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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The conventional methods for mRNA quantitation such as Northern blotting or ribonuclease protection assay sometimes lack enough sensitivity to study low abundance mRNAs or to work with limited amounts of biological samples. The sensitivity of the polymerase chain reaction (PCR) linked to reverse transcription (RT-PCR) has proven useful in amplifying specific mRNAs, especially those present in low copy number. Though, the quantitation of nucleic acids by means of PCR has proven problematic. The main constraint in obtaining quantitative data is inherent in the amplification reaction. Because amplification is an exponential process, small variations in the efficiency of amplification may significantly affect the final yield of the PCR product. The variables that influence the rate of the PCR include the abundance of the mRNA present in the starting material, the concentrations of the Taq DNA polymerase, dNTPs and magnesium ions, the annealing and elongation conditions, the ramping temperatures and the formation of primer secondary structures. Moreover, with the progression of the PCR cycles, reagents are consumed and inhibitors generated, leading to non-linear synthesis of DNA. Finally, tube-to-tube variations sometimes preclude accurate quantitation. Most of the above-mentioned problems can be overcome by the choice of adequate internal controls. The present report reviews two recently developed methods for RNA quantitation, the semi-quantitative PCR and the quantitative PCR illustrated for the measurement of monoamine oxidase (MAO) A and B mRNAs and the estrogen receptor (ER) mRNA respectively, with a particular emphasis on the design of appropriate internal controls to compensate for the intra- and inter-assay variability inherent to RT-PCR.
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PMID:Quantitation of low abundance mRNAs in glial cells using different polymerase chain reaction (PCR)-based methods. 938 56

Previous experiments have demonstrated that C-type natriuretic peptide (CNP) expression in the uterus varies during the estrous cycle with maximal expression at proestrus. The present study was designed to determine whether exogenous steroid hormones regulate uterine CNP expression in ovariectomized mice. Estradiol increased significantly (3-fold) uterine immunoreactive CNP (irCNP) rapidly and dose dependently in ovariectomized mice as measured by radioimmunoassay. Other steroids produced either no significant change (testosterone, 1 mg; 2-methoxyestradiol, 1 microgram) or weak induction (estriol, 1 microgram) from vehicle controls. Progesterone (1 mg) significantly attenuated the estrogen-stimulated irCNP response by 50% when injected 30 min before estrogen (1 microgram) in estrogen-primed ovariectomized mice. Estrogen-stimulated increases in uterine CNP transcripts detected by ribonuclease protection analyses were blocked by actinomycin D (160 micrograms) or ICI-164,384 (20 micrograms), a specific nuclear estrogen receptor antagonist. These results indicate that a nuclear estrogen receptor is required for estrogen to stimulate uterine CNP transcription and that progesterone negatively regulates estrogen-stimulated CNP expression.
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PMID:Estradiol induces C-type natriuretic peptide gene expression in mouse uterus. 943 3

Environmental contaminants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), cause alterations in gene expression. In this study, we measured the regulation of estrogen receptor (ER) mRNA in female CD-1 mice by competitive RT-PCR. Previous work suggests that ER protein levels are affected by TCDD, but how this is regulated is uncertain. These studies found no significant changes in ER mRNA levels, but the methods used (Northern blot analysis and RNase protection assays) lack sensitivity for measuring the low levels of RNA transcript, such as ER mRNA. The method described here offers an excellent alternative for quantifying the changes in mRNA levels. Internal competitors were created with gene-specific primers for ER and beta-actin by PCR reactions at low annealing temperatures. For each sample, the mRNA levels of ER and beta-actin were determined. Using competitive RT-PCR, the relative changes in ER mRNA from TCDD-treated and control animals were determined after normalization with the levels of beta-actin mRNA. The ER mRNA from female CD-1 mice treated with TCDD (single dose 5 micrograms/kg, i.p., 4 days) was found to be significantly suppressed as compared with the vehicle control in all tissues examined. TCDD decreased ER mRNA in the liver (30.1%) as expected. However, the greatest effect was in the reproductive tissues, with a 64.2% reduction in ER mRNA in the ovary. This is the first demonstration that TCDD causes tissue-specific downregulation of ER mRNA. These effects may contribute to the tissue-specific toxicity of TCDD.
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PMID:Regulation of estrogen receptor mRNA by 2,3,7,8-tetrachlorodibenzo-p-dioxin as measured by competitive RT-PCR. 944 63

The effects of estradiol treatment, which stimulates cell division in rat uterine epithelial cells, on the in vivo expression of heparin-binding epidermal growth factor (HB-EGF), cyclin D1, and cyclin B1 messenger RNA (mRNA) in these cells have been examined using ribonuclease protection assays. Estradiol gave rise to significant increases in steady state levels of HB-EGF 2 and 24 h after treatment. Cyclin D1 mRNA levels were elevated 8 and 10 h after estradiol administration, corresponding to the G1 phase of the mitotic cycle, and cyclin B1 mRNA was only expressed 16-24 h after estradiol treatment, which corresponds to the G2 and M phases of the rat uterine epithelial cell cycle. Estradiol-stimulated increases in HB-EGF mRNA were not affected by treatment with cycloheximide, but were inhibited by the estrogen antagonist compound, ICI 164,384, demonstrating that the estrogen-stimulated increase in HB-EGF mRNA is a primary, estrogen receptor-mediated response of rat uterine epithelium to estradiol. Progesterone treatment, which blocks epithelial cells in G1 of the cycle, suppressed levels of HB-EGF mRNA below those observed in ovariectomized rats. These results indicate that HB-EGF mediates the regulatory effects of both estradiol and progesterone on rat uterine epithelial cell proliferation through an effect on the production of G1 phase molecules such as cyclin D1.
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PMID:Mediators of estradiol-stimulated mitosis in the rat uterine luminal epithelium. 949 26

The prognosis in ovarian carcinoma, the most lethal of the gynecologic neoplasms, is poor and has changed little in the last three decades. Only a small number respond to antiestrogen therapy, although the classic estrogen receptor, ER-alpha, has been identified in ovarian surface epithelium, from which approximately 90% of ovarian cancers originate. We have previously shown that ER-beta mRNA is most abundant in human fetal ovaries, suggesting that it might play an important role in ovarian development. Therefore, we investigated the mRNA levels of both ERs in normal ovaries, ovarian serous cystadenocarcinomas, granulosa cells from patients undergoing in vitro fertilization (IVF), the ovarian surface epithelium cell line IOSE-Van, and the ovarian cancer cell lines SKOV3, HEY and OCC1. Northern blots of normal and neoplastic ovaries were hybridized with an ER-beta riboprobe that spans the A/B domain. We detected two major hybridizing bands at approximately 8 and 10 kb. An RNase protection assay using the same probe revealed a single band of the expected size. Hybridizing the same blot with an ER-alpha riboprobe showed a strong hybridizing band at approximately 6.5 kb. In ovarian cancer samples, ER-beta mRNA level was decreased when compared to normal ovaries. Using 25 cycles of RT-PCR followed by Southern blotting, we found equal amounts of ER-alpha and -beta mRNAs in normal ovaries in all age groups from 33 to 75 years; however, in ovarian cancer tissue, the level of ER-alpha mRNA was similar or slightly higher, comparable to 10(3) to 10(4) copies of plasmid DNA, but ER-beta mRNA levels were markedly decreased. Granulosa cells from IVF patients expressed high levels of ER-beta mRNA. The OSE cell line expressed a low level of ER-alpha, detectable after 40 cycles of RT-PCR and no ER-beta mRNA. SKOV3 showed a low level of ER-alpha and -beta mRNAs, whereas OCC1 showed a low level of ER-beta and a relatively high level of ER-alpha. HEY did not contain detectable amounts of either ER after 40 cycles of RT-PCR. We found no evidence of differential splicing or major deletions in almost the entire coding region of ER-beta in either normal ovaries or tumor samples.
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PMID:Estrogen receptor alpha (ER-alpha) and beta (ER-beta) mRNAs in normal ovary, ovarian serous cystadenocarcinoma and ovarian cancer cell lines: down-regulation of ER-beta in neoplastic tissues. 950 68

Increasing evidence suggests that estrogen deficiency in women promotes the expansion of populations of bone marrow cells that differentiate into osteoclasts under the influence of osteotropic hormones and local factors. A progressive cytoplasmic accumulation of osteoclastic bone resorbing enzymes, such as tartrate-resistant acid phosphatase (TRACP) and carbonic anhydrase II (CA II), characterizes osteoclast differentiation. To evaluate the possibility that estrogen may have a direct effect on osteoclast precursor cells, we investigated the mRNA levels of estrogen receptor a (ERa), TRACP and CA II genes in neonatal rat bone imprints by fluorescence in situ hybridization and confocal microscopy. Morphological assessment of bone imprints has shown that the putative mononuclear osteoclast precursor cells (MOPC) display strongly basophilic cytoplasm and a low nuclear/cytoplasmic ratio, while some of these cells possess pale-staining ruffled border regions similar to those observed in osteoclasts. Both CA II and TRACP mRNAs were detected in putative MOPC as well as multinuclear osteoclasts. The gene transcripts were mainly located in the cytoplasm of these cells. To determine whether these putative MOPC possess ER mRNA, a 637 base pair antisense ER riboprobe was used. The results indicated that MOPC which show TRACP reactivity express high levels of ER gene transcripts in their cytoplasm. In contrast, only a few multinuclear osteoclasts in the bone imprints possessed ER gene transcripts. Interestingly, the levels of ER mRNA in these multinuclear osteoclasts were very low compared with those in the putative MOPC. Treatment with RNase prior to hybridization resulted in a significant loss of signal in these cells. The results of these studies suggest that estrogen may have a direct role in modulating the recruitment of osteoclast precursor cells during osteoclastogenesis.
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PMID:Detection of estrogen receptor alpha, carbonic anhydrase II and tartrate-resistant acid phosphatase mRNAs in putative mononuclear osteoclast precursor cells of neonatal rats by fluorescence in situ hybridization. 958 36

To gain insight into the possible biological role of variant estrogen receptor (ER) expression in human breast cancer, we have undertaken a study to determine if the expression of the clone 4 variant ER mRNA was associated with markers of either reduced endocrine sensitivity [i.e., progesterone receptor (PgR) negativity] or a poor prognosis (node positivity, large tumor size, and high percentage S-phase fraction). mRNA levels of clone 4 variant ER and wild-type (WT) ER were assayed by RNase protection assay in 106 breast cancer specimens. The tumors comprised two major groups: "good" prognosis and "poor" prognosis based on several conventional biological prognostic features. Each group was divided into three subgroups (ER+/PgR+, ER+/PgR-, and ER-/PgR-). WT and clone 4 variant ER mRNAs were undetected in ER-/PgR- tumors. We determined that clone 4 variant ER mRNA levels varied proportionately with WT mRNA levels, and regression analysis was used to determine if the amount of clone 4 variant ER mRNA relative to WT was associated with prognosis or PgR content. Significantly higher levels of clone 4 variant ER mRNA relative to WT were found in tumors with markers of poor prognosis compared to those with markers of good prognosis (P = 0.0004). Significantly higher levels of clone 4 variant ER mRNA relative to WT were found in PgR- tumors compared to PgR+ tumors (P = 0.011). Such data are consistent with an association of clone 4 variant ER mRNA expression with progression of human breast cancer from hormone dependence to independence.
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PMID:Relationship of clone 4 estrogen receptor variant messenger RNA expression to some known prognostic variables in human breast cancer. 981 68

Estradiol imprinting plays an important role in the regulation of galanin (GAL) gene expression in the rat. In the anterior pituitary gland, GAL gene expression is greatly induced by estrogen. The relative involvement that the two estrogen receptor subtypes, alpha and beta, have in regulating this induction is not known. We have utilized ER alpha-knock-out (ER alphaKO) mice to discriminate the roles of ER alpha and ER beta in the regulation of GAL gene expression in the anterior pituitary gland. Our goals were to measure the effects of estradiol on GAL gene expression by solution hybridization ribonuclease protection assay in wild-type mice and to determine the roles of ER alpha and, indirectly, ER beta by measuring the same response in the ER alphaKO mice. Estradiol treatment for one week elevated GAL gene expression 30-40 fold in the wild-type mouse pituitary. Evaluation of estrogen effects on GAL gene expression in the anterior pituitary of ER alphaKO animals revealed that ER alpha is essential, because no response to estrogen was observed in these animals. Since ER beta mRNA was identified in the anterior pituitary by RT-PCR, but estrogen had no effects on GAL gene expression in the ER alphaKO mice, the beta subtype of ER does not appear to participate in estrogen-evoked GAL gene expression in the mouse anterior pituitary.
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PMID:Estradiol induces galanin gene expression in the pituitary of the mouse in an estrogen receptor alpha-dependent manner. 1034 50

Targeted disruption of the mouse estrogen receptor-alpha gene (estrogen receptor-alpha knockout; ERKO) results in a highly novel ovarian phenotype in the adult. The ERKO mouse model was used to characterize ER alpha-dependent processes in the ovary. Visualization of the ovaries of 10-, 20-, and 50-day-old wild-type (WT) and ERKO mice showed that the ERKO phenotype developed between 20 and 50 days of age. Developmental progression through the primordial, primary, and antral follicle stages appeared normal, but functional maturation of preovulatory follicles was arrested resulting in atresia or in anovulatory follicles, which in many cases formed large, hemorrhagic cysts. Corpora lutea were absent, which also indicates that the normal biochemical and mechanical processes that accomplish ovulation were compromised. Northern and ribonuclease protection analyses indicated that ERKO ovary FSH receptor (FSHR) messenger RNA (mRNA) expression was approximately 4-fold greater than in WT controls. Ovarian LH receptor (LHR) mRNA expression was also higher in the ERKO animals. Cellular localization studies by in situ hybridization analysis of ERKO ovaries showed a high level of LHR mRNA expression in the granulosa and thecal layers of virtually all the antral follicles. Ribonuclease protection analyses showed that ovarian progesterone receptor and androgen receptor mRNA expression were similar in the two groups. These results indicated that ER alpha action was not a prerequisite for LHR mRNA expression by thecal or granulosa cells or for ovarian expression of progesterone receptor mRNA. Ovarian estrogen receptor beta (ER beta) was detected immunohistochemically, was sharply compartmentalized to the granulosa cells, and was expressed approximately equally in the ERKO animals and the WT controls. In contrast, ER alpha staining was present in the thecal cells but not the granulosa cells of the WT animals. The summary findings indicate that in the adult the major cause of the ERKO phenotype is high circulating LH interacting with functional LHR of the theca and granulosa cells. These features result in a failure of the normal maturational events leading to successful ovulation and luteinization and presumably involve both hypothalamic-pituitary and intraovarian mechanisms dependent upon ER alpha action. The presence of ER beta in the granulosa cells did not rescue the phenotype of the ovary.
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PMID:Targeted disruption of the estrogen receptor-alpha gene in female mice: characterization of ovarian responses and phenotype in the adult. 1034 64

Glucosidase I initiates the processing of asparagine (N-) linked glycoproteins by removing the distal alpha1,2-linked glucosyl residue of the tetradecasaccharide Glc(3)Man(9)GlcNAc(2). The gene encoding this enzyme was isolated and its structural organization and promoter activity determined. The major transcript for glucosidase I on northern blot appeared to be 3.1 kb; Southern blotting and DNA sequencing indicated the size of the gene to be 6.8 kb, comprising four exons separated by three introns. The first exon encodes the cytoplasmic tail and transmembrane domain; the fourth encodes the putative catalytic domain of the enzyme. Exon-intron junctions are flanked by consensus splice donor and acceptor sequences. Transcription initiation sites were mapped by primer extension, ribonuclease protection assay and RT-PCR analysis. Primer extension results showed multiple initiation sites at -150, -156, and -272 bp relative to the translation initiation codon ATG. Sequence analysis of 5' flanking region showed no canonical TATA box, a high GC content, Sp1 and ETF binding sites (typical of a housekeeping gene promoter). Also noteworthy, the promoter region contains several generic STAT factor binding sites, one nearly perfect, and two half GR binding elements. Other cis- acting elements recognized by transcription factors such as AP-2, NF-kappaB, estrogen receptor, and progesterone receptor (PR) were also present in the putative promoter region. To determine the promoter activity, a construct encompassing the region between -2114 to -5 bp of the putative promoter was ligated to the chloramphenicol acetyltransferase (CAT) reporter plasmid and transiently transfected into COS 7 cells. CAT assay results clearly show transcriptional activity of the promoter.
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PMID:Genomic organization and promoter activity of glucosidase I gene. 1040 45


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