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)

HGF is a rare oral condition characterized by a slow, progressive enlargement of the gingiva, involving both the maxilla and mandible. HGF provides a model for the study of regulatory features of conditions characterized by connective tissue hyperplasia. In this study, the culture characteristics of gingival fibroblasts derived from patients of the same family with HGF (n = 4) were similar with regard to cell cycle analysis. Flow cytometric DNA content analysis revealed uniform DNA diploidy for fibroblasts cultured from NG and HGF. NG cells showed a low S-phase fraction (19.8%) and G2/M fraction (5.8%) and a relatively high G1 phase fraction (74%). In contrast, HGF cells from all members of the tested kindred, exhibited diploid cells with a higher S-phase (40.9%) and G2/M (10.1%) fraction and a relatively low G1 phase fraction (40.9%). Furthermore, we demonstrated that the expression and production of Hsp47 parallels the increased levels of collagen secretion observed in HGF. In addition, we show that Hsp47 and collagen are coordinately regulated following stress via a feedback mechanism mediated by N-terminal procollagen propeptides. Utilizing confocal microscopy and antibodies directed against GST-fusion proteins encompassing the pro alpha1(I) N-propeptide globular domain (NP1) (residues 23-108), it was apparent that this regulatory mechanism does not involve significant interaction with Hsp47's chaperoning of procollagen.
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PMID:Increase in expression of Hsp47 and collagen in hereditary gingival fibromatosis is modulated by stress and terminal procollagen N-propeptides. 1075 12

The Src homology (SH)2 domain-containing protein-tyrosine phosphatase SHP-1 is tyrosine phosphorylated in platelets in response to the glycoprotein VI (GPVI)-selective agonist collagen-related peptide (CRP), collagen, and thrombin. Two major unidentified tyrosine-phosphorylated bands of 28 and 32 kDa and a minor band of 130 kDa coprecipitate with SHP-1 in response to all three agonists. Additionally, tyrosine-phosphorylated proteins of 50-55 and 70 kDa specifically associate with SHP-1 following stimulation by CRP and collagen. The tyrosine kinases Lyn, which exists as a 53 and 56-kDa doublet, and Syk were identified as major components of these bands, respectively. Kinase assays on SHP-1 immunoprecipitates performed in the presence of the Src family kinase inhibitor PP1 confirmed the presence of a Src kinase in CRP- but not thrombin-stimulated cells. Lyn, Syk, and SLP-76, along with tyrosine-phosphorylated 28-, 32-, and 130-kDa proteins, bound selectively to a glutathione S-transferase protein encoding the SH2 domains of SHP-1, suggesting that this is the major site of interaction. Platelets isolated from motheaten viable mice (mev/mev) revealed the presence of a heavily tyrosine-phosphorylated 26-kDa protein that was not found in wild-type platelets. CRP-stimulated mev/mev platelets manifested hypophosphorylation of Syk and Lyn and reduced P-selectin expression relative to controls. These observations provide evidence of a functional role for SHP-1 in platelet activation by GPVI.
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PMID:Evidence of a role for SHP-1 in platelet activation by the collagen receptor glycoprotein VI. 1087 5

During oxidative stress, reactive aldehydes, including trans-4-hydroxy-2-nonenal (4-HNE), are generated by peroxidation of membrane lipids and purportedly stimulate hepatic stellate cells to produce excessive extracellular matrix, including type I collagen. An important question concerning the ability of 4-HNE to modulate collagen production by stellate cells is the potential of these specialized cells to detoxify 4-HNE. The objective of the present study was to characterize the ability of stellate cell lines, derived from normal (NFSC) and cirrhotic (CFSC) rat livers, to metabolize 4-HNE by oxidative, reductive and conjugative pathways. These two stellate cell lines were noted to have differing susceptibilities to the cytotoxic effect of 4-HNE. Treatment of both stellate cell lines with a range of 4-HNE doses demonstrated that the concentration which was cytotoxic to 50% of CFSC (TD(50)) was 25% greater than that for NFSC (967.57+/-9.26 nmol/10(6) cells vs. 769.90+/-5.32 nmol/10(6) cells respectively). The capacity of these cell lines to metabolizes 4-HNE was determined by incubating them in suspension with 50 microM 4-HNE (10 nmol/10(6) cell); 4-HNE elimination and metabolite formation were quantified over a 20 min time course. Both stellate cell lines rapidly metabolized 4-HNE, with the CFSC line eliminating 4-HNE at a rate that was approx. 2-fold greater than the NFSC line. The rate of 4-HNE metabolism attributable to glutathione S-transferase (GST) was similar in both cell lines, though differential cell specific expressions of GST isoforms GSTP1-1 and GSTA4-4 were observed. The greater rate of 4-HNE elimination by CFSC was attributable to its aldehyde dehydrogenase (ALDH) activity which accounted for approx. 50% of 4-HNE metabolism in CFSC but was insignificant in NFSC. Neither cell line had detectable alcohol dehydrogenase activity or protein levels. Measurement of cellular GSH concentrations revealed that NFSC contain approx. 2-fold greater concentrations of GSH when compared to CFSC and that following 4-HNE treatment, GSH levels were rapidly depleted from both cell lines. Concomitant with 4-HNE mediated GSH depletion, a corresponding increase in the 4-HNE-glutathione adduct formation was observed with the NFSC line forming greater amounts of the glutathione adduct than did the CFSC line. Taken together, these data demonstrate that both stellate cell lines have the capacity to metabolize 4-HNE but that CFSC have a greater rate of metabolism which is attributable to their greater ALDH activity, suggesting that the stellate cells isolated from cirrhotic liver may be differentially responsive to the biologic effects of 4-HNE.
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PMID:Characterization of 4-hydroxy-2-nonenal metabolism in stellate cell lines derived from normal and cirrhotic rat liver. 1101 74

L-Proline supplementation of the medium for collagen gel cultures of hepatocytes has been shown to improve albumin secretion. A study was made as to whether L-proline is also essential for the maintenance of xenobiotic biotransformation capacities in collagen gel sandwich and immobilisation cultures of rat and human hepatocytes. Key phase I (cytochrome P450-dependent monooxygenase [CYP)] and microsomal epoxide hydrase [mEH]) and phase II (glutathione S-transferase [GST]) biotransformation enzyme activities and the secretion of albumin in the culture medium were assessed in the absence and presence of L-proline. CYP and mEH activities were not affected by the addition of L-proline, whereas phase II alpha-Class GST activity of rat hepatocytes in collagen cultures was decreased. Species differences were demonstrated, as human hepatocytes showed a better maintenance of GST activities than their rat counterparts in the presence of L-proline. Albumin secretion, often considered to be a marker for differentiated cell function, does not parallel the biotransformation capacities of the hepatocytes in culture. Additional results demonstrated an L-proline-mediated enhancement of the proliferation rate of contaminating stellate cells in conventional monolayer culture. Transdifferentiation of stellate cells to proliferating myofibroblasts, along with an increased albumin secretion and collagen synthesis, are characteristic of fibrotic liver. Since the last two phenomena have been observed in L-proline-supplemented collagen gel cultures, it can be concluded that when stable collagen gel cultures of rat hepatocytes are needed for long-term pharmacotoxicological studies, it is preferable to use an L-proline-free culture medium. Further studies on medium optimisation are required for hepatocytes from species other than rat.
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PMID:Effects of L-proline on phase I and phase II xenobiotic biotransformation capacities of rat and human hepatocytes in long-term collagen gel cultures. 1117 73

Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia are caused by mutations of the WAS protein (WASP) gene. WASP may be involved in the regulation of podosome, an actin-rich dynamic cell adhesion structure formed by various types of cells. The molecular links between WASP and podosomes or other cell adhesion structures are unknown. Platelets express an SH2-SH3 adapter molecule, CrkL, that can directly associate with paxillin, which is localized in podosomes. The hypothesis that CrkL binds to WASP was, therefore, tested. Results from coprecipitation experiments using anti-CrkL and GST-fusion proteins suggest that CrkL binds to WASP through its SH3 domain and that the binding was not affected by WASP tyrosine phosphorylation. The binding of GST-fusion SH3 domain of PSTPIP1 in vitro was also not affected by WASP tyrosine phosphorylation, suggesting that the binding of the SH3 domains to WASP is not inhibited by tyrosine phosphorylation of WASP. Anti-CrkL also coprecipitates a 72-kd protein, which was identified as syk tyrosine kinase, critical for collagen induced-platelet activation. CrkL immunoprecipitates contain kinase-active syk, as evidenced by an in vitro kinase assay. Coprecipitation experiments using GST-fusion CrkL proteins suggest that both SH2 and SH3 domains of CrkL are involved in the binding of CrkL to syk. WASP, CrkL, syk, and paxillin-like Hic-5 incorporated to platelet cytoskeleton after platelet aggregation. Thus, CrkL is a novel molecular adapter for WASP and syk and may potentially transfer these molecules to the cytoskeleton through association with cytoskeletal proteins such as Hic-5.
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PMID:CrkL is an adapter for Wiskott-Aldrich syndrome protein and Syk. 1131 52

Linear IgA disease (LAD) is an autoimmune subepidermal blistering skin disease characterized by the linear deposition of IgA at the dermoepidermal junction. Serum from patients with LAD most commonly contains autoantibodies that are directed against the hemidesmosomal transmembrane glycoprotein BP180 (type XVII collagen). Various antigenic sites on the extracellular domain of this anchoring filament protein have been shown to be targeted by autoantibodies in different autoimmune bullous skin diseases, including bullous pemphigoid and cicatricial pemphigoid (CP). However, little is known about epitopes on BP180 recognized by autoantibodies in LAD. In this study, we used three recombinant GST fusion proteins, together roughly covering the entire BP180 ectodomain, to characterize the autoimmune response in serum from patients with LAD. Interestingly, we found both IgA and IgG reactivity to all three portions of the BP180 ectodomain. The strongest reactivity was observed with the C-terminal portion of BP180. This is also the major region recognized by autoantibodies in patients with CP. This finding correlates with the observation that there may be significant overlap of the clinical and immunopathological findings in LAD and CP.
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PMID:Mapping of epitopes on the BP180 ectodomain targeted by IgA and IgG autoantibodies in patients with the lamina lucida-type of linear IgA disease. 1135 23

We evaluated the antioxidant status, namely cellular lipid peroxidation, by measuring thiobarbituric acid reactive substances (TBARS), cellular reduced glutathione (GSH) content, glutathione reductase (GSSG-R), glutathione transferase (GST), glutathione peroxidase (GSH-Px) and catalase activities in rat liver, hepatocytes immediately after isolation and in two-dimensional (2D) culture (on non-coated or collagen-coated dishes, as collagen-collagen or collagen-Matrigel sandwich cultures) or three-dimensional (3D) culture on Matrigel-coated dishes. Microsomal cytochrome P450 (CYP)- and UDP-glucuronosyl transferase (UGT)- dependent activities were also assessed in rat livers and hepatocyte cultures. The overall antioxidant status of rat hepatocytes immediately after isolation was not significantly different from that of rat livers. During culture, GSH was increased in 2D but not in 3D cultures in accordance with morphological observations; that is that matrix-cell interactions involving GSH, important in 2D, are minimal in 3D cultures. While UGT- and GST-dependent activities were equivalent in cultured hepatocytes and in rat livers, both catalase and GSH-Px activities decreased with time in all culture configurations. Constitutive CYP-dependent activities were drastically decreased in hepatocytes after isolation and attachment and did not recover in any culture configuration tested. Our results highlight that, although 2D sandwich cultures and 3D cultures on Matrigel allow longevity of rat hepatocyte cultures and optimal induction of CYPs, an imbalance in phase I/phase II detoxication processes in cultured rat hepatocytes occurs, whatever the culture configuration.
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PMID:Evaluation of the effect of culture configuration on morphology, survival time, antioxidant status and metabolic capacities of cultured rat hepatocytes. 1181 44

Trimucytin is a powerful platelet aggregation inducer isolated from the venom of Taiwan habu snake (Trimeresurus mucrosquamatus). In this study, we found that the snake venom protein, crovidisin, which prevents collagen-platelet interaction through its high-affinity binding to collagen, inhibited competitively trimucytin-induced aggregation of washed human platelets with a pA(2) value of 6.65. The ability of trimucytin in triggering platelet aggregation was suppressed by a monoclonal antibody (A2-IIE10) raised against the alpha2 subunit of alpha2beta1 integrin (glycoprotein Ia/IIa), indicating that platelet alpha2beta1 integrin plays a central role in trimucytin's platelet reactivity. Many studies have localized the major reactive site of alpha2beta1 integrin to the I-domain of alpha2 subunit. However, Escherichia coli-produced recombinant alpha2 I-domain (GST-alpha2 fusion protein) blocking collagen-induced platelet aggregation failed to inhibit aggregation of platelets in response to trimucytin. Based on these findings, it is concluded that the platelet reactivity of trimucytin is alpha2beta1 integrin-dependent, while the I-domain present in the alpha2 subunit is not involved. This novel snake venom protein would be useful for mapping the functional domain of alpha2beta1 integrin.
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PMID:Trimucytin, a collagen-like snake venom protein, activates platelets independent of I-domain within alpha2 subunit of alpha2beta1 integrin. 1195 6

Discoidin domain receptor 2 (DDR2) is a new type of receptor tyrosine kinases, and was thought to be involved in the metastasis of some tumors. Its ligand is fibrillar collagen. The activation of DDR2 induced by collagen mediates the over-expression of matrix metalloproteinase 1 (MMP-1) in cells. A specific inhibitor of DDR2 was necessary for the study of DDR2 function. Theoretically, a soluble receptor could possibly be used as specific inhibitor for the native receptor on cell membrane. In this report, a fragment (DB) of extracellular part of DDR2 was cloned and expressed for the use as potential inhibitor. This DB fragment corresponded to the polypeptide from the 23rd amino acid residue to the 293rd amino acid residue of DDR2. The fragment was amplified by RT-PCR from human lung cancer tissue, and the product was cloned into pMD18-T vector. After identification by sequence analysis, the fragment was sub-cloned into pGEX-4T-1 vector. Fusion protein of GST-DB was expressed in JM109 E.coli cells as expected and the soluble part accounted for about 13% of the total fusion protein. The soluble fusion protein was then purified with glutathione affinity resin, and GST-DB with purity of 86.1% was obtained. Competitive combination inhibitory test showed that the purified GST-DB inhibited the interaction between collagen II and DDR2 on the surface of RA synovial fibroblasts. Zymography analysis showed that the level of MMP-1 of both NIH 3T3 cell and RA synovial fibroblasts with collagen II-stimulation decreased after adding GST-DB fusion protein. The results indicated that the fusion protein GST-DB could inhibit the function of DDR2 on cells, and DDR2 might mediate collagen II-induced over-expression of MMP-1 in these cells.
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PMID:Expression, Purification and Functional Identification of Extracellular Part of Discoidin Domain Receptor 2. 1203 56

White spot syndrome virus (WSSV) is at present one of the major pathogens in shrimp culture worldwide. The complete genome of this virus has been sequenced recently. To identify the structural and functional proteins of WSSV, the purified virions were separated by SDS-PAGE. Twenty-four protein bands were excised, in-gel digested with trypsin, and subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry, respectively. Eighteen proteins matching the open reading frames of WSSV genome were identified. Except for three known structural proteins and collagen, the functions of the remaining 14 proteins were unknown. Temporal analysis revealed that all the genes were transcribed in the late stage of WSSV infection except for vp121. Of the newly identified proteins, VP466 (derived from band 16) was further characterized. The cDNA encoding VP466 was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Specific antibody was generated with the purified GST-VP466 fusion protein. Western blot showed that the mouse anti-GST-VP466 antibody bound specifically to a 51-kDa protein of WSSV. Immunogold labeling revealed that VP466 protein is a component of the viral envelope. Results in this investigation thus proved the effectiveness of proteomic approaches for discovering new proteins of WSSV.
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PMID:Proteomic analysis of shrimp white spot syndrome viral proteins and characterization of a novel envelope protein VP466. 1209 22


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