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)

We developed a one-step real-time duplex reverse transcription PCR (RT-PCR) method using the LightCycler platform. This method allows simultaneous reverse transcription and real-time PCR amplification of two mRNAs of specific genes of interest (analyte genes) and mRNA of constantly transcribed genes (housekeeping genes) in a single-tube reaction. Specimen total nucleic acids were used because eukaryotic cDNA is discriminated from genomic DNA using exon-spanning primers and/or fluorescence resonance energy transfer (FRET) probes. Transcripts of murine arginase I and hypoxanthine-phosphoribosyl transferase (HPRT; housekeeping gene) or murine arginase II analyte and porphobilinogen deaminase (PBGD; housekeeping gene) were quantified in such duplex RT-PCRs. Twenty-minute reverse transcription reactions at 55 degrees C followed by 18 high-stringency step-down thermal cycles and 25 relaxed-stringency fluorescence acquisition cycles produced sensitive and accurate RT-PCR results. Fluorescent signal spillover between channels was fully compensated. A matrix of duplex PCRs at variable ratios of target standards yielded equations for factors that correct PCR-specific target ratio-dependent deviations in quantification. The one-step real-time duplex RT-PCRs reliably and accurately determined 10-10,000 copies of each target over a 100,000-fold range of target copy ratios (analyte to housekeeping mRNA = 10(-2.5)-10(2.5)) in a single assay.
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PMID:One-step real-time duplex reverse transcription PCRs simultaneously quantify analyte and housekeeping gene mRNAs. 1503 67

Reliable housekeeping gene controls are critical for measuring and comparing gene expression at the transcription level by Northern blot and RT-PCR. In order to develop such controls for studying cytokine mRNA expression in dogs, DNA sequence encoding a full-length canine HPRT protein has been obtained. Numerous primer pairs derived from the canine HPRT sequence have been tested on canine genomic DNA as well as cDNA. The data from the present study suggest that there may be processed HPRT pseudogenes in dogs. Three pairs of canine HPRT primers designed and tested in the present study were able to differentiate between cDNA and genomic DNA under specific PCR conditions. These primers would be useful controls for measurement of mRNA expression by RT-PCR in the dog.
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PMID:Selection of HPRT primers as controls for determination of mRNA expression in dogs by RT-PCR. 1511 53

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

Preeclampsia and diabetes are complications of pregnancy that contribute to maternal and perinatal mortality worldwide. Results emerging from molecular studies of placentae may elucidate etiologically important genomic alterations. Appropriate application of real time reverse transcription (RT) PCR in comparative gene expression studies requires endogenous housekeeping genes to normalize between sample variations. Ideal housekeeping genes must have stable tissue expression, but few have been specifically studied in the placenta. We sought to identify candidate control genes by analyzing seven functionally distinct housekeeping genes (B2M, GAPDH, HMBS, HPRT, SDHA, TBP, YWHAZ) for their expression stability and level in the placenta. mRNA isolated from 20 placentae was analyzed for gene expression using RT-PCR. Expression stability (M) was assessed using normalization strategies previously used for other tissues. TBP and SDHA were the most stable, with an average expression stability of M = 0.43, followed by YWHAZ (M = 0.44) > HPRT (M = 0.53) > HMBS (M = 0.57) > GAPDH (M = 0.61) > B2M (M = 0.69). The genes tested ranged in abundance, with an approximately 300-fold increase from the lowest (HMBS) to the highest (B2M). By using TBP, SDHA and YWHAZ, with greater expression stability than those housekeeping genes commonly used in placenta studies, gene expression profile comparisons will have more sensitivity and specificity.
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PMID:Evaluation of housekeeping genes in placental comparative expression studies. 1608 39

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

Valid housekeeping genes (HKG) are a prerequisite for accurate gene quantification. We performed real-time reverse transcription-polymerase chain reaction to investigate the gene expression of five commonly used HKGs (beta-actin, glyceraldehyde-3-phosphate dehydrogenase [GAPDH], ubiquitin C [UBC], hypoxanthine phosphoribosyl-transferase [HPRT], and cyclophilin A [CYPa]) and antioxidant enzymes in the liver of young and old male Fischer rats. A wide variation in HKG expression existed during the aging process, and HPRT was identified as the most stable HKG in rat liver aging. When Cu/Zn-superoxide dismutase gene expression was normalized to HPRT, there was no detectable difference between young and old rats; however, a significant difference was seen when it was normalized to UBC. The variation of UBC caused the misinterpretation of Cu/Zn-superoxide dismutase expression. Catalase expression was significantly decreased, whereas glutathione peroxidase expression was not altered with age. We demonstrated that HPRT was an appropriate HKG, validation of HKGs was vital for accurate quantification, and decreased catalase expression might be involved in the decline of antioxidant defenses during rat liver aging.
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PMID:Identification of valid housekeeping genes and antioxidant enzyme gene expression change in the aging rat liver. 1645 91

In order to quantify in vivo the mRNAs of cytokines which play important roles in leptospirosis, we have developed quantitative real-time PCR assays for interleukin-2 (IL-2), IL-4, IL-10, IL-12p40, tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), transforming growth factor beta, and two housekeeping genes (encoding beta-actin and hypoxanthine phosphoribosyltransferase). We used a lethal hamster model reflecting severe leptospirosis in humans. The LightCycler system was used to quantify the gene expression levels with the SYBR green I detection format using external standard curves for each target. We compared the expression levels of cytokine mRNA in the peripheral blood mononuclear cells of both control (uninfected) hamsters and Leptospira interrogans-inoculated hamsters from 1 to 24 h and then 1 to 4 days postinfection. In this kinetic study, there was pronounced expression of Th1 cytokine mRNA (TNF-alpha, IFN-gamma, and IL-12), with transcripts being detected as early as 1 h postinfection. Expression of anti-inflammatory cytokines, such as IL-4 and IL-10, was prominent in delayed samples from 1 to 4 days postinfection in response to infection with Leptospira interrogans. Our data are the first to establish that pathogenic leptospires can stimulate in vivo the production of type 1 cytokines involved in cellular immunity by using this informative animal model. Measuring and assessing cytokine profiles may provide a useful method for accurate study of the mechanisms of anti-Leptospira immunity, indications of prognosis factors, and prospective evaluation of leptospirosis vaccine efficacy in humans.
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PMID:Proinflammatory and immunomodulatory cytokine mRNA time course profiles in hamsters infected with a virulent variant of Leptospira interrogans. 1679 Jul 92

Only limited amounts of peripheral blood samples can be obtained from small children. Therefore, a polymerase chain reaction (PCR) aided analysis of cytokine gene expression by PBMC or T cells is a valuable tool. We present a combination of procedures to obtain an accurate estimation of the expression of the cytokines IL-4 and IFN-gamma. This can be performed on T cells purified from blood samples of up to 5 ml in volume from children aged 0-4 years with allergic asthma and atopic dermatitis. This procedure includes multiple sampling of PCR products to determine the linear phase of the PCR; inter-experiment correction using a helper T-cell clone, expressing both IL-4 and IFN-gamma; interpatient correction by comparing the expression of a housekeeping gene (HPRT); and finally the development of specific software to analyse densitometric data obtained by scanning photographs of agarose gels, separating PCR products. In this way it is possible to study cytokine gene expression from a very small amount of material.
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PMID:Analysis of cyt0kine gene expression in stimulated T cells of small children by semi-quantitative PCR. 1847 39

Metabolic activation of 17beta-estradiol (E2) to catechols and quinones together with lack of deactivation constitute risk factors in human breast carcinogenesis. E2-catchols are generated by cytochrome P450-dependent monooxygenases (CYPs). Deactivation of E2, E2-catechols, and E2-quinones is mediated by UDP-glucuronosyltransferase (UGT), sulfotransferase (SULT), catechol-O-methyltransferase (COMT), glutathione-S-transferase (GST), and NADPH-quinone-oxidoreductase (QR) isozymes, respectively. The aim of the present study was to quantify mRNA levels of E2-metabolizing isozymes expressed in MCF-7 cells cultured in the presence/absence of steroids by reverse transcription/competitive PCR in relation to the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase and compare them with expression levels in normal human mammary gland (MG) and liver tissue. CYP1A1, 1B1, SULT1A1, 1A2, membrane-bound and soluble COMT, GSTT1, QR1, and UGT2B7 were detected in both tissues and MCF-7 cells; however, most enzymes were expressed at least tenfold higher in liver. Yet, CYP1B1 was expressed as high in breast as in liver and UGTs were not detected in MCF-7 cells cultured with steroids. MCF-7 cells cultured steroid-free additionally expressed CYP1A2 as well as UGT1A4, 1A8, and 1A9. Normal human liver but not MG expressed CYP1A2, 3A4, UGT1A1, 1A3, 1A4, 1A9, and SULT2A1. UGT1A8 was only detected in MCF7 cells but was not found in human liver. Thus, our study provides a comprehensive overview of expression levels of E2-metabolizing enzymes in a popular in vitro model and in human tissues, which will contribute to the interpretation of in vitro studies concerning the activation/deactivation of E2.
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PMID:Gene expression of 17beta-estradiol-metabolizing isozymes: comparison of normal human mammary gland to normal human liver and to cultured human breast adenocarcinoma cells. 1849 89

Neuronal transcription factors play vital roles in the specification and development of neurons, including dopaminergic (DA) neurons. Mutations in the gene encoding the purine biosynthetic enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) cause the resulting intractable and largely untreatable neurological impairment of Lesch-Nyhan disease (LND). The disorder is associated with a defect in basal ganglia DA pathways. The mechanisms connecting the purine metabolic defect and the central nervous system (CNS) phenotype are poorly understood but have been presumed to reflect a developmental defect of DA neurons. We have examined the effect of HPRT deficiency on the differentiation of neurons in the well-established human (NT2) embryonic carcinoma neurogenesis model. We have used a retrovirus expressing a small hairpin RNA (shRNA) to knock down HPRT gene expression and have examined the expression of a number of transcription factors essential for neuronal differentiation and marker genes involved in DA biosynthetic pathway. HPRT-deficient NT2 cells demonstrate aberrant expression of several transcription factors and DA markers. Although differentiated HPRT-deficient neurons also demonstrate a striking deficit in neurite outgrowth during differentiation, resulting neurons demonstrate wild-type electrophysiological properties. These results represent direct experimental evidence for aberrant neurogenesis in HPRT deficiency and suggest developmental roles for other housekeeping genes in neurodevelopmental disease.
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PMID:Deficiency of the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT) dysregulates neurogenesis. 1967 49


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