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:3.1.27.4 (ribonuclease)
6,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Fetal rat liver expresses a 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/Fru-2,6-Pase2) form that differs from the adult liver enzyme in the inhibition by phosphorylation by the adenosine 3',5'-cyclic monophosphate-dependent protein kinase and in the recognition by an antibody specific for the NH2-terminal domain of the adult liver enzyme. Northern blot analysis shows that fetal hepatocytes contain a species of mRNA that is 2.2 kb in size and that exhibits the maximal levels after delivery. PFK-2/Fru-2,6-Pase2 mRNA analysis using a sensitive ribonuclease protection assay reveals the presence of nearly similar amounts of adult liver-specific and skeletal muscle-specific mRNA in fetal liver and hepatocytes during the last days of gestation, as well as a 233-bp protected fragment present in fetal liver. These results were confirmed by polymerase chain reaction using specific oligonucleotide pairs. Primer extension of fetal liver cDNA suggests the presence of two initiation sites of transcription. Analysis of the adult liver PFK-2/Fru-2,6-Pase2 protein during the perinatal transition using a specific antibody shows a marked accumulation of this form immediately after birth.
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PMID:Multiple forms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase are expressed in perinatal rat liver. 877 45

Thrombin is one of the first regulatory molecules present at sites of CNS trauma or injury. Exposure of neuronal and glial cells to thrombin produces potent morphological as well as cytoprotective and cytotoxic effects, but little is known about how this important modulator affects neurotransmitter signaling. In astrocyte cultures that have been morphologically differentiated by exposure to transforming growth factor-alpha, addition of thrombin induced a retraction of astrocytic processes and suppressed the stimulation of phosphoinositide hydrolysis by the selective metabotropic glutamate receptor (mGluR) agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid. In addition to the suppression of phosphoinositide hydrolysis, thrombin treatment produced a corresponding reduction in level of mGluR5 mRNA as demonstrated with ribonuclease protection assay and reduced content of mGluR5 receptor protein as seen with western blotting. In contrast, thrombin exposure up-regulated astrocyte beta-actin mRNA levels. A synthetic hexapeptide with a sequence corresponding to the amino-terminus of the thrombin receptor's tethered ligand also mimicked the ability of thrombin to suppress mGluR5 levels and to increase beta-actin mRNA content, suggesting that these effects of thrombin are mediated by proteolytically activated cell surface thrombin receptors. Thrombin's suppressive effect on mGluR5 was resistant to pretreatment with pertussis toxin or various protein kinase and protein phosphatase inhibitors. However, the serine/threonine protein kinase inhibitor H-7 did prevent thrombin-induced reversal of astrocyte stellation and induction of beta-actin mRNA levels, indicating that these effects of thrombin involve a signaling pathway distinct from the one that mediates the suppressive effects of thrombin on mGluR5.
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PMID:Exposure of astrocytes to thrombin reduces levels of the metabotropic glutamate receptor mGluR5. 885 25

In humans the last steps in the synthesis of aldosterone and cortisol rely on the activity of two cytochrome P450 genes termed CYP11B2 (aldosterone synthase; P450aldo) and CYP11B1 (11 beta hydroxylase; P450cl1). The mechanisms which lead to differential expression of these two genes within the adrenal cortex are not well-defined. The human adrenocortical cell line. H295R, was utilized in this study to examine the intracellular second messenger pathways regulating expression of P450aldo and P450c11. using specific ribonuclease protection assays. Treatment of H295R cells with angiotensin II or potassium (K+) caused a time-dependent induction in the level of P450aldo transcripts. While K+ treatment was more specific for the induction of P450aldo mRNA, treatment with angiotensin II increased levels of both P450aldo and P450c11 transcripts. To define the second messenger systems which influence transcript levels for these enzymes, the effects of agonists of the protein kinase A, protein kinase C, and calcium pathways were tested on the expression of P450aldo and P450c11. Activation of the protein kinase A pathway by the agonists, dibutyryl cAMP or forskolin, preferentially increased the P450c11 transcript to a greater degree than P450aldo. Interestingly, activation of the protein kinase C pathway by tetradecanoylphorbol acetate (TPA) did not alter transcripts for either P450aldo or P450c11. The calcium channel agonist BAYK 8644 mimicked the effects of K+ by increasing the transcript for P450aldo. However, the calcium channel blocker nifedipine attenuated the stimulatory effects of angiotensin II and K+ on the levels of P450aldo. However, the calcium channel blocker nifedipine attenuated the stimulatory effects of angiotensin II and K+ on the levels of P450aldo transcripts without affecting the stimulatory effect of dbcAMP. This study demonstrates that the protein kinase A pathway preferentially induces P450c11 mRNA over that of P450aldo. In addition, pharmacologic agents that affect calcium levels provide evidence for an additional regulatory mechanism in modulating the expression of P450aldo. This is of importance since the major physiologic regulators of aldosterone secretion, angiotensin II and K+ are able to increase intracellular calcium but have little effect on intracellular cAMP levels.
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PMID:Differential regulation of 11 beta-hydroxylase and aldosterone synthase in human adrenocortical H295R cells. 886 69

The nuclear gene NUC1 encodes the major mitochondrial (mt) ribonuclease in the yeast Saccharomyces cerevisiae. We describe an in vitro mt transcription assay system based on lysates of purified mitochondria from a petite (rho-, mt deletion mutant) yeast strain in which NUC1 has been insertionally inactivated. Control in vitro run-on transcription assays using intact mitochondria demonstrate that the rate of incorporation of labeled precursor into mt RNA is identical in organelles from the nuc1 rho- mutant and its otherwise isochromosomal NUC1 parent strain. Brij-35 lysates of mitochondria from the nuc1 strain incorporate precursor into mt RNA at nearly the same rate as do intact organelles from that strain, while similar mt lysates from NUC1 cells show no such incorporation. Other control studies show that mt lysates from the nuc1 strain retain functional mt cAMP-dependent protein kinase and other critical activities. When the cloned template DNA encoding the yeast mt 21S rRNA gene, which is not retained in the nuc1 rho- strain, is added to mt lysates from that strain, transcripts are produced from the template under standard assay conditions.
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PMID:An in vitro transcription assay for yeast mitochondria using organellar lysates. 895 60

The principal mechanism of homologous desensitization of the beta-adrenergic receptor (beta2AR) is phosphorylation of the receptor by the betaAR kinase (betaARK) or other closely related G protein-coupled receptor kinases (GRKs). However, within a single organ such as the lung where many cell types express the receptor, the presence or extent of beta2AR desensitization in different cells has been noted to be highly variable. We hypothesized that such variability in desensitization is due to significant cell-type differences in betaARK expression and/or function. To approach this, in situ hybridization was carried out in the lung and indeed revealed heterogeneity in betaARK gene expression. Quantitative studies using ribonuclease protection assays with cell lines revealed that the level of betaARK mRNA in airway smooth muscle cells was approximately 20% of that in bronchial epithelial cells and approximately 11% of that in mast cells (6.65 +/- 0.96 versus 32.6 +/- 4.0 and 60.7 +/- 1.5 relative units, respectively, p < 0. 001). betaARK2 gene expression was not detected in any of these cells. At the protein level, betaARK expression in airway smooth muscle cells was nearly undetectable, being approximately 10-fold less than that expressed on mast cells. The activities of the GRKs in cell extracts were assessed in vitro by quantitating their ability to phosphorylate rhodopsin in the presence of light. Consistent with the gene and protein expression results, a marked discrepancy in activities was observed between extracts derived from mast cells (90.7 +/- 0.5 relative units) as compared to airway smooth muscle cells (9.28 +/- 0.6 relative units, p < 0.001). In contrast, the activities of protein kinase A (the other kinase that phosphorylates beta2AR) in these extracts were not different. We predicted, then, that airway smooth muscle beta2AR would undergo minimal short-term (5 min) agonist-promoted desensitization as compared to the beta2AR expressed on mast cells. Mast cell cAMP reached maximal levels after 90 s and did not further increase over time, indicative of receptor desensitization in this cell. In contrast, cAMP levels of airway smooth muscle cells did not plateau, increasing at a rate of 103 +/- 9% per min, consistent with little desensitization over the study period. We conclude that there is significant cell-type variation in expression of betaARK and that such variation is directly related to the extent of short-term agonist-promoted desensitization of the beta2AR.
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PMID:Heterogeneity in beta-adrenergic receptor kinase expression in the lung accounts for cell-specific desensitization of the beta2-adrenergic receptor. 905 32

The cyclin-dependent kinase (CDK) inhibitor p18 blocks progression of the cell cycle by associating with the cyclin D-dependent kinases CDK6 and CDK4. To better understand the regulation of p18 gene expression, we isolated full-length cDNA clones from a human BT-20 breast cancer cell cDNA library. These clones were then used to isolate the human gene from a human genomic DNA library. The human p18 gene spans at least 7.5 kb and is composed of three exons, two of which encode the p18 protein. The genomic clone we isolated contained 5 kb of putative promotor sequence which directed expression of the luciferase reporter gene in transient transfection experiments. The longest cDNA that we isolated from BT-20 cells contained 2103 nucleotides which corresponds to the size of the major RNA transcript detected by Northern analysis in these cells. Transcription start sites mapping to the 5' end of the putative full-length cDNA were identified by ribonuclease protection assays. A novel polymorphism was identified in the 3' untranslated region of BT-20 cell cDNA clones that contained the previously described codon 72 mutation. The codon 72 mutation was also detected in 3 of 35 breast tumors analyzed using a mismatch PCR/RFLP strategy.
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PMID:Structure of the gene encoding the human cyclin-dependent kinase inhibitor p18 and mutational analysis in breast cancer. 963 70

RNase L is the 2',5'-oligoadenylate (2-5A)-dependent endoribonuclease that functions in interferon action and apoptosis. One of the intriguing, albeit unexplained, features of RNase L is its significant homology to protein kinases. Despite the homology, however, no protein kinase activity was detected during activation and RNA cleavage reactions with human RNase L. Similarly, the kinase plus ribonuclease domains of RNase L produced no detectable protein kinase activity in contrast to the phosphorylation obtained with homologous domains of the related kinase and endoribonuclease, yeast IRE1p. In addition, neither ATP nor pA(2'p5'A)3was hydrolyzed by RNase L. To further investigate the function of the kinase homology in RNase L, the conserved lysine at residue 392 in protein kinase-like domain II was replaced with an arginine residue. The resulting mutant, RNase LK392R, showed >100-fold decreases in 2-5A-dependent ribonuclease activity without reducing 2-5A- or RNA-binding activities. The greatly reduced activity of RNase LK392Rwas correlated to a defect in the ability of RNase L to dimerize. These results demonstrate a critical role for lysine 392 in the activation and dimerization of RNase L, thus suggesting that these two activities are intimately linked.
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PMID:Alternative function of a protein kinase homology domain in 2', 5'-oligoadenylate dependent RNase L. 986 63

To define protein domains important for activation of the interferon (IFN)-induced enzyme 2-5A-dependent RNaseL, we have generated vaccinia virus (VV) recombinants able to express in cultured cells truncated forms of this protein and compared their biologic activities with those producing the wild-type enzyme, with and without coexpression of 2-5A synthetase. Our results show that full activation of RNaseL requires binding of 2-5A oligonucleotides within amino acid positions 212-339, corresponding to ankyrin repeats 6 to 9. The protein kinase and ribonuclease domains of RNaseL, amino acids 340-741, are sufficient for a constitutively active enzyme that is unresponsive to excess 2-5A. These results demonstrate in vivo the importance of the ankyrin domains in the biologic function of RNaseL. We suggest that ankyrin repeats act as key modulators of RNaseL activity.
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PMID:Full activation of RNaseL in animal cells requires binding of 2-5A within ankyrin repeats 6 to 9 of this interferon-inducible enzyme. 1009 Mar 96

cAMP mediates many of the effects of vasopressin, prostaglandin E2, and beta-adrenergic agents upon salt and water transport in the renal collecting duct. The present studies examined the role of cAMP-dependent protein kinase (PKA) in mediating these effects. PKA is a heterotetramer comprised of two regulatory (R) subunits and two catalytic (C) subunits. The four PKA isoforms may be distinguished by their R subunits that have been designated RIalpha, RIbeta, RIIalpha, and RIIbeta. Three regulatory subunits, RIalpha, RIIalpha, and RIIbeta, were detected by immunoblot and ribonuclease protection in both primary cultures and fresh isolates of rabbit cortical collecting ducts (CCDs). Monolayers of cultured CCDs grown on semipermeable supports were mounted in an Ussing chamber, and combinations of cAMP analogs that selectively activate PKA type I vs. PKA type II were tested for their effect on electrogenic ion transport. Short-circuit current (Isc) was significantly increased by the PKA type II-selective analog pairs N6-monobutyryl-cAMP plus 8-(4-chlorophenylthio)-cAMP or N6-monobutyryl-cAMP plus 8-chloro-cAMP. In contrast the PKA type I-selective cAMP analog pair [N6-monobutyryl-cAMP plus 8-(6-aminohexyl)-amino-cAMP] had no effect on Isc. These results suggest PKA type II is the major isozyme regulating electrogenic ion transport in the rabbit collecting duct.
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PMID:Type II cAMP-dependent protein kinase regulates electrogenic ion transport in rabbit collecting duct. 1019 23

The molecular mechanisms involved in regulation of CRH-binding protein (CRH-BP) gene expression were examined using primary rat astrocyte cultures. The cells were treated with various regulators, and CRH-BP messenger RNA (mRNA) levels were determined using ribonuclease protection assays. Forskolin (Fsk, 10 microM) or 12-O-tetradecanoyl-phorbol 13-acetate (TPA, 100 nM) increases CRH-BP mRNA levels up to 30 times control level, and together they act synergistically to increase CRH-BP gene expression up to 100 times control levels. CRH can also positively regulate CRH-BP gene expression to 6.1 times control levels. All of these increases in steady-state CRH-BP mRNA levels can be repressed by dexamethasone, a synthetic glucocorticoid. To determine whether these changes in steady-state CRH-BP mRNA levels are caused by altered transcription or RNA stability, heteronuclear (hn) CRH-BP species were examined using ribonuclease protection assays. CRH-BP hnRNA transcripts can be detected transiently after the addition of Fsk or TPA, and dexamethasone can repress Fsk- or TPA-induced CRH-BP hnRNA levels in this assay. These results demonstrate that CRH, glucocorticoids, and the protein kinase A and protein kinase C signaling pathways are involved in regulation of CRH-BP gene expression in astrocyte cultures, and that this regulation is caused, at least in part, by altered transcription of the gene.
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PMID:Transcriptional regulation of corticotropin-releasing hormone-binding protein gene expression in astrocyte cultures. 1046 81


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