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Query: UNIPROT:P43026 (lipopolysaccharide)
 62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Treatment of Escherichia coli K-12 strain S15, containing a normal amount of phospholipase A, with ethylenediaminetetraacetate (EDTA) resulted in an increase in sensitivity of the organism to actinomycin D. Strain S17, a mutant deficient in both detergent-resistant phospholipase A and detergent-sensitive phospholipase A, was considerably less sensitive to the antibiotic after the treatment. Both strains released lipopolysaccharide after EDTA treatment, indicating that this outer membrane component alone is not the barrier to actinomycin in these organisms. The phospholipase A-deficient strain released less alkaline phosphatase, a periplasmic enzyme. EDTA treatment of S15 resulted in the accumulation of free fatty acids, indicative of phospholipase A activation. Cells briefly treated with EDTA regained the barrier to actinomycin when incubated in growth media, and the cessation of the accumulation of free fatty acids was in approximate temporal agreement with restoration of the barrier. Cells in which phospholipase A was activated by brief exposure to EDTA synthesized relatively more phosphatidylethanolamine than did untreated cells in the initial period after dilution into growth media. These experiments suggest that the EDTA-induced loss of outer membrane barrier function of E. coli K-12 is mediated through the activation of phospholipase A.
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PMID:Effect of ethylenediaminetetraacetate on phospholipids and outer membrane function in Escherichia coli. 10 74

A simple preparative method is described for isolation of the cytoplasmic and outer membranes from E. coli. The characteristics of both membrane fractions were studied chemically, biologically, and morphologically. Spheroplasts of E. coli K-12 strain W3092, prepared by treating cells with EDTA-lysozyme [EC 3.2.1.17], were disrupted in a French press. The crude membrane fraction was washed with 3 mM EDTA-10% (w/v) sucrose, pH 7.2, and the cytoplasmic membranes and outer membranes were separated by sucrose isopycnic density gradient centrifugation. The crude membrane fraction contained approximately 10% of the protein of the whole cells, 0.3% of the DNA, 0.7% of the RNA, 0.3% of the peptidoglycan, and about 30% of the lipopolysaccharide. The cytoplasmic membrane fraction was rich in phospholipid, while the outer membrane fraction contained much lipopolysaccharide and carbohydrate; the relative contents of lipopolysaccharide and carbohydrate per mg protein in the cytoplasmic membrane fraction were 12 and 40%, respectively, of the contents in the outer membrane fraction. Cytochrome b1, NADH oxidase, D-lactate dehydrogenase [EC 1.1.1.28], succinate dehydrogenase [EC 1.3.99.1], ATPase [EC 3.5.1.3], and activity for concentrative uptake of proline were found to be localized mainly in the cytoplasmic membranes; their specific activities in the outer membrane fraction were 1.5 to 3% of those in the cytoplasmic membrane fraction. In contrast, a phospholipase A appeared to be localized mainly in the outer membranes and its specific activity in the cytoplasmic membrane fraction was only 5% of that in the outer membrane fraction. The cytoplasmic and outer membrane fractions both appeared homogeneous in size and shape and show vesicular structures by electron microscopy. The advantages of this method for large scale preparation of the cytoplasmic and outer membrane fractions are discussed.
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PMID:Cytoplasmic membrane vesicles of Escherichia coli. A simple method for preparing the cytoplasmic and outer membranes. 12 74

The cell envelope of Neisseria gonorrhoeae, colony type 4, was studied. Outer membrane was isolated by lysozyme and ethylenediaminetetraacetic acid treatment of plasmolyzed cells according to Wolf-Watz et al. (1973). The degree of purity of the membrane preparations was checked by electron microscopy. The membrane fraction obtained had a density of 1.25 g/cm(3), was rich in phospholipase A and lysophospholipase, and contained only 10% of the total membrane activity of succinate dehydrogenase and d-lactate dehydrogenase. The outer membrane protein profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed at least six major proteins. The predominating protein showed a molecular weight of 35,000. The lipopolysaccharide component was characterized by gas chromatography. The carbohydrates found were galactose, glucose, and glucosamine. d-Glycero-l-manno-heptose was present in very low amounts. Lipid A contained lauric acid, stearic acid, and beta-hydroxy-myristic acid. About 20% of the fatty acids in the outer membrane was derived from lipid A. The phospholipids were characterized as phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. There was no evidence for a lipoprotein anchored to the peptidoglycan. The peptidoglycan of N. gonorrhoeae was of the chemotype I. The cell envelope of N. gonorrhoeae was found to be highly permeable to gentian violet. Cell envelopes of one penicillin-resistant and two penicillin-sensitive strains were compared. Only moderate differences in fatty acid composition were found.
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PMID:Cell envelope of Neisseria gonorrhoeae: outer membrane and peptidoglycan composition of penicillin-sensitive and-resistant strains. 80 26

Recently we reported that rapid killing of Escherichia coli by granulocytes or granulocyte fractions is accompanied by an equally rapid and discrete increase in permeability of the microbial envelope (Beckerdite, Mooney, Weiss, Franson, and Elsbach. 1974. J. Exp. Med. 140: 396-409). Most of this permeability-increasing activity (PI) is found in a crude granule preparation. PI is quantitatively recovered in a 23,000-g supernatant fraction (Sup II) after sulfuric acid extraction of granulocyte homogenates prepared in water. PI is nondialyzable, destroyed by pronase and trypsin, stable at 4degreesC for at least 2 mo, and destroyed by heating at 94degreesC. Anionic substances, such as heparin sulfate and isolated E. coli lipopolysaccharide, bind to and inhibit PI. PI has been purified up to 1,000-fold from homogenate in a yield of 50percent by acid extraction and carboxymethyl-Sephadex chromatography. Such purified fractions have bactericidal activity that equals that of disrupted granulocytes and Sup II, are similarly enriched with respect to granule-associated phospholipase, and protease activities. Whereas E. coli, sensitive to PI, binds or inactivates solubilized PI, a resistant strain of Serratia marcescens does not. Binding of PI to sensitive microorganisms seems to be necessary for expression of its biological activity since both the apparent binding to and the biological effect of PI on E. coli are completely blocked by 10-20 mM Mg2+ or Ca2+. Mg2+ or Ca2+ can reverse the effect on E. coli permeability produced by Sup II or the carboxymethyl-Sephadex fraction but not that produced by granulocyte homogenate. The close association of bactericidal, phospholipase A2, and permeability-increasing activities towards several gram-negative bacterial species suggests that they may be related.
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PMID:Partial characterization and purification of a rabbit granulocyte factor that increases permeability of Escherichia coli. 108 9

The extractable and bound lipids of a moderately halophilic gram-negative rod, strain No. 101 (wild type) grown in a medium containing 2 M NaC1, were examined. The extractable lipids were separated into at least 8 components by using thin-layer chromatography. The major phospholipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an unidentified phosphoglycolipid in the whole cells, cell envelopes and outer membrane preparations, commonly. Judging from mild alkaline hydrolysis and exzymatic treatment with phospholipase A2, C and D, the unidentified phosphoglycolipid possessing Pi, glycerol, fatty acids and glucose in a molar ratio of 1 : 2 : 2 : 1, appeared likely to be a glucosyl derivative of phosphatidylglycerol. No glucuronic acid containing lipid was detected. The exractable lipid composition varied greatly with the concentrations of NaC1 in the medium and the stages of bacterial growth. The most characteristic phosphoglycolipid in this organism increased up to 25% of the total phospholipids with the addition of 1% glucose in the medium. The major fatty acids of the extractable lipids were C16:0, C16:1, C18:0, C18:1 and cyclopropanoic C17 and C19 acids and these compositions were very similar for each phospholipid. The cyclopropanoic fatty acids predominated as growth proceeded. The fatty acids of the bound lipids comprised a high concentration of 3-hydroxydodecanoic acid. The esterified fatty acids of the lipopolysaccharide molecule seemed to contain a wide variety of hydroxy and non-hydroxy shorter chain fatty acids, while the amide-linked fatty acids consisted almost entirely of 3-hydroxydodecanoic acid.
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PMID:Lipids and fatty acids of a moderately halophilic bacterium, No. 101. 125 64

Porins, a family of hydrophobic proteins located in the outer membrane of the cell wall of gram-negative bacteria and lipopolysaccharide (LPS), were shown to stimulate the synthesis of platelet activating factor (PAF), a phospholipid mediator of inflammation and endotoxic shock, by cultured human glomerular mesangial cells (MC). The synthesis of PAF induced by porins was rapid (peak at 20 min) and independent either from contamination by LPS or from generation of an endotoxin-induced cytokine such as tumor necrosis factor (TNF) since it was not prevented by cycloheximide, an inhibitor of protein synthesis or anti-TNF blocking antibodies. LPS also stimulated PAF synthesis by MC. However, the kinetic of PAF synthesis induced by LPS was biphasic with an early and transient peak at 10 minutes and a second and sustained peak at three to six hours. This second peak required an intact protein synthesis and was prevented by anti-TNF antibodies, suggesting the dependency on LPS-induced synthesis of TNF. Experiments with labeled precursors demonstrated that in MC, either after stimulation with porins or LPS, PAF was synthesized via the remodeling pathway that involves acetylation of 1-0-alkyl-sn-glyceryl-3-phosphorylcholine (2-lyso-PAF) generated from 1-0-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase A2 (PLA2) activity. Porins and LPS, indeed, induced PLA2-dependent mobilization of [14C]-arachidonic acid that was inhibited by p-bromodiphenacylbromide (PBDB). PBDB, an inhibitor of PLA2, also blocked PAF synthesis by preventing the mobilization of 2-lyso-PAF, the substrate for PAF-specific acetyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Porins and lipopolysaccharide stimulate platelet activating factor synthesis by human mesangial cells. 133 27

Body reaction to injury comprises two major pathways: the immune response, predominantly mediated by IL-1 and other cytokines, and neuroendocrine mechanisms, resulting in an increased glucocorticoid production. Each has distinct effects on prostaglandin (PG) production, which may in turn mediate both systemic and local inflammatory responses. The interactions, if any, between the two systems on PG synthesis have not been studied. Bovine aortic endothelial cell cultures were used and prostacyclin (PGI2) production was monitored. Cells were treated with dexamethasone (Dex) 10(-6) M and IL-1 10-30 U/ml in one experiment, and lipopolysaccharide (LPS, 0.1-1.0 micrograms/ml) in another experiment, separately or in combination, for either 2 or 24 + 2 h. While Dex was without effect, IL-1 and LPS stimulated PGI2 in a concentration- and time-dependent manner. Short exposure to Dex (2 h) enhanced the stimulation by IL-1 and LPS. On the contrary, more prolonged exposure (24 + 2 h) reversed the effects of IL-1 and LPS, resulting in PGI2 levels below the baseline. A biphasic regulation by Dex was also observed with increasing concentrations of LPS. Dex was actually ineffective by itself, but it enhanced PGI2 production in combination with lower concentrations of LPS, while abolishing the influence of higher concentrations of this agonist. The data suggest that Dex may initially stimulate phospholipase A2 (PLA2) activity, while inhibiting it later. This biphasic behavior may be attributed to different concentrations of a PLA2-modulating protein, possibly lipocortin, that accumulate during exposure to Dex.
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PMID:Biphasic regulation by dexamethasone of IL-1- and LPS-stimulated endothelial prostacyclin production. 141 91

We describe here and partially characterize a Ca(2+)-independent phospholipase A2 that acts on phosphatidylinositol in normal human peripheral blood neutrophils. Neutrophils incubated with myo-[3H]inositol to form [3H]phosphatidylinositol and then stimulated with the calcium ionophore A23187 produced [3H]lysophosphatidylinositol. This deacylation was further characterized in cell sonicates by the specific release of [3H]arachidonic acid from exogenous [1-14C]stearoyl-2-[3H]arachidonyl-phosphatidylinositol. This phospholipase A2 is Ca2+ independent, retaining full activity in the presence of 10 mM EDTA, and is optimally active at alkaline pH (pH 9). A phosphatidylinositol-hydrolyzing phospholipase C activity was characterized by the production of [3H]-/[14C]-diglycerides. This phospholipase C activity is dependent on the presence of exogenous Ca2+ and is optimally active at neutral pH (pH 7.5). The lipoxygenase/cyclooxygenase inhibitors eicosatetraenoic acid and nordihydroguaiaretic acid and the calmodulin antagonist trifluoperazine were the only compounds tested that showed significant inhibition of phospholipase A2 activity. However, none of these phosphatidylinositol-hydrolyzing phospholipase A2 inhibitory compounds resulted in the accumulation of any radiolabeled diglyceride, monoglyceride, or phosphatidic acid intermediates. Following subcellular fractionation on sucrose density gradients, it was found that the plasma membrane-enriched fractions contained the highest specific activity for phospholipase A2; however, the cytosolic fraction contained a large part of the total phospholipase A2 activity. Furthermore, when neutrophils were first exposed to several agents, including lipopolysaccharide, phorbol myristate acetate, or N-formyl-methionyl-leucyl- phenylalanine, and then subfractionated, there was a significant translocation of the enzyme activity from the cytosolic fraction to the membrane-enriched fractions. These data suggest that this Ca(2+)-independent, phosphatidylinositol-hydrolyzing phospholipase A2 may play an important role in early cell activation, providing free arachidonic acid for subsequent metabolism into biologically active eicosanoids.
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PMID:Phosphatidylinositol hydrolysis by phospholipase A2 and C activities in human peripheral blood neutrophils. 146 38

Body reaction to injury comprises two major pathways: the immune response is predominantly mediated by IL-1 and other cytokines, and neuroendocrine mechanisms, resulting in increased glucocorticoid production. Each has distinct effects on prostaglandin (PG) production, which may in turn mediate both systemic and local inflammatory responses. The interactions, if any, between the two systems on PG synthesis have not been studied. Bovine aortic endothelial cell cultures were used and prostacyclin (PGI2) production was monitored. Cells were treated with dexamethasone (Dex) 10(-6) M and IL-1 10-30 U/ml in one experiment, and lipopolysaccharide (LPS, 0.1-1.0 microgram/ml) in another experiment, separately or in combination, for either 2 or 24 + 2 h. While Dex was without effect, IL-1 and LPS stimulated PGI2 in a concentration and time dependent manner. Short exposure to Dex (2 h) enhanced the stimulation by IL-1 and LPS. On the contrary, more prolonged exposure (24 + 2 h) reversed the effects of IL-1 and LPS, resulting in PGI2 levels below baseline. A biphasic regulation by Dex was also observed with increasing concentrations of LPS. Dex was actually ineffective by itself, but it enhanced PGI2 production in combination with lower concentrations of LPS, while abolishing the influence of higher concentrations of this agonist. The data suggest that Dex may initially stimulate phospholipase A2 (PLA2) activity, while inhibiting it later. This biphasic behavior may be attributed to different concentrations of a PLA2-modulating protein, possibly lipocortin, that accumulate during exposure to Dex.
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PMID:Biphasic regulation by dexamethasone of IL-1- and LPS-stimulated endothelial prostacyclin production. 152 96

Two potent inflammatory mediators, interleukin 1 (IL-1) and tumor necrosis factor (TNF) as well as lipopolysaccharide (LPS) increased group II phospholipase A2 (PLA2) mRNA levels, which resulted in enhanced secretion of the PLA2 enzyme from rat smooth muscle cells. cAMP-elevating agents also stimulated the release of PLA2 and increased the mRNA, but IL-1, TNF and LPS did not affect cAMP levels. Furthermore, the effects of TNF and cAMP-elevating agents were not additive but synergistic. Therefore, we concluded that the level of rat group II PLA2 mRNA is controlled at least by two distinct mechanisms, one involves cAMP and the other is mediated by TNF, IL-1 and LPS. This study also suggests important roles of group II PLA2 in pathogenesis of vascular inflammation.
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PMID:Group II phospholipase A2 mRNA synthesis is stimulated by two distinct mechanisms in rat vascular smooth muscle cells. 168 72


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