Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.1 (RNase)
 16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A factor preventing spontaneous dissolution of germinal vesicle (GVBD) of mouse oocytes in vitro was isolated from bovine granulosa cells and designated as granulosa cell factor (GCF). Granulosa cells from medium sized (2-5 mm) follicles were suspended in 10 mM Tris-HCl buffer, pH 8.3, containing 1 M urea and 5 mM EDTA. GCF was separated from the extract by gel filtration on Sephadex G-25 column. The molecular weight (Mr) of GCF was estimated to be less than 6,000 daltons. Oocytes treated with GCF and resuspended in control medium resumed GVBD. The partially purified GCF lost GVBD-preventing activity when digested with pronase but retained its activity when treated with DNase, RNase, or glycosidase. GCF was stable to heating at 100 degrees C for 15 min, not absorbed by charcoal, and did not bind to Concanavalin A-Sepharose 4B. The present findings suggest that GCF is a heat-stable polypeptide and its action is reversible.
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PMID:A factor from bovine granulosa cells preventing oocyte maturation. 671 45

Unfixed, isolated metaphase chromosomes were used as template in an in vitro RNA synthesis assay. In these conditions, no RNA synthesis was observed by autoradiography. Transcription was effective after treatment with methanol-acetic acid, HCl 0,2 N, NaCl 0,35 M or pancreatic RNase. Transcription is the most important after treatment with RNase. This result points out the problem of the role of RNA in inhibition of chromosome during mitosis.
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PMID:[Role of RNA in the inactivation of chromosomes during the metaphase]. 680 64

Antibodies to the RNase-sensitive RNP and to the RNase-resistant Sm nuclear antigens were used to affinity purify these antigens from a saline extract of rabbit thymus acetone powder. Determination of the protein subunits recovered by either glycine-HCl, pH 2.8, or 2.5 M MgCl2 elution on gradient sodium dodecyl sulfate-polyacrylamide electrophoresis containing mercaptoethanol revealed that RNP was composed of five proteins with mol. wts from 10,000 to 15,000 whereas Sm contained the same or similar five chains plus six additional subunits with mol. wts from 21,000 to 42,000. RNase treatment of the thymus extract increased the recovery in Sm of the same bands compared to untreated extract. Thus, RNP and Sm appear to have different numbers of protein components and RNP may be a subset of Sm. Sucrose gradient centrifugation of the 125I-labeled, pH 2.8 eluted antigens gave peaks of 3 and 6S for RNP and Sm, respectively. Sucrose gradient centrifugation of the crude untreated thymus extract followed by quantitative single radial immunodiffusion analysis of each fraction produced a broad peak from 16S to the top of the gradient while pretreatment of the extract with RNase resulted in a discrete 6S peak. These results indicate that in rabbit thymus acetone powder native RNP and Sm exist as larger polydisperse complexes with additional material including RNA and that after acid elution or RNase treatment the antigens are found in a smaller monodisperse form.
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PMID:Characterization of RNP and Sm ribonucleoprotein nuclear antigens. 681 Jan 1

Bovine retinas contain a factor that stimulates proliferation of aortic endothelial cells in culture as well as neovascularization on the chicken chorioallantoic membrane. The stimulatory activity has been partially purified from a balanced salt solution extract of bovine retinas. The stability of the activity to acid pH was utilized as the first step in purification. The acid-treated material was subjected to ion-exchange chromatography on DEAE Bio-Gel A. The material that was eluted with 0.1 M NaCl/50 mM Tris.HCl (pH 7.6) stimulated both the proliferation of vascular endothelial cells in culture and angiogenesis in vivo. A molecular weight of between 50,000 and 100,000 for the native molecule is suggested by ultrafiltration and gel chromatography; gel electrophoresis of partially purified material under reducing and denaturing conditions revealed the presence of two major components with apparent molecular weights of 50,000 and 70,000. The endothelial cell stimulatory activity was stable to pH 4-9 and to heating at up to 60 degrees C for 30 min, to incubating for 1 hr with DNase or RNase, to incubating for 2 hr with immobilized Pronase or immobilized or soluble trypsin, and to treating with 1 mM 2-mercaptoethanol, 4 M urea, or 2 M guanidine. Heating at 65-75 degrees C for 30 min, boiling for 2 min, extreme acidic (pH 2) or basic (pH 12) conditions, treating with 0.02% NaDodSO4, or incubating (5 hr) with soluble Pronase destroyed the activity. The significance of an angiogenic factor derived from retina stems from the fact that neovascularization is a serious complication in a number of ocular diseases.
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PMID:Angiogenic activity from bovine retina: partial purification and characterization. 694 16

A rapid inexpensive method is presented for detecting peripheral blood lymphocyte chromatin activation by the neutral red "topo-optical" reaction, which causes strong and easily measurable birefringence in the lymphocyte nuclei. This reaction can be enhanced by fixing the cells with 150 mM/l NaCl in 70% ethanol and/or by treating the unfixed cellular suspensions with 0.2 M/l HCl to remove histones. In histone-removed preparations, 30 min DNase I treatment almost completely abolished the birefringent reaction, whereas RNase treatment resulted in only 18% loss. Chromatin activation induced by enzyme inhibition increased chromatin birefringence significantly. The same phenomenon could be induced in sensitive subjects' lymphocytes by specific antigens or haptens much more rapidly. The monocytes were not activated to a significant extent. In non-sensitive subjects different kinetics of antigen or hapten-dependent activation and no cytotoxic effects have been observed. Depletion of T-lymphocytes in vivo in SLE patients or by in vitro treatment with 0.5 mM/l KCN as well as with 0.02% trypsin has caused a significant drop in the mean chromatin birefringence. The effect of trypsin was reversible.
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PMID:Measurement of lymphocyte activation by a chromatin topo-optical reaction. Mechanism and specificity of the test. 723 31

Based on the finding that the nuclear DNA of cancerous cells is much more unstable than that of non-cancerous cells, yielding a larger amount of single-stranded DNA by acid hydrolysis, we developed a new method of cancer cell detection in ordinary pathological sections by immunohistochemical staining with anti-single-stranded DNA antiserum after acid hydrolysis. Methylated bovine serum albumin was conjugated with heat-denatured calf thymus DNA and used as the antigen of single-stranded DNA, and white male rabbits were immunized with the antigen to obtain the polyclonal antiserum. Ordinary paraffin-embedded sections were prepared from the formalin-fixed biopsy specimens taken from 482 malignant tumors and 73 benign tumors of human epithelial and non-epithelial origins. Additional 82 biopsy specimens of borderline malignancy were also examined. The sections were immunohistochemically stained with the antiserum after RNase digestion and DNA denaturation by hydrolysis with 2 N HCl at 30 degrees C. The acid hydrolysis for 20-30 min was optimum for the specimens fixed with 10% buffered formalin at room temperature for 16-24 hrs, and all cancerous cells were specifically stained positive, in sharp contrast to the negative stainability of all non-cancerous cells including inflammatory cells. This new method gives us decisive help in making diagnosis of malignancy in daily pathological examination. The possibility of malignancy of borderline lesion was discussed.
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PMID:Differential immunohistochemical staining of cancerous cells with anti-single-stranded DNA antiserum in ordinary pathological paraffin section after DNA-denaturation by acid hydrolysis. 751 May 37

Although the hepatitis delta virus genome contains multiple open reading frames, only one of these reading frames is known to be expressed during replication of the virus. This open reading frame encodes two distinct molecular species of hepatitis delta antigen (HDAg), p24 delta and p27 delta, depending on the location of the stop codon which terminates translation. We found antibody specific for p27 delta to be capable of precipitating p24 delta in extracts of infected liver, indicating that p27 delta and p24 delta form heterologous complexes in vivo. After cross-linking with 0.05% glutaraldehyde, specific HDAg dimers were detected in antigen prepared from both the liver and serum of an HDV-infected woodchuck carrier of woodchuck hepatitis virus. Guanidine HCl-denatured HDAg extracted from liver and dialyzed against phosphate-buffered saline sedimented in rate-zonal sucrose density gradients as 15S multimeric complexes. These 15S multimers were stable in the presence of 1.2% Nonidet P-40. After RNase digestion, the 15S complex was reduced to a 12S complex without associated RNA, while boiling for 3 min in 1% sodium dodecyl sulfate-0.5% 2-mercaptoethanol further reduced the 15S complex to 3S HDAg monomers. In the absence of glutaraldehyde cross-linking, HDAg extracted from liver migrated as monomer species in reducing and nonreducing gels, suggesting that the conserved cysteine residue present in p27 delta does not play a role in the formation of either dimers or multimers. On the other hand, an amino-terminal chymotrypsin-digested HDAg fragment, with a predicted length of 81 or less amino acids, retained the ability to form dimers, consistent with the hypothesis that a coiled-coil motif present between residues 27 and 58 may play a role in HDAg protein interactions in vivo.
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PMID:Hepatitis delta virus antigen forms dimers and multimeric complexes in vivo. 767 57

A novel noncollagenous protein of the mineralized matrix of bovine bone was isolated by ion exchange and gel permeation chromatography. The apparent M(r) of the protein is 63,000 as determined by SDS-polyacrylamide gel electrophoresis. The protein is a rather minor constituent in bone and could not be detected in other connective tissues by enzyme-linked immunosorbent assay of guanidine HCl extracts. The 63-kDa protein was detected in the osteoid and around the osteocytes upon immuno-histochemical staining of bovine compact bone. The sequence of the 63-kDa protein was deduced from cDNA clones isolated from a rat calvaria lambda gt11 expression library. The protein contains two centrally located EF-hand Ca(2+)-binding domains. Seven heptad repeats are present indicating the ability of the protein for coiled-coil interactions. Ability to bind calcium was confirmed by 45Ca2+ binding to protein blotted onto nitrocellulose membrane. The protein was synthesized in calvaria explants as detected by immunoprecipitation of radiolabeled protein from the culture medium. Although the protein can be detected in biochemical amounts in bone only, varying amounts of mRNA for this protein were detected in several rat tissues by RNase protection assay with highest levels in rat calvaria. This extracellular protein corresponds to a mouse protein called nucleobindin.
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PMID:Isolation, characterization, and primary structure of a calcium-binding 63-kDa bone protein. 789 Jul 46

We and others have described methods to label specific nucleic acid sequences in fixed cells by reverse in situ transcription (IST). They are simple alternatives to the tedious steps of in situ hybridization with labeled probes. We have favored use of thermostable DNA polymerases after heat denaturation of template secondary structure, accompanied by synthesis of cDNA from an annealed primer, but the approach has been limited by the low reverse transcriptase (RT) activity of Taq polymerase and delayed detection methods. We have improved the technique by the use of recombinant Thermus thermophilus (rTth) DNA polymerase and fluorescein-12-dUTP (FIST). Jurkat T lymphocytes were stimulated with ionomycin + phorbol myristate acetate to produce interleukin-2 (IL-2) mRNA in vitro overnight. They were cytospun onto slides and fixed in 70% ethanol + 30% DEPC-treated water, acetone, and air-dried. The slides were placed on a temperature-controlled heating block, and the cell spot was covered with a plastic coverslip. The temperature was raised to 95 degrees C, and 5-10 microliters of modified Perkin-Elmer/Cetus rTth RT reaction mix was injected under the edge of the coverslip. Each 10 microliters of mix in DEPC-water contained 10 mM Tris-HCl, pH 8.3, 90 mM KCl, 1 mM MnCl2, 1 mM dithiothreitol, 10 U placental ribonuclease inhibitor, 0.125 mM dA,C,GTPs, 0.1 mM fluorescein-12-dUTP, 2 U rTth DNA polymerase, and 4 pM 22-mer oligonucleotide primer, which spanned the second intron of IL-2. After 3 min at 95 degrees C, 1 min at 50 degrees C and 10 min at 72 degrees C, the slides were washed in 0.5 x phosphate-buffered saline, pH 7.0, at 42 degrees C, in 70% ethanol, 100% ethanol, and air-dried. The cells were mounted in antifade solution (2% n-propyl gallate in 70% glycerol), and could be viewed immediately by fluorescence microscopy. Image analysis showed that stimulated Jurkat cells were brighter than uninduced controls or those treated with RNase or without polymerase or primer. FIST appears to be useful for the detection of specific mRNAs in single cells.
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PMID:In situ transcription with Tth DNA polymerase and fluorescent nucleotides. 798 81

The slow refolding of guanidine-HCl-denatured ribonuclease-A was studied by volume change and by kinetic CD at 222 and 276 nm. Dilatometric measurements revealed that on refolding there is a fast volume change of +232 mL/mol of protein. This is followed by a very slow nonexponential change that takes about 25 min to reach equilibrium. By adding varying amounts of (NH4)2SO4, the slow volume change curve was resolved into 2 concurrent reactions. The faster of the 2 slow events entails a negative volume change of -64 mL/mol of protein and appears to arise from proline isomerization. The slower process, attended by a positive change of +53 mL/mol of protein, has properties consistent with the "XY" reaction of Lin and Brands (1983, Biochemistry 22:563-573). This reaction is so named because the conformational nature of neither its initial (Y) nor its final state (X) is known; the transition is characterized solely by its absorbance and fluorescence kinetics. These are the first direct physical measures attributable to the "XY" process. The early formation of a compact structure in the event responsible for the rapid +232-mL/mol volume change, however, is consistent with the sequential model of folding (Cook KH, Schmid FX, Baldwin RL, 1979, Proc Natl Acad Sci USA 76:6157-6161; Kim PS, Baldwin RL, 1980, Biochemistry 19:6124-6129). The usefulness of volume change measurements as a method of detecting structural rearrangements was confirmed by finding agreement between time constants obtained from parallel volume change and kinetic CD experiments. The measured volume changes arise from both changes in hydration and changes in the packing of atoms in the interior of the protein.
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PMID:Slow-folding kinetics of ribonuclease-A by volume change and circular dichroism: evidence for two independent reactions. 800 82


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