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PubMed1993

Glycoconjugates as components of receptors for Bordetella avium on the tracheal mucosa of turkeys.

Bordetella avium is an important respiratory tract pathogen of turkeys. In common with other pathogenic bordetellae, B avium manifests a tissue tropism for cilia of the respiratory tract epithelium. To determine the molecular characteristics of the host cell receptors for B avium, we used hemagglutination and in vivo adherence assays. Carbohydrates, mucus, sialic acid-specific lectin, and other glycoconjugates were evaluated for their ability to competitively inhibit binding of B avium to host cells. The gangliosides, GD1a and GT1b, completely inhibited hemagglutination, whereas N-acetylneuraminic acid (sialic acid) partially inhibited hemagglutination. Adherence to turkey tracheal mucosa in vivo was significantly (P < 0.01) inhibited by GD1a and GT1b gangliosides, N-acetylneuraminic acid, bovine submaxillary mucin, and horseshoe crab (Limulus polyphemus) lectin. Treatment of the tracheal mucosa with neuraminidase also inhibited adherence of B avium. We conclude that N-acetylneuraminic acid and the gangliosides, GD1a and GT1b, may be important components of the tracheal mucosa receptor for B avium in turkeys.

Animals↗

PubMed1994

Biosynthesis of N-glycolylneuraminic acid-containing glycoconjugates. Purification and characterization of the key enzyme of the cytidine monophospho-N-acetylneuraminic acid hydroxylation system.

We have proposed that cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) hydroxylation is carried out by a multienzyme system involving CMP-NeuAc hydroxylase (the terminal enzyme of the system), cytochrome b5, and an NADH-dependent cytochrome b5-reducing factor (Kozutsumi, Y., Kawano, T., Yamakawa, T., and Suzuki, A. (1990) J. Biochem. 108, 704-706). The CMP-NeuAc hydroxylase was purified to homogeneity from the cytosolic fraction of mouse liver, using ion exchange columns, a Red-Sepharose column, and a soluble cytochrome b5-immobilized Sepharose column. The purified enzyme exhibited a single band (64 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a single peak (58 kDa) on gel permeation chromatography, indicating that it is composed of a single polypeptide chain. The absorption spectrum did not indicate the presence of a heme prosthetic group in the enzyme. Atomic absorption spectrometry and an inhibition test using an iron chelator indicate that the enzyme contains non-heme iron as an electron acceptor. A reconstitution experiment with the purified CMP-NeuAc hydroxylase, soluble cytochrome b5, and recombinant NADH-cytochrome b5 reductase revealed that these three factors are essential for the reaction. The hydroxylase exhibited high affinity to CMP-NeuAc (Km = 5 microM) and was greatly stabilized by CMP-NeuAc. The molecular activity of the enzyme (approximately 500/min) is much lower than that reported for NADH-cytochrome b5 reductase, suggesting that the activity or amount of hydroxylase is rate-limiting in CMP-N-glycolylneuraminic acid (NeuGc) biosynthesis. These results, together with the previous observation that the level of CMP-NeuAc hydroxylase activity was associated with the expression of NeuGc in various tissues, support the notion that the enzyme is the key for regulation of the overall velocity of CMP-NeuAc hydroxylation and consequently for the expression of NeuGc-containing glycoconjugates.

Animals↗

PubMed1993

Lectin histochemistry of cell-surface glycoconjugates in the primary olfactory projections of the newt.

The binding of 14 lectins were performed on paraffin-embedded sections of the olfactory bulb of Triturus to identify specific glycoconjugates on the cell surface of primary olfactory projections. The histochemical lectin staining patterns indicate that the membrane of olfactory neurons terminating in the main olfactory bulb contained prevalently oligosaccharides with alpha-acetyl-D-galactosamine as terminal residues. In the accessory olfactory bulb, instead, the primary olfactory projections possess a high density of alpha-D-galactose as sugar residues. The selective lectin binding on the surface of primary olfactory axons suggests that specific cell surface glycoproteins may have a role in the axonal growth due to the continual cycle of proliferation and death of olfactory receptors.

Animals↗

PubMed1993

[Histochemical studies on the glycoconjugates of experimental alkali burned cornea in rabbit].

The model of experimental alkali burn of cornea in rabbit was obtained using 1 mol/L NaOH solution. After being burned for 1, 3, 7 and 14 days, the histochemical changes of these models were studied by using labelled concanavalin A (ConA-FITC) and wheat germ agglutinin (WGA-FITC). The results showed that after being burned, the amount of glycoconjugates interacted with these lectins decreased, and increased gradually after 7 days, then reached the level higher than that in the normal cornea. These changes were related to the recovery of alkali burned cornea.

Animals↗

PubMed1993

Characterization of the glycoconjugate sugar residues in developing chick esophageal epithelium.

The development of the esophagus in the chick embryo is characterized by remarkable morphological changes especially at the level of the epithelium. Using horseradish peroxidase-conjugated lectins (DBA, PNA, SBA, WGA, ConA, LTA, UEAI) we have studied, at the level of the esophagus of chick embryos from the 8th to the 21st day of incubation and of 1- and 2-day-old chicken, the evolution of the saccharidic moieties of glycoconjugates, which precedes and/or is concomitant with the epithelial morphological transformations. We have found differences in content and cellular distribution of oligosaccharides during the histogenetic processes which characterize the lining and glandular epithelium. Before the appearance of cilia and mucus secretion at the bathyprismatic epithelial cells, the sugar residues D-galactose-(beta 1-->3)-N-acetyl-D-galactosamine, beta-N-acetyl-D-galactosamine and alpha-L-fucose were detected only at the luminal cell surface. These oligosaccharides were probably involved in giving rise to the polarization of the esophageal epithelial cells. The esophageal gland mucus was first characterized by the presence of alpha-L-fucose and afterwards also by the presence of D-galactose-(beta 1-->3)-N-acetyl-D-galactosamine, D-glucosamine and sialic acid.

Animals↗

PubMed1995

Chronic administration of epidermal growth factor to pigs induces growth, especially of the urinary tract with accumulation of epithelial glycoconjugates.

BACKGROUND: Epidermal growth factor (EGF) receptor hyperstimulation induced by systemically administered EGF or by the development of transgenic mice overexpressing transforming growth factor alpha (TGF alpha) or other EGF-related ligands is known to induce various effects, such as acceleration of developmental processes like incisor eruption, inhibition of gastric acid secretion, morphologic changes in the pancreas resembling pancreatitis, and malignancies in mammary glands and the liver. The present investigation was initiated to explore the effects of systemic EGF administration to the mature organism in a species with greater anatomic resemblance to humans than rodents. EXPERIMENTAL DESIGN: Eleven Goettingen Minipigs underwent 4 weeks of treatment either with placebo (n = 5) or human recombinant EGF 30 micrograms/kg/day (n = 6) administered subcutaneously. At the end of Week 4, the animals were sacrificed, autopsy was performed, and tissue samples were collected for histologic examination. RESULTS: EGF treatment caused macroscopic enlargement of the ureters, kidney, and heart. The ureters increased 4-fold in cross sectional area due to growth of all wall layers. The urothelium was hyperplastic with intracellular accumulations of material staining with Periodic acid-Schiff. Similar but less pronounced changes were found in the pancreas, lungs, salivary glands and esophagus. CONCLUSIONS: The most important observation of the present study is that systemic treatment with EGF for 4 weeks induces considerable growth to the urinary tract. We suggest new biologic effects of the EGF family in promoting growth of the urinary tract and in stimulating epithelial glycoconjugate biosynthesis in the urothelium and excretory ducts of the pancreas.

Animals↗

PubMed1996

Lectin histochemical evaluation of glycoconjugates in dog efferent ductules.

Glycoconjugates in the epithelial cells of the efferent ductules in the dog were investigated using lectin histochemistry. These ductules connect the extratesticular rete with the epididymis. The epithelium of the ductules consisted both of ciliated and nonciliated cells. Whereas the apical zone of ciliated cells showed selective binding with WGA, SWGA, SNA, MAA and neuraminidase-PNA, that of nonciliated cells bound to all lectins used in the present study: WGA, SWGA, SNA, MAA, PNA, neuraminidase-PNA, RCA1, DBA and SBA. The nonciliated cells were divided into 3 types: type A cells which lacked both specific granules and vacuoles, type B cells which were characterised by a few specific apical vacuoles and many large specific granules, and type C cells which were characterised by some specific apical vacuoles and small basal granules. The specific granules and vacuoles of type B cells showed binding with WGA, SWGA and MAA. The specific granules of type C cells showed binding with WGA, SWGA, SNA, MAA, PNA and neuraminidase-PNA, while their specific vacuoles showed binding with WGA, SWGA, SNA and MAA. The Golgi zone both of ciliated and type A cells did not bind with any lectins used in this study, while type B and C cells showed similar lectin binding patterns between the Golgi zone and their specific granules. Specific lectin binding patterns revealed a different carbohydrate composition of each type of cell, indicating a biological difference between the ciliated cells and the 3 types of nonciliated cells in dog efferent ductules.

Animals↗

PubMed1995

Glycoconjugates and keratin 18 define subsets of taste cells.

Sections of neonatal, normal adult and denervated adult rat tongue were examined with lectin histochemistry. Attention was focused upon intragemmal cells (cells within the taste bud) and the surrounding perigemmal cells. Informative staining patterns were observed with four of 12 lectins: Ulex europaeus (UEA-I), Bauhinia purpurea (BPA), Helix pomatia (HPA) and Lotus tetragonolobus (LTA) agglutinins. In normal adult tongues, BPA bound to those lingual epithelial cells lacking contact with the basal lamina. After they formed, vallate taste buds were laterally surrounded by distinctive BPA-positive cells. HPA reacted selectively with 28% and LTA with 23% of the intragemmal cells in vallate/foliate taste buds. In double-stained taste buds there was a statistically significant overlap of LTA-positive cells and keratin 18-positive cells. The overlap between HPA binding and keratin 18 was more marked: double-stained cells comprized 67% of all stained cells. During taste bud development in neonates keratin 18 synthesis preceded HPA binding. In contrast, during the replacement of adult taste cells, keratin 18 synthesis and HPA binding were generally concurrent. Keratin 18 and HPA probably identify the same subset of older taste receptor cells. HPA may bind to glycoconjugates on the surface of keratin 18-positive cells. In denervated adult tongue the loss of all UEA-I-positive or BPA-positive perigemmal cells suggests that perigemmal as well as intragemmal cells are nerve-dependent.

Animals↗

PubMed1995

Lectin histochemical study of glycoconjugates in the chick embryo proventriculus.

A lectin histochemical study was performed to investigate the glycoconjugate saccharidic moieties of the lining and glandular epithelium of the chick embryo proventriculus from day 7 to hatching. The sugar residues D-galactose-(beta 1-->3)-N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, alpha/beta-N-acetyl-D-galactosamine and alpha-D-mannose were present at the lining epithelium of the proventriculus from the 7th day onwards; alpha-D-fucosyl residues were never detected. The same sugar residues were detected in the cells of the deep glands from the 7th day; alpha-L-fucose appeared later. The sugar residues appeared in the cells of the superficial glands at different times during the development of the proventriculus. In comparison to that of the deep glands, the secretion of the superficial glands was characterized, by the absence of N-acetylgalactosamine and alpha-D-mannose. From day 20 onwards, the composition of the mucus released by the two types of glands was identical from a qualitative point of view. The histological and histochemical results showed that the superficial glands are well defined morphological structures and not "an artefact of contraction", as claimed by other authors.

Animals↗

PubMed1996

CD15-containing glycoconjugates in the central nervous system.

CD15-containing glycoconjugates have a common trisaccharide residue, 3-fucosyl-N-acetyllactosamine, which can be recognized by a panel of monoclonal antibodies. Immunohistochemical studies revealed a widespread distribution of CD15 in several epithelial non-neural tissues as well as in the CNS. In the mature mammalian brain CD15-containing glycolipids and glycoproteins are constantly present in astrocytes, whereas oligodendrocytes and particular subpopulations of neurons are variably immunostained. CD15 immunoreactive astrocytes are spatially distributed in some brain regions, which points to specialized functions of astroglial subpopulations. The expression of CD15 follows a timely ordered pattern during the development of glial cells and neurons of certain brain areas, such as the human and rat cerebellum and the mouse visual system. During morphogenesis, CD15 may exert either growth-promoting or growth-repulsive activities to guide cell migration. In CNS lesions altered expression patterns of CD15 may occur. For example, in human gliomas the staining intensity for CD15 inversely correlates with the grade of malignancy. In degenerative brain diseases reactive astrocytes may reveal an increased labelling intensity on their cell surface as well as an abnormal cytosolic accumulation of the epitope. The functional significance of CD15 in the CNS is not exactly known yet. The carbohydrate could be involved in cellular adhesion and/or as receptor molecule in signal transduction pathways, as has recently been demonstrated for leukocyte-platelet or leukocyte-endothelial cell interactions.

Adult↗

PubMed1996

[Glycoconjugates on the basis of polyacrylamide--tools for studying lectins, antibodies, and glycosyltransferases in glycobiology, cytochemistry, and histochemistry].

The synthesis, physicochemical characteristics, and application for studying the carbohydrate-binding molecules of some analogues of human cell glycoconjugates, neoglycoconjugates, were reviewed. An approach to the synthesis of the polyacrylamide derivatives of carbohydrates based on the interaction of fully activated polyacrylic acid with omega-aminoalkyl glycosides was described. It provides highly reproducible results, is more simple than the previously known methods of the synthesis of such derivatives, and expands the range of synthetic possibilities because it can provide both the sugar-polymer type molecules and conjugates bearing various labels and effectors, sorbents, glycosurfaces, etc. In the first part of the review, the synthesis of polyacrylamide conjugates and their physicochemical properties were described. In its second part, the synthesis of some complex constructions, such as pseudoglycoproteins, pseudomucins, glycoparticles, and glycosurfaces, was outlined. Some examples of the application of the described conjugates in various fields of glycobiology were simultaneously discussed. Prospects of the further development of the presented approach in glycotechnology and medicine were also described.

Acrylic Resins↗

PubMed1995

[Histochemical characterization of glycoconjugates in the tongue of the toad Bufo marinus L. (Amphibian, anuran) with conventional techniques for light microscopy].

Histochemical stains with and without enzymatic digestions such as alpha-amylase, neuraminidase and hyaluronidase, made possible to localize and differentiate various types of glycoconjugates (GCs) in the tongue of the toad Bufo marinus. In the dorsal mucosae the covering epithelium of the filiform papillae, of the central folds and in the marginal cells of the fungiform papillae there were present large amounts of neutral GCs with little or no galactose and/or N-acetylgalactosamine and scanty carboxylic acid GCs while the superficial strata of the taste organs showed a mixture of neutral an acid GCs with a predominance of sulfated and carboxylic acid GCs. The glandular secretory cells showed neutral GCs almost exclusively with a gradient of concentrations increasing from the base to the apex being galactose or N-acetyl-galactosamine one of the component sugars. The ventral epithelium showed two types of mucous cells, one with neutral GCs and the other with neutral and acidic GCs. The connective tissue contained many mast cells showing highly acid GCs both sulfated and carboxylic with some neutral GCs. The extracellular connective matrix showed scanty neutral and acid GCs. Glycogen was present in the cytoplasm of glandular epithelial cells and of the striated muscle fibers. Additionally, the obtained results suggest the presence of a type of GC with a carboxylic acid (sialic acid) resistant to neuraminidase of Clostridium perfringens used in this study.

Animals↗

PubMed1997

[ELISA of IgM antibodies to Thomsen-Friedenreich (TF) hapten in cancer diagnostics: comparison of data obtained with four TF-glycoconjugates].

The level of IgM antibodies to the Thomsen-Friedenreich hapten (TF) relative to the total IgM level in the blood sera of gastric and breast carcinoma patients and healthy persons was determined using enzyme-linked immunosorbent assay. The following TF glycoconjugates were tested: TF-polyacrylamide (PAA)(with 10 mol.% of TF hapten Gal beta 1-3GalNAc alpha 1-O(CH2)3NH per number of monomeric units in the polyacrylamide), TF-human serum albumin (HSA)(Gal beta 1-3GalNAc alpha 1-O-p-C6H4-HSA containing approximately 15 carbohydrate residues per HSA molecule), asialo-kappa-caseinoglycopeptide, and asialoglycophorin. The total IgM level was determined using antibodies to the mu-chain of human IgM. The statistically significant difference between cancer patients and healthy donors was revealed with two conjugates: TF-PAA and TF-HSA. In the case of TF-PPA, the sensitivity of the assay was 75-83%, and the specificity was 77%. Thus, TF-PAA is the most suitable conjugate for measuring the level of serum anti-TF-IgM antibodies.

Acrylic Resins↗

PubMed1991

Monoclonal antibodies against Gal alpha 1-4Gal beta 1-4Glc (Pk, CD77) produced with a synthetic glycoconjugate as immunogen: reactivity with carbohydrates, with fresh frozen human tissues and hematopoietic tumors.

The purpose of this study was to isolate mouse monoclonal antibodies (MAbs) recognizing Gal alpha 1-4Gal beta 1-4Glc (Pk, CD77), which has been described as a Burkitt's-lymphoma-associated antigen. Three IgGI MAbs reactive with Pk were developed using a synthetic glycoconjugate as immunogen and a filter immunoplaque screening assay. One MAb (PK67) was characterized by immuno-thin-layer chromatography, ELISA and competition assays using neutral glycolipids from various sources and a variety of carbohydrates and glycoproteins. Epitope analysis showed that all 3 carbohydrates moieties are required in PK67 binding. No cross-reactivity was observed with closely related carbohydrate structures, with the exception of Gal alpha 1-4Gal beta 1-4GlcNAc. However, the reactivity with this structure was several orders of magnitude lower than with Pk. Immunohistochemical analysis of fresh-frozen tissue specimens showed that Gb3 is widely expressed: prominent staining was observed in many normal tissues, e.g., kidney and gastric tissue and on capillary endothelial cells. In lymph nodes, very weak staining of a few B cells was observed. Flow cytometric analysis of hematopoietic neoplasms showed Gb3 expression on B-cell neoplasms, particularly those with committed B-cell phenotype indicating that, in addition to earlier reports of Gb3 expression on Burkitt's lymphoma, Gb3 is present on other committed B-cell neoplasms.

Animals↗

PubMed1993

Stimulatory effect of TGF-beta on anionic glycoconjugate synthesis by rat calvarial cells: specificity, uncoupling of cell density dependence, and modulation by chondroitin sulfate.

Anchorage-dependent cultures of a population of cells derived from the outer part of the rat calvaria demonstrated decreased net accumulation of radiolabeled chondroitin sulfate (CS) and hyaluronic acid (HA) per cell as the cell density of the cultures increased. The addition of TGF-beta 1 resulted in large stimulations of the net CS, but not of the net HA, accumulating in the medium at all cell densities and an abolition of the density-dependent effect. These effects were largely due to increases in newly synthesized CS appearing in the medium. Supplementation of the culture media with CS had complex but relatively small effects on the stimulation of the net accumulation of radiolabeled medium CS by TGF-beta 1. The addition of TGF-beta 1 also resulted in a biphasic effect on cell growth that depended on the plating density, but cell growth differences could not account for the marked stimulation of CS synthesis by TGF-beta 1. Experiments with cycloheximide and beta-xyloside and isolation of the intact anionic glycoconjugates (AG) indicated that although synthesis of core protein was the limiting factor in CS synthesis, TGF-beta 1 stimulated the synthesis of CS chain when sufficient beta-xyloside acceptor was available. The overall results suggest that, in this cell system, the action of TGF-beta 1 on the synthesis of the major extracellular AGs is characterized by a relatively specific upregulation of CS proteoglycan (PG) synthesis and an uncoupling of the inhibitory effect of high cell density on CS PG synthesis.

Animals↗

PubMed1981

The role of glycoconjugates in metastatic melanoma blood-borne arrest and cell surface properties.

The role of glycoconjugates in cell surface and blood-borne implantation properties of murine metastatic melanoma sublines of low (B16-F1) or high (B16-F10) potential to colonize lungs was investigated by treating melanoma cells with the antibiotic tunicamycin. This drug prevents glycosylation of glycoproteins by inhibiting the formation of lipid-linked oligosaccharide precursors. The degree of tunicamycin-mediated modifications in glycoproteins was assessed by monitoring the decrease in cell surface sialogalactoproteins by binding of 125I-labeled Ricinus communis agglutinin I. Scanning electron microscopy of tunicamycin-treated B16-F1 and B16-F10 cells showed morphologic changes such as cell rounding and formation of numerous surface blebs. Tunicamycin-treated B16-F1 and B16-F10 cells lost their lung colonization abilities when injected intravenously into C57BL/6 mice, concomitant with lowered rates of adhesion to endothelial cell monolayers, endothelial extracellular matrix (basal lamina), and polyvinyl-immobilized fibronectin in vitro, suggesting that this drug inhibits experimental metastasis by modifying the surface glycoproteins involved in determining the adhesive properties of malignant cells.

Animals↗

PubMed1986

Lectin histochemistry of galactose and N-acetyl-galactosamine glycoconjugates in normal gastric mucosa and gastric cancer and the relationship with ABO and secretor status.

The histochemical binding of four lectin-peroxidase conjugates to normal human gastric mucosa and gastric carcinoma is described. The lectins were peanut agglutinin (PNA) which is specific for galactose residues and soy bean agglutinin (SBA), Dolichos biflorus agglutinin (DBA) and Helix pomatia agglutinin (HPA) which are specific for N-acetylgalactosamine. Binding of PNA to surface mucous cells or normal gastric mucosa occurred in non-secretors but not secretors and was independent of ABO blood group at all sites. PNA binding was unrelated to the immunohistochemical demonstration of Thomsen-Friedenreich (T) antigen. DBA and HPA bound selectively to surface mucous cells in normal gastric mucosa from group A secretors but binding at other sites was independent of ABO status. SBA binding showed no relationship with blood group or secretor status. In gastric cancers the major finding was the occurrence of extensive masking of lectin binding sites by sialic acid which was not seen in normal mucosa. Sialic acid masking was most marked with PNA and least marked with DBA. There was no correlation between lectin binding patterns and the stage or differentiation of tumours. Results are consistent with in vitro studies demonstrating increased sialation of membrane glycoproteins following malignant transformation. Difficulties in interpreting the histochemical demonstration of lectin binding in terms of specific glycoconjugates are discussed.

ABO Blood-Group System↗

PubMed1986

Distribution of glycoconjugates in normal rat ventral prostate and their use as markers of androgen-controlled secretory function in culture.

The distribution of glycoconjugates in uncultured and cultured rat ventral prostate was studied by using eight fluorescent lectins. The prostate pieces were cultured in defined medium with or without testosterone for 1-14 days. Each lectin revealed a characteristic binding pattern. Con A, LCA, WGA, and RCA I stained both epithelial and interstitial components. SBA and PNA were specific for the epithelium: SBA stained the region of the Golgi complex; PNA showed the brightest fluorescence in the apical part of the cells representing the region of secretory granules. In culture without testosterone the epithelial cells gradually lost their fluorescence, whereas the stromal fluorescence increased. The basement membrane was disorganized. With testosterone the integrity of the epithelium and stroma was maintained, and the staining pattern of the lectins was in the main similar as in vivo. However, at 14 days a change in the staining pattern of apical cytoplasm with PNA was noted, indicating that in long-term cultures, in addition to testosterone, other hormones and growth factors are necessary to complete especially the last stages of the secretory process in the epithelial cells.

Animals↗