Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.1 (RNase)
 16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of estrogen and estrogen agonists can be mediated by estrogen receptor alpha (ER alpha) and estrogen receptor beta (ER beta). We now report the identification and initial characterization of several novel isoforms of rat ER beta messenger RNA (mRNA). The most abundant of these mRNA variants we have called ER beta2. ER beta2 had an in-frame insertion of 54 nucleotides that resulted in the predicted insertion of 18 amino acids within the ligand binding domain. We demonstrated by semiquantitative RT-PCR and RNase protection that ER beta2 mRNA was expressed at levels equal to those of the previously published ER beta (ER beta1) in ovary, prostate, pituitary, and muscle. In tissues of the nervous system, including frontal cortex, hippocampus, and hypothalamus, ER beta1 was present in a 2- to 6-fold greater abundance than ER beta2. We have also detected variants of both ER beta1 and ER beta2 mRNAs that contained deletions of 117 bp encompassing the region encoding the second zinc finger of the DNA binding domain. All four mRNA species were efficiently translated into functional protein in a heterologous system. ER beta2 bound estradiol with a lower affinity (Kd 5.1 nM) than either ER alpha (0.19 nM) or ER beta1 (0.14 nM). The binding of ER beta2 was selective in that cortisol, testosterone, aldosterone, and progesterone among other agents did not compete for estradiol binding. However, a variety of known estrogenic agents, including physiological estrogens (estrone and estriol), plant and environmental estrogens (genistein, coumestrol, bisphenol A, methoxychlor), and pharmocological agents (tamoxifen, 4-hydroxytamoxifen) did effectively compete for estradiol binding to both ER beta1 and ER beta2. Interestingly, the binding pharmacology differed among the agents tested. For example, genistein competed effectively for estradiol binding to ER beta1 but was > 150-fold weaker at competing from ER beta2. In contrast, 4-hydroxytamoxifen competed equally well at both receptors. We have also demonstrated by a gel shift assay that both ER beta1 and ER beta2 bound specifically to DNA containing a consensus estrogen response element. ER beta1 and ER beta2 could heterodimerize with each other and with ER alpha. Both ER beta1 and ER beta2 activated transcription in response to estradiol, however, ER beta2 required a 1000-fold greater estradiol concentration for activity than did ER beta1. Cotransfection of ER beta2 had no effect on ER beta1 activation when used in a equal ratio. A 10-fold excess of ER beta2 did raise the half-maximal dose of estradiol required for transcriptional activation, whereas the maximal level of induction did not change. The ER beta complementary DNAs deleted within the DNA binding domain could not bind to DNA or activate transcription from this reporter in the cell backgrounds tested. In conclusion, although the physiological significance of these ER beta variants warrants further investigation, ER beta2 mRNA encodes a specific, functional receptor for estradiol and estrogenic agents. We propose that ER beta2 should also be considered in addition to ER beta1 and ER alpha when describing the effects of estrogen, estrogen agonists/antagonists, or environmental estrogens.
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PMID:Identification of estrogen receptor beta2, a functional variant of estrogen receptor beta expressed in normal rat tissues. 949 41

The expression time course of estrogen receptor alpha (ER alpha) was analyzed by RT-PCR in fetal and newborn rat pituitaries. In addition to the classical ER alpha messenger RNA (mRNA), three shorter transcripts were detected and subsequently cloned. Sequence analysis showed that they corresponded to ER alpha mRNAs lacking exon 3 (which encodes a zinc finger in the DNA-binding domain), exon 4 (which encodes the nuclear localization signal and part of the steroid-binding domain), or both exons 3 and 4. As analyzed by RT-PCR and ribonuclease protection assay, the respective expression levels of the different transcripts varied dramatically during pituitary development; short forms appeared 4 days before full-length ER alpha mRNA. On Western blots from rat pituitaries of different ages, an ER alpha-specific antiserum labeled four protein bands of the expected molecular weights, revealing that all four ER alpha mRNAs are translated in vivo. Immunocytochemistry, using the same antiserum, showed the ER alpha to be present first in the cytosol of intermediate lobe cells (around embryonic day 16). Only 5 days later, nuclear staining became detectable in the anterior lobe. We argue that the observed cytosolic staining will be essentially due to short ER alpha isoforms, which are indeed more abundantly expressed in the intermediate lobe. These data suggest that during pituitary development, the activity of the ER alpha might be specifically regulated by differential splicing of its primary transcript, resulting in a differential subcellular localization of the isoforms.
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PMID:Stage- and region-specific expression of estrogen receptor alpha isoforms during ontogeny of the pituitary gland. 1034 69

Peroxisome proliferator-activated receptor binding protein (PBP), a nuclear receptor coactivator, interacts with estrogen receptor alpha (ERalpha) in the absence of estrogen. This interaction was enhanced in the presence of estrogen but was reduced in the presence of antiestrogen, tamoxifen. Transfection of PBP in CV-1 cells resulted in enhancement of estrogen-dependent transcription, indicating that PBP serves as a coactivator in ER signaling. To examine whether overexpression of PBP plays a role in breast cancer because of its coactivator function in ER signaling, we determined the levels of PBP expression in breast tumors. High levels of PBP expression were detected in approximately 50% of primary breast cancers and breast cancer cell lines by ribonuclease protection analysis, in situ hybridization, and immunoperoxidase staining. Fluorescence in situ hybridization of human chromosomes revealed that the PBP gene is located on chromosome 17q12, a region that is amplified in some breast cancers. We found PBP gene amplification in approximately 24% (6/25) of breast tumors and approximately 30% (2/6) of breast cancer cell lines, implying that PBP gene overexpression can occur independent of gene amplification. This gene comprises 17 exons that, together, span >37 kilobases. The 5'-flanking region of 2.5 kilobase pairs inserted into a luciferase reporter vector revealed that the promoter activity in CV-1 cells increased by deletion of nucleotides from -2,500 to -273. The -273 to +1 region, which exhibited high promoter activity, contains a typical CCAT box and multiple cis-elements such as C/EBPbeta, YY1, c-Ets-1, AP1, AP2, and NFkappaB binding sites. These observations, in particular PBP gene amplification, suggest that PBP, by its ability to function as ERalpha coactivator, might play a role in mammary epithelial differentiation and in breast carcinogenesis.
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PMID:Amplification and overexpression of peroxisome proliferator-activated receptor binding protein (PBP/PPARBP) gene in breast cancer. 1048 14

In rodent uterus, both up- and down-regulation of estrogen receptor alpha (ERalpha) messenger ribonucleic acid (mRNA) and protein levels by estradiol has been demonstrated; however, it is not known which of the uterine compartments (endometrial epithelium, stroma, myometrium) respond to estradiol with autoregulation of ERalpha. The purpose of the present study was to investigate and compare the kinetics and cell type-specific effects of estradiol on uterine ERalpha expression in immature and adult rats. Ovariectomized female rats were injected s.c. with sesame oil or estradiol-17beta. Uteri were collected and analyzed for changes in ERalpha mRNA using RNase protection assays (RPA) and in situ hybridization using radiolabeled probes specific for ERalpha. Immunohistochemical analysis was performed with a polyclonal antibody specific to ERalpha. Expression of ERalpha in the uterine epithelial cells decreased at 3 and 6 h after estradiol administration to immature and adult rats, respectively. At 24 h, ERalpha mRNA levels in the immature and mature rat uterus were higher than pretreatment levels but returned to baseline by 72 h. Pretreatment with cycloheximide did not block the 3-h repressive effect of estradiol, suggesting that the estradiol-induced decrease in ERalpha mRNA occurs independent of new protein synthesis. A decrease in ERalpha mRNA and protein was also observed in uterine epithelia at 3 and 6 h after an estradiol injection to immature and adult rats, and intensity of both the in situ hybridization signal and the immunostaining in the epithelium increased at 24 and 72 h. However, the periluminal stromal cells in the adult uterus and the majority of stromal cells of the immature uterus appeared to have increased ERalpha expression. The results indicate that down-regulation of ERalpha in the epithelia and up-regulation of stromal ERalpha play a role in early events associated with estradiol-induced cell proliferation of the uterine epithelia.
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PMID:Effect of estradiol on estrogen receptor expression in rat uterine cell types. 1061 Oct 82

The presence of an exon 1' sequence in the estrogen receptor alpha (ERalpha) mRNA was detected in different stocks of ER-positive MCF-7 human breast cancer cells by reverse transcriptase polymerase chain reaction (RT-PCR) and ribonuclease protection analysis (RPA), but not by Northern blot analysis. This mRNA, however, was not detectable in ERalpha-positive ZR-75-1 or ERalpha-negative MDA-MB-231 breast cancer cells, suggesting that exon 1' ER mRNA is differentially expressed in some but not all ER-positive cell lines, and then, only at very low levels.
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PMID:Differential expression of an estrogen receptor messenger RNA containing exon 1' sequences in MCF-7 breast cancer cell line stocks. 1068 May 97

The fact that male estrogen receptor alpha (ERalpha) knockout mice are infertile indicates a role for this receptor in male reproduction. Here, objectives were to evaluate ERalpha expression in male goat reproductive tissues at the transcriptional level using RNase protection assay (RPA) and in situ hybridization (ISH), and to clone a partial cDNA for caprine ERalpha using reverse transcription-polymerase chain reaction (RT-PCR). For RPA and ISH procedures, a radiolabeled antisense cRNA probe, generated in vitro from the ovine oER8 cDNA template, was employed. Evaluations were made on individual samples obtained from adult goats. Labeled cRNA sense probe was used as a negative control in ISH. A 530-base pair amplicon was generated by RT-PCR from efferent ductules (EDs), epididymis (EP), and testis, cloned from the ED and EP, and sequenced. The caprine ERalpha (cERalpha) cDNA displayed 81%-96% sequence identity with that of other species. A signal indicative of ERalpha mRNA was identified by both RT-PCR and RPA in all tissues, but was strongest in the ED. Compared with ED, ERalpha signal was sixfold lower in the EP, and 66-fold lower in the testis. Similarly, strong ERalpha expression was observed in ED epithelium, whereas little or no signal was detected in EP or testis by ISH. Thus, among different segments of the male reproductive tract and testis, the highest level of ERalpha mRNA expression was found in epithelium of the ED.
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PMID:Expression and molecular characterization of estrogen receptor alpha messenger RNA in male reproductive organs of adult goats. 1131 48

Long term exposure to estradiol increases the risk of breast cancer in a variety of animal species, as well as in women. The mechanisms responsible for this effect have not been firmly established. The prevailing theory proposes that estrogens increase the rate of cell proliferation by stimulating estrogen receptor-mediated transcription and thereby the number of errors occurring during DNA replication. An alternative hypothesis proposes that estradiol can be metabolized to quinone derivatives which can react with DNA and then remove bases from DNA through a process called depurination. Error prone DNA repair then results in point mutations. We postulate that these two processes, increased cell proliferation and genotoxic metabolite formation, act in an additive or synergistic fashion to induce cancer. If correct, aromatase inhibitors would block both processes whereas anti-estrogens would only inhibit receptor-mediated effects. Accordingly, aromatase inhibitors would be more effective in preventing breast cancer than use of anti-estrogens. Our studies initially demonstrated that catechol estrogen (CE) quinone metabolites are formed in MCF-7 human breast cancer cells in culture. Measurement of estrogen metabolites and conjugates involved utilization of an HPLC separation coupled with an electrochemical detector. We then utilized an animal model that allows dissociation of estrogen receptor-mediated function from that of the effects of estradiol metabolites. Wnt-1 transgenic mice harboring a knock-out of ERalpha provides a means of examining the effect of estrogen deprivation in the absence of the ER in animals with a high incidence of breast tumors. ERbeta was shown to be absent in the breast tissue of these animals by RNase protection assay. In the breast tissue of these estrogen receptor alpha knock-out (ERKO)/Wnt-1 transgenic mice, we demonstrated formation of genotoxic estradiol metabolites. The ERKO/Wnt-1 breast extracts contained picomole amounts of the 4-catechol estrogens, but not their methoxy conjugates nor the 2-CE or their methoxy conjugates. The 4-CE conjugates with glutathione or its hydrolytic products (cysteine and N-acetylcysteine) were detected in picomole amounts in both tumors and hyperplastic mammary tissue, demonstrating the formation of CE-3,4-quinones. These results are consistent with the hypothesis that mammary tumor development is primarily initiated by metabolism of estrogens to 4-CE and, then, to CE-3,4-quinones, which may react with DNA to induce oncogenic mutations. The next set of experiments examined the incidence of tumors formed in Wnt-1 transgenic mice bearing wild type ERalpha (ER+/+), the heterozygous combination of genes (ER+/ER-) or ERalpha knock-out (ER-/-). To assess the effect of estrogens in the absence of ER, half of the animals were oophorectomized on day 15 and the other half were sham operated. Castration reduced the incidence of breast tumors in all animal groups and demonstrated the dependence of tumor formation upon estrogens. A trend toward reduction in tumor number (not statistically significant at this interim analysis) occurred in the absence of functional ER since the number of tumors was markedly reduced in ERKO animals which were castrated early in life. In aggregate, our results support the concept that metabolites of estradiol may act in concert with ER mediated mechanisms to induce breast cancer.
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PMID:Genotoxic metabolites of estradiol in breast: potential mechanism of estradiol induced carcinogenesis. 1462 47

The objective of this study was to examine the expression of receptors for androgen, estrogen, and progesterone in the fetal sheep brain during the critical period for sexual differentiation. We isolated mRNA from the hypothalamus-preoptic area (HPOA), amygdala (AMYG), medulla (MD), frontal cortex (FCTX) and olfactory bulbs (OB) of fetal sheep that were delivered on day 64 of gestation. Using a ribonuclease protection assay and species-specific cRNA probes, we measured mRNA expression levels of androgen receptor (AR), estrogen receptor alpha (ERalpha) and progesterone receptor (PR). ERalpha and AR mRNA were expressed in all of the tissues tested and highest in the HPOA. PR mRNA was measured in HPOA and AMYG only and was significantly higher in male than in female fetuses. We conclude that the fetal brain is a target site for circulating steroid hormones. These data have implications for the steroid dependent development of sexually dimorphic brain functions in sheep.
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PMID:Expression of steroid hormone receptors in the fetal sheep brain during the critical period for sexual differentiation. 1687 81

A rapid method of 5-bromo-2'-deoxyuridine (BrdU) immunostaining was developed in cryosections of bovine mammary tissue while preserving RNA quality of the stained section. A thymidine analog that is incorporated into DNA of proliferating cells, BrdU serves as a proliferation marker. Immunostaining of BrdU-labeled cells within a histological section requires heat, enzymatic or chemical-mediated antigen retrieval to open double-stranded DNA, and exposure to the BrdU antigen. Although these established treatments permit staining, they preclude use of cells within the tissue section for further gene expression experiments. Additionally, long antibody incubations and washing steps lead to extensive RNA degradation and elution. A protocol was developed for immunolocalization of BrdU-labeled cells in cryosections of bovine mammary tissue, which does not require harsh DNA denaturation and preserved RNA integrity and quantity. This protocol used an initial acetone:polyethylene glycol 300 [9:1 (vol/vol)] fixation (2 min) followed by staining with methyl green (0.5% aqueous; 2 min) to stabilize macromolecules, antigen retrieval with deionized formamide (70% in nuclease-free phosphate buffered saline; 4 min incubation), antibody incubation in the presence of RNase inhibitors (5 min), and minimal washing to facilitate recovery of RNA from cells from the stained sections. Applicability of this protocol to other nuclear antigens was evaluated by testing its suitability for staining estrogen receptor alpha and Ki-67 antigen. In both cases, use of the protocol provided good immunostaining and tissue morphology. The RNA quality of estrogen receptor alpha- and Ki-67-stained sections was not evaluated. Quality of the isolated RNA from BrdU-stained sections was evaluated by micro-fluidic electrophoresis and its utility was confirmed using quantitative reverse transcription-PCR. Staining intensity obtained with this labeling protocol was similar to that obtained using conventional immunohistochemistry protocols. When coupled with laser microdissection and RNA or cDNA amplification, this immunostaining protocol provided a means for future transcriptome analysis of BrdU-labeled cells within a complex tissue.
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PMID:Technical note: A rapid method for 5-bromo-2'-deoxyuridine (BrdU) immunostaining in bovine mammary cryosections that retains RNA quality. 2049 66