Development of cleaved amplified polymorphic sequence (CAPS)-based markers for identification of sweetpotato cultivars

To develop a simple and reliable method to identify sweetpotato cultivars, we designed cleaved amplified polymorphic sequence (CAPS)-based markers and used them to perform genotyping of Japanese sweetpotato cultivars. In order to screen the CAPS-based markers, 13 primer pairs were designed from the exon sequences of 11 sweetpotato genes to amplify fragments containing an intron. By digesting the amplified products with 8 restriction enzymes having different recognition sites, a total of 27 polymorphic marker fragments were obtained. Genotyping of 60 Japanese sweetpotato cultivars using these markers suggested that the markers can effectively distinguish sweetpotato cultivars. Among the genes used for primer design, the gene encoding the dihydroflavonol 4-reductase (DFR) showed the largest degree of polymorphism. To our knowledge, this is the first report on the development of CAPS-based markers in sweetpotato.     

Evaluation of a new eastern blotting technique for the analysis of ginsenoside Re in American ginseng berry pulp extracts

A new eastern blotting technique has been established for ginsenoside Re (G-Re) contained in American ginseng berry pulp extracts. G-Re in American ginseng berry pulp was extracted using 100% methanol, 100% ethanol, 50% aqueous methanol, and 50% aqueous ethanol. The combined crude extracts were applied onto a polyethersulfone membrane and developed using the methanol–water–acetic acid solvent system (45:55:1 v/v). Separated components were immunostained using anti-G-Re monoclonal antibody. G-Re was first specifically detected and then quantitatively analyzed using NIH Imaging software. We also confirmed that the most suitable solvent was 50% aqueous methanol for extracting G-Re from American ginseng berry pulp.     

3-Prenyl luteolin,a new prenylated flavone with melanin biosynthesis inhibitory activity from wood of Artocarpus heterophyllus

In our efforts to find new whitening agent from natural resources, we focused on wood of Artocarpus heterophyllus which shows anti-melanogenesis activity. By activity-guided fractionation of A. heterophyllus wood extract, a new prenylated flavonoid, 3-prenyl luteolin (1) was isolated. The IC₅₀ of mushroom tyrosinase inhibitotry activity of 1 was 76.3 µM. The results of the comparison with that of luteolin showed the prenyl substituent at C-3 position of 1 play an important role for revealing tyrosinase inhibition. In melanin formation inhibition on B16 melanoma cells, IC₅₀ of 1 was 56.7 µM with less cytotoxicity.     

Ontogenetic changes in water-use efficiency (δC) and leaf traits differ among tree species growing in a semiarid region of the Loess Plateau,China

On the Loess Plateau, China, several planted tree species such as Populus hopeinsis and Robinia pseudoacacia suffer occasional diebacks in the top shoots, reducing growth rates after maturation. However, this does not usually occur in other species, e.g., Ulmus pumila and Zizyphus jujuba. We compared stable carbon isotope ratios (δ¹³C) as indicators of leaf water-use efficiency (WUE), leaf mass per unit area (LMA), and leaf area- and mass-based nitrogen content (N_area and N_mass) by tree height (1, 5, and 10 m) in P. hopeinsis, R. pseudoacacia, U. pumila, and Z. jujuba. In P. hopeinsis and R. pseudoacacia, leaf δ¹³C and N_area were significantly lower in 10-m trees than in 1-m saplings, indicating that leaf WUE and photosynthetic rates of both species decreased with tree height. In contrast, δ¹³C in Z. jujuba varied little with tree height. The δ¹³C of 10-m-tall U. pumila trees was significantly higher than that of 1-m plants, demonstrating an increase in WUE with tree height. Decreasing WUE, leaf N_area, and N_mass with height increases in P. hopeinsis and R. pseudoacacia may be related to water and nutrient limitations for these species in semiarid regions. In contrast, stable or increasing δ¹³C with tree height in U. pumila and Z. jujuba may account (to some extent) for successful production of these species under identical environmental conditions. Diameter growth rate also decreased with maturity in P. hopeinsis and R. pseudoacacia, but increased or was stable for U. pumila and Z. jujuba. The differences in leaf WUE and LMA among species with tree maturity may be related to species’ growth patterns and susceptibility to drought stress, and are likely to be important new criteria for plantation species selection on the Loess Plateau.     

Expression of HIF-1α ODD domain fused canine caspase 3 by EGFR promoter-driven adenovirus vector induces cytotoxicity in canine breast tumor cells under hypoxia

Adenovirus (Ad) vectors are widely used in cancer gene therapies. However, compared to human patients, relatively limited information is available on gene transduction efficiency or cell-specific cytotoxicity in canine tumor cells transduced with Ad vectors. Since epidermal growth factor receptor (EGFR) is highly expressed on canine breast tumor cells, we sought to develop an Ad vector based on the RGD fiber-mutant adenovirus vector (AdRGD) that expresses canine caspase 3 under the control of EGFR promoter. The aims of this study were to achieve high transduction efficiency with transgene expression restricted to canine breast tumor cells. Using EGFR promoter-driven AdRGD, we were able to restrict transgene expression to canine breast tumor cells with no evidence of expression in normal cells. Canine breast tumor cells transduced with EGFR promoter-driven AdRGD carrying canine caspase 3 gene showed cytotoxic activity. We constructed a second AdRGD vector that expressed oxygen-dependent degradation (ODD)-caspase 3 under the control of the EGFR promoter; the fusion protein contains a core part of the ODD domain of hypoxia inducible factor-1 alpha (HIF-1α) fused to caspase 3. Transduction of canine breast tumor cells with EGFR promoter-driven AdRGD expressing ODD-caspase 3 induced a higher rate of cell death under hypoxic conditions compared with under normoxia. The results indicate that the EGFR promoter-driven AdRGD vectors will be of value for tumor-specific transgene expression and safe cancer gene therapy in dogs.     

Anti type I allergic property of Japanese butterbur extract and its mast cell degranulation inhibitory ingredients

Pollenosis is a disease that affects 1 in 10 of the Japanese population. During the season of cedar pollen dispersal, many patients suffer from symptoms such as sniffling, sternutation, and itching of the eyes. Japanese butterbur is a popular vegetable and is one of the few domestic vegetables in Japan. The anti type I allergic effects of an aqueous ethanol extract from aerial parts of Japanese butterbur (JBE) were evaluated in rats and RBL-2H3 mast cells. In the passive cutaneous anaphylaxis reaction in rats, a single oral treatment of JBE (1000 mg/kg) was found to suppress the reaction. In IgE-sensitized RBL-2H3 cells, JBE (10-100 microg/mL) inhibited beta-hexosaminidase release, leukotriene C(4)/D(4)/E(4) synthesis, and TNF-alpha production. Moreover, a high concentration of JBE (1000 microg/mL) suppressed smooth muscle constriction induced by histamine (10 microM) and leukotriene D(4) (10 nM) in a guinea pig trachea strip. The search for components in JBE with an inhibitory activity on mast cell degranulation was guided by inhibition of beta-hexsosaminidase release. Two eremophilane-type sesquiterpenes, six polyphenolic compounds, and two triterpene glycosides were isolated. Of these compounds, fukinolic acid, a principal polyphenol constituent, showed potent inhibitory activity (IC(50) value = 2.1 microg/mL). Consequently, On the basis of its inhibition of mast cell activation and direct smooth muscle reaction induced by released mediators, JBE was found to suppress the type I allergic reaction.     

Isolation of pathogenicity- and gregatin-deficient mutants of <Emphasis Type="Italic">Phialophora gregata</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis> through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation

Phialophora gregata f. sp. adzukicola, a causal agent of brown stem rot in adzuki beans, produces phytotoxic compounds: gregatins A, B, C, D, and E. Gregatins A, C, and D cause wilting and vascular browning in adzuki beans, which resemble the disease symptoms. Thus, gregatins are considered to be involved in pathogenicity. However, molecular analyses have not been conducted, and little is known about other pathogenic factors. We sought to isolate nonpathogenic and gregatin-deficient mutants through Agrobacterium tumefaciens-mediated transformation (ATMT) for cloning of pathogenicity-related genes. The co-cultivation of P. gregata and A. tumefaciens for 48 h at 20°C with 200 μM acetosyringone resulted in approximately 80 transformants per 10⁶ conidia. The presence of acetosyringone in the A. tumefaciens pre-cultivation period led to an increase in T-DNA copy number per genome. Of 420 and 110 transformants tested for their pathogenicity and productivity of gregatins, one nonpathogenic and three gregatin-deficient mutants were obtained, respectively. The nonpathogenic mutant produced gregatins, whereas the gregatin-deficient mutants had pathogenicity comparable to the wild-type strain. This is the first report of ATMT of P. gregata. Further analysis of these mutants will help reveal the nature of the pathogenicity of this fungus including the role of gregatin in pathogenesis.     

Immunoperoxidase staining characteristics of Dirofilaria immitis in the dog

The immunoperoxidase staining characteristics of Dirofilaria immitis and pulmonary tissues from infected dogs were studied by using the following sera: anti-fresh D immitis, anti-processed D immitis, anti-dog IgG, anti-dog IgG Fc, anti-dog IgM and anti-dog C3. Marked staining was observed using anti-fresh D immitis serum. Body cavity fluid and cuticle were strongly stained and hypodermis, muscle, lateral cord, testis, vas deferens, ovary, oviduct and uterus were moderately stained. Oesophagus and intestine were mildly stained. Degenerate worms were stained by all antisera. The intact and cut surfaces of microfilariae and eggs and sperm present in filariae were stained, but not their internal contents. Circulating and stored immotile microfilariae did not stain. Excreted eggs, presumed to be unfertilized and, or, degenerate, stained positively. Immunoperoxidase staining of routinely processed histological samples provides a means of assessing D immitis antigen.