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)

To study cell proliferation in different cell types and segments of the mammary gland, we devised a dual staining procedure, combining nuclear labeling by 5-bromo-2'-deoxy-uridine (BrdU) uptake (revealed by a dark-brown precipitate) and an alternative (red or blue) cytoplasmic labeling by antibodies specific for the differentiation proteins of epithelial, myoepithelial, and secretory cell types. The following markers, revealed by APAAP or beta-galactosidase procedure, were selected: alpha-smooth muscle actin for the myoepithelial cells, keratin (detected by AE1 monoclonal) for the luminal epithelial cells, alpha-lactalbumin and beta-casein for the secretory cells. To follow the full process of organogenesis, the study was conducted in mouse mammary glands from virgin, primed, and lactating animals and from glands cultured in vitro under specific hormone stimulation. Cell proliferation was localized mainly in focal areas (end buds), and mostly corresponded to "null" undifferentiated cells. Estrogen and progestin stimulation induced a relative increase of proliferating differentiated cells of either epithelial or myoepithelial type, localized in ducts and alveolar structures. Prolactin stimulation induced proliferation in secretory cells.
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PMID:Immunocytochemical identification of proliferating cell types in mouse mammary gland. 221 15

There is a concern that chemicals in our environment are affecting human health by disrupting normal endocrine function. Much of the concern has focused on chemicals that can interact directly with steroid hormone receptors. We have used a yeast-based assay to assess chemical interactions with the estrogen, androgen, and progesterone receptors. The yeast transformants used in this study contained the human estrogen, androgen, or progesterone receptor along with the appropriate steroid responsive elements upstream of the beta-galactosidase reporter gene. Chemicals were added to yeast cultures in doses ranging from 10(-12) to 10(-4) M and following incubation, the yeasts were then lysed and assayed for beta-galactosidase activity. Diethylstilbesterol and 17-beta estradiol were most active in the estrogen receptor assay, followed by the phytoestrogen, coumestrol. p-Nonylphenol and bisphenol A were approximately 5000- and 15,000-fold less active, respectively, than estradiol. Methoxychlor, DDT and its metabolites, o,p'-DDD, and o,p'-DDE ranged in potency from 5 to 24 X 10(6) less potent than estradiol. Testosterone and dihydrotestosterone were most potent in the androgen receptor assay, followed by estradiol and progesterone. p,p'-DDE was approximately 10(6)-fold less potent than testosterone. None of the industrial chemicals tested interacted with the progesterone receptor. These data demonstrate the utility of using yeast-based receptor assays for detecting chemical interaction with steroid receptors and these assays should serve as a useful component of an in vitro-in vivo strategy to assess the effects of chemicals on endocrine function.
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PMID:Evaluation of chemicals with endocrine modulating activity in a yeast-based steroid hormone receptor gene transcription assay. 907 9

The estrogenic activity of dieldrin, toxaphene, and an equimolar mixture of both compounds (dieldrin/toxaphene) was investigated in the 21-day-old B6C3F1 mouse uterus, MCF-7 human breast cancer cells, and in yeast-based reporter gene assays. Treatment of the animals with 17beta-estradiol (E2) (0.0053 kg/day x3) resulted in a 3.1-, 4.8-, and 7.8-fold increase in uterine wet weight, peroxidase activity, and progesterone receptor binding, respectively. In contrast, treatment with 2.5, 15 and 60 micromol/kg (x3) doses of toxaphene, dieldrin, or dieldrin/toxaphene (equimolar) did not significantly induce a dose-dependent increase in any of the E2-induced responses. The organochlorine pesticides alone and the binary mixture did not bind to the mouse uterine estrogen receptor (ER) in a competitive binding assay using [3H]E2 as the radioligand. In parallel studies, estrogenic activities were determined in MCF-7 cells by using a cell proliferation assay and by determining induction of chloramphenicol acetyl transferase (CAT) activity in MCF-7 cells transiently transfected with plasmids containing estrogen-responsive 5'-promoter regions from the rat creatine kinase B and human cathepsin D genes. E2 caused a 24-fold increase in CAT activity in MCF-7 cells transiently transfected with creatine kinase B and a 3.8-fold increase in cells transiently transfected with the human cathepsin D construct. Treatment of MCF-7 cells with dieldrin, toxaphene, or an equimolar mixture of dieldrin plus toxaphene (10(-8)-10(-5) M) did not significantly induce cell proliferation or CAT activity in the transient transfection experiment with both plasmids. The relative competitive binding of the organochlorine pesticides was determined by incubating MCF-7 cells with 10(-9) M [3H]E2 in the presence or absence of 2 x 10(-7) M unlabeled E2 (to determine nonspecific binding), toxaphene (10(-5) M), dieldrin (10(-5) M), and equimolar concentrations of the dieldrin plus toxaphene mixture (10(-5) M). The binding observed for [3H]E2 in the whole cell extracts was displaced by unlabeled E2, whereas the organochlorine pesticides and binary mixture exhibited minimal to nondetectable competitive binding activity. E2 caused a 5000-fold induction of beta-galactosidase (beta-gal) activity in yeast transformed with the human ER and a double estrogen responsive element upstream of the beta-gal reporter gene. Treatment with 10(-6)-10(-4) M chlordane, dieldrin, toxaphene, or an equimolar mixture of dieldrin/toxaphene did not induce activity, whereas 10(-4) M endosulfan caused a 2000-fold increase in beta-gal activity. Diethylstilbestrol caused a 20-fold increase in activity in yeast transformed with the mouse ER and a single estrogen responsive element upstream of the beta-gal reporter gene. Dieldrin, chlordane, toxaphene, and endosulfan induced a 1.5- to 4-fold increase in activity at a concentration of 2.5 x 10(-5) M. Synergistic transactivation was not observed for any equimolar binary mixture of the pesticides at concentrations of either 2.5 x 10(-5) M or 2.5 x 10(-4) M. The results of this study demonstrate that for several estrogen-responsive assays in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based reporter gene assays, the activities of both dieldrin and toxaphene were minimal, and no synergistic interactions were observed with a binary mixture of the two compounds.
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PMID:Estrogenic activity of a dieldrin/toxaphene mixture in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based estrogen receptor assays: no apparent synergism. 907 11

Gonadal steroids and physiological stressors affect the regulation of proenkephalin (PPE) gene expression in the paraventricular (PVN) and ventromedial (VMH) hypothalamic nuclei. To examine the effects of these modulators at the cellular level, the current study utilized a transgenic mouse line that expresses a human proenkephalin promoter/bacterial beta-galactosidase fusion gene (ENK-1). Previous studies have demonstrated that the regulatory sequences included in this transgene are sufficient to support appropriate transcriptional regulation of the reported gene in the PVN of male ENK-1 mice in response to stress. The present experimental paradigm was designed to examine possible interactions of sex and circulating estrogen levels with the opioid responses to acute systemic stressors, an intraperitoneal injection of hypertonic (1.5 M) or isotonic (0.15 M) saline. Adult ENK-1 mice were gonadally intact, gonadectomized, or 21 days postpartum. Forty-eight hours before perfusion, castrated males and ovariectomized females received either 10 micrograms estradiol benzoate or oil vehicle and 4 animals per group received no further treatment. Six h before perfusion, remaining animals received a single intraperitoneal injection of either hypertonic or isotonic saline. Tissues were sectioned through the hypothalamus and processed for X-gal histochemistry. In the VMH of ovariectomized females that received isotonic saline, estrogen significantly elevated transgene expression. This effect was not seen in females that only received estrogen or in those that received the severe systemic stressor of a injection of hypertonic saline. Estrogen and stress did not interact to elevate transgene expression in the VMH of males. A different pattern of expression was observed in the PVN; injection of hypertonic saline induced transgene expression only in gonadally intact males and in castrated males given estrogen. These findings demonstrate that stress and estrogen have sex-specific and site-specific regulatory effects on the expression of a PPE promoter transgene in hypothalamic neurons.
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PMID:Estrogen and stress interact to regulate the hypothalamic expression of a human proenkephalin promoter-beta-galactosidase fusion gene in a site-specific and sex-specific manner. 914 95

Estrogen regulates proliferation and morphogenesis of mammary ductal epithelium by interacting with a specific intracellular estrogen receptor (ER) that acts as a hormone-dependent transcriptional regulator of gene expression. The mechanisms by which ER regulates transcription in response to estrogen have been analyzed extensively in tissue culture and in cell-free systems. These studies have demonstrated that the transcriptional activity of ER is strongly influenced by cellular context and highlight the need to address ER transcriptional activity in an appropriate cellular background. Thus, to gain insight into the mechanistic role of ER in mammary epithelial morphogenesis, we have used an adenoviral gene delivery strategy to introduce an estrogen-responsive reporter gene into the mammary epithelium and to monitor the activity of endogenous ERs in their natural environment where cellular context including stromal-epithelial interactions can be taken into account. Using this approach, we first demonstrated highly efficient adenoviral delivery throughout the mammary epithelium using a beta-galactosidase (betagal) reporter gene under the control of the constitutively active cytomegalovirus (CMV) promoter. Next, we constructed an adenoviral vector by substituting the CMV promoter with an estrogen-dependent promoter fragment-linked betagal (Ad-ERE-tk-betagal). This adenoviral reporter system provides evidence that ER positive mammary epithelial cells display a differential sensitivity in a region-specific manner toward estrogen induction. Our data suggest that the availability of factor(s) other than ER is necessary for ER-mediated gene activation and may be important in modulating the differential responses of mammary epithelial cells to estrogen.
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PMID:Reconstitution of estrogen-dependent transcriptional activation of an adenoviral target gene in select regions of the rat mammary gland. 960 2

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

The mechanisms through which gonadal steroids exert feedback actions on the activity of the GnRH neurons are not understood. Using a series of GnRH-LacZ transgenic mice we have examined the manner in which gonadal steroids suppress GnRH mRNA expression in male and female mice. The long-term gonadectomy of 5.5-GNZ-3.5 transgenic mice resulted in significant increases in cellular GnRH mRNA expression (P < 0.05) and plasma LH concentrations (P < 0.01) in both sexes. However, cellular levels of LacZ mRNA and beta-galactosidase, which provide an index of GnRH gene transcription, were only elevated in males after gonadectomy. This sexually differentiated response was also observed in mice gonadectomized for 2 wk. Estrogen replacement in gonadectomized males returned transgene expression to intact levels. Experiments in transgenic mice with 3' and 5' deleted GnRH-LacZ constructs revealed that the suppressive influence of estrogen on LacZ transcription in the male required a critical element located between -5.2 and -1.7 kb of the GnRH promoter. These studies show that the suppression of GnRH mRNA expression by estrogen in the male involves a decrease in GnRH gene transcription that is dependent on a distal GnRH promoter element. The same mechanism does not exist in females, indicating that gonadal steroids suppress GnRH mRNA levels in a sexually dimorphic manner.
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PMID:Sex differences in estrogen-dependent transcription of gonadotropin-releasing hormone (GnRH) gene revealed in GnRH transgenic mice. 1286 13

The functional impairment associated with atherogenic factors, including hypertension, constitutes a limitation to the ability of endothelial progenitor cells (EPCs) to repair. In addition, estrogens have been shown to play a role in reendothelialization after vascular injury. We investigated the effects of estrogens on differentiation and senescence of EPCs derived from bone marrow (BM-EPCs) in spontaneously hypertensive rats (SHR/Izm). Bone marrow (BM) cells were obtained from the tibias and femurs of age-matched, male SHR/Izm and Wistar-Kyoto rats (WKY/Izm). The number of differentiated, adherent BM-EPCs derived from SHR/Izm was significantly smaller than the number derived from WKY/Izm. 17beta-Estradiol (E2) significantly increased the number of adherent BM-EPCs from SHR/Izm, and this effect was significantly attenuated by pharmacological phosphatidylinositol 3-kinase (PI3-K) blockers. Immunoblotting analysis revealed that E2 treatment led to phosphorylation of Akt. Senescence, as assessed by acidic beta-galactosidase staining, occurred at a significantly greater rate in the BM-EPCs from SHR/Izm than in those from WKY/Izm, but E2 treatment dramatically delayed the senescence of BM-EPCs from SHR/Izm. A polymerase chain reaction (PCR)-ELISA based assay revealed that telomerase activity in BM-EPCs from SHR/Izm was significantly lower than in those from WKY/Izm, but that E2 treatment significantly augmented it. Both MTS and colony forming unit assay revealed that E2 treatment significantly augmented the functional activity in BM-endothelial cell (EC)-like cells from SHR/Izm compared to that in control BM-EC-like cells (no treatment). In conclusion, the differentiation of BM-EPCs derived from SHR/Izm was significantly decreased compared with that of BM-EPCs from WKY/Izm. In addition, the rate of senescence was significantly greater in the BM-EPCs from SHR/Izm than in those from WKY/Izm. Estrogen was shown to augment differentiation and delay the onset of senescence in BM-EPCs from SHR/Izm.
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PMID:Effect of estrogen on differentiation and senescence in endothelial progenitor cells derived from bone marrow in spontaneously hypertensive rats. 1641 50

Estrogen has been demonstrated to promote therapeutic reendothelialization after vascular injury by bone marrow (BM)-derived endothelial progenitor cell (EPC) mobilization and phenotypic modulation. We investigated the primary hypothesis that estrogen regulates physiological postnatal vasculogenesis by modulating bioactivity of BM-derived EPCs through the estrogen receptor (ER), in cyclic hormonally regulated endometrial neovascularization. Cultured human EPCs from peripheral blood mononuclear cells (PB-MNCs) disclosed consistent gene expression of ER alpha as well as downregulated gene expressions of ER beta. Under the physiological concentrations of estrogen (17beta-estradiol, E2), proliferation and migration were stimulated, whereas apoptosis was inhibited on day 7 cultured EPCs. These estrogen-induced activities were blocked by the receptor antagonist, ICI182,780 (ICI). In BM transplanted (BMT) mice with ovariectomy (OVX) from transgenic mice overexpressing beta-galactosidase (lacZ) regulated by an endothelial specific Tie-2 promoter (Tie-2/lacZ/BM), the uterus demonstrated a significant increase in BM-derived EPCs (lacZ expressing cells) incorporated into neovasculatures detected by CD31 immunohistochemistry after E2 administration. The BM-derived EPCs that were incorporated into the uterus dominantly expressed ER alpha, rather than ER beta in BMT mice from BM of transgenic mice overexpressing EGFP regulated by Tie-2 promoter with OVX (Tie-2/EGFP/BMT/OVX) by ERs fluorescence immunohistochemistry. An in vitro assay for colony forming activity as well as flow cytometry for CD133, CD34, KDR, and VE-cadherin, using human PB-MNCs at 5 stages of the female menstrual-cycle (early-proliferative, pre-ovulatory, post-ovulatory, mid-luteal, late-luteal), revealed cycle-specific regulation of EPC kinetics. These findings demonstrate that physiological postnatal vasculogenesis involves cyclic, E2-regulated bioactivity of BM-derived EPCs, predominantly through the ER alpha.
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PMID:Estrogen-mediated endothelial progenitor cell biology and kinetics for physiological postnatal vasculogenesis. 1765 79