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PubMedDate not supplied

The cut-off criterion for a positive hydrogen breath test in children: a reappraisal.

Seventy-three preschool children with adequate nutritional status underwent interval-sampling, 3-hour breath-hydrogen carbohydrate absorption tests after consuming either 240 ml of intact milk (containing 12 g of lactose) or the same volume of milk with 90-95% of its lactose prehydrolyzed in vitro (containing less than 1 g lactose, with the remaining sugar as glucose and galactose). Results were examined in a reappraisal of the cut-off criterion for the rise of breath H2 concentration signifying biologically incomplete absorption. If the greater than or equal to 10-ppm criterion advocated by some investigators is used, 83% of our subjects would have been classified as incomplete lactose digesters and 30% would have their tests with the monosaccharide-rich milk classified as positive. With the greater than or equal to 20 ppm criterion used in our laboratory and others, the prevalence of lactose maldigestion in the sample becomes 60% and only 4% of subjects have apparent monosaccharide absorption, 96% having a rise below the cut-off level with prehydrolyzed milk. At least in Guatemalan preschoolers, the 20-ppm criterion for a positive breath H2 test provides a superior specificity-sensitivity balance and more reasonable diagnostic conclusions.

Animals↗
PubMed2002

Defining tympanostomy tube plugs.

OBJECTIVES: To define the composition of tympanostomy tube plugs because selecting or developing effective solvents depends on such knowledge. STUDY DESIGN: Prospective, in vitro laboratory study. METHODS: Luminal contents of 105 plugged, microscopically removed tympanostomy tubes were expressed, pooled, acid hydrolyzed, and passed through a high-performance liquid chromatography column. Retention times were compared with high-performance liquid chromatography standards to develop free amino acid and monosaccharide profiles. Cerumen, blood, and chronic mucoid effusion (collected during myringotomy) were pooled and subjected to the same analysis. The elution profiles of each substance were compared to determine which substance most closely matched the plugs. RESULTS: High-performance liquid chromatography amino acid and monosaccharide analysis demonstrated greatest similarity between tympanostomy tube plugs and mucoid effusion. CONCLUSIONS: Tympanostomy tube plug composition is more similar to mucoid effusion than to blood or cerumen. Solvents to open plugged tympanostomy tubes should be directed against the components of mucoid effusion.

Amino Acids↗
PubMed2005

Terminal sialic acid residues on human glycophorin A are recognized by porcine kupffer cells.

BACKGROUND: We have previously shown that recognition of human erythrocytes by porcine Kupffer cells is mediated by a carbohydrate-dependent mechanism. The present study explores the possible ligands existing on human glycophorin A and tests their ability to inhibit erythrocyte rosette formation. METHODS: Human erythrocytes were tested for ABO and MN specificity and used as targets in a 51Chromium quantitative erythrocyte rosette assay. Monosaccharides present on human glycophorin A, neuraminyl lactoses, bovine and porcine submaxillary mucins (BSM and PSM), and hyaluronic acid as well as proteoglycan N-linked glycosidase F(PNGaseF)- and sialidase A-treated human erythrocyte glycoproteins (hEGP) and human erythrocytes were all tested for inhibitory potential in the rosetting assay. RESULTS: Porcine Kupffer-cell recognition of human erythrocytes was insensitive to differences in blood groups A, B, O, or MN. At 30 mM, the monosaccharide, N-acetylneuraminic acid, and the trisaccharide mixture, neuraminyl lactoses, disrupted human erythrocyte recognition by 25% and 30%, respectively. A dilution of BSM but not PSM inhibited the rosetting assay by 17% (.2 mg/mL), 33% (1 mg/mL), and 53% (2 mg/mL). The same dilution of hyaluronic acid had no effect on rosetting. Removal of N-linked oligosaccharides from hEGP with PNGaseF did not impair its ability to inhibit the rosetting assay. In contrast, removal of sialic acid completely abrogated its inhibitory ability. Treatment of whole human erythrocytes with sialidase A likewise prevented recognition by porcine Kupffer cells. CONCLUSIONS: Terminal sialic acid on human erythrocytes is a target recognized by porcine Kupffer cells, suggesting a role for a sialic-acid receptor in innate cellular recognition of xenogeneic epitopes. Inasmuch as this work reveals a carbohydrate-recognition mechanism for cellular rejection, we shed light on a potential new boundary that will need to be overcome within xenotransplantation.

ABO Blood-Group System↗
PubMed2004

Evidence for apoplasmic phloem unloading in developing apple fruit.

The phloem unloading pathway remains unclear in fleshy fruits accumulating a high level of soluble sugars. A structural investigation in apple fruit (Malus domestica Borkh. cv Golden Delicious) showed that the sieve element-companion cell complex of the sepal bundles feeding the fruit flesh is symplasmically isolated over fruit development. 14C-autoradiography indicated that the phloem of the sepal bundles was functional for unloading. Confocal laser scanning microscopy imaging of carboxyfluorescein unloading showed that the dye remained confined to the phloem strands of the sepal bundles from the basal to the apical region of the fruit. A 52-kD putative monosaccharide transporter was immunolocalized predominantly in the plasma membrane of both the sieve elements and parenchyma cells and its amount increased during fruit development. A 90-kD plasma membrane H(+)-ATPase was also localized in the plasma membrane of the sieve element-companion cell complex. Studies of [14C]sorbitol unloading suggested that an energy-driven monosaccharide transporter may be functional in phloem unloading. These data provide clear evidence for an apoplasmic phloem unloading pathway in apple fruit and give information on the structural and molecular features involved in this process.

Biological Transport↗
PubMed2004

Production of exopolysaccharides by Antarctic marine bacterial isolates.

AIMS: This study was undertaken to examine and characterize Antarctic marine bacterial isolates and the exopolysaccharides (EPS) they produce in laboratory culture. METHODS AND RESULTS: Two EPS-producing bacterial strains CAM025 and CAM036 were isolated from particulate material sampled from seawater and sea ice in the southern ocean. Analyses of 16S rDNA sequences placed these isolates in the genus Pseudoalteromonas. In batch culture, both strains produced EPS. The yield of EPS produced by CAM025 was 30-fold higher at -2 and 10 degrees C than at 20 degrees C. Crude chemical analyses showed that these EPS were composed primarily of neutral sugars and uronic acids with sulphates. Gas chromatographic analysis of monosaccharides confirmed these gross compositional findings and molar ratios of monosaccharides revealed differences between the two EPS. CONCLUSIONS: The EPS produced by Antarctic bacterial isolates examined in this study appeared to be polyanionic and, therefore, 'sticky' with respect to cations such as trace metals. SIGNIFICANCE AND IMPACT OF THE STUDY: As the availability of iron as a trace metal is of critical importance in the southern ocean where it is know to limit primary production, the role of these bacterial EPS in the Antarctic marine environment has important ecological implications.

Antarctic Regions↗
PubMed2004

A new Ralstonia solanacearum high-affinity mannose-binding lectin RS-IIL structurally resembling the Pseudomonas aeruginosa fucose-specific lectin PA-IIL.

The plant pathogen Ralstonia solanacearum produces two lectins, each with different affinity to fucose. We described previously the properties and sequence of the first lectin, RSL (subunit M(r) 9.9 kDa), which is related to fungal lectins (Sudakevitz, D., Imberty, A., and Gilboa-Garber, N., 2002, J Biochem 132: 353-358). The present communication reports the discovery of the second one, RS-IIL (subunit M(r) 11.6 kDa), a tetrameric lectin, with high sequence similarity to the fucose-binding lectin PA-IIL of Pseudomonas aeruginosa. RS-IIL recognizes fucose but displays much higher affinity to mannose and fructose, which is opposite to the preference spectrum of PA-IIL. Determination of the crystal structure of RS-IIL complexed with a mannose derivative demonstrates a tetrameric structure very similar to the recently solved PA-IIL structure (Mitchell, E., et al., 2002, Nature Struct Biol 9: 918-921). Each monomer contains two close calcium cations that mediate the binding of the monosaccharide and explain the outstandingly high affinity to the monosaccharide ligand. The binding loop of the cations is fully conserved in RS-IIL and PA-IIL, whereas the preference for mannose versus fucose can be attributed to the change of a three-amino-acid sequence in the 'specificity loop'.

Adhesins, Bacterial↗
PubMed1979

Alternation of low and high affinities of secreted and cell-bound antibodies during the anamnestic response of rabbits to Salmonella senftenberg microorganisms.

We have investigated at the cellular level the variations in affinities of secreted antibodies and of cell receptors during the anamnestic response of rabbits, by using an immunization schedule made up of administrations, over successive intervals of 40 days, of Salmonella senftenberg vaccine. Affinities of free and membrane-bound antibodies biosynthesized at a given time were measured by inhibition of haemolytic plaque-forming cells (PFC) and rosette-forming cells (RFC) with spleen, lymph node, and peripheral blood cells. The immunodominant monosaccharide, methyl-alpha-D-glucopyranoside (alpha-Me-Glc), was used as inhibitor, and the target cells were coated with the O-specific polysaccharide of the immunogenic microorganisms. We observed that the monosaccharide concentration causing a 50% inhibition (I50) alternated between low and high values in both PFC and RFC assays. Furthermore, when sheep erythrocytes were coated with polysaccharides from other strains of Salmonella bearing the determinants for serotypes 1 or 3 the percentage of cross-reactive PFC obtained exhibited a reverse oscillating pattern of variation. These results demonstrated that affinity of secreted antibodies or of antigen-receptors of cells, directed against different determinants of the antigenic polysaccharide, alternated between low and high values after each new vaccine administration. Possible mechanisms accounting for this phenomenon are discussed.

Animals↗
PubMed1985

Biosynthesis of the O9 antigen of Escherichia coli. Synthetic glycosyldiphosphomoraprenols as probes for requirement of mannose acceptors.

Synthetic monosaccharide derivatives (alpha-glucosyl, beta-glucosyl, alpha-mannosyl) and disaccharide derivatives (alpha-mannosyl-1,2-alpha-glucosyl, alpha-mannosyl-1,3-alpha-glucosyl, alpha-mannosyl-1,4-alpha-glucosyl, alpha-mannosyl-1,6-alpha-glucosyl) of diphosphomoraprenol were used as putative mannose acceptors in the biosynthesis of Escherichia coli O9 antigen. Membranes of E. coli O9 derived from the rfe mutant F 1357 were reconstituted with these compounds and then incubated with different concentrations of GDP-[14C]mannose. Of the monosaccharide derivatives tested, only alpha-glucodiphosphomoraprenol was a mannose acceptor and the only disaccharide derivative which accepted mannose was alpha-mannosyl-1,3-alpha-glucosyldiphosphomoraprenol. The alpha-glucosyl derivative accepted only one mannose unit at 4 microM GDP-[14C]mannose, and above 50 microM GDP-[14C]mannose about 25% of the product had a minimum size of about 30 mannose units. The alpha-mannosyl-1,3-alpha-glucosyl derivative was only a mannose acceptor at a GDP-[14C]mannose concentration of 50 microM and higher, and the product had a minimum size of about 30 mannose units. The results are discussed with respect to requirement of mannose acceptors.

Antigens, Bacterial↗
PubMed1982

Glycosylation of proteins in developing human brain.

The carbohydrate content of human brain glycoproteins was studied during development from the age of 12 fetal weeks to 8 postnatal months. The concentration of all the glycoprotein monosaccharides increased with age. The total amount of glycoprotein monosaccharides per lipid-free dry tissue increased by about 150% from the end of the third fetal month to the time of delivery. The increase leveled off around term, and only minor increase occurred after birth. The adult level was reached by the fifth postnatal month.

Adult↗
PubMed1994

Determination of specificities of rabbit antisera against the O-antigenic polysaccharide from Escherichia coli O126.

Rabbit polyclonal antibodies against the lipopolysaccharide of Escherichia coli O126 were serologically characterized by ELISA. The antibody specificities were determined by studying the inhibitory effects of the methyl glycosides of both anomeric configurations of the constituent monosaccharides and the oligosaccharides derived from the O-antigenic polysaccharide of E. coli O126. It was found that, amongst the monosaccharides, beta-D-N-acetyl glucosamine was the most effective inhibitory sugar in the O126 polysaccharide and the major specificity of the polyclonal antibodies was found to be directed against the trisaccharide having the structure alpha-D-Galp (1-->3)-beta-D-GlcpNAc(1-->2)-D-Manp.

Animals↗
PubMed1980

Fatty acids and neutral sugars present in lipopolysaccharides isolated from Fusobacterium species.

Lipopolysaccharides (LPS) extracted from cells of Fusobacterium necrophorum, F. mortiferum, F. Gonidiaformans. varium and single strains of F. naviforme and F. russi were analysed for sugars and fatty acids. All preparations contained a monosaccharide tentatively identified as L-glycero-D-mannoheptose, but showed variation with respect to the presence of glucose, galactose, rhamnose and another heptose isomer, tentatively identified as L-glycero-D-mannoheptose. L-glycero-D-mannoheptose was a monosaccharide of great abundance in all LPS examined. 2-keto-3-deoxy-octonate and glucosamine were present in all LPS preparations. The fatty acids present were 3-hydroxy-tetradecanoic acid as the main component, and n-tetradecanoic acid. Some LPS contained n-hexadecanoic acid. All the strains of F. nucleatum contained 3-hydroxy-hexadecanoic acid as a group-specific LPS constituent.

Calorimetry↗
PubMed2000

Factors affecting exocellular polysaccharide production by Lactobacillus delbrueckii subsp. bulgaricus grown in a chemically defined medium.

We developed a chemically defined medium (CDM) containing lactose or glucose as the carbon source that supports growth and exopolysaccharide (EPS) production of two strains of Lactobacillus delbrueckii subsp. bulgaricus. The factors found to affect EPS production in this medium were oxygen, pH, temperature, and medium constituents, such as orotic acid and the carbon source. EPS production was greatest during the stationary phase. Composition analysis of EPS isolated at different growth phases and produced under different fermentation conditions (varying carbon source or pH) revealed that the component sugars were the same. The EPS from strain L. delbrueckii subsp. bulgaricus CNRZ 1187 contained galactose and glucose, and that of strain L. delbrueckii subsp. bulgaricus CNRZ 416 contained galactose, glucose, and rhamnose. However, the relative proportions of the individual monosaccharides differed, suggesting that repeating unit structures can vary according to specific medium alterations. Under pH-controlled fermentation conditions, L. delbrueckii subsp. bulgaricus strains produced as much EPS in the CDM as in milk. Furthermore, the relative proportions of individual monosaccharides of EPS produced in pH-controlled CDM or in milk were very similar. The CDM we developed may be a useful model and an alternative to milk in studies of EPS production.

Culture Media↗
PubMed1977

Cell wall studies of Histoplasma capsulatum and Blastomyces dermatitidis using autologous and heterologous enzymes.

Enzymes capable of hydrolyzing cell walls of Blastomyces dermatitidis and chemotypes I and II of Histoplasma capsulatum were prepared in the laboratory or obtained from commercial sources. They included chitinases, beta-1,3-glucanases, beta-1,6-glucanase, and Pronase. Monosaccharides and disaccharides of glucose released from the cell walls by the enzymes were determined qualitatively by paper and gas-liquid chromatography, and monosaccharides were quantitated by the latter technique as well. An enzyme system isolated from Streptomyces sp. containing both chitinase and glucanase released maximum amounts of glucose and N-acetylglucosamine from the cell walls of H. capsulatum chemotype I. A chitinase preparation, free of glucanase, from Serratia marcescens released only chitobiose and N-acetylglucosamine from chemotype I cell walls, but the total quantity of N-acetylglucosamine released was about 60% less than that released by the Streptomyces system. A beta-1,3-glucanase from Bacillus circulans hydrolyzed the cell walls of H. capsulatum chemotype I, but a beta-1,6-glucanase failed to release glucose from the same walls. Autolytic enzymes, viz., beta-1,3-glucanases and several glycosidases were detected as constitutive enzymes in both yeast and mycelial phases of B. dermatitidis and H. capsulatum chemotypes I and II. No difference in the amount of activity was found between cell sap and culture filtrate preparations. The beta-glucanases prepared from the Histoplasma and Blastomyces strains were active on the cell walls of the yeast phases of H. capsulatum chemotypes I and II, releasing laminaribiose and glucose, but were essentially inactive on the cell walls of B. dermatitidis. Chitinase, beta-1,6-glucanase, alpha-glucanase, and alpha-glucosidase activities were absent from these fungal enzyme preparations.

Acetylglucosamine↗
PubMed1980

Lectin-mediated induction of human neutrophil chemotaxis, chemokinesis, and cap formation.

Six lectins, including concanavalin A, phytohemagglutinin P, castor bean I, wheat germ agglutinin, peanut agglutinin, and pokeweed mitogen, were studied for their ability to stimulate human neutrophil locomotion and cap formation. Five of these lectins with known monosaccharide specificities, including concanavalin A, phytohemagglutinin, P, castor bean I, wheat germ agglutinin, and peanut agglutinin, were found to stimulate human neutrophil migration in a modified Boyden assay. Pokeweed mitogen showed negligible activity in the locomotion assay as compared with other lectins. Tests were performed to determine if the observed neutrophil migration in response to lectins was directional, and it was found that concanavalin A, phytohemagglutinin P, and peanut agglutinin were both chemokinetic and chemotactic, whereas castor bean I was only chemokinetic. Wheat germ agglutinin could not be declared chemotactic or chemokinetic due to its tendency to agglutinate neutrophils. Studies with fluoresceinated lectins demonstrated that lectins which stimulate neutrophil migration also bind to neutrophil surfaces. Preincubation with specific monosaccharide ligands blocked both stimulated locomotion and fluorescence, suggesting that an available lectin-binding site was required both for lectin binding and the stimulation of migration. Additional experiments indicated that fluoresceinated concanavalin A, phytohemagglutinin P, castor bean I, wheat germ agglutinin, and peanut agglutinin all induce cap formation on the neutrophil.

Binding Sites↗
PubMed1987

The immunogenicity and antigenicity of lipid A are influenced by its physicochemical state and environment.

We investigated the immunogenicity and antigenicity of synthetic lipid A and partial structures thereof. Included in the study were compounds which varied in the position of phosphate (1-mono-, 4'-mono-, and 1,4'-bisphosphates) and in the acylation (type, number, and distribution of fatty acids) and, in the case of monosaccharide compounds, the nature of the backbone sugar (D-glucosamine, D-glucose, 3-amino-3-deoxy-D-glucose, and 2,3-diamino-2,3-dideoxy-D-glucose). With the aid of the passive-hemolysis and passive-hemolysis-inhibition assays and by absorption experiments, five distinct antibody specificities were detected in polyclonal rabbit antisera raised against sheep erythrocyte-coated lipid A and lipid A incorporated into the membrane of liposomes (liposome-incorporated immunogens). Three antibody specificities reacted with disaccharide antigens specific for a 1-mono-, 4'-mono-, and 1,4'-bisphosphorylated beta-1,6-linked D-glucosamine disaccharide. Two antibodies reacted with either 1- or 4-phosphates of acylated D-gluco-configured monosaccharides and exhibited no cross-reaction with each other. However, they cross-reacted with disaccharide antigens with phosphate groups in the appropriate positions. We found that the physicochemical state and the environment of lipid A modulated its immunoreactivity. The immunogenicity was best expressed by erythrocyte-coated and liposome-incorporated immunogens. The antigenicity of lipid A was also greatly influenced by its physical surroundings. The reaction pattern of the above antibodies was highly specific in the hemolysis assay and in absorption experiments (the antibody reacted with antigen embedded in a cell membrane), whereas some cross-reactivities were observed in inhibition studies (the antibody reacts with antigen in aqueous solution). By using liposome-incorporated antigens as inhibitors, nonspecific reactions were avoided and specific ones were enhanced. Thus the antibodies described above against lipid A recognize epitopes in the hydrophilic backbone, the exposure of which depends on the intrinsic physicochemical properties of lipid A on the one hand and the physical environment on the other.

Animals↗
PubMed1972

Composition of fatty acids and carbohydrates in Leptospira.

The fatty acid and monosaccharide composition of four pathogenic and two saprophytic strains of Leptospira was analyzed by gas chromatography (GC) and GC-mass spectrometry. Among the fatty acids, palmitic acid was most abundant and constituted 30 to 50% of the total fatty acids. Even-numbered unsaturated acids including octadecenoic, hexadecenoic, octadecadienoic, and tetradecadienoic acids comprised 40 to 60% of the total fatty acids. Tetradecanoic acid was about 5% in saprophytic strains, but 1% or less in pathogenic strains. The amount of chloroform-methanol extract of L. biflexa strain Ancona was 14 to 20% of the dry weight of the cell. Tetradecadienoic acid was found in the chloroform-methanol insoluble fraction, suggesting the presence of the acid in a bound form. GC analysis of monosaccharides revealed the existence of arabinose, xylose, rhamnose, mannose, galactose, glucose, glucosamine, and muramic acid in the cells. Among the neutral sugars, glucose was a minor component and was especially low in pathogenic strains. Total pentose content was about two to three times greater than total hexose.

Amino Sugars↗
PubMed1994

Biochemical and structural analyses of the extracellular matrix fibrils of Myxococcus xanthus.

It is characteristic of myxobacteria to produce large amounts of extracellular material. This report demonstrates that this material in Myxococcus xanthus is fibrillar and describes the structure and chemical composition of the fibrils. The extracellular matrix fibrils are the mediators of cell-cell cohesion in M. xanthus. As such, the fibrils play an important role in the cell-cell interactions that form the basis for the social and developmental lifestyle of this organism. The fibrils are composed of protein and carbohydrate in a 1.0:1.2 ratio. Combined, the two fractions accounted for greater than 85% of the mass of isolated fibrils, and the fibrils were found to compose up to 10% of the dry weight of cells grown at high density on a solid surface. The polysaccharide portion of the fibrils was shown to be composed of five different monosaccharides: galactose, glucosamine, glucose, rhamnose, and xylose. Glucosamine, one of the component monosaccharides of the fibrils and a known morphogen for M. xanthus, inhibited cohesion to a level near that of Congo red (the positive control for cohesion inhibition). Glucose and xylose also inhibited cohesion but less than did glucosamine. Analysis of the morphology of the fibrils, the periodicities within the distribution of fibril diameters observed by field emission scanning electron microscopy, and the observation of fibrils on hydrated cells strongly suggested that the extracellular matrix of M. xanthus was indeed arranged as fibrils. Furthermore, results suggested that the fibrils were constructed as carbohydrate structures with associated proteins.

Bacterial Adhesion↗
PubMed1985

Chemical and enzymatic variation in the cell walls of pathogenic Candida species.

Cell walls, isolated from seven pathogenic species of Candida, were lipid extracted and fractionated by treatment with ethylenediamine or enzymatically hydrolyzed using chitinase and laminarinase. Two different chitinase preparations were used, one from Streptomyces sp. which had some beta-1,3-glucanase activity, and another from Serratia marcescens which did not have glucanase activity. Laminarinase was a commercial preparation. The monosaccharide constituents of whole cell walls and the fractions derived from them were determined qualitatively and quantitatively by gas-liquid chromatography of the products of a mild acid hydrolysis and by the phenol - sulfuric acid assay of the products of a stronger acid hydrolysis. The monomeric constituents of the enzymatic hydrolyses were analyzed using gas-liquid chromatography. Approximately 50% of all walls was soluble in ethylenediamine. Glucose and mannose were the only monosaccharides found in all of the fractions derived from ethylenediamine extraction examined. Similarities among the strains, based upon relative amounts of glucose and mannose, were more apparent than differences, but statistical analyses of the data revealed a general trend of decreasing similarity in the following order, C. albicans and C. stellatoidea, C. tropicalis and C. parapsilosis, and C. pseudotropicalis, C. guilliermondii, and C. krusei. In the enzymatic assays, mannose and glucose were released by laminarinase, whereas glucose and N-acetyl-D-glucosamine or N-acetyl-D-glucosamine alone were released by the chitinases. These assays supported the trend in relationships cited above, with the data being somewhat more definitive.

Acetylglucosamine↗