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The effect of saccharides on the post-thaw recovery of cane toad (Bufo marinus) spermatozoa.

The effect of monosaccharides (glucose, fructose) and disaccharides (maltose, sucrose, trehalose) as diluents, in cryoprotective additives containing 15% (v/v) DMSO or glycerol as cryoprotectants, were investigated on the recovery of sperm motility after cryopreservation of cane toad (Bufo marinus) spermatoazoa at low (approximately 5 degrees C/min(-1)) and high cooling rates (approximately 35 degrees C/min(-1)). The results show that: 1. recovery of percentage motility was higher with slow cooling than with high cooling rates (37.0 +/- 2.5%, 15.3 +/- 1.6%, P<0.001, respectively), 2. disaccharides were more effective than monosaccharides in protecting spermatozoa with slow cooling (43.9 +/- 1.2%, 26.8 +/- 2.5%, P<0.02, respectively), 3. glycerol was more effective than DMSO with fast cooling (18.3 +/- 2.2%, 12.6 +/- 2.3%, P<0.02, respectively), 4. trehalose with glycerol was the most effective cryoprotective additive with fast cooling (31.0 +/- 3.2%, P<0.05), and 5. overall the recovery of degree (vigour) of motility (range, 1.9 - 3.2) was more resilient to cryopreservation than recovery of percentage motility (range, 8.9 - 51.5 %). Comparison of post-thaw percentage and vigour of sperm motility up to 24 minutes after activation showed disaccharides supported greater duration sperm motility than monosaccharides This result and the recovery of spermatozoa immediately after freeze-thaw, show the main effect of saccharides are as cryoprotectants and not as exogenous energy substrates.

Animals↗
PubMed1992

Ligand binding characteristics of the major mistletoe lectin.

Carbohydrate binding specificity of the galactose-specific, major lectin of mistletoe extract (ML-1) was studied by an inhibition assay using monosaccharides, monosaccharide derivatives, disaccharides, and compounds containing multiple galactosyl terminals. The results indicate that 1) both alpha- and beta-galactosyl residues are recognized equally well; 2) each of the hydroxyl groups of galactose contributes to varying degrees to the binding process, the 4-OH being the most important and the 6-OH the least important hydroxyl group; 3) disaccharide sequences of Gal beta 2Gal and Gal beta 3Gal have much higher affinity than galactose, whereas affinity of all other Gal-disaccharides is only slightly better than galactose; 4) macromolecular ligands having 10 or more terminal galactosyl residues have 500-fold higher affinity than Gal; and 5) a group on ML-1 with pK alpha of 4.8 appears to be involved in the binding of ligand.

Binding Sites↗
PubMed1992

Continuous release of secretory granule proteoglycans from a cell strain derived from the bone marrow of a patient with diffuse cutaneous mastocytosis.

A human cell strain (designated HBM-M) that was derived from the bone marrow of a child with diffuse cutaneous mastocytosis was previously found to possess features that suggested it belonged in the mast cell/monocyte lineage. HBM-M cells synthesized approximately 150-Kd Pronase-resistant proteoglycans that were recognized by an antihuman secretory granule proteoglycan peptide core antibody. These cells also contained in relatively high abundance the same sized mRNA transcript that encodes the peptide core of proteoglycans that are normally localized to secretory granules of hematopoietic cells. However, unlike most other hematopoietic cells, HBM-M cells continuously released their newly synthesized 35S-labeled proteoglycans rather than retaining them in an intracellular storage compartment. Chondroitinase ABC, nitrous acid, and heparinase degraded approximately 76%, 17%, and 7%, respectively, of the HBM-M cell-derived 35S-labeled proteoglycans. As assessed by high performance liquid chromatography, 91% of the unsaturated 35S-labeled disaccharides generated by treatment with chondroitinase ABC were delta Di-4S. The remaining chondroitin sulfate 35S-labeled disaccharides appeared to be primarily a complex mixture of disulfated disaccharides. The 35S-labeled glycosaminoglycans that were not degraded by chondroitinase ABC migrated in two-dimensional cellulose acetate electrophoresis as if they were heparan sulfate or under-sulfated heparin. Thus, although the HBM-M cell-derived proteoglycans had some of the features of proteoglycans produced by normal human mast cells, the heparin-like and chondroitin sulfate glycosaminoglycans bound to the HBM-M cell proteoglycans were considerably less sulfated. Because the only human cell types that have so far been shown to synthesize proteoglycans that have heparin-like glycosaminoglycans bound to a protease-resistant peptide core are mast cells and basophilic leukocytes from patients with myelogenous leukemia, it is possible that the HBM-M cell is a mast cell progenitor cell.

Bone Marrow↗
PubMed1992

The human blood fluke Schistosoma mansoni synthesizes a novel type of glycosphingolipid.

Previous studies have shown that the glycoprotein oligosaccharides synthesized by adult Schistosoma mansoni, the organism responsible for human schistosomiasis, are unusual in that they contain terminal beta-GalNAc residues and lack sialic acid. These observations and other studies indicating that schistosome glycoproteins and glycolipids are antigenic in infected animals led us to investigate the structures of the glycosphingolipids synthesized by these organisms and to determine whether they are structurally related to those synthesized by their vertebrate hosts. For our studies, adult schistosomes were metabolically radiolabeled with either [3H]galactose or [3H]glucosamine, and the newly synthesized glycosphingolipids were isolated and characterized. The major glycosphingolipids synthesized by adult schistosomes were found to be galactosylceramide and glucosylceramide. The adult worms synthesized no lactosylceramide (Gal beta 1-4Glc-ceramide), a common constituent of vertebrate cells; however, another disaccharide-containing glycosphingolipid cleavable by ceramide glycanase was found. The results of compositional and methylation analyses and exoglycosidase treatments demonstrated that this ceramide-disaccharide has the structure GalNAc beta 1-4Glc-ceramide. We also found that extracts of adult schistosomes are unable to transfer Gal from UDP-Gal to glucosylceramide, whereas extracts of Chinese hamster ovary cells, as a control, are able to do so, confirming that schistosomes are unable to synthesize lactosylceramide. Low levels of higher molecular weight glycosphingolipids were also found to be synthesized by adult schistosomes, and although their levels were too small to allow definitive characterization, compositional analyses indicated that they also contained GalNAc. We have tentatively designated the new disaccharide structure GalNAc beta 1, 4Glc- the "schistocore", which may represent a new type of glycosphingolipid core series.

Animals↗
PubMed1990

Purification and substrate specificity of heparitinase I and heparitinase II from Flavobacterium heparinum. Analyses of the heparin and heparan sulfate degradation products by 13C NMR spectroscopy.

The purification of two heparitinases and a heparinase, in high yields from Flavobacterium heparinum was achieved by a combination of molecular sieving and cation-exchange chromatography. Heparinase acts upon N-sulfated glucosaminido-L-iduronic acid linkages of heparin. Substitution of N-sulfate by N-acetyl groups renders the heparin molecule resistant to degradation by the enzyme. Heparitinase I acts on N-acetylated or N-sulfated glucosaminido-glucuronic acid linkages of the heparan sulfate. Sulfate groups at the 6-position of the glucosamine moiety of the heparan sulfate chains seem to be impeditive for heparitinase I action. Heparitinase II acts upon heparan sulfate producing disulfated, N-sulfated and N-acetylated-6-sulfated disaccharides, and small amounts of N-acetylated disaccharide. These and other results suggest that heparitinase II acts preferentially upon N,6-sulfated glucosaminido-glucuronic acid linkages. The total degradation of heparan sulfate is only achieved by the combined action of both heparitinases. The 13C NMR spectra of the disaccharides formed from heparan sulfate and a heparin oligosaccharide formed by the action of the heparitinases are in accordance to the proposed mode of action of the enzymes. Comparative studies of the enzymes with the commercially available heparinase and heparitinase are described.

Carbohydrate Sequence↗
PubMed1985

[Monospecific antibodies against synthetic T-antigen. Characteristics of their specificity and use in the identification of T-antigenic determinants on the cell surface].

Monospecific antibodies directed to a Thomsen-Friedenreich antigen (T-antigen) were obtained using artificial antigen. T-antigen immunodominant alpha-disaccharide Galbeta (1----3) GalNAc alpha 1-(T alpha) and its beta-anomer Gal beta (1----3) GalNAc beta 1-(T beta) were bound to bovine serum albumin (BSA) and cytochrome C (CCC) through a spacer (sp = -O(CH2)3NHCO (CH2)4CO-) by the azide method to give neoglycoproteins T alpha-sp-BSA, T alpha-sp-CCC and T beta-sp-BSA. Anti-T alpha antiserum was obtained by immunization of rabbits with T alpha-sp-BSA and then purified by sequential affinity chromatography on BSA-Sepharose and T alpha-sp-BSA-Sepharose to yield monospecific anti-T IgG antibodies. As elucidated by ELISA method, binding T alpha-sp-BSA to the antibodies was inhibited by T alpha-sp-CCC, T alpha-sp-OEt, asialofetuin, T alpha-OBzl, the activity of the inhibitors decreasing in the above order. Methyl beta-galactopyranoside, benzyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside, disaccharide Gal beta (1----3) GalNAc and H-sp-BSA were inactive. The inhibitory analysis suggests that both disaccharide moiety T alpha- and a definite part of the spacer are important for the binding and that T alpha-OCH2 seems to be the minimal recognized structure. In immunoprecipitation tests the antibodies react with T alpha-sp-BSA but not with T beta-sp-BSA, whereas peanut (Arachis hypogaea) lectin (PNL) precipitated both T alpha- and T beta-sp-BSA. These data suggest the significance of the alpha-galactosaminide bond for the antibody recognition. Desialylated human erithrocites (natural T-antigen) were effectively agglutinated with the antibodies. Murine cortical thymocytes (obtained by agglutination-sedimentation method using PNL) were agglutinated with the antibodies only partially (67%), while these cells as well as the cells unaffected by the antibodies were completely agglutinated with PNL. These results indicate to different contents of glycoproteins (T alpha) and glycolipids (T beta) oligosaccharide determinants on the surface of cortical thymocytes species.

Agglutination Tests↗
PubMed1989

Isolation and characterization of amaranthin, a lectin present in the seeds of Amaranthus caudatus, that recognizes the T- (or cryptic T)-antigen.

A lectin (Amaranthin) present in the seeds of Amaranthus caudatus has been isolated by fractionation on DEAE-cellulose followed by affinity chromatography on Synsorb-T beads (Gal beta 1,3GalNAc alpha-O-R-Synsorb). The lectin appeared homogeneous by gel electrophoresis at pH 4.3 and gave a single protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Mr = 33,000-36,000. A native Mr = 54,000 was determined by gel filtration suggesting that amaranthin exists as a homodimer. Compositional analysis revealed high amounts of acidic and hydroxyamino acids and relatively large amounts of lysine, methionine, and tryptophan for a plant protein. Amaranthin formed a precipitate with asialo-bovine submaxillary mucin, asialo-ovine submaxillary, porcine submaxillary mucin, asialo-fetuin and asialoglycophorin. Hapten inhibition of precipitate formation between amaranthin and asialo-ovine submaxillary indicated that the T-disaccharide and its alpha-linked glycosides (Gal beta 1,3GalNAc alpha-O-R; R = OH, methyl, -(CH2)8-COOCH3, allyl, o-nitrophenyl, or benzyl) were the best inhibitors. N-Acetylgalactosamine, the only monosaccharide which inhibited precipitation, was 350-fold less effective than Gal beta 1,3GalNAc alpha-O-R. Hapten inhibition with derivatives of the T-disaccharide suggested that the C'-4 axial hydroxyl group of the galactosyl moiety, and the C-4 axial hydroxyl group, and the C-2 acetamido group of the GalNAc unit are the most important loci for lectin interaction. NeuAc alpha 2,3Gal beta 1,3GalNAc alpha-O-(CH2)8CO2CH3 was as potent an inhibitor as Gal beta 1,3GalNAc alpha-O-(CH2)8CO2-CH3, and amaranthin was precipitated by NeuAc alpha 2,3Gal beta 1,3GalNAc alpha-O-BSA (where BSA is bovine serum albumin), indicating that the amaranthin-combining site tolerates substitutions at the C'-3 hydroxyl group. Amaranthin was precipitated by a Gal beta 1,3GalNAc alpha-O-BSA glycoconjugate but not by the anomeric Gal beta 1,3GalNAc beta-O-BSA glycoconjugate illustrating that the disaccharide must be linked alpha in order to interact with the lectin. Metal ions do not appear to be required for lectin activity. A study of pH dependence showed significant precipitate formation between pH 4 to 9 with a maximum at pH 5. Hapten inhibition and glycoconjugate precipitation assays were also conducted for peanut (Arachis hypogaea) agglutinin. A comparison between the carbohydrate-binding specificities of amaranthin and peanut (Arachis hypogaea) agglutinin is discussed.

Antigens, Neoplasm↗
PubMed1978

Immunochemistry of streptococcal group C polysaccharide and the nature of its crossreaction with the Forssman glycolipid.

Acid hydrolysis of streptococal Group C polysaccharide yields a disaccharide, 3-O-alpha-N-acetylgalactosaminosyl-N-acetylgalactosamine (3-O-alpha-GalNAc-GalNAc) which expresses Group C antigenic activity. This disaccharide, which exists as a side chain in the intact polysaccharide, can completely inhibit the binding between Group C polysaccharide and most Group C antibodies, indicating that this unit is the immunodominant feature of the intact polysaccharide. The alpha anomeric configuration and N-acetylation are required for the expression of the antigenic activity by the haptenic disaccharide. Also obtained from the acid hydrolysis of the Group C polysaccharide are rhamnose oligosaccharides with structural identity to the Group A-variant polysaccharide and with Group A-variant antigenic activity. It is inferred from these data that the Group A-varient polysaccharide structure is the core unit of the Group C polysaccharide. The nature of the immunologic crossreactivity between the Forssman glycolipid and Group C polysaccharide, which possess identical nonreducing terminal digalactosamine units, was investigated. Rabbit anti-Group C antibodies bound the Forssman glycolipid with approximately the same affinity as 3-O-alpha-GalNAc-GalNAc and were capable of mediating lysis of sheep red blood cells (SRBC). Antibody fractions isolated from anti-sheep hemolysin were likewise able to bind Group C polysaccharide. The heterologous reactions were in most assay systems weaker than reactions with the immunizing antigen.

Amino Sugars↗
PubMed1986

Secretory granules of heparin-containing rat serosal mast cells also possess highly sulfated chondroitin sulfate proteoglycans.

Rat serosal mast cells, which synthesize only heparin proteoglycans as detected by intrinsic labeling with [35S]sulfate, were analyzed for the presence of intracellular chondroitin sulfate proteoglycans by chemical and immunochemical means. Rat serosal mast cells of greater than 99% purity were treated with Zwittergent 3-12 and 4 M guanidine HCl, and the extracted nonradiolabeled proteoglycans were purified by density gradient centrifugation. As assessed by quantification of the unsaturated disaccharides released from the proteoglycans by chondroitinase ABC treatment, 10(6) rat serosal mast cells contained 2.4-4.5 micrograms of chondroitin sulfate proteoglycans. Analysis of the chondroitinase ABC digests by high performance liquid chromatography revealed the unsaturated disaccharides delta Di-4S, delta Di-diSB, and delta Di-diSE which were derived from GlcA----GalNAc-4-SO4, iduronic acid-2-SO4----GalNAc-4-SO4, and GlcA----GalNAc-4,6-diSO4, respectively. The molar ratio of the monosulfated to disulfated disaccharides was approximately 2:1 with delta Di-diSE greater than delta Di-diSB. When analyzed with a mouse anti-chondroitin sulfate monoclonal antibody and fluorescein-labeled F(ab')2 goat anti-mouse IgG, approximately 91% of permeabilized and chondroitinase ABC-treated cells in the mast cell preparations exhibited intracellular fluorescence, and the pattern of staining indicated that the chondroitin sulfate molecules were located in the secretory granules. The specificity of the monoclonal antibody for the unsaturated double bond created by chondroitinase ABC treatment of the proteoglycan in situ was established by the absence of fluorescence when the chondroitinase ABC step was omitted or when heparinase digestion was substituted for chondroitinase ABC. Furthermore, the ability of the anti-chondroitin sulfate monoclonal antibody to mediate fluorescence in situ was markedly reduced by absorption with solid-phase chondroitin sulfate proteoglycan that had been chondroitinase ABC-treated, but not by absorption with undigested proteoglycan or with solid-phase heparin. The highly sulfated chondroitin sulfate proteoglycans of rat serosal mast cells are the same type synthesized by the rat mucosal mast cell subclass.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Analysis of the major antigenic determinants of the characteristic phenolic glycolipid from Mycobacterium leprae.

Antibodies to the major phenolic glycolipid purified from Mycobacterium leprae have been demonstrated previously in sera of leprosy but not tuberculosis patients using an ELISA. The major antigenic determinants on this molecule were investigated using antisera raised in rabbits to the purified glycolipid and with a pool of sera from human lepromatous leprosy patients. A small, but significant cross-reaction was observed with the glycolipids from M. bovis and M. kansasii, which contain the phenolphthiocerol dimycocerosate part of the molecule but have different sugars, and also with a semi-synthetic 'attenuation indicator lipid' which shares the phenolphthiocerol but has no sugars. There was however no cross-reaction with phthiocerol dimycocerosate. The disaccharide, corresponding to the two terminal sugars of the M. leprae glycolipid has been chemically synthesized and shown to inhibit the reaction between glycolipid and antibody in the ELISA. The cross-reactivity observed with the M. bovis and M. kansasii glycolipids was not inhibited by the synthetic disaccharide. These findings suggest that the cross-reactivity is associated with the phenol ring and implies the disaccharide may be a unique antigenic determinant of M. leprae.

Animals↗
PubMed1980

Immunochemistry of Salmonella O-antigens: specificity of rabbit antibodies against the O-antigen 2 determinant elicited by whole bacteria and 3-O-alpha-paratopyranosyl-D-mannopyranosyl conjugated to bovine serum albumin.

The binding specificities of antibodies directed against the Salmonella sero-group A-specific O-antigen 2 determinant were characterized by precipitation-inhibition and enzyme-linked immunosorbent assay inhibition tests. Two different antigen O-2-specific antisera were investigated: one conventional factor O-2 serum (elicited by whole heat-killed Salmonella paratyphi A bacteria) and another elicited by the synthetic disaccharide 3-O-a-paratopyranosyl-D-mannopyranosyl (formula: see text) covalently linked via a p-isothiocyanatophenyl aglycon to bovine serum albumin (PM-BSA). The inhibition data showed that factor O-2 antibodies have combining sites which recognize structures larger than the (formula: see text) disaccharide and equal to or smaller than an O-antigen O-2-specific octasaccharide derived from the S. paratyphi A O-polysaccharide. Although the factor O-2 serum exhibited a high specificity for the homologous S. paratyphi A O-antigen it still precipitated, though weakly, a heterologous Salmonella typhimurium O-antigen. In contrast, anti-PM-BSA antibodies were exclusively specific for the O-2 determinant of the native polysaccharide antigen. The combining sites of these antibodies best recognized the (formula: see text) disaccharide, including the linkage arm and the lysyl residue of the BSA carrier protein molecule. These data extend earlier findings as to the superior specificity of anti-PM-BSA antibodies as compared to conventional factor O-2 antibodies.

Animals↗
PubMed1994

Sequencing of heparan sulfate proteoglycans: identification of variable and constant oligosaccharide regions in eight heparan sulfate proteoglycans of different origins.

The sequence of the disaccharide units of eight heparan sulfate proteoglycans of different origins is described. All heparan sulfates contain 5 variable regions made of oligosaccharide blocks of disaccharides, namely, GlcUA(1-4)GlcNAc, GlcUA(1-4)GlcNS, IdoUA (1-4) GlcNS,6S,IdoUA-GlcNAc,6S, and IdoUA,2S(1-4)GlcNS,6S, besides two constant regions made of an internal tetrasaccharide (IdoUA-GlcNAc-IdoUA-GlcNS) and monosaccharides (GlcNS, and GlcNS,6S) at the non-reducing terminal. The N-acetylated region of the heparan sulfates is linked to the serine of the protein core through a trisaccharide of Xyl-Gal-Gal. Heparan sulfates differ from one another in terms of the number of disaccharides that compose each block.

Acetylation↗
PubMed1994

Oligosaccharide sequences of endothelial cell surface heparan sulfate proteoglycan with affinity for lipoprotein lipase.

Lipoprotein lipase (LpL) catalyzes the hydrolysis of triglycerides in plasma lipoproteins at the luminal surface of the vascular endothelium. This enzyme is bound via electrostatic interactions to heparan sulfate (HS). The specific endothelial cell surface HS oligosaccharide sequences that are necessary for binding of LpL to HS have not been characterized. To identify this LpL-binding oligosaccharide sequence, oligosaccharides were isolated from bovine aortic endothelial cell-derived HS and assessed for LpL binding properties. Endothelial HS chains that were isolated from endothelial total cell-associated proteoglycans were deacetylated by complete hydrazinolysis, cleaved with nitrous acid (pH 4.5), and reduced with [3H]NaBH4. The resulting fragments composed of N-sulfated glucosamine-rich oligosaccharides terminating with [3H]2,5-anhydromannitol (AManR) were chromatographed on a LpL-Sepharose column. A high affinity decasaccharide was isolated and characterized. Disaccharide analysis of this decasaccharide indicated that it yielded only the disaccharide IdceA(2-SO4)-->AManR(6-SO4) on treatment with nitrous acid at low pH. Therefore, the sequence of the LpL-binding decasaccharide is [IdceA(2-SO4) alpha 1-4GlcNSO4(6-S0(4)) alpha 1-4]4-IdceA(2-SO4) alpha 1-4AManR(6-SO4) and is distinct from those that bind antithrombin and basic fibroblast growth factor. Partial depolymerization of endothelial HS chains with hydrazine/high pH nitrous acid treatment gave rise to lipase-binding oligosaccharides larger than decasaccharide. However, further complete depolymerization of these oligosaccharides resulted in only a high affinity decasaccharide composed of repeating disaccharide units of [IdceA(2-SO4) alpha 1-4GlcNSO4(6-S0(4))]. These results indicate that the decasaccharide is the active fragment that binds to LpL with high affinity. Molecular modeling studies of the decasaccharide indicate that it presents a linear array of negatively charged sulfate groups that may adopt a favorable disposition to bind to peptide region(s) comprised of basic amino acid residues of LpL with high affinity.

Animals↗
PubMed1998

Changes in sulfation patterns of chondroitin sulfate in equine articular cartilage and synovial fluid in response to aging and osteoarthritis.

OBJECTIVES: To determine effects of aging on sulfation of chondroitin sulfate (CS) in articular cartilage and synovial fluid from normal equine middle carpal joints, and to determine whether CS compositional analysis can be used to assess alterations in proteoglycan turnover in degenerative cartilage obtained from horses with carpal osteochondral fractures. SAMPLE POPULATION: Carpal articular cartilage and synovial fluid from 44 cadavers with normal joints and from 16 Thoroughbred racehorses during routine carpal arthroscopic surgery. PROCEDURE: After papain/chondroitinase digestion of cartilage, CS disaccharides (unsulfated disaccharide delta Di0S, and monosulfated disaccharides delta Di4S and delta Di6S) were quantified by capillary zone electrophoresis. The CS was purified from synovial fluid chondroitinase digested, and analyzed. The CS nonreducing terminal residues, N-acetylgalctosamine (galNAc) or glucuronic acid adjacent to a 4-sulfated or 6-sulfated galNAc, were quantified. RESULTS: In cartilage, the delta Di6S-to-delta Di4S ratio increased with age; in degenerative cartilage, this ratio was not significantly different from the normal value. Percentage of delta Di0S decreased with age and was significantly higher in degenerative than in normal cartilage. The galNAc4S and galNAc4,6S represented > or = 96% of the terminal residues. There was a significant decrease in 6-sulfation of the terminal residues in degenerative cartilage. CONCLUSIONS: 6-Sulfation of internal and terminal CS residues increased with age. Cartilage degeneration in racehorses was accompanied by deposition of CS chains with altered sulfation patterns, in normal and diseased joints of horses > 2 years old, synovial fluid CS was not indicative of cartilage CS and may represent turnover products of a subpopulation of proteoglycan within the matrix.

Aging↗
PubMed1998

Pulmonary metastases of breast carcinomas: ligandohistochemical, nuclear, and structural analysis of primary and metastatic tumors with emphasis on period of occurrence of metastases and survival.

BACKGROUND AND OBJECTIVES: Pulmonary metastases of breast carcinomas have a high frequency and are often subject to surgical intervention. To contribute to advances in the knowledge about morphometric and biochemical parameters of primary tumors and their metastatic lesions, analysis of syntactic structure and thermodynamic aspects as well as of expression of distinct glycohistochemical features with respect to period of metastasis occurrence and patient survival is desirable. METHODS: Clinical history, surgical findings, histopathological reports, survival of the patients with a maximum follow-up of 15 years, and paraffin blocks of 32 breast carcinoma specimens and their pulmonary metastases were examined. Only potentially curative resections of both the breast carcinoma and their metastases have been included for analysis. The following markers were applied: neoglycoconjugates with histoblood group A- and H-trisaccharides, lactose, alpha-N-acetyl-D-galactosamine and the Forssman disaccharide, a polyclonal immunoglobulin G fraction from human serum with specificity for 9-0-acetylated sialic acid, which is a tumor marker for melanomas, the serum lectins serum amyloid P component and mannan-binding lectin, the mannose-specific plant lectin concanavalin A, and monoclonal antibodies specific for estrogen and progesterone receptors, respectively. In addition, measurements of the integrated optical density (IOD) and tissue structure were performed. RESULTS: The frequency of expression of hormone receptors and expression of binding capacities to most of the applied probes was similar between the primary and metastatic tumors; however, it varied markedly between different patients. For the IOD parameters, a close association between the primary tumors and their metastases was seen, especially a rather low S-phase-related tumor cell fraction and a high percentage of tumor cells with an IOD >5C. The mean time for the development of intrapulmonary metastases measured 43 months. It was considerably longer in progesterone receptor-negative tumors (49.7 months) and those with a lack of expression of sites with specificity for the Forssman disaccharide (48.7 months). The survival was positively correlated with the presence of binding capacity of histoblood group A-trisaccharide and certain structural parameters, especially the structural entropy and its current. The presence of estrogen and progesterone receptors was not associated with the total survival at a statistically significant level. CONCLUSIONS: Histochemical features between the primary breast carcinoma and their intrapulmonary metastases can evidently vary. Analysis of the hormone receptor status in metastatic lesions seems to be useful for diagnostic purposes only in rare cases, i.e., distinguishing metastases from primary lung carcinoma. Nonetheless, the survival of patients with metastasizing breast carcinoma is associated with features of the primary tumors, especially the detection of binding capacities for the Forssman disaccharide and the histoblood group A-trisaccharide. Extent of lymph node involvement of the breast carcinoma is not prognostic for later pulmonary involvement.

Adult↗
PubMed1996

Characterisation of the tumour-associated carbohydrate epitope recognised by monoclonal antibody 4D3.

The tumour-associated epitope recognised by monoclonal antibody (MAb) 4D3 is expressed on a high m.w. mucin glycoprotein preparation known as small intestinal mucin antigen (SIMA). This epitope is detected in tissue from a high proportion of patients with colorectal cancer, and elevated levels occur in serum from a significant number of such patients, highlighting the potential clinical utility of MAb 4D3. In the present study, insight into the composition and structure of the carbohydrate epitope recognised by MAb 4D3 was gained following characterisation of 2 glycopeptides that co-purified with SIMA. Sequence analysis of 1 of these glycopeptides revealed that it was identical to the glycoprotein alpha-1-anti-chymotrypsin. This glycoprotein was subsequently deglycosylated to yield 5 forms corresponding to alpha-1-anti-chymotrypsin substituted with 4, 3, 2, 1 or no branched glycans. MAb 4D3 was reactive with each of the glycosylated forms, including the form carrying only 1 branched glycan, but did not react with fully deglycosylated alpha-1-anti-chymotrypsin. MAb 4D3 also reacted to different extents with ovine, bovine or porcine submaxillary mucins, each of which has a different amount of the O-linked sialylated disaccharide known as sialosyl Tn. Of these mucins, MAb 4D3 was most reactive with ovine submaxillary mucin, in which almost all of the carbohydrate chains are sialosyl Tn. Reactivity of MAb 4D3 towards isolated glycans, sialosyl Tn and related structures led to the conclusion that the preferred MAb 4D3 epitope involves the sialylated N-acetyl galactosamine disaccharide as well as an additional monosaccharide present on a neighbouring carbohydrate chain. Although the preferred epitope recognised by MAb 4D3 involves this sialylated disaccharide, the specificity of MAb 4D3 was different from that of other MAbs with a reported specificity for sialosyl Tn.

Amino Acid Sequence↗
PubMed2000

Synthesis of the sialyl Lewis X epitope attached to glycolipids with different core structures and their selectin-binding characteristics in a dynamic test system.

Sialyl Lewis X (sLeX)/selectin-mediated leukocyte rolling along endothelial cells has recently gained wide interest. In this paper the influence of the spacer length of laterally clustered neoglycolipids 1a-d on cell rolling in a dynamic test system is investigated. The required di-O-hexadecyl glycerols with none, and with three, six, or nine ethylene glycol units as spacer groups (compounds 4a-d) could be readily obtained. The synthesis of 1-O-thexyldimethylsilyl-protected sLeX 24 was based on sialylation of 2,3,4-O-unprotected galactose derivative 11 with sialyl phosphite 8 as donor; this afforded the desired disaccharide 12, which was transformed into trichloroacetimidate 14 as disaccharide donor. Reaction of 3-O-unprotected glucosamine derivative 18 with fucosyl donor 20 afforded disaccharide 21, which was transformed into the 4-O-unprotected derivative 23. Reaction of 14 with 23 furnished the desired tetrasaccharide 24 in good yield. Transformation of 24 into the trichloroacetimidate 26 as donor, followed by the reaction with 4a-d as acceptor gave, after deprotection, the target molecules 1a-d. For comparison, 4d was also connected with a sialyl residue (-->31) and with an N-acetylglucosamine residue (-->34). Compounds 1c and 1d with a hexaethylene glycol and a nonaethylene glycol spacer, respectively, were much more efficient in mediating selectin-dependent cell rolling in the dynamic test system than compounds 1a and 1b, which had no spacer (1a), or only a triethylene glycol spacer (1b).

Animals↗
PubMed1994

Molecular dynamics simulation of oligosaccharides containing N-acetyl neuraminic acid.

alpha D-N-acetyl neuraminic acid (Neu5Ac, sialic acid) is a commonly occurring carbohydrate residue in various cell surface glycolipids and glycoproteins. This residue is linked terminally or internally to Gal residues via an alpha(2-->3) or alpha(2-->6) linkage. In the cell surface receptor, sialyl-LewisX, a terminal alpha(2-->3) linkage is present. Previous studies from our laboratory have shown that in solution LewisX adopts a relatively rigid structure. In order to model the Neu5Ac residue, vacuum molecular dynamics of this monosaccharide were compared with simulations that explicitly include solvent water. The dynamical average of the monosaccharide conformation obtained from the two simulations was similar. Vacuum calculations for the disaccharide Neu5Ac alpha(2-->3) Gal beta-O-methyl show that a number of low energy minima are accessible to this disaccharide. Molecular dynamics simulations starting from the low energy minima show conformational transitions with a time scale of 10-50 ps among several of the minima while large barriers between other minima prevent transitions on the time scale studied. Simulations of this disaccharide in the presence of solvent show fewer conformational transitions, illustrating a dampening effect of the solvent that has been observed in some other studies. Our results are most consistent with an equilibrium among multiple conformations for the Neu5Ac alpha(2-->3) Gal beta linkage.

Carbohydrate Conformation↗