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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Friend erythroleukemia cell line T3-C12, which produces Friend murine leukemia virus (F-MuLV) and can be induced to synthesize hemoglobin by dimethyl sulfoxide (DMSO), was monitored for viral RNA-dependent DNA polymerase reverse transcriptase (RT) activity. The amount of viral 60-70S RNA released from DMSO-treated cells was unaffected or increased compared to that from control cells, while RT activity from treated cells was decreased. Accordingly, the specific activity in F-MuLV from DMSO-treated cells expressed as RT/70S RNA was decreased to 8% of the control activity. The 5-bromo-2'-deoxyuridine added to cultures containing DMSO reversed the differentiation process, and the F-MuLV thus treated did not exhibit the reduced RT activity normally observed in DMSO-treated virus. Cell-free F-MuLV incubated with and without DMSO showed the same RT activity, indicating that DMSO itself did not inhibit RT activity. However, when F-MuLV-containing pellets from control and DMSO-treated culture fluids were mixed, there was marked inhibition of the control RT activity, suggesting that RNase hybrid activity was stimulated or that an inhibitor was produced. Assays of F-MuLV-RNase hybrid released from control and DMSO-treated cells showed no difference in activity, indicating that a specific inhibitor of RT was produced or activated. Additions of certain nucleotide triphosphates to RT incubation mixtures did not result in any stimulation of RT activity in DMSO-treated F-MuLV, suggesting that phosphatase was not responsible for the observed inhibition. The results suggested that DMSO treatment of T3-C12 cells caused a reduction in viral RT activity by stimulating the production of an inhibitor, the nature of which is unknown.
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PMID:Viral reverse transcriptase suppression associated with erythroid differentiation of Friend leukemia cells. 6 77

Replicative intermediate (RI), replicative form (RF) and single-stranded (SS) RNA have been isolated from BHK cells infected with a bovine enterovirus by salt precipitation and gel filtration techniques. Kinetic experiments showed that at no time up to 16 h post-infection (p.i.) did the amount of RF exceed that of RI or SS RNA. Electrophoresis of RF on 1.5% polyacrylamide-agarose gels showed that at least three species of double-stranded RNA were present, one of which was associated with an accessible poly(A)-containing tract. All of the RF was denatured by 99% dimethylsulphoxide (DMSO), although reannealling occurred rapidly when samples were returned to aqueous conditions. No evidence for circular structures in the RF molecular population was found by use of caesium sulphate density gradients containing ethidium bromide. Treatment of RI with ribonuclease produced double-stranded RNA molecules, some of which were smaller in size than intact RF. Denaturation with DMSO and analysis on 99% DMSO sucrose gradients showed that the RI did not contain single strands of greater length than virion RNA. A portion of the RI bound to poly(U)-Sepharose 4B columns. The poly(A) tracts involved were present only in the nascent RNA strands with greatest sedimentation coefficients (30 to 35S). Bovine enterovirus induced SS RNA was heterogeneous with regard to both sedimentation through sucrose gradients and mobility on acrylamide gels compared to purified virion RNA. The reason for this difference has never been satisfactorily resolved. Sedimentation through 99% DMSO-sucrose gradients showed that the heterogeneity was due to aggregation rather than any variation in chain length or conformational differences. Our results support the single-stranded template model rather than a circular model for picornavirus RNA replication.
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PMID:Studies of the replication of a bovine enterovirus RNA. 22 21

Treatment of tobacco mosaic virus (TMV) RNA with T1 RNase under mild conditions cuts the RNA molecule into a large number of fragments, only a few of which may be specifically recognized by disks of TMV protein. It has been shown elsewhere that these specifically recognized RNA fragments are a part of the coat protein cistron, the portion coding for amino acids 95 to 129 of the coat protein. It is reported that different size classes of partially uncoated virus particles were prepared by limited reconstitution between TMV RNA and protein or by partial stripping of intact virus with DMSO. Both procedures produce nucleoprotein rods in which the 5'-terminal portion of the RNA is encapsidated and the 3'-terminal region is free. The free and the encapsidated portions of the RNA were each tested for the ability to give rise to the aforesaid specifically recognized fragments of the coat protein cistron upon partial T1 RNase digestion. It was found that only the 3'-terminal third of the virus particle need to be uncoated in order to expose the portion of the RNA molecule from which these fragments are derived. We conclude, therefore, that the coat protein cistron is situated upon the 3'-terminal third of the RNA chain, i.e. within 2000 nucleotides of the 3'-end.
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PMID:Location of the cistron of the tobacco mosaic virus coat protein. 120 19

We have isolated a 725-bp full-length cDNA clone for the human eosinophil cationic protein (ECP). ECP is a small, basic protein found in the matrix of the eosinophil's large specific granule that has cytotoxic, helminthotoxic, and ribonuclease activity, and is a member of the ribonuclease multigene family. The cDNA sequence shows 89% sequence identity with that reported for the related granule protein, eosinophil-derived neurotoxin (EDN). The open reading frame encodes a previously unidentified 27-amino acid leader sequence preceding a 133-residue mature ECP polypeptide with a molecular mass of 15.6 kD. The encoded amino acid sequence of ECP shows 66% identity to that of EDN and 31% identity to that of human pancreatic ribonuclease, including conservation of the essential structural cysteine and cataytic lysine and histidine residues. mRNA for ECP was detected in eosinophil-enriched peripheral granulocytes and in a subclone of the promyelocytic leukemia line, HL-60, induced toward eosinophilic differentiation with IL-5. No ECP mRNA was detected in uninduced HL-60 cells, or in HL-60 cells induced toward monocytic differentiation with vitamin D3 or toward neutrophilic differentiation with DMSO. In contrast, mRNA for EDN was detected in uninduced HL-60 cells and was upregulated in HL-60 cells induced with DMSO. Despite similarities in sequence and cellular localization, these results suggest that ECP and EDN are subject to different regulatory mechanisms.
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PMID:Human eosinophil cationic protein. Molecular cloning of a cytotoxin and helminthotoxin with ribonuclease activity. 247 57

Thrombospondin (TSP), a multifunctional homotrimeric glycoprotein of approximately 450,000 M(r), is a component of the extracellular matrix that mediates the adhesive interactions of several different cell types including hematopoietic progenitor cells. We have used the promyelocytic leukemia HL-60 cell line to examine TSP receptor expression during differentiation of leukocytes along either the monocyte/macrophage or the polymorphonuclear leukocyte (PMN) pathway. 125I-labeled TSP binding to undifferentiated or differentiated HL-60 cells was time-dependent reaching saturation by 45 min. Undifferentiated HL-60 cells expressed a single class of heparin-inhibitable TSP receptors. Treating HL-60 cells with PMA induced their differentiation to macrophage-like cells and resulted in a concomitant 10-fold increase in TSP receptor expression. As with undifferentiated cells, a single class of heparin-inhibitable receptors was observed. Treating HL-60 cells with DMSO induced their differentiation to PMN-like cells and resulted in a fivefold increase in TSP receptor expression. However, in this case two classes of binding sites were apparent on PMN-like cells, only 40% of which were heparin inhibitable. This is reminiscent of TSP binding to normal peripheral blood PMN (S.J. Suchard, L.A. Boxer, and V.M. Dixit. 1991. J. Immunol. 147:651). In parallel studies, we also examined TSP synthesis during HL-60 cell differentiation. Undifferentiated HL-60 cells synthesized and secreted TSP as assessed by immunoprecipitation. TSP synthesis increased about fourfold when cells were differentiated toward PMN-like cells. In contrast, TSP was not detected in macrophage-like cells. RNase protection assays showed that TSP transcript levels paralleled TSP protein expression during differentiation. These findings suggest that expression of both TSP and TSP receptors are differentially regulated during blood cell maturation.
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PMID:Modulation of thrombospondin receptor expression during HL-60 cell differentiation. 750 39

The Ba2+ currents and mRNA levels of four members of the rat brain family of alpha 1-subunit Ca2+ channel genes were examined and compared in the rat cell lines GH3 and PC-12 and in the mouse lines NIE-115 and AtT-20. The RNA was measured with ribonuclease protection assays using probes derived from rat brain (rb) Ca2+ channel cDNAs (rbA, rbB, rbC, and rbD), and the Ba2+ currents were studied by whole cell patch-clamp recording. L-, N-, P-, and T-type currents were discriminated by the voltage dependence and pharmacological properties of Ba2+ currents. All cell lines expressed all four rat brain Ca2+ channel genes, except GH3 cells, which lacked rbB. The functional diversity of Ba2+ currents, however, was quite different among the cell lines. GH3 cells showed evidence of L- and T-type currents, undifferentiated PC-12 cells of L-type currents, AtT-20 cells of L-, N-, and P-type currents, and undifferentiated NIE-115 cells of a T-type current that was partially blocked by both nifedipine and BAY K 8644. Dimethyl sulfoxide-differentiated NIE-115 cells also had an L-type current. Differentiation of NIE-115 cells caused an increase in the levels of rbB, rbC, and rbD RNAs. Differentiation by nerve growth factor caused an increase in levels of all four genes in PC-12. Our data give further support for the assignment of rbA, rbB, and rbC/rbD gene products as components of P-, N-, and L-type Ca2+ channels, respectively.
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PMID:Calcium channels in excitable cells: divergent genotypic and phenotypic expression of alpha 1-subunits. 752 Nov 26

Gaseous CO2 was used as an antisolvent to induce the fractional precipitation of alkaline phosphatase, insulin, lysozyme, ribonuclease, trypsin, and their mixtures from dimethylsulfoxide (DMSO). Compressed CO2 was added continuously and isothermally to stationary DMSO solutions (gaseous antisolvent, GAS). Dissolution of CO2 was accompanied by a pronounced, pressure-dependent volumetric expansion of DMSO and a consequent reduction in solvent strength of DMSO towards dissolved proteins. View cell experiments were conducted to determine the pressures at which various proteins precipitate from DMSO. The solubility of each protein in CO2-expanded DMSO was different, illustrating the potential to separate and purify proteins using gaseous antisolvents. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) was used to quantify the separation of lysozyme from ribonuclease, alkaline phosphatase from insulin, and trypsin from catalase. Lysozyme biological activity assays were also performed to determine the composition of precipitates from DMSO initially containing lysozyme and ribonuclease. SDS-PAGE characterizations suggest that the composition and purity of solid-phase precipitated from a solution containing multiple proteins may be accurately controlled through the antisolvent's pressure. Insulin, lysozyme, ribonuclease, and trypsin precipitates recovered substantial amounts of biological activity upon redissolution in aqueous media. Alkaline phosphatase, however, was irreversibly denaturated. Vapor-phase antisolvents, which are easily separated and recovered from proteins and liquid solvents upon depressurization, appear to be a reliable and effective means of selectively precipitating proteins.
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PMID:Protein purification with vapor-phase carbon dioxide. 1009 36

The hematopoietic growth factor erythropoietin (Epo) triggers changes in the expression of genes that encode important regulators of erythroid cell growth and differentiation. We now report that Epo markedly upregulates chop (gadd153) expression and that this transcription factor plays a role in erythropoiesis. Using a differential hybridization assay, we isolated a full-length cDNA of chop as an Epo upregulated gene in Rauscher murine erythroleukemia cells. RNase protection assays demonstrated that Epo or dimethyl sulfoxide induction increased steady-state mRNA levels 10- to 20-fold after 24 to 48 hours. Western blot analysis confirmed a marked increase in CHOP protein. Among the other c/ebp family members, only c/ebp beta was also upregulated during erythroid differentiation. Among normal hematopoietic cells examined, steady-state mRNA levels were highest in erythroid cells, with levels peaking during terminal differentiation. Transient overexpression of chop in Rauscher cells resulted in a significant increase in Epo- or dimethyl sulfoxide (DMSO)-induced hemoglobinization, further linking chop upregulation to erythroid differentiation. Artificial downregulation of chop in normal murine bone marrow cells with antisense oligodeoxynucleotides inhibited colony-forming unit-erythroid (CFU-E)-derived colony growth in a concentration-dependent manner. Burst-forming unit-erythroid (BFU-E)-derived colony growth was not affected. Using a Far Western type of analysis, we detected several potential CHOP binding partners among the nuclear proteins of Rauscher cells. Importantly, the number and relative abundance of these proteins changed with differentiation. The results strongly suggest that CHOP plays a role in erythropoiesis, possibly through interactions with both C/EBP and non-C/EBP family members.
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PMID:Regulated expression and functional role of the transcription factor CHOP (GADD153) in erythroid growth and differentiation. 1023 89

After the first histochemical demonstration by Chayen and Gahan of the presence of phospholipids and especially of sphingomyelin in chromatin, this became the object of long debate and of contradictory results. The general conclusion was that the presence of phospholipids may due to contamination during the isolation of chromatin. More recently the existence of a phospholipid chromatin fraction was confirmed by demonstrating that isolated hepatocyte nuclei, labelled by saturated and unsaturated radioiodination method, showed the presence of radioactivity only in the membrane and not in the isolated chromatin. The phospholipid composition showed an enrichment in sphingomyelin which increased during hepatocyte maturation or erythroleukemic cell differentiation induced by DMSO. A decrease in sphingomyelin was observed at the beginning of the S-phase in regenerating liver or in cultured proliferating cells. These changes were due to the presence of sphingomyelinase and sphingomyelin synthase in the chromatin, the activity of which paralleled the variation in sphingomyelin content. The sphingomyelin was co-localized with RNA as shown by biochemical and electron microscopy methods. Using bromo-uridine it was demonstrated that labelled RNA and sphingomyelin were present in actively transcribing nuclear regions. Isolated nuclear complexes after DNase and RNase digestion contained not only protein, but also RNA and sphingomyelin. After hydrolysis of sphingomyelin the RNAse-resistant RNA becomes RNAse sensitive. It can therefore be concluded that sphingomyelin and the related enzymes are present in the chromatin; sphingomyelin may have a role in RNA transcription protecting RNA by RNAse digestion before its transfer to the cytoplasm.
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PMID:Chromatin-associated sphingomyelin: metabolism in relation to cell function. 1291 Apr 72

Solvent conditions play a major role in a wide range of physical properties of proteins in solution. Organic solvents, including dimethyl sulfoxide (DMSO), have been used to precipitate, crystallize and denature proteins. We have studied here the interactions of DMSO with proteins by differential refractometry and amino acid solubility measurements. The proteins used, i.e., ribonuclease, lysozyme, beta-lactoglobulin and chymotrypsinogen, all showed negative preferential DMSO binding, or preferential hydration, at low DMSO concentrations, where they are in the native state. As the DMSO concentration was increased, the preferential interaction changed from preferential hydration to preferential DMSO binding, except for ribonuclease. The preferential DMSO binding correlated with structural changes and unfolding of these proteins observed at higher DMSO concentrations. Amino acid solubility measurements showed that the interactions between glycine and DMSO are highly unfavorable, while the interactions of DMSO with aromatic and hydrophobic side chains are favorable. The observed preferential hydration of the native protein may be explained from a combination of the excluded volume effects of DMSO and the unfavorable interaction of DMSO with a polar surface, as manifested by the unfavorable interactions of DMSO with the polar uncharged glycine molecule. Such an unfavorable interaction of DMSO with the native protein correlates with the enhanced self-association and precipitation of proteins by DMSO. Conversely, the observed conformational changes at higher DMSO concentration are due to increased binding of DMSO to hydrophobic and aromatic side chains, which had been newly exposed on protein unfolding.
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PMID:Protein precipitation and denaturation by dimethyl sulfoxide. 1790 24


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