The life cycle of Hymenoscyphus fraxineus on Manchurian ash,Fraxinus mandshurica,in Japan

Hymenoscyphus fraxineus causes a lethal disease known as “ash dieback” in the common ash, Fraxinus excelsior, in Europe. It is hypothesized that the fungus originated from East Asia. This fungus is found on the leaf litter of the Manchurian ash, Fraxinus mandshurica, in Japan and is reported to produce apothecia on pseudosclerotial plates formed mainly on decomposing rachises. However, dieback disease has not been reported in Japan, and little is known about the life cycle of H. fraxineus. This study was conducted to explore the behavior and life cycle of this fungus. It was revealed that, after infection by ascospores, H. fraxineus endophytically inhabits the living leaves of F. mandshurica. On fallen leaves, the fungus behaves saprophytically, producing apothecia on pseudosclerotial plates formed mainly on the decomposing rachises. Analysis by real-time quantitative polymerase chain reaction (qPCR) revealed that the amount of H. fraxineus DNA sharply increased in rachises, while such sharp increase of DNA was not found in leaflets.     

Functional Analyses of LONELY GUY Cytokinin-Activating Enzymes Reveal the Importance of the Direct Activation Pathway in Arabidopsis

Cytokinins play crucial roles in diverse aspects of plant growth and development. Spatiotemporal distribution of bioactive cytokinins is finely regulated by metabolic enzymes. LONELY GUY (LOG) was previously identified as a cytokinin-activating enzyme that works in the direct activation pathway in rice (Oryza sativa) shoot meristems. In this work, nine Arabidopsis thaliana LOG genes (At LOG1 to LOG9) were predicted as homologs of rice LOG. Seven At LOGs, which are localized in the cytosol and nuclei, had enzymatic activities equivalent to that of rice LOG. Conditional overexpression of At LOGs in transgenic Arabidopsis reduced the content of N⁶-(Δ²-isopentenyl)adenine (iP) riboside 5′-phosphates and increased the levels of iP and the glucosides. Multiple mutants of At LOGs showed a lower sensitivity to iP riboside in terms of lateral root formation and altered root and shoot morphology. Analyses of At LOG promoter:β-glucuronidase fusion genes revealed differential expression of LOGs in various tissues during plant development. Ectopic overexpression showed pleiotropic phenotypes, such as promotion of cell division in embryos and leaf vascular tissues, reduced apical dominance, and a delay of leaf senescence. Our results strongly suggest that the direct activation pathway via LOGs plays a pivotal role in regulating cytokinin activity during normal growth and development in Arabidopsis.     

Molecular cloning and overexpression of fleA gene encoding a fucose-specific lectin of Aspergillus oryzae

A protein from the cell lysate of Aspergillus oryzae was purified by column chromatography immobilized with a ferrichrysin (Fcy), which is one of the siderophores of A. oryzae. It is produced only in an iron-deficient culture and its molecular weight is estimated as 35,000 by SDS-PAGE. Two internal amino acid sequences of the protein obtained by lysylendopeptidase digestion were analyzed. Molecular cloning shows that it encodes 310 putative amino acid residues separated by 4 introns and is designated as fleA. It shows approximately 26% similarity with the gene encoding a fucose-specific lectin of Aleuria aurantia (AAL). The gene was overexpressed under control of the melO promoter in a submerged culture of A. oryzae. The fleA gene product showed hemagglutination activity against rabbit erythrocytes. A hemagglutination inhibition assay of monosaccharides showed that this lectin specifically binds to L-fucose and weakly reacts with mannose and N-acetyl-neuraminic acid.     

Alterations in hepatitis B virus nucleotide sequences in a chronic virus carrier from immunotolerant to immunoactive phase

Factors involved in transition from the immunotolerant to immunoactive phase in chronic hepatitis B virus (HBV) infection remain unclear. We investigated viral mutations occurring during transition and elucidated their virological and immunological significance. Full-length HBV DNA sequences were serially determined in a chronic HBV carrier from the immunotolerant to immunoactive phase. Viral replicative competence was examined by transfection analysis. HBV-specific CD8⁺ T cell response was evaluated by coculture of CD8⁺ T cells with autologous dendritic cells followed by interferon-γ Elispot assay. Eleven point mutations and two deletions appeared around the onset of the immunoactive phase. Viral replicative competence declined significantly after the onset of active hepatitis. Examination of the CD8⁺ T cell response against two putative T-cell epitopes, which contained substituted amino acids from the immunotolerant to immunoactive phase, showed that mutant HBV epitopes gave a lesser T cell response than wild-type HBV ones. In summary, point mutations and deletions may occur prior to or concurrent with the onset of the immunoactive phase during chronic HBV infection. These mutations may result in a significant decrease in both viral replicative competence and HBV-specific CD8⁺ T cell response, suggesting a possible adaptation for the maintenance of viral persistence.     

Characterization of Methanosarcina mazeii TMA isolated from a paddy field soil

We isolated a methanogenic strain, designated as strain TMA (=DSM 9195), from an enrichment culture inoculated with a Japanese paddy field soil. Strain TMA was Gram positive and strictly anaerobic. Cell shape was pseudosarcina-like, and cells were nonmotile. The strain was able to use methylamines, methanol, H₂–CO₂, and acetate as substrates for methanogenesis, but did not utilize formate. The optimum temperature and optimum pH were 30–37°C and 6.5–7.5 respectively. The G+C content of the DNA was 42.1 mol %. Strain TMA had DNA-DNA hybridization values of more than 80% with Methanosarcina mazeii S-6^T (T = type strain). On the basis of phenotypic and genotypic characteristics, we identified strain TMA as M. mazeii. This is the first methylotrophic methanogen isolated from a paddy field soil and identified to the species level.     

Increased Serum LIGHT Levels Correlate with Disease Progression and Severity of Interstitial Pneumonia in Patients with Dermatomyositis: A Case Control Study

BackgroundActivated CD8+ T cells play an important role in the pathogenesis of dermatomyositis (DM) with interstitial pneumonia (IP). Serum CD8+ T-cell activator, LIGHT, and Th1/Th2/Th17 cytokines were measured in DM-IP patients and compared with clinical parameters to investigate their usefulness.MethodsThe correlations between the clinical findings and serum LIGHT and Th1/Th2/Th17 cytokine levels were investigated in 21 patients with DM-IP (14 with rapidly progressive IP [RPIP] and 7 with chronic IP [CIP], including 4 fatal cases of IP).ResultsThe median serum LIGHT level was 119 (16–335.4) pg/ml, which was higher than that in healthy control subjects and DM patients without IP. The median serum IL–6 level was 14.7 (2.4–154.5) pg/ml (n = 13). The other cytokines were detected in only a few patients. The median serum LIGHT level in DM-RPIP patients (156 [49.6–335.4] pg/ml) was significantly higher than that in DM-CIP patients (94.3 [16–164.2] pg/ml) (P = 0.02). The serum IL–6 level did not correlate with either progression or outcome of DM-IP. ROC curve analysis determined a serum LIGHT level of ≥120 pg/ml to be the cut-off value for the rapid progression of DM-IP. Serum LIGHT levels correlated significantly with %DLco (R = 0.55, P = 0.04) and total ground-glass opacity scores (R = 0.72, P = 0.0002). The serum LIGHT level significantly decreased to 100.5 (12.4–259.3) pg/ml 4 weeks after treatment initiation (P = 0.04).ConclusionsThe serum LIGHT level may be a promising marker of disease progression and severity in patients with DM-IP.     

A single amino acid substitution converts cytochrome P450(14DM) to an inactive form, cytochrome P450SG1: complete primary structures deduced from cloned DNAS

Genes for lanosterol 14-demethylase, cytochrome P450(14DM), and a mutated inactive cytochrome P450SG1 were cloned from S. cerevisiae strains D587 and SG1, respectively. A single nucleotide change resulting in substitution of Asp for Gly-310 of cytochrome P450(14DM) was found to have occurred in cytochrome P450SG1. In this protein the 6th ligand to heme iron is a histidine residue instead of a water molecule, which may be the ligand for the active cytochrome P450(14DM). Molecular models of the active sites of the cytochrome P450(14DM) and cytochrome P450SG1 were built by computer modeling on the basis of the known structure of that of cytochrome P450CAM whose crystallographic data are available. The mechanisms which may cause a histidine residue to gain access to the heme iron are discussed.