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)

We describe pOp/LhGR, a dexamethasone-inducible derivative of the pOp/LhG4 transcription activation system, and its use in tobacco to regulate expression of uidA (encoding beta-glucuronidase; GUS) and the cytokinin-biosnythetic gene ipt. The pOp/LhGR system exhibited stringent regulation and strong induced phenotypes in soil and tissue culture. In conjunction with an improved target promoter, pOp6, that carries six copies of an optimized lac operator sequence the pOp6/LhGR system directed induced GUS activities that exceeded those obtained with pOp/LhG4 or the CaMV 35S promoter but without increased uninduced activity. A single dose of dexamethasone was sufficient to direct cytotoxic levels of ipt expression in soil-grown plants although uninduced plants grew normally throughout a complete life cycle. In vitro, induced transcripts were detectable within an hour of dexamethasone application and 1 nM dexamethasone was sufficient for half maximal induction of GUS activity. Various methods of dexamethasone application were successfully applied under tissue culture and greenhouse conditions. We observed no inhibitory effects of dexamethasone or LhGR on plant development even with the highest concentrations of inducer, although tobacco seedlings were adversely affected by ethanol used as a solvent for dexamethasone stock solutions. The pOp/LhGR system provides a highly sensitive, efficient, and tightly regulated chemically inducible transgene expression system for tobacco plants.
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PMID:pOp6/LhGR: a stringently regulated and highly responsive dexamethasone-inducible gene expression system for tobacco. 1574 54

The efficacy of the ethanol-inducible alc transgene expression system, derived from the filamentous fungus Aspergillus nidulans, has been demonstrated in transgenic tomato. Two direct comparisons have been made. First, this study has utilized two transgenic lines carrying distinct reporter genes (chloramphenicol acetyltransferase and beta-glucuronidase) to distinguish aspects of induction determined by the nature of the gene/gene product rather than that of the plant. Second, comparisons have been made to data generated in other species in order to identify any species-specific effects. The induction profiles for different genes in different species have shown remarkable similarity indicating the broad applicability of this gene switch. While there are minor differences observed between species, these probably arise from diversity in their metabolism. A series of potential alternative inducers have also been tested, revealing that ethanol (through metabolism to acetaldehyde) is better than other alcohols and ketones included in this study. Expression driven by alc was demonstrated to vary spatially, the upper younger leaves having higher activity than the lower older leaves; this will be important for some applications, and for experimental design. The highest levels of activity from ethanol-inducible transgene expression were determined to be the equivalent of those from the constitutive Cauliflower Mosaic Virus 35S promoter. This suggests that the alc system could be an important tool for plant functional genomics.
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PMID:Characterization of the ethanol-inducible alc gene expression system in tomato. 1585 14

The ALCR/alcA (alc) two-component, ethanol-inducible gene expression system provides stringent control of transgene expression in genetically modified plants. ALCR is an ethanol-activated transcription factor that can drive expression from the ALCR-responsive promoter (alcA). However, the alc system has been shown to have constitutive expression when used in plant callus or cell suspension cultures, possibly resulting from endogenous inducer produced in response to lowered oxygen availability. To widen the use of the alc system in plant cell culture conditions, the receptor domain of the rat glucocorticoid receptor (GR) was translationally fused to the C terminus of ALCR to produce ALCR-GR, which forms the basis of a glucocorticoid-inducible system (alc-GR). The alc-GR switch system was tested in tobacco (Nicotiana tabacum) Bright Yellow-2 suspension cells using a constitutively expressed ALCR-GR with four alternative alcA promoter-driven reporter genes: beta-glucuronidase, endoplasmic reticulum-targeted green fluorescent protein, haemagglutinin, and green fluorescent protein-tagged Arabidopsis (Arabidopsis thaliana) Arath;CDKA;1 cyclin-dependent kinase. Gene expression was shown to be stringently dependent on the synthetic glucocorticoid dexamethasone and, in cell suspensions, no longer required ethanol for induction. Thus, the alc-GR system allows tight control of alcA-driven genes in cell culture and complements the conventional ethanol switch used in whole plants.
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PMID:The alc-GR system: a modified alc gene switch designed for use in plant tissue culture. 1601

5-[4-Acridin-9-ylamino]phenyl]-5-methyl-3-methylenedihydrofuran-2-one (CYL-26z) inhibited the formyl-Met-Leu-Phe (fMLP)-stimulated phospholipase D (PLD) activity, which was assessed by the production of phosphatidylethanol (PEt) in the presence of ethanol, in rat neutrophils (IC50 1.2+/-0.2 microM). CYL-26z caused a slight but significant attenuation of the global protein tyrosine phosphorylation stimulated by fMLP only at concentrations of CYL-26z up to 30 microM. CYL-26z blocked the membrane recruitment of protein kinase C-alpha (PKC-alpha) at concentrations of CYL-26z > or =3 microM, but failed to affect the membrane association of PKC-betaI and -betaII. The translocation of RhoA to the membrane was attenuated by CYL-26z (IC50 3.8+/-0.8 microM) in fMLP-stimulated neutrophils, whereas CYL-26z caused no significant inhibition of the membrane recruitment of ADP-ribosylation factor (Arf). CYL-26z inhibited the activation of RhoA and dissociation of the RhoA-Rho guanine nucleotide dissociation inhibitor (GDI) complex in fMLP-stimulated neutrophils (IC50 1.8+/-1.0 microM and 1.8+/-0.9 microM, respectively). In a cell-free system, CYL-26z effectively attenuated the membrane association of RhoA in response to GTPgammaS (IC50 1.3+/-0.5 microM). In contrast, the GTPgammaS-stimulated translocation of Arf to membrane was suppressed only at concentrations of CYL-26z up to 30 microM. CYL-26z inhibited the fMLP-stimulated membrane expression of CD11b, CD45 and CD63, and the release of lysozyme and beta-glucuronidase. These results indicate that CYL-26z inhibited the fMLP-stimulated PLD activity, mainly through the blockade of RhoA activation, and degranulation in rat neutrophils.
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PMID:Inhibition of phospholipase D activation by CYL-26z in formyl peptide-stimulated neutrophils involves the blockade of RhoA activation. 1602 1

We tested the efficiency and optimized the conditions for controlled alcohol-inducible transgene expression in Populus using gus as a reporter gene. Specificity of induction, efficiency in different organs, effect of three chemical inducers, and induction methods were tested using up to 10 independent transgenic events generated in two different Populus genotypes. The optimal inducer concentration and the duration of induction period were determined in dose-response and in time-course experiments. Under in vitro conditions, beta-glucuronidase (GUS) induction was efficient both in the aerial parts and in the roots of regenerated plantlets. Among the chemical inducers tested, ethanol was the most effective activator with no apparent phytotoxicity when concentrations were at or below 2%. After 5 days of treatment, fluorometrically-determined the GUS activity could be detected when inducing with ethanol at concentrations as low as 0.5%. Prolonged induction by ethanol vapors significantly increased the GUS activity in leaves from both the tissue culture plants and greenhouse-grown plants.
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PMID:Alcohol-inducible gene expression in transgenic Populus. 1649 53

Ethyl glucuronide (ethyl-beta-D-6-glucosiduronic acid, EtG), a unique metabolite of ethanol, has received much recent attention as a sensitive and specific biological marker of ethanol consumption. Formed in the liver via conjugation of ethanol with activated glucuronate, EtG remains detectable in serum, plasma, and hair for days after ethanol abuse. Thus far, gas chromatography-mass spectrometry and enzyme-linked immunosorbent assays have been developed to detect trace quantities of EtG for forensic purposes, but reports of the nuclear magnetic resonance (NMR) properties of EtG have been scarce. Herein we present the first report of EtG determination using proton NMR spectroscopy. We collected 700-MHz proton spectra of liver extracts from rats treated with a 4-day binge ethanol protocol (average ethanol dose: 8.6g/kg/day). An unexpected signal (triplet, 1.24 ppm) appeared in ethanol-treated liver extracts but not in control samples; based on chemical shift and multiplicity, we suspected EtG. We observed quantitative hydrolysis of the unknown species to ethanol while incubating our samples with beta-glucuronidase, confirming that the methyl protons of EtG were responsible for the triplet at 1.24 ppm. This study demonstrates that proton NMR spectroscopy is capable of detecting EtG and that future NMR-based metabolomic studies may encounter this metabolite of ethanol.
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PMID:Proton nuclear magnetic resonance spectroscopic determination of ethanol-induced formation of ethyl glucuronide in liver. 1702 4

Kava, like a number of herbals, has been associated with causing liver damage based on limited evidence. In contrast, the present study found that in rats, 3 mo feedings of two types of kava extracts (an acetone extract and an ethanol extract of the Samoan kava cultivar Ava Laau) at three different doses (31.25, 62.5 and 133 mg/kg diet) produced no liver injury based on serum markers of liver damage (sorbitol dehydrogenase activities, bile acid concentrations, and beta-glucuronidase activities) and serum lipid peroxide readings. In fact, for some measurements and some kava doses, the injury marker readings were below control values. Moreover, for these same parameters, kava feeding did not enhance the effects of the hepatotoxin galacatosamine (500 mg/kg ip); some kava doses even showed modest protection against liver injury. Liver histology analysis showed no signs of kava causing or enhancing liver injury. Thus, this study does not support the concept that kava produces or aggravates liver injury.
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PMID:Kava feeding in rats does not cause liver injury nor enhance galactosamine-induced hepatitis. 1735 74

The experimental control of gene expression in specific tissues or cells at defined time points is a useful tool for the analysis of gene function. GAL4/VP16-UAS enhancer trap lines can be used to selectively express genes in specific tissues or cells, and an ethanol-inducible system can help to control the time of expression. In this study, the combination of the two methods allowed the successful regulation of gene expression in both time and space. For this purpose, a binary vector, 962-UAS::GUS, was constructed in which the ALCR activator and beta-glucuronidase (GUS) reporter gene were placed under the control of upstream activator sequence (UAS) elements and the alcA response element, respectively. Three different GAL4/VP16-UAS enhancer trap lines of Arabidopsis were transformed, resulting in transgenic plants in which GUS activity was detected only on ethanol induction and exclusively in the predicted tissues of the enhancer trap lines. As a library of different enhancer trap lines with distinct green fluorescent protein (GFP) patterns exist, transformation with a similar vector, in which GUS is replaced by another gene, would enable the control of the time and place of transgene expression. We have constructed two vectors for easy cloning of the gene of interest, one with a polylinker site and one that is compatible with the GATEWAY vector conversion system. The method can be extended to other species when enhancer trap lines become available.
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PMID:Combination of the ALCR/alcA ethanol switch and GAL4/VP16-UAS enhancer trap system enables spatial and temporal control of transgene expression in Arabidopsis. 1744 66

The purpose of this study was to perform a comprehensive analysis of hepatic gene expression in a standard model of an alcohol-induced fatty liver using the cDNA microarray analysis. Male Sprague-Dawley rats were randomly divided into two groups and were given either an ethanol diet (ED), or a control diet (CD) for eight weeks. The ED rats showed significantly elevated levels of plasma total and HDL cholesterol as well as hepatic cholesterol and triglyceride compared to the pair-fed control rats. Among the 5185 genes on the rat cDNA microarray used in the current study, 74 genes were up-regulated and 108 genes were down-regulated greater than 2.0-fold in the liver of ED rats compared with those in the CD rats. The microarray results were verified by conducting real-time RT-PCR on the fourteen selected genes with varied expression ratios. After clustering the regulated genes based on their biological function, it was found that chronic ethanol consumption regulated mainly the genes implicated in the processes of signal transduction, transcription, immune response, and protein/amino acid metabolism. The microarray results obtained in this study revealed, for the first time, that several genes, including beta-glucuronidase, UDP-glycosyltransferase 1, UDP-glucose dehydrogenase, apoC-III, and gonadotropin-releasing hormone receptor, were regulated by chronic ethanol exposure in the rat liver.
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PMID:Changes in the hepatic gene expression profile in a rat model of chronic ethanol treatment. 1792 Jul 46

The uptake of five fluorescein labeled cell-penetrating peptides (Tat, Tat(2), mutated-Tat, peptide vascular endothelial-cadherin and transportan) was studied in wheat immature embryos. Interestingly, permeabilization treatment of the embryos with toluene/ethanol (1 : 20, v/v with permeabilization buffer) resulted in a remarkably higher uptake of cell-penetrating peptides, whereas nonpermeabilized embryos failed to show significant cell-penetrating peptide uptake, as observed under fluorescence microscope and by fluorimetric analysis. Among the cell-penetrating peptides investigated, Tat monomer (Tat) showed highest fluorescence uptake (4.2-fold greater) in permeabilized embryos than the nonpermeabilized embryos. On the other hand, mutated-Tat serving as negative control did not show comparable fluorescence levels even in permeabilized embryos. A glucuronidase histochemical assay revealed that Tat peptides can efficiently deliver functionally active beta-glucuronidase (GUS) enzyme in permeabilized immature embryos. Tat(2)-mediated GUS enzyme delivery showed the highest number of embryos with GUS uptake (92.2%) upon permeabilization treatment with toluene/ethanol (1 : 40, v/v with permeabilization buffer) whereas only 51.8% of nonpermeabilized embryos showed Tat(2)-mediated GUS uptake. Low temperature, endocytosis and macropinocytosis inhibitors reduced delivery of the Tat(2)-GUS enzyme cargo complex. The results suggest that more than one mechanism of cell entry is involved simultaneously in cell-penetrating peptide-cargo uptake in wheat immature embryos. We also studied Tat(2)-plasmid DNA (carrying Act-1GUS) complex formation by gel retardation assay, DNaseI protection assay and confocal laser microscopy. Permeabilized embryos transfected with Tat(2)-plasmid DNA complex showed 3.3-fold higher transient GUS gene expression than the nonpermeabilized embryos. Furthermore, addition of cationic transfecting agent Lipofectamine 2000 to the Tat(2)-plasmid DNA complex resulted in 1.5-fold higher transient GUS gene expression in the embryos. This is the first report demonstrating translocation of various cell-penetrating peptides and their potential to deliver macromolecules in wheat immature embryos in the presence of a cell membrane permeabilizing agent.
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PMID:Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos. 1839 18


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