Transfer of the Rsv3 locus from ‘Harosoy’ for resistance to soybean mosaic virus strains C and D in Japan

Resistance to soybean mosaic virus (SMV) is imperative for soybean (Glycine max (L.) Merr.) production in the Tohoku region. Molecular markers for SMV resistance were previously reported for U.S. SMV strains, but they cannot be applied because of the differences in strain classification between Japan and the U.S. A U.S. variety ‘Harosoy’ has been used mainly as a donor of resistance to SMV strains C and D in a Japanese breeding program, resulting in resistant varieties such as ‘Fukuibuki.’ Because ‘Harosoy’ harbors the Rsv3 gene conferring resistance to the virulent SMV strain groups, G5 through G7, it appears that the Rsv3 gene confers resistance to strains C and D. In this study, we introduced resistance to the two strains from ‘Fukuibuki’ into a leading variety ‘Ohsuzu’ by recurrent backcrossing with marker-assisted selection. All lines selected with markers near Rsv3 showed resistance to the strains, suggesting that the Rsv3 locus is responsible for the resistance. Three years of trials showed that one of the breeding lines, ‘Tohoku 169,’ was equivalent to ‘Ohsuzu’ with respect to agricultural characteristics such as seed size, maturity date, and seed yield, except for the SMV resistance.     

Immunohistochemical localization of enzymes related to lignin biosynthesis in the primary xylem of hybrid aspen

We have investigated the spatial regulation of the accumulation of enzymes involved in the biosynthesis of shikimate and lignin during differentiation of primary xylem from the apical meristem via procambium in hybrid aspen (Populus sieboldii x Populus grandidentata). Immuohistochemical staining revealed that, in the top part of shoots, lignification began in a single or just a few adjacent vessel elements and subsequently spread to neighboring cells. The spatial localization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS), which is one of the key enzymes in the shikimate pathway, was tightly correlated with the cell-specific deposition of lignin in the primary xylem. We also found that the spatial localization of enzymes in the general phenylpropanoid pathway and in the lignin-specific pathway was closely associated with the cell-specific deposition of lignin and the accumulation of DAHPS. Our data suggest that enzymes that act in the shikimate, general phenylpropanoid, and lignin-specific pathways are initially produced and function coordinately in a single or a few adjacent elements at the start of primary xylem development.     

Technique for screening immune-enhancing polysaccharides in food using 1,25-dihydroxyvitamin D3-differentiated HL60 cells

A technique for screening immune-enhancing polysaccharides in food using the phagocytotic activity of 1,25-dihydroxyvitamin D3 (VD3)-differentiated HL60 cells is presented. HL60 cells, a human acute promyelocytic cell line, can differentiate along the monocytic lineage following exposure to VD3 or phorbol-12-myristate-13-acetate (PMA). For differentiated cells along the monocytic pathway, HL60 cells were maintained in RPMI 1640 medium supplemented with 10% FBS and 120 nM VD3 for more than 1 week. VD3-differentiated HL60 cells were seeded into 48-well plates, YG-labeled microspheres and polysaccharides were added and mixed using a plate shaker at 1100 rpm for 30 s, and then the mixture was incubated overnight at 37 degrees C in 5% CO2. The cells were fixed with 2% formaldehyde and resuspended in phosphate-buffered saline. The rate of phagocytosis was measured with a flow cytometer. VD3-differentiated cells but not non- and PMA-differentiated cells resulted in an elevation of phagocytic activity by various immune-enhancing polysaccharides in foods.     

2'-epi-orobanchol and solanacol, two unique strigolactones, germination stimulants for root parasitic weeds, produced by tobacco

Germination stimulants for root holoparasitic weeds broomrapes ( Orobanche and Phelipanche spp.) produced by tobacco ( Nicotiana tabacum L.) were purified and characterized. The root exudates of tobacco contained at least five different stimulants, and LC-MS/MS analyses revealed that four of them were strigolactones; a tetradehydrostrigol isomer, a didehydrostrigol isomer, and two strigol isomers. The two isomers of strigol were identified as (+)-orobanchol and its 2'-epimer by comparison of NMR and GC- and LC-MS data with those of synthetic standards. The structure of the tetradehydrostrigol isomer, the major stimulant of the bright yellow tobacco cultivars, was determined as 4-alpha-hydroxy-5,8-dimethyl-GR24 [( E)-4-alpha-hydroxy-5,8-dimethyl-3-(4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)methylene)-3a,4-dihydro-3 H-indeno[1,2- b]furan-2(8b H)-one] and named solanacol. 2'-Epi-orobanchol and solanacol are the first natural strigolactones having a 2'-epi stereochemistry and a benzene ring, respectively.     

Establishment and characterization of a cell line, MCO-Y4, derived from canine mammary gland osteosarcoma

A cell line, MCO-Y4, was established from a mammary gland osteosarcoma of a 16-year-old female mongrel dog. Histopathologically the tumor was composed of osteoblastic cells with an osteoid meshwork and chondroid matrix. The mean doubling time of the cells at the 93rd passage was 32.39+/-4.66 hr. Immunohistochemically, the osteoblastic and chondroblastic cells were positive for bone morphogenetic protein (BMP)-2/4 and BMP receptor (BMPR) II. The cultured cells were spindle in shape during the growth and the confluent phases. No tumor matrix was detected in the culture dish by alcian blue staining or von-Kossa silver impregnation. MCO-Y4 cells on the chamber slides showed intense immunoreactivity for BMP-2/4 and BMPR II. Noggin, an antagonist for BMP-2/4, showed the growth inhibition on MCO-Y4 cells. In addition, fibronectin might be potential for stimulating growth of MCO-Y4 cells. When transplanted into severe combined immunodeficiency mice, the cells formed tumors consisting of solid proliferation of osteoblastic and fibroblastic cells with woven-bone trabeculae. These tumor cells were intensely positive for BMP-2/4 and BMPR II. Our results suggested that the cell line might be useful for studying the role of BMPs in canine osteosarcoma and the mechanism of ossification.     

Cytokinin signaling and its inhibitor AHP6 regulate cell fate during vascular development

The cell lineages that form the transporting tissues (xylem and phloem) and the intervening pluripotent procambial tissue originate from stem cells near the root tip. We demonstrate that in Arabidopsis, cytokinin phytohormones negatively regulate protoxylem specification. AHP6, an inhibitory pseudophosphotransfer protein, counteracts cytokinin signaling, allowing protoxylem formation. Conversely, cytokinin signaling negatively regulates the spatial domain of AHP6 expression. Thus, by controlling the identity of cell lineages, the reciprocal interaction of cytokinin signaling and its spatially specific modulator regulates proliferation and differentiation of cell lineages during vascular development, demonstrating a previously unrecognized regulatory circuit underlying meristem organization.     

Ethanol Does Not Promote MeIQx-initiated Rat Colon Carcinogenesis Based on Evidence from Analysis of a Colon Cancer Surrogate Marker

Epidemiological studies suggest that alcohol consumption increases the risk of developing colorectal cancer. However, the data are confounded by numerous cosegregating variables. To cast further light on the relationships between alcohol intake and colon cancer development, 21-day-old male F344/DuCrj rats were fed 200 ppm 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in their diet for 8 weeks and doses of 0, 0.1, 0.3, 1, 3, 10 and 20% of ethanol in their drinking water ad libitum for 16 weeks thereafter. The rats were sacrificed after 24 weeks of experiment, and aberrant crypt foci (ACF), surrogate lesions for colon cancer, were examined under a light microscope at low magnification. Ethanol was found not to affect the ACF formation at any dose compared with the initiated-controls. Furthermore, ethanol did not alter colon epithelial cell proliferation. These data, obtained by analysis of a colon cancer surrogate marker lesion, indicate that ethanol lacks promotion activity for MeIQx-initiated rat colon carcinogenesis.     

Equine pyoderma associated with malnutrition and unhygienic conditions due to neglect in a herd

Twelve horses kept at a riding club suffered from pyoderma. All the horses displayed crusting, scaling and alopecia. The lesions were distributed in the chest, back, rump and limbs. Some of the horse patients also showed epilation with an attached crust similar to a 'paintbrush lesion' of dermatophilosis, but normal skin flora or opportunistic pathogenic bacteria were only isolated from the lesions. Some patients clearly showed weight loss, anemia and low levels of serum protein and cholesterol. General condition and skin lesions of the patients were improved gradually with improvement of feed and environment after being moved to new stables. Malnutrition under conditions of poor hygiene and poor management due to neglect might be associated with these equine cases of pyoderma in the herd.     

Sex identification of Japanese black bear,Ursus thibetanus japonicus,by PCR based on amelogenin gene

A method for sex identification of the Japanese black bear was examined using a polymerase chain reaction (PCR) and sequencing of a part of the amelogenin gene. This gene is located on the X and Y chromosomes, and there are 54 nucleotide deletions on the Y chromosome-specific gene. Forty-seven (26 male and 21 female) DNA samples and 23 (13 male and 10 female) DNA samples, respectively extracted from white blood cells and hairs of Japanese black bears were analyzed. The primers SE47 and SE48 from this X-Y homologous region were used in sex identification by PCR amplification. These primers amplified X- and Y-specific bands, which could be used to discriminate between sexes by a length polymorphism in all samples. We suggest that PCR amplification using the primers SE47 and SE48 is useful for sex determination of the Japanese black bear and could be applied to DNA analysis of small samples such as hairs.     

Establishment of a canine lens epithelial cell line

Some immortalized lens epithelial cell lines have been established and are useful for molecular analysis. The establishment of additional cell lines must, however, enable a variety of in-vitro examinations. The objective of this study was to establish a new canine lens epithelial cell line by isolating CLC-1 cells from the lens tissue of a dog with cataracts. In CLC-1 cells, transforming growth factor beta (TGF-β) treatment significantly decreased gene expression of an epithelial marker and elevated that of mesenchymal markers; these characteristics are similar to those of a human lens epithelial cell line. Interestingly, CLC-1 cells exhibited lower expression of an epithelial marker and higher expression of mesenchymal markers than an anterior lens capsule. These results suggest that CLC-1 cells were derived from a cell population that was committed to epithelial-mesenchymal transition in cataract lens tissue. In conclusion, CLC-1 cells could be useful for analyzing molecular pathogenesis in canine cataracts.