Diversity and ubiquity of xylariaceous endophytes in live and dead leaves of temperate forest trees

To test the hypothesis that xylariaceous endophytes were ubiquitous on live and dead leaves of various tree species in the field, xylariaceous fungi were isolated from live leaves and bleached and nonbleached portions of dead leaves of a total of 94 tree species in a cool temperate forest in Japan. The biodiversity of xylariaceous endophytes was evaluated as the richness of operational taxonomic units (OTUs) determined by phylogenetic analysis of the nucleotide sequence of the D1/D2 region of the LSU rDNA of fungal isolates. A total of 326 isolates of xylariaceous fungi were isolated from live and dead leaves and classified into 15 OTUs. The three major OTUs, Xylaria sp.1, Nemania sp., and Biscogniauxia sp., accounted for 94% (308 isolates) of the total number of isolates, and were isolated from various live and dead leaves. Xylaria sp.1 was frequently encountered on bleached portions (which were produced due to the selective decomposition of lignin) of dead leaves of broad-leaved deciduous tree species. The results suggest that xylariaceous endophytes did not show host specificity and had a saprobic phase on dead leaves in their life cycles and that Xylaria sp.1 was capable of decomposing lignin in the field conditions.     

Characterization of the reaction of decoupling ubiquinone with bovine mitochondrial respiratory complex I

We previously produced the unique ubiquinone QT (“decoupling” quinone), the catalytic reduction of which in NADH-quinone oxidoreduction with bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I) is completely decoupled from proton translocation across the membrane domain. This feature is markedly distinct from those of typical short-chain quinones such as ubiquinone-1. To further characterize the features of the QT reaction with complex I, we herein synthesized three QT analogs, QT2–QT4, and characterized their electron transfer reactions. We found that all aspects of electron transfer (e.g. electron-accepting activity and membrane potential formation) vary significantly among these analogs. The features of QT2 as decoupling quinone were slightly superior to those of original QT. Based on these results, we conclude that the bound positions of QTs within the quinone binding cavity susceptibly change depending on their side-chain structures, and the positions, in turn, govern the behavior of QTs as electron acceptors.     

Three new <Emphasis Type="Italic">Ophiostoma</Emphasis> species with <Emphasis Type="Italic">Pesotum</Emphasis> anamorphs associated with bark beetles infesting <Emphasis Type="Italic">Abies</Emphasis> species in Nikko,Japan

Three species of Ophiostoma possessing Pesotum anamorphs isolated from bark beetles and their galleries infesting Abies species in Nikko, Japan, are described as new species. Ophiostoma nikkoense is characterized by brush-shaped synnemata producing long septate clavate conidia, perithecia with neck, and allantoid ascospores. Ophiostoma microcarpum has smaller perithecia with hyphoid ostiolar hyphae on the neck, and the ascospores are cylindrical or ossiform in side and face views. Ophiostoma abieticola has perithecia without ostiolar hyphae on the neck and produces orange-section-shaped or reniform ascospores.Contribution no. 187, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba     

Relationship between the Decomposition Process of Coarse Woody Debris and Fungal Community Structure as Detected by High-Throughput Sequencing in a Deciduous Broad-Leaved Forest in Japan

We examined the relationship between the community structure of wood-decaying fungi, detected by high-throughput sequencing, and the decomposition rate using 13 years of data from a forest dynamics plot. For molecular analysis and wood density measurements, drill dust samples were collected from logs and stumps of Fagus and Quercus in the plot. Regression using a negative exponential model between wood density and time since death revealed that the decomposition rate of Fagus was greater than that of Quercus. The residual between the expected value obtained from the regression curve and the observed wood density was used as a decomposition rate index. Principal component analysis showed that the fungal community compositions of both Fagus and Quercus changed with time since death. Principal component analysis axis scores were used as an index of fungal community composition. A structural equation model for each wood genus was used to assess the effect of fungal community structure traits on the decomposition rate and how the fungal community structure was determined by the traits of coarse woody debris. Results of the structural equation model suggested that the decomposition rate of Fagus was affected by two fungal community composition components: one that was affected by time since death and another that was not affected by the traits of coarse woody debris. In contrast, the decomposition rate of Quercus was not affected by coarse woody debris traits or fungal community structure. These findings suggest that, in the case of Fagus coarse woody debris, the fungal community structure is related to the decomposition process of its host substrate. Because fungal community structure is affected partly by the decay stage and wood density of its substrate, these factors influence each other. Further research on interactive effects is needed to improve our understanding of the relationship between fungal community structure and the woody debris decomposition process.     

Phenosite: a web database integrating the mouse phenotyping platform and the experimental procedures in mice

Recently, a number of collaborative large-scale mouse mutagenesis programs have been launched. These programs aim for a better understanding of the roles of all individual coding genes and the biological systems in which these genes participate. In international efforts to share phenotypic data among facilities/institutes, it is desirable to integrate information obtained from different phenotypic platforms reliably. Since the definitions of specific phenotypes often depend on a tacit understanding of concepts that tends to vary among different facilities, it is necessary to define phenotypes based on the explicit evidence of assay results. We have developed a website termed PhenoSITE (Phenome Semantics Information with Terminology of Experiments: http://www.gsc.riken.jp/Mouse/), in which we are trying to integrate phenotype-related information using an experimental-evidence-based approach. The site's features include (1) a baseline database for our phenotyping platform; (2) an ontology associating international phenotypic definitions with experimental terminologies used in our phenotyping platform; (3) a database for standardized operation procedures of the phenotyping platform; and (4) a database for mouse mutants using data produced from the large-scale mutagenesis program at RIKEN GSC. We have developed two types of integrated viewers to enhance the accessibility to mutant resource information. One viewer depicts a matrix view of the ontology-based classification and chromosomal location of each gene; the other depicts ontology-mediated integration of experimental protocols, baseline data, and mutant information. These approaches rely entirely upon experiment-based evidence, ensuring the reliability of the integrated data from different phenotyping platforms.     

Novel scaffold for cathepsin K inhibitors

Pyrrolopyrimidine, a novel scaffold, allows to adjust interactions within the S3 subsite of cathepsin K. The core intermediate 10 facilitated the P3 optimization and identified highly potent and selective cathepsin K inhibitors 11-20.     

Mutations in the helix termination motif of mouse type I IRS keratin genes impair the assembly of keratin intermediate filament

Two classical mouse hair coat mutations, Rex (Re) and Rex wavy coat (Re(wc)), are linked to the type I inner root sheath (IRS) keratin genes of chromosome 11. An N-ethyl-N-nitrosourea-induced mutation, M100573, also maps close to the type I IRS keratin genes. In this study, we demonstrate that Re and M100573 mice bear mutations in the type I IRS gene Krt25; Re(wc) mice bear an additional mutation in the type I IRS gene Krt27. These three mutations are located in the helix termination motif of the 2B alpha-helical rod domain of a type I IRS keratin protein. Immunohistological analysis revealed abnormal foam-like immunoreactivity with an antibody raised to type II IRS keratin K71 in the IRS of Re/+ mice. These results suggest that the helix termination motif is essential for the proper assembly of types I and II IRS keratin protein complexes and the formation of keratin intermediate filaments.     

A series of ENU-induced single-base substitutions in a long-range cis-element altering Sonic hedgehog expression in the developing mouse limb bud

Mammal-fish-conserved-sequence 1 (MFCS1) is a highly conserved sequence that acts as a limb-specific cis-acting regulator of Sonic hedgehog (Shh) expression, residing 1 Mb away from the Shh coding sequence in mouse. Using gene-driven screening of an ENU-mutagenized mouse archive, we obtained mice with three new point mutations in MFCS1: M101116, M101117, and M101192. Phenotype analysis revealed that M101116 mice exhibit preaxial polydactyly and ectopic Shh expression at the anterior margin of the limb buds like a previously identified mutant, M100081. In contrast, M101117 and M101192 show no marked abnormalities in limb morphology. Furthermore, transgenic analysis revealed that the M101116 and M100081 sequences drive ectopic reporter gene expression at the anterior margin of the limb bud, in addition to the normal posterior expression. Such ectopic expression was not observed in the embryos carrying a reporter transgene driven by M101117. These results suggest that M101116 and M100081 affect the negative regulatory activity of MFCS1, which suppresses anterior Shh expression in developing limb buds. Thus, this study shows that gene-driven screening for ENU-induced mutations is an effective approach for exploring the function of conserved, noncoding sequences and potential cis-regulatory elements.