The ensemble of hetero-proteins in inorganic nanochannels

The assembly and proper alignment of two heterofluorescent proteins (sGFP and DsRed) in the mesoporous channels of ethanol-treated FSM6.2 (a folded-sheet mesoporous material with a pore diameter of 6.2 nm) was confirmed using a fluorescence resonance energy transfer (FRET) technique. The sGFP-DsRed-FSM6.2 conjugate showed a large decrease in the emission of donor (sGFP) fluorescence, indicating that the conjugate functions as an energy transfer system through the combination of the two heteroproteins, due to the successful encapsulation of the sGFP-DsRed pairs in the mesopores. Fluorescence spectral analysis demonstrated that the proteins were highly dispersed and homogeneously encapsulated in the mesopores of FSM6.2, even at high concentration, although they spontaneously aggregated and showed a red shift in solution at the concentration corresponding to that in the conjugate. Furthermore, an increase in the amount of sGFP and DsRed adsorbed to the pores of FSM6.2 led to a decrease in the distance between these proteins, resulting in enhancement of FRET efficiency.     

Identification and characterization of ten polymorphic microsatellite loci in the red panda Ailurus fulgens

Ten polymorphic microsatellite loci were characterized from two genomic DNA-enriched libraries of the red panda (Ailurus fulgens). The number of observed alleles among 35 samples of red pandas ranged from five to 12. Observed and expected heterozygosities were 0.286–0.971 and 0.443–0.894, and the mean polymorphic information content was 0.712. All loci followed Hardy–Weinberg expectations except Aifu-14 and Aifu-16, which may due to the presence of inbreeding or null alleles. Three pairs of loci exhibited significant linkage disequilibrium after Bonferroni correction for multiple comparisons. These microsatellites would be useful to strengthen population management, genetic diversity exploration, and demographic history speculation of this species.     

Hyperosmotic stress up-regulates the expression of major vault protein in SW620 human colon cancer cells

The major vault protein (MVP) is the major constituent of the vault particle, the largest ribonuclear protein complex described to date and is identical to lung resistance-related protein (LRP). Although MVP is also expressed in several normal tissues, little is known about its physiological role. MVP played a protective role against some xenobiotics and other stresses. We thus investigated the effect of osmotic stress on MVP expression by treating human colon cancer SW620 cells with sucrose or NaCl. The expression level of both MVP protein and MVP mRNA was increased by the osmostress. Sucrose or sodium chloride could also enhance MVP promoter activity. Inhibition of p38 MAPK in SW620 cells by SB203580 inhibited the expression of MVP under hyperosmotic stress. These findings suggested that osmotic stress up-regulated the MVP expression through p38 MAPK pathway. Down-regulation of MVP expression by MVP interfering RNA (RNAi) in SW620 cells increased the sensitivity of the cells to hyperosmotic stress and enhanced apoptosis. Furthermore, MVP RNAi prevented the osmotic stress-induced, time-dependent increase in phosphorylated Akt. These findings suggest that the PI3K/Akt pathway might be implicated in the cytoprotective effect of MVP.Our data demonstrate that exposure of cells to hyperosmotic stress induces MVP that might play an important role in the protection of the cells from the adverse effects of osmotic stress.     

Crystal structure of Tk-subtilisin folded without propeptide: requirement of propeptide for acceleration of folding

Tk-subtilisin (a subtilisin homologue from Thermococcus kodakaraensis) is matured from Pro-Tk-subtilisin upon autoprocessing and degradation of Tk-propeptide. To analyze the folding mechanism of Tk-subtilisin, the crystal structure of the active site mutant of Tk-subtilisin (S324A-subtilisin^∗), which was refolded in the presence of Ca²⁺ and absence of Tk-propeptide, was determined at 2.16 Å resolution. This structure is essentially the same as that of Tk-subtilisin matured from Pro-Tk-subtilisin. S324A-subtilisin^∗ was refolded with a rate constant of 0.17 and 1.8 min⁻¹ at 30 °C in the absence and presence of Tk-propeptide, respectively, indicating that Tk-subtilisin does not require Tk-propeptide for folding but requires it for acceleration of folding.     

Food shortage affects flight migration of the giant water bug Lethocerus deyrolli in the prewintering season

The endangered giant water bug Lethocerus deyrolli (Vuillefroy) is frequently attracted in large numbers to artificial lights in Japan. To examine factors enhancing flight migration for L. deyrolli, we carried out field work in western Hyogo Prefecture, central Japan, in September during the nonreproductive and prewintering season. The body weight of specimens collected under flight migration (flight bugs) was significantly less than that of those collected in ponds (pond bugs). A field experiment using open cages in a rice paddy field was carried out with two treatments, with and without a food supply. The remaining rate of L. deyrolli for the food present treatment was significantly higher than that for the food absent treatment for the first two days. These results suggest that L. deyrolli would fly in search of food when the food supply of the present habitat becomes unsuitable.     

Development of high-throughput screening system for osteogenic drugs using a cell-based sensor

To effectively treat osteoporosis and other bone-loss disorders, small compounds that potently induce bone formation are needed. The present study initially attempted to establish a monitoring system that could detect osteogenic differentiation easily, precisely, and noninvasively. For this purpose, we established pre-osteoblastic MC3T3E1 cells stably transfected with the GFP reporter gene driven by a 2.3 kb fragment of rat type I collagen promoter (Col1a1GFP-MC3T3E1). Among these cells, we selected a clone that fluoresced upon osteogenic stimulation by BMP2. The GFP fluorescence intensity corresponded well to the intensity of alkaline phosphatase (ALP) staining and to the level of osteocalcin (Oc) mRNA. Using this system, we screened natural and synthetic compound libraries and thus identified an isoflavone derivative, glabrisoflavone (GI). GI induced ALP staining and Oc mRNA in a dose-dependent manner. The Col1a1GFP-MC3T3E1 system may be useful for identifying novel osteogenic drugs.     

A pelagic larva of <Emphasis Type="Italic">Plagiopsetta glossa</Emphasis> (Pleuronectiformes,Samaridae), characterized by a trailing gut and S-shaped pelvic bone: description and phylogenetic considerations

 The pelagic larval stage of a pleuronectiform samarid, Plagiopsetta glossa, is described and illustrated, for the first time, from a 8.4-mm BL postflexion specimen collected off Tosa Bay, southern Japan. The larva is distinctive in having broad, semitransparent pterygiophore zones on the dorsal and anal fins, a trailing gut, flexible S-shaped pelvic bone, and a skin fold under the throat. These characteristics are shared with poecilopsettid larvae, suggesting a close relationship between the two families.     

Larval development of Pseudorhombus oculocirris and P. arsius (Pleuronectiformes, Paralichthyidae) from off southern Japan

 Larvae of two paralichthyids, Pseudorhombus oculocirris and P. arsius, are described and illustrated from specimens collected off Tosa Bay, southern Japan. Peudorhombus oculocirris larvae (5 specimens, 4.5–7.8 mm BL) are characteristic in having 6 or 7 elongated anterior dorsal fin rays and poorly developed head spines and melanophores on the tail. Pseudorhombus arsius larvae (3 specimens, 5.3–8.4 mm BL) are distinctive in having 11 or 12 elongated anterior dorsal fin rays and well-developed head spines, including a row of spines on the sphenotic. Received: June 28, 2001 / Revised: November 2, 2001 / Accepted: November 22, 2001