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PubMed2005

Non-invasive imaging of the endocrine pancreas (review).

The non-invasive imaging of the endocrine pancreas is currently considered by concerned institutions as a priority theme of research. Because the endocrine pancreas represents only about one percent of the pancreatic gland, highly specific tools are required for labelling the insulin-producing cells. The present review deals with the possible use of D-mannoheptulose for the non-invasive quantification of insulin-producing cells in the pancreas. This heptose is transported into hepatocytes and insulin-producing cells, but not other cell types, at the intervention of GLUT2. Its uptake coincides with the intracellular accumulation of acidic metabolites generated by phosphorylation of D-mannoheptulose. These metabolites remain in the islet cells after prolonged washing, suggesting that imaging of the pancreas at a relatively late time after intravenous administration of D-mannoheptulose could avoid significant extracellular contamination. Using tritiated D-mannoheptulose as tracer, a new method was designed for quantification of the total mass of insulin-producing cells in either isolated perfused rat pancreata or isolated pancreatic islets. Likewise, a preferential labelling of hepatocytes and insulin-producing transplanted cells can be achieved in vivo after administration of tritiated D-mannoheptulose. In the light of results obtained with 6-deoxy-6-iodo-D-glucose, it is proposed that 7-deoxy-7-iodo-D-mannoheptulose could be used for the non-invasive imaging of the endocrine pancreas.

Animals↗

PubMed1992

Bacteroides and Staphylococcus glycocalyx: chemical analysis, and the effects on chemiluminescence and chemotaxis of human polymorphonuclear leucocytes.

Glycocalyx (or slime), which is an important virulence factor of many pathogenic bacteria, was isolated from Bacteroides fragilis, Bacteroides thetaiotaomicron and Staphylococcus epidermidis. Organisms were grown for 24 h in a chemically defined, dialysable liquid medium. Bacteria were centrifuged and the supernatant was concentrated and dialysed against distilled water. Total carbohydrate and protein were estimated using standard methods. Thin layer and gas-liquid chromatography of trifluoro acetic acid hydrolysed and non-hydrolysed samples provided evidence for the presence of polysaccharide, the absence of nucleic acids and lipopolysaccharide and for the identification of the individual sugar residues. Glucose, mannose and galactose (B. fragilis), glucose (B. thetaiotaomicron), and glucose and heptose (S. epidermidis) were the sugar residues detected. Uronic acid and hexosamine were detected in all species. Glycocalyx preparations (1 mg/ml) from Bacteroides and Staphylococcus significantly inhibited the chemiluminescence and chemotactic responses of viable human polymorphonuclear leucocytes (PMNL), but were not toxic for PMNL.

Bacteroides↗

PubMedDate not supplied

[Characterization of lipopolysaccharides from Pseudomonas fluorescens IMB 2108 (biovar II) and IMB 2111 (biovar IV) with O-chains represented by alpha-glucan].

Results of studies of the structurally unique O-chains of lipopolysaccharides, which were isolated from the dry biomass of Pseudomonas fluorescens IMB 2108 (biovar II) and IMB 2111 (biovar IV) by the Westphal technique and purified by repeated ultracentrifugation, are reported. The bulk of the lipopolysaccharide preparations contained S- and R-molecules at an average molar ratio of 1: 2. The main components of the hydrophobic moiety of lipid A were 3-hydroxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, hexadecanoic, and octadecanoic acids, as well as hexadecenoic and octadecenoic acids. Glucosamine and phosphoethanolamine were identified as components of the hydrophilic moiety of lipid A. The degree of lipid A phosphorylation amounted to 3-4%. Fractions of the core oligosaccharide contained glucose, galactose, mannose, rhamnose, arabinose, glucosamine (only in strain IMB 2108), alanine, phosphoethanolamine, phosphorus, and 2-keto-3-deoxyoctulosonic acid (KDO). Heptose was present in trace amounts. O-specific polysaccharide chains were represented by a linear polymer of D-glucose units, which were linked together via alpha-(1,4) glycoside bonds. The existence of P. fluorescens strains that have alpha-1,4-glucan as the O-chain of their lipopolysaccharides has not been described before.

Chromatography↗

PubMed1991

Structural and biological comparison of lipopolysaccharides (LPS) from Bordetella species.

Lipopolysaccharides (LPS) isolated from Bordetella pertussis (Bp), B. parapertussis (Bpp), and B. bronchiseptica (Bbs) were analysed for their chemical composition, molecular heterogeneity, and immunological and biological properties. All LPS contained heptose, KDO, GlcN, uronic acid, phosphate, and fatty acids. The fatty acids C14:0, C16:0 and 3-OHC14:0 were common to all LPS preparations. By SDS-PAGE, Bp-LPS had two bands of low molecular mass, and Bpp- and Bbs-LPS showed a low molecular mass band together with ladder bands of high molecular mass. Immunological assays demonstrated that Bp-LPS reacted with antisera prepared from Bp and Bpp; Bpp-LPS reacted with antisera against Bpp and Bbs, and Bbs-LPS reacted with antisera against any of the three species. Bp-LPS showed biological activities comparable to those of E. coli LPS in terms of lethal toxicity, pyrogenicity, mitogenicity, macrophage activation, and induction of tumor necrosis factor. All activities of Bpp-LPS, except mitogenicity, were lower than those of E. coli LPS. Biological activities stronger or comparable to those of E. coli LPS were observed for Bbs-LPS.

Animals↗

PubMed1991

Mapping of the binding specificity for five monoclonal antibodies recognizing 3-deoxy-D-manno-octulosonic acid in bacterial lipopolysaccharides.

Mouse mAb were produced against the deep rough strains Salmonella minnesota R 595, Salmonella typhimurium SL 1102, and Escherichia coli D21f2 and screened by enzyme immunoassay against LPS of several chemotypes. Five antibodies were selected for their ability to bind to chemotype deep rough (Re) LPS which has two 3-deoxy-D-manno-octulosonic acid (Kdo) residues in its nonreducing end. Structurally verified oligosaccharides isolated from rough LPS and synthetic analogues of Kdo were used in an enzyme immunoassay inhibition test to determine the binding epitopes for the antibodies. According to their specificities, the antibodies could be divided into three groups. For two of the groups, the recognized structure was the alpha-Kdo (2----4) Kdo disaccharide and for one group the alpha-Kdo (2----4) alpha-Kdo beta-D-GlcN (1----6) alpha-D-GlcN tetrasaccharide, representing a partial structure of the Re LPS. Inhibition studies with synthetic analogues of Kdo showed that the anomeric configuration and the free carboxyl group of the Kdo residue are important features for antibody binding. Changes in the C-1 to C-6 region of the Kdo molecule do influence the antibody recognition considerably whereas changes in the exocyclic C-7 to C-8 region are of secondary importance. Calculation of the conformation of the inner core region showed that the alpha-Kdo (2----4) alpha-Kdo (2---- disaccharide was free and accessible in chemotype Re LPS, but that linkage of a L-glycero-D-manno-heptose to O-5 of the subterminal Kdo both changes the conformation of the Kdo-disaccharide and covers it thereby making it less accessible.

Animals↗

PubMed1985

Immunological study of Pseudomonas aeruginosa extracellular slime.

Crude extracellular slime (C-ES) was isolated from 15 laboratory strains of Pseudomonas aeruginosa. Glycolypoprotein (GLP) was prepared from partially purified C-ES by the phenol treatment method. Lipopolysaccharides (LPS) were isolated from P. aeruginosa and Salmonella typhi-murium. Some correlation has been found between the quantity of the produced C-ES and virulence of P. aeruginosa for mice. Toxicity (LD50 value) and the yield of GLP did not correlate with the strain virulence. C-ES and GLP contained hexose, protein and hexosamine. Heptoses (characteristic LPS components) were present in C-ES and practically absent from GLP. Paper chromatographic analysis of GLP showed the presence of uronic acids, galactose, glucose and ribose in all strains; GLP of most strains contained rhamnose and a low amount of mannose and several strains xylose. Uronic acids, galactose and, probably, ribose and mannose were characteristic ES components. C-ES and GLP were studied in active and passive mouse protection. ES isolated from P. aeruginosa strains of different O serogroups or immunotypes induced a marked cross protective response.

Antigens, Bacterial↗

PubMed1988

The heterogeneity of lipopolysaccharides from Citrobacter 023 serotype.

Chemical studies on carbohydrate part of lipopolysaccharides isolated from Citrobacter 87/57 and Citrobacter 1556 strains (serotype 023) were carried out. Lipopolysaccharides (LPS) were hydrolyzed with 1% acetic acid and the carbohydrate material was fractionated on Sephadex G-50 and Bio-Gel P-4 columns. All fractions obtained were subjected to chemical analysis by colorimetric methods and by gas-liquid chromatography. It was found that O-specific polysaccharide from LPS 87/57 is composed of mannose and N-acetylgalactosamine in the molar ratio of 3:1. Both lipopolysaccharides contain unsubstituted core oligosaccharides (built of heptose, glucose and galactose in molar ratio of 3:3:1) as well as core oligosaccharides substituted with shorter (LPS 87/57 and LPS 1556) and longer (LPS 87/57) O-specific oligosaccharides. Strain 87/57 is of S (smooth) type and strain 1556 lacking O-specific polysaccharide is of SR (semi-rough) type. The data of Sephadex G-100 chromatography showed that O-antigen from Citrobacter 87/57 is heterogeneous; it contains polysaccharide chains of different length but the same composition. The heterogeneity of Citrobacter 023 lipopolysaccharides has been also detected by SDS-polyacrylamide gel electrophoresis.

Acetylgalactosamine↗

PubMedDate not supplied

[Immunochemical characteristics of a lipopolysaccharide from Pseudomonas aurantiaca].

A lipopolysaccharide was isolated from Pseudomonas aurantiaca IMB 31 by extraction with aqueous phenol and purified by ultracentrifugation. The lipopolysaccharide was confined to the phenol phase. Fucosamine (2-amino-2,6-dideoxygalactose) (36%) and bacillosamine (2,4-diamino-3,4,6-trideoxyglucose) (23%) were identified as hypothetic components of the O-chain in the carbohydrate moiety of the macromolecule using the techniques of paper chromatography, gas-liquid chromatography and ion-exchange chromatography on an amino acid analyser. Rhamnose, glucose, galactose, glucosamine and galactosamine were detected as hypothetical components of the core in the lipopolysaccharide composition, as well as 2-keto-3-deoxyoctonic acid, heptose, alpha-alanine and phosphorus, usual components of the core in Pseudomonas. The following predominant fatty acids were identified in the composition of lipid A using the techniques of gas-liquid chromatography with standard compounds and gas-liquid mass spectrometry: 3-OH C10:0 (14.4%), C12:0 (30.5%), 2-OH C12:0 (14.9%), 3-OH C12:0 (17.4%), C16:0 (9.9%). The serological relationship between P. aurantiaca strains was studied, and their phylogenetic relationship with P. fluorescens is discussed.

Immunochemistry↗

PubMed1986

Modification of the chemical composition and structure of the U.S. Reference Standard Endotoxin (RSE) by 60Co radiation.

A highly purified bacterial lipopolysaccharide (LPS) preparation was exposed in water to megadoses of ionizing radiation from a 60Co source. As evidenced by electrophoresis, the radiation treatment progressively degraded the lipopolysaccharide molecules by removing first the O-side chain units and then components of the R-core. Chemical analysis of the irradiated (LPS) preparations showed that, in accord with the structural changes, the most profound effects of ionizing radiation occurred in the hydrophilic oligo/polysaccharide moieties (R-core and O-side chain). Progressively higher doses of radiation degraded the simple sugars in decreasing order of galactose, galactosamine, glucosamine, glucose, and heptose. The R-core component 2-keto-3-deoxyoctonate was the most "resistant" sugar derivative to ionizing radiation. Due to its central position in the LPS aggregates in water, even at comparatively high doses of radiation the hydrophobic lipid A moiety of endotoxin was less affected than the sugar components. Of the fatty acids of lipid A, however, either partial conversion of beta-hydroxymyristic acid into myristic acid or selective loss of the former occurred. The observed structural and chemical changes of LPS are consistent with the effect of active oxygen radicals of radiolysis. In addition, the extensive physicochemical changes explain the altered biological reactivity of radiation-treated endotoxins.

Cobalt Radioisotopes↗

PubMed1986

Mechanism of human platelet activation by endotoxic glycolipid-bearing mutant Re595 of Salmonella minnesota.

The mechanism through which human blood platelets interact with gram-negative bacteria with well-defined structural variations in endotoxic lipopolysaccharide was studied. Secretion of 14C-serotonin and aggregation of platelets separated from plasma proteins were observed on challenge with rough mutant Re595 of Salmonella minnesota possessing a glycolipid outer layer composed of Lipid A and 2-keto-3-deoxyoctonate (KDO) but lacking heptose phosphate in the core and O-polysaccharide in its outer portion. Both 14C-serotonin secretion and platelet aggregation were concentration-dependent, with a half-maximum response at the ratio of one bacterial colony-forming unit (CFU) to two platelets. The aggregation of human platelets induced by mutant Re595 was divalent cation-dependent and required secretion of ADP and fibrinogen from platelet storage granules because it was inhibited by chelators, by the ADP-splitting enzyme apyrase, and by monospecific antifibrinogen Fab fragments. The synthetic peptide analog of the platelet receptor recognition site on the gamma chain of fibrinogen, gamma 400-411, inhibited platelet aggregation induced by mutant Re595 (IC50 160 mumol/L), whereas serotonin secretion was unaffected. Tetrapeptide, RGDS, analogous to human fibrinogen alpha chain (alpha 572-575) and to the cell adhesion site of fibronectin, also inhibited aggregation induced by mutant Re595 (IC50 60 mumol/L). Secretion of 14C-serotonin was preceded by a very rapid phosphorylation of a platelet protein of mol wt 47,000, which is associated with protein kinase C activation. Myosin light chain (mol wt 20,000) was also phosphorylated. Both phosphoproteins were dephosphorylated while secretion was reaching maximum. Furthermore, release of 3H-arachidonic acid from platelet phospholipids and generation of thromboxane B2 via the cyclooxygenase pathway were observed. Inhibition of this pathway with acetylsalicylic acid (10(-4) mol/L) or indomethacin (5 X 10(-4) mol/L) reduced 14C-serotonin secretion and platelet aggregation. The role of Lipid A in the interaction of mutant Re595 with human platelets was deduced from the inhibitory effect of the Lipid A-binding protein present in Limulus amebocyte lysate. Likewise, polymyxin B, known to complex with Lipid A, was inhibitory. The reactivity of mutant Re595 toward platelets was attenuated by mild acid hydrolysis, during which KDO was dissociated from the glycolipid, and by alkaline hydrolysis, which breaks ester-linked fatty acids in Lipid A. In contrast to mutant Re595, strain S218 of S minnesota bearing "complete" endotoxic lipopolysaccharide did not induce secretion and aggregation of human platelets.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗

PubMed1977

Biochemical and biological studies on the lipopolysaccharide of Bacteroides nodosus.

Lipopolysaccharides (LPS) were extracted from three Bacteroides nodosus isolates by the aqueous phenol method and purified by ultracentrifugation. The structure of B nodosus LPS appears to be similar to enterobacterial LPS, with the polysaccharide component joined to the lipid A moiety through an acid-labile 2-keto-3-deoxyoctonic acid (KDO) linkage. B nodosus LPS contained glucose, galactose, hexosamine, KDO and heptose and no sugars apart from ribose, which was possibly a nucleic acid contaminant, were unique to any of the isolates. Electron microscopic examination indicated a similar morphology to LPS derived from other Gram-negative bacteria. B nodosus LPS was found to exhibit biological properties characteristic of endotoxins, such as pyrogenicity, leucopenic and leucocytotic activity, production of the primary inflammatory response in rabbit skin and Shwartzman reactivity. However, the toxicity of B nodosus LPS was low.

Animals↗

PubMed1981

Serological analysis of core oligosaccharides of Shigella flexneri serotype 6 and its R mutants lipopolysaccharides.

The core oligosaccharides originating from Shigella flexneri 6 S strains and R mutants were examined by the complement fixation inhibition and passive hemolysis inhibition tests using antisera against the complete and partially degraded core regions. Strong cross reactivity between Shigella flexneri 6, Shigella sonnei phase II and E. coli C core fractions which all are of R1 type was observed. Terminal beta-D-glucosyl groups and alpha-D-galactosyl-1,2-alpha-D-galactosyl sequences are essential elements of R1 type immunodeterminants. Heptose region of the R1 cores seems not be involved in their serological specificity.

Antigens, Bacterial↗

PubMed1983

Characterization of the lipopolysaccharide from the nod mutant of Rhizobium trifolii.

Lipopolysaccharides (LPS) from the non-nodulating Rhizobium trifolii 24SM 15 and from the nodulating R. trifolii 24SM 13 were isolated and examined by means of gas-liquid chromatography and mass spectrometry. Analysis of LPS showed these preparations from both strains examined contained Lipid A, 2-keto-3-deoxyoctonate, neutral sugars, amino sugars, and trace amounts of amino acids. In 24SM 13 LPS prevailed glucose and rhamnose whereas LPS from the non-nodulating strain SM 15 contained mainly mannose, galactose and heptose. Quinovosamine and mannosamine were detected only in the nodulating strain. The ratio of glucosamine phosphate to glucosamine was higher in the LPS of the non-nodulating strain SM 15 than in the corresponding material of the nodulating one. An unknown component producing a peak at the position of glyceryl-S-cysteine on amino acid analysis profiles was detected in SM 15 LPS. The differences in LPS composition were associated with the alterations in the sensitivity to phage 3H, and nodulation ability.

Amino Acids↗

PubMed1984

[Structure of O-specific polysaccharide isolated from the Yersinia pseudotuberculosis serotype 1A lipopolysaccharide].

An O-specific polysaccharide from the lipopolysaccharide Yersinia pseudotuberculosis 1A serovar has been isolated and characterized. This compound was shown to contain residues of paratose, 6-deoxy-D-manno-heptose, D-galactose and 2-amino-2-deoxy-D-glucose in equimolar ratios. Using methylation studies, partial acid hydrolysis and 13C NMR spectroscopy, the following structure was proposed for the repeating unit of the O-specific polysaccharide: (Formula: see text).

Antigens, Bacterial↗

PubMed1982

Arrangement of bacteriophage lambda receptor protein (LamB) in the Escherichia coli cell surface.

The lambda receptor protein (LamB) was assembled into an ordered hexagonal lattice structure with a lattice constant of about 7.8 nm in the presence of lipopolysaccharide. Both the heptose-containing polysaccharide region and the fatty acid region are suggested to be involved in the interaction with LamB. The lattice structure was preferably formed on the peptidoglycan layer when the lipoprotein was covalently bound to this layer. In the presence of chloroform, the ordered hexagonal lattice structure was active in the receptor function for lambda, resulting in phage adsorption and DNA ejection.

Bacterial Outer Membrane Proteins↗

PubMed1984

Responses of equine neutrophils to contagious equine metritis organism and its lipopolysaccharides.

Morphology and function of equine neutrophils were evaluated after combination with contagious equine metritis organism (CEMO) or 1 of 2 CEMO lipopolysaccharides (LPS). The 2 LPS (LPS-a; LPS-p) isolated from the CEMO contained 14- and 16-carbon fatty acids, ketodeoxyoctanate, hexose, and heptose, but were morphologically distinct. Neutrophils exposed to LPS had fewer granules, whereas those exposed to CEMO had more granules than did the controls (phosphate-buffered saline solution). Neutrophil iodination was significantly increased with 10 and 25 micrograms of LPS-a, but not significantly altered by LPS-p or CEMO. Staphylococcus aureus ingestion was not influenced by CEMO and was mildly decreased by LPS-a. These results indicate that CEMO may have at least 2 functionally and morphologically distinct, but chemically similar, LPS and that 1 of these LPS (LPS-a) may enhance neutrophil killing by stimulating neutrophil iodinating mechanisms.

Animals↗

PubMed1982

Purification and structural determination of nontoxic lipid A obtained from the lipopolysaccharide of Salmonella typhimurium.

Endotoxin extracted from the heptose-less mutant of Salmonella typhimurium was hydrolyzed in 0.1 N HCl in methanol/water (1:1, v/v) at 100 degrees C to yield lipid A, which was then fractionated on a Sephadex LH-20 column to yield a major monophosphoryl lipid A fraction. The monophosphoryl lipid A was further fractionated by preparative thin layer chromatography. This process yielded three major bands (TLC-1, -3, and -5) and two minor bands (TLC-7 and -9). The purity of these fractions was established by ion exchange and reverse phase high performance liquid chromatography. The thin layer fractions were analyzed by fast atom bombardment mass spectrometry. TLC-1 and -3 gave molecular ions (M-H)- at m/e 1730 and 1716, respectively. Both of these fractions contained beta-hydroxymyristic, lauric, and 3-myristoxymyristic acids in O-acyl linkages. The molecular formula and Mr of TLC-1 are C95H179O22N2P and 1731.16; those of TLC-3 are C94H177O22N2P and 1717.15. TLC-1 was a methyl homolog of TLC-3. The major component of TLC-5 (C80H151O22N2P and Mr = 1506.99) gave a molecular ion at m/e 1506 and contained two beta-hydroxymyristic acids and a lauric acid in the O-acyl linkages. The major component of TLC-7 (C66H125O19N2P and Mr = 1280.83) and the single component of TLC-9 gave molecular ions at m/e 1280 and 1098, respectively. TLC-7 contained lauric and beta-hydroxymyristic acids in the O-acyl linkages. TLC-9 (C54H103O18N2P and Mr = 1098.69) contained a single O-acylated beta-hydroxymyristate group. TLC-1 and -3 were nontoxic in the chick embryo lethality test and regressed established tumors in the syngeneic guinea pigs.

Chromatography, High Pressure Liquid↗

PubMed1980

Killing of the S and Re forms of Salmonella minnesota via the classical pathway of complement activation in guinea-pig and human sera.

The S (wildtype) and Re form (heptose-deficient, core-defective mutant) of Salmonella minnesota were killed by treatment with normal guinea-pig serum (GPS). Using C4-deficient GPS and serum containing 0.02 M ethyleneglycol-bis-(beta-aminoethylether)-tetraacetic acid and 0.02 M MgCl2 (EGTA-Mg2+) a reduced killing rate was observed. In normal GPS diluted 1:10 containing 0.02 M EGTA-Mg2+ or in C4-deficient GPS diluted 1:10 no killing occurred, whereas the same serum dilution without EGTA-Mg2+ showed a strong bactericidal effect indicating a dependency upon C4 and Ca2+ ions. Furthermore, in contrast to normal human serum (NHS) no killing occurred in a selective complete C1q-deficient human serum. The bactericidal effect, however, could be restored by addition of highly purified C1q; this is a further indication for a dependency upon the classical pathway of C activation. The C-dependent bactericidal activity was totally abolished when phosphate buffer was used, partially reduced in the presence of veronal-buffered saline (VBS), and not affected by tris-(hydroxymethyl)-aminomethane(Tris) or thioglycollate-buffered system EGTA-Mg2+ alone slightly reduced the growth rate of the bacteria whereas disodium ethylene diaminetetraacetate (EDTA) had a bacteriostatic effect on the S-form. The inhibition of the growth of the Re-form by EDTA was amplified by the addition of serum. Pre-incubation of bacteria with serum for absorption of antibodies did not increase the killing rate of such pre-treated bacteria excluding an antibody-mediated bactericidal reaction. Furthermore, pre-treatment of the bacteria with GPS at 0 degrees reduced the serum sensitivity of both types of bacteria.

Animals↗