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.     

Effects of amino nitrogen on fermentation parameters by mixed ruminal microbes when energy or nitrogen is limited

Ruminal microbes harvested from a ruminally fistulated cow were incubated in simple batch and semicontinuous cultures with NH₃‐N or amino‐N on nitrogen‐ or energy‐excess diets in quantity (HN and LN diets, respectively, consisting of timothy hay plus soybean meal, or corn grain), based on evaluation with the National Research Council and Cornell Net Carbohydrate and Protein System models. In a batch culture experiment, supplementation with amino‐N promoted digestion and fermentation in the course of incubation (4–24 h) on both diets, but these effects mostly disappeared when the diets were sufficiently digested (at 48 h). In a semicontinuous culture experiment using Rusitec, no effect of amino‐N was exhibited after sufficient fermentation and digestion, but significant promotion of digestion was shown in the course of incubation on the HN diet, while no such effect was detected on the LN diet. The microbial yield for 24 h did not show a significant difference between the N sources of either of the two diets. These results suggest that the stimulatory effects of amino‐N are diminished when the diets are sufficiently digested after a long retention and incubation, and also that the effectiveness of amino‐N does not require a quantitatively energy‐excess status.     

Immunohistological distributions of fibronectin, tenascin, type I, III and IV collagens, and laminin during tooth development and degeneration in fetuses of minke whale, Balaenoptera acutorostrata.

The immunohistological distributions of fibronectin, tenascin, type I, III and IV collagens, and laminin were observed in the tooth buds of fetuses of minke whale, Balaenoptera acutorostrata. Distributions of extracellular matrices (ECMs) examined in this study except for tenascin were generally similar to those of terrestrial mammalian species during development of the tooth bud. Tenascin in the fetuses of minke whale showed characteristic distributions in the dental lamina and the enamel organ in the early tooth developmental stage. In the physiological degeneration stage of tooth bud development, immunoreactivity of the ECMs were very weakly and limitedly detected in the dental papilla and the surrounding mesenchyme. Immunoreactivity of tenascin and type I and III collagens were positively detected in the developing baleen plate germ which was associated with the degenerating tooth bud. These findings suggested that expressions of the ECMs were related to the formation of the tooth bud and baleen plate germ, and that the lack of the ECMs was related to the degeneration of the tooth bud in the fetal minke whale.     

Daily incremental lines in sika deer (Cervus nippon) dentine

This work was designed to observe the dentine incremental lines of the sika deer (Cervus nippon) fawns and to investigate their periodicity using the chronological labeling method with fluorochromes. The incremental lines were observed in decalcified specimens stained by Bodian's silver technique, and the fluorescence-labeled lines were observed in undecalcified and ground specimens. In the silver stained specimens, there were two types of lines, deeply stained thick lines and faintly stained minute regular incremental lines. The intervals and staining intensities of the deeply stained thick lines were very similar to those of the fluorescence-labeled lines in the ground specimens obtained from the same tooth, and hence, it appeared that the both lines were identical. The number of minute incremental lines between the deeply stained thick lines was the same as that of days between the time when each fluorescent labeling injection was made. Therefore, it seemed that each minute incremental line was formed each day. The possibility of age estimation in days using diurnal dentine increments was discussed.     

Isolation of myxoviruses from migratory waterfowls in San-in district,western Japan in winters of 1997-2000

Between November 1997 and February 2000, winter migratory waterfowls of several species staying in San-in district, western Japan were surveyed for influenza A virus and paramyxovirus at four stations. A total of 18 influenza A viruses was isolated from 1,404 fecal samples of whistling swans, pintails, mallards, and white-fronted geese. Five different hemagglutinins and eight neuraminidases were identified in the viruses isolated, in 11 different combinations, including H7N8 related to a subtype of a highly pathogenic chicken virus. In 2000, five lentogenic (non-pathogenic) Newcastle disease viruses were also isolated from white-fronted geese. These results suggested that possible precursor viruses for highly pathogenic avian myxoviruses are still brought into Japan by migratory waterfowls. The results also support the contention that continued surveillance of wild waterfowl population should be an integral part of control policies for these serious poultry diseases.     

Immunohistochemical localization of steroidogenic enzymes in corpus luteum of wild sika deer during early mating season

We analyzed the localization of steroidogenic enzymes (P450 scc, 3 beta HSD, P450 arom and P450 c17) in the corpora lutea of two Hokkaido sika deer (Cervus nippon yesoensis) during the early mating season. Two corpora lutea were found in each female and the timing of formation of the corpora lutea seemed different. P450 scc, and 3 beta HSD, positive luteal cells were found in both corpora lutea. The existence of two functional corpora lutea from the early mating season through pregnancy suggests that progesterone secreted by two or more corpora lutea is necessary for maintenance of pregnancy in sika deer.     

Improved survival of vitrified in vivo-derived porcine embryos

An efficient cryopreservation protocol for porcine morulae was investigated with three types of vitrification having different cooling rates (Exp. 1). Survival of embryos vitrified after removal of cytoplasmic lipid droplets was also examined by means of the minimum volume cooling (MVC) method (Exp. 2). In Exp. 1, the morula stage embryos were vitrified with a 0.25 ml plastic straw (ST-method), gel loading tip (GLT-method) and the MVC-method, respectively, and stored in liquid nitrogen after which they were warmed in sucrose solutions with cryoprotectants being subsequently removed in a stepwise manner. In Exp. 2, morulae were centrifuged with 7.5 microg/ml cytocharasin B at 12000 x g for 20 min to polarize the cytoplasmic lipid droplets that were then removed from the embryos by micromanipulation (delipation). Both those delipated at the morula stage and the intact embryos at the morula to blastocyst stages were vitrified by the MVC-method. In vitro survival of the vitrified embryos was assessed in both experiments by culturing in NCSU-23 + 10% FCS for 48 h. In vitro developments of vitrified embryos after warming to blastocysts were 20% (6/30) for the ST-method, 39% (18/46) for the GLT-method, and 60% (26/43) for the MVC-method. Embryo survival was further improved by vitrification after delipation (95%, 35/37) compared to intact vitrified morulae (24/42, 57%, P<0.001) and blastocysts (23/31, 74%, P<0.05). Moreover, the number of cells in blastocysts (92 +/- 25) derived from the delipated-vitrified morulae was comparable to those derived from intact control non-vitrified embryos (103 +/- 31). Our results demonstrate that vitrified porcine morulae have the highest survival when using the MVC-method in conjunction with delipation.