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)

A ribonucleoprotein fraction that contains most of the rapidly labelled hnRNA has been isolated from gently ruptured oocytes of Triturus cristatus. This fraction consists of large aggregates of ribonucleoprotein and has a high (30:1) ratio of protein to RNA. The labelled RNA is contained in ribonucleoprotein particles that have a density of 1.27 g/cm3 in Cs2SO4 gradients (1.39 g/cm3 after formaldehyde fixation in CSCl gradients). Evidence is presented that the particles are associated in vivo with a fibrillar protein network. When the ribonucleoprotein aggregates are treated with ribonuclease, high salt concentration and nonionic detergent, a fibrillar protein residue is produced which contains many species of protein but a few that have electrophoretic characteristics that are identical to major ribonucleoprotein particle proteins. Isolated labelled hnRNA has been shown to bind specifically polypeptides of molecular weight 60 000 and 54 000 that are found in both particle and fibril preparations. In binding assays in vitro, these polypeptides are found to interact with mRNA to a lesser extent and not with rRNA. The isolated 60 000-Mr and 54 000-Mr proteins have the dual ability of forming ribonucleoprotein 'particles' with hnRNA and of polymerizing to generate 10-nm fibrillar structures in the absence of RNA. The possible cellular functions of these proteins are discussed.
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PMID:Interaction of the hnRNA of amphibian oocytes with fibril-forming proteins. 714 Jul 70

In situ hybridization analysis of shrimp histological sections, utilizing Taura syndrome virus (TSV) specific cDNA probes, is the most sensitive diagnostic technique presently available for the detection of this penaeid shrimp viral disease. However, false negative genomic probe results are obtained frequently from samples of Pacific white shrimp, Penaeus vannamei, that have been preserved with Davidson's AFA (acetic acid, formaldehyde, alcohol) fixative and that, otherwise, demonstrate pathognomonic TSV lesions by routine histology. This problem was linked to prolonged storage of shrimp samples in Davidson's fixative, which is highly acidic (pH approximately 3.5-4). Degradation of TSV genomic RNA was hypothesized to be due to either fixative- induced acid hydrolysis and/or acidophilic endogenous ribonuclease activity. Routine H and E histology and in situ hybridization analyses were conducted on equal numbers of TSV infected P. vannamei juveniles that were preserved for four different time periods (2, 6, 10 and 14 days) with either Davidson's fixative or a new, near neutral (pH approximately 6.0-7.0), RNA-friendly fixative (R-F) that was developed by the authors. In situ hybridization assays were conducted with and without R Nase precautions and all of the samples tested contained moderate to severe TSV lesions by routine histology. Davidson's preserved samples produced weak TSV probe signals after 2 days fixation, but did not react with the probes in those samples that were stored for > 6 days in the fixative. In contrast, TSV was detectable by gene probe in all of the time treatment samples preserved with the new R-F fixative. Equivalent in situ hybridization results were obtained when the same samples were analyzed in the absence of RNase-free conditions. These findings suggest that TSV RNA is degraded when samples are stored in an acidic fixative, such as Davidson's, for more than 2 days and that this problem can be prevented through preservation of shrimp samples with R-F fixative. The efficacy of this new fixative is demonstrated and the results show that RNase-free conditions are not necessary for conducting TSV in situ hybridization analyses.
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PMID:A new RNA-friendly fixative for the preservation of penaeid shrimp samples for virological detection using cDNA genomic probes. 925 34

Gene expression is one key mechanism to regulate cell growth and differentiation. It is usually determined by Northern blotting or RT-PCR. However, studies with primary cell cultures are frequently hampered due to contaminating cells such as fibroblasts. We have developed a method to isolate intact full-size mRNA from sorted cells. In many cell types, e.g. cardiac myocytes, cell sorting without prior fixation revealed complete RNA breakdown. Based on a murine fibroblast cell line (AKR-2B), ethanol and formaldehyde at various concentrations and pre-treatment with ribonuclease inactivating DEPC were compared with each other. Fixation with 75% ice-cold DEPC-pre-treated ethanol for 5 min yielded mostly intact RNA. In contrast, antibody staining prior to sorting required 15 min fixation. Addition of RNAse-free BSA (0.5%) and 2 mM CaCl2 optimised the cell recovery ratio and thus a better RNA yield (60% compared to control) after sorting than former studies. Northern blotting and RT-PCR show the intact mRNA species beta-actin. Furthermore, dependent on the cellular PCNA content, we have demonstrated the cell cycle dependent cdk2 and cyclin A expression. This fast and reliable method allows to isolate intact full-size mRNA species appropriate for Northern blotting and RT-PCR to monitor gene expression.
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PMID:Isolation of full-size mRNA from cells sorted by flow cytometry. 1048 62

The effects of proteolytic enzymes, ribonuclease, and deoxyribonuclease upon a fibrous component of chick embryo mitochondria, which was previously shown to have many fixation and staining properties characteristic of the bacterial nucleoplasm, are reported. Pepsin digestion of formaldehyde-fixed tissues removed the membranes and matrices of mitochondria, but a pepsin-resistant fibrous material remained which was heavily stained by uranyl and lead ions. Experiments on a DNA "model system" showed that DNA treated with osmium tetroxide can be depolymerized by deoxyribonuclease. Zinc ions strongly inhibited the depolymerization of DNA. Digestion of osmium tetroxide-fixed tissues (fixed only briefly) with deoxyribonuclease for 1 hour greatly reduced the Feulgen staining of the nuclei, and after 4 hours the Feulgen reaction was completely abolished. The reduction and the disappearance of the Feulgen reaction in nuclei was paralleled by partial to complete digestion of the mitochondrial fibers in the regions studied (after 1 and 4 hours, respectively), without any other obvious changes in cellular structures. When deoxyribonuclease was inhibited by the addition of zinc ions, the nuclear Feulgen reaction was not diminished, nor were the mitochondrial fibers removed. Buffer control incubations for deoxyribonuclease and ribonuclease did not alter the structure or staining properties of the mitochondrial fibers, nor did incubation with ribonuclease. The latter reaction digested the cytoplasmic and nucleolar ribosomes after a 4-hour incubation period, in parallel with the abolishment of toluidine blue staining. The results contribute further evidence that these mitochondria contain deoxyribonucleic acid.
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PMID:INTRAMITOCHONDRIAL FIBERS WITH DNA CHARACTERISTICS. II. ENZYMATIC AND OTHER HYDROLYTIC TREATMENTS. 1408 39

A strategy is presented for comparative analysis of glycoproteins in which the variation of protein concentration, variation of glycosylation site occupancy and variation of glycoform profile can be determined. A comparative study was performed using stable isotope labeling of glycopeptides and peptides by formaldehyde-H(2) and formaldehyde-D(2) and analysis by ESI-MS analysis. The relative intensity of the nonglycosylated peptide provided information about protein concentration variation. Variation of the glycoform profile was obtained by comparing the glycoform profile of d(0)- and d(4)-dimethyl labeled glycopeptides. By knowing the variation of protein concentration and the variation of glycoform profile, the variation of glycosylation site occupancy could be calculated. The utility of the proposed strategy was demonstrated with ribonuclease B with different protein concentrations, different levels of glycosylation site occupancy and different glycoform profiles.
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PMID:A comparative study of glycoprotein concentration, glycoform profile and glycosylation site occupancy using isotope labeling and electrospray linear ion trap mass spectrometry. 2256 Feb 80


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