Quantitative analysis of the Tricholoma ustale-derived toxin, ustalic acid, in mushroom and food samples by LC-MS/MS.

Tricholoma ustale, a poisonous member of the Tricholomataceae family, causes gastrointestinal symptoms such as diarrhea and vomiting. In Japan, 86 cases (affecting a total of 347 patients) of poisoning with Tricholoma ustale have been reported between 1989 and 2010. Ustalic acid is one of the primary toxic components in Tricholoma ustale. In the present study, the quantitative analysis of the ustalic acid content in mushroom and food samples was conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mushroom and food samples were extracted using methanol containing 0.5% formic acid and 50% aqueous methanol, respectively. Purification using SAX solid-phase extraction (SPE) was conducted prior to LC-MS/MS analysis, which was performed in the ESI negative mode using a C18 column. The method developed for the LC-MS/MS analysis of ustalic acid was extremely sensitive. The limits of quantitation calculated at a signal-to-noise ratio of 10 were 10ng/g (shiitake mushroom) and 0.40ng/g (miso soup). The accuracies of quantitation in the shiitake mushroom and miso soup samples ranged from 99.8%-105% and 98.8%-102%, respectively. This method was applied to leftover mushroom samples from a food poisoning case; here, ustalic acid was detected at 0.57, 3.7µg/g. This analytical method using LC-MS/MS could be useful in food poisoning cases involving mushrooms. This is the first report in which the ustalic acid content was determined using the leftovers of a food poisoning case.

The Mosses of Crocker Range Park, Malaysian Borneo.

This paper reports the mosses from Crocker Range Park (CRP) in Sabah, Malaysian Borneo. In total, 293 species, three subspecies and eight varieties belonging to 118 genera and 36 families are reported. This represents about 40% and 47% of the species and infra-specific taxa reported from Borneo and Sabah, respectively. Out of these, six species are new records for Borneo, namely Barbella horridula, Chaetomitrium lancifolium, Distichophyllum leiopogon, Rhaphidostichum luzonense, Rosulabryum capillare and Taxiphyllum taxirameum and 12 species and one variety are new to Sabah. With these additions, the current number of mosses in Sabah and Borneo are 651 and 766, respectively. The largest family of mosses is Calymperaceae with 35 species and one subspecies, followed by Sematophyllaceae with 32 species and two varieties and Pylaisiadelphaceae with 21 species and one variety. In conclusion, CRP has a very high species richness of mosses which is the second highest in Borneo, after Mount Kinabalu.

Adaptive evolution of rbcL in Conocephalum (Hepaticae, bryophytes).

An excess of nonsynonymous substitutions over synonymous ones has been regarded as an important indicator of adaptive evolution or positive selection at the molecular level. We now report such a case for rbcL sequences among cryptic species in Conocephalum (Hepaticae, Bryophytes). This finding can be regarded as evidence of adaptive evolution in several cryptic species (especially in F and JN types) within the genus. Bryophytes are small land plants with simple morphology. We can therefore expect the existence of several biologically distinct units or cryptic species within each morphological species. In our previous study, we found three rbcL types in Asian Conocephalum japonicum (Thunb.) Grolle and also found evidence strongly suggesting that the three types are reproductively isolated cryptic species. Additionally, we examined rbcL sequence variation in six cryptic species of C. conicum (L.) Dumort. previously recognized by allozyme analyses. As a result, we were able to discriminate the six cryptic species based only on their rbcL sequences. We were able to show that rbcL sequence variation is also useful in finding cryptic species of C. conicum.

Morphology and phylogenetic position of a mat-forming green plant from acidic rivers in Japan.

A green plant, which we have named "Misuzugoke", was found in acidic rivers in Nagano Prefecture, Japan, where it forms macroscopic mats or aggregates. The field-collected aggregates were nearly hemispherical and were composed of compact branched filaments radiating from a central base. The cells of the plant contained a single nucleus and numerous discoid chloroplasts lacking pyrenoids. Due to the plant's simple filamentous organization and absence of reproductive organs, its classification as an alga or a higher plant could not be determined from the field-collected material. When grown for more than 3 months on neutral agar medium (pH 7.0) containing hormones, leafy shoot buds characteristic of bryophytes appeared on the plant. A partial sequence of the plant's rbcL gene, which encodes the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase was 98% similar to that of Dicranella heteromalla (Hedw.) Schimp. (Haplolepideae, Bryopsida). Phylogenetic analyses based on rbcL gene sequences strongly indicate that Misuzugoke is positioned within the Haplolepideae. This mat-forming green plant is therefore considered to be a reduced form of moss in the Haplolepideae.

An outbreak of fulminant hepatitis B in immunocompromised hemodialysis patients.

BACKGROUND: We aimed to clarify the pathogenesis of an outbreak of fulminant hepatitis B in hemodialysis (HD) patients whose compromised cell-mediated immunity in turn contributed to chronic hepatitis B virus (HBV) carriage. METHODS: Five consecutive adult HD patients with acute hepatitis B were evaluated. Viral genotype, mutations, and HBV-DNA levels were studied in relation to viral clearance, liver disease severity, and liver histology by immunostaining. RESULTS: All five patients had hepatitis B surface antigen (HBsAg) genotype C, a G-to-A stop codon mutation at nucleotide (nt) 1896 in the precore region, an A-to-T mutation at nt 1762 and an G-to-A mutation at nt 1764 in the basal core promoter. The possible index patient, who suffered from liver cirrhosis, had HBsAg genotype C, anti-hepatitis B envelope (HBe), and these mutations. The level of HBV-DNA declined by about 10 percent per week and no difference in viral kinetics between the patients who died and the survivor was found, irrespective of therapies. The amount of liver cell apoptosis, as assessed by single-stranded DNA, was scarce. The risk of fulminant hepatic failure did not correlate with the preexistent liver histopathological changes. Acute HBV superinfection was associated with hepatitis C virus (HCV) elimination and increased mortality. CONCLUSIONS: This outbreak of fulminant hepatitis B suggests that HD patients can foster highly virulent HBV strains (possibly owing to their compromised immune responses), which may place others at risk of severe, life-threatening acute liver damage and at increased risk of mortality if chronic carriers of HCV should be infected. We aimed to clarify the pathogenesis of an outbreak of fulminant hepatitis B in hemodialysis (HD) patients whose compromised cell-mediated immunity in turn contributed to chronic hepatitis B virus (HBV) carriage.