Scale calcification in the goldfish in vitro: histological and quantitative analysis

The external layer of a teleost fish scale is composed of type I collagen, an amorphous matrix substance and hydroxyapatite crystals. Calcification of this layer can be inhibited in the scale-regenerating process under calcium- and phosphate-deficient (CaDPD) conditions, and can be facilitated by incubation in physiological saline. The aim of this study was to evaluate this model of calcification using histological and quantitative analysis in order to promote further understanding of the mechanism of calcification in fish scales. We found that the external layer of the scales produced under CaDPD conditions contained more densely aligned collagen fibrils with a small amount of the amorphous matrix substance. The CaDPD scale contained only one-third of the amount of calcium and phosphate present in the control fish. After 4 hours of incubation, a two- to threefold increase in calcium content and a 1.5-fold increase in phosphate content were observed. Calcification proceeded in the external layer, and mineral deposits grew only in the electron-dense matrix substance. We conclude that this model would be suitable for studying the early process of fish scale calcification that occurs in the noncollagenous substance. The electron-dense substance may contain key molecules that promote calcification.     

Characterization of the morphological and physiological traits of rice cultivars with adaptation to unflooded condition during early vegetative growth

This study aimed to characterize the physiological and morphological traits that are associated with adaptation to unflooded soil conditions in rice. Four indica rice cultivars (Puluik Arang, Badari Dhan, Shwe Nang Gyi, and Ratul), which were previously identified as highly or less adaptable to unflooded soil conditions, were grown under flooded and unflooded (soil water potential; －0.10 MPa) soil conditions. Water uptake was measured every day for three weeks, and then the leaf water potential, the stomatal conductance, the dry matter weight, shoot and root morphological traits were measured. Puluik Arang and Badari Dhan exhibited greater leaf area expansion and higher maintenance of root development under the unflooded condition than that by other cultivars. The leaf water potential and stomatal conductance of fully expanded highest leaf in Puluik Arang and Badari Dhan were not affected by unflooded soil regime. Leaf area and root morphological traits were significantly correlated with water uptake regardless of soil moisture regimes. These results suggested that Puluik Arang and Badari Dhan exhibited great water uptake capacity through physiological and morphological adaptation of shoot and root traits to unflooded condition, resulting in great biomass productivity under the condition.     

Infection dynamics of Neoheterobothrium hirame (Monogenea) on juvenile olive flounder, Paralichthys olivaceus (Temminck & Schlegel), in coastal waters of Japan

A recent epidemic of the invasive monogenean Neoheterobothrium hirame is suspected to have caused the significant decline of the commercial catch of olive flounder, Paralichthys olivaceus, in Japan. To clarify the causal link between parasite invasion and host depletion, we monitored the infections of N. hirame on juvenile flounder for 2 years at two widely separate bays, Obama Bay, experiencing a devastating reduction in flounder catches, and Miyako Bay with a stable catch. Prevalence, mean intensity and abundance of developmental stages of N. hirame were compared between the bays and between fish year classes. Fishes captured at Obama Bay harboured three times more worms than those at Miyako Bay and had a significantly higher overall prevalence of infection. In addition, there was a negative correlation between parasite intensity and host condition factor at Obama Bay, suggesting a causal link between the N. hirame epidemic and the reduction of local flounder populations.     

Attenuated mutants of Potato virus Y necrotic strain produced by nitrous acid treatment and mutagenesis-in-tissue culture methods

We produced attenuated mutants of Potato virus Y necrotic strain not only by nitrous acid treatment but also by a novel method, probably unique to plant viruses, which we call the “mutagenesis-in-tissue culture method”. This relies on the natural or experimental generation of virus sequence variants within an infected plant, and then isolating the mutants by serially cloning them in plants. A total of fifteen attenuated mutants were obtained and studied. Nucleotide and amino acid sequences of the genomes of the attenuated mutant populations were compared with those parental severe isolates, and the amino acid changes in relevant genomic regions for viral attenuation were inferred. Many of the mutations were located in the 5’ half of the genome; 65 % were located in the protein 1 (P1) and helper component proteinase protein (HC-Pro) encoding regions. Amino acid changes mostly involved simultaneous changes in two or more protein encoding regions, one of which was often in the HC-Pro encoding region. The attenuated mutants M-MY10 and N-NA10 were effective in cross-protection against the original severe isolate NTND6.     

Genetic relationships of Japanese potato cultivars assessed by RAPD analysis

Seventy-three breeding lines and cultivars, which included all Japanese cultivars, were assessed by random amplified polymorphic DNA (RAPD) analysis. Thirty-one of 40 decamer primers revealed 84 scorable RAPDs. Using these RAPDs, 67 of 73 cultivars were distinguished from each other. One of the cultivars released as a protoclonal variant was able to distinguish from the parent by increasing the number of primers surveyed. Based on the number of different RAPDs between each pair of accessions, cluster analysis was performed to exploit genetic diversity in Japanese cultivars. Closely related cultivars deduced from their pedigrees were clustered closely, indicating that similarities based on the presence vs. absence of RAPDs were reflections of genetic similarities. Old Japanese cultivars, as suggested by their similarity of chloroplast DNA toS. tuberosum ssp.andigena, were indeed clustered with ssp.andigena accessions, supporting these were relic cultivars of early European potatoes. The present study suggests that Japanese potatoes are an interesting mass of genetic diversity including North American, European, and Andean germplasms and relic potatoes.     

Effects of polyphenols from seed shells of Japanese horse chestnut (Aesculus turbinata BLUME) on methotrexate-induced intestinal injury in rats

The purpose of this study was to evaluate the effects of polyphenols from seed shells of Japanese horse chestnut (JHP) on methotrexate (MTX)-induced intestinal injury in rats. MTX application caused intestinal morphological injury and increase in malondialdehyde (MDA) levels, decrease in levels of glutathione (GSH) and glutathione peroxidase (GSH-Px) activities in small intestine. However, oral administration of JHP ameliorated MTX-induced intestinal injury and inhibited the increase in MDA and the decrease in GSH and GSH-Px activity in small intestine. In conclusion, our results indicated that oral administration of JHP alleviated MTX-induced intestinal injury through its antioxidant properties.     

Proventricular dilatation disease associated with avian bornavirus infection in a Citron-crested Cockatoo that was born and hand-reared in Japan

A 5-month-old female Citron-crested Cockatoo (Cacatua sulphurea citrinocristata) that was born and hand-reared in Japan died with suspected proventricular dilatation disease (PDD). Macroscopic and microscopic examinations of the bird revealed characteristic features of PDD, i.e., distention of the proventriculus and infiltration of lymphocytes and plasma cells in ganglia of various organs and in central and peripheral nerves. A linkage of this PDD case to infection with avian bornavirus (ABV) was documented by RT-PCR amplification of the virus genomes from the affected bird. Phylogenetic analysis revealed that the ABV identified in this study clustered into the genotype 2, which is one of the dominant ABV genotypes worldwide. To the best of our knowledge, this is the first report of a natural case of PDD associated with ABV infection in Japan.     

Cloned porcine embryos can maintain developmental ability after cryopreservation at the morula stage

The aim of the present study was to clarify the overall efficiency of porcine somatic cell nuclear transfer (SCNT) by incorporating cryopreservation of the cloned embryos before transfer. The SCNT embryos reconstructed with preadipocytes and in vitro-matured (IVM) oocytes were cultured to harvest morula stage embryos; they were then subjected to delipation (removal of cytoplasmic lipid droplets) and vitrification. After warming and culture, the embryos developing to blastocysts were transferred to recipients to obtain cloned piglets. From 372 reconstructed embryos, 188 (50.5%) reached the morula stage and 117 (31.5%) developed to blastocysts after vitrification. Transfer of 98 (26.3%) morphologically normal blastocysts gave rise to 6 (1.6%) piglets, including 1 stillborn. The efficiency of the cloned piglet production was comparable with that obtained using SCNT embryos without cryopreservation (2.7%, 17/635). Here, we demonstrate that porcine somatic cell cloning can be performed without a significant reduction in efficiency even when the SCNT embryos are cryopreserved before transfer.