Polysaccharide Moiety Research Articles

Enhancing sustainability of a processing tomato cultivation system by using bioactive compost teas

Compost teas are compost-derived liquid products that may potentially induce positive effects on treated crops, including disease suppressiveness and biostimulation of the overall improved plant status. In this work four teas, produced from agricultural and municipal waste compost, were applied in a processing tomato cropping system to gain sustainable diseases management. Field trials showed that teas had a positive impact on health and vegetative status of the plants subjected to natural leaf blights, and induced significant increases of yields. The occurrence of combined suppressive and biostimulation mechanisms, sustained by microbial communities, nutrients supply and carbon-based bioactive compounds, was assumed to underlie the observed effects of tea on plants. Furthermore, NMR spectra suggested that the supramolecular organic structures contained in compost teas may be involved in the inductive processes of biostimulation, including an auxin-like activity. Molecular profiles of organic matter dissolved in teas indicated that the interaction between hydrophobic groups and polysaccharidic moieties could be related to vegetative responses of treated plants. This study provides new insights concerning the comprehension of the overall functionality of compost teas and their applicability in a timely disease management of crop systems.

Activation of Macrophages: Establishing a Role for Polysaccharides in Drug Delivery Strategies Envisaging Antibacterial Therapy.

Infectious diseases are an important health concern, as several pathogens have developed the ability to survive inside phagocytic cells (mostly macrophages), encountered at early infection stages, using these cells as trojan horses. In fact, in several cases macrophages have become a nutrient reservoir that helps pathogens to grow in number and spread. It is frequent that conventional therapeutic schedules include long periods of drug intake, at high doses, in some cases leading to severe side effects and clinical relapses due to prolonged intake, along with an increased risk for the development of antibiotic resistances. Therefore, there is a compelling need to develop new therapeutic strategies providing a targeted drug delivery to macrophages. These cells have unique surface receptors that might recognise preferentially several polysaccharide moieties present on the surface of infecting organisms, including in the bacterial cell wall. Benefiting from a similar composition regarding the referred moieties, polysaccharides might be good candidates to compose the matrix of drug carriers aimed at macrophage targeting, as they can use the same recognition pathways of the infecting organisms. This review describes the features and the role of macrophages in infectious conditions, while addressing their potential as therapeutic targets and unravelling the prominent role of polysaccharides as matrix materials of drug delivery systems developed for the therapy of infectious diseases.

Separation of acid-soluble constituents of soil humic acids by dissolution in alkaline urea solution and precipitation with acid

Humic substances are considered to be composed of relatively small, heterogeneous molecules bound by weak linkages. The dissociation of acid-soluble constituents from soil humic acids (HAs) during the preparative polyacrylamide gel electrophoresis in the presence of concentrated urea has been previously demonstrated. Moreover, the dissociation of acid-soluble constituents has been attributed to the action of concentrated urea. The aim of this study was to investigate the effects of concentrated urea on the dissociation of acid-soluble constituents of soil HAs. Three types of soil HAs were solubilized in 0.1 M NaOH containing 7 M urea and precipitated after 16 h by acidifying the samples to pH 1.0. The acid-soluble constituents were separated from the dark-colored precipitates by concentrated urea treatment and accounted for 16–45 % of the total organic carbon in HAs. Approximately half of the acid-soluble constituents was recovered in the DAX-8-adsorbed fraction. The humification degree of the DAX-8-adsorbed fraction was considerably lower than that of the corresponding unfractionated HA. In contrast, the humification degree of the precipitated fraction increased due to the separation of acid-soluble constituents. The molecular sizes of the DAX-8-adsorbed and DAX-8-non-adsorbed fractions, estimated by high-performance size exclusion chromatography, were similar and smaller than the precipitated fraction. Three-dimensional excitation-emission matrix fluorescence spectroscopy revealed that the acid-soluble constituents exhibited fluorescence similar to that of fulvic acid (FA), added to which the DAX-8-non-adsorbed fraction exhibited protein-like fluorescence. Diffuse reflectance infrared Fourier transform spectroscopy showed that the DAX-8-adsorbed fraction contained proteinous moieties and the DAX-8-non-adsorbed fraction was rich in proteinous and polysaccharide moieties. The present findings suggest that soil HAs are formed by the molecular associations between dark-colored acid-insoluble constituents, FA-like acid-soluble constituents, protein-like constituents, and polysaccharides bound by weak linkages.

Temperature-Tunable Nanoparticles for Selective Biointerface

Drugs can be delivered by a temperature change-driven shrinking of the nanocarrier followed by the cargo release. This paper describes a different structural response to temperature, performed by nanoparticles of poly(N-isopropylacrylamide) and hyaluronic acid. Around 35 °C, the hydrophobicity of the vinyl polymer drives a core-shell rearrangement with the acrylamide chains confined in the core and the polysaccharide moiety forming the shell. In this arrangement, the nanoparticles enable the active targeting of tumor cells, due to the specific interaction of hyaluronic acid with the CD44 receptors. When doxorubicin-loaded nanoparticles are up-taken, the polysaccharide part degrades in the cytoplasm and the cytotoxic effect of the anticancer drug in colon adenocarcinoma cells has a 2-fold increase with respect to healthy fibroblasts. These core-shell particles have hyaluronic acid as the key factor for the specific targeting of tumor cells and drug release with poly(N-isopropylacrylamide) driving the transition.

B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses (IRM10P.608)

Abstract CSR in B cells is crucial to the maturation of antibody responses. It is induced by engagement of CD40 or dual engagement of a TLR and BCR. Here, we have addressed cross-regulation between two different TLRs or between a TLR and CD40 in CSR induction by using a B cell stimulation system involving LPS. LPS mediated long-term primary class-switched antibody responses and induced generation of class-switched B cells and plasma cells. Consistent with the requirement for dual TLR and BCR engagement in CSR induction, LPS, which engages TLR4 through its lipid A moiety, triggered cytosolic Ca2+ flux in B cells through its BCR-engaging polysaccharidic moiety. In the presence of BCR crosslinking, LPS synergized with a TLR1/2 ligand (Pam3CSK4) in CSR induction, but much less efficiently with a TLR7 (R-848) or TLR9 (CpG) ligand. In the absence of BCR crosslinking, R-848 and CpG, virtually abrogated CSR, as induced by LPS or CD154, and reduced secretion of class-switched Igs, without affecting B cell proliferation or IgM expression. The CSR inhibition by TLR9 was associated with reduced AID expression and/or IgH germline IH-S-CH transcription, and required co-stimulation of B cells by CpG with LPS or CD154. Unexpectedly, B cells also failed to undergo CSR when co-stimulated by LPS and CD154. Our study suggests the complexity of how different stimuli cross-regulate an important B cell differentiation process and an important role of TLRs in inducing T-independent antibody responses.

Evidence of glycoproteins and sulphated proteoglycan-like presence in extracellular polymeric substance from anaerobic granular sludge

The protein fraction of extracellular polymeric substance (EPS) from two anaerobic granular sludge samples was characterized with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and a far western blotting method. SDS-PAGE was used with various staining applications to obtain a protein (silver), glycoprotein [periodic acid–Shiff's (PAS)] or proteoglycan-like (Alcian blue at pH 2.5 (carboxylic group) or 1 (sulphated group)) fingerprint. The fingerprints of the EPS denatured protein from the two sludge samples differed. Some proteins are specific to Soluble (S) or Bound (B)-EPS (20–100 kDa). Denatured proteins with a polysaccharide moieties characterization are more present in B-EPS. Glycoproteins with α-d-mannosyl and/or α-d-glucosyl (90, 50, 40 kDa) were detected. Proteoglycan-like and sulphated proteoglycan-like substances are also detected, mainly in B-EPS. A 68 kDa sulphated proteoglycan-like substance contains two glucidic residue types: α-d-mannosyl and/or α-d-glucosyl and N-acetyl-β-d-glucosamine. Such heteroproteins are present around the membrane as well as the surface-layer from Archaea and from some bacteria. The glycoprotein and sulphated proteoglycan-like substance are assumed to contribute to anaerobic granule strength, thanks to their ability to perform interactions of various nature (ionic, hydrophobic, Ca2+ as divalent cation bridging, etc.).

Type, density, and presentation of grafted adhesion peptides on polysaccharide-based hydrogels control preosteoblast behavior and differentiation.

In this work, cell-responsive polysaccharide hydrogels were prepared by a simple procedure based on the sequential bioconjugation and cross-linking of the polysaccharide backbone with bioactive peptides and poly(ethylene glycol)-bis(thiol) (PEG-(SH)2), respectively. Using thiol-ene reactions, we successfully functionalized hyaluronic acid (HA) and carboxymethylcellulose (CMC) with short and long peptides (5-mer and 15-mer derivatives, respectively) derived from adhesive proteins of bone extracellular matrix. The resulting HA-peptide and CMC-peptide conjugates with varying degrees of substitution were then carefully characterized by (1)H NMR spectroscopy to precisely control the peptide density into the hydrogels cross-linked with PEG-(SH)2. Preosteoblast seeded on the hydrogels with controlled identical stiffness spread in a manner that was strongly dependent on ligand density. Surprisingly, increasing the density of the adhesive peptide anchors did not result in a plateau of initial cell spreading but rather in a bell-shaped cell response that varies with the nature of both polysaccharide backbone and functional peptide. Placing the cells under optimal conditions for cell/hydrogel interaction, we showed that in HA hydrogels, the polysaccharide moiety is not solely a passive scaffold that presents the active peptides but is an active player in cell microenvironment to control and sustain cell activity.

Structure-activity relationships of sulfated glycoproteins from Codium fragile on nitric oxide releasing capacity from RAW264.7 Cells.

The effects of sulfate and protein contents as well as molecular weights of the sulfated glycoproteins (NF2) from Codium fragile on the immunomodulation were systematically investigated. The obtained NF2 derivatives displayed various amounts of proteins (2.3-8.7 %) and sulfates (4.3-8.1 %) as well as different molecular weights (47.3-128.0 × 10(3) g/mol). NF2 was not able to stimulate RAW264.7 cells to release NO without its protein moiety, which was essential to activate NF-κB pathway through the degradation and phosphorylation of IκB-α and the subsequent translocation of p65/p50 complex in the cell nucleus. In addition, the proteins in NF2 were required to trigger MAPK pathway for the phosphorylation of ERK1/2, p38, and JNK1/2 as well as the nuclear translocation of c-JUN and c-FOS. However, the protein moiety itself could not activate RAW264.7 cells, thus the complex formation of the polysaccharide and protein moieties in NF2 was pivotal to stimulate macrophage cells.

B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: Modulation by BCR and CD40, and relevance to T-independent antibody responses

Ig class switch DNA recombination (CSR) in B cells is crucial to the maturation of antibody responses. It requires IgH germline IH-CH transcription and expression of AID, both of which are induced by engagement of CD40 or dual engagement of a Toll-like receptor (TLR) and B cell receptor (BCR). Here, we have addressed cross-regulation between two different TLRs or between a TLR and CD40 in CSR induction by using a B cell stimulation system involving lipopolysaccharides (LPS). LPS-mediated long-term primary class-switched antibody responses and memory-like antibody responses in vivo and induced generation of class-switched B cells and plasma cells in vitro. Consistent with the requirement for dual TLR and BCR engagement in CSR induction, LPS, which engages TLR4 through its lipid A moiety, triggered cytosolic Ca2+ flux in B cells through its BCR-engaging polysaccharidic moiety. In the presence of BCR crosslinking, LPS synergized with a TLR1/2 ligand (Pam3CSK4) in CSR induction, but much less efficiently with a TLR7 (R-848) or TLR9 (CpG) ligand. In the absence of BCR crosslinking, R-848 and CpG, which per se induced marginal CSR, virtually abrogated CSR to IgG1, IgG2a, IgG2b, IgG3 and/or IgA, as induced by LPS or CD154 (CD40 ligand) plus IL-4, IFN-γ or TGF-β, and reduced secretion of class-switched Igs, without affecting B cell proliferation or IgM expression. The CSR inhibition by TLR9 was associated with the reduction in AID expression and/or IgH germline IH-S-CH transcription, and required co-stimulation of B cells by CpG with LPS or CD154. Unexpectedly, B cells also failed to undergo CSR or plasma cell differentiation when co-stimulated by LPS and CD154. Overall, by addressing the interaction of TLR1/2, TLR4, TLR7 and TLR9 in the induction of CSR and modulation of TLR-dependent CSR by BCR and CD40, our study suggests the complexity of how different stimuli cross-regulate an important B cell differentiation process and an important role of TLRs in inducing effective T-independent antibody responses to microbial pathogens, allergens and vaccines.

Native lysozyme and dry-heated lysozyme interactions with membrane lipid monolayers: Lateral reorganization of LPS monolayer, model of the Escherichia coli outer membrane

Lysozyme is mainly described active against Gram-positive bacteria, but is also efficient against some Gram-negative species. Especially, it was recently demonstrated that lysozyme disrupts Escherichia coli membranes. Moreover, dry-heating changes the physicochemical properties of the protein and increases the membrane activity of lysozyme. In order to elucidate the mode of insertion of lysozyme into the bacterial membrane, the interaction between lysozyme and a LPS monolayer mimicking the E. coli outer membrane has been investigated by tensiometry, ellipsometry, Brewster angle microscopy and atomic force microscopy. It was thus established that lysozyme has a high affinity for the LPS monolayer, and is able to insert into the latter as long as polysaccharide moieties are present, causing reorganization of the LPS monolayer. Dry-heating increases the lysozyme affinity for the LPS monolayer and its insertion capacity; the resulting reorganization of the LPS monolayer is different and more drastic than with the native protein.

Polysaccharides from Ferula sinkiangensis and Potent Inhibition of Protein Tyrosine Phosphatase 1B

Ferula sinkiangensis is known as a medicinal plant for thousands of years in China. It is a perennial plant species endemic in Xinjiang and is used in traditional Uyghur medicine for a long time 1 . There are many reports on the pharmacological activities of Ferula, which include treating dysentery, counteracting toxins killing parasitic worms, and resolving phlegm. It is also used as a deodorant. Previous investigations of the chemical composition of Ferula have identified essential oils, flavonoids, minerals, and ferulic acid in the extract [2]. Most reports of Ferula have concentrated on the pharmacological aspects of these compounds. Only a few reports have described methods for the extraction of the polysaccharides, and none has described the isolation or identification of these compounds. To the best of our knowledge, there is no report about polysaccharides from roots of Ferula.This study will provide a basis for future research and application of these polysaccharides. The goal of the current work was to establish a method to isolate and quantitate the polysaccharides from roots of Ferula, as well as to determine their monosaccharide compositions, elucidate their structures, and evaluate water-soluble polysaccharides (FSPs), a potent inhibitor of protein tyrosine phosphatase 1B (PTP1B) in vitro. Lipids, pigments, and low-molecular-weight compounds were removed from air-dried raw material by extraction with petroleum ether, CHCl3, and MeOH [3]. The remaining raw material was dried and then extracted with hot water to obtain FSPs. The yield was 9.5%. The total sugar and protein were measured by anthrone-sulfuric acid and Bradford methods [4, 5] with D-glucose and bovine serum albumin (BSA) as standard. The analysis of total sugar and protein contents indicated that FSPs was composed of 45.1 3% polysaccharide and 12.5 3% protein. Furthermore, uronic acid measurement by the m-hydroxydiphenol method [6] with D-galacturonic acid as standard indicated that 28.1 3% of the polysaccharide moiety in FSPs was acidic. Consequently, the preliminary results demonstrated that FSPs was an acid proteoglycan. After removing proteins by the Savage method [7], the crude polysaccharides (FSPs) gave a negative response to the Bradford test, and no absorption was detected at 280 and 260 nm, indicating the absence of protein and nucleic acids. The neutral polysaccharide, eluted with distilled water, was named FSPs-n. The acidic polysaccharides, eluted by the stepwise addition of 0–1.0 M NaCl solutions, was named FSPs-a. The yields of the two fractions were 6.8 and 14.8% in the crude polysaccharides [8, 9]. According to the results of HPLC analysis [10, 11], the carbohydrate portion of FSPs contained ribose, arabinose, glucose, fucose, and galactose in a ratio of 8.9:3.3:2.1:1.5:0.3. FSPs-n was composed of glucose, xylose, arabinose, galactose, and mannose in a ratio of 3.9:4.0:1.8:1.4:0.8, and FSPs-a contained glucose, xylose, mannose, and arabinose in a ratio of 6.5:4.0:1.7:1.0. Furthermore, FSPs and FSPs-a were composed of glucuronic acid and galacturonic acid in a ratio of 1.1:0.3 and 2.3:5.5, respectively.

Structural characterization of the polysaccharide moiety of an aqueous glycopeptide from mannatide

A homogeneous glycopeptide with a molecular weight of 1.61×105Da, termed as MT2-A, was isolated from crude mannatide by DEAE-52 cellulose column and Sephacryl S-300 gel column. The polysaccharide moiety of MT2-A was mainly composed of mannose and trace amount of glucose. Besides, MT2-A contained 16 kinds of amino acids and the total amino acid content was 4.41%. The detailed chemical structure of MT2-A was elucidated using methylation analysis, Fourier transform infrared spectroscopy (FT-IR), partial acid hydrolysis, selective acetolysis and 1D/2D nuclear magnetic resonance (NMR) spectroscopy. Based on the experimental results, it was concluded that the polysaccharide moiety of MT2-A had a backbone of (1→6)-α-D-mannopyranose residues, which highly branched at O-2 position of (1→2,6)-α-D-mannopyranose residues. The side chains were mainly composed of (1→)-α-D-mannopyranose, (1→2)-α-D-mannopyranose and (1→3)-α-D-mannopyranose residues. The length of the side chains could not be longer than four mannose residues.

Analysis of lignin–carbohydrate and lignin–lignin linkages after hydrolase treatment of xylan–lignin, glucomannan–lignin and glucan–lignin complexes from spruce wood

Xylan-lignin (XL), glucomannan-lignin (GML) and glucan-lignin (GL) complexes were isolated from spruce wood, hydrolyzed with xylanase or endoglucanase/β-glucosidase, and analyzed by analytical pyrolysis and 2D-NMR. The enzymatic hydrolysis removed most of the polysaccharide moieties in the complexes, and the lignin content and relative abundance of lignin-carbohydrate linkages increased. Analytical pyrolysis confirmed the action of the enzymatic hydrolysis, with strong decreases of levoglucosane and other carbohydrate-derived products. Unexpectedly it also revealed that the hydrolase treatment alters the pattern of lignin breakdown products, resulting in higher amounts of coniferyl alcohol. From the anomeric carbohydrate signals in the 2D-NMR spectra, phenyl glycoside linkages (undetectable in the original complexes) could be identified in the hydrolyzed GML complex. Lower amounts of glucuronosyl and benzyl ether linkages were also observed after the hydrolysis. From the 2D-NMR spectra of the hydrolyzed complexes, it was concluded that the lignin in GML is less condensed than in XL due to its higher content in β-O-4' ether substructures (62 % of side chains in GML vs 53 % in XL) accompanied by more coniferyl alcohol end units (16 vs 13 %). In contrast, the XL lignin has more pinoresinols (11 vs 6 %) and dibenzodioxocins (9 vs 2 %) than the GML (and both have ~13 % phenylcoumarans and 1 % spirodienones). Direct 2D-NMR analysis of the hydrolyzed GL complex was not possible due to its low solubility. However, after sample acetylation, an even less condensed lignin than in the GML complex was found (with up to 72 % β-O-4' substructures and only 1 % pinoresinols). The study provides evidence for the existence of structurally different lignins associated to hemicelluloses (xylan and glucomannan) and cellulose in spruce wood and, at the same time, offers information on some of the chemical linkages between the above polymers.

Fractionation of soil humic acids by preparative polyacrylamide gel electrophoresis in the presence of concentrated urea

To separate soil humic acids (HAs) into their constituents and characterize them, polyacrylamide gel electrophoresis (PAGE) was carried out in the presence of 7 M urea using a preparative electrophoresis system. Two types of soil HAs were fractionated into nine fractions by PAGE. The dark-colored constituents were recovered from the electrophoretic fractions by precipitation on acidification, and the brown-colored constituents dissolved in the acidic solution of fast-moving fractions were recovered by adsorption onto DAX-8 resin. High-performance size-exclusion chromatography (HPSEC) confirmed that the constituents of the HAs were separated based on their molecular size by PAGE. The dark-colored constituents exhibited higher degrees of humification than did the corresponding unfractionated HAs, except for the constituents remaining in the electrophoretic gels at the end of electrophoresis. Diffuse reflectance infrared spectroscopy revealed that the chemical properties of the dark-colored constituents changed regularly: the content of carboxyl groups decreased and the proportions of proteinous, aliphatic and polysaccharide moieties increased with increasing molecular size. The humification degrees of the constituents adsorbed onto DAX-8 resin were considerably lower than those of the corresponding unfractionated HAs. The chemical properties of the DAX-8-adsorbed constituents were different from those of the dark-colored constituents. Observation of electrophoretic fractions under blue light (470 nm) and HPSEC with fluorescence detection at an excitation wavelength of 460 nm and an emission wavelength of 520 nm showed that green fluorescent substances were largely concentrated in the smallest molecular size fractions and were partitioned into both the dark-colored precipitates and DAX-8-adsorbed fractions. The proportion of organic carbon recovered by precipitation and adsorption onto DAX-8 resin was 45–63%, indicating that substantial parts of the HA constituents were missing. The unrecovered constituents were considered to be acid-soluble, nearly colorless substances. The dissociation of the acid-soluble constituents from the acid-insoluble dark-colored constituents during the preparative PAGE of soil HAs was ascribed to disruption of hydrogen bonding and hydrophobic interactions caused by concentrated urea.

Interactions between Rotavirus and Natural Organic Matter Isolates with Different Physicochemical Characteristics

Interaction forces between rotavirus and Suwanee River natural organic matter (SRNOM) or Colorado River NOM (CRNOM) were studied by atomic force microscopy (AFM) in NaCl solutions and at unadjusted pH (5.7-5.9). Compared to CRNOM, SRNOM has more aromatic carbon and phenolic/carboxylic functional groups. CRNOM is characterized with aliphatic structure and considerable presence of polysaccharide moieties rich in hydroxyl functional groups. Strong repulsive forces were observed between rotavirus and silica or mica or SRNOM. The interaction decay length derived from the approaching curves for these systems involving rotavirus in high ionic strength solution was significantly higher than the theoretical Debye length. While no adhesion was observed for rotavirus and SRNOM, attraction was observed between CRNOM and rotavirus during approach and adhesion during retraction. Moreover, these adhesion forces decreased with increasing ionic strength. Interactions due to ionic hydrogen bonding between deprotonated carboxyl groups on rotavirus and hydroxyl functional groups on CRNOM were suggested as the dominant interaction mechanisms between rotavirus and CRNOM.

Both LCCL-domains of human CRISPLD2 have high affinity for lipid A

The LCCL-domain is a recently defined protein module present in diverse extracellular multidomain proteins. Practically nothing is known about the molecular function of these domains; based on functional features of proteins harboring LCCL-domains it has been suggested that these domains might function as lipopolysaccharide-binding domains. Here we show that the two LCCL-domains of human CRISPLD2 protein, a lipopolysaccharide-binding serum protein involved in defense against endotoxin shock, have higher affinity for the lipid A, the toxic moiety of lipopolysaccharides than for ipopolysaccharide. Our observation that the LCCL-domains of CRISPLD2 are specific for the toxic lipid A moiety of the endotoxin suggests that it may block the interaction between endotoxins and the host endotoxin receptors without interfering with the development of antibacterial immunity against the polysaccharide moiety of LPS. We suggest that the anti-inflammatory function of CRISPLD2 protein may account for its role in various pathological and developmental processes.

Characterization of polysaccharides with marked inhibitory effect on lipid accumulation in Pleurotus eryngii

Mushrooms have a great potential for the production of useful bioactive metabolites. To explore the bioactive compounds from edible mushrooms for interfering with the development of macrophage-derived foam cells, which is recognized as the hallmark of early atherosclerosis, eight types of mushrooms polysaccharides had been selected to be tested. Consequently, different mushrooms polysaccharides displayed diverse component profiles. Of polysaccharides that we tested, the Pleurotus eryngii polysaccharide had the strongest inhibitory effect on lipid accumulation. Furthermore, through fractionation of DEAE-52 and Sephadex G-100, the polysaccharide from P. eryngii had been successfully purified and identified. By the analysis of IR, GC, and HPLC, the purified polysaccharide was estimated to be 30–38kDa for the average molecular weight with the monosaccharide composition mainly composed of D-types of mannose, glucose and galactose. Findings presented in this report firstly provide direct evidence, which links the purified polysaccharide moiety with the biological function in foam-cell model.

Structural characterization and immunostimulatory activity of a novel protein-bound polysaccharide produced by Hirsutella sinensis Liu, Guo, Yu & Zeng

HS002-II, a novel protein-bound polysaccharide with 44kDa molecular weight, was fractionated from submerged cultures of Hirsutella sinensis Liu, Guo, Yu & Zeng by DEAE-Sepharose and Sephacryl S200 chromatography. Based on the results of infrared spectroscopy, high performance liquid chromatography, methylation, amino acid analysis, NMR spectroscopy and atomic force microscopy, the polysaccharide moieties of HS002-II mainly contained a long backbone of (1→3)-linked α-d-ribofuranosyl units (1→4)-linked α-d-xylopyranosyl units and (1→4)-linked β-d-glucopyranosyl units, which was substituted at C-6. The two branches were β-d-mannopyranosyl residues and β-d-galactopyranosyl residues terminated with α-l-arabinopyranosyl residues, respectively. HS002-II consisted of 57.9% polysaccharide and 42.1% protein with the existence of N-type carbohydrate–protein linkage. In terms of the pro-inflammatory cytokines assay (NO, TNF-α, IL-1β and NF-κB) using murine macrophages cell line (RAW264.7), HS002-II exhibited significant immunomodulatory activity by stimulating the IκB-NF-κB pathway.

Separation and characterization of the constituents of compost and soil humic acids by two-dimensional electrophoresis

To investigate the chemical heterogeneity of humic acids (HAs), we applied two-dimensional (2-D) electrophoresis to HAs from a compost and two types of soils. In this method, HAs are first separated by isoelectric focusing (IEF) and then separated by polyacrylamide gel electrophoresis (PAGE). IEF and PAGE were carried out in the presence of 7 M urea. Upon 2-D electrophoresis of HAs, dark-colored substances were spread out across the gel mainly in the isoelectric point (pI) range of 3.0–4.5. Green fluorescence was observed in the smaller molecular size region of the gel, especially in the pI range of 3.0–4.5, and the most intense fluorescence was found at the moving front. The gels were divided into 36 sections, and then HA constituents were extracted from the individual sections and recovered by precipitation with acid. The distribution of organic carbon (C) among the gel sections coincided with that of the dark-colored substances on the gel. The total C recoveries were only 43–50%, suggesting that a considerable amount of HA constituents was lost during the extraction from the gels and purification. High-performance size-exclusion chromatography confirmed that the constituents of HAs were separated based on their molecular sizes by PAGE. The measurement of diffuse reflectance infrared Fourier transform (DRIFT) spectra indicated that the chemical properties of the HA constituents differed depending on the position on the gels and were affected by the molecular size rather than the pI. The fractions of the compost HA were characterized by higher proportions of aliphatic, proteinous and polysaccharide moieties and by the presence of lignin-derived structures. For the soil HAs, the fractions were characterized by a high proportion of the carboxyl group and a low proportion of aliphatic moieties. The proportion of proteinous and polysaccharide moieties in the fractions of soil HAs decreased with decreasing molecular size. The chemical properties of the green fluorescent substances remained unclear, since there was not enough of the substances to measure the DRIFT spectra. The present study showed that 2-D electrophoresis in the presence of concentrated urea offers an effective method for fractionating and isolating the constituents of HAs.

Separation and structural elucidation of a polysaccharide CC30w-1 from the fruiting body of Coprinus comatus

Abstract In this study, a water soluble polysaccharide (CC30w-1), with a molecular mass of 1.03×104, was isolated from the fruiting body of Coprinus comatus. The polysaccharide moiety was composed of L-Fuc and D-Gal in the ratio of 1:3.84. The structure of CC30w-1was elucidated by methylated analysis, GC–MS, 1H and 13C NMR spectroscopy, together with 1H–1H COSY, TOCSY, HMQC and HMBC. Results showed that the polysaccharide consists of the following repeating units: α − L − Fuc p 1 ↓ 2 → 6 ) − α − D − Gal p − ( 1 → 6 ) − α − D − Gal p − ( 1 → 6 ) − α − D − Gal p − ( 1 → 6 ) − α − D − Gal p ( 1 →