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:2.4.2.8 (hypoxanthine-guanine phosphoribosyltransferase)
2,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

2fTGH is a human cell line containing the selectable marker guanine phosphoribosyltransferase regulated by alpha interferon (IFN-alpha). Two IFN-alpha-unresponsive mutants were isolated previously at a low frequency (ca. 10(-8)) by selecting mutagenized 2fTGH cells in selective medium containing 6-thioguanine and IFN-alpha. By using five rounds of mutagenesis, mutants can be isolated at an appreciably higher frequency, greater than 3 x 10(-7). Five new mutants have been isolated, and all are recessive, as are the two mutants we described previously. The seven mutants are in four complementation groups (U1-U4). Since several different types of mutants unresponsive to IFN-alpha have been isolated with high frequency, related approaches may succeed with other cytokines or growth factors. Mutants in the two new complementation groups U3 and U4 are unresponsive to IFN-alpha and, surprisingly, also unresponsive to IFN-gamma. They are also partially defective in response to double-stranded RNA. These results indicate that the signaling pathways for the two types of IFN and double-stranded RNA share common components or that their function depends on common enzymes or transcription factors. IFN receptors are unaffected in mutants U3A and U4A. A major defect appears to be in the synthesis or activation of E, the transcription factor mediating the primary response to type I (alpha/beta) IFNs. Band-shift complementation assays show that U3A contains the E gamma subunit but does not contain an active E alpha subunit after treatment with IFN-alpha.
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PMID:High-frequency mutagenesis of human cells and characterization of a mutant unresponsive to both alpha and gamma interferons. 183 50

Previously we described human cell line 2fTGH, in which expression of guanine phosphoribosyltransferase is tightly controlled by the upstream region of interferon (IFN)-stimulated human gene 6-16. After mutagenesis of 2fTGH and selection with 6-thioguanine and IFN-alpha, we isolated 11.1, a recessive mutant that does not respond to IFN-alpha. We now describe U2, a second recessive mutant, selected similarly, that complements 11.1. U2 had no response to IFN-alpha or IFN-beta, and its response to IFN-gamma was partially defective. Although many genes did respond to IFN-gamma in U2, the 9-27 gene did not and the antiviral response of U2 cells to IFN-gamma was greatly reduced. Band shift assays showed that none of the transcription factors normally induced in 2fTGH cells by IFN-alpha (E and M) or IFN-gamma (G) were induced in U2. However, extracts of untreated U2 cells gave rise to a novel band that was increased by treatment with IFN-gamma but not IFN-alpha. Band shift complementation assays revealed that untreated and IFN-gamma-treated U2 cells lack the functional E gamma subunit of transcription factor E and that IFN-alpha-treated U2 cells do contain the functional E alpha subunit.
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PMID:Isolation and characterization of a new mutant human cell line unresponsive to alpha and beta interferons. 190 77

We have selected mutations in genes encoding components of the signaling pathway for alpha interferon (IFN-alpha) by using a specially constructed cell line. The upstream region of the IFN-regulated human gene 6-16 was fused to the Escherichia coli guanine phosphoribosyltransferase (gpt) gene and transfected into hypoxanthine-guanine phosphoribosyltransferase-negative human cells. These cells express gpt only in the presence of IFN-alpha. They grow in medium containing hypoxanthine, aminopterin, and thymidine plus IFN and are killed by 6-thioguanine plus IFN. Two different types of mutants were obtained after treating the cells with mutagens. A recessive mutant, selected in 6-thioguanine plus IFN, was completely resistant to IFN-alpha but responded normally to IFN-gamma and, unexpectedly, partially to IFN-beta. A constitutive mutant, selected in hypoxanthine-aminopterin-thymidine alone, was abnormal in expressing endogenous genes in the absence of IFN. Both types revert infrequently, allowing selection for complementation of the defects by transfection.
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PMID:Use of a selectable marker regulated by alpha interferon to obtain mutations in the signaling pathway. 251 75

Sezary's syndrome is a leukemic proliferation of OKT4+ lymphocytes. Sezary cells were isolated by differential centrifugation and fused to CEM.8azar.C, and HGPRTase-lacking clone of CEM. The hybrid cells were studied for their ability to produce soluble mediators of human monocyte cytotoxicity. The product of a single clone, FtF3, which bore the surface phenotype of Sezary cells, was characterized. Monocyte cytotoxicity-inducing factor (MCF) was found to be stable at pH 2 for 1 hr, unlike IFN-gamma, and was found to be more heat stable as well. Moreover, treatment of MCF with antisera to IFN-gamma, IFN-alpha or a combination of IFN-gamma and IFN-alpha failed to neutralize its biologic activity. MCF binds to matrix gel Red A. MCF eluted from this dye-ligand was found to have an apparent m.w. of 11,500 by gel filtration and 14,700 by SDS-polyacrylamide gel electrophoresis. MCF produced by hybridized Sezary cells appear to be neither IFN-gamma nor an altered molecular form of IFN-gamma, yet is a potent inducer of human monocyte cytotoxicity.
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PMID:Identification of a human monocyte cytotoxicity-inducing factor from T cell hybridomas produced from Sezary's cells. 308 3

A synthetic DNA construct has been developed as a standard molecule whereby murine cytokine mRNA molecules can be quantified by the reverse transcription-polymerase chain reaction (RT-PCR). The construct, designated Cytoquant 1, allows the quantification of murine IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IFN-gamma, TNF-alpha, TGF-beta, GM-CSF, CD4, CD8, HPRT and beta-actin mRNA levels. This technique is based on the amplification of a transcribed RNA molecule from Cytoquant 1 as an internal standard control in both the RT and PCR reactions. The quantification data from these analyses are expressed in absolute values, i.e. molecules/cell, which allows the data derived from separate experiments to be compared. In this study, mRNAs encoding beta-actin, IL-10, IFN-gamma and GM-CSF have been quantitated in both Th1 and Th2 cell clones with, and without, stimulation. The quantitative analysis data are highly reproducible and cytokine mRNA concentrations are reflective of restricted cytokine secretion patterns. Furthermore, constitutive cytokine mRNA levels are detectable in resting cells, eliminating the need for exogenous stimulation. The high degree of sensitivity and accuracy make this methodology uniquely suited for the study of T-cell subset cytokine expression in both in vivo and in vitro biological models.
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PMID:A synthetic standard DNA construct for use in quantification of murine cytokine mRNA molecules. 747 5

Rapamycin (RAP) disrupts signaling events implicated in cytokine-dependent proliferation of lymphocytes and other cells. This action is known to involve the formation of molecular complexes between the drug and intracellular binding proteins, termed FKBPs. However, the biochemical target(s) for the effector RAP-FKBP complexes remain uncharacterized. As an approach to explore the mechanism of action of RAP, we have isolated three independent sets of somatic mutants of the YAC-1 murine T cell line with markedly reduced sensitivity to the drug's inhibitory effects on proliferation and on IL-1-induced IFN-gamma production. These mutants were still fully sensitive to FK-506, an immunosuppressant structurally related to RAP whose mode of action also involves an interaction with FKBPs. Furthermore, the 12-kDa FKBP, FKBP12, was detectable in immunoblots from cytosolic extracts and eluates from RAP-affinity matrix in the mutants as in wild-type cells, suggesting that the resistance to RAP in the mutants is not due to a lack of FKBP12 expression. Cell fusion experiments were conducted to further define the nature of the alterations imparting RAP resistance in these mutants. Clones deficient in either thymidine kinase or hypoxanthine-guanine phosphoribosyltransferase, suitable as fusion partners for aminopterin-based selection of hybrids were generated from the wild-type or mutant lines. In most instances, the hybrids derived from the fusion between RAP-sensitive clones and RAP-resistant clones exhibited a RAP-resistant phenotype. Similar results were obtained with hybrids between RAP-resistant YAC-1 clones and the RAP-sensitive EL-4 cell line. Therefore, the mutations that confer resistance to RAP in the present system are dominant. Altogether, our observations are consistent with a model where pharmacologically relevant targets for the RAP-FKBP complex, rather than FKBP, might be altered in the mutants such that the inactivation of these targets by the effector complex is prevented.
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PMID:Dominant mutations confer resistance to the immunosuppressant, rapamycin, in variants of a T cell lymphoma. 753 11

Competitive polymerase chain reaction (PCR) is a sensitive method for quantification of cytokine mRNA expression. Co-amplification of an internal standard serves as control for comparing the efficiency of PCR in different samples. We have developed a novel control fragment for multiple analyses of rat cytokine gene expression containing primers for IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, TNF-alpha, TGF-beta 1, IFN-gamma and MIP-2. Additional primers were incorporated to analyse the content of T cells (CD3), activated T cells (CD25) and housekeeping genes (beta-actin and HPRT). As an example we demonstrate analysis of IL-2 mRNA expression in small pieces of kidney tissue obtained from rats after kidney allotransplantation. The IL-2 expression decreased tenfold during treatment with an anti-rat CD4 monoclonal antibody as compared to untreated animals.
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PMID:A novel multispecific competitor fragment for quantitative PCR analysis of cytokine gene expression in rats. 782 31

It has been postulated that HIV-infected patients undergo an active production of virus and CD4+ T cell destruction from the early stages of the disease, and that an extensive postthymic expansion of CD4+ T cells prevents a precipitous decline in CD4+ T cell number. Based on the rebound of the CD4+ T cell number observed in patients undergoing antiretroviral therapy with protease inhibitors, it has been calculated that, on average, 5% of T cells are replaced every day in HIV-infected patients. To obtain an independent estimate of the recycling rate of T cells in the patients, we measured the frequency of cells carrying a loss-of-function mutation at the hypoxanthine guanine phosphoribosyl transferase (hprt) locus. Assuming a recycling rate of 5%/d, an accumulation of 2.6 mutations/10(6)/yr over the physiological accumulation was predicted. Indeed, we observed an elevated frequency of HPRT mutants in the CD4+ T cells of most patients with < 300 CD4+ T cells/mm3 of blood and in the CD8+ T cells of most patients with < 200 CD4+ T cells/mm3, consistent with an elevated and protracted increased division rate in both subsets. However, in earlier stages of the disease the mutant frequency in both CD4+ and CD8+ T cells was lower than in healthy controls. The cytokine production profile of most HPRT mutant CD4+ T cell clones from both healthy and HIV-infected patients was typical of T helper cells type 2 (high IL-4 and IL-10, low IFN-gamma), whereas the cytokine production pattern of wild-type clones was heterogeneous. The cytokine profile of CD8+ clones was indistinguishable between HPRT mutants and wild type. Our data provide evidence of increased CD4+ and CD8+ T cell recycling in the HIV-infected patients.
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PMID:Frequency and cytokine profile of HPRT mutant T cells in HIV-infected and healthy donors: implications for T cell proliferation in HIV disease. 904 68

A variety of commercial DNA arrays specific for humans and rodents are widely available; however, microarrays containing well-characterized genes to study pathway-specific gene expression are not as accessible for domestic animals, such as cattle, sheep and pigs. Therefore, a small-scale application-targeted bovine immune-endocrine cDNA array was developed to evaluate genetic pathways involved in the immune-endocrine axis of cattle during periods of altered homeostasis provoked by physiological or environmental stressors, such as infection, vaccination or disease. For this purpose, 167 cDNA sequences corresponding to immune, endocrine and inflammatory response genes were collected and categorized. Positive controls included 5 housekeeping genes (glyceraldehydes-3-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase, ribosomal protein L19, beta-actin, beta2-microglobulin) and bovine genomic DNA. Negative controls were a bacterial gene (Rhodococcus equi 17-kDa virulence-associated protein) and a partial sequence of the plasmid pACYC177. In addition, RNA extracted from un-stimulated, as well as superantigen (Staphylococcus aureus enterotoxin-A, S. aureus Cowan Pansorbin Cells) and mitogen-stimulated (LPS, ConA) bovine blood leukocytes was mixed, reverse transcribed and PCR amplified using gene-specific primers. The endocrine-associated genes were amplified from cDNA derived from un-stimulated bovine hypothalamus, pituitary, adrenal and thyroid gland tissues. The array was constructed in 4 repeating grids of 180 duplicated spots by coupling the PCR amplified 213-630 bp gene fragments onto poly-l-lysine coated glass slides. The bovine immune-endocrine arrays were standardized and preliminary gene expression profiles generated using Cy3 and Cy5 labelled cDNA from un-stimulated and ConA (5 microg/ml) stimulated PBMC of 4 healthy Holstein cows (2-4 replicate arrays/cow) in a time course study. Mononuclear cell-derived cytokine and chemokine (IL-2, IL-1alpha, TNFalpha, IFN-gamma, TGFbeta-1, MCP-1, MCP-2 and MIP-3alpha) mRNA exhibited a repeatable and consistently low expression in un-stimulated cells and at least a two-fold increased expression following 6 and 24 h ConA stimulation as compared to 0 h un-stimulated controls. In contrast, expression of antigen presenting molecules, MHC-DR, MHC-DQ and MHC-DY, were consistently at least two-fold lower following 6 and 24 h ConA stimulation. The only endocrine gene with differential expression following ConA stimulation was prolactin. Additionally, due to the high level of genetic homology between ovine, swine and bovine genes, RNA similarly acquired from sheep and pigs was evaluated and similar gene expression patterns were noted. These data demonstrate that this application-targeted array containing a set of well characterized genes can be used to determine the relative gene expression corresponding to immune-endocrine responses of cattle and related species, sheep and pigs.
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PMID:Construction and application of a bovine immune-endocrine cDNA microarray. 1526 89

We have established an easy real-time PCR assay, which allows the precise quantification of changes in the expression level of 6 relevant porcine cytokines, and 3 housekeeping genes. This assay simultaneously detects 9 sequences by measuring 3 x 3 targets in a triplex-format. The mRNA of the lymphokines IL-2, IL-4, IL-10, and IFN-gamma, of the proinflammatory cytokines IL-1alpha and IL-6, and of the housekeeping genes are quantified using TaqMan-probes by means of standard dilution series on the iCycler iQ. The standard consists of equal aliquots of the experimental cDNAs under investigation. Simultaneously the most suitable combination of 3 out of the four housekeeping genes beta-actin, HPRT, GAPDH, and cyclophilin can be selected, and their averaged expression values constitute a normalisation factor. The raw data of all targets of interest is then calculated relative to this normalisation factor, making eventual changes of the relative expression level of the single housekeeping genes controllable and quantifiable. We have applied this assay to quantify changes in the cytokine mRNA levels of porcine stimulated with various concentrations of LPS and ConA, known to induce different cytokine expression patterns. We have shown, that even small differences in the expression level (less than 2-fold) can be precisely quantified, and reveal statistically significant changes, when using the normalisation factor. This assay will be useful for studying changes in the expression of relevant porcine cytokines and will help to further improve the investigation of immune responses in the pig.
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PMID:Quantitative simultaneous multiplex real-time PCR for the detection of porcine cytokines. 1622 7


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