Japanese black cattle with ateliosis showed lower insulin responses during glucose tolerance test

In order to determine insulin secretability and glucose utilization, a glucose tolerance test was performed in ateliotic cattle of 2 paternal strains; MHO and HSK cattle. MHO and HSK cattle showed different endocrine patterns in our previous study. Area under the insulin concentration curves (insulin-AUC) in the ateliotic cattle were significantly lower (122.3 +/- 59.4 ng.min/ml and 99.2 +/- 24.8 ng.min/ml for MHO and HSK cattle, respectively) than the control cattle (420.2 +/- 175.2 ng.min/ml). These low insulin responses to GTT may have an influence on growth retardation in MHO and HSK cattle.     

Chromosome constitution of hybrid strains constructed by protoplast fusion between the tomato and strawberry pathotypes of <Emphasis Type="Italic">Alternaria alternata</Emphasis>

To analyze the genetics of host-specific toxin production and its relation to the specific pathogenicity of a mitosporic fungus Alternaria alternata, we developed a protoplast fusion system. Protoplasts of drug-resistant transformants of the A. alternata tomato pathotype (AAL-toxin producer) and A. alternata strawberry pathotype (AF-toxin producer) were fused by electrofusion. Of five fusion strains examined, two strains were pathogenic on both tomato and strawberry host plants, whereas the rest of the fusion strains were pathogenic only on tomato. Pulsed-field gel electrophoresis analysis demonstrated that the hybrid strains pathogenic on both tomato and strawberry carry 1.0- and 1.05-Mb conditionally dispensable (CD) chromosomes derived, respectively, from the parental strains of the tomato and strawberry pathotypes. On the other hand, the fusion strains appeared to maintain only a single homologous chromosome derived from one of the parental strain in the case of essential chromosomes (A chromosomes). The results suggest that fusion strains between two different pathotypes of A. alternata might be haploid resulting from the deletion of extra sets of essential chromosomes in the fused nuclei, whereas the CD chromosomes derived from each parental strain could be maintained stably in a new genetic background with an expanded range of pathogenicity. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank database under the accession numbers AB469331to AB469354.     

Bacterial community in plant residues in a Japanese paddy field estimated by RFLP and DGGE analyses

Plant residues (PRs) are “hot spots” of microbial activities in soil. PRs with the size more than 0.5 mm were collected from a Japanese paddy field during rice cultivation period (from May to September) and fractionated into four categories by size (>4, 2-4, 1-2, and 0.5-1 mm) using sieves. Restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE) patterns were compared among the fractions after DNA extraction from the PRs and PCR amplification. The total amount of PRs with the size over 0.5 mm decreased in the field with the first-order kinetics (r²=0.810, p<0.01) with time from rice transplanting to harvest. RFLP analysis showed that the bacterial community structure in PRs with the 0.5-2 mm fraction was different from that in PRs with the >2 mm fraction and the latter community structure changed after the midseason drainage. In contrast, the DGGE patterns of the bacterial community in the PRs indicated the succession from June to September during rice cultivation forming three major groups irrespective of the fraction size. Sequence analysis of DGGE bands showed that Firmicutes (clostridia), α-, γ-, δ-Proteobacteria (myxobacteria), Nitrospira, Acidobacteria, Bacteroidetes, Verrucomicrobia and Spirochaetes were predominant members in the PRs irrespective of fraction size.     

Comparison of phospholipid fatty acid composition in percolating water,floodwater, and the plow layer soil during the rice cultivation period in a Japanese paddy field

Azolla has been used as a green manure for rice in Vietnam and southern China (3). Recently it was adopted by farmers in the Koronadal area in South Cotabato Province, the Philippines (2). In that area Azolla pinnata grows reasonably well without phosphate fertilizer, which is generally used for stimulating Azolla growth (3, 5).     

ATP depletion inhibits retraction of chromatophores induced by GABA in cephalopod Todarodes pacificus

Color changes in cephalopods are generated by the expansion or retraction of chromatophores located under the dermis. The behavior of the chromatophores is regulated by neurotransmitters; l-glutamate (l-Glu) is an excitatory transmitter that causes the chromatophores to expand. To date, serotonin [5-hydroxytryptamine (5-HT)] is the only neurotransmitter known to stimulate retraction of chromatophores. We found that the chromatophores in the Japanese squid Todarodes pacificus were regulated by γ-aminobutyric acid (GABA), an inhibitory neurotransmitter, and that GABA caused expanded chromatophores to retract. We also found that adenosine 5′-triphosphate (ATP) concentrations in skin samples remained stable at their initial values for more than 24 h after the death of each squid; therefore, the chromatophores could respond to both l-Glu and GABA during that period. Furthermore, we attempted to reduce the levels of ATP by storing skin sample in sodium azide solution. The chromatophores in sodium azide-treated skin samples were induced to expand by l-Glu, but these expanded chromatophores could not be induced to retract by GABA. Based on these observations, we conclude that ATP is essential for retraction, but not expansion, of chromatophores.     

Promotion of initial swimbladder inflation in Pacific bluefin tuna,Thunnus orientalis (Temminck and Schlegel), larvae

Initial swimbladder inflation (ISI) of Pacific bluefin tuna (PBT), Thunnus orientalis, larvae was studied to increase the survival of cultured larvae. Experiment 1 was conducted to explore promotion and inhibition of ISI under different water surface conditions; including the use of surface skimmer to remove autogenous surface substances (SS), covering the water surface with liquid‐paraffin‐layer (LP) and oil film (OF), and a control (non‐treatment, NT). Significantly higher inflation frequency was observed in SS (62.2%) than NT (11.9%), LP (2.7%) and OF (3.9%). This indicates that ISI in PBT larvae can be promoted by removal of surface substances on rearing water which inhibit larval air gulping. Experiment 2 aimed to elucidate proper day of larval age to start skimming for promoting ISI with four different periods of oil film removal: from 3 to 8 (SF3D), 4 to 8 (SF4D), 5 to 8 (SF5D), 6 to 8 (SF6D) days‐post‐hatch (dph). Significant improvement in ISI frequency was observed in SF3D (80.2%) but the frequency was very poor in SF4D, SF5D, and SF6D (17.8–7.5%). This implies the need of oil film removal without missing a narrow window, 1 day of 3 dph, to promote ISI in practical PBT larviculture.     

Identification of genes required for the fitness of Rhodococcus equi during the infection of mice via signature-tagged transposon mutagenesis

Rhodococcus equi is a Gram-positive facultative intracellular bacterium that causes pyogranulomatous pneumonia in foals and immunocompromised people. In the present study, signature-tagged transposon mutagenesis was applied for the negative selection of R. equi mutants that cannot survive in vivo. Twenty-five distinguishable plasmid-transposon (plasposon) vectors by polymerase chain reaction (PCR), each containing a unique oligonucleotide tag, were constructed and used to select the transposon mutants that have in vivo fitness defects using a mouse systemic infection model. Of the 4,560 transposon mutants, 102 mutants were isolated via a real-time PCR-based screening as the mutants were unable to survive in the mouse model. Finally, 50 single transposon insertion sites were determined via the self-cloning strategy. The insertion of the transposon was seen on the virulence plasmid in 15 of the 50 mutants, whereas the remaining 35 mutants had the insertion of transposon on the chromosome. The chromosomal mutants contained transposon insertions in genes involved in cellular metabolism, DNA repair and recombination, gene regulation, non-ribosomal peptide synthesis, and unknown functions. Additionally, seven of the chromosomal mutants showed a reduced ability to multiply in the macrophages in vitro. In this study, we have identified several biosynthetic pathways as fitness factors associated with the growth within macrophages and survival in mice.