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G F Clark

Publications and source records attributed to G F Clark.

At least 37 records · Page 2Linked to original sources
PubMed1993

A revised structure for fucoidan may explain some of its biological activities.

Fucoidan from Fucus vesiculosus inhibits human sperm-zona pellucida binding and blocks the zona pellucida-induced acrosome reaction in human sperm. Fucoidan also potently inhibits selectin-mediated adhesion of leukocytes to vascular endothelium. To understand the molecular basis for fucoidan's inhibition of specific cell adhesion events, we have investigated the structure of this fucan using definitive methods of carbohydrate structural analysis. We report the revised average structure for fucoidan. [formula: see text] This average structure differs from the previous model for fucoidan in two respects. First, the core region of the fucan is composed primarily of a polymer of alpha 1-3-linked fucose with sulfate groups substituted at the 4 position on some of the fucose residues. Secondly, fucose is also attached to this polymer to form branch points, one for every 2-3 fucose residues within the chain. This revised average structure is consistent with previous studies suggesting a branched random coil as the best model for this polysaccharide. The proposed model is also a closer structural analogue of the sulfated carbohydrate ligands that bind to selectins. This information should be useful for determining whether a relationship exists between selectin-mediated adhesion of leukocytes and human sperm-egg binding.

Carbohydrate Conformation↗
PubMed1993

Fucoidin binding activity and its localization on human spermatozoa.

We previously reported that fucoidin (a polymer of predominantly sulfated L-fucose) significantly inhibits: (1) tight binding of human sperm to human zona pellucida in vitro and (2) stimulation of the acrosome reaction by acid solubilized human zona pellucida. Here, we determined fucoidin binding activity on human spermatozoa and its localization on both live and permeabilized human sperm populations. A typical binding curve was demonstrated with biotinylated fucoidin. In competitive inhibition assays with unlabelled fucoidin or human sperm membrane extracts, IC50's were 4.0 micrograms/ml and 31.4 micrograms/ml, respectively. Fucoidin binding was localized over the acrosomal region of methanol-fixed human sperm and this pattern of binding significantly decreased from 92 +/- 3% to 74 +/- 6% with calcium ionophore A23187 treatment (p < 0.01). Binding of fucoidin-coated beads to live (non-permeabilized) human sperm was less than 1%. Addition of the detergent, Triton-X, to permeabilize sperm membranes resulted in a significant increase in binding (p = 0.001). These results provide evidence for the presence of a fucoidin binding compound in human spermatozoa that is localized to the membranes of the acrosomal region and can be extracted by a mild detergent extraction. Absence of binding by fucoidin to intact but not permeabilized spermatozoa suggests that the heteropolysaccharide binds to a receptor within the acrosomal matrix. However, further investigation is warranted to determine whether a fucoidin binding site is present both at the sperm's surface for the initial contact with the zona pellucida, and also for secondary binding after exposure of the acrosomal membranes.

Acrosome↗
PubMed1992

Polyglycosylceramides with branched N-acetyllactosamine sequences are synthesized by the human pancreatic carcinoma cell line PANC-1.

We have metabolically labeled the human pancreatic tumor cell line PANC-1 with high specific activity tritiated sugar precursors to study the expression of glycosphingolipids by this cell type. We have used a combination of detergent solubilization, exhaustive protease digestion, ceramide glycanase digestion, and reverse-phase chromatography to isolate glycosphingolipid-derived oligosaccharides specifically labeled in their component sugars. A significant proportion of the oligosaccharides derived from polar glycosphingolipids were of high molecular mass (greater than 2000 Da). The results of compositional studies, lectin affinity chromatography, and methylation analysis suggested that this high molecular weight fraction consists of lactosaminoglycan type oligosaccharides derived from polyglycosylceramides. There are on average three beta 1-6 linked N-acetyllactosamine branches attached to the polylactosamine backbone in this type of glycosphingolipid-derived oligosaccharide. The majority of the oligosaccharides also contain 1-2 mol of sialic acid that are linked alpha 2-3 to penultimate galactose. The results indicate that PANC-1 cells, like human colorectal tumor cells, express highly extended neolacto type glycosphingolipids. However, the lactosaminoglycan sequences are highly branched, unlike those associated with colorectal tumor cells.

Amino Sugars↗
PubMed1991

Decreased biosynthesis of Forssman glycolipid after retinoic acid-induced differentiation of mouse F9 teratocarcinoma cells. Lectin-affinity chromatography of the glycolipid-derived oligosaccharide.

Glycolipids synthesized by the mouse teratocarcinoma F9 cells and F9 cells (RA/F9 cells) induced to differentiate by a 3-day treatment with 0.1 microM all-trans-retinoic acid were analyzed. Both F9 cells and RA/F9 cells were incubated in media containing either D-[6-3H]galactose or D-[6-3H]glucosamine; the metabolically-radiolabeled glycolipids were isolated and the oligosaccharides were released from the glycolipids by ozonolysis and alkali fragmentation. From both cells, a single major pentasaccharide was isolated from the mixture of neutral [3H]oligosaccharides by affinity chromatography on a column of immobilized Helix pomatia agglutinin. The structure of this oligosaccharide was analyzed by methylation analysis and specific exoglycosidase treatments and identified as the Forssman pentasaccharide alpha-D-GalpNAc-(1----3)-beta-D-GalpNAc-(1----4)-alpha-D-Galp-(1----4)-b eta-D- Galp-(1----4)-D-Glc. There was a 3-4-fold decreased amount of the Forssman pentasaccharide from RA/F9 cells relative to F9 cells. In contrast, there were no major differences between these cells in the levels of globoside, the precursor to Forssman glycolipid. To investigate the basis for the decline in Forssman glycolipid synthesis upon differentiation, the activity of UDP-D-Gal-NAc:GbOse4Cer alpha-(1----3)-N-acetyl-D-galactosaminyltransferase (Forssman synthase) was determined in extracts of both the F9 and RA/F9 cells. The specific activity of Forssman synthase was approximately 70% lower in differentiated relative to the nondifferentiated cells. These data demonstrated that F9 cells synthesize authentic Forssman glycolipid, and that its expression and the activity of Forssman synthase were decreased following induced cellular differentiation.

Animals↗
PubMed1991

Fucoidin inhibits the zona pellucida-induced acrosome reaction in human spermatozoa.

We recently reported that fucoidin (a polymer of predominantly sulfated L-fucose) significantly inhibits tight binding of human sperm to the human zona pellucida in vitro and that several oligosaccharides obtained after acid hydrolysis possess sperm-zona pellucida binding inhibitory activity equal to the original fucoidin. This inhibition may be specific to sperm-zona interactions or may be the consequence of the interruption of capacitation, a series of biochemical and physiological events leading to final sperm maturation, that must occur for successful fertilization. Completion of capacitation is most often determined by assessing two end-points of the process: acquisition of hyperactivated motility and ability to complete the acrosome reaction. Here, we examined the effects of fucoidin on these two end-points of capacitation in vitro. Fucoidin did not affect the proportion of sperm with hyperstimulated motility. Neither did fucoidin cause an increase in sperm that had spontaneously acrosome-reacted at 4.5 hours compared to controls as evaluated by indirect immunofluorescence using the acrosomal marker, monoclonal antibody, T-6. Comparable percentages of sperm had completed the acrosome reaction when exogenously stimulated by calcium ionophore A23187 with and without the addition of fucoidin. However, in the presence of fucoidin, stimulation of the acrosome reaction by acid solubilized human zonae pellucidae was significantly inhibited. These data indicate that fucoidin does not impede the normal progression of capacitation. These results provide strong evidence to support the hypothesis is that the inhibitory effect of fucoidin is at the level of the sperm membrane since inhibition can be bypassed by increasing intracellular calcium directly with a calcium ionophore.

Acrosome↗
PubMed1991

Nature of the inhibitory effect of complex saccharide moieties on the tight binding of human spermatozoa to the human zona pellucida.

Fucoidin and heparin sulfate inhibit binding of human sperm to the human zona pellucida under hemizona assay (HZA) conditions. Here we used the HZA to further assess tight sperm binding with/without preincubation of the sperm with other sulfated and nonsulfated glycoconjugates and charged polymers. Fucoidin significantly inhibited binding compared with controls (greater than 75% inhibition), even if sperm were washed after preincubation with the saccharide. Dextran sulfate also produced significant inhibition, although to a lesser extent (54% inhibition). Chondroitin sulfates A and B, heparin, and dextran did not affect binding. Sodium sulfate and polyglutamic acid did not affect HZA results; polyphosphates produced only moderate inhibition. The potent inhibitory effect of the sulfated carbohydrates fucoidin and dextran is probably competitive (receptor-ligand type) in nature. However, the lack of significant effects of simple charged molecules (nonspecific effects) suggests that the degree of sulfation (charge) may not be crucial to its inhibitory action.

Adult↗
PubMed1990

Detection of serotype-specific antibodies or capsular antigen of Actinobacillus pleuropneumoniae by a double-label radioimmunoassay.

Diagnostic tests for Actinobacillus pleuropneumoniae have been problematic because current tests do not use a purified antigen and in most cases measure either antibody or antigen, but not both. We describe a Farr-type double-label radioimmunoassay that utilizes purified, serotype-specific, 3H-capsule to measure antibody to capsule directly or that can measure capsule in a sample indirectly by inhibition of antibody binding. The assay could detect about 1 ng of serotype-specific antibody in serum or at least 100 pg of capsule in a sample. Due to the sensitivity of the assay, false-positive results were common with neat sera (probably due to cross-reacting antibodies to unrelated antigens), but the specificity was improved when the sera were diluted 1:100. The radioimmunoassay should prove to be a useful reference method for research and diagnostic testing and for comparison of new assays for detection of capsule or antibodies to capsule.

Actinobacillus↗
PubMed1989

Mouse monoclonal antibodies against human sperm: evidence for immunodominant glycosylated antigenic sites.

Thirty mouse monoclonal antibodies (MoAbs) raised against human sperm detect common antigenic determinants on human lymphocytes, erythrocytes, bacteria and endotoxin. Specific chemical, enzymatic and lectin blocking studies indicate that the sperm-associated antigens defined by these MoAbs are glycoconjugates. Further studies including reactivity of these MoAbs with organic sperm extracts indicate that the predominant carriers of these carbohydrate antigens are glycolipids and that the terminal immunodominant monosaccharide may be N-acetyl-glucosamine or N-acetylgalactosamine.

Animals↗
PubMed1988

Separation of oligosaccharides containing terminal alpha-linked galactose residues by affinity chromatography on Griffonia simplicifolia I bound to concanavalin A-sepharose.

The seeds of Griffonia simplicifolia contain a family of five isolectins (GS-I) (L. A. Murphy and I. J. Goldstein (1977) J. Biol. Chem. 252, 4739-4742) that bind with high affinity to glycoconjugates containing terminal nonreducing alpha-linked galactose residues. Here, we report that GS-I itself is bound via its high mannose-type, Asn-linked sugar chains to immobilized concanavalin A (Con A-Sepharose). The GS-I in the GS-I-Con A-Sepharose complex retains its ability to bind glycoconjugates containing terminal alpha-linked galactose residues. This convenient method to immobilize GS-I is rapid and quantitative. We have exploited this affinity system to separate oligosaccharides based on their number of terminal alpha-linked D-galactose residues.

Animals↗
PubMed1987

Toxin A from Clostridium difficile binds to rabbit erythrocyte glycolipids with terminal Gal alpha 1-3Gal beta 1-4GlcNAc sequences.

The binding of Toxin A isolated from Clostridium difficile to rabbit erythrocyte glycolipids has been studied. Total lipid extracts from rabbit erythrocytes were subjected to thin-layer chromatography and toxin-binding glycolipids detected by using 125I-labeled Toxin A in a direct binding overlay technique. Two major and several minor toxin-binding glycolipids were detected in rabbit erythrocytes by this method. The results of structural analyses of the major toxin-binding glycolipids were consistent with a pentasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) and a branched decasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3[Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) previously identified as the two most abundant glycolipids in rabbit erythrocytes. 125I-Toxin A binding to these glycolipids could be inhibited by bovine thyroglobulin, monospecific antiserum to the toxin, or by treatment of the glycolipids with alpha-galactosidase. The absence of toxin interaction with isoglobotriaosylceramide (Gal alpha 1-3Gal beta 1-4Glc-Cer) isolated from canine intestine suggested that the GlcNAc residue present in the terminal Gal alpha 1-3Gal beta 1-4GLcNAc sequence common to all known toxin binding glycoconjugates is required for carbohydrate-specific recognition by Toxin A. These observations are consistent with the proposed carbohydrate binding specificity of Toxin A for the nonreducing terminal sequence, Gal alpha 1-3Gal beta 1-4GlcNAc.

Animals↗
PubMed1986

Structural and conformational features that affect the interaction of polylactosaminoglycans with immobilized wheat germ agglutinin.

We examined the interaction between immobilized wheat germ agglutinin and the large, polylactosamine-containing glycans from human erythrocytes and human K-562 erythroleukemic cells. Three classes of interaction were identified. One class of glycan was merely retarded during chromatography. The other two classes were retained and could be distinguished by their ease of displacement with N-acetylglucosamine (GlcNAc); one was a moderate-affinity fraction displaced by 0.1 M GlcNAc and the other was a high-affinity fraction subsequently displaced by 1.0 M GlcNAc. A relatively small fraction of the K-562 polylactosamines were in the high-affinity class. We explored the role that fucose and sialic acid substitutions play in the strength of the lectin-glycan interaction. Although sialic acid is recognized by wheat germ agglutinin, sialylation was not required for the high-affinity interaction, and the presence of sialic acids actually prevented some glycans from binding with high affinity. In contrast, fucose is not part of the binding determinant, yet the removal of fucose resulted in decreased affinity. The possibility that some of these changes in affinity were the result of conformational changes was explored using matrices that had wheat germ agglutinin immobilized at different densities. At low wheat germ agglutinin densities, adult and fetal erythroglycans and K-562 glycophorin-like glycans were not retained by the matrix. As the density increased, the proportion of glycans that were retarded, and ultimately retained, increased. While these increases in the proportions retained occurred in parallel for the three different glycans, the apparent affinities of the glycan-lectin interactions differed. The glycophorin-like glycans were always readily displaced by 0.1 M GlcNAc, even at higher wheat germ agglutinin densities. In contrast, as the wheat germ agglutinin density increased, the proportion of erythroglycans that could be displaced by 0.1 M GlcNAc decreased; at 10 mg/ml immobilized wheat germ agglutinin, greater than 80% of the erythroglycans exhibited this tighter interaction. We suggest that this higher affinity interaction is the result of the large glycans spanning adjacent wheat germ agglutinin molecules, and is determined by the proximity of these molecules and the conformation of the glycans.

Amino Sugars↗
PubMed1986

Cell surface binding site for Clostridium difficile enterotoxin: evidence for a glycoconjugate containing the sequence Gal alpha 1-3Gal beta 1-4GlcNAc.

This study was undertaken to determine whether a binding site for Clostridium difficile enterotoxin (toxin A) exists in the brush border membranes (BBMs) of the hamster, an animal known to be extremely sensitive to the action of the toxin. Toxin A was the only antigen adsorbed by the BBMs from the culture filtrate of C. difficile. The finding that binding activity could not be destroyed by heat indicated that a carbohydrate moiety might be involved. We therefore examined erythrocytes from various animal species for binding activity since erythrocytes provide a variety of carbohydrate sequences on their cell surfaces. Only rabbit erythrocytes bound the toxin, and the cells agglutinated. A binding assay based on an enzyme-linked immunosorbent assay method for quantifying C. difficile toxin A was used to compare binding of the toxin to hamster BBMs, rabbit erythrocytes, and BBMs from rats, which are less susceptible to the action of C. difficile toxin A than hamsters. Results of this comparison indicated the following order of toxin-binding frequency: rabbit erythrocytes greater than hamster BBMs greater than rat BBMs. Binding of toxin A to hamster BBMs at 37 degrees C was comparable to what has been observed with cholera toxin, but binding was enhanced at 4 degrees C. A similar binding phenomenon was observed with rabbit erythrocytes. Examination of the cell surfaces of hamster BBMs and rabbit erythrocytes with lectins and specific glycosidases revealed a high concentration of terminal alpha-linked galactose. Treatment of both membrane types with alpha-galactosidase destroyed the binding activity. The glycoprotein, calf thyroglobulin, also bound the toxin and inhibited toxin binding to cells. Toxin A did not bind to human erythrocytes from blood group A, B, or O donors. However, after fucosidase treatment of human erythrocytes, only blood group B erythrocytes, which possess the blood group B structure Gal alpha 1-3[Fuc alpha 1-2]Gal beta 1-4GlcNAc-R, bound the toxin. This indicated that toxin A was likely binding to Gal alpha 1-3Gal beta 1-4GlcNAc, a carbohydrate sequence also found on calf thyroglobulin and rabbit erythrocytes. All of the results indicate that hamster BBMs contain a carbohydrate-binding site for toxin A that has at least a Gal alpha 1-3Gal beta 1-4GlcNAc nonreducing terminal sequence.

Animals↗
PubMed1983

Formation of dolichol-linked sugar intermediates during the postnatal development of skeletal muscle.

The postnatal development of skeletal muscle is characterized by changes in membrane function associated with N-linked glycoproteins. In the present study, early reactions involved in the synthesis of the dolichol-linked core oligosaccharide were examined in neonatal and adult rabbit skeletal muscle sarcoplasmic reticulum membranes. The initial rate of N-acetylglucosamine incorporation in the presence of exogenous dolichol phosphate was similar between neonate and adult (3.5-4.1 pmol of GlcNAc/min/mg). The Km values for UDP-GlcNAc and exogenous dolichol phosphate were similar. Tunicamycin (0.04-0.08 micrograms/ml) inhibited N-acetylglucosamine incorporation by 50%. UDP-GlcNAc pyrophosphatase activity was greater in neonatal membranes than adult (840 versus 350 pmol of GlcNAc-1-P/min/mg), explaining, in part, the greater enhancement of neonatal GlcNAc incorporation by pyrophosphatase inhibitors. Nucleotide-sugar pyrophosphatase inhibitors (alpha, beta-methylene ATP and dimercaptopropanol) increased the capacity of neonatal activity 4-fold and adult enzyme 2-fold. Analysis of dolichol-linked products by mild acid hydrolysis however, revealed that neonate had higher capacity for N,N'-diacetylchitobiosyl(pyro)phosphoryldolichol synthesis than adult. Mannosyltransferase and glucosyltransferase were elevated 6- and 5-fold in neonate compared to adult membranes. Neonate exhibited 4-fold greater GDP-Man pyrophosphatase activity than adult (500 versus 125 pmol of Man-1-P/min/mg). The Km for GDP-Man increased in the presence of exogenous dolichol phosphate. Increasing concentrations of exogenous dolichol phosphate did not equalize neonate and adult mannosyltransferase activity, indicating that the decline in activity during development was not due to a decrease in a pool of dolichol phosphate accessible to mannosyltransferase. Glucosyltransferase for the synthesis of glucosylphosphoryldolichol was also elevated 5-fold in neonatal compared to adult sarcoplasmic reticulum (7 versus 1.4 pmol of Glc/min/mg). In a previous study, it was reported that glycoprotein sialyltransferase activity decreased by a factor of 6.5 during the postnatal maturation and that total membrane hexose content of sarcoplasmic reticulum decreased by a factor of 8. Together, these results suggest that the postnatal development of skeletal muscle is characterized by coordinated changes in the expression of enzymes involved in both the "early" and "late" reactions of N-linked oligosaccharide biosynthesis.

Acetylglucosamine↗
PubMed1983

Studies on glycoconjugate metabolism in developing skeletal muscle membranes.

The postnatal development of mammalian skeletal muscle is characterized by changes in the properties of several key membrane glycoprotein enzymes and receptors. In the present study, CMP-sialic acid: fetuin sialyltransferase and CMP-sialic acid: lactosylceramide sialyltransferase activity was characterized in sarcolemma and sarcoplasmic reticulum membranes isolated from neonatal (0-1 week) and adult (8 week) rabbit skeletal muscle. CMP-sialic acid: fetuin sialyltransferase decreased by a factor of 10 in sarcolemma and 6 in sarcoplasmic reticulum during development, whereas CMP-sialic acid: lactosylceramide sialyltransferase activity decreased by a factor of 6 in sarcolemma and 18 in sarcoplasmic reticulum. The Km for CMP-sialic acid using the lipid acceptor declined during the development of sarcoplasmic reticulum (neonate vs. adult: 538 vs. 33 microM), but not in sarcolemma. The carbohydrate composition of sarcolemma was changed only with respect to total sialic acid content (neonate vs. adult: 67 vs. 44 nmol/mg). Similar analysis of sarcoplasmic reticulum carbohydrates showed decreases in total sialic acid, lipid-bound sialic acid, hexosamines and hexoses. The major ganglioside was GM3 for both types of membrane. No qualitative changes were observed in ganglioside composition comparing neonatal and adult membranes.

Aging↗
PubMed1980

Beta-adrenergic receptor-adenylate cyclase alterations during the postnatal development of skeletal muscle.

The postnatal development of mammalian skeletal muscle is associated with an increased capacity for glycogenolysis. In the present study rabbit skeletal muscle underwent a 7-fold increase in glycogen synthase and glycogen phosphorylase activity over the postnatal period of 0--8 weeks. An enriched fraction of sarcolemma was prepared from neonatal and adult muscle to examine the development of the beta-adrenergic receptor-adenylate cyclase system. Adult membranes possessed a 2-fold greater Na+K+(Mg2+)-ATPase activity and a 6--8 fold greater sodium fluoride- and epinephrine-stimulated adenylate cyclase activity. The activation ratio (effector activity/basal activity) increased 2--3 fold for epinephrine and sodium fluoride in adult sarcolemma. The activation by catecholamines conformed to the physiological beta 2 type response with isoproterenol (1.8 . 10(-8) M) > epinephrine (1.1 . 10(-7) M) > norinephrine (3.2 . 10(-6) M). In contrast, binding studies employing (-)-[3H]dihydroalprenolol showed little difference between neonatal and adult membranes with respect to (1) number of binding sites, (2) equilibrium dissociation constant and (3) displacement of (-)-[3H]dihydroalprenolol by catecholamine agonists. Protein and lipid components of the sarcolemma were also modified during development. Neonatal membranes possessed two glycopeptides of Mr 80000 and 86000, whereas in the adult only a single Mr 113000 species was evident. The total lipid phosphorus and phospholipid composition was unchanged during development. The content of linoleic acid increased approx. 3-fold during development in the phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine phospholipids. The cholesterol content of adult membranes was decreased by 29% compared to neonatal membranes.

Adenylyl Cyclases↗
PubMedDate not supplied

Involvement of selectin-like carbohydrate binding specificity in human gamete interaction.

The recognition of carbohydrate epitopes by complimentary protein receptors has been shown to be a critical factor in gamete interaction in many different animal species. In this study it was hypothesized that, in the human, gamete binding requires an interaction between selectin ligands on the zona pellucida and putative egg binding proteins on the sperm surface. The hemizona assay (a unique internally controlled bioassay that evaluates tight binding of sperm to the zona) and advanced methods of carbohydrate analysis were used to test this hypothesis. From these tests it was shown that oligosaccharide recognition is also required for initial human gamete binding. This study suggests the existence of distinct egg binding proteins on human sperm that can bind to selectin ligands. Additionally, the results suggest a possible convergence in the types of carbohydrate sequences recognized during initial human gamete binding and immune/inflammatory cell interactions. Glycoconjugates that manifest selectin-ligand activity and that express specific carbohydrate epitopes have potent contraceptive and immunosuppressive effects. Such specific oligosaccharide sequences may provide an appropriate recognition signal for embryo development and protection.

Carbohydrate Metabolism↗