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PubMed1996

Expression of the human cell surface glycoconjugates Lewis x and Lewis y by Helicobacter pylori isolates is related to cagA status.

Monoclonal antibodies were used in an enzyme-linked immunosorbent assay for the detection of human Lewis immunodeterminants in the lipopolysaccharide of Helicobacter pylori. In 94 H. pylori isolates, expression of Lewis(x) (Le(x)) and Le(y) was a stable phenotypic marker independent of the growth medium and cell age; 46 (49%) of the isolates expressed both and 34 (36%) of the isolates expressed either Le(x) or Le(y); 14 (15%) were negative for both determinants. Twelve (13%) isolates expressed Le(b), 3 (3%) expressed Le(a), and 2 (2%) expressed sialyl-Le(x). H. pylori isolates positive for both Le(x) and Le(y) were predominantly cagA+ (P < 0.001) and possessed the s1 signal sequence (P < 0.05) and the m1 midregion type (P = 0.033) of vacA. Isogenic mutants of H. pylori CPY3401 were created by interruption of the cagA, picB, or ureA gene. The cagA-ablated strain (but not the picB- and ureA-ablated mutant strains) had significantly (P < 0.01) diminished expression of Le(y) compared with that of the wild-type strain; for all four strains, expression of Le(x) was similar. In conclusion, 89% of H. pylori isolates express Le determinants in their lipopolysaccharide, mimicking human cell surface glycoconjugates. Strong expression of Le(x) and Le(y) by cagA+ isolates could counterbalance their enhanced proinflammatory activities and thereby facilitate persistence.

Antibodies, Monoclonal↗

PubMed1984

Specific identification of Salmonella serogroup E antigen O3 by immunofluorescence and coagglutination with antiserum elicited by a synthetic trisaccharide-bovine serum albumin glycoconjugate.

Antiserum specific for Salmonella O3 antigen was raised by immunization of rabbits with an artificial glycoconjugate consisting of the synthetic trisaccharide beta-D- Manp (1----4)-alpha-L- Rhap (1----3)-alpha-D-Galp covalently linked to bovine serum albumin (beta- MRG -BSA). Enzyme immunoassays showed that only lipopolysaccharides extracted from Salmonella serogroup E (O3 antigen-containing) bacteria bound the antiserum. The usefulness of the beta- MRG -BSA antiserum for rapid and accurate identification of Salmonella isolates of serogroup E was shown by indirect immunofluorescence tests in which 50 Salmonella strains of serogroup E 1-4 were correctly identified from among 651 intestinal strains investigated. The finding that one strain of Citrobacter freundii was positive in immunofluorescence tests with this antiserum is readily explained by the known cross-reactivity between some C. freundii strains and Salmonella spp. strains of serogroup E. As expected, the specificity of the antiserum for the O3 antigen could further be demonstrated in coagglutination tests with staphylococci sensitized with beta- MRG -BSA antiserum.

Agglutination Tests↗

PubMed1994

Heterologous expression of delta F508 CFTR results in decreased sialylation of membrane glycoconjugates.

The cystic fibrosis transmembrane conductance regulator (CFTR) is commonly mutated in cystic fibrosis to the delta F508 CFTR. CFTR has been shown to function as a adenosine 3',5'-cyclic monophosphate-dependent Cl- channel at the cell surface, and there is evidence to suggest that CFTR may also have a role in transmembrane Cl- conductance in intracellular membrane compartments. Studies using cells from cystic fibrosis patients or heterologous expression systems have demonstrated that defective Cl- conductance at the cell surface and defective acidification of the Golgi compartment are associated with the presence of mutant forms of CFTR. It is possible that mutation of CFTR could also result in altered Golgi function, consistent with reports of changes in the glycosylation of cell surface and secreted glycoproteins in cystic fibrosis. Glycosylation of cell surface glycoproteins, particularly levels of sialylation, may also be related to the increased binding of Pseudomonas to cystic fibrosis cells. The current study was undertaken to compare the sialylation of cell surface glycoconjugates in heterologous cells overexpressing normal CFTR or delta F508 CFTR. The presence of sialylated residues on cells was assessed by the surface binding of the specific lectins, wheat germ agglutinin and elderberry bark lectin. A fluorescent cholera toxin B subunit probe was used to measure surface binding to sialylated gangliosides in transfected cells. Our studies show that cells lacking CFTR and cells expressing normal CFTR have unaltered levels of sialylation. In contrast, cells expressing the delta F508 CFTR have significantly decreased amounts of sialylated glycoproteins and gangliosides on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Localization of glycoconjugates recognized by the HNK-1 antibody in mouse and chick embryos during early neural development.

The monoclonal antibody HNK-1 recognizes a carbohydrate epitope present on a host of glycoconjugates which include the glycoproteins neural cell adhesion molecules (N-CAM) myelin-associated glycoprotein and ependymins, and two glycolipids. Other antibodies, including NC-1, L2 and IgM paraproteins from neuropathy patients share similar binding specificity to the same or related sulfated glucuronyl-containing antigen (SGA). To further investigate its possible significance in early development, the distribution of SGA was studied in the head region of early developing chick (S13-S18) and mouse (E8.5-E11.5) embryos by immunocytochemistry. A striking species difference was found in the apparent distribution of immunodetectable SGA. In chick, migrating neural crest cells and their related cell types were heavily stained by HNK-1; whereas no stain was detectable on mouse neural crest cells at comparable stages, except perhaps in a restricted area adjacent to the otic placode and immediately adjacent to the neural tube. Within the developing CNS, the distribution of SGA was similar in both species. It was first expressed on neuroepithelial cells prior to axonal outgrowth, and was distributed in a continuous zone along the entire lateral walls of the early neural tube. Little or no SGA was detectable along most of the floor and roof plates. SGA appeared during this same period in the lateral basal lamina and within the adjacent mesenchyme and nearby cells. SGA was particularly evident on neuroepithelial endfeet at this stage. Early developing longitudinal axons were subsequently found to grow in association with the endfeet of SGA-positive neuroepithelial cells. These findings, in conjunction with previous studies, suggest that SGA is associated as a marker, and perhaps functionally, with the organization of early neuronal settling and axonal growth patterns within the developing vertebrate CNS.

Animals↗

PubMed1983

Deposition of alpha 1-antitrypsin and loss of glycoconjugate carrying Ulex europaeus agglutinin-I binding sites in the glomerular sclerotic process. Phenomena common to chronic pyelonephritis and chronic diffuse proliferative glomerulonephritis.

Sclerotic processes of glomeruli in chronic pyelonephritis (CPN) and chronic diffuse proliferative glomerulonephritis (CDPGN) were investigated in a lectin binding study in connection with an immunofluorescent examination of protease inhibitor deposition. Ulex europaeus agglutinin-I (UEA-I), which is specific to a certain terminal alpha-L-fucosyl residue of glycoconjugates, specifically labelled intact endothelia of glomerular capillaries, peritubular capillaries and blood vessels in human kidneys. Segmental or global loss of the UEA-I binding with glomerular capillaries was observed in the sclerotic areas where alpha 1-antitrypsin (alpha 1AT) deposits were always detected in the glomeruli with segmental or global sclerosis of CPN. This high correlation between loss of UEA-I binding and alpha 1AT deposition was also observed in the affected glomeruli of CDPGN. In considering glomerular sclerosis, it is significant that loss of UEA-I binding and alpha 1AT deposition are common to both CPN and CDPGN, although their original etiologies are quite different.

Animals↗

PubMed1986

Ultrastructural localization of glycoconjugates in the fungus Ascocalyx abietina, the Scleroderris canker agent of conifers, using lectin-gold complexes.

Different glycoconjugates were revealed in the fungus Ascocalyx abietina (Lagerberg.) Schlaepfer-Bernhard, by using various lectin-gold complexes. N-acetylglucosamine, N-acetylgalactosamine, and D-mannose were specifically localized in cell walls of fungal cells. N-acetylneuraminic acid (sialic acid) and L-fucose were detected in structures corresponding to lipid bodies, whereas they were totally absent in the cell wall. This is the first report on the occurrence of sialic acid in fungi and of fucose in Ascomycetes. The great advantage of using lectin-gold complexes for ultrastructural localization of sugars in phytopathogenic fungi, as well as in studies concerning host-pathogen interactions, is discussed.

Acetylgalactosamine↗

PubMed1986

An improved periodic acid-thiosemicarbazide-osmium technique to reveal glycoconjugates at the molecular level in situ.

The periodic acid-thiocarbohydrazide or thiosemicarbazide-OsO4 method (Seligman AM, Hanker JS, Wasserkrug H, Katzoff L: J Histochem Cytochem 13:629, 1965) has been modified in order to obtain a periodic acid-Schiff (PAS)-like reaction for electron microscopy capable of visualizing structures at the molecular level in situ. Thiocarbohydrazide (TCH) and thiosemicarbazide (TSC) have been used dissolved in distilled water and bubbled with SO2. Treatment of previously oxidized thin sections with TCH (SO2) or TSC (SO2), followed by osmification, resulted in selective and very good staining of all the PAS-positive structures examined: glycogen, intestinal mucopolysaccharides, plasma membrane glycoproteins, basement membranes, Golgi apparatus, and collagen. The staining reaction was highly specific when TSC was used on thin sections from paraformaldehyde-fixed samples. The non-particulate end-reaction product made possible visualization of a periodic distribution of sugar residues in the 64-nm unit of collagen and the structural organization of the PAS-positive glycoconjugate components in the glomerular basement membrane.

Animals↗

PubMed1986

Cell surface glycoconjugates and carbohydrate-binding proteins: possible recognition signals in sensory neurone development.

Dorsal root ganglion (DRG) neurones transmit cutaneous sensory information from the periphery to the dorsal horn of the spinal cord. Subpopulations of DRG neurones that subserve distinct sensory modalities project to discrete regions in the dorsal horn. The formation of specific sensory connections during development may involve cell-surface interactions with spinal cord cells. Molecules that are expressed on the surface of functional subpopulations of DRG and dorsal horn neurones have therefore been identified. Distinct subsets of DRG neurones express globo- or lactoseries carbohydrate differentiation antigens. The expression of defined carbohydrate structures correlates with the embryonic lineage, peptide phenotype and the central termination site of DRG neurones. Similar or identical glycoconjugates have been implicated in cellular interactions that contribute to preimplantation embryonic development. Small-diameter DRG neurones that project to the superficial dorsal horn express N-acetyllactosamine backbone structures that are potential ligands for beta-galactoside-specific binding proteins (lectins). Two lectins have been identified that are expressed early in development in the superficial dorsal horn. These complementary molecules may contribute to the development of sensory afferent projections in the spinal cord.

Aging↗

PubMed1994

Synthesis of artificial glycoconjugates of arginine-vasopressin and their antidiuretic activities.

Arginine-vasopressin (AVP) derivatives modified at the glutamine side chain amide with carbohydrate via an alkylene spacer (1a--d) were synthesized from new glycosylated glutamine derivatives (3a--d) by solid-phase synthesis. Glycoconjugates of AVP modified at the C-terminal amide (2a--d) were also synthesized from vasopressionic acid. All of them exhibited antidiuretic activity.

Amino Acid Sequence↗

PubMed2000

A new method for radioiodination of polysaccharides and its use in biodistribution studies of an immunomodulating glycoconjugate (Immunoferon).

A new method for the labeling of polysaccharides by using tyrosine derivatives after CNBr activation is presented, along with its use for pharmacokinetic studies of an orally active immunomodulating noncovalent glycoconjugate, which was administered labeled either in the polysaccharide or the protein moiety. The relationship between the obtained results and the mechanism of action are also discussed.

Adjuvants, Immunologic↗

PubMed2003

Glycoconjugate vaccines to prevent group B streptococcal infections.

Group B Streptococcus (GBS) is an opportunistic pathogen of humans. At-risk populations include neonates born to colonised mothers, peripartum women, diabetics, and the elderly with underlying illnesses. Vaccines to prevent GBS disease have been developed by coupling purified capsular polysaccharide (CPS) antigen of GBS with an immunogenic protein carrier. Glycoconjugate vaccines against all nine currently identified GBS serotypes have been synthesised and shown to be immunogenic in mice, rabbits and baboons in preclinical trials. Healthy adults have safely received conjugate vaccines prepared with GBS types Ia, Ib, II, III, and V CPSs in Phase I and II clinical trials. These vaccines elicited CPS-specific antibody that opsonised GBS for in vitro killing by human peripheral blood leukocytes in the presence of complement. Results from these preclinical and clinical studies strongly suggest that GBS conjugate vaccines will be effective in preventing diseases caused by GBS.

Animals↗

PubMed2003

Glycosylation of the Arg-gingipains of Porphyromonas gingivalis and comparison with glycoconjugate structure and synthesis in other bacteria.

Post-translational modification of proteins by covalent attachment of sugars to the protein backbone (protein glycosylation) is the most common post-translational modification in the eucaryotic cell. However, the addition of carbohydrates to proteins of Eubacteria and Archaea has been demonstrated and accepted only recently. There is now a rapidly expanding list of bacterial glycoproteins that have been characterised from a variety of different organisms including many important pathogens. The Arg-gingipains of Porphyromonas gingivalis are recent additions to this list. In this review we present a summary of our investigations on the structure of the glycan additions to these proteolytic enzymes, the genetics of the glycosylation process and some of the effects on enzyme function and recognition. These findings are placed in the context of the current status of understanding of glycoconjugate structure and synthesis in other bacteria. Given the importance of glycosylation of eucaryotic proteins to their stability, structure, resistance to proteolysis and recognition, the modifications to the proteases described in the present report are likely to have a functional role in the properties of these enzymes in periodontal disease.

Adhesins, Bacterial↗

PubMed1998

Relationships between the structure and the roles of lipoarabinomannans and related glycoconjugates in tuberculosis pathogenesis.

The mechanisms and the molecular basis of the mycobacteria virulence remain obscure. However, recent findings provide evidences that, among the glycoconjugates which compose the mycobacterial envelope, lipoglycans including lipoarabinomannan, lipomannan and phosphatidyl-myo-inositol mannosides, are involved in the major steps of the tuberculosis immunopathogenesis. These steps are the mycobacterial phagocytosis process and the macrophage activation via the regulation of the production and secretion of cytokines. In this article, we examine recent observations about comparative structural models of the lipoglycans from the pathogenic Mycobacterium tuberculosis strain, and the vaccinal M. bovis BCG strain, and finally avirulent mycobacteria strains. We also consider the role of the lipoglycans in the mycobacterial phagocytosis process and in the regulation of the macrophage microbicidal activity.

Antigens, Bacterial↗

PubMed1999

Use of animal testing for evaluating glycoconjugate vaccine immunogenicity.

Most animal species respond with high antibody levels to polysaccharide antigens after they have been covalently linked to a protein carrier, converting a T-cell independent to a T-cell dependent antigen. This chemical modification has enabled the development of glycoconjugate vaccines for Haemophilus influenzae type b, Neisseria meningitidis, and multivalent Streptococcus pneumoniae. This new generation of vaccines can be well characterized physically and chemically to ensure consistent vaccine manufacture. Such analytical tests provide an alternative to animal models for Quality Control Laboratories; biological models can be difficult and costly to develop and use on a routine basis. If animal tests are used, they need to be refined, defined, and validated for their intended purpose.

Animal Testing Alternatives↗

PubMed2000

Oral immunization of mice with a glycoconjugate vaccine containing the O157 antigen of Escherichia coli O157:H7 admixed with cholera toxin fails to elicit protection against subsequent colonization by the pathogen.

It has been postulated that a humoral immune response directed against the O157 antigen of Escherichia coli O157:H7, and expressed in the intestine, might afford protection from colonization and consequent infection by this enteric pathogen. The present study was conducted to determine whether such an immune response can be experimentally generated in mice. To this end, mice were orally immunized with a glycoconjugate vaccine consisting of horse serum albumin and the O157 polysaccharide admixed with the mucosal adjuvant, cholera toxin. Mice consistently developed robust local and systemic immune responses to the cholera toxin adjuvant, but were far from uniformly reactive to the test vaccine. Moreover, vaccinated mice were as susceptible to transient intestinal colonization following challenge with an isolate of E. coli O157:H7 as unvaccinated control mice. These results indicate that this vaccination approach is unlikely to be straightforward in target bovine or human hosts.

Animals↗

PubMed2003

Histochemical detection of glycoconjugates in the male reproductive system of the horse.

Lectins are glycoproteins of plant and animal origin that have the ability to bind specific carbohydrate residues of cell glycoconjugates, particularly in terminal positions. In this study, the binding of lectins, Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA), Bandeiraea simplicifolia BS-1 (isolectin B4), Triticum vulgaris (WGA), Arachis hypogaea (PNA), and Ulex europaeus (UEA-I), was studied in the reproductive systems of male thoroughbred horses.DBA was detected in the stereocilia of the caput and corpus epididymis, and in the vas deferens. It was weakly detected in connective tissue of the corpus epididymis. Strong SBA staining was seen in epithelial cells in the testis, stereocilia of the corpus and cauda epididymis, and in the vas deferens. There were intense positive reactions for isolectin B4 in interstitial cells in all tissue and serosa of the vas deferens. PNA staining was seen only in stereocilia in the caput and corpus epididymis, and in the vas deferens. Strong WGA staining was seen throughout the testis, except in Sertoli cells, stereocilia, and connective tissue. UEA-I was detected in secondary spermatids, stereocilia, and epithelial cells of the cauda epididymis. These results show that degenerating cells in the testis, epididymal tubules, and vas deferens have differential affinities for lectins, and suggest that lectins play a role in the reproductive system of the horse. The heterogeneity of the lectin staining pattern in the reproductive tubules of adult horses suggests that the carbohydrate composition of each cell type is region specific.

Animals↗

PubMed2003

Application of fluorescein-labelled lectins with different glycan-binding specificities to the studies of cellular glycoconjugates in human full-term placenta.

In the present study 13 lectins of plant, fungal and animal origin, characterised by different glycan-binding specificities were used to examine the structure and distribution of specific glycans in the tissues of human placenta. Histochemical analysis was focused on villi (villous syncytiotrophoblast and stroma), cytotrophoblast of the basal plate and amniotic epithelium. It was found that glycoconjugates containing mannose and N-acetyl glucosamine were widely expressed while external fucosyl residues were absent on all studied structures of placenta. Lectins GNA (revealing non-reducing terminal mannosyl residues) and LPA (revealing sialic acid) bound selectively to the villus structures. The presence of sialic acid residues was observed in the superficial plasmalemma of syncytiotrophoblast and on the surface of the foetal capillary endothelium. Lectins LCA and PSA showed specific affinity to the plasmalemma of the cytotrophoblast and to the amniotic epithelium basement membrane. N-acetyl lactosamine-specific fungal lectin PSL selectively labelled amniotic epithelial and cytotrophoblast cell membranes and superficial plasmalemma of the syncytiotrophoblast.

Animals↗

PubMed1992

[Effect of the tumor cell associated glycoconjugate (TCA) derived Kato III, human gastric cancer cells on autologous mixed lymphocyte reaction in patients with rheumatoid arthritis].

We have been developing a new treatment for patients with rheumatoid arthritis (RA) by using intradermal injection of carbohydrate molecule complex. Among them, tumor cell associated glycoconjugate (TCA), the membrane structure of Kato III is one of the effective molecules. We studied the immunomodulatory effect of TCA on the autologous mixed lymphocyte reaction (AMLR) using PWM-mitogen induced lymphoblasts as stimulator cells and peripheral blood mononuclear cells (PBMC) as responder cells. In the kinetic study of the AMLR, its maximum proliferation was observed on days five through seven and responding CD4 cells highly expressed HLA-DR antigen. Studied AMLR in 10 patients with RA, proliferative responses of AMLR in these patients were divided into two types, high and low AMLR types. In vitro examination of TCA on AMLR showed that TCA at a concentration of 250 ng/ml significantly suppressed the AMLR response (p less than 0.01, paired T-test) and this phenomenon was found more frequently in high AMLR type patients than in low AMLR type patients. The suppressive effect of TCA on AMLR had a tendency to correlate with the efficacy of TCA therapy in patients studied. These results suggest that TCA may play a role in regulating the function of autoreactive lymphocytes of patients with RA.

Aged↗