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PubMed1992

[Reproducibility of orocecal transit time and hydrogen production measured by breath tests].

The hydrogen breath test after a lactulose oral load in the fasting period is currently used to measure mouth to cecum transit time (MCTT). However, the reproducibility of this test is poor, and normal values are very scattered. The aim of the study was to determine the reproducibility of hydrogen breath test for MCTT measurement and hydrogen production after administration of 2 disaccharides: lactulose and lactitol ingested in the fasting state and postprandial period. Twelve healthy volunteers (6 men and 6 women; mean age = 34.6 +/- 9.6 years) were studied eight times in a random order, each disaccharide being studied twice in the fasting state and twice in the postprandial period. In the later, lactulose or lactitol was ingested 30 min after a liquid meal completely absorbed (400 kcal; glucide: 55 p. 100, lipid: 30 p. 100, protein: 15 p. 100; 400 ml of Inkopeptide). The MCTT was significantly increased with both disaccharides in the postprandial period as compared with the fasting state (P less than 0.0001). There was nos significant correlation between the 2 measurements of the MCTT in the fasting state, in contrast, the 2 measurements of the MCTT were closely related in the fed state (r = 0.62, P less than 0.05, et r = 0.79, P less than 0.003 for lactulose and lactitol respectively). During both periods no significant difference was found in the MCTT between lactulose and lactitol. As well, hydrogen production did not differ between the 2 disaccharides, but was significantly increased in the postprandial period, and in non methane producers.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

[Synthesis of spaced trisaccharides with blood group A and B specificity, their fragments, and structural analogs].

Trisaccharides GalNAc alpha 1----3(Fuc alpha 1----2)Gal and Gal alpha 1----3(Fuc alpha 1----2)Gal, which are the determinant fragments of the human blood group specific antigens A and B, respectively, were synthesized as R-glycosides (R = beta-OCH2CH2CH2NHCOCF3). 4,6-BdGal-R was acetylated selectively at 3-OH, the 3-O-acetate was alpha-fucosylated and then deacetylated to give a protected H-disaccharide bearing a free 3-OH. The disaccharide was alpha-glycosylated with 2-azido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-galactopyranosyl chloride (GCl) or 2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl bromide (GBr) to give protected spacered trisaccharides A and B, respectively. Disaccharides GalNAc alpha 1----3Gal-R and Gal alpha 1----3Gal-R were synthesized in two ways. 1. Hydrogenolysis followed by benzylidenation of Bzl3-2-O-Ac-Gal-R gave 4,6-Bd-2-O-Ac-Gal-R, which was alpha-glycosylated with GCl and GBr. 2. The required disaccharides were isolated from the mixture of di- and trisaccharides which was obtained by selective glycosylation of 4,6-Bd-Gal-R with GCl and GBr. Synthesis of GalNAc alpha 1----3(GalNAc alpha 1----2)Gal and Gal alpha 1----3(Gal alpha 1----2)Gal, non-natural analogues of A and B trisaccharides, is also described. Deprotected R-glycosides were converted to OCH2CH2CH2NH2 (R1) derivatives, N-biotinyl derivatives were synthesized from GalNAc alpha 1----3(Fuc alpha 1----2)Gal-R1 and Gal alpha 1----3(Fuc alpha 1----2)Gal-R1 glycosides. The oligosaccharides, their macromolecular forms, and affinity sorbents obtained from them were used in the epitope specificity studies of the monoclonal antibodies and blood group typing.

ABO Blood-Group System↗
PubMed1990

Structure of a dermatan sulfate hexasaccharide that binds to heparin cofactor II with high affinity.

Dermatan sulfate increases the rate of inhibition of thrombin by heparin cofactor II (HCII) approximately 1000-fold by providing a catalytic template to which both the inhibitor and the protease bind. Dermatan sulfate is a linear polymer of D-glucuronic acid (GlcA) or L-iduronic acid (IdoA) alternating with N-acetyl-D-galactosamine (GalNAc) residues. Heterogeneity in dermatan sulfate results from varying degrees of O-sulfation and from the presence of the two types of uronic acid residues. To characterize the HCII-binding site in dermatan sulfate, we isolated the smallest fragment of dermatan sulfate that bound to HCII with high affinity. Dermatan sulfate was partially N-deacetylated by hydrazinolysis, cleaved with nitrous acid at pH 4, and reduced with [3H]NaBH4. The resulting fragments, containing an even number of monosaccharide units with the reducing terminal GalNAc converted to [3H]2,5-anhydro-D-talitol (ATalR), were size-fractionated and then chromatographed on an HCII-Sepharose column. The smallest HCII-binding fragments were hexasaccharides, of which approximately 6% bound. Based on ion-exchange chromatography, the bound material appeared to comprise a heterogeneous mixture of molecules possessing four, five, or six sulfate groups per hexasaccharide. Subsequently, hexasaccharides with the highest affinity for HCII were isolated by overloading the HCII-Sepharose column. The high-affinity hexasaccharides were fractionated by strong anion-exchange chromatography, and one major peak representing approximately 2% of the starting hexasaccharides was isolated. The high-affinity hexasaccharide was cleaved to disaccharides that were analyzed by anion-exchange chromatography, paper electrophoresis, and paper chromatography. A single disulfated disaccharide, IdoA(2-SO4)----ATalR(4-SO4) was observed, indicating that the hexasaccharide has the following structure: IdoA(2-SO4)----GalNAc(4-SO4)----IdoA(2-SO4)---- GalNAc(4-SO4)----IdoA(2-SO4)----ATalR(4-SO4). Since IdoA(2-SO4)----GalNAc(4-SO4) comprises only approximately 5% of the disaccharides present in intact dermatan sulfate, clustering of these disaccharides must occur during biosynthesis to form the high-affinity binding site for HCII.

Animals↗
PubMed1985

Purification and characterization of protease-resistant secretory granule proteoglycans containing chondroitin sulfate di-B and heparin-like glycosaminoglycans from rat basophilic leukemia cells.

Proteoglycans were extracted from nuclease-digested sonicates of 10(9) rat basophilic leukemia (RBL-1) cells by the addition of 0.1% Zwittergent 3-12 and 4 M guanidine hydrochloride and were purified by sequential CsCl density gradient ultracentrifugation, DE52 ion exchange chromatography, and Sepharose CL-6B gel filtration chromatography under dissociative conditions. Between 0.3 and 0.8 mg of purified proteoglycan was obtained from approximately 1 g initial dry weight of cells with a purification of 200-800-fold. The purified proteoglycans had a hydrodynamic size range of Mr 100,000-150,000 and were resistant to degradation by a molar excess of trypsin, alpha-chymotrypsin, Pronase, papain, chymopapain, collagenase, and elastase. Amino acid analysis of the peptide core revealed a preponderance of Gly (35.4%), Ser (22.5%), and Ala (9.5%). Approximately 70% of the glycosaminoglycan side chains of RBL-1 proteoglycans were digested by chondroitinase ABC and 27% were hydrolyzed by treatment with nitrous acid. Sephadex G-200 chromatography of glycosaminoglycans liberated from the intact molecule by beta-elimination demonstrated that both the nitrous acid-resistant (chondroitin sulfate) and the chondroitinase ABC-resistant (heparin/heparan sulfate) glycosaminoglycans were of approximately Mr 12,000. Analysis of the chondroitin sulfate disaccharides in different preparations by amino-cyano high performance liquid chromatography revealed that 9-29% were the unusual disulfated disaccharide chondroitin sulfate di-B (IdUA-2-SO4----GalNAc-4-SO4); the remainder were the monosulfated disaccharide GlcUA----GalNAc-4-SO4. Subpopulations of proteoglycans in one preparation were separated by anion exchange high performance liquid chromatography and were found to contain chondroitin sulfate glycosaminoglycans whose disulfated disaccharides ranged from 9-49%. However, no segregation of subpopulations without both chondroitin sulfate di-B and heparin/heparan sulfate glycosaminoglycans was achieved, suggesting that RBL-1 proteoglycans might be hybrids containing both classes of glycosaminoglycans. Sepharose CL-6B chromatography of RBL-1 proteoglycans digested with chondroitinase ABC revealed that less than 7% of the molecules in the digest chromatographed with the hydrodynamic size of undigested proteoglycans, suggesting that at most 7% of the proteoglycans lack chondroitin sulfate glycosaminoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗
PubMed1985

Histochemical examination of glycoconjugates of epithelial cells in the human fallopian tube.

In human fallopian tubes, secretory cells made up about 70% and ciliated cells most of the remainder of the epithelium. At the light microscopic level, lectin-peroxidase conjugates and other methods demonstrated on secretory cells a surface glycocalyx containing glycoconjugates with fucose residues, terminal sialic acid-beta-galactose disaccharides, and sulfate esters. Secretory cells also expressed ABO blood group antigens, on their surface, staining for terminal alpha-N-acetylgalactosamine and alpha-galactose in blood group A and B subjects, respectively. The glycocalyx coating the cilia of all ciliated cells contained an acidic glycoconjugate with terminal sialic acid-beta-galactose disaccharides. Ciliated cells ranged from strongly positive to negative in staining for blood group ABO antigens, fucose, and terminal beta-galactose. The glycocalyx on the surface of cilia differed from that on the secretory cell surface in lacking sulfate esters and possessing terminal sialic acid-beta-galactose disaccharides cytochemically unlike those on secretory cells. Cytoplasmic granules, presumably secretory in nature, stained for blood group antigen in secretory cells of some specimens. Two lectin conjugates with affinity for the core region of N-glycosidically linked oligosaccharides revealed bodies interpreted as lysosomes in ciliated and secretory cells of most specimens. The latter lectins also stained mast cells intensely, presumably because of affinity for N-acetylglucosamine. The luminal surface of endothelial cells stained heavily for fucose and for a terminal sialic acid-beta-galactose disaccharide like that on the surface of cilia. The staining for carbohydrate did not vary qualitatively, and the ratio of secretory to ciliated cells did not differ with the phase of the menstrual cycle. The salpingeal epithelium in two patients with inactive endometrium did not differ from the others. The epithelium in a specimen from a patient with uterine fibroids showed foci of mucous metaplasia, containing a nonsulfated sialylated glycoconjugate. At the electron microscopic level, fucosylated glycoconjugate was visualized with a specific lectin conjugate on the surface of the secretory cells but not on the surface of ciliated and endothelial cells. Infrequent cytoplasmic granules containing mucosubstance apparently provided the cell mechanism in secretory cells for collecting and transporting material from biosynthetic sites to the glycocalyx.(ABSTRACT TRUNCATED AT 400 WORDS)

ABO Blood-Group System↗
PubMed1988

Differential binding properties of Ga1NAc and/or Ga1 specific lectins.

Grouping of lectin binding properties, based on determinant structure rather than monosaccharide inhibition pattern, should facilitate the selection of lectins as structural probes for glycans as well as for the interpretation of the distribution and the properties of the carbohydrate chains on the cell surface. Based on the binding specificities studied with glycan by precipitin-inhibition, competitive-binding and hemagglutinin-inhibition assays, twenty Ga1 and/or Ga1NAc specific lectins have been divided into six classes according to their specificity for the disaccharide as all or part of the determinants and Ga1NAc alpha 1----Ser(Thr) of the peptide chain. The differential affinities of these lectins were characterized by quantitative precipitin assay. Abbreviation of the following six lectin determinants can also be used to classify these lectins. (1) F determinant (GalNAc alpha 1----3GalNAc, Forssman specific disaccharide). (2) A (Af) determinant (GalNAc alpha 1----3Gal, Human blood group A specific disaccharide; Af, fucosylated A, (GalNAc alpha 1----3 [LFuc alpha 1----2]Gal). (3) Tn determinant (GalNAc alpha 1----0 to Ser (Thr) of the protein core, Tn antigen). (4) T determinant (T antigen, Gal beta 1----3GalNAc alpha 1----0 to Ser (Thr) of the protein core, the mucin type sugar sequence on the human erythrocyte membrane or Gal beta 1----3GalNAc beta 1---- at the nonreducing end of ganglioside). (5) I and II determinants (human blood group type I and II carbohydrate sequences). Most of the lectins reactive to Gal beta 1----4GlcNAc (II) are also reactive to Gal beta 1----3GlcNAc (I). Lectin I (II) determinants (i.e. Gal beta 1----3 (4) GlcNAc residues) can be found at the nonreducing end of the carbohydrate chains derived from either N-glycosidic or O-glycosidic linkages. (6) B determinant (Gal alpha 1----3Gal, Human blood group B specific disaccharide). Their carbohydrate specificities are classified as following: (Table see text). The differential binding properties of lectins can be defined from comparisons of their carbohydrate specificities listed above.

Acetylgalactosamine↗
PubMed1979

Carbohydrate transport in Moniliformis dubius (Acanthocephala). III. Post-absorptive fate of fructose, mannose, and galactose.

The initial metabolism of fructose, mannose, and galactose in Moniliformis dubius (M. moniliformis; Acanthocephala) was examined following brief aerobic incubations in 14C-labeled substrate. The pattern of incorporation of radioactivity from 14C-fructose and 14C-mannose into intermediates of hexose metabolism was indistinguishable from that reported elsewhere for the initial post-absorptive metabolism of 14C-glucose under comparable conditions; these hexoses were phosphorylated rapidly following their absorption, and much of the radioactivity absorbed as mannose or fructose was recovered in the nonreducing disaccharide trehalose [alpha-D-glucopyranosyl-(1 leads to 1)-alpha-D-glucopyranoside]. 14C-Galactose was phosphorylated less readily than the other hexoses. More than half of the radioactivity absorbed as galactose was incorporated into disaccharide; some of the galactose-derived disaccharide had properties suggesting the presence of a galactosyl moiety. Incorporation of radioactivity from any of the hexoses into glycogen was minimal. The extensive incorporation of hexose moieties into trehalose or a trehalose-like disaccharide rather than glycogen underscores the probable importance of trehalose metabolism to carbohydrate assimilation in Moniliformis. Physiological factors which might favor trehalose biosynthesis over glycogenesis are considered.

Absorption↗
PubMed1984

Isolation and structural studies of heparan sulfates and chondroitin sulfates from three species of molluscs.

The isolation and some structural features of heparan sulfates and chondroitin sulfates from three species of molluscs (Pomacea sp., Tagelus gibbus, and Anomalocardia brasiliana) are reported. It is shown that heparan sulfates with structural similarities to the ones found in mammals are present in the three molluscs analyzed. All the heparan sulfates were degraded by heparitinases I and II to four distinct unsaturated disaccharides with the same properties as the ones present in heparan sulfates of mammalian origin. This suggests that these four disaccharide units are maintained through the evolution. Furthermore, the proportion of these units varied in the heparan sulfates according to the species of origin. The chondroitin sulfates, on the other hand, exhibit different structural features according to the species of origin. For instance, by the action of chondroitinase AC, 4- and nonsulfated disaccharides are produced from Pomacea chondroitin, whereas 4- and 6-sulfated disaccharides are formed from Tagelus and Anomalocardia. The possible role of these compounds in cell recognition and/or adhesiveness is discussed in view of the present findings.

Animals↗
PubMed1982

Culture from mouse bone marrow of a subclass of mast cells possessing a distinct chondroitin sulfate proteoglycan with glycosaminoglycans rich in N-acetylgalactosamine-4,6-disulfate.

A differentiated population of cells with metachromatically staining granules and surface IgE receptors was obtained from mouse bone marrow cultured for 2 weeks in the presence of conditioned medium derived from concanavalin A-stimulated splenocytes. The cells were found to incorporate large amounts of [35S]sulfate into an intracellular 35S-labeled proteoglycan of Mr approximately 200,000 containing a maximum of seven glycosaminoglycan side chains (Mr = 25,000). After chondroitinase ABC treatment of density gradient-purified [3H] serine-labeled proteoglycan, the resulting core was Mr approximately 26,000 as assessed by gel filtration. Two-dimensional cellulose acetate electrophoresis of beta-eliminated 35S-labeled glycosaminoglycan revealed a single type of glycosaminoglycan that migrated at the position of oversulfated chondroitin sulfate E from squid cartilage. Chondroitinase ABC degradation of the 35S-labeled glycosaminoglycan yielded two cleavage products in approximately equal molar amounts which co-migrated in both descending paper chromatography and high voltage paper electrophoresis with a monosulfated disaccharide, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose, and a disulfated disaccharide, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-6-di-O-sulfo-D-galactose. The release of some free [35S]sulfate from the oversulfated disaccharide with either chondro-4-sulfatase or chondro-6-sulfatase and the complete desulfation by their combined action established that the oversulfated disaccharide contained N-acetylgalactosamine-4,6-disulfate. The 35S]labeled proteoglycan of these unique IgE receptor-bearing and histamine-containing cells, therefore, is composed of chondroitin sulfate E rather than heparin glycosaminoglycan, and thus is the first identification of such an intracellular localized proteoglycan in a mammalian cell.

Acetylgalactosamine↗
PubMed1994

Oligosaccharide-receptor interaction of the Gal alpha 1-4Gal binding adhesin of Streptococcus suis. Combining site architecture and characterization of two variant adhesin specificities.

The sugar binding specificities of two groups of Streptococcus suis, a pig pathogen that causes meningitis also in man, were determined. Both the group represented by a recently characterized strain inhibitable by galactose and N-acetylgalactosamine (type PN) and the group inhibitable by galactose (type PO) were found by hemagglutination and solid-phase binding inhibition experiments to recognize the disaccharide Gal alpha 1-4Gal of the P1 and Pk blood group antigens. Both types preferred the disaccharide in terminal position. PN showed some, whereas PO showed almost no, binding to the globoside oligosaccharide containing an additional GalNAc beta 1-3 residue. The complete hydrogen bonding patterns were determined by using deoxy and other synthetic derivatives of the receptor disaccharide, and the constructed models of the interactions were compared with that of Escherichia coli PapG396 adhesin. The essential hydroxyls for binding were the HO-4', HO-6', HO-2, and HO-3 hydroxyls on the beta' alpha-side of the Gal alpha 1-4Gal molecule. Type PO adhesin also formed weak interactions with the hydroxyls HO-6 and HO-3'. The mechanism differed from that of E. coli, which binds to a cluster of five hydroxyls (HO-6, HO-2', HO-3', HO-4', and HO-6') and thus to a different part of the receptor disaccharide. These results represent the first example of the comparison of the saccharide receptor hydrogen bonding patterns of two bacterial organisms of different origin and show that the same saccharide may be recognized by two different binding mechanisms.

Adhesins, Bacterial↗
PubMed1996

[Isolation and characteristics of galactose-binding lectins from human blood serum].

Two galactose-binding lectins, SL1 and SL2, were isolated from human serum by two-step affinity chromatography on sorbents with immobilized disaccharides Gal beta 1-3GlcNAc beta (Lec) and Fuc alpha 1-2Gal beta (Hdi). The purification degree of the SL1 and SL2 preparations was 4000-6000 times and yields were 6.9 and 4.7 micrograms/ml of serum, respectively. Electrophoresis showed that both lectins are oligomers with molecular masses of about 440 kDa, which are composed of ca. 67 kDa subunits. The carbohydrate specificity of the lectins was assayed by hemagglutination inhibition using mono- and oligosaccharides. Both lectins showed specificity for Gal and GalNAc residues but differed in the interactions with oligosaccharides. In particular, SL1 showed maximum affinity for disaccharide Gal beta 1-3GalNAc beta (in the form of 4-nitrophenyl glycoside), 6'-sialyllactose, and disaccharide residues Fuc alpha 1-3Gal beta and Gal alpha 1-3Gal beta, whereas SL2 was specific for GalNAc alpha residue and its derivatives as well as for disaccharide Hdi.

Carbohydrate Sequence↗
PubMed1997

Epitope mapping of twelve monoclonal antibodies against the phenolic glycolipid-I of M. leprae.

Epitope mapping of 12 monoclonal antibodies (MAbs) directed to the trisaccharide part of the phenolic glycolipid-I (PGL-I) of Mycobacterium leprae was carried out by using the set of chemically synthesized sugar-BSA conjugates. The results can be summarized as follows: mAb (1-21), mAb (1-24) and mAb (1-25) recognized the outer (nonreducing end) monosaccharide of the trisaccharide chain of PGL-I. However, the affinity of these MAbs to the outer monosaccharide was weak. They required the contributions of some parts of the second sugar for enough affinity. MAbs ml 6A12, ml 8A2, ml 8B2, and PG2 B8F recognized the outer disaccharide. MAb F47-21-3 recognized the outer disaccharide and some parts of the third sugar. MAb SF 1 recognized the trisaccharide of PGL-I. MAb 3D1-A9 recognized the phenol group and the structure around the branching point on the carrier protein in addition to the trisaccharide. MAbs DZ 1 and 2G3-A8 had unique characters which recognized the inner part of the sugar chain. MAb DZ 1 recognized the inner (reducing end) disaccharide. MAb 2G3-A8 recognized the inner monosaccharide, phenol group and the structure around the branching point on the carrier protein. All of the MAbs tested, except for ml 6A12, recognized the anomeric configurations in the sugar parts they recognized; ml 6A12 recognized the anomeric configuration only within the outer disaccharide. This set of MAbs, which were well defined on their binding specificity, promises to be an effective tool for the immunological study of PGL-I and the clinical assessment of leprosy.

Antibodies, Bacterial↗
PubMed1998

Analysis of hyaluronic acid in human gingival crevicular fluid using high-performance liquid chromatography.

We determined the hyaluronic acid disaccharides, delta Di-HA, in the gingival crevicular fluid (GCF) and whole saliva of patients with periodontal disease, and in the peri-implant sulcus fluid (PISF) from sites around titanium osseointegrated implants, and compared these values with those in the GCF and whole saliva of controls. We also determined values for chondroitin sulfate disaccharide isomers at the same time. Glycosaminoglycans were extracted by digestion with Pronase E, followed by digestion of GAGs with hyaluronidase SD and chondroitinase ACII. Unsaturated disaccharide isomers produced from hyaluronic acid and chondroitin sulfate were analyzed by high-performance liquid chromatography (HPLC). The hyaluronic acid disaccharide delta Di-HA was found in all samples of GCF, PISF and whole saliva. The concentration of delta Di-HA in both GCF and whole saliva of the periodontitis group was greater than that in the controls. There was no difference in the concentration of delta Di-HA between the PISF and GCF of the controls. The ratios of hyaluronic acid to chondroitin sulfate in the GCF and in the whole saliva of the periodontitis group were significantly lower than that of the controls. There was no difference between the ratios in PISF and those in GCF of the controls. These results indicate that checking hyaluronic acid in GCF and whole saliva using HPLC is a useful means of assessing the condition of periodontal tissues, and that assaying hyaluronic acid in PISF may also be effective for monitoring the condition of tissues around dental implants.

Adult↗
PubMed2000

Analysis of neutral isomeric low molecular weight carbohydrates using ferrocenyl boronate derivatization and tandem electrospray mass spectrometry.

A new analytical technique for small carbohydrates utilizing the cyclic ferrocenyl boronic esters (FcBors) of several neutral mono- and disaccharides is demonstrated. Distinction between the diastereomers of mono- and disaccharides is obtained. Analysis is by tandem electrospray-mass spectrometry (ES-MS) using a modified ion-source that promotes the preformation of ions. Selection of the molecular ion produced during single-electron oxidation of the ferrocene moiety of a specific population of saccharide isomers permits a variety of collisionally induced dissociation (CID) experiments. The resultant MS2/MS3 spectra reflect the ensemble of possible cyclic esters in equilibrium. An array of stable cross pyranose ring fragment ions representing sequential carbon loss as 30 u is observed. Consequently, the system provides an information-rich set of MSn spectra containing large amounts of structural information. Identification of D-glucose (D-Glc), its two commonly found epimers (D-mannose and D-galactose), and the two major L-diastereomers of 6-dideoxymonosaccharides, L-fucose (L-Fuc) and L-rhamnose (L-Rhm), are demonstrated. Selected pairs of disaccharides can be distinguished in terms of their anomeric linkage by reference to their MS3 spectra.

Carbohydrate Sequence↗
PubMed2000

Regioselectivity of enzymatic glycosylation of 6-O-acyl glycosides in supersaturated solutions.

The regioselectivity of enzymatic transglycosylation of 6-O-acetyl glycosides in supersaturated solutions was investigated using a range of commercially available enzymes, Escherichia coli, barley, and Kluyveromyces spp. beta-galactosidase, green coffee bean alpha-galactosidase, jack bean alpha-mannosidase, rice alpha-glucosidase, and almond beta-glucosidase. It has been shown that 6-O-acetyl glycosides serve as good substrates for these enzymes, which, under the reaction conditions, are "forced" to transfer monosaccharide units to the secondary hydroxyl groups of the acceptors. In a variety of transglycosylations studied the (1-3)-linked disaccharide products were the predominant regioisomers isolated. The selectivity of the reaction varied significantly depending on the acceptor glycosides and the enzyme used. Exquisite specificity was observed in some cases, but in others approximately equal quantities of two disaccharides products were isolated. In the best transfers the yield approached 30%. The methodology described offers a quick and facile route to disaccharides that may be difficult and/or time consuming to make by conventional chemical synthesis.

Carbohydrate Conformation↗
PubMed2003

MM3 potential energy surfaces of trisaccharides. II. Carrageenan models containing 3,6-anhydro-D-galactose.

The adiabatic potential energy surfaces (PES) of six trisaccharides-namely 3,6-An-alpha-D-Galp-(1-->3)-beta-D-Galp-(1-->4)-3,6-An-alpha-D-Galp, beta-D-Galp-(1-->4)-3,6-An-alpha-D-Galp-(1-->3)-beta-D-Galp, and their derivatives sulfated on positions 2 and 4 of the beta-galactose unit-were obtained using the MM3 force field. Each PES was described by a single contour map for which the energy is plotted against the two psi glycosidic angles, given the small variations of the phi glycosidic torsional angle in the low-energy regions of disaccharide maps. In five of the six examples, the surfaces are those expected from the maps of the disaccharidic repeating units of carrageenans, with less important factors altering the additive effect of both linkages. However, when a sulfate group is present on C2 of a beta-galactose reducing end, a new low-energy minimum in a different region is produced, originated in a hydrogen bond between the first and third monosaccharidic moieties of the trisaccharide. The flexibility of the beta-linkages is nearly identical to that in their disaccharide counterparts, while that of the alpha-linkages is slightly reduced, independent of their presence closer or further away from the reducing end. A fair agreement is observed between the x-ray fiber diffraction analysis for a kappa-carrageenan double helix and the surfaces obtained for the trisaccharide analogs of that polymer.

Carbohydrate Conformation↗
PubMed2005

Application of polyacrylamide gel electrophoresis of fluorophore-labeled saccharides for analysis of hyaluronan and chondroitin sulfate in human and animal tissues and cell cultures.

Hyaluronan (HA) and chondroitin sulfate (CS) are glycosaminoglycans (GAGs) with great importance in biological events, since they participate in and regulate cell adhesion, migration and proliferation. Quantitation and analysis of the fine structure of GAGs are increasingly important for understanding many biological processes, among which are many critical aspects of pathology development and specific phenotype descriptions. Human umbilical cord and human synovial fluid are connective tissues containing high amounts of GAGs and change in the quantity and structure of these macromolecules is described in tissue development and is commonly associated with disease. Moreover, also in Xenopus laevis embryo development and chondrocyte cultures, the GAG content and structure play a critical role. A rapid analysis of hyaluronan and chondroitin sulfate Delta-disaccharides derived from the above human and animal samples, derivatized with 2-aminoacridone and analyzed by polyacrylamide gel electrophoresis, is described in this report. Qualitative and quantitative analysis were performed by comparing their migration and the pixel density with standard Delta-disaccharides, running in the same gel. Since this method allows the analysis of large numbers of samples simultaneously in one gel and has a relatively high sensitivity (less than 25 pmol), it is suggested as a cost-effective and useful tool for the fast screening of small amounts of hyaluronan and chondroitin sulfate disaccharides.

Aminoacridines↗
PubMed2004

A hydration study of (1-->4) and (1-->6) linked alpha-glucans by comparative 10 ns molecular dynamics simulations and 500-MHz NMR.

The hydration behavior of two model disaccharides, methyl-alpha-D-maltoside (1) and methyl-alpha-D-isomaltoside (2), has been investigated by a comparative 10 ns molecular dynamics study. The detailed hydration of the two disaccharides was described using three force fields especially developed for modeling of carbohydrates in explicit solvent. To validate the theoretical results the two compounds were synthesized and subjected to 500 MHz NMR spectroscopy, including pulsed field gradient diffusion measurements (1: 4.0. 10(-6) cm(2). s(-1); 2: 4.2. 10(-6) cm(2). s(-1)). In short, the older CHARMM-based force field exhibited a more structured carbohydrate-water interaction leading to better agreement with the diffusional properties of the two compounds, whereas especially the alpha-(1-->6) linkage and the primary hydroxyl groups were inaccurately modeled. In contrast, the new generation of the CHARMM-based force field (CSFF) and the most recent version of the AMBER-based force field (GLYCAM-2000a) exhibited less structured carbohydrate-water interactions with the result that the diffusional properties of the two disaccharides were underestimated, whereas the simulations of the alpha-(1-->6) linkage and the primary hydroxyl groups were significantly improved and in excellent agreement with homo- and heteronuclear coupling constants. The difference between the two classes of force field (more structured and less structured carbohydrate-water interaction) was underlined by calculation of the isotropic hydration as calculated by radial pair distributions. At one extreme, the radial O em leader O pair distribution function yielded a peak density of 2.3 times the bulk density in the first hydration shell when using the older CHARMM force field, whereas the maximum density observed in the GLYCAM force field was calculated to be 1.0, at the other extreme.

Algorithms↗