A high-precision Li isotope analysis was developed for determining the Li+ ion emitted from lithium phosphate as an ion source material by a Re double-filament ionization method in the thermal ionization mass spectrometry. In this method, Li isotopic fractionation is distinctly less sensitive to the filament temperature than those in the previous methods, and stable and high ion beam intensity of more than 8×10-11 A for 7Li is obtained. These advantages in determining the 7Li/6Li ratio result in the analytical reproducibility of 0.26‰ (1σ). Furthermore, the sample preparation is simple and the low temperature (850°C) required by this method ensures the analysis of the isotopic composition of small amounts of Li with less influence of Li contamination to the sample, compared with the previous methods.
Molecular and in vivo studies of renin-angiotensin system (RAS) have been developed by introducing new techniques.1) First, human renin was isolated and characterized after about half million-fold purification by introducing the new affinity column, pepstatin column. Secondly, molecular biology including recombinant DNA technology has made great progress on studies of renin and angiotensinogen as well as their genes. The cDNA of human renin precursor was isolated and the primary structure of human renin precursor was deduced from its nucleotide sequences. Genomic DNAs of human renin precursor and human angiotensinogen were also isolated and the structures of their exons and introns in addition to 5'- and 3'-flanking regions were determined. Thirdly, transgenic mice carrying both human renin and human angiotensinogen genes were produced as the first animal model for human hypertension which had simple and clear human genetic background. The hypertensive transgenic mice were named as “Tsukuba Hypertensive Mice”.
Duration of pupal stage of Bombyx mandarina is 2 to 4 times longer than that of Bombyx mori. We attempted to investigate whether this represents a state of diapause or not. Based on the pupal duration and oxygen consumption, developmental pattern of pupae in B. mandarina was mainly classified to three types, A, B and C, though these were dependent on the rearing seasons. In the spring season, typical type-A pupae had the shortest duration the same as in B. mori. Type-B pupae were intermediate of type-A and C. Pupae that had the longest duration were classified as type-C. Oxygen consumption decreased quickly soon after pupation and each type showed a typical U-shape respiration curve during pupal stage. Oxygen consumption in type-C animals was lowest of all 3 types. It reached extremely low levels of 5μl/hr/g, which went on for more than 2 weeks. When the type-A pupae were maintained in low temperature, pupal duration was elongated in proportion to the temperature decrease. However, in this experiment the respiratory activity curve in the lowest temperature of 20°C was clearly different from that of type-C pupae. In another experiment, type-A and B pupae showed increased respiration, proportionally to the temperature increase, while there was no increase in respiration of the C-type animals when subjected to the different temperatures. Finally, we can conclude that the low level of oxygen consumption in the C-type animals is characteristic to diapausing insects and this is in good accordance with the insensitivity of these animals to different temperatures. It was consequently determined that in B. mandarina there is a kind of facultative diapause in pupal stage.
In the silkworm, Bombyx mandarina, duration of pupal-adult development was mainly divided into three different types among the Japanese population: the first one was 2 weeks duration, the second was about 3 weeks and in the third type more than 30 to 40 days were necessary for the adult development, although these were dependent on the rearing seasons. However, the average number of days required for adult development was 14 in the majority of the Chinese population. In the third type of pupal-adult development of Japanese population, the development of pupal stage was arrested. Oxygen consumption of the animals of which adult eclosion occurred at 34 days after pupation was very low during the pupal development. Ovarian development of these animals was also suppressed considerably. The fluctuation patterns of ecdysteroid titer in haemolymph of Japanese population were quite different from that of the Chinese population. Ecdysteroid titer was undetectable at 4 to 13 days after pupation in the Japanese population, but the titer increased by day-15 and reached a maximum level of 4.5 μg at day-20. The reinitiation of ovarian development was coincident to the increase of ecdysteroids in haemolymph of these animals. It was suggested that the delay of pupal-adult development may be due to the low levels of ecdysteroids in the Japanese population of Bombyx mandarina.
Populations of Chinese origin of the silkworm, Bombyx mandarina, do not show arrested pupal stage, however populations of Japanese origin undergo pupal summer diapause. Hormonal control of this pupal summer diapause in Bombyx mandarina of Japanese origin was investigated using KK-42 treated pupae of Chinese origin. When an imidazole compound (KK-42:1-benzyl-5-[(E) 2, 6-dimethyl-1, 5-heptadienyl] imidazole) was injected into the newly ecdysed pupae of Chinese populations, the pupal-adult development was arrested for as long as 40 days. Resumption of pupal-adult development of the KK-42 treated pupae was induced by injection of 20-hydroxyecdysone (20-HE). Oxygen consumption of the KK-42 treated animals was very low comparing to the control but it increased gradually about 35 to 37 days after treatment. Abrupt increase of oxygen consumption was observed about 12hr after the application of 20-HE in the KK-42 treated animals. These results strongly indicate that the pupal summer diapause in Japanese populations of Bombyx mandarina is due to the low level of ecdysteroids in their body thus preventing them from proceeding to the pupal-adult development.
In order to elucidate the induction mechanism of summer diapause in Bombyx mandarina of the Japanese populations, the authors mainly focused on the role of the brain. Brain extirpation, which was performed within 12hr after pupation, caused the prolongation of pupal-adult development, although no delay of development was observed when the surgical operation was carried out 72hr after pupation. The brains derived from Bombyx mori or from non-diapausing Bombyx mandarina of Chinese populations had the potency to restore the development in diapausing pupae of Japanese population. Resumption of pupal-adult development could be induced by injection of prothracicotropic hormone (PTTH) from Bombyx mori or 20-hydroxyecdysone. Restoration of oxygen consumption was also observed by the application of these hormones. The results presented here strongly suggest that summer diapause in the Japanese population of Bombyx mandarina is due to the brain's inability to synthesize PTTH.