Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.5 (RNase)
 17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ovarian-derived estradiol plays a critical endocrine role in the regulation of gonadotropin synthesis and secretion from the hypothalamic-pituitary axis. In turn, several para/autocrine effects of estrogen within the ovary are known, including increased ovarian weight, stimulation of granulosa cell growth, augmentation of FSH action, and attenuation of apoptosis. The estrogen receptor-alpha (ERalpha) is present in all three components of the hypothalamic-pituitary-ovarian axis of the mouse. In contrast, estrogen receptor-beta (ERbeta) is easily detectable in ovarian granulosa cells but is low to absent in the pituitary of the adult mouse. This distinct expression pattern for the two ERs suggests the presence of separate roles for each in the regulation of ovarian function. Herein, we definitively show that a lack of ERalpha in the hypothalamic-pituitary axis of the ERalpha-knockout (alphaERKO) mouse results in chronic elevation of serum LH and is the primary cause of the ovarian phenotype of polycystic follicles and anovulation. Prolonged treatment with a GnRH antagonist reduced serum LH levels and prevented the alphaERKO cystic ovarian phenotype. To investigate a direct role for ERalpha within the ovary, immature alphaERKO females were stimulated to ovulate with exogenous gonadotropins. Ovulatory capacity in the immature alphaERKO female was reduced compared with age-matched wild-type (14.5+/-2.9 vs. 40.6+/-2.6 oocytes/animal, respectively); however, oocytes collected from the alphaERKO were able to undergo successful in vitro fertilization. A similar discrepancy in oocyte yield was observed after superovulation of peripubertal (42 days) wild-type and alphaERKO females. In addition, ovaries from immature superovulated alphaERKO females possessed several ovulatory but unruptured follicles. Investigations of the possible reasons for the reduced number of ovulations in the alphaERKO included ribonuclease protection assays to assess the mRNA levels of several markers of follicular maturation and ovulation, including ERbeta, LH-receptor, cyclin-D2, P450-side chain cleavage enzyme, prostaglandin synthase-2, and progesterone receptor. No marked differences in the expression pattern for these mRNAs during the superovulation regimen were observed in the immature alphaERKO ovary compared with that of the wild-type. Serum progesterone levels just before ovulation were slightly lower in the alphaERKO compared with wild-type. These studies indicate that treatment of alphaERKO females with a GnRH antagonist decreased the serum LH levels to within the wild-type range and concurrently prevented development of the characteristic ovarian phenotype of cystic and hemorrhagic follicles. Furthermore, a lack of functional ERalpha within the ovary had no effect on the regulation of several genes required for follicular maturation and ovulation. However, the reduced numbers of ovulations following the administration of exogenous gonadotropins in the alphaERKO suggests an intraovarian role for ERalpha in follicular development and ovulation.
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PMID:Prevention of the polycystic ovarian phenotype and characterization of ovulatory capacity in the estrogen receptor-alpha knockout mouse. 1057 51

Growth of ovarian Graafian follicles and cytodifferentiation of granulosa and theca cells are regulated by gonadotropins, sex steroids and peptidyl growth factors. For example insulin and intraovarian insulin-like growth factor type I (IGF-I) may amplify the actions of both follicle stimulating hormone (FSH) and luteinizing hormone (LH) in promoting biochemical luteinization and enhancing steroidogenesis. To explore further the notion of interactions between insulinomimetic peptides and LH and to examine the associated mechanisms, we have established porcine granulosa cells in monolayer culture for 48 h in 3% serum with insulin (1 microg/ml), estradiol (0.5 microg/ml), and follicle stimulating hormone (FSH, 5 ng/ml) to allow cell anchorage, facilitate in vitro cytodifferentiation and confer LH responsiveness. To limit any carry-over effects of serum, granulosa cells were stabilized overnight in serum-free medium. Studies were then initiated to assess the impact of insulin on the dose-responsive actions of LH. A maximally effective concentration of insulin (1 microg/ml) synergistically augmented LH's dose-dependent ampilification of progesterone and cAMP accumulation; viz. by approximately twofold (progesterone) and approximately 2.5-fold (cAMP) above that observed in maximally LH-stimulated cultures (P < 0.001). Mechanistically, insulin significantly enhanced the sensitivity of granulosa cells to LH's drive of cAMP accumulation [ED50 for LH 61 +/- 14 ng/ml (control) vs. 10 +/- 1.0 ng/ml (insulin) (P < 0.01)]. Insulin also augmented the maximal stimulatory effect of LH; i.e. LH efficacy rose from 6.5 +/- 0.4 to 17 +/- 1.4 (pmole cAMP/microg DNA/48 h; P < 0.001). Insulin dose-response analysis showed that insulin alone minimally elevated basal, but significantly heightened LH's stimulation of progesterone and cAMP accumulation at (insulin) concentrations as low as 3-10 ng/ml. The molecular mechanisms underlying insulin and LH's synergy were assessed by RNase protection assays with (porcine) cRNA probes encoding the low density lipoprotein receptor (LDL-R), Steroidogenic Acute Regulatory Protein (StAR), P450 cholesterol sidechain cleavage enzyme (P450scc) and (as a possible negative control) Sterol Carrier Protein 2 (SCP-2) [data normalized to constitutive 18S rRNA]. Non linear least-squares analysis was applied to confirm or refute an hypothesis of interactive synergy between LH and insulin on gene expression. LH and insulin alone exerted no effect on StAR message accumulation, and LH alone minimally stimulated P450scc and LDL-R mRNA's accumulation at 48 h. In contrast, insulin in combination with LH augmented StAR mRNA concentrations by approximately 5-10-fold and stimulated LDL-R message levels by threefold above the respective maximally LH-driven values (P < 0.01). Maximal P450scc mRNA expression was enhanced twofold by cotreatment with LH and insulin compared with maximal LH-treated cultures. In contrast SCP-2 mRNA accumulation remained unaffected by any treatment. In summary, we have used a serum-free, in vitro differentiated porcine granulosa cell culture system to assess regulatory interactions between the disparate first messengers, LH and insulin. We observe marked LH-insulin steroidogenic synergy after 48 h of joint hormonal stimulation, and further clarify that the mechanism(s) of synergy include augmentation of cAMP production and increased steady-state concentrations of transcripts of key sterol-regulatory genes; namely, LDL-R, StAR, and P450scc, but not SCP-2. Since the encoded products of these genes variously control sterol substrate uptake, delivery to and utilization in mitochondrial steroidogenesis, we speculate that the concerted actions of insulin-like peptides and LH may contribute to steroidogenic differentiation during the later stages of follicular maturation and the granulosa-luteal cell transition.
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PMID:Mechanisms underlying the steroidogenic synergy of insulin and luteinizing hormone in porcine granulosa cells: joint amplification of pivotal sterol-regulatory genes encoding the low-density lipoprotein (LDL) receptor, steroidogenic acute regulatory (stAR) protein and cytochrome P450 side-chain cleavage (P450scc) enzyme. 1068 49

Altered expression of hepatic CYP2E1 by xenobiotic or physiological stimuli is largely mediated through post-transcriptional mechanisms that may include altered CYP2E1 mRNA translation and/or protein degradation. Examination of the polyribosomal distribution of rat hepatic P450 mRNAs indicated that, whereas nearly all of the CYP2B, CYP3A, and CYP4A mRNAs were recovered in the polysomal fractions, indicating active translation, approximately 30-40% of CYP2E1 mRNA was not associated with polysomes and therefore not actively engaged in protein synthesis. To examine the CYP2E1 mRNA molecule for sequences that might affect its translational efficiency, a series of CYP2E1 recombinant RNAs (rcRNAs) with modified 5' or 3' untranslated regions (UTRs) was translated in vitro using the rabbit reticulocyte lysate system. Deletion of most of the CYP2E1 5' UTR, which was predicted to contain secondary structure, increased in vitro CYP2E1 protein synthesis. Polysomal distribution analyses of 5'-modified rcRNAs demonstrated that, as seen for hepatic CYP2E1 mRNA, a substantial fraction of each CYP2E1 rcRNA was not associated with polysomes. The polysomal distribution analyses of the CYP2E1 rcRNAs also confirmed that the observed changes in CYP2E1 protein synthesis were associated with altered ribosomal loading. Deletion of the poly(A) tail, and partial or complete deletion of the 3' UTR, decreased CYP2E1 protein synthesis. These changes in protein synthesis were accompanied by increased degradation of the CYP2E1 rcRNAs. Incubation with translational inhibitors, but not increased levels of RNase inhibitor, decreased the degradation of the rcRNAs during in vitro translation. In conclusion, these studies suggest that secondary structure in the 5' UTR of CYP2E1 mRNA is at least partially responsible for the inefficient translation of this mRNA. The poly(A) tail and sequences contained within the 3' UTR appear to be important for protecting CYP2E1 mRNA from RNase activity associated with the translation machinery.
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PMID:Post-transcriptional regulation of rat CYP2E1 expression: role of CYP2E1 mRNA untranslated regions in control of translational efficiency and message stability. 1072 4

Reverse transcriptase-polymerase chain reaction was used to amplify a partial cDNA from rabbit lung mRNA that shared 77% protein sequence identity with the mouse pregnane X receptor (PXR). Rapid amplification of cDNA ends from a rabbit kidney lambdaZAP expression library resulted in the isolation of overlapping cDNAs spanning the complete coding sequence. The deduced amino acid sequence of 411 residues exhibited 79% overall amino acid identity with human PXR and 77% identity with mouse PXR. Based on this protein sequence relationship and a similar degree of conservation exhibited by the mouse and human PXR orthologs, the cDNA appears to encode the rabbit PXR ortholog. 5'-rapid amplification of cDNA ends performed on an adaptor-ligated cDNA library from rabbit liver revealed the presence of an alternate mRNA, which differed at the 5'-terminus. RNase protection assays indicated that the alternate mRNA was expressed at >50-fold lower levels in rabbit kidney and liver. Rifampicin treatment of CV-1 cells cotransfected with a rabbit PXR expression plasmid and a luciferase reporter construct containing two copies of the DR3 enhancer from CYP3A23 produced a 6-fold induction of luciferase activity. In contrast, rat PXR was not responsive to this antibiotic under the same conditions. Pregnenolone 16alpha-carbonitrile was an efficacious activator of rat PXR, but failed to significantly activate rabbit PXR at equivalent concentrations. These results indicate that the ligand activation profile of rabbit PXR is distinct from rat PXR and more closely resembles that of human PXR. The rabbit PXR activation profile is consistent with the cytochrome P450 (P450) 3A6 induction profile in rabbits.
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PMID:Rabbit pregnane X receptor is activated by rifampicin. 1077 31

Adrenocortical carcinoma manifesting pure hyperaldosteronism is extremely rare. We report here a 61-year-old woman with biochemically proven primary aldosteronism due to right adrenocortical carcinoma. Computed tomographic scan showed 4.5x5.3 cm lobulated mass with tiny calcification, while there was no significant uptake of 131I-iodomethyl norcholesterol in the tumor. Immunohistochemical analysis demonstrated expression of steroidogenic enzymes in the tumor tissue: P-450scc, P-45c21, 3beta-hydroxysteroid dehydrogenase, P450(17alpha), and P-450(11beta). In addition, we could demonstrate mRNA expression of aldosterone synthase (P-450aldo:CYP11B2) in the tumor by specific ribonuclease protection assay. This is the first report of a case of primary aldosteronism due to adrenocortical carcinoma, in which expression of all sets of steroidogenic enzymes required for aldosterone synthesis was proven.
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PMID:Adrenocortical carcinoma manifesting pure primary aldosteronism: a case report and analysis of steroidogenic enzymes. 1080 Jul 65

We demonstrated previously that testosterone regulates aromatase activity in the anterior/dorsolateral hypothalamus of male rhesus macaques. To determine the level of the androgen effect, we developed a ribonuclease protection assay to study the effects of testosterone or dihydrotestosterone (DHT) on aromatase (P450(AROM)) mRNA in selected brain areas. Adult male rhesus monkeys were treated with testosterone or DHT. Steroids in serum were quantified by RIA. Fourteen brain regions were analyzed for P450(AROM) mRNA. Significant elevations of its message over controls (P<0.05) were found in the medial preoptic area/anterior hypothalamus of both androgen treatment groups and the medial basal hypothalamus of the testosterone-treated males. Other brain areas were not affected by androgen treatment. We conclude that testosterone and DHT regulate P450(AROM) mRNA in brain regions that mediate reproductive behaviors and gonadotropin release. The P450(AROM) mRNA of other brain areas is not androgen dependent. Brain-derived estrogens may also be important for maintaining neural circuitry in brain areas not related to reproduction. The control of P450(AROM) mRNA in these areas may differ from what we report here, but it is equally important to understand the function of in situ estrogen formation in these areas.
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PMID:Region-specific regulation of cytochrome P450 aromatase messenger ribonucleic acid by androgen in brains of male rhesus monkeys. 1081 87

Differential expression of two rainbow trout CYP1A genes was measured in vivo and in vitro in response to treatment with the model CYP1A inducers beta-naphthoflavone (BNF), 3-methylcholanthrene (3-MC), isosafrole (ISF), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, only in vitro). Originally described by Berndtson and Chen (Arch. Biochem. Biophys. 310, 187-195, 1994) as CYP1A1 and CYP1A2, these genes were renamed CYP1A3 and CYP1A1, respectively, by the P450 nomenclature committee. A significant, differential, inducer-dependent induction of the two CYP1A mRNAs, as measured by RNase protection assay, was observed in vivo. CYP1A3 and CYP1A1 mRNA levels in liver were significantly induced 50- and 18-fold, respectively, following ip injection with BNF. Conversely, CYP1A3 and CYP1A1 mRNA levels were significantly induced 5- and 66-fold, respectively, following ip injection with 3-MC. Isosafrole had no significant effect on in vivo induction of CYP1A mRNA levels. In primary cultures of hepatocytes, BNF, 3-MC, ISF, as well as TCDD all significantly induced CYP1A3 and CYP1A1 mRNA levels compared to controls. The differential induction of the two CYP1A genes was not as evident in vitro as in vivo. In addition, reanalysis and sequence comparison of the these two trout CYP1A genes with the first trout CYP1A cDNA described by Heilmann et al. (DNA 7, 379-387, 1988) indicate that the Heilmann cDNA is a hybrid of the two trout genes. The 5' portion of the cDNA sequence (212 bp) was determined by sequencing of a genomic clone and is 100% identical to the trout CYP1A3 gene. The majority of the cDNA sequence (2377 bp), however, was sequenced from a partial cDNA clone and is 99.2% identical to trout CYP1A1. Although the nomenclature of these two trout CYP1A genes is undergoing revision, these results demonstrate a differential, inducer-dependent response to model mammalian CYP1A inducers.
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PMID:Differential expression of two CYP1A genes in rainbow trout (Oncorhynchys mykiss). 1086 Aug 69

In vertebrates, the growth and maturation of the ovarian follicle is dependent on the appropriate dynamics of sex steroid secretion, which is dictated by gene expression of the steroidogenic enzymes. The molecular aspects of steroid regulation are poorly understood in fishes, so as a first step we determined the pattern of expression of four key steroidogenic genes throughout the ovarian cycle in an annually spawning teleost, the channel catfish (Ictalurus punctatus). The abundance of transcripts encoding 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and cholesterol side chain cleavage (P450(scc)), 17 alpha-hydroxylase/lyase (P450(c17)), and aromatase (P450(arom)) were determined by rtqRT-PCR or ribonuclease protection assay and correlated to ovarian growth and plasma titers of estradiol (E(2)) and testosterone (T) in two populations of catfish. Elevations in transcript abundance for P450(c17), P450(scc), and P450(arom) were observed at the onset of ovarian recrudescence and during early vitellogenic growth of the oocytes; however, all three decreased precipitously with the completion of vitellogenesis. Changes in the expression of these genes strongly suggest a direct correlation to E(2) and T titers. Alternatively 3 beta-HSD transcript abundance was relatively stable throughout the year. This study suggests that the genes encoding the three steroidogenic cytochrome P450s have a similar regulatory mechanism.
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PMID:Changes in the expression of genes encoding steroidogenic enzymes in the channel catfish (Ictalurus punctatus) ovary throughout a reproductive cycle. 1109 Apr 35

The expression, in adult human skin, of genes encoding flavin-containing monooxygenases (FMOs) 1, 3, 4, and 5 and cytochromes P450 (CYPs) 2A6, 2B6, and 3A4 was determined by RNase protection. Each FMO and CYP exhibits inter-individual variation in expression in this organ. Of the individuals analysed, all contained CYP2B6 mRNA in their skin, 90% contained FMO5 mRNA and about half contained mRNAs encoding FMOs 1, 3, and 4, and CYPs 2A6 and 3A4. The amount of each of the FMO and CYP mRNAs in skin is much lower than in the organ in which it is most highly expressed, namely the kidney (for FMO1) and the liver (for the others). In contrast to the latter organs, in the skin FMO mRNAs are present in amounts similar to, or greater than, CYP mRNAs. Only the mRNA encoding CYP2B6 decreased in abundance in skin with increasing age of the individual. All of the mRNAs were substantially less abundant in cultures of keratinocytes than in samples of skin from which the cells were derived. In contrast, an immortalized human keratinocyte cell line, HaCaT, expressed FMO3, FMO5, and CYP2B6 mRNAs in amounts that fall within the range detected in the whole skin samples analysed. FMO1, CYP2A6, and CYP3A4 mRNAs were not detected in HaCaT cells, whereas FMO4 expression was markedly increased in this cell line compared to whole skin. In situ hybridization showed that the expression of each of the FMOs and CYPs analysed was localized to the epidermis, sebaceous glands and hair follicles.
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PMID:Quantification and cellular localization of expression in human skin of genes encoding flavin-containing monooxygenases and cytochromes P450. 1155 24

We have previously established the presence of a functional bone morphogenetic protein (BMP) system in the ovary by demonstrating the expression of BMP ligands and receptors as well as novel cellular functions. Specifically, BMP-4 and BMP-7 are expressed in theca cells, and their receptors by granulosa cells. These BMPs enhanced and attenuated the stimulatory action of FSH on estradiol and progesterone production, respectively. To investigate the underlying mechanism of the differential regulation, we analyzed mRNA levels for key regulators in the steroid biosynthetic pathways by RNase protection assay. BMP-7 enhanced P450 aromatase (P450(arom)) but suppressed steroidogenic acute regulatory protein (StAR) mRNAs induced by FSH, whereas mRNAs encoding further-downstream steroidogenic enzymes, including P450 side-chain cleavage enzyme and 3beta-hydroxysteroid dehydrogenase, were not significantly altered. These findings suggest that BMP-7 stimulation and inhibition of P450(arom) and StAR mRNA expression, respectively, may play a role in the mechanisms underlying the differential regulation of estradiol and progesterone production. To establish the physiological relevance of BMP functions, we investigated the in vivo effects of injections of recombinant BMP-7 into the ovarian bursa of rats. Ovaries treated with BMP-7 had decreased numbers of primordial follicles, yet had increased numbers of primary, preantral, and antral follicles, suggesting that BMP-7 may act to facilitate the transition of follicles from the primordial stage to the pool of primary, preantral, and antral follicles. In this regard, we have also found that BMP-7 caused an increase in DNA synthesis and proliferation of granulosa cells from small antral follicles in vitro. In contrast to the stimulatory activity, BMP-7 exhibited pronounced inhibitory effects on ovulation rate and serum progesterone levels. These findings establish important new biological activities of BMP-7 in the context of ovarian physiology, including folliculogenesis and ovulation.
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PMID:Effect of bone morphogenetic protein-7 on folliculogenesis and ovulation in the rat. 1156 18


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