A Decrease in the Hardness of Feces with Added Glucosylceramide Extracted from Koji In Vitro-A Working Hypothesis of Health Benefits of Dietary Glucosylceramide.

Skin barrier function, prevent colon cancer, head and neck cancer, and decrease liver cholesterol. However, the mechanism of action has not yet been elucidated. In this study, we propose a new working hypothesis regarding the health benefits and functions of glucosylceramide: decreased fecal hardness. This hypothesis was verified using an in vitro hardness test. The hardness of feces supplemented with glucosylceramide was significantly lower than that of the control. Based on these results, a new working hypothesis of dietary glucosylceramide was conceived: glucosylceramide passes through the small intestine, interacts with intestinal bacteria, increases the tolerance of these bacteria toward secondary bile acids, and decreases the hardness of feces, and these factors synergistically result in in vivo effects. This hypothesis forms the basis for further studies on the health benefits and functions of dietary glucosylceramides.

Starmerella kisarazuensis f.a., sp. nov., a novel yeast isolated from Trifolium pratense flowers.

Three yeast isolates, NBRC 115909T, NBRC 115910 and NBRC 116270, were isolated from Trifolium pratense (red clover) flowers collected from Kisarazu, Chiba, Japan. Analysis of the sequences of the D1/D2 domains of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) regions revealed that these isolates represent a single novel species within the genus Starmerella. Also, no ascospore formation was observed. The yeast isolates were closely related to Starmerella vitae UWOPS 00-107.2T and Starmerella bombi NRRL Y-17081T. They differed from S. vitae, the most closely related species with a validly published name, by ten nucleotide substitutions with two gaps in the D1/D2 domains and 20 nucleotide substitutions in the ITS region. Moreover, the three isolates exhibited distinct phenotypic characteristics from the closely related species. Therefore, we suggest that these three isolates represent a novel species, designated as Starmerella kisarazuensis f.a., sp. nov. The holotype is NBRC 115909T (isotype: CBS 18485T).

Vermiculite as a culture substrate greatly improves the viability of frozen cultures of ectomycorrhizal basidiomycetes.

We assessed a new cryopreservation protocol that uses vermiculite as a culture substrate, called the vermiculite protocol (VP), by assessing the viability, recovery time of hyphae after revival, and colony diameter of cryosensitive ectomycorrhizal basidiomycete strains after storage for 2 weeks or 1 year in a vapour-phase liquid nitrogen tank. Twelve difficult-to-preserve strains of nine species (Amanita citrina, A. pantherina, A. rubescens, A. spissa, Kobayasia nipponica, Lactarius akahatsu, L. hatsudake, Sarcodon aspratus, and Tricholoma flavovirens) that did not achieve good revival after cryopreservation with our previous Homolka's perlite protocol and modified perlite protocol (MPP) experiments were used to assess the new methodology. Vermiculite and liquid medium were put into a cryotube and inoculated with an agar plug containing mycelia. The cryotube was cultured for various incubation times. After adequate mycelial growth, a mixture of cryoprotectants (5% dimethyl sulfoxide and 10% trehalose [5D10T] or 5% glycerol and 10% trehalose [5G10T]) was placed into the cryotube. The cryotube was frozen in a freezing container in a -80 °C freezer and then stored in vapour-phase liquid nitrogen. In the recovery test, 10 of 12 strains showed 100% revival after 2 weeks of storage in the 5G10T cryoprotectant, and all 12 strains showed 100% revival after 2 weeks of storage in the 5D10T cryoprotectant. Furthermore, all strains were viable after 1 year of storage in a vapour-phase liquid nitrogen tank. Thus, the VP is applicable to a wide range of ectomycorrhizal basidiomycete cultures, including highly cryosensitive strains.

A modified perlite protocol with a mixed dimethyl sulfoxide and trehalose cryoprotectant improves the viability of frozen cultures of ectomycorrhizal basidiomycetes.

Homolka's perlite protocol (HPP) was evaluated for cryopreservation of a wide range of ectomycorrhizal basidiomycete cultures, then a modified perlite protocol (MPP), in which cryoprotectant was added just before freezing rather than during the culturing process, was applied to cryosensitive strains that failed to survive when HPP was used. Further modifications of MPP with various cryoprotectants were explored to improve the cryopreservation of hard-to-preserve strains. The efficacy of HPP was assessed in 111 strains of 38 species of basidiomycetes of various cryosensitivities. After freezing strains using HPP, the viability and colony diameter of the strains were examined after 2 wk, 6 mo, and 1 y of storage at -80 C. Of the 111 strains tested, 91 survived after 1 y of storage with high viability of 80% or more, whereas the remaining 20 strains exhibited low and unstable viability. For those selected cryosensitive strains that did not survive well when HPP was used, MPP was applied with a mixture of cryoprotectants, dimethyl sulfoxide (DMSO), glycerol, and trehalose, at different concentrations and combinations. Toxicity testing of the cryoprotectants in the nonfrozen state revealed that 12% (v/v) glycerol was highly toxic for six strains (four species), whereas DMSO (5% and 10% [v/v]) was less toxic than glycerol. The viability of the cryosensitive strains after freezing demonstrated that DMSO was more efficient than glycerol, and trehalose enhanced the cryoprotective effects of both glycerol and DMSO when MPP was used for cryopreservation. Our comparative analysis of MPP with various combinations and concentrations of cryoprotectants revealed that a mixture of 5% DMSO and 10% trehalose was the most effective cryoprotectant, and that using MPP with this cryoprotectant was applicable to many cryosensitive strains.

A new method for determining the mycelial weight of the koji-mold Aspergillus oryzae by measuring its glycosylceramide content.

At present, the quantitation of the mycelial weight of the industrially important non-pathogenic fungus Aspergillus oryzae, which is used for manufacturing koji, is performed by quantitating N-acetylglucosamine. However, since N-acetylglucosamine is a cell wall component, the extraction procedure is costly and tedious, and its quantitative performance is poor. Here, we report a novel method for the quantitation of A. oryzae mycelial weight. The amount of glycosylceramide significantly correlated with both the mycelial weight of A. oryzae and the amount of N-acetylglucosamine, an established index of the mycelial weight of A. oryzae in koji. This new method is simple and efficient and can be used in the brewing and food industries to determine the mycelial weight of A. oryzae.

Structural determination of glucosylceramides in the distillation remnants of shochu, the Japanese traditional liquor, and its production by Aspergillus kawachii.

Shochu is traditional Japanese liquor produced from various crops and fungi Aspergillus kawachi or A. awamorii . The amount of unutilized shochu distillation remnants is increasing because of the recent prohibition of ocean dumping of these remnants. In this Article, we first describe the structures of glucosylceramides contained in shochu distillation remnants by fragment ion analysis using ESI-tandem mass spectrometry. Shochu distillation remnant produced from barley contained glucosylceramides d18:2/C16:0h, d18:2/C20:0h, d19:2/C18:1h, and d18:2/C18:0h. Koji (barley fermented with A. kawachii) contained the same glucosylceramides. Shochu distillation remnants produced from rice contained glucosylceramides d18:2/C18:0h and d19:2/C18:1h. The culture broth of A. kawachii contained glucosylceramides d19:2/C18:1h and d19:2/C18:0h. These results indicate that the glucosylceramides contained in crops and those produced by A. kawachii transfer through the processes of fermentation with yeast and distillation to the shochu distillation remnant. This information will enable utilization of shochu distillation remnants and koji as novel sources of sphingolipids.

Unadjuvanted pandemic H1N1 influenza vaccine in HIV-1-infected adults.

We evaluated the immune response to a 2009 influenza A (H1N1) unadjuvanted vaccine in HIV-infected patients and assessed the boosting effect of a second dose. HIV-infected adults were enrolled and scheduled to receive the H1N1 unadjuvanted vaccine containing 15µg of A/California/7/2009 haemagglutinin. Anti-H1N1 antibody titers were measured at enrollment and 4-8 weeks after each vaccination by using haemagglutination inhibition (HI) and virus neutralization (NT) assays. One hundred and four patients were analyzed. Seroconversion, as measured by using HI and NT assays, was observed in 52 (50.0%) patients and 49 (47.1%) patients, respectively, after the first dose. Seroconversion rate evaluated by using NT, but not HI, antibody titers was associated with HIV RNA levels of <400copies/ml (odds ratio, 3.21; 95% CI, 1.15-8.96). Other parameters, including CD4 cell count, were not associated with seroconversion. In a cohort that received two vaccine doses at a 4-8-week interval (n=54), the seroconversion rate and geometric mean titer for HI antibodies were 44.4% (95% CI, 30.8-58.1%) and 30.5 (95% CI, 19.9-46.9) after the first dose, respectively, and 48.1% (95% CI, 34.4-61.9%) and 39.0 (95% CI, 26.1-58.2) after the second dose, respectively. Among HIV-infected patients, the seroconversion rate was around 50% after the first dose of unadjuvanted vaccine. A second dose of vaccine had a limited boosting effect on immunity in this patient cohort.

Amphibian chytridiomycosis in Japan: distribution, haplotypes and possible route of entry into Japan.

A serious disease of amphibians caused by the chytrid fungus Batrachochytrium dendrobatidis was first found in Japan in December 2006 in imported pet frogs. This was the first report of chytridiomycosis in Asia. To assess the risk of pandemic chytridiomycosis to Japanese frogs, we surveyed the distribution of the fungus among captive and wild frog populations. We established a nested PCR assay that uses two pairs of PCR primers to amplify the internal transcribed spacer (ITS) region of a ribosomal RNA cassette to detect mild fungal infections from as little as 0.001 pg (1 fg) of B. dendrobatidis DNA. We collected swab samples from 265 amphibians sold at pet shops, 294 bred at institutes and 2103 collected at field sites from northern to southwestern Japan. We detected infections in native and exotic species, both in captivity and in the field. Sequencing of PCR products revealed 26 haplotypes of the B. dendrobatidis ITS region. Phylogenetic analysis showed that three of these haplotypes were specific to the Japanese giant salamander (Andrias japonicus) and appeared to have established a commensal relationship with this native amphibian. Many other haplotypes were carried by alien amphibians. The highest genetic diversity of B. dendrobatidis was found in the American bullfrog (Rana catesbeiana). Some strains of B. dendrobatidis appeared to be endemic to Japanese native amphibians, but many alien strains are being introduced into Japan via imported amphibians. To improve chytridiomycosis risk management, we must consider the risk of B. dendrobatidis changing hosts as a result of anthropogenic disturbance of the host-specific distribution of the fungus.

JBIR-37 and -38, novel glycosyl benzenediols, isolated from the sponge-derived fungus, Acremonium sp. SpF080624G1f01.

In the course of our chemical screening program for new secondary metabolites, we isolated new compounds JBIR-37 (1) and -38 (2) from a culture broth of the marine sponge-derived fungus, Acremonium sp. SpF080624G1f01. The structures of 1 and 2 were determined to be di- and mono-O-beta-D-glucopyranosyloxy-4-(1,1-dimethyl-2-propenyl)benzene on the basis of extensive NMR and MS spectroscopic data, respectively.

JBIR-15, a new aspochracin derivative, isolated from a sponge-derived fungus, Aspergillus sclerotiorum Huber Sp080903f04.

In the course of our chemical screening program for novel metabolites by LC-MS monitoring, we isolated a new aspochracin derivative, JBIR-15 (1), together with aspochracin, from the culture broth of a sponge-derived fungus, Aspergillus sclerotiorum Huber Sp080903f04. The structure of 1 was determined to be N-demethyl aspochracin at the alanyl residue on the basis of extensive NMR and MS analyses.

New sesquiterpenes, JBIR-27 and -28, isolated from a tunicate-derived fungus, Penicillium sp. SS080624SCf1.

In the course of our screening program for novel metabolites from tunicate-derived fungi, novel sesquiterpenoids, named JBIR-27 (1) and -28 (2), together with known sporogen-AO1 and phomenone, were isolated from the culture broth of Penicillium sp. SS080624SCf1. The structures of 1 and 2 were determined to be eremophilane analogs on the basis of extensive NMR and MS analyses. Sporogen-AO1, phomenone and 2 showed cytotoxicity against human cervical carcinoma cell line HeLa at IC(50) values of 8.3, 19 and 92 microM, respectively, whereas 1 was inactive at a concentration of 80 microM.

Novel 24-membered macrolides, JBIR-19 and -20 isolated from Metarhizium sp. fE61.

In the course of our screening program for active compounds that induce cell morphological changes of Saccharomyces cerevisiae, the culture broth of an entomopathogenic fungus Metarhizium sp. fE61 exhibited a unique morphological phenotype. We conducted an activity-guided isolation from the fermentation broth of Metarhizium sp. fE61 to yield two new macrolide compounds named JBIR-19 (1) and -20 (2) as active substances. Their structures were determined to be 24-membered macrolide analogs containing a 2-aminoethyl phosphate ester on the basis of NMR and other spectroscopic data. Compounds 1 and 2 induced striking elongated morphology of S. cerevisiae at concentrations of 3.1 and 13 microM, but showed weak antiyeast activity at MICs of 200 and >200 microM, respectively.

Production of bioactive compounds based on phylogeny in the genus Penicillium preserved at NBRC.

Penicillium strains (n=394) preserved at NBRC (the NITE Biological Resource Center) were compared as to groupings (11 species-clusters) based on phylogeny and the production of bioactive compounds. The strains in two clusters, of which P. chrysogenum and P. citrinum are representative, showed higher rates of positive strains with multi-biological activities.

Comparison of groupings among members of the genus Aspergillus based on phylogeny and production of bioactive compounds.

Three hundred sixty-six Aspergillus strains preserved at the National Institute of Technology and Evaluation (NITE) were compared as to phylogenetic relationships (11 species-clusters) based on the DNA sequences of the D1/D2 domains of LSU rRNA and ITS regions, including the 5.8S rRNA and biological activities of their secondary metabolites. The results showed relatively well correlation between the phylogenetic distribution and the production of bioactive compounds, especially, antimicrobial activities.