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:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

[1,2,6,7-3H]Testosterone (250 muCi) was administered to castrated male rats; after 30 min a labelled testosterone-receptor protein complex with a pI of 5.1 was recovered from the pancreatic cytosol. A labelled testosterone-receptor complex with an identical pI was also extracted from the nuclear fraction of rat pancreas after incubation of minced pancreatic tissue with 0.1 muM-]1,2,6,7-3H]testosterone for 30 min at 37 degrees C. Studies in vitro showed that [1,2,6,7-3H]testosterone was bound to a receptor protein focusing at a pI of 5.1 and with a Kd of 2 nM and a number of binding sites of 4.7 fmol/mg of protein in castrated male rats. The testosterone-receptor complex sedimented at 3.5 S in high-salt sucrose-density gradients, was excluded from Sephadex G-200 and Ultragel ACA-34, was stable towards treatment with dextran-coated charcoal, was relatively sensitive to heat, and was stable to treatment with deoxyribonuclease and ribonuclease, but was sensitive to treatment which proteinase. It is suggested that the pancreatic androgen receptor, which was also present in castrated female rats, may play a role in sex-steroid regulation of pancreatic function.
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PMID:Demonstration of an androgen receptor in rat pancreas. 18 42

We have investigated the ability of dehydroepiandrosterone (DHEA) to alter the production of interleukin-2 (IL-2) and to bind to a specific binding complex in antiCD3 epsilon activated T cells. Binding activity correlated with the presence of a specific DHEA binding complex in the cytosol and nuclei of DHEA-responsive T-cell hybridomas, as well as in CD4+ and CD8+ cells isolated from peripheral lymph nodes of normal mice. Scatchard analysis determined that intact lymphocytes and cytosolic fractions contained high affinity binding for [3H]DHEA (approx. 2.6 nM) with 1000-7000 binding sites existing per cell. Five of the T-cell hybridomas tested both responded to DHEA treatment with increased production of IL-2 and also contained specific high affinity [3H]DHEA binding. Four additional T-cell hybridomas were found to contain no specific [3H]DHEA binding and were also unresponsive to DHEA influences on IL-2 production. Sucrose density gradients demonstrated a 3-4s [3H]DHEA binding complex in high salt and a 7-8s binding complex in low salt. Specific binding was inhibited by preincubation of the cytosol fractions with either trypsin or chymotrypsin, or by heating to 60 degrees C for 1 h (less than 15% of control). [3H]DHEA binding was unaffected by preincubation of the cytosol fractions with ribonuclease, deoxyribonuclease, or phospholipase A. The DHEA-protein complexes bound to DNA-cellulose with the amount of binding being slightly increased by preincubation at 25 degrees C as compared to 4 degrees C. As expected, [3H]DHEA binding was inhibited by the addition of unlabeled DHEA, but was also modestly inhibited by dihydrotestosterone and cortisol. Binding of DHEA was unaffected by progesterone, dexamethasone, estradiol, androsterone, DHEAS, and beta-etiocholanolone at all concentrations tested. DHEA was incapable of inhibiting the binding of [3H]DHT to the androgen receptor or [3H]dexamethasone to the glucocorticoid receptor. Collectively, these findings suggest that murine T cells contain a specific DHEA receptor. We believe that DHEA is a steroid hormone that is directly involved in the regulation of IL-2 production by both normal and some T-cell hybridomas.
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PMID:The presence of a dehydroepiandrosterone-specific receptor binding complex in murine T cells. 135 1

Much clinical evidence indicates that androgens have beneficial effects in the treatment of breast cancer in women. Physiological concentrations of androgens strongly inhibit both basal and estrogen-induced cell proliferation in the human breast cancer cell line ZR-75-1 through their interaction with the androgen receptor. The present study shows that androgens strongly suppress estrogen receptor (ER) and progesterone receptor contents in this model, as measured by radioligand binding and anti-ER monoclonal antibodies. Similar inhibitory effects are observed on the levels of ER messenger RNA (mRNA) measured by ribonuclease protection assay. The androgenic effect is observed at subnanomolar concentrations of the nonaromatizable androgen 5 alpha-dihydrotestosterone, regardless of the presence of estrogens, and is competitively reversed by the antiandrogen hydroxyflutamide. Such data on ER expression provide an explanation for at least part of the antiestrogenic effects of androgens on breast cancer cell growth and moreover suggest that the specific inhibitory effects of androgen therapy could be additive to the standard treatment limited to blockade of estrogens by antiestrogens.
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PMID:Down-regulation of estrogen receptors by androgens in the ZR-75-1 human breast cancer cell line. 266 Dec 9

Androgens are known to exert a variety of effects on an organism while follicle-stimulating hormone (FSH) seems to act specifically on the gonads. To investigate whether these effects are reflected by the expression pattern of the androgen receptor (AR) or the FSH receptor (FSHR) we screened 38 different tissues and organs of one intact and one castrated male non-human primate (Macaca fascicularis). By means of a highly sensitive ribonuclease protection assay (RPA) we demonstrated AR mRNA expression in all tissues of the intact monkey investigated. Immunohistochemistry of selected organs from this monkey revealed a good correlation between AR mRNA and protein expression. In the castrated monkey, the overall AR mRNA expression was markedly lower compared with the intact monkey, although higher expression was present in the pituitary, thyroid and prostate glands. FSHR mRNA was only detected in testicular tissue. This study has revealed, for the first time, ubiquitious expression of the AR mRNA in a non-human primate. The testis-specific expression of the FSHR highlights the importance of FSH for spermatogenesis with the testis being apparently the only target organ.
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PMID:Ubiquitous expression of the androgen receptor and testis-specific expression of the FSH receptor in the cynomolgus monkey (Macaca fascicularis) revealed by a ribonuclease protection assay. 757 19

A sensitive, solution-hybridization ribonuclease-protection assay (RPA) was established to quantify the expression of mRNA for the androgen receptor (AR) and follicle-stimulating hormone receptor (FSHR) in total RNA samples isolated from tissues of the cynomolgous monkey, human testes obtained from elderly patients undergoing orchidectomy because of prostatic carcinoma or from transsexual men undergoing gender reassignment as well as human cell lines DU 145, REP and RVP. Sensitivity experiments revealed that, in the human and monkey, 1-2 micrograms of total RNA were sufficient to achieve quantifiable signals of the different receptor mRNA species. Quantification of AR and FSHR mRNA levels showed a 1.7-fold higher expression of AR mRNA and a 2.4-fold higher expression of FSHR mRNA in the monkey testes compared to human testes from patients with prostatic carcinoma. Normal spermatogenesis in both human and monkey testes indicated no relationship between spermatogenic status and receptor expression. The significantly lower expression of AR and FSHR mRNA in humans than in monkeys might therefore be either age- or species-related. Quantification of mRNA for AR and FSHR in the testis of the transsexual patients undergoing oestrogen and antiandrogen treatment displayed a drastic increase (4.5-fold) in mRNA for the AR, whereas mRNA for the FSHR was barely detectable. Due to its high sensitivity, reproducibility and its ability to quantify mRNA transcripts, the RPA is a useful tool for investigating expression of low abundant receptor genes and their regulation when only very small amounts of tissue are available. Furthermore, it is suitable for use in clinical and experimental studies in which accurate quantification of transcripts is necessary.
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PMID:Quantification of androgen receptor and follicle-stimulating hormone receptor mRNA levels in human and monkey testes by a ribonuclease-protection assay. 766 15

Androgen insensitivity is an X-linked disorder of sexual differentiation resulting from mutations in the androgen receptor (AR) gene. In this paper, we report the clinical phenotype and molecular analysis of two siblings with severe partial androgen insensitivity due to a novel mutation in the ligand-binding domain of the AR gene. Binding studies using cultured genital skin fibroblasts demonstrated reduced AR affinity and binding capacity. Nucleotide sequence analysis of the AR gene of both siblings revealed a point mutation causing a glycine to arginine amino acid substitution at position 907 within a conserved region of the ligand-binding domain. A silent guanine to adenine substitution was also identified in the protein-coding region of exon 1. Using an expression vector in which the identified mutation was recreated by site-directed mutagenesis, the mutant receptor was found to have a reduced binding affinity (Kd = 3.06 nmol/L) for mibolerone compared with that of normal AR (Kd = 1.71 nmol/L) when expressed in COS-7 cells. In cotransfection experiments using CV-1 cells and a mouse mammary tumor virus-chloramphenicol acetyltransferase reporter system, the concentration of dihydrotestosterone required to induce half-maximal chloramphenicol acetyltransferase gene expression was 50-fold higher in cells transfected with the mutant AR complementary DNA than in cells transfected with normal AR complementary DNA. AR messenger ribonucleic acid levels in genital skin fibroblasts determined by both competitive PCR amplification and ribonuclease protection assay were decreased compared with normal values. Our studies demonstrate the importance of this region of the AR gene in normal AR function and AR gene expression.
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PMID:Partial androgen insensitivity caused by an androgen receptor mutation at amino acid 907 (Gly-->Arg) that results in decreased ligand binding affinity and reduced androgen receptor messenger ribonucleic acid levels. 855 Jul 58

Sertoli cell lines have been established from H-2K(b)-tsA58 transgenic mice carrying an inducible temperature-sensitive SV40 T antigen in their germline. All cell lines tested for expression of Sertoli cell products by reverse transcription-polymerase chain reaction were shown to express mRNAs for alpha-inhibin, Steel factor, SGP-2, and transferrin as well as for androgen receptor and the orphan nuclear receptor SF-1. Selected cell lines were shown by immunocytochemistry to express the established Sertoli cell-specific pattern of cytoskeletal markers. The FSH receptor gene was also expressed, though downregulated by comparison with in vivo levels of expression. In some lines low expression of the luteinizing hormone receptor gene could also be detected. The gene for the transcription factor GATA-1, which is expressed specifically in Sertoli cells, was expressed only in a subset of the cell lines. Quantitative analysis of SGP-2 transcript levels by ribonuclease protection assays showed an increase at the nonpermissive temperature, whereas using a similar assay, Steel factor mRNA was shown to be expressed in amounts comparable to the in vivo situation only in two cell lines during permanent growth. In summary, cell lines that exhibit distinct Sertoli cell characteristics have been established, which may resemble different stages of phenotypic development.
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PMID:Sertoli cell lines established from H-2Kb-tsA58 transgenic mice differentially regulate the expression of cell-specific genes. 866 Sep 30

gamma-Aminobutyric acid (GABA)ergic neurons terminating in the rostral hypothalamus are stimulated by testosterone. To investigate whether this action is mediated locally through androgen receptors in the rostral hypothalamus, bilateral microcannulas (28 gauge) containing the androgen receptor antagonist, hydroxyflutamide (HF), were stereotaxically implanted into the rostral medial preoptic area (rMPA) just dorsal to the major population of GnRH cell bodies. Two days later, blood samples were collected for assay of LH, and animals were killed for determination of GABAergic neuronal activity in tissue dissected from the site of the implanted cannulas. Animals were decapitated either without treatment or 60 min after inhibition of GABA degradation by aminooxyacetic acid (100 mg/kg, ip). The rate of GABA accumulation in the tissue after aminooxyacetic acid treatment was used as a measure of GABA turnover. Levels of messenger RNA for both forms of glutamic acid decarboxylase (GAD65 and GAD67), the rate-limiting enzyme responsible for GABA synthesis also were measured by a microlysate ribonuclease protection assay. LH levels were significantly increased (1.8-fold) in HF-treated animals compared with controls. In the MPA, beneath the implant cannulas, GABA turnover was significantly reduced in HF-treated rats. There was no effect of treatment in the frontal cortex, which was used as a control region. Surprisingly, levels of messenger RNA for both GAD65 and GAD67 were significantly increased in HF-treated rats. The results indicate that GABAergic neurons terminating in the rostral hypothalamus are tonically stimulated by testosterone acting by means of androgen receptors localized in this region. These findings support the working hypothesis that androgen-sensitive GABAergic neurons in the rMPA mediate the negative feedback action of testosterone on GnRH secretion in the male rat.
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PMID:Antiandrogen microimplants into the rostral medial preoptic area decrease gamma-aminobutyric acidergic neuronal activity and increase luteinizing hormone secretion in the intact male rat. 882 73

In rat ovary, androgen receptor (AR) is predominantly expressed in granulosa cells and is developmentally regulated. However, the exact mechanism that is responsible for the regulation of AR in granulosa cells has not been elucidated. The aim of this study was to examine 1) the levels of AR messenger RNA (mRNA) expression in granulosa cells from follicles of different size and 2) the effects of FSH, 8-bromo-cAMP, androgen, and estrogen on AR mRNA levels in granulosa cells in vitro. The abundance of AR mRNA was examined by ribonuclease protection assay using 32P-labeled AR complementary RNA probe and related to that of P450aromatase (P450arom) mRNA, a well established maker of granulosa cell differentiation. In large follicles (> 400 microns in diameter), the abundance of AR mRNA was decreased to 51% of that in small follicles (< 200 microns; P < 0.01), whereas the abundance of P450arom mRNA increased to 277% (P < 0.01). In medium follicles (200-400 microns), the abundance of AR mRNA was maintained (101%), whereas the abundance of P450arom mRNA increased to 202% of that in small follicles (P < 0.05). Treatment with FSH (0-300 ng/ml) or 8-bromo-cAMP (0-4 mM) induced P450arom mRNA in the cultured granulosa cells in a dose-dependent manner; however, it did not affect the levels of AR mRNA expression. Treatment with 5 alpha-dihydrotestosterone (1 microM) resulted in a significant reduction in the abundance of AR mRNA to 67% of the control value (P < 0.05). This effect was reversed by the addition of FSH (100 ng/ml; P < 0.01). Treatment with diethylstilbestrol (1 microM), alone or in combination with FSH (100 ng/ml), did not have any significant effect, although these treatments tended to decrease the abundance of AR mRNA to 81% and 85%, respectively. Both 5 alpha-dihydrotestosterone and diethylstilbestrol dramatically enhanced the abundance of FSH-induced P450arom mRNA compared to the effect of FSH alone. These results indicate that 1) the down-regulation of AR mRNA expression takes place in granulosa cells of preovulatory follicles; 2) FSH is not directly responsible for this event; and 3) androgen down-regulates AR mRNA expression in immature granulosa cells, and this effect is reversed by FSH. We conclude that androgen and FSH jointly regulate AR mRNA expression in rat granulosa cells.
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PMID:Androgen receptor gene expression in rat granulosa cells: the role of follicle-stimulating hormone and steroid hormones. 882

The expression of the androgen receptor in the human epididymis was analysed by ribonuclease protection, in-situ hybridization and immunohistochemistry. Androgen receptor mRNA and protein could be detected throughout the entire organ, albeit in different quantities, in the caput, corpus and cauda regions, respectively. Also positive, though only weakly, was the ductus deferens, while the efferent ducts were devoid of specific signals. In-situ transcript hybridization and immunocytochemistry localized androgen receptor mRNA and protein primarily to the epithelium of the epididymal duct. In the ductal epithelial cells androgen receptor immunoreactivity showed a distinct nuclear distribution. While peritubular cells occasionally displayed weak signals, interstitial cells as well as blood vessels were consistently negative throughout the entire organ. The observed pattern of androgen receptor expression in the human epididymis supports the notion that the structure and function of the epididymis is differentially controlled by androgens in a region-specific manner, whereas it would not seem compatible with a direct role for androgens in the regulation of epididymal blood flow.
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PMID:Region-specific expression of the androgen receptor in the human epididymis. 943 17


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