Genetic and geographical differentiation of <Emphasis Type="Italic">Pandaka</Emphasis> gobies in Japan

The mitochondrial DNA (mtDNA) phylogeny of Japanese Pandaka species (Perciformes: Gobiidae) was inferred from partial nucleotide sequences of the mitochondrial 12S and 16S rRNA genes (1083bp). The resultant mtDNA tree showed two major clades (clade I and clade II), which were inconsistent with the present taxonomic classification. One of the major clades was further divided into two geographical groups, distributed on the Japanese Major Islands (clade I-A) and from Amami-oshima Island to Iriomote Island (clade I-B). The mtDNA haplotypes in clade II were found only on Iriomote Island. The mtDNA divergences in clade I indicated that the Japanese Major Island (clade I-A) and Ryukyu (clade I-B) groups have been geographically isolated from each other for millions of years, based on the putative molecular divergence rate. The geographical distributions of mtDNA haplotypes in clade I-A and clade I-B also suggested that Pandaka gobies had not dispersed to distant offshore islands, indicating that their geographical differentiation may be closely associated with the geological history of the Japanese and Ryukyu Archipelagos.This revised version was published online in January 2005 with corrections to the repetition of the 1st authors name.     

The influence of floral symmetry and pollination systems on flower size variation

We compared the amount of variation in flower size between autogamous and insect-pollinated species to examine the hypothesis that pollinator-mediated selection stabilizes flower size in plant populations. One would expect the flower size variation to be larger in selfing species that are less affected by pollinator-mediated stabilizing selection than in insect-pollinated species. The results of phylogenetic comparisons between autogamous and insect-pollinated flowers supported the pollinator-mediated stabilizing selection hypothesis, although the non-phylogenetic comparison did not. According to our results, we discuss the factors influencing the flower size variation.     

Imaging gene expression in regional brain ischemia in vivo with a targeted [111in]-antisense radiopharmaceutical

The gene encoding glial fibrillary acidic protein (GFAP) is downregulated 24 hr after reversible brain ischemia, such as with a middle cerebral artery occlusion (MCAO). The in vivo imaging of decreased GFAP gene expression in cerebral ischemia was examined in the present studies using a targeted peptide nucleic acid (PNA), which was labeled with (111)In, and which hybridized to nucleotides 20-37 of the rat GFAP mRNA. The PNA was monobiotinylated, and was attached to a monoclonal antibody (MAb) to the transferrin receptor (TfR) via a biotin-streptavidin linkage. The TfR MAb enables trans-membrane transport of the PNA antisense radiopharmaceutical from blood to the cytosol of brain cells. The decreased GFAP gene expression at 24 hr after a 1-hr reversible MCAO was confirmed by immunocytochemistry. The [(111)In]-labeled PNA - MAb conjugate was administered intravenously to anesthetized rats at 24 hr after the 1-hr reversible MCAO, and the brain uptake of the targeted antisense imaging agent was decreased relative to brain regions outside of the infarct zone. These studies provide evidence that decreased expression of a target gene in brain can be imaged in vivo with a sequence-specific PNA, provided the antisense radiopharmaceutical is delivered across cell membranes with a receptor-specific targeting agent.     

NTH201, a novel class II KNOTTED1-like protein, facilitates the cell-to-cell movement of Tobacco mosaic virus in tobacco

NTH201, a novel class II KNOTTED1-like protein gene, was cloned from tobacco (Nicotiana tabacum cv. Xanthi) and its role in Tobacco mosaic virus (TMV) infection was analyzed. Virus-induced gene silencing of NTH201 caused a delay in viral RNA accumulation as well as virus spread in infected tobacco plants. Overexpression of the gene in a transgenic tobacco plant (N. tabacum cv. Xanthi nc) infected by TMV showed larger local lesions than those of the nontransgenic plant. NTH201 exhibited no intercellular trafficking ability but did exhibit colocalization with movement protein (MP) at the plasmodesmata. When NTH201-overexpressing tobacco BY-2 cultured cells were infected with TMV, the accumulation of MP but not of viral genomic and subgenomic RNA clearly was accelerated compared with those in nontransgenic cells at an early infection period. The formation of virus replication complexes (VRC) also was accelerated in these transgenic cells. Conversely, NTH201-silenced cells showed less MP accumulations and fewer VRC formations than did nontransgenic cells. These results suggested that NTH201 might indirectly facilitate MP accumulation and VRC formation in TMV-infected cells, leading to rapid viral cell-to-cell movement in plants at an early infection stage.     

Successions of bacterial community in composting cow dung wastes with or without hyperthermophilic pre-treatment

Comparative analyses of bacterial community successions in the composting materials were done for a conventional windrow post-treatment (WPOT) process with the hyperthermophilic pre-treatment (HTPRT) and simple windrow composting (SWC; without the HTPRT). Multidimensional scaling profiles based on data of terminal restriction fragment length polymorphisms of the bacterial population in the samples of every 7 days composting material and analyses of the 16S rRNA gene-based clone library of the 7 and 21 days composting materials suggested that bacterial communities of the composting materials differed much between these two processes until the 35 days of composting, whereas that they were closely related to each other at the final composting stage (42 days of composting). Detailed phylogenetic analysis clarified that all WPOT clone libraries contained many clones of the lineages of aerobic bacteria (for example, bacilli). However, the most abundant clones retrieved from all SWC materials were affiliated with a clone cluster closely related to identified and classified members of the phylum Firmicutes that have strictly anaerobic metabolism pathways. From these results, we conclude that the HTPRT process contributed to easily establish an aerobic ecosystem from the early stage to the final stage of WPOT composting with plowing the materials only once a week.     

Thermodynamic characterization of specific interactions between the human Lon protease and G-quartet DNA

Lon is an ATP-powered protease that binds DNA. However, the function of DNA binding by Lon remains elusive. Studies suggest that human Lon (hLon) binds preferentially to a G-rich single-stranded DNA (ssDNA) sequence overlapping the light strand promoter of mitochondrial DNA. This sequence is contained within a 24-base oligonucleotide referred to as LSPas. Here, we use biochemical and biophysical approaches to elucidate the structural properties of ssDNAs bound by hLon, as well as the thermodynamics of DNA binding by hLon. Electrophoretic mobility shift assay and circular dichroism show that ssDNAs with a propensity for forming parallel G-quartets are specifically bound by hLon. Isothermal titration calorimetry demonstrates that hLon binding to LSPas is primarily driven by enthalpy change associated with a significant reduction in heat capacity. Differential scanning calorimetry pinpoints an excess heat capacity upon hLon binding to LSPas. By contrast, hLon binding to an 8-base G-rich core sequence is entropically driven with a relatively negligible change in heat capacity. A considerable enhancement of thermal stability accompanies hLon binding to LSPas as compared to the G-rich core. Taken together, these data support the notion that hLon binds G-quartets through rigid-body binding and that binding to LSPas is coupled with structural adaptation.     

Risk of accidental invasion and expansion of allochthonous mice in Tokyo metropolitan coastal areas in Japan

House mouse (Mus musculus) is one of the perilous animal vectors for imported zoonosis such as a lymphocytic choriomeningitis (LCMV) infectious disease, and probably unknown emerging and/or re-emerging infectious diseases as well. It is necessary to prevent such diseases by regular surveys for behavioral trends of these allochthonous mice. However, such a trial has never been attempted in Japan. From 1998 to 2002, we analyzed partial sequences of the D-loop region in mtDNA, which provides powerful diagnostic SNPs for subspecies identification in the Mus musculus species, from 301 individuals of mice collected in 23 international bays or airports in Japan. We found that invasion of many allochthonous mice, which were identified as European subspecies, Mus musculus domesticus, occurred in Tokyo metropolitan coastal area. Based on the evidence, we warn that extensive invasion of allochthonous mice has occurred recently and, therefore, the risk of emerging and/or re-emerging infectious diseases invasion might be high in Tokyo metropolitan area.     

Rat organic solute carrier protein 1 (rOscp1) mediated the transport of organic solutes in Xenopus laevis oocytes: isolation and pharmacological characterization of rOscp1

Rat organic solute carrier protein 1 (rOscp1) was isolated from a rat testis cDNA library. Isolated rOscp1 cDNA consisted of 1089 base pairs that encoded a 363-amino acid protein, and the amino acid sequence was 88% and 93% identical to that of human OSCP1 (hOSCP1) and mouse Oscp1 (mOscp1), respectively. The message for rOscp1 is highly detected in rat testis. When expressed in X. oocytes, rOscp1 mediated the high affinity transport of p-aminohippurate (PAH) with a Km value of 15.7+/-1.9 microM, and rOscp1-mediated organic solutes were exhibited in time- and Na+-independent manners. rOscp1 also transported various structurally heterogenous compounds such as testosterone, dehydroepiandrosterone sulfate (DHEA-S), and taurocholate with some differences in substrate specificity compared with hOSCP1. Immunohistochemical analysis revealed that the rOscp1 protein is localized in the basal membrane side of Sertoli cells as observed in mouse testis [Kobayashi et al., 2007; Kobayashi, Y., Tsuchiya, A., Hayashi, T., Kohyama, N., Ohbayashi, M., Yamamoto, T., 2007. Isolation and characterization of polyspecific mouse organic solute carrier protein 1 (mOscp1). Drug Metabolism and Disposition 35 (7), 1239-1245]. Thus, the present results indicate that a newly isolated cDNA clone, rOscp1, is a polyspecific organic solute carrier protein with some differences in substrate specificity compared with human and mouse OSCP1.     

Design and synthesis of 6-fluoro-2-naphthyl derivatives as novel CCR3 antagonists with reduced CYP2D6 inhibition

In our previous study on discovering novel types of CCR3 antagonists, we found a fluoronaphthalene derivative (1) that exhibited potent CCR3 inhibitory activity with an IC(50) value of 20 nM. However, compound 1 also inhibited human cytochrome P450 2D6 (CYP2D6) with an IC(50) value of 400 nM. In order to reduce its CYP2D6 inhibitory activity, we performed further systematic structural modifications on 1. In particular, we focused on reducing the number of lipophilic moieties in the biphenyl part of 1, using ClogD(7.4) values as the reference index of lipophilicity. This research led to the identification of N-{(3-exo)-8-[(6-fluoro-2-naphthyl)methyl]-8-azabicyclo[3.2.1]oct-3-yl}-3-(piperidin-1-ylcarbonyl)isonicotinamide 1-oxide (30) which showed comparable CCR3 inhibitory activity (IC(50)=23 nM) with much reduced CYP2D6 inhibitory activity (IC(50)=29,000 nM) compared with 1.