Linkage disequilibrium analysis of allelic heterogeneity in DNA methylation

Heterogeneity of DNA methylation status among alleles is observed in various cell types and is involved in epigenetic gene regulation and cancer biology. However, the individual methylation profile within each allele has not yet been examined at the whole-genome level. In the present study, we applied linkage disequilibrium analysis to the DNA methylation data obtained from whole-genome bisulfite sequencing studies in mouse germline and other types of cells. We found that the methylation status of 2 consecutive CpG sites showed deviation from equilibrium frequency toward concordant linkage (both methylated or both unmethylated) in germline cells. In the imprinting loci where methylation of constituent alleles is known, our analysis detected the deviation toward the concordant linkage as expected. In addition, we applied this analysis to the transitional zone between methylated and unmethylated regions and to the cells undergoing epigenetic reprogramming. In both cases, deviation to the concordant-linked alleles was conspicuous, indicating that the methylation pattern is not random but rather concordant within each allele. These results will provide the key to understanding the mechanism underlying allelic heterogeneity.     

Reactive oxygen species production and activation mechanism of the rice NADPH oxidase OsRbohB

Reactive oxygen species (ROS) produced by plant NADPH oxidases (NOXes) are important in plant innate immunity. The Oryza sativa respiratory burst oxidase homologue B (OsRbohB) gene encodes a NOX the regulatory mechanisms of which are largely unknown. Here, we used a heterologous expression system to demonstrate that OsRbohB shows ROS-producing activity. Treatment with ionomycin, a Ca(2+) ionophore, and calyculin A, a protein phosphatase inhibitor, activated ROS-producing activity; it was thus OsRbohB activated by both Ca(2+) and protein phosphorylation. Mutation analyses revealed that not only the first EF-hand motif but also the upstream amino-terminal region were necessary for Ca(2+)-dependent activation, while these regions are not required for phosphorylation-induced ROS production.     

Design and synthesis of novel p38α MAP kinase inhibitors: discovery of pyrazole-benzyl ureas bearing 2-molpholinopyrimidine moiety

The discovery that pyrazole-benzyl urea derivatives bearing a 2-molpholinopyrimidine moiety are novel p38α inhibitors is described. A comparative view of the binding modes of SB-203580 and BIRB-796 by structural alignment of two X-ray co-crystal structures was utilized to identify this novel series. Modification of the benzyl group led to compound 2b, a highly potent p38α inhibitor. In in vivo studies, 2b inhibited the production of tumor necrosis factor-alpha in lipopolysaccharide-treated mouse in a dose-dependent manner. Furthermore, the results of a 5-day repeated oral dose toxicity study suggest that 2b has low hepatotoxicity.     

Discovery of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221) as a functional antagonist of the apelin (APJ) receptor

The recently discovered apelin/APJ system has emerged as a critical mediator of cardiovascular homeostasis and is associated with the pathogenesis of cardiovascular disease. A role for apelin/APJ in energy metabolism and gastrointestinal function has also recently emerged. We disclose the discovery and characterization of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221), a potent APJ functional antagonist in cell-based assays that is >37-fold selective over the closely related angiotensin II type 1 (AT1) receptor. ML221 was derived from an HTS of the ～330,600 compound MLSMR collection. This antagonist showed no significant binding activity against 29 other GPCRs, except to the κ-opioid and benzodiazepinone receptors (<50/<70%I at 10 μM). The synthetic methodology, development of structure–activity relationship (SAR), and initial in vitro pharmacologic characterization are also presented.     

Improvements in ethanol production from xylose by mating recombinant xylose-fermenting Saccharomyces cerevisiae strains

To improve the ability of recombinant Saccharomyces cerevisiae strains to utilize the hemicellulose components of lignocellulosic feedstocks, the efficiency of xylose conversion to ethanol needs to be increased. In the present study, xylose-fermenting, haploid, yeast cells of the opposite mating type were hybridized to produce a diploid strain harboring two sets of xylose-assimilating genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase. The hybrid strain MN8140XX showed a 1.3- and 1.9-fold improvement in ethanol production compared to its parent strains MT8-1X405 and NBRC1440X, respectively. The rate of xylose consumption and ethanol production was also improved by the hybridization. This study revealed that the resulting improvements in fermentation ability arose due to chromosome doubling as well as the increase in the copy number of xylose assimilation genes. Moreover, compared to the parent strain, the MN8140XX strain exhibited higher ethanol production under elevated temperatures (38 °C) and acidic conditions (pH 3.8). Thus, the simple hybridization technique facilitated an increase in the xylose fermentation activity.     

Whole-brain mapping of direct inputs to midbrain dopamine neurons

Recent studies indicate that dopamine neurons in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) convey distinct signals. To explore this difference, we comprehensively identified each area's monosynaptic inputs using the rabies virus. We show that dopamine neurons in both areas integrate inputs from a more diverse collection of areas than previously thought, including autonomic, motor, and somatosensory areas. SNc and VTA dopamine neurons receive contrasting excitatory inputs: the former from the somatosensory/motor cortex and subthalamic nucleus, which may explain their short-latency responses to salient events; and the latter from the lateral hypothalamus, which may explain their involvement in value coding. We demonstrate that neurons in the striatum that project directly to dopamine neurons form patches in both the dorsal and ventral striatum, whereas those projecting to GABAergic neurons are distributed in the matrix compartment. Neuron-type-specific connectivity lays a foundation for studying how dopamine neurons compute outputs.     

Production of quorum-sensing-related signal molecules by epiphytic bacteria inhabiting wheat heads

The production of quorum-sensing-related signal molecules (QSRMs) among culturable bacteria comprising the community on wheat heads was investigated. The taxonomic position of 186 bacterial isolates obtained from ten heads was inferred based on 16S rRNA gene sequences, and their QSRM production was determined using two bioreporter strains of N-acylhomoserine lactones. Approximately 33% of isolates produced QSRMs, though the proportion of QSRM-producing isolates on a wheat head was significantly negatively correlated with population size. Most of the producing isolates were Pantoea species, most commonly Pantoea ananatis. Furthermore, the proportion of Pantoea ananatis that produced QSRMs was significantly negatively correlated with the number of bacterial genera (community richness) on each head. Finally, community richness was positively correlated with population size. Qualitative analysis using thin-layer-chromatography revealed that the QSRMs of Pantoea isolates were composed of at least two compounds. This is the first report indicating that Pantoea ananatis isolates inhabiting wheat heads are capable of producing QSRMs. QSRM production by Pantoea spp. may contribute to the predominance of this genus on wheat heads, particularly at relatively low population densities and community diversity.