Biomedical subjects

Toru Shimada

Publications and source records attributed to Toru Shimada.

At least 19 recordsLinked to original sources

PubMed2006

N+NO reaction on Rh(111) surfaces studied with fast near-edge X-ray absorption fine structure spectroscopy: role of NO dimer as an extrinsic precursor.

We studied the mechanism of the N+NO reaction on Rh(111) surfaces by means of fast near-edge X-ray absorption fine structure spectroscopy. This reaction is important as a basis of NOx reduction reactions on platinum-group metal surfaces. Atomic nitrogen layers on Rh(111) were titrated with NO at various temperatures. N2O is exclusively formed and desorbs into the gas phase below 350 K. The consumption rate of atomic nitrogen exhibits strange temperature dependence between 100 and 350 K; the reaction proceeds slower with increasing temperature. Reaction kinetics analyses and isotope-controlled experiments have revealed that the surface N atoms do not react with chemisorbed NO molecules but with NO dimers weakly bound on top of the chemisorbed layer, which play a role as an extrinsic precursor. The present results may support the possibility that NO dimers participate in various NO-related synthetic, biochemical, and surface reactions as an intermediate.

Journal Article↗

PubMed2006

Mutational analysis of active site residues of chitinase from Bombyx mori nucleopolyhedrovirus.

Infection of Bombyx mori larvae with B. mori nucleopolyhedrovirus (BmNPV) results in liquefaction of the host. This process is attributed to the synergistic action of two virus-encoded genes, chitinase (v-chiA) and cathepsin (v-cath). Previous studies have suggested that Autographa californica nucleopolyhedrovirus (AcMNPV) CATH cannot be processed within infected cells in the absence of AcMNPV CHIA. To investigate the interactions between V-CHIA and V-CATH, we generated a recombinant BmNPV (103ChiAmut) in which the residues of the active site of BmNPV chiA were mutated (D302NE306Q) and the gene was driven by its own promoter at the native locus. Mutation at the active site of BmNPV CHIA resulted in complete loss of chitinolytic activity. Bombyx mori larvae infected with 103ChiAmut survived longer than larvae infected with wild-type BmNPV and did not undergo terminal liquefaction after death. Cysteine protease activity and Western blot analysis showed that, in cells infected with v-chiA-deleted BmNPV (ChiAD), BmNPV CATH was not processed properly and was accumulated as a detergent-insoluble form, suggesting that BmNPV CHIA plays a crucial role in V-CATH processing. In cells infected with 103ChiAmut, BmNPV CATH formed insoluble aggregates, suggesting that active site-mutated BmNPV CHIA loses its additional role as a molecular chaperon during V-CATH processing.

Amino Acid Substitution↗

PubMed2006

Structural study of NO adsorbed on the reconstructed Pt(110)-(1 x 2) surface with X-ray photoelectron diffraction and near-edge X-ray absorption fine structure spectroscopy.

The adsorption structure of NO on the reconstructed Pt(110)-(1 x 2) surface was studied with X-ray photoelectron spectroscopy (XPS), X-ray photoelectron diffraction (XPD), low-energy scanned-angle photoelectron diffraction (LESA-PD), and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy. The experiments were performed at 180 K, where no surface lifting from (1 x 2) to (1 x 1) takes place after NO adsorption. XPS indicates that the (1 x 2) unit cell of the Pt(110) surface contains 1.5 NO molecules at the saturated coverage. XPD and LESA-PD analyses allow us to propose a structural model for the NO adlayer, where two-thirds of the NO molecules in the (1 x 2) unit cell are adsorbed on the atop site of the close-packed Pt rows (ridges) along the [10] direction with an inclined geometry and one-third of the NO molecules adsorb on the bridge site between the Pt ridges with an upright configuration. This model is supported by the N K-edge NEXAFS experiments and is consistent with the recently reported model based on the density functional theory (Orita, H.; Nakamura, I.; Fujitani, T. J. Phys. Chem. B 2005, 109, 10312).

Journal Article↗

PubMed2006

N-linked glycans of Bombyx mori nucleopolyhedrovirus fibroblast growth factor are crucial for its secretion.

Bombyx mori nucleopolyhedrovirus (BmNPV) fibroblast growth factor (BmFGF) is a glycosylated protein that is efficiently secreted into the medium. Here, we constructed mutant NPVs expressing His-tagged wild-type (wt) or mutant BmFGFs and showed that the two residues, asparagine 44 and 171, are the glycosylation sites of BmFGF. Also, removal of N-linked glycans from BmFGF resulted in almost complete inhibition of the secretion into the medium, suggesting that N-linked glycans of BmFGF are required for its secretion. These residues are not conserved in closely related Autographa californica NPV (AcMNPV)-encoded vFGF (AcFGF). Western blot analysis suggested that AcFGF is not glycosylated and is poorly secreted. A mutant AcFGF possessing two N-linked glycosylation sites was secreted into the medium more abundantly than that which occurred for wt AcFGF. This is the first direct evidence showing the role of N-linked glycans in the secretion process of a baculovirus protein.

Amino Acid Sequence↗

PubMed2006

In vivo and in vitro analyses of a Bombyx mori nucleopolyhedrovirus mutant lacking functional vfgf.

All lepidopteran baculovirus genomes sequenced to date encode a viral fibroblast growth factor homolog (vfgf), suggesting that vfgf may play an important role in the infection cycle of lepidopteran baculoviruses. Here, we describe the characterization of a Bombyx mori nucleopolyhedrovirus (BmNPV) mutant lacking functional vfgf. We constructed a vfgf deletion mutant (BmFGFD) and characterized it in BmN cells and B. mori larvae. We observed that budded virus (BV) production was reduced in BmFGFD-infected BmN cells and B. mori larvae. The larval bioassays also revealed that deletion of vfgf did not reduce the infectivity; however, the mutant virus did take 20 h longer to kill B. mori larvae than wild-type BmNPV, when tested either by BV injection or by polyhedrin-inclusion body ingestion. These results suggest that BmNPV vfgf is involved in efficient virus production in BmN cells and B. mori larvae.

Animals↗

PubMed2006

Mechanism of CO oxidation reaction on O-covered Pd(111) surfaces studied with fast x-ray photoelectron spectroscopy: change of reaction path accompanying phase transition of O domains.

We studied the mechanism of CO oxidation on O-precovered Pd(111) surfaces by means of fast x-ray photoelectron spectroscopy (XPS). The oxygen overlayer is compressed upon CO coadsorption from a p(2 x 2) structure into a (square root(3) x square root(3))R30 degrees structure and then into a p(2 x 1) structure with increasing CO coverage. These three O phases exhibit distinctly different reactivities. (1) The p(2 x 2) phase does not react with CO unless the surface temperature is sufficiently high (<290 K). (2) In the square root(3) x square root(3))R30 degrees phase, the reaction occurs exclusively at island peripheries. CO molecules in a high-density phase formed under CO exposure react with oxygen atoms, leading to quite a small apparent activation energy. (3) The reaction proceeds uniformly over the islands in the p(2 x 1) phase.

Journal Article↗

PubMed2006

Comparative studies of Bombyx mori nucleopolyhedrovirus chitinase and its host ortholog, BmChi-h.

Baculovirus-encoded chitinases (V-CHIAs) were first proposed to be acquired from a bacterium via horizontal gene transfer. However, we have recently reported that lepidopteran hosts also encode v-chiA orthologs. Here we describe comparative studies of Bombyx mori nucleopolyhedrovirus (BmNPV) chitinase and its host ortholog, BmChi-h. We constructed recombinant BmNPVs in which native and modified forms of BmChi-h were driven under the polyhedrin promoter and the authentic v-chiA was deleted. Western blot analysis indicated that BmCHI-h was rapidly secreted from virus-infected BmN cells whereas BmNPV CHIA was localized within the virus-infected cells; probably because of the presence of a C-terminal endoplasmic reticulum retention motif on BmNPV CHIA. Enzymological studies showed that BmNPV CHIA was able to retain much higher chitinolytic activity under alkaline conditions. For B. mori larvae infected with v-chiA-deleted BmNPV, the terminal liquefaction of dead larvae and the activation of baculovirus-encoded cysteine protease were not observed, and the introduction of BmChi-h did not rescue these defects. Our findings show that BmNPV chiA possesses unique features that are not shared by host orthologs, which may reflect functional specialization of baculovirus chitinases.

Animals↗

PubMed2006

Role of the silkworm argonaute2 homolog gene in double-strand break repair of extrachromosomal DNA.

The argonaute protein family provides central components for RNA interference (RNAi) and related phenomena in a wide variety of organisms. Here, we isolated, from a Bombyx mori cell, a cDNA clone named BmAGO2, which is homologous to Drosophila ARGONAUTE2, the gene encoding a repressive factor for the recombination repair of extrachromosomal double-strand breaks (DSBs). RNAi-mediated silencing of the BmAGO2 sequence markedly increased homologous recombination (HR) repair of DSBs in episomal DNA, but had no effect on that in chromosomes. Moreover, we found that RNAi for BmAGO2 enhanced the integration of linearized DNA into a silkworm chromosome via HR. These results suggested that BmAgo2 protein plays an indispensable role in the repression of extrachromosomal DSB repair.

Amino Acid Sequence↗

PubMed2006

Lepidopteran ortholog of Drosophila breathless is a receptor for the baculovirus fibroblast growth factor.

The Bombyx mori nucleopolyhedrovirus (BmNPV) encodes a gene homologous to the mammalian fibroblast growth factor (FGF) family. We report the cloning of B. mori and Spodoptera frugiperda orthologous genes (Bmbtl and Sfbtl, respectively) of Drosophila melanogaster breathless (btl) encoding a receptor for Branchless/FGF and show that these genes encode the receptor for a baculovirus-encoded FGF (vFGF). Sequence analysis showed that BmBtl is composed of 856 amino acid residues, which potentially encodes a 97.3-kDa polypeptide and shares structural features and sequence similarities with the FGF receptor family. Reverse transcription-PCR experiments showed that Bmbtl was abundantly expressed in the trachea and midgut in B. mori larvae, with moderate expression observed in the hemocytes and the B. mori cultured cell line BmN. We generated Sf-9 cells that stably expressed His-tagged BmBtl. Western blot analysis revealed that BmBtl was an approximately 110-kDa protein. Immunoprecipitation experiments showed that BmNPV vFGF markedly phosphorylated BmBtl in Sf-9 cells. In addition, we found that BmBtl overexpression enhanced the migration activity for BmNPV vFGF. Furthermore, we generated Sf-9 cells in which Sfbtl was knocked down by transfection with double-strand RNA-expressing plasmids. In these cells, cell motility triggered by vFGF was markedly reduced. These results strongly suggest that the Btl orthologs, BmBtl and SfBtl, are the receptors for vFGF, which mediate vFGF-induced host cell chemotaxis.

Animals↗

PubMed2005

Simple sequence repeat-based consensus linkage map of Bombyx mori.

We established a genetic linkage map employing 518 simple sequence repeat (SSR, or microsatellite) markers for Bombyx mori (silkworm), the economically and culturally important lepidopteran insect, as part of an international genomics program. A survey of six representative silkworm strains using 2,500 (CA)n- and (CT)n-based SSR markers revealed 17-24% polymorphism, indicating a high degree of homozygosity resulting from a long history of inbreeding. Twenty-nine SSR linkage groups were established in well characterized Dazao and C108 strains based on genotyping of 189 backcross progeny derived from an F(1) male mated with a C108 female. The clustering was further focused to 28 groups by genotyping 22 backcross progeny derived from an F(1) female mated with a C108 male. This set of SSR linkage groups was further assigned to the 28 chromosomes (established linkage groups) of silkworm aided by visible mutations and cleaved amplified polymorphic sequence markers developed from previously mapped genes, cDNA sequences, and cloned random amplified polymorphic DNAs. By integrating a visible mutation p (plain, larval marking) and 29 well conserved genes of insects onto this SSR-based linkage map, a second generation consensus silkworm genetic map with a range of 7-40 markers per linkage group and a total map length of approximately 3431.9 cM was constructed and its high efficiency for genotyping and potential application for synteny studies of Lepidoptera and other insects was demonstrated.

Animals↗

PubMed2005

Glycine-rich protein genes, which encode a major component of the cuticle, have different developmental profiles from other cuticle protein genes in Bombyx mori.

Three types of GRP (glycine-rich proteins) cDNAs were identified in the EST database of Bombyx mori. These came from 21 ESTs in the W3-stage wing disc EST library. We named them BmGRP1, BmGRP2 and BmGRP3. BmGRP1 and BmGRP2 had 57% identity in deduced amino acid sequences. Expression of all BmGRPs was observed in the epidermis at the fourth molting stage, and in the wing at pupation and mid-pupal stage. It is suggested that BmGRPs contribute to larval, pupal and adult cuticles together with other cuticle proteins. Transcripts of BmGRP2 increased after 7 days of pupal stage. BmGRP2 is suggested to construct adult trachea in the wing. Hormonal response of BmGRPs was compared with that of another group of cuticular protein genes, BMWCPs. BmGRPs were induced by a pulse of 20E. Induction of BmGRP3 was observed in W1 wing discs in the presence of JHA which was added with 20E, whereas that of BMWCP2 was inhibited in the presence of JHA. Induction of BmGRPs was observed in the wing discs of V3 and W1 stages, while that of BMWCP2 was not observed in the V3 wing discs. These differences between BMWCPs and BmGRPs in response to hormones at different developmental stages are discussed.

Amino Acid Sequence↗

PubMed2005

The Bmdsx transgene including trimmed introns is sex-specifically spliced in tissues of the silkworm, Bombyx mori.

Bmdsx is an orthologue of the sex-determining gene doublesex (dsx) and known to be sex-specifically expressed in various tissues of the silkworm, Bombyx mori. Its pre-mRNA is sex-specifically spliced and encodes female-specific or male-specific polypeptides. The open reading frame of Bmdsx consists of 5 exons, of which exons 3 and 4 are female-specific and its pre-mRNA was known to undergo default processing to generate the female-type mRNA. Previous reports have shown that the mechanism of splicing of the doublesex gene is different in Drosophila melanogaster and Bombyx mori. However, intron 4 is so long that it is difficult to identify the intronic cis-element(s) required for male-specific splicing of Bmdsx pre-mRNA using Bmdsx minigenes whose introns are shortened in various manners. As a first step toward discovery of the cis-element, the Bmdsx mini gene, which consisted of exon 1 and 5 and internally shortened introns 2 to 4, was constructed, and transgenic silkworms expressing this construct were generated. Bmdsx pre-mRNA transcribed derived from transgene was sex-specifically spliced. This result shows that the mini gene contained the information necessary for the correct regulation of alternative splicing.

Alternative Splicing↗

PubMed2005

A baculovirus-encoded protein tyrosine phosphatase gene induces enhanced locomotory activity in a lepidopteran host.

Enhanced locomotory activity (ELA), such as wandering, is a normal behavior that occurs at the end of the larval stage in lepidopteran (butterflies and moths) insects. Baculovirus infection can also induce ELA in lepidopteran larvae. The belief is that the virus induces this behavior to increase its transmission [Goulson, D. (1997) Oecologia 109, 219-228]. Here we show that a baculovirus-encoded protein tyrosine phosphatase (PTP) gene (ptp) induces ELA that is activated by light. ELA was induced in silkworm Bombyx mori infected with the baculovirus B. mori nucleopolyhedrovirus (BmNPV) beginning at approximately 3.75 days postinfection (p.i.) and continued until 4.75 days p.i. The intensity of the ELA was dramatically reduced immediately before death at 5.25 days p.i. Light activated the intensity of the ELA by approximately 3-fold, and larvae with ELA showed positive phototropism. ELA was not induced in larvae of B. mori infected with a BmNPV ptp knockout mutant (BmPTPD). However, when a silkworm-derived ptp gene (Bmptp-h) was inserted into BmPTPD, ELA was partially recovered. Bmptp-h was identified from silkworms at 2 days after the start of the natural wandering stage. The deduced amino acid sequence of Bmptp-h showed 48.2% identify (80.7% similarity) to the deduced amino acid sequence of BmNPV ptp. On the basis of the high homology and larval stage at which Bmptp-h was isolated, we postulate that the modern baculovirus may have acquired its ptp gene from an ancestral host and that this gene was selectively maintained because it increases virus transmission.

Amino Acid Sequence↗

PubMed2005

Characteristics of two genes encoding proteins with an ADAM-type metalloprotease domain, which are induced during the molting periods in Bombyx mori.

By microarray analyses, we identified two genes (BmADAMTS-1 and BmADAMTS-like) encoding a protein, which are induced during the pupal ecdysis in the wing discs of Bombyx mori; these genes are homologous to ADAMTS family members (a disintegrin and metalloproteinase domain, with thrombospondin type-1 repeats). A complete metal-binding motif of the ADAM-type metalloprotease domain (HEXXHXXGXXHD) was contained in both amino acid sequences. However, thrombospondin type 1 (TSP-1) repeats were observed only in BmADAMTS-1. The BmADAMTS-1 gene was expressed in the hemocyte and midgut of the larvae at day 2 of wandering stage (W2), and strongly induced during the pupal ecdysis in the hemolymph. The BmADAMTS-like gene was expressed in the epithelial tissues of the larvae at W2, and had expression peaks slightly later than the BmADAMTS-1 gene. Our results indicate that BmADAMTS-1 and BmADAMTS-like may cleave the extracellular matrix (ECM) in the degenerating and remodeling tissues during the molting periods.

ADAM Proteins↗

PubMed2005

Identification of molting fluid carboxypeptidase A (MF-CPA) in Bombyx mori.

Using microarray analyses, we identified carboxypeptidase A (MF-CPA), which was induced during pupal ecdysis in the wing discs of Bombyx mori. Here, we report the functional characterization of MF-CPA. MF-CPA has amino acid sequence similarities with the proteins in the carboxypeptidase A/B subfamily, from human to nematode. The MF-CPA gene is expressed during the molting periods in the epithelial tissues. MF-CPA is detected in the molting fluid, which fills the space between the old and new cuticle during molting. By Western blot analysis, we show that MF-CPA is secreted as a zymogen and processed in the molting fluid. Recombinant MF-CPA expressed in the insect cells has carboxypeptidase A activity. We propose that MF-CPA degrades the proteins from the old cuticle during the molting periods and contributes to recycling of the amino acids.

Amino Acid Sequence↗

PubMed2005

Accurate pre-mRNA splicing using a nuclear extract from Bombyx mori fat body.

A cell-free pre-mRNA splicing system was developed from the nuclear extract of Bombyx mori fat bodies. The synthetic pre-mRNA from the cytoplasmic actin gene of B. mori (BmA3 ) was used as a substrate for in vitro splicing. The pre-mRNA was accurately spliced in the nuclear extract prepared from the fat body. Sequencing of the amplicon by RT-PCR revealed that the spliced site in vitro was identical to the site in vivo. We found that a part of the input capped pre-mRNA was accurately spliced after 2h of incubation. In addition, the RT-PCR analysis precisely mapped the branch point to an adenine located at 30 nucleotides before the 3' end of the first intron in BmA3. This system was a first example that differentiated tissue in insects yielded active extracts for in vitro splicing.

Actins↗

PubMed2005

The BmChi-h gene, a bacterial-type chitinase gene of Bombyx mori, encodes a functional exochitinase that plays a role in the chitin degradation during the molting process.

The silkworm, Bombyx mori, has been recently demonstrated to contain a bacterial-type chitinase gene (BmChi-h) in addition to a well-characterized endochitinase gene (BmChitinase). The deduced amino acid sequence of BmChi-h showed extensive structural similarities with chitinases from bacteria such as Serratia marcescens chiA and baculoviruses (v-CHIA). Bacterial-type chitinase genes have not been found from any eukaryotes and viruses except for lepidopteran insects and lepidopteran baculoviruses. Thus, it was suggested that BmChi-h may be derived from a bacterial or baculovirus chitinase gene via horizontal gene transfer. In this report, we investigated the biological function of BmChi-h. Our enzymological study indicated that a chitinase encoded by BmChi-h has exo-type substrate preference, which is the same as S. marcescens chiA and v-CHIA, and different from BmChitinase, which has endo-type substrate preference. An immunohistochemical study revealed that BmChi-h localizes in the chitin-containing tissues during the molting stages, indicating that it plays a role in chitin degradation during molting. These results suggest that BmChi-h (exochitinase) and BmChitinase (endochitinase) may catalyze a native chitin by a concerted mechanism. Cloning and comparison of BmChi-h orthologues revealed that bacterial-type chitinase genes are highly conserved among lepidopteran insects, suggesting that the utilization of a bacterial-type chitinase during the molting process may be a general feature of lepidopteran insects.

Amino Acid Sequence↗

PubMed2005

Novel macula-like virus identified in Bombyx mori cultured cells.

We identified a novel, 6,513-bp-long RNA, termed Bombyx mori macula-like latent virus (BmMLV) RNA, which abundantly expressed in B. mori cultured BmN cells. BmMLV RNA potentially encodes two proteins, putative RNA replicase and coat protein, which share structural features and sequence similarities with those of a plant RNA virus, the genus Maculavirus. Northern blot analysis showed that two transcripts were expressed in BmN cells: a 6.5-kb-long RNA, which contains both putative RNA replicase and coat protein genes, and a 1.2-kb-long RNA, which contains only a coat protein gene. Southern blot analysis showed that BmMLV RNA is not carried by the B. mori genome. RT-PCR analysis also revealed the presence of BmMLV RNA in several B. mori cell lines other than BmN cells, suggesting that BmMLV RNA latently exists in B. mori cultured cells. Infection studies showed that BmMLV virions were able to infect BmMLV-negative Spodoptera frugiperda Sf-9 cells and B. mori larvae. Electron microscopy and Northern blot analysis of a purified BmMLV revealed that isometric virions appear to be 28 to 30 nm in diameter and contain a 6.5-kb genomic RNA. These results showed that BmMLV is a novel macula-like virus infectious to and replicable in B. mori-derived cells.

Amino Acid Sequence↗