Manipulated Mouse Embryos as Bioassay System for Water Quality Control

Mouse pronuclear stage embryos with intact slit zona pellucida (manipulated) were cultured in vitro until the hatched blastocyst stage in simplex optimized medium with higher K⁺ concentration (KSOM) prepared with three different water types: tap, deionized reverse osmosis (D‐O) water and Milli‐Q system (M‐Q) water. The culture media were supplemented with or without protein and ethylenediaminetetraacetic acid (EDTA, disodium salt). The rates of hatched blastocysts were significantly affected (p < 0.01) by micromanipulation, protein supplement and water source. The water source has no influence (p > 0.05) on development in EDTA‐supplemented protein‐free culture media, whereas in EDTA‐ and protein‐free culture media, the water quality significantly (p < 0.001) affected the rates of development, with higher rates in media prepared with M‐Q water. The micromanipulated embryos showed higher sensitivity to the water quality (p < 0.01). It worth mentioning that the rates of hatched blastocysts in protein‐free culture media were very low (0–7.5%). Furthermore, the three different water types were analysed by measuring the electrical conductivity, inorganic ions, total organic carbon and endotoxins to evaluate the purity. M‐Q water showed the lowest levels of inorganic ion, total organic carbon and endotoxin concentrations. We concluded that manipulated mouse embryos are good system to evaluate the quality of water used in biological system.     

Development and characterization of monoclonal antibodies against canine trypsin

Canine cationic trypsin was purified by salting-out, gel filtration and affinity chromatography. Purity was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was ca. 28 kDa by SDS-PAGE.

Thirty hybridomas were obtained which produced mAb to canine cationic trypsin by the cell fusion technique. Twenty-two of these recognized cationic trypsin only, while eight hybridomas recognized both cationic and anionic trypsin. Several of the anti-canine cationic trypsin mAb were purified by salting-out and DEAE ion-change chromatography using ascites fluid of immunized BALB/c mice. The mAb proved to have very high specificity to canine cationic trypsin as shown by immunoblotting and it may be possible to use them to develop clinical assays.     

Oxidative damage to the membrane of canine erythrocytes with inherited high Na, K-ATPase activity.

Oxidative damage to the membrane in canine erythrocytes with inherited high Na, K-ATPase activity (HK cells) was compared with that in normal canine cells (LK cells). When 30 mM beta-acetylphenylhydrazine (APH) was applied to HK and LK cells, lipid peroxidation and hemoglobin denaturation occurred. Lipid peroxidation determined from malondialdehyde (MDA) formation was significantly lower in HK than in LK cells so far as endogenous glutathione (GSH) concentration was maintained at appropriate levels. With the depletion of GSH, MDA formation was accelerated and difference between HK and LK cells was not significant. Denatured hemoglobin bound to the membrane protein was less in HK than in LK cells. During incubation with APH, osmotic fragility increased markedly in LK cells, while HK cells showed very little change. The amounts of total lipid, total and free cholesterol, glycolipid, phospholipid and fatty acids were essentially the same in both cell types. Fatty acid compositions showed very small differences. The membrane of HK cells thus appear to have greater protection against oxidative damage induced by APH, owing to the presence of excess GSH in HK cells. The capability of HK cells to withstand oxidative damage would not be due to differences in membrane lipid compositions.     

In vitro binding of germanium to proteins of rice shoots

The possibility of in vitro binding between proteins of rice shoots and germanium (Ge) was investigated. The proteins in mixtures of aqueous extracts of rice shoots and radioactive germanium (⁶⁸GeO₂) were fractionated. The binding of radioactivity to the proteins was observed even after 5 successive fractionation steps from the original mixtures. At the final fractionation step using polyacrylamide gel electrophoresis, a constant proportionality between protein concentration and associated radioactivity was found in most samples although not all. These results indicate that the binding of ⁸⁸Ge to proteins is not due to the simple adsorption by proteins.     

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.     

Characteristic of Inflammatory Airway Disease in Japanese Thoroughbred Racehorses

Inflammatory airway disease (IAD) is a common cause of poor performance, interruption of training and premature retirement in racehorses. It is also reported that up to 80% of horses are affected at some point in the first years of training in UK and Australia. However, no studies with regard to the information on occurrence of IAD in Japanese Thoroughbred racehorses have been reported. To investigate the occurrence and the characteristics of IAD, epidemic research including endoscopic examination of the airway tract and trachea wash was conducted for Thoroughbred racehorses presenting coughs or poor performance which airway tract disease was suspected stalled in training facility managed by Japan Racing Association. Fifty-six out of 76 Thoroughbred racehorses (73.7%) presenting coughing or poor performance were diagnosed as IAD. Mean incidence rate of IAD was 0.3% and it has been confirmed that constant number of IAD exists in Japan. Up to 35.7% of IAD horses showed upper airway abnormalities in some extent. There was a trend for IAD horses to use wood shavings for bedding and fed hay from the ground compared with the control group. Therefore, improvement of stabling environment may aid in preventing IAD. This study demonstrated that Japanese Thoroughbred racehorses are affected by IAD likewise other countries as well as demonstrated the characteristics of IAD which may contribute to the clarification of the pathogenesis of IAD.