Isolation and monolayer culture of bovine oviduct epithelial cells

Oviduct epithelia obtained from 32 cows were cultured. The oviducts were classified into follicular and luteal phases and divided into ampulla and isthmus regions. The epithelial cells were dissociated by enzyme digestion and cultured in plastic dishes with Dulbecco's modified Eagle's medium/Ham's F12 (1:1) containing 10% calf serum. After enzyme treatment, the epithelial suspension showed free ciliated and non-ciliated cells, and cell mass. The non-ciliated cells contained secretory granules in the cytoplasm. The cell mass was composed of ciliated and secretory cells. The cell mass adhered to the dish within 12-24 hr, while the free ciliated cells attached on Day 2 of the culture. The cells grew into confluent monolayers on Day 4. The cell monolayers contained ciliated and non-ciliated cells. The monolayered non-ciliated cells showed a few secretory granules. When the cells were further cultured without subculturing, ciliary activity diminished on Day 5 and was rarely detected on Day 9. When the cells were subcultured on Day 3, ciliary movement was detected on the monolayers for only 2 days. Cell mass that did not adhere to the dish and remained floating in the medium formed ball-like structures on Day 2. Active ciliary beating was observed on the cells that were cultured in the medium supplemented with 10(-5) and 10(-9) M estradiol-17 beta, however, the ciliary activity diminished on Day 5. No difference in the cell growth was observed between the follicular and luteal phases or between the ampulla and isthmus regions.     

Immunohistochemical analysis of pseudorabies virus spread through neurons innervating the eyeball

Pseudorabies virus (PRV) was inoculated intraocularly into BALB/c mice, and the distribution pattern of cells positive for several neurotransmitters and viral antigens in the eyeball, trigeminal nerve ganglia, and superior cervical ganglia was examined immunohistochemically to clarify the neural route of the virus spread. In the eyeball, substance P (SP)- and calcitonin gene-related peptide (CGRP)-positive cells were detected in the ipsilateral iris and ciliary body, neuropeptide tyrosine (NPY)-positive cells were detected in the choloid membrane, and tyrosine hydroxylase (TH)-positive cells were detected in the ipsilateral inner nuclear layer of the retina; all these cells contained viral antigens. In the superior cervical ganglia, viral antigen-positive cells containing TH or NPY were found at bilateral sites. In the trigeminal nerve ganglia, viral antigen-positive cells containing SP or CGRP were found at bilateral sites. These findings indicated that the SP- and CGRP-positive ganglion cells of the trigeminal nerve ganglia innervating the iris or ciliary body, and the NPY-positive ganglion cells of the superior cervical ganglia innervating the iris, ciliary body, and choroid membrane served as the route for the virus spread. These findings also suggested that dopaminergic neurons, such as the TH-positive retinal cells and TH-positive ganglion cells of the superior cervical ganglia, served as the route for virus spread.     

Radiocarbon Content in Urban Atmospheric Aerosols

We attempted to elucidate seasonal variations in fossil-fuel-derived carbon (FC) and biomass-derived carbon (BC) in urban atmospheric aerosols. We undertook continuous measurements of the composition of fine particle (PM_2.1) in central Tokyo, including the ¹⁴C/¹²C ratio. The percent modern carbon (pMC) contained in all samples averaged 43, and the highest was 54 in late December and the lowest was 31 in early August. From the observed carbonaceous component concentrations and the pMC we could calculate the content ratio of FC and BC in PM_2.1 and investigate their seasonal variations. Although there was almost no seasonal variation in the ratio of FC, the ratio of BC was observed to rise in early winter. This indicates that FC is influenced by the emission sources without seasonal variations (such as automobiles driven in urban areas). Furthermore, there is significant correlation between BC and organic carbon (OC), and even for urban areas, it is considered that the contribution of biomass carbon to OC in PM_2.1 is high.     

Improving the antibacterial activity against Staphylococcus aureus of composite sheets containing wasted tea leaves by roasting

We used various kinds of wasted tea leaves to develop composite sheets with antibacterial properties. Antibacterial tests showed that the number of viable bacterial cells for the sheet containing wasted green tea leaves was around 10:⁶–10⁷ CFU/ml after 18 h culture compared to 108 CFU/ml for a tea-free sheet. This indicates that cell growth was signifi cantly inhibited. For sheets containing other types of tea leaves (oolong, black, hojicha, and pu-erh), living cells were not found, indicating that these sheets had superior antibacterial effects. Living cells were also not found in sheets containing wasted black tea leaves or roasted tea leaves at a concentration of 60% by weight after 6 h cultivation. Therefore, roasting treatment of wasted green tea leaves was examined to improve the antibacterial activity of the sheet. In particular, the focus was on structural conversion of catechins by heating.     

Seasonal stream habitat of adult Sakhalin taimen,Parahucho perryi,in the Bekanbeushi River system,eastern Hokkaido,Japan

Behavioural tracking of endangered adult Sakhalin taimen (Parahucho perryi) by acoustic telemetry was conducted in the Bekanbeushi River system that flows through eastern Hokkaido in 2008–2010. A total of 39 tracked P. perryi showed extensive use of the river system, both upstream and downstream, regardless of the month or year. No tracked adult P. perryi stayed in the ocean for more than 1 day. Some of the fish (20%) dwelled in upstream habitat consistently from spring to autumn, while 40% of individuals showed wide utilisation of the entire upstream and downstream systems. Although 39.4% of tracked fish utilised two or more primary tributaries to the main river, 6.1% used only the main river system. Distinct variability was observed between individuals based on the proportion of time spent in each stream reach (up‐, mid‐, and downstream). A trend was observed in 2008 and 2010 showing that tracked fish moved to the upper stream reaches when water temperatures downstream increased. This trend was not observed in 2009 when the summer water temperature downstream was more than 2 °C below average when compared to 2008 and 2010. Our results suggest that adult P. perryi in the Bekanbeushi River system exhibit high behavioural plasticity, that is, the fish can select habitats based on their own individual requirements. Our results also demonstrate the importance of physical continuity among river reaches both upstream and downstream such that P. perryi does not experience barriers in moving to optimal habitats.     

Restriction endonuclease analysis of canine herpesviruses isolated in Japan

Eighteen canine herpesvirus (CHV) isolates from Japan and two reference strains were compared by restriction endonuclease analysis technique using total DNA extracts from cells infected with the viruses. In order to select the suitable restriction endonucleases for differentiation of CHV isolates, ten enzymes were used and three of them, HindIII, XbaI, and PvuII, were found to be useful for strain differentiation. With these enzymes, CHV isolates from unrelated individuals were readily differentiated from each other. In contrast, all the isolates derived from the same litter were not distinguishable on the basis of restriction cleavage patterns. However, slight mobility shifts were observed among the isolates from the same litter or the same individual. The results showed that this method provides a powerful tool for epidemiological surveys of CHV infection.     

An enzyme-linked immunosorbent assay for detection of antibodies to exogenous avian leukosis virus

A microplate enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to avian leukosis virus (ALV) of subgroups A and B in infected chickens was developed with the use of Rous-associated virus (RAV)-1 (subgroup A) and RAV-2 (subgroup B) antigens purified by sucrose-gradient centrifugation. The antigen was used for ELISA after treatment with Triton X-100. In the ELISA, the subgroup viral antigen reacted strongly with homologous antiserum but also reacted with heterologous antiserum. Tests with serum absorbed with purified homologous and heterologous virus and tests for antigen-blocking by group-specific antibodies to ALV revealed that the reaction was caused mainly by subgroup-specific antibodies. The ELISA was 8 to 32 times more sensitive than the virus-neutralization (VN) test and detected antibodies to ALV earlier than the VN test in chickens infected experimentally with RAV-1 and RAV-2. In field application of the ELISA, 44.2% of 484 chicken sera were positive for RAV-1 and/or RAV-2 antigen, and 80.4% of flocks were positive. These findings indicate that ELISA is superior to the VN test in sensitivity, simplicity, rapidity, and applicability for large-scale field surveys for ALV infection.