DEMONSTRATION OF ALKALINE PHOSPHATASE PARTICIPATION IN THE MINERALIZATION OF OSTEOBLASTS BY ANTISENSE RNA APPROACH

MC3T3-E1 cells grown with ascorbic acid express sequentially osteoblastic marker proteins such as alkaline phosphatase (ALPase) and then form a mineralized extracellular matrix (ECM) as a consequence of osteoblastic differentiation. To explore the functional roles of ALPase in the process of osteoblastic maturation, an inducible expression vector for antisense ALPase RNA was constructed and stably transfected into MC3T3-E1 cells. The expression of antisense ALPase RNA in the differentiated MC3T3-E1 transfectants reduced markedly the ALPase activity, which resulted in a significant decrease in the deposition of minerals upon prolonged culture. These findings demonstrated directly that ALPase participated in the mineralizationof ECM.     

Mesophyll conductance decreases in the wild type but not in an ABA‐deficient mutant (aba1) of Nicotiana plumbaginifolia under drought conditions

Under drought conditions, leaf photosynthesis is limited by the supply of CO₂. Drought induces production of abscisic acid (ABA), and ABA decreases stomatal conductance (g_s). Previous papers reported that the drought stress also causes the decrease in mesophyll conductance (g_m). However, the relationships between ABA content and g_m are unclear. We investigated the responses of g_m to the leaf ABA content [(ABA)_L] using an ABA‐deficient mutant, aba1, and the wild type (WT) of Nicotiana plumbaginifolia. We also measured leaf water potential (Ψ_L) because leaf hydraulics may be related to g_m. Under drought conditions, g_m decreased with the increase in (ABA)_L in WT, whereas both (ABA)_L and g_m were unchanged by the drought treatment in aba1. Exogenously applied ABA decreased g_m in both WT and aba1 in a dose‐dependent manner. Ψ_L in WT was decreased by the drought treatment to ?0.7 MPa, whereas Ψ_L in aba1 was around ?0.8 MPa even under the well‐watered conditions and unchanged by the drought treatment. From these results, we conclude that the increase in (ABA)_L is crucial for the decrease in g_m under drought conditions. We discuss possible relationships between the decrease in g_m and changes in the leaf hydraulics.     

Comparative foraging ecology of Madagascar vangids (Vangidae)

The Vangidae, an endemic family in Madagascar, contains 14 species in 11 genera. The foraging behaviour of 13 species of vangids was investigated during the periods August-November 1989 and September-November 1990. These vangids are grouped into six bill types according to their bill shape, using the analogy of gripping tools: (1) forceps. (2) radio pliers, (3) strong pinchers, (4) pliers, (5) standard and (6) flat forceps types. We recognized seven foraging niches: (A) canopy-gleaning, (B) general, (C) ground-snatching, (D) trunk/ branch-probing, (E) leaf and twig/branch-gleaning/probing, (F) leaf and twig/branch-gleaning and (G) trunk-gleaning niches. Two or more coexisting species with the same foraging niche differed in their bill types. Those vangids that exploited similar resources segregated in their bill types, geographic ranges and/or foraging niches, presumably to avoid severe competition. Several vangid species partially occupied the ecological niche that would be filled by woodpeckers elsewhere.     

Polymorphism of axillary bud galls induced by Rhopalomyia longitubifex (Diptera: Cecidomyiidae) on Artemisia princeps and A. montana (Asteraceae) in Japan and Korea, with designation of new synonyms

In the past, Rhopalomyia longitubifex, Rhopalomyia shinjii, and Rhopalomyia sp. (Diptera: Cecidomyiidae) have been regarded as independent species based on differences in the sizes and shapes of axillary bud galls induced on Artemisia montana (Asteraceae) in Japan and A. princeps in Japan and Korea. However, comparison of morphological features and molecular sequencing data indicate that these Rhopalomyia gall midges are identical and that the differences in gall shape are polymorphisms, although the measurements of gall height and diameter overlap slightly. This finding suggests that although galls have frequently been regarded as extensions of the phenotype of a species, differences in gall shape may not always be reliable for identifying gall‐inducing cecidomyiids. The older name, R. longitubifex, is applied to these gall midges, and the names that were applied to this species on later occasions are revised or synonymized. The mature and immature stages of R. longitubifex are redescribed and information on the distribution, host range, and gall size of this species is provided. We also discuss the role of gall polymorphism in the early stages of speciation.     

The composition and foraging behaviour of mixed-species flocks of forest-living birds in Madagascar

Mixed-species flocks of forest living birds were investigated in a rainforest at Perinet/ Analamazaotra (Andasibe), Madagascar. Most insectivorous birds in the area participated in mixed-species flocks. Flocks were composed of foliage gleaners, foliage-branch gleaners, trunk gleaners and flycatchers. Species whose foraging techniques and foraging locations in vegetation were similar were different from each other in foraging heights. The nuclear species often occurred in small conspecific flocks in and out of mixed-species flocks.     

Development of a novel pink-eyed dilution mouse model showing progressive darkening of the eyes and coat hair with aging

Oca2^p-cas (oculocutaneous albinism II; pink-eyed dilution castaneus) is a coat color mutant gene on mouse chromosome 7 that arose spontaneously in wild Mus musculus castaneus mice. Mice homozygous for Oca2^p-cas usually exhibit pink eyes and gray coat hair on the non-agouti genetic background, and this ordinary phenotype remains unchanged throughout life. During breeding of a mixed strain carrying this gene on the C57BL/6J background, we discovered a novel spontaneous mutation that causes darkening of the eyes and coat hair with aging. In this study, we developed a novel mouse model showing this unique phenotype. Gross observations revealed that the pink eyes and gray coat hair of the novel mutant young mice became progressively darker in color by approximately 3 months after birth. Light and transmission-electron microscopic observations revealed a marked increase in melanin pigmentation of coat hair shafts and choroid of the eye in the novel mice compared to that in the ordinary mice. Sequence analysis of Oca2^p-cas revealed a 4.1-kb deletion involving exons 15 and 16 of its wild-type gene. However, there was no sequence difference between the two types of mutant mice. Mating experiments suggested that the novel mutant phenotype was not inherited in a simple fashion, due to incomplete penetrance. The novel spontaneous mutant mouse is the first example of progressive hair darkening animals and is an essential animal model for understanding of the regulation mechanisms of melanin biosynthesis with aging.     

Emergence of the Extrachromosomal Circular DNA Complexes as One of the Earliest Signals of Cellular Differentiation in the Early Development of Mouse Embryo

A small number of mouse embryos and embryonal carcinoma cells were pressed by mica sheet; then the extruded DNA complexes were adsorbed to mica and processed for electron microscopy. Extrachromosomal circular DNA complexes longer than 1 μm emerged during the compaction process of mouse embryos and during the differentiation of embryonal carcinoma cells induced with retinoic acid. These DNA molecules are discussed as possible products of developmental gene rearrangements occurring in the chromosomal DNA.     

Difference in the distribution and speciation of cellular nickel between nickel‐tolerant and non‐tolerant Nicotiana tabacum L. cv. BY‐2 cells

To evaluate Ni dynamics at the subcellular level, the distribution and speciation of Ni were determined in wild‐type (WT) and Ni‐tolerant (NIT) tobacco BY‐2 cell lines. When exposed to low but toxic levels of Ni, NIT cells were found to contain 2.5‐fold more Ni (14% of whole‐cell Ni values) in their cell walls than WT cells (6% of whole‐cell Ni values). In addition to higher levels of Ni in the apoplast, a higher proportion (94%) of symplastic Ni was localized in the vacuoles of NIT cells than in the vacuoles of WT cells (81%). The concentration of cytosolic Ni in the NIT cells was significantly lower (18 nmol g^?1 FW) than that in the WT cells (85 nmol g^?1 FW). In silico simulation showed that 95% of vacuolar Ni was in the form of Ni‐citrate complexes, and that free Ni²⁺ was virtually absent in the NIT cells. On the other hand, the amount of free metal ions was markedly increased in WT cells because free citrate was depleted by chelation of Ni. A protoplast viability assay using BCECF‐AM further demonstrated that the main mechanism that confers strong Ni tolerance was present in the symplast as opposed to the cell wall.