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The distribution of Vibrio species in samples of surface water, bottom water (water 2 m above the sediment), and sediment from the Seto Inland Sea was studied. A simple technique using a membrane filter and short preenrichment in alkaline peptone water was developed to resuscitate the injured cells, followed by plating them onto TCBS agar. In addition, a survey was conducted to determine the incidence of Clostridium botulinum in sediment samples. Large populations of heterotrophs were found in surface water, whereas large numbers of total vibrios were found in bottom water. In samples from various water sampling regions, high counts of all bacterial populations were found in the inner regions having little exchange of seawater when compared with those of the open region of the inland sea. In the identification of 463 isolates, 23 Vibrio spp. and 2 Listonella spp. were observed. V. harveyi was prevalent among the members of the Vibrio genus. Vibrio species were categorized into six groups; an estimated 20% of these species were in the so-called "pathogenic to humans" group. In addition, a significant proportion of this group was hemolytic and found in the Bisan Seto region. V. vulnificus, V. fluvialis, and V. cholerae non-O1 predominated in the constricted area of the inland sea, which is eutrophic as a result of riverine influence. It was concluded that salinity indirectly governs the distribution of total vibrios and analysis of variance revealed that all bacterial populations were distributed homogeneously and the variance values were found to be significant in some water sampling regions.(ABSTRACT TRUNCATED AT 250 WORDS)

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In animal regeneration, control of position-dependent cell proliferation is crucial for the complete restoration of patterned appendages in terms of both, shape and size. However, detailed mechanisms of this process are largely unknown. In this study, we identified leucine/glutamine and v-ATPase/lysosomal acidification, via mechanistic target of rapamycin complex 1 (mTORC1) activation, as effectors of amputation plane-dependent zebrafish caudal fin regeneration. mTORC1 activation, which functions in cell proliferation, was regulated by lysosomal acidification possibly via v-ATPase activity at 3 h post amputation (hpa). Inhibition of lysosomal acidification resulted in reduced growth factor-related gene expression and suppression of blastema formation at 24 and 48 hpa, respectively. Along the proximal-distal axis, position-dependent lysosomal acidification and mTORC1 activation were observed from 3 hpa. We also report that Slc7a5 (L-type amino acid transporter), whose gene expression is position-dependent, is necessary for mTORC1 activation upstream of lysosomal acidification during fin regeneration. Furthermore, treatment with leucine and glutamine, for both proximal and distal fin stumps, led to an up-regulation in cell proliferation via mTORC1 activation, indicating that leucine/glutamine signaling possesses the ability to change the position-dependent regeneration. Our findings reveal that leucine/glutamine and v-ATPase/lysosomal acidification via mTORC1 activation are required for position-dependent zebrafish fin regeneration.

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CpG methylation in genomic DNA is well known as a repressive epigenetic marker in eukaryotic transcription, and DNA methylation of the promoter regions is correlated with silencing of gene expression. In contrast to the promoter regions, the function of DNA methylation during transcription termination remains to be elucidated. A recent study has revealed that mouse DNA methyltransferase 3a (Dnmt3a) mainly functions in de novo methylation in the promoter and gene body regions (including transcription termination sites (TTSs)) during development. To investigate the relationship between DNA methylation overlapping the TTSs and transcription termination, we employed two strategies: informatic analysis using already deposited datasets of Dnmt3a-/- mouse cells and the zebrafish model system. Bioinformatic analysis using methylome and transcriptome data showed that hypomethylated differentially methylated regions overlapping the TTSs were associated with increased read counts and chimeric transcripts downstream of TTSs in Dnmt3a-/- Agouti-related protein neurons, but not in Dnmt3a-/- ES cells and MEFs. We experimentally detected increased read-through and chimeric transcripts downstream of hypomethylated TTSs in zebrafish maternal-zygotic dnmt3aa-/- mutants. This study is the first to identify transcription termination defects in DNA hypomethylated TTSs in Dnmt3a-/- vertebrates.

CpG methylation of genomic DNA is a well-known repressive epigenetic marker in eukaryotic transcription, and DNA methylation of promoter regions is correlated with gene silencing. In contrast to the promoter regions, the function of DNA methylation during transcription termination remains to be elucidated. A recent study revealed that mouse DNA methyltransferase 3a (Dnmt3a) mainly functions in de novo methylation in the promoter and gene body regions, including transcription termination sites (TTSs), during development. To investigate the relationship between DNA methylation overlapping the TTSs and transcription termination, we performed bioinformatics analysis using six pre-existing Dnmt -/mouse cell datasets: four types of neurons (three Dnmt3a -/and one Dnmt1 -/mutants) and two types of embryonic fibroblasts (MEFs) (Dnmt3a -/and Dnmt3b -/mutants). Combined analyses using methylome and transcriptome data revealed that read counts downstream of hypomethylated TTSs were increased in three types of neurons (two Dnmt3a -/and one Dnmt1 -/mutants). Among these, an increase in chimeric transcripts downstream of the TTSs was observed in Dnmt3a -/mature olfactory sensory neurons and Dnmt3a -/agoutirelated peptide (protein)-producing neurons, thereby indicating that read-through occurs in hypomethylated TTSs at specific gene loci in these two mutants. Conversely, in Dnmt3a -/-MEFs, we detected reductions in read counts downstream of hypomethylated TTSs. These results indicate that the hypomethylation of TTSs can both positively and negatively regulate transcription termination, dependent on Dnmt and cell types. This study is the first to identify the aberrant termination of transcription at specific gene loci with DNA hypomethylated TTSs attributable to Dnmt deficiency.