Effects of polysaccharides from abalone viscera (Haliotis discus hannai Ino) on MGC 803 cells proliferation.

The polysaccharides (AVP) was obtained from abalone (Haliotis discus hannai Ino) viscera, using the alkaline protease to enzymolysis, sevage method and repeated freezing and thawing method to remove protein and hydrogen peroxide method to depigment. The total sugar content was 46.27±1.5% and uronic acid, sulfate radical, hexosamine and protein contents were 17.44±0.22%, 16.98±0.15%, 0.65±0.02% and 1.64±0.13% in AVP respectively. The main monosaccharide compositions of AVP were d-galactose, d-xylose, d-mannose, d-glucose and d-glucuronic acid. MTT assay showed AVP had a significant anti-tumor activity to gastric carcinoma cells, especially to MGC 803, while it had no influence upon proliferation of normal stomach cells GES 1. The results of Morphological changes, cell migration ability and AO/EB staining indicated that MGC803 cells underwent apoptosis in a dose-dependent manner induced by AVP. Moreover, the western blotting results showed that the expressions of survivin, Bcl-2 and VEGF were decreased, while the expression of Bax and p53 were increased in a dose-dependent manner of AVP. The results suggested that AVP might be a potential anti-tumor agent securely and naturally.

A sensitive and efficient method for determination of N-acetylhexosamines and N-acetylneuraminic acid in breast milk and milk-based products by high-performance liquid chromatography via UV detection and mass spectrometry identification.

A sensitive and efficient method of high performance liquid chromatography using 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP) as pre-column derivatization reagent coupled with UV detection (HPLC-UV) and online mass spectrometry identification was established for determination of the most common N-Acetylhexosamines (N-acetyl-d-glucosamine (GlcNAc) and N-acetyl-d-galactosamine (GalNAc)) and N-acetylneuraminic acid (Neu5Ac). In order to obtain the highest liberation level of the three monosaccharides without destruction of Neu5Ac or conversion of GlcNAc/GalNAc to GlcN/GalN in the hydrolysis procedure, the pivotal parameters affecting the liberation of N-acetylhexosamines/Neu5Ac from sample were investigated with response surface methodology (RSM). Under the optimized condition, maximum yield was obtained. The effects of key parameters on derivatization, separation and detection were also investigated. At optimized conditions, three monosaccharides were labeled fast and entirely, and all derivatives exhibited a good baseline resolution and high detection sensitivity. The developed method was linear over the calibration range 0.25-12µM, with R(2)>0.9991. The detection limits of the method were between 0.48 and 2.01pmol. Intra- and inter-day precisions for the three monosaccharides (GlcNAc, GalNAc and Neu5Ac) were found to be in the range of 3.07-4.02% and 3.69-4.67%, respectively. Individual monosaccharide recovery from spiked milk was in the range of 81%-97%. The sensitivity of the method, the facility of the derivatization procedure and the reliability of the hydrolysis conditions suggest the proposed method has a high potential for utilization in routine trace N-acetylhexosamines and Neu5Ac analysis in biological samples.

Structure and antioxidant activity of a novel poly-N-acetylhexosamine produced by a medicinal fungus.

A novel poly-N-acetylhexosamine (polyhexNAc) about 6 kDa average molecular weight (MW) was isolated from the low-MW fraction of exopolysaccharide produced by liquid fermentation of a medicinal fungus Cordyceps sinensis Cs-HK1. The composition and linkage of sugar residues were determined by mass spectrometry and methylation analysis, and the anomeric configuration and chain linkage were confirmed by NMR. From the analytical results, the molecular structure was elucidated as a [-4-ß-D-ManNAc-(1→3)-ß-D-GalNAc-(1→] disaccharide repeating unit in the main chain with a Gal branch occurring randomly at the 3-position of ManNAc. This polyhexNAc showed notable antioxidant activities with a Trolox equivalent antioxidant capacity of 330 µmol Trolox/g, a ferric reducing ability of plasma of 45.7 µmol Fe(II)/g, and significant cytoprotective effect against H2O2-induced PC12 cell injury. This is the first report on the structure and bioactivity of an extracellular amino-polysaccharide from the Cordyceps species.

Heterologous production and detection of recombinant directing 2-deoxystreptamine (DOS) in the non-aminoglycoside-producing host Streptomyces venezuelae YJ003.

2-Deoxystreptamine is a core aglycon that is vital to backbone formation in various aminoglycosides. This core structure can be modified to develop hybrid types of aminoglycoside antibiotics. We obtained three genes responsible for 2-deoxystreptamine production, neo7, neo6, and neo5, which encode 2-deoxy-scyllo-inosose synthase, L-glutamine: 2-deoxy-scyllo-inosose aminotransferase, and dehydrogenase, respectively, from the neomycin gene cluster. These genes were cloned into pIBR25, a Streptomyces expression vector, resulting in pNDOS. The recombinant pNDOS was transformed into a non-aminoglycoside-producing host, Streptomyces venezuelae YJ003, for heterologous expression. Based on comparisons of the retention time on LC-ESI/MS and ESIMS data with those of the 2-deoxystreptamine standard, a compound produced by S. venezuelae YJ003/pNDOS was found to be 2-deoxystreptamine.

The characterization and molecular structure of hepatoproliferin: a liver regeneration factor from rat hepatocytes.

Hepatoproliferin (HPF) was purified from regenerating rat livers as an oligomeric entity (big-HPF) from which the monomeric form (small-HPF) could be obtained using disaggregating conditions. By using a solid-phase ion-exchange method, small-HPF was forced to dissociate into two charged ionic species, namely norepinephrine (NE) and a sulfonated disaccharide with a molecular structure consisting of D-glucuronic acid bound to glucosamine 2,6-disulfate by a beta-glycosidic linkage having a beta, 1 --> 4 configuration. Monomeric HPF stemmed from the formation of three electrostatic bonds between the protonated amine groups of three norepinephrines, of which two bind to the deprotonated sulfonic groups of glucosamine 2,6-disulfate and one to the deprotonated carboxylic group of glucuronic acid, to constitute a tightly associated complex with a molecular mass of 1046 Da. This represents one of the two purified isoforms of small-HPF. The other isoform, which has a lower molecular mass of 877 Da, lack one NE, leaving the weaker carboxylic group of glucuronic acid unoccupied, to constitute a more acidic form of HPF.

Preparative production and separation of 2-acetamido-2-deoxymannopyranoside-containing saccharides using borate-saturated polyolic exclusion gels.

A new separation method based on the combination of exclusion and ion exchange chromatography in borate buffer was developed. It allows semi-preparatory and preparatory separation of isobaric N-acylhexosamines (C-2 epimers) and corresponding methyl glycosides (anomers and tautomers). Three types of polyolic gels were tested for these separations. Ion-exchange HPLC was used as a rapid and reliable method for the quantification of the respective analytes. NMR studies of the interactions of N-acetylhexosamines with borate confirmed the importance of a proper stereochemical arrangement of acetamido sugars for their interactions with borate anions.

Oligomeric hepatoproliferin disaggregates into an active monomer that was purified and forcefully dissociated into two different ionic species.

Hepatoproliferin (HPF), a liver regeneration factor, was isolated initially as an aggregated molecule (big-HPF) and was purified into two homogeneous, bioactive species of 14 kDa and 18.5 kDa. These two big-HPFs were disaggregated to completion into two monomeric forms (small-HPFs) when incubated for 10 days in 0.15 M ammonium bicarbonate at 25 degrees C. Both monomeric forms were purified to homogeneity as active entities, one with a molecular mass of 944 Da and one with a molecular mass of 1066 Da. Each of the two (35)S-labelled small-HPFs was found, by enzymic analysis, to contain a charged sulfonated saccharide, which was neutralized by a specific amine. Monomeric HPF is therefore a stable ionic complex formed between these two ionic species. So strong was the electrostatic association that small-HPF remained intact in solution and no amine was displaced by the ammonium ions of the buffer. Small-HPF remained unimpaired during purification, since all activity was retained despite alternating acidic and basic conditions. However, when small-HPF was brought into contact with either a cationic or an anionic resin, it was dissociated to completion when mixed continuously with the resin for 4 days. The ionic entity that was released had no bio-activity and was either a pure radioactively labeled saccharide or a non-labeled amine, depending on the kind of resin used. When incubated together, the separated counterions combine to regain full activity after 2 days of reassociation. However, with incubation for longer, this reassociated small-HPF formed different oligomeric HPFs by aggregation. Small-HPF is therefore a new kind of growth enhancer, consisting of an acidic sulfonated saccharide and a basic amine assembled into a stable active ionic complex that has a tendency to aggregate.

Inhibition of porcine small intestinal sucrase by valienamine.

Valienamine, an aminocyclitol, has been isolated from the enzymolysis broth of validamycins. The absolute configuration of valienamine is similar to that of alpha-D-glucose. The inhibitory effect of this amino-sugar analog of alpha-D-glucose, valienamine, on porcine small intestinal sucrase was examined. Valienamine was found to be potent, competitive reversible inhibitor of porcine small intestinal sucrase in vitro with an IC50 value of 1.17 x 10(-3)M. Valienamine also exhibited dose-dependent, instantaneous inhibition of porcine small intestinal sucrase. The inhibition of porcine small intestinal sucrase by valienamine was pH-independent.

A rapid formaldehyde assay using purpald reagent: application under periodation conditions.

Measurement of formaldehyde is encountered in a broad range of applications including the wine and alcohol industry and environmental pollution surveillance. In carbohydrate structural chemistry, frequent use is made of formaldehyde by periodate oxidation of terminal vicinal diols. Popular methods for the detection of formaldehyde use reagents such as chromotropic acid (4,5-dihydroxynaphthalene-2,7-disulfonic acid) or acetylacetone. The chromotropic acid method requires heating of the sample under strongly acidic conditions, which is undesirable in many applications. The acetylacetone method yields a yellow color product, and is less specific and sensitive (Mimura et al., J. Hyg. Chem. 22, 39-41, 1976). The reaction of formaldehyde with Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole) works under alkaline conditions at room temperature, and the sensitivity is superior to other methods. The color development by this reagent, however, requires oxidation of the adduct with hydrogen peroxide, air oxygen, or dilute periodate. We found that low levels of periodate, commonly used to oxidize specifically terminal vicinal diols to yield formaldehyde, are compatible with color development with the Purpald reagent. We have investigated the conditions required for use of the Purpald reagent, especially in conjunction with periodate oxidation reactions. We have used the assay either in test tubes or with microplates, attaining sensitivity of as little as 1 nmol formaldehyde.

Separation of sugars by ion-exclusion chromatography on a cation-exchange resin.

A method for the separation of N-acetylmannosamine and N-acetylglucosamine is described, which consists of chromatography of the two sugars on a column (30 x 1 cm) of the cation-exchange resin, Dowex 50W-X2, in borate buffer at pH 7.8. N-Acetylmannosamine is eluted near the void volume, while N-acetylglucosamine emerges in a more retarded position. It is postulated that the separation occurs as a result of the combined effects of ion exclusion and gel permeation. Thus, in borate solution, N-acetylmannosamine presumably exists largely as a negatively charged complex and is therefore excluded from the sulfonated polystyrene matrix, while N-acetylglucosamine occurs mainly as the free sugar in the equilibrium mixture and, being a neutral compound, has free access to the porous resin. The proposed mechanism for the separation was supported by the finding that glucose and glucose 6-phosphate could also be separated on a column of the same resin, with water as the eluent.

Separation of N-acetylglucosamine and N-acetylmannosamine by chromatography on Sephadex in borate buffer.

Several procedures have been used previously for the separation of N-acetylglucosamine and N-acetylmannosamine, which are based on the difference in strength of the borate complexes of the two N-acetylhexosamines and include paper chromatography on borate-treated paper, paper electrophoresis in borate buffer, and anion-exchange chromatography of the borate complexes. In the present study, we have observed that the two sugars, despite their identical size in noncomplexed form, may also be separated by gel chromatography in borate buffer. Nearly complete resolution was obtained by chromatography on a column (1.5 x 117 cm) of Sephadex G-15, which was eluted at room temperature with 0.27 M sodium borate, pH 7.8 (prepared from H3BO3 by addition of NaOH), at a flow rate of 10 ml/h. This procedure complements existing methods for the separation of N-acetylmannosamine and N-acetylglucosamine and has the advantage that it can be carried out on a relatively large preparative scale.

The compositional analysis of bacterial extracellular polysaccharides by high-performance anion-exchange chromatography.

A high-performance liquid chromatography (HPLC) method with pulsed-amperometric detection (PAD) was developed for the compositional analysis of the acidic, neutral, and basic monosaccharides recovered from the acid hydrolysis of bacterial cell wall polysaccharides. This HPLC-PAD method involved the chromatography of the acid hydrolysis products on a CarboPac PA-1 anion-exchange column of pellicular resin, with PAD detection following postcolumn addition of alkali. Complete resolution of a mixture of 19 monosaccharides, comprising 9 neutral, 3 basic, and 7 acidic sugars, frequently found in bacterial polysaccharides was achieved within 60 min by the system. The presence of amino acids in the mixture was shown not to affect the analysis. This protocol was applied to the compositional analysis of 2 extracellular polysaccharides produced by Escherichia coli, colanic acid, and K30 antigen, which share constituent monosaccharides. The overproduction of extracellular polysaccharide in E. coli CWG56 was shown to be a consequence of deregulation of K30 biosynthesis and not of coexpression of an additional polymer.

Synthesis of fluorescent oligosaccharides for covalent attachment to living cells.

Asparagine-linked oligosaccharides were liberated from glycoproteins by hydrazinolysis. The treatment resulted in de-N-acetylation of the amino sugars. After isolation of the oligosaccharides free amino groups were labeled with fluorescein isothiocyanate and remaining amino groups reacetylated. The fluorescent oligosaccharides were used to label living cells. They were converted to hydrazine derivatives and covalently attached to cell surface oligosaccharides, which had been treated with periodate or neuraminidase and galactose oxidase. This enabled the visualization of the attached oligosaccharides at the external aspect of the plasma membrane by fluorescence microscopy.

Incorporation of sialic acid into Trypanosoma cruzi macromolecules. A proposal for a new metabolic route.

Sialo- and asialoglycoconjugates were isolated from Trypanosoma cruzi epimastigotes and their composition determined. Sialoglycoconjugates bound to wheat germ agglutinin (WGA)-Sepharose and were precipitated by concanavalin A, Wistaria floribunda hemagglutinin and WGA. Asialoglycoconjugate bound to concanavalin A-Sepharose and precipitated with concanavalin-A and W. floribunda hemagglutinin but not with WGA. Cells grown in the presence of fetal calf serum were agglutinated by WGA but not by peanut agglutinin. The reverse was true for cells grown without fetal calf serum. Neuraminidase-treated cells incorporated sialic acid or its 7-carbon analog, 5-acetamido-3,5-dideoxy-L-arabino-2-heptulosonic acid (AcNeu7) from sialylated compounds such as fetuin or sialyl-lactose but did not incorporate free sialic acid. Restoration of the WGA sialylreceptors in neuraminidase-treated cells, as determined by cell agglutination with WGA, was also obtained by incubation with fetuin or sialyl-lactose but not with free sialic acid. Moreover, restoration of agglutinability by WGA in neuraminidase-treated cells or cells grown in medium without fetal calf serum occurred equally well in energy-rich or energy-depleted cells. A transglycosilase reaction for sialic acid incorporation in T. cruzi epimastigotes is suggested.

Sera from patients with poststreptococcal glomerulonephritis contain antibodies to glomerular heparan sulfate proteoglycan.

Antibodies, found in human sera from patients with poststreptococcal glomerulonephritis, against proteoglycans (PG) derived from bovine and human glomeruli were investigated. PG were isolated by 4 M guanidine-HCl extraction of whole glomeruli, followed by DEAE-Sepharose CL-6B ion exchange chromatography. The anionic fractions were further purified by chromatography on Sepharose CL-4B. Biochemical analysis of the two resulting peaks revealed the presence of high molecular weight anionic material containing protein, uronic acid, glucosamine, and galactosamine. Enzymatic and chemical susceptibilities indicated the presence of heparan sulfate PG and a galactosamine-containing PG. Immunologic studies revealed the presence of anti-PG antibodies to both PG peaks of the Sepharose CL-4B column in glomerulonephritis sera. Inhibition studies using an ELISA demonstrated that heparan sulfate was a major antigenic determinant. Cross-reactivity with both mammalian and streptococcal hyaluronate was noted. Inhibition studies also indicated the presence of a second antigenic site containing N-acetylgalactosamine, possibly representing chondroitin or dermatan sulfate PG.

Actaplanin, new glycopeptide antibiotics produced by Actinoplanes missouriensis. The isolation and preliminary chemical characterization of actaplanin.

Actaplanin (A4696), a new complex of broad spectrum Gram-positive antibiotics is produced by Actinoplanes missouriensis. High performance liquid chromatography was used to show that this complex is composed of several actaplanins. Hydrolytic experiments with acetaplanins A, B1, B2, B3, C1 and G showed that these actaplanins were composed of the same peptide core, an amino sugar and varying amounts of glucose, mannose and rhamnose. The neutral sugar content was determined for each actaplanin. A bioautographic study of aglycone formation during hydrolysis of the actaplanin complex showed that within a short time a simple mixture of two antimicrobially active hydrolysis products was obtained. These substances retained the antimicrobial spectrum and a high percentage of the antibiotic activity of the parent actaplanin complex. Methanolysis of the acetaplanin complex as well as the individual actaplanins resulted in the selective loss of the neutral sugar moieties and the isolation of actaplanin psi (pseudo)-aglycone--the core peptide which still retained an amino sugar group. The 1H NMR spectrum of this substance indicated a similarity to many features of ristocetin psi-aglycone. Hydrolytic studies showed that the amino sugar present in actaplanin was identical with L-ristosamine. It is concluded that the aglycone of actaplanin is a complex peptide composed of aromatic amino acids, and that the actaplanins each possess this aglycone and L-ristosamine but are differentiated by their neutral sugar composition.

Simultaneous determination of uronic acids, hexosamines, and galactose of glycosaminoglycans by gas-liquid chromatography.

A quantitative gas-liquid chromatographic method has been developed for the simultaneous determination of the several monosaccharides present in glycosaminoglycans from animal tissues. In order to achieve a high degree of depolymerization of the glycosaminoglycans, it was found necessary to make them more susceptible to methanolysis by re-N-acetylation during the methanolysis procedure. Good resolution of all common monosaccharides, such as pertrimethylsilyl methyl glycosides, was achieved by the use of a capillary column of fused silica with the liquid phase CPtm leads to Sil 5. The method described was tested on glycosaminoglycans isolated from bovine periodontal ligament and the sensitivity (down to 3 micrograms monosaccharide) makes this method useful in the analysis of small amounts of soft connective tissues with low glycosaminoglycan contents.