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)

Corticotrophin-releasing hormone (CRH) is a 41 amino acid neuropeptide which is cleaved at a pair of dibasic amino acids from a larger precursor molecule (pre-proCRH) by the action of endopeptidases. In cells possessing a regulated secretory pathway, sorting of proneuropeptides and prohormones occurs within the trans-Golgi network, where they are finally packaged into secretory vesicles to be released in response to an external stimulus. Such cells also possess a constitutive secretory pathway, and neuropeptides are also translocated into this subcellular compartment. We have recently established stably transfected CHO-K1 cells expressing the rat pre-proCRH cDNA, and shown that proCRH was localized within the secretory pathway and the nucleus of transfected cells. Both the cytoplasmic and nuclear species of IR-CRH displayed an apparent molecular weight approximately 19 kDa, consistent with the size of the uncleaved CRH precursor molecule. In this paper, we further characterized the bitopological, i.e. nuclear and cytoplasmic localization of proCRH within transfected CHO-K1 cells. Immunoreactive nuclear CRH was not extractable using detergents (Triton X-100 and CHAPS), 10 mM salt washes or RNase digestion but could be abolished by digestion with DNase I. These results therefore suggest that nuclear proCRH is in close association with DNA/chromatin. Treatment of transfected cells with inhibitors of protein and RNA synthesis for up to 24 h had no effect upon immunoreactive nuclear CRH, indicating that it is very stable with a long half life. Brefeldin A treatment had no effect upon the nuclear translocation of newly synthesized proCRH, suggesting that late stages of the secretory pathway (i.e. post rough endoplasmic reticulum compartments) of the transfected cells do not play a role in proCRH nuclear transport. We also demonstrate that proCRH synthesized within stably transfected CHO-K1 cells is capable of stimulating ACTH release from primary cultures of anterior pituitary cells, therefore showing for the first time that the intact precursor is also biologically active and could act as an ACTH secretagogue in-vivo.
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PMID:Expression of biologically active procorticotrophin-releasing hormone (proCRH) in stably transfected CHO-K1 cells: characterization of nuclear proCRH. 764 68

Brefeldin A (BFA), a unique fungal metabolite of a 13-membered lactone ring, exhibits various biological actions, including antitumor, antifungal and antiviral activities. In the present study, mouse LB cells were treated with various concentrations of interferon (IFN) and/or BFA overnight and infected with encephalomyocarditis virus (EMCV) after removal of IFN and BFA. Doses of BFA which neither inhibit the metabolism of the cell nor the infectivity of EMCV, decreased the IFN-induced antiviral activity against EMCV as demonstrated by virus titer from supernatants. Since 2-5A synthetase and double-stranded RNA (dsRNA)-dependent protein kinase (PKR) have been suggested to be involved in the antiviral action of IFN against EMCV, their activities were investigated in LB cells after BFA treatment. Northern blot analysis and in situ hybridization showed a decrease (2-3-fold) in the mRNA of 2'-5' oligoadenylate (2-5A) synthetase after BFA treatment. BFA also inhibited the activity of 2-5A synthetase, 2-5A dependent RNase and phosphorylation of PKR in cellular extracts, indicating that BFA may be exerting its inhibitory effect both at the transcriptional and post-transcriptional levels. This study reports a new biological action of BFA, demonstrating that BFA antagonized the antiviral action of IFN by inhibiting IFN-induced enzymatic pathways. These studies also suggest that both 2-5A and PKR are important in the antiviral activity of IFN against EMCV.
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PMID:Brefeldin A inhibits the antiviral action of interferon against encephalomyocarditis virus. 872 8

Onconase is a cytotoxic ribonuclease with antitumor properties. A semisynthetic gene encoding the entire protein sequence was constructed by fusing oligonucleotides coding for the first 15 and the last 6 of the 104 amino acids to a genomic clone that encoded the remaining amino acid residues [Newton, D. L., et al. (1997) Protein Eng. 10, 463-470]. The resulting protein product expressed in Escherichia coli exhibited little enzymatic or cytotoxic activity due to the unprocessed N-terminal Met amino acid residue. In this study, we demonstrate that modification of the 5'-region of the gene to encode [Met-(-1)]Ser or [Met-(-1)]Tyr instead of the native pyroglutamate results in recombinant onconase derivatives with restored activities. [Met-(-1)]rOnc(E1S) was more active than [Met-(-1)]rOnc(E1Y) in all assays tested. Consistent with the action of native onconase, [Met-(-1)]rOnc(E1S) was a potent inhibitor of protein synthesis in the cell-free rabbit reticulocyte lysate assay, degrading tRNA at concentrations that correlated with inhibition of protein synthesis. An interesting difference between the recombinant onconase derivatives and the native protein was their susceptibility to inhibition by the major intracellular RNase inhibitor, PRI (onconase is refractory to PRI inhibition). [Met-(-1)]rOnc(E1S) and [Met-(-1)]rOnc(E1Y) inhibited protein synthesis in intact SF539 neuroblastoma cells with IC50's very similar to that of onconase (IC50 3.5, 10, and 10 microg/mL after 1 day and 0.16, 0.35, and 2.5 microg/mL after 5 days for onconase, [Met-(-1)]rOnc(E1S), and [Met-(-1)]rOnc(E1Y), respectively). Similar to that of onconase, cytotoxic activity of the recombinant derivatives was potentiated by monensin, NH4Cl, and retinoic acid. Brefeldin A completely blocked the enhancement of cytotoxicity caused by retinoic acid with all three proteins. Thus, drug-induced alterations of the intracellular trafficking of the recombinant derivatives also resembles that of onconase. Stability studies as assessed in serum-containing medium in the presence or absence of cells at 37 degreesC showed that the recombinant proteins were as stable to temperature and cell culture conditions as the native protein. Therefore, exchanging the Glu amino acid residue at the amino terminus of onconase with an amino acid residue containing a hydroxyl group produces recombinant proteins with ribonuclease and cytotoxic properties similar to native onconase.
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PMID:Single amino acid substitutions at the N-terminus of a recombinant cytotoxic ribonuclease markedly influence biochemical and biological properties. 954 48

Adenoviral p53 gene transfer (Ad-p53) induces apoptosis in glioma cells expressing mutant p53, but fails in cells with wild-type p53. Endogenously, gliomas express varied levels of Fas/CD95, yet constitutively high levels of Fas/CD95 ligand. Because the mechanism behind the differential apoptotic response to Ad-p53 infection remains elusive, we examined how the Fas/CD95 pathway is involved in U87MG (wt-p53), D54 (wt-p53), U251MG (mutant-p53), and U373MG (mutant-p53) glioma cell lines. Ad-p53 infection did not alter the levels of Fas/CD95 ligand in either wild-type or mutant p53-expressing cell lines. In contrast, Ad-p53 infection led to an approximately 3-fold increase in Fas/CD95 mRNA expression in mutant p53-bearing cell lines but not in their wild-type (wt) counterparts, as assessed in an RNase protection assay. Fas/CD95 mRNA induction appeared to be regulated at the transcriptional level because Ad-p53 infection resulted in up to a 4-fold increase in Fas/CD95 promoter reporter activity. Subsequently, flow cytometric analysis revealed a 2- to 4-fold increase in surface Fas/CD95 expression following Ad-p53 infection in mutant-p53-containing cell lines. Use of the protein transport inhibitor Brefeldin A significantly inhibited Ad-p53-induced surface Fas/CD95 expression, but only partially inhibited apoptosis in mutant-p53 cell lines. These results suggest that p53 regulates Fas/CD95 expression at the transcriptional level and through protein trafficking in mutant-p53 cell lines. Fluorogenic activity assays demonstrated that induction of caspase-8 activity following Ad-p53 infection correlated with increases in Fas/CD95 expression. Incubating cells with a caspase-8-specific inhibitor Ac-IETD-CHO prior to Ad-p53 infection inhibited caspase-8 activity and apoptosis. Together, our results suggest that regulation of the Fas/CD95 pathway is partly responsible for Ad-p53-induced apoptosis in glioma cells, which depends on the p53 status of the involved cells. Additionally, the inability of Ad-p53 to activate the Fas/CD95 pathway in wt-p53 glioma cells coincides with their apoptotic-resistant phenotype. Further elucidation of the nature of this resistance could ultimately augment the efficacy of Ad-p53 gene therapy.
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PMID:Differential activation of the Fas/CD95 pathway by Ad-p53 in human gliomas. 1471 18