Melting behaviour of schizophyllan extracellular polysaccharide gels in the temperature range between 5 and 20°C

Gels of the glucan schizophyllan, consisting of a 1,3-β- -linked backbone of glucose residues with 1,6-β- -glucosyl side groups, were found to show melting behaviour in the temperature range between 5 and 20°C, depending on the glucose concentration in the solvent (0–50 wt% glucose). While the qualitative features of the modulus-versus-concentration and modulus-versus-temperature rheological data for the gels can be modelled using modified cascade theory (which implicitly assumes that no sub-level of organisation exists in the gel structure), a consistent quantitative fit cannot be achieved. The inconsistencies found are consistent with the idea that the gel is composed of bundles (consisting of many triple helices of schizophyllan) with strong intra-bundle attraction and weak inter-bundle forces. Transmission Electron Microscopy (TEM) micrographs of diluted samples indicate that schizophyllan polymers engage in lateral aggregation of triple helical strands at temperatures below the melting temperature, suggesting that indeed bundles of polymers will be present in the gel state.     

深层培养裂褶菌胞外多糖的提取及结构研究

对深层培养裂褶菌 (Schizophyllumcommune)胞外多糖的提取工艺及多糖结构进行了初步研究。将等电点法与Sevag法相结合可高效的去除多糖中的蛋白 ,其方法简单有效。纯多糖经凝胶柱层析 ,聚丙烯酰胺凝胶电泳 ,高效液相色谱分析为均一组分 ,分子量 4×1 0 4D。通过完全水解 ,纸层析 ,气相色谱分析单糖组分 ,红外光谱 ,酶解反应 ,高碘酸氧化分析结构 ,证明了裂褶菌多糖是以葡萄糖为单一组分 ,β (1 3)和β (1 6)糖苷键组成的β D葡聚糖。     

Removal of the side-chain glucose groups from schizophyllan improves the thermal stability of the polycytidylic acid complexes under the physiological conditions

Thermal stabilization of the complex between polycytidylic acid [poly(C)] and the modified schizophyllan (SPG) whose hydrophilic side-chain glucose groups are selectively removed utilizing mild Smith-degradation has been investigated. With the decrease in the side-chain glucose groups of schizophyllan, the complex with poly(C) can be considerably stabilized compared with unmodified SPG; for example, the T(m) value after the removal of the side-chain glucose groups from 33.3 (unmodified) to 1.0 is enhanced by 14 degrees C. In addition, the thermal stabilization effect is even operative under the physiological conditions ([NaCl] = 0.15 mol dm(-3)). This effect is exerted owing to the construction of the hydrophobic atmosphere around the complex. Although schizophyllan lost the side-chain glucose groups, it still kept the protection effect of the bound poly(C) chain against RNaseA-mediated hydrolysis as observed for unmodified schizophyllan. The assessment of the cytotoxicity for A375:human malignant melanoma, and HL60:human promyelocytic leukemia revealed that the modified schizophyllan scarcely increases the cytotoxicity. These results indicate that the present modification for schizophyllan is of great significance in a viewpoint to develop the practical gene carriers operative even under the physiological conditions.     

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.     

Preparation and antimicrobial activity of scleraldehyde from Schizophyllum commune

The present study investigates the antimicrobial activity of oxidized schizophyllan (scleraldehyde) against Gram-positive and Gram-negative bacteria by diffusion and tube dilution analysis. Schizophyllan is a natural polysaccharide produced by fungi of the genus Schizophyllum. Periodate oxidation specifically cleaves the vicinal glycols in scleraldehyde to form their dialdehyde derivatives. The antibacterial activity exhibited by scleraldehyde was defined using various tests such as the disc diffusion assay, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). MIC and MBC values were found to be in the range of 3.0-8.0 mg/mL. Hence, the present studies establish that the scleraldehyde possesses effective antibacterial properties and can be used as a biopreservative for preservation of raw hides and skins.     

Polysaccharide-polynucleotide complexes. Part 7. Hydrogen-ion and salt concentration dependence of complexation between schizophyllan and single-stranded homo RNAs

Schizophyllan belongs to a beta-1,3-D-glucan family, which exists as a random coil in dimethyl sulfoxide (DMSO) and as a triple helix in water, respectively. The schizophyllan single chain forms a complex with single-stranded homo RNAs in water/DMSO mixed solvents. Using circular dichroism, we studied the complexation and its stability as a function of apparent pH (pH(*)) in a mixed solvent system and as a function of the salt concentration. The complex is formed in the pH(*) range 6.5-10, and dissociated in the pH(*) range 4-6. Both poly(A) and poly(C) adopt a double strand in the pH(*) range 4-6 and a single strand in the pH(*) range 6.5-10. Therefore, the conformational change of each polynucleotide is responsible for dissociation/association of the complex, i.e., the single strand of the polynucleotides can form complexes, whereas the double one cannot. This result indicates that hydrogen bonding and similarity of the helix parameters are essential for the complex formation. The melting temperature of the complex reaches the maximum around 0.05 M of NaCl and KCl, and the value of the maximum temperature depends on the cation species.     

β-1,3-Glucan/antisense oligonucleotide complex stabilized with phosphorothioation and its gene suppression

Most of antisense oligonucleotides (ASOs) subjected to current clinical evaluation belong to phosphorothioate (PS) analogues. Although PS has great advantage in DNase resistance, it can induce nonspecific side-effects. Thus it is important to investigate the influence of ASOs with different PS contents. In this paper, we prepared the complex consisting of schizophyllan (SPG) and ASOs attached a dA₄₀ tail with different PS contents to the 3′ end of the ODN, which is introduced to stabilize the complex with SPG. With increase of PS content in the dA₄₀, its complexation ability with SPG was improved and the complex showed high thermal stability. The thermal stability of the fully phosphorothioated ASOs was obtained by only replacing 20% of the oxygen of the phosphodiester moiety. The ability of gene suppression between PS and phosphodiester for antisense sequences was almost the same, indicating that the antisense sequences need not to be PS backbone. These data may provide new insight for the interaction between β-1,3-glucan and DNA and help to deliver therapeutic ODNs.     

The effect of (1-->3)-beta-D-glucans, carboxymethylglucan and schizophyllan on human leukocytes in vitro

(1-->3)-beta-D-glucans are known as potent inductors of humoral and cell-mediated immunity in humans and animals. (1-->3)-beta-D-glucans isolated from various sources differ in their chemical structure and physical parameters and consequently in their immunomodulatory potential. In this study the immunomodulatory activity of two (1-->3)-beta-D-glucans schizophyllan (SPG) and carboxymethylglucan (CMG) was determined and compared on human blood leukocytes in vitro. Both SPG and CMG activated blood phagocytes and lymphocytes as demonstrated by increased whole blood production of reactive oxygen species, by increased production of pro-inflammatory cytokines IL-6, IL-8, and TNF-alpha, by increased surface expression of CD69 on lymphocytes, and by altered expression of CD11b and CD62L on polymorphonuclear leukocytes and monocytes. SPG demonstrated a significantly higher potential to stimulate blood phagocytes and production of selected pro-inflammatory cytokines than CMG. The higher potency of SPG to stimulate human blood phagocytes in vitro could be caused by factors such as higher branching frequencies or neutral polymer charge of SPG or different conformation in solution if compared with CMG.     

Induction of potent cell growth inhibition by schizophyllan/K-ras antisense complex in combination with gemcitabine

Antisense oligonucleotides (AS-ODNs) specifically hybridize with target mRNAs, resulting in interference with the splicing mechanism or the regulation of protein translation. In our previous reports, we demonstrated that β-glucan schizophyllan (SPG) can form a complex with AS-ODNs attached with oligo deoxyadenosine dA₄₀ (AS-ODN-dA₄₀/SPG), and that this complex can be recognized by β-glucan receptor Dectin-1 on antigen presenting cells and lung cancer cells. In many types of cancer cell, activating K-ras mutations related to malignancy are frequently observed. In this study, we first designed 78 AS-ODNs for K-ras to optimize the sequence for highly efficient gene suppression. The selected AS-ODN (K-AS07) having dA₄₀ made a complex with SPG. The resultant complex (K-AS07-dA₄₀/SPG) showed an effect of silencing the ras gene in the cells (PC9: human adenocarcinoma differentiated from lung tissue) expressing Dectin-1, leading to the suppression of cell growth. Furthermore, the cytotoxic effect was enhanced when used in combination with the anticancer drug gemcitabine. Gemcitabine, a derivative of cytidine, was shown to interact with dA₄₀ in a sequence-dependent manner. This interaction did not appear to be so strong, with the gemcitabine being released from the complex after internalization into the cells. SPG and the dA₄₀ part of K-AS07-dA₄₀ play roles in carriers for K-AS07 and gemcitabine, respectively, resulting in a strong cytotoxic effect. This combination effect is a novel feature of the AS-ODN-dA₄₀/SPG complexes. These results could facilitate the clinical application of these complexes for cancer treatment.     

Naphthalene-hydrophobized β-1,3-glucan nanogel for doxorubicin delivery to immunocytes

A water soluble β-1,3-glucan schizophyllan (SPG) can be recognized by an immunocyte receptor called dectin-1. When we introduced naphthalene into the side chain of SPG (nSPG), it formed nanogel by physical cross-link and gained capability to ingest hydrophobic compounds such as doxorubicin. Our in vitro assay revealed that this nanogel can be used as specific delivery of anti-cancer drugs to immunocytes.