Molecular characterization of ENU mouse mutagenesis and archives

The large-scale mouse mutagenesis with ENU has provided forward-genetic resources for functional genomics. The frozen sperm archive of ENU-mutagenized generation-1 (G1) mice could also provide a "mutant mouse library" that allows us to conduct reverse genetics in any particular target genes. We have archived frozen sperm as well as genomic DNA from 9224 G1 mice. By genome-wide screening of 63 target loci covering a sum of 197 Mbp of the mouse genome, a total of 148 ENU-induced mutations have been directly identified. The sites of mutations were primarily identified by temperature gradient capillary electrophoresis method followed by direct sequencing. The molecular characterization revealed that all the identified mutations were point mutations and mostly independent events except a few cases of redundant mutations. The base-substitution spectra in this study were different from those of the phenotype-based mutagenesis. The ENU-based gene-driven mutagenesis in the mouse now becomes feasible and practical.     

Identification of five Orius species in Japan by multiplex polymerase chain reaction

We developed multiplex polymerase chain reaction methods to identify five Orius (Heteroptera: Anthocoridae) species that occur commonly in Japan: Orius sauteri, Orius minutus, Orius strigicollis, Orius nagaii, and Orius tantillus. The method amplified internal transcribed spacer 1 of the nuclear ribosomal DNA by using five primers simultaneously and produced species-specific banding patterns upon agarose gel electrophoresis. Reliability of the method was tested for 350 individuals of 23 strains, and consistent results were obtained. Dichotomous keys are also provided for easy and quick species identification.     

Role of internalization in the pathogenicity of Shiga toxin-producing Escherichia coli infection in a gnotobiotic murine model

We investigated the role of bacterial internalization in the killing caused by Shiga toxin-producing Escherichia coli (STEC) infection using a gnotobiotic murine model. A high number of internalized STEC was found in the colonic epithelial cells of STEC-infected mice by both an ex vivo assay and transmission electron microscopy. Most of these mice were killed within 10 days after infection. However, the implantation of lactic acid bacteria in such mice before infection markedly decreased the number of internalized STECs and also completely protected these hosts from killing by a STEC infection. The inhibition of such internalization by immunoglobulin also prevented the hosts from being killed. The Shiga toxin levels in these hosts indicated an inhibition of the penetration of Shiga toxins produced in the colon to the underlying tissue. These results suggested that the internalization plays an important role in the pathogenicity caused by STEC infection in a gnotobiotic murine model.     

Larval dispersal dampens population fluctuation and shapes the interspecific spatial distribution patterns of rocky intertidal gastropods

Many marine benthic invertebrates pass through a planktonic larval stage whereas others spend their entire lifetimes in benthic habitats. Recent studies indicate that non‐planktonic species show relatively greater fine‐scale patchiness than do planktonic species, but the underlying mechanisms remain unknown. One hypothesis for such a difference is that larval dispersal enhances the connectivity of populations and buffers population fluctuations and reduces local extinction risk, consequently increasing patch occupancy rate and decreasing spatial patchiness. If this mechanism does indeed play a significant role, then the distribution of non‐planktonic species should be more aggregated – both temporally and spatially – than the distribution of species with a planktonic larval stage. To test this prediction, we compared 1) both the spatial and the temporal abundance–occupancy relationships and 2) both the spatial and the temporal mean–variance relationships of population size across species of rocky intertidal gastropods with differing dispersive traits from the Pacific coast of Japan. We found that, compared to planktonic species, non‐planktonic species exhibited 1) a smaller occupancy rate for any given level of mean population size and 2) greater variations in population size, both spatially and temporally. This suggests that the macroecological patterns observed in this study (i.e. the abundance–occupancy relationships and mean–variance relationships of population size across species) were shaped by the effect of larval dispersal dampening population fluctuation, which works over both space and time. While it has been widely assumed that larval dispersal enhances population fluctuations, larval dispersal may in fact enhance the connectively of populations and buffer population fluctuations and reduce local extinction risks.     

Competition for Mitogens Regulates Spermatogenic Stem Cell Homeostasis in an Open Niche

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Induction of L-lysine-2-oxoglutarate reductase by glucagon and glucocorticoid in developing and adult rats: in vivo and in vitro studies

L-Lysine-2-oxoglutarate reductase (EC 1.5.1.8, NADP) in the liver of adult rats increased 4-5 times when the animals were treated with alloxan. In diabetic rats injection of insulin or adrenalectomy prevented the increase in enzyme activity. The activity of the similar enzyme in kidney was not changed by these treatments. The enzyme activity in primary cultured adult rat hepatocytes was also induced by addition of dexamethasone and glucagon together, and glucagon could be replaced by dibutyryl cyclic AMP. Insulin inhibited the induction. The hormonal induction was also inhibited by actinomycin D and by cycloheximide. During development of rats, fetal liver showed very low activity, but the activity appeared on day 1 after birth and then increased rapidly, reaching the adult level by day 5. The activity of the kidney enzyme increased more slowly and reached adult level 1 month after birth. Intra-uterine injection of glucagon caused precocious induction of the liver enzyme in fetuses. These results indicate that the activity of L-lysine-2-oxoglutarate reductase in the adult liver and in part in neonatal liver also, in controlled by both glucagon and glucocorticoid.     

Superoxide production at phagosomal cup/phagosome through beta I protein kinase C during Fc gamma R-mediated phagocytosis in microglia

Protein kinase C (PKC) plays a prominent role in immune signaling. To elucidate the signal transduction in a respiratory burst and isoform-specific function of PKC during FcgammaR-mediated phagocytosis, we used live, digital fluorescence imaging of mouse microglial cells expressing GFP-tagged molecules. betaI PKC, epsilonPKC, and diacylglycerol kinase (DGK) beta dynamically and transiently accumulated around IgG-opsonized beads (BIgG). Moreover, the accumulation of p47(phox), an essential cytosolic component of NADPH oxidase and a substrate for betaI PKC, at the phagosomal cup/phagosome was apparent during BIgG ingestion. Superoxide (O(2)(-)) production was profoundly inhibited by G?6976, a cPKC inhibitor, and dramatically increased by the DGK inhibitor, R59949. Ultrastructural analysis revealed that BIgG induced O(2)(-) production at the phagosome but not at the intracellular granules. We conclude that activation/accumulation of betaI PKC is involved in O(2)(-) production, and that O(2)(-) production is primarily initiated at the phagosomal cup/phagosome. This study also suggests that DGKbeta plays a prominent role in regulation of O(2)(-) production during FcgammaR-mediated phagocytosis.