Nucleic acids of Enterococcus faecalis strain EC-12 are potent Toll-like receptor 7 and 9 ligands inducing interleukin-12 production from murine splenocytes and murine macrophage cell line J774.1

In experiment 1 of this study, the interleukin-12 (IL-12)-inducing ability of six Enterococcus strains was evaluated in comparison with that of five Lactobacillus strains using murine splenocytes. At the same time, the involvement of Toll-like receptor (TLR) ligands in IL-12-inducing ability was assessed using splenocytes from TLR2-, TLR4- and MyD88-deficient mice. Most Enterococcus strains, especially Enterococcus faecalis strain EC-12, exerted higher IL-12-inducing ability compared with the Lactobacillus strains evaluated. Almost the same amount of IL-12 protein was produced by all lactic acid bacteria strains in splenocytes from TLR2- and TLR4-deficient mice, whereas splenocytes from MyD88-deficient mice showed no IL-12 production against all bacteria evaluated. In experiment 2, the role of TLR7, 8 and 9 ligands of E. faecalis strain EC-12 in the induction of IL-12 production was evaluated using murine macrophage cell line J774.1. A drastic decrease in IL-12-inducing ability was observed when heat-killed E. faecalis strain EC-12 was treated with nuclease, particularly RNase. In addition, less than one-tenth of IL-12 was produced by heat-killed E. faecalis strain EC-12 when both TLR7 and 9 were antagonized. These facts indicate that the nucleic acids of E. faecalis strain EC-12, particularly its RNA, are the potent TLR7 and 9 ligands that induce IL-12 production from antigen-presenting cells.     

QTL-based analysis of leaf senescence in an indica/japonica hybrid in rice (Oryza sativa L.)

In order to identify quantitative trait loci (QTLs) for leaf senescence and related traits in rice (Oryza sativa L.), we developed two backcross populations, indica/japonica// japonica and indica/japonica//indica, using IR36 as the indica parent and Nekken-2 as the japonica parent. The QTLs were mapped using a set of simple sequence-repeat markers (SSRs) in the BC₁F₁ population. Senescence was characterized in these plants by measuring the leaf chlorophyll content 25 days after flowering (DAF), the reduction in chlorophyll content (the difference between the chlorophyll content at flowering and at 25 DAF), and the number of late-discoloring leaves per panicle at 25 DAF in five plants from each BC₁F₂ line. These plants were moved into a temperature-controlled growth cabinet at the time of flowering and allowed to mature under identical conditions. Eleven QTLs were detected in the two populations. The major of QTLs for senescence were found on the short arm of chromosome 6 and on the long arm of chromosome 9. Of these, one QTL on chromosome 6 and two on chromosome 9 were verified by confirming the effects of the genotypes on the phenotypes of the BC₁F₃ lines. The japonica parent was found to contribute to late senescence at all but one QTL. Based on a comparison of the effects of heterozygotes and homozygotes on the phenotypic values of each QTL genotype, we concluded that the differential senescence observed in the indica-japonica hybrid was not due to over-dominance; rather, it was the result of partial-dominance genes that were donated from either of the parents.     

A database of recombinant viruses and recombinant viral vectors available from the RIKEN DNA bank

BACKGROUND: Viral vectors are required as gene-delivery systems for gene therapy and basic research. Recombinant adenoviruses (rAds) expressing genes of interest are being developed as research tools and many studies in vitro and in vivo have already been performed with such rAds. METHODS: Shuttle vectors for rAds were constructed with full-length cDNAs and rAds were generated in HEK293 cells by the COS-TPC method. The rAds and shuttle vectors were developed by the Japanese research community and deposited in the RIKEN DNA Bank (RDB; http://www.brc.riken.jp/lab/dna/en/) for distribution to the scientific community. The Recombinant Virus Database (RVD; http://www.brc.riken.jp/lab/dna/rvd/) was established at the RIKEN BioResource Center (BRC) in Japan as the source of information about and distribution of the various resources. RESULTS: The RIKEN BRC is releasing more than 300 recombinant viruses (RVs) and 500 shuttle vectors, as well as all related information, which is included in a newly established database, the RVD. The RVD consists of (i) information about the RVs, the inserted cDNAs and the shuttle vectors; (ii) data about sequence-tagged sites (STSs) that are markers of viral DNAs; and (iii) experimental protocols for the use of RVs. CONCLUSIONS: The new database and available resources should be very useful to scientists who are studying human gene therapy and performing related basic research. It is a web-interfaced flat-file database that can be accessed through the internet. Moreover, all of the resources deposited in the RDB, which is a public facility in Japan, are available to researchers around the world.     

Techniques of SNP genotyping and remote sensing applied for elucidation of the contribution of polygene and environmental factors to inter-individual variation in skin pigmentation phenotype

We present a conceptual framework for applying techniques of SNP genotyping as a molecular biological approach and remote sensing as an ecological approach to elucidation of the contribution of polygene and environmental factors to inter-individual variation in skin pigmentation phenotype. Additionally, we discuss the obstacles that frustrate our efforts to identify how the human genome encodes the complex phenotype and suggest the use of computational methods designed for knowledge discovery within hereditary database.     

Aglycon specificity profiling of alpha-glucosidases using synthetic probes

We designed and synthesized hydrogen bond based probes 1-8 with the exception of known glycosidase inhibition mechanisms, and aglycon specificity of 11 different sources of alpha-glucosidases were investigated using their probes. Probe 4 (2,6-anhydro-1-deoxy-1-[(1-oxopentyl-5-hydroxy)amino]-D-glycero-D-ido-heptitol) showed a potent inhibition of S. cerevisiae alpha-glucosidase among all alpha-glucosidases. Probe 4 was found to be a competitive inhibitor for S. cerevisiae alpha-glucosidase with Ki 0.13 mM.     

Nanogel-quantum dot hybrid nanoparticles for live cell imaging

We report here a novel carrier of quantum dots (QDs) for intracellular labeling. Monodisperse hybrid nanoparticles (38 nm in diameter) of QDs were prepared by simple mixing with nanogels of cholesterol-bearing pullulan (CHP) modified with amino groups (CHPNH2). The CHPNH2-QD nanoparticles were effectively internalized into the various human cells examined. The efficiency of cellular uptake was much higher than that of a conventional carrier, cationic liposome. These hybrid nanoparticles could be a promising fluorescent probe for bioimaging.     

Genome-wide application of RNAi to the discovery of potential drug targets

Progress is being made in the development of RNA interference-based (RNAi-based) strategies for the control of gene expression. It has been demonstrated that small interfering RNAs (siRNAs) can silence the expression of target genes in a sequence-specific manner in mammalian cells. Various groups, including our own, have developed systems for vector-mediated specific RNAi. Vector-based siRNA- (or shRNA) expression libraries directed against the entire human genome and siRNA libraries based on chemically synthesized oligonucleotides now allow the rapid identification of functional genes and potential drug targets. Use of such libraries will enhance our understanding of numerous biological phenomena and contribute to the rational design of drugs against heritable, infectious and malignant diseases.