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.     

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.     

Development of a docosahexaenoic acid (DHA)-rich yellowtail <Emphasis Type="Italic">Seriola quinqueradiata</Emphasis> using tuna by-product oil

Because a diet rich in docosahexaenoic acid (DHA) is beneficial to human health, it is desirable for yellowtail Seriola quinqueradiata intended for human consumption to contain a high amount of DHA. We investigated the effects on the growth and fatty acid composition of yellowtail of replacing some of the pollack liver oil (PO) in the yellowtail diet with a DHA-rich tuna oil (TO). Experimental diets were prepared in which PO was replaced with 0, 15, 30, and 60% TO. Replacement of PO with TO did not negatively affect growth in a 16-week trial. In contrast, the feed efficiency of the group that received 60% TO was significantly higher than that of the control group after 6 weeks. The DHA level in the whole fish body increased with increasing dietary TO level. The groups that received 30 and 60% TO replacement diets showed significantly higher DHA levels than those of the control group. DHA level in the fillet was significantly higher in the group that received 60% TO than in the control group. DHA retention tended to increase with increasing TO level. These results show that utilizing TO as a fat source can increase DHA accumulation without incurring any adverse effects on the growth of yellowtail.     

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.     

Glycine max and Glycine soja are capable of cold acclimation

Soybean has been considered a cold intolerant species; based largely upon seed germination and soil emergent evaluations. This study reports a distinct acquisition of cold tolerance, in seedlings, following short acclimation periods. Diversity in cold responses was assessed in eight cultivars of Glycine max and six accessions of G. soja. All varieties of soybean significantly increased in freezing tolerance following acclimation. This study indicates soybean seedlings are indeed capable of sensing cold and acquiring cold tolerance. Germination rates after cold imbibition were negatively correlated with maturity group, but positively correlated with cold acclimation potential in G. soja. Seed fatty acid composition was varied between the species, with Glycine soja accessions containing about 2‐times more linolenic acid (18:3) than G. max. Furthermore, high levels of linoleic acid (18:2) in seeds were positively correlated with germination rates following cold imbibition in G. soja only. We suggest that domestication has not impacted the overall ability of soybean to cold acclimate at the seedling stage and that there is little variation within the domesticated species for ability to cold acclimate. Thus, this brief comparative study reduces the enthusiasm for the “wild” species as an additional source of genetic diversity for cold tolerance.     

Characterization of a novel nicotinamide adenine dinucleotide-cytochrome b5 reductase mutation associated with canine hereditary methemoglobinemia

Hereditary methemoglobinemia associated with nicotinamide adenine dinucleotide-cytochrome b5 reductase (b5R) deficiency is a rare autosomal recessive disorder in animals. Recently, nonsynonymous b5R gene (CYB5R3) variants have been reported to be associated with canine and feline hereditary methemoglobinemia. However, the underlying molecular mechanisms of canine and feline methemoglobinemia caused by these nonsynonymous variants have not yet been reported. Previously, we reported a Pomeranian dog family with hereditary methemoglobinemia, carrying CYB5R3 mutation of an A>C transition at codon 194 in exon 7, replacing an isoleucine residue with leucine (p.Ile194Leu). In this study, we investigated the enzymatic and structural properties of the soluble form of wild-type and Ile194Leu canine b5Rs to characterize the effects of this missense mutation. Our results showed that the kinetic properties of the mutant enzyme were not affected by this amino acid substitution. The secondary structure of the wild-type and Ile194Leu b5Rs detected by circular dichroism showed a similar pattern. However, the mutant enzyme exhibited decreased heat stability and increased susceptibility to trypsin hydrolysis. Moreover, the thermostability and unfolding measurements indicated that the mutant enzyme was more sensitive to temperature-dependent denaturation than the wild-type b5R. We concluded from these results that unstable mutant enzyme properties with normal enzymatic activity would be associated with hereditary methemoglobinemia in the Pomeranian dog family.     

Genetic diversity of Pinus densiflora pollen flowing over fragmented populations during a mating season

We attempted to evaluate the genetic diversity of long-distance transported pollen flowing over fragmented Pinus densiflora populations during a mating season. A P.?densiflora clonal seed orchard, which was located in a rural area where many fragmented populations exist, was selected for pollen capture. Immigrant pollen captured by three clones having different flowering times was regarded as the pollen flowing over fragmented populations during a mating season. The genetic diversity (H _e) values of the immigrant pollen captured by the three clones were high (H _e?>?0.894). The correlation of paternity (r _p) values of the seeds having immigrant parent generated from the three clones were calculated to be negative. From these parameters, the pollen cloud is considered to have maintained high genetic diversity during the mating season. The genetic composition of the pollen cloud showed slight variation. The pollen captured by different trees (i.e., clonal ramets of the three clones) was significantly different based on analysis of molecular variance. Especially, the pollen pools captured by trees planted in the western side of the orchard were significantly different from the gene pool of the surrounding populations. Factors affecting this differentiation could be that the donors of the pollen transported to the orchard vary with time, as well as nonuniform dispersal of the pollen. From these results, the pollen flowing over fragmented P.?densiflora populations is considered to have high genetic diversity, compensating to some extent for fragmentation.     

Comparison of dendritic cell-mediated immune responses among canine malignant cells

Dendritic cell (DC) vaccination is one of the most attractive immunotherapies for malignancies in dogs. To examine the differences in DC-mediated immune responses from different types of malignancies in dogs, we vaccinated dogs using autologous DCs pulsed with keyhole limpet hemocyanin (KLH) and cell lysate prepared from squamous cell carcinoma SCC2/88 (SCC-KLH-DC), histiocytic sarcoma CHS-5 (CHS-KLH-DC), or B cell leukemia GL-1 (GL-KLH-DC) in vitro. In vivo inductions of immune responses against these tumor cells were compared by the delayed-type hypersensitivity (DTH) skin test. The DTH response against SCC2/88 cells were observed in dogs vaccinated with autologous SCC-KLH-DC, while the response was undetectable against CHS-5 and GL-1 cells in dogs vaccinated with autologous CHS-KLH-DC and GL-KLH-DC. Skin biopsies taken from DTH challenge sites were then examined for immunohistochemistry, and recruitment of CD8 and CD4 T cells was detected at the site where SCC2/88 cells were inoculated in dogs vaccinated with SCC-KLH-DC. By contrast, neither CD8 nor CD4 T cell infiltration was found at the DTH challenge site in the dogs vaccinated with CHS-KLH-DC or GL-KLH-DC. These findings may reflect that the efficacy of immune induction by DC vaccination varies among tumor types and that immune responses could be inducible in squamous cell carcinoma. Our results encouraged further investigation of therapeutic vaccination for dogs with advanced squamous cell carcinoma in clinical trials.