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)

Thirty-nine strains of gram-positive microaerophilic cocci isolated from cases of heifer and dry-cow mastitis were biochemically characterized with the API 50E and API-ZYM test kit systems, gas-liquid chromatography for analysis of end products of glucose metabolism, and anaerobic biochemical tests (L. V. Holdeman, E. P. Cato, and W. E. C. Moore, Anaerobe Laboratory Manual, Virginia Polytechnic Institute, Blacksburg, 1977). Strains were screened for production of a variety of extracellular enzymes on substrate-containing agar plates and for hemolysin and coagulase production. Antibiotic susceptibility and sensitivity tests were also performed. The microaerophilic cocci displayed homogeneity with respect to the majority of the biochemical tests used; i.e., greater than or equal to 90% of the strains were consistently positive or negative in any one test and probably represent one species. All produced deoxyribonuclease, ribonuclease, and hyaluronidase, and 92% were positive for chondroitin sulfatase. Catalase and coagulase tests were negative. Greening was observed on bovine blood agar. Acetic and succinic acids were produced by all strains as the only detectable products of glucose metabolism. The strains were susceptible to penicillin G, cefoxitin, doxycycline, and chloramphenicol and were resistant to clindamycin, novobiocin, and metronidazole. Their taxonomic position remains unclear.
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PMID:Biochemical characterization of unidentified microaerophilic cocci isolated from heifer and dry-cow mastitis. 39 19

Intact RNA from various rat organs was isolated by an efficient and rapid method. This method of RNA isolation is a modification of an earlier method that uses guanidinium isothiocynate followed by extraction in the presence of sarcosyl, acetate and phenol. The RNA obtained by the method reported here was comparable with the RNA prepared by the CsCl2 ultracentrifugation method and the commercially available kit based on published methods. The quality of RNA was found suitable for Northern blotting analysis, RNase protection assays and reverse transcriptase-polymerase chain reaction (RT-PCR). Since reverse transcriptase is active in the buffer used for Taq DNA polymerase, only one reaction needs to be set up. We also found that the use of aurintricarboxylic acid in the RNA preparation prevents the degradation of RNA during storage. Expression of low density lipoprotein (LDL) receptor, apolipoprotein (apo) AI, AII and AIV mRNAs were quantified in various rat organs. Our results indicated that rat LDL receptor mRNA is expressed in several organs whereas apoAI and AIV mRNAs were expressed mainly in the liver and intestine. However, apo AII mRNA is expressed mainly in the liver. Unlike mice and some species of monkeys, in the rat apoAI mRNA is expressed at 5-6 times higher levels in the intestine compared to liver. Apo AIV mRNA abundance was also found to be several fold higher in intestine compared to hepatic tissues. We present here, for the first time, data on the absolute amounts of LDL receptor, apoAI, AII and AIV mRNA in various rat organs which were quantified by a novel RNase protection/solution hybridization assay.
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PMID:Expression of low density lipoprotein receptor, apolipoprotein AI, AII and AIV in various rat organs utilizing an efficient and rapid method for RNA isolation. 137 76

A dot blotting assay using digoxigenin hydrazide (Glycan detection kit, Boehringer Mannheim Biochemicals) was used to screen an endoproteinase Lys-C peptide map of ribonuclease B for the presence of glycopeptides. The carbohydrate content of the identified glycopeptide fraction was then further characterized by monosaccharide analysis using high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). The tandem use of a hydrazide dot blotting technique to screen peptide maps for glycopeptides and subsequent use of HPAE-PAD to identify the monosaccharide composition of glycopeptide hydrolyzates proved to be a quick, sensitive and reliable method for identifying glycopeptides and analyzing their glycan composition without derivatization of the carbohydrate.
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PMID:Sensitive blotting assay for the detection of glycopeptides in peptide maps. 220 15

Studies were conducted with the goal of developing a kit for assaying anti-Chlamydia pneumoniae antibodies in human serum which would enable judging positive cases with high specificity by means of an objective numerical index. Thus, an enzyme-linked immunosorbent assay (ELISA) method employing a C. pneumoniae outer membrane complex protein was established. Elementary bodies (EB) were purified from the YK-41 strain of C. pneumoniae, and subsequent treatment with Sarkosyl, DNase and RNase yielded chlamydial outer membrane complex (COMC). COMC was employed as the antigen and immobilized on 96-well microplates for ELISA method. This ELISA method was used to test 51 serum specimens from patients who had been demonstrated to be positive for C. pneumoniae antigen (throat swab: PCR positive), and the levels of IgG, IgA and IgM antibodies were assayed. For each specimen, comparison was made with the antibody titers determined by the micro immunofluorescence test (Micro-IF method). The results showed good correlation coefficients of 0.950 for IgG, 0.852 for IgA and 0.866 for IgM. In addition, the two assay methods showed the following high agreement rates: 90.2% for IgG, 84.3% for IgA and 82.4% for IgM. Specimens which did not yield the same results with the ELISA method and Micro-IF method were subjected to analysis by Western blot method, and the rates of agreement with the ELISA results were 80% for IgG, 87.5% for IgA and 88.9% for IgM. These data indicate the efficacy of this new ELISA method. Moreover, COMC was reacted with mouse antisera to three Chlamydia species, and the mouse IgG antibody was assayed. Anti-C. pneumoniae antiserum showed the strongest reactivity, whereas weaker reactivity was shown by anti-C. trachomatis antiserum (1/32nd of the reactivity of the anti-C. pneumoniae antiserum) and anti-C. psittaci antiserum (1/4th). In addition, sera from patients infected with C. trachomatis or C. psittaci (Psittacosis) were subjected to the ELISA method using COMC from C. pneumoniae. It was found that the correlation between the ELISA and Micro-IF methods was higher in relation to the anti-C. pneumoniae antibody titer than either the anti-C. trachomatis antibody titer or anti-C. psittaci antibody titer. These findings indicate this new assay kit based on the ELISA method has high specificity for C. pneumoniae.
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PMID:[Assay of specific anti-Chlamydia pneumoniae antibodies by ELISA method. 1. Evaluation of ELISA kit using outer membrane complex]. 889 May 50

The aim of the present study was to test the hypothesis that the vasoconstrictive peptide endothelin-1 is upregulated in ischemia and reperfusion in skeletal muscle. Sixty-eight Wistar rats were included in the series: 12 served as controls that did not undergo the procedure, 16 underwent sham operations, and 40 were subjected to a modified tourniquet ischemia for 3 hours and 20 minutes. Of the 40 rats, 16 were killed at the end of the ischemic period, 16 underwent reperfusion for 2 hours, and eight underwent reperfusion for 72 hours. Areas of necrosis were measured by morphometry in hematoxylin and eosin-stained cross sections of the anterior tibial muscles that had been reperfused for 72 hours. Sections from the controls, the muscles that had not been reperfused, and the reperfused muscles were immunostained for endothelin-1. Serum endothelin-1 levels in blood samples from the aorta were determined with a commercial enzyme immunoassay kit. The anterior tibial muscle was harvested for preproendothelin-1 mRNA analysis with RNase protection assay. The hematoxylin and eosin-stained sections showed extensive necrosis with an acellular core of no reperfusion. The muscular core demonstrated weak immunostaining for endothelin-1 in all sections, a subfascial narrow brim of fibers showed enhanced immunoreactivity at the end of ischemia, and all fibers outside the core stained by 2 hours after the start of reperfusion. After 72 hours of reperfusion, the fibers outside the core stained positive in a checkerboard-like pattern. There were no differences in serum endothelin-1 levels between the groups. Preproendothelin-1 mRNA analysis with RNase protection assay showed 2-fold upregulation at the end of ischemia and 4-fold upregulation after 2 hours of reperfusion (p = 0.001). This study supports the hypothesis that both ischemia and reperfusion upregulate endothelin-1 in skeletal muscle.
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PMID:Endothelin-1 is upregulated during skeletal muscle ischemia and reperfusion. 956 85

The ribonuclease protection assay (RPA) represents a sensitive method to detect and quantify RNA levels. It can be adapted to allow the simultaneous analysis of more than 10 different mRNAs. The multiprobe RPA kit mCK-5 from PharMingen was used to analyze the expression of chemokines in CCR5 chemokine receptor knockout mice. Upon careful analysis it was found that the mCK-5 kit is defective and can lead to false results for the chemokines IP-10 and MCP-1. The problem is caused by a long-known sequence polymorphism within the 3'-untranslated region of the murine IP-10 gene. This polymorphism leads to a protected IP-10 fragment approximately 20 nucleotides shorter than expected, yielding a length similar to the protected MCP-1 fragment from the mCK-5 kit. Since the identification of specific transcripts with this kit is based exclusively on the size of the various protected fragments, false-negative results for IP-10 together with false-positive results for MCP-1 can be obtained. Interestingly, the polymorphism was found not only in 129/CD-1 mice, but also in MRL and SJL/J mice. To facilitate troubleshooting in the future, all templates from the mCK-5 set were isolated and sequenced.
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PMID:The mCK-5 multiprobe RNase protection assay kit can yield erroneous results for the murine chemokines IP-10 and MCP-1. 1106 40

Towards a goal of detecting scaled-up DNA adducts as altered deoxynucleotides by mass spectrometry, we have set up a practical and general method for isolating DNA-derived deoxyribonucleoside-5'-monophosphates devoid of ribonucleotides starting with a 1 g sample of mammalian tissue. The method is practical because costs have been minimized, and it is general because it can be applied to a more difficult sample such as mouse skin or non-fresh calf liver. The procedure, consisting of a series of steps that were largely gleaned and tuned from prior literature, proceeds as follows: (1) homogenize the tissue in sodium dodecyl sulfate; (2) digest with ribonuclease A, ribonuclease TI, alpha-amylase and proteinase K; (3) partition between water and phenol; (4) precipitate the DNA with ethanol followed by redissolving and dialysis; and (5) digest with nuclease P1 and phosphodiesterase I followed by ultrafiltration and boric acid gel chromatography. The yellow to brown color of DNA from difficult tissues only persisted up to the ultrafiltration step. Apparently this DNA was contaminated with iron-containing proteins. Residual ribonucleotides were not observable (<0.1%) by HPLC in the final sample. Without boric acid gel chromatography, residual contamination by ribonucleotides was about 1% even when the DNA was purified before digestion by phenol partitioning followed by use of a Genomic Tip kit from Qiagen.
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PMID:Phenolic extraction of DNA from mammalian tissues and conversion to deoxyribonucleoside-5'-monophosphates devoid of ribonucleotides. 1554 80

E(rns) is an envelope glycoprotein of classical swine fever virus (CSFV) with RNase activity. The purpose of this study was to produce an active E(rns) for further applications using the yeast secreted expression system. The E(rns) gene was cloned into the expression vector pGAPZalphaC which was introduced into Pichia pastoris. Expression of E(rns) protein in culture supernatant was confirmed by Western blot analysis using both the monoclonal antibody against CSFV E(rns) and CSFV-positive swine serum. The yeast-expressed E(rns) (yE(rns)) was shown to have N-linked glycosylation and to form homodimer of 74 kDa molecules. All monomer, homodimer, and deglycosylated forms of yE(rns) demonstrated intrinsic ribonuclease activity and a clear preference for uridine-rich sequence. A direct sandwich blocking enzyme-linked immunosorbent assay (ELISA) based on the yE(rns) was developed with a high sensitivity and specificity. The yE(rns) which possesses enzymatic activity and retains antigenicity may provide a useful material for developing a diagnostic kit.
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PMID:Secreted expression of the classical swine fever virus glycoprotein E(rns) in yeast and application to a sandwich blocking ELISA. 1621

In this chapter, we describe a simple and relatively rapid technique for detecting low-abundance slug mRNA in cultured cells. The procedure uses nonradioactive digoxigenin-labeled RNA probes that are more sensitive than deoxyribonucleic probes and simpler to detect than radioactively labeled probes. Cells are grown in glass chamber slides, fixed in an acidic fixative, dehydrated through ethanol and xylene, permeabilized in pepsin, and post-fixed. Slides are then incubated overnight at 37 degrees C in a buffer containing 50% formamide and 5-10 ng/gL probe. Excess probe is removed by washing at high temperature in low-salt buffer and by treatment with RNase. Probe is detected immunohistochemically with an anti-digoxigenin Fab fragment, using a tyramide amplification kit to enhance signal and Fast Red for visualization. The technique has the advantages of probe stability and sensitivity, hybridization at low temperature, rapidity and sensitivity of probe detection, and the production of permanent specimens.
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PMID:An in situ hybridization technique to detect low-abundance slug mRNA in adherent cultured cells. 1678 Feb 1

The proteomic study on human temporal lobe can help us to understand the physiological function of CNS in normal as well as in pathological state. Proteomic tools are potent for the assessment of protein stability post mortem. In this pilot study, the human temporal lobe biopsy specimen with chronic pharmacoresistant temporal lobe epilepsy (TLE) and autopsy specimen in control were separated by 2-DE. Using MALDI-TOF-MS and MS/MS, 375 protein spots were identified which were the products of 267 genes. Six down-regulated and 23 up-regulated protein spots in the autopsy specimen were ascertained after the gel image analysis with the ImageMaster software. A number of proteins that include neurotransmitter metabolic and glycolytic enzymes, cytoprotective proteins and cytoskeleton were found decreased while the precursor of apolipoprotein A-I increased in the TLE brain. We tried several methods to prepare the protein samples and found that DNase and RNase treatment, ultracentrifugation and Amersham clean-up kit purification can improve gel separation quality. This work optimized the sample preparation method and constructed a primary protein database of human temporal lobe and found some proteins with remarkable level change probably involved in the post-mortem process and chronic pharmacoresistant TLE pathogenesis.
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PMID:Proteomic analysis and comparison of the biopsy and autopsy specimen of human brain temporal lobe. 1691 69


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