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)

Biochemical mechanisms underlying acrylamide induced neurotoxicity were examined using an in vitro model consisting of sagittal slices of rat brain. Incubation of brain slices under oxygen in artificial cerebrospinal fluid containing acrylamide produced a dose and time dependent inhibition of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Lysosomal enzymes, acid phosphatase, N-acetyl glucosaminidase and beta-glucuronidase decreased in a similar manner, while no changes were observed in the activity of Na+K+ATPase, cytochrome c oxidase and lactate dehydrogenase. Incubation of slices with two structurally related compounds, acetamide (a non-neurotoxic amide) and methylene bis-acrylamide (a weak neurotoxin), indicated that acrylamide selectively inhibited GAPDH, enolase and N-acetyl glucosaminidase at low concentration; similar doses of acetamide and methylene bis-acrylamide did not have the same effect on brain slices. Incubation with acrylamide depleted glutathione levels in slices, and the addition of glutathione to the incubation medium prevented acrylamide induced inhibition of GAPDH and lysosomal enzymes. Time dependent inhibition of lysosomal enzymes was also observed in vivo, in the brain and sciatic nerve of rats following a single dose of acrylamide. These results demonstrate that both in vitro and in vivo, lysosomal enzymes are also inhibited following acrylamide exposure. The rat brain slice model exhibits both selectivity and sensitivity towards neurotoxicants and hence, may prove to be an useful in vitro model for the mechanistic evaluation of neurotoxicity.
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PMID:The use of rat brain slices as an in vitro model for mechanistic evaluation of neurotoxicity-studies with acrylamide. 195 83

A chimaeric beta-glucuronidase (GUS) gene has been created by ligating the Aspergillus nidulans glyceraldehyde 3-phosphate dehydrogenase promoter to the coding sequence of the E. coli uidA gene. Co-transformation of this vector into A. nidulans, A. niger and the tomato pathogen Fulvia fulva (syn. Cladosporium fulvum (Cooke] resulted in the expression of beta-glucuronidase. GUS activity was detected by growth on agar media containing X-gluc and by enzyme assays of mycelial extracts. Expression of the gene in F. fulva transformants was also easily detectable during growth in plants and did not affect pathogenicity. These results form the basis for a versatile and sensitive reporter gene system for industrial and phytopathogenic filamentous fungi.
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PMID:Expression of the Escherichia coli beta-glucuronidase gene in industrial and phytopathogenic filamentous fungi. 250 1

We report here the identification of a cis-acting region involved in light regulation of the nuclear gene (GapB) encoding the B subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase from Arabidopsis thaliana. Our results show that a 664-bp GapB promoter fragment is sufficient to confer light induction and organ-specific expression of the Escherichia coli beta-glucuronidase reporter gene (Gus) in transgenic tobacco (Nicotiana tabacum) plants. Deletion analysis indicates that the -261 to -173 upstream region of the GapB gene is essential for light induction. This region contains four direct repeats with the consensus sequence 5'-ATGAA(A/G)A-3' (Gap boxes). Deletion of all four repeats abolishes light induction completely. In addition, we have linked a 109-bp (-263 to -152) GapB upstream fragment containing the four direct repeats in two orientations to the -92 to +6 upstream sequence of the cauliflower mosaic virus 35S basal promoter. The resulting chimeric promoters are able to confer light induction and to enhance leaf-specific expression of the Gus reporter gene in transgenic tobacco plants. Based on these results we conclude that Gap boxes are essential for light regulation and organ-specific expression of the GapB gene in A. thaliana. Using gel mobility shift assays we have also identified a nuclear factor from tobacco that interacts with GapA and GapB DNA fragments containing these Gap boxes. Competition assays indicate that Gap boxes are the binding sites for this factor. Although this binding activity is present in nuclear extracts from leaves and roots of light-grown or dark-treated tobacco plants, the activity is less abundant in nuclear extracts prepared from leaves of dark-treated plants or from roots of greenhouse-grown plants. In addition, our data show that this binding factor is distinct from the GT-1 factor, which binds to Box II and Box III within the light-responsive element of the RbcS-3A gene of pea.
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PMID:Identification of a light-responsive region of the nuclear gene encoding the B subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase from Arabidopsis thaliana. 802 58

We report the characterization of cis-acting elements involved in light regulation of the nuclear gene (GapA) that encodes the A subunit of glyceraldehyde 3-phosphate dehydrogenase in Arabidopsis thaliana. Our previous deletion analyses indicate that the -277 to -195 upstream region of GapA is essential for light induction of the beta-glucuronidase reporter gene in transgenic tobacco (Nicotiana tabacum) plants. This region contains three direct repeats with the consensus sequence 5'-CAAATGAA(A/G)A-3' (Gap boxes). Our results show that 2-bp substitutions of the last four nucleotides (AA or GA) of the Gap boxes by CC abolish light induction of the beta-glucuronidase reporter gene in vivo and affect binding of the Gap box binding factor in vitro. We have also identified an additional cis-acting element, AE (Activation Element) box, that is involved in regulation of GapA. A combination of a Gap box trimer and an AE box dimer can confer light responsiveness of the cauliflower mosaic virus 35S promoter containing the -92 to +6 upstream sequence, whereas oligomers of Gap boxes or AE boxes alone cannot confer light responsiveness on the same promoter. These results suggest that Gap boxes and AE boxes function together as the light-responsive element of GapA.
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PMID:Cis-acting elements essential for light regulation of the nuclear gene encoding the A subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase in Arabidopsis thaliana. 897

Human adenocarcinoma cells of the line WiDr have been treated with 2 mM 5-aminolaevulinic acid (5-ALA) in the presence of 10% foetal calf serum. The treatment induces a linear accumulation of protoporphyrin IX (PpIX) for at least 7.5 h. After 7.5 h of incubation about 45% of the PpIX accumulated is cell-bound, while the rest is found in the medium (25%) or lost from the cells during washing with phosphate-buffered saline (30%). Exposure to white light at an intensity of 30 W/m2 for 18 min results in 95% reduction of clonogenicity in cells treated with 2 mM 5-ALA for 3.5 h. The enzymatic activities of enzymes located in cytosol (glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase) and lysosomes (acid phosphatase and beta-glucuronidase) are not influenced by a 5-ALA and light treatment inactivating about 35% of the cells. The MTT assay, which reflects mitochondrial dehydrogenase activity, but not succinate dehydrogenase, is partly inhibited by the same treatment. Treatment with 5-ALA in the absence of light increases O2 consumption by a factor of two, while the O2 consumption is inhibited when 5-ALA treatment is combined with exposure to light. In addition, 5-ALA and light exposure enhance accumulation of rhodamine 123 by 40% and reduce the intracellular ATP level by 25%. Confocal laser scanning microscopical analysis indicates granular perinuclear localization of the PpIX formed by 5-ALA treatment. In conclusion, photodynamic treatment using 5-ALA as a prodrug induces damage to mitochondrial function without inhibiting lysosomal and cytosolic marker enzymes.
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PMID:Photodynamically induced effects in colon carcinoma cells (WiDr) by endogenous photosensitizers generated by incubation with 5-aminolaevulinic acid. 1039 65

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

Obtaining reliable gene expression data using real-time quantitative polymerase chain reaction (qPCR) is highly dependent on the choice of normalization method. We tested the expression stability of multiple candidate genes in the salivary glands (SG) and synganglia (SYN) of female Ixodes scapularis (Say) ticks in multiple blood-feeding phases. We found that the amount of total RNA in both the SG and SYN increases dramatically during tick feeding, with 34x and 5.8x increases from 62 and 7.1 ng of unfed tick, respectively. We tested candidate genes that were predicted from I. scapularis genome data to encode glyceraldehyde 3-phosphate dehydrogenase (gapdh), ribosomal protein L13A (l13a), TATA box-binding protein (tbp), ribosomal protein S4 (rps4), glucose 6-phosphate dehydrogenase (gpdh), and beta-glucuronidase (gusb). The geNorm and NormFinder algorithms were used to analyze data from different feeding phases (i.e., daily samples from unfed to fully engorged females over a 7-d period in three replicate experiments). We found that the rps4 and l13a genes showed highly stable expression patterns over the feeding duration in both the SG and SYN. Furthermore, the highly expressed rps4 gene makes it useful as a normalization factor when we perform studies using minute amounts of dissected tissue for qPCR. We conclude that rps4 and l13a, whether individually or as a pair, serve as suitable internal reference genes for qRT-PCR studies in the SG and SYN of I. scapularis.
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PMID:Validation of internal reference genes for real-time quantitative polymerase chain reaction studies in the tick, Ixodes scapularis (Acari: Ixodidae). 2342 55