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Query: UNIPROT:P06889 (Mol)
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CAR and C1, two carrot (Daucus carota L.) suspension cultures of different genotypes, were subjected to stepwise selection for tolerance to the herbicide glyphosate [(N-phosphonomethyl)glycine]. The specific activity of the target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), as well as the mRNA level and copy number of the structural gene increased with each glyphosate selection step. Therefore, the tolerance to glyphosate is due to stepwise amplification of the EPSPS genes. During the amplification process, DNA rearrangement did not occur within the EPSPS gene of the CAR cell line but did occur during the selection step from 28 to 35 mM glyphosate for the C1 cell line, as determined by Southern hybridization of selected cell DNA following EcoRI restriction endonuclease digestion. Two cell lines derived from a previously selected glyphosate-tolerant cell line (PR), which also had undergone EPSPS gene amplification but have been maintained in glyphosate-free medium for 2 and 5 years, have lost 36 and 100% of the increased EPSPS activity, respectively. Southern blot analysis of these lines confirms that the amplified DNA is relatively stable in the absence of selection. These studies demonstrate that stepwise selection for glyphosate resistance reproducibly produces stepwise amplification of the EPSPS genes. The relative stability of this amplification indicates that the amplified genes are not extrachromosomal.
Mol Gen Genet 1992 Apr
PMID:Glyphosate selected amplification of the 5-enolpyruvylshikimate-3-phosphate synthase gene in cultured carrot cells. 137 13

Expression of the RNA replicase domain of tobacco mosaic virus (TMV) and certain protein-coding regions in other plant viruses, is mediated by translational readthrough of a leaky UAG stop codon. It has been proposed that normal tobacco tyrosine tRNAs are able to read the UAG codon of TMV by non-conventional base-pairing but recent findings that stop codons can also be bypassed as a result of extended translocational shifts (tRNA hopping) have encouraged a re-examination. In light of the alternatives, we investigated the sequences flanking the leaky UAG codon using an in vivo assay in which bypass of the stop codon is coupled to the transient expression of beta-glucuronidase (GUS) reporter genes in tobacco protoplasts. Analysis of GUS constructions in which codons flanking the stop were altered allowed definition of the minimal sequence required for read through as UAG-CAA-UUA. The effects of all possible single-base mutations in the codons flanking the stop indicated that 3' contexts of the form CAR-YYA confer leakiness and that the 3' context permits read through of UAA and UGA stop codons as well as UAG. Our studies demonstrate a major role for the 3' context in the read through process and do not support a model in which teh UAG is bypassed exclusively as a result of anticodon-codon interactions. No evidence for tRNA hopping was obtained. The 3' context apparently represents a unique sequence element that affects translation termination.
J Mol Biol 1991 Mar 20
PMID:The signal for a leaky UAG stop codon in several plant viruses includes the two downstream codons. 201 Sep 14

Human and animal cell cultures were evaluated for their susceptibility to two environmental toxins found as contaminants in human food supplies: aflatoxin B1, a hepatotoxin produced by the mold Aspergillus flavus, and saxitoxin, a paralytic neurotoxin produced by the marine dinoflagellate Gonyaulax catenella. Both toxins cause food poisoning in humans and other animals. The acute cytotoxicity of both toxins was measured and compared by inhibition of cell growth and by progressive cytopathogenicity resulting in cell destruction. Aflatoxin B1 was cytotoxic to all of the 11 primary kidney cultures derived from susceptible animals. The cell growth inhibition 10% values (TD10) ranged from 0.02 to 6.0 micrograms/ml: mouse (TD10 = 0.02 micrograms), guinea pig (0.03 micrograms), rat (0.07 micrograms), hamster (0.16 micrograms), monkey (0.1 microgram), human (0.7-1.5 micrograms), chick (0.05 micrograms), and duck (6.0 micrograms). The corresponding TD50 levels were about 10 times higher concentrations and caused cell destruction within 2 d. Saxitoxin did not induce cytotoxicity manifestations in cultures derived from susceptible species--mouse kidney, human carcinoma HeLa line, chick embryo, and goldfish fin (CAR) cell line--at high concentration levels up to 5 micrograms/ml. When the same toxin preparation at only 1 microgram was injected into mice, the animals died immediately. The results indicate that animal cell cultures are useful for studies of general cytotoxins that affect common essential metabolism but cannot be used to detect environmental toxins that cause toxic manifestations by an interference with specific physiological functions of organ systems.
Mol Toxicol
PMID:Comparative cytotoxicity of aflatoxin B1 and saxitoxin in cell cultures. 313 May 68

PBREM, the phenobarbital-responsive enhancer module of the cytochrome P-450 Cyp2b10 gene, contains two potential nuclear receptor binding sites, NR1 and NR2. Consistent with the finding that anti-retinoid X receptor (RXR) could supershift the NR1-nuclear protein complex, DNA affinity chromatography with NR1 oligonucleotides enriched the nuclear orphan receptor RXR from the hepatic nuclear extracts of phenobarbital-treated mice. In addition to RXR, the nuclear orphan receptor CAR was present in the same enriched fraction. In the phenobarbital-treated mice, the binding of both CAR and RXR was rapidly increased before the induction of CYP2B10 mRNA. In vitro-translated CAR bound to NR1, but only in the presence of similarly prepared RXR. PBREM was synergistically activated by transfection of CAR and RXR in HepG2 and HEK293 cells when the NR1 site was functional. A CAR-RXR heterodimer has thus been characterized as a trans-acting factor for the phenobarbital-inducible Cyp2b10 gene.
Mol Cell Biol 1998 Oct
PMID:The nuclear orphan receptor CAR-retinoid X receptor heterodimer activates the phenobarbital-responsive enhancer module of the CYP2B gene. 974 82

Adenoviruses (Ad) are a significant cause of acute infections in humans; however, replication-defective forms of this virus are currently under investigation for human gene therapy. Approximately 20 to 25% of all the gene therapy trials (phases I to III) conducted over the past 10 years involve the use of Ad gene delivery for treatment inherited or acquired diseases. At present, the most promising applications involve the use of Ad vectors to irradicate certain nonmetastatic tumors and to promote angiogenesis in order to alleviate cardiovascular disease. While specific problems of using Ad vectors remain to be overcome (as is true for almost all viral and nonviral delivery methods), a distinct advantage of Ad is the extensive knowledge of its macromolecular structure, genome organization, sequence, and mode of replication. Moreover, significant information has also been acquired on the interaction of Ad particles with distinct host cell receptors, events which strongly affect virus tropism. This review provides an overview of the structure and function of Ad attachment (coxsackievirus and Ad receptor [CAR]) and internalization (alpha(v) integrins) receptors and discusses their precise role in virus infection and gene delivery. Recent structure studies of integrin-Ad complexes by cryoelectron microscopy are also highlighted. Finally, unanswered questions arising from the current state of knowledge of Ad-receptor interactions are presented in the context of improving Ad vectors for future human gene therapy applications.
Microbiol Mol Biol Rev 1999 Sep
PMID:Role of alpha(v) integrins in adenovirus cell entry and gene delivery. 1047 14

We tested the hypothesis that the folding, assembly and insertion of neuronal nicotinic receptors are critically dependent on the host cell line. We used recombinant adenoviruses encoding either the rat alpha7, alpha4 or beta2 subunits in which expression of the subunit is controlled by a tetracycline-dependent promoter to screen five cell lines (GH4C1, SH-EP1, CV1, SN-56, and CHO-CAR). All five lines do not express detectable nicotinic receptor but do express receptor for human adenovirus, and all expressed mRNA for alpha7, alpha4 and beta2 subunits when infected with viruses. Each cell line expressed varying levels of alpha4beta2 receptors that bound [3H]cytisine, but only the GH4C1 and SH-EP1 cell lines expressed either surface or internal alpha7 receptors that bound [125I]alpha-bungarotoxin ([125I]alpha-BGT). All five cell lines expressed a 60 kDa protein immunoblotted by anti-alpha7 antibodies when infected with the alpha7 virus, presumably representing unassembled alpha7 subunits. In addition, GH4C1 cells expressed over 10-fold more surface alpha7 receptor than SH-EP1 cells, even though the total alpha7 receptor in the two cell lines was similar. Sedimentation experiments indicate that SH-EP1 cells only partially assemble alpha7 receptors compared with GH4C1 cells and control alpha7 from rat brain. These data suggest that not only is surface alpha7 receptor expression a multistep process, but that each step may involve cell-specific assembly factors.
Brain Res Mol Brain Res 2000 Feb 22
PMID:Multistep expression and assembly of neuronal nicotinic receptors is both host-cell- and receptor-subtype-dependent. 1068 51

A wide range of xenobiotic compounds are metabolized by cytochrome P450 (CYP) enzymes, and the genes that encode these enzymes are often induced in the presence of such compounds. Here, we show that the nuclear receptor CAR can recognize response elements present in the promoters of xenobiotic-responsive CYP genes, as well as other novel sites. CAR has previously been shown to be an apparently constitutive transactivator, and this constitutive activity is inhibited by androstanes acting as inverse agonists. As expected, the ability of CAR to transactivate the CYP promoter elements is blocked by the inhibitory inverse agonists. However, CAR transactivation is increased in the presence of 1,4-bis[2-(3, 5-dichloropyridyloxy)]benzene (TCPOBOP), the most potent known member of the phenobarbital-like class of CYP-inducing agents. Three independent lines of evidence demonstrate that TCPOBOP is an agonist ligand for CAR. The first is that TCPOBOP acts in a dose-dependent manner as a direct agonist to compete with the inhibitory effect of the inverse agonists. The second is that TCPOBOP acts directly to stimulate coactivator interaction with the CAR ligand binding domain, both in vitro and in vivo. The third is that mutations designed to block ligand binding block not only the inhibitory effect of the androstanes but also the stimulatory effect of TCPOBOP. Importantly, these mutations do not block the apparently constitutive transactivation by CAR, suggesting that this activity is truly ligand independent. Both its ability to target CYP genes and its activation by TCPOBOP demonstrate that CAR is a novel xenobiotic receptor that may contribute to the metabolic response to such compounds.
Mol Cell Biol 2000 May
PMID:The xenobiotic compound 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene is an agonist ligand for the nuclear receptor CAR. 1075 80

Efficient adenovirus vector-mediated gene transfer depends on the presence of sufficient amounts of the high-affinity coxsackie-adenovirus (Ad) receptor (CAR) on the surface of the target cell leading to receptor-mediated endocytosis of the vector. The present study evaluates the effect of free cholesterol, a lipid component of endocytic vesicles, on Ad uptake into CAR-deficient cells. Infection in the presence of free cholesterol at its maximum solubility in water led to increased binding, uptake, and expression of Ad in human skin fibroblasts and alveolar macrophages, two primary human cells known to be deficient in CAR. The effect of free cholesterol was maximal at its solubility maximum in aqueous solution. Increase of Ad vector-mediated gene transfer with cholesterol was dependent on the lack of CAR receptor expression on the surface and was diminished by overexpression of CAR in CAR-deficient cells. Cholesterol-mediated increase of Ad-mediated gene expression was dependent on coincubation of both cholesterol and Ad and was not dependent on the cholesterol content of the cell. Increased Ad vector-mediated gene expression in the presence of free cholesterol was also observed in murine skin in vivo. Structural analysis of the Ad-cholesterol mixture showed complexation between Ad particles leading to formation of multivirus aggregates due to hydrophobic interaction. The addition of free cholesterol with Ad vectors may be a simple way to increase Ad-mediated gene transfer to cells that are poor targets due to their lack of a sufficient number of Ad receptors.
Mol Ther 2000 Jan
PMID:Free cholesterol enhances adenoviral vector gene transfer and expression in CAR-deficient cells. 1093 10

The nuclear orphan receptor CAR (constitutively active receptor or constitutive androstane receptor) can be activated in response to xenochemical exposure, such as activation by phenobarbital of a response element called NR1 found in the CYP2B gene. Here various steroids were screened for potential endogenous chemicals that may activate CAR, using the NR1 enhancer and Cyp2b10 induction in transfected HepG2 cell and/or in mouse primary hepatocytes as the experimental criteria. 17beta-Estradiol and estrone activated NR1, whereas estriol, estetrol, estradiol sulfate, and the synthetic estrogen diethylstilbestrol did not. On the other hand, progesterone and androgens repressed NR1 activity in HepG2 cells, and the repressed NR1 activity was fully restored by estradiol. Moreover, estrogen treatment elicited nuclear accumulation of CAR in the mouse livers, as well as primary hepatocytes, and induced the endogenous Cyp2b10 gene. Ovariectomy did not affect either the basal or induced level of CAR in the nucleus of the female livers, while castration slightly increased the basal and greatly increased the induced levels in the liver nucleus of male mice. Thus, endogenous estrogen appears not to regulate CAR in female mice, whereas endogenous androgen may be the repressive factor in male mice. Estrogen at pharmacological levels is an effective activator of CAR in both female and male mice, suggesting a biological and/or toxicological role of this receptor in estrogen metabolism. In addition to mouse CAR, estrogens activated rat CAR, whereas human CAR did not respond well to the estrogens under the experimental conditions.
Mol Endocrinol 2000 Nov
PMID:Estrogen activation of the nuclear orphan receptor CAR (constitutive active receptor) in induction of the mouse Cyp2b10 gene. 1107 20

The barbiturate phenobarbital induces the transcription of cytochromes P450 (CYPs) 2B through the constitutive androstane receptor (CAR; NR1I3). CAR is a member of the nuclear receptor family (NR1) mostly expressed in the liver, which heterodimerizes with retinoid X receptor (RXR) and was shown to transactivate both the phenobarbital responsive element module of the human CYP2B6 gene and the CYP3A4 xenobiotic response element. Because previous studies in rodent hepatocyte cultures have shown that the phenobarbital-mediated induction of CYP2B genes is potentiated by glucocorticoids, we examined the role of activated glucocorticoid receptor in this process. We show that submicromolar concentrations of dexamethasone enhance phenobarbital-mediated induction of CYP3A4, CYP2B6, and CYP2C8 mRNA in cultured human hepatocytes. In parallel, we observed that glucocorticoid agonists, such as dexamethasone, prednisolone, or hydrocortisone, specifically increase human car (hCAR) mRNA expression. Accumulation of hCAR mRNA parallels that of tyrosine aminotransferase: both mRNAs reach a maximum at a concentration of 100 nM dexamethasone and are down-regulated by concomitant treatment with the glucocorticoid antagonist RU486. Moreover, the effect of dexamethasone on hCAR mRNA accumulation appears to be of transcriptional origin because the addition of protein synthesis inhibitor cycloheximide has no effect, and dexamethasone does not affect the degradation of hCAR mRNA. Furthermore, dexamethasone increases both basal and phenobarbital-mediated nuclear translocation of CAR immunoreactive protein in human hepatocytes. The up-regulation of CAR mRNA and protein in response to dexamethasone explains the synergistic effect of this glucocorticoid on phenobarbital-mediated induction of CYP2B genes and the controversial role of the glucocorticoid receptor on phenobarbital-mediated CYP gene inductions.
Mol Pharmacol 2000 Dec
PMID:Dexamethasone enhances constitutive androstane receptor expression in human hepatocytes: consequences on cytochrome P450 gene regulation. 1109 84


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