Detection of carbohydrate terminals in the enteric parasite Enteromyxum scophthalmi (Myxozoa) and possible interactions with its fish host Psetta maxima

The existence and localisation of carbohydrate terminals in Enteromyxum scophthalmi stages was investigated at light (LM) and transmission electron microscopes (TEM) using lectin histochemistry techniques, with the aim of contributing to elucidate the participation of carbohydrate–lectin interactions in the parasite invasion and relationships with the fish host. The presence of abundant mannose and/or glucose residues was demonstrated by the intense staining by concanavalin A at both LM and TEM. The staining pattern obtained with soybean agglutinin and Bandeiraea simplicifolia I (BSI) indicated the abundance of N-acetyl-galactosamine and D-galactose at a lesser extent. The lectins wheat germ agglutinin, BSI and Ulex europaeus agglutinin produced weaker marks. Most lectins recognised structures present in both pre-sporogonic and sporogonic stages, though the glycosidic pattern and/or staining intensity varied between developmental stages. No staining was obtained with Sambucus nigra agglutinin. The TEM studies demonstrated glucose–mannose, N-acetyl-glucosamine, N-acetyl-galactosamine and α-D-galactose as dominant structures at the parasite membrane and host–parasite interface, suggesting a role in host–parasite interactions. All these terminals were also detected in the mitochondria of P cells and were scant in the S cells and nuclei. In turbot intestine, mannose–glucose terminals and N-acetyl-glucosamine were labelled on the epithelial brush border and in the mucous cells and rodlet cells. The relevance of these findings in relation to the host–parasite interaction is discussed.     

Structural analysis and mucosal immune regulation of exopolysaccharide fraction from Bifidobacterium animalis RH

An exopolysaccharide fraction (EPSa) produced by Bifidobacterium animalis RH was purified to dissect its chemical structure and to investigate its ability to modulate the mucosal immunity. Our results showed that EPSa had a backbone of (1→6)-linked glucose, (1→3)-linked galactose, (1→4, 6)-linked mannose, (1→3, 4)-linked mannose and (1→4)-linked glucose, branched with glucose and arabinose, and terminated with glucose residues. Scanning electron microscopy identified spherical particle to be the basic structural component of EPSa. The Congo red test indicated that EPSa was not in triple-helical conformation. Our in vitro results showed that EPSa was able to stimulate the proliferation of peripheral blood mononuclear cells and intestinal Peyer’s patch lymphocytes in the presence of concanavalin A. Our in vivo results demonstrated that EPSa can enhance the mucosal immunity by maintaining the Peyer’s patch number in small intestine and the IFN-γ level in serum, and balancing the Th1/Th2 responses.     

PstS‐1, the 38‐kDa Mycobacterium tuberculosis Glycoprotein,is an Adhesin,Which Binds the Macrophage Mannose Receptor and Promotes Phagocytosis

Mycobacterium tuberculosis, the primary causative agent of tuberculosis, infects macrophages and transforms the hostile intracellular environment into a permissive niche. M. tuberculosis infects macrophages using a variety of microbial ligand/cell receptor systems. In this study, binding assays with biotin‐labelled mycobacterial cell wall proteins revealed five Concanavalin A‐reactive proteins that bind macrophages. Among these proteins, we identified PstS‐1, a 38‐kDa M. tuberculosis mannosylated glycolipoprotein, and characterized it as an adhesin. Inhibition assays with mannan and immunoprecipitation demonstrated that PstS‐1 binds the mannose receptor. We purified PstS‐1 to 95.9% purity using ion exchange chromatography. The presence of mannose in purified PstS‐1 was demonstrated by Concanavalin A interaction, which was abolished in the presence of sodium m‐periodate and α‐D‐mannosidase. Gas chromatography revealed that purified PstS‐1 contained 1% of carbohydrates by weight, which was mainly mannose. Finally, we used fluorescent microbeads coated with purified PstS‐1 in phagocytosis assays and discovered that microbead uptake was inhibited by the pre‐incubation of cells with GlcNAc, mannan and α‐methyl mannoside. The interaction of PstS‐1 coated beads with the mannose receptor was confirmed by confocal colocalization studies that showed high Pearson and Manders's colocalization coefficients. Our findings contribute to a better understanding of the strategies M. tuberculosis uses to infect host cells, the critical first step in the pathogenesis of tuberculosis.     

Mannose prevents lipopolysaccharide-induced acute lung injury in rats

Objective: To investigate the effect of mannose on lipopolysaccharide (LPS) induced acute lung injury (ALI) in rats. Methods: Ten groups of Sprague–Dawley rats were used: 1) the control group received an intratracheal instillation of saline, 2) the LPS group received an intratracheal instillation of LPS (3 mg/kg), 3–6) the mannose groups were injected i.v. with 15, 45, 135, and 405 mg/kg mannose, 7–9) the glucose, galactose, and fructose groups were injected with different hexoses (135 mg/kg), and 10) the dexamethasone (DXM) group was injected with DXM (2 mg/kg). In groups 2–8, LPS was administered after injection of drugs. Lung wet/dry weight ratio, permeability index (PPI), total leukocytes and polymorphonuclear neutrophils (PMNs) counts in bronchoalveolar lavage fluid (BALF), myeloperoxidase (MPO) and superoxide dismutase (SOD) activity, tumor necrosis factor (TNF)-α and interleukin (IL)-10 in lung and BALF were determined. Results: Pre-treatment with mannose attenuated pulmonary edema and protein exudation in a dose-dependent manner, the maximal effect was similar to or greater than that of DXM. Mannose also prevented the inflammatory cell accumulation, although the maximal effect was weaker than that of DXM. Mannose was more effective than DXM in inhibiting MPO activity and restoring SOD activity. Moreover, it inhibited production of TNF-α and IL-10. Histological changes of the lungs were also ameliorated by mannose. There were no significant improvements observed in rats pre-treated with glucose, galactose or fructose. Conclusions: Mannose is effective in reducing LPS-induced ALI. Project supported by the National Natural Science Foundation of China, No. 30670930 and the Science and Technology Department of Zhejiang Province (No.2004C23011) Received 4 March 2007; returned for revision 13 May 2007; returned for final revision 17 July 2007; accepted by M. Katori 12 September 2007     

Localization of glycoconjugates at the tegument of the tapeworms Hymenolepis nana and H. microstoma with gold labelled lectins

Gold labelled lectins were used for electron microscopic localization of carbohydrate components of the tegument surface of two tapeworm species, Hymenolepis nana and H. microstoma. WGA, succinylated WGA, SBA, APA, PNA and, to a lesser extent, Con A were preferentially bound to the spines of the microtrichs. UEA-I and DBA were not adsorbed. The results indicate that the surface coat of both species has exposed N-acetylglucosamine, galactose and perhaps glucose and/or mannose residues.The location of lectin-binding glycoconjugates within the tegument and parenchyma was found using the light microscope on sections of material embedded in Lowikryl K4M after lectin-gold labelling and silver enhancement of the gold grains. The tegument selectively adsorbs WGA and SBA and strongly; adsorbtion of PNA and Con A is less intense. Strong adsorbtion of DBA and PNA was confined to the basal lamina. The parenchyma adsorbed Con A, PNA and DBA, but little WGA and SBA.The results indicate that many glycoconjugates are present in the tegument. They have similar terminal sugar residues to those of the surface coat. The significance of these carbohydrates for hostparasite interactions is discussed.     

Biochemical studies on the juvenile form of ceroid-lipofuscinosis

Accumulation of oligosaccharyl diphosphodolichols (oligo-PP-Dol) in brains of patients with various forms of ceroid-lipofuscinoses (CL) is one of the most reproducible biochemical changes known so far. The objective of this study is to understand the biochemical basis of this observation. The biosynthesis of oligo-PP-Dol was studied by the incorporation of labelled glucose from UDP [¹⁴C] glucose into oligo-PP-Dol in cultured skin fibroblasts, and showed no changes in the level of synthesis. The level of labelled glucose incorporated into glycoproteins was also unchanged, suggesting that there is no decrease in the oligosaccharide transfer to proteins in this disorder. Since the biosynthesis and utilization of oligo-PP-dol are unaffected, a defect in the catabolism may be the only possibility for the storage of this compound in CL. Since terminal mannose residues are present in the accumulating oligo-PP-Dol, mannosidase activities at pH 4.4 and 6.0 were determined in cultured skin fibroblasts. Both mannosidase activities were unchanged in skin fibroblasts of juvenile CL. Endo-β-N-acetylglucosaminidase-1 activities were determined in cultured skin fibroblasts using dansylated Man₆GlcNAcGlcNAc-Asn as substrate. In three patients, a drastic reduction in the level of the pH 4.5 enzyme was shown, while the neutral pH enzyme activity was unaffected. A deficiency of the endo-β-N-acetylglucosaminidase-1 will not only explain the accumulation of oligo-PP-Dol but also the known storage of high-mannose glycoproteins.     

Complementing mutant alleles define three loci involved in mannosylation of Man5-GlcNAc2-P-P-dolichol in Chinese hamster ovary cells

Dolichol-linked oligosaccharides consisting of twoN-acetylglucosamine, nine mannose, and three glucose residues (Glc₃Man₉GlcNAc₂) are transferred to proteins that contain the consensus sequence Asn-X-Ser/Thr. This transfer occurs upon protein import into the lumen of the endoplasmic reticulum. An intermediate in the biosynthesis of the Glc₃Man₉GlcNAc₂ lipid-linked oligosaccharide contains two GlcNAc and five mannose residues. This intermediate serves as a substrate for further mannosylation and glucosylation before transfer to protein. The addition of the sixth mannose residue to this intermediate requires the enzyme mannosyltransferase VI and the mannose donor, mannose-P-dolichol. Several different CHO cell line mutants that fail to efficiently catalyze this transfer have been described. In this report, we examine seven independent mutant cell lines with various biochemical phenotypes and demonstrate that all can be assigned to one of three genetic complementation groups. One mutation affects mannose-P-dolichol biosynthesis (Lec15), three affect dolichol phosphate biosynthesis (Lec9), and three appear to affect the functional orientation of enzyme substrates (PIR).     

Oxidation of exogenous carbohydrate during prolonged exercise: the effects of the carbohydrate type and its concentration

Summary We studied rates of exogenous carbohydrate (CHO) oxidation during 90 min of cycling exercise in trained cyclists exercising at 70% of maximal oxygen consumption (VO_2max) when they ingested glucose, sucrose, or glucose polymer solutions at concentrations of 7.5%, 10% or 15%. Drinks were labelled with [U-¹⁴C]glucose or sucrose and were ingested at a rate of 100 ml · 10 min^–1. Rates of oxidation of the ingested CHO were calculated from the specific radio-activity of the labelled CHO, expired¹⁴CO₂ and carbon dioxide output (VCO₂). Total CHO oxidation, determined from oxygen consumption andVCO₂ was not influenced by CHO type or concentration. Gastric emptying (P=0.01) and the rate of exogenous CHO oxidation (P=0.028) was greatest for the glucose polymer solutions, and least for glucose. Although gastric emptying (P=0.006) decreased with increasing CHO concentration, CHO delivery to the intestine and exogenous CHO oxidation increased linearly with increasing CHO concentration. The percentage of the CHO delivered to the intestine that was oxidized ranged from 30.0% for 7.5% CHO to 38.1% for 15% CHO. Our results indicated that the rate of gastric emptying for CHO was not controlled to provide a constant rate of energy delivery as is commonly believed and that factors subsequent to gastric emptying limit the rate of exogenous CHO oxidation from the ingested solution.     

Radiorespirometric analysis of glucose dissimilation in Rhodococcus sp. An 117 and An 213

Radiorespirometric experiments were carried out by incubating various types of resting cells of either Rhodococcus sp. An 117 or Rhodococcus sp. An 213 under aerobic conditions with differentially labelled glucose. The results obtained indicate the constitutive nature of glucose catabolism in both these strains, i.e. there was no principle shift in glucose dissimilation during cell growth with different carbon substrates. On the basis of the observed ratios of percentage (cumulative) ¹⁴CO₂-formation from (6-¹⁴C)glucose and (1-¹⁴)glucose, the glycolytic EMP-pathway was calculated to be the predominant catabolic route being operative to an extent of about 95% (strain An 117) and 78% (strain An 213).     

Saccharomyces cerevisiae strains sensitive to inorganic mercury

Summary Saccharomyces cerevisiae strains sensitive to inorganic mercury (Ono and Sakamoto 1985) did not grow well on the medium rich in glucose and poor in peptone. This growth inhibition, like growth inhibition caused by inorganic mercury, was relieved by exogenous tyrosine. Sugars such as fructose and mannose were as inhibitory as glucose, but glycerol was not at all. Galactose was inhibitory but not so much as glucose. Agal2l mutation (defective in galactose uptake) partly relieved growth inhibition caused by excess galactose. Moreover, it was found that some of revertants which gained ability to grow well in the presence of excess glucose were defective in the glucose uptake. From these observations, we conclude that growth inhibition of the inorganic mercury sensitive strains by excess sugar is a consequence of the catabolite regulation. In other words, the inorganic mercury sensitive strains are hyper-sensitive to the catabolite regulation due to the presence of theHGS2-1 allele.     

Characterization of Aspergillus umbrosus carbohydrate antigens by biotinylated lectins and IgG response to mannan/mannoprotein antigens in patients with farmer's lung

Twenly-one different biotinylated lectins were used to recognize the carbohydrates of Aspergillus umbrosus, one of the most common microbes that patients with farmer's lung in Finland are exposed to. The glycoprotein fraction of A. umbrosus was bound especially well by Concanavalin A and consisted mainly of carbohydrates mannose and glucose. The carbohydrate fraction of A. umbrous antigens were isolated from the crude extract of A. umbrosus with Con A-Sepharose affinity chromatography. Serum IgG antibodies to A. umhrosus mannoprotein fraction were determined in 57 patients with farmer's lung, 10 asymptomatic exposed farmers and 10 healthy controls by enzyme linked immunosorbent assay. Candida albicans and Saccharomyces cerevisiae mannan were used as controls. Patients had high levels, whereas asymptomatic exposed farmers showed moderate, and healthy controls low levels, of IgG antibodies to the A. umbrosus mannan/mannoprotein fraction. There were no significant differences in the mean antibody levels between the patients and controls against C. albicans or S. cerevisiae mannose fraction although in all groups more antibodies were detected against C. albicans than S. cerevisiae.     

A comparison of the toxic effects of 2-deoxy-D-glucose and 2-deoxy-2-fluoro-D-hexoses on Saccharomyces cerevisiae cells and protoplasts

The toxicity to the cells and protoplasts of Saccharomyces cerevisiae of the sugar analogues modified at carbon 2 increases in the order 2-deoxy-D-glucose (DG), 2-deoxy-2-fluoro-D-glucose (FG) and 2-deoxy-2-fluoro-D-mannose (FM). The fluorohexoses, similarly as DG, behave generally as analogues of both glucose and mannose, depending on the hexose used as a carbon source in the medium. Relative inhibitions of glucan and mannan synthesis in protoplasts were found to be dependent more on glucose and mannose used as the growth support than on the type of the sugar analogue. Certain degree of structural relationship of fluorohexoses to the corresponding natural hexoses was reflected in their effects on growth of intact cells. Growth on glucose was inhibited most effectively by FM, growth on mannose by FG. The data obtained support the view that the sugar analogues interfere mainly with the glucose-mannose interconversion catalyzed by hexosephosphateisomerases. A comparison of the effects of fluorohexoses and DG on the synthesis of extracellular invertase and intracellular α-glucosidase and alkaline phosphatase in protoplasts pointed to the fact that all three sugar analogues tested also participate in metabolic control of enzyme synthesis.     

Tunicamycin does not inhibit transport of phosphatidylinositol toXenopus rod outer segments

Summary Tunicamycin inhibits the dolichol pathway forN-linked glycosylation of proteins, including photoreceptor opsin, and causes a buildup of tubulo-vesicular profiles in the intersegmental space between photoreceptor rod inner and outer segments associated with disruption of new disc assembly. We tested the hypothesis that a tunicamycin lesion in photoreceptors would block lipid transport into the outer segment. AdultXenopus retinas were preincubated in dim red light with 20 g ml^–1 of tunicamycin for one hour followed by incubation in the light for 2–6 h with tunicamycin plus either [³H]mannose, [³H]leucine, [2-³H]glycerol or [³H]myo-inositol. Tunicamycin caused accumulation of tubulo-vesicular membranes in the intersegmental space and significantly reduced both [³H]leucine and [³H]mannose incorporation into the basal region of rod outer segments. However, tunicamycin had no effect on [³H]glycerol incorporation into the rod outer segment phospholipids. After 5 h incubation with [³H]glycerol, radiolabel in outer segment fractions was associated primarily with phosphatidylinositol in both control and tunicamycin treated retinas. Quantitative light microscope autoradiography of both [³H]glycerol and [³H]inositol labelled retinas showed diffuse labelling over the entire rod outer segment in both control and tunicamycin treated retinas with no accumulation of radioactivity in the basal discs of control retinas or in the tubulo-vesicular structures in the intersegmental space of tunicamycin treated retinas. Our results indicate that despite the morphological disruption and inhibition of glycoprotein transport to outer segments after tunicamycin treatment, transport of labelled phosphatidylinositol occurs normally. These data add to a growing body of evidence separating the lipid and protein transport pathways to the outer segment.     

Effects of glucose ingestion or glucose infusion on fuel substrate kinetics during prolonged exercise

To determine if bypassing both intestinal absorption and hepatic glucose uptake by intravenous glucose infusion might increase the rate of muscle glucose oxidation above 1 g · min^–1, ten endurance-trained subjects were studied during 125 min of cycling at 70% of peak oxygen uptake (VO_{2 peak}). During exercise the subjects ingested either a 15 g · 100 ml^–1 U-¹⁴C labelled glucose solution or H₂O labelled with a U-¹⁴C glucose tracer for the determination of the rates of plasma glucose oxidation (Rox) and exogenous carbohydrate (CHO) oxidation from plasma¹⁴C glucose and¹⁴CO₂ specific activities, and respiratory gas exchange. Simultaneously, 2-³H glucose was infused at a constant rate to measure glucose turnover, while unlabelled glucose (25% dextrose) was infused into those subjects not ingesting glucose to maintain plasma glucose concentration at 5 mmol · l^–1. Despite similar plasma glucose concentrations [ingestion 5.3 (SEM 0.13) mmol · l^–1; infusion 5.0 (0.09) mmol · l^–1], compared to glucose infusion, CHO ingestion significantly increased plasma insulin concentrations [12.9 (1.0) vs 4.8 (0.5) mU · l^–1;P<0.05], raised total Rox values [9.5 (1.2) vs 6.2 (0.7) mmol · 125 min^–1 kg fat free mass^–1 (FFM);P<0.05] and rates of CHO oxidation [37.2 (2.8)vs 24.1 (3.9) mmol · 125 min^–1 kg FFM^–1;P<0.05]. An increased reliance on CHO metabolism with CHO ingestion was associated with a decrease in fat oxidation. Whereas the contribution from fat oxidation to energy production increased to 51 (10)% with glucose infusion, it only reached 18 (4)% with glucose ingestion (P<0.05). Despite these differences in plasma insulin concentration and rates of fat oxidation, the rates of glucose oxidation by muscle were similar after 125 min of exercise for both trials [ingestion 93 (8); infusion 85 (5) mol · min^–1 kg FFM^–1], suggesting that peak rates of muscle glucose oxidation were primarily dependent on blood glucose concentration which, in turn, regulated the hepatic appearance of ingested CHO.     

Analysis of the relationship between bacterial adherence and extracellular production of mannose,galactose, glucose and ribose inStaphylococcus epidermidis andStaphylococcus hominis

Gas-liquid chromatography-mass spectrometry was used to analyze the extracellular extracts of 108 coagulase-negative staphylococcal strains for the presence of mannose, galactose, glucose and ribose, in order to determine whether production of these four monosaccharides, regarded as potential staphylococcal slime components, was associated with the adherence capacity of the individual strains. A total of 90Staphylococcus epidermidis and 18Staphylococcus hominis strains were studied. Using the quantitative spectrophotometric assay, 21Staphylococcus epidermidis strains were classified as strongly adherent, 12 as moderately adherent, 11 as weakly adherent, and 46 as nonadherent. All 18Staphylococcus hominis strains were nonadherent. Mannose, galactose, glucose and ribose were detected as the main monosaccharide components in the extracellular extracts of all strains examined. Moreover, the mean relative concentrations of these monosaccharides were essentially the same for the different adherence phenotypes within the speciesStaphylococcus epidermidis. These results showed that there was no causal connection between the adherence of coagulase-negative staphylococci and the extracellular production of any of the four monosaccharides analyzed.     

Incorporation of [14C] hexoses into the carbohydrate of gamma2-globulin in vitro

A method for the labelling and isolation of specific γ₂-globulin antibody formed in vitro by intact rabbit lymphoid cells has been described. This method has been applied to the study of the synthesis of the carbohydrate portion of γ₂-globulin using [¹⁴C] mannose, [¹⁴C] galactose as labelled precursors. Metabolic alterations of all three sugars led to redistribution of much of the ¹⁴C-label into the amino acids of the protein portion, but a 6- to 18-fold relative enrichment, on a weight basis, was observed in the carbohydrate fraction. Labelling was greatest with mannose and least with galactose under the conditions used in this study.     

Exopolysaccharide production by nitrogen-fixing bacteria within nodules of Medicago plants exposed to chronic radiation in the Chernobyl exclusion zone

Nitrogen-fixing bacteria isolated from root nodules of Medicago plants growing in the 10 km zone around the Chernobyl nuclear power plant were screened for the production of new water-soluble acidic exopolysaccharides (EPSs). The different strains belonged to the Enteriobacteriaceae family (Enterobacter ludwigii, Raoultella terrigena, Klebsiella oxytoca), except for one which belonged to the Rhizobiaceae family (Sinorhizobium meliloti). All of the bacteria produced highly viscous EPS with an average molecular weight comprised between 1 × 10⁶ and 3 × 10⁶ Da. Five different compositions of EPS were characterized by physico-chemical analyses and ¹H NMR spectroscopy: galactose/mannose (2/1), galactose/glucose (1/1), galactose/glucose/mannose (1/2/1), fucose/galactose/glucose (2/1/1) and fucose/galactose/glucose/mannose (2/2/1/1 or 1/1/2/4). Glucuronic acid, a charged monosaccharide, was also recovered in most of the different EPSs.     

Evidence for the involvement of carbohydrate moieties in the adhesion of U937 cells to tumor necrosis factor-α (TNFα)-stimulated vascular endotheliumin vitro

Treatment of U937 cells with fructose 1-phosphate (P) and fucoidan dose-dependently inhibited the adhesion of these monocytic cells to TNF-stimulated human umbilical vein eindothelial cells (HUVEC) (IC₅₀=1 mM and 10 g/ml respectively). These carbohydrates (CHO) failed to inhibit U937 adhesion to unstimulated (basal) HUVEC or phorbol 12, 13 dibutyrate (PdBu)-stimulated HUVEC. At 10 mM concentration, both fucose 1-P and lactose 1-P inhibited TNF-stimulated adhesion while the latter also inhibited basal adhesion. Fructose 6-P, fucose, galactose 1-P, glucose 1-P, glucose 6-P, glucuronic acid,-glycerol 1-P, mannose 1-P, mannose 6-P, ribose 1-P and ribose 5-P tested at 10 mM did not inhibit U937 cells adhesion to basal or TNF-stimulated HUVEC. These data suggest that CHO may play an important role in modulating monocytes adhesion to cytokine-induced adhesion molecule(s) on the surface of HUVEC.     

Physicochemical Properties of Neisseria meningitidis Group C and Y Polysaccharide Antigens

Chemical and physical studies of the Neisseria meningitidis group C and Y antigens indicated that these preparations are acidic polysaccharides. The group C antigen contains predominately n-acetyl neuraminic acid, with small amounts of glucose, glucosamine, and mannose, whereas the group Y antigen contains glucose, glucosamine, and mannose. The molecular weights of the group C and Y antigens were found to be 88,000 and 197,000, respectively. The group C antigen was susceptible to neuraminidase digestion. Finally, the immunological reactivity of neither antigen was inhibited in hemagglutination or immunodiffusion systems by neutral or substituted sugars.