Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Fujita Ryosuke Last modified date:2021.06.11

Associate Professor / Department of Bioresource Sciences / Faculty of Agriculture


Papers
1. Ryosuke Fujita, Maki N. Inoue, Takumi Takamatsu, Hiroshi Arai, Mayu Nishino, Nobuhiko Abe, Kentaro Itokawa, Madoka Nakai, Syun-ichi Urayama, Yuto Chiba, Michael Amoa-Bosompem, Yasuhisa Kunimi, Late Male-Killing Viruses in Homona magnanima Identified as Osugoroshi Viruses, Novel Members of Partitiviridae, Frontiers in Microbiology, 10.3389/fmicb.2020.620623, 11, 2021.01, Late male-killing, a male-specific death after hatching, is a unique phenotype found in Homona magnanima, oriental tea tortrix. The male-killing agent was suspected to be an RNA virus, but details were unknown. We herein successfully isolated and identified the putative male-killing virus as Osugoroshi viruses (OGVs). The three RNA-dependent RNA polymerase genes detected were phylogenetically related to Partitiviridae, a group of segmented double-stranded RNA viruses. Purified dsRNA from a late male-killing strain of H. magnanima revealed 24 segments, in addition to the RdRps, with consensus terminal sequences. These segments included the previously found male-killing agents MK1068 (herein OGV-related RNA16) and MK1241 (OGV-related RNA7) RNAs. Ultramicroscopic observation of purified virions, which induced late male-killing in the progeny of injected moths, showed sizes typical of Partitiviridae. Mathematical modeling showed the importance of late male-killing in facilitating horizontal transmission of OGVs in an H. magnanima population. This study is the first report on the isolation of partiti-like virus from insects, and one thought to be associated with late male-killing, although the viral genomic contents and combinations in each virus are still unknown..
2. Takayuki Nonoyama, Lei Wang, Masumi Tsuda, Yuki Suzuki, Ryuji Kiyama, Kazunori Yasuda, Shinya Tanaka, Kousuke Nagata, Ryosuke Fujita, Naoya Sakamoto, Noriyuki Kawasaki, Hisayoshi Yurimoto, Jian Ping Gong, Isotope Microscopic Observation of Osteogenesis Process Forming Robust Bonding of Double Network Hydrogel to Bone., Advanced healthcare materials, 10.1002/adhm.202001731, e2001731-2001731, 2020.11, Tough double network (DN) hydrogels are promising substitutes of soft supporting tissues such as cartilage and ligaments. For such applications, it is indispensable to robustly fix the hydrogels to bones with medically feasible methods. Recently, robustly bonding the DN hydrogels to defected bones of rabbits in vivo has been proved successful. The low crystalline hydroxyapatite (HAp) of calcium-phosphate-hydroxide salt coated on the surface layer of the DN hydrogels induced spontaneous osteogenesis penetrating into the semi-permeable hydrogels to form a gel/bone composite layer. In this work, the 44 Ca isotope-doped HAp/DN hydrogel is implanted in a defect of rabbit femoral bone and the dynamic osteogenesis process at the gel/bone interface is analyzed by tracing the calcium isotope ratio using isotope microscopy. The synthetic HAp hybridized on the surface layer of DN gel dissolves rapidly in the first two weeks by inflammation, and then the immature bone with a gradient structure starts to form in the gel region, reutilizing the dissolved Ca ions. These results reveal, for the first time, that synthetic HAp is reutilized for osteogenesis. These facts help to understand the lifetime of bone absorbable materials and to elucidate the mechanism of spontaneous, non-toxic, but strong fixation of hydrogels to bones..
3. How to overcome the issues related to medical insects.
4. Ryosuke Fujita, Masato Hino, Takeru Ebihara, Takumi Nagasato, Akitsu Masuda, Jae Man Lee, Tsuguru Fujii, Hiroaki Mon, Kohei Kakino, Ryo Nagai, Miyu Tanaka, Yoshino Tonooka, Takato Moriyama, Takahiro Kusakabe, Efficient production of recombinant SARS-CoV-2 spike protein using the baculovirus-silkworm system., Biochemical and biophysical research communications, 10.1016/j.bbrc.2020.06.020, 529, 2, 257-262, 2020.08, In the case of a new viral disease outbreak, an immediate development of virus detection kits and vaccines is required. For COVID-19, we established a rapid production procedure for SARS-CoV-2 spike protein (S protein) by using the baculovirus-silkworm expression system. The baculovirus vector-derived S proteins were successfully secreted to silkworm serum, whereas those formed insoluble structure in the larval fat body and the pupal cells. The ectodomain of S protein with the native sequence was cleaved by the host furin-protease, resulting in less recombinant protein production. The S protein modified in furin protease-target site was efficiently secreted to silkworm serum and was purified as oligomers, which showed immunoreactivity for anti-SARS-CoV-2 S2 antibody. By using the direct transfection of recombinant bacmid to silkworms, we achieved the efficient production of SARS-CoV-2 S protein as fetal bovine serum (FBS)-free system. The resultant purified S protein would be useful tools for the development of immunodetection kits, antigen for immunization for immunoglobulin production, and vaccines..
5. Michael Amoa-Bosompem, Daisuke Kobayashi, Katsunori Murota, Astri Nur Faizah, Kentaro Itokawa, Ryosuke Fujita, Joseph Harold Nyarko Osei, Esinam Agbosu, Deborah Pratt, Shohei Kimura, Kofi Dadzie Kwofie, Mitsuko Ohashi, Joseph H Kofi Bonney, Samuel Dadzie, Toshinori Sasaki, Nobuo Ohta, Haruhiko Isawa, Kyoko Sawabe, Shiroh Iwanaga, Entomological Assessment of the Status and Risk of Mosquito-borne Arboviral Transmission in Ghana., Viruses, 10.3390/v12020147, 12, 2, 2020.01, Entomological surveillance is one of the tools used in monitoring and controlling vector-borne diseases. However, the use of entomological surveillance for arboviral infection vector control is often dependent on finding infected individuals. Although this method may suffice in highly endemic areas, it is not as effective in controlling the spread of diseases in low endemic and non-endemic areas. In this study, we examined the efficiency of using entomological markers to assess the status and risk of arbovirus infection in Ghana, which is considered a non-endemic country, by combining mosquito surveillance with virus isolation and detection. This study reports the presence of cryptic species of mosquitoes in Ghana, demonstrating the need to combine morphological identification and molecular techniques in mosquito surveillance. Furthermore, although no medically important viruses were detected, the importance of insect-specific viruses in understanding virus evolution and arbovirus transmission is discussed. This study reports the first mutualistic relationship between dengue virus and the double-stranded RNA Aedes aegypti totivirus. Finally, this study discusses the complexity of the virome of Aedes and Culex mosquitoes and its implication for arbovirus transmission..
6. Expression and Purification of Vaccinia Virus DNA Topoisomerase IB Produced in the Silkworm-Baculovirus Expression System..
7. Yurie Kinoshita, Jian Xu, Akitsu Masuda, Kosuke Minamihata, Noriho Kamiya, Hiroaki Mon, Ryosuke Fujita, Takahiro Kusakabe, Jae 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..
8. Expression of the thermostable Moloney murine leukemia virus reverse transcriptase by silkworm-baculovirus expression system
© 2019 Korean Society of Applied Entomology 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..
9. Expression, purification, and characterization of highly active endo-α-N-acetylgalactosaminidases expressed by silkworm-baculovirus expression system
© 2019 Korean Society of Applied Entomology 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..
10. Ryosuke Fujita, Fumihiro Kato, Daisuke Kobayashi, Katsunori Murota, Tomohiko Takasaki, Shigeru Tajima, Chang-Kweng Lim, Masayuki Saijo, Haruhiko Isawa, Kyoko Sawabe, Persistent viruses in mosquito cultured cell line suppress multiplication of flaviviruses., Heliyon, 10.1016/j.heliyon.2018.e00736, 4, 8, e00736, 2018.08, In the growth kinetics analysis of flaviviruses in Aedes albopictus C6/36 cell lines obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank and the European Collection of Authenticated Cell Culture (ECACC), these two cells line showed different viral susceptibility for Zika virus (ZIKV), Dengue virus (DENV), and Japanese encephalitis virus (JEV). Next-generation sequencing (NGS) analysis revealed that the C6/36 JCRB strain was persistently infected with two viruses without showing any cytopathic effects. The complete sequence analysis demonstrated that the one virus was Menghai rhabdovirus (MERV), which has been found from Aedes albopictus mosquito. The other virus was a novel virus, designated as Shinobi tetravirus (SHTV). Interestingly, the viral susceptibility of these two strains was almost even for Sindbis virus and Getah virus. We cloned SHTV and MERV from JCRB C6/36 cell line and then re-infected them into another C6/36 cell line, resulting in the reproduction of persistent infection with each virus. ZIKV growth was suppressed in SHTV and/or MERV re-infected C6/36 cells also. To our knowledge, this is the first demonstration that persistent infection with rhabdovirus and/or permutotetravirus suppressed flavivirus replication in mosquito cells..
11. Daisuke Kobayashi, Katsunori Murota, Ryosuke Fujita, Kentaro Itokawa, Akira Kotaki, Meng Ling Moi, Hiroko Ejiri, Yoshihide Maekawa, Kohei Ogawa, Yoshio Tsuda, Toshinori Sasaki, Mutsuo Kobayashi, Tomohiko Takasaki, Haruhiko Isawa, Kyoko Sawabe, Dengue Virus Infection in Aedes albopictus during the 2014 Autochthonous Dengue Outbreak in Tokyo Metropolis, Japan., The American journal of tropical medicine and hygiene, 10.4269/ajtmh.17-0954, 98, 5, 1460-1468, 2018.05, In 2014 in Japan, 162 autochthonous dengue cases were reported for the first time in nearly 70 years. Here, we report the results of the detection and isolation of dengue virus (DENV) from mosquitoes collected in Tokyo Metropolis in 2014 and 2015. The phylogenetic relationship among DENV isolates from mosquitoes and from patients based on both the entire envelope gene and whole coding sequences was evaluated. Herein, 2,298 female and 956 male Aedes albopictus mosquitoes were collected at six suspected locations of DENV infection in Tokyo Metropolis from August to October in 2014 and grouped into 124 and 35 pools, respectively, for viral genome detection and DENV isolation. Dengue virus RNA was detected using reverse transcription polymerase chain reaction and TaqMan assays from 49 female pools; 16 isolates were obtained using C6/36 and Vero cells. High minimum infection rates (11.2-66.7) persisted until mid-September. All DENV isolates belonged to the genotype I in serotype 1 (DENV-1), and its sequences demonstrated > 99% homology to the sequence of the DENV isolated from a patient in the vicinity of Tokyo Metropolis in 2014. Therefore, Ae. albopictus was a major DENV vector, and a single DENV-1 strain circulated in Tokyo Metropolis in 2014. Dengue virus was not detected from male mosquitoes in 2014 and wild larvae in April 2015. Thus, the possibility of both vertical transmission and overwintering of DENV was extremely low, even in dengue-epidemic areas. This study reports the first entomological information on a dengue outbreak in a temperate region, where no Aedes aegypti mosquitoes are distributed..
12. Hiroko Ejiri, Chang-Kweng Lim, Haruhiko Isawa, Ryosuke Fujita, Katsunori Murota, Tomomi Sato, Daisuke Kobayashi, Miki Kan, Masashi Hattori, Toshiya Kimura, Yukie Yamaguchi, Mutsuyo Takayama-Ito, Madoka Horiya, Guillermo Posadas-Herrera, Shohei Minami, Ryusei Kuwata, Hiroshi Shimoda, Ken Maeda, Yukie Katayama, Tetsuya Mizutani, Masayuki Saijo, Koki Kaku, Hiroto Shinomiya, Kyoko Sawabe, Characterization of a novel thogotovirus isolated from Amblyomma testudinarium ticks in Ehime, Japan: A significant phylogenetic relationship to Bourbon virus., Virus research, 10.1016/j.virusres.2018.03.004, 249, 57-65, 2018.04, The genus Thogotovirus, as represented by Thogoto virus and Dhori virus, comprises a group of arthropod-borne viruses, most members of which are transmitted by ticks. Here we report the genetic and biological characterization of a new thogotovirus, designated Oz virus (OZV), isolated from the hard tick Amblyomma testudinarium in Ehime, Japan. OZV efficiently replicated and induced a cytopathic effect in Vero cells, from which enveloped pleomorphic virus particles were formed by budding. OZV could also replicate in BHK-21 and DH82 cells and caused high mortality in suckling mice after intracerebral inoculation. Phylogenetic analyses of six viral proteins indicated that OZV is clustered with Dhori and related viruses, and is most closely related in glycoprotein (GP) and matrix protein (M) sequences to Bourbon virus, a human-pathogenic thogotovirus discovered recently in the United States. Our findings emphasize the need for understanding the geographic distribution and ecology of OZV and related viruses and for reevaluation of the medical and public health importance of thogotoviruses..
13. Hiroko Ejiri, Chang-Kweng Lim, Haruhiko Isawa, Yukie Yamaguchi, Ryosuke Fujita, Mutsuyo Takayama-Ito, Ryusei Kuwata, Daisuke Kobayashi, Madoka Horiya, Guillermo Posadas-Herrera, Itoe Iizuka-Shiota, Satsuki Kakiuchi, Yukie Katayama, Toshihiko Hayashi, Toshinori Sasaki, Mutsuo Kobayashi, Shigeru Morikawa, Ken Maeda, Tetsuya Mizutani, Koki Kaku, Masayuki Saijo, Kyoko Sawabe, Isolation and characterization of Kabuto Mountain virus, a new tick-borne phlebovirus from Haemaphysalis flava ticks in Japan., Virus research, 10.1016/j.virusres.2017.11.030, 244, 252-261, 2018.01, In Japan, indigenous tick-borne phleboviruses (TBPVs) and their associated diseases first became evident in 2013 by reported human cases of severe fever with thrombocytopenia syndrome (SFTS). In this study, we report a novel member of the genus Phlebovirus designated as Kabuto Mountain virus (KAMV), which was isolated from the ixodid tick Haemaphysalis flava in Hyogo, Japan. A complete viral genome sequencing and phylogenetic analyses showed that KAMV is a novel member of TBPVs, which is closely related to the Uukuniemi and Kaisodi group viruses. However, unlike the Uukuniemi group viruses, the 165-nt intergenic region (IGR) in the KAMV S segment was highly C-rich in the genomic sense and not predicted to form a secondary structure, which are rather similar to those of the Kaisodi group viruses and most mosquito/sandfly-borne phleboviruses. Furthermore, the NSs protein of KAMV was highly divergent from those of other TBPVs. These results provided further insights into the genetic diversity and evolutionary relationships of TBPVs. KAMV could infect and replicate in some rodent and primate cell lines. We evaluated the infectivity and pathogenicity of KAMV in suckling mice, where we obtained a virulent strain after two passages via intracerebral inoculation. This is the first report showing the existence of a previously unrecognized TBPV in Japan, other than the SFTS virus..
14. Ejiri H, Lim CK, Isawa H, Yamaguchi Y, Fujita R, Takayama-Ito M, Kuwata R, Kobayashi D, Horiya M, Posadas-Herrera G, Iizuka-Shiota I, Kakiuchi S, Katayama Y, Hayashi T, Sasaki T, Kobayashi M, Morikawa S, Maeda K, Mizutani T, Kaku K, Saijo M, Sawabe K, Isolation and characterization of Kabuto Mountain virus, a new tick-borne phlebovirus from Haemaphysalis flava ticks in Japan., Virus research, 10.1016/j.virusres.2017.11.030, 244, 252-261, 2017.11.
15. Daisuke Kobayashi, Haruhiko Isawa, Ryosuke Fujita, Katsunori Murota, Kentaro Itokawa, Yukiko Higa, Yukie Katayama, Toshinori Sasaki, Tetsuya Mizutani, Shiroh Iwanaga, Nobuo Ohta, Arlene Garcia-Bertuso, Kyoko Sawabe, Isolation and characterization of a new iflavirus from Armigeres spp. mosquitoes in the Philippines., The Journal of general virology, 10.1099/jgv.0.000929, 98, 11, 2876-2881, 2017.11, During an entomological surveillance for arthropod-borne viruses in the Philippines, we isolated a previously unrecognized virus from female Armigeres spp. mosquitoes. Whole-genome sequencing, genetic characterization and phylogenetic analysis revealed that the isolated virus, designated Armigeres iflavirus (ArIFV), is a novel member of the iflaviruses (genus Iflavirus, family Iflaviridae) and phylogenetically related to Moku virus, Hubei odonate virus 4, slow bee paralysis virus and Graminella nigrifrons virus 1. To our knowledge, this is the first successful isolation of iflavirus from a dipteran insect. Spherical ArIFV particles of approximately 30 nm in diameter contained at least three major structural proteins. ArIFV multiplied to high titres (~109 p.f.u. ml-1) and formed clear plaques in a mosquito cell line, C6/36. Our findings provide new insights into the infection mechanism, genetic diversity and evolution of the Iflaviridae family..
16. Fujita R, Ejiri H, Lim CK, Noda S, Yamauchi T, Watanabe M, Kobayashi D, Ito MT, Murota K, Herrera GP, Minami S, Kuwata R, Yamaguchi Y, Horiya M, Katayama Y, Shimoda H, Saijo M, Maeda K, Mizutani T, Isawa H, Sawabe K, Isolation and characterization of Tarumizu tick virus: a new coltivirus from Haemaphysalis flava ticks in Japan, Virus Research, 10.1016/j.virusres.2017.09.017, 242, 131-140, 2017.10.
17. Kobayashi D, Isawa H, Fujita R, Murota K, Itokawa K, Higa Y, Katayama Y, Sasaki T, Mizutani T, Iwanaga S, Ohta N, Bertuso AG, Sawabe K, Isolation and characterization of a new iflavirus from Armigeres spp. mosquitoes in the Philippines, The Journal of general virology, 10.1099/jgv.0.000929, 2876-2881, 2017.10.
18. Ryosuke Fujita, Hiroko Ejiri, Chang-Kweng Lim, Shinichi Noda, Takeo Yamauchi, Mamoru Watanabe, Daisuke Kobayashi, Mutsuyo Takayama-Ito, Katsunori Murota, Guillermo Posadas-Herrera, Shohei Minami, Ryusei Kuwata, Yukie Yamaguchi, Madoka Horiya, Yukie Katayama, Hiroshi Shimoda, Masayuki Saijo, Ken Maeda, Tetsuya Mizutani, Haruhiko Isawa, Kyoko Sawabe, Isolation and characterization of Tarumizu tick virus: A new coltivirus from Haemaphysalis flava ticks in Japan., Virus research, 10.1016/j.virusres.2017.09.017, 242, 131-140, 2017.10, During the course of tick-borne virus surveillance in Japan, three independent isolates of probably the same virus were obtained from three geographically distant populations of the hard tick Haemaphysalis flava. Genome analyses of the three isolates demonstrated that they were closely related but distinct strains of a novel virus, designated Tarumizu tick virus (TarTV), which has a genome of 12 double-stranded RNA segments. The development of the virus-induced cytopathic effects on BHK cells significantly varied according to virus strains. Ten out of 12 segments of TarTV appeared to encode putative orthologs or functional equivalents of viral proteins of Colorado tick fever virus (CTFV) and Eyach virus, suggesting that TarTV is the third member of the genus Coltivirus in the family Reoviridae. This was supported by the facts that the 5'- and 3'-terminal consensus sequences of coltivirus genomes were found also in TarTV genome, and segment 9 of TarTV had sequence and structural features that may mediate a stop codon read-through as observed in that of CTFV. However, segment 7 and 10 of TarTV had no significant sequence similarities to any other proteins of known coltiviruses. Electron microscopic analysis demonstrated that TarTV particle had a non-enveloped bilayer icosahedral structure, and viral inclusion bodies were formed in infected cells. TarTV could infect and replicate in several mammalian cell lines tested, but show no clinical symptoms in intracerebrally inoculated mice. Taken together, our findings provide new insights into genetic diversity and evolution of the genus Coltivirus..
19. Ryosuke Fujita, Ryusei Kuwata, Daisuke Kobayashi, Arlene Garcia Bertuso, Haruhiko Isawa, Kyoko Sawabe, Bustos virus, a new member of the negevirus group isolated from a Mansonia mosquito in the Philippines, ARCHIVES OF VIROLOGY, 10.1007/s00705-016-3068-4, 162, 1, 79-88, 2017.01, We isolated two distinct viruses from mosquitoes collected in Bustos, Bulacan province, Philippines, in 2009. These viruses show rapid replication and strong cytopathic effects in mosquito C6/36 cells. Whole-genome analysis of these viruses demonstrated that both viruses belong to the negevirus group. One of the viruses, from Culex vishunui mosquitoes, is a new strain of Negev virus. The other virus, from a Mansonia sp. mosquito, is a new negevirus designated Bustos virus. Gene expression analysis of the Bustos virus revealed that infected cells contain viral subgenomic RNAs that probably include open reading frame (ORF) 2 or ORF3. Purified Bustos virus particles contained at least three proteins, and the major component (a probable major capsid protein) is encoded by ORF3. Bustos virus did not show infectivity in mammalian BHK-21 cells, suggesting that it is an insect-specific virus, like other known negeviruses..
20. Shigenori Ota, Miyuki Nishimura, Yuya Murakami, Naoko Kubo Birukawa, Akihiro Yoneda, Hiroki Nishita, Ryosuke Fujita, Yasushi Sato, Kenjiro Minomi, Keiko Kajiwara, Miyono Miyazaki, Maki Uchiumi, Shintaro Mikuni, Yasuaki Tamura, Toru Mizuguchi, Masafumi Imamura, Makoto Meguro, Yasutoshi Kimura, Koichi Hirata, Yoshiro Niitsu, Involvement of Pancreatic Stellate Cells in Regeneration of Remnant Pancreas after Partial Pancreatectomy, PLOS ONE, 10.1371/journal.pone.0165747, 11, 12, e0165747, 2016.12, Background and objectives
Mechanism of regeneration of remnant pancreas after partial pancreatectomy (PX) is still unknown. In this study, effect of siRNA against the collagen specific chaperone, HSP47, which inhibits collagen secretion from activated pancreas stellate cells (aPSCs), and induces their apoptosis, on regeneration of remnant pancreas was determined.
Methods
Pancreatectomy was performed according to established methods. Proliferation of cells was assessed by BrdU incorporation. Immunostaining of HSP47 was employed to identify PSCs. Progenitor cells were identified by SOX9 staining. Acinar cells were immunostained for amylase. Co-culture of acinar cells with aPSCs were carried out in a double chamber with a cell culture insert. siRNA HSP47 encapsulated in vitamin A-coupled liposome (VA-lip siRNA HSP47) was delivered to aPSCs by iv injection.
Results
In remnant pancreas of 90% PX rat, new areas of foci were located separately from duodenal areas with normal pancreatic features. After PX, BrdU uptake of acinar cells and islet cells significantly increased, but was suppressed by treatment with VA-lip siRNA HSP47. BrdU uptake by acinar cells was augmented by co-culturing with aPSCs and the augmentation was nullified by siRNA HSP47. BrdU uptake by progenitor cells in foci area was slightly enhanced by the same treatment. New area which exhibited intermediate features between those of duodenal and area of foci, emerged after the treatment.
Conclusion
aPSCs play a crucial role in regeneration of remnant pancreas, proliferation of acinar and islet cells after PX through the activity of secreted collagen. Characterization of new area emerged by siRNA HSP47 treatment as to its origin is a future task..
21. Fujita R, Kuwata R, Kobayashi D, Bertuso A, Isawa H, Sawabe K, Characterization of Bustos virus, a new member of the Negevirus group isolated from Mansonia mosquito in the Phillippines, The Internatinal Congress on Invertebrate Pathology and Microbial Control and the 49th Annual Meeting of the Society for Invertebrate Pathology, 71-71, 2016.07.
22. 小林 大介, 伊澤 晴彦, 藤田 龍介, 糸川 健太郎, Osei Joseph H.N., Opoku Millicent, Agbekudzi Alfred, Joannides Jay, Agbosu Esinam, Dadzie Samuel, Bonney Kofi, 佐々木 年則, 沢辺 京子, 大橋 光子, 太田 伸生, 2015年ガーナ共和国アクラ市各所における疾病媒介蚊およびマダニの採集と保有ウイルスの調査(Entomological surveillance for arbovirus infection in Accra, Ghana in 2015), 衛生動物, 67, Suppl., 92-92, 2016.04.
23. Kota Kawakami, Yudistira Wahyu Kurnia, Ryosuke Fujita, Toshiaki Ito, Haruhiko Isawa, Shin-ichiro Asano, Ngo Dinh Binh, Hisanori Bando, Characterization of a novel negevirus isolated from Aedes larvae collected in a subarctic region of Japan, ARCHIVES OF VIROLOGY, 10.1007/s00705-015-2711-9, 161, 4, 801-809, 2016.04, We isolated and characterized a novel positive-sense, single-stranded RNA virus from Aedes larvae collected on Okushiri Island, Hokkaido, Japan. This virus, designated Okushiri virus (OKV), replicated in the Aedes albopictus cell line C6/36 with severe cytopathic effects and produced a large number of spherical viral particles that were 50-70 nm in diameter and released into the cell culture medium. The OKV genome consisted of 9,704 nucleotides, excluding the poly(A) tail at the 3'-terminus, and contained three major open reading frames (ORF1, ORF2, and ORF3). ORF1 encoded a putative protein of approximately 268 kDa that included a methyltransferase domain, FtsJ-like methyltransferase domain, helicase domain, and RNA-dependent RNA polymerase domain. The genome organization and results of a phylogenetic analysis based on the amino acid sequence predicted from the nucleotide sequence indicated that OKV is a member of a new insect virus group of negeviruses with a possible evolutionary relationship to some plant viruses. ORF2 and ORF3 were suggested to encode hypothetical membrane-associated proteins of approximately 45 kDa and 22 kDa, respectively. This is the first study on a novel negevirus isolated from mosquito larvae in Japan..
24. Ryosuke Fujita, Chikako Ono, Isamu Ono, Shin-ichiro Asano, Hisanori Bando, Analysis of the Bombyx mori nucleopolyhedrovirus ie-1 promoter in insect, mammalian, plant, and bacterial cells, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2015.07.126, 464, 4, 1297-1301, 2015.09, The Bombyx mori nucleopolyhedrovirus (BmNPV) ie-1 promoter exhibits strong transcriptional activity and is used in transient foreign gene expression systems in insect cells. In a reporter assay experiment using the BmNPV ie-1 promoter, we found that it exhibited activity even in non-host mammalian BHK cells, plant BY-2 cells, and also bacterial Escherichia coli cells. An analysis using a deletion series of the BmNPV ie-1 promoter demonstrated that the core promoter region of this promoter was sufficient to display promoter activity in BHK cells, BY-2 cells, and E. coli cells, whereas upstream elements were required for higher activity in insect cells. Furthermore, we found that the BmNPV ie-1 promoter exhibited sufficient activity for a beta-galactosidase assay in E. coil cells. The results obtained here suggest that the BmNPV ie-1 promoter has potential as a universal promoter for transient expression systems in insect, mammalian, plant, and bacterial cells. (C) 2015 Elsevier Inc. All rights reserved..
25. Naoko Kubo Birukawa, Kazuyuki Murase, Yasushi Sato, Akemi Kosaka, Akihiro Yoneda, Hiroki Nishita, Ryosuke Fujita, Miyuki Nishimura, Takafumi Ninomiya, Keiko Kajiwara, Miyono Miyazaki, Yusuke Nakashima, Sigenori Ota, Yuya Murakami, Yasunobu Tanaka, Kenjiro Minomi, Yasuaki Tamura, Yoshiro Niitsu, Activated Hepatic Stellate Cells Are Dependent on Self-collagen, Cleaved by Membrane Type 1 Matrix Metalloproteinase for Their Growth, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M113.544494, 289, 29, 20209-20221, 2014.07, Stellate cells are distributed throughout organs, where, upon chronic damage, they become activated and proliferate to secrete collagen, which results in organ fibrosis. An intriguing property of hepatic stellate cells (HSCs) is that they undergo apoptosis when collagen is resolved by stopping tissue damage or by treatment, even though the mechanisms are unknown. Here we disclose the fact that HSCs, normal diploid cells, acquired dependence on collagen for their growth during the transition from quiescent to active states. The intramolecular RGD motifs of collagen were exposed by cleavage with their own membrane type 1 matrix metalloproteinase (MT1-MMP). The following evidence supports this conclusion. When rat activated HSCs (aHSCs) were transduced with siRNA against the collagen-specific chaperone gp46 to inhibit collagen secretion, the cells underwent autophagy followed by apoptosis. Concomitantly, the growth of aHSCs was suppressed, whereas that of quiescent HSCs was not. These in vitro results are compatible with the in vivo observation that apoptosis of aHSCs was induced in cirrhotic livers of rats treated with siRNAgp46. siRNA against MT1-MMP and addition of tissue inhibitor of metalloproteinase 2 (TIMP-2), which mainly inhibits MT1-MMP, also significantly suppressed the growth of aHSCs in vitro. The RGD inhibitors echistatin and GRGDS peptide and siRNA against the RGD receptor alpha V beta 1 resulted in the inhibition of aHSCs growth. Transduction of siRNAs against gp46, alpha V beta 1, and MT1-MMP to aHSCs inhibited the survival signal of PI3K/AKT/I kappa B. These results could provide novel antifibrosis strategies..
26. Masako Yokoo, Ryosuke Fujita, Yumiko Nakajima, Mamoru Yoshimizu, Hisae Kasai, Shin-ichiro Asano, Hisanori Bando, Mos1 transposon-based transformation of fish cell lines using baculoviral vectors, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2013.08.037, 439, 1, 18-22, 2013.09, Drosophila Mos1 belongs to the mariner family of transposons, which are one of the most ubiquitous transposons among eukaryotes. We first determined nuclear transportation of the Drosophila Mos1-EGFP fusion protein in fish cell lines because it is required for a function of transposons. We next constructed recombinant baculoviral vectors harboring the Drosophila Mos1 transposon or marker genes located between Mos1 inverted repeats. The infectivity of the recombinant virus to fish cells was assessed by monitoring the expression of a fluorescent protein encoded in the viral genome. We detected transgene expression in CHSE-214, HINAE, and EPC cells, but not in GF or RTG-2 cells. In the co-infection assay of the Mos1-expressing virus and reporter gene-expressing virus, we successfully transformed CHSE-214 and HINAE cells. These results suggest that the combination of a baculovirus and Mos1 transposable element may be a tool for transgenesis in fish cells. © 2013 Elsevier Inc..
27. Hirotoshi Ishiwatari, Yasushi Sato, Kazuyuki Murase, Akihiro Yoneda, Ryosuke Fujita, Hiroki Nishita, Naoko Kubo Birukawa, Tsuyoshi Hayashi, Tsutomu Sato, Koji Miyanishi, Rishu Takimoto, Masayoshi Kobune, Shigenori Ota, Yasutoshi Kimura, Koichi Hirata, Junji Kato, Yoshiro Niitsu, Treatment of pancreatic fibrosis with siRNA against a collagen-specific chaperone in vitamin A-coupled liposomes, GUT, 10.1136/gutjnl-2011-301746, 62, 9, 1328-1339, 2013.09, Background and objective
Fibrosis associated with chronic pancreatitis is an irreversible lesion that can disrupt pancreatic exocrine and endocrine function. Currently, there are no approved treatments for this disease. We previously showed that siRNA against collagen-specific chaperone protein gp46, encapsulated in vitamin A-coupled liposomes (VA-lip-siRNAgp46), resolved fibrosis in a model of liver cirrhosis. This treatment was investigated for pancreatic fibrosis induced by dibutyltin dichloride (DBTC) and cerulein in rats.
Methods
Specific uptake of VA-lip-siRNAgp46, conjugated with 6-carboxyfluorescein (FAM) by activated pancreatic stellate cells (aPSCs), was analysed by fluorescence activated cell sorting (FACS). Intracellular distribution of VA-lip-siRNAgp46-FAM was examined by fluorescent microscopy. Suppression of gp46 expression by VA-lip-siRNAgp46 was assessed by immunoblotting. Collagen synthesis in aPSCs was assayed by dye-binding. Specific delivery of VA-lip-siRNAgp46 to aPSCs in DBTC rats was verified following intravenous VA-lip-siRNA-FAM and H-3-VA-lip-siRNAgp46. The effect of VA-lip-siRNA on pancreatic histology in DBTC- and cerulein-treated rats was determined by Azan-Mallory staining and hydroxyproline content.
Results
FACS analysis revealed specific uptake of VA-lip-siRNAgp46-FAM through the retinol binding protein receptor by aPSCs in vitro. Immunoblotting and collagen assay verified knockdown of gp46 and suppression of collagen secretion, respectively, by aPSCs after transduction of VA-lip-siRNAgp46. Specific delivery of VA-lip-siRNAgp46 to aPSCs in fibrotic areas in DBTC rats was confirmed by fluorescence and radioactivity 24h after the final injection. 10 systemic VA-lip-siRNAgp46 treatments resolved pancreatic fibrosis, and suppressed tissue hydroxyproline levels in DBTC- and cerulein-treated rats.
Conclusion
These data suggest the therapeutic potential of the present approach for reversing pancreatic fibrosis..
28. Ryosuke Fujita, Daisuke Ohtsuka, Ken Sahara, Shinichiro Asano, Hisanori Bando, An HDAC inhibitor increases AcMNPV gene expression in mammalian cells, ARCHIVES OF VIROLOGY, 10.1007/s00705-010-0614-3, 155, 4, 577-581, 2010.04, The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is used as a safer viral vector in mammalian cells with potential applications in gene therapy. However, the mechanism for the insusceptibility of mammalian cells to proliferative infection by entomopathogenic viruses is not well understood. Here, we studied the significance of epigenetic modifications such as histone acetylation, histone methylation and HP1 accumulation for AcMNPV gene expression in mammalian BHK cells. Real-time PCR and chromatin immunoprecipitation with sodium butyrate revealed an important relationship between viral gene expression and histone acetylation, with implications for a mechanism of suppression of AcMNPV gene expression in BHK cells..
29. Ohtsuka D, Nakatsukasa T, Fujita R, Asano S, Sahara K, Bando H, Use of Bombyx mori U6 Promoter for Inducing Gene-Silencing in Silkworm Cells, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.77.3_125, 77, 3, 125-131, 2009.03.
30. Fujita R, Asano S, Sahara K, Bando H, Supression of AcMNPV gene expression in mammalian cells, 41th Annual meeting of the society for invertebrate pathology and 9th international conference on Bacillus thuringiensis, 19-19, 2008.08.
31. Fujita R, Asano S, Sahara K, Bando H, Restricted gene transcription of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in mammalian cells, 40th Annual meeting of the society for invertebrate pathology and 1st international forum on entomopathogenic nematodes and symbiotic bacteria, 95-95, 2007.08.
32. Fujita R, Asano S, Sahara K, Bando H, Marked decrease of ribosomal RNA in BmN cells infected with AcMNPV, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.74.125, 74, 3, 125-128, 2006.11.
33. R Fujita, T Matsuyama, J Yamagishi, K Sahara, S Asano, H Bando, Expression of Autographa californica multiple nucleopolyhedrovirus genes in mammalian cells and Upregulation of the host beta-actin gene, JOURNAL OF VIROLOGY, 10.1128/JVI.80.5.2390-2395.2006, 80, 5, 2390-2395, 2006.03, The gene expression of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was examined in two types of mammalian cells, human HeLa14 and hamster BHK cells. DNA microarray analysis followed by reverse transcription-PCR identified at least 12 viral genes transcribed in both HeLa14 cells and BHK cells inoculated with AcMNPV. 5' rapid amplification of cDNA ends was carried out to examine the transcriptional fidelity of these genes in HeLa14 cells. The transcription of ie-1, ie-0 and gp64 was initiated at a baculovirus early gene motif, CAGT, accompanied by a TATA motif. In addition, the same splicing observed for ie-0 mRNA in Sf9 cells occurred in HeLa14 cells. While the transcription initiation sites for pe38 and p6.9 were not located in the CAGT motif, most of them were in a typical eukaryotic RNA polymerase II promoter structure (a conventional TATA motif and/or an initiator). Interestingly, the expression of beta-actin was upregulated in the mammalian cells inoculated with AcMNPV. Subsequent experiments using UV-inactivated virus confirmed the upregulation, suggesting that de novo synthesis of viral products is not required for the event. These results indicated that the AcMNPV genome acts as a template for transcription in mammalian cells through the usual infection pathway, though there is no evidence for the functional expression of viral genes at present..
34. T Abe, N Miyake, Y Nishijima, R Fujita, K Sahara, S Asano, H Bando, Enhancement of cauliflower mosaic virus 35S promoter in insect cells infected with baculovirus, VIRUS RESEARCH, 10.1016/j.virusres.2005.03.019, 112, 1-2, 38-41, 2005.09, We happened to discover that the cauliflower mosaic virus (CaMV) 35S promoter inserted into a recombinant Autographa californica multicapsid nucleopolyhedrovirus (rAcMNPV) was strongly activated during the replication of the recombinant virus in Spodoptera frugiperda (Sf9) cells. The expression of the luciferase gene from the 35S promoter in rAcMNPV was remarkably increased late in infection and was resistant to alpha-amanitin treatment. Primer extension indicated that transcriptional initiation from the 35S promoter in Sf9 cells occurred within one of the two baculoviral late promoter TAAG motifs located in the vicinity of the transcription start site in plant cells. These observations suggested that the CaMV 35S promoter served as a transcription start site for AcMNPV-induced RNA polymerase. (c) 2005 Elsevier B.V. All rights reserved..