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)

Nucleolar organizer region (NOR) silver staining was applied to sections of fixed material. A positive reaction on cryo-ultrathin sections was found as well as on semithin and ultrathin Lowicryl sections. Repeatable staining that was easy to control was obtained by a one-step procedure after aldehyde-Carnoy fixation. Fixation of the material by formaldehyde and glutaraldehyde alone in cacodylate buffer also maintained reaction selectivity when ammonium chloride was used after fixation. Enzymatic digestion by pronase, RNase A, DNase I, or micrococcal nuclease was applied to ultrathin Lowicryl sections. Pronase digestion removed the silver-stained proteins, whereas digestion by the nucleases did not. A routine procedure is proposed for easy NOR silver staining of sections that preserves a good tissue ultrastructure and is also compatible with cytochemical and immunological investigations.
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PMID:Silver staining of the nucleolar organizer regions (NORs) on Lowicryl and cryo-ultrathin sections. 258 Aug 79

Previous studies have documented nuclear insulin accumulation in a variety of cell types. The present investigation extends these observations by demonstrating that insulin associates with the matrix fraction of H35 rat hepatoma cell nuclei. Nuclei were isolated from [125I]insulin-loaded cells and extracted with DNase I, RNase A and high salt. The resulting matrix fraction was found to contain greater than 75% of the radiolabel initially present. Ultrastructural studies to confirm these findings were carried out using an agarose-encapsulated nuclear matrix preparation. Electron microscopic immunocytochemistry specifically detected insulin in matrices prepared from insulin-treated cells. No reaction was observed in matrices obtained from non-insulin-treated (control) cells. Further biochemical analysis revealed that matrix-associated insulin could be solubilized with 1% sodium dodecyl sulfate (SDS) or in the presence of high urea concentrations. Gel filtration analysis of urea-solubilized matrix material revealed the presence of apparently intact [125I]insulin and a higher molecular weight peak. It is hypothesized that the latter may represent a tightly associated complex of insulin with some matrix protein(s).
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PMID:Intranuclear localization of insulin in rat hepatoma cells: insulin/matrix association. 269 59

We have devised a method to measure the protein and nucleic acid content of the nuclear matrix using flow cytometry. Nuclear matrices were prepared from nuclei by DNase I digestion followed by 3 M NaCl extraction. The resulting particles were stained with fluorescein isothiocyanate (FITC) for protein and propidium iodide (PI) for double-stranded nucleic acids, and fluorescence as well as forward angle light scatter was detected. The matrices were also subjected to additional chemical or enzymatic perturbations, and changes in the above parameters were measured. Results showed that matrices from heat-shocked cells not only retained the majority of heat-induced excess nuclear protein, but also exhibited higher PI signals than controls after RNase A digestion. This observation did not hold if RNase A digestion preceded high-salt extraction, suggesting that a salt-extractable moiety had been replaced or altered by heat so that double-stranded RNA was protected from the nucleolytic attack. The residual PI fluorescence in matrices from heated cells bore a linear relationship to the increased protein content in those matrices, indicating that the excess protein sequesters matrix-associated RNA. Polyacrylamide gel electrophoresis of matrix polypeptides revealed increased amounts of many proteins as a result of heat as well as the appearance of several new proteins, one of which comigrates with the HSP72/73 heat-shock proteins. The results of these studies show that flow cytometry can be used to study the nuclear matrix and is capable of detecting changes that result from alterations in its protein composition.
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PMID:Flow cytometric studies of the nuclear matrix. 271 14

Phosphodiesterase I [EC 3.1.4.1] was purified from normal human urine in a highly purified state free from phosphodiesterase II, RNase, DNase I, DNase II, and phosphatase by column chromatographies of DEAE-Toyopearl, butyl-Toyopearl, Affi-Gel blue, and Sephadex G-150. The molecular weight of the enzyme was 1.9 x 10(5) and the pH optimum around 9.0 with p-nitrophenyl deoxythymidine 5'-phosphate as the substrate. The enzyme hydrolyzed the 3'-5' linkage of various dinucleoside monophosphates at approximately the same rate and the phosphodiester bonds of cyclic 3',5'-mononucleotides to produce mononucleoside 5'-phosphate. The enzyme also hydrolyzed ADP to 5'-AMP and Pi, ATP to 5'-AMP and PPi, and NAD+ to 5'-AMP and NMN. The enzyme activity was abolished by removal of metal ions with EDTA, and the metal-free enzyme was reactivated on the addition of Zn2+. The enzyme activity was also abolished by some reducing agents and the inhibition was reversed by Zn2+. The metal-free enzyme was less stable than the native enzyme, and Zn2+ and Co2+ restored the stability of the metal-free enzyme to the level of the native enzyme. The enzyme degraded oligonucleotides and high molecular nucleotides stepwise from the 3'-termini to give 5'-mononucleotides. The enzyme hydrolyzed single-stranded DNA more preferentially than double-stranded DNA. The enzyme also nicked superhelical covalently closed circular phi X174 DNA to yield first open circular DNA and then linear DNA.
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PMID:Phosphodiesterase I in human urine: purification and characterization of the enzyme. 282 85

The upstream regulatory region of the human papilloma virus-16 (HPV-16) genomic DNA contains a sequence element with a large degree of homology to the partially palindromic sequence GGTACANNNTGTTCT, which is the consensus sequence of the glucocorticoid responsive elements of known genes regulated by this steroid hormone. DNase I and dimethylsulfate protection experiments reveal the binding of this sequence by rat glucocorticoid receptor protein. A 400-bp DNA segment centrally containing this sequence confers strong inducibility by dexamethasone to the promoter p97 of HPV-16 and to the Herpes simplex virus thymidine kinase promoter, as judged by chloramphenicol acetyltransferase activity and RNase protection assays. The same DNA segment, that does not contain the consensus sequences of all papilloma viruses relevant for E2 protein-mediated transcription enhancement, functions in an enhancer-like fashion in addition to its glucocorticoid responsive action. This hormone-independent transcription enhancement is absent in human MCF7 cells, but is strong in human HeLa cells where the combined activity of the constitutive and the steroid hormone-dependent enhancer elements stimulate transcription by a factor of 500. This cell type specificity of the HPV-16 enhancer may be responsible for the tissue tropism of the virus. These observations and the presence of numerous homologies to known enhancers of cellular and viral genes suggest a complex pattern of activation of the human papilloma virus-16 promoters.
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PMID:The upstream regulatory region of the human papilloma virus-16 contains an E2 protein-independent enhancer which is specific for cervical carcinoma cells and regulated by glucocorticoid hormones. 282 35

In order to study hepatocellular carcinoma-associated antigens, screening of sera and ascites was done from hepatocellular carcinoma patients having antibodies reactive with three hepatocellular carcinoma cell lines (PLC/PRF/5, Hep 3B and HA22T/VGH). The indirect immunofluorescent antibody test was used. Ten of 86 (11.6%) sera and 3 of 14 (21.4%) ascites from hepatocellular carcinoma patients showed positive bindings, whereas only 1 of 35 (2.8%) sera, none of 4 (0%) ascites from chronic hepatitis patients and 3 of 60 (5%) normal human sera had positive immunofluorescent antibody activity. The binding specificities of these positive specimens were further defined by other human cancer cell lines and mouse NIH/3T3 fibroblasts. The antinuclear antibody test against mouse liver sections was also performed. The results suggested that antigens identified by the two tests may not be identical. The nature of nuclear antigens reactive with one of the serum samples, S83, and ascites A83 were characterized. These antigens were sensitive to trypsin but not to RNase A and DNase I. Further studies by radioimmunoprecipitation and two-dimensional gel electrophoresis with serum S83 and ascites A83 showed two acidic phosphorylated antigens with molecular weights of 77 and 79 kd, which had a pI around pH 5.2. The presence of a large amount of these two phosphorylated proteins in 5 of 7 human hepatocellular carcinoma cell lines suggests that these two antigens might play some roles in the carcinogenesis or progression of human hepatocellular carcinoma.
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PMID:Nuclear antigens reacted with sera and ascites of hepatocellular carcinoma patients. 283 90

Autoimmune serum from a patient with scleroderma was shown by indirect immunofluorescence to label nucleoli in a variety of cells tested including: rat kangaroo PtK2, Xenopus A6, 3T3, HeLa, and human peripheral blood lymphocytes. Immunoblot analysis of nucleolar proteins with the scleroderma antibody resulted in the labeling of a single protein band of 34 kD molecular weight with a pI of 8.5. Electron microscopic immunocytochemistry demonstrated that the protein recognized by the scleroderma antiserum was localized exclusively in the fibrillar region of the nucleolus which included both dense fibrillar and fibrillar center regions. Therefore, we have named this protein "fibrillarin". Fibrillarin was found on putative chromosomal nucleolar organizer regions (NORs) in metaphase and anaphase, and during telophase fibrillarin was found to be an early marker for the site of formation of the newly forming nucleolus. Double label indirect immunofluorescence and immunoelectron microscopy on normal, actinomycin D-segregated, and DRB-treated nucleoli showed that fibrillarin and nucleolar protein B23 were predominantly localized to the fibrillar and granular regions of the nucleolus, respectively. RNase A and DNase I digestion of cells in situ demonstrated that fibrillarin was partially removed by RNase and completely removed by DNase. These results suggest that fibrillarin is a widely occurring basic nonhistone nucleolar protein whose location and nuclease sensitivity may indicate some structural and/or functional role in the rDNA-containing dense fibrillar and fibrillar center regions of the nucleolus.
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PMID:Fibrillarin: a new protein of the nucleolus identified by autoimmune sera. 293 2

A DNA-binding nonhistone protein, protein BA, was previously demonstrated to co-localize with U-snRNPs within discrete nuclear domains (Bennett, F. C., and L. C. Yeoman, 1985, Exp. Cell Res., 157:379-386). To further define the association of protein BA and U-snRNPs within these discrete nuclear domains, cells were fractionated in situ and the localization of the antigens determined by double-labeled immunofluorescence. Protein BA was extracted from the nucleus with the 2.0 M NaCl soluble chromatin fraction, while U-snRNPs were only partially extracted from the 2.0 M NaCl-resistant nuclear structures. U-snRNPs were extracted from the residual nuclear material by combined DNase I/RNase A digestions. Using an indirect immunoperoxidase technique and electron microscopy, protein BA was localized to interchromatinic regions of the cell nucleus. Protein BA was noted to share a number of chemical and physical properties with a family of cytoplasmic enzymes, the glutathione S-transferases. Comparison of the published amino acid composition of protein BA and glutathione S-transferases showed marked similarities. Nonhistone protein BA isolated from saline-EDTA nuclear extracts exhibited glutathione S-transferase activity with a variety of substrates. Substrate specificity and subunit analysis by SDS polyacrylamide gel electrophoresis revealed that it was a mixture of several glutathione S-transferase isoenzymes. Protein BA isolated from rat liver chromatin was shown by immunoblotting and peptide mapping techniques to be two glutathione S-transferase isoenzymes composed of the Yb and Yb' subunits. Glutathione S-transferase Yb subunits were demonstrated to be both nuclear and cytoplasmic proteins by indirect immunolocalization on rat liver cryosections. The identification of protein BA as glutathione S-transferase suggests that this family of multifunctional enzymes may play an important role in those nuclear domains containing U-snRNPs.
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PMID:Nonhistone protein BA is a glutathione S-transferase localized to interchromatinic regions of the cell nucleus. 293 45

Earlier studies suggested that the 70K (70 X 10(3) Mr) polypeptide is a nuclear matrix (associated) protein since it is the only U1 RNP-associated antigen that is not released from the nucleus after treatment of the cell with, successively, detergents, DNase I and/or RNase A and high salt. The possibility that the 70K protein functions in the binding of U1 RNP to the nuclear matrix is now further substantiated by the finding that U1 RNP particles that did or did not contain the 70K protein could be isolated, depending on the method of isolation. When U1 RNP particles were obtained by means of sonic disruption of the nucleus they contained the 70K polypeptide, whereas particles that were isolated by extraction at room temperature and a slightly alkaline pH lacked the 70K protein but contained the intact U1 RNA and the other U1 RNA-associated proteins. During interphase the localization of the 70K protein is restricted to the nucleus, giving a dot-like distribution pattern with exclusion of the nucleoli. During prophase to late anaphase the protein is dispersed throughout the entire cytoplasm with the exception of the chromatin regions. Immunofluorescence studies, using a monoclonal anti-70K antibody in combination with human autoimmune sera that react with U1 RNA-associated proteins, demonstrate that the 70K protein is localized in those areas of the cell where other U RNP proteins occur, also during mitosis. Topoisomerase I and nuclear lamins, typical nuclear matrix proteins, show completely different distribution patterns in all phases of the cell cycle. Assembly of the nuclear envelope is attended by the re-formation of the clustered appearance of the 70K antigen. These results suggest that, although associated with the nuclear matrix fraction in interphase cells, the 70K protein remains associated with the U1 RNP particles during cell division.
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PMID:Distribution of the 70K U1 RNA-associated protein during interphase and mitosis. Correlation with other U RNP particles and proteins of the nuclear matrix. 295 89

Retinol (vitamin A alcohol), which plays an important role in the differentiation of epithelia, can be transferred to chromatin in vitro. Rat liver chromatin can accept retinol in a specific and saturable manner only when the retinol is presented as a complex with cellular retinol-binding protein (CRBP). A partial characterization of the nuclear components responsible for accepting retinol is reported here. A preparation of solubilized chromatin isolated from liver nuclei was able to accept retinol from its complex with CRBP as described previously for nuclei and chromatin. The binding of retinol to chromatin was noncovalent. However, chromatin prepared from nuclei which were incubated with DNase I or micrococcal nuclease did not accept retinol specifically. Chromatin in the form of mono and dinucleosomes also did not accept retinol. However, treatment of nuclei with RNase did not affect the specific binding of retinol. Furthermore, it has been found that retinol was not transferred to purified double or single stranded DNA. These results are interpreted to indicate that the transfer of retinol to specific nuclear binding sites requires a higher order of chromatin structure than that occurring in nucleosome preparations.
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PMID:Partial characterization of nuclear binding sites for retinol delivered by cellular retinol binding protein. 298 10


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