Multiple tolerance of Rhodotorula glutinis R-1 to acid, aluminum ion and manganese ion, and its unusual ability of neutralizing acidic medium.

A red yeast isolated from the acidic water of Kusatsu hot spring could grow in an acidic medium of pH 1.5 and was identified as Rhodotorula glutinis. Electron microscope observations (scanning electron microscopy [SEM] and transmission electron microscopy [TEM]) showed that cell envelope became wrinkled and thick as the pH values of media became lower. The cell membrane grown at pH 1.5 was about four times as thick as that grown at pH 6.0. It was suggested that the change of cell envelope plays an important role in the acid tolerance. Cellular proteins at pH 1.5 appeared to be different from those at pH 6.0 and the amounts of phospholipids and non-phospholipids increased and decreased under low pH conditions, respectively. The acid-tolerant yeast also showed strong resistance to both aluminum and manganese ions. An acidic medium (pH 3.0) containing these ions (100 mM) was shifted to neutral pH by long-term cultivation of the red yeast, suggesting the potential of using this yeast in the bioremediation of acidic soil containing these ions at a high level.

Antitumor effects of (1-->3)-beta-D-glucan and (1-->6)-beta-D-glucan purified from newly cultivated mushroom, Hatakeshimeji (Lyophyllum decastes Sing.).

Eleven polysaccharides were isolated from a hot-water extract of fruiting bodies of Lyophyllum decastes Sing. by ion-exchange chromatography and gel permeation chromatography. Three polysaccharides (IV-1, IV-2, and IV-3) composed mainly of glucose showed marked antitumor activities against Sarcoma 180 and their average molecular weights were 305 kDa, 130 kDa and 14 kDa, respectively. From methylation analyses and 13C-NMR spectra, it was suggested that IV-1 was a (1-->3)-beta-D-glucan-type polysaccharide, IV-3 was a (1-->6)-beta-D-glucan-type polysaccharide, and IV-2 was a (1-->3, 1-->6)-beta-d-glucan-type polysaccharide or a mixture of both polysaccharides. Increases in the number of peritoneal macrophages and third component of complement (C3)-positive fluorescent cells in mice treated with IV-1 suggested that the inhibitory effect on tumor growth is due to immunological host-mediated mechanisms.

Investigation of cyclomaltooligosaccharide-bound 6-(4-methoxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one for enhanced chemiluminescence.

The chemiluminescence compound 2-methyl-6-(4-methoxyphenyl)imidazo[1, 2-a]pyrazin-3(7H)-one (MCLA) was attached to cyclomaltooligosaccharides (cyclodextrins) through a single spacer by the formation of an amide bond. The properties of oxygen-induced chemiluminescence of the synthesized cyclodextrin-bound MCLA were investigated in an aqueous phosphate buffer, pH 8.0. The light-emitting efficiency was remarkably dependent on the kind of bound cyclodextrin, spacer length between the MCLA and cyclodextrin, and the binding site in cyclodextrin. The light-emitting efficiencies of cyclomaltooctaose (gamma-cyclodextrin)-bound compounds were higher than those of cyclomaltohexaose- or cyclomaltoheptaose-bound compounds. Especially, compounds in which MCLA attached to the secondary side of gamma-cyclodextrin through a short chain showed an up to 44-fold enhancement over that of a non-cyclodextrin compound. In the current case, the efficiency of single excited-state formation was 23 times greater than that of the non-cyclodextrin compound and significantly responsible for greater light-emitting efficiency. The chemiluminescence spectra indicated the wide entrance of the secondary side of gamma-cyclodextrin, and the short spacer allowed suitable intramolecular affinity between the singlet excited-state chromophore moiety and the cyclodextrin.

Chemiluminescence of 2-methyl-6-arylimidazo-[1,2-a]pyrazin-3(7H)-one in protic solvents: electron-donating substituent effect on the formation of the neutral singlet excited-state molecule.

2-Methyl-6-arylimidazo[1,2-a]pyrazin-3(7H)-ones with a substituent such as phenyl, 4-methoxyphenyl or 4-trifluoromethoxyphenyl at the 6-position of the imidazo[1,2-a]pyrazin-3(7H)-one ring system, produced chemiluminescence emission in mixtures of water and DMF and in several mixtures of MeOH and DMF under neutral conditions. Under these protic luminescence conditions, the respective light emissions were generated from neutral singlet excited-state molecules. The electron-donating effect of the 4-methoxy substituent on the phenyl group increased the efficiency of the neutral singlet excited state formation, whereas non-substitution and a 4-trifluoromethoxy group having no electron donating ability decreased the efficiency. The compound having the electron-donating methoxy group substituent showed two chemiluminescence emitters, which generated light at lambda(max) 410-420 nm and 460 nm. It was determined that the neutral molecules in the excited state generating light emission at the shorter wavelengths are neutral singlet excited-state molecules suitable for highly efficient singlet excited-state formation. A role of the electron-donating effect of the methoxy group is postulated to be generation of the special neutral singlet excited-state molecules.

Adhesion of polysaccharides to intact cells and protoplasts of Nicotiana tabacum BY-2 and its stimulative effects on protoplast growth.

The adhesion of water-soluble beta-d-glycans, including cellulose-adhesive schizophyllan, xyloglucan, and locust bean gum to intact cells and protoplasts of Nicotiana tabacum BY-2 was examined using their fluorescent derivatives. Fluorescence microscopy showed that schizophyllan, xyloglucan, locust bean gum, and xanthan gum bound to the intact cells, and that schizophyllan, xanthan gum, and succinoglycan bound to the protoplasts. These adhesive beta-d-glycans raised considerably the number of the cells regenerated from protoplasts. The results suggest that some water soluble beta-d-glycans showing affinity for intact cells and/or protoplasts will be suitable for use as stabilizers of protoplast division.

Convenient Regioselective mono-2-O-Sulfonation of Cyclomaltooctaose.

Regioselective mono-2-O-sulfonation of cyclomaltooctaose was conveniently achieved by using the combination of sulfonyl imidazole and molecular sieves in DMF. In this reaction, no 3-O- or 6-O-sulfonation products were produced. The reactions do not require strict anhydrous or basic conditions, or specific sulfonyl groups.

The correlation between adhesion of schizophyllan to yeast glucan and its effect on regeneration of yeast protoplast.

Schizophyllan, a water-soluble (1-->3)-beta-D-glucan with a triple-helical conformation, adheres to yeast glucan and curdlan gel. As the molecular weight of schizophyllan decreases, both its adhesion to the water-insoluble glucans and its ability to promote the regeneration of yeast protoplasts are reduced. Therefore, we hypothesize that schizophyllan can surround yeast protoplasts by adhering to a fragment of yeast glucan remaining or/and resynthesized on the cell surface and that this encapsulation allows regeneration of the protoplast cells to occur at very high frequency.

Effects of degraded schizophyllans on regeneration of protoplast cells of Saccharomyces cerevisiae.

Schizophyllan was heated at 100 degrees C in 85% dimethyl sulfoxide (DMSO) containing 0.01 M H2SO4 for various times, and fractionated by gel-permeation chromatography. Molecular weights (M(r)) of the depolymerized products thus obtained were measured in water and DMSO by GPC-LALLS to estimate their conformations in water. The products with triple helical structure stimulated regeneration of Saccharomyces cerevisiae protoplast cells, while those of single chain conformation were totally inactive.

Degradation of humoral host defense by Candida albicans proteinase.

The effect of an extracellular proteinase from the pathogenic yeast Candida albicans on the bactericidal and opsonizing activities of human serum was studied. The ability of human polymorphonuclear leukocytes to kill Staphylococcus aureus was greatly reduced when the bacteria were opsonized with human serum treated with the proteinase. The reduction in the opsonizing activity of human serum was attributed to degradation of the Fc portion of immunoglobulin G by the action of C. albicans proteinase as determined by immunoprecipitation reaction. However, the Fab portion of immunoglobulin G was resistant to proteolysis by the proteinase. A clear reduction in the bactericidal activity of human serum against Escherichia coli was observed when the serum was treated with C. albicans proteinase. The reduction of serum bactericidal activity was attributed to the degradation of complement C3 by proteolysis by the proteinase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while C5 resisted the action of the proteinase. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteinase also degrades endogenous proteinase inhibitors, such as alpha 2 macroglobulin and alpha 1 proteinase inhibitor, which are involved in regulating inflammation. These results suggest that destruction of a host's defense-oriented or regulatory proteins facilitates debilitation of the infected host.

Eleven newly characterized xyloglucan oligoglycosyl alditols: the specific effects of sidechain structure and location on 1H NMR chemical shifts.

Eleven previously uncharacterized oligosaccharides, each containing from seventeen to twenty glycosyl residues, were isolated from the xyloglucan produced by suspension-cultured Acer pseudo-platanus cells and characterized by 1H NMR spectroscopy, fast-atom bombardment mass spectrometry, and matrix-assisted laser-desorption mass spectrometry. The complex mixture of xyloglucan oligosaccharides released by endo-(1-->4)-beta-glucanase (Trichoderma reesei) treatment of cell walls was similar to that released by digestion of the soluble xyloglucan present in the culture medium. The oligosaccharides were converted to oligoglycosyl alditols by borohydride reduction and purified by a combination of gel-permeation (Bio-Gel P-2) chromatography, normal-phase HPLC, reversed-phase HPLC, and high-performance anion-exchange (HPAE) chromatography. Eleven new oligoglycosyl alditols (along with several others that had been previously characterized) were isolated and characterized, allowing additional correlations between xyloglucan structure and specific chemical shift effects in the 1H NMR spectra to be determined. The correlations between structural and spectral features deduced in this study will facilitate the structural determination of a wide range of xyloglucans and their subunit oligosaccharides.

Cyclic (1----2)-beta-D-glucans (cyclosophorans) produced by Agrobacterium and Rhizobium species.

Neutral and acidic cyclic (1----2)-beta-D-glucans (cyclosophorans), obtained from culture filtrates and cells of Agrobacterium and Rhizobiun, are synthesised on the cell surface and then secreted. Eight cyclosophorans with dp 17-24 were isolated; all of the strains of Agrobacterium showed almost the same distribution pattern, whereas there were three other distribution patterns for the strains of Rhizobium.

Characterization of seven xyloglucan oligosaccharides containing from seventeen to twenty glycosyl residues.

The complete primary structures of seven oligosaccharide subunits of the xyloglucan secreted by suspension-cultured Acer pseudoplatanus cells were determined. The oligosaccharides, ranging in size from 17 to 20 glycosyl residues, were generated by treatment of the xyloglucan with an endo-beta-(1----4)-glucanase. The oligosaccharide components of a fraction obtained by Bio-Gel P-2 chromatography of enzyme-treated xyloglucan were further purified by normal-phase h.p.l.c. and then converted to the corresponding oligoglycosyl alditols by reduction with NaBH4. The oligoglycosyl alditols, after purification to near homogeneity by reversed-phase h.p.l.c., were structurally characterized by 1H-n.m.r. spectroscopy, fast-atom bombardment mass spectrometry (f.a.b.-m.s.), and analysis of their glycosyl-residue and glycosyl-linkage compositions. Novel structural elements of xyloglucans were observed in this study, including beta-D-xylopyranosyl and alpha-L-arabinofuranosyl-(1----3)-beta-D-xylopyranosyl sidechains. The results also extend our list of correlations between 1H-n.m.r. resonances and specific structural features of xyloglucans and thus enhance our ability to determine the structures of xyloglucans from various sources.

A new undecasaccharide subunit of xyloglucans with two alpha-L-fucosyl residues.

A new oligosaccharide subunit of xyloglucan was isolated from the beta-(1----4)-endoglucanase digestion products of the xyloglucan in what is referred to as "sycamore extracellular polysaccharides" and found to be an undecasaccharide having two terminal alpha-L-fucopyranosyl residues. The undecasaccharide was structurally characterized by 1H-n.m.r. spectroscopy, fast-atom bombardment mass spectrometry (f.a.b.-m.s.), and glycosyl-residue and glycosyl-linkage composition analyses. The structure of the undecasaccharide was confirmed by digesting it with a hydrolase that releases alpha-D-Xylp-(1----6)-D-Glc from the non-reducing end of xyloglucan oligosaccharides.

Spontaneous mutation of polysaccharide production in Alcaligenes faecalis var. myxogenes 10C3.

Alcaligenes faecalis var. myxogenes 10C3, which produces large amounts of succinoglucan and small amounts of curdlan, was genetically unstable and mutated spontaneously to a form producing more curdland than succinoglucan when stocked on nutrient agar slants. The mutation occurred in the absence of cell division when the cells were incubated in saline and was enhanced by treatment with N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methane sulfonate, or ultraviolet light. Mutant strains were genetically stable and did not revert spontaneously for at least 1 year when stocked on nutrient agar slants.