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:3.1.27.4 (ribonuclease)
6,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A nonradioactive modification of the traditional ribonuclease protection assay is described and applied to the detection of two messenger RNAs. The biotinylated probes are stable and easy to handle, which permits the processing of large numbers of samples. Chemiluminescence is generated with streptavidin-conjugated alkaline phosphatase and provides sensitivity equal to or greater than that achieved with the radioactive detection method.
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PMID:Ribonuclease protection assay: use of biotinylated probes for the detection of two messenger RNAs. 837 93

Stable, nonradioactive riboprobes were used in ribonuclease protection assays to monitor the changes in cyclin mRNA expression during cell cycle progression in human mammary epithelial cells. Probes labeled with biotin demonstrated sufficient sensitivity (comparable to 32P) to detect these low-abundance mRNAs and thus offer a safe and easy alternative to traditional radioactive assays. Three detection systems based on chemiluminescence generated by horseradish peroxidase or alkaline phosphatase were compared for sensitivity, background and ease of use.
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PMID:Detection of cyclin messenger RNAs by nonradioactive ribonuclease protection assay: a comparison of four detection methods. 858 15

An advantage of exporting a recombinant protein to the periplasm of Escherichia coli is decreased proteolysis in the periplasm compared with that in the cytoplasm. However, protein degradation in the periplasm also occurs. It has been widely accepted that the thermodynamic stability of a protein is an important factor for protein degradation in the cytoplasm of E.coli. To investigate the effect of the thermodynamic stability of an exported protein on the extent of proteolysis in the periplasm, barnase (an extracellular ribonuclease from Bacillus amyloliquefaciens) fused to alkaline phosphatase leader peptide was used as a model protein. A set of singly or doubly mutated barnase variants were constructed for export to the E.coli periplasm. It was found that the half-life of the barnase variants in vivo increased with their thermodynamic stability in vitro. A dominant factor for the final yield of exported barnase was not exportability but the turnover rate of the barnase variant. The yield of a stabilized mutant was up to 50% higher than that of the wild type. This suggests that exporting a protein to the periplasm and using protein engineering to enhance the stability can be combined as a strategy to optimize the production of recombinant proteins.
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PMID:Relationship between thermal stability, degradation rate and expression yield of barnase variants in the periplasm of Escherichia coli. 901 Sep 33

Alterations of the renal function in the isolated perfused rat kidney system after application of two bacterial RNases, Bacillus intermedius RNase (binase) and ribonuclease produced by Bacillus amyloliquefaciens (barnase), were investigated with two different treatment regimens in comparison with catalytically inactive derivates of the enzymes, photooxidated at the active site His101 binase and inactive mutant His102Gln barnase. For the in vitro approach the test enzymes were dissolved in the perfusion media and applied to the kidney after removal from the animal. Alternatively, the test ribonucleases were administered to rats in vivo and the renal effects were assessed in the isolated perfused rat kidney 1 and 6 h after treatment. In the in vitro regimen both active enzymes induced time- and concentration-dependent nephrotoxicity reflected in enhancement of urinary protein excretion, decline of glucose reabsorption, increase of gamma-glutamyltranspeptidase and alkaline phosphatase activities in urine. In vivo administration of active binase induced functional impairment of the isolated perfused organ in a similar way. None of the inactive RNases in both regimens and at all concentrations tested altered any renal parameter. The results suggest that RNA degradation may be involved in the nephrotoxic effects of bacillar RNases.
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PMID:Nephrotoxic effects of bacterial ribonucleases in the isolated perfused rat kidney. 916 Jan 9

Cytokine-driven activation of hepatic stellate cells (HSC) in tissue injury and inflammation is a key pathogenetic event in liver fibrogenesis leading to an expanded pool of matrix producing myofibroblasts (MFB) which represent the transformed counterpart of HSC. We hypothesize that expansion of the pool of MFB might also be accomplished by modulation of apoptosis, which plays an opposite and complementary role to mitosis in the cellular homeostasis. We characterized the susceptibility of HSC in primary culture and of MFB in secondary culture to apoptosis induced by the soluble Fas ligand (sFasL) and related the effects to the expression levels of Fas (APO-1/CD95) and some major proapoptotic and contra-apoptotic protooncogenes. MFB showed a dose-dependent apoptotic reaction upon exposure to sFasL as evidenced by a strong increase of nucleosomal DNA fragments, loss of cellular DNA, positive TUNEL reaction, and annexin staining. The effect was found only if protein synthesis (cycloheximide) or RNA synthesis (actinomycin D) were arrested. HSC maintained for various times in primary culture were completely resistant to sFasL in combination with cycloheximide, but in late primary cultures (day 7 onward) an increasing susceptibility to sFasL-mediated apoptosis was developed. By semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis and alkaline phosphatase-anti-alkaline phosphatase staining Fas receptor was identified both in HSC and MFB at comparable expression levels. The expression of the contra-apoptotic protooncogenes bcl-2 and bcl-xl was found to be much stronger in early HSC than in late HSC and MFB as shown by ribonuclease protection assay. The expression of bcl-2 was additionally confirmed by semiquantitative RT-PCR and immunoblotting. Proapoptotic bax was found in comparable quantities at the RNA level in HSC and MFB but at the protein level MFB showed increased bax expression. It is concluded that transformation of HSC to MFB is paralleled by an increasing sensitivity to sFasL-mediated apoptosis, which might be related to a strong decrease of bcl-2 and bcl-xl expression, leading to a preponderance of proapoptotic gene expression in MFB. Modulation of apoptotic susceptibility of transforming HSC could be an important complementary pathway in the pathogenesis of fibrosis.
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PMID:Transformation-dependent susceptibility of rat hepatic stellate cells to apoptosis induced by soluble Fas ligand. 969 16

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

We describe a method for obtaining radioactive fingerprints from nonradioactive ribonucleic acid. Fragments derived by T1 ribonuclease digestion of RNA are dephosphorylated with bacterial alkaline phosphatase. When these fragments are used as primers for the reaction of primer dependent polynucleotide phosphorylase with [alpha-(32)P]GDP in the presence of T1 ribonuclease the 3'-hydroxyl group of each fragment becomes phosphorylated. The degree of phosphorylation is reasonably uniform. The method has been applied to T1 ribonuclease digests of Escherichia coli tRNA(Met) (f); the oligonucleotides were further analyzed by spleen phosphodiesterase digestion. In a similar manner fingerprints of pancreatic ribonuclease digests of RNA can be obtained, when [alpha-(32)P]UDP, polynucleotide phosphorylase and pancreatic ribonuclease are used.
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PMID:Fingerprinting nonradioactive ribonucleic acid with the aid of polynucleotide phosphorylase. 1079 69

RNase U2 is an endoribonuclease secreted by the fungus Ustilago sphaerogena. Its genomic DNA (rnu2), containing an intron of 116 bp, has been isolated and cloned. The corresponding cDNA has also been synthesized. The recombinant RNase U2 was successfully produced in Pichia pastoris, fused to the yeast alkaline phosphatase signal peptide. The recombinant RNase U2, purified by affinity chromatography, contains three extra amino acids at its amino-terminal end and retains the enzymatic and spectroscopic properties of the natural fungal protein.
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PMID:Ribonuclease U2: cloning, production in Pichia pastoris and affinity chromatography purification of the active recombinant protein. 1093 Jul 32

A method has been described for isolation of the specific cytoplasmic granules of rabbit polymorphonuclear leucocytes. Homogeneous suspensions of leucocytes were disrupted by lysis in 0.34 M sucrose. This procedure liberated the cytoplasmic contents of the cell and dissolved a considerable proportion of the nuclei. Following disruption, the sucrose lysate was separated into three fractions by differential centrifugation, i.e. 400 g or nuclear pellet, 8,200 g or granule pellet and the postgranule supernate. Microscopic examination revealed that the 8,200 g pellet was composed of intact granules as well as occasional mitochondria. The other two fractions were morphologically heterogeneous. Studies with isolated granules demonstrated their lysis by a variety of weak acids and surface-active agents. When buffered solutions were employed between the ranges of pH 2.0 and 9.0, granule lysis began at pH 5.5 and was complete at pH 4.0. Chemical analysis disclosed that the granule pellet contained protein and phospholipid with only traces of nucleic acids. Approximately 70 to 80 per cent of the total cellular antimicrobial agent phagocytin was present in the granule fraction. This material was liberated from the granules by acid (pH 5.0 or lower). Studies on selected enzymes showed that acid phosphatase, alkaline phosphatase, nucleotidase, ribonuclease, deoxyribonuclease, and beta glucuronidase were predominantly localized in the granule fraction. Approximately 50 per cent of total cellular lysozyme and cathepsin were also present in the 8,200 g pellet. Disruption of the granules was associated with the release of the majority of granule protein and enzymes in a non-sedimentable form. The properties and composition of rabbit polymorphonuclear leucocyte granules seem to be analogous to those of liver lysosomes.
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PMID:The isolation and properties of the specific cytoplasmic granules of rabbit polymorphonuclear leucocytes. 1369 90

Fibroblast growth factors (FGFs) are involved in stimulation of angiogenesis in tumors and other pathological circumstances. Increased activity of normal skeletal muscles resulting from chronic electrical stimulation is a very potent stimulus for capillary growth but a relationship between the initiation of this angiogenesis and the involvement of autocrine growth factors has yet to be established. Although FGF expression has been reported in muscles stimulated for 3 weeks, capillary growth is underway significantly earlier, beginning around 3 days. The present experiments have therefore studied the possible involvement of basic fibroblast growth factor (FGF-2) in stimulated rat fast skeletal muscles prior to, and coincident with, capillary growth. Muscle contractions were induced via electrodes implanted in the vicinity of the peroneal nerve and maintained for 8h/day for 2, 4 or 7 days. Capillary/fiber ratio (C/F), based on staining of capillary endothelium for alkaline phosphatase, was not changed in either extensor digitorum longus (EDL) or tibialis anterior (TA) after 2 days stimulation, but increased in TA stimulated for 4 days and in both muscles after 7 days. The expression of mRNA for FGF-2, detected by ribonuclease protection assay, was decreased in all stimulated muscles compared with control or contralateral muscles; immunohistochemistry showed FGF-2 gene product in nerves and larger blood vessels but not in capillaries. There was no evidence from immunohistochemistry for up-regulation of receptors flg and bek for FGF-2. The presence of FGF-2, flg and bek in arterioles may indicate a possible role for FGF-2 in the regulation of blood flow since we have previously shown it to be a dilator of small arterioles. However, based on the lack of correlation between changes in capillary density and the expression of mRNA and protein for FGF-2 and its receptors, it is unlikely that it is directly linked with the initiation of angiogenesis resulting from chronic activity in skeletal muscles.
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PMID:Lack of involvement of basic fibroblast growth factor (FGF-2) in capillary growth in skeletal muscles exposed to long-term contractile activity. 1451 78


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