Kazuaki Takahashi

A novel method for viral display of ER membrane proteins on budded baculovirus.

The baculovirus expression system has been used to express large quantities of various proteins, including membrane receptors. Here, we reveal a novel property of this expression system to be that certain membrane proteins can be displayed on the budded virus itself. We introduced the genes encoding sterol regulatory element-binding protein-2 (SREBP-2) or SREBP cleavage-activating protein (SCAP), important integral membrane proteins of the endoplasmic reticulum (ER) and/or the Golgi apparatus related to cellular cholesterol regulation, into a baculovirus vector. When insect cells were infected with SREBP-2 or SCAP recombinant viruses, it was found that these ER membrane proteins appeared on the budded baculovirus in addition to the host cell membrane fraction. Compared to proteins expressed on the cell membrane, membrane proteins displayed on virus exhibited both less aggregation and less degradation upon immunoblotting. Using this viral displayed SCAP as the screening antigen, we then generated a new monoclonal antibody specific against SCAP, which was useful for immunological localization studies. This system, which takes advantage of the viral display of membrane proteins, should prove to be a powerful additional tool for postgenomic protein analysis.

Fleshy fruit characteristics in a temperate deciduous forest of Japan: how unique are they?

This study investigated the fleshy fruit characteristics of 28 woody species in a Japanese temperate forest where large sedentary seed-dispersing mammals are present. We tested whether the findings in previous studies in temperate forests of Europe and North America are universal or not. Results have suggested that fruits of all species were eaten both by birds and mammals except for four species with larger fruits, which were eaten only by mammals. A gradient was found from a syndrome characterized by small, oily, and large-seeded fruits to a syndrome characterized by large, succulent, non-oily, and small-seeded fruits. The sizes and colors of the fruits were not conspicuously different from previous findings in Europe and North America. On the other hand, nitrogen and lipids in the fleshy part did not show seasonally increasing trends, or even seasonally decreasing trends in terms of dry weight. This result, suggesting the absence of community-level adaptation of fruit traits to migratory bird dispersers, contrasted with findings in Europe and North America. Large sedentary arboreal or tree-climbing mammals may have a greater effect on the evolution of fruit-disperser relations than opportunistic migratory birds.

Factors affecting seed rain beneath fleshy-fruited plants.

We investigated factors affecting seed rain beneath nine fleshy-fruited fruiting plant species growing in a 1-ha plot of planted Pinus thunbergii in central Japan. We tested whether the numbers of seeds and seed species dropped by birds beneath fruiting plants were correlated with the number of fruits removed by birds from the plants. Most of fruiting plant species with high fruit removal had significantly high seed rain. Both the numbers of seeds and seed species dropped were significantly, positively correlated with the number of fruits removed across for all fruiting plant species. Therefore, fruit removal predicted the difference among heterospecific fruiting plants in seed rain. We also tested whether the number of fruits removed from fruiting plants by birds was related with fruit crop size, fruit size, and height of the plants, and the numbers of fruits and fruit species of neighboring plants near the plants. Most of fruiting plant species with high fruit crop size had significantly high fruit removal. The number of fruits removed was significantly, positively correlated with both the fruit crop size and the number of neighboring fruits across the nine fruiting plant species. However, the effect of the neighboring fruit density on fruit removal was lower remarkably than that of fruit crop size. Therefore, fruit crop size best predicted the differences among heterospecific fruiting plants in fruit removal. We suggest that fruiting plant species with high fruit crop size and high fruit removal contribute to intensive seed rain beneath them.

Seasonal variation in fleshy fruit use and seed dispersal by the Japanese black bear (Ursus thibetanus japonicus)

Fruit use by the Japanese black bear (Ursus thibetanus japonicus) and seed clumping in bear scat were studied in central Japan using fecal analyses. Between May and November 2003 and 2004, the life form and fruit size of plants consumed by bears and the species composition and intactness of seeds contained in scat were examined in five transects (approximately 10xa0kmxa0×xa010xa0m) in broad-leaved deciduous forests. In 2003, scats with seeds were found only in the autumn, when fruiting trees and shrubs were abundant. In 2004, scats with seeds occurred intermittently from the summer, when fruiting plants were rare, up to the autumn. Yearly and seasonal variation in fruit use reflects the opportunistic foraging behavior of Japanese black bears. Seven of the nine plant species detected in scats had medium-sized fruits (6–15xa0mm width), whereas the other two species had relatively large fruits (20–100xa0mm width). In total, 14,492 seeds were detected, of which 97.6% were intact; the remainder were damaged. Intact seeds of one or two species were found in each scat. The number of intact seeds per scat ranged from 1 to 5476. Japanese black bears seldom digest ingested seeds, thereby contributing to the seed dispersal of their food plants, including species with fruits that are too large to be swallowed by frugivorous birds.

Functional reconstitution of olfactory receptor complex on baculovirus.

Despite that recent progress in genomics has elucidated the genomic structure of the olfactory receptors (ORs), most of them are still orphan receptors. The low expression level of ORs in heterologous cells has hampered many attempts to establish cell biological OR assay systems. Recently, we demonstrated that certain G protein-coupled receptors, such as the leukotriene B4 receptor or the dopamine D1 receptor, were efficiently reconstituted on baculovirus budding from infected Sf9 cells. The budded virus (BV) was shown to be mostly free of exogenous proteins other than those related to viral infection, resulting in low-noise assay conditions. Taking advantage of these conditions, we attempted to reconstitute OR complexes on BV. Sf9 cells were coinfected with recombinant baculoviruses harboring the cDNAs encoding adenylyl cyclase, trimeric G-protein, and the receptor: mOR-EG or S6. The coexpression of these proteins was detected by western blot, and the agonist- or antagonist-dependent receptor response was confirmed using ligand-dependent cyclic AMP production. These results demonstrated the successful reconstitution of functional OR complex on BV. Additionally, the expression of OR8B3 on BV, one of human orphan ORs, was also confirmed. This BV expression system is expected to be a highly effective tool for screening unknown ligands for ORs.

Spatial distribution and size of small canopy gaps created by Japanese black bears: estimating gap size using dropped branch measurements

BackgroundJapanese black bears, a large-bodied omnivore, frequently create small gaps in the tree crown during fruit foraging. However, there are no previous reports of black bear-created canopy gaps. To characterize physical canopy disturbance by black bears, we examined a number of parameters, including the species of trees in which canopy gaps were created, gap size, the horizontal and vertical distribution of gaps, and the size of branches broken to create gaps. The size of black bear-created canopy gaps was estimated using data from branches that had been broken and dropped on the ground.ResultsThe disturbance regime was characterized by a highly biased distribution of small canopy gaps on ridges, a large total overall gap area, a wide range in gap height relative to canopy height, and diversity in gap size. Surprisingly, the annual rate of bear-created canopy gap formation reached 141.3xa0m2xa0ha–1xa0yr–1 on ridges, which were hot spots in terms of black bear activity. This rate was approximately 6.6 times that of tree-fall gap formation on ridges at this study site. Furthermore, this rate was approximately two to three times that of common tree-fall gap formation in Japanese forests, as reported in other studies.ConclusionsOur findings suggest that the ecological interaction between black bears and fruit-bearing trees may create a unique light regime, distinct from that created by tree falls, which increases the availability of light resources to plants below the canopy.

Do Small Canopy Gaps Created by Japanese Black Bears Facilitate Fruiting of Fleshy-Fruited Plants?

Japanese black bears often break branches when climbing trees and feeding on fruit in canopies, thereby creating small canopy gaps. However, the role of black bear-created canopy gaps has not been evaluated in the context of multiple forest dynamics. Our hypothesis was that small canopy gaps created by black bears improve light conditions, which facilitates fruiting of adult fleshy-fruited plants located beneath the gaps, and also that this chain interaction depends on interactions among the size of gaps, improved light conditions, forest layers, and life form of plants. The rPPFD, size of black bear-created canopy gaps, and fruiting/non-fruiting of fleshy-fruited plants were investigated in five forest layers beneath black-bear-created canopy gaps and closed canopies of Mongolian oak (Quercus crispula). We found that light conditions improved beneath black bear-disturbed trees with canopy gaps of large size, and the effect of improvement of light conditions was reduced with descending forest layers. Fruiting of fleshy-fruited plants, especially woody lianas and trees, was facilitated by the improvement of light conditions accompanied by an increase in the size of black-bear-created gaps. Data from this study revealed that canopy disturbance by black bears was key for improving light conditions and accelerating fruiting of fleshy-fruited trees and woody lianas in the canopy layers in particular. Therefore, our hypothesis was mostly supported. Our results provide evidence that Japanese black bears have high potential as ecosystem engineers that increase the availability of resources (light and fruit in this study) to other species by causing physical state changes in biotic materials (branches of Q. crispula in this study).

Is there a risk-dilution effect of naturally fallen fruits on post-dispersal seed predation by wood mice?

AimsThe aim of this study was to test the hypothesis that heavy predation of fallen fruits reduces the predation of neighboring post-dispersed seeds.MethodsWe performed a seed/fruit removal cafeteria experiment to assess the effects of seed species, of fruit species, and of density of fallen fruits on seed predation by wood mice (Apodemus sp.). In total, 6,930 seeds and 9,660 fruits from seven species were used. A generalized linear mixed model procedure was used to test these effects.ResultsSeed predation was significantly affected by the species of both seeds and neighboring fruits, but not by their density. Multiple comparison tests showed that most of treatments tested had no significant effects of species and density of neighboring fruits on seed removal. However, seed removal was significantly lower in some treatments with fruits than in the treatments without fruits for one of the seven seed species tested. Seed removal of two species tested was significantly differed among some treatments with different fruit species.ConclusionMost of the results in this study do not support our overall hypothesis. However, a part of results supported our hypothesis, suggesting that seed removal is affected by complex interactions between the preference of wood mice for fruits and fruiting environment.