Epitope analysis of lipid A preparations from Pseudomonas diminuta and Pseudomonas vesicularis

In vitro antigenic reactivity of lipid A from Pseudomonas diminuta and Pseudomonas vesicularis with homologous and heterologous lipid A antibodies including monoclonal antibodies was studied by inhibition test of enzyme-linked immunosorbent assay (ELISA). The results suggest that both Pseudomonas lipid As have very similar epitopes, including species-specific and cross-reactive epitopes as compared with enterobacterial lipid A.     

Epitope analysis of lipid A preparations from Pseudomonas diminuta and Pseudomonas vesicularis

Abstract In vitro antigenic reactivity of lipid A from Pseudomonas diminuta and Pseudomonas vesicularis with homologous and heterologous lipid A antibodies including monoclonal antibodies was studied by inhibition test of enzyme-linked immunosorbent assay (ELISA). The results suggest that both Pseudomonas lipid As have very similar epitopes, including species-specific and cross-reactive epitopes as compared with enterobacterial lipid A.     

Isolation of 9-hydroxy-delta-tetradecalactone from lipid A of Pseudomonas diminuta and Pseudomonas vesicularis

A lipid component was isolated from the fatty acid fraction of acid hydrolysates of lipid A derived from Pseudomonas diminuta JCM 2788 and Pseudomonas vesicularis JCM 1477 lipopolysaccharides. By structural analysis of the lipid and its trimethylsilyl and acetyl derivatives by thin-layer chromatography, gas chromatography-mass spectrometry, mass spectrometry, infrared spectrometry and 13C-NMR, it was identified as 9-hydroxy-delta-tetradecalactone.     

Isolation of 9-hydroxy-δ-tetradecalactone from lipid A of Pseudomonas diminuta and Pseudomonas vesicularis

Abstract A lipid component was isolated from the fatty acid fraction of acid hydrolysates of lipid A derived from Pseudomonas diminuta JCM 2788 and Pseudomonas vesicularis JCM 1477 lipopolysaccharide. By structural analysis of the lipid and its trimethylsilyl and acetyl derivatives by thin-layer chromatography, gas chromatography-mass spectrometry, mass spectrometry, infrared spectrometry and ¹³C-NMR, it was identified as 9-hydroxy-δ-tetradecalactone.     

Interaction of endotoxic lipid A and lipid X with purified human platelet protein kinase C

Lipid A, the toxic principle of endotoxic lipopolysaccharide, and its precursor, Lipid X, interact with human platelets and modulate protein kinase C therein (Grabarek, J., Timmons, S., and Hawiger, J. (1988) J. Clin. Invest. 82, 964-971). We have now purified protein kinase C from human platelets and studied its interaction with endotoxic Lipids A and X. Protein kinase C-dependent phosphorylation of histone III-S was increased 15 times in the presence of Lipid A and 300 microM Ca2+. The Ca2+ requirement for such activation was lower when 4 beta-phorbol 12-myristate 13-acetate (PMA) or 1,2-diolein were added. Lipid A also induced autophosphorylation of protein kinase C, and its activation was enhanced by phosphatidylserine without reducing the Ca2+ requirement. Kinetic analysis of protein kinase C activation induced by Lipid A, in regard to ATP as a substrate, demonstrated that Lipid A increased the rate of the reaction (Vmax) without modifying the affinity of the enzyme (Km) for the substrate. Lipid X inhibited the activation of the enzyme induced by Lipid A. Lipid X also inhibited protein kinase C activation by phosphatidylserine, 1,2-diolein, and PMA. However, 10 times more of Lipid X was required for 50% inhibition (IC50) when PMA was used as an activator of protein kinase C in the presence of phosphatidylserine than when Lipid A and 1,2-diolein were used. These results support the hypothesis that endotoxic Lipid A and Lipid X exert their biological effect in platelets through direct interactions with protein kinase C.     

Production of Glutaric Acid: a Useful Criterion for Differentiating Pseudomonas diminuta from Pseudomonas vesiculare

A gas-liquid chromatographic procedure was used to determine short-chain acids produced by Pseudomonas diminuta and P. vesiculare after growth on Trypticase soy agar. Each of nine strains of P. diminuta produced glutaric acid, whereas none of the strains of P. vesiculare produced this acid.     

Synthetic and natural Escherichia coli free lipid A express identical endotoxic activities

The recently chemically synthesized Escherichia coli lipid A and the natural free lipid A of E. coli were compared with respect to their endotoxic activities in the following test systems: lethal toxicity, pyrogenicity, local Shwartzman reactivity, Limulus amoebocyte lysate gelation capacity, tumour necrotizing activity, B cell mitogenicity, induction of prostaglandin synthesis in macrophages, and antigenic specificity. It was found that synthetic and natural free lipid A exhibit identical activities and are indistinguishable in all tests.     

Partial Purification and Some Properties of a Benzhydrylamide-bond Hydrolyzing Enzyme in Pseudomonas diminuta

An enzyme in Pseudomonas diminuta showed hydrolyzing activity of a benzhydrylamide ( = diphenylmethylamide) bond in S-benzylcysteinylglycine benzhydrylamide. The enzyme was purified 225-fold by precipitation with ammonium sulfate, and column chromatography with ECTEOLA-cellulose, DEAE-cellulose and hydroxyapatite. It showed an optimum pH of 6 to 8 and it was markedly inhibited by Hg^{2 +} or p-chloromercuribenzoate. The preparation was more specific against S-benzylcysteinylglycine benzhydrylamide than other substrates tested.     

Change in membrane lipid fluidity induced by phospholipase A activation: a mechanism of endotoxic shock

The effects of endotoxin administration on the fluidity of dog liver plasma membranes and their relationship with changes in phospholipase A2 activity were studied. Endotoxin administration decreased the fluidity of liver plasma membranes and this decrease was reversible by phosphatidylcholine. The endotoxin-induced decrease in membrane fluidity could be mimicked by digesting control liver membranes with exogenous phospholipase A2. Endotoxin administration also increased the endogenous phospholipase A2 activity. Endotoxin in vitro had no phospholipase A2-like activity but it activated the hydrolytic activity of exogenous phospholipase A2. Based on these data, it is concluded that endotoxin administration decreased the fluidity of canine liver plasma membranes by acting through activation of phospholipase A2. The decrease in membrane lipid fluidity induced by endotoxin administration may play a significant role in the development of the pathophysiology of endotoxic shock at the cellular level.     

Identification of a plasmid-borne parathion hydrolase gene from Flavobacterium sp. by southern hybridization with opd from Pseudomonas diminuta

Parathion hydrolases have been previously described for an American isolate of Pseudomonas diminuta and a Philippine isolate of Flavobacterium sp. (ATCC 27551). The gene which encodes the broad-spectrum organophosphate phosphotriesterase in P. diminuta has been shown by other investigators to be located on a 66-kilobase (kb) plasmid. The intact gene (opd, organophosphate-degrading gene) from this degradative plasmid was cloned into M13mp10 and found to express parathion hydrolase under control of the lac promoter in Escherichia coli. In Flavobacterium sp. strain ATCC 27551, a 43-kb plasmid was associated with the production of parathion hydrolase by curing experiments. The M13mp10-cloned fragment of the opd gene from P. diminuta was used to identify a homologous genetic region from Flavobacterium sp. strain ATCC 27551. Southern hybridization experiments demonstrated that a genetic region from the 43-kb Flavobacterium sp. plasmid possessed significant homology to the opd sequence. Similar hybridization did not occur with three other native Flavobacterium sp. plasmids (approximately 23, 27, and 51 kb) present within this strain or with genomic DNA from cured strains. Restriction mapping of various recombinant DNA molecules containing subcloned fragments of both opd plasmids revealed that the restriction maps of the two opd regions were similar, if not identical, for all restriction endonucleases tested thus far. In contrast, the restriction maps of the cloned plasmid sequences outside the opd regions were not similar. Thus, it appears that the two discrete bacterial plasmids from parathion-hydrolyzing soil bacteria possess a common but limited region of sequence homology within potentially nonhomologous plasmid structures.     

Identification of a plasmid-borne parathion hydrolase gene from Flavobacterium sp. by southern hybridization with opd from Pseudomonas diminuta.

Parathion hydrolases have been previously described for an American isolate of Pseudomonas diminuta and a Philippine isolate of Flavobacterium sp. (ATCC 27551). The gene which encodes the broad-spectrum organophosphate phosphotriesterase in P. diminuta has been shown by other investigators to be located on a 66-kilobase (kb) plasmid. The intact gene (opd, organophosphate-degrading gene) from this degradative plasmid was cloned into M13mp10 and found to express parathion hydrolase under control of the lac promoter in Escherichia coli. In Flavobacterium sp. strain ATCC 27551, a 43-kb plasmid was associated with the production of parathion hydrolase by curing experiments. The M13mp10-cloned fragment of the opd gene from P. diminuta was used to identify a homologous genetic region from Flavobacterium sp. strain ATCC 27551. Southern hybridization experiments demonstrated that a genetic region from the 43-kb Flavobacterium sp. plasmid possessed significant homology to the opd sequence. Similar hybridization did not occur with three other native Flavobacterium sp. plasmids (approximately 23, 27, and 51 kb) present within this strain or with genomic DNA from cured strains. Restriction mapping of various recombinant DNA molecules containing subcloned fragments of both opd plasmids revealed that the restriction maps of the two opd regions were similar, if not identical, for all restriction endonucleases tested thus far. In contrast, the restriction maps of the cloned plasmid sequences outside the opd regions were not similar. Thus, it appears that the two discrete bacterial plasmids from parathion-hydrolyzing soil bacteria possess a common but limited region of sequence homology within potentially nonhomologous plasmid structures.     

A new yeast gene with a myosin-like heptad repeat structure

Summary We isolated a gene encoding a 218 kDa myosin-like protein from Saccharomyces cerevisiae using a monoclonal antibody directed against human platelet myosin as a probe. The protein sequence encoded by the MLP1 gene (for myosin-like protein) contains extensive stretches of a heptad-repeat pattern suggesting that the protein can form coiled coils typical of myosins. Immunolocalization experiments using affinity-purified antibodies raised against a TrpE-MLP1 fusion protein showed a dot-like structure adjacent to the nucleus in yeast cells bearing the MLP1 gene on a multicopy plasmid. In mouse epithelial cells the yeast anti-MLP1 antibodies stained the nucleus. Mutants bearing disruptions of the MLP1 gene were viable, but more sensitive to ultraviolet light than wild-type strains, suggesting an involvement of MLP1 in DNA repair. The MLP1 gene was mapped to chromosome 11, 25 cM from met1.     

Fatty acid composition of lipid A in Pseudomonas fluorescens

The fatty acid composition of lipid A was studied using gas-liquid chromatography (GLC) and GLC-mass spectrometry in Pseudomonas fluorescens strains of biovars A, B, C, i, F and G, the type strain ATCC 13525 (biovar A) inclusive. The following fatty acids were identified as predominant in the composition of lipid A in the strains representing biovars A, B, C, i, F and G: 3-hydroxydecanoic (3-OH C10:0), 2-hydroxydodecanoic (2-OH C12:0), 3-hydroxydodecanoic (3-OH C12:0), dodecanoic (C12:0), hexadecanoic (C16:0), octadecanoic (C18:0), hexadecenoic (C16:1) and octadecenoic (C18:1) acids. Lipid A of a biovar G strain differed noticeably from other strains in its fatty acid composition. Its main components were as follows: 3-hydroxytetradecanoic (3-OH C14:0), 3-hydroxypentadecanoic (3-OH C15:0) and dodecanoic (C12:0) fatty acids. The coefficients of similarity were determined for lipid A specimens isolated from the studied strains of P. fluorescens by calculating their fatty acid composition with a computer.     

Studies of lipid A fractions from the lipopolysaccharides of Pseudomonas aeruginosa and Pseudomonas alcaligenes

Lipid A fractions from Pseudomonas aeruginosa and Pseudomonas alcaligenes have similar compositions and structural features. By means of hydrazinolysis of the parent lipopolysaccharides and partial hydrolysis of the deacylation products, it was established that both lipids are derived from the β-(1→6)-linked disaccharide of glucosamine. Phosphorylated derivatives of the disaccharide from Ps. aeruginosa were also characterized. The lipids differ mainly in the absence of hexadecanoic acid and 2-hydroxydodecanoic acid from the lipid from Ps. alcaligenes. Evidence that in Ps. aeruginosa these acids are ester-linked to residues of 3-hydroxyalkanoic acids (including 3-hydroxydecanoic acid) was obtained. Heterogeneity of lipid A fractions was indicated by t.l.c., and by gel filtration of de-O-acylation products from mild alkaline methanolysis of the lipids.