Presence of O-glycosidically linked oligosaccharides in the cell wall mannan of Candida krusei purified with Benanomicin A.

Cell wall mannan of the pathogenic yeast Candida krusei was prepared using the antibiotic Benanomicin A, which has a lectin-like function. The chemical structure of this molecule was found to be similar to that of mannan prepared from the same yeast by the conventional method using Fehling reagent. Only a few degradation products were detected when the mannan prepared using Fehling reagent was subjected to alkali treatment (ß-elimination), but multiple α-1,2-linked oligosaccharides were detected when the mannan purified with Benanomicin A was treated with alkali. These results indicate that most of the O-linked sugar chains in mannan were lost under conventional conditions when exposed to the strongly alkaline Fehling reagent. In contrast, the O-glycosidic bond in mannan was not cleaved and the O-linked sugar chains were maintained and almost intact following treatment with the mild novel preparation method using Benanomicin A. Therefore, we argue that the new mannan preparation method using Benanomicin A is superior to conventional methods. In addition, our study suggests that some yeast mannans, whose overall structure has already been reported, may contain more O-linked sugar chains than previously recognized.

Liquid phase immunoassays utilizing magnetic markers and SQUID magnetometer.

BACKGROUND: Immunoassays are one main detection system used in the field of clinical chemistry. Recent developments of a new detection method utilizing a magnetic marker and magnetic sensor have enabled rapid and sensitive immunoassay without the need for bound/free (BF) separation. METHODS: Newly-synthesized conjugated avidin was used as the magnetic marker for quantitative analysis of human interleukin-8 (hIL-8) and immunoglobulin E (hIgE) in several media. A superconducting quantum interference device sensor detected the magnetic fields from markers fixed to antigens by the sandwich method. Magnetic signals from unbound markers were nearly zero due to Brownian rotation. RESULTS: Our magnetic immunoassay could detect four attomoles of model proteins (hIL-8, hIgE) in phosphate buffer without BF separation. Using our standard curve, the range of protein detected ranged from 40 femtomoles to 4 attomoles, and we observed a strong association between protein amounts and magnetic signals from the bound markers. The homogeneous immunoassay could also quantify three hundred cells from the fungus Candida albicans in phosphate buffer. CONCLUSIONS: The present study demonstrates the ability of magnetic markers for measuring biological targets without BF separation. This detection system has great potential for use as the next generation's analytical system.

Structural analysis of cell wall mannan of Candida sojae, a new yeast species isolated from defatted soybean flakes.

We investigated the structural and immunochemical characteristics of cell wall mannan obtained from Candida sojae JCM 1644, which is a new yeast species isolated from defatted soybean flakes. The results of a slide-agglutination test and of an enzyme-linked immunosorbent assay using anti-factor sera to the pathogenic Candida species indicated that the cells and the C. sojae mannan were cross-reactive to the specific anti-factor sera against Candida albicans serotype A (FAb 6) and Candida guilliermondii (FAb 9). Two-dimensional homonuclear Hartmann-Hahn analysis indicated that the mannan consisted of various linked oligomannosyl side chains containing alpha-1,2-, alpha-1,3-, alpha-1,6- and beta-1,2-linked mannose residues. However, although the determinants of antigenic factors 6 and 9 could be not found in this mannan, branched side chains, Manbeta1-2Manalpha1-3[Manalpha1-6]Manalpha1-(2Manalpha1-)n2Man and a linear alpha-1,6-linked polymannosyl backbone, which are cross-reacted by FAbs 6 and 9, respectively, were identified. The mannan was subjected to acetolysis in order to determine the polymerization length of the alpha-1,2-linked oligomannosyl residue in the side chains. The result of (1)H-nuclear magnetic resonance analysis of the released oligosaccharides showed that the remarkable regularity in the length of alpha-1,2-linked oligomannosyl side chains, which were previously found in mannans of other Candida species, is not observed in this mannan.

Distribution of antigenic oligomannosyl side chains in the cell wall mannans of several strains of Candida tropicalis.

In order to clarify the distribution of antigenic oligomannosyl side chains in the cell wall mannans of the pathogenic yeast Candida tropicalis, the chemical structure of mannans isolated from four C. tropicalis strains was investigated using nuclear magnetic resonance, two-dimensional homonuclear Hartmann-Hahn (2D-HOHAHA) spectroscopy. Two-dimensional maps of the 2D-HOHAHA clearly showed the distribution of oligomannosyl side chains in the mannans. The linear side chain Manalpha1-3Manalpha1-(2Manalpha1-)(n)2Man [n> or =2] is present in the mannans from C. tropicalis IFO 0589 and IFO 1400, but not in the mannans from IFO 0199 and IFO 1647. The mannan of IFO 0589 is the only mannan with the branched side chains, Manalpha1-3[Manalpha1-6]Manalpha1-(2Manalpha1-)(n)2Man and Manalpha1-2Manalpha1-3[Manalpha1-6]Manalpha1-(2Manalpha1-)(n)2Man [n> or =2]. However, this mannan lacked the phosphate group and the beta-1,2-linked oligomannosyl side chain which are features of this group. The mannans of the C. tropicalis strains IFO 0589 and IFO 1400 possessed the side chains containing an alpha-1,3-linked mannose residue previously observed in Candida albicans.