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:3.2.1.31 (beta-glucuronidase)
7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An assay system for transcriptional profile analysis of cultured eukaryotic cells has been developed to simultaneously handle multiple samples in a rapid, sensitive, and internally controlled manner. The methodology incorporates a microtiter plate assay system, a rapid cell-harvest enzyme-assay technique, and the bacterial reporter genes beta-glucuronidase and beta-galactosidase. We demonstrate, using beta-actin and SV40 (late) transcription promoting sequences, that this technically refined microtiter-triton-lysate (MTL) assay methodology can readily differentiate between the transcriptional states of human melanocytes before and after pharmacologic stimulation and malignantly transformed versus normal cell environments. Differences in the transcriptional environments are revealed by the relative expression of transcription element probes. The transcriptional activity ratio of the beta-actin compared to the SV40 late transcription promoting sequences was approximately 1:2 in primary cultured melanocytes, 2:1 in 12-0-tetradecanoyl phorbol-13-acetate (TPA)-treated melanocytes and 1:4 in the Tang melanoma cell line. Because this MTL assay methodology can accommodate a panel of transcription element probes, we anticipate that the resultant transcriptional profiles will prove useful in deciphering the diverse transcriptional changes that occur within normally regulated and malignantly transformed cells.
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PMID:A novel approach to analysis of transcriptional regulation in human cells: initial application to melanocytes and melanoma cells. 185 Jul 74

A method was developed for measuring in vivo rates of mRNA synthesis in mice by pulse-labeling with the RNA precursor [3H]orotate and then using hybridization to recover specific mRNAs. The efficiency of recovery is determined with synthetic RNAs as internal hybridization standards. The method is particularly applicable to the kidney since this organ shows a strong preferential uptake of the label. Rates of synthesis, expressed as a fraction of total RNA synthesis, were measured for the androgen-inducible mRNAs coding for beta-glucuronidase (GUS), ornithine decarboxylase (ODC), the protein coded by the RP-2 gene, and the so-called kidney androgen-regulated protein (KAP). Control mRNAs coded for beta-actin, phosphoenolpyruvate carboxykinase, and major urinary protein. Testosterone markedly increased the synthesis of the androgen-inducible mRNAs, but not the control mRNAs. Induction was not seen in mutant mice lacking functional androgen receptor protein. For GUS, ODC, and RP-2 mRNAs, the fold induction of synthesis was less than the fold induction of concentration, suggesting that mRNA stabilization also plays a part in the response to androgen. For GUS, ODC, and RP-2 mRNAs, but not KAP mRNA, induction of synthesis was rapidly reversed after testosterone removal. KAP mRNA was also exceptional in that its concentration was disproportionately high compared with its rate of synthesis, implying that it is a particularly stable mRNA.
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PMID:mRNA synthesis rates in vivo for androgen-inducible sequences in mouse kidney. 338 33

When cDNAs for human and rodent beta-glucuronidases were expressed in COS-7 cells using several different promoters, rodent beta-glucuronidases were produced three times more than human beta-glucuronidase, although their transcriptional levels were similar. Similar observations were also recorded in LMTK- cells using SV40 early or chicken beta-actin promoters. In hopes of enhancing yields of recombinant human beta-glucuronidase for enzyme replacement therapy, we sought to determine the region within the linear sequences responsible for the higher levels of expression of the rodent cDNAs. To do so, we made various rat-human chimeric cDNAs utilizing conserved restriction enzyme sites. The levels of products expressed from these chimeric cDNAs in COS cells were assessed by activity assay and by metabolic labeling of the proteins followed by immunoprecipitation and SDS-PAGE. From the results of these expression studies, we identified a 155-bp ClaI (643)-AflII (797) fragment in the rat open reading frame responsible for the increased rate of translation of the rat beta-glucuronidase (RBG) cDNA. Replacement of the homologous ClaI (683)-AflII (838) fragment in human beta-glucuronidase (HBG) with this 155-bp fragment from RBG increased the translation level of the resulting chimeric HRaH. Conversely, substitution of the 155-bp human fragment for that of rat in RBG cDNA reduced the total synthesis of the resulting chimeric HHaR. Placement of the 155-bp segment between the initiation ATG and the promoter has only negative effects on the expression of either cDNA. A more stable secondary structure of the human cDNA in this region might explain a reduced rate of translation. However, secondary structure analysis of mRNAs from the 155-bp fragment of rat and human cDNAs predicted that, while both can form stem-loops, the rat fragment (delta Gzero = -42.1 kcal/ mol) is actually more stable than the human fragment (delta Gzero = -32.1 kcal/mol). In fact, the free energy of stability of the first 50 bp within this ClaI-AflII fragment from rat (-10.3 kcal/mol) indicates that the secondary structure is considerably more stable than the corresponding 50 bp from human (-2.1 kcal/mol). This segment of the rat sequence also contains a tar-like sequence in a stem-loop. Although tar-like sequences can enhance rates of translation, altering this sequence by mutagenesis had no effect on the rate of synthesis of rat beta-glucuronidase. Thus, although the region conferring enhanced rate of synthesis from rat cDNA has been identified, the mechanism by which it does so is not yet clear.
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PMID:Enhanced translation of rat beta-glucuronidase cDNA is conferred by 155-bp segment of internal coding sequence. 880 77

Quantitative gene expression protocols require adequate controls to monitor intersample variation. Quantitative approaches to describe relative changes in gene expression use endogenous controls--"housekeeping" genes. Given the low amounts of mRNA in fat cells, RT-PCR is the method of choice, and housekeeping genes are widely used as endogenous controls. However, literature reports suggest changes in gene expression of typical housekeeping genes (e. g. GAPDH, beta-actin, 18S rRNA) upon hormonal stimulation or during adipogenic differentiation. Thus, we tested the influence of 6 hormones and adipogenic differentiation on gene expression levels of 11 commonly used housekeeping genes in primary cultured mature human adipocytes and preadipocytes. Using the TaqMan RT-PCR technique and "Human Endogenous Control Assays" (PE Biosystems), we found several housekeeping genes with at least twice the difference in expression levels between stimulated and unstimulated cells (such as acidic ribosomal protein, beta-actin, beta(2)-microglobulin and beta-glucuronidase). Only GAPDH and transferrin receptor gene expression levels did not change under any of the stimuli tested, thus appeared best suited for gene expression studies in human adipose cells across a wide range of experimental settings.
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PMID:Validation of endogenous controls for gene expression studies in human adipocytes and preadipocytes. 1160 84

For interpretation of quantitative gene expression measurements in clinical tumor samples, a normalizer is necessary to correct expression data for differences in cellular input, RNA quality, and RT efficiency between samples. In many studies, a single housekeeping gene is used for normalization. However, no unequivocal single reference gene (with proven invariable expression between cells) has been identified yet. As the best alternative, the mean expression of multiple housekeeping genes can be used for normalization. In this study, no attempt was made to determine the gold-standard gene for normalization, but to identify the best single housekeeping gene that could accurately replace the measurement of multiple genes. Expression patterns of 13 frequently used housekeeping genes were determined in 80 normal and tumor samples from colorectal, breast, prostate, skin, and bladder tissues with real-time quantitative RT-PCR. These genes included, large ribosomal protein, beta-actin, cyclophilin A, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerokinase 1, beta-2-microglobin, beta-glucuronidase, hypoxanthine ribosyltransferase (HPRT), TATA-box-binding protein, transferrin receptor, porphobilinogen deaminase, ATP synthase 6, and 18S ribosomal RNA. Principal component analysis was used to analyze these expression patterns, independent of the level of expression. Our approach identified HPRT as the single best reference gene that could be used as an accurate and economic alternative for the measurement of multiple housekeeping genes. We recommend this gene for future studies to standardize gene expression measurements in cancer research and tumor diagnostics until a definite gold standard has been determined.
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PMID:Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes. 1554 3

Real-time reverse transcription PCR has greatly improved the ease and sensitivity of quantitative gene expression studies. However, measurement of gene expression generally requires selection of a valid reference (housekeeping gene) for data normalization to compensate for inherent variations. Given the dynamic nature of early embryonic development, application of this technology to studies of oocyte and early embryonic development is further complicated due to limited amounts of starting material and a paucity of information on constitutively expressed genes for data normalization. We have validated quantitative procedures for real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA abundance during bovine meiotic maturation and early embryogenesis and utilized this technology to determine temporal changes in mRNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, beta-glucuronidase, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, and histone H2A. Quantification of amounts of specific exogenous RNAs added to samples revealed acceptable rates of RNA recovery and efficiency of reverse transcription with minimal variation. Progression of bovine oocytes to metaphase II resulted in reduced abundance of polyadenylated, but not total transcripts for majority of above genes; however phosphoglycerokinase exhibited a significant decline in both RNA populations. Abundance of mRNAs for above genes in early embryos generally remained low until the blastocyst stage, but abundance of ribosomal protein L-15 mRNA was increased at the morula stage and histone H2A mRNA showed dynamic changes prior to embryonic genome activation. Results demonstrate a valid approach for quantitative analysis of mRNA abundance in oocytes and embryos, but do not support constitutive expression of above genes during early embryonic development.
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PMID:Quantitative analysis of messenger RNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, beta-glucuronidase, glyceraldehyde 3-phosphate dehydrogenase, beta-actin, and histone H2A during bovine oocyte maturation and early embryogenesis in vitro. 1626 7

The brain tissue obtained after death is subjected to several circumstances that can affect RNA integrity. The present study has been directed to reveal possible pitfalls and to control RNA normalization in post-mortem samples in order to recognize the limitations and minimize errors when using TaqMan PCR technology. This has been carried out in samples of the frontal cortex in a series of control and diseased cases covering Parkinson's disease, dementia with Lewy bodies pure form and common form, and Alzheimer's disease. Special attention has been paid to the value of the agonal state, post-mortem delay and pH of the nervous tissue as approximate predictors of the quality of RNA, as well as to the use of the Bioanalyzer to confirm RNA preservation. In addition, since possible disease-modified mRNAs have to be normalized with ideal unaltered RNAs, TaqMan human endogenous control plates have been used to determine the endogenous control most appropriate for the study. beta-glucuronidase (GUS) and beta-actin were good endogenous controls because their expression levels showed a small variation across a representative number of control and pathological cases. RNA stability was also analysed in a paradigm mimicking cumulative delay in tissue processing. GUS mRNA levels were not modified although beta-actin mRNA levels showed degradation at 22 h. Finally, the control of RNA degradation for the normalization of genes of interest was also tested. mRNA expression levels for superoxide dismutase 1 (SOD1) and metalloproteinase domain 22 (ADAM22) were examined at several artificial post-mortem times, and their expression levels compared with those for putative controls beta-actin and GUS. In our paradigm, the expressions of SOD1 and ADAM22 were apparently not modified when normalized with beta-actin. Yet their expression levels were reduced with post-mortem delay when values were normalized with GUS. Taken together, these observations point to practical consequences in TaqMan PCR studies. Short post-mortem delays and acceptable pH of the brain are not sufficient to rule out RNA degradation. The selection of adequate endogenous controls is pivotal in the study. beta-actin and GUS are found to be good endogenous controls in these pathologies, although GUS but not beta-actin expression levels are preserved in samples with long post-mortem delay.
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PMID:TaqMan PCR assay in the control of RNA normalization in human post-mortem brain tissue. 1652 42

Quantitative real-time RT-PCR was used to investigate the effects of prototypical drug-metabolizing enzyme inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) on mRNA expression levels of the housekeeping genes beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-glucuronidase (GUSB), hypoxanthine phosphoribosyltransferase 1 (HPRT1), peptidylprolylisomerase A (PPIA), TATA box binding protein (TBP), and transferrin receptor (TFRC) in primary cultures of cryopreserved human and rat hepatocytes. The mRNA levels of ACTB, GAPDH, GUSB, PPIA, TBP, and TFRC relative to HPRT1 in human hepatocytes were constant at all concentrations of inducers. However, the mRNA level of GAPDH relative to HPRT1 in rat hepatocytes was markedly increased by Rif. The mRNA levels of GAPDH, GUSB, PPIA, TBP, and TFRC relative to HPRT1 in rat hepatocytes were significantly increased by Dex. ACTB and HPRT1 are suitable internal controls for evaluating mRNA expression levels in primary cultures of human and rat hepatocytes after Rif, Dex, or Ome exposure.
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PMID:Effects of prototypical drug-metabolizing enzyme inducers on mRNA expression of housekeeping genes in primary cultures of human and rat hepatocytes. 1677 70

Selection of appropriate housekeeping genes (HKG) for normalization of quantitative PCR data for genes of interest is critical for interpretation of results. Ideally, copy number of the chosen HKG mRNA will not vary with experimental treatments or physiological state in the tissue studied, which improves accuracy in detecting changes in genes of interest. Because of the liver's dynamic role in metabolism, physiological state or dietary treatments could alter mRNA expression of commonly used HKG. Therefore, the objective of this study was to evaluate stability of mRNA expression for a number of candidate HKG in bovine liver across different physiological and dietary experimental conditions during the periparturient period. A publicly available program (geNorm) was used to evaluate expression stability of 8 HKG (beta-actin, glyceraldehyde 3-phosphate dehydrogenase, beta-glucuronidase, peptidylprolyl isomerase A, polyubiquitin, ribosomal protein S9, ribosomal protein L32, and 18S ribosomal RNA) in 91 liver RNA samples. Screened samples included liver from cows in 3 groups: 1) cows receiving a dietary supplement pre- and postpartum (n = 10); 2) cows with clinical or subclinical ketosis (n = 7); and 3) cows consuming different amounts of energy prepartum (n = 74). In group 3, samples from d -65, -30, -14, 1, 14, 28, and 49 relative to parturition were included to enable characterization of HKG mRNA expression across different physiological states. Initial analyses indicated that mRNA for ribosomal protein S9 (RPS9) was one of the most stably expressed across different experiment types. To determine the best gene, 200 bootstrap replications of the original data set were performed to determine if the ranking of RPS9 was superior to the other 7 genes evaluated. Average ranks and estimated standard errors for the top 3 genes were 1.64 +/- 0.06, 3.27 +/- 0.10, and 3.71 +/- 0.12 for RPS9, GAPDH, and beta-actin, respectively. Ribosomal protein S9 was ranked first 59% of the time and was never ranked lower than fifth. The lowest-ranked gene was polyubiquitin, ranked last 46.5% of the time (average rank = 6.85 +/- 0.10). In this study, physiological state, amount of intake, or dietary treatment influenced the mRNA expression of commonly used HKG in bovine liver. Ideally, expression stability should be tested before collection of data in all experiments; however, we have shown that RPS9 mRNA is stable across several physiological and diet-related experimental conditions for dairy cows, making it a good HKG in liver quantitative PCR experiments.
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PMID:Housekeeping gene expression in bovine liver is affected by physiological state, feed intake, and dietary treatment. 1743 Sep 24

Gene expression changes are used with increasing frequency to assess the effects of exposure to environmental agents. Housekeeping (Hk) genes are essential in these analyses as internal controls for normalizing expression levels evaluated with Real-Time PCR (RT-PCR). Ideal Hk genes are constitutively expressed, do not respond to external stimuli and exhibit little or no sample-to-sample or run-to-run variation. Previous studies indicate that some commonly used Hk genes including glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin have differential expression in various cell lines. Here we examine the expression of 11 Hk genes in four normal human lymphoblastoid cell lines and one T-cell leukemia (Jurkat) cell line following exposure to graded doses of ionizing radiation or to varying ratio concentrations of phytohemagglutinin (PHA) and phorbol myristate acetate (PMA). PHA and PMA are known to have synergistic effects on the expression of some genes and have very different effects from those of radiation. There has been no systematic study performed to ascertain the best control genes for radiation and/or PHA/PMA exposures in lymphoblastoid cells. Using a two-step reverse-transcriptase RT-PCR protocol we show that following radiation doses ranging from 0 to 400 cGy, 18S rRNA, acidic ribosomal protein, beta-actin, cyclophilin, GAPDH, phosphoglycerokinase, beta-2 microglobulin (B2M), beta-glucuronidase, hypoxanthine phosphoribosyltransferase and transferrin receptor showed no significant variation in expression in normal lymphoblastoid cells. In contrast, only 18S rRNA levels were unchanged in Jurkat cells. After PHA/PMA treatment of the same normal cell lines, B2M showed no significant variation and 18S rRNA, GAPDH and transcription binding protein (TBP) were minimally responsive, whereas in Jurkat cells all these genes were unresponsive. While our results suggest that the utility of a particular Hk gene should be determined for each experimental condition, 18S rRNA and B2M appear to be excellent candidates for use as internal controls in RT-PCR in human lymphoblastoid cells because they have the most constant levels of expression across cell lines following exposure to ionizing radiation as well as to PHA/PMA.
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PMID:Evaluation and validation of housekeeping genes in response to ionizing radiation and chemical exposure for normalizing RNA expression in real-time PCR. 1790 13


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