Comparative aspects on the role of polypyrimidine tract-binding protein in internal initiation of hepatitis C virus and picornavirus RNAs

We compared the effects of polypyrimidine tract-binding protein (PTB) on hepatitis C virus (HCV genotype IIa), encephalomyocarditis virus (EMCV) and poliovirus internal ribosome entry site (IRES) activities in vitro. It bound strongly to EMCV IRES, but weakly to PV and HCV RNAs. PV IRES showed the strongest dependency to PTB and it showed less than one-tenth of IRES activity after the immuno-depletion of PTB from HeLa S10 lysate with pre-coated anti-PTB IgG beads, comparing to the normal IgG beads-treated S10 lysate. EMCV IRES activity was approximately 40% of that of normal control after PTB depletion. Especially, HCV IRES activity was approximately 95%, and most weekly affected by the depletion of PTB. Repletion of PTB to depleted S10 lysate restored activities of PV and EMCV IRESs. The data suggest that PTB plays an important role in picornaviral IRESs, but not in HCV IRES.     

Effects of resveratrol treatment on mitochondria and subsequent embryonic development of bovine blastocysts cryopreserved by slow freezing

This study evaluated the effects of cryopreservation by slow freezing on the mitochondrial function, DNA integrity, and developmental ability of bovine embryos and examined whether resveratrol treatment of the frozen‐thawed blastocysts improved embryonic viability. In vitro produced bovine embryos were subjected to slow freezing. After thawing, the ATP content and mitochondrial DNA integrity (mtDNA), determined by real‐time PCR targeting short and long mitochondrial sequences, was found to be lower in frozen‐thawed embryos than in fresh embryos, and mtDNA copy number was significantly reduced during the 24‐hr incubation post warming. Furthermore, immunostaining against double‐strand DNA revealed DNA damage in frozen‐thawed embryos. When frozen‐thawed embryos were incubated in the medium containing 0.5 µM resveratrol, SIRT1 expression, and survival rate of the embryos significantly improved compared with the vehicle‐treated embryos. In addition, cell‐free mtDNA content in medium was higher in case of resveratrol‐treated embryos than of vehicle‐treated embryos. In conclusion, slow freezing affects mitochondrial integrity and function in the blastocysts. In the frozen‐thawed embryos, mitochondria were removed during post‐thawing incubation and resveratrol enhanced the process, resulting in improved survivability of the embryos.     

Cytological and genetical studies on male sterility inCryptomeria japonica D. Don

Genetic male sterility is a useful trait in plant breeding, especially in angiosperm crops such as corn, onion and carrot. We found a male sterile sugi (Cryptomeria japonica D. Don) tree in Toyama, Japan. Pollen of sugi is one of the major causes of pollinosis in Japan. We carried out this research in an attempt to make clear the characteristics and inheritance of this male sterility. Microsporogenesis of the male sterile tree proceeded meiosis, however, the microspores collapsed after they were separated from pollen tetrads in locules, resulting in complete male sterility. Most likely, ethylene evolution was responsible for male sterility expression. Full seed setting in the male sterile tree indicated normal macrosporogenesis. Seeds obtained from crossing between male sterile and normal lines showed relatively high level of germination and their seedlings grew vigorously. The somatic chromosome numbers of 241 germinated seeds, derived from the male sterile tree, were mostly 22, euploid. These results indicated that male sterile tree was different from other similar previously reported trees with low pollen fertility, resulting from triploid or trisomics. Probably, male sterility in sugi is either nuclear genetic male sterility or cytoplasmic male sterility. The study was partially supported by Program for Promotion of Basic Research Actives for Innovative Biosciences.     

Intraradical hyphae phosphatase of the arbuscular mycorrhizal fungus, Gigaspora margarita

An alkaline phosphatase in the intraradical hyphae of arbuscular mycorrhizal fungi was found to be closely related to an improvement of plant growth. To detect the phosphatase activity in a crude extract of mycorrhizal roots, phosphatase isozymes in mycorrhizal and non-mycorrhizal onion roots were compared with those in Gigaspora margarita by electrophoresis. A mycorrhiza-specific band was found when the phosphatase was stained under alkaline conditions. To clarify the origin of this phosphatase, the phosphatase extracted from intraradical hyphae was also compared with the phosphatase from mycorrhizal roots by electrophoresis. The intraradical hyphae was isolated from mycorrhizal roots by enzyme digestion followed by Percoll gradient centrifugation. The soluble protein was extracted from the hyphae by ultra-sonication after treatment with chitinase. A phosphatase in the hyphal soluble protein showed a similar, but slightly higher, relative mobility on the gel, compared with the mycorrhiza-specific phosphatase from roots. By adding the hyphal extract to the root extract, the relative mobility of the mycorrhiza-specific phosphatase was slightly changed and became identical to that of the phosphatase in the hyphae. This indicated that the specific band of phosphatase found in the crude extract from mycorrhizal roots was of intraradical hyphal origin. Received: 16 April 1997     

First isolation of Sarcocystis hominis from cattle in Japan.

Sarcocystis hominis was first isolated from slaughtered cattle raised in Japan. Cysts were 1,220-4,460 x 80-384 microns in size and their wall was 3 to 6 microns thick and appeared radially striated in the histopathological sections because of the presence of palisade-like villar protrusions on the surface. The protrusions were 3.1-4.3 x 0.7-1.1 microns in size and had many microtubules in the core. Two cynomolgus monkeys, Macaca fascicularis, fed with the Sarcocystis cysts began to pass sporocysts, which measured a size of 14.3-15 x 9.5-10 microns, in the feces 10 days after ingestion.     

Estimating the origins of adult chum salmon <Emphasis Type="Italic">Oncorhynchus keta</Emphasis> in the Okhotsk and Sea of Japan regions using discriminant analysis of scale characteristics

Linear discriminant analysis (LDA) based on scale patterns was used to develop a methodology of estimating regional origins of chum salmon. Age-4 fish were sampled in 2004–2006 from 12 river stocks of the Okhotsk and Sea of Japan (SJ) regions from Hokkaido to Honshu. The scale radius at the first annulus of each fish was separated into i intervals and the radius of each interval was divided by the number of scale circuli within the interval to quantify scale patterns. The i variables and five other morphometric measurements were used in a stepwise LDA to classify the following regional groups: Hokkaido and Honshu (I), Okhotsk and SJ (II), Okhotsk, Hokkaido SJ and Honshu SJ (III). Percentages of correctly classified fish (hit rates) improved with increased i but tended to be close to asymptotic values in all cases. Hit rates for each river stock in case (I) ranged from 74.3% to 100% (mean 97.2%), estimated by direct maximum likelihood methods using predictor variable sets from the best models for LDAs. Hit rates were lower in cases (II) and (III). This study demonstrated that scale patterns are useful for classifying the origins of chum salmon, at least between Hokkaido and Honshu.     

Low-fidelity homing behaviour of Biwa salmon <Emphasis Type="Italic">Oncorhynchus</Emphasis> sp. landlocked in Lake Biwa as inferred from otolith elemental and Sr isotopic compositions

Biwa salmon Oncorhynchus sp. is endemic to Lake Biwa, Japan, where it is an important commercial and recreational fisheries species. However, no information is currently available on its population structure and migration ecology. Therefore, here we evaluated whether otolith Sr/Ca, Ba/Ca and ⁸⁷Sr/⁸⁶Sr ratios can be used as natural signatures in Biwa salmon and then used these to determine the natal origins of lake-migration-phase individuals and spawning adults, and the homing ability of spawning adults in the Lake Biwa water system. Quadratic discriminant function analysis demonstrated that the lake-migration school comprised individuals with multiple origins, including rivers to the east, west and north of Lake Biwa, and that the homing rate of spawning adults was low (18 out of 80 individuals), with ca. 78% of fish straying into non-natal rivers. However, this straying behaviour was not spatially random, with fish tending to migrate upstream in rivers neighbouring their natal rivers. The high rate of straying in spawning adults is considered important for establishing and maintaining this species, which is highly adapted to life in the Lake Biwa water system where environmental disturbances often occur.     

Fine mapping of the gene for susceptibility to black spot disease in Japanese pear (Pyrus pyrifolia Nakai)

Black spot disease, which is caused by the Japanese pear pathotype of the filamentous fungus Alternaria alternata (Fries) Keissler, is one of the most harmful diseases in Japanese pear cultivation. We mapped a gene for susceptibility to black spot disease in the Japanese pear (Pyrus pyrifolia Nakai) cultivar ‘Kinchaku’ (Aki gene) at the top of linkage group 11, similar to the positions of the susceptibility genes Ani in ‘Osa Nijisseiki’ and Ana in ‘Nansui’. Using synteny-based marker enrichment, we developed novel apple SSR markers in the target region. We constructed a fine map of linkage group 11 of ‘Kinchaku’ and localized the Aki locus within a 1.5-cM genome region between SSR markers Mdo.chr11.28 and Mdo.chr11.34. Marker Mdo.chr11.30 co-segregated with Aki in all 621 F₁ plantlets of a ‘Housui’ × ‘Kinchaku’ cross. The physical size of the Aki region, which includes three markers (Mdo.chr11.28, Mdo.chr11.30, and Mdo.chr11.34), was estimated to be 250 Kb in the ‘Golden Delicious’ apple genome and 107 Kb in the ‘Dangshansuli’ Chinese pear genome. Our results will help to identify the candidate gene for susceptibility to black spot disease in Japanese pear.