Characterization of lipopolysaccharides present in settled house dust

The 3-hydroxy fatty acids (3-OHFAs) in lipopolysaccharides (LPS) play an important role in determining endotoxin activity, and childhood exposure to endotoxin has recently been associated with reduced risk of atopic diseases. To characterize the 3-OHFAs in house dust (HD), we used gas chromatography-mass spectrometry to assay 190 HD samples. Dust from beds, bedroom floors, family rooms, and kitchen floors was collected as part of a birth cohort study of childhood asthma (study 1) and a longitudinal study of home allergen and endotoxin (study 2). We also measured endotoxin activity with a Limulus assay and computed specific activity (endotoxin activity per nanomole of LPS). Longer-chain (C(16:0) and C(18:0)) 3-OHFAs were predominant in HD compared with short-chain (C(10:0), C(12:0), and C(14:0)) acids. Endotoxin activity was positively correlated with short-chain 3-OHFAs in both studies. In study 2, 3-OH C(16:0) was negatively correlated and 3-OH C(18:0) was not correlated with endotoxin activity, consistent with previous findings that the Limulus assay responds preferentially to LPS containing short-chain 3-OHFAs. Kitchen dust contained the highest concentrations of 3-OH C(10:0), the highest endotoxin activities, and the highest specific activities (P < 0.03). Bed dust contained the largest amounts of long-chain 3-OHFAs, the highest concentrations of LPS, and the lowest specific activities. Apartments had significantly different types of LPS (P = 0.03) compared with single-family homes in study 2. These data suggest that the Limulus assay may underestimate exposure to certain types of LPS. Because nontoxic LPS may have immune modulating effects, analysis of 3-OHFAs may be useful in epidemiologic studies.     

Use of 3-hydroxy fatty acid concentrations in a murine air pouch infection model as a surrogate marker for LPS activity: a feasibility study using environmental Burkholderia cenocepacia isolates

Using a murine hypodermic air pouch infection model designed to mimic the release of bacterial products at physiological levels, 3-hydroxy fatty acid (3-OH FA) and endotoxin unit levels from Burkholderia cenocepacia isolates were assessed. The B. cenocepacia environmental isolates (n = 35) survived in the hypodermic air pouch but did not invade across the peritoneal epithelial layer during a 72-h infection. For all 35 strains, when the molar ratio of C_14:0 3-OH FA to C_16:0 3-OH FA in the air pouch fluid wash samples was between 1.4 and 2.5, the concentrations of C_14:0 3-OH FA were correlated with the endotoxin unit levels. However, both surrogate markers exhibited different correlations to the inflammatory response. The linear regression coefficient was 0.4234 for C_14:0 3-OH FA concentrations vs. NO productions, 0.223 for endotoxin unit levels vs. NO productions, 0.5008 for C_14:0 3-OH FA concentrations vs. TNF-alpha productions and 0.2869 for endotoxin unit levels vs. TNF-alpha productions. Therefore, C_14:0 3-OH FA concentrations, rather than endotoxin unit levels, acted as an immunostimulatory indicator for LPS in the B. cenocepacia isolates.     

Use of 3-hydroxy fatty acid concentrations in a murine air pouch infection model as a surrogate marker for LPS activity: A feasibility study using environmental Burkholderia cenocepacia isolates

Using a murine hypodermic air pouch infection model designed to mimic the release of bacterial products at physiological levels, 3-hydroxy fatty acid (3-OH FA) and endotoxin unit levels from Burkholderia cenocepacia isolates were assessed. The B. cenocepacia environmental isolates (n = 35) survived in the hypodermic air pouch but did not invade across the peritoneal epithelial layer during a 72-h infection. For all 35 strains, when the molar ratio of C_14:0 3-OH FA to C_16:0 3-OH FA in the air pouch fluid wash samples was between 1.4 and 2.5, the concentrations of C_14:0 3-OH FA were correlated with the endotoxin unit levels. However, both surrogate markers exhibited different correlations to the inflammatory response. The linear regression coefficient was 0.4234 for C_14:0 3-OH FA concentrations vs. NO productions, 0.223 for endotoxin unit levels vs. NO productions, 0.5008 for C_14:0 3-OH FA concentrations vs. TNF-alpha productions and 0.2869 for endotoxin unit levels vs. TNF-alpha productions. Therefore, C_14:0 3-OH FA concentrations, rather than endotoxin unit levels, acted as an immunostimulatory indicator for LPS in the B. cenocepacia isolates.     

The Release and Detection of Endotoxin from Liposomes

Incorporation of lipopolysaccharide (LPS) into liposomes dramatically reduces its ability to coagulateLimulusamebocyte lysate (LAL). The coagulation of LAL is commonly used to signal the presence of endotoxinin vitro.This study demonstrates a simple method to release masked endotoxin from liposomal dispersions using moderate amounts of detergent to form mixed micelles containing lipid, detergent, and LPS. Several parameters were found to affect the degree of liposome solubilization and/or the sensitivity of the LAL assay. These included detergent type and concentration, temperature for solubilization, lipid composition, liposome morphology, and time for test incubation. The nonionic detergent polyoxyethylene 10 lauryl ether (C₁₂E₁₀) proved to be unique in its ability to solubilize liposomes and minimally interfere with endotoxin detection. The LAL endotoxin detection limit for samples dispersed in C₁₂E₁₀varied with the phospholipid component; the sensitivity decreased in the order DSPC > DPPC = EPC DMPC. Cholesterol lowered the solubility limit of the liposomes, but did not appear to affect the LAL assay sensitivity once the liposomes were completely solubilized. The presence of negatively charged phospholipids, DSPG and Pops, also lowered the solubility limit. Pops, but not DSPG, at 10 mol% further decreased the LAL endotoxin detection limit. This detergent-solubilization method should be useful in liposomal LPS immunological studies or in other situations where accurate determination of endotoxin concentration is important.     

Use of gas chromatography-mass spectrometry versus the limulus amebocyte lysate test for the determination of airborne endotoxin in confined swine buildings

Airborne endotoxin (lipopolysaccharide, LPS) in filter samples collected in three different swine confinement buildings was determined by using the Limulus amebocyte lysate test and by applying gas chromatography-mass spectrometry (GC-MS) to analyze 3-hydroxy fatty acids (3-OH-FAs). The amounts of LPS as shown by GC-MS were 30–50 times larger than the amounts detected by the Limulus test. GC-MS revealed that 21% of the LPS collected on cellulose acetate filters and 26% on polycarbonate filters remained on the filters after buffer extraction. Better correlation with the Limulus test and the 3-OH-FA measurements was achieved when considering the sum of 3-OH C12:0 and 3-OH C14:0 rather than the sum of all of the detected 3-OH-FAs (i.e. those with 12–18-carbon chains), indicating that the bioactivity of the LPS was dependent upon the relative distribution of the 3-OH-FAs. Linear regression analysis between air concentrations of dust and endotoxin/LPS gaveR ²values that varied from 0.407 to 0.739. The air concentrations of LPS were lower in the uninsulated swine house (a green house with an alternative housing system) than in the two insulated buildings (conventional housing systems; one designed as a climate chamber, i.e. climatic parameters could be controlled), whereas the opposite was found for the concentrations of LPS in airborne dust from the three buildings. The numbers of viable bacteria and fungi were highest in the uninsulated swine house. Moreover the relative distribution of 3-OH-FAs in that building differed from the distribution in the insulated buildings, which reflects differences in the microflora, probably mainly due to differences between the housing systems and the design of the buildings.     

Salegentibacter echinorum sp. nov., isolated from the sea urchin Hemicentrotus pulcherrimus

A novel Gram-negative, strictly aerobic, heterotrophic, non-motile and yellow-pigmented bacterial strain, designated HD4^T, was isolated from the sea urchin Hemicentrotus pulcherrimus collected from the Yellow Sea in China. Optimal growth of the strain was observed at 28–30 °C, pH 6.8–7.3, and in the presence of 3–5 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain HD4^T exhibited high similarity with the members of Salegentibacter (92.3–95.4 %). The DNA G+C content was 37.0 mol%, MK-6 was the main respiratory quinone and summed feature 3 (comprising iso-C_15:0 2-OH/C_16:1ω7c), iso-C_15:0, iso-C_17:0 3-OH and anteiso-C_15:0 were the major cellular fatty acids. The predominant polar lipids in strain HD4^T were phosphatidylethanolamine and two unknown lipids (L2, L4). Based on the phylogenetic, physiological and biochemical characteristics, strain HD4^T should be classified as a novel species within the genus Salegentibacter, for which the name Salegentibacter echinorum sp. nov. is proposed. The type strain is HD4^T (=CICC 10466^T = NRRL B-59666^T).     

Rapid Determination of Bacteriological Water Quality by Using Limulus Lysate

The Limulus lysate assay was used to measure the endotoxin content in stream water and was found to reflect the degree of bacterial contamination as measured by coliform, enteric, gram-negative, and heterotrophic bacteria. The firm-clot method was found to be a less sensitive and reproducible technique for the detection of endotoxin than was the spectrophotometric modification of the Limulus lysate assay. Bound endotoxin, as determined by the spectrophotometric modification of the Limulus lysate assay, was found to be a better measure of the endotoxin associated with bacterial cells than was total endotoxin.     

Neutralization of bacterial endotoxins by frog antimicrobial peptides

The ability of skin antimicrobial peptides of the southern bell frog, Litoria raniformis, to neutralize in vitro the endotoxin, proinflammatory lipopolysaccharide (LPS) complex, from two different gram‐negative bacterial pathogens, human pathogen Escherichia coli (0111:B4) and frog pathogen Klebsiella pneumoniae, was investigated. The LPS neutralization activity of the natural mixture of skin antimicrobial peptides was measured using chromogenic Limulus amebocyte lysate assays. These skin antimicrobial peptides neutralized the LPSs from both pathogens at physiologically relevant concentrations (IC₅₀ < 100 µg/mL) showing their potential for non‐specific LPS neutralization in vivo in the skin of infected frogs and for development of anti‐endotoxin agents.     

Isolation and Characterization of Marine Nonylphenol-Degrading Bacteria and Description of Pseudomaricurvus alkylphenolicus gen. nov., sp. nov.

Two novel aerobic p-n-nonylphenol-degrading bacterial strains were isolated from seawater obtained from the coastal region of Ogasawara Islands, Japan. The 16S rRNA gene sequence analysis indicated that the strains are affiliated with the order Alteromonadales within the class Gammaproteobacteria. One isolate, strain KU41G2, is most closely related to Maricurvus nonylphenolicus (99.2 % similarity), and is tentatively identified as M. nonylphenolicus. The other isolate, strain KU41G^T, is also most closely related to M. nonylphenolicus; however, the 16S rRNA gene sequence similarity was only 94.7 %. Cells of strain KU41G^T are Gram-negative rods with a single polar flagellum. The predominant respiratory lipoquinone was ubiquinone-8, and the major cellular fatty acids were C_17:1 ω8c (24.2 %); C_15:0 iso 2-OH; and/or C_16:1 ω7c (16.3 %), C_15:0 (10.3 %), C_11:0 3-OH (9.5 %), C_9:0 3-OH (6.7 %), C_10:0 3-OH (6.4 %), and C_18:1 ω7c (5.5 %). The DNA G+C content was 53.3 mol%. On the basis of physiological, chemotaxonomic, and phylogenetic data, strain KU41G^T is suggested to represent a novel species of a new genus, for which we propose the name Pseudomaricurvus alkylphenolicus gen. nov., sp. nov. The type strain of P. alkylphenolicus is KU41G^T (=JCM 19135^T = KCTC 32386^T).     

Investigation of Lipopolysaccharides from Sinorhizobium meliloti SKHM1-188 and Two of Its Mutants with Decreased Nodulation Competitiveness

A comparative study of the lipopolysaccharides (LPS) isolated from Sinorhizobium meliloti SKHM1-188 and two of its LPS mutants (Tb29 and Ts22) with sharply decreased nodulation competitiveness was conducted. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate revealed two forms of LPS in all three strains: a higher molecular weight LPS1, containing O-polysaccharide (O-PS), and a lower molecular weight LPS2, without O-PS. However, the LPS1 content in mutants was significantly smaller than in the parent strain. The LPS of the strains studied contained glucose, galactose, mannose, xylose, three nonidentified sugars (X ₁ (TGlc 0.53), X ₂ (TGlc 0.47), and X ₃ (TGlc 0.43)), glucosamine, and ethanolamine, while the LPS of S. meliloti SKHM1-188 additionally contained galactosamine, glucuronic and galacturonic acids, and 2-keto-3-deoxyoctulosonic acid (KDO), as well as such fatty acids as 3-OH C14:0, 3-OH C15:0, 3-OH C16:0, 3-OH C18:0, nonidentified hydroxy X (T_{3-OH C14:0} 1.33), C18:0, and unsaturated C18:1 fatty acids. The LPS of both mutants were similar in the component composition but differed from the LPS of the parent strain by lower X ₂, X ₃, and 3-OH C14:0 contents and higher KDO, C18:0, and hydroxy X contents. The LPS of all the strains were subjected to mild hydrolysis with 1% acetic acid and fractionated on a column with Sephadex G-25. The higher molecular weight fractions (2500–4000 Da) contained a set of sugars typical of intact LPS and, supposedly, corresponded to the LPS polysaccharide portion (PS1). In the lower molecular weight fractions (600–770 Da, PS2), glucose and uronic acids were the major components; galactose, mannose, and X ₁ were present in smaller amounts. The PS1/PS2 ratio for the two mutants was significantly lower than for strain SKHM1-188. The data obtained show that the amount of O-PS–containing molecules (LPS1) in the heterogeneous lipopolysaccharide complex of the mutants was smaller than in the SKHM1-188 LPS; this increases the hydrophobicity of the cell surface of the mutant bacteria, which supposedly contributes to their nonspecific adhesion to the roots of the host plant, thus decreasing their nodulation competitiveness.     

An in vitro method for evaluating endotoxic activity using prostaglandin E2 induction in bovine peripheral blood

Severe side effects of veterinary vaccines, in particular Histophilus somni-containing vaccines for cows, have frequently been reported in Japan. These side effects are probably caused by endotoxins. Contamination levels of endotoxins could be monitored using the Limulus amebocyte lysate (LAL) test; however, the LAL test is not completely adequate for evaluation of in vivo endotoxic activities. In this study, we established a method for evaluating endotoxic activities using prostaglandin E₂ (PGE₂) induction in bovine peripheral blood. Blood and standard endotoxin, derived from Escherichia coli, were mixed and incubated. The concentration of induced PGE₂ in the culture supernatant reached a maximum after 24-h incubation. A linear dose-response curve was observed for PGE₂ concentration and the logarithmic transformed standard endotoxin concentration (5–5000 ng/ml). The endotoxic activity of H. somni in cows was the highest among those of several tested endotoxins. However, the LAL activities of H. somni were not as high as those of the other tested endotoxins. These results may provide a reason for the many report of side effects of H. somni-containing vaccines. The PGE₂ detection assay described here could be a valuable method for evaluating the endotoxic activities of vaccines in cows.     

Composition,structure, and biological properties of lipopolysaccharides from different strains of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">atrofaciens</Emphasis>

The composition, structure, and certain biological properties of lipopolysaccharides (LPS) isolated from six strains of bacteria Pseudomonas syringae pv. atrofaciens pathogenic for grain-crops (wheat, rye) are presented. The LPS-protein complexes were isolated by a sparing procedure (extraction from microbial cells with a weak salt solution). They reacted with the homologous O sera and contained one to three antigenic determinants. Against the cells of warm-blooded animals (mice, humans) they exhibited the biological activity typical of endotoxins (stimulation of cytokine production, mitogenetic activity, etc.). The LCD of the biovar type strain was highly toxic to mice sensitized with D-galactosamine. The structural components of LPS macromolecules obtained by mild acidic degradation were characterized: lipid A, core oligosaccharide, and O-specific polysaccharide (OPS). Fatty acids 3-HO-C_10:0, C_12:0, 2-HO-C_12:0, 3-HO-C_12:0, C_16:0, C_16:1, C_18:0, and C_18:1 were identified in lipid A of all the strains, as well as the components of the hydrophilic part: glucosamine (GlcN), ethanolamine (EtN), phosphate, and phosphoethanolamine (EtN-P). In the core LPS, glucose (Glc), rhamnose (Rha), L-glycero-D-manno-heptose (Hep), GlcN, galactosamine (GalN), 2-keto-3-deoxy-D-mannooctonoic acid (KDO), alanine (Ala), and phosphate were present. The O chain of all the strains consisted of repeated elements containing a linear chain of three to four L-(two strains) or D-Rha (four strains) residues supplemented with a single residue of 3-acetamido-3,6-dideoxy-D-galactose (D-Fucp3Nac), N-acetyl-D-glucosamine (D-GlcpNAc), D-fucose (D-Fucf), or D-Rhap (strain-dependent) as a side substituent. In different strains the substitution position for Rha residues in the repeated components of the major rhamnan chain was also different. One strain exhibited a unique type of O-chain heterogeneity. Immunochemical investigation of the LPS antigenic properties revealed the absence of close serological relations between the strains of one pathovar; this finding correlates with the differences in their OPS structure. Resemblance between the investigated strains and other P. syringae strains with similar LPS structures was revealed. The results of LPS analysis indicate the absence of correlation between the OPS structure and the pathovar affiliation of the strains.     

Inactivation of endotoxin by Limulus amoebocyte lysate.

The inactivation of bacterial endotoxin by aqueous extracts (Limulus amoebocyte lysate) of the circulating blood cells (amoebocytes) of the horseshoe crab, Limulus polyphemus, is described. Active extracts were obtained by heating Limulus amoebocyte lystate (LAL) to 60°C for 20 min to denature the clotting enzyme, rendering the LAL incapable of gel formation in the presence of endotoxin. Endotoxin inactivation was assayed using the Limulus amoebocyte lysate test and by rabbit bioassay. Inactivation of endotoxin with heated extracts of LAL was suggestive of enzymatic mediation, as indicated by dependence on time, temperature, pH, and the kinetics of inactivation. Endotoxin inactivation occurred over a broad pH range, 4.5–8.5, with the optimum at a pH of 6.1. Temperature optima were between 37° and 50°C, with observed activity between 0° and 65°C. Ionized calcium was inhibitory to endotoxin inactivation with heated extracts of LAL, with partial inhibition at 0.001 m calcium and complete inhibition at 0.02 m calcium. Other divalent cations (Mg, Ba, Mn, and Cu) were also found to inhibit the inactivation of endotoxin. Similarities between the endotoxin-inactivating system of L. polyphemus and those described to be present in mammalian and lower vertebrate sera are discussed.     

Enhancement of endotoxin activity by pulmonary surfactant

The modification of the activity of lipopolysaccharide (LPS) in the pulmonary lavage fluid (PLF) of guinea pigs was assessed by a chromogenic Limulus assay. The activity of the lPS bound to Escherichia coli or of LPS extracted from the bacteria was observed to increase significantly in PLF. This increase in activity was amplified after heating at 75°C for 5 min. Pulmonary surfactant (PS) obtained from PLF showed a similar increase in the activity of LPS, indicating that PS is most probably the key agent in this modification.     

Structural modification of Escherichia coli lipid A by myristoyltransferase gene from Klebsiella pneumoniae

Lipid A in lipopolysaccharide (LPS) of Escherichia coli mutant strains was modified by the introduction of myristoyltransferase gene cloned from Klebsiella pneumoniae. When the gene was introduced into the mutant having lipid A containing only 3‐hydroxymyristic acids, it produced lipid A with two additional myristic acids (C_14:0). When the same gene was introduced into the mutant with pentaacylated lipid A containing one lauric acid (C_12:0), C_12:0 was replaced by C_14:0. IL‐6‐inducing activity of LPS with modified lipid A structure suggested that C_12:0 in lipid A could be replaced by C_14:0 without changing the immunostimulating activity.     

Chemical Structure of Lipid A Isolated from Flavobacterium meningosepticum Lipopolysaccharide

The chemical structure of the lipid A of the lipopolysaccharide component isolated from Flavobacterium meningosepticum IFO 12535 was elucidated. Methylation and nuclear magnetic resonance analyses showed that two kinds of hydrophilic backbone exist in the free lipid A: a β (1→6)-linked 2-amino-2-deoxy-d-glucose, which is usually present in enterobacterial lipid A’s, and a 2-amino-6-O-(2,3-diamino-2,3-dideoxy-β-d-glucopyranosyl)-2-deoxy-d-glucose, in a molar ratio of 1.00:0.35. Both backbones were α-glycosidically phosphorylated in position 1, and the hydroxyl groups at positions 4, 4′, and 6′ were unsubstituted. Liquid secondary ion-mass spectrometry revealed a pseudomolecular ion at m/z 1673 [M-H]⁻ as a major monophosphoryl lipid A component carrying five acyl groups. Fatty acid analysis showed that the lipid A contained 1 mol each of amide-linked (R)-3-OH iC_17:0, ester-linked (R)-3-OH iC_15:0, amide-linked (R)-3-O-(iC_15:0)-iC_17:0, and both amide- and ester-linked (R)-3-OH C_16:0. Fatty acid distribution analyses using several mass spectrometry determinations demonstrated that the former two constituents were distributed on positions 2 and 3 of the reducing terminal unit of the backbones and that the latter two were attached to the 2′ and 3′ positions in the nonreducing terminal residue.Lipopolysaccharide (LPS) is known to act as an endotoxin that mediates pathophysiological changes such as fever and shock which occur in the course of severe gram-negative bacterial infection (5, 18, 27). The pathophysiological activity of LPS depends on the chemical structure of the hydrophobic portion called lipid A, the biologically active center of LPS (12, 14), which generally consists of a β(1→6)-linked 2-amino-2-deoxy-d-glucose (GlcN) disaccharide carrying phosphate and fatty acid residues; many fine structural variations are observed in different bacterial families (38).Since many of the LPSs from various gram-negative bacteria cause similar endotoxic effects despite differences in chemical composition and positions of substitution, the chemical structure required for the activity does not seem to be very strict. It has been reported, however, that several lipid A forms, isolated from the LPSs of Porphyromonas gingivalis (16), Rhodobacter sphaeroides (22, 26) and Rhodobacter capsulatus (20), as well as chemically synthesized lipid A analogs (6, 12), which are structurally similar to the active-type lipid A, exhibit dramatically low endotoxicity. Biologically active lipid A has been found to be changed to completely nontoxic derivatives by simple chemical modifications (28, 29). These findings indicate that the biological activity of lipid A is controlled by the fine structural variations. The nontoxic or low-toxicity lipid A preparations are very important for the determination of the relationship between the chemical structure and biological activity of lipid A, and also for the systematic development of LPS antagonists. However, the essential structural requirements for the complete activity or nontoxicity of lipid A are still uncertain. It is, therefore, meaningful to study the chemical and biological properties of naturally occurring lipid A’s which possess a unique structure.Flavobacterium meningosepticum is an aerobic gram-negative rod which is known to cause meningitis and septicemia in newborn infants (4, 21). Interestingly, the bacterium does not induce Limulus gelation activity when tested with whole cells (32), strongly suggesting that the LPS is of low toxicity or nontoxic and that the lipid A must have a unique structure relative to other enterobacterial lipid A’s.In the present study, the chemical structure of lipid A isolated from F. meningosepticum LPS was characterized by compositional study, methylation analysis, mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy.     

Structure–activity relationships of lipopolysaccharide sequestration in guanylhydrazone-bearing lipopolyamines

The toxicity of Gram-negative bacterial endotoxin (lipopolysaccharide, LPS) resides in its structurally highly conserved glycolipid component called lipid A. Our major goal has been to develop small-molecules that would sequester LPS by binding to the lipid A moiety, so that it could be useful for the prophylaxis or adjunctive therapy of Gram-negative sepsis. We had previously identified in rapid-throughput screens several guanylhydrazones as potent LPS binders. We were desirous of examining if the presence of the guanylhydrazone (rather than an amine) functionality would afford greater LPS sequestration potency. In evaluating a congeneric set of guanylhydrazone analogues, we find that C₁₆ alkyl substitution is optimal in the N-alkylguanylhydrazone series; a homospermine analogue with the terminal amine N-alkylated with a C₁₆ chain with the other terminus of the molecule bearing an unsubstituted guanylhydrazone moiety is marginally more active, suggesting very slight, if any, steric effects. Neither C₁₆ analogue is significantly more active than the N-C₁₆-alkyl or N-C₁₆-acyl compounds that we had characterized earlier, indicating that basicity of the phosphate-recognizing cationic group, is not a determinant of LPS sequestration activity.     

Quantitative Limulus lysate assay for endotoxin activity: Aggregation of radioiodinated coagulogen monomers

This communication describes a modification of the Limulus lysate assay which allows precise quantitation of picograms of bacterial lipopolysaccharide activity. The method measures the incorporation of ¹²⁵I-labeled coagulogen monomers into the lysate clot as a function of lipopolysaccharide concentration. The method is more precise and requires less lysate than the previously described quantitative assays for endotoxin activity.     

The effect of endotoxin on the lipoxygenase-mediated conversion of exogenous and endogenous arachidonic acid in mouse peritoneal macrophages

The influence of lipopolysaccharide (LPS, endotoxin) or its lipid A component (bacterial and synthetic) on the synthesis of zymosan induced leukotriene C₄, prostaglandin E₂ and prostacyclin and on the conversion of exogenous arachidonic acid was studied in mouse peritoneal macrophages. It was found that following preincubation with LPS the amount of leukotriene C₄ released during phagocytosis of zymosan was substantially decreased. The levels of prostaglandin E₂ and prostacyclin, however, were the same in LPS-treated cells and controls. Likewise, pretreatment with LPS impaired the capacity to convert exogenously added arachidonic acid to mono- and di-HETE's. Lipid A (bacterial and synthetic) exhibited the same activity as LPS. LPS had no effect on macrophages of the endotoxin low responder mouse strain (C3H/ HeJ). Several explanations could be possible for the observed LPS effect. The finding that low doses of α-tocopheryl acetate prevented the LPS-induced decrease of LTC₄ synthesis indicates a protective role of this agent. We would, therefore, favour the idea that lipoxygenases undergo oxidative selfinactivation during LPS action.