Nucleocapsid-like structures of Ebola virus reconstructed using electron tomography

Electron tomography (ET) is a new technique for high resolution, three-dimensional (3D) reconstruction of pleiomorphic macromolecular complexes, such as virus components. By employing this technique, we resolved the 3D structure of Ebola virus nucleocapsid-like (NC-like) structures in the cytoplasm of cells expressing NP, VP24, and VP35: the minimum components required to form these NC-like structures. Reconstruction of these tubular NC-like structures of Ebola virus showed them to be composed of left-handed helices spaced at short intervals, which is structurally consistent with other non-segmented negative-strand RNA viruses.     

Enzyme-linked immunosorbent assay for detection of canine chromogranin A by use of immunological cross-reactivity of rabbit anti-bovine chromogranin A antibody

Bovine and canine chromogranin A were extracted and purified from each specie's adrenal glands. Isolated bovine 70 kDa protein showed 100% identity to bovine CgA reported previously, whereas isolated canine 68 kDa protein showed 83.3% identity to bovine CgA by the NH(2)-terminal amino acid sequence analysis. Rabbit antibody to purified bovine protein (CgA) was found to immunologically cross-reacted with purified canine protein (CgA). In sandwich ELISA with anti-bovine CgA, concentration-dependent curves were obtained ranging from 0.3 to 20 mug/ml for canine CgA. From these findings, sandwich ELISA with anti-bovine CgA is found to be useful to determine the concentration of canine CgA.     

Molecular characterization of the major outer membrane protein of Haemophilus somnus

The major outer membrane protein (MOMP) of Haemophilus somnus shows antigenic and molecular mass diversity that forms the basis of a preliminary grouping system for H. somnus strains. In this study, the gene encoding MOMP of H. somnus strain 8025 was cloned in three overlapping fragments by PCR techniques, and then sequenced. The gene consists of a 1164-bp open reading frame encoding a deduced 380-amino acid protein with a 19-amino acid signal sequence, giving a mature protein with a calculated molecular mass of 39,913 Da. Significant homology was found between MOMP and porin protein sequences of bacteria in Pasteurellaceae species. When expressed in Escherichia coli, the protein from the MOMP gene directed by the T7 promoter was identical in size (approximately 40 kDa) to native MOMP and reacted with MOMP-specific antibodies. Comparisons of the MOMP gene sequences from six unrelated strains of H. somnus to that of strain 8025 revealed that the genes of three MOMP type 1 strains were highly conserved with that of strain 8025 in length and sequence. However, two MOMP type 3c strains and one MOMP type 3a strain differed markedly from the MOMP of strain 8025 in their 3'-terminal halves. Their deduced MOMP amino acid sequences differed in sequence (3c, 80.5 and 82.7% identity; 3a, 62.4% identity) and in length (3c, 384 and 376; 3a, 316), indicating that the molecular differences are the basis of antigenicity and molecular mass differences of H. somnus MOMP. In the predicted MOMP secondary structure, the variable sequences primarily mapped to putative surface-exposed loops, and a variable and surface-exposed epitope of MOMP-specific antibody was identified in the seventh-largest loop. These findings are useful for understanding the structural and immunological characteristics of H. somnus.     

Transstadial and intrastadial experimental transmission of Ehrlichia canis by male Rhipicephalus sanguineus

The acquisition and transmission of rickettsial pathogens by different tick developmental stages has important epidemiological implications. The purpose of this study was to determine if male Rhipicephalus sanguineus can experimentally acquire and transmit Ehrlichia canis in the absence of female ticks. Two trials were performed where nymphal and male R. sanguineus were simultaneously acquisition fed on the same infected donor hosts, and transstadially or intrastadially exposed male ticks were fed on separate pathogen-free dogs as a test for transmission. A single-step p30-based PCR assay was used to test canine and tick hosts for E. canis infections before and after tick feeding. E. canis was detected after either intrastadial or transstadial passage in male ticks, the organism remained detectable in both tick groups after transmission feeding, and both tick groups transmitted the rickettsia to susceptible dogs. Infection of dogs via tick feeding resulted in milder clinical signs and lower antibody titers than intravenous inoculation of carrier blood, but further investigation is needed to understand the mechanisms responsible for this observation. These results demonstrate that male R. sanguineus can take multiple feedings, and that they can both acquire and transmit E. canis in the absence of female ticks. This tick development stage could be important in transmission of E. canis, and perhaps related pathogens, between vertebrate hosts under natural and experimental conditions.     

Inhibitory effects of Zingiber officinale Roscoe derived components on aldose reductase activity in vitro and in vivo

Ginger (Zingiber officinale Roscoe) continues to be used as an important cooking spice and herbal medicine around the world. Scientific research has gradually verified the antidiabetic effects of ginger. Especially gingerols, which are the major components of ginger, are known to improve diabetes including the effect of enhancement against insulin-sensitivity. Aldose reductase inhibitors have considerable potential for the treatment of diabetes, without increased risk of hypoglycemia. The assay for aldose reductase inhibitors in ginger led to the isolation of five active compounds including 2-(4-hydroxy-3-methoxyphenyl)ethanol (2) and 2-(4-hydroxy-3-methoxyphenyl)ethanoic acid (3). Compounds 2 and 3 were good inhibitors of recombinant human aldose reductase, with IC50 values of 19.2 +/- 1.9 and 18.5 +/- 1.1 microM, respectively. Furthermore, these compounds significantly suppressed not only sorbitol accumulation in human erythrocytes but also lens galactitol accumulation in 30% of galactose-fed cataract rat model. A structure-activity relationship study revealed that the applicable side alkyl chain length and the presence of a C3 OCH3 group in the aromatic ring are essential features for enzyme recognition and binding. These results suggested that it would contribute to the protection against or improvement of diabetic complications for a dietary supplement of ginger or its extract containing aldose reductase inhibitors.     

Genetic analyses of agronomic traits in Tartary buckwheat (Fagopyrum tataricum (L.) Gaertn.)

The consumption of products made from Tartary buckwheat (Fagopyrum tataricum (L.) Gaertn.) has increased in recent years in Japan. Increased consumer demand has led to recognition of the need for early varieties of this crop with high and stable yields. In order to accomplish this, more information is needed on the genetic mechanisms affecting earliness and yield. We conducted genetic analysis of 3 agronomic traits (days to flowering, plant height and total seed weight per plant) to segregate F₂ and F₃ populations derived from a cross between Tartary buckwheat cultivars ‘Hokuriku No. 4’ and ‘Ishisoba’. Broad-sense heritability estimates for days to flowering, plant height and total seed weight were 0.70, 0.62 and 0.75, respectively, in F₃ population. Narrow-sense heritability for total seed weight (0.51) was highest, followed by heritability for days to flowering (0.37), with heritability for plant height (0.26) lowest. Later flowering was associated with increased plant height and higher yields. From the F₄ generation, we identified twelve candidate plants with earlier maturity and reduced plant height compared to ‘Hokuriku No. 4’, but almost the same total seed weight. These results suggest that hybridization breeding using the single seed descent (SSD) method is an effective approach for improving agronomic characteristics of Tartary buckwheat.     

Traits of shattering resistant buckwheat ‘W/SK86GF’

Seed shattering is a significant problem with buckwheat, especially at harvesting time. Several reports have shown that a green-flower mutant of buckwheat, such as W/SK86GF, has a strong pedicel. Although a strong pedicel may provide some resistance to shattering in the field, no study has thoroughly examined this issue. In this paper, we demonstrate that a W/SK86GF has shattering resistance by comparing the degrees of shattering of W/SK86GF and Kitawasesoba (leading variety of Hokkaido with non-green-flower traits) through a test for four years, including a typhoon hit year in the field. In a non-typhoon year, the shattering seed ratio (shattering seed weight/(yield + shattering seed weight) × 100) of W/SK86GF at maturing time +15 days (+15D) was lower than that of Kitawasesoba. In a typhoon hit year, the shattering seed ratios of Kitawasesoba at maturing time and +15D were surprisingly high, 14.4 and 21.1%, respectively. On the other hand, those of W/SK86GF were only 3.08% and 2.57%, respectively; indicating W/SK86GF is promising as a shattering resistant line even in a typhoon hit year. From these results, shattering resistance of W/SK86GF can be evaluated after maturing time such as +15D and pedicel strength would confer W/SK86GF a shattering resistant trait.     

Seasonal and interannual variation in net ecosystem production of an evergreen needleleaf forest in Japan

Carbon dioxide (CO₂) flux was measured above the forest at the Fujiyoshida site on the northern slope of Mount Fuji in Japan in 2000?C2008 using an eddy covariance technique. The forest mainly consists of Japanese red pine (Pinus densiflora) and Japanese holly (Ilex pedunculosa). The 9-year average of monthly mean net ecosystem production (NEP) ranged from ?0.1?g?C?m^?2?day^?1 in January to 2.5?g?C?m^?2?day^?1 in May. The maximum net uptake was observed in May, although gross primary production (GPP) was highest in July. Variation in the leaf amount did not notably affect seasonal variation in GPP. This site was characterized by carbon uptake even in winter, if the meteorological conditions were conducive for photosynthesis and a resulting long period of carbon uptake. The 9-year averages of annual NEP, GPP, and ecosystem respiration (RE) were 388, 1,802, and 1,413?g?C?m^?2?year^?1, respectively. The annual NEP was lowest in 2003 and highest in 2004 over the 9?years. Year-to-year variability of NEP mainly depended on air temperature and photosynthetically active radiation in summer, and the dependence of the deviation of annual NEP on that of GPP was greater than that of RE. Long-term observational data indicated that the carbon uptake ability at the study site was at a moderate level in comparison with other temperate humid evergreen forests around the world. These data also indicated that the site had a high carbon uptake ability compared with other deciduous forests in Japan because of the duration of carbon uptake.     

Detection of γ-H2AX,a Biomarker for DNA Double-strand Breaks,in Urinary Bladders of N -Butyl- N -(4-Hydroxybutyl)-Nitrosamine-Treated Rats

To evaluate the potential role of DNA repair in bladder carcinogenesis, we performed an immunohistochemical analysis of expression of various DNA repair enzymes and γ-H2AX, a high-sensitivity marker of DNA double-strand breaks, in the urothelium of male F344 rats treated with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN), a bladder-specific carcinogen. Our results clearly demonstrated that γ-H2AX aggregation was specifically generated in nuclei of bladder epithelial cells of BBN-treated rats, which was not found in untreated controls or mesenchymal cells. γ-H2AX-positive cells were detected not only in hyperplastic and neoplastic areas but also in the normal-like urothelium after BBN treatment. These data indicate that γ-H2AX has potential as a useful biomarker for early detection of genotoxicity in the rat urinary bladder. To the best of our knowledge, this is the first report demonstrating expression of γ-H2AX during bladder carcinogenesis.