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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Estrogen-related receptor (ERR) alpha-1 shares a high amino acid sequence homology with estrogen receptor alpha. Although estrogens are not ligands of ERR alpha-1, our recent results suggest that toxaphene and chlordane, two organochlorine pesticides with estrogen-like activity, behave as antagonists for this orphan nuclear receptor. The two compounds increased ERR alpha-1-mediated expression of the reporter enzyme beta-galactosidase in a yeast-based assay. The screen was developed by expressing the hERR alpha-1-yeast Gal 4 activation domain fusion protein in yeast cells carrying the beta-galactosidase reporter plasmid, which contains an ERR alpha-1-binding element. In transfection experiments using mammalian cell lines, such as the SK-BR-3 breast cancer cell line, the compounds were found to have an antagonist activity against ERR alpha-1-mediated expression of the reporter chloramphenicol acetyltransferase. In contrast to the findings with ERR alpha-1, the two compounds were found to slightly induce the estrogen receptor a-mediated expression of chloramphenicol acetyltransferase in SK-BR-3 cells. In a ligand-independent manner, the ERR alpha-1 activity in SK-BR-3 cells was induced 3-fold by cotransfection with the GRIP1 coactivator expression plasmid. Toxaphene was found to be capable of suppressing the GRIP1 coactivator-induced ERR alpha-1 activity in SK-BR-3 cells. In addition, a stable ERR alpha-1 expressing HepG2 hepatoma cell line was generated, and the aromatase activity in the transfected cell line was found to be twice that in the untransfected cell line. The enzyme aromatase converts androgens to estrogens, and aromatase expression in HepG2 cells is regulated in part by an ERR alpha-1-modulating promoter. A 24-h incubation of an ERR alpha-1-transfected HepG2 cell line with 10 microM toxaphene reduced its aromatase activity to the level in the untransfected cell line. Because toxaphene is not an inhibitor of aromatase, it is thought that the decrease of the aromatase activity in ERR alpha-1 transfected HepG2 cells following toxaphene treatment resulted from a suppression of the aromatase expression by toxaphene acting as the antagonist of ERR alpha-1. Toxaphene and chlordane are among the 12 persistent organic pollutants identified by the United Nations Environment Programme as requiring urgent attention. Their antagonistic effects on ERR alpha-1 should not be overlooked.
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PMID:Two organochlorine pesticides, toxaphene and chlordane, are antagonists for estrogen-related receptor alpha-1 orphan receptor. 1049 99

Intracistronic complementation of N-terminally truncated beta-galactosidase mutants such as M15 by coexpressed alpha-peptide was originally discovered in Escherichia coli and exploited for plasmid cloning as the well-known blue-white screening method. We show here that alpha-complementation also works in the budding yeast Saccharomyces cerevisiae, and that it can be used as a simple nonselective enzymatic marker for a variety of in vivo studies, for example, on the role of molecular chaperones in protein folding and assembly. To be able to induce alpha-complementation post-translationally, we have constructed a hormone-inducible alpha-peptide by fusion of the DNA encoding the alpha-peptide to that of to the hormone binding domain of the estrogen receptor. The accumulation of both subunits, the alpha-peptide and M15, is severely compromised when they are expressed separately, presumably because their hydrophobic surfaces remain exposed. Moreover, alpha-complementation is defective in a strain of S. cerevisiae carrying a point mutant of the molecular chaperone heat-shock protein 90. Heat-shock protein 90, which coprecipitates with M15, might be required in vivo to prevent the degradation of unassembled M15 and to hold it in an interaction-competent conformation.
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PMID:Alpha-complemented beta-galactosidase. An in vivo model substrate for the molecular chaperone heat-shock protein 90 in yeast. 1056 93

Although melatonin is believed to mediate many seasonal and circadian effects of photoperiod on reproduction in salmonids, the precise mechanisms underlying such effects are still largely unknown. Recent data of the literature indicate a relationship between melatonin and expression of estrogen receptors (ER) in various tissues. In this study, the effects of melatonin on estrogen receptor and/or vitellogenin expression were studied by a combination of in vivo and in vitro experiments. In yeast stably expressing ER and transfected with an estrogen-responsive element-beta-galactosidase reporter gene, melatonin had no effect on basal or E2-stimulated ER expression. Incubation of hepatocyte aggregates with melatonin (10(-8) to 10(-4)) for 16 or 48 h did not modify the E2-stimulated ER and vitellogenin mRNA, as measured by dot blots. Finally, neither pinealectomy nor melatonin implants caused any effect on basal or E2-stimulated ER and vitellogenin mRNA contents in the liver. Altogether, these results suggest that, although we cannot exclude potential effects at the brain or pituitary levels, melatonin has no or little effects on estrogen receptor in the liver.
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PMID:Effects of melatonin on liver estrogen receptor and vitellogenin expression in rainbow trout: an in vitro and in vivo study. 1089 May 73

Estrogenic and anti-estrogenic activities of diesel exhaust particles (DEP) were evaluated using yeast cells expressing the human estrogen receptor and the responsive element regulating the expression of the receptor gene for beta-galactosidase (Routledge and Sumpter, 1996). It was found that a suspension of whole DEP suspension is not estrogenic but that this preparation possesses the ability to reduce the estrogen-dependent reporter activity. DEP were serially extracted with hexane, benzene, dichloromethane, methanol, and 1 M ammonia, and the estrogenic and anti-estrogenic activities of these preparations were determined. None of the extracts of DEP were estrogenic, but the extracts of benzene, dichloromethane and methanol possessed anti-estrogenic activity, and the activity of estrogen in the presence of hexane extract was slightly decreased. These results indicated that DEP contain heterologous compounds having anti-estrogenic activity. It is thought that those compounds in DEP can modulate the activity of estrogen, leading to the distruption of balance between estrogen and androgen. In this paper, the environmental effects of DEP in relation to the endocrine disrupting effect of organic compounds in DEP are discussed.
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PMID:Anti-estrogenic activity of diesel exhaust particles. 1114 81

We have developed a universally applicable system for conditional gene expression in embryonic stem (ES) cells that relies on tamoxifen-dependent Cre recombinase-loxP site-mediated recombination and bicistronic gene-trap expression vectors that allow transgene expression from endogenous cellular promoters. Two vectors were introduced into the genome of recipient ES cells, successively: (i) a bicistronic gene-trap vector encoding the beta-galactosidase/neo(R) fusion protein and the Cre-ER(T2) (Cre recombinase fused to a mutated ligand-binding domain of the human estrogen receptor) and (ii) a bicistronic gene-trap vector encoding the hygro(R) protein and the human alkaline phosphatase (hAP), the expression of which is prevented by tandemly repeated stop-of-transcription sequences flanked by loxP sites. In selected clones, hAP expression was shown to be regulated accurately by 4'hydroxy-tamoxifen. Strict hormone-dependent expression of hAP was achieved (i) in vitro in undifferentiated ES cells and embryoid bodies, (ii) in vivo in virtually all the tissues of the 10-day-old chimeric fetus (after injection of 4'hydroxy-tamoxifen to foster mothers), and (iii) ex vivo in primary embryonic fibroblasts isolated from chimeric fetuses. Therefore, this approach can be applied to drive conditional expression of virtually any transgene in a large variety of cell types, both in vitro and in vivo.
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PMID:An efficient system for conditional gene expression in embryonic stem cells and in their in vitro and in vivo differentiated derivatives. 1122 62

The efficient introduction of somatic mutations in a given gene, at a given time, in a specific cell type, will facilitate studies of gene function and the generation of animal models for human diseases. We have established a conditional site-specific recombination system in mice using a new version of the Cre/lox system. The Cre recombinase has been fused to a mutated ligand binding domain of the human estrogen receptor (ER), resulting in a tamoxifen-dependent Cre recombinase, Cre-ER(T), that is activated by tamoxifen, but not by estradiol. Transgenic mice were generated expressing Cre-ER(T) under the control of a cytomegalovirus promoter. Administration of tamoxifen to these transgenic mice induced excision of a chromosomally integrated gene flanked by loxP sites in a number of tissues, whereas no excision could be detected in untreated animals. However, the efficiency of excision varied between tissues, and the highest level (approximately 40%) was obtained in the skin. To determine the efficiency of excision mediated by Cre-ER(T) in a given cell type, Cre-ER(T)-expressing mice were crossed with reporter mice in which expression of Escherichia coli beta-galactosidase can be induced through Cre-mediated recombination. The efficiency and kinetics of this recombination were analyzed at the cellular level in the epidermis of 6- to 8-week-old double transgenic mice. Site-specific excision occurred within a few days of tamoxifen treatment in essentially all epidermis cells expressing Cre-ER(T). These results indicate that cell-specific expression of Cre-ER(T) in transgenic mice can be used for efficient tamoxifen-dependent Cre-mediated recombination at loci containing loxP sites, to generate site-specific somatic mutations in a spatiotemporally controlled manner. This conditional site-specific recombination system should allow the analysis of knockout phenotypes that cannot be addressed by conventional gene targeting.
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PMID:Site- and time-specific gene targeting in the mouse. 1132 5

Here, we describe a rapid, convenient, and quantitative beta-galactosidase assay in liquid culture of recombinant yeast that expresses the estrogen receptor. This assay allows large-scale screening of chemicals (more than 600 samples/day) for the evaluation of their direct estrogenic potency and accurate determination of their EC50 with minimal manipulations. The assay, which is based on digestion of the yeast cell wall by lyticase (zymolase), a beta-glucanase isolated from Arthrobacter luteus, followed by a hypoosmotic shock lysis, is performed completely in 96-well plates. This protocol for using recombinant yeast with the two-hybrid technology significantly advances recombinant yeast manipulation.
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PMID:Streamlined beta-galactosidase assay for analysis of recombinant yeast response to estrogens. 1135 34

The use of the site-specific DNA recombinases FLP and Cre is well-established in a broad range of organisms. Here we investigate the applicability of both recombinases to the Xenopus system where they have not been analyzed yet. We show that injection of FLP mRNA triggers the excision of an FLP recombination target (FRT)-flanked green fluorescent protein (GFP) sequence in a coinjected reporter construct inducing the expression of a downstream beta-galactosidase gene (lacZ). The FLP-mediated gene activation can be controlled in Xenopus embryos by injecting a mRNA encoding a fusion of FLP to the mutant ligand binding domain of the human estrogen receptor whose activity is dependent on 4-hydroxytamoxifen. We also demonstrate that a Cre reporter injected into fertilized eggs is fully recombined by Cre recombinase before zygotic gene transcription initiates. Our results indicate that in Xenopus embryos Cre is more effective than FLP in recombining a given quantity of reporter molecules. Finally, we present FLP-inducible double reporter systems encoding two fluorescence proteins (EYFP, ECFP, DsRed or GFP). These novel gene expression systems enable the continuous analysis of two reporter activities within living embryos and are expected to allow cell-lineage studies based on recombinase-mediated DNA rearrangement in transgenic Xenopus lines.
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PMID:FLP and Cre recombinase function in Xenopus embryos. 1137 65

There is a growing concern that environmental xenobiotics may be affecting human and wildlife health by disrupting normal endocrine function via interaction with steroid hormone receptors. Several of these persistent contaminants are chiral and may have enantiomer-specific biological properties. Previous experiments have demonstrated that (-)-o,p'-DDT enantiomer is a more active estrogen-mimic than the (+)-enantiomer in rats. However, these results have not been extrapolated to other biological systems. This study used a yeast-based assay to assess the enantiomer-specific transcriptional activity of DDT with the human estrogen receptor (hER). (+)-17beta-estradiol, racemic DDT and individual DDT enantiomers were added to yeast cultures and hER activity was measured by quantification of beta-galactosidase. The relative activity of o,p'-DDT was weak compared to estradiol. For o,p'-DDT, the (-)-enantiomer was the active estrogen mimic whereas the hER activity of (+)-o,p'-DDT was negligible. The presence of the (+)-enantiomer at relatively greater concentration decreased the transcriptional activity of (-)-o,p'-DDT. This data demonstrates the need to consider stereochemistry of environmental contaminants and their potential influence on biological responses.
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PMID:Enantiomer-specific activity of o,p'-DDT with the human estrogen receptor. 1170 Dec 25

Oviduct-specific glycoprotein (OGP) is a high molecular weight glycoprotein belonging to the chitinase protein family. OGPs are found to associate with the zona pellucida and plasma membrane of the oocyte and developing embryo. Recent studies have shown that OGP plays an important role in pre-fertilization reproductive events (i.e. sperm capacitation, sperm-zona binding and zona penetration). To have a better understanding of human OGP (HuOGP) gene expression, a 3.3 kb DNA fragment containing the 5'-flanking and the intron I region of the HuOGP gene was isolated. DNA sequence analysis of the HuOGP putative promoter revealed little homology with its hamster and mouse ogp gene counterparts. One transcription initiation site was found 12 nucleotides upstream of the first ATG codon of the HuOGP gene. The HuOGP gene promoter lacked typical CAAT or GC boxes, but contained eight half estrogen-responsive elements (ERE) and an imperfect ERE (iERE; 5'-GGTCANNNTGACT-3') site. Deletion mutants of the 3.3 kb DNA fragment were generated and fused to a promoterless beta-galactosidase (beta-Gal) gene. Transfection studies revealed that in the presence of 100 nmol/l estradiol-17 beta (E(2)), a minimal 0.3 kb promoter construct (pH-298/+25 beta Gal) mediated a high level of beta-Gal expression in immortalized human oviductal epithelial OE-E6/E7 cells, but not in MCF-7 and CHO-K1 cells. By electromobility shift assay and specific estrogen receptor antibodies, we demonstrated that estrogen receptor beta present in the OE-E6/E7 cells binds to the iERE. These findings allow a better understanding of the regulation of OGP gene expression in the human oviduct.
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PMID:Cloning and characterization of the human oviduct-specific glycoprotein (HuOGP) gene promoter. 1181 19


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