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)

Basal expression of the human plasminogen activator inhibitor-1 (PAI-1) is mediated by a promoter element named B box that binds the helicase-like transcription factor (HLTF), homologous to SNF/SWI proteins. Electrophoretic mobility shift assays performed on a set of B box point mutants demonstrated two HLTF sites flanking and partially overlapping with a GT box binding Sp1 and Sp3. Mutations affecting either the Sp1/Sp3 or the two HLTF sites inhibited by 6- and 2.5-fold, respectively, transient expression in HeLa cells of a reporter gene fused to the PAI-1 promoter. In Sp1/Sp3-devoid insect cells, co-expression of PAI-1-lacZ with Sp1 or Sp3 led to a 14-26-fold induction while HLTF had no effect. Simultaneous presence of Sp1 or Sp3 and the short HLTF form (initiating at Met-123) provided an additional 2-3-fold synergistic activation suppressed by mutations that prevented HLTF binding. Moreover, a DNA-independent interaction between HLTFMet123 and Sp1/Sp3 was demonstrated by co-immunoprecipitation from HeLa cell extracts and glutathione S-transferase pull-down experiments. The interaction domains were mapped to the carboxyl-terminal region of each protein; deletion of the last 85 amino acids of HLTFMet123 abolished the synergy with Sp1. This is the first demonstration of a functional interaction between proteins of the Sp1 and SNF/SWI families.
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PMID:Functional interactions between Sp1 or Sp3 and the helicase-like transcription factor mediate basal expression from the human plasminogen activator inhibitor-1 gene. 1039 91

Even small increases in the frequency of thrombotic disease in users of OCs have general health impact because of their widespread use, which is currently expanding to potential risk groups. The present investigations were launched to study the effects of OCs containing 20-40 micrograms of EE combined with the latest developed gonane progestogens on biochemical risk markers within metabolic systems involved in the development of arterial thrombotic disease. The studies included evaluation of carbohydrate and lipid metabolism as well as the haemostatic system and were performed in non-diabetic women and in women with IDDM, who are prone to the development of arterial thrombosis. In the evaluation of the carbohydrate metabolism in non-diabetic women, we found no effect on fasting glucose or insulin and no effect on the insulin response to oral glucose in women using monophasic OCs containing EE combined with DSG or GST. This contrasts the evaluation of triphasic OCs containing EE combined with GST or NGT, which increased fasting insulin and reduced insulin sensitivity without affecting the glucose-effectiveness or the beta-cell function. Impaired glucose tolerance developed in 10% of the women after 6 months. These finding suggest that OCs are able to induce a state of insulin resistance, which should be considered in the prescription for women with potential disturbed insulin sensitivity or reduced beta-cell secretory capacity e.g. women with ovarian hyperandrogenism, obesity, previous GDM or perimenopausal women. We found no change in glycaemic control in 22 women with well-regulated IDDM treated with a monophasic combination of EE and GST for one year and none of the women developed microalbuminuria during treatment. In the women with diabetes we observed an increase in fasting levels of triglycerides, a decrease in LDL-cholesterol, and unchanged concentrations of total cholesterol and HDL-cholesterol during treatment. In non-diabetic women treated with the same compound or an OC containing EE and DSG we found similar changes in triglycerides and total cholesterol, but increased levels of HDL-cholesterol and unchanged LDL-cholesterol concentrations. In the women with IDDM there was a negative correlation between daily insulin requirement and HDL-cholesterol before and during treatment, but no other statistically significant correlation between estimates of glycaemic control and lipids and lipoproteins were observed. In the non-diabetic women, changes in the haemostatic system included an increase in the procoagulant factors fibrinogen and Factor VIIc; the concentration of active t-PA increased, mainly because of decreased inhibition by PAI-1. The ratio between molecular markers of the activity of the coagulation system and the efficacy of fibrinolysis was unchanged. This was also found in the women with IDDM, who showed evidence of increased fibrin formation and an attenuated fibrinolytic response during treatment. The regulation of the t-PA/PAI system was studied in non-diabetic women in order to elucidate if the effects of OCs are caused by a direct effect on synthesis or clearance of these variables or if they are secondary to changed insulin sensitivity, as described in individuals with atherosclerosis. We found no indications that insulin resistance is involved in the regulation of t-PA and PAI-1 antigen levels, neither before nor during intake of OCs. We showed, however, that the decreased t-PA antigen concentration observed in OC users is caused by reduced synthesis outside the splanchnic circulation. The studies indicate that low-dose OCs containing newer gonane progestogens are able to induce insulin resistance and to impair glucose tolerance. Lipoproteins were not adversely influenced by the OCs neither in the diabetic nor the non-diabetic women; on the contrary, there was a tendency towards increased plasma levels of HDL-cholesterol and decreased LDL-cholesterol which are associated with a decreased risk of atherosclerosis. The changes observed within the haemostatic system were in accordance with a maintained balance between coagulation and fibrinolysis although the rate of fibrin formation may be increased in the women with IDDM. Irrespective of OC use, the interrelationships between metabolic systems in young non-diabetic women are different from those reported in individuals with atherosclerosis or insulin resistance. The effects of OCs on the t-PA/PAI system seem to be mediated by a direct effect on the vessel wall and not by changes in the hepatic clearance. The present findings were obtained in diabetic women without vascular complications, so the conclusion that women with IDDM can use OCs without metabolic alterations of known clinical significance is therefore restricted to those without evidence of diseased vessels. When evaluating the results obtained in the non-diabetic women, it should be remembered that women with recognised risk factors were excluded. The results may therefore be of limited value when evaluating the risk of arterial thrombosis in predisposed populations. In healthy individuals, the present integrated evaluation of biochemical markers does not indicate an increased risk of arterial thrombosis during use of low-dose OCs containing newer gonane progestogens; thus, the findings are in accordance with the recent epidemiological studies on these compounds. The application of relevant biochemical markers facilitate the understanding of the non-reproductive effects of sex steroids which have increasing importance because of their expanding use, not only as contraceptives, but also in the treatment of benign gynaecological disorders, as hormone replacement therapy and as prophylactic agents against specific degenerative conditions. Moreover, they may prove to be helpful in the future identification of women, who have increased susceptibility to the metabolic effects of sex steroids due to genetic predisposition.
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PMID:Pharmacodynamic effects of oral contraceptive steroids on biochemical markers for arterial thrombosis. Studies in non-diabetic women and in women with insulin-dependent diabetes mellitus. 1189 23

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD), a ubiquitous environmental pollutant, elicits a variety of toxicities and is a well-known carcinogen. TCDD alters the expression of many genes including CYP1A1/2, CYP1B1, glutathione S-transferase Ya, aldehyde-3-dehydrogenase, NAD(P)H:quinone oxidoreductase, transforming growth factor (TGF)-alpha and TGF-beta. The present study was aimed at characterization of TCDD to induce plasminogen activator inhibitor-1 (PAI-1) in mouse hepatoma cell lines. A Hepa1c1c7 wild-type cell [H1(wt)], an aryl hydrocarbon receptor (AhR)-deficient mutant [H1(AhR(-))] and an AhR nuclear translocator (Arnt)-deficient mutant [H1(Arnt(-))] were used for this study. TCDD induced PAI-1 in H1(wt) cells, but not in H1(AhR(-)) and H1(Arnt(-)) mutants, indicating a functional role of the AhR-Arnt complex in this effect. Cycloheximide (CHX) treatment resulted in increased PAI-1 mRNA induction, indicating that this response to TCDD is a direct effect on transcription and not a secondary effect mediated by other TCDD-induced proteins. Transfection with PAI-1 promoter led to increased PAI-1 promoter activity in H1(wt) cells treated with TCDD, but no such effect occurred in H1(AhR(-)) or H1(Arnt(-)) cells, implying involvement of the AhR and Arnt. In addition, alpha-naphthoflavone and phenanthroline, two AhR antagonists, each blocked the enhancing effect of TCDD on PAI-1 promoter-coupled luciferase activity in H1(wt) cells. PAI-1 promoter deletion analysis indicated that TCDD-induced PAI-1 transcription was distinctly different from TGF-beta-dependent PAI-1 transcription, particularly in the region between -161 to +73. In summary, TCDD induced the PAI-1 gene directly via an AhR- and Arnt-dependent mechanism, which was distinctly different from TGF-beta-driven PAI-1 transcription.
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PMID:2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces plasminogen activator inhibitor-1 through an aryl hydrocarbon receptor-mediated pathway in mouse hepatoma cell lines. 1211 Oct 5

Licorice is a commonly used herbal medicine for treatment of liver disorders. Its biological activities have been widely studied. However, little information on its transcriptional regulation has been reported. In the present study, the effect of an aqueous extract of licorice on the gene expression in rat liver cells (Clone 9) was investigated. The results show the expression of GST-pi, DT-diaphorase, PAI-1, fosl-1 and uPAR were over two-fold increased. Northern blot analysis revealed that the over-expression of these genes was concentration-dependent (0.25-3 mg/ml) but the temporal expression profile (8-48 h) of each individual gene varied. The over-expression of fosl-1 could be related to the event in the induction process leading to the expression of GST-pi, DT-diaphorase, uPAR and PAI-1 through AP-1. Induction of the over-expression of GST-pi and DT-diaphorase genes may contribute to the hepatoprotective properties of licorice whereas activation of uPAR and PAI-1 together with down-regulation of TIMP-3 suggest a role of LE in the regulation of cell mobility.
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PMID:Transcriptional regulation of fosl-1 by licorice in rat Clone 9 cells. 1455 Aug 51

We developed a sensitive immunoassay to determine the concentration of mouse plasminogen activator inhibitor-1. The assay was a non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) based on the production of a specific polyclonal antibody against mouse plasminogen activator inhibitor type-1 (PAI-1) used both as a trapping and detecting antibody. This antibody was raised in a rabbit by direct introduction of the expression vector plasmid DNA encoding mouse PAI-1, instead of conventional immunization with the purified protein. The standard curve was constructed with a recombinant glutathione S-transferase (GST)-mouse PAI-1 fusion protein (GST-mPAI-1) and dose-response of the assay was linear for GST-mPAI-1 between 6.25 and 100 pM. In order to assess the consistency of the assay, we measured PAI-1 antigen in normal mouse pooled plasma several times. We found that the intra-assay and inter-assay coefficients of variation (CV) were 4.8% and 9.2%, respectively, indicating that the ELISA would be sufficiently repeatable and reproducible. In this assay, lipopolysaccharide (LPS)-injected mice showed substantially higher levels (22-fold) of plasma PAI-1 antigen than did control mice (12.5+/-2.4 vs. 0.58+/-0.16 nM), similar to results reported elsewhere. Taken together, the DNA vaccine method is extremely useful for preparing specific antibodies against mouse PAI-1, which can be utilized to establish the ELISA and analyze the profile of PAI-1 distributions in mice under various conditions. This approach might also be useful for immunological investigation of other coagulation factors and related proteins.
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PMID:Enzyme immunoassay for measurement of murine plasminogen activator inhibitor-1, employing a specific antibody produced by the DNA vaccine method. 1469 77

PAI-1 modulates many biological processes involving fibrinolysis, cell migration or tissue remodelling. In addition to inhibiting serine proteases (mainly tPA and uPA), PAI-1 interacts with vitronectin (Vn), fibrin or alpha(1)-acid glycoprotein, interactions which are important for PAI-1-mediated effects in inflammation, tumor invasion and metastasis. To further identify proteins interacting with PAI-1, the yeast two-hybrid strategy was employed. Screening of a human placenta cDNA library identified--in addition to the C-terminal region of cytokeratin 18 (CK18(182-430))--a large C-terminal fragment of alpha-actinin-4 (Act-4) as a binding partner for PAI-1. Two different cDNA clones encoding Act-4(287-911) and Act-4(330-911) respectively, were isolated. An Act-4(330-911)/GST-fusion protein, but not GST alone, was immunoprecipitated together with active PAI-1. In solid phase binding assays, active wild-type PAI-1 as well as the PAI-1 variant Q123K (which does not interact with multimeric Vn) was found to bind to Act-4(330-911)/GST. Latent PAI-1, latent Q123K, and the inactive PAI-1 variant Q55P did not display any binding activity. Act-4 is mainly present intracellularly and is involved in cellular motility via interaction with the actin cytoskeleton, thus probably affecting the metastatic potential of tumor cells. However, an extracellular Act-4-derived fragment (mactinin) has previously been identified, which (i) is generated by proteolytic action of uPA, (ii) displays significant chemotactic activity for monocytes, and (iii) promotes monocyte/macrophage maturation. We suggest that PAI-1, via interaction with both Act-4 and uPA, may function as a modulator of this mononuclear phagocyte response, not only in inflammation but also in tumor invasion and metastasis.
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PMID:Non-muscle alpha-actinin-4 interacts with plasminogen activator inhibitor type-1 (PAI-1). 1549 75

Transforming growth factor beta (TGF-beta) stimulation results in the assembly of Smad-containing protein complexes that mediate activation or repression of TGF-beta responsive genes. To determine if disruption of specific Smad protein-protein interactions would selectively inhibit responses to TGF-beta or generally interfere with Smad-dependent signaling, we developed three Smad-binding peptide aptamers by introducing Smad interaction motifs from Smad-binding proteins CBP, FoxH1 and Lef1 into the scaffold protein E. coli thioredoxin A (Trx). All three classes of aptamers bound to Smads by GST pulldown assays and co-immunoprecipitation from mammalian cells. Expression of the aptamers in HepG2 cells did not generally inhibit Smad-dependent signaling as evaluated using seven TGF-beta responsive luciferase reporter genes. The Trx-xFoxH1b aptamer inhibited TGF-beta-induced expression from a reporter dependent on the Smad-FoxH1 interaction, A3-lux, by 50%. Trx-xFoxH1b also partially inhibited two reporters not dependent on a Smad-FoxH1 interaction, 3TP-lux and Twntop, and endogenous PAI-1 expression. Trx-Lef1 aptamer only inhibited expression of the Smad-Lef1 responsive reporter gene TwnTop. The Trx-CBP aptamer had no significant effect on reporter gene expression. The results suggest that Smad-binding peptide aptamers can be developed to selectively inhibit TGF-beta-induced gene expression.
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PMID:Selective inhibition of TGF-beta responsive genes by Smad-interacting peptide aptamers from FoxH1, Lef1 and CBP. 1575 Jun 22

PI3K (phosphoinositide 3-kinase) activity is involved in Ang (angiotensin) II-stimulated VSMC (vascular smooth muscle cell) growth and hypertrophy. In the present study, we demonstrate that the inhibition of PI3K in VSMCs by expression of a dominant-negative p85alpha mutant lacking the p110-binding domain (Deltap85), or by treatment of cells with LY294002, inhibited Ang II-stimulated PAI-1 (plasminogen activator inhibitor-1) mRNA expression. Using a GST (glutathione S-transferase) fusion protein containing the p85 N-terminal SH2 (Src homology 2) domain as 'bait' followed by MS/MS (tandem MS), we identified a 70 kDa fragment of the p70 PDGFR-beta (platelet-derived growth factor receptor-beta) as a signalling adapter that is phosphorylated and recruits the p85 subunit of PI3K after Ang II stimulation of AT1 (Ang II subtype 1) receptors on VSMCs. This fragment of the PDGFR-beta, which has a truncation of its extracellular domain, accounted for approx. 15% of the total PDGFR-beta detected in VSMCs with an antibody against its cytoplasmic domain. Stimulation of VSMCs with Ang II increased tyrosine-phosphorylation of p70 PDGFR-beta at Tyr751 and Tyr1021 and increased its binding to p85. PDGF also induced phosphorylation of p70 PDGFR-beta, a response inhibited by the PDGF tyrosine kinase selective inhibitor, AG1296. By contrast, Ang II-induced phosphorylation of the 70 kDa receptor was not affected by AG1296. Ang II-stimulated phosphorylation of the p70 PDGFR-beta was blocked by the AT1 receptor antagonist, candesartan (CV 11974) and was partially inhibited by PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine}, an Src family kinase inhibitor. Our result suggests that the p70 PDGFR-beta functions as an adapter that recruits PI3K to the membrane upon AT1 receptor stimulation.
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PMID:Angiotensin II stimulates phosphorylation of an ectodomain-truncated platelet-derived growth factor receptor-beta and its binding to class IA PI3K in vascular smooth muscle cells. 1656 13

Orphan nuclear receptor small heterodimer partner (SHP) is an atypical member of the nuclear receptor superfamily; SHP regulates the nuclear receptor-mediated transcription of target genes but lacks a conventional DNA binding domain. In this study, we demonstrate that SHP represses transforming growth factor-beta (TGF-beta)-induced gene expression through a direct interaction with Smad, a transducer of TGF-beta signaling. Transient transfection studies demonstrate that SHP represses Smad3-induced transcription. In vivo and in vitro protein interaction assays revealed that SHP directly interacts with Smad2 and Smad3 but not with Smad4. Mapping of domains mediating the interaction between SHP and Smad3 showed that the entire N-terminal domain (1-159 amino acids) of SHP and the linker domain of Smad3 are involved in this interaction. In vitro glutathione S-transferase pulldown competition experiments revealed the SHP-mediated repression of Smad3 transactivation through competition with its co-activator p300. SHP also inhibits the activation of endogenous TGF-beta-responsive gene promoters, the p21, Smad7, and plasminogen activator inhibitor-1 (PAI-1) promoters. Moreover, adenovirus-mediated overexpression of SHP decreases PAI-1 mRNA levels, and down-regulation of SHP by a small interfering RNA increases both the transactivation of Smad3 and the PAI-1 mRNA levels. Finally, the PAI-1 gene is expressed in SHP(-/-) mouse hepatocytes at a higher level than in normal hepatocytes. Taken together, these data indicate that SHP is a novel co-regulator of Smad3, and this study provides new insights into regulation of TGF-beta signaling.
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PMID:Orphan nuclear receptor small heterodimer partner inhibits transforming growth factor-beta signaling by repressing SMAD3 transactivation. 1707 65

The mechanisms involved in regulating mammary cell turnover during the pregnancy-lactation cycle in dairy cows are unclear. The objective of present experiment was to describe expression of genes encoding proteins known to be involved in pathways regulating mammary cell proliferation, apoptosis, differentiation, cell survival, and tissue remodeling. Mammary gland biopsies were taken 7 times during the pregnancy-lactation cycle of 10 dairy cows, and samples were analyzed by immunohistochemistry and real-time PCR. Cell proliferation was greatest during the dry period and apoptosis was high in early dry period and early lactation. Based on Fas (tumor necrosis factor receptor superfamily member 6), Fas ligand, and caspase-3, caspase-8, and caspase-9 gene expression, no indication was found of a stage-dependent shift between the extrinsic and intrinsic pathways leading to apoptosis. Gene expression of microsomal glutathione S-transferase (mGST) did not vary significantly, whereas B-cell leukemia/lymphoma 2 (Bcl-2) and BCL2-associated X protein (Bax) gene expression was greatest during the dry period and early lactation and coincided with high cell turnover. Gene expression of early response genes c-Fos, c-Jun, and c-Myc correlated to neither rate of cell proliferation nor plasma concentration of insulin-like growth factor (IGF)-I and insulin. Gene expression of nuclear factor of kappa light chain gene enhancer in B-cells (NFkappaB) and NFkappaB inhibitor alpha was greatest in the periparturient period, and NFkappaB gene expression coincided with an anticipated need for cell survival factors. Expression of transforming growth factor beta (TGF-beta) receptor 1 and 2 mRNA was greatest in early lactation, whereas TGF-beta1 did not vary significant during the pregnancy-lactation cycle. Even though our results on the TGF-beta system did not comply with other studies, the gene expression pattern of the TGF-beta receptors indicates a role in regulating apoptosis in early lactation. Signal transducer and activator of transcription 5 (STAT5) gene expression was high in the periparturient period, which suggests a role for STAT5 in regulation of mammary cell proliferation and differentiation in dairy cows. Expression of tissue-plasminogen activator, plasminogen activator inhibitor-1, and IGF binding protein 5 genes was greatest in early lactation, suggesting a role for IGF binding protein 5 in coordinating regulation of apoptosis and tissue remodeling.
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PMID:Cellular mechanisms in regulating mammary cell turnover during lactation and dry period in dairy cows. 1848 54


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