Use of refractometer to determine soluble solids of astringent fruits of Japanese persimmon (Diospyros kaki L.)

A method was developed to determine soluble solids refractometrically in astringent Japanese persimmon fruits with a high soluble tannin content. Adding 5% PEG # 6000 to the fruit juice in equal amount followed by centrifugation or filtration removed interfering tannins effectively from the mixture by forming precipitates. The refractometer readings were taken with the resultant solution and 2.5% PEG # 6000 and then the refractometer sugar value of the original juice was calculated by multiplying by two the difference of the two readings. The calculated value approximated closely to the total sugar content of juice.     

Generation of canine dendritic cells from peripheral blood monocytes without using purified cytokines

Dendritic cells (DCs), which differentiate in vitro from peripheral blood monocytes (PBMOs) or bone marrow precursors, are a promising candidate for immunotherapy against cancer. The dog, which suffers common types of cancers along with humans, make an ideal large animal model for cancer studies. Monocyte-derived DCs in the dog have not been well characterized, however, since the appropriate condition for in vitro differentiation has not been established. To tackle this problem, we have developed a conditioned media by culturing T cells with immobilized anti-canine CD3 antibody, and sought to induce differentiation of DCs from PBMOs. When purified CD14+ PBMOs were cultured in the presence of 25% T cell conditioned medium (TCCM), the PBMOs increased size and had extended dendritic processes by day 12 of the culture. The cultured PBMOs were found to increase the expression of MHC class II and CD1a molecules, and significantly increased stimulatory activity for allogeneic T cells in the mixed leukocyte reaction. Moreover, the cells significantly increased their expression of IL-18 and IFN-gamma when stimulated with polyinosinic-polycytidylic acid (Poly (I:C)). The cells have a reduced phagocytic activity, which is a common defect in mature DCs. It follows from these results that TCCM does induce the differentiation of DCs from PBMOs.     

Indirect hemagglutination test in equine infectious anemia.

An indirect hemagglutination was developed for the diagnosis of equine infectious anemia using sheep red blood cells coated with group specific virus antigen which had been highly purified by affinity chromatography. The presence of indirect hemagglutination antibodies was demonstrated in horses with equine infectious anemia since the cells were specifically agglutinated by all the serum samples obtained from experimentally infected horses. Antibodies appeared within 35 days after inoculation, and development of which coincided well with that of precipitating and complement fixing antibodies. Titer of indirect hemagglutination antibodies were ten to 320 times greater than those of precipitating antibodies. Test results could be read more clearly by the indirect hemagglutination test especially in weakly positive cases. Ninety-six samples from suspected field cases collected from every region of Japan which were positive on the immunodiffusion test were also positive on indirect hemagglutination test. Serum samples from 420 horses in one race track were examined by both the indirect hemagglutination and immunodiffusion tests to determine the reliability of the indirect hemagglutination test for diagnosis of equine infectious anemia. The same result was obtained on both tests. Based on this evidence, the indirect hemagglutination test can be employed as a very sensitive serological test for the diagnosis of equine infectious anemia.     

Seasonal fluctuation of invasive flatworm predation pressure on land snails: Implications for the range expansion and impacts of invasive species

Introduction of the snail-eating flatworm Platydemus manokwari has caused extinction and decline of native land snails on tropical and subtropical islands. As the factors influencing flatworm predation pressure on land snails remain unclear, I examined the effects of seasonal variation in flatworm predation pressure on land snail survival in the wild on a subtropical island. I also examined the feeding activities of P. manokwari under laboratory conditions. Survival rates of land snails experimentally placed on the forest floor for 7 days ranged from 0% to 100% among seasons on the oceanic island Chichijima [Ogasawara (Bonin) Islands]. More than 90% of snails were killed by P. manokwari within 7 days in the period from July to November, while less than 40% of snails were killed in other months. Snail mortality rate (0-100%) attributable to P. manokwari was positively correlated with mean temperature (17.1-27.3 °C) in the study area. Laboratory experiments showed that low temperature influenced snail survival and regulated feeding activity of P. manokwari. Laboratory experiments also suggested that high densities of P. manokwari may cause a rapid decline in snail survival. Therefore, ambient temperature and density of P. manokwari may regulate seasonal variations in predation pressure on land snails. Recent global warming may increase the probability of invasion and population establishment, and elevate the impacts of P. manokwari in temperate regions.     

Testing the possibility of horizontal transfer of introduced neomycin phosphotransferase （nptⅡ） gene of transgenic Eucalyptus camaldulensis into soil bacteria

The possible horizomal transfer of transgenes is of great concern when the transgenic plants are released imo the field. To test the possible transfer of nptII of transgenic trees into soil bacteria, we have used a stool DNA preparation kit to isolate the DNA from the soils in the rhizospheres of two non- and eight transgenic Eucalyptus camaldulensis trees. All the samples have provided the corresponding PCR products in the amplification with bacterial 16S RNA specific sequences, which indicates that the quality of the isolated DNA is adequate for amplification. The nptⅡ specific band has been amplified in three soil samples from the transgenic trees and even treated with filtration before the DNA isolation. This indicates that nptII DNA exists in the soil, although it is still unclear whether the DNA was in the soil particles, in the soil bacteria or in the Agrobacterium comamination which was used for the E. camaldulensis transformation. Two approaches on isolation of bacterial DNA have been suggested for testing the possibility of this event in the future.     

Determination of the Chemical Structures of Tandyukisins B–D,Isolated from a Marine Sponge-Derived Fungus

Tandyukisins B–D (1–3), novel decalin derivatives, have been isolated from a strain of Trichoderma harzianum OUPS-111D-4 originally derived from the marine sponge Halichondria okadai, and their structures have been elucidated on the basis of spectroscopic analyses using 1D and 2D NMR techniques. In addition, their chemical structures were established by chemical transformation. They exhibited weak cytotoxicity, but selective growth inhibition on panel screening using 39 human cancer cell lines.     

Rapid estimation of species-specific DNA digestibility based on differential qPCR

Digestibility of DNA in selected prey species was estimated by an in vivo feeding trial using six test diets. Each diet contained one of the following: (a) goldfish, (b) goldfish + citric acid, (c) goldfish + CaCO₃, (d) shrimp, (e) snail, and (f) goldfish + shrimp + snail. The diets were fed to rainbow trout and fecal samples were collected. Mitochondrial DNA was extracted from both diet and fecal samples and quantified by qPCR using two sets of species-specific primers for each prey species. The first set of primers amplified a short stretch of DNA (51–80 bp), whereas another set amplified a longer stretch (126–162 bp). DNA digestibility was estimated based on the ratio between short and long amplicons using the following formula: Digestibility (%) = log_{((Mt?S)/(Mt?L))} ([S]/[L])/(0.01 × Mt), where Mt: 16,000 (bp), S: length of short amplicon (bp), L: length of long amplicon (bp), [S]: relative quantity of short amplicon, [L]: relative quantity of long amplicon. Calculated fecal digestibility of goldfish DNA ranged from ?1.05 % (f) to 2.07 % (a). Digestibility of shrimp DNA was 10.79 % (d) and 12.61 % (f). Digestibility of snail DNA was 1.88 % (e) and 2.06 % (f). These results suggest that the digestibility of dietary DNA can be estimated based on the ratio between long and short fragments.     

Comparison of prevalence of Felis catus papillomavirus type 2 in squamous cell carcinomas in cats between Taiwan and Japan

Felis catus papillomavirus (FcaPV), especially type 2 (FcaPV2) is considered as one of the causative agents in squamous cell carcinoma (SCC) in cats. However, our previous study detected FcaPV3 and FcaPV4, but not FcaPV2 in feline SCCs collected in Japan, suggesting that the prevalence of FcaPV2 in SCC may vary depending on geographic locations. To evaluate this hypothesis, two conventional PCR reactions targeting E1 and E7 genes were performed to detect FcaPV2 in feline SCC samples collected in Taiwan and Japan. While 46.9% (23/49) of feline SCC cases from Taiwan were PCR positive for FcaPV2, only 8.6% (3/35) cases from Japan were positive. Our result suggests that the prevalence of FcaPV2 in feline SCCs may depend on the region.     

Monitoring genetic stability inQuercus serrata Thunb. somatic embryogenesis using RAPD markers

Genetic stability of propagules regeneratedvia somatic embryogenesis is of paramount importance for its application to clonal forestry. Random amplified polymorphic DNA (RAPD) markers were used to determine the genetic stability in somatic embryogenesis ofQuercus serrata Thunb. (Japanese white oak). Forty samples from an embryogenic line, consisting of regenerated plantlets, somatic embryos, and embryogenic calli, were examined using 54 decanucleotide primers. A total of 6520 clear reproducible bands obtained from these studies exhibited no aberration in RAPD banding pattern among the tested samples. Our results show that somaclonal variation is absent in our plant propagation system. The genetic stability is discussed in terms of the origin of somatic embryos.