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 cellular localization of glutathione-requiring PGD synthetase, which catalyzes the predominant formation of PGD2 in various peripheral tissues, was investigated in adult rats by immunoperoxidase-staining with a polyclonal antibody specific for this enzyme. Although the 25 N-terminal amino acid residues of synthetase are 56% identical and 76% similar to those of several rat glutathione S-transferase subunits, the antibody cross-reacted only with synthetase in dot blotting and was nearly completely inactive with all transferase isozymes thus far purified. In Western blotting after SDS-PAGE of crude extracts of rat spleen, the antibody showed a single positive band at the same position as that of the purified enzyme (Mr = 26,000). The positive immunocytochemical stain was found in a number of histiocytes and/or dendritic cells in spleen, thymus, and Peyer's patch of intestine. The immunostain was also observed in such cells in lamina propria of the villus in small intestine and colon, in submucosal layer of stomach, and in Kupffer cells in liver. Immunoelectron microscopy confirmed that immunoreactivity of this enzyme was distributed in cytoplasm of those cells. Such immunoreactive cells were not observed in brain, spinal cord, kidney, heart, testis, and skeletal muscle. These observations suggest that PGD2 is produced by glutathione-requiring PGD synthetase localized in these types of APC in various tissues and may play a critical role in dictating the progression of immune responses.
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PMID:The major source of endogenous prostaglandin D2 production is likely antigen-presenting cells. Localization of glutathione-requiring prostaglandin D synthetase in histiocytes, dendritic, and Kupffer cells in various rat tissues. 250 61

Colon cancer provides an attractive setting for chemoprevention trials because of the frequency and variation of familial predisposition that is observed in this malignancy. Additionally, the adenomatous polyp, the precursor of colon cancer, is a valuable intermediate marker for judging the effectiveness of candidate chemopreventive agents. Inherited colon cancer susceptibility varies from mild to severe. Conditions with extreme susceptibility include the autosomal dominantly inherited syndromes of familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC). These are highly penetrant syndromes with extreme cancer risk. FAP arises from mutations of the APC gene and HNPCC from mutations of the mismatch repair genes. Specific and individual genetic diagnosis is now possible in both syndromes, thus allowing identification of genetically affected individuals for chemoprevention trials. FAP accounts for less than 1% of colon cancers, while HNPCC may be present in up to 5% of cases. Familial clustering is common in the remainder of cases, which are often referred to as sporadic, but probably arise in part from inherited susceptibility. Epidemiologic studies have shown that first-degree relatives have a two- to four-fold increased risk of acquiring colon cancer compared to the general population. Ten percent of individuals in the U.S. have a first-degree with colon cancer. This clinically identifiable higher risk group thus constitutes a large potential cohort for chemoprevention trials. The common familial cases of colon cancer can be further stratified by severity. A relative diagnosed under the age of 50 or two first-degree relatives affected with colon cancer confers an even greater risk for this malignancy, estimated to be four to six times that of the general population. Adenomatous polyps also precede the development of colon cancer in these categories, thereby providing a readily identifiable clinical endpoint to judge the effectiveness of chemoprevention. It is expected that genetic markers will soon be available for more precise identification of common colon cancer susceptibility. Candidate markers include mild mutations of the APC and mismatch repair genes, glutathione transferase isoenzymes, acetylator status, and phospholipase A2 expression. Bile acid concentrations of the bowel may be genetically and/or environmentally determined and likely have a role in colon cancer susceptibility. We recently identified a large kindred with polyp and cancer susceptibility arising from a mild mutation of the APC gene. There are over 4,000 kindred members and mutational testing has demonstrated 140 gene carriers to date. We expect to institute chemoprevention trials in this kindred using adenomatous polyp number as an endpoint of effectiveness.
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PMID:Cohorts with familial disposition for colon cancers in chemoprevention trials. 902 9

GRA4, a dense granule protein of Toxoplasma gondii elicits both mucosal and systemic immune responses following oral infection of mice with cysts. We studied the antigenicity and immunogenicity of truncated and soluble forms of GRA4 expressed as glutathione S-transferase fusion proteins in Escherichia coli. Protein C (amino-acids 297-345) was particularly well recognized by serum IgG antibodies, milk IgA antibodies and intestinal IgA antibodies from T. gondii infected mice and by serum IgG antibodies from T. gondii infected humans and T. gondii infected sheep. One major B epitope was localized within the last 11 C-terminal residues of GRA4. A second epitope, recognized with lower frequency, was mapped within the region 318-334. In contrast, the N domain of GRA4 (amino acids 25-276) was poorly recognized. Oral immunization of C57BL/6 mice with N, C or NC (amino acids 25-276 fused to 297-345) in association with cholera toxin induced a significant production of serum anti-GRA4 IgG antibodies but a weak and inconsistent intestinal anti-GRA4 IgG antibody response and afforded partial resistance to oral infection with T. gondii. These results provide new molecular and immunological understanding of GRA4 and indicate that it is a potential candidate for oral vaccination against T. gondii.
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PMID:Mapping of B epitopes in GRA4, a dense granule antigen of Toxoplasma gondii and protection studies using recombinant proteins administered by the oral route. 961 29

Immunodominance or cripticity of a peptide-borne determinant may be influenced by the protein context in which the epitope is embedded. In this frame, we previously showed that certain human T cell clones, derived from different donors, may differentially recognize the RT248-262 helper determinant depending on whether it is provided to the presenting cells as a synthetic peptide or as a recombinant carrier protein to which the sequence of interest is fused. We now report that, upon in vitro immunization of human PBL with autologous APC, the epitope-specific TCRVB repertoire obtained when selection is applied by pulsing the APC with the cognate synthetic peptide is different from that found when a recombinant protein is used in which the antigenic sequence is placed at either a N-terminal or C-terminal location of the GST carrier. As the TCRVB distribution is not a function of the APC used, we propose that processing of different recombinant molecules containing the same epitope may generate MHC/peptide complexes which, being antigenically diverse, may recruit distinct TCR specificities. These findings may be relevant for evaluating and predicting the immunogenic potential of subunit vaccines based on synthetic peptides or on recombinant proteins as compared to the native antigen.
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PMID:In vitro immunization with a recombinant antigen carrying the HIV-1 RT248-262 determinant inserted at different locations results in altered TCRVB region usage. 1052 82

An assay based on fluorescence resonance energy transfer (FRET) has been developed to screen for ubiquitination inhibitors. The assay measures the transfer of ubiquitin from Ubc4 to HECT protein Rsc 1083. Secondary reagents (streptavidin and antibody to glutathione-S-transferase [GST]), pre-labeled with fluorophores (europium chelate, Eu(3+), and allophycocyanin [APC]), are noncovalently attached via tags (biotin and GST) to the reactants (ubiquitin and Rsc). When Rsc is ubiquitinated, Eu(3+) and APC are brought into close proximity, permitting energy transfer between the two fluorescent labels. FRET was measured as time-resolved fluorescence at the emission wavelength of APC, almost entirely free of nonspecific fluorescence from Eu(3+) and APC. The FRET assay generated a lower ratio of signal to background (8 vs. 31) than an assay for the same ubiquitination step that was developed as a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). However, compared to the DELFIA method, use of FRET resulted in higher precision (4% vs. 11% intraplate coefficient of variation). Quenching of fluorescence was minimal when compounds were screened at 10 microg/ml using FRET. Employing a quick and simple homogeneous method, the FRET assay for ubiquitin transfer is ideally suited for high throughput screening.
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PMID:Development of a ubiquitin transfer assay for high throughput screening by fluorescence resonance energy transfer. 1108 Jun 90

The C-terminal regions of several DNA repair and cell cycle checkpoint proteins are homologous to the breast-cancer-associated BRCA-1 protein C-terminal region. These regions, known as BRCT domains, have been found to mediate important protein-protein interactions. We produced the BRCT domain of DNA ligase IIIalpha (L3[86]) for biophysical and structural characterization. A glutathione S-transferase (GST) fusion with the L3[86] domain (residues 837-922 of ligase IIIalpha) was expressed in Escherichia coli and purified by glutathione affinity chromatography. The GST fusion protein was removed by thrombin digestion and further purification steps. Using this method, (15)N-labeled and (13)C/(15)N-double-labeled L3[86] proteins were prepared to enable a full determination of structure and dynamics using heteronuclear NMR spectroscopy. To obtain evidence of binding activity to the distal BRCT of the repair protein XRCC1 (X1BRCTb), as well as to provide insight into the interaction between these two BRCT binding partners, the corresponding BRCT heterocomplexes were also prepared and studied. Changes in the secondary structures (amount of helix and sheet components) of the two constituents were not observed upon complex formation. However, the melting temperature of the complex was significantly higher relative to the values obtained for the L3[86] or X1BRCTb proteins alone. This increased thermostability imparted by the interaction between the two BRCT domains may explain why cells require XRCC1 to maintain ligase IIIalpha activity.
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PMID:Expression, purification, and biophysical characterization of the BRCT domain of human DNA ligase IIIalpha. 1128 14

Methylobacterium dichloromethanicum DM4 is able to grow with dichloromethane as the sole carbon and energy source by using a dichloromethane dehalogenase/glutathione S-transferase (GST) for the conversion of dichloromethane to formaldehyde. Mammalian homologs of this bacterial enzyme are also known to catalyze this reaction. However, the dehalogenation of dichloromethane by GST T1-1 from rat was highly mutagenic and toxic to methylotrophic bacteria. Plasmid-driven expression of rat GST T1-1 in strain DM4-2cr, a mutant of strain DM4 lacking dichloromethane dehalogenase, reduced cell viability 10(5)-fold in the presence of dichloromethane. This effect was exploited to select dichloromethane-resistant transconjugants of strain DM4-2cr carrying a plasmid-encoded rGSTT1 gene. Transconjugants that still expressed the GST T1 protein after dichloromethane treatment included rGSTT1 mutants encoding protein variants with sequence changes from the wild-type ranging from single residue exchanges to large insertions and deletions. A structural model of rat GST T1-1 suggested that sequence variation was clustered around the glutathione activation site and at the protein C-terminus believed to cap the active site. The enzymatic activity of purified His-tagged GST T1-1 variants expressed in Escherichia coli was markedly reduced with both dichloromethane and the alternative substrate 1,2-epoxy-3-(4'-nitrophenoxy)propane. These results provide the first experimental evidence for the involvement of Gln102 and Arg107 in catalysis, and illustrate the potential of in vivo approaches to identify catalytic residues in GSTs whose activity leads to toxic effects.
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PMID:Dichloromethane mediated in vivo selection and functional characterization of rat glutathione S-transferase theta 1-1 variants. 1145 94

Aberrant signalling activities of beta-catenin, originally identified as a component of cell-adhesion complexes, are now considered to be an important factor in colorectal carcinogenesis. However, recently it was shown that also gamma- as well as p120 catenins have a dual role either in cell adhesion or in affecting some gene activation. Therefore, the levels and interactions of these three catenins in human colorectal carcinoma cell lines were analysed. A great heterogeneity in the expression of all catenins tested was found in colorectal carcinoma cell lines HT29 and LS174T. Detailed analysis of beta-catenin interactions was done. GST-APC fragment-fused proteins were used to absorb beta-catenin and its complexes from cell lysates. Similarly, the E-cadherin binding capacity of the residual pool of beta-catenin was analysed using the GST-ECT construct. It was found that the level of beta-catenin does not necessarily depend either on the APC or beta-catenin gene mutations and that co-precipitation of beta-, gamma-, and p120 catenins is not limited to cells that express E-cadherin.
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PMID:Expression and interaction of different catenins in colorectal carcinoma cells. 1171 88

Several classes of cytoplasmic proteins have been found to interact specifically with the carboxyl-terminal cytoplasmic region of the angiotensin II type 1 (AT(1)) receptor to regulate different aspects of AT(1) receptor physiology. The murine Angiotensin II Receptor-Associated Protein (Agtrap) is a new member of them. We have recently cloned a new human gene cDNA that codes for a homolog of the murine Agtrap protein from a human fetal brain cDNA library. The deduced polypeptide product of the cDNA is 22 kDa in size, and its DNA and amino acid sequences are 85 and 77% identical to those of the mouse Agtrap gene, respectively. Hence we have named it the human Angiotensin II Receptor-Associated Protein (AGTRAP) gene. The mRNA of AGTRAP was most abundantly expressed in kidney, heart, pancreas and thyroid. Using the yeast two-hybrid screening of a human fetal brain cDNA library, we have identified a new interaction partner of the human AGTRAP protein, RACK1 (Receptor of Activated Protein C Kinase). The AGTRAP-RACK1 interaction was confirmed by GST fusion protein pull-down assays, co-immunoprecipitation and surface plasmon resonance. We suggest that the AGTRAP-RACK1 interaction may help to recruit signaling complex to the AT(1) receptor to affect AT(1) receptor signaling.
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PMID:Identification and characterization of AGTRAP, a human homolog of murine Angiotensin II Receptor-Associated Protein (Agtrap). 1173 89

Spindle checkpoint proteins, such as Mad2 and BubR1, and the motors dynein/dynactin and CENP-E usually leave kinetochores prior to anaphase onset by microtubule-dependent mechanisms. Likewise, 'chromosome passenger proteins' including INCENP are depleted from the centromeres after anaphase onset and then move to the midzone complex, an event that is essential for cytokinesis. Here we test whether the cell cycle changes that occur at anaphase onset require or contribute to the depletion of kinetochore and centromere proteins independent of microtubules. This required the development of a novel non-antibody method to induce precocious anaphase onset in vivo by using a bacterially expressed fragment of the spindle checkpoint protein Mad1 capable of activating the APC/C, called GST-Mad1F10. By injecting PtK1 cells in nocodazole with GST-Mad1F10 and processing the cells for immunofluorescence microscopy after anaphase sister chromatid separation in nocodazole we found that Mad2, BubR1, cytoplasmic dynein, CENP-E and the 3F3/2 phosphoepitope remain on kinetochores. Thus depletion of these proteins (or phosphoepitope) at kinetochores is not required for anaphase onset and anaphase onset does not produce their depletion independent of microtubules. In contrast, both microtubules and anaphase onset are required for depletion of the 'chromosome passenger' protein INCENP from centromeres, as INCENP does not leave the chromosomes prior to anaphase onset in the presence or absence of microtubules, but does leave the centromeres after anaphase onset in the presence of microtubules.
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PMID:Anaphase onset does not require the microtubule-dependent depletion of kinetochore and centromere-binding proteins. 1223 89


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