Shigeo Imanishi

National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan

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Publications (34)75.43 Total impact

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    ABSTRACT: Juvenile hormone (JH) regulates many physiological processes in insects. However, the signal cascades in which JH is active have not yet been fully elucidated, particularly in comparison to another major hormone ecdysteroid. Here we identified two JH inducible transcription factors as candidate components of JH signaling pathways in the silkworm, Bombyx mori. DNA microarray analysis showed that expression of two transcription factor genes, E75 and Enhancer of split mβ (E(spl)mβ), was induced by juvenile hormone I (JH I) in NIAS-Bm-aff3 cells. Real time RT-PCR analysis confirmed that expression of four E75 isoforms (E75A, E75B, E75C and E75D) and E(spl)mβ was 3 to 8 times greater after JH I addition. Addition of the protein synthesis inhibitor cycloheximide did not suppress JH-induced expression of the genes, indicating that they were directly induced by JH. JH-induced expression of E75 and E(spl)mβ was also observed in four other B. mori cell lines and in larval hemocytes of final instar larvae. Notably, E75A expression was induced very strongly in larval hemocytes by topical application of the JH analog fenoxycarb; the level of induced expression was comparable to that produced by feeding larvae with 20-hydroxyecdysone. These results suggest that E75 and E(spl)mβ are general and direct target genes of JH and that the transcription factors encoded by these genes play important roles in JH signaling. Copyright © 2015. Published by Elsevier Ltd.
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    ABSTRACT: We previously established the first Bombyx mori macula-like virus (BmMLV)-free cell line (BmVF cells) from a B. mori embryo. In this study, we evaluated the expression of recombinant proteins in BmVF cells using a B. mori nucleopolyhedrovirus (BmNPV)-derived expression vector. Our results showed that BmVF cells are susceptible to BmNPV, and both the promoter activity of the polyhedrin gene and the post-translated modifications of a recombinant protein are equivalent between BmMLV-negative BmVF and -positive BmN4 cells. These findings indicate that persistent infection with BmMLV has no discernible effect on BmNPV-mediated protein production in B. mori cells.
    Journal of Invertebrate Pathology 11/2014; DOI:10.1016/j.jip.2014.09.004 · 2.60 Impact Factor
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    ABSTRACT: Three distinct classes of nuclear receptors, EcR, E75, and HR3, are key regulators in the ecdysone-inducible gene activation cascade in insects. The transcription of these genes is induced by ecdysone (20E) differently, although the detailed mechanisms underlying their responses to 20E are largely unknown. We identified ecdysone response elements (EcREs) present in the promoters of genes coding BmEcR-B1, BmE75-A, and BHR3-B isoforms from Bombyx mori employing luciferase reporter assays in an ecdysteroid-responsive cultured cell line, NIAS-Bm-aff3 (aff3). The EcRE of BmEcR-B1 at -2800 comprises of two adjacent elements separated by 5 bp, E1 (15 bp) and E2 (21 bp), both of which are required for the 20E response. Further analysis using electrophoretic mobility shift assays showed that E1 binds to the EcR/USP heterodimer and that E2 may bind to the E-box (CACGTG) binding factor such as bHLH protein. The unique E1+E2-type EcRE is also detected in the promoter upstream regions of EcR-B1 from seven lepidopteran species studied. In contrast, both a 20 bp EcRE identified in the promoter of BmE75-A and a 18 bp EcRE identified in the BHR3-B promoter, contained only E1-type EcR/USP binding element but the E2 type element was not in the promoter regions of these genes. The combination of presence of the E2 element or other cis-regulatory elements in promoter regions explains the different 20E response of each class of nuclear receptor genes. Furthermore, the E1+E2 structure for EcR-B1 can be involved in a possible cross-talk between ecdysteroid and other regulatory pathways.
    PLoS ONE 11/2012; 7(11):e49348. DOI:10.1371/journal.pone.0049348 · 3.53 Impact Factor
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    ABSTRACT: The Krüppel homolog 1 gene (Kr-h1) has been proposed to play a key role in the repression of insect metamorphosis. Kr-h1 is assumed to be induced by juvenile hormone (JH) via a JH receptor, methoprene-tolerant (Met), but the mechanism of induction is unclear. To elucidate the molecular mechanism of Kr-h1 induction, we first cloned cDNAs encoding Kr-h1 (BmKr-h1) and Met (BmMet1 and BmMet2) homologs from Bombyx mori. In a B. mori cell line, BmKr-h1 was rapidly induced by subnanomolar levels of natural JHs. Reporter assays identified a JH response element (kJHRE), comprising 141 nucleotides, located ∼2 kb upstream from the BmKr-h1 transcription start site. The core region of kJHRE (GGCCTCCACGTG) contains a canonical E-box sequence to which Met, a basic helix-loop-helix Per-ARNT-Sim (bHLH-PAS) transcription factor, is likely to bind. In mammalian HEK293 cells, which lack an intrinsic JH receptor, ectopic expression of BmMet2 fused with Gal4DBD induced JH-dependent activity of an upstream activation sequence reporter. Meanwhile, the kJHRE reporter was activated JH-dependently in HEK293 cells only when cotransfected with BmMet2 and BmSRC, another bHLH-PAS family member, suggesting that BmMet2 and BmSRC jointly interact with kJHRE. We also found that the interaction between BmMet2 and BmSRC is dependent on JH. Therefore, we propose the following hypothesis for the mechanism of JH-mediated induction of BmKr-h1: BmMet2 accepts JH as a ligand, JH-liganded BmMet2 interacts with BmSRC, and the JH/BmMet2/BmSRC complex activates BmKr-h1 by interacting with kJHRE.
    Proceedings of the National Academy of Sciences 07/2012; 109(29):11729-34. DOI:10.1073/pnas.1204951109 · 9.81 Impact Factor
  • Shigeo Imanishi, Jun Kobayashi, Toshiaki Sekine
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    ABSTRACT: We established the first continuous cell line that uses a serum-free culture from the embryo of Bombyx mori (Lepidoptera: Bombycidae), designated as NIAS-Bm-Ke17. This cell line was serially subcultured in the SH-Ke-117 medium. The cells adhere weakly to the culture flask, and most cells have an oval shape. The cell line was subcultured 154 times, and the population doubling time is 83.67±5.22 h. Random amplification of polymorphic DNA-polymerase chain reaction with a tenmar single primer for discrimination of insect cell lines recognized the NIAS-Bm-Ke1 cell line as B. mori. This cell line does not support the growth of the B. mori nuclear polyhedrosis virus (BmNPV) in the absence of the heat-inactivated hemolymph of B. mori. However, the heat-inactivated hemolymph in 1% volume of the medium supported a high level of susceptibility to BmNPV. In addition, the cooling treatment of the cells at 2.5°C also enhanced the susceptibility. We report a new serum-free culture system of the B. mori cell line for the baculovirus expression vector system.
    In Vitro Cellular & Developmental Biology - Animal 02/2012; 48(3):137-42. DOI:10.1007/s11626-011-9465-9 · 1.00 Impact Factor
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    ABSTRACT: Cabbage butterfly, Pieris rapae, contains a unique DNA ADP-ribosylating protein, pierisin-1, which transfers ADP-ribose moiety of NAD to guanine bases of DNA. Pierisin-like proteins are only distributed in subtribes Pierina, Aporiina and Appiadina of the family Pieridae. In this study, we obtained genomic clones carrying the pierisin-1 gene from adult samples of P. rapae by plaque hybridization. The pierisin-1 gene was found to consist of two exons, 0.1-kb exon 1 and 3.9-kb exon 2, and a 2.3-kb intron. In addition, we could demonstrate that the putative promoter in the about 3-kb upstream region from the transcription start site of the gene include a transcriptional activating motif involved in immune pathways and hormonal regulation. We also examined chromosomal localization of the pierisin-1 gene. Fluorescence in situ hybridization (FISH) analysis using Cy3-labeled pierisin-1 genomic clone demonstrated the localization of the gene near the kinetochore in chromosome 9. Thus, we confirmed that the pierisin-1 gene is located in the genome of P. rapae.
    Genetica 01/2012; 139(10):1251-8. DOI:10.1007/s10709-011-9626-5 · 1.75 Impact Factor
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    ABSTRACT: Previously, a novel macula-like virus was identified from Bombyx mori cultured cell line BmN and termed B. mori macula-like virus (BmMLV). BmMLV encodes a 6.5-kb-long positive, single-strand RNA genome, which contains putative RNA-dependent RNA polymerase (RdRp), coat protein (cp) and p15 genes. In this study, CP expression in several B. mori-derived cell lines was examined by using the CP antibody. Surprisingly, Western blot analysis revealed that all of the cell lines tested have already been infected with BmMLV. To perform reverse genetic studies in BmMLV, a new BmMLV-negative cell line, designated as BmVF from the embryos of B. mori was established. Infection studies showed that BmVF cells were permissive to BmMLV persistent infection. In addition, a full-length infectious cDNA clone of BmMLV, termed pHMLV was developed. Upon transfection of pHMLV into BmMLV-negative BmVF cells, viral CP was detected in both cells and conditioned medium. When the cDNA-derived virus in conditioned medium was inoculated onto BmVF cells, efficient propagation of BmMLV was observed. Collectively, these results indicate that the new BmMLV-negative cell line and the infectious cDNA clone of BmMLV will be useful for elucidation of the mechanism of BmMLV replication and the functional roles of BmMLV genes.
    Journal of virological methods 11/2011; 179(2):316-24. DOI:10.1016/j.jviromet.2011.11.016 · 1.88 Impact Factor
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    ABSTRACT: Transfection of an expression plasmid possessing inverted repeat (IR) DNA into cultured cells leads to the overexpression of hairpin RNA and efficient suppression of target gene expression. Such DNA vector-based RNA interference (RNAi) is widely used for characterizing genes of interest in cultured cell lines. In this study, we developed a new method to convert an inserted DNA fragment (IDF) in specially designed plasmid vectors into an IR structure by using nicking endonucleases and BcaBEST DNA polymerase. This method consists of the following steps: (1) linearization of the plasmid with a nick by using a restriction enzyme and a nicking endonuclease, (2) formation of the hairpin-loop DNA at the end near the IDF of the linearized plasmid, (3) insertion of a nick at the other end of the IDF by a nicking endonuclease, (4) execution of the strand displacement reaction from the nick to synthesize IR DNA, and (5) self-ligation of the linear double-stranded DNA. The IR DNA containing expression plasmids constructed by this method effectively induced target-specific RNAi in a silkworm cell line. We further established a method to purify expression plasmids containing IR DNA. Our new methods provide techniques for the construction of long hairpin RNA (lhRNA) expression plasmids for silencing specific genes in silkworms and other organisms, and offer a fundamental methodology for constructing an lhRNA expression library from a cDNA plasmid library.
    Molecular Biotechnology 04/2011; 50(1):18-27. DOI:10.1007/s12033-011-9408-4 · 2.28 Impact Factor
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    ABSTRACT: Wolbachia and Cardinium are bacterial endosymbionts that are widely distributed amongst arthropods. Both cause reproductive alterations, such as cytoplasmic incompatibility, parthenogenesis and feminization. Here we studied differentially expressed genes in Wolbachia- and Cardinium-infected Bm-aff3 silkworm cells using a silkworm microarray. Wolbachia infection did not alter gene expression or induce or suppress immune responses. In contrast, Cardinium infection induced many immune-related genes, including antimicrobial peptides, pattern recognition receptors and a serine protease. Host immune responses differed, possibly because of the different cell wall structures of Wolbachia and Cardinium because the former lacks genes encoding lipopolysaccharide components and two racemases for peptidoglycan formation. A few possibly non-immune-related genes were differentially expressed, but their involvement in host reproductive alteration was unclear.
    Insect Molecular Biology 02/2011; 20(3):279-89. DOI:10.1111/j.1365-2583.2010.01056.x · 2.98 Impact Factor
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    ABSTRACT: Bmdsx is a sex-determining gene in the silkworm and is alternatively spliced in males and females. CE1 is a splicing silencer element responsible for the sex-specific splicing of Bmdsx. To identify sex-specific factors implicated in the sex-specific splicing of Bmdsx, we performed RNA affinity chromatography using CE1 RNA as a ligand. We have identified BmIMP, a Bombyx homolog of IGF-II mRNA binding protein (IMP), as a male-specific factor that specifically binds to CE1. The gene encoding BmIMP is localized on the Z chromosome and is male-specifically expressed in various tissues. Antisense inhibition of BmIMP expression increased female-specific splicing of Bmdsx pre-mRNA. Coimmunoprecipitation and glutathione S-transferase (GST) pulldown analyses demonstrated that BmIMP physically interacts with BmPSI, which has been identified as a factor implicated in the sex-specific splicing of Bmdsx, through the KH domains of BmIMP. The functional consequence of this interaction was examined using RNA mobility shift analysis. BmIMP increased BmPSI-CE1 RNA binding activity by decreasing the rate of BmPSI dissociation from CE1 RNA. Truncation analysis of BmIMP suggested that the KH domains are responsible for enhancing BmPSI-CE1 RNA binding activity. These results suggest that BmIMP may enhance the male-specific splicing of Bmdsx pre-mRNA by increasing RNA binding activity of BmPSI.
    Molecular and Cellular Biology 10/2010; 30(24):5776-86. DOI:10.1128/MCB.00444-10 · 5.04 Impact Factor
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    ABSTRACT: A palmitoyl conjugate of an insect pentapeptide that occurs in diapausing insects causes a reversible cell-cycle arrest and suppresses mitochondrial respiration. This peptide compound also causes growth arrest in murine leukemic cell line expressing human gene Bcr/Abl and a farnesoyl peptide induces embryonic diapause in Bombyx mori. These results demonstrate that the insect peptide compounds can lead to the understanding of a common pathway in developmental arrest in animals and may provide a new peptidominetic analog in the development of biopharmaceuticals and pest management.
    Peptides 03/2010; 31(5):827-33. DOI:10.1016/j.peptides.2010.02.017 · 2.61 Impact Factor
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    ABSTRACT: In this study, we have newly identified three bacteria-induced genes from the silkworm Bombyx mori by quantitative reverse transcriptase-polymerase chain reaction. One of these, eukaryotic initiation factor 4E-1 (eIF4E-1), is assumed to encode an eIF4E family, which plays a role in the initiation of translation as a mRNA cap-binding protein. The second gene is BmFOXG1, belonging to a family of forkhead transcription factors, FOXG1. The third gene is MBF2-related (MBF2-R) whose product has high homology to a co-activator protein MBF2 from B. mori. Although BmFOXG1 was up-regulated in the fat body in response to three kinds of bacteria, Escherichia coli, Staphylococcus aureus, and Bacillus subtilis, eIF4E-1 and MBF2-R were up-regulated by E. coli and B. subtilis, but not S. aureus, suggesting that bacteria possessing meso-diaminopimelic acid-containing peptidoglycan but not lysine-containing peptidoglycan activate eIF4E-1 and MBF2-R, probably through a conserved immune deficiency pathway. We further profiled the expression of three genes in different tissues and a silkworm cell line, NIAS-Bm-aff3, in response to bacteria, and at different times after bacterial challenge in the fat body.
    Archives of Insect Biochemistry and Physiology 01/2010; 73(3):148-62. DOI:10.1002/arch.20347 · 1.16 Impact Factor
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    ABSTRACT: A cDNA encoding an IkappaB family protein was identified and the full nucleotide sequence was determined in the silkworm Bombyx mori. The IkappaB gene, designated BmCactus, was constitutively expressed mainly in the fat body and hemocytes. Transfection experiments on a B. mori cell line, NIAS-Bm-aff3, with expression vectors containing BmCactus, BmRelA, BmRelB, or the active portion of BmRelish1 showed that activation of the CecB1 gene promoter by either BmRelA or BmRelB, but not the active portion of BmRelish1, was strongly inhibited by BmCactus. In addition, activation of CecB1 gene by autoclaved E. coli in the cultured cells was observed regardless of the presence or absence of BmCactus. A glutathione S-transferase pull-down assay and analysis using a yeast two-hybrid system demonstrated that BmCactus interacted with the BmRel Rel homology domain, but not with the BmRelish Rel homology domain. These results suggest that BmCactus is involved in the Toll signal transduction pathway in B. mori.
    Bioscience Biotechnology and Biochemistry 12/2009; 73(12):2665-70. DOI:10.1271/bbb.90511 · 1.21 Impact Factor
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    ABSTRACT: Gene-knockdown technology using RNA interference (RNAi) is widely used to characterize gene functions in many organisms. In this study, we analyzed the conditions for employing DNA vector-based RNAi in silkworm cell lines using long-hairpin RNA-expressing plasmid DNA. We found that NIAS-Bm-oyanagi2 was the most effective cell line for RNAi. Expression of long-hairpin RNA containing an approximately 500 base-pair stem region suppressed expression of a reporter target gene by more than 99% in this cell line. Furthermore, the loop sequence of hairpin RNA was not as important to RNAi efficiency as previously observed in Drosophila melanogaster. DNA vector-based RNAi also induced significant suppression of endogenous clathrin in NIAS-Bm-oyanagi2. Luciferase activity from recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) containing luciferase in the clathrin-knockdown cells was significantly less than in the control cells, suggesting that clathrin is indispensable for the entry of BmNPV into silkworm cell lines.
    Bioscience Biotechnology and Biochemistry 10/2009; 73(9):2026-31. DOI:10.1271/bbb.90223 · 1.21 Impact Factor
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    ABSTRACT: The global transcriptional profile of host genes in the silkworm cell line during the early phase of Bombyx mori nucleopolyhedrovirus (BmNPV) infection was analyzed by oligonucleotide microarray. Our analysis showed 35 genes were significantly up-regulated and 17 genes were significantly down-regulated. This is the first report of changes in the expression of these genes in response to NPV infection. We further quantified the levels of mRNA expression by quantitative reverse transcriptase-polymerase chain reaction and confirmed that the expression of 13 (such as BmEts and BmToll10-3) genes significantly increased and 7 genes (such as Hsp20-1) significantly decreased after BmNPV infection. However, the expression levels of most genes were not dramatically changed except BmEts expression increased approximately 8.0-fold 12h after BmNPV infection.
    Virus Research 10/2009; 147(2):166-75. DOI:10.1016/j.virusres.2009.10.015 · 2.83 Impact Factor
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    ABSTRACT: Dry-preservation of nucleated cells from multicellular animals represents a significant challenge in life science. As anhydrobionts can tolerate a desiccated state, their cells and organs are expected to show high desiccation tolerance in vitro. In the present study, we established cell lines derived from embryonic tissues of an anhydrobiotic chironomid, Polypedilum vanderplanki, designated as Pv11 and Pv210. Salinity stress induced the expression of a set of anhydrobiosis-related genes in both Pv11 and Pv210 cells, suggesting that at least a part of cells can autonomously control the physiological changes for the entry into anhydrobiosis. When desiccated with medium supplemented with 300 mM trehalose or sucrose and stored for 4 weeks in dry air (approximately 5% relative humidity), a small percentage of the cells was found to be viable upon rehydration, although surviving cells seemed not to be able to multiply. We also attempted dry-preservation of organs isolated from P. vanderplanki larvae, and found that a proportion of cells in some organs, including fat body, testis, nerve and dorsal vessel, tolerated in vitro desiccation.
    Cryobiology 10/2009; 60(2):138-46. DOI:10.1016/j.cryobiol.2009.10.004 · 1.64 Impact Factor
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    ABSTRACT: A new cell line, designated as NIAS-Boma-529b, was established from the larval fat bodies of Bombyx mandarina (B. mandarina), which is believed to be an ancestor of Bombyx mori (B. mori). This cell line has been cultured for approximately 150 passages during 2years in an IPL-41 medium supplemented with 10% fetal bovine serum at a constant temperature of 26 degrees C. The morphology of this line includes adhesive round and spindle-shaped cells. Random-amplified polymorphic DNA analysis (RAPD) using 7 primers and a statistical analysis based on Nei's genetic distance revealed that this cell line was closely related to B. mori-derived cell lines. An infection study also revealed that this cell line was susceptible to B. mori nucleopolyhedrovirus (BmNPV); however, it had no apparent susceptibility to Autographa californica NPV (AcNPV), which is closely related to BmNPV. Nevertheless, cells infected with AcNPV showed an extensive cytopathic effect (CPE), including a rough cell surface, rounding, nuclear expansion, and cell blebbing. These results suggest that this cell line can be useful to clarify the mechanism of host range determination of BmNPV and AcNPV.
    Journal of Invertebrate Pathology 06/2009; 101(2):124-9. DOI:10.1016/j.jip.2009.05.004 · 2.60 Impact Factor
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    ABSTRACT: In the silkworm, Bombyx mori, antimicrobial peptide (AMP) genes are upregulated in the larval fat body by injection of bacteria and peptidoglycans (PGNs). The DAP-type PGN from Escherichia coli and Bacillus subtilis exhibited stronger elicitor activity for expression of AMP genes in B. mori than Lys-type PGN from Staphylococcus aureus, suggesting that differences in bacterial influence on the induction levels of these genes depend on the differences in types of PGN. BmRelish1 mRNA was more abundant than BmRel mRNAs in the larval fat bodies. Moreover, the ability of the BmRelish1 active form to enhance the promoter activity of AMP genes was higher than that of BmRels. The difference was related to the binding affinity of Rel family proteins to kappaB sites. Our results suggest that different amounts and different transcriptional activities of Rel family proteins result in differential activation of AMP genes by PGN type and bacterium species.
    Bioscience Biotechnology and Biochemistry 04/2009; 73(3):599-606. DOI:10.1271/bbb.80685 · 1.21 Impact Factor
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    ABSTRACT: Lipopolysaccharide (LPS), a major cell wall component of gram-negative bacteria, was found to be unable to activate immune-related genes in Drosophila melanogaster. In contrast, highly purified LPS elicited immune-related gene expression in the fat body of Bombyx mori. However, the level of activation by highly purified LPS was lower than crude LPS and peptidoglycan. Furthermore, synthetic lipid A also activated these genes, suggesting that B. mori possesses unknown signal pathways to activate immune-related genes by LPS. Up-regulation of antimicrobial peptide genes by highly purified LPS was not confirmed in the immune-responsive cell line, NIAS-Bm-aff3, suggesting that some factors necessary for signal transduction activated by LPS are deficient in this cell line.
    Insect Molecular Biology 03/2009; 18(1):71-5. DOI:10.1111/j.1365-2583.2009.00851.x · 2.98 Impact Factor