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)

The present study was designed to determine the antibody specificity for the human immunodeficiency virus type 1 (HIV-1) V3 domains of infectious and noninfectious virions present in the serum of AIDS patients. To accomplish this, HIV-1 was isolated in the presence of autologous antibodies from the serum samples of six AIDS patients in HIV-1-negative donor peripheral blood mononuclear cells by short-term cultivation. The isolated virus, defined as the infectious cell-free virus (iCFV), was characterized by sequence analysis of the proviral DNA coding for the third hypervariable (V3) region of the external glycoprotein gp120. This was carried out by amplifying and cloning the V3 region. In all six cases studied, 20 randomly selected V3 clones derived from the proviral DNA of the iCFV, 20 clones from patient cell-free virus, and 20 clones from cell-integrated virus were sequenced to study the distribution and frequency of the intrapatient virus population. The number of major virus variants in the six patients ranged from three to nine. The various V3 sequences found in the AIDS patients showed the typical amino acid pattern of the syncytium-inducing and non-syncytium-inducing viral phenotypes characteristic for the late stage of infection. However, only one patient-specific iCFV variant was detected within the 20 V3 clones analyzed per virus isolation. For the six patients a total of 34 V3-loop variants, either iCFV or non-iCFV, was observed. All 34 V3-loop sequences were expressed as glutathione-S-transferase fusion proteins (V3-GST). The autologous antibody response to the V3-GST fusion proteins was studied by Western immunoblot analysis. A strong antibody response to almost all non-iCFV V3-GST proteins was found in the sera of the six patients. In contrast, the autologous antibody response to the six iCFV V3 loops was undetectable (in four patients) or very faint (in two patients) compared with that to the non-iCFV V3 loops. Five of the six iCFV loops showed positively charged amino acids at positions strongly associated with the syncytium-inducing phenotype. These findings suggest that our in vitro isolation system selects for virions which are not recognized by V3-specific antibodies and are infectious both in vitro and in vivo.
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PMID:Antibodies of symptomatic human immunodeficiency virus type 1-infected individuals are directed to the V3 domain of noninfectious and not of infectious virions present in autologous serum. 818 27

The N-terminal fragment comprising residues +1 to +50 (gB1-50) of equine herpesvirus type 1 (EHV-1) glycoprotein B was expressed as a glutathione S-transferase fusion protein in Escherichia coli. Recombinant gB1-50 (rgB1-50) was recognized in immunoblots by sera from rabbits immunized with EHV-1 and by convalescent-phase sera from horses with natural EHV-1 infections. An enzyme-linked immunosorbent assay (ELISA) for monitoring antibody levels against EHV-1 was developed by using rgB1-50, and its specificity was assessed with a panel of reference antisera against other equine viruses. A specific cross-reaction was detected with EHV-4, which was confirmed by inhibition ELISA. Convalescent-phase sera from horses with natural EHV-1 or EHV-4 infections possessed antibody titers against rgB1-50 ranging from 1:2,000 to 1:64,000, indicating the presence of an immunodominant antigenic site. The study demonstrated the potential application of rgB1-50 as a diagnostic antigen and highlights the glutathione S-transferase fusion system as a simple and effective method of producing purified milligram quantities of antigen.
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PMID:Detection of antibodies against equine herpesvirus types 1 and 4 by using recombinant protein derived from an immunodominant region of glycoprotein B. 838 9

Alkylating agents, natural products and platinum complexes are the primary chemotherapeutic agents used in the treatment of patients with ovarian cancer. Resistance frequently develops to all three classes of drugs and can be functionally separated into distinct biochemical pathways: (1) relative dose intensity plays a role in resistance to platinum complexes and to a lesser degree with alkylating agents; (2) induction of the membrane P-170 glycoprotein confers resistance to natural products and due to the potential usefulness of Taxol (a natural product extracted from the bark of yew trees), this mechanism of resistance may become more clinically relevant in the future; (3) increased levels of cellular glutathione (GSH) and glutathione S-transferases are important in the detoxification of alkylating agents and platinum complexes; and (4) increased DNA repair also is characteristic of resistance to platinum complexes and alkylating agents. Clinical trials have been initiated with agents that may inhibit the biochemical mechanisms of acquired drug resistance. Clinical trials are already in progress with alkylating agents combined with inhibition of GSH biosynthesis (i.e., buthionine sulfoximine) or enzymatic inhibitors of glutathione S-transferase activity (i.e., ethacrynic acid). Furthermore, the combination of aphidicolin, an inhibitor of DNA repair, together with platinum complexes also soon will be clinically tested based on promising results in preclinical models of ovarian cancer. Ovarian cancer is a disease of the elderly. Advances in the pharmacology of platinum compounds and in our understanding of the mechanisms of drug resistance should permit these patients to receive increasingly more effective chemotherapy.
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PMID:Mechanisms of drug resistance in ovarian cancer. 842 Jun 89

A series of truncated equine herpesvirus 1 (EHV1) glycoprotein C (gC) molecules was examined for use as serodiagnostic antigens for EHV1 and EHV4. Small regions of EHV1 glycoprotein C, an immunodominant EHV1 glycoprotein, were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins using the bacterial expression vector pGEX-2T. Sera obtained from horses, including sera from specific-pathogen-free (SPF) foals, following exposure to either EHV1, EHV4 or both viruses were used. Several of the fusion proteins were shown to encompass EHV1 specific epitopes while others encompassed strong, cross-reactive epitopes. One clone, termed pEC-3, produced a soluble and stable fusion protein which encompassed amino acids 107-275 of EHV1 gC. Strong cross-reactive epitopes on pEC-3 were localised to a region encompassed by amino acids 137 to approximately 152 while EHV1 specific epitope(s) were identified downstream of this region, i.e., approximately amino acids 152 to 275. E. coli expressed EHV1 gC polypeptides showed clear potential for use as diagnostic reagents for the detection of cross-reactive and type-specific EHV1 and EHV4 antibodies present in convalescent equine sera.
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PMID:Expression of small regions of equine herpesvirus 1 glycoprotein C in Escherichia coli. 854 55

Sequence analysis of BamHI fragment 1 of the pseudorabies virus (PrV) genome identified a novel PrV gene located upstream of the UL50 gene encoding PrV dUTPase. The deduced protein product displayed homology to the product of the herpes simplex virus type 1 UL49.5 protein. The predicted PrV UL49.5 protein consists of 98 amino acids with a calculated molecular mass of 10,155 Da. It contains putative signal peptide and transmembrane domains but lacks a consensus sequence for N glycosylation. PrV UL49.5 was expressed as a fusion protein with glutathione S-transferase in Escherichia coli, and a rabbit antiserum was generated. In Western blots (immunoblots) of purified virions, the antiserum detected a protein with an apparent molecular mass of 14 kDa. After fractionation of the virions, the 14-kDa protein was detected in the envelope fraction. Localization of the UL49.5 protein in the viral envelope was confirmed by immunoelectron microscopy. The treatment of purified virions with glycosidases led to a reduction of the apparent molecular mass in Western blots by approximately 2 kDa following digestion with neuraminidase and O-glycosidase. Our results demonstrate that the PrV UL49.5 protein is an O-glycosylated structural component of the viral envelope. It represents the 10th PrV glycoprotein described. According to the unified nomenclature for alphaherpesvirus glycoproteins, we propose to designate it glycoprotein N (gN).
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PMID:The UL49.5 gene of pseudorabies virus codes for an O-glycosylated structural protein of the viral envelope. 855 87

An analysis of linear antibody-binding sites of the glycoprotein B (gB) molecule of murine cytomegalovirus (MCMV) and of genetic variation within these regions was performed. To achieve this, a series of overlapping fragments spanning the entire coding sequence of the gB gene of the K181 strain of MCMV was expressed in E. coli as fusion proteins with glutathione S-transferase (GST) using the pGEX expression system. Four antibody-binding regions were mapped to locations spanning amino acid residues 17-79 (BS), 155-278 (BE2), 809-926 (SS) and 347-508 (BB and EE), based on reactivity in Western blot analysis of GST-gB fusion proteins with murine polyclonal antiserum raised against MCMV. Only the antibody-binding region BE2 (155-278) elicited an antiserum that exhibited complement-dependent neutralizing activity, and immunization of mice with the fusion protein BE2 led to moderate but significant reductions in the level of MCMV replication in the spleen. Polyclonal antisera raised against the GST-gB fusion proteins detected purified virion proteins of 105 kDa (anti-BS and anti-BE2) and 52 kDa (anti-SS) and are therefore likely to recognize the N-terminal and C-terminal portions of the gB molecule, respectively. The antibody-binding region within amino acid residues 17-79 was found to be MCMV strain-specific, whereas antibody-binding regions within residues 155-278 and 809-926 were found to be conserved among MCMV field isolates. Comparative sequence analysis of the corresponding regions of MCMV gB revealed a level and extent of sequence of sequence heterogeneity consistent with these findings.
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PMID:Assessment of antigenicity and genetic variation of glycoprotein B of murine cytomegalovirus. 855 28

Clones expressing a partial human cytomegalovirus putative fusion receptor were selected by binding specifically to monoclonal anti-idiotypic antibodies that mimic glycoprotein H. cDNA was isolated from 2 of the clones (131 and 611) and fused in frame with the glutathione S-transferase gene in a pGEX-4T-1 vector. Two purified peptides (FR131 and FR611) were produced: both were shown to bind specifically to the monoclonal anti-idiotypic antibodies and inhibit virus/cell fusion and viral plaque formation in a specific and dose-dependent manner. This is the first demonstration of cloned peptides encoding a putative cell membrane receptor that are able to block cytomegalovirus infection.
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PMID:Molecular cloning and expression of receptor peptides that block human cytomegalovirus/cell fusion. 860 45

To further investigate the immunological properties of the stage-specific 82-kDa glycoprotein (gp82) of Trypanosoma cruzi metacyclic trypomastigotes, previously shown to induce antigen-specific humoral and T-cell responses in mice, we performed a series of experiments with recombinant proteins containing sequences of gp82 fused to glutathione S-transferase. Of five fusion proteins tested, only J18b and J18b1, the carboxyproximal peptides containing amino acids 224 to 516 and 303 to 516, respectively, were recognized by monoclonal antibody 3F6 as well as by various anti-T. cruzi antisera and, when administered to mice, were capable of eliciting antibodies directed to the native gp82. The amino-terminal peptide and other carboxyterminal recombinant proteins lacking the central domain of gp82 (amino acids 224 to 356), which is exposed on the surface of live metacyclic forms, did not display any of these properties. Spleen cells derived from mice immunized with any of the five recombinant proteins proliferated in vitro in the presence of native gp82.J18b was the most stimulatory, whereas J18b3, the peptide containing amino acids 408 to 516, elicited the weakest response. When BALB/c mice immunized with J18b antigen plus A1(OH)3 as adjuvant were challenged 10 5 metacyclic trypomastigotes, 85% of them resisted acute infection, in comparison with control mice that received glutathione S-transferase plus adjuvant. Antibodies induced by J18b protein lacked agglutinating or complement-dependent lytic activity and failed to neutralize parasite infectivity. On the other hand, CD4+T cells from the spleens of J18b-immunized mice displayed an intense proliferative activity upon stimulation with 1.25 microgram of native gp82 per ml, which resulted in increased production of gamma interferon, a cytokine associated with resistance to T. cruzi infection.
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PMID:A recombinant protein based on the Trypanosoma cruzi metacyclic trypomastigote 82-kilodalton antigen that induces and effective immune response to acute infection. 860 64

Sialyltransferase activities, SAT-3 (CMP-NeuAc:nLcOse4Cer alpha 2-3sialyltransferase) and SAT-4 (CMP-NeuAc:GgOse4Cer alpha 2-3sialyltransferase), in Colo 205 cells catalyze the transfer of sialic acid to the terminal galactose of GlcNc-- and GalNAc-containing glycolipid substrates, respectively. Competition kinetic studies with nLcOse4Cer and GM1 as substrates in a sialyltransferase assay show that these two activities are catalyzed by two different catalytic entities. The two enzymes were co-solubilized with taurochlorate and resolved by DEAE--Cibacron Blue--Sepharose column chromatography into two elution peaks. The column eluent with SAT-3 activity failed to transfer sialic acid to asialo alpha(1)-acid glycoprotein, indicating that this enzyme is different from the sialyltransferase (ST3N) that synthesizes NeuAc alpha 2-3Gal linkage in asparagine-linked oligosaccharides of glycoprotein. However, SAT-3 activity can be immunoprecipitated with a polyclonal antibody produced against a protein expressed in Escherichia coli as GST-fusion protein from an ECB cDNA homolog of an alpha 2-3 sialyltransferase SAT-3 or STZ) the has been cloned from human melanoma cell and human placenta. Thus a concentration-dependent decrease in the residual SAT-3 activity relative to SAT-4 activity was observed in the supernatant after precipitation of the immune complex. Expression of SAT-3 (STZ) cDNA was also detected in Colo 205 cell by RT-PCR, followed by sequence analysis of the RT-PCR product. Characterization of the catalytic reaction products of SAT-3 and SAT-4 with thin-layer chromatography, sialidase treatment, and binding to specific antibodies indicates that both SAT-3 and SAT-4 catalyze the formation of alpha 2-3 linkage between sialic acid and terminal galactose of glycolipid substrates.
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PMID:Characterization of two glycolipid: alpha 2-3sialyltransferases, SAT-3 (CMP-NeuAc:nLcOse4Cer alpha 2-3sialyltransferase) and SAT-4 (CMP-NeuAc:GgOse4Cer alpha 2-3sialyltransferase), from human colon carcinoma (Colo 205) cell line. 861

Expression of the glutathione S-transferase (GST) subclasses alpha, mu and pi was investigated immunohistochemically in 20 normal or hyperplastic mesothelium and in 57 malignant mesothelioma cases. These results were correlated with survival and also with P-170 glycoprotein expression. Nearly all the non-neoplastic mesothelium cases were positive for GST alpha and pi. About half of the non-neoplastic cases were positive for mu. Twenty-nine (51%) malignant mesotheliomas were positive for at least one of the GST species; 21 (37%) showed immunoreactivity for alpha, 18 (31.5%) for mu and 21 (37%) for pi. A total of 54 mesothelioma cases displayed immunoreactivity for the P-170 glycoprotein. For GST pi and GST mu, a statistical significance between expression and increased survival was found (respectively P = 0.012 and 0.024) while for GST alpha no significance was found. The results of this study demonstrate that expression of GST pi correlates positively with increased survival in malignant mesothelioma. It is also concluded that, in mesothelioma, GST and P-170 glycoprotein may contribute to the resistance to cytotoxic drugs frequently observed in these tumours. No correlation between GST and P-170 expression was demonstrated.
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PMID:Glutathione S-transferase expression in malignant mesothelioma and non-neoplastic mesothelium: an immunohistochemical study. 887 60


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