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:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A procedure for the purification of Neisseria meningitidis lipopolysaccharide (LPS) from outer membrane vesicles (OMV) in spent growth media was developed. Five different LPS strains of group A N. meningitidis were grown in tryptic soy broth with vigorous aeration for 36-48 h, and centrifuged to collect both cells and supernatants. The amount of LPS in the OMV in the supernatants was higher or at least equal to that in the cells. The OMV in each supernatant were concentrated, pelleted by ultracentrifugation, and treated with 2% sodium deoxycholate to dissociate LPS from OMV. The LPS was then separated from capsular polysaccharides, proteins and phospholipids by gel filtration on Sephacryl S-300 column in 1% sodium deoxycholate, and precipitated from the column fractions in 70% ethanol. In addition, LPS was also extracted from cells with hot phenol-water, ultracentrifuged once after treatment with ribonuclease, and purified on Sephacryl S-300. When compared with an improved phenol-water extraction method, the LPS obtained from either OMV or cells by the above methods gave a 40-180% increase in yield. The LPS also had much higher activities in limulus amebocyte lysate assay, rabbit pyrogenic test, and enzyme-linked immunosorbent assay. The LPS purified from cells and from OMV were indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.
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PMID:Purification of rough-type lipopolysaccharides of Neisseria meningitidis from cells and outer membrane vesicles in spent media. 177 80

Along with classical lipopolysaccharide (LPS), O-specific material not precipitated by ultracentrifugation has been isolated from the water-phenol extract of S. sonnei avirulent strain 9090 possessing complete antigenic properties. The purification of O-antigen contained in the supernatant fluid has been carried out by the gel filtration of the fluid, previously treated with ribonuclease, in a column packed with Sephadex G-100. The polysaccharide nature of O-antigen thus obtained, the absence of lipid A and KDO and the low content of hexoses, or core-specific saccharides of S. sonnei LPS, in this antigen make it possible to classify this material with O-components of microbial cells, described by different authors as "native protoplasmic polysaccharide" or "L-hapten" and formed by polymers of LPS O-side chains. The content of this component in S. sonnei strains under study is, on the average, 2.5% of the weight of dry microbial substance. L-hapten preparations obtained in the course of our investigations have been found to contain two O-specific antigens detected by immunoelectrophoresis and immunodiffusion, as well as by sedimentation in saccharose gradient, where they form peaks corresponding to 4.3 S and 10.8 S. This polysaccharide O-antigen is supposed to be capable of interaction with ribosomal particles and suitable for use as a component of ribosomal dysentery vaccines.
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PMID:[O-specific L-hapten in the composition of Shigella sonnei]. 248 42

A new approach has been developed to circumvent the problems of false positive reactions in the Limulus Amoebocyte Lysate (LAL) assay for lipopolysaccharide (LPS) in root surface materials. These LAL-reactive materials include thrombin, thromboplastin, ribonuclease, ribonucleic acid, lipoteichoic acid and peptidoglycan fragments. In the present study, hot phenol/water extraction of these substances followed by ultracentrifugation of the resulting aqueous phases reduced their concentrations to very low levels. Furthermore, the application of Polymyxin B/Sepharose 4B affinity chromatography to these extracts enabled their intrinsic LAL-activity to be determined. Use of these techniques to assay root surface materials has identified LPS as being the major LAL-reactive material present. The mean LPS yield for the periodontally involved teeth was 4.13 micrograms/tooth, representing 2.82 micrograms/root. In contrast, the mean yield of LPS for the periodontally uninvolved teeth was 3.12 ng/tooth.
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PMID:Identity of limulus amoebocyte lysate-active root surface materials from periodontally involved teeth. 346 18

A lipopolysaccharide (LPS) fraction was extracted from Nichols, nonpathogenic Treponema pallidum by the hot, phenol-water procedure. The LPS was freed of nucleic acids and water-soluble proteins by successive exposures to ribonuclease, deoxyribonuclease, and Pronase. Purified LPS responded positively in a colorimetric assay for lipopolysaccharide. Electron microscope examination of the LPS both before and after purification demonstrated a heterogeneous mixture of forms including spheres, doughnuts, and ribbons. The trilaminar nature of the ribbon forms was observed by both negative staining and thin sectioning. Lyophilization of the LPS caused an increase in the number and length of ribbon forms seen. Results suggest that the surface layers of treponemes are similar to those of gram-negative bacteria.
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PMID:Ultrastructure of lipopolysaccharide isolated from Treponema pallidum. 412 67

In mice, active protection against Pseudomonas aeruginosa could be induced with two fractions derived from a crude preparation of ribosomes from P. aeruginosa. The two fractions were obtained by gel filtration chromatography of the crude ribosomal preparation on Sepharose CL-2B. In fraction I, less than 1% of the ribonucleic acid (RNA) applied to the column was recovered. Fraction II contained RNA and protein in a ratio of 1.94. The presence of ribosomes in this fraction was confirmed by analysis on a sucrose density gradient. The protection by fraction I was not affected by treatment with ribonuclease; in contrast, incubation of fraction II with ribonuclease completely abolished active protection. Fraction I contained lipopolysaccharide (LPS) as was indicated by the presence of 2-keto-3-deoxyoctonic acid. No LPS was found in fraction II. The adjuvant dimethyl dioctadecyl ammonium bromide enhanced the protection by fraction II; however, immunity by a low dose of fraction I was abolished by dimethyl dioctadecyl ammonium bromide. Protection by fractions I and II appeared to be restricted to the homologous serotype of P. aeruginosa. These results indicate that RNA is required for protection by fraction II. Active protection by fraction I is likely due to LPS.
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PMID:Ribonuclease-sensitive ribosomal vaccine of Pseudomonas aeruginosa. 615 37

Ribosomal and ribonucleic acid (RNA)-rich preparations derived from Salmonella typhimurium were examined for their ability to enhance the primary in vitro antibody response of normal mouse spleen cell cultures to sheep erythrocytes. Both of these fractions were consistently more active in elevating the antibody response of normal mouse splenocytes from lipopolysaccharide (LPS) responder mice than was LPS. Furthermore, injection of mice with either the ribosomal or RNA-rich fraction induced antibody response helper factor activity in 2-h post-treatment serum similar to that induced by LPS. Endotoxin low-responding C3H/HeJ mice were stimulated to release helper factors by ribosomes and the RNA extracts but not by LPS. Treatment of the ribosomes and RNA fractions with ribonuclease destroyed their ability to stimulate the production of the helper factor in serum of treated mice. Therefore, it appears likely that ribosomes and RNA-rich fractions stimulated an intermediate helper factor due to the presence of RNA and not LPS.
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PMID:Induction of immunoenhancing factors for murine splenocyte cultures by Salmonella typhimurium ribosome and ribonucleic acid extracts. 616 65

To obtain information about the nature of the immunogens in the ribosomal vaccine (fraction II) of Pseudomonas aeruginosa, we studied the specificity of rabbit antibodies to fraction II. Crossed immunoelectrophoresis demonstrated the presence of antibodies which precipitated with ribosomal antigens, but not with lipopolysaccharide (LPS). By means of an enzyme-linked immunosorbent assay, antibodies to LPS were detected in antibodies to fraction II. Antibodies to fraction II could protect mice against a lethal challenge with P. aeruginosa. Absorption experiments demonstrated that the protective ability of antibodies to fraction II was due to antibodies to cell envelope antigens, whereas antibodies to ribosomal antigens did not contribute to the protection. Antibodies to LPS could be detected in mice 1 week after a single vaccination with fraction II. It was concluded that the protective activity of fraction II was due, at least in part, to the presence of LPS in the ribosomal vaccine. Treatment of fraction II with ribonuclease decreased the protective activity of the ribosomal vaccine. Addition of synthetic polyadenylic acid-polyuridylic acid restored the protective activity of ribonuclease-treated fraction II, indicating that RNA in the ribosomal vaccine might act as an adjuvant or a carrier in the presentation of the of the contaminating cell envelope antigens. The protective activity and the toxicity of fraction II were compared with the protective activity and the toxicity of fraction I, which contained cell envelope components, including LPS, and of purified LPS. The results indicated that protection by the ribosomal vaccine was associated with a slightly higher toxicity than was protection by fraction I, whereas purified LPS was the most toxic vaccine.
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PMID:Evidence for the presence of lipopolysaccharide in a ribonuclease-sensitive ribosomal vaccine of Pseudomonas aeruginosa. 678 42

In many diseases, tissue hypoxia occurs in conjunction with other inflammatory processes. Since previous studies have demonstrated a role for leukocytes in ischemia/reperfusion injury, we hypothesized that endothelial hypoxia may "superinduce" expression of an important leukocyte adhesion molecule, E-selectin (ELAM-1, CD62E). Bovine aortic endothelial monolayers were exposed to hypoxia in the presence or absence of tumor-necrosis factor alpha (TNF-alpha) or lipopolysaccharide (LPS). Cell surface E-selectin was quantitated by whole cell ELISA or by immunoprecipitation using polyclonal anti-E-selectin sera. Endothelial mRNA levels were assessed using ribonuclease protection assays. Hypoxia alone did not induce endothelial E-selectin expression. However, enhanced induction of E-selectin was observed with the combination of hypoxia and TNF-alpha (270% increase over normoxia and TNF-alpha) or hypoxia and LPS (190% increase over normoxia and LPS). These studies revealed that a mechanism for such enhancement may be hypoxia-elicited decrements in endothelial intracellular levels of cAMP (<50% compared with normoxia). Addition of forskolin and isobutyl-methyl-xanthine during hypoxia resulted in reversal of cAMP decreases and a loss of enhanced E-selectin surface expression with the combination of TNF-alpha and hypoxia. We conclude that endothelial hypoxia may provide a novel signal for superinduction of E-selectin during states of inflammation.
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PMID:Hypoxia enhances stimulus-dependent induction of E-selectin on aortic endothelial cells. 869 47

Systemic exposure to gram-negative bacterial substances such as lipopolysaccharide (LPS, or endotoxin) induces an uncontrolled, massive inflammatory reaction which culminates in multiple system organ failure and death. Septic shock often does not respond to corticosteroids; however, certain low-molecular-weight antioxidant compounds have been discovered to possess potent anti-inflammatory action, and some of these novel compounds can rescue animals from experimentally induced septic shock. Phenyl-N-tert-butylnitrone (PBN) is the archetype of the nitrone class of antioxidants which we have previously shown to suppress LPS-induced cytokine biosynthesis in vivo. Using a multiprobe ribonuclease protection assay, we now demonstrate the ability of PBN to suppress proapoptotic gene expression in the LPS-induced model of endotoxic shock. The broad-spectrum gene-suppressive affects of PBN are discussed in the context of inflammatory signal transduction and models are proposed to explain why certain antioxidants may also possess anti-inflammatory and antiapoptotic properties.
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PMID:Phenyl-N-tert-butylnitrone demonstrates broad-spectrum inhibition of apoptosis-associated gene expression in endotoxin-treated rats. 1022 40

Bone marrow-culture-derived macrophages activated with interferon-gamma and lipopolysaccharide produced less nitric oxide (NO) when cultured with vesicular stomatitis virus (VSV)-infected BALB/c3T3 (3T3-VSV) than macrophages activated in an identical manner and cultured alone, with uninfected BALB/c3T3 (3T3), or with P815. However, all four groups of macrophages produced nearly the same amount of interleukin-6 (IL-6). Addition of VSV to activated macrophages did not change the amount of NO produced. The amount of NO generated by two non-macrophage sources of NO was not affected by the presence of either P815 or 3T3-VSV. Reverse transcriptase-polymerase chain reaction showed a decrease in the amount of inducible nitric oxide synthase (iNOS) but not IL-6 mRNA from macrophages cocultured with 3T3-VSV compared with macrophages cocultured with P815. The reduction in iNOS mRNA was confirmed by ribonuclease protection assay. When RAW 264.7 transfected with an iNOS regulatory construct were activated and incubated with 3T3-VSV there was a decrease in the expression of the reporter luciferase gene and NO production but not IL-6 production compared with cells incubated with either medium alone or with P815.
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PMID:Interaction with vesicular stomatitis virus-infected BALB/c3T3 cells inhibits the synthesis of nitric oxide in activated murine bone marrow culture-derived macrophages. 1033 88


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