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)

Glucocorticoid-specific binding macromolecules were measured and characterized in L2C cells, a B-lymphocyte guinea pig leukemia that is resistant to administered cortisol or adrenocorticotrophic hormone. L2C cells were harvested by cardiac puncture followed by Ficoll:Hypaque separation techniques. The cells were lysed by sonication, and the cytosol was obtained after centrifugation at 106,000 x g for 60 min at 0 degrees. Cytosol glucocorticoid receptor measurements were obtained by hydroxylapatite assay or column chromatography using Sephadex G-25. Maximal specifically bound [3H]triamcinolone acetonide in the cytosol fraction was 300 fmol/10(8) cells. Scatchard plot of specific L2C cytosol binding gave a Kd of 18 nM and an estimate of 2000 binding sites/cell. The specificity of binding in L2C cytosol was triamcinolone acetonide > dexamethasone > cortisol > progesterone > testosterone = estradiol. Binding of [3H]triamcinolone acetonide to cytosol glucocorticoid receptors was maximal at 22 hr of incubation at 19 degrees, and the receptor complex was stable for 48 hr. The receptor complex was not affected by DNase or RNase, but the receptor complex was lost with pronase or heat denaturation. Whole-cell binding assays were also performed, which resulted in similar quantities of maximally bound glucocorticoid receptor as well as the specificity of binding of various hormone analogs as found in the cytosol receptor assays. Transferring the whole cells from 0 to 22 degrees resulted in the appearance in nuclei of approximately 65% bound receptors. However, these translocated receptor complexes do not appear to affect the viability of the L2C cells as measured by trypan blue exclusion.
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PMID:Characterization of glucocorticoid-specific binding in the L2C leukemia. 693 47

Cytosols of whole testicular homogenates from the Syrian golden hamster contained specific binding sites for [3H]triamcinolone acetonide that exhibited limited capacity and high affinity binding characteristic of glucocorticoid receptors in other target tissues. The receptor complex sedimented as an 8.6S binder in low salt 5-20% linear sucrose gradients and as 6.2S and 4.0S moieties in 0.15M and 0.4 M KCl, respectively. The Ka at equilibrium was 3.1-3.3 X 10(9) M-1 at 4 C in intact and adrenalectomized males. The testicular glucocorticoid binder was vulnerable to proteolytic degradation while being completely resistant to the action of RNase and DNase. In addition the binding protein exhibited the usual steroid specificities for type I glucocorticoid receptor: triamcinolone acetonide greater than dexamethasone greater than cortisol greater than corticosterone greater than progesterone greater than aldosterone greater than prednisone greater than 5 alpha-dihydrotestosterone greater than diethylstilbestrol. Unexpectedly, 17 beta-estradiol competed for receptor binding to the same extent as prednisone. A 3.2 S nuclear receptor was extracted from purified testicular nuclei after incubation of whole suspensions in culture media containing 5 nm radiolabeled triamcinolone acetonide at 32 C. Although the glucocorticoid receptor concentrations in prepubertal, adrenalectomized, and hypophysectomized animals were markedly higher in the testis compared to the concentration in the normal adult hamster (52 +/- 4 fmol/mg cytosol protein), the greatest total amount of receptor per testis was found in the mature intact animal. Moreover, under the conditions studied, the concentration of glucocorticoid receptor substantially exceeded the levels of either androgen or estrogen receptor when determined simultaneously. In contrast, no measurable cytoplasmic [3H]triamcinolone acetonide binding was detected in adjacent urogenital organs such as the epididymis and seminal vesicle. It is therefore unlikely that the testicular glucocorticoid receptor is associated with the spermatid or present as a secretory product in the seminiferous tubule lumen.
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PMID:Characterization of specific glucocorticoid receptor in the Syrian hamster testis. 729 80

The effects of long term treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) on estrogen receptor (ER) expression in the human breast cancer cell line, MCF-7, were studied. This study demonstrates that treatment of cells with the phorbol ester blocked estrogen receptor activity. Treatment of cells with 100 nM TPA resulted in an 80% decrease in the level of ER protein and a parallel decrease in ER mRNA and binding capacity. Following removal of TPA from the medium, the level of ER protein and mRNA returned to control values; however, the receptor failed to bind estradiol. These cells also failed to induce progesterone receptor in response to estradiol. In addition, TPA treatment blocked transcription from an estrogen response element in transient transfection assays and inhibited ER binding to its response element in a DNA mobility shift assay. The estrogen receptor in treated cells was recognized by two monoclonal anti-ER antibodies and was not quantitatively different from ER in control cells. RNase protection analysis failed to detect any qualitative changes in the ER mRNA transcript. Mixing experiments suggest that TPA induces/activates a factor which interacts with the ER to block binding of estradiol. The effects of TPA on ER levels and binding capacity were concentration-dependent. Low concentrations of TPA inhibited estradiol binding without a decrease in the level of protein, whereas higher concentrations were required to decrease the level of ER protein. The effects of TPA appear to be mediated by activation of protein kinase C since the protein kinase C inhibitors, H-7 and bryostatin, block the effects of TPA on estradiol induction of progesterone receptor. TPA treatment had no effect on the level or binding capacity of the glucocorticoid receptor, indicating that the effects are not universal for steroid receptors. These data demonstrate that activation of the protein kinase C signal transduction pathway modulates the estrogen receptor pathway. The long term effect of protein kinase C activation is to inhibit estrogen receptor function through induction/activation of a factor which interacts with the receptor.
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PMID:Effects of 12-O-tetradecanoylphorbol-13-acetate on estrogen receptor activity in MCF-7 cells. 755 63

The effects of different steroids on the expression of angiotensin AT1 receptors by the human hepatoma cell line, PLC-PRF-5 was studied. Dexamethasone and aldosterone decreased the specific binding of [3H]angiotensin II to intact PLC-PRF-5 cells by 57 +/- 4% and 54 +/- 2%, respectively, compared to control, untreated cells. EC50 values for dexamethasone, cortisol and aldosterone were 1.8 +/- 0.6, 40 +/- 6, and 310 +/- 20 nM, respectively, suggesting that these effects were mediated via a glucocorticoid receptor. Scatchard analysis revealed that dexamethasone decreased the number of angiotensin AT1 receptors expressed (50 +/- 4% relative to control) with no change in receptor affinity. Treating cells with dexamethasone in the presence of either an angiotensin converting enzyme inhibitor or an angiotensin II receptor antagonist did not prevent the reduction in angiotensin AT1 receptor expression, ruling out a mechanism involving a dexamethasone induced increase in endogenous angiotensin II production. A ribonuclease protection assay established that the steady state level of angiotensin AT1 receptor mRNA in dexamethasone treated cells was reduced to 34.7 +/- 8.4% of untreated cells. The decrease in the number of angiotensin AT1 receptors expressed on the cell surface after treatment with dexamethasone therefore seems likely to reflect the decreased steady state level of the mRNA coding for this receptor.
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PMID:Glucocorticoids regulate the expression of angiotensin AT1 receptors, in the human hepatoma cell line, PLC-PRF-5. 777 81

Using deletion analysis and site-specific mutagenesis to map the 5' regulatory region of the DNA methyltransferase (MeTase) gene, we show that a 106-bp sequence (at -1744 to -1650) bearing three AP-1 sites is responsible for induction of DNA MeTase promoter activity. Using transient cotransfection chloramphenicol acetyl-transferase assays in P19 cells, we show that the DNA MeTase promoter is induced by c-Jun or Ha-Ras but not by a dominant negative mutant of Jun, delta 9. The activation of the DNA MeTase promoter by Jun is inhibited in a ligand dependent manner by the glucocorticoid receptor. Stable expression of Ha-Ras in P19 cells results in induction of transcription of the DNA MeTase mRNA as determined by nuclear run-on assays and the steady state levels of DNA MeTase mRNA as determined by an RNase protection assay. These experiments establish a potential molecular link between nodal cellular signaling pathways and the control of expression of the DNA MeTase gene. This provides us with a possible molecular explanation for the hyperactivation of DNA MeTase in many cancer cells and suggests that DNA MeTase is one possible downstream effector of Ras.
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PMID:Regulation of the DNA methyltransferase by the Ras-AP-1 signaling pathway. 782 90

The hippocampus plays an important role in mediating glucocorticoid effects on the brain. Glucocorticoids are also implicated in neurogenesis and age-related neuronal death in the hippocampus. The effects of glucocorticoids in the hippocampus are elicited through two receptors with high-affinity for corticosterone, the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). In this study, we used a sensitive RNase protection assay to quantify the ontogeny of GR mRNA and MR mRNA in hippocampus from embryonic day 18 (E18) to postnatal day 60 (P60). GR mRNA and MR mRNA are expressed at approximately equal levels in the E18 hippocampus. However, by birth, the level of MR mRNA is three-fold that of GR mRNA and remains elevated up to P60. The levels of both mRNAs increase gradually during the period of postnatal neurogenesis after which they markedly increase to adult levels. In addition, the levels of hippocampal MR mRNA are the same in male and female rats, whereas the levels of GR mRNA are significantly higher in the P60 female rat hippocampus, but not in younger female rats. Our data on the development of mRNA levels do not parallel the levels of glucocorticoid and mineralocorticoid receptors as reported in a number of binding studies. Therefore, our studies, when considered together with previous reports, suggest that posttranscriptional mechanisms play a major role in regulating the levels of glucocorticoid-binding sites in the hippocampus.
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PMID:Development of mRNAs for glucocorticoid and mineralocorticoid receptors in rat hippocampus. 817 25

We have cloned several novel sequences upstream from the first coding exon of the rat glucocorticoid receptor (GR) mRNA using PCR. Analysis of these sequences in RNase protection assays showed that one of the cloned sequences represents the major GR 5' non-coding exon which is expressed in all tissues studied, both at different stages of development and under different hormonal conditions. This major exon is homologous to the human GR 5' untranslated region (UTR). Three other sequences were cloned, but could not be detected in the RNase protection assay, suggesting that they are only minor transcripts, at least under the varying conditions of GR expression studied. One of these sequences is identical to a previously described rat GR cDNA sequence, while another was shown to be contiguous with the rat genomic DNA sequence.
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PMID:Heterogeneity in the 5' untranslated region of the rat glucocorticoid receptor mRNA. 824 Sep 87

Regulation of glucocorticoid receptor (GR) expression by its cognate ligand was examined in the glucocorticoid-sensitive human leukemic T-cell line 6TG1.1 and in the human B-cell line IM-9. In contrast to the decrease in GR mRNA seen in IM-9 cells after treatment with 1 microM dexamethasone for 16-18 h, treatment of 6TG1.1 cells resulted in an 8-fold increase in GR mRNA, as determined by Northern blot and RNase protection analysis, with a corresponding 3- to 4-fold increase in GR protein. Half-maximal induction of GR mRNA and protein in 6TG1.1 cells was observed between 10-100 nM dexamethasone, and inclusion of 1 microM RU 38486 completely blocked the effects of 100 nM dexamethasone, demonstrating that positive autoregulation of GR expression in 6TG1.1 cells is a receptor-mediated response. Positive autoregulation of GR expression was also observed in glucocorticoid-resistant CEM-C1 cells, which contain functional GR, but whose growth is unaffected by glucocorticoids. Thus, positive autoregulation is neither a consequence nor the sole cause of growth arrest. The degree of negative autoregulation in IM-9 cells and positive autoregulation in 6TG1.1 cells was unaffected by inhibition of protein synthesis with cycloheximide. Measurement of GR mRNA turnover in 6TG1.1 cells treated with actinomycin-D revealed a half-life of 2.5 h, which was unaffected by dexamethasone treatment. A similar half-life was determined in IM-9 cells and was also unaffected by steroid treatment. These results are consistent with the interpretation that glucocorticoid-mediated autoregulation of GR expression is a tissue-specific primary transcriptional response.
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PMID:Differential autoregulation of glucocorticoid receptor expression in human T- and B-cell lines. 831 74

The establishment of transformation of primary baby rat kidney epithelial cells by human papillomavirus type 16 DNA requires glucocorticoid hormones (Pater et al., Nature (Lond.), 335: 832-835, 1988). In this report we provide evidence that growth of transformed baby rat kidney cells in culture also requires glucocorticoids. However, transformed cells for which growth does not require hormone readily arise after a brief period of crisis, if cultured without added hormone. No reduction of glucocorticoid receptor was evident in non-hormone-requiring cells. The expression of human papillomavirus 16 RNA in these cells was analyzed by Northern blot, primer extension, and RNase protection analysis. Cells that do not require hormone had greatly reduced levels of transcripts initiated from the viral P97 promoter. However, there is evidence for compensating alterations to allow more efficient expression of E7 mRNA, since the growth of these cells is correlated with altered patterns of viral RNA expression and processing.
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PMID:Glucocorticoid requirement for growth of human papillomavirus 16-transformed primary rat kidney epithelial cells: correlation of development of hormone resistance with viral RNA expression and processing. 839 83

Recent studies have shown that corticotropin releasing factor (CRF) stimulates c-fos gene expression in the AtT-20 corticotroph cell line, and that overexpression of c-Fos results in activation of POMC gene transcription. Since transactivation by c-Fos requires dimerization with a Jun family member to form the active transcription factor AP-1, we have examined the expression of multiple fos and jun related genes and have correlated their expression with AP-1 DNA binding activity in AtT-20 nuclear extracts after stimulation with CRF. Although basal expression of c-fos mRNA was extremely low, it was rapidly and transiently stimulated in AtT-20 cells following administration of either constant or a single pulse of CRF. In contrast, basal expression of c-jun mRNA was slightly higher and underwent little or no change in response to CRF. Specific ribonuclease protection analysis showed that in addition to c-fos, mRNA transcripts encoding fos B and jun B were rapidly stimulated in response to CRF, though levels of induced fos B mRNA were 20-40 times lower than c-fos or jun B, respectively. Gel shift analysis demonstrated that CRF caused a sustained increase in AP-1 DNA binding to both a canonical AP-1 element as well as to the POMC exon-1 AP-1 site. Studies with specific antisera directed against c-Fos revealed that although no c-Fos could be detected in AP-1 complexes in basal cell extracts, c-Fos became a prominent component of AP-1 following CRF stimulation, reaching maximal levels by 4 h. Despite the fact that AP-1 DNA binding activity remained elevated for at least 24 h after CRF, c-Fos was most prominent during the early phase of the response. Similarly, JunB was shown to be a major component of AP-1 DNA binding activity in CRF-stimulated AtT-20 nuclear extracts that persisted for at least 24h after stimulation. Despite the obvious induction of fos B mRNA in response to CRF, FosB protein was not detected in DNA bound AP-1 complexes. These data demonstrate that CRF is a potent stimulus for corticotroph expression of c-fos, jun B and fos B, and suggest that the subsequent increase in AP-1 may play a role in activation of gene expression and/or as a modulator of glucocorticoid receptor function.
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PMID:CRF stimulates expression of multiple fos and jun related genes in the AtT-20 corticotroph cell. 879 51


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