Gordonan,an Acidic Polysaccharide with Cell Aggregation-Inducing Activity in Insect BM-N4 Cells,Produced by Gordonia sp.

An acidic polysaccharide, termed gordonan, was isolated from the culture medium of Gordonia sp. as an inducer of cell aggregation in an insect cell line, BM-N4. Gordonan had an average molecular weight of 5×10⁶ and its structure was identified as →3)-4-O-(1-carboxyethyl)-β-D-Manp-(1→4)-β-D-GlcAp-(1→4)-β-D-Glcp-(1→ mainly by acid hydrolysis experiments and NMR analysis. It induces cell aggregation at the concentration of 4 μg/ml. A partially hydrolyzed polysaccharide derived from gordonan with a molecular weight of 5×10⁵ showed weak activity, while any fragment molecules with lower molecular weights prepared from gordonan showed no activity.     

Structural characteristics of a bioactive polysaccharide from Sorghum arundinaceum

A polysaccharide, an alpha-D-glucan with an apparent molecular weight of 6.85 x 10(4), called PSa glucan, was isolated from fresh seeds of Sorghum arundinaceum by fractionation on Sephacryl S-300 HR and Sephadex G-25. Chemical and spectroscopic studies indicated that it has a highly branched glucan type structure composed of alpha-(1-->4) linked D-glucopyranose residues with (1-->3), (1-->6) branching points, and a significant amount of alpha-(1-->6) branching to alpha-(1-->3) linked D-glucopyranose residues. The anti-inflammatory activity of the polysaccharide was performed using the capillary permeability assay.     

Purification, characterization and enzymatic degradation of YCP, a polysaccharide from marine filamentous fungus Phoma herbarum YS4108

YCP, a mitogenic polysaccharide with its molecular weight (MW) of 2.4 x 10(3) kDa, was isolated from the mycelium of the marine filamentous fungus Phoma herbarum YS4108 by a combination of ion-exchange chromatography on DEAE-32 and gel permeation over Sephacryl S-400. The detailed compositional, spectroscopic and methylation analyses of the polysaccharide demonstrated that its backbone possessed most likely a linear alpha-(1 --> 4) bonded glucopyranoside main chain co-bearing through side alpha-(1 --> 6)-linkage. The alpha-(1 --> 4) bondage of the glucopyranoside building blocks in YCP was confirmed by the observation that it could be hydrolyzed by the alpha-amylase produced by Bacillus licheniformis. A reliable concentration monitoring experimentation highlighted that the reducing sugars released continuously from YCP during its incubation with the enzyme, and the MW of the main resulting fragment weighed 0.8 x 10(4) Da with approximately 10% of YCP converted to maltose, maltotriose and glucose after a 120-min enzymatic degradation. Finally, YCP was found to be able to increase phagocytic activity of mice in vitro and in vivo, indicating that it may be looked up as a potent immunomodulator that could activate macrophages.     

Characterization of an arabinogalactan-protein isolated from pressed juice of Echinacea purpurea by precipitation with the beta-glucosyl Yariv reagent

An arabinogalactan-protein (AGP) from pressed juice of Echinacea purpurea herb was isolated from a high molecular weight fraction by precipitation with the beta-glucosyl Yariv reagent, followed by gel-permeation chromatography. It revealed characteristic features of other AGPs: i.e., a high amount of polysaccharide (83%) with a ratio of galactose to arabinose of 1.8:1, some uronic acids (4-5%), and a low protein content (7%) with high levels of serine, alanine and hydroxyproline. The molecular weight was estimated to be 1.2 x 10(6) Da. Linkage and 13C NMR analyses showed that the AGP is composed of a highly branched core polysaccharide of 3-, 6-, and 3,6-linked Galp residues with terminal Araf, GlcAp and terminal units of Araf-(1-->5)-Araf-(1-->. Partial acid hydrolysis resulted in loss of Araf residues at the periphery of the molecule. Complete loss of reactivity toward the beta-glucosyl Yariv antigen was then noticed.     

Purification, structure and immunobiological activity of a new water-soluble polysaccharide from the mycelium of Polyporus albicans (Imaz.) Teng

A new water-soluble intracellular polysaccharide named as PTP, with a molecular mass of 3.7x10(4) Da, was obtained from the mycelium of Polyporus albicans (Imaz.) Teng. Structure features of the purified polysaccharide were investigated by a combination of chemical and instrumental analysis. The results indicated that PTP consisted of a backbone composed of (1-->3)-linked-beta-d-mannopyranosyl, (1-->3,6)-linked-beta-d-mannopyranosyl and (1-->6)-linked-alpha-d-galactopyranosyl residues in the ratio of 3:1:1, and terminated with a single non-reducing terminal (1-->)-beta-d-mannopyranosyl residues at the C-6 position of (1-->3,6)-linked-beta-d-mannopyranosyl, along the main chain. This is the first report describing the isolation and structure elucidation of a new intracellular polysaccharide produced from the mycelium of P. albicans (Imaz.) Teng. Preliminary tests in vitro showed PTP have potent stimulating effects on murine lymphocyte proliferation induced by concanavalin A or lipopolysaccharide and its branches are extremely important for the expression of the enhancement of the immunological activity.     

Experimental study on anticoagulant and antiplatelet aggregation activity of a chemically sulfated marine polysaccharide YCP

A polysaccharide YCP was prepared from a marine filamentous fungus Keissleriella sp. YS4108, which exhibited as a molecular weight (Mw) of 2.4x10(3) kDa and its three sulfated derivatives (YCP-SL, YCP-SM and YCP-SH) were synthesized, the degree of substitution (DS) of which were determined to be 0.13, 0.99 and 1.3, with the average molecular weight 0.64x10(3), 0.57x10(3) and 0.45x10(3) kDa, respectively. Anticoagulant activity and antiplatelet aggregation activity of these sulfated derivates were evaluated by activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and platelet aggregation assay. The results showed that YCP sulfates significantly prolonged APTT, TT and PT. The derivates showed no effects on thrombin in the presence or in the absence of antithrombin III (AT III) or heparin cofactor II (HC II), while the derivates effectively inhibited factor Xa in the presence of AT III. At the same time, YCP-SH also possessed potent antiplatelet aggregation activity in vitro compared with aspirin. YCP sulfates specifically interfered with different stages of the coagulation cascade, and the anticoagulant activity improved with the increasing DS and decreased Mw.     

A beta-D-glucan from the sclerotia of Pleurotus tuber-regium (Fr.) Sing

An alkali-soluble polysaccharide (Hunai polysaccharide, 1) from the fruit body of Pleurotus tuber-regium (Fr.) Sing was shown to be homogeneous by gel permeation chromatography and its molecular weight was approximately 4.3 x 10(5). Complete acid hydrolysis, periodate oxidation, Smith degradation, methylation, FT-IR and 13C NMR analysis, complex formation with Congo Red, indicated that 1 has a beta-(1 --> 3)-linked D-glucopyranosyl backbone with a single beta-D-glucopyranosyl group at O-6 of every third glucose residue.     

Purification, structure and immunobiological activity of a water-soluble polysaccharide from the fruiting body of Pleurotus ostreatus

The water-soluble polysaccharide (POP), with a molecular mass of 2.4x10(4)Da, was obtained from the fruiting body of Pleurotus ostreatus. Structure features of the purified polysaccharide were investigated by a combination of chemical and instrumental analysis, such as methylation analysis, Smith degradation, GC-MS, (13)C and (1)H NMR and FTIR. The results indicated that the backbone of POP was composed of (1-->6)-linked-alpha-D-galactopyranosyl and (1-->2,6)-linked-alpha-D-galactopyranosyl residues, which were terminated with a single terminal (1-->)-beta-D-glucose residue at the O-2 position of galactosyl along the main chain in the ratio of 1:1:1. Preliminary tests in vitro showed POP is capable of enhancing concanavalin A (ConA)- or lipopolysaccharide (LPS)-induced lymphocyte proliferation, which suggested that POP could be a potential immunostimulating agent for use in functional foods or medicine against both pathogens and cancer.     

Structural characterization and immunomodulating activity of a complex glucan from spores of Ganoderma lucidum.

A polysaccharide with a molecular weight of 1.26 x 10(5), obtained from the sporoderm-broken spores of Ganoderma lucidum, was purified by anion-exchange and gel-permeation chromatography. This polysaccharide had a strong effect on suppressing the antibody production and the Con A or LPS induced lymphocyte proliferation in mice. Chemically, the structure of the polysaccharide was identified by methylation analysis, 1 D, 2 D NMR and ESI-MS spectroscopic studies of the native one and of the oligosaccharide fragments generated by partial acid hydrolysis, Smith degradation, and acetolysis. It was concluded that the intact polysaccharide was a complex beta-D-glucan consisting of a (1-->6)-linked backbone chain, in which every other glucosyl residue was substituted at C-3 or C-4 with mono-, di- and trisaccharide branches.     

Correlation of structure to antitumor activities of five derivatives of a beta-glucan from Poria cocos sclerotium

A water-insoluble (1-->3)-beta-D-glucan isolated from fresh sclerotium of Poria cocos was, respectively, sulfated, carboxymethylated, methylated, hydroxyethylated, and hydroxypropylated, to afford five water-soluble derivatives. Their weight-average molecular masses (Mw) and intrinsic viscosities ([eta]) were determined by size-exclusion chromatography combined with laser light scattering (SEC-LLS), LLS, and viscometry in phosphate buffer solution (PBS) at 37 degrees C. The antitumor activities, against Sarcoma 180 tumor cell (S-180) and gastric carcinoma cell strain (MKN-45 and SGC-7901) of the native beta-glucan and the five derivatives, were tested in vitro and in vivo. The Mw values of the five derivatives in PBS were determined to be 3.8 x 10(4), 18.9 x 10(4), 16.0 x 10(4), 76.8 x 10(4), and 224.3 x 10(4), respectively. The high Mw values of the hydroxyethylated and hydroxypropylated derivatives in aqueous solution resulted from aggregation, and their true Mw values obtained in dimethyl sulfoxide were 20.1 x 10(4) and 19.1 x 10(4). The sulfated and carboxymethylated derivatives having DS of 1.0-1.3 show good water solubility, and exist as relatively expanded chains in aqueous solution. Interestingly, the native beta-glucan did not show antitumor activity, whereas the sulfated and carboxymethylated derivatives exhibit significant antitumor activities against S-180 and gastric carcinoma tumor cells. This work showed that good water solubility, relatively high chain stiffness, and moderate molecular mass of the derivatives in aqueous solution contribute beneficial to enhancement of antitumor activity.     

Structural characterization of a water-soluble polysaccharide from the roots of Codonopsis pilosula and its immunity activity

The water-soluble polysaccharide (CPP), with a molecular mass of 1.1x10(4) Da, was obtained from the roots of Codonopsis pilosula. Structure feature investigation by a combination of chemical and instrumental analysis revealed that CPP had a backbone consisting of (1-->3)-linked-beta-D-galactopyranosyl, (1-->2, 3)-linked-beta-D-galactopyranosyl and (1-->3)-linked-alpha-D-rhamnopyranosyl residues, which were branched with two glycosyl residues composed of alpha-L-arabinose-(1-->5)-alpha-L-arabinose(1-->linked residues at the O-2 position of galactosyl along the main chain in the ratio of 1:1:2:1:1. Preliminary immunological tests in vitro showed CPP could stimulate concanavalin A (ConA)- or lipopolysaccharide (LPS)-induced lymphocyte proliferation in a dose-dependent manner.     

A new model of pneumococcal lipoteichoic acid structure resolves biochemical, biosynthetic, and serologic inconsistencies of the current model

Lipoteichoic acid (LTA) is an essential bacterial membrane polysaccharide (cell wall component) that is attached to the membrane via a lipid anchor. According to the currently accepted structure of pneumococcal LTA, the polysaccharide is comprised of several repeating units, each of which starts with glucose and ends with ribitol, with the lipid anchor predicted to be Glc(beta1-->3)AATGal(beta1-->3)Glc(alpha1-->3)-acyl(2)Gro, where AATGal is 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose. However, this lipid anchor has not been detected in pneumococcal membranes. Furthermore, the currently accepted structure does not explain the Forssman antigen properties of LTA and predicts a molecular weight for LTA that is larger than its actual observed molecular weight. To resolve these problems, we used mass spectrometry to analyze the structure of LTA isolated from several pneumococcal strains. Our study found that the R36A pneumococcal strain produces LTA that is more representative of pneumococci than that previously characterized from the R6 strain. Analysis of LTA fragments obtained after hydrofluoric acid and nitrous treatments showed that the fragments were consistent with an LTA nonreducing terminus consisting of GalNAc(alpha1-->3)GalNAc(beta1-->, which is the minimal structure for the Forssman antigen. Based on these data, we propose a revised model of LTA structure: its polysaccharide repeating unit begins with GalNAc and ends with AATGal, and its lipid anchor is Glc(alpha1-->3)-acyl(2)Gro, a common lipid anchor found in pneumococcal membranes. This new model accurately predicts the observed molecular weights. The revised model should facilitate investigation of the relationship between LTA's structure and its function.     

Determination of molecular size and shape of hyperbranched polysaccharide in solution

The chemical structure of a water-soluble polysaccharide, coded as TM3b, extracted from sclerotia of Pleurotus tuber-rigium was analyzed to be a hyperbranched beta-D-glucan with beta-(1-->6), beta-(1-->4), and beta-(1-->3)-linked residues, with degree of branching (DB) of 57.6%. The results from size-exclusion chromatography combined with laser light scattering (SEC-LLS) revealed that the hyperbranched polysaccharide easily aggregated in 0.15 M aqueous NaCl, whereas it dispersed as individual chains in DMSO. The weight-average molecular weight (M(w)), radius of gyration, intrinsic viscosity, and chain density of TM3b in DMSO and in 0.15 M aqueous NaCl were measured with SEC-LLS, LLS, and viscometry. The results indicated that single chains and aggregates with aggregation number of 12 coexisted in the aqueous solution, whereas individual molecules of TM3b occurred in DMSO. In view of the molecular parameters, the aggregates in aqueous solution exhibited more compact chain structure than the individual molecules in DMSO. Furthermore, transmission electron microscopy and atomic force microscopy showed that all of the aggregates and individual molecules exhibited spherical particles in the solutions. This work provided the valuable information of chain conformation and molecular morphology of the hyperbranched polysaccharide in different solvents.     

虫草多糖的分离、纯化和初步药效活性研究

以虫草发酵菌丝体为原料提取虫草多糖并对多糖进行结构鉴定和初步药效活性研究。采用水提醇沉和离子交换纤维素进行分离得到两种虫草多糖,HPLC测定相对分子质量,紫外光谱、红外光谱、部分酸水解、甲基化分析和高效液相色谱等方法研究单糖组成及连接方式,四甲基偶氮唑盐微量酶反应比色法（MTT）检测虫草多糖的活性。经分离得到中性（CPSl）、酸性（CPS2）两种成分,相对分子质量分别为2．56×10^4,9．91×10^;单糖组成：CPSln,（葡萄糖）：n（甘露糖）：n（半乳糖）：n（阿拉伯糖）=46：36：18：l;CPS2／n,（葡萄糖）：n,（甘露糖）：n（半乳糖）：n（半乳糖酸）：n（木糖）：n（阿拉伯糖）：n（鼠李糖）=30：25：14：4：3：3：1。红外谱揭示均含-a-键。CPSl主链含（1→6）糖苷键的高度分支的葡甘露半乳聚糖,另有少量葡萄糖分支点在2,3,4位。CPS2主链含葡萄糖（Glc）、甘露糖（Man）、半乳糖（Gal）,分支点主要在3位,侧链含多种单糖。CPSl和CPS2都对顺铂（CDDP）损伤的vero细胞有一定保护作用。     

Structure determination,apoptosis induction,and telomerase inhibition of CFP-2, a novel lichenin from Cladonia furcata

A great deal of experimental evidence has accumulated in the past several decades, suggesting that polysaccharides have wide bioactivities. Cladonia furcata polysaccharide, CFP-2, a water-soluble lichenin with a mean Mr 7.6 x 10(4), was first obtained by 0.25 M NaOH solution extraction, ethanol precipitation, DEAE-cellulose, and Sephadex G-200 column chromatography. Gas chromatography of acid hydrolyzate of CFP-2 suggested that it was composed of D-glucose, D-galactose, and D-mannose in the molar ratios of 8:1:1. Periodate oxidation, Smith degradation, IR, and NMR spectroscopy analysis revealed that CFP-2 had a backbone consisting of alpha-(1-->3) and alpha-(1-->4)-linked D-glucopyranosyl residues substituted at O-6 with beta-(1-->6)-linked D-galactopyranosyl residue and alpha-(1-->6)-linked D-mannopyranosyl residue. CFP-2 was able to reduce viability of cultured HL-60 and K562 cells. The antiproliferative properties of CFP-2 appeared to be attributable to its induction of apoptotic cell death as determined by ultrastructural change, internucleosomal DNA fragmentation, and increased proportion of the subdiploid cell population. To elucidate molecular events in the apoptosis, protein expressions of Bcl-2, Bax, Fas, and FasL were measured by Western blotting using specific antibodies in HL-60 cells. The level of Bcl-2 remained largely unchanged, but the Bax, Fas, and FasL expression showed up-regulation. Moreover, the telomerase activity analyzed by TRAP-ELISA assay in HL-60 cells treated with CFP-2 decreased as compared with the untreated control cells. These results suggest that CFP-2 could have a possible cancer therapeutic potential.     

Mechanism of type 3 capsular polysaccharide synthesis in Streptococcus pneumoniae

The glycosidic linkages of the type 3 capsular polysaccharide of Streptococcus pneumoniae ([3)-beta-D-GlcUA-(1-->4)-beta-D-Glc-(1-->](n)) are formed by the membrane-associated type 3 synthase (Cps3S), which is capable of synthesizing polymer from UDP sugar precursors. Using membrane preparations of S. pneumoniae in an in vitro assay, we observed type 3 synthase activity in the presence of either Mn(2+) or Mg(2+) with maximal levels seen with 10-20 mM Mn(2+). High molecular weight polymer synthesized in the assay was composed of Glc and glucuronic acid and could be degraded to a low molecular weight product by a type 3-specific depolymerase from Bacillus circulans. Additionally, the polymer bound specifically to an affinity column made with a type 3 polysaccharide-specific monoclonal antibody. The polysaccharide was rapidly synthesized from smaller chains and remained associated with the enzyme-containing membrane fraction throughout its synthesis, indicating a processive mechanism of synthesis. Release of the polysaccharide was observed, however, when the level of one of the substrates became limiting. Finally, addition of sugars to the growing type 3 polysaccharide was shown to occur at the nonreducing end of the polysaccharide chain.     

Correlation between the antitumor activity of a polysaccharide schizophyllan and its triple-helical conformation in dilute aqueous solution

Eight samples of a polysaccharide schizophyllan ranging in weight-average molecular weight Mw (in water) from 5 x 10(3) to 1.3 x 10(5) were prepared and their antitumor activity (expressed in terms of the tumor inhibition ratio) against Sarcoma 180 ascites, intrinsic viscosities [eta], and gel-filtration chromatograms in aqueous solution were determined. The tumor inhibition ratio was essentially unity for samples with Mw higher than 9 x 10(4), but reduced to zero or even to a negative value when Mw was lower than 10(4). The [eta] data combined with the chromatographic data showed that above Mw approximately 9 x 10(4) the predominant species of schizophyllan in aqueous solution is the previously found rigid triple helix, whereas below Mw approximately 9 x 10(4) both triple helices and single chains coexist in the solution and the fraction of triple helices decreases monotonically to zero as Mw is decreased to 5 x 10(3). From these findings it was concluded that the antitumor potency of schizophyllan in water is related to the amount of triple helices relative to that of single chains.     

Production of polysaccharides from Ganoderma lucidum (CCRC 36041) under limitations of nutrients

This research studied the production of polysaccharides from Ganoderma lucidum under the various limitations of nutrients, including carbon-source, nitrogen-source, phosphate-source, magnesium-source, and dissolved oxygen. The different responses of polysaccharide production were observed under different limitations of nutrients. The concentration of polysaccharides was from 1.79 g/L decreasing to 0.91 g/L when the concentration of glucose was from 60 g/L decreasing to 20 g/L. The highest specific polysaccharide production was found at 60 g/L glucose media with 0.299 g/g-cell, and the lowest molecular weight was found in carbon-source limitation. Under nitrogen-source limitation the concentration of cells was low, but both polysaccharide production (1.61 g/L) and specific polysaccharide production (0.492 g/g-cell) were the highest. The lowest molecular weight of polysaccharides was found under nitrogen-source limitation. Both the phosphate-source and magnesium-source limitations showed low cell growth. With the phosphate-source limitation both low polysaccharide production and a lower molecular weight of polysaccharides was found. In the magnesium-source limitation low polysaccharide production, but a higher molecular weight of polysaccharides, was found. For the factor of oxygen supply the best polysaccharide production was found with sufficient oxygen for the first 5 days’ cultivation and then, after changing to oxygen limitation for another 5 days’ cultivation. On the other hand, the highest molecular weight of polysaccharides was found from the beginning with oxygen limitation throughout the process of fermentation.     

Components and antioxidant activity of the polysaccharide from Streptomyces virginia H03

A polysaccharide was isolated from the broth of cultured Streptomyces virginia H03 which was treated by ethanol deposition and savage method to remove the protein, and was purified using Sephadex G-150 column chromatography. The components of the polysaccharide were determined by gas chromatography. The purified polysaccharide was made up of mannose, glucose and galactose, in a 2:1:1 proportion. Its average apparent molecular weight was 3.76 x 10(4) Da which was determined by gel permeation chromatography. In addition, several antioxidant assays were adopted to investigate the antioxidant activity of the polysaccharide in vitro. The results indicated that the purified polysaccharide showed significant antioxidant activity against superoxide anion, hydrogen peroxide and 1,1-diphenyl-2-picrylhydrazyl radical, and lipid peroxidation as with standard antioxidants such as vitamin C. Furthermore, the polysaccharide had a better heat stability than vitamin C, which suggested that the polysaccharide might be a potent useful antioxidant.     

Production of a glucoglucuronan by a rhizobia strain infecting alfalfa. Structure of the repeating unit

The Rhizobium sp. T1 strain. which induces nodule formation on alfalfa and clover roots, produces, during growth, an extracellular polysaccharide composed of D-glucose and D-glucuronic acid noted glucoglucuronan. During the bacterial growth, the pH of the medium decreases slightly. The control of pH in the growth medium slightly reduces the glucoglucuronan production. Under the conditions tested in the present work, the weight-average molecular weight of the polymers produced with or without pH control are similar: Mw approximately 2 x 10(6); the repeating unit determined by chemical and NMR analyses corresponds to the disaccharide: --> 3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1 -->.