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PubMed1986

In situ localization of lectin-binding glycoconjugates in the matrix of growth-plate cartilage.

In the distal hypertrophic zone of growth-plate cartilage, the pericellular matrix surrounding individual chondrocytes and the territorial matrix uniting chondrocytes into columnar groups are invaded by metaphyseal endothelial cells prior to osteogenesis. In the present study, lectin-binding glycoconjugates were analyzed in these two matrix compartments of growth-plate cartilage from Yucatan swine. Nine lectin-fluorescein conjugates were tested by a postembedment method on 1-micron-thick, nondecalcified, Epon-embedded sections. Chondrocytes in all cellular zones were surrounded by a pericellular matrix which showed positive binding for peanut agglutinin (PNA), ricin agglutinin (RCA-I), and soybean agglutinin (SBA). Binding by these lectins was sensitive to digestion with hyaluronidase, chondroitinase, and trypsin. Pericellular glyconconjugtes that bind RCA-I and concanvalin A (CONA) after periodic acid oxidation, and which were sensitive to trypsin but not to chondroitinase or hyaluronidase, were present in the hypertrophic cell zone. Within the territorial matrix, binding of lectins specific for galactose, N-acetylgalactosamine, and fucose showed gradients of intensity which became maximal at the last transverse septum. Lectin-binding histochemistry more precisely differentiated the microheterogeneity of glycoconjugate distribution within these two matrix compartments than has been possible with other histochemical techniques. Lectin-binding affinity is a potentially useful technique by which to isolate cartilage matrix macromolecules unique to specific cellular zones of the growth plate.

Animals↗
PubMed1987

Histochemical evaluation of glycoconjugates in the male reproductive tract with lectin-horseradish peroxidase conjugates: II. Staining of ciliated cells, basal cells, flask cells, and clear cells in the mouse.

Efferent reproductive ducts of male mice, including ductuli efferentes, epididymis, and vas deferens, were fixed and embedded in paraffin, and sections were stained with a battery of lectin-horseradish peroxidase conjugates to localize specific sugars or sugar sequences in glycoconjugates. Cilia and the apical surfaces of ciliated cells in the ductuli efferentes stained intensely with lectin specific for sialic acid and terminal alpha-N-acetyl-D-galactosamine. Flask cells and clear cells in the epididymis reacted positively and similarly with most lectins used, providing evidence that these cell types are related. In contrast, disparities in lectin staining suggest that flask cells and clear cells are a cell type distinct from principal cells. Basal cells were not present in the ductuli efferentes but formed a continuous layer in the epididymis and vas deferens. Basal cells contained oligosaccharides terminated by sialic acid and alpha-D-galactose and varying amounts of terminal beta-D-galactose and alpha-N-acetyl-D-galactosamine. Basal cells also stained variably with lectins specific for the core region of complex type N-glycosidic side chains. The basal cells varied structurally, having long spinous apical processes approaching or reaching the lumen in region I of the epididymis and being low cuboidal or squamoid and lacking apical processes in epididymal regions II-V and in the vas deferens. The contiguous nature of the basal cells and the presence of glycoconjugates bearing terminal alpha-galactosyl residues in all basal cells suggest a possible role for these cells in a regulatory influence on transepithelial movement of fluid and/or ions in the epididymis and vas deferens.

Animals↗
PubMed1993

The spectrum of immune responses to Campylobacter jejuni and glycoconjugates in Guillain-Barré syndrome and in other neuroimmunological disorders.

An acute infectious illness frequently precedes the Guillain-Barré syndrome. Recently, Campylobacter jejuni was claimed to be a predominant precipitating agent that may also trigger a humoral immune response to glycoconjugates of peripheral myelin in Guillain-Barré syndrome. Because of conflicting reports, we determined the frequency of a recent infection with C. jejuni in 38 patients with Guillain-Barré syndrome using a highly sensitive and specific immunoblot technique, and of the presence of circulating antibodies to gangliosides. We detected IgM and/or IgG C. jejuni directed antibodies in 15 of 38 patients with Guillain-Barré syndrome. In contrast, only 7 of 39 healthy control subjects, 3 of 20 patients with multiple sclerosis, and 2 of 72 patients with neuroborreliosis showed IgA or IgM antibody responses to C. jejuni. In Guillain-Barré syndrome, C. jejuni-specific antibodies were predominantly directed to outer membrane proteins of one specific serotype, Lior 11, whereas the most common serotype associated with enteritis in Germany is Lior 4. Two of 27 patients with Guillain-Barré syndrome had ganglioside-specific IgA antibodies; 1 of 32 patients, antibodies of IgM; and 4 of 31 patients, antibodies of IgG class. There was no correlation between severity, type (axonal versus demyelinating), and outcome of the disease and the presence or absence of a humoral immune response to C. jejuni or to glycoconjugates. Our findings do not support previous suggestions that a preceding C. jejuni infection heralds a poorer outcome or that antibodies to gangliosides carry prognostic significance.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Postnatal development and localization of an N-acetylgalactosamine containing glycoconjugate associated with nonpyramidal neurons in cat visual cortex.

We have analyzed the appearance of N-acetylgalactosamine containing glycoconjugates by staining sections of fixed cat visual cortex with the lectins from Vicia villosa (VVA) and Glycine max (SBA) conjugated to fluorescent labels, horseradish peroxidase, or biotin. The appearance of lectin staining during postnatal development followed an inside-out gradient starting in the deep cortical layers at the fourth postnatal week, successively including more of the superficial layers during the first three postnatal months until labelled cells were present throughout layers II-VI at the seventh postnatal month. Staining was associated with the soma and proximal dendrites of bipolar and multipolar nonpyramidal neurons. Outside layer IV large neurons with basket cell and neurogliaform morphology predominated, whereas small multipolar cells were stained in layer IV. High power observation revealed a lattice-like staining on neuronal surfaces. In the cortical white matter staining was found at the nodes of Ranvier. The ultrastructural localization of lectin binding sites was assessed by a pre-embedding histochemical procedure with biotinylated lectin visualized with HRP-conjugated avidin and diaminobenzidine as the chromogen. The reaction product was found exclusively in close association with synaptic terminals on somata and proximal dendrites of nonpyramidal neurons. There was no preference with respect to the morphology of the synaptic structure, i.e., symmetric synapses with ovoid or asymmetric synapses with round vesicles. In the vicinity of synaptic contacts the staining was associated with membranes of astrocytic processes ensheathing the synapses. In the cortical white matter astrocytic processes running along myelinated axons were strongly labelled at the nodes of Ranvier. Comparison of Western blots from 4-week-old and adult cat cortex membranes revealed one lectin-positive protein band with an apparent molecular weight of about 24 kD in conjunction with the histochemical expression of perisynaptic staining in the adult tissue. It is concluded that the employed lectins selectively visualize distal astroglial processes ensheathing synapses on a subpopulation of cortical interneurons and nodes of Ranvier. On the basis of the developmental appearance and localization we consider and discuss the possibility that the N-acetylgalactosamine containing glycoconjugate is involved in the stabilization of synaptic contacts on GABA-ergic interneurons.

Acetylgalactosamine↗
PubMed1987

An autoradiographic analysis of N-linked glycoconjugates in embryonic salivary gland morphogenesis.

The synthesis, deposition, and loss of mannose-bearing glycoconjugates during branching morphogenesis of embryonic mouse salivary glands has been evaluated. Day 13 embryonic mouse salivary glands were cultured for 44 hr, pulse labeled 4 hr with [3H]mannose, then fixed after 0, 2, 4, 8, or 24 hr of chase in nonradioactive medium, and processed for autoradiography. Light microscopic autoradiograms of sectioned rudiments reveal extensive label within the epithelium, little label over the mesenchyme, and a concentration of radioactivity at the basal surface of the epithelium. Autoradiograms of "chased" rudiments reveal a) no detectable loss of label from the epithelium or the basal epithelial surface over the first 8 hr, and b) significant label loss by 24 hr of chase at the basal epithelial surface, while moderate amounts of radioactivity remain throughout the rest of the epithelium. The [3H]bound material is insensitive to chondroitinase ABC, a glycosaminoglycan degradative enzyme, but is sensitive to tunicamycin presence in the culture medium. Earlier studies showed that embryonic mouse salivary glands cultured in medium containing tunicamycin (25 ng/ml) continued normal epithelial branching while epithelial growth was inhibited. The present autoradiographic studies of [3H]mannose-labeled rudiments demonstrate that tunicamycin causes a significant decrease in radioactivity, relative to controls. Thus, our results suggest that epithelial branching activity is independent of control levels of mannose-containing/tunicamycin-sensitive, glycoconjugate deposition.

Animals↗
PubMed1987

Fucosylated glycoconjugates appear on mouse embryos during blastocyst formation.

Mouse embryos were analysed for expression of surface glycoconjugates using a panel of fluorescein-labelled lectins. Morulae and blastocysts but not early cleavage stages stained brightly with fucose binding lectins. By contrast, cleavage stages stained brightly with all the other lectins tested. These findings provide evidence for substantial reorganisation of the cell surface during blastocyst formation and are consistent with a role for fucosylated glycoconjugates in compaction.

Animals↗
PubMed1977

Distribution of glycoconjugates in mouse fibroblasts with varying degrees of tumorigenicity.

Analysis of glucosamine labeled glycoconjugates in cultured cells has been made comparing 2 clones and the parent embryonic mouse cell line. Hyaluronic acid, heparan sulphate, and chondrotin sulphate as well as a complex mixture of glycopeptides were found in the medium, the trypsinate, and the trypsinized cells, although the distribution was not uniform. The 3 cell lines had very similar in vitro growth properties, including their plating efficiency in viscous medium. However, the tumorigenicity of the cells, determined syngeneic mice, was found to differ. All 3 cell lines were found to have similar glycoconjugate distributions, although a slight relative increase in labeled hyaluronic acid was found in the more tumorigenic mass cell line than either of the clones. The possible significance of this increase is discussed.

Animals↗
PubMed1989

Characterization of protein-linked glycoconjugates produced by identified neurons of Aplysia californica.

The biosynthetic capabilities of individual neurons of the abdominal ganglion of the marine mollusc Aplysia californica have been analyzed after intrasomatic injection of 3H-monosaccharides. Glycopeptides prepared from the metabolically labeled cells were fractionated using serial lectin affinity and gel filtration chromatography. The fractionation procedure yielded eight populations of glycopeptides, and comparison of two different neurons (R2 and R14) showed that the quantity of the individual species produced is cell-dependent. Structural analysis indicated that the glycoconjugates produced by the Aplysia neuron constitute both O- and N-linked structures as well as an unusual class of oligosaccharide whose linkage to protein is unknown. The O-linked units are small and consist only of N-acetylglucosamine or N-acetylgalactosamine attached to protein. High-mannose-type asparagine-linked units are produced by the neurons, and some of these appear to be processed to biantennary complex-type units that bind to lentil lectin-agarose. Overall, although the Aplysia neurons produce oligosaccharides of a nature similar to that produced by higher eucaryotes, the N- and O-linked structures produced by the neurons do not achieve the complexity of the comparable structures produced by mammalian cells. The results provide a basis for further studies aimed at understanding the role of glycoconjugates in the development of the nervous system.

Animals↗
PubMed1995

Conjugation in the ciliate Euplotes octocarinatus: comparison of ciliary and cell body-associated glycoconjugates of non-mating-competent, mating-competent, and conjugating cells.

Pair formation in the hypotrichous ciliate Euplotes octocarinatus is a poorly understood phenomenon. In order to obtain information about the molecules involved in this process, we compared ciliary and cell body-associated glycoconjugates of non-mating-competent, mating-competent, and conjugating cells. Detection of glycoconjugates was carried out on Western blots by immunostaining of oxidized, digoxigenin-labeled carbohydrate moieties. Using this method, in both of two complementary mating types a 130-kDa glycoprotein was identified, which appeared on cilia during acquisition of mating competence and was reduced during cell pairing. This suggests an active role of this glycoprotein in ciliary adhesion during pair formation. Additionally, in both of the two mating types a cell body-associated 135-kDa glycoprotein was detected, which is present in non-mating-competent cells as well as in mating-competent cells, but is strongly reduced in conjugating cells. In contrast to the ciliary 130-kDa glycoprotein, the cell body-associated 135-kDa glycoprotein is not surface-exposed [8]. We therefore propose that the cell body-associated glycoprotein is either involved in the preparation for cell fusion or meiosis or that it serves as a cytoplasmic pool for the ciliary 130-kDa glycoprotein.

Animals↗
PubMed1999

Possible gonadotropin cells in the lamprey pituitary: colocalization of mammalian LH-like immunoreactivity and glycoconjugate in adult sea lampreys (Petromyzon marinus).

In lampreys, although gonadotropin (GTH) has not yet been isolated from the pituitary gland, the presence of GTH has been strongly suggested. To detect possible GTH in the sea lamprey (Petromyzon marinus) pituitary, two different cytochemical probes were tested: One was the use of antibodies to GTHs, and the other was the use of lectin-screening kits for demonstration of glycoconjugate in hormonal molecules. GTH-like immunoreactivity was found in cells distributed in the ventral half of the proximal pars distalis. These cells were stained intensely by all four lots of anti-ovine LH including LHbeta, and were stained moderately or weakly by several other antibodies to LH-related GTHs, such as human LHbeta, hCGbeta, amphibian LH, and sturgeon GTH IIbeta. On the other hand, there were no positive reactions in the sea lamprey pituitary using the antibodies to FSH-related GTHs, thyrotropin (TSH), or pituitary glycoprotein hormones of teleost origin. Thus, GTH-like material in the sea lamprey pituitary seems to be more closely related to mammalian-like LH, rather than to FSH or TSH, as far as immunocytochemical determinants. A total of 21 kinds of lectins was tested. Among those, GTH-positive cells were also stained positively by concanavalin A and Vicia villosa agglutinin. Thus, the present study demonstrates colocalization of LH-like immunoreactivity and glycoconjugate in cells in the ventral half of the proximal pars distalis of the sea lamprey pituitary. It is suggested that those cells are most likely to be GTH cells in the sea lamprey pituitary.

Animals↗
PubMed1992

Distribution of type I collagen, type II collagen and PNA binding glycoconjugates during chondrogenesis of three distinct embryonic cartilages.

Previous studies of chondrogenesis have been focused on limb bud cartilage, whereas little is known about chondrogenic processes of other cartilages with different developmental fates. We hypothesize that cartilages with various developmental fates might show identical characteristics of chondrogenesis. The chondrogenic processes in the nasal septum, the mandible, and the limb bud of the mouse were examined by means of PNA-binding glycoconjugate, and types I and II collagen expression. Swiss-Webster mouse embryos of 11 days (E11) to 14 days (E14) gestation were fixed and processed for immuno- and lectin histochemistry. The blastema of mesenchymal cell aggregates stained positively with anti-type I collagen, but very weakly with anti-type II collagen in all three models at E12, whereas PNA bound to the blastema in the limb bud but not in nasal septum or mandible. Types I and II collagens coexisted in cartilages at E13. Type II collagen was predominant in E14; type I collagen was confined to the peripheral region. The synchronized transitional expression of the collagen phenotypes in all three embryonic cartilages may be systemically regulated. The presence or absence of the PNA-binding glycoconjugates may be involved in characterizing the nature of the cartilages.

Animals↗
PubMed1990

Detection and mapping of endogenous receptors for carrier-immobilized constituents of glycoconjugates (lectins) by labelled (neo)glycoproteins and by affinity chromatography in human adult mesencephalon, pons, medulla oblongata and cerebellum.

Different carrier-immobilized carbohydrate moieties were employed as tools to detect respective binding sites glycohistochemically and glycobiochemically. Besides ascertaining their presence the pattern of endogenous sugar receptors (lectins) in different regions of the human central nervous system was mapped to reveal any non-uniform expression. A strong and specific staining with biotinylated neoglycoproteins, exposing different sugar moieties as ligands, indicated the presence of sugar receptors in the nuclei, neuronal pathways and accessory structures such as ependyma cells, plexus chorioideus, intra- and extracerebral vessels and leptomeninges localized in the mesencephalon, in the pons, in the medulla oblongata and in the cerebellum. Significant differences were seen for various neuroanatomical regions like nerve cells in the basal and central regions of the nuclei pontis in the glycohistochemically detected level of expression of endogenous sugar receptors (lectins). The used approach with carbohydrate constituents of cellular glycoconjugates as ligands in search of specific receptors complemented studies on the localization of glycoconjugates with sugar-specific tools like plant lectins. Exemplary glycobiochemical investigations on the medulla oblongata and cerebellum were performed to investigate the molecular nature of sugar receptors detected glycohistochemically. Despite notable overall similarities, carbohydrate-binding proteins of differing molecular weight can be isolated from these two regions of the central nervous system, namely in the case of receptors with specificity to beta-galactoside termini, to N-acetyl-D-galactosamine and N-acetyl-D-glucosamine and to D-xylose. These combined glycohistochemical and glycobiochemical results serve as a guideline for exploring the physiological relevance of the detected regional differences.

Adult↗
PubMed1993

Immunocytochemical approach to the structure of human blood group B and H glycoconjugate antigens in the three days old rat cochlea.

The possible structure of human blood-group antigens, as found in cochlear hair cells of 3-day-old rats, is suggested. Data were obtained from immunocytochemical studies using 77 antibodies against the major human blood group antigens of the ABO, H, I and Lewis genetic systems. Neither the anti-A-related nor the anti-Lewis-related antibodies showed any positive immunoreaction on hair cells. In contrast, anti-B, anti-AB and anti-H antibodies displayed specific positive immunoreactive patterns on the hair cells. The results suggest that, in immature hair cells, two main glycoconjugate structures of the lactoseries are present: H type 2 antigen, which is the precursor of the B type 2 antigen, and the B type 2 antigen itself. Similar H and B carbohydrate structures have been reported in rat olfactory receptors. The type 2 glycoconjugates carrying these H and B antigens of auditive and olfactory receptors are resistant to fixation and paraffin embedding, suggesting that they might be glycoproteins. These auditive and olfactory H and B antigens must be different from the B-related antigens that are expressed by pseudo-unipolar neurons of rat posterior root ganglia, that are built from type 4 core chains, and that are destroyed by routine paraffin embedding procedures.

ABO Blood-Group System↗
PubMed1986

Glycoconjugate with terminal alpha galactose. A property common to basal cells and a subpopulation of columnar cells of numerous epithelia in mouse and rat.

Glycoconjugates associated with the basal cell layer of various types of epithelia in the mouse and rat were examined histochemically with a battery of lectin-horseradish peroxidase (HRP) conjugates of differing sugar binding specificities. Basal cells in paraffin sections of composite tissue blocks stained with an isolectin from Griffonia simplicifolia (GSA I-B4) specific for terminal alpha-galactose residues but failed to react with the other lectins. Basal cells in epithelium lining striated and excretory ducts of salivary and lacrimal glands, tongue, esophagus, trachea, renal calyx, ureter, urinary bladder, urethra, epididymis and vas deferens stained selectively and intensely for content of a glycoconjugate with terminal alpha-galactose. This galacto-conjugate appeared associated with the plasmalemma of basal cells. Basal cells with a galactocalyx formed an intermittent to continuous layer generally increasing in prevalence distally in glandular duct systems. A minor population of pyramido-columnar cells with cytosolic GSA I-B4 reactivity occurred in striated ducts and appeared less numerous in intralobular excretory ducts and more prevalent in extraglandular ducts. In trachea and renal pelvis, the GSA I-B4 positive cell profiles ranged from low cuboidal to tall pyramidal in contour, but the latter appeared not to reach the lumen. In contrast, no GSA I-B4 positive basal cells were seen in any segment of the pancreatic or bile ducts or in the epithelium of the gastrointestinal tract. These findings suggest that the basal cells found in similar sites in different epithelia and possessing in common a unique alpha-galactoconjugate may function in a manner common to all and not simply in providing progenitor cells for epithelial renewal.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1975

The histochemical application of dansylhydrazine as a fluorescent labeling reagent for sialic acid in cellular glycoconjugates.

A new method for the fluorescent staining of stalic acid-containing glycoconjugates in fixed tissues is described. The procedure uses mild periodate oxidation, followed by condensation with dansylhydrazine and reduction of the hydrazones to hydrazines. The specificity of the reaction for sialic acid is tested on model glycoconjugates. The procedure gives superior resolution in comparison to the standard periodate Schiff procedure for cellular carbohydrates.

Animals↗
PubMed1987

Evaluation of structural and secretory glycoconjugates in normal human jejunum by means of lectin histochemistry.

The labelling pattern of eight lectins was studied in jejunal samples from ten normal subjects, in order to define the normal distribution of structural and secretory glycoconjugates in the small bowel. The following lectins were studied by means of a peroxidase technique on formalin-fixed samples: Arachis hypogaea, Ricinus communis, Canavalia ensiformis, Lens culinaris, Phaseolus vulgaris, Triticum vulgaris, Ulex europaeus, Dolichos biflorus. Phaseolus vulgaris reacted with goblet cell mucus throughout the villus-crypt axis. Conversely Ulex europaeus, Dolichos biflorus and Triticum vulgaris lectin labelling of goblet cells appeared to be confined to the upper part of the villi. This finding suggests that during cell migration from crypt to villus tip, the continuing maturation of goblet cells is associated with the differentiation of secretory carbohydrates, which probably parallels the cell maturation cycle. Lectin histochemistry appears to be a reliable tool for the study of structural and secretory glycoconjugates in the jejunal mucosa, and might be of value in the study of diseases in which the cell-maturation cycle in the small bowel is altered.

Concanavalin A↗
PubMed1987

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 host-parasite interactions is discussed.

Animals↗
PubMed1976

[Disturbed distribution of surface glycoconjugates of the plasma cell membrane in psoriasis. Cytochemical detection using the alcianblue-lanthan-reaction (author's transl)].

Lanthanum has been used as a tracer for the intercellular space and the surface coats in different epithelia including human epidermis, however, lanthanum compounds may or may not penetrate into skin specimes and reaction product may or may not be present in the intercellular space. After treatment with alcian blue prior exposure to lanthanum the trilaminar plasma membrane was more regularly stained and a thin coat of reaction product corresponding to superficial glycoconjugates outlined the cell surface and filled the narrow intercellular space in normal epidermis. In psoriatic epidermis the quantity of superficial membrane-bound glycoconjugates was rather reduced, their distribution and compartmentalization was profoundly changed and the outer leaflet of the trilaminar plasma membrane was not always clearly seen, having a "moth-eaten appearance" in some places. These findings show once more that alterations of the cell membranes and their surface architecture are present in psoriasis. They may play a major role in the pathogenesis of the disease, influencing the activities of membrane-bound enzymes and the flow of the epidermal growth control mechanism. The glycocalyx may serve in this mechanism as an accumulator of energy rich compounds or as a receptor site of membrane-bound ezymes. According to our views it represents a "ground substance" of epidermal origin, which controls the social behaviour of the keratinocytes in tissue.

Adult↗