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R L Schnaar

Publications and source records attributed to R L Schnaar.

89 records · Page 5Linked to original sources
PubMed1984

Agonists and cations regulate the glutamic acid receptors on intact neuroblastoma hybrid cells.

Glutamate is thought to be a major excitatory neurotransmitter in the vertebrate brain. In the preceding paper (Malouf, A. T., Schnaar, R. L., and Coyle, J. T. (1984) J. Biol. Chem. 259, 12756-12762), we demonstrated specific binding of [3H]glutamate to membranes from a neuroblastoma hybrid cell line, N18-RE-105. These sites are pharmacologically and kinetically similar to those seen on rat brain membranes and are regulated by ions added to the isolated membranes. In the current paper, we describe an additional level of regulation for the glutamate receptor in this cell line. Long-term incubation (72 h) of intact N18-RE-105 cells with glutamate (10 mM) results in a 2- to 3-fold increase in [3H]glutamate binding. Scatchard analysis reveals that the increase in binding is due to an increase in the number of glutamate receptors without significant change in their affinity. The ability of glutamate analogs to induce such up-regulation mirrors their ability to compete for [3H]glutamate binding to isolated membranes, suggesting that up-regulation is receptor-mediated. Binding of [3H]glutamate to membranes isolated from cells grown in the presence of glutamate can be further up-regulated by brief exposure (10 min) of the isolated membranes to calcium ions. This suggests that agonist-induced and calcium-induced up-regulation occur via independent mechanisms. The short-term ion-induced up-regulation and the long-term agonist-induced up-regulation described in this paper may model two levels of synaptic potentiation reported to occur in the vertebrate hippocampus. The N18-RE-105 cell line may offer a homogeneous cell type in which to study the molecular mechanisms underlying these phenomena.

Amino Acids↗
PubMed1984

Carbohydrate-specific adhesion of alveolar macrophages to mannose-derivatized surfaces.

The ability of rabbit alveolar macrophages to specifically recognize and adhere to surfaces derivatized with carbohydrates was examined. Otherwise inert polyacrylamide gels were derivatized with aminohexylglycosides as previously described (Guarnaccia, S. P., and Schnaar, R. L. (1982) J. Biol. Chem. 257, 14288-14292). Intact viable rabbit alveolar macrophages, isolated by lung lavage, were placed in contact with surfaces derivatized with different glycosides. Only those surfaces derivatized with alpha-D-mannose residues were capable of supporting rabbit alveolar macrophage adhesion. Adhesion was rapid, obtaining maximal levels within 10 min, and occurred readily at either 0 or 37 degrees C. The carbohydrate specificity of the cell adhesion was investigated by the use of soluble carbohydrate inhibitors. The potency of various saccharides to block the adhesion correlated with that demonstrated for blocking the uptake or binding of radiolabeled soluble glycoproteins (Shepherd, V. L., Lee, Y. C., Schlesinger, P. H., and Stahl, P. D. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 1019-1022). Thus, the order of potency observed was: D-Man congruent to L-Fuc greater than D-GlcNAc congruent to D-Glc much greater than D-Gal congruent to D-GalNAc congruent to L-rhamnose. While soluble monosaccharides were capable of blocking adhesion when added in millimolar concentrations, polymannosylated neoglycoproteins were able to block adhesion in the nanomolar concentration range. Adhesion to the mannose-derivatized surfaces was a dynamic event even at 0 degrees C, since adhesion was less susceptible to monosaccharide inhibition at later incubation times. Surfaces derivatized with aminohexyl S-mannoside ligands were more effective in supporting adhesion than those derivatized with the corresponding O-mannosides. Soluble inhibitor studies suggest that this was due to a more favorable conformation of the S-glycoside for binding to the cell surface receptor. The results reported here demonstrate that the previously reported alveolar macrophage mannose/fucose receptor can mediate carbohydrate-specific cell adhesion.

Animals↗
PubMed1983

Carbohydrate-specific cell adhesion is mediated by immobilized glycolipids.

We describe a technique for examining the ability of one important class of cell surface complex carbohydrates, glycosphingolipids, to mediate carbohydrate-specific cell recognition and adhesion. Analogs of natural glycosphingolipids were synthesized, consisting of 1-glycosyl derivatives of 3-deoxyceramide (N-palmitoyl-2-aminostearol) radiolabeled in the fatty acid portion. Methods were developed to efficiently adsorb both these synthetic glycolipids and natural glycosphingolipids (including gangliosides) from aqueous ethanol solution onto plastic wells. The glycolipids remained firmly attached to the surface in aqueous solutions, but could be recovered using detergents or organic solvents. The ability of the adsorbed glycolipids to elicit specific adhesion of intact hepatocytes was tested using specific adhesion of intact hepatocytes was tested using a cell adhesion assay based on that of McClay, D. R., Wessel, G. M., and Marchase, R. B. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4975-4979. When otherwise nonadhesive plastic surfaces were adsorbed with N-acetylglucosaminyl 3-deoxyceramide, they supported adhesion of 80-95% of the chicken hepatocytes added to the well. No adhesion above background levels (10-25%) was observed to surfaces adsorbed with other synthetic glycolipids including glucosyl, galactosyl, mannosyl, or lactosyl 3-deoxyceramide, 3-deoxyceramide, or to the naturally occurring glycosphingolipids, lactosyl ceramide or ganglioside GM1. Chicken hepatocyte adhesion to surfaces adsorbed with N-acetylglucosaminyl 3-deoxyceramide was inhibited by soluble N-acetylglucosamine (IC50 = 3 m M), but not by other soluble sugars. Rat hepatocytes adhered preferentially to surfaces adsorbed with lactosyl 3-deoxyceramide, but not to surfaces adsorbed with the N-acetylglucosaminyl derivative. These studies demonstrate the ability of adsorbed glucolipids to mediate carbohydrate- and cell-specific adhesion from intact cells. Using these techniques, the ability of naturally occurring complex glycosphingolipids to elicit specific cellular responses from a variety of cell types can be examined.

Animals↗
PubMed1983

Tunicamycin inhibits ganglioside biosynthesis in neuronal cells.

The antibiotic tunicamycin blocks the transfer of GlcNAc-1-P from UDP-GlcNAc to dolichol phosphate, thereby blocking the synthesis of N-linked oligosaccharide chains on glycoproteins. Its effect on the biosynthesis of gangliosides has not been reported. We report that tunicamycin caused a 70-80% reduction in incorporation of [(3)H]GlcN into gangliosides and neutral glycosphingolipids of the neuroblastoma-glioma hybrid cell line NG 108-15 at antibiotic concentrations that caused a 90% reduction of the radiolabel incorporation into glycoproteins. The effect of tunicamycin on ganglioside biosynthesis was apparent after only 4 hr of incubation, and maximum inhibition was seen within 6 hr. When control or tunicamycin-treated (5 mug/ml) cells were collected and fractionated to separate glycoproteins, neutral glycosphingolipids, gangliosides, and nucleotide sugar-precursor pools, the following results were obtained: (i) UDP-GlcNAc and UDP-GalNAc pool sizes increased >3-fold, and specific activities decreased 50% upon treatment with tunicamycin; (ii) when corrected for this value, the percentage inhibition of GlcN incorporation into various glycoconjugates by tunicamycin in these cells was 82% for glycoproteins, 54% for neutral glycosphingolipids, and 50% for gangliosides; and (iii) the different gangliosides were affected differentially, with the most striking inhibition apparent in GM(3) biosynthesis, which was decreased 78% in the presence of tunicamycin. These data suggest that the effects of tunicamycin on glycosphingolipids as well as on glycoproteins must be considered when interpreting its effects on intact cells and organisms.

Acetylglucosamine↗
PubMed1983

Ganglioside composition is regulated during differentiation in the neuroblastoma X glioma hybrid cell line NG108-15.

Cellular differentiation of the neuroblastoma X glioma hybrid cell line NG108-15 was measured and correlated with quantitative changes in the cells' ganglioside composition. The degree of differentiation was measured using an enzymatic marker, choline acetyltransferase (CAT), which is responsible for neurotransmitter synthesis in this cell line. Differentiation of these cells is commonly induced by agents such as dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP). However, in our studies, we observed that these cells "self-differentiated," in the absence of chemical inducers, when the cells became dense in culture. The differentiation marker, CAT specific activity, rose from 150 to more than 400 pmol/min/mg of protein as cell density increased, attaining a level higher than that achieved by treatment with Bt2cAMP. Differentiation of sparse cultures could be induced by conditioned medium removed from dense cultures. This effect was not due to depletion of a serum component from the medium by the cells, since it was not mimicked by serum depletion or inhibited by addition of fresh serum to the conditioned medium. These data suggest that cell density-dependent differentiation was caused by release of a factor from the cells which induced differentiation in a concentration-dependent manner. Gangliosides, therefore, were purified from sparse control cultures, dense cultures, and cultures treated with the differentiating agents Bt2cAMP, prostaglandin E1 (plus theophylline), or butyric acid. Quantitative thin layer chromatography revealed that all of the cultures contained the four gangliosides GM3, GM2, GM1, and GD1a. The concentration of one of the gangliosides, GM2, increased markedly (up to 12-fold) during differentiation. The GM2 concentration correlated closely with the level of CAT activity in the different cultures (r = 0.99). These data demonstrate that the ganglioside concentration in these cells is regulated during differentiation, a finding consistent with a possible role for gangliosides in the differentiated phenotype.

Alprostadil↗
PubMed1982

Hepatocyte adhesion to immobilized carbohydrates. I. Sugar recognition is followed by energy-dependent strengthening.

Cell-cell recognition and adhesion are thought to be complex, multistep phenomena, and may involve cell surface carbohydrates and their receptors on apposing cell surfaces. We have modeled such interactions using hepatocytes and polymer (gel) surfaces derivatized with carbohydrate ligands, and have demonstrated carbohydrate-specific cell adhesion (Schnaar, R. L., Weigel, P. H., Kuhlenschmidt, M. S., Lee, Y. C., and Roseman, S. (1978) J. Biol. Chem. 253, 7940-7951). In the present studies, we have developed a method to quantitate the forces involved in cell-gel adhesion. Our method is based on that of McClay et al. (McClay, D. R., Wessel, G. M., and Marchase, R. B. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4975-4979) which was designed to measure the forces involved in intercellular adhesion. The following results were obtained. 1) Chicken and rat hepatocytes adhere specifically to gels derivatized with N-acetylglucosamine and galactose, respectively, at either 4 degrees C or 37 degrees C. 2) At 37 degrees C, after a lag of 10-20 min, stabilization of the adhesion occurs, resulting in a 15-fold (or greater) increase in the force of adhesion. 3) This marked strengthening of adhesion does not occur at 4 degrees C and is blocked by inhibitors of oxidative phosphorylation. These data demonstrate that cells can recognize and adhere to specific carbohydrates on apposing surfaces, and can then respond by mobilizing cellular energy to strengthen that adhesion. This series of events is strikingly similar to that shown for cell-cell adhesion between hepatocytes or between neural retina cells (Umbreit, J., and Roseman, S. (1975) J. Biol. Chem. 250, 9360-9368; McClay, D. R., Wessel, G. M., and Marchase, R. B. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4975-4979). The present results suggest that the cell surface analogs described may provide a well controlled experimental system to probe the molecular events involved in cell-cell adhesion.

Animals↗
PubMed1978

Adhesion of chicken hepatocytes to polyacrylamide gels derivatized with N-acetylglucosamine.

Complex carbohydrates on the surfaces of eukaryotic cells are thought to participate in a wide variety of cell-cell interactions. A model system has therefore been developed to study these processes. In the present experiments, the ability of chicken hepatocytes to recognize and adhere to sugars covalently linked to polyacrylamide gels was investigated. The gels were snythesized by two methods. Type I gels were prepared from a co-polymer of an active ester of acrylic acid (N-succinimidyl acrylate), acrylamide, and bisacrylamide. The "activated" polyacrylamide gel was then treated with the desired ligand containing an amino group, such as 6-aminohexyl O- or S-glycoside. Type II gels were formed by treating similar ligands with acryloyl chloride, followed by co-polymerization of the resulting N-substituted acrylamide with acrylamide and N,N'-methylenebisacrylamide. These polyacrylamide derivatives offer many advantages for studies with intact cells. They are not toxic to any cell type studied, can be cast in any desired shape, are transparent and stable over a wide range of pH values, and contain no cationic and low to negligible levels of anionic charge (charged groups can be introduced if desired), and the polyacrylamide matrix is stable to common biological agents such as bacteria and enzymes. In addition, type I gels can be synthesized using a broad range of molecules containing amino groups, such as glycopeptides, proteins, etc. The hepatocytes were prepared by collagenase perfusion of intact chicken livers. The rate and extent of adhesion of the cells to the derivatized gels was determined by measuring lactate dehydrogenase in these cells. This enzyme was also used to assay viability and cell "leakiness." At 37 degrees C, 70 to 100% of the cells adhered within 60 min to gels derivatized with N-acetylglucosamine, i.e. gels derivatized with 6-aminohexyl 2-acetamido-2-deoxy-beta-D-glucopyranoside (or the corresponding thioglycoside). By contrast, less than 5% of the cells adhered to polyacrylamide or to gels derivatized with 6-aminohexanol or the 6-aminohexyl glycosides of beta-D-glucose, beta-D-galactose, alpha-D-mannose, beta-D-maltose, beta-D-melibiose, beta-D-cellobiose, and (alpha or beta)-D-lactose. Kinetic studies with the chicken hepatocytes and N-acetylglucosamine gels showed that cell-gel binding was dependent upon Ca2+ and was decreased at low temperatures. Binding was inhibited by N-acetylglucosamine or by glycosides of this sugar, the most effective inhibitor being orosomucoid (alpha1-acid glycoprotein) pretreated with sialidase and beta-galactosidase. The cell surface receptor(s) involved in this interaction is not known, but may be related or identical to the chicken liver binding protein described by Lunney and Ashwell (Lunney, J., and Ashwell, G. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 341--343). The present results suggest that this model system should prove useful in delineating cell surface interactions with carbohydrates.

Acetylglucosamine↗
PubMed1975

Polyacrylamide gels copolymerized with active esters. A new medium for affinity systems.

A new and versatile method for linking biologically active ligands to a polyacrylamide matrix is reported. Active esters of acrylic acid (N-succinimicyl acrylate and N-phthalimidyl acrylate) were synthesized, then copolymerized with acrylamide and N,N'-methylenebisacrylamide. Displacement of the active ester in the gel thus formed by various ligands containing aliphatic amino groups resulted in the formation of stable amid bonds between the ligands and the polyacrylamide gel. The affinity gel thus prepared has the following advantages: (i) resistance to chemical and microbiological degradation, (ii) ease of control of ligand level and higher levels of ligand possible, (iii) ease of control of porosity, and (iv) total displacement of the active ester under suitable conditions. Efficacy of this system was tested by preparation of 6-aminohexyl 2-acetamido-2-deoxy-beta-D-glucopyranoside derivative polyacrylamide gel by the described method. It was found to be more effective for purification of wheat germ agglutinin than the previously published affinity chromatography systems and the wheat germ hemagglutinin was obtained in crystalline form. In addition, partial resolution of isolectins was obtained from the affinity gel witha pH gradient.

Acrylamides↗