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)

Previous studies reported that, in the absence of drug exposure, multidrug resistance, including resistance to Adriamycin (ADR), could develop in primary rat hepatocyte cultures (B. Carr, Proc. Am. Assoc. Cancer Res., 29:1158, 1988). However, the hepatocytes in that report were cultured on plastic without the benefit of an extracellular matrix (ECM). Because the ECM regulates hepatic gene expression, we have critically evaluated in primary cultures of rat hepatocytes how the ECM affects hepatic ADR resistance, the level of the drug efflux transporter associated with MDR, P-glycoprotein (pgp), and transport of a prototypical pgp substrate, vincristine. Hepatocytes cultured on type I collagen (Vitrogen) had greater resistance to ADR toxicity accompanied by parallel increases in the level of pgp mRNA, decreased drug accumulation, and enhanced drug efflux when compared with the hepatocytes maintained on the basement membrane matrix Matrigel. The development of ADR resistance coincided with the time course of increased pgp mRNA but was not coincident with the time course of expression of either the placental isozyme of glutathione S-transferase or P-450 reductase, proteins associated with MDR in some resistance models. Southern blot analysis revealed neither gross changes in pgp gene structure or gene copy number to account for the increase in pgp RNA levels for hepatocytes cultured on Vitrogen. ECM also regulated xenobiotic-inducible expression of hepatic pgp, since chemotherapeutic agents, including vincristine and colchicine, induced pgp mRNA exclusively in hepatocytes cultured on Vitrogen. The critical matrix proteins in Matrigel responsible for regulation of pgp were determined by the selective addition of its components to the culture environment. The presentation of the individual matrix elements as a rigid substratum to the hepatocyte did not decrease pgp mRNA. In contrast, the presentation to the same hepatocytes of either laminin or type IV collagen in a nonrigid state (solubly in the medium) selectively decreased hepatocellular pgp mRNA. We conclude that primary rat hepatocytes develop ADR resistance with time in culture due to increased expression of pgp and that ECM proteins represent endogenous physiological modulators of both basal and chemotherapeutically inducible expression of hepatic P-glycoprotein.
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PMID:Extracellular matrix regulation of multidrug resistance in primary monolayer cultures of adult rat hepatocytes. 842 4

BP180 is a 180kDa hemidesmosomal protein recognized by bullous pemphigoid (BP) and pemphigoid gestationis (PG) autoantibodies. Recent cloning and sequence analysis performed by our laboratory have revealed that BP180 is a transmembrane protein with a long extracellular collagen-like region. A rabbit polyclonal antibody has been generated against a recombinant protein, designated GST-N delta 1, containing a segment of the BP180 ectodomain. The resulting antiserum, RN delta 1A, was shown to specifically react with BP180 on immunoblot, and labelled the extracellular region of the epidermal hemidesmosome on immunoelectron microscopy. A panel of normal and neoplastic human tissues were analysed by indirect immunofluorescence (IF) and RN delta 1A, to determine the distribution of BP180. A total of nine basal cell carcinomas (BCCs) and four squamous cell carcinomas (SCCs) of the skin were also studied. Intense IF staining was seen along the basement membrane zone (BMZ) of the epidermis, hair follicles, and the periphery of sebaceous gland lobules. The sebaceous lobules showed more intense staining in areas close to the duct. The epithelial BMZ of the following tissues also reacted with RN delta 1A: cornea, ocular conjunctiva, buccal mucosa, upper oesophagus, placenta (amnion placentum), umbilical cord and transitional epithelium of the bladder. The epithelium of the jejunum and ovary failed to react with RN delta 1A. Staining of the BCCs and SCCs was variable. Five of six nodular BCCs showed some anti-BP180 staining at the tumour-stromal interface, although the level of staining was less intense than that observed in the overlying normal epidermis. All three morphoeic BCCs analysed in this investigation did not show any staining with RN delta 1A. Three of four SCCs showed weak staining at the tumour-stromal interface. Thus, the tissue distribution of BP180 paralleled that of hemidesmosomes, and expression of this protein was found to be decreased or absent in cutaneous neoplasms.
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PMID:Expression pattern of the bullous pemphigoid-180 antigen in normal and neoplastic epithelia. 854 92

Hsp47, an endoplasmic reticulum-resident heat shock protein in fibroblasts, has gelatin-binding properties. It had been hypothesized that it functions as a chaperone regulating procollagen chain folding and/or assembly, but the mechanism of the hsp47-procollagen I interaction was not clear. Hsp47 could bind to both denatured and native procollagen I. A series of competition studies were carried out in which various collagens and collagen domain peptides were incubated with 35[S]-methionine-labeled murine 3T6 cell lysates prior to mixing with gelatin-Sepharose 4B beads. The gelatin-bound proteins were collected and analyzed by gel electrophoresis and autoradiography. Collagenase digested procollagen I had the same effect as denatured intact procollagen, indicating that the propeptides were the major interaction sites. The addition of intact pro alpha 1(I)-N-propeptide at 25 micrograms/ml completely inhibited hsp47 binding to the gelatin-Sepharose. Even the pentapeptide VPTDE, residues 86-90 of the pro alpha 1(I)-N-propeptide, inhibits hsp47-gelatin binding. These data implicating the pro alpha 1(I)-N-propeptide domain were confirmed by examination of polysome-associated pro alpha chains. The nascent pro alpha 1(I)-chains with intact N-propeptide regions could be precipitated by monoclonal hsp47 antibody 11D10, but could not be precipitated by monoclonal anti-pro alpha 1 (I)-N-propeptide antibody SP1.D8 unless dissociated from the hsp47. GST-fusion protein constructs of residues 23-108 (NP1), 23-151 (NP2), and 23-178 (NP3) within the pro alpha 1 (I)- N-propeptide were coupled to Sepharose 4B and used as affinity beads for collection of hsp47 from 3T6 cell lysates. NP1 and NP2 both showed strong specific binding for lysate hsp47. Finally, the interaction was studied in membrane-free in vitro cotranslation systems in which the complete pro alpha 1(I)- and pro alpha 2(I)-chain RNAs were translated alone and in mixtures with each other and with hsp47 RNA. There was no interaction evident between pro alpha 2(I)-chains and hsp47, whereas there was strong interaction between pro alpha 1(I)-chains and nascent hsp47. SP1.D8 could not precipitate pro alpha 1(I)-chains from the translation mix if nascent hsp47 was present. These data all suggest that if hsp47 has a "chaperone" role during procollagen chain processing and folding it performs this specific role via its preferential interaction with the pro alpha 1 (I) chain, and the pro alpha 1(I) amino-propeptide region in particular.
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PMID:Endoplasmic reticulum protein Hsp47 binds specifically to the N-terminal globular domain of the amino-propeptide of the procollagen I alpha 1 (I)-chain. 856 53

The role of Shc as a substrate of receptors for growth factors and cytokines is well established. To gain further insight into the function of Shc in signal transduction, we used an affinity method to identify potential Shc-binding proteins. Incubation of bovine brain lysates with a glutathione S-transferase (GST)-Shc fusion protein immobilized on glutathione-Sepharose beads resulted in the binding of cellular proteins of approximately 115, 110, and 100 kDa as well as those of 50 and 17 kDa. Amino acid sequencing of tryptic peptides revealed that the 100-kDa protein was almost identical to beta-adaptin and that the 110- and 115-kDa proteins were almost identical to alphaA-adaptin. Using immunoblot analysis, anti-alpha-adaptin antibody recognized several proteins of 100 approximately 115 kDa, and anti-beta-adaptin antibody recognized a 100-kDa protein, suggesting that alphaA-, alphaC-, and beta-adaptins are bound to the GST-Shc fusion protein. Immunoblot analysis with anti-alpha-adaptin antibody revealed that alpha-adaptin was coimmunoprecipitated with Shc from PC12, KB, and COS cell lysates, suggesting a specific interaction of Shc and adaptins in intact cells. A binding study using mutant GST-Shc fusion proteins revealed that the collagen homologous region (amino acids 233-377) of Shc was required for adaptin binding. Conversely, the collagen homologous region of Shc inhibited the binding of adaptins to GST-Shc. In addition, adaptin was able to bind mutant fusion proteins containing amino acids 233-369, 233-355, 346-369, and 346-355 of Shc, but failed to bind a mutant containing amino acids 233-345, suggesting that amino acids 346-355 (RDLFDMKPFE) in the collagen homologous region of Shc are required for adaptin binding. Thus, this study indicates the specific interaction of Shc with alpha- and beta-adaptin components of plasma membrane adaptor proteins that are thought to be involved in receptor endocytosis.
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PMID:Interaction of Shc with adaptor protein adaptins. 861 12

The ability of kininogens to modulate thrombin-induced aggregation of human platelets has been assigned to domain 3 (D3) in the common heavy chain coded for by exons 7, 8, and 9 of kininogen gene. We expressed each of the exons 7, 8, and 9, and various combinations as glutathione S-transferase fusion proteins in Escherichia coli. Each of the exon products 7 (Lys236-Gln292), 9 (Val293-Gly328), and 8 (Gln329-Met357), and their combinations were evaluated for the ability to inhibit thrombin induced platelet aggregation. Only products containing exon 7 inhibited platelet aggregation induced by thrombin with an IC50 of > 20 microM. A deletion mutant of exon 7 product, polypeptide 7A product (Lys236-Lys270) did not block thrombin-induced platelet aggregation, while 7B product (Thr255-Gln292) and 7C product (Leu271-Gln292) inhibited aggregation. These findings indicated that the inhibitory activity is localized to residues Leu271-Gln292. Peptides Phe279-Ile283 and Phe281-Gln292 did not block thrombin, and Asn275-Phe279 had only minimal inhibitory activity. A heptapeptide Leu271-Ala277 inhibited thrombin-induced aggregation of platelets with an IC50 of 65 microM. The effect is specific for the activation of platelets by thrombin but not ADP or collagen. No evidence for a thrombin-kininogen complex was found, and neither HK nor its derivatives directly inhibited thrombin activity. Knowledge of the critical sequence of kininogen should allow design of compounds that can modulate thrombin activation of platelets.
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PMID:Thrombin-induced platelet aggregation is inhibited by the heptapeptide Leu271-Ala277 of domain 3 in the heavy chain of high molecular weight kininogen. 862 72

Shc has two distinct domains, amino-terminal and SH2 domain, which can interact with activated growth factor receptors. Shc interacts with insulin receptor via Shc-amino-terminal (N) domain, whereas Shc associates with epidermal growth factor (EGF) receptor through both Shc-N and -SH2 domains. In accordance with the different functional roles between insulin and EGF receptors, EGF stimulated tyrosine phosphorylation of Shc faster than insulin. To clarify the functional importance of three distinct Shc domains on insulin and EGF signaling, we microinjected glutathione S-transferase (GST) fusion proteins containing the amino terminus plus collagen homology domain (NCH), collagen homology domain (CH), and Src homology 2 domain (SH2) into Rat1 fibroblasts expressing insulin receptors (HIRc). Bromodeoxyuridine (BrdUrd) incorporation into newly synthesized DNA was subsequently studied to assess the importance of the three distinct domains of Shc. Microinjection of the NCH-GST fusion protein inhibited BrdUrd incorporation induced by both EGF and insulin, whereas microinjection of the SH2-GST fusion protein inhibited EGF, but not insulin stimulation of DNA synthesis. Neither EGF- nor insulin-induced BrdUrd incorporation was inhibited by the CH-GST fusion protein. Following EGF or insulin stimulation, Shc is phosphorylated on single Tyr-317 residue serving as a docking site for Grb2. Microinjection of Shc-N+CH GST fusion protein with Tyr-317 --> Phe replacement (Y317F) also inhibited insulin stimulation of DNA synthesis. Next, we stably overexpressed wild-type Shc or Y317F mutant Shc into HIRc cells. Insulin-induced tyrosine phosphorylation of IRS-1 was compared among the transfected cell lines, since IRS-1 and Shc could competitively interact with insulin receptor. Insulin-stimulated tyrosine phosphorylation of IRS-1 was decreased in both WT-Shc and Y317F-Shc cells compared with that in HIRc cells. Furthermore, overexpression of the Shc-SH2 domain or Shc-N+CH domain with Y317F mutation interfered with EGF-stimulated endogenous Shc phosphorylation. These results suggest that the amino terminus domain of Shc is functionally important in insulin- and EGF-induced cell cycle progression and that the phosphorylation of Shc Tyr-317 residue is independent of Shc interaction with these receptors.
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PMID:Functional importance of amino-terminal domain of Shc for interaction with insulin and epidermal growth factor receptors in phosphorylation-independent manner. 870 28

Mitogen-activated protein kinases (MAPKs), a family of protein serine/threonine kinases regulating cell growth and differentiation, are activated by a dual-specificity kinase through phosphorylation at threonine and tyrosine. We used a recently described selective inhibitor of the p42/p44mapk-activating enzyme, PD 98059 [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one], to investigate the role of the p42/p44mapk pathway in human platelets. PD 98059 inhibited p42/p44mapk activation in thrombin-, collagen- and phorbol esterstimulated platelets, as determined from in-gel renaturation kinase assays, with an IC50 of approx. 5 microM (thrombin stimulation). It also prevented activation of MAPK kinase, which was measured in whole-cell lysates with glutathione S-transferase/p42mapk fusion protein (GST-MAPK) as substrate. Inhibition of p42/p44mapk did not affect platelet responses to thrombin or collagen such as aggregation, 5-hydroxytryptamine release and protein kinase C activation. In addition, PD 98059 did not interfere with release of arachidonic acid, a response mediated by cytosolic phospholipase A2 (cPLA2), or with cPLA2 phosphorylation. This suggests that platelet cPLA2 is not regulated by p42/p44mapk after stimulation with physiological agonists. In contrast, phorbol ester-induced phosphorylation of cPLA2 and potentiation of arachidonic acid release stimulated by Ca2+ ionophore A23187 were inhibited by PD 98059, indicating that p42/p44mapk phosphorylates cPLA2 after activation of protein kinase C by the non-physiological tumour promoter.
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PMID:Inhibition of mitogen-activated protein kinase kinase does not impair primary activation of human platelets. 876 73

Alpha 1 beta 1 heterodimer is a member of the integrin receptor superfamily that has been described to be involved in cell-matrix binding through its interaction with collagens, fibronectin and laminin. The alpha 1 integrin belongs to a subset of I-domain containing integrins that includes alpha M, alpha L, alpha X and alpha 2. In this study we describe an anti-alpha 1 mAb (FB12) that recognizes an epitope located in the human alpha 1 I-domain, since the mAb can bind to human, but not to rat, recombinant I-domain GST fusion protein. FB12 mAb efficiently and specifically inhibits the binding of activated human lymphocytes to laminin, collagen and fibronectin. These data support the notion that the alpha 1 I-domain itself has an important role in receptor-ligand binding. In particular, we show that alpha 1 integrin-dependent lymphocyte adhesion to fibronectin is I-domain mediated, at variance with the RGD-dependent adhesion which seems to be mediated by the beta 1 rather than the alpha 1 integrin chain. Lastly, the overexpression of the alpha 1-integrin by stromal cells and blood vessels of solid tumors may suggest a role for this integrin in tumor biology.
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PMID:A functional monoclonal antibody recognizing the human alpha 1-integrin I-domain. 886 74

In order to explore the protective function of human glutathione S-transferase pi (GST-pi) in vitro and in vivo, transfected NIH 3T3 clones were examined in cytotoxicity assays using the carcinogen (+/-)anti-benzo(a)pyrene 7,8-diol-9,10-epoxide (BPDE) or inoculated into nude mice and treated with the carcinogen benzo(a)pyrene (BP) to induce tumor formation. The human GST-pi cDNA under the control of the murine alpha 2(I)collagen promoter was transfected into NIH 3T3 cells and G418 resistant clones were analyzed by Southern, northern, western, and two-dimensional analysis. Clone A2 stably expressed human GST-pi and has 2.5-fold greater activity toward the substrate 1-chloro-2,4-dinitrobenzene and a 1.7-fold increase in LD50 for BPDE in vitro when compared to control-transfected clone G3. This increase in protection, however, did not prevent the formation of BP-induced tumors in vivo.
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PMID:Overexpression of human glutathione S-transferase pi protects NIH 3T3 cells against (+/-)anti BPDE cytotoxicity but not tumor formation. 886 91

The cuticle of parasitic nematodes is composed of extracellular structural proteins. Over 90% of these proteins are collagenous molecules in the basal and median layers of the cuticle. The outermost layers of the cuticle, the epicuticle, is composed of non-collagenous proteins, that represent the structural surface of nematodes. In Ascaris these proteins have been termed 'cuticlins'. While cuticular collagens have been well studied by both biochemical and genetic means, knowledge of the molecular structure of cuticlin components of parasitic nematodes is scarce. In the present paper we report on the production of monoclonal antibody 8.1, which is specific for cuticlin, but does not recognize collagen epitopes. We have screened a cDNA library derived from adult Ascaris suum mRNA of the hypodermal tissue underlying and synthesizing the cuticle. One positive cDNA clone encodes alanine-rich repetitive motifs, which are part of the insoluble cuticlin of the outermost layers of the epicuticle of Ascaris suum. This was shown in immunocytochemical experiments using specific polyclonal antisera raised against these motifs, expressed as fusion protein with glutathione S-transferase of the helminth Schistosoma japonicum. Comparison of the repetitive amino acid sequence with structural proteins of the nematode Caenorhabditis elegans and the insects Locusta migratoria and Ceratitis capitata revealed a minimal consensus motif.
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PMID:Repetitive peptide motifs in the cuticlin of Ascaris suum. 888 22


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