Kyushu University [takahiro kusakabe (Professor) Faculty of Agriculture, Department of Bioresource Sciences]

takahiro kusakabe

Last modified date：2023.11.27

Professor / Agricultural Bioresource Sciences / Department of Bioresource Sciences / Faculty of Agriculture

Papers

1.	Yuri Kato, Kazuhiro Nishiyama, Jae Man Lee, Yuko Ibuki, Yumiko Imai, Takamasa Noda, Noriho Kamiya, Takahiro Kusakabe, Yasunari Kanda, Motohiro Nishida, TRPC3-Nox2 Protein Complex Formation Increases the Risk of SARS-CoV-2 Spike Protein-Induced Cardiomyocyte Dysfunction through ACE2 Upregulation., International journal of molecular sciences, 10.3390/ijms24010102, 24, 1, 2022.12, Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of the key determinants of COVID-19 severity and mortality. SARS-CoV-2 entry to host cells is initiated by binding with its receptor, angiotensin-converting enzyme (ACE) 2, and the ACE2 abundance is thought to reflect the susceptibility to infection. Here, we report that ibudilast, which we previously identified as a potent inhibitor of protein complex between transient receptor potential canonical (TRPC) 3 and NADPH oxidase (Nox) 2, attenuates the SARS-CoV-2 spike glycoprotein pseudovirus-evoked contractile and metabolic dysfunctions of neonatal rat cardiomyocytes (NRCMs). Epidemiologically reported risk factors of severe COVID-19, including cigarette sidestream smoke (CSS) and anti-cancer drug treatment, commonly upregulate ACE2 expression level, and these were suppressed by inhibiting TRPC3-Nox2 complex formation. Exposure of NRCMs to SARS-CoV-2 pseudovirus, as well as CSS and doxorubicin (Dox), induces ATP release through pannexin-1 hemi-channels, and this ATP release potentiates pseudovirus entry to NRCMs and human iPS cell-derived cardiomyocytes (hiPS-CMs). As the pseudovirus entry followed by production of reactive oxygen species was attenuated by inhibiting TRPC3-Nox2 complex in hiPS-CMs, we suggest that TRPC3-Nox2 complex formation triggered by panexin1-mediated ATP release participates in exacerbation of myocardial damage by amplifying ACE2-dependent SARS-CoV-2 entry..
2.	Hiroaki Mon, Masanao Sato, Jae Man Lee, Takahiro Kusakabe, Construction of gene co-expression networks in cultured silkworm cells and identification of previously uncharacterized lepidopteran-specific genes required for chromosome dynamics., Insect biochemistry and molecular biology, 10.1016/j.ibmb.2022.103875, 151, 103875-103875, 2022.12, Advances in sequencing technology and bioinformatics have accelerated gene discovery and homology-based functional annotation in many species, and numerous targeted gene studies have greatly expanded the understanding of gene functions. Nevertheless, there are still many genes that lack homology with genes in other evolutionary lineages and are left as genes with unknown functions. We constructed a gene co-expression network from the Bombyx mori ovary-derived cell line, BmN4, and attempted to infer the biological roles of uncharacterized genes based on the correlation between the function-known and unknown genes. Within this network, we focused on the co-expression modules involved in chromosome architecture, dynamics, and integrity, and selected the uncharacterized genes for subsequent RNAi-based phenotypic screening. This approach enabled the identification of 5 genes whose knockdown led to abnormalities in chromosome dynamics and spindle morphology in mitosis. One of them was a recently characterized gene, BmCenp-T, which plays a central role in building the kinetochore protein complex on the silkworm holocentric chromosomes. In this study, we suggest a method for constructing the gene co-expression network and selecting candidate genes for small-scale RNAi screening. This approach is complementary to homology-based annotation and may be useful for the analysis of lineage-specific uncharacterized genes such as orphan genes..
3.	Akihiro Morio, Jae Man Lee, Tsuguru Fujii, Hiroaki Mon, Akitsu Masuda, Kohei Kakino, Jian Xu, Yutaka Banno, Takahiro Kusakabe, The biological role of core 1β1-3galactosyltransferase (T-synthase) in mucin-type O-glycosylation in Silkworm, Bombyx mori, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2023.103936, 156, 103936-103936, 2023.05.
4.	Akitsu Masuda, Jae Man Lee, Takeshi Miyata, Shintaro Sato, Atsushi Masuda, Masahiro Taniguchi, Ryosuke Fujita, Hiroshi Ushijima, Keisuke Morimoto, Takeru Ebihara, Masato Hino, Kohei Kakino, Hiroaki Mon, Takahiro Kusakabe, High yield production of norovirus GII.4 virus-like particles using silkworm pupae and evaluation of their protective immunogenicity., Vaccine, 10.1016/j.vaccine.2022.12.015, 41, 3, 766-777, 2023.01, Noroviruses (NoVs) are one of the major causes of acute viral gastroenteritis in humans. Virus-like particles (VLPs) without genomes that mimic the capsid structure of viruses are promising vaccine candidates for the prevention of NoVs infection. To produce large amounts of recombinant protein, including VLPs, the silkworm-expression vector system (silkworm-BEVS) is an efficient and powerful tool. In this study, we constructed a recombinant baculovirus that expresses VP1 protein, the major structural protein of NoV GII.4. Expression analysis showed that the baculovirus-infected silkworm pupae expressed NoV VP1 protein more efficiently than silkworm larval fat bodies. We obtained about 4.9 mg of purified NoV VP1 protein from only five silkworm pupae. The purified VP1 protein was confirmed by dynamic light scattering and electron microscopy to form VLPs of approximately 40 nm in diameter. Antisera from mice immunized with the antigen blocked NoV VLPs binding to histo-blood group antigens of pig gastric mucin and also blocked NoV infection in intestinal epithelial cells derived from human induced pluripotent stem (iPS) cells. Our findings demonstrated that NoV VLP eliciting protective antibodies could be obtained in milligram quantities from a few silkworm pupae using the silkworm-BEVS..
5.	Akitsu Masuda, Jae Man Lee, Takeshi Miyata, Hiroaki Mon, Keita Sato, Kosuke Oyama, Yasuteru Sakurai, Jiro Yasuda, Daisuke Takahashi, Tadashi Ueda, Yuri Kato, Motohiro Nishida, Noriko Karasaki, Kohei Kakino, Takeru Ebihara, Takumi Nagasato, Masato Hino, Ayaka Nakashima, Kengo Suzuki, Yoshino Tonooka, Miyu Tanaka, Takato Moriyama, Hirokazu Nakatake, Ryosuke Fujita, Takahiro Kusakabe, Optimization of SARS-CoV-2 Spike Protein Expression in the Silkworm and Induction of Efficient Protective Immunity by Inoculation With Alum Adjuvants., Frontiers in immunology , 10.3389/fimmu.2021.803647, 12, 803647, 2021.08, カイコ-バキュロウイルス発現系を用いて新型コロナウイルスワクチン抗原の大量生産に成功し、人への投与が認可されているAlumアジュバントを用いて十分な中和抗体誘導のうがあることを動物実験等で確認した。.
6.	Hirofumi Ohga, Kosuke Ito, Kohei Kakino, Hiroaki Mon, Takahiro Kusakabe, Jae Man Lee, Michiya Matsuyama, Leptin Is an Important Endocrine Player That Directly Activates Gonadotropic Cells in Teleost Fish, Chub Mackerel., Cells, 10.3390/cells10123505, 10, 12, 2021.12, Leptin, secreted by adipocytes, directly influences the onset of puberty in mammals. Our previous study showed that leptin stimulation could promote the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from pituitary cells in primary culture and ovarian development in chub mackerel. This study aimed to elucidate the detailed mechanism of leptin-induced effects on gonadotropin hormone-producing cells. We produced recombinant leptin using silkworm pupae and investigated the effects of leptin on FSH and LH secretion and gene expression in the primary culture of pituitary cells from chub mackerel. The presence or absence of co-expression of lepr mRNA, FSH and LH b-subunit mRNA in gonadotropic cells was examined by double-labeled in situ hybridization. The addition of leptin significantly increased the secretion and gene expression of FSH and LH from male and female pituitary cells in primary culture. In contrast, gonadotropin-releasing hormone 1 affected neither FSH secretion in cells from females nor fshb and lhb expression in cells from males and females. The expression of lepr was observed in FSH- and LH-producing cells of both males and females. The results indicate that leptin directly regulates gonadotropin synthesis and secretion and plays an important role in the induction of puberty in teleost fish..
7.	Miyu Tanaka, Tsuguru Fujii, Hiroaki Mon, Jae Man Lee, Kohei Kakino, Hisayoshi Fukumori, Takeru Ebihara, Takumi Nagasato, Masato Hino, Yoshino Tonooka, Takato Moriyama, Ryosuke Fujita, Yutaka Banno, Takahiro Kusakabe, Silkworm FoxL21 plays important roles as a regulator of ovarian development in both oogenesis and ovariole development., Insect biochemistry and molecular biology, 10.1016/j.ibmb.2022.103737, 143, 103737-103737, 2022.04, The ovary is an important organ in reproduction. In insects, especially lepidopteran insects, the oocytes and reproductive organs develop rapidly during the pupal stage. Despite their drastic morphological changes, the molecular mechanisms of ovary development are not fully understood. In this study, it is found that forkhead box transcription factor L2, member 1 (FoxL21), which is known to be involved in ovarian differentiation and maintenance in vertebrates, is required for the development of the ovary in the silkworm, Bombyx mori. FoxL21 was expressed in the ovary and ovariole during the larval and pupal stage, respectively. In silkworms in which FoxL21 was knocked out by genome editing, multiple ovarian dysfunctions, such as, abnormal egg formation, thinning of the ovariole sheaths, and defective connection of the oviductus geminus with the ovariole were observed. Finally, ovarian transplantation experiments using the knockout silkworms revealed that FoxL21 functions in the ovariole, but not in the oviductus geminus..
8.	Takeshi Miyata, Kosuke Minamihata, Koichi Kurihara, Yui Kamizuru, Mari Gotanda, Momoka Obayashi, Taiki Kitagawa, Keita Sato, Momoko Kimura, Kosuke Oyama, Yuta Ikeda, Yukihiro Tamaki, Jae Man Lee, Kozue Sakao, Daisuke Hamanaka, Takahiro Kusakabe, Mayumi Tachibana, Hisham R Ibrahim, Highly efficient protein expression of Plasmodium vivax surface antigen, Pvs25, by silkworm and its biochemical analysis., Protein expression and purification, 10.1016/j.pep.2022.106096, 195-196, 106096-106096, 2022.08, Plasmodium vivax ookinete surface protein, Pvs25, is a candidate for a transmission-blocking vaccine (TBV) for malaria. Pvs25 has four EGF-like domains containing 22 cysteine residues forming 11 intramolecular disulfide bonds, a structural feature that makes its recombinant protein expression difficult. In this study, we report the high expression of recombinant Pvs25 as a soluble form in silkworm, Bombyx mori. The Pvs25 protein was purified from hemolymphs of larvae and pupae by affinity chromatography. In the Pvs25 expressed by silkworm, no isoforms with inappropriate disulfide bonds were found, requiring no further purification step, which is necessary in the case of Pichia pastoris-based expression systems. The Pvs25 from silkworm was confirmed to be molecularly uniform by sodium dodecyl sulfate gel electrophoresis and size-exclusion chromatography. To examine the immunogenicity, the Pvs25 from B. mori was administered to BALB/c mice subcutaneously with oil adjuvant. The Pvs25 produced by silkworm induced potent and robust immune responses, and the induced antisera correctly recognized P. vivax ookinetes in vitro, demonstrating the potency of Pvs25 from silkworm as a candidate for a malaria TBV. To the best of our knowledge, this is the first study to construct a system for mass-producing malaria TBV antigens using silkworm..
9.	Yuri Kato, Kazuhiro Nishiyama, Akiyuki Nishimura, Takamasa Noda, Kaori Okabe, Takahiro Kusakabe, Yasunari Kanda, Motohiro Nishida, Drug repurposing for the treatment of COVID-19., Journal of pharmacological sciences, 10.1016/j.jphs.2022.04.007, 149, 3, 108-114, 2022.07, Coronavirus disease 2019 (COVID-19) remains prevalent worldwide since its onset was confirmed in Wuhan, China in 2019. Vaccines against the causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have shown a preventive effect against the onset and severity of COVID-19, and social and economic activities are gradually recovering. However, the presence of vaccine-resistant variants has been reported, and the development of therapeutic agents for patients with severe COVID-19 and related sequelae remains urgent. Drug repurposing, also called drug repositioning or eco-pharma, is the strategy of using previously approved and safe drugs for a therapeutic indication that is different from their original indication. The risk of severe COVID-19 and mortality increases with advancing age, cardiovascular disease, hypertension, diabetes, and cancer. We have reported three protein-protein interactions that are related to heart failure, and recently identified that one mechanism increases the risk of SARS-CoV-2 infection in mammalian cells. This review outlines the global efforts and outcomes of drug repurposing research for the treatment of severe COVID-19. It also discusses our recent finding of a new protein-protein interaction that is common to COVID-19 aggravation and heart failure..
10.	Daigo Tsubokawa, Taisei Kikuchi, Jae Man Lee, Takahiro Kusakabe, Yasuhiko Yamamoto, Haruhiko Maruyama, Venestatin from parasitic helminths interferes with receptor for advanced glycation end products (RAGE)-mediated immune responses to promote larval migration., PLoS pathogens, 10.1371/journal.ppat.1009649, 17, 6, e1009649, 2021.06, Parasitic helminths can reside in humans owing to their ability to disrupt host protective immunity. Receptor for advanced glycation end products (RAGE), which is highly expressed in host skin, mediates inflammatory responses by regulating the expression of pro-inflammatory cytokines and endothelial adhesion molecules. In this study, we evaluated the effects of venestatin, an EF-hand Ca2+-binding protein secreted by the parasitic helminth Strongyloides venezuelensis, on RAGE activity and immune responses. Our results demonstrated that venestatin bound to RAGE and downregulated the host immune response. Recombinant venestatin predominantly bound to the RAGE C1 domain in a Ca2+-dependent manner. Recombinant venestatin effectively alleviated RAGE-mediated inflammation, including footpad edema in mice, and pneumonia induced by an exogenous RAGE ligand. Infection experiments using S. venezuelensis larvae and venestatin silencing via RNA interference revealed that endogenous venestatin promoted larval migration from the skin to the lungs in a RAGE-dependent manner. Moreover, endogenous venestatin suppressed macrophage and neutrophil accumulation around larvae. Although the invasion of larvae upregulated the abundance of RAGE ligands in host skin tissues, mRNA expression levels of tumor necrosis factor-α, cyclooxygenase-2, endothelial adhesion molecules vascular cell adhesion protein-1, intracellular adhesion molecule-1, and E-selectin were suppressed by endogenous venestatin. Taken together, our results indicate that venestatin suppressed RAGE-mediated immune responses in host skin induced by helminthic infection, thereby promoting larval migration. The anti-inflammatory mechanism of venestatin may be targeted for the development of anthelminthics and immunosuppressive agents for the treatment of RAGE-mediated inflammatory diseases..
11.	Akitsu Masuda, Jae Man Lee, Takeshi Miyata, Takeru Ebihara, Kohei Kakino, Masato Hino, Ryosuke Fujita, Hiroaki Mon, Takahiro Kusakabe, Stable trimer formation of spike protein from porcine epidemic diarrhea virus improves the efficiency of secretory production in silkworms and induces neutralizing antibodies in mice., Veterinary research, 10.1186/s13567-021-00971-5, 52, 1, 102-102, 2021.07, Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen of watery diarrhea that causes serious economic loss to the swine industry worldwide. Especially because of the high mortality rate in neonatal piglets, a vaccine with less production cost and high protective effect against PEDV is desired. The intrinsically assembled homotrimer of spike (S) protein on the PEDV viral membrane contributing to the host cell entry is a target of vaccine development. In this study, we designed trimerized PEDV S protein for efficient production in the silkworm-baculovirus expression vector system (silkworm-BEVS) and evaluated its immunogenicity in the mouse. The genetic fusion of the trimeric motif improved the expression of S protein in silkworm-BEVS. A small-scale screening of silkworm strains to further improve the S protein productivity finally achieved the yield of about 2 mg from the 10 mL larval serum. Mouse immunization study demonstrated that the trimerized S protein could elicit strong humoral immunity, including the S protein-specific IgG in the serum. These sera contained neutralizing antibodies that can protect Vero cells from PEDV infection. These results demonstrated that silkworm-BEVS provides a platform for the production of trimeric S proteins, which are promising subunit vaccines against coronaviruses such as PEDV..
12.	Tsuguru Fujii, Kohei Kakino, Hisayoshi Fukumori, Masato Hino, Jae Man Lee, Takahiro Kusakabe, Yutaka Banno, Non-molting dwarf (nm-d) as a mutant of Bombyx mori with a defect in purine synthesis., Insect biochemistry and molecular biology, 10.1016/j.ibmb.2021.103636, 138, 103636-103636, 2021.08, There are several known non-molting mutations of the silkworm, Bombyx mori, including non-molting dwarf (nm-d). Larvae with this mutation hatch normally and start eating leaves, but die before the completion of the first ecdysis. Genetic analysis of the nm-d mutation would contribute to the isolation of essential genes for the larval development of lepidopteran insects. To identify the causative gene of the nm-d locus, we conducted RNA-seq based rough mapping. Using two sets of RNA-seq data, one from a pooled sample of normal larvae, and one from a pooled sample of nm-d larvae, the nm-d locus was narrowed to a 500 kb region. Among the genes located in this region, an nm-d-specific exon loss was identified in the Bombyx homolog of the ATIC (5-aminoimidazole-4-carboxamide ribonucleotide transformylase/Inosine 5'-monophosphate cyclohydrolase) (BmATIC) gene, which catalyzes the final two steps of the de novo purine biosynthetic pathway in mammals. PCR and subsequent sequencing analysis revealed that a region containing exon 9 of the BmATIC gene is deleted in the nm-d larvae. A knockout allele of the BmATIC gene (BmATICKO), that was generated using the CRISPR/Cas9 system, revealed that first instar knockout larvae died while exhibiting the dark brown larval body that is a typical feature of mutants that lack uric acid in the integument. Lethal larvae resulted from crosses between +/BmATICKO moths. The uric acid content in the whole-body of the first instar was drastically reduced in the nm-d larvae compared to normal larvae. These results indicated that the BmATIC gene is responsible for the nm-d phenotype, and that nm-d larvae have a defect in purine biosynthesis, including uric acid. We also discuss the possibility that the BmATIC mRNA is maternally transmitted to eggs. Our results indicated that RNA-seq based mapping using pooled samples is a practical method for the identification of the causative genes of lethal mutations..
13.	Masahiko Kobayashi, Jian Xu, Kohei Kakino, Akitsu Masuda, Masato Hino, Naoki Fujimoto, Kosuke Minamihata, Noriho Kamiya, Hiroaki Mon, Hiroshi Iida, Masateru Takahashi, Takahiro Kusakabe, Jae Man Lee, Optimal silkworm larva host for high-level production of Mus musculus IL-4 using a baculovirus expression vector system, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2019.12.014, 23, 1, 268-273, 2020.04, Interleukine-4 (IL-4) is a cytokine that plays an important role in the immune system and recognized as a biological medicine. Therefore, there is a demand for the production of IL-4 with high performance. The expression of a recombinant IL-4 protein in the prokaryotic system usually results in the formation of an inclusion body. To date, the solution to obtain those active products without the refolding process remains to be established. In this study, we tried to acquire a biologically active recombinant Mus musculus IL-4 (rMmIL-4) using a silkworm-baculovirus expression vector system (silkworm-BEVS). We constructed two recombinant baculoviruses coding rMmIL-4 with the distinct location of affinity purification tags and succeeded in the expression and purification of rMmIL-4 proteins directly without the refolding process. Both purified proteins displayed comparable biological activity to the commercial proteins produced by the E. coli expression system. Besides, we performed screening of silkworm strains to seek optimal hosts for the mass-production of rMmIL-4. Intriguingly, we found that some silkworm strains showed significantly higher secretion levels of rMmIL-4 in silkworm sera. Our study provides meaningful insights into the industrial-scale production of rMmIL-4 with high productivity for pharmaceutical applications in the future..
14.	Masato Hino, Noriko Karasaki, Tsuneyuki Tatsuke, Daisuke Morokuma, Akitsu Masuda, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Mild inactivation and inhibition of phenoloxidase in silkworm serum for xeno-free culture of insect cells, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.89.1_009, 89, 1, 9-16, 2020.01, In insect cell culture, a fetal bovine serum is often used to maintain the cellular physiological conditions, irre-spective of its relatively high cost and unstable supply. Several serum-free media for insects were developed, but it is challenging to adopt some insect cells to these serum-free media. Silkworm serum was used as a re-placement of fetal bovine serum for a long time but not used widely due to the melanization of insect serum catalyzed by phenoloxidase (PO). PO inhibitors reported for insect serum preparation have a significantly nega-tive effect on the long-term culture of healthy insect cells. In this study, we evaluated several kinds of PO inhibi-tors and found the method to prepare silkworm serum, which is suitable for the maintenance of insect cells and the production of the recombinant proteins by the baculovirus expression system. When L-Glutathione (reduced form) or L-Cysteine is used as a PO inhibitor at the time of recovery of silkworm serum, it is possible to sup-press the browning of the silkworm serum. However, in this state of silkworm serum, browning is observed in long-term cell culture use. Therefore, it is preferable for cell culture to add 10% of silkworm serum treated at 50°C for 30 minutes to the medium with 2.7 mM L-Glutathione. Our results suggest that silkworm serum can be an alternative of mammalian serum for cell culture and used for producing the proteins for clinical application without any risk of contamination of zoonotic infectious disease viruses..
15.	Bingqian Li, Zhiqing Li, Chenchen Lu, Li Chang, Dongchao Zhao, Guanwang Shen, Takahiro Kusakabe, Qingyou Xia, Ping Zhao, Heat shock cognate 70 functions as a chaperone for the stability of kinetochore protein cenp-n in holocentric insect silkworms, International journal of molecular sciences, 10.3390/ijms20235823, 20, 23, 2019.12, The centromere, in which kinetochore proteins are assembled, plays an important role in the accurate congression and segregation of chromosomes during cell mitosis. Although the function of the centromere and kinetochore is conserved from monocentric to holocentric, the DNA sequences of the centromere and components of the kinetochore are varied among different species. Given the lack of core centromere protein A (CENP-A) and CENP-C in the lepidopteran silkworm Bombyx mori, which possesses holocentric chromosomes, here we investigated the role of CENP-N, another important member of the centromere protein family essential for kinetochore assembly. For the first time, cellular localization and RNA interference against CENP-N have confirmed its kinetochore function in silkworms. To gain further insights into the regulation of CENP-N in the centromere, we analyzed the affinity-purified complex of CENP-N by mass spectrometry and identified 142 interacting proteins. Among these factors, we found that the chaperone protein heat shock cognate 70 (HSC70) is able to regulate the stability of CENP-N by prohibiting ubiquitin–proteasome pathway, indicating that HSC70 could control cell cycle-regulated degradation of CENP-N at centromeres. Altogether, the present work will provide a novel clue to understand the regulatory mechanism for the kinetochore activity of CENP-N during the cell cycle..
16.	Daigo Tsubokawa, Jae Man Lee, Takeshi Hatta, Fusako Mikami, Haruhiko Maruyama, Takeshi Arakawa, Takahiro Kusakabe, Naotoshi Tsuji, Characterization of the RAGE-binding protein, Strongyloides venestatin, produced by the silkworm-baculovirus expression system, Infection, Genetics and Evolution, 10.1016/j.meegid.2019.103964, 75, 2019.11, The receptor for advanced glycation end products (RAGE) recognizes Ca⁺⁺-binding proteins, such as members of the S100 protein family released by dead or devitalized tissues, and plays an important role in inflammatory responses. We recently identified the Ca⁺⁺-binding protein, venestatin, secreted from the rodent parasitic nematode, Strongyloides venezuelensis. We herein characterized recombinant venestatin, which is abundantly produced by the silkworm-baculovirus expression system (silkworm-BES), particularly in its interaction with RAGE. Venestatin from silkworm-BES possessed a binding capacity with Ca⁺⁺ ions and vaccine immunogenicity against S. venezuelensis larvae in mice, which is similar to venestatin produced by the E. coli expression system (EES). Venestatin from silkworm-BES had a higher affinity for human recombinant RAGE than that from EES, and their affinities were Ca⁺⁺-dependent. RAGE in the mouse lung co-immunoprecipitated with venestatin from silkworm-BES administered intranasally, indicating that it bound endogenous mouse RAGE. The present results suggest that venestatin from silkworm-BES affects RAGE-mediated pathological processes..
17.	Sun Mee Hong, Ji Hyun Choi, Sun Jung Jo, Kwan Sik Min, Dae Jung Kim, Jae Man Lee, Takahiro Kusakabe, Heterologous Production and Glycosylation of Japanese Eel Follitropin Using Silkworm, Biotechnology and Bioprocess Engineering, 10.1007/s12257-019-0045-2, 24, 5, 745-753, 2019.09, Follitropin, an important gonadotropin hormone, participates in vitellogenesis and spermatogenesis. Equine chorionic gonadotropin (eCG) can induce gonadotropin hormone activity in non-equid species and exhibits a long biological half-life. Here, we report the production, using silkworm larval and pupal systems, of biologically active recombinant hybrid-type follitropins based on the coding sequence of the eCG C-terminal peptide (CTP) between the mature β- and α-chains of eel. The three constructs, rJeFSH, rJeFSH·eCG, and rJeFSH·2xeCG were produced and verified to be N- or O-glycosylated and secreted mature peptides. Although rJeFSH·eCG contains more elaborate O-linked carbohydrate chains than rJeFSH, it elicited no significant in vitro oocyte maturation, which may be a result of insufficient terminal sialylation of its N-and O-linked carbohydrate chains. Then, a hybrid of rJeFSH·2xeCG extended with two eCG CTP. Furthermore, the receptor binding assay revealed potency of rJeFSH and rJeFSH·2xeCG to be a few folds greater than that of rJeFSH·eCG. The findings of this study will be useful for the development of more efficient GTHs in teleosts, including eels, when various modifications with two or more extended eCG CTP produced by silkworm are included..
18.	Jian Xu, Jae Man Lee, Tuneyuki Tatsuke, Takeru Ebihara, Akitsu Masuda, Masato Hino, Daisuke Morokuma, Ryosuke Fujita, Hiroaki Mon, Takahiro Kusakabe, Masateru Takahashi, Expression and Purification of Vaccinia Virus DNA Topoisomerase IB Produced in the Silkworm–Baculovirus Expression System, Molecular Biotechnology, 10.1007/s12033-019-00184-4, 61, 8, 622-630, 2019.08, Type IB DNA topoisomerases are enzymes to change the topological state of DNA molecules and are essential in studying replication, transcription, and recombination of nucleic acids in vitro. DNA topoisomerase IB from Vaccinia virus (vTopIB) is a 32 kDa, type I eukaryotic topoisomerase, which relaxed positively and negatively supercoiled DNAs without Mg²⁺ and ATP. Although vTopIB has been effectively produced in E. coli expression system, no studies remain available to explore an alternative platform to express recombinant vTopIB (rvTopIB) in a higher eukaryote, where the one can expect post-translational modifications that affect the activity of rvTopIB. Here in this study, rvTopIB with N-terminal tags was constructed and expressed in a silkworm–baculovirus expression vector system (silkworm–BEVS). We developed a simple two consecutive chromatography purification to obtain highly pure rvTopIB. The final yield of rvTopIB obtained from a baculovirus-infected silkworm larva was 83.25 μg. We also evaluated the activity and function of rvTopIB by the DNA relaxation activity assays using a negatively supercoiled pUC19 plasmid DNA as a substrate. With carefully assessing optimized conditions for the reaction buffer, we found that divalent ions, Mg²⁺, Mn²⁺, Ca²⁺, as well as ATP stimulate the DNA relaxation activity by rvTopIB. The functional and active form of rvTopIB, together with the yields of the protein we obtained, suggests that silkworm–BEVS would be a potential alternative platform to produce eukaryotic topoisomerases on an industrial scale..
19.	Yurie Kinoshita, Jian Xu, Akitsu Masuda, Kosuke Minamihata, Noriho Kamiya, Hiroaki Mon, Ryosuke Fujita, Takahiro Kusakabe, Man Lee, Expression and purification of biologically active human granulocyte-macrophage colony stimulating factor (hGM-CSF) using silkworm-baculovirus expression vector system, Protein Expression and Purification, 10.1016/j.pep.2019.03.010, 159, 69-74, 2019.07, Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a hematopoietic growth factor. It is widely employed as a therapeutic agent targeting neutropenia in cancer patients undergoing chemotherapy and in patients with AIDS or after bone marrow transplantation. In this study, we constructed the recombinant baculoviruses for the expression of recombinant hGM-CSF (rhGM-CSF) with two small affinity tags (His-tag and Strep-tag) at the N or C-terminus. Compared to N-tagged rhGM-CSF, C-tagged rhGM-CSF was highly recovered from silkworm hemolymph. The purified rhGM-CSF proteins migrated as a diffuse band and were confirmed to hold N-glycosylations. A comparable activity was achieved when commercial hGM-CSF was tested as a control. Considering the high price of hGM-CSF in the market, our results and strategies using silkworm-baculovirus system can become a great reference for mass production of the active rhGM-CSF at a lower cost..
20.	Takumi Yano, Man Lee, Jian Xu, Yoshiki Morifuji, Akitsu Masuda, Masato Hino, Daisuke Morokuma, Ryosuke Fujita, Masateru Takahashi, Takahiro Kusakabe, Hiroaki Mon, Expression of the thermostable Moloney murine leukemia virus reverse transcriptase by silkworm-baculovirus expression system, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2019.02.008, 22, 2, 453-457, 2019.06, Reverse transcriptase from Moloney murine leukemia virus (MMLVRT) is an RNA dependent DNA polymerase, which has been used as a fundamental tool for molecular biology and biotechnology. The secondary structures formed in the RNA templates decrease the accessibility of the reverse transcriptase to the RNA templates; it is important to unfold the RNA secondary structure by increasing the reaction temperature to perform the successful transcription. In this study, we applied silkworm baculovirus expression vector system (silkworm-BEVS) to mass-produce and purify the recombinant MMLVRTs with the N- or C- terminal tandem tags. We confirmed that both of the recombinant MMLVRT enzymes have intact DNA polymerase activity. It is notable that C- terminal tagged MMLVRT outperformed MMLVRT obtained from the E. coli expression system in terms of thermostability and sensitivity to low quantities of RNA template. Taken together, these results demonstrate that silkworm-BEVS is a promising alternative strategy to produce the functional and thermostable reverse transcriptase..
21.	Akihiro Morio, Jian Xu, Akitsu Masuda, Yurie Kinoshita, Masato Hino, Daisuke Morokuma, Hatsumi M. Goda, Nozomu Okino, Makoto Ito, Hiroaki Mon, Ryosuke Fujita, Takahiro Kusakabe, Man Lee, Expression, purification, and characterization of highly active endo-α-N-acetylgalactosaminidases expressed by silkworm-baculovirus expression system, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2019.01.009, 22, 2, 404-408, 2019.06, The O-glycosidase, endo-α-N-acetylgalactosaminidase from Enterococcus faecalis (endoEF) catalyzes the cleavage of core 1 and core 3 type O-linked disaccharides between GalNAc and serine or threonine residues from glycoproteins. The endoEF has broad substrate specificity and thus is extensively utilized for the structural and functional analysis of the O-linked glycans. In this study, we expressed and purified the recombinant endoEF (rEndoEF) by using the silkworm-baculovirus expression vector system (Silkworm-BEVS) and confirmed the deglycosylation activity of rEndoEF targeting reporter glycoproteins, which was equivalent to the commercial O-glycosidase. Thus, our study provides important clues to produce highly active rEndoEF O-glycosidases employing silkworm-BEVS as an alternative..
22.	Patmawati, Kosuke Minamihata, Tsuneyuki Tatsuke, Man Lee, Takahiro Kusakabe, Noriho Kamiya, Functional horseradish peroxidase−streptavidin chimeric proteins prepared using a silkworm-baculovirus expression system for diagnostic purposes, Journal of Biotechnology, 10.1016/j.jbiotec.2019.03.007, 297, 28-31, 2019.05, Rapid, convenient, sensitive detection methods are of the utmost importance in analytical tools. Enzyme-based signal amplification using horseradish peroxidase (HRP) is commonly implemented in clinical diagnostics kits based on enzyme-linked immunosorbent assay (ELISA), by which the limit of detection is greatly improved. Herein we report the design and preparation of recombinant fusion proteins comprising HRP and streptavidin (Stav), in which HRP was fused to either the N- or C-terminus of Stav ((HRP) ₄ –Stav or Stav–(HRP) ₄ , respectively) using a baculovirus-silkworm expression system. Both (HRP) ₄ –Stav and Stav–(HRP) ₄ were secreted in the apo form but they were easily converted to the holo form and activated by simple incubation with hemin overnight at 4 °C. The activated (HRP) ₄ –Stav and Stav–(HRP) ₄ could be combined with a commercial biotinylated anti-OVA IgG antibody to detect ovalbumin (OVA) as the antigen in ELISA. The enzymatic activity of (HRP) ₄ –Stav was twofold higher than that of Stav–(HRP) ₄ , and the sensitivity of (HRP) ₄ -Stav in ELISA was higher than that of a commercial HRP–Stav chemical conjugate. The successful use of (HRP) ₄ –Stav chimeric protein as a molecular probe in ELISA shows that the baculovirus-silkworm expression system is promising to produce enzyme–Stav conjugates to substitute for those prepared by chemical methods..
23.	Mai Morishita, Akitsu Masuda, Hiroaki Mon, Man Lee, Takahiro Kusakabe, Kosuke Tashiro, Chisa Yasunaga-Aoki, Kazuhiro Iiyama, Identification of an insecticidal toxin produced by Enterobacter sp. strain 532 isolated from diseased Bombyx mori silkworms, FEMS microbiology letters, 10.1093/femsle/fny295, 366, 2, 2019.01, Although Enterobacter sp. 532 shows pathogenicity in Bombyx mori, the insecticidal mechanisms are unclear. Here, we identified and characterised an insecticidal protein from Enterobacter. The insecticidal protein was purified from the strain and inoculated into B. mori larvae. Intracellular proteins were prepared, purified and separated by preparative native polyacrylamide gel electrophoresis (PAGE); one protein band had insecticidal activity. Sodium dodecyl sulfate-PAGE showed the presence of several bands, indicating that the insecticidal protein formed a complex. Peptide mass fingerprinting of a prominent 255.3-kDa band revealed 64 peptides that matched one protein with 33.0% sequence coverage. This protein was a homologue of the A component of the toxin complex (Tc), and the VRP1 domain was conserved; thus, the gene was named itcA (insecticidal toxin complex A). In the itcA downstream region, B and C component gene homologues were found, and these genes were located on an 86.2-kb contig sequence. Two repA genes and 27 genes related to conjugation transfer of plasmids were located on the contig, suggesting that the contig originated from a mobilisable plasmid. Therefore, these findings suggested that the strain may have acquired the Tc genes by horizontal transfer. This is the first description of Tc produced by the genus Enterobacter..
24.	Dani Permana, Kosuke Minamihata, Tsuneyuki Tatsuke, Jae M. Lee, Takahiro Kusakabe, Masahiro Goto, Noriho Kamiya, Polymerization of Horseradish Peroxidase by a Laccase-Catalyzed Tyrosine Coupling Reaction, Biotechnology Journal, 10.1002/biot.201800531, 2019.01, The polymerization of proteins can create newly active and large bio-macromolecular assemblies that exhibit unique functionalities depending on the properties of the building block proteins and the protein units in polymers. Herein, the first enzymatic polymerization of horseradish peroxidase (HRP) is reported. Recombinant HRPs fused with a tyrosine-tag (Y-tag) through a flexible linker at the N- and/or C-termini are expressed in silkworm, Bombyx mori. Trametes sp. laccase (TL) is used to activate the tyrosine of Y-tagged HRPs with molecular O ₂ to form a tyrosyl-free radical, which initiates the tyrosine coupling reaction between the HRP units. A covalent dityrosine linkage is also formed through a HRP-catalyzed self-crosslinking reaction in the presence of H ₂ O ₂ . The addition of H ₂ O ₂ in the self-polymerization of Y-tagged HRPs results in lower activity of the HRP polymers, whereas TL provides site-selectivity, mild reaction conditions and maintains the activity of the polymeric products. The cocrosslinking of Y-tagged HRPs and HRP-protein G (Y-HRP-pG) units catalyzed by TL shows a higher signal in enzyme-linked immunosorbent assay (ELISA) than the genetically pG-fused HRP, Y-HRP-pG, and its polymers. This new enzymatic polymerization of HRP promises to provide highly active and functionalized polymers for biomedical applications and diagnostics probes..
25.	Mai Morishita, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Maximiano Corrêa Cassal, Chisa Yasunaga-Aoki, Kazuhiro Iiyama, Toxin complex is a major virulence determinant of Enterobacter sp. 532 against the silkworm, Bombyx mori, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.88.1_021, 88, 1, 1_021-1_025, 2019.01.
26.	Jian Xu, Akihiro Morio, Daisuke Morokuma, Yudai Nagata, Masato Hino, Akitsu Masuda, Zhiqing Li, Hiroaki Mon, Takahiro Kusakabe, Man Lee, A functional polypeptide N-acetylgalactosaminyltransferase (PGANT) initiates O-glycosylation in cultured silkworm BmN4 cells, Applied Microbiology and Biotechnology, 10.1007/s00253-018-9309-6, 102, 20, 8783-8797, 2018.10, Mucin-type O-glycosylation is initiated by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts or PGANTs), attaching GalNAc to serine or threonine residue of a protein substrate. In the insect model from Lepidoptera, silkworm (Bombyx mori), however, O-glycosylation pathway is totally unexplored and remains largely unknown. In this study, as the first report regarding protein O-glycosylation analysis in silkworms, we verified the O-glycan profile that a common core 1 Gal (β1-3) GalNAc disaccharide branch without terminally sialylated structure is mainly formed for a baculovirus-produced human proteoglycan 4 (PRG4) protein. Intriguingly, functional screenings in cultured silkworm BmN4 cells for nine Bmpgants reveal that Bmpgant2 is the solo functional BmPGANT for PRG4, implying that Bmpgants may have unique cell/tissue or protein substrate preferences. Furthermore, a recombinant BmPGANT2 protein was successfully purified from silkworm-BEVS and exhibited a high ability to transfer GalNAc for both peptide and protein substrates. Taken together, the present results clarified the functional BmPGANT2 in cultured silkworm cells, providing crucial fundamental insights for future studies dissecting the detailed silkworm O-glycosylation pathways and productions of glycoproteins with O-glycans..
27.	Akitsu Masuda, Jian Xu, Kosuke Minamihata, Genki Kagawa, Yusei Hamada, Yoshiki Morifuji, Takumi Yano, Masato Hino, Daisuke Morokuma, Noriko Karasaki, Hiroaki Mon, Noriho Kamiya, Takahiro Kusakabe, Jae Man Lee, Production of a biologically active human basic fibroblast growth factor using silkworm-baculovirus expression vector system, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2018.05.002, 21, 2, 716-720, 2018.06, As a therapeutic treatment, recombinant human basic fibroblast growth factor (rhbFGF) is usually employed in tissue regeneration, and as an essential component in culture medium for maintaining the induced pluripotent stem (iPS) cell and embryonic stem (ES) cell in an undifferentiated state. Therefore, a large amount of biologically active rhbFGF is required. In this study, silkworm-baculovirus expression vector system (silkworm-BEVS) is employed to achieve a high productivity of recombinant rhbFGF with two small affinity tags (His-tag and STREP-tag) at the N or C-terminus. It is observed that rhbFGF with 30 K signal peptide of silkworm were successfully expressed but are not sufficiently secreted into the culture medium of cultured insect cells. Then we purified the N- or C-tagged intracellular rhbFGF protein and obtained a yield of about 0.7 mg/larva and 1.2 mg/larva, respectively. Although the final yield of the C-tagged rhbFGF is higher than that of the N-tagged, rhbFGF with N-tag demonstrated promising and comparable biological activity, which is evaluated through a mammalian cell proliferation assay. Taken together, these results indicate that silkworm-BEVS could contribute to the mass-production of the biologically active rhbFGF for medical uses..
28.	Patmawati xxxx, Kosuke Minamihata, Tsuneyuki Tatsuke, Man Lee, Takahiro Kusakabe, Noriho Kamiya, Expression and Activation of Horseradish Peroxidase–Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes, Biotechnology Journal, 10.1002/biot.201700624, 13, 6, 2018.06, Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio-probe for high-quality HRP-based diagnostic systems. A HRP-protein A/G fusion protein (HRP-pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP-pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP-pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP-pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP-pAG is conjugated with streptavidin to form a HRP-pAG multimer and the multimeric HRP-pAG produced higher signals in the ELISA system than monomeric HRP-pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP-based bioconjugates as well as further functionalization of HRP by applying enzymatic post-translational modifications..
29.	S. Jin, T. Cheng, Y. Guo, P. Lin, P. Zhao, C. Liu, Takahiro Kusakabe, Q. Xia, Bombyx mori epidermal growth factor receptor is required for nucleopolyhedrovirus replication, Insect Molecular Biology, 10.1111/imb.12386, 2018.01, Baculovirus-host interactions are important models for studying the biological control of lepidopteran pests. Research on baculovirus-host interactions has focussed on baculovirus manipulation of cellular signalling pathways, including the extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3-kinases/protein kinase B (PI3K/Akt) signalling pathways. However, the mechanism underlying ERK and PI3K/Akt activation and function in response to baculovirus infection remains poorly understood. Here, we demonstrated that baculovirus activated the Bombyx mori ERK and PI3K/Akt signalling pathways via the B. mori epidermal growth factor receptor (BmEGFR). To further characterize the function of the BmEGFR/ERK signalling pathway in baculovirus replication, we calculated genome-wide changes in kinase-chromatin interactions for ERK after baculovirus infection using chromatin immunoprecipitation followed by high-throughput sequencing. A Gene Ontology analysis showed that virus infection had effects on the biological regulation, cellular process and metabolic process pathways. Moreover, ERK was shown to regulate the transcription of late viral genes. Taken together, our results suggest that baculoviruses manipulate components of the host cell machinery for replication via modulation of the BmEGFR signalling pathway..
30.	Koki Mukai, Ayano Teramoto, Xuchun Qiu, Yohei Shimasaki, Yoko Kato-Unoki, Man Lee, Naohiro Mizoguchi, Mst Ruhina Margia Khanam, Hina Satone, Tsuneyuki Tatsuke, Takahiro Kusakabe, Yuji Oshima, Gene structure and cDNA sequence of 2-Cys peroxiredoxin in the harmful algal bloom species Chattonella marina and its gene transcription under different light intensities, European Journal of Phycology, 10.1080/09670262.2017.1346206, 53, 1, 29-38, 2018.01, We investigated the gene structure and predicted amino acid sequence of the antioxidant enzyme 2-Cys peroxiredoxin (2-Cys Prx) in the raphidophyte Chattonella marina, which is a harmful algal bloom (HAB) species. The open reading frame of 2-Cys Prx was 585 bp long and encoded a protein consisting of 195 amino acids. The putative amino acid sequence contained two cysteine residues located at the 49th and 170th amino acid positions from the N-terminal methionine residue. The sequence also possessed 2-Cys Prx characteristic motifs, F (FFYPLDFTFVCPTEI) and EVCP. The position of the 2-Cys Prx gene relative to several others (ycf59–2-CysPrx–rpl35–rpl20) was the same as that found in the chloroplast genome in the raphidophyte Heterosigma akashiwo. Upstream of the 2-Cys Prx gene, possible TATA and GGA motifs recognized by nuclear-encoded plastid RNA polymerase (NEP), and a possible -10 box and -35 box recognized by plastid-encoded plastid RNA polymerase (PEP) were observed. We measured the transcript levels of 2-Cys Prx in C. marina cells grown under three different light intensities (0, 100, 1000 µmol photons m^–2 s^–1, 14-h light/8-h dark photoperiod) by quantitative PCR. The 2-Cys Prx transcript level in cells grown under the highest light intensity on day 3 was threefold that on day 0 but two lower light intensities resulted in relatively stable transcription levels. The 2-Cys Prx transcript level was significantly positively related to the H₂O₂ concentration per cell and the H₂O₂ scavenging activity per cell. These results suggest that C. marina 2-Cys Prx functions in the chloroplast and its transcription could be regulated by both NEP and PEP. Moreover, the 2-Cys Prx transcript level might increase to remove excessive H₂O₂ produced under strong light conditions in order to maintain cell proliferation activity..
31.	Mitsunori Shiroishi, Yuji Ito, Kenta Shimokawa, Jae Man Lee, Takahiro Kusakabe, Tadashi Ueda, Structure–function analyses of a stereotypic rheumatoid factor unravel the structural basis for germline-encoded antibody autoreactivity, Journal of Biological Chemistry, 10.1074/jbc.M117.814475, 293, 18, 7008-7016, 2018.01, 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..
32.	Yoshiki Morifuji, Jian Xu, Noriko Karasaki, Kazuhiro Iiyama, Daisuke Morokuma, Masato Hino, Akitsu Masuda, Takumi Yano, Hiroaki Mon, Takahiro Kusakabe, Man Lee, Expression, Purification, and Characterization of Recombinant Human α ₁ -Antitrypsin Produced Using Silkworm–Baculovirus Expression System, Molecular Biotechnology, 10.1007/s12033-018-0127-y, 2018.01, Human α ₁ -antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α ₁ -antitrypsin is mainly purified from human plasma. There is a high demand for a stable and low-cost supply of recombinant AAT (rAAT). In this study, the baculovirus expression vector system using silkworm larvae as host was employed and a large amount of highly active AAT was recovered from the silkworm serum (~ 15 mg/10 ml) with high purity. Both the enzymatic activity and stability of purified rAAT were comparable with those of commercial product. Our results provide an alternative method for mass production of the active rAAT in pharmaceutical use..
33.	Akitsu Masuda, Kosuke Minamihata, Masato Hino, Daisuke Morokuma, Noriko Karasaki, Hiroaki Mon, Noriho Kamiya, Takahiro Kusakabe, Man Lee, Modular surface display for porcine circovirus type 2 virus-like particles mediated by microbial transglutaminase, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.87.2_053, 87, 2, 53-60, 2018.01, Virus-like particles (VLPs) are multi-protein complex, which mimic viral particles without viral genomes. Recently various applications of VLPs for medical and pharmaceutical fields are developed. Furthermore, surface modification of VLPs is expected to extend their functional versatility. As an enzymatic strategy for the modification, microbial transglutaminase (MTG) that catalyzes the covalent bond between a glutamine residue and a ly-sine residue or a primary amine is suitable for efficient protein ligation. In the previous study, we demonstrated the efficient production of immunogenic VLPs of porcine circovirus type 2 (PCV2) using silkworm-baculovirus expression vector system (silkworm-BEVS). Herein, we established the display system of exotic molecules to VLPs by enzymatic covalent cross-linking using MTG. For the target modification, Q-tag (YPLQMRG) that is MTG reactive sequence were introduced into the N-terminus, C-terminus, or loop BC of capsid protein of PCV2 and successfully expressed as VLPs in silkworm pupae. Of these, PCV2 VLPs with Q-tag at the loop BC and C-terminus were efficiently conjugated with FITC-cadaverine and K-tagged (MRHKGS) EGFP by MTG. These results showed that flexible surface modification of VLPs mediated by MTG could be used for development of several therapeutic tools such as multivalent vaccines..
34.	Akitsu Masuda, Man Lee, Takeshi Miyata, Tetsuo Sato, Shizuka Hayashi, Masato Hino, Daisuke Morokuma, Noriko Karasaki, Hiroaki Mon, Takahiro Kusakabe, Purification and characterization of immunogenic recombinant virus-like particles of porcine circovirus type 2 expressed in silkworm pupae, Journal of General Virology, 10.1099/jgv.0.001087, 99, 7, 917-926, 2018.01, Porcine circovirus type 2 (PCV2) is a primary causative agent of postweaningmultisystemic wasting syndrome (PMWS), which has a significant economic impact on the swine industry. The capsid protein (Cap) encoded by ORF2 of the viral genome has been used effectively as a vaccine against PCV2 infection. The Cap protein can spontaneously assemble into virus-like particles (VLPs) that are safe and highly immunogenic for vaccine applications. Several expression systems, including bacteria, yeast and insect cells, have been utilized to produce PCV2 VLPs. However, in some cases, the recombinant Cap (rCap) proteins produced in bacteria and yeast do not assemble spontaneously. In this study, we expressed rCap protein using a silkworm–baculovirus expression vector system (silkworm–BEVS) for mass production of PCV2 VLPs and established a simple three-step protocol for its purification from pupae: extraction by detergent, ammonium sulfate precipitation and anion exchange column chromatography. Size-exclusion chromatography (SEC) analysis and transmission electron microscope (TEM) observation showed that purified rCap proteins formed VLPs with a similar morphology to that of the original virus. Furthermore, the VLPs produced in silkworms were capable of inducing neutralizing antibodies against PCV2 in mice. Our results demonstrated that the silkworm system is a powerful tool for the production of PCV2 VLPs and will be useful for the development of a reliable and cost-effective PCV2 vaccine..
35.	Zhiqing Li, Qixin Cui, Jian Xu, Daojun Cheng, Xiaoyan Wang, Bingqian Li, Man Lee, Qingyou Xia, Takahiro Kusakabe, Ping Zhao, SUMOylation regulates the localization and activity of Polo-like kinase 1 during cell cycle in the silkworm, Bombyx mori, Scientific Reports, 10.1038/s41598-017-15884-7, 7, 1, 2017.12, Polo-like kinase 1 (Plk1) is a crucial cell cycle regulator by its specific localization and activity during cell cycle. It has been shown that the phosphorylation and ubiquitylation of Plk1 are required for its own activation and localization. Here, we report that SUMOylation regulates the activity of Plk1 in the lepidopteran insect of Bombyx mori. In the absence of SUMOylation, it causes the lost localization of Plk1 on centrosomes and kinetochores, as well as an uneven distribution in midzone. We further identify that the putative SUMOylation site of Bombyx Plk1 at lysine 466 is required for its localization on centrosomes, and K466 mutation in Plk1 could influence its interaction with Smt3/Ubc9 complex. These findings are also confirmed by Drosophila Polo and human Plk1, which together reveals a conserved role of Plk1 SUMOylation in mammals. Moreover, conjugation of Smt3 to Plk1 SUMOylation mutant promotes its localization on centrosomes and kinetochores, and rescues functional defects of chromosome alignment in cells depleted of endogenous Plk1. Altogether, the present data indicate that the SUMOylation of Plk1 could participate in proper chromosome alignment and segregation during mitosis, and provides a novel layer for the regulation of Plk1 localization and activity throughout cell cycle..
36.	Anandrao Ashok Patil, Tsuneyuki Tatsuke, Hiroaki Mon, Man Lee, Daisuke Morokuma, Masato Hino, Takahiro Kusakabe, Molecular characterization of mitochondrial Zucchini and its relation to nuage-piRNA pathway components in Bombyx mori ovary-derived BmN4 cells, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2017.09.107, 493, 2, 971-978, 2017.11, Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs that associate with PIWI subfamily proteins, which play an important role in transposon silencing in animal germ cell. The piRNAs biogenesis is divided into two major pathways: primary and secondary, and both pathways are independent of double-stranded RNA-processing enzyme Dicer, which processes the single-stranded RNA transcripts in microRNA (miRNA) and siRNA (small interfering RNA) pathway. Primary piRNAs are processed from long non-coding RNA precursors transcribed from piRNA clusters. Zucchini (Zuc), a mitochondrial phospholipase D (PLD) superfamily protein is conserved among the animals and involved in piRNA biogenesis. Recent studies showed that the Zucchini is an endoribonuclease essential for primary piRNA maturation and production of phased piRNA in secondary piRNA biogenesis of drosophila germ cell. Based on these reports, here we identified and studied the silkworm Zucchini (BmZuc) at subcellular level in ovary-derived BmN4 cell. The silkworm Zuc specifically expressed in germ-related tissues and localized on mitochondria and partially co-localized with perinuclear nuage-piRNA pathway components and nuage marker protein BmVasa. Molecular dissection analyses revealed that the conserved mitochondrial localization sequence, RGV motif, PLDc 2 domain and HKD motif are important for the BmZuc mitochondrial localization. Moreover, the knockdown analyses showed that the piRNA pathway components are independent on BmZuc for their nuage localization, whereas BmZuc depend on piRNA pathway components for the proper localization. Our data provides vital information on mitochondrial BmZuc and its relationship to “nuage” in ovary-derived BmN4 cell..
37.	Ming Ming Ji, Man Lee, Hiroaki Mon, Kazuhiro Iiyama, Tsuneyuki Tatsuke, Daisuke Morokuma, Masato Hino, Mami Yamashita, Kazuma Hirata, Takahiro Kusakabe, Lipidation of BmAtg8 is required for autophagic degradation of p62 bodies containing ubiquitinated proteins in the silkworm, Bombyx mori, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2017.08.006, 89, 86-96, 2017.10, p62/Sequestosome-1 (p62/SQSTM1, hereafter referred to as p62) is a major adaptor that allows ubiquitinated proteins to be degraded by autophagy, and Atg8 homologs are required for p62-mediated autophagic degradation, but their relationship is still not understood in Lepidopteran insects. Here it is clearly demonstrated that the silkworm homolog of mammalian p62, Bombyx mori p62 (Bmp62), forms p62 bodies depending on its Phox and Bem1p (PB1) and ubiquitin-associated (UBA) domains. These two domains are associated with Bmp62 binding to ubiquitinated proteins to form the p62 bodies, and the UBA domain is essential for the binding, but Bmp62 still self-associates without the PB1 or UBA domain. The p62 bodies in Bombyx cells are enclosed by BmAtg9-containing membranes and degraded via autophagy. It is revealed that the interaction between the Bmp62 AIM motif and BmAtg8 is critical for the autophagic degradation of the p62 bodies. Intriguingly, we further demonstrate that lipidation of BmAtg8 is required for the Bmp62-mediated complete degradation of p62 bodies by autophagy. Our results should be useful in future studies of the autophagic mechanism in Lepidopteran insects..
38.	Anandrao Ashok Patil, Tsuneyuki Tatsuke, Hiroaki Mon, Man Lee, Daisuke Morokuma, Masato Hino, Takahiro Kusakabe, Characterization of Armitage and Yb containing granules and their relationship to nuage in ovary-derived cultured silkworm cell, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2017.06.008, 490, 2, 134-140, 2017.08, PIWI-interacting RNAs (piRNAs) are a class of endogenous small non-coding RNAs, which are mostly 24–32 nucleotides in length and interact specifically with PIWI subfamily of argonaute proteins. Despite the significant research progress in germ line piRNA pathway, its role in somatic cell is not well known. In Drosophila ovarian somatic cell, maturation of primary piRNA and its loading onto Piwi occurs at perinuclear Yb body. The Armitage (Armi) and Yb proteins are the major components of Yb body and specially expressed in ovarian somatic cell. Based on the reports, here we studied the BmArmi and BmYb in Bombyx mori ovary-derived BmN4 cells expressing BmVasa. In this study, we show that BmArmi and BmYb co-localized with BmVasa at nuage. The helicase domains of BmArmi and BmYb are important for nuage localization. Moreover, RNAi of piRNA components reveal that BmArmi depend on BmAgo3 for nuage localization, and BmArmi and BmYb form cytoplasmic granules independently in the absence of BmVasa. Our results provide evidence that the BmArmi and BmYb coexist with BmVasa and play an important role in perinuclear nuage granules formation in ovary-derived BmN4 cell..
39.	Hiroaki Mon, Man Lee, Masanao Sato, Takahiro Kusakabe, Identification and functional analysis of outer kinetochore genes in the holocentric insect Bombyx mori, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2017.04.005, 86, 1-8, 2017.07, The kinetochore creates chromosomal attachment sites for microtubules. The kinetochore-microtubule interface plays an important role in ensuring accurate transmission of genetic information to daughter cells. Bombyx mori is known to possess holocentric chromosomes, where spindle microtubules attach along the entire length of the chromosome. Recent evidence suggests that CENP-A and CENP-C, which are essential for centromere structure and function in other species, have lost in holocentric insects, implying that B. mori is able to build its kinetochore regardless of the lack of CENP-A and CENP-C. Here we report the identification of three outer kinetochore genes in the silkworm B. mori by using bioinformatics and RNA interference-based screening. While the homologs of Ndc80 and Mis12 have strong similarity with those of other organisms, the five encoded proteins (BmNuf2, BmSpc24, BmSpc25, BmDsn1 and BmNnf1) are highly diverged from their counterparts in other species. Microscopic studies show that the outer kinetochore protein is distributed along the entire length of the chromosomes, which is a key feature of holocentric chromosomes. We also demonstrate that BmDsn1 forms a heterotrimeric complex with BmMis12 and BmNnf1, which acts as a receptor of the Ndc80 complex. In addition, our study suggests that a small-scale RNAi-based candidate screening is a useful approach to identify genes which may be highly divergent among different species..
40.	Mami Yamashita, Jian Xu, Daisuke Morokuma, Kazuma Hirata, Masato Hino, Hiroaki Mon, Masateru Takahashi, Samir M. Hamdan, Kosuke Sakashita, Kazuhiro Iiyama, Yutaka Banno, Takahiro Kusakabe, Man Lee, Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System, Molecular Biotechnology, 10.1007/s12033-017-0008-9, 59, 6, 221-233, 2017.06, The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively. Interestingly, we found that rKOD-C shows higher thermostability at 95 °C than that of rKOD-N, while that rKOD-N is significantly unstable after exposing to long period of heat-shock. We also assessed the polymerase activity as well as the fidelity of purified rKODs under various conditions. Compared with commercially available rKOD, which is expressed in E. coli expression system, rKOD-C exhibited almost the same PCR performance as the commercial rKOD did, while rKOD-N did lower performance. Taken together, our results suggested that silkworm-BEVS can be used to express and purify efficient rKOD in a commercial way..
41.	Daisuke Morokuma, Jian Xu, Masato Hino, Hiroaki Mon, Jasmeen S. Merzaban, Masateru Takahashi, Takahiro Kusakabe, Man Lee, Expression and Characterization of Human β-1, 4-Galactosyltransferase 1 (β4GalT1) Using Silkworm–Baculovirus Expression System, Molecular Biotechnology, 10.1007/s12033-017-0003-1, 59, 4-5, 151-158, 2017.05, Baculovirus expression vector system (BEVS) is widely known as a mass-production tool to produce functional recombinant glycoproteins except that it may not be always suitable for medical practice due to the differences in the structure of N-linked glycans between insects and mammalian. Currently, various approaches have been reported to alter N-linked glycan structures of glycoproteins derived from insects into terminally sialylated complex-type N-glycans. In the light of those studies, we also proposed in vitro maturation of N-glycan with mass-produced and purified glycosyltransferases by silkworm–BEVS. β-1,4-Galactosyltransferase 1 (β4GalT1) is known as one of type II transmembrane enzymes that transfer galactose in a β-1, 4 linkage to accepter sugars, and a key enzyme for further sialylation of N-glycans. In this study, we developed a large-scale production of recombinant human β4GalT1 (rhβ4GalT1) with N- or C-terminal tags in silkworm–BEVS. We demonstrated that rhβ4GalT1 is N-glycosylated and without mucin-type glycosylation. Interestingly, we found that purified rhβ4GalT1 from silkworm serum presented higher galactosyltransferase activity than that expressed from cultured mammalian cells. We also validated the UDP-galactose transferase activity of produced rhβ4GalT1 proteins by using protein subtracts from silkworm silk gland. Taken together, rhβ4GalT1 from silkworms can become a valuable tool for producing high-quality recombinant glycoproteins with mammalian-like N-glycans..
42.	Kazuhiro Iiyama, Eigo Takahashi, Man Lee, Hiroaki Mon, Mai Morishita, Takahiro Kusakabe, Chisa Yasunaga-Aoki, Alkaline protease contributes to pyocyanin production in Pseudomonas aeruginosa, FEMS Microbiology Letters, 10.1093/femsle/fnx051, 364, 7, 2017.04, The role of the alkaline protease (AprA) in pyocyanin production in Pseudomonas aeruginosa was investigated. AprA was overproduced when a plasmid carrying the aprA gene was introduced to an aprA-deletion mutant strain, EG03; thus, aprA-complemented EG03 was used as an overproducing strain. The complemented strain produced higher pyocyanin than the mutant strain in all commercially available media evaluated. Particularly, pyocyanin production was higher in the complemented than in the parental strain in brain-heart infusion and tryptic soy broths. These results suggested that protein degradation products by AprA were utilized for pyocyanin production. Protein-rich media were used in subsequent validation studies. Similar results were obtained when the basal medium was supplemented with casein or skim milk as the sole organic nitrogen source. However, gelatin failed to induce abundant pyocyanin production in the complemented strain, despite the presence of protein degradation products by AprA as assessed by SDS-PAGE. Thus, gelatin degradation products may not be suitable for pyocyanin synthesis. In conclusion, AprA could contribute to pyocyanin production in the presence of several proteins or peptides..
43.	takahiro kusakabe, Expression and characterization of human β-1, 4-galactosyltransferase 1 (β4GalT1) using silkworm-baculovirus expression system, 59, 151-158, 2017.01.
44.	Kazuhiro Iiyama, Mai Morishita, Man Lee, Hiroaki Mon, Takahiro Kusakabe, Kosuke Tashiro, Taiki Akasaka, Chisa Yasunaga-Aoki, Kazuhisa Miyamoto, A reconsideration of the taxonomic position of two bacterial strains isolated from Flacherie-Diseased silkworms in 1965, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.86.2_035, 86, 2, 35-41, 2017.01, Recent advances in bacterial characterization methodologies have made taxonomic categorization significantly more accurate. Here, we re-evaluated the position of bacterial strains (532 and 652) belonging to the genus Hafnia, isolated from flacherie-diseased silkworms in 1965. Phylogenetic analysis based on the 16S rRNA gene sequences of these strains suggests that they belong to genus Enterobacter. Using multilocus sequence analysis (MLSA), these strains were further classified to MLSA group A, which is a “core” group of Enterobacter containing E. cloacae (the type species of the genus). Although these strains were closely related to E. mori, E. tabaci, and E. asburiae, they also had other MLSA characteristics that distinguished them from these neighboring bacterial species. These data were supported by further biochemical analysis. Thus, it appears that the 532 and 652 strains isolated almost half a century ago belong to genus Enterobacter, and their unique characteristics strongly suggest that they are a novel bacterial species..
45.	Hina Satone, Shohei Nonaka, Man Lee, Yohei Shimasaki, Takahiro Kusakabe, Shun-Ichiro Kawabata, Yuji Oshima, Tetrodotoxin- and tributyltin-binding abilities of recombinant pufferfish saxitoxin and tetrodotoxin binding proteins of Takifugu rubripes, Toxicon, 10.1016/j.toxicon.2016.11.245, 125, 50-52, 2017.01, We investigated the ability of recombinant pufferfish saxitoxin and tetrodotoxin binding protein types 1 and 2 of Takifugu rubripes (rTrub.PSTBP1 and rTrub.PSTBP2) to bind to tetrodotoxin (TTX) and tributyltin. Both rTrub.PSTBPs bound to tributyltin in an ultrafiltration binding assay but lost this ability on heat denaturation. In contrast, only rTrub.PSTBP2 bound to TTX even heat denaturation. This result suggests that the amino acid sequence of PSTBP2 may be contributed for its affinity for TTX..
46.	takahiro kusakabe, In vitro screening for inhibitor of cloned Drosophila melanogaster tyramine-β-hydroxylase and docking studies, International Journal of Biological Macromolecules, 93, 889-895, 2016.12.
47.	takahiro kusakabe, Proteasome inhibitor MG132 impairs autophagic flux through compromising formation of autophagosomes in Bombyx cells, 479, 690-696, 2016.11.
48.	takahiro kusakabe, Co-expression of silkworm allatostatin-C receptor BNGR-A1 with its cognate G protein subunits enhances the GPCR display on the budding baculovirus, JOURNAL OF ASIA-PACIFIC ENTOMOLOGY, 10.1016/j.aspen.2016.07.007, 19, 3, 753-760, 2016.09.
49.	takahiro kusakabe, Effect of antibiotics on extracellular protein level in Pseudomonas aeruginosa, Plasmid, 84, 44-50, 2016.06.
50.	takahiro kusakabe, Expression and Characterization of Recombinant Serratia liquefaciens Nucleases Produced with Baculovirus-mediated Silkworm Expression System, 10.1007/s12033-016-9937-y, 58, 6, 393-403, 2016.06.
51.	takahiro kusakabe, Characterization of the roles of DNA polymerases, clamp, and clamp loaders during S-phase progression and cell cycle regulation in the silkworm, Bombyx mori, 85, 21-29, 2016.06.
52.	takahiro kusakabe, Molecular analysis and bioactivity of luteinizing hormone from Japanese eel, Anguilla japonica, produced in silkworm pupae, BIOTECHNOLOGY AND BIOPROCESS ENGINEERING, 10.1007/s12257-016-0042-7, 21, 3, 381-388, 2016.06.
53.	takahiro kusakabe, High-level expression and purification of biologically active human IL-2 using silkworm-baculovirus expression vector system, JOURNAL OF ASIA-PACIFIC ENTOMOLOGY, 10.1016/j.aspen.2016.03.014, 19, 2, 313-317, 2016.06.
54.	Daisuke Morokuma, 門宏明, YUTAKA BANNO, takahiro kusakabe, JAE MAN LEE, Differential N-Glycan Modifications of Human Alpha 1-Acid Glycoprotein (α1AGP) Produced in Different Silkworm Strains using the Baculovirus Expression System, 84, 2, 49-53, 2016.05.
55.	takahiro kusakabe, Virulence of lipopolysaccharide-deficient mutants of Serratia liquefaciens toward the silkworm, Bombyx mori., Journal of Insect Biotechnology and Sericology, 10.11416/jibs.85.1_007, 85, 7-14, 2016.04.
56.	takahiro kusakabe, CRISPR/Cas9-mediated knockout of factors in non-homologous end joining pathway enhances gene targeting in silkworm cells, SCIENTIFIC REPORTS, 10.1038/srep18103, 5, 2015.12.
57.	takahiro kusakabe, Comparative proteomic analysis of hemolymph proteins from Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-sensitive or -resistant silkworm strains during infections, COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY D-GENOMICS & PROTEOMICS, 10.1016/j.cbd.2015.07.003, 16, 36-47, 2015.12.
58.	takahiro kusakabe, Roles of silkworm endoplasmic reticulum chaperones in the secretion of recombinant proteins expressed by baculovirus system, MOLECULAR AND CELLULAR BIOCHEMISTRY, 10.1007/s11010-015-2529-5, 409, 1-2, 255-262, 2015.11.
59.	takahiro kusakabe, Amyloidogenic lysozyme accumulates in the endoplasmic reticulum tangling with GRP78/BiP and evokes ER stress, PROTEIN SCIENCE, 24, 135-136, 2015.10.
60.	takahiro kusakabe, Heme precursor injection is effective for Arthromyces ramosus peroxidase fusion protein production by a silkworm expression system, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 10.1016/j.jbiosc.2015.02.013, 120, 4, 384-386, 2015.10.
61.	Li Zhu, Tsuneyuki Tatsuke, Jian Xu, Zhiqing Li, 門宏明, JAE MAN LEE, takahiro kusakabe, Loqs depends on R2D2 to localize in D2 body-like granules and functions in RNAi pathways in silkworm cells, 64, 78-90, 2015.09.
62.	takahiro kusakabe, Mass Production of an Active Peptide-N-Glycosidase F Using Silkworm-Baculovirus Expression System, MOLECULAR BIOTECHNOLOGY, 10.1007/s12033-015-9866-1, 57, 8, 735-745, 2015.08.
63.	takahiro kusakabe, Expression of Recombinant Viscum Album Coloratum lectin B-chain in the Silkworm Expression System and Evaluation of Antioxidant Activity, BIOTECHNOLOGY AND BIOPROCESS ENGINEERING, 10.1007/s12257-014-0806-x, 20, 3, 515-522, 2015.06.
64.	takahiro kusakabe, Amyloidogenic lysozymes accumulate in the endoplasmic reticulum accompanied by the augmentation of ER stress signals, BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 10.1016/j.bbagen.2015.01.018, 1850, 6, 1107-1119, 2015.06.
65.	takahiro kusakabe, Improvement of Endo-β-N-acetylglucosaminidase H production using silkworm–baculovirus protein expression system, Journal of Asia-Pacific Entomology, 18, 2, 175-180, 2015.06.
66.	takahiro kusakabe, Human alpha 1-acid glycoprotein as a model protein for glycoanalysis in baculovirus expression vector system, JOURNAL OF ASIA-PACIFIC ENTOMOLOGY, 10.1016/j.aspen.2015.03.006, 18, 2, 303-309, 2015.06.
67.	takahiro kusakabe, Characterization and evolutionary analysis of tributyltin‐binding protein and pufferfish saxitoxin and tetrodotoxin‐binding protein genes in toxic and nontoxic pufferfishes, 28, 5, 1103-1108, 2015.05.
68.	Y. Hashiguchi, Man Lee, M. Shiraishi, S. Komatsu, S. Miki, Yohei Shimasaki, Noritaka Mochioka, Takahiro Kusakabe, Yuji Oshima, Characterization and evolutionary analysis of tributyltin-binding protein and pufferfish saxitoxin and tetrodotoxin-binding protein genes in toxic and nontoxic pufferfishes, Journal of Evolutionary Biology, 10.1111/jeb.12634, 28, 5, 1103-1118, 2015.01, Understanding the evolutionary mechanisms of toxin accumulation in pufferfishes has been long-standing problem in toxicology and evolutionary biology. Pufferfish saxitoxin and tetrodotoxin-binding protein (PSTBP) is involved in the transport and accumulation of tetrodotoxin and is one of the most intriguing proteins related to the toxicity of pufferfishes. PSTBPs are fusion proteins consisting of two tandem repeated tributyltin-binding protein type 2 (TBT-bp2) domains. In this study, we examined the evolutionary dynamics of TBT-bp2 and PSTBP genes to understand the evolution of toxin accumulation in pufferfishes. Database searches and/or PCR-based cDNA cloning in nine pufferfish species (6 toxic and 3 nontoxic) revealed that all species possessed one or more TBT-bp2 genes, but PSTBP genes were found only in 5 toxic species belonging to genus Takifugu. These toxic Takifugu species possessed two or three copies of PSTBP genes. Phylogenetic analysis of TBT-bp2 and PSTBP genes suggested that PSTBPs evolved in the common ancestor of Takifugu species by repeated duplications and fusions of TBT-bp2 genes. In addition, a detailed comparison of Takifugu TBT-bp2 and PSTBP gene sequences detected a signature of positive selection under the pressure of gene conversion. The complicated evolutionary dynamics of TBT-bp2 and PSTBP genes may reflect the diversity of toxicity in pufferfishes..
69.	takahiro kusakabe, Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706 T, Meta gene, 4, 29-44, 2014.06.
70.	takahiro kusakabe, Middle region of FancM interacts with Mhf and Rmi1 in silkworms, a species lacking the Fanconi anaemia (FA) core complex, INSECT MOLECULAR BIOLOGY, 10.1111/imb.12072, 23, 2, 185-198, 2014.04.
71.	takahiro kusakabe, Characterization and recombinant protein expression of ferritin light chain homologue in the silkworm, Bombyx mori, INSECT SCIENCE, 10.1111/1744-7917.12031, 21, 2, 135-146, 2014.04.
72.	takahiro kusakabe, Establishment of Caenorhabditis elegans SID-1-Dependent DNA Delivery System in Cultured Silkworm Cells, MOLECULAR BIOTECHNOLOGY, 10.1007/s12033-013-9694-0, 56, 3, 193-198, 2014.03.
73.	takahiro kusakabe, Roles of Piwi Proteins in Transcriptional Regulation Mediated by HP1s in Cultured Silkworm Cells, PLOS ONE, 10.1371/journal.pone.0092313, 9, 3, 2014.03.
74.	takahiro kusakabe, Baculovirus-mediated gene transfer systems in silkworm larvae using constitutive host promoters, JOURNAL OF ASIA-PACIFIC ENTOMOLOGY, 10.1016/j.aspen.2013.10.009, 17, 1, 73-78, 2014.03.
75.	takahiro kusakabe, Chromatin-induced spindle assembly plays an important role in metaphase congression of silkworm holocentric chromosomes, INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY, 10.1016/j.ibmb.2013.11.007, 45, 40-50, 2014.02.
76.	takahiro kusakabe, Establishment of a soaking RNA interference and Bombyx mori nucleopolyhedrovirus (BmNPV)-hypersensitive cell line using Bme21 cell, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 10.1007/s00253-013-5279-x, 97, 24, 10435-10444, 2013.12.
77.	takahiro kusakabe, Soaking RNAi in Bombyx mori BmN4-SID1 cells arrests cell cycle progression, JOURNAL OF INSECT SCIENCE, 13, 2013.12.
78.	takahiro kusakabe, Characterization of Tudor-sn-containing granules in the silkworm, Bombyx mori, INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY, 10.1016/j.ibmb.2013.04.004, 43, 8, 664-674, 2013.08.
79.	takahiro kusakabe, phiC31-integrase-mediated, site-specific integration of transgenes in the silkworm, Bombyx mori (Lepidoptera: Bombycidae), APPLIED ENTOMOLOGY AND ZOOLOGY, 10.1007/s13355-013-0182-6, 48, 3, 265-273, 2013.08.
80.	takahiro kusakabe, Production and characterization of the celery mismatch endonuclease CEL II using baculovirus/silkworm expression system, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 10.1007/s00253-012-4583-1, 97, 15, 6813-6822, 2013.08.
81.	takahiro kusakabe, PRODUCTION OF SMALL ANTIBACTERIAL PEPTIDES USING SILKWORM-BACULOVIRUS PROTEIN EXPRESSION SYSTEM, PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY, 10.1080/10826068.2012.762717, 43, 6, 565-576, 2013.08.
82.	日下部宜宏, Soaking RNAi-mediated modification of Sf9 cells for baculovirus expression system by ectopic expression of Caenorhabditis elegans SID-1, 10.1007/s00253-013-4785-1, 97, 13, 5921-5931, 2013.07.
83.	takahiro kusakabe, RNAi suppression of beta-N-acetylglucosaminidase (BmFDL) for complex-type N-linked glycan synthesis in cultured silkworm cells, BIOTECHNOLOGY LETTERS, 10.1007/s10529-013-1183-9, 35, 7, 1009-1016, 2013.07.
84.	takahiro kusakabe, A MC motif in silkworm Argonaute 1 is indispensible for translation repression, INSECT MOLECULAR BIOLOGY, 10.1111/imb.12023, 22, 3, 320-330, 2013.06.
85.	takahiro kusakabe, Characterization, Localization, and Stage-Dependent Gene Expression of Gonadotropin Receptors in Chub Mackerel (Scomber japonicus) Ovarian Follicles, BIOLOGY OF REPRODUCTION, 10.1095/biolreprod.112.107292, 88, 6, 2013.06.
86.	takahiro kusakabe, AMINO ACID DEPRIVATION-INDUCED EXPRESSION OF ASPARAGINE SYNTHETASE REGULATES THE GROWTH AND SURVIVAL OF CULTURED SILKWORM CELLS, ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, 10.1002/arch.21091, 83, 2, 57-68, 2013.06.
87.	Lee, J., Mon, H., Banno, Y., Iiyama, K., Kusakabe, T., Bombyx mori strains useful for efficient recombinant protein production using a baculovirus vector., J. Biotech. Biomater., 2012.06.
88.	Zhu, L., Tatsuke, T., Li, Z., Mon, H., Xu, Z., Lee, J., and Kusakabe T.,, Molecular cloning of BmTUDOR-SN and analysis of its role in RNAi pathway in the silkworm, Bombyx mori (Lepidoptera: Bombycidae). , Appl. Entomol. Zool., 2012.06.
89.	Sugahara, R., Mon, H., Lee, J., and Kusakabe, T.,, Monoubiquitination-Dependent Chromatin Loading of FancD2 in Silkworms, a Species Lacking the FA Core Complex., Gene, 2012.05.
90.	Li, Z., Cheng, D., Mon, H., Tatsuke, T., Zhu, L., Xu, J., Lee, J., Xia, Q., and Kusakabe, T., Genome-wide identification of Polycomb target genes reveals a functional association of Pho with Scm in Bombyx mori., PLoS ONE, , 7, e34330, 2012.04.
91.	Nagata, Y., Sakashita, K., Imanishi, S., Lee, J., and Kuskabe T. , Expression of glycosylated mucin-like domain using baculovirus expression system in silkworm, Bombyx mori., J. Fac. Agr. Kyushu Univ., , 57, 83-86, 2012.04.
92.	Fujii, M., Takahashi, M., Mon, H., Tatsuke, T., Lee, J., and Kuskabe T., Molecular cloning of the silkworm p53R2 homolog gene. , J. Fac. Agr. Kyushu Univ., , 57, 79-82, 2012.04.
93.	Li, Z., Tatsuke, T., Sakashita, K., Zhu, L., Xu, J., Mon, H., Lee, J., and Kusakabe, T., Identification and characterization of Polycomb group genes in the silkworm, Bombyx mori., Mol. Biol. Report, , 39, 5575-5588, 2012.01.
94.	Kobayashi, I., Tsukioka, H., Kômoto, N., Uchino, K., Sezutsu, H., Tamura, T., Kusakabe, T., and Tomita, S., SID-1 protein of Caenorhabditis elegans mediates uptake of dsRNA into Bombyx cells. , Insect Biochem. Mol. Biol., , 42, 148-154, 2012.01.
95.	Mitsunobu, H., Izumi, H., Mon, H., Tatsuke, T., Lee, J., Kusakabe, T., Molecular Characterization of heterochromatin proteins 1a and 1b from the Silkworm, Bombyx mori., Insect Mol. Biol. , 21, 9-20, 2012.01.
96.	Mon, H., Kobayashi, I., Ohkubo, S. Tomita, S., Lee, J., Sezutsu, H., Tamura, T., Kusakabe, T., Effective RNA interference in cultured silkworm cells mediated by overexpression of Caenorhabditis elegans SID-1., RNA Biol., , 9, 40-46, 2012.01.
97.	Iiyama, K., Lee, J., Kusakabe, T., Yasunaga-Aoki, C., and Shimizu, S., Improvement in pHERD vectors to express recombinant proteins tagged with hexahistidine at either the NH2- or COOH- terrminal in Pseudomonas aeruginosa., J. Insect Biotech. Seric., , 80, 57-61, 2011.10.
98.	Chieda, Y., Iiyama, K., Lee, J., Kusakabe, T., Yasunaga-Aoki, C., and Shimizu, S., Virulence of an exotoxin A-deficient strain of Pseudomonas aeruginosa toward the silkworm, Bombyx mori., Microbial. Pathogenesis,, 51, 407-414, 2011.10.
99.	Mon, H., Lee, J., Kawaguchi, Y., Kusakabe, T., Double-strand breaks repair by gene conversion in silkworm holocentric chromosomes, Mol. Genet. Genomics, 2011.09.
100.	Sugimoto, Y., Kamada, Y., Tokunag, Y., Shinohara, H., Matsumoto, M., Kusakabe, T., Ohkuri, T., Ueda, T., Aggregates with lysozyme and ovalbumin show features of amyloid-like fibrils., Biochem. Cell Biol.,, 2011.09.
101.	Mon, H., Izumi, M., Mitsunobu, H., Tatsuke, T., Iiyama, K., Jikuya, H., Lee, J., Kusakabe, T., Post-translational modifications of the N-terminal tail of histone H3 in holocentric chromosomes of bombyx mori. Insect Biochem. , Mol. Genet. Genomics, 2011.09.
102.	Mitsunobu, H., Izumi, H., Mon, H., Tatsuke, T., Lee, J., Kusakabe, T., Molecular Characterization of heterochromatin proteins 1a and 1b from the Silkworm, Bombyx mori. , Insect Mol. Biol. , 2011.04.
103.	Satone, H., Lee, J., Oba, Y., Kusakabe, T., Akahoshi, E., Miki, S., Suzuki, N., Sasayama, Y., Nassef, M., Shimasaki, Y., Kawabata, S., Honjo, T., Oshima, Y., Tributyltin-binding protein type 1, a lipocalin, prevents inhibition of osteoblastic activity by tributyltin in fish scales. , Aquat. Toxicol. , 103, 79-84, 2011.02.
104.	Mitsunobu, H., Izumi, M., Iiyama, K., Jikuya, H., Lee, J., Mon, H., Kawaguchi, Y., Kusakabe, T. , Molecular Characterization of Core Histones in the Silkworm, Bombyx mori. , J. Insect Biotech. Seric., 79, 75-83, 2010.10.
105.	Sugiura, N., Baba, Y., Kawaguchi, Y., Iwatani, T., Suzuki, K., Kusakabe, T., Yamagishi, K., Kimata, K., Kakuta, Y., Watanabe, H. , Glucuronyltransferase activity of KfiC from Escherichia coli strain K5 requires association of KfiA: KfiC and KfiA are essential enzymes for production of K5 polysaccharide, N-acetylheparosan. , J Biol Chem., 285, 1597-1606, 2010.04.
106.	Hong, S.M., Yamashita, J., Mitsunobu, H., Uchino, K., Kobayashi, I., Tamura, T., Nakajima, H., Miyagawa, H., Lee, J.M., Mon, H., Miyata, T., Kawaguchi, Y., Kusakabe, T., Efficient Soluble Protein Production on Transgenic Silkworms Expressing Cytoplasmic Chaperones using Baculovirus Expression System. , Appl. Microbiol. Biotech, 2010.04.
107.	Hong, S.M., Noh, S.K., Kim, K.A., Mitsunobu, H., Mon, H., Lee, J.M., Kawaguchi, Y., Kusakabe, T. , Molecular Characterization, Localization, and Distribution of Innexins in the Silkworm, Bombyx mori. , Mol. Biotechnol. , 43, 52-58., 2009.09.
108.	Mon, H., Sugahara, R., Hong, S.M., Lee, J., Kamachi, Y., Kawaguchi, Y., Kusakabe, T. , Analysis of protein interactions with two-hybrid system in cultured insect cells. , Anal. Biochem. , 392, 180-182., 2009.09.
109.	Karasaki, N., Mon, H., Takahashi, M., Lee, J., Koga, K., Kawaguchi, Y., Kusakabe, T. , Establishment of Tetracycline-inducible Gene Expression Systems in the Silkworm, Bombyx mori. , Biotechnol. Lett. , 31, 495-500., 2009.06.
110.	Sakashita, K., Tatsuke, T., Lee, J., Kawaguchi, Y., and Kusakabe, T. , Sequence-Nonspecific Suppression of Gene Expression by Double-stranded RNA in Silkworm Cultured Cells. , J. Insect Biotech. Seric. , 78, 33-37., 2009.06.
111.	Sakashita, K., Tatsuke, T., Masaki, Y., Lee, J., Kawaguchi, Y., Kusakabe, T. , dsRNA Binding Activity of Silkworm Larval Hemolymph is Mediated by Lipophorin Complex , J. Fac. Agr., Kyushu Univ. , 54, 401-406, 2009.04.
112.	Tatsuke, T., Hong, S.M., Tobata, H., Mon, H., Lee, J., Kawaguchi, Y., Kusakabe, T., Construction of piggyBac-based Vectors Using Visible and Drug-resistance Marker for Introducing Foreign Genes into Silkworm Cultured Cells. , J. Fac. Agr., Kyushu Univ. , 54, 397-400, 2009.04.
113.	Egami, I., Iiyama, K., Zhang, P., Chieda, Y., Ino, N., Hasegawa, K., Lee, J., Kusakabe, T., Yasunaga-Aoki, C., Shimizu, S. , Insecticidal bacterium isolated from an ant lion larva from Munakata, Japan. , J. Appl. Entomol. , 133 117-124., 2009.03.
114.	Lee, J., Takahashi, M., Mon, H., Nakajima, Y., Koga, K., Kawaguchi, Y., Kusakabe, T., , Construction of gene expression systems in insect cell lines using promoters from the silkworm, Bombyx mori. , J. Biotechnol. , 133, 9-17., 2008.06.
115.	Kawakami, N., Lee, J., Mon, H., Kubo, Y., Banno, Y., Kawaguchi, Y., Maenaka, K., Park, E.Y., Koga, K., Kusakabe, T., , Efficient Protein Expression in Bombyx mori Larvae of the Strain d17 Highly Sensitive to B. mori Nucleopolyhedrovirus., Mol. Biotechnol. , 40, 180-185., 2008.06.
116.	Chieda,Y., Iiyama,K., Lee, J., Kusakabe, T., Yasunaga-Aoki, C., Shimizu, S., Inactivation of pyocyanin synthesis genes has no effect on the virulence of Pseudomonas aeruginosa PAO1 toward the silkworm, Bombyx mori. , FEMS Microbiol. Lett. , 278, 101–107., 2008.03.
117.	Kawaguchi Y., Tatsuke T., Kusakabe T., Lee J., and Koga K., Singular compound eye architecture of the varnished eye mutation, ve, in Bombyx mori. , J. Insect Biotech. Seric. , 77, 53-58., 2008.03.
118.	Hong, S.M., Kusakabe, T., Lee, J., Tatsuke, T., Kawaguchi, Y., Kang, M.W., Kim, K.A., Nho, S.K., Structure and Expression Analysis of the Cecropin-E Gene from the Silkworm, Bombyx mori. , Biosci. Biotechnol. Biochem. , 72, 992-1998., 2008.03.
119.	Kawaguchi, Y., Tatsuke, T., Oike, Y., Taniguchi, A., Kusakabe, T., and Lee, J., and Koga, K., Fertility and Hatching of the vit Mutant Eggs in Bombyx mori. , J. Insect Biotech. Seric. , 77, 121-124., 2008.03.
120.	Iiyama, K., Chieda, Y., Lee, J., Kusakabe, T., Yasunaga-Aoki, C., and Shimizu, S. , Virulence of Phospholipase C Mutants of Pseudomonas aeruginosa PAO1 against the Silkworm, Bombyx mori., J. Insect Biotech. Seric. , 77, 115-120., 2008.03.
121.	Zhang, P., Aso, Y., Jikuya, H., Kusakabe, T., Lee, J., Kawaguchi, Y., Yamamoto, K., Banno, Y., Fujii, H., , Proteomic profiling of the silkworm skeletal muscle proteins during larval-pupal metamorphosis. , J. Proteome res. , 6, 2295-2303., 2007.07.
122.	Kawaguchi Y., Yoshida Y., Kusakabe T., Lee J., and Koga K. , Characteristic of Se, the white-sided egg mutation in Bombyx mori. , J. Insect Biotech. Seric. , 76, 71-78., 2007.06.
123.	Chieda,Y., Iiyama,K., Lee, J., Kusakabe, T., Yasunaga-Aoki, C., Lee, J., Kusakabe, T., Shimizu, S. , The gacA gene of Pseudomonas aeruginosa PAO1 is not required for full virulence in Bombyx mori. J. Insect Biotechnol. Sericol. , J. Insect Biotech. Seric., 76, 89-96., 2007.06.
124.	Lee, J., Mon, H., Takahashi, M., Kawakami, N. Yoshida, Y., Banno, Y., Koga, K., Kawaguchi, Y., Kusakabe, T., , Screening of high-permissive silkworm strains for efficient recombinant protein production in Autographa californica nuclear polyhedrosis virus (AcNPV). , J. Insect Biotech. Seric., 76, 101-106., 2007.06.
125.	Yamashita, J., Miyagawa, Y., Sugahara, R., Mon, H., Mitsunobu, H., Lee, J., Kawaguchi, Y., and Kusakabe, T., , Molecular Cloning of Silkworm Cdc37 and its Interaction with Hsp90 Chaperon. , J. Insect Biotech. Seric., 76, 137-143., 2007.06.
126.	Sugahara, R., Mon, H., Yamashita, J., Mitsunobu, H., Lee, J., Kawaguchi, Y., Koga, K., and Kusakabe, T.,, Heterotrimeric Complex of Replication Protein A, a Single-Stranded DNABinding Protein, from the Silkworm, Bombyx mori., J. Insect Biotech. Seric., 76, 129-135., 2007.06.
127.	Iiyama,K., Chieda,Y., Lee, J., Kusakabe, T., Yasunaga-Aoki, C., Lee, J., Kusakabe, T., Shimizu, S. , Effect of inactivation of the superoxide dismutase genes on the virulence of Pseudomonas aeruginosa PAO1 to silkworm, Bombyx mori. , Appl. Environ. Microbiol. , 73, 1569-1575., 2007.03.
128.	Shinohara H., Horiuchi M., Sato M., Kurisaki J., Kusakabe T., Koga K., Minami Y., AokiT., Kato I., and Sugimoto Y., Transition of ovalbumin to thermostable structure entails conformational changes involving the reactive center loop. , Biochim. Biophys. Acta, 1770, 5-11., 2007.01.
129.	Takahashi M., Lee J., Mon H., Yoshida H., Kawaguchi Y., Maekawa H., Koga K., and Kusakabe T. , Radiation resistance and its inheritance in the silkworm, Bombyx mori., J. Fac. Agr., Kyushu Univ. , 51, 261-264., 2006.10.
130.	Mitsunobu H., Sakashita K., Mon H., Lee J., Kawaguchi Y., Koga K., and Kusakabe T. , Construction of gateway-based destination vectors for detecting subcellular localization of proteins in the silkworm, Bombyx mori. , J. Insect Biotech. Seric. , 75, 141-146., 2006.10.
131.	Kawaguchi Y., Kusakabe T., Lee J., Nakajima Y., and Koga K. (2006) , Micropylar structure of chorion of the female sterile mutation, bd, in, Bombyx mori. , J. Insect Biotech. Seric. , 75, 9-14., 2006.03.
132.	Tsukioka H., Takahashi M., Mon H., Okano K., Mita K., Shimada T., Lee J., Kawaguchi Y., Koga K., and Kusakabe T. , Role of the Silkworm Argonaute2 Homologue Gene in Double-Strand Break Repair of Extrachromosomal DNA. , Nucleic Acids Res. , 34. 1092-1101., 2006.02.
133.	Takahashi M., Lee J., Mon H., Kawaguchi Y., Koga K., and Kusakabe T., Cell Cycle Arrest induced by Radiation in Cultured Silkworm Cells, J. Insect Biotech. Seric., 75, 75-78., 2006.01.
134.	Nakayama G., Kawaguchi Y., Koga K., and Kusakabe T. , Site-specific gene integration in cultured silkworm cells mediated by φC31 integrase., Mol. Genet. Genomics , 275, 1-8., 2006.01.
135.	Maeda T., Kusakabe T., Lee J., Miyagawa Y., Kawaguchi Y., Koga K., Efficient nonviral gene transfer mediated by polyethylenimine in an insect cell line., J. Insect Biotech. Seric., 74, 21-26., 2005.01.
136.	Kawaguchi, Y., Doira, H., Ohta, K., Kusakabe, T., and Koga, K., Inheritance of gon, a body color mutation of newly hatched larvae in Bombyx mori., J. Insect Biotech. Seric., 74, 35-37., 2005.01.
137.	Chieda,Y., Iiyama,K., Yasunaga-Aoki, C., Lee, J., Kusakabe, T., Shimizu, S., Pathogenicity of gacA mutant of Pseudomonas aeruginosa PA01 in the silkworm, Bombyx mori., FEMS Microbiol. Lett., 10.1016/j.femsle.2005.01.032, 244, 1, 181-186, 244, 181?186., 2005.01.
138.	Shinohara H., Iwasaki T., Miyazaki Y., Matsuo K., Aoki T., Matsumoto M., Oka T., Kurisaki J.I., Mizumachi K., Kusakabe T., Koga K., and Sugimoto Y., Thermostabilized ovalbumin that occurs naturally during development accumulates in embryonic tissues., Biochim. Biophys. Acta, 10.1016/j.bbagen.2005.02.016, 1723, 1-3, 106-113, 1723, 106-113., 2005.01.
139.	Miyagawa Y., Lee J., Maeda T., Koga K. Kawaguchi Y., Kusakabe T., Differential expression of a Bombyx mori AHA1 homologue during spermatogenesis., Insect Mol. Biol., 10.1111/j.1365-2583.2004.00553.x, 14, 3, 245-253, 14, 245-253., 2005.01.
140.	Maeda T., Kusakabe T., Lee J., Miyagawa Y., Kawaguchi Y., Koga K., Cloning and Characterization of a Ribonuclease L Inhibitor from the Silkworm, Bombyx mori., DNA Sequence, 10.1080/10425170400028871, 16, 1, 21-27, 16, 21-27., 2005.01.
141.	Ohsaki A., Iiyama K., Miyagawa Y., Kawaguchi Y., Koga K., and Kusakabe T., Nonhomologous end-joining in a cell-free extract from the cultured silkworm cell line BmN4., Mol. Biol. Reports, 10.1007/s11033-004-2474-y, 32, 1, 25-34, 32, 25-34., 2005.01.
142.	Lee J., Takahashi M., Mon H., Koga K., Kawaguchi Y., and Kusakabe T., Efficient gene transfer into silkworm larval tissues by a combination of sonoporation and lipofection., Cell Biol. Int., 10.1016/j.cellbi.2005.07.007, 29, 11, 976-979, 2005.01.
143.	Sakaguchi B., Kawaguchi Y., Koga K., and Kusakabe T., Evidence of defect in fibroin secretion in the posterior silk gland cells of the flc mutant of Bombyx mori., J. Insect Biotech. Seric., 74, 75-78., 2005.01.
144.	Kawaguchi, Y., Kawakami, K., Kusakabe, T., and Koga, K., Histological observation of eggs of the embryonic lethal mutation, Set, Bombyx mori., J. Insect Biotech. Seric., 43, 101-106., 2004.01.
145.	Lee J., Kusakabe T., Kawaguchi Y., Takahashi M., Mon H., Nho S., and Koga K., Molecular cloning and characterization of the translationally controlled tumor protein (TCTP) gene in Bombyx mori., Comp. Biochem. Phys., 10.1016/j.cbpc.2004.06.004, 139, 1, 35-43, 139, 35-43., 2004.01.
146.	Mon H., Kusakabe, T., Lee, J., Kawaguch, Y., and Koga, K., A method for measuring the efficiency of gene targeting in cultured silkworm cells., Comp. Biochem. Phys., 139, 99-106., 2004.01.
147.	Miyagawa Y., Kusakabe T., Lee J., Maeda T., Kawaguchi Y., and Koga K., Isolation and characterization of differently expressed cDNAs in a meiotic recombination strain of Bombyx mori., J. Insect Biotech. Seric., 73, 117-127, 2004.01.
148.	He N., Fujii H., Kusakabe T., Aso Y., Banno Y., and Yamamoto Y., Overexpression in Escherichia coli and purification of recombinant CI-b1, a Kunitz-type chymotrypsin inhibitor of silkworm., Protein Expr. Purif., 10.1016/j.pep.2004.07.010, 38, 1, 9-16, 38, 9-16., 2004.01.
149.	Mon, H., Kusakabe, T., Bando, H., Kojima, K., Kawaguch, Y., and Koga, K., Analysis of extrachromosomal homologous recombination in cultured silkworm cells, Biochem. Biophys. Res. Commun., 10.1016/j.bbrc.2003.10.169, 312, 3, 684-690, 312, 684-690., 2003.12.
150.	Kawaguchi, Y., Kusakabe, T., and Koga, K., Influence of N-metyl-N-nitrosourea treatment on embryogenesis of Bombyx mori, J. Fac. Agr., Kyushu Univ., 48, 1-2, 59-64, 48, 59-64., 2003.09.
151.	Kawaguchi, Y., Ichida, M., Kusakabe, T., and Koga, K., Chorion architecture in the Japanese giant silkmoth, Caligula japonica japonica moore, Sericologia, 43, 29-39., 2003.06.
152.	Fujikawa, T., Kawaguchi, Y., Kusakabe, T., and Koga, K., Histological studies on the yolk granule formation in the egg character mutant, vit, of Bombyx mori, J. Insect Biotech. Seric., 72, 111-115., 2003.05.
153.	Lee, J., Kusakabe, T., Kawaguchi, Y., Aoki, C., Nho, S., and Koga, K., Molecular characterization of a Heat Shock Cognate 70-4 promoter from the silkworm, Bombyx mori, J. Insect Biotech. Seric., 72, 33-39., 2003.02.
154.	Kawaguchi, Y., Kusakabe, T., and Koga, K., Morphological variation of micropylar apparatus in Bombyx mori eggs, J. Insect Biotech. Seric., 71, 49-54., 2002.09.
155.	Ota, A., Kusakabe, T., Yasushi Sugimoto, Takahashi, T., Nakajima, Y., Y Kawaguchi, Y., and Koga, K., Cloning and characterization of testis-specific tektin in Bombyx mori, Comp. Biochem. Phys., 10.1016/S1096-4959(02)00153-7, 133, 3, 371-382, 133, 371-382., 2002.03.
156.	Sugimoto, Y., Sanuki, S., Ibrahim, R. H., Aoki, T., Kusakabe, T., and Koga K., Occurrence of ovalbumin in ovarian yolk of chicken during oogenesis., Biochim. Biophys. Acta, 10.1016/S0304-4165(01)00095-2, 1526, 1, 1-4, 20367, 1-4., 2001.01.
157.	Kusakabe, T., Maeda, T., Kawaguchi, Y., and Koga, K., Role of Interaction between Two Silkworm RecA Homologs in Homologous DNA Pairing., Arch. Biochem. Biophys., 10.1006/abbi.2001.2275, 388, 1, 39-44, 388, 39-44., 2001.01.
158.	Yamamoto, T., Fujii, H., Kusakabe, T., Koga, K., Aso, Y., and Ishiguro, M., Change in phenoloxidase and its precursor during silkworm (a80 strain) development., J. Fac. Agr., Kyushu Univ., 45, 2, 487-493, 45, 487-493., 2001.01.
159.	Ogura, I., Kusakabe, T., Kawaguchi, Y., Maeda, T., and Koga, K., Molecular clonong and expression of cDNAs encording testis-specific and non-specific ATPase inhibitor-like proteins in Bombyx mori., J. Insect Biotech. Seric., 70, 121-128., 2001.01.
160.	Kusakabe, T., Sugimoto, Y., Maeda, T., Miyano, M., Nishikawa, J., Tone, S., Kawaguchi, Y., Koga, K., and Ohyama, T., Linearization and integration of DNA into cells preferentially occurs at intrinsically curvedregions from human LINE-1 repetitive element., Gene., 10.1016/S0378-1119(01)00631-X, 274, 1-2, 271-281, 274, 271-281., 2001.01.
161.	Kawaguchi, Y., Doira, H., Kusakabe, T., and Koga, K., Genetic analysis of the maternally conditioned "mosaic of Tanaka "mutation in Bombyx mori., J. Insect Biotech. Seric., 70, 193-197., 2001.01.
162.	Xia Q., Fujii H., Kusakabe T., and Banno Y., Identification of three annexin IX isoforms generated by alternative splicing of the carboxyl-terminal exon in silkworm, Bombyx mori., Insect Biochem. Mol. Biol., 10.1016/S0965-1748(01)00074-1, 32, 1, 9-14, 32, 9-14., 2001.01.
163.	Miyano, M., Okabe, T., Kusakabe, T., and Ohyama, T., Effect of upstream DNA architecture on transcription of a human LINE-1., J. Adv. Sci., 13, 1-6., 2001.01.
164.	Matsuo, H., Moriguchi, T., Takagi, T., Kusakabe, T., Buratowski, S., Sekine, M., Kyogoku, Y., and Wagner, G., Efficient synthesis of 13C, 15N labeled RNA containing the cap structure m7GpppA., J. Am.Chem.Soc., 122, 2417-2412., 2000.01.
165.	Yamamoto, T., Yakiyama, M., Fujii, H., Kusakabe, T., Koga, K., Aso, Y., and Ishiguro, M., Expression of Prophenoloxidase mRNA during silkworm hemocyte development., Biosci. Biotechnol. Biochem., 10.1271/bbb.64.1197, 64, 6, 1197-1202, 64, 1197-1202., 2000.01.
166.	Kusakabe, T., Sugimoto, Y., Hirota, Y., Tone, S., Kawaguchi, Y., Koga, K., and Ohyama, T., Isolation of replicational cue elements from a library of bent DNAs of Aspergillus oryzae., Mol. Biol. Reports, 10.1023/A:1007076511814, 27, 1, 13-19, 27, 13-19., 2000.01.
167.	Tone, S., Kubo, T., Ohyama, T., Kusakabe, T., and Minatogawa, Y., Quantitation of mRNA using in vitro RNA amplification and Northern hybridization., Anal. Biochem., 10.1006/abio.2000.4745, 284, 2, 420-422, 284, 420-422., 2000.01.
168.	Kawaguchi, Y., Kusakabe, T., and Koga, K., Characteristicof tsg, the low-temperature sensitive gray-egg mutation in Bombyx mori. J. Seric. Sci. Jpn., J. Seric. Sci. Jpn., 69, 289-295., 2000.01.
169.	Kawaguchi, Y., Kusakabe, T., and Koga, K., Chorion morphology of the Eri-silkworm, Samia cynthia ricini (Donovan) (Lepidoptera:Saturniidae)., Appl. Entomol. Zool., 10.1303/aez.2000.427, 35, 4, 427-434, 35, 419-426., 2000.01.
170.	Sugimoto, Y., Sanuki, S., Ohsako, S., Higashimoto, Y., Kondo, M., Kurawaki, J., Ibrahim, R. H., Aoki, T., Kusakabe, T., and Koga K., Ovalbumin in developing chicken eggs migrates from egg white to embryonic organs while changing its conformation and thermal stability., J. Biol. Chem., 10.1074/jbc.274.16.11030, 274, 16, 11030-11037, 274, 11030-11037., 1999.01.
171.	Kusakabe, T., Hine. A., Hubert, S., Wargner, G., and Richardson, C. C., Cys4 zinc-binding motifs in sequence-specific, single-stranded DNA recognition., Proc. Natl. Aca. Sci. USA., 96, 4295-4300., 1999.01.
172.	Kawaguchi, Y., Kusakabe, T., Banno, Y., and Koga, K., Effect of ooplasmic size on the larval development as observed in the small egg mutant emi of Bombyx mori., J. Sericult. Sci. JPN., 68, 245-250., 1999.01.
173.	Kawaguchi, Y., Kusakabe, T., and Koga, K., Unique chorion structure of the mottled gray (mgr) egg, an egg-character mutation of Bombyx mori (Lepidoptera: Bombycidae)., Appl. Entomol. Zool., 34, 3, 359-364, 34, 359-364., 1999.01.
174.	Kusakabe, T., Baradaran, K., Lee, J., and Richardson, C. C., Roles of the helicase and primase domain of the gene 4 protein of bacteriophage T7 in accessing primase recognition site., EMBO J., 17, 1542-1552., 1998.01.
175.	Lee, J., Chastain, P. D., Kusakabe, T., Griffith, J. D., and Richardson, C. C., Coordinated leading and lagging strand DNA synthesis on a mini-circular template., Mol. Cell, 1, 1001-1010., 1998.01.
176.	Takagi, T., Taylor, G. S., Kusakabe, T., Charbonneau, H., and Buratowski, S., A PTP-like protein from baculovirus has RNA 5'-triphosphatase and diphosphatase activity., Proc. Natl. Aca. Sci. USA., 95, 9808-9812., 1998.01.
177.	Kusakabe, T., and Richardson, C. C., Template recognition and ribonucleotide specificity of the DNA primase of bacteriophage T7., J. Biol. Chem., 272, 5943-5951., 1997.01.
178.	Inoue, T., Yatsuki, H., Kusakabe, T., Joh, K., Takasaki, Y., Nikoh, N., Miyata, T., and Hori, K., Caenorhabditis elegans has two isozymic forms, CE-1 and CE-2, of fructose-1,6-bisphosphate aldolases which are encoded by different genes., Arch. Biochem. Biophys., 339, 226-234., 1997.01.
179.	Kusakabe, T., and Richardson, C. C., Gene 4 DNA primase of bacteriophage T7 mediates the annealing and extension of oligonucleotides at primase recognition sites., J. Biol. Chem., 272, 12446-12453., 1997.01.
180.	Zhang, R., Kusakabe, T., Yatsuki, H., and Hori, K., Lamprey fructose-1, 6-bisphosphate aldolases - Characterization of the muscle-type and non-muscle-type isozymes., Arch. Biochem. Biophys., 341, 170-176., 1997.01.
181.	Kusakabe, T., Motoki, K., and Hori, K., Mode of interactions of human aldolase isozymes with cytoskeletons., Arch. Biochem. Biophys., 344, 184-193., 1997.01.
182.	Kuba, M, Yatsuki, H., Kusakabe. T., Takasaki. Y., Nikoh. N., Miyata. T., Yamaguchi. T., and Hori, K., Molecular evolution of amphioxus fructose-1,6-bisphosphate aldolases., Arch. Biochem. Biophys., 348, 329-336., 1997.01.
183.	Kaihara, R., Matsuhashi, S., Kusakabe, T., Kondo, T., Iwanaga, A., and Hori, K., Monoclonal anti-human aldolase C antibodies that react to the isozyme group-specific sequences and generally conserved sequences of human aldolase C., J. Biochem., 119, 281-290., 1996.01.
184.	Sugimoto, Y., Kusakabe, T., Nagaoka, S., Nirasawa, T., Tatsuguchi, K., Fujii, S., Aoki. T., and Koga, K., A proteinase inhibitor from egg yolk of hen is an ovoinhibitor analog., Biochim. Biophys. Acta, 1295, 1, 96-102, 1295, 96-102., 1996.01.
185.	Kusakabe, T., and Richardson, C. C., The role of zinc finger motif in DNA primase., J. Biol. Chem., 271, 19563-19570., 1996.01.
186.	Kadokawa, Y., Kusakabe, T., Kamachi, Y., Isobe, K., Kondoh, H., and Ohyama, T., A murine Thy-1.2 reporter vector containing SV40 ori for rapid cloning and analysis of eukaryotic promoters. Gene 153, 277-278, Gene, 153, 277-278 ., 1995.01.
187.	Zhang, R., Yatsuki, H., Kusakabe, T., Iwabe, N., Miyata, T., Imai, T., Yoshida, M., and Hori, K., The structures of cDNAs encoding the muscle type and non muscle type isozymes of lamprey fructose bisphosphate aldolases and the evolution of aldolase gene., J. Biochem., 117, 545-553 ., 1995.01.
188.	Zhang, R., Sugimoto, Y., Kai, T., Kusakabe, T., Takasaki, Y., Koga, K., and Hori, K., Drosophila melanogaster aldolase-Characterization of the isozymes alpha, beta and gamma generated from a single gene., J. Biochem., 118, 1, 183-188, 118, 183-188., 1995.01.
189.	Sugimoto, Y., Kusakabe, T., Kai, T., Okamura, T., Koga, K., and Hori, K., Analysis of the in vitro translation products of a novel type Drosophila melanogaster aldolase mRNA in which two carboxyl-terminal exon remain unspliced., Arch. Biochem. Biophys., 10.1006/abbi.1995.9953, 323, 2, 361-366, 323, 361-366, 1995.01.
190.	Sone, T., Komiyama, N., Simizu, K., Kusakabe, T., Morikubo, K., and Kino, K., Cloning and sequencing of cDNA coding for Cri J I, a major allergen of Japanese cedar pollen, Biochim. Biophys. Res. Commun., 199, 619-625., 1994.10.
191.	Kusakabe, T., Koga, K., and Sugimoto, Y., Isolation and characterization of cDNA and genomic promoter region for a heat shock protein 30 from Aspergillus nidulans, Biochim. Biophys. Acta, 10.1016/0167-4781(94)90088-4, 1219, 2, 555-558, 1219, 555-558., 1994.10.
192.	Kusakabe, T., Motoki, K., and Hori, K., Human aldolase C: Characterization of the recombinant enzyme expressed in Escherichia coli, J. Biochem., 115, 1172-1177., 1994.06.
193.	Kusakabe, T., Motoki, K., Sugimoto, Y., Takasaki, Y., and Hori, K., Human aldolase B: Liver-specific properties of the isozyme depend on type-B isozyme group-specific sequences., Protein Enginieer., 7, 1387-1393., 1994.01.
194.	Ohyama, T., and Kusakabe, T., High efficiency shotgun cloning of curved DNA segments from chromosomal DNA, Anal. Biochem., 212, 287-289, 1993.06.
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