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Immunostimulant potential of eight marine yeast glucans (YG) from Candida parapsilosis R20, Hortaea werneckii R23, Candida spencermartinsiae R28, Candida haemulonii R63, Candida oceani R89, Debaryomyces fabryi R100, Debaryomyces nepalensis R305 and Meyerozyma guilliermondii R340 were tested against WSSV challenge in Penaeus monodon post larvae (PL). Structural characterization of these marine yeast glucans by proton nuclear magnetic resonance (NMR) indicated structures containing (1-6)-branched (1-3)-β-D-glucan. PL were fed 0.2% glucan incorporated diet once in seven days for a period of 45 days and the animals were challenged with white spot syndrome virus (WSSV). The immunostimulatory activity of yeast glucans were assessed pre- and post-challenge WSSV by analysing the expression profile of six antimicrobial peptide (AMP) genes viz., anti-lipopolysaccharide factor (ALF), crustin-1, crustin-2, crustin-3, penaeidin-3 and penaeidin-5 and 13 immune genes viz., alpha-2-macroglobulin (α-2-M), astakine, caspase, catalase, glutathione peroxidase, glutathione-s-transferase, haemocyanin, peroxinectin, pmCathepsinC, prophenol oxidase (proPO), Rab-7, superoxide dismutase and transglutaminase. Expression of seven WSSV genes viz., DNA polymerase, endonuclease, protein kinase, immediate early gene, latency related gene, thymidine kinase and VP28 were also analysed to detect the presence and intensity of viral infection in the experimental animals post-challenge. The study revealed that yeast glucans (YG) do possess immunostimulatory activity against WSSV and also supported higher survival (40-70 %) post-challenge WSSV. Among the various glucans tested, YG23 showed maximum survival (70.27%), followed by YG20 (66.66%), YG28 (60.97%), YG89 (58.53%), YG100 (54.05%), YG63 (48.64%), YG305 (45.7%) and YG340 (43.24%)., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The human pathogen Candida albicans encodes at least three putative two-component histidine kinase signal transduction proteins, including Chk1p and a response regulator protein (Cssk1p). Strains deleted in CHK1 are avirulent in a murine model of hematogenously disseminated disease. The specific function of Chk1p has not been established, but hyphae of the chk1 mutant exhibit extensive flocculation while yeast forms are less adherent to reconstituted human esophageal tissue, indicating that this protein may regulate cell surface properties. Herein, we analyze glucan, mannan and chitin profiles in strains deleted in chk1 (CHK21) compared to a gene-reconstituted strain (CHK23) and a parental strain CAF2. Total alkali-soluble hexose from the cell wall of the chk1 mutant (strain CHK21) was significantly reduced. Western blots of cell wall extracts from CHK21, CHK23 and CAF2 reacted with a Mab to the acid-stable mannan fraction revealed extensive staining of lower molecular mass species in strain CHK21 only. FACE (fluorophore assisted carbohydrate electrophoresis) was used to characterize the oligosaccharide side chains of beta-eliminated (O-linked), acid-hydrolyzed (acid-labile phosphomannan) and acetolysis (acid-stable mannan) extracted fractions of total mannan. The profiles of O-linked as well as the acid-labile oligosaccharides were similar in both CAF2 and CHK21, but the acid-stable oligosaccharide side chains were significantly truncated. We also characterized the beta-glucan from each strain using NMR, and found that both the degree of polymerization and the ratio of (1-3)/(1-6) linkages was lower in CHK21 relative to wild-type cells. The sensitivity of CHK21 to antifungal drugs and inhibitors was unaffected. In summary, our data have identified a new function for a histidine kinase two-component signal protein in a human pathogenic fungus.

Health benefits of the polysaccharide (1-->3)-beta-D-glucan, reported to induce immunobiological, hypocholesterolemic, and hypoglycemic effects in humans and animals, have made the isolation, characterization, and assay of a viable glucan product critical. A new analytical method, based on internal standard proton NMR analysis, for the assay of solvent-wet samples containing (1-->3)-beta-D-glucan is presented. The method enables glucan identification, provides a solvent-free assay, and improves upon the previous multistep extraction and lyophilization procedure by reducing the 1-2 day analysis time to 1-2 h. NMR offers a rapid method for quantifying the glucan in commercial samples, such as nutraceuticals, as well as industrial samples enabling better evaluation of the efficacy of these carbohydrates in health-related applications.

Glucans are fungal cell wall polysaccharides which stimulate innate immune responses. We determined the minimum unit ligand that would bind to glucan receptors on human U937 cells using laminarin-derived pentaose, hexaose, and heptaose glucan polymers. When U937 membranes were pretreated with the oligosaccharides and passed over a glucan surface, only the heptasaccharide inhibited the interaction of glucan with membrane receptors at a K(d) of 31 microM (95% CI 20-48 microM) and 100% inhibition. However, the glucan heptasaccharide did not stimulate U937 monocyte NFkappaB signaling, nor did it increase survival in a murine model of polymicrobial sepsis. Laminarin, a larger and more complex glucan polymer (M(w) = 7700 g/mol), only partially inhibited binding (61 +/- 4%) at a K(d) of 2.6 microM (99% CI 1.7-4.2 microM) with characteristics of a single binding site. These results indicate that a heptasaccharide is the smallest unit ligand recognized by macrophage glucan receptors. The data also indicate the presence of at least two glucan-binding sites on U937 cells and that the binding sites on human monocyte/macrophages can discriminate between glucan polymers. The heptasaccharide and laminarin were receptor antagonists, but they were not receptor agonists with respect to activation of NFkappaB-dependent signaling pathways or protection against experimental sepsis.

Glucans are (1-->3)-beta-linked glucose polymers which have immune-stimulating capability. The extraction of water-insoluble (1-->3)-beta-D-glucan form Saccharomyces cerevisiae employs hydrochloric acid. Hydrochloric acid is difficult to employ in the large-scale pharmaceutical extraction of glucans due to its corrosive nature and toxicity. To address these concerns, we determined whether acetic, formic or phosphoric acid can be substituted for hydrochloric acid in the process for the isolation of (1-->3)-beta-D-glucan. The resulting microparticulate glucans were employed as the starting material for the production of (1-->3)-beta-D-glucan phosphate. 13C NMR analysis of the glucan phosphates derived from the acetic, formic or phosphoric acid-extracted microparticulate glucan show excellent correspondence to hydrochloric acid extracted glucan and laminarin, a (1-->3)-beta-D-glucan standard, indicating that the primary structure is not altered by the acid used for extraction. Glucan phosphate prepared from hydrochloric acid had a Mw of 7.2 x 10(4) g/mol, rmsz of 17.7 nm, of 1.50 and (eta) of 49.0 mL/g. Glucan phosphate prepared from acetic acid had a primary polymer peak with a Mw of 1.4 x 10(6) g/mol, rmsz of 23.6 nm, I of 1.93 and (eta) of 62.4 mL/g. Glucan phosphate prepared from formic acid had a main polymer peak with a Mw of 1.2 x 10(6) g/mol, rmsz 27.1 nm, I of 1.56 and (eta) of 89.0 mL/g. Glucan phosphate prepared from phosphoric acid had a primary polymer peak with a Mw of 6.6 x 10(5) g/mol, rmsz of 32.3 nm, I of 2.70 and (eta) of 91.3 mL/g. These data indicate that the molecular mass, size, polydispersity and intrinsic viscosity of the glucan phosphate obtained is influenced by the pKa of protic acid employed to extract the microparticulate glucan. However, the primary structure and side-chain branching are not substantially altered regardless of the acid employed.