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PubMed1985

The acrosomal membrane of boar sperm: a Golgi-derived membrane poor in glycoconjugates.

The acrosome is a large secretory vesicle of the sperm head that carries enzymes responsible for the digestion of the oocyte's investments. The event leads to sperm penetration and allows fertilization to occur. Release of the acrosomal enzymes is mediated by the interaction between sperm acrosomal and plasma membranes (acrosome reaction). Biochemical characterization of the acrosomal membrane has been restrained by a lack of methods to isolate uncontaminated fractions of the membrane. Here, we use new methods to expose the membrane to in situ cytochemical labeling by lectin-gold complexes. We study the topology and relative density of glycoconjugates both across and along the plane of the acrosomal membrane of boar sperm. Detachment of the plasma membrane from glutaraldehyde-fixed cells exposed the cytoplasmic surface of the acrosome to the lectin markers; freeze-fractured halves of the acrosomal membrane were marked by "fracture-label" (Aguas, A. P., and P. Pinto da Silva, 1983, J. Cell Biol. 97:1356-1364). We show that the cytoplasmic surface of the intact acrosome is devoid of binding sites for both concanavalin A (Con A) and wheat germ agglutinin (WGA). By contrast, it contains a high density of neuraminidase-resistant anionic sites detected by cationic ferritin. On freeze-fractured sperm, the receptors for Con A partitioned with the exoplasmic membrane half of the acrosomal membrane. The Con A-binding glycoconjugates were accumulated on the equatorial segment of the membrane. A low density of WGA receptors, as well as of intramembrane particles, was found on the freeze-fracture halves of the acrosomal membrane. The plasma membrane displayed, in the same preparations, a high density of receptors for both Con A and WGA. We conclude that the acrosome is limited by a membrane poor in glycoconjugates, which are exclusively exposed on the exoplasmic side of the bilayer. Regionalization of Con A receptors on the acrosome shows that sperm intracellular membranes, like the sperm surface, express domain distribution of glycocomponents.

Acrosome↗
PubMed1995

Sialyl-Tn-KLH, glycoconjugate analysis and stability by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD).

The quantitation of sialyl-Tn (STn) conjugated to keyhole limpet haemocyanin (KLH) can be determined by quantitating the amount of N-acetylneuraminic acid (NANA) released by acid or enzymatic digestion. An optimal 0.1 N H2SO4 acid hydrolysis at 80 degrees C results in quantitative release of NANA with minimal loss. A rapid isocratic method for the quantitation and separation of NANA is described using high-pH anion-exchange chromatography and pulsed amperometric detection (PAD). Multiple injection of NANA standard and/or samples containing protein led to a decrease in the PAD response which was corrected by addition of internal standard, alpha-2-keto-3-deoxyoctonate (KDO). The ratio of NANA/KDO peak area or peak height gives a linear response with increasing amount of NANA in the range 2.5-20 micro g/ml (r2 = 0.99). The limit of quantitation (LOQ) for NANA using this isocratic method is 1.9 micro g/ml (approximately 160 pmol/25 micro l injection). Based on the multiple determination the glycoconjugate, STn-KLH, showed a NANA content of 2.9% (w/w). Acid hydrolysis and the sialidase treatment of STn-KLH both yielded a similar NANA content. The carrier protein, KLH, showed the absence of NANA. The stability of glycoconjugate STn-KLH was monitored by a gradient method which separated possible degradation products STn-crotyl, NANA and GalNAc. Subjecting the glycoconjugate STn-KLH to various stress conditions of temperature, pH and oxidation does not result in any release of sialic acid, GalNac and STn-crotyl group.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed1994

The effect of cell confluency state on the expression of neural cell surface glycoconjugates.

The expression of neural cell surface glycoconjugates during development reflects the precise control of cellular adhesivity that is required for the exact positioning of the developing cells. We have investigated the effect of cell confluency state on the expression of key cell surface glycoconjugates using the mouse neuroblastoma cell line, neuro-2A. There was an up-regulation of expression of the neural cell adhesion molecule (NCAM) coincident with an increase in cell confluency state and a parallel decrease in the expression of the amyloid precursor protein, beta APP. The expression pattern of cell surface glycoconjugates in neuro-2A cells is therefore similar to that observed during the early embryonic period of development during which cells aggregate to form collectives.

Amyloid beta-Protein Precursor↗
PubMed1986

The glycoconjugate derived from a Leishmania major receptor for macrophages is a suppressogenic, disease-promoting antigen in murine cutaneous leishmaniasis.

Lymphoid cells from genetically-susceptible BALB/c mice immunized against a glycoconjugate of the protozoan parasite, Leishmania major, promote chronic cutaneous disease in BALB/c nude mice. This cell population therefore differs from cells harvested from non-immunized BALB/c mice that are known to be potent mediators of protection against cutaneous leishmaniasis in minimally-reconstituted, syngeneic nude mice. The glycoconjugate when injected into genetically-resistant C57BL/6 mice will increase the size and persistence of cutaneous lesions. Recent studies have established that the water soluble glycoconjugate is derived from a membrane-bound glycolipid that is a receptor used by the parasite in the attachment to macrophages. This glycolipid can protectively immunize mice against cutaneous leishmaniasis. Identification of a vaccinating glycolipid antigen and a suppressogenic component derived from it will greatly facilitate analysis of disease-promoting and resistance-promoting immunity in cutaneous leishmaniasis. However, the fact that a host-protective antigen contains a disease-promoting component may militate against the immediate use of this molecular vaccine in man.

Animals↗
PubMedDate not supplied

Distribution of glycoconjugates in human testis. A histochemical study using fluorescein- and rhodamine-conjugated lectins.

Differentiation in the seminiferous epithelium involves the orderly transformation of germ cells into spermatozoa. We have employed ten fluorescein- and rhodamine-labeled lectins to visualize distinctive changes in the distribution of carbohydrate containing compounds during spermatogenesis and noticed the increase in RCA I, PNA, SBA and HPA binding sites during germ cell differentiation, suggesting the appearance of certain galactose and N-acetylgalactosamine containing glycoconjugates. Besides, in the cytoplasm of all germ cell types the positive reactions with Con A, LCA, WGA, LPA and UEA I indicate the presence of mannose, N-acetylglucosamine, sialic acid and fucose containing glycosubstances. Developing acrosomes demonstrated binding sites for most lectins, and particular HPA binding glycoconjugates were expressed in the equatorial segment region of late spermatids and testicular spermatozoa. In addition, the characteristic staining patterns of other testicular compartments are described. Our results suggest that human germ cells are rich in various carbohydrate containing compounds and there are specific alterations in cellular glycoconjugates during germ cell differentiation.

Acrosome↗
PubMed1987

Myelin-associated glycoprotein and related glycoconjugates in developing cat peripheral nerve: a correlative biochemical and morphometric study.

The expression and accumulation of the myelin-associated glycoprotein (MAG) and other glycoconjugates have been studied during myelination in the developing cat peripheral nervous system. The glycoconjugates studied have in common a similar carbohydrate determinant which is bound by many antibodies, including the mouse monoclonal antibody HNK-1, and human IgM paraproteins from patients with neuropathy. In addition to MAG, the reactive glycoconjugates include a 60-kilodalton (kD) glycoprotein and a group of 20-26 kD glycoproteins, as well as a group of recently identified acidic glycolipids, the major one of which is sulfate-3-glucuronyl paragloboside (SGPG). The accumulation of these glycoproteins and glycolipids is compared with the established myelin proteins P0, P1, and P2 and with morphometric indices of myelin volume and axonal perimeter. The study demonstrates that MAG appears and accumulates very early during myelination, being present at 15% of the maximum level prior to the appearance of P0, and at 80% of the maximum level when P0 is at 30% of its maximum level. In the adult, the level of MAG falls to 60% maximum. The 60 kD and 20-26 kD glycoproteins accumulate at the same time as or later than P0, suggesting that they are either compact myelin proteins or in membranes closely associated with compact myelin. SGPG accumulates with P0 early in myelination, but falls to 60% of maximum in the adult. By comparing biochemical and morphometric data, we demonstrate that P0 and other compact myelin proteins accumulate synchronously with the increase in myelin area. MAG accumulation, however, is closely related to changes in axonal perimeter, consistent with a predominant localization of MAG to the periaxonal membranes in the peripheral nervous system.

Aging↗
PubMed1983

Association of glycoconjugates with the cytoskeletal framework.

The association of glycoconjugates with the cytoskeletal framework was examined in detergent-extracted cells. Sparse cultures of fibroblasts that assemble only minimal amounts of extracellular matrix were extracted under mild conditions with Triton X-100 which remove most of the lipids and soluble cellular proteins. The detergent-resistant framework retains lectin binding sites in the nucleus, in the perinuclear area occupied by the rough endoplasmic reticulum-Golgi system of the intact cell, and in a network throughout the cytoskeletal framework. Fluorescent-antibody staining with antibody against collagen type I and fibronectin reveals extensive perinuclear staining of the remnant rough endoplasmic reticulum-Golgi system. In contrast, only sporadic staining of the pericellular area is obtained with these antibodies, in sparse cultures of whole cells. Lectin binding sites were detected in the nucleus and are attributed to chromatin-associated glycoconjugates. They can be removed by DNase under conditions that preserve the cytoplasmic lectin binding sites and the nuclear matrix. The results suggest a high degree of integration of the membrane residues of the cytoplasmic elements and the nuclear matrix with the skeletal framework and indicate a possible role for the glycoconjugates in this structural integration.

Animals↗
PubMed1984

Abnormal goblet cell glycoconjugates in rectal biopsies associated with an increased risk of neoplasia in patients with ulcerative colitis: early results of a prospective study.

A group of 18 patients with stable ulcerative colitis involving the entire colon for at least eight years was subjected to a biopsy of normal appearing rectal mucosa and followed prospectively over four years for the development of either dysplasia or cancer. Goblet cell glycoconjugate structure was examined in the rectal biopsies using fluorescein conjugated lectins. At the beginning of the study, 13 of the 18 patients had abnormalities of goblet cell mucin or cytoplasmic glycoconjugates in the rectal biopsies. Dysplasia subsequently developed in six and carcinoma in one of these patients. Among the five patients with normal lectin binding studies in the initial rectal biopsies, colonic dysplasia has subsequently developed in one. The abnormalities seen in the rectal goblet cells resembled in part those previously seen in immature and neoplastic colonic cells. The dysplastic tissues all contained the form of mucin which has been found in other neoplastic colonic tissues. This preliminary report after four years of prospective study suggests that abnormalities of glycoconjugate structure may be associated with, and may precede, neoplastic events in the setting of chronic ulcerative colitis.

Colitis, Ulcerative↗
PubMed1988

Lectin-gold labeling of glycoconjugates in normal and Brattleboro rat papilla: effect of vasopressin.

Some reports suggest that the plasma membrane glycocalyx of collecting duct epithelial cells, as well as interstitial glycoconjugates, may be involved in vasopressin action and urinary concentration. In view of this, we have used the lectin-gold technique to map and quantify Helix pomatia lectin (HPL)-binding sites in the inner medulla of kidneys from normal Long-Evans rats, vasopressin-deficient Brattleboro rats, and Brattleboro rats treated for up to 5 wk with exogenous vasopressin. The results show that the labeling of epithelial cell plasma membranes from collecting ducts and thin limbs of Henle is not different between normal and Brattleboro rats, and the labeling is not modified by chronic vasopressin treatment. In contrast, the heavy interstitial labeling seen in normal rats is virtually absent from Brattleboro rats, but it is progressively restored by chronic vasopressin treatment of Brattleboro rats. These results show that vasopressin does not modify HPL-binding glycoconjugates on epithelial cell plasma membranes, but that vasopressin treatment has a major effect on HPL-binding glycoconjugates in the medullary interstitium.

Animals↗
PubMed1987

Postnatal appearance of glycoconjugate with terminal N-acetylgalactosamine on the surface of selected neurons in mouse brain.

Selected neurons in the cortex and other regions of the brain of adult mice stain with Vicia villosa agglutinin (VVA) conjugated to horseradish peroxidase (HRP). This staining demonstrates glycoconjugate with terminal N-acetylgalactosamine (GalNAc) in periodic foci on the neuronal surface. However, brains from mice at 1 week of age lacked neurons with surface reactivity for VVA-HRP but showed neurons with faint cytoplasmic staining. Two weeks postnatally, infrequent cortical neurons located mostly in layer IV disclosed light surface affinity for VVA-HRP. The deep cerebellar nucleus at 2 weeks revealed VVA-positive neurons that were less abundant than in adults but exceeded in number the stained neurons in the cortex of 2-week-old mice. By 3 weeks of age the prevalence of positive neurons in cortical layers II-VI and the deep cerebellar nucleus and the intensity of the surface staining approximated that observed in adult mice. No staining of fiber tracts was observed 1 week postnatally. However, certain tracts exemplified by decussating fibers in the basal pontine region showed strong VVA-HRP reactivity in brains of 2-week-old mice as in adults. Acquisition of VVA surface affinity 3 weeks postnatally coincided with the appearance of cytosolic immunostaining for glutamic acid decarboxylase in a population of GABAergic neurons in the cortex. These findings demonstrate the postnatal development of GalNAc-containing glycoconjugate associated with selected neurons and the variable acquisition of the glycoconjugate among these neurons possibly related to onset of function.

Acetylgalactosamine↗
PubMed1987

Histochemistry of glycoconjugates in the secretory epithelium of the goat bulbourethral gland.

To investigate the histochemical nature of the secretory epithelium lining the goat bulbourethral gland, glycoconjugates contained in these secretory epithelial cells have been studied by means of light- and electron-microscopic histochemistry. The methods employed involved a series of conventional staining and peroxidase-labeled lectin diaminobenzidine procedures together with combined selective methods such as digestion with enzymes. According to the results obtained in the present work, the secretory cells of the goat bulbourethral gland can be grouped into two types: cells with glycoconjugate-rich granules and those with less amounts of glycoconjugates. The gland is at least dual in the nature of its secretory activities.

Animals↗
PubMed1983

Lectin-defined cell surface glycoconjugates of pancreatic cancer cells and their nonmalignant counterparts.

Alterations of cell surface carbohydrates of human pancreatic cancer cells from long-term cultures (COLO 357, RPMI 7451, PC 103, PC 107) were assessed ultrastructurally by use of an array of lectin-enzyme conjugates, and compared with lectin-defined changes of glycoconjugates on human pancreatic tissue sections of normal and various pathological conditions. Ulex europeus and, to a lesser degree, Lotus tetragonolobus lectin binding indicate that L-fucose-containing glycoconjugates are expressed predominantly on pancreatic cancer cell surfaces, but not, or restricted to intracytoplasmic structures, on nonmalignant pancreas cells. A comparable binding pattern to pancreatic carcinoma cells is found for Phaseolus vulgaris lectin. This is in contrast to the results with soy bean lectin, the reactivity of which was not restricted to cancer cell surfaces, and with Helix pomatia lectin, which did not bind to pancreatic cancer cells at all, although the latter three lectins possess similar sugar specificities. Between the long-term-cultured malignant pancreas cells differences were observed concerning the binding of wheat germ and pokeweed lectin. Besides, qualitative assets of lectin-binding absorption analyses elaborated quantitative differences in the expression of lectin-defined glycoconjugates on pancreatic cancer cell surfaces.

Carbohydrates↗
PubMed1985

Characterization of sulfated glycoconjugates in human gastric mucosa.

Radiolabeled glycoconjugates were prepared by incorporation of [35S]sulfate and [3H]glucosamine into the gastric mucosa of human gastric resection specimens, using organ culture techniques. Specimens which incorporated higher amounts of [35S]sulfate into high molecular glycoconjugates (Fr-1) were designated as group I. The average specific activity in Fr-1 of group I was 30 times that of group II, but the glucosamine uptake remained about the same in all cases. All specimens in group I were positive to intestinal metaplasia in their adjacent mucosal area. The sulfated glycoproteins in Fr-1 of group I were found to incorporate 80% of the 35S and the remaining 20% by sulfated glycosaminoglycans. But the glycoconjugates constituting all the Fr-1 of group II were identified as glycoproteins and incorporating 35S only to a small extent were identified as sulfated glycoproteins. The susceptibility of the glycoproteins from group I mucosa to pronase digestion was different from that of group II. The glycoproteins synthesized in group I mucosa were as sulfated glycoproteins as well as nonsulfated glycoproteins and glycosaminoglycans, while the group II glycoproteins consisted primarily of nonsulfated glycoproteins.

Adult↗
PubMed1976

Cytochemical localization of glycoconjugate in mitochondria.

Complex carbohydrates and cations have been localized by cytochemical methods in mitochondria of mammalian leukocytes, hepatocytes and oyster gill epithelium. Glycoconjugate of acidic nature was visualized with the dialyzed iron method in or on the outer membrane and inner boundary membrane and in the outer intermembrane space but not on the membranes of cristae or in the intracristate space, or matrix. Sulfated glycoconjugate was demonstrated with the high iron diamine method in a similar distribution and in, on, or between, membranes of cristae as well. Intermittent aggregates of dialyzed iron and high iron diamine stained material were often found in the outer intermembrane space. The periphery of isolated rat liver mitochondria also stained with a Concanavalin A horseradish peroxidase technique, indicating the presence of macromolecules, presumably glycoproteins containing mannose or glucose. The distribution of antimonate reactive cation in mitochondria of leukocytes resembled that of the acidic glycoconjugate, indicating binding of cations to anionic groups of the latter. The complex carbohydrates and cations demonstrated cytochemically in mitochondria are considered in relation to previous biochemical studies.

Animals↗
PubMed1982

Ultrastructural localization of acidic glycoconjugates with the low iron diamine method.

The present study has applied the low iron diamine (LID) method at the ultrastructural level to demonstrate acid glycoconjugates. We have examined rat epiphyseal cartilage, human bone marrow, rat tracheal glands, and mouse sublingual glands stained with LID prior to embedment. The LID staining appeared to require postosmication for adequate visualization at the electron microscope level. Thiocarbohydrazide-silver proteinate (TCH-SP) staining of thin sections variably enhanced LID reactive sites. LID-TCH-SP stained carboxyl and sulfate groups of glycosaminoglycans in the extracellular cartilage matrix, secretory granules, and expanded Golgi saccules of chondrocytes. In human bone marrow, LID-TCH-SP variably stained the cytoplasmic granules, known to contain sulfated glycosaminoglycans, and the external surface of the plasma membrane of leukocytes. Moderately strong LID staining was observed in secretory granules in mucous tubules of rat tracheal glands, known to contain sulfated glycoproteins, and in acinar cells of mouse sublingual glands, known to contain a sialoglycoprotein. The lack of sulfated glycoconjugates in acinar cells of the mouse sublingual gland was confirmed by their failure to stain with the high iron diamine method. Thus these studies indicate that the LID and LID-TCH-SP methods are useful for the ultrastructural localization of carboxylated and sulfated glycoconjugates in extracellular and intracellular sites.

Animals↗
PubMed1985

Histochemical localization of galactose-containing glycoconjugate at peripheral nodes of Ranvier in the rat.

Lectin-horseradish peroxidase conjugates were used to study glycoconjugates in paraffin sections of dorsal roots of the rat spinal cord. Griffonia simplicifolia-B4 isolectin (GSA I-B4) and peanut agglutinin (PNA) stained strongly the nodes of Ranvier, localizing, respectively, terminal alpha- and beta-D-galactose. Sialidase digestion did not increase staining with PNA at the node of Ranvier, suggesting the presence of a neutral glycoconjugate. Staining of the nodal but not the internodal axolemma was observed with PNA. The outer surface of the myelin sheath in axons of the dorsal root stained strongly with GSA I-B4 but only weakly with PNA, demonstrating an abundance of terminal alpha-galactose. PNA staining was enhanced in this site by sialidase digestion, showing terminal sialic acid-beta-galactose dimers. The presence of sialic acid here was further evidenced by labeling of these membranes with the lectin derived from the slug, Limax flavus (LFA). Affinity for a high iron diamine-Alcian blue (pH 2.5) sequence demonstrated, in addition, the presence of sulfate esters in glycoconjugates on the outer myelin membrane. GSA I-B4 imparted strong reactivity to nonmyelinated fibers in the dorsal root and the spinal nerve. The present findings appear to reflect several localizations of biochemically described nervous system glycoproteins containing O-glycosidically linked side chains terminated by alpha- and beta-D-galactose.

Animals↗
PubMed1998

Synthesis and antidiuretic activities of novel glycoconjugates of arginine-vasopressin.

Arginine-vasopressin (AVP) was acylated with various acyl azides (2a-j) in pH 9.1 buffer to give AVP derivatives (11a-j) modified at the tyrosine side chain with a carbohydrate via a spacer arm. Glycoconjugates of AVP modified at the N-terminal amide (12a-e) were also synthesized from AVP and carboxylic acids (3a-e) using dicyclohexylcarbodiimide and 1-hydroxybenzotriazole as coupling agent. Analogues (11a-j) exhibited greater in vivo antidiuretic activity than AVP. AVP and glycoconjugates (12a-e) were stable in rat plasma. On the other hand, glycoconjugates (11a-i) were found to readily convert to AVP according to first order kinetics. Hence, 11a-j are considered to be prodrugs of AVP.

Animals↗
PubMed2001

Glycoconjugate in rat taste buds.

The taste buds of the fungiform papillae, circumvallate papilla, foliate papillae, soft palate and epiglottis of the rat oral cavity were examined by lectin histochemistry to elucidate the relationships between expression of glycoconjugates and innervation. Seven out of 21 lectins showed moderate to intense staining in at least more than one taste bud. They were succinylated wheat germ agglutinin (s-WGA). Dolichos biflorus agglutinin (DBA), Bandeiraea simplicifolia lectin-I (BSL-I), Ricinus communis agglutinin-I (RCA-I), peanut agglutinin (PNA), Ulex europaeus agglutinin-I (UEA-I) and Phaseolus vulgaris agglutinin-L (PHA-L). UEA-I and BSL-I showed moderate to intense staining in all of the taste buds examined. They strongly stained the taste buds of the epiglottis, which are innervated by the cranial nerve X. UEA-I intensely stained the taste buds of the fungiform papillae and soft palate, both of which are innervated by the cranial nerve VII. The taste buds of circumvallate papilla and foliate papillae were innervated by the cranial nerve IX and strongly stained by BSL-I. Thus, UEA-I and BSL-I binding glycoconjugates, probably alpha-linked fucose and alpha-D-galactose, respectively, might be specific for taste buds. Although the expression of these glycoconjugates would be related to the innervation of the cranial nerve X, the differential expression of alpha-linked fucose and alpha-D-galactose might be related to the innervation of the cranial nerve VII and IX, respectively.

Animals↗