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PubMed1983

Homogeneity of cell envelope protein subtypes, lipopolysaccharide serotypes, and biotypes among Haemophilus influenzae type b from patients with meningitis in The Netherlands.

Eighty strains of Haemophilus influenzae type b were randomly selected from 531 strains collected between 1975 and 1982 from patients with meningitis in The Netherlands. Subtyping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that 67 of 80 isolates had identical major outer membrane protein patterns (subtype 1). Among the 13 other isolates four different polyacrylamide gel electrophoresis patterns were observed, two of which closely resembled subtype 1. Lipopolysaccharides were characterized immunologically by immunoprecipitation (Ouchterlony technique) and the gel-immuno-radio-assay. Four serotypes were found among the 80 selected strains, leaving one strain not typable. Seventy-four strains (93%) belonged to the same lipopolysaccharide serotype; 77 (97%) of 80 of the strains belonged to biotype I. Sixty strains (75%) had identical major outer membrane protein patterns (subtype 1), lipopolysaccharide serotypes (serotype 1), and biotypes (I).

Animals↗

PubMed1992

Lipopolysaccharide heterogeneity in Escherichia coli J5 variants: analysis by flow cytometry.

A lipopolysaccharide O-side chain-producing phenotypic variant was isolated from a uridine 5'-diphosphogalactose 4-epimeraseless Escherichia coli J5 rough mutant strain by fluorescence-activated cell sorting with a monoclonal antibody (MAb) specific for the O-side chain of E. coli O111:B4 smooth parent lipopolysaccharide. The variant (J5-2) was recognized by both core- and O-side chain-specific MAbs, while the "original" rough mutant (J5-1) and smooth parent strains stained only with core- and O-side chain-specific MAb, respectively. J5-2 produced complete and incomplete (Rc chemotype) core and O polysaccharide in the presence of galactose. Three other E. coli J5 variants were either phenotypically similar to J5-1 (J5-UK) or distinct from J5-1 and J5-2 (J5-A, -B). The latter phenotype had a lower-molecular-weight core, compared with J5-1 and J5-2, and distinct MAb specificities. Various J5 phenotypes also differed in galactokinase levels, the ability to use galactose, and susceptibility to core-specific MAb binding on solid media. The J5-2 strain showed reciprocal changes in O-side chain and core expression during log-phase growth. E. coli J5 thus undergoes spontaneous alterations in lipopolysaccharide phenotype.

Antibodies, Monoclonal↗

PubMed1991

Specific incorporation of glycine into bacterial lipopolysaccharide. Novel function of specific transfer ribonucleic acids.

It has been found that the bacterial endotoxins (lipopolysaccharides, LPSs) contain some amino acids and glycine is the most abundant amino acid in the polysaccharide core preparations of LPSs of gram-negative bacteria. Until now nothing was known about the mechanism of amino acid incorporation into the lipopolysaccharide core. We found that one out of three glycyl-tRNAs(Gly) from Escherichia coli is the donor of amino acid and is the substrate for a putative aminoacyl-tRNA:LPS transferase. We have isolated, purified this tRNA and determined its nucleotide sequence to be major E.coli tRNA(3Gly). This tRNA(Gly) (anticodon GCC) conserved the tRNA structural features. The assay for determination of the specific incorporation of glycine into the lipopolysaccharide was also invented and described.

Acylation↗

PubMed2006

Midazolam inhibits proinflammatory mediators in the lipopolysaccharide-activated macrophage.

BACKGROUND: Midazolam, a benzodiazepine, has a hypnotic effect and is widely used as a sedative. The role of midazolam in activation of macrophages during sepsis is not known. The aim of this study was to evaluate the antiinflammatory actions of midazolam in cultured macrophages. METHODS: Using a macrophage cell line, RAW264.7 cells, the effect of midazolam on proinflammatory mediators and activation of mitogen-activated protein kinase was measured by Western blot. Nuclear factor-kappaB (NF-kappaB) activation and translocation of p65 subunit of NF-kappaB was measured using luciferase assay and immunocytochemistry. Superoxide production was measured by lucigenin chemiluminescence. RESULTS: Midazolam significantly inhibited lipopolysaccharide-induced up-regulation of both cyclooxygenase 2 and inducible nitric oxide synthase in a dose-dependent manner (approximately 3-30 microm). IkappaB-alpha degradation and NF-kappaB transcriptional activity induced by lipopolysaccharide were also suppressed by the midazolam. Nuclear translocation of the p65 subunit of NF-kappaB was inhibited by midazolam. Furthermore, midazolam suppressed phosphorylation of p38 mitogen-activated protein kinase and also inhibited lipopolysaccharide-induced superoxide production in macrophages. CONCLUSIONS: These results suggest that midazolam has an antiinflammatory action by inhibiting inducible nitric oxide synthase and cyclooxygenase-2 expression, possibly through suppression of NF-kappaB and p38 mitogen-activated protein kinase activation.

Animals↗

PubMed2003

Lipopolysaccharide and TNFalpha regulate the expression of GDNF, neurturin and their receptors.

Inflammatory processes in the brain may trigger specific neuroprotective responses in glial cells. Here, we show that bacterial lipopolysaccharide strongly up-regulates glial derived neurotrophic factor (GDNF) mRNA while it down-regulates that of neurturin. Tumor necrosis factor alpha (TNFalpha) had different effects since it stimulated neurturin expression without enhancing GDNF mRNA. Interestingly, both lipopolysaccharide and TNFalpha triggered a significant decrease in the expression of the GDNF receptor, GFRalpha1, in glial cells. While the significance of such down-regulation during inflammatory processes remains to be characterised, the differential regulation of GDNF and neurturin following lipopolysaccharide and TNFalpha treatments suggest specific neuroprotective responses of glial cells in case of bacterial infection, trauma, transplantation or neurodegenerative diseases.

Animals↗

PubMed2006

Microvascular effects following treatment with polyethylene glycol-albumin in lipopolysaccharide-induced endotoxemia.

OBJECTIVE: To determine whether resuscitation with polyethylene glycol conjugated bovine serum albumin (2.5% weight/volume) infused at 16 mL/kg/hr (PEG-BSA-16) or at 24 mL/kg/hr (PEG-BSA-24) for 1 hr improves microcirculatory conditions in endotoxemia compared with dextran 70 (6% weight/volume) infused at 24 mL/kg/hr (Dex). DESIGN: Prospective study. SETTING: University research laboratory. SUBJECTS: Male Golden Syrian hamsters. INTERVENTIONS: Hamsters implemented with a skinfold window chamber were given an intravenous injection of lipopolysaccharide and resuscitated within 10 mins with Dex, PEG-BSA-16, or PEG-BSA-24. MEASUREMENTS AND MAIN RESULTS: Hamsters were observed during 24 hrs after lipopolysaccharide injection. Systemic variables measured included mean arterial pressure, heart rate, and systemic arterial blood gas. Microvascular function was characterized by measuring vessel diameter; red blood cell velocity; functional capillary density (FCD); P(O2) in arterioles, venules, and tissue; and perivascular nitric oxide concentration 6 hrs after lipopolysaccharide injection. At 6 hrs, animals with no treatment had the lowest FCD (6.7 +/- 5.7% of baseline). PEG-BSA provided significantly improved microvascular conditions as shown by restoration of FCD. Recovery of FCD was related to improved microvascular flow and perivascular and tissue P(O2), normalization of shear rate, and decreased perivascular nitric oxide concentration. These effects were related to improved fluid retention using PEG-BSA-24 as evidenced by the significantly lower hematocrit at 24 hrs after resuscitation. Nitric oxide at 6 hrs after induction of sepsis achieved perivascular millimolar concentrations, which were reduced to normal values by PEG-BSA-24 treatment. At 6 hrs there were significant differences in FCD, tissue P(O2), and perivascular nitric oxide concentration following PEG-BSA treatment by comparison with Dex treatment, although there were no differences in systemic variables between Dex and PEG-BSA groups. CONCLUSIONS: PEG-BSA produces improved microcirculatory conditions in the treatment of endotoxemia when compared with dextran 70.

Animals↗

PubMed2003

Endotoxemia and elevation of lipopolysaccharide-binding protein after hematopoietic stem cell transplantation.

BACKGROUND: Hematopoietic stem cell transplantation (SCT) carries a significant risk of severe therapy-associated complications chief among which is acute graft vs.host disease (aGVHD). Animal models indicate that myeloablative chemotherapy compromises the mucosal barrier, thereby allowing translocation of intestinal flora-derived lipopolysaccharides (or endotoxin) that subsequently trigger aGVHD, but there are no comparable data in humans. Our aim was to gain insight into the potential role of endotoxin and endotoxin-induced acute phase proteins in children undergoing SCT. METHODS: Plasma concentrations of C-reactive protein (CRP) and lipopolysaccharide-binding protein (LBP) were measured in 57 pediatric patients undergoing SCT. In addition plasma endotoxin levels were measured in 25 patients. RESULTS: The previously described rise in CRP was confirmed, and a marked elevation of LBP was observed that peaked at Day 7 (median value, 6.6 microg/ml; P < 0.03 for all pairwise comparisons). CRP and LBP values were significantly correlated (r = 0.77, P < 0.001). A significant but complex relationship was noted between LBP concentrations at Day 0 and severity of subsequent aGVHD (P = 0.02). Of the 25 patients assayed, 11 (44%) had detectable endotoxemia, including 4 who were endotoxin-positive at Day 0. CONCLUSIONS: The detection of endotoxemia coupled with marked elevations in LBP at Day 7 raises the possibility that inflammatory responses early after SCT may be driven in part by the entry of lipopolysaccharide into the bloodstream.

Acute-Phase Proteins↗

PubMed1990

Multivariate analyses of carbohydrate data from lipopolysaccharides of Actinobacillus (Haemophilus) actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus.

The taxonomic distinction between Actinobacillus (Haemophilus) actinomycetemcomitans and Haemophilus aphrophilus and the taxonomic distinction between H. aphrophilus and Haemophilus paraphrophilus have been questioned. This study was done to determine whether multivariate statistical analyses of carbohydrate data from lipopolysaccharides could be used to distinguish between these closely related species. Lipopolysaccharides were extracted with phenol-water and purified. Carbohydrates were assessed by using gas chromatography and gas chromatography-mass spectrometry after methanolysis and derivatization with trifluoroacetic acid anhydride. The lipopolysaccharides from all of the species contained rhamnose, fucose, galactose, glucose, L-glycero-D-mannoheptose, and glucosamine plus galactosamine, but in varying amounts. A. actinomycetemcomitans and H. paraphrophilus also contained D-glycero-D-mannoheptose, while H. aphrophilus did not. Sample- and variable-oriented principal-component analyses of the carbohydrate data clearly distinguished among A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus. Soft independent modelling of class analogy showed that no sample in the A. actinomycetemcomitans class fell within the 95% confidence limits of the H. aphrophilus class. H. paraphrophilus fell outside both classes.

Actinobacillus↗

PubMed1978

The effect of lipopolysaccharide composition on the ultrastructure of Pseudomonas aeruginosa.

The surface structure of Pseudomonas aeruginosa PACl and PAClR and of lipopolysaccharide-defective mutants derived from them was studied by negative-staining and thin-section electron microscopy and compared with that of a rough mutant with wild-type lipopolysaccharide. The rough mutant and the parent strains had fairly smooth outer layers. Negatively stained preparations of all the mutants lacking polymerized O-antigenic sidechains, including a semi-rough mutant, showed numerous blebs on the surface. In thin sections of these mutants occasional extrusions from the surface were seen. They appeared to consist of material extruded from the outer membrane, but there was no evidence to suggest they were complete unit membranes. Polymerized O-antigenic side-chains in the lipopolysaccharide appear to be required to produce the wild-type appearance of the outer membrane in P. aeruginosa.

Antigens, Bacterial↗

PubMed1986

Isolation and characterization of an O-methylglucose-containing lipopolysaccharide produced by Nocardia otitidis-caviarum.

The low-Mr lipopolysaccharide produced by Nocardia otitidis-cavirum is composed of 6-O-methyl-D-glucose (11 mol), D-glucose (8 mol) and 3-O-methyl-D-glucose (1 mol). Glyceric acid was also found as a constituent. Methylation and periodate oxidation analyses suggested that the backbone was formed by (1----4)-linked glucose and O-methylglucose residues. Glucose (1 mol) and 6-O-methylglucose (1 mol) served as branching points. Acetic acid was the major acyl substituent esterifying glucose residues. The lipopolysaccharide was isolated from the cytoplasmic fraction. Several other Nocardia strains were examined; all possessed the same lipopolysaccharide in their extracts.

3-O-Methylglucose↗

PubMed1975

Isolation of lipopolysaccharide from the walls of Micropolyspora faeni: chemical composition and serological reactivity.

Extraction of mycelium or walls of Micropolyspora faeni with cold or hot aqueous phenol yielded a lipopolysaccharide consisting of lipid A, phosphate, galactose, arabinose, glucose, glucosamine, and a dideoxy sugar. Extraction with trichloroacetic acid (TCA) yielded an incomplete molecule lacking lipid A. Part of an O-chain was secreted into the culture medium. Phenol and TCA extracts gave three lines of precipitation with human serum from cases of farmer's lung disease, and one of these was given by the culture medium polysaccharide. Serologically-reactive sugars were arabinose, galactose and glucose. The lipopolysaccharide fixed on to red cells which agglutinated in the presence of specific antibody and lysed on the addition of complement. The lipopolysaccharide appeared to elicit mainly IgM antibodies in animals, but IgM and IgG antibodies in humans.

Actinomycetales↗

PubMed1984

The effect of capsular polysaccharide and lipopolysaccharide of Bacteroides fragilis on polymorph function and serum killing.

The determinant responsible for the ability of Bacteroides spp. to inhibit polymorph phagocytic killing of aerobic organisms has not yet been identified. Therefore, the roles of lipopolysaccharide and capsular polysaccharide of B. fragilis were investigated. Serum-resistant and serum-sensitive strains of Proteus mirabilis were used to indicate inhibition of phagocytic killing and serum killing of aerobes. Whole organisms of B. fragilis, purified lipopolysaccharide and capsular polysaccharide were added to an in-vitro phagocytosis system. Results showed that greater than 10(7) bacteroides/ml inhibited both serum and phagocytic killing. Concentrations below 10(7)/ml had little effect on either process. Purified capsular polysaccharide (10 or 100 micrograms/ml), either alone in the system or in combination with sub-inhibitory concentrations of B. fragilis also markedly inhibited serum and phagocytic killing. Lipopolysaccharide (9 micrograms/ml) appeared relatively inert. B. ovatus, reputedly non-capsulated, produced identical results to those obtained with B. fragilis, but an encapsulated strain of Streptococcus pneumoniae did not inhibit serum or phagocytic killing.

Bacteroides fragilis↗

PubMed2002

Lipopolysaccharide phosphorylating enzymes encoded in the genomes of Gram-negative bacteria are related to the eukaryotic protein kinases.

By means of profile-matching procedures, conservation of functionally important residues, and fold-recognition techniques, we show that two distinct families of lipopolysaccharide kinases encoded in the genomes of Gram-negative bacteria are related to each other and to two distinct classes of proteins, namely eukaryotic protein kinases and right open reading frame (RIO1). Members of one of the lipopolysaccharide kinase families are identified only in pathogenic bacteria. Phosphorylation by these enzymes is relevant in the construction of outer membrane, immune response, and pathogenic virulence. The class of proteins called RIO1, also related to eukaryotic protein kinases and previously known to occur only in archaea and eukaryotes, are now identified in eubacteria as well. It has been suggested here that RIO1 proteins are intermediately related to lipopolysaccharide kinases and eukaryotic protein kinases implying an evolutionary relationship between the three classes of proteins.

Amino Acid Sequence↗

PubMed1980

Cell growth and antimicrobial activity of mouse peritoneal macrophages in response to glucocorticoids, choleragen and lipopolysaccharide.

Normal, thioglycollate-stimulated and BCG-activated mouse peritoneal macrophages were cultivated in vitro with the conditioned medium of mouse L-929 cells. The thioglycollate- and BCG-macrophages rapidly proliferated, whereas normal macrophages grew more slowly. A clear morphological difference between the three types of macrophages in the culture was observed. Glucocorticoids inhibited the growth of the macrophages at pharmacological concentrations. Other steroids, progesterone, diethylstilbestrol and testosterone in that order, had a far lower growth-inhibiting effect. Macrophages cultured with 10(-6) M dexamethasone had a reduced antimicrobial effect on Candida parapsilosis compared with that of the untreated cells. Choleragen had the same effect on the macrophages as glucocorticoids. The toxin inhibited growth at a concentration as low as 10 pg/ml and cells treated with 1 ng of choleragen per ml had decreased antifungal activity. Similarly, Escherichia coli lipopolysaccharide at 10 ng/ml inhibited the growth of thioglycollate-macrophages. However, macrophages incubated with the lipopolysaccharide had enhanced anticandida activity. Thus, the immunosuppressors glucocorticoid and choleragen inhibited both the increase in the number of macrophages and the microbicidal activity of the phagocytes. Lipopolysaccharide, an immunostimulant, stimulated macrophage activity, but was toxic for cell growth.

Animals↗

PubMed1990

The occurrence of alpha (1----2) linked N-acetylperosamine--homopolymer in lipopolysaccharides of non-O1 Vibrio cholerae possessing an antigenic factor in common with O1 V. cholerae.

Chemical analysis was carried out on lipopolysaccharides from Vibrio cholerae bio-serogroup Hakata 487-85. The O-specific chain of the phenol-soluble lipopolysaccharides was demonstrated by 13C-NMR spectroscopy and methylation analysis to contain a linear homopolymer of alpha(1----2) linked N-acetylperosamine (4-acetamido-4,6-dideoxy-D-mannopyranose), which was closely similar to but not identical to a linear alpha(1----2) linked N-3-deoxy-L-glycerotetronyl (S-2,4-dihydroxybutyryl) perosamine-homopolymer constituting that of O1 Vibrio cholerae lipopolysaccharides.

Amino Sugars↗

PubMed1992

Helicobacter pylori lipopolysaccharide stimulates gastric mucosal pepsinogen secretion.

The effect of Helicobacter pylori lipopolysaccharide on guinea pig gastric mucosal pepsinogen secretion has been examined using an Ussing chamber technique. Luminal addition of H. pylori lipopolysaccharide resulted in a fifty-fold stimulation of pepsinogen secretion compared to a twelve-fold increase with E. coli lipopolysaccharide. Electron microscopy showed marked degranulation of zymogen granules but no evidence of chief cell disruption.

Animals↗

PubMed1992

Synthesis of lipopolysaccharide by Escherichia coli cells recovering from sublethal heat stress.

Escherichia coli cells exposed to a sublethal heat treatment at 55 degrees C for 15s synthesized lipopolysaccharide during their recovery period after heat stress. As chloramphenicol at least partly inhibited the synthesis of lipopolysaccharide, it is suggested that its synthesis might be in part due to the lipopolysaccharide-synthesizing enzymes produced de novo. The results obtained coincided with our previous finding that the permeability barrier function was repaired by heat-stressed cells.

Adaptation, Biological↗

PubMed1994

Detection of lipopolysaccharide in the picogram range of tissue culture media by a kinetic chromogenic Limulus amebocyte lysate assay.

A kinetic chromogenic Limulus amebocyte lysate assay for detection of lipopolysaccharide in the picogram range is described. The kinetic Limulus assay was used to detect lipopolysaccharide in a tissue culture media containing interfering low-molecular-weight substances. The interfering substances were removed by ultrafiltration. The recovery of lipopolysaccharide from the ultrafiltrated tissue culture media was 61.6 +/- 21.4%. The kinetic Limulus assay was compared with the traditional end-point chromogenic Limulus assay. The results in the two assays showed a good correlation. The detection limit was 1 pg/ml in the chromogenic assay and 0.25 pg/ml in the kinetic assay.

Amino Acid Sequence↗