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Sample records for hypoxanthine-guanine phosphoribosyl transferase

  1. Molecular and clonal analysis of in vivo hprt (hypoxanthine-guanine phosphoribosyl-transferase) mutations in human cells

    SciTech Connect

    Albertini, R.J.; O'Neill, J.P.; Nicklas, J.A.; Allegretta, M. . Genetics Lab.); Recio, L.; Skopek, T.R. )

    1989-08-08

    There is no longer doubt that gene mutations occur in vivo in human somatic cells, and that methods can be developed to detect, quantify and study them. Four assays are now available for such purpose; two detecting mutations that arise in bone marrow erythroid stem cells and two defining mutations that occur in T-lymphocytes. The red cell assays measure changes in mature red blood cells that involve either the blood group glycophorin-A locus or the hemoglobin loci; the lymphocyte assays score for genetic events at either the X-chromosomal hypoxanthine-guanine phosphoribosyl-transferase (hprt) locus. We describe here our attempts in studying in vivo gene mutations in human T-lymphocytes. 35 refs., 3 figs., 3 tabs.

  2. Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines.

    PubMed

    López-Cruz, Roberto I; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal, Jaime A; Real-Valle, Roberto A; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

    2016-01-01

    Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements. PMID:27375492

  3. Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines

    PubMed Central

    López-Cruz, Roberto I.; Crocker, Daniel E.; Gaxiola-Robles, Ramón; Bernal, Jaime A.; Real-Valle, Roberto A.; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

    2016-01-01

    Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements. PMID:27375492

  4. Radioprotector WR1065 reduces radiation-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in V79 cells

    SciTech Connect

    Grdina, D.J.; Hill, C.K.; Peraino, C. ); Biserka, N. ); Wells, R.L. . Dept. of Radiology and Radiation Biology)

    1985-06-01

    N-(2-mercaptoethyl)-1,3-diaminopropane (WR1065) protects against radiation-induced cell killing and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 Chinese hamster lung fibroblast cells. WR1065 (4 mm) was found to be effective in protecting against radiation-induced cell lethality only if present during irradiation. No protective effect was observed if the protector was added within 5 min after irradiation or 3 h later. The effect of WR1065 on radiation-induced mutation, expressed as resistance to the cytotoxic purine analogue 6-thioguanine (HGPRT), was also investigated. This agent was effective in reducing radiation-induced mutations regardless of when it was administered. Following 10 Gy of /sup 60/Co ..gamma..-rays, the mutation frequencies observed per 10/sup 6/ survivors were 77 +- 8, 27 +- 6, 42 +- 7, and 42 +- 7 for radiation only, and WR1065 present during, immediately after, or 3 h after irradiation. These data suggest that although a segment of radiation-induced damage leading to reproductive death cannot be modulated through the postirradiation action of WR1065, processes leading to the fixation of gross genetic damage and mutation induction in surviving cells can be effectively altered and interfered with leading to a marked reduction in mutation frequency.

  5. Quantifaction of mutagens at the Na/sup +/-K/sup +/-ATPase and hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene loci in Chinese hamster ovary cells

    SciTech Connect

    Li, A.P.

    1982-01-01

    The Chinese hamster ovary (CHO) cell/hypoxanthine guanine phosphoribosyl transferase (HGPRT) mutagen assay developed by Hsie et al., was simplified by culturing the cells as unattached cultures, and also modified to include mutation at the Na/sup +/-K/sup +/ ATPase (ouabain resistance) gene locus. The cost and time involved were decreased by culturing the CHO cells unattached on nontissue culture plates during the expression period. The inclusion of a second gene locus ensures that mutagenicities observed were not due to the peculiar properties of a specific gene locus. These procedures are now used in our laboratory for routine testing of environmental chemicals and complex mixtures.

  6. Assay of SF/sub 6/ and spark-decomposed SF/sub 6/ for mutagenic activity in the CHO/HGPRT (Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase) mammalian cell system

    SciTech Connect

    Kurka, K.; Griffin, G.D.

    1987-01-01

    The potential mutagenic (and cytotoxic) activity of SF/sub 6/ and spark-decomposed SF/sub 6/ was investigated in an in vitro mammalian cell culture system using Chinese Hamster Ovary (CHO) cells. The CHO cells were exposed to the gases in vacutainer tubes which were constantly rotated. After a 4 h exposure the mutagenic and cytotoxic activity was assayed with the CHO/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system. Results indicated that SF/sub 6/ was neither cytotoxic nor mutagenic to CHO cells. Spark-decomposed SF/sub 6/ was found to be strongly cytotoxic (-80% cell death following 4 h exposure to 2 kJ spark discharge in 60 cm/sup 3/ at 1000 torr of SF/sub 6/) but not mutagenic. Increasing spark energy increased cytotoxicity but the spark samples remained nonmutagenic. The CHO/HGPRT system was coupled with a metabolic activation (S9 fraction) system used for detecting promutagens. When exposures were carried out in the presence of S9 fraction, SF/sub 6/ was still neither cytotoxic nor mutagenic; spark-decomposed SF/sub 6/ was again strongly cytotoxic but not mutagenic. It appears that SF/sub 6/ and sparked SF/sub 6/ are neither promutagens nor direct acting mutagens in the CHO/HGPRT system. Studies have begun using a more mutagenically sensitive subclone of the CHO cells known as CHO-AS/sub 52/. The results of initial experiments using the CHO-AS/sub 52/ cells remain unchanged. 9 refs., 1 tab.

  7. Determination of Activity of the Enzymes Hypoxanthine Phosphoribosyl Transferase (HPRT) and Adenine Phosphoribosyl Transferase (APRT) in Blood Spots on Filter Paper.

    PubMed

    Auler, Kasie; Broock, Robyn; Nyhan, William L

    2015-01-01

    Hypoxanthine-guanine phosphoribosyl-transferase (HPRT) deficiency is the cause of Lesch-Nyhan disease. Adenine phosphoribosyl-transferase (APRT) deficiency causes renal calculi. The activity of each enzyme is readily determined on spots of whole blood on filter paper. This unit describes a method for detecting deficiencies of HPRT and APRT. PMID:26132002

  8. Hydrophilic-interaction liquid chromatography-tandem mass spectrometric determination of erythrocyte 5-phosphoribosyl 1-pyrophosphate in patients with hypoxanthine-guanine phosphoribosyltransferase deficiency.

    PubMed

    Hasegawa, Hiroshi; Shinohara, Yoshihiko; Nozaki, Sayako; Nakamura, Makiko; Oh, Koei; Namiki, Osamu; Suzuki, Kiyotaka; Nakahara, Akihiko; Miyazawa, Mari; Ishikawa, Ken; Himeno, Takahiro; Yoshida, Sayaka; Ueda, Takanori; Yamada, Yasukazu; Ichida, Kimiyoshi

    2015-01-22

    Mutations in the gene encoding hypoxanthine-guanine phosphoribosyltransferase (HPRT) cause Lesch-Nyhan disease (LND) and its variants (LNV). Due to the technical problems for measuring the HPRT activity in vitro, discordances between the residual HPRT activity and the clinical severity were found. 5-Phosphoribosyl 1-pyrophosphate (PRPP) is a substrate for HPRT. Since increased PRPP concentrations were observed in erythrocytes from patients with LND and LNV, we have turned our attention to erythrocyte PRPP as a biomarker for the phenotype classification. In the present work, a method for determination of PRPP concentration in erythrocyte was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM). Packed erythrocyte samples were deproteinized by heating and the supernatants were injected into the LC-MS/MS system. All measurement results showed good precision with RSD <6%. PRPP concentrations of nine normal male subjects, four male patents with LND and six male patients with LNV were compared. The PRPP concentrations in erythrocyte from patients with LND were markedly increased compared with those from normal subjects, and those from patients with LNV were also increased but the degree was smaller than those with LND. The increase pattern of PRPP concentration in erythrocyte from patients with HPRT deficiency was consistent with the respective phenotypes and was correlated with the disease severity. PRPP concentration was suggested to give us supportive information for the diagnosis and the phenotype classification of LND and LNV. PMID:25482009

  9. INDUCTION OF MUTATIONS BY CHEMICAL AGENTS AT THE HYPOXANTHINE-GUANINE PHOSPHORIBOSYL TRANSFERASE LOCUS IN HUMAN EPITHELIAL TERATOMA CELLS

    EPA Science Inventory

    Induction of 6-thioguanine (TG) resistance by chemical mutagens was examined in a line of cells derived from a human epithelial teratocarcinoma cell clone. The cells, designated as P3 cells, have a stable diploid karyotype with 46(XX) chromosomes, including a translocation betwee...

  10. Kinetic mechanism of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase.

    PubMed

    Roy, Sourav; Nagappa, Lakshmeesha K; Prahladarao, Vasudeva S; Balaram, Hemalatha

    2015-12-01

    Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT) exhibits a kinetic mechanism that differs from that of the human homolog. Human HGPRT follows a steady-state ordered mechanism, wherein PRPP binding precedes the binding of hypoxanthine/guanine and release of product IMP/GMP is the rate limiting step. In the current study, initial velocity kinetics with PfHGXPRT indicates a steady-state ordered mechanism, wherein xanthine binding is conditional to the binding of PRPP. The value of the rate constant for IMP dissociation is greater by 183-fold than the kcat for hypoxanthine phosphoribosylation and this results in the absence of burst in progress curves from pre-steady-state kinetics. Further, IMP binding is 1000 times faster (4s(-1) at 0.5μM IMP) when compared to the kcat (3.9±0.2×10(-3)s(-1)) for the reverse IMP pyrophosphorolysis reaction. These results lend support to the fact that in both forward and reverse reactions, the process of chemical conversion (formation of IMP/hypoxanthine) is slow and the events of ligand association and dissociation are faster. PMID:26902413

  11. The use of primary rat hepatocytes to achieve metabolic activation of promutagens in the Chinese hamster ovary/hypoxantine-guanine phosphoribosyl transferase mutational assay

    SciTech Connect

    Bermudez, E.; Couch, D.B.; Tillery, D.

    1982-01-01

    A method is described in which primary rat hepatocytes have been cocultured with chinese hamster ovary (CHO) cells to provide metabolic activation of promutgens in the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) mutational assay. Single cell hepatocyte suspensions were prepared from male Fisher-344 rats using the in situ collagenase perfusion technique. Hepatocytes were allowed to attach for 1.5 hours in tissue culture dishes containing an approximately equal number of CHO cells in log growth. The cocultures were exposed to promutagens for up to 20 hours in serum-free medium. The survival and 6-thioguanine-resistant fraction of treated CHO cells were then determined as in the standard CHO/HGPRT assay. Aflatoxin B/sub 1/ (AFB/sub 1/) 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (B(a)P) were found to produce increases in the mutant fractions of treated CHO cells as a function of concentration. The time required for optimum expression of the mutant phenotype following exposure to DMBA and AFB/sub 1/ was approximately 8 days. Primary cell-mediated mutagenesis may be useful in elucidating methobolic pathways important in the production and detoxification of genotoxic products in vivo.

  12. Crystal structures of Apo and GMP bound hypoxanthine-guanine phosphoribosyltransferase from Legionella pneumophila and the implications in gouty arthritis.

    PubMed

    Zhang, Nannan; Gong, Xiaojian; Lu, Min; Chen, Xiaofang; Qin, Ximing; Ge, Honghua

    2016-06-01

    Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) reversibly catalyzes the transfer of the 5-phophoribosyl group from 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) to hypoxanthine or guanine to form inosine monophosphate (IMP) or guanosine monophosphate (GMP) in the purine salvage pathway. To investigate the catalytic mechanism of this enzyme in the intracellular pathogen Legionella pneumophila, we determined the crystal structures of the L. pneumophila HGPRT (LpHGPRT) both in its apo-form and in complex with GMP. The structures reveal that LpHGPRT comprises a core domain and a hood domain which are packed together to create a cavity for GMP-binding and the enzymatic catalysis. The binding of GMP induces conformational changes of the stable loop II. This new binding site is closely related to the Gout arthritis-linked human HGPRT mutation site (Ser103Arg). Finally, these structures of LpHGPRT provide insights into the catalytic mechanism of HGPRT. PMID:26968365

  13. Differential Distortion of Purine Substrates by Human and Plasmodium falciparum Hypoxanthine-Guanine Phosphoribosyltransferase to Catalyse the Formation of Mononucleotides.

    PubMed

    Karnawat, Vishakha; Gogia, Spriha; Balaram, Hemalatha; Puranik, Mrinalini

    2015-07-20

    Plasmodium falciparum (Pf) hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a potential therapeutic target. Compared to structurally homologous human enzymes, it has expanded substrate specificity. In this study, 9-deazapurines are used as in situ probes of the active sites of human and Pf HGPRTs. Through the use of these probes it is found that non-covalent interactions stabilise the pre-transition state of the HGPRT-catalysed reaction. Vibrational spectra reveal that the bound substrates are extensively distorted, the carbonyl bond of nucleobase moiety is weakened and the substrate is destabilised along the reaction coordinate. Raman shifts of the human and Pf enzymes are used to quantify the differing degrees of hydrogen bonding in the homologues. A decreased Raman cross-section in enzyme-bound 9-deazaguanine (9DAG) shows that the phenylalanine residue (Phe186 in human and Phe197 in Pf) of HGPRT stacks with the nucleobase. Differential loss of the Raman cross-section suggests that the active site is more compact in human HGPRT as compared to the Pf enzyme, and is more so in the phosphoribosyl pyrophosphate (PRPP) complex 9DAG-PRPP-HGPRT than in 9-deazahypoxanthine (9DAH)-PRPP-HGPRT. PMID:25944719

  14. Absence of hypoxanthine:guanine phosphoribosyltransferase activity in murine Dunn osteosarcoma

    SciTech Connect

    Abelson, H.T.; Gorka, C.

    1983-09-01

    The transplantable murine Dunn osteosarcoma has no detectable hypoxanthine:guanine phosphoribosyltransferase (EC 2.4.2.8) activity. This was established from the tumors directly and from tissue culture cell lines derived from the tumor using a variety of assays: e.g., no (3H)hypoxanthine uptake into tumor or tissue culture cells, no conversion of (3H)hypoxanthine to (3H)IMP by cell extracts from tumors or tissue culture cells, no growth of tissue culture cells in hypoxanthine:aminopterin:thymidine medium, and normal growth of these cells in 10 microM 6-mercaptopurine. Ten human osteosarcomas have been assayed, and two have no apparent hypoxanthine:guanine phosphoribosyltransferase enzyme activity. After high-dose methotrexate treatment in vivo, murine tumors could be selectively killed and normal tissues could be spared by using a rescue regimen of hypoxanthine-thymidine-allopurinol.

  15. Effect of salicylates on histamine and L-histidine metabolism. Inhibition of imidazoleacetate phosphoribosyl transferase.

    PubMed Central

    Moss, J; De Mello, M C; Vaughan, M; Beaven, M A

    1976-01-01

    In man and other animals, urinary excretion of the histidine and histamine metabolite, imidazoleacetate, is increased and that of its conjugated metabolite, ribosylimidazoleacetate, decreased by salicylates. Imidazoleacetate has been reported to produce analgesia and narcosis. Its accumulation as a result of transferase inhibition could play a part in the therapeutic effects of salicylates. To determine the locus of salicylate action, we have investigated the effect of anti-inflammatory drugs on imidazoleacetate phosphoribosyl transferase, the enzyme that catalyzes the ATP-dependent conjugation of imidazoleacetate with phosphoribosylpyrophosphate. As little as 0.2 mM aspirin produced 50% inhibition of the rat liver transferase. In vivo, a 30% decrease in the urinary excretion of ribosylimidazoleacetate has been observed with plasma salicylate concentrations of 0.4 mM. The enzyme was also inhibited by sodium salicylate but not by salicylamide, sodium gentisate, aminopyrine, phenacetin, phenylbutazone, or indomethacin. The last four drugs have been shown previously not to alter the excretion of ribosylimidazoleacetate when administered in vivo. Since both the drug specificity and inhibitory concentrations are similar in vivo and in vitro, it seems probable that the effect of salicylates on imidazoleacetate conjugation results from inhibition of imidazoleacetate phosphoribosyl transferase. PMID:180057

  16. Acyclic phosph(on)ate inhibitors of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase

    PubMed Central

    Clinch, Keith; Crump, Douglas R.; Evans, Gary B.; Hazleton, Keith Z.; Mason, Jennifer M.; Schramm, Vern L.

    2013-01-01

    The pathogenic protozoa responsible for malaria lack enzymes for the de novo synthesis of purines and rely on purine salvage from the host. In Plasmodium falciparum (Pf), hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) converts hypoxanthine to inosine monophosphate and is essential for purine salvage making the enzyme an anti-malarial drug target. We have synthesized a number of simple acyclic aza-C- nucleosides and shown that some are potent inhibitors of Pf HGXPRT while showing excellent selectivity for the Pf versus the human enzyme. PMID:23810424

  17. Herpes simplex virus-mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells

    SciTech Connect

    Palella, T.D.; Silverman, L.J.; Schroll, C.T.; Homa, F.L.; Levine, M.; Kelley, W.N.

    1988-01-01

    The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolation of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.

  18. Acyclic Immucillin Phosphonates. Second-Generation Inhibitors of Plasmodium falciparum Hypoxanthine- Guanine-Xanthine Phosphoribosyltransferase

    SciTech Connect

    Hazelton, Keith Z.; Ho, Meng-Chaio; Cassera, Maria B.; Clinch, Keith; Crump, Douglas R.; Rosario Jr., Irving; Merino, Emilio F.; Almo, Steve C.; Tyler, Peter C.; Schramm, Vern L.

    2012-06-22

    We found that Plasmodium falciparum is the primary cause of deaths from malaria. It is a purine auxotroph and relies on hypoxanthine salvage from the host purine pool. Purine starvation as an antimalarial target has been validated by inhibition of purine nucleoside phosphorylase. Hypoxanthine depletion kills Plasmodium falciparum in cell culture and in Aotus monkey infections. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) from P. falciparum is required for hypoxanthine salvage by forming inosine 5'-monophosphate, a branchpoint for all purine nucleotide synthesis in the parasite. We present a class of HGXPRT inhibitors, the acyclic immucillin phosphonates (AIPs), and cell permeable AIP prodrugs. The AIPs are simple, potent, selective, and biologically stable inhibitors. The AIP prodrugs block proliferation of cultured parasites by inhibiting the incorporation of hypoxanthine into the parasite nucleotide pool and validates HGXPRT as a target in malaria.

  19. Hypoxanthine-Guanine Phosphoribosyltransferase Deficiency: Activity in Normal, Mutant, and Heterozygote-Cultured Human Skin Fibroblasts

    PubMed Central

    Fujimoto, Wilfred Y.; Seegmiller, J. Edwin

    1970-01-01

    Cultured skin fibroblasts from patients deficient for the enzyme hypoxanthine-guanine phosphoribosyltransferase (PRT) activity show very low but nevertheless significant levels of apparent PRT enzyme despite absence of detectable activity (<0.004% of normal) in erythrocytes of the same patients. In fibroblasts this mutant enzyme is more heat labile than the normal enzyme. These findings indicate that PRT deficiency in this disorder is not due to a deletion mutation of the PRT locus. Individual cultured skin fibroblasts from heterozygote females for PRT deficiency show normal, intermediate, or very low levels of PRT activity. The mosaicism demonstrated in the heterozygotes for this X-linked disorder accounts for the cells with normal and very low activities of PRT. Intermediate activity can best be explained by the phenomenon of metabolic cooperation presumably from the transfer of either PRT enzyme or messenger RNA, from normal to mutant cells. Images PMID:5267139

  20. Comprehensive X-Ray Structural Studies of the Quinolinate Phosphoribosyl Transferase (BNA6) From Saccharomyces Cerevisiae

    SciTech Connect

    di Luccio, E.; Wilson, D.K.

    2009-05-14

    Quinolinic acid phosphoribosyl transferase (QAPRTase, EC 2.4.2.19) is a 32 kDa enzyme encoded by the BNA6 gene in yeast and catalyzes the formation of nicotinate mononucleotide from quinolinate and 5-phosphoribosyl-1-pyrophosphate (PRPP). QAPRTase plays a key role in the tryptophan degradation pathway via kynurenine, leading to the de novo biosynthesis of NAD{sup +} and clearing the neurotoxin quinolinate. To improve our understanding of the specificity of the eukaryotic enzyme and the course of events associated with catalysis, we have determined the crystal structures of the apo and singly bound forms with the substrates quinolinate and PRPP. This reveals that the enzyme folds in a manner similar to that of various prokaryotic forms which are {approx}30% identical in sequence. In addition, the structure of the Michaelis complex is approximated by PRPP and the quinolinate analogue phthalate bound to the active site. These results allow insight into the kinetic mechanism of QAPRTase and provide an understanding of structural diversity in the active site of the Saccharomyces cerevisiae enzyme when compared to prokaryotic homologues.

  1. Discovery of a novel nicotinamide phosphoribosyl transferase (NAMPT) inhibitor via in silico screening.

    PubMed

    Takeuchi, Mikio; Niimi, Tatsuya; Masumoto, Mari; Orita, Masaya; Yokota, Hiroyuki; Yamamoto, Tomoko

    2014-01-01

    Nicotinamide phosphoribosyl transferase (NAMPT) is a key enzyme in the salvage pathway of mammalian nicotinamide adenine dinucleotide (NAD) biosynthesis, catalyzing the synthesis of nicotinamide mononucleotide from nicotinamide (Nam). The diverse functions of NAD suggest that NAMPT inhibitors are potential drug candidates as anticancer agents, immunomodulators, or other agents. However, difficulty in conducting high-throughput NAMPT assay with good sensitivity has hampered the discovery of novel anti-NAMPT drugs with improved profiles. We combined an in silico screening strategy with a radioisotope (RI)-based enzyme assay and rationally identified promising NAMPT inhibitors with novel structures. AS1604498 was the most potent inhibitor, with an IC50 of 44 nM, and inhibited THP-1 and K562 cell line growth with the IC50 of 198 nM and 673 nM, respectively. The mode of action was found to reduce intracellular NAD following apoptosis, suggesting that these compounds inhibit NAMPT in cell-based assay. This strategy can be used to discover new drug candidates with targets which are difficult to assess through high-throughput screening. Our hit compounds may be used as seed compounds for developing new therapeutics with NAMPT. PMID:24389478

  2. AMP-activated protein kinase regulates nicotinamide phosphoribosyl transferase expression in skeletal muscle

    PubMed Central

    Brandauer, Josef; Vienberg, Sara G; Andersen, Marianne A; Ringholm, Stine; Risis, Steve; Larsen, Per S; Kristensen, Jonas M; Frøsig, Christian; Leick, Lotte; Fentz, Joachim; Jørgensen, Sebastian; Kiens, Bente; Wojtaszewski, Jørgen F P; Richter, Erik A; Zierath, Juleen R; Goodyear, Laurie J; Pilegaard, Henriette; Treebak, Jonas T

    2013-01-01

    Deacetylases such as sirtuins (SIRTs) convert NAD to nicotinamide (NAM). Nicotinamide phosphoribosyl transferase (Nampt) is the rate-limiting enzyme in the NAD salvage pathway responsible for converting NAM to NAD to maintain cellular redox state. Activation of AMP-activated protein kinase (AMPK) increases SIRT activity by elevating NAD levels. As NAM directly inhibits SIRTs, increased Nampt activation or expression could be a metabolic stress response. Evidence suggests that AMPK regulates Nampt mRNA content, but whether repeated AMPK activation is necessary for increasing Nampt protein levels is unknown. To this end, we assessed whether exercise training- or 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (AICAR)-mediated increases in skeletal muscle Nampt abundance are AMPK dependent. One-legged knee-extensor exercise training in humans increased Nampt protein by 16% (P < 0.05) in the trained, but not the untrained leg. Moreover, increases in Nampt mRNA following acute exercise or AICAR treatment (P < 0.05 for both) were maintained in mouse skeletal muscle lacking a functional AMPK α2 subunit. Nampt protein was reduced in skeletal muscle of sedentary AMPK α2 kinase dead (KD), but 6.5 weeks of endurance exercise training increased skeletal muscle Nampt protein to a similar extent in both wild-type (WT) (24%) and AMPK α2 KD (18%) mice. In contrast, 4 weeks of daily AICAR treatment increased Nampt protein in skeletal muscle in WT mice (27%), but this effect did not occur in AMPK α2 KD mice. In conclusion, functional α2-containing AMPK heterotrimers are required for elevation of skeletal muscle Nampt protein, but not mRNA induction. These findings suggest AMPK plays a post-translational role in the regulation of skeletal muscle Nampt protein abundance, and further indicate that the regulation of cellular energy charge and nutrient sensing is mechanistically related. PMID:23918774

  3. Mouse model for somatic mutation at the HPRT (hypoxanthine phosphoribosyl-transferase) gene: Molecular and cellular analyses

    SciTech Connect

    Burkhart-Schultz, K.; Strout, C.L.; Jones, I.M.

    1989-07-11

    Our goal is to use the mouse to model the organismal, cellular and molecular factors that affect somatic mutagenesis in vivo. A fundamental tenet of genetic toxicology is that the principles of mutagenesis identified in one system can be used to predict the principles of mutagenesis in another system. The validity of this tenet depends upon the comparability of the systems involved. To begin to achieve an understanding of somatic mutagenesis in vivo, we have been studying mutations that occur in the hypoxanthine phosphoribosyl-transferase (HPRT) gene of lymphocytes of mice. Our in vivo model for somatic mutation allows us to analyse factors that affect somatic mutation. Having chosen the mouse, we are working with cells in which the karyotype is normal, and metabolic and DNA repair capacity are defined by the mouse strain chosen. At the organismal level, we can vary sex, age, the exposure history, and the tissue source of cells analysed. (All studies reported here have, however, used male mice.) At the cellular level, T lymphocytes and their precursors are the targets and reporters of mutation. 26 refs., 1 fig., 1 tab.

  4. Orotate phosphoribosyl transferase MoPyr5 is involved in uridine 5'-phosphate synthesis and pathogenesis of Magnaporthe oryzae.

    PubMed

    Qi, Zhongqiang; Liu, Muxing; Dong, Yanhan; Yang, Jie; Zhang, Haifeng; Zheng, Xiaobo; Zhang, Zhengguang

    2016-04-01

    Orotate phosphoribosyl transferase (OPRTase) plays an important role in de novo and salvage pathways of nucleotide synthesis and is widely used as a screening marker in genetic transformation. However, the function of OPRTase in plant pathogens remains unclear. In this study, we characterized an ortholog of Saccharomyces cerevisiae Ura5, the OPRTase MoPyr5, from the rice blast fungus Magnaporthe oryzae. Targeted gene disruption revealed that MoPyr5 is required for mycelial growth, appressorial turgor pressure and penetration into plant tissues, invasive hyphal growth, and pathogenicity. Interestingly, the ∆Mopyr5 mutant is also involved in mycelial surface hydrophobicity. Exogenous uridine 5'-phosphate (UMP) restored vegetative growth and rescued the defect in pathogenicity on detached barley and rice leaf sheath. Collectively, our results show that MoPyr5 is an OPRTase for UMP biosynthesis in M. oryzae and indicate that UTP biosynthesis is closely linked with vegetative growth, cell wall integrity, and pathogenicity of fungus. Our results also suggest that UMP biosynthesis would be a good target for the development of novel fungicides against M. oryzae. PMID:26810198

  5. The Housekeeping Gene Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) Regulates Multiple Developmental and Metabolic Pathways of Murine Embryonic Stem Cell Neuronal Differentiation

    PubMed Central

    Bader, Joel S.; Friedmann, Theodore

    2013-01-01

    The mechanisms by which mutations of the purinergic housekeeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT) cause the severe neurodevelopmental Lesch Nyhan Disease (LND) are poorly understood. The best recognized neural consequences of HPRT deficiency are defective basal ganglia expression of the neurotransmitter dopamine (DA) and aberrant DA neuronal function. We have reported that HPRT deficiency leads to dysregulated expression of multiple DA-related developmental functions and cellular signaling defects in a variety of HPRT-deficient cells, including human induced pluripotent stem (iPS) cells. We now describe results of gene expression studies during neuronal differentiation of HPRT-deficient murine ESD3 embryonic stem cells and report that HPRT knockdown causes a marked switch from neuronal to glial gene expression and dysregulates expression of Sox2 and its regulator, genes vital for stem cell pluripotency and for the neuronal/glial cell fate decision. In addition, HPRT deficiency dysregulates many cellular functions controlling cell cycle and proliferation mechanisms, RNA metabolism, DNA replication and repair, replication stress, lysosome function, membrane trafficking, signaling pathway for platelet activation (SPPA) multiple neurotransmission systems and sphingolipid, sulfur and glycan metabolism. We propose that the neural aberrations of HPRT deficiency result from combinatorial effects of these multi-system metabolic errors. Since some of these aberrations are also found in forms of Alzheimer's and Huntington's disease, we predict that some of these systems defects play similar neuropathogenic roles in diverse neurodevelopmental and neurodegenerative diseases in common and may therefore provide new experimental opportunities for clarifying pathogenesis and for devising new potential therapeutic targets in developmental and genetic disease. PMID:24130677

  6. Fine structure mapping of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene region of the human X chromosome (Xq26).

    PubMed Central

    Nicklas, J A; Hunter, T C; O'Neill, J P; Albertini, R J

    1991-01-01

    The Xq26-q27 region of the X chromosome is interesting, as an unusually large number of genes and anonymous RFLP probes have been mapped in this area. A number of studies have used classical linkage analysis in families to map this region. Here, we use mutant human T-lymphocyte clones known to be deleted for all or part of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene, to order anonymous probes known to map to Xq26. Fifty-seven T-cell clones were studied, including 44 derived from in vivo mutation and 13 from in vitro irradiated T-lymphocyte cultures. Twenty anonymous probes (DXS10, DXS11, DXS19, DXS37, DXS42, DXS51, DXS53, DXS59, DXS79, DXS86, DXS92, DXS99, DXS100d, DXS102, DXS107, DXS144, DXS172, DXS174, DXS177, and DNF1) were tested for codeletion with the hprt gene by Southern blotting methods. Five of these probes (DXS10, DXS53, DXS79, DXS86 and DXS177) showed codeletion with hprt in some mutants. The mutants established the following unambiguous ordering of the probes relative to the hprt gene: DXS53-DXS79-5'hprt3'-DXS86-DXS10-DXS177 . The centromere appears to map proximal to DXS53. These mappings order several closely linked but previously unordered probes. In addition, these studies indicate that rather large deletions of the functionally haploid X chromosome can occur while still retaining T-cell viability. Images Figure 1 PMID:1678246

  7. Genetics Home Reference: Lesch-Nyhan syndrome

    MedlinePlus

    ... HA. Hypoxanthine-guanine phosphoribosyl transferase regulates early developmental programming of dopamine neurons: implications for Lesch-Nyhan disease pathogenesis. Hum Mol Genet. 2009 Jul 1;18(13):2317-27. doi: 10.1093/hmg/ ...

  8. Allopurinol enhances the activity of hypoxanthine-guanine phosphoribosyltransferase in inflammatory bowel disease patients during low-dose thiopurine therapy: preliminary data of an ongoing series.

    PubMed

    Seinen, Margien L; de Boer, Nanne K H; Smid, Kees; van Asseldonk, Dirk P; Bouma, Gerd; van Bodegraven, Adriaan A; Peters, Godefridus J

    2011-12-01

    Thiopurines are crucial in the treatment of inflammatory bowel disease. The phenotype of pivotal metabolic enzymes determines whether thioguanine nucleotides (6-TGN) are generated in clinically sufficiently high levels. The first step in activation of thiopurine prodrugs to 6-TGN is catalysis by hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Often, patients exhibit a clinically unfavorable metabolism, leading to discontinuation of conventional thiopurine therapy. The combination of allopurinol and low-dose thiopurine therapy may optimize this variant metabolism, presumably by affecting enzyme activities. We performed a prospective pharmacodynamic study to determine the effect of combination therapy on the activity of HGPRT. The activity of HGPRT and 6-TGN concentrations was measured in red blood cells during thiopurine monotherapy and after 4 weeks of combination therapy. The activity of HGPRT was also measured after 12 weeks of combination therapy. From the results, we conclude that combination therapy increases the activity of HGPRT and subsequently 6-TGN concentrations. PMID:22132961

  9. Effect of glutathione S-transferase M1 polymorphisms on biomarkers of exposure and effects.

    PubMed Central

    Srám, R J

    1998-01-01

    Genotypes responsible for interindividual differences in ability to activate or detoxify genotoxic agents are recognized as biomarkers of susceptibility. Among the most studied genotypes are human glutathione transferases. The relationship of genetic susceptibility to biomarkers of exposure and effects was studied especially in relation to the genetic polymorphism of glutathione S-transferase M1 (GSTM1). For this review papers reporting the effect of GSTM1 genotype on DNA adducts, protein adducts, urine mutagenicity, Comet assay parameters, chromosomal aberrations, sister chromatid exchanges (SCE), micronuclei, and hypoxanthine-guanine phosphoribosyl transferase mutations were assessed. Subjects in groups occupationally exposed to polycyclic aromatic hydrocarbons, benzidine, pesticides, and 1,3-butadiene were included. As environmentally exposed populations, autopsy donors, coal tar-treated patients, smokers, nonsmokers, mothers, postal workers, and firefighters were followed. From all biomarkers the effect of GSTM1 and N-acetyl transferase 2 was seen in coke oven workers on mutagenicity of urine and of glutathione S-transferase T1 on the chromosomal aberrations in subjects from 1,3-butadiene monomer production units. Effects of genotypes on DNA adducts were found from lung tissue of autopsy donors and from placentas of mothers living in an air-polluted region. The GSTM1 genotype affected mutagenicity of urine in smokers and subjects from polluted regions, protein adducts in smokers, SCE in smokers and nonsmokers, and Comet assay parameters in postal workers. A review of all studies on GSTM1 polymorphisms suggests that research probably has not reached the stage where results can be interpreted to formulate preventive measures. The relationship between genotypes and biomarkers of exposure and effects may provide an important guide to the risk assessment of human exposure to mutagens and carcinogens. PMID:9539016

  10. The role of the C-terminal region on the oligomeric state and enzymatic activity of Trypanosoma cruzi hypoxanthine phosphoribosyl transferase.

    PubMed

    Valsecchi, Wanda M; Cousido-Siah, Alexandra; Defelipe, Lucas A; Mitschler, André; Podjarny, Alberto; Santos, Javier; Delfino, José M

    2016-06-01

    Hypoxanthine phosphoribosyl transferase from Trypanosoma cruzi (TcHPRT) is a critical enzyme for the survival of the parasite. This work demonstrates that the full-length form in solution adopts a stable and enzymatically active tetrameric form, exhibiting large inter-subunit surfaces. Although this protein irreversibly aggregates during unfolding, oligomerization is reversible and can be modulated by low concentrations of urea. When the C-terminal region, which is predicted as a disordered stretch, is excised by proteolysis, TcHPRT adopts a dimeric state, suggesting that the C-terminal region acts as a main guide for the quaternary arrangement. These results are in agreement with X-ray crystallographic data presented in this work. On the other hand, the C-terminal region exhibits a modulatory role on the enzyme, as attested by the enhanced activity observed for the dimeric form. Bisphosphonates act as substrate-mimetics, uncovering long-range communications among the active sites. All in all, this work contributes to establish new ways applicable to the design of novel inhibitors that could eventually result in new drugs against parasitic diseases. PMID:26969784

  11. A survey of splice variants of the human hypoxanthine phosphoribosyl transferase and DNA polymerase beta genes: products of alternative or aberrant splicing?

    PubMed Central

    Skandalis, Adonis; Uribe, Elke

    2004-01-01

    Errors during the pre-mRNA splicing of metazoan genes can degrade the transmission of genetic information, and have been associated with a variety of human diseases. In order to characterize the mutagenic and pathogenic potential of mis-splicing, we have surveyed and quantified the aberrant splice variants in the human hypoxanthine phosphoribosyl transferase (HPRT) and DNA polymerase β (POLB) in the presence and the absence of the Nonsense Mediated Decay (NMD) pathway, which removes transcripts with premature termination codons. POLB exhibits a high frequency of splice variants (40–60%), whereas the frequency of HPRT splice variants is considerably lower (∼1%). Treatment of cells with emetine to inactivate NMD alters both the spectrum and frequency of splice variants of POLB and HPRT. It is not certain at this point, whether POLB and HPRT splice variants are the result of regulated alternative splicing processes or the result of aberrant splicing, but it appears likely that at least some of the variants are the result of splicing errors. Several mechanisms that may contribute to aberrant splicing are discussed. PMID:15601998

  12. Amplification of Adenine Phosphoribosyltransferase Suppresses the Conditionally Lethal Growth and Virulence Phenotype of Leishmania donovani Mutants Lacking Both Hypoxanthine-guanine and Xanthine Phosphoribosyltransferases*

    PubMed Central

    Boitz, Jan M.; Ullman, Buddy

    2010-01-01

    Leishmania donovani cannot synthesize purines de novo and obligatorily scavenge purines from the host. Previously, we described a conditional lethal Δhgprt/Δxprt mutant of L. donovani (Boitz, J. M., and Ullman, B. (2006) J. Biol. Chem. 281, 16084–16089) that establishes that L. donovani salvages purines primarily through hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine phosphoribosyltransferase (XPRT). Unlike wild type L. donovani, the Δhgprt/Δxprt knock-out cannot grow on 6-oxypurines and displays an absolute requirement for adenine or adenosine and 2′-deoxycoformycin, an inhibitor of parasite adenine aminohydrolase activity. Here, we demonstrate that the ability of Δhgprt/Δxprt parasites to infect mice was profoundly compromised. Surprisingly, mutant parasites that survived the initial passage through mice partially regained their virulence properties, exhibiting a >10-fold increase in parasite burden in a subsequent mouse infection. To dissect the mechanism by which Δhgprt/Δxprt parasites persisted in vivo, suppressor strains that had regained their capacity to grow under restrictive conditions were cloned from cultured Δhgprt/Δxprt parasites. The ability of these suppressor clones to grow in and metabolize 6-oxypurines could be ascribed to a marked amplification and overexpression of the adenine phosphoribosyltransferase (APRT) gene. Moreover, transfection of Δhgprt/Δxprt cells with an APRT episome recapitulated the suppressor phenotype in vitro and enabled growth on 6-oxypurines. Biochemical studies further showed that hypoxanthine, unexpectedly, was an inefficient substrate for APRT, evidence that could account for the ability of the suppressors to metabolize hypoxanthine. Subsequent analysis implied that APRT amplification was also a potential contributory mechanism by which Δhgprt/Δxprt parasites displayed persistence and increased virulence in mice. PMID:20363738

  13. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5'-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris), semi-aquatic (Lontra longicaudis annectens) and terrestrial (Sus scrofa).

    PubMed

    Barjau Pérez-Milicua, Myrna; Zenteno-Savín, Tania; Crocker, Daniel E; Gallo-Reynoso, Juan P

    2015-01-01

    Aquatic and semiaquatic mammals have the capacity of breath hold (apnea) diving. Northern elephant seals (Mirounga angustirostris) have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens) can hold their breath for about 30 s. Such periods of apnea may result in reduced oxygen concentration (hypoxia) and reduced blood supply (ischemia) to tissues. Production of adenosine 5'-triphosphate (ATP) requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa), are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal) (n = 11), semiaquatic (neotropical river otter) (n = 4), and terrestrial (domestic pig) (n = 11). Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) was determined by spectrophotometry, and activity of inosine 5'-monophosphate dehydrogenase (IMPDH) and the concentration of hypoxanthine (HX), inosine 5'-monophosphate (IMP), adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), ATP, guanosine 5'-diphosphate (GDP), guanosine 5'-triphosphate (GTP), and xanthosine 5'-monophosphate (XMP) were determined by high-performance liquid chromatography (HPLC). The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP, and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise), aquatic, and semiaquatic mammals have less purine mobilization than their terrestrial counterparts. PMID:26283971

  14. Temporal order of replication of genes responsible for hypoxanthine phosphoribosyl transferase and Na/sup +//K/sup +/ ATPase in chemically transformed human fibroblasts

    SciTech Connect

    Tsutsui, T.; Suzuki, N.; Elmore, E.; Maizumi, H.

    1986-06-01

    The cytotoxic and mutagenic effects of a direct perturbation of DNA during various portions of the DNA synthetic period (S phase) of a chemically induced, transformed line (Hut-11A cells) derived from diploid human skin fibroblasts were examined. The cells were synchronized by a period of growth in low serum with a subsequent blockage of the cells at the G1/S boundary by hydroxyurea. This method resulted in over 90% synchrony, although approximately 20% of the cells were noncycling. Synchronized cells were treated for each of four 2-h periods during the S phase with 5-bromodeoxyuridine (BrdU) followed by irradiation with near-ultraviolet (UV). The BrdU-plus-irradiation treatment was cytotoxic and mutagenic, while treatment with BrdU alone or irradiation alone was neither cytotoxic nor mutagenic. The cytotoxicity was dependent upon the periods of S phase during which treatment was administered. The highest lethality was observed for treatment in early to middle S phase, particularly in the first 2 h of S phase, whereas scare lethality was observed in late S phase. The BrdU-plus-irradiation treatment induced ouabain- and 6-thioguanine-resistant mutants, while BrdU alone or irradiation alone was not mutagenic. Ouabain-resistant mutants were induced during early S phase by the BrdU-plus-irradiation treatment. 6-Thioguanine-resistant mutants, however, were induced during middle to late S phase. These results suggest that a certain region or regions in the DNA of Hut-11A cells, as designated by their specific temporal relationship in the S phase, may be more sensitive to the DNA perturbation by BrdU treatment plus near-UV irradiation for cell survival and that gene(s) responsible for Na/sup +//K/sup +/ ATPase is replicated during early S phase and gene(s) for hypoxanthine phosphoribosyl transferase is replicated during middle to late S phase.

  15. Method for protection against genotoxic mutagenesis

    DOEpatents

    Grdina, David J.

    1996-01-01

    A method and pharmaceutical for protecting against genotoxic damage in irradiated cells. Reduction of mutations at the hypoxanthine-guanine phosphoribosyl transferase locus is accomplished by administering an effective dose of a compound having protected sulfhydryl groups which metabolize in vivo to produce both free sulfhydryl groups and disulfides.

  16. Method for protection against genotoxic mutagenesis

    DOEpatents

    Grdina, D.J.

    1996-01-30

    A method and pharmaceutical for protecting against genotoxic damage in irradiated cells are disclosed. Reduction of mutations at the hypoxanthine-guanine phosphoribosyl transferase locus is accomplished by administering an effective dose of a compound having protected sulfhydryl groups which metabolize in vivo to produce both free sulfhydryl groups and disulfides. 10 figs.

  17. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5′-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris), semi-aquatic (Lontra longicaudis annectens) and terrestrial (Sus scrofa)

    PubMed Central

    Barjau Pérez-Milicua, Myrna; Zenteno-Savín, Tania; Crocker, Daniel E.; Gallo-Reynoso, Juan P.

    2015-01-01

    Aquatic and semiaquatic mammals have the capacity of breath hold (apnea) diving. Northern elephant seals (Mirounga angustirostris) have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens) can hold their breath for about 30 s. Such periods of apnea may result in reduced oxygen concentration (hypoxia) and reduced blood supply (ischemia) to tissues. Production of adenosine 5′-triphosphate (ATP) requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa), are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal) (n = 11), semiaquatic (neotropical river otter) (n = 4), and terrestrial (domestic pig) (n = 11). Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) was determined by spectrophotometry, and activity of inosine 5′-monophosphate dehydrogenase (IMPDH) and the concentration of hypoxanthine (HX), inosine 5′-monophosphate (IMP), adenosine 5′-monophosphate (AMP), adenosine 5′-diphosphate (ADP), ATP, guanosine 5′-diphosphate (GDP), guanosine 5′-triphosphate (GTP), and xanthosine 5′-monophosphate (XMP) were determined by high-performance liquid chromatography (HPLC). The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP, and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise), aquatic, and semiaquatic mammals have less purine mobilization than their terrestrial counterparts. PMID:26283971

  18. Morin, a dietary bioflavonol suppresses monosodium urate crystal-induced inflammation in an animal model of acute gouty arthritis with reference to NLRP3 inflammasome, hypo-xanthine phospho-ribosyl transferase, and inflammatory mediators.

    PubMed

    Dhanasekar, Chitra; Rasool, Mahaboobkhan

    2016-09-01

    The anti-inflammatory effect of morin, a dietary bioflavanol was explored on monosodium urate (MSU) crystal-induced inflammation in rats, an experimental model for acute gouty arthritis. Morin treatment (30mg/kg b.wt) significantly attenuated the ankle swelling and the levels of lipid peroxidation, nitric oxide, serum pro-inflammatory cytokines (tumor necrosis factor (TNF) -α, interleukin (IL)-1β, and IL-6), monocyte chemoattractant protein (MCP)-1, vascular endothelial growth factor (VEGF), prostaglandin E2 (PGE2), and articular elastase along with an increased anti-oxidant status (catalase (CAT) and superoxide dismutase (SOD)) in the joint homogenate of MSU crystal-induced rats. Histological assessment revealed that morin limited the diffusion of joint space, synovial hyperplasia, and inflammatory cell infiltrations. The mRNA expression of NLRP3 (nucleotide oligomerization domain (NOD)-like receptor family, pyrin domain containing 3) inflammasome, caspase-1, pro-inflammatory cytokines, MCP-1, inflammatory enzymes (inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2)), and nuclear factor-kappa B (NF-κB) p65 was found downregulated and HPRT (hypo-xanthine phospho-ribosyl transferase) mRNA expression was upregulated in morin treated MSU crystal-induced rats. In addition, morin treatment reduced the protein expression of NF-κB p65, p-NF-κB p65, iNOS, COX-2, and TNF-α. The results clearly demonstrated that morin exert a potent anti-inflammatory effect on MSU crystal-induced inflammation in rats. PMID:27268719

  19. The development of in vitro mutagenicity testing systems using T-lymphocytes

    SciTech Connect

    Albertini, R.J.

    1992-05-01

    This work has focused on the development of in vitro T-cell mutation assays. Conditions have been defined to measure the in vitro induction of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in human T-lymphocytes. This assay is a parallel to our in vivo hprt assay, in that the same cells are utilized. However, the in vitro assay allows for carefully controlled dose response studies. 21 refs., 16 figs., 13 tabs.

  20. Human somatic mutation assays as biomarkers of carcinogenesis

    SciTech Connect

    Compton, P.J.E.; Smith, M.T. ); Hooper, K. )

    1991-08-01

    This paper describes four assays that detect somatic gene mutations in humans: the hypoxanthine-guanine phosphoribosyl transferase assay, the glycophorin A assay, the HLA-A assay, and the sickle cell hemoglobin assay. Somatic gene mutations can be considered a biomarker of carcinogenesis, and assays for somatic mutation may assist epidemiologists in studies that attempt to identify factors associated with increased risks of cancer. Practical aspects of the use of these assays are discussed.

  1. The development of in vitro mutagenicity testing systems using T-lymphocytes. Research progress report, November 1, 1989--April 30, 1992

    SciTech Connect

    Albertini, R.J.

    1992-05-01

    This work has focused on the development of in vitro T-cell mutation assays. Conditions have been defined to measure the in vitro induction of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in human T-lymphocytes. This assay is a parallel to our in vivo hprt assay, in that the same cells are utilized. However, the in vitro assay allows for carefully controlled dose response studies. 21 refs., 16 figs., 13 tabs.

  2. Quantitative assay for mutation in diploid human lymphoblasts using microtiter plates

    SciTech Connect

    Furth, E.A.; Thilly, W.G.; Penman, B.W.; Liber, H.L.; Rand, W.M.

    1981-01-01

    A microtiter plating technique which eliminates the need for soft agar and fibroblast feeder layers to determine the colony-forming ability of diploid human lymphoblast lines was described. The calculation of cloning efficiency is based on the Poisson distribution, and a statistical method for calculating confidence intervals is presented. This technique has been applied to the comcomitant examination of induced mutation at the putative loci for hypoxanthine guanine phosphoribosyl transferase, thymidine, kinase, and Na/sup +//K/sup +/ adenosine triphosphatase.

  3. Application of an in vivo mutagenesis system to assess aminothiol effects on neutron-induced genotoxic damage in mouse spleenocytes

    SciTech Connect

    Basic, I. . Dept. of Animal Physiology); Grdina, D.J.; Lyons, T. )

    1989-01-01

    A cloning technique has been developed to quantitate and study {ital in vivo} somatic mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in human lymphocytes. In this paper we describe a modification of this assay to quantify HGPRT mutations in mouse spleenocytes. In particular, we have investigated the effects of the aminothiol on mutagenesis induced by single doses of whole body exposures to fission-spectrum neutrons from the JANUS reactor at Argonne National Laboratory. 7 refs., 3 tabs.

  4. Cytotoxicity and mutagenicity of the fungicides captan and folpet in cultured mammalian cells (CHO/HGPRT system)

    SciTech Connect

    O'Neill, J.P.; Forbes, N.L; Hsie, A.W.

    1981-01-01

    The cytotoxicity and mutagenicity of the fungicides captan and folpet were determined in the CHO/HGPRT system which utilizes Chinese hamster ovary cells and resistance to 6-thioguanine to estimate mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus. Treatment of cultures with each compound for 5 hr in serum-free medium resulted in reproducible, significant, concentration-dependent increases in the frequency of 6-thioguanine-resistant mutants.

  5. Quantitative mutagenesis and mutagen screening with Chinese hamster ovary cells

    SciTech Connect

    Hsie, A.W.; San Sebastian, J.R.; Tan, E.L.

    1980-01-01

    A summary is presented on the development of a specific gene mutation assay, the Chinese hamster ovary cells/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system, and the utilization of this system to study structure-activity relationship affecting cytotoxicity and gene mutation by various carcinogens. Then, preliminary development and validation of a Multiplex CHO System for the simultaneous determination of chromosome aberration, sister chromatid exchange in addition to cytotoxicity and gene mutation is presented. The potential use of a CHO/human cell hybrid system for measuring chromosomal deletion and loss is discussed.

  6. X-ray induction of persistent hypersensitivity to mutation

    SciTech Connect

    Frank, J.P.; Williams, J.R.

    1982-04-16

    The progeny of x-irradiated V79 cells are hypersensitive to PUVA-(8-methoxypsoralen plus longwave ultraviolet light) induced mutation at the locus for hypoxanthine-guanine phosphoribosyl transferase. This hypersensitivity is most evident at low doses of pUVA that do not induce mutation in non-x-irradiated cells. The hypersensitivity is evoked by x-irradiation delivered as a single dose or as multiple fractions over a long period and persists for at least 108 days of exponential growth. This radiation-induced hypersensitivity to subsequent mutation is a new phenomenon that may be relevant to multistage carcinogenesis.

  7. Mutagenicity and cytotoxicity of ethylene oxide in the CHO/HGPRT system

    SciTech Connect

    Tan, E.L.; Cumming, R.B.; Hsie, A.W.

    1981-01-01

    Ethylene oxide (EO) is made on an industrial scale (2.5 billion pounds per year) and is used not only to prepare a number of commercially important compounds but also as a widely used fumigant to sterilize foodstuffs, textiles, and medical instruments. EO is an alkylating agent that has been shown to interact with proteins and nucleic acids and is mutagenic in a number of test systems, eg, rodents, bacteria, rice, and Drosophila. Reported is the cytotoxicity and mutagenicity of EO in a mammalian cel culture system, the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system.

  8. Pre-thymic somatic mutation leads to high mutant frequency at hypoxanthine-guanine phosphoribosyltransferase gene

    SciTech Connect

    Jett, J.

    1994-12-01

    While characterizing the background mutation spectrum of the Hypoxathine-guanine phosphoribosyltransferase (HPRT) gene in a healthy population, an outlier with a high mutant frequency of thioguanine resistant lymphocytes was found. When studied at the age of 46, this individual had been smoking 60 cigarettes per day for 38 years. His mutant frequency was calculated at 3.6 and 4.2x10{sup {minus}4} for two sampling periods eight months apart. Sequencing analysis of the HPRT gene in his mutant thioguanine resistant T lymphocytes was done to find whether the cells had a high rate of mutation, or if the mutation was due to a single occurrence of mutation and, if so, when in the T lymphocyte development the mutation occurred. By T-cell receptor analysis it has been found that out of 35 thioguanine resistant clones there was no dominant gamma T cell receptor gene rearrangement. During my appointment in the Science & Engineering Research Semester, I found that 34 of those clones have the same base substitution of G{yields}T at cDNA position 197. Due to the consistent mutant frequency from both sampling periods and the varying T cell receptors, the high mutant frequency cannot be due to recent proliferation of a mature mutant T lymphocyte. From the TCR and DNA sequence analysis we conclude that the G{yields}T mutation must have occurred in a T lymphocyte precursor before thymic differentiation so that the thioguanine resistant clones share the same base substitution but not the same gamma T cell receptor gene.

  9. Relative photomutagenicity of furocoumarins and limettin in the hypoxanthine phosphoribosyl transferase assay in V79 cells.

    PubMed

    Raquet, Nicole; Schrenk, Dieter

    2009-09-01

    Furocoumarins are phototoxic and photomutagenic natural plant constituents found in many medicinal plants and food items. Because plants contain mixtures of several furocoumarins, there is a need for a comparative risk assessment of a large number of furocoumarins. Little is known about the photomutagenicity of the structurally related family of coumarins, which are also abundant in many plant species. In this study, we analyzed the photomutagenic potency of the linear furocoumarins 5-methoxypsoralen (5-MOP) and 8-methoxypsoralen (8-MOP), the angular furocoumarin angelicin, and the coumarin limettin. Above certain concentrations, all test compounds were more or less phototoxic in the presence of UVA doses between 50 and 200 mJ/cm(2), 5-MOP being the most phototoxic compound. At nonphototoxic concentrations, linear correlations were found between concentration and mutagenicity at a UVA dose of 125 mJ/cm(2) for all test compounds including limettin. For 5-MOP, strictly linear correlations were also found for the relationships of mutagenicity vs concentration at various UVA doses or vs UVA dose at given concentrations, respectively. These data indicate that the photomutagenicity of 5-MOP is proportional to the UVA dose x concentration product for noncytotoxic combinations of both factors. They also suggest that the slope of the concentration-photomutagenicity correlation at a given UVA dose may provide a basis for comparison between individual compounds. Applying this concept, in vitro photomutagenicity equivalency factors at 125 mJ/cm(2) were as follows: 1.0 (5-MOP, reference compound), 0.25 (8-MOP), and 0.02 (angelicin and limettin, respectively). These findings provide a new concept for the description of the relative photomutagenic potency of coumarins and furocoumarins and indicate that, in V79 cells, 8-MOP is less photomutagenic and limettin and angelicin are much less photomutagenic than 5-MOP. PMID:19725558

  10. Generation of Hprt-disrupted rat through mouse←rat ES chimeras

    PubMed Central

    Isotani, Ayako; Yamagata, Kazuo; Okabe, Masaru; Ikawa, Masahito

    2016-01-01

    We established rat embryonic stem (ES) cell lines from a double transgenic rat line which harbours CAG-GFP for ubiquitous expression of GFP in somatic cells and Acr3-EGFP for expression in sperm (green body and green sperm: GBGS rat). By injecting the GBGS rat ES cells into mouse blastocysts and transplanting them into pseudopregnant mice, rat spermatozoa were produced in mouse←rat ES chimeras. Rat spermatozoa from the chimeric testis were able to fertilize eggs by testicular sperm extraction combined with intracytoplasmic sperm injection (TESE-ICSI). In the present paper, we disrupted rat hypoxanthine-guanine phosphoribosyl transferase (Hprt) gene in ES cells and produced a Hprt-disrupted rat line using the mouse←rat ES chimera system. The mouse←rat ES chimera system demonstrated the dual advantages of space conservation and a clear indication of germ line transmission in knockout rat production. PMID:27062982

  11. Basal Ganglia Dopamine Loss Due to Defect in Purine Recycling

    PubMed Central

    Egami, Kiyoshi; Yitta, Silaja; Kasim, Suhail; Lewers, J. Chris; Roberts, Rosalinda C.; Lehar, Mohamed; Jinnah, H. A.

    2007-01-01

    Several rare inherited disorders have provided valuable experiments of nature highlighting specific biological processes of particular importance to the survival or function of midbrain dopamine neurons. In both humans and mice, deficiency of hypoxanthine-guanine phosphoribosyl transferase (HPRT) is associated with profound loss of striatal dopamine, with relative preservation of other neurotransmitters. In the current studies of knockout mice, no morphological signs of abnormal development or degeneration were found in an exhaustive battery that included stereological and morphometric measures of midbrain dopamine neurons, electron microscopic studies of striatal axons and terminals, and stains for degeneration or gliosis. A novel culture model involving HPRT-deficient dopaminergic neurons also exhibited significant loss of dopamine without a morphological correlate. These results suggest dopamine loss in HPRT deficiency has a biochemical rather than anatomical basis, and imply purine recycling to be a biochemical process of particular importance to the function of dopaminergic neurons. PMID:17374562

  12. Estimates of cellular mutagenesis from cosmic rays

    NASA Technical Reports Server (NTRS)

    Cucinotta, Francis A.; Wilson, John W.

    1994-01-01

    A parametric track structure model is used to estimate the cross section as a function of particle velocity and charge for mutations at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus in human fibroblast cell cultures. Experiments that report the fraction of mutations per surviving cell for human lung and skin fibroblast cells indicate small differences in the mutation cross section for these two cell lines when differences in inactivation rates between these cell lines are considered. Using models of cosmic ray transport, the mutation rate at the HGPRT locus is estimated for cell cultures in space flight and rates of about 2 to 10 x 10(exp -6) per year are found for typical spacecraft shielding. A discussion of how model assumptions may alter the predictions is also presented.

  13. Use of PCR amplification of cDNA to study mechanisms of human cell mutagenesis and malignant transformation

    SciTech Connect

    Maher, V.M.; Yang, Jialing; Chen, Rueyhwa; McGregor, W.G.; Lukash, L.; Scheid, J.M.; Reinhold, D.S.; McCormick, J.J. )

    1991-01-01

    PCR is widely employed to amplify short segments of genomic DNA to determine if a specific change has occurred. But some investigators need to sequence the entire coding region of mammalian genes to determine what specific changes have occurred. In 1989, the authors described a method to copy mRNA of the hypoxanthine (guanine) phosphoribosyl transferase (HPRT) gene directly from the lysate of a clone of 6-thioguanine-resistant mutant diploid human fibroblasts without the need for RNA extraction or DNA template purification. The consensus sequence of the cDNA is determined by direct nucleotide sequencing. Using this method, they have investigated the kinds of mutations induced by carcinogens in the coding region of the HPRT gene and their location in the gene and examined the role of DNA repair were exposed to mutagens in exponential growth or synchronized and exposed at the beginning of S phase or in G{sub 1} phase several hr prior to DNA replication.

  14. Lesch-Nyhan Syndrome in an Indian Child.

    PubMed

    Chandekar, Priyanka; Madke, Bhushan; Kar, Sumit; Yadav, Nidhi

    2015-01-01

    Hypoxanthine guanine phosphoribosyl transferase-1 (HGPRT-1) leading to Lesch-Nyhan syndrome (LNS) is one of the important causes of self-mutilation. Hereby, we report a case of LNS in a three and half-year-old male child, who presented with characteristic self-mutilating behavior. He had history of developmental delay, difficulty in social interaction, attention deficit and features of autism. His serum blood biochemistry was normal except for low hemoglobin levels and raised serum uric acid levels. With a diagnosis of LNS, the child was treated with allopurinol. With various modalities of physical restraint, his self-mutilating behavior came under control and currently the patient is being followed up. PMID:26120162

  15. Dependence of antimutagenic activity of simple phenols on the number of hydroxyl groups

    SciTech Connect

    Pashin, Yu.V.; Bakhitova, L.M.; Bentkhen, T.I.

    1987-01-01

    The authors seek to establish the antioxidative and antimutagenetic effects of three phenols--phenol itself, resorcinol, and pyrogallol--on benzopyrene and its metabolic activation both in vivo and in vitro. In the in vivo system the mutagenic activity of the chemicals and their mixtures was tested relative to induction of micronuclei in polychromatophylic bone marrow erythrocytes of mice. The action of the phenols on the mutagenic activity of benzopyrene in an in vitro system was studied by counting induced direct gene mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in cultures of Chinese hamster V-70 somatic cells. It is found that the inhibition of the mutagenic activity of benzopyrene by polyhydric phenols is evidently connected with the presence of reactable hydrogen atoms in these compounds which inhibit free-radical self-oxidation of the mutagen.

  16. Genotoxicity of alpha particles in human embryonic skin fibroblasts

    SciTech Connect

    Chen, D.J.; Strniste, G.F.; Tokita, N.

    1984-11-01

    Cell inactivation and induced mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus have been measured in cultured human fibroblasts (GM10) exposed to ..cap alpha.. particles from /sup 238/ Pu and 250 kVp X rays. The survival curves resulting from exposure to ..cap alpha.. particles are exponential. The mean lethal dose, D/sub 0/, is approximately 1.3 Gy for X rays and 0.25 Gy for ..cap alpha.. particles. As a function of radiation dose, mutation induction at the HGPRT locus was linear for ..cap alpha.. particles whereas the X-ray-induced mutation data were better fitted by a quadratic function. When mutation frequencies were plotted against the log of survival, mutation frequency at a given survival level was greater in cells exposed to ..cap alpha.. particles than to X rays.

  17. Molecular analysis of mutations in the human HPRT gene.

    PubMed

    Keohavong, Phouthone; Xi, Liqiang; Grant, Stephen G

    2014-01-01

    The HPRT assay uses incorporation of toxic nucleotide analogues to select for cells lacking the purine scavenger enzyme hypoxanthine-guanine phosphoribosyl transferase. A major advantage of this assay is the ability to isolate mutant cells and determine the molecular basis for their functional deficiency. Many types of analyses have been performed at this locus: the current protocol involves generation of a cDNA and multiplex PCR of each exon, including the intron/exon junctions, followed by direct sequencing of the products. This analysis detects point mutations, small deletions and insertions within the gene, mutations affecting RNA splicing, and products of illegitimate V(D)J recombination within the gene. Establishment of and comparisons with mutational spectra hold the promise of identifying exposures to mutation-inducing genotoxicants from their distinctive pattern of gene-specific DNA damage at this easily analyzed reporter gene. PMID:24623237

  18. Generation of Hprt-disrupted rat through mouse←rat ES chimeras.

    PubMed

    Isotani, Ayako; Yamagata, Kazuo; Okabe, Masaru; Ikawa, Masahito

    2016-01-01

    We established rat embryonic stem (ES) cell lines from a double transgenic rat line which harbours CAG-GFP for ubiquitous expression of GFP in somatic cells and Acr3-EGFP for expression in sperm (green body and green sperm: GBGS rat). By injecting the GBGS rat ES cells into mouse blastocysts and transplanting them into pseudopregnant mice, rat spermatozoa were produced in mouse←rat ES chimeras. Rat spermatozoa from the chimeric testis were able to fertilize eggs by testicular sperm extraction combined with intracytoplasmic sperm injection (TESE-ICSI). In the present paper, we disrupted rat hypoxanthine-guanine phosphoribosyl transferase (Hprt) gene in ES cells and produced a Hprt-disrupted rat line using the mouse←rat ES chimera system. The mouse←rat ES chimera system demonstrated the dual advantages of space conservation and a clear indication of germ line transmission in knockout rat production. PMID:27062982

  19. Sodium arsenite potentiates the clastogenicity and mutagenicity of DNA cross linking agents

    SciTech Connect

    Lee, T.C.; Lee, K.C.; Tzeng, Y.J.; Huang, R.Y.; Jan, K.Y.

    1986-01-01

    To see if sodium arsenite enhances the clastogenicity and the mutagenicity of DNA crosslinking agents, Chinese hamster ovary (CHO) cells and human skin fibroblasts were exposed to cis-diamminedichloroplatinum (II) (cis-Pt(II)) or 8-methoxypsoralen (8-MOP) plus long-wave ultraviolet light (UVA) and then to sodium arsenite. The results indicate that the clastogenicity of cis-Pt(II) and 8-MOP pllus UVA are enhanced by the post-treatment with sodium arsenite. Chromatid breaks and exchanges are predominantly increased in doubly treated cells. Furthermore, the mutagenicity of cis-Pt(II) at the hypoxanthine-guanine phosphoribosyl transferase locus is also potentiated by sodium arsenite in CHO cells

  20. Use of Aroclor 1254-induced rat liver homogenate in the assaying of promutagens in Chinese hamster ovary cells

    SciTech Connect

    Li, A.P.

    1984-01-01

    Seven promutagens belonging to two chemical classes - polycyclic aromatic hydrocarbons (PAHs) and alkyl nitrosamines - were studied in Chinese hamster ovary (CHO) cells. Findings of practical importance in the use of Aroclor 1254-induced rat liver homogenate (S9) in the CHO/hypoxanthine-guanine phosphoribosyl transferase (HGPRT) mutation assay were made. Novel findings are (1) the inclusion of CaCl/sub 2/ in the S9 cofactor mixture dramatically decreased the cytotoxicity of S9, and (2) different S9 optimum concentrations were observed for struturally similar promutagens. The inclusion of CaCl/sub 2/ in the S9 cofactor mixture and the testing of each chemical of unknown S9 requirement at several S9 concentrations are therefore recommended for assaying promutagens in the CHO/HGPRT mutation assay.

  1. Quantitative and molecular analyses of radiation-induced mutation in AS52 cells

    SciTech Connect

    Stankowski, L.F. Jr.; Hsie, A.W.

    1986-01-01

    pSV2gpt-Transformed and wild-type Chinese hamster ovary (CHO) cell lines have been used to study radiation-induced mutation at the molecular level. The transformant, designated AS52, was constructed from a hypoxanthine-guanine phosphoribosyl transferase (HPRT)-deficient CHO cell line and contains a single, functional copy of the Escherichia coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) stably integrated into the Chinese hamster genome. AS52 and wild-type CHO-K1-BH4 cells exhibit similar cytotoxic responses to uv light and X rays; however, significant differences occur in mutation induction at the gpt and hprt loci. A number of HPRT and XPRT mutants which arose following irradiation were analyzed by Southern-blot hybridization. Most XPRT (21/26) and all HPRT (23/23) mutants induced by uv light exhibited hybridization patterns indistinguishable from their parental cell lines. In contrast, all XPRT (26/26) and most HPRT mutants (15/21) induced by X irradiation contained deletion mutations affecting some or all of the gpt and hprt loci, respectively. These results indicate that X rays induce predominantly deletion mutations, while uv light is likely to induce point mutations at both loci.

  2. Corynebacterium glutamicum ATP-phosphoribosyl transferases suitable for L-histidine production--Strategies for the elimination of feedback inhibition.

    PubMed

    Kulis-Horn, Robert K; Persicke, Marcus; Kalinowski, Jörn

    2015-07-20

    L-Histidine biosynthesis in Corynebacterium glutamicum is mainly regulated by L-histidine feedback inhibition of the ATP-phosphoribosyltransferase HisG that catalyzes the first step of the pathway. The elimination of this feedback inhibition is the first and most important step in the development of an L-histidine production strain. For this purpose, a combined approach of random mutagenesis and rational enzyme redesign was performed. Mutants spontaneously resistant to the toxic L-histidine analog β-(2-thiazolyl)-DL-alanine (2-TA) revealed novel and unpredicted mutations in the C-terminal regulatory domain of HisG resulting in increased feedback resistance. Moreover, deletion of the entire C-terminal regulatory domain in combination with the gain of function mutation S143F in the catalytic domain resulted in a HisG variant that is still highly active even at L-histidine concentrations close to the solubility limit. Notably, the S143F mutation on its own provokes feedback deregulation, revealing for the first time an amino acid residue in the catalytic domain of HisG that is involved in the feedback regulatory mechanism. In addition, we investigated the effect of hisG mutations for L-histidine production on different levels. This comprised the analysis of different expression systems, including plasmid- and chromosome-based overexpression, as well as the importance of codon choice for HisG mutations. The combination of domain deletions, single amino acid exchanges, codon choice, and chromosome-based overexpression resulted in production strains accumulating around 0.5 g l(-1) L-histidine, demonstrating the added value of the different approaches. PMID:25892668

  3. The 1.25 Å resolution structure of phosphoribosyl-ATP pyrophosphohydrolase from Mycobacterium tuberculosis

    SciTech Connect

    Javid-Majd, Farah; Yang, Dong; Ioerger, Thomas R.; Sacchettini, James C.

    2008-06-01

    The crystal structure of M. tuberculosis phosphoribosyl-ATP pyrophosphohydrolase, the second enzyme in the histidine-biosynthetic pathway, is presented. The structural and inferred functional relationships between M. tuberculosis phosphoribosyl-ATP pyrophosphohydrolase and other members of the nucleoside-triphosphate pyrophosphatase-fold family are described. Phosphoribosyl-ATP pyrophosphohydrolase is the second enzyme in the histidine-biosynthetic pathway, irreversibly hydrolyzing phosphoribosyl-ATP to phosphoribosyl-AMP and pyrophosphate. It is encoded by the hisE gene, which is present as a separate gene in many bacteria and archaea but is fused to hisI in other bacteria, fungi and plants. Because of its essentiality for growth in vitro, HisE is a potential drug target for tuberculosis. The crystal structures of two native (uncomplexed) forms of HisE from Mycobacterium tuberculosis have been determined to resolutions of 1.25 and 1.79 Å. The structure of the apoenzyme reveals that the protein is composed of five α-helices with connecting loops and is a member of the α-helical nucleoside-triphosphate pyrophosphatase superfamily. The biological unit of the protein is a homodimer, with an active site on each subunit composed of residues exclusively from that subunit. A comparison with the Campylobacter jejuni dUTPase active site allowed the identification of putative metal- and substrate-binding sites in HisE, including four conserved glutamate and glutamine residues in the sequence that are consistent with a motif for pyrophosphohydrolase activity. However, significant differences between family members are observed in the loop region between α-helices H1 and H3. The crystal structure of M. tuberculosis HisE provides insights into possible mechanisms of substrate binding and the diversity of the nucleoside-triphosphate pyrophosphatase superfamily.

  4. Crystallization and preliminary X-ray diffraction study of phosphoribosyl pyrophosphate synthetase from E. Coli

    NASA Astrophysics Data System (ADS)

    Timofeev, V. I.; Abramchik, Yu. A.; Zhukhlistova, N. E.; Kuranova, I. P.

    2015-09-01

    Enzymes of the phosphoribosyl pyrophosphate synthetase family (PRPPS, EC 2.7.6.1) catalyze the formation of 5-phosphoribosyl pyrophosphate (5-PRPP) from adenosine triphosphate and ribose 5-phosphate. 5-Phosphoribosyl pyrophosphate is an important intermediate in the synthesis of purine, pyrimidine, and pyridine nucleotides, as well as of the amino acids histidine and tryptophan. The crystallization conditions for E. coli PRPPS were found by the vapor-diffusion technique and were optimized to apply the capillary counter-diffusion technique. The X-ray diffraction data set was collected from the crystals grown by the counter-diffusion technique using a synchrotron radiation source to 3.1-Å resolution. The crystals of PRPPS belong to sp. gr. P6322 and have the following unit-cell parameters: a = b = 104.44 Å, c = 124.98 Å, α = β = 90°, γ = 120°. The collected X-ray diffraction data set is suitable for the solution of the three-dimensional structure of PRPPS at 3.1-Å resolution.

  5. The 1.25 Å resolution structure of phosphoribosyl-ATP pyrophosphohydrolase from Mycobacterium tuberculosis

    SciTech Connect

    Javid-Majd, Farah; Yang, Dong; Ioerger, Thomas R.; Sacchettini, James C.

    2008-06-23

    Phosphoribosyl-ATP pyrophosphohydrolase is the second enzyme in the histidine-biosynthetic pathway, irreversibly hydrolyzing phosphoribosyl-ATP to phosphoribosyl-AMP and pyrophosphate. It is encoded by the hisE gene, which is present as a separate gene in many bacteria and archaea but is fused to hisI in other bacteria, fungi and plants. Because of its essentiality for growth in vitro, HisE is a potential drug target for tuberculosis. The crystal structures of two native (uncomplexed) forms of HisE from Mycobacterium tuberculosis have been determined to resolutions of 1.25 and 1.79 {angstrom}. The structure of the apoenzyme reveals that the protein is composed of five -helices with connecting loops and is a member of the {alpha}-helical nucleoside-triphosphate pyrophosphatase superfamily. The biological unit of the protein is a homodimer, with an active site on each subunit composed of residues exclusively from that subunit. A comparison with the Campylobacter jejuni dUTPase active site allowed the identification of putative metal- and substrate-binding sites in HisE, including four conserved glutamate and glutamine residues in the sequence that are consistent with a motif for pyrophosphohydrolase activity. However, significant differences between family members are observed in the loop region between {alpha}-helices H1 and H3. The crystal structure of M. tuberculosis HisE provides insights into possible mechanisms of substrate binding and the diversity of the nucleoside-triphosphate pyrophosphatase superfamily.

  6. Crystallization and preliminary X-ray diffraction study of phosphoribosyl pyrophosphate synthetase from E. Coli

    SciTech Connect

    Timofeev, V. I. Abramchik, Yu. A. Zhukhlistova, N. E. Kuranova, I. P.

    2015-09-15

    Enzymes of the phosphoribosyl pyrophosphate synthetase family (PRPPS, EC 2.7.6.1) catalyze the formation of 5-phosphoribosyl pyrophosphate (5-PRPP) from adenosine triphosphate and ribose 5-phosphate. 5-Phosphoribosyl pyrophosphate is an important intermediate in the synthesis of purine, pyrimidine, and pyridine nucleotides, as well as of the amino acids histidine and tryptophan. The crystallization conditions for E. coli PRPPS were found by the vapor-diffusion technique and were optimized to apply the capillary counter-diffusion technique. The X-ray diffraction data set was collected from the crystals grown by the counter-diffusion technique using a synchrotron radiation source to 3.1-Å resolution. The crystals of PRPPS belong to sp. gr. P6{sub 3}22 and have the following unit-cell parameters: a = b = 104.44 Å, c = 124.98 Å, α = β = 90°, γ = 120°. The collected X-ray diffraction data set is suitable for the solution of the three-dimensional structure of PRPPS at 3.1-Å resolution.

  7. Coupled optical assay for adenine phosphoribosyltransferase and its extension for the spectrophotometric and radioenzymatic determination of 5-phosphoribosyl-1-pyrophosphate in mixtures and in tissue extracts

    SciTech Connect

    Ipata, P.L.; Mura, U.; Camici, M.; Giovannitti, M.P.

    1987-08-01

    A reliable assay was developed to characterize crude cell homogenates with regard to their adenine phosphoribosyltransferase activities. The 5-phosphoribosyl-1-pyrophosphate (PRPP)-dependent formation of AMP from adenine is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: AMP + H/sub 2/O----adenosine + Pi (5'-nucleotidase); adenosine + H/sub 2/O----inosine + NH/sub 3/ (adenosine deaminase). The same principle was applied to develop a spectrophotometric and a radioenzymatic assay for PRPP. The basis of the spectrophotometric assay is the absorbance change at 265 nm associated with the enzymatic conversion of PRPP into inosine, catalyzed by the sequential action of partially purified adenine phosphoribosyltransferase, commercial 5'-nucleotidase, and commercial adenosine deaminase, in the presence of excess adenine. In the radiochemical assay PRPP is quantitatively converted into (/sup 14/C)inosine via the same combined reaction. Tissue extracts are incubated with excess (/sup 14/C)adenine. The radioactivity of inosine, separated by a thin-layer chromatographic system, is a measure of PRPP present in tissue extracts. The radioenzymatic assay is at least as sensitive as other methods based on the use of adenine phosphoribosyltransferase. However, it overcomes the reversibility of the reaction and the need to use transferase preparations free of any phosphatase and adenosine deaminase activities.

  8. Calculation of Heavy Ion Inactivation and Mutation Rates in Radial Dose Model of Track Structure

    NASA Technical Reports Server (NTRS)

    Cucinotta, Francis A.; Wilson, John W.; Shavers, Mark R.; Katz, Robert

    1997-01-01

    In the track structure model, the inactivation cross section is found by summing an inactivation probability over all impact parameters from the ion to the sensitive sites within the cell nucleus. The inactivation probability is evaluated by using the dose response of the system to gamma rays and the radial dose of the ions and may be equal to unity at small impact parameters. We apply the track structure model to recent data with heavy ion beams irradiating biological samples of E. Coli, B. Subtilis spores, and Chinese hamster (V79) cells. Heavy ions have observed cross sections for inactivation that approach and sometimes exceed the geometric size of the cell nucleus. We show how the effects of inactivation may be taken into account in the evaluation of the mutation cross sections in the track structure model through correlation of sites for gene mutation and cell inactivation. The model is fit to available data for HPRT (hypoxanthine guanine phosphoribosyl transferase) mutations in V79 cells, and good agreement is found. Calculations show the high probability for mutation by relativistic ions due to the radial extension of ions track from delta rays. The effects of inactivation on mutation rates make it very unlikely that a single parameter such as LET (linear energy transfer) can be used to specify radiation quality for heavy ion bombardment.

  9. The mutagenic potential of high flash aromatic naphtha.

    PubMed

    Schreiner, C A; Edwards, D A; McKee, R H; Swanson, M; Wong, Z A; Schmitt, S; Beatty, P

    1989-06-01

    Catalytic reforming is a refining process that converts naphthenes to aromatics by dehydrogenation to make higher octane gasoline blending components. A portion of this wide boiling range hydrocarbon stream can be separated by distillation and used for other purposes. One such application is a mixture of predominantly 9-carbon aromatic molecules (C9 aromatics, primarily isomers of ethyltoluene and trimethylbenzene), which is removed and used as a solvent--high-flash aromatic naphtha. A program was initiated to assess the toxicological properties of high-flash aromatic naphtha since there may be human exposure through inhalation or external body contact. The current study was conducted partly to assess the potential for mutagenic activity and also to assist in an assessment of carcinogenic potential. The specific tests utilized included the Salmonella/mammalian microsome mutagenicity assay, the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) forward mutation assay in CHO cells, in vitro chromosome aberration and sister chromatid exchange (SCE) assays in CHO cells, and an in vivo chromosome aberration assay in rat bone marrow. PMID:2670086

  10. Locations and contexts of sequences that hybridize to poly(dG-dT).(dC-dA) in mammalian ribosomal DNAs and two X-linked genes.

    PubMed Central

    Braaten, D C; Thomas, J R; Little, R D; Dickson, K R; Goldberg, I; Schlessinger, D; Ciccodicola, A; D'Urso, M

    1988-01-01

    Sequences located several kilobases both 5' and 3' of the stably transcribed portion of several genes hybridize to radio-labeled pure fragments of the alternating sequence poly (dG-dT) (dC-dA) ["poly(GT)"]. The genes include the ribosomal DNA of mouse, rat, and human, and also human glucose-6-phosphate dehydrogenase (G6PD) and mouse hypoxanthine-guanine phosphoribosyl transferase (HPRT). HPRT has additional hybridizing sequences in introns. Fragments that include the hybridizing sequences and up to 300 bp of adjoining DNA show perfect runs of poly(GT) (greater than 30bp) in all but the human 5' region of rDNA, which shows a somewhat different alternating purine:pyrimidine sequence, poly(GTAT) (36bp). Within 150 bp of these sequences in various instances are found a number of other sequences reported to affect DNA conformation in model systems. Most marked is an enhancement of sequences matching at least 67% to the consensus binding sequence for topoisomerase II. Two to ten-fold less of such sequences were found in other sequenced portions of the nontranscribed spacer or in the transcribed portion of rDNA. The conservation of the locations of tracts of alternating purine:pyrimidine between evolutionarily diverse species is consistent with a possible functional role for these sequences. Images PMID:3267216

  11. Effects of 2.45 GHz electromagnetic fields with a wide range of SARs on bacterial and HPRT gene mutations.

    PubMed

    Koyama, Shin; Takashima, Yoshio; Sakurai, Tomonori; Suzuki, Yukihisa; Taki, Masao; Miyakoshi, Junji

    2007-01-01

    Present day use of mobile phones is ubiquitous. This causes some concern for human health due to exposure to high-frequency electromagnetic fields (HFEMF) from mobile phones. Consequently, we have examined the effects of 2.45 GHz electromagnetic fields on bacterial mutations and the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutations. Using the Ames test, bacteria were exposed to HFEMF for 30 min at specific absorption rates (SARs) from 5 to 200 W/kg. In all strains, there was no significant difference in the frequency of revertant colonies between sham exposure and HFEMF-exposed groups. In examination of mutations of the HPRT gene, Chinese hamster ovary (CHO)-K1 cells were exposed to HFEMF for 2 h at SARs from 5 to 200 W/kg. We detected a combination effect of simultaneous exposure to HFEMF and bleomycin at the respective SARs. A statistically significant difference was observed between the cells exposed to HFEMF at the SAR of 200 W/kg. Cells treated with the combination of HFEMF at SARs from 50 to 200 W/kg and bleomycin exhibited increased HPRT mutations. As the exposure to HFEMF induced an increase in temperature, these increases of mutation frequency may be a result of activation of bleomycin by heat. We consider that the increase of mutation frequency may be due to a thermal effect. PMID:17179647

  12. Mutation induction by charged particles of defined linear energy transfer.

    PubMed

    Hei, T K; Chen, D J; Brenner, D J; Hall, E J

    1988-07-01

    The mutagenic potential of charged particles of defined linear energy transfer (LET) was assessed using the hypoxanthine-guanine phosphoribosyl transferase locus (HGPRT) in primary human fibroblasts. Exponentially growing cultures of early passaged fibroblasts were grown as monolayers on thin mylar sheets and were irradiated with accelerated protons, deuterons or helium-3 ions. The mutation rates were compared with those generated by 137Cs gamma-rays. LET values for charged particles accelerated at the Radiological Research Accelerator Facility, using the track segment mode, ranged from 10 to 150 keV/micron. After irradiation, cells were trypsinized, subcultured and assayed for both cytotoxicity and 6-thioguanine resistance. For gamma-rays, and for the charged particles of lower LET, the dose-response curves for cell survival were characterized by a marked initial shoulder, but approximated to an exponential function of dose for higher LETs. Mutation frequencies, likewise, showed a direct correlation to LET over the dose range examined. Relative biological effectiveness (RBE) for mutagenesis, based on the initial slopes of the dose-response curves, ranged from 1.30 for 10 keV/micron protons to 9.40 for 150 keV/micron helium-3 ions. Results of the present studies indicate that high-LET radiations, apart from being efficient inducers of cell lethality, are even more efficient in mutation induction as compared to low-LET ionizing radiation. These data are consistent with results previously obtained with both rodent and human fibroblast cell lines. PMID:3383341

  13. A Modified Intraoral Resin Mouthguard to Prevent Self-Mutilations in Lesch-Nyhan Patients

    PubMed Central

    Delucchi, Alessia; Calcagno, Enrico; Servetto, Roberto

    2014-01-01

    Lesch-Nyhan syndrome, described in 1964 by Lesch and Nyhan, is a X-linked recessive disorder, occurring in 1 : 100000 to 1 : 380000 live births. LNS is characterized by a decrease in activity of hypoxanthine guanine phosphoribosyl transferase, an enzyme involved in purine metabolism, resulting in overproduction of uric acid. Hyperuricemia and neurological features including choreoathetoid spasticity, self-mutilation, and mental retardation clinically characterize this syndrome. In LNS patients the typical feature is loss of tissue from biting themselves with partial or complete amputation of fingers, lips, and tongue. The self-mutilation compares with the eruption of the deciduous teeth. Several drugs trials have been administered to improve self-destructive behavior and invasive treatment approaches, such as extractions of teeth and orthognathic surgery, have been suggested with variable effectiveness. Nowadays prevention is, therefore, the standard of care. The role of dentistry is essential in the management of the self-mutilating behavior, because the teeth represent the main self-injury instrument. This report presents a revision of various therapeutic approaches to manage self-destruction, highlighting the effectiveness of a preventive treatment. It describes a new technique: a resin mouthguard, realized at Gaslini Hospital, to obtain immediate healing of the oral lesions, confirmed in the follow-up period. PMID:25101126

  14. Selection of Reliable Reference Genes for Real-time qRT-PCR Analysis of Zi Geese (Anser anser domestica) Gene Expression

    PubMed Central

    Ji, Hong; Wang, Jianfa; Liu, Juxiong; Guo, Jingru; Wang, Zhongwei; Zhang, Xu; Guo, Li; Yang, Huanmin

    2013-01-01

    Zi geese (Anser anser domestica) belong to the white geese and are excellent layers with a superior feed-to-egg conversion ratio. Quantitative gene expression analysis, such as Real-time qRT-PCR, will provide a good understanding of ovarian function during egg-laying and consequently improve egg production. However, we still don’t know what reference genes in geese, which show stable expression, should be used for such quantitative analysis. In order to reveal such reference genes, the stability of seven genes were tested in five tissues of Zi geese. Methodology/Principal Findings: The relative transcription levels of genes encoding hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1), β-actin (ACTB), β-tubulin (TUB), glyceraldehyde-3-phosphate-dehydrogenase (GADPH), succinate dehydrogenase flavoprotein (SDH), 28S rRNA (28S) and 18S rRNA (18S) have been quantified in heart, liver, kidney, muscle and ovary in Zi geese respectively at different developmental stages (1 d, 2, 4, 6 and 8 months). The expression stability of these genes was analyzed using geNorm, NormFinder and BestKeeper software. Conclusions: The expression of 28S in heart, GAPDH in liver and ovary, ACTB in kidney and HPRT1 in muscle are the most stable genes as identified by the three different analysis methods. Thus, these genes are recommended for use as candidate reference genes to compare mRNA transcription in various developmental stages of geese. PMID:25049806

  15. Metabolism and toxicological evaluation of the aromatic amide herbicide propanil and its derivatives

    SciTech Connect

    McMillian, D.C.

    1989-01-01

    Since propanil is structurally similar to other carcinogenic arylamides, the potential chronic toxicity of propanil and its derivatives were examined in short-term assays for genotoxicity. Propanil, 3,4-dichloroaniline, and their N-oxidized derivatives were inactive in the Salmonella typhimurium reversion, Chinese hamster ovary/hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT), and rat hepatocyte/DNA repair assays. The metabolism of propanil and 3,4-dichloroaniline was subsequently examined in liver microsomes from males Sprague-Dawley rats to identify metabolites that may be involved in the acute toxicity of propanil. The major pathway of propanil metabolism was acylamidase-catalyzed hydrolysis to 3,4-dichloroaniline. Oxidized metabolites were isolated by high performance liquid chromatography, and identified as 2{prime}-hydroxy-propanil and 6-hydroxy-propanil by comparison of their mass and nuclear magnetic resonance spectra to synthetic standards. Experiments were performed to determine if propanil exposure could be monitored by the analysis of hemoglobin binding. Administration of (ring-U-{sup 14}C)propanil to rats increased methemoglobin formation in a dose-dependent manner. Concomitant with methemoglobin formation, dose-dependent covalent binding of radiolabeled propanil to hemoglobin was detected. HPLC analysis indicated that the hemoglobin adducts were sulfinic acid esters, and these data suggest that human exposure to propanil may be monitored by the analysis of propanil metabolites bound to hemoglobin.

  16. SERS internship: Fall 1994 abstracts and research papers

    SciTech Connect

    Goldman, B.

    1994-12-01

    This publication is a collection of articles generated as a result of the fall 1994 Science and Engineering Research Semester program at Lawrence Livermore Laboratory. Research titles include: electrochemical cells in the reduction of hexavalent chromium; an automated system for studying the power distribution of electron beams; the mapping of novel genes to human chromosome 19; bolometer analysis comparisons; design and implementation of the LLNL Gigabit Testbed; in vitro synthesis and purification of PhIP-Deoxyguanosine and PhIP-DNA Covalent Complexes; pre-thymic somatic mutation leads to high mutant frequency hypoxanthine-guanine phosphoribosyl transferase gene; characterization of thin film multi-layers with magnetization curves and modeling of low angle X-ray diffraction data; total least squares; determining the water content of the Geysers Graywacke of northern California; a general approach to sharing data between scientific representations; nanomechanical properties of SiC thin films grown from C{sub 60} precursors; advanced information technology, a tool set for building clean database applications; the design of an automated electrolytic enrichment procedure for tritium; fluvial terrace dating using in-situ cosmogenic {sup 21}Ne; computer- aided mapping of stream channels beneath the Lawrence Livermore National Laboratory, Livermore, CA; X-ray spectroscopic technique for energetic electron transport studies in short-pulse laser/plasma interactions. Separate entries have been put in the energy data base for articles from this report. Selected papers are indexed separately for inclusion in the Energy Science and Technology Database.

  17. The genotoxicity of lucidin, a natural component of Rubia tinctorum L., and lucidinethylether, a component of ethanolic Rubia extracts.

    PubMed

    Westendorf, J; Poginsky, B; Marquardt, H; Groth, G; Marquardt, H

    1988-06-01

    The genotoxic activity of lucidin (1,3-dihydroxy-2-hydroxymethyl-9,10-anthraquinone), a natural component of Rubia tinctorum L., was tested in a battery of short-term tests. The compound was mutagenic in five Salmonella typhimurium strains without metabolic activation, but the mutagenicity was increased after addition of rat liver S9 mix. In V79 cells, lucidin was mutagenic at the hypoxanthine-guanine phosphoribosyl transferase gene locus and active at inducing DNA single-strand breaks and DNA-protein cross-links as assayed by the alkaline elution method. Lucidin also induced DNA repair synthesis in primary rat hepatocytes and transformed C3H/M2-mouse fibroblasts in culture. We also investigated lucidinethylether, which is formed from lucidin by extraction of madder roots with boiling ethanol. This compound was also mutagenic in Salmonella, but only after addition of rat liver S9 mix. Lucidinethylether was weakly mutagenic to V79 cells which were cocultivated with rat hepatocytes. The compound did not induce DNA repair synthesis in hepatocytes from untreated rats, but positive results were obtained when hepatocytes from rats pretreated with phenobarbital were used. We conclude that lucidin and its derivatives are genotoxic. PMID:3069188

  18. Phenotypic expression time of mutagen-induced 6-thioguanine resistance in Chinese hamster ovary cells (CHO/HGPRT system): expression in division-arrested cell cultures

    SciTech Connect

    O'Neill, J.P.; Machanoff, R.; Hsie, A.W.

    1982-01-01

    The phenotypic expression time of ethyl methanesulfonate (EMS) induced 6-thioguanine-resistant mutants was studied with Chinese hamster ovary cells in culture (CHO/HGPRT system). After mutagen treatment of exponential phase cultures, the cells were maintained either in the exponential phase through subculture in medium containing 5% dialyzed fetal bovine serum (FBS) or in a nondividing viable state by use of medium containing 0-1% dialyzed FBS. The time course of expression of the 6-thioguanine-resistant phenotype was similar with both exponential phase division-arrested cultures showing maximum expression by 9 days after mutagen event is fixed, the expression of the mutant phenotype does not require continued cell division since it occurs in division-arrested cultures. These results also suggest that both dilution of pre-existing hypoxanthine-guanine phosphoribosyl transferase (HGPRT) enzyme by cell division and turnover by protein degradation are involved in the phenotypic expression. These results separate genetic damage and phenotypic expression in a temporal sense, and point out the need to consider the mechanisms responsible for each process involved in the induction and expression of mutations.

  19. Evaluation of the genotoxicity of process stream extracts from a coal gasification system

    SciTech Connect

    Shimizu, R.W.; Benson, J.M.; Li, A.P.; Henderson, R.F.; Brooks, A.L.

    1984-01-01

    Extracts of three complex organic environmental mixtures, two from an experimental coal gasifier (a raw gas and a clean gas sample) and one from a coke oven main, were examined for genotoxicity. Three short-term genotoxicity assay systems were used: Ames Salmonella typhimurium reverse mutation assay, Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) gene locus mutation assay, and the Chinese hamster lung primary culture/sister chromatid exchange (CHL/SCE) assay. Aroclor-1254-induced rat liver homogenate fraction (S-9) was required to observe genotoxicity in both gene locus mutation assays (CHO/HGPRT and Ames). The relative survival of CHO cells exposed to extracts was highest in cells exposed to clean gas samples, with the raw gas sample being the most cytotoxic either with or without the addition of S-9. All three complex mixtures induced sister chromatid exchanges in primary lung cell cultures without the addition of S-9. The relative genotoxicity ranking of the samples varied between the mammalian and prokaryotic assay systems. The results of all three assays indicate that the cleanup process used in the experimental gasifier was effective in decreasing the genotoxic materials in the process stream. These data also reemphasize the necessity of evaluating genotoxicity of complex mixtures in a variety of short-term systems.

  20. Cytotoxic and mutagenic properties of shale oil byproducts. I. Activation of retort process waters with near ultraviolet light

    SciTech Connect

    Strniste, G.F.; Chen, D.J.

    1981-01-01

    Cultured Chinese hamster ovary (CHO) cells were exposed to dilutions of shale oil retort process waters obtained from three different retorting processes located in the Green River oil shale formations in the western part of the United States. Although the intensity of the response was dictated by the process water used, all induced a cytotoxic (reduction in colony-forming ability) and mutagenic (induced at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus) response in cells pretreated with dilutions of the waters and subsequent exposure to near ultraviolet light (NUV). Combinations of process water plus NUV yielded mutation frequencies as great as 50% that witnessed for the mutation frequency induced by the potent carcinogen far ultraviolet light. NUV alone was nontoxic and nonmutagenic at the doses of radiation used. Exposure of CHO cells in the dark to nontoxic dilutions of the process waters resulted in small but significant increases in 6-thioguanine resistent mutants (1-2 times background rates). The biological consequences resulting from the disposal of retort process waters into the delcate environment present in this oil shale region could be further complicated by this photoactivating process.

  1. Biological availability of nickel arsenides: cellular response to soluble Ni/sub 5/As/sub 2/

    SciTech Connect

    Gurley, L.R.; Valdez, J.G.; Miglio, J.J.; Cox, S.H.; Tobey, R.A.

    1986-01-01

    It has been determined that particulate Ni/sub 5/As/sub 2/ that might be produced during oil-shale retorting could be mobilized to the environment and made available to the cells of living organisms, including humans. Particulate Ni/sub 5/As/sub 2/ was found to be 12 times more soluble in culture growth medium than in distilled water, and much more soluble in solutions of amino acids, inorganic salts, organic constituents of culture medium, and 15% calf serum. These observations suggest Ni/sub 5/As/sub 2/ particles in airborne dust would be dissolved when they came in contact with the biological fluids of the lung and gastrointestinal tract. The availability to cells of the soluble products of Ni/sub 5/As/sub 2/ was demonstrated by measuring its effects on cell proliferation. As little as 1 ppm soluble Ni/sub 5/As/sub 2/ retarded Chinese hamster (CHO) cell proliferation in culture, and 4 ppm resulted in cell death. Flow cytometry measurements indicated there was a preferential cytotoxic effect on S-phase cells. Despite this, many cells survived to form colonies, causing cell generations. This did not appear to be the case, however, for no mutations could be detected at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in cells that survived the cytotoxic effects.

  2. Mutagenesis testing with mammalian cells: validating and adapting a multiple-marker bioassay to activate and detect mutagens in crude samples for energy technology. Progress report, July 1975-September 1980

    SciTech Connect

    Carver, J.H.; Hatch, F.T.

    1980-11-05

    The Chinese hamster ovary (CHO) assay developed during this period offers the unique capability of measuring forward mutation at four gene loci within a single cell line - the autosomal adenine phosphoribosyltransferase (aprt) and thymidine kinase (tk) loci quantified by mutant resistance to azaadenine and fluorodeoxyuridine, as well as the genes involved in resistance to ouabain (Na-K-ATPase) and thioguanine (hypoxanthine-guanine phosphoribosyl transferase, hgprt). This multiple-marker system combines and expands the attributes of the two major mammalian mutagenesis assays currently in use: CHO systems employing the single-locus hgprt assay and mouse L5178Y systems assaying mutation only at the tk locus. Extensive validation carried out during the course of this study indicates that using a combination of loci may increase the reliability and generality of in vitro mammalian mutagenesis assays to detect a variety of mutagens. In particular, the aprt locus offers rapid expression kinetics and minimal technical problems with cell density artifacts and dilution procedures and provides a data base obtained with a known marker for single gene mutation. Preliminary evidence from experiments with plant flavonols suggests that these clastogens producing chromosome aberrations and tetraploidy are detected as mutagens at the tk locus but not at the other three markers. The rapid expression of tk mutants and the possibility that this locus detects a broader spectrum of genetic lesions than do the other markers argues for using tk and aprt loci in combination.

  3. The effect of phorbols on metabolic cooperation between human fibroblasts

    SciTech Connect

    Mosser, D.D.; Bols, N.C.

    1982-01-01

    Autoradiography has been used to study the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), 4-O-methyl TPA, and phorbol on metabolic cooperation between human diploid fibroblasts. When the donors, hypoxanthine-guanine phosphoribosyl transferase+ (HGPRT+) cells, and recipients, HGPRT- cells, were plated together in the presence of (/sup 3/H)hypoxanthine and either 4-O-methyl TPA or phorbol, nearly all interactions that developed in 4 h were positive for metabolic cooperation whereas when high concentrations of TPA were used, the number of positive interactions was significantly less than the control. If the phorbol analogs were added after the donors and recipients had made contact, the number of positive interactions was the same as the control in all cases. However, although primary recipients in the cultures that had been treated with phorbol had the same number of grains as those in the control, primary recipients in cultures that had been treated with TPA or high concentrations of 4-O-methyl TPA had significantly fewer grains than those in the control. TPA treatment for 4 h had no effect on total (/sup 3/H)hypoxanthine incorporation or incorporation into acid-soluble and acid-insoluble fractions. Thus, the effect of TPA on metabolic cooperation is interpreted as a reduction in the transfer of (/sup 3/H)nucleotides and is an indication of an interference with intercellular communication.

  4. A modified intraoral resin mouthguard to prevent self-mutilations in lesch-nyhan patients.

    PubMed

    Ragazzini, Giulia; Delucchi, Alessia; Calcagno, Enrico; Servetto, Roberto; Denotti, Gloria

    2014-01-01

    Lesch-Nyhan syndrome, described in 1964 by Lesch and Nyhan, is a X-linked recessive disorder, occurring in 1 : 100000 to 1 : 380000 live births. LNS is characterized by a decrease in activity of hypoxanthine guanine phosphoribosyl transferase, an enzyme involved in purine metabolism, resulting in overproduction of uric acid. Hyperuricemia and neurological features including choreoathetoid spasticity, self-mutilation, and mental retardation clinically characterize this syndrome. In LNS patients the typical feature is loss of tissue from biting themselves with partial or complete amputation of fingers, lips, and tongue. The self-mutilation compares with the eruption of the deciduous teeth. Several drugs trials have been administered to improve self-destructive behavior and invasive treatment approaches, such as extractions of teeth and orthognathic surgery, have been suggested with variable effectiveness. Nowadays prevention is, therefore, the standard of care. The role of dentistry is essential in the management of the self-mutilating behavior, because the teeth represent the main self-injury instrument. This report presents a revision of various therapeutic approaches to manage self-destruction, highlighting the effectiveness of a preventive treatment. It describes a new technique: a resin mouthguard, realized at Gaslini Hospital, to obtain immediate healing of the oral lesions, confirmed in the follow-up period. PMID:25101126

  5. Lack of evidence for an association between the frequency of mutants or translocations in circulating lymphocytes and exposure to radon gas in the home

    SciTech Connect

    Cole, J.; Green, M.H.L.; Bridges, B.A.

    1996-01-01

    Radon measurements in the living room and main bedroom of 41 houses in the town of Street, Somerset, England have been made. Exposure levels, weighted using the formula of the UK National Radiological Protection Board, of 19-484 Bq m{sup -3} (about half >100 Bq m{sup -3}) were found. Blood samples were obtained from a total of 66 occupants in these homes, and the frequency of genetic alterations in lymphocytes was estimated using two different end points. Gene mutations at the hypoxanthine guanine phosphoribosyl transferase locus were determined in T lymphocytes for 65 subjects using a clonal assay, and the frequency of the BCL-2 t(14;18) translocation, a chromosomal event associated with leukemia/lymphoma, was estimated in lymphocytes using a polymerase chain reaction-based technique for 64 subjects. In neither case was a significant correlation with radon levels in the home found, in contrast to our earlier observation with a smaller series. 52 refs., 5 figs., 2 tabs.

  6. Cytotoxic and mutagenic properties of shale oil byproducts. I. Activation of retort process waters with near ultraviolet light

    SciTech Connect

    Strniste, G.F.; Chen, D.J.

    1981-01-01

    Cultured Chinese hamster ovary (CHO) cells were exposed to dilutions of shale oil retort process waters obtained from three different retorting processes located in the Green River oil shale formations in the western part of the United States. Although the intensity of the response was dictated by thd process water used, all induced a cytotoxic (reduction in colony-forming ability) and mutagenic (induced at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus) response in cells pretreated with dilutions of the waters and subsequent exposure to near ultraviolet light (NUV). Combinations of process water plus NUV yielded mutation frequencies as great as 50% that witnessed for the mutation frequency induced by the potent carcinogen far ultraviolet light. NUV alone was nontoxic and nonmutagenic at the doses of radiation used. Exposure of CHO cells in the dark to nontoxic dilutions of the process waters resulted in small but significant increases in 6-thioguanine resistant mutants. (1-2 time background rates). The biological consequences resulting from the disposal of retort process waters into the delicate environment present in this oil shale region could be further complicated by this photoactivating process.

  7. Selection of suitable reference genes for expression analysis in human glioma using RT-qPCR.

    PubMed

    Grube, Susanne; Göttig, Tatjana; Freitag, Diana; Ewald, Christian; Kalff, Rolf; Walter, Jan

    2015-05-01

    In human glioma research, quantitative real-time reverse-transcription PCR is a frequently used tool. Considering the broad variation in the expression of candidate reference genes among tumor stages and normal brain, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. This study aimed at testing a panel of nine reference genes [beta-2-microglobulin, cytochrome c-1 (CYC1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase, hypoxanthine guanine phosphoribosyl transferase 1, ribosomal protein L13a (RPL13A), succinate dehydrogenase, TATA-box binding protein and 14-3-3 protein zeta] to identify and validate the most suitable reference genes for expression studies in human glioma of different grades (World Health Organization grades II-IV). After analysis of the stability values calculated using geNorm, NormFinder, and BestKeeper algorithms, GAPDH, RPL13A, and CYC1 can be indicated as reference genes applicable for accurate normalization of gene expression in glioma compared with normal brain and anaplastic astrocytoma or glioblastoma alone within this experimental setting. Generally, there are no differences in expression levels and variability of candidate genes in glioma tissue compared to normal brain. But stability analyses revealed just a small number of genes suitable for normalization in each of the tumor subgroups and across these groups. Nevertheless, our data show the importance of validation of adequate reference genes prior to every study. PMID:25862007

  8. Low-radiation environment affects the development of protection mechanisms in V79 cells.

    PubMed

    Fratini, E; Carbone, C; Capece, D; Esposito, G; Simone, G; Tabocchini, M A; Tomasi, M; Belli, M; Satta, L

    2015-05-01

    Very little is known about the influence of environmental radiation on living matter. In principle, important information can be acquired by analysing possible differences between parallel biological systems, one in a reference-radiation environment (RRE) and the other in a low-radiation environment (LRE). We took advantage of the unique opportunity represented by the cell culture facilities at the Gran Sasso National Laboratories of the Istituto Nazionale di Fisica Nucleare, where environment dose rate reduction factors in the underground (LRE), with respect to the external laboratory (RRE), are as follows: 10(3) for neutrons, 10(7) for directly ionizing cosmic rays and 10 for total γ-rays. Chinese hamster V79 cells were cultured for 10 months in both RRE and LRE. At the end of this period, all the cultures were kept in RRE for another 6 months. Changes in the activities of antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPX) and spontaneous mutation frequency at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus were investigated. The results obtained suggest that environmental radiation might act as a trigger of defence mechanisms in V79 cells, specifically those in reference conditions, showing a higher degree of defence against endogenous damage as compared to cells grown in a very low-radiation environment. Our findings corroborate the hypothesis that environmental radiation contributes to the development of defence mechanisms in today living organisms/systems. PMID:25636513

  9. Analysis of in vivo mutation in the Hprt and Tk genes of mouse lymphocytes.

    PubMed

    Dobrovolsky, Vasily N; Shaddock, Joseph G; Heflich, Robert H

    2014-01-01

    Assays measuring mutant frequencies in endogenous reporter genes are used for identifying potentially genotoxic environmental agents and discovering phenotypes prone to genomic instability and diseases, such as cancer. Here, we describe methods for identifying mouse spleen lymphocytes with mutations in the endogenous X-linked hypoxanthine guanine phosphoribosyl transferase (Hprt) gene and the endogenous autosomal thymidine kinase (Tk) gene. The selective clonal expansion of mutant lymphocytes is based upon the phenotypic properties of HPRT- and TK-deficient cells. The same procedure can be utilized for quantifying Hprt mutations in most strains of mice (and, with minor changes, in other mammalian species), while mutations in the Tk gene can be determined only in transgenic mice that are heterozygous for inactivation of this gene. Expanded mutant clones can be further analyzed to classify the types of mutations in the Tk gene (small intragenic mutations vs. large chromosomal mutations) and to determine the nature of intragenic mutation in both the Hprt and Tk genes. PMID:24623234

  10. Comparative mutagenicity of a coal combustion fly ash extract in Salmonella typhimurium and Chinese hamster ovary cells

    SciTech Connect

    Li, A.P.; Clark, C.R.; Hanson, R.L.; Henderson, T.R.; Hobbs, C.H.

    1983-01-01

    The dichloromethane extract of a coal combustion fly ash sample obtained from an experimental fluidized bed coal combustor was tested for mutagenicity in Salmonella typhimurium and cultured Chinese hamster ovary (CHO) cells. The extract was directly mutagenic in S typhimurium strain TA98 and the nitroreductase deficient strains TA98NR and TA98/1,8DNP/sub 6/. The mutagenicity observed in TA98NR and TA98/1,8DNP/sub 6/ was lower than that in TA98. Addition of exogenous Aroclor 1254-induced rat liver supernatant (liver S9) decreased the bacterial mutagenicity of the extract. A different mutagenic response was observed in CHO cells. In the absence of liver S9, although the extract was cytotoxic to CHO cells, no significant mutagenicity was observed. Addition of exogenous liver S9 decreased the cytotoxicity and increased the mutagenicity at both Na/sup +/-K/sup +/-ATPase and hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene loci in CHO cells. Using gas chromatography/mass spectrometry (GC/MS) and tandem quadruple mass spectrometry, a number of polynuclear aromatic hydrocarbons (PAHs) and nitrated PAHs (nitro-PAHs) were tentatively identified and quantitated. A possible explanation of the difference in bacterial and mammalian mutagenicity of the extract is that the bacterial mutagenicity was induced by the nitro-PAHs that are potent bacterial mutagens and mammalian mutagenicity was induced by both PAHs and nitro-PAHs that are promutagens.

  11. Effects of cell cycle position on ionizing radiation mutagenesis. I. Quantitative assays of two genetic loci in a human lymphoblastoid cell line

    SciTech Connect

    Chuang, Yao-Yu; Liber, H.L.

    1996-11-01

    Relatively little work has been done on the influence of the position of the cell in the cell cycle on ionizing radiation-induced mutagenesis. We synchronized WTK1 human lymphoblastoid cells with 200 {mu}M lovastatin for 48 h; under these conditions more than 80% of the cells were arrested in G{sub 1} phase. Upon release, there was a 12-15-h lag followed by movement of a large fraction into S phase. We irradiated cells with either 1.5 Gy X rays at 1, 15, 18, 21 or 24 h or 1.5 Gy {gamma} rays at 1, 5, 10, 15 or 24 h after release from lovastatin. We showed that WTK1 cells were most sensitive to ionizing radiation-induced toxicity in G{sub 1} and into S phase, and more resistant in mid to late S and G{sub 2}/M phase. Somewhat surprisingly, we found that the two different gene loci had different sensitivities to radiation-induced mutation through the cell cycle. Cells in late G{sub 1} through mid-S phase were most sensitive to radiation-induced mutations at the autosomal thymidine kinase (TK) locus, whereas G{sub 1} phase was the most sensitive phase at the X-linked hypoxanthine guanine phosphoribosyl transferase (HPRT) locus. 29 refs., 6 figs., 1 tab.

  12. Genotype-phenotype correlations in neurogenetics: Lesch-Nyhan disease as a model disorder.

    PubMed

    Fu, Rong; Ceballos-Picot, Irene; Torres, Rosa J; Larovere, Laura E; Yamada, Yasukazu; Nguyen, Khue V; Hegde, Madhuri; Visser, Jasper E; Schretlen, David J; Nyhan, William L; Puig, Juan G; O'Neill, Patrick J; Jinnah, H A

    2014-05-01

    Establishing meaningful relationships between genetic variations and clinical disease is a fundamental goal for all human genetic disorders. However, these genotype-phenotype correlations remain incompletely characterized and sometimes conflicting for many diseases. Lesch-Nyhan disease is an X-linked recessive disorder that is caused by a wide variety of mutations in the HPRT1 gene. The gene encodes hypoxanthine-guanine phosphoribosyl transferase, an enzyme involved in purine metabolism. The fine structure of enzyme has been established by crystallography studies, and its function can be measured with very precise biochemical assays. This rich knowledge of genetic alterations in the gene and their functional effect on its protein product provides a powerful model for exploring factors that influence genotype-phenotype correlations. The present study summarizes 615 known genetic mutations, their influence on the gene product, and their relationship to the clinical phenotype. In general, the results are compatible with the concept that the overall severity of the disease depends on how mutations ultimately influence enzyme activity. However, careful evaluation of exceptions to this concept point to several additional genetic and non-genetic factors that influence genotype-phenotype correlations. These factors are not unique to Lesch-Nyhan disease, and are relevant to most other genetic diseases. The disease therefore serves as a valuable model for understanding the challenges associated with establishing genotype-phenotype correlations for other disorders. PMID:23975452

  13. Protection against radiation-induced mutations at the hprt locus by spermine and N,N{double_prime}-(dithiodi-2,1-ethanediyl)bis-1,3-propanediamine (WR-33278)

    SciTech Connect

    Grdina, D.J.; Schwartz, J.L. |; Shigematsu, N.

    1993-06-01

    The polyamine spermine and the disulfide NN{double_prime}-(dithiodi-2,1-ethanediyl)bis-1,3-propanediamine (WR-33278) are structurally similar agents capable of binding to DNA. WR-33278 is the disulfide moiety of the clinically studied radioprotective agent (WR-2721). Because of their structural similarities, it was of interest to characterize and compare their radioprotective properties using the endpoints of cell survival and mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in Chinese hamster AA8 cells. In order to facilitate both the uptake of VM-33278 into cells and the direct comparison between the protective properties of WR-33278 and spermine, these agents were electroporated into cells. Electroporation alone reduced cell survival to 75% but had no effect on hprt mutation frequency. The electroporation of either spermine or WR-33278 at concentrations greater than 0.01 mM was extremely toxic. The exposure of cells to both electroporation and irradiation gave rise to enhanced cell killing and mutation induction. Cell survival values at a radiation dose of 750 cGy were enhanced by factors of 1.3 and 1.8 following electroporation of 0.01 mM of spermine and WR-33278, respectively, 30 min prior to irradiation. Neither agent was protective at a concentration of 0.001 mM. Protection against radiation-induced hprt mutations was observed for both spermine and WR-33278 under all experimental conditions tested.

  14. Induction of sister chromatid exchanges by direct and indirect chemical agents in a human teratoma cell line

    SciTech Connect

    Murison, G. . Dept. of Biological Sciences)

    1989-01-01

    In the present work, we have extended the characterization of the P3 cell line, derived from a human epithelial teratocarcinoma, by studying the induction of sister chromatid exchanges (SCEs) by direct and indirect carcinogens. Several direct-acting carcinogens produce a dose-dependent increase in SCEs. Most notably, N-methyl-N{prime}-nitro-N-nitrosoguanidine and 7{beta}, 8{alpha}-dihydroxy-9 {alpha},10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene produce increases in SCEs at dosages comparable to those used to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase locus. The indirect carcinogens elicit SCEs only when the P3 cells are cocultured with cells capable of metabolizing the indirect carcinogens to the active form. Human breast carcinoma (BJ-015) and rat hepatoma (RL12) cells are equally efficient in activating polycyclic aromatic hydrocarbons to the active form. This cell-mediated induction of SCEs is obtained when P3 cells are incubated with live, x-irradiated, or UV-irradiated BJ or RL cells. This P3 cell line is thus equally suitable to study the induction of mutations or the induction of SCEs with direct and indirect carcinogens. 35 refs., 3 tabs.

  15. Validation of housekeeping genes in the brains of rats submitted to chronic intermittent hypoxia, a sleep apnea model.

    PubMed

    Julian, Guilherme Silva; de Oliveira, Renato Watanabe; Perry, Juliana Cini; Tufik, Sergio; Chagas, Jair Ribeiro

    2014-01-01

    Obstructive sleep apnea (OSA) is a syndrome characterized by intermittent nocturnal hypoxia, sleep fragmentation, hypercapnia and respiratory effort, and it has been associated with several complications, such as diabetes, hypertension and obesity. Quantitative real-time PCR has been performed in previous OSA-related studies; however, these studies were not validated using proper reference genes. We have examined the effects of chronic intermittent hypoxia (CIH), which is an experimental model mainly of cardiovascular consequences of OSA, on reference genes, including beta-actin, beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine guanine phosphoribosyl transferase and eukaryotic 18S rRNA, in different areas of the brain. All stability analyses were performed using the geNorm, Normfinder and BestKeeper software programs. With exception of the 18S rRNA, all of the evaluated genes were shown to be stable following CIH exposure. However, gene stability rankings were dependent on the area of the brain that was analyzed and varied according to the software that was used. This study demonstrated that CIH affects various brain structures differently. With the exception of the 18S rRNA, all of the tested genes are suitable for use as housekeeping genes in expression analyses. PMID:25289636

  16. Ionizing radiation-induced 6-thioguanine-resistant clones in synchronous CHO cells

    SciTech Connect

    Burki, J.

    1980-01-01

    When cultured Chinese hamster ovary (CHO) cells are exposed to acute doses of ionizing radiation at different times during the cell division cycle, there is a characteristic cell-cycle response for radiation-induced cell killing and induced resistance to 6-thio-guanine (6TG). For cell killing the sensitive periods of the cell cycle are the G1, G2, M, and early S periods, as others have reported. For mutation induction the sensitive stage is the G1 period with the maximum sensitivity near the boundary between the G1 and the S period. Cells appear to be very refractile to induction of 6TG resistance in other periods of the cell cycle. These results suggest that chromosomal rearrangements of the X chromosome are most likely to occur in the G1 period before the gene for hypoxanthine-guanine-phosphoribosyl-transferase replicates, most likely due to genetic recombination. Clones resistant to 6TG after exposure to x rays are most likely induced by a different mutagenic pathway than ones stimulated by ultraviolet (uv) or ethylnitrosourea treatments, since the mutation induction patterns in the cell cycle are quite different.

  17. The human T-cell cloning assay: identifying genotypes susceptible to drug toxicity and somatic mutation.

    PubMed

    Hou, Sai-Mei

    2014-01-01

    Humans exhibit marked genetic polymorphisms in drug metabolism that contribute to high incidence of adverse effects in susceptible individuals due to altered balance between metabolic activation and detoxification. The T-cell cloning assay, which detects mutations in the gene for hypoxanthine-guanine phosphoribosyl transferase (HPRT), is the most well-developed reporter system for studying specific locus mutation in human somatic cells. The assay is based on a mitogen- and growth factor-dependent clonal expansion of peripheral T-lymphocytes in which the 6-thioguanine-resistant HPRT mutants can be selected, enumerated, and collected for molecular analysis of the mutational nature. The assay provides a unique tool for studying in vivo and in vitro mutagenesis, for investigating the functional impact of common polymorphism in metabolism and repair genes, and for identifying risk genotypes for drug-induced toxicity and mutagenicity. This chapter presents a simple and reliable method for the enumeration of HPRT mutant frequency induced in vitro without using any source of recombinant interleukin-2. The other main feature is that only truly induced and unique mutants are collected for further analysis. PMID:24623236

  18. Comparative mutagenicity of a coal combustion fly ash extract in Salmonella typhimurium and chinese hamster ovary cells

    SciTech Connect

    Li, A.P.; Clark, C.R.; Hanson, R.L.; Henderson, T.R.; Hobbs, C.H.

    1983-01-01

    The dichloromethane extract of a coal combustion fly ash sample obtained from an experimental fluidized bed coal combustor was tested for mutagenicity in Salmonella typhimurium and cultured Chinese hamster ovary (CHO) cells. The extract was directly mutagenic in S typhimurium strain TA98 and the nitroreducatase deficient strains TA98NR and TA98/1,8DNP/sub 6/. The mutagenicity observed in TA98NR and TA98/1,8DNP/sub 6/ was lower than that in TA98. Addition of exogenous Aroclor 1254-induced rat liver supernatant (liver S9) decreased the bacterial mutagenicity of the extract. A different mutagenic response was observed in CHO cells. In the absence of liver S9, although the extract was cytotoxic to CHO cells, no significant mutagenicity was observed. Addition of exogenous liver S9 decreased the cytotoxicity and increased the mutagenicity at both Na/sup +/ -K/sup +/ -ATPase and hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene loci in CHO cells. Using gas chromatography/mass spectrometry (GC/MS) and tandem quadruple mass spectrometry, a number of polynuclear aromatic hydrocarbons (PAHs) and nitrated PAHs (nitro-PAHs) were tentatively identified and quantitated. A possible explanation of the difference in bacterial and mammalian mutagenicity of the extract is that the bacterial mutagenicity was induced by the nitro-PAHs that are potent bacterial mutagens and mammalian mutagenicity was induced by both PAHs and nitro-PAHs that are promutagens.

  19. Comparison of 6-thioguanine-resistant mutation and sister chromatid exchanges in Chinese hamster V79 cells with forty chemical and physical agents

    SciTech Connect

    Nishi, Y.; Hasegawa, M.M.; Taketomi, M.; Ohkawa, Y.; Inui, N.

    1984-08-01

    The induction of sister chromatid exchanges (SCE) and mutation at the hypoxanthine-guanine phosphoribosyl transferase locus and toxicities of 40 different chemical and physical agents were examined on Chinese hamster V79 cells. These agents included mono-, di-, tri-, and polyfunctional alkylating agents, intercalators, gamma-rays, and UV light irradiation. Mutation was measured as resistance to 6-thioguanine and toxicity as loss of cell-plating efficiency. SCE were examined 29 hr after treatment. With the agents examined, a highly positive correlation existed between SCE-inducing and mutagenic potencies, when expressed as increase in the number per a unit dose over the control values. But the great difference of the ratios of mutagenic potencies versus SCE-inducing potencies among agents was observed, the maximal difference in the ratios being about 200-fold. The agents that showed the higher values of the ratio (agents producing more mutations than SCE) were bleomycin, cobalt-60 gamma-rays, all ethylating agents (N-ethyl-N-nitrosourea, N-ethyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, and diethylsulfate), N-propyl-N-nitrosourea, N-butyl-N-nitrosourea, isopropyl methanesulfonate, intercalating acridine compounds (2-methoxy-6-chloro-9-(3-(ethyl-2-chloroethyl)aminopropylamino)-acridine X 2HCl and 2-methoxy-6-chloro-9-(3-(chloroethyl)-aminopropylamino)acridine 2HCl) and UV light at 254 nm.

  20. Toxicologic responses to a complex coal conversion by-product: mammalian cell mutagenicity and dermal carcinogenicity

    SciTech Connect

    Cunningham, M.L.; Haugen, D.A.; Kirchner, F.R.; Reilly, C.A. Jr.

    1984-01-01

    In the present study, we measured mutagenicity and cytotoxicity in hamster and human cells in vitro and tumorigenicity in mouse skin in vivo. The Chinese hamster ovary cell/hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT) assay has proven useful in estimating the mutagenic activity of pure compounds but has been used only to a limited extent with complex mixtures. The human teratocarcinoma cell line, designated P/sub 3/, used in these studies has recently been adapted for use in mutagenesis assays of individual compounds but has not previously been used to evaluate mutagenesis by complex mixtures. In this report, we compare the responses of the hamster and human cell lines and the mouse skin to chemical class fractions of a complex organic by-product condensate (tar) of coal gasification. The composition of this complex tar is chemically similar to that of petroleum-derived tars and products of fossil fuel combustion. By testing basic, acidic, and neutral chemical class fractions of the complex tar, we demonstrated that the predominant genotoxic components were present in the neutral fraction as measured both in the CHO/HGPRT and dermal carcinogenicity assays. The human P/sub 3/ cells were less sensitive for mutagenesis and cytotoxicity than were the rodent cells. Furthermore, fractionation and bioassay provided evidence for interactive effects that indicate the importance of combining chemical characterization and toxicologic evaluation of complex mixtures. 25 references, 1 figure, 2 tables.

  1. Genotoxicity of phthalates.

    PubMed

    Erkekoglu, Pınar; Kocer-Gumusel, Belma

    2014-12-01

    Many of the environmental, occupational and industrial chemicals are able to generate reactive oxygen species (ROS) and cause oxidative stress. ROS may lead to genotoxicity, which is suggested to contribute to the pathophysiology of many human diseases, including inflammatory diseases and cancer. Phthalates are ubiquitous environmental chemicals and are well-known peroxisome proliferators (PPs) and endocrine disruptors. Several in vivo and in vitro studies have been conducted concerning the carcinogenic and mutagenic effects of phthalates. Di(2-ethylhexyl)-phthalate (DEHP) and several other phthalates are shown to be hepatocarcinogenic in rodents. The underlying factor in the hepatocarcinogenesis is suggested to be their ability to generate ROS and cause genotoxicity. Several methods, including chromosomal aberration test, Ames test, micronucleus assay and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutation test and Comet assay, have been used to determine genotoxic properties of phthalates. Comet assay has been an important tool in the measurement of the genotoxic potential of many chemicals, including phthalates. In this review, we will mainly focus on the studies, which were conducted on the DNA damage caused by different phthalate esters and protection studies against the genotoxicity of these chemicals. PMID:25174766

  2. Escape from Het-6 Incompatibility in Neurospora Crassa Partial Diploids Involves Preferential Deletion within the Ectopic Segment

    PubMed Central

    Smith, M. L.; Yang, C. J.; Metzenberg, R. L.; Glass, N. L.

    1996-01-01

    Self-incompatible het-6(OR)/het-6(PA) partial diploids of Neurospora crassa were selected from a cross involving the translocation strain, T(IIL -> IIIR)AR18, and a normal sequence strain. About 25% of the partial diploids exhibited a marked increase in growth rate after 2 weeks, indicating that ``escape'' from het-6 incompatibility had occurred. Near isogenic tester strains with different alleles (het-6(OR) and het-6(PA)) were constructed and used to determine that 80 of 96 escape strains tested were het-6(PA), retaining the het-6 allele found in the normal-sequence LGII position; 16 were het-6(OR), retaining the allele in the translocated position. Restriction fragment length polymorphisms in 45 escape strains were examined with probes made from cosmids that spanned the translocated region. Along with electrophoretic analysis of chromosomes from three escape strains, RFLPs showed that escape is associated with deletion of part of one or the other of the duplicated DNA segments. Deletions ranged in size from ~70 kbp up to putatively the entire 270-kbp translocated region but always included a 35-kbp region wherein we hypothesize het-6 is located. The deletion spectrum at het-6 thus resembles other cases where mitotic deletions occur such as of tumor suppressor genes and of the hprt gene (coding for hypoxanthine-guanine phosphoribosyl-transferase) in humans. PMID:8889517

  3. A straightforward radiometric technique for measuring IMP dehydrogenase.

    PubMed

    Cooney, D A; Wilson, Y; McGee, E

    1983-04-15

    [2-3H]Inosinic acid ([2-3H]IMP) has been biosynthesized in good yield from [2-3H]hypoxanthine and PRPP via the action of a partially purified preparation of hypoxanthine/guanine phosphoribosyl transferase from mouse brain. The product was purified in one step by ascending paper chromatography, and used to assess the activity of IMP dehydrogenase. To conduct the assay, tritiated substrate is admixed with enzyme in a final volume of 10 microliters; NAD is present to serve as cofactor for the reaction, and allopurinol to inhibit the oxidation of any hypoxanthine generated as a consequence of side reactions. After an appropriate period of incubation, the 3H2O arising from the oxidation of tritiated IMP via [3H]NAD is isolated by quantitative microdistillation. Performed as described, the assay is facile, sensitive, and accurate, with the capability of detecting the dehydrogenation of as little as 1 pmol of [3H]IMP. Using it, measurements have been made of IMP dehydrogenase in a comprehensive array of mouse organs. Of these, pancreas contained the enzyme at the highest specific activity. PMID:6135372

  4. Quantitative and molecular analyses of mutation in a pSV2gpt transformed CHO cell line

    SciTech Connect

    Stankowski, L.F. Jr.; Tindall, K.R.; Hsie, A.W.

    1983-01-01

    Following NDA-mediated gene transfer we have isolated a cell line useful for studying gene mutation at the molecular level. This line, AS52, derived from a hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficient Chinese hamster ovary (CHO) cell line, carries a single copy of the E. coli xanthine-guanine phosphoribosyl transferase (XGPRT) gene (gpt) and exhibits a spontaneous mutant frequency of 20 TG/sup r/ mutants/10/sup 6/ clonable cells. As with HGPRT/sup -/ mutants, XGPRT/sup -/ mutants can be selected in 6-thioguanine. AS52 (XGPRT/sup +/) and wild type CHO (HGPRT/sup +/) cell exhibit almost identical cytotoxic responses to various agents. We observed significant differences in mutation induction by UV light and ethyl methanesulfonate (EMS). Ratios of XGPRT/sup -/ to HGPRT/sup -/ mutants induced per unit dose (J/m/sup 2/ for UV light and ..mu..g/ml for EMS) are 1.4 and 0.70, respectively. Preliminary Southern blot hybridization analyses has been performed on 30 XGPRT/sup -/ AS52 mutants. A majority of spontaneous mutants have deletions ranging in size from 1 to 4 kilobases (9/19) to complete loss of gpt sequences (4/19); the remainder have no detectable (5/19) or only minor (1/19) alterations. 5/5 UV-induced and 5/6 EMS-induced mutants do not show a detectable change. Similar analyses are underway for mutations induced by x-irradiation and ICR 191 treatment.

  5. Structural and biochemical analyses of the catalysis and potency impact of inhibitor phosphoribosylation by human nicotinamide phosphoribosyltransferase.

    PubMed

    Oh, Angela; Ho, Yen-Ching; Zak, Mark; Liu, Yongbo; Chen, Xukun; Yuen, Po-Wai; Zheng, Xiaozhang; Liu, Yichin; Dragovich, Peter S; Wang, Weiru

    2014-05-26

    Prolonged inhibition of nicotinamide phosphoribosyltransferase (NAMPT) is a strategy for targeting cancer metabolism. Many NAMPT inhibitors undergo NAMPT-catalyzed phosphoribosylation (pRib), a property often correlated with their cellular potency. To understand this phenomenon and facilitate drug design, we analyzed a potent cellularly active NAMPT inhibitor (GNE-617). A crystal structure of pRib-GNE-617 in complex with NAMPT protein revealed a relaxed binding mode. Consistently, the adduct formation resulted in tight binding and strong product inhibition. In contrast, a biochemically equipotent isomer of GNE-617 (GNE-643) also formed pRib adducts but displayed significantly weaker cytotoxicity. Structural analysis revealed an altered ligand conformation of GNE-643, thus suggesting weak association of the adducts with NAMPT. Our data support a model for cellularly active NAMPT inhibitors that undergo NAMPT-catalyzed phosphoribosylation to produce pRib adducts that retain efficient binding to the enzyme. PMID:24797455

  6. Three-dimensional structure of phosphoribosyl pyrophosphate synthetase from E. coli at 2.71 Å resolution

    NASA Astrophysics Data System (ADS)

    Timofeev, V. I.; Abramchik, Yu. A.; Zhukhlistova, N. E.; Muravieva, T. I.; Esipov, R. S.; Kuranova, I. P.

    2016-01-01

    Phosphoribosyl pyrophosphate synthetase from Escherichia coli was cloned, purified, and crystallized. Single crystals of the enzyme were grown under microgravity. The X-ray diffraction data set was collected at the Spring-8 synchrotron facility and used to determine the three-dimensional structure of the enzyme by the molecular-replacement method at 2.71 Å resolution. The active and regulatory sites in the molecule of E. coli phosphoribosyl pyrophosphate synthetase were revealed by comparison with the homologous protein from Bacillus subtilis, the structure of which was determined in a complex with functional ligands. The conformations of polypeptide-chain fragments surrounding and composing the active and regulatory sites were shown to be identical in both proteins.

  7. Phosphoribosyl pyrophosphate synthetase activity affects growth and riboflavin production in Ashbya gossypii

    PubMed Central

    Jiménez, Alberto; Santos, María A; Revuelta, José L

    2008-01-01

    Background Phosphoribosyl pyrophosphate (PRPP) is a central compound for cellular metabolism and may be considered as a link between carbon and nitrogen metabolism. PRPP is directly involved in the de novo and salvage biosynthesis of GTP, which is the immediate precursor of riboflavin. The industrial production of this vitamin using the fungus Ashbya gossypii is an important biotechnological process that is strongly influenced by substrate availability. Results Here we describe the characterization and manipulation of two genes of A. gossypii encoding PRPP synthetase (AGR371C and AGL080C). We show that the AGR371C and AGL080C gene products participate in PRPP synthesis and exhibit inhibition by ADP. We also observed a major contribution of AGL080C to total PRPP synthetase activity, which was confirmed by an evident growth defect of the Δagl080c strain. Moreover, we report the overexpression of wild-type and mutant deregulated isoforms of Agr371cp and Agl080cp that significantly enhanced the production of riboflavin in the engineered A. gossypii strains. Conclusion It is shown that alterations in PRPP synthetase activity have pleiotropic effects on the fungal growth pattern and that an increase in PRPP synthetase enzymatic activity can be used to enhance riboflavin production in A. gossypii. PMID:18782443

  8. In vivo footprint analysis and genomic sequencing of the human hypoxanthine-phosphoribosyl transferase (HPRT) 5 prime region on the active and inactive X chromosome

    SciTech Connect

    Hornstra, I.K.; Yang, T.P. )

    1991-03-11

    In female placental mammals, one of the two X chromosome in each somatic cell is randomly inactivated during female embryogenesis as a mechanism for dosage compensation. Once a given X chromosome is inactivated, all mitotic progeny maintain the same X chromosome in the inactive state. DNA-protein interactions and DNA methylation are hypothesized to maintain this allele-specific system of differential gene expression. Ligation-mediated polymerase chain reaction (LMPCR) in vivo footprinting and genomic sequencing were used to study DNA-protein interactions and DNA-methylation within the 5{prime} region of the X-linked human HPRT gene on the active and inactive X chromosomes. In vivo footprint analysis reveals at least one DNA-protein interaction specific to the active HPRT allele in human male fibroblast cells and hamster-human hybrid cells containing only the active human X chromosome. In the region examined, all CpG dinucleotides are methylated on the inactive HPRT allele and unmethylated on the active X allele in hamster-human hybrid cells carrying either the inactive or active human X chromosome, respectively. Thus, DNA-methylation may be mediating the differential binding of sequence-specific DNA-binding proteins to the active or inactive HPRT alleles.

  9. [Effect of co-expression of nicotinic acid phosphoribosyl transferase and pyruvate carboxylase on succinic acid production in Escherichia coli BA002].

    PubMed

    Cao, Weijia; Gou, Dongmei; Liang, Liya; Liu, Rongming; Chen, Kequan; Ma, Jiangfeng; Jiang, Min

    2013-12-01

    Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD+. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate was accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E. coli BA016 was investigated. Results in 3 L fermentor showed that OD600 is 4.64 and BA016 consumed 35.00 g/L glucose and produced 25.09 g/L succinate after 112 h under anaerobic conditions. Overexpression of pncB and pyc in BA016, the accumulation of pyruvic acid was further decreased, and the formation of succinic acid was further increased. PMID:24660633

  10. Identification and analysis of residues contained on β → α loops of the dual-substrate (βα)8 phosphoribosyl isomerase A specific for its phosphoribosyl anthranilate isomerase activity

    PubMed Central

    Noda-García, Lianet; Camacho-Zarco, Aldo R; Verdel-Aranda, Karina; Wright, Helena; Soberón, Xavier; Fülöp, Vilmos; Barona-Gómez, Francisco

    2010-01-01

    A good model to experimentally explore evolutionary hypothesis related to enzyme function is the ancient-like dual-substrate (βα)8 phosphoribosyl isomerase A (PriA), which takes part in both histidine and tryptophan biosynthesis in Streptomyces coelicolor and related organisms. In this study, we determined the Michaelis–Menten enzyme kinetics for both isomerase activities in wild-type PriA from S. coelicolor and in selected single-residue monofunctional mutants, identified after Escherichia coli in vivo complementation experiments. Structural and functional analyses of a hitherto unnoticed residue contained on the functionally important β → α loop 5, namely, Arg139, which was postulated on structural grounds to be important for the dual-substrate specificity of PriA, is presented for the first time. Indeed, enzyme kinetics analyses done on the mutant variants PriA_Ser81Thr and PriA_Arg139Asn showed that these residues, which are contained on β → α loops and in close proximity to the N-terminal phosphate-binding site, are essential solely for the phosphoribosyl anthranilate isomerase activity of PriA. Moreover, analysis of the X-ray crystallographic structure of PriA_Arg139Asn elucidated at 1.95 Å herein strongly implicates the occurrence of conformational changes in this β → α loop as a major structural feature related to the evolution of the dual-substrate specificity of PriA. It is suggested that PriA has evolved by tuning a fine energetic balance that allows the sufficient degree of structural flexibility needed for accommodating two topologically dissimilar substrates—within a bifunctional and thus highly constrained active site—without compromising its structural stability. PMID:20066665

  11. Functional specialization of one copy of glutamine phosphoribosyl pyrophosphate amidotransferase in ureide production from symbiotically fixed nitrogen in Phaseolus vulgaris.

    PubMed

    Coleto, Inmaculada; Trenas, Almudena T; Erban, Alexander; Kopka, Joachim; Pineda, Manuel; Alamillo, Josefa M

    2016-08-01

    Purines are essential molecules formed in a highly regulated pathway in all organisms. In tropical legumes, the nitrogen fixed in the nodules is used to generate ureides through the oxidation of de novo synthesized purines. Glutamine phosphoribosyl pyrophosphate amidotransferase (PRAT) catalyses the first committed step of de novo purine synthesis. In Phaseolus vulgaris there are three genes coding for PRAT. The three full-length sequences, which are intron-less genes, were cloned, and their expression levels were determined under conditions that affect the synthesis of purines. One of the three genes, PvPRAT3, is highly expressed in nodules and protein amount and enzymatic activity in these tissues correlate with nitrogen fixation activity. Inhibition of PvPRAT3 gene expression by RNAi-silencing and subsequent metabolomic analysis of the transformed roots shows that PvPRAT3 is essential for the synthesis of ureides in P. vulgaris nodules. PMID:27004600

  12. Use of HeLa cell guanine nucleotides by Chlamydia psittaci.

    PubMed Central

    Ceballos, M M; Hatch, T P

    1979-01-01

    Exogenous guanine was found to be incorporated into the nucleic acids of Chlamydia psittaci when the parasite was grown in HeLa cells containing hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8) activity but not when the parasite was grown in transferase-deficient HeLa cells. No evidence for a chlamydia-specific transferase activity was found in either transferase-containing or transferase-deficient infected HeLa cells. It is concluded that C. psittaci is incapable of metabolizing guanine, but that the parasite can use host-generated guanine nucleotides as precursors for nucleic acid synthesis. Images PMID:478649

  13. WR-2721 protects against cytoxan-induced hprt mutagenesis without affecting therapeutic effectiveness

    SciTech Connect

    Kataoka, Yasushi; Perrin, J.; Hunter, N.; Milas, L.; Grdina, D. ||

    1995-12-31

    The radioprotector S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) was evaluated for its ability to protect against cytoxan-induced mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in mouse splenocytes under conditions that would not interfere with the therapeutic effectiveness of cytoxan in the treatment of fibrosarcoma lung tumors. Mutations at the hprt locus increase in frequency as a function of the dose of cytoxan used. With a spontaneous mutation frequency in C3H mice of 1.5 {times} 10{sup {minus}6}, mutation frequencies increased from 6.2 {times} 10{sup {minus}6} to 2.0 {times} 10{sup {minus}5} as the dose of cytoxan increased from 50 to 200 mg/kg. C3H male mice were injected in their tail veins with 3.5 {times} 10{sup 5} viable fibrosarcoma (FSa) cells. This protocol gave rise to an average of 68 tumor colonies per mouse. Four days following injection animals were treated with cytoxan at a dose of 100 mg/kg, which gave rise to significant tumor cell killing and a reduction in tumor colony number to less than an average of one per animal. WR-2721 at a concentration of 100 mg/kg did not affect on cytoxan`s therapeutic effectiveness. However, a 100 mg/kg dose of WR-2721 was effective in reducing the cytoxan induced hprt mutation frequency in mice from 160 to 35 per 10{sup 5} viable cells regardless of whether it was administered 30 min before or 2 h following cytoxan treatment.

  14. Charged-particle mutagenesis II. Mutagenic effects of high energy charged particles in normal human fibroblasts

    NASA Astrophysics Data System (ADS)

    Chen, D. J.; Tsuboi, K.; Nguyen, T.; Yang, T. C.

    1994-10-01

    The biological effects of high LET charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/μm to 975 KeV/gmm with particle energy (on the cells) between 94 - 603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/μm. The inactivation cross-section (αi) and the action-section for mutant induction (αm) ranged from 2.2 to 92.0 μm2 and 0.09 to 5.56 × 10-3 μm2, respectively. The maximum values were obtained by 56Fe with an LET of 200 keV/μm. The mutagenicity (αm/αi) ranged from 2.05 to 7.99 × 10-5 with the maximum value at 150 keV/μm. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.

  15. Chemical characterization and mutagenic properties of polycyclic aromatic compounds in sediment from tributaries of the Great Lakes

    USGS Publications Warehouse

    Fabacher, David L.; Schmitt, Christopher J.; Besser, John M.; Mac, Michael J.

    1988-01-01

    Sediments from four inshore industrial sites and a reference site in the Great Lakes were extracted with solvents and characterized chemically for polycyclic aromatic compounds (PACs). An aqueous phase and a crude organic extract were obtained. The crude organic extract was further resolved into fractions A-2 (polycyclic aromatic hydrocarbons) and A-3 (nitrogen-containing polycyclic aromatic compounds), which were analyzed for PACs by gas chromatography and gas chromatography-mass spectrometry. The extracts and fractions were tested for mutagenicity in three assays: Ames, rat hepatocyte unscheduled DNA synthesis, and Chinese hamster ovary hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT). Sediments from the industrial sites contained 27 to 363 μg/g total PACs; the reference site, less than 1 μg/g. Qualitative differences in the residue profiles among the sites were attributable to the probable sources of the PACs (petroleum versus combustion). Only one industrial site yielded measurable (0.1 μg/g or more) concentrations of individual nitrogen-containing PACs. In the Ames assay, only the highest doses of the A-2 fractions from two sites approached positive results. Conversely, the crude organic extract and A-2 and A-3 fractions from all sites induced unscheduled DNA synthesis. Crude organic extracts and the A-2 and A-3 fractions from all industrial sites gave well-defined dose-response relations in the CHO/HGPRT assay. We established the presence of chemical mutagens in sediment that could be correlated with neoplasms in fish from many of the sites; however, the mutagenicity of the sediment extracts was not completely related to the degree of contamination by PACs. We also discuss the utility of mutagenicity assays in the evaluation of complex chemical mixtures and recommend the use of a CHO/HGPRT-type assay in which cells are not required to proliferate in the presence of potential interfering chemicals.

  16. Evaluation of the genotoxicity of process stream extracts from a coal gasification system.

    PubMed

    Shimizu, R W; Benson, J M; Li, A P; Henderson, R F; Brooks, A L

    1984-01-01

    Extracts of three complex organic environmental mixtures, two from an experimental coal gasifier (a raw gas and a clean gas sample) and one from a coke oven main, were examined for genotoxicity. Three short-term genotoxicity assay systems were used: Ames Salmonella typhimurium reverse mutation assay, Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) gene locus mutation assay, and the Chinese hamster lung primary culture/sister chromatid exchange (CHL/SCE) assay. Aroclor-1254-induced rat liver homogenate fraction (S-9) was required to observe genotoxicity in both gene locus mutation assays (CHO/HGPRT and Ames). The relative survival of CHO cells exposed to extracts was highest in cells exposed to clean gas samples, with the raw gas sample being the most cytotoxic either with or without the addition of S-9. All three complex mixtures induced sister chromatid exchanges in primary lung cell cultures without the addition of S-9. The relative genotoxicity ranking of the samples varied between the mammalian and prokaryotic assay systems. Coke oven main extract produced fewer revertants in bacteria than the raw gas sample. However, the coke oven main extract was more genotoxic in the two eukaryotic systems (CHL/SCE and CHO/HGPRT) than was the raw gas sample. The results of all three assays indicate that the cleanup process used in the experimental gasifier was effective in decreasing the genotoxic materials in the process stream. These data also reemphasize the necessity of evaluating genotoxicity of complex mixtures in a variety of short-term systems. PMID:6389110

  17. Charged-particle mutagenesis II. Mutagenic effects of high energy charged particles in normal human fibroblasts

    NASA Technical Reports Server (NTRS)

    Chen, D. J.; Tsuboi, K.; Nguyen, T.; Yang, T. C.

    1994-01-01

    The biological effects of high LET charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/micrometer to 975 KeV/micrometer with particle energy (on the cells) between 94-603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/micrometer. The inactivation cross-section (alpha i) and the action cross-section for mutant induction (alpha m) ranged from 2.2 to 92.0 micrometer2 and 0.09 to 5.56 x 10(-3) micrometer2, respectively. The maximum values were obtained by 56Fe with an LET of 200 keV/micrometer. The mutagenicity (alpha m/alpha i) ranged from 2.05 to 7.99 x 10(-5) with the maximum value at 150 keV/micrometer. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.

  18. Charged-particle mutagenesis 2. Mutagenic effects of high energy charged particles in normal human fibroblasts

    NASA Technical Reports Server (NTRS)

    Chen, D. J.; Tsuboi, K.; Nguyen, T.; Yang, T. C.

    1994-01-01

    The biological effects of high Linear Energy Transfer (LET) charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/micrometer to 975 KeV/micrometer with particle energy (on the cells) between 94-603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/micrometer. The inactivation cross-section (alpha i) and the action cross-section for mutant induction (alpha m) ranged from 2.2 to 92.0 sq micrometer and 0.09 to 5.56 x 10(exp -3) sq micrometer respectively. The maximum values were obtained by Fe-56 with an LET of 200 keV/micrometer. The mutagenicity (alpha m/alpha i) ranged from 2.05 to 7.99 x 10(exp -5) with the maximum value at 150 keV/micrometer. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.

  19. Fluorescent light irradiation and its mutagenic potential in cultured mammalian cells

    SciTech Connect

    Pant, K.; Thilager, A.

    1994-12-31

    The photobiological effect of light is characterized by its energy emission at different wave lengths. Therefore by studying the energy emission spectra at different light sources and their photobiological activities, one can relate wavelength range(s) of the spectrum to a particular photobiological effect. We studied the potential of light irradiation from standard fluorescent bulbs (Sylvania 34WT-12) used in offices and laboratories to induce unscheduled DNA Synthesis (UDS) and mutations in cultured mammalian cells. The energy emission spectrum of the bulbs was determined at every 10 nanometers from 300nM to 700nM. The Chinese hamster ovary (CHO) cells were used to study the induction of mutations at the Hypoxanthine Guanine Phosphoribosyl Transferase (HGPRT) locus. Primary rat hepatocyte cultures were used to study the effect of light irradiation on UDS. The CHO cells were cultured in tissue culture flasks in minimum light conditions (.02mw/cm{sup 2}) and exposed to light irradiations with durations from 0 to 40 minutes. The cultures were maintained in darkness during the expression period and evaluated for HGPRT mutant frequencies. Similarly, the primary rat hepatocyte cultures were cultured on cover slips under minimal light conditions except for light irradiation and evaluated for UDS using 3H-thymidine labelled auto-radiography. The results of the study indicate that irradiation from fluorescent lights caused a slight elevation in the HGPRT mutant frequency in CHO cells. However a significant increase in UDS was not observed even at the maximum light irradiation dose. These results were compared to data obtained from similar experiments conducted with fluorescent bulbs with different energy emission spectra.

  20. Comparative investigation of antimutagenic activity of sterically hindered phenols

    SciTech Connect

    Pashin, Yu.V.; Bakhitova, L.M.; Bentkhen, T.I.

    1985-07-01

    Mutagenic properties of primarily inactive carcinogenic polycyclic aromatic hydrocarbons (PAH) are manifested after metabolic oxidation by microsomal enzymes. It has been established that activation of carcinogens in biological systems is accompanied by intensification of free-radical processes, effective inhibition of which is achieved by sterically hindered phenols (SHP). The authors studied the effect of SHP on the mutagenic activity of benzo(a)pyrene (BP) using estimation of induced direct gene mutation at the locus for hypoxanthine-guanine-phosphoribosyl transferase (HGPRT) in somatic Chinese hamster cells of line V-79 cultured in vitro and with estimation of the induction of micronuclei in polychromatophilic erythrocytes of mouse bone marrow in vivo. The reference mutagen was BP from Fluka and the following SHP were used: dibunol, F-800, and F-804. Genetic activity of each substance tested and their combination was studied in an in vitro system under conditions of metabolic activation by mouse liver microsomes and in vivo according to induction of micronuclei in polychromatophilic bone marrow erythrocytes in (CBA x C57B1/6J)F/sub 1/ mice 60-80 days old, which reflects gross defects of chromosomes at the erythroblast stage. In order to establish optimal time for recording the frequency of induction of micronuclei, bone marrow samples were taken from the animals, 24, 48, 72, and 96 h after a single intraperitoneal injection of the agents. The BP was dissolved in sunflower oil and used in a concentration constituting 1/3 of the lowest lethal dose in mice. The SHP was then dissolved in water or dimethyl sulfoxide and administered in a ratio with BP of 1:1 or 1:0.5. The smears were then stained in methanol, washed with twice-distilled water, and stained in 7% Giemsa solution.

  1. Effects of asbestos fibers on cell division, cell survival, and formation of thioguanine-resistant mutants in Chinese hamster ovary cells

    SciTech Connect

    Kenne, K.; Ljungquist, S.; Ringertz, N.R.

    1986-04-01

    The ability of crocidolite fibers to induce point mutations and mitotic abnormalities in Chinese hamster ovary (CHO) cells was examined in cell cultures. The purpose has been to study the possibilities for establishing in vitro test methods to quantify genetic damage induced by asbestos and other mineral fibers. Results obtained with the CHO/hypoxanthine guanine phosphoribosyl transferase system indicated that crocidolite fibers per se do not significantly increase the number of thioguanine-resistant mutants. Crocidolite fibers also failed to potentiate the mutagenicity of benzo(a)pyrene. Time-lapse cinematography and microscopy showed that asbestos (crocidolite) fibers were markedly cytotoxic. Among surviving cells some underwent abnormal cell divisions which resulted in multi- and micronucleate cells. Many cells that contained a few asbestos fibers, however, underwent mitosis and successfully formed two mononucleate daughter cells capable of further divisions. Individual, fiber-containing cells were examined by time-lapse television recordings for 4-5 days. During this time period some cells underwent six divisions and generated an almost normal number of daughter cells. Cells which contained fibers that were longer or equivalent to the diameter of the mitotic cell (20 ..mu..m), showed different forms of mitotic abnormalities. The frequency of multinucleate cells was drastically increased following exposure to asbestos fibers. Only rarely, however, did these cells divide to produce viable daughter cells capable of continued cell multiplication. The frequency of multinucleate cells was dependent on the dose of exposure to asbestos fibers and could possible be used as an index of the degree of mitotic disturbances induced by mineral fibers.

  2. Genotoxicity of titanium dioxide nanoparticles.

    PubMed

    Chen, Tao; Yan, Jian; Li, Yan

    2014-03-01

    Titanium dioxide nanoparticles (TiO(2)-NPs, <100 nm) are increasingly being used in pharmaceuticals and cosmetics due to the unique properties derived from their small sizes. However, their large surface-area to mass ratio and high redox potential may negatively impact human health and the environment. TiO(2)-NPs can cause inflammation, pulmonary damage, fibrosis, and lung tumors and they are possibly carcinogenic to humans. Because cancer is a disease involving mutation, there are a large number of studies on the genotoxicity of TiO(2)-NPs. In this article, we review the results that have been reported in the literature, with a focus on data generated from the standard genotoxicity assays. The data include genotoxicity results from the Ames test, in vitro and in vivo Comet assay, in vitro and in vivo micronucleus assay, sister chromatid exchange assay, mammalian cell hypoxanthine-guanine phosphoribosyl transferase gene assay, the wing somatic mutation and recombination assay, and the mouse phosphatidylinositol glycan, class A gene assay. Inconsistent results have been found in these assays, with both positive and negative responses being reported. The in vitro systems for assessing the genotoxicity of TiO(2)-NPs have generated a greater number of positive results than the in vivo systems, and tests for DNA and chromosome damage have produced more positive results than the assays measuring gene mutation. Nearly all tests for measuring the mutagenicity of TiO(2)-NPs were negative. The current data indicate that the genotoxicity of TiO(2)-NPs is mediated mainly through the generation of oxidative stress in cells. PMID:24673907

  3. Genotoxic evaluation of titanium dioxide nanoparticles in vivo and in vitro.

    PubMed

    Chen, Zhangjian; Wang, Yun; Ba, Te; Li, Yang; Pu, Ji; Chen, Tian; Song, Yanshuang; Gu, Yongen; Qian, Qin; Yang, Jinglin; Jia, Guang

    2014-05-01

    With the extensive application of titanium dioxide (TiO2) nanoparticles (NPs) in food industry, there is a rising debate concerning the possible risk associated with exposure to TiO2 NPs. The purpose of this study is to evaluate the genotoxicity of TiO2 NPs using in vivo and in vitro test systems. In vivo study, the adult male Sprague-Dawley rats were exposed to anatase TiO2 NPs (75 ± 15 nm) through intragastric administration at 0, 10, 50 and 200mg/kg body weight every day for 30 days. The γ-H2AX assay showed TiO2 NPs could induce DNA double strand breaks in bone marrow cells after oral administration. However, the micronucleus test revealed that the oral-exposed TiO2 NPs did not cause damage to chromosomes or mitotic apparatus observably in rat bone marrow cells. In vitro study, Chinese hamster lung fibroblasts (V79 cells) were exposed to TiO2 NPs at the dose of 0, 5, 10, 20, 50 and 100 μg/mL. Significant decreases in cell viability were detected in all the treated groups after 24h and 48h exposure. Significant DNA damage was only observed at the concentration of 100 μg/mL after 24h treatment using the comet assay. The obvious gene mutation was observed at the concentration of 20 and 100 μg/mL after 2h treatment using hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutation assay. This study presented a comprehensive genotoxic evaluation of TiO2 NPs, and TiO2 NPs were shown to be genotoxic both in vivo and in vitro tests. The gene mutation and DNA strand breaks seem to be more sensitive genetic endpoints for the detection of TiO2 NPs induced genotoxic effects. PMID:24594277

  4. Protection against radiation-induced mutations at the hprt locus by spermine and N,N{double_prime}-(dithiodi-2,1-ethanediyl)bis-1,3-propanediamine (WR-33278). WR-33278 and spermine protect against mutation induction

    SciTech Connect

    Grdina, D.J.; Shigematsu, N.; Schwartz, J.L.

    1994-08-01

    The polyamine spermine and the disulfide N,N{double_prime}-(dithiodi-2,1-ethanediyl)bis-1,3-propanediamine (WR-33278) are structurally similar agents capable of binding to DNA. WR-33278 is the disulfide moiety of the clinically studied radioprotective agent S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721). Because of their reported structural and functional similarities, it was of interest to characterize and compare their radioprotective properties using the endpoints of cell survival and mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in Chinese hamster AA8 cells. In order to facilitate both the uptake of WR-33278 into cells and the direct comparison between the protective properties of WR-33278 and spermine, these agents (at concentrations of 0.01 mM and 0.001 mM) were electroporated into cells. The exposure of cells to both electroporation and irradiation gave rise to enhanced cell killing and mutation induction, with the sequence of irradiation followed 3 h later by electroporation being the more toxic protocol. Enhanced cell survival was observed following electroporation of 0.01 mM of spermine and WR-33278 30 min prior to irradiation; protection factors (PF) of 1.3 and 1.8, respectively. Neither agent was protective at a concentration of 0.001 mM. Protection against radiation-induced hprt mutations was observed for both spermine and WR-33278 under all experimental conditions tested. These data suggest that the properties of radioprotection and chemoprevention exhibited by the phosphorothioate (WR-2721) and associated aminothiol (WR-1065) and disulfide (WR-33278) metabolites may be mediated via endogenous spermine-like polyamine processes. Such a mechanism would have important implications with respect to the design and development of new generation drugs for use in radioprotection and chemoprevention.

  5. Low dopamine activity in Lesch Nyhan Disease. An 18-fluorodopa PET study

    SciTech Connect

    Ernst, M.; Zametkin, A.; Matochik, J.

    1996-05-01

    Lesch-Nyhan Disease (LND) is a rare devastating X-linked recessive disorder characterized by the virtual absence of hypoxanthine guanine phosphoribosyl transferase (HPRT), a major enzyme of the salvage pathway of purine metabolism. The clinical presentation includes hyperuricemia choreoathetosis, dystonia, aggression and self-injurious behavior. The genetic and biochemical abnormalities are fully identified. However, the neuropathophysiological process by which the lack of HPRT produces the neuropsychiatric syndrome of LND in unclear. Presynaptic uptake of 18-Fluorodopa (FD) in basal ganglia, substantia nigra, and frontal and occipital cortices was measured by PET in 12 patients with LND, 10 to 20 years old, and 15 health controls, 12 to 23 years old. Radioactive counts (mCi/cc), recorded between 90 and 130 minutes after tracer injection, were measured in regions of interest by a rater blind to subjects` identities. Results were expressed as ratios of FD uptake in specific to non-specific (occipital cortex) brain areas. Presynaptic dopamine activity was significantly lower by 69% in putamen (p<0.0001), 61% in caudate (p<0.0001), 56% in frontal cortex (p=0.003) and 43% in substantiat nigra (p<0.016) in LND patients than in control subjects. Absolute FD measures in occipital regions did not differ between the two groups. Activity of FD in the basal ganglia was stable over time in the LND group and tended to increase in the control group (r=0.50, n=15, p=0.060). In the LND group, aggressive behavior was worse as FD activity was higher (r=0.60, n=12, p=0.40). LND is associated with a striking reduction of presynaptic dopamine activity that is not region-specific. The temporal stability of FD measures and of the severity of LND symptomatology is consistent with a developmental rather than degenerative process.

  6. Evaluation of the genotoxicity of process stream extracts from a coal gasification system

    SciTech Connect

    Shimizu, R.W.; Benson, J.M.; Li, A.P.; Henderson, R.F.; Brooks, A.L.

    1984-01-01

    Extracts of three complex organic environmental mixtures, two from an experimental coal gasifier (a raw gas and a clean gas sample) and one from a coke oven main, were examined for genotoxicity. Three short-term genotoxicity assay systems were used: Ames Salmonella typhimurium reverse mutation assay, Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) gene locus mutation assay, and the Chinese hamster lung primary culture/sister chromatid exchange (CHL/SCE) assay. Aroclor-1254-induced rat liver homogenate fraction (S-9) was required to observe genotoxicity in both gene locus mutation assays. The relative survival of CHO cells exposed to extracts was highest in cells exposed to clean gas samples, with the raw gas sample being the most cytotoxic either with or without the addition of S-9. All three complex mixtures induced sister chromatid exchanges in primary lung cell cultures without the addition of S-9. The relative genotoxicity ranking of the samples varied between the mammalian and prokaryotic assay systems. Coke oven main extract produced fewer revertants in bacteria than the raw gas sample. However, the coke oven main extract was more genotoxic in the two eukaryotic systems (CHL/SCE and CHO/HGPRT) than was the raw gas sample. The results of all three assays indicate that the cleanup process used in the experimental gasifier was effective in decreasing the genotoxic materials in the process stream. These data also reemphasize the necessity of evaluating genotoxicity of complex mixtures in a variety of short-term systems. 24 references, 3 figures, 2 tables.

  7. Assessment of mercaptopurine (6MP) metabolites and 6MP metabolic key-enzymes in childhood acute lymphoblastic leukemia.

    PubMed

    Wojtuszkiewicz, Anna; Barcelos, Ana; Dubbelman, Boas; De Abreu, Ronney; Brouwer, Connie; Bökkerink, Jos P; de Haas, Valerie; de Groot-Kruseman, Hester; Jansen, Gerrit; Kaspers, Gertjan L; Cloos, Jacqueline; Peters, G J

    2014-01-01

    Pediatric acute lymphoblastic leukemia (ALL) is treated with combination chemotherapy including mercaptopurine (6MP) as an important component. Upon its uptake, 6MP undergoes a complex metabolism involving many enzymes and active products. The prognostic value of all the factors engaged in this pathway still remains unclear. This study attempted to determine which components of 6MP metabolism in leukemic blasts and red blood cells are important for 6MP's sensitivity and toxicity. In addition, changes in the enzymatic activities and metabolite levels during the treatment were analyzed. In a cohort (N=236) of pediatric ALL patients enrolled in the Dutch ALL-9 protocol, we studied the enzymes inosine-5'-monophosphate dehydrogenase (IMPDH), thiopurine S-methyltransferase (TPMT), hypoxanthine guanine phosphoribosyl transferase (HGPRT), and purine nucleoside phosphorylase (PNP) as well as thioguanine nucleotides (TGN) and methylthioinosine nucleotides (meTINs). Activities of selected enzymes and levels of 6MP derivatives were measured at various time points during the course of therapy. The data obtained and the toxicity related parameters available for these patients were correlated with each other. We found several interesting relations, including high concentrations of two active forms of 6MP--TGN and meTIN--showing a trend toward association with better in vitro antileukemic effect of 6MP. High concentrations of TGN and elevated activity of HGPRT were found to be significantly associated with grade III/IV leucopenia. However, a lot of data of enzymatic activities and metabolite concentrations as well as clinical toxicity were missing, thereby limiting the number of assessed relations. Therefore, although a complex study of 6MP metabolism in ALL patients is feasible, it warrants more robust and strict data collection in order to be able to draw more reliable conclusions. PMID:24940700

  8. Metabolism of 2-acetylaminofluorene in the Chinese hamster ovary cell mutation assay

    SciTech Connect

    Heflich, R.H.; Djuric, Z.; Zhuo, Z.; Fullerton, N.F.; Casciano, D.A.; Beland, F.A.

    1988-01-01

    Chinese hamster ovary (CHO) cells were exposed to 2-acetylaminofluorene (2-AAF) and 2-aminofluorene (2-AF), and several of their N-oxidized metabolites in order to study the mechanisms by which arylamides and arylamines produce mutations in mammalian cells. The number of mutations induced at the hypoxanthine-guanine phosphoribosyl transferase locus by each compound was estimated to be: N-acetoxy-2-AAF, 310; N-hydroxy-2-AF, 3; N-hydroxy-2-AAF, 0.7; 2-AAF, 0.1; and 2-AF, 0.09. With each compound, DNA adducts were also identified and quantified, and in all cases the major adduct was N-(deoxyguanosin-8-yl)-2-AF. The relationship between mutation induction and adduct formation for each of the derivatives was similar to that previously reported for N-hydroxy-2-AF. Inclusion of the deacetylase inhibitor, paraoxon, reduced the mutagenicity of 2-AFF, N-hydroxy-2-AAF and N-acetoxy-2-AAF, and the DNA adducts produced by N-acetoxy-2-AAF to background levels. Taken together, these data indicate that CHO cells metabolized N-acetoxy-2-AAF to a reactive derivative by N-deacetylation to N-acetoxy-2-AF, while N-hydroxy-2-AF reacted directly with DNA. The major pathway of N-hydroxy-2-AAF activation appeared to be an initial O-acetylation to N-acetoxy-2-AAF and this occurred to only a limited extent in the CHO cells. The initial step in the activation of 2-AAF and 2-AF was an N-oxidation to N-hydroxy-2-AAF and N-hydroxy-2-AF, respectively. The limited O-acetylase activity in CHO cells appeared to contribute to the low sensitivity of these cells toward mutation induction by arylamines and arylamides.

  9. The induction of sister chromatid exchanges by environmental pollutants: relationship of SCE to other measures of genetic damage

    SciTech Connect

    Brooks, A.L.; Shimizu, R.W.; Li, A.P.; Benson, J.M.; Dutcher, J.S.

    1984-01-01

    Sister chromatid exchanges (SCEs), induced by environmental pollutants from fossil fuel use, were measured in 2 cell systems, Chinese hamster ovary (CHO) cells and Chinese hamster primary lung cell cultures. The frequency of SCEs induced in these cell systems was related to other measures of genetic damage, namely mutations in CHO cells at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene locus and in bacteria (Salmonella mutagenicity test TA-98), produced by the same pollutants. The pollutants were divided into 2 classes: those produced in oxidizing combustion environments--extracts of particles from light-duty diesel cars, spark-ignition cars, and an automotive tunnel; and those produced in reducing environments--extracts from coke oven mains and condensates from a low BTU coal gasifier obtained either before or after cleanup of the process stream. Sister chromatid exchanges were induced by all pollutants without the addition of a rat liver microsomal fraction (S-9 mix), whereas S-9 mix was required to induce a positive response in the CHO/HGPRT assay for all pollutants. The pollutants produced in a reducing environment required metabolic activation by S-9 mix to be mutagenic in the Salmonella mutation assay. The addition of S-9 mix to pollutants produced in an oxidizing environment reduced the response in the Salmonella test. The relative genotoxic potency for each pollutant was determined for all 3 endpoints. The slopes of dose-response curves for each pollutant were plotted for each assay to compare relative potency. When the bacterial mutagenicity test was compared to either mammalian cell assay, SCE or CHO/HGPRT, there was little correlation between relative potencies. However, the data indicated that the responses in the 2 mammalian cell assays, SCE and CHO/HGPRT, showed similar relative responses to the pollutants.

  10. Mutagenic effects of alpha particles in normal human skin fibroblasts

    SciTech Connect

    Chen, D.J.; Carpenter, S.; Hanks, T.

    1992-12-31

    Alpha-irradiation to the bronchial airways from inhaled radon progeny increases the risk of developing lung cancer. The molecular mechanism of radon-induced lung cancer is not clear, but one of the most important genetic effects of ionizing radiation is the induction of gene mutation. Mutations, especially those associated with visible chromosome abnormalities in humans, have been associated with cancer. Therefore, our objective is to use a well-defined model system to determine the mutagenic potential of alpha particles in normal human skin cells and to define this action at the molecular level. Normal human skin fibroblasts were irradiated with alpha particles (3.59 MeV, LET 115 keV {mu}m{sup {minus}1}) emitted from the decay of {sup 238}Pu. Mutagenicity was determined at the X-linked hypoxanthine guanine phosphoribosyl transferase (HPRT) locus. Results from this study indicate that beta particles were more efficient in mutation induction than gamma rays. Based on the initial slopes of the dose-response curves, the RBE for mutation is about 8 for alpha particles. HPRT-deficient mutants which are resistant to 6-thioguanine have been isolated and analyzed by the Southern blot technique. To date, we have characterized 69 gamma-ray-induced and 195 alpha-particle-induced HPRT-deficient mutants. Our data indicate that more than 50% of all gamma-ray-induced mutants have band patterns identical to that observed for the normal structural HPRT gene, whereas the remaining mutants (45%) contain either a rearrangement, partial deletion, or total deletion of the HPRT gene. In contrast, only 30% of alpha-particle-induced human HPRT mutants contain a normal Southern blot pattern, and about 50% indicate total deletion of the HPRT gene. Our results support the notion that high-LET radiation produces more unrepaired or misrepaired DNA damage than do gamma rays.

  11. Purine metabolism in response to hypoxic conditions associated with breath-hold diving and exercise in erythrocytes and plasma from bottlenose dolphins (Tursiops truncatus).

    PubMed

    del Castillo Velasco-Martínez, Iris; Hernández-Camacho, Claudia J; Méndez-Rodríguez, Lía C; Zenteno-Savín, Tania

    2016-01-01

    In mammalian tissues under hypoxic conditions, ATP degradation results in accumulation of purine metabolites. During exercise, muscle energetic demand increases and oxygen consumption can exceed its supply. During breath-hold diving, oxygen supply is reduced and, although oxygen utilization is regulated by bradycardia (low heart rate) and peripheral vasoconstriction, tissues with low blood flow (ischemia) may become hypoxic. The goal of this study was to evaluate potential differences in the circulating levels of purine metabolism components between diving and exercise in bottlenose dolphins (Tursiops truncatus). Blood samples were taken from captive dolphins following a swimming routine (n=8) and after a 2min dive (n=8). Activity of enzymes involved in purine metabolism (hypoxanthine guanine phosphoribosyl transferase (HGPRT), inosine monophosphate deshydrogenase (IMPDH), xanthine oxidase (XO), purine nucleoside phosphorylase (PNP)), and purine metabolite (hypoxanthine (HX), xanthine (X), uric acid (UA), inosine monophosphate (IMP), inosine, nicotinamide adenine dinucleotide (NAD(+)), adenosine, adenosine monophosphate (AMP), adenosine diphosphate (ADP), ATP, guanosine diphosphate (GDP), guanosine triphosphate (GTP)) concentrations were quantified in erythrocyte and plasma samples. Enzymatic activity and purine metabolite concentrations involved in purine synthesis and degradation, were not significantly different between diving and exercise. Plasma adenosine concentration was higher after diving than exercise (p=0.03); this may be related to dive-induced ischemia. In erythrocytes, HGPRT activity was higher after diving than exercise (p=0.007), suggesting an increased capacity for purine recycling and ATP synthesis from IMP in ischemic tissues of bottlenose dolphins during diving. Purine recycling and physiological adaptations may maintain the ATP concentrations in bottlenose dolphins after diving and exercise. PMID:26506131

  12. Prolonged fasting increases purine recycling in post-weaned northern elephant seals

    PubMed Central

    Soñanez-Organis, José Guadalupe; Vázquez-Medina, José Pablo; Zenteno-Savín, Tania; Aguilar, Andres; Crocker, Daniel E.; Ortiz, Rudy M.

    2012-01-01

    SUMMARY Northern elephant seals are naturally adapted to prolonged periods (1–2 months) of absolute food and water deprivation (fasting). In terrestrial mammals, food deprivation stimulates ATP degradation and decreases ATP synthesis, resulting in the accumulation of purines (ATP degradation byproducts). Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) salvages ATP by recycling the purine degradation products derived from xanthine oxidase (XO) metabolism, which also promotes oxidant production. The contributions of HGPRT to purine recycling during prolonged food deprivation in marine mammals are not well defined. In the present study we cloned and characterized the complete and partial cDNA sequences that encode for HGPRT and xanthine oxidoreductase (XOR) in northern elephant seals. We also measured XO protein expression and circulating activity, along with xanthine and hypoxanthine plasma content in fasting northern elephant seal pups. Blood, adipose and muscle tissue samples were collected from animals after 1, 3, 5 and 7 weeks of their natural post-weaning fast. The complete HGPRT and partial XOR cDNA sequences are 771 and 345 bp long and encode proteins of 218 and 115 amino acids, respectively, with conserved domains important for their function and regulation. XOR mRNA and XO protein expression increased 3-fold and 1.7-fold with fasting, respectively, whereas HGPRT mRNA (4-fold) and protein (2-fold) expression increased after 7 weeks in adipose tissue and muscle. Plasma xanthine (3-fold) and hypoxanthine (2.5-fold) levels, and XO (1.7- to 20-fold) and HGPRT (1.5- to 1.7-fold) activities increased during the last 2 weeks of fasting. Results suggest that prolonged fasting in elephant seal pups is associated with increased capacity to recycle purines, which may contribute to ameliorating oxidant production and enhancing the supply of ATP, both of which would be beneficial during prolonged food deprivation and appear to be adaptive in this species. PMID

  13. Substitutions in hamster CAD carbamoyl-phosphate synthetase alter allosteric response to 5-phosphoribosyl-alpha-pyrophosphate (PRPP) and UTP.

    PubMed Central

    Simmons, Christine Q; Simmons, Alan J; Haubner, Aaron; Ream, Amber; Davidson, Jeffrey N

    2004-01-01

    CPSase (carbamoyl-phosphate synthetase II), a component of CAD protein (multienzymic protein with CPSase, aspartate transcarbamylase and dihydro-orotase activities), catalyses the regulated steps in the de novo synthesis of pyrimidines. Unlike the orthologous Escherichia coli enzyme that is regulated by UMP, inosine monophosphate and ornithine, the mammalian CPSase is allosterically inhibited by UTP, and activated by PRPP (5-phosphoribosyl-a-pyrophosphate) and phosphorylation. Four residues (Thr974, Lys993, Lys954 and Thr977) are critical to the E. coli inosine monophosphate/UMP-binding pocket. In the present study, three of the corresponding residues in the hamster CPSase were altered to determine if they affect either PRPP activation or UTP inhibition. Substitution of the hamster residue, positionally equivalent to Thr974 in the E. coli enzyme, with alanine residue led to an enzyme with 5-fold lower activity and a near loss of PRPP activation. Whereas replacement of the tryptophan residue at position 993 had no effect, an Asp992-->Asn substitution yielded a much-activated enzyme that behaved as if PRPP was present. The substitution Lys954-->Glu had no effect on PRPP stimulation. Only modest decreases in UTP inhibitions were observed with each of the altered CPSases. The results also show that while PRPP and UTP can act simultaneously, PRPP activation is dominant. Apparently, UTP and PRPP have distinctly different associations within the mammalian enzyme. The findings of the present study may prove relevant to the neuropathology of Lesch-Nyhan syndrome PMID:14651476

  14. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase: a bent dimer defining the adenine specificity of the substrate ATP.

    PubMed

    Andersen, Rune W; Leggio, Leila Lo; Hove-Jensen, Bjarne; Kadziola, Anders

    2015-03-01

    The enzyme 5-phosphoribosyl-1-α-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg(2+)-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP synthase was synthesised in vitro with optimised codon usage for expression in Escherichia coli. Following expression of the gene in E. coli PRPP synthase was purified by heat treatment and ammonium sulphate precipitation and the structure of S. solfataricus PRPP synthase was determined at 2.8 Å resolution. A bent dimer oligomerisation was revealed, which seems to be an abundant feature among PRPP synthases for defining the adenine specificity of the substrate ATP. Molecular replacement was used to determine the S. solfataricus PRPP synthase structure with a monomer subunit of Methanocaldococcus jannaschii PRPP synthase as a search model. The two amino acid sequences share 35 % identity. The resulting asymmetric unit consists of three separated dimers. The protein was co-crystallised in the presence of AMP and ribose 5-phosphate, but in the electron density map of the active site only AMP and a sulphate ion were observed. Sulphate ion, reminiscent of the ammonium sulphate precipitation step of the purification, seems to bind tightly and, therefore, presumably occupies and blocks the ribose 5-phosphate binding site. The activity of S. solfataricus PRPP synthase is independent of phosphate ion. PMID:25605536

  15. Roles for glutathione transferases in antioxidant recycling

    PubMed Central

    Dixon, David P; Steel, Patrick G

    2011-01-01

    Uniquely among the plant glutathione transferases, two classes possess a catalytic cysteine capable of performing glutathione-dependent reductions. These are the dehydroascorbate reductases (DHARs) and the lambda-class glutathione transferases (GSTLs). Using immobilized GSTLs probed with crude plant extracts we have identified flavonols as high affinity ligands and subsequently demonstrated a novel glutathione-dependent role for these enzymes in recycling oxidized quercetin. By comparing the activities of DHARs and GSTLs we now propose a unified catalytic mechanism that suggests oxidized anthocyanidins and tocopherols may be alternative polyphenolic substrates of GSTLs. PMID:21778824

  16. Purification and characterization of the Oligosaccharyl transferase

    SciTech Connect

    Kapoor, T.M.

    1990-11-01

    Oligosaccharyl transferase was characterized to be a glycoprotein with at least one saccharide unit that had a D-manno or D- glucopyranose configuration with unmodified hydroxy groups at C-3, C-4 and C-6, using a Concanavalin A affinity column. This afforded a 100 fold increase in the transferase purity in the solubilized microsomal sample and also removed over 90% of the microsomal proteins (the cytosolic ones being removed before solubilization). The detergent, N,N-Dimethyldodecylamine N-oxide (LDAO) was used for solubilization and it yielded a system compatible with the assay and the purification steps. An efficient method for detergent extraction without dilution of sample or protein precipitation was also developed.

  17. Nomenclature for mammalian soluble glutathione transferases.

    PubMed

    Mannervik, Bengt; Board, Philip G; Hayes, John D; Listowsky, Irving; Pearson, William R

    2005-01-01

    The nomenclature for human soluble glutathione transferases (GSTs) is extended to include new members of the GST superfamily that have been discovered, sequenced, and shown to be expressed. The GST nomenclature is based on primary structure similarities and the division of GSTs into classes of more closely related sequences. The classes are designated by the names of the Greek letters: Alpha, Mu, Pi, etc., abbreviated in Roman capitals: A, M, P, and so on. (The Greek characters should not be used.) Class members are distinguished by Arabic numerals and the native dimeric protein structures are named according to their subunit composition (e.g., GST A1-2 is the enzyme composed of subunits 1 and 2 in the Alpha class). Soluble GSTs from other mammalian species can be classified in the same manner as the human enzymes, and this chapter presents the application of the nomenclature to the rat and mouse GSTs. PMID:16399376

  18. Single prenyl-binding site on protein prenyl transferases

    PubMed Central

    Desnoyers, Luc; Seabra, Miguel C.

    1998-01-01

    Three distinct protein prenyl transferases, one protein farnesyl transferase (FTase) and two protein geranylgeranyl transferases (GGTase), catalyze prenylation of many cellular proteins. One group of protein substrates contains a C-terminal CAAX motif (C is Cys, A is aliphatic, and X is a variety of amino acids) in which the single cysteine residue is modified with either farnesyl or geranylgeranyl (GG) by FTase or GGTase type-I (GGTase-I), respectively. Rab proteins constitute a second group of substrates that contain a C-terminal double-cysteine motif (such as XXCC in Rab1a) in which both cysteines are geranylgeranylated by Rab GG transferase (RabGGTase). Previous characterization of CAAX prenyl transferases showed that the enzymes form stable complexes with their prenyl pyrophosphate substrates, acting as prenyl carriers. We developed a prenyl-binding assay and show that RabGGTase has a prenyl carrier function similar to the CAAX prenyl transferases. Stable RabGGTase:GG pyrophosphate (GGPP), FTase:GGPP, and GGTase-I:GGPP complexes show 1:1 (enzyme:GGPP) stoichiometry. Chromatographic analysis of prenylated products after single turnover reactions by using isolated RabGGTase:GGPP complex revealed that Rab is mono-geranylgeranylated. This study establishes that all three protein prenyl transferases contain a single prenyl-binding site and suggests that RabGGTase transfers two GG groups to Rabs in independent and consecutive reactions. PMID:9770475

  19. The Genetic Architecture of Murine Glutathione Transferases

    PubMed Central

    Lu, Lu; Pandey, Ashutosh K.; Houseal, M. Trevor; Mulligan, Megan K.

    2016-01-01

    Glutathione S-transferase (GST) genes play a protective role against oxidative stress and may influence disease risk and drug pharmacokinetics. In this study, massive multiscalar trait profiling across a large population of mice derived from a cross between C57BL/6J (B6) and DBA2/J (D2)—the BXD family—was combined with linkage and bioinformatic analyses to characterize mechanisms controlling GST expression and to identify downstream consequences of this variation. Similar to humans, mice show a wide range in expression of GST family members. Variation in the expression of Gsta4, Gstt2, Gstz1, Gsto1, and Mgst3 is modulated by local expression QTLs (eQTLs) in several tissues. Higher expression of Gsto1 in brain and liver of BXD strains is strongly associated (P < 0.01) with inheritance of the B6 parental allele whereas higher expression of Gsta4 and Mgst3 in brain and liver, and Gstt2 and Gstz1 in brain is strongly associated with inheritance of the D2 parental allele. Allele-specific assays confirmed that expression of Gsto1, Gsta4, and Mgst3 are modulated by sequence variants within or near each gene locus. We exploited this endogenous variation to identify coexpression networks and downstream targets in mouse and human. Through a combined systems genetics approach, we provide new insight into the biological role of naturally occurring variants in GST genes. PMID:26829228

  20. Detection of glutathione transferase activity on polyacrylamide gels.

    PubMed

    Ricci, G; Lo Bello, M; Caccuri, A M; Galiazzo, F; Federici, G

    1984-12-01

    A simple and sensitive assay for glutathione transferase activity on polyacrylamide gel is described. The method is based on the fast reduction of nitroblue tetrazolium salt by glutathione. Blue insoluble formazan colors the gel except in the glutathione transferase area. The stable and defined colorless zone is still detectable with 0.005 unit enzyme. This technique has been successfully applied with enzyme preparations of human heart and other tissues. PMID:6532239

  1. N-(5′-Phosphoribosyl)anthranilate isomerase–indol-3-ylglycerol phosphate synthetase of tryptophan biosynthesis. Relationship between the two activities of the enzyme from Escherichia coli

    PubMed Central

    Creighton, Thomas E.

    1970-01-01

    Further evidence is presented to confirm the previous conclusion that the enzyme from Escherichia coli catalysing the two sequential reactions in tryptophan biosynthesis, N-(5′-phosphoribosyl)anthranilic acid (PRA) → 1-(o-carboxyphenyl-amino)-1-deoxyribulose 5-phosphate (CdRP) → indol-3-ylglycerol phosphate (InGP)+CO2+H2O, consists of a single polypeptide chain. The kinetic properties of the enzyme demonstrate that intermediate CdRP formed from PRA must dissociate from the enzyme before it can be converted into InGP. It is concluded that there are two distinct and non-overlapping catalytic sites on the enzyme for the two reactions. The expected complementation between a mutationally altered form of the enzyme lacking the first reaction and a mutationally altered form lacking the second reaction has been demonstrated in vitro by InGP formation from PRA. This system thus exhibits intracistronic complementation with a non-oligomeric protein gene product. PMID:4924490

  2. Expression of phosphoribosyl pyrophosphate synthetase genes in U87 glioma cells with ERN1 knockdown: effect of hypoxia and endoplasmic reticulum stress.

    PubMed

    Minchenko, O H; Garmash, I A; Kovalevska, O V; Tsymbal, D O; Minchenko, D O

    2014-01-01

    Activation of pentose phosphate pathway is an important factor of enhanced cell proliferation and tumor growth. Phosphoribosyl pyrophosphate synthetase (PRPS) is a key enzyme of this pathway and plays a central role in the synthesis of purines and pyrimidines. Hypoxia as well as ERN1 (from endoplasmic reticulum to nuclei-1) mediated endoplasmic reticulum stress response-signalling pathway is linked to the proliferation because the blockade of ERN1 suppresses tumor growth, including glioma. We studied the expression of different PRPS genes in glioma cells with ERN1 knockdown under hypoxic condition. It was shown that hypoxia decreases the expression of PRPS1 and PRPS2 genes in both types of glioma cells, being more pronounced in cells without ERN1 function, but PRPSAP1 and PRPSAP2 gene expressions are suppressed by hypoxia only in glioma cells with blockade of ERN1. Moreover, the blockade of endoribonuclease activity of ERN1 does not affect the expression of PRPS1 and PRPS2 as well as PPRS-associated protein genes in U87 glioma cells. At the same time, the induction of endoplasmic reticulum stress by tunicamycin in glioma cells with suppressed activity of ERN1 endoribonuclease decreases the expression level of PRPS1 and PRPS2 genes only. Results of this investigation clearly demonstrated that the expression of different genes encoding subunits of PRPS enzyme is affected by hypoxia in U87 glioma cells, but the effect of hypoxia is modified by suppression of endoplasmic reticulum stress signaling enzyme ERN1. PMID:25816608

  3. Identification and sequence analysis of Escherichia coli purE and purK genes encoding 5'-phosphoribosyl-5-amino-4-imidazole carboxylase for de novo purine biosynthesis.

    PubMed Central

    Watanabe, W; Sampei, G; Aiba, A; Mizobuchi, K

    1989-01-01

    It has been shown that the Escherichia coli purE locus specifying 5'-phosphoribosyl-5-amino-4-imidazole carboxylase in de novo purine nucleotide synthesis is divided into two cistrons. We cloned and determined a 2,449-nucleotide sequence including the purE locus. This sequence contains two overlapped open reading frames, ORF-18 and ORF-39, encoding proteins with molecular weights of 18,000 and 39,000, respectively. The purE mutations of CSH57A and DCSP22 were complemented by plasmids carrying ORF-18, while that of NK6051 was complemented by plasmids carrying ORF-39. Thus, the purE locus consists of two distinct genes, designated purE and purK for ORF-18 and ORF-39, respectively. These genes constitute a single operon. A highly conserved 16-nucleotide sequence, termed the PUR box, was found in the upstream region of purE by comparing the sequences of the purF and purMN operons. We also found three entire and one partial repetitive extragenic palindromic (REP) sequences in the downstream region of purK. Roles of the PUR box and REP sequences are discussed in relation to the genesis of the purEK operon. Images PMID:2644189

  4. Bilirubin UDP-Glucuronosyltransferase 1A1 (UGT1A1) Gene Promoter Polymorphisms and HPRT, Glycophorin A, and Micronuclei Mutant Frequencies in Human Blood

    SciTech Connect

    Grant, D; Hall, I J; Eastmond, D; Jones, I M; Bell, D A

    2004-10-06

    A dinucleotide repeat polymorphism (5-, 6-, 7-, or 8-TA units) has been identified within the promoter region of UDP-glucuronosyltransferase 1A1 gene (UGT1A1). The 7-TA repeat allele has been associated with elevated serum bilirubin levels that cause a mild hyperbilirubinemia (Gilbert's syndrome). Studies suggest that promoter transcriptional activity of UGT1A1 is inversely related to the number of TA repeats and that unconjugated bilirubin concentration increases directly with the number of TA repeat elements. Because bilirubin is a known antioxidant, we hypothesized that UGT1A1 repeats associated with higher bilirubin may be protective against oxidative damage. We examined the effect of UGT1A1 genotype on somatic mutant frequency in the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) gene in human lymphocytes and the glycophorin A (GPA) gene of red blood cells (both N0, NN mutants), and the frequency of lymphocyte micronuclei (both kinetochore (K) positive or micronuclei K negative) in 101 healthy smoking and nonsmoking individuals. As hypothesized, genotypes containing 7-TA and 8-TA displayed marginally lower GPA{_}NN mutant frequency relative to 5/5, 5/6, 6/6 genotypes (p<0.05). In contrast, our analysis showed that lower expressing UGT1A1 alleles (7-TA and 8-TA) were associated with modestly increased HPRT mutation frequency (p<0.05) while the same low expression genotypes were not significantly associated with micronuclei frequencies (K-positive or K-negative) when compared to high expression genotypes (5-TA and 6-TA). We found weak evidence that UGT1A1 genotypes containing 7-TA and 8-TA were associated with increased GPA{_}N0 mutant frequency relative to 5/5, 5/6, 6/6 genotypes (p<0.05). These data suggest that UGT1A1 genotype may modulate somatic mutation of some types, in some cell lineages, by a mechanism not involving bilirubin antioxidant activity. More detailed studies examining UGT1A1 promoter variation, oxidant/antioxidant balance and genetic

  5. Inhibition of Mycoplasma pneumoniae growth by FDA-approved anticancer and antiviral nucleoside and nucleobase analogs

    PubMed Central

    2013-01-01

    Background Mycoplasma pneumoniae (Mpn) is a human pathogen that causes acute and chronic respiratory diseases and has been linked to many extrapulmonary diseases. Due to the lack of cell wall, Mpn is resistant to antibiotics targeting cell wall synthesis such as penicillin. During the last 10 years macrolide-resistant Mpn strains have been frequently reported in Asian countries and have been spreading to Europe and the United States. Therefore, new antibiotics are needed. In this study, 30 FDA-approved anticancer or antiviral drugs were screened for inhibitory effects on Mpn growth and selected analogs were further characterized by inhibition of target enzymes and metabolism of radiolabeled substrates. Results Sixteen drugs showed varying inhibitory effects and seven showed strong inhibition of Mpn growth. The anticancer drug 6-thioguanine had a MIC (minimum inhibitory concentration required to cause 90% of growth inhibition) value of 0.20 μg ml-1, whereas trifluorothymidine, gemcitabine and dipyridamole had MIC values of approximately 2 μg ml-1. In wild type Mpn culture the presence of 6-thioguanine and dipyridamole strongly inhibited the uptake and metabolism of hypoxanthine and guanine while gemcitabine inhibited the uptake and metabolism of all nucleobases and thymidine. Trifluorothymidine and 5-fluorodeoxyuridine, however, stimulated the uptake and incorporation of radiolabeled thymidine and this stimulation was due to induction of thymidine kinase activity. Furthermore, Mpn hypoxanthine guanine phosphoribosyl transferase (HPRT) was cloned, expressed, and characterized. The 6-thioguanine, but not other purine analogs, strongly inhibited HPRT, which may in part explain the observed growth inhibition. Trifluorothymidine and 5-fluorodeoxyuridine were shown to be good substrates and inhibitors for thymidine kinase from human and Mycoplasma sources. Conclusion We have shown that several anticancer and antiviral nucleoside and nucleobase analogs are potent

  6. Characterizing mutagenesis in the hprt gene of rat alveolar epithelial cells

    SciTech Connect

    Driscoll, K.E.; Deyo, L.C.; Howard, B.W.

    1995-12-31

    A clonal selection assay was developed for mutation in the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene of rat alveolar epithelial cells. Studies were conducted to establish methods for isolation and long-term culture of rat alveolar epithelial cells. When isolated by pronase digestion purified on a Nycodenz gradient and cultured in media containing 7.5% fetal bovine serum (FBS), pituitary extract, EGF, insulin, and IGF-1, rat alveolar epithelial cells could be maintained in culture for several weeks with cell doubling times of 2-4 days. The rat alveolar epithelial cell cultures were exposed in vitro to the mutagens ethylnitrosourea (ENU) and H{sub 2}O{sub 2}, and mutation in the hprt gene was selected for by culture in the presence of the toxic purine analog, 6-thioguanine (6TG). In vitro exposure to ENU or H{sub 2}O produced a dose-dependent increase in hprt mutation frequency in the alveolar epithelial cells. To determine if the assay system could be used to evaluate mutagenesis in alveolar type II cells after in vivo mutagen or carcinogen exposure, cells were isolated from rats treated previously with ENU or {alpha}-quartz. A significant increase in hprt mutation frequency was detected in alveolar epithelial cells obtained from rats exposed to ENU or {alpha}-quartz; the latter observation is the first demonstration that crystalline silica exposure is mutagenic in vivo. In summary, these studies show that rat alveolar epithelial cells isolated by pronase digestion and Nycodenz separation techniques and cultured in a defined media can be used in a clonal selection assay for mutation in the hprt gene. This assay demonstrates that ENU and H{sub 2}O{sub 2} in vitro and ENU and {alpha}-quartz in vivo are mutagenic for rat alveolar epithelial cells. This model should be useful for investigating the genotoxic effects of chemical and physical agents on an important lung cell target for neoplastic transformation. 41 refs., 4 figs., 3 tabs.

  7. On the quality of mutations in mammalian cells induced by high LET radiations

    NASA Astrophysics Data System (ADS)

    Baumstark-Khan, Christa; Rosendahl, Ilja M.; Rink, Hermann

    The deleterious effects of accelerated heavy ions as component of the space radiation environment on living cells are of increasing importance for long duration human space flight activities. The most important aspect of such densely ionizing particle radiation is attributed to the type and quality of biological damage induced by them. This issue is addressed by investigating cell inactivation and mutation induction at the Hprt locus (coding for hypoxanthine-guanine-phosphoribosyl-transferase) of cultured V79 Chinese hamster cells exposed to densely ionizing radiation (accelerated heavy ions with different LETs from oxygen to gold, specific energies ranging from 1.9 to 69.7 MeV/u, corresponding LET values range from 62 to 13,223 keV/μm) and to sparsely ionizing radiation (200 kV X-rays). 30 spontaneous, 40 X-ray induced and 196 heavy ion induced 6-thioguanine resistant Hprt mutant colonies were characterized by Southern technique using the restriction enzymes EcoRI, PstI and BglII and a full length Hprt cDNA probe isolated from the plasmid pHPT12. Restriction patterns of the spontaneous Hprt mutants were indistinguishable from the wild type pattern, as these mutants probably contain only small deletions or even point mutations in the Hprt locus. In contrast, the overall spectrum of heavy ion induced mutations revealed a majority of partial or total deletions of the Hprt gene. With constant particle fluence (3 × 10 6 particles/cm 2) the quality of heavy ion induced mutations in the Hprt locus depends on physical parameters of the beam (atomic number, specific energy, LET). This finding suggests a relationship between the type of DNA damage and track structure. The fraction of mutants with severe deletions in the Hprt locus after exposure to oxygen ions increases from 65% at 60 keV/μm up to a maximum (100%) at 300 keV/μm and declines with higher LET values to 75% at 750 keV/μm. With heavier ions (Ca- and Au-ions) and even higher LET-values this mutant fraction

  8. Increased frequency of in vivo hprt gene-mutated T cells in the peripheral blood of patients with systemic sclerosis.

    PubMed Central

    Sfikakis, P P; Tesar, J; Theocharis, S; Klipple, G L; Tsokos, G C

    1994-01-01

    OBJECTIVE--Activated T lymphocytes are involved in the pathogenesis of scleroderma (systemic sclerosis, SSc); such cells rapidly divide in vivo and are thus theoretically subject to random mutation more frequently than resting cells. To study whether SSc is associated with rapidly expanding T cell clones the frequency was determined of in vivo mutated T cells (MF) at the hypoxanthine guanine phosphoribosyl transferase (hprt) gene in the peripheral blood from patients with SSc. Specific clinical or serological associations were also investigated. METHODS--Peripheral blood lymphocytes from 16 healthy individuals and 20 patients with SSc were cultured using an hprt clonal assay; mutated and wild T cell clones were established to assess individual values of T cell MF. T cell clones were further expanded in vitro and their phenotype was determined by standard immunofluorescence technique. Enzyme-linked immunosorbent assays were used for simultaneous measurements of plasma levels of soluble Interleukin-2 receptors (s-IL-2R) and Intercellular adhesion molecule-1 (s-ICAM-1). RESULT--Mean (SD) value of T cell MF in patients with SSc was 2.5-fold higher than the normal mean (SD) value [10.6 (6.6) x 10(-6) v [4.4 (2.8) x 10(-6), p = 0.0007]. Eleven of 20 patients with SSc (55%) had T cell MF values greater than two SD above the normal mean value. The majority (84%) of mutated T cells had a helper/inducer, memory phenotype while 12% were cytotoxic/suppressor T cells. There was no association between T cell MF and the extent of skin involvement or the duration of Raynaud's phenomenon. High individual T cell MF values were not related to a possible concurrent immune overactivity as assessed by plasma levels of s-IL-2R and s-ICAM-1. Patients with long standing skin disease, however, had almost double T cell MF values than patients with early skin disease [(13.6 (7.4)) x 10(-6) v (7.5 (4.3)) x 10(-6), p = 0.03], suggesting that increased T cell MF in SSc may reflect an ongoing

  9. Effect of carbon monoxide on gene expression in cerebrocortical astrocytes: Validation of reference genes for quantitative real-time PCR.

    PubMed

    Oliveira, Sara R; Vieira, Helena L A; Duarte, Carlos B

    2015-09-15

    Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a widely used technique to characterize changes in gene expression in complex cellular and tissue processes, such as cytoprotection or inflammation. The accurate assessment of changes in gene expression depends on the selection of adequate internal reference gene(s). Carbon monoxide (CO) affects several metabolic pathways and de novo protein synthesis is crucial in the cellular responses to this gasotransmitter. Herein a selection of commonly used reference genes was analyzed to identify the most suitable internal control genes to evaluate the effect of CO on gene expression in cultured cerebrocortical astrocytes. The cells were exposed to CO by treatment with CORM-A1 (CO releasing molecule A1) and four different algorithms (geNorm, NormFinder, Delta Ct and BestKeeper) were applied to evaluate the stability of eight putative reference genes. Our results indicate that Gapdh (glyceraldehyde-3-phosphate dehydrogenase) together with Ppia (peptidylpropyl isomerase A) is the most suitable gene pair for normalization of qRT-PCR results under the experimental conditions used. Pgk1 (phosphoglycerate kinase 1), Hprt1 (hypoxanthine guanine phosphoribosyl transferase I), Sdha (Succinate Dehydrogenase Complex, Subunit A), Tbp (TATA box binding protein), Actg1 (actin gamma 1) and Rn18s (18S rRNA) genes presented less stable expression profiles in cultured cortical astrocytes exposed to CORM-A1 for up to 60 min. For validation, we analyzed the effect of CO on the expression of Bdnf and bcl-2. Different results were obtained, depending on the reference genes used. A significant increase in the expression of both genes was found when the results were normalized with Gapdh and Ppia, in contrast with the results obtained when the other genes were used as reference. These findings highlight the need for a proper and accurate selection of the reference genes used in the quantification of qRT-PCR results

  10. The induction of sister chromatid exchanges by environmental pollutants: relationship of SCE to other measures of genetic damage.

    PubMed

    Brooks, A L; Shimizu, R W; Li, A P; Benson, J M; Dutcher, J S

    1984-01-01

    Sister chromatid exchanges (SCEs), induced by environmental pollutants from fossil fuel use, were measured in 2 cell systems, Chinese hamster ovary (CHO) cells and Chinese hamster primary lung cell cultures. The frequency of SCEs induced in these cell systems was related to other measures of genetic damage, namely mutations in CHO cells at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene locus and in bacteria (Salmonella mutagenicity test TA-98), produced by the same pollutants. The pollutants were divided into 2 classes: those produced in oxidizing combustion environments--extracts of particles from light-duty diesel cars, spark-ignition cars, and an automotive tunnel; and those produced in reducing environments--extracts from coke oven mains and condensates from a low BTU coal gasifier obtained either before or after cleanup of the process stream. Sister chromatid exchanges were induced by all pollutants without the addition of a rat liver microsomal fraction (S-9 mix), whereas S-9 mix was required to induce a positive response in the CHO/HGPRT assay for all pollutants. The pollutants produced in a reducing environment required metabolic activation by S-9 mix to be mutagenic in the Salmonella mutation assay. The addition of S-9 mix to pollutants produced in an oxidizing environment reduced the response in the Salmonella test. The relative genotoxic potency for each pollutant was determined for all 3 endpoints. The slopes of dose-response curves for each pollutant were plotted for each assay to compare relative potency. When the bacterial mutagenicity test was compared to either mammalian cell assay, SCE or CHO/HGPRT, there was little correlation between relative potencies. However, the data indicated that the responses in the 2 mammalian cell assays, SCE and CHO/HGPRT, showed similar relative responses to the pollutants. Differences in the requirement for S-9 mix seem to be related to both the chemical nature of the mixture and the endpoint

  11. Interrelationship between anionic and cationic forms of glutathione S-transferases of human liver.

    PubMed Central

    Awasthi, Y C; Dao, D D; Saneto, R P

    1980-01-01

    Human liver glutathione S-transferases (GSH S-transferases) were fractionated into cationic and anionic proteins. During fractionation with (NH4)2SO4 the anionic GSH S-transferases are concentrated in the 65%-saturated-(NH4)2SO4 fraction, whereas the cationic GSH S-transferases separate in the 80%-saturated-(NH4)2SO4 fraction. From the 65%-saturated-(NH4)2SO4 fraction two new anionic GSH S-transferases, omega and psi, were purified to homogeneity by using ion-exchange chromatography on DEAE-cellulose, Sephadex G-200 gel filtration, affinity chromatography on GSH bound to epoxy-activated Sepharose and isoelectric focusing. By a similar procedure, cationic GSH S-transferases were purified from the 80%-saturated-(NH4)2SO4 fraction. Isoelectric points of GSH S-transferases omega and psi are 4.6 and 5.4 respectively. GSH S-transferase omega is the major anionic GSH S-transferase of human liver, whereas GSH S-transferase psi is present only in traces. The subunit mol.wt. of GSH S-transferase omega is about 22500, whereas that of cationic GSH S-transferases is about 24500. Kinetic and structural properties as well as the amino acid composition of GSH S-transferase omega are described. The antibodies raised against cationic GSH S-transferases cross-react with GSH S-transferase omega. There are significant differences between the catalytic properties of GSH S-transferase omega and the cationic GSH S-transferases. GSH peroxidase II activity is displayed by all five cationic GSH S-transferases, whereas both anionic GSH S-transferases do not display this activity. Images Fig. 3. PMID:7470087

  12. Terminal Deoxynucleotidyl Transferase: The Story of a Misguided DNA Polymerase

    PubMed Central

    Motea, Edward A.; Berdis, Anthony J.

    2009-01-01

    Nearly every DNA polymerase characterized to date exclusively catalyzes the incorporation of mononucleotides into a growing primer using a DNA or RNA template as a guide to direct each incorporation event. There is, however, one unique DNA polymerase designated terminal deoxynucleotidyl transferase that performs DNA synthesis using only single-stranded DNA as the nucleic acid substrate. In this chapter, we review the biological role of this enigmatic DNA polymerase and the biochemical mechanism for its ability to perform DNA synthesis in the absence of a templating strand. We compare and contrast the molecular events for template-independent DNA synthesis catalyzed by terminal deoxynucleotidyl transferase with other well-characterized DNA polymerases that perform template-dependent synthesis. This includes a quantitative inspection of how terminal deoxynucleotidyl transferase binds DNA and dNTP substrates, the possible involvement of a conformational change that precedes phosphoryl transfer, and kinetic steps that are associated with the release of products. These enzymatic steps are discussed within the context of the available structures of terminal deoxynucleotidyl transferase in the presence of DNA or nucleotide substrate. In addition, we discuss the ability of proteins involved in replication and recombination to regulate the activity of the terminal deoxynucleotidyl transferase. Finally, the biomedical role of this specialized DNA polymerase is discussed focusing on its involvement in cancer development and its use in biomedical applications such as labeling DNA for detecting apoptosis. PMID:19596089

  13. Inhibition of hepatic glutathione transferases by propylthiouracil and its metabolites.

    PubMed

    Kariya, K; Sawahata, T; Okuno, S; Lee, E

    1986-05-01

    The effects of propylthiouracil (PTU) and its metabolites on the activity of GSH transferases were examined using rat liver cytosol. PTU inhibited the enzyme activity toward both CDNB and DCNB in a concentration-dependent manner. At the concentration of 10 mM, PTU caused 25% inhibition, which was the maximum effect. PTU derivatives such as propyluracil and thiouracil showed the same effect as the parent compound. On the other hand, S-oxides of PTU such as PTU-SO2 and PTU-SO3, which were chemically synthesized by the oxidation of PTU, were more potent inhibitors of GSH transferases than the parent PTU. A significant inhibition was observed at a concentration of 0.1 mM of PTU S-oxides. At a concentration of 10 mM the S-oxides caused an 80% inhibition of the enzyme activity. PTU inhibited the transferase activity by competing with GSH but the S-oxides of PTU acted by another mechanism. In contrast to the effect on GSH transferases, PTU-SO3 had a weak inhibitory effect on GSH peroxidase activity. Thus, oxidation of PTU leads to products which are potent inhibitors of GSH transferases. PMID:3707612

  14. 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

  15. Thioltransferase activity of bovine lens glutathione S-transferase.

    PubMed Central

    Dal Monte, M; Cecconi, I; Buono, F; Vilardo, P G; Del Corso, A; Mura, U

    1998-01-01

    A Mu-class glutathione S-transferase purified to electrophoretic homogeneity from bovine lens displayed thioltransferase activity, catalysing the transthiolation reaction between GSH and hydroxyethyldisulphide. The thiol-transfer reaction is composed of two steps, the formation of GSSG occurring through the generation of an intermediate mixed disulphide between GSH and the target disulphide. Unlike glutaredoxin, which is only able to catalyse the second step of the transthiolation process, glutathioneS-transferase catalyses both steps of the reaction. Data are presented showing that bovine lens glutathione S-transferase and rat liver glutaredoxin, which was used as a thioltransferase enzyme model, can operate in synergy to catalyse the GSH-dependent reduction of hydroxyethyldisulphide. PMID:9693102

  16. METAL-INDUCED INHIBITION OF GLUTATHIONE S-TRANSFERASES

    EPA Science Inventory

    The glutathione S-transferases comprise a group of multi-functional enzymes involved in the biotransformation/detoxication of a broad spectrum of hydrophobic compounds bearing an electrophilic center. The enzymes facilitate the nucleophilic attack of the -SH group of reduced glut...

  17. Rational design of an organometallic glutathione transferase inhibitor

    SciTech Connect

    Ang, W.H.; Parker, L.J.; De Luca, A.; Juillerat-Jeanneret, L.; Morton, C.J.; LoBello, M.; Parker, M.W.; Dyson, P.J.

    2010-08-17

    A hybrid organic-inorganic (organometallic) inhibitor was designed to target glutathione transferases. The metal center is used to direct protein binding, while the organic moiety acts as the active-site inhibitor. The mechanism of inhibition was studied using a range of biophysical and biochemical methods.

  18. GLUTATHIONE S-TRANSFERASE-MEDIATED METABOLISM OF BROMODICHLOROMETHANE

    EPA Science Inventory

    GLUTATHIONE s-TRANSFERASE-MEDIATED METABOLISM OF BROMODICHLOROMETHANE. M K Ross1 and R A Pegram2. 1Curriculum in Toxicology, University of North Carolina at Chapel Hill; 2Experimental Toxicology Division, NHEERL/ORD, United States Environmental Protection Agency, Research Triangl...

  19. 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 Test Systems § 862.1535 Ornithine...

  20. Genetics Home Reference: succinyl-CoA:3-ketoacid CoA transferase deficiency

    MedlinePlus

    ... CoA:3-ketoacid CoA transferase deficiency succinyl-CoA:3-ketoacid CoA transferase deficiency Enable Javascript to view ... PDF Open All Close All Description Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is an inherited ...

  1. Developmental aspects of glutathione S-transferase B (ligandin) in rat liver.

    PubMed Central

    Hales, B F; Neims, A H

    1976-01-01

    The postnatal development in male Sprague-Dawley rats of hepatic glutathione S-transferase B (ligandin) in relation to the other glutathione S-transferases is described. The concentration of glutathione S-transferase B in 1-day-old male rats is about one-fifth of that in adult animals. The enzyme reaches adult concentrations 4-5 weeks later. When assessed by substrate specificity or immunologically, the proportion of transferase B relative to the other glutathione S-transferases is high during the first week after birth. At this age, 67.5% of the transferase activity towards 1-chloro-2,4-dinitrobenzene is immunoprecipitable by anti-(transferase B), compared with about 50% in adults and older pups. Between the second and the fifth postnatal week, the fraction of transferase B increases in parallel fashion with the other transferases in hepatic cytosol. Neither L-thyroxine nor cortisol induce a precocious increase in glutathione S-transferase activity. Phenobarbital did induce transferase activity towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene in both pups and adults. The extent of induction by phenobarbital was a function of basal activity during development such that the percentage stimulation remained constant from 5 days postnatally to adulthood. PMID:1008852

  2. Purification and characterization of a DNA strand transferase from broccoli.

    PubMed

    Tissier, A F; Lopez, M F; Signer, E R

    1995-05-01

    A protein with DNA binding, renaturation, and strand-transfer activities has been purified to homogeneity from broccoli (Brassica oleracea var italica). The enzyme, broccoli DNA strand transferase, has a native molecular mass of at least 200 kD and an apparent subunit molecular mass of 95 kD and is isolated as a set of isoforms differing only in charge. All three activities are saturated at very low stoichiometry, one monomer per approximately 1000 nucleotides of single-stranded DNA. Strand transfer is not effected by nuclease activity and reannealing, is only slightly dependent on ATP, and is independent of added Mg2+. Transfer requires homologous single- and double-stranded DNA and at higher enzyme concentrations results in very high molecular mass complexes. As with Escherichia coli RecA, transfer by broccoli DNA strand transferase depends strongly on the presence of 3' homologous ends. PMID:7784508

  3. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1315 Galactose-1-phosphate uridyl transferase test system. (a)...

  4. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1315 Galactose-1-phosphate uridyl transferase test system. (a)...

  5. Glutathione transferase mimics: micellar catalysis of an enzymic reaction.

    PubMed Central

    Lindkvist, B; Weinander, R; Engman, L; Koetse, M; Engberts, J B; Morgenstern, R

    1997-01-01

    Substances that mimic the enzyme action of glutathione transferases (which serve in detoxification) are described. These micellar catalysts enhance the reaction rate between thiols and activated halogenated nitroarenes as well as alpha,beta-unsaturated carbonyls. The nucleophilic aromatic substitution reaction is enhanced by the following surfactants in descending order: poly(dimethyldiallylammonium - co - dodecylmethyldiallylammonium) bromide (86/14) >>cetyltrimethylammonium bromide>zwittergent 3-16 (n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate)>zwittergent+ ++ 3-14 (n-tetradecyl-N,N-dimethyl - 3 - ammonio -1 - propanesulphonate) approximately N,N - dimethyl - laurylamine N-oxide>N,N-dimethyloctylamine N-oxide. The most efficient catalyst studied is a polymeric material that incorporates surfactant properties (n-dodecylmethyldiallylammonium bromide) and opens up possibilities for engineering sequences of reactions on a polymeric support. Michael addition to alpha,beta-unsaturated carbonyls is exemplified by a model substance, trans-4-phenylbut-3-en-2-one, and a toxic compound that is formed during oxidative stress, 4-hydroxy-2-undecenal. The latter compound is conjugated with the highest efficiency of those tested. Micellar catalysts can thus be viewed as simple models for the glutathione transferases highlighting the influence of a positive electrostatic field and a non-specific hydrophobic binding site, pertaining to two catalytic aspects, namely thiolate anion stabilization and solvent shielding. PMID:9173899

  6. Nucleotidyl transferase assisted DNA labeling with different click chemistries

    PubMed Central

    Winz, Marie-Luise; Linder, Eva Christina; André, Timon; Becker, Juliane; Jäschke, Andres

    2015-01-01

    Here, we present a simple, modular and efficient strategy that allows the 3′-terminal labeling of DNA, regardless of whether it has been chemically or enzymatically synthesized or isolated from natural sources. We first incorporate a range of modified nucleotides at the 3′-terminus, using terminal deoxynucleotidyl transferase. In the second step, we convert the incorporated nucleotides, using either of four highly efficient click chemistry-type reactions, namely copper-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, Staudinger ligation or Diels-Alder reaction with inverse electron demand. Moreover, we create internal modifications, making use of either ligation or primer extension, after the nucleotidyl transferase step, prior to the click reaction. We further study the influence of linker variants on the reactivity of azides in different click reactions. We find that different click reactions exhibit distinct substrate preferences, a fact that is often overlooked, but should be considered when labeling oligonucleotides or other biomolecules with click chemistry. Finally, our findings allowed us to extend our previously published RNA labeling strategy to the use of a different copper-free click chemistry, namely the Staudinger ligation. PMID:26013812

  7. [Selective N-heterylazimine inhibition of reactions catalyzed by rat liver glutathione transferase].

    PubMed

    Stulovskiĭ, A V; Voznyĭ, I V; Rozengart, E V; Suvorov, A A; Khovanskikh, A E

    1992-01-01

    Three reactions (nucleophile substitution, thiolysis and N-deoxygenation) catalyzed by rat liver glutathione transferase have been studied using several N-heterylazimine inhibitors. The inhibitors are sharply different in their effectiveness in the transferase reactions. Their efficiency depends on their structure. The mechanism which underlies the found regularities is suggested. PMID:1413125

  8. Glutathione S-transferase class {pi} polymorphism in baboons

    SciTech Connect

    Aivaliotis, M.J.; Cantu, T.; Gilligan, R.

    1995-02-01

    Glutathione S-transferase (GST) comprises a family of isozymes with broad substrate specificities. One or more GST isozymes are present in most animal tissues and function in several detoxification pathways through the conjugation of reduced glutathione with various electrophiles, thereby reducing their potential toxicity. Four soluble GST isozymes encoded by genes on different chromosomes have been identified in humans. The acidic class pi GST, GSTP (previously designated GST-3), is widely distributed in adult tissues and appears to be the only GST isozyme present in leukocytes and placenta. Previously reported electrophoretic analyses of erythrocyte and leukocyte extracts revealed single bands of activity, which differed slightly in mobility between the two cell types, or under other conditions, a two-banded pattern. To our knowledge, no genetically determined polymorphisms have previously been reported in GSTP from any species. We now report a polymorphism of GSTP in baboon leukocytes, and present family data that verifies autosomal codominant inheritance. 14 refs., 2 figs., 1 tab.

  9. Recombinant baculovirus vectors expressing glutathione-S-transferase fusion proteins.

    PubMed

    Davies, A H; Jowett, J B; Jones, I M

    1993-08-01

    Recombinant baculoviruses are a popular means of producing heterologous protein in eukaryotic cells. Purification of recombinant proteins away from the insect cell background can, however, remain an obstacle for many developments. Recently, prokaryotic fusion protein expression systems have been developed allowing single-step purification of the heterologous protein and specific proteolytic cleavage of the affinity tag moiety from the desired antigen. Here we report the introduction of these attributes to the baculovirus system. "Baculo-GEX" vectors enable baculovirus production of fusion proteins with the above advantages, but in a eukaryotic post-translational processing environment. Glutathione-S-transferase (GST) fusions are stable cytoplasmic proteins in insect cells and may therefore be released by sonication alone, avoiding the solubility problems and detergent requirements of bacterial systems. Thus large amounts of authentic antigen may be purified in a single, non-denaturing step. PMID:7763917

  10. Functional analysis and localisation of a delta-class glutathione S-transferase from Sarcoptes scabiei.

    PubMed

    Pettersson, Eva U; Ljunggren, Erland L; Morrison, David A; Mattsson, Jens G

    2005-01-01

    The mite Sarcoptes scabiei causes sarcoptic mange, or scabies, a disease that affects both animals and humans worldwide. Our interest in S. scabiei led us to further characterise a glutathione S-transferase. This multifunctional enzyme is a target for vaccine and drug development in several parasitic diseases. The S. scabiei glutathione S-transferase open reading frame reported here is 684 nucleotides long and yields a protein with a predicted molecular mass of 26 kDa. Through phylogenetic analysis the enzyme was classified as a delta-class glutathione S-transferase, and our paper is the first to report that delta-class glutathione S-transferases occur in organisms other than insects. The recombinant S. scabiei glutathione S-transferase was expressed in Escherichia coli via three different constructs and purified for biochemical analysis. The S. scabiei glutathione S-transferase was active towards the substrate 1-chloro-2,4-dinitrobenzene, though the positioning of fusion partners influenced the kinetic activity of the enzyme. Polyclonal antibodies raised against S. scabiei glutathione S-transferase specifically localised the enzyme to the integument of the epidermis and cavities surrounding internal organs in adult parasites. However, some minor staining of parasite intestines was observed. No staining was seen in host tissues, nor could we detect any antibody response against S. scabiei glutathione S-transferase in sera from naturally S. scabiei infected dogs or pigs. Additionally, the polyclonal sera raised against recombinant S. scabiei glutathione S-transferase readily detected a protein from mites, corresponding to the predicted size of native glutathione S-transferase. PMID:15619514

  11. Crystal structure of E. coli lipoprotein diacylglyceryl transferase.

    PubMed

    Mao, Guotao; Zhao, Yan; Kang, Xusheng; Li, Zhijie; Zhang, Yan; Wang, Xianping; Sun, Fei; Sankaran, Krishnan; Zhang, Xuejun C

    2016-01-01

    Lipoprotein biogenesis is essential for bacterial survival. Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) is an integral membrane enzyme that catalyses the first reaction of the three-step post-translational lipid modification. Deletion of the lgt gene is lethal to most Gram-negative bacteria. Here we present the crystal structures of Escherichia coli Lgt in complex with phosphatidylglycerol and the inhibitor palmitic acid at 1.9 and 1.6 Å resolution, respectively. The structures reveal the presence of two binding sites and support the previously reported structure-function relationships of Lgt. Complementation results of lgt-knockout cells with different mutant Lgt variants revealed critical residues, including Arg143 and Arg239, that are essential for diacylglyceryl transfer. Using a GFP-based in vitro assay, we correlated the activities of Lgt with structural observations. Together, the structural and biochemical data support a mechanism whereby substrate and product, lipid-modified lipobox-containing peptide, enter and leave the enzyme laterally relative to the lipid bilayer. PMID:26729647

  12. Inactivation of Anopheles gambiae Glutathione Transferase ε2 by Epiphyllocoumarin

    PubMed Central

    Marimo, Patience; Hayeshi, Rose; Mukanganyama, Stanley

    2016-01-01

    Glutathione transferases (GSTs) are part of a major family of detoxifying enzymes that can catalyze the reductive dehydrochlorination of dichlorodiphenyltrichloroethane (DDT). The delta and epsilon classes of insect GSTs have been implicated in conferring resistance to this insecticide. In this study, the inactivation of Anopheles gambiae GSTε2 by epiphyllocoumarin (Tral 1) was investigated. Recombinant AgGSTε2 was expressed in Escherichia coli cells containing a pET3a-AGSTε2 plasmid and purified by affinity chromatography. Tral 1 was shown to inactivate GSTε2 both in a time-dependent manner and in a concentration-dependent manner. The half-life of GSTε2 in the presence of 25 μM ethacrynic acid (ETA) was 22 minutes and with Tral 1 was 30 minutes, indicating that Tral 1 was not as efficient as ETA as an inactivator. The inactivation parameters kinact and KI were found to be 0.020 ± 0.001 min−1 and 7.5 ± 2.1 μM, respectively, after 90 minutes of incubation. Inactivation of GSTε2 by Tral 1 implies that Tral 1 covalently binds to this enzyme in vitro and would be expected to exhibit time-dependent effects on the enzyme in vivo. Tral 1, therefore, would produce irreversible effects when used together with dichlorodiphenyltrichloroethane (DDT) in malaria control programmes where resistance is mediated by GSTs. PMID:26925266

  13. Crystal structure of E. coli lipoprotein diacylglyceryl transferase

    PubMed Central

    Mao, Guotao; Zhao, Yan; Kang, Xusheng; Li, Zhijie; Zhang, Yan; Wang, Xianping; Sun, Fei; Sankaran, Krishnan; Zhang, Xuejun C.

    2016-01-01

    Lipoprotein biogenesis is essential for bacterial survival. Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) is an integral membrane enzyme that catalyses the first reaction of the three-step post-translational lipid modification. Deletion of the lgt gene is lethal to most Gram-negative bacteria. Here we present the crystal structures of Escherichia coli Lgt in complex with phosphatidylglycerol and the inhibitor palmitic acid at 1.9 and 1.6 Å resolution, respectively. The structures reveal the presence of two binding sites and support the previously reported structure–function relationships of Lgt. Complementation results of lgt-knockout cells with different mutant Lgt variants revealed critical residues, including Arg143 and Arg239, that are essential for diacylglyceryl transfer. Using a GFP-based in vitro assay, we correlated the activities of Lgt with structural observations. Together, the structural and biochemical data support a mechanism whereby substrate and product, lipid-modified lipobox-containing peptide, enter and leave the enzyme laterally relative to the lipid bilayer. PMID:26729647

  14. Benzene oxide is a substrate for glutathione S-transferases.

    PubMed

    Zarth, Adam T; Murphy, Sharon E; Hecht, Stephen S

    2015-12-01

    Benzene is a known human carcinogen which must be activated to benzene oxide (BO) to exert its carcinogenic potential. BO can be detoxified in vivo by reaction with glutathione and excretion in the urine as S-phenylmercapturic acid. This process may be catalyzed by glutathione S-transferases (GSTs), but kinetic data for this reaction have not been published. Therefore, we incubated GSTA1, GSTT1, GSTM1, and GSTP1 with glutathione and BO and quantified the formation of S-phenylglutathione. Kinetic parameters were determined for GSTT1 and GSTP1. At 37 °C, the putative Km and Vmax values for GSTT1 were 420 μM and 450 fmol/s, respectively, while those for GSTP1 were 3600 μM and 3100 fmol/s. GSTA1 and GSTM1 did not exhibit sufficient activity for determination of kinetic parameters. We conclude that GSTT1 is a critical enzyme in the detoxification of BO and that GSTP1 may also play an important role, while GSTA1 and GSTM1 seem to be less important. PMID:26554337

  15. Modulation of Rab GTPase function by a protein phosphocholine transferase.

    PubMed

    Mukherjee, Shaeri; Liu, Xiaoyun; Arasaki, Kohei; McDonough, Justin; Galán, Jorge E; Roy, Craig R

    2011-09-01

    The intracellular pathogen Legionella pneumophila modulates the activity of host GTPases to direct the transport and assembly of the membrane-bound compartment in which it resides. In vitro studies have indicated that the Legionella protein DrrA post-translationally modifies the GTPase Rab1 by a process called AMPylation. Here we used mass spectrometry to investigate post-translational modifications to Rab1 that occur during infection of host cells by Legionella. Consistent with in vitro studies, DrrA-mediated AMPylation of a conserved tyrosine residue in the switch II region of Rab1 was detected during infection. In addition, a modification to an adjacent serine residue in Rab1 was discovered, which was independent of DrrA. The Legionella effector protein AnkX was required for this modification. Biochemical studies determined that AnkX directly mediates the covalent attachment of a phosphocholine moiety to Rab1. This phosphocholine transferase activity used CDP-choline as a substrate and required a conserved histidine residue located in the FIC domain of the AnkX protein. During infection, AnkX modified both Rab1 and Rab35, which explains how this protein modulates membrane transport through both the endocytic and exocytic pathways of the host cell. Thus, phosphocholination of Rab GTPases represents a mechanism by which bacterial FIC-domain-containing proteins can alter host-cell functions. PMID:21822290

  16. Glutathione S-transferase activity and glutathione S-transferase mu expression in subjects with risk for colorectal cancer.

    PubMed

    Szarka, C E; Pfeiffer, G R; Hum, S T; Everley, L C; Balshem, A M; Moore, D F; Litwin, S; Goosenberg, E B; Frucht, H; Engstrom, P F

    1995-07-01

    The glutathione S-transferases (alpha, mu, and pi), a family of Phase II detoxication enzymes, play a critical role in protecting the colon mucosa by catalyzing the conjugation of dietary carcinogens with glutathione. We investigated the efficacy of using the glutathione S-transferase (GST) activity of blood lymphocytes and GST-mu expression as biomarkers of risk for colorectal cancer. GST activity was measured in the blood lymphocytes of control individuals (n = 67) and in the blood lymphocytes (n = 60) and colon tissue (n = 34) of individuals at increased risk for colon cancer. Total GST activity was determined spectrophotometrically with the use of 1-chloro-2,4-dinitrobenzene as a substrate. The ability to express the um subclass of GST was determined with the use of an ELISA. Although interindividual variability in the GST activity of blood lymphocytes was greater than 8-fold (range, 16.7-146.8 nmol/min/mg), the GST activity of blood lymphocytes and colon tissue within an individual was constant over time and was unrelated to sex, age, or race. The GST activity of blood lymphocytes from high-risk individuals was significantly lower than that of blood lymphocytes from control individuals (P < or = 0.004). No association was observed between the frequency of GST-mu phenotype and risk for colorectal cancer. Blood lymphocytes from high-risk individuals unable to express GST-mu had lower levels of GST activity than did those from control subjects with the GST-mu null phenotype; however, this difference was significant in male subjects only (P < or = 0.006). Analysis of paired samples of blood lymphocytes and colon tissue indicated a strong correlation between the GST activity of the two tissue types (Spearman's rank correlation, r = 0.87; P < or = 0.0001). The GST activity of blood lymphocytes may be used to identify high-risk individuals with decreased protection from this Phase II detoxication enzyme who may benefit from clinical trials evaluating GST modulators

  17. Analysis of Arabidopsis glutathione-transferases in yeast.

    PubMed

    Krajewski, Matthias P; Kanawati, Basem; Fekete, Agnes; Kowalski, Natalie; Schmitt-Kopplin, Philippe; Grill, Erwin

    2013-07-01

    The genome of Arabidopsis thaliana encodes 54 functional glutathione transferases (GSTs), classified in seven clades. Although plant GSTs have been implicated in the detoxification of xenobiotics, such as herbicides, extensive redundancy within this large gene family impedes a functional analysis in planta. In this study, a GST-deficient yeast strain was established as a system for analyzing plant GSTs that allows screening for GST substrates and identifying substrate preferences within the plant GST family. To this end, five yeast genes encoding GSTs and GST-related proteins were simultaneously disrupted. The resulting yeast quintuple mutant showed a strongly reduced conjugation of the GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl). Consistently, the quintuple mutant was hypersensitive to CDNB, and this phenotype was complemented by the inducible expression of Arabidopsis GSTs. The conjugating activity of the plant GSTs was assessed by in vitro enzymatic assays and via analysis of exposed yeast cells. The formation of glutathione adducts with dinitrobenzene was unequivocally verified by stable isotope labeling and subsequent accurate ultrahigh-resolution mass spectrometry (ICR-FTMS). Analysis of Arabidopsis GSTs encompassing six clades and 42 members demonstrated functional expression in yeast by using CDNB and NBD-Cl as model substrates. Subsequently, the established yeast system was explored for its potential to screen the Arabidopsis GST family for conjugation of the fungicide anilazine. Thirty Arabidopsis GSTs were identified that conferred increased levels of glutathionylated anilazine. Efficient anilazine conjugation was observed in the presence of the phi, tau, and theta clade GSTs including AtGSTF2, AtGSTF4, AtGSTF6, AtGSTF8, AtGSTF10, and AtGSTT2, none of which had previously been known to contribute to fungicide detoxification. ICR-FTMS analysis of yeast extracts allowed the simultaneous detection and

  18. Selection of antisense oligodeoxynucleotides against glutathione S-transferase Mu.

    PubMed Central

    't Hoen, Peter A C; Out, Ruud; Commandeur, Jan N M; Vermeulen, Nico P E; van Batenburg, F H D; Manoharan, Muthiah; van Berkel, Theo J C; Biessen, Erik A L; Bijsterbosch, Martin K

    2002-01-01

    The aim of the present study was to identify functional antisense oligodeoxynucleotides (ODNs) against the rat glutathione S-transferase Mu (GSTM) isoforms, GSTM1 and GSTM2. These antisense ODNs would enable the study of the physiological consequences of GSTM deficiency. Because it has been suggested that the effectiveness of antisense ODNs is dependent on the secondary mRNA structures of their target sites, we made mRNA secondary structure predictions with two software packages, Mfold and STAR. The two programs produced only marginally similar structures, which can probably be attributed to differences in the algorithms used. The effectiveness of a set of 18 antisense ODNs was evaluated with a cell-free transcription/translation assay, and their activity was correlated with the predicted secondary RNA structures. Four phosphodiester ODNs specific for GSTM1, two ODNs specific for GSTM2, and four ODNs targeted at both GSTM isoforms were found to be potent, sequence-specific, and RNase H-dependent inhibitors of protein expression. The IC50 value of the most potent ODN was approximately 100 nM. Antisense ODNs targeted against regions that were predicted by STAR to be predominantly single stranded were more potent than antisense ODNs against double-stranded regions. Such a correlation was not found for the Mfold prediction. Our data suggest that simulation of the local folding of RNA facilitates the discovery of potent antisense sequences. In conclusion, we selected several promising antisense sequences, which, when synthesized as biologically stable oligonucleotides, can be applied for study of the physiological impact of reduced GSTM expression. PMID:12515389

  19. Phosphonocarboxylates Inhibit the Second Geranylgeranyl Addition by Rab Geranylgeranyl Transferase*

    PubMed Central

    Baron, Rudi A.; Tavaré, Richard; Figueiredo, Ana C.; Błażewska, Katarzyna M.; Kashemirov, Boris A.; McKenna, Charles E.; Ebetino, Frank H.; Taylor, Adam; Rogers, Michael J.; Coxon, Fraser P.; Seabra, Miguel C.

    2009-01-01

    Rab geranylgeranyl transferase (RGGT) catalyzes the post-translational geranylgeranyl (GG) modification of (usually) two C-terminal cysteines in Rab GTPases. Here we studied the mechanism of the Rab geranylgeranylation reaction by bisphosphonate analogs in which one phosphonate group is replaced by a carboxylate (phosphonocarboxylate, PC). The phosphonocarboxylates used were 3-PEHPC, which was previously reported, and 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid ((+)-3-IPEHPC), a >25-fold more potent related compound as measured by both IC50 and Ki.(+)-3-IPEHPC behaves as a mixed-type inhibitor with respect to GG pyrophosphate (GGPP) and an uncompetitive inhibitor with respect to Rab substrates. We propose that phosphonocarboxylates prevent only the second GG transfer onto Rabs based on the following evidence. First, geranylgeranylation of Rab proteins ending with a single cysteine motif such as CAAX, is not affected by the inhibitors, either in vitro or in vivo. Second, the addition of an -AAX sequence onto Rab-CC proteins protects the substrate from inhibition by the inhibitors. Third, we demonstrate directly that in the presence of (+)-3-IPEHPC, Rab-CC and Rab-CXC proteins are modified by only a single GG addition. The presence of (+)-3-IPEHPC resulted in a preference for the Rab N-terminal cysteine to be modified first, suggesting an order of cysteine geranylgeranylation in RGGT catalysis. Our results further suggest that the inhibitor binds to a site distinct from the GGPP-binding site on RGGT. We suggest that phosphonocarboxylate inhibitors bind to a GG-cysteine binding site adjacent to the active site, which is necessary to align the mono-GG-Rab for the second GG addition. These inhibitors may represent a novel therapeutic approach in Rab-mediated diseases. PMID:19074143

  20. 40 CFR 158.230 - Experimental use permit data requirements for toxicology.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ..., thymidine kinase (tk) gene locus, maximizing assay conditions for small colony expression or detection; ii... phosphoribosyl transferase (hgprt) gene locus, accompanied by an appropriate in vitro test for clastogenicity; or iii. CHO cells strains AS52, xanthine-guanine phosphoribosyl transferase (xprt) gene locus. 11....

  1. 40 CFR 158.230 - Experimental use permit data requirements for toxicology.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ..., thymidine kinase (tk) gene locus, maximizing assay conditions for small colony expression or detection; ii... phosphoribosyl transferase (hgprt) gene locus, accompanied by an appropriate in vitro test for clastogenicity; or iii. CHO cells strains AS52, xanthine-guanine phosphoribosyl transferase (xprt) gene locus. 11....

  2. 40 CFR 158.230 - Experimental use permit data requirements for toxicology.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ..., thymidine kinase (tk) gene locus, maximizing assay conditions for small colony expression or detection; ii... phosphoribosyl transferase (hgprt) gene locus, accompanied by an appropriate in vitro test for clastogenicity; or iii. CHO cells strains AS52, xanthine-guanine phosphoribosyl transferase (xprt) gene locus. 11....

  3. 40 CFR 158.230 - Experimental use permit data requirements for toxicology.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ..., thymidine kinase (tk) gene locus, maximizing assay conditions for small colony expression or detection; ii... phosphoribosyl transferase (hgprt) gene locus, accompanied by an appropriate in vitro test for clastogenicity; or iii. CHO cells strains AS52, xanthine-guanine phosphoribosyl transferase (xprt) gene locus. 11....

  4. 40 CFR 158.230 - Experimental use permit data requirements for toxicology.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ..., thymidine kinase (tk) gene locus, maximizing assay conditions for small colony expression or detection; ii... phosphoribosyl transferase (hgprt) gene locus, accompanied by an appropriate in vitro test for clastogenicity; or iii. CHO cells strains AS52, xanthine-guanine phosphoribosyl transferase (xprt) gene locus. 11....

  5. Post-transcriptional regulation of chloramphenicol acetyl transferase.

    PubMed Central

    Byeon, W H; Weisblum, B

    1984-01-01

    The +1 site for initiation of inducible chloramphenicol acetyl transferase (CAT) mRNA encoded by plasmid pC194 was determined experimentally by using [alpha-32P]ATP-labeled runoff transcripts partially digested with T1 RNase. By partial digestion of the in vitro transcripts with S1, T1, and cobra venom nucleases as probes of mRNA conformation, single- and double-stranded regions, respectively, were also identified. Thus, a prominent inverted complementary repeat sequence was demonstrated spanning the +14 to +50 positions, which contain the complementary sequences CCUCC and GGAGG (the Shine and Dalgarno sequence for synthesis of CAT) symmetrically apposed and paired as part of a perfect 12-base-pair inverted complementary repeat sequence (-19.5 kcal [ca. -81.7 kJ] per mol). The CAT mRNA was stable to digestion by T1 RNase at the four guanosine residues in the Shine and Dalgarno sequence GGAGG , even at 60 degrees C, suggesting that nascent CAT mRNA allows ribosomes to initiate protein synthesis inefficiently and that induction involves post-transcriptional unmasking of the Shine and Dalgarno sequence. Consistent with this model of regulation, we found that cells carrying pC194 , induced with chloramphenicol, contain about the same concentration of pulse-labeled CAT-specific RNA as do uninduced cells. Induction of CAT synthesis by the non- acetylatable chloramphenicol analog fluorothiamphenicol was tested by using minicells of Bacillus subtilis carrying pC194 as well as minicells containing the cloned pC194 derivatives in which parts of the CAT structural gene were deleted in vitro with BAL 31 exonuclease. Optimal induction of both full-length (active) and deleted (inactive) CAT required similar concentrations of fluorothiamphenicol, whereas induction by chloramphenicol required a higher concentration for the wild-type full-length (active) CAT than for the (inactive) deleted CAT. Because synthesis of deleted CAT was inducible, we infer that CAT plays no direct role

  6. Glucomannan synthesis in pea epicotyls: the mannose and glucose transferases.

    PubMed

    Piro, G; Zuppa, A; Dalessandro, G; Northcote, D H

    1993-01-01

    Membrane fractions and digitonin-solubilized enzymes prepared from stem segments isolated from the third internode of etiolated pea seedlings (Pisum sativum L. cv. Alaska) catalyzed the synthesis of a beta-1,4-[14C]mannan from GDP-D-[U-14C]-mannose, a mixed beta-1,3- and beta-1,4-[14C]glucan from GDP-D-[U-14C]-glucose and a beta-1,4-[14C]-glucomannan from both GDP-D-[U-14C]mannose and GDP-D-[U-14C]glucose. The kinetics of the membrane-bound and soluble mannan and glucan synthases were determined. The effects of ions, chelators, inhibitors of lipid-linked saccharides, polyamines, polyols, nucleotides, nucleoside-diphosphate sugars, acetyl-CoA, group-specific chemical probes, phospholipases and detergents on the membrane-bound mannan and glucan synthases were investigated. The beta-glucan synthase had different properties from other preparations which bring about the synthesis of beta-1,3-glucans (callose) and mixed beta-1,3- and beta-1,4- glucans and which use UDP-D-glucose as substrate. It also differed from xyloglucan synthase because in the presence of several concentrations of UDP-D-xylose in addition to GDP-D-glucose no xyloglucan was formed. Using either the membrane-bound or the soluble mannan synthase, GDP-D-glucose acted competitively in the presence of GDP-D-mannose to inhibit the incorporation of mannose into the polymer. This was not due to an inhibition of the transferase activity but was a result of the incorporation of glucose residues from GDP-D-glucose into a glucomannan. The kinetics and the composition of the synthesized glucomannan depended on the ratio of the concentrations of GDP-D-glucose and GDP-D-mannose that were available. Our data indicated that a single enzyme has an active centre that can use both GDP-D-mannose and GDP-D-glucose to bring about the synthesis of the heteropolysaccharide. PMID:7685647

  7. Characterization of prenyl protein transferase enzymes in a human keratinocyte cell line.

    PubMed

    MacNulty, E E; Ryder, N S

    1996-02-01

    Prenylation is a post-translational modification of proteins that involves the attachment of an isoprenoid group derived from mevalonic acid, either 15-carbon farnesyl or 20-carbon geranylgeranyl, to a specific carboxy-terminal domain of acceptor proteins. Three prenyl transferase enzymes have been identified so far. In this paper we report the presence of two prenyl transferases in the HaCaT human keratinocyte cell line. Chromatography of a cytosolic extract from these cells resolved a farnesyl protein transferase (FPT) and geranylgeranyl protein transferase-I (GGPT-I) whose activities were measured using a novel peptide-based assay. Both enzymes were inhibited dose dependently by zaragozic acids A and C. Zaragozic acid C was more active towards the FPT than GGPT-I while zaragozic acid A inhibited both enzymes with similar potency. Incubation of HaCaT cell homogenates with [3H] prenyl precursors resulted in the labelling of a number of proteins which was increased when the cells were pretreated with an inhibitor of hydroxymethylglutaryl CoA reductase. Given the role of prenylated proteins in proliferative and inflammatory processes, our finding that prenyl transferases capable of prenylating endogenous substrates are also present in keratinocytes suggests that these enzymes might provide novel therapeutic targets of dermatological importance. PMID:8605230

  8. Mutation Induction in Mammalian Cells by Accelerated Heavy Ions

    NASA Astrophysics Data System (ADS)

    Rosendahl, I. M.; Baumstark-Khan, C.; Rink, H.

    The deleterious effects of accelerated heavy ions on living cells are of increasing importance for long duration human space flight activities. An important aspect of this field is attributed to the type and quality of biological damage induced by these densely ionizing particles. To address this aspect, cell inactivation and mutation induction at the hprt locus (coding for hypoxanthine-guanine-phosphoribosyl-transferase) was investigated in cultured V79 Chinese Hamster Cells irradiated with accelerated heavy ions (8-O, 20-Ca, 79-Au, and 92-U) and X-rays. Specific energies of the ions ranged from 1.9 to 69.7 MeV/u and corresponding LET values were between 62 band 15,580 keV/μ m. 30 spontaneous and 196 heavy-ion induced 6-thioguanine resistant hprt mutant colonies were characterized by Southern technique using the restriction enzymes EcoRI, PstI and BglII and a full length hprt cDNA probe isolated from the plasmid pHpt12 (kindly provided by Dr. J. Thacker). While inactivation cross sections (σ i) rise over the whole LET range, mutation induction cross sections (σ m) increase up to approximately 300 keV/μ m (O-ions) but decline with heavier ions and more extreme LET values. A similar behaviour is seen with mutation frequency dependent on particle fluence. After irradiation with accelerated uranium ions (8.8 MeV/u, 15,580 keV/μ m) a significant decrease of mutation frequency was found with higher particle fluences (3× 106 particles cm-2). Nearly no mutants were recovered with 8× 106 particles cm-2. All restriction patterns of the spontaneous hprt mutants were indistinguishable from the wild type pattern. These mutants probably contain small deletions or point mutations in the hprt locus. In contrast, the overall spectrum of heavy ion induced mutations revealed a majority (67%) of partial or complete deletions of the hprt gene. With constant particle fluence (3× 106 particles cm-2) the quality of heavy ion induced mutations in the hprt locus depends on physical

  9. O-GlcNAc transferase invokes nucleotide sugar pyrophosphate participation in catalysis

    PubMed Central

    Schimpl, Marianne; Zheng, Xiaowei; Borodkin, Vladimir S.; Blair, David E.; Ferenbach, Andrew T.; Schüttelkopf, Alexander W.; Navratilova, Iva; Aristotelous, Tonia; Albarbarawi, Osama; Robinson, David A.; Macnaughtan, Megan A.; van Aalten, Daan M.F.

    2012-01-01

    Protein O-GlcNAcylation is an essential post-translational modification on hundreds of intracellular proteins in metazoa, catalyzed by O-GlcNAc transferase using unknown mechanisms of transfer and substrate recognition. Through crystallographic snapshots and mechanism-inspired chemical probes, we define how human O-GlcNAc transferase recognizes the sugar donor and acceptor peptide and employs a novel catalytic mechanism of glycosyl transfer, involving the sugar donor α-phosphate as the catalytic base, as well as an essential lysine. This mechanism appears to be a unique evolutionary solution to the spatial constraints imposed by a bulky protein acceptor substrate, and explains the unexpected specificity of a recently reported metabolic O-GlcNAc transferase inhibitor. PMID:23103942

  10. The yeast WBP1 is essential for oligosaccharyl transferase activity in vivo and in vitro.

    PubMed Central

    te Heesen, S; Janetzky, B; Lehle, L; Aebi, M

    1992-01-01

    Asparagine-linked N-glycosylation is a highly conserved and functionally important modification of proteins in eukaryotic cells. The central step in this process is a cotranslational transfer of lipid-linked core oligosaccharides to selected Asn-X-Ser/Thr-sequences of nascent polypeptide chains, catalysed by the enzyme N-oligosaccharyl transferase. In this report we show that the essential yeast protein WBP1 (te Heesen et al., 1991) is required for N-oligosaccharyl transferase in vivo and in vitro. Depletion of WBP1 correlates with a defect in transferring core oligosaccharides to carboxypeptidase Y and proteinase A in vivo. In addition, in vitro N-glycosylation of the acceptor peptide Tyr-Asn-Leu-Thr-Ser-Val using microsomal membranes from WBP1 depleted cells is reduced as compared with membranes from wild-type cells. We propose that WBP1 is an essential component of the oligosaccharyl transferase in yeast. Images PMID:1600939

  11. Terminal Deoxynucleotidyl Transferase in a Case of Childhood Acute Lymphoblastic Leukemia

    PubMed Central

    McCaffrey, Ronald; Smoler, Donna F.; Baltimore, David

    1973-01-01

    Cells from a patient with childhood acute lymphoblastic leukemia contain an apparent DNA polymerase activity that was not found in any other cells except thymus cells. The enzyme has the properties of terminal transferase, an enzyme known to be found in thymocytes. The cells also contain the three major DNA polymerases found in growing cells. The results suggest that these tumor cells arose from a block in the differentiation of thymocytes. Terminal transferase may be a marker for the origin of leukemic cells. PMID:4346893

  12. Type II Hydride Transferases from Different Microorganisms Yield Nitrite and Diarylamines from Polynitroaromatic Compounds▿ †

    PubMed Central

    van Dillewijn, Pieter; Wittich, Rolf-Michael; Caballero, Antonio; Ramos, Juan-Luis

    2008-01-01

    Homogenous preparations of XenB of Pseudomonas putida, pentaerythritol tetranitrate reductase of Enterobacter cloacae, and N-ethylmaleimide reductase of Escherichia coli, all type II hydride transferases of the Old Yellow Enzyme family of flavoproteins, are shown to reduce the polynitroaromatic compound 2,4,6-trinitrotoluene (TNT). The reduction of this compound yields hydroxylaminodinitrotoluenes and Meisenheimer dihydride complexes, which, upon condensation, yield stoichiometric amounts of nitrite and diarylamines, implying that type II hydride transferases are responsible for TNT denitration, a process with important environmental implications for TNT remediation. PMID:18791007

  13. 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…

  14. [(1)H] magnetic resonance spectroscopy of urine: diagnosis of a guanidinoacetate methyl transferase deficiency case.

    PubMed

    Tassini, Maria; Zannolli, Raffaella; Buoni, Sabrina; Engelke, Udo; Vivi, Antonio; Valensin, Gianni; Salomons, Gajja S; De Nicola, Anna; Strambi, Mirella; Monti, Lucia; Morava, Eva; Wevers, Ron A; Hayek, Joseph

    2010-01-01

    For the first time, the use of urine [(1)H] magnetic resonance spectroscopy has allowed the detection of 1 case of guanidinoacetate methyl transferase in a database sample of 1500 pediatric patients with a diagnosis of central nervous system impairment of unknown origin. The urine [(1)H] magnetic resonance spectroscopy of a 9-year-old child, having severe epilepsy and nonprogressive mental and motor retardation with no apparent cause, revealed a possible guanidinoacetic acid increase. The definitive assignment of guanidinoacetic acid was checked by addition of pure substance to the urine sample and by measuring [(1)H]-[(1)H] correlation spectroscopy. Diagnosis of guanidinoacetate methyl transferase deficiency was further confirmed by liquid chromatography-mass spectrometry, brain [(1)H] magnetic resonance spectroscopy, and mutational analysis of the guanidinoacetate methyl transferase gene. The replacement therapy was promptly started and, after 1 year, the child was seizure free. We conclude that for this case, urine [(1)H] magnetic resonance spectroscopy screening was able to diagnose guanidinoacetate methyl transferase deficiency. PMID:19461121

  15. Maize white seedling 3 results from disruption of homogentisate solanesyl transferase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize white seedling 3 (w3) has served as a model albino-seedling mutant since its discovery in 1923. We show here 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 re...

  16. 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...

  17. 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...

  18. 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...

  19. 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...

  20. 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...

  1. 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...

  2. 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...

  3. 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...

  4. 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...

  5. 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...

  6. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... genetically modified cotton, oilseed rape, and tomatoes in accordance with the following prescribed conditions... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Aminoglycoside 3â²-phospho- transferase II. 573.130 Section 573.130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  7. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... genetically modified cotton, oilseed rape, and tomatoes in accordance with the following prescribed conditions... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Aminoglycoside 3â²-phospho- transferase II. 573.130 Section 573.130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  8. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... genetically modified cotton, oilseed rape, and tomatoes in accordance with the following prescribed conditions... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Aminoglycoside 3â²-phospho- transferase II. 573.130 Section 573.130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  9. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... genetically modified cotton, oilseed rape, and tomatoes in accordance with the following prescribed conditions... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Aminoglycoside 3â²-phospho- transferase II. 573.130 Section 573.130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  10. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... genetically modified cotton, oilseed rape, and tomatoes in accordance with the following prescribed conditions... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Aminoglycoside 3â²-phospho- transferase II. 573.130 Section 573.130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  11. Structural and biochemical analyses reveal how ornithine acetyl transferase binds acidic and basic amino acid substrates.

    PubMed

    Iqbal, Aman; Clifton, Ian J; Chowdhury, Rasheduzzaman; Ivison, David; Domene, Carmen; Schofield, Christopher J

    2011-09-21

    Structural and biochemical analyses reveal how ornithine acetyl-transferases catalyse the reversible transfer of an acetyl-group from a basic (ornithine) to an acidic (glutamate) amino acid by employing a common mechanism involving an acetyl-enzyme intermediate but using different side chain binding modes. PMID:21796301

  12. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Galactose-1-phosphate uridyl transferase test system. 862.1315 Section 862.1315 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES...

  13. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Galactose-1-phosphate uridyl transferase test system. 862.1315 Section 862.1315 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... hereditary disease galactosemia (disorder of galactose metabolism) in infants. (b) Classification. Class II....

  14. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Galactose-1-phosphate uridyl transferase test system. 862.1315 Section 862.1315 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... hereditary disease galactosemia (disorder of galactose metabolism) in infants. (b) Classification. Class II....

  15. Preliminary X-ray crystallographic analysis of glutathione transferase zeta 1 (GSTZ1a-1a)

    SciTech Connect

    Boone, Christopher D.; Zhong, Guo; Smeltz, Marci; James, Margaret O. McKenna, Robert

    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°.

  16. 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...

  17. 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...

  18. 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. PMID:27113843

  19. Glycosyl transferases in chondroitin sulphate biosynthesis. Effect of acceptor structure on activity.

    PubMed Central

    Gundlach, M W; Conrad, H E

    1985-01-01

    The D-glucuronosyl (GlcA)- and N-acetyl-D-galactosaminyl (GalNAc)-transferases involved in chondroitin sulphate biosynthesis were studied in a microsomal preparation from chick-embryo chondrocytes. Transfer of GlcA and GalNAc from their UDP derivatives to 3H-labelled oligosaccharides prepared from chondroitin sulphate and hyaluronic acid was assayed by h.p.l.c. of the reaction mixture. Conditions required for maximal activities of the two enzymes were remarkably similar. Activities were stimulated 3.5-6-fold by neutral detergents. Both enzymes were completely inhibited by EDTA and maximally stimulated by MnCl2 or CoCl2. MgCl2 neither stimulated nor inhibited. The GlcA transferase showed a sharp pH optimum between pH5 and 6, whereas the GalNAc transferase gave a broad optimum from pH 5 to 8. At pH 7 under optimal conditions, the GalNAc transferase gave a velocity that was twice that of the GlcA transferase. Oligosaccharides prepared from chondroitin 4-sulphate and hyaluronic acid were almost inactive as acceptors for both enzymes, whereas oligosaccharides from chondroitin 6-sulphate and chondroitin gave similar rates that were 70-80-fold higher than those observed with the endogenous acceptors. Oligosaccharide acceptors with degrees of polymerization of 6 or higher gave similar Km and Vmax. values, but the smaller oligosaccharides were less effective acceptors. These results are discussed in terms of the implications for regulation of the overall rates of the chain-elongation fractions in chondroitin sulphate synthesis in vivo. PMID:3921015

  20. 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. PMID:27242998

  1. 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

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

    NASA Astrophysics Data System (ADS)

    Murphy, Jesse; Mullins, Elwood; Kappock, T.

    2016-05-01

    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 less than 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) analogue dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analogue 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.

  3. Radiographic changes and lung function in relation to activity of the glutathione transferases theta and mu among asbestos cement workers.

    PubMed

    Jakobsson, K; Rannug, A; Alexandrie, A K; Warholm, M; Rylander, L; Hagmar, L

    1995-05-01

    Experimental data indicate that active oxygen species may be casually involved in the development of asbestos-related disease. Thus, it was hypothesized that individual differences in glutathione transferase activity, which may affect the ability to inactivate molecules formed in relation to oxidative stress, could influence the biological response to asbestos exposure. We could, however, not demonstrate an increased risk for radiographic changes or reduced lung function among asbestos cement workers deficient for glutathione transferase theta (GSTT1), glutathione transferase mu (GSTM1), or having a combined deficiency of enzyme activity. PMID:7618163

  4. Regiospecificity of placental metabolism by cytochromes P450 and glutathione S-transferase.

    PubMed

    McRobie, D J; Glover, D D; Tracy, T S

    1996-01-01

    The placenta possesses the ability to metabolize numerous xenobiotics and endogenous steroids. However, it is unknown whether regional differences in these enzymatic reactions exist in the human placenta. To this end, we undertook a study of four regions of the placenta, the chorionic plate, maternal surface, placental margin and whole tissue, to assess the activities of cytochrome P450 1A1 and 19A1 (aromatase) and glutathione S-stransferase in these fractions. No differences in either P450 1A1 or glutathione S-transferase activities were noted among any of the placental fractions. However, with respect to P450 19A1 activity, the placental margin differed significantly from all other fractions (p < 0.05). This study demonstrates that whole tissue samples of the human placenta are adequate for placental cytochrome P450 and glutathione S-transferase metabolism studies. PMID:8938464

  5. Identification of a diazinon-metabolizing glutathione S-transferase in the silkworm, Bombyx mori.

    PubMed

    Yamamoto, Kohji; Yamada, Naotaka

    2016-01-01

    The glutathione S-transferase superfamily play key roles in the metabolism of numerous xenobiotics. We report herein the identification and characterization of a novel glutathione S-transferase in the silkworm, Bombyx mori. The enzyme (bmGSTu2) conjugates glutathione to 1-chloro-2,4-dinitrobenzene, as well as metabolizing diazinon, one of the organophosphate insecticides. Quantitative reverse transcription-polymerase chain reaction analysis of transcripts demonstrated that bmGSTu2 expression was induced 1.7-fold in a resistant strain of B. mori. Mutagenesis of putative amino acid residues in the glutathione-binding site revealed that Ile54, Glu66, Ser67, and Asn68 are crucial for enzymatic function. These results provide insights into the catalysis of glutathione conjugation in silkworm by bmGSTu2 and into the detoxification of organophosphate insecticides. PMID:27440377

  6. Identification of a diazinon-metabolizing glutathione S-transferase in the silkworm, Bombyx mori

    PubMed Central

    Yamamoto, Kohji; Yamada, Naotaka

    2016-01-01

    The glutathione S-transferase superfamily play key roles in the metabolism of numerous xenobiotics. We report herein the identification and characterization of a novel glutathione S-transferase in the silkworm, Bombyx mori. The enzyme (bmGSTu2) conjugates glutathione to 1-chloro-2,4-dinitrobenzene, as well as metabolizing diazinon, one of the organophosphate insecticides. Quantitative reverse transcription–polymerase chain reaction analysis of transcripts demonstrated that bmGSTu2 expression was induced 1.7-fold in a resistant strain of B. mori. Mutagenesis of putative amino acid residues in the glutathione-binding site revealed that Ile54, Glu66, Ser67, and Asn68 are crucial for enzymatic function. These results provide insights into the catalysis of glutathione conjugation in silkworm by bmGSTu2 and into the detoxification of organophosphate insecticides. PMID:27440377

  7. Glutathione and gamma-glutamyl transferases are involved in the formation of cadmium-glutathione complex.

    PubMed

    Adamis, Paula Daniela Braga; Mannarino, Sérgio Cantú; Eleutherio, Elis Cristina Araújo

    2009-05-01

    In a wild-type strain of Saccharomyces cerevisiae, cadmium induces the activities of both gamma-glutamyl transferase (gamma-GT) and glutathione transferase 2 (Gtt2). However, Gtt2 activity did not increase under gamma-GT or Ycf1 deficiencies, suggesting that the accumulation of glutathione-cadmium in the cytosol inhibits Gtt2. On the other hand, the balance between the cytoplasmic and vacuolar level of glutathione seems to regulate gamma-GT activity, since this enzyme was not activated in a gtt2 strain. Taken together, these results suggest that gamma-GT and Gtt2 work together to remove cadmium from the cytoplasm, a crucial mechanism for metal detoxification that is dependent on glutathione. PMID:19345220

  8. Glutathione transferase activity and formation of macromolecular adducts in two cases of acute methyl bromide poisoning.

    PubMed Central

    Garnier, R; Rambourg-Schepens, M O; Müller, A; Hallier, E

    1996-01-01

    OBJECTIVES: To determine the activity of glutathione transferase and to measure the S-methylcysteine adducts in blood proteins, after acute inhalation exposure to methyl bromide. To examine the influence of the polymorphism of glutathione-S-transferase theta (GSTT1) on the neurotoxicity of methyl bromide. METHODS: Two workers acutely exposed to methyl bromide with inadequate respiratory protective devices were poisoned. Seven weeks after the accident, blood samples were drawn from both patients, for measurement of glutathione transferase activity in erythrocytes (conjugator status--that is, GSTT1 phenotype) and measurement of binding products of methyl bromide with blood proteins. Conjugator status was determined by a standard procedure. The binding product of methyl bromide, S-methylcysteine, was measured in globin and albumin. RESULTS: Duration and intensity of exposure were identical for both patients as they worked together with the same protective devices and with similar physical effort. However, one patient had very severe poisoning, whereas the other only developed mild neurotoxic symptoms. The first patient was a "conjugator" with normal glutathone transferase activity, whereas this activity was undetectable in the erythrocytes of the second patient, who consequently had higher concentrations of S-methylcysteine adduct in albumin (149 v 91 nmol/g protein) and in globin (77 v 30 nmol/g protein). CONCLUSIONS: Methyl bromide is genotoxic and neurotoxic. Its genotoxicity seems to be the consequence of the alkylating activity of the parent compound, and conjugation to glutathione has a protective effect. The data presented here suggest a different mechanism for methyl bromide neurotoxicity which could be related to the transformation of methylglutathione into toxic metabolites such as methanethiol and formaldehyde. If such metabolites are the ultimate toxic species, N-acetylcysteine treatment could have a toxifying rather than a detoxifying effect. PMID:8704864

  9. A comparison of erythrocyte glutathione S-transferase activity from human foetuses and adults.

    PubMed Central

    Strange, R C; Johnston, J D; Coghill, D R; Hume, R

    1980-01-01

    Glutathione S-transferase activity was measured in partially purified haemolysates of erythrocytes from human foetuses and adults. Enzyme activity was present in erythrocytes obtained between 12 and 40 weeks of gestation. The catalytic properties of the enzyme from foetal cells were similar to those of the enzyme from adult erythrocytes, indicating that probably only one form of the erythrocytes enzyme exists throughout foetal and adult life. PMID:7396875

  10. Homology between O-linked GlcNAc transferases and proteins of the glycogen phosphorylase superfamily.

    PubMed

    Wrabl, J O; Grishin, N V

    2001-11-30

    The O-linked GlcNAc transferases (OGTs) are a recently characterized group of largely eukaryotic enzymes that add a single beta-N-acetylglucosamine moiety to specific serine or threonine hydroxyls. In humans, this process may be part of a sugar regulation mechanism or cellular signaling pathway that is involved in many important diseases, such as diabetes, cancer, and neurodegeneration. However, no structural information about the human OGT exists, except for the identification of tetratricopeptide repeats (TPR) at the N terminus. The locations of substrate binding sites are unknown and the structural basis for this enzyme's function is not clear. Here, remote homology is reported between the OGTs and a large group of diverse sugar processing enzymes, including proteins with known structure such as glycogen phosphorylase, UDP-GlcNAc 2-epimerase, and the glycosyl transferase MurG. This relationship, in conjunction with amino acid similarity spanning the entire length of the sequence, implies that the fold of the human OGT consists of two Rossmann-like domains C-terminal to the TPR region. A conserved motif in the second Rossmann domain points to the UDP-GlcNAc donor binding site. This conclusion is supported by a combination of statistically significant PSI-BLAST hits, consensus secondary structure predictions, and a fold recognition hit to MurG. Additionally, iterative PSI-BLAST database searches reveal that proteins homologous to the OGTs form a large and diverse superfamily that is termed GPGTF (glycogen phosphorylase/glycosyl transferase). Up to one-third of the 51 functional families in the CAZY database, a glycosyl transferase classification scheme based on catalytic residue and sequence homology considerations, can be unified through this common predicted fold. GPGTF homologs constitute a substantial fraction of known proteins: 0.4% of all non-redundant sequences and about 1% of proteins in the Escherichia coli genome are found to belong to the GPGTF

  11. 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. PMID:27213958

  12. Chemoenzymatic synthesis of glycopeptides with PglB, a bacterial oligosaccharyl transferase from Campylobacter jejuni.

    PubMed

    Glover, Kerney Jebrell; Weerapana, Eranthie; Numao, Shin; Imperiali, Barbara

    2005-12-01

    The gram-negative bacterium Campylobacter jejuni has a general N-linked glycosylation pathway encoded by the pgl gene cluster. One of the proteins in this cluster, PgIB, is thought to be the oligosaccharyl transferase due to its significant homology to Stt3p, a subunit of the yeast oligosaccharyl transferase complex. PgIB has been shown to be involved in catalyzing the transfer of an undecaprenyl-linked heptasaccharide to the asparagine side chain of proteins at the Asn-X-Ser/Thr motif. Using a synthetic disaccharide glycan donor (GaINAc-alpha1,3-bacillosamine-pyrophosphate-undecaprenyl) and a peptide acceptor substrate (KDFNVSKA), we can observe the oligosaccharyl transferase activity of PgIB in vitro. Furthermore, the preparation of additional undecaprenyl-linked glycan variants reveals the ability of PgIB to transfer a wide variety of saccharides. With the demonstration of PgIB activity in vitro, fundamental questions surrounding the mechanism of N-linked glycosylation can now be addressed. PMID:16356848

  13. Subfunctionality of Hydride Transferases of the Old Yellow Enzyme Family of Flavoproteins of Pseudomonas putida▿

    PubMed Central

    van Dillewijn, Pieter; Wittich, Rolf-Michael; Caballero, Antonio; Ramos, Juan-Luis

    2008-01-01

    To investigate potential complementary activities of multiple enzymes belonging to the same family within a single microorganism, we chose a set of Old Yellow Enzyme (OYE) homologs of Pseudomonas putida. The physiological function of these enzymes is not well established; however, an activity associated with OYE family members from different microorganisms is their ability to reduce nitroaromatic compounds. Using an in silico approach, we identified six OYE homologs in P. putida KT2440. Each gene was subcloned into an expression vector, and each corresponding gene product was purified to homogeneity prior to in vitro analysis for its catalytic activity against 2,4,6-trinitrotoluene (TNT). One of the enzymes, called XenD, lacked in vitro activity, whereas the other five enzymes demonstrated type I hydride transferase activity and reduced the nitro groups of TNT to hydroxylaminodinitrotoluene derivatives. XenB has the additional ability to reduce the aromatic ring of TNT to produce Meisenheimer complexes, defined as type II hydride transferase activity. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite; the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo. PMID:18791012

  14. Characterization of affinity-purified isoforms of Acinetobacter calcoaceticus Y1 glutathione transferases.

    PubMed

    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

  15. 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

  16. Complementary DNA cloning, messenger RNA expression, and induction of alpha-class glutathione S-transferases in mouse tissues.

    PubMed

    Buetler, T M; Eaton, D L

    1992-01-15

    Glutathione S-transferases (EC 2.5.1.18) are a multigene family of related proteins divided into four classes. Each class has multiple isoforms that exhibit tissue-specific expression, which may be an important determinant of susceptibility of that tissue to toxic injury or cancer. Recent studies have suggested that alpha-class glutathione S-transferase isoforms may play an important role in the development of cancers. Several alpha-class glutathione S-transferase isozymes have been characterized, purified, and cloned from a number of species, including rats, mice, and humans. Here we report on the cloning, sequencing, and mRNA expression of two alpha-class glutathione S-transferases from mouse liver, termed mYa and mYc. While mYa was shown to be identical to the known alpha-class glutathione S-transferase complementary DNA clone pGT41 (W. R. Pearson et al., J. Biol. Chem., 263: 13324-13332, 1988), the other clone, mYc, was demonstrated to be a novel complementary DNA clone encoding a glutathione S-transferase homologous to rat Yc (subunit 2). The mRNA for this novel complementary DNA is expressed constitutively in mouse liver. It also is the major alpha-class glutathione S-transferase isoform expressed in lung. The levels of expression of the butylated hydroxyanisole-inducible form (mYa) are highest in kidney and intestine. Treatment of mice with butylated hydroxyanisole had little effect on the expression levels of mYc but strongly induced mYa expression in liver. Butylated hydroxyanisole treatment increased expression levels for both mYa and mYc to varying degrees in kidney, lung, and intestine. The importance of the novel mouse liver alpha-class glutathione S-transferase isoform (mYc) in the metabolism of aflatoxin B1 and other carcinogens is discussed. PMID:1728405

  17. Crystallographic trapping of the glutamyl-CoA thioester intermediate of family I CoA transferases

    SciTech Connect

    Rangarajan,E.; Li, Y.; Ajamian, E.; Iannuzzi, P.; Kernaghan, S.; Fraser, M.; Cygler, M.; Matte, A.

    2005-01-01

    Coenzyme A transferases are involved in a broad range of biochemical processes in both prokaryotes and eukaryotes, and exhibit a diverse range of substrate specificities. The YdiF protein from Escherichia coli O157:H7 is an acyl-CoA transferase of unknown physiological function, and belongs to a large sequence family of CoA transferases, present in bacteria to humans, which utilize oxoacids as acceptors. In vitro measurements showed that YdiF displays enzymatic activity with short-chain acyl-CoAs. The crystal structures of YdiF and its complex with CoA, the first co-crystal structure for any Family I CoA transferase, have been determined and refined at 1.9 and 2.0 Angstrom resolution, respectively. YdiF is organized into tetramers, with each monomer having an open {alpha}/{beta} structure characteristic of Family I CoA transferases. Co-crystallization of YdiF with a variety of CoA thioesters in the absence of acceptor carboxylic acid resulted in trapping a covalent {gamma}-glutamyl-CoA thioester intermediate. The CoA binds within a well defined pocket at the N- and C-terminal domain interface, but makes contact only with the C-terminal domain. The structure of the YdiF complex provides a basis for understanding the different catalytic steps in the reaction of Family I CoA transferases.

  18. Probing the leucyl/phenylalanyl tRNA protein transferase active site with tRNA substrate analogues.

    PubMed

    Fung, Angela Wai Shan; Ebhardt, H Alexander; Krishnakumar, Kollappillil S; Moore, Jack; Xu, Zhizhong; Strazewski, Peter; Fahlman, Richard P

    2014-07-01

    Aminoacyl-tRNA protein transferases post-translationally conjugate an amino acid from an aminoacyl-tRNA onto the N-terminus of a target polypeptide. The eubacterial aminoacyl-tRNA protein transferase, L/F transferase, utilizes both leucyl-tRNA(Leu) and phenylalanyl-tRNA(Phe) as substrates. X-ray crystal structures with substrate analogues, the minimal substrate phenylalanyl adenosine (rA-Phe) and inhibitor puromycin, have been used to characterize tRNA recognition by L/F transferase. However analyses of these two X-ray crystal structures reveal significant differences in binding. Through structural analyses, mutagenesis, and enzymatic activity assays, we rationalize and demonstrate that the substrate analogues bind to L/F transferase with similar binding affinities using a series of different interactions by the various chemical groups of the analogues. Our data also demonstrates that enlarging the hydrophobic pocket of L/F transferase selectively enhances puromycin inhibition and may aid in the development of improved inhibitors for this class of enzymes. PMID:24521222

  19. Immunolabeling of Gamma-glutamyl transferase 5 in Normal Human Tissues Reveals Expression and Localization Differs from Gamma-glutamyl transferase 1

    PubMed Central

    Hanigan, Marie H.; Gillies, Elizabeth M.; Wickham, Stephanie; Wakeham, Nancy; Wirsig-Wiechmann, Celeste R.

    2014-01-01

    Gamma-glutamyl transferase (GGT5) was discovered due to its ability to convert leukotriene C4 (LTC4, a glutathione S-conjugate) to LTD4 and may have an important role in the immune system. However, it was not known which cells express the enzyme in humans. We have developed a sensitive and specific antibody that can be used to detect human GGT5 on western blots and in fixed tissue sections. We localized GGT5 expression in normal human tissues. We observed GGT5 expressed by macrophages present in many tissues, including tissue-fixed macrophages such as Kupffer cells in the liver and dust cells in the lung. GGT5 was expressed in some of the same tissues that have been shown to express gamma-glutamyl transferase (GGT1), the only other enzymatically active protein in this family. But, the two enzymes were often expressed by different cell types within the tissue. For example, GGT5 was expressed by the interstitial cells of the kidney; whereas, GGT1 is expressed on the apical surface of the renal proximal tubules. Other tissues with GGT5-positive cells included: adrenal gland, salivary gland, pituitary, thymus, spleen, liver, bone marrow, small intestine, stomach, testis, prostate and placenta. GGT5 and GGT1 are cell surface enzymes. The different pattern of expression results in their access to different extracellular fluids and therefore different substrates. GGT5 has access to substrates in blood and intercellular fluids, while GGT1 has access primarily to fluids in ducts and glands throughout the body. These data provide new insights into the different functions of these two related enzymes. PMID:25377544

  20. Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis

    PubMed Central

    Sato, Mitsunari; Yoshida, Yasuo; Nagano, Keiji; Hasegawa, Yoshiaki; Takebe, Jun; Yoshimura, Fuminobu

    2016-01-01

    Butyryl-CoA:acetate CoA transferase, which produces butyrate and acetyl-CoA from butyryl-CoA and acetate, is responsible for the final step of butyrate production in bacteria. This study demonstrates that in the periodontopathogenic bacterium Porphyromonas gingivalis this reaction is not catalyzed by PGN_1171, previously annotated as butyryl-CoA:acetate CoA transferase, but by three distinct CoA transferases, PGN_0725, PGN_1341, and PGN_1888. Gas chromatography/mass spectrometry (GC-MS) and spectrophotometric analyses were performed using crude enzyme extracts from deletion mutant strains and purified recombinant proteins. The experiments revealed that, in the presence of acetate, PGN_0725 preferentially utilized butyryl-CoA rather than propionyl-CoA. By contrast, this preference was reversed in PGN_1888. The only butyryl-CoA:acetate CoA transferase activity was observed in PGN_1341. Double reciprocal plots revealed that all the reactions catalyzed by these enzymes follow a ternary-complex mechanism, in contrast to previously characterized CoA transferases. GC-MS analysis to determine the concentrations of short chain fatty acids (SCFAs) in culture supernatants of P. gingivalis wild type and mutant strains revealed that PGN_0725 and PGN_1888 play a major role in the production of butyrate and propionate, respectively. Interestingly, a triple deletion mutant lacking PGN_0725, PGN_1341, and PGN_1888 produced low levels of SCFAs, suggesting that the microorganism contains CoA transferase(s) in addition to these three enzymes. Growth rates of the mutant strains were mostly slower than that of the wild type, indicating that many carbon compounds produced in the SCFA synthesis appear to be important for the biological activity of this microorganism. PMID:27486457

  1. Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis.

    PubMed

    Sato, Mitsunari; Yoshida, Yasuo; Nagano, Keiji; Hasegawa, Yoshiaki; Takebe, Jun; Yoshimura, Fuminobu

    2016-01-01

    Butyryl-CoA:acetate CoA transferase, which produces butyrate and acetyl-CoA from butyryl-CoA and acetate, is responsible for the final step of butyrate production in bacteria. This study demonstrates that in the periodontopathogenic bacterium Porphyromonas gingivalis this reaction is not catalyzed by PGN_1171, previously annotated as butyryl-CoA:acetate CoA transferase, but by three distinct CoA transferases, PGN_0725, PGN_1341, and PGN_1888. Gas chromatography/mass spectrometry (GC-MS) and spectrophotometric analyses were performed using crude enzyme extracts from deletion mutant strains and purified recombinant proteins. The experiments revealed that, in the presence of acetate, PGN_0725 preferentially utilized butyryl-CoA rather than propionyl-CoA. By contrast, this preference was reversed in PGN_1888. The only butyryl-CoA:acetate CoA transferase activity was observed in PGN_1341. Double reciprocal plots revealed that all the reactions catalyzed by these enzymes follow a ternary-complex mechanism, in contrast to previously characterized CoA transferases. GC-MS analysis to determine the concentrations of short chain fatty acids (SCFAs) in culture supernatants of P. gingivalis wild type and mutant strains revealed that PGN_0725 and PGN_1888 play a major role in the production of butyrate and propionate, respectively. Interestingly, a triple deletion mutant lacking PGN_0725, PGN_1341, and PGN_1888 produced low levels of SCFAs, suggesting that the microorganism contains CoA transferase(s) in addition to these three enzymes. Growth rates of the mutant strains were mostly slower than that of the wild type, indicating that many carbon compounds produced in the SCFA synthesis appear to be important for the biological activity of this microorganism. PMID:27486457

  2. Prediction of function for the polyprenyl transferase subgroup in the isoprenoid synthase superfamily

    PubMed Central

    Wallrapp, Frank H.; Pan, Jian-Jung; Ramamoorthy, Gurusankar; Almonacid, Daniel E.; Hillerich, Brandan S.; Seidel, Ronald; Patskovsky, Yury; Babbitt, Patricia C.; Almo, Steven C.; Jacobson, Matthew P.; Poulter, C. Dale

    2013-01-01

    The number of available protein sequences has increased exponentially with the advent of high-throughput genomic sequencing, creating a significant challenge for functional annotation. Here, we describe a large-scale study on assigning function to unknown members of the trans-polyprenyl transferase (E-PTS) subgroup in the isoprenoid synthase superfamily, which provides substrates for the biosynthesis of the more than 55,000 isoprenoid metabolites. Although the mechanism for determining the product chain length for these enzymes is known, there is no simple relationship between function and primary sequence, so that assigning function is challenging. We addressed this challenge through large-scale bioinformatics analysis of >5,000 putative polyprenyl transferases; experimental characterization of the chain-length specificity of 79 diverse members of this group; determination of 27 structures of 19 of these enzymes, including seven cocrystallized with substrate analogs or products; and the development and successful application of a computational approach to predict function that leverages available structural data through homology modeling and docking of possible products into the active site. The crystallographic structures and computational structural models of the enzyme–ligand complexes elucidate the structural basis of specificity. As a result of this study, the percentage of E-PTS sequences similar to functionally annotated ones (BLAST e-value ≤ 1e−70) increased from 40.6 to 68.8%, and the percentage of sequences similar to available crystal structures increased from 28.9 to 47.4%. The high accuracy of our blind prediction of newly characterized enzymes indicates the potential to predict function to the complete polyprenyl transferase subgroup of the isoprenoid synthase superfamily computationally. PMID:23493556

  3. The DinB Superfamily Includes Novel Mycothiol, Bacillithiol and Glutathione S-transferases

    PubMed Central

    Newton, Gerald L.; Leung, Stephan S.; Wakabayashi, Judy I.; Rawat, Mamta; Fahey, Robert C.

    2011-01-01

    The superfamily of glutathione S-transferases has been the subject of extensive study but Actinobacteria produce mycothiol (MSH) in place of glutathione and no mycothiol S-transferase (MST) has been identified. Using mycothiol and monochlorobimane as substrates a MST activity was detected in extracts of Mycobacterium smegmatis and purified sufficiently to allow identification of MSMEG_0887, a member the DUF664 family of the DinB superfamily, as the MST. The identity of the M. smegmatis and homologous Mycobacterium tuberculosis (Rv0443) enzymes was confirmed by cloning and the expressed proteins were found to be active with MSH but not bacillithiol (BSH) or glutathione (GSH). Bacillus subtilis YfiT is another member of the DinB superfamily but this bacterium produces BSH. The YfiT protein was shown to have S-transferase activity with monochlorobimane when assayed with BSH but not with MSH or GSH. Enterococcus faecalis EF_3021 shares some homology with MSMEG_0887 but this organism produces GSH but not MSH or BSH. Cloned and expressed EF_0321 was active with monochlorobimane and GSH but not with MSH or BSH. MDMPI_2 is another member of the DinB superfamily and has been previously shown to have mycothiol-dependent maleylpyruvate isomerase activity. Three of the eight families of the DinB superfamily include proteins shown to catalyze thiol-dependent metabolic or detoxification activities. Since more than two-thirds of the sequences assigned to the DinB superfamily are members of these families it seems likely that such activity is dominant in the DinB superfamily. PMID:22059487

  4. MIF protein are theta-class glutathione S-transferase homologs.

    PubMed Central

    Blocki, F. A.; Ellis, L. B.; Wackett, L. P.

    1993-01-01

    MIF proteins are mammalian polypeptides of approximately 13,000 molecular weight. This class includes human macrophage migration inhibitory factor (MIF), a rat liver protein that has glutathione S-transferase (GST) activity (TRANSMIF), and the mouse delayed early response gene 6 (DER6) protein. MIF proteins were previously linked to GSTs by demonstrating transferase activity and observing N-terminal sequence homology with a mu-class GST (Blocki, F.A., Schlievert, P.M., & Wackett, L.P., 1992, Nature 360, 269-270). In this study, MIF proteins are shown to be structurally related to the theta class of GSTs. This is established in three ways. First, unique primary sequence patterns are developed for each of the GST gene classes. The patterns identify the three MIF proteins as theta-like transferase homologs. Second, pattern analysis indicates that GST members of the theta class contain a serine residue in place of the N-terminal tyrosine that is implicated in glutathione deprotonation and activation in GSTs of known structure (Liu, S., et al., 1992, J. Biol. Chem. 267, 4296-4299). The MIF proteins contain a threonine at this position. Third, polyclonal antibodies raised against recombinant human MIF cross-react on Western blots with rat theta GST but not with alpha and mu GSTs. That MIF proteins have glutathione-binding ability may provide a common structural key toward understanding the varied functions of this widely distributed emerging gene family. Because theta is thought to be the most ancient evolutionary GST class, MIF proteins may have diverged early in evolution but retained a glutathione-binding domain. PMID:8298459

  5. Induction of glutathione-S-transferase activity by antioxidants in hepatocyte culture.

    PubMed

    Chen, L H; Shiau, C C

    1989-01-01

    Twelve male Sprague-Dawley rats were used for the study. Six rats were injected with benzo(a)pyrene (BP); the other six rats served as the control. Twenty-four hours after injection, hepatocytes were isolated and cultured. The cultured plates were divided into 5 groups and treated with absolute ethanol (control), butylated hydroxytoluene, vitamin E, ascorbic acid or vitamin Elascorbic acid. After 48 hours, the hepatocytes were harvested for enzyme activation determination. With both control and BP-injected rats, each antioxidant treatment significantly increased glutathione-S-transferase activity. The results suggest that antioxidants may have a detoxifying effect against BP-induced carcinogenesis. PMID:2817788

  6. The Phosphopantetheinyl Transferases: Catalysis of a Posttranslational Modification Crucial for Life

    PubMed Central

    Beld, Joris; Sonnenschein, Eva C.; Vickery, Christopher R.; Noel, Joseph P.; Burkart, Michael D.

    2014-01-01

    Although holo-acyl carrier protein synthase, AcpS, a phosphopantetheinyl transferase (PPTase), was characterized in the 1960s, it was not until the publication of the landmark paper by Lambalot et al. in 1996 that PPTases garnered wide-spread attention being classified as a distinct enzyme superfamily. In the past two decades an increasing number of papers has been published on PPTases ranging from identification, characterization, structure determination, mutagenesis, inhibition, and engineering in synthetic biology. In this review, we comprehensively discuss all current knowledge on this class of enzymes that post-translationally install a 4′-phosphopantetheine arm on various carrier proteins. PMID:24292120

  7. Fucosylation of xyloglucan: localization of the transferase in dictyosomes of pea stem cells. [Pisum sativum

    SciTech Connect

    Camirand, A.; Brummell, D.; MacLachlan, G.

    1987-07-01

    Microsomal membranes from elongating regions of etiolated Pisum sativum stems were separated by rate-zonal centrifugation on Renografin gradients. The transfer of labeled fucose and xylose from GDP-(/sup 14/C) fucose and UDP-(/sup 14/C)xylose to xyloglucan occurred mainly in dictyosome-enriched fractions. No transferase activity was detected in secretory vesicle fractions. Pulse-chase experiments using pea stem slices incubated with (/sup 3/H)fucose suggest that xyloglucan chains are fucosylated and their structure completed within the dictyosomes, before being transported to the cell wall by secretory vesicles.

  8. Photoactivation of hypericin down-regulates glutathione S-transferase activity in nasopharyngeal cancer cells.

    PubMed

    Du, H Y; Olivo, M; Tan, B K H; Bay, B H

    2004-04-30

    Photodynamic therapy (PDT) is a new modality of treatment for cancer. Hypericin is a photosensitizer, which is known to generate reactive oxygen species upon activation with light. We observed that photoactivated hypericin induces the generation of reactive oxygen intermediates in nasopharyngeal cancer (NPC) cells in vitro. There was also significant reduction of Glutathione S-transferase (GST) activity in HK1 and CNE-2 NPC cells and in tumor tissues from the NPC/HK1 murine tumor model by hypericin-mediated PDT. As antioxidants protect cells against phototoxicity, down-regulation of GST activity would potentiate the efficacy of hypericin-PDT treatment. PMID:15072826

  9. Glutathion S-transferase activity and DDT-susceptibility of Malaysian mosquitos.

    PubMed

    Lee, H L; Chong, W L

    1995-03-01

    Comparative DDT-susceptibility status and glutathion s-transferase (GST) activity of Malaysian Anopheles maculatus, Culex quinquefasciatus and Aedes aegypti was investigated to ascertain the role of this enzyme in DDT resistance. The standardised WHO dose-mortality bioassay tests were used to determine DDT susceptibility in these mosquitos, whilst GST microassay (Brogdon and Barber, 1990) was conducted to measure the activity of this enzyme in mosquito homogenate. It appeared that DDT susceptibility status of Malaysian mosquitos was not correlated with GST activity. PMID:8525405

  10. Structural plasticity of Cid1 provides a basis for its distributive RNA terminal uridylyl transferase activity.

    PubMed

    Yates, Luke A; Durrant, Benjamin P; Fleurdépine, Sophie; Harlos, Karl; Norbury, Chris J; Gilbert, Robert J C

    2015-03-11

    Terminal uridylyl transferases (TUTs) are responsible for the post-transcriptional addition of uridyl residues to RNA 3' ends, leading in some cases to altered stability. The Schizosaccharomyces pombe TUT Cid1 is a model enzyme that has been characterized structurally at moderate resolution and provides insights into the larger and more complex mammalian TUTs, ZCCHC6 and ZCCHC11. Here, we report a higher resolution (1.74 Å) crystal structure of Cid1 that provides detailed evidence for uracil selection via the dynamic flipping of a single histidine residue. We also describe a novel closed conformation of the enzyme that may represent an intermediate stage in a proposed product ejection mechanism. The structural insights gained, combined with normal mode analysis and biochemical studies, demonstrate that the plasticity of Cid1, particularly about a hinge region (N164-N165), is essential for catalytic activity, and provide an explanation for its distributive uridylyl transferase activity. We propose a model clarifying observed differences between the in vitro apparently processive activity and in vivo distributive monouridylylation activity of Cid1. We suggest that modulating the flexibility of such enzymes-for example by the binding of protein co-factors-may allow them alternatively to add single or multiple uridyl residues to the 3' termini of RNA molecules. PMID:25712096

  11. Selective inhibition of farnesyl-protein transferase blocks ras processing in vivo.

    PubMed

    Gibbs, J B; Pompliano, D L; Mosser, S D; Rands, E; Lingham, R B; Singh, S B; Scolnick, E M; Kohl, N E; Oliff, A

    1993-04-15

    The ras oncogene product, Ras, is synthesized in vivo as a precursor protein that requires post-translational processing to become biologically active and to be capable of transforming mammalian cells. Farnesylation appears to be a critical modification of Ras, and thus inhibitors of the farnesyl-protein transferase (FPTase) that catalyzes this reaction may block ras-dependent tumorigenesis. Three structural classes of FPTase inhibitors were identified: (alpha-hydroxyfarnesyl)phosphonic acid, chaetomellic acids, and zaragozic acids. By comparison, these compounds were weaker inhibitors of geranylgeranyl-protein transferases. Each of these inhibitors was competitive with respect to farnesyl diphosphate in the FPTase reaction. All compounds were assayed for inhibition of Ras processing in Ha-ras-transformed NIH3T3 fibroblasts. Ras processing was inhibited by 1 microM (alpha-hydroxyfarnesyl)phosphonic acid. Neither chaetomellic acid nor zaragozic acid were active in this assay. These results are the first demonstration that a small organic chemical selected for inhibition of FPTase can inhibit Ras processing in vivo. PMID:8463291

  12. Structure of Human O-GlcNAc Transferase and its Complex with a Peptide Substrate

    SciTech Connect

    M Lazarus; Y Nam; J Jiang; P Sliz; S Walker

    2011-12-31

    The essential mammalian enzyme O-linked {beta}-N-acetylglucosamine transferase (O-GlcNAc transferase, here OGT) couples metabolic status to the regulation of a wide variety of cellular signalling pathways by acting as a nutrient sensor. OGT catalyses the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine (UDP-GlcNAc) to serines and threonines of cytoplasmic, nuclear and mitochondrial proteins, including numerous transcription factors, tumour suppressors, kinases, phosphatases and histone-modifying proteins. Aberrant glycosylation by OGT has been linked to insulin resistance, diabetic complications, cancer and neurodegenerative diseases including Alzheimer's. 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 {angstrom} resolution) and as a ternary complex with UDP and a peptide substrate (1.95 {angstrom}). 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; it will also help the design of inhibitors for use as cellular probes and help to assess its potential as a therapeutic target.

  13. Two Active Forms of UDP-N-Acetylglucosamine Enolpyruvyl Transferase in Gram-Positive Bacteria

    PubMed Central

    Du, Wensheng; Brown, James R.; Sylvester, Daniel R.; Huang, Jianzhong; Chalker, Alison F.; So, Chi Y.; Holmes, David J.; Payne, David J.; Wallis, Nicola G.

    2000-01-01

    Gene sequences encoding the enzymes UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from many bacterial sources were analyzed. It was shown that whereas gram-negative bacteria have only one murA gene, gram-positive bacteria have two distinct genes encoding these enzymes which have possibly arisen from gene duplication. The two murA genes of the gram-positive organism Streptococcus pneumoniae were studied further. Each of the murA genes was individually inactivated by allelic replacement. In each case, the organism was viable despite losing one of its murA genes. However, when attempts were made to construct a double-deletion strain, no mutants were obtained. This indicates that both genes encode active enzymes that can substitute for each other, but that the presence of a MurA function is essential to the organism. The two genes were further cloned and overexpressed, and the enzymes they encode were purified. Both enzymes catalyzed the transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetylglucosamine, confirming they are both active UDP-N-acetylglucosamine enolpyruvyl transferases. The catalytic parameters of the two enzymes were similar, and they were both inhibited by the antibiotic fosfomycin. PMID:10894720

  14. Selective inhibitors of glutathione transferase P1 with trioxane structure as anticancer agents.

    PubMed

    Bräutigam, Maria; Teusch, Nicole; Schenk, Tobias; Sheikh, Miriam; Aricioglu, Rocky Z; Borowski, Swantje H; Neudörfl, Jörg-Martin; Baumann, Ulrich; Griesbeck, Axel G; Pietsch, Markus

    2015-04-01

    The response to chemotherapy in cancer patients is frequently compromised by drug resistance. Although chemoresistance is a multifactorial phenomenon, many studies have demonstrated that altered drug metabolism through the expression of phase II conjugating enzymes, including glutathione transferases (GSTs), in tumor cells can be directly correlated with resistance against a wide range of marketed anticancer drugs. In particular, overexpression of glutathione transferase P1 (GSTP1) appears to be a factor for poor prognosis during cancer therapy. Former and ongoing clinical trials have confirmed GSTP1 inhibition as a principle for antitumor therapy. A new series of 1,2,4-trioxane GSTP1 inhibitors were designed via a type II photooxygenation route of allylic alcohols followed by acid-catalyzed peroxyacetalization with aldehydes. A set of novel inhibitors exhibit low micromolar to high nanomolar inhibition of GSTP1, revealing preliminary SAR for further lead optimization. Importantly, high selectivity over another two human GST classes (GSTA1 and GSTM2) has been achieved. The trioxane GSTP1 inhibitors may therefore serve as a basis for the development of novel drug candidates in overcoming chemoresistance. PMID:25694385

  15. STT3, a highly conserved protein required for yeast oligosaccharyl transferase activity in vivo.

    PubMed Central

    Zufferey, R; Knauer, R; Burda, P; Stagljar, I; te Heesen, S; Lehle, L; Aebi, M

    1995-01-01

    N-linked glycosylation is a ubiquitous protein modification, and is essential for viability in eukaryotic cells. A lipid-linked core-oligosaccharide is assembled at the membrane of the endoplasmic reticulum and transferred to selected asparagine residues of nascent polypeptide chains by the oligosaccharyl transferase (OTase) complex. Based on the synthetic lethal phenotype of double mutations affecting the assembly of the lipid-linked core-oligosaccharide and the OTase activity, we have performed a novel screen for mutants in Saccharomyces cerevisiae with altered N-linked glycosylation. Besides novel mutants deficient in the assembly of the lipid-linked oligosaccharide (alg mutants), we identified the STT3 locus as being required for OTase activity in vivo. The essential STT3 protein is approximately 60% identical in amino acid sequence to its human homologue. A mutation in the STT3 locus affects substrate specificity of the OTase complex in vivo and in vitro. In stt3-3 cells very little glycosyl transfer occurs from incomplete lipid-linked oligosaccharide, whereas the transfer of full-length Glc3Man9GlcNAc2 is hardly affected as compared with wild-type cells. Depletion of the STT3 protein results in loss of transferase activity in vivo and a deficiency in the assembly of OTase complex. Images PMID:7588624

  16. Structural and thermodynamic properties of kappa class glutathione transferase from Camelus dromedarius.

    PubMed

    Malik, Ajamaluddin; Fouad, Dalia; Labrou, Nikolaos E; Al-Senaidy, Abdulrahman M; Ismael, Mohamed A; Saeed, Hesham M; Ataya, Farid S

    2016-07-01

    The Arabian camel, Camelus dromedarius is naturally adapted to extreme desert climate and has evolved protective mechanisms to limit oxidative stress. The mitochondrial kappa class glutathione transferase enzyme is a member of GST supergene family that represents an important enzyme group in cellular Phase II detoxification machinery and is involved in the protection against oxidative stress and xenobiotics. In the present study, C. dromedarius kappa class glutathione transferase (CdGSTK1-1) was cloned, expressed in E. coli BL21, purified and its structural, thermodynamic and unfolding pathway was investigated. The results showed that CdGSTK1-1 has unique trimeric structure, exhibits low thermostability and a complex equilibrium unfolding profile. It unfolds through three folding states with formation of thinly populated intermediate species. The melting points (Tm) of the first unfolding transition was 40.3±0.2°C and Tm of the second unfolding transition was 49.1±0.1°C. The van't Hoff enthalpy of the first and second transition were 298.7±13.2 and 616.5±2.4kJ/mol, respectively. Moreover, intrinsic fluorescence and near-UV CD studies indicates that substrate binding does not leads to major conformational changes in CdGSTK1-1. PMID:27044344

  17. Expression of glutathione transferases in corneal cell lines, corneal tissues and a human cornea construct.

    PubMed

    Kölln, Christian; Reichl, Stephan

    2016-06-15

    Glutathione transferase (GST) expression and activity were examined in a three-dimensional human cornea construct and were compared to those of excised animal corneas. The objective of this study was to characterize phase II enzyme expression in the cornea construct with respect to its utility as an alternative to animal cornea models. The expression of the GSTO1-1 and GSTP1-1 enzymes was investigated using immunofluorescence staining and western blotting. The level of total glutathione transferase activity was determined using 1-chloro-2,4- dinitrobenzene as the substrate. Furthermore, the levels of GSTO1-1 and GSTP1-1 activity were examined using S-(4-nitrophenacyl)glutathione and ethacrynic acid, respectively, as the specific substrates. The expression and activity levels of these enzymes were examined in the epithelium, stroma and endothelium, the three main cellular layers of the cornea. In summary, the investigated enzymes were detected at both the protein and functional levels in the cornea construct and the excised animal corneas. However, the enzymatic activity levels of the human cornea construct were lower than those of the animal corneas. PMID:27113863

  18. Functional Diversification of Fungal Glutathione Transferases from the Ure2p Class

    PubMed Central

    Thuillier, Anne; Ngadin, Andrew A.; Thion, Cécile; Billard, Patrick; Jacquot, Jean-Pierre; Gelhaye, Eric; Morel, Mélanie

    2011-01-01

    The glutathione-S-transferase (GST) proteins represent an extended family involved in detoxification processes. They are divided into various classes with high diversity in various organisms. The Ure2p class is especially expanded in saprophytic fungi compared to other fungi. This class is subdivided into two subclasses named Ure2pA and Ure2pB, which have rapidly diversified among fungal phyla. We have focused our analysis on Basidiomycetes and used Phanerochaete chrysosporium as a model to correlate the sequence diversity with the functional diversity of these glutathione transferases. The results show that among the nine isoforms found in P. chrysosporium, two belonging to Ure2pA subclass are exclusively expressed at the transcriptional level in presence of polycyclic aromatic compounds. Moreover, we have highlighted differential catalytic activities and substrate specificities between Ure2pA and Ure2pB isoforms. This diversity of sequence and function suggests that fungal Ure2p sequences have evolved rapidly in response to environmental constraints. PMID:22164343

  19. Glutathione Transferases Superfamily: Cold-Inducible Expression of Distinct GST Genes in Brassica oleracea.

    PubMed

    Vijayakumar, Harshavardhanan; Thamilarasan, Senthil Kumar; Shanmugam, Ashokraj; Natarajan, Sathishkumar; Jung, Hee-Jeong; Park, Jong-In; Kim, HyeRan; Chung, Mi-Young; Nou, Ill-Sup

    2016-01-01

    Plants, as sessile organisms, can suffer serious growth and developmental consequences under cold stress conditions. Glutathione transferases (GSTs, EC 2.5.1.18) are ubiquitous and multifunctional conjugating proteins, which play a major role in stress responses by preventing oxidative damage by reactive oxygen species (ROS). Currently, understanding of their function(s) during different biochemical and signaling pathways under cold stress condition remain unclear. In this study, using combined computational strategy, we identified 65 Brassica oleracea glutathione transferases (BoGST) and characterized them based on evolutionary analysis into 11 classes. Inter-species and intra-species duplication was evident between BoGSTs and Arabidopsis GSTs. Based on localization analyses, we propose possible pathways in which GST genes are involved during cold stress. Further, expression analysis of the predicted putative functions for GST genes were investigated in two cold contrasting genotypes (cold tolerance and susceptible) under cold condition, most of these genes were highly expressed at 6 h and 1 h in the cold tolerant (CT) and cold susceptible (CS) lines, respectively. Overall, BoGSTU19, BoGSTU24, BoGSTF10 are candidate genes highly expressed in B. oleracea. Further investigation of GST superfamily in B. oleracea will aid in understanding complex mechanism underlying cold tolerance in plants. PMID:27472324

  20. Nucleoside Diphosphate Sugar-Starch Glucosyl Transferase Activity of wx Starch Granules 1

    PubMed Central

    Nelson, Oliver E.; Chourey, Prem S.; Chang, Ming Tu

    1978-01-01

    Starch granule preparations from the endosperm tissue of all waxy maize (Zea mays L.) mutants tested have low and approximately equal capability to incorporate glucose from adenosine diphosphate glucose into starch. As the substrate concentration is reduced, however, the activity of waxy preparations relative to nonmutant increases until, at the lowest substrate concentration utilized (0.1 μM), the activity of the waxy preparations is nearly equal to that of the nonmutant preparation. The apparent Km (adenosine diphosphate glucose) for starch granule preparations from wx-C/wx-C/wx-C endosperms was 7.1 × 10−5 M, which is compared to 3 × 10−3 M for preparations from nonwaxy endosperms. Starch granule preparations from three other waxy mutants of independent mutational origin have levels of enzymic activity approximately equal to wx-C at a given substrate concentration giving rise to similar apparent Km estimates. We conclude that there is in maize endosperm starch granules a second starch granule-bound glycosyl transferase, whose presence is revealed when mutation eliminates activity of the more active glucosyl transferase catalyzing the same reaction. PMID:16660522

  1. Glutathione Transferases Superfamily: Cold-Inducible Expression of Distinct GST Genes in Brassica oleracea

    PubMed Central

    Vijayakumar, Harshavardhanan; Thamilarasan, Senthil Kumar; Shanmugam, Ashokraj; Natarajan, Sathishkumar; Jung, Hee-Jeong; Park, Jong-In; Kim, HyeRan; Chung, Mi-Young; Nou, Ill-Sup

    2016-01-01

    Plants, as sessile organisms, can suffer serious growth and developmental consequences under cold stress conditions. Glutathione transferases (GSTs, EC 2.5.1.18) are ubiquitous and multifunctional conjugating proteins, which play a major role in stress responses by preventing oxidative damage by reactive oxygen species (ROS). Currently, understanding of their function(s) during different biochemical and signaling pathways under cold stress condition remain unclear. In this study, using combined computational strategy, we identified 65 Brassica oleracea glutathione transferases (BoGST) and characterized them based on evolutionary analysis into 11 classes. Inter-species and intra-species duplication was evident between BoGSTs and Arabidopsis GSTs. Based on localization analyses, we propose possible pathways in which GST genes are involved during cold stress. Further, expression analysis of the predicted putative functions for GST genes were investigated in two cold contrasting genotypes (cold tolerance and susceptible) under cold condition, most of these genes were highly expressed at 6 h and 1 h in the cold tolerant (CT) and cold susceptible (CS) lines, respectively. Overall, BoGSTU19, BoGSTU24, BoGSTF10 are candidate genes highly expressed in B. oleracea. Further investigation of GST superfamily in B. oleracea will aid in understanding complex mechanism underlying cold tolerance in plants. PMID:27472324

  2. Identification of a Mycoloyl Transferase Selectively Involved in O-Acylation of Polypeptides in Corynebacteriales

    PubMed Central

    Huc, Emilie; de Sousa-D'Auria, Célia; de la Sierra-Gallay, Inès Li; Salmeron, Christophe; van Tilbeurgh, Herman; Bayan, Nicolas; Houssin, Christine

    2013-01-01

    We have previously described the posttranslational modification of pore-forming small proteins of Corynebacterium by mycolic acid, a very-long-chain α-alkyl and β-hydroxy fatty acid. Using a combination of chemical analyses and mass spectrometry, we identified the mycoloyl transferase (Myt) that catalyzes the transfer of the fatty acid residue to yield O-acylated polypeptides. Inactivation of corynomycoloyl transferase C (cg0413 [Corynebacterium glutamicum mytC {CgmytC}]), one of the six Cgmyt genes of C. glutamicum, specifically abolished the O-modification of the pore-forming proteins PorA and PorH, which is critical for their biological activity. Expectedly, complementation of the cg0413 mutant with either the wild-type gene or its orthologues from Corynebacterium diphtheriae and Rhodococcus, but not Nocardia, fully restored the O-acylation of the porins. Consistently, the three-dimensional structure of CgMytC showed the presence of a unique loop that is absent from enzymes that transfer mycoloyl residues onto both trehalose and the cell wall arabinogalactan. These data suggest the implication of this structure in the enzyme specificity for protein instead of carbohydrate. PMID:23852866

  3. Nicotinamide Mononucleotide Adenylyl Transferase 2: A Promising Diagnostic and Therapeutic Target for Colorectal Cancer

    PubMed Central

    Cui, Chunhui; Qi, Jia; Deng, Quanwen; Chen, Rihong; Zhai, Duanyang; Yu, Jinlong

    2016-01-01

    Colorectal cancer (CRC) is one of the most common cancers all over the world. It is essential to search for more effective diagnostic and therapeutic methods for CRC. Abnormal nicotinamide adenine dinucleotide (NAD) metabolism has been considered as a characteristic of cancer cells. In this study, nicotinamide mononucleotide adenylyl transferases (NMNATs) as well as p53-mediated cancer signaling pathways were investigated in patients with colorectal cancer. The CRC tissues and adjacent normal tissues were obtained from 95 untreated colorectal cancer patients and were stained for expression of nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) and p53. The survival rate was analyzed by the Kaplan-Meier method and the log-rank test. The multivariate Cox proportional hazard regression analysis was conducted as well. Our data demonstrated that expression of NMNAT2 and p53 was significantly higher in CRC tissues, while NMNAT2 expression is in correlation with the invasive depth of tumors and TNM stage. Significant positive correlation was found between the expression of NMNAT2 and the expression of p53. However, NMNAT2 expression was not a statistically significant prognostic factor for overall survival. In conclusion, our results indicated that NMNAT2 might participate in tumorigenesis of CRC in a p53-dependent manner and NMNAT2 expression might be a potential therapeutic target for CRC. PMID:27218101

  4. Characterisation of the Candida albicans Phosphopantetheinyl Transferase Ppt2 as a Potential Antifungal Drug Target

    PubMed Central

    Dobb, Katharine S.; Kaye, Sarah J.; Beckmann, Nicola; Thain, John L.; Stateva, Lubomira; Birch, Mike; Oliver, Jason D.

    2015-01-01

    Antifungal drugs acting via new mechanisms of action are urgently needed to combat the increasing numbers of severe fungal infections caused by pathogens such as Candida albicans. The phosphopantetheinyl transferase of Aspergillus fumigatus, encoded by the essential gene pptB, has previously been identified as a potential antifungal target. This study investigated the function of its orthologue in C. albicans, PPT2/C1_09480W by placing one allele under the control of the regulatable MET3 promoter, and deleting the remaining allele. The phenotypes of this conditional null mutant showed that, as in A. fumigatus, the gene PPT2 is essential for growth in C. albicans, thus fulfilling one aspect of an efficient antifungal target. The catalytic activity of Ppt2 as a phosphopantetheinyl transferase and the acyl carrier protein Acp1 as a substrate were demonstrated in a fluorescence transfer assay, using recombinant Ppt2 and Acp1 produced and purified from E.coli. A fluorescence polarisation assay amenable to high-throughput screening was also developed. Therefore we have identified Ppt2 as a broad-spectrum novel antifungal target and developed tools to identify inhibitors as potentially new antifungal compounds. PMID:26606674

  5. Glucose-induced expression of MIP-1 genes requires O-GlcNAc transferase in monocytes

    SciTech Connect

    Chikanishi, Toshihiro; Fujiki, Ryoji; Hashiba, Waka; Sekine, Hiroki; Yokoyama, Atsushi; Kato, Shigeaki

    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} 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.

  6. Acyl carrier protein-specific 4'-phosphopantetheinyl transferase activates 10-formyltetrahydrofolate dehydrogenase.

    PubMed

    Strickland, Kyle C; Hoeferlin, L Alexis; Oleinik, Natalia V; Krupenko, Natalia I; Krupenko, Sergey A

    2010-01-15

    4'-Phosphopantetheinyl transferases (PPTs) catalyze the transfer of 4'-phosphopantetheine (4-PP) from coenzyme A to a conserved serine residue of their protein substrates. In humans, the number of pathways utilizing the 4-PP post-translational modification is limited and may only require a single broad specificity PPT for all phosphopantetheinylation reactions. Recently, we have shown that one of the enzymes of folate metabolism, 10-formyltetrahydrofolate dehydrogenase (FDH), requires a 4-PP prosthetic group for catalysis. This moiety acts as a swinging arm to couple the activities of the two catalytic domains of FDH and allows the conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. In the current study, we demonstrate that the broad specificity human PPT converts apo-FDH to holoenzyme and thus activates FDH catalysis. Silencing PPT by small interfering RNA in A549 cells prevents FDH modification, indicating the lack of alternative enzymes capable of accomplishing this transferase reaction. Interestingly, PPT-silenced cells demonstrate significantly reduced proliferation and undergo strong G(1) arrest, suggesting that the enzymatic function of PPT is essential and nonredundant. Our study identifies human PPT as the FDH-modifying enzyme and supports the hypothesis that mammals utilize a single enzyme for all phosphopantetheinylation reactions. PMID:19933275

  7. Structural plasticity of Cid1 provides a basis for its distributive RNA terminal uridylyl transferase activity

    PubMed Central

    Yates, Luke A.; Durrant, Benjamin P.; Fleurdépine, Sophie; Harlos, Karl; Norbury, Chris J.; Gilbert, Robert J.C.

    2015-01-01

    Terminal uridylyl transferases (TUTs) are responsible for the post-transcriptional addition of uridyl residues to RNA 3′ ends, leading in some cases to altered stability. The Schizosaccharomyces pombe TUT Cid1 is a model enzyme that has been characterized structurally at moderate resolution and provides insights into the larger and more complex mammalian TUTs, ZCCHC6 and ZCCHC11. Here, we report a higher resolution (1.74 Å) crystal structure of Cid1 that provides detailed evidence for uracil selection via the dynamic flipping of a single histidine residue. We also describe a novel closed conformation of the enzyme that may represent an intermediate stage in a proposed product ejection mechanism. The structural insights gained, combined with normal mode analysis and biochemical studies, demonstrate that the plasticity of Cid1, particularly about a hinge region (N164–N165), is essential for catalytic activity, and provide an explanation for its distributive uridylyl transferase activity. We propose a model clarifying observed differences between the in vitro apparently processive activity and in vivo distributive monouridylylation activity of Cid1. We suggest that modulating the flexibility of such enzymes—for example by the binding of protein co-factors—may allow them alternatively to add single or multiple uridyl residues to the 3′ termini of RNA molecules. PMID:25712096

  8. Structural Determinants Allowing Transferase Activity in SENSITIVE TO FREEZING 2, Classified as a Family I Glycosyl Hydrolase*

    PubMed Central

    Roston, Rebecca L.; Wang, Kun; Kuhn, Leslie A.; Benning, Christoph

    2014-01-01

    SENSITIVE TO FREEZING 2 (SFR2) is classified as a family I glycosyl hydrolase but has recently been shown to have galactosyltransferase activity in Arabidopsis thaliana. Natural occurrences of apparent glycosyl hydrolases acting as transferases are interesting from a biocatalysis standpoint, and knowledge about the interconversion can assist in engineering SFR2 in crop plants to resist freezing. To understand how SFR2 evolved into a transferase, the relationship between its structure and function are investigated by activity assay, molecular modeling, and site-directed mutagenesis. SFR2 has no detectable hydrolase activity, although its catalytic site is highly conserved with that of family 1 glycosyl hydrolases. Three regions disparate from glycosyl hydrolases are identified as required for transferase activity as follows: a loop insertion, the C-terminal peptide, and a hydrophobic patch adjacent to the catalytic site. Rationales for the effects of these regions on the SFR2 mechanism are discussed. PMID:25100720

  9. Partial characterization of glutathione S-transferases from wheat (Triticum spp.) and purification of a safener-induced glutathione S-transferase from Triticum tauschii.

    PubMed Central

    Riechers, D E; Irzyk, G P; Jones, S S; Fuerst, E P

    1997-01-01

    Hexaploid wheat (Triticum aestivum L.) has very low constitutive glutathione S-transferase (GST) activity when assayed with the chloroacetamide herbicide dimethenamid as a substrate, which may account for its low tolerance to dimethenamid in the field. Treatment of seeds with the herbicide safener fluxofenim increased the total GST activity extracted from T. aestivum shoots 9-fold when assayed with dimethenamid as a substrate, but had no effect on glutathione levels. Total GST activity in crude protein extracts from T. aestivum, Triticum durum, and Triticum tauschii was separated into several component GST activities by anion-exchange fast-protein liquid chromatography. These activities (isozymes) differed with respect to their activities toward dimethenamid or 1-chloro-2,4-dinitrobenzene as substrates and in their levels of induction by safener treatment. A safener-induced GST isozyme was subsequently purified by anion-exchange and affinity chromatography from etiolated shoots of the diploid wheat species T. tauschii (a progenitor of hexaploid wheat) treated with the herbicide safener cloquintocet-mexyl. The isozyme bound to a dimethenamid-affinity column and had a subunit molecular mass of 26 kD based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme (designated GST TSI-1) was recognized by an antiserum raised against a mixture of maize (Zea mays) GSTs. Amino acid sequences obtained from protease-digested GST TSI-1 had significant homology with the safener-inducible maize GST V and two auxin-regulated tobacco (Nicotiana tabacum) GST isozymes. PMID:9276955

  10. Zaragozic acids D and D2: potent inhibitors of squalene synthase and of Ras farnesyl-protein transferase.

    PubMed

    Dufresne, C; Wilson, K E; Singh, S B; Zink, D L; Bergstrom, J D; Rew, D; Polishook, J D; Meinz, M; Huang, L; Silverman, K C

    1993-11-01

    Two new zaragozic acids, D and D2, have been isolated from the keratinophilic fungus Amauroascus niger. Zaragozic acids D [4] and D2 [5] are related to the previously described zaragozic acids A [1], B [2], and C [3] and are potent inhibitors of squalene synthase. Furthermore, all the zaragozic acids (A, B, C, D, and D2) are also active against farnesyl transferase. Zaragozic acids D and D2 inhibit farnesyl transferase with IC50 values of 100 nM, while zaragozic acids A and B are less potent. PMID:8289063

  11. Differential expression patterns of N-acetylglucosaminyl transferases and polylactosamines in uterine lesions.

    PubMed

    Clark, A T R; Guimarães da Costa, V M L; Bandeira Costa, L; Bezerra Cavalcanti, C L; De Melo Rêgo, M J B; Beltrão, E I C

    2014-01-01

    Polylactosamine (polyLacNAc) is a fundamental structure in glycoconjugates and it is expressed in specific cells/tissues associated with the development and carcinogenesis. β1,3-N-acetylglucosaminyl transferases (β3GnTs) play an important role in polyLacNAc synthesis, however the roles of these glycosyltransferases and their products in cancer progression are still unclear. In this sense, this work aimed to evaluate differential expression pattern of the N-acetylglucosaminyl transferases and polylactosamines in invasive and premalignant lesions of the uterus cervix. The expression of β3GnT2 and β3GnT3 were evaluated in normal (n=10) and uterine cervix lesions (n= 120) malignant (squamous carcinoma - SC) and premalignant (cervical intraepithelial neoplasia - CIN - grades 1, 2 and 3) using immunohistochemistry. Besides, lectin histochemistry with Phytolacca americana lectin (PWM) and Wheat germ agglutinin (WGA) was also carried out to observe the presence of polyLacNAc chains and N-acetylglucosamine (GlcNAc), respectively. The β3GnT3 was expressed in almost all samples (99%) and β3GnT2 was higher expressed in disease samples mainly in CIN 3, when compared with normal (P=0.002), CIN 1 (P=0.009) and CIN 2 (P=0.03). The expression of polyLacNAc was higher is SC samples, when compared with normal (P=0.03), CIN 1 (P=0.02) and CIN 3 (P=0.004), and was observed only nuclear expression in nearly 50% of the SC samples, showing a statistically significant when compared with normal (P=0.01), CIN 1 (P=0.002), CIN 2 (P=0.007) and CIN 3 (P=0.04). Deferring from transferases and polyLacNAc chains, GlcNAc (WGA ligand) reveals a gradual staining pattern decrease with the increase of the lesion degree, being more expressed in CIN 1 lesions when compared with normal (P<0.0001), CIN 2 (P<0.0001), SC (P<0.0001) and CIN 3 (P=0.0003). Our data reveals β3GnT2 and polyLacNAc may be involved in the progression of the pre-malignant lesions of human the uterine cervix. In addition, poly

  12. 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

  13. DNA sequencing and expression of the formyl coenzyme A transferase gene, frc, from Oxalobacter formigenes.

    PubMed Central

    Sidhu, H; Ogden, S D; Lung, H Y; Luttge, B G; Baetz, A L; Peck, A B

    1997-01-01

    Oxalic acid, a highly toxic by-product of metabolism, is catabolized by a limited number of bacterial species utilizing an activation-decarboxylation reaction which yields formate and CO2. frc, the gene encoding formyl coenzyme A transferase, an enzyme which transfers a coenzyme A moiety to activate oxalic acid, was cloned from the bacterium Oxalobacter formigenes. DNA sequencing revealed a single open reading frame of 1,284 bp capable of encoding a 428-amino-acid protein. A presumed promoter region and a rho-independent termination sequence suggest that this gene is part of a monocistronic operon. A PCR fragment containing the open reading frame, when overexpressed in Escherichia coli, produced a product exhibiting enzymatic activity similar to the purified native enzyme. With this, the two genes necessary for bacterial catabolism of oxalate, frc and oxc, have now been cloned, sequenced, and expressed. PMID:9150242

  14. 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

  15. Structures of aminoarabinose transferase ArnT suggest a molecular basis for lipid A glycosylation.

    PubMed

    Petrou, Vasileios I; Herrera, Carmen M; Schultz, Kathryn M; Clarke, Oliver B; Vendome, Jérémie; Tomasek, David; Banerjee, Surajit; Rajashankar, Kanagalaghatta R; Belcher Dufrisne, Meagan; Kloss, Brian; Kloppmann, Edda; Rost, Burkhard; Klug, Candice S; Trent, M Stephen; Shapiro, Lawrence; Mancia, Filippo

    2016-02-01

    Polymyxins are antibiotics used in the last line of defense to combat multidrug-resistant infections by Gram-negative bacteria. Polymyxin resistance arises through charge modification of the bacterial outer membrane with the attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose to lipid A, a reaction catalyzed by the integral membrane lipid-to-lipid glycosyltransferase 4-amino-4-deoxy-L-arabinose transferase (ArnT). Here, we report crystal structures of ArnT from Cupriavidus metallidurans, alone and in complex with the lipid carrier undecaprenyl phosphate, at 2.8 and 3.2 angstrom resolution, respectively. The structures show cavities for both lipidic substrates, which converge at the active site. A structural rearrangement occurs on undecaprenyl phosphate binding, which stabilizes the active site and likely allows lipid A binding. Functional mutagenesis experiments based on these structures suggest a mechanistic model for ArnT family enzymes. PMID:26912703

  16. Pharmacogenetics of azathioprine in inflammatory bowel disease: a role for glutathione-S-transferase?

    PubMed

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

    2014-04-01

    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

  17. Biochemical properties of an omega-class glutathione S-transferase of the silkmoth, Bombyx mori.

    PubMed

    Yamamoto, Kohji; Nagaoka, Sumiharu; Banno, Yutaka; Aso, Yoichi

    2009-05-01

    A cDNA encoding an omega-class glutathione S-transferase of the silkmoth, Bombyx mori (bmGSTO), was cloned by reverse transcriptase-polymerase chain reaction. The resulting clone was sequenced and deduced for amino acid sequence, which revealed 40, 40, and 39% identities to omega-class GSTs from human, pig, and mouse, respectively. A recombinant protein (rbmGSTO) was functionally overexpressed in Escherichia coli cells in a soluble form and purified to homogeneity. rbmGSTO was able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a model substrate for GST, as well as with 4-hydroxynonenal, a product of lipid peroxidation. This enzyme was shown to have high affinity for organophosphorus insecticide and was present abundantly in silkmoth strain exhibiting fenitrothion resistance. These results indicate that bmGSTO could be involved in the increase in level of insecticide resistance for lepidopteran insects. PMID:19022397

  18. Modulating the activity of the peptidyl transferase center of the ribosome

    PubMed Central

    Beringer, Malte

    2008-01-01

    The peptidyl transferase (PT) center of the ribosome catalyzes two nucleophilic reactions, peptide bond formation between aminoacylated tRNA substrates and, together with release factor, peptide release. Structure and function of the PT center are modulated by binding of aminoacyl-tRNA or release factor, thus providing the basis for the specificity of catalysis. Another way by which the function of the PT center is controlled is signaling from the peptide exit tunnel. The SecM nascent peptide induces ribosome stalling, presumably by inhibition of peptide bond formation. Similarly, the release factor-induced hydrolytic activity of the PT center can be suppressed by the TnaC nascent peptide contained in the exit tunnel. Thus, local and long-range conformational rearrangements can lead to changes in the reaction specificity and catalytic activity of the PT center. PMID:18369182

  19. Review: Human guanidinoacetate n-methyl transferase (GAMT) deficiency: A treatable inborn error of metabolism.

    PubMed

    Iqbal, Furhan

    2015-11-01

    The creatine biosynthetic pathway is essential for cellular phosphate associated energy production and storage, particularly in tissues having higher metabolic demands. Guanidinoacetate N-Methyl transferase (GAMT) is an important enzyme in creatine endogenous biosynthetic pathway, with highest expression in liver and kidney. GAMT deficiency is an inherited autosomal recessive trait that was the first among creatine deficiency syndrome to be reported in 1994 having characteristic features of no comprehensible speech development, severe mental retardation, muscular hypotonia, involuntary movements and seizures that partly cannot be treated with anti-epileptic drugs. Due to problematic endogenous creatine biosynthesis, systemic depletion of creatine/phosphocreatine and accumulation of guanidinoacetate takes place that are the diagnostic features of this disease. Dietary creatine supplementation alone or along with arginine restriction has been reported to be beneficial for all treated patients, although to various extent. However, none of the GAMT deficient patient has been reported to return to complete normal developmental level. PMID:26639513

  20. MicroRNA Regulating Glutathione S-Transferase P1 in Prostate Cancer

    PubMed Central

    Singh, Savita; Shukla, Girish C; Gupta, Sanjay

    2015-01-01

    Glutathione S-transferase P1 (GSTP1), an enzyme involved in detoxification process, is frequently inactivated in prostate cancer due to epigenetic modifications. Through in silico analysis we identified a subset of miRNAs that are putative targets in regulating GSTP1. miRNAs are small endogenous non-coding RNA that are critical regulators of various physiologic and pathologic processes and their level of expression may play a precise role in early diagnosis and prognosis of cancer. These small molecules have been detected in a wide variety of human biological specimens including blood, serum, urine, ejaculate and tissues, which could be utilized as clinically useful biomarker in early detection and prognosis of prostate cancer. The chapter summarizes the current knowledge about miRNA involved in GSTP1 regulation in prostate cancer and their potential as useful biomarkers of disease for early detection and prognosis, along with challenges and limitations in this development. PMID:25774339

  1. Single-Molecule Study of Ribosome Hierarchic Dynamics at the Peptidyl Transferase Center

    PubMed Central

    Altuntop, Mediha Esra; Ly, Cindy Tu; Wang, Yuhong

    2010-01-01

    During protein biosynthesis the ribosome moves along mRNA in steps of precisely three nucleotides. The mechanism for this ribosome motion remains elusive. Using a classification algorithm to sort single-molecule fluorescence resonance energy transfer data into subpopulations, we found that the ribosome dynamics detected at the peptidyl transferase center are highly inhomogeneous. The pretranslocation complex has at least four subpopulations that sample two hybrid states, whereas the posttranslocation complex is mainly static. We observed transitions among the ribosome subpopulations under various conditions, including 1), in the presence of EF-G; 2), spontaneously; 3), in different buffers, and 4), bound to antibiotics. Therefore, these subpopulations represent biologically active ribosomes. One key observation indicates that the Hy2 hybrid state only exists in a fluctuating ribosome subpopulation, which prompts us to propose that ribosome dynamics are hierarchically arranged. This proposal may have important implications for the regulation of cellular translation rates. PMID:21044598

  2. 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. PMID:26862193

  3. Structural basis for the interaction of antibiotics with peptidyl transferase center in eubacteria

    SciTech Connect

    Schlunzen, Frank; Zarivach, Raz; Harms, Jörg; Bashan, Anat; Tocilj, Ante; Albrecht, Renate; Yonath, Ada; Franceschi, Francois

    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 cavity 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.

  4. Crystallization and preliminary X-ray analysis of glutathione transferases from cyanobacteria

    SciTech Connect

    Feil, Susanne C.; Tang, Julian; Hansen, Guido; Gorman, Michael A.; Wiktelius, Eric; Stenberg, Gun; Parker, Michael W.

    2009-05-08

    Glutathione S-transferases (GSTs) are a group of multifunctional enzymes that are found in animals, plants and microorganisms. Their primary function is to remove toxins derived from exogenous sources or the products of metabolism from the cell. Mammalian GSTs have been extensively studied, in contrast to bacterial GSTs which have received relatively scant attention. A new class of GSTs called Chi has recently been identified in cyanobacteria. Chi GSTs exhibit a high glutathionylation activity towards isothiocyanates, compounds that are normally found in plants. Here, the crystallization of two GSTs are presented: TeGST produced by Thermosynechococcus elongates BP-1 and SeGST from Synechococcus elongates PCC 6301. Both enzymes formed crystals that diffracted to high resolution and appeared to be suitable for further X-ray diffraction studies. The structures of these GSTs may shed further light on the evolution of GST catalytic activity and in particular why these enzymes possess catalytic activity towards plant antimicrobial compounds.

  5. Functional Identification of Proteus mirabilis eptC Gene Encoding a Core Lipopolysaccharide Phosphoethanolamine Transferase

    PubMed Central

    Aquilini, Eleonora; Merino, Susana; Knirel, Yuriy A.; Regué, Miguel; Tomás, Juan M.

    2014-01-01

    By comparison of the Proteus mirabilis HI4320 genome with known lipopolysaccharide (LPS) phosphoethanolamine transferases, three putative candidates (PMI3040, PMI3576, and PMI3104) were identified. One of them, eptC (PMI3104) was able to modify the LPS of two defined non-polar core LPS mutants of Klebsiella pneumoniae that we use as surrogate substrates. Mass spectrometry and nuclear magnetic resonance showed that eptC directs the incorporation of phosphoethanolamine to the O-6 of l-glycero-d-mano-heptose II. The eptC gene is found in all the P. mirabilis strains analyzed in this study. Putative eptC homologues were found for only two additional genera of the Enterobacteriaceae family, Photobacterium and Providencia. The data obtained in this work supports the role of the eptC (PMI3104) product in the transfer of PEtN to the O-6 of l,d-HepII in P. mirabilis strains. PMID:24756091

  6. Design of a monomeric human glutathione transferase GSTP1, a structurally stable but catalytically inactive protein.

    PubMed

    Abdalla, Abdel-Monem; Bruns, Christopher M; Tainer, John A; Mannervik, Bengt; Stenberg, Gun

    2002-10-01

    By the introduction of 10 site-specific mutations in the dimer interface of human glutathione transferase P1-1 (GSTP1-1), a stable monomeric protein variant, GSTP1, was obtained. The monomer had lost the catalytic activity but retained the affinity for a number of electrophilic compounds normally serving as substrates for GSTP1-1. Fluorescence and circular dichroism spectra of the monomer and wild-type proteins were similar, indicating that there are no large structural differences between the subunits of the respective proteins. The GSTs have potential as targets for in vitro evolution and redesign with the aim of developing proteins with novel properties. To this end, a monomeric GST variant may have distinct advantages. PMID:12468717

  7. Glutathione S-transferases variants as risk factors in Alzheimer's disease.

    PubMed

    Wang, Tengfei

    2015-10-01

    Glutathione S-transferase (GST) was suggested as an important contributor to Alzheimer's disease (AD). The GSTs polymorphisms have been investigated as candidate genetic risk factors for AD, yet results remained uncertain. Therefore, we performed a meta-analysis to clarify the relationship of GSTs polymorphisms with the occurrence of AD. PubMed, Embase, Cochrane library and Alzgene databases were searched and potential literatures were selected. Pooled analyses and subgroup analyses were conducted, and also publication bias tests and cumulative meta-analysis. This meta-analysis suggested null associations between polymorphisms of GSTM1, GSTT1, GSTM3, GSTP1, GSTO1 and AD risk. GSTs variants may not have an impact on the morbidity of Alzheimer's disease. Further well designed researches are required to confirm these findings of the current study. PMID:25981226

  8. Detection and quantification of flavivirus NS5 methyl-transferase activities.

    PubMed

    Lim, Siew Pheng; Bodenreider, Christophe; Shi, Pei-Yong

    2013-01-01

    Flavivirus NS5 is the most conserved protein amongst the flavivirus proteins and is an essential enzyme for viral mRNA capping and replication. It encodes a methyl-transferase (MTase) domain at its N-terminal region which carries out sequential N7 and 2'-O methylation, resulting in the formation of the cap1 structure on its viral RNA genome. Two key methods have been established to measure these activities in vitro: thin-layer chromatography (TLC) and scintillation proximity assays (SPA). TLC offers the advantage of direct visualization of the amounts and types of cap structures formed whilst the SPA assay is more sensitive and quantitative. It is also amenable to high-throughput compound screening. The drawback of both assays is the need for radioisotope usage. We further describe the adaptation of a nonradioactive immune-competitive fluorescence polarization assay for detection of dengue virus MTase activity. PMID:23821274

  9. Terminal deoxynucleotidyl transferase in human lymphomas: possible existence of forms with high and low molecular weights.

    PubMed Central

    Vezzoni, P.; Campagnari, F.; Di Fronzo, G.; Clerici, L.

    1981-01-01

    Optimized methods for extraction and enzyme assay in crude tissue preparations were used to determine the amounts of terminal deoxnucleotidyl transferase (TdT) in malignant lymphomas. The TdT concentration was increased only in lymphoblastic lymphomas (LL) and was as high in these tumours as in the white blood cells from untreated patients with acute lymphoblastic leukaemia (ALL). The enzymes extracted from such lymphomas and from the leukaemic lymphoblasts had the same properties. Moreover, forms of TdT with low and high mol. wt were found in the LL tumours, similar to other reports of TdT-positive leukaemias. The overall study points at some basic biochemical identity of certain lymphoblastic malignancies, irrespective of whether the transformed cells are in solid tumours or are disseminated in the blood. PMID:6939447

  10. Structural flexibility modulates the activity of human glutathione transferase P1-1. Influence of a poor co-substrate on dynamics and kinetics of human glutathione transferase.

    PubMed

    Caccuri, A M; Ascenzi, P; Antonini, G; Parker, M W; Oakley, A J; Chiessi, E; Nuccetelli, M; Battistoni, A; Bellizia, A; Ricci, G

    1996-07-01

    Presteady-state and steady-state kinetics of human glutathione transferase P1-1 (EC 2.5.1.18) have been studied at pH 5.0 by using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, a poor co-substrate for this isoenzyme. Steady-state kinetics fits well with the simplest rapid equilibrium random sequential bi-bi mechanism and reveals a strong intrasubunit synergistic modulation between the GSH-binding site (G-site) and the hydrophobic binding site for the co-substrate (H-site); the affinity of the G-site for GSH increases about 30 times at saturating co-substrate and vice versa. Presteady-state experiments and thermodynamic data indicate that the rate-limiting step is a physical event and, probably, a structural transition of the ternary complex. Similar to that observed with 1-chloro-2, 4-dinitrobenzene (Ricci, G., Caccuri, A. M., Lo Bello, M., Rosato, N. , Mei, G., Nicotra, M., Chiessi, E., Mazzetti, A. P., and Federici, G.(1996) J. Biol. Chem. 271, 16187-16192), this event may be related to the frequency of enzyme motions. The observed low, viscosity-independent kcat value suggests that these motions are slow and diffusion-independent for an increased internal viscosity. In fact, molecular modeling suggests that the hydroxyl group of Tyr-108, which resides in helix 4, may be in hydrogen bonding distance of the oxygen atom of this new substrate, thus yielding a less flexible H-site. This effect might be transmitted to the G-site via helix 4. In addition, a new homotropic behavior exhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole is found in Cys-47 mutants revealing a structural intersubunit communication between the two H-sites. PMID:8663073

  11. Functional promiscuity correlates with conformational heterogeneity in A-class glutathione S-transferases.

    PubMed

    Hou, Liming; Honaker, Matthew T; Shireman, Laura M; Balogh, Larissa M; Roberts, Arthur G; Ng, Kei-Cheuk; Nath, Abhinav; Atkins, William M

    2007-08-10

    The structurally related glutathione S-transferase isoforms GSTA1-1 and GSTA4-4 differ greatly in their relative catalytic promiscuity. GSTA1-1 is a highly promiscuous detoxification enzyme. In contrast, GSTA4-4 exhibits selectivity for congeners of the lipid peroxidation product 4-hydroxynonenal. The contribution of protein dynamics to promiscuity has not been studied. Therefore, hydrogen/deuterium exchange mass spectrometry (H/DX) and fluorescence lifetime distribution analysis were performed with glutathione S-transferases A1-1 and A4-4. Differences in local dynamics of the C-terminal helix were evident as expected on the basis of previous studies. However, H/DX demonstrated significantly greater solvent accessibility throughout most of the GSTA1-1 sequence compared with GSTA4-4. A Phe-111/Tyr-217 aromatic-aromatic interaction in A4-4, which is not present in A1-1, was hypothesized to increase core packing. "Swap" mutants that eliminate this interaction from A4-4 or incorporate it into A1-1 yield H/DX behavior that is intermediate between the wild type templates. In addition, the single Trp-21 residue of each isoform was exploited to probe the conformational heterogeneity at the intrasubunit domain-domain interface. Excited state fluorescence lifetime distribution analysis indicates that this core residue is more conformationally heterogeneous in GSTA1-1 than in GSTA4-4, and this correlates with greater stability toward urea denaturation for GSTA4-4. The fluorescence distribution and urea sensitivity of the mutant proteins were intermediate between the wild type templates. The results suggest that the differences in protein dynamics of these homologs are global. The results suggest also the possible importance of extensive conformational plasticity to achieve high levels of functional promiscuity, possibly at the cost of stability. PMID:17561509

  12. Kinetics and catalytic properties of coenzyme A transferase from Peptostreptococcus elsdenii.

    PubMed Central

    Schulman, M; Valentino, D

    1976-01-01

    Coenzyme A (CoA) transferase from Peptostreptococcus elsdenii was purified to homogeneity, and some of its physical and catalytic properties were determined. The native enzyme has a molecular weight of 181,000 and is composed of two alpha subunits (molecular weight, 65,000) and one beta subunit (molecular weight 50,000). Heat treatment of the crude cell extract to 58 degrees C causes proteolysis of the native enzyme and yields a catalytically active enzyme with an approximate molecular weight of 120,000. The native CoA transferase is specific for CoA esters of short-chain alkyl monocarboxylic acids. With acetate as CoA acceptor the enzyme is active with propionyl-, butyryl-, isobutyryl-, valeryl-, isovaleryl,- and hexanoyl-CoA but not with heptanoyl or longer-chain CoA esters. There is no activity with acetoacetyl-CoA or the CoA esters of dicarboxylic acids. Steady-state kinetics indicated that the reaction proceeds via a classical bi-, bi-ping-pong mechanism. Maximal activity is obtained with propionyl- or butyryl-CoA, and both the Vmax and Km decrease as the alkyl chain length of the CoA ester increases. All CoA esters apompetitive inhibitor although it is not active as a substrate. Evidence for an enzyme CoA intermediate was provided by demonstration of an exchange between 14C-free acids (acetate and butyrate) and their corresponding CoA esters and by isolation of a 3H-labeled CoA enzyme after incubation of the enzyme with 3H-labeled acetyl-CoA. Approximately 2 mol of CoA was bound per mol of enzyme. Images PMID:977540

  13. Glutathione S-transferase GSTT1 and GSTM1 allozymes: beyond null alleles.

    PubMed

    Agúndez, José A G; Ladero, José M

    2008-03-01

    Moyer AM, Salavaggione OE, Hebbring SJ et al.: Glutathione S-transferase T1 and M1: gene sequence variation and functional genomics. Clin. Cancer Res. 13, 7207-7216 (2007). Genetic variations in the glutathione S-transferases GSTT1 and GSTM1 have been studied in many human populations, and association of these variations with environmentally-related cancers, drug-induced hepatotoxicity and even chronification of viral hepatitis has been shown. However, studies carried out to date have been limited to gene deletion, designated as null alleles, and no extensive studies on other types of genetic variations have been carried out. This study is of great importance, as it describes the occurrence and the allele frequencies for 18 SNPs in the GSTT1 gene, including four nonsynonymous SNPs, and 69 SNPs, two of which are nonsynonymous, in the GSTM1 gene. The GSTT1 SNPs leading to the amino acid substitutions Asp43Asn, Thr65Met, Thr104Pro and a single nucleotide deletion in exon 4 cause a decrease in immunoreactive protein. Interestingly, the previously described nonsynonymous GSTT1 SNPs rs2266635 (Ala21Thr), rs11550606 (Leu30Pro), rs17856199 (Phe45Cys), rs11550605 (Thr104Pro), rs2266633 (Asp141Asn) and rs2234953 (Glu173Lys) were not identified in 400 subjects, thus indicating that these variant alleles are expected to occur at extremely low frequencies. This study reinforces the need to combine SNP databases and resequencing. On combining the data reported in this study with SNP databases, the most promising target SNPs for GSTT1 association studies are those causing the amino acid changes Asp43Asn, Thr65Met, Thr104Pro and the single nucleotide deletion in exon 4. These gene variants should be analyzed in African-American and Hispanic subjects to increase the predictive capacity of genetic tests. For Caucasians and Oriental subjects, testing for null alleles seems to be sufficient. PMID:18303971

  14. Effect of three xenobiotic compounds on Glutathione S-Transferase in the clam Ruditapes decussatus.

    PubMed

    Hoarau, Pascal; Garello, Ginette; Gnassia-Barelli, Mauricette; Roméo, Michèle; Girard, Jean-Pierre

    2004-05-28

    The effects of 4,4'DDE, methoxychlor and imidazole were studied on glutathione S-transferase activities in the gills and hepatopancreas of the clam Ruditapes decussatus. The contamination doses were 0.14 microM for 4,4'DDE, 0.14 microM for methoxychlor and 25 microM for imidazole. GST activities were spectrophotometrically measured. SDS-PAGE and isoelectric focusing (IEF) were used to separate the different GST isoforms in control and treated animals, followed by Western blotting performed with anti-alpha, anti-mu and anti-pi GST anti-sera. In the hepatopancreas, GST-CDNB activities were always two to five-fold lower than in the gills where the activities were significantly increased after exposure to 4,4'DDE (ca. 1.6-fold) and to methoxychlor (ca. 1.3-fold) compared to the controls (ca. 3 micromolmin(-1)mg(-1)protein) whereas they remained unchanged after treatment with imidazole. When using glutathione S-transferase anti-alpha, anti-mu and anti-pi anti-sera, a single 26 kDa polypeptide was observed in the hepatopancreas and in the gills in all the tested conditions. Five GST subunits were observed after IEF showing greater immuno-reactivity with the anti-pi mammalian class antiserum than with the anti-alpha or anti-mu mammalian anti-sera. One isoform of pI 5.77 was particularly induced by 4,4'DDE and methoxychlor; it was recognized by the three anti-sera tested and seemed to be more efficient in the gills than in the hepatopancreas. This isoform may play a role in organochlorine detoxication. PMID:15110472

  15. 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)...

  16. Dimethyl adenosine transferase (KsgA) deficiency in Salmonella Enteritidis confers susceptibility to high osmolarity and virulence attenuation in chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    : 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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  18. Polymerase θ is a robust terminal transferase that oscillates between three different mechanisms during end-joining.

    PubMed

    Kent, Tatiana; Mateos-Gomez, Pedro A; Sfeir, Agnel; Pomerantz, Richard T

    2016-01-01

    DNA polymerase θ (Polθ) promotes insertion mutations during alternative end-joining (alt-EJ) by an unknown mechanism. Here, we discover that mammalian Polθ transfers nucleotides to the 3' terminus of DNA during alt-EJ in vitro and in vivo by oscillating between three different modes of terminal transferase activity: non-templated extension, templated extension in cis, and templated extension in trans. This switching mechanism requires manganese as a co-factor for Polθ template-independent activity and allows for random combinations of templated and non-templated nucleotide insertions. We further find that Polθ terminal transferase activity is most efficient on DNA containing 3' overhangs, is facilitated by an insertion loop and conserved residues that hold the 3' primer terminus, and is surprisingly more proficient than terminal deoxynucleotidyl transferase. In summary, this report identifies an unprecedented switching mechanism used by Polθ to generate genetic diversity during alt-EJ and characterizes Polθ as among the most proficient terminal transferases known. PMID:27311885

  19. CHARACTERIZATION OF A p-COUMAROYL TRANSFERASE RESPONSIBLE FOR THE INCORPORATION OF pCA INTO GRASS CELL WALLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Grasses form unique acylated lignins involving p-coumarate (pCA) residues primarily linked to syringyl units in lignin. A p-coumaroyl transferase (pCAT) is responsible for incorporation of pCA into cell walls as pCA-monolignol conjugates. Conjugates are synthesized in the cytoplasm, shuttled out int...

  20. The association of glutathione S-transferase polymorphisms in patients with osteosarcoma: evidence from a meta-analysis.

    PubMed

    Wang, Z; Xu, H; He, M; Wu, H; Zhu, Y; Su, Z

    2015-05-01

    Osteosarcoma is a life-threatening malignancy that often occurs in teenagers. Numerous studies have reported glutathione S-transferase polymorphisms are associated with osteosarcoma, but the results are inconclusive, partially because the sample size in each of published studies is relatively small. Therefore, we performed a meta-analysis of the published studies to estimate the association more accurately. To preciously examine the association between the glutathione S-transferase polymorphisms and osteosarcoma, we undertook a meta-analysis of six case-control studies. The association between the glutathione S-transferase polymorphisms and osteosarcoma risk was assessed by odds ratios together with their 95% confidence intervals using a fixed-effects model or random-effects model. In addition, hazard ratio was used to measure the relationship between glutathione S-transferase polymorphisms and prognosis in patients with osteosarcoma. We found that there was significant association between the polymorphisms in GSTT1 or GSTM3 (AA versus BB) and osteosarcoma risk. In addition, there is no evidence of association on GSTM1, GSTT1, GSTP1 (IIe/IIe versus IIe/Val) or GSTP1 (IIe/IIe versus Val/Val) polymorphisms with prognosis in osteosarcoma. In conclusion, the GSTT1 and GSTM3 polymorphisms might influence osteosarcoma risk. PMID:24689813

  1. Polymerase θ is a robust terminal transferase that oscillates between three different mechanisms during end-joining

    PubMed Central

    Kent, Tatiana; Mateos-Gomez, Pedro A; Sfeir, Agnel; Pomerantz, Richard T

    2016-01-01

    DNA polymerase θ (Polθ) promotes insertion mutations during alternative end-joining (alt-EJ) by an unknown mechanism. Here, we discover that mammalian Polθ transfers nucleotides to the 3’ terminus of DNA during alt-EJ in vitro and in vivo by oscillating between three different modes of terminal transferase activity: non-templated extension, templated extension in cis, and templated extension in trans. This switching mechanism requires manganese as a co-factor for Polθ template-independent activity and allows for random combinations of templated and non-templated nucleotide insertions. We further find that Polθ terminal transferase activity is most efficient on DNA containing 3’ overhangs, is facilitated by an insertion loop and conserved residues that hold the 3’ primer terminus, and is surprisingly more proficient than terminal deoxynucleotidyl transferase. In summary, this report identifies an unprecedented switching mechanism used by Polθ to generate genetic diversity during alt-EJ and characterizes Polθ as among the most proficient terminal transferases known. DOI: http://dx.doi.org/10.7554/eLife.13740.001 PMID:27311885

  2. The TIP GROWTH DEFECTIVE1 S-Acyl Transferase Regulates Plant Cell Growth in ArabidopsisW⃞

    PubMed Central

    Hemsley, Piers A.; Kemp, Alison C.; Grierson, Claire S.

    2005-01-01

    TIP GROWTH DEFECTIVE1 (TIP1) of Arabidopsis thaliana affects cell growth throughout the plant and has a particularly strong effect on root hair growth. We have identified TIP1 by map-based cloning and complementation of the mutant phenotype. TIP1 encodes an ankyrin repeat protein with a DHHC Cys-rich domain that is expressed in roots, leaves, inflorescence stems, and floral tissue. Two homologues of TIP1 in yeast (Saccharomyces cerevisiae) and human (Homo sapiens) have been shown to have S-acyl transferase (also known as palmitoyl transferase) activity. S-acylation is a reversible hydrophobic protein modification that offers swift, flexible control of protein hydrophobicity and affects protein association with membranes, signal transduction, and vesicle trafficking within cells. We show that TIP1 binds the acyl group palmitate, that it can rescue the morphological, temperature sensitivity, and yeast casein kinase2 localization defects of the yeast S-acyl transferase mutant akr1Δ, and that inhibition of acylation in wild-type Arabidopsis roots reproduces the Tip1− mutant phenotype. Our results demonstrate that S-acylation is essential for normal plant cell growth and identify a plant S-acyl transferase, an essential research tool if we are to understand how this important, reversible lipid modification operates in plant cells. PMID:16100337

  3. 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...

  4. LIGNIFICATION IN TRANSGENICS DEFICIENT IN P-COUMARATE 3-HYDROXYLASE (C3H) AND THE ASSOCIATED HYDROXYCINNAMOYL TRANSFERASE (HCT)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects on lignification of downregulating most of the genes for enzymes on the monolignol biosynthetic pathway have been reasonably well studied in angiosperms. The exception to this is the crucial hydroxylase, cinnamate 3-hydroxylase (C3H), and its associated hydroxycinnamyl transferase (HCT),...

  5. COMPARATIVE EXPRESSION OF TWO ALPHA CLASS GLUTATHIONE S-TRANSFERASES IN HUMAN ADULT AND PRENATAL LIVER TISSUES. (R827441)

    EPA Science Inventory

    Abstract

    The ability of the fetus to detoxify transplacental drugs and chemicals can be a critical determinant of teratogenesis and developmental toxicity. Developmentally regulated expression of alpha class glutathione S-transferases (GSTs) is of particular int...

  6. 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...

  7. Bilberry (Vaccinium myrtillus) Anthocyanins Modulate Heme Oxygenase-1 and Glutathione S-Transferase-pi Expression in the ARPE-19 Cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PURPOSE. To determine whether anthocyanin-enriched bilberry extracts modulate pre- or post-translational levels of oxidative stress defense enzymes heme-oxygenase (HO)-1 and glutathione S-transferase-pi (GST-pi) in cultured human retinal pigment epithelial (RPE) cells. METHODS. Confluent ARPE-19 c...

  8. Genetic and functional analyses of PptA, a phospho-form transferase targeting type IV pili in Neisseria gonorrhoeae.

    PubMed

    Naessan, Cecilia L; Egge-Jacobsen, Wolfgang; Heiniger, Ryan W; Wolfgang, Matthew C; Aas, Finn Erik; Røhr, Asmund; Winther-Larsen, Hanne C; Koomey, Michael

    2008-01-01

    The PilE pilin subunit protein of Neisseria gonorrhoeae undergoes unique covalent modifications with phosphoethanolamine (PE) and phosphocholine (PC). The pilin phospho-form transferase A (PptA) protein, required for these modifications, shows sequence relatedness with and architectural similarities to lipopolysaccharide PE transferases. Here, we used regulated expression and mutagenesis as means to better define the relationships between PptA structure and function, as well as to probe the mechanisms by which other factors impact the system. We show here that pptA expression is coupled at the level of transcription to its distal gene, murF, in a division/cell wall gene operon and that PptA can act in a dose-dependent fashion in PilE phospho-form modification. Molecular modeling and site-directed mutagenesis provided the first direct evidence that PptA is a member of the alkaline phosphatase superfamily of metalloenzymes with similar metal-binding sites and conserved structural folds. Through phylogenetic analyses and sequence alignments, these conclusions were extended to include the lipopolysaccharide PE transferases, including members of the disparate Lpt6 subfamily, and the MdoB family of phosphoglycerol transferases. Each of these enzymes thus likely acts as a phospholipid head group transferase whose catalytic mechanism involves a trans-esterification step generating a protein-phospho-form ester intermediate. Coexpression of PptA with PilE in Pseudomonas aeruginosa resulted in high levels of PE modification but was not sufficient for PC modification. This and other findings show that PptA-associated PC modification is governed by as-yet-undefined ancillary factors unique to N. gonorrhoeae. PMID:17951381

  9. Modelling and bioinformatics studies of the human Kappa-class glutathione transferase predict a novel third glutathione transferase family with similarity to prokaryotic 2-hydroxychromene-2-carboxylate isomerases.

    PubMed Central

    Robinson, Anna; Huttley, Gavin A; Booth, Hilary S; Board, Philip G

    2004-01-01

    The Kappa class of GSTs (glutathione transferases) comprises soluble enzymes originally isolated from the mitochondrial matrix of rats. We have characterized a Kappa class cDNA from human breast. The cDNA is derived from a single gene comprising eight exons and seven introns located on chromosome 7q34-35. Recombinant hGSTK1-1 was expressed in Escherichia coli as a homodimer (subunit molecular mass approximately 25.5 kDa). Significant glutathione-conjugating activity was found only with the model substrate CDNB (1-chloro-2,4-ditnitrobenzene). Hyperbolic kinetics were obtained for GSH (parameters: K(m)app, 3.3+/-0.95 mM; V(max)app, 21.4+/-1.8 micromol/min per mg of enzyme), while sigmoidal kinetics were obtained for CDNB (parameters: S0.5app, 1.5+/-1.0 mM; V(max)app, 40.3+/-0.3 micromol/min per mg of enzyme; Hill coefficient, 1.3), reflecting low affinities for both substrates. Sequence analyses, homology modelling and secondary structure predictions show that hGSTK1 has (a) most similarity to bacterial HCCA (2-hydroxychromene-2-carboxylate) isomerases and (b) a predicted C-terminal domain structure that is almost identical to that of bacterial disulphide-bond-forming DsbA oxidoreductase (root mean square deviation 0.5-0.6 A). The structures of hGSTK1 and HCCA isomerase are predicted to possess a thioredoxin fold with a polyhelical domain (alpha(x)) embedded between the beta-strands (betaalphabetaalpha(x)betabetaalpha, where the underlined elements represent the N and C motifs of the thioredoxin fold), as occurs in the bacterial disulphide-bond-forming oxidoreductases. This is in contrast with the cytosolic GSTs, where the helical domain occurs exclusively at the C-terminus (betaalphabetaalphabetabetaalphaalpha(x)). Although hGSTK1-1 catalyses some typical GST reactions, we propose that it is structurally distinct from other classes of cytosolic GSTs. The present study suggests that the Kappa class may have arisen in prokaryotes well before the divergence of the

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

    SciTech Connect

    Cary, J.W.; Petersen, D.J.; Bennett, G.N. ); Papoutsakis, E.T. )

    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 defect 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.

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

    SciTech Connect

    Torres, Rodrigo; Lan, Benson; Latif, Yama; Chim, Nicholas; Goulding, Celia W.

    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 NO 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

  12. Contribution of liver mitochondrial membrane-bound glutathione transferase to mitochondrial permeability transition pores

    SciTech Connect

    Hossain, Quazi Sohel; Ulziikhishig, Enkhbaatar; Lee, Kang Kwang; Yamamoto, Hideyuki; Aniya, Yoko

    2009-02-15

    We recently reported that the glutathione transferase in rat liver mitochondrial membranes (mtMGST1) is activated by S-glutathionylation and the activated mtMGST1 contributes to the mitochondrial permeability transition (MPT) pore and cytochrome c release from mitochondria [Lee, K.K., Shimoji, M., Quazi, S.H., Sunakawa, H., Aniya, Y., 2008. Novel function of glutathione transferase in rat liver mitochondrial membrane: role for cytochrome c release from mitochondria. Toxcol. Appl. Pharmacol. 232, 109-118]. In the present study we investigated the effect of reactive oxygen species (ROS), generator gallic acid (GA) and GST inhibitors on mtMGST1 and the MPT. When rat liver mitochondria were incubated with GA, mtMGST1 activity was increased to about 3 fold and the increase was inhibited with antioxidant enzymes and singlet oxygen quenchers including 1,4-diazabicyclo [2,2,2] octane (DABCO). GA-mediated mtMGST1 activation was prevented by GST inhibitors such as tannic acid, hematin, and cibacron blue and also by cyclosporin A (CsA). In addition, GA induced the mitochondrial swelling which was also inhibited by GST inhibitors, but not by MPT inhibitors CsA, ADP, and bongkrekic acid. GA also released cytochrome c from the mitochondria which was inhibited completely by DABCO, moderately by GST inhibitors, and somewhat by CsA. Ca{sup 2+}-mediated mitochondrial swelling and cytochrome c release were inhibited by MPT inhibitors but not by GST inhibitors. When the outer mitochondrial membrane was isolated after treatment of mitochondria with GA, mtMGST1 activity was markedly increased and oligomer/aggregate of mtMGST1 was observed. These results indicate that mtMGST1 in the outer mitochondrial membrane is activated by GA through thiol oxidation leading to protein oligomerization/aggregation, which may contribute to the formation of ROS-mediated, CsA-insensitive MPT pore, suggesting a novel mechanism for regulation of the MPT by mtMGST1.

  13. 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

  14. Effects of curcumin on cytochrome P450 and glutathione S-transferase activities in rat liver.

    PubMed

    Oetari, S; Sudibyo, M; Commandeur, J N; Samhoedi, R; Vermeulen, N P

    1996-01-12

    The stability of curcumin, as well as the interactions between curcumin and cytochrome P450s (P450s) and glutathione S-transferases (GSTs) in rat liver, were studied. Curcumin is relatively unstable in phosphate buffer at pH 7.4. The stability of curcumin was strongly improved by lowering the pH or by adding glutathione (GSH), N-acetyl L-cysteine (NAC), ascorbic acid, rat liver microsomes, or rat liver cytosol. Curcumin was found to be a potent inhibitor of rat liver P450 1A1/1A2 measured as ethoxyresorufin deethylation (EROD) activity in beta-naphthoflavone (beta NF)-induced microsomes, a less potent inhibitor of P450 2B1/2B2, measured as pentoxyresorufin depentylation (PROD) activity in phenobarbital (PB)-induced microsomes and a weak inhibitor of P450 2E1, measured as p-nitrophenol (PNP) hydroxylation activity in pyrazole-induced microsomes. Ki values were 0.14 and 76.02 microM for the EROD- and PROD-activities, respectively, and 30 microM of curcumin inhibited only 9% of PNP-hydroxylation activity. In ethoxyresorufin deethylation (EROD) and pentoxyresorufin depentylation (PROD) experiments, curcumin showed a competitive type of inhibition. Curcumin was also a potent inhibitor of glutathione S-transferase (GST) activity in cytosol from liver of rats treated with phenobarbital (PB), beta-naphthoflavone (beta NF) and pyrazole (Pyr), when measured towards 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. In liver cytosol from rats treated with phenobarbital (PB), curcumin inhibited GST activity in a mixed-type manner with a Ki of 5.75 microM and Ki of 12.5 microM. In liver cytosol from rats treated with pyrazole (Pyr) or beta-naphthoflavone (beta NF), curcumin demonstrated a competitive type of inhibition with Ki values of 1.79 microM and 2.29 microM, respectively. It is concluded that these strong inhibitory properties of curcumin towards P450s and GSTs, in addition to its well-known antioxidant activity, may help explain the previously observed anticarcinogenic

  15. Effects of some metal ions on rainbow trout erythrocytes glutathione S-transferase enzyme: an in vitro study.

    PubMed

    Comakli, Veysel; Ciftci, Mehmet; Kufrevioglu, O Irfan

    2013-12-01

    Glutathione S-transferase enzyme (GST) (EC 2.5.1.18) was purified from rainbow trout erythrocytes, and some characteristics of the enzyme and effects of some metal ions on enzyme activity were investigated. For this purpose, erythrocyte glutathione S-transferase enzyme which has 16.54 EU/mg protein specific activities was purified 11,026-fold by glutathione-agarose affinity chromatography with a yield of 59%. Temperature was kept under control (+4°C) during purification. Enzyme purification was checked by performing SDS-PAGE. Optimal pH, stable pH, optimal temperature, and K(M) and Vmax values for GSH and 1-chloro-2, 4-dinitrobenzene (CDNB) were also determined for the enzyme. In addition, IC50 values, Ki constants and the type of inhibition were determined by means of Line-Weaver-Burk graphs obtained for such inhibitors as Ag(+); Cd(2+), Cr(2+) and Mg(2+). PMID:23057421

  16. Crystal Structure of Human ADP-ribose Transferase ARTD15/PARP16 Reveals a Novel Putative Regulatory Domain*

    PubMed Central

    Karlberg, Tobias; Thorsell, Ann-Gerd; Kallas, Åsa; Schüler, Herwig

    2012-01-01

    ADP-ribosylation is involved in the regulation of DNA repair, transcription, and other processes. The 18 human ADP-ribose transferases with diphtheria toxin homology include ARTD1/PARP1, a cancer drug target. Knowledge of other family members may guide therapeutics development and help evaluate potential drug side effects. Here, we present the crystal structure of human ARTD15/PARP16, a previously uncharacterized enzyme. ARTD15 features an α-helical domain that packs against its transferase domain without making direct contact with the NAD+-binding crevice or the donor loop. Thus, this novel domain does not resemble the regulatory domain of ARTD1. ARTD15 displays auto-mono(ADP-ribosylation) activity and is affected by canonical poly(ADP-ribose) polymerase inhibitors. These results add to a framework that will facilitate research on a medically important family of enzymes. PMID:22661712

  17. Inhibition of liver glutathione S-transferase activity in rats by hypolipidemic drugs related or unrelated to clofibrate.

    PubMed

    Foliot, A; Touchard, D; Mallet, L

    1986-05-15

    The effects of in vivo administration of six hypolipidemic drugs on rat liver glutathione S-transferase activity were compared. This activity was measured with sulfobromophthalein (BSP), 1,2-dichloro-4-nitrobenzene (DCNB) or 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. Except for the nicotinic acid derivative ethanolamine oxiniacate, all the compounds tested significantly reduced it, whether or not they were related to clofibrate. The hepatic glutathione concentration either remained unchanged or only increased slightly after treatment with the various drugs. When measured, the maximal excretion rate of bile BSP dropped significantly, but not that of phenol-3,6-dibromophthalein (DBSP). Hepatic dye uptake and storage were not impaired. These results show that hypolipidemic drugs of the peroxisome proliferator type inhibit rat liver glutathione S-transferase activity and may reduce transport of anions conjugated with glutathione before excretion. PMID:3707598

  18. Frequencies of glutathione s-transferase (GSTM1, GSTM3 AND GSTT1) polymorphisms in a Malaysian population

    PubMed Central

    Alshagga, Mustafa A.; Mohamed, Norazlina; Nazrun Suhid, Ahmad; Abdel Aziz Ibrahim, Ibrahim; Zulkifli Syed Zakaria, Syed

    2011-01-01

    Introduction Glutathione S-transferase (GST) is a xenobiotic metabolising enzyme (XME), which may modify susceptibility in certain ethnic groups, showing ethnic dependent polymorphism. The aim of this study was to determine GSTM1, GSTM3 and GSTT1 gene polymorphisms in a Malaysian population in Kuala Lumpur. Material and methods Blood or buccal swab samples were collected from 137 Form II students from three schools in Wilayah Persekutuan Kuala Lumpur. Genotyping was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results Glutathione-S-transferase GSTM3 gene frequencies were 89% for AA, 10% for AB and 1% for BB. The gene frequencies for deleted GSTM1 and GSTT1 were 66% and 18% respectively. Conclusions This study suggested that the Malay population is at risk for environmental diseases and provides the basis for gene-environment association studies to be carried out. PMID:22291790

  19. Glutathione transferase from Plasmodium falciparum--interaction with malagashanine and selected plant natural products.

    PubMed

    Mangoyi, Rumbidzai; Hayeshi, Rose; Ngadjui, Bonventure; Ngandeu, Francois; Bezabih, Merhatibebe; Abegaz, Berhanu; Razafimahefa, Solofoniaina; Rasoanaivo, Philippe; Mukanganyama, Stanley

    2010-12-01

    A glutathione transferase (PfGST) isolated from Plasmodium falciparum has been associated with chloroquine resistance. A range of natural products including malagashanine (MG) were screened for inhibition of PfGST by a GST assay with 1-chloro-2,4-dinitrobenzene as a substrate. Only the sesquiterpene (JBC 42C), the bicoumarin (Tral-1), ellagic acid and curcumin, were shown to be potent inhibitors of PfGST with IC(50) values of 8.5, 12, 50 and 69 μM, respectively. Kinetic studies were performed on PfGST using ellagic acid as an inhibitor. Uncompetitive and mixed types of inhibition were obtained for glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB). The K(i) for GSH and CDNB were -0.015 μM and 0.011 μM, respectively. Malagashanine (100 µM) only reduced the activity of PfGST to 80% but showed a time-dependent inactivation of PfGST with a t(1/2) of 34 minutes compared to >120 minutes in the absence of MG or in the presence of 5 mM GSH. This work facilitates the understanding of the interaction of PfGST with some plant derived compounds. PMID:20521884

  20. Assembly of Multi-tRNA Synthetase Complex via Heterotetrameric Glutathione Transferase-homology Domains.

    PubMed

    Cho, Ha Yeon; Maeng, Seo Jin; Cho, Hyo Je; Choi, Yoon Seo; Chung, Jeong Min; Lee, Sangmin; Kim, Hoi Kyoung; Kim, Jong Hyun; Eom, Chi-Yong; Kim, Yeon-Gil; Guo, Min; Jung, Hyun Suk; Kang, Beom Sik; Kim, Sunghoon

    2015-12-01

    Many multicomponent protein complexes mediating diverse cellular processes are assembled through scaffolds with specialized protein interaction modules. The multi-tRNA synthetase complex (MSC), consisting of nine different aminoacyl-tRNA synthetases and three non-enzymatic factors (AIMP1-3), serves as a hub for many signaling pathways in addition to its role in protein synthesis. However, the assembly process and structural arrangement of the MSC components are not well understood. Here we show the heterotetrameric complex structure of the glutathione transferase (GST) domains shared among the four MSC components, methionyl-tRNA synthetase (MRS), glutaminyl-prolyl-tRNA synthetase (EPRS), AIMP2 and AIMP3. The MRS-AIMP3 and EPRS-AIMP2 using interface 1 are bridged via interface 2 of AIMP3 and EPRS to generate a unique linear complex of MRS-AIMP3:EPRS-AIMP2 at the molar ratio of (1:1):(1:1). Interestingly, the affinity at interface 2 of AIMP3:EPRS can be varied depending on the occupancy of interface 1, suggesting the dynamic nature of the linear GST tetramer. The four components are optimally arranged for maximal accommodation of additional domains and proteins. These characteristics suggest the GST tetramer as a unique and dynamic structural platform from which the MSC components are assembled. Considering prevalence of the GST-like domains, this tetramer can also provide a tool for the communication of the MSC with other GST-containing cellular factors. PMID:26472928

  1. Transcriptomic Responses of Phanerochaete chrysosporium to Oak Acetonic Extracts: Focus on a New Glutathione Transferase

    PubMed Central

    Thuillier, Anne; Chibani, Kamel; Belli, Gemma; Herrero, Enrique; Dumarçay, Stéphane; Gérardin, Philippe; Kohler, Annegret; Deroy, Aurélie; Dhalleine, Tiphaine; Bchini, Raphael; Jacquot, Jean-Pierre; Gelhaye, Eric

    2014-01-01

    The first steps of wood degradation by fungi lead to the release of toxic compounds known as extractives. To better understand how lignolytic fungi cope with the toxicity of these molecules, a transcriptomic analysis of Phanerochaete chrysosporium genes was performed in the presence of oak acetonic extracts. It reveals that in complement to the extracellular machinery of degradation, intracellular antioxidant and detoxification systems contribute to the lignolytic capabilities of fungi, presumably by preventing cellular damages and maintaining fungal health. Focusing on these systems, a glutathione transferase (P. chrysosporium GTT2.1 [PcGTT2.1]) has been selected for functional characterization. This enzyme, not characterized so far in basidiomycetes, has been classified first as a GTT2 compared to the Saccharomyces cerevisiae isoform. However, a deeper analysis shows that the GTT2.1 isoform has evolved functionally to reduce lipid peroxidation by recognizing high-molecular-weight peroxides as substrates. Moreover, the GTT2.1 gene has been lost in some non-wood-decay fungi. This example suggests that the intracellular detoxification system evolved concomitantly with the extracellular ligninolytic machinery in relation to the capacity of fungi to degrade wood. PMID:25107961

  2. Inhibition of O-GlcNAc transferase activity reprograms prostate cancer cell metabolism

    PubMed Central

    Itkonen, Harri M.; Gorad, Saurabh S.; Duveau, Damien Y.; Martin, Sara E.S.; Barkovskaya, Anna; Bathen, Tone F.; Moestue, Siver A.; Mills, Ian G.

    2016-01-01

    Metabolic networks are highly connected and complex, but a single enzyme, O-GlcNAc transferase (OGT) can sense the availability of metabolites and also modify target proteins. We show that inhibition of OGT activity inhibits the proliferation of prostate cancer cells, leads to sustained loss of c-MYC and suppresses the expression of CDK1, elevated expression of which predicts prostate cancer recurrence (p=0.00179). Metabolic profiling revealed decreased glucose consumption and lactate production after OGT inhibition. This decreased glycolytic activity specifically sensitized prostate cancer cells, but not cells representing normal prostate epithelium, to inhibitors of oxidative phosphorylation (rotenone and metformin). Intra-cellular alanine was depleted upon OGT inhibitor treatment. OGT inhibitor increased the expression and activity of alanine aminotransferase (GPT2), an enzyme that can be targeted with a clinically approved drug, cycloserine. Simultaneous inhibition of OGT and GPT2 inhibited cell viability and growth rate, and additionally activated a cell death response. These combinatorial effects were predominantly seen in prostate cancer cells, but not in a cell-line derived from normal prostate epithelium. Combinatorial treatments were confirmed with two inhibitors against both OGT and GPT2. Taken together, here we report the reprogramming of energy metabolism upon inhibition of OGT activity, and identify synergistically lethal combinations that are prostate cancer cell specific. PMID:26824323

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

    PubMed

    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. PMID:27126403

  4. 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. PMID:27044684

  5. Potent and selective inhibitors of glutathione S-transferase omega 1 that impair cancer drug resistance.

    PubMed

    Tsuboi, Katsunori; Bachovchin, Daniel A; Speers, Anna E; Spicer, Timothy P; Fernandez-Vega, Virneliz; Hodder, Peter; Rosen, Hugh; Cravatt, Benjamin F

    2011-10-19

    Glutathione S-transferases (GSTs) are a superfamily of enzymes that conjugate glutathione to a wide variety of both exogenous and endogenous compounds for biotransformation and/or removal. Glutathione S-tranferase omega 1 (GSTO1) is highly expressed in human cancer cells, where it has been suggested to play a role in detoxification of chemotherapeutic agents. Selective inhibitors of GSTO1 are, however, required to test the role that this enzyme plays in cancer and other (patho)physiological processes. With this goal in mind, we performed a fluorescence polarization activity-based protein profiling (fluopol-ABPP) high-throughput screen (HTS) with GSTO1 and the Molecular Libraries Small Molecule Repository (MLSMR) 300K+ compound library. This screen identified a class of selective and irreversible α-chloroacetamide inhibitors of GSTO1, which were optimized to generate an agent KT53 that inactivates GSTO1 with excellent in vitro (IC(50) = 21 nM) and in situ (IC(50) = 35 nM) potency. Cancer cells treated with KT53 show heightened sensitivity to the cytotoxic effects of cisplatin, supporting a role for GSTO1 in chemotherapy resistance. PMID:21899313

  6. Potent and Selective Inhibitors of Glutathione S-transferase Omega 1 that Impair Cancer Drug Resistance

    PubMed Central

    Tsuboi, Katsunori; Bachovchin, Daniel A.; Speers, Anna E.; Spicer, Timothy P.; Fernandez-Vega, Virneliz; Hodder, Peter; Rosen, Hugh; Cravatt, Benjamin F.

    2011-01-01

    Glutathione S-transferases (GSTs) are a superfamily of enzymes that conjugate glutathione to a wide variety of both exogenous and endogenous compounds for biotransformation and/or removal. Glutathione S-tranferase omega 1 (GSTO1) is highly expressed in human cancer cells, where it has been suggested to play a role in detoxification of chemotherapeutic agents. Selective inhibitors of GSTO1 are, however, required to test the role that this enzyme plays in cancer and other (patho)physiological processes. With this goal in mind, we performed a fluorescence polarization activity-based protein profiling (fluopol-ABPP) high-throughput screen (HTS) with GSTO1 and the Molecular Libraries Small Molecule Repository (MLSMR) 300K+ compound library. This screen identified a class of selective and irreversible α-chloroacetamide inhibitors of GSTO1, which were optimized to generate an agent KT53 that inactivates GSTO1 with excellent in vitro (IC50 = 21 nM) and in situ (IC50 = 35 nM) potency. Cancer cells treated with KT53 show heightened sensitivity to the cytotoxic effects of cisplatin, supporting a role for GSTO1 in the detoxification of chemo-therapeutic agents PMID:21899313

  7. A phosphopantetheinyl transferase that is essential for mitochondrial fatty acid biosynthesis.

    PubMed

    Guan, Xin; Chen, Hui; Abramson, Alex; Man, Huimin; Wu, Jinxia; Yu, Oliver; Nikolau, Basil J

    2015-11-01

    In this study we report the molecular genetic characterization of the Arabidopsis mitochondrial phosphopantetheinyl transferase (mtPPT), which catalyzes the phosphopantetheinylation and thus activation of mitochondrial acyl carrier protein (mtACP) of mitochondrial fatty acid synthase (mtFAS). This catalytic capability of the purified mtPPT protein (encoded by AT3G11470) was directly demonstrated in an in vitro assay that phosphopantetheinylated mature Arabidopsis apo-mtACP isoforms. The mitochondrial localization of the AT3G11470-encoded proteins was validated by the ability of their N-terminal 80-residue leader sequence to guide a chimeric GFP protein to this organelle. A T-DNA-tagged null mutant mtppt-1 allele shows an embryo-lethal phenotype, illustrating a crucial role of mtPPT for embryogenesis. Arabidopsis RNAi transgenic lines with reduced mtPPT expression display typical phenotypes associated with a deficiency in the mtFAS system, namely miniaturized plant morphology, slow growth, reduced lipoylation of mitochondrial proteins, and the hyperaccumulation of photorespiratory intermediates, glycine and glycolate. These morphological and metabolic alterations are reversed when these plants are grown in a non-photorespiratory condition (i.e. 1% CO2 atmosphere), demonstrating that they are a consequence of a deficiency in photorespiration due to the reduced lipoylation of the photorespiratory glycine decarboxylase. PMID:26402847

  8. Solution Structure of Alg13: The Sugar Donor Subunit of a Yeast N-Acetylglucosamine Transferase

    PubMed Central

    Wang, Xu; Weldeghorghis, Thomas; Zhang, Guofeng; Imperiali, Barbara; Prestegard, James H.

    2008-01-01

    Summary The solution structure of Alg13, the glycosyl-donor binding domain of an important bipartite glycosyltransferase in the yeast S. cerevisiae, is presented. This glycosyl transferase is unusual in that it is only active in the presence of a binding partner, Alg14. Alg13 is found to adopt a unique topology amongst glycosyltransferases. Rather than the conventional Rossmann fold found in all GT-B type enzymes, the N-terminal half of the protein is a Rossmann-like fold with a mixed parallel and anti-parallel β sheet. The Rossmann fold of the C-terminal half of Alg13 is conserved. However, while conventional GT-B type enzymes usually possess three helices at the C-terminus, only two helices are present in Alg13. Titration of Alg13 with both UDP-GlcNAc, the native glycosyl donor, and a paramagnetic mimic, UDP-TEMPO, shows that the interaction of Alg13 with the sugar donor is primarily through the residues in the C-terminal half of the protein. PMID:18547528

  9. 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. PMID:17593756

  10. Functional analysis of N-linking oligosaccharyl transferase enzymes encoded by deep-sea vent proteobacteria.

    PubMed

    Mills, Dominic C; Jervis, Adrian J; Abouelhadid, Sherif; Yates, Laura E; Cuccui, Jon; Linton, Dennis; Wren, Brendan W

    2016-04-01

    Bacterial N-linking oligosaccharyl transferases (OTase enzymes) transfer lipid-linked glycans to selected proteins in the periplasm and were first described in the intestinal pathogen Campylobacter jejuni, a member of the ε-proteobacteria-subdivision of bacteria. More recently, orthologues from other ε-proteobacterial Campylobacter and Helicobacter species and a δ-proteobacterium, Desulfovibrio desulfuricans, have been described, suggesting that these two subdivisions of bacteria may be a source of further N-linked protein glycosylation systems. Whole-genome sequencing of both ε- and δ-proteobacteria from deep-sea vent habitats, a rich source of species from these subdivisions, revealed putative ORFs encoding OTase enzymes and associated adjacent glycosyltransferases similar to the C. jejuni N-linked glycosylation locus. We expressed putative OTase ORFs from the deep-sea vent species Nitratiruptor tergarcus, Sulfurovum lithotrophicum and Deferribacter desulfuricans in Escherichia coli and showed that they were able to functionally complement the C. jejuni OTase, CjPglB. The enzymes were shown to possess relaxed glycan specificity, transferring diverse glycan structures and demonstrated different glycosylation sequon specificities. Additionally, a permissive D. desulfuricans acceptor protein was identified, and we provide evidence that the N-linked glycan synthesized by N. tergarcus and S. lithotrophicum contains an acetylated sugar at the reducing end. This work demonstrates that deep-sea vent bacteria encode functional N-glycosylation machineries and are a potential source of biotechnologically important OTase enzymes. PMID:26610891

  11. Functional analysis of N-linking oligosaccharyl transferase enzymes encoded by deep-sea vent proteobacteria

    PubMed Central

    Mills, Dominic C.; Jervis, Adrian J.; Abouelhadid, Sherif; Yates, Laura E.; Cuccui, Jon; Linton, Dennis; Wren, Brendan W.

    2016-01-01

    Bacterial N-linking oligosaccharyl transferases (OTase enzymes) transfer lipid-linked glycans to selected proteins in the periplasm and were first described in the intestinal pathogen Campylobacter jejuni, a member of the ε-proteobacteria-subdivision of bacteria. More recently, orthologues from other ε-proteobacterial Campylobacter and Helicobacter species and a δ-proteobacterium, Desulfovibrio desulfuricans, have been described, suggesting that these two subdivisions of bacteria may be a source of further N-linked protein glycosylation systems. Whole-genome sequencing of both ε- and δ-proteobacteria from deep-sea vent habitats, a rich source of species from these subdivisions, revealed putative ORFs encoding OTase enzymes and associated adjacent glycosyltransferases similar to the C. jejuni N-linked glycosylation locus. We expressed putative OTase ORFs from the deep-sea vent species Nitratiruptor tergarcus, Sulfurovum lithotrophicum and Deferribacter desulfuricans in Escherichia coli and showed they were able to functionally complement the C. jejuni OTase, CjPglB . The enzymes were shown to possess relaxed glycan specificity, transferring diverse glycan structures and demonstrated different glycosylation sequon specificities. Additionally a permissive D. desulfuricans acceptor protein was identified, and we provide evidence that the N-linked glycan synthesised by N. tergarcus and S. lithotrophicum contains an acetylated sugar at the reducing end. This work demonstrates that deep-sea vent bacteria encode functional N-glycosylation machineries and are a potential source of biotechnologically important OTase enzymes. PMID:26610891

  12. Effects of gestational and overt diabetes on placental cytochromes P450 and glutathione S-transferase.

    PubMed

    Glover; McRobie; Tracy

    1998-07-01

    Objective: Animal and in vivo human studies have observed that diabetes alters the expression of hepatic metabolizing cytochrome P450 (CYP) and glutathione S-transferase (GST) enzymes. The placenta has the ability to metabolize a number of xenobiotic and endogenous compounds by processes similar to those seen in the liver. Our objective was to compare placental xenobiotic metabolizing activity in diabetics to matched non-diabetic controls to determine if the presence of diabetes alters placental xenobiotic metabolizing activity.Methods: The catalytic activities of 7-ethoxyresorufin-O-deethylation [EROD] (CYP1A1), chlorzoxazone 6-hydroxylation (CYP2E1), dextromethorphan N-demethylation (CYP3A4), dextromethorphan O-demethylation (CYP2D6), and 1-chloro-2,4-dinitrobenzene (CDNB) conjugation with glutathione (GST) from placentas of diet controlled (class A1) and insulin-dependent (class A2) gestational diabetics and overt diabetics were compared to matched controls.Results: No differences in EROD activity were observed among overt or gestational diabetics and their respectively matched controls. CYP2E1, 2D6, and 3A4 enzyme activity were not detected in human placentas. In contrast, GST activity was significantly reduced by 30% (P <.05) in overt diabetics as compared to their matched controls and gestational diabetics.Conclusion: Pregnant women with overt diabetes have reduced GST activity in the placenta, which could potentially result in exposure of the fetus to harmful reactive electrophilic metabolites. PMID:10838356

  13. Purification and kinetic mechanism of the major glutathione S-transferase from bovine brain.

    PubMed Central

    Young, P R; Briedis, A V

    1989-01-01

    The major glutathione S-transferase isoenzyme from bovine brain was isolated and purified approx. 500-fold. The enzyme has a pI of 7.39 +/- 0.02 and consists of two non-identical subunits having apparent Mr values of 22,000 and 24,000. The enzyme is uniformly distributed in brain, and kinetic data at pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate suggest a random rapid-equilibrium mechanism. The kinetics of inhibition by product, by GSH analogues and by NADH are consistent with the suggested mechanism and require inhibitor binding to several different enzyme forms. Long-chain fatty acids are excellent inhibitors of the enzyme, and values of 1nKi for hexanoic acid, octanoic acid, decanoic acid and lauric acid form a linear series when plotted as a function of alkyl chain length. A free-energy change of -1900 J/mol (-455 cal/mol) per CH2 unit is calculated for the contribution of hydrophobic binding energy to the inhibition constants. The turnover number of the purified enzyme dimer is approx. 3400/min. When compared with the second-order rate constant for the reaction between CDNB and GSH, the enzyme is providing a rate acceleration of about 1000-fold. The role of entropic contributions to this small rate acceleration is discussed. PMID:2930465

  14. Effects of antioxidants on glutathione-S-transferase activities in hepatocyte culture

    SciTech Connect

    Chen, L.H. )

    1991-03-15

    Hepatocyte cultures from control rats and rats injected with 3-methylcholanthrene(3-MC) were used to study the effects of antioxidants on the activity of glutathione-S-transferases (GSH-S-T). This group of enzymes catalyzes conjugation of xenobiotics or their metabolites with reduced glutathione and plays an important role in detoxification of xenobiotics. In Experiment 1, treatment of hepatocyte cultures from both control and 3-MC-injected rats with 25 {mu}M or 50 {mu}M butylated hydroxyanisole (BHA) for 24 hours or 48 hours significantly increased GSH-S-T activity with I-chloro-2,4-dinitrobenzene (CDNB) as the substrate. In Experiment 2, treatment of hepatocytes from both control and 3-MC-treated rats with 25 {mu}M ethoxyquin or vitamin E, but not vitamin A or ascorbic acid, significantly increased GSH-S-T activity when CDNB, 1,2-dichloro-4-nitrobenzene or p-nitrobenzyl chloride was used as the substrate, respectively. The results suggested that BHA, ethoxyquin and vitamin E may have detoxification effects against 3-MC-induced carcinogenesis.

  15. The role of glutathione-S-transferase polymorphisms in ovarian cancer survival.

    PubMed

    Nagle, Christina M; Chenevix-Trench, Georgia; Spurdle, Amanda B; Webb, Penelope M

    2007-01-01

    Resistance to chemotherapy represents one of the most important causes of treatment failure in patients with ovarian cancer. Common polymorphisms in the glutathione-S-transferase (GSTM1, GSTP1 and GSTT1) family have been implicated in chemoresistence and ovarian cancer survival. In this study, we have analysed Australian women diagnosed with primary invasive epithelial ovarian cancer between 1985 and 1997, using DNA extracted from peripheral blood and archival uninvolved (normal) tissues. GSTP1 genotypes were determined using ABI Prism 7700 Sequence Detection System methodology (n=448) and GSTT1 and GSTM1 genotypes using PCR-agarose methodology (n=239). We observed a significant survival advantage among carriers of GSTP1 Ile105Val GG/GA genotype (HR 0.77, 95% confidence interval (CI) 0.61-0.99,p=0.04) and a non-significant survival advantage among women who were homozygous for the GSTM1 and GSTT1 deletion variants. There was also evidence of an additive effect, with a stronger survival benefit in women carrying three low function GST genotypes (GSTM1 null, GSTT1 null and GSTP1 GA/GG) (HR 0.47, 95% CI 0.22-1.02). The results of this study, the largest to date, are consistent with a number of previous smaller studies which have also observed that reduced GST function was associated with better survival outcomes in patients with ovarian cancer. PMID:17084623

  16. Prolactin confers resistance against cisplatin in breast cancer cells by activating glutathione-S-transferase.

    PubMed

    LaPensee, Elizabeth W; Schwemberger, Sandy J; LaPensee, Christopher R; Bahassi, El Mustapha; Afton, Scott E; Ben-Jonathan, Nira

    2009-08-01

    Resistance to chemotherapy is a major obstacle for successful treatment of breast cancer patients. Given that prolactin (PRL) acts as an anti-apoptotic/survival factor in the breast, we postulated that it antagonizes cytotoxicity by chemotherapeutic drugs. Treatment of breast cancer cells with PRL caused variable resistance to taxol, vinblastine, doxorubicin and cisplatin. PRL prevented cisplatin-induced G(2)/M cell cycle arrest and apoptosis. In the presence of PRL, significantly less cisplatin was bound to DNA, as determined by mass spectroscopy, and little DNA damage was seen by gamma-H2AX staining. PRL dramatically increased the activity of glutathione-S-transferase (GST), which sequesters cisplatin in the cytoplasm; this increase was abrogated by Jak and mitogen-activated protein kinase inhibitors. PRL upregulated the expression of the GSTmu, but not the pi, isozyme. A GST inhibitor abrogated antagonism of cisplatin cytotoxicity by PRL. In conclusion, PRL confers resistance against cisplatin by activating a detoxification enzyme, thereby reducing drug entry into the nucleus. These data provide a rational explanation for the ineffectiveness of cisplatin in breast cancer, which is characterized by high expression of both PRL and its receptor. Suppression of PRL production or blockade of its actions should benefit patients undergoing chemotherapy by allowing for lower drug doses and expanded drug options. PMID:19443905

  17. Highly ordered protein nanorings designed by accurate control of glutathione S-transferase self-assembly.

    PubMed

    Bai, Yushi; Luo, Quan; Zhang, Wei; Miao, Lu; Xu, Jiayun; Li, Hongbin; Liu, Junqiu

    2013-07-31

    Protein self-assembly into exquisite, complex, yet highly ordered architectures represents the supreme wisdom of nature. However, precise manipulation of protein self-assembly behavior in vitro is a great challenge. Here we report that by taking advantage of the cooperation of metal-ion-chelating interactions and nonspecific protein-protein interactions, we achieved accurate control of the orientation of proteins and their self-assembly into protein nanorings. As a building block, we utilized the C2-symmetric protein sjGST-2His, a variant of glutathione S-transferase from Schistosoma japonicum having two properly oriented His metal-chelating sites on the surface. Through synergic metal-coordination and non-covalent interactions, sjGST-2His self-assembled in a fixed bending manner to form highly ordered protein nanorings. The diameters of the nanorings can be regulated by tuning the strength of the non-covalent interaction network between sjGST-2His interfaces through variation of the ionic strength of the solution. This work provides a de novo design strategy that can be applied in the construction of novel protein superstructures. PMID:23865524

  18. Inhibition of insect glutathione S-transferase (GST) by conifer extracts.

    PubMed

    Wang, Zhiling; Zhao, Zhong; Abou-Zaid, Mamdouh M; Arnason, John T; Liu, Rui; Walshe-Roussel, Brendan; Waye, Andrew; Liu, Suqi; Saleem, Ammar; Cáceres, Luis A; Wei, Qin; Scott, Ian M

    2014-12-01

    Insecticide synergists biochemically inhibit insect metabolic enzyme activity and are used both to increase the effectiveness of insecticides and as a diagnostic tool for resistance mechanisms. Considerable attention has been focused on identifying new synergists from phytochemicals with recognized biological activities, specifically enzyme inhibition. Jack pine (Pinus banksiana Lamb.), black spruce (Picea mariana (Mill.) BSP.), balsam fir (Abies balsamea (L.) Mill.), and tamarack larch (Larix laricina (Du Roi) Koch) have been used by native Canadians as traditional medicine, specifically for the anti-inflammatory and antioxidant properties based on enzyme inhibitory activity. To identify the potential allelochemicals with synergistic activity, ethanol crude extracts and methanol/water fractions were separated by Sephadex LH-20 chromatographic column and tested for in vitro glutathione S-transferase (GST) inhibition activity using insecticide-resistant Colorado potato beetle, Leptinotarsa decemlineata (Say) midgut and fat-body homogenate. The fractions showing similar activity were combined and analyzed by ultra pressure liquid chromatography-mass spectrometry. A lignan, (+)-lariciresinol 9'-p-coumarate, was identified from P. mariana cone extracts, and L. laricina and A. balsamea bark extracts. A flavonoid, taxifolin, was identified from P. mariana and P. banksiana cone extracts and L. laricina bark extracts. Both compounds inhibit GST activity with taxifolin showing greater activity compared to (+)-lariciresinol 9'-p-coumarate and the standard GST inhibitor, diethyl maleate. The results suggested that these compounds can be considered as potential new insecticide synergists. PMID:25270601

  19. Ghrelin O-Acyl Transferase in Zebrafish Is an Evolutionarily Conserved Peptide Upregulated During Calorie Restriction.

    PubMed

    Hatef, Azadeh; Yufa, Roman; Unniappan, Suraj

    2015-10-01

    Ghrelin is a multifunctional orexigenic hormone with a unique acyl modification enabled by ghrelin O-acyl transferase (GOAT). Ghrelin is well-characterized in nonmammals, and GOAT sequences of several fishes are available in the GenBank. However, endogenous GOAT in non-mammals remains poorly understood. In this research, GOAT sequence comparison, tissue-specific GOAT expression, and its regulation by nutrient status and exogenous ghrelin were studied. It was found that the bioactive core of zebrafish GOAT amino acid sequence share high identity with that of mammals. GOAT mRNA was most abundant in the gut. GOAT-like immunoreactivity (i.r.) was found colocalized with ghrelin in the gastric mucosa. Food deprivation increased, and feeding decreased GOAT and preproghrelin mRNA expression in the brain and gut. GOAT and ghrelin peptides in the gut and brain showed corresponding decrease in food-deprived state. Intraperitoneal injection of acylated fish ghrelin caused a significant decrease in GOAT mRNA expression, suggesting a feedback mechanism regulating its abundance. Together, these results provide the first in-depth characterization of GOAT in a non-mammal. Our results demonstrate that endogenous GOAT expression is responsive to metabolic status and availability of acylated ghrelin, providing further evidences for GOAT in the regulation of feeding in teleosts. PMID:26226634

  20. Glutathione transferase A4-4 resists adduction by 4-hydroxynonenal☆

    PubMed Central

    Shireman, Laura M.; Kripps, Kimberly A.; Balogh, Larissa M.; Conner, Kip P.; Whittington, Dale; Atkins, William M.

    2010-01-01

    4-Hydroxy-2-trans-nonenal (HNE) is a lipid peroxidation product that contributes to the pathophysiology of several diseases with components of oxidative stress. The electrophilic nature of HNE results in covalent adduct formation with proteins, fatty acids and DNA. However, it remains unclear whether enzymes that metabolize HNE avoid inactivation by it. Glutathione transferase A4-4 (GST A4-4) plays a significant role in the elimination of HNE by conjugating it with glutathione (GSH), with catalytic activity toward HNE that is dramatically higher than the homologous GST A1-1 or distantly related GSTs. To determine whether enzymes that metabolize HNE resist its covalent adduction, the rates of adduction of these GST isoforms were compared and the functional effects of adduction on catalytic properties were determined. Although GST A4-4 and GST A1-1 have striking structural similarity, GST A4-4 was insensitive to adduction by HNE under conditions that yield modest adduction of GST A1-1 and extensive adduction of GST P1-1. Furthermore, adduction of GST P1-1 by HNE eliminated its activity toward the substrates 1-chloro- 2,4-dinitrobenzene (CDNB) and toward HNE itself. HNE effects on GST A4-4 and A1-1 were less significant. The results indicate that enzymes that metabolize HNE may have evolved structurally to resist covalent adduction by it. PMID:20836986

  1. Glutathione transferase A4-4 resists adduction by 4-hydroxynonenal.

    PubMed

    Shireman, Laura M; Kripps, Kimberly A; Balogh, Larissa M; Conner, Kip P; Whittington, Dale; Atkins, William M

    2010-12-15

    4-Hydroxy-2-trans-nonenal (HNE) is a lipid peroxidation product that contributes to the pathophysiology of several diseases with components of oxidative stress. The electrophilic nature of HNE results in covalent adduct formation with proteins, fatty acids and DNA. However, it remains unclear whether enzymes that metabolize HNE avoid inactivation by it. Glutathione transferase A4-4 (GST A4-4) plays a significant role in the elimination of HNE by conjugating it with glutathione (GSH), with catalytic activity toward HNE that is dramatically higher than the homologous GST A1-1 or distantly related GSTs. To determine whether enzymes that metabolize HNE resist its covalent adduction, the rates of adduction of these GST isoforms were compared and the functional effects of adduction on catalytic properties were determined. Although GST A4-4 and GST A1-1 have striking structural similarity, GST A4-4 was insensitive to adduction by HNE under conditions that yield modest adduction of GST A1-1 and extensive adduction of GST P1-1. Furthermore, adduction of GST P1-1 by HNE eliminated its activity toward the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and toward HNE itself. HNE effects on GST A4-4 and A1-1 were less significant. The results indicate that enzymes that metabolize HNE may have evolved structurally to resist covalent adduction by it. PMID:20836986

  2. Erythrocyte glutathione transferase: a general probe for chemical contaminations in mammals

    PubMed Central

    Bocedi, A; Fabrini, R; Lai, O; Alfieri, L; Roncoroni, C; Noce, A; Pedersen, JZ; Ricci, G

    2016-01-01

    Glutathione transferases (GSTs) are enzymes devoted to the protection of cells against many different toxins. In erythrocytes, the isoenzyme (e-GST) mainly present is GSTP1-1, which is overexpressed in humans in case of increased blood toxicity, as it occurs in nephrophatic patients or in healthy subjects living in polluted areas. The present study explores the possibility that e-GST may be used as an innovative and highly sensitive biomarker of blood toxicity also for other mammals. All distinct e-GSTs from humans, Bos taurus (cow), Sus scrofa (pig), Capra hircus (goat), Equus caballus (horse), Equus asinus (donkey) and Ovis aries (sheep), show very similar amino acid sequences, identical kinetics and stability properties. Reference values for e-GST in all these mammals reared in controlled farms span from 3.5±0.2 U/gHb in the pig to 17.0±0.9 U/gHb in goat; such activity levels can easily be determined with high precision using only a few microliters of whole blood and a simple spectrophotometric assay. Possibly disturbing factors have been examined to avoid artifact determinations. This study provides the basis for future screening studies to verify if animals have been exposed to toxicologic insults. Preliminary data on cows reared in polluted areas show increased expression of e-GST, which parallels the results found for humans. PMID:27551520

  3. Inhibition of various isoforms of rat liver glutathione S-transferases by tannic acid and butein.

    PubMed

    Zhang, K; Mack, P; Wong, K P

    1997-07-01

    Glutathione S-transferases (EC.2.5.1.18, GSTs) were purified from rat liver by S-hexylglutathione affinity chromatography and six isoforms, namely C-1, C-2, C-3, C-4, A-2 and A-1, were isolated by CM-cellulose and DEAE-cellulose ion-exchange columns. Tannic acid and butein showed varying degrees of inhibition on the six individual GST isoforms. When 1-chloro-2,4-dinitrobenzene (CDNB) was used as a substrate, butein exerted significantly more potent inhibition on the cationic isoforms C-2, C-3 and C-4 with IC50 values of 6.8, 8.5 and 8.0 muM respectively. All the isoforms showed lower activity towards p-nitrobenzyt chloride when compared to CDNB and inhibition of the p-nitrobenzyl chloride-activity by tannic acid and butein was also weaker. The inhibitory effects of tannic acid and butein on each isoform decreased generally with increasing pH in the range of 6.0 to 8.0. The optimum pHs for inhibitions by tannic acid and butein on the six individual isoforms lie in the pH range of 6.0 to 6.5. PMID:19856286

  4. Allyl isothiocyanate depletes glutathione and upregulates expression of glutathione S-transferases in Arabidopsis thaliana

    PubMed Central

    Øverby, Anders; Stokland, Ragni A.; Åsberg, Signe E.; Sporsheim, Bjørnar; Bones, Atle M.

    2015-01-01

    Allyl isothiocyanate (AITC) is a phytochemical associated with plant defense in plants from the Brassicaceae family. AITC has long been recognized as a countermeasure against external threats, but recent reports suggest that AITC is also involved in the onset of defense-related mechanisms such as the regulation of stomatal aperture. However, the underlying cellular modes of action in plants remain scarcely investigated. Here we report evidence of an AITC-induced depletion of glutathione (GSH) and the effect on gene expression of the detoxification enzyme family glutathione S-transferases (GSTs) in Arabidopsis thaliana. Treatment of A. thaliana wild-type with AITC resulted in a time- and dose-dependent depletion of cellular GSH. AITC-exposure of mutant lines vtc1 and pad2-1 with elevated and reduced GSH-levels, displayed enhanced and decreased AITC-tolerance, respectively. AITC-exposure also led to increased ROS-levels in the roots and loss of chlorophyll which are symptoms of oxidative stress. Following exposure to AITC, we found that GSH rapidly recovered to the same level as in the control plant, suggesting an effective route for replenishment of GSH or a rapid detoxification of AITC. Transcriptional analysis of genes encoding GSTs showed an upregulation in response to AITC. These findings demonstrate cellular effects by AITC involving a reversible depletion of the GSH-pool, induced oxidative stress, and elevated expression of GST-encoding genes. PMID:25954298

  5. Erythrocyte glutathione transferase: a general probe for chemical contaminations in mammals.

    PubMed

    Bocedi, A; Fabrini, R; Lai, O; Alfieri, L; Roncoroni, C; Noce, A; Pedersen, J Z; Ricci, G

    2016-01-01

    Glutathione transferases (GSTs) are enzymes devoted to the protection of cells against many different toxins. In erythrocytes, the isoenzyme (e-GST) mainly present is GSTP1-1, which is overexpressed in humans in case of increased blood toxicity, as it occurs in nephrophatic patients or in healthy subjects living in polluted areas. The present study explores the possibility that e-GST may be used as an innovative and highly sensitive biomarker of blood toxicity also for other mammals. All distinct e-GSTs from humans, Bos taurus (cow), Sus scrofa (pig), Capra hircus (goat), Equus caballus (horse), Equus asinus (donkey) and Ovis aries (sheep), show very similar amino acid sequences, identical kinetics and stability properties. Reference values for e-GST in all these mammals reared in controlled farms span from 3.5±0.2 U/gHb in the pig to 17.0±0.9 U/gHb in goat; such activity levels can easily be determined with high precision using only a few microliters of whole blood and a simple spectrophotometric assay. Possibly disturbing factors have been examined to avoid artifact determinations. This study provides the basis for future screening studies to verify if animals have been exposed to toxicologic insults. Preliminary data on cows reared in polluted areas show increased expression of e-GST, which parallels the results found for humans. PMID:27551520

  6. Role of oxidative stress mediated by glutathione-s-transferase in thiopurines' toxic effects.

    PubMed

    Pelin, Marco; De Iudicibus, Sara; Fusco, Laura; Taboga, Eleonora; Pellizzari, Giulia; Lagatolla, Cristina; Martelossi, Stefano; Ventura, Alessandro; Decorti, Giuliana; Stocco, Gabriele

    2015-06-15

    Azathioprine (AZA), 6-mercaptopurine (6-MP), and 6-thioguanine (6-TG) are antimetabolite drugs, widely used as immunosuppressants and anticancer agents. Despite their proven efficacy, a high incidence of toxic effects in patients during standard-dose therapy is recorded. The aim of this study is to explain, from a mechanistic point of view, the clinical evidence showing a significant role of glutathione-S-transferase (GST)-M1 genotype on AZA toxicity in inflammatory bowel disease patients. To this aim, the human nontumor IHH and HCEC cell lines were chosen as predictive models of the hepatic and intestinal tissues, respectively. AZA, but not 6-MP and 6-TG, induced a concentration-dependent superoxide anion production that seemed dependent on GSH depletion. N-Acetylcysteine reduced the AZA antiproliferative effect in both cell lines, and GST-M1 overexpression increased both superoxide anion production and cytotoxicity, especially in transfected HCEC cells. In this study, an in vitro model to study thiopurines' metabolism has been set up and helped us to demonstrate, for the first time, a clear role of GST-M1 in modulating AZA cytotoxicity, with a close dependency on superoxide anion production. These results provide the molecular basis to shed light on the clinical evidence suggesting a role of GST-M1 genotype in influencing the toxic effects of AZA treatment. PMID:25928802

  7. Purification and characterization of a glutathione S-transferase from Mucor mucedo.

    PubMed

    Hamed, Ragaa R; Abu-Shady, Mohamed R; El-Beih, Fawkia M; Abdalla, Abdel-Monem A; Afifi, Ola M

    2005-01-01

    An intracellular glutathione transferase was purified to homogenity from the fungus, Mucor mucedo, using DEAE-cellulose ion-exchange and glutathione affinity chromatography. Gel filtration chromatography and SDS-PAGE revealed that the purified GST is a homodimer with approximate native and subunit molecular mass of 53 kDa and 23.4 kDa, respectively. The enzyme has a pI value of 4.8, a pH optimum at pH 8.0 and apparent activation energy (Ea) of 1.42 kcal mol(-1). The purified GST acts readily on CDNB with almost negligible peroxidase activity and the activity was inhibited by Cibacron Blue (IC50 0.252 microM) and hematin (IC50 3.55 microM). M. mucedo GST displayed a non-Michaelian behavior. At low (0.1-0.3 mM) and high (0.3-2 mM) substrate concentration, Km (GSH) was calculated to be 0.179 and 0.65 mM, whereas Km(CDNB) was 0.531 and 11 mM and k(cat) was 39.8 and 552 s(-1), respectively. The enzyme showed apparent pKa values of 6-6.5 and 8.0. PMID:16209109

  8. Inhibition of human placenta glutathione transferase P1-1 by calvatic acid.

    PubMed

    Caccuri, A M; Ricci, G; Desideri, A; Buffa, M; Fruttero, R; Gasco, A; Ascenzi, P

    1994-04-01

    The inhibition mechanism of the dimeric human placenta glutathione transferase (GST P1-1) by the antibiotic p-carboxyphenylazoxycyanide (calvatic acid) has been investigated at pH 7.0 and 30.0 degrees C. Experiments performed at different calvatic acid/GST P1-1 molar ratios indicate that one mole of calvatic acid inactivates one mole of the homodimeric enzyme molecule, containing two catalytically equivalent active sites. The apparent second order rate constant for GST P1-1 inactivation is 2.4 +/- 0.3 M-1 s-1. The recovery of all the 5,5'-dithio-bis(2-nitro-benzoic acid)-titratable thiol groups as well as the original catalytic activity of GST P1-1 after treatment of the inhibited enzyme with dithiothreitol indicates that two disulfide bridges per dimer, likely between Cys47 and Cys101, have been formed during the reaction with calvatic acid. To the best of the authors knowledge, calvatic acid represents a unique case of enzyme inhibitor acting also throughout its reaction product(s). PMID:8069231

  9. Characterization of Discrete Phosphopantetheinyl Transferases in Streptomyces tsukubaensis L19 Unveils a Complicate Phosphopantetheinylation Network

    PubMed Central

    Wang, Yue-Yue; Zhang, Xiao-Sheng; Luo, Hong-Dou; Ren, Ni-Ni; Jiang, Xin-Hang; Jiang, Hui; Li, Yong-Quan

    2016-01-01

    Phosphopantetheinyl transferases (PPTases) play essential roles in both primary metabolisms and secondary metabolisms via post-translational modification of acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs). In this study, an industrial FK506 producing strain Streptomyces tsukubaensis L19, together with Streptomyces avermitilis, was identified to contain the highest number (five) of discrete PPTases known among any species thus far examined. Characterization of the five PPTases in S. tsukubaensis L19 unveiled that stw ACP, an ACP in a type II PKS, was phosphopantetheinylated by three PPTases FKPPT1, FKPPT3, and FKACPS; sts FAS ACP, the ACP in fatty acid synthase (FAS), was phosphopantetheinylated by three PPTases FKPPT2, FKPPT3, and FKACPS; TcsA-ACP, an ACP involved in FK506 biosynthesis, was phosphopantetheinylated by two PPTases FKPPT3 and FKACPS; FkbP-PCP, an PCP involved in FK506 biosynthesis, was phosphopantetheinylated by all of these five PPTases FKPPT1-4 and FKACPS. Our results here indicate that the functions of these PPTases complement each other for ACPs/PCPs substrates, suggesting a complicate phosphopantetheinylation network in S. tsukubaensis L19. Engineering of these PPTases in S. tsukubaensis L19 resulted in a mutant strain that can improve FK506 production. PMID:27052100

  10. Trichinella spiralis: low vaccine potential of glutathione S-transferase against infections in mice.

    PubMed

    Li, Ling Ge; Wang, Zhong Quan; Liu, Ruo Dan; Yang, Xuan; Liu, Li Na; Sun, Ge Ge; Jiang, Peng; Zhang, Xi; Zhang, Gong Yuan; Cui, Jing

    2015-06-01

    We have previously reported that Trichinella spiralis glutathione-S-transferase (TsGST) gene is an up-regulated gene in intestinal infective larvae (IIL) compared to muscle larvae (ML). In this study, the TsGST gene was cloned, and recombinant TsGST (rTsGST) was produced. Anti-rTsGST serum recognized the native TsGST by Western blotting in crude antigens of ML, adult worm (AW) and newborn larvae (NBL) of T. spiralis, but not in ML excretory-secretory (ES) antigens. Expression of TsGST was observed in all different developmental stages (IIL, AW, NBL and ML). An immunolocalization analysis identified TsGST in the cuticle, stichosome and genital primordium of the parasite. The rTsGST had GST enzymatic activity. After a challenge infection with T. spiralis larvae, mice immunized with rTsGST displayed a 35.71% reduction in adult worms and a 38.55% reduction in muscle larvae. The vaccination of mice with rTsGST induced the Th1/Th2-mixed type of immune response with Th2 predominant (high levels of IgG1) and partial protective immunity against T. spiralis infection. PMID:25757368

  11. Activity-regulated trafficking of the palmitoyl-acyl transferase DHHC5.

    PubMed

    Brigidi, G Stefano; Santyr, Brendan; Shimell, Jordan; Jovellar, Blair; Bamji, Shernaz X

    2015-01-01

    Synaptic plasticity is mediated by the dynamic localization of proteins to and from synapses. This is controlled, in part, through activity-induced palmitoylation of synaptic proteins. Here we report that the ability of the palmitoyl-acyl transferase, DHHC5, to palmitoylate substrates in an activity-dependent manner is dependent on changes in its subcellular localization. Under basal conditions, DHHC5 is bound to PSD-95 and Fyn kinase, and is stabilized at the synaptic membrane through Fyn-mediated phosphorylation of a tyrosine residue within the endocytic motif of DHHC5. In contrast, DHHC5's substrate, δ-catenin, is highly localized to dendritic shafts, resulting in the segregation of the enzyme/substrate pair. Neuronal activity disrupts DHHC5/PSD-95/Fyn kinase complexes, enhancing DHHC5 endocytosis, its translocation to dendritic shafts and its association with δ-catenin. Following DHHC5-mediated palmitoylation of δ-catenin, DHHC5 and δ-catenin are trafficked together back into spines where δ-catenin increases cadherin stabilization and recruitment of AMPA receptors to the synaptic membrane. PMID:26334723

  12. Induction of epoxide hydrolase, glucuronosyl transferase, and sulfotransferase by phenethyl isothiocyanate in male Wistar albino rats.

    PubMed

    Abdull Razis, Ahmad Faizal; Mohd Noor, Noramaliza; Konsue, Nattaya

    2014-01-01

    Phenethyl isothiocyanate (PEITC) is an isothiocyanate found in watercress as the glucosinolate (gluconasturtiin). The isothiocyanate is converted from the glucosinolate by intestinal microflora or when contacted with myrosinase during the chopping and mastication of the vegetable. PEITC manifested protection against chemically-induced cancers in various tissues. A potential mechanism of chemoprevention is by modulating the metabolism of carcinogens so as to promote deactivation. The principal objective of this study was to investigate in rats the effect of PEITC on carcinogen-metabolising enzyme systems such as sulfotransferase (SULT), N-acetyltransferase (NAT), glucuronosyl transferase (UDP), and epoxide hydrolase (EH) following exposure to low doses that simulate human dietary intake. Rats were fed for 2 weeks diets supplemented with PEITC at 0.06 µmol/g (low dose, i.e., dietary intake), 0.6 µmol/g (medium dose), and 6.0 µmol/g (high dose), and the enzymes were monitored in rat liver. At the Low dose, no induction of the SULT, NAT, and EH was noted, whereas UDP level was elevated. At the Medium dose, only SULT level was increased, whereas at the High dose marked increase in EH level was observed. It is concluded that PEITC modulates carcinogen-metabolising enzyme systems at doses reflecting human intake thus elucidating the mechanism of its chemoprevention. PMID:24592387

  13. Identification and characterization of GSTT3, a third murine Theta class glutathione transferase.

    PubMed Central

    Coggan, Marjorie; Flanagan, Jack U; Parker, Michael W; Vichai, Vanicha; Pearson, William R; Board, Philip G

    2002-01-01

    A novel Theta class glutathione transferase (GST) isoenzyme from mouse termed mGSTT3 has been identified by analysis of the expressed sequence tag database. The gene encoding mGSTT3 is clustered with the mGSTT1 and mGSTT2 genes on chromosome 10 and has an exon/intron structure that is similar to that of the other Theta class genes. mGSTT3 is expressed strongly in the liver and to a decreasing extent in the kidney and testis. Recombinant mGSTT3-3 expressed in Escherichia coli had a substrate-specificity profile that differed significantly from that of GSTT1-1 and GSTT2-2 isoenzymes. A molecular model of mGSTT3 suggested that, in comparison with GSTT2, a decrease in volume of the hydrophobic substrate-binding site and the loss of the sulphate-binding pocket prevents its use of the GSTT2 substrate 1-menaphthyl sulphate. PMID:12038961

  14. Isothiocyanate exposure, glutathione S-transferase polymorphisms, and colorectal cancer risk1234

    PubMed Central

    Gao, Yu-Tang; Shu, Xiao-Ou; Cai, Qiuyin; Li, Guo-Liang; Li, Hong-Lan; Ji, Bu-Tian; Rothman, Nathaniel; Dyba, Marcin; Xiang, Yong-Bing; Chung, Fung-Lung; Chow, Wong-Ho; Zheng, Wei

    2010-01-01

    Background: Isothiocyanates, compounds found primarily in cruciferous vegetables, have been shown in laboratory studies to possess anticarcinogenic activity. Glutathione S-transferases (GSTs) are involved in the metabolism and elimination of isothiocyanates; thus, genetic variations in these enzymes may affect in vivo bioavailability and the activity of isothiocyanates. Objective: The objective was to prospectively evaluate the association between urinary isothiocyanate concentrations and colorectal cancer risk as well as the potential modifying effect of GST genotypes on the association. Design: A nested case-control study of 322 cases and 1251 controls identified from the Shanghai Women's Health Study was conducted. Results: Urinary isothiocyanate concentrations were inversely associated with colorectal cancer risk; the inverse association was statistically significant or nearly significant in the GSTM1-null (P for trend = 0.04) and the GSTT1-null (P for trend = 0.07) genotype groups. The strongest inverse association was found among individuals with both the GSTM1-null and the GSTT1-null genotypes, with an adjusted odds ratio of 0.51 (95% CI: 0.27, 0.95), in a comparison of the highest with the lowest tertile of urinary isothiocyanates. No apparent associations between isothiocyanate concentration and colorectal cancer risk were found among individuals who carried either the GSTM1 or GSTT1 gene (P for interaction < 0.05). Conclusion: This study suggests that isothiocyanate exposure may reduce the risk of colorectal cancer, and this protective effect may be modified by the GSTM1 and GSTT1 genes. PMID:20042523

  15. Expression profiling of selected glutathione transferase genes in Zea mays (L.) seedlings infested with cereal aphids.

    PubMed

    Sytykiewicz, Hubert; Chrzanowski, Grzegorz; Czerniewicz, Paweł; Sprawka, Iwona; Łukasik, Iwona; Goławska, Sylwia; Sempruch, Cezary

    2014-01-01

    The purpose of this report was to evaluate the expression patterns of selected glutathione transferase genes (gst1, gst18, gst23 and gst24) in the tissues of two maize (Zea mays L.) varieties (relatively resistant Ambrozja and susceptible Tasty Sweet) that were colonized with oligophagous bird cherry-oat aphid (Rhopalosiphum padi L.) or monophagous grain aphid (Sitobion avenae L.). Simultaneously, insect-triggered generation of superoxide anion radicals (O2•-) in infested Z. mays plants was monitored. Quantified parameters were measured at 1, 2, 4, 8, 24, 48 and 72 h post-initial aphid infestation (hpi) in relation to the non-infested control seedlings. Significant increases in gst transcript amounts were recorded in aphid-stressed plants in comparison to the control seedlings. Maximal enhancement in the expression of the gst genes in aphid-attacked maize plants was found at 8 hpi (gst23) or 24 hpi (gst1, gst18 and gst24) compared to the control. Investigated Z. mays cultivars formed excessive superoxide anion radicals in response to insect treatments, and the highest overproduction of O2•- was noted 4 or 8 h after infestation, depending on the aphid treatment and maize genotype. Importantly, the Ambrozja variety could be characterized as having more profound increments in the levels of gst transcript abundance and O2•- generation in comparison with the Tasty Sweet genotype. PMID:25365518

  16. Expression Profiling of Selected Glutathione Transferase Genes in Zea mays (L.) Seedlings Infested with Cereal Aphids

    PubMed Central

    Sytykiewicz, Hubert; Chrzanowski, Grzegorz; Czerniewicz, Paweł; Sprawka, Iwona; Łukasik, Iwona; Goławska, Sylwia; Sempruch, Cezary

    2014-01-01

    The purpose of this report was to evaluate the expression patterns of selected glutathione transferase genes (gst1, gst18, gst23 and gst24) in the tissues of two maize (Zea mays L.) varieties (relatively resistant Ambrozja and susceptible Tasty Sweet) that were colonized with oligophagous bird cherry-oat aphid (Rhopalosiphum padi L.) or monophagous grain aphid (Sitobion avenae L.). Simultaneously, insect-triggered generation of superoxide anion radicals (O2•−) in infested Z. mays plants was monitored. Quantified parameters were measured at 1, 2, 4, 8, 24, 48 and 72 h post-initial aphid infestation (hpi) in relation to the non-infested control seedlings. Significant increases in gst transcript amounts were recorded in aphid-stressed plants in comparison to the control seedlings. Maximal enhancement in the expression of the gst genes in aphid-attacked maize plants was found at 8 hpi (gst23) or 24 hpi (gst1, gst18 and gst24) compared to the control. Investigated Z. mays cultivars formed excessive superoxide anion radicals in response to insect treatments, and the highest overproduction of O2•− was noted 4 or 8 h after infestation, depending on the aphid treatment and maize genotype. Importantly, the Ambrozja variety could be characterized as having more profound increments in the levels of gst transcript abundance and O2•− generation in comparison with the Tasty Sweet genotype. PMID:25365518

  17. Serum gamma glutamyl transferase as a specific indicator of bile duct lesions in the rat liver.

    PubMed Central

    Leonard, T. B.; Neptun, D. A.; Popp, J. A.

    1984-01-01

    Serum gamma-glutamyl transferase (GGT), a marker of hepatic injury used extensively in humans, has been used rarely in rats because its specificity has not been previously defined. Studies were designed for investigation of the specificity of serum GGT activity with the use of cell type specific hepatotoxicants in Fischer 344 rats. Single necrogenic doses of CCl4, allyl alcohol (AA), and alpha-naphthylisothiocyanate (ANIT) were used to produce cell specific injury in centrilobular hepatocytes, periportal hepatocytes, and bile duct cells, respectively. Administration of CCl4 markedly increased serum activities of alanine aminotransferase (ALT), alkaline phosphatase (AP), and serum bile acid concentrations within 24 hours but had no effect on serum GGT activity. ANIT treatment increased serum GGT and AP activities and bile acid concentration 24 hours following administration. Allyl alcohol administration increased serum ALT activity but had no effect on GGT activity. Administration of ANIT in the diet at 0.01%, 0.022%, 0.047%, and 0.1% for 2, 4, and 6 weeks produced dose- and time-dependent increases in serum GGT activity which strongly correlated with quantitative increases in hepatic bile duct volume, which was determined morphometrically. These observations support the use of serum GGT activity in the rat as diagnostic of bile duct cell necrosis when increases are detected shortly after the insult and as an indicator of possible bile duct hyperplasia. Images Figure 1 Figure 3 PMID:6147091

  18. Serum gamma glutamyl transferase as a specific indicator of bile duct lesions in the rat liver.

    PubMed

    Leonard, T B; Neptun, D A; Popp, J A

    1984-08-01

    Serum gamma-glutamyl transferase (GGT), a marker of hepatic injury used extensively in humans, has been used rarely in rats because its specificity has not been previously defined. Studies were designed for investigation of the specificity of serum GGT activity with the use of cell type specific hepatotoxicants in Fischer 344 rats. Single necrogenic doses of CCl4, allyl alcohol (AA), and alpha-naphthylisothiocyanate (ANIT) were used to produce cell specific injury in centrilobular hepatocytes, periportal hepatocytes, and bile duct cells, respectively. Administration of CCl4 markedly increased serum activities of alanine aminotransferase (ALT), alkaline phosphatase (AP), and serum bile acid concentrations within 24 hours but had no effect on serum GGT activity. ANIT treatment increased serum GGT and AP activities and bile acid concentration 24 hours following administration. Allyl alcohol administration increased serum ALT activity but had no effect on GGT activity. Administration of ANIT in the diet at 0.01%, 0.022%, 0.047%, and 0.1% for 2, 4, and 6 weeks produced dose- and time-dependent increases in serum GGT activity which strongly correlated with quantitative increases in hepatic bile duct volume, which was determined morphometrically. These observations support the use of serum GGT activity in the rat as diagnostic of bile duct cell necrosis when increases are detected shortly after the insult and as an indicator of possible bile duct hyperplasia. PMID:6147091

  19. 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.

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

    SciTech Connect

    Low, Wai Yee; Feil, Susanne C.; Ng, Hooi Ling; Gorman, Michael A.; Morton, Craig J.; Pyke, James; McConville, Malcolm J.; Bieri, Michael; Mok, Yee-Foong; Robin, Charles; Gooley, Paul R.; Parker, Michael W.; Batterham, Philip

    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 of 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.

  1. Septins guide microtubule protrusions induced by actin-depolymerizing toxins like Clostridium difficile transferase (CDT).

    PubMed

    Nölke, Thilo; Schwan, Carsten; Lehmann, Friederike; Østevold, Kristine; Pertz, Olivier; Aktories, Klaus

    2016-07-12

    Hypervirulent Clostridium difficile strains, which are associated with increased morbidity and mortality, produce the actin-ADP ribosylating toxin Clostridium difficile transferase (CDT). CDT depolymerizes actin, causes formation of microtubule-based protrusions, and increases pathogen adherence. Here, we show that septins (SEPT) are essential for CDT-induced protrusion formation. SEPT2, -6, -7, and -9 accumulate at predetermined protrusion sites and form collar-like structures at the base of protrusions. The septin inhibitor forchlorfenuron or knockdown of septins inhibits protrusion formation. At protrusion sites, septins colocalize with the GTPase Cdc42 (cell division control protein 42) and its effector Borg (binder of Rho GTPases), which act as up-stream regulators of septin polymerization. Precipitation and surface plasmon resonance studies revealed high-affinity binding of septins to the microtubule plus-end tracking protein EB1, thereby guiding incoming microtubules. The data suggest that CDT usurps conserved regulatory principles involved in microtubule-membrane interaction, depending on septins, Cdc42, Borgs, and restructuring of the actin cytoskeleton. PMID:27339141

  2. Increased skin tumorigenesis in mice lacking pi class glutathione S-transferases

    PubMed Central

    Henderson, Colin J.; Smith, Austin G.; Ure, Jan; Brown, Ken; Bacon, E. Jane; Wolf, C. Roland

    1998-01-01

    The activity of chemical carcinogens is a complex balance between metabolic activation by cytochrome P450 monooxygenases and detoxification by enzymes such as glutathione S-transferase (GST). Regulation of these proteins may have profound effects on carcinogenic activity, although it has proved impossible to ascribe the observed effects to the activity of a single protein. GstP appears to play a very important role in carcinogenesis, although the precise nature of its involvement is unclear. We have deleted the murine GstP gene cluster and established the effects on skin tumorigenesis induced by the polycyclic aromatic hydrocarbon 7,12-dimethylbenz anthracene and the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate. After 20 weeks, a highly significant increase in the number of papillomas was found in the GstP1/P2 null mice [GstP1/P2(−/−) mice, 179 papillomas, mean 9.94 per animal vs. GstP1/P2(+/+) mice, 55 papillomas, mean 2.89 per animal, (P < 0.001)]. This difference in tumor incidence provides direct evidence that a single gene involved in drug metabolism can have a profound effect on tumorigenicity, and demonstrates that GstP may be an important determinant in cancer susceptibility, particularly in diseases where exposure to polycyclic aromatic hydrocarbons is involved, for instance in cigarette smoke-induced lung cancer. PMID:9560266

  3. Dual functionality of O-GlcNAc transferase is required for Drosophila development.

    PubMed

    Mariappa, Daniel; Zheng, Xiaowei; Schimpl, Marianne; Raimi, Olawale; Ferenbach, Andrew T; Müller, H-Arno J; van Aalten, Daan M F

    2015-12-01

    Post-translational modification of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc) catalysed by O-GlcNAc transferase (OGT) has been linked to regulation of diverse cellular functions. OGT possesses a C-terminal glycosyltransferase catalytic domain and N-terminal tetratricopeptide repeats that are implicated in protein-protein interactions. Drosophila OGT (DmOGT) is encoded by super sex combs (sxc), mutants of which are pupal lethal. However, it is not clear if this phenotype is caused by reduction of O-GlcNAcylation. Here we use a genetic approach to demonstrate that post-pupal Drosophila development can proceed with negligible OGT catalysis, while early embryonic development is OGT activity-dependent. Structural and enzymatic comparison between human OGT (hOGT) and DmOGT informed the rational design of DmOGT point mutants with a range of reduced catalytic activities. Strikingly, a severely hypomorphic OGT mutant complements sxc pupal lethality. However, the hypomorphic OGT mutant-rescued progeny do not produce F2 adults, because a set of Hox genes is de-repressed in F2 embryos, resulting in homeotic phenotypes. Thus, OGT catalytic activity is required up to late pupal stages, while further development proceeds with severely reduced OGT activity. PMID:26674417

  4. Microinjection of recombinant O-GlcNAc transferase potentiates Xenopus oocytes M-phase entry

    SciTech Connect

    Dehennaut, Vanessa; Hanoulle, Xavier; Bodart, Jean-Francois; Vilain, Jean-Pierre; Michalski, Jean-Claude; Landrieu, Isabelle; Lippens, Guy; Lefebvre, Tony

    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 to 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.

  5. The Biochemistry of O-GlcNAc Transferase: Which Functions Make It Essential in Mammalian Cells?

    PubMed

    Levine, Zebulon G; Walker, Suzanne

    2016-06-01

    O-linked N-acetylglucosamine transferase (OGT) is found in all metazoans and plays an important role in development but at the single-cell level is only essential in dividing mammalian cells. Postmitotic mammalian cells and cells of invertebrates such as Caenorhabditis elegans and Drosophila can survive without copies of OGT. Why OGT is required in dividing mammalian cells but not in other cells remains unknown. OGT has multiple biochemical activities. Beyond its well-known role in adding β-O-GlcNAc to serine and threonine residues of nuclear and cytoplasmic proteins, OGT also acts as a protease in the maturation of the cell cycle regulator host cell factor 1 (HCF-1) and serves as an integral member of several protein complexes, many of them linked to gene expression. In this review, we summarize current understanding of the mechanisms underlying OGT's biochemical activities and address whether known functions of OGT could be related to its essential role in dividing mammalian cells. PMID:27294441

  6. Ataxin-10 interacts with O-GlcNAc transferase OGT in pancreatic {beta} cells

    SciTech Connect

    Andrali, Sreenath S.; Maerz, Pia; Oezcan, Sabire . E-mail: sozcan@uky.edu

    2005-11-11

    Several nuclear and cytoplasmic proteins in metazoans are modified by O-linked N-acetylglucosamine (O-GlcNAc). This modification is dynamic and reversible similar to phosphorylation and is catalyzed by the O-linked GlcNAc transferase (OGT). Hyperglycemia has been shown to increase O-GlcNAc levels in pancreatic {beta} cells, which appears to interfere with {beta}-cell function. To obtain a better understanding of the role of O-linked GlcNAc modification in {beta} cells, we have isolated OGT interacting proteins from a cDNA library made from the mouse insulinoma MIN6 cell line. We describe here the identification of Ataxin-10, encoded by the SCA10 (spinocerebellar ataxia type 10) gene as an OGT interacting protein. Mutations in the SCA10 gene cause progressive cerebellar ataxias and seizures. We demonstrate that SCA10 interacts with OGT in vivo and is modified by O-linked glycosylation in MIN6 cells, suggesting a novel role for the Ataxin-10 protein in pancreatic {beta} cells.

  7. Inhibition of O-GlcNAc transferase activity reprograms prostate cancer cell metabolism.

    PubMed

    Itkonen, Harri M; Gorad, Saurabh S; Duveau, Damien Y; Martin, Sara E S; Barkovskaya, Anna; Bathen, Tone F; Moestue, Siver A; Mills, Ian G

    2016-03-15

    Metabolic networks are highly connected and complex, but a single enzyme, O-GlcNAc transferase (OGT) can sense the availability of metabolites and also modify target proteins. We show that inhibition of OGT activity inhibits the proliferation of prostate cancer cells, leads to sustained loss of c-MYC and suppresses the expression of CDK1, elevated expression of which predicts prostate cancer recurrence (p=0.00179). Metabolic profiling revealed decreased glucose consumption and lactate production after OGT inhibition. This decreased glycolytic activity specifically sensitized prostate cancer cells, but not cells representing normal prostate epithelium, to inhibitors of oxidative phosphorylation (rotenone and metformin). Intra-cellular alanine was depleted upon OGT inhibitor treatment. OGT inhibitor increased the expression and activity of alanine aminotransferase (GPT2), an enzyme that can be targeted with a clinically approved drug, cycloserine. Simultaneous inhibition of OGT and GPT2 inhibited cell viability and growth rate, and additionally activated a cell death response. These combinatorial effects were predominantly seen in prostate cancer cells, but not in a cell-line derived from normal prostate epithelium. Combinatorial treatments were confirmed with two inhibitors against both OGT and GPT2. Taken together, here we report the reprogramming of energy metabolism upon inhibition of OGT activity, and identify synergistically lethal combinations that are prostate cancer cell specific. PMID:26824323

  8. Glutathione S-transferase polymorphisms in varicocele patients: a meta-analysis.

    PubMed

    Zhu, B; Yin, L; Zhang, J Y

    2015-01-01

    The glutathione S-transferase (GST) family represents a major group of detoxification and antioxidant enzymes. Studies have shown that high oxidative stress levels are associated with varicocele. The objective of this study was to assess the relationship between GSTM1 and GSTT1 null polymorphisms and varicocele using a study group of 497 varicocele patients and 476 control subjects. A systematic literature search (for articles published up to September 2014) utilizing Google Scholar and PubMed was conducted. The chi-square-based Q test and I(2) index were used to evaluate data from retrieved studies. The possible publication bias was evaluated by Begg funnel plot and the Egger test. No statistically significant association was found between GSTM1 or GSTT1 null genotypes and varicocele in the overall data analysis. In a subgroup analysis, only the null GSTM1 genotype was observed at a significantly higher frequency in Caucasian varicocele patients. In the Chinese subgroup, no association was established between the GSTM1 and GSTT1 null genotypes and this condition. More attention should be drawn to oxidative stress-related pathological manifestations for Caucasian varicocele patients. PMID:26782535

  9. Protein S-ACYL Transferase10 is critical for development and salt tolerance in Arabidopsis.

    PubMed

    Zhou, Liang-Zi; Li, Sha; Feng, Qiang-Nan; Zhang, Yu-Ling; Zhao, Xinying; Zeng, Yong-lun; Wang, Hao; Jiang, Liwen; Zhang, Yan

    2013-03-01

    Protein S-acylation, commonly known as palmitoylation, is a reversible posttranslational modification that catalyzes the addition of a saturated lipid group, often palmitate, to the sulfhydryl group of a Cys. Palmitoylation regulates enzyme activity, protein stability, subcellular localization, and intracellular sorting. Many plant proteins are palmitoylated. However, little is known about protein S-acyl transferases (PATs), which catalyze palmitoylation. Here, we report that the tonoplast-localized PAT10 is critical for development and salt tolerance in Arabidopsis thaliana. PAT10 loss of function resulted in pleiotropic growth defects, including smaller leaves, dwarfism, and sterility. In addition, pat10 mutants are hypersensitive to salt stresses. We further show that PAT10 regulates the tonoplast localization of several calcineurin B-like proteins (CBLs), including CBL2, CBL3, and CBL6, whose membrane association also depends on palmitoylation. Introducing a C192S mutation within the highly conserved catalytic motif of PAT10 failed to complement pat10 mutants, indicating that PAT10 functions through protein palmitoylation. We propose that PAT10-mediated palmitoylation is critical for vacuolar function by regulating membrane association or the activities of tonoplast proteins. PMID:23482856

  10. Functional Analyses of Mycobacterial Lipoprotein Diacylglyceryl Transferase and Comparative Secretome Analysis of a Mycobacterial lgt Mutant

    PubMed Central

    Tschumi, Andreas; Grau, Thomas; Albrecht, Dirk; Rezwan, Mandana; Antelmann, Haike

    2012-01-01

    Preprolipopoprotein diacylglyceryl transferase (Lgt) is the gating enzyme of lipoprotein biosynthesis, and it attaches a lipid structure to the N-terminal part of preprolipoproteins. Using Lgt from Escherichia coli in a BLASTp search, we identified the corresponding Lgt homologue in Mycobacterium tuberculosis and two homologous (MSMEG_3222 and MSMEG_5408) Lgt in Mycobacterium smegmatis. M. tuberculosis lgt was shown to be essential, but an M. smegmatis ΔMSMEG_3222 mutant could be generated. Using Triton X-114 phase separation and [14C]palmitic acid incorporation, we demonstrate that MSMEG_3222 is the major Lgt in M. smegmatis. Recombinant M. tuberculosis lipoproteins Mpt83 and LppX are shown to be localized in the cell envelope of parental M. smegmatis but were absent from the cell membrane and cell wall in the M. smegmatis ΔMSMEG_3222 strain. In a proteomic study, 106 proteins were identified and quantified in the secretome of wild-type M. smegmatis, including 20 lipoproteins. All lipoproteins were secreted at higher levels in the ΔMSMEG_3222 mutant. We identify the major Lgt in M. smegmatis, show that lipoproteins lacking the lipid anchor are secreted into the culture filtrate, and demonstrate that M. tuberculosis lgt is essential and thus a validated drug target. PMID:22609911

  11. Fluorometric microplate assay to measure glutathione S-transferase activity in insects and mites using monochlorobimane.

    PubMed

    Nauen, Ralf; Stumpf, Natascha

    2002-04-15

    Elevated levels of glutathione S-transferases (GSTs) play a major role as a mechanism of resistance to insecticides and acaricides in resistant pest insects and mites, respectively. Such compounds are either detoxicated directly via phase I metabolism or detoxicated by phase II metabolism of metabolites as formed by microsomal monooxygenases. Here we used monochlorobimane (MCB) as an artificial substrate and glutathione to determine total GST activity in equivalents of single pest insects and spider mites in a sensitive 96-well plate-based assay system by measuring the enzymatic conversion of MCB to its fluorescent bimane-glutathione adduct. The differentiation by their GST activity between several strains of the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae), with different degrees of resistance to numerous acaricides was more sensitive with MCB compared to the commonly used substrate 1-chloro-2,4-dinitrobenzene (CDNB). Compared to an acaricide-susceptible reference strain, one field population of T. urticae showed a more than 10-fold higher GST activity measured with MCB, in contrast to a less than 2-fold higher activity when CDNB was used. Furthermore, we showed that GST activity can be sensitively assessed with MCB in homogenates of pest insects such as Heliothis virescens, Spodoptera frugiperda (Lepidoptera: Noctuidae), Plutella xylostella (Lepidoptera: Yponomeutidae), and Myzus persicae (Hemiptera: Aphididae). PMID:11950219

  12. Identification of terminal adenylyl transferase activity of the poliovirus polymerase 3Dpol.

    PubMed Central

    Neufeld, K L; Galarza, J M; Richards, O C; Summers, D F; Ehrenfeld, E

    1994-01-01

    A terminal adenylyl transferase (TATase) activity has been identified in preparations of purified poliovirus RNA-dependent RNA polymerase (3Dpol). Highly purified 3Dpol is capable of adding [32P]AMP to the 3' ends of chemically synthesized 12-nucleotide (nt)-long RNAs. The purified 52-kDa polypeptide, isolated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renatured, retained the TATase activity. Two 3Dpol mutants, purified from Escherichia coli expression systems, displayed no detectable polymerase activity and were unable to catalyze TATase activity. Likewise, extracts from the parental E. coli strain that harbored no expression plasmid were unable to catalyze formation of the TATase products. With the RNA oligonucleotide 5'-CCUGCUUUUGCA-3' used as an acceptor, the products formed by wild-type 3Dpol were 9 and 18 nt longer than the 12-nt oligomer. GTP, CTP, and UTP did not serve as substrates for transfer to this RNA, either by themselves or when all deoxynucleoside triphosphates were present in the reaction. Results from kinetic and stoichiometric analyses suggest that the reaction is catalytic and shows substrate and enzyme dependence. The 3'-terminal 13 nt of poliovirus minus-strand RNA also served as an acceptor for TATase activity, raising the possibility that this activity functions in poliovirus RNA replication. The efficiency of utilization and the nature of the products formed during the reaction were dependent on the acceptor RNA. Images PMID:8057462

  13. Staphylococcus aureus formyl-methionyl transferase mutants demonstrate reduced virulence factor production and pathogenicity.

    PubMed

    Lewandowski, Thomas; Huang, Jianzhong; Fan, Frank; Rogers, Shannon; Gentry, Daniel; Holland, Reannon; Demarsh, Peter; Aubart, Kelly; Zalacain, Magdalena

    2013-07-01

    Inhibitors of peptide deformylase (PDF) represent a new class of antibacterial agents with a novel mechanism of action. Mutations that inactivate formyl methionyl transferase (FMT), the enzyme that formylates initiator methionyl-tRNA, lead to an alternative initiation of protein synthesis that does not require deformylation and are the predominant cause of resistance to PDF inhibitors in Staphylococcus aureus. Here, we report that loss-of-function mutations in FMT impart pleiotropic effects that include a reduced growth rate, a nonhemolytic phenotype, and a drastic reduction in production of multiple extracellular proteins, including key virulence factors, such as α-hemolysin and Panton-Valentine leukocidin (PVL), that have been associated with S. aureus pathogenicity. Consequently, S. aureus FMT mutants are greatly attenuated in neutropenic and nonneutropenic murine pyelonephritis infection models and show very high survival rates compared with wild-type S. aureus. These newly discovered effects on extracellular virulence factor production demonstrate that FMT-null mutants have a more severe fitness cost than previously anticipated, leading to a substantial loss of pathogenicity and a restricted ability to produce an invasive infection. PMID:23571548

  14. Glutathione S-transferases of Aulacorthum solani and Acyrthosiphon pisum: partial purification and characterization.

    PubMed

    Francis, F; Haubruge, E; Gaspar, C; Dierickx, P J

    2001-05-01

    Glutathione S-transferases (GST) play an important role in the detoxification of many substances including allelochemicals from plants. Brassicaceae plants contain glucosinolates and emit volatile isothiocyanates which affect the GST system. A comparison of the GST of two aphid species, the generalist Aulacorthum solani found on Brassicaceae and the Fabaceae specialist Acyrthosiphon pisum, was made to try to explain their respective feeding behaviour. Differences of GST were determined among the two aphid species based on purification by affinity chromatography, SDS-PAGE and on kinetic studies. Purification yields using an epoxy-activated Sepharose 6B column were highly different for the two aphid species (18% and 34% for A. solani and A. pisum, respectively). These variations were confirmed by SDS-PAGE. While only a 27-kDa band was observed for A. pisum, two bands of approximately 25-kDa were visualized for the generalist aphid, A. solani. Considering the kinetic results, differences of Km and Vmax were observed following the aphid species when a range of substrates (CDNB and DCNB) and GSH concentrations were tested. Studies on the detoxification enzymes of generalist and specialist herbivores would be undertaken to determine accurately the effect of the host plant on the organisms eating them, particularly in terms of biochemical and ecological advantages. PMID:11337260

  15. Use of heterologously-expressed cytochrome P450 and glutathione transferase enzymes in toxicity assays.

    PubMed

    Guengerich, F Peter; Wheeler, James B; Chun, Young-Jin; Kim, Donghak; Shimada, Tsutomu; Aryal, Pramod; Oda, Yoshimitsu; Gillam, Elizabeth M J

    2002-12-27

    Our groups have had a long-term interest in utilizing bacterial systems in the characterization of bioactivation and detoxication reactions catalyzed by cytochrome P450 (P450) and glutathione transferase (GST) enzymes. Bacterial systems remain the first choice for initial screens with new chemicals and have advantages, including high-throughput capability. Most human P450s of interest in toxicology have been readily expressed in Escherichia coli with only minor sequence modification. These enzymes can be readily purified and used in assays of activation of chemicals. Bicistronic systems have been developed in order to provide the auxiliary NADPH-P450 reductase. Alternative systems involve these enzymes expressed together within bacteria. In one approach, a lac selection system is used with E. coli and has been applied to the characterization of inhibitors of P450s 1A2 and 1B1, as well as in basic studies involving random mutagenesis. Another approach utilizes induction of the SOS (umu) response in Salmonella typhimurium, and systems have now been developed with human P450s 1A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4, which have been used to report responses from heterocyclic amines. S. typhimurium his reporter systems have also been used with GSTs, first to demonstrate the role of rat GST 5-5 in the activation of dihalomethanes. These systems have been used to compare these GSTs with regard to activation of dihaloalkanes and potential toxicity. PMID:12505322

  16. Optical biosensor consisting of glutathione-S-transferase for detection of captan.

    PubMed

    Choi, Jeong-Woo; Kim, Young-Kee; Song, Sun-Young; Lee, In-ho; Lee, Won-Hong

    2003-10-15

    The optical biosensor consisting of a glutathione-S-transferase (GST)-immobilized gel film was developed to detect captan in contaminated water. The sensing scheme was based on the decrease of yellow product, s-(2,4-dinitrobenzene) glutathione, produced from substrates, 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH), due to the inhibition of GST reaction by captan. Absorbance of the product as the output of enzyme reaction was detected and the light was guided through the optical fibers. The enzyme reactor of the sensor system was fabricated by the gel entrapment technique for the immobilized GST film. The immobilized GST had the maximum activity at pH 6.5. The optimal concentrations of substrates were determined with 1 mM for both of CDNB and GSH. The optimum concentration of enzyme was also determined with 100 microg/ml. The activity of immobilized enzyme was fairly sustained during 30 days. The proposed biosensor could successfully detect the captan up to 2 ppm and the response time to steady signal was about 15 min. PMID:12941561

  17. Glutathione-S-Transferases As Determinants of Cell Survival and Death

    PubMed Central

    Townsend, Danyelle M.

    2012-01-01

    Abstract Significance: The family of glutathione S-transferases (GSTs) is part of a cellular Phase II detoxification program composed of multiple isozymes with functional human polymorphisms that have the capacity to influence individual response to drugs and environmental stresses. Catalytic activity is expressed through GST dimer-mediated thioether conjugate formation with resultant detoxification of a variety of small molecule electrophiles. Recent Advances: More recent work indicates that in addition to the classic catalytic functions, specific GST isozymes have other characteristics that impact cell survival pathways in ways unrelated to detoxification. These characteristics include the following: regulation of mitogen-activated protein kinases; facilitation of the addition of glutathione to cysteine residues in certain proteins (S-glutathionylation); as a novel cellular partner of the human papilloma virus-16 E7 oncoprotein playing a pivotal role in preventing cell death in infected human cells; mitogenic influence in myeloproliferative pathways; participant in the process of cocaine addiction. Critical Issues: Some of these functions have provided a platform for targeting GST with novel small molecule therapeutics, particularly in cancer where evidence of clinical applications is emerging. Future Directions: Our evolving understanding of the GST superfamily and their divergent expression patterns in individuals make them attractive candidates for translational studies in a variety of human pathologies. In addition, their role in regulating cell fate in signaling and cell death pathways has opened up a significant functional complexity that extends well beyond standard detoxification reactions. Antioxid. Redox Signal. 17, 1728–1737. PMID:22540427

  18. Association between herbivore stress and glutathione S-transferase expression in Pinus brutia Ten.

    PubMed

    Semiz, A; Çelik-Turgut, G; Semiz, G; Özgün, Ö; Şen, A

    2016-01-01

    Plants have developed mechanisms to defend themselves against many factors including biotic stress such as herbivores and pathogens. Glutathione S-transferase (GST) is a glutathione-dependent detoxifying enzyme and plays critical roles in stress tolerance and detoxification metabolism in plants. Pinus brutia Ten. is a prominent native forest tree species in Turkey, due to both its economic and ecological assets. One of the problems faced by P. brutia afforestation sites is the attacks by pine processionary moth (Thaumetopoea wilkinsoni Tams.). In this study, we investigated the changes in activity and mRNA expression of GST in pine samples taken from both resistant and susceptible clones against T. wilkinsoni over a nine month period in a clonal seed orchard. It was found that the average cytosolic GST activities of trees in March and July were significantly higher than the values obtained in November. November was considered to be the control since trees were not under stress yet. In addition, RT-PCR results clearly showed that levels of GST transcripts in March and July samples were significantly higher as compared to the level seen in November. These findings strongly suggest that GST activity from P. brutia would be a valuable marker for exposure to herbivory stress. PMID:27064879

  19. Staphylococcus aureus Formyl-Methionyl Transferase Mutants Demonstrate Reduced Virulence Factor Production and Pathogenicity

    PubMed Central

    Lewandowski, Thomas; Huang, Jianzhong; Fan, Frank; Rogers, Shannon; Gentry, Daniel; Holland, Reannon; DeMarsh, Peter; Zalacain, Magdalena

    2013-01-01

    Inhibitors of peptide deformylase (PDF) represent a new class of antibacterial agents with a novel mechanism of action. Mutations that inactivate formyl methionyl transferase (FMT), the enzyme that formylates initiator methionyl-tRNA, lead to an alternative initiation of protein synthesis that does not require deformylation and are the predominant cause of resistance to PDF inhibitors in Staphylococcus aureus. Here, we report that loss-of-function mutations in FMT impart pleiotropic effects that include a reduced growth rate, a nonhemolytic phenotype, and a drastic reduction in production of multiple extracellular proteins, including key virulence factors, such as α-hemolysin and Panton-Valentine leukocidin (PVL), that have been associated with S. aureus pathogenicity. Consequently, S. aureus FMT mutants are greatly attenuated in neutropenic and nonneutropenic murine pyelonephritis infection models and show very high survival rates compared with wild-type S. aureus. These newly discovered effects on extracellular virulence factor production demonstrate that FMT-null mutants have a more severe fitness cost than previously anticipated, leading to a substantial loss of pathogenicity and a restricted ability to produce an invasive infection. PMID:23571548

  20. Role of glutathione S-transferases in the spinocerebellar ataxia type 2 clinical phenotype.

    PubMed

    Almaguer-Gotay, D; Almaguer-Mederos, L E; Aguilera-Rodríguez, R; Estupiñán-Rodríguez, A; González-Zaldivar, Y; Cuello-Almarales, D; Laffita-Mesa, J M; Vázquez-Mojena, Y

    2014-06-15

    Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative and incurable hereditary disorder caused by a CAG repeat expansion mutation on ATXN2 gene. The identification of reliable biochemical markers of disease severity is of paramount significance for the development and assessment of clinical trials. In order to evaluate the potential use of glutathione-S-transferase (GST) activity as a biomarker for SCA2, a case-control study in 38 affected, presymptomatic individuals or healthy controls was conducted. An enlarged sample of 121 affected individuals was set to assess the impact of GST activity on SCA2 clinical expression. There was a significant increase in GST activity in affected individuals relative to controls, although sensibility and specificity were not high. GST activity was not significantly influenced by sex, age, disease duration or CAG repeat size and did not significantly influence disease severity markers. These findings show a disruption of in vivo GST activity in SCA2, suggesting a role for oxidative stress in the neurodegenerative process. PMID:24780439

  1. The Impact of Nitric Oxide Toxicity on the Evolution of the Glutathione Transferase Superfamily

    PubMed Central

    Bocedi, Alessio; Fabrini, Raffaele; Farrotti, Andrea; Stella, Lorenzo; Ketterman, Albert J.; Pedersen, Jens Z.; Allocati, Nerino; Lau, Peter C. K.; Grosse, Stephan; Eltis, Lindsay D.; Ruzzini, Antonio; Edwards, Thomas E.; Morici, Laura; Del Grosso, Erica; Guidoni, Leonardo; Bovi, Daniele; Lo Bello, Mario; Federici, Giorgio; Parker, Michael W.; Board, Philip G.; Ricci, Giorgio

    2013-01-01

    Glutathione transferases (GSTs) are protection enzymes capable of conjugating glutathione (GSH) to toxic compounds. During evolution an important catalytic cysteine residue involved in GSH activation was replaced by serine or, more recently, by tyrosine. The utility of these replacements represents an enigma because they yield no improvements in the affinity toward GSH or in its reactivity. Here we show that these changes better protect the cell from nitric oxide (NO) insults. In fact the dinitrosyl·diglutathionyl·iron complex (DNDGIC), which is formed spontaneously when NO enters the cell, is highly toxic when free in solution but completely harmless when bound to GSTs. By examining 42 different GSTs we discovered that only the more recently evolved Tyr-based GSTs display enough affinity for DNDGIC (KD < 10−9 m) to sequester the complex efficiently. Ser-based GSTs and Cys-based GSTs show affinities 102–104 times lower, not sufficient for this purpose. The NO sensitivity of bacteria that express only Cys-based GSTs could be related to the low or null affinity of their GSTs for DNDGIC. GSTs with the highest affinity (Tyr-based GSTs) are also over-represented in the perinuclear region of mammalian cells, possibly for nucleus protection. On the basis of these results we propose that GST evolution in higher organisms could be linked to the defense against NO. PMID:23828197

  2. The HTLV-1-encoded protein HBZ directly inhibits the acetyl transferase activity of p300/CBP

    PubMed Central

    Wurm, Torsten; Wright, Diana G.; Polakowski, Nicholas; Mesnard, Jean-Michel; Lemasson, Isabelle

    2012-01-01

    The homologous cellular coactivators p300 and CBP contain intrinsic lysine acetyl transferase (termed HAT) activity. This activity is responsible for acetylation of several sites on the histones as well as modification of transcription factors. In a previous study, we found that HBZ, encoded by the Human T-cell Leukemia Virus type 1 (HTLV-1), binds to multiple domains of p300/CBP, including the HAT domain. In this study, we found that HBZ inhibits the HAT activity of p300/CBP through the bZIP domain of the viral protein. This effect correlated with a reduction of H3K18 acetylation, a specific target of p300/CBP, in cells expressing HBZ. Interestingly, lower levels of H3K18 acetylation were detected in HTLV-1 infected cells compared to non-infected cells. The inhibitory effect of HBZ was not limited to histones, as HBZ also inhibited acetylation of the NF-κB subunit, p65, and the tumor suppressor, p53. Recent studies reported that mutations in the HAT domain of p300/CBP that cause a defect in acetylation are found in certain types of leukemia. These observations suggest that inhibition of the HAT activity by HBZ is important for the development of adult T-cell leukemia associated with HTLV-1 infection. PMID:22434882

  3. The active site of O-GlcNAc transferase imposes constraints on substrate sequence

    PubMed Central

    Rafie, Karim; Blair, David E.; Borodkin, Vladimir S.; Albarbarawi, Osama; van Aalten, Daan M. F.

    2016-01-01

    O-GlcNAc transferase (OGT) glycosylates a diverse range of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc), an essential and dynamic post-translational modification in metazoa. Although this enzyme modifies hundreds of proteins with O-GlcNAc, it is not understood how OGT achieves substrate specificity. In this study, we describe the application of a high-throughput OGT assay on a library of peptides. The sites of O-GlcNAc modification were mapped by ETD-mass spectrometry, and found to correlate with previously detected O-GlcNAc sites. Crystal structures of four acceptor peptides in complex with human OGT suggest that a combination of size and conformational restriction defines sequence specificity in the −3 to +2 subsites. This work reveals that while the N-terminal TPR repeats of hOGT may play a role in substrate recognition, the sequence restriction imposed by the peptide-binding site makes a significant contribution to O-GlcNAc site specificity. PMID:26237509

  4. Association study of Glutathione S-Transferase polymorphisms and risk of endometriosis in an Iranian population

    PubMed Central

    Hassani, Mina; Saliminejad, Kioomars; Heidarizadeh, Masood; Kamali, Koorosh; Memariani, Toktam; Khorram Khorshid, Hamid Reza

    2016-01-01

    Background: Endometriosis influenced by both genetic and environmental factors. Associations of glutathione S-transferases (GSTs) genes polymorphisms in endometriosis have been investigated by various researchers; however, the results are not consistent. Objective: We examined the associations of GSTM1 and GSTT1 null genotypes and GSTP1 313 A/G polymorphisms with endometriosis in an Iranian population. Materials and Methods: In this case-control study, 151 women with diagnosis of endometriosis and 156 normal healthy women as control group were included. The genotyping was determined using multiplex PCR and PCR- RFLP methods. Results: The GSTM1 null genotype was significantly higher (p=0.027) in the cases (7.3%) than the control group (1.3%). There was no significant difference between the frequency of GSTT1 genotypes between the cases and controls. The GSTP1 313 AG genotype was significantly lower (p=0.048) in the case (33.1%) than the control group (44.4%). Conclusion: Our results showed that GSTM1 and GSTP1 polymorphisms may be associated with susceptibility of endometriosis in Iranian women. PMID:27351025

  5. Dual functionality of O-GlcNAc transferase is required for Drosophila development

    PubMed Central

    Mariappa, Daniel; Zheng, Xiaowei; Schimpl, Marianne; Raimi, Olawale; Ferenbach, Andrew T.; Müller, H.-Arno J.; van Aalten, Daan M. F.

    2015-01-01

    Post-translational modification of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc) catalysed by O-GlcNAc transferase (OGT) has been linked to regulation of diverse cellular functions. OGT possesses a C-terminal glycosyltransferase catalytic domain and N-terminal tetratricopeptide repeats that are implicated in protein–protein interactions. Drosophila OGT (DmOGT) is encoded by super sex combs (sxc), mutants of which are pupal lethal. However, it is not clear if this phenotype is caused by reduction of O-GlcNAcylation. Here we use a genetic approach to demonstrate that post-pupal Drosophila development can proceed with negligible OGT catalysis, while early embryonic development is OGT activity-dependent. Structural and enzymatic comparison between human OGT (hOGT) and DmOGT informed the rational design of DmOGT point mutants with a range of reduced catalytic activities. Strikingly, a severely hypomorphic OGT mutant complements sxc pupal lethality. However, the hypomorphic OGT mutant-rescued progeny do not produce F2 adults, because a set of Hox genes is de-repressed in F2 embryos, resulting in homeotic phenotypes. Thus, OGT catalytic activity is required up to late pupal stages, while further development proceeds with severely reduced OGT activity. PMID:26674417

  6. Structure, function and disease relevance of Omega-class glutathione transferases.

    PubMed

    Board, Philip G; Menon, Deepthi

    2016-05-01

    The Omega-class cytosolic glutathione transferases (GSTs) have distinct structural and functional attributes that allow them to perform novel roles unrelated to the functions of other GSTs. Mammalian GSTO1-1 has been found to play a previously unappreciated role in the glutathionylation cycle that is emerging as significant mechanism regulating protein function. GSTO1-1-catalyzed glutathionylation or deglutathionylation of a key signaling protein may explain the requirement for catalytically active GSTO1-1 in LPS-stimulated pro-inflammatory signaling through the TLR4 receptor. The observation that ML175 a specific GSTO1-1 inhibitor can block LPS-stimulated inflammatory signaling has opened a new avenue for the development of novel anti-inflammatory drugs that could be useful in the treatment of toxic shock and other inflammatory disorders. The role of GSTO2-2 remains unclear. As a dehydroascorbate reductase, it could contribute to the maintenance of cellular redox balance and it is interesting to note that the GSTO2 N142D polymorphism has been associated with multiple diseases including Alzheimer's disease, Parkinson's disease, familial amyotrophic lateral sclerosis, chronic obstructive pulmonary disease, age-related cataract and breast cancer. PMID:26993125

  7. Glutathione-S-transferase in Nereis succinea (Polychaeta) and its induction by xeno-estrogen.

    PubMed

    Ayoola, James A O; García-Alonso, Javier; Hardege, Jörg D

    2011-10-01

    The need to replace or at least to reduce the use of vertebrates in toxicity tests is a timely major concern in research and industry but to date, efforts made to minimize their use are still far from complete. Increasing demands for toxicity tests put considerable pressures upon the development of future fast and efficient test methods using invertebrates. In fact, to date, few studies provide links between biochemical and cellular effects of xeno-estrogens in aquatic invertebrates. Glutathione-S-transferase (GST) activity, as a biomarker of stress exposure, was measured in the population of clamworms (Nereis succinea) from Cardiff Bay. In addition, we examined the effect of single exposure to nonylphenol (NP) on this enzymatic activity. Field study results showed a relationship between the worm's size, reproductive status, and GST activity from the field population. In addition, we show a significant increase in the GST activity at 100 μg/L NP with sex-specific responses. The xeno-estrogens, which could affect reproduction of nereid by interfering in normal endocrinological pathways, are eliminated through GST by conjugation with glutathione. This work shows for the first time that GST activity depends on sex and stage of the clamworms and also that the xeno-estrogen NP induces its activity. This study supports the use of this species as a bioindicator of aquatic pollution and lays the foundation to causally link toxic exposure with reproductive output. PMID:20549611

  8. 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

  9. Solution Structural Studies of GTP:Adenosylcobinamide-Phosphateguanylyl Transferase (CobY) from Methanocaldococcus jannaschii

    PubMed Central

    Singarapu, Kiran K.; Otte, Michele M.; Tonelli, Marco; Westler, William M.; Escalante-Semerena, Jorge C.; Markley, John L.

    2015-01-01

    GTP:adenosylcobinamide-phosphate (AdoCbi-P) guanylyl transferase (CobY) is an enzyme that transfers the GMP moiety of GTP to AdoCbi yielding AdoCbi-GDP in the late steps of the assembly of Ado-cobamides in archaea. The failure of repeated attempts to crystallize ligand-free (apo) CobY prompted us to explore its 3D structure by solution NMR spectroscopy. As reported here, the solution structure has a mixed α/β fold consisting of seven β-strands and five α-helices, which is very similar to a Rossmann fold. Titration of apo-CobY with GTP resulted in large changes in amide proton chemical shifts that indicated major structural perturbations upon complex formation. However, the CobY:GTP complex as followed by 1H-15N HSQC spectra was found to be unstable over time: GTP hydrolyzed and the protein converted slowly to a species with an NMR spectrum similar to that of apo-CobY. The variant CobYG153D, whose GTP complex was studied by X-ray crystallography, yielded NMR spectra similar to those of wild-type CobY in both its apo- state and in complex with GTP. The CobYG153D:GTP complex was also found to be unstable over time. PMID:26513744

  10. 3-Methyleneoxindole: an affinity label of glutathione S-transferase pi which targets tryptophan 38.

    PubMed

    Pettigrew, N E; Brush, E J; Colman, R F

    2001-06-26

    The compound 3-methyleneoxindole (MOI), a photooxidation product of the plant auxin indole-3-acetic acid, functions as an affinity label of the dimeric pi class glutathione S-transferase (GST) isolated from pig lung. MOI inactivates the enzyme to a limit of 14% activity. The k for inactivation by MOI is decreased 20-fold by S-hexylglutathione but only 2-fold by S-methylglutathione, suggesting that MOI does not react entirely within the glutathione site. The striking protection against inactivation provided by S-(hydroxyethyl)ethacrynic acid indicates that MOI reacts in the active site region involving both the glutathione and the xenobiotic substrate sites. Incorporation of [(3)H]MOI up to approximately 1 mol/mol of enzyme dimer concomitant with maximum inactivation suggests that there are interactions between subunits. Fractionation of the proteolytic digest of [(3)H]MOI-modified GST pi yielded Trp38 as the only labeled amino acid. The crystal structure of the human GST pi-ethacrynic acid complex (2GSS) shows that the indole of Trp38 is less than 4 A from ethacrynic acid. Similarly, MOI may bind in this substrate site. In contrast to its effect on the pi class GST, MOI inactivates much less rapidly and extensively alpha and mu class GSTs isolated from the rat. These results show that MOI reacts preferentially with GST pi. Such a compound may be useful in novel combination chemotherapy to enhance the efficacy of alkylating cancer drugs while minimizing toxic side effects. PMID:11412109

  11. Induction of Epoxide Hydrolase, Glucuronosyl Transferase, and Sulfotransferase by Phenethyl Isothiocyanate in Male Wistar Albino Rats

    PubMed Central

    Mohd Noor, Noramaliza; Konsue, Nattaya

    2014-01-01

    Phenethyl isothiocyanate (PEITC) is an isothiocyanate found in watercress as the glucosinolate (gluconasturtiin). The isothiocyanate is converted from the glucosinolate by intestinal microflora or when contacted with myrosinase during the chopping and mastication of the vegetable. PEITC manifested protection against chemically-induced cancers in various tissues. A potential mechanism of chemoprevention is by modulating the metabolism of carcinogens so as to promote deactivation. The principal objective of this study was to investigate in rats the effect of PEITC on carcinogen-metabolising enzyme systems such as sulfotransferase (SULT), N-acetyltransferase (NAT), glucuronosyl transferase (UDP), and epoxide hydrolase (EH) following exposure to low doses that simulate human dietary intake. Rats were fed for 2 weeks diets supplemented with PEITC at 0.06 µmol/g (low dose, i.e., dietary intake), 0.6 µmol/g (medium dose), and 6.0 µmol/g (high dose), and the enzymes were monitored in rat liver. At the Low dose, no induction of the SULT, NAT, and EH was noted, whereas UDP level was elevated. At the Medium dose, only SULT level was increased, whereas at the High dose marked increase in EH level was observed. It is concluded that PEITC modulates carcinogen-metabolising enzyme systems at doses reflecting human intake thus elucidating the mechanism of its chemoprevention. PMID:24592387

  12. Methionine sulfoxide reductase regulates brain catechol-O-methyl transferase activity.

    PubMed

    Moskovitz, Jackob; Walss-Bass, Consuelo; Cruz, Dianne A; Thompson, Peter M; Bortolato, Marco

    2014-10-01

    Catechol-O-methyl transferase (COMT) plays a key role in the degradation of brain dopamine (DA). Specifically, low COMT activity results in higher DA levels in the prefrontal cortex (PFC), thereby reducing the vulnerability for attentional and cognitive deficits in both psychotic and healthy individuals. COMT activity is markedly reduced by a non-synonymous single-nucleotide polymorphism (SNP) that generates a valine-to-methionine substitution on the residue 108/158, by means of as-yet incompletely understood post-translational mechanisms. One post-translational modification is methionine sulfoxide, which can be reduced by the methionine sulfoxide reductase (Msr) A and B enzymes. We used recombinant COMT proteins (Val/Met108) and mice (wild-type (WT) and MsrA knockout) to determine the effect of methionine oxidation on COMT activity and COMT interaction with Msr, through a combination of enzymatic activity and Western blot assays. Recombinant COMT activity is positively regulated by MsrA, especially under oxidative conditions, whereas brains of MsrA knockout mice exhibited lower COMT activity (as compared with their WT counterparts). These results suggest that COMT activity may be reduced by methionine oxidation, and point to Msr as a key molecular determinant for the modulation of COMT activity in the brain. The role of Msr in modulating cognitive functions in healthy individuals and schizophrenia patients is yet to be determined. PMID:24735585

  13. Activated Drosophila Ras1 is selectively suppressed by isoprenyl transferase inhibitors.

    PubMed Central

    Kauffmann, R C; Qian, Y; Vogt, A; Sebti, S M; Hamilton, A D; Carthew, R W

    1995-01-01

    Ras CAAX (C = cysteine, A = aliphatic amino acid, and X = any amino acid) peptidomimetic inhibitors of farnesyl protein transferase suppress Ras-dependent cell transformation by preventing farnesylation of the Ras oncoprotein. These compounds are potential anticancer agents for tumors associated with Ras mutations. The peptidomimetic FTI-254 was tested for Ras1-inhibiting activity in whole animals by injection of activated Ras1val12 Drosophila larvae. FTI-254 decreased the ability of Ras1val12 to form supernumerary R7 photoreceptor cells in the compound eye of transformed flies. In contrast, it had no effect on the related supernumerary R7 phenotypes of flies transformed with either the activated sevenless receptor tyrosine kinase, Raf kinase, or a chimeric Ras1val12 protein that is membrane associated through myristylation instead of isoprenylation. Therefore, FTI-254 acts as an isoprenylation inhibitor to selectively inhibit Ras1val12 signaling activity in a whole-animal model system. Images Fig. 2 PMID:7479910

  14. Glutathione S-transferase K1 genotype and overweight status in schizophrenia patients: A pilot study.

    PubMed

    Oniki, Kentaro; Kamihashi, Ryoko; Tomita, Tetsu; Ishioka, Masamichi; Yoshimori, Yuki; Osaki, Natsumi; Tsuchimine, Shoko; Sugawara, Norio; Kajiwara, Ayami; Morita, Kazunori; Miyata, Keishi; Otake, Koji; Nakagawa, Kazuko; Ogata, Yasuhiro; Saruwatari, Junji; Yasui-Furukori, Norio

    2016-05-30

    Elevated oxidative stress in mitochondria and mitochondrial dysfunction are associated with weight gain in schizophrenia (SCZ) patients. Glutathione S-transferase kappa 1 (GSTK1) protects cells against exogenous and endogenous oxidative stress in the mitochondria. This exploratory study investigated the possible effects of a common GSTK1 polymorphism (rs1917760, G-1308T) on the risk for overweight status among 329 SCZ patients and 305 age- and gender-matched controls and on the GSTK1 mRNA level in peripheral blood mononuclear cells among 14 SCZ patients. The GSTK1 T/T genotype was associated with having a higher BMI value among SCZ male patients, whereas this genotype tended to be associated with a lower BMI value among female patients. Conversely, these associations were not observed among the controls. The GSTK1 T/T genotype was associated with decreased GSTK1 mRNA level among SCZ patients. The GSTK1 T/T genotype may be a novel risk factor for the prediction of overweight status in SCZ male patients, although the results of this pilot study should be verified by a larger study. PMID:27010189

  15. Distinct and cooperative activities of HESO1 and URT1 nucleotidyl transferases in microRNA turnover in Arabidopsis.

    PubMed

    Tu, Bin; Liu, Li; Xu, Chi; Zhai, Jixian; Li, Shengben; Lopez, Miguel A; Zhao, Yuanyuan; Yu, Yu; Ramachandran, Vanitharani; Ren, Guodong; Yu, Bin; Li, Shigui; Meyers, Blake C; Mo, Beixin; Chen, Xuemei

    2015-04-01

    3' uridylation is increasingly recognized as a conserved RNA modification process associated with RNA turnover in eukaryotes. 2'-O-methylation on the 3' terminal ribose protects micro(mi)RNAs from 3' truncation and 3' uridylation in Arabidopsis. Previously, we identified HESO1 as the nucleotidyl transferase that uridylates most unmethylated miRNAs in vivo, but substantial 3' tailing of miRNAs still remains in heso1 loss-of-function mutants. In this study, we found that among nine other potential nucleotidyl transferases, UTP:RNA uridylyltransferase 1 (URT1) is the single most predominant nucleotidyl transferase that tails miRNAs. URT1 and HESO1 prefer substrates with different 3' end nucleotides in vitro and act cooperatively to tail different forms of the same miRNAs in vivo. Moreover, both HESO1 and URT1 exhibit nucleotidyl transferase activity on AGO1-bound miRNAs. Although these enzymes are able to add long tails to AGO1-bound miRNAs, the tailed miRNAs remain associated with AGO1. Moreover, tailing of AGO1-bound miRNA165/6 drastically reduces the slicing activity of AGO1-miR165/6, suggesting that tailing reduces miRNA activity. However, monouridylation of miR171a by URT1 endows the miRNA the ability to trigger the biogenesis of secondary siRNAs. Therefore, 3' tailing could affect the activities of miRNAs in addition to leading to miRNA degradation. PMID:25928405

  16. 23S rRNA nucleotides in the peptidyl transferase center are essential for tryptophanase operon induction.

    PubMed

    Yang, Rui; Cruz-Vera, Luis R; Yanofsky, Charles

    2009-06-01

    Distinct features of the ribosomal peptide exit tunnel are known to be essential for recognition of specific amino acids of a nascent peptidyl-tRNA. Thus, a tryptophan residue at position 12 of the peptidyl-tRNA TnaC-tRNA(Pro) leads to the creation of a free tryptophan binding site within the ribosome at which bound tryptophan inhibits normal ribosome functions. The ribosomal processes that are inhibited are hydrolysis of TnaC-tRNA(Pro) by release factor 2 and peptidyl transfer of TnaC of TnaC-tRNA(Pro) to puromycin. These events are normally performed in the ribosomal peptidyl transferase center. In the present study, changes of 23S rRNA nucleotides in the 2585 region of the peptidyl transferase center, G2583A and U2584C, were observed to reduce maximum induction of tna operon expression by tryptophan in vivo without affecting the concentration of tryptophan necessary to obtain 50% induction. The growth rate of strains with ribosomes with either of these changes was not altered appreciably. In vitro analyses with mutant ribosomes with these changes showed that tryptophan was not as efficient in protecting TnaC-tRNA(Pro) from puromycin action as wild-type ribosomes. However, added tryptophan did prevent sparsomycin action as it normally does with wild-type ribosomes. These findings suggest that these two mutational changes act by reducing the ability of ribosome-bound tryptophan to inhibit peptidyl transferase activity rather than by reducing the ability of the ribosome to bind tryptophan. Thus, the present study identifies specific nucleotides within the ribosomal peptidyl transferase center that appear to be essential for effective tryptophan induction of tna operon expression. PMID:19329641

  17. QUANTITATIVE IMAGE CYTOMETRY OF HEPATOCYTES EXPRESSING GAMMA-GLUTAMYL TRANSPEPTIDASE AND GLUTATHIONE S-TRANSFERASE IN DIETHYLNITROSAMINE-INITIATED RATS TREATED WITH PHENOBARBITAL AND/OR PHTHALATE ESTERS

    EPA Science Inventory

    Image cytometry was used to quantify the volume of liver tissue expressing two widely accepted biochemical markers of neoplasia, gammaglutamyl transpeptidase (GGT) and the placental isozyme of glutathione s-transferase (GST-P). ats were treated with hepatocarcinogen, diethylnitro...

  18. Identification of a hard surface contact-induced gene in Colletotrichum gloeosporioides conidia as a sterol glycosyl transferase, a novel fungal virulence factor.

    PubMed

    Kim, Yeon-Ki; Wang, Yuhuan; Liu, Zhi-Mei; Kolattukudy, Pappachan E

    2002-04-01

    Hard surface contact has been known to be necessary to induce infection structure (appressorium) formation in many phytopathogenic fungi. However, the molecular basis of this requirement is unknown. We have used a differential display approach to clone some of the genes induced in the conidia by hard surface contact. We report that one of the genes induced by hard-surface contact of the conidia of Colletotrichum gloeosporioides, chip6, encodes a protein with homology to sterol glycosyl transferases. chip6 expressed in E. coli catalyses glucosyl transfer from UDP-glucose to cholesterol. Disruption of chip6 causes a marked decrease in the transferase activity and a drastic reduction in virulence on its natural host, avocado fruits, although the mutant is capable of normal growth and appressorium formation. The requirement for sterol glycosyl transferase for pathogenicity suggests a novel biological function for this transferase. PMID:12000454

  19. 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...

  20. SIAH-mediated ubiquitination and degradation of acetyl-transferases regulate the p53 response and protein acetylation.

    PubMed

    Grishina, Inna; Debus, Katherina; García-Limones, Carmen; Schneider, Constanze; Shresta, Amit; García, Carlos; Calzado, Marco A; Schmitz, M Lienhard

    2012-12-01

    Posttranslational modification of proteins by lysine acetylation regulates many biological processes ranging from signal transduction to chromatin compaction. Here we identify the acetyl-transferases CBP/p300, Tip60 and PCAF as new substrates for the ubiquitin E3 ligases SIAH1 and SIAH2. While CBP/p300 can undergo ubiquitin/proteasome-dependent degradation by SIAH1 and SIAH2, the two other acetyl-transferases are exclusively degraded by SIAH2. Accordingly, SIAH-deficient cells show enhanced protein acetylation, thus revealing SIAH proteins as indirect regulators of the cellular acetylation status. Functional experiments show that Tip60/PCAF-mediated acetylation of the tumor suppressor p53 is antagonized by the p53 target gene SIAH2 which mediates ubiquitin/proteasome-mediated degradation of both acetyl-transferases and consequently diminishes p53 acetylation and transcriptional activity. The p53 kinase HIPK2 mediates hierarchical phosphorylation of SIAH2 at 5 sites, which further boosts its activity as a ubiquitin E3 ligase for several substrates and therefore dampens the late p53 response. PMID:23044042

  1. A shotgun lipidomics study of a putative lysophosphatidic acid acyl transferase (PlsC) in Sinorhizobium meliloti.

    PubMed

    Basconcillo, Libia Saborido; Zaheer, Rahat; Finan, Turlough M; McCarry, Brian E

    2009-09-15

    A shotgun lipidomics approach was used to study the knockout mutant of a putative lysophosphatidic acyl acid transferase (PlsC) in order to delineate the function of this enzyme in Sinorhizobium meliloti. In plsC knockout mutant lipids that contained 16:0 and 16:1 fatty acids and their biosynthetically related cyclopropane fatty acid (cis-9,10-methylene hexadecanoic acid) decreased up to 93%. Tandem mass spectrometry experiments in the presence of added Li(+) showed that the putative PlsC (SMc00714) functioned as a lysophosphatidic acid acyl transferase specific for the transfer of C16 fatty acids to the sn-2 position of lipids. The levels of lipids containing C18 fatty acids were unaffected in plsC mutant, suggesting the presence of one or more fatty acyl transferases in the genome of S. meliloti with selectivity towards C18 fatty acids. Two non-phosphorus containing lipid classes, sulfoquinovosyldiacylglycerol and 1,2-diacylglyceryl-trimethylhomoserine lipids, showed similar decreases in C16 fatty acid content as phospholipids in plsC knockout mutant; these non-phosphorus containing lipids share a common biosynthetic origin with phospholipids, most likely involving phosphatidic acid. Ornithine lipids containing C16 fatty acids also showed decreased levels in PlsC knockout mutant, suggesting that PlsC is also involved in their biosynthesis. PMID:19525157

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

    SciTech Connect

    Conklin, Daniel J.; Haberzettl, Petra; Jagatheesan, Ganapathy; Baba, Shahid; Merchant, Michael L.; Prough, Russell A.; Williams, Jessica D.; Prabhu, Sumanth D.; Bhatnagar, Aruni

    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 than 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 and

  3. Genomic cloning and characterization of the rat glutathione S-transferase-A3-subunit gene.

    PubMed Central

    Fotouhi-Ardakani, N; Batist, G

    1999-01-01

    The rat glutathione S-transferase-A3-subunit (GSTA3) gene is a member of the class Alpha GSTs, which we have previously reported to be overexpressed in anti-cancer-drug-resistant cells. In this study, we report the isolation and characterization of the entire rat GSTA3 (rGST Yc1) subunit gene. The rat GSTA3 subunit gene is approximately 15 kb in length and consists of seven exons interrupted by introns of different lengths. Exon 1, with a length of 219 bp, contains only the 5'-untranslated region of the gene. Each exon-intron splicing junction exhibited the consensus sequence for a mammalian splice site. The transcription start site and exon 1 of rat GSTA3 were characterized by a combination of primer extension and rapid amplification of the cDNA ends. Position +1 was identified 219 bp upstream of the first exon-intron splicing junction. The proximal promoter region of the rat GSTA3 subunit gene does not contain typical TATA or CAAT boxes. A computer-based search for potential transcription-factor binding sites revealed the existence of a number of motifs such as anti-oxidant-responsive element, ras-response element, activator protein-1, nuclear factor-kappaB, cAMP-response-element-binding protein, Barbie box and E box. The functional activity of the regulatory region of the rat GSTA3 subunit gene was shown by its ability to drive the expression of a chloramphenicol acetyltransferase reporter gene in rat mammary carcinoma cells, and its activity was greater in melphalan-resistant cells known to have transcriptional activation of this gene by previous studies. The structure of the gene, with a large intron upstream of the translation-initiation site, may explain why the isolation of this promoter has been so elusive. This information will provide the opportunity to examine the involvement of the rat GSTA3 subunit gene in drug resistance and carcinogenesis. PMID:10215608

  4. Identification of glutathione S-transferase genes responding to pathogen infestation in Populus tomentosa.

    PubMed

    Liao, Weihua; Ji, Lexiang; Wang, Jia; Chen, Zhong; Ye, Meixia; Ma, Huandi; An, Xinmin

    2014-09-01

    Stem blister canker, caused by Botryosphaeria dothidea, is becoming the most serious disease of poplar in China. The molecular basis of the poplar in response to stem blister canker is not well understood. To reveal the global transcriptional changes of poplar to infection by B. dothidea, Solexa paired-end sequencing of complementary DNAs (cDNAs) from control (NB) and pathogen-treated samples (WB) was performed, resulting in a total of 339,283 transcripts and 183,881 unigenes. A total of 206,586 transcripts were differentially expressed in response to pathogen stress (false discovery rate ≤0.05 and an absolute value of log2Ratio (NB/WB) ≥1). In enrichment analysis, energy metabolism and redox reaction-related macromolecules were accumulated significantly in Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analyses, indicating components of dynamic defense against the fungus. A total of 852 transcripts (575 upregulated and 277 downregulated transcripts) potentially involved in plant-pathogen interaction were also differentially regulated, including genes encoding proteins linked to signal transduction (putative leucine-rich repeat (LRR) protein kinases and calcium-binding proteins), defense (pathogenesis-related protein 1), and cofactors (jasmonate-ZIM-domain-containing proteins and heat shock proteins). Moreover, transcripts encoding glutathione S-transferase (GST) were accumulated to high levels, revealing key genes and proteins potentially related to pathogen resistance. Poplar RNA sequence data were validated by quantitative real-time PCR (RT-qPCR), which revealed a highly reliability of the transcriptomic profiling data. PMID:24870810

  5. Activities of the peptidyl transferase center of ribosomes lacking protein L27

    PubMed Central

    Maracci, Cristina; Wohlgemuth, Ingo; Rodnina, Marina V.

    2015-01-01

    The ribosome is the molecular machine responsible for protein synthesis in all living organisms. Its catalytic core, the peptidyl transferase center (PTC), is built of rRNA, although several proteins reach close to the inner rRNA shell. In the Escherichia coli ribosome, the flexible N-terminal tail of the ribosomal protein L27 contacts the A- and P-site tRNA. Based on computer simulations of the PTC and on previous biochemical evidence, the N-terminal α-amino group of L27 was suggested to take part in the peptidyl-transfer reaction. However, the contribution of this group to catalysis has not been tested experimentally. Here we investigate the role of L27 in peptide-bond formation using fast kinetics approaches. We show that the rate of peptide-bond formation at physiological pH, both with aminoacyl-tRNA or with the substrate analog puromycin, is independent of the presence of L27; furthermore, translation of natural mRNAs is only marginally affected in the absence of L27. The pH dependence of the puromycin reaction is unaltered in the absence of L27, indicating that the N-terminal α-amine is not the ionizing group taking part in catalysis. Likewise, L27 is not required for the peptidyl-tRNA hydrolysis during termination. Thus, apart from the known effect on subunit association, which most likely explains the phenotype of the deletion strains, L27 does not appear to be a key player in the core mechanism of peptide-bond formation on the ribosome. PMID:26475831

  6. Increased transcription of Glutathione S-transferases in acaricide exposed scabies mites

    PubMed Central

    2010-01-01

    Background Recent evidence suggests that Sarcoptes scabiei var. hominis mites collected from scabies endemic communities in northern Australia show increasing tolerance to 5% permethrin and oral ivermectin. Previous findings have implicated detoxification pathways in developing resistance to these acaricides. We investigated the contribution of Glutathione S-transferase (GST) enzymes to permethrin and ivermectin tolerance in scabies mites using biochemical and molecular approaches. Results Increased in vitro survival following permethrin exposure was observed in S. scabiei var. hominis compared to acaricide naïve mites (p < 0.0001). The addition of the GST inhibitor diethyl maleate restored in vitro permethrin susceptibility, confirming GST involvement in permethrin detoxification. Assay of GST enzymatic activity in mites demonstrated that S. scabiei var. hominis mites showed a two-fold increase in activity compared to naïve mites (p < 0.0001). Increased transcription of three different GST molecules was observed in permethrin resistant S. scabiei var. canis- mu 1 (p < 0.0001), delta 1 (p < 0.001), and delta 3 (p < 0.0001). mRNA levels of GST mu 1, delta 3 and P-glycoprotein also significantly increased in S. scabiei var. hominis mites collected from a recurrent crusted scabies patient over the course of ivermectin treatment. Conclusions These findings provide further support for the hypothesis that increased drug metabolism and efflux mediate permethrin and ivermectin resistance in scabies mites and highlight the threat of emerging acaricide resistance to the treatment of scabies worldwide. This is one of the first attempts to define specific genes involved in GST mediated acaricide resistance at the transcriptional level, and the first application of such studies to S. scabiei, a historically challenging ectoparasite. PMID:20482766

  7. Characterization and Functional Analysis of Four Glutathione S-Transferases from the Migratory Locust, Locusta migratoria

    PubMed Central

    Qin, Guohua; Jia, Miao; Liu, Ting; Zhang, Xueyao; Guo, Yaping; Zhu, Kun Yan; Ma, Enbo; Zhang, Jianzhen

    2013-01-01

    Glutathione S-transferases (GSTs) play an important role in detoxification of xenobiotics in both prokaryotic and eukaryotic cells. In this study, four GSTs (LmGSTd1, LmGSTs5, LmGSTt1, and LmGSTu1) representing different classes were identified from the migratory locust, Locusta migratoria. These four proteins were heterologously expressed in Escherichia coli as soluble fusion proteins, purified by Ni2+-nitrilotriacetic acid agarose column and biochemically characterized. LmGSTd1, LmGSTs5, and LmGSTu1 showed high activities with 1-chloro-2, 4-dinitrobenzene (CDNB), detectable activity with p-nitro-benzyl chloride (p-NBC) and 1, 2-dichloro-4-nitrobenzene (DCNB), whereas LmGSTt1 showed high activity with p-NBC and detectable activity with CDNB. The optimal pH of the locust GSTs ranged between 7.0 to 9.0. Ethacrynic acid and reactive blue effectively inhibited all four GSTs. LmGSTs5 was most sensitive to heavy metals (Cu2+ and Cd2+). The maximum expression of the four GSTs was observed in Malpighian tubules and fat bodies as evaluated by western blot. The nymph mortalities after carbaryl treatment increased by 28 and 12% after LmGSTs5 and LmGSTu1 were silenced, respectively. The nymph mortalities after malathion and chlorpyrifos treatments increased by 26 and 18% after LmGSTs5 and LmGSTu1 were silenced, respectively. These results suggest that sigma GSTs in L. migratoria play a significant role in carbaryl detoxification, whereas some of other GSTs may also involve in the detoxification of carbaryl and chlorpyrifos. PMID:23505503

  8. Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin

    PubMed Central

    Ralat, Luis A.; Misquitta, Stephanie A.; Manevich, Yefim; Fisher, Aron B.; Colman, Roberta F.

    2016-01-01

    Glutathione S-transferase pi has been shown to reactivate 1-cysteine peroxiredoxin (1-Cys Prx) by formation of a complex. A model of the complex was proposed based on the crystal structures of the two enzymes. We have now characterized the complex of GST pi/1-Cys Prx by determining the Mw of the complex, by measuring the catalytic activity of the GST pi monomer, and by identifying the interaction sites between GST pi and 1-Cys Prx. The Mw of the purified GST pi/1-Cys Prx complex is 50,200 at pH 8.0 in the presence of 2.5 mM glutathione, as measured by light scattering, providing direct evidence that the active complex is a heterodimer composed of equimolar amounts of the two proteins. In the presence of 4 M KBr, GST pi is dissociated to monomer and retains catalytic activity, but the Km value for GSH is increased substantially. To identify the peptides of GST pi that interact with 1-Cys Prx, GST pi was digested with V8 protease and the peptides were purified. The binding by 1-Cys Prx of each of four pure GST pi peptides (residues 41–85, 115–124, 131–163, and 164–197) was investigated by protein fluorescence titration. An apparent stoichiometry of 1 mol/subunit 1-Cys Prx was measured for each peptide and the formation of the heterodimer is decreased when these peptides are included in the incubation mixture. These results support our proposed model of the heterodimer. PMID:18358825

  9. Genetic polymorphism for glutathione-S-transferase mu in asbestos cement workers.

    PubMed Central

    Jakobsson, K; Rannug, A; Alexandrie, A K; Rylander, L; Albin, M; Hagmar, L

    1994-01-01

    OBJECTIVE--To investigate whether a lack of glutathione-S-transferase mu (GSTM1) activity was related to an increased risk for adverse outcome after asbestos exposure. METHODS--A study was made of 78 male former asbestos cement workers, with retrospective cohort data on exposure, radiographical findings, and lung function. Venous blood samples were obtained for the analysis of GSTM1 polymorphism by the polymerase chain reaction technique. Chest x ray films were classified according to the International Labour Organisation (ILO) 1980 classification. Vital capacity (VC) and forced expiratory volume during 1 s (FEV1) were determined. Individual estimates of asbestos exposure were calculated, and expressed as duration of exposure, average exposure intensity, and cumulative dose. Data on smoking were obtained from interviews. RESULTS--The lung function in the study group was reduced, compared with reference equations. 23% of the workers had small opacities > or = 1/0, 29% circumscribed pleural thickenings, 14% diffuse thickenings, and 12% obliterated costophrenic angles. 54% of the workers were GSTM1 deficient. They were comparable with the other workers in age, follow up time (median 30 years), and duration of exposure (median 18 years), but had a slightly higher cumulated dose (median 18 v 10 fibre-years) than the others. Neither in radiographical changes nor lung function variables were there any differences between the different GSTM1 groups. The findings were similar when smoking habits and estimated asbestos exposure were taken into account. CONCLUSIONS--We could not show that lack of GSTM1 activity was related to an increased risk for radiographical or lung function changes in a group of asbestos cement workers, followed up for a long period after the end of exposure. PMID:7849864

  10. Urinary π-glutathione S-transferase Predicts Advanced Acute Kidney Injury Following Cardiovascular Surgery.

    PubMed

    Shu, Kai-Hsiang; Wang, Chih-Hsien; Wu, Che-Hsiung; Huang, Tao-Min; Wu, Pei-Chen; Lai, Chien-Heng; Tseng, Li-Jung; Tsai, Pi-Ru; Connolly, Rory; Wu, Vin-Cent

    2016-01-01

    Urinary biomarkers augment the diagnosis of acute kidney injury (AKI), with AKI after cardiovascular surgeries being a prototype of prognosis scenario. Glutathione S-transferases (GST) were evaluated as biomarkers of AKI. Urine samples were collected in 141 cardiovascular surgical patients and analyzed for urinary alpha-(α-) and pi-(π-) GSTs. The outcomes of advanced AKI (KDIGO stage 2, 3) and all-cause in-patient mortality, as composite outcome, were recorded. Areas under the receiver operator characteristic (ROC) curves and multivariate generalized additive model (GAM) were applied to predict outcomes. Thirty-eight (26.9%) patients had AKI, while 12 (8.5%) were with advanced AKI. Urinary π-GST differentiated patients with/without advanced AKI or composite outcome after surgery (p < 0.05 by generalized estimating equation). Urinary π-GST predicted advanced AKI at 3 hrs post-surgery (p = 0.033) and composite outcome (p = 0.009), while the corresponding ROC curve had AUC of 0.784 and 0.783. Using GAM, the cutoff value of 14.7 μg/L for π-GST showed the best performance to predict composite outcome. The addition of π-GST to the SOFA score improved risk stratification (total net reclassification index = 0.47). Thus, urinary π-GST levels predict advanced AKI or hospital mortality after cardiovascular surgery and improve in SOFA outcome assessment specific to AKI. PMID:27527370

  11. Heterologous expression, purification and characterization of rat class theta glutathione transferase T2-2.

    PubMed Central

    Jemth, P; Stenberg, G; Chaga, G; Mannervik, B

    1996-01-01

    Rat glutathione transferase (GST) T2-2 of class Theta (rGST T2-2), previously known as GST 12-12 and GST Yrs-Yrs, has been heterologously expressed in Escherichia coli XLI-Blue. The corresponding cDNA was isolated from a rat hepatoma cDNA library, ligated into and expressed from the plasmid pKK-D. The sequence is the same as that of the previously reported cDNA of GST Yrs-Yrs. The enzyme was purified using ion-exchange chromatography followed by affinity chromatography with immobilized ferric ions, and the yield was approx. 200 mg from a 1 litre bacterial culture. The availability of a stable recombinant rGST T2-2 has paved the way for a more accurate characterization of the enzyme. The functional properties of the recombinant rGST T2-2 differ significantly from those reported earlier for the enzyme isolated from rat tissues. These differences probably reflect the difficulties in obtaining fully active enzyme from sources where it occurs in relatively low concentrations, which has been the case in previous studies. 1-Chloro-2,4-dinitrobenzene, a substrate often used with GSTs of classes Alpha, Mu and Pi, is a substrate also for rGST T2-2, but the specific activity is relatively low. The Km value for glutathione was determined with four different electrophiles and was found to be in the range 0.3 mM-0.8 mM. The Km values for some electrophilic substrates were found to be in the micromolar range, which is low compared with those determined for GSTs of other classes. The highest catalytic efficiency was obtained with menaphthyl sulphate, which gave a Kcat/Km value of 2.3 x 10(6) s-1.M-1 and a rate enhancement over the uncatalysed reaction of 3 x 10(10). PMID:8645195

  12. Busulfan conjugation by glutathione S-transferases alpha, mu, and pi.

    PubMed

    Czerwinski, M; Gibbs, J P; Slattery, J T

    1996-09-01

    Busulfan is eliminated by glutathione S-transferase (GST)-catalyzed conjugation with glutathione (GSH). We have characterized the busulfan-conjugating activity of purified human liver GSTA1-1, GSTA1-2, GSTA2-2, GSTM1-1, and placental GSTP1-1. Isoforms were purified from cytosol by GSH-affinity chromatography and chromatofocusing. In addition, the busulfan-conjugating activity of cDNA-expressed GTH1 and GTH2, corresponding to GSTA1-1 and GSTA2-2, were characterized. The major product of busulfan conjugation, a thiophenium ion (THT+), was assayed by GC/MS after conversion to tetrahydrothiophene (THT). THT+ formation rate increased linearly with busulfan concentration up to its solubility limit for all GST isoforms. Because Vmax and KM could not be determined separately, the slope of the velocity vs. substrate concentration plot, Vmax/KM was used to compare isoform activities. Vmax/KM for GSTA1-1 was 7.95 microliters/min/mg protein, the highest busulfan-conjugating activity of all human liver and placenta isoforms evaluated. GSTM1-1 and GSTP1-1, respectively, had 46% and 18% of the activity of GSTA1-1. Since the polymorphic mu-class GST catalyzed busulfan conjugation, we examined busulfan clearance in 50 patients undergoing high-dose busulfan before bone marrow transplantation. Busulfan clearance was normally distributed, suggesting that GSTM1-1 does not contribute significantly to the elimination of busulfan from the body. We conclude that GSTA1-1 is the major isoform catalyzing busulfan conjugation, whereas GSTM1-1 and GSTP1-1 may be important in the protection of specific cells. PMID:8886613

  13. Comparison of human liver and small intestinal glutathione S-transferase-catalyzed busulfan conjugation in vitro.

    PubMed

    Gibbs, J P; Yang, J S; Slattery, J T

    1998-01-01

    The apparent oral clearance of busulfan has been observed to vary as much as 10-fold in the population of children and adults receiving high-dose busulfan. The only identified elimination pathway for busulfan involves glutathione conjugation. The reaction is predominantly catalyzed by glutathione S-transferase (GST) A1-1, which is present in both liver and intestine. The purpose of this study was to compare busulfan Vmax/Km in cytosol prepared from adult human liver and small intestine. Tetrahydrothiophenium ion formation rate per milligram of cytosolic protein was constant along the length (assessed in 30-cm segments) of three individual small intestines. A 30-cm-long intestinal segment 90-180 cm from the pylorus was chosen to be representative of intestinal cytosolic busulfan conjugating activity. Busulfan Vmax/Km (mean +/- SD) in cytosol prepared from 23 livers and 12 small intestines was 0.166 +/- 0.066 and 0.176 +/- 0.085 microl/min/mg cytosolic protein, respectively, in incubations with 5 microM busulfan, 1 mM glutathione, and 2 mg of cytosolic protein. The relative content of GSTalpha (A1-1, A1-2, and A2-2) was compared for human liver and intestinal cytosol using Western blot. The levels of GSTalpha in liver and intestinal cytosol were 1.12 +/- 0.56 and 1.36 +/- 0.32 integrated optimal density units/5 microg cytosolic protein, respectively. Busulfan conjugation in vitro was comparable per milligram of cytosolic protein in liver and intestinal cytosol. PMID:9443852

  14. 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

  15. The Sigma Class Glutathione Transferase from the Liver Fluke Fasciola hepatica

    PubMed Central

    LaCourse, E. James; Perally, Samirah; Morphew, Russell M.; Moxon, Joseph V.; Prescott, Mark; Dowling, David J.; O'Neill, Sandra M.; Kipar, Anja; Hetzel, Udo; Hoey, Elizabeth; Zafra, Rafael; Buffoni, Leandro; Pérez Arévalo, José; Brophy, Peter M.

    2012-01-01

    Background Liver fluke infection of livestock causes economic losses of over US$ 3 billion worldwide per annum. The disease is increasing in livestock worldwide and is a re-emerging human disease. There are currently no commercial vaccines, and only one drug with significant efficacy against adult worms and juveniles. A liver fluke vaccine is deemed essential as short-lived chemotherapy, which is prone to resistance, is an unsustainable option in both developed and developing countries. Protein superfamilies have provided a number of leading liver fluke vaccine candidates. A new form of glutathione transferase (GST) family, Sigma class GST, closely related to a leading Schistosome vaccine candidate (Sm28), has previously been revealed by proteomics in the liver fluke but not functionally characterised. Methodology/Principal Findings In this manuscript we show that a purified recombinant form of the F. hepatica Sigma class GST possesses prostaglandin synthase activity and influences activity of host immune cells. Immunocytochemistry and western blotting have shown the protein is present near the surface of the fluke and expressed in eggs and newly excysted juveniles, and present in the excretory/secretory fraction of adults. We have assessed the potential to use F. hepatica Sigma class GST as a vaccine in a goat-based vaccine trial. No significant reduction of worm burden was found but we show significant reduction in the pathology normally associated with liver fluke infection. Conclusions/Significance We have shown that F. hepatica Sigma class GST has likely multi-functional roles in the host-parasite interaction from general detoxification and bile acid sequestration to PGD synthase activity. PMID:22666515

  16. The Carnitine Palmitoyl Transferase (CPT) System and Possible Relevance for Neuropsychiatric and Neurological Conditions.

    PubMed

    Virmani, Ashraf; Pinto, Luigi; Bauermann, Otto; Zerelli, Saf; Diedenhofen, Andreas; Binienda, Zbigniew K; Ali, Syed F; van der Leij, Feike R

    2015-10-01

    The carnitine palmitoyl transferase (CPT) system is a multiprotein complex with catalytic activity localized within a core represented by CPT1 and CPT2 in the outer and inner membrane of the mitochondria, respectively. Two proteins, the acyl-CoA synthase and a translocase also form part of this system. This system is crucial for the mitochondrial beta-oxidation of long-chain fatty acids. CPT1 has two well-known isoforms, CPT1a and CPT1b. CPT1a is the hepatic isoform and CPT1b is typically muscular; both are normally utilized by the organism for metabolic processes throughout the body. There is a strong evidence for their involvement in various disease states, e.g., metabolic syndrome, cardiovascular diseases, and in diabetes mellitus type 2. Recently, a new, third isoform of CPT was described, CPT1c. This is a neuronal isoform and is prevalently localized in brain regions such as hypothalamus, amygdala, and hippocampus. These brain regions play an important role in control of food intake and neuropsychiatric and neurological diseases. CPT activity has been implicated in several neurological and social diseases mainly related to the alteration of insulin equilibrium in the brain. These pathologies include Parkinson's disease, Alzheimer's disease, and schizophrenia. Evolution of both Parkinson's disease and Alzheimer's disease is in some way linked to brain insulin and related metabolic dysfunctions with putative links also with the diabetes type 2. Studies show that in the CNS, CPT1c affects ceramide levels, endocannabionoids, and oxidative processes and may play an important role in various brain functions such as learning. PMID:26041663

  17. Glutathione-supported arsenate reduction coupled to arsenolysis catalyzed by ornithine carbamoyl transferase

    SciTech Connect

    Nemeti, Balazs; Gregus, Zoltan

    2009-09-01

    Three cytosolic phosphorolytic/arsenolytic enzymes, (purine nucleoside phosphorylase [PNP], glycogen phosphorylase, glyceraldehyde-3-phosphate dehydrogenase) have been shown to mediate reduction of arsenate (AsV) to the more toxic arsenite (AsIII) in a thiol-dependent manner. With unknown mechanism, hepatic mitochondria also reduce AsV. Mitochondria possess ornithine carbamoyl transferase (OCT), which catalyzes phosphorolytic or arsenolytic citrulline cleavage; therefore, we examined if mitochondrial OCT facilitated AsV reduction in presence of glutathione. Isolated rat liver mitochondria were incubated with AsV, and AsIII formed was quantified. Glutathione-supplemented permeabilized or solubilized mitochondria reduced AsV. Citrulline (substrate for OCT-catalyzed arsenolysis) increased AsV reduction. The citrulline-stimulated AsV reduction was abolished by ornithine (OCT substrate inhibiting citrulline cleavage), phosphate (OCT substrate competing with AsV), and the OCT inhibitor norvaline or PALO, indicating that AsV reduction is coupled to OCT-catalyzed arsenolysis of citrulline. Corroborating this conclusion, purified bacterial OCT mediated AsV reduction in presence of citrulline and glutathione with similar responsiveness to these agents. In contrast, AsIII formation by intact mitochondria was unaffected by PALO and slightly stimulated by citrulline, ornithine, and norvaline, suggesting minimal role for OCT in AsV reduction in intact mitochondria. In addition to OCT, mitochondrial PNP can also mediate AsIII formation; however, its role in AsV reduction appears severely limited by purine nucleoside supply. Collectively, mitochondrial and bacterial OCT promote glutathione-dependent AsV reduction with coupled arsenolysis of citrulline, supporting the hypothesis that AsV reduction is mediated by phosphorolytic/arsenolytic enzymes. Nevertheless, because citrulline cleavage is disfavored physiologically, OCT may have little role in AsV reduction in vivo.

  18. The still mysterious roles of cysteine-containing glutathione transferases in plants

    PubMed Central

    Lallement, Pierre-Alexandre; Brouwer, Bastiaan; Keech, Olivier; Hecker, Arnaud; Rouhier, Nicolas

    2014-01-01

    Glutathione transferases (GSTs) represent a widespread multigenic enzyme family able to modify a broad range of molecules. These notably include secondary metabolites and exogenous substrates often referred to as xenobiotics, usually for their detoxification, subsequent transport or export. To achieve this, these enzymes can bind non-substrate ligands (ligandin function) and/or catalyze the conjugation of glutathione onto the targeted molecules, the latter activity being exhibited by GSTs having a serine or a tyrosine as catalytic residues. Besides, other GST members possess a catalytic cysteine residue, a substitution that radically changes enzyme properties. Instead of promoting GSH-conjugation reactions, cysteine-containing GSTs (Cys-GSTs) are able to perform deglutathionylation reactions similarly to glutaredoxins but the targets are usually different since glutaredoxin substrates are mostly oxidized proteins and Cys-GST substrates are metabolites. The Cys-GSTs are found in most organisms and form several classes. While Beta and Omega GSTs and chloride intracellular channel proteins (CLICs) are not found in plants, these organisms possess microsomal ProstaGlandin E-Synthase type 2, glutathionyl hydroquinone reductases, Lambda, Iota and Hemerythrin GSTs and dehydroascorbate reductases (DHARs); the four last classes being restricted to the green lineage. In plants, whereas the role of DHARs is clearly associated to the reduction of dehydroascorbate to ascorbate, the physiological roles of other Cys-GSTs remain largely unknown. In this context, a genomic and phylogenetic analysis of Cys-GSTs in photosynthetic organisms provides an updated classification that is discussed in the light of the recent literature about the functional and structural properties of Cys-GSTs. Considering the antioxidant potencies of phenolic compounds and more generally of secondary metabolites, the connection of GSTs with secondary metabolism may be interesting from a pharmacological

  19. The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center.

    PubMed

    Englander, Michael T; Avins, Joshua L; Fleisher, Rachel C; Liu, Bo; Effraim, Philip R; Wang, Jiangning; Schulten, Klaus; Leyh, Thomas S; Gonzalez, Ruben L; Cornish, Virginia W

    2015-05-12

    The cellular translational machinery (TM) synthesizes proteins using exclusively L- or achiral aminoacyl-tRNAs (aa-tRNAs), despite the presence of D-amino acids in nature and their ability to be aminoacylated onto tRNAs by aa-tRNA synthetases. The ubiquity of L-amino acids in proteins has led to the hypothesis that D-amino acids are not substrates for the TM. Supporting this view, protein engineering efforts to incorporate D-amino acids into proteins using the TM have thus far been unsuccessful. Nonetheless, a mechanistic understanding of why D-aa-tRNAs are poor substrates for the TM is lacking. To address this deficiency, we have systematically tested the translation activity of D-aa-tRNAs using a series of biochemical assays. We find that the TM can effectively, albeit slowly, accept D-aa-tRNAs into the ribosomal aa-tRNA binding (A) site, use the A-site D-aa-tRNA as a peptidyl-transfer acceptor, and translocate the resulting peptidyl-D-aa-tRNA into the ribosomal peptidyl-tRNA binding (P) site. During the next round of continuous translation, however, we find that ribosomes carrying a P-site peptidyl-D-aa-tRNA partition into subpopulations that are either translationally arrested or that can continue translating. Consistent with its ability to arrest translation, chemical protection experiments and molecular dynamics simulations show that P site-bound peptidyl-D-aa-tRNA can trap the ribosomal peptidyl-transferase center in a conformation in which peptidyl transfer is impaired. Our results reveal a novel mechanism through which D-aa-tRNAs interfere with translation, provide insight into how the TM might be engineered to use D-aa-tRNAs, and increase our understanding of the physiological role of a widely distributed enzyme that clears D-aa-tRNAs from cells. PMID:25918365

  20. The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center

    PubMed Central

    Englander, Michael T.; Avins, Joshua L.; Fleisher, Rachel C.; Liu, Bo; Effraim, Philip R.; Wang, Jiangning; Schulten, Klaus; Leyh, Thomas S.; Gonzalez, Ruben L.; Cornish, Virginia W.

    2015-01-01

    The cellular translational machinery (TM) synthesizes proteins using exclusively L- or achiral aminoacyl-tRNAs (aa-tRNAs), despite the presence of D-amino acids in nature and their ability to be aminoacylated onto tRNAs by aa-tRNA synthetases. The ubiquity of L-amino acids in proteins has led to the hypothesis that D-amino acids are not substrates for the TM. Supporting this view, protein engineering efforts to incorporate D-amino acids into proteins using the TM have thus far been unsuccessful. Nonetheless, a mechanistic understanding of why D-aa-tRNAs are poor substrates for the TM is lacking. To address this deficiency, we have systematically tested the translation activity of D-aa-tRNAs using a series of biochemical assays. We find that the TM can effectively, albeit slowly, accept D-aa-tRNAs into the ribosomal aa-tRNA binding (A) site, use the A-site D-aa-tRNA as a peptidyl-transfer acceptor, and translocate the resulting peptidyl-D-aa-tRNA into the ribosomal peptidyl-tRNA binding (P) site. During the next round of continuous translation, however, we find that ribosomes carrying a P-site peptidyl-D-aa-tRNA partition into subpopulations that are either translationally arrested or that can continue translating. Consistent with its ability to arrest translation, chemical protection experiments and molecular dynamics simulations show that P site-bound peptidyl-D-aa-tRNA can trap the ribosomal peptidyl-transferase center in a conformation in which peptidyl transfer is impaired. Our results reveal a novel mechanism through which D-aa-tRNAs interfere with translation, provide insight into how the TM might be engineered to use D-aa-tRNAs, and increase our understanding of the physiological role of a widely distributed enzyme that clears D-aa-tRNAs from cells. PMID:25918365

  1. The poplar Phi class glutathione transferase: expression, activity and structure of GSTF1

    PubMed Central

    Pégeot, Henri; Koh, Cha San; Petre, Benjamin; Mathiot, Sandrine; Duplessis, Sébastien; Hecker, Arnaud; Didierjean, Claude; Rouhier, Nicolas

    2014-01-01

    Glutathione transferases (GSTs) constitute a superfamily of enzymes with essential roles in cellular detoxification and secondary metabolism in plants as in other organisms. Several plant GSTs, including those of the Phi class (GSTFs), require a conserved catalytic serine residue to perform glutathione (GSH)-conjugation reactions. Genomic analyses revealed that terrestrial plants have around ten GSTFs, eight in the Populus trichocarpa genome, but their physiological functions and substrates are mostly unknown. Transcript expression analyses showed a predominant expression of all genes both in reproductive (female flowers, fruits, floral buds) and vegetative organs (leaves, petioles). Here, we show that the recombinant poplar GSTF1 (PttGSTF1) possesses peroxidase activity toward cumene hydroperoxide and GSH-conjugation activity toward model substrates such as 2,4-dinitrochlorobenzene, benzyl and phenetyl isothiocyanate, 4-nitrophenyl butyrate and 4-hydroxy-2-nonenal but interestingly not on previously identified GSTF-class substrates. In accordance with analytical gel filtration data, crystal structure of PttGSTF1 showed a canonical dimeric organization with bound GSH or 2-(N-morpholino)ethanesulfonic acid molecules. The structure of these protein-substrate complexes allowed delineating the residues contributing to both the G and H sites that form the active site cavity. In sum, the presence of GSTF1 transcripts and proteins in most poplar organs especially those rich in secondary metabolites such as flowers and fruits, together with its GSH-conjugation activity and its documented stress-responsive expression suggest that its function is associated with the catalytic transformation of metabolites and/or peroxide removal rather than with ligandin properties as previously reported for other GSTFs. PMID:25566286

  2. 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

  3. 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. PMID:23499941

  4. Cloning, expression and analysis of the olfactory glutathione S-transferases in coho salmon

    PubMed Central

    Espinoza, Herbert M.; Shireman, Laura M.; McClain, Valerie; Atkins, William; Gallagher, Evan P.

    2013-01-01

    The glutathione S-transferases (GSTs) provide cellular protection by detoxifying xenobiotics, maintaining redox status, and modulating secondary messengers, all of which are critical to maintaining olfaction in salmonids. Here, we characterized the major coho salmon olfactory GSTs (OlfGSTs), namely omega, pi, and rho subclasses. OlfGST omega contained an open reading frame of 720 bp and encoded a protein of 239 amino acids. OlfGST pi and OlfGST rho contained open reading frames of 727 and 681 bp, respectively, and encoded proteins of 208 and 226 amino acids. Whole-protein mass spectrometry yielded molecular weights of 29,950, 23,354, and 26,655 Da, respectively, for the GST omega, pi, and rho subunits. Homology modeling using four protein-structure prediction algorithms suggest that the active sites in all three OlfGST isoforms resembled counterparts in other species. The olfactory GSTs conjugated prototypical GST substrates, but only OlfGST rho catalyzed the demethylation of the pesticide methyl parathion. OlfGST pi and rho exhibited thiol oxidoreductase activity towards 2-hydroxyethyl disulfide (2-HEDS) and conjugated 4-hydroxynonenal (HNE), a toxic aldehyde with neurodegenerative properties. The kinetic parameters for OlfGST pi conjugation of HNE were KM = 0.16 ± 0.06 mM and Vmax = 0.5 ± 0.1 μmol min−1 mg−1 for OlfGST pi, whereas OlfGST rho was more efficient at catalyzing HNE conjugation (KM = 0.022 ± 0.008 mM and Vmax = 0.47 ± 0.05 μmol min−1 mg−1). Our findings indicate that the peripheral olfactory system of coho expresses GST isoforms that detoxify certain electrophiles and pesticides and that help maintain redox statusand signal transduction. PMID:23261526

  5. Cloning, expression and analysis of the olfactory glutathione S-transferases in coho salmon.

    PubMed

    Espinoza, Herbert M; Shireman, Laura M; McClain, Valerie; Atkins, William; Gallagher, Evan P

    2013-03-15

    The glutathione S-transferases (GSTs) provide cellular protection by detoxifying xenobiotics, maintaining redox status, and modulating secondary messengers, all of which are critical to maintaining olfaction in salmonids. Here, we characterized the major coho salmon olfactory GSTs (OlfGSTs), namely omega, pi, and rho subclasses. OlfGST omega contained an open reading frame of 720bp and encoded a protein of 239 amino acids. OlfGST pi and OlfGST rho contained open reading frames of 627 and 681nt, respectively, and encoded proteins of 208 and 226 amino acids. Whole-protein mass spectrometry yielded molecular weights of 29,950, 23,354, and 26,655Da, respectively, for the GST omega, pi, and rho subunits. Homology modeling using four protein-structure prediction algorithms suggest that the active sites in all three OlfGST isoforms resembled counterparts in other species. The olfactory GSTs conjugated prototypical GST substrates, but only OlfGST rho catalyzed the demethylation of the pesticide methyl parathion. OlfGST pi and rho exhibited thiol oxidoreductase activity toward 2-hydroxyethyl disulfide (2-HEDS) and conjugated 4-hydroxynonenal (HNE), a toxic aldehyde with neurodegenerative properties. The kinetic parameters for OlfGST pi conjugation of HNE were K(M)=0.16 ± 0.06mM and V(max)=0.5 ± 0.1μmolmin⁻¹mg⁻¹, whereas OlfGST rho was more efficient at catalyzing HNE conjugation (K(M)=0.022 ± 0.008 mM and V(max)=0.47 ± 0.05μmolmin⁻¹mg⁻¹). Our findings indicate that the peripheral olfactory system of coho expresses GST isoforms that detoxify certain electrophiles and pesticides and that help maintain redox status and signal transduction. PMID:23261526

  6. The stereochemical course of 4-hydroxy-2-nonenal metabolism by glutathione S-transferases.

    PubMed

    Balogh, Larissa M; Roberts, Arthur G; Shireman, Laura M; Greene, Robert J; Atkins, William M

    2008-06-13

    4-Hydroxy-2-nonenal (HNE) is a toxic aldehyde generated during lipid peroxidation and has been implicated in a variety of pathological states associated with oxidative stress. Glutathione S-transferase (GST) A4-4 is recognized as one of the predominant enzymes responsible for the metabolism of HNE. However, substrate and product stereoselectivity remain to be fully explored. The results from a product formation assay indicate that hGSTA4-4 exhibits a modest preference for the biotransformation of S-HNE in the presence of both enantiomers. Liquid chromatography mass spectrometry analyses using the racemic and enantioisomeric HNE substrates explicitly demonstrate that hGSTA4-4 conjugates glutathione to both HNE enantiomers in a completely stereoselective manner that is not maintained in the spontaneous reaction. Compared with other hGST isoforms, hGSTA4-4 shows the highest degree of stereoselectivity. NMR experiments in combination with simulated annealing structure determinations enabled the determination of stereochemical configurations for the GSHNE diastereomers and are consistent with an hGSTA4-4-catalyzed nucleophilic attack that produces only the S-configuration at the site of conjugation, regardless of substrate chirality. In total these results indicate that hGSTA4-4 exhibits an intriguing combination of low substrate stereoselectivity with strict product stereoselectivity. This behavior allows for the detoxification of both HNE enantiomers while generating only a select set of GSHNE diastereomers with potential stereochemical implications concerning their effects and fates in biological tissues. PMID:18424441

  7. Cloning, expression and identification of two glutathione S-transferase isoenzymes from Perna viridis.

    PubMed

    Li, Zhenzhen; Chen, Rong; Zuo, Zhenghong; Mo, Zhengping; Yu, Ang

    2013-08-01

    Glutathione S-transferases (GSTs; EC 2.5.1.18) are phase II enzymes involved in major detoxification reactions of xenobiotic in many organisms. In the present study, two classes of GSTs (PvGST1 and PvGST2) were cloned from P. viridis by rapid amplification of cDNA ends method. Sequence alignments and phylogenetic analysis together supported that PvGST1 and PvGST2 belonged to the pi and omega classes, respectively. The PvGST1 cDNA was 1214 nucleotides (nt) in length and contained a 618 nt open reading frame (ORF) encoding 206 amino acid residues, and had 46 nt of 5'-untranslated region (UTR) and a 3' UTR of 550 nt including a tailing signal (AATAAA) and a poly (A) tail. The molecular mass of the predicted PvGST1 was 23.815kDa, with the calculated isoelectric point being 5.39. PvGST2 was 1093bp, consisting of a 5' UTR of 13bp, a 3' UTR of 246bp and an ORF of 834bp. The deduced protein was composed of 278 amino acids, with an estimated molecular mass of 32.476kDa and isoelectric point of 8.88. Tissue distribution analysis of the PvGST1 and PvGST2 mRNA revealed that the GST expression level was higher in digestive gland and gonad, while lower in gill and mantle in both genders. Molecular modeling analysis of two GSTs implicated their various functions account for their different enzymatic features. PMID:23711756

  8. Nuclear morphometry and glutathione S-transferase pi expression in breast cancer.

    PubMed

    Huang, J; Bay, B H; Tan, P H

    2000-01-01

    Glutathione S-transferase pi (GST-pi) is a phase II detoxification enzyme whose expression is increased in estrogen receptor (ER)-poor breast cancers and in breast cancers resistant to certain chemotherapeutic agents. The aim of this study was to investigate the immunohistochemical expression of GST-pi in invasive breast carcinoma and to correlate the findings with those of nuclear morphometry. Formalin-fixed paraffin-embedded tissue specimens obtained from 21 invasive breast cancers and 16 adjacent (benign) tissues were immunohistochemically stained using polyclonal anti-human GST-pi antibody. There was positive (defined as >10% immunoreactive tumor cells) but variable expression of GST-pi in 10 (48%) cases. Nuclear morphometry in these 10 tumors revealed immunoreactive malignant cells to be larger (mean area 41.7+/-1.0 microm2) and more rounded in form when compared with non-staining cancer cells (mean area 28.7+/-0.7 microm2). It was also observed that GST-pi immunonegative tumor cells in GST-pi expressing tumors had different morphologies from malignant cells in the remaining 11 (52%) cancers that were regarded as GST-pi negative. Increased GST-pi expression determined by the percentage of positively staining tumor cells, was found to be significantly correlated with increased variability in nuclear area and perimeter (Spearman's rho=0.821, p=0.044 for both) in the subset of node-positive tumors. Our findings suggest that there exists two sub-populations of cancer cells with distinct nuclear morphologies in GST-pi positive tumors; factors other than GST-pi expression are likely to have a phenotypic effect on breast cancer cells; and there may be a special significance of this enzyme in axillary node-positive breast tumors. PMID:10767377

  9. Inhibitory effects of plant polyphenols on rat liver glutathione S-transferases.

    PubMed

    Zhang, K; Das, N P

    1994-06-01

    Several novel naturally occurring flavonoids and other polyphenols exerted varying degrees of concentration-dependent inhibition on uncharacterized rat liver glutathione S-transferase (EC 2.5.1.18, GST) isoforms. The order of inhibitory potencies of the five most potent polyphenols was tannic acid > 2-hydroxyl chalcone > butein > morin > quercetin, and their IC50 values were 1.044, 6.758, 9.033, 13.710 and 18.732 microM, respectively. Their inhibitions were reversible, as indicated by dialysis experiments. The optimum pH for the inhibitions by four of the compounds (tannic acid, butein, 2-hydroxyl chalcone and morin) was in the range of pH 6.0 to 6.5, but for quercetin the optimum pH was 8.0. These potent inhibitors possess one or more of the following chemical structural features: (a) polyhydroxylation substitutions, (b) absence of a sugar moiety, (c) for the chalcones, the presence of an open C-ring and hydroxylation at either the C-2 or C-3 position, (d) for the flavonoids, the attachment of the B-ring to C-2, and (e) a double bond between C-2 and C-3. Butein exhibited a non-competitive inhibition toward both glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Interestingly, tannic acid showed a non-competitive inhibition toward CDNB but a competitive inhibition toward GSH. The inhibitory potency of tannic acid on rat liver GSTs was concentration and substrate dependent. Using CDNB, p-nitrobenzyl chloride, 4-nitropyridine-N-oxide, and ethacrynic acid as substrates, the IC50 values for tannic acid were 1.044, 11.151, 20.206, and 57.664 microM, respectively. PMID:8010991

  10. Exploiting the Substrate Promiscuity of Hydroxycinnamoyl-CoA:Shikimate Hydroxycinnamoyl Transferase to Reduce Lignin.

    PubMed

    Eudes, Aymerick; Pereira, Jose H; Yogiswara, Sasha; Wang, George; Teixeira Benites, Veronica; Baidoo, Edward E K; Lee, Taek Soon; Adams, Paul D; Keasling, Jay D; Loqué, Dominique

    2016-03-01

    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 to 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. PMID:26858288

  11. The poplar Phi class glutathione transferase: expression, activity and structure of GSTF1.

    PubMed

    Pégeot, Henri; Koh, Cha San; Petre, Benjamin; Mathiot, Sandrine; Duplessis, Sébastien; Hecker, Arnaud; Didierjean, Claude; Rouhier, Nicolas

    2014-01-01

    Glutathione transferases (GSTs) constitute a superfamily of enzymes with essential roles in cellular detoxification and secondary metabolism in plants as in other organisms. Several plant GSTs, including those of the Phi class (GSTFs), require a conserved catalytic serine residue to perform glutathione (GSH)-conjugation reactions. Genomic analyses revealed that terrestrial plants have around ten GSTFs, eight in the Populus trichocarpa genome, but their physiological functions and substrates are mostly unknown. Transcript expression analyses showed a predominant expression of all genes both in reproductive (female flowers, fruits, floral buds) and vegetative organs (leaves, petioles). Here, we show that the recombinant poplar GSTF1 (PttGSTF1) possesses peroxidase activity toward cumene hydroperoxide and GSH-conjugation activity toward model substrates such as 2,4-dinitrochlorobenzene, benzyl and phenetyl isothiocyanate, 4-nitrophenyl butyrate and 4-hydroxy-2-nonenal but interestingly not on previously identified GSTF-class substrates. In accordance with analytical gel filtration data, crystal structure of PttGSTF1 showed a canonical dimeric organization with bound GSH or 2-(N-morpholino)ethanesulfonic acid molecules. The structure of these protein-substrate complexes allowed delineating the residues contributing to both the G and H sites that form the active site cavity. In sum, the presence of GSTF1 transcripts and proteins in most poplar organs especially those rich in secondary metabolites such as flowers and fruits, together with its GSH-conjugation activity and its documented stress-responsive expression suggest that its function is associated with the catalytic transformation of metabolites and/or peroxide removal rather than with ligandin properties as previously reported for other GSTFs. PMID:25566286

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

    SciTech Connect

    Woldegiorgis, S.; Ahmed, R.C.; Zhen, Y.; Erdmann, C.A.; Russell, M.L.; Goth-Goldstein, R.

    2002-04-01

    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 have 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.

  13. Structural analysis of an epsilon-class glutathione transferase from housefly, Musca domestica.

    PubMed

    Nakamura, Chihiro; Yajima, Shunsuke; Miyamoto, Toru; Sue, Masayuki

    2013-01-25

    Glutathione transferases (GSTs) play an important role in the detoxification of insecticides, and as such, they are a key contributor to enhanced resistance to insecticides. In the housefly (Musca domestica), two epsilon-class GSTs (MdGST6A and MdGST6B) that share high sequence homology have been identified, which are believed to be involved in resistance against insecticides. The structural determinants controlling the substrate specificity and enzyme activity of MdGST6s are unknown. The aim of this study was to crystallize and perform structural analysis of the GST isozyme, MdGST6B. The crystal structure of MdGST6B complexed with reduced glutathione (GSH) was determined at a resolution of 1.8 Å. MdGST6B was found to have a typical GST folding comprised of N-terminal and C-terminal domains. Arg113 and Phe121 on helix 4 were shown to protrude into the substrate binding pocket, and as a result, the entrance of the substrate binding pocket was narrower compared to delta- and epsilon-class GSTs from Africa malaria vector Anopheles gambiae, agGSTd1-6 and agGSTe2, respectively. This substrate pocket narrowing is partly due to the presence of a π-helix in the middle of helix 4. Among the six residues that donate hydrogen bonds to GSH, only Arg113 was located in the C-terminal domain. Ala substitution of Arg113 did not have a significant effect on enzyme activity, suggesting that the Arg113 hydrogen bond does not play a crucial role in catalysis. On the other hand, mutation at Phe108, located just below Arg113 in the binding pocket, reduced the affinity and catalytic activity to both GSH and the electrophilic co-substrate, 1-chloro-2,4-dinitrobenzene. PMID:23268341

  14. Effects of Local Heart Irradiation in a Glutathione S-Transferase Alpha 4-Null Mouse Model

    PubMed Central

    Boerma, Marjan; Singh, Preeti; Sridharan, Vijayalakshmi; Tripathi, Preeti; Sharma, Sunil; Singh, Sharda P.

    2015-01-01

    Glutathione S-transferase alpha 4 (GSTA4-4) is one of the enzymes responsible for the removal of 4-hydroxynonenal (4-HNE), an electrophilic product of lipid peroxidation in cellular membranes during oxidative stress. 4-HNE is a direct activator of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a transcription factor with many target genes encoding antioxidant and anti-electrophile enzymes. We have previously shown that Gsta4-null mice on a 129/Sv background exhibited increased activity of Nrf2 in the heart. Here we examined the sensitivity of this Gsta4-null mouse model towards cardiac function and structure loss due to local heart irradiation. Male Gsta4-null and wild-type mice were exposed to a single X-ray dose of 18 Gy to the heart. Six months after irradiation, immunohistochemical staining for respiratory complexes 2 and 5 indicated that radiation exposure had caused most pronounced alterations in mitochondrial morphology in Gsta4-null mice. On the other hand, wild-type mice showed a decline in cardiac function and an increase in plasma levels of troponin-I, while no such changes were observed in Gsta4-null mice. Radiation-induced Nrf2-target gene expression only in Gsta4-null mice. In conclusion, although loss of GSTA4-4 led to enhanced susceptibility of cardiac mitochondria to radiation-induced loss of morphology, cardiac function was preserved in Gsta4-null mice. We propose that this protection against cardiac function loss may occur, at least in part, by upregulation of the Nrf2 pathway. PMID:26010708

  15. Genomic cloning and characterization of the rat glutathione S-transferase-A3-subunit gene.

    PubMed

    Fotouhi-Ardakani, N; Batist, G

    1999-05-01

    The rat glutathione S-transferase-A3-subunit (GSTA3) gene is a member of the class Alpha GSTs, which we have previously reported to be overexpressed in anti-cancer-drug-resistant cells. In this study, we report the isolation and characterization of the entire rat GSTA3 (rGST Yc1) subunit gene. The rat GSTA3 subunit gene is approximately 15 kb in length and consists of seven exons interrupted by introns of different lengths. Exon 1, with a length of 219 bp, contains only the 5'-untranslated region of the gene. Each exon-intron splicing junction exhibited the consensus sequence for a mammalian splice site. The transcription start site and exon 1 of rat GSTA3 were characterized by a combination of primer extension and rapid amplification of the cDNA ends. Position +1 was identified 219 bp upstream of the first exon-intron splicing junction. The proximal promoter region of the rat GSTA3 subunit gene does not contain typical TATA or CAAT boxes. A computer-based search for potential transcription-factor binding sites revealed the existence of a number of motifs such as anti-oxidant-responsive element, ras-response element, activator protein-1, nuclear factor-kappaB, cAMP-response-element-binding protein, Barbie box and E box. The functional activity of the regulatory region of the rat GSTA3 subunit gene was shown by its ability to drive the expression of a chloramphenicol acetyltransferase reporter gene in rat mammary carcinoma cells, and its activity was greater in melphalan-resistant cells known to have transcriptional activation of this gene by previous studies. The structure of the gene, with a large intron upstream of the translation-initiation site, may explain why the isolation of this promoter has been so elusive. This information will provide the opportunity to examine the involvement of the rat GSTA3 subunit gene in drug resistance and carcinogenesis. PMID:10215608

  16. Biochemical studies on glutathione S-transferase from the bovine filarial worm Setaria digitata.

    PubMed

    Srinivasan, Lakshmy; Mathew, Nisha; Karunan, Twinkle; Muthuswamy, Kalyanasundaram

    2011-07-01

    Setaria digitata is a filarial worm of the cattle used as a model system for antifilarial drug screening, due to its similarity to the human filarial parasites Wuchereria bancrofti and Brugia malayi. Since filarial glutathione S-transferase (GST) is a good biochemical target for antifilarial drug development, a study has been undertaken for the biochemical characterization of GST from S. digitata. Cytosolic fraction was separated from the crude S.digitata worm homogenate by ultracentrifugation at 100,000 g and subjected to ammonium sulfate precipitation followed by affinity chromatography using GSH-agarose column. The kinetic parameters K (m) and V (max) values with respect to GSH were 0.45 mM and 0.105 μmol min(-1) mL(-1) respectively. With respect to 1-chloro-2,4-dinitrobenzene, the K (m) and V (max) values were 1.21 and 0.117 μmol min(-1) mL(-1) respectively. The effect of temperature and pH on GST enzyme activity was studied. The protein retained its enzyme activity between 0°C and 40°C, beyond which it showed a decreasing tendency, and at 80°C, the activity was lost completely. The enzyme activity was varying with change in pH, and the maximum GST activity was observed at pH 7.5. Gel filtration chromatographic studies indicated that the protein has a native molecular mass of about 54 kDa. The single band of GST subunit appeared in sodium dodecyl sulfate polyacrylamide gel electrophoresis was found to have molecular mass of ∼27 kDa. This shows that cytosolic S. digitata GST protein is homodimeric in nature. PMID:21207063

  17. Multiple isoforms of mitochondrial glutathione S-transferases and their differential induction under oxidative stress.

    PubMed Central

    Raza, Haider; Robin, Marie-Anne; Fang, Ji-Kang; Avadhani, Narayan G

    2002-01-01

    The mitochondrial respiratory chain, which consumes approx. 85-90% of the oxygen utilized by cells, is a major source of reactive oxygen species (ROS). Mitochondrial genetic and biosynthetic systems are highly susceptible to ROS toxicity. Intramitochondrial glutathione (GSH) is a major defence against ROS. In the present study, we have investigated the nature of the glutathione S-transferase (GST) pool in mouse liver mitochondria, and have purified three distinct forms of GST: GSTA1-1 and GSTA4-4 of the Alpha family, and GSTM1-1 belonging to the Mu family. The mitochondrial localization of these multiple GSTs was confirmed using a combination of immunoblot analysis, protease protection assay, enzyme activity, N-terminal amino acid sequencing, peptide mapping and confocal immunofluorescence analysis. Additionally, exogenously added 4-hydroxynonenal (HNE), a reactive byproduct of lipid peroxidation, to COS cells differentially affected the cytosolic and mitochondrial GSH pools in a dose- and time-dependent manner. Our results show that HNE-mediated mitochondrial oxidative stress caused a decrease in the GSH pool, increased membrane lipid peroxidation, and increased levels of GSTs, glutathione peroxidase and Hsp70 (heat-shock protein 70). The HNE-induced oxidative stress persisted for longer in the mitochondrial compartment, where the recovery of GSH pool was slower than in the cytosolic compartment. Our study, for the first time, demonstrates the presence in mitochondria of multiple forms of GSTs that show molecular properties similar to those of their cytosolic counterparts. Our results suggest that mitochondrial GSTs may play an important role in defence against chemical and oxidative stress. PMID:12020353

  18. Heterologous expression, purification and characterization of rat class theta glutathione transferase T2-2.

    PubMed

    Jemth, P; Stenberg, G; Chaga, G; Mannervik, B

    1996-05-15

    Rat glutathione transferase (GST) T2-2 of class Theta (rGST T2-2), previously known as GST 12-12 and GST Yrs-Yrs, has been heterologously expressed in Escherichia coli XLI-Blue. The corresponding cDNA was isolated from a rat hepatoma cDNA library, ligated into and expressed from the plasmid pKK-D. The sequence is the same as that of the previously reported cDNA of GST Yrs-Yrs. The enzyme was purified using ion-exchange chromatography followed by affinity chromatography with immobilized ferric ions, and the yield was approx. 200 mg from a 1 litre bacterial culture. The availability of a stable recombinant rGST T2-2 has paved the way for a more accurate characterization of the enzyme. The functional properties of the recombinant rGST T2-2 differ significantly from those reported earlier for the enzyme isolated from rat tissues. These differences probably reflect the difficulties in obtaining fully active enzyme from sources where it occurs in relatively low concentrations, which has been the case in previous studies. 1-Chloro-2,4-dinitrobenzene, a substrate often used with GSTs of classes Alpha, Mu and Pi, is a substrate also for rGST T2-2, but the specific activity is relatively low. The Km value for glutathione was determined with four different electrophiles and was found to be in the range 0.3 mM-0.8 mM. The Km values for some electrophilic substrates were found to be in the micromolar range, which is low compared with those determined for GSTs of other classes. The highest catalytic efficiency was obtained with menaphthyl sulphate, which gave a Kcat/Km value of 2.3 x 10(6) s-1.M-1 and a rate enhancement over the uncatalysed reaction of 3 x 10(10). PMID:8645195

  19. Biochemical Warfare on the Reef: The Role of Glutathione Transferases in Consumer Tolerance of Dietary Prostaglandins

    PubMed Central

    Whalen, Kristen E.; Lane, Amy L.; Kubanek, Julia; Hahn, Mark E.

    2010-01-01

    Background Despite the profound variation among marine consumers in tolerance for allelochemically-rich foods, few studies have examined the biochemical adaptations underlying diet choice. Here we examine the role of glutathione S-transferases (GSTs) in the detoxification of dietary allelochemicals in the digestive gland of the predatory gastropod Cyphoma gibbosum, a generalist consumer of gorgonian corals. Controlled laboratory feeding experiments were used to investigate the influence of gorgonian diet on Cyphoma GST activity and isoform expression. Gorgonian extracts and semi-purified fractions were also screened to identify inhibitors and possible substrates of Cyphoma GSTs. In addition, we investigated the inhibitory properties of prostaglandins (PGs) structurally similar to antipredatory PGs found in high concentrations in the Caribbean gorgonian Plexaura homomalla. Principal Findings Cyphoma GST subunit composition was invariant and activity was constitutively high regardless of gorgonian diet. Bioassay-guided fractionation of gorgonian extracts revealed that moderately hydrophobic fractions from all eight gorgonian species examined contained putative GST substrates/inhibitors. LC-MS and NMR spectral analysis of the most inhibitory fraction from P. homomalla subsequently identified prostaglandin A2 (PGA2) as the dominant component. A similar screening of commercially available prostaglandins in series A, E, and F revealed that those prostaglandins most abundant in gorgonian tissues (e.g., PGA2) were also the most potent inhibitors. In vivo estimates of PGA2 concentration in digestive gland tissues calculated from snail grazing rates revealed that Cyphoma GSTs would be saturated with respect to PGA2 and operating at or near physiological capacity. Significance The high, constitutive activity of Cyphoma GSTs is likely necessitated by the ubiquitous presence of GST substrates and/or inhibitors in this consumer's gorgonian diet. This generalist's GSTs may

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

    PubMed Central

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

    2009-01-01

    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. PMID:19270193

  1. Relation of Gamma-Glutamyl Transferase to Cardiovascular Events in Patients With Acute Coronary Syndromes.

    PubMed

    Ndrepepa, Gjin; Braun, Siegmund; Cassese, Salvatore; Fusaro, Massimiliano; Laugwitz, Karl-Ludwig; Schunkert, Heribert; Kastrati, Adnan

    2016-05-01

    The prognostic value of gamma-glutamyl transferase (GGT) in patients with acute coronary syndromes (ACS) has been incompletely investigated. We investigated this clinically relevant question in 2,534 consecutive patients with ACS who underwent percutaneous coronary intervention (PCI). GGT activity was measured before PCI procedure in all patients. Statin therapy at hospital discharge was prescribed in 94% of the patients. The primary outcome was 3-year mortality. Patients were divided into 3 groups: the group with GGT in the first tertile (GGT <28 U/L; n = 848 patients), the group with GGT in the second tertile (GGT 28 to <50 U/L; n = 843 patients), and the group with GGT in the third tertile (GGT ≥50 U/L; n = 843 patients). The primary outcome (all-cause deaths) occurred in 250 patients: 70 deaths (9.7%) among patients of the first, 69 deaths (9.0%) among patients of the second, and 111 deaths (14.8%) among patients of the third GGT tertile (adjusted hazard ratio [HR] 1.24, 95% CI 1.08 to 1.42, p = 0.002) and cardiac and noncardiac deaths occurred in 157 (63%) and 93 patients (37%), respectively. GGT was associated with the increased risk of noncardiac mortality (adjusted HR 1.35 [1.09 to 1.66], p = 0.005) but not cardiac mortality (adjusted HR 1.16 [0.97 to 1.38], p = 0.098; all 3 risk estimates were calculated per SD increase in the logarithmic scale of GGT activity). In conclusion, in contemporary patients with ACS treated with PCI and on statin therapy, elevated GGT activity was associated with the increased risk of all-cause and noncardiac mortality but not with the risk of cardiac mortality. PMID:26956636

  2. Glutathione S-transferases in rat olfactory epithelium: purification, molecular properties and odorant biotransformation.

    PubMed Central

    Ben-Arie, N; Khen, M; Lancet, D

    1993-01-01

    The olfactory epithelium is exposed to a variety of xenobiotic chemicals, including odorants and airborne toxic compounds. Recently, two novel, highly abundant, olfactory-specific biotransformation enzymes have been identified: cytochrome P-450olf1 and olfactory UDP-glucuronosyltransferase (UGT(olf)). The latter is a phase II biotransformation enzyme which catalyses the glucuronidation of alcohols, thiols, amines and carboxylic acids. Such covalent modification, which markedly affects lipid solubility and agonist potency, may be particularly important in the rapid termination of odorant signals. We report here the identification and characterization of a second olfactory phase II biotransformation enzyme, a glutathione S-transferase (GST). The olfactory epithelial cytosol shows the highest GST activity among the extrahepatic tissues examined. Significantly, olfactory epithelium had an activity 4-7 times higher than in other airway tissues, suggesting a role for this enzyme in chemoreception. The olfactory GST has been affinity-purified to homogeneity, and shown by h.p.l.c. and N-terminal amino acid sequencing to constitute mainly the Yb1 and Yb2 subunits, different from most other tissues that have mixtures of more enzyme classes. The identity of the olfactory enzymes was confirmed by PCR cloning and restriction enzyme analysis. Most importantly, the olfactory GSTs were found to catalyse glutathione conjugation of several odorant classes, including many unsaturated aldehydes and ketones, as well as epoxides. Together with UGT(olf), olfactory GST provides the necessary broad coverage of covalent modification capacity, which may be crucial for the acuity of the olfactory process. Images Figure 1 Figure 4 Figure 5 PMID:8503873

  3. Glutathione S-Transferase Regulation in Calanus finmarchicus Feeding on the Toxic Dinoflagellate Alexandrium fundyense

    PubMed Central

    Roncalli, Vittoria; Jungbluth, Michelle J.; Lenz, Petra H.

    2016-01-01

    The effect of the dinoflagellate, Alexandrium fundyense, on relative expression of glutathione S-transferase (GST) transcripts was examined in the copepod Calanus finmarchicus. Adult females were fed for 5-days on one of three experimental diets: control (100% Rhodomonas spp.), low dose of A. fundyense (25% by volume, 75% Rhodomonas spp.), and high dose (100% A. fundyense). Relative expression of three GST genes was measured using RT-qPCR on days 0.5, 1, 2 and 5 in two independent experiments. Differential regulation was found for the Delta and the Sigma GSTs between 0.5 to 2 days, but not on day 5 in both experiments. The third GST, a microsomal, was not differentially expressed in either treatment or day. RT-qPCR results from the two experiments were similar, even though experimental females were collected from the Gulf of Maine on different dates and their reproductive output differed. In the second experiment, expression of 39 GSTs was determined on days 2 and 5 using RNA-Seq. Global gene expression analyses agreed with the RT-qPCR results. Furthermore, the RNA-Seq measurements indicated that only four GSTs were differentially expressed under the experimental conditions, and the response was small in amplitude. In summary, the A. fundyense diet led to a rapid and transient response in C. finmarchicus in three cytosolic GSTs, while a fourth GST (Omega I) was significantly up-regulated on day 5. Although there was some regulation of GSTs in response the toxic dinoflagellate, the tolerance to A. fundyense by C. finmarchicus is not dependent on the long-term up-regulation of specific GSTs. PMID:27427938

  4. A critical perspective of the diverse roles of O-GlcNAc transferase in chromatin.

    PubMed

    Gambetta, Maria Cristina; Müller, Jürg

    2015-12-01

    O-linked β-N-Acetylglucosamine (O-GlcNAc) is a posttranslational modification that is catalyzed by O-GlcNAc transferase (Ogt) and found on a plethora of nuclear and cytosolic proteins in animals and plants. Studies in different model organisms revealed that while O-GlcNAc is required for selected processes in Caenorhabditis elegans and Drosophila, it has evolved to become required for cell viability in mice, and this has challenged investigations to identify cellular functions that critically require this modification in mammals. Nevertheless, a principal cellular process that engages O-GlcNAcylation in all of these species is the regulation of gene transcription. Here, we revisit several of the primary experimental observations that led to current models of how O-GlcNAcylation affects gene expression. In particular, we discuss the role of the stable association of Ogt with the transcription factors Hcf1 and Tet, the two main Ogt-interacting proteins in nuclei of mammalian cells. We also critically evaluate the evidence that specific residues on core histones, including serine 112 of histone 2B (H2B-S112), are O-GlcNAcylated in vivo and discuss possible physiological effects of these modifications. Finally, we review our understanding of the role of O-GlcNAcylation in Drosophila, where recent studies suggest that the developmental defects in Ogt mutants are all caused by lack of O-GlcNAcylation of a single transcriptional regulator, the Polycomb repressor protein Polyhomeotic (Ph). Collectively, this reexamination of the experimental evidence suggests that a number of recently propagated models about the role of O-GlcNAcylation in transcriptional control should be treated cautiously. PMID:25894967

  5. Controversial role of gamma-glutamyl transferase activity in cisplatin nephrotoxicity.

    PubMed

    Fliedl, Lukas; Wieser, Matthias; Manhart, Gabriele; Gerstl, Matthias P; Khan, Abdulhameed; Grillari, Johannes; Grillari-Voglauer, Regina

    2014-01-01

    Nephrotoxicity of chemotherapeutics is a major hindrance in the treatment of various tumors. Therefore, test systems that reflect mechanisms of human kidney toxicity are necessary, and to reduce animal testing cell culture based systems have to be developed. One cell type that is of specific interest in this regard are renal proximal tubular epithelial cells, as they reabsorb substances from human primary urine filtrates and thus are exposed to urinary excreted xenobiotics and are a major target of cisplatin toxicity. While animal studies using gamma glutamyl transferase (GGT) knock-out mice or GGT inhibitors show that GGT activity increases kidney toxicity of cisplatin, the use of various cell models gives contradictory results. We therefore used a cell panel of immortalized human renal proximal tubular epithelial (RPTECs) cell lines differing in GGT activity. Low GGT activity resulted in high cisplatin sensitivity, as observed in RPTEC-SV40 cells or after siRNA mediated knock-down of GGT in RPTEC/TERT1 cells that have high GGT activity. However, the addition of GGT did not rescue, but also increased cisplatin sensitivity and adding GGT inhibitor as well as substrate (glutathione) or product (cysteinyl-glycine) of GGT resulted in decreased sensitivity. While our data suggest that the use of cell panels are of value in toxicology and toxicogenomics, they also emphasize on the complex interplay of toxins with the intracellular and extracellular microenvironment. In addition, we hypothesize that especially epithelial barrier formation and polarity of RPTECs need to be considered in toxicity models to validly predict the in vivo situation. PMID:24664430

  6. Methods for purification of glutathione transferases in the earthworm genus Eisenia, and their characterization.

    PubMed

    Borgeraas, J; Nilsen, K; Stenersen, J

    1996-06-01

    Isoenzymes of glutathione transferase (GST) were partially purified from the earthworm species Eisenia andrei and E. veneta using affinity chromatography followed by ion exchange chromatography and reversed-phase HPLC. In E. veneta, five activity peaks, named EvGST Ia, Ib, II, III and IV, were separated by anion exchange chromatography. The GSTs in E. andrei were resolved by cation exchange chromatography into six groups, named EaGST I-VI. Using reversed-phase HPLC, the affinity-purified GSTs from E. andrei and E. veneta were resolved into 14 subunits, named Ea1-Ea14 and Ev1-Ev14, respectively. EaGST I, II, IV and EvGST Ia were further characterized. These forms displayed different substrate specificity towards the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene, ethacrynic acid (ETHA) and cumene hydroperoxide, as well as different subunit composition determined by SDS-PAGE and reversed-phase HPLC. EaGST IV and EvGST Ia showed exceptionally high ETHA activity compared with the other forms. EaGST IV consisted of a homodimeric protein involving subunit Ea6 with an apparent molecular weight of 26.5 kDa, whereas EvGST Ia is composed of two different subunits (Ev9 and Ev10). Amino acid composition and N-terminal analysis of the first 33 residues of Ea6 indicated that the enzyme is most related to the pi class. Subunit Ev10 had 67% identity with Ea6, over the region sequenced (12 residues), but up to 90% identity with GSTs from several nematodes. Exposure of both species to trans-stilbene oxide, 3-methylcholanthrene and phenobarbital for three weeks did not elevate the activity of GST measured with CDNB and ETHA. PMID:8760608

  7. O-GlcNAc Transferase Directs Cell Proliferation in Idiopathic Pulmonary Arterial Hypertension

    PubMed Central

    Barnes, Jarrod W.; Tian, Liping; Heresi, Gustavo A.; Farver, Carol F.; Asosingh, Kewal; Comhair, Suzy A. A.; Aulak, Kulwant S.; Dweik, Raed A.

    2015-01-01

    Background Idiopathic Pulmonary arterial Hypertension (IPAH) is a cardiopulmonary disease characterized by cellular proliferation and vascular remodeling. A more recently recognized characteristic of the disease is dysregulation of glucose metabolism. The primary link between altered glucose metabolism and cell proliferation in IPAH has not been elucidated. We aimed to determine the relationship between glucose metabolism and smooth muscle cell proliferation in IPAH. Methods and Results Human IPAH and control patient lung tissues and pulmonary artery smooth muscle cells (PASMCs) were used to analyze a specific pathway of glucose metabolism, the hexosamine biosynthetic pathway (HBP). We measured the levels of O-linked N-acetylglucosamine modification (O-GlcNAc), O-GlcNAc transferase (OGT), and O-GlcNAc hydrolase (OGA) in control and IPAH cells and tissues. Our data suggests that the activation of the HBP directly increased OGT levels and activity triggering changes in glycosylation and PASMC proliferation. Partial knockdown of OGT in IPAH PASMCs resulted in reduced global O-GlcNAc levels and abrogated PASMC proliferation. The increased proliferation observed in IPAH PASMCs was directly impacted by proteolytic activation of the cell cycle regulator, host cell factor-1 (HCF-1). Conclusions Our data demonstrate that HBP flux is increased in IPAH and drives OGT-facilitated PASMC proliferation through specific proteolysis and direct activation of HCF-1. These findings establish a novel regulatory role for OGT in IPAH, shed a new light on our understanding of the disease pathobiology, and provide opportunities to design novel therapeutic strategies for IPAH. PMID:25663381

  8. 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

  9. Association of γ-glutamyl transferase with premature coronary artery disease

    PubMed Central

    GHATGE, MADANKUMAR; SHARMA, ANKIT; VANGALA, RAJANI KANTH

    2016-01-01

    Accumulating evidence from epidemiological studies suggests that higher γ-glutamyl transferase (GGT) levels in the blood are associated with the incident of cardiovascular disease (CVD), including atherosclerosis, and have prognostic importance. However, to the best of our knowledge, the association of the GGT level with premature coronary artery disease (CAD) in an Asian Indian population has not been evaluated. In the present study, 240 (120 unaffected and 120 CAD affected) young subjects (males, ≤45 years and females, ≤50 years) were selected. The markers assayed were GGT, high-sensitivity C-reactive protein, lipids, secretory phospholipase A2, neopterin, myeloperoxidase, interleukin-6, cystatin-C, tumor necrosis factor-like weak inducer of apoptosis and lipoprotein (a). The plasma GGT levels in these subjects showed a positive correlation with quantitative variables, such as waist circumference, triglycerides, neopterin levels and cross-sectional correlation with qualitative variable smoking. The findings suggest that the subjects in the highest tertile of GGT had a 2.1-fold [odds ratio (OR), 2.104; 95% confidence interval (CI), 1.063–4.165; P=0.033] higher risk of developing premature CAD in comparison with the reference tertile. Furthermore, a 1 U/l increase of GGT (on a log scale) increased the OR by 5.2-fold (OR, 5.208; 95% CI, 1.018–24.624; P=0.048) and 7.4-fold (OR, 7.492; 95% CI, 1.221–45.979; P=0.030) on addition of associated risk factors. In conclusion, the elevated plasma GGT levels potentially indicate increased oxidative stress and the risk of developing premature CAD. Therefore, these findings could be potentially used in the risk stratification of premature CAD following further evaluation. PMID:26998267

  10. Deficiency of glutathione transferase zeta causes oxidative stress and activation of antioxidant response pathways.

    PubMed

    Blackburn, Anneke C; Matthaei, Klaus I; Lim, Cindy; Taylor, Matthew C; Cappello, Jean Y; Hayes, John D; Anders, M W; Board, Philip G

    2006-02-01

    Glutathione S-transferase (GST) zeta (GSTZ1-1) plays a significant role in the catabolism of phenylalanine and tyrosine, and a deficiency of GSTZ1-1 results in the accumulation of maleylacetoacetate and its derivatives maleylacetone (MA) and succinylacetone. Induction of GST subunits was detected in the liver of Gstz1(-/-) mice by Western blotting with specific antisera and high-performance liquid chromatography analysis of glutathione affinity column-purified proteins. The greatest induction was observed in members of the mu class. Induction of NAD(P)H:quinone oxidoreductase 1 and the catalytic and modifier subunits of glutamate-cysteine ligase was also observed. Many of the enzymes that are induced in Gstz1(-/-) mice are regulated by antioxidant response elements that respond to oxidative stress via the Keap1/Nrf2 pathway. It is significant that diminished glutathione concentrations were also observed in the liver of Gstz1(-/-) mice, which supports the conclusion that under normal dietary conditions, the accumulation of electrophilic intermediates such as maleylacetoacetate and MA results in a high level of oxidative stress. Elevated GST activities in the livers of Gstz1(-/-) mice suggest that GSTZ1-1 deficiency may alter the metabolism of some drugs and xenobiotics. Gstz1(-/-) mice given acetaminophen demonstrated increased hepatotoxicity compared with wild-type mice. This toxicity may be attributed to the increased GST activity or the decreased hepatic concentrations of glutathione, or both. Patients with acquired deficiency of GSTZ1-1 caused by therapeutic exposure to dichloroacetic acid for the clinical treatment of lactic acidosis may be at increased risk of drug- and chemical-induced toxicity. PMID:16278372

  11. Functional characterization of glutathione S-transferases associated with insecticide resistance in Tetranychus urticae.

    PubMed

    Pavlidi, Nena; Tseliou, Vasilis; Riga, Maria; Nauen, Ralf; Van Leeuwen, Thomas; Labrou, Nikolaos E; Vontas, John

    2015-06-01

    The two-spotted spider mite Tetranychus urticae is one of the most important agricultural pests world-wide. It is extremely polyphagous and develops resistance to acaricides. The overexpression of several glutathione S-transferases (GSTs) has been associated with insecticide resistance. Here, we functionally expressed and characterized three GSTs, two of the delta class (TuGSTd10, TuGSTd14) and one of the mu class (TuGSTm09), which had been previously associated with striking resistance phenotypes against abamectin and other acaricides/insecticides, by transcriptional studies. Functional analysis showed that all three GSTs were capable of catalyzing the conjugation of both 1-chloro-2,4 dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene(DCNB) to glutathione (GSH), as well as exhibiting GSH-dependent peroxidase activity toward Cumene hydroperoxide (CumOOH). The steady-state kinetics of the T. urticae GSTs for the GSH/CDNB conjugation reaction were determined and compared with other GSTs. The interaction of the three recombinant proteins with several acaricides and insecticides was also investigated. TuGSTd14 showed the highest affinity toward abamectin and a competitive type of inhibition, which suggests that the insecticide may bind to the H-site of the enzyme. The three-dimensional structure of the TuGSTd14 was predicted based on X-ray structures of delta class GSTs using molecular modeling. Structural analysis was used to identify key structural characteristics and to provide insights into the substrate specificity and the catalytic mechanism of TuGSTd14. PMID:26047112

  12. Immunohistochemical localization and activity of glutathione transferase zeta (GSTZ1-1) in rat tissues.

    PubMed

    Lantum, Hoffman B M; Baggs, Raymond B; Krenitsky, Daria M; Board, Philip G; Anders, M W

    2002-06-01

    Glutathione transferase zeta (GSTZ1-1) catalyzes the biotransformation of a range of alpha-haloacids, including dichloroacetic acid (DCA), and the penultimate step in the tyrosine degradation pathway. DCA is a rodent carcinogen and a common drinking water contaminant. DCA also causes multiorgan toxicity in rodents and dogs. The objective of this study was to determine the expression and activities of GSTZ1-1 in rat tissues with maleylacetone and chlorofluoroacetic acid as substrates. GSTZ1-1 protein was detected in most tissues by immunoblot analysis after immunoprecipitation of GSTZ1-1 and by immunohistochemical analysis; intense staining was observed in the liver, testis, and prostate; moderate staining was observed in the brain, heart, pancreatic islets, adrenal medulla, and the epithelial lining of the gastrointestinal tract, airways, and bladder; and sparse staining was observed in the renal juxtaglomerular regions, skeletal muscle, and peripheral nerve tissue. These patterns of expression corresponded to GSTZ1-1 activities in the different tissues with maleylacetone and chlorofluoroacetic acid as substrates. Specific activities ranged from 258 +/- 17 (liver) to 1.1 +/- 0.4 (muscle) nmol/min/mg of protein with maleylacetone as substrate and from 4.6 +/- 0.89 (liver) to 0.09 +/- 0.01 (kidney) nmol/min/mg of protein with chlorofluoroacetic acid as substrate. Rats given DCA had reduced amounts of immunoreactive GSTZ1-1 protein and activities of GSTZ1-1 in most tissues, especially in the liver. These findings indicate that the DCA-induced inactivation of GSTZ1-1 in different tissues may result in multiorgan disorders that may be associated with perturbed tyrosine metabolism. PMID:12019185

  13. Urinary π-glutathione S-transferase Predicts Advanced Acute Kidney Injury Following Cardiovascular Surgery

    PubMed Central

    Shu, Kai-Hsiang; Wang, Chih-Hsien; Wu, Che-Hsiung; Huang, Tao-Min; Wu, Pei-Chen; Lai, Chien-Heng; Tseng, Li-Jung; Tsai, Pi-Ru; Connolly, Rory; Wu, Vin-Cent

    2016-01-01

    Urinary biomarkers augment the diagnosis of acute kidney injury (AKI), with AKI after cardiovascular surgeries being a prototype of prognosis scenario. Glutathione S-transferases (GST) were evaluated as biomarkers of AKI. Urine samples were collected in 141 cardiovascular surgical patients and analyzed for urinary alpha-(α-) and pi-(π-) GSTs. The outcomes of advanced AKI (KDIGO stage 2, 3) and all-cause in-patient mortality, as composite outcome, were recorded. Areas under the receiver operator characteristic (ROC) curves and multivariate generalized additive model (GAM) were applied to predict outcomes. Thirty-eight (26.9%) patients had AKI, while 12 (8.5%) were with advanced AKI. Urinary π-GST differentiated patients with/without advanced AKI or composite outcome after surgery (p < 0.05 by generalized estimating equation). Urinary π-GST predicted advanced AKI at 3 hrs post-surgery (p = 0.033) and composite outcome (p = 0.009), while the corresponding ROC curve had AUC of 0.784 and 0.783. Using GAM, the cutoff value of 14.7 μg/L for π-GST showed the best performance to predict composite outcome. The addition of π-GST to the SOFA score improved risk stratification (total net reclassification index = 0.47). Thus, urinary π-GST levels predict advanced AKI or hospital mortality after cardiovascular surgery and improve in SOFA outcome assessment specific to AKI. PMID:27527370

  14. Exploiting the Substrate Promiscuity of Hydroxycinnamoyl-CoA:Shikimate Hydroxycinnamoyl Transferase to Reduce Lignin

    PubMed Central

    Eudes, Aymerick; Pereira, Jose H.; Yogiswara, Sasha; Wang, George; Teixeira Benites, Veronica; Baidoo, Edward E.K.; Lee, Taek Soon; Adams, Paul D.; Keasling, Jay D.; Loqué, Dominique

    2016-01-01

    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 to 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. PMID:26858288

  15. Genetic Deficiency of Glutathione S-Transferase P Increases Myocardial Sensitivity to Ischemia-Reperfusion Injury

    PubMed Central

    Conklin, Daniel J.; Guo, Yiru; Jagatheesan, Ganapathy; Kilfoil, Peter; Haberzettl, Petra; Hill, Bradford G.; Baba, Shahid P.; Guo, Luping; Wetzelberger, Karin; Obal, Detlef; Rokosh, D. Gregg; Prough, Russell A.; Prabhu, Sumanth D.; Velayutham, Murugesan; Zweier, Jay L.; Hoetker, David; Riggs, Daniel W.; Srivastava, Sanjay; Bolli, Roberto; Bhatnagar, Aruni

    2016-01-01

    Rationale Myocardial ischemia-reperfusion (I/R) results in the generation of oxygen-derived free radicals and the accumulation of lipid peroxidation-derived unsaturated aldehydes. However, the contribution of aldehydes to myocardial I/R injury has not been assessed. Objective We tested the hypothesis that removal of aldehydes by glutathione S-transferase P (GSTP) diminishes I/R injury. Methods and Results In adult male C57BL/6 mouse hearts, Gstp1/2 was the most abundant GST transcript followed by Gsta4 and Gstm4.1, and GSTP activity was a significant fraction of the total GST activity. mGstp1/2 deletion reduced total GST activity, but no compensatory increase in GSTA and GSTM or major antioxidant enzymes was observed. Genetic deficiency of GSTP did not alter cardiac function, but in comparison with hearts from wild-type (WT) mice, the hearts isolated from GSTP-null mice were more sensitive to I/R injury. Disruption of the GSTP gene also increased infarct size after coronary occlusion in situ. Ischemia significantly increased acrolein in hearts, and GSTP deficiency induced significant deficits in the metabolism of the unsaturated aldehyde, acrolein, but not in the metabolism 4-hydroxy-trans-2-nonenal (HNE) or trans-2-hexanal; and, upon ischemia, the GSTP-null hearts accumulated more acrolein-modified proteins than WT hearts. GSTP-deficiency did not affect I/R-induced free radical generation, JNK activation or depletion of reduced glutathione. Acrolein-exposure induced a hyperpolarizing shift in INa, and acrolein-induced cell death was delayed by SN-6, a Na+/Ca++ exchange inhibitor. Cardiomyocytes isolated from GSTP-null hearts were more sensitive than WT myocytes to acrolein-induced protein crosslinking and cell death. Conclusions GSTP protects the heart from I/R injury by facilitating the detoxification of cytotoxic aldehydes such as acrolein. PMID:26169370

  16. Expression of glutathione, glutathione peroxidase and glutathione S-transferase pi in canine mammary tumors

    PubMed Central

    2014-01-01

    Background Glutathione (GSH) is one of the most important agents of the antioxidant defense system of the cell because, in conjunction with the enzymes glutathione peroxidase (GSH-Px) and glutathione S transferase pi (GSTpi), it plays a central role in the detoxification and biotransformation of chemotherapeutic drugs. This study evaluated the expression of GSH and the GSH-Px and GSTpi enzymes by immunohistochemistry in 30 canine mammary tumors, relating the clinicopathological parameters, clinical outcome and survival of the bitches. In an in vitro study, the expression of the genes glutamate cysteine ligase (GCLC) and glutathione synthetase (GSS) that synthesize GSH and GSH-Px gene were verified by qPCR and subjected to treatment with doxorubicin, to check the resistance of cancer cells to chemotherapy. Results The immunohistochemical expression of GSH, GSH-Px and GSTpi was compared with the clinical and pathological characteristics and the clinical outcome in the bitches, including metastasis and death. The results showed that high immunoexpression of GSH was correlated to the absence of tumor ulceration and was present in dogs without metastasis (P < 0.05). There was significant correlation of survival with the increase of GSH (P < 0.05). The expression of the GSH-Px and GSTpi enzymes showed no statistically significant correlation with the analyzed variables (p > 0.05). The analysis of the relative expression of genes responsible for the synthesis of GSH (GCLC and GSS) and GSH-Px by quantitative PCR was done with cultured cells of 10 tumor fragments from dogs with mammary tumors. The culture cells showed a decrease in GCLC and GSS expression when compared with no treated cells (P < 0.05). High GSH immunoexpression was associated with better clinical outcomes. Conclusion Therefore, high expression of the GSH seems to play an important role in the clinical outcome of patients with mammary tumors and suggest its use as prognostic marker. The in

  17. Inhibition characteristics of hypericin on rat small intestine glutathione-S-transferases.

    PubMed

    Tuna, Gamze; Kulaksiz Erkmen, Gulnihal; Dalmizrak, Ozlem; Dogan, Arin; Ogus, I Hamdi; Ozer, Nazmi

    2010-10-01

    Glutathione-S-transferases constitute a family of enzymes involving in the detoxification of xenobiotics, signalling cascades and serving as ligandins or/and catalyzing the conjugation of various chemicals and drugs. The widely expressed cytosolic GST-pi is a marker protein in various cancers and its increased concentration is linked to drug resistance. GST-pi is autoregulated by S-glutathionylation and it catalyzes the S-glutathionylation of other proteins in response to oxidative or nitrosative stress. S-glutathionylation of GST-pi results in multimer formation and the breakage of ligand binding interactions with c-Jun NH(2)-terminal kinase (JNK). Another widely expressed GST enzyme, GST-alpha is assumed as a marker in hepatocellular damage, is implicated in cancer, asthma, cardiovascular disease and response to chemotherapy. Although, it was shown that hypericin binds and inhibits GST-alpha and GST-pi, the inhibition characteristics have not been investigated in detail. The aim of this study was to investigate the effects of hypericin on major GSTs; GST-alpha and GST-pi purified from rat small intestine. When GSH used as varied substrate the inhibition pattern with hypericin was uncompetitive for GST-alpha (K(i)=0.16 + or - 0.02 microM) and noncompetitive for GST-pi (K(i) = 2.46 + or - 0.43 microM). While using CDNB (1-chloro-2,4-dinitrobenzene) as the varied substrate, the inhibition patterns were noncompetitive for GST-alpha and competitive for GST-pi; K(i) values for GST-alpha and GST-pi were 1.91 + or - 0.21 and 0.55 + or - 0.07 microM, respectively. Since hypericin accumulated in cancer cells and important in photodynamic therapy (PDT), inhibition of GST-alpha and GST-pi by hypericin might increase the effectivity of the treatment. Considering that GST-pi is responsible for the drug resistance its inhibition might increase the benefit obtained from chemotherapy. PMID:20637187

  18. Detection and adequacy evaluation of erythrocyte glutathione transferase on levels of circulating toxins in hemodialysis patients.

    PubMed

    Yin, Rui; Qiu, Hui; Zuo, Huaiyun; Cui, Min; Zhai, Nailiang; Zheng, Hongguang; Zhang, Dewei; Huo, Ping; Hong, Min

    2016-08-01

    To explore detection and adequacy evaluation of erythrocyte glutathione S transferase (GST) on levels of circulating toxins in hemodialysis patients in Qinhuangdao region in China, this study divided 84 cases of long-term, end-stage hemodialysis patients into 2 groups: one group of 33 cases of adequate hemodialysis (spKt/V ≥ 1.3) and another group of 51 cases of inadequate hemodialysis (spKt/V < 1.3), according to the urea index value of the unit chamber model (spKt/V). Another 50 cases of subjects found healthy by a physical examination were taken as the control group, and the differences in the related clinical and biochemical indexes of the 3 groups were compared and analyzed. The levels of GST, creatinine, high sensitivity C-reactive protein (hs-CRP), transferrin saturation (TSAT), parathyroid hormone (PTH), interleukin-2,6,8 (IL-2,6,8) and tumor necrosis factor-a (TNF-a) in the hemodialysis group were significantly higher than those in the control group (P < 0.05), and GST, IL-2, 6, 8, and TNF-a levels in the inadequate hemodialysis group were significantly higher than in the adequate hemodialysis group (P < 0.05). Pearson's relevant analysis showed that the levels of GST and spKt/V, IL-2, IL-6, IL-8, and TNF-a have a positive correlation (P < 0.05), and they have no correlation with levels of creatinine, hs-CRP, TSAT, and PHT (P > 0.05). There were 23 patients with levels of spKt/V ≥ 1.3 after adjusting the dialysis solution for 51 cases of inadequate hemodialysis patients, and the GST level after the adjustment was significantly lower than that before the adjustment, but still higher than that in the adequate dialysis group. This concludes that the maintenance of hemodialysis in patients has certain relevance on spKt/V and associated inflammatory factors. Through the study, it can be determined that GST can effectively respond to adequate hemodialysis, which has a guiding significance on adjusting the blood dialysis solution in clinical practice. PMID

  19. Habitual consumption of fruits and vegetables: associations with human rectal glutathione S-transferase.

    PubMed

    Wark, Petra A; Grubben, Marina J A L; Peters, Wilbert H M; Nagengast, Fokko M; Kampman, Ellen; Kok, Frans J; van 't Veer, Pieter

    2004-11-01

    The glutathione (GSH)/glutathione S-transferase (GST) system is an important detoxification system in the gastrointestinal tract. A high activity of this system may benefit cancer prevention. The aim of the study was to assess whether habitual consumption of fruits and vegetables, especially citrus fruits and brassica and allium vegetables, is positively associated with parameters reflecting the activity of the GSH/GST enzyme system in human rectal mucosa. GST enzyme activity, GST isoenzyme levels of GST-alpha (A1-1, A1-2 and A2-2), -mu (M1-1) and -pi (P1-1), and GSH levels were measured in rectal biopsies from 94 subjects. Diet, lifestyle, GSTM1 and GSTT1 null polymorphisms were assessed. Mean GST enzyme activity was 237 nmol/min/mg protein (SD = 79). Consumption of citrus fruits was positively associated with GST enzyme activity [difference between high and low consumption: 28.9 (95% confidence interval (CI) = 9.3-48.6) nmol/min/mg protein], but was not associated with the other parameters. A positive association with brassica vegetables was found among carriers of the GSTM1-plus genotype [difference between high and low consumption: 22.6 (95% CI = 0.2-45.0) nmol/min/mg protein], but not among GSTM1-null individuals (-25.8 nmol/min/mg protein, 95% CI = -63.3-11.8). This is in line with a positive association between consumption of brassica vegetables and GSTM isoenzyme level [difference between high and low consumption: 67.5%, 95% CI = (6.8-162.7)]. Consumption of allium vegetables was not associated with GST enzyme activity, but negatively with GSTP1-1 levels [difference between high and low consumption: -23.3%, 95% CI = (-35.5; -8.6)]. Associations were similar among those with the GSTT1-plus and GSTT1-null genotype. In conclusion, variations in habitual consumption of fruits, particularly citrus fruits, and of vegetables, in particular brassica vegetables, among those with the GSTM1-plus genotype, may contribute to variations in human rectal GST enzyme

  20. Proteomic and immunochemical characterization of glutathione transferase as a new allergen of the nematode Ascaris lumbricoides.

    PubMed

    Acevedo, Nathalie; Mohr, Jens; Zakzuk, Josefina; Samonig, Martin; Briza, Peter; Erler, Anja; Pomés, Anna; Huber, Christian G; Ferreira, Fatima; Caraballo, Luis

    2013-01-01

    Helminth infections and allergy have evolutionary and clinical links. Infection with the nematode Ascaris lumbricoides induces IgE against several molecules including invertebrate pan-allergens. These antibodies influence the pathogenesis and diagnosis of allergy; therefore, studying parasitic and non-parasitic allergens is essential to understand both helminth immunity and allergy. Glutathione transferases (GSTs) from cockroach and house dust mites are clinically relevant allergens and comparative studies between them and the GST from A. lumbricoides (GSTA) are necessary to evaluate their allergenicity. We sought to analyze the allergenic potential of GSTA in connection with the IgE response to non-parasitic GSTs. IgE to purified GSTs from Ascaris (nGSTA and rGSTA), house dust mites (rDer p 8, nBlo t 8 and rBlo t 8), and cockroach (rBla g 5) was measured by ELISA in subjects from Cartagena, Colombia. Also, multidimensional proteomic approaches were used to study the extract of A. lumbricoides and investigate the existence of GST isoforms. We found that among asthmatics, the strength of IgE levels to GSTA was significantly higher than to mite and cockroach GSTs, and there was a strong positive correlation between IgE levels to these molecules. Specific IgE to GSTA was found in 13.2% of controls and 19.5% of asthmatics. In addition nGSTA induced wheal and flare in skin of sensitized asthmatics indicating that it might be of clinical relevance for some patients. Frequency and IgE levels to GSTA were higher in childhood and declined with age. At least six GST isoforms in A. lumbricoides bind human IgE. Four isoforms were the most abundant and several amino acid substitutions were found, mainly on the N-terminal domain. In conclusion, a new allergenic component of Ascaris has been discovered; it could have clinical impact in allergic patients and influence the diagnosis of mite and cockroach allergy in tropical environments. PMID:24223794

  1. Prognostic significance of glutathione S-transferase-pi in invasive breast cancer.

    PubMed

    Huang, Jingxiang; Tan, Puay-Hoon; Thiyagarajan, Jayabaskar; Bay, Boon-Huat

    2003-06-01

    Glutathione S-transferase pi (GST-pi), a Phase II detoxification enzyme, has recently been implicated in protection against apoptosis. Expression of GST-pi and Bcl-2 protein, an established apoptosis marker, was analyzed by immunohistochemistry in 116 cases of infiltrative ductal breast carcinomas in Singapore women. The markers were correlated with apoptosis detected by the TUNEL method and clinico-pathological parameters. There were 67 (58%) GST-pi-positive breast tumors and 43 (37%) Bcl-2-positive tumors. In a large proportion of GST-pi-positive/Bcl-2-positive tumors, there was a distinct accumulation of the GST-pi enzyme within the nucleus of cancer cells when examined by double immunofluorescence labeling under confocal microscopy. GST-pi immunoreactivity was not significantly correlated with any of the traditional histologic factors known to influence prognosis, whereas Bcl-2 overexpression was associated with reduced size of primary tumor (P =.021) and positive estrogen receptor status (P =.001). Univariate analysis revealed that GST-pi-positive, Bcl-2-positive, and lower histological grade tumors had decreased levels of apoptosis (P =.024, P =.011, and P =.029, respectively). However, multivariate analysis showed that histological grade and Bcl-2, but not GST-pi, immunoreactivity were correlated with apoptotic status. The Kaplan-Meier disease-free survival curves showed a significant difference between GST-pi-positive and GST-pi-negative breast cancer cases (P =.002). Disease-free survival in patients with GST-pi-positive tumors was also worse than that in patients with GST-pi-negative tumors in the group who had adjuvant chemotherapy (P =.04). In patients who were lymph node positive, GST-pi immunopositivity was found to influence disease-free survival. Recurrence of tumors was also significantly affected by GST-pi immunoreactivity (relative risk of 8.1). The findings indicate that GST-pi-positive tumors are more aggressive and have a poorer prognosis than

  2. Infection with Salmonella typhimurium modulates the immune response to Schistosoma mansoni glutathione-S-transferase.

    PubMed Central

    Comoy, E E; Vendeville, C; Capron, A; Thyphronitis, G

    1997-01-01

    Immune response polarization is controlled by several factors, including cytokines, antigen-presenting cells, antigen dose, and others. We have previously shown that adjuvants and live vectors play a critical role in polarization. Thus, immunization with the Schistosoma mansoni 28-kDa glutathione-S-transferase (Sm28-GST) in aluminum hydroxide induced a type 2 cytokine profile and the production of immunoglobulin G1 (IgG1)- and IgE-specific antibodies. In contrast, mice infected with recombinant Salmonella typhimurium expressing Sm28-GST developed a type 1 cytokine profile and produced IgG2a-specific antibodies against Sm28-GST and Salmonella antigens. In this study, to determine if S. typhimurium not expressing Sm28-GST would still influence the type of the response against this antigen, we compared the profiles of the immune responses generated against Sm28-GST administered in alum in mice infected and not infected with S. typhimurium. Infected mice generated both IgG1 and IgG2a antibodies against Sm28-GST, while noninfected mice produced only IgG1 anti-Sm28-GST antibodies. Moreover, interleukin-4 (IL-4) mRNA expression in infected mice was near background levels, while gamma interferon (IFN-gamma) mRNA expression in coinfected mice was significantly higher than in mice immunized with Sm28-GST in alum only. However, after antigen-specific stimulation in vitro with Sm28-GST, levels of IL-4 and IFN-gamma cytokine production were similar in the two groups of mice. These results suggest that (i) the immune milieu produced during an infection may modify the response against an irrelevant antigen and (ii) isotype switching may be influenced by the cytokine environment of a bystander immune response, even though the specific antigen-driven cytokine production is not modified. Thus, the isotypic profile is not always an absolute reflection of the cytokines produced by antigen-specific Th cells. PMID:9234784

  3. Chemical Reactivity Window Determines Prodrug Efficiency toward Glutathione Transferase Overexpressing Cancer Cells.

    PubMed

    van Gisbergen, Marike W; Cebula, Marcus; Zhang, Jie; Ottosson-Wadlund, Astrid; Dubois, Ludwig; Lambin, Philippe; Tew, Kenneth D; Townsend, Danyelle M; Haenen, Guido R M M; Drittij-Reijnders, Marie-José; Saneyoshi, Hisao; Araki, Mika; Shishido, Yuko; Ito, Yoshihiro; Arnér, Elias S J; Abe, Hiroshi; Morgenstern, Ralf; Johansson, Katarina

    2016-06-01

    Glutathione transferases (GSTs) are often overexpressed in tumors and frequently correlated to bad prognosis and resistance against a number of different anticancer drugs. To selectively target these cells and to overcome this resistance we previously have developed prodrugs that are derivatives of existing anticancer drugs (e.g., doxorubicin) incorporating a sulfonamide moiety. When cleaved by GSTs, the prodrug releases the cytostatic moiety predominantly in GST overexpressing cells, thus sparing normal cells with moderate enzyme levels. By modifying the sulfonamide it is possible to control the rate of drug release and specifically target different GSTs. Here we show that the newly synthesized compounds, 4-acetyl-2-nitro-benzenesulfonyl etoposide (ANS-etoposide) and 4-acetyl-2-nitro-benzenesulfonyl doxorubicin (ANS-DOX), function as prodrugs for GSTA1 and MGST1 overexpressing cell lines. ANS-DOX, in particular, showed a desirable cytotoxic profile by inducing toxicity and DNA damage in a GST-dependent manner compared to control cells. Its moderate conversion of 500 nmol/min/mg, as catalyzed by GSTA1, seems hereby essential since the more reactive 2,4-dinitrobenzenesulfonyl doxorubicin (DNS-DOX) (14000 nmol/min/mg) did not display a preference for GSTA1 overexpressing cells. DNS-DOX, however, effectively killed GSTP1 (20 nmol/min/mg) and MGST1 (450 nmol/min/mg) overexpressing cells as did the less reactive 4-mononitrobenzenesulfonyl doxorubicin (MNS-DOX) in a MGST1-dependent manner (1.5 nmol/min/mg) as shown previously. Furthermore, we show that the mechanism of these prodrugs involves a reduction in GSH levels as well as inhibition of the redox regulatory enzyme thioredoxin reductase 1 (TrxR1) by virtue of their electrophilic sulfonamide moiety. TrxR1 is upregulated in many tumors and associated with resistance to chemotherapy and poor patient prognosis. Additionally, the prodrugs potentially acted as a general shuttle system for DOX, by overcoming resistance

  4. Steady-state kinetics and chemical mechanism of octopus hepatopancreatic glutathione transferase.

    PubMed Central

    Tang, S S; Chang, G G

    1995-01-01

    The kinetic mechanism of glutathione S-transferase (GST) from Octopus vulgaris hepatopancreas was investigated by steady-state analysis. Initial-velocity studies showed an intersecting pattern, which suggests a sequential kinetic mechanism for the enzyme. Product-inhibition patterns by chloride and the conjugate product were all non-competitive with respect to glutathione or 1-chloro-2,4-dinitrobenzene (CDNB), which indicates that the octopus digestive gland GST conforms to a steady-state sequential random Bi Bi kinetic mechanism. Dead-end inhibition patterns indicate that ethacrynic acid ([2,3-dichloro-4-(2-methyl-enebutyryl) phenoxy]acetic acid) binds at the hydrophobic H-site, norophthalmic acid (gamma-glutamylalanylglycine) binds at the glutathione G-site, and glutathione-ethacrynate conjugate occupied both H- and G-sites of the enzyme. The chemical mechanism of the enzyme was examined by pH and kinetic solvent-isotope effects. At pH (and p2H) = 8.011, in which kcat. was independent of pH or p2H, the solvent isotope effects on V and V/KmGSH were near unity, in the range 1.069-1.175. An inverse isotope effect was observed for V/KmCDNB (0.597), presumably resulting from the hydrogen-bonding of enzyme-bound glutathione, which has pKa of 6.83 +/- 0.04, a value lower by 2.34 pH units than the pKa of glutathione in aqueous solution. This lowering of the pKa value for the sulphydryl group of the bound glutathione was presumably due to interaction with the active site Tyr7, which had a pKa value of 8.46 +/- 0.09 that was raised to 9.63 +/- 0.08 in the presence of glutathione thiolate. Subsequent chemical reaction involves attacking of thiolate anion at the electrophilic substrate with the formation of a negatively charged Meisenheimer complex, which is the rate-limiting step of the reaction. Images Scheme 2 PMID:7619078

  5. In vitro kinetics of hepatic glutathione s-transferase conjugation in largemouth bass and brown bullheads

    SciTech Connect

    Gallagher, E.P.; Sheehy, K.M.; Lame, M.W.; Segall, H.J.

    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 broad 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 concentrations

  6. 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). PMID:26461371

  7. Catalytic characterization of human microsomal glutathione S-transferase 2: identification of rate-limiting steps.

    PubMed

    Ahmad, Shabbir; Niegowski, Damian; Wetterholm, Anders; Haeggström, Jesper Z; Morgenstern, Ralf; Rinaldo-Matthis, Agnes

    2013-03-12

    Microsomal glutathione S-transferase 2 (MGST2) is a 17 kDa trimeric integral membrane protein homologous to leukotriene C4 synthase (LTC4S). MGST2 has been suggested to catalyze the biosynthesis of the pro-inflammatory mediator leukotriene C4 (LTC4) in cells devoid of LTC4S. A detailed biochemical study of MGST2 is critical for the understanding of its cellular function and potential role as an LTC4-producing enzyme. Here we have characterized the substrate specificity and catalytic properties of purified MGST2 by steady-state and pre-steady-state kinetic experiments. In comparison with LTC4S, which has a catalytic efficiency of 8.7 × 10(5) M(-1) s(-1), MGST2, with a catalytic efficiency of 1.8 × 10(4) M(-1) s(-1), is considerably less efficient in producing LTC4. However, the two enzymes display a similar KM(LTA4) of 30-40 μM. While LTC4S has one activated glutathione (GSH) (forming a thiolate) per enzyme monomer, the MGST2 trimer seems to display only third-of-the-sites reactivity for thiolate activation, which in part would explain its lower catalytic efficiency. Furthermore, MGST2 displays GSH-dependent peroxidase activity of ∼0.2 μmol min(-1) mg(-1) toward several lipid hydroperoxides. MGST2, but not LTC4S, is efficient in catalyzing conjugation of the electrophilic substrate 1-chloro-2,4-dinitrobenzene (CDNB) and the lipid peroxidation product 4-hydroxy-2-nonenal with GSH. Using stopped-flow pre-steady-state kinetics, we have characterized the full catalytic reaction of MGST2 with CDNB and GSH as substrates, showing an initial rapid equilibrium binding of GSH followed by thiolate formation. Burst kinetics for the CDNB-GSH conjugation step was observed only at low GSH concentrations (thiolate anion formation becoming rate-limiting under these conditions). Product release is rapid and does not limit the overall reaction. Therefore, in general, the chemical conjugation step is rate-limiting for MGST2 at physiological GSH concentrations. MGST2 and LTC4S

  8. Proteomic and Immunochemical Characterization of Glutathione Transferase as a New Allergen of the Nematode Ascaris lumbricoides

    PubMed Central

    Acevedo, Nathalie; Mohr, Jens; Zakzuk, Josefina; Samonig, Martin; Briza, Peter; Erler, Anja; Pomés, Anna; Huber, Christian G.; Ferreira, Fatima; Caraballo, Luis

    2013-01-01

    Helminth infections and allergy have evolutionary and clinical links. Infection with the nematode Ascaris lumbricoides induces IgE against several molecules including invertebrate pan-allergens. These antibodies influence the pathogenesis and diagnosis of allergy; therefore, studying parasitic and non-parasitic allergens is essential to understand both helminth immunity and allergy. Glutathione transferases (GSTs) from cockroach and house dust mites are clinically relevant allergens and comparative studies between them and the GST from A. lumbricoides (GSTA) are necessary to evaluate their allergenicity. We sought to analyze the allergenic potential of GSTA in connection with the IgE response to non-parasitic GSTs. IgE to purified GSTs from Ascaris (nGSTA and rGSTA), house dust mites (rDer p 8, nBlo t 8 and rBlo t 8), and cockroach (rBla g 5) was measured by ELISA in subjects from Cartagena, Colombia. Also, multidimensional proteomic approaches were used to study the extract of A. lumbricoides and investigate the existence of GST isoforms. We found that among asthmatics, the strength of IgE levels to GSTA was significantly higher than to mite and cockroach GSTs, and there was a strong positive correlation between IgE levels to these molecules. Specific IgE to GSTA was found in 13.2% of controls and 19.5% of asthmatics. In addition nGSTA induced wheal and flare in skin of sensitized asthmatics indicating that it might be of clinical relevance for some patients. Frequency and IgE levels to GSTA were higher in childhood and declined with age. At least six GST isoforms in A. lumbricoides bind human IgE. Four isoforms were the most abundant and several amino acid substitutions were found, mainly on the N-terminal domain. In conclusion, a new allergenic component of Ascaris has been discovered; it could have clinical impact in allergic patients and influence the diagnosis of mite and cockroach allergy in tropical environments. PMID:24223794

  9. O-linked-N-acetylglucosamine modification of mammalian Notch receptors by an atypical O-GlcNAc transferase Eogt1

    SciTech Connect

    Sakaidani, Yuta; Ichiyanagi, Naoki; Saito, Chika; Nomura, Tomoko; Ito, Makiko; Nishio, Yosuke; Nadano, Daita; Matsuda, Tsukasa; Furukawa, Koichi; Okajima, Tetsuya

    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 shown 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.

  10. An Entamoeba histolytica ADP-ribosyl transferase from the diphtheria toxin family modifies the bacterial elongation factor Tu.

    PubMed

    Avila, Eva E; Rodriguez, Orlando I; Marquez, Jaqueline A; Berghuis, Albert M

    2016-06-01

    ADP-ribosyl transferases are enzymes involved in the post-translational modification of proteins; they participate in multiple physiological processes, pathogenesis and host-pathogen interactions. Several reports have characterized the functions of these enzymes in viruses, prokaryotes and higher eukaryotes, but few studies have reported ADP-ribosyl transferases in lower eukaryotes, such as parasites. The locus EHI_155600 from Entamoeba histolytica encodes a hypothetical protein that possesses a domain from the ADP-ribosylation superfamily; this protein belongs to the diphtheria toxin family according to a homology model using poly-ADP-ribosyl polymerase 12 (PARP12 or ARTD12) as a template. The recombinant protein expressed in Escherichia coli exhibited in vitro ADP-ribosylation activity that was dependent on the time and temperature. Unlabeled βNAD(+), but not ADP-ribose, competed in the enzymatic reaction using biotin-βNAD(+) as the ADP-ribose donor. The recombinant enzyme, denominated EhToxin-like, auto-ADP-ribosylated and modified an acceptor from E. coli that was identified by MS/MS as the elongation factor Tu (EF-Tu). To the best of our knowledge, this is the first report to identify an ADP-ribosyl transferase from the diphtheria toxin family in a protozoan parasite. The known toxins from this family (i.e., the diphtheria toxin, the Pseudomonas aeruginosa toxin Exo-A, and Cholix from Vibrio cholerae) modify eukaryotic elongation factor two (eEF-2), whereas the amoeba EhToxin-like modified EF-Tu, which is another elongation factor involved in protein synthesis in bacteria and mitochondria. PMID:27234208

  11. Phosphoethanolamine Transferase LptA in Haemophilus ducreyi Modifies Lipid A and Contributes to Human Defensin Resistance In Vitro

    PubMed Central

    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. PMID:25902140

  12. Maternally supplied S-acyl-transferase is required for crystalloid organelle formation and transmission of the malaria parasite.

    PubMed

    Santos, Jorge M; Duarte, Neuza; Kehrer, Jessica; Ramesar, Jai; Avramut, M Cristina; Koster, Abraham J; Dessens, Johannes T; Frischknecht, Friedrich; Chevalley-Maurel, Séverine; Janse, Chris J; Franke-Fayard, Blandine; Mair, Gunnar R

    2016-06-28

    Transmission of the malaria parasite from the mammalian host to the mosquito vector requires the formation of adequately adapted parasite forms and stage-specific organelles. Here we show that formation of the crystalloid-a unique and short-lived organelle of the Plasmodium ookinete and oocyst stage required for sporogony-is dependent on the precisely timed expression of the S-acyl-transferase DHHC10. DHHC10, translationally repressed in female Plasmodium berghei gametocytes, is activated translationally during ookinete formation, where the protein is essential for the formation of the crystalloid, the correct targeting of crystalloid-resident protein LAP2, and malaria parasite transmission. PMID:27303037

  13. Glutathione S-transferase and gamma-glutamyl transpeptidase activities in cultured rat hepatocytes treated with tocotrienol and tocopherol.

    PubMed

    Ong, F B; Wan Ngah, W Z; Shamaan, N A; Md Top, A G; Marzuki, A; Khalid, A K

    1993-09-01

    1. The effect of tocotrienol and tocopherol on glutathione S-transferase (GST) and gamma-glutamyl transpeptidase (GGT) activities in cultured rat hepatocytes were investigated. 2. Tocotrienol and tocopherol significantly decreased GGT activities at 5 days in culture but tocotrienol also significantly decreased GGT activities at 1-2 days. 3. Tocotrienol and tocopherol treatment significantly decreased GST activities at 3 days compared to the control but tocotrienol also decreased GST activities at 1-3 days. 4. Tocotrienol showed a more pronounced effect at a dosage of greater than 50 microM tocotrienol at 1-3 days in culture compared to the control. PMID:7903615

  14. The molecular basis for the post-translational addition of amino acids by L/F transferase in the N-end rule pathway.

    PubMed

    Fung, Angela Wai S; Fahlman, Richard P

    2015-01-01

    The N-end rule pathway is a conserved targeted proteolytic process observed in organisms ranging from eubacteria to mammals. The N-end rule relates the metabolic stability of a protein to its N-terminal amino acid residue. The identity of the N-terminal amino acid residue is a primary degradation signal, often referred to as an N-degron, which is recognized by the components of the N-end rule when it is a destabilizing N-terminus. N-degrons may be exposed by non-processive proteolytic cleavages or by post-translational modifications. One modification is the post-translational addition of amino acids to the N-termini of proteins, a reaction catalyzed by aminoacyl-tRNA protein transferases. The aminoacyl-tRNA protein transferase in eubacteria like Escherichia coli is L/F transferase. Recent investigations have reported unexpected observations regarding the L/F transferase catalytic mechanism and its mechanisms of substrate recognition. Additionally, recent proteome-wide identification of putative in vivo substrates facilitates hypothesis into the yet elusive biological functions of the prokaryotic N-end rule pathway. Here we summarize the recent findings on the molecular mechanisms of catalysis and substrate recognition by the E. coli L/F transferase in the prokaryotic N-end rule pathway. PMID:25692952

  15. Isolation and identification of kahweol palmitate and cafestol palmitate as active constituents of green coffee beans that enhance glutathione S-transferase activity in the mouse.

    PubMed

    Lam, L K; Sparnins, V L; Wattenberg, L W

    1982-04-01

    Glutathione (GSH) S-transferase is a major detoxification enzyme system that catalyzes the binding of a variety of electrophiles, including reactive forms of chemical carcinogens, to GSH. Green coffee beans fed in the diet induced increased GSH S-transferase activity in the mucosa of the small intestine and in the liver of mice. A potent compound that induces increased GSH S-transferase activity was isolated from green coffee beans and identified as kahweol palmitate. The corresponding free alcohol, kahweol, and its synthetic monoacetate are also potent inducers of the activity of GSH S-transferase. A similar diterpene ester, cafestol palmitate, isolated from green coffee beans was active but less so than was kahweol palmitate. Likewise, the corresponding alcohol, cafestol, and its monoacetate showed moderate potency as inducers of increased GSH S-transferase activity. Kahweol palmitate and cafestol palmitate were extracted from green coffee beans into petroleum ether. The petroleum ether extract was fractionated by preparative normal-phase and reverse-phase liquid chromatographies successively. Final purification with silver nitrate-impregnated thin-layer chromatography yielded the pure palmitates of cafestol and kahweol. The structures were determined by examination of the spectroscopic data of the esters and their parent alcohols and by derivative comparison. PMID:7059995

  16. Influence of glutathione S-transferase B (ligandin) on the intermembrane transfer of bilirubin. Implications for the intracellular transport of nonsubstrate ligands in hepatocytes.

    PubMed Central

    Zucker, S D; Goessling, W; Ransil, B J; Gollan, J L

    1995-01-01

    To examine the hypothesis that glutathione S-transferases (GST) play an important role in the hepatocellular transport of hydrophobic organic anions, the kinetics of the spontaneous transfer of unconjugated bilirubin between membrane vesicles and rat liver glutathione S-transferase B (ligandin) was studied, using stopped-flow fluorometry. Bilirubin transfer from glutathione S-transferase B to phosphatidylcholine vesicles was best described by a single exponential function, with a rate constant of 8.0 +/- 0.7 s-1 (+/- SD) at 25 degrees C. The variations in transfer rate with respect to acceptor phospholipid concentration provide strong evidence for aqueous diffusion of free bilirubin. This finding was verified using rhodamine-labeled microsomal membranes as acceptors. Bilirubin transfer from phospholipid vesicles to GST also exhibited diffusional kinetics. Thermodynamic parameters for bilirubin dissociation from GST were similar to those for human serum albumin. The rate of bilirubin transfer from rat liver basolateral plasma membranes to acceptor vesicles in the presence of glutathione S-transferase B declined asymptotically with increasing GST concentration. These data suggest that glutathione S-transferase B does not function as an intracellular bilirubin transporter, although expression of this protein may serve to regulate the delivery of bilirubin, and other nonsubstrate ligands, to sites of metabolism within the cell. Images PMID:7560084

  17. Novel Hydroxycinnamoyl-Coenzyme A Quinate Transferase Genes from Artichoke Are Involved in the Synthesis of Chlorogenic Acid1[W

    PubMed Central

    Sonnante, Gabriella; D'Amore, Rosalinda; Blanco, Emanuela; Pierri, Ciro L.; De Palma, Monica; Luo, Jie; Tucci, Marina; Martin, Cathie

    2010-01-01

    Artichoke (Cynara cardunculus subsp. scolymus) extracts have high antioxidant capacity, due primarily to flavonoids and phenolic acids, particularly chlorogenic acid (5-caffeoylquinic acid [CGA]), dicaffeoylquinic acids, and caffeic acid, which are abundant in flower bracts and bioavailable to humans in the diet. The synthesis of CGA can occur following different routes in plant species, and hydroxycinnamoyl-coenzyme A transferases are important enzymes in these pathways. Here, we report on the isolation and characterization of two novel genes both encoding hydroxycinnamoyl-coenzyme A quinate transferases (HQT) from artichoke. The recombinant proteins (HQT1 and HQT2) were assayed after expression in Escherichia coli, and both showed higher affinity for quinate over shikimate. Their preferences for acyl donors, caffeoyl-coenzyme A or p-coumaroyl-coenzyme A, were examined. Modeling and docking analyses were used to propose possible pockets and residues involved in determining substrate specificities in the HQT enzyme family. Quantitative real-time polymerase chain reaction analysis of gene expression indicated that HQT1 might be more directly associated with CGA content. Transient and stable expression of HQT1 in Nicotiana resulted in a higher production of CGA and cynarin (1,3-dicaffeoylquinic acid). These findings suggest that several isoforms of HQT contribute to the synthesis of CGA in artichoke according to physiological needs and possibly following various metabolic routes. PMID:20431089

  18. Effect of municipal waste water effluent upon the expression of Glutathione S-transferase isoenzymes of brine shrimp Artemia.

    PubMed

    Grammou, Athina; Papadimitriou, Chrisa; Samaras, Peter; Vasara, Eleni; Papadopoulos, Athanasios I

    2011-06-01

    Multiple isoenzymes of the detoxification enzyme family Glutathione S-transferase are expressed in the brine shrimp Artemia. The number of the major ones detected in crude extract by means of chromatofocusing varied between three and four, depending on the age. Two isoenzymes, one alkaline and one neutral (with corresponding isoelectric points of 8.5 and 7.2) appear to be dominant in all three developmental stages studied, (24, 48, and 72 h after hatching). Culturing Artemia for 48 h after hatching, in artificial sea water prepared by municipal wastewater effluent resulted to significant alterations of the isoenzyme profile. In comparison to organisms cultured for the same period of time in artificial sea water prepared by filtered tap water, the expression of the alkaline isoenzyme decreased by 62% while that of the neutral isoenzyme increased by 58%. Furthermore, the enzyme activity of the major isoenzyme of the acidic area increased by more than two folds. It is worth mentioning that although the specific activity of the total enzyme in the whole body homogenate was elevated, no statistically significant alteration of the Km value was observed. These findings suggest that study of the isoenzyme profile of Glutathione S-transferase may offer high sensitivity in detecting environmental pollution and needs to be further investigated. PMID:21429555

  19. Cantharidin Impedes Activity of Glutathione S-Transferase in the Midgut of Helicoverpa armigera Hübner.

    PubMed

    Khan, Rashid Ahmed; Liu, Ji Yuan; Rashid, Maryam; Wang, Dun; Zhang, Ya Lin

    2013-01-01

    Previous investigations have implicated glutathione S-transferases (GSTs) as one of the major reasons for insecticide resistance. Therefore, effectiveness of new candidate compounds depends on their ability to inhibit GSTs to prevent metabolic detoxification by insects. Cantharidin, a terpenoid compound of insect origin, has been developed as a bio-pesticide in China, and proves highly toxic to a wide range of insects, especially lepidopteran. In the present study, we test cantharidin as a model compound for its toxicity, effects on the mRNA transcription of a model Helicoverpa armigera glutathione S-transferase gene (HaGST) and also for its putative inhibitory effect on the catalytic activity of GSTs, both in vivo and in vitro in Helicoverpa armigera, employing molecular and biochemical methods. Bioassay results showed that cantharidin was highly toxic to H. armigera. Real-time qPCR showed down-regulation of the HaGST at the mRNA transcript ranging from 2.5 to 12.5 folds while biochemical assays showed in vivo inhibition of GSTs in midgut and in vitro inhibition of rHaGST. Binding of cantharidin to HaGST was rationalized by homology and molecular docking simulations using a model GST (1PN9) as a template structure. Molecular docking simulations also confirmed accurate docking of the cantharidin molecule to the active site of HaGST impeding its catalytic activity. PMID:23528854

  20. Conversion of melphalan to 4-(glutathionyl)phenylalanine. A novel mechanism for conjugation by glutathione-S-transferases.

    PubMed

    Dulik, D M; Fenselau, C

    1987-01-01

    One of the conjugates of melphalan, characterized following incubation with glutathione (GSH) and immobilized microsomal glutathione-S-transferases, has been identified as 4-(glutathionyl)-phenylalanine. This conjugate is formed by displacement of the mustard moiety. The structure was confirmed by reaction of the corresponding 4-halophenylalanines with GSH as well as by TLC, HPLC, and FAB mass spectrometry. Evidence is presented here to support the hypothesis that this novel reaction occurs via a cyclic aziridinium ion. To test this proposed mechanism, N,N-dimethyl-p-toluidine and its corresponding quaternary ammonium iodide salt were incubated with GSH in the presence of immobilized glutathione-S-transferases at 37 degrees C for 1 hr at pH 7.4. The tertiary amine did not react, whereas the quaternary compound produced 4-(glutathionyl)toluene. The effect of ring substituent requirements for the reaction was evaluated. The formation of GSH adducts of alkylating agents may be a factor in the development of resistance to these drugs. PMID:2882977

  1. Cantharidin Impedes Activity of Glutathione S-Transferase in the Midgut of Helicoverpa armigera Hübner

    PubMed Central

    Khan, Rashid Ahmed; Liu, Ji Yuan; Rashid, Maryam; Wang, Dun; Zhang, Ya Lin

    2013-01-01

    Previous investigations have implicated glutathione S-transferases (GSTs) as one of the major reasons for insecticide resistance. Therefore, effectiveness of new candidate compounds depends on their ability to inhibit GSTs to prevent metabolic detoxification by insects. Cantharidin, a terpenoid compound of insect origin, has been developed as a bio-pesticide in China, and proves highly toxic to a wide range of insects, especially lepidopteran. In the present study, we test cantharidin as a model compound for its toxicity, effects on the mRNA transcription of a model Helicoverpa armigera glutathione S-transferase gene (HaGST) and also for its putative inhibitory effect on the catalytic activity of GSTs, both in vivo and in vitro in Helicoverpa armigera, employing molecular and biochemical methods. Bioassay results showed that cantharidin was highly toxic to H. armigera. Real-time qPCR showed down-regulation of the HaGST at the mRNA transcript ranging from 2.5 to 12.5 folds while biochemical assays showed in vivo inhibition of GSTs in midgut and in vitro inhibition of rHaGST. Binding of cantharidin to HaGST was rationalized by homology and molecular docking simulations using a model GST (1PN9) as a template structure. Molecular docking simulations also confirmed accurate docking of the cantharidin molecule to the active site of HaGST impeding its catalytic activity. PMID:23528854

  2. 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

  3. Structure and expression of a cluster of glutathione S-transferase genes from a marine fish, the plaice (Pleuronectes platessa).

    PubMed Central

    Leaver, M J; Wright, J; George, S G

    1997-01-01

    Glutathione S-transferases are involved in the detoxification of reactive electrophilic compounds, including intracellular metabolites, drugs, pollutants and pesticides. A cluster of three glutathione S-transferase genes, designated GSTA, GSTA1 and GSTA2, was isolated from the marine flatfish, plaice (Pleuronectes platessa). GSTA and GSTA1 code for protein products with 76% amino acid identity. GSTA2 appears to contain a single nucleotide deletion which would render any product non-functional. All of these genes consist of six exons of similar sizes and greater than 70% nucleotide identity, and are interrupted by five introns of differing sizes. GSTA and GSTA1 mRNAs were present in a range of tissues, while GSTA2 mRNA was no detected. Expression of GSTA mRNA was increased in plaice intestine and spleen by pretreatment with beta-naphthoflavone, and expression of both GSTA and GSTA1 mRNAs was increased in plaice liver and gill by pretreatment with the peroxisome proliferating agent perfluoro-octanoic acid. PMID:9020873

  4. Carnitine palmitoyl transferase-1A (CPT1A): a new tumor specific target in human breast cancer

    PubMed Central

    Zonetti, Maria Josè; Fisco, Tommaso; Polidoro, Chiara; Bocchinfuso, Gianfranco; Palleschi, Antonio; Novelli, Giuseppe; Spagnoli, Luigi G.

    2016-01-01

    Transcriptional mechanisms epigenetically-regulated in tumoral tissues point out new targets for anti-cancer therapies. Carnitine palmitoyl transferase I (CPT1) is the rate-limiting enzyme in the transport of long-chain fatty acids for β-oxidation. Here we identified the tumor specific nuclear CPT1A as a product of the transcript variant 2, that doesn't retain the classical transferase activity and is strongly involved in the epigenetic regulation of cancer pro-survival, cell death escaping and tumor invasion pathways. The knockdown of CPT1A variant 2 by small interfering RNAs (siRNAs), was sufficient to induce apoptosis in MCF-7, SK-BR3 and MDA-MB-231 breast cancer cells. The cell death triggered by CPT1A silencing correlated with reduction of HDAC activity and histone hyperacetylation. Docking experiments and molecular dynamics simulations confirmed an high binding affinity of the variant 2 for HDAC1. The CPT1A silenced cells showed an up-regulated transcription of pro-apoptotic genes (BAD, CASP9, COL18A1) and down-modulation of invasion and metastasis related-genes (TIMP-1, PDGF-A, SERPINB2). These findings provide evidence of the CPT1 variant 2 involvement in breast cancer survival, cell death escape and invasion. Thus, we propose nuclear CPT1A as a striking tumor specific target for anticancer therapeutics, more selective and effective as compared with the well-known HDAC inhibitors. PMID:26799588

  5. Mitogen-activated protein kinase p38b interaction with delta class glutathione transferases from the fruit fly, Drosophila melanogaster.

    PubMed

    Wongtrakul, Jeerang; Sukittikul, Suchada; Saisawang, Chonticha; Ketterman, Albert J

    2012-01-01

    Glutathione transferases (GSTs) are a family of multifunctional enzymes involved in xenobiotic biotransformation, drug metabolism, and protection against oxidative damage. The p38b mitogen-activated protein kinase is involved in cellular stress response. This study screened interactions between Drosophila melanogaster Meigen (Diptera: Drosophilidae) Delta class glutathione transferases (DmGSTs) and the D. melanogaster p38b MAPK. Therefore, 12 DmGSTs and p38b kinase were obtained as recombinant proteins. The study showed that DmGSTD8 and DmGSTD11b significantly increased p38b activity toward ATF2 and jun, which are transcription factor substrates. DmGSTD3 and DmGSTD5 moderately increased p38b activity for jun. In addition, GST activity in the presence of p38b was also measured. It was found that p38b affected substrate specificity toward CDNB (1-chloro-2,4-dinitrobenzene) and DCNB (1,2-dichloro-4-nitrobenzene) of several GST isoforms, i.e., DmGSTD2, DmGSTD5, DmGSTD8, and DmGSTD11b. The interaction of a GST and p38b can affect the substrate specificity of either enzyme, which suggests induced conformational changes affecting catalysis. Similar interactions do not occur for all the Delta enzymes and p38b, which suggests that these interactions could be specific. PMID:23438069

  6. Crystallographic study of the phosphoethanolamine transferase EptC required for polymyxin resistance and motility in Campylobacter jejuni

    PubMed Central

    Fage, Christopher D.; Brown, Dusty B.; Boll, Joseph M.; Keatinge-Clay, Adrian T.; Trent, M. Stephen

    2014-01-01

    The foodborne enteric pathogen Campylobacter jejuni decorates a variety of its cell-surface structures with phosphoethanolamine (pEtN). Modifying lipid A with pEtN promotes cationic antimicrobial peptide resistance, whereas post-translationally modifying the flagellar rod protein FlgG with pEtN promotes flagellar assembly and motility, which are processes that are important for intestinal colonization. EptC, the pEtN transferase required for all known pEtN cell-surface modifications in C. jejuni, is a predicted inner-membrane metalloenzyme with a five-helix N-terminal transmembrane domain followed by a soluble sulfatase-like catalytic domain in the periplasm. The atomic structure of the catalytic domain of EptC (cEptC) was crystallized and solved to a resolution of 2.40 Å. cEptC adopts the α/β/α fold of the sulfatase protein family and harbors a zinc-binding site. A phosphorylated Thr266 residue was observed that was hypothesized to mimic a covalent pEtN–enzyme intermediate. The requirement for Thr266 as well as the nearby residues Asn308, Ser309, His358 and His440 was ascertained via in vivo activity assays on mutant strains. The results establish a basis for the design of pEtN transferase inhibitors. PMID:25286856

  7. 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. PMID:25682008

  8. Regulation of aflatoxin B1-metabolizing aldehyde reductase and glutathione S-transferase by chemoprotectors.

    PubMed Central

    McLellan, L I; Judah, D J; Neal, G E; Hayes, J D

    1994-01-01

    Ingestion of aflatoxin B1 (AFB1) represents a major risk factor in the aetiology of human hepatocellular carcinoma. In the rat, the harmful effects of AFB1 can be prevented by the administration of certain drugs which induce hepatic detoxification enzymes. We have previously shown that treatment of rats with the chemoprotector ethoxyquin (EQ) results in a marked increase in expression of the Alpha-class glutathione S-transferase (GST) Yc2 subunit which has high activity towards AFB1-8,9-epoxide [Hayes, Judah, McLellan, Kerr, Peacock and Neal (1991) Biochem. J. 279, 385-398]. To allow an assessment of whether the increased expression of GST Yc2 represents a general adaptive resistance mechanism to chemical stress, that is invoked by both chemoprotectors and carcinogens, we have examined the effects of EQ, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), phenobarbital (PB), AFB1, 3-methylcholanthrene (3-MC) and clofibrate on the AFB1-glutathione-conjugating activity and the GST subunit levels in rat liver. In addition, the effect of these drugs on the hepatic levels of an aldehyde reductase (AFB1-AR) that metabolizes the cytotoxic dialdehydic form of AFB1 has been studied as this enzyme also appears to be important in chemoprotection. Administration of the antioxidants EQ, BHA or BHT, as well as PB, led to a marked increase in levels of the GST Yc2 subunit in rat liver, and this increase coincided with a substantial rise in the GST activity towards AFB1-8,9-epoxide; neither AFB1, 3-MC nor clofibrate caused induction of Yc2 or any of the GST subunits examined. Among the xenobiotics studied, EQ was found to be the most effective inducing agent for the Yc2 subunit as well as Yc1, Yb1 and Yf. However, PB was equally as effective as EQ in increasing levels of the Ya-type subunits, although it was not found to be as potent an inducer of the other GST subunits, including Yc2. In addition to induction of GST, EQ caused a substantial increase in the hepatic

  9. Properties of succinyl-coenzyme A:D-citramalate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus.

    PubMed

    Friedmann, Silke; Alber, Birgit E; Fuchs, Georg

    2006-09-01

    The phototrophic bacterium Chloroflexus aurantiacus uses the 3-hydroxypropionate cycle for autotrophic CO(2) fixation. This cycle starts with acetyl-coenzyme A (CoA) and produces glyoxylate. Glyoxylate is an unconventional cell carbon precursor that needs special enzymes for assimilation. Glyoxylate is combined with propionyl-CoA to beta-methylmalyl-CoA, which is converted to citramalate. Cell extracts catalyzed the succinyl-CoA-dependent conversion of citramalate to acetyl-CoA and pyruvate, the central cell carbon precursor. This reaction is due to the combined action of enzymes that were upregulated during autotrophic growth, a coenzyme A transferase with the use of succinyl-CoA as the CoA donor and a lyase cleaving citramalyl-CoA to acetyl-CoA and pyruvate. Genomic analysis identified a gene coding for a putative coenzyme A transferase. The gene was heterologously expressed in Escherichia coli and shown to code for succinyl-CoA:d-citramalate coenzyme A transferase. This enzyme, which catalyzes the reaction d-citramalate + succinyl-CoA --> d-citramalyl-CoA + succinate, was purified and studied. It belongs to class III of the coenzyme A transferase enzyme family, with an aspartate residue in the active site. The homodimeric enzyme composed of 44-kDa subunits was specific for succinyl-CoA as a CoA donor but also accepted d-malate and itaconate instead of d-citramalate. The CoA transferase gene is part of a cluster of genes which are cotranscribed, including the gene for d-citramalyl-CoA lyase. It is proposed that the CoA transferase and the lyase catalyze the last two steps in the glyoxylate assimilation route. PMID:16952935

  10. Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site.

    PubMed

    Wongsantichon, Jantana; Robinson, Robert C; Ketterman, Albert J

    2015-01-01

    Epsilon class glutathione transferases (GSTs) have been shown to contribute significantly to insecticide resistance. We report a new Epsilon class protein crystal structure from Drosophila melanogaster for the glutathione transferase DmGSTE6. The structure reveals a novel Epsilon clasp motif that is conserved across hundreds of millions of years of evolution of the insect Diptera order. This histidine-serine motif lies in the subunit interface and appears to contribute to quaternary stability as well as directly connecting the two glutathiones in the active sites of this dimeric enzyme. PMID:26487708

  11. Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site

    PubMed Central

    Wongsantichon, Jantana; Robinson, Robert C.; Ketterman, Albert J.

    2015-01-01

    Epsilon class glutathione transferases (GSTs) have been shown to contribute significantly to insecticide resistance. We report a new Epsilon class protein crystal structure from Drosophila melanogaster for the glutathione transferase DmGSTE6. The structure reveals a novel Epsilon clasp motif that is conserved across hundreds of millions of years of evolution of the insect Diptera order. This histidine-serine motif lies in the subunit interface and appears to contribute to quaternary stability as well as directly connecting the two glutathiones in the active sites of this dimeric enzyme. PMID:26487708

  12. Genotoxicity of 1,3-butadiene and its epoxy intermediates.

    PubMed

    Walker, Vernon E; Walker, Dale M; Meng, Quanxin; McDonald, Jacob D; Scott, Bobby R; Seilkop, Steven K; Claffey, David J; Upton, Patricia B; Powley, Mark W; Swenberg, James A; Henderson, Rogene F

    2009-08-01

    Current risk assessments of 1,3-butadiene (BD*) are complicated by limited evidence of its carcinogenicity in humans. Hence, there is a critical need to identify early events and factors that account for the heightened sensitivity of mice to BD-induced carcinogenesis and to deter-mine which animal model, mouse or rat, is the more useful surrogate of potency for predicting health effects in BD-exposed humans. HEI sponsored an earlier investigation of mutagenic responses in mice and rats exposed to BD, or to the racemic mixture of 1,2-epoxy-3-butene (BDO) or of 1,2,3,4-diepoxybutane (BDO2; Walker and Meng 2000). In that study, our research team demonstrated (1) that the frequency of mutations in the hypoxanthine-guanine phosphoribosyl transferase (Hprt) gene of splenic T cells from BD-exposed mice and rats could be correlated with the species-related differences in cancer susceptibility; (2) that mutagenic-potency and mutagenic-specificity data from mice and rats exposed to BD or its individual epoxy intermediates could provide useful information about the BD metabolites responsible for mutations in each species; and (3) that our novel approach to measuring the mutagenic potency of a given chemical exposure as the change in Hprt mutant frequencies (Mfs) over time was valuable for estimating species-specific differences in mutagenic responses to BD exposure and for predicting the effect of BD metabolites in each species. To gain additional mode-of-action information that can be used to inform studies of human responses to BD exposure, experiments in the current investigation tested a new set of five hypotheses about species-specific patterns in the mutagenic effects in rodents of exposure to BD and BD metabolites: 1. Repeated BD exposures at low levels that approach the occupational exposure limit for BD workers (set by the U.S. Occupational Safety and Health Administration) are mutagenic in female mice. 2. The differences in mutagenic responses of the Hprt gene to BD

  13. Final report on the safety assessment of octoxynol-1, octoxynol-3, octoxynol-5, octoxynol-6, octoxynol-7, octoxynol-8, octoxynol-9, octoxynol-10, octoxynol-11, octoxynol-12, octoxynol-13, octoxynol-16, octoxynol-20, octoxynol-25, octoxynol-30, octoxynol-33, octoxynol-40, octoxynol-70, octoxynol-9 carboxylic acid, octoxynol-20 carboxylic acid, potassium octoxynol-12 phosphate, sodium octoxynol-2 ethane sulfonate, sodium octoxynol-2 sulfate, sodium octoxynol-6 sulfate, and sodium octoxynol-9 sulfate.

    PubMed

    Johnson, Wilbur

    2004-01-01

    autoimmune response in mice. In the Ames test, Octoxynol-1 was not mutagenic with and without metabolic activation nor was Octoxynol-9 clastogenic. Results for Octoxynol-9 were negative in the following assays: unscheduled DNA synthesis, hypoxanthine guanine phosphoribosyl transferase mutation assay, malignant transformation assay, DNA alkaline unwinding test, and mouse lymphoma thymidine kinase locus forward mutation assay. Ethoxylated alkylphenols are generally considered to be estrogenic in that they mimic the effects of estradiol. Dermal exposure at three dose levels of rats to Octoxynol-9 failed to induce any malformations by category (external, visceral, or skeletal) or by individual anatomical location that were different from controls at statistically significant level. An increased incidence of a vestigial thoracic rib was observed in all dose groups. Octoxynol-9 also did not induce developmental toxicity (number of viable litters, liveborn per litter, percentage survival, birth weight per pup, and weight gain per pup) in female specific pathogen-free CD-1 mice dosed daily by gavage on gestation days 6 through 13. No reproductive toxicity was seen in male albino rats which received 5% Octoxynol-40 in the diet daily for 3 months; however, in an in vitro test, Octoxynol-9 (0.24 mg/ml) totally immobilized all human spermatozoa within 20 s. Women who used Nonoxynol-9 or Octoxynol-9 as spermicides, but who did become pregnant, did not have an increase in the overall risk of fetal malformations. In a human skin irritation study, formulations containing 2.0% Octoxynol-9 were classified as moderately irritating and minimally irritating, respectively, in a 24-h single-insult, occlusive patch test. Octoxynol-9 (1.0%) was classified as a nonirritant in a clinical study of nine subjects patch tested for 4 consecutive days. The skin sensitization potential of Octoxynols-1, -3, -5, -9, and -13 was evaluated using 50 subjects. Octoxynol-1 induced sensitization in two subjects; all

  14. 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. PMID:27086966

  15. Glutathione S-transferase in the midgut tissue of gypsy moth (Lymantria dispar) caterpillars exposed to dietary cadmium.

    PubMed

    Vlahović, Milena; Ilijin, Larisa; Mrdaković, Marija; Todorović, Dajana; Matić, Dragana; Lazarević, Jelica; Mataruga, Vesna Perić

    2016-06-01

    Activity of glutathione S-transferase (GST) in midgut of gypsy moth caterpillars exposed to 10 and 30μg Cd/g dry food was examined. Based on the enzyme reaction through conjugation with glutathione, overall activity remained unaltered after acute and chronic treatment. No-observed-effect-concentration (10μg Cd/g dry food) significantly increased activity only after 3-day recovery following cadmium administration. Almost all comparisons of the indices of phenotypic plasticity revealed statistically significant differences. Despite the facts that GST has important role in xenobiotic biotransformation, our results indicate that this enzyme in insect midgut does not represent the key factor in cadmium detoxification. PMID:27084993

  16. Glutathione S-transferase (GST) genes in the red flour beetle, Tribolium castaneum, and comparative analysis with five additional insects.

    PubMed

    Shi, Houxia; Pei, Lianghong; Gu, Shasha; Zhu, Shicheng; Wang, Yanyun; Zhang, Yi; Li, Bin

    2012-11-01

    Glutathione S-transferases are important detoxification enzymes involved in insecticide resistance. Sequencing the Tribolium castaneum genome provides an opportunity to investigate the structure, function, and evolution of GSTs on a genome-wide scale. Thirty-six putative cytosolic GSTs and 5 microsomal GSTs have been identified in T. castaneum. Furthermore, 40, 35, 13, 23, and 32 GSTs have been discovered the other insects, Drosophila, Anopheles, Apis, Bombyx, and Acyrthosiphon, respectively. Phylogenetic analyses reveal that insect-specific GSTs, Epsilon and Delta, are the largest species-specific expanded GSTs. In T. castaneum, most GSTs are tandemly arranged in three chromosomes. Particularly, Epsilon GSTs have an inverted long-fragment duplication in the genome. Other four widely distributed classes are highly conserved in all species. Given that GSTs specially expanded in Tribolium castaneum, these genes might help to resist poisonous chemical environments and produce resistance to kinds of different insecticides. PMID:22824654

  17. Versatile O-GlcNAc transferase assay for high-throughput identification of enzyme variants, substrates, and inhibitors.

    PubMed

    Kim, Eun J; Abramowitz, Lara K; Bond, Michelle R; Love, Dona C; Kang, Dong W; Leucke, Hans F; Kang, Dae W; Ahn, Jong-Seog; Hanover, John A

    2014-06-18

    The dynamic glycosylation of serine/threonine residues on nucleocytoplasmic proteins with a single N-acetylglucosamine (O-GlcNAcylation) is critical for many important cellular processes. Cellular O-GlcNAc levels are highly regulated by two enzymes: O-GlcNAc transferase (OGT) is responsible for GlcNAc addition and O-GlcNAcase (OGA) is responsible for removal of the sugar. The lack of a rapid and simple method for monitoring OGT activity has impeded the efficient discovery of potent OGT inhibitors. In this study we describe a novel, single-well OGT enzyme assay that utilizes 6 × His-tagged substrates, a chemoselective chemical reaction, and unpurified OGT. The high-throughput Ni-NTA Plate OGT Assay will facilitate discovery of potent OGT-specific inhibitors on versatile substrates and the characterization of new enzyme variants. PMID:24866374

  18. The substrate promiscuity of a phosphopantetheinyl transferase SchPPT for coenzyme A derivatives and acyl carrier proteins.

    PubMed

    Wang, Yue-Yue; Luo, Hong-Dou; Zhang, Xiao-Sheng; Lin, Tao; Jiang, Hui; Li, Yong-Quan

    2016-03-01

    Phosphopantetheinyl transferases (PPTases) catalyze the posttranslational modification of acyl carrier proteins (ACPs) in fatty acid synthases (FASs), ACPs in polyketide synthases, and peptidyl carrier proteins (PCPs) in nonribosomal peptide synthetases (NRPSs) in all organisms. Some bacterial PPTases have broad substrate specificities for ACPs/PCPs and/or coenzyme A (CoA)/CoA analogs, facilitating their application in metabolite production in hosts and/or labeling of ACPs/PCPs, respectively. Here, a group II PPTase SchPPT from Streptomyces chattanoogensis L10 was characterized to accept a heterologous ACP and acetyl-CoA. Thus, SchPPT is a promiscuous PPTase and may be used on polyketide production in heterologous bacterial host and labeling of ACPs. PMID:26748983

  19. Transcriptome-wide identification and expression analysis of glutathione S-transferase genes involved in flavonoids accumulation in Dracaena cambodiana.

    PubMed

    Zhu, Jia-Hong; Li, Hui-Liang; Guo, Dong; Wang, Ying; Dai, Hao-Fu; Mei, Wen-Li; Peng, Shi-Qing

    2016-07-01

    Dragon's blood is a traditional medicine widely used in the world, and the main components of which are flavonoids. However, little is known about its formation mechanism. Previous studies indicate that plant glutathione S-transferase (GST) genes are involved in transportation of flavonoids from cytosolic synthesis to vacuolar accumulation. In this study, 20 Dracaena cambodiana GST genes (DcGSTs) were identified based on transcriptome database. Phylogenetic analysis revealed that 20 DcGSTs belonged to seven different classes. Tissue-specific expression analysis suggested that DcGSTs displayed differential expressions either in their transcript abundance or expression patterns under normal growth conditions. The transcript profiles of three DcGSTs in response to the inducer of dragon's blood were strongly correlated with flavonoids biosynthetic genes, consistent with dragon's blood accumulation. Our survey provides useful information for future studies on GST genes involved in dragon's blood formation in D. cambodiana. PMID:27208821

  20. Micro-Plasticity of Genomes As Illustrated by the Evolution of Glutathione Transferases in 12 Drosophila Species

    PubMed Central

    Saisawang, Chonticha; Ketterman, Albert J.

    2014-01-01

    Glutathione transferases (GST) are an ancient superfamily comprising a large number of paralogous proteins in a single organism. This multiplicity of GSTs has allowed the copies to diverge for neofunctionalization with proposed roles ranging from detoxication and oxidative stress response to involvement in signal transduction cascades. We performed a comparative genomic analysis using FlyBase annotations and Drosophila melanogaster GST sequences as templates to further annotate the GST orthologs in the 12 Drosophila sequenced genomes. We found that GST genes in the Drosophila subgenera have undergone repeated local duplications followed by transposition, inversion, and micro-rearrangements of these copies. The colinearity and orientations of the orthologous GST genes appear to be unique in many of the species which suggests that genomic rearrangement events have occurred multiple times during speciation. The high micro-plasticity of the genomes appears to have a functional contribution utilized for evolution of this gene family. PMID:25310450