Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Progress over the past 30 years has revealed many strengths of the rainbow trout as an alternative model for environmental carcinogenesis research. These include low rearing costs, an early life-stage ultrasensitive bioassay, sensitivity to many classes of carcinogen, a well-described tumor pathology, responsiveness to tumor promoters and inhibitors, and a mechanistically informative nonmammalian comparative status. Low-cost husbandry, for example, has permitted statistically challenging tumor study designs with up to 10,000 trout to investigate the quantitative interrelationships among carcinogen dose, anticarcinogen dose, DNA adduct formation, and final tumor outcome. The basic elements of the trout carcinogen bioassay include multiple exposure routes, carcinogen response, husbandry requirements, and pathology. The principal known neoplasms occur in liver (mixed hepatocellular/cholangiocellular adenoma and carcinoma, hepatocellular carcinoma), kidney (nephroblastoma), swim bladder (adenopapilloma), and stomach (adenopapilloma). Trout possess a complex but incompletely characterized array of cytochromes P450, transferases, and other enzymic systems for phase I and phase II procarcinogen metabolism. In general, trout exhibit only limited capacity for DNA repair, especially for removal of bulky DNA adducts. This factor, together with a high capacity for P450 bioactivation and negligible glutathione transferase-mediated detoxication of the epoxide, accounts for the exceptional sensitivity of trout to aflatoxin B1 carcinogenesis. At the gene level, all trout tumors except nephroblastoma exhibit variable and often high incidences of oncogenic Ki-ras gene mutations. Mutations in the trout p53 tumor suppressor gene have yet to be described. There are many aspects of the trout model, especially the lack of complete organ homology, that limit its application as a surrogate for human cancer research. Within these limitations, however, it is apparent that trout and other fish models can serve as highly useful adjuncts to conventional rodent models in the study of environmental carcinogenesis and its modulation. For some problems, fish models can provide wholly unique approaches.
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PMID:Fish models for environmental carcinogenesis: the rainbow trout. 872 7

Tyrosine phosphorylation of cellular proteins is an early and key step after activation of the insulin receptor kinase (IRK). The study of the properties of these proteins should contribute to our understanding of insulin action. In rat hepatoma cells overexpressing human insulin receptors (HTC-IR), insulin treatment resulted in rapid tyrosine phosphorylation of proteins of 180, 94, 68, and 60 kDa. When lysates from insulin-treated cells were immunoprecipitated with anti-Syp antibody, subsequent immunoblotting identified p65 and p68, which reacted with anti-Syp, and p6O and p68, which reacted with antiphosphotyrosine antibody. Thus, insulin treatment yielded tyrosine phosphorylation of both Syp and a Syp-associated p6O molecule. When lysates from insulin-treated cells were adsorbed with a glutathione S-transferase (GST)-Syp-Src homology-2 (SH2) fusion protein, tyrosine- phosphorylated p6O was sequestered. After subjecting lysates to SDS-PAGE, the GST-SypSH2 fusion protein was found to bind to p18O, p94, and p6O. Thus, Syp associates directly with a 60-kDa IRK substrate via its SH2 domains. Syp-associated p6O differed from the 60- to 62-kDa proteins, associating with ras guanosine triphosphatase-activating protein, which also underwent modest tyrosine phosphorylation in response to insulin. Preadsorption of cell lystates with antibody against the 85-kDa subunit (p85) of phosphatidylinositol 3-kinase substantially reduced the amount of p60 subsequently immunoprecipitated by anti-Syp. Thus, p60 associates with both Syp and p85. The amount of tyrosine-phosphorylated p60 exceeded that of p180 in anti-Syp immunoprecipitates, whereas their proportion was comparable in anti-p85 immunoprecipitates. Grb2 was also observed in the anti-Syp immunoprecipitates. When lysates from insulin-treated cells were adsorbed with GST-p85SH2 domains or GST-Grb2, the subsequent eluates contained tyrosine-phosphorylated p60, as determined by immunoblotting with antiphosphotyrosine. Membrane binding assays using GST fusion proteins showed that these associations were direct. Studies in rat liver, muscle, and adipose tissue identified insulin-dependent association of Syp, Grb2, and p85 with tyrosine-phosphorylated p60 in adipose tissue only. We conclude that insulin treatment of HTC-IR cells and rat adipose tissue results in the tyrosine phosphorylation of p60, which might participate in the recruitment of downstream effectors involved in insulin signal transduction.
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PMID:A 60-kilodalton protein in rat hepatoma cells overexpressing insulin receptor was tyrosine phosphorylated and associated with Syp, phophatidylinositol 3-kinase, and Grb2 in an insulin-dependent manner. 877 Aug 81

We previously identified a novel src- and ras-suppressed gene, 322, encoding a mitogenic regulatory function (Lin, X., Nelson, P. J., Frankfort, B., Tombler, E., Johnson, R., and Gelman, I. H. (1995) Mol. Cell. Biol. 15, 2754-2762). Here, we characterize the 322 gene product as an in vivo and in vitro substrate of protein kinase C (PKC). Hence, we named this product SSeCKS (pronounced essex) for Src Suppressed C Kinase Substrate. Rabbit polyclonal sera raised against glutathione S-transferase (GST)-SSeCKS recognized a myristylated 280/290-kDa doublet in Rat-6 fibroblasts. SSeCKS levels in src- and ras-transformed Rat-6 cells were 15- and 8-fold less, respectively, than those in untransformed cells. Short-term addition of phorbol ester resulted in a 5-fold increase in SSeCKS phosphorylation which was inhibited by bis-indolylmaleimide. In vitro phosphorylation of GST-SSeCKS by purified rabbit brain PKC-alpha was enhanced by phosphatidylserine and blocked by excess PKC pseudosubstrate inhibitor peptide. GST-SSeCKS bound purified PKC-alpha or PKC from Rat-6 lysates in a phosphatidylserine-dependent manner. Four SSeCKS domains containing Lys/Arg-rich motifs similar to the PKC phosphorylation site in MARCKS were phosphorylated in vitro by PKC. Immunofluorescence analysis showed SSeCKS present throughout the cytoplasm with enrichment in podosomes and at the cell edge. Short-term addition of phorbol esters caused the movement of SSeCKS from plasma membrane sites to the perinucleus coincident with a loss of actin stress fibers. These data suggest a role for SSeCKS in the control of cellular cytoskeletal architecture.
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PMID:A novel src- and ras-suppressed protein kinase C substrate associated with cytoskeletal architecture. 891 Apr 68

We have developed a polyclonal antibody that activates the heterodimeric p85-p110 phosphatidylinositol (PI) 3'-kinase in vitro and in microinjected cells. Affinity purification revealed that the activating antibody recognized the N-terminal SH2 (NSH2) domain of p85, and the antibody increased the catalytic activity of recombinant p85-p110 dimers threefold in vitro. To study the role of endogenous PI 3'-kinase in intact cells, the activating anti-NSH2 antibody was microinjected into GRC + LR73 cells, a CHO cell derivative selected for tight quiescence during serum withdrawal. Microinjection of anti-NSH2 antibodies increased bromodeoxyuridine (BrdU) incorporation fivefold in quiescent cells and enhanced the response to serum. These data reflect a specific activation of PI 3'-kinase, as the effect was blocked by coinjection of the appropriate antigen (glutathione S-transferase-NSH2 domains from p85 alpha), coinjection of inhibitory anti-p110 antibodies, or treatment of cells with wortmannin. We used the activating antibodies to study signals downstream from PI 3'-kinase. Although treatment of cells with 50 nM rapamycin only partially decreased anti-NSH2-stimulated BrdU incorporation, coinjection with an anti-p70 S6 kinase antibody effectively blocked anti-NSH2-stimulated DNA synthesis. We also found that coinjection of inhibitory anti-ras antibodies blocked both serum- and anti-NSH2-stimulated BrdU incorporation by approximately 60%, and treatment of cells with a specific inhibitor of MEK abolished antibody-stimulated BrdU incorporation. We conclude that selective activation of physiological levels of PI 3'-kinase is sufficient to stimulate DNA synthesis in quiescent cells. PI 3'-kinase-mediated DNA synthesis requires both p70 S6 kinase and the P21ras/MEK pathway.
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PMID:Specific activation of p85-p110 phosphatidylinositol 3'-kinase stimulates DNA synthesis by ras- and p70 S6 kinase-dependent pathways. 897 5

Peptide specific polyclonal antibodies directed against C-termini of ras p21 related GTP-binding proteins, ralA and ralB, were generated. To assess antibody specificity, cDNAs coding for full length ralA and ralB were expressed in Escherichia coli as GST fusion proteins. Western blotting analysis using enhanced chemiluminescence technique confirmed that ralA and ralB antibodies were specific for their respective protein. To determine the concentration and distribution, varying amounts of GST-ralA and GST-ralB and, human platelet particulate and cytosolic proteins were loaded during Western blotting. The amount of ralA and ralB proteins in the platelet particulate fraction was determined to be 0.16 +/- 0.017 microgram/mg protein (n = 3) and 0.15 +/- 0.009 microgram/mg protein (n = 3) respectively. In the cytosol, only ralB protein was detected and its concentration was estimated to be 0.03 +/- 0.009 microgram/mg protein (n = 3). Both ralA and ralB proteins were isoprenylated in the presence of [3H] mevalonolactone plus rabbit reticulocyte lysate although radioactivity incorporated into ralA was three times higher than that associated with the ralB protein. Addition of geranylgeranyl pyrophosphate to the reaction mixture inhibited incorporation of radioactivity into ralA and ralB but not cH-ras suggesting that both ralA and ralB proteins are geranylgeranylated. Differential distribution of ralA and ralB GTP-binding proteins in human platelets suggests a distinct role for each of these proteins in platelet function.
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PMID:Generation of antibodies specific for the RalA and RalB GTP-binding proteins and determination of their concentration and distribution in human platelets. 897 29

Protein phosphorylation is one of several representative post-translational modifications. Cyclic AMP-dependent protein kinase (PKA) plays the crucial and varying role of signal transduction. On the other hand, ras proteins plays an important role in cell proliferation and growth. Although a previous report showed that H-ras protein was phosphorylated by PKA, the stoichiometry was not determined, so we investigated the stoichiometry of phosphorylation of the protein by PKA. H-ras cDNA inserted into a pGEX-2T expressing vector produced high levels of recombinant H-ras (rH-ras) in a fusion protein with glutathione S-transferase. rH-ras was obtained after cleavage by thrombin. Phosphorylation of ras protein by the catalytic subunit of PKA was performed, and the radioactivity was counted after SDS-PAGE and autoradiography. The results indicate that less than 0.1 mol of phosphate was incorporated per mol of H-ras protein, and suggest that H-ras protein could not be a physiologically meaningful substrate for PKA.
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PMID:Phosphorylation of H-ras proteins by protein kinase A. 906 27

The A-G polymorphism at codon 104 in the glutathione S-transferase P1 (GSTP1) gene was examined in 138 male lung cancer patients and 297 healthy controls. The patients had significantly higher frequency of the GG genotype (15.9%) and a lower frequency of AA (38.4%) than the controls (9.1% and 51.5%, respectively). The level of hydrophobic DNA-adducts were determined in lung tissue from 70 current smokers. Patients with the GG genotype had a significantly higher adduct level than patients with AA (15.5 +/- 10.2 vs 7.9 +/- 5.1 per 10(8) nucleotides, P = 0.006). We also analyzed the deletion polymorphism in the GSTM1 gene in 135 male patients and 342 controls. The patients were stratified according to histology, smoking dose, age, adduct level and mutational types found in the tumors (Ki-ras and p53 genes). The results consistently indicated that the GSTM1 null genotype was associated with a slightly increased lung cancer risk. When the combined GST M1 and P1 genotypes were examined, patients with the combination null and AG or GG had significantly higher adduct levels than all other genotype combinations (P = 0.011). The distribution of combined genotypes was also significantly different in cases and controls, mainly due to increased frequency of the combination GSTM1 null and GSTP1 AG or GG among patients.
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PMID:Genotypes of glutathione transferase M1 and P1 and their significance for lung DNA adduct levels and cancer risk. 923 Feb 69

We have investigated the modulating effects of low amounts of dietary oils rich in polyunsaturated fatty acids (PUFA) on diethylnitrosamine (DEN)-induced hepatocarcinogenesis in male F344 rats. A total of 112 animals were divided into eight groups. Groups 1-4 were given drinking water containing 40 ppm DEN for five weeks. Groups 5-8 served as controls without DEN treatment. Groups 1 and 5 were fed a basal diet containing 5% beef tallow, Groups 2 and 6 were fed a 5% olive oil diet, Groups 3 and 7 were fed a 5% safflower oil diet, and Groups 4 and 8 were fed a 5% perilla oil diet for 21 weeks, starting 1 week before DEN exposure. Beef tallow, olive oil, safflower oil, and perilla oil are rich in saturated fatty acids, a monounsaturated fatty acid, n-6 PUFA, and n-3 PUFA, respectively. All rats were killed 20 weeks after the start of the experiment. Incidences of hepatocellular adenoma and carcinoma were 100% in DEN-treated groups, irrespective of dietary oils. Multiplicities of adenomas in Groups 3 and 4 were significantly (p < 0.05) lower than in Groups 1 and 2. Multiplicity of carcinoma in Group 3 was significantly (p < 0.05) lower than in Group 1. Mean volumes of placental glutathione S-transferase-positive foci per liver and the number of argyrophilic nucleolar organizer region proteins per nucleus in the liver tumors were significantly (p < 0.05) lower in Groups 3 and 4 than in Groups 1 and 2. ras mRNA expression in liver neoplasms was also suppressed significantly (p < 0.05) in Groups 3 and 4 compared with Groups 1 and 2. Significantly (p < 0.05) higher levels of n-6 and n-3 PUFA in the phospholipid fraction of the liver were found in Groups 3 and 4, respectively, than in the other groups. In contrast, a significantly (p < 0.05) decrease in monounsaturated fatty acid was observed in Groups 3 and 4 compared with Groups 1 and 2. These results suggest that safflower oil and perilla oil, rich in n-6 and n-3 PUFA, respectively, alter the membrane fatty acid composition of the liver and suppress the development of liver cell carcinoma in rats.
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PMID:Suppressive effect of low amounts of safflower and perilla oils on diethylnitrosamine-induced hepatocarcinogenesis in male F344 rats. 963 89

In a previous study on the replication of Kunjin virus using immunoelectron microscopy (E. G. Westaway, J. M. Mackenzie, M. T. Kenney, M. K. Jones, and A. A. Khromykh, 1997, J. Virol. 71, 6650-6661), NS1 and NS3 were found associated with double-stranded RNA (dsRNA) within vesicle packets (VP) in infected Vero cells, suggesting that these induced membrane structures may be the cytoplasmic sites of RNA replication. NS2B and NS3 (comprising the virus-encoded protease) were colocalized within distinct paracrystalline (PC) or convoluted membranes (CM), also induced in the cytoplasm, suggesting that these membranes are the sites of proteolytic cleavage. In this study we found by immunofluorescence (IF) that the small hydrophobic nonstructural proteins NS2A and NS4A were located in discrete foci in the cytoplasm of infected cells at both 16 and 24 h postinfection, partially coincident with dsRNA foci. In cryosections of infected cells at 24 h, NS2A was located by immunogold labeling primarily within VP, associated with labeled dsRNA. NS2A fused to glutathione S-transferase (GST) bound strongly to the 3' untranslated region of Kunjin RNA and also to the proposed replicase components NS3 and NS5 in cell lysates. NS4A was localized by immunogold labeling within a majority of the virus-induced membranes, including VP, CM, and PC. GST-NS4A bound weakly to the 3' untranslated region of Kunjin RNA but was bound to NS4A strongly and to most of the other viral nonstructural proteins, including NS3 and NS5. Taken together the results indicate that the flavivirus replication complex includes NS2A and NS4A in the VP in addition to the previously identified NS1 and NS3.
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PMID:Subcellular localization and some biochemical properties of the flavivirus Kunjin nonstructural proteins NS2A and NS4A. 963 60

The full-length dengue virus NS3 protein has been successfully expressed as a 94-kDa GST fusion protein in Escherichia coli. Treatment of the purified fusion protein with thrombin released a 68-kDa protein which is the expected molecular mass for the DEN1 NS3 protein. The identity of this protein was confirmed by Western blotting using dengue virus antisera. Two related activities of the recombinant NS3 protein were characterized, which were the binding of the protein to the 3'-noncoding region of the dengue virus RNA genome and NTPase activity. We demonstrated using a band shift assay that the DEN1 NS3 protein could form a complex with the stem-loop structure in the 3'-noncoding region (3'-NCR), although sites outside the stem-loop may also participate in binding. Using various unlabeled homopolymeric and heteropolymeric RNAs as competitors for binding, it was further shown that the DEN1 NS3 protein exhibits preferential binding to a 94-nt RNA transcript from the 3'-NCR of the dengue virus. The NTPase activity of the recombinant DEN1 NS3 protein was characterized using a thin-layer chromatography assay. We found that the DEN1 NS3 protein possesses some aspects of NTPase activity, which are distinct from those found in other flaviviruses. Although the NS3 protein was able to utilize all four ribonucleoside triphosphates as its substrates, the NS3 protein showed a distinct preference for purine triphosphates (i.e., ATP and GTP). The addition of poly(U) did not stimulate NTPase activity in DEN1 NS3 protein, which contrasts with the reports for other flaviviral NS3 proteins. However, NTPase activity was specifically stimulated by the viral NS5 protein, which was manifested by a more than twofold increase in the rate of ATP hydrolysis and a 25% increase in the yield of ADP at the end of a 120-min reaction. These data suggest that the NTPase activity of the NS3 protein may be regulated by the viral NS5 protein during virus replication.
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PMID:Recombinant dengue virus type 1 NS3 protein exhibits specific viral RNA binding and NTPase activity regulated by the NS5 protein. 965 59


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