A new variety ofMyxotrichum ochraceum from Japanese soil

Myxotrichum ochraceum var.frigidum, a new variety ofMyxotrichum, is described and illustrated from the material from Japanese soil. It is morphologically characterized by dull green to black ascomata with a yellow-orange centrum, peridium consisting of a fragile hyphal network, peridial hyphae with internal coiled branches and short spine-like appendages, pale yellow fusiform striate ascospores, and the absence of an anamorph. The new variety primarily differs from the type variety in the absence of elongate appendages. A key to all accepted species of the genus is revised.     

No reduction of hatching rates among F1 hybrids of naturally hybridizing three Far Eastern daces,genus <Emphasis Type="Italic">Tribolodon</Emphasis> (Cypriniformes,Cyprinidae)

Egg hatching rates of F1 hybrids among three fishes, Japanese dace Tribolodon hakonensis, Pacific redfin T. brandtii, and rosyface dace T. sachalinensis, were compared with pure crosses. They are highly divergent (diverged 10–20 million years ago) but naturally hybridizing. Although crosses involving female rosyface dace were not available, hatching success was high among all combinations of hybrids (76 ± 23%) and comparable to pure crosses (86 ± 8%), implying ecological or genetic isolation mechanisms maintaining each species in later life stages.     

Effects of Pleistocene climate change on genetic structure and diversity of <Emphasis Type="Italic">Shorea macrophylla</Emphasis> in Kalimantan Rainforest

The island of Borneo is the diversity center of the Dipterocarpaceae, the most important family of tropical rainforest trees in Southeast Asia. However, changes in land use and climate have affected dipterocarp distributions on the island, raising concerns about the vulnerability (inter alia) of the endemic riparian species Shorea macrophylla. Thus, to aid conservation efforts, we have investigated the genetic diversity, structure, and demographic history of S. macrophylla. The species’ genetic diversity and structure in Kalimantan (part of Indonesia, covering 75% of the island) were explored by examining genotypes of 377 individuals representing 13 populations in three regions (Northeast, Central, and West Kalimantan) using 14 newly developed microsatellite loci. Higher genetic diversity was found, at all loci, in samples from Northeast Kalimantan than in samples from the other regions. Moderate genetic differentiation between populations was detected (F_ST 0.093). Bayesian clustering, principal coordinate, and neighbor joining tree analyses of the population structure consistently identified two genetically distinct groups, one in the Northeast and the other in the Central and West regions. The higher diversity of the diverged populations in Northeast Kalimantan indicates that the region may have hosted rainforest refugia during the ice age. Accordingly, analysis using DIY ABC software indicated that the Northeast and Central-West Kalimantan groups diverged 194,000 years ago. We conclude that global climate change during the Pleistocene strongly influenced the genetic diversity and structure of S. macrophylla populations in Kalimantan.     

Multidrug resistant cancer cells susceptibility to cytotoxic taxane diterpenes from Taxus yunnanensis and Taxus chinensis

Twelve taxane diterpenes (1-12), which were isolated previously from the EtOH extract of the aerial parts of Taxus yunnanensis or Taxus chinensis, were evaluated for cytotoxicity against the multidrug resistant cancer cells KB-VIN and KB-7d. Compounds and showed significant cytotoxicity in these cell lines. Compounds and also demonstrated significant activity against KB-7d. The biflavonoid isolated from T. yunnanensis was only marginally cytotoxic against the A549 (lung) cell line, but a simple methoxylated analogue (14) was inactive.     

Discovery of a muscarinic M3 receptor antagonist with high selectivity for M3 over M2 receptors among 2-[(1S,3S)-3-sulfonylaminocyclopentyl]phenylacetamide derivatives

In the course of developing a metabolically stable M3 receptor antagonist from the prototype antagonist, J-104129 (1), introduction of certain substituents into the cyclopentane ring of 1 was found to be effective not only in improving metabolic stability but also in greatly enhancing the subtype selectivity. Among the cyclopentane analogues, sulfonamide derivatives (10f) and (10g) displayed 160- and 310-fold selectivity for M3 over M2 receptors, and both were significantly more selective than the prototype antagonist (120-fold). Subsequent derivatization of the sulfonamide series led to the highly selective M3 receptor antagonists (10h, 10i and 10j) with >490-fold selectivity for M3 over M2 receptors. Among them, p-nitrophenylsulfonamide (J-107320, 10h) exhibited 1100-fold selectivity for M3 receptors (Ki = 2.5 nM) over M2 receptors (Ki = 2800 nM) in the human muscarinic receptor binding assay using [3H]-NMS as a radio ligand.     

Redirection of the Reaction Specificity of a Thermophilic Acetolactate Synthase toward Acetaldehyde Formation

Acetolactate synthase and pyruvate decarboxylase are thiamine pyrophosphate-dependent enzymes that convert pyruvate into acetolactate and acetaldehyde, respectively. Although the former are encoded in the genomes of many thermophiles and hyperthermophiles, the latter has been found only in mesophilic organisms. In this study, the reaction specificity of acetolactate synthase from Thermus thermophilus was redirected to catalyze acetaldehyde formation to develop a thermophilic pyruvate decarboxylase. Error-prone PCR and mutant library screening led to the identification of a quadruple mutant with 3.1-fold higher acetaldehyde-forming activity than the wild-type. Site-directed mutagenesis experiments revealed that the increased activity of the mutant was due to H474R amino acid substitution, which likely generated two new hydrogen bonds near the thiamine pyrophosphate-binding site. These hydrogen bonds might result in the better accessibility of H⁺ to the substrate-cofactor-enzyme intermediate and a shift in the reaction specificity of the enzyme.     

Human nonsense-mediated mRNA decay factor UPF2 interacts directly with eRF3 and the SURF complex

Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that regulates gene expression and mRNA quality. A complex network of macromolecular interactions regulates NMD initiation, which is only partially understood. According to prevailing models, NMD begins by the assembly of the SURF (SMG1–UPF1–eRF1–eRF3) complex at the ribosome, followed by UPF1 activation by additional factors such as UPF2 and UPF3. Elucidating the interactions between NMD factors is essential to comprehend NMD, and here we demonstrate biochemically and structurally the interaction between human UPF2 and eukaryotic release factor 3 (eRF3). In addition, we find that UPF2 associates with SURF and ribosomes in cells, in an UPF3-independent manner. Binding assays using a collection of UPF2 truncated variants reveal that eRF3 binds to the C-terminal part of UPF2. This region of UPF2 is partially coincident with the UPF3-binding site as revealed by electron microscopy of the UPF2–eRF3 complex. Accordingly, we find that the interaction of UPF2 with UPF3b interferes with the assembly of the UPF2–eRF3 complex, and that UPF2 binds UPF3b more strongly than eRF3. Together, our results highlight the role of UPF2 as a platform for the transient interactions of several NMD factors, including several components of SURF.     

Epidemiological study on chronotype among preschool children in Japan: Prevalence,sleep–wake patterns,and associated factors

Our current 24-h society and the weekday–weekend switch of our social clocks may affect young children’s sleep and circadian rhythms. However, such evidence is scarce. We conducted a nationwide epidemiological study of sleep and health in preschool children aged 3–5 years attending kindergarten or childcare centers in Japan, using stratified one-stage cluster sampling. The target population was 2 969 627 individuals (as of 1 April 2013). The Children’s ChronoType Questionnaire was used to measure chronotypes (morning (M)-type, neither (N)-type and evening (E)-type), and weekday and weekend sleep–wake parameters. Randomly sampled population estimates were obtained via respondents with a person-level weight, which accounted for survey responses and poststratification. Standard errors and 95% confidence intervals were adjusted for the complex survey design using jackknife estimation. A linear regression model of the correlation between chronotype and sleep–wake parameters and a multivariate logistic regression model for the links between chronotype and putative associated factors were used for statistical analyses. The estimated prevalence of M-, N- and E-types were 31.6%, 55.9% and 10.0%, respectively. The corresponding numbers of children were 937 910, 1 659 574 and 296 083. The remaining 2.5% was not specified. The proportions of children who woke up by themselves during the weekdays were 55.1%, 43.0% and 1.9% for M-, N- and E-types, respectively. Overall, bedtime, sleep onset time, wake-up time and get-up time during the weekdays were 21:04, 21:26, 6:55 and 6:59, respectively. Nocturnal sleep period, time in bed (TIB) and 24 h TIB (TIB and nap) during the weekdays were 9.49, 9.93 and 10.55 h, respectively. Sleep–wake timings were significantly and linearly delayed from M-, N-, to E-types (p < 0.001). The weekday 24 h TIB (10.47–10.66 h) and weekend nocturnal sleep period (9.58–9.76 h) did not differ significantly among chronotypes. For E-types, socially advanced weekdays rising times (approximately 1 h) caused nocturnal sleep deficit (0.57 h). Children’s socially scheduled times (e.g. start and finish times, mealtimes and daytime nap) and their parents’ diurnal preferences had significant adjusted odds ratios among E-types, while the significant unadjusted odds ratios for morning sunlight and multimedia exposure disappeared. These results suggest the importance of chronobiologically planned sleep discipline at home as well as assessment of socially scheduled times in children.     

Microbial diversity and methanogenic potential in a high temperature natural gas field in Japan

Microbial diversity and methanogenic potential in formation water samples from a dissolved-in-water type gas field were investigated by using 16S rRNA gene libraries and culture-based methods. Two formation water samples (of 46 and 53°C in temperature) were obtained from a depth of 700 to 800 m. Coenzyme F₄₂₀-autofluorescence indicated that 10³–10⁴ cells per ml of active methanogens were present, accounting for at least 10% of the total cell count. The 16S rRNA gene sequence analysis indicated that the diversity of Archaea and Bacteria of the two samples was quite limited; i.e., the archaeal libraries were dominated by the sequences related to Methanobacterium formicicum and Methanothermobacter thermautotrophicus, and the bacterial libraries were dominated by the sequences related to Hydrogenophilus and Deferribacter. Of the methanogenic substrates tested using the formation water-based medium, only H₂–CO₂ gave rise to methane formation. Those dominant archaeal and bacterial genera have the potential to use hydrogen for growth at the in situ temperatures, suggesting that the formation water of the Pliocene strata in the gas field has been provided with hydrogen, probably from underneath the strata, and thus on-going active methanogenesis has been occurring to date. An erratum to this article can be found at     

Roles of a short connecting disulfide bond in the stability and function of psychrophilic Shewanella violacea cytochrome c 5*

Cys-59 and Cys-62, forming a disulfide bond in the four-residue loop of Shewanella violacea cytochrome c ₅ (SV cytc ₅), contribute to protein stability but not to redox function. These Cys residues were substituted with Ala in SV cytc ₅, and the structural and functional properties of the resulting C59A/C62A variant were determined and compared with those of the wild-type. The variant had similar features to those of the wild-type in absorption, circular dichroic, and paramagnetic ¹H NMR spectra. In addition, the redox potentials of the wild-type and variant were essentially the same, indicating that removal of the disulfide bond from SV cytc ₅ does not affect the redox function generated in the vicinity of heme. However, calorimetric analysis of the wild-type and variant showed that the mutations caused a drastic decrease in the protein stability through enthalpy, but not entropy. Four residues are encompassed by the SV cytc ₅ disulfide bond, which is the shortest one that has been proved to affect protein stability. The protein stability of SV cytc ₅ can be controlled without changing the redox function, providing a new strategy for regulating the stability and function of cytochrome c.