Non-sulfated Polysaccharide Research Articles

Characterization and cytotoxic activity of sulfated derivatives of polysaccharides from Agaricus brasiliensis

Agaricus brasiliensis cell-wall polysaccharides isolated from fruiting body (FR) and mycelium (MI) and their respective sulfated derivatives (FR-S and MI-S) were chemically characterized using elemental analysis, TLC, FT-IR, NMR, HPLC, and thermal analysis. Cytotoxic activity was evaluated against A549 tumor cells by MTT and sulforhodamine assays. The average molecular weight (Mw) of FR and MI was estimated to be 609 and 310kDa, respectively. FR-S (127kDa) and MI-S (86kDa) had lower Mw, probably due to hydrolysis occurring during the sulfation reaction. FR-S and MI-S presented ~14% sulfur content in elemental analysis. Sulfation of samples was characterized by the appearance of two new absorption bands at 1253 and 810cm−1 in the infrared spectra, related to SO and CSO sulfate groups, respectively. Through 1H and 13C NMR analysis FR-S was characterized as a (1→6)-(1→3)-β-d-glucan fully sulfated at C-4 and C-6 terminal and partially sulfated at C-6 of (1→3)-β-d-glucan moiety. MI-S was shown to be a (1→3)-β-d-gluco-(1→2)-β-d-mannan, partially sulfated at C-2, C-3, C-4, and C-6, and fully sulfated at C-6 of the terminal residues. The combination of high degree of sulfation and low molecular weight was correlated with the increased cytotoxic activity (48h of treatment) of both FR-S (EC50=605.6μg/mL) and MI-S (EC50=342.1μg/mL) compared to the non-sulfated polysaccharides FR and MI (EC50>1500μg/mL).

Size determination of hyaluronan using a gas-phase electrophoretic mobility molecular analysis

Hyaluronan (HA) is a linear non-sulfated polysaccharide mainly found in the extracellular matrix. The size of HA can vary from a few disaccharides up to at least 25,000 units, reaching molecular weights of 1010(3)kDa. HA has many biological functions, and both its size and tissue concentration play an important role in many physiological and pathological processes. It is relatively easy to determine the HA concentration using enzyme-linked binding protein assays, but the molecular weight of HA has so far been shown to be a more challenging task to measure. Here, we present a method for size determination of HA using gas-phase electrophoretic mobility molecular analysis (GEMMA), which utilizes the electrophoretic mobility of molecules in air to estimate the molecular weight of the analyte. We show that this method gives reliable molecular weight estimations of HA in the range of 30-2400kDa, which covers almost its whole biological range. The average measuring time for one GEMMA spectrum is between 5 and 10min using only 6pg of HA. In addition, the peak area in a GEMMA spectrum can be used to estimate the HA concentration in the sample. The high sensitivity and small sample volumes make GEMMA an excellent tool for both size determinations and estimation of concentration of samples with low HA concentration, as is the case for HA extracted from small tissue samples.

In Vitro Antioxidant Activities of Sulfated Derivatives of Polysaccharides Extracted from Auricularia auricular

In this research, two types of sulfated polysaccharide derivatives were successfully synthesized. Their antioxidant activities were investigated by employing various established in vitro systems. In addition, the degree of sulfation was evaluated using ion-chromatography and IR spectra. The results verify that, when employing scavenging superoxide radical tests, both the sulfation of acid Auricularia auricular polysaccharides (SAAAP) and the sulfation of neutral Auricularia auricular polysaccharides (SNAAP) derivatives possessed considerable antioxidant activity and had a more powerful antioxidant competence than that of the native non-sulfated polysaccharides (AAAP and NAAP). On the other hand, AAAP and NAAP exhibited stronger activity on scavenging both the hydroxyl radical and lipid peroxidation. Available data obtained with in vitro measurements indicates that the sulfated groups of AAAP and NAAP played an important role on antioxidant activity. In sum, the research demonstrates that the antioxidant activity of sulfated polysaccharide derivatives in vitro has a potential significance for seeking new natural antioxidant protective agents.

Hyaluronidases, a group of glycosidases: Current and future perspectives

Given the constant synthesis and degradation of hyaluronan (hyaluronic acid, HA) in tissues, it is remarkable that the human body maintains precise levels of hyaluronan as tightly as it does. Hyaluronidases represent a group of glycosidases, which mainly degrade hyaluronan, a linear, non-sulfated polysaccharide composed of repeating disaccharide units [D-glucuronic acid (1-β-3) N-acetyl-D-glucosamine (1-β-4)]n. They are widely distributed in nature, being found in mammals, insects, leeches and bacteria. There has been a plethora of review articles on hyaluronidases, either as a subject on their own or as part of a review on glycosidases enzymes. The present review summarizes the current research on their classification, sources, activity assays, bio-physical and chemical properties, crystal structural features and its catalytic mechanism. Special emphasis is given to the importance role of that type of enzymes in biotechnological processes, as well as its medicinal and bio-industrial applications.

Sulfation of a polysaccharide obtained from Phellinus ribis and potential biological activities of the sulfated derivatives

The paper reports the preparation, characterization and potential biological activities of a chemically sulfated polysaccharide isolated from Phellinus ribis. Four sulfated derivatives (PRP-SI–IV) with variable degrees of substitution were obtained by the chlorosulfonic acid method, without degradation of the polysaccharide (PRP). The sulfate groups were not regularly distributed along the polysaccharide chain with a multiple substitution pattern as determined by 13C NMR. The sulfated derivatives except for PRP-SI showed significant inhibition effects on HepG2 cells in comparison with the native non-sulfated polysaccharide (PRP). All sulfated derivatives could block new angiogenic vessel formation in zebrafish assay, however, the effects were less than PRP.

Sequence determination of a non-sulfated glycosaminoglycan-like polysaccharide from melanin-free ink of the squid Ommastrephes bartrami by negative-ion electrospray tandem mass spectrometry and NMR spectroscopy

A non-sulfated polysaccharide was isolated from the ink sac of squid Ommastrephes bartrami after removal of the melanin granules. The carbohydrate sequence of this polysaccharide was assigned by negative-ion electrospray tandem mass spectrometry with collision-induced dissociation of the oligosaccharide fractions produced by partial acid hydrolysis of the polysaccharide. The structural determination was completed by NMR for assignment of anomeric configuration and confirmation of linkage information and it was unambiguously identified as a glycosaminoglycan-like polysaccharide containing a glucuronic acid-fucose (GlcA-Fuc) disaccharide repeat in the main chain and a N-acetylgalactosamine (GalNAc) branch at Fuc position 3: -[3GlcAbeta1-4(GalNAcalpha1-3)Fucalpha1](n)-. Partial hydrolysis of the polysaccharide to obtain several oligosaccharide fractions with different numbers of the repeating unit assisted the assignment. In the negative-ion tandem mass spectrometric analysis, the unique (0,2)A type fragmentation was important to establish the presence of a 4-linked fucose in the main polysaccharide chain and a GalNAc branch at the Fuc position-3 of the disaccharide repeat.

Structure and Antiviral Activity of an Acidic Polysaccharide from an Edible Blue-green Alga, Nostoc flagelliforme

Recently, the development of antiviral agents with novel mechanisms of action has been required since many types of infectious disease have become a serious problem in our society. In the present study, we isolated a novel acidic polysaccharide, nostoflan (NSF), from a terrestrial blue-green alga, Nostoc flagelliforme, and examined its structure and antiviral activity. The sugar composition and methylation analyses of NSF revealed that it is mainly composed of (-->4)- D-Glcp-(1-->, -->6,4)-D-Glcp-(1-->, -->4)-D-Galp-(1-->, -->4)-D-Xylp-(1-->, D-GlcAp-(1-->, D-Manp-(1-->) with a ratio of ca. 1:1:1:1:0.8:0.2. Oligosaccharide analysis after partial acid hydrolysis of NSF revealed that this polysaccharide might be mainly composed of the sugar sequences of (-->4)-beta-D-Glcp-(1-->4)-D-Xylp-(1 and-->4)-[beta-D-GlcAp-(1-->6)-]-beta-D-Glcp-(1-->4)-D-Galp-(1-->). NSF showed potent antiviral activities against several enveloped viruses including herpes simplex virus type 1, type 2 (HSV-1, HSV-2), human cytomegalovirus, and influenza A virus (IFV). NSF selectively inhibited the attachment of HSV-1 to host cells but not its penetration phase. In an experimental animal study where IFV-infected mice received NSF intranasally, the mortality of mice was significantly decreased. Neutralizing titers in sera of mice treated with NSF were higher than in those treated with oseltamivir. From these results, NSF was found to be a novel polysaccharide that shows antiviral activity in vitro and in vivo in spite of a nonsulfated polysaccharide.

The binding of human betacellulin to heparin, heparan sulfate and related polysaccharides

Recombinant human betacellulin binds strongly to heparin, requiring of the order of 0.8 M NaCl for its elution from a heparin affinity matrix. This is in complete contrast to the prototypic member of its cytokine superfamily, epidermal growth factor, which fails to bind to the column at physiological pH and strength. We used a well-established heparin binding ELISA to demonstrate that fucoidan and a highly sulfated variant of heparan sulfate compete strongly for heparin binding. Low sulfated heparan sulfates and also chondroitin sulfates are weaker competitors. Moreover, although competitive activity is reduced by selective desulfation, residual binding to extensively desulfated heparin remains. Even carboxyl reduction followed by extensive desulfation does not completely remove activity. We further demonstrate that both hyaluronic acid and the E. coli capsular polysaccharide K5, both of which are unsulfated polysaccharides with unbranched chains of alternating N-acetylglucosamine linked beta(1-4) to glucuronic acid, are also capable of a limited degree of competition with heparin. Heparin protects betacellulin from proteolysis by LysC, but K5 polysaccharide does not. Betacellulin possesses a prominent cluster of basic residues, which is likely to constitute a binding site for sulfated polysaccharides, but the binding of nonsulfated polysaccharides may take place at a different site.

Characterization of extracellular polysaccharides fromNostoc flagelliforme cells in liquid suspension culture

Nostoc flagelliforme cells were studied with regard to the physico-chemical characterization of the extracellular polysaccharides (EPS) secreted in a liquid suspension culture. The hydrolyzed EPS were determined to be composed of four neutral sugars, which were glucose (43.2%), xylose (20.6%), galactose (29.9%), and mannose (6.3%). The glucuronic acid was the only uronic acid identified in the residue. The apparent molecular weight was estimated at 2.79×105. The Fourier transform infrared spectra showed that the EPS evidenced characteristics typical of non-sulfated polysaccharides. The UV spectrum and Bradford reaction indicated that there were no nucleic acids and proteins in them. The thermal analysis showed a decomposition peak at 245°C on the thermogravimetric (TG) curves. The scanning electron microscope (SEM) analysis indicated that the EPS possessed a porous structure. The observed microstructural irregularities indicated that the polysaccharide was a type of amorphous solid. These results showed that the EPS ofN. flagelliforme cells might be ernployed as a substitute for those normally derived from field colonies. The results of this study may prove to be beneficial to the protection of the natural resource represented byN. flagelliforme.

Enzymatically Active N-Deacetylase/N-Sulfotransferase-2 Is Present in Liver but Does Not Contribute to Heparan Sulfate N-Sulfation

Heparan sulfate (HS) proteoglycans influence embryonic development through interactions with growth factors and morphogens. The interactions depend on HS structure, which is largely determined during biosynthesis by Golgi enzymes. NDST (glucosaminyl N-deacetylase/N-sulfotransferase), responsible for HS N-sulfation, is a key enzyme directing further modifications including O-sulfation. To elucidate the roles of the different NDST isoforms in HS biosynthesis, we took advantage of mice with targeted mutations in NDST1 and NDST2 and used liver as our model organ. Of the four NDST isoforms, only NDST1 and NDST2 transcripts were shown to be expressed in control liver. The absence of NDST1 or NDST2 in the knock-out mice did not affect transcript levels of other NDST isoforms or other HS modification enzymes. Although the sulfation level of HS synthesized in NDST1-/- mice was drastically lowered, liver HS from wild-type mice, from NDST1+/-, NDST2-/-, and NDST1+/- / NDST2-/- mice all had the same structure despite greatly reduced NDST enzyme activity (30% of control levels in NDST1+/- / NDST2-/- embryonic day 18.5 embryos). Enzymatically active NDST2 was shown to be present in similar amounts in wild-type, NDST1-/-, and NDST1+/- embryonic day 18.5 liver. Despite the substantial contribution of NDST2 to total NDST enzyme activity in embryonic day 18.5 liver (approximately 40%), its presence did not appear to affect HS structure as long as NDST1 was also present. In NDST1-/- embryonic day 18.5 liver, in contrast, NDST2 was responsible for N-sulfation of the low sulfated HS. A tentative model to explain these results is presented.

Despite differences between dendritic cells and Langerhans cells in the mechanism of papillomavirus-like particle antigen uptake, both cells cross-prime T cells

As human papillomavirus-like particles (HPV-VLP) represent a promising vaccine delivery vehicle, delineation of the interaction of VLP with professional APC should improve vaccine development. Differences in the capacity of VLP to signal dendritic cells (DC) and Langerhans cells (LC) have been demonstrated, and evidence has been presented for both clathrin-coated pits and proteoglycans (PG) in the uptake pathway of VLP into epithelial cells. Therefore, we compared HPV-VLP uptake mechanisms in human monocyte-derived DC and LC, and their ability to cross-present HPV VLP-associated antigen in the MHC class I pathway. DC and LC each took up virus-like particles (VLP). DC uptake of and signalling by VLP was inhibited by amiloride or cytochalasin D (CCD), but not by filipin treatment, and was blocked by several sulfated and non-sulfated polysaccharides and anti-CD16. In contrast, LC uptake was inhibited only by filipin, and VLP in LC were associated with caveolin, langerin, and CD1a. These data suggest fundamentally different routes of VLP uptake by DC and LC. Despite these differences, VLP taken up by DC and LC were each able to prime naive CD8 + T cells and induce cytolytic effector T cells in vitro.

Dipeptidyl peptidase IV (CD26) activity in the hematopoietic system: differences between the membrane-anchored and the released enzyme activity.

Dipeptidyl peptidase IV (DPP-IV; CD26) (EC 3.4.14.5) is a membrane-anchored ectoenzyme with N-terminal exopeptidase activity that preferentially cleaves X-Pro-dipeptides. It can also be spontaneously released to act in the extracellular environment or associated with the extracellular matrix. Many hematopoietic cytokines and chemokines contain DPP-IV-susceptible N-terminal sequences. We monitored DPP-IV expression and activity in murine bone marrow and liver stroma cells which sustain hematopoiesis, myeloid precursors, skin fibroblasts, and myoblasts. RT-PCR analysis showed that all these cells produced mRNA for DPP-IV. Partially purified protein reacted with a commercial antibody to CD26. The K M values for Gly-Pro-p-nitroanilide ranged from 0.43 to 0.98 mM for the membrane-associated enzyme of connective tissue stromas, and from 6.76 to 8.86 mM for the enzyme released from the membrane, corresponding to a ten-fold difference, but only a two-fold difference in K M was found in myoblasts. K M of the released soluble enzyme decreased in the presence of glycosaminoglycans, nonsulfated polysaccharide polymers (0.8-10 micro g/ml) or simple sugars (320-350 micro g/ml). Purified membrane lipid rafts contained nearly 3/4 of the total cell enzyme activity, whose K M was three-fold decreased as compared to the total cell membrane pool, indicating that, in the hematopoietic environment, DPP-IV activity is essentially located in the lipid rafts. This is compatible with membrane-associated events and direct cell-cell interactions, whilst the long-range activity depending upon soluble enzyme is less probable in view of the low affinity of this form.

Milligram-scale preparation and purification of oligosaccharides of defined length possessing the structure of chondroitin from defructosylated capsular polysaccharide K4.

Escherichia coli K4 bacterium synthesizes a nonsulfated capsule polysaccharide (K4) composed of a repeating disaccharide subunit of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) to which beta-fructofuranose units are linked to C-3 of D-glucuronic acid residues. The K4 polyanion is easily defructosylated under acid conditions with no fragmentation of the polymer to produce a polysaccharide having a repeated disaccharide unit of chondroitin consisting of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) (K4d). K4 and K4d were depolymerized by partial digestion with testicular hyaluronidase and separated into uniform-size oligosaccharides from 4-mers to 16-mers by preparative anion-exchange chromatography after removal of the hyaluronidase. The purity and size of each oligosaccharide was confirmed by using anion-exchange HPLC, HPSEC analysis, and FACE. Mg-scale K4d oligosaccharides were obtained from 50 mg K4d starting material. Under the conditions used to degrade the K4 polysaccharide by testicular hyaluronidase, fructose is slowly liberated forming the defructosylated K4. As a consequence, a mixture of uniform- size K4 and K4d oligosaccharide species, from approximately 4- to 20-mers, are generated and size-separated by anion-exchange chromatography. These pure, uniform-size, and large ranges of K4d oligosaccharides having the structure of a chondroitin, -->4)-GlcUA-beta(1-->3)GalNAc-beta(1-->, will be available for investigating important biological functions of this polymer.

Preventive effects of Cladosiphon fucoidan against Helicobacter pylori infection in Mongolian gerbils.

Recently, the acquisition by Helicobacter pylori of resistance to antibiotics has become a serious problem. Therefore, nonantibiotic substances are required to diminish H. pylori-induced gastric lesions. In the present study, the effects of Cladosiphon fucoidan were examined in terms of H. pylori attachment to porcine gastric mucin in vitro and Helicobacter pylori-induced gastritis in vivo. The inhibitory effect of Cladosiphon fucoidan and other polysaccharides on H. pylori attachment to porcine gastric mucin was assayed in vitro with mucin-coated microtiter plates. The effect of Cladosiphon fucoidan on H. pylori-induced gastritis was examined in vivo using Mongolian gerbils. H. pylori-inoculated gerbils were given fucoidan in drinking water. Six weeks after H. pylori-inoculation, gerbils were sacrificed for macroscopic and microscopic examination of gastric lesions and counting of viable H. pylori in the gastric mucosa. Cladosiphon fucoidan inhibited the H. pylori attachment to porcine gastric mucin at pH 2.0 and 4.0. Two other sulfated polysaccharides, Fucus fucoidan and dextran sulfate sodium, also inhibited the attachment but only at pH 2.0. Inhibitory effects of these three sulfated polysaccharides were not observed at pH 7.2 and nonsulfated polysaccharides, such as mannan and dextran, exerted no influence at any pH. In the in vivo experiment, the H. pylori-induced gastritis and the prevalence of H. pylori infected animals were markedly reduced by fucoidan in a dose-dependent manner, at doses of 0.05 and 0.5% in the drinking water. Cladosiphon fucoidan may deserve particular attention as a safe agent that can prevent H. pylori infection and reduce the risk of associated gastric cancer.

Glycosaminoglycan blotting on nitrocellulose membranes treated with cetylpyridinium chloride after agarose-gel electrophoretic separation.

We describe a method for blotting and immobilizing several nonsulfated and sulfated complex polysaccharides on membranes made hydrophilic and positively charged by a cationic detergent after their separation by conventional agarose gel electrophoresis. Nitrocellulose membranes were derivatized with the cationic detergent cetylpyridinium chloride (CPC) and mixtures of glycosaminoglycans (GAGs) were capillary-blotted after their separation in agarose gel electrophoresis in barium acetate/1,2-diaminopropane. Single purified species of variously sulfated polysaccharides were transferred onto the derivatized membranes after electrophoresis with an efficiency of 100% and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining) permitting about 0.1 nug threshold of detection. Nonsulfated polyanions, hyaluronic acid, a fructose-containing polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41, and its defructosylated product, were also electrophoretically separated and transferred onto membranes. The limit of detection for desulfated GAGs was about 0.1-0.5 nug after irreversible or reversible staining. GAG extracts from bovine, lung and aorta, and human aorta and urine were separated by agarose gel electrophoresis and blotted on CPC-treated nitrocellulose membranes. The polysaccharide composition of these extracts was determined. The membrane stained with toluidine blue (reversible staining) was destained and the same lanes used for immunological detection or other applications. Reversible staining was also applied to recover single species of polysaccharides after electrophoretic separation of mixtures of GAGs and their transfer onto membranes. Single bands were released from the membrane with an efficiency of 70-100% for further biochemical characterization.

The medicinal plant Porana volubilis contains polysaccharides with anticoagulant activity mediated by heparin cofactor II

We searched for polysaccharides with anticoagulant activity and inhibitory action on platelet aggregation induced by collagen in 59 species of medicinal plants. We then concentrated our studies on the polysaccharide from the species Porana volubilis, which showed the highest anticoagulant activity among the plants tested. The polysaccharide from this species has an average molecular mass of ∼10 kDa, contains mainly galactose, galacturonic acid, and mannose but no sulfate esters. Its anticoagulant activity is mediated by the enhancement of thrombin inhibition that in turn is mediated by heparin cofactor II but not by antithrombin. The galacturonic acid residues are essential for activity since after reduction of its carboxyl groups the anticoagulant activity disappears. Our report is the first description of a natural nonsulfated polysaccharide from higher plants with anticoagulant activity, which may constitute a new source of compounds with action on coagulation and, perhaps, on thrombosis.

Influence of charge density, sulfate group position and molecular mass on adsorption of chondroitin sulfate onto coral

The adsorption of chondroitin sulfate onto granules of natural coral of specific diameter, between 100 and 500 μm, having high calcium content (>98%) and a homogeneous surface was investigated. Several chondroitin sulfate samples desulfated to various extents, with a sulfate to disaccharide ratio (charge density) of 0.98–0.07 and non-sulfated polysaccharide possessing a chondroitin sulfate backbone structure were tested for their ability to adsorb onto coral. Adsorption of chondroitin sulfate onto coral depends on its charge density, as the removal of sulfate groups totally abolishes this capacity. Various chondroitin sulfates of molecular mass from 26,950 to 1140 were also tested. No appreciable effect depending on the molecular mass was evident. Also, chondroitin sulfate fractions with molecular mass of about 3530 (formed by about 6 disaccharide units) and 1140 (formed by about 2 disaccharide units) retain their full capacity to adsorb onto coral. Furthermore, the position of sulfate groups inside the polysaccharide chains does not influence the ability of chondroitin sulfate to adsorb onto coral. In fact, chondroitin sulfate derivatives almost completely sulfated (>90%) in position 4 of galactosamine and chondroitin almost completely (>90%) sulfated in position 6 show a full adsorbtion onto coral. Thus, large amounts of chondroitin sulfate are adsorbed onto coral, and sulfate groups are of paramount importance in the adsorption process. On the other hand, the capacity of chondroitin sulfate to adsorb onto coral is quite aspecific. In fact, it does not depend on the presence of sulfate groups esterified in a specific position or sulfated sequences arranged in blocks but rather on the presence of sulfate groups, and this ability increases with increasing charge density, as indicated by the values of the Langmuir constant, the adsorption capacity, that decreases with decreasing chondroitin sulfate charge density reaching very low values for the totally desulfated polymer.

The molecular mechanism of sulfated carbohydrate recognition by the cysteine-rich domain of mannose receptor

The mannose receptor (MR) binds foreign and host ligands through interactions with their carbohydrates. Two portions of MR have distinct carbohydrate recognition properties. One is conferred by the amino-terminal cysteine-rich domain (Cys-MR), which plays a critical role in binding sulfated glycoproteins including pituitary hormones. The other is achieved by tandemly arranged C-type lectin domains that facilitate carbohydrate-dependent uptake of infectious microorganisms. This dual carbohydrate binding specificity enables MR to bind ligands by interacting with both sulfated and non-sulfated polysaccharide chains. We previously determined crystal structures of Cys-MR complexed with 4-SO4-N-acetylglucosamine and with an unidentified ligand resembling Hepes (N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]). In continued efforts to elucidate the mechanism of sulfated carbohydrate recognition by Cys-MR, we characterized the binding affinities between Cys-MR and potential carbohydrate ligands using a fluorescence-based assay. We find that Cys-MR binds sulfated carbohydrates with relatively high affinities (KD=0.1 mM to 1.0 mM) compared to the affinities of other lectins. Cys-MR also binds Hepes with a KD value of 3.9 mM, consistent with the suggestion that the ligand in the original Cys-MR crystal structure is Hepes. We also determined crystal structures of Cys-MR complexed with 3-SO4-Lewisx, 3-SO4-Lewisa, and 6-SO4-N-acetylglucosamine at 1.9 Å, 2.2 Å, and 2.5 Å resolution, respectively, and the 2.0 Å structure of Cys-MR that had been treated to remove Hepes. The conformation of the Cys-MR binding site is virtually identical in all Cys-MR crystal structures, suggesting that Cys-MR does not undergo conformational changes upon ligand binding. The structures are used to rationalize the binding affinities derived from the biochemical studies and to elucidate the molecular mechanism of sulfated carbohydrate recognition by Cys-MR.

Properties of fucoidan from Cladosiphon okamuranus tokida in gastric mucosal protection.

To elucidate the anti-ulcer potential of Cladosiphon fucoidan, anti-peptic activity, bFGF stabilizing activity and inflammatory properties of this and related substances were investigated. Anti-peptic activity was observed with this and other sulfated polysaccharides such as dextran sulfate, carrageenan, and Fucus fucoidan. However, non-sulfated polysaccharides such as mannan and dextran did not exert the anti-peptic activity. The loss of bFGF bioactivity was prevented by all sulfated polysaccharides tested except chondroitin sulfate, at pH 7.4 and at pH 4.0. At pH 2.0, only heparin protected the bFGF activity. The generation of superoxide by macrophages and PMNs was stimulated by dextran sulfate, carrageenan, and Fucus fucoidan, whereas Cladosiphon fucoidan, heparin and chondroitin did not. Dextran sulfate, carrageenan, and Fucus fucoidan also stimulated the secretion of TNFalpha from macrophages, while Cladosiphon fucoidan did not. Thus, Cladosiphon fucoidan is a sulfated polysaccharide without inflammatory action. These results suggest that Cladosiphon fucoidan is a safe substance with potential for gastric protection.

Sulfated Polysaccharides Are Required for Collagen-Induced Vascular Tube Formation

We have previously shown that soluble type I collagen can induce vascular tube formation when it contacts the apical side of a confluent endothelial monolayer. In this study we have examined which soluble agent(s) are required for collagen-induced tube formation. Human neo-natal foreskin microvascular endothelial cells, maintained in basal medium, were preincubated with each test agent for 2 h prior to the addition of solubilised type I collagen (100 μg/ml). After 6 h, tube formation was quantitated using image analysis and expressed as the mean area of tube formation (mm2) per microscopic field of view. Collagen-induced tube formation did not occur in the presence of endothelial cell growth supplement, basic fibroblast growth factor, or normal pooled human serum. In contrast, the addition of heparin at 5 or 50 μg/ml caused extensive tube formation (0.22 ± 0.07 and 0.30 ± 0.12 mm2, respectively) whereas at 500 μg/ml little tube formation occurred (0.03 ± 0.02 mm2). Protamine sulfate, an antagonist of heparin, inhibited collagen-induced tube formation in a dose-dependent manner. Pentosan polysulfate, dextran sulfate, heparan sulfate, and chondroitin sulfate mimicked the action of heparin. Partially sulfated heparin (de-N-sulfated heparin) stimulated less tube formation compared to heparin (0.15 ± 0.06 mm2 at 50 μg/ml). The nonsulfated polysaccharides, xylan and dextran, had no effect on tube formation. In summary, sulfated polysaccharides are required for collagen-induced vascular tube formation in vitro. The sulfation of these molecules appears to be vital for collagen-induced tube formation.