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PubMed1981

Lectin labeling of sprouting neurons. I. Regional distribution of surface glycoconjugates.

Well-defined ferritin-conjugated lectins were used to map glycoconjugates on the surface of sprouting neurons from rat superior cervical ganglion (SCG) and spinal cord (SC). The cultured neurons were exposed to the markers and processed for electron microscopy, and the number of ferritin particles per unit area of plasmalemma was measured in three different regions: perikaryon, neuritic shaft, and growth cone. Three different binding patterns are observed for different lectin: equal receptor density throughout the plasmalemma of the growing neuron (e.g., Ricinus communis agglutinin I in SCG neurons), gradual decrease (e.g., wheat-germ agglutinin in SCG and SC neurons) and gradual increase (e.g., Ricinus communis agglutinin II in SC neurons) in the density of lectin receptors as one moves from the perikaryon to the growth cone. Furthermore, lectin receptor densities differ in the two types of neurons analyzed. We can conclude that the plasmalemma of the growth cone has biochemical properties different from those of the perikaryon, and that the neuron's structural polarity is expressed in its surface glycoconjugates. This phenomenon may be related to the growth cone's special functional properties and to the process of expansion of the plasma membrane.

Animals↗
PubMed1983

Isolation and characterization of a large, neurite-associated glycoconjugate from neuroblastoma cells.

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.

Animals↗
PubMed2006

Serogroup C meningococcal glycoconjugate vaccine in adolescents: persistence of bactericidal antibodies and kinetics of the immune response to a booster vaccine more than 3 years after immunization.

BACKGROUND: The persistence of protection from meningococcal disease following immunization with serogroup C meningococcal (MenC) glycoconjugate vaccines in infancy is short-lived. The duration of protective immunity afforded by these vaccines in other at-risk age groups (i.e., adolescents and young adults) is not known. We evaluated the persistence of bactericidal antibodies following immunization with a MenC glycoconjugate vaccine (MenCV) in adolescents and the kinetics of immune response to a meningococcal AC plain polysaccharide vaccine (MenPS) challenge or a repeat dose of MenCV. METHODS: We conducted a randomized comparative trial of 274 healthy 13-15-year-olds from whom a total of 4 blood samples were obtained (prior to administration of a dose of MenPS or MenCV, again on 2 further occasions at varying times from days 2-7 after vaccination, and finally on day 28 after vaccination. The correlate of protection was a serum bactericidal assay titer > or = 8 (with a serum bactericidal assay using human complement). RESULTS: A serum bactericidal assay using human complement titer > or = 8 was observed in 75% of participants at baseline (mean age, 14.5 years; mean time since routine MenCV vaccination, 3.7 years). No increase in serum bactericidal assay geometric mean titers was detected until day 5 after administration of MenPS. Geometric mean titers following administration of MenCV were significantly higher than those observed following administration of MenPS, at days 5, 7, and 28. CONCLUSIONS: This study showed sustained levels of bactericidal antibodies for at least 3 years after immunization of adolescents with MenCV. After challenge of immunized adolescents with MenPS, there was no increase in serum bactericidal assay observed until day 5 after vaccination, indicating that immunological memory may be too slow to generate protection against this potentially rapidly invasive organism.

Adolescent↗
PubMed1997

Structure of a glycoconjugate in solution and in complex with an antibody Fv fragment.

By use of heteronuclear (13C, 1H) NMR methods, the three-dimensional structure and dynamics of the glycoconjugate estrone-3-glucuronide (E3G) uniformly 13C enriched in the glucuronic acid moiety has been probed both in free solution and in association with an anti-E3G antibody single-chain Fv fragment. The glycan is found to exist in multiple conformations in free solution, with particularly large torsional fluctuations about the glycosidic linkage psi. Resonance assignments and distance restraints for the glycoconjugate in the bound state were obtained from heteronuclear proton-carbon-carbon-proton-COSY and isotope-edited NOESY techniques, respectively. Quantitation of the NOE data with a full-relaxation matrix approach showed that the antibody selects a conformation from the solution repertoire which does not correspond with either of the two lowest energy conformations of the free glycan, and the internal energy of the glycan in the bound state is estimated to be at most approximately 15 kcal/mol higher than the global minimum energy conformation. The glucuronide moiety undergoes a stacking interaction with an aromatic ring in the binding site, and both ring-current shifts and nuclear Overhauser effects computed from the predicted bound-state conformation are in good agreement with experiment. The bound-state conformation is also in good agreement with preliminary x-ray data on a related complex.

Carbohydrate Conformation↗
PubMed1984

Effect of retinoic acid and low calcium conditions on surface glycoconjugates defined by differential lectin labelling in mouse epidermal cell culture.

The appearance of cell surface glycoconjugates (detected by fluorescein-isothiocyanate-conjugated lectins and bullous pemphigoid antibody) was serially examined in mouse epidermal cell cultures treated with trans-retinoic acid and aromatic retinoic acid (etretinate) and in cultures maintained under low calcium conditions. The changes in lectin staining occurred in concert with the process of differentiation as assessed by cell morphology and colony growth characteristics, and they correlated with the patterns observed in whole mouse skin. The keratocyte cultures treated with retinoic acid showed delayed and reduced differentiation and stratification, and this was associated with markedly reduced binding of lectins specific for N-acetyl-glucosamine and fucose. The low calcium concentration produced similar changes. Thus, the loss of surface glycoconjugates in the epidermal cell culture system was not specific for either retinoic acid or low calcium, but correlated with the degree of cell differentiation.

Animals↗
PubMed1992

Isolation and characterization of a novel glycosyl-phosphatidylinositol-anchored glycoconjugate expressed by developing neurons.

In search of new markers for studying thymic and nervous system ontogeny, we raised rat monoclonal antibodies against glycosyl-phosphatidylinositol-anchored molecules among which larger groupings have been shown to be ectoenzymes and adhesion molecules. Two of these monoclonal antibodies (H193-4 and H194-563, IgG) were found to recognize glycosyl-phosphatidylinositol-anchored glycoconjugates of 28-33 kDa (P31) and 50-70 kDa in developing mouse brain and thymus respectively, when these tissues were analysed by immunoblot experiments. P31 antigen was found to be transiently expressed by neurons in neural primary cultures [Rougon, G., Alterman, L., Dennis, K., Guo, X. J. & Kinnon, K. (1991) Eur. J. Immunol. 21, 1397-1402]. We show in this report that, in developing mouse brain, a maximal expression occurred between embryonic day 17 and post-natal day 5, a period that corresponds to the formation of neuronal networks. P31 antigen was immunopurified and found to possess the following properties: (a) it was soluble in alkaline solvents; (b) it bound to DEAE-cellulose and was eluted by a salt gradient of 0-1 M NaCl; (c) it was sensitive to endoglycosidase F digestion; (d) it was insensitive to heparinase, hyaluronidase, chondroitinase ABC, endo-beta-galactosidase and sialidase treatment; (e) it was labile to mild acid hydrolysis without loss of immunoreactivity; (f) it contained phosphate; (g) it lost its immunoreactivity after treatment with phosphatidylinositol phospholipase C and treatment. These characteristics combine to suggest that P31 is an anionic glycoconjugate sharing similarities with Leishmania donovani lipophosphoglycan and with the heat-stable antigen recognized by J11d antibody on murine hematopoïetic cells. This last hypothesis was further confirmed by the observation that oligonucleotide probes derived from the heat-stable antigen-encoding cDNA detect, in developing brain, a 1.8-kb mRNA species similar in size to that reported for the heat-stable antigen mRNA and following the same developmental expression as P31 antigen.

Animals↗
PubMed1996

Changes in glycoconjugate-lectin binding characteristics of in vitro and in vivo mouse blastocysts.

OBJECTIVE: To investigate the influence of embryo culture, on blastocysts cultured from the zygote stage, on blastocyst cell surface glycoconjugates as an indicator of cellular metabolism. METHOD: Eleven fluorescein isothiocyanate (FITC)-labelled lectins, which recognize different oligosaccharide structures, were applied to zona pellucida intact and zona pellucida free blastocysts which had developed either in vivo or in vitro. The differential binding patterns for the zona pellucida, trophectoderm and inner cell mass were determined using fluorescence microscopy. RESULTS: With the exception of wheat germ agglutinin (WGA) and Limax flavus agglutinin (LFA) the remaining nine lectins bound less intensely to the zona pellucida of the in vitro blastocysts compared with the in vivo blastocysts. The trophectoderm from both groups bound seven lectins with equal intensity, of the remaining four Lotus tetragonolbus agglutinin (LTA), Bandeiraea simplicifolia 4 (BSB4), Phaseolus vulgaris agglutinin P (PHA-P) and Limulus polyphemus agglutinin (LPA) demonstrated differential binding. The inner cell mass from the two blastocyst groups bound only three lectins identically, peanut agglutinin (PNA), Bandeiraea simplicifolia 4 (BSB4) and Dolichos biflorus agglutinin (DBA). CONCLUSION: Oviductal glycoproteins contribute to the zona pellucida glycoprotein content during early development, however embryo culture from the zygote stage may not provide adequate substrates for normal inner cell mass glycoconjugate biosynthesis.

Animals↗
PubMed1986

Detection of mucus glycoconjugates in human conjunctiva by using the lectin-colloidal gold technique in TEM. II. A quantitative study in dry-eye patients.

The mucus glycoconjugates produced by conjunctival goblet cells in dry-eye patients were studied by a specific cytochemical reaction in Transmission Electron Microscopy (TEM). Four lectins, proteins of vegetal origin which specifically bind glycosidic residues, (WGA, PNA, SBA and ConA) were used conjugated with colloidal gold as ultrastructural marker. We performed a quantitative analysis by counting the colloidal gold particles present on mucus granules. The results were compared with normal conditions. We found a decrease in sialic acid, N-acetyl-glucosamine, N-acetyl-galactosamine and galactose-N-acetyl-galactosamine and an increase in mannose. The different content of glycoconjugates in goblet cells may reflect in the change of physical and functional properties of mucus. We think these data may be useful in the search for a therapeutic mucomimetic drug.

Carbohydrate Metabolism↗
PubMed1986

Characterization of cellular immune response to chemically defined glycoconjugates from Leishmania mexicana subsp. amazonensis.

Two defined glycoconjugates (GP-10/20 and FR II Phe) purified from Leishmania mexicana subsp. amazonensis were analyzed with respect to their ability to induce cellular responses in immunized and infected mice. Each glycoconjugate was recognized by specific immune cells, as assessed by the proliferative response of lymph node cells of immunized mice. The response to GP-10/20 depended on helper T cells and antigen-presenting cells and was restricted by a major histocompatibility complex class II gene product. A specific anti-GP-10/20 T-cell line was established, and it was able to transfer a delayed-type hypersensitivity (DTH) response to normal mice. Both antigens were also recognized during an ongoing disease, as assessed by DTH response of infected mice. By this response, it was possible to distinguish susceptible from resistant strains of mice. In the course of the disease in resistant mice a correlation between the size of the primary lesion and the DTH response to GP-10/20 was observed. The presence of the glycoproteins on both promastigote and amastigote forms of the parasite, the antigenic similarities between both fractions, and the distribution of the GP-10/20 antigen in other trypanosomatids were studied. The results showed that both antigens were present on promastigotes and amastigotes. GP-10/20 shared no epitopes with FR II Phe, was included as part of the crude preparation leishmanin, and had some cross-reactive determinants with Leishmania donovani and Crithidia deanei.

Animals↗
PubMed2002

A glycoconjugate vaccine for Neisseria meningitidis induces antibodies in human infants that afford protection against meningococcal bacteremia in a neonate rat challenge model.

The functional activities of serum samples from human infants immunized with a glycoconjugate vaccine for Neisseria meningitidis serogroup C were assessed in a complement-mediated antibody-dependent serum bactericidal assay (SBA) and in a neonate rat model of protection from bacteremia. Selective serum samples from individual human infants were combined to make a panel of 11 serum pools to obtain a sufficient volume for testing. Each pool was assayed (i) for the anti-N. meningitidis serogroup C capsular polysaccharide (PS) immunoglobulin G (IgG) concentration as determined by reactivity in a direct-binding enzyme-linked immunosorbent assay, (ii) for bactericidal activity against N. meningitidis serogroup C strain C11, and (iii) for the ability to reduce bacteremia after passive transfer into a neonate rat model. Representative serum samples from infants who were not previously immunized with any N. meningitidis serogroup C vaccine served as a negative control. The prepared serum pools ranged in antibody concentration from 0.18 to 17.31 micro g of IgG specific for N. meningitidis serogroup C PS per ml. For this serum panel, a direct relationship between concentrations of anti-N. meningitidis serogroup C PS-specific IgG and serum SBA titers (r = 0.9960) was observed. Passive transfer to neonate rats demonstrated the ability of postimmunization serum samples to significantly reduce (> or =2-log(10) reduction compared to control animals) the level of bacteremia following a challenge. Of 79 neonate rats that received > or =0.031 micro g of human infant anti-N. meningitidis serogroup C PS IgG, 75 (94.9%) had a > or =2-log(10) reduction in bacteremia, whereas of the animals that received <0.031 micro g of antigen-specific IgG, 10.3% (4 of 39 rats) showed a > or =2-log(10) reduction in bacteremia. It was concluded that the anti-N. meningitidis serogroup C PS IgG antibody induced by this glycoconjugate vaccine had in vitro functional activity (as determined by a SBA) and also afforded protection against meningococcal bacteremia in an animal model.

Animals↗
PubMed1979

Cytochemical characterization of secretory and cell surface glycoconjugates by light and electron microscopy.

Lectin methods have increased the capacity for histochemical characterization and differentiation of glycoproteins and have demonstrated, for example, greater reactivity of gastrointestinal than of respiratory tract secretions with the periodate-concanavalin A-horseradish peroxidase method for localizing mannose-rich glycoprotein. Application of a battery of ultrastructural cytochemical methods with specificity for the constituents characteristically present in the complex carbohydrates provides knowledge of the distribution of the various recognizable types of glycoconjugates in tissues and cells showing, for example, marked differences in glycoconjugates of the apical compared with the basolateral plasmalemma in a given cell type and differences between apical plasmalemmas or basement membranes of different cell types. Such information raises questions as to the biologic significance of the different complex carbohydrates in various sites and, hopefully, will lead to a clearer understanding of their physiologic roles.

Animals↗
PubMed1982

Sponge glycoconjugates: immunological properties and localization by fluorescent antibodies and lectins.

By immunodiffusion and immunoelectrophoresis, glycoconjugates previously isolated from a sponge, Spongia officinalis, and fractionated on lectins, showed identical immunological behaviour which was species specific. By fluorescent antiserum and lectins, these glycoconjugates were located on sections of two sponges, S. officinalis and Chondrosia reniformis. A strong pericellular staining occurred together with a diffused labeling.

Animals↗
PubMed1983

Application of lectin--gold complexes for electron microscopic localization of glycoconjugates on thin sections.

A method is described for the electron microscopic detection of lectin-binding sites in different cellular compartments and extracellular structures that uses thin sections from resin-embedded tissues. Various lectins (Ricinus communis lectin I and II, peanut lectin, Lotus tetragonolobus lectin, Ulex europeus lectin I, Lens culinaris lectin, Helix pomatia lectin, and soybean lectin) were bound to particles of colloidal gold and used for direct staining of thin sections or glycoprotein--gold complexes were prepared and applied in an indirect technique (concanavalin A and horseradish peroxidase--gold complex; wheat germ lectin and ovomucoid--gold complex). The details for preparation of such complexes from 14 nm gold particles are reported. The conditions of tissue processing that gave satisfactory staining results and good fine structure preservation were mild aldehyde fixation without osmification and low temperature embedding with the hydrophilic resin Lowicryl K4M. None of the so-called etching procedures was necessary prior to labeling of Lowicryl K4M thin sections. Examples of the use of this approach for detection of glycoconjugates in the rough endoplasmic reticulum, Golgi apparatus, and mucin of intestinal goblet cells as well as plasma membrane and various intracellular structures of absorptive intestinal and renal tubular cells are shown. A comparison is made with preembedding staining results on Concanavalin A-binding site localization in rat liver which shows that problems of penetration common in such a technique are circumvented by the postembedding approach described here. Concanavalin A-binding sites were not only consistently found in nuclear envelope, rough and smooth endoplasmic reticulum, plasma membranes, and collagen fibers, but also in mitochondria, glycogen, ribosomes, and nucleus. These data and those of a previous investigation (Roth J, Cytochem 31:547, 1983) prove the applicability of this cytochemical technique for postembedding localization of glycoconjugates by light and electron microscopy.

Animals↗
PubMed1978

Desialylation of glycoconjugates using immobilized vibrio cholerae neuraminidase. Preparation, properties and use of the bound enzyme.

Neuraminidase from Vibrio cholerae was immobilized on Sepharose 4B using the cyanogen bromide technique. The properties of the bound enzyme were found to be similar to those of the soluble form, except for an appreciably improved stability on storage at equivalent dilution and a reduction in recovered activity. Evidence was obtained that the binding of large molecular weight substrates to the bound enzyme is modified due to the immobilized state of the enzyme. The use of the enzyme gel for desialylation of glycoconjugates in a closed circuit system was investigated and optimal conditions delineated using fetuin as a model substrate. The bound enzyme could be used repeatedly with only low loss of activity. The value of the desialylation circuit in the preparation of partially and completely desialylated glycoconjugates is discussed. The use of immobilized neuraminidase for the desialylation of cells is described.

Drug Stability↗
PubMed1980

Glycoconjugates (glycosaminoglycans and glycoproteins) and glycogen in the human cervix uteri.

Glycoconjugates and glycogen obtained from the cervix uteri of nonpregnant women of proliferative phase and secretory phase, and from the postpartum cervix uteri were determined by electrophoresis on cellulose acetate membrane and chemical analysis before and after enzymatic digestion and nitrous acid treatment. Although the contents of neutral glycopeptide fraction in these tissues were similar, the glycogen content in the postpartum tissue greatly decreased. The contents in terms of hexosamine of hyaluronic acid, chondroitin sulfate, heparan sulfate and acidic glycopeptides in the postpartum tissue greatly increased in comparison with those in the nonpregnant tissues, whereas that of dermatan sulfate slightly decreased. In addition, dermatan sulfate in the postpartum tissue contained a small portion of non-sulfate disaccharide units, which was not detected in the nonpregnant tissues. The results indicate that significant changes in the contents of glycoconjugates and glycogen of human cervix uteri occur in ripening. The roles of these substances in the cervical ripening are discussed.

Cell Division↗
PubMed1981

Hormonal effects on the sulfation of sulfated glycoconjugates in the uterine endometrium of non-ovariectomized rabbit.

A particulate fraction was separated from endometrial scrapings of uterus of hormone-treated non-ovariectomized or intact rabbit. The effects of ovarial hormones on the incorporation of [35S]sulfate from 3'-[35S]phosphoadenosine 5'-phosphosulfate (PAPs) into endogenous acceptors in the particulate fractions were studied. Treatment of the animal with estrogen elevated the incorporation of [35S]sulfate, but exogenous progesterone suppressed this effect. The results of DEAE-Sephadex A-25 (Cl- form) column chromatography of the pronase digest of the 35S-labeled substances indicated that all the subfractions were sensitive to the hormones. The present observations, together with previous findings, suggest that the sulfation of sulfated glycoconjugates in the particulate fractions is stimulated by exogenous estrogen, but exogenous progesterone suppressed this effect, and also that endogenous ovarial hormones exert little influence on the effects of the present doses of these exogenous hormones on the sulfation. Therefore, non-ovariectomized rabbit can be used for rough evaluation of the effects of regular doses of these exogenous hormones on the sulfation of sulfated glycoconjugates in the uterine endometrium.

Adenine Nucleotides↗
PubMed1991

Glycoconjugates in the otolithic organ of the developing chick embryo.

The presence of glycoconjugates in the otolithic organ of developing chick embryos was investigated histochemically using lectins. On the 6-day-old chick embryo, intense labelling with lectins was observed in the sensory epithelium, on the surface of the epithelium and on the immature otoconia. The otoconia were intensely labelled with lectins at every stage of the chick embryos, while the labelling with lectins in the sensory epithelium became weaker with the maturing of the otoconia. In TEM observation, the secretory granules of the supporting cells of the sensory epithelium were labelled with lectin. The reaction of lectin was more intense in the electronic dense zone of the otoconium than in the electronic lucent zone at every stage of the chick embryos. These findings indicate that the precursors of the otoconia are secreted by the supporting cells of the sensory epithelium and that glycoconjugates play an important role in otoconial formation.

Animals↗
PubMed2004

Haemophilus influenzae type b-outer membrane protein complex glycoconjugate vaccine induces cytokine production by engaging human toll-like receptor 2 (TLR2) and requires the presence of TLR2 for optimal immunogenicity.

Conjugate vaccines consisting of the capsular polysaccharide (PS) of Haemophilus influenzae type b (Hib) covalently linked to carrier proteins, unlike pure PS, are immunogenic in infants and have significantly reduced Hib infections in the United States, but require multiple doses to induce protective anti-PS Ab titers. Hib-meningococcal outer membrane protein complex (OMPC) conjugate vaccine, however, elicits protective anti-PS Ab titers after one dose. We found that OMPC and Hib-OMPC engaged human Toll-like receptor 2 (TLR2) expressed in human embryonic kidney (HEK) cells, inducing IL-8 production, and engaged mouse TLR2 on bone marrow-derived dendritic cells, inducing TNF release. Hib conjugated to the carrier proteins CRM(197) and tetanus toxoid did not engage TLR2 on HEK or dendritic cells. Engagement of TLR2 by Hib-OMPC was MyD88 dependent, as Hib-OMPC-induced TNF production was ablated in MyD88 knockout (KO) mice. Hib-OMPC was significantly less immunogenic in TLR2 KO mice, inducing lower Hib PS IgG and IgM titers compared with those in wild-type mice. Splenocytes from OMPC-immunized TLR2 KO mice also produced significantly less IL-6 and TNF-alpha than those from wild-type mice. Hib-OMPC is unique among glycoconjugate vaccines by engaging TLR2, and the ability of Hib-OMPC to elicit protective levels of Abs after one dose may be related to TLR2-mediated induction and regulation of cytokines produced by T cells and macrophages in addition to the peptide/MHC II-dependent recruitment of T cell help commonly afforded by carrier proteins. TLR2 engagement by an adjuvant or carrier protein may be a useful strategy for augmentation of the anti-PS Ab response induced by glycoconjugate vaccines.

Adaptor Proteins, Signal Transducing↗