrocytes, and carbonic anhydrase in both oligodendrocyte precursors and astrocytes. In forebrains of 6-week-old mice the neurotoxicant, cuprizone, induced oligodendrocyte degeneration, gliosis, and partial demyelination. Degeneration, gliosis, and partial demyelination. Degeneration of oligodendrocytes, and astrocytosis, began during the initial week of cuprizone feeding, and by the end of the eighth week some demyelination was observed. After mice were fed cuprizone for 4 to 7 weeks, Pi appeared in some of the reactive astrocytes, and Pi-positive astrocytes were present for at least 7 additional weeks. Normally, Pi appeared only in oligodendrocytes. Very few Pi-positive oligodendrocytes remained after the second week. During the eighth week healthy-looking carbonic anhydrase-positive oligodendrocytes reappeared and began to accumulate, and a few small patches of Pi-positive oligodendrocytes were also observed. In summary, some novel findings about glial cells were the observation of an enzyme (Pi) that is lost earlier from oligodendrocytes than is carbonic anhydrase, the apparently unique shift in Pi expression from oligodendrocytes to astrocytes and the greater temporal dissociation between loss of oligodendrocytes and demyelination in the older mice.

Glutathione S-transferase iso-enzymes in perfusate from pumped kidneys are associated with delayed graft function.

PubMed

Hall, I E; Bhangoo, R S; Reese, P P; Doshi, M D; Weng, F L; Hong, K; Lin, H; Han, G; Hasz, R D; Goldstein, M J; Schröppel, B; Parikh, C R

2014-04-01

Accurate and reliable assessment tools are needed in transplantation. The objective of this prospective, multi-center study was to determine the associations of the alpha and pi iso-enzymes of glutathione S-transferase (GST), measured from perfusate solution at the start and end (base and post) of kidney allograft machine perfusion, with subsequent delayed graft function (DGF). We also compared GST iso-enzyme perfusate levels from discarded versus transplanted kidneys. A total of 428 kidneys were linked to outcomes as recorded by the United Network of Organ Sharing. DGF, defined as any dialysis in the first week of transplant, occurred in 141 recipients (32%). Alpha- and pi-GST levels significantly increased during machine perfusion. The adjusted relative risks (95% confidence interval) of DGF with each log-unit increase in base and post pi-GST were 1.14 (1.0-1.3) and 1.36 (1.1-1.8), respectively. Alpha-GST was not independently associated with DGF. There were no significant differences in GST values between discarded and transplanted kidneys, though renal resistance was significantly higher in discarded kidneys. We found pi-GST at the end of machine perfusion to be independently associated with DGF. Further studies should elucidate the utility of GST for identifying injured kidneys with regard to organ allocation, discard and recipient management decisions. © Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.

Effects of imidacloprid on detoxifying enzyme glutathione S-transferase on Folsomia candida (Collembola).

PubMed

Sillapawattana, Panwad; Schäffer, Andreas

2017-04-01

Chemical analyses of the environment can document contamination by various xenobiotics, but it is also important to understand the effect of pollutants on living organisms. Thus, in the present work, we investigated the effect of the pesticide imidacloprid on the detoxifying enzyme glutathione S-transferase (GST) from Folsomia candida (Collembola), a standard test organism for estimating the effects of pesticides and environmental pollutants on non-target soil arthropods. Test animals were treated with different concentrations of imidacloprid for 48 h. Changes in steady-state levels of GST messenger RNA (mRNA) and GST enzyme activity were investigated. Extracted proteins were separated according to their sizes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved protein bands were detected by silver staining. The size of the glutathione (GSH) pool in Collembola was also determined. A predicted protein sequence of putative GSTs was identified with animals from control group. A 3-fold up-regulation of GST steady-state mRNA levels was detected in the samples treated with 5.0 mg L -1 imidacloprid compared to the control, while a 2.5- and 2.0- fold up-regulation was found in organisms treated with 2.5 and 7.5 mg L -1 imidacloprid, respectively. GST activity increased with increasing imidacloprid amounts from an initial activity of 0.11 μmol min -1  mg -1 protein in the control group up to 0.25 μmol min -1  mg -1 protein in the sample treated with the 5.0 mg L -1 of pesticide. By contrast, the total amount of GSH decreased with increasing imidacloprid concentration. The results suggest that the alteration of GST activity, steady-state level of GST mRNA, and GSH level may be involved in the response of F. candida to the exposure of imidacloprid and can be used as biomarkers to monitor the toxic effects of imidacloprid and other environmental pollutants on Collembola.

Glutathione-S-transferase polymorphisms (GSTM1, GSTT1 and GSTP1) and acute leukemia risk in Asians: a meta-analysis.

PubMed

Tang, Zhen-Hai; Zhang, Chi; Cheng, Pan; Sun, Hong-Min; Jin, Yu; Chen, Yuan-Jing; Huang, Fen

2014-01-01

The association between glutathione-S-transferase polymorphisms (GSTM1, GSTT1 and GSTP1) and risk of acute leukemia in Asians remains controversial. This study was therefore designed to evaluate the precise association in 23 studies identified by a search of PubMed and several other databases, up to December 2013. Using random or fixed effects models odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were calculated. Heterogeneity across studies was assessed, and funnel plots were constructed to test for publication bias. The meta-analysis showed positive associations between GST polymorphisms (GSTM1 and GSTT1 but not GSTP1) and acute leukemia risk [(OR=1.47, 95% CI 1.18-1.83); (OR=1.32, 95% CI 1.07-1.62); (OR=1.01, 95% CI 0.84-1.23), respectively] and heterogeneity between the studies. The results suggested that the GSTM1 null genotype and GSTT1null genotype, but not the GSTP1 polymorphism, might be a potential risk factors for acute leukemia. Further well-designed studies are needed to confirm our findings.

Glutathione S-transferase gene polymorphisms (GSTM1, GSTT1, and GSTP1) in Egyptian pediatric patients with sickle cell disease.

PubMed

Shiba, Hala Fathy; El-Ghamrawy, Mona Kamal; Shaheen, Iman Abd El-Mohsen; Ali, Rasha Abd El-Ghani; Mousa, Somaia Mohammed

2014-01-01

Sickle cell disease (SCD) complications are associated with oxidative stress. Glutathione S-transferases (GSTs) are a group of enzymes that protect against oxidative stress. The aims of this study was to evaluate the prevalence of GSTM1, GSTT1, and GSTP1 gene polymorphisms among homozygous sickle cell anemia patients and to investigate the possible association between the presence of these polymorphisms and SCD severity and complications. Genotyping the polymorphisms in GSTT1 and GSTM1 genes was performed using the multiplex polymerase chain reaction (PCR) method. The GSTP1 ILe105Val polymorphism was determined using PCR-restriction fragment length polymorphism. GSTM1 null genotype was significantly associated with increased risk of severe vaso-occlusive crises (VOC) (odds ratio  =  1.52, 95% confidence interval  =  0.42-5.56, P  =  0.005). We found no significant association between GST genotypes and frequency of sickle cell-related pain, transfusion frequency, disease severity, or hydroxyurea treatment. GSTM1 gene polymorphism may be associated with risk of severe VOC among Egyptian SCD patients.

The Importance of Sulfate Adenylyl Transferase in S and O Fractionation by Sulfate Reducing Bacteria

NASA Astrophysics Data System (ADS)

Smith, D. A.; Johnston, D. T.; Bradley, A. S.

2016-12-01

Microbial sulfate reduction (MSR) is critical to the oxidation of organic matter in modern and ancient oceans, and plays an important role in regulating the redox state of the Earth's surface. The sulfur and oxygen isotopic composition of seawater sulfate and of sulfate minerals reflect the biogeochemical processes that cycle sulfur, of which MSR is among the most important. MSR is a multi-enzymatic reaction network that partitions the isotopes of sulfur and oxygen as a consequence of both the flux of sulfate through this biochemical network and the fractionation imposed by each individual enzyme. MSR affects the δ18O of residual, extracellular sulfate mainly by the equilibration of the MSR intermediate sulfite with extracellular water (Antler et al., 2013 GCA, Wankel et al., 2013 Geobiol). A series of oxidative and exchange reactions catalyzed by APS reductase (APSr), sulfate adenylyl transferase (Sat), and sulfate transporters promote the conversion of water-equilibrated intracellular sulfite to extracellular sulfate. The flux of sulfoxy anions via these proteins will be, at least in part, dependent on the activity of these enzymes. To test this, we examined sulfur and oxygen isotope fractionation in genetically engineered mutants of the sulfate reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH). In these mutants, the activity of Sat has been artificially increased by perturbing the (i) transcriptional repressor Rex and (ii) its binding site upstream of the gene encoding Sat (Christensen et al., 2015 J. Bacteriol). It was predicted that this would minimize the back reaction of Sat, enhance the intracellular pool of APS, and minimize the equilibration between sulfite and adenosine monophosphate (AMP). Both mutants, along with the wild type DvH were grown in batch culture made with water enriched in 18O. Samples were collected throughout batch growth, and we report the evolution of the S and O isotopic composition of sulfate, and of the S isotopic

Glutathione S-transferase M1 (GSTM1) null genotype and coronary artery disease risk: a meta-analysis.

PubMed

Zhang, Zhen-Xian; Zhang, Ye

2014-01-01

The Glutathione S-Transferase M1 (GSTM1) null genotype has been indicated to be correlated with coronary artery disease (CAD) susceptibility, but study results are still debatable. Thus, a meta-analysis was conducted. Databases including PubMed, Embase, Web of Science, and Chinese National Knowledge Infrastructure (CNKI) were searched. Data were extracted and pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated. Twenty-six studies with 10595 cases and 13782 controls were included in this meta-analysis. The association between GSTM1 null genotype and CAD risk was significant (OR = 1.35; 95% CI, 1.09 - 1.67; P < 0.01). When stratified by ethnicity, the significantly elevated risk were observed in Caucasians (OR = 1.39; 95% CI, 1.07 - 1.81; P = 0.01) but not in Asians (OR = 1.27; 95% CI, 0.87 - 1.86; P = 0.22). No significantly increased myocardial infarction risk was observed (OR = 0.96; 95% CI, 0.78 - 1.18; P = 0.68). Subgroup analysis on the smoking status showed that the increased risk was found in smokers (OR = 1.66; 95% CI, 1.14 - 2.42; P < 0.01) but not in non-smokers (OR = 1.30; 95% CI, 1.74 - 2.28; P = 0.37). In conclusion, this meta-analysis suggested that GSTM1 null genotype was a risk factor for CAD, especially in Caucasians and smokers.

Lack of association between the glutathione-s-transferase genes (GSTT1 and GSTM1) and nasal polyposis.

PubMed

Arbag, Hamdi; Cora, Tulin; Acar, Hasan; Ozturk, Kayhan; Sari, Fatih; Ulusoy, Bulent

2006-03-01

To evaluate the glutation-S-transferase (GST) polymorphisms (GSTM1 and GSTT1) in nasal polyposis (NP). The study population consisted of 102 unrelated healthy individuals and 98 patients with NP (67 without asthma, 31 with asthma). Genotyping of the polymorphism in the GSTM1 and GSTT1 genes was performed using the multiplex polymerase chain reaction (PCR)-based method. GSTM1 and GSTT1 null-genotypes were found in 46.1% and 23.5% of the controls, and in 43.9% and 33.7% of the NP patients, respectively. These differences were not significant (for GSTM1 null odds ratio (OR) = 0.92; 95% confidence interval (CI) = 0.52-1.6 and for GSTT1, OR = 1.65; 95% CI = 0.89-3.07). Although no significant difference for combined GSTM1 and GSTT1 null genotypes between control (8.8%) and NP patients (17.3%) was found, there was a 2.16-fold increased proportion in the NP with the combined GSTM1-null and GSTT1-null genotype (OR = 2.16; 95% CI = 0.91-5.13). These results suggest that there is lack of association between GSTM1 and GSTT1 polymorphisms and NP. The GSTM1 or GSTT1 polymorphisms had also no relevant developing effect on NP patients without or with asthma.

Thio-Linked UDP–Peptide Conjugates as O-GlcNAc Transferase Inhibitors

PubMed Central

2018-01-01

O-GlcNAc transferase (OGT) is an essential glycosyltransferase that installs the O-GlcNAc post-translational modification on the nucleocytoplasmic proteome. We report the development of S-linked UDP–peptide conjugates as potent bisubstrate OGT inhibitors. These compounds were assembled in a modular fashion by photoinitiated thiol–ene conjugation of allyl-UDP and optimal acceptor peptides in which the acceptor serine was replaced with cysteine. The conjugate VTPVC(S-propyl-UDP)TA (Ki = 1.3 μM) inhibits the OGT activity in HeLa cell lysates. Linear fusions of this conjugate with cell penetrating peptides were explored as prototypes of cell-penetrant OGT inhibitors. A crystal structure of human OGT with the inhibitor revealed mimicry of the interactions seen in the pseudo-Michaelis complex. Furthermore, a fluorophore-tagged derivative of the inhibitor works as a high affinity probe in a fluorescence polarimetry hOGT assay. PMID:29723473

The effects of endosulfan on cytochrome P450 enzymes and glutathione S-transferases in zebrafish (Danio rerio) livers.

PubMed

Dong, Miao; Zhu, Lusheng; Shao, Bo; Zhu, Shaoyuan; Wang, Jun; Xie, Hui; Wang, Jinhua; Wang, Fenghua

2013-06-01

Endosulfan, an organochlorine pesticide, has been used worldwide in the past decades. The present study was performed to investigate the effect of endosulfan on liver microsomal cytochrome P450 (CYP) enzymes and glutathione S-transferases (GST) in zebrafish. Male and female zebrafish were separated and exposed to a control and four concentrations of endosulfan (0.01, 0.1, 1, and 10μgL(-1)) and were sampled on days 7, 14, 21, and 28. After exposure to endosulfan, the content of CYP increased and later gradually fell back to control level in most sampling time intervals. A similar tendency was also found in the activities of NADPH-P450 reductase (NCR), aminopyrine N-demethylase (APND) and erythromycin N-demethylase (ERND). GST activities were generally higher in treatment groups than control groups. Regarding sex-based differences, the induction degree of the activity of NCR was generally higher in males than females. Similar differences were also found on the 28th day in the activities of APND and ERND, as well as GST activity on the 7th day. Overall, the present results demonstrate the toxicity at low doses of endosulfan and indicated marked induction of CYP and GST enzymes in zebrafish liver. Copyright © 2012 Elsevier Inc. All rights reserved.

Structural and Biochemical Analyses Reveal the Mechanism of Glutathione S-Transferase Pi 1 Inhibition by the Anti-cancer Compound Piperlongumine.

PubMed

Harshbarger, Wayne; Gondi, Sudershan; Ficarro, Scott B; Hunter, John; Udayakumar, Durga; Gurbani, Deepak; Singer, William D; Liu, Yan; Li, Lianbo; Marto, Jarrod A; Westover, Kenneth D

2017-01-06

Glutathione S-transferase pi 1 (GSTP1) is frequently overexpressed in cancerous tumors and is a putative target of the plant compound piperlongumine (PL), which contains two reactive olefins and inhibits proliferation in cancer cells but not normal cells. PL exposure of cancer cells results in increased reactive oxygen species and decreased GSH. These data in tandem with other information led to the conclusion that PL inhibits GSTP1, which forms covalent bonds between GSH and various electrophilic compounds, through covalent adduct formation at the C7-C8 olefin of PL, whereas the C2-C3 olefin of PL was postulated to react with GSH. However, direct evidence for this mechanism has been lacking. To investigate, we solved the X-ray crystal structure of GSTP1 bound to PL and GSH at 1.1 Å resolution to rationalize previously reported structure activity relationship studies. Surprisingly, the structure showed that a hydrolysis product of PL (hPL) was conjugated to glutathione at the C7-C8 olefin, and this complex was bound to the active site of GSTP1; no covalent bond formation between hPL and GSTP1 was observed. Mass spectrometry (MS) analysis of the reactions between PL and GSTP1 confirmed that PL does not label GSTP1. Moreover, MS data also indicated that nucleophilic attack on PL at the C2-C3 olefin led to PL hydrolysis. Although hPL inhibits GSTP1 enzymatic activity in vitro, treatment of cells susceptible to PL with hPL did not have significant anti-proliferative effects, suggesting that hPL is not membrane-permeable. Altogether, our data suggest a model wherein PL is a prodrug whose intracellular hydrolysis initiates the formation of the hPL-GSH conjugate, which blocks the active site of and inhibits GSTP1 and thereby cancer cell proliferation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Expression Patterns of Glutathione Transferase Gene (GstI) in Maize Seedlings Under Juglone-Induced Oxidative Stress

PubMed Central

Sytykiewicz, Hubert

2011-01-01

Juglone (5-hydroxy-1,4-naphthoquinone) has been identified in organs of many plant species within Juglandaceae family. This secondary metabolite is considered as a highly bioactive substance that functions as direct oxidant stimulating the production of reactive oxygen species (ROS) in acceptor plants. Glutathione transferases (GSTs, E.C.2.5.1.18) represent an important group of cytoprotective enzymes participating in detoxification of xenobiotics and limiting oxidative damages of cellular macromolecules. The purpose of this study was to investigate the impact of tested allelochemical on growth and development of maize (Zea mays L.) seedlings. Furthermore, the effect of juglone-induced oxidative stress on glutathione transferase (GstI) gene expression patterns in maize seedlings was recorded. It was revealed that 4-day juglone treatment significantly stimulated the transcriptional activity of GstI in maize seedlings compared to control plants. By contrast, at the 6th and 8th day of experiments the expression gene responses were slightly lower as compared with non-stressed seedlings. Additionally, the specific gene expression profiles, as well as the inhibition of primary roots and coleoptile elongation were proportional to juglone concentrations. In conclusion, the results provide strong molecular evidence that allelopathic influence of juglone on growth and development of maize seedlings may be relevant with an induction of oxidative stress in acceptor plants. PMID:22174645

Characterization and expression profiling of glutathione S-transferases in the diamondback moth, Plutella xylostella (L.).

PubMed

You, Yanchun; Xie, Miao; Ren, Nana; Cheng, Xuemin; Li, Jianyu; Ma, Xiaoli; Zou, Minming; Vasseur, Liette; Gurr, Geoff M; You, Minsheng

2015-03-05

Glutathione S-transferases (GSTs) are multifunctional detoxification enzymes that play important roles in insects. The completion of several insect genome projects has enabled the identification and characterization of GST genes over recent years. This study presents a genome-wide investigation of the diamondback moth (DBM), Plutella xylostella, a species in which the GSTs are of special importance because this pest is highly resistant to many insecticides. A total of 22 putative cytosolic GSTs were identified from a published P. xylostella genome and grouped into 6 subclasses (with two unclassified). Delta, Epsilon and Omega GSTs were numerically superior with 5 genes for each of the subclasses. The resulting phylogenetic tree showed that the P. xylostella GSTs were all clustered into Lepidoptera-specific branches. Intron sites and phases as well as GSH binding sites were strongly conserved within each of the subclasses in the GSTs of P. xylostella. Transcriptome-, RNA-seq- and qRT-PCR-based analyses showed that the GST genes were developmental stage- and strain-specifically expressed. Most of the highly expressed genes in insecticide resistant strains were also predominantly expressed in the Malpighian tubules, midgut or epidermis. To date, this is the most comprehensive study on genome-wide identification, characterization and expression profiling of the GST family in P. xylostella. The diversified features and expression patterns of the GSTs are inferred to be associated with the capacity of this species to develop resistance to a wide range of pesticides and biological toxins. Our findings provide a base for functional research on specific GST genes, a better understanding of the evolution of insecticide resistance, and strategies for more sustainable management of the pest.

21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

Code of Federal Regulations, 2012 CFR

2012-04-01

... SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.130 Aminoglycoside 3′-phospho- transferase II. The food additive aminoglycoside 3′-phosphotransferase II may be safely used in the development of...

21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

Code of Federal Regulations, 2010 CFR

2010-04-01

... SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.130 Aminoglycoside 3′-phospho- transferase II. The food additive aminoglycoside 3′-phosphotransferase II may be safely used in the development of...

21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

Code of Federal Regulations, 2013 CFR

2013-04-01

... SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.130 Aminoglycoside 3′-phospho- transferase II. The food additive aminoglycoside 3′-phosphotransferase II may be safely used in the development of...

21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

Code of Federal Regulations, 2011 CFR

2011-04-01

... SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.130 Aminoglycoside 3′-phospho- transferase II. The food additive aminoglycoside 3′-phosphotransferase II may be safely used in the development of...

21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

Code of Federal Regulations, 2014 CFR

2014-04-01

... SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.130 Aminoglycoside 3′-phospho- transferase II. The food additive aminoglycoside 3′-phosphotransferase II may be safely used in the development of...

Succinyl-CoA:(R)-Benzylsuccinate CoA-Transferase: an Enzyme of the Anaerobic Toluene Catabolic Pathway in Denitrifying Bacteria†

PubMed Central

Leutwein, Christina; Heider, Johann

2001-01-01

Anaerobic microbial toluene catabolism is initiated by addition of fumarate to the methyl group of toluene, yielding (R)-benzylsuccinate as first intermediate, which is further metabolized via β-oxidation to benzoyl-coenzyme A (CoA) and succinyl-CoA. A specific succinyl-CoA:(R)-benzylsuccinate CoA-transferase activating (R)-benzylsuccinate to the CoA-thioester was purified and characterized from Thauera aromatica. The enzyme is fully reversible and forms exclusively the 2-(R)-benzylsuccinyl-CoA isomer. Only some close chemical analogs of the substrates are accepted by the enzyme: succinate was partially replaced by maleate or methylsuccinate, and (R)-benzylsuccinate was replaced by methylsuccinate, benzylmalonate, or phenylsuccinate. In contrast to all other known CoA-transferases, the enzyme consists of two subunits of similar amino acid sequences and similar sizes (44 and 45 kDa) in an α2β2 conformation. Identity of the subunits with the products of the previously identified toluene-induced bbsEF genes was confirmed by determination of the exact masses via electrospray-mass spectrometry. The deduced amino acid sequences resemble those of only two other characterized CoA-transferases, oxalyl-CoA:formate CoA-transferase and (E)-cinnamoyl-CoA:(R)-phenyllactate CoA-transferase, which represent a new family of CoA-transferases. As suggested by kinetic analysis, the reaction mechanism of enzymes of this family apparently involves formation of a ternary complex between the enzyme and the two substrates. PMID:11418570

Analysis of Protein Adduction Kinetics by Quantitative Mass Spectrometry. Competing Adduction Reactions of Glutathione-S-Transferase P1-1 with Electrophiles

PubMed Central

Orton, Christopher R.; Liebler, Daniel C.

2007-01-01

Defining the mechanisms and consequences of protein adduction is crucial to understanding the toxicity of reactive electrophiles. Application of tandem mass spectrometry and data analysis algorithms enables detection and mapping of chemical adducts at the level of amino acid sequence. Nevertheless, detection of adducts does not indicate relative reactivity of different sites. Here we describe a method to measure the kinetics of competing adduction reactions at different sites on the same protein. Adducts are formed by electrophiles at Cys14 and Cys47 on the metabolic enzyme glutathione-S-transferase P1-1 and modification is accompanied by a loss of enzymatic activity. Relative quantitation of protein adducts was done by tagging N-termini of peptide digests with isotopically labeled phenyl isocyanate and tracking the ratio of light-tagged peptide adducts to heavy-tagged reference samples in liquid chromatography-tandem mass spectrometry analyses using a multiple reaction monitoring method. This approach was used to measure rate constants for adduction at both positions with two different model electrophiles, N-iodoacetyl-N-biotinylhexylenediamine and 1-biotinamido-4-(4′-[maleimidoethyl-cyclohexane]-carboxamido)butane. The results indicate that Cys47 was approximately 2–3-fold more reactive toward both electrophiles than was Cys14. This result was consistent with the relative reactivity of these electrophiles in a complex proteome system and with previously reported trends in reactivity of these sites. Kinetic analyses of protein modification reactions provide a means of evaluating the selectivity of reactive mediators of chemical toxicity. PMID:17433278

Impacts on silkworm larvae midgut proteomics by transgenic Trichoderma strain and analysis of glutathione S-transferase sigma 2 gene essential for anti-stress response of silkworm larvae.

PubMed

Li, Yingying; Dou, Kai; Gao, Shigang; Sun, Jianan; Wang, Meng; Fu, Kehe; Yu, Chuanjin; Wu, Qiong; Li, Yaqian; Chen, Jie

2015-08-03

Lepidoptera is a large order of insects that have major impacts on humans as agriculture pests. The midgut is considered an important target for insect control. In the present study, 10 up-regulated, 18 down-regulated, and one newly emerged protein were identified in the transgenic Trichoderma-treated midgut proteome. Proteins related to stress response, biosynthetic process, and metabolism process were further characterized through quantitative real-time PCR (qPCR). Of all the identified proteins, the glutathione S-transferase sigma 2 (GSTs2) gene displayed enhanced expression when larvae were fed with Trichoderma wild-type or transgenic strains. Down regulation of GSTs2 expression by RNA interference (RNAi) resulted in inhibition of silkworm growth when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. Weight per larva decreased by 18.2%, 11.9%, and 10.7% in the untreated control, ddH2O, and GFP dsRNA groups, respectively, at 24h, while the weight decrease was higher at 42.4%, 28.8% and 32.4% at 72 h after treatment. Expression of glutathione S-transferase omega 2 (GSTo2) was also enhanced when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. These results indicated that there was indeed correlation between enhanced expression of GSTs2 and the anti-stress response of silkworm larvae against Trichoderma. This study represents the first attempt at understanding the effects of transgenic organisms on the midgut proteomic changes in silkworm larvae. Our findings could not only broaden the biological control targets of insect at the molecular level, but also provide a theoretical foundation for biological safety evaluation of the transgenic Trichoderma strain. Copyright © 2015 Elsevier B.V. All rights reserved.

Association of glutathione S-transferase P1 (GSTP1) polymorphism with Tourette syndrome in Taiwanese patients.

PubMed

Shen, Che-Piao; Chou, I-Ching; Liu, Hsin-Ping; Lee, Cheng-Chun; Tsai, Yuhsin; Wu, Bor-Tsang; Hsu, Ban-Dar; Lin, Wei-Yong; Tsai, Fuu-Jen

2014-01-01

The etiology of Tourette syndrome (TS) is multifactorial. TS vulnerability may be associated with genetic and environmental factors. From the genetic point of view, TS is heterogeneous. Previous studies showed that some single-nucleotide polymorphisms (SNPs) of the glutathione-S-transferase P1 (GSTP1) gene can affect cellular proliferation and apoptotic activity and TS is a neurodevelopmental disorder. We guessed that there was a relationship between TS and genetic variants of the GSTP1 gene. Therefore, in this study, we aimed to test the hypothesis that GSTP1 SNPs were associated with TS. We performed a case-control study. One hundred twenty-one TS children and 105 normal children were included in the study. Polymerase chain reaction was used to identify the GSTP1 gene polymorphism at position rs6591256 (A/G, promoter polymorphism) in TS patients and normal children. The polymorphism at position rs6591256 in the GSTP1 gene revealed significant differences in the allele (p=0.0135) and genotype (p=0.0159) distributions between the TS patients and the control group. The A allele was present at a higher frequency than the G allele in the TS patients compared with the control group (odds ratio [OR]=1.91, 95% confidence interval [CI]: 1.14-3.21). The AA genotype was associated with susceptibility to TS with an OR of 2.38 for the AA versus AG genotype (95% CI: 1.29-4.41). These findings suggest that variants in the GSTP1 gene may play a role in susceptibility to TS.

Variants of glutathione s-transferase pi 1 exhibit differential enzymatic activity and inhibition by heavy metals.

PubMed

Goodrich, Jaclyn M; Basu, Niladri

2012-06-01

Nonsynonymous single nucleotide polymorphisms in glutathione s-transferase pi 1 (GSTP1; Ile/Val 105, Ala/Val 114) have been associated with altered toxicant metabolism in epidemiological cohorts. We explored the impact of GSTP1 genotype on enzyme kinetics and heavy metal inhibition in vitro. Four GSTP1 allozymes (105/114: Ile/Ala, Val/Ala, Ile/Val, Val/Val) were expressed in and purified from Escherichia coli. Enzyme activity assays quantifying the rate of glutathione conjugation with 1-chloro-2,4-dinitrobenzene (CDNB) revealed significant differences in kinetic parameters depending on genotype (p<0.01). Allozymes with Ile105 had better catalytic efficiency and greater affinity for CDNB (mean ± SEM: Ile105 Ala114 K(m)=0.33 ± 0.07 mM vs. Val105 Ala114 K(m)=1.15 ± 0.07 mM). Inhibition of GSTP1 activity by heavy metals was assessed following treatment with mercury (inorganic-HgCl(2), methylmercury-MeHg), selenium, cadmium, lead, arsenic, and manganese. All allozymes were inhibited by HgCl(2) (IC(50) range: 24.1-172 μM), MeHg (93.9-480 μM), and selenium (43.7-62.8 μM). Genotype significantly influenced the potency of mercury with GSTP1 Ile105 Val114 the least sensitive and Val105 Ala114 the most sensitive to inhibition by HgCl(2) and MeHg. Overall, genotype of two nonsynonymous polymorphisms in GSTP1 influenced enzyme kinetics pertaining to an electrophilic substrate and inhibition by two mercury species. Published by Elsevier Ltd.

Expression of thymidylate synthase and glutathione-s-transferase pi in patients with esophageal squamous cell carcinoma.

PubMed

Huang, Jun-Xing; Li, Feng-Yue; Xiao, Wei; Song, Zheng-Xiang; Qian, Rong-Yu; Chen, Ping; Salminen, Eeva

2009-09-14

To investigate the expression of thymidylate synthase (TS) and glutathione-s-transferase pi (GST-pi) in esophageal squamous cell carcinoma and their association with the clinicopathologic characteristics. Immunohistochemical methods were used to detect the expression of TS and GST-pi in surgically resected formalin-fixed, paraffin-embedded esophageal squamous cell carcinoma (ESCC) tissue sections from 102 patients (median age, 58 years) and in 28 normal esophageal mucosa (NEM) samples. The relationship between TS and GST-pi expression and clinicopathologic factors was examined. The expression of TS and GST-pi was not statistically significantly associated with age of the patients, tumor size, lymph node metastasis, depth of invasion or tumor stage. TS staining was positive in 17.86% of normal esophageal mucosa and in 42.16% of ESCC samples (P < 0.05). The expression level of TS was not only significantly lower in well-differentiated (21.88%) than in poorly-differentiated carcinomas (51.43%, P < 0.05), but was also significantly higher in samples from male patients (46.51%) than from female patients (18.75%, P < 0.05). GST-pi was positively stained in 78.57% of normal esophageal mucosa and in 53.92% of ESCC samples (P < 0.05). The expression level of GST-pi was also significantly higher in well-differentiated carcinomas (65.63%) than in poorly-differentiated carcinomas (35.00%, P < 0.05). The expression of TS and of GST-pi may be used as molecular markers for the characterization of ESCC. Poorly-differentiated cells showed increased expression of TS and reduced expression of GST-pi.

Variants of glutathione s-transferase pi 1 exhibit differential enzymatic activity and inhibition by heavy metals

PubMed Central

Goodrich, Jaclyn M.; Basu, Niladri

2012-01-01

Nonsynonymous single nucleotide polymorphisms in glutathione s-transferase pi 1 (GSTP1; Ile/Val 105, Ala/Val 114) have been associated with altered toxicant metabolism in epidemiological cohorts. We explored the impact of GSTP1 genotype on enzyme kinetics and heavy metal inhibition in vitro. Four GSTP1 allozymes (105/114: Ile/Ala, Val/Ala, Ile/Val, Val/Val) were expressed in and purified from E. coli. Enzyme activity assays quantifying the rate of glutathione conjugation with 1-chloro-2,4-dinitrobenzene (CDNB) revealed significant differences in kinetic parameters depending on genotype (p<0.01). Allozymes with Ile105 had better catalytic efficiency and greater affinity for CDNB (mean ±SEM: Ile105 Ala114 Km= 0.33±0.07 mM vs. Val105 Ala114 Km=1.15±0.07 mM). Inhibition of GSTP1 activity by heavy metals was assessed following treatment with mercury (inorganic- HgCl2, methylmercury- MeHg), selenium, cadmium, lead, arsenic, and manganese. All allozymes were inhibited by HgCl2 (IC50 range: 24.1–172 μM), MeHg (93.9–480 μM), and selenium (43.7–62.8 μM). Genotype significantly influenced the potency of mercury with GSTP1 Ile105 Val114 the least sensitive and Val105 Ala114 the most sensitive to inhibition by HgCl2 and MeHg. Overall, genotype of two nonsynonymous polymorphisms in GSTP1 influenced enzyme kinetics pertaining to an electrophilic substrate and inhibition by two mercury species. PMID:22401947

Analysis of glutathione S-transferase allergen cross-reactivity in a North American population: Relevance for molecular diagnosis.

PubMed

Mueller, Geoffrey A; Pedersen, Lars C; Glesner, Jill; Edwards, Lori L; Zakzuk, Josefina; London, Robert E; Arruda, Luisa Karla; Chapman, Martin D; Caraballo, Luis; Pomés, Anna

2015-11-01

It is not clear whether cross-reactivity or cosensitization to glutathione S-transferases (GSTs) occurs in tropical and subtropical environments. In the United States, Bla g 5 is the most important GST allergen and lack of coexposure to GSTs from certain species allows a better assessment of cross-reactivity. To examine the molecular structure of GST allergens from cockroach (Bla g 5), dust mites (Der p 8 and Blo t 8), and helminth (Asc s 13) for potential cross-reactive sites, and to assess the IgE cross-reactivity of sensitized patients from a temperate climate for these allergens for molecular diagnostic purposes. Four crystal structures were determined. Sera from patients allergic to cockroach and mite were tested for IgE reactivity to these GSTs. A panel of 6 murine anti-Bla g 5 mAb was assessed for cross-reactivity with the other 3 GSTs using antibody binding assays. Comparisons of the allergen structures, formed by 2-domain monomers that dimerize, revealed few contiguous regions of similar exposed residues, rendering cross-reactivity unlikely. Accordingly, anti-Bla g 5 or anti-Der p 8 IgE from North American patients did not recognize Der p 8 or Bla g 5, respectively, and neither showed binding to Blo t 8 or Asc s 13. A weaker binding of anti-Bla g 5 IgE to Der p 8 versus Bla g 5 (∼ 100-fold) was observed by inhibition assays, similar to a weak recognition of Der p 8 by anti-Bla g 5 mAb. Patients from tropical Colombia had IgE to all 4 GSTs. The lack of significant IgE cross-reactivity among the 4 GSTs is in agreement with the low shared amino acid identity at the molecular surface. Each GST is needed for accurate molecular diagnosis in different geographic areas. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. All rights reserved.

Matrin 3 co-immunoprecipitates with the heat shock proteins glucose-regulated protein 78 (GRP78), GRP75 and glutathione S-transferase π isoform 2 (GSTπ2) in thymoma cells.

PubMed

Osman, Ahmed M; van Loveren, Henk

2014-06-01

Here, we report evidence that matrin 3 (MATR3), a highly conserved inner nuclear matrix phosphoprotein, whose function is largely unknown, interacts specifically with the heat shock proteins glucose-regulated protein 78 (GRP78), GRP75 and glutathione S-transferase π isoform 2 (GSTπ2). Using immunoprecipitation experiments of lysates obtained from control and tributyltin oxide (TBTO)-treated thymoma cell line (EL4), we identified MATR3 and its partners by MS/MS analysis and confirmed by immunoblot. We also show that MATR3 undergoes degradation as reported before and that this cleavage process, which is inhibited by the broad-spectrum caspase inhibitor, z-VAD-FMK, is more marked in TBTO-treated cells. Further, we found that the heat shock protein glucose-regulated protein 78 was downregulated in the TBTO-treated cells. The GRP78 protein is known to protect cells from apoptosis by complexing with procaspase 7 thereby preventing caspase activation cascade. By immunoblot analysis, we found that the levels of procaspases-3 and -7 were lower in TBTO-treated cells; in contrast, the level of p20, the active form of caspase 3, was relatively higher in the treated cells compared to that of control cells. We propose that the TBTO-mediated downregulation of GRP78 triggers the caspase cascade pathway leading to MATR3 degradation. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Cyclopentenone prostaglandins as potential inducers of phase II detoxification enzymes. 15-deoxy-delta(12,14)-prostaglandin j2-induced expression of glutathione S-transferases.

PubMed

Kawamoto, Y; Nakamura, Y; Naito, Y; Torii, Y; Kumagai, T; Osawa, T; Ohigashi, H; Satoh, K; Imagawa, M; Uchida, K

2000-04-14

Exposure of cells to a wide variety of chemoprotective compounds confers resistance to a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of protective enzymes, such as glutathione S-transferases (GSTs). In the present study, we developed a cell culture system that potently responds to phenolic antioxidants and found that antitumor prostaglandins (PGs) are potential inducers of GSTs. We screened primary hepatocytes and multiple cell lines for inducing GST activity upon incubation with the phenolic antioxidant (tert-butylhydroquinone) and found that rat liver epithelial RL34 cells most potently responded. Based on an extensive screening of diverse chemical agents on the induction of GST activity in RL34 cells, the J2 series of PGs, 15-deoxy-Delta(12,14)-prostaglandin J2 (15-deoxy-Delta(12,14)-PGJ2) in particular, were found to be potential inducers of GST. Enhanced gene expression of Class pi GST isozyme (GSTP1) by 15-deoxy-Delta(12,14)-PGJ2 was evident as a drastic elevation of the mRNA level. Hence, we examined the molecular mechanism underlying the 15-deoxy-Delta(12, 14)-PGJ2-induced GSTP1 gene expression. From functional analysis of various deletion mutant genes, we found that the 15-deoxy-Delta(12, 14)-PGJ2 reponse element was localized in a region containing a GSTP1 enhancer I (GPEI) that consists of two imperfect phorbol 12-O-tetradecanoylphorbol-13-acetate response elements. When the GPEI was combined with the minimum GSTP1 promoter, the element indeed showed an enhancer activity in response to 15-deoxy-Delta(12, 14)-PGJ2. Point mutations of either of the two imperfect 12-O-tetradecanoylphorbol-13-acetate response elements in GPEI completely abolished the enhancer activity. Gel mobility shift assays demonstrated that 15-deoxy-Delta(12,14)-PGJ2 specifically stimulated the binding of nuclear proteins including the transcription factor c-Jun, but not Nrf2, to GPEI. These results

The relationship between changes in functional cardiac parameters following anthracycline therapy and carbonyl reductase 3 and glutathione S transferase Pi polymorphisms.

PubMed

Volkan-Salanci, Bilge; Aksoy, Hakan; Kiratli, Pınar Özgen; Tülümen, Erol; Güler, Nilüfer; Öksüzoglu, Berna; Tokgözoğlu, Lale; Erbaş, Belkıs; Alikaşifoğlu, Mehmet

2012-10-01

The aim of this prospective clinical study is to evaluate the relationship between changes in functional cardiac parameters following anthracycline therapy and carbonyl reductase 3 (CBR3p.V244M) and glutathione S transferase Pi (GSTP1p.I105V) polymorphisms. Seventy patients with normal cardiac function and no history of cardiac disease scheduled to undergo anthracycline chemotherapy were included in the study. The patients' cardiac function was evaluated by gated blood pool scintigraphy and echocardiography before and after chemotherapy, as well as 1 year following therapy. Gene polymorphisms were genotyped in 70 patients using TaqMan probes, validated by DNA sequencing. A deteriorating trend was observed in both systolic and diastolic parameters from GG to AA in CBR3p.V244M polymorphism. Patients with G-allele carriers of GSTP1p.I105V polymorphism were common (60%), with significantly decreased PFR compared to patiens with AA genotype. Variants of CBR3 and GSTP1 enzymes may be associated with changes in short-term functional cardiac parameters.

Transmutation of human glutathione transferase A2-2 with peroxidase activity into an efficient steroid isomerase.

PubMed

Pettersson, Par L; Johansson, Ann-Sofie; Mannervik, Bengt

2002-08-16

A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.

Overexpression of a glutathione S-transferase (Mdgst) and a galactosyltransferase-like gene (Mdgt1) is responsible for imidacloprid resistance in house flies.

PubMed

Reid, William R; Sun, Haina; Becnel, James J; Clark, Andrew G; Scott, Jeffrey G

2018-06-21

Neonicotinoids are the largest class of insecticides and are used for control of house fly populations at animal production facilities throughout the world. There have been several reports of neonicotinoid resistance in house fly populations, but identification of the factors involved in resistance has proven challenging. The KS8S3 population of house flies is highly resistant to the neonicotinoid insecticide imidacloprid due to two factors: one on chromosome 3 and one on chromosome 4. A comparative transcriptomic approach was used, followed by validation using transgenic Drosophila melanogaster to investigate the genes responsible for resistance in the KS8S3 strain. Overexpression of a microsomal glutathione S-transferase (Mdgst) was identified as the factor likely responsible for resistance on chromosome 3. Resistance on chromosome 4 appears to be due to an unidentified trans-regulatory gene which causes overexpression of a galactosyltransferase-like gene (Mdgt1). No single nucleotide polymorphisms were found that could be associated with imidacloprid resistance. Identification of the underlying processes that cause imidacloprid resistance is an important first step towards the development of novel and sensitive resistance monitoring techniques. It will be valuable to investigate if overexpression of Mdgst and Mdgt1 are found in other imidacloprid resistant populations. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Glutathione S-transferase M1 (GSTM1) null genotype and coronary artery disease risk: a meta-analysis

PubMed Central

Zhang, Zhen-Xian; Zhang, Ye

2014-01-01

Background: The Glutathione S-Transferase M1 (GSTM1) null genotype has been indicated to be correlated with coronary artery disease (CAD) susceptibility, but study results are still debatable. Thus, a meta-analysis was conducted. Materials and methods: Databases including PubMed, Embase, Web of Science, and Chinese National Knowledge Infrastructure (CNKI) were searched. Data were extracted and pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated. Results: Twenty-six studies with 10595 cases and 13782 controls were included in this meta-analysis. The association between GSTM1 null genotype and CAD risk was significant (OR = 1.35; 95% CI, 1.09 - 1.67; P < 0.01). When stratified by ethnicity, the significantly elevated risk were observed in Caucasians (OR = 1.39; 95% CI, 1.07 - 1.81; P = 0.01) but not in Asians (OR = 1.27; 95% CI, 0.87 - 1.86; P = 0.22). No significantly increased myocardial infarction risk was observed (OR = 0.96; 95% CI, 0.78 - 1.18; P = 0.68). Subgroup analysis on the smoking status showed that the increased risk was found in smokers (OR = 1.66; 95% CI, 1.14 - 2.42; P < 0.01) but not in non-smokers (OR = 1.30; 95% CI, 1.74 - 2.28; P = 0.37). Conclusion: In conclusion, this meta-analysis suggested that GSTM1 null genotype was a risk factor for CAD, especially in Caucasians and smokers. PMID:25419371

Modulatory influence of sandalwood oil on mouse hepatic glutathione S-transferase activity and acid soluble sulphydryl level.

PubMed

Banerjee, S; Ecavade, A; Rao, A R

1993-02-01

The effect of the oil from the wood of Santalum album on glutathione S-transferase (GST) activity and acid soluble sulphydryl (SH) levels in the liver of adult male Swiss albino mice was investigated. Oral feeding by gavage to mice each day with 5 and 15 microliters sandalwood oil for 10 and 20 days exhibited an increase in GST activity in time- and dose-responsive manners. Feeding a dose of 5 microliters sandalwood oil for 10 and 20 days caused, respectively, a 1.80-fold (P < 0.001) and 1.93-fold (P < 0.001) increase in GST enzyme activity, while feeding a dose of 15 microliters of the oil per day for 10 and 20 days induced, respectively, 4.73-fold (P < 0.001) and 6.10-fold (P < 0.001) increases in the enzyme's activity. In addition, there were 1.59-fold (P < 0.001) and 1.57 (P < 0.001) increases in acid-soluble SH levels in the hepatic tissue of the mice following feeding of the oil at the dose levels of 5 and 15 microliters for 10 days. Furthermore, mice fed on a diet containing 1% 2(3)-butyl-4-hydroxyanisole (positive control) also showed an increase in hepatic GST activity and SH levels. Enhancement of GST activity and acid-soluble SH levels are suggestive of a possible chemopreventive action of sandalwood oil on carcinogenesis through a blocking mechanism.

Prostate cancer molecular detection in plasma samples by glutathione S-transferase P1 (GSTP1) methylation analysis.

PubMed

Dumache, Raluca; Puiu, Maria; Motoc, Marilena; Vernic, Corina; Dumitrascu, Victor

2014-01-01

Prostate cancer (PCa) represents the most commonly diagnosed type of malignancy among men in Western European countries and the second cause of cancer-related deaths among men worldwide. Methylation of the CpG island has an important role in prostate carcinogenesis and progression. The purpose of the study was to analyse the diagnostic value of aberrant promoter hypermethylation of the gene for glutathione S-transferase P1 (GSTP1) in plasma DNA to discriminate between prostate cancer (PCa) and benign prostatic hyperplasia (BPH) patients by minimally invasive methods. Aberrant promoter hypermethylation was investigated in DNA isolated from plasma samples of 31 patients with diagnostic of PCa and 44 cancer-free males (control subjects). Extracted genomic DNA was bisulfite treated and analyzed using methylation-specific polymerase chain reaction (MS-PCR) technique. Hypermethylation of the GSTP1 gene was detected in plasma samples from 27 of 31 (92.86%) patients with PCa. Genomic DNA from plasma samples from the 44 controls without genitourinary cancer revealed promoter hypermethylation of GSTP1 gene in 3 (10.6%) of the 44 patients. Receiver operating curve (ROC) included clinico-pathological parameters such as: serum PSA levels, pathological stage, Gleason score, hypermethylation status of GSTP1 gene, and it gave a predictive accuracy of 93% with a sensitivity and specificity of 95% and 87%, respectively. In this study, we have evaluated the ability of GSTP1 gene to discriminate between PCa and BPH patients in genomic DNA from plasma samples by non-invasive methods.

Analysis of glutathione S-transferase Pi isoform (GSTP1) single-nucleotide polymorphisms and macular telangiectasia type 2.

PubMed

Szental, Joshua A; Baird, Paul N; Richardson, Andrea J; Islam, F M Amirul; Scholl, Hendrik P N; Charbel Issa, Peter; Holz, Frank G; Gillies, Mark; Guymer, Robyn H

2010-12-01

Recent imaging studies have suggested that macular pigment is decreased centrally in macular telangiectasia type 2 (MT2). The uptake of xanthophyll pigment into the macula is thought to be facilitated by a xanthophyll-binding protein (XBP). The Pi isoform of glutathione S-transferase (GSTP1) represents one such XBP with high binding affinity. This case-control study aimed to determine whether two common single-nucleotide polymorphisms (SNPs) of GSTP1 were associated with MT2. DNA samples from 39 cases and 21 controls were collected. Two polymorphic sites of Ile105Val and Ala114Val in exons 5 and 6 respectively, of the GSTP1 gene were analysed. Comparison of alleles and genotypes between cases and controls indicated that there were no statistically significant differences for either the Ile105Val SNP (P=0.43) or the Ala114Val SNP (P=0.85), or for any combinations; however, the homozygous at-risk genotype (GG) of the Ile105Val SNP was present in 8% of cases but absent in controls. This study found no statistically significant association between two common GSTP1 SNPs and MT2; however, a trend towards a greater frequency of the GG genotype of the Ile105Val SNP in cases is of great interest. The biological plausibility of disturbed macular pigment uptake in MT2 makes GSTP1 an excellent candidate gene. Further investigation is warranted in future studies of MT2.

Mechanism of Gene Expression of Arabidopsis Glutathione S-Transferase, AtGST1, and AtGST11 in Response to Aluminum Stress1

PubMed Central

Ezaki, Bunichi; Suzuki, Masakatsu; Motoda, Hirotoshi; Kawamura, Masako; Nakashima, Susumu; Matsumoto, Hideaki

2004-01-01

The gene expression of two Al-induced Arabidopsis glutathione S-transferase genes, AtGST1 and AtGST11, was analyzed to investigate the mechanism underlying the response to Al stress. An approximately 1-kb DNA fragment of the 5′-upstream region of each gene was fused to a β-glucuronidase (GUS) reporter gene (pAtGST1::GUS and pAtGST11::GUS) and introduced into Arabidopsis ecotype Landsberg erecta. The constructed transgenic lines showed a time-dependent gene expression to a different degree in the root and/or leaf by Al stress. The pAtGST1::GUS gene was induced after a short Al treatment (maximum expression after a 2-h exposure), while the pAtGST11::GUS gene was induced by a longer Al treatment (approximately 8 h for maximum expression). Since the gene expression was observed in the leaf when only the root was exposed to Al stress, a signaling system between the root and shoot was suggested in Al stress. A GUS staining experiment using an adult transgenic line carrying the pAtGST11::GUS gene supported this suggestion. Furthermore, Al treatment simultaneously with various Ca depleted conditions in root region enhanced the gene expression of the pAtGST11::GUS in the shoot region. This result suggested that the degree of Al toxicity in the root reflects the gene response of pAtGST11::GUS in the shoot via the deduced signaling system. Both transgenic lines also showed an increase of GUS activity after cold stress, heat stress, metal toxicity, and oxidative damages, suggesting a common induction mechanism in response to the tested stresses including Al stress. PMID:15047894

Chronically sun-damaged melanomas express low levels of nuclear glutathione-S-transferase-π: an epidemiological and clinicopathological study in Italy.

PubMed

Campione, Elena; Medda, Emanuela; Paternò, Evelin J; Diluvio, Laura; Ricozzi, Ilaria; Carboni, Isabella; Costanza, Gaetana; Rossi, Piero; Rapanotti, Cristina; Di Stefani, Alessandro; Chimenti, Sergio; Bianchi, Luca; Orlandi, Augusto

2015-01-01

The detoxifying enzyme glutathione-s-transferase pi (GST-π) is present in keratinocytes and melanocytes and exerts a protective role against tumour progression. Melanomas close to melanocytic naevus remnants occur less frequently on sun-exposed areas, whereas solar dermal elastosis, hallmark of chronic sun-damage, characterise melanomas on sun-exposed skin. We evaluated the expression of GST-π in 113 melanomas associated to melanocytic naevus remnants or to solar dermal elastosis, classified according to clinical characteristics, history of sun exposure, histological subtypes and AJCC staging. Chronically sun-damaged melanomas, identified by moderate-severe solar dermal elastosis, showed a lower nuclear GST-π expression and a higher thickness than those related to melanocytic naevus remnants (p < 0.03). Multivariate logistic regression analysis demonstrated that male gender and chronic sun-exposure are independent risk factors significantly associated to melanomas localised on the trunk (OR = 3.36, 95% CI: 1.31-8.65; OR = 5.97, 95% CI: 1.71-20.87). If confirmed on a larger series, lower expression of nuclear GST-π in melanoma cells could represent a possible marker of chronically sun-damaged melanoma pathogenesis.

Nuclear translocation of glutathione S-transferase {pi} is mediated by a non-classical localization signal

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawakatsu, Miho; Goto, Shinji, E-mail: sgoto@nagasaki-u.ac.jp; Yoshida, Takako

2011-08-12

Highlights: {yields} Nuclear translocation of GST{pi} is abrogated by the deletion of the last 16 amino acid residues in the carboxy-terminal region, indicating that residues 195-208 of GST{pi} are required for nuclear translocation. {yields} The lack of a contiguous stretch of positively charged amino acid residues within the carboxy-terminal region of GST{pi}, suggests that the nuclear translocation of GST{pi} is mediated by a non-classical nuclear localization signal. {yields} An in vitro transport assay shows that the nuclear translocation of GST{pi} is dependent on cytosolic factors and ATP. -- Abstract: Glutathione S-transferase {pi} (GST{pi}), a member of the GST family ofmore » multifunctional enzymes, is highly expressed in human placenta and involved in the protection of cellular components against electrophilic compounds or oxidative stress. We have recently found that GST{pi} is expressed in the cytoplasm, mitochondria, and nucleus in some cancer cells, and that the nuclear expression of GST{pi} appears to correlate with resistance to anti-cancer drugs. Although the mitochondrial targeting signal of GST{pi} was previously identified in the amino-terminal region, the mechanism of nuclear translocation remains completely unknown. In this study, we find that the region of GST{pi}195-208 is critical for nuclear translocation, which is mediated by a novel and non-classical nuclear localization signal. In addition, using an in vitro transport assay, we demonstrate that the nuclear translocation of GST{pi} depends on the cytosolic extract and ATP. Although further experiments are needed to understand in depth the precise mechanism of nuclear translocation of GST{pi}, our results may help to establish more efficient anti-cancer therapy, especially with respect to resistance to anti-cancer drugs.« less

Human glutathione S-transferase P1-1 functions as an estrogen receptor α signaling modulator

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Xiyuan; An, Byoung Ha; Kim, Min Jung

2014-09-26

Highlights: • GSTP induces the classical ERα signaling event. • The functional GSTP is a prerequisite for GSTP-induced ERα transcription activity. • The expression of RIP140, a transcription cofactor, was inhibited by GSTP protein. • We propose the novel non-enzymatic role of GSTP. - Abstract: Estrogen receptor α (ERα) plays a crucial role in estrogen-mediated signaling pathways and exerts its action as a nuclear transcription factor. Binding of the ligand-activated ERα to the estrogen response element (ERE) is a central part of ERα-associated signal transduction pathways and its aberrant modulation is associated with many disease conditions. Human glutathione S-transferase P1-1more » (GSTP) functions as an enzyme in conjugation reactions in drug metabolism and as a regulator of kinase signaling pathways. It is overexpressed in tumors following chemotherapy and has been associated with a poor prognosis in breast cancer. In this study, a novel regulatory function of GSTP has been proposed in which GSTP modulates ERE-mediated ERα signaling events. Ectopic expression of GSTP was able to induce the ERα and ERE-mediated transcriptional activities in ERα-positive but GSTP-negative MCF7 human breast cancer cells. This inductive effect of GSTP on the ERE-transcription activity was diminished when the cells express a mutated form of the enzyme or are treated with a GSTP-specific chemical inhibitor. It was found that GSTP inhibited the expression of the receptor interacting protein 140 (RIP140), a negative regulator of ERα transcription, at both mRNA and protein levels. Our study suggests a novel non-enzymatic role of GSTP which plays a significant role in regulating the classical ERα signaling pathways via modification of transcription cofactors such as RIP140.« less

Glutathione S-transferase pi isoform (GSTP1) expression in murine retina increases with developmental maturity.

PubMed

Lee, Wen-Hsiang; Joshi, Pratibha; Wen, Rong

2014-01-01

Glutathione S-transferase pi isoform (GSTP1) is an intracellular detoxification enzyme that catalyzes reduction of chemically reactive electrophiles and is a zeaxanthin-binding protein in the human macula. We have previously demonstrated that GSTP1 levels are decreased in human age-related macular degeneration (AMD) retina compared to normal controls (Joshi et al., Invest Ophthalmol Vis Sci, e-abstract, 2009). We also showed that GSTP1 levels parallel survival of human retinal pigment epithelial (RPE) cells exposed to ultraviolet (UV) light, and GSTP1 over-expression protects them against UV light damage (Joshi et al., Invest Ophthalmol Vis Sci, e-abstract, 2010). In the present work, we determined the developmental time course of GSTP1 expression in murine retina and in response to light challenge. Eyes from BALB/c mice at postnatal day 20, 1 month, and 2 months of age were prepared for retinal protein extraction and cryo sectioning, and GSTP1 levels in the retina were analyzed by Western blot and immunohistochemistry (IHC). Another group of BALB/c mice with the same age ranges was exposed to 1000 lx of white fluorescent light for 24 h, and their retinas were analyzed for GSTP1 expression by Western blot and IHC in a similar manner. GSTP1 levels in the murine retina increased in ascending order from postnatal day 20, 1 month, and 2 months of age. Moreover, GSTP1 expression in murine retina at postnatal day 20, 1 month, and 2 months of age increased in response to brief light exposure compared to age-matched controls under normal condition. GSTP1 expression in retina increases with developmental age in mice and accompanies murine retinal maturation. Brief exposure to light induces GSTP1 expression in the murine retina across various developmental ages. GSTP1 induction may be a protective response to light-induced oxidative damage in the murine retina.

Glutathione S-Transferase Pi Isoform (GSTP1) Expression in Murine Retina Increases with Developmental Maturity

PubMed Central

Lee, Wen-Hsiang; Joshi, Pratibha; Wen, Rong

2014-01-01

Background and Aims Glutathione S-transferase pi isoform (GSTP1) is an intracellular detoxification enzyme that catalyzes reduction of chemically reactive electrophiles and is a zeaxanthin-binding protein in the human macula. We have previously demonstrated that GSTP1 levels are decreased in human age-related macular degeneration (AMD) retina compared to normal controls [1]. We also showed that GSTP1 levels parallel survival of human retinal pigment epithelial (RPE) cells exposed to UV light, and GSTP1 over-expression protects them against UV light damage [2]. In the present work, we determined the developmental time course of GSTP1 expression in murine retina and in response to light challenge. Methods Eyes from BALB/c mice at post-natal day 20, 1 month, and 2 months of age were prepared for retinal protein extraction and cryo sectioning, and GSTP1 levels in the retina were analyzed by Western blot and immunohistochemistry (IHC). Another group of BALB/c mice with the same age ranges was exposed to 1000 lux of white fluorescent light for 24 hours, and their retinas were analyzed for GSTP1 expression by Western blot and IHC in a similar manner. Results GSTP1 levels in the murine retina increased in ascending order from post-natal day 20, 1 month, and 2 months of age. Moreover, GSTP1 expression in murine retina at post-natal day 20, 1 month, and 2 months of age increased in response to brief light exposure compared to age-matched controls under normal condition. Conclusions GSTP1 expression in retina increases with developmental age in mice and accompanies murine retinal maturation. Brief exposure to light induces GSTP1 expression in the murine retina across various developmental ages. GSTP1 induction may be a protective response to light-induced oxidative damage in the murine retina. PMID:24664677

Green tea consumption and glutathione S-transferases genetic polymorphisms on the risk of adult leukemia.

PubMed

Liu, Ping; Zhang, Min; Xie, Xing; Jin, Jie; Holman, C D'Arcy J

2017-03-01

Green tea may have a beneficial role of inhibiting leukemia. Glutathione S-transferases (GSTs) are known to detoxify certain carcinogens. We investigated the roles of green tea consumption and polymorphisms of GSTM1, GSTT1 and GSTP1 on the risk of adult leukemia, and to determine whether the associations varied within GSTs genotypes. A multicenter case-control study was conducted in China, 2008-2013. It comprised 442 incident, hematologically confirmed adult leukemia cases and 442 outpatient controls, individually matched to cases by gender, birth quinquennium and study site. Data were collected by face-to-face interview using a validated questionnaire. Genetic polymorphisms were assayed by PCR. An inverse association between green tea consumption and adult leukemia risk was observed. Compared with non-tea drinkers, the adjusted odds ratios (95 % confidence intervals) were 0.50 (0.27-0.93), 0.31 (0.17-0.55) and 0.53 (0.29-0.99) for those who, respectively, consumed green tea >20 years, ≥2 cups daily and dried tea leaves >1000 g annually. In assessing the associations by GSTs genotypes, risk reduction associated with green tea consumption was stronger in individuals with the GSTT1-null genotype (OR 0.24; 95 % CI 0.11-0.53) than GSTT1-normal carriers (OR 0.67; 95 % CI 0.42-1.05; P interaction = 0.02). GSTM1 and GSTP1 did not significantly modify the inverse association of leukemia with green tea. The results suggest that regular daily green tea consumption may reduce leukemia risk in Chinese adults regardless of GSTM1 and GSTP1 polymorphic status. The association between green tea and adult leukemia risk varied with GSTT1 genotype and highlights further study.

Glutathione S-transferase M1 genotypes and the risk of vulvar cancer: a population-based case-control study.

PubMed

Chen, C; Madeleine, M M; Weiss, N S; Daling, J R

1999-09-01

Glutathione S-transferases (GSTs) facilitate the excretion of a variety of potential carcinogens. Some 50-60% of Caucasians are homozygous for the null allele of GSTM1, a gene responsible for the presence of one of these enzymes. The authors examined whether women with the GSTM1 null genotype are at altered risk of vulvar cancer. They obtained peripheral blood specimens from 18- to 79-year-old residents of King, Pierce, and Snohomish counties of western Washington who were diagnosed with vulvar cancer between April 1991 and June 1994. Blood specimens were also obtained from controls identified via random digit telephone dialing of western Washington households. The authors determined the GSTM1 genotype of 137 cases (120 in situ and 17 invasive cases) and 248 controls. The frequency of the GSTM1 null genotype was 46.7% among cases and 57.3% among controls. The age-adjusted odds ratio associated with the GSTM1 null genotype was 0.7 (95% confidence interval: 0.4, 1.0). Among current smokers of cigarettes, the age-adjusted odds ratio associated with the GSTM1 null genotype was 0.5 (95% confidence interval: 0.2, 0.9), differing little between heavy and light smokers. Our data suggest that women with the GSTM1 null genotype are not at increased risk of vulvar cancer.

The NEXT-A (N-terminal EXtension with Transferase and ARS) reaction.

PubMed

Taki, Masumi; Kuroiwa, Hiroyuki; Sisido, Masahiko

2009-01-01

L/F-transferase is known to catalyze transfer of hydrophobic amino acids from aminoacyl tRNA to the N-terminus of a protein possessing lysine or arginine as the N-terminus. Combining L/F-transferase with E. coli phenylalanyl-tRNA synthetase (ARS), we achieved non-ribosomal N-terminal-specific introduction of various kinds of nonnatural amino acids to a protein. A nonnatural amino acid is once charged onto an E. coli tRNA(Phe) by a mutant ARS in situ, and successively transferred from the tRNA to a target protein, namely the NEXT-A reaction. Besides alphaA294G mutation on the ARS, alphaT251A, betaG318W, or betaA356W double-mutation were effective to increase the introduction efficiency through the NEXT-A reaction. Protein specific fluorescence labelling via the NEXT-A reaction followed by Huisgen cycloaddition was also demonstrated.

Aberrant Epigenetic Alterations of Glutathione-S-Transferase P1 in Age-Related Nuclear Cataract.

PubMed

Chen, Jia; Zhou, Jing; Wu, Jian; Zhang, Guowei; Kang, Lihua; Ben, Jindong; Wang, Yong; Qin, Bai; Guan, Huaijin

2017-03-01

Oxidative damage of lens tissue contributes to the formation of age-related cataract. Pi-class glutathione-S-transferase (GSTP1) plays a role in the removal of oxidative adducts by transferring them to glutathione. To assess epigenetic regulation of GSTP1 and its potential role in age-related nuclear cataract (ARNC) pathogenesis, we evaluated GSTP1 mRNA expression, methylation, and chromatin modifications in lenses from ARNC patients. The mRNA and protein of lens GSTP1 were assayed by relative quantitative real-time polymerase chain reaction (qRT-PCR) and Western blots. Methylation of the GSTP1 promoter was determined by bisulfite genomic sequencing. Chromatin modification was detected by chromatin immunoprecipitation. DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities were also assayed by enzyme-linked immunosorbent assay (ELISA)-like reaction. To assess the effect of DNA methylation on the mRNA expression of GSTP1, human lens epithelium HLE-B3 cells were treated with the demethylation compound 5-aza-dC, followed by qRT-PCR assay. GSTP1 mRNA and protein levels were significantly reduced in lens epithelium and cortex of ARNC cases versus age-matched controls. The changes corresponded to hypermethylation of the GSTP1 promoter CpG islands. The loss of GSTP1 mRNA and protein and the increased DNA promoter methylation might be correlated with the severity of the ARNC. ARNC lenses also had lower acetylation of histone proteins H3, H4, and lower methylation of H3K4, and higher methylation of H3K9. Histone modifications were not correlated with the severity of the ARNCs. DNMT and HDAC were elevated in lenses from ARNCs compared with controls. Demethylation treatment of HLE-B3 cells with 5-aza-dC enhanced the expression of GSTP1. Epigenetic alteration of GSTP1 regulates its expression in lens epithelial and cortical tissues. These changes likely contribute to the pathogenesis of ARNC.

Glutathione S-transferase pi expression regulates the Nrf2-dependent response to hormetic diselenides.

PubMed

Bartolini, D; Commodi, J; Piroddi, M; Incipini, L; Sancineto, L; Santi, C; Galli, F

2015-11-01

Glutathione S-transferase pi (GSTP), a phase II gene downstream of the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant-responsive element (ARE)/electrophile response element (EpRE) transcription pathway, plays a key role in both the signaling and detoxification response to Se-organic compounds with thiol peroxidase activity. We here investigated the role of GSTP on the Nrf2 activation response of cells challenged with a new class of diselenides derived from the basic structure of diphenyl diselenide [(PhSe)2]. These diselenides, and particularly 2,2'-diselenyl dibenzoic acid (DSBA), behave as mild thiol peroxidases leading to a moderate generation of H2O2 and NOx, and signaling of stress-activated and survival-promoting MAPKs, which ultimately control the mitochondrial pathway of apoptosis. Used in murine embryonic fibroblasts (MEFs) and HepG2 human hepatocarcinoma cells to produce submaximal conditions of stress, the diselenide compounds stimulated Nrf2 nuclear translocation and then the transcription of the same Nrf2 gene as well as of GSTP and other phase II genes. This resulted in a higher degree of protection against H2O2 cytotoxicity (hormetic effect). Diselenide toxicity increased in GSTP knockout MEFs by a higher generation of NOx and stress activated protein kinase (SAPK)/JNK activation. A lowered hormetic potential of these cells was observed in association with an abnormal expression and nuclear translocation of Nrf2 protein. Immunoprecipitation and affinity purification experiments revealed the existence of an Nrf2/GSTP complex in MEFs and HepG2 cells. Covalent oligomers of GSTP subunits were observed in DSBA-treated HepG2 cells. In conclusion, GSTP gene expression influences the Nrf2-dependent response to hormetic diselenides. Mechanistic interpretation for this GSTP-dependent effect may include a direct and redox-sensitive interaction of GSTP with Nrf2 protein. Copyright © 2015 Elsevier Inc. All rights reserved.

Glutathione S-transferase expression and isoenzyme composition during cell differentiation of Caco-2 cells.

PubMed

Scharmach, E; Hessel, S; Niemann, B; Lampen, A

2009-11-30

The human colon adenocarcinoma cell line Caco-2 is frequently used to study human intestinal metabolism and transport of xenobiotica. Previous studies have shown that both Caco-2 cells and human colon cells constitutively express the multigene family of detoxifying enzymes glutathione S-transferases (GSTs), particularly GST alpha and GST pi. GSTs may play a fundamental role in the molecular interplay between phase I, II enzymes and ABC-transporters. The gut fermentation product, butyrate, can modulate the potential for detoxification. The aim of this study was to investigate the basal expression of further cytosolic GSTs in Caco-2 cells during cell differentiation. In addition, a comparison was made with expression levels in MCF-7 and HepG2, two other cell types with barrier functions. Finally, the butyrate-mediated modulation of gene and protein expression was determined by real time PCR and western blot analysis. In Caco-2, gene and protein expression levels of GST alpha increased during cell differentiation. High levels of GSTO1 and GSTP1 were constantly expressed. No expression of GSTM5 and GSTT1 was detected. HepG2 expressed GSTO1 and MCF-7 GSTZ1 most intensively. No expression of GSTA5, GSTM5, or GSTP1 was detected in either cell. Incubation of Caco-2 cells with butyrate (5 mM) significantly induced GSTA1 and GSTM2 in proliferating Caco-2 cells. In differentiated cells, butyrate tended to increase GSTO1 and GSTP1. The results of this study show that a differentiation-dependent expression of GSTs in Caco-2 cells may reflect the in vivo situation and indicate the potential of butyrate to modify intestinal metabolism. GSTA1-A4 have been identified as good markers for cell differentiation. The Caco-2 cell line is a useful model for assessing the potential of food-related substances to modulate the GST expression pattern.

Glutathione transferases, regulators of cellular metabolism and physiology.

PubMed

Board, Philip G; Menon, Deepthi

2013-05-01

The cytosolic glutathione transferases (GSTs) comprise a super family of proteins that can be categorized into multiple classes with a mixture of highly specific and overlapping functions. The review covers the genetics, structure and function of the human cytosolic GSTs with particular attention to their emerging roles in cellular metabolism. All the catalytically active GSTs contribute to the glutathione conjugation or glutathione dependant-biotransformation of xenobiotics and many catalyze glutathione peroxidase or thiol transferase reactions. GSTs also catalyze glutathione dependent isomerization reactions required for the synthesis of several prostaglandins and steroid hormones and the catabolism of tyrosine. An increasing body of work has implicated several GSTs in the regulation of cell signaling pathways mediated by stress-activated kinases like Jun N-terminal kinase. In addition, some members of the cytosolic GST family have been shown to form ion channels in intracellular membranes and to modulate ryanodine receptor Ca(2+) channels in skeletal and cardiac muscle. In addition to their well established roles in the conjugation and biotransformation of xenobiotics, GSTs have emerged as significant regulators of pathways determining cell proliferation and survival and as regulators of ryanodine receptors that are essential for muscle function. This article is part of a Special Issue entitled Cellular functions of glutathione. Copyright © 2012 Elsevier B.V. All rights reserved.

Association of Angiotensin-Converting Enzyme and Glutathione S-Transferase Gene Polymorphisms with Body Mass Index among Hypertensive North Indians

PubMed Central

Rizvi, Saliha; Raza, Syed T.; Siddiqi, Zeba; Abbas, Shania; Mahdi, Farzana

2015-01-01

Objectives: This study aimed to examine the association of angiotensin-converting enzyme (ACE) and glutathione S-transferase (GST) gene polymorphisms with body mass index (BMI) in hypertensive North Indians. Methods: This case-control study was carried out between May 2013 and November 2014 at the Era’s Lucknow Medical College & Hospital, Lucknow, India, and included 378 subjects divided into three groups. One group constituted 253 hypertensive individuals (sustained diastolic blood pressure of >90 mmHg and systolic blood pressure of >140 mmHg) who were subcategorised according to normal (<25 kg/m2) or high (≥25 kg/m2) BMI. The third group consisted of 125 age-, gender- and ethnically-matched normotensive controls with a normal BMI. Gene polymorphisms were evaluated by polymerase chain reaction. The genotypic and allelic frequency distribution among both groups were analysed. Results: A significant difference was found between GST theta 1-null and GST mu 1-positive genotype frequencies among the hypertensive overweight/obese individuals and controls (P = 0.014 and 0.033, respectively). However, no difference was observed in the frequency of ACE polymorphisms. ACE insertion/insertion genotype (P = 0.006), insertion and deletion alleles (P = 0.007 each) and GST theta 1-null and GST theta 1-positive genotypes (P = 0.006 each) were found to differ significantly between hypertensive cases and controls, regardless of BMI. Conclusion: ACE and GST gene polymorphisms were not associated with BMI but were significantly associated with hypertension among the studied group of North Indians. PMID:26629373

Identification of aldo-keto reductase (AKR7A1) and glutathione S-transferase pi (GSTP1) as novel renal damage biomarkers following exposure to mercury.

PubMed

Shin, Y-J; Kim, K-A; Kim, E-S; Kim, J-H; Kim, H-S; Ha, M; Bae, O-N

2017-01-01

The kidney is one of the main targets for toxicity induced by xenobiotics. Sensitive detection of early impairment is critical to assess chemical-associated renal toxicity. The aim of this study was to identify potential nephrotoxic biomarkers in rat kidney tissues after exposure to mercury (Hg), a representative nephrotoxicant, and to evaluate these new biomarkers employing in vivo and in vitro systems. Mercuric chloride was administered orally to Sprague-Dawley rats for 2 weeks. Proteomic analysis revealed that aldo-keto reductase (AKR7A1) and glutathione S-transferase pi (GSTP1) were significantly elevated in kidney after Hg exposure. While the levels of conventional nephrotoxic clinical markers including blood urea nitrogen and serum creatinine were not elevated, the mRNA and protein levels of AKR7A1 and GSTP1 were increased upon Hg exposure in a dose-dependent manner. The increases in AKR7A1 and GSTP1 were also observed in rat kidneys after an extended exposure for 6 weeks to low-dose Hg. In in vitro rat kidney proximal tubular cells, changes in AKR7A1 and GSTP1 levels correlated well with the extent of cytotoxicity induced by Hg, cadmium, or cisplatin. AKR7A1 and GSTP1 were identified as new candidates for Hg-induced nephrotoxicity, suggesting that these biomarkers have potential for evaluating or predicting nephrotoxicity.

Mechanistic insights into EgGST1, a Mu class glutathione S-transferase from the cestode parasite Echinococcus granulosus.

PubMed

Arbildi, Paula; Turell, Lucía; López, Verónica; Alvarez, Beatriz; Fernández, Verónica

2017-11-01

Glutathione transferases (GSTs) comprise a major detoxification system in helminth parasites, displaying both catalytic and non-catalytic activities. The kinetic mechanism of these enzymes is complex and depends on the isoenzyme which is being analyzed. Here, we characterized the kinetic mechanism of rEgGST1, a recombinant form of a cytosolic GST from Echinococcus granulosus (EgGST1), which is related to the Mu-class of mammalian enzymes, using the canonical substrates glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Initial rate and product inhibition studies were consistent with a steady-state random sequential mechanism, where both substrates are bound to the enzyme before the products are released. Kinetic constants were also determined (pH 6.5 and 30 °C). Moreover, rEgGST1 lowered the pK a of GSH from 8.71 ± 0.07 to 6.77 ± 0.08, and enzyme-bound GSH reacted with CDNB 1 × 10 5 times faster than free GSH at pH 7.4. Finally, the dissociation of the enzyme-GSH complex was studied by means of intrinsic fluorescence, as well as that of the complex with the anthelminth drug mebendazole. This is the first report on mechanistic issues related to a helminth parasitic GST. Copyright © 2017 Elsevier Inc. All rights reserved.

Dual Lifetimes for Complexes between Glutathione-S-transferase (hGSTA1-1) and Product-like Ligands Detected by Single-Molecule Fluorescence Imaging.

PubMed

Pettersson, John R; Lanni, Frederick; Rule, Gordon S

2017-08-08

Single-molecule fluorescence techniques were used to characterize the binding of products and inhibitors to human glutathione S-transferase A1-1 (hGSTA1-1). The identification of at least two different bound states for the wild-type enzyme suggests that there are at least two conformations of the protein, consistent with the model that ligand binding promotes closure of the carboxy-terminal helix over the active site. Ligand induced changes in ensemble fluorescence energy transfer support this proposed structural change. The more predominant state in the ensemble of single molecules shows a significantly faster off-rate, suggesting that the carboxy-terminal helix is delocalized in this state, permitting faster exit of the bound ligand. A point mutation (I219A), which is known to interfere with the association of the carboxy-terminal helix with the enzyme, shows increased rates of interconversion between the open and closed state. Kinematic traces of fluorescence from single molecules show that a single molecule readily samples a number of different conformations, each with a characteristic off-rate.

Driving carbon flux through exogenous butyryl-CoA: Acetate CoA-transferase to produce butyric acid at high titer in Thermobifida fusca.

PubMed

Deng, Yu; Mao, Yin; Zhang, Xiaojuan

2015-12-20

Butyric acid, a 4-carbon short chain fatty acid, is widely used in chemical, food, and pharmaceutical industries. The low activity of butyryl-CoA: acetate CoA-transferase in Thermobifida fusca muS, a thermophilic actinobacterium whose optimal temperature was 55°C, was found to hinder the accumulation of high yield of butyric acid. In order to solve this problem, an exogenous butyryl-CoA: acetate CoA-transferase gene (actA) from Thermoanaerobacterium thermosaccharolyticum DSM571 was integrated into the chromosome of T. fusca muS by replacing celR gene, forming T. fusca muS-1. We demonstrated that on 5g/L cellulose, the yield of butyric acid by the engineered muS-1 strain was increased by 42.9 % compared to the muS strain. On 100g/L of cellulose, the muS-1 strain could consume 90.5% of total cellulose in 144h, with 33.2g/L butyric acid produced. Furthermore, on the mix substrates including the major components of biomass: cellulose, xylose, mannose and galactose, 70.4g/L butyric acid was produced in 168h by fed-batch fermentation. To validate the ability of fermenting biomass, the muS-1 strain was grown on the milled corn stover ranging from 200 to 250μm. The muS-1 strain had the highest butyrate titer 17.1g/L on 90g/L corn stover. Copyright © 2015 Elsevier B.V. All rights reserved.

Dietary isothiocyanates, glutathione S-transferase M1 (GSTM1), and lung cancer risk in African Americans and Caucasians from Los Angeles County, California.

PubMed

Carpenter, Catherine L; Yu, Mimi C; London, Stephanie J

2009-01-01

Isothiocyanates, found in cruciferous vegetables, are anticarcinogenic. Racial differences in smoking do not fully account for the African-American excess lung cancer incidence. African Americans consume more cruciferous vegetables than U.S. Whites. Impact on lung cancer risk is unknown. The glutathione S transferase M1 (GSTM1) gene promotes urinary isothiocyanate excretion. We evaluated dietary isothiocyanates and lung cancer using a population-based case-control study of 933 African Americans and Caucasians (non-Hispanic U.S. White) from Los Angeles County, California (311 cases; 622 controls). Broccoli, cauliflower, greens, and cabbage food-frequency variables represented isothiocyanates. Isothiocyanates were protective for lung cancer risk. Adjusted odds ratio (OR) for the uppermost quartile > 80 micro mol isothiocyanates/wk, compared to lowest, was 0.65 [95% confidence interval (CI) = 0.41-1.00, trend P = 0.02]. Association was stronger among subjects with homozygous deletion of GSTM1 (OR = 0.52, 95% CI = 0.31-0.86) than subjects with at least one GSTM1 copy (OR = 0.77, 95% CI = 0.49-1.21). The difference was not statistically significant (P = 0.16). Despite African Americans consuming more cruciferous vegetables, the isothiocyanate association did not vary by race (P = 0.52). Reduced lung cancer risk with higher isothiocyanate intake may be slightly stronger among subjects with deletion of GSTM1.

The Glutathione-S-Transferase, Cytochrome P450 and Carboxyl/Cholinesterase Gene Superfamilies in Predatory Mite Metaseiulus occidentalis

PubMed Central

Hoy, Marjorie A.

2016-01-01

Pesticide-resistant populations of the predatory mite Metaseiulus (= Typhlodromus or Galendromus) occidentalis (Arthropoda: Chelicerata: Acari: Phytoseiidae) have been used in the biological control of pest mites such as phytophagous Tetranychus urticae. However, the pesticide resistance mechanisms in M. occidentalis remain largely unknown. In other arthropods, members of the glutathione-S-transferase (GST), cytochrome P450 (CYP) and carboxyl/cholinesterase (CCE) gene superfamilies are involved in the diverse biological pathways such as the metabolism of xenobiotics (e.g. pesticides) in addition to hormonal and chemosensory processes. In the current study, we report the identification and initial characterization of 123 genes in the GST, CYP and CCE superfamilies in the recently sequenced M. occidentalis genome. The gene count represents a reduction of 35% compared to T. urticae. The distribution of genes in the GST and CCE superfamilies in M. occidentalis differs significantly from those of insects and resembles that of T. urticae. Specifically, we report the presence of the Mu class GSTs, and the J’ and J” clade CCEs that, within the Arthropoda, appear unique to Acari. Interestingly, the majority of CCEs in the J’ and J” clades contain a catalytic triad, suggesting that they are catalytically active. They likely represent two Acari-specific CCE clades that may participate in detoxification of xenobiotics. The current study of genes in these superfamilies provides preliminary insights into the potential molecular components that may be involved in pesticide metabolism as well as hormonal/chemosensory processes in the agriculturally important M. occidentalis. PMID:27467523

Establishing an Evidence-Based Adult Education System. NCSALL Occasional Paper.

ERIC Educational Resources Information Center

Comings, John P.; Beder, Hal; Bingman, Beth; Reder, Stephen; Smith, Cristine

To benefit from the support of public and private sector leaders and to ensure that all students receive effective services, the adult education system must identify program models that have empirical evidence to support claims of effectiveness. The U.S. Department of Education's Institute of Education Sciences defines evidence-based education as…

Association of N-acetyltransferase-2 and glutathione S-transferase polymorphisms with idiopathic male infertility in Vietnam male subjects.

PubMed

Trang, Nguyen Thi; Huyen, Vu Thi; Tuan, Nguyen Thanh; Phan, Tran Duc

2018-04-25

N-acetyltransferase-2 (NAT2) and Glutathione S-transferases (GSTs) are phase-II xenobiotic metabolizing enzymes participating in detoxification of toxic arylamines, aromatic amines, hydrazines and reactive oxygen species (ROS), which are produced under oxidative and electrophile stresses. The purpose of this research was to investigate whether two common single-nucleotide polymorphisms (SNP) of NAT2 (rs1799929, rs1799930) and GSTP1 (rs1138272, rs1695) associated with susceptibility to idiopathic male infertility. A total 300 DNA samples (150 infertile patients and 150 healthy control) were genotyped for the polymorphisms by ARMS - PCR. We revealed a significant association between the NAT2 variant genotypes (CT + TT (rs1799929), (OR: 3.74; p < 0.001)) and (GA + AA (rs1799930), (OR: 3.75; p < 0.001)) or GSTP1 variant genotypes (GA + AA (rs1695), (OR: 5.11; p < 0,001)) and (CT + TT (rs1138272), (OR: 7.42; p < 0,001) with idiopathic infertility risk. Our findings rate the effect of single-nucleotide polymorphisms of GSTP1 and/or NAT2 in modulation of the risk of male infertility in subjects from Vietnam. This pilot study is the first (as far as we know) to reveal that polymorphisms of NAT2 (rs1799929, rs1799930) and GSTP1 (rs1138272, rs1695) are some novel genetic markers for susceptibility to idiopathic male infertility. Copyright © 2018 Elsevier B.V. All rights reserved.

Inhibition of P-glycoprotein and glutathione S-transferase-pi mediated resistance by fluoxetine in MCF-7/ADM cells.

PubMed

Zhang, Ye; Zhou, Ting; Duan, Jingjing; Xiao, Zhijun; Li, Guihua; Xu, Feng

2013-10-01

Chemotherapy is important in the systematic treatment of breast cancer. While multidrug resistance (MDR) is the main obstacle in chemotherapy, a reversal reagent with high reversal effect but low toxicity is the hotspot issue at present to overcome MDR. Antidepressant fluoxetine (FLX) is a potential new highly effective chemosensitizer, however, the possible mechanism is unclear. In this study, the effect of FLX on multidrug resistance mediated by P-glycoprotein (P-gp) and glutathione S-transferase-pi (GST-π) were researched in resistant/sensitive breast cancer cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to determine the cells viability after being incubated with FLX/Adriamycin (ADM)/Paclitaxel (PTX) alone or FLX-ADM, FLX-PTX combination. Western blot was performed to assay the expression of P-gp and GST-π proteins. Reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) were performed to assay the level of MDR1 mRNA. The results showed that pre-treatment with FLX enhance cytotoxicity significantly both on resistant and sensitive cells, downregulated the expression of P-gp and GST-π proteins in resistance cells, decreased the MDR1 mRNA by FLX-PTX combination only. No P-gp and GST-π were detected in sensitive cells. Our research thus indicated that FLX reverse the breast cancer cell's resistance and enhance the chemosensitivity by regulating P-gp and GST-π levels. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Nuclear glutathione S-transferase pi prevents apoptosis by reducing the oxidative stress-induced formation of exocyclic DNA products.

PubMed

Kamada, Kensaku; Goto, Shinji; Okunaga, Tomohiro; Ihara, Yoshito; Tsuji, Kentaro; Kawai, Yoshichika; Uchida, Koji; Osawa, Toshihiko; Matsuo, Takayuki; Nagata, Izumi; Kondo, Takahito

2004-12-01

We previously found that nuclear glutathione S-transferase pi (GSTpi) accumulates in cancer cells resistant to anticancer drugs, suggesting that it has a role in the acquisition of resistance to anticancer drugs. In the present study, the effect of oxidative stress on the nuclear translocation of GSTpi and its role in the protection of DNA from damage were investigated. In human colonic cancer HCT8 cells, the hydrogen peroxide (H(2)O(2))-induced increase in nuclear condensation, the population of sub-G(1) peak, and the number of TUNEL-positive cells were observed in cells pretreated with edible mushroom lectin, an inhibitor of the nuclear transport of GSTpi. The DNA damage and the formation of lipid peroxide were dependent on the dose of H(2)O(2) and the incubation time. Immunological analysis showed that H(2)O(2) induced the nuclear accumulation of GSTpi but not of glutathione peroxidase. Formation of the 7-(2-oxo-hepyl)-substituted 1,N(2)-etheno-2'-deoxyguanosine adduct by the reaction of 13-hydroperoxyoctadecadienoic acid (13-HPODE) with 2'-deoxyguanosine was inhibited by GSTpi in the presence of glutathione. The conjugation product of 4-oxo-2-nonenal, a lipid aldehyde of 13-HPODE, with GSH in the presence of GSTpi, was identified by LS/MS. These results suggested that nuclear GSTpi prevents H(2)O(2)-induced DNA damage by scavenging the formation of lipid-peroxide-modified DNA.

Structural snapshots along the reaction pathway of Yersinia pestis RipA, a putative butyryl-CoA transferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Torres, Rodrigo; Lan, Benson; Latif, Yama

2014-04-01

The crystal structures of Y. pestis RipA mutants were determined to provide insights into the CoA transferase reaction pathway. Yersinia pestis, the causative agent of bubonic plague, is able to survive in both extracellular and intracellular environments within the human host, although its intracellular survival within macrophages is poorly understood. A novel Y. pestis three-gene rip (required for intracellular proliferation) operon, and in particular ripA, has been shown to be essential for survival and replication in interferon γ-induced macrophages. RipA was previously characterized as a putative butyryl-CoA transferase proposed to yield butyrate, a known anti-inflammatory shown to lower macrophage-produced NOmore » levels. RipA belongs to the family I CoA transferases, which share structural homology, a conserved catalytic glutamate which forms a covalent CoA-thioester intermediate and a flexible loop adjacent to the active site known as the G(V/I)G loop. Here, functional and structural analyses of several RipA mutants are presented in an effort to dissect the CoA transferase mechanism of RipA. In particular, E61V, M31G and F60M RipA mutants show increased butyryl-CoA transferase activities when compared with wild-type RipA. Furthermore, the X-ray crystal structures of E61V, M31G and F60M RipA mutants, when compared with the wild-type RipA structure, reveal important conformational changes orchestrated by a conserved acyl-group binding-pocket phenylalanine, Phe85, and the G(V/I)G loop. Binary structures of M31G RipA and F60M RipA with two distinct CoA substrate conformations are also presented. Taken together, these data provide CoA transferase reaction snapshots of an open apo RipA, a closed glutamyl-anhydride intermediate and an open CoA-thioester intermediate. Furthermore, biochemical analyses support essential roles for both the catalytic glutamate and the flexible G(V/I)G loop along the reaction pathway, although further research is required to fully

The Drosophila protein palmitoylome: Characterizing palmitoyl-thioesterases and DHHC palmitoyl-transferases

PubMed Central

Bannan, Barbra A.; Van Etten, Jamie; Kohler, John A.; Tsoi, Yui; Hansen, Nicole M.; Sigmon, Stacey; Fowler, Elizabeth; Buff, Haley; Williams, Tiffany S.; Ault, Jeffrey G.; Glaser, Robert L.; Korey, Christopher A.

2010-01-01

Palmitoylation is the post-translational addition of a palmitate moiety to a cysteine residue through a covalent thioester bond. The addition and removal of this modification is controlled by both palmitoyl acyl-transferases and thioesterases. Using bioinformatic analysis, we identified 22 DHHC family palmitoyl acyl-transferase homologs in the Drosophila genome. We used in situ hybridization, RT-PCR, and published FlyAtlas microarray data to characterize the expression patterns of all 22 fly homologs. Our results indicate that all are expressed genes, but several, including CG1407, CG4676, CG5620, CG6017/dHIP14, CG6618, CG6627, and CG17257 appear to be enriched in neural tissues suggesting that they are important for neural function. Furthermore, we have found that several may be expressed in a sex-specific manner with adult male-specific expression of CG4483 and CG17195. Using tagged versions of the DHHC genes, we demonstrate that fly DHHC proteins are primarily located in either the Golgi Apparatus or Endoplasmic Reticulum in S2 cells, except for CG1407, which was found on the plasma membrane. We also characterized the subcellular localization and expression of the three known thioesterases: Palmitoyl-protein Thioesterase 1 (Ppt1), Palmitoyl-protein Thioesterase 2 (Ppt2), and Acyl-protein Thioesterase 1 (APT1). Our results indicate that Ppt1 and Ppt2 are the major lysosomal thioesterases while APT1 is the likely cytoplasmic thioesterase. Finally, in vivo rescue experiments show that Ppt2 expression cannot rescue the neural inclusion phenotypes associated with loss of Ppt1, further supporting distinct functions and substrates for these two thioesterases. These results will serve as the basis for a more complete understanding of the protein palmitoylome's normal cellular functions in the fly and will lead to further insights into the molecular etiology of diseases associated with the mis-regulation of palmitoylation. PMID:18719403

An analysis of Methylenetetrahydrofolate reductase and Glutathione S-transferase omega-1 genes as modifiers of the cerebral response to ischemia

PubMed Central

Peddareddygari, Leema Reddy; Dutra, Ana Virginia; Levenstien, Mark A; Sen, Souvik; Grewal, Raji P

2009-01-01

Background Cerebral ischemia involves a series of reactions which ultimately influence the final volume of a brain infarction. We hypothesize that polymorphisms in genes encoding proteins involved in these reactions could act as modifiers of the cerebral response to ischemia and impact the resultant stroke volume. The final volume of a cerebral infarct is important as it correlates with the morbidity and mortality associated with non-lacunar ischemic strokes. Methods The proteins encoded by the methylenetetrahydrofolate reductase (MTHFR) and glutathione S-transferase omega-1 (GSTO-1) genes are, through oxidative mechanisms, key participants in the cerebral response to ischemia. On the basis of these biological activities, they were selected as candidate genes for further investigation. We analyzed the C677T polymorphism in the MTHFR gene and the C419A polymorphism in the GSTO-1 gene in 128 patients with non-lacunar ischemic strokes. Results We found no significant association of either the MTHFR (p = 0.72) or GSTO-1 (p = 0.58) polymorphisms with cerebral infarct volume. Conclusion Our study shows no major gene effect of either the MTHFR or GSTO-1 genes as a modifier of ischemic stroke volume. However, given the relatively small sample size, a minor gene effect is not excluded by this investigation. PMID:19624857

Glutathione S-transferase Pi expression predicts response to adjuvant chemotherapy for stage C colon cancer: a matched historical control study.

PubMed

Jankova, Lucy; Robertson, Graham; Chan, Charles; Tan, King L; Kohonen-Corish, Maija; Fung, Caroline L-S; Clarke, Candice; Lin, Betty P C; Molloy, Mark; Chapuis, Pierre H; Bokey, Les; Dent, Owen F; Clarke, Stephen J

2012-05-28

This study examined the association between overall survival and Glutathione S-transferase Pi (GST Pi) expression and genetic polymorphism in stage C colon cancer patients after resection alone versus resection plus 5-fluourouracil-based adjuvant chemotherapy. Patients were drawn from a hospital registry of colorectal cancer resections. Those receiving chemotherapy after it was introduced in 1992 were compared with an age and sex matched control group from the preceding period. GST Pi expression was assessed by immunohistochemistry. Overall survival was analysed by the Kaplan-Meier method and Cox regression. From an initial 104 patients treated with chemotherapy and 104 matched controls, 26 were excluded because of non-informative immunohistochemistry, leaving 95 in the treated group and 87 controls. Survival did not differ significantly among patients with low GST Pi who did or did not receive chemotherapy and those with high GST Pi who received chemotherapy (lowest pair-wise p = 0.11) whereas patients with high GST Pi who did not receive chemotherapy experienced markedly poorer survival than any of the other three groups (all pair-wise p <0.01). This result was unaffected by GST Pi genotype. Stage C colon cancer patients with low GST Pi did not benefit from 5-fluourouracil-based adjuvant chemotherapy whereas those with high GST Pi did.

Assessment of cumulative evidence for the association between glutathione S-transferase polymorphisms and lung cancer: application of the Venice interim guidelines

PubMed Central

Langevin, Scott M.; Ioannidis, John P.A.; Vineis, Paolo; Taioli, Emanuela

2010-01-01

There is an overwhelming abundance of genetic association studies available in the literature, which often can be collectively difficult to interpret. To address this issue, the Venice interim guidelines were established for determining the credibility of the cumulative evidence. The objective of this report is to evaluate the literature on the association of common GST variants (GSTM1 null, GSTT1 null and GSTP1 Ile105Val polymorphism) and lung cancer, and to assess the credibility of the associations using the newly proposed cumulative evidence guidelines. Information from the literature was enriched with an updated meta-analysis and a pooled analysis using data from the Genetic Susceptibility to Environmental Carcinogens (GSEC) database. There was a significant association between GSTM1 null and lung cancer for the meta- (meta OR = 1.17, 95% CI: 1.10–1.25) and pooled analysis (adjusted OR = 1.10, 95% CI: 1.04–1.16), although substantial heterogeneity was present. No overall association between lung cancer and GSTT1 null or GSTP1 Ile105Val was found. When the Venice criteria was applied, cumulative evidence for all associations were considered “weak”, with the exception of East Asian carriers of the G allele of GSTP1 Ile105Val, which was graded as “moderate” evidence. In spite of large amounts of studies, and several statistically significant summary estimates produced by meta-analyses, the application of the Venice criteria suggests extensive heterogeneity and susceptibility to bias for the studies on association of common genetic polymorphisms, such as with GST variants and lung cancer. PMID:20729793

Glutathione S-transferase genes and the risk of type 2 diabetes mellitus: Role of sexual dimorphism, gene-gene and gene-smoking interactions in disease susceptibility.

PubMed

Azarova, Iuliia; Bushueva, Olga; Konoplya, Alexander; Polonikov, Alexey

2018-05-01

Compromised defense against reactive oxygen species (ROS) is considered important in the pathogenesis of type 2 diabetes mellitus (T2DM); therefore, genes encoding antioxidant defense enzymes may contribute to disease susceptibility. This study investigated whether polymorphisms in genes encoding glutathione S-transferase M1 (GSTM1), T1 (GSTT1), and P1 (GSTP1) jointly contribute to the risk of T2DM. In all, 1120 unrelated Russian subjects (600 T2DM patients, 520 age- and sex-matched healthy subjects), were recruited to the study. Genotyping was performed by multiplex polymerase chain reaction (PCR; del/del polymorphisms of GSTM1 and GSTT1) and TaqMan-based PCR (polymorphisms I105V and A114V of GSTP1). Plasma ROS and glutathione levels in study subjects were analyzed by fluorometric and colorimetric assays, respectively. Genotype del/del GSTT1 was significantly associated with the risk of T2DM (odds ratio [OR] 1.60, 95% confidence interval [CI] 1.17-2.21, P = 0.003). Gender-stratified analysis showed that the deletion genotypes of GSTM1 (OR 1.99, 95% CI 1.30-3.05; P = 0.0002, Q = 0.016) and GSTT1 (OR 2.23, 95% CI 1.22-4.09; P = 0.008, Q = 0.0216), as well as genotype 114A/V of GSTP1 (OR 2.85, 95% CI 1.44-5.62; P = 0.005, Q = 0.02) were associated with an increased risk of T2DM exclusively in males. Three genotype combinations (i.e. GSTM1+ × GSTT1+, GSTM1+ × GSTP1 114A/A and GSTT1+ × GSTP1 114A/A) showed significant associations with a decreased risk of T2DM in males. This study demonstrates, for the first time, that genes encoding glutathione S-transferases jointly contribute to the risk of T2DM, and that their effects on disease susceptibility are gender specific. © 2017 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

Effect of Patulin from Penicillium vulpinum on the Activity of Glutathione-S-Transferase and Selected Antioxidative Enzymes in Maize

PubMed Central

Ismaiel, Ahmed A.

2017-01-01

The mycotoxin patulin (PAT) was purified from Penicillium vulpinum CM1 culture that has been isolated from a soil cultivated with maize. The effect of PAT and of a fungal culture filtrate on the activities of glutathione-S-transferase (GST) and some antioxidant enzymes viz. ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) was investigated in roots and shoots of 8-day-old maize seedlings. PAT and culture filtrate caused significant reduction effects in a dose-related manner on the total GST activity. Upon application of the high PAT concentration (25 μg·mL−1) and of the concentrated fungal filtrate (100%, v/v), the reduction in GST activity of roots was 73.8–76.0% and of shoots was 60–61.7%. Conversely, significant increases in the activities of antioxidant enzymes were induced. Application of 25 μg·PAT·mL−1 increased APX, GR, DHAR, and MDHAR activity of root by 2.40-, 2.00-, 1.24-, and 2.16-fold, respectively. In shoots, the enzymatic activity was increased by 1.57-, 1.45-, 1.45-, and 1.61-fold, respectively. Similar induction values of the enzymatic activity were obtained upon application of the concentrated fungal filtrate. This is the first report describing the response of GST and antioxidant enzyme activities of plant cells to PAT toxicity. PMID:28737668

Identification of glutathione S-transferases in Bemisia tabaci (Hemiptera: Aleyrodidae) and evidence that GSTd7 helps explain the difference in insecticide susceptibility between B. tabaci Middle East-Minor Asia 1 and Mediterranean.

PubMed

He, C; Xie, W; Yang, X; Wang, S-L; Wu, Q-J; Zhang, Y-J

2018-02-01

The Bemisia tabaci (Gennadius) (Hemiptera:Aleyrodidae) species complex includes invasive and destructive pests of field crops, and the sibling species MEAM1 and MED are its two most damaging members. Previous research indicated that the replacement of Middle East-Minor Asia 1 (MEAM1) by Mediterranean (MED) as the dominant B. tabaci species in China can be mainly attributed to MED's greater tolerance to insecticides. Glutathione S-transferases (GSTs) play important roles in the detoxification of hydrophobic toxic compounds. To increase our understanding of differences in insecticide resistance between B. tabaci MEAM1 and MED, we searched the genomic and transcriptomic databases and identified 23 putative GSTs in both B. tabaci MEAM1 and MED. Through measuring mRNA levels of 18 of the GSTs after B. tabaci MEAM1 and MED adults were exposed to the insecticide imidacloprid, we found that the expression levels were increased more in B. tabaci MED than in MEAM1 (in particular, the expression level of GST-d7 was increased by 4.39-fold relative to the control). Knockdown of GST-d7 in B. tabaci MED but not in B. tabaci MEAM1 resulted in a substantial increase in the mortality of imidacloprid-treated adults. These results indicate that differences in GST-d7 may help explain why insecticide tolerance is greater in B. tabaci MED than in B. tabaci MEAM1. © 2017 The Royal Entomological Society.

Effect of recombinant glutathione S-transferase as vaccine antigen against Rhipicephalus appendiculatus and Rhipicephalus sanguineus infestation.

PubMed

Sabadin, Gabriela Alves; Parizi, Luís Fernando; Kiio, Irene; Xavier, Marina Amaral; da Silva Matos, Renata; Camargo-Mathias, Maria Izabel; Githaka, Naftaly Wang'ombe; Nene, Vish; da Silva Vaz, Itabajara

2017-12-04

The ticks Rhipicephalus appendiculatus and Rhipicephalus sanguineus are the main vectors of Theileria parva and Babesia spp. in cattle and dogs, respectively. Due to their impact in veterinary care and industry, improved methods against R. appendiculatus and R. sanguineus parasitism are under development, including vaccines. We have previously demonstrated the induction of a cross-protective humoral response against Rhipicephalus microplus following vaccination with recombinant glutathione S-transferase from Haemaphysalis longicornis tick (rGST-Hl), suggesting that this protein could control tick infestations. In the present work, we investigated the effect of rGST-Hl vaccine against R. appendiculatus and R. sanguineus infestation in rabbits. In silico analysis revealed that GST from H. longicornis, R. appendiculatus and R. sanguineus have >80% protein sequence similarity, and multiple conserved antigenic sites. After the second vaccine dose, rGST-Hl-immunized rabbits showed elevated antibody levels which persisted until the end of experiment (75 and 60 days for R. appendiculatus and R. sanguineus, respectively). Western blot assays demonstrated cross-reactivity between anti-rGST-Hl antibodies and native R. appendiculatus and R. sanguineus GST extracts from ticks at different life stages. Vaccination with rGST-Hl decreased the number, weight, and fertility of engorged R. appendiculatus adults, leading to an overall vaccine efficacy of 67%. Interestingly, histological analysis of organ morphology showed damage to salivary glands and ovaries of R. appendiculatus adult females fed on vaccinated animals. In contrast, rGST-Hl vaccination did not affect R. appendiculatus nymphs, and it was ineffective against R. sanguineus across the stages of nymph and adult. Taken together, our results show the potential application of rGST-Hl as an antigen in anti-tick vaccine development, however indicating a broad difference in efficacy among tick species. Copyright © 2017 Elsevier

A novel biomarker for marine environmental pollution of pi-class glutathione S-transferase from Mytilus coruscus.

PubMed

Liu, Huihui; He, Jianyu; Zhao, Rongtao; Chi, Changfeng; Bao, Yongbo

2015-08-01

Glutathione S-transferases (GSTs) are the superfamily of phase II detoxification enzymes that play crucial roles in innate immunity. In this study, a pi-class GST homolog was identified from Mytilus coruscus (named as McGST1, KC525103). The full-length cDNA sequence of McGST1 was 621bp with a 5' untranslated region (UTR) of 70bp and a 3'-UTR of 201bp. The deduced amino acid sequence was 206 residues in length with theoretical pI/MW of 5.60/23.72kDa, containing the conserved G-site and diversiform H-site. BLASTn analysis and phylogenetic relationship strongly suggested that this cDNA sequence was a member of pi class GST family. The prediction of secondary structure displayed a preserved N-terminal and a C-terminal comprised with α-helixes. Quantitative real time RT-PCR showed that constitutive expression of McGST1 was occurred, with increasing order in mantle, muscle, gill, hemocyte, gonad and hepatopancreas. The stimulation of bacterial infection, heavy metals and 180CST could up-regulate McGST1 mRNA expression in hepatopancreas with time-dependent manners. The maximum expression appeared at 6h after pathogenic bacteria injected, with 10-fold in Vibrio alginolyticus and 16-fold in Vibrio harveyi higher than that of the control. The highest point of McGST1 mRNA appeared at different time for exposure to copper (10-fold at day 15), cadmium (9-fold at day10) and 180 CST (10-fold at day 15). These results suggested that McGST1 played a significant role in antioxidation and might potentially be used as indicators and biomarkers for detection of marine environmental pollution. Copyright © 2015 Elsevier Inc. All rights reserved.

Protection against oxidative DNA damage and stress in human prostate by glutathione S-transferase P1

PubMed Central

Kanwal, Rajnee; Pandey, Mitali; Bhaskaran, Natarajan; MacLennan, Gregory T; Fu, Pingfu; Ponsky, Lee E; Gupta, Sanjay

2014-01-01

The pi-class glutathione S-transferase (GSTP1) actively protect cells from carcinogens and electrophilic compounds. Loss of GSTP1 expression via promoter hypermethylation is the most common epigenetic alteration observed in human prostate cancer. Silencing of GSTP1 can increase generation of reactive oxygen species (ROS) and DNA damage in cells. In this study we investigated whether loss of GSTP1 contributes to increased DNA damage that may predispose men to a higher risk of prostate cancer. We found significantly elevated (103%; P<0.0001) levels of 8-oxo-2′-deoxogunosine (8-OHdG), an oxidative DNA damage marker, in adenocarcinomas, compared to benign counterparts, which positively correlated (r=0.2) with loss of GSTP1 activity (34%; P<0.0001). Silencing of GSTP1 using siRNA approach in normal human prostate epithelial RWPE1 cells caused increased intracellular production of ROS and higher susceptibility of cells to H2O2-mediated oxidative stress. Additionally, human prostate carcinoma LNCaP cells, which contain a silenced GSTP1 gene, were genetically modified to constitutively express high levels of GSTP1. Induction of GSTP1 activity lowered endogenous ROS levels in LNCaP-pLPCX-GSTP1 cells, and when exposed to H2O2, these cells exhibited significantly reduced production of ROS and 8-OHdG levels, compared to vector control LNCaP-pLPCX cells. Furthermore, exposure of LNCaP cells to green tea polyphenols caused re-expression of GSTP1, which protected the cells from H2O2-mediated DNA damage through decreased ROS production compared to non-exposed cells. These results suggest that loss of GSTP1 expression in human prostate cells, a process that increases their susceptibility to oxidative stress-induced DNA damage, may be an important target for primary prevention of prostate cancer. PMID:22833520

The relationship of glutathione-S-transferases copy number variation and indoor air pollution to symptoms and markers of respiratory disease.

PubMed

Hersoug, Lars-Georg; Brasch-Andersen, Charlotte; Husemoen, Lise Lotte Nystrup; Sigsgaard, Torben; Linneberg, Allan

2012-07-01

Exposure to particulate matter (PM) may induce inflammation and oxidative stress in the airways. Carriers of null polymorphisms of glutathione S-transferases (GSTs), which detoxify reactive oxygen species, may be particularly susceptible to the effects of PM. To investigate whether deletions of GSTM1 and GSTT1 modify the potential effects of exposure to indoor sources of PM on symptoms and objective markers of respiratory disease. We conducted a population-based, cross-sectional study of 3471 persons aged 18-69 years. Information about exposure to indoor sources of PM and respiratory symptoms was obtained by a self-administered questionnaire. In addition, measurements of lung function (spirometry) and fractional exhaled nitric oxide were performed. Copy number variation of GSTM1 and GSTT1 was determined by polymerase chain reaction-based assays. We found that none of the symptoms and objective markers of respiratory disease were significantly associated with the GST null polymorphisms. An increasing number of positive alleles of the GSTM1 polymorphism tended to be associated lower prevalence of wheeze, cough, and high forced expiratory volume in 1 s (FEV(1) ), but these trends were not statistically significant. Furthermore, we did not observe any statistically significant interactions between GST copy number variation and exposure to indoor sources of PM in relation to respiratory symptoms and markers. In this adult population, GST copy number variations were not significantly associated with respiratory outcomes and did not modify the effects of self-reported exposure to indoor sources of PM on respiratory outcomes. © 2011 Blackwell Publishing Ltd.

Purification, molecular cloning and heterologous expression of a glutathione S-transferase involved in insecticide resistance from the rice brown planthopper, Nilaparvata lugens.

PubMed

Vontas, John G; Small, Graham J; Nikou, Dimitra C; Ranson, Hilary; Hemingway, Janet

2002-03-01

A novel glutathione S-transferase (GST)-based pyrethroid resistance mechanism was recently identified in Nilaparvata lugens [Vontas, Small and Hemingway (2001) Biochem. J. 357, 65-72]. To determine the nature of GSTs involved in conferring this resistance, the GSTs from resistant and susceptible strains of N. lugens were partially purified by anion exchange and affinity chromatography. The majority of peroxidase activity, previously correlated with resistance, was confined to the fraction that bound to the affinity column, which was considerably elevated in the resistant insects. A cDNA clone encoding a GST (nlgst1-1) - the first reported GST sequence from Hemiptera with up to 54% deduced amino-acid identity with other insect class I GSTs - was isolated from a pyrethroid-resistant strain. Northern analysis showed that nlgst1-1 was overexpressed in resistant insects. nlgst1-1 was expressed in Escherichia coli, purified and characterized. The ability of the recombinant protein to bind to the S-hexylglutathione affinity matrix, its substrate specificities and its immunological properties confirmed that this GST was one from the elevated subset of N. lugens GSTs. Peroxidase activity of the recombinant nlgst1-1 indicated that it had a role in resistance, through detoxification of lipid peroxidation products induced by pyrethroids. Southern analysis of genomic DNA from the resistant and susceptible strains indicated that GST-based insecticide resistance may be associated with gene amplification in N. lugens.

Testing GSTP1 genotypes and haplotypes interactions in Slovenian post-/pre-menopausal women: novel involvement of glutathione S-transferases in bone remodeling process.

PubMed

Mlakar, Simona Jurkovic; Prezelj, Janez; Marc, Janja

2012-02-01

Osteoporosis (OP) is an age-related disease associated with increased production of reactive oxygen species (ROS) and a reduction in antioxidant defense system, such as low activity of glutathione S-transferase (GST) family. The enzyme activity of the member of GSTs, GSTP1, depends on gene polymorphisms such as: Ala114Val and Ile105Val. The aim of this study was to evaluate the association between genetic polymorphisms of the GSTP1 gene and BMD variation and biochemical bone remodeling markers in 523 Slovenian pre- and post-menopausal women. Observational pilot study in a representative cohort of Slovenian patients with adjustment for potential confounders (age, height, weight, years since menopause, smoking status and glucocorticoid use) using univariate one-way and two-way analyses. Ala114Val and Ile105Val polymorphisms genotypes of GSTP1 gene, bone mineral density (BMD) values of total hip (_th), femoral neck (_fn) and lumbar spine (_ls), plasma osteocalcin (OC), serum bone alkaline phosphatase (BALP), free soluble RANKL and serum osteoprotegerin (sOPG) concentrations were determined. Our results show that the Ala114Val heterozygotes are (borderline) significantly associated with higher concentrations of pOC (p=0.052) and decreased BMD_fn values (p=0.053) and the same trend is shown for BMD_th and BMD_ls values in osteopenic postmenopausal women. Furthermore, significantly higher concentrations of pOC were determined among Val allele carriers of Ile105Val gene polymorphism (p=0.037) and in carriers with the absent 114Ala-105Ile haplotype combination, again in osteopenic post-menopausal women. In addition, in pre-menopausal women the significant associations between sOPG and Ala114Val genotypes subgroups and between sBALP and Ile105Val genotypes subgroups, alone or in combination with Ala114Val, were determined (0.032, 0.026 and 0.008, respectively). Since significant associations existed in Ala114Val genotype and 114Ala-105Ile haplotype subgroups, these

Interactions of GST Polymorphisms in Air Pollution Exposure and Respiratory Diseases and Allergies.

PubMed

Bowatte, Gayan; Lodge, Caroline J; Perret, Jennifer L; Matheson, Melanie C; Dharmage, Shyamali C

2016-11-01

The purpose of this review is to summarize the evidence from recently published original studies investigating how glutathione S-transferase (GST) gene polymorphisms modify the impact of air pollution on asthma, allergic diseases, and lung function. Current studies in epidemiological and controlled human experiments found evidence to suggest that GSTs modify the impact of air pollution exposure on respiratory diseases and allergies. Of the nine articles included in this review, all except one identified at least one significant interaction with at least one of glutathione S-transferase pi 1 (GSTP1), glutathione S-transferase mu 1 (GSTM1), or glutathione S-transferase theta 1 (GSTT1) genes and air pollution exposure. The findings of these studies, however, are markedly different. This difference can be partially explained by regional variation in the exposure levels and oxidative potential of different pollutants and by other interactions involving a number of unaccounted environment exposures and multiple genes. Although there is evidence of an interaction between GST genes and air pollution exposure for the risk of respiratory disease and allergies, results are not concordant. Further investigations are needed to explore the reasons behind the discordancy.

Polymorphism in the intron 20 of porcine O-linked N-acetylglucosamine transferase

USDA-ARS?s Scientific Manuscript database

Objective: O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) catalyzes the addition of O-GlcNAc and GlcNAcylation has extensive crosstalk with phosphorylation to regulate signaling and transcription. Pig OGT is located near the region of chromosome X that affects follicle stimulating hormone...

Interaction between mercury (Hg), arsenic (As) and selenium (Se) affects the activity of glutathione S-transferase in breast milk; possible relationship with fish and sellfish intake.

PubMed

Gaxiola-Robles, Ramón; Labrada-Martagón, Vanessa; Celis de la Rosa, Alfredo de Jesús; Acosta-Vargas, Baudilio; Méndez-Rodríguez, Lía Celina; Zenteno-Savín, Tania

2014-08-01

Breast milk is regarded as an ideal source of nutrients for the growth and development of neonates, but it can also be a potential source of pollutants. Mothers can be exposed to different contaminants as a result of their lifestyle and environmental pollution. Mercury (Hg) and arsenic (As) could adversely affect the development of fetal and neonatal nervous system. Some fish and shellfish are rich in selenium (Se), an essential trace element that forms part of several enzymes related to the detoxification process, including glutathione S-transferase (GST). The goal of this study was to determine the interaction between Hg, As and Se and analyze its effect on the activity of GST in breast milk. Milk samples were collected from women between day 7 and 10 postpartum. The GST activity was determined spectrophotometrically; total Hg, As and Se concentrations were measured by atomic absorption spectrometry. To explain the possible association of Hg, As and Se concentrations with GST activity in breast milk, generalized linear models were constructed. The model explained 44% of the GST activity measured in breast milk. The GLM suggests that GST activity was positively correlated with Hg, As and Se concentrations. The activity of the enzyme was also explained by the frequency of consumption of marine fish and shellfish in the diet of the breastfeeding women. Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.

Glutathione S-transferase activity in follicular fluid from women undergoing ovarian stimulation: role in maturation.

PubMed

Meijide, Susana; Hernández, M Luisa; Navarro, Rosaura; Larreategui, Zaloa; Ferrando, Marcos; Ruiz-Sanz, José Ignacio; Ruiz-Larrea, M Begoña

2014-10-01

Female infertility involves an emotional impact for the woman, often leading to a state of anxiety and low self-esteem. The assisted reproduction techniques (ART) are used to overcome the problem of infertility. In a first step of the in vitro fertilization therapy women are subjected to an ovarian stimulation protocol to obtain mature oocytes, which will result in competent oocytes necessary for fertilization to occur. Ovarian stimulation, however, subjects the women to a high physical and psychological stress, thus being essential to improve ART and to find biomarkers of dysfunction and fertility. GSH is an important antioxidant, and is also used in detoxification reactions, catalysed by glutathione S-transferases (GST). In the present work, we have investigated the involvement of GST in follicular maturation. Patients with fertility problems and oocyte donors were recruited for the study. From each woman follicles at two stages of maturation were extracted at the preovulatory stage. Follicular fluid was separated from the oocyte by centrifugation and used as the enzyme source. GST activity was determined based on its conjugation with 3,4-dichloronitrobenzene and the assay was adapted to a 96-well microplate reader. The absorbance was represented against the incubation time and the curves were adjusted to linearity (R(2)>0.990). Results showed that in both donors and patients GST activity was significantly lower in mature oocytes compared to small ones. These results suggest that GST may play a role in the follicle maturation by detoxifying xenobiotics, thus contributing to the normal development of the oocyte. Supported by FIS/FEDER (PI11/02559), Gobierno Vasco (Dep. Educación, Universiades e Investigación, IT687-13), and UPV/EHU (CLUMBER UFI11/20 and PES13/58). The work was approved by the Ethics Committee of the UPV/EHU (CEISH/96/2011/RUIZLARREA), and performed according to the UPV/EHU and IVI-Bilbao agreement (Ref. 2012/01). Copyright © 2014. Published by

Dual protective role for Glutathione S-transferase class pi against VCD-induced ovotoxicity in the rat ovary

DOE Office of Scientific and Technical Information (OSTI.GOV)

Keating, Aileen F.; Sen, Nivedita; Sipes, I. Glenn

2010-09-01

The occupational chemical 4-vinylcyclohexene diepoxide (VCD) selectively destroys ovarian small pre-antral follicles in rats and mice via apoptosis. Detoxification of VCD can occur through glutathione conjugation, catalyzed by glutathione S-transferase (GST) enzymes. Further, GST class pi (GSTp) can negatively regulate JNK activity through protein:protein interactions in extra-ovarian tissues. Dissociation of this protein complex in the face of chemical exposure releases the inhibition of pro-apoptotic JNK. Increased JNK activity during VCD-induced ovotoxicity has been shown in isolated ovarian small pre-antral follicles following in vivo dosing of rats (80 mg/kg/day; 15 days, i.p.). The present study investigated the pattern of ovarian GSTpmore » expression during VCD exposure. Additionally, the effect of VCD on an ovarian GSTp:JNK protein complex was investigated. PND4 F344 rat ovaries were incubated in control medium {+-} VCD (30 {mu}M) for 2-8 days. VCD increased ovarian GSTp mRNA (P < 0.05) relative to control on d4-d8; whereas GSTp protein was increased (P < 0.05) on d6-d8. A GSTp:JNK protein complex was detected by immunoprecipitation and Western blotting in ovarian tissues. Relative to control, the amount of GSTp-bound JNK was increased (P = 0.09), while unbound JNK was decreased (P < 0.05) on d6 of VCD exposure. The VCD-induced decrease in unbound JNK was preceded by a decrease in phosphorylated c-Jun which occurred on d4. These findings are in support of a possible dual protective role for GSTp in the rat ovary, consisting of metabolism of VCD and inhibition of JNK-initiated apoptosis.« less

Glutathione S-transferase pi mediates MPTP-induced c-Jun N-terminal kinase activation in the nigrostriatal pathway.

PubMed

Castro-Caldas, Margarida; Carvalho, Andreia Neves; Rodrigues, Elsa; Henderson, Colin; Wolf, C Roland; Gama, Maria João

2012-06-01

Parkinson's disease (PD) is a progressive movement disorder resulting from the death of dopaminergic neurons in the substantia nigra. Neurotoxin-based models of PD using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) recapitulate the neurological features of the disease, triggering a cascade of deleterious events through the activation of the c-Jun N-terminal kinase (JNK). The molecular mechanisms underlying the regulation of JNK activity under cellular stress conditions involve the activation of several upstream kinases along with the fine-tuning of different endogenous JNK repressors. Glutathione S-transferase pi (GSTP), a phase II detoxifying enzyme, has been shown to inhibit JNK-activated signaling by protein-protein interactions, preventing c-Jun phosphorylation and the subsequent trigger of the cell death cascade. Here, we use C57BL/6 wild-type and GSTP knockout mice treated with MPTP to evaluate the regulation of JNK signaling by GSTP in both the substantia nigra and the striatum. The results presented herein show that GSTP knockout mice are more susceptible to the neurotoxic effects of MPTP than their wild-type counterparts. Indeed, the administration of MPTP induces a progressive demise of nigral dopaminergic neurons together with the degeneration of striatal fibers at an earlier time-point in the GSTP knockout mice when compared to the wild-type mice. Also, MPTP treatment leads to increased p-JNK levels and JNK catalytic activity in both wild-type and GSTP knockout mice midbrain and striatum. Moreover, our results demonstrate that in vivo GSTP acts as an endogenous regulator of the MPTP-induced cellular stress response by controlling JNK activity through protein-protein interactions.

Decreased glutathione S-transferase expression and activity and altered sex steroids in Lake Apopka brown bullheads (Ameriurus nebulosus)

USGS Publications Warehouse

Gallagher, E.P.; Gross, T.S.; Sheehy, K.M.

2001-01-01

A number of freshwater lakes and reclaimed agricultural sites in Central Florida have been the receiving waters for agrochemical and municipal runoff. One of these sites, Lake Apopka, is also a eutrophic system that has been the focus of several case studies reporting altered reproductive activity linked to bioaccumulation of persistent organochlorine chemicals in aquatic species. The present study was initiated to determine if brown bullheads (Ameriurus nebulosus) from the north marsh of Lake Apopka (Lake Apopka Marsh) exhibit an altered capacity to detoxify environmental chemicals through hepatic glutathione S-transferase (GST)-mediated conjugation as compared with bullheads from a nearby reference site (Lake Woodruff). We also compared plasma sex hormone concentrations (testosterone, 17-?? estradiol, and 11 keto-testosterone) in bullheads from the two sites. Female bullheads from Lake Apopka had 40% lower initial rate GST conjugative activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 50% lower activity towards p-nitrobutyl chloride (NBC), 33% lower activity toward ethacrynic acid (ECA), and 43% lower activity toward ??5-androstene-3,17-dione (??5-ADI), as compared with female bullheads from Lake Woodruff. Enzyme kinetic analyses demonstrated that female bullheads from Lake Apopka had lower GST-catalyzed CDNB clearance than did female Lake Woodruff bullheads. Western blotting studies of bullhead liver cytosolic proteins demonstrated that the reduced GST catalytic activities in female Lake Apopka bullheads were accompanied by lower expression of hepatic GST protein. No site differences were observed with respect to GST activities or GST protein expression in male bullheads. Female Lake Apopka bullheads also had elevated concentrations of plasma androgens (testosterone and 11-ketotestosterone) as compared with females from Lake Woodruff. In contrast, male Lake Apopka bullheads had elevated levels of plasma estrogen but similar levels of androgens as compared with

Structural basis for the interaction of antibiotics with peptidyl transferase center in eubacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schlunzen, Frank; Zarivach, Raz; Harms, Jörg

2009-10-07

Ribosomes, the site of protein synthesis, are a major target for natural and synthetic antibiotics. Detailed knowledge of antibiotic binding sites is central to understanding the mechanisms of drug action. Conversely, drugs are excellent tools for studying the ribosome function. To elucidate the structural basis of ribosome-antibiotic interactions, we determined the high-resolution X-ray structures of the 50S ribosomal subunit of the eubacterium Deinococcus radiodurans, complexed with the clinically relevant antibiotics chloramphenicol, clindamycin and the three macrolides erythromycin, clarithromycin and roxithromycin. We found that antibiotic binding sites are composed exclusively of segments of 23S ribosomal RNA at the peptidyl transferase cavitymore » and do not involve any interaction of the drugs with ribosomal proteins. Here we report the details of antibiotic interactions with the components of their binding sites. Our results also show the importance of putative Mg{sup +2} ions for the binding of some drugs. This structural analysis should facilitate rational drug design.« less

Influence of glutathione S-transferase polymorphisms (GSTT1, GSTM1, GSTP1) on type-2 diabetes mellitus (T2D) risk in an endogamous population from north India.

PubMed

Mastana, Sarabjit S; Kaur, Antarpreet; Hale, Rachel; Lindley, Martin R

2013-12-01

Glutathione S-transferases (GSTs) belong to a group of multigene and multifunctional detoxification enzymes, which defend cells against a wide variety of toxic insults and oxidative stress. Oxidative stress leads to cellular dysfunction which contributes to the pathophysiology of diseases such as cancer, atherosclerosis, and diabetes mellitus. It is important to assess whether the glutathione S-Transferase (GSTT1, GSTM1 and GSTP1) genotypes are associated with type 2 diabetes mellitus as deletion polymorphisms have an impaired capability to counteract the oxidative stress which is a feature of diabetes. GSTT1, GSTM1 and GSTP1 gene polymorphisms were analysed in 321 patients and 309 healthy controls from an endogamous population from north India. An association analysis was carried out at two levels (a) individual genes and (b) their double and triple combinations. The proportion of GSTT1 and GSTM1 null genotypes was higher in diabetics compared to controls (GSTT1 30.8 vs. 21.0 %; GSTM1 49.5 vs. 27.2 %). The frequency of the null genotype at both loci was higher in diabetics (19.6 vs. 7.8 %) leading to an odds ratio of 2.90 (CI 1.76-4.78, P < 0.0001). At GSTP1locus, patients had a higher frequency of the V/V genotype (15.6 vs. 7.5 %) and significant susceptible odds ratio (2.56, CI 1.47-4.48, P < 0.001). A combination of null genotypes at GSTT1 and GSTM1 loci and V/V genotype of GSTP1 locus showed highest odds ratio (9.64, CI 1.53-60.63, P < 0.01). Overall this study highlights that GST genes may play an important role in the pathogenesis of type 2 diabetes. The risk is higher in individuals carrying more than one susceptible genotype at these loci. The potential role of GST polymorphisms as markers of susceptibility to type 2 diabetes needs further investigations in a larger number of patients and populations.

O-GlcNAc transferase inhibitors: current tools and future challenges.

PubMed

Trapannone, Riccardo; Rafie, Karim; van Aalten, Daan M F

2016-02-01

The O-linked N-acetylglucosamine (O-GlcNAc) post-translational modification (O-GlcNAcylation) is the dynamic and reversible attachment of N-acetylglucosamine to serine and threonine residues of nucleocytoplasmic target proteins. It is abundant in metazoa, involving hundreds of proteins linked to a plethora of biological functions with implications in human diseases. The process is catalysed by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) that add and remove sugar moieties respectively. OGT knockout is embryonic lethal in a range of animal models, hampering the study of the biological role of O-GlcNAc and the dissection of catalytic compared with non-catalytic roles of OGT. Therefore, selective and potent chemical tools are necessary to inhibit OGT activity in the context of biological systems. The present review focuses on the available OGT inhibitors and summarizes advantages, limitations and future challenges. © 2016 Authors; published by Portland Press Limited.

The link between antioxidant enzymes catalase and glutathione S-transferase and physiological condition of a control population of terrestrial isopod (Porcellio scaber).

PubMed

Jemec, Anita; Lešer, Vladka; Drobne, Damjana

2012-05-01

The aim of this work was to investigate if the activities of catalase and glutathione S-transferase in a control population of terrestrial isopods (Porcellio scaber) are correlated with the physiological condition of the isopods. For this purpose, the activities of these enzymes were analysed in isopods from a stock population and in parallel, the physiological condition of the same specimens was assessed using a histological approach based on epithelial thickness and lipid droplets. We found a correlation between antioxidant enzymes and the physiological condition of the isopods. This implies that these enzymes could be used as predictive indicators of the physiological condition in a stock population before comprehensive toxicological studies are conducted and also in control group after the experiment. When a control group is found to be very heterogeneous in terms of physiological condition, the experiment should be repeated with a larger number of experimental animals. The findings of this study will contribute to more accurate experimental design of toxicity tests when using biomarkers. This should encourage other researchers to increase their effort to know the physiological state of their test organisms. Copyright © 2011 Elsevier Inc. All rights reserved.

Conformational stability of pGEX-expressed Schistosoma japonicum glutathione S-transferase: a detoxification enzyme and fusion-protein affinity tag.

PubMed Central

Kaplan, W.; Hüsler, P.; Klump, H.; Erhardt, J.; Sluis-Cremer, N.; Dirr, H.

1997-01-01

A glutathione S-transferase (Sj26GST) from Schistosoma japonicum, which functions in the parasite's Phase II detoxification pathway, is expressed by the Pharmacia pGEX-2T plasmid and is used widely as a fusion-protein affinity tag. It contains all 217 residues of Sj26GST and an additional 9-residue peptide linker with a thrombin cleavage site at its C-terminus. Size-exclusion HPLC (SEC-HPLC) and SDS-PAGE studies indicate that purification of the homodimeric protein under nonreducing conditions results in the reversible formation of significant amounts of 160-kDa and larger aggregates without a loss in catalytic activity. The basis for oxidative aggregation can be ascribed to the high degree of exposure of the four cysteine residues per subunit. The conformational stability of the dimeric protein was studied by urea- and temperature-induced unfolding techniques. Fluorescence-spectroscopy, SEC-HPLC, urea- and temperature-gradient gel electrophoresis, differential scanning microcalorimetry, and enzyme activity were employed to monitor structural and functional changes. The unfolding data indicate the absence of thermodynamically stable intermediates and that the unfolding/refolding transition is a two-state process involving folded native dimer and unfolded monomer. The stability of the protein was found to be dependent on its concentration, with a delta G degree (H2O) = 26.0 +/- 1.7 kcal/mol. The strong relationship observed between the m-value and the size of the protein indicates that the amount of protein surface area exposed to solvent upon unfolding is the major structural determinant for the dependence of the protein's free energy of unfolding on urea concentration. Thermograms obtained by differential scanning microcalorimetry also fitted a two-state unfolding transition model with values of delta Cp = 7,440 J/mol per K, delta H = 950.4 kJ/mol, and delta S = 1,484 J/mol. PMID:9041642

Conformational stability of pGEX-expressed Schistosoma japonicum glutathione S-transferase: a detoxification enzyme and fusion-protein affinity tag.

PubMed

Kaplan, W; Hüsler, P; Klump, H; Erhardt, J; Sluis-Cremer, N; Dirr, H

1997-02-01

A glutathione S-transferase (Sj26GST) from Schistosoma japonicum, which functions in the parasite's Phase II detoxification pathway, is expressed by the Pharmacia pGEX-2T plasmid and is used widely as a fusion-protein affinity tag. It contains all 217 residues of Sj26GST and an additional 9-residue peptide linker with a thrombin cleavage site at its C-terminus. Size-exclusion HPLC (SEC-HPLC) and SDS-PAGE studies indicate that purification of the homodimeric protein under nonreducing conditions results in the reversible formation of significant amounts of 160-kDa and larger aggregates without a loss in catalytic activity. The basis for oxidative aggregation can be ascribed to the high degree of exposure of the four cysteine residues per subunit. The conformational stability of the dimeric protein was studied by urea- and temperature-induced unfolding techniques. Fluorescence-spectroscopy, SEC-HPLC, urea- and temperature-gradient gel electrophoresis, differential scanning microcalorimetry, and enzyme activity were employed to monitor structural and functional changes. The unfolding data indicate the absence of thermodynamically stable intermediates and that the unfolding/refolding transition is a two-state process involving folded native dimer and unfolded monomer. The stability of the protein was found to be dependent on its concentration, with a delta G degree (H2O) = 26.0 +/- 1.7 kcal/mol. The strong relationship observed between the m-value and the size of the protein indicates that the amount of protein surface area exposed to solvent upon unfolding is the major structural determinant for the dependence of the protein's free energy of unfolding on urea concentration. Thermograms obtained by differential scanning microcalorimetry also fitted a two-state unfolding transition model with values of delta Cp = 7,440 J/mol per K, delta H = 950.4 kJ/mol, and delta S = 1,484 J/mol.

Decreased expression of glutathione S-transferase pi correlates with poorly differentiated grade in patients with oral squamous cell carcinoma.

PubMed

Ma, Hai-long; Yu, Cong; Liu, Ying; Tan, Yi-ran; Qiao, Jin-ke; Yang, Xi; Wang, Li-zhen; Li, Jiang; Chen, Qiong; Chen, Fu-xiang; Zhang, Zhi-yuan; Zhong, Lai-ping

2015-03-01

Glutathione S transferase pi (GSTP1) is a member of phase II detoxification enzymes as a major regulator of cell signaling in response to stress, hypoxia, growth factors, and other stimuli. The clinical role of GSTP1 in cancer is still unclear. The aim of this study was to investigate the serum GSTP1 level in patients with oral squamous cell carcinoma (OSCC) and the GSTP1 expression in tissue samples from patients with OSCC and OSCC lines. One hundred and sixty-six patients with OSCC and 120 normal persons were used to screen potential serum peptide biomarkers using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Serum GSTP1 concentration was detected in 18 patients with OSCC and 18 normal persons using ELISA. Immunohistochemistry was used to detect GSTP1 expression in tissue samples from twenty-eight OSCC patients. Western blot and real-time PCR were used to detect GSTP1 expression in nine OSCC lines. Decreased GSTP1 concentration was found in the patients with OSCC compared with the normal persons by MALDI-TOF-MS, which was then confirmed by ELISA (P = 0.019). Decreased GSTP1 mRNA level and protein expression were also found in the OSCC lines. Decreased GSTP1 expression was found correlating with pathological differentiation grade in the tissue samples from OSCC patients, a lower GSTP1 expression indicating a poorer pathological differentiation grade (P = 0.041). These results suggest that decreased GSTP1 expression in patients with OSCC and a lower GSTP1 expression indicating a poorer pathological differentiation grade in OSCC tissue samples. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A Framework for Evidence-Based Licensure of Adaptive Autonomous Systems: Technical Areas

DTIC Science & Technology

2016-03-01

subjective judgments by human experts . They are design dependent, but address questions of whether the system is performing as needed, as opposed...I N S T I T U T E F O R D E F E N S E A N A L Y S E S A Framework for Evidence-Based Licensure of Adaptive Autonomous Systems : Technical Areas...and Licensure of Autonomous Systems ,” for the Air Force Research Laboratory (AFRL). The views, opinions, and findings should not be construed as

Isolation and characterization of a cDNA clone coding for a glutathione S-transferase class delta enzyme from the biting midge Culicoides variipennis sonorensis Wirth and Jones.

PubMed

Abdallah, M A; Pollenz, R S; Droog, F N; Nunamaker, R A; Tabachnick, W J; Murphy, K E

2000-12-01

Culicoides variipennis sonorensis is the primary vector of bluetongue viruses in North America. Glutathione S-transferases (GSTs) are enzymes that catalyze nucleophilic substitutions, converting reactive lipophilic molecules into soluble conjugates. Increased GST activity is associated with development of insecticide resistance. Described here is the isolation of the first cDNA encoding a C. variipennis GST. The clone consists of 720 translated bases encoding a protein with a M(r) of approximately 24,800 composed of 219 amino acids. The deduced amino acid sequence is similar (64%-74%) to class Delta (previously named Theta) GSTs from the dipteran genera Musca, Drosophila, Lucilia and Anopheles. The cDNA was subcloned into pET-11b, expressed in Epicurian coli BL21 (DE3) and has a specific activity of approximately 28,000 units/mg for the substrate 1-chloro-2,4-dinitrobenzene.

Recombinant human dihydroxyacetonephosphate acyl-transferase characterization as an integral monotopic membrane protein.

PubMed

Piano, Valentina; Nenci, Simone; Magnani, Francesca; Aliverti, Alessandro; Mattevi, Andrea

2016-12-02

Although the precise functions of ether phospholipids are still poorly understood, significant alterations in their physiological levels are associated either to inherited disorders or to aggressive metastatic cancer. The essential precursor, alkyl-dihydroxyacetone phosphate (DHAP), for all ether phospholipids species is synthetized in two consecutive reactions performed by two enzymes sitting on the inner side of the peroxisomal membrane. Here, we report the characterization of the recombinant human DHAP acyl-transferase, which performs the first step in alkyl-DHAP synthesis. By exploring several expression systems and designing a number of constructs, we were able to purify the enzyme in its active form and we found that it is tightly bound to the membrane through the N-terminal residues. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Association of glutathione S-transferase pi (GSTP1) Ile105Val polymorphism with the risk of skin cancer: a meta-analysis.

PubMed

Zhou, Cheng-Fan; Ma, Tai; Zhou, Deng-Chuan; Shen, Tong; Zhu, Qi-Xing

2015-08-01

Numerous epidemiological studies have evaluated the association of Glutathione S-transferase P1 (GSTP1) Ile105Val polymorphism with the risk of skin cancer. However, the results remain inconclusive. To derive a more precise estimation of the association between the GSTP1 Ile105Val polymorphism and skin cancer risk, a meta-analysis was performed. A comprehensive search was conducted to identify the eligible studies. We used odds ratios (ORs) with 95 % confidence intervals (CIs) to assess the association of GSTP1 Ile105Val polymorphism with skin cancer risk. Thirteen case-control studies in nine articles, which included a total of 1504 cases and 2243 controls. Overall, we found that GSTP1 Ile105Val polymorphism was not associated with skin cancer risk. Furthermore, subgroup analysis by histological types showed that GSTP1 Ile105Val polymorphism was associated with risks of malignant melanoma under the dominant model (Val/Val + Val/Ile vs. Ile/Ile: OR 1.230, 95 % CI 1.017-1.488, P = 0.033). However, lack of association between GSTP1 Ile105Val polymorphism and BCC and SCC risk in all genetic models. Our meta-analysis suggested that the GSTP1 Ile105Val polymorphism might be associated with increased risk of malignant melanoma in Caucasian population.

Identification and characterization of an Apis cerana cerana Delta class glutathione S-transferase gene ( AccGSTD) in response to thermal stress

NASA Astrophysics Data System (ADS)

Yan, Huiru; Jia, Haihong; Wang, Xiuling; Gao, Hongru; Guo, Xingqi; Xu, Baohua

2013-02-01

Glutathione S-transferases (GSTs) are members of a multifunctional enzyme super family that plays a pivotal role in both insecticide resistance and protection against oxidative stress. In this study, we identified a single-copy gene, AccGSTD, as being a Delta class GST in the Chinese honey bee ( Apis cerana cerana). A predicted antioxidant response element, CREB, was found in the 1,492-bp 5'-flanking region, suggesting that AccGSTD may be involved in oxidative stress response pathways. Real-time PCR and immunolocalization studies demonstrated that AccGSTD exhibited both developmental- and tissue-specific expression patterns. During development, AccGSTD transcript was increased in adults. The AccGSTD expression level was the highest in the honey bee brain. Thermal stress experiments demonstrated that AccGSTD could be significantly upregulated by temperature changes in a time-dependent manner. It is hypothesized that high expression levels might be due to the increased levels of oxidative stress caused by the temperature challenges. Additionally, functional assays of the recombinant AccGSTD protein revealed that AccGSTD has the capability to protect DNA from oxidative damage. Taken together, these data suggest that AccGSTD may be responsible for antioxidant defense in adult honey bees.

A study of the association of glutathione S-transferase M1/T1 polymorphisms with susceptibility to vitiligo in Egyptian patients.

PubMed

Aly, Dalia Gamal; Salem, Samar Abdallah; Amr, Khalda Sayed; El-Hamid, Mahmoud Fawzy Abd

2018-01-01

The association of glutathione S-transferases M1/T1 (GSTM1/T1) null polymorphisms with vitiligo was proposed in several studies including two Egyptian studies with contradictory results. The aim here was to assess the association between GSTM1/T1 null polymorphisms and the susceptibility to vitiligo in a larger sample of Egyptian patients with generalized vitiligo. This study included 122 vitiligo patients and 200 healthy controls that were age, and gender matched. Assessment of GSTM1/T1 gene polymorphisms was done using a multiplex polymerase chain reaction (PCR). Increased odds of generalized vitiligo was observed with the null genotypes of GSTM1 and GSTT1 polymorphisms (P<0.05). Controls with GSTM1 null/GSTT1+ heterozygosis presented with a 2.97 odds protection from having generalized vitiligo (OR=2.97, 95%CI=1.1-7.7) (P=0.02) compared with patients. Small sample size of patients. This study showed a significant trend towards an association with the combination of the GSTM1/GSTT1 double null polymorphism and generalized vitiligo. Individuals with GSTM1 null/GSTT1+ heterozygosis have a 2.97 odds protection from having generalized vitiligo compared with patients. It was is the first time, to our knowledge, that such an association has been reported.

Thiazolides inhibit growth and induce glutathione-S-transferase Pi (GSTP1)-dependent cell death in human colon cancer cells.

PubMed

Müller, Joachim; Sidler, Daniel; Nachbur, Ueli; Wastling, Jonathan; Brunner, Thomas; Hemphill, Andrew

2008-10-15

Thiazolides are a novel class of broad-spectrum anti-infective drugs with promising in vitro and in vivo activities against intracellular and extracellular protozoan parasites. The nitrothiazole-analogue nitazoxanide (NTZ; 2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide) represents the thiazolide parent compound, and a number of bromo- and carboxy-derivatives with differing activities have been synthesized. Here we report that NTZ and the bromo-thiazolide RM4819, but not the carboxy-thiazolide RM4825, inhibited proliferation of the colon cancer cell line Caco2 and nontransformed human foreskin fibroblasts (HFF) at or below concentrations the compounds normally exhibit anti-parasitic activity. Thiazolides induced typical signs of apoptosis, such as nuclear condensation, DNA fragmentation and phosphatidylserine exposure. Interestingly, the apoptosis-inducing effect of thiazolides appeared to be cell cycle-dependent and induction of cell cycle arrest substantially inhibited the cell death-inducing activity of these compounds. Using affinity chromatography and mass spectrometry glutathione-S-transferase P1 (GSTP1) from the GST class Pi was identified as a major thiazolide-binding protein. GSTP1 expression was more than 10 times higher in the thiazolide-sensitive Caco2 cells than in the less sensitive HFF cells. The enzymatic activity of recombinant GSTP1 was strongly inhibited by thiazolides. Silencing of GSTP1 using siRNA rendered cells insensitive to RM4819, while overexpression of GSTP1 increased sensitivity to RM4819-induced cell death. Thiazolides may thus represent an interesting novel class of future cancer therapeutics.

Polymorphisms of glutathione S-transferase π 1 and toll-like receptors 2 and 9: Association with breast cancer susceptibility

PubMed Central

AL-HARRAS, MOHAMMAD F.; HOUSSEN, MAHA E.; SHAKER, MOHAMED E.; FARAG, KAMEL; FAROUK, OMAR; MONIR, REHAN; EL-MAHDY, RASHA; ABO-HASHEM, EKBAL M.

2016-01-01

Polymorphisms in antioxidant enzymes and innate immune receptors have been implicated in the development of various types of cancer. The present study aimed to investigate whether polymorphisms of glutathione S-transferase π 1 (GSTP1) and toll-like receptors (TLRs) 2 and 9 are associated with susceptibility to breast cancer among females. The study was conducted on 72 Egyptian female patients with breast cancer, along with 100 healthy volunteers. Polymorphisms of GSTP1 (codon 105 Ile/Val) and TLR9 rs187084 (1237T/C) genes were assessed by polymerase chain reaction (PCR)-restriction fragment length polymorphism, while the −196 to −174 deletion/insertion (del/ins) polymorphism of TLR2 was detected by PCR. The results indicated a decrease in GSTP1 Val allele frequency in breast cancer patients compared with healthy controls, at rates of 22.9 vs. 32.5%, respectively. In addition, the breast cancer group demonstrated a decreased TLR9 C allele frequency compared with the control group, at rates of 36.1 vs. 51.5%, respectively (P=0.0047). A non-significant difference was detected in the frequency of the TLR2 −196 to −174 del allele in breast cancer patients when compared to normal controls. In conclusion, these results suggested that the GSTP1 Val and TLR9 1237C alleles, but not TLR2 −196 to −174 del, are likely to be associated with breast cancer development among females. PMID:26998146

Polymorphisms of glutathione S-transferase π 1 and toll-like receptors 2 and 9: Association with breast cancer susceptibility.

PubMed

Al-Harras, Mohammad F; Houssen, Maha E; Shaker, Mohamed E; Farag, Kamel; Farouk, Omar; Monir, Rehan; El-Mahdy, Rasha; Abo-Hashem, Ekbal M

2016-03-01

Polymorphisms in antioxidant enzymes and innate immune receptors have been implicated in the development of various types of cancer. The present study aimed to investigate whether polymorphisms of glutathione S-transferase π 1 (GSTP1) and toll-like receptors (TLRs) 2 and 9 are associated with susceptibility to breast cancer among females. The study was conducted on 72 Egyptian female patients with breast cancer, along with 100 healthy volunteers. Polymorphisms of GSTP1 (codon 105 Ile/Val) and TLR9 rs187084 (1237T/C) genes were assessed by polymerase chain reaction (PCR)-restriction fragment length polymorphism, while the -196 to -174 deletion/insertion (del/ins) polymorphism of TLR2 was detected by PCR. The results indicated a decrease in GSTP1 Val allele frequency in breast cancer patients compared with healthy controls, at rates of 22.9 vs. 32.5%, respectively. In addition, the breast cancer group demonstrated a decreased TLR9 C allele frequency compared with the control group, at rates of 36.1 vs. 51.5%, respectively (P=0.0047). A non-significant difference was detected in the frequency of the TLR2 -196 to -174 del allele in breast cancer patients when compared to normal controls. In conclusion, these results suggested that the GSTP1 Val and TLR9 1237C alleles, but not TLR2 -196 to -174 del, are likely to be associated with breast cancer development among females.

Molecular cloning, expression and enzymatic characterization of glutathione S-transferase from Antarctic sea-ice bacteria Pseudoalteromonas sp. ANT506.

PubMed

Shi, Yonglei; Wang, Quanfu; Hou, Yanhua; Hong, Yanyan; Han, Xiao; Yi, Jiali; Qu, Junjie; Lu, Yi

2014-01-01

A glutathione S-transferase (GST) gene from Antarctic sea-ice bacteria Pseudoalteromonas sp. ANT506 (namely PsGST), was cloned and expressed in Escherichia coli. The open reading frame of PsGST comprised 654 bp encoding a protein of 217 amino acids with a calculated molecular size of 24.3 kDa. The rPsGST possesses the conserved amino acid defining the binding sites of glutathione (G-site) and substrate binding pocket (H-site) in GST N_3 family. PsGST was expressed in E. coli and the recombinant PsGST (rPsGST) was purified by Ni-affinity chromatography with a high specific activity of 74.21 U/mg. The purified rPsGST showed maximum activity at 40 °C and exhibited 14.2% activity at 0 °C. It was completely inactivated at 50 °C for 40 min. These results indicated that rPsGST was a typical cold active GST with low thermostability. The enzyme was little affected by H2O2 and Triton X-100, and 50.2% of the remaining activity was detected in the presence of high salt concentrations (2M NaCl). The enzymatic Km values for CDNB and GSH was 0.22 mM and 1.01 mM, respectively. These specific enzyme properties may be related to the survival environment of Antarctic sea ice bacteria. Copyright © 2013 Elsevier GmbH. All rights reserved.

Glutathione S-transferase Pi expression predicts response to adjuvant chemotherapy for stage C colon cancer: a matched historical control study

PubMed Central

2012-01-01

Background This study examined the association between overall survival and Glutathione S-transferase Pi (GST Pi) expression and genetic polymorphism in stage C colon cancer patients after resection alone versus resection plus 5-fluourouracil-based adjuvant chemotherapy. Methods Patients were drawn from a hospital registry of colorectal cancer resections. Those receiving chemotherapy after it was introduced in 1992 were compared with an age and sex matched control group from the preceding period. GST Pi expression was assessed by immunohistochemistry. Overall survival was analysed by the Kaplan-Meier method and Cox regression. Results From an initial 104 patients treated with chemotherapy and 104 matched controls, 26 were excluded because of non-informative immunohistochemistry, leaving 95 in the treated group and 87 controls. Survival did not differ significantly among patients with low GST Pi who did or did not receive chemotherapy and those with high GST Pi who received chemotherapy (lowest pair-wise p = 0.11) whereas patients with high GST Pi who did not receive chemotherapy experienced markedly poorer survival than any of the other three groups (all pair-wise p <0.01). This result was unaffected by GST Pi genotype. Conclusion Stage C colon cancer patients with low GST Pi did not benefit from 5-fluourouracil-based adjuvant chemotherapy whereas those with high GST Pi did. PMID:22639861

Glutathione S-transferase polymorphisms (GSTM1, GSTT1, GSTP1) and male factor infertility risk: a pooled analysis of studies.

PubMed

Safarinejad, Mohammad Reza; Dadkhah, Farid; Ali Asgari, Majid; Hosseini, Seyed Yousef; Kolahi, Ali Asgar; Iran-Pour, Elham

2012-01-01

To determine the role of glutathione S-transferases (GSTs; GSTM1, GSTT1, and GSTP1) gene polymorphisms in susceptibility to male factor infertility. We report a pooled analysis of 11 studies on the association of GSTM1, GSTT1, and GSTP1 polymorphisms and male factor infertility, including 1323 cases and 1054 controls. An overall significant association was determined between the GSTM1 null genotype [odds ratio (OR), 2.74; 95% confidence interval (CI), 1.72 to 3.84; P = .003], GSTT1 null genotype (OR, 1.54; 95% CI, 1.43 to 3.47; P = .02), and male factor infertility. The GSTP1 Ile/Val genotype had overall protective effect against development of infertility (OR, 0.48; 95% CI, 0.27 to 0.77), while there was significant heterogeneity between studies. In sensitivity analysis, two studies were excluded; the association and direction between GSTM1 and GSTT1 null genotypes and GSTP1 Ile/Val genotype and male infertility remained unchanged. There was no significant interaction between smoking status and studied genotypes on male infertility risk (P = .26). These results demonstrated that amongst populations studied to date, GSTM1 and GSTT1 null genotypes are associated with strong and modest increase in the risk of male infertility, respectively. On the contrary, GSTP1 Ile/Val genotype has protective effect.

An ethylene-responsive enhancer element is involved in the senescence-related expression of the carnation glutathione-S-transferase (GST1) gene.

PubMed

Itzhaki, H; Maxson, J M; Woodson, W R

1994-09-13

The increased production of ethylene during carnation petal senescence regulates the transcription of the GST1 gene encoding a subunit of glutathione-S-transferase. We have investigated the molecular basis for this ethylene-responsive transcription by examining the cis elements and trans-acting factors involved in the expression of the GST1 gene. Transient expression assays following delivery of GST1 5' flanking DNA fused to a beta-glucuronidase receptor gene were used to functionally define sequences responsible for ethylene-responsive expression. Deletion analysis of the 5' flanking sequences of GST1 identified a single positive regulatory element of 197 bp between -667 and -470 necessary for ethylene-responsive expression. The sequences within this ethylene-responsive region were further localized to 126 bp between -596 and -470. The ethylene-responsive element (ERE) within this region conferred ethylene-regulated expression upon a minimal cauliflower mosaic virus-35S TATA-box promoter in an orientation-independent manner. Gel electrophoresis mobility-shift assays and DNase I footprinting were used to identify proteins that bind to sequences within the ERE. Nuclear proteins from carnation petals were shown to specifically interact with the 126-bp ERE and the presence and binding of these proteins were independent of ethylene or petal senescence. DNase I footprinting defined DNA sequences between -510 and -488 within the ERE specifically protected by bound protein. An 8-bp sequence (ATTTCAAA) within the protected region shares significant homology with promoter sequences required for ethylene responsiveness from the tomato fruit-ripening E4 gene.

Purification, molecular cloning and heterologous expression of a glutathione S-transferase involved in insecticide resistance from the rice brown planthopper, Nilaparvata lugens.

PubMed Central

Vontas, John G; Small, Graham J; Nikou, Dimitra C; Ranson, Hilary; Hemingway, Janet

2002-01-01

A novel glutathione S-transferase (GST)-based pyrethroid resistance mechanism was recently identified in Nilaparvata lugens [Vontas, Small and Hemingway (2001) Biochem. J. 357, 65-72]. To determine the nature of GSTs involved in conferring this resistance, the GSTs from resistant and susceptible strains of N. lugens were partially purified by anion exchange and affinity chromatography. The majority of peroxidase activity, previously correlated with resistance, was confined to the fraction that bound to the affinity column, which was considerably elevated in the resistant insects. A cDNA clone encoding a GST (nlgst1-1) - the first reported GST sequence from Hemiptera with up to 54% deduced amino-acid identity with other insect class I GSTs - was isolated from a pyrethroid-resistant strain. Northern analysis showed that nlgst1-1 was overexpressed in resistant insects. nlgst1-1 was expressed in Escherichia coli, purified and characterized. The ability of the recombinant protein to bind to the S-hexylglutathione affinity matrix, its substrate specificities and its immunological properties confirmed that this GST was one from the elevated subset of N. lugens GSTs. Peroxidase activity of the recombinant nlgst1-1 indicated that it had a role in resistance, through detoxification of lipid peroxidation products induced by pyrethroids. Southern analysis of genomic DNA from the resistant and susceptible strains indicated that GST-based insecticide resistance may be associated with gene amplification in N. lugens. PMID:11853540

Photoaffinity labelling of the active site of the rat glutathione transferases 3-3 and 1-1 and human glutathione transferase A1-1.

PubMed

Cooke, R J; Björnestedt, R; Douglas, K T; McKie, J H; King, M D; Coles, B; Ketterer, B; Mannervik, B

1994-09-01

The glutathione transferases (GSTs) form a group of enzymes responsible for a wide range of molecular detoxications. The photoaffinity label S-(2-nitro-4-azidophenyl)glutathione was used to study the hydrophobic region of the active site of the rat liver GST 1-1 and 2-2 isoenzymes (class Alpha) as well as the rat class-Mu GST 3-3. Photoaffinity labelling was carried out using a version of S-(2-nitro-4-azidophenyl)glutathione tritiated in the arylazido ring. The labelling occurred with higher levels of radioisotope incorporation for the Mu than the Alpha families. Taking rat GST 3-3, 1.18 (+/- 0.05) mol of radiolabel from S-(2-nitro-4-azidophenyl)glutathione was incorporated per mol of dimeric enzyme, which could be blocked by the presence of the strong competitive inhibitor, S-tritylglutathione (Ki = 1.4 x 10(-7) M). Radiolabelling of the protein paralleled the loss of enzyme activity. Photoaffinity labelling by tritiated S-(2-nitro-4-azidophenyl)glutathione on a preparative scale (in the presence and absence of S-tritylglutathione) followed by tryptic digestion and purification of the labelled peptides indicated that GST 3-3 was specifically photolabelled; the labelled peptides were sequenced. Similarly, preparative photoaffinity labelling by S-(2-nitro-4-azidophenyl)glutathione of the rat liver 1-1 isoenzyme, the human GST A1-1 and the human-rat chimaeric GST, H1R1/1, was carried out with subsequent sequencing of radiolabelled h.p.l.c.-purified tryptic peptides. The results were interpreted by means of molecular-graphics analysis to locate photoaffinity-labelled peptides using the X-ray-crystallographic co-ordinates of rat GST 3-3 and human GST A1-1. The molecular-graphical analysis indicated that the labelled peptides are located within the immediate vicinity of the region occupied by S-substituted glutathione derivatives bound in the active-site cavity of the GSTs investigated.

Characterization of liver injury, oval cell proliferation and cholangiocarcinogenesis in glutathione S-transferase A3 knockout mice.

PubMed

Crawford, Dana R; Ilic, Zoran; Guest, Ian; Milne, Ginger L; Hayes, John D; Sell, Stewart

2017-07-01

We recently generated glutathione S-transferase (GST) A3 knockout (KO) mice as a novel model to study the risk factors for liver cancer. GSTA3 KO mice are sensitive to the acute cytotoxic and genotoxic effects of aflatoxin B1 (AFB1), confirming the crucial role of GSTA3 in resistance to AFB1. We now report histopathological changes, tumor formation, biochemical changes and gender response following AFB1 treatment as well as the contribution of oxidative stress. Using a protocol of weekly 0.5 mg AFB1/kg administration, we observed extensive oval (liver stem) cell (OC) proliferation within 1-3 weeks followed by microvesicular lipidosis, megahepatocytes, nuclear inclusions, cholangiomas and small nodules. Male and female GSTA3 KO mice treated with 12 and 24 weekly AFB1 injections followed by a rest period of 12 and 6 months, respectively, all had grossly distorted livers with macro- and microscopic cysts, hepatocellular nodules, cholangiomas and cholangiocarcinomas and OC proliferation. We postulate that the prolonged AFB1 treatment leads to inhibition of hepatocyte proliferation, which is compensated by OC proliferation and eventually formation of cholangiocarcinoma (CCA). At low-dose AFB1, male KO mice showed less extensive acute liver injury, OC proliferation and AFB1-DNA adducts than female KO mice. There were no significant compensatory changes in KO mice GST subunits, GST enzymatic activity, epoxide hydrolase, or CYP1A2 and CYP3A11 levels. Finally, there was a modest increase in F2-isoprostane and isofuran in KO mice that confirmed putative GSTA3 hydroperoxidase activity in vivo for the first time. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Glutathione S-transferases in human renal cortex and neoplastic tissue: enzymatic activity, isoenzyme profile and immunohistochemical localization.

PubMed

Rodilla, V; Benzie, A A; Veitch, J M; Murray, G I; Rowe, J D; Hawksworth, G M

1998-05-01

1. Glutathione S-transferase (GST) activity in the cytosol of renal cortex and tumours from eight men and eight women was measured using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. GST activities ranged from 685 to 2192 nmol/min/mg protein in cortex (median 1213) and from non-detectable (minimum 45) to 2424 nmol/min/mg protein in tumours (median 469). The activities in the tumours were lower than those in the normal cortices (p < 0.05). 2. In men, the activity in the cortical cytosol was in all cases higher than that measured in the corresponding tumours (p < 0.05). In women, the difference in activity between cortices and tumours was not significantly different (p > 0.05). 3. The age of the patients ranged from 42 to 81 years (median 62) and was not found to play a role in the levels of GST activity observed in cortex or in renal tumours from either sex. 4. Immunoblotting and immunohistochemical studies confirmed that GST-alpha was the predominant form expressed both in normal cortex and tumour and probably accounted for most of the GST activity present in these samples. GST-mu and GST-phi were expressed in both tumours and normal cortex and, while in some cases the level of expression in the cortices was higher than that found in the tumours, the reverse was also observed. Within the GST-mu class, GST M1/M2 was only detected in one sample (tumour), which showed the highest overall expression of GST-mu. GSTM3 was the predominant isoenzyme of the mu class in normal and tumour tissue, whereas GTM4 and GSTM5 were not detected. 5. These differences could have functional significance where xenobiotics or cytotoxic drugs are specific substrates for the different classes of GSTs.

Interaction of Ferulic Acid with Glutathione S-Transferase and Carboxylesterase Genes in the Brown Planthopper, Nilaparvata lugens.

PubMed

Yang, Jun; Sun, Xiao-Qin; Yan, Shu-Ying; Pan, Wen-Jun; Zhang, Mao-Xin; Cai, Qing-Nian

2017-07-01

Plant phenolics are crucial defense phytochemicals against herbivores and glutathione S-transferase (GST) and carboxylesterase (CarE) in herbivorous insects are well-known detoxification enzymes for such xenobiotics. To understand relationship between a plant phenolic and herbivore GST or CarE genes, we evaluated the relationship between a rice phenolic ferulic acid and resistance to brown planthopper (BPH, Nilaparvata lugens), and investigated the interaction of ferulic acid with GST or CarE genes in BPH. The results indicate that ferulic acid content in tested rice varieties was highly associated with resistance to BPH. Bioassays using artificial diets show that the phenolic acid toxicity to BPH was dose dependent and the LC 25 and LC 50 were 5.81 and 23.30 μg/ml at 72 hr, respectively. Activities of the enzymes BPH GST and CarE were increased at concentrations below the LC 50 of ferulic acid. Moreover, low ferulic acid concentrations (< LC 25 ) upregulated the transcriptional levels of NlGSTD1 and NlGSTE1 of the GST family and NlCE of the CarE family. By using dsRNA-induced gene silencing (DIGS) of GST or CarE, it was shown that suppressed expression levels of NlGSTD1, NlGSTE1 and NlCE were 14.6%-21.2%, 27.8%-34.2%, and 10.5%-19.8%, respectively. Combination of NlGSTD1, NlGSTE1 or NlCE knockdown with ferulic acid increased nymph mortality by 92.9%, 119.9%, or 124.6%, respectively. These results suggest that depletion of detoxification genes in herbivorous insects by plant-mediated RNAi technology might be a new potential resource for improving rice resistance to BPH.

Endothelial cell palmitoylproteomics identifies novel lipid modified targets and potential substrates for protein acyl transferases

PubMed Central

Marin, Ethan P.; Derakhshan, Behrad; Lam, TuKiet T.; Davalos, Alberto; Sessa, William C.

2012-01-01

Rationale Protein S-palmitoylation is the post-translational attachment of a saturated 16-carbon palmitic acid to a cysteine side chain via a thioester bond. Palmitoylation can affect protein localization, trafficking, stability, and function. The extent and roles of palmitoylation in endothelial cell (EC) biology is not well understood, in part due to technological limits on palmitoylprotein detection. Objective To develop a method using acyl-biotinyl exchange (ABE) technology coupled with mass spectrometry to globally isolate and identify palmitoylproteins in EC. Methods and Results More than 150 putative palmitoyl proteins were identified in EC using ABE and mass spectrometry. Among the novel palmitoylproteins identified is superoxide dismutase 1 (SOD1), an intensively studied enzyme that protects all cells from oxidative damage. Mutation of cysteine 6 prevents palmitoylation, leads to reduction in SOD1 activity in vivo and in vitro, and inhibits nuclear localization, thereby supporting a functional role for SOD1 palmitoylation. Moreover, we used ABE to search for substrates of particular protein acyl transferases in EC. We found that palmitoylation of the cell adhesion protein PECAM1 is dependent on the protein acyl transferase ZDHHC21. We show that knockdown of ZDHHC21 leads to reduced levels of PECAM1 at the cell surface. Conclusions Our data demonstrate the utility of EC palmitoylproteomics to reveal new insights into the role of this important post-translational lipid modification in EC biology. PMID:22496122

Response of detoxification system genes on Chironomus riparius aquatic larvae after antibacterial agent triclosan exposures.

PubMed

Martínez-Paz, Pedro

2018-05-15

Triclosan (TCS) is an antimicrobial agent used in a range of personal care and consumer products and is commonly detected in aquatic ecosystems. In the present study, the effects of TCS at the molecular level on the detoxification system of Chironomus riparius aquatic larvae, a test organism widely used for the assessment of aquatic toxicology, were evaluated. The obtained results show that this xenobiotic was able to induce significant changes in transcripts from different cytochrome P450s and glutathione s-transferases genes, involved in phase I and phase II of detoxification system, respectively. In contrast, TCS did not affect the glutathione S-transferase enzyme activity nor the expression pattern of multidrug resistance-associated protein 1, which belongs to phase III of detoxification system. These results provide information about the effects of TCS on the detoxification mechanism of C. riparius and offers different biomarker genes that could be useful in ecotoxicological studies, risk assessment and bioremediation. Copyright © 2017 Elsevier B.V. All rights reserved.

The role of endoxyloglucan transferase in the organization of plant cell walls.

PubMed

Nishitani, K

1997-01-01

The plant cell wall plays a central role in morphogenesis as well as responsiveness to environmental signals. Xyloglucans are the principal component of the plant cell wall matrix and serve as cross-links between cellulose microfibrils to form the cellulose-xyloglucan framework. Endoxyloglucan transferase (EXGT), which was isolated and characterized in 1992, is an enzyme that mediates molecular grafting reaction between xyloglucan molecules. Structural studies on cDNAs encoding EXGT and its related proteins have disclosed the ubiquitous presence in the plant kingdom of a large multigene family of xyloglucan-related proteins (XRPs). Each XRP functions as either hydrolase or transferase acting on xyloglucans and is considered to be responsible for rearrangement of the cellulose-xyloglucan framework, the processes essential for the construction, modification, and degradation of plant cell walls. Different XRP genes exhibit potentially different expression profiles with respect to tissue specificity and responsiveness to hormonal and mechanical signals. The molecular approach to individual XRP genes will open a new path for exploring the controlling mechanisms by which the plant cell wall is constructed and reformed during plant growth and development.

Role of induced glutathione-S-transferase from Helicoverpa armigera (Lepidoptera: Noctuidae) HaGST-8 in detoxification of pesticides.

PubMed

Labade, Chaitali P; Jadhav, Abhilash R; Ahire, Mehul; Zinjarde, Smita S; Tamhane, Vaijayanti A

2018-01-01

The present study deals with glutathione-S-transferase (GST) based detoxification of pesticides in Helicoverpa armigera and its potential application in eliminating pesticides from the environment. Dietary exposure of a pesticide mixture (organophosphates - chlorpyrifos and dichlorvos, pyrethroid - cypermethrin; 2-15ppm each) to H. armigera larvae resulted in a dose dependant up-regulation of GST activity and gene expression. A variant GST from H. armigera (HaGST-8) was isolated from larvae fed with 10ppm pesticide mixture and it was recombinantly expressed in yeast (Pichia pastoris HaGST-8). HaGST-8 had a molecular mass of 29kDa and was most active at pH 9 at 30°C. GC-MS and LC-HRMS analysis validated that HaGST-8 was effective in eliminating organophosphate type of pesticides and partially reduced the cypermethrin content (53%) from aqueous solutions. Unlike the untransformed yeast, P. pastoris HaGST-8 grew efficiently in media supplemented with pesticide mixtures (200 and 400ppm each pesticide) signifying the detoxification ability of HaGST-8. The amino acid sequence of HaGST-8 and the already reported sequence of HaGST-7 had just 2 mismatches. The studies on molecular interaction strengths revealed that HaGST-8 had stronger binding affinities with organophosphate, pyrethroid, organochloride, carbamate and neonicotinoid type of pesticides. The abilities of recombinant HaGST-8 to eliminate pesticides and P. pastoris HaGST-8 to grow profusely in the presence of high level of pesticide content can be applied for removal of such residues from food, water resources and bioremediation. Copyright © 2017 Elsevier Inc. All rights reserved.

Preferential glutathione conjugation of a reverse diol epoxide compared to a bay region diol epoxide of phenanthrene in human hepatocytes: relevance to molecular epidemiology studies of glutathione-s-transferase polymorphisms and cancer.

PubMed

Hecht, Stephen S; Berg, Jeannette Zinggeler; Hochalter, J Bradley

2009-03-16

Bay region diol epoxides are recognized ultimate carcinogens of polycyclic aromatic hydrocarbons (PAH), and in vitro studies have demonstrated that they can be detoxified by conjugation with glutathione, leading to the widely investigated hypothesis that individuals with low activity forms of glutathione-S-transferases are at higher risk of PAH induced cancer, a hypothesis that has found at most weak support in molecular epidemiology studies. A weakness in this hypothesis was that the mercapturic acids resulting from the conjugation of PAH bay region diol epoxides had never been identified in human urine. We recently analyzed smokers' urine for mercapturic acids derived from phenanthrene, the simplest PAH with a bay region. The only phenanthrene diol epoxide-derived mercapturic acid in smokers' urine was produced from the reverse diol epoxide, anti-phenanthrene-3,4-diol-1,2-epoxide (11), not the bay region diol epoxide, anti-phenanthrene-1,2-diol-3,4-epoxide (10), which does not support the hypothesis noted above. In this study, we extended these results by examining the conjugation of phenanthrene metabolites with glutathione in human hepatocytes. We identified the mercapturic acid N-acetyl-S-(r-4,t-2,3-trihydroxy-1,2,3,4-tetrahydro-c-1-phenanthryl)-L-cysteine (14a), (0.33-35.9 pmol/mL at 10 microM 8, 24 h incubation, N = 10) in all incubations with phenanthrene-3,4-diol (8) and the corresponding diol epoxide 11, but no mercapturic acids were detected in incubations with phenanthrene-1,2-diol (7), and only trace amounts were observed in incubations with the corresponding bay region diol epoxide 10. Taken together with our previous results, these studies clearly demonstrate that glutathione conjugation of a reverse diol epoxide of phenanthrene is favored over conjugation of a bay region diol epoxide. Since reverse diol epoxides of PAH are generally weakly or nonmutagenic/carcinogenic, these results, if generalizable to other PAH, do not support the widely held

Nuclear factor erythroid-derived 2-related factor 2 activates glutathione S-transferase expression in the midgut of Spodoptera litura (Lepidoptera: Noctuidae) in response to phytochemicals and insecticides.

PubMed

Chen, S; Lu, M; Zhang, N; Zou, X; Mo, M; Zheng, S

2018-05-10

Detoxication enzymes play an important role in insect resistance to xenobiotics such as insecticides and phytochemicals. We studied the pathway for activating the expression of glutathione S-transferases (GSTs) in response to selected xenobiotics. An assay of the promoter activity of GST epsilon 1 (Slgste1) of Spodoptera litura led to the discovery of a cis-regulating element. An antioxidant response element was activated in response to indole-3-carbinol (I3C) and chlorpyrifos (CPF) and was able to bind with the xenobiotic sensor protein nuclear factor erythroid-derived 2-related factor 2 (SlNrf2). SlNrf2 and Slgste1 were responsive to reactive oxygen species induced by I3C and CPF in a S. litura cell line, as well as in S. litura midguts. SlNrf2 RNA interference (RNAi) reduced the message RNA levels of Slgste1 and the peroxidase activity of GSTs in response to I3C, xanthotoxin, CPF and deltamethrin. SlNrf2 RNAi and inhibitor treatment of GST activity decreased the viability of I3C-treated cells. These results indicate that SlNrf2 activates the expression of GSTs in response to oxidative stresses caused by exposure to xenobiotics. © 2018 The Royal Entomological Society.

Human cytosolic glutathione-S-transferases: quantitative analysis of expression, comparative analysis of structures and inhibition strategies of isozymes involved in drug resistance.

PubMed

Mohana, Krishnamoorthy; Achary, Anant

2017-08-01

Glutathione-S-transferase (GST) inhibition is a strategy to overcome drug resistance. Several isoforms of human GSTs are present and they are expressed in almost all the organs. Specific expression levels of GSTs in various organs are collected from the human transcriptome data and analysis of the organ-specific expression of GST isoforms is carried out. The variations in the level of expressions of GST isoforms are statistically significant. The GST expression differs in diseased conditions as reported by many investigators and some of the isoforms of GSTs are disease markers or drug targets. Structure analysis of various isoforms is carried out and literature mining has been performed to identify the differences in the active sites of the GSTs. The xenobiotic binding H site is classified into H1, H2, and H3 and the differences in the amino acid composition, the hydrophobicity and other structural features of H site of GSTs are discussed. The existing inhibition strategies are compared. The advent of rational drug design, mechanism-based inhibition strategies, availability of high-throughput screening, target specific, and selective inhibition of GST isoforms involved in drug resistance could be achieved for the reversal of drug resistance and aid in the treatment of diseases.

Association of glutathione S-transferase pi isoform single-nucleotide polymorphisms with exudative age-related macular degeneration in a Chinese population.

PubMed

Gu, Hong; Sun, Erdan; Cui, Lei; Yang, Xiufen; Lim, Apiradee; Xu, Jun; Snellingen, Torkel; Liu, Xipu; Wang, Ningli; Liu, Ningpu

2012-10-01

To investigate the association between single-nucleotide polymorphisms in the pi isoform of glutathione S-transferase (GSTP1) gene and the risk of exudative age-related macular degeneration (AMD) in a Chinese case-control cohort. A total of 131 Chinese patients with exudative AMD and 138 control individuals were recruited. Genomic DNA was extracted from venous blood leukocytes. Two common nonsynonymous single-nucleotide polymorphisms in GSTP1 (rs1695 and rs1138272) were genotyped by polymerase chain reaction followed by allele-specific restriction enzyme digestion and direct sequencing. Significant association with exudative AMD was detected for single-nucleotide polymorphism, rs1695 (P = 0.019). The risk G allele frequencies were 21.8% in AMD patients and 12.7% in control subjects (P = 0.007). Compared with the wild-type AA genotype, odds ratio for the risk of AMD was 1.91 (95% confidence interval, 1.09-3.35) for the heterozygous AG genotype and 2.52 (95% confidence interval, 0.6-10.61) for the homozygous GG genotype. In contrast, rs1138272 was not associated with exudative AMD (P = 1.00). The risk G allele frequencies of rs1138272 were 0.4% in AMD patients and 0.4% in control subjects (P = 1.00). Our data suggest that the GSTP1 variant rs1695 moderately increases the risk of exudative AMD. The variant rs1138272 was rare and was not associated with exudative AMD in this Chinese cohort.

Fluorescein diacetate (FDA) and its analogue as substrates for Pi-class glutathione S-transferase (GSTP1) and their biological application.

PubMed

Fujikawa, Yuuta; Nampo, Taiki; Mori, Masaya; Kikkawa, Manami; Inoue, Hideshi

2018-03-01

Pi class glutathione S-transferase (GSTP1) is highly expressed in various cancerous cells and pre-neoplastic legions, where it is involved in apoptotic resistance or metabolism of several anti-tumour chemotherapeutics. Therefore, GSTP1 is a marker of malignant and pre-malignant cells and is a promising target for visualization and drug development. Here we demonstrate that fluorescein diacetate (FDA), a fluorescent probe used for vital staining, is a fluorescently activated by esterolytic activity of human GSTP1 (hGSTP1) selectively among various cytosolic GSTs. Fluorescence activation of FDA susceptible to GST inhibitors was observed in MCF7 cells exogenously overexpressing hGSTP1, but not in cells overexpressing hGSTA1 or hGSTM1. Inhibitor-sensitive fluorescence activation was also observed in several cancer cell lines endogenously expressing GSTP1, suggesting that GSTP1 is involved in FDA esterolysis in these cells. Among the FDA derivatives examined, FOMe-Ac, the acetyl ester of fluorescein O-methyl ether, was found to be a potential reporter for GSH-dependent GSTP1 activity as well as for carboxylesterase activity. Since GSTP1 is highly expressed in various types of cancer cells compared to their normal counterparts, improving the fluorogenic substrates to be more selective to the esterolysis activity of GSTP1 rather than carboxylesterases should lead to development of tools for detecting GSTP1-overexpressing cancer cells and investigating the biological functions of GSTP1. Copyright © 2017 Elsevier B.V. All rights reserved.

Functional Role of Tyr12 in the Catalytic Activity of Novel Zeta-like Glutathione S-transferase from Acidovorax sp. KKS102.

PubMed

Shehu, Dayyabu; Alias, Zazali

2018-05-19

Glutathione S-transferases (GSTs) are a family of enzymes that function in the detoxification of variety of electrophilic substrates. In the present work, we report a novel zeta-like GST (designated as KKSG9) from the biphenyl/polychlorobiphenyl degrading organism Acidovorax sp. KKS102. KKSG9 possessed low sequence similarity but similar biochemical properties to zeta class GSTs. Functional analysis showed that the enzyme exhibits wider substrate specificity compared to most zeta class GSTs by reacting with 1-chloro-2,4-dinitrobenzene (CDNB), p-nitrobenzyl chloride (NBC), ethacrynic acid (EA), hydrogen peroxide, and cumene hydroperoxide. The enzyme also displayed dehalogenation function against dichloroacetate, permethrin, and dieldrin. The functional role of Tyr12 was also investigated by site-directed mutagenesis. The mutant (Y12C) displayed low catalytic activity and dehalogenation function against all the substrates when compared with the wild type. Kinetic analysis using NBC and GSH as substrates showed that the mutant (Y12C) displayed a higher affinity for NBC when compared with the wild type, however, no significant change in GSH affinity was observed. These findings suggest that the presence of tyrosine residue in the motif might represent an evolutionary trend toward improving the catalytic activity of the enzyme. The enzyme as well could be useful in the bioremediation of various types of organochlorine pollutants.

Analyses of Genetic Variations of Glutathione S-Transferase Mu1 and Theta1 Genes in Bangladeshi Tannery Workers and Healthy Controls.

PubMed

Akther, Jobaida; Ebihara, Akio; Nakagawa, Tsutomu; Islam, Laila N; Suzuki, Fumiaki; Hosen, Md Ismail; Hossain, Mahmud; Nabi, A H M Nurun

2016-01-01

Glutathione S-transferases (GSTs) belong to a group of multigene detoxification enzymes, which defend cells against oxidative stress. Tannery workers are at risk of oxidative damage that is usually detoxified by GSTs. This study investigated the genotypic frequencies of GST Mu1 (GSTM1) and GST Theta1 (GSTT1) in Bangladeshi tannery workers and healthy controls followed by their status of oxidative stress and total GST activity. Of the 188 individuals, 50.0% had both GSTM1 and GSTT1 (+/+), 12.2% had GSTM1 (+/-), 31.4% had GSTT1 (-/+) alleles, and 6.4% had null genotypes (-/-) with respect to both GSTM1 and GSTT1 alleles. Among 109 healthy controls, 54.1% were double positive, 9.2% had GSTM1 allele, 32.1% had GSTT1 allele, and 4.6% had null genotypes. Out of 79 tannery workers, 44.3% were +/+, 16.8% were +/-, 30.5% were -/+, and 8.4% were -/-. Though the polymorphic genotypes or allelic variants of GSTM1 and GSTT1 were distributed among the study subjects with different frequencies, the differences between the study groups were not statistically significant. GST activity did not vary significantly between the two groups and also among different genotypes while level of lipid peroxidation was significantly higher in tannery workers compared to controls irrespective of their GST genotypes.

Analyses of Genetic Variations of Glutathione S-Transferase Mu1 and Theta1 Genes in Bangladeshi Tannery Workers and Healthy Controls

PubMed Central

Akther, Jobaida; Ebihara, Akio; Nakagawa, Tsutomu; Islam, Laila N.; Suzuki, Fumiaki; Hosen, Md. Ismail; Hossain, Mahmud; Nabi, A. H. M. Nurun

2016-01-01

Glutathione S-transferases (GSTs) belong to a group of multigene detoxification enzymes, which defend cells against oxidative stress. Tannery workers are at risk of oxidative damage that is usually detoxified by GSTs. This study investigated the genotypic frequencies of GST Mu1 (GSTM1) and GST Theta1 (GSTT1) in Bangladeshi tannery workers and healthy controls followed by their status of oxidative stress and total GST activity. Of the 188 individuals, 50.0% had both GSTM1 and GSTT1 (+/+), 12.2% had GSTM1 (+/−), 31.4% had GSTT1 (−/+) alleles, and 6.4% had null genotypes (−/−) with respect to both GSTM1 and GSTT1 alleles. Among 109 healthy controls, 54.1% were double positive, 9.2% had GSTM1 allele, 32.1% had GSTT1 allele, and 4.6% had null genotypes. Out of 79 tannery workers, 44.3% were +/+, 16.8% were +/−, 30.5% were −/+, and 8.4% were −/−. Though the polymorphic genotypes or allelic variants of GSTM1 and GSTT1 were distributed among the study subjects with different frequencies, the differences between the study groups were not statistically significant. GST activity did not vary significantly between the two groups and also among different genotypes while level of lipid peroxidation was significantly higher in tannery workers compared to controls irrespective of their GST genotypes. PMID:27294127

Enhanced phytoremediation of mixed heavy metal (mercury)-organic pollutants (trichloroethylene) with transgenic alfalfa co-expressing glutathione S-transferase and human P450 2E1.

PubMed

Zhang, Yuanyuan; Liu, Junhong; Zhou, Yuanming; Gong, Tingyun; Wang, Jing; Ge, Yinlin

2013-09-15

Soil contamination is a global environmental problem and many efforts have been made to find efficient remediation methods over the last decade. Moreover, remediation of mixed contaminated soils are more difficult. In the present study, transgenic alfalfa plants pKHCG co-expressing glutathione S-transferase (GST) and human P450 2E1 (CYP2E1) genes were used for phytoremediation of mixed mercury (Hg)-trichloroethylene (TCE) contaminants. Simultaneous expression of GST and CYP2E1 may produce a significant synergistic effect, and leads to improved resistance and accumulation to heavy metal-organic complex contaminants. Based on the tolerance and accumulation assays, pKHCG transgenic plants were more resistant to Hg/TCE complex pollutants and many folds higher in Hg/TCE-accumulation than the non-transgenic control plants in mixed contaminated soil. It is confirmed that GST and CYP2E1 co-expression may be a useful strategy to help achieve mixed heavy metal-organic pollutants phytoremediation. Copyright © 2013 Elsevier B.V. All rights reserved.

O-GlcNAc Transferase Is Essential for Sensory Neuron Survival and Maintenance

PubMed Central

Su, Cathy

2017-01-01

O-GlcNAc transferase (OGT) regulates a wide range of cellular processes through the addition of the O-GlcNAc sugar moiety to thousands of protein substrates. Because nutrient availability affects the activity of OGT, its role has been broadly studied in metabolic tissues. OGT is enriched in the nervous system, but little is known about its importance in basic neuronal processes in vivo. Here, we show that OGT is essential for sensory neuron survival and maintenance in mice. Sensory neuron-specific knock-out of OGT results in behavioral hyposensitivity to thermal and mechanical stimuli accompanied by decreased epidermal innervation and cell-body loss in the dorsal root ganglia. These effects are observed early in postnatal development and progress as animals age. Cultured sensory neurons lacking OGT also exhibit decreased axonal outgrowth. The effects on neuronal health in vivo are not solely due to disruption of developmental processes, because inducing OGT knock-out in the sensory neurons of adult mice results in a similar decrease in nerve fiber endings and cell bodies. Significant nerve-ending loss occurs before a decrease in cell bodies; this phenotype is indicative of axonal dieback that progresses to neuronal death. Our findings demonstrate that OGT is important in regulating axonal maintenance in the periphery and the overall health and survival of sensory neurons. SIGNIFICANCE STATEMENT We show the importance of O-GlcNAc transferase (OGT) for sensory neuron health and survival in vivo. This study is the first to find that loss of OGT results in neuronal cell death. Moreover, it suggests that aberrant O-GlcNAc signaling can contribute to the development of neuropathy. The sensory neurons lie outside of the blood–brain barrier and therefore, compared to central neurons, may have a greater need for mechanisms of metabolic sensing and compensation. Peripheral sensory neurons in particular are subject to degeneration in diabetes. Our findings provide a

Sirtuin 3 enhanced drug sensitivity of human hepatoma cells through glutathione S-transferase pi 1/JNK signaling pathway

PubMed Central

Cai, Xue-Fei; Zhang, Wen-Lu; Ren, Ji-Hua; Zhou, Li; Chen, Xiang; Chen, Ke; Li, Wan-Yu; Liu, Bo; Yang, Qiu-Xia; Cheng, Sheng-Tao; Huang, Li-Xia; Huang, Ai-Long; Chen, Juan

2016-01-01

SIRT3, a class III histone deacetylase, has been implicated in various cancers as a novel therapeutic target. In hepatocellular carcinoma (HCC), we previously reported that SIRT3 induced cell apoptosis by regulating GSK-3β/Bax signaling pathway. Downregulation of SIRT3 in HCC cells facilitates tumor cell survival. In this study, we found that chemotherapeutic agents (doxorubicin, cisplatin and epirubicin) and sorafenib treatment downregulated SIRT3 mRNA and protein levels in three HCC cell lines. MTS assay found that SIRT3 overexpression sensitized liver cancer cells to chemotherapeutic agents and sorafenib in SMMC-7721, Huh-7 and PLC/PRF/5 cell lines. Moreover, SIRT3 overexpression promoted chemotherapeutic agents-induced or sorafenib-induced apoptosis as evidenced by flow cytometry, enhanced PARP cleavage and enhanced Caspase-9 cleavage in three HCC cells. In contrast, SIRT3 silencing increased drug resistance of HCC cells to chemotherapeutic agents. Mechanistic study found that SIRT3 downregulated the mRNA and protein levels of glutathione S-transferase pi 1 (GSTP1), which is a member of phase II detoxification enzymes families involved in metabolizing for chemotherapeutic agents. Moreover, SIRT3 decreased the amount of GSTP1 that was associated with JNK, which finally contributed the activation of JNK activity and activation of downstream target c-Jun and Bim. Importantly, GSTP1 overexpression or JNK inhibitor abolished SIRT3-induced apoptosis in HCC cells exposed to chemotherapeutic agents. Finally, there was a negative correlation between SIRT3 expression and GSTP1 expression in human HCC tissues. Together, our findings revealed SIRT3 could enhance the drug sensitivity of HCC cells to an array of chemotherapeutic agents. SIRT3 may serve as a potential target for improving the chemosensitivity of HCC patients. PMID:27367026

Sirtuin 3 enhanced drug sensitivity of human hepatoma cells through glutathione S-transferase pi 1/JNK signaling pathway.

PubMed

Tao, Na-Na; Zhou, Hong-Zhong; Tang, Hua; Cai, Xue-Fei; Zhang, Wen-Lu; Ren, Ji-Hua; Zhou, Li; Chen, Xiang; Chen, Ke; Li, Wan-Yu; Liu, Bo; Yang, Qiu-Xia; Cheng, Sheng-Tao; Huang, Li-Xia; Huang, Ai-Long; Chen, Juan

2016-08-02

SIRT3, a class III histone deacetylase, has been implicated in various cancers as a novel therapeutic target. In hepatocellular carcinoma (HCC), we previously reported that SIRT3 induced cell apoptosis by regulating GSK-3β/Bax signaling pathway. Downregulation of SIRT3 in HCC cells facilitates tumor cell survival. In this study, we found that chemotherapeutic agents (doxorubicin, cisplatin and epirubicin) and sorafenib treatment downregulated SIRT3 mRNA and protein levels in three HCC cell lines. MTS assay found that SIRT3 overexpression sensitized liver cancer cells to chemotherapeutic agents and sorafenib in SMMC-7721, Huh-7 and PLC/PRF/5 cell lines. Moreover, SIRT3 overexpression promoted chemotherapeutic agents-induced or sorafenib-induced apoptosis as evidenced by flow cytometry, enhanced PARP cleavage and enhanced Caspase-9 cleavage in three HCC cells. In contrast, SIRT3 silencing increased drug resistance of HCC cells to chemotherapeutic agents. Mechanistic study found that SIRT3 downregulated the mRNA and protein levels of glutathione S-transferase pi 1 (GSTP1), which is a member of phase II detoxification enzymes families involved in metabolizing for chemotherapeutic agents. Moreover, SIRT3 decreased the amount of GSTP1 that was associated with JNK, which finally contributed the activation of JNK activity and activation of downstream target c-Jun and Bim. Importantly, GSTP1 overexpression or JNK inhibitor abolished SIRT3-induced apoptosis in HCC cells exposed to chemotherapeutic agents. Finally, there was a negative correlation between SIRT3 expression and GSTP1 expression in human HCC tissues. Together, our findings revealed SIRT3 could enhance the drug sensitivity of HCC cells to an array of chemotherapeutic agents. SIRT3 may serve as a potential target for improving the chemosensitivity of HCC patients.

Risks, Outcomes, and Evidence-based Interventions for Girls in the U.S. Juvenile Justice System

PubMed Central

Leve, Leslie D.; Chamberlain, Patricia; Kim, Hyoun K.

2015-01-01

The proportion of the juvenile justice population that is comprised of females is increasing, yet few evidence-based models have been evaluated and implemented with girls in the juvenile justice system. Although much is known about the risk and protective factors for girls who participate in serious delinquency, significant gaps in the research base hamper the development and implementation of theoretically based intervention approaches. In this review, we first summarize the extant empirical work about the predictors and sequelae of juvenile justice involvement for girls. Identified risk and protective factors that correspond to girls’ involvement in the juvenile justice system have been shown to largely parallel those of boys, although exposure rates and magnitudes of association sometimes differ by sex. Second, we summarize findings from empirically validated, evidence-based interventions for juvenile justice–involved youths that have been tested with girls. The interventions include Functional Family Therapy, Multisystemic Therapy, Multidimensional Family Therapy, and Treatment Foster Care Oregon (formerly known as Multidimensional Treatment Foster Care). We conclude that existing evidence-based practices appear to be effective for girls. However, few studies have been sufficiently designed to permit conclusions about whether sex-specific interventions would yield any better outcomes for girls than would interventions that already exist for both sexes and that have a strong base of evidence to support them. Third, we propose recommendations for feasible, cost-efficient next steps to advance the research and intervention agendas for this under-researched, under-served population of highly vulnerable youths. PMID:26119215

Imidazopyridine and Pyrazolopiperidine Derivatives as Novel Inhibitors of Serine Palmitoyl Transferase.

PubMed

Genin, Michael J; Gonzalez Valcarcel, Isabel C; Holloway, William G; Lamar, Jason; Mosior, Marian; Hawkins, Eric; Estridge, Thomas; Weidner, Jeffrey; Seng, Thomas; Yurek, David; Adams, Lisa A; Weller, Jennifer; Reynolds, Vincent L; Brozinick, Joseph T

2016-06-23

To develop novel treatments for type 2 diabetes and dyslipidemia, we pursued inhibitors of serine palmitoyl transferase (SPT). To this end compounds 1 and 2 were developed as potent SPT inhibitors in vitro. 1 and 2 reduce plasma ceramides in rodents, have a slight trend toward enhanced insulin sensitization in DIO mice, and reduce triglycerides and raise HDL in cholesterol/cholic acid fed rats. Unfortunately these molecules cause a gastric enteropathy after chronic dosing in rats.

Effects of chlorpyrifos on life cycle parameters, cytochrome P450S expression, and antioxidant systems in the monogonont rotifer Brachionus koreanus.

PubMed

Kim, Ryeo-Ok; Kim, Bo-Mi; Jeong, Chang-Bum; Lee, Jae-Seong; Rhee, Jae-Sung

2016-06-01

Chlorpyrifos is a widely used organophosphorus insecticide for controlling diverse insect pests of crops. In the monogonont rotifer Brachionus koreanus, population growth retardation with the inhibition of lifespan, fecundity, and individual body size of ovigerous females was shown over 10 d in response to chlorpyrifos exposure. At the molecular and biochemical levels, the rotifer B. koreanus defensome, composed of cytochrome P450 complements, heat shock protein 70, and antioxidant enzymatic systems (i.e., glutathione, glutathione peroxidase, glutathione reductase, and glutathione S-transferase), was significantly induced in response to different concentrations of chlorpyrifos. Thus, chlorpyrifos strongly induced a defensome system to mitigate the deleterious effects of chlorpyrifos at in vivo and in vitro levels as a trade-off in fitness costs. Environ Toxicol Chem 2016;35:1449-1457. © 2015 SETAC. © 2015 SETAC.

Influence of ethacrynic acid on glutathione S-transferase pi transcript and protein half-lives in human colon cancer cells.

PubMed

Shen, H; Ranganathan, S; Kuzmich, S; Tew, K D

1995-10-12

Ethacrynic acid (EA) is a plant phenolic acid that is both an inhibitor and an inducer of glutathione S-transferase (GST) activity. To determine contributory factors in the increased GST activity caused by EA treatment, human colon carcinoma HT29 cells were compared with a cloned EA-resistant population (HT6-8) maintained in medium containing 72 microM EA. Several factors are involved in the increased expression of GST pi in HT6-8. For example, nuclear run-on experiments showed an approximately 2-fold increase in the rate of transcription of GST pi. In addition, the half-life of GST pi transcript was increased from 4.1 (wild type, HT29, HT4-1) to 8.4 hr. The half-life of GST pi protein was 1-2 hr in HT4-1 cells versus 8-9 hr in HT6-8 cells. When either human ovarian carcinoma cells (SKOV3) or human prostatic carcinoma cells (DU145) were treated with EA, the half-life of the GST pi transcript was also increased. The transcript half-lives of another thiol-metabolism enzyme, gamma-glutamylcysteine synthetase (gamma-GCS), and a phase II detoxification enzyme, dihydrodiol dehydrogenase (DDH), were also increased in HT6-8, SKOV3 and DU145 cells treated with EA. However, the half-lives of transcripts from "housekeeping genes," such as glyceraldehyde 3-phosphate dehydrogenase (G3PDH), beta-actin and beta-tubulin, were not changed in these cell lines following EA. Apparently, a number of coordinated factors are involved in EA-enhanced expression of GST pi and other detoxification enzymes.

Clinicopathological correlates and prognostic significance of glutathione S-transferase Pi expression in 468 patients after potentially curative resection of node-positive colonic cancer.

PubMed

Tan, King L; Jankova, Lucy; Chan, Charles; Fung, Caroline L-S; Clarke, Candice; Lin, Betty P C; Robertson, Graham; Molloy, Mark; Chapuis, Pierre H; Bokey, Les; Dent, Owen F; Clarke, Stephen J

2011-12-01

This study investigated the association between glutathione S-transferase Pi (GST Pi) expression, histopathology and overall survival in 468 patients after resection of stage C colonic adenocarcinoma. Data were drawn from a prospective hospital registry of consecutive bowel cancer resections with a minimum follow-up of 5 years. Nuclear and cytoplasmic GST Pi expression, assessed by both intensity of staining and percentage of stained cells at both the central part of the tumour and the invasive tumour front, were evaluated retrospectively by tissue microarray immunohistochemistry on archival specimens. The most effective measure of GST Pi expression was the percentage of immunostained nuclei in central tumour tissue, where >40% stained was associated significantly with high grade, invasion beyond the muscularis propria, involvement of a free serosal surface or apical node, and invasion into an adjacent organ or structure. After adjustment of other predictors, GST Pi expression remained independently prognostic for reduced overall survival (hazard ratio 1.4, P = 0.002). In patients with clinicopathological stage C colonic cancer, GST Pi expression is associated with features of tumour aggressiveness and with reduced overall survival. Further appropriately designed studies should aim to discover whether GST Pi can predict response to adjuvant chemotherapy. © 2011 Blackwell Publishing Limited.

The Potato Aphid Salivary Effector Me47 Is a Glutathione-S-Transferase Involved in Modifying Plant Responses to Aphid Infestation.

PubMed

Kettles, Graeme J; Kaloshian, Isgouhi

2016-01-01

Polyphagous aphid pests cause considerable economic damage to crop plants, primarily through the depletion of photoassimilates and transfer of viruses. The potato aphid (Macrosiphum euphorbiae) is a notable pest of solanaceous crops, however, the molecular mechanisms that underpin the ability to colonize these hosts are unknown. It has recently been demonstrated that like other aphid species, M. euphorbiae injects a battery of salivary proteins into host plants during feeding. It is speculated that these proteins function in a manner analagous to secreted effectors from phytopathogenic bacteria, fungi and oomycetes. Here, we describe a novel aphid effector (Me47) which was identified from the potato aphid salivary secretome as a putative glutathione-S-transferase (GST). Expression of Me47 in Nicotiana benthamiana enhanced reproductive performance of green peach aphid (Myzus persicae). Similarly, delivery of Me47 into leaves of tomato (Solanum lycopersicum) by Pseudomonas spp. enhanced potato aphid fecundity. In contrast, delivery of Me47 into Arabidopsis thaliana reduced GPA reproductive performance, indicating that Me47 impacts the outcome of plant-aphid interactions differently depending on the host species. Delivery of Me47 by the non-pathogenic Pseudomonas fluorescens revealed that Me47 protein or activity triggers defense gene transcriptional upregulation in tomato but not Arabidopsis. Recombinant Me47 was purified and demonstrated to have GST activity against two specific isothiocyanates (ITCs), compounds implicated in herbivore defense. Whilst GSTs have previously been associated with development of aphid resistance to synthetic insecticides, the findings described here highlight a novel function as both an elicitor and suppressor of plant defense when delivered into host tissues.

Identification and characterisation of seventeen glutathione S-transferase genes from the cabbage white butterfly Pieris rapae.

PubMed

Liu, Su; Zhang, Yu-Xing; Wang, Wen-Long; Zhang, Bang-Xian; Li, Shi-Guang

2017-11-01

Insect glutathione S-transferases (GSTs) play essential roles in the detoxification of insecticides and other xenobiotic compounds. The cabbage white butterfly, Pieris rapae, is an economically important agricultural pest. In this study, 17 cDNA sequences encoding putative GSTs were identified in P. rapae. All cDNAs include a complete open reading frame and were designated PrGSTd1-PrGSTz2. Based on phylogenetic analysis, PrGSTs were divided into six classes (delta, epsilon, omega, sigma, theta and zeta). The exon-intron organizations of these PrGSTs were also analysed. Recombinant proteins of eight PrGSTs (PrGSTD1, PrGSTD2, PrGSTE1, PrGSTE2, PrGSTO1, PrGSTS1, PrGSTT1 and PrGSTZ1) were heterologously expressed in Escherichia coli, and all of these proteins displayed glutathione-conjugating activity towards 1-chloro-2,4-dinitrobenzene (CDNB). Expression patterns in various larval tissues, at different life stages, and following exposure to sublethal doses of abamectin, chlorantraniliprole or lambda-cyhalothrin were determined by reverse transcription-quantitative PCR. The results showed that PrGSTe3, PrGSTs1, PrGSTs2, and PrGSTs4 were mainly transcribed in the fat body, while PrGSTe2 was expressed predominantly in the Malpighian tubules. Four genes (PrGSTe2, PrGSTo4, PrGSTs4 and PrGSTt1) were mainly expressed in fourth-instar larvae, while others were ubiquitously expressed in egg, larval, pupa and/or adult stages. Abamectin treatment significantly upregulated ten genes (PrGSTd1, PrGSTd3, PrGSTe1, PrGSTe2, PrGSTo1, PrGSTo3, PrGSTs1, PrGSTs3, PrGSTs4 and PrGSTt1). Chlorantraniliprole and lambda-cyhalothrin treatment significantly upregulated nine genes (PrGSTd1, PrGSTd2, PrGSTe1, PrGSTe2, PrGSTe3, PrGSTs1, PrGSTs3, PrGSTs4 and PrGSTz1) and ten genes (PrGSTd1, PrGSTd3, PrGSTe1, PrGSTe2, PrGSTo1, PrGSTo2, PrGSTs1, PrGSTs2, PrGSTs3 and PrGSTz2), respectively. These GSTs are potentially involved in the detoxification of insecticides. Copyright © 2017 Elsevier Inc. All

Glutathione S-transferase pi polymorphism contributes to the treatment outcomes of advanced non-small cell lung cancer patients in a Chinese population.

PubMed

Chen, J B; Wang, F; Wu, J J; Cai, M

2016-07-25

We analyzed the association between polymorphisms in three glutathione S-transferase genes (GSTP1, GSTM1, and GSTT1) and the treatment outcome for advanced non-small cell lung cancer (NSCLC). We recruited 284 NSCLC patients at advanced stage from Department of Radiotherapy in Peace Hospital Attached to Changzhi Medical College between May 2009 and May 2011, who had received cisplatin-based chemotherapy. The GSTP1, GSTM1, and GSTT1 genotyping for was determined using DNA pyrosequencing on an ABI Prism 3100 DNA analyzer. In the Cox proportional hazards model, the IIe/Val and Val/Val genotypes of GSTP1 were associated with lower risk of disease progression compared with the IIe/IIe genotype, and the HRs (95%CIs) were 0.37 (0.18-0.74) and 0.15 (0.06-0.35), respectively. The IIe/Val and Val/Val genotypes significantly decreased risk of death from all causes in patients with NSCLC, and the HRs (95%CIs) were 0.52 (0.29-0.92) and 0.37 (0.17- 0.79), respectively No significant association was observed between GSTM1 and GSTT1 polymorphisms and progression-free survival and overall survival in the NSCLC patients. In summary, we suggest that GSTP1 polymorphisms might influence the treatment outcome of advanced NSCLC patients, and our results could help improve individualized therapy.

Glutathione S-Transferase Pi-Ile 105 Val Polymorphism and Susceptibility to T2DM in Population from Turabah Region of Saudi Arabia.

PubMed

Mergani, Adil; Mansour, Ahmed Abdelkhalik; Askar, Tamer; Zahran, Rasha Nabeel; Mustafa, Adil Musa; Mohammed, Mukhtar Ahmed; Saleh, Osama Mosailhy

2016-08-01

Type 2 diabetes mellitus is characterized by chronic hyperglycemia and associated with oxidative stress resulting from accumulation of free radicals in body's tissues, which especially affects beta cells in pancreas and is an important factor in the development of diabetes and its complications. Glutathione S-transferases (GSTs) are a family of antioxidant enzymes that play important roles in decreasing ROS species and act as a kind of antioxidant defense. In a case-control study, we investigated the role of GSTP1 Ile105Val polymorphism in predisposition to T2DM in patients from Tarabah province, Saudi Arabia. The polymorphism was screened by PCR-RFLP in 90 T2DM patients and 87 healthy controls. The genotypes and alleles frequencies in cases and controls were assessed using Cochran-Armitage trend test and odds ratios (ORs), and 95 % confidence intervals (CIs) in different genetic models of inheritance were calculated. Our data indicate that G allele (Val) is associated with an increased risk for T2DM in this population in any combination (OR 4.101, 95 % CI 1.986-8.469, P = 0.00008). This indicates that individuals who are carriers for the mutant allele, either in homozygous (GG) or heterozygous (AG) state, are at fourfold higher risk for development of T2DM than other subjects in this population.

Glutathione-S-transferase pi (GSTP1) codon 105 polymorphism is not associated with oxaliplatin efficacy or toxicity in advanced colorectal cancer patients.

PubMed

Kweekel, Dina M; Gelderblom, Hans; Antonini, Ninja F; Van der Straaten, Tahar; Nortier, Johan W R; Punt, Cornelis J A; Guchelaar, Henk-Jan

2009-03-01

Oxaliplatin is detoxified by conjugation to glutathione via the enzyme Glutathione-S-transferase pi (GSTP1). The aim of this study is to investigate the association of GSTP1 Ile105Val genetic polymorphism with oxaliplatin efficacy and toxicity in advanced colorectal cancer (ACC) patients. A total of 91 ACC patients received capecitabine and oxaliplatin (CAPOX) as a part of a multicentre phase-III study of the Dutch Colorectal Cancer Group. Tumour response was evaluated according to RECIST, toxicity was graded using CTC, and GSTP1 Ile105Val was determined by pyrosequencing. Overall survival after CAPOX was similar for patients with the Ile/Ile (11.5 mo), Ile/Val (11.6 mo) and Val/Val (12.6 mo) genotypes (p=0.602). Likewise, there were no statistically significant differences in progression-free survival (p=0.252). Overall grades 3-4 toxicity was not related to genotype (p=0.313). There were no differences in any grade or grades 3-4 neurotoxicity amongst the patients who received > or =500 mg/m(2) of oxaliplatin (p-values of 0.376 and 0.772, respectively). The results of this study indicate that the GSTP1 genotype is not predictive for progression-free survival or overall survival in ACC patients treated with CAPOX. Moreover, overall neurotoxicity and neurotoxicity in patients receiving 500 mg/m(2) of oxaliplatin was not associated with GSTP1 genotype.

Association of polymorphisms in glutathione S-transferase genes (GSTM1, GSTT1, GSTP1) with idiopathic azoospermia or oligospermia in Sichuan, China.

PubMed

Xiong, Da-Ke; Chen, Hong-Han; Ding, Xian-Ping; Zhang, Shao-Hong; Zhang, Jian-Hui

2015-01-01

The reported effects of the glutathione S-transferase (GSTs) genes (GSTM1, GSTT1, and GSTP1) on male factor infertility have been inconsistent and even contradictory. Here, we conducted a case-control study to investigate the association between functionally important polymorphisms in GST genes and idiopathic male infertility. The study group consisted of 361 men with idiopathic azoospermia, 118 men with idiopathic oligospermia, and 234 age-matched healthy fertile male controls. Genomic DNA was extracted from the peripheral blood, and analyzed by polymerase chain reaction and restriction fragment length polymorphism analysis. There was a significant association between the GSTP1 variant genotype (Ile/Val + Val/Val) with idiopathic infertility risk (odds ratio [OR]: 1.53; 95% confidence interval [CI]: 1.11-2.11; P = 0.009). Similarly, a higher risk of infertility was noted in individuals carrying a genotype combination of GSTT1-null and GSTP1 (Ile/Val + Val/Val) (OR: 2.17; 95% CI: 1.43-3.31; P = 0.0002). These results suggest an increased risk of the GSTP1 variant genotype (Ile/Val + Val/Val) for developing male factor infertility. Our findings also underrate the significance of the effect of GSTM1 and/or GSTT1 (especially the former) in modulating the risk of male infertility in males from Sichuan, Southwest China.

Identification of a novel transposon-associated phosphoethanolamine transferase gene, mcr-5, conferring colistin resistance in d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B.

PubMed

Borowiak, Maria; Fischer, Jennie; Hammerl, Jens A; Hendriksen, Rene S; Szabo, Istvan; Malorny, Burkhard

2017-12-01

Plasmid-mediated mobilized colistin resistance is currently known to be caused by phosphoethanolamine transferases termed MCR-1, MCR-2, MCR-3 and MCR-4. However, this study focuses on the dissection of a novel resistance mechanism in mcr-1-, mcr-2- and mcr-3-negative d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B (Salmonella Paratyphi B dTa+) isolates with colistin MIC values >2 mg/L. A selected isolate from the strain collection of the German National Reference Laboratory for Salmonella was investigated by WGS and bioinformatical analysis to identify novel phosphoethanolamine transferase genes involved in colistin resistance. Subsequently PCR screening, S1-PFGE and DNA-DNA hybridization were performed to analyse the prevalence and location of the identified mcr-5 gene. Cloning and transformation experiments in Escherichia coli DH5α and Salmonella Paratyphi B dTa+ control strains were carried out and the activity of MCR-5 was determined in vitro by MIC testing. In this study, we identified a novel phosphoethanolamine transferase in 14 mcr-1-, mcr-2- and mcr-3-negative Salmonella Paratyphi B dTa+ isolates with colistin MIC values >2 mg/L that were received during 2011-13. The respective gene, further termed as mcr-5 (1644 bp), is part of a 7337 bp transposon of the Tn3 family and usually located on related multi-copy ColE-type plasmids. Interestingly, in one isolate an additional subclone with a chromosomal location of the mcr-5 transposon was observed. Our findings suggest that the transfer of colistin-resistance-mediating phosphoethanolamine transferase genes from bacterial chromosomes to mobile genetic elements has occurred in multiple independent events raising concern regarding their variety, prevalence and impact on public health. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Exploiting the Substrate Promiscuity of Hydroxycinnamoyl-CoA:Shikimate Hydroxycinnamoyl Transferase to Reduce Lignin

DOE PAGES

Eudes, Aymerick; Pereira, Jose H.; Yogiswara, Sasha; ...

2016-02-08

Lignin poses a major challenge in the processing of plant biomass for agro-industrial applications. For bioengineering purposes, there is a pressing interest in identifying and characterizing the enzymes responsible for the biosynthesis of lignin. Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (HCT; EC 2.3.1.133) is a key metabolic entry point for the synthesis of the most important lignin monomers: coniferyl and sinapyl alcohols. In this study, we investigated the substrate promiscuity of HCT from a bryophyte (Physcomitrella) and from five representatives of vascular plants (Arabidopsis, poplar, switchgrass, pine and Selaginella) using a yeast expression system. We demonstrate for these HCTs a conserved capacity tomore » acylate with p-coumaroyl-CoA several phenolic compounds in addition to the canonical acceptor shikimate normally used during lignin biosynthesis. Using either recombinant HCT from switchgrass (PvHCT2a) or an Arabidopsis stem protein extract, we show evidence of the inhibitory effect of these phenolics on the synthesis of p-coumaroyl shikimate in vitro, which presumably occurs via a mechanism of competitive inhibition. A structural study of PvHCT2a confirmed the binding of a non-canonical acceptor in a similar manner to shikimate in the active site of the enzyme. Finally, we exploited in Arabidopsis the substrate flexibility of HCT to reduce lignin content and improve biomass saccharification by engineering transgenic lines that overproduce one of the HCT non-canonical acceptors. Our results demonstrate conservation of HCT substrate promiscuity and provide support for a new strategy for lignin reduction in the effort to improve the quality of plant biomass for forage and cellulosic biofuels.« less

Dishevelled3 is a novel arginine methyl transferase substrate.

PubMed

Bikkavilli, Rama Kamesh; Avasarala, Sreedevi; Vanscoyk, Michelle; Sechler, Marybeth; Kelley, Nicole; Malbon, Craig C; Winn, Robert A

2012-01-01

Dishevelled, a phosphoprotein scaffold, is a central component in all the Wnt-sensitive signaling pathways. In the present study, we report that Dishevelled is post-translationally modified, both in vitro and in vivo, via arginine methylation. We also show protein arginine methyl transferases 1 and 7 as the key enzymes catalyzing Dishevelled methylation. Interestingly, Wnt3a stimulation of F9 teratocarcinoma cells results in reduced Dishevelled methylation. Similarly, the methylation-deficient mutant of Dishevelled, R271K, displayed spontaneous membrane localization and robust activation of Wnt signaling; suggesting that differential methylation of Dishevelled plays an important role in Wnt signaling. Thus arginine methylation is shown to be an important switch in regulation of Dishevelled function and Wnt signaling.

Menadione stress in Saccharomyces cerevisiae strains deficient in the glutathione transferases.

PubMed

Castro, F A V; Herdeiro, R S; Panek, A D; Eleutherio, E C A; Pereira, M D

2007-02-01

Using S. cerevisiae as a eukaryotic cell model we have analyzed the involvement of both glutathione transferase isoforms, Gtt1 and Gtt2, in constitutive resistance and adaptive response to menadione, a quinone which can exert its toxicity as redox cycling and/or electrophiles. The detoxification properties, of these enzymes, have also been analyzed by the appearance of S-conjugates in the media. Direct exposure to menadione (20 mM/60 min) showed to be lethal for cells deficient on both Gtt1 and Gtt2 isoforms. However, after pre-treatment with a low menadione concentration, cells deficient in Gtt2 displayed reduced ability to acquire tolerance when compared with the control and the Gtt1 deficient strains. Analyzing the toxic effects of menadione we observed that the gtt2 mutant showed no reduction in lipid peroxidation levels. Moreover, measuring the levels of intracellular oxidation during menadione stress we have shown that the increase of this oxidative stress parameter was due to the capacity menadione possesses in generating reactive oxygen species (ROS) and that both GSH and Gtt2 isoform were required to enhance ROS production. Furthermore, the efflux of the menadione-GSH conjugate, which is related with detoxification of xenobiotic pathways, was not detected in the gtt2 mutant. Taken together, these results suggest that acquisition of tolerance against stress generated by menadione and the process of detoxification through S-conjugates are dependent upon Gtt2 activity. This assessment was corroborated by the increase of GTT2 expression, and not of GTT1, after menadione treatment.

Effect of a glutathione S-transferase inhibitor on oxidative stress and ischemia-reperfusion-induced apoptotic signalling of cultured cardiomyocytes

PubMed Central

Röth, E; Marczin, N; Balatonyi, B; Ghosh, S; Kovács, V; Alotti, N; Borsiczky, B; Gasz, B

2011-01-01

Oxidative stress and ischemia-reperfusion (I/R) injury are crucial in the pathogenesis of cardiovascular diseases. The antioxidant glutathione S-transferase (GST) is responsible for the high-capacity metabolic inactivation of electrophilic compounds and toxic substrates. The main objective of the present study was to examine the effect of GST inhibition (with the administration of ethacrynic acid [EA]) on the viability and apoptosis of cardiomyocytes when these cells are exposed to various stress components of I/R and mitogen-activated protein kinase (c-Jun N-terminal kinase, p38 and extracellular signal-regulated kinase [ERK]) inhibitors. The primary culture of neonatal rat cardiomyocytes was divided into six experimental groups: control group of cells (group 1), cells exposed to H2O2 (group 2), I/R (group 3), I/R and EA (group 4), H2O2 coupled with EA (group 5), and EA alone (group 6). The viability of cardiomyocytes was determined using a colorimetric MTT assay. The apoptosis ratio was evaluated via fluorescein isothiocyanate-labelled annexin V and propidium iodide staining. c-Jun N-terminal kinase, p38, Akt/protein kinase B and ERK/p42-p44 transcription factors were monitored with flow cytometry. c-Jun N-terminal kinase activation increased due to GST inhibition during I/R. EA administration led to a significant increase in p38 activation following both H2O2 treatment and I/R. ERK phosphorylation increased when GST was exposed to I/R. A pronounced decrease in Akt phosphorylation was observed when cells were cotreated with EA and H2O2. GST plays an important role as a regulator of mitogen-activated protein kinase pathways in I/R injury. PMID:22065940

Genetic polymorphisms in glutathione S-transferase (GST) superfamily and arsenic metabolism in residents of the Red River Delta, Vietnam

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agusa, Tetsuro; Center for Marine Environmental Studies; Iwata, Hisato, E-mail: iwatah@agr.ehime-u.ac.j

To elucidate the role of genetic factors in arsenic metabolism, we investigated associations of genetic polymorphisms in the members of glutathione S-transferase (GST) superfamily with the arsenic concentrations in hair and urine, and urinary arsenic profile in residents in the Red River Delta, Vietnam. Genotyping was conducted for GST omega1 (GSTO1) Ala140Asp, Glu155del, Glu208Lys, Thr217Asn, and Ala236Val, GST omega2 (GSTO2) Asn142Asp, GST pi1 (GSTP1) Ile105Val, GST mu1 (GSTM1) wild/null, and GST theta1 (GSTT1) wild/null. There were no mutation alleles for GSTO1 Glu208Lys, Thr217Asn, and Ala236Val in this population. GSTO1 Glu155del hetero type showed higher urinary concentration of As{sup V} thanmore » the wild homo type. Higher percentage of DMA{sup V} in urine of GSTM1 wild type was observed compared with that of the null type. Strong correlations between GSTP1 Ile105Val and arsenic exposure level and profile were observed in this study. Especially, heterozygote of GSTP1 Ile105Val had a higher metabolic capacity from inorganic arsenic to monomethyl arsenic, while the opposite trend was observed for ability of metabolism from As{sup V} to As{sup III}. Furthermore, other factors including sex, age, body mass index, arsenic level in drinking water, and genotypes of As (+ 3 oxidation state) methyltransferase (AS3MT) were also significantly co-associated with arsenic level and profile in the Vietnamese. To our knowledge, this is the first study indicating the associations of genetic factors of GST superfamily with arsenic metabolism in a Vietnamese population.« less

Immunoreactivities for glutathione S-transferases and glutathione peroxidase in the lateral wall of pigmented and albino guinea pig cochlea.

PubMed

Fujimura, Takeyuki; Suzuki, Hideaki; Udaka, Tsuyoshi; Shiomori, Teruo; Mori, Takanori; Inaba, Tsuyoshi; Hiraki, Nobuaki; Kayashima, Kotaro; Doi, Yoshiaki

2008-09-01

Dark-skinned people are known to be more tolerant of ototraumatic noise than are light-skinned people, and pigmented animals are more tolerant of ototraumatic noise and aminoglycoside ototoxicity than are albino animals. Such tolerance may be dependent on the local ability of detoxification and antioxidant enzymes, including glutathione S-transferase (GST) and glutathione peroxidase (GSPx). In the present study, we examined the difference in GST/GSPx expression in the lateral wall of the cochlea between pigmented and albino guinea pigs. Eight-week-old male pigmented and albino guinea pigs were killed by transcardiac perfusion with 2% paraformaldehyde. The cochlear ducts were isolated, further fixed with 4% paraformaldehyde, decalcified, and then embedded in paraffin. Sections prepared at 5-microm thickness were incubated with anti-GST-alpha,-mu,-pi, or anti-GSPx antibody, reacted with Alexa Fluorconjugated secondary antibody, and examined under a Carl Zeiss Axioskop 2 plus fluorescence microscope. The cochlea ducts were also subjected to immunoelectron microscopy for GST-pi by the postembedment method. The stria vascularis of pigmented guinea pigs was strongly immunoreactive for GST-alpha,-mu,-pi, and GSPx, whereas no or only weak immunoreactivities were seen in the stria vascularis of albino guinea pigs. The spiral ligament showed positive but different immunoreactivities for these enzymes between the strains. Double-stained immunofluorescence micrographs for GST-pi and GSPx showed a close resemblance of localization between the two enzymes in both pigmented and albino guinea pigs. At the ultrastructural level, immunoreactivity for GST-pi was localized preferentially in the melanin cells of pigmented guinea pigs. These results suggest that correlation between pigmentation and inner ear susceptibility is, at least partially, attributed to the different distribution of GST/GSPx in the stria vascularis.

Genetic polymorphisms in glutathione S-transferase (GST) superfamily and arsenic metabolism in residents of the Red River Delta, Vietnam.

PubMed

Agusa, Tetsuro; Iwata, Hisato; Fujihara, Junko; Kunito, Takashi; Takeshita, Haruo; Minh, Tu Binh; Trang, Pham Thi Kim; Viet, Pham Hung; Tanabe, Shinsuke

2010-02-01

To elucidate the role of genetic factors in arsenic metabolism, we investigated associations of genetic polymorphisms in the members of glutathione S-transferase (GST) superfamily with the arsenic concentrations in hair and urine, and urinary arsenic profile in residents in the Red River Delta, Vietnam. Genotyping was conducted for GST omega1 (GSTO1) Ala140Asp, Glu155del, Glu208Lys, Thr217Asn, and Ala236Val, GST omega2 (GSTO2) Asn142Asp, GST pi1 (GSTP1) Ile105Val, GST mu1 (GSTM1) wild/null, and GST theta1 (GSTT1) wild/null. There were no mutation alleles for GSTO1 Glu208Lys, Thr217Asn, and Ala236Val in this population. GSTO1 Glu155del hetero type showed higher urinary concentration of As(V) than the wild homo type. Higher percentage of DMA(V) in urine of GSTM1 wild type was observed compared with that of the null type. Strong correlations between GSTP1 Ile105Val and arsenic exposure level and profile were observed in this study. Especially, heterozygote of GSTP1 Ile105Val had a higher metabolic capacity from inorganic arsenic to monomethyl arsenic, while the opposite trend was observed for ability of metabolism from As(V) to As(III). Furthermore, other factors including sex, age, body mass index, arsenic level in drinking water, and genotypes of As (+3 oxidation state) methyltransferase (AS3MT) were also significantly co-associated with arsenic level and profile in the Vietnamese. To our knowledge, this is the first study indicating the associations of genetic factors of GST superfamily with arsenic metabolism in a Vietnamese population. Copyright 2009 Elsevier Inc. All rights reserved.

Enzymatic Activity of Glutathione S-Transferase and Dental Fluorosis Among Children Receiving Two Different Levels of Naturally Fluoridated Water.

PubMed

Bonola-Gallardo, Irvin; Irigoyen-Camacho, María Esther; Vera-Robles, Liliana; Campero, Antonio; Gómez-Quiroz, Luis

2017-03-01

This study was conducted to measure the activity of the enzyme glutathione S-transferase (GST) in saliva and to compare the activity of this enzyme in children with and without dental fluorosis in communities with different concentrations of naturally fluoridated water. A total of 141 schoolchildren participated in this cross-sectional study. Children were selected from two communities: one with a low (0.4 ppm) and the other with a high (1.8 ppm) water fluoride concentration. Dental fluorosis was evaluated by applying the Thylstrup and Fejerskov Index (TFI) criteria. Stimulated saliva was obtained, and fluoride concentration and GST activity were measured. The GST activity was compared among children with different levels of dental fluorosis using multinomial logistic regression models and odds ratios (OR). The mean age of the children was 10.6 (±1.03) years. Approximately half of the children showed dental fluorosis (52.5 %). The average GST activity was 0.5678 (±0.1959) nmol/min/μg. A higher concentration of fluoride in the saliva was detected in children with a higher GST activity (p = 0.039). A multinomial logistic regression model used to evaluate the GST activity and the dental fluorosis score identified a strong association between TFI = 2-3 (OR = 15.44, p = 0.007) and TFI ≥ 4 (OR = 55.40, p = 0.026) and the GST activity level, compared with children showing TFI = 0-1, adjusted for age and sex. Schoolchildren with higher levels of dental fluorosis and a higher fluoride concentration in the saliva showed greater GST activity. The increased GST activity most likely was the result of the body's need to inactivate free radicals produced by exposure to fluoride.

A prototype system to support evidence-based practice.

PubMed

Demner-Fushman, Dina; Seckman, Charlotte; Fisher, Cheryl; Hauser, Susan E; Clayton, Jennifer; Thoma, George R

2008-11-06

Translating evidence into clinical practice is a complex process that depends on the availability of evidence, the environment into which the research evidence is translated, and the system that facilitates the translation. This paper presents InfoBot, a system designed for automatic delivery of patient-specific information from evidence-based resources. A prototype system has been implemented to support development of individualized patient care plans. The prototype explores possibilities to automatically extract patients problems from the interdisciplinary team notes and query evidence-based resources using the extracted terms. Using 4,335 de-identified interdisciplinary team notes for 525 patients, the system automatically extracted biomedical terminology from 4,219 notes and linked resources to 260 patient records. Sixty of those records (15 each for Pediatrics, Oncology & Hematology, Medical & Surgical, and Behavioral Health units) have been selected for an ongoing evaluation of the quality of automatically proactively delivered evidence and its usefulness in development of care plans.

A Prototype System to Support Evidence-based Practice

PubMed Central

Demner-Fushman, Dina; Seckman, Charlotte; Fisher, Cheryl; Hauser, Susan E.; Clayton, Jennifer; Thoma, George R.

2008-01-01

Translating evidence into clinical practice is a complex process that depends on the availability of evidence, the environment into which the research evidence is translated, and the system that facilitates the translation. This paper presents InfoBot, a system designed for automatic delivery of patient-specific information from evidence-based resources. A prototype system has been implemented to support development of individualized patient care plans. The prototype explores possibilities to automatically extract patients’ problems from the interdisciplinary team notes and query evidence-based resources using the extracted terms. Using 4,335 de-identified interdisciplinary team notes for 525 patients, the system automatically extracted biomedical terminology from 4,219 notes and linked resources to 260 patient records. Sixty of those records (15 each for Pediatrics, Oncology & Hematology, Medical & Surgical, and Behavioral Health units) have been selected for an ongoing evaluation of the quality of automatically proactively delivered evidence and its usefulness in development of care plans. PMID:18998835

Directed evolution of glutathione transferases towards a selective glutathione-binding site and improved oxidative stability.

PubMed

Axarli, Irine; Muleta, Abdi W; Chronopoulou, Evangelia G; Papageorgiou, Anastassios C; Labrou, Nikolaos E

2017-01-01

Glutathione transferases (GSTs) are a family of detoxification enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic compounds. A library of alpha class GSTs was constructed by DNA shuffling using the DNA encoding the human glutathione transferase A1-1 (hGSTA1-1) and the rat glutathione transferase A1-1 (rGSTA1-1). Activity screening of the library allowed the selection of a chimeric enzyme variant (GSTD4) that displayed high affinity towards GSH and GSH-Sepharose affinity adsorbent, higher k cat /K m and improved thermal stability, compared to the parent enzymes. The crystal structures of the GSTD4 enzyme in free form and in complex with GSH were determined to 1.6Šand 2.3Šresolution, respectively. Analysis of the GSTD4 structure showed subtle conformational changes in the GSH-binding site and in electron-sharing network that may contribute to the increased GSH affinity. The shuffled variant GSTD4 was further optimized for improved oxidative stability employing site-saturation mutagenesis. The Cys112Ser mutation confers optimal oxidative stability and kinetic properties in the GSTD4 enzyme. DNA shuffling allowed the creation of a chimeric enzyme variant with improved properties, compared to the parent enzymes. X-ray crystallography shed light on how recombination of a specific segment from homologous GSTA1-1 together with point mutations gives rise to a new functionally competent enzyme with improved binding, catalytic properties and stability. Such an engineered GST would be useful in biotechnology as affinity tool in affinity chromatography as well as a biocatalytic matrix for the construction of biochips or enzyme biosensors. Copyright © 2016 Elsevier B.V. All rights reserved.

The Potato Aphid Salivary Effector Me47 Is a Glutathione-S-Transferase Involved in Modifying Plant Responses to Aphid Infestation

PubMed Central

Kettles, Graeme J.; Kaloshian, Isgouhi

2016-01-01

Polyphagous aphid pests cause considerable economic damage to crop plants, primarily through the depletion of photoassimilates and transfer of viruses. The potato aphid (Macrosiphum euphorbiae) is a notable pest of solanaceous crops, however, the molecular mechanisms that underpin the ability to colonize these hosts are unknown. It has recently been demonstrated that like other aphid species, M. euphorbiae injects a battery of salivary proteins into host plants during feeding. It is speculated that these proteins function in a manner analagous to secreted effectors from phytopathogenic bacteria, fungi and oomycetes. Here, we describe a novel aphid effector (Me47) which was identified from the potato aphid salivary secretome as a putative glutathione-S-transferase (GST). Expression of Me47 in Nicotiana benthamiana enhanced reproductive performance of green peach aphid (Myzus persicae). Similarly, delivery of Me47 into leaves of tomato (Solanum lycopersicum) by Pseudomonas spp. enhanced potato aphid fecundity. In contrast, delivery of Me47 into Arabidopsis thaliana reduced GPA reproductive performance, indicating that Me47 impacts the outcome of plant–aphid interactions differently depending on the host species. Delivery of Me47 by the non-pathogenic Pseudomonas fluorescens revealed that Me47 protein or activity triggers defense gene transcriptional upregulation in tomato but not Arabidopsis. Recombinant Me47 was purified and demonstrated to have GST activity against two specific isothiocyanates (ITCs), compounds implicated in herbivore defense. Whilst GSTs have previously been associated with development of aphid resistance to synthetic insecticides, the findings described here highlight a novel function as both an elicitor and suppressor of plant defense when delivered into host tissues. PMID:27536306

Molecular cloning and expression of five glutathione S-transferase (GST) genes from Banana (Musa acuminata L. AAA group, cv. Cavendish).

PubMed

Wang, Zhuo; Huang, Suzhen; Jia, Caihong; Liu, Juhua; Zhang, Jianbin; Xu, Biyu; Jin, Zhiqiang

2013-09-01

Three tau class MaGSTs responded to abiotic stress, MaGSTF1 and MaGSTL1 responded to signaling molecules, they may play an important role in the growth of banana plantlet. Glutathione S-transferases (GST) are multifunctional detoxification enzymes that participate in a variety of cellular processes, including stress responses. In this study, we report the molecular characteristics of five GST genes (MaGSTU1, MaGSTU2, MaGSTU3, MaGSTF1 and MaGSTL1) cloned from banana (Musa acuminate L. AAA group, cv. Cavendish) using a RACE-PCR-based strategy. The predicted molecular masses of these GSTs range from 23.4 to 27.7 kDa and their pIs are acidic. At the amino acid level, they share high sequence similarity with GSTs in the banana DH-Pahang (AA group) genome. Phylogenetic analysis showed that the deduced amino acid sequences of MaGSTs also have high similarity to GSTs of other plant species. Expression analysis by semi-quantitative RT-PCR revealed that these genes are differentially expressed in various tissues. In addition, their expression is regulated by various stress conditions, including exposure to signaling molecules, cold, salinity, drought and Fusarium oxysporum f specialis(f. Sp) cubense Tropical Race 4 (Foc TR4) infection. The expression of the tau class MaGSTs (MaGSTU1, MaGSTU2 and MaGSTU3) mainly responded to cold, salinity and drought while MaGSTF1 and MaGSTL1 expressions were upregulated by signaling molecules. Our findings suggest that MaGSTs play a key role in both development and abiotic stress responses.

Placental biomarkers of PAH exposure and glutathione-S-transferase biotransformation enzymes in an obstetric population from Tijuana, Baja California, Mexico.

PubMed

Dodd-Butera, Teresa; Quintana, Penelope J E; Ramirez-Zetina, Martha; Batista-Castro, Ana C; Sierra, Maria M; Shaputnic, Carolyn; Garcia-Castillo, Maura; Ingmanson, Sonja; Hull, Stacy

2017-01-01

Environmental exposures along the US-Mexico border have the potential to adversely affect the maternal-fetal environment. The purpose of this study was to assess placental biomarkers of environmental exposures in an obstetric population at the California-Baja California border in relation to detoxifying enzymes in the placenta and nutritional status. This study was conducted on consenting, full-term, obstetric patients (n=54), delivering in a hospital in Tijuana, Baja California (BC), Mexico. Placental polyaromatic hydrocarbon (PAH)-DNA adducts were measured in addition to placental glutathione-S-transferase (GST) activity and genotype, maternal serum folate, and maternal and umbilical cord blood lead and cadmium levels. A questionnaire was administered to the mothers to determine maternal occupation in a maquiladora, other exposures, and obstetric indicators. In univariate analysis, maternal serum folate levels were inversely correlated with total PAH-DNA adducts (rho=-0.375, p=0.007); adduct #1 (rho=-0.388, p=0.005); and adduct #3 (rho =-0.430, p=0.002). Maternal lead levels were significantly positively correlated with cord blood lead levels (rho=0.512, p<0.001). Cadmium levels were generally very low but significantly higher in mothers exposed to environmental tobacco smoke (ETS) (either at work or at home, n=10). In multivariate analysis, only maternal serum folate levels remained as a significant negative predictor of total DNA-PAH adducts levels in placenta. These findings affirm that placental tissue is a valuable and readily available source of human tissue for biomonitoring; and indicate that further study of the role of nutrition in detoxification and mitigation of environmental exposures in pregnant women is warranted. Copyright © 2016 Elsevier Inc. All rights reserved.

Expression of ovarian microsomal epoxide hydrolase and glutathione S-transferase during onset of VCD-induced ovotoxicity in B6C3F{sub 1} mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Keating, Aileen F.; Sipes, I. Glenn; Hoyer, Patricia B.

2008-07-01

4-vinylcyclohexene diepoxide (VCD) specifically destroys small pre-antral follicles in the rodent ovary. VCD can be detoxified to an inactive tetrol by microsomal epoxide hydrolase (mEH), or by conjugation to glutathione (GSH) by glutathione S-transferase (GST). Formation of VCD-GSH adducts in the mouse ovary 4 h after VCD exposure (0.57 mmol/kg/day) has been demonstrated. Because the mouse ovary expresses both mEH and GST, expression of mEH and GST pi and mu during a time-course of VCD-induced ovotoxicity was evaluated in a neonatal mouse ovarian culture system. Ovaries from postnatal day 4 (PND4) B6C3F{sub 1} mice were incubated with VCD (15 {mu}M)more » for 2, 4, 6, 8, 10, 12, or 15 days. Following incubation, ovaries were histologically evaluated, or assessed for mRNA or protein expression. VCD did not cause follicle loss (p > 0.05) on days 2, 4, or 6 of culture. At days 8, 10, 12, and 15, VCD reduced (p < 0.05) both primordial and primary follicle numbers. Increased (p < 0.05) expression of mEH, GST pi and GST mu mRNA was detected after 4 days of VCD exposure. This expression was reduced on days 6 and 8, when follicle loss was underway, but increased (p < 0.05) after 10 days of exposure. mEH and GST pi proteins were elevated (p < 0.05) following 8 days of VCD-exposure however there was no increase in GST mu protein. These findings suggest that with continuous exposure to VCD, increased expression of detoxification enzymes may participate in retarding the onset of follicle loss, but that this loss cannot ultimately be prevented.« less

Genome-Wide Identification, Characterization, and Expression Profiling of Glutathione S-Transferase (GST) Family in Pumpkin Reveals Likely Role in Cold-Stress Tolerance

PubMed Central

Abdul Kayum, Md.; Nath, Ujjal Kumar; Park, Jong-In; Choi, Eung Kyoo; Song, Jae-Young; Kim, Hoy-Taek; Nou, Ill-Sup

2018-01-01

Plant growth and development can be adversely affected by cold stress, limiting productivity. The glutathione S-transferase (GST) family comprises important detoxifying enzymes, which play major roles in biotic and abiotic stress responses by reducing the oxidative damage caused by reactive oxygen species. Pumpkins (Cucurbita maxima) are widely grown, economically important, and nutritious; however, their yield can be severely affected by cold stress. The identification of putative candidate genes responsible for cold-stress tolerance, including the GST family genes, is therefore vital. For the first time, we identified 32 C. maxima GST (CmaGST) genes using a combination of bioinformatics approaches and characterized them by expression profiling. These CmaGST genes represent seven of the 14 known classes of plant GSTs, with 18 CmaGSTs categorized into the tau class. The CmaGSTs were distributed across 13 of pumpkin’s 20 chromosomes, with the highest numbers found on chromosomes 4 and 6. The large number of CmaGST genes resulted from gene duplication; 11 and 5 pairs of CmaGST genes were segmental- and tandem-duplicated, respectively. In addition, all CmaGST genes showed organ-specific expression. The expression of the putative GST genes in pumpkin was examined under cold stress in two lines with contrasting cold tolerance: cold-tolerant CP-1 (C. maxima) and cold-susceptible EP-1 (Cucurbita moschata). Seven genes (CmaGSTU3, CmaGSTU7, CmaGSTU8, CmaGSTU9, CmaGSTU11, CmaGSTU12, and CmaGSTU14) were highly expressed in the cold-tolerant line and are putative candidates for use in breeding cold-tolerant crop varieties. These results increase our understanding of the cold-stress-related functions of the GST family, as well as potentially enhancing pumpkin breeding programs. PMID:29439434

Dietary Isothiocyanates, Glutathione S-Transferase M1 (GSTM1), and Lung Cancer Risk in African Americans and Caucasians from Los Angeles County, California

PubMed Central

Carpenter, Catherine L.; Yu, Mimi C.; London, Stephanie J.

2013-01-01

Isothiocyanates, found in cruciferous vegetables, are anti-carcinogenic. Racial differences in smoking do not fully account for the African American excess lung cancer incidence. African Americans consume more cruciferous vegetables than US Whites. Impact on lung cancer risk is unknown. Glutathione S transferase M1 (GSTM1) gene promotes urinary isothiocyanate excretion. We evaluated dietary isothiocyanates and lung cancer using a population-based case-control study of 933 African Americans and Caucasians (non-Hispanic US White) from Los Angeles County, California (311 cases; 622 controls). Broccoli, cauliflower, greens and cabbage food-frequency variables represented isothiocyanates. Isothiocyanates were protective for lung cancer risk. Adjusted odds ratio (OR) for the uppermost quartile, > 80 μMol isothiocyanates/week, compared to lowest, was 0.65 (95% confidence interval (CL) = 0.41 – 1.00, trend p = 0.02). Association was stronger among subjects with homozygous deletion of GSTM1 (OR=0.52; 95% CL = 0.31 – 0.86), than subjects with at least one GSTM1 copy (OR = 0.77; 95% CL = 0.49 – 1.21). Difference was not statistically significant (p = 0.16). Despite African Americans consuming more cruciferous vegetables, the isothiocyanate association did not vary by race (p=0.52). Reduced lung cancer risk with higher isothiocyanate intake may be slightly stronger among subjects with deletion of GSTM1. PMID:19838921

Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV

NASA Technical Reports Server (NTRS)

Lim, Kap; Ho, Joseph X.; Keeling, Kim; Gilliland, Gary L.; Ji, Xinhua; Rueker, Florian; Carter, Daniel C.

1994-01-01

The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-1) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class was used as a molecular replacement model. The structure consists of a 4-stranded beta-sheet and 3 alpha-helices in domain 1 and 5 alpha-helices in domain 2. The space group of the Sj GST crystal is P4(sub 3)2(sub 1)2 with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes mu, alpha, and pi. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.

Regulation of chlorogenic acid biosynthesis by hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase in Lonicera japonica.

PubMed

Zhang, Jingru; Wu, Minlin; Li, Weidong; Bai, Genben

2017-12-01

For many centuries, Lonicera japonica has been used as an effective herb for the treatment of inflammation and swelling because of the presence of bioactive components such as chlorogenic acid (CGA). To clarify the relationship between L. japonica hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (HQT) gene expression and CGA content, an HQT eukaryotic expression system was constructed using Gateway cloning. L. japonica callus transformed with HQT was obtained using Agrobacterium tumefaciens-mediated transformation. We found a positive correlation between CGA content, determined by High-Performance Liquid Chromatography (HPLC), and the expression of HQT, analyzed by semi-quantitative RT-PCR. This study demonstrates that the HQT gene positively regulates CGA synthesis and lays the foundation for further study into enhancing efficacious components of medicinal plants. Copyright © 2017. Published by Elsevier Masson SAS.

Superoxide dismutase, catalase, glutathione peroxidase and gluthatione S-transferases M1 and T1 gene polymorphisms in three Brazilian population groups.

PubMed

de Oliveira Hiragi, Cássia; Miranda-Vilela, Ana Luisa; Rocha, Dulce Maria Sucena; de Oliveira, Silviene Fabiana; Hatagima, Ana; de Nazaré Klautau-Guimarães, Maria

2011-01-01

Antioxidants such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX1) reduce the oxidation rates in the organism. Gluthatione S-transferases (GSTs) play a vital role in phase 2 of biotransformation of many substances. Variation in the expression of these enzymes suggests individual differences for the degree of antioxidant protection and geographical differences in the distribution of these variants. We described the distribution frequency of CAT (21A/T), SOD2 (Ala9Val), GPX1 (Pro198Leu), GSTM1 and GSTT1 polymorphisms in three Brazilian population groups: Kayabi Amerindians (n = 60), Kalunga Afro-descendants (n = 72), and an urban mixed population from Federal District (n = 162). Frequencies of the variants observed in Kalunga (18% to 58%) and Federal District (33% to 63%) were similar to those observed in Euro and Afro-descendants, while in Kayabi (3% to 68%), depending on the marker, frequencies were similar to the ones found in different ethnic groups. Except for SOD2 in all population groups studied here, and for GPX1 in Kalunga, the genotypic distributions were in accordance with Hardy-Weinberg Equilibrium. These data can clarify the contribution of different ethnicities in the formation of mixed populations, such as that of Brazil. Moreover, outcomes will be valuable resources for future functional studies and for genetic studies in specific populations. If these studies are designed to comprehensively explore the role of these genetic polymorphisms in the etiology of human diseases they may help to prevent inconsistent genotype-phenotype associations in pharmacogenetic studies.

Observational Evidence for a Dark Side to NGC 5128's Globular Cluster System

NASA Astrophysics Data System (ADS)

Taylor, Matthew A.; Puzia, Thomas H.; Gomez, Matias; Woodley, Kristin A.

2015-05-01

We present a study of the dynamical properties of 125 compact stellar systems (CSSs) in the nearby giant elliptical galaxy NGC 5128, using high-resolution spectra (R ≈ 26, 000) obtained with Very Large Telescope/FLAMES. Our results provide evidence for a new type of star cluster, based on the CSS dynamical mass scaling relations. All radial velocity (vr) and line-of-sight velocity dispersion (σlos) measurements are performed with the penalized pixel fitting (ppxf ) technique, which provided σppxf estimates for 115 targets. The σppxf estimates are corrected to the 2D projected half-light radii, σ1/2, as well as the cluster cores, σ0, accounting for observational/aperture effects and are combined with structural parameters, from high spatial resolution imaging, in order to derive total dynamical masses ({{M}dyn}) for 112 members of NGC 5128’s star cluster system. In total, 89 CSSs have dynamical masses measured for the first time along with the corresponding dynamical mass-to-light ratios (\\Upsilon Vdyn). We find two distinct sequences in the \\Upsilon Vdyn-{{M}dyn} plane, which are well approximated by power laws of the forms \\Upsilon Vdyn\\propto Mdyn0.33+/- 0.04 and \\Upsilon Vdyn\\propto Mdyn0.79+/- 0.04. The shallower sequence corresponds to the very bright tail of the globular cluster luminosity function (GCLF), while the steeper relation appears to be populated by a distinct group of objects that require significant dark gravitating components such as central massive black holes and/or exotically concentrated dark matter distributions. This result would suggest that the formation and evolution of these CSSs are markedly different from the “classical” globular clusters in NGC 5128 and the Local Group, despite the fact that these clusters have luminosities similar to the GCLF turnover magnitude. We include a thorough discussion of myriad factors potentially influencing our measurements. Based on observations collected under program 081.D-0651 (PI

Preliminary X-ray crystallographic analysis of glutathione transferase zeta 1 (GSTZ1a-1a)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boone, Christopher D.; Zhong, Guo; Smeltz, Marci

2014-01-21

Crystals of glutathione transferase zeta 1 were grown and shown to diffract X-rays to 3.1 Å resolution. They belonged to space group P1, with unit-cell parameters a = 42.0, b = 49.6, c = 54.6 Å, α = 82.9, β = 69.9, γ = 73.4°.

Protective role for ovarian glutathione S-transferase isoform pi during 7,12-dimethylbenz[a]anthracene-induced ovotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhattacharya, Poulomi, E-mail: poulomib@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

2012-04-15

7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles at all developmental stages. This study investigated a role for the glutathione S-transferase (Gst) isoforms alpha (a), mu (m) and pi (p) and the transcription factors, Ahr and Nrf2, during DMBA-induced ovotoxicity, and their regulation by phosphatidylinositol-3 kinase (PI3K) signaling. Negative regulation of JNK by GSTP during DMBA exposure was also studied. Post-natal day (PND) 4 Fischer 344 rat ovaries were exposed to vehicle control (1% DMSO) ± DMBA (1 μM) or vehicle control (1% DMSO) ± LY294002 (PI3K inhibitor; 20 μM) for 1, 2, 4, or 6 days. Total RNA or protein was isolated,more » followed by RT-PCR or Western blotting to determine mRNA or protein level, respectively. Immunoprecipitation using an anti-GSTP antibody was performed to determine interaction between GSTP and JNK, followed by Western blotting to determine JNK and p-c-Jun protein level. DMBA had no impact on Gsta, Gstm or Nrf2 mRNA level, but increased Gstp mRNA and protein after 2 days. Ahr mRNA and protein increased after 2 and 4 days of DMBA exposure, respectively and DMBA increased NRF2 protein level after 4 days. JNK bound to GSTP was increased during DMBA exposure, with a concomitant decrease in unbound JNK and p-c-Jun. Ahr and Gstp mRNA were decreased (2 days) and increased (4 days) by PI3K inhibition, while Gstm mRNA increased (P < 0.05) after both time points, and there was no effect on Nrf2 mRNA. PI3K inhibition increased AHR, NRF2 and GSTP protein level. These findings support involvement of ovarian GSTP during DMBA exposure, and indicate a regulatory role for the PI3K signaling pathway on ovarian xenobiotic metabolism gene expression. -- Highlights: ► Ovarian GSTP is activated in response to DMBA exposure. ► AhR and Nrf2 transcription factors are up-regulated by DMBA. ► PI3K signaling regulates Ahr, Nrf2 and Gstp expression. ► GSTP negatively regulates ovarian JNK in response to DMBA exposure.« less

Single nucleotide polymorphisms and microsatellites in the canine glutathione S-transferase pi 1 (GSTP1) gene promoter.

PubMed

Sacco, James; Mann, Sarah; Toral, Keller

2017-01-01

Genetic polymorphisms within the glutathione S-transferase P1 ( GSTP1 ) gene affect the elimination of toxic xenobiotics by the GSTP1 enzyme. In dogs, exposure to environmental chemicals that may be GSTP1 substrates is associated with cancer. The objectives of this study were to investigate the genetic variability in the GSTP1 promoter in a diverse population of 278 purebred dogs, compare the incidence of any variants found between breeds, and predict their effects on gene expression. To provide information on ancestral alleles, a number of wolves, coyotes, and foxes were also sequenced. Fifteen single nucleotide polymorphisms (SNPs) and two microsatellites were discovered. Three of these loci were only polymorphic in dogs while three other SNPs were unique to wolves and coyotes. The major allele at c.-46 is T in dogs but is C in the wild canids. The c.-185 delT variant was unique to dogs. The microsatellite located in the 5' untranslated region (5'UTR) was a highly polymorphic GCC tandem repeat, consisting of simple and compound alleles that varied in size from 10 to 22-repeat units. The most common alleles consisted of 11, 16, and 17-repeats. The 11-repeat allele was found in 10% of dogs but not in the other canids. Unequal recombination and replication slippage between similar and distinct alleles may be the mechanism for the multiple microsatellites observed. Twenty-eight haplotypes were constructed in the dog, and an additional 8 were observed in wolves and coyotes. While the most common haplotype acrossbreeds was the wild-type *1A(17), other prevalent haplotypes included *3A(11) in Greyhounds, *6A(16) in Labrador Retrievers, *9A(16) in Golden Retrievers, and *8A(19) in Standard Poodles. Boxers and Siberian Huskies exhibited minimal haplotypic diversity. Compared to the simple 16*1 allele, the compound 16*2 allele (found in 12% of dogs) may interfere with transcription factor binding and/or the stability of the GSTP1 transcript. Dogs and other canids exhibit

Partial purification and characterization of a mannosyl transferase involved in O -linked mannosylation of glycoproteins in Candida albicans.

PubMed

Arroyo-Flores, Blanca L; Calvo-Méndez, Carlos; Flores-Carreón, Arturo; López-Romero, Everardo

2004-04-01

Incubation of a mixed membrane fraction of C. albicans with the nonionic detergents Nonidet P-40 or Lubrol solubilized a fraction that catalyzed the transfer of mannose either from endogenously generated or exogenously added dolichol-P-[14C]Man onto endogenous protein acceptors. The protein mannosyl transferase solubilized with Nonidet P-40 was partially purified by a single step of preparative nondenaturing electrophoresis and some of its properties were investigated. Although transfer activity occurred in the absence of exogenous mannose acceptors and thus depended on acceptor proteins isolated along with the enzyme, addition of the protein fraction obtained after chemical de-mannosylation of glycoproteins synthesized in vitro stimulated mannoprotein labeling in a concentration-dependent manner. Other de-mannosylated glycoproteins, such as yeast invertase or glycoproteins extracted from C. albicans, failed to increase the amount of labeled mannoproteins. Mannosyl transfer activity was not influenced by common metal ions such as Mg(2+), Mn(2+) and Ca(2+), but it was stimulated up to 3-fold by EDTA. Common phosphoglycerides such as phosphatidylglycerol and, to a lower extent, phosphatidylinositol and phosphatidylcholine enhanced transfer activity. Interestingly, coupled transfer activity between dolichol phosphate mannose synthase, i.e., the enzyme responsible for Dol-P-Man synthesis, and protein mannosyl transferase could be reconstituted in vitro from the partially purified transferases, indicating that this process can occur in the absence of cell membranes.

Studer Group® ' s evidence-based leadership initiatives.

PubMed

Schuller, Kristin A; Kash, Bita A; Gamm, Larry D

2015-01-01

The purpose of this paper is to analyze the implementation of an organizational change initiative--Studer Group®'s Evidence-Based Leadership (EBL)--in two large, US health systems by comparing and contrasting the factors associated with successful implementation and sustainability of the EBL initiative. This comparative case study assesses the responses to two pairs of open-ended questions during in-depth qualitative interviews of leaders and managers at both health systems. Qualitative content analysis was employed to identify major themes. Three themes associated with success and sustainability of EBL emerged at both health systems: leadership; culture; and organizational processes. The theme most frequently identified for both success and sustainability of EBL was culture. In contrast, there was a significant decline in salience of the leadership theme as attention shifts from success in implementation of EBL to sustaining EBL long term. Within the culture theme, accountability, and buy-in were most often cited by interviewees as success factors, while sense of accountability, buy-in, and communication were the most reported factors for sustainability. Cultural factors, such as accountability, staff support, and communication are driving forces of success and sustainability of EBL across both health systems. Leadership, a critical factor in several stages of implementation, appears to be less salient as among factors identified as important to longer term sustainability of EBL.

Quantification of butyryl CoA:acetate CoA-transferase genes reveals different butyrate production capacity in individuals according to diet and age.

PubMed

Hippe, Berit; Zwielehner, Jutta; Liszt, Kathrin; Lassl, Cornelia; Unger, Frank; Haslberger, Alexander G

2011-03-01

The gastrointestinal microbiota produces short-chain fatty acids, especially butyrate, which affect colonic health, immune function and epigenetic regulation. To assess the effects of nutrition and aging on the production of butyrate, the butyryl-CoA:acetate CoA-transferase gene and population shifts of Clostridium clusters lV and XlVa, the main butyrate producers, were analysed. Faecal samples of young healthy omnivores (24 ± 2.5 years), vegetarians (26 ± 5 years) and elderly (86 ± 8 years) omnivores were evaluated. Diet and lifestyle were assessed in questionnaire-based interviews. The elderly had significantly fewer copies of the butyryl-CoA:acetate CoA-transferase gene than young omnivores (P=0.014), while vegetarians showed the highest number of copies (P=0.048). The thermal denaturation of the butyryl-CoA:acetate CoA-transferase gene variant melting curve related to Roseburia/Eubacterium rectale spp. was significantly more variable in the vegetarians than in the elderly. The Clostridium cluster XIVa was more abundant in vegetarians (P=0.049) and in omnivores (P<0.01) than in the elderly group. Gastrointestinal microbiota of the elderly is characterized by decreased butyrate production capacity, reflecting increased risk of degenerative diseases. These results suggest that the butyryl-CoA:acetate CoA-transferase gene is a valuable marker for gastrointestinal microbiota function. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Characterization of spermidine hydroxycinnamoyl transferases from eggplant (Solanum melongena L.) and its wild relative Solanum richardii Dunal

USDA-ARS?s Scientific Manuscript database

Eggplant produces a variety of hydroxycinnamic acid amides (HCAAs) that play an important role in plant development and adaptation to environmental changes. However, the HCAA pathway remains largely uncharacterized in Solanaceae. In this study, a spermidine hydroxycinnamoyl transferase (SHT) from eg...

Hierarchy of evidence: a simple system for orthopaedic research?

PubMed

Pemberton, Julia; Kraeva, Juliana; Bhandari, Mohit

2007-01-01

To be able to make a sound recommendation for a treatment based on the best available evidence, it is necessary to follow specific steps in acquiring literature, appraising the study design and quality, and assessing the results. Evidence-based medicine is founded on the concepts of using best evidence, levels of evidence, and grades of recommendation, and aims to provide clinicians with standardized rules to help them appraise the validity of published research. A number of systems have been developed to categorize research studies into consistent levels of evidence. These systems are based primarily on consensus expert opinion, and have not been validated to any extent. The use of different systems does not allow for effective communication between users; there is a lack of accord even between users of the same system. The GRADE working group has devised a new rating system that attempts to address deficiencies seen within other systems.

Development of a cell transducible RhoA inhibitor TAT-C3 transferase and its encapsulation in biocompatible microspheres to promote survival and enhance regeneration of severed neurons.

PubMed

Tan, Elaine Y M; Law, Janice W S; Wang, Chi-Hwa; Lee, Alan Y W

2007-12-01

Neurons in post-traumatized mammalian central nervous system show only limited degree of regeneration, which can be attributed to the presence of neurite outgrowth inhibitors in damaged myelin and glial scar, and to the apoptosis of severed central neurons and glial cells during secondary Wallerian degeneration. RhoA GTPase has been implicated as the common denominator in these counter-regeneration events, which shows significant and persistent up-regulation for weeks in injured spinal cord and cerebral infarct after stroke. While the exoenzyme C3 transferase is a potent RhoA inhibitor, its extremely low efficiency of cell entry and degradation in vivo has restricted the therapeutic value. This study aims to circumvent these problems by developing a membrane-permeating form of C3 transferase and a biopolymer-based microsphere depot system for sustainable controlled release of the protein. A membrane-permeating form of C3 transferase was developed by fusing a Tat (trans-activating transcription factor) transduction domain of human immunodeficiency virus to its amino terminal using standard molecular cloning techniques. After confirming efficient cell entry into epithelial and neuroblastoma cells, the resulting recombinant protein TAT-C3 was encapsulated in biocompatible polymer poly(D,L -lactide-co-glycolide) in the form of microspheres by a water-in-oil-in-water (W/O/W) emulsion method. By blending capped and uncapped form of the polymer at different ratios, TAT-C3 protein release profile was modified to suit the expression pattern of endogenous RhoA during CNS injuries. Bioactivity of TAT-C3 released from microspheres was assessed by RhoA ribosylation assay. In contrast to wild-type C3 transferase, the modified TAT-C3 protein was found to efficiently enter NIH3T3 and N1E-115 neuroblastoma cells as early as 6 hours of incubation. The fusion of TAT sequence to C3 transferase imposed no appreciable effects on its biological activity in promoting neurite outgrowth

Molecular cloning, biochemical characterization, and expression analysis of two glutathione S-transferase paralogs from the big-belly seahorse (Hippocampus abdominalis).

PubMed

Tharuka, M D Neranjan; Bathige, S D N K; Lee, Jehee

2017-12-01

Glutathione S-transferases (GSTs, EC 2.5.1.18) are important Phase II detoxifying enzymes that catalyze hydrophobic, electrophilic xenobiotic substance with the conjugation of reduced glutathione (GSH). In this study, GSTμ and GSTρ paralogs of GST in the big belly seahorse (Hippocampus abdominalis; HaGSTρ, HaGSTμ) were biochemically, molecularly, functionally characterized to determine their detoxification range and protective capacities upon different pathogenic stresses. HaGSTρ and HaGSTμ are composed of coding sequences of 681bp and 654bp, which encode proteins 225 and 217 amino acids, with predicted molecular masses of 26.06kDa and 25.74kDa respectively. Sequence analysis revealed that both HaGSTs comprise the characteristic GSH-binding site in the thioredoxin-like N-terminal domain and substrate binding site in the C-terminal domain. The recombinant HaGSTρ and HaGSTμ proteins catalyzed the model GST substrate 1-chloro-2, 4-dinitrobenzene (CDNB). Enzyme kinetic analysis revealed different K m and V max values for each rHaGST, suggesting that they have different conjugation rates. The optimum conditions (pH, temperature) and inhibitory assays of each protein demonstrated different optimal ranges. However, HaGSTμ was highly expressed in the ovary and gill, whereas HaGSTρ was highly expressed in the gill and pouch. mRNA expression of HaGSTρ and HaGSTμ was significantly elevated upon lipopolysaccharide, Poly (I:C), and Edwardsiella tarda challenges in liver and in blood cells as well as with Streptococcus iniae challenge in blood cells. From these collective experimental results, we propose that HaGSTρ and HaGSTμ are effective in detoxifying xenobiotic toxic agents, and importantly, their mRNA expression could be stimulated by immunological stress signals in the aquatic environment. Copyright © 2017 Elsevier Inc. All rights reserved.

Microinjection of recombinant O-GlcNAc transferase potentiates Xenopus oocytes M-phase entry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dehennaut, Vanessa; EA 4020, Laboratoire de Regulation des Signaux de Division, USTL, IFR147, Villeneuve d'Ascq; Hanoulle, Xavier

2008-05-02

In order to understand the importance of the cytosolic and nuclear-specific O-linked N-acetylglucosaminylation (O-GlcNAc) on cell cycle regulation, we recently reported that inhibition of O-GlcNAc transferase (OGT) delayed or blocked Xenopus laevis oocyte germinal vesicle breakdown (GVBD). Here, we show that increased levels of the long OGT isoform (ncOGT) accelerate X. laevis oocyte GVBD. A N-terminally truncated isoform (sOGT) with a similar in vitro catalytic activity towards a synthetic CKII-derived peptide had no effect, illustrating the important role played by the N-terminal tetratrico-peptide repeats. ncOGT microinjection in the oocytes increases both the speed and extent of O-GlcNAc addition, leads tomore » a quicker activation of the MPF and MAPK pathways and finally results in a faster GVBD. Microinjection of anti-OGT antibodies leads to a delay of the GVBD kinetics. Our results hence demonstrate that OGT is a key molecule for the timely progression of the cell cycle.« less

Evidence for a $$B_s^0 \\pi^\\pm$$ state

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abazov, V. M.

Here, we report evidence for a narrow structure, X(5568), in the decay sequence X(5568)→more » $$B_s^0 \\pi^\\pm$$, B$$_s^0$$ →J/ψΦ, J/ψ→μ+μ-, Φ→K+K-. This is evidence for the first instance of a hadronic state with valence quarks of four different flavors. The mass and natural width of this state are measured to be m=5567.8±2.9(stat) $$+0.9\\atop{-1.9}$$ (syst) MeV/c 2 and Γ=21.9±6.4(stat)$$+5.0\\atop{-2.5}$$ (syst) MeV/c 2. If the decay is X(5568)→B$$*\\atop{s}$$ π ±→B$$0\\atop{s}$$ γπ ± with an unseen γ, m(X(5568)) will be shifted up by m(B$$*\\atop{s}$$ )-m(B$$0\\atop{s}$$)~49 MeV/c 2. This measurement is based on 10.4 fb -1 of $$p\\bar{p}$$ collision data at $$\\sqrt{s}$$ =1.96 TeV collected by the D0 experiment at the Fermilab Tevatron collider.« less

Evidence for a $$B_s^0 \\pi^\\pm$$ state

DOE PAGES

Abazov, V. M.

2016-07-07

Here, we report evidence for a narrow structure, X(5568), in the decay sequence X(5568)→more » $$B_s^0 \\pi^\\pm$$, B$$_s^0$$ →J/ψΦ, J/ψ→μ+μ-, Φ→K+K-. This is evidence for the first instance of a hadronic state with valence quarks of four different flavors. The mass and natural width of this state are measured to be m=5567.8±2.9(stat) $$+0.9\\atop{-1.9}$$ (syst) MeV/c 2 and Γ=21.9±6.4(stat)$$+5.0\\atop{-2.5}$$ (syst) MeV/c 2. If the decay is X(5568)→B$$*\\atop{s}$$ π ±→B$$0\\atop{s}$$ γπ ± with an unseen γ, m(X(5568)) will be shifted up by m(B$$*\\atop{s}$$ )-m(B$$0\\atop{s}$$)~49 MeV/c 2. This measurement is based on 10.4 fb -1 of $$p\\bar{p}$$ collision data at $$\\sqrt{s}$$ =1.96 TeV collected by the D0 experiment at the Fermilab Tevatron collider.« less

Single 23S rRNA mutations at the ribosomal peptidyl transferase centre confer resistance to valnemulin and other antibiotics in Mycobacterium smegmatis by perturbation of the drug binding pocket.

PubMed

Long, Katherine S; Poehlsgaard, Jacob; Hansen, Lykke H; Hobbie, Sven N; Böttger, Erik C; Vester, Birte

2009-03-01

Tiamulin and valnemulin target the peptidyl transferase centre (PTC) on the bacterial ribosome. They are used in veterinary medicine to treat infections caused by a variety of bacterial pathogens, including the intestinal spirochetes Brachyspira spp. Mutations in ribosomal protein L3 and 23S rRNA have previously been associated with tiamulin resistance in Brachyspira spp. isolates, but as multiple mutations were isolated together, the roles of the individual mutations are unclear. In this work, individual 23S rRNA mutations associated with pleuromutilin resistance at positions 2055, 2447, 2504 and 2572 (Escherichia coli numbering) are introduced into a Mycobacterium smegmatis strain with a single rRNA operon. The single mutations each confer a significant and similar degree of valnemulin resistance and those at 2447 and 2504 also confer cross-resistance to other antibiotics that bind to the PTC in M. smegmatis. Antibiotic footprinting experiments on mutant ribosomes show that the introduced mutations cause structural perturbations at the PTC and reduced binding of pleuromutilin antibiotics. This work underscores the fact that mutations at nucleotides distant from the pleuromutilin binding site can confer the same level of valnemulin resistance as those at nucleotides abutting the bound drug, and suggests that the former function indirectly by altering local structure and flexibility at the drug binding pocket.

Biosynthesis of glycoproteins in the human pathogenic fungus Sporothrix schenckii: synthesis of dolichol phosphate mannose and mannoproteins by membrane-bound and solubilized mannosyl transferases.

PubMed

Ruiz-Baca, Estela; Villagómez-Castro, Julio C; Leal-Morales, Carlos A; Sabanero-López, Myrna; Flores-Carreón, Arturo; López-Romero, Everardo

2005-01-01

A membrane fraction obtained from the filamentous form of Sporothrix schenckii was able to transfer mannose from GDP-Mannose into dolichol phosphate mannose and from this inTermediate into mannoproteins in coupled reactions catalyzed by dolichol phosphate mannose synthase and protein mannosyl transferase(s), respectively. Although the transfer reaction depended on exogenous dolichol monophosphate, membranes failed to use exogenous dolichol phosphate mannose for protein mannosylation to a substantial extent. Over 95% of the sugar was transferred to proteins via dolichol phosphate mannose and the reaction was stimulated several fold by Mg2+ and Mn2+. Incubation of membranes with detergents such as Brij 35 and Lubrol PX released soluble fractions that transferred the sugar from GDP-Mannose mostly into mannoproteins, which were separated by affinity chromatography on Concanavilin A-Sepharose 4B into lectin-reacting and non-reacting fractions. All proteins mannosylated in vitro eluted with the lectin-reacting proteins and analytical electrophoresis of this fraction revealed the presence of at least nine putative mannoproteins with molecular masses in the range of 26-112 kDa. The experimental approach described here can be used to identify and isolate specific glycoproteins mannosylated in vitro in studies of O-glycosylation.

Peroxiredoxin 6 homodimerization and heterodimerization with glutathione S-transferase pi are required for its peroxidase but not phospholipase A2 activity.

PubMed

Zhou, Suiping; Sorokina, Elena M; Harper, Sandra; Li, Haitao; Ralat, Luis; Dodia, Chandra; Speicher, David W; Feinstein, Sheldon I; Fisher, Aron B

2016-05-01

Peroxiredoxin 6 (Prdx6) is a unique 1-Cys member of the peroxiredoxin family with both GSH peroxidase and phospholipase A2 (PLA2) activities. It is highly expressed in the lung where it plays an important role in antioxidant defense and lung surfactant metabolism. Glutathionylation of Prdx6 mediated by its heterodimerization with GSH S-transferase π (πGST) is required for its peroxidatic catalytic cycle. Recombinant human Prdx6 crystallizes as a homodimer and sedimentation equilibrium analysis confirmed that this protein exists as a high affinity dimer in solution. Based on measurement of molecular mass, dimeric Prdx6 that was oxidized to the sulfenic acid formed a sulfenylamide during storage. After examination of the dimer interface in the crystal structure, we postulated that the hydrophobic amino acids L145 and L148 play an important role in homodimerization of Prdx6 as well as in its heterodimerization with πGST. Oxidation of Prdx6 also was required for its heterodimerization. Sedimentation equilibrium analysis and the Duolink proximity ligation assay following mutation of the L145 and L148 residues of Prdx6 to Glu indicated greatly decreased dimerization propensity reflecting the loss of hydrophobic interactions between the protein monomers. Peroxidase activity was markedly reduced by mutation at either of the Leu sites and was essentially abolished by the double mutation, while PLA2 activity was unaffected. Decreased peroxidase activity following mutation of the interfacial leucines presumably is mediated via impaired heterodimerization of Prdx6 with πGST that is required for reduction and re-activation of the oxidized enzyme. Copyright © 2016 Elsevier Inc. All rights reserved.

Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV

NASA Technical Reports Server (NTRS)

Lim, K.; Ho, J. X.; Keeling, K.; Gilliland, G. L.; Ji, X.; Ruker, F.; Carter, D. C.

1994-01-01

The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-1) (Muster T et al., 1993, J Virol 67:6642-6647) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992, Biochemistry 31:10169-10184) was used as a molecular replacement model. The structure consists of a 4-stranded beta-sheet and 3 alpha-helices in domain 1 and 5 alpha-helices in domain 2. The space group of the Sj GST crystal is P4(3)2(1)2, with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes mu, alpha, and pi. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.

Identification and expression profiles of fifteen delta-class glutathione S-transferase genes from a stored-product pest, Liposcelis entomophila (Enderlein) (Psocoptera: Liposcelididae).

PubMed

Jing, Tian-Xing; Wu, Yu-Xian; Li, Ting; Wei, Dan-Dan; Smagghe, Guy; Wang, Jin-Jun

2017-04-01

Glutathione S-transferases (GSTs) comprise a diverse family of enzymes found ubiquitously in aerobic organisms and they play important roles in insecticide resistance. In this study, we tested the sensitivities of Liposcelis entomophila, collected from four different field populations, to three insecticides. The results showed that the insects from Tongliang population had a relatively higher tolerance to malathion and propuxor than insects from other field populations. The insecticide sensitivities of different populations detected in psocids may be due to the different control practices. Through sequence mining and phylogenetic analyses, we identified 15 delta class GST genes that contained the conserved motifs of the GSTs. Quantitative real-time PCR (Q-PCR) analysis indicated that the 15 GST genes were expressed at all tested developmental stages, and 12 GST genes had significantly higher expression levels in adulthood than in egg stage. The expression levels of 15 GST genes in different field populations showed that 9 GST genes were significantly higher in Tongliang population compared to other populations. Furthermore, Q-PCR confirmed that the expression of several delta class GSTs was upregulated at different times after malathion, propuxor and deltamethrine exposure with the LC 50 concentration of insecticide. Taken together, these findings showed that delta class GST genes have various expression levels in different developmental stages and different field populations, and they were up-regulated in response to insecticide exposure, which suggested that these GSTs may be associated with insecticide metabolism in psocids. Copyright © 2017 Elsevier Inc. All rights reserved.

Differential expression of glutathione s-transferase enzyme in different life stages of various insecticide-resistant strains of Anopheles stephensi: a malaria vector.

PubMed

Sanil, D; Shetty, V; Shetty, N J

2014-06-01

Interest in insect glutathione s-transferases (GSTs) has primarily focused on their role in insecticide resistance. These play an important role in biotransformation and detoxification of many different xenobiotic and endogenous substances including insecticides. The GST activity among 10 laboratory selected insecticide resistant and susceptible/control strains of Anopheles stephensi was compared using the substrates 1-chloro-2,4-dinitrobenzene (CDNB). The difference in the GST activities of different life stages of diverse insecticide resistant strains was compared and presented. About 100 larvae, pupae, adult males, adult females and eggs (100 μg in total weight) were collected and used for the experiment. The extracts were prepared from each of the insecticide-resistant strains and control. Protein contents of the enzyme homogenate and GST activities were determined. Deltamethrin and cyfluthrin-resistant strains of An. stephensi showed significantly higher GST activity. Larvae and pupae of DDT-resistant strain showed peak GST activity followed by the propoxur-resistant strain. On contrary, the GST activity was found in reduced quantity in alphamethrin, bifenthrin, carbofuran and chloropyrifos resistant strains. Adults of either sexes showed higher GST activity in mosquito strain resistant to organophosphate group of insecticides namely, temephos and chloropyrifos. The GST activity was closely associated with almost all of the insecticides used in the study, strengthening the fact that one of the mechanisms associated with resistance includes an increase of GST activity. This comparative data on GST activity in An. stephensi can be useful database to identify possible underlying mechanisms governing insecticide-resistance by GSTs.

Cloning and characterization of a novel sigma-like glutathione S-transferase from the giant panda parasitic nematode, Baylisascaris schroederi.

PubMed

Xie, Yue; Zhou, Xuan; Chen, Lin; Zhang, Zhihe; Wang, Chengdong; Gu, Xiaobin; Wang, Tao; Peng, Xuerong; Yang, Guangyou

2015-01-23

Baylisascaris schroederi, an intestinal nematode of the giant panda, is the cause of the often fatal disease, baylisascariasis. Glutathione S-transferases (GSTs) are versatile enzymes that can affect parasite survival and parasite-host interactions and, are therefore, potential targets for the development of diagnostic tests and vaccines. In this study, we identified a full-length cDNA that encoded a novel, secretory sigma-like GST (Bsc-GSTσ) from a B. schroederi-omic dataset. Following cloning and sequencing, sequence and structural analyses and comparative modeling were performed using online-bioinformatics and proteomics tools. The recombinant Bsc-GSTσ (rBsc-GSTσ) protein was prokaryotically expressed and then used to detect antigenicity and reactivity using immunoblotting assays. In addition, the native protein in female adult B. schroederi was located via immunofluorescence techniques, while the preliminary ELISA-based serodiagnostic potential of rBsc-GSTσ was assessed in native and infected mouse sera. Bsc-GSTσ contained a 621-bp open reading frame that encoded a polypeptide of 206 amino acids with two typical sigma GST domain profiles, including a GST_N_Sigma_like at the N-terminus and a GST_C_Sigma_like at the C-terminus. The presence of an N-terminal signal sequence indicated that Bsc-GSTσ was a secretory protein. Sequence alignment and phylogenetic analyses showed that Bsc-GSTσ was a nematode-specific member of the Sigma class GSTs and shared the closest genetic distance with its homologue in Ascaris suum. Further comparative structure analyses indicated that Bsc-GSTσ possessed the essential structural motifs (e.g., βαβαββα) and the consensus secondary or tertiary structure that is typical for other characterized GSTσs. Immunolocalization revealed strong distributions of native Bsc-GSTσ in the body hypodermis, lateral chords, gut epithelium, gut microvilli, oviduct epithelium, and ovaries of adult female worms, similar to its homologue in

Glutathione-S-transferase A3 knockout mice are sensitive to acute cytotoxic and genotoxic effects of aflatoxin B1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ilic, Zoran, E-mail: zxi01@health.state.ny.u; Crawford, Dana, E-mail: crawfod@mail.amc.ed; Egner, Patricia A., E-mail: pegner@jhsph.ed

Aflatoxin B1 (AFB1) is a major risk factor for hepatocellular carcinoma (HCC) in humans. However, mice, a major animal model for the study of AFB1 carcinogenesis, are resistant, due to high constitutive expression, in the mouse liver, of glutathione S-transferase A3 subunit (mGSTA3) that is lacking in humans. Our objective was to establish that a mouse model for AFB1 toxicity could be used to study mechanisms of toxicity that are relevant for human disease, i.e., an mGSTA3 knockout (KO) mouse that responds to toxicants such as AFB1 in a manner similar to humans. Exons 3-6 of the mGSTA3 were replacedmore » with a neomycin cassette by homologous recombination. Southern blotting, RT-PCR, Western blotting, and measurement of AFB1-N{sup 7}-DNA adduct formation were used to evaluate the mGSTA3 KO mice. The KO mice have deletion of exons 3-6 of the mGSTA3 gene, as expected, as well as a lack of mGSTA3 expression at the mRNA and protein levels. Three hours after injection of 5 mg/kg AFB1, mGSTA3 KO mice have more than 100-fold more AFB1-N{sup 7}-DNA adducts in their livers than do similarly treated wild-type (WT) mice. In addition, the mGSTA3 KO mice die of massive hepatic necrosis, at AFB1 doses that have minimal toxic effects in WT mice. We conclude that mGSTA3 KO mice are sensitive to the acute cytotoxic and genotoxic effects of AFB1, confirming the crucial role of GSTA3 subunit in protection of normal mice against AFB1 toxicity. We propose the mGSTA3 KO mouse as a useful model with which to study the interplay of risk factors leading to HCC development in humans, as well as for testing of additional possible functions of mGSTA3.« less

Cost of Tuberculosis Treatment: Evidence from Iran’s Health System

PubMed Central

Bay, Vahid; Tabarsi, Payam; Rezapour, Aziz; Marzban, Sima; Zarei, Ehsan

2017-01-01

Objectives This study aimed to estimate the cost of smear-positive drug-susceptible pulmonary tuberculosis (TB) treatment of the patients in the Azadshahr district, Golestan Province, Iran. Methods In this retrospective study, all new smear positive pulmonary TB patients who had been registered at the district’s health network between April, 2013 and December, 2015 and had successfully completed their treatment were entered into the study (45 patients). Treatment costs were estimated from the provider’s perspective using an activity-based costing (ABC) method. Results The cost of treating a new smear-positive pulmonary TB patient was US dollar (USD) 1,409.00 (Iranian Rial, 39,438,260), which can be divided into direct and indirect costs (USD 1,226.00 [87%] and USD 183.00 [13%], respectively). The highest cost (58.1%) was related to care and management of TB patients (including 46.1% human resources costs and 12% directly-observed treatment, short course implementation) and then respectively related to hospitalization (12.1%), supportive activity centers (11.4%), transportation (6.5%), medicines (5.3%), and laboratory tests and radiography (3.2%). Conclusion Using disease-specific cost studies can help the healthcare system management to have correct insight into the financial burden created by the disease. This can subsequently be used in prioritization, planning, operational budgeting, economic evaluation of programs, interventions, and ultimately in disease management. PMID:29164047

Structure of human O-GlcNAc transferase and its complex with a peptide substrate

PubMed Central

Lazarus, Michael B.; Nam, Yunsun; Jiang, Jiaoyang; Sliz, Piotr; Walker, Suzanne

2010-01-01

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that couples metabolic status to the regulation of a wide variety of cellular signaling pathways by acting as a nutrient sensor1. OGT catalyzes the transfer of N-acetyl-glucosamine from UDP-GlcNAc to serines and threonines of cytoplasmic, nuclear and mitochondrial proteins2,3, including numerous transcription factors4, tumor suppressors, kinases5, phosphatases1, and histone-modifying proteins6. Aberrant O-GlcNAcylation by OGT has been linked to insulin resistance7, diabetic complications8, cancer9 and neurodegenerative diseases including Alzheimer’s10. Despite the importance of OGT, the details of how it recognizes and glycosylates its protein substrates are largely unknown. We report here two crystal structures of human OGT, as a binary complex with UDP (2.8 A) and a ternary complex with UDP and a peptide substrate (1.95 A). The structures provide clues to the enzyme mechanism, show how OGT recognizes target peptide sequences, and reveal the fold of the unique domain between the two halves of the catalytic region. This information will accelerate the rational design of biological experiments to investigate OGT’s functions and the design of inhibitors for use as cellular probes and to assess its potential as a therapeutic target. PMID:21240259

Glucose-induced expression of MIP-1 genes requires O-GlcNAc transferase in monocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chikanishi, Toshihiro; ERATO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi-shi, Saitama 332-0012; Fujiki, Ryoji

2010-04-16

O-glycosylation has emerged as an important modification of nuclear proteins, and it appears to be involved in gene regulation. Recently, we have shown that one of the histone methyl transferases (MLL5) is activated through O-glycosylation by O-GlcNAc transferase (OGT). Addition of this monosaccharide is essential for forming a functional complex. However, in spite of the abundance of OGT in the nucleus, the impact of nuclear O-glycosylation by OGT remains largely unclear. To address this issue, the present study was undertaken to test the impact of nuclear O-glycosylation in a monocytic cell line, THP-1. Using a cytokine array, MIP-1{alpha} and -1{beta}more » genes were found to be regulated by nuclear O-glycosylation. Biochemical purification of the OGT interactants from THP-1 revealed that OGT is an associating partner for distinct co-regulatory complexes. OGT recruitment and protein O-glycosylation were observed at the MIP-1{alpha} gene promoter; however, the known OGT partner (HCF-1) was absent when the MIP-1{alpha} gene promoter was not activated. From these findings, we suggest that OGT could be a co-regulatory subunit shared by functionally distinct complexes supporting epigenetic regulation.« less

Glutathione Transferase from Trichoderma virens Enhances Cadmium Tolerance without Enhancing Its Accumulation in Transgenic Nicotiana tabacum

PubMed Central

Dixit, Prachy; Mukherjee, Prasun K.; Ramachandran, V.; Eapen, Susan

2011-01-01

Background Cadmium (Cd) is a major heavy metal pollutant which is highly toxic to plants and animals. Vast agricultural areas worldwide are contaminated with Cd. Plants take up Cd and through the food chain it reaches humans and causes toxicity. It is ideal to develop plants tolerant to Cd, without enhanced accumulation in the edible parts for human consumption. Glutathione transferases (GST) are a family of multifunctional enzymes known to have important roles in combating oxidative stresses induced by various heavy metals including Cd. Some GSTs are also known to function as glutathione peroxidases. Overexpression/heterologous expression of GSTs is expected to result in plants tolerant to heavy metals such as Cd. Results Here, we report cloning of a glutathione transferase gene from Trichoderma virens, a biocontrol fungus and introducing it into Nicotiana tabacum plants by Agrobacterium-mediated gene transfer. Transgenic nature of the plants was confirmed by Southern blot hybridization and expression by reverse transcription PCR. Transgene (TvGST) showed single gene Mendelian inheritance. When transgenic plants expressing TvGST gene were exposed to different concentrations of Cd, they were found to be more tolerant compared to wild type plants, with transgenic plants showing lower levels of lipid peroxidation. Levels of different antioxidant enzymes such as glutathione transferase, superoxide dismutase, ascorbate peroxidase, guiacol peroxidase and catalase showed enhanced levels in transgenic plants expressing TvGST compared to control plants, when exposed to Cd. Cadmium accumulation in the plant biomass in transgenic plants were similar or lower than wild-type plants. Conclusion The results of the present study suggest that transgenic tobacco plants expressing a Trichoderma virens GST are more tolerant to Cd, without enhancing its accumulation in the plant biomass. It should be possible to extend the present results to crop plants for developing Cd tolerance and

Cloning, identification and functional characterization of a pi-class glutathione-S-transferase from the freshwater mussel Cristaria plicata.

PubMed

Hu, Baoqing; Deng, Lirong; Wen, Chungen; Yang, Xilan; Pei, Pengzu; Xie, Yanhai; Luo, Shaoqing

2012-01-01

Glutathione-S-transferases (GSTs) are multifunctional phase II detoxification enzymes that catalyze the attachment of electrophilic substrates to glutathione and play an important role in protecting organisms against the toxicity of reactive oxygen species (ROS). The piGST cDNA was cloned and sequenced after rapid amplification of cDNA ends (RACE) from the freshwater mussel Cristaria plicata. The comparison of the deduced amino acid sequences with GSTs from other species showed that the enzymes belonged to the pi-class and the amino acids defining the binding sites of glutathione (G-site) and for xenobiotic substrates (H-site) were highly conserved. The Cp-piGST cDNA is 816 nucleotides (nt) in length and contained a 615 nt open reading frame (ORF) encoding 205 amino acid residues, and has 19 nt of 5' untranslated region (UTR) and a 3' UTR of 182 nt including a tailing signal (AATAAA) and a poly (A) tail. The molecular weight of the predicted piGST is 23.4 kDa, with the calculated PI being 5.2. The mRNA transcript of Cp-piGST could be detected in all the examined tissues with highest expression level in hepatopancreas. The expression level of Cp-piGST in hepatopancreas and gill showed similar trend that were significantly increased after bacterial challenge compared to the control group at 12 h. Furthermore, the recombinant Cp-piGST with high enzyme activity was induced to be expressed as a soluble form by IPTG at 20°C for 8 h, and then was purified by using the native Ni(2+) affinity chromatography. The specific activity of the purified soluble Cp-piGST enzyme into pET30 was 2.396 μmol/min/mg, and which into pET32 was 1.706 μmol/min/mg. The recombinant Cp-piGST had a maximum activity at approximately pH 8.0, and its optimum temperature was 37°C. The recombinant Cp-piGST enzyme activity became lower gradually with the denaturant concentration increasing. Copyright © 2011 Elsevier Ltd. All rights reserved.

33 CFR 157.148 - COW system: Evidence for inspections.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false COW system: Evidence for... CARRYING OIL IN BULK Crude Oil Washing (COW) System on Tank Vessels Inspections § 157.148 COW system... inspector evidence that the COW system has been installed in accordance with the plans accepted under § 157...

How arbuscular mycorrhizal fungi influence the defense system of sunflower during different abiotic stresses.

PubMed

Mayer, Zoltán; Duc, Nguyen Hong; Sasvári, Zita; Posta, Katalin

2017-12-01

The association between terrestrial plants and arbuscular mycorrhizal (AM) fungi is one of the most common and widespread mutualistic plant-fungi interaction. AM fungi are of beneficial effects on the water and nutrient uptake of plants and increase plant defense mechanisms to alleviate different stresses. The aim of this study was to determine the level of polyphenol oxidase (PPO), guaiacol peroxidase (POX) and glutathione S-transferase (GST) enzyme activities and to track the expression of glutathione S-transferase (GST) gene in plant-arbuscular mycorrhizal system under temperature- and mechanical stress conditions. Our results suggest that induced tolerance of mycorrhizal sunflower to high temperature may be attributed to the induction of GST, POX and PPO enzyme activities as well as to the elevated expression of GST. However, the degree of tolerance of the plant is significantly influenced by the age which is probably justified by the energy considerations.

The identification and characterisation of a functional interaction between arginyl-tRNA-protein transferase and topoisomerase II.

PubMed

Barker, Catherine R; Mouchel, Nathalie A P; Jenkins, John R

2006-04-07

Topoisomerase II is required for the viability of all eukaryotic cells. It plays important roles in DNA replication, recombination, chromosome segregation, and the maintenance of the nuclear scaffold. Proteins that interact with and regulate this essential enzyme are of great interest. To investigate the role of proteins interacting with the N-terminal domain of the Saccharomyces cerevisiae topoisomerase II, we used a yeast two-hybrid protein interaction screen. We identified an interaction between arginyl-tRNA-protein transferase (Ate1) and the N-terminal domain of the S. cerevisiae topoisomerase II, including the potential site of interaction. Ate1 is a component of the N-end rule protein degradation pathway which targets proteins for degradation. We also propose a previously unidentified role for Ate1 in modulating the level of topoisomerase II through the cell cycle.

Prevalence of Null Genotypes of Glutathione S-Transferase T1 (GSTT1) and M1 (GSTM1) in Seven Iranian Populations

PubMed Central

NASSERI, Gholamreza; ZAHEDI, Tahereh; MOUSAVI-KAZEROONI, Fatemeh; SAADAT, Mostafa

2015-01-01

Background: Previous studies have revealed significant differences between populations for genotypic frequencies of glutathione S-transferase T1 (GSTT1) and M1 (GSTM1) polymorphisms. In order to find the frequency of the null genotypes of GSTM1 and GSTT1 in Iranian populations, the present study was carried out. Methods: The total study subjects consisted of 1340 unrelated healthy Muslims/Iranian. From these 297, 200, 123, 168, 152, 200, and 200 individuals from Tabriz (East Azerbaijan Province; belong to Azaris), Yasuj (Kohgiluyeh-va-Boyerahmad Province; belong to Lurs), Abarku (Yazd Province; belong to Persians), Zahedan (Sistan-va-Balouchestan Province; belong to Balouchis), Zahedan (Sistan-va-Balouchestan Province; belong to Sistanis), Kermanshah (Kermanshah Province; belong to Kurds), and Gorgan (Golestan Province; belong to Turkmen) respectively. The genotypes were detected by multiplex PCR. Results: The frequency of GSTM1 null genotype among Azaris, Lurs, Persians, Balouchis, Sistanis, Kurds, and Turkmen was 43.8, 50.0, 52.0, 50.0, 51.3, 56.0, and 53.0%, respectively. There was no significant difference between these populations for the genotypic distribution of the GSTM1 polymorphism (χ2=8.47, df=6, P=0.206). The frequency of GSTT1 null genotype among Azaris, Lurs, Persians, Balouchis, Sistanis, Kurds, and Turkmen was 18.2, 17.0, 29.3, 20.8, 17.8, 18.5, and 23.0%, respectively. There was very similarity between Azaris, Kurds and Lurs for the frequency of GSTT1 genotypes (χ2=0.17, df=2, P=0.916). Conclusion: By comparing the frequency of GSTT1 genotypes among Iranian populations, Caucasians and Asians, it is concluded that Azaris, Kurds and Lurs were similar to each other. Taken together, it is suggested that although Azaris are Turkish speaking belong to Caucasians. PMID:26811816

Genomic Insights into the Glutathione S-Transferase Gene Family of Two Rice Planthoppers, Nilaparvata lugens (Stål) and Sogatella furcifera (Horváth) (Hemiptera: Delphacidae)

PubMed Central

Zhou, Wen-Wu; Liang, Qing-Mei; Xu, Yi; Gurr, Geoff M.; Bao, Yan-Yuan; Zhou, Xue-Ping; Zhang, Chuan-Xi; Cheng, Jiaan; Zhu, Zeng-Rong

2013-01-01

Background Glutathione S-transferase (GST) genes control crucial traits for the metabolism of various toxins encountered by insects in host plants and the wider environment, including insecticides. The planthoppers Nilaparvata lugens and Sogatella furcifera are serious specialist pests of rice throughout eastern Asia. Their capacity to rapidly adapt to resistant rice varieties and to develop resistance to various insecticides has led to severe outbreaks over the last decade. Methodology/Principal Findings Using the genome sequence of N. lugens, we identified for the first time the complete GST gene family of a delphacid insect whilst nine GST gene orthologs were identified from the closely related species S. furcifera. Nilaparvata lugens has 11 GST genes belonging to six cytosolic subclasses and a microsomal class, many fewer than seen in other insects with known genomes. Sigma is the largest GST subclass, and the intron–exon pattern deviates significantly from that of other species. Higher GST gene expression in the N. lugens adult migratory form reflects the higher risk of this life stage in encountering the toxins of non-host plants. After exposure to a sub-lethal dose of four insecticides, chlorpyrifos, imidacloprid, buprofezin or beta-cypermethrin, more GST genes were upregulated in S. furcifera than in N. lugens. RNA interference targeting two N. lugens GST genes, NlGSTe1 and NlGSTm2, significantly increased the sensitivity of fourth instar nymphs to chlorpyrifos but not to beta-cypermethrin. Conclusions/Significance This study provides the first elucidation of the nature of the GST gene family in a delphacid species, offering new insights into the evolution of metabolic enzyme genes in insects. Further, the use of RNA interference to identify the GST genes induced by insecticides illustrates likely mechanisms for the tolerance of these insects. PMID:23457591

Genomic insights into the glutathione S-transferase gene family of two rice planthoppers, Nilaparvata lugens (Stål) and Sogatella furcifera (Horváth) (Hemiptera: Delphacidae).

PubMed

Zhou, Wen-Wu; Liang, Qing-Mei; Xu, Yi; Gurr, Geoff M; Bao, Yan-Yuan; Zhou, Xue-Ping; Zhang, Chuan-Xi; Cheng, Jiaan; Zhu, Zeng-Rong

2013-01-01

Glutathione S-transferase (GST) genes control crucial traits for the metabolism of various toxins encountered by insects in host plants and the wider environment, including insecticides. The planthoppers Nilaparvata lugens and Sogatella furcifera are serious specialist pests of rice throughout eastern Asia. Their capacity to rapidly adapt to resistant rice varieties and to develop resistance to various insecticides has led to severe outbreaks over the last decade. Using the genome sequence of N. lugens, we identified for the first time the complete GST gene family of a delphacid insect whilst nine GST gene orthologs were identified from the closely related species S. furcifera. Nilaparvata lugens has 11 GST genes belonging to six cytosolic subclasses and a microsomal class, many fewer than seen in other insects with known genomes. Sigma is the largest GST subclass, and the intron-exon pattern deviates significantly from that of other species. Higher GST gene expression in the N. lugens adult migratory form reflects the higher risk of this life stage in encountering the toxins of non-host plants. After exposure to a sub-lethal dose of four insecticides, chlorpyrifos, imidacloprid, buprofezin or beta-cypermethrin, more GST genes were upregulated in S. furcifera than in N. lugens. RNA interference targeting two N. lugens GST genes, NlGSTe1 and NlGSTm2, significantly increased the sensitivity of fourth instar nymphs to chlorpyrifos but not to beta-cypermethrin. This study provides the first elucidation of the nature of the GST gene family in a delphacid species, offering new insights into the evolution of metabolic enzyme genes in insects. Further, the use of RNA interference to identify the GST genes induced by insecticides illustrates likely mechanisms for the tolerance of these insects.

Phosphoethanolamine Transferase LptA in Haemophilus ducreyi Modifies Lipid A and Contributes to Human Defensin Resistance In Vitro.

PubMed

Trombley, Michael P; Post, Deborah M B; Rinker, Sherri D; Reinders, Lorri M; Fortney, Kate R; Zwickl, Beth W; Janowicz, Diane M; Baye, Fitsum M; Katz, Barry P; Spinola, Stanley M; Bauer, Margaret E

2015-01-01

Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, β-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. The triple mutant was significantly more susceptible to both α- and β-defensins; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin LL-37 may be more important than defensin resistance to H. ducreyi pathogenesis.

Induction of the pi class of glutathione S-transferase by carnosic acid in rat Clone 9 cells via the p38/Nrf2 pathway.

PubMed

Lin, Chia-Yuan; Wu, Chi-Rei; Chang, Shu-Wei; Wang, Yu-Jung; Wu, Jia-Jiuan; Tsai, Chia-Wen

2015-06-01

Induction of phase II enzymes is important in cancer chemoprevention. We compared the effect of rosemary diterpenes on the expression of the pi class of glutathione S-transferase (GSTP) in rat liver Clone 9 cells and the signaling pathways involved. Culturing cells with 1, 5, 10, or 20 μM carnosic acid (CA) or carnosol (CS) for 24 h in a dose-dependent manner increased the GSTP expression. CA was more potent than CS. The RNA level and the enzyme activity of GSTP were also enhanced by CA treatment. Treatment with 10 μM CA highly induced the reporter activity of the enhancer element GPEI. Furthermore, CA markedly increased the translocation of nuclear factor erythroid-2 related factor 2 (Nrf2) from the cytosol to the nucleus after 30 to 60 min. CA the stimulated the protein induction of p38, nuclear Nrf2, and GSTP was diminished in the presence of SB203580 (a p38 inhibitor). In addition, SB203580 pretreatment or silencing of Nrf2 by siRNA suppressed the CA-induced GPEI-DNA binding activity and GSTP protein expression. Knockdown of p38 or Nrf2 by siRNA abolished the activation of p38 and Nrf2 as well as the protein induction and enzyme activity of GSTP by CA. These results suggest that CA up-regulates the expression and enzyme activity of GSTP via the p38/Nrf2/GPEI pathway.

Effects of detergents on the properties of 4-hydroxybenzoate. Polyprenyl transferase and the specificity of the polyprenyl pyrophosphate synthetic system in mitochondria.

PubMed

Nishino, T; Rudney, H

1977-02-22

The properties of 4-hydroxybenzoate:polyprenyl transferase and the system synthesizing polyprenyl pyrophosphate have been studied in mitochondria from rat and guinea pig livers. With solanesyl pyrophosphate and 4-hydroxybenzoate as substrates the formation of 3-nonaprenyl-4-hydroxybenzoate was linear with time, concentration of protein, and concentration of solanesyl pyrophosphate. Solanesyl monophosphate is inactive as a substrate and is noninhibitory. Conversion of solanesyl monophosphate to the pyrophosphate could not be detected. Detergents such as Triton X-100, Tween-80, and sodium deoxycholate activated the enzyme in mitochondria which were aged by freezing at -20 degrees C for periods ranging from 1 h to several days. Maximum activation also required Mg2+. In agreement with previous observation the effect of Mg2+ and Triton X-100 on fresh mitochondria was quite variable; however, activation with aged preparations was very consistent. Treatment with TritonX-100 causes al alteration in the biosynthetic pattern of rat liver mitochondria so that rather than nonaprenyl, decaprenyl, pyrophosphate is preferentially made in the presence of solanesyl pyrophosphate and isopentenyl pyrophosphate. In the presence of Triton X-100 and added pool of solanesyl pyrophosphate appears to exert a feedback inhibition on the incorporation of isopentenyl pyrophosphate into solanesyl pyrophosphate. In the case of guinea pig liver mitochondria a different pattern is observed with Triton X-100 in contrast to the rat. The de novo formation of decaprenyl pyrophosphate from isopentenyl pyrophosphate appears to be inhibited by Triton X-100, but the synthesis of decaprenyl pyrophosphate from isopentenyl pyrophosphate and nonaprenyl pyrophosphate is not inhibited. The data also indicate that in guinea pig liver in a system synthesizing decaprenyl pyrophosphate from isopentenyl pyrophosphate, there does not appear to be a detectable pool of nonaprenyl pyrophosphate. These results show that

Reverted glutathione S-transferase-like genes that influence flower color intensity of carnation (Dianthus caryophyllus L.) originated from excision of a transposable element.

PubMed

Momose, Masaki; Itoh, Yoshio; Umemoto, Naoyuki; Nakayama, Masayoshi; Ozeki, Yoshihiro

2013-12-01

A glutathione S-transferase-like gene, DcGSTF2, is responsible for carnation (Dianthus caryophyllus L.) flower color intensity. Two defective genes, DcGSTF2mu with a nonsense mutation and DcGSTF2-dTac1 containing a transposable element dTac1, have been characterized in detail in this report. dTac1 is an active element that produces reverted functional genes by excision of the element. A pale-pink cultivar 'Daisy' carries both defective genes, whereas a spontaneous deep-colored mutant 'Daisy-VPR' lost the element from DcGSTF2-dTac1. This finding confirmed that dTac1 is active and that the resulting reverted gene, DcGSTF2rev1, missing the element is responsible for this color change. Crosses between the pale-colored cultivar '06-LA' and a deep-colored cultivar 'Spectrum' produced segregating progeny. Only the deep-colored progeny had DcGSTF2rev2 derived from the 'Spectrum' parent, whereas progeny with pale-colored flowers had defective forms from both parents, DcGSTF2mu and DcGSTF2-dTac1. Thus, DcGSTF2rev2 had functional activity and likely originated from excision of dTac1 since there was a footprint sequence at the vacated site of the dTac1 insertion. Characterizing the DcGSTF2 genes in several cultivars revealed that the two functional genes, DcGSTF2rev1 and DcGSTF2rev2, have been used for some time in carnation breeding with the latter in use for more than half a century.

Identification of Glutathione S-Transferase (GST) Genes from a Dark Septate Endophytic Fungus (Exophiala pisciphila) and Their Expression Patterns under Varied Metals Stress

PubMed Central

Qiao, Qin; Liu, Lei; Wang, Jun-Ling; Cao, Guan-Hua; Li, Tao; Zhao, Zhi-Wei

2015-01-01

Glutathione S-transferases (GSTs) compose a family of multifunctional enzymes that play important roles in the detoxification of xenobiotics and the oxidative stress response. In the present study, twenty four GST genes from the transcriptome of a metal-tolerant dark septate endophyte (DSE), Exophiala pisciphila, were identified based on sequence homology, and their responses to various heavy metal exposures were also analyzed. Phylogenetic analysis showed that the 24 GST genes from E. pisciphila (EpGSTs) were divided into eight distinct classes, including seven cytosolic classes and one mitochondrial metaxin 1-like class. Moreover, the variable expression patterns of these EpGSTs were observed under different heavy metal stresses at their effective concentrations for inhibiting growth by 50% (EC50). Lead (Pb) exposure caused the up-regulation of all EpGSTs, while cadmium (Cd), copper (Cu) and zinc (Zn) treatments led to the significant up-regulation of most of the EpGSTs (p < 0.05 to p < 0.001). Furthermore, although heavy metal-specific differences in performance were observed under various heavy metals in Escherichia coli BL21 (DE3) transformed with EpGSTN-31, the over-expression of this gene was able to enhance the heavy metal tolerance of the host cells. These results indicate that E. Pisciphila harbored a diverse of GST genes and the up-regulated EpGSTs are closely related to the heavy metal tolerance of E. pisciphila. The study represents the first investigation of the GST family in E. pisciphila and provides a primary interpretation of heavy metal detoxification for E. pisciphila. PMID:25884726

Evidence Acquisition and Evaluation for a U.S. Government Evidence Summit on Protecting Children outside Family Care

ERIC Educational Resources Information Center

Higgs, Elizabeth S.; Zlidar, Vera M.; Balster, Robert L.

2012-01-01

Recognizing the need for evidence to inform policies, strategies, and programs to care for vulnerable children, the U.S. Government convened an Evidence Summit on Protecting Children Outside of Family Care on December 12-13, 2011, in Washington, DC, USA. This paper summarizes the background and methods for the acquisition and evaluation of the…

Succinyl-CoA:Mesaconate CoA-Transferase and Mesaconyl-CoA Hydratase, Enzymes of the Methylaspartate Cycle in Haloarcula hispanica.

PubMed

Borjian, Farshad; Johnsen, Ulrike; Schönheit, Peter; Berg, Ivan A

2017-01-01

Growth on acetate or other acetyl-CoA-generating substrates as a sole source of carbon requires an anaplerotic pathway for the conversion of acetyl-CoA into cellular building blocks. Haloarchaea (class Halobacteria ) possess two different anaplerotic pathways, the classical glyoxylate cycle and the novel methylaspartate cycle. The methylaspartate cycle was discovered in Haloarcula spp. and operates in ∼40% of sequenced haloarchaea. In this cycle, condensation of one molecule of acetyl-CoA with oxaloacetate gives rise to citrate, which is further converted to 2-oxoglutarate and then to glutamate. The following glutamate rearrangement and deamination lead to mesaconate (methylfumarate) that needs to be activated to mesaconyl-C1-CoA and hydrated to β-methylmalyl-CoA. The cleavage of β-methylmalyl-CoA results in the formation of propionyl-CoA and glyoxylate. The carboxylation of propionyl-CoA and the condensation of glyoxylate with another acetyl-CoA molecule give rise to two C 4 -dicarboxylic acids, thus regenerating the initial acetyl-CoA acceptor and forming malate, its final product. Here we studied two enzymes of the methylaspartate cycle from Haloarcula hispanica , succinyl-CoA:mesaconate CoA-transferase (mesaconate CoA-transferase, Hah_1336) and mesaconyl-CoA hydratase (Hah_1340). Their genes were heterologously expressed in Haloferax volcanii , and the corresponding enzymes were purified and characterized. Mesaconate CoA-transferase was specific for its physiological substrates, mesaconate and succinyl-CoA, and produced only mesaconyl-C1-CoA and no mesaconyl-C4-CoA. Mesaconyl-CoA hydratase had a 3.5-fold bias for the physiological substrate, mesaconyl-C1-CoA, compared to mesaconyl-C4-CoA, and virtually no activity with other tested enoyl-CoA/3-hydroxyacyl-CoA compounds. Our results further prove the functioning of the methylaspartate cycle in haloarchaea and suggest that mesaconate CoA-transferase and mesaconyl-CoA hydratase can be regarded as

The dyad palindromic glutathione transferase P enhancer binds multiple factors including AP1.

PubMed Central

Diccianni, M B; Imagawa, M; Muramatsu, M

1992-01-01

Glutathione Transferase P (GST-P) gene expression is dominantly regulated by an upstream enhancer (GPEI) consisting of a dyad of palindromically oriented imperfect TPA (12-O-tetradecanoyl-phorbol-13-acetate)-responsive elements (TRE). GPEI is active in AP1-lacking F9 cells as well in AP1-containing HeLa cells. Despite GPEI's similarity to a TRE, c-jun co-transfection has only a minimal effect on transactivation. Antisense c-jun and c-fos co-transfection experiments further demonstrate the lack of a role for AP1 in GPEI mediated trans-activation in F9 cells, although endogenously present AP1 can influence GPEI in HeLa cells. Co-transfection of delta fosB with c-jun, which forms an inactive c-Jun/delta FosB heterodimer that binds TRE sequences, inhibits GPEI-mediated transcription in AP1-lacking F9 cells as well as AP1-containing HeLa cells. These data suggest novel factor(s) other than AP1 are influencing GPEI. Binding studies reveal multiple nucleoproteins bind to GPEI. These factors are likely responsible for the high level of GPEI-mediated transcription observed in the absence of AP1 and during hepatocarcinogenesis. Images PMID:1408831

The dyad palindromic glutathione transferase P enhancer binds multiple factors including AP1.

PubMed

Diccianni, M B; Imagawa, M; Muramatsu, M

1992-10-11

Glutathione Transferase P (GST-P) gene expression is dominantly regulated by an upstream enhancer (GPEI) consisting of a dyad of palindromically oriented imperfect TPA (12-O-tetradecanoyl-phorbol-13-acetate)-responsive elements (TRE). GPEI is active in AP1-lacking F9 cells as well in AP1-containing HeLa cells. Despite GPEI's similarity to a TRE, c-jun co-transfection has only a minimal effect on transactivation. Antisense c-jun and c-fos co-transfection experiments further demonstrate the lack of a role for AP1 in GPEI mediated trans-activation in F9 cells, although endogenously present AP1 can influence GPEI in HeLa cells. Co-transfection of delta fosB with c-jun, which forms an inactive c-Jun/delta FosB heterodimer that binds TRE sequences, inhibits GPEI-mediated transcription in AP1-lacking F9 cells as well as AP1-containing HeLa cells. These data suggest novel factor(s) other than AP1 are influencing GPEI. Binding studies reveal multiple nucleoproteins bind to GPEI. These factors are likely responsible for the high level of GPEI-mediated transcription observed in the absence of AP1 and during hepatocarcinogenesis.

DEVELOPMENT OF THE SIGMA-1 RECEPTOR IN C-TERMINALS OF MOTONEURONS AND COLOCALIZATION WITH THE N,N’-DIMETHYLTRYPTAMINE FORMING ENZYME, INDOLE-N-METHYL TRANSFERASE

PubMed Central

Mavlyutov, Timur A.; Epstein, Miles L.; Liu, Patricia; Verbny, Yakov I.; Ziskind-Conhaim, Lea; Ruoho, Arnold E.

2012-01-01

The function of the sigma-1 receptor (S1R) has been linked to modulating the activities of ion channels and G-protein coupled receptors (GPCR). In the CNS the S1R is expressed ubiquitously but is enriched in mouse motoneurons (MN), where it is localized to subsurface cisternae of cholinergic postsynaptic densities, also known as C-terminals. We found that S1R is enriched in mouse spinal MN at late stages of embryonic development when it is first visualized in the endoplasmic reticulum. S1Rs appear to concentrate at C-terminals of mouse MN only on the second week of postnatal development. We found that Indole-N-methyl transferase (INMT), an enzyme that converts tryptamine into the sigma-1 ligand dimethyltryptamine (DMT), is also localized to postsynaptic sites of C-terminals in close proximity to the S1R. This close association of INMT and SIRs suggest that DMT is synthesized locally to effectively activate S1R in MN. PMID:22265729

Hypermethylation of the Human Glutathione S-Transferase-π Gene (GSTP1) CpG Island Is Present in a Subset of Proliferative Inflammatory Atrophy Lesions but Not in Normal or Hyperplastic Epithelium of the Prostate

PubMed Central

Nakayama, Masashi; Bennett, Christina J.; Hicks, Jessica L.; Epstein, Jonathan I.; Platz, Elizabeth A.; Nelson, William G.; De Marzo, Angelo M.

2003-01-01

Somatic inactivation of the glutathione S-transferase-π gene (GSTP1) via CpG island hypermethylation occurs early during prostate carcinogenesis, present in ∼70% of high-grade prostatic intraepithelial neoplasia (high-grade PIN) lesions and more than 90% of adenocarcinomas. Recently, there has been a resurgence of the concept that foci of prostatic atrophy (referred to as proliferative inflammatory atrophy or PIA) may be precursor lesions for the development of prostate cancer and/or high-grade PIN. Many of the cells within PIA lesions contain elevated levels of GSTP1, glutathione S-transferase-α (GSTA1), and cyclooxygenase-II proteins, suggesting a stress response. Because not all PIA cells are positive for GSTP1 protein, we hypothesized that some of the cells within these regions acquire GSTP1 CpG island hypermethylation, increasing the chance of progression to high-grade PIN and/or adenocarcinoma. Separate regions (n =199) from 27 formalin-fixed paraffin-embedded prostates were microdissected by laser-capture microdissection (Arcturus PixCell II). These regions included normal epithelium (n = 48), hyperplasticepithelium from benign prostatic hyperplasia nodules (n = 22), PIA (n = 64), high-grade PIN (n = 32), and adenocarcinoma (n = 33). Genomic DNA was isolated and assessed for GSTP1 CpG island hypermethylation by methylation-specific polymerase chain reaction. GSTP1 CpG island hypermethylation was not detected in normal epithelium (0 of 48) or in hyperplastic epithelium (0 of 22), but was found in 4 of 64 (6.3%) PIA lesions. The difference in the frequency of GSTP1 CpG island hypermethylation between normal or hyperplastic epithelium and PIA was statistically significant (P = 0.049). Similar to studies using nonmicrodissected cases, hypermethylation was found in 22 of 32 (68.8%) high-grade PIN lesions and in 30 of 33 (90.9%) adenocarcinoma lesions. Unlike normal or hyperplastic epithelium, GSTP1 CpG island hypermethylation can be detected in some PIA

Identification and expression profiles of nine glutathione S-transferase genes from the important rice phloem sap-sucker and virus vector Laodelphax striatellus (Fallén) (Hemiptera: Delphacidae).

PubMed

Zhou, Wen-Wu; Li, Xi-Wang; Quan, Yin-Hua; Cheng, Jiaan; Zhang, Chuan-Xi; Gurr, Geoff; Zhu, Zeng-Rong

2012-09-01

Glutathione S-transferases (GSTs) have received considerable attention in insects for their roles in insecticide resistance. Laodelphax striatellus (Fallén) is a serious rice pest. L. striatellus outbreaks occur frequently throughout eastern Asia. A key problem in controlling this pest is its rapid adaptation to numerous insecticides. In this research, nine cDNAs encoding GSTs in L. striatellus were cloned and characterised. The cloned GSTs of L. striatellus belonged to six cytosolic classes and a microsomal subgroup. Exposure to sublethal concentrations of each of the six insecticides, DDT, chlorpyrifos, fipronil, imidacloprid, buprofezin and beta-cypermethrin, quickly induced (6 h) up-expression of LsGSTe1. The expression of LsGSTs2 was increased by chlorpyrifos, fipronil and beta-cypermethrin. Furthermore, exposure of L. striatellus to fipronil, imidacloprid, buprofezin and beta-cypermethrin increased the expression of the LsGSTm gene after 24 or 48 h. This work is the first identification of GST genes from different GST groups in Auchenorrhyncha species and their induction characteristics with insecticide types and time. The elevated expression of GST genes induced by insecticides might be related to the enhanced tolerance of this insect to insecticides and xenobiotics. Copyright © 2012 Society of Chemical Industry.

Rab geranylgeranyl transferase β subunit is essential for male fertility and tip growth in Arabidopsis

PubMed Central

Gutkowska, Malgorzata; Wnuk, Marta; Nowakowska, Julita; Lichocka, Malgorzata; Stronkowski, Michal M.; Swiezewska, Ewa

2015-01-01

Rab proteins, key players in vesicular transport in all eukaryotic cells, are post-translationally modified by lipid moieties. Two geranylgeranyl groups are attached to the Rab protein by the heterodimeric enzyme Rab geranylgeranyl transferase (RGT) αβ. Partial impairment in this enzyme activity in Arabidopsis, by disruption of the AtRGTB1 gene, is known to influence plant stature and disturb gravitropic and light responses. Here it is shown that mutations in each of the RGTB genes cause a tip growth defect, visible as root hair and pollen tube deformations. Moreover, FM 1–43 styryl dye endocytosis and recycling are affected in the mutant root hairs. Finally, it is demonstrated that the double mutant, with both AtRGTB genes disrupted, is non-viable due to absolute male sterility. Doubly mutated pollen is shrunken, has an abnormal exine structure, and shows strong disorganization of internal membranes, particularly of the endoplasmic reticulum system. PMID:25316062

Mimicking Insect Communication: Release and Detection of Pheromone, Biosynthesized by an Alcohol Acetyl Transferase Immobilized in a Microreactor

PubMed Central

Muñoz, Lourdes; Dimov, Nikolay; Carot-Sans, Gerard; Bula, Wojciech P.; Guerrero, Angel; Gardeniers, Han J. G. E.

2012-01-01

Infochemical production, release and detection of (Z,E)-9,11-tetradecadienyl acetate, the major component of the pheromone of the moth Spodoptera littoralis, is achieved in a novel microfluidic system designed to mimic the final step of the pheromone biosynthesis by immobilized recombinant alcohol acetyl transferase. The microfluidic system is part of an “artificial gland”, i.e., a chemoemitter that comprises a microreactor connected to a microevaporator and is able to produce and release a pre-defined amount of the major component of the pheromone from the corresponding (Z,E)-9,11-tetradecadienol. Performance of the entire chemoemitter has been assessed in electrophysiological and behavioral experiments. Electroantennographic depolarizations of the pheromone produced by the chemoemitter were ca. 40% relative to that evoked by the synthetic pheromone. In a wind tunnel, the pheromone released from the evaporator elicited on males a similar attraction behavior as 3 virgin females in most of the parameters considered. PMID:23155372

Placental biomarkers of PAH exposure and glutathione-S-transferase biotransformation enzymes in an obstetric population from Tijuana, Baja California, Mexico

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dodd-Butera, Teresa, E-mail: tdbutera@csusb.edu

Environmental exposures along the US-Mexico border have the potential to adversely affect the maternal-fetal environment. The purpose of this study was to assess placental biomarkers of environmental exposures in an obstetric population at the California-Baja California border in relation to detoxifying enzymes in the placenta and nutritional status. This study was conducted on consenting, full-term, obstetric patients (n=54), delivering in a hospital in Tijuana, Baja California (BC), Mexico. Placental polyaromatic hydrocarbon (PAH)-DNA adducts were measured in addition to placental glutathione-S-transferase (GST) activity and genotype, maternal serum folate, and maternal and umbilical cord blood lead and cadmium levels. A questionnaire wasmore » administered to the mothers to determine maternal occupation in a maquiladora, other exposures, and obstetric indicators. In univariate analysis, maternal serum folate levels were inversely correlated with total PAH-DNA adducts (rho=−0.375, p=0.007); adduct #1 (rho=−0.388, p=0.005); and adduct #3 (rho =−0.430, p=0.002). Maternal lead levels were significantly positively correlated with cord blood lead levels (rho=0.512, p<0.001). Cadmium levels were generally very low but significantly higher in mothers exposed to environmental tobacco smoke (ETS) (either at work or at home, n=10). In multivariate analysis, only maternal serum folate levels remained as a significant negative predictor of total DNA-PAH adducts levels in placenta. These findings affirm that placental tissue is a valuable and readily available source of human tissue for biomonitoring; and indicate that further study of the role of nutrition in detoxification and mitigation of environmental exposures in pregnant women is warranted. - Highlights: • Maternal-fetal environment susceptible to toxic exposures at US-Mexico border. • Lower serum folate was correlated with higher PAH-DNA adduct levels at birth. • Placental DNA adducts in GST mu (-) cord

Gene-knockdown in the honey bee mite Varroa destructor by a non-invasive approach: studies on a glutathione S-transferase

PubMed Central

2010-01-01

Background The parasitic mite Varroa destructor is considered the major pest of the European honey bee (Apis mellifera) and responsible for declines in honey bee populations worldwide. Exploiting the full potential of gene sequences becoming available for V. destructor requires adaptation of modern molecular biology approaches to this non-model organism. Using a mu-class glutathione S-transferase (VdGST-mu1) as a candidate gene we investigated the feasibility of gene knockdown in V. destructor by double-stranded RNA-interference (dsRNAi). Results Intra-haemocoelic injection of dsRNA-VdGST-mu1 resulted in 97% reduction in VdGST-mu1 transcript levels 48 h post-injection compared to mites injected with a bolus of irrelevant dsRNA (LacZ). This gene suppression was maintained to, at least, 72 h. Total GST catalytic activity was reduced by 54% in VdGST-mu1 gene knockdown mites demonstrating the knockdown was effective at the translation step as well as the transcription steps. Although near total gene knockdown was achieved by intra-haemocoelic injection, only half of such treated mites survived this traumatic method of dsRNA administration and less invasive methods were assessed. V. destructor immersed overnight in 0.9% NaCl solution containing dsRNA exhibited excellent reduction in VdGST-mu1 transcript levels (87% compared to mites immersed in dsRNA-LacZ). Importantly, mites undergoing the immersion approach had greatly improved survival (75-80%) over 72 h, approaching that of mites not undergoing any treatment. Conclusions Our findings on V. destructor are the first report of gene knockdown in any mite species and demonstrate that the small size of such organisms is not a major impediment to applying gene knockdown approaches to the study of such parasitic pests. The immersion in dsRNA solution method provides an easy, inexpensive, relatively high throughput method of gene silencing suitable for studies in V. destructor, other small mites and immature stages of ticks

ROLE OF STEROID HORMONES AND DECIDUAL INDUCTION IN THE REGULATION OF ADENOSINE DIPHOSPHORIBOSYL TRANSFERASE ACTIVITY IN RAT ENDOMETRIUM

EPA Science Inventory

To assess the effect of ovarian steroid hormones on enzyme activity, adenosine diphosphoribosyl transferase (ADPRT) was measured in endometrial nuclei isolated on estrus and on d 4 from rats ovariectomized on estrus (d 0) and treated d 0-3 with (a) vehicle, (b) 1 ug estrone/d (E)...

BIOTRANSFORMATION AND GENOTOXICITY OF THE DRINKING WATER DISINFECTION BYPRODUCT BROMODICHLOROMETHANE: DNA BINDING MEDIATED BY GLUTATHIONE TRANSFERASE THETA 1-1

EPA Science Inventory

The drinking water disinfection byproduct bromodichloromethane (CHBrCl2) was
previously shown to be mutagenic in Salmonella typhimurium that overexpress rat glutathione
transferase theta 1-1 (GSTT1-1). Several experimental approaches were undertaken in this study
to inve...

Dimethyl adenosine transferase (KsgA) deficiency in Salmonella Enteritidis confers susceptibility to high osmolarity and virulence attenuation in chickens

USDA-ARS?s Scientific Manuscript database

: Dimethyladenosine transferase (KsgA) performs diverse roles in bacteria including ribosomal maturation, DNA mismatch repair, and synthesis of KsgA is responsive to antibiotics and cold temperature. We previously showed that ksgA mutation in Salmonella Enteritidis results in impaired invasiveness i...

Problem-Based Test: Functional Analysis of Mutant 16S rRNAs

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2010-01-01

Terms to be familiar with before you start to solve the test: ribosome, ribosomal subunits, antibiotics, point mutation, 16S, 5S, and 23S rRNA, Shine-Dalgarno sequence, mRNA, tRNA, palindrome, hairpin, restriction endonuclease, fMet-tRNA, peptidyl transferase, initiation, elongation, termination of translation, expression plasmid, transformation,…

Evidence-based management of ambulatory electronic health record system implementation: an assessment of conceptual support and qualitative evidence.

PubMed

McAlearney, Ann Scheck; Hefner, Jennifer L; Sieck, Cynthia; Rizer, Milisa; Huerta, Timothy R

2014-07-01

While electronic health record (EHR) systems have potential to drive improvements in healthcare, a majority of EHR implementations fall short of expectations. Shortcomings in implementations are often due to organizational issues around the implementation process rather than technological problems. Evidence from both the information technology and healthcare management literature can be applied to improve the likelihood of implementation success, but the translation of this evidence into practice has not been widespread. Our objective was to comprehensively study and synthesize best practices for managing ambulatory EHR system implementation in healthcare organizations, highlighting applicable management theories and successful strategies. We held 45 interviews with key informants in six U.S. healthcare organizations purposively selected based on reported success with ambulatory EHR implementation. We also conducted six focus groups comprised of 37 physicians. Interview and focus group transcripts were analyzed using both deductive and inductive methods to answer research questions and explore emergent themes. We suggest that successful management of ambulatory EHR implementation can be guided by the Plan-Do-Study-Act (PDSA) quality improvement (QI) model. While participants did not acknowledge nor emphasize use of this model, we found evidence that successful implementation practices could be framed using the PDSA model. Additionally, successful sites had three strategies in common: 1) use of evidence from published health information technology (HIT) literature emphasizing implementation facilitators; 2) focusing on workflow; and 3) incorporating critical management factors that facilitate implementation. Organizations seeking to improve ambulatory EHR implementation processes can use frameworks such as the PDSA QI model to guide efforts and provide a means to formally accommodate new evidence over time. Implementing formal management strategies and incorporating

Phylogenetic characterization of Clonorchis sinensis proteins homologous to the sigma-class glutathione transferase and their differential expression profiles.

PubMed

Bae, Young-An; Kim, Jeong-Geun; Kong, Yoon

2016-01-01

Glutathione transferase (GST) is one of the major antioxidant proteins with diverse supplemental activities including peroxidase, isomerase, and thiol transferase. GSTs are classified into multiple classes on the basis of their primary structures and substrate/inhibitor specificity. However, the evolutionary routes and physiological environments specific to each of the closely related bioactive enzymes remain elusive. The sigma-like GSTs exhibit amino acid conservation patterns similar to the prostaglandin D synthases (PGDSs). In this study, we analyzed the phylogenetic position of the GSTs of the biocarcinogenic liver fluke, Clonorchis sinensis. We also observed induction profile of the GSTs in association with the parasite's maturation and in response to exogenous oxidative stresses, with special attention to sigma-class GSTs and PGDSs. The C. sinensis genome encoded 12 GST protein species, which were separately assigned to cytosolic (two omega-, one zeta-, two mu-, and five sigma-class), mitochondrial (one kappa-class), and microsomal (one membrane-associated proteins in eicosanoid and glutathione metabolism-like protein) GST families. Multiple sigma GST (or PGDS) orthologs were also detected in Opisthorchis viverrini. Other trematode species possessed only a single sigma-like GST gene. A phylogenetic analysis demonstrated that one of the sigma GST lineages duplicated in the common ancestor of trematodes were specifically expanded in the opisthorchiids, but deleted in other trematodes. The induction profiles of these sigma GST genes along with the development and aging of C. sinensis, and against various exogenous chemical stimuli strongly suggest that the paralogous sigma GST genes might be undergone specialized evolution to cope with the diverse hostile biochemical environments within the mammalian hepatobiliary ductal system. Copyright © 2016 Elsevier B.V. All rights reserved.

O-GlcNAc site-mapping of liver X receptor-α and O-GlcNAc transferase.

PubMed

Fan, Qiong; Moen, Anders; Anonsen, Jan Haug; Bindesbøll, Christian; Sæther, Thomas; Carlson, Cathrine Rein; Grønning-Wang, Line M

2018-05-05

The Liver X Receptor α (LXRα) belongs to the nuclear receptor superfamily and plays an essential role in regulating cholesterol, lipid and glucose metabolism and inflammatory responses. We have previously shown that LXRα is post-translationally modified by O-linked β-N-acetyl-glucosamine (O-GlcNAc) with increased transcriptional activity. Moreover, we showed that LXRα associates with O-GlcNAc transferase (OGT) in vitro and in vivo in mouse liver. In this study, we report that human LXRα is O-GlcNAc modified in its N-terminal domain (NTD) by identifying a specific O-GlcNAc site S49 and a novel O-GlcNAc modified peptide 20 LWKPGAQDASSQAQGGSSCILRE 42 . However, O-GlcNAc site-mutations did not modulate LXRα transactivation of selected target gene promoters in vitro. Peptide array and co-immunoprecipitation assays demonstrate that LXRα interacts with OGT in its NTD and ligand-binding domain (LBD) in a ligand-independent fashion. Moreover, we map two new O-GlcNAc sites in the longest OGT isoform (ncOGT): S437 in the tetratricopeptide repeat (TPR) 13 domain and T1043 in the far C-terminus, and a new O-GlcNAc modified peptide (amino acids 826-832) in the intervening region (Int-D) within the catalytic domain. We also map four new O-GlcNAc sites in the short isoform sOGT: S391, T393, S399 and S437 in the TPRs 11-13 domain. Future studies will reveal the biological role of identified O-GlcNAc sites in LXRα and OGT. Copyright © 2018 Elsevier Inc. All rights reserved.

Measurement of glutathione S-transferase and its class-pi in plasma and tissue biopsies obtained after laparoscopy and endoscopy from subjects with esophagus and gastric cancer.

PubMed

Mohammadzadeh, G S; Nasseri Moghadam, S; Rasaee, M J; Zaree, A B; Mahmoodzadeh, H; Allameh, A

2003-06-01

To develop an indirect enzyme-linked immunosorbent assay (ELISA) for measuring class-pi glutathione S-transferase (GST) in plasma, and tissue biopsies obtained from upper gastrointestinal cancer (UGI Ca) patients. GST activity and GST-pi concentration were detected in normal human squamous esophageal epithelium, normal gastric cardia and their corresponding malignant tumor biopsies. Plasma GST was significantly higher (p < 0.05) in UGI Ca patients as compared to those obtained from normal individuals. Plasma GST-pi concentration in normal subjects was 6.6 +/- 1.9 ng/mg protein, whereas it was higher in UGI Ca patients (esophageal, 10.0 +/- 1.8; gastric, 10.7 +/- 1.7 ng/mL, p

A review of CARE’s Community Score Card experience and evidence

PubMed Central

Gullo, Sara; Galavotti, Christine; Altman, Lara

2016-01-01

The global community’s growing enthusiasm for the potential of social accountability approaches to improve health system performance and accelerate health progress makes it imperative that we learn from social accountability intervention implementation experience and results. To this end, we carried out a review of Cooperative for Assistance and Relief Everywhere, Inc. (CARE)’s experience with the Community Score Card© (CSC)—a social accountability approach CARE developed in Malawi. We reviewed projects that CARE implemented between 2002 and 2013 that employed the CSC and that had at least one evaluation in English. We systematically collected and synthesized information from evaluations on the projects’ characteristics, CSC-related outcomes and challenges. Eight projects, spanning five countries, met our inclusion criteria. The projects applied the CSC to various focus areas, mostly health. We identified one to three evaluations, mostly qualitative, for each project. While the evaluations had many limitations, consistency of the results, as well as the range of outcomes, suggests that the CSC is contributing to significant changes. All projects reported CSC-related governance outcomes and service outcomes. There is promising evidence that the CSC can contribute to citizen empowerment, service provider and power-holder effectiveness, accountability and responsiveness and spaces for negotiation between the two that are expanded, effective and inclusive. There is also evidence that the CSC may contribute to improvements in service availability, access, utilization and quality. The CSC seems particularly suited to building trust and strengthening relationships between the community and service providers and to improving the user-centred dimension of quality. All of the projects reported challenges, with ensuring national responsiveness and inclusion of marginalized groups in the CSC process proving to be the most intractable. To improve health system

MicroRNA-133b targets glutathione S-transferase π expression to increase ovarian cancer cell sensitivity to chemotherapy drugs.

PubMed

Chen, Shuo; Jiao, Jin-Wen; Sun, Kai-Xuan; Zong, Zhi-Hong; Zhao, Yang

2015-01-01

Accumulating studies reveal that aberrant microRNA (miRNA) expression can affect the development of chemotherapy drug resistance by modulating the expression of relevant target proteins. The aim of this study was to investigate the role of miR-133b in the development of drug resistance in ovarian cancer cells. We examined the levels of miR-133b expression in ovarian carcinoma tissues and the human ovarian carcinoma cell lines (A2780, A2780/DDP and A2780/Taxol, respectively). We determined the cell viability of these cell lines treated with cisplatin or paclitaxel in the presence or absence of miR-133b or anti-miR-133b transfection using the MTT assay. Reverse transcription polymerase chain reaction and Western blotting were used to assess the mRNA and protein expression levels of two drug-resistance-related genes: glutathione S-transferase (GST)-π and multidrug resistance protein 1 (MDR1). The dual-luciferase reporter assay was used to detect the promoter activity of GST-π in the presence and absence of miR-133b. The expression of miR-133b was significantly lower in primary resistant ovarian carcinomas than in the chemotherapy-sensitive carcinomas (P<0.05), and the same results were found in primary resistant ovarian cell lines (A2780/Taxol and A2780/DDP) compared to the chemotherapy-sensitive cell line (A2780; P<0.05). Following miR-133b transfection, four cell lines showed increased sensitivity to paclitaxel and cisplatin, while anti-miR-133b transfection reduced cell sensitivity to paclitaxel and cisplatin. Dual-luciferase reporter assay showed that miR-133b interacted with the 3'-untranslated region of GST-π. Compared to controls, the mRNA and protein levels of MDR1 and GST-π were downregulated after miR-133b transfection and upregulated after anti-miR-133b transfection. MicroRNA-133b may reduce ovarian cancer drug resistance by silencing the expression of the drug-resistance-related proteins, GST-π and MDR1. In future, the combination of miR-133b with

Size effect of elemental selenium nanoparticles (Nano-Se) at supranutritional levels on selenium accumulation and glutathione S-transferase activity.

PubMed

Peng, Dungeng; Zhang, Jinsong; Liu, Qingliang; Taylor, Ethan Will

2007-10-01

It has been shown that 36 nm Nano-Se has lower toxicity than selenite or selenomethionine, but these forms of selenium (Se) all possess similar ability to increase selenoenzyme levels. The size of nanoparticles plays an important role in their biological activity: as expected, 5-200 nm Nano-Se can directly scavenge free radicals in vitro in a size-dependent fashion. However, in Se-deficient cells and Se-deficient mice, the size effect of Nano-Se on increasing selenoenzymes and liver Se disappears unexpectedly. We hypothesize that under conditions of Se deficiency, the avidity of Se uptake mechanisms may be increased to maintain the biosynthesis of selenoenzymes, which are fundamental for redox homeostasis. This increased avidity may override the potential advantage of small size Nano-Se seen under Se-replete conditions, thereby eliminating the size effect. Once selenoenzymes have been saturated, Se uptake mechanisms may downregulate; accordingly, the size effect of Nano-Se can then reappear. To test this hypothesis, Se-deficient mice were administered either 36 or 90 nm Nano-Se at supranutritional doses, in both a short-term model and a single-dose model. Under these conditions, Nano-Se showed a size effect on Se accumulation and glutathione S-transferase (GST) activity. A size effect of Nano-Se was found in 15 out of 18 total comparisons between sizes at the same dose and time in the two models. Furthermore, the magnitude of the size effect was more prominent on Se accumulation than on GST activity. GST is strictly regulated by transcriptional and translational mechanisms, so its increase in activity normally does not exceed 3-fold. In contrast, the homeostasis of Se accumulation is not as tightly controlled. In the present experiments, GST activity had reached or was approaching saturation, but liver Se was far below saturation. Therefore, our results strongly suggest that the saturation profile of the tested biomarker has an impact on the size effect of Nano

MOF Acetyl Transferase Regulates Transcription and Respiration in Mitochondria.

PubMed

Chatterjee, Aindrila; Seyfferth, Janine; Lucci, Jacopo; Gilsbach, Ralf; Preissl, Sebastian; Böttinger, Lena; Mårtensson, Christoph U; Panhale, Amol; Stehle, Thomas; Kretz, Oliver; Sahyoun, Abdullah H; Avilov, Sergiy; Eimer, Stefan; Hein, Lutz; Pfanner, Nikolaus; Becker, Thomas; Akhtar, Asifa

2016-10-20

A functional crosstalk between epigenetic regulators and metabolic control could provide a mechanism to adapt cellular responses to environmental cues. We report that the well-known nuclear MYST family acetyl transferase MOF and a subset of its non-specific lethal complex partners reside in mitochondria. MOF regulates oxidative phosphorylation by controlling expression of respiratory genes from both nuclear and mtDNA in aerobically respiring cells. MOF binds mtDNA, and this binding is dependent on KANSL3. The mitochondrial pool of MOF, but not a catalytically deficient mutant, rescues respiratory and mtDNA transcriptional defects triggered by the absence of MOF. Mof conditional knockout has catastrophic consequences for tissues with high-energy consumption, triggering hypertrophic cardiomyopathy and cardiac failure in murine hearts; cardiomyocytes show severe mitochondrial degeneration and deregulation of mitochondrial nutrient metabolism and oxidative phosphorylation pathways. Thus, MOF is a dual-transcriptional regulator of nuclear and mitochondrial genomes connecting epigenetics and metabolism. Copyright © 2016 Elsevier Inc. All rights reserved.

Transgenic expression of a maize geranyl geranyl transferase gene sequence in maize callus increases resistance to ear rot pathogens

USDA-ARS?s Scientific Manuscript database

Determining the genes responsible for pest resistance in maize can allow breeders to develop varieties with lower losses and less contamination with undesirable toxins. A gene sequence coding for a geranyl geranyl transferase-like protein located in a fungal ear rot resistance quantitative trait loc...

Inhibition of JNK by pi class of glutathione S-transferase through PKA/CREB pathway is associated with carnosic acid protection against 6-hydroxydopamine-induced apoptosis.

PubMed

Lin, Chia-Yuan; Fu, Ru-Huei; Chou, Ruey-Hwang; Chen, Jing-Hsien; Wu, Chi-Rei; Chang, Shu-Wei; Tsai, Chia-Wen

2017-05-01

Pi class of glutathione S-transferase (GST) is known to suppress c-Jun N-terminal kinase (JNK)-related apoptosis through protein-protein interactions. Moreover, signaling by PKA/cAMP response element binding protein (CREB) is necessary for GSTP up-regulation. This study explored whether carnosic acid (CA) from rosemary prevents 6-hydroxydopamine (6-OHDA)-induced neurotoxicity by inhibition of JNK through GSTP via PKA/CREB signaling. Results indicated that the GSTP protein was increased in SH-SY5Y cells treated with CA for 18 and 24 h. However, CA had no significant effect on alpha or mu class of GST. Treatment of CA increased the induction of p-PKAα, nuclear p-CREB, and CRE-DNA binding activity. These effects of CA were attenuated in cells pretreated with the PKA inhibitor H89. CA pretreatment suppressed 6-OHDA-induced apoptosis by inhibition of JNK phosphorylation, poly(ADP)-ribose polymerase cleavage, and nuclear condensation. Pretreatment with H89 and GSTP siRNA attenuated the ability of CA to reverse 6-OHDA-induced apoptosis. By use of immunoprecipitation with JNK antibody to examine the interaction of GSTP-JNK with CA, we showed that CA pretreatment increased the immunoprecipitation of GSTP after 6-OHDA treatment, which suggests that CA promoted the interaction between GSTP and JNK. CA prevents 6-OHDA-induced apoptosis via inhibition of JNK by GSTP through the PKA/CREB pathway. Copyright © 2017 Elsevier Ltd. All rights reserved.

GSTP1 Polymorphisms and their Association with Glutathione Transferase and Peroxidase Activities in Patients with Motor Neuron Disease.

PubMed

Gajewska, Beata; Kaźmierczak, Beata; Kuźma-Kozakiewicz, Magdalena; Jamrozik, Zygmunt; Barańczyk-Kuźma, Anna

2015-01-01

Glutathione S-transferase pi (GSTP1) is a crucial enzyme in detoxification of electrophilic compounds and organic peroxides. Together with Se-dependent glutathione peroxidase (Se-GSHPx) it protects cells against oxidative stress which may be a primary factor implicated in motor neuron disease (MND) pathogenesis. We investigated GSTP1 polymorphisms and their relationship with GST and Se-GSTPx activities in a cohort of Polish patients with MND. Results were correlated with clinical phenotypes. The frequency of genetic variants for GSTP1 exon 5 (I105V) and exon 6 (A114V) was studied in 104 patients and 100 healthy controls using real-time polymerase chain reaction. GST transferase activity was determined in serum with 1-chloro-2,4-dinitrobenzene, its peroxidase activity with cumene hydroperoxide, and Se-GSHPx activity with hydrogen peroxide. There were no differences in the prevalence of GSTP1 polymorphism I105V and A114V between MND and controls, however the occurrence of CT variant in codon 114 was associated with a higher risk for MND. GSTP1 polymorphisms were less frequent in classic ALS than in progressive bulbar palsy. In classic ALS C* (heterozygous I /V and A /V) all studied activities were significantly lower than in classic ALS A* (homozygous I /I and A/A). GST peroxidase activity and Se-GSHPx activity were lower in classic ALS C* than in control C*, but in classic ALS A* Se-GSHPx activity was significantly higher than in control A*. It can be concluded that the presence of GSTP1 A114V but not I105V variant increases the risk of MND, and combined GSTP1 polymorphisms in codon 105 and 114 may result in lower protection of MND patients against the toxicity of electrophilic compounds, organic and inorganic hydroperoxides.

Simulation of interindividual differences in inactivation of reactive para-benzoquinone imine metabolites of diclofenac by glutathione S-transferases in human liver cytosol.

PubMed

den Braver, Michiel W; Zhang, Yongjie; Venkataraman, Harini; Vermeulen, Nico P E; Commandeur, Jan N M

2016-07-25

Diclofenac is a widely prescribed NSAID that causes severe idiosyncratic drug induced liver injury (IDILI) in a small part of the patient population. Formation of protein-reactive metabolites is considered to play a role in the development of diclofenac-induced IDILI. Therefore, a high hepatic activity of enzymes involved in bioactivation of diclofenac is expected to increase the risk for liver injury. However, the extent of covalent protein binding may also be determined by activity of protective enzymes, such as glutathione S-transferases (GSTs). This is supported by an association study in which a correlation was found between NSAID-induced IDILI and the combined null genotypes of GSTM1 and GSTT1. In the present study, the activity of 10 different recombinant human GSTs in inactivation of protein-reactive quinoneimine (QI) metabolites of diclofenac was tested. Both at low and high GSH concentrations, high activities of GSTA1-1, A2-2, A3-3, M1-1, M3-3 and P1-1 in the inactivation of these QIs were found. By using the expression levels of GSTs in livers of 22 donors, a 6-fold variation in GST-dependent inactivation of reactive diclofenac metabolites was predicted. Moreover, it was shown in vitro that GSTs can strongly increase the efficiency of GSH to protect against the alkylation of the model thiol N-acetylcysteine by reactive diclofenac metabolites. The results of this study demonstrate that variability of GST expression may significantly contribute to the inter-individual differences in susceptibility to diclofenac-induced liver injury. In addition, expression levels of GSTs in in vitro models for hepatotoxicity may be important factors determining sensitivity to diclofenac cytotoxicity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Cholinesterase and glutathione S-transferase activities of three mollusc species from the NW Portuguese coast in relation to the 'Prestige' oil spill.

PubMed

Tim-Tim, Ana L S; Morgado, Fernando; Moreira, Susana; Rangel, Rui; Nogueira, António J A; Soares, Amadeu M V M; Guilhermino, Lúcia

2009-12-01

In November 2002, the tanker 'Prestige' released about 19,000 tonnes of a heavy fuel oil (no. 6) before sinking with about 58,000 tonnes of its cargo, 135 miles from Cabo Finisterra (Spain). A considerable part of the released fuel oil reached the Galician coast, causing a heavy black tide and an ecological disaster. Although the black tide did not reach the NW coast of Portugal, it is possible that some of the fuel oil or its components also arrived to this area directly through the sea water and/or indirectly through the food chain. Therefore, the aim of this study was to investigate possible changes in two widely used biomarkers, the activity of the enzymes cholinesterases (ChE) and glutathione S-transferases (GST), of three molluscs (Mytilus galloprovincialis, Nucella lapillus and Monodonta lineata) from wild populations of the NW Portuguese coast in relation to the 'Prestige' oil spill. Molluscs were collected seasonally before (autumn 2002) and after (winter 2002/2003), spring and summer 2003) the oil spill at several sites along the Portuguese NW coast. Enzymatic activities determined before the accident were compared with those determined at different times after the oil spill taking into consideration abiotic factors. Information from different parameters was integrated by Redundancy Analysis and Principal Response Curves (PRC). Results show that GST and ChE activities were influenced by abiotic factors. Despite this influence, the results of PRC analysis also suggest that some of the fuel oil reached the NW Portuguese coast changing the patterns of ChE and GST activities of local populations of rocky shore species. Furthermore, the present study highlights the need of long-term monitoring with wild populations to assess both historical and punctual effects of pollution in the marine environment.

Quantification and visualization of glutathione S-transferase omega 1 in cells using inductively coupled plasma mass spectrometry (ICP-MS) and fluorescence microscopy.

PubMed

Liang, Yong; Jiang, Xin; Tang, Nannan; Yang, Limin; Chen, Haifeng; Wang, Qiuquan

2015-03-01

We report a novel activity-based and Cu-free click chemistry (CC) mediated methodology for glutathione S-transferase omega 1 (GSTO1) quantification using species-unspecific isotope dilution inductively coupled plasma mass spectrometry (SUID ICP-MS), in which dibenzylcyclooctyne-modified 2-chloroacetamide (DBCO-ChAcA) was designed and synthesized, meanwhile, as a navigator towards GSTO1 for subsequent N3-DOTA-Eu-tagging via Cu-free CC. Using (153)Eu-SUID ICP-MS coupled with size exclusion chromatography (SEC), the LOD (3σ) of GSTO1 reached 6.9 fmol with an RSD of 2.4% at the 0.1 μM level (n = 5) considering the recovery of GSTO1 on the SEC was 96.5 ± 2.4%. The GSTO1 contents in the cells of human hepatocellular carcinoma C7721 and breast carcinoma MCF-7 as well as normal hepatic C7701 without or with cis-platin administration were quantified to be from 1.2 μg/10,000 cells (n = 3, RSD = 4.5%) corresponding to 1.2 × 10(-2) ng per cell to 4.76 μg/10,000 cells (n = 3, RSD = 2.9%) corresponding to 4.76 × 10(-2) ng per cell. For a comparative study, DBCO-ChAcA-fluor 488-based fluorescence microscopy could not alone visualize GSTO1 in the cells but could together with those from the small SH-containing molecules such as GSH and that from extra N3-fluor 488 in the cells. This activity-based CC-mediated tagging/labeling strategy provided an opportunity for ICP-MS-based targeted protein quantification, and is very much expected to find its applications in biological mechanism study and the subsequent drug design.

A Knowledge-based System for Intelligent Support in Pharmacogenomics Evidence Assessment: Ontology-driven Evidence Representation and Retrieval.

PubMed

Lee, Chia-Ju; Devine, Beth; Tarczy-Hornoch, Peter

2017-01-01

Pharmacogenomics holds promise as a critical component of precision medicine. Yet, the use of pharmacogenomics in routine clinical care is minimal, partly due to the lack of efficient and effective use of existing evidence. This paper describes the design, development, implementation and evaluation of a knowledge-based system that fulfills three critical features: a) providing clinically relevant evidence, b) applying an evidence-based approach, and c) using semantically computable formalism, to facilitate efficient evidence assessment to support timely decisions on adoption of pharmacogenomics in clinical care. To illustrate functionality, the system was piloted in the context of clopidogrel and warfarin pharmacogenomics. In contrast to existing pharmacogenomics knowledge bases, the developed system is the first to exploit the expressivity and reasoning power of logic-based representation formalism to enable unambiguous expression and automatic retrieval of pharmacogenomics evidence to support systematic review with meta-analysis.

Reverted glutathione S-transferase-like genes that influence flower color intensity of carnation (Dianthus caryophyllus L.) originated from excision of a transposable element

PubMed Central

Momose, Masaki; Itoh, Yoshio; Umemoto, Naoyuki; Nakayama, Masayoshi; Ozeki, Yoshihiro

2013-01-01

A glutathione S-transferase-like gene, DcGSTF2, is responsible for carnation (Dianthus caryophyllus L.) flower color intensity. Two defective genes, DcGSTF2mu with a nonsense mutation and DcGSTF2-dTac1 containing a transposable element dTac1, have been characterized in detail in this report. dTac1 is an active element that produces reverted functional genes by excision of the element. A pale-pink cultivar ‘Daisy’ carries both defective genes, whereas a spontaneous deep-colored mutant ‘Daisy-VPR’ lost the element from DcGSTF2-dTac1. This finding confirmed that dTac1 is active and that the resulting reverted gene, DcGSTF2rev1, missing the element is responsible for this color change. Crosses between the pale-colored cultivar ‘06-LA’ and a deep-colored cultivar ‘Spectrum’ produced segregating progeny. Only the deep-colored progeny had DcGSTF2rev2 derived from the ‘Spectrum’ parent, whereas progeny with pale-colored flowers had defective forms from both parents, DcGSTF2mu and DcGSTF2-dTac1. Thus, DcGSTF2rev2 had functional activity and likely originated from excision of dTac1 since there was a footprint sequence at the vacated site of the dTac1 insertion. Characterizing the DcGSTF2 genes in several cultivars revealed that the two functional genes, DcGSTF2rev1 and DcGSTF2rev2, have been used for some time in carnation breeding with the latter in use for more than half a century. PMID:24399917

Sphingobium sp. SYK-6 LigG involved in lignin degradation is structurally and biochemically related to the glutathione transferase ω class.

PubMed

Meux, Edgar; Prosper, Pascalita; Masai, Eiji; Mulliert, Guillermo; Dumarçay, Stéphane; Morel, Mélanie; Didierjean, Claude; Gelhaye, Eric; Favier, Frédérique

2012-11-16

SpLigG is one of the three glutathione transferases (GSTs) involved in the process of lignin breakdown in the soil bacterium Sphingobium sp. SYK-6. Sequence comparisons showed that SpLigG and several proteobacteria homologues form an independent cluster within cysteine-containing GSTs. The relationship between SpLigG and other GSTs was investigated. The X-ray structure and biochemical properties of SpLigG indicate that this enzyme belongs to the omega class of glutathione transferases. However, the hydrophilic substrate binding site of SpLigG, together with its known ability to stereoselectively deglutathionylate the physiological substrate α-glutathionyl-β-hydroxypropiovanillone, argues for broadening the definition of the omega class. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

From Augustine of Hippo’s Memory Systems to Our Modern Taxonomy in Cognitive Psychology and Neuroscience of Memory: A 16-Century Nap of Intuition before Light of Evidence

PubMed Central

Cassel, Jean-Christophe; Cassel, Daniel; Manning, Lilianne

2012-01-01

Over the last half century, neuropsychologists, cognitive psychologists and cognitive neuroscientists interested in human memory have accumulated evidence showing that there is not one general memory function but a variety of memory systems deserving distinct (but for an organism, complementary) functional entities. The first attempts to organize memory systems within a taxonomic construct are often traced back to the French philosopher Maine de Biran (1766–1824), who, in his book first published in 1803, distinguished mechanical memory, sensitive memory and representative memory, without, however, providing any experimental evidence in support of his view. It turns out, however, that what might be regarded as the first elaborated taxonomic proposal is 14 centuries older and is due to Augustine of Hippo (354–430), also named St Augustine, who, in Book 10 of his Confessions, by means of an introspective process that did not aim at organizing memory systems, nevertheless distinguished and commented on sensible memory, intellectual memory, memory of memories, memory of feelings and passion, and memory of forgetting. These memories were envisaged as different and complementary instances. In the current study, after a short biographical synopsis of St Augustine, we provide an outline of the philosopher’s contribution, both in terms of questions and answers, and focus on how this contribution almost perfectly fits with several viewpoints of modern psychology and neuroscience of memory about human memory functions, including the notion that episodic autobiographical memory stores events of our personal history in their what, where and when dimensions, and from there enables our mental time travel. It is not at all meant that St Augustine’s elaboration was the basis for the modern taxonomy, but just that the similarity is striking, and that the architecture of our current viewpoints about memory systems might have preexisted as an outstanding intuition in the

Cytokine Responses to the Anti-schistosome Vaccine Candidate Antigen Glutathione-S-transferase Vary with Host Age and Are Boosted by Praziquantel Treatment

PubMed Central

Bourke, Claire D.; Nausch, Norman; Rujeni, Nadine; Appleby, Laura J.; Trottein, François; Midzi, Nicholas; Mduluza, Takafira; Mutapi, Francisca

2014-01-01

Background Improved helminth control is required to alleviate the global burden of schistosomiasis and schistosome-associated pathologies. Current control efforts rely on the anti-helminthic drug praziquantel (PZQ), which enhances immune responses to crude schistosome antigens but does not prevent re-infection. An anti-schistosome vaccine based on Schistosoma haematobium glutathione-S-transferase (GST) is currently in Phase III clinical trials, but little is known about the immune responses directed against this antigen in humans naturally exposed to schistosomes or how these responses change following PZQ treatment. Methodology Blood samples from inhabitants of a Schistosoma haematobium-endemic area were incubated for 48 hours with or without GST before (n = 195) and six weeks after PZQ treatment (n = 107). Concentrations of cytokines associated with innate inflammatory (TNFα, IL-6, IL-8), type 1 (Th1; IFNγ, IL-2, IL-12p70), type 2 (IL-4, IL-5, IL-13), type 17 (IL-17A, IL-21, IL-23p19) and regulatory (IL-10) responses were quantified in culture supernatants via enzyme-linked immunosorbent assay (ELISA). Factor analysis and multidimensional scaling were used to analyse multiple cytokines simultaneously. Principal Findings A combination of GST-specific type 2 (IL-5 and IL-13) and regulatory (IL-10) cytokines was significantly lower in 10–12 year olds, the age group at which S. haematobium infection intensity and prevalence peak, than in 4–9 or 13+ year olds. Following PZQ treatment there was an increase in the number of participants producing detectable levels of GST-specific cytokines (TNFα, IL-6, IL-8, IFNγ, IL-12p70, IL-13 and IL-23p19) and also a shift in the GST-specific cytokine response towards a more pro-inflammatory phenotype than that observed before treatment. Participant age and pre-treatment infection status significantly influenced post-treatment cytokine profiles. Conclusions/Significance In areas where schistosomiasis is endemic host

Function and phylogeny of bacterial butyryl-CoA:acetate transferases and their diversity in the proximal colon of swine

USDA-ARS?s Scientific Manuscript database

Studying the host-associated butyrate-producing bacterial community is important because butyrate is essential for colonic homeostasis and gut health. Previous research has identified the butyryl-coA:acetate transferase (2.3.8.3) as a the main gene for butyrate production in intestinal ecosystems; h...

Systems vaccinology: Probing humanity’s diverse immune systems with vaccines

PubMed Central

Pulendran, Bali

2014-01-01

Homo sapiens are genetically diverse, but dramatic demographic and socioeconomic changes during the past century have created further diversification with respect to age, nutritional status, and the incidence of associated chronic inflammatory disorders and chronic infections. These shifting demographics pose new challenges for vaccination, as emerging evidence suggests that age, the metabolic state, and chronic infections can exert major influences on the immune system. Thus, a key public health challenge is learning how to reprogram suboptimal immune systems to induce effective vaccine immunity. Recent advances have applied systems biological analysis to define molecular signatures induced early after vaccination that correlate with and predict the later adaptive immune responses in humans. Such “systems vaccinology” approaches offer an integrated picture of the molecular networks driving vaccine immunity, and are beginning to yield novel insights about the immune system. Here we discuss the promise of systems vaccinology in probing humanity’s diverse immune systems, and in delineating the impact of genes, the environment, and the microbiome on protective immunity induced by vaccination. Such insights will be critical in reengineering suboptimal immune systems in immunocompromised populations. PMID:25136102

Impact of the Ile105Val Polymorphism of the Glutathione S-transferase P1 (GSTP1) Gene on Obesity and Markers of Cardiometabolic Risk in Young Adult Population.

PubMed

Chielle, E O; Trott, A; da Silva Rosa, B; Casarin, J N; Fortuna, P C; da Cruz, I B M; Moretto, M B; Moresco, R N

2017-05-01

The aim of the study was to investigate the association between Glutathione S-transferase P1 (GSTP1) gene polymorphism with obesity and markers of cardiometabolic risk. A cross-sectional study was carried out in individuals aged≥18 and ≤30 years. The study included 54 normal weight, 27 overweight and 68 obese volunteers. Anthropometric measurements and biochemical parameters were evaluated, the DNA was extracted from blood samples and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to measure GSTP1 Ile 105 Val gene polymorphism of the study participants. Also, biochemical analysis and hormone assays were carried out. A positive association between GSTP1 polymorphism and obesity was observed on subjects carrying at least one G allele (AG and GG). GG genotype was found only in the obese group. The G allele carriers presented 2.4 times higher chance of obesity when compared to those with the AA genotype. These results were independent of sex and age. We suggest that despite a study in population regional (south of Brazil), the GSTP1 gene polymorphism may play a significant role in the increase of susceptibility of obesity and contribute to identify the cardiovascular risk in young adults. © Georg Thieme Verlag KG Stuttgart · New York.

Development of the sigma-1 receptor in C-terminals of motoneurons and colocalization with the N,N'-dimethyltryptamine forming enzyme, indole-N-methyl transferase.

PubMed

Mavlyutov, T A; Epstein, M L; Liu, P; Verbny, Y I; Ziskind-Conhaim, L; Ruoho, A E

2012-03-29

The function of the sigma-1 receptor (S1R) has been linked to modulating the activities of ion channels and G-protein-coupled receptors (GPCR). In the CNS, the S1R is expressed ubiquitously but is enriched in mouse motoneurons (MN), where it is localized to subsurface cisternae of cholinergic postsynaptic densities, also known as C-terminals. We found that S1R is enriched in mouse spinal MN at late stages of embryonic development when it is first visualized in the endoplasmic reticulum. S1Rs appear to concentrate at C-terminals of mouse MN only on the second week of postnatal development. We found that indole-N-methyl transferase (INMT), an enzyme that converts tryptamine into the sigma-1 ligand dimethyltryptamine (DMT), is also localized to postsynaptic sites of C-terminals in close proximity to the S1R. This close association of INMT and S1Rs suggest that DMT is synthesized locally to effectively activate S1R in MN. Published by Elsevier Ltd.

O-linked-N-acetylglucosamine modification of mammalian Notch receptors by an atypical O-GlcNAc transferase Eogt1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakaidani, Yuta; Ichiyanagi, Naoki; Saito, Chika

2012-03-02

Highlights: Black-Right-Pointing-Pointer We characterized A130022J15Rik (Eogt1)-a mouse gene homologous to Drosophila Eogt. Black-Right-Pointing-Pointer Eogt1 encodes EGF domain O-GlcNAc transferase. Black-Right-Pointing-Pointer Expression of Eogt1 in Drosophila rescued the cell-adhesion defect in the Eogt mutant. Black-Right-Pointing-Pointer O-GlcNAcylation reaction in the secretory pathway is conserved through evolution. -- Abstract: O-linked-{beta}-N-acetylglucosamine (O-GlcNAc) modification is a unique cytoplasmic and nuclear protein modification that is common in nearly all eukaryotes, including filamentous fungi, plants, and animals. We had recently reported that epidermal growth factor (EGF) repeats of Notch and Dumpy are O-GlcNAcylated by an atypical O-GlcNAc transferase, EOGT, in Drosophila. However, no study has yet shownmore » whether O-GlcNAcylation of extracellular proteins is limited to insects such as Drosophila or whether it occurs in other organisms, including mammals. Here, we report the characterization of A130022J15Rik, a mouse gene homolog of Drosophila Eogt (Eogt 1). Enzymatic analysis revealed that Eogt1 has a substrate specificity similar to that of Drosophila EOGT, wherein the Thr residue located between the fifth and sixth conserved cysteines of the folded EGF-like domains is modified. This observation is supported by the fact that the expression of Eogt1 in Drosophila rescued the cell-adhesion defect caused by Eogt downregulation. In HEK293T cells, Eogt1 expression promoted modification of Notch1 EGF repeats by O-GlcNAc, which was further modified, at least in part, by galactose to generate a novel O-linked-N-acetyllactosamine structure. These results suggest that Eogt1 encodes EGF domain O-GlcNAc transferase and that O-GlcNAcylation reaction in the secretory pathway is a fundamental biochemical process conserved through evolution.« less

In vitro kinetics of hepatic glutathione s-transferase conjugation in largemouth bass and brown bullheads

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gallagher, E.P.; Sheehy, K.M.; Lame, M.W.

2000-02-01

The kinetics of glutathione 5-transferase (GST) catalysis were investigated in largemouth bass (Micropterus salmoides) and brown bullheads (Amerius nebulosus), two freshwater fish species found in a variety of polluted waterways in the eastern US. The initial rates of hepatic GST activity toward four GST substrates, including 1-chloro-2,4-dinitrobenzene, ethacrynic acid, {Delta}5-androstene-17-dione, and nitrobutyl chloride, were significantly higher in brown bullheads than in largemouth bass. Hepatic GST activity toward 1,2-dichloro-4-nitrobenzene, a {mu}-class GST substrate in rodents, was not detectable in either species. Liver cytosolic GSTs were more efficient in bullheads than in bass at catalyzing 1-chloro-2,4-dinitrobenzene-reduced glutathione (CDNB-GSH) conjugation over a broadmore » range of electrophile (CDNB) concentrations, including those representative of environmental exposure. In contrast, largemouth bass maintained higher ambient concentrations of GSH, the nucleophilic cofactor for GST-mediated conjugation, than brown bullheads. Biphasic kinetics for GST-CDNB conjugation under conditions of variable GSH concentration were apparent in Eadie-Hofstee plots of the kinetic data, suggesting the presence of at least two hepatic GST isozymes with markedly different K{sub m} values for GSH in both species. The GST-CDNB reaction rate data obtained under conditions of variable GSH were well fitted (R{sup 2} = 0.999) by the two-enzyme Michaelis-Menten equation. In addition, Western blotting experiments confirmed the presence of two different hepatic GST-like proteins in both largemouth bass and brown bullhead liver. Collectively, these findings indicate that largemouth bass and brown bullhead GSTs catalyze the conjugation of structurally diverse, class-specific GST substrates, and that brown bullheads exhibit higher initial rates of GST activity than largemouth bass. The relatively higher rates of in vitro liver GST activity at the low substrate

2,2'-Dihydroxychalcone, a glutathione transferase inhibitor, sensitises human colon adenocarcinoma cells to chlorambucil and melphalan, but not to actinomycin D.

PubMed

Goh, Kenneth; Chen, Yufan; Zheng, Lin; Ong, Laichun; Jin, Yi; Chow, Pierce; Zhang, Kai

2008-01-01

2,2'-Dihydroxychalcone (2,2'DHC) is a potent inhibitor of glutathione S-transferases (GSTs). Pre-treatment of human colon cancer cells by a non-toxic concentration of this GST inhibitor significantly sensitised cancer cells to chlorambucil and melphalan, which are substrates of glutathione (GSH) conjugation. However, sensitisation to actinomycin D, which has not been shown to be detoxified by GSH-related mechanisms, was not observed. These results further confirm the contribution of GSH-related mechanisms to drug resistance by increased detoxification of drugs. 2,2'DHC inhibited GST activity and the transport of GSH conjugates by cancer cells. Its combined effects on GST and glutathione conjugate export (GS-X) pump may provide more potent sensitisation of cancer cells to chemotherapeutic drugs.

Checkpoint kinase 1-induced phosphorylation of O-linked β-N-acetylglucosamine transferase regulates the intermediate filament network during cytokinesis.

PubMed

Li, Zhe; Li, Xueyan; Nai, Shanshan; Geng, Qizhi; Liao, Ji; Xu, Xingzhi; Li, Jing

2017-12-01

Checkpoint kinase 1 (Chk1) is a kinase instrumental for orchestrating DNA replication, DNA damage checkpoints, the spindle assembly checkpoint, and cytokinesis. Despite Chk1's pivotal role in multiple cellular processes, many of its substrates remain elusive. Here, we identified O- linked β- N -acetylglucosamine ( O -GlcNAc)-transferase (OGT) as one of Chk1's substrates. We found that Chk1 interacts with and phosphorylates OGT at Ser-20, which not only stabilizes OGT, but also is required for cytokinesis. Phospho-specific antibodies of OGT-pSer-20 exhibited specific signals at the midbody of the cell, consistent with midbody localization of OGT as reported previously. Moreover, phospho-deficient OGT (S20A) cells attenuated cellular O -GlcNAcylation levels and also reduced phosphorylation of Ser-71 in the cytoskeletal protein vimentin, a modification critical for severing vimentin filament during cytokinesis. Consequently, elongated vimentin bridges were observed in cells depleted of OGT via an si OGT- based approach. Lastly, expression of plasmids resistant to si OGT efficiently rescued the vimentin bridge phenotype, but the OGT-S20A rescue plasmids did not. Our results suggest a Chk1-OGT-vimentin pathway that regulates the intermediate filament network during cytokinesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Expression and significance of glucose transporter-1, P-glycoprotein, multidrug resistance-associated protein and glutathione S-transferase-π in laryngeal carcinoma.

PubMed

Mao, Zhong-Ping; Zhao, Li-Jun; Zhou, Shui-Hong; Liu, Meng-Qin; Tan, Wei-Feng; Yao, Hong-Tian

2015-02-01

Increasing glucose transporter-1 (GLUT-1) activity is one of the most important ways to increase the cellular influx of glucose. We previously demonstrated that increased GLUT-1 expression was an independent predictor of survival in patients with laryngeal carcinoma. Thus, GLUT-1 may present a novel therapeutic target in laryngeal carcinoma. In this study, the expression of GLUT-1, P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and glutathione S-transferase-π (GST-π) in laryngeal carcinomas was investigated by immunohistochemistry. Additionally, possible correlations between GLUT-1 and P-gp, MRP1 and GST-π and various clinicopathological parameters were analyzed. In this study, 52.9% (18/34), 58.8% (20/34), 20.6% (7/34) and 58.8% (20/34) of the laryngeal carcinomas were positive for GLUT-1, P-gp, MRP1 and GST-π, respectively. The expression of GLUT-1, P-gp, MRP1 and GST-π was higher in laryngeal carcinoma specimens when compared with laryngeal precancerous lesions (P<0.05). Pearson's correlation analysis showed correlations between GLUT-1 and P-gp (r=0.364; P=0.034), GLUT-1 and MRP1 (r=0.359; P=0.037) and P-gp and GST-π (r=0.426; P=0.012). GLUT-1 expression was found to significantly correlate with tumor-node-metastasis classification (P=0.02) and clinical stage (P=0.037). Furthermore, P-gp was found to significantly correlate with clinical stage (P=0.026). Univariate analysis showed that MRP1 expression was significantly associated with poor survival (c 2 =5.16; P=0.023). Multivariate analysis revealed that lymph node metastasis (P=0.009) and MRP1 overexpression (P=0.023) were significant predictors of poor survival. In the present study, the expression of GLUT-1, P-gp, MRP1 and GST-π in laryngeal carcinomas was investigated, as well as the correlations between these proteins. P-gp was found to significantly correlate with clinical stage, while MRP1 overexpression was significantly associated with poor survival.

Scaling up Evidence-based Practices for Children and Families in New York State: Towards Evidence-based Policies on Implementation for State Mental Health Systems

PubMed Central

Hoagwood, Kimberly Eaton; Olin, S. Serene; Horwitz, Sarah; McKay, Mary; Cleek, Andrew; Gleacher, Alissa; Lewandowski, Eric; Nadeem, Erum; Acri, Mary; Chor, Ka Ho Brian; Kuppinger, Anne; Burton, Geraldine; Weiss, Dara; Frank, Samantha; Finnerty, Molly; Bradbury, Donna M.; Woodlock, Kristin M.; Hogan, Michael

2014-01-01

Dissemination of innovations is widely considered the sine qua non for system improvement. At least two dozen states are rolling-out evidence-based mental health practices targeted at children and families using trainings, consultations, webinars, and learning collaboratives to improve quality and outcomes. In New York State (NYS) a group of researchers, policy-makers, providers and family support specialists have worked in partnership since 2002 to redesign and evaluate the children’s mental health system. Five system strategies driven by empirically-based practices and organized within a state-supported infrastructure have been used in the child and family service system with over 2,000 providers: (a) business practices; (b) use of health information technologies in quality improvement; (c) specific clinical interventions targeted at common childhood disorders; (d) parent activation; and (e) quality indicator development. The NYS system has provided a laboratory for naturalistic experiments. We describe these initiatives, key findings and challenges, lessons learned for scaling, and implications for creating evidence-based implementation policies in state systems. PMID:24460518

O-linked N-acetylglucosamine transferase enhances secretory clusterin expression via liver X receptors and sterol response element binding protein regulation in cervical cancer.

PubMed

Kim, Min Jun; Choi, Mee Young; Lee, Dong Hoon; Roh, Gu Seob; Kim, Hyun Joon; Kang, Sang Soo; Cho, Gyeong Jae; Kim, Yoon Sook; Choi, Wan Sung

2018-01-12

O-linked N-acetylglucosamine transferase (OGT) expression is increased in various cancer types, indicating the potential importance of O-GlcNAcylation in tumorigenesis. Secretory clusterin (sCLU) is involved in cancer cell proliferation and drug resistance, and recently, liver X receptors (LXRs) and sterol response element binding protein-1 (SREBP-1) were reported to regulate sCLU transcription. Here, we found that sCLU is significantly increased in cervical cancer cell lines, which have higher expression levels of O-GlcNAc and OGT than keratinocytes. OGT knockdown decreased expression of LXRs, SREBP-1 and sCLU through hypo-O-GlcNAcylation of LXRs. Additionally, treatment with Thiamet G, O-GlcNAcase OGA inhibitor, increased expression of O-GlcNAcylation and sCLU, and high glucose increased levels of LXRs, SREBP-1 and sCLU in HeLa cells. Moreover, OGT knockdown induced G 0 /G 1 phase cell cycle arrest and late apoptosis in cisplatin-treated HeLa cells, and decreased viability compared to OGT intact HeLa cells. Taken together, these findings suggest that OGT, O-GlcNAcylated LXRs, and SREBP-1 increase sCLU expression in cervical cancer cells, which contributes to drug resistance.

Comprehensive genome-wide analysis of Glutathione S-transferase gene family in potato (Solanum tuberosum L.) and their expression profiling in various anatomical tissues and perturbation conditions.

PubMed

Islam, Md Shiful; Choudhury, Mouraj; Majlish, Al-Nahian Khan; Islam, Tahmina; Ghosh, Ajit

2018-01-10

Glutathione S-transferases (GSTs) are ubiquitous enzymes which play versatile functions including cellular detoxification and stress tolerance. In this study, a comprehensive genome-wide identification of GST gene family was carried out in potato (Solanum tuberosum L.). The result demonstrated the presence of at least 90 GST genes in potato which is greater than any other reported species. According to the phylogenetic analyses of Arabidopsis, rice and potato GST members, GSTs could be subdivided into ten different classes and each class is found to be highly conserved. The largest class of potato GST family is tau with 66 members, followed by phi and lambda. The chromosomal localization analysis revealed the highly uneven distribution of StGST genes across the potato genome. Transcript profiling of 55 StGST genes showed the tissue-specific expression for most of the members. Moreover, expression of StGST genes were mainly repressed in response to abiotic stresses, while largely induced in response to biotic and hormonal elicitations. Further analysis of StGST gene's promoter identified the presence of various stress responsive cis-regulatory elements. Moreover, one of the highly stress responsive StGST members, StGSTU46, showed strong affinity towards flurazole with lowest binding energy of -7.6kcal/mol that could be used as antidote to protect crop against herbicides. These findings will facilitate the further functional and evolutionary characterization of GST genes in potato. Copyright © 2017 Elsevier B.V. All rights reserved.

Maternal Belief Systems: The Discourse of Cultural Practice as Evidence

ERIC Educational Resources Information Center

Mastergeorge, Ann M.

2007-01-01

Fully developed definitions of evidence-based practice incorporate evidence from family perspectives, as well as evidence from research on the effectiveness of particular interventions. Systems for appraising research evidence typically place qualitative analyses at lower levels. The argument in this article is that qualitative data offer a…

Indigofera suffruticosa Mill extracts up-regulate the expression of the π class of glutathione S-transferase and NAD(P)H: quinone oxidoreductase 1 in rat Clone 9 liver cells.

PubMed

Chen, Chun-Chieh; Liu, Chin-San; Li, Chien-Chun; Tsai, Chia-Wen; Yao, Hsien-Tsung; Liu, Te-Chung; Chen, Haw-Wen; Chen, Pei-Yin; Wu, Yu-Ling; Lii, Chong-Kuei; Liu, Kai-Li

2013-09-01

Because induction of phase II detoxification enzyme is important for chemoprevention, we study the effects of Indigofera suffruticosa Mill, a medicinal herb, on the expression of π class of glutathione S-transferase (GSTP) and NAD(P)H: quinone oxidoreductase 1 (NQO1) in rat Clone 9 liver cells. Both water and ethanolic extracts of I. suffruticosa significantly increased the expression and enzyme activities of GSTP and NQO1. I. suffruticosa extracts up-regulated GSTP promoter activity and the binding affinity of nuclear factor erythroid 2-related factor 2 (Nrf2) with the GSTP enhancer I oligonucleotide. Moreover, I. suffruticosa extracts increased nuclear Nrf2 accumulation as well as ARE transcriptional activity. The level of phospho-ERK was augmented by I. suffruticosa extracts, and the ERK inhibitor PD98059 abolished the I. suffruticosa extract-induced ERK activation and GSTP and NQO-1 expression. Moreover, I. suffruticosa extracts, especially the ethanolic extract increased the glutathione level in mouse liver and red blood cells as well as Clone 9 liver cells. The efficacy of I. suffruticosa extracts in induction of phase II detoxification enzymes and glutathione content implies that I. suffruticosa could be considered as a potential chemopreventive agent. Copyright © 2013 Elsevier Ltd. All rights reserved.

Modulation of ethoxyresorufin O-deethylase and glutathione S-transferase activities in Nile tilapia (Oreochromis niloticus) by polycyclic aromatic hydrocarbons containing two to four rings: implications in biomonitoring aquatic pollution.

PubMed

Pathiratne, Asoka; Hemachandra, Chamini K

2010-08-01

Despite ubiquity of polycyclic aromatic hydrocarbons (PAHs) in the tropical environments, little information is available concerning responses of tropical fish to PAHs and associated toxicity. In the present study, effects of five PAHs containing two to four aromatic rings on hepatic CYP1A dependent ethoxyresorufin O-deethylase (EROD), glutathione S-transferase (GST) and serum sorbitol dehydrogenase (SDH) activities in Nile tilapia, a potential fish species for biomonitoring pollution in tropical waters, were evaluated. Results showed that EROD activities were induced by the PAHs containing four aromatic rings (pyrene and chrysene) in a dose dependent manner. However PAHs with two to three aromatic rings (naphthalene, phenanthrene and fluoranthene) caused no effect or inhibition of EROD activities depending on the dose and the duration. Fluoranthene was the most potent inhibitor. SDH results demonstrated that high doses of fluoranthene induced hepatic damage. GST activity was induced by the lowest dose of phenanthrene, fluoranthene and chrysene but high doses had no effect. The results indicate that induction of EROD enzyme in Nile tilapia is a useful biomarker of exposure to PAHs such as pyrene and chrysene. However EROD inhibiting PAHs such as fluoranthene in the natural environment may modulate the EROD inducing potential of other PAHs thereby influencing PAH exposure assessments.

Activation of Nrf2 is required for up-regulation of the π class of glutathione S-transferase in rat primary hepatocytes with L-methionine starvation.

PubMed

Lin, Ai-Hsuan; Chen, Haw-Wen; Liu, Cheng-Tze; Tsai, Chia-Wen; Lii, Chong-Kuei

2012-07-04

Numerous genes expression is regulated in response to amino acid shortage, which helps organisms adapt to amino acid limitation. The expression of the π class of glutathione (GSH) S-transferase (GSTP), a highly inducible phase II detoxification enzyme, is regulated mainly by activates activating protein 1 (AP-1) binding to the enhancer I of GSTP (GPEI). Here we show the critical role of nuclear factor erythroid-2-related factor 2 (Nrf2) in up-regulating GSTP gene transcription. Primary rat hepatocytes were cultured in a methionine-restricted medium, and immunoblotting and RT-PCR analyses showed that methionine restriction time-dependently increased GSTP protein and mRNA expression over a 48 h period. Nrf2 translocation to the nucleus, nuclear proteins binding to GPEI, and antioxidant response element (ARE) luciferase reporter activity were increased by methionine restriction as well as by l-buthionine sulfoximine (BSO), a GSH synthesis inhibitor. Transfection with Nrf2 siRNA knocked down Nrf2 expression and reversed the methionine-induced GSTP expression and GPEI binding activity. Chromatin immunoprecipitation assay confirmed the binding of Nrf2 to the GPEI. Phosphorylation of extracellular signal-regulated kinase 2 (ERK2) was increased in methionine-restricted and BSO-treated cells. ERK2 siRNA abolished methionine restriction-induced Nrf2 nuclear translocation, GPEI binding activity, ARE-luciferase reporter activity, and GSTP expression. Our results suggest that the up-regulation of GSTP gene transcription in response to methionine restriction likely occurs via the ERK-Nrf2-GPEI signaling pathway.

Sulforaphane and alpha-lipoic acid upregulate the expression of the pi class of glutathione S-transferase through c-jun and Nrf2 activation.

PubMed

Lii, Chong-Kuei; Liu, Kai-Li; Cheng, Yi-Ping; Lin, Ai-Hsuan; Chen, Haw-Wen; Tsai, Chia-Wen

2010-05-01

The anticarcinogenic effect of dietary organosulfur compounds has been partly attributed to their modulation of the activity and expression of phase II detoxification enzymes. Our previous studies indicated that garlic allyl sulfides upregulate the expression of the pi class of glutathione S-transferase (GSTP) through the activator protein-1 pathway. Here, we examined the modulatory effect of sulforaphane (SFN) and alpha-lipoic acid (LA) or dihydrolipoic acid (DHLA) on GSTP expression in rat Clone 9 liver cells. Cells were treated with LA or DHLA (50-600 micromol/L) or SFN (0.2-5 micromol/L) for 24 h. Immunoblots and real-time PCR showed that SFN, LA, and DHLA dose dependently induced GSTP protein and mRNA expression. Compared with the induction by the garlic organosulfur compound diallyl trisulfide (DATS), the effectiveness was in the order of SFN > DATS > LA = DHLA. The increase in GSTP enzyme activity in cells treated with 5 micromol/L SFN, 50 micromol/L DATS, and 600 micromol/L LA and DHLA was 172, 75, 122, and 117%, respectively (P < 0.05). A reporter assay showed that the GSTP enhancer I (GPEI) was required for GSTP induction by the organosulfur compounds. Electromobility gel shift assays showed that the DNA binding of GPEI to nuclear proteins reached a maximum at 0.5-1 h after SFN, LA, and DHLA treatment. Super-shift assay revealed that the transcription factors c-jun and nuclear factor erythroid-2 related factor 2 (Nrf2) were bound to GPEI. These results suggest that SFN and LA in either its oxidized or reduced form upregulate the transcription of the GSTP gene by activating c-jun and Nrf2 binding to the enhancer element GPEI.

2-Phenethyl Isothiocyanate, Glutathione S-transferase M1 and T1 Polymorphisms, and Detoxification of Volatile Organic Carcinogens and Toxicants in Tobacco Smoke.

PubMed

Yuan, Jian-Min; Murphy, Sharon E; Stepanov, Irina; Wang, Renwei; Carmella, Steven G; Nelson, Heather H; Hatsukami, Dorothy; Hecht, Stephen S

2016-07-01

Cigarette smoke contains relatively large quantities of volatile organic toxicants or carcinogens such as benzene, acrolein, and crotonaldehyde. Among their detoxification products are mercapturic acids formed from glutathione conjugation, catalyzed in part by glutathione S-transferases (GST). A randomized phase II clinical trial with a crossover design was conducted to evaluate the effect of 2-phenethyl isothiocyanate (PEITC), a natural product formed from gluconasturtiin in certain cruciferous vegetables, on the detoxification of benzene, acrolein, and crotonaldehyde in 82 cigarette smokers. Urinary mercapturic acids of benzene, acrolein, and crotonaldehyde at baseline and during treatment were quantified. Overall, oral PEITC supplementation increased the mercapturic acid formed from benzene by 24.6% (P = 0.002) and acrolein by 15.1% (P = 0.005), but had no effect on crotonaldehyde. A remarkably stronger effect was observed among subjects with the null genotype of both GSTM1 and GSTT1: in these individuals, PEITC increased the detoxification metabolite of benzene by 95.4% (P < 0.001), of acrolein by 32.7% (P = 0.034), and of crotonaldehyde by 29.8% (P = 0.006). In contrast, PEITC had no effect on these mercapturic acids in smokers possessing both genes. PEITC had no effect on the urinary oxidative stress biomarker 8-iso-prostaglandin F2α or the inflammation biomarker prostaglandin E2 metabolite. This trial demonstrates an important role of PEITC in detoxification of environmental carcinogens and toxicants which also occur in cigarette smoke. The selective effect of PEITC on detoxification in subjects lacking both GSTM1 and GSTT1 genes supports the epidemiologic findings of stronger protection by dietary isothiocyanates against the development of lung cancer in such individuals. Cancer Prev Res; 9(7); 598-606. ©2016 AACR. ©2016 American Association for Cancer Research.

Elevated Levels of Urinary 8-Hydroxy-2′-deoxyguanosine, Lymphocytic Micronuclei, and Serum Glutathione S-Transferase in Workers Exposed to Coke Oven Emissions

PubMed Central

Liu, Ai-Lin; Lu, Wen-Qing; Wang, Zeng-Zhen; Chen, Wei-Hong; Lu, Wen-Hong; Yuan, Jing; Nan, Pei-Hong; Sun, Jian-Ya; Zou, Ya-Lin; Zhou, Li-Hong; Zhang, Chi; Wu, Tang-Chun

2006-01-01

To investigate associations among occupational exposure to coke oven emissions (COEs), oxidative stress, cytogenotoxic effects, change in the metabolizing enzyme glutathione S-transferase (GST), and internal levels of polycyclic aromatic hydrocarbons (PAHs) in coke oven workers, we recruited 47 male coke oven workers and 31 male control subjects from a coke oven plant in northern China. We measured the levels of 1-hydroxypyrene (1-OHP) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in urine, micronucleated binucleated cells (BNMNs) in peripheral blood lymphocyte, and GST in serum. Our results showed that the group exposed to COEs had significantly increased levels of 1-OHP [median 5.7; interquartile range (IQR), 1.4–12.0 μmol/mol creatinine] compared with the control group (3; 0.5–6.4 μmol/mol creatinine). In addition, the median levels (IQR) of 8-OHdG, BNMNs, and GST were markedly increased in the exposed [1.9 (1.4–15.4) μmol/mol creatinine; 6 (2–8) per thousand; 22.1 (14.9–31.2) U/L, respectively] compared with controls [1.3 (1.0–4.0) μmol/mol creatinine, 2 (0–4) per thousand; and 13.1 (9.5–16.7) U/L, respectively]. These results appeared to be modified by smoking. However, multivariate logistic regression analysis revealed that exposure to COEs had the highest odds ratio among variables analyzed and that smoking was not a significant confounder of the levels of studied biomarkers. Overall, the present findings suggest that COE exposure led to increased internal PAH burden, genetic damage, oxidative stress, and GST activity. The consequences of the changes in these biomarkers, such as risk of cancer, warrant further investigations. PMID:16675419

Germline glutathione S-transferase variants in breast cancer: Relation to diagnosis and cutaneous long-term adverse effects after two fractionation patterns of radiotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Edvardsen, Hege; Kristensen, Vessela N.; Medical Faculty, University of Oslo, Oslo

Purpose: To explore whether certain glutathione S-transferase (GST) polymorphisms are associated with an increased risk of breast cancer or the level of radiation-induced adverse effects after two fractionation patterns of adjuvant radiotherapy. Methods and Materials: The prevalence of germline polymorphic variants in GSTM1, GSTP1, and GSTT1 was determined in 272 breast cancer patients and compared with that in a control group of 270 women from the general population with no known history of breast cancer. The genetic variants were determined using multiplex polymerase chain reaction followed by restriction enzyme fragment analysis. In 253 of the patients surveyed for radiotherapy-induced sidemore » effects after a median observation time of 13.7 years (range, 7-22.8 years), the genotypes were related to the long-term effects observed after two fractionation patterns (treatment A, 4.3 Gy in 10 fractions for 156 patients; and treatment B, 2.5 Gy in 20 fractions for 97; both administered within a 5-week period). Results: None of the GST polymorphisms conferred an increased risk of breast cancer, either alone or in combination. Compared with treatment B, treatment A was followed by an increased level of moderate to severe radiation-induced side effects for all the endpoints studied (i.e., degree of telangiectasia, subcutaneous fibrosis and atrophy, lung fibrosis, costal fractures, and pleural thickening; p <0.001 for all endpoints). A significant association was found between the level of pleural thickening and the GSTP1 Ile105Val variant. Conclusion: The results of this study have illustrated the impact of hypofractionation on the level of adverse effects and indicated that the specific alleles of GSTP1, M1, and T1 studied here may be significant in determining the level of adverse effects after radiotherapy.« less

Testing the Effects of SIAH Ubiquitin E3 Ligases on Lysine Acetyl Transferases.

PubMed

Hagenbucher, Jan; Stekman, Hilda; Rodriguez-Gil, Alfonso; Kracht, Michael; Schmitz, M Lienhard

2017-01-01

The family of seven-in-absentia (SIAH) ubiquitin E3 ligases functions in the control of numerous key signaling pathways. These enzymes belong to the RING (really interesting new gene) group of E3 ligases and mediate the attachment of ubiquitin chains to substrates, which then leads to their proteasomal degradation. Here, we describe a protocol that allows measuring SIAH-mediated ubiquitination and degradation of its client proteins as exemplified by acetyl transferases using simple overexpression experiments. The impact of SIAH expression on the relative amounts of target proteins and their mRNAs can be quantified by Western blotting and quantitative PCR (qPCR) as described here.

Parkinson’s disease as a system-level disorder

PubMed Central

Caligiore, Daniele; Helmich, Rick C; Hallett, Mark; Moustafa, Ahmed A; Timmermann, Lars; Toni, Ivan; Baldassarre, Gianluca

2016-01-01

Traditionally, the basal ganglia have been considered the main brain region implicated in Parkinson’s disease. This single area perspective gives a restricted clinical picture and limits therapeutic approaches because it ignores the influence of altered interactions between the basal ganglia and other cerebral components on Parkinsonian symptoms. In particular, the basal ganglia work closely in concert with cortex and cerebellum to support motor and cognitive functions. This article proposes a theoretical framework for understanding Parkinson’s disease as caused by the dysfunction of the entire basal ganglia–cortex–cerebellum system rather than by the basal ganglia in isolation. In particular, building on recent evidence, we propose that the three key symptoms of tremor, freezing, and impairments in action sequencing may be explained by considering partially overlapping neural circuits including basal ganglia, cortical and cerebellar areas. Studying the involvement of this system in Parkinson’s disease is a crucial step for devising innovative therapeutic approaches targeting it rather than only the basal ganglia. Possible future therapies based on this different view of the disease are discussed. PMID:28725705