Putative virulence-related genes in Vibrio anguillarum identified by random genome sequencing

The genome of Vibrio anguillarum strain H775-3 was partially determined by a random sequencing procedure. A total of 2,300 clones, 2,100 from a plasmid library and 200 from a cosmid library, were sequenced and subjected to homology search by the BLAST algorithm. The total length of the sequenced clones is 1.5 Mbp. The nucleotide sequences were classified into 17 broad functional categories. Forty putative virulence-related genes were identified, 36 of which are novel in V. anguillarum, including a repeat in toxin gene cluster, haemolysin genes, enterobactin gene, protease genes, lipopolysaccharide biosynthesis genes, capsule biosynthesis gene, flagellar genes and pilus genes.     

Inhibition of hirame rhabdovirus growth by RNA aptamers

RNA aptamers are artificial nucleic acids that specifically bind to a wide variety of targets. They are an effective tool for pharmaceutical research and development of antiviral agents. Here, we describe four Hirame rhabdovirus (HIRRV)‐RNA aptamers (H1, H2, H3 and H4) that we obtained from an in vitro process called the systematic evolution of ligands by exponential enrichment (SELEX). The HIRRV‐RNA aptamers specifically bind to HIRRV. Hirame natural embryo (HINAE) cells treated with virus and the RNA aptamer showed a decrease in appearance of cytopathic effect when compared with control (treated only with virus). Rhodovulum sulfidophilum was transformed with genes for the RNA aptamers, and the aptamers were detected in the culture medium, indicating that they were secreted from the cells. Thus, the recombinant R. sulfidophilum might be a powerful tool for the prevention of HIRRV in aquaculture.     

Seed germination and seedling establishment of the monocarpic dwarf bamboo Sasa veitchii var. hirsuta

In order to determine the influences of environmental factors on seed germination and to identify the effects of germination characteristics on seedling establishment for Sasa veitchii var. hirsuta, germination experiments and a survey on current seedling dynamics were conducted in a natural habitat after mass flowering. The results of the germination experiments revealed that the seeds require a low-temperature environment for germination; those exposed to low-temperature conditions for a time similar to the length of winter (4 months of low-temperature conditions) germinated gradually, while those exposed for longer (>4 months of low-temperature conditions) germinated rapidly. These results were compatible with field observations indicating that seedlings emerged from June to October in the year after mass flowering, and they suggested this germination characteristic (i.e., variation of germination timing due to seed dormancy) may play an ecological function in spreading risk (although it may also increase the potential for seed predation). The results also revealed that stronger light and alternating temperature conditions had no effect on germination, whereas high temperatures and drying had negative effects. The outcomes of the survey on current seedling dynamics revealed that the seedling establishment ratio was high in a forest understory compared to that seen at a site where solar radiation was strong and soil water content was low. This implies that germination characteristics may promote seedling establishment in forest understory.     

Detection of quinolone-resistance genes in Photobacterium damselae subsp. piscicida strains by targeting-induced local lesions in genomes

Quinolone-resistant strains of the fish-pathogenic bacterium, Photobacterium damselae subsp. piscicida are distributed widely in cultured yellowtail, Seriola quinqueradiata (Temminck & Schlegel), in Japan. The quinolone resistance-determining region (QRDR) was amplified with degenerate primers, followed by cassette ligation-mediated PCR. Open reading frames encoding proteins of 875 and 755 amino acid residues were detected in the gyrA and parC genes, respectively. Resistant strains of P. damselae subsp. piscicida carried a point mutation only in the gyrA QRDR leading to a Ser-to-Ile substitution at residue position 83. No amino acid alterations were discovered in the ParC sequence. A mutation in the gyrA gene was also detected in nalidixic acid-resistant mutants of strain SP96002 obtained from agar medium containing increased levels of quinolone. These results suggest that GyrA, as in other Gram-negative bacteria, is a target of quinolone in P. damselae subsp. piscicida. Furthermore, we attempted to detect a point mutation using targeting-induced local lesions in genomes (TILLING), which is a general strategy used for the detection of a variety of induced point mutations and naturally occurring polymorphisms. We developed a new detection method for the rapid and large-scale identification of quinolone-resistant strains of P. damselae subsp. piscicida using TILLING.     

Regulation of mammalian tooth cusp patterning by ectodin

Mammalian tooth crowns have precise functional requirements but cannot be substantially remodeled after eruption. In developing teeth, epithelial signaling centers, the enamel knots, form at future cusp positions and are the first signs of cusp patterns that distinguish species. We report that ectodin, a secreted bone morphogenetic protein (BMP) inhibitor, is expressed as a "negative" image of mouse enamel knots. Furthermore, we show that ectodin-deficient mice have enlarged enamel knots, highly altered cusp patterns, and extra teeth. Unlike in normal teeth, excess BMP accelerates patterning in ectodin-deficient teeth. We propose that ectodin is critical for robust spatial delineation of enamel knots and cusps.     

Expressed sequence tag analysis of phyllosomas and hemocytes of Japanese spiny lobster Panulirus japonicus

Two complementary DNA (cDNA) libraries were constructed from phyllosomas and hemocytes of adult Japanese spiny lobster Panulirus japonicus and a total of 2,673 expressed sequence tags (ESTs) were obtained. After assembly and clustering, 450 and 458 unique sequences were found from the phyllosoma and hemocyte cDNA libraries, respectively. Of these, 114 and 220 ESTs showed significant homologies with known genes in the National Centre for Biotechnology Information (NCBI) database. The remaining sequences were of unknown function. Immune-related genes found in this study include lectin, proteinase inhibitor, prophenoloxidase, heat-shock protein, antimicrobial peptide, and a few putative defense-related proteins.     

Gene transfer for Japanese flounder fertilized eggs by particle gun bombardment

ABSTRACT:   Fish transgenesis has progressed considerably. However, the technique of gene transfer for most marine aquaculture species has not been established. A method to introduce foreign genes into fertilized eggs of Japanese flounder Paralichthys olivaceus by particle gun bombardment was developed by the authors. A recombinant plasmid which contains the Japanese flounder keratin gene promoter linked to the green fluorescence protein (GFP) gene was introduced into fertilized eggs by particle gun bombardments twice at 250 psi. In each experimental group, 25 000–35 000 eggs were treated. However, the survival rate was 12.8% which is lower than that of the control groups (56.8%). Of 2606–5205 the embryos survived for 24 h, 43–61 GFP positive embryos were obtained in one experiment, giving a final gene transfer efficiency of 1.4%. All GFP-positive embryos developed and hatched normally. GFP expression was observed in epithelial tissue throughout the developmental stages. At 3 months after gene transfer, foreign DNA was detected by genome polymerase chain reaction analysis in 37 of 69 fry (53.6%). These results suggest that the particle gun method is an effective method to use with fertilized Japanese flounder eggs.     

Histopathological changes in the pancreas due to decreased pancreatic blood flow in a canine tachycardia-induced cardiomyopathy model

In dogs, pancreatic acinar cell injury is thought to be caused by decreased pancreatic blood flow due to heart failure. In previous our report, it demonstrated that decreased heart function causes a significant decrease in pancreatic blood flow in heart failure dog model caused by rapid ventricular pacing (RVP). However, the types of histopathological changes remain unclear. We aimed to verify the types of histopathological changes occurring in the pancreatic tissue due to decreased heart function. After RVP for 4 weeks, atrophy of pancreatic acinar cells, characterized by a decrease in zymogen granules, was observed in all areas of the pancreas. In conclusion, the result of this study suggests that attention should be paid to ischemia/hypoperfusion injury in the pancreas.