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The article discusses a study which reports the draft genome sequence of Paenibacillus popilliae, a rod-shaped endospore-forming bacterium, which causes milky disease in Japanese beetle Popillia japonica and related species. Topics discussed include preparation of the bacterial culture to extract genomic DNA from bacterial cells, phylogenetic analysis using previous data sets on microbial population, and comparison of the analysis with other entomo-pathogenic bacteria.

Human bone morphogenetic protein 4 (BMP4) fused to the collagen-binding domain (CBD) of fibronectin was constructed and expressed in a silkworm-baculovirus expression system. When recombinant BmNPV was injected into the silkworm larvae and harvested after approximately 4 days, 0.22 mg/ml (0.1 mg/larva) of recombinant CBD-BMP4 was secreted into the silkworm haemolymph. Interestingly, cultured silkworm cells infected with the same recombinant virus could not secrete the recombinant CBD-BMP4 into culture media. The purified rCBDBMP4 showed Smad- and MAPK-stimulating activities in human UET-13 cells. [ABSTRACT FROM AUTHOR]

The article discusses a study which reports the draft genome sequence of Paenibacillus popilliae, a rod-shaped endospore-forming bacterium, which causes milky disease in Japanese beetle Popillia japonica and related species. Topics discussed include preparation of the bacterial culture to extract genomic DNA from bacterial cells, phylogenetic analysis using previous data sets on microbial population, and comparison of the analysis with other entomo-pathogenic bacteria.

Using a cDNA microarray, we monitored global gene expression profiles in silkworm BmN4-SID1 cells after the knockdown of BmAGOI and BmAG02 genes. Interestingly, there was a significant overlap between the target genes up-regulated by BmAgol (292 out of 481) and BmAgo2 (292 out of 361). Of these, the Replicase gene of macula-like virus (BmMLV) was highly induced in both of BmAgol - and BmAgo2-depleted cells. RT-PCR analysis confirmed that the knockdown of silkworm RNAi pathway-related genes increased the expression of the Replicase gene. These data strongly suggested that the RNAi pathway negatively regulated BmMLV proliferation in silkworm BmN4 cells. [ABSTRACT FROM AUTHOR]

Four housekeeping genes including gapA, groEL, gyrA and pgi were used in order to reveal a phylogenetic relationship among Paenibacillus species. Phylogenetic analysis and end-to-end pairwise analysis were carried out using nucleotide and amino acid sequences of the housekeeping genes. A monophylogenetic clade including P. popilliae, P. thiaminolyticus and P. dendritiformis formed in all phylogenetic trees. The results in both analyses indicated that the relationship between P. larvae subsp. larvae and P. popilliae was comparatively far. The result of the end-to-end pairwise analysis also showed that, among the Paenibacillus species used in this study, the closest species to P. popilliae was P. dendritiformis. [ABSTRACT FROM AUTHOR]

Epigenetic modifiers and transcription factors contribute to developmentally programmed gene expression. Here, we establish a functional link between epigenetic regulation by Polycomb group (PcG) proteins and transcriptional regulation by C/ebp that orchestrates the correct expression of Bombyx mori asparagine synthetase (BmASNS), a gene involved in the biosynthesis of asparagine. We show that the cis-regulatory elements of YY1-binding motifs and the CpG island present on the BmASNS promoter are required for the recruitment of PcG proteins and the subsequent deposition of the epigenetic repression mark H3K27me3. RNAi-mediated knockdown of PcG genes leads to derepression of the BmASNS gene via the recruitment of activators, including BmC/ebp, to the promoter. Intriguingly, we find that PcG proteins and BmC/ebp can dynamically modulate the transcriptional output of the BmASNS target in a cell cycle-dependent manner. It will be essential to suppress BmASNS expression by PcG proteins at the G2/M phase of the cell cycle in the presence of BmC/ebp activator. Thus, our results provide a novel insight into the molecular mechanism underlying the recruitment and regulation of the PcG system at a discrete gene locus in Bombyx mori. [ABSTRACT FROM AUTHOR]

Serratia liquefaciens FK01 produces two serralysin-like metalloproteases. One of the genes, serf, has been cloned before. Another gene, designated as ser2, was cloned in this study. The protein encoded by ser2 was a typical serralysin-like metalloprotease, since a zinc binding motif (HEXXHXUGUXH), glycine-rich repeats (GGXGXD), a Met-turn (SXMXY) and an ABC exporter motif (DXXX) were found in the deduced amino acid se-quence. In order to investigate the contribution of these proteases to the virulence in an insect, protease- defi-cient mutants were created. The mutant strains were inoculated to the silkworm, Bombyx mori. The result of the mortality test suggested that we could not show that deficiency in serl and ser2 caused a virulence attenuation in the silkworm under our experimental conditions used in this study. It also suggested that other virulence fac-tors existed in the infection in the silkworm. [ABSTRACT FROM AUTHOR]

We constructed pHERDHis vectors to express polyhistidine-tagged proteins based on the pHERD vectors. The oligonucleotides containing the hexahistidine sequence were designed to be able to introduce the tag at either the NH2- or COOH- terminal. To demonstrate the usefulness of the vectors, a pyocyanin biosynthetic protein, PhzS, was expressed in a pyocyanin-deficient mutant of Pseudomonas aeruginosa. In the mutant strain, both HisPhzS (tag at the NH2- terminal) and PhzSHis (tag at the COOH- terminal) worked as a functional protein and pyocyanin was produced. It was also demonstrated that HisPhzS was purified by standard affinity chromatography. [ABSTRACT FROM AUTHOR]

The article discusses research on the epicuticular wax crystal (EWC) development in the Campbell Early Grape. Researchers Kyoung-Hee Shin et al. collected samples during the early growth period for the EWC morphology and then after harvest to observe the morphological changes in EWC through a scanning electron microscope (SEM). Findings showed little changes in EWC basic forms 18 days after bloom (DAFB) and after 34 days, changes in EWC pattern include size enlargment and change of shape.

Tetracycline-inducible gene expression (Tet-on) system has become one of the first choices for the control of transgenes expression in mammal and drosophila. However, the Tet-on systems that have been established in mammalian system or tuned into drosophila do not function in the silkworm, Bombyx mori. To construct a functional Tet-on system in B. mori, we modified rtTA by introducing a transcription activation domain of immediate-early gene 1 of Autographa californica nuclear polyhedrosis virus and nuclear localization signal of SV40 large T-antigen. The modified rtTA can activate the transcription from 9 × tetO promoter in the silkworm cells up to 250-fold in the presence of doxycycline. [ABSTRACT FROM AUTHOR]

The ribonuclease L (RNase L) pathway plays an important role in the response of cells to double-stranded RNA (dsRNA) during the events such as virus infection. Ribonuclease L inhibitor (RLI) belonging to the ABC transporter family is known as a regulator of the RNase L pathway. The homologs of RLI were reported in many organisms including the fruit fly and mosquito, but their functions in insects and arthropods have not been elucidated to date. In the present study, we cloned a cDNA of a silkworm RLI homolog, termed BmRLI, and its nucleotide sequence was determined. RT-PCR analysis revealed that the expression of BmRLI mRNA was marked in the testis, ovary and fat body. From the cDNA, recombinant protein with an apparent molecular mass of 69?kDa was expressed in Escherichia coli and cultured insect cells. Although no obvious effect of up-regulation of the BmRLI expression on RNAi was observed, its down-regulation slightly reduced RNAi efficiency. [ABSTRACT FROM AUTHOR]

Abstract The phase formation and electrical properties of (Bi, La)4Ti3O12 (BLT) thin film and V-, Sm-doped BLT thin films prepared by the chemical solution deposition method on Pt/TiO2/SiO2/Si substrates have been investigated. It was observed that the microstructure and electrical properties of BLT thin films dramatically varied with V- and Sm-doping. The crystallinity and grain size of BLT thin films were definitely increased by V- and Sm-doping into BLT films, which resulted in the enhancement of remanent polarization in doped BLT films. The remanent polarization (Pr) of Sm-doped BLT films annealed for 3 min by an RTA system was about 9 µC/cm2. The V- and Sm-doped BLT films also exhibited good fatigue characteristics under bipolar stressing to 1010 cycles. [ABSTRACT FROM AUTHOR]

The article discusses the comparison of elementary-school science curricula in Japan, Thailand, Republic of Korea, and Philippines. Science curriculum in Japan was revised in 1977, 1980 in Thailand, and 1973 in Korea and Philippines. The basic structure of the educational system in Japan, Thailand, and Korea is based on a 6-3-3 pattern, while a 6-4 pattern is used in the Philippines. The general characteristic of science curricular design in Japan is humanistic. Thailand is having an integrated type of curricular design, scientific concept type in Korea, and process-skill type in Philippines.

Selectins are key to mediating interactions involved in cellular adhesion and migration, underlying processes such as immune responses, metastasis, and transplantation. Selectins are composed of a lectin domain, an epidermal growth factor (EGF)-like domain, multiple short consensus repeats (SCRs), a transmembrane domain, and a cytoplasmic tail. It is well-established that the lectin and EGF domains are required to mediate interactions with ligands; however, the contributions of the other domains in mediating these interactions remain obscure. Using various E-selectin constructs produced in a newly developed silkworm-based expression system and several assays performed under both static and physiological flow conditions, including flow cytometry, glycan array analysis, surface plasmon resonance, and cell-rolling assays, we show here that a reduction in the number of SCR domains is correlated with a decline in functional E-selectin binding to hematopoietic cell E- and/or L-selectin ligand (HCELL) and P-selectin glycoprotein ligand-1 (PSGL-1). Moreover, the binding was significantly improved through E-selectin dimerization and by a substitution (A28H) that mimics an extended conformation of the lectin and EGF domains. Analyses of the association and dissociation rates indicated that the SCR domains, conformational extension, and dimerization collectively contribute to the association rate of E-selectin-ligand binding, whereas just the lectin and EGF domains contribute to the dissociation rate. These findings provide the first evidence of the critical role of the association rate in functional E-selectin-ligand interactions, and they highlight that the SCR domains have an important role that goes beyond the structural extension of the lectin and EGF domains. [ABSTRACT FROM AUTHOR]

To investigate the role of superoxide dismutase (SOD) in virulence against the silkworm, Bombyx mori, mutants of Pseudomonas aeruginosa PAO1 lacking manganese-SOD (PAO1sodM), iron-SOD (PAO1sodB), or both (PAO1sodMB) were generated. The mutants were injected into the hemocoel of B. mori. The virulence decreased in the order PAO1 = PAO1sodM > PAO1sodB > PAO1sodMB. In particular, PAO1sodMB was avirulent at a dose of 105 cells or less. The sod double mutant PAO1sodMB was then complemented with either pSodM or pSodB in trans. In both the complemented strains, the virulence was partially restored. Of the two plasmids, pSodB contributed more to the virulence of P. aeruginosa against B. mori. The results of growth in B. mori hemolymph broth and microscopic analysis suggested that a longer lag phase and superoxide sensitivity correlated with decreased virulence in sod mutants. In conclusion, the SODs are required for full virulence of P. aeruginosa against B. mori and Fe-SOD is more important than Mn-SOD in the infection process. [ABSTRACT FROM AUTHOR]

This paper describes the whole procedures to determine ice-induced global loads on the ship using measured full-scale data in accordance with the method proposed by the Canadian Hydraulics Centre of the National Research Council of Canada. Ship motions of 6 degrees of freedom (dof) are found by processing the commercial sensor signals named Motion Pak II under the assumption of rigid body motion. Linear accelerations as well as angular rates were measured by Motion Pak II data. To eliminate the noise of the measured data and the staircase signals due to the resolution of the sensor, a band pass filter that passes frequencies between 0.001 and 0.6 Hz and cubic spline interpolation resampling had been applied. 6 dof motions were computed by the integrating and/or differentiating the filtered signals. Added mass and damping force of the ship had been computed by the 3-dimensional panel method under the assumption of zero frequency. Once the coefficients of hydrodynamic and hydrostatic data as well as all the 6 dof motion data had been obtained, global ice loads can be computed by solving the fully coupled 6 dof equations of motion. Full-scale data were acquired while the ARAON rammed old ice floes in the high Arctic. Estimated ice impact forces for two representative events showed 7–15 MN when ship operated in heavy ice conditions.

Rheumatoid factors (RFs) are autoantibodies against the fragment- crystallizable (Fc) region of IgG. In individuals with hematological diseases such as cryoglobulinemia and certain B cell lymphoma forms, the RFs derived from specific heavy- and light-chain germline pairs, so-called "stereotypic RFs," are frequently produced in copious amounts and form immune complexes with IgG in serum. Of note, many structural details of the antigen recognition mechanisms in RFs are unclear. Here we report the crystal structure of the RF YES8c derived from the IGHV1-69/IGKV3-20 germline pair, the most common of the stereotypic RFs, in complex with human IgG1-Fc at 2.8 Å resolution. We observed that YES8c binds to the CH2-CH3 elbow in the canonical antigen-binding manner involving a large antigen-antibody interface. On the basis of this observation, combined with mutational analyses, we propose a recognition mechanism common to IGHV1-69/IGKV3-20 RFs: (1) the interaction of the Leu432-His435 region of Fc enables the highly variable complementarity-determining region (CDR)-H3 to participate in the binding, (2) the hydrophobic tip in the CDR-H2 typical of IGHV1-69 antibodies recognizes the hydrophobic patch on Fc, and (3) the interaction of the highly conserved RF light chain with Fc is important for RF activity. These features may determine the putative epitope common to the IGHV1-69/IGKV3-20 RFs. We also showed that some mutations in the binding site of RF increase the affinity to Fc, which may aggravate hematological diseases. Our findings unravel the structural basis for germline-encoded antibody autoreactivity. [ABSTRACT FROM AUTHOR]

N -glycosylation is an important posttranslational modification that results in a variety of biological activities, structural stability, and protein-protein interactions. There are still many mysteries in the structure and function of N-glycans, and detailed elucidation is necessary. Baculovirus expression system (BES) is widely used to produce recombinant glycoproteins, but it is not suitable for clinical use due to differences in N-glycan structure between insects and mammals. It is necessary to develop adequate model glycoproteins for analysis to efficiently alter the insect-type N-glycosylation pathway to human type. The previous research shows the recombinant alpha 1-acid glycoprotein (α1AGP) secreted from silkworm cultured cells or larvae is highly glycosylated and expected to be an excellent research candidate for the glycoprotein analysis expressed by BES. Therefore, we improved the α1AGP to be a better model for studying glycosylation. The modified α1AGP (α1AGPΔ) recombinant protein was successfully expressed and purified by using BES, however, the expression level in silkworm cultured cells and larvae were lower than that of the α1AGP. Subsequently, we confirmed the detailed profile of N-glycan on the α1AGPΔ by LS/MS analysis the N-glycan structure at each glycosylation site. These results indicated that the recombinant α1AGPΔ could be usable as a better model glycoprotein of N-glycosylation research in BES. [ABSTRACT FROM AUTHOR]

Recombinant proteins can be generated quickly and easily in large amounts and at low-cost in silkworm larvae by using Bombyx mori nuclear polyhedrosis virus (BmNPV). We searched for high-permissive silkworm strains that have high production levels of heterologous proteins and are thus suitable for use as biofactories. In this study, we performed the analysis using a BmNPV vector expressing luciferase as a marker, and we confirmed protein expression by evaluating luciferase activity, determined by western blotting and luciferase ELISA, and confirmed transcription expression by semi- and quantitative real time PCR. For the selection of host silkworm strains, we first chose 52 domestic BmNPV sensitive strains and then identified 10 high-permissive and 5 low-permissive strains. In addition, to determine which hybrid of the high-permissive strains would show heterosis, nine strains derived through threeway crossing were tested for luciferase activity by western blotting, and luciferase ELISA. We found a correlation between luciferase activity and luciferase protein expression, but not transcription. There was no noticeable difference in protein expression levels between Jam313 as the high-permissive control strain and the three-way hybrid strains; however, the three-way cross strains showed lower luciferase activity compared with Jam313. In this study, luciferase protein production in the larvae of 52 domestic silkworm strains was elucidated using BmNPV. [ABSTRACT FROM AUTHOR]

Demonstrates that the over-expression of wild-type calsequestrin (CSQ) or a CSQ mutant lacking the junction binding region in mouse skeletal C2C12 myotube enhanced caffeine- and voltage-induced calcium[sup 2+] release by increasing the calcium[sup 2+] load in sarcoplasmic reticulum. Role of the aspartate-rich segment of CSQ in sustaining structural interactions that interfere with store-operated calcium[sup 2+] entry mechanism; Implications on membrane transport and structure and biogenesis.