No abstract

Silk fibroin produced by the silkworm Bombyx mori consists of a heavy chain, a light chain, and a glycoprotein, P25. The heavy and light chains are linked by a disulfide bond, and P25 associates with disulfide-linked heavy and light chains by noncovalent interactions. Quantitative enzyme-linked immunosorbent assay revealed that molar ratios of the heavy chain, light chain, and P25 were 6:6:1, both in cocoons and in fibroin secreted into the lumen of posterior silk gland. Trace amounts of fibroin produced by three "naked pupa" mutants of B. mori lacked the light chain, but the molar ratio of heavy chain and P25 was also 6:1. Gel filtration chromatography and sedimentation equilibrium analysis demonstrated that a large protein complex of approximately 2.3 MDa, designated an elementary unit of fibroin having 6:6:1 molar ratios of the heavy chain, light chain, and P25, existed in posterior silk gland cells. Inaccessibility of biotinylated concanavalin A to the native elementary unit and partial dissociation of the elementary unit after incubation with excess N-glycosidase F or endoglycosidase H suggest that a single molecule of P25 is located internally and plays an important role in maintaining integrity of the complex.A vast amount of silk fibroin is synthesized within the cells of a pair of PSGs 1 of the silkworm, Bombyx mori, during the larval fifth instar, secreted into the lumen of PSG, and transported through the middle silk gland, where heterogeneous molecules of sericin are added, and further toward the anterior part of the silk gland, where the silk fiber is formed and spun. Both the efficient secretion of the vast amount of fibroin from PSG cells into the lumen and the maintenance of solubility of fibroin during the luminal transport have been speculated to be facilitated by the formation of a molecular complex consisting of fibroin heavy chain (H-chain) of 350 kDa and two lower molecular mass protein components: fibroin light chain (Lchain) of 26 kDa (1) and P25, which is a glycoprotein of about 30 kDa (2).H-chain and L-chain are linked by a single disulfide bond between Cys-172 of L-chain and Cys-c20 (twentieth residue from the C terminus) of H-chain (3). The H-L linkage is essential for the secretion of a vast amount of fibroin, because the silkworm carrying the Nd-s or Nd-s D mutation of the L-chain gene (fibL) could not form the disulfide linkage with H-chain, and less than 1% of the normal level fibroin is secreted (4).This tremendous reduction of fibroin secretion has been suggested to be caused by the appearance of a free sulfhydryl group of Cys-c20 on H-chain, which prevents the transport of H-chain from ER to Golgi (4).On the other hand, P25 has been shown to associate with the H-L complex by noncovalent interactions, i.e. mainly by hydrophobic interactions with the H-chain moiety (2, 5). However, the role of P25 in the formation of the fibroin molecular complex has not been elucidated, mainly because a silkworm carrying a mutation of the P25 gene has not been available.In the present study, a m...

No abstract

No abstract

A new forced-swimming apparatus for determining maximum swimming time in mice was devised for use in the evaluation of the endurance capacity of Std and ddY and CDF1 mice after various diet and drug treatments. With the apparatus, a water current is generated by circulating water with a pump in a swimming pool. A spout and suction slit were contrived to generate a constant current while the strength of the current is regulated by a valve. The decrease in the leg-kicking intervals of mice accompanying the increase in the current speed confirmed that the workload is adjustable by regulation of the current speed. Compared with the number of forelimb strokes, that of the hindlimb kicks was greater. The swimming time until fatigue was observed to decrease with increasing current speed in the two strains of mice. As biochemical indexes, the blood lactate and muscle glycogen levels corroborated the correlation between current speed and increase in workload. These results indicate that the apparatus employed in the present study is suitable for the evaluation of the endurance capacity of mice and that is useful for detecting the effects of dietary differences and drug pretreatments on this capacity.

Background and Purpose Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with no effective treatment. Fasudil hydrochloride (fasudil), a potent rho kinase (ROCK) inhibitor, is useful for the treatment of ischaemic diseases. In previous reports, fasudil improved pathology in mouse models of Alzheimer's disease and spinal muscular atrophy, but there is no evidence in that it can affect ALS. We therefore investigated its effects on experimental models of ALS. Experimental Approach In mice motor neuron (NSC34) cells, the neuroprotective effect of hydroxyfasudil (M3), an active metabolite of fasudil, and its mechanism were evaluated. Moreover, the effects of fasudil, 30 and 100 mg·kg−1, administered via drinking water to mutant superoxide dismutase 1 (SOD1G93A) mice were tested by measuring motor performance, survival time and histological changes, and its mechanism investigated. Key Results M3 prevented motor neuron cell death induced by SOD1G93A. Furthermore, M3 suppressed both the increase in ROCK activity and phosphorylated phosphatase and tensin homologue deleted on chromosome 10 (PTEN), and the reduction in phosphorylated Akt induced by SOD1G93A. These effects of M3 were attenuated by treatment with a PI3K inhibitor (LY294002). Moreover, fasudil slowed disease progression, increased survival time and reduced motor neuron loss, in SOD1G93A mice. Fasudil also attenuated the increase in ROCK activity and PTEN, and the reduction in Akt in SOD1G93A mice. Conclusions and Implications These findings indicate that fasudil may be effective at suppressing motor neuron degeneration and symptom progression in ALS. Hence, fasudil may have potential as a therapeutic agent for ALS treatment.

Multiferroic CuFe1−xAlxO2 (x = 0.02) exhibits a ferroelectric ordering accompanied by a proper helical magnetic ordering below T = 7K under zero magnetic field. By polarized neutron diffraction and pyroelectric measurements, we have revealed a one-to-one correspondence between the spin helicity and the direction of the spontaneous electric polarization. This result indicates that the spin helicity of the proper helical magnetic ordering is essential for the ferroelectricity in CuFe1−xAlxO2. The induction of the electric polarization by the proper helical magnetic ordering is, however, cannot be explained by the Katsura-Nagaosa-Balatsky model, which successfully explains the ferroelectricity in the recently explored ferroelectric helimagnets, such as TbMnO3. We thus conclude that CuFe1−xAlxO2 is a new class of magnetic ferroelectrics.PACS numbers: 75.80.+q, 75.25.+z, 77.80.-e Novel types of couplings between dielectric property and magnetism, which produce colossal magnetoelectric (ME) effects, have been extensively investigated since a gigantic ME effect was discovered in RMnO 3 (R is a rare earth material) [1]. Among several types of couplings between spins and electric polarizations, a ferroelectricity induced by noncollinear spin arrangements has been most widely investigated experimentally and theoretically [2,3,4,5,6,7,8]. Katsura, Nagaosa and Baratsky (KNB) proposed that the local electric dipole moment p, which arises between neighboring two spins S i and S i+1 , can be described in the form of p ∝ e i,i+1 × (S i × S i+1 ), where e i,i+1 is a unit vector connecting two spins [2]. This formula successfully explains the ferroelectric property in cycloidal or conical magnetic orderings of some transition metal oxides, such as RMnO 3 (R=Tb, Tb 1−x Dy x ), Ni 3 V 2 O 8 , MnWO 4 and CoCr 2 O 4 [4,5,6,7,8]. Moreover, a recent polarized neutron diffraction study on TbMnO 3 demonstrated that the spin helicity, clockwise or counterclockwise, correlates with the direction of the electric polarization, as predicted in the formula [9]. It is, however, recently reported that ferroelectricity in a helical magnetic ordering of a delafossite multiferroic CuFe 1−x Al x O 2 cannot be explained by the above formula [10]. Therefore, CuFe 1−x Al x O 2 provides an opportunity to explore an another type of spin-polarization coupling.CuFeO 2 , which is one of model materials of a triangular lattice antiferromagnet, has been extensively investigated as a geometrically frustrated spin system for last fifteen years [11,12,13]. The ground state of CuFeO 2 is a collinear commensurate 4-sublattice (↑↑↓↓) state with the magnetic moments along the c axis, which is normal to the triangular lattice layers, in spite of the Heisenberg spin character expected from the electronic configuration of Fe. When a magnetic field is applied along the c axis at low temperature, CuFeO 2 exhibits a multi-step magnetization process consisting of several magnetization plateaus and slopes, which is accompanied by stepwise changes of lattice constants [14...

Huntington's disease (HD) is a neurodegenerative disease caused by a CAG repeat expansion in exon 1 of the HD gene, and the expression level of either normal or mutant huntingtin is implicated in the pathogenesis of HD. However, a molecular base of the HD gene transcription has not been elucidated as yet. In this study, we identified two proteins, HDBP1 and HDBP2, which bind to the promoter region for the HD gene using a yeast one-hybrid system. Amino acid sequence analysis of the proteins deduced the presence of nuclear localization signal, nuclear export signal, zinc finger, serine/ proline-rich region, and highly conserved C-terminal region. In vitro DNA binding assay indicated that the C-terminal conserved regions of the proteins were responsible for binding to the unique promoter DNA sequences of the HD gene. The DNA sequence protected from DNase I digestion was a 7-bp consensus sequence (GCCGGCG), which resides in triplicate at intervals of 13 bp within and proximal to the 20-bp direct repeat sequences of the HD promoter region. The mutation of 7-bp consensus sequence abolishes the HD promoter function in a neuronal cell line (IMR32). In human cultured cells, ectopically expressed green fluorescent protein-fused HDBP1 and HDBP2 localized in the cytoplasm, but both proteins totally shift from cytoplasm to nucleus by the treatment with an inhibitor of the nuclear export, leptomycin B, and mutagenesis of the putative nuclear export signals. Taken together, HDBP1 and HDBP2 are novel transcription factors shuttling between nucleus and cytoplasm and bind to the specific GCCGGCG, which is an essential cis-element for HD gene expression in neuronal cells.