Researcher Database

Hirofumi Sawa
Research Center for Zoonosis Control Division of Molecular Pathobiology
Professor

Researcher Profile and Settings

Affiliation

  • Research Center for Zoonosis Control Division of Molecular Pathobiology

Job Title

  • Professor

URL

J-Global ID

Research Interests

  • 感染症疫学   モデルマウス   細胞内輸送   ウイルス学   animal model   trafficking   Cellular Biology   Molecular Pathology   Virology   

Research Areas

  • Life sciences / Molecular biology
  • Life sciences / Experimental pathology
  • Life sciences / Cell biology
  • Life sciences / Virology

Academic & Professional Experience

  • 2012/12 - Today Research cdenter for Zoonosis Control, Hokkaido University Profesor

Association Memberships

  • Global Virus network   分子生物学会   日本病理学会   日本内科学会   日本生化学会   日本神経病理学会   日本循環器学会   日本ウイルス学会   American Society of MicrobiologyAmerican Society for Biochemistry and Molecular BiologyInternational Society for Neurovirology   

Research Activities

Published Papers

  • Michihito Sasaki, Paulina D. Anindita, Wallaya Phongphaew, Michael Carr, Shintaro Kobayashi, Yasuko Orba, Hirofumi Sawa
    VIRUS RESEARCH 243 69 - 74 0168-1702 2018/01 [Refereed][Not invited]
     
    Subviral particles (SVPs) self-assemble and are released from cells transfected with expression plasmids encoding flavivirus structural proteins. Flavivirus-like particles (VLPs), consisting of flavivirus structural proteins and a subgenomic replicon, can enter cells and cause single-round infections. Neither SVPs or VLPs possess complete viral RNA genomes, therefore are replication-incompetent systems; however, they retain the capacity to fuse and bud from target cells and follow the same maturation process as whole virions. SVPs and VLPs have been previously employed in studies analyzing entry and release steps of viral life cycles. In this study, we have developed quantitative methods for the detection of cellular entry and release of SVPs and VLPs by applying a luciferase complementation assay based on the high affinity interaction between the split NanoLuc luciferase protein, LgBiT and the small peptide, HiBiT. We introduced HiBiT into the structural protein of West Nile virus and generated SVPs and VLPs harboring HiBiT (SVP-HiBiT and VLP-HiBiT, respectively). As SVP-HiBiT emitted strong luminescence upon exposure to LgBiT and its substrate, the nascently budded SVP-HiBiT in the supernatant was readily quantified by luminometry. Similarly, the cellular entry of VLP-HiBiT generated luminescence when VLP-HiBiT was infected into LgBiT-expressing cells. These methods utilizing SVP-HiBiT and VLP-HiBiT will facilitate research into life cycles of flaviviruses, including WNV.
  • Hirohito Ogawa, Masahiro Kajihara, Naganori Nao, Asako Shigeno, Daisuke Fujikura, Bernard M Hang'ombe, Aaron S Mweene, Alisheke Mutemwa, David Squarre, Masao Yamada, Hideaki Higashi, Hirofumi Sawa, Ayato Takada
    Viruses 9 (12) 2017/12/04 [Refereed][Not invited]
     
    Bats are important reservoirs for emerging zoonotic viruses. For extensive surveys of potential pathogens in straw-colored fruit bats (Eidolon helvum) in Zambia, a total of 107 spleen samples of E. helvum in 2006 were inoculated onto Vero E6 cells. The cell culture inoculated with one of the samples (ZFB06-106) exhibited remarkable cytopathic changes. Based on the ultrastructural property in negative staining and cross-reactivity in immunofluorescence assays, the virus was suspected to be an adenovirus, and tentatively named E. helvum adenovirus 06-106 (EhAdV 06-106). Analysis of the full-length genome of 30,134 bp, determined by next-generation sequencing, showed the presence of 28 open reading frames. Phylogenetic analyses confirmed that EhAdV 06-106 represented a novel bat adenovirus species in the genus Mastadenovirus. The virus shared similar characteristics of low G + C contents with recently isolated members of species Bat mastadenoviruses E, F and G, from which EhAdV 06-106 diverged by more than 15% based on the distance matrix analysis of DNA polymerase amino acid sequences. According to the taxonomic criteria, we propose the tentative new species name "Bat mastadenovirus H". Because EhAdV 06-106 exhibited a wide in vitro cell tropism, the virus might have a potential risk as an emerging virus through cross-species transmission.
  • Michael Carr, Gabriel Gonzalez, Michihito Sasaki, Serena E. Dool, Kimihito Ito, Akihiro Ishii, Bernard M. Hang'ombe, Aaron S. Mweene, Emma C. Teeling, William W. Hall, Yasuko Orba, Hirofumi Sawa
    JOURNAL OF GENERAL VIROLOGY 98 (11) 2771 - 2785 0022-1317 2017/11 [Refereed][Not invited]
     
    Polyomaviruses (PyVs) are considered to be highly host-specific in different mammalian species, with no well-supported evidence for host-switching events. We examined the species diversity and host specificity of PyVs in horseshoe bats (Rhinolophus spp.), a broadly distributed and highly speciose mammalian genus. We annotated six PyV genomes, comprising four new PyV species, based on pairwise identity within the large T antigen (LTAg) coding region. Phylogenetic comparisons revealed two instances of highly related PyV species, one in each of the Alphapolyomavirus and Betapolyomavirus genera, present in different horseshoe bat host species (Rhinolophus blasii and R. simulator), suggestive of short-range host-switching events. The two pairs of Rhinolophus PyVs in different horseshoe bat host species were 99.9 and 88.8% identical with each other over their respective LTAg coding sequences and thus constitute the same virus species. To corroborate the species identification of the bat hosts, we analysed mitochondrial cytb and a large nuclear intron dataset derived from six independent and neutrally evolving loci for bat taxa of interest. Bayesian estimates of the ages of the most recent common ancestors suggested that the near-identical and more distantly related PyV species diverged approximately 9.1 E4 (5E3-2.8E5) and 9.9E6 (4E6-18E6) years before the present, respectively, in contrast to the divergence times of the bat host species: 12.4E6 (10.4E6-15.4E6). Our findings provide evidence that short-range host-switching of PyVs is possible in horseshoe bats, suggesting that PyV transmission between closely related mammalian species can occur.
  • Youhei Takagi, Kouhei Matsui, Haruaki Nobori, Haruka Maeda, Akihiko Sato, Takeshi Kurosu, Yasuko Orba, Hirofumi Sawa, Kazunari Hattori, Kenichi Higashino, Yoshito Numata, Yutaka Yoshida
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 27 (15) 3586 - 3590 0960-894X 2017/08 [Refereed][Not invited]
     
    NS2B-NS3 protease is an essential enzyme for the replication of dengue virus (DENV), which continues to be a serious threat to worldwide public health. We designed and synthesized a series of cyclic peptides mimicking the substrates of this enzyme, and assayed their activity against the DENV-2 NS2B-NS3 protease. The introduction of aromatic residues at the appropriate positions and conformational restriction generated the most promising cyclic peptide with an IC50 of 0.95 mu M against NS2B-NS3 protease. Cyclic peptides with proper positioning of additional arginines and aromatic residues exhibited antiviral activity against DENV. Furthermore, replacing the C-terminal amide bond of the polybasic amino acid sequence with an amino methylene moiety stabilized the cyclic peptides against hydrolysis by NS2B-NS3 protease, while maintaining their enzyme inhibitory activity and antiviral activity. (C) 2017 Elsevier Ltd. All rights reserved.
  • Ramesh Akkina, Heinz Ellerbrok, William Hall, Hideki Hasegawa, Yasushi Kawaguchi, Harold Kleanthous, Edward McSweegan, Natalia Mercer, Victor Romanowski, Hirofumi Sawa, Anders Vahlne
    ANTIVIRAL RESEARCH 142 21 - 29 0166-3542 2017/06 [Refereed][Not invited]
     
    The Global Virus Network (GVN) was established in 2011 in order to strengthen research and responses to current viral causes of human disease and to prepare against new viral pandemic threats. There are now 38 GVN Centers of Excellence and 6 Affiliate laboratories in 24 countries. GVN scientists meet annually to learn about each other's current research, address collaborative priorities and plan future programs. The 2016 meeting was held from October 23-25 in Hokkaido, Japan, in partnership with the Japanese Society for Virology, the National Institute of Infectious Diseases of Japan and the Research Center for Zoonosis Control of Hokkaido University. This report highlights the accomplishments of GVN researchers in many priority areas of medical virology, including the current Zika epidemic, infections by human papillomavirus, influenza, Ebola, Lassa, dengue, HIV, hepatitis C, and chikungunya viruses, and the development of improved diagnostics and new vaccines. Published by Elsevier B.V.
  • Rie Hasebe, Ryo Nakao, Aiko Ohnuma, Takeshi Yamasaki, Hirofumi Sawa, Shinji Takai, Motohiro Horiuchi
    JOURNAL OF VETERINARY MEDICAL SCIENCE 79 (6) 962 - 969 0916-7250 2017/06 [Refereed][Not invited]
     
    We analyzed the pathogenicity of various serotypes of Listeria monocytogenes using a Balb/c mouse intravenous injection model. The survival rates of mice inoculated with strains NS1/2b (serotype 1/2b), NS3b (serotype 3b) and NS 4b (serotype 4b) were 60, 63.6 and 63.6%, respectively. Although the survival rates were similar, the bacterial growth in the liver of NS3b-infected mice was 144.5-fold higher than that in the liver of NS4b-infected mice. Histopathological analyses suggest that the NS4b strain replicated more in monocytes/macrophages, whereas the NS3b strain replicated more in hepatocytes. These results raise a possibility that the serotype 4b strains replicated more in monocytes/macrophages compared to the other serotype strains. To assess this, we isolated CD11b-positive cells from mouse livers infected with EGDe (serotype 1/2a), NS1/2b, NS3b, NS4b and the serotype 4b strains 51414 and F17 and counted the number of live bacteria in these cells. CD11b-positive cells from the NS4b-, 51414- and F17-infected mice possessed 24.4- to 42.7-fold higher numbers of live bacteria than those from mice infected with EGDe and NS3b strains. These results suggest that serotype 4b strains replicated more in monocytes/macrophages than the other serotypes, and this may be involved in the pathogenicity of serotype 4b strains, particularly in the dissemination of L. monocytogenes through the host body.
  • Yuji Wada, Yasuko Orba, Michihito Sasaki, Shintaro Kobayashi, Michael J. Carr, Haruaki Nobori, Akihiko Sato, William W. Hall, Hirofumi Sawa
    VIROLOGY 505 102 - 112 0042-6822 2017/05 [Refereed][Not invited]
     
    Chikungunya fever (CHIKF) is caused by chikungunya virus (CHIKV) infection which is a re-emerging mosquito-borne zoonosis. At present, there are no approved therapeutics for CHIKF. Herein, we have investigated candidate compounds which can inhibit CHIKV infection. Screening of chemical compound libraries were performed and one candidate, a benzimidazole-related compound designated Compound-A was found to inhibit infection by several CHIKV strains and a Sindbis virus strain at nanomolar concentrations. To investigate the inhibitory mechanism of action, a Compound-A resistant CHIKV (res-CHIKV) was isolated and a key mutation associated with resistance was identified by reverse-genetic recombinant CHIICVs containing amino acid substitutions present in res-CHIKV. These results demonstrated that the target site of Compound-A was the M2295 residue in the nonstructural protein 4 (nsP4), which is located in one of the functional domains of RNA-dependent RNA-polymerase (RdRp). We also confirmed that Compound-A inhibits RdRp function of CHIKV by using CHIKV replicons.
  • Michael Carr, Gabriel Gonzalez, Michihito Sasaki, Kimihito Ito, Akihiro Ishii, Bernard M. Hang'ombe, Aaron S. Mweene, Yasuko Orba, Hirofumi Sawa
    JOURNAL OF GENERAL VIROLOGY 98 (4) 726 - 738 0022-1317 2017/04 [Refereed][Not invited]
     
    Bat species represent natural reservoirs for a number of high-consequence human pathogens. The present study investigated the diversity of polyomaviruses (PyVs) in Zambian insectivorous and fruit bat species. We describe the complete genomes from four newly proposed African bat PyV species employing the recently recommended criteria provided by the Polyomaviridae Study Group of the International Committee on Taxonomy of Viruses. A comprehensive phylogenetic and recombination analysis was performed to determine genetic relationships and the distribution of recombination events in PyV from mammalian and avian species. The novel species of PyV from Zambian bats segregated with members of the genera Alphapolyomavirus and Betapolyomavirus, forming monophyletic clades with bat and non-human primate PyVs. Miniopterus schreibersii polyomavirus 1 and 2 segregated in a clade with South American bat PyV species, Old World monkey and chimpanzee PyVs and Human polyomavirus 13 (New Jersey PyV). Interestingly, the newly described Egyptian fruit bat PyV, tentatively named Rousettus aegyptiacus polyomavirus 1, had the highest nucleotide sequence identity to species of PyV from Indonesian fruit bats, and Rhinolophus hildebrandtii polyomavirus 1 was most closely related to New World monkey PyVs. The distribution of recombination events in PyV genomes was non-random: recombination boundaries existed in the intergene region between VP1 and LTAg and also at the 3' end of VP2/3 in the structural genes, whereas infrequent recombination was present within the LTAg gene. These findings indicate that recombination within the LTAg gene has been negatively selected against during polyomaviral evolution and support the recent proposal for taxonomic classification based on LTAg to define novel PyV species.
  • Michael Carr, Akira Kawaguchi, Michihito Sasaki, Gabriel Gonzalez, Kimihito Ito, Yuka Thomas, Bernard M. Hang'ombe, Aaron S. Mweene, Guoyan Zhao, David Wang, Yasuko Orba, Akihiro Ishii, Hirofumi Sawa
    ARCHIVES OF VIROLOGY 162 (2) 543 - 548 0304-8608 2017/02 [Refereed][Not invited]
     
    To investigate the diversity of simian immunodeficiency virus (SIV) among nonhuman primates (NHPs) in Zambia, next-generation sequencing was performed to determine the complete genome sequence of a novel SIV recovered by co-culturing African green monkey (AGM) peripheral blood lymphocytes with human CD4(+) T-cell lines. We report the first described SIV (SIVagmMAL-ZMB) from a malbrouck (Chlorocebus cynosuros). SIVagmMAL-ZMB was detected by real-time PCR analysis of splenic RNA in 3.2% (3/94) of AGMs and was undetectable in baboons (0/105). SIVagmMAL-ZMB possessed < 80% nucleotide sequence identity to known SIV isolates and was located basally to vervet monkey SIV strains in all phylogenies.
  • Ryo Nakao, Keita Matsuno, Yongjin Qiu, Junki Marilyama, Nao Eguchi, Naganori Nao, Masahiro Kajihara, Kentaro Yoshii, Hirofumi Sawa, Ayato Takada, Chihiro Sugimoto
    TICKS AND TICK-BORNE DISEASES 8 (1) 103 - 111 1877-959X 2017 [Refereed][Not invited]
     
    Ticks harbour various microorganisms, some of which act as pathogens of humans and animals. The recent advancement of genome sequencing technologies revealed that a wide range of previously unrecognised microorganisms exist in ticks. Continuous cell lines established from ticks could play a key role in the isolation of such microorganisms; however, tick cells themselves have been known to harbour symbiotic microorganisms. The present study aimed to characterise putative RNA viral sequences detected in the culture supernatant of one of the most frequently used tick cell lines, ISE6, which was derived from embryos of the blacklegged tick Ixodes scapularis. Viral particles purified from the culture supernatant were used for RNA extraction, followed by Illumina sequencing. The reads were de novo assembled and the resulting contigs were annotated by tBLASTx search. The results suggested that there were at least five putative viral sequences of four phylogenetically distinct lineages in ISE6 cells. The predominant viral sequence found in ISE6 cells, designated L scapularis iflavirus, was a member of the family Iflaviridae, which is an arthropod-infecting virus group. We also identified L and M segments of the family Bunyaviridae, which could not be classified into any of the five known genera, and a potential capsid protein related to Drosophila A virus. In addition to these previously unrecognised viruses, ISE6 was revealed to harbour a putative genome sequence of L scapularis-associated virus-1, which was reported in a recent metagenomic study of L scapularis itself. All the five putative viral sequences were detected by RT-PCR in both ISE6 cells and the culture supernatant. Electron microscopic analysis showed the existence of spherical virions with a varying diameter of 50-70 nm in the culture supernatant of ISE6 cells. Further studies are required to investigate the potential roles of ISE6-associated viruses in ticks. (C) 2016 Elsevier GmbH. All rights reserved.
  • Wallaya Phongphaew, Shintaro Kobayashi, Michihito Sasaki, Michael Carr, William W. Hall, Yasuko Orba, Hirofumi Sawa
    VIRUS RESEARCH 228 114 - 123 0168-1702 2017/01 [Refereed][Not invited]
     
    Valosin-containing protein (VCP) is classified as a member of the type II AAA(+) ATPase protein family. VCP functions in several cellular processes, including protein degradation, membrane fusion, vesicular trafficking and disassembly of stress granules. Moreover, VCP is considered to play a role in the replication of several viruses, albeit through different mechanisms. In the present study, we have investigated the role of VCP in West Nile virus (WNV) infection. Endogenous VCP expression was inhibited using either VCP inhibitors or by siRNA knockdown. It could be shown that the inhibition of endogenous VCP expression significantly inhibited WNV infection. The entry assay revealed that silencing of endogenous VCP caused a significant reduction in the expression levels of WNV-RNA compared to control siRNA-treated cells. This indicates that VCP may play a role in early steps either the binding or entry steps of the WNV life cycle. Using WNV virus like particles and WNV-DNA-based replicon, it could be demonstrated that perturbation of VCP expression decreased levels of newly synthesized WNV genomic RNA. These findings suggest that VCP is involved in early steps and during genome replication of the WNV life cycle. (C) 2016 Elsevier B.V. All rights reserved.
  • Gabriel Gonzalez, Michihito Sasaki, Lucy Burkitt-Gray, Tomonori Kamiya, Noriko M. Tsuji, Hirofumi Sawa, Kimihito Ito
    SCIENTIFIC REPORTS 7 40447  2045-2322 2017/01 [Refereed][Not invited]
     
    Advances in Next Generation Sequencing technologies have enabled the generation of millions of sequences from microorganisms. However, distinguishing the sequence of a novel species from sequencing errors remains a technical challenge when the novel species is highly divergent from the closest known species. To solve such a problem, we developed a new method called Optimistic Protein Assembly from Reads (OPAR). This method is based on the assumption that protein sequences could be more conserved than the nucleotide sequences encoding them. By taking advantage of metagenomics, bioinformatics and conventional Sanger sequencing, our method successfully identified all coding regions of the mouse picobirnavirus for the first time. The salvaged sequences indicated that segment 1 of this virus was more divergent from its homologues in other Picobirnaviridae species than segment 2. For this reason, only segment 2 of mouse picobirnavirus has been detected in previous studies.
  • Michihito Sasaki, Yasuko Orba, Satoko Sasaki, Gabriel Gonzalez, Akihiro Ishii, Bernard M. Hang'ombe, Aaron S. Mweene, Kimihito Ito, Hirofumi Sawa
    JOURNAL OF GENERAL VIROLOGY 97 (10) 2488 - 2493 0022-1317 2016/10 [Refereed][Not invited]
     
    Group A rotavirus is a major cause of diarrhoea in humans, especially in young children. Bats also harbour group A rotaviruses, but the genetic backgrounds of bat rotavirus strains are usually distinct from those of human rotavirus strains. We identified a new strain of group A rotavirus in the intestinal contents of a horseshoe bat in Zambia. Whole genome sequencing revealed that the identified virus, named RVA/Bat-wt/ZMB/LUS12-14/2012/G3P[3], possessed the genotype constellation G3-P[3]-I3-R2-C2-M3-A9-N2-T3-E2-H3. Several genome segments of LUS12-14 were highly similar to those of group A rotaviruses identified from humans, cows and antelopes, indicating interspecies transmission of rotaviruses between bats and other mammals with possible multiple genomic reassortment events.
  • Paulina Duhita Anindita, Michihito Sasaki, Haruaki Nobori, Akihiko Sato, Michael Carr, Naoto Ito, Makoto Sugiyama, Yasuko Orba, Hirofumi Sawa
    VIRUS RESEARCH 215 121 - 128 0168-1702 2016/04 [Refereed][Not invited]
     
    Rabies is an invariably fatal disease caused by Rabies virus (RABV), a member of the family Rhabdoviridae, genus Lyssavirus. Once central nervous infection occurs and symptoms develop, the case fatality rate approaches 100% despite availability of post-exposure prophylaxis. Therefore, new antiviral therapies for rabies are urgently required. Antivirals which can inhibit virus replication can be identified through screening of small compounds, however, as RABV infection does not generate easily discernible cytopathic effects in vitro, cell viability assays may not be feasible to observe antiviral activity of small compounds against RABV. In this study, recombinant RABVs (rRABVs) encoding NanoLuc luciferase (NanoLuc) were generated to facilitate the screening of small compound libraries. NanoLuc expression was confirmed in single-step growth cures of virus infection and showed that the rRABVs were capable of viral replication without decrease of luciferase activity through ten serial passages. Furthermore, the rRABVs were able to quantify the antiviral activity of the nucleoside analogue ribavirin against RABV in vitro. These findings confirm the potential of the rRABV encoding NanoLuc system to facilitate screening of small compounds to inhibit RABV infection. (C) 2016 Elsevier B.V. All rights reserved.
  • Michihito Sasaki, Gabriel Gonzalez, Yuji Wada, Agus Setiyono, Ekowati Handharyani, Ibenu Rahmadani, Siswatiana Taha, Sri Adiani, Munira Latief, Zainal Abidin Kholilullah, Mawar Subangkit, Shintaro Kobayashi, Ichiro Nakamura, Takashi Kimura, Yasuko Orba, Kimihito Ito, Hirofumi Sawa
    SCIENTIFIC REPORTS 6 24257  2045-2322 2016/04 [Refereed][Not invited]
     
    Bufavirus is a recently recognized member of the genus Protoparvovirus in the subfamily Parvovirinae. It has been reported that human bufavirus was detected predominantly in patients with diarrhoea in several countries. However, little is known about bufavirus or its close relatives in nonhuman mammals. In this study, we performed nested-PCR screening and identified bufavirus from 12 megabats of Pteropus spp. in Indonesia. Furthermore, we determined nearly the full genome sequence of a novel megabat-borne bufavirus, tentatively named megabat bufavirus 1. Phylogenetic analyses showed that megabat bufavirus 1 clustered with known protoparvoviruses, including human bufavirus but represented a distinct lineage of bufavirus. Our analyses also inferred phylogenetic relationships among animal-borne bufaviruses recently reported by other studies. Recombination analyses suggested that the most common recent ancestor of megabat bufavirus 1 might have arisen from multiple genetic recombination events. These results characterized megabat bufavirus 1 as the first protoparvovirus discovered from megabats and indicates the high genetic divergence of bufavirus.
  • Shintaro Kobayashi, Tadaki Suzuki, Akira Kawaguchi, Wallaya Phongphaew, Kentaro Yoshii, Tomohiko Iwano, Akihiro Harada, Hiroaki Kariwa, Yasuko Orba, Hirofumi Sawa
    JOURNAL OF BIOLOGICAL CHEMISTRY 291 (12) 6559 - 6568 0021-9258 2016/03 [Refereed][Not invited]
     
    West Nile virus (WNV) particles assemble at and bud into the endoplasmic reticulum (ER) and are secreted from infected cells through the secretory pathway. However, the host factor related to these steps is not fully understood. Rab proteins, belonging to the Ras superfamily, play essential roles in regulating many aspects of vesicular trafficking. In this study, we sought to determine which Rab proteins are involved in intracellular trafficking of nascent WNV particles. RNAi analysis revealed that Rab8b plays a role in WNV particle release. We found that Rab8 and WNV antigen were colocalized in WNV-infected human neuroblastoma cells, and that WNV infection enhanced Rab8 expression in the cells. In addition, the amount of WNV particles in the supernatant of Rab8b-deficient cells was significantly decreased compared with that of wild-type cells. We also demonstrated that WNV particles accumulated in the recycling endosomes in WNV-infected cells. In summary, these results suggest that Rab8b is involved in trafficking of WNV particles from recycling endosomes to the plasma membrane.
  • Hirohito Ogawa, Hiroko Miyamoto, Eri Nakayama, Reiko Yoshida, Ichiro Nakamura, Hirofumi Sawa, Akihiro Ishii, Yuka Thomas, Emiko Nakagawa, Keita Matsuno, Masahiro Kajihara, Junki Maruyama, Naganori Nao, Mieko Muramatsu, Makoto Kuroda, Edgar Simulundu, Katendi Changula, Bernard Hang'ombe, Boniface Namangala, Andrew Nambota, Jackson Katampi, Manabu Igarashi, Kimihito Ito, Heinz Feldmann, Chihiro Sugimoto, Ladslav Moonga, Aaron Mweene, Ayato Takada
    JOURNAL OF INFECTIOUS DISEASES 212 S101 - S108 0022-1899 2015/10 [Refereed][Not invited]
     
    Fruit bats are suspected to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Using an enzyme-linked immunosorbent assay based on the viral glycoprotein antigens, we detected filovirus-specific immunoglobulin G antibodies in 71 of 748 serum samples collected from migratory fruit bats (Eidolon helvum) in Zambia during 2006-2013. Although antibodies to African filoviruses (eg, Zaire ebolavirus) were most prevalent, some serum samples showed distinct specificity for Reston ebolavirus, which that has thus far been found only in Asia. Interestingly, the transition of filovirus species causing outbreaks in Central and West Africa during 2005-2014 seemed to be synchronized with the change of the serologically dominant virus species in these bats. These data suggest the introduction of multiple species of filoviruses in the migratory bat population and point to the need for continued surveillance of filovirus infection of wild animals in sub-Saharan Africa, including hitherto nonendemic countries.
  • Michihito Sasaki, Yasuko Orba, Paulina D. Anindita, Akihiro Ishii, Keisuke Ueno, Bernard M. Hang'ombe, Aaron S. Mweeile, Kimihito Ito, Hirofumi Sawa
    EMERGING INFECTIOUS DISEASES 21 (7) 1230 - 1233 1080-6040 2015/07 [Refereed][Not invited]
     
    Viral metagenomic analysis identified a new parvovirus genome in the intestinal contents of wild shrews in Zambia. Related viruses were detected in spleen tissues from wild shrews and nonhuman primates. Phylogenetic analyses showed that these viruses are related to human bufaviruses, highlighting the presence and genetic diversity of bufaviruses in wildlife.
  • Yoshinori Makino, Masumi Tsuda, Yusuke Ohba, Hiroshi Nishihara, Hirofumi Sawa, Kazuo Nagashima, Shinya Tanaka
    CELL COMMUNICATION AND SIGNALING 13 35  1478-811X 2015/07 [Refereed][Not invited]
     
    Background: The complex of Dock180/ELMO1 that functions as a bipartite guanine nucleotide exchange factor for Rac is essential for diverse physiological and pathological processes of cells such as cell migration, phagocytosis, and invasion of cancer cells. Among the Src-family tyrosine kinases (SFKs), it has been reported that Hck directly phosphorylates ELMO1, regulating phagocytosis by promoting activation of Rac1; however, the involvement of other SFKs in ELMO1 phosphorylation has remained unknown. Here, we identified novel tyrosine (Y) residues of ELMO1 phosphorylated by SFKs, and examined the effects on Rac1 activity, cell adhesion, spreading, and cell motility on extracellular matrix (ECM). Results: In this study, we unveiled that Src and Fyn can induce tyrosine phosphorylation of ELMO1 in in vivo and in vitro phosphorylation assays. Mutational analyses identified both Y720 and Y724 residues of ELMO1 as Src-mediated phosphorylation sites, preferentially on Y724. Single substitution of Y724 to Phe abrogated Rac1 activation triggered by Src. To elucidate the biological function of pY724, we established NIH3T3 cells stably expressing wild-type ELMO1 or its Y724F mutant together with Dock180. Among them, Y724-deficient cells exhibited a depletion of Rac1 activity with diminished phosphorylation of ELMO1 even upon the ECM-stimulation. It is noteworthy that NIH3T3 cells with ELMO1 Y724F were strikingly defective to promote cell spreading on fibronectin-coated dish, concomitantly exhibiting immature assemblies of actin stress fibers and focal adhesions. Eventually, ELMO1 Y724F significantly impaired cell migration. Conclusion: These results define that Src-mediated Y724 phosphorylation in ELMO1 plays a critical role for cell spreading via activation of Rac1, leading to promotion of cell migration. As the overexpression and/or hyperactivation of Src have been shown in a wide variety of human cancers, Src-mediated phosphorylation of Y724 in ELMO1 may regulate cancer cell adhesion to the ECM, invasion into surrounding tissues, and subsequent distant metastasis.
  • Keisuke Aoshima, Erina Inoue, Hirofumi Sawa, Yuki Okada
    EMBO REPORTS 16 (7) 803 - 812 1469-221X 2015/07 [Refereed][Not invited]
     
    Epigenetic modifications, such as DNA methylation and histone modifications, are dynamically altered predominantly in paternal pronuclei soon after fertilization. To identify which histone modifications are required for early embryonic development, we utilized histone K-M mutants, which prevent endogenous histone methylation at the mutated site. We prepared four single K-M mutants for histone H3.3, K4M, K9M, K27M, and K36M, and demonstrate that overexpression of H3.3 K4M in embryos before fertilization results in developmental arrest, whereas overexpression after fertilization does not affect the development. Furthermore, loss of H3K4 methylation decreases the level of minor zygotic gene activation (ZGA) predominantly in the paternal pronucleus, and we obtained similar results from knockdown of the H3K4 methyltransferase Mll3/4. We therefore conclude that H3K4 methylation, likely established by Mll3/4 at the early pronuclear stage, is essential for the onset of minor ZGA in the paternal pronucleus, which is necessary for subsequent preimplantation development in mice.
  • Hirohito Ogawa, Nobuo Koizumi, Aiko Ohnuma, Alisheke Mutemwa, Bernard M. Hang'ombe, Aaron S. Mweene, Ayato Takada, Chihiro Sugimoto, Yasuhiko Suzuki, Hiroshi Kida, Hirofumi Sawa
    INFECTION GENETICS AND EVOLUTION 32 143 - 147 1567-1348 2015/06 [Refereed][Not invited]
     
    The role played by bats as a potential source of transmission of Leptospira spp. to humans is poorly understood, despite various pathogenic Leptospira spp. being identified in these mammals. Here, we investigated the prevalence and diversity of pathogenic Leptospira spp. that infect the straw-colored fruit bat (Eidolon helvum). We captured this bat species, which is widely distributed in Africa, in Zambia during 2008-2013. We detected the flagellin B gene (flaB) from pathogenic Leptospira spp. in kidney samples from 79 of 529 E. helvum (14.9%) bats. Phylogenetic analysis of 70 flaB fragments amplified from E. helvum samples and previously reported sequences, revealed that 12 of the fragments grouped with Leptospira borgpetersenii and Leptospira kirschneri; however, the remaining 58 flaB fragments appeared not to be associated with any reported species. Additionally, the 16S ribosomal RNA gene (rrs) amplified from 27 randomly chosen flaB-positive samples was compared with previously reported sequences, including bat-derived Leptospira spp. All 27 rrs fragments clustered into a pathogenic group. Eight fragments were located in unique branches, the other 19 fragments were closely related to Leptospira spp. detected in bats. These results show that rrs sequences in bats are genetically related to each other without regional variation, suggesting that Leptospira are evolutionarily well-adapted to bats and have uniquely evolved in the bat population. Our study indicates that pathogenic Leptospira spp. in E. helvum in Zambia have unique genotypes. (C) 2015 The Authors. Published by Elsevier B.V.
  • Paulina Duhita Anindita, Michihito Sasaki, Agus Setiyono, Ekowati Handharyani, Yasuko Orba, Shintaro Kobayashi, Ibnu Rahmadani, Siswatiana Taha, Sri Adiani, Mawar Subangkit, Ichiro Nakamura, Hirofumi Sawa, Takashi Kimura
    ARCHIVES OF VIROLOGY 160 (4) 1113 - 1118 0304-8608 2015/04 [Refereed][Not invited]
     
    Bats have been shown to serve as natural reservoirs for numerous emerging viruses including severe acute respiratory syndrome coronavirus (SARS-CoV). In the present study, we report the discovery of bat CoV genes in Indonesian Moluccan naked-backed fruit bats (Dobsonia moluccensis). A partial RNA-dependent RNA polymerase gene sequence was detected in feces and tissues samples from the fruit bats, and the region between the RdRp and helicase genes could also be amplified from fecal samples. Phylogenetic analysis suggested that these bat CoVs are related to members of the genus Betacoronavirus.
  • Shintaro Kobayashi, Michihito Sasaki, Ryo Nakao, Agus Setiyono, Ekowati Handharyani, Yasuko Orba, Ibnu Rahmadani, Siswatiana Taha, Sri Adiani, Mawar Subangkit, Ichiro Nakamura, Takashi Kimura, Hirofumi Sawa
    ARCHIVES OF VIROLOGY 160 (4) 1075 - 1082 0304-8608 2015/04 [Refereed][Not invited]
     
    Bats are an important natural reservoir for a variety of viral pathogens, including polyomaviruses (PyVs). The aims of this study were: (i) to determine which PyVs are present in bats in Indonesia and (ii) to analyze the evolutionary relationships between bat PyVs and other known PyVs. Using broad-spectrum polymerase chain reaction (PCR)-based assays, we screened PyV DNA isolated from spleen samples from 82 wild fruit bats captured in Indonesia. Fragments of the PyV genome were detected in 10 of the 82 spleen samples screened, and eight full-length viral genome sequences were obtained using an inverse PCR method. A phylogenetic analysis of eight whole viral genome sequences showed that BatPyVs form two distinct genetic clusters within the proposed genus Orthopolyomavirus that are genetically different from previously described BatPyVs. Interestingly, one group of BatPyVs is genetically related to the primate PyVs, including human PyV9 and trichodysplasia spinulosa-associated PyV. This study has identified the presence of novel PyVs in fruit bats in Indonesia and provides genetic information about these BatPyVs.
  • Yasuko Orba, Michihito Sasaki, Hiroki Yamaguchi, Akihiro Ishii, Yuka Thomas, Hirohito Ogawa, Bernard M. Hang'ombe, Aaron S. Mweene, Shigeru Morikawa, Masayuki Saijo, Hirofumi Sawa
    JOURNAL OF GENERAL VIROLOGY 96 (Pt 2) 390 - 394 0022-1317 2015/02 [Refereed][Not invited]
     
    Human monkeypox is a viral zoonosis caused by monkeypox virus, an orthopoxvirus (OPXV). The majority of human monkeypox cases have been reported in moist forested regions in West and Central Africa, particularly in the Democratic Republic of the Congo (DRC). In this study we investigated zoonotic OPXV infection among wild animals in Zambia, which shares a border with DRC, to assess the geographical distribution of OPXV. We screened for OPXV antibodies in sera from non-human primates (NHPs), rodents and shrews by ELISA, and performed real-time PCR to detect OPXV DNA in spleen samples. Serological analysis indicated that 38 of 259 (14.7 %) rodents, 14 of 42 (33.3%) shrews and 4 of 188 (2.1 %) NHPs had antibodies against OPXV. The OPXV DNA could not be detected in spleens from any animals tested. Our results indicated that wild animals living in rural human habitation areas of Zambia have been infected with OPXV.
  • Michihito Sasaki, Yasuko Orba, Keisuke Ueno, Akihiro Ishii, Ladslav Moonga, Bernard M. Hang'ombe, Aaron S. Mweene, Kimihito Ito, Hirofumi Sawa
    JOURNAL OF GENERAL VIROLOGY 96 (Pt 2) 440 - 452 0022-1317 2015/02 [Refereed][Not invited]
     
    Shrews are small insectivorous mammals that are distributed worldwide. Similar to rodents, shrews live on the ground and are commonly found near human residences. In this study, we investigated the enteric virome of wild shrews in the genus Crocidura using a sequence-independent viral metagenomics approach. A large portion of the shrew enteric virome was composed of insect viruses, whilst novel viruses including cyclovirus, picornavirus and picorna-like virus were also identified. Several cycloviruses, including variants of human cycloviruses detected in cerebrospinal fluid and stools, were detected in wild shrews at a high prevalence rate. The identified picornavirus was distantly related to human parechovirus, inferring the presence of a new genus in this family. The identified picorna-like viruses were characterized as different species of calhevirus 1, which was discovered previously in human stools. Complete or nearly complete genome sequences of these novel viruses were determined in this study and then were subjected to further genetic characterization. Our study provides an initial view of the diversity and distinctiveness of the shrew enteric virome and highlights unique novel viruses related to human stool-associated viruses.
  • John Yabe, Pharaoh Hamambulu, Edgar Simulundu, Hirohito Ogawa, Masahiro Kajihara, Akina Mori-Kajihara, Katendi Changula-Chitanga, Max Mwase, Mutinta Mweemba-Muwowo, Herman Moses Chambaro, Liywalii Mataa, Bernard Hang'ombe, Bonniface Namangala, Paul Fandamu, Hirofumi Sawa, Ayato Takada, Hideaki Higashi, Aaron Simanyengwe Mweene
    TROPICAL ANIMAL HEALTH AND PRODUCTION 47 (2) 459 - 463 0049-4747 2015/02 [Refereed][Not invited]
     
    African swine fever (ASF) is a highly contagious and fatal hemorrhagic viral disease of domestic pigs. The disease is widespread in sub-Saharan Africa and has repeatedly been introduced into other continents. The current study describes the diagnostic investigations of a hemorrhagic disease that was reported in pigs in Lusaka (October 2013), Zambia. Necropsy, histopathology, and molecular diagnosis using polymerase chain reaction and sequence analysis confirmed the disease to be ASF. The sequences obtained showed high similarity to previously isolated ASF viruses. Consistent surveillance and rapid diagnosis of the disease is recommended to prevent future outbreaks and economic losses as there is currently no vaccine against the disease.
  • Akihiro Ishii, Keisuke Ueno, Yasuko Orba, Michihito Sasaki, Ladslav Moonga, Bernard M. Hang'ombe, Aaron S. Mweene, Takashi Umemura, Kimihito Ito, William W. Hall, Hirofumi Sawa
    NATURE COMMUNICATIONS 5 5651  2041-1723 2014/12 [Refereed][Not invited]
     
    Bats can carry important zoonotic pathogens. Here we use a combination of next-generation sequencing and classical virus isolation methods to identify novel nairoviruses from bats captured from a cave in Zambia. This nairovirus infection is highly prevalent among giant leaf-nosed bats, Hipposideros gigas (detected in samples from 16 individuals out of 38). Whole-genome analysis of three viral isolates (11SB17, 11SB19 and 11SB23) reveals a typical bunyavirus tri-segmented genome. The strains form a single phylogenetic clade that is divergent from other known nairoviruses, and are hereafter designated as Leopards Hill virus (LPHV). When i.p. injected into mice, the 11SB17 strain causes only slight body weight loss, whereas 11SB23 produces acute and lethal disease closely resembling that observed with Crimean-Congo Haemorrhagic Fever virus in humans. We believe that our LPHV mouse model will be useful for research on the pathogenesis of nairoviral haemorrhagic disease.
  • Edgar Simulundu, Naganori Nao, John Yabe, Nilton A. Muto, Thami Sithebe, Hirofumi Sawa, Rashid Manzoor, Masahiro Kajihara, Mieko Muramatsu, Akihiro Ishii, Hirohito Ogawa, Aaron S. Mweene, Ayato Takada
    ARCHIVES OF VIROLOGY 159 (10) 2633 - 2640 0304-8608 2014/10 [Refereed][Not invited]
     
    Whilst remarkable progress in elucidating the mechanisms governing interspecies transmission and pathogenicity of highly pathogenic avian influenza viruses (AIVs) has been made, similar studies focusing on low-pathogenic AIVs isolated from the wild waterfowl reservoir are limited. We previously reported that two AIV strains (subtypes H6N2 and H3N8) isolated from wild waterfowl in Zambia harbored some amino acid residues preferentially associated with human influenza virus proteins (so-called human signatures) and replicated better in the lungs of infected mice and caused more morbidity than a strain lacking such residues. To further substantiate these observations, we infected chickens and mice intranasally with AIV strains of various subtypes (H3N6, H3N8, H4N6, H6N2, H9N1 and H11N9) isolated from wild waterfowl in Zambia. Although some strains induced seroconversion, all of the tested strains replicated poorly and were nonpathogenic for chickens. In contrast, most of the strains having human signatures replicated well in the lungs of mice, and one of these strains caused severe illness in mice and induced lung injury that was characterized by a severe accumulation of polymorphonuclear leukocytes. These results suggest that some strains tested in this study may have the potential to infect mammalian hosts directly without adaptation, which might possibly be associated with the possession of human signature residues. Close monitoring and evaluation of host-associated signatures may help to elucidate the prevalence and emergence of AIVs with potential for causing zoonotic infections.
  • Shintaro Kobayashi, Yasuko Orba, Hiroki Yamaguchi, Kenta Takahashi, Michihito Sasaki, Rie Hasebe, Takashi Kimura, Hirofumi Sawa
    VIRUS RESEARCH 191 83 - 91 0168-1702 2014/10 [Refereed][Not invited]
     
    Autophagy is a lysosomal degradation pathway that is implicated in many viral infections. However, its role in West Nile virus (WNV) infection remains controversial. In the present study, we examined the relationship between WNV infection and autophagy in infected cells. We demonstrated that LC3-II expression, a molecular marker for autophagosomal membranes, was enhanced in WNV-infected cells 6 h post-infection. LC3-II expression was further enhanced in WNV-inoculated cells when treated with a lysosomal protease inhibitor. Meanwhile, WNV replication in cells lacking Atg5, an essential factor for autophagy, was increased compared with replication in wild-type cells. In addition, WNV replication was inhibited in cells lacking Atg5 when they were transfected with an ATG5 expression plasmid. These results suggest an antiviral role for autophagy in WNV-infected cells. We also examined which viral replication stages were affected by autophagy by using a Tat-beclin 1 peptide to induce autophagy and pseudo-infectious WNV reporter virus particles (WNV-RVPs) that monitor viral genome replication and gene expression stages via GFP expression. We found that autophagy induction in HeLa cells by Tat-beclin 1 peptide 3 h after WNV inoculation inhibited viral replication, and GFP expression was significantly inhibited in wild-type cells when compared with cells lacking Atg5. Taken together, these results suggest that autophagy is induced by WNV infection, and that this induction inhibits WNV replication at the viral genome replication and gene expression stages. (C) 2014 Elsevier B.V. All rights reserved.
  • Jesca Nakayima, Kyoko Hayashida, Ryo Nakao, Akihiro Ishii, Hirohito Ogawa, Ichiro Nakamura, Ladslav Moonga, Bernard M. Hang'ombe, Aaron S. Mweene, Yuka Thomas, Yasuko Orba, Hirofumi Sawa, Chihiro Sugimoto
    PARASITES & VECTORS 7 490  1756-3305 2014/10 [Refereed][Not invited]
     
    Background: Wildlife may harbor infectious pathogens that are of zoonotic concern acting as a reservoir of diseases transmissible to humans and domestic animals. This is due to human-wildlife conflicts that have become more frequent and severe over recent decades, competition for the available natural habitats and resources leading to increased human encroachment on previously wild and uninhabited areas. Methods: A total of 88 spleen DNA samples from baboons and vervet monkeys from Zambia were tested for zoonotic pathogens using genus or species-specific PCR. The amplified products were then subjected to sequencing analysis. Results: We detected three different pathogenic agents, including Anaplasma phagocytophilum in 12 samples (13.6%), Rickettsia spp. in 35 samples (39.8%) and Babesia spp. in 2 samples (2.3%). Conclusion: The continuously increasing contacts between humans and primate populations raise concerns about transmission of pathogens between these groups. Therefore, increased medical and public awareness and public health surveillance support will be required to detect and control infections caused by these agents at the interface between humans and wildlife.
  • Michihito Sasaki, Agus Setiyono, Ekowati Handharyani, Shintaro Kobayashi, Ibenu Rahmadani, Siswatiana Taha, Sri Adiani, Mawar Subangkit, Ichiro Nakamura, Hirofumi Sawa, Takashi Kimura
    JOURNAL OF VIROLOGY 88 (17) 9819 - 9829 0022-538X 2014/09 [Refereed][Not invited]
     
    Bats are known to harbor emerging RNA viruses. Recent studies have used high-throughput sequencing technology to identify various virus species, including DNA viruses that are harbored by bats; however, little is known about the nature of these potentially novel viruses. Here, we report the characterization of a novel herpesvirus isolated from an Indonesian pteropodid bat. The virus, tentatively named fruit bat alphaherpesvirus 1 (FBAHV1), has a double-stranded DNA genome of 149,459 bp. The phylogenetic analyses suggested that FBAHV1 is phylogenetically grouped with simplexviruses within the subfamily Alphaherpesvirinae. Inoculation of FBAHV1 into laboratory mice caused a lethal infection. Virus infection was observed in lung, liver, and brain tissue. Serological and PCR screening revealed that fruit bats infected with FBAHV1 or its related virus are widely distributed in Indonesia. The identification of FBAHV1 makes a considerable contribution to our understanding of simplexviruses associated with bats. IMPORTANCE Bats are known to harbor emerging viruses, such as lyssaviruses, henipaviruses, severe acute respiratory syndrome-like coronaviruses, and filoviruses. Although alphaherpesviruses are disseminated in humans and other animals, there is little information about their distribution in bats. Here, we isolated a previously unknown alphaherpesvirus from an Indonesian fruit bat. Genome sequence analysis suggested that the virus is a member of the genus Simplexvirus within the subfamily Alphaherpesvirinae, which also includes common human viruses, such as herpes simplex virus 1 and herpes simplex virus 2. FBAHV1 is the first batderived alphaherpesvirus whose complete genome has been sequenced.
  • Sawang Kesdangsakonwut, Yuji Sunden, Keisuke Aoshima, Yoshimi Iwaki, Masahiro Okumura, Hirofumi Sawa, Takashi Umemura
    NEUROPATHOLOGY 34 (3) 277 - 283 0919-6544 2014/06 [Refereed][Not invited]
     
    Rabies is a fatal zoonotic disease for which no effective treatment measures are currently available. Rabies virus (RABV) has anti-apoptotic and anti-inflammatory properties that suppress nerve cell damage and inflammation in the CNS. These features imply that the elimination of RABV from the CNS by appropriate treatment could lead to complete recovery from rabies. Ten rabbits showing neuromuscular symptoms of rabies after subcutaneous (SC) immunization using commercially available vaccine containing inactivated whole RABV particles and subsequent fixed RABV (CVS strain) inoculation into hind limb muscles were allocated into three groups. Three rabbits received no further treatment (the SC group), three rabbits received three additional SC immunizations using the same vaccine, and four rabbits received three intrathecal (IT) immunizations, in which the vaccine was inoculated directly into the cerebrospinal fluid (the SC/IT group). An additional three naive rabbits were inoculated intramuscularly with RABV and not vaccinated. The rabbits exhibited neuromuscular symptoms of rabies within 4-8 days post-inoculation (dpi) of RABV. All of the rabbits died within 8-12 dpi with the exception of one rabbit in the SC group and all four rabbits in SC/IT group, which recovered and started to respond to external stimuli at 11-18 dpi and survived until the end of the experimental period. RABV was eliminated from the CNS of the surviving rabbits. We report here a possible, although still incomplete, therapy for rabies using IT immunization. Our protocol may rescue the life of rabid patients and prompt the future development of novel therapies against rabies.
  • Hiroki Yamaguchi, Shintaro Kobayashi, Junki Maruyama, Michihito Sasaki, Ayato Takada, Takashi Kimura, Hirofumi Sawa, Yasuko Orba
    JOURNAL OF VETERINARY MEDICAL SCIENCE 76 (5) 637 - 644 0916-7250 2014/05 [Refereed][Not invited]
     
    Recently, we detected novel vervet monkey polyomavirus 1 (VmPyV) in a vervet monkey. Among amino acid sequences of major capsid protein VP1s of other polyomaviruses, VmPyV VP1 is the longest with additional amino acid residues in the C-terminal region. To examine the role of VmPyV VP1 in virion formation, we generated virus-like particles (VLPs) of VmPyV VP1, because VLP is a useful tool for the investigation of the morphological characters of polyomavirus virions. After the full-length VmPyV VP1 was subcloned into a mammalian expression plasmid, the plasmid was transfected into human embryonic kidney 293T (HEK293T) cells. Thereafter, VmPyV VLPs were purified from the cell lysates of the transfected cells via sucrose gradient sedimentation. Electron microscopic analyses revealed that VmPyV VP1 forms VLPs with a diameter of approximately 50 nm that are exclusively localized in cell nuclei. Furthermore, we generated VLPs consisting of the deletion mutant VmPyV VP1 (Delta C VP1) lacking the C-terminal 116 amino acid residues and compared its VLP formation efficiency and morphology to those of VLPs from wild-type VmPyV VP1 (WT VP1). WT and Delta C VP1 VLPs were similar in size, but the number of Delta C VP1 VLPs was much lower than that of WT VP1 VLPs in VP1-expressing HEK293T cells. These results suggest that the length of VP1 is unrelated to virion morphology; however, the C-terminal region of VmPyV VP1 affects the efficiency of its VLP formation.
  • Walter Muleya, Michihito Sasaki, Yasuko Orba, Akihiro Ishii, Yuka Thomas, Emiko Nakagawa, Hirohito Ogawa, Bernard Hang'ombe, Boniface Namangala, Aaron Mweene, Ayato Takada, Takashi Kimura, Hirofumi Sawa
    JOURNAL OF VETERINARY MEDICAL SCIENCE 76 (4) 611 - 614 0916-7250 2014/04 [Refereed][Not invited]
     
    In this study, we describe the detection of novel paramyxoviruses from the Eidolon helvum species of fruit bats. We extracted RNA from 312 spleen samples from bats captured in Zambia over a period of 4 years (2008-2011). Semi-nested RT-PCR detected a total of 25 (8%) positive samples for paramyxoviruses which were then directly sequenced and analyzed using phylogenetic analysis. Among the positive samples, seven novel paramyxoviruses were detected. Five viruses were closely related to the genus Henipavirus, while two viruses were related to the unclassified Bat paramyxoviruses from Ghana and Congo Brazzaville. Our study identified novel Henipavirus-related and unrelated viruses using RT-PCR in fruit bats from Kansaka National Park and indicated the presence of similar Bat paramyxoviruses originating from wide geographic areas, suggesting the ability of bats to harbor and transmit viruses. The presence of these viruses in fruit bats might pose a public health risk.
  • Michihito Sasaki, Walter Muleya, Akihiro Ishii, Yasuko Orba, Bernard M. Hang'ombe, Aaron S. Mweene, Ladslav Moonga, Yuka Thomas, Takashi Kimura, Hirofumi Sawa
    JOURNAL OF GENERAL VIROLOGY 95 (Pt 2) 325 - 330 0022-1317 2014/02 [Refereed][Not invited]
     
    Rodents and shrews are known to harbour various viruses. Paramyxoviruses have been isolated from Asian and Australian rodents, but little is known about them in African rodents. Recently, previously unknown paramyxovirus sequences were found in South African rodents. To date, there have been no reports related to the presence and prevalence of paramyxoviruses in shrews. We found a high prevalence of paramyxoviruses in wild rodents and shrews from Zambia. Seminested reverse transcription-PCR assays were used to detect paramyxovirus RNA in 21% (96/ 462) of specimens analysed. Phylogenetic analysis revealed that these viruses were novel paramyxoviruses and could be classified as morbillivirus- and henipavirus-related viruses, and previously identified rodent paramyxovirus-related viruses. Our findings suggest the circulation of previously unknown paramyxoviruses in African rodents and shrews, and provide new information regarding the geographical distribution and genetic diversity of paramyxoviruses.
  • Yoshinori Makino, Tadaki Suzuki, Rie Hasebe, Takashi Kimura, Akihiko Maeda, Hidehiro Takahashi, Hirofumi Sawa
    JOURNAL OF VIROLOGICAL METHODS 195 250 - 257 0166-0934 2014/01 [Refereed][Not invited]
     
    West Nile virus (WNV) is one of flaviviruses and has emerged recently in the United States as a significant cause of viral encephalitis. Although cellular entry of WNV is important for viral pathogenesis, its mechanisms have not been elucidated fully. To explore the entry mechanisms, a virus-particle tracking system in live cells by using fluorescently labeled subviral particles (SVPs) and time-lapse epifluorescence microscopy was established. This study revealed that, following cellular entry, SVP movements could be divided into two phases: early (slow movement) and late (fast movement) phase. Moreover, fast viral particle movements at the late phase correlated with SVP-microtubule association. (C) 2013 Elsevier B.V. All rights reserved.
  • Sawa H, Kobayashi S, Suzuki T, Orba Y
    Uirusu 64 (1) 25 - 34 0042-6857 2014 [Refereed][Not invited]
  • Tadaki Suzuki, Yasuko Orba, Yoshinori Makino, Yuki Okada, Yuji Sunden, Hideki Hasegawa, William W. Hall, Hirofumi Sawa
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 110 (46) 18668 - 18673 0027-8424 2013/11 [Refereed][Not invited]
     
    Viroporins, which are encoded by a wide range of animal viruses, oligomerize in host cell membranes and form hydrophilic pores that can disrupt a number of physiological properties of the cell. Little is known about the relationship between host cell proteins and viroporin activity. The human JC polyomavirus (JCV) is the causative agent of progressive multifocal leukoencephalopathy. The JCV-encoded agnoprotein, which is essential for viral replication, has been shown to act as a viroporin. Here we demonstrate that the JCV agnoprotein specifically interacts with adaptor protein complex 3 through its delta subunit. This interaction interrupts adaptor protein complex 3-mediated vesicular trafficking with suppression of the targeting of the protein to the lysosomal degradation pathway and instead permits the transport of agnoprotein to the cell surface with resulting membrane permeabilization. The findings demonstrate a previously undescribed paradigm in virus-host interactions allowing the host to regulate viroporin activity and suggest that the viroporins of other viruses may also be highly regulated by specific interactions with host cell proteins.
  • Takashi Kimura, Megumi Okumura, Eunmi Kim, Michihito Sasaki, Yasuko Orba, Hirofumi Sawa
    MICROBIOLOGY AND IMMUNOLOGY 57 (10) 723 - 731 0385-5600 2013/10 [Refereed][Not invited]
     
    Neurons are the major target cell of Japanese encephalitis virus (JEV). Rats intracerebrally inoculated with JEV show an age-dependent pattern of resistance to infection in which resistance is closely associated with neuronal maturation. However, because there is no reliable and convenient cell culture system that mimics the in vivo properties of JEV infection of immature and mature neurons, the mechanisms underlying this association remain poorly understood. The aim of the present study was to examine JEV infection in immortalized CSM14.1 rat neuronal cells, which can be induced to differentiate into neurons by culture under non-permissive conditions. JEV infected undifferentiated CSM14.1 cells more efficiently than differentiated cells, resulting in production of more progeny virus in the former setting than in the latter. An infectious virus recovery assay detected more internalized virions in undifferentiated cells. On the other hand, JEV infection of differentiated cells induced more rapid and stronger expression of interferon- gene, along with smaller amounts of JEV RNA. Taken together, these results show that the initial phase of viral infection and the later IFN response play roles in the viral susceptibility of undifferentiated and differentiated CSM14.1 cells. Because CSM14.1 cells became more resistant to JEV infection as they mature, this culture system can be used as an in vitro model for studying age-dependent resistance of neurons to JEV infection.
  • Michihito Sasaki, Akihiro Ishii, Yasuko Orba, Yuka Thomas, Bernard M. Hang'ombe, Ladslav Moonga, Aaron S. Mweene, Hirohito Ogawa, Ichiro Nakamura, Takashi Kimura, Hirofumi Sawa
    EMERGING INFECTIOUS DISEASES 19 (9) 1500 - 1503 1080-6040 2013/09 [Refereed][Not invited]
     
    Human parainfluenza virus type 3 (HPIV3) genome was detected in 4 baboons in Zambia. Antibody for HPIV3 was detected in 13 baboons and 6 vervet monkeys in 2 distinct areas in Zambia. Our findings suggest that wild nonhuman primates are susceptible to HPIV3 infection.
  • Yusuke K. Kawai, Kensuke P. Watanabe, Akihiro Ishii, Aiko Ohnuma, Hirofumi Sawa, Yoshinori Ikenaka, Mayumi Ishizuka
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY D-GENOMICS & PROTEOMICS 8 (3) 201 - 208 1744-117X 2013/09 [Refereed][Not invited]
     
    The cytochrome P450 (CYP) 1-3 families are involved in xenobiotic metabolism, and are expressed primarily in the liver. Ostriches (Struthio camelus) are members of Palaeognathae with the earliest divergence from other bird lineages. An understanding of genes coding for ostrich xenobiotic metabolizing enzyme contributes to knowledge regarding the xenobiotic metabolisms of other Palaeognathae birds. We investigated CYP1-3 genes expressed in female ostrich liver using a next-generation sequencer. We detected 10 CYP genes: CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2W2, CYP2AC1, CYP2AC2, CYP2AF1, and CYP3A37. We compared the gene expression levels of CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2AF1, and CYP3A37 in ostrich liver and determined that CYP2G19 exhibited the highest expression level. The mRNA expression level of CYP2G19 was approximately 2-10 times higher than those of other CYP genes. The other CYP genes displayed similar expression levels. Our results suggest that CYP2G19, which has not been a focus of previous bird studies, has an important role in ostrich xenobiotic metabolism. (C) 2013 Elsevier Inc. All rights reserved.
  • Ichiro Nakamura, Bernard Mudenda Hang'ombe, Hirofumi Sawa, Shintaro Kobayashi, Yasuko Orba, Akihiro Ishii, Yuka Thomas, Rie Isozumi, Kumiko Yoshimatsu, Aaron S. Mweene, Ayato Takada, Chihiro Sugimoto, Jiro Arikawa
    JOURNAL OF VETERINARY MEDICAL SCIENCE 75 (6) 819 - 825 0916-7250 2013/06 [Refereed][Not invited]
     
    A total of 466 rodents were captured in the Republic of Zambia from 2006 to 2010. Based on morphological observations and phylogenetic analyses of mitochondrial gene sequences, rodents were divided into 10 groups consisting of 39 Rattus rodents, 263 multimammate rats, 18 other Murinae rodents, 95 gerbils, 11 pouched mice, 1 giant-pouched rat, 38 fat mice and 1 dormouse. Rodent antibodies except that from Rattus were examined for their cross-reactivity to commercially available antibody detection reagents. Anti-mouse immunoglobulin G (IgG) was most cross-reactive to heterologous antibodies including multimammate rat, gerbil, pouched mouse and fat mouse. Thus, anti-mouse IgG would be a useful detection tool in serological examination of the Zambian rodent population. Preliminary sero-surveillance for plague, leptospirosis and hantavirus infection was performed by ELISA.
  • Yamaguchi H, Kobayashi S, Ishii A, Ogawa H, Nakamura I, Moonga L, Hang'ombe BM, Mweene AS, Thomas Y, Kimura T, Sawa H, Orba Y
    The Journal of general virology Pt 6 94 (Pt 6) 1357 - 1364 0022-1317 2013/06 [Refereed][Not invited]
  • Kenichi Niikura, Tatsuya Matsunaga, Tadaki Suzuki, Shintaro Kobayashi, Hiroki Yamaguchi, Yasuko Orba, Akira Kawaguchi, Hideki Hasegawa, Kiichi Kajino, Takafumi Ninomiya, Kuniharu Ijiro, Hirofumi Sawa
    ACS NANO 7 (5) 3926 - 3938 1936-0851 2013/05 [Refereed][Not invited]
     
    This paper demonstrates how the shape and size of gold nanoparticles (AuNPs) affect immunological responses in vivo and in vitro for the production of antibodies for West Nile virus (WNV). We prepared spherical (20 and 40 nm in diameter), rod (40 x 10 nm), and cubic (40 x 40 x 40 nm) AuNPs as adjuvants and coated them with WNV envelope (E) protein. We measured anti-WNVE antibodies after inoculation of these WNVE-coated AuNPs (AuNP-Es) into mice. The 40 nm spherical AuNP-Es (Sphere40-Es) induced the highest level of WNVE-specific antibodies, while rod AuNP-Es (Rod-Es) induced only 50% of that of Sphere40-E. To examine the mechanisms of the shape-dependent WNVE antibody production, we next measured the efficiency of cellular uptake of AuNP-Es into RAW264.7 macrophage cells and bone-marrow-derived dendritic cells (BMDCs) and the subsequent cytokine secretion from BMDCs. The uptake of Rod-Es into the cells proceeded more efficiently than those of Sphere-Es or cubic WNVE-coated AuNPs (Cube-Es), suggesting that antibody production was not dependent on the uptake efficiency of the different AuNP-Es. Cytokine production from BMDCs treated with the AuNP-Es revealed that only Rod-E-treated cells produced significant levels of interleukin-1 beta (IL-1 beta) and interleukin-18 (IL-18), indicating that Rod-Es activated inflammasome-dependent cytokine secretion. Meanwhile, Sphere40-Es and Cube-Es both significantly induced inflammatory cytokine production, including tumor necrosis factor-alpha (TNF-alpha), IL-6, IL-12, and granulocyte macrophage colony-stimulating factor (GM-CSF). These results suggested that AuNPs are effective vaccine adjuvants and enhance the immune response via different cytokine pathways depending on their sizes and shapes.
  • Niikura K, Sugimura N, Musashi Y, Mikuni S, Matsuo Y, Kobayashi S, Nagakawa K, Takahara S, Takeuchi C, Sawa H, Kinjo M, Ijiro K
    Molecular bioSystems 3 9 (3) 501 - 507 1742-206X 2013/03 [Refereed][Not invited]
  • Kenta Takahashi, Yasuko Orba, Taichi Kimura, Lei Wang, Shinji Kohsaka, Masumi Tsuda, Mishie Tanino, Hiroshi Nishihara, Kazuo Nagashima, Hirofumi Sawa, Shinya Tanaka
    JAPANESE JOURNAL OF INFECTIOUS DISEASES 66 (2) 126 - 132 1344-6304 2013/03 [Refereed][Not invited]
     
    JC virus (JCV) is a causative agent of progressive multifocal leukoencephalopathy (PML). Methyl CpG binding protein 2 (MeCP2) is a transcriptional control nuclear protein that is abundantly expressed in neurons. We previously observed that the MeCP2 protein is expressed in JCV large T antigen (TAg)-expressing glial cells in PML brains. To investigate the relationship between MeCP2 and JCV TAg, we examined the promoter activity and mRNA and protein expression levels of MeCP2 in JCV TAg-expressing cells. We found that JCV TAg enhances the promoter activity of MeCP2, but does not enhance the mRNA and protein levels of MeCP2. These results suggest that post-transcriptional mechanisms may play a role in MeCP2 expression.
  • Kobayashi S, Suzuki T, Igarashi M, Orba Y, Ohtake N, Nagakawa K, Niikura K, Kimura T, Kasamatsu H, Sawa H
    PloS one 10 8 (10) e76668  2013 [Refereed][Not invited]
  • Walter Muleya, Boniface Namangala, Martin Simuunza, Ryo Nakao, Noboru Inoue, Takashi Kimura, Kimihito Ito, Chihiro Sugimoto, Hirofumi Sawa
    PARASITES & VECTORS 5 255  1756-3305 2012/11 [Refereed][Not invited]
     
    Background: Theileriosis, caused by Theileria parva, is an economically important disease in Africa. It is a major constraint to the development of the livestock industry in some parts of eastern, central and southern Africa. In Zambia, theileriosis causes losses of up to 10,000 cattle annually. Methods: Cattle blood samples were collected for genetic analysis of Theileria parva from Isoka and Petauke districts in Zambia. Microsatellite analysis was then performed on all Theileria parva positive samples for PCR using a panel of 9 microsatellite markers. Microsatellite data was analyzed using microsatellite toolkit, GenAlEx ver. 6, Fstat ver. 2.9.3.2, and LIAN computer softwares. Results: The combined percentage of positive samples in both districts determined by PCR using the p104 gene primers was 54.9% (95% CI: 46.7 - 63.1%, 78/142), while in each district, it was 44.8% (95% CI: 34.8 - 54.8%) and 76.1% (95% CI = 63.9 - 88.4%) for Isoka and Petauke districts, respectively. We analyzed the population genetic structure of Theileria parva from a total of 61 samples (33 from Isoka and 28 from Petauke) using a panel of 9 microsatellite markers encompassing the 4 chromosomes of Theileria parva. Wright's F index (F-ST = 0.178) showed significant differentiation between the Isoka and Petauke populations. Linkage disequilibrium was observed when populations from both districts were treated as a single population. When analyzed separately, linkage disequilibrium was observed in Kanyelele and Kalembe areas in Isoka district, Isoka district overall and in Petauke district. Petauke district had a higher multiplicity of infection than Isoka district. Conclusion: Population genetic analyses of Theileria parva from Isoka and Petauke districts showed a low level of genotype exchange between the districts, but a high level of genetic diversity within each district population, implying genetic and geographic sub-structuring between the districts. The sub-structuring observed, along with the lack of panmixia in the populations, could have been due to low transmission levels at the time of sampling. However, the Isoka population was less diverse than the Petauke population.
  • Akihiro Ishii, Yuka Thomas, Ladslav Moonga, Ichiro Nakamura, Aiko Ohnuma, Bernard M. Hang'ombe, Ayato Takada, Aaron S. Mweene, Hirofumi Sawa
    JOURNAL OF GENERAL VIROLOGY 93 (10) 2247 - 2251 0022-1317 2012/10 [Refereed][Not invited]
     
    In order to survey arenaviruses in the Republic of Zambia, we captured 335 rodents from three cities between 2010 and 2011. Eighteen Luna virus (LUNV) and one lymphocytic choriomeningitis virus (LCMV)-related virus RNAs were detected by one-step RT-PCR from Mastomys natalensis and Mus minutoides, respectively. Four LUNV strains and one LCMV-related virus were isolated, and the whole genome nucleotide sequence was determined by pyrosequencing. Phylogenetic analyses revealed that the LUNV clade consists of two branches that are distinguished by geographical location and that the LCMV-related virus belongs to the LCMV clade, but diverges from the typical LCMVs. Comparison of nucleoprotein amino acid sequences indicated that the LCMV-related virus could be designated a novel arenavirus, which was tentatively named as the Lunk virus. Amino acid sequences of the GP, NP, Z and L proteins showed poor similarity among the three Zambian arenavirus strains, i.e. Luna, Lunk and Lujo virus.
  • Michihito Sasaki, Agus Setiyono, Ekowati Handharyani, Ibenu Rahmadani, Siswatiana Taha, Sri Adiani, Mawar Subangkit, Hirofumi Sawa, Ichiro Nakamura, Takashi Kimura
    VIROLOGY JOURNAL 9 240  1743-422X 2012/10 [Refereed][Not invited]
     
    Background: Fruit bats are known to harbor zoonotic paramyxoviruses including Nipah, Hendra, and Menangle viruses. The aim of this study was to detect the presence of paramyxovirus RNA in fruit bats from Indonesia. Methods: RNA samples were obtained from the spleens of 110 fruit bats collected from four locations in Indonesia. All samples were screened by semi-nested broad spectrum reverse transcription PCR targeting the paramyxovirus polymerase (L) genes. Results: Semi-nested reverse transcription PCR detected five previously unidentified paramyxoviruses from six fruit bats. Phylogenetic analysis showed that these virus sequences were related to henipavirus or rubulavirus. Conclusions: This study indicates the presence of novel paramyxoviruses among fruit bat populations in Indonesia.
  • Tadaki Suzuki, Shingo Semba, Yuji Sunden, Yasuko Orba, Shintaro Kobayashi, Kazuo Nagashima, Takashi Kimura, Hideki Hasegawa, Hirofumi Sawa
    MICROBIOLOGY AND IMMUNOLOGY 56 (9) 639 - 646 0385-5600 2012/09 [Refereed][Not invited]
     
    JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and causes progressive multifocal leukoencephalopathy in humans. JCV encodes early proteins (large T antigen, small T antigen, and T' antigen) and four late proteins (agnoprotein, and three viral capsid proteins, VP1, VP2, and VP3). In the current study, a novel function for JCV agnoprotein in the morphogenesis of JC virion particles was identified. It was found that mature virions of agnoprotein-negative JCV are irregularly shaped. Sucrose gradient sedimentation and cesium chloride gradient ultracentrifugation analyses revealed that the particles of virus lacking agnoprotein assemble into irregularly sized virions, and that agnoprotein alters the efficiency of formation of VP1 virus-like particles. An in vitro binding assay and immunocytochemistry revealed that agnoprotein binds to glutathione S-transferase fusion proteins of VP1 and that some fractions of agnoprotein colocalize with VP1 in the nucleus. In addition, gel filtration analysis of formation of VP1-pentamers revealed that agnoprotein enhances formation of these pentamers by interacting with VP1. The present findings suggest that JCV agnoprotein plays a role, similar to that of SV40 agnoprotein, in facilitating virion assembly.
  • Nilton Akio Muto, Yuji Sunden, Tomoe Hattori, Daisuke Fujikura, Yosuke Nakayama, Tadaaki Miyazaki, Mitsuo Maruyama, Takashi Kimura, Hirofumi Sawa
    JAPANESE JOURNAL OF INFECTIOUS DISEASES 65 (5) 383 - 391 1344-6304 2012/09 [Refereed][Not invited]
     
    This study examined pathological changes in the lung tissues of young and aged mice infected with influenza virus. Young mice inoculated with influenza virus showed body weight loss at 4 days post-infection (dpi), meanwhile body weight decrease started from 9 dpi in the aged mice. We histopathologically examined the lungs of these mice. Immunohistochemical examination revealed that viral antigen-positive bronchiolar and alveolar epithelial cell numbers at 3 dpi were significantly higher in young mice than in the aged ones. Further, viral antigen-positive cells were observed at 9 dpi in the aged mice, but not in the young ones. Diffuse and severe bronchointerstitial pneumonia characterized by the accumulation of polymorphonuclear leukocytes (PMNs) was observed in young mice at 6 dpi. Histopathological changes in the aged mice were milder than those in the young mice. Moreover, T cell and macrophage accumulation in the lungs was significantly higher in the young mice than in the aged mice at 9 dpi. These results suggest that there may be a correlation between the relatively low level of infiltration of PMNs, macrophages, and T lymphocytes and the delayed body weight loss and longer lasting infections observed in the lungs of the aged mice. These findings provide detailed insights into the age-specific course of infection in young and aged populations with associated differences in lung pathology.
  • Shintaro Kobayashi, Yasuko Orba, Hiroki Yamaguchi, Takashi Kimura, Hirofumi Sawa
    NEUROPATHOLOGY 32 (4) 398 - 405 0919-6544 2012/08 [Refereed][Not invited]
     
    West Nile virus (WNV) belongs to the Flaviviridae family of viruses and has emerged as a significant cause of viral encephalitis in humans, animals and birds. It has been reported that WNV replication directly induces neuronal injury, followed by neuronal cell death proven as apoptosis. Therefore, it is important to understand the mechanism of neuronal apoptosis caused by this virus to develop strategies to control its pathogenicity. Accumulation of ubiquitinated abnormal proteins has been reported to be associated with neuronal apoptosis in some pathological conditions. A lot of cellular stresses prevent cellular protein quality control mechanisms, resulting in the accumulation of ubiquitinated abnormal proteins. To obtain a better understanding of the mechanisms of WNV-induced neuronal apoptosis, we evaluated the accumulation of ubiquitinated proteins in the WNV-infected neuronal cells. We have observed that WNV infection caused massive neuronal injury in the brains of mice. Viral antigen was detected in the neuronal cytoplasm of the cells exhibiting neuronal apoptosis. Notably, ubiquitinated proteins were detected in WNV-infected neuronal cells. In addition, accumulation of ubiquitinated proteins was markedly enhanced in mouse neuroblastoma, Neuro-2a cells after WNV infection. Our histopathological and in vitro studies suggest that accumulation of ubiquitinated proteins in neuronal cells might be associated with neuronal apoptosis caused by WNV infection.
  • Inhibitory effects of an M2-specific monoclonal antibody on different strains of influenza A virus
    Nilton Akio Muto, Reiko Yoshida, Tadaki Suzuki, Shintaro Kobayashi, Hiroichi Ozaki, Daisuke Fujikura, Rashid Manzoor, Mieko Muramatsu, Ayato Takada, Takashi Kimura, Hirofumi Sawa
    JAPANESE JOURNAL OF VETERINARY RESEARCH 60 (2-3) 71 - 83 0047-1917 2012/08 [Refereed][Not invited]
     
    New approaches to the treatment of influenza have been designed based on the highly conserved antigenicity of the M2 envelope protein among influenza A virus strains. The present study examined the anti-viral activities of an anti-M2 ectodomain (M2e) monoclonal antibody (clone rM2ss23), which binds to the M2 proteins of the influenza A virus strains A/Aichi/2/68 (H3N2) (Aichi) and A/PR/8/34 (H1N1) (PR8). The results showed that rM2ss23 bound to both Aichi and PR8 M2 proteins expressed on the cell surface. While the antibody did not prevent virus entry into cells, it significantly inhibited plaque formation by the Aichi strain in a dose-dependent manner when infected cells were cultured in the presence of the antibody. By contrast, the growth of PR8 (H1N1) was not affected by the antibody. A reverse genetics approach revealed that the inhibitory effect of rM2ss23 on the Aichi virus was abolished by replacing the genes encoding the HA and/or M proteins with those of the PR8 strain. These results suggest that rM2ss23 prevents virus release from infected cells and further suggest that the mechanisms underlying the virus budding mediates by HA and M2 proteins might differ between the Aichi and PR8 strains.
  • Mudenda B. Hang'ombe, James C. L. Mwansa, Sergio Muwowo, Phillip Mulenga, Muzala Kapina, Eric Musenga, David Squarre, Liywali Mataa, Suzuki Y. Thomas, Hirohito Ogawa, Hirofumi Sawa, Hideaki Higashi
    TROPICAL DOCTOR 42 (3) 136 - 139 0049-4755 2012/07 [Refereed][Not invited]
     
    There has been a reduction of incidences of anthrax in the developed countries but it is still a public health problem in the developing countries where communities live in interface areas with wildlife. An outbreak of anthrax in Hippopotamus amphibious was observed in Zambia. Following the death of hippopotamuses, suspected human cases were reported. The objective of this study was to isolate and confirm Bacillus anthracis and to determine the antimicrobial susceptibility for the management of the disease. Of the specimens collected, 29.4% (95% confidence interval [CI], 11.4-56.0) were from humans, 42.1% (95% CI, 21.1-66.0) were from hippopotamuses and 20.0% (95% CI, 6.61-44.3) from the soil were found to be positive were for B. anthracis. An antimicrobial susceptibility test revealed that all the isolates were found to be sensitive to the recommended antibiotics. The disease control was achieved by case management and by explaining to the communities that they should avoid contact with animals that die from unknown causes.
  • Yasushi Furuta, Nobuhiko Oridate, Norihito Takeichi, Satoshi Fukuda, Hirofumi Sawa
    ANNALS OF OTOLOGY RHINOLOGY AND LARYNGOLOGY 121 (6) 419 - 425 0003-4894 2012/06 [Refereed][Not invited]
     
    Objectives: To investigate the biological factors related to the onset of Bell's palsy, we sought to identify differentially expressed genes in peripheral blood mononuclear cells (PBMCs) and plasma of patients with Bell's palsy and Ramsay Hunt syndrome (RHS). Methods: We carried out DNA microarray analyses using PBMCs taken from patients with Bell's palsy at their initial visit and 2 to 4 weeks later. To validate these analyses, we measured the relative messenger RNA levels of alpha-defensin in paired PBMCs by reverse transcription-polymerase chain reaction. The plasma concentrations of alpha-defensin in patients and healthy volunteers were quantified by enzyme-linked immunosorbent assay. Results: The DNA microarray analysis identified alpha-defensin as a candidate gene related to the onset of Bell's palsy. Reverse transcription polymerase chain reaction analysis showed that the relative alpha-defensin messenger RNA levels in PBMCs from the later visit were increased at least twofold in 9 of 13 patients (69%) with Bell's palsy and in 4 of 6 patients (67%) with RHS. The plasma alpha-defensin concentrations in the patients with RHS were significantly higher than those in healthy volunteers (p = 0.0013) and in the patients with Bell's palsy (p = 0.0306). Elevations of plasma alpha-defensin were observed in 5 of the 9 patients with Bell's palsy who demonstrated alpha-defensin overexpression in PBMCs. Conclusions: alpha-Defensin may be one of the biological factors related to the onset of Bell's palsy and RHS.
  • Shigetomo Fukuhara, Szandor Simmons, Shunsuke Kawamura, Asuka Inoue, Yasuko Orba, Takeshi Tokudome, Yuji Sunden, Yuji Arai, Kazumasa Moriwaki, Junji Ishida, Akiyoshi Uemura, Hiroshi Kiyonari, Takaya Abe, Akiyoshi Fukamizu, Masanori Hirashima, Hirofumi Sawa, Junken Aoki, Masaru Ishii, Naoki Mochizuki
    JOURNAL OF CLINICAL INVESTIGATION 122 (4) 1416 - 1426 0021-9738 2012/04 [Refereed][Not invited]
     
    The bioactive lysophospholipid mediator sphingosine-l-phosphate (SIP) promotes the egress of newly formed T cells from the thymus and the release of immature B cells from the bone marrow. It has remained unclear, however, where and how SIP is released. Here, we show that in mice, the SIP transporter spinster homolog 2 (Spns2) is responsible for the egress of mature T cells and immature B cells from the thymus and bone marrow, respectively. Global Spns2-KO mice exhibited marked accumulation of mature T cells in thymi and decreased numbers of peripheral T cells in blood and secondary lymphoid organs. Mature recirculating B cells were reduced in frequency in the bone marrow as well as in blood and secondary lymphoid organs. Bone marrow reconstitution studies revealed that Spns2 was not involved in SW release from blood cells and suggested a role for Spns2 in other cells. Consistent with these data, endothelia-specific deletion of Spns2 resulted in defects of lymphocyte egress similar to those observed in the global Spns2-KO mice. These data suggest that Spns2 functions in ECs to establish the S1P gradient required for T and B cells to egress from their respective primary lymphoid organs. Furthermore, Spns2 could be a therapeutic target for a broad array of inflammatory and autoimmune diseases.
  • Xin Dang, Christian Wuethrich, Jennifer Gordon, Hirofumi Sawa, Igor J. Koralnik
    PLOS ONE 7 (4) e35793  1932-6203 2012/04 [Refereed][Not invited]
     
    JC virus encephalopathy (JCVE) is a newly described gray matter disease of the brain caused by productive infection of cortical pyramidal neurons. We characterized the full length sequence of JCV isolated from the brain of a JCVE patient, analyzed its distribution in various compartments by PCR, and determined viral gene expression in the brain by immunohistochemistry(IHC). We identified a novel JCV variant, JCV(CPN1), with a unique 143 bp deletion in the Agno gene encoding a truncated 10 amino acid peptide, and harboring an archetype-like regulatory region. This variant lacked one of three nuclear protein binding regions in the Agno gene. It was predominant in the brain, where it coexisted with an Agno-intact wild-type strain. Double immunostaining with anti-Agno and anti-VP1 antibodies demonstrated that the truncated JCVCPN1 Agno peptide was present in the majority of cortical cells productively infected with JCV. We then screened 68 DNA samples from 8 brain, 30 CSF and 30 PBMC samples of PML patients, HIV+and HIV-control subjects. Another JCV(CPN) strain with a different pattern of Agno-deletion was found in the CSF of an HIV+/PML patient, where it also coexisted with wildtype, Agno-intact JCV. These findings suggest that the novel tropism for cortical pyramidal neurons of JCV(CPN1,) may be associated with the Agno deletion. Productive and lytic infection of these cells, resulting in fulminant JCV encephalopathy and death may have been facilitated by the co-infection with a wild-type strain of JCV.
  • Walter Muleya, Boniface Namangala, Aaron Mweene, Luke Zulu, Paul Fandamu, Douglas Banda, Takashi Kimura, Hirofumi Sawa, Akihiro Ishii
    VIRUS RESEARCH 163 (1) 160 - 168 0168-1702 2012/01 [Refereed][Not invited]
     
    The National Livestock Epidemiology and Information Center (NALEIC) in Zambia reported over 132 cases of canine rabies diagnosed by the direct fluorescent antibody test (DFAT) from 2004 to 2009. In this study, the lineage of rabies virus (RABV) in Zambia was determined by phylogenetic analysis of the nucleoprotein (N) and glycoprotein (G) gene sequences. Total RNA was extracted from 87-DFAT brain specimens out of which only 35 (40%) were positive on nested reverse transcription polymerase chain reaction (RT-PCR) for each gene, and 26 being positive for both genes. Positive specimens for the N (n =33) and G (n = 35) genes were used for phylogenetic analysis. Phylogenetic analysis of the N gene showed two phylogenetic clusters in Zambia belonging to the Africa 1b lineage present in eastern and southern Africa. While one cluster exclusively comprised Zambian strains, the other was more heterogeneous regarding the RABV origins and included strains from Tanzania, Mozambique and Zambia. Phylogenetic analysis of the G gene revealed similar RABV strains in different hosts and regions of Zambia. We designed primers for reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay from the consensus sequence of the N gene in an attempt to improve the molecular diagnosis of RABV in Zambia. The specificity and reproducibility of the RI-LAMP assay was confirmed with actual clinical specimens. Therefore, the RI-LAMP assay presented in this study may prove to be useful for routine diagnosis of rabies in Zambia. (C) 2011 Elsevier B.V. All rights reserved.
  • Hang'ombe BM, Nakamura I, Samui KL, Kaile D, Mweene AS, Kilonzo BS, Sawa H, Sugimoto C, Wren BW
    BMC research notes 5 72  2012/01 [Refereed][Not invited]
  • Michihito Sasaki, Eunmi Kim, Manabu Igarashi, Kimihito Ito, Rie Hasebe, Hideto Fukushi, Hirofumi Sawa, Takashi Kimura
    JOURNAL OF BIOLOGICAL CHEMISTRY 286 (45) 39370 - 39378 0021-9258 2011/11 [Refereed][Not invited]
     
    Equine herpesvirus-1 (EHV-1), an alpha-herpesvirus of the family Herpesviridae, causes respiratory disease, abortion, and encephalomyelitis in horses. EHV-1 utilizes equine MHC class I molecules as entry receptors. However, hamster MHC class I molecules on EHV-1-susceptible CHO-K1 cells play no role in EHV-1 entry. To identify the MHC class I molecule region that is responsible for EHV-1 entry, domain exchange and site-directed mutagenesis experiments were performed, in which parts of the extracellular region of hamster MHC class I (clone C5) were replaced with corresponding sequences from equine MHC class I (clone A68). Substitution of alanine for glutamine at position 173 (Q173A) within the alpha 2 domain of the MHC class I molecule enabled hamster MHC class I C5 to mediate EHV-1 entry into cells. Conversely, substitution of glutamine for alanine at position 173 (A173Q) in equine MHC class I A68 resulted in loss of EHV-1 receptor function. Equine MHC class I clone 3.4, which possesses threonine at position 173, was unable to act as an EHV-1 receptor. Substitution of alanine for threonine at position 173 (T173A) enabled MHC class I 3.4 to mediate EHV-1 entry into cells. These results suggest that the amino acid residue at position 173 of the MHC class I molecule is involved in the efficiency of EHV-1 entry.
  • Tadaki Suzuki, Akira Ainai, Noriyo Nagata, Tetsutaro Sata, Hirofumi Sawa, Hideki Hasegawa
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 414 (4) 719 - 726 0006-291X 2011/11 [Refereed][Not invited]
     
    Transcription and replication of the negative-sense single-stranded influenza A virus genomic viral RNA are catalyzed by the viral RNA polymerase, which is a trimeric complex encoded by the three largest segments of the influenza virus genome: PB1, PB2, and PA. Numerous studies of the trimeric polymerase complex assembly have substantially contributed to current understanding of influenza virus replication. However, the dynamics of spatial and temporal macromolecular interactions involving virus and host proteins during the formation of the trimeric polymerase complex (PA-PB1-PB2) are still not completely understood. In this study, bimolecular fluorescence complementation (BiFC) and Raster image correlation spectroscopy (RICS) were applied to monitor the interactions between PB1, PB2, and PA. The BiFC probes of PA-PB1 and PB1-PB2 could monitor the trimeric polymerase complex as well as the binary complex. Furthermore, the C-terminal domain of PA (PAC) promoted interaction between PB1 and PB2 in the cytoplasm and that the N-terminal domain of PA (PAN) inhibited the aberrant trimeric complex formation and assembly of higher-order oligomers induced by PAC in the cytoplasm. Taken together, these results revealed a novel function of PAN in the formation of the trimeric polymerase complexes of influenza A virus. (C) 2011 Elsevier Inc. All rights reserved.
  • Akihiro Ishii, Yuka Thomas, Ladslav Moonga, Ichiro Nakamura, Aiko Ohnuma, Bernard Hang'ombe, Ayato Takada, Aaron Mweene, Hirofumi Sawa
    EMERGING INFECTIOUS DISEASES 17 (10) 1921 - 1924 1080-6040 2011/10 [Refereed][Not invited]
     
    To investigate arenavirus in Zambia, we characterized virus from the kidneys of 5 arenavirus RNA-positive rodents (Mastomys natalensis) among 263 captured. Full-genome sequences of the viruses suggested that they were new strains similar to Lassa virus-related arenaviruses. Analyzing samples from additional rodents and other species can elucidate epizootiologic aspects of arenaviruses.
  • Akihiro Ishii, Yuka Thomas, Ladslav Moonga, Ichiro Nakamura, Aiko Ohnuma, Bernard Hang'ombe, Ayato Takada, Aaron Mweene, Hirofumi Sawa
    EMERGING INFECTIOUS DISEASES 17 (10) 1921 - 1924 1080-6040 2011/10 [Refereed][Not invited]
     
    To investigate arenavirus in Zambia, we characterized virus from the kidneys of 5 arenavirus RNA-positive rodents (Mastomys natalensis) among 263 captured. Full-genome sequences of the viruses suggested that they were new strains similar to Lassa virus-related arenaviruses. Analyzing samples from additional rodents and other species can elucidate epizootiologic aspects of arenaviruses.
  • Shinobu Teramoto, Miki Kaiho, Yasuo Takano, Rika Endo, Hideaki Kikuta, Hirofumi Sawa, Tadashi Ariga, Nobuhisa Ishiguro
    MICROBIOLOGY AND IMMUNOLOGY 55 (7) 525 - 530 0385-5600 2011/07 [Not refereed][Not invited]
     
    Polyomaviruses KI (KIPyV) and WU (WUPyV) were detected from 7 (3.0%) and 38 (16.4%) of 232 children with respiratory tract infections by real-time PCR. The rates of infection by KIPyV and WUPyV alone were 3 of 7 (42.9%) and 20 of 38 (52.6%), respectively. In the other samples, various viruses (human respiratory syncytial virus, human metapneumovirus, human rhinovirus, parainfluenza virus 1 and human bocavirus) were detected simultaneously. One case was positive for KIPyV, WUPyV and hMPV. There was no obvious difference in clinical symptoms between KIPyV-positive and WUPyV-positive patients with or without coinfection. KIPyV was detected in one of 30 specimens of lung tissue (3.3%). Neither of the viruses was detected in 30 samples of lung adenocarcinoma tissue.
  • Shinobu Teramoto, Miki Kaiho, Yasuo Takano, Rika Endo, Hideaki Kikuta, Hirofumi Sawa, Tadashi Ariga, Nobuhisa Ishiguro
    MICROBIOLOGY AND IMMUNOLOGY 55 (7) 525 - 530 0385-5600 2011/07 [Refereed][Not invited]
     
    Polyomaviruses KI (KIPyV) and WU (WUPyV) were detected from 7 (3.0%) and 38 (16.4%) of 232 children with respiratory tract infections by real-time PCR. The rates of infection by KIPyV and WUPyV alone were 3 of 7 (42.9%) and 20 of 38 (52.6%), respectively. In the other samples, various viruses (human respiratory syncytial virus, human metapneumovirus, human rhinovirus, parainfluenza virus 1 and human bocavirus) were detected simultaneously. One case was positive for KIPyV, WUPyV and hMPV. There was no obvious difference in clinical symptoms between KIPyV-positive and WUPyV-positive patients with or without coinfection. KIPyV was detected in one of 30 specimens of lung tissue (3.3%). Neither of the viruses was detected in 30 samples of lung adenocarcinoma tissue.
  • Edgar Simulundu, Akihiro Ishii, Manabu Igarashi, Aaron S. Mweene, Yuka Suzuki, Bernard M. Hang'ombe, Boniface Namangala, Ladslav Moonga, Rashid Manzoor, Kimihito Ito, Ichiro Nakamura, Hirofumi Sawa, Chihiro Sugimoto, Hiroshi Kida, Chuma Simukonda, Wilbroad Chansa, Jack Chulu, Ayato Takada
    JOURNAL OF GENERAL VIROLOGY 92 (6) 1416 - 1427 0022-1317 2011/06 [Refereed][Not invited]
     
    Although the quest to clarify the role of wild birds in the spread of the highly pathogenic H5N1 avian influenza virus (AIV) has yielded considerable data on AIVs in wild birds worldwide, information regarding the ecology and epidemiology of AIVs in African wild birds is still very limited. During AIV surveillance in Zambia (2008-2009), 12 viruses of distinct subtypes (H3N8, H4N6, H6N2, H9N1 and H11N9) were isolated from wild waterfowl. Phylogenetic analyses demonstrated that all the isolates were of the Eurasian lineage. Whilst some genes were closely related to those of AIVs isolated from wild and domestic birds in South Africa, intimating possible AIV exchange between wild birds and poultry in southern Africa, some gene segments were closely related to those of AIVs isolated in Europe and Asia, thus confirming the inter-regional AIV gene flow among these continents. Analysis of the deduced amino acid sequences of internal proteins revealed that several isolates harboured particular residues predominantly observed in human influenza viruses. Interestingly, the isolates with human-associated residues exhibited higher levels of virus replication in the lungs of infected mice and caused more morbidity as measured by weight loss than an isolate lacking such residues. This study stresses the need for continued monitoring of AIVs in wild and domestic birds in southern Africa to gain a better understanding of the emergence of strains with the potential to infect mammals.
  • Eunmi Kim, Megumi Okumura, Hirofumi Sawa, Tadaaki Miyazaki, Daisuke Fujikura, Shuhei Yamada, Kazuyuki Sugahara, Michihito Sasaki, Takashi Kimura
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 409 (4) 717 - 722 0006-291X 2011/06 [Refereed][Not invited]
     
    Glycosaminoglycans (GAGs) have diverse functions in the body and are involved in viral infection. The purpose of this study was to evaluate the possible roles of the E-disaccharide units GlcA beta 1-3Gal-NAc(4,6-O-disulfate) of chondroitin sulfate (CS), a GAG involved in neuritogenesis and neuronal migration, in Japanese encephalitis virus (JEV) infection. Soluble CS-E (sCS-E) derived from squid cartilage inhibited JEV infection in African green monkey kidney-derived Vero cells and baby hamster kidney-derived BHK cells by interfering with viral attachment. In contrast, sCS-E enhanced viral infection in the mouse neuroblastoma cell line Neuro-2a, despite the fact that viral attachment to Neuro-2a cells was inhibited by sCS-E. This enhancement effect in Neuro-2a cells seemed to be related to increased viral RNA replication and was also observed in a rat infection model in which intracerebral coadministration of sCS-E with JEV in 17-day-old rats resulted in higher brain viral loads than in rats infected without sCS-E administration. These results show the paradoxical effects of sCS-E on JEV infection in different cell types and indicate that potential use of sCS-E as an antiviral agent against JEV infection should be approached with caution considering its effects in the neuron, the major target of JEV. (C) 2011 Elsevier Inc. All rights reserved.
  • Yasuko Orba, Shintaro Kobayashi, Ichiro Nakamura, Akihiro Ishii, Bernard M. Hang'ombe, Aaron S. Mweene, Yuka Thomas, Takashi Kimura, Hirofumi Sawa
    JOURNAL OF GENERAL VIROLOGY 92 (4) 789 - 795 0022-1317 2011/04 [Refereed][Not invited]
     
    To investigate polyomavirus infection in wild rodents, we analysed DNA samples from the spleens of 100 wild rodents from Zambia using a broad-spectrum PCR-based assay. A previously unknown polyomavirus genome was identified in a sample from a multimammate mouse (Mastomys species) and the entire viral genome of 4899 bp was :subsequently sequenced. This viral genome contained potential ORFs for the capsid proteins, VP1, VP2 and VP3, and early proteins, small t antigen and large T antigen. Phylogenetic analysis showed that it was a novel member of the family Polyomaviridae, and thus the virus was tentatively named mastomys polyomavirus. After transfection of the viral genome into several mammalian cell lines, transient expression of the VP1 and large T antigen proteins was confirmed by immunoblotting and immunocytochemical analyses. Comparison of large T antigen function in mastomys polyomavirus with that in rhesus monkey polyomavirus SV40 and human polyomavirus JC virus revealed that the large T antigen from mastomys polyomavirus interacted with the tumour suppressor protein pRb, but not with p53.
  • Michihito Sasaki, Rie Hasebe, Yoshinori Makino, Tadaki Suzuki, Hideto Fukushi, Minoru Okamoto, Kazuya Matsuda, Hiroyuki Taniyama, Hirofumi Sawa, Takashi Kimura
    GENES TO CELLS 16 (4) 343 - 357 1356-9597 2011/04 [Refereed][Not invited]
     
    The endotheliotropism of equine herpesvirus-1 (EHV-1) leads to encephalomyelitis secondary to vasculitis and thrombosis in the infected horse central nervous system (CNS). To identify the host factors involved in EHV-1 infection of CNS endothelial cells, we performed functional cloning using an equine brain microvascular endothelial cell cDNA library. Exogenous expression of equine major histocompatibility complex (MHC) class I heavy chain genes conferred susceptibility to EHV-1 infection in mouse NIH3T3 cells, which are not naturally susceptible to EHV-1 infection. Equine MHC class I molecules bound to EHV-1 glycoprotein D (gD), and both anti-gD antibodies and a soluble form of gD blocked viral entry into NIH3T3 cells stably expressing the equine MHC class I heavy chain gene (3T3-A68 cells). Treatment with an anti-equine MHC class I monoclonal antibody blocked EHV-1 entry into 3T3-A68 cells, equine dermis (E. Derm) cells and equine brain microvascular endothelial cells. In addition, inhibition of cell surface expression of MHC class I molecules in E. Derm cells drastically reduced their susceptibility to EHV-1 infection. These results suggest that equine MHC class I is a functional gD receptor that plays a pivotal role in EHV-1 entry into equine cells.
  • Saya Shirai, Kenta Takahashi, Shinji Kohsaka, Tetsu Tsukamoto, Hiroshi Isogai, Shinichi Kudo, Hirofumi Sawa, Kazuo Nagashima, Shinya Tanaka
    NEUROPATHOLOGY 31 (1) 38 - 41 0919-6544 2011/02 [Refereed][Not invited]
     
    Mutations of the methyl CpG binding protein 2 (MeCP2) gene are a major cause of Rett syndrome. To investigate whether the expression of this gene was related to JC virus (JCV) infection, we examined brains of four progressive multifocal leukoencephalopathy (PML) patients. JCV infection was confirmed by immunohistochemical labeling with antibodies against JCV VP1, agnoprotein and large T antigen. MeCP2 expression was examined by immunohistochemistry using a specific polyclonal antibody against MeCP2. In normal brains and uninfected cortices of PML brains, MeCP2 expression was observed in the nuclei of neurons, but not observed in glial and endothelial cell nuclei. However, in PML brains intense immunolabeling was observed in abnormally enlarged glial nuclei of JCV-infected cells. Double immunolabeling using antibodies against large T antigen (visualized as blue) and MeCP2 (visualised as red) revealed dark red JCV-infected nuclei, which confirmed that the JCV infected nuclei expressed MeCP2. We conclude that MeCP2 is highly expressed in the JCV-infected nuclei of PML brain and these results may provide a new insight into the mechanism which regulates the MeCP2 expression in glial cells by the infection of JCV.
  • 臨床神経学 51 (11) 1051 - 1057 2011 [Not refereed][Not invited]
  • T. Kimura, M. Sasaki, M. Okumura, E. Kim, H. Sawa
    VETERINARY PATHOLOGY 47 (5) 806 - 818 0300-9858 2010/09 [Not refereed][Not invited]
     
    Encephalitic flaviviruses are important arthropod-borne pathogens of humans and other animals. In particular, the recent emergence of the West Nile virus (WNV) and Japanese encephalitis virus (JEV) in new geographic areas has caused a considerable public health alert and international concern. Among the experimental in vivo models of WNV and JEV infection, mice and other laboratory rodents are the most thoroughly studied and well-characterized systems, having provided data that are important for understanding the infectious process in humans. Macaca monkeys have also been used as a model for WNV and JEV infection, mainly for the evaluation of vaccine efficacy, although a limited number of published studies have addressed pathomorphology. These animal models demonstrate the development of encephalitis with many similarities to the human disease; however, the histological events that occur during infection, especially in peripheral tissues, have not been fully characterized.
  • Akira Kawaguchi, Tadaki Suzuki, Takashi Kimura, Naoki Sakai, Tokiyoshi Ayabe, Hirofumi Sawa, Hideki Hasegawa
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 398 (4) 778 - 784 0006-291X 2010/08 [Refereed][Not invited]
     
    Gene-encoded antimicrobial peptides (AMPS) are an essential component of the innate immune system in many species. Analysis of beta-defensin gene expression in mouse tissue using primers that were specific for conserved sequences located outside of the beta-defensin translated region identified a novel small gene. The novel gene had an open reading frame of 114 bp and encoded a predicted protein of 37 amino acid residues. A search of the genome database revealed that the gene locus and the sequence of exon 1 of this novel gene were similar to subgroup 1 mouse beta-defensins. A small peptide, K17 (FSPQMLQDIIEKKTKIL), derived from the amino acid sequence of this novel gene was synthesized. Circular dichroism (CD) spectroscopic analysis of chemically synthesized peptide demonstrated that the peptide exhibited random coil conformation in aqueous solution, but the peptide adopted helical conformation in the presence of trifluoroethanol or sodium dodecyl sulfate, a membrane-mimicking environment. The peptide exhibited bactericidal activity against Salmonella enterica serovar Typhimurium (Gram negative) and Staphylococcus aureus (Gram positive); it was not cytotoxic in cultures of mammalian cells or hemolytic in cultures of erythrocytes. These results suggested that K17 may be a candidate therapeutic for the treatment of bacterial infection. (C) 2010 Elsevier Inc. All rights reserved.
  • Akira Kawaguchi, Tadaki Suzuki, Takashi Kimura, Naoki Sakai, Tokiyoshi Ayabe, Hirofumi Sawa, Hideki Hasegawa
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 398 (4) 778 - 784 0006-291X 2010/08 [Not refereed][Not invited]
     
    Gene-encoded antimicrobial peptides (AMPS) are an essential component of the innate immune system in many species. Analysis of beta-defensin gene expression in mouse tissue using primers that were specific for conserved sequences located outside of the beta-defensin translated region identified a novel small gene. The novel gene had an open reading frame of 114 bp and encoded a predicted protein of 37 amino acid residues. A search of the genome database revealed that the gene locus and the sequence of exon 1 of this novel gene were similar to subgroup 1 mouse beta-defensins. A small peptide, K17 (FSPQMLQDIIEKKTKIL), derived from the amino acid sequence of this novel gene was synthesized. Circular dichroism (CD) spectroscopic analysis of chemically synthesized peptide demonstrated that the peptide exhibited random coil conformation in aqueous solution, but the peptide adopted helical conformation in the presence of trifluoroethanol or sodium dodecyl sulfate, a membrane-mimicking environment. The peptide exhibited bactericidal activity against Salmonella enterica serovar Typhimurium (Gram negative) and Staphylococcus aureus (Gram positive); it was not cytotoxic in cultures of mammalian cells or hemolytic in cultures of erythrocytes. These results suggested that K17 may be a candidate therapeutic for the treatment of bacterial infection. (C) 2010 Elsevier Inc. All rights reserved.
  • Tadaki Suzuki, Satoko Yamanouchi, Yuji Sunden, Yasuko Orba, Takashi Kimura, Hirofumi Sawa
    JOURNAL OF MEDICAL VIROLOGY 82 (7) 1229 - 1235 0146-6615 2010/07 [Refereed][Not invited]
     
    The human polyomavirus JC virus (JCV) infects 70-80% of humans and establishes latent infection in the kidney. In immunosuppressed patients, JCV reactivates and causes a fatal and progressive neurological disease known as progressive multifocal leukoencephalopathy (PML). Over the past three decades, PML has become an important neurological complication in acquired immunodeficiency syndrome (AIDS) patients. Recently, it was reported that patients treated with therapeutics that target the integrin receptor very late antigen (VLA)-4 are at increased risk of developing PML. However, the relationship between Natalizumab and this unexpected onset of PML has yet to be elucidated. Here, we investigated the effect of Natalizumab on the growth of JCV in the permissive human neural cell line IMR-32 following viral inoculation. Natalizumab had no effect either on the expression levels of viral proteins as determined by immunoblot analysis using specific antibodies or on the hemagglutination activity of cellular lysates from infected cells. These results suggest that there is no direct effect of Natalizumab on JCV infectivity in IMR-32 cells in vitro. J. Med. Virol. 82:1229-1235, 2010. (C) 2010 Wiley-Liss, Inc.
  • Rie Hasebe, Tadaki Suzuki, Yoshinori Makino, Manabu Igarashi, Satoko Yamanouchi, Akihiko Maeda, Motohiro Horiuchi, Hirofumi Sawa, Takashi Kimura
    BMC MICROBIOLOGY 10 165  1471-2180 2010/06 [Refereed][Not invited]
     
    Background: West Nile virus (WNV) causes viremia after invasion to the hosts by mosquito bite. Endothelial cells could play an important role in WNV spread from the blood stream into the central nervous system and peripheral tissues. Here, we analyzed the capacity of virus-like particles (VLPs) of the highly virulent NY99 6-LP strain (6-LP VLPs) and the low virulence Eg101 strain (Eg VLPs) to cross cultured human endothelial cells. Results: 6-LP VLPs were transported from the apical to basolateral side of endothelial cells, whereas Eg VLPs were hardly transported. The localization of tight junction marker ZO-1 and the integrity of tight junctions were not impaired during the transport of 6-LP VLPs. The transport of 6-LP VLPs was inhibited by treatment with filipin, which prevents the formation of cholesterol-dependent membrane rafts, suggesting the involvement of raft-associated membrane transport. To determine the amino acid residues responsible for the transport of VLPs, we produced mutant VLPs, in which residues of E protein were exchanged between the 6-LP and Eg strains. Double amino acid substitution of the residues 156 and 159 greatly impaired the transport of VLPs. Conclusion: Our results suggest that a transcellular pathway is associated with 6-LP VLPs transport. We also showed that the combination of the residues 156 and 159 plays an important role in the transport of VLPs across endothelial cells.
  • Karen M. Watters, Jonathan Dean, Hideki Hasegawa, Hirofumi Sawa, William Hall, Noreen Sheehy
    AIDS RESEARCH AND HUMAN RETROVIRUSES 26 (5) 593 - 603 0889-2229 2010/05 [Refereed][Not invited]
     
    The Tax protein encoded by HTLV-1 plays a key role in the development of ATL in infected individuals. Our previous studies showed that tax transgenic mice develop disease that is almost identical to human ATL, with widespread organ invasion by lymphomatous cells and the development of leukemia. The same pathology develops rapidly in SCID mice engrafted with cells from the transgenic animals. In the present study, we used this SCID model to analyze the expression levels of several cytokines, growth factors, and adhesion molecules to determine their possible involvement in the development of disease. We showed that Tax expression was undetectable at the protein level in the tax-transformed cells used to inoculate the SCID mice and that these cells displayed constitutive NF-kappa B and Akt activity. We demonstrated significant differences in the levels of circulating PDGF-BB, TNF-alpha, sICAM-1, and sVCAM-1 in inoculated animals. Cell-surface staining of the tax transgenic cells showed that they do not express receptors for any of the upregulated growth factors. Significant differences were not found in the secreted levels of bFGF, MMP9, VEGF, or E-selectin, whereas IL-2, IL-15, IL-6, IL-1 beta, and IFN-gamma expression was undetectable. Even though the number of factors analyzed is limited, our study identified TNF-alpha, PDGF-BB, and the adhesion molecules sICAM-1 and sVCAM-1 as factors that may contribute to the high levels of organ infiltration by leukemic cells in this tax transgenic SCID model.
  • Noriko Ohtake, Kenichi Niikura, Tadaki Suzuki, Keita Nagakawa, Shintaro Mikuni, Yasutaka Matsuo, Masataka Kinjo, Hirofumi Sawa, Kuniharu Ijiro
    CHEMBIOCHEM 11 (7) 959 - 962 1439-4227 2010/05 [Refereed][Not invited]
  • Tadaki Suzuki, Yasuko Orba, Yuki Okada, Yuji Sunden, Takashi Kimura, Shinya Tanaka, Kazuo Nagashima, William W. Hall, Hirofumi Sawa
    PLOS PATHOGENS 6 (3) e1000801  1553-7366 2010/03 [Refereed][Not invited]
     
    Virus infections can result in a range of cellular injuries and commonly this involves both the plasma and intracellular membranes, resulting in enhanced permeability. Viroporins are a group of proteins that interact with plasma membranes modifying permeability and can promote the release of viral particles. While these proteins are not essential for virus replication, their activity certainly promotes virus growth. Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease resulting from lytic infection of oligodendrocytes by the polyomavirus JC virus (JCV). The genome of JCV encodes six major proteins including a small auxiliary protein known as agnoprotein. Studies on other polyomavirus agnoproteins have suggested that the protein may contribute to viral propagation at various stages in the replication cycle, including transcription, translation, processing of late viral proteins, assembly of virions, and viral propagation. Previous studies from our and other laboratories have indicated that JCV agnoprotein plays an important, although as yet incompletely understood role in the propagation of JCV. Here, we demonstrate that agnoprotein possesses properties commonly associated with viroporins. Our findings demonstrate that: (i) A deletion mutant of agnoprotein is defective in virion release and viral propagation; (ii) Agnoprotein localizes to the ER early in infection, but is also found at the plasma membrane late in infection; (iii) Agnoprotein is an integral membrane protein and forms homo-oligomers; (iv) Agnoprotein enhances permeability of cells to the translation inhibitor hygromycin B; (v) Agnoprotein induces the influx of extracellular Ca(2+); (vi) The basic residues at amino acid positions 8 and 9 of agnoprotein key are determinants of the viroporin activity. The viroporin-like properties of agnoprotein result in increased membrane permeability and alterations in intracellular Ca(2+) homeostasis leading to membrane dysfunction and enhancement of virus release.
  • Takeshi Ichinohe, Akira Ainai, Tomoyuki Nakamura, Yukihito Akiyama, Jun-ichi Maeyama, Takato Odagiri, Masato Tashiro, Hidehiro Takahashi, Hirofumi Sawa, Shin-ichi Tamura, Joe Chiba, Takeshi Kurata, Tetsutaro Sata, Hideki Hasegawa
    JOURNAL OF MEDICAL VIROLOGY 82 (1) 128 - 137 0146-6615 2010/01 [Refereed][Not invited]
     
    The identification of a safe and effective adjuvant that is able to enhance mucosal immune responses is necessary for the development of an efficient inactivated intranasal influenza vaccine, The present study demonstrated the effectiveness of extracts of mycelia derived from edible mushrooms as adjuvants for intranasal influenza vaccine. The adjuvant effect of extracts of mycelia was examined by intranasal co-administration of the extracts and inactivated A/PR8 (H1N1) influenza virus hemagglutinin (HA) vaccine in BALB/c mice. The inactivated vaccine in combination with mycelial extracts induced a high anti-A/PR8 HA-specific IgA and IgG response in nasal washings and serum, respectively. Virus-specific cytotoxic T-lymphocyte responses were also induced by administration of the vaccine with extract of mycelia, resulting in protection against lethal lung infection with influenza virus A/PR8. In addition, intranasal administration of NIBRG14 vaccine derived from the influenza A/Vietnam/1194/2004 (H5N1) virus strain administered in conjunction with mycelial extracts from Phellinus linteus conferred cross-protection against heterologous influenza A/Indonesia/6/2005 virus challenge in the nasal infection model. In addition, mycelial extracts induced proinflammatory cytokines and CD40 expression in bone marrow-derived dendritic cells. These results suggest that mycelial extract-adjuvanted vaccines can confer cross-protection against variant H5N1 influenza viruses. The use of extracts of mycelia derived from edible mushrooms is proposed as a new safe and effective mucosal adjuvant for use for nasal vaccination against influenza virus infection. J. Med. Virol. 82:128-137, 2010. (C) 2009 Wiley-Liss, Inc.
  • Yasuko Orba, Tadaki Suzuki, Yoshinori Makino, Kanako Kubota, Shinya Tanaka, Takashi Kimura, Hirofumi Sawa
    JOURNAL OF BIOLOGICAL CHEMISTRY 285 (2) 1544 - 1554 0021-9258 2010/01 [Refereed][Not invited]
     
    Large T antigen (TAg) of the human polyomavirus JC virus (JCV) possesses DNA binding and helicase activities, which, together with various cellular proteins, are required for replication of the viral genome. We now show that JCV-infected cells expressing TAg accumulate in the G(2) phase of the cell cycle as a result of the activation of ATM- and ATR-mediated G(2) checkpoint pathways. Transient transfection of cells with a TAg expression vector also induced G(2) checkpoint signaling and G(2) arrest. Analysis of TAg mutants with different subnuclear localizations suggested that the association of TAg with cellular DNA contributes to the induction of G(2) arrest. Abrogation of G(2) arrest by inhibition of ATM and ATR, Chk1, and Wee1 suppressed JCV genome replication. In addition, abrogation of the G(2)-M transition by Cdc2 depletion disabled Wee1 depletion induced suppression of JCV genome replication, suggesting that JCV replication is facilitated by G(2) arrest resulting from G(2) checkpoint signaling. Moreover, inhibition of ATM and ATR by caffeine suppressed JCV production. The observation that oligodendrocytes productively infected with JCV in vivo also undergo G(2) arrest suggests that G(2) checkpoint inhibitors such as caffeine are potential therapeutic agents for JCV infection.
  • Hidehiro Takahashi, Naohiro Ohtaki, Masae Maeda-Sato, Michiko Tanaka, Keiko Tanaka, Hirofumi Sawa, Toyokazu Ishikawa, Akihisa Takamizawa, Tomohiko Takasaki, Hideki Hasegawa, Tetsutaro Sata, William W. Hall, Takeshi Kurata, Asato Kojima
    MICROBES AND INFECTION 11 (13) 1019 - 1028 1286-4579 2009/11 [Refereed][Not invited]
     
    Expression of genes for precursor M (prM) and envelope (E) proteins of West Nile virus (WNV) leads to the production of small. capsidless, and non-infectious virus-like particles (VLPs) possessing the E antigen which is responsible for viral entry and immune protection It has been reported that processing of the secretion signal affects viral release We examined the secretion efficiency of VLPs into the culture medium front RK13 or 293 T cells transfected with expression vectors for prM and E proteins of WNV which were constructed to comprise different lengths of signal peptides upstream of the prM-E domain The number of amino acid residues present in the segment markedly affected the production, processing, and secretion of VLPs Secreted VLPs possessed both the processed M protein and the glycosylated E protein In addition, immunization with VLPs induced neutralizing, antibodies in C3H/HeN mice. These results indicate that the number of amino acid residues comprising the N-terminus of the signal segment controls the efficiency of assembly, maturation, and release of VLPs; in the absence of viral protease, which in turn indicates the potential of VLPs as a candidate for an effective WNV subunit vaccine. (C) 2009 Elsevier Masson SAS. All rights reserved
  • Yoshinori Kitagawa, Masae Maeda-Sato, Keiko Tanaka, Minoru Tobiume, Hirofumi Sawa, Hideki Hasegawa, Asato Kojima, William W. Hall, Takeshi Kurata, Tetsutaro Sata, Hidehiro Takahashi
    MICROBIOLOGY AND IMMUNOLOGY 53 (11) 609 - 620 0385-5600 2009/11 [Refereed][Not invited]
     
    The oligomerization of HIV-1 Gag and Gag-Pol proteins, which are assembled at the plasma membrane, leads to viral budding. The budding generally places the viral components under non-reducing conditions. Here the effects of non-reducing conditions on Gag structures and viral RNA protection were examined. Using different reducing conditions and SDS-PAGE, it was shown that oligomerized Gag possesses intermolecular covalent bonds under non-reducing conditions. In addition, it was demonstrated that the mature viral core contains a large amount of covalent bonded Gag multimers, as does the immature core. Viral genomic RNA becomes sensitive to ribonuclease in reducing conditions. These results suggest that, under non-reducing conditions, covalent bonded Gag multimers are formed within the viral particles and play a role in protection of the viral genome.
  • Akira Kawaguchi, Yasuko Orba, Takashi Kimura, Hidekatsu Iha, Masao Ogata, Takahiro Tsuji, Akira Ainai, Tetsutaro Sata, Takashi Okamoto, William W. Hall, Hirofumi Sawa, Hideki Hasegawa
    BLOOD 114 (14) 2961 - 2968 0006-4971 2009/10 [Refereed][Not invited]
     
    Adult T-cell leukemia (ATL) is a T-cell malignancy caused by human T lymphotropic virus type I, and presents as an aggressive leukemia with characteristic widespread leukemic cell infiltration into visceral organs and skin. The molecular mechanisms associated with leukemic cell infiltration are poorly understood. We have used mouse models of ATL to investigate the role of chemokines in this process. Transfer of splenic lymphomatous cells from transgenic to SCID mice reproduces a leukemia and lymphoma that is histologically identical to human disease. It could be shown that lymphomatous cells exhibit specific chemotactic activity in response to stromal cell-derived factor-1 alpha (SDF-1 alpha). Lymphomatous cells exhibited surface expression of CXCR4, the specific receptor of SDF-1 alpha. AMD3100, a CXCR4 antagonist, was found to inhibit both SDF-1 alpha-induced migration and phosphorylation of extracellular signal-related kinase 1/2. Investigation of cultured cells from human ATL patients revealed identical findings. Using the SCID mouse model, it could be demonstrated that AMD3100 inhibited infiltration of lymphomatous cells into liver and lung tissues in vivo. These results demonstrate the involvement of the SDF-1 alpha/CXCR4 interaction as one mechanism of leukemic cell migration and this may provide a novel target as part of combination therapy for ATL. (Blood. 2009; 114: 2961-2968)
  • Rie Hasebe, Michihito Sasaki, Hirofumi Sawa, Ryuichi Wada, Takashi Umemura, Takashi Kimura
    VIROLOGY 393 (2) 198 - 209 0042-6822 2009/10 [Refereed][Not invited]
     
    We investigated the mechanism by which equine herpesvirus-1 (EHV-1) enters primary Cultured equine brain microvascular endothelial cells (EBMECs) and equine dermis (E. Derm) cells. EHV-1 colocalized with caveolin in EBMECs and the infection was greatly reduced by the expression of a dominant negative form of equine caveolin-1 (ecavY14F), suggesting that EHV-1 enters EBMECs via caveolar endocytosis. EHV-1 entry into E. Derm cells was significantly reduced by ATP depletion and treatments with lysosomotropic agents. Enveloped virions were detected from E. Derm cells by infectious virus recovery assay after vital internalization, suggesting that EHV-1 enters E. Derm cells via energy- and pH-dependent endocytosis. These results suggest that EHV-1 utilizes multiple endocytic pathways in different cell types to establish productive infection. (C) 2009 Elsevier Inc. All rights reserved.
  • Niikura K, Nagakawa K, Ohtake N, Suzuki T, Matsuo Y, Sawa H, Ijiro K
    Bioconjugate chemistry 82 (1) 128 - 137 1043-1802 2009/10 [Refereed][Not invited]
  • Edgar Simulundu, Aaron S. Mweene, Daisuke Tomabechi, Bernard M. Hang'ombe, Akihiro Ishii, Yuka Suzuki, Ichiro Nakamura, Hirofumi Sawa, Chihiro Sugimoto, Kimihito Ito, Hiroshi Kida, Lewis Saiwana, Ayato Takada
    ARCHIVES OF VIROLOGY 154 (9) 1517 - 1522 0304-8608 2009/09 [Refereed][Not invited]
     
    We characterized an influenza virus isolated from a great white pelican in Zambia. Phylogenetic analysis showed that all of its gene segments belonged to the Eurasian lineage and that they appear to have evolved in distinct geographical regions in Europe, Asia, and Africa, suggesting reassortment of virus genes maintained in wild aquatic birds whose flyways overlap across these continents. It is notable that this virus might possess some genes of the same origin as those of highly pathogenic H7 and H5 viruses isolated in Eurasia. The present study underscores the need for continued monitoring of avian influenza viruses in Eurasia and Africa.
  • Takuya Watanabe, Masumi Tsuda, Shinya Tanaka, Yusuke Ohba, Hideaki Kawaguchi, Tokifumi Majima, Hirofumi Sawa, Akio Minami
    MOLECULAR CANCER RESEARCH 7 (9) 1582 - 1592 1541-7786 2009/09 [Refereed][Not invited]
     
    The adaptor protein Crk mediates intracellular signaling related to cell motility and proliferation and is implicated in human tumorigenesis. The role of Crk in the growth of human sarcoma has remained unclear, however. The present study shows that Crk-induced activation of Src and subsequent signaling by p38 mitogen-activated protein kinase (MAPK) contribute to the enhanced proliferation of human synovial sarcoma cells. Depletion of Crk by RNA interference markedly inhibited proliferation of the synovial sarcoma cell lines HS-SYII, SYO-1, and Fuji as well as prevented anchorage-independent growth. Conversely, reconstitution with CrkII by authentic small interfering RNA-resistant Crk gene restored proliferation in Crk-silenced SYO-1 cells. Crk-depleted synovial sarcoma cells manifested enhanced transcriptional activity and expression of the p16(INK4A) gene, resulting in their accumulation in G, phase of the cell cycle. In response to hepatocyte growth factor stimulation, Crk prominently induced the tyrosine phosphorylation of Grb2-associated binder 1 through activation of Src and focal adhesion kinase, and the Src family kinase inhibitor PP2 almost completely inhibited the proliferation of SYO-1 cells. Crk also induced the phosphorylation of p38 MAPK, and SB203580, a p38 MAPK-specific inhibitor, increased expression of p16(INK4A) gene in SYO-1 cells. Furthermore, SB203580 or depletion of p38 MAPK by small interfering RNA suppressed both the phosphorylation of Akt triggered by hepatocyte growth factor and the proliferation of SYO-1 cells. These results suggest that Crk promotes proliferation of human synovial sarcoma cells through activation of Src and its downstream signaling by a novel p38 MAPK-Akt pathway, with these signaling molecules providing potent new targets for molecular therapeutics. (Mol Cancer Res 2009;7(9):1582-92)
  • Taichi Kimura, Mieko Sakai, Kouichi Tabu, Lei Wang, Ryosuke Tsunematsu, Masumi Tsuda, Hirofumi Sawa, Kazuo Nagashima, Hiroshi Nishihara, Shigetsugu Hatakeyama, Keiko Nakayama, Marc Ladanyi, Shinya Tanaka, Keiichi I. Nakayama
    LABORATORY INVESTIGATION 89 (6) 645 - 656 0023-6837 2009/06 [Refereed][Not invited]
     
    SYT-SSX protein, resulted from chromosomal translocation, causes synovial sarcoma, which is a malignant tumor accounting for 10% of soft tissue sarcoma. However, biological functions of SYT (synovial sarcoma translocation), also known as SS18, are largely unclear, whereas it has been proven that Syt-null mice die at early stages of embryonic development. Here, we generated Syt-deficient mice and confirmed the reported phenotypes, including growth retardation, open neural tube and haplo-insufficient lethality, and therefore, there is no doubt that Syt is essential for embryonic development. However, placental defects, described in the earlier report, were rarely seen in our mice and we frequently observed cardiac defect in Syt-deficient mice. As the mechanisms responsible for embryonic lethality seem to be complicate, we performed additional experiments. By using primary cultured embryonic fibroblasts, we showed that Syt(-/-) MEFs deregulate actin organization and suppressed cell migration. These observations suggest that Syt may contribute to the signaling pathway important for various cellular functions in vivo and in vitro, and we propose that Syt-deficient MEFs would be a powerful means to understand the biological roles of SYT in vitro. Laboratory Investigation (2009) 89, 645-656; doi:10.1038/labinvest.2009.25; published online 30 March 2009
  • Hidehiro Takahashi, Yoshinori Kitagawa, Masae Maeda-Satoh, Hideki Hasegawa, Hirofumi Sawa, Tetsutaro Sata
    HYBRIDOMA 28 (1) 63 - 65 1554-0014 2009/02 [Refereed][Not invited]
     
    Telomerase, a ribonucleoprotein enzyme, is a cellular RNA-dependent DNA polymerase that serves to maintain the tandem arrays of telomeric repeats at the eukaryotic chromosomal ends. We previously reported that topoisomerase I dissociates HIV-1 reverse transcriptase from genomic RNAs, and binding of topoisomerase I to RNA template regulates cDNA synthesis. We also found that a monoclonal antibody (MAb) against topoisomerase I, designated as MAb 1, suppresses the reverse transcription efficiency using a detergent-disrupted HIV-1 virion. In this study, we describe how MAb 1 suppresses telomerase activity in cellular lysates. In addition, siRNAs of topoisomerase I has attenuated telomerase activity in culture cells. These results suggest that topoisomerase I is involved in telomerase activity, as well as HIV-1 reverse transcription.
  • Hidehiro Takahashi, Yoshinori Kitagawa, Masae Maeda-Satoh, Hideki Hasegawa, Hirofumi Sawa, Tetsutaro Sata
    HYBRIDOMA 28 (1) 63 - 65 1554-0014 2009/02 [Not refereed][Not invited]
     
    Telomerase, a ribonucleoprotein enzyme, is a cellular RNA-dependent DNA polymerase that serves to maintain the tandem arrays of telomeric repeats at the eukaryotic chromosomal ends. We previously reported that topoisomerase I dissociates HIV-1 reverse transcriptase from genomic RNAs, and binding of topoisomerase I to RNA template regulates cDNA synthesis. We also found that a monoclonal antibody (MAb) against topoisomerase I, designated as MAb 1, suppresses the reverse transcription efficiency using a detergent-disrupted HIV-1 virion. In this study, we describe how MAb 1 suppresses telomerase activity in cellular lysates. In addition, siRNAs of topoisomerase I has attenuated telomerase activity in culture cells. These results suggest that topoisomerase I is involved in telomerase activity, as well as HIV-1 reverse transcription.
  • Tomoko Matoba, Yasuko Orba, Tadaki Suzuki, Yoshinori Makino, Hideo Shichinohe, Satoshi Kuroda, Takahiro Ochiya, Hiroshi Itoh, Shinya Tanaka, Kazuo Nagashima, Hirofumi Sawa
    NEUROPATHOLOGY 28 (3) 286 - 294 0919-6544 2008/06 [Refereed][Not invited]
     
    JC virus (JCV) is the etiological agent of the demyelinating disease progressive multifocal leukoencephalopathy (PML). Because JCV has a very narrow host range, it has been difficult to develop an animal model of JCV infection; as a result, no effective therapy for PML has been established. In this study, we have tried to create an animal model that replaces an in vivo JCV infection. As a result, we have obtained a stable persistence of JCV-infected human cells in the mouse brain by inoculating the virus-infected cells into the nude mice brains. In this model, the JCV-infected cells were well preserved in the nude mouse brains for 2 weeks. We then treated JCV-injected brains with an siRNA against the JCV agnoprotein that is known to be an effective inhibitor of JCV infection in vitro. A highly purified type I collagen, atelocollagen, was used as a carrier for the siRNA. The siRNA inhibited the expression of JCV protein in inoculated JCV-infected cells in the mouse brain, compared to the medium containing only atelocollagen used as a placebo. Thus, the combination of siRNA and atelocollagen might be a candidate therapeutic agent for the treatment of JCV infection.
  • Misato Hirano, Randeep Rakwal, Junko Shibato, Hirofumi Sawa, Kazuo Nagashima, Yoko Ogawa, Yasukazu Yoshida, Hitoshi Wahashi, Etsuo Niki, Yoshinori Masuo
    JOURNAL OF PROTEOME RESEARCH 7 (6) 2471 - 2489 1535-3893 2008/06 [Refereed][Not invited]
     
    Two global omics approaches were applied to develop an inventory of differentially expressed proteins and genes in Wig rat, a promising animal model of attention-deficit hyperactivity disorder (ADHD). The frontal cortex, striatum, and midbrain of Wig rat at 4 weeks of age were dissected for proteomics and transcriptomics analyses. Two-dimensional gel electrophoresis detected 13, 1, and 16 differentially expressed silver nitrate-stained spots in the frontal cortex, striatum, and midbrain, respectively. Peptide mass fingerprinting/tandem mass spectrometry identified 19 nonredundant proteins, belonging to 7 functional categories, namely, signal transduction, energy metabolism, cellular transport, protein with binding function, protein synthesis, cytoskeleton, and cell rescue. Interestingly, 10 proteins that were indentified in the present study were also previously reported in studies involving neurodegenerative diseases and psychiatric disorders, such as Alzheimer's disease (AD), Parkinson's disease, and Schizophrenia. Moreover, some of the proteins identified in the midbrain were involved in synaptic vesicular transport, suggesting abnormality in neurotransmitter release in this region. On the other hand, transcriptomics analysis of combined frontal cortex, striatum, and midbrain by rat whole genome 44K DNA oligo microarray revealed highly up-regulated (28) and down-regulated (33) genes. Functional categorization of these genes showed cellular transport, metabolism, protein fate, signal transduction, and transcription as the major categories, with 26% genes of unknown function. Some of the identified genes were related to AD, fragile X syndrome, and ADHD. This is a first comprehensive study providing insight into molecular components in Wig rat brain, and will help to elucidate the roles of identified proteins and genes in Wig rat brain, hopefully leading to uncovering the pathogenesis of ADHD.
  • Yuichi Hayashi, Akio Kimura, Shimon Kato, Akihiro Koumura, Takeo Sakurai, Yuji Tanaka, Isao Hozumi, Yuji Sunden, Yasuko Orba, Hirofumi Sawa, Hitoshi Takahashi, Takashi Inuzuka
    JOURNAL OF THE NEUROLOGICAL SCIENCES 1-2 268 (1-2) 195 - 198 0022-510X 2008/05 [Refereed][Not invited]
     
    We report progressive multifocal leukoencephalopathy (PML) and CD4+ T-lymphocytopenia in a 71-year-old man with Sjogren syndrome (SjS). The patient was admitted to our hospital because of progressive dementia and gait disturbance. T2-weighted MR images showed high-intensity lesions in his left frontal white matter thalamus, cerebellum and brainstem. A pathological diagnosis of PML was made by brain biopsy. SjS is frequently accompanied with immunological complications; however, there are few reports on PML in patients with SjS. Recently, isolated CD4+ T-lymphocytopenia is reported to be one of the based immunological conditions associated with the development of PML. In the present case, CD4+ T-lymphocytopenia was also observed on admission, which is also associated with SjS. (C) 2007 Elsevier B.V. All rights reserved.
  • Jun Nakae, Yongheng Cao, Miyo Oki, Yasuko Orba, Hirofumi Sawa, Hiroshi Kiyonari, Kristy Iskandar, Koji Suga, Marc Lombes, Yoshitake Hayashi
    DIABETES 57 (3) 563 - 576 0012-1797 2008/03 [Refereed][Not invited]
     
    OBJECTIVE-Adipose tissue serves as an integrator of various physiological pathways, energy balance, and glucose homeostasis. Forkhead box-containing protein O subfamily (FoxO) 1 mediates insulin action at the transcriptional level. However, physiological roles of FoxO1 in adipose tissue remain unclear. RESEARCH DESIGN AND METHODS-In the present study, we generated adipose tissue-specific FoxO1. transgenic mice (adipocyte protein 2 [aP(2)]-FLAG-Delta 256) using an aP(2) promoter/enhancer and a mutant FoxO1 (FLAG Delta 256) in which the carboxyl terminal transactivation domain was deleted. Using these mice, we analyzed the effects of the overexpression of FLAG Delta 256 on glucose metabolism and energy homeostasis. RESULTS-The aP(2)-FLAG-Delta 256 mice showed improved glucose tolerance and insulin sensitivity accompanied with smaller-sized adipocytes and increased adiponectin (adipoq) and Glut 4 (Slc2a4) and decreased tumor necrosis factor alpha (Tnf) and chemokine (C-C motif) receptor 2 (Ccr2) gene expression levels in white adipose tissue (WAT) under a high-fat diet. Furthermore, the aP(2)-FLAG-Delta 256 mice had increased oxygen consumption accompanied with increased expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1 alpha protein and uncoupling protein (UCP)-1 (Ucp1), UCP-2 (Ucp2), and beta 3-AR (Adrb3) in brown adipose tissue (BAT). Overexpression of FLAG Delta 256 in T37i cells, which are derived from the hibernoma of SV40 large T antigen transgenic mice, increased expression of PGC-1 alpha protein and Ucp1. Furthermore, knockdown of endogenous FoxO1 in T37i cells increased Pgc1 alpha (Ppargc1a), Pgc1 beta (Ppargc1b), Ucp1, and Adrb3 gene expression. CONCLUSIONS-These data suggest that FoxO1 modulates energy homeostasis in WAT and BAT through regulation of adipocyte size and adipose tissue-specific gene expression in response to excessive calorie intake.
  • Jun Nakae, Yongheng Cao, Miyo Oki, Yasuko Orba, Hirofumi Sawa, Hiroshi Kiyonari, Kristy Iskandar, Koji Suga, Marc Lombes, Yoshitake Hayashi
    DIABETES 57 (3) 563 - 576 0012-1797 2008/03 [Not refereed][Not invited]
     
    OBJECTIVE-Adipose tissue serves as an integrator of various physiological pathways, energy balance, and glucose homeostasis. Forkhead box-containing protein O subfamily (FoxO) 1 mediates insulin action at the transcriptional level. However, physiological roles of FoxO1 in adipose tissue remain unclear. RESEARCH DESIGN AND METHODS-In the present study, we generated adipose tissue-specific FoxO1. transgenic mice (adipocyte protein 2 [aP(2)]-FLAG-Delta 256) using an aP(2) promoter/enhancer and a mutant FoxO1 (FLAG Delta 256) in which the carboxyl terminal transactivation domain was deleted. Using these mice, we analyzed the effects of the overexpression of FLAG Delta 256 on glucose metabolism and energy homeostasis. RESULTS-The aP(2)-FLAG-Delta 256 mice showed improved glucose tolerance and insulin sensitivity accompanied with smaller-sized adipocytes and increased adiponectin (adipoq) and Glut 4 (Slc2a4) and decreased tumor necrosis factor alpha (Tnf) and chemokine (C-C motif) receptor 2 (Ccr2) gene expression levels in white adipose tissue (WAT) under a high-fat diet. Furthermore, the aP(2)-FLAG-Delta 256 mice had increased oxygen consumption accompanied with increased expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1 alpha protein and uncoupling protein (UCP)-1 (Ucp1), UCP-2 (Ucp2), and beta 3-AR (Adrb3) in brown adipose tissue (BAT). Overexpression of FLAG Delta 256 in T37i cells, which are derived from the hibernoma of SV40 large T antigen transgenic mice, increased expression of PGC-1 alpha protein and Ucp1. Furthermore, knockdown of endogenous FoxO1 in T37i cells increased Pgc1 alpha (Ppargc1a), Pgc1 beta (Ppargc1b), Ucp1, and Adrb3 gene expression. CONCLUSIONS-These data suggest that FoxO1 modulates energy homeostasis in WAT and BAT through regulation of adipocyte size and adipose tissue-specific gene expression in response to excessive calorie intake.
  • Noriko Ohtake, Kenichi Niikura, Tadaki Suzuki, Keita Nagakawa, Hirofumi Sawa, Kuniharu Ijiro
    BIOCONJUGATE CHEMISTRY 19 (2) 507 - 515 1043-1802 2008/02 [Refereed][Not invited]
     
    Herein, we present the efficient cellular uptake of immobilized virus-like particles (VLPs) made of recombinant JC virus capsid proteins. VLPs expressed in Escherichia coli were labeled with fluorescein isothiocyanate (FITC). We compared two approaches for the cellular uptake of the FITC-VLPs. In the first approach, FITC-VLPs were immobilized on a polystyrene substrate, and then NIH3T3 cells were cultured on the same substrate. In the second approach, cells were cultured on a polystyrene substrate, and FITC-VLPs were then added to the cell culture medium. Flow cytometric analysis and confocal laser microscopic observation revealed that immobilized VLPs were incorporated into the cells with higher efficiency than were the diffusive VLPs suspended in solution. The cellular uptake of VLPs on the substrate was increased in a VLP density-dependent manner. As a control, disassembling VLPs to form VP1 pentamers abolished incorporation into the cells. Displaying sialic acid on the substrate enhanced VLP density through the specific affinities between the VLPs and sialic acid, resulting in efficient incorporation into the seeded cells. These techniques can be applied to the development of novel drug delivery systems and cell microarrays not only of nucleic acids but also of small molecules and proteins through their encapsulation in VLPs.
  • Naomi Ohnishi, Hitomi Yuasa, Shinya Tanaka, Hirofumi Sawa, Motohiro Miura, Atsushi Matsui, Hideaki Higashi, Manabu Musashi, Kazuya Lwabuchi, Misao Suzuki, Gen Yamada, Takeshi Azuma, Masanori Hatakeyama
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 (3) 1003 - 1008 0027-8424 2008/01 [Refereed][Not invited]
     
    Infection with cagA-positive Helicobacter pylori is associated with gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma of B cell origin. The cagA-encoded CagA protein is delivered into gastric epithelial cells via the bacterial type IV secretion system and, upon tyrosine phosphorylation by Src family kinases, specifically binds to and aberrantly activates SHIP-2 tyrosine phosphatase, a bona fide oncoprotein in human malignancies. CagA also elicits junctional and polarity defects in epithelial cells by interacting with and inhibiting partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) independently of CagA tyrosine phosphorylation. Despite these CagA activities that contribute to neoplastic transformation, a causal link between CagA and in vivo oncogenesis remains unknown. Here, we generated transgenic mice expressing wild-type or phosphorylation-resistant CagA throughout the body or predominantly in the stomach. Wild-type CagA transgenic mice showed gastric epithelial hyperplasia and some of the mice developed gastric polyps and adenocarcinomas of the stomach and small intestine. Systemic expression of wild-type CagA further induced leukocytosis with IL-3/GM-CSF hypersensitivity and some mice developed myeloid leukemias and B cell lymphomas, the hematological malignancies also caused by gain-of-function SHIP-2 mutations. Such pathological abnormalities were not observed in transgenic mice expressing phosphorylation-resistant CagA. These results provide first direct evidence for the role of CagA as a bacterium-derived oncoprotein (bacterial oncoprotein) that acts in mammals and further indicate the importance of CagA tyrosine phosphorylation, which enables CagA to deregulate SHIP-2, in the development of H. pylori-associated neoplasms.
  • Yasuko Orba, Yuji Sunden, Tadaki Suzuki, Kazuo Nagashima, Takashi Kimura, Shinya Tanaka, Hirofumi Sawa
    VIROLOGY 370 (1) 173 - 183 0042-6822 2008/01 [Refereed][Not invited]
     
    The human Polyomavirus JC virus (JCV) utilizes cellular proteins for viral replication and transcription in the host cell nucleus. These cellular proteins represent potential targets for antiviral drugs against the JCV. In this study, we examined the antiviral effects of the pharmacological cyclin-dependent kinase (cdk) inhibitor R-Roscovitine, which has been shown to have antiviral activity against other viruses. We found that Roscovitine significantly inhibited the viral production and cytopathic effects of the JCV in a JCV-infected cell line. Roscovitine attenuated the transcriptional activity of JCV late genes, but not early genes, and also prevented viral replication via inhibiting phosphorylation of the viral early protein, large T antigen. These data suggest that the JCV requires cdks to transcribe late genes and to replicate its own DNA. That Roscovitine exhibited antiviral activity in JCV-infected cells suggests that Roscovitine might have therapeutic utility in the treatment of progressive multifocal leukoencephalopathy (PML). (c) 2007 Elsevier Inc. All rights reserved.
  • Takeshi Ichinohe, Shin-ichi Tamura, Akira Kawaguchi, Ai Ninomiya, Masaki Imai, Shigeyuki Itamura, Takato Odagiri, Masato Tashiro, Hidehiro Takahashi, Hirofumi Sawa, William M. Mitchell, David R. Strayer, William A. Carter, Joe Chiba, Takeshi Kurata, Tetsutaro Sata, Hideki Hasegawa
    JOURNAL OF INFECTIOUS DISEASES 196 (9) 1313 - 1320 0022-1899 2007/11 [Refereed][Not invited]
     
    Background. Avian H5N1 influenza A virus is an emerging pathogen with the potential to cause substantial human morbidity and mortality. We evaluated the ability of currently licensed seasonal influenza vaccine to confer cross-protection against highly pathogenic H5N1 influenza virus in mice. Methods. BALB/c mice were inoculated 3 times, either intranasally or subcutaneously, with the trivalent inactivated influenza vaccine licensed in Japan for the 2005-2006 season. The vaccine included A/NewCaledonia/20/99 (H1N1), A/NewYork/55/2004 (H3N2), and B/Shanghai/361/2002 viral strains and was administered together with poly(I): poly(C 12 U) (Ampligen) as an adjuvant. At 14 days after the final inoculation, the inoculated mice were challenged with either the A/HongKong/483/97, the A/Vietnam/1194/04, or the A/Indonesia/6/05 strain of H5N1 influenza virus. Results. Compared with noninoculated mice, those inoculated intranasally manifested cross-reactivity of mucosal IgA and serum IgG with H5N1 virus, as well as both a reduced H5N1 virus titer in nasal-wash samples and increased survival, after challenge with H5N1 virus. Subcutaneous inoculation did not induce a cross-reactive IgA response and did not afford protection against H5N1 viral infection. Conclusions. Intranasal inoculation with annual influenza vaccine plus the Toll-like receptor -3 agonist, poly(I): poly(C-12 U), may overcome the problem of a limited supply of H5N1 virus vaccine by providing cross-protective mucosal immunity against H5N1 viruses with pandemic potential.
  • Takeshi Ichinohe, Shin-ichi Tamura, Akira Kawaguchi, Ai Ninomiya, Masaki Imai, Shigeyuki Itamura, Takato Odagiri, Masato Tashiro, Hidehiro Takahashi, Hirofumi Sawa, William M. Mitchell, David R. Strayer, William A. Carter, Joe Chiba, Takeshi Kurata, Tetsutaro Sata, Hideki Hasegawa
    JOURNAL OF INFECTIOUS DISEASES 196 (9) 1313 - 1320 0022-1899 2007/11 [Not refereed][Not invited]
     
    Background. Avian H5N1 influenza A virus is an emerging pathogen with the potential to cause substantial human morbidity and mortality. We evaluated the ability of currently licensed seasonal influenza vaccine to confer cross-protection against highly pathogenic H5N1 influenza virus in mice. Methods. BALB/c mice were inoculated 3 times, either intranasally or subcutaneously, with the trivalent inactivated influenza vaccine licensed in Japan for the 2005-2006 season. The vaccine included A/NewCaledonia/20/99 (H1N1), A/NewYork/55/2004 (H3N2), and B/Shanghai/361/2002 viral strains and was administered together with poly(I): poly(C 12 U) (Ampligen) as an adjuvant. At 14 days after the final inoculation, the inoculated mice were challenged with either the A/HongKong/483/97, the A/Vietnam/1194/04, or the A/Indonesia/6/05 strain of H5N1 influenza virus. Results. Compared with noninoculated mice, those inoculated intranasally manifested cross-reactivity of mucosal IgA and serum IgG with H5N1 virus, as well as both a reduced H5N1 virus titer in nasal-wash samples and increased survival, after challenge with H5N1 virus. Subcutaneous inoculation did not induce a cross-reactive IgA response and did not afford protection against H5N1 viral infection. Conclusions. Intranasal inoculation with annual influenza vaccine plus the Toll-like receptor -3 agonist, poly(I): poly(C-12 U), may overcome the problem of a limited supply of H5N1 virus vaccine by providing cross-protective mucosal immunity against H5N1 viruses with pandemic potential.
  • Kouichi Tabu, Yusuke Ohba, Tadaki Suzuki, Yoshinori Makino, Taichi Kimura, Akiko Ohnishi, Mieko Sakai, Takuya Watanabe, Shinya Tanaka, Hirofurni Sawa
    MOLECULAR CANCER RESEARCH 5 (10) 1099 - 1109 1541-7786 2007/10 [Refereed][Not invited]
     
    The basic helix-loop-helix transcription factor, oligodendrocyte lineage transcription factor 2 (OLIG2), is specifically expressed in the developing and mature central nervous system and plays an important role in oligodendrogenesis from neural progenitors. It is also expressed in various types of glial tumors, but rarely in glioblastoma. Although we previously showed that OLIG2 expression inhibits glioma cell growth, its role in tumorigenesis remains incompletely understood. Here, we investigated the effect of OLIG2 expression on the migration of the human glioblastoma cell line U12-1. In these cells, OLIG2 expression is controlled by the Tet-off system. Induction of OLIG2 expression inhibited both the migration and invasiveness of U12-1 cells. OLIG2 expression also increased the activity of the GTPase RhoA as well as inducing the cells to form stress fibers and focal adhesions. Experiments using short interfering RNA against p27(KiP1) revealed that up-regulation of the p27(Kip1) protein was not essential for RhoA activation, rather it contributed independently to the decreased motility of OLIG2-expressing U12-1 cells. Alternatively, semiquantitative reverse transcription-PCR analysis revealed that mRNA expression of RhoGAP8, which regulates cell migration, was decreased by OLIG2 expression. Furthermore, expression of C3 transferase, which inhibits Rho via ADP ribosylation, attenuated the OLIG2-induced inhibition of cell motility. Imaging by fluorescence resonance energy transfer revealed that in U12-1 cells lacking OLIG2, the active form of RhoA was localized to protrusions of the cell membrane. In contrast, in OLIG2-expressing cells, it lined almost the entire plasma membrane. Thus, OLIG2 suppresses the motile phenotype of glioblastoma cells by activating RhoA.
  • [Case of highly active anti-retroviral therapy-induced immune reconstitution inflammatory syndrome in AIDS-related progressive multifocal leukoencephalopathy].
    Imamura E, Yamashita H, Fukuhara T, Sawa H, Nagashima K, Kuwabara M, Tokinobu H
    Rinsho shinkeigaku = Clinical neurology 10 47 650 - 656 0009-918X 2007/10 [Refereed][Not invited]
  • Kouichi Tabu, Yusuke Ohba, Tadaki Suzuki, Yoshinori Makino, Taichi Kimura, Akiko Ohnishi, Mieko Sakai, Takuya Watanabe, Shinya Tanaka, Hirofurni Sawa
    MOLECULAR CANCER RESEARCH 5 (10) 1099 - 1109 1541-7786 2007/10 [Not refereed][Not invited]
     
    The basic helix-loop-helix transcription factor, oligodendrocyte lineage transcription factor 2 (OLIG2), is specifically expressed in the developing and mature central nervous system and plays an important role in oligodendrogenesis from neural progenitors. It is also expressed in various types of glial tumors, but rarely in glioblastoma. Although we previously showed that OLIG2 expression inhibits glioma cell growth, its role in tumorigenesis remains incompletely understood. Here, we investigated the effect of OLIG2 expression on the migration of the human glioblastoma cell line U12-1. In these cells, OLIG2 expression is controlled by the Tet-off system. Induction of OLIG2 expression inhibited both the migration and invasiveness of U12-1 cells. OLIG2 expression also increased the activity of the GTPase RhoA as well as inducing the cells to form stress fibers and focal adhesions. Experiments using short interfering RNA against p27(KiP1) revealed that up-regulation of the p27(Kip1) protein was not essential for RhoA activation, rather it contributed independently to the decreased motility of OLIG2-expressing U12-1 cells. Alternatively, semiquantitative reverse transcription-PCR analysis revealed that mRNA expression of RhoGAP8, which regulates cell migration, was decreased by OLIG2 expression. Furthermore, expression of C3 transferase, which inhibits Rho via ADP ribosylation, attenuated the OLIG2-induced inhibition of cell motility. Imaging by fluorescence resonance energy transfer revealed that in U12-1 cells lacking OLIG2, the active form of RhoA was localized to protrusions of the cell membrane. In contrast, in OLIG2-expressing cells, it lined almost the entire plasma membrane. Thus, OLIG2 suppresses the motile phenotype of glioblastoma cells by activating RhoA.
  • Toshitsugu Ftijita, Andres D. Maturana, Junko Ikuta, Juri Hamada, Sebastien Walchli, Tadaki Suzuki, Hirofurni Sawa, Marie W. Wooten, Toshihide Okajima, Kenji Taternatsu, Katsuyuki Tanizawa, Shun'ichi Kuroda
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 361 (3) 605 - 610 0006-291X 2007/09 [Refereed][Not invited]
     
    Fasciculation and elongation protein zeta-1 (FEZ1) promotes efficiently the neurite elongation of rat phaeochromocytoma PC12 cells. We here characterized FEZ1 in PC12 cells. Nerve growth factor (NGF) stimulation induces significant expression of endogenous FEZ1 in PC12 cells. Upon NGF stimulation FEZ1 localizes in both cytoplasm and neuritis, co-localizing with mitochondria. Silencing of FEZ1 by RNA interference efficiently reduces NGF-induced neurite elongation and the anterograde motility of mitochondria in PC12 cells. immunoprecipitation and pulldown assay shows that FEZ1 interacts with kinesin superfamily protein 5 (KIF5) and tubulin. Thus, our results suggest that the FEZ1/kinesin complex functions for the transport of mitochondria along microtubules toward the extending neurites in differentiating PC12 cells. (c) 2007 Elsevier Inc. All rights reserved.
  • Takeshi Ichinohe, Akira Kawaguchi, Shin-ichi Tamura, Hidehiro Takahashi, Hirofumi Sawa, Ai Ninomiya, Masaki Imai, Shigeyuki Itamura, Takato Odagiri, Masato Tashiro, Joe Chiba, Tetsutaro Sata, Takeshi Kurata, Hideki Hasegawa
    MICROBES AND INFECTION 9 (11) 1333 - 1340 1286-4579 2007/09 [Refereed][Not invited]
     
    The avian H5N1 influenza virus has the potential to cause a new pandemic. Since it is difficult to predict which strain of influenza will cause a pandemic, it is advantageous to produce vaccines that confer cross-protective immunity. Mucosal vaccine administration was reported to induce cross-protective immunity by inducing secretion of IgA at the mucosal surface. Adjuvants can also enhance the development of fully protective mucosal immunity. Here we show that a new mucosal adjuvant, polyI:polyC(12)U (Ampligen (R)), a Toll-like receptor 3 agonist proven to be safe in a Phase III human trial, is an effective adjuvant for H5N1 influenza vaccination. Intranasal administration of a candidate influenza vaccine with Ampligen resulted in secretion of IgA, and protected mice that were subsequently challenged with homologous A/Vietnam/1194/2004 and heterologous A/HK/483/97 and A/Indonesia/6/2005 virus. (C) 2007 Elsevier Masson SAS. All rights reserved.
  • [Etiological agent and pathogenicity mechanism of PML].
    Suzuki T, Nagashima K, Sawa H
    Nihon rinsho. Japanese journal of clinical medicine 8 65 1495 - 1500 0047-1852 2007/08 [Refereed][Not invited]
  • Yoshikazu Nakaoka, Keigo Nishida, Masahiro Narimatsu, Atsunori Kamiya, Takashi Minami, Hirofumi Sawa, Katsuya Okawa, Yasushi Fujio, Tatsuya Koyama, Makiko Maeda, Manami Sone, Satoru Yamasaki, Yuji Arai, Gou Young Koh, Tatsuhiko Kodama, Hisao Hirota, Kinya Otsu, Toshio Hirano, Naoki Mochizuki
    JOURNAL OF CLINICAL INVESTIGATION 117 (7) 1771 - 1781 0021-9738 2007/07 [Refereed][Not invited]
     
    Grb2-associated binder (Gab) family of scaffolding adaptor proteins coordinate signaling cascades downstream of growth factor and cytokine receptors. In the heart, among EGF family members, neuregulin-1 beta (NRG-1 beta, a paracrine factor produced from endothelium) induced remarkable tyrosine phosphorylation of Gab 1 and Gab2 via erythroblastic leukemia viral oncogene (ErbB) receptors. We examined the role of Gab family proteins in NRG-1 beta/ErbB-mediated signal in the heart by creating cardiomyocyte-specific Gab1/Gab2 double knockout mice (DKO mice). Although DKO mice were viable, they exhibited marked ventricular dilatation and reduced contractility with aging. DKO mice showed high mortality after birth because of heart failure. In addition, we noticed remarkable endocardial fibroelastosis and increase of abnormally dilated vessels in the ventricles of DKO mice. NRG-1 beta induced activation of both ERK and AKT in the hearts of control mice but not in those of DKO mice. Using DNA microarray analysis, we found that stimulation with NRG-1 beta upregulated expression of an endothelium-stabilizing factor, angiopoietin 1, in the hearts of control mice but not in those of DKO mice, which accounted for the pathological abnormalities in the DKO hearts. Taken together, our observations indicated that in the NRG-1 beta/ErbB signaling, Gab1 and Gab2 of the myocardium are essential for both maintenance of myocardial function and stabilization of cardiac capillary and endocardial endothelium in the postnatal heart.
  • Takeshi Ichinohe, Noriyo Nagata, Peter Strong, Shin-ichi Tamura, Hidehiro Takahashi, Ai Ninomiya, Masaki Imai, Takato Odagiri, Masato Tashiro, Hirofumi Sawa, Joe Chiba, Takeshi Kurata, Tetsutaro Sata, Hideki Hasegawa
    JOURNAL OF MEDICAL VIROLOGY 79 (6) 811 - 819 0146-6615 2007/06 [Refereed][Not invited]
     
    Highly pathogenic avian influenza virus (H5N1) is an emerging pathogen with the potential to cause great harm to humans, and there is concern about the potential for a new influenza pandemic. This virus is resistant to the antiviral effects of interferons and tumor necrosis factor-of.. However, the mechanism of interferon-independent protective innate immunity is not well understood. The prophylactic effects of chitin microparticles as a stimulator of innate mucosal immunity against a recently obtained strain of H5N1 influenza virus infection were examined in mice. Clinical parameters and the survival rate of mice treated by intranasal application of chitin microparticles were significantly improved compared to non-treated mice after a lethal influenza virus challenge. Flow cytometric analysis revealed that the number of natural killer cells that expressed tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and that had migrated into the cervical lymph node was markedly increased (26-fold) after intranasal treatment with chitin microparticles. In addition, the level of IL-6 and interferon-gamma-inducible protein-10 (IP-10) in the nasal mucosa after H5N1 influenza virus challenge was decreased by prophylactic treatment with chitin microparticles. These results suggest that prophylactic intranasal administration of chitin microparticles enhanced the local accumulation of natural killer cells and suppressed hyper-induction of cytokines, resulting in an innate immune response to prevent pathogenesis of H5N1 influenza virus. J. Med. Virol. 79:811-819, 2007. (C) 2007 Wiley-Liss, Inc.
  • Yoshinori Masuo, Masami Ishido, Masatoshi Morita, Hirofumi Sawa, Kazuo Nagashima, Etsuo Niki
    EUROPEAN JOURNAL OF NEUROSCIENCE 25 (12) 3659 - 3666 0953-816X 2007/06 [Refereed][Not invited]
     
    Recently, congenic wiggling (Wig) rats were described as a good model for attention-deficit hyperactivity disorder; 12- to 14-week-old animals demonstrated hyperactivity, impulsive behaviour and an impaired working memory. Here, we show that 4- to 5-week-old Wig rats displayed significantly greater spontaneous motor activity than control rats during a period of darkness. Subcutaneous injection of 4 mg/kg methamphetamine exacerbated hyperactivity, the reverse of its effect in rats with neonatally induced 6-hydroxydopamine lesions. Immunohistochemistry showed low levels of tyrosine hydroxylase in the ventral midbrain, similar to 6-hydroxydopamine-treated rats. In cDNA macroarrays, 4-week-old Wig rats showed increased expression of the adenosine A2a receptor in the dorsal striatum, macrophage migration inhibitory factor in the frontal cortex, ventral striatum and midbrain, and calbindin 2 in the dorsal and ventral midbrain. Expression of the gamma-aminobutyric acid (GABA) transporter and sterol carrier protein 2 genes was reduced in all regions. Dopamine transporter gene expression was increased in the dorsal midbrain but decreased in the ventral midbrain, a pattern distinct from that induced by 6-hydroxydopamine. Although abnormal development of dopaminergic neurons may underlie motor hyperactivity, other mechanisms may control responsiveness to methamphetamine. Wig rats may provide a model of attention-deficit hyperactivity disorder in which treatment with psychostimulants accelerate the hyperactivity.
  • Takahiro Tsuji, Noreen Sheehy, Virginie W. Gautier, Hitoshi Hayakawa, Hirofumi Sawa, William W. Hall
    JOURNAL OF BIOLOGICAL CHEMISTRY 282 (18) 13875 - 13883 0021-9258 2007/05 [Refereed][Not invited]
     
    HTLV-1 is the etiologic agent of the adult T cell leukemia-lymphoma (ATLL). The viral regulatory protein Tax plays a central role in leukemogenesis as a transcriptional transactivator of both viral and cellular gene expression, and this requires Tax activity in both the cytoplasm and the nucleus. In the present study, we have investigated the mechanisms involved in the nuclear localization of Tax. Employing a GFP fusion expression system and a range of Tax mutants, we could confirm that the N-terminal 60 amino acids, and specifically residues within the zinc finger motif in this region, are important for nuclear localization. Using an in vitro nuclear import assay, it could be demonstrated that the transportation of Tax to the nucleus required neither energy nor carrier proteins. Specific and direct binding between Tax and p62, a nucleoporin with which the importin beta family of proteins have been known to interact was also observed. The nuclear import activity of wild type Tax and its mutants and their binding affinity for p62 were also clearly correlated, suggesting that the entry of Tax into the nucleus involves a direct interaction with nucleoporins within the nuclear pore complex (NPC). The nuclear export of Tax was also shown to be carrier independent. It could be also demonstrated that Tax it self may have a carrier function and that the NF-kappa B subunit p65 could be imported into the nucleus by Tax. These studies suggest that Tax could alter the nucleocytoplasmic distribution of cellular proteins, and this could contribute to the deregulation of cellular processes observed in HTLV-1 infection.
  • Akiyuki Takaya, Takahiro Kamio, Michitaka Masuda, Naoki Mochizuki, Hirofumi Sawa, Mami Sato, Kazuo Nagashima, Akiko Mizutani, Akira Matsuno, Etsuko Kiyokawa, Michiyuki Matsuda
    MOLECULAR BIOLOGY OF THE CELL 18 (5) 1850 - 1860 1059-1524 2007/05 [Refereed][Not invited]
     
    R-Ras is a Ras-family small GTPase that regulates various cellular functions such as apoptosis and cell adhesion. Here, we demonstrate a role of R-Ras in exocytosis. By the use of specific anti-R-Ras antibody, we found that R-Ras was enriched on both early and recycling endosomes in a wide range of cell lines. Using a fluorescence resonance energy transfer-based probe for R-Ras activity, R-Ras activity was found to be higher on endosomes than on the plasma membrane. This high R-Ras activity on the endosomes correlated with the accumulation of an R-Ras effector, the Rgl2/RIf guanine nucleotide exchange factor for RalA, and also with high RalA activity. The essential role played by R-Ras in inducing high levels of RalA activity on the endosomes was evidenced by the short hairpin RNA (shRNA)-mediated suppression of R-Ras and by the expression of R-Ras GAP. In agreement with the reported role of RalA in exocytosis, the shRNA of either R-Ras or RalA was found to suppress calcium-triggered exocytosis in PC12 pheochromocytoma cells. These data revealed that R-Ras activates RalA on endosomes and that it thereby positively regulates exocytosis.
  • [Recent research on the JC virus].
    Sawa H, Suzuki T, Orba Y, Sunden Y, Nagashima K
    Brain and nerve = Shinkei kenkyu no shinpo 2 59 101 - 108 1881-6096 2007/02 [Refereed][Not invited]
  • Lamprey TLRs with properties distinct from those of the variable lymphocyte receptors.
    Ishii A, Matsuo A, Sawa H, Tsujita T, Shida K, Matsumoto M, Seya T
    Journal of immunology (Baltimore, Md. : 1950) 1 178 (1) 397 - 406 0022-1767 2007/01 [Refereed][Not invited]
  • Identification of DDX1 as a JC virus transcriptional control region-binding protein
    Yuji Sunden, Shingo Semba, Tadaki Suzuki, Yuki Okada, Yasuko Orba, Kazuo Nagashima, Takashi Umemura, Hirofumi Sawa
    MICROBIOLOGY AND IMMUNOLOGY 51 (3) 327 - 337 0385-5600 2007 [Refereed][Not invited]
     
    To investigate the mechanism behind JC virus (JCV) cell specificity we performed electrophoretic mobility shift assays (EMSA) using probes derived from the JCV transcriptional control region (JCV-TCR). Using nuclear extracts from the JCV-susceptible neuroblastoma cell line IMR-32, EMSA revealed a 670 kDa JCV-TCR-binding protein complex designated as #3-bp. This complex could not be detected in nuclear extracts from non-susceptible cell lines. Using column chromatographic purification and microsequencing, we identified cleavage stimulation factor (CstF) as a component of #3-bp. However, as CstF is present in many cell types, we speculated that the IMR-32-specific component(s) of #3bp bind CAR We performed a yeast two-hybrid assay using CstF-77 as the bait against a HeLa cDNA-subtracted IMR-32 cDNA library. This analysis detected binding between CstF-77 and the RNA helicase DDX1. Subsequently, biotinylated DNA affinity precipitation and chromatin immunoprecipitation assays also confirmed that DDX1 binds specifically to JCV-TCR. Our findings indicate that an association between DDX1 and the JCV-TCR may play a significant role in JCV infection in IMR-32 cells.
  • DDX1 promotes proliferation of the JC virus through transactivation of its promoter
    Yuji Sunden, Shingo Semba, Tadaki Suzuki, Yuki Okada, Yasuko Orba, Kazuo Nagashima, Takashi Umemura, Hirofumi Sawa
    MICROBIOLOGY AND IMMUNOLOGY 51 (3) 339 - 347 0385-5600 2007 [Refereed][Not invited]
     
    Recently, we demonstrated that the DEAD box protein 1 (DDX1), an RNA helicase, and the cleavage stimulation factor (CstF) form a complex that binds to the JC virus transcriptional control region (JCV-TCR). Here, we examined the function of DDX1, which is expressed at much higher levels in the JCV-susceptible cell line IMR-32 than in non-susceptible cell lines. DDX1 had no effect on the replication efficiency of JCV, but overexpression of DDX1 significantly increased transactivation of the JCV promoter. Furthermore, DDX1 enhanced the expression of JCV proteins in JCV infected cells, and knockdown of DDX1 using small interfering (si) RNA suppressed the expression of JCV proteins. Our results clearly demonstrate that DDX1 regulates proliferation of JCV in vitro through transcriptional activation.
  • 高活性レトロウイルス療法により免疫再構築症候群をきたしたAIDSにともなう進行性多巣性白質脳症の1剖検例
    臨床神経学 65 1495 - 1500 2007 [Not refereed][Not invited]
  • 進行性多巣性白質脳症 (PML):原因ウイルスと発病機構。
    日本臨床 65 1495 - 1500 2007 [Not refereed][Not invited]
  • Yasuko Asahi-Ozaki, Shigeyuki Itamura, Takeshi Ichinohe, Peter Strong, Shin-ichi Tamura, Hidehiro Takahashi, Hirofumi Sawa, Masami Moriyama, Masato Tashiro, Tetsutaro Sata, Takeshi Kurata, Hideki Hasegawa
    MICROBES AND INFECTION 8 (12-13) 2706 - 2714 1286-4579 2006/10 [Refereed][Not invited]
     
    Attenuated recombinant H5N1 influenza virus was constructed to develop a safe H5N1 influenza vaccine. The immunogenicity and protective effect of the vaccine prepared from haemagglutinin-modified recombinant H5N1 influenza virus was evaluated in mice intranasally co-administered with cholera toxin B subunit containing a trace amount of holotoxin (CTB*), synthetic double-stranded RNA, poly (I:C) or chitin microparticles (CMP) as adjuvants. Intranasal administration of recombinant H5 HA split vaccine with CTB* or poly(I:C) and/or CMP elicited an immunological response with both anti-H5 HA IgA in the nasal wash and anti-H5 HA IgG antibody in the serum, and showed a protective against lethal H5N1 A/Hong Kong/483/97 (HK483) infection. We also demonstrated that intranasal co-administration of antigen with both poly (I:C) and CMP enhanced the expression of Toll-like receptor (TLR) 3, TLR7 in the spleen. These results indicate that poly (I:C) and CMP are highly effective as mucosal adjuvants for use with the nasal H5N1 vaccine. (c) 2006 Elsevier Masson SAS. All rights reserved.
  • Masae Maeda, Hirofumi Sawa, Minoru Tobiume, Kenzo Tokunaga, Hideki Hasegawa, Takeshi Ichinohe, Tetsutaro Sata, Masami Moriyama, William W. Hall, Takeshi Kurata, Hidehiro Takahashi
    MICROBES AND INFECTION 8 (11) 2647 - 2656 1286-4579 2006/09 [Refereed][Not invited]
     
    HIV-1 genome has an AU-rich sequence and requires rapid nuclear export by Rev activity to prevent multiple splicing. HIV-1 infection occurs in activated CD4(+) T cells where the decay of mRNAs of cytokines and chemokines is regulated by the binding of AU-rich elements to the mRNA-destabilizing protein tristetraprolin. We here investigated the influence of tristetraprolin on the replication of HIV-1. Treatment of siRNA against tristetraprolin in a latently HIV-1 infected cell line increases HIV-1 production following stimulation. A chloramphenicol acetyltransferase and luciferase assay revealed that exogenous tristetraprolin reduced HIV-1 virion production and in contrast increased the multiply spliced products. Furthermore, quantitative RT-PCR analysis showed tristetraprolin increases the ratio of multiple-spliced RNAs to un-, single-spliced RNA. Moreover. an electrophoretic mobility shift assay showed that tristetraprolin binds to synthesized HIV-1 RNA with AU-rich sequence but not to RNA with less AU sequence. These results suggest that tristetraprolin is a regulator of HIV-1 replication and enhances splicing by direct binding to AU-rich sequence of HIV-1 RNAs. (c) 2006 Elsevier Masson SAS. All rights reserved.
  • T Ichinohe, Watanabe, I, E Tao, S Ito, A Kawaguchi, S Tamura, H Takahashi, H Sawa, M Moriyama, J Chiba, K Komase, Y Suzuki, T Kurata, T Sata, H Hasegawa
    JOURNAL OF MEDICAL VIROLOGY 78 (7) 954 - 963 0146-6615 2006/07 [Refereed][Not invited]
     
    A safe and effective adjuvant is necessary to enhance mucosal immune responses for the development of an inactivated intranasal influenza vaccine. The present study demonstrated the effectiveness of surf clam microparticles (SMP) derived from natural surf clams as an adjuvant for an intranasal influenza vaccine. The adjuvant effect of SMP was examined when co-administered intranasally with inactivated A/PR8 (H1N1) influenza virus hemagglutinin vaccine in BALB/c mice. Administration of the vaccine with SMP induced a high anti-PR8 haemagglutinin (HA)specific immunoglobulin A (IgA) response in the nasal wash and immunoglobulin G (IgG) response in the serum, resulting in protection against both nasal-restricted infection and lethal lung infection by A/PR8 virus. In addition, administration of SMP with A/Yamagata (H1N1), A/Beijing (H1N1), or A/Guizhou (H3N2) vaccine conferred complete protection against A/PR8 virus challenge in the nasal infection model, suggesting that SMP adjuvanted vaccine can confer cross-protection against variant influenza viruses. The use of SMP is suggested as a new safe and effective mucosal adjuvant for nasal vaccination against influenza virus infection.
  • Takuya Watanabe, Masumi Tsuda, Yoshinori Makino, Shin Ichihara, Hirofumi Sawa, Akio Minami, Naoki Mochizuki, Kazuo Nagashima, Shinya Tanaka
    MOLECULAR CANCER RESEARCH 4 (7) 499 - 510 1541-7786 2006/07 [Refereed][Not invited]
     
    Activation of the c-Met receptor tyrosine kinase through its ligand, hepatocyte growth factor (HGF), promotes mitogenic, motogenic, and morphogenic cellular responses. Aberrant HGF/c-Met signaling has been strongly implicated in tumor cell invasion and metastasis. Both HGF and its receptor c-Met have been shown to be overexpressed in human synovial sarcoma, which often metastasizes to the lung; however, little is known about HGF-mediated biological effects in this sarcoma. Here, we provide evidence that Crk adaptor protein is required for the sustained phosphorylation of c-Met-docking protein Grb2-associated binder 1 (Gab1) in response to HGF, leading to the enhanced cell motility of human synovial sarcoma cell lines SY0-1, HS-SY-II, and Fuji. HGF stimulation induced the sustained phosphorylation on Y307 of Gab1 where Crk was recruited. Crk knockdown by RNA interference disturbed this HGF-induced tyrosine phosphorylation of Gab1. By mutational analysis, we identified that Src homology 2 domain of Crk is indispensable for the induction of the phosphorylation on multiple Tyr-X-X-Pro motifs containing Y307 in Gab1. HGF remarkably stimulated cell motility and scattering of synovial sarcoma cell lines, consistent with the prominent activation of Rac1, extreme filopodia formation, and membrane ruffling. Importantly, the elimination of Crk in these cells induced the disorganization of actin cytoskeleton and complete abolishment of HGF-mediated Rac1 activation and cell motility. Time-lapse microscopic analysis revealed the significant attenuation in scattering of Crk knockdown cells following HGF treatment. Furthermore, the depletion of Crk remarkably inhibited the tumor formation and its invasive growth in vivo. These results suggest that the sustained phosphorylation of Gab1 through Crk in response to HGF contributes to the prominent activation of Rac1 leading to enhanced cell motility, scattering, and cell invasion, which may support the crucial role of Crk in the aggressiveness of human synovial sarcoma.
  • H Linghu, M Tsuda, Y Makino, M Sakai, T Watanabe, S Ichihara, H Sawa, K Nagashima, N Mochizuki, S Tanaka
    ONCOGENE 25 (25) 3547 - 3556 0950-9232 2006/06 [Not refereed][Not invited]
     
    Signaling adaptor protein Crk regulates cell motility and growth through its targets Dock180 and C3G, those are the guanine-nucleotide exchange factors (GEFs) for small GTPases Rac and Rap, respectively. Recently, overexpression of Crk has been reported in various human cancers. To de. ne the role for Crk in human cancer cells, Crk expression was targeted in the human ovarian cancer cell line MCAS through RNA interference, resulting in the establishment of three Crk knockdown cell lines. These cell lines exhibited disorganized actin fibers, reduced number of focal adhesions, and abolishment of lamellipodia formation. Decreased Rac activity was demonstrated by pull-down assay and FRET-based time-lapse microscopy, in association with suppression of both motility and invasion by phagokinetic track assay and transwell assay in these cells. Furthermore, Crk knockdown cells exhibited slow growth rates in culture and suppressed anchorage-dependent growth in soft agar. Tumor forming potential in nude mice was attenuated, and intraperitoneal dissemination was not observed when Crk knockdown cells were injected into the peritoneal cavity. These results suggest that the Crk is a key component of focal adhesion and involved in cell growth, invasion, and dissemination of human ovarian cancer cell line MCAS.
  • Y Sunden, T Suzuki, Y Orba, T Umemura, M Asamoto, K Nagashima, S Tanaka, H Sawa
    ACTA NEUROPATHOLOGICA 111 (4) 379 - 387 0001-6322 2006/04 [Refereed][Not invited]
     
    Polyomavirus JC virus (JCV) is the causative agent of progressive multifocal leukoencephalopathy, a fatal demyelinating disease of the central nervous system. Similar to other polyomaviruses, such as simian vacuolating virus 40 (SV40) and BK virus (BKV), JCV is also associated with human tumours. The Polyomavirus early protein large T antigen (TAg) plays a crucial role in tumour pathogenesis. An antibody to SV40 TAg (PAb416), which cross-reacts with TAgs of both JCV and BKV, has been used widely for the detection of JCV and BKV TAgs. As a consequence, it is difficult to discriminate between the TAgs of SV40, BKV and JCV by immunohistochemical analyses. In the present study, we generated JCV TAg-specific polyclonal antibodies (JCT629 and JCT652) by immunization of New Zealand white rabbits with synthetic peptides reproducing the JCV TAg carboxyl-terminal region as immunogens. Immunoblotting analyses indicated that the new antibodies bind specifically to JCV TAg, and not to those of SV40 or BKV. We also demonstrated that these antibodies can be used for immunoprecipitation, immunocytochemical analyses and immunohistochemical staining of routinely processed specimens. In conclusion, the newly generated JCV-specific TAg antibodies may be useful both in the investigation of the pathophysiological function of JCV TAg and in discriminating between Polyomavirus-related clinical samples.
  • H Hasegawa, H Sawa, MJ Lewis, Y Orba, N Sheehy, Y Yamamoto, T Ichinohe, Y Tsunetsugu-Yokota, H Katano, H Takahashi, J Matsuda, T Sata, T Kurata, K Nagashima, WW Hall
    NATURE MEDICINE 12 (4) 466 - 472 1078-8956 2006/04 [Refereed][Not invited]
     
    Adult T-cell leukemia-lymphoma (ATLL) is a group of T-cell malignancies caused by infection with human T-lymphotropic virus type I (HTLV-I). Although the pathogenesis of ATLL remains incompletely understood, the viral regulatory protein Tax is centrally involved in cellular transformation. Here we describe the generation of HTLV-I Tax transgenic mice using the Lck proximal promoter to restrict transgene expression to developing thymocytes. After prolonged latency periods, transgenic mice developed diffuse large-cell lymphomas and leukemia with clinical, pathological and immunological features characteristic of acute ATLL. Transgenic mice were functionally immunocompromised and they developed opportunistic infections. Fulminant disease also developed rapidly in SCID mice after engraftment of lymphomatous cells from transgenic mice. Flow cytometry showed that the cells were CD4(-) and CD8(-), but CD44(+), CD25(+) and cytoplasmic CD3(+). This phenotype is indicative of a thymus-derived pre-T-cell phenotype, and disease development was associated with the constitutive activation of NF-kappa B. Our model accurately reproduces human disease and will provide a tool for analysis of the molecular events in transformation and for the development of new therapeutics.
  • K Tabu, A Ohnishi, Y Sunden, T Suzuki, M Tsuda, S Tanaka, T Sakai, K Nagashima, H Sawa
    JOURNAL OF CELL SCIENCE 119 (7) 1433 - 1441 0021-9533 2006/04 [Refereed][Not invited]
     
    The basic helix-loop-helix transcription factor OLIG2 is specifically expressed in cells of the oligodendrocyte lineage. It is also expressed in various tumors originating from glial cells; however, the expression of OLIG2 is rare or weak in glioblastomas, the most malignant gliomas. The role of OLIG2 in glioma remains unclear. To investigate the function of OLIG2 in glial tumor cells, we have established a glioblastoma cell line, U12-1, in which the expression of OLIG2 is induced by the Tet-off system. Induction of OLIG2 resulted in suppression of both the proliferation and anchorage-independent growth of U12-1. It also resulted in an increase in the expression of p27(Kip1). A luciferase assay revealed that the CTF site of the p27(Kip1) gene promoter was essential for OLIG2-dependent activation of p27(Kip1) gene transcription. Electrophoretic mobility shift assays confirmed that a nuclear extract of OLIG2-expressing U12-1 cells contained a protein complex that binds to the CTF site of the p27(Kip1) gene promoter. Furthermore, siRNA against p27(Kip1) rescued the OLIG2-mediated growth and DNA synthesis inhibition of U12-1 cells. These results indicate that OLIG2 suppresses the proliferation of U12-1 and that this effect is mediated by transactivation of the p27(Kip1) gene, and low expression of OLIG2 may be related to the malignant behavior of human glioblastoma.
  • Y Makino, M Tsuda, S Ichihara, T Watanabe, M Sakai, H Sawa, K Nagashima, S Hatakeyama, S Tanaka
    JOURNAL OF CELL SCIENCE 119 (5) 923 - 932 0021-9533 2006/03 [Refereed][Not invited]
     
    Dock180, a member of the CDM family of proteins, plays roles in biological processes such as phagocytosis and motility through its association with the signalling adaptor protein Crk. Recently, the complex formation between Dock180 and Elmo1 was reported to function as a bipartite guanine nucleotide exchange factor for Rac. In this study, we demonstrated that the amount of Dock180 increased when Elmo1 was co-expressed. Dock180 was found to be ubiquitylated and Dock180 protein levels could be augmented by treatment with proteasome inhibitor. The ubiquitylation of Dock180 was enhanced by epidermal growth factor (EGF), Crk and adhesion-dependent signals. Furthermore, Elmo1 inhibited labiquitylation of Dock180, resulting in the increase in Dock180 levels. The Elmo1 mutant Delta 531, which encompasses amino acids required for Dock180 binding, preserved the inhibitory effects on ubiquitylation of Dock180. Upon EGF stimulation, both Dock180 and ubiquitin were demonstrated to translocate to the cell periphery by immunofluorescence, and we found ubiquitylation of Dock180 and its inhibition by Elmo1 to occur in cellular membrane fractions by in vivo ubiquitylation assay. These data suggest that Dock180 is ubiquitylated on the plasma membrane, and also that Elmo1 functions as an inhibitor of ubiquitylation of Dock180. Therefore, an ubiquitin-proteasome-dependent protein degradation mechanism might contribute to the local activation of Rac on the plasma membrane.
  • H Takahashi, M Maeda, H Sawa, H Hasegawa, M Moriyama, T Sata, WW Hall, T Kurata
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 340 (3) 807 - 814 0006-291X 2006/02 [Refereed][Not invited]
     
    AU-rich elements (AREs) in the 3'-untranslated region of mRNAs promote rapid decay of the mRNAs for certain cytokines, including that encoding granulocyte-macrophage colony-stimulating factor (GM-CSF). We show that,in RNA molecule based on the ARE of GM-CSF mRNA is cleaved between U and A residues in the presence of bovine serum albumin Of Which cleavage effect is attenuated by acetylation. Furthermore, the expression of RNA molecule containing the ARE of GM-CSF mRNA in human cell lines was increased by inhibition of histone deacetylase activity and attenuation of Dicer expression. These findings suggest that degradation of mRNAs containing an ARE might be regulated by positive charge of polypeptides and Dicer. (c) 2005 Elsevier Inc. All rights reserved.
  • M Tsuda, T Watanabe, T Seki, T Kimura, H Sawa, A Minami, T Akagi, K Isobe, K Nagashima, S Tanaka
    ONCOGENE 24 (54) 7984 - 7990 0950-9232 2005/12 [Refereed][Not invited]
     
    Oncogenic protein provokes cell cycle arrest termed premature senescence. In this process Ras has been known to induce cyclin-dependent kinase inhibitor (CKI) p16(INK4A) in primary fibroblasts. Here, we present a novel finding that human chimeric oncoprotein SYT-SSX1 induces CKI p21(WAF1/CIP1) (p21) for suppression of cell growth. In human synovial sarcoma cell lines, the expression levels of p21 were high and the transcriptional activity of the p21 gene promoter was significantly elevated. The transient expression of SYT- SSX1-induced activation of the p21 gene promoter in human diploid fibroblasts. The N-terminus deletion form of SYT-SSX1, which failed to bind to hBRM one of the chromatin remodeling factors, preserved the p21 induction ability. This effect of SYT-SSX1 was similar in extent in both wild-type and p53-deficient HCT116 cell lines. Furthermore, the introduction of mutation in Sp1/Sp3 binding sites of the p21 gene promoter abolished the SYT-SSX1-induced transcriptional activity of its promoter. In SW13 cells, the stable expression of SYT-SSX1 suppressed cell growth in culture. These results suggest that SYT-SSX1 is able to induce p21 in a manner independent on hBRM and p53 but dependent on Sp1/Sp3.
  • Akazawa S, Igarashi M, Sawa H, Tamashiro H
    Environmental health and preventive medicine 5 10 (5) 282 - 285 1342-078X 2005/09 [Refereed][Not invited]
  • K Khalili, MK White, H Sawa, K Nagashima, M Safak
    JOURNAL OF CELLULAR PHYSIOLOGY 204 (1) 1 - 7 0021-9541 2005/07 [Refereed][Not invited]
     
    The late region of the three primate polyomaviruses (JCV, BKV, and SV40) encodes a small, highly basic protein known as agnoprotein. While much attention during the last two decades has focused on the transforming proteins encoded by the early region (small and large T-antigens), it has become increasingly evident that agnoprotein has a critical role in the regulation of viral gene expression and replication, and in the modulation of certain important host cell functions including cell cycle progression and DNA repair. The importance of agnoprotein is underscored by its expression during lytic infection of glial cells by JCV that occurs in progressive multifocal leukoencephalopathy (PML), and also in some JCV-associated human neural tumors particularly medulloblastoma. In this review, we will discuss the structure and function of agnoprotein in the viral life cycle during the course of lytic infection and the consequences of agnoprotein expression for the host cell.
  • T Suzuki, Y Okada, S Semba, Y Orba, S Yamanouchi, S Endo, S Tanaka, T Fujita, S Kuroda, K Nagashima, H Sawa
    JOURNAL OF BIOLOGICAL CHEMISTRY 280 (26) 24948 - 24956 0021-9258 2005/07 [Refereed][Not invited]
     
    The human polyomavirus JC virus (JCV) is the causative agent of a fatal demyelinating disease, progressive multifocal leukoencephalopathy, and encodes six major proteins, including agnoprotein. Agnoprotein colocalizes with microtubules in JCV-infected cells, but its function is not fully understood. We have now identified fasciculation and elongation protein zeta 1 (FEZ1) as a protein that interacted with JCV agnoprotein in a yeast two-hybrid screen of a human brain cDNA library. An in vitro binding assay showed that agnoprotein interacted directly with FEZ1 and microtubules. A microtubule co-sedimentation assay revealed that FEZ1 also associates with microtubules and that agnoprotein induces the dissociation of FEZ1 from microtubules. Agnoprotein inhibited the promotion by FEZ1 of neurite outgrowth in PC12 cells. Conversely, overexpression of FEZ1 suppressed JCV protein expression and intracellular trafficking in JCV-infected cells. These results suggest that FEZ1 promotes neurite extension through its interaction with microtubules, and that agnoprotein facilitates JCV propagation by inducing the dissociation of FEZ1 from microtubules.
  • Y Okada, T Suzuki, Y Sunden, Y Orba, S Kose, N Imamoto, H Takahashi, S Tanaka, WW Hall, K Nagashima, H Sawa
    EMBO REPORTS 6 (5) 452 - 457 1469-221X 2005/05 [Refereed][Not invited]
     
    The nuclear envelope is one of the chief obstacles to the translocation of macromolecules that are larger than the diameter of nuclear pores. Heterochromatin protein 1 (HP1) bound to the lamin B receptor (LBR) is thought to contribute to reassembly of the nuclear envelope after cell division. Human polyomavirus agnoprotein ( Agno) has been shown to bind to HP1 alpha and to induce its dissociation from LBR, resulting in destabilization of the nuclear envelope. Fluorescence recovery after photobleaching showed that Agno increased the lateral mobility of LBR in the inner nuclear membrane. Biochemical and immunofluorescence analyses showed that Agno is targeted to the nuclear envelope and facilitates the nuclear egress of polyomavirus-like particles. These results indicate that dissociation of HP1a from LBR and consequent perturbation of the nuclear envelope induced by polyomavirus Agno promote the translocation of virions out of the nucleus.
  • H Sawa, T Nagashima, K Nagashima, T Shinohara, T Chuma, Y Mano, N Tachi, WW Hall
    JOURNAL OF NEUROVIROLOGY 11 (2) 199 - 207 1355-0284 2005/04 [Refereed][Not invited]
     
    Human T-lymphotropic virus type I (HTLV-I) is known to be the causative agent of the chronic myelopathy, HTLV- I-associated myelopathy ( HAM), and on rare occasions infection is also associated with the development of polyneuropathy. Here the authors present an HTLV-I-positive family of whom four members developed a chronic demyelinating polyneuropathy without HAM. Four female patients in a family from Hokkaido in Japan developed distal dominant paresthesia and muscle weakness in the second and third decades of their life. Neurological findings at ages ranging from 50 to 65 years included mild painful sensorimotor disturbances with atrophy of the distal parts of the extremities but without pyramidal signs or hyperactive tendon reflexes. Magnetic resonance imaging (MRI) findings of brain and spinal cord were unremarkable. Serum HTLV- I antibody levels were elevated at 1: 8,192 to 1: 32,768, whereas those in cerebrospinal fluid were low at 1: 4 to 1: 8. Electrophysiological studies revealed polyphasic compound muscle action potentials with denervation potentials on nerve conduction studies and neurogenic patterns by electromyography, which were consistent with signs of chronic motor dominant demyelinating polyneuropathy. Sural nerve biopsy showed decreased myelinated fibers, occurrence of globule formation, myelin ovoid and remyelinated fibers, and an infiltration of CD68-positive macrophages with occasional CD4-positive T cells in the nerve fascicles. The polyneuropathy was responsive to steroid therapy. Analyses of serological human leukocyte antigen (HLA) types indicated that none of the patients possessed a high-risk HLA type known to be associated with adult T-cell leukemia (ATL), whereas they did have high responsive alleles to HTLV- I env similar to that observed in HAM. Nucleotide sequence analysis of the HTLV- I tax region demonstrated the B subgroup in all patients. This study suggests that HTLV- I infection can result in the development of a familial form of polyneuropathy that is associated with distinct HLA class I alleles, which might possibly involve a distinct virus subtype.
  • T Ichinohe, Watanabe, I, S Ito, H Fujii, M Moriyama, S Tamura, H Takahashi, H Sawa, J Chiba, T Kurata, T Sata, H Hasegawa
    JOURNAL OF VIROLOGY 79 (5) 2910 - 2919 0022-538X 2005/03 [Refereed][Not invited]
     
    The mucosal adjuvant effect of synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [poly(I:C)] against influenza virus was examined under intranasal coadministration with inactivated hemagglutinin (RA) vaccine in BALB/c mice and was shown to have a protective effect against both nasal-restricted infection and lethal lung infection. Intranasal administration of vaccine from PR8 (H1N1) with poly(I:C) induced a high anti-HA immunoglobulin A (IgA) response in the nasal wash and IgG antibody response in the serum, while vaccination without poly(I:C) induced little response. Intracerebral injection confirmed the safety of poly(I:C). In addition, we demonstrated that administration of poly(I:C) with either A/Beijing (H1N1) or A/Yamagata (H1N1) vaccine conferred complete protection against PR8 challenge in this mouse nasal infection model, suggesting that poly(I:C) possessed cross-protection ability against variant viruses. To investigate the mechanism of the protective effect of poly(I:C), mRNA levels of Toll-like receptors and cytokines were examined in the nasal-associated lymphoid tissue after vaccination or virus challenge. Intranasal administration of HA vaccine with poly(I:C) up-regulated expression of Toll-like receptor 3 and alpha/beta interferons as well as Th1- and Th2-related cytokines. We propose that poly(I:C) is a new effective intranasal adjuvant for influenza virus vaccine.
  • C Henmi, H Sawa, H Iwata, Y Orba, S Tanaka, K Nagashima
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 327 (1) 242 - 251 0006-291X 2005/02 [Refereed][Not invited]
     
    Polyomavirus JC (JCV) infection causes the fatal human demyelinating disease, progressive multifocal leukoencephalopathy. Although the initial interaction of JCV with host cells occurs through direct binding of the major viral capsid protein (VP1) with cell-surface molecules possessing sialic acid, these molecules have not yet been identified. In order to isolate monoclonal antibodies which inhibit attachment of JCV, we established an immunoscreening system using virus-like particles consisting of the VP1. Using this system, among monoclonal antibodies against the cell membrane fraction from JCV-permissive human neuroblastoma IMR-32 cells, we isolated a monoclonal antibody designated as 24132 that specifically inhibited attachment and infection of JCV to IMR-32 cells. The antibody 24132 recognized a single molecule of around 60 kDa in molecular weight in the IMR-32 membrane fraction. Immunohistochemical staining with 24132 demonstrated immunoreactivity in the cell membrane of JCV-permissive cell lines and glial cells of the human brain. These results suggested that the molecule recognized by 24132 plays a role in JCV infection, and that it might participate as a receptor or a co-receptor in JCV attachment and entry into the cells. (C) 2004 Elsevier Inc. All rights reserved.
  • The JC virus-like particle overlay assay.
    Sawa H, Komagome R
    Methods in molecular biology (Clifton, N.J.) 292 175 - 186 1064-3745 2005 [Refereed][Not invited]
  • H Hasegawa, T Ichinohe, P Strong, Watanabe, I, S Ito, S Tamura, H Takahashi, H Sawa, J Chiba, T Kurata, T Sata
    JOURNAL OF MEDICAL VIROLOGY 75 (1) 130 - 136 0146-6615 2005/01 [Refereed][Not invited]
     
    Chitin in the form of microparticles (chitin microparticles, CMP) has been demonstrated to be a potent stimulator of macrophages, promoting T-helper-1 (Th1) activation and cytokine response. In order to examine the mucosal adjuvant effect of CMP co-administered with influenza hemagglutinin (HA) vaccine against influenza infection, CMP were intranasally co-administered with influenza HA vaccine prepared from PR8 (H1N1) virus. Inoculation of the vaccine with CMP induced primary and secondary anti-HA IgA responses in the nasal wash and anti-HA IgG responses in the serum, which were significantly higher than those of nasal vaccination without CMP, and provided a complete protection against a homologous influenza virus challenge in the nasal infection influenza model. In addition, CMP-based immunization using A/Yamagata (H1N1) and A/Guizhou (H3N2) induced PR8 HA-reactive IgA in the nasal washes and specific-IgG in the serum. The immunization with A/Yamagata and CMP resulted in complete protection against a PR8 (H1N1) challenge in A/Yamagata (H1N1)-vaccinated mice, while that with A/Guizhou (H3H2) and CMP exhibited a 100-fold reduction of nasal virus titer, demonstrating the cross-protective effect of CMP and influenza vaccine. It is suggested that CMP provide a safe and effective adjuvant for nasal vaccination with inactivated influenza vaccine. (C) 2005 Wiley-Liss, Inc.
  • Sawa H, Nagashima T, Nagashima K, Shinohara T, Chuma T, Mano Y, Tachi N, Hall WW. Clinicopathological and virological analyses of familial HTLV-I associated polyneuropathy. J Neurovirol 11: 199-207, 2005*
    2005 [Not refereed][Not invited]
  • Hasegawa H, Ichinohe T, Strong P, Watanabe I, Ito S, Tamura S-i, Takahashi H, Sawa H, Chiba J, Kurata T, Sata T: Protection against influenza virus infection by intranasal administration of HA vaccine with chitin microparticles as an adjuvant. J Med V・・・
    2005 [Not refereed][Not invited]
     
    Hasegawa H, Ichinohe T, Strong P, Watanabe I, Ito S, Tamura S-i, Takahashi H, Sawa H, Chiba J, Kurata T, Sata T: Protection against influenza virus infection by intranasal administration of HA vaccine with chitin microparticles as an adjuvant. J Med Virol 75: 130-136, 2005
  • Henmi C, Sawa H*, Iwata H, Orba Y, Tanaka S, Nagashima K: Isolation of a monoclonal antibody recognizing a cell-surface molecule as a receptor for JC virus. Biochem Biophys Res Commun 327: 242-251, 2005 (* corresponding author)*
    2005 [Not refereed][Not invited]
  • Ichinohe T, Watanabe I, Ito S, Moriyama M, Tamura S, Takahashi H, Sawa H, Chiba J, Kurata T, Sata T, Hasegawa H: Synthetic double-stranded RNA [poly (I:C)] combined with mucosal vaccine protects against influenza virus infection. J Virol 79: 2910-2919・・・
    2005 [Not refereed][Not invited]
     
    Ichinohe T, Watanabe I, Ito S, Moriyama M, Tamura S, Takahashi H, Sawa H, Chiba J, Kurata T, Sata T, Hasegawa H: Synthetic double-stranded RNA [poly (I:C)] combined with mucosal vaccine protects against influenza virus infection. J Virol 79: 2910-2919, 2005*
  • Khalili K, White MK, Sawa H, Nagashima K, Safak M: The agnoprotein of polyomaviruses: A multifunctional auxiliary protein. J Cell Physiol 204:1-7, 2005*
    2005 [Not refereed][Not invited]
  • Okada Y, Suzuki T, Sunden Y, Orba Y, Kose S, Imamoto N, Takahashi H, Tanaka S, Hall WW, Nagashima K, Sawa H*: Dissociation of heterochromatin protein 1 from lamin B receptor induced by human polyomavirus agnoprotein: role in nuclear egress of viral par・・・
    2005 [Not refereed][Not invited]
     
    Okada Y, Suzuki T, Sunden Y, Orba Y, Kose S, Imamoto N, Takahashi H, Tanaka S, Hall WW, Nagashima K, Sawa H*: Dissociation of heterochromatin protein 1 from lamin B receptor induced by human polyomavirus agnoprotein: role in nuclear egress of viral particles. EMBO Rep 6: 452-457, 2005 (* corresponding author)*
  • Akazawa S, Igarashi M, Sawa H, Tamashiro H: Strategic approach to information security and assurance in health research. Environ Health Prev Med 10: 282-285, 2005
    2005 [Not refereed][Not invited]
  • Tsuda M, Watanabe T, Seki T, Kimura T, Sawa H, Minami A, Akagi T, Isobe KI, Nagashima K, Tanaka S: Induction of p21(WAF1/CIP1) by human synovial sarcoma-associated chimeric oncoprotein SYT-SSX1. Oncogene. 24: 7984-7990, 2005*
    2005 [Not refereed][Not invited]
  • 2005 [Not refereed][Not invited]
     
    Suzuki T, Okada Y, Semba S, Orba Y, Yamanouchi S, Endo S, Tanaka S, Fujita T, Kuroda S, Nagashima K, Sawa H*: Identification of FEZ1 as a protein that interacts with JC virus agnoprotein and microtubules: role of agnoprotein-induced dissociation of FEZ1 from microtubules in viral propagation. J Biol Chem 280, 24948-24956, 2005 (* corresponding author)*
  • M Jin, H Sawa, T Suzuki, K Shimizu, Y Makino, S Tanaka, T Nojima, Y Fujioka, M Asamoto, N Suko, M Fujita, K Nagashima
    JOURNAL OF MEDICAL VIROLOGY 74 (4) 668 - 676 0146-6615 2004/12 [Refereed][Not invited]
     
    It has been reported that Simian virus 40 (SV40) is linked to human beings by inoculation of contaminated poliovaccines and may have a role in the etiology of malignant mesothelioma. However, there have been no reports describing the relationship between SV40 and malignant mesothelioma in Japan. A study was undertaken to investigate whether SV40 was related to patients of malignant mesothelioma in Japan by the polymerase chain reaction (PCR) assay, DNA sequence analysis, and immunohistochemical methods. Paraffin-embedded samples of the 18 autopsied patients with pleural malignant mesothelioma were collected from five hospitals in Japan. After isolation of DNA from paraffin blocks, PCR analyses followed by sequencing were performed using three different sets of primers for detection of SV40 large T antigen (TAg) gene. All 18 malignant mesothelioma samples were also immunohistochemically evaluated for expression of SV40 TAg protein with two different anti-SV40 TAg antibodies. SV40 TAg genome was detected in eight malignant mesothelioma cases. Only one of three primer pairs successfully amplified SV40 genome in the samples, whereas all pairs yielded a PCR product in the controls, suggesting a low content of virus DNA. No immunopositive staining for SV40 TAg was found in any of the samples. This study shows that SV40 genome was present in a subset of Japanese malignant mesothelioma patients who were unlikely to have received a contaminated polio vaccine based on their age.
  • Nagashima T, Chuma T, Mano Y, Goto Y, Hayashi YK, Minami N, Nishino I, Nonaka I, Takahashi T, Sawa H, Aoki M, Nagashima K
    Neuropathology : official journal of the Japanese Society of Neuropathology 4 24 (4) 341 - 346 0919-6544 2004/12 [Refereed][Not invited]
  • M Tsuda, Y Makino, T Iwahara, H Nishihara, H Sawa, K Nagashima, H Hanafusa, S Tanaka
    JOURNAL OF BIOLOGICAL CHEMISTRY 279 (45) 46843 - 46850 0021-9258 2004/11 [Refereed][Not invited]
     
    Cell migration is a well organized process regulated by the extracellular matrix-mediated cytoskeletal reorganization. The signaling adaptor protein Crk has been shown to regulate cell motility, but its precise role is still under investigation. Herein, we report that Crk associates with ERM family proteins ( including ezrin, radixin, and moesin), activates RhoA, and promotes cell motility toward hyaluronic acid. The binding of Crk with ERMs was demonstrated both by transient and stable protein expression systems in 293T cells and 3Y1 cells, and it was shown that v-Crk translocated the phosphorylated form of ERMs to microvilli in 3Y1 cells by immunofluorescence and immunoelectron microscopy. This v-Crk-dependent formation of microvilli was suppressed by inhibitors of Rho-associated kinase, and the activity of RhoA was elevated by coexpression of c-Crk-II and ERMs in 3Y1 cells. In concert with the activation of RhoA by Crk, Crk was found to associate with Rho-GDI, which has been shown to bind to ERMs. Furthermore, upon hyaluronic acid treatment, coexpression of c-Crk-II and ERMs enhanced cell motility, whereas the sole expression of c-Crk-II or either of the ERMs decreased the motility of 3Y1 cells. These results suggest that Crk may be involved in regulation of cell motility by a hyaluronic acid-dependent mechanism through an association with ERMs.
  • H Aizawa, F Ohtani, Y Furuta, H Sawa, S Fukuda
    JOURNAL OF MEDICAL VIROLOGY 74 (2) 355 - 360 0146-6615 2004/10 [Refereed][Not invited]
     
    The mechanism by which reactivation of varicella-zoster virus (VZV) causes facial paralysis in Ramsay Hunt syndrome remains unclear. The relationship between VZV load and the onset of facial paralysis was analyzed in 42 patients with Ramsay Hunt syndrome. The patients were divided into three groups according to the times of appearance of zoster and of facial paralysis; group I (zoster preceding, n = 13), group II (simultaneous, n = 22), group III (paralysis preceding, n = 7). A real-time quantitative PCR assay was used to measure VZV DNA copy number in saliva, and paired sera were assayed for anti-VZV IgG and IgM antibodies. In group I, the VZV DNA-positive rate was low and virus load decreased gradually after the initial hospital visit around the time of onset of paralysis. The level of anti-VZV antibodies had in most cases already increased at that time. In group III, viral load tended to increase after the onset of paralysis and peaked around the time of appearance of zoster. The level of anti-VZV antibodies was low at the onset of paralysis but showed a significant increase when paired sera were tested. In group II, virus load and changes in level of anti-VZV antibodies either resembled group I or group III behavior. These results indicate that facial paralysis in Ramsay Hunt syndrome can occur at various times between the early and the regression phase of VZV reactivation, suggesting that there are variable patterns of development of facial nerve dysfunction caused by VZV reactivation and the progression of neuritis.
  • Y Kamioka, S Fukuhara, H Sawa, J Nagashima, M Masuda, M Matsuda, N Mochizuki
    JOURNAL OF BIOLOGICAL CHEMISTRY 279 (38) 40091 - 40099 0021-9258 2004/09 [Refereed][Not invited]
     
    Dynamin associates with a variety of SH3 domain-containing molecules via a C-terminal proline-rich motif and takes part, with them, in endocytic processes. Here, we have investigated a new dynamin-associating molecule, formin-binding protein 17 (FBP17), involved in deforming the plasma membrane and in endocytosis. FBP17 formed tubular invaginations originating from the plasma membrane. Its N-terminal Fer/CIP4 homology domain, a coiled-coil domain, and a proline-rich motif were required for tubular invagination and self-assembly, by which tubular invagination might be induced. Using anti-FBP17 antibody, we detected positive immunoreactions in the testis that were restricted to the germ cells. We also detected FBP17 in the brain by immunoblotting and in situ hybridization. When COS cells expressing enhanced green fluorescent protein-tagged FBP17 were incubated with fluorescently labeled transferrin, epidermal growth factor, and cholera toxin, these molecules co-localized with FBP17-induced tubular invaginations, suggesting that FBP17 is involved in dynamin-mediated endocytosis in both a clathrin-dependent and -independent manner. These observations therefore indicate that FBP17 interacts with dynamin and regulates endocytosis by forming vesicotubular structures.
  • Y Furuta, H Aizawa, F Ohtani, H Sawa, S Fukuda
    ANNALS OF OTOLOGY RHINOLOGY AND LARYNGOLOGY 113 (9) 700 - 705 0003-4894 2004/09 [Refereed][Not invited]
     
    We have investigated whether the copy number of varicella-zoster virus (VZV) in saliva correlates with the clinical symptoms in patients with Ramsay Hunt syndrome. A real-time quantitative polymerase chain reaction assay was used to examine the VZV DNA copy number in saliva samples from 37 patients. We detected VZV DNA in 6 of the 7 patients with oropharyngeal zoster lesions (86%) and in 17 of the 30 patients who had zoster lesions only on the skin (57%). Patients with oropharyngeal zoster lesions had a high VZV load in their saliva, and the difference between the copy number in patients with oropharyngeal zoster lesions and those without was around 10,000 copies per 50 muL. In addition, patients with oropharyngeal zoster lesions showed worse recovery of facial function than those without. It seems that the VZV DNA level in saliva reflects the kinetics of viral reactivation in the facial nerve, as well as in the oropharyngeal epithelium, in patients with Ramsay Hunt syndrome.
  • K Chikai, A Ohnishi, T Kato, J Ikeda, Y Sawamura, Y Iwasaki, T Itoh, H Sawa, K Nagashima
    ACTA NEUROPATHOLOGICA 108 (2) 109 - 114 0001-6322 2004/08 [Refereed][Not invited]
     
    Five cases of pilomyxoid astrocytoma (PmA) characterized by a monophasic pattern with a myxoid background were selected for a clinicopathological study from 23 cases previously diagnosed as pilocytic astrocytoma (PA). All PmA patients were either infants or young children (mean age 2.1 years), and all tumors were located in the optic chiasm/hypothalamus region. All cases received chemotherapy, which reduced tumor size, and the location of the tumor became confined to the optic chiasm. In two cases, tumor recurrence occurred 3 and 7 years after chemotherapy. Histology of the recurrent tumors showed the biphasic pattern of classical PA. Hence, we conclude that PmA might be an infantile form of PA and speculate that a subset of PmA in the optic pathway/hypothalamus originates from the optic chiasm, possibly derived from radial glia existing in the embryonic optic chiasm.
  • T Minamitani, T Ikuta, Y Saito, G Takebe, M Sato, H Sawa, T Nishimura, F Nakamura, K Takahashi, H Ariga, K Matsumoto
    EXPERIMENTAL CELL RESEARCH 298 (1) 305 - 315 0014-4827 2004/08 [Refereed][Not invited]
     
    Tenascin-X (TNX) is an extracellular matrix glycoprotein. We previously demonstrated that TNX regulates the expression of type VI collagen. In this study, we investigated the binding of TNX to type I collagen as well as to type VI collagen and the effects of these proteins on fibrillogenesis of type I collagen. Full-length recombinant TNX, which is expressed in and purified from mammalian cell cultures, and type VI collagen purified from bovine placenta were used. Solid-phase assays revealed that TNX or type VI collagen bound to type I collagen, although TNX did not bind to type VI collagen, fibronectin, or laminin. The rate of collagen fibril formation and its quantity, measured as increased turbidity, was markedly increased by the presence of TNX, whereas type VI collagen did not increase the quantity but accelerated the rate of collagen fibril formation. Combined treatment of both had an additive effect on the rate of collagen fibril formation. Furthermore, deletion of the epidermal growth factor-like (EGF) domain or fibrinogen-like domain of TNX attenuated the initial rate of collagen fibril formation. Finally, we observed abnormally large collagen fibrils by electron microscopy in the skin from TNX-deficient (TNX-/-) mice during development. These findings demonstrate a fundamental role for TNX and type VI collagen in regulation of collagen fibrillogenesis in vivo and in vitro. (C) 2004 Elsevier Inc. All rights reserved.
  • Matsumoto K, Sato T, Oka S, Orba Y, Sawa H, Kabayama K, Inokuchi J, Ariga H
    Genes to cells : devoted to molecular & cellular mechanisms 8 9 (8) 737 - 748 1356-9597 2004/08 [Refereed][Not invited]
  • Y Orba, H Sawa, H Iwata, S Tanaka, K Nagashima
    JOURNAL OF VIROLOGY 78 (13) 7270 - 7273 0022-538X 2004/07 [Refereed][Not invited]
     
    RNA interference has been applied for the prevention of virus infections in mammalian cells but has not succeeded in eliminating infections from already infected cells. We now show that the transfection of JC virus-infected SVG-A human glial cells with small interfering RNAs that target late viral proteins, including agnoprotein and VP1, results in a marked inhibition both of viral protein expression and of virus production. RNA interference directed against JC virus genes may thus provide a basis for the development of new strategies to control infections with this polyomavirus.
  • K Matsumoto, T Minamitani, Y Orba, M Sato, H Sawa, H Ariga
    EXPERIMENTAL CELL RESEARCH 297 (2) 404 - 414 0014-4827 2004/07 [Refereed][Not invited]
     
    The results of our previous study showed that tumor invasion and metastasis are promoted in extracellular matrix (ECM) tenascin-X-deficient (TNX-/-) mice via increased expression of matrix metalloproteinases (MMPs). However, little is known about the relationship between TNX deficiency and activation of MMP genes. In this study, we investigated the molecular mechanism by which TNX deficiency activates the MMP-2 gene. We examined the intracellular signaling pathways that regulate gene expression of the proteinase in isolated fibroblasts. Results of gelatin zymography showed that MMP-2 was induced to a greater extent in TNX-/- fibroblasts embedded in type I collagen than in wild-type fibroblasts. RT-PCR analysis revealed that the increased level of MMP-2 expression was caused at the transcription level. Conversely, stable overexpression of TNX in a fibroblast cell line reduced MMP-2 expression and suppressed MMP-2 promoter activity. In addition, treatment of TNX-/- fibroblasts with SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, and genistein, a tyrosine kinase inhibitor, suppressed the increased level of proMMP-2 and increased MMP-2 promoter activity in TNX-/- fibroblasts. Furthermore, increased activation of JNK and tyrosine phosphorylation of certain proteins were observed in TNX-/- fibroblasts. These findings suggest that induction of MMP-2 by TNX deficiency is mediated, at least in part, through the JNK and protein tyrosine kinase phosphorylation pathway. (C) 2004 Elsevier Inc. All rights reserved.
  • QM Qu, H Sawa, T Suzuki, S Semba, C Henmi, Y Okada, M Tsuda, S Tanaka, WJ Atwood, K Nagashima
    JOURNAL OF BIOLOGICAL CHEMISTRY 279 (26) 27735 - 27742 0021-9258 2004/06 [Refereed][Not invited]
     
    JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and causes progressive multifocal leukoencephalopathy in humans. Although transport of virions to the nucleus is an important step in JCV infection, the mechanism of this process has remained unclear. The outer shell of the JCV virion comprises the major capsid protein VP1, which possesses a putative nuclear localization signal (NLS), and virus-like particles (VLPs) consisting of recombinant VP1 exhibit a virion-like structure and physiological functions ( cellular attachment and intracytoplasmic trafficking) similar to those of JCV virions. We have now investigated the mechanism of nuclear transport of JCV with the use of VLPs. Wild-type VLPs (wtVLPs) entered the nucleus of most HeLa or SVG cells. The virion structure of VLPs was preserved during transport to the nucleus as revealed by confocal microscopy of cells inoculated with fluorescein isothiocyanate-labeled wtVLPs containing packaged Cy3. The nuclear transport of wtVLPs in digitonin-permeabilized cells was dependent on the addition of importins alpha and beta and was prevented by wheat germ agglutinin or by antibodies to the nuclear pore complex. The nuclear entry of VLPs composed of VP1 with a mutated NLS was greatly inhibited, compared with that of wtVLPs, in both intact and permeabilized cells. Unlike wtVLPs, the mutant VLPs did not bind to importins alpha or beta. Limited proteolysis analysis revealed that the NLS of VP1 was exposed on the surface of wtVLPs. These results suggest that JCV VLPs bind to cellular importins via the NLS of VP1 and are transported into the nucleus through the nuclear pore complex.
  • H Takahashi, H Sawa, H Hasegawa, K Nagashima, T Sata, T Kurata
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 313 (4) 1073 - 1078 0006-291X 2004/01 [Refereed][Not invited]
     
    Both HIV-1 reverse transcriptase (RT) and topoisomerase I bind to structural RNAs and they cooperate to synthesize cDNA during the replication of HIV-1. In this study, we find that human topoisomerase I exclusively dissociated HIV-1 reverse transcriptase, which strongly binds to structural RNAs. Meanwhile, topoisomerase I did not dissociate either HIV-1 nucleocapsid proteins or murine leukemia virus RT which was bound to RNA. We propose that human topoisomerase I may regulate the binding of RT to RNAs and play a pivotal role in HIV-1 replication. (C) 2003 Elsevier Inc. All rights reserved.
  • Nucleolin and the packaging signal, psi, promote the budding of human immunodeficiency virus type-1 (HIV-1)
    T Ueno, K Tokunaga, H Sawa, M Maeda, J Chiba, A Kojima, H Hasegawa, Y Shoya, T Sata, T Kurata, H Takahashi
    MICROBIOLOGY AND IMMUNOLOGY 48 (2) 111 - 118 0385-5600 2004 [Refereed][Not invited]
     
    Gag proteins of human immunodeficiency virus type 1 (HIV-1) play a pivotal role in the budding of the virion, in which the zinc finger motifs of the gag proteins recognize the packaging signal of genomic RNA. Nucleolin, an RNA-binding protein, is identified as a cellular protein that binds to murine leukemia virus (MuLV) gag proteins and regulates the viral budding, suggesting that HIV-1 gag proteins, the packaging signal, psi and nucleolin affect the budding of HIV-1. Here we report that nucleolin enhances the release of HIV-1 virions which contain psi. Furthermore, nucleolin and gag proteins form a complex incorporated into virions, and nucleolin promotes the infectivity of HIV-1. Our results suggest that an empty particle which contains neither nucleolin nor the genomic RNA is eliminated during the budding process, and this mechanism is beneficial for escape from the host immune response against HIV-1.
  • Qu Q, Sawa H*, Suzuki T, Semba S, Henmi C, Okada Y, Tsuda M, Tanaka S, Nagashima K: Nuclear entry mechanism of the human polyomavirus JC virus like particle: role of importins and the nuclear pore complex. J Biol Chem 279: 27735-27742, 2004 (* corresp・・・
    2004 [Not refereed][Not invited]
     
    Qu Q, Sawa H*, Suzuki T, Semba S, Henmi C, Okada Y, Tsuda M, Tanaka S, Nagashima K: Nuclear entry mechanism of the human polyomavirus JC virus like particle: role of importins and the nuclear pore complex. J Biol Chem 279: 27735-27742, 2004 (* corresponding author)*
  • Minamitani T, Ikuta T, Saito Y, Takebe G, Sato M, Sawa H, Nishimura T, Nakamura F, Takahashi K, Ariga H, Matsumoto K: Modulation of collagen fibrillogenesis by tenascin-X and type VI collagen. Exp Cell Res 298: 305-15, 2004*
    2004 [Not refereed][Not invited]
  • Chikai K, Ohnishi A, kato T, Ikeda J, Sawamura Y, Iwasaki Y, Itoh T, Sawa H, Nagashima K: Clinico-pathological features of piloymyxoid astrocytoma of the optic pathway. Acta Neuropathol 108: 109-114, 2004*
    2004 [Not refereed][Not invited]
  • Kamioka Y, Fukuhara S, Sawa H, Nagashima K, Masuda M, Matsuda M, Mochizuki N: A novel dynamin-associating molecule, formin-binding Protein 17, induces tubular membrane invaginations and participates in endocytosis. J Biol Chem 279: 40091-40099, 2004*
    2004 [Not refereed][Not invited]
  • Matsumoto K, Minamitani T, Orba Y, Sato M, Sawa H, Ariga H: Induction of matrix metalloproteinase-2 by tenascin-X deficiency is mediated through the c-Jun N-terminal kinase and protein tyrosine kinase phosphorylation pathway. Exp Cell Res 297: 404-414・・・
    2004 [Not refereed][Not invited]
     
    Matsumoto K, Minamitani T, Orba Y, Sato M, Sawa H, Ariga H: Induction of matrix metalloproteinase-2 by tenascin-X deficiency is mediated through the c-Jun N-terminal kinase and protein tyrosine kinase phosphorylation pathway. Exp Cell Res 297: 404-414, 2004*
  • Tsuda M, Makino Y, Iwahara T, Nishihara H, Sawa H, Nagashima K, Hanafusa H, Tanaka S: Crk associates with ERM proteins and promotes cell motility toward hyaluronic acid. J Biol Chem 279: 46843-46850, 2004*
    2004 [Not refereed][Not invited]
  • Orba Y, Sawa H*, Iwata H, Tanaka S, Nagashima K: Inhibition of virus production in JC virus-infected cells by postinfection RNA interference. J Virol 78: 7270-7273, 2004 (* corresponding author)*
    2004 [Not refereed][Not invited]
  • Furuta Y, Aizawa H, Ohtani F, Sawa H, Fukuda S: Varicella-zoster virus DNA level and facial paralysis in Ramsay Hunt syndrome. Ann Otol Rhinol Laryngol 113: 700-705, 2004.*
    2004 [Not refereed][Not invited]
  • Jin M, Sawa H*, Suzuki T, Shimizu K, Makino Y, Tanaka S, Nojima T, Fujioka Y, Asamoto M, Suko N, Nagashima K: Investigation of simian virus 40 large T antigen in 18 autopsied malignant mesothelioma patients in Japan. J Med Virol 74: 668-676, 2004 (* ・・・
    2004 [Not refereed][Not invited]
     
    Jin M, Sawa H*, Suzuki T, Shimizu K, Makino Y, Tanaka S, Nojima T, Fujioka Y, Asamoto M, Suko N, Nagashima K: Investigation of simian virus 40 large T antigen in 18 autopsied malignant mesothelioma patients in Japan. J Med Virol 74: 668-676, 2004
    (* corresponding author)*
  • Aizawa H, Ohtani F, Furuta Y, Sawa H, Fukuda S: Variable patterns of varicella-zoster virus reactivation in Ramsay Hunt syndrome. J Med Virol 74: 355-360, 2004.*
    2004 [Not refereed][Not invited]
  • Ueno T, Tokunaga K, Sawa H, Maeda M, Chiba J, Kojima A, Hasegawa H, Shoya Y, Sata T, Kutrata T, Takahashi H: Nucleolin and the packaging signal,  promote the budding of human immunodeficiency virus type-1 (HIV-1). Microbiol Immunol 48: 111-118, 2004*
    2004 [Not refereed][Not invited]
  • Takahashi H, Sawa H, Hasegawa H, Nagashima K, Sata T, Kurata T: Topoisomerase I dissociates human immunodeficiency virus type 1 reverse transcriptase from genomic RNAs. Biochem Biophys Res Commun. 313: 1073-1078, 2004.*
    2004 [Not refereed][Not invited]
  • Nagashima T, Chuma T, Mano Y, Goto Y-i, Hayashi KY, Minami N, Nishino I, Nonaka I, Takahashi T, Sawa H, Aoki M, Nagashima K: Dysferlinopathy associated with rigid spine syndrome. Neuropathology 24: 341-346, 2004.*
    2004 [Not refereed][Not invited]
  • Chikai K, Ohnishi A, kato T, Ikeda J, Sawamura Y, Iwasaki Y, Itoh T, Sawa H, Nagashima K: Clinico-pathological features of piloymyxoid astrocytoma of the optic pathway. Acta Neuropathol 108: 109-114, 2004*
    2004 [Not refereed][Not invited]
  • T Teramoto, H Kaneko, M Funato, H Sawa, K Nagashima, Y Hirose, N Kondo
    SCANDINAVIAN JOURNAL OF INFECTIOUS DISEASES 35 (11-12) 909 - 910 0036-5548 2003/12 [Refereed][Not invited]
     
    To our knowledge, this is the first case report describing progressive multifocal leukoencephalopathy (PML) associated with X-linked agammaglobulinemia. The JC virus was confirmed at autopsy and PML was diagnosed. Humoral immunodeficiency with normal cellular immunity could be infected with JCV.
  • Watanabe, I, TM Ross, S Tamura, T Ichinohe, S Ito, H Takahashi, H Sawa, J Chiba, T Kurata, T Sata, H Hasegawa
    VACCINE 21 (31) 4532 - 4538 0264-410X 2003/11 [Refereed][Not invited]
     
    For the induction of mucosal immune responses by intranasal vaccination, cholera toxin B subunits (CTB) and Escherichia coli heat-labile toxin (I-T) are often administered as mucosal adjuvants in order to enhance immune responses to mucosally co-administered bystander antigens. However, these toxin also are the causative agents of diarrhea. There is a demand for the establishment of an effective and safer adjuvant or vaccine that elicits mucosal immunity, but does not require the use of CTB or LT adjuvants. In order to induce protective mucosal immune responses in the nasal area against influenza virus infection, we have examined the recombinant protein composed of the complement component, CM, which is fused to the secreted form of hemagglutinin (sHA-mC3d(3)) in the influenza-BALB/c mouse model. The fusion protein sHA-mC3d(3), the secretory form of hemagglutinin, and the transmembrane form of HA (tmHA) from the influenza virus were intranasally administered to the mice with or without CTB containing a trace amount of holotoxin (CTB*) as an adjuvant. After intranasal administration of these proteins with CTB*, all mice produced nasal IgA and serum IgG antibodies (Abs) against the viral HA. In addition, viral infection was completely inhibited in these mice. In contrast, in the absence of the adjuvant, only sHA-mC3d(3)-induced locally secreted IgA and serum IgG Abs and provided complete protection against the influenza virus challenge. Thus, C3d fused to the influenza HA antigen is an effective and safe tool for mucosal vaccination. (C) 2003 Elsevier Ltd. All rights reserved.
  • Induction of chromosomal instability in colonic cells by the human polyomavirus JC virus
    L Ricciardiello, M Baglioni, C Giovannini, M Pariali, G Cenacchi, A Ripalti, MP Landini, H Sawa, K Nagashima, RJ Frisque, A Goel, CR Boland, M Tognon, E Roda, F Bazzoli
    CANCER RESEARCH 63 (21) 7256 - 7262 0008-5472 2003/11 [Refereed][Not invited]
     
    Most colorectal cancers display chromosomal instability, which is characterized by gross chromosomal rearrangements, loss of heterozygosity and aneuploidy. We have previously demonstrated a link between JC virus strains Mad-1 and Delta98 and colorectal cancer. Others have also associated the virus to the induction of colon cancer and aneuploid brain tumors by producing a highly tumorigenic protein named T antigen (TAg), which binds to beta-catenin and inactivates key proteins such as p53. The aim is to demonstrate that JC virus is capable of inducing chromosomal instability in colonic cells. We used the human colon cancer cell line RKO as a model. The cell line has wild-type p53, wild-type beta-catenin and APC and is diploid. Neuroblastoma JCI cells, which are infected with the virus, VA13 fibroblasts, which are transformed by the SV40 TAg, were used as positive controls. HCT116, which has mutated P-catenin, and SW480, which is a model of CIN, were also used as controls. The genomes of the Mad-1 and Delta98 strains were transfected into cells. As negative controls we used pUC or no plasmids. Cells were collected at 0, 7, 14, and 21 days after transfection. PCR was used for the detection of TAg and the regulatory region DNA sequences at different time frames and Southern blot of whole genomic extracts for viral DNA integration into the host genome. Immuntifluorescence and Western blot were performed for TAg, viral capsid proteins, and nuclear beta-catenin expressions, whereas coimmunoprecipitation was used to detect protein interactions. Karyotype analysis and electron microscopy were performed to seek chromosomal instability and cell abnormalities, respectively. Retention of viral sequences was observed for Mad-1- and Delta98-transfected RKO cells at all time frames with PCR only, whereas Southern blot analysis showed nonintegrated sequences at T7 alone. TAg and capsid protein expressions, as well as increased p53 and nuclear beta-catenin, were observed between TO and T7 for Mad-l and Delta98 alone. Also, interaction between TAg and both p53 and beta-catenin was also observed between TO and T7. Chromosomal instability, characterized by chromosomal breakage, dicentric chromosomes, and increasing ploidy, was observed at all time frames for Mad-l and Delta98, as well as cell abnormalities. In conclusion, we demonstrate that JC virus Mad-1 and Delta98 are able to induce chromosomal instability in colonic cells with a hit and run mechanism that involves an early interaction with beta-catenin and p53.
  • Expression of the oligodendroglial lineage-associated markers Olig1 and Olig2 in different types of human gliomas
    A Ohnishi, H Sawa, M Tsuda, Y Sawamura, T Itoh, Y Iwasaki, K Nagashima
    JOURNAL OF NEUROPATHOLOGY AND EXPERIMENTAL NEUROLOGY 62 (10) 1052 - 1059 0022-3069 2003/10 [Refereed][Not invited]
     
    Because a specific group of oligodendrogliomas is susceptible to adjuvant therapy, it is important to elucidate the biological characteristics of these tumors. In situ hybridization analyses have revealed that Olig genes are expressed in oligodendroglial lineage cells and are highly expressed in oligodendrogliomas. To clarify whether OLIG is a tumor-specific marker for oligodendrogliomas, we have investigated the expression of Olig transcripts by semiquantitative RT-PCR assay and OLIG2 protein with a new antibody in a variety of glial tumors. The semiquantitative RT-PCR revealed that high levels of expression of Olig1 and Olig2 mRNAs were present in anaplastic oligodendrogliomas and anaplastic astrocytomas, while expression of these mRNAs in grade IV glioblastomas was lower than in grade II and grade III gliomas (p < 0.01). Immunohistochemical analyses demonstrated that the mean immunopositive proportion of OLIG2 was 82% in anaplastic oligodendrogliomas but only 34% in anaplastic astrocytomas. Therefore, although OLIG2 expression was detected in a range of gliomas not specific for oligodendrogliomas, the expression level in anaplastic oligodendrogliomas was more uniform and intense than that in other glial tumors. In conclusion, combining Olig mRNA expression and immunohistochemistry of OLIG2 enables oligodendrogliomas to be distinguished from glioblastomas and other astrocytic glial tumors.
  • Nagashima T, Mizutani Y, Kawahara H, Maguchi S, Terayama Y, Shinohara T, Orba Y, Chuma T, Mano Y, Itoh T, Sawa H, Sakai K, Motomura M, Nagashima K
    Neuropathology : official journal of the Japanese Society of Neuropathology 3 23 (3) 230 - 238 0919-6544 2003/09 [Refereed][Not invited]
  • Y Orba, S Tanaka, H Nishihara, N Kawamura, T Itoh, M Shimizu, H Sawa, K Nagashima
    CANCER CYTOPATHOLOGY 99 (4) 198 - 204 0008-543X 2003/08 [Refereed][Not invited]
     
    BACKGROUND. The demonstration of the monoclonality of immunoglobulin heavy chain (IgH) gene rearrangement is an indispensable method for the diagnosis of B-cell lymphoma as well as histocytochemical analysis. For the detection of IgH gene rearrangement, the extraction of DNA from a homogenous cell population is necessary. Recently, the laser capture microdissection (LCM) technique was shown to isolate specific cells from histopathologic specimens for molecular analysis. However, to the authors' knowledge the applicability of LCM to cytologic specimens has not yet been well established. METHODS. Using LCM, a homogenous population of B-cell lymphoma cells as both histologic sections and cytologic specimens was captured, and genomic DNA was extracted from the captured cells. IgH gene rearrangement was analyzed by the polymerase chain reaction (PCR)-based single-strand conformational polymorphism (SSCP) method. RESULTS. Genomic DNAs were extracted successfully from ethanol-fixed cytologic specimens, but cells were not captured from air-dried specimens. Using PCR-SSCP analysis, the monoclonality of the IgH gene rearrangement was detected in five cases of tissue sections among nine analyzed cases of malignant lymphoma diagnosed immunohistochemically. However, analysis of the cytologic specimens with LCM demonstrated the monoclonality of the IgH gene rearrangement in seven cases of lymphoma. CONCLUSIONS. The results of the current study suggest that the novel application of LCM to cytologic specimens occasionally exhibits high sensitivity for the detection of IgH gene rearrangement monoclonality compared with the use of histologic sections.
  • H Ogita, S Kunimoto, Y Kamioka, H Sawa, M Masuda, N Mochizuki
    CIRCULATION RESEARCH 93 (1) 23 - 31 0009-7330 2003/07 [Refereed][Not invited]
     
    Rho-kinase, an effector of Rho GTPase, increases the contractility of vascular smooth muscle by phosphorylating myosin light chain ( MLC) and by inactivating MLC phosphatase. A wide variety of extracellular stimuli activate RhoA via G protein-coupled receptors. In the present study, we demonstrate a novel cell-cell interaction - mediated Rho activation signaling pathway in vascular smooth muscle cells (VSMCs). Among many receptor tyrosine kinases, the Eph family receptors are unique in that they require cell-cell interaction to engage their ligands, ephrin. We found that a novel VSMC-specific guanine nucleotide exchange factor (GEF) for Rho ( Vsm-RhoGEF/KIAA0915) was expressed specifically in VSMCs of several organs including the heart, aorta, liver, kidney, and spleen, as examined by the immunohistochemical analysis using a specific antibody against Vsm-RhoGEF. Based on the association of Vsm-RhoGEF with EphA4 in quiescent cells, we tested whether EphA4 and Vsm-RhoGEF were expressed in the same tissue and further studied the molecular mechanism of Vsm-RhoGEF regulation by EphA4. Immunohistochemical analysis showed that EphA4 and Vsm-RhoGEF expression overlapped in VSMCs. Additionally, tyrosine phosphorylation of Vsm-RhoGEF induced by EphA4 upon ephrin-A1 stimulation enhanced the Vsm-RhoGEF activity for RhoA. The requirement of Vsm-RhoGEF for ephrin-A1 - induced assembly of actin stress fibers in VSMCs was shown by the overexpression of a dominant-negative form of VSM-RhoGEF and by the depletion of Vsm-RhoGEF using RNA interference. These results suggested that ephrin-A1 - triggered EphA4-Vsm-RhoGEF-RhoA pathway is involved in the cell-cell interaction - mediated RhoA activation that regulates vascular smooth muscle contractility.
  • Y Shoya, K Tokunaga, H Sawa, M Maeda, T Ueno, T Yoshikawa, H Hasegawa, T Sata, T Kurata, WW Hall, BR Cullen, H Takahashi
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 100 (14) 8442 - 8447 0027-8424 2003/07 [Refereed][Not invited]
     
    Although HIV type 1 (HIV-1) cannot efficiently replicate in simian cells, the mechanism(s) involved in the restriction of virus tropism remain unclear. To investigate this, we have focused on the identification of human cellular factors that can influence the infectivity of HIV-1 derived from African green monkey producer cells. Whereas the infectivity of HIV-1 derived from such cells was only 10-15% of that of human cell-derived virus, expression of human topoisomerase I in the African green monkey cells resulted in a 5-fold increase of the infectivity of progeny HIV-1 virions. Replacement of glutamate-236 and asparagine-237 of human topoisomerase I with the corresponding residues (aspartate and serine, respectively) of the African green monkey enzyme abolished this enhancement of HIV-1 infectivity. This positive effect of human topoisomerase I expression in the African green monkey producer cells seemed to result from the promotion of HIV-1 cDNA synthesis. Thus, human topoisomerase I plays an important role in HIV-1 replication and infectivity, and differences in the species specificity of HIV-1 infection can at least in part be attributed to differences in topoisomerase I activities.
  • R Ishikawa, H Kikuchi, ML Jin, M Fujita, T Itoh, H Sawa, K Nagashima
    PATHOLOGY INTERNATIONAL 53 (6) 401 - 406 1320-5463 2003/06 [Refereed][Not invited]
     
    Desmoplastic mesothelioma is a rare subtype of diffuse malignant mesothelioma, and is often difficult to distinguish from reactive pleural fibrosis because of associated extensive collagen fibrosis. An 82-year-old woman with a severe cough was revealed to have pleural effusion and diffuse pleural thickening on the right side. Antibiotics were ineffective, and a compression fracture of the ninth and tenth thoracic vertebral bodies was recognized on X-ray. Autopsy revealed a diffuse pleural thickening with hyalinized collagen tissue in the central part of the pleura. However, the peripheral part of the fibrous tissue was composed of spindle and polygonal cell proliferation that were immunohistochemically positive for antibodies against cytokeratin and vimentin. In addition, the ninth and tenth thoracic spines were infiltrated by similar cells. The condition was diagnosed as desmoplastic mesothelioma with bone metastases. Asbestos bodies were detected in the thickened pleura and fibrosed alveolar septa, and it was suggested retrospectively that the patient had been exposed to asbestos. Thus, autopsy analyses of fibrous pleurisy are necessary to detect a desmoplastic variant of mesothelioma of the pleura and its association with asbestos exposure.
  • E Higuchi, N Oridata, Y Furuta, S Suzuki, H Hatakeyama, H Sawa, K Sunayashiki-Kusuzaki, K Yamazaki, Y Inuyama, S Fukuda
    HEAD AND NECK-JOURNAL FOR THE SCIENCES AND SPECIALTIES OF THE HEAD AND NECK 25 (3) 187 - 193 1043-3074 2003/03 [Refereed][Not invited]
     
    Background. The mechanism by which cancer cells become resistant to cis-Diamminedichloroplatinum (II) (cDDP) is not completely understood. To investigate the molecular markers involved in the cDDP resistance, we compared the gene expression profiles between a head and neck squamous cell carcinoma (HNSCC) line sensitive to cDDP and its cDDP-resistant variant. Methods. Both a fluorescent differential display and a cDNA microarray analysis were applied to distinguish the gene profiles between KB, a human HNSCC line, and its cDDP-resistant variant (KB/cDDP). These results were confirmed by Northern blot analysis. Results. One up-regulated gene, glycoprotein hormone alpha-subunit, and two down-regulated genes coding membrane proteins, human folate receptor and tumor-associated antigen L6, were identified in KB/cDDP cells. Conclusions. Our findings suggest that development of the cDDP-resistant phenotype is accompanied by alternations of gene expression including a glycoprotein hormone and membrane proteins. These gene products could be new molecular markers for resistance to cDDP. (C) 2003 Wiley Periodicals, Inc.
  • Ishikawa R, Kikuchi H, Jin M, Fujita M, Itoh T, Sawa H, Nagashima K: Desmoplastic malignant mesothelioma of the pleura: Autopsy reveals asbestos exposure. Pathol Int 53: 401-406, 2003*
    2003 [Not refereed][Not invited]
  • Orba Y, Nishihara H, Sawa H, Itoo T, Shimizu M, Tanaka S, Nagashima K: Application of laser capture microdissection on cytological specimens for detection of immunoglobulin heavy chain gene rearrangement of malignant lymphoma. Cancer (Cancer Cytopatho・・・
    2003 [Not refereed][Not invited]
     
    Orba Y, Nishihara H, Sawa H, Itoo T, Shimizu M, Tanaka S, Nagashima K: Application of laser capture microdissection on cytological specimens for detection of immunoglobulin heavy chain gene rearrangement of malignant lymphoma. Cancer (Cancer Cytopathol) 99: 198-204, 2003*
  • Nagashima T, Mizutani Y, Kawahara H, Maguchi S, Terayama Y, Shinohara T, Orba Y, Chuma T, Mano Y, Itoh T, Sawa H, Sakai K, Motomura M, Nagashima K. Anti-Hu paraneoplastic syndrome presenting with brainstem-cerebellar symptoms and Lambert-Eaton myasthen・・・
    2003 [Not refereed][Not invited]
     
    Nagashima T, Mizutani Y, Kawahara H, Maguchi S, Terayama Y, Shinohara T, Orba Y, Chuma T, Mano Y, Itoh T, Sawa H, Sakai K, Motomura M, Nagashima K. Anti-Hu paraneoplastic syndrome presenting with brainstem-cerebellar symptoms and Lambert-Eaton myasthenic syndrome. Neuropathology 23: 230-238, 2003*
  • Endo S, Okada Y, Orba Y, Nishihara H, Tanaka S, Nagashima K, Sawa H*: JC virus (JCV) agnoprotein colocalizes with tubulin. J Neurovirol 9 (Suppl 1): 10-14, 2003 (*corresponding author)*
    2003 [Not refereed][Not invited]
  • Higuchi E, Oridate N, Furuta Y, Suzuki S, Hatakeyama H, Sawa H, Sunayashiki-Kusazaki K, Yamazaki-k, Inuyama-Y, Fukuda S: Differentially expressed genes associated with cis-diammeinedichloroplatinum (II) resistance in head and neck cancer using differen・・・
    2003 [Not refereed][Not invited]
     
    Higuchi E, Oridate N, Furuta Y, Suzuki S, Hatakeyama H, Sawa H, Sunayashiki-Kusazaki K, Yamazaki-k, Inuyama-Y, Fukuda S: Differentially expressed genes associated with cis-diammeinedichloroplatinum (II) resistance in head and neck cancer using differential display and cDNA microarray. Head Neck 25:187-93, 2003.*
  • Ricciardiello L, Baglioni M, Giovannini C, Pariali M, Cenacchi G, Ripalti A, Landini MP, Sawa H, Nagashima K, Frisque RJ, Goel A, Boland CR, Tognon M, Roda E, Bazzoli F: Induction of Chromosomal Instability in Colonic Cells by the Human Polyomavirus JC・・・
    2003 [Not refereed][Not invited]
     
    Ricciardiello L, Baglioni M, Giovannini C, Pariali M, Cenacchi G, Ripalti A, Landini MP, Sawa H, Nagashima K, Frisque RJ, Goel A, Boland CR, Tognon M, Roda E, Bazzoli F: Induction of Chromosomal Instability in Colonic Cells by the Human Polyomavirus JC Virus. Cancer Res 63:7256-62, 2003*
  • Ohnishi A, Sawa H, Tsuda M, Sawamura Y, Marukawa K, Iwasaki Y, Nagashima K: Expression of the oligodendroglial lineage-associated markers Olig1 and Olig2 in different types of human gliomas. J Neuropathol Exp Neurol 62: 1052-1059, 2003*
    2003 [Not refereed][Not invited]
  • Shoya Y, Tokunaga T, Sawa H, Maeda M, Ueno T, Yoshikawa T, Sata T, Kurata T, Hall WW, Cullen BR, Takahashi H: Human topoisomerase I promotes HIV-1 proviral DNA synthesis: implications for the species specificity and cellular tropism of HIV-1 infection.・・・
    2003 [Not refereed][Not invited]
     
    Shoya Y, Tokunaga T, Sawa H, Maeda M, Ueno T, Yoshikawa T, Sata T, Kurata T, Hall WW, Cullen BR, Takahashi H: Human topoisomerase I promotes HIV-1 proviral DNA synthesis: implications for the species specificity and cellular tropism of HIV-1 infection. Proc Natl Acad Sci USA 100: 8442-8447, 2003*
  • Yamamoto S, Furukawa H, Kitamoto T, Takamaru Y, Morita N, Yasuda M, Okada Y, Sawa H, Nagashima K: An atypical form of sporadic panencephalopathic Cleutzfeldt-Jakob disease in Japan. Neuropathol Appl Neurobiol 29: 77-80, 2003*
    2003 [Not refereed][Not invited]
  • Teramoto T, Kaneko H, Futano M, Sawa H, Nagashima K, Hirose Y, Kondo N: Progressive multifocal leukoencephalopathy in a patient with X-linked agammaglobulinemia. Scand J Infect Dis 35: 910-911, 2003*
    2003 [Not refereed][Not invited]
  • S Endo, Y Okada, Y Orba, H Nishibara, S Tanaka, K Nagashima, H Sawa
    JOURNAL OF NEUROVIROLOGY 9 10 - 14 1355-0284 2003 [Refereed][Not invited]
     
    The human polyomavirus JC (JCV) encodes an agnoprotein that consists of 71 amino acid residues, with a molecular weight of approximately 8 kDa, from the late protein coding region. The agnoprotein of JCV shares 50% to 60% homology with those of simian virus 40 (SV40) and BK virus (BKV), and the carboxyl-terminal region of JCV agnoprotein is relatively unique. By using specific antibody to the carboxyl-terminal region of JCV agnoprotein, the authors have demonstrated that JCV agnoprotein expressed in the JCV-infected cells, where it localized predominantly in the perinuclear region of the cytoplasm, and colocalizes with the cellular cytoskeletal protein, tubulin. The results suggest that JCV agnoprotein may play a role in the stability of microtubules and the preservation of JCV infected cells via an interaction with tubulin.
  • H Nishihara, M Maeda, A Oda, M Tsuda, H Sawa, K Nagashima, S Tanaka
    BLOOD 100 (12) 3968 - 3974 0006-4971 2002/12 [Refereed][Not invited]
     
    The CDM (ced-5 of Caenorhabditis elegans, DOCK180 [downstream of Crk with molecular weight of 180 kDa] of humans, and myoblast city of Drosophila melanogaster) family of proteins has been shown to play a pivotal role in the integrin-mediated signaling pathway under the regulation of an adaptor molecule c-CT10- related kinase II (c-Crk-II) in adherent cells. Recently, hematopoietic cell-specific CDM protein DOCK2 has been shown to be indispensable for lymphocyte migration. However, the regulatory mechanism for DOCK2 is still unknown because DOCK2 lacks a c-Crk-II binding consensus motif. In this study, we demonstrated that DOCK2 bound to CrkL, which is present exclusively in hematopoietic cells both in vivo and in vitro, and we also found that 2 separate regions of DOCK2 contributed to its binding to Src homology 3 (SH3) domain of CrkL. Colocalization of DOCK2 with Crk-like (CrkL) and F-actin was shown by immunocytochemical analysis with the use of Jurkat cells. We also found that CrkL-induced activation of small guanine triphosphatase (GTPase) Rac1 was significantly inhibited by the DOCK2-dCS mutant in 293T cells. Furthermore, the association of DOCK2 and Vav, the guanine-nucleotide exchanging factor (GEF) for Rac1, was demonstrated in Jurkat cells. Finally, the stable expression of DOCK2-dCS mutant in Jurkat cells was shown to reduce cell attachment. These data suggest the presence of a novel protein complex of CrkL, DOCK2, and Vav to regulate Rac1 in leukemia cell lines. (C) 2002 by The American Society of Hematology.
  • R Komagome, H Sawa, T Suzuki, Y Suzuki, S Tanaka, WJ Atwood, K Nagashima
    JOURNAL OF VIROLOGY 76 (24) 12992 - 13000 0022-538X 2002/12 [Refereed][Not invited]
     
    JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and in humans causes a demyelinating disease of the central nervous system, progressive multifocal leukoencephalopathy. Its hemagglutination activity and entry into host cells have been reported to depend on an N-linked glycoprotein containing sialic acid. In order to identify the receptors of JCV, we generated virus-like particles (VLP) consisting of major viral capsid protein VP1. We then developed an indirect VLP overlay assay to detect VLP binding to glycoproteins and a panel of glycolipids. We found that VLP bound to sialoglycoproteins, including alpha1-acid glycoprotein, fetuin, and transferrin receptor, and that this binding depended on alpha2-3-linked sialic acids and N-linked sugar chains. Neoglycoproteins were synthesized by using ovalbumin and conjugation with oligosaccharides containing the terminal alpha2-3- or alpha2-6-linked sialic acid or the branched alpha2-6-linked sialic acid. We show that the neoglycoprotein containing the terminal alpha2-6-linked sialic acid had the highest affinity for VLP, inhibited the hemagglutination activity of VLP and JCV, and inhibited the attachment of VLP to cells. We also demonstrate that VLP bound to specific glycolipids, such as lactosylceramide, and gangliosides, including GM3, GD2, GD3, GD1b, GT1b, and GQ1b, and that VLP bound weakly to GD1a but did not bind to GM1a, GM2, or galactocerebroside. Furthermore, the neoglycoprotein containing the terminal alpha2-6-linked sialic acid and the ganglioside GT1b inhibited JCV infection in the susceptible cell line IMR-32. These results suggest that the oligosaccharides of glycoproteins and glycolipids work as JCV receptors and may be feasible as anti-JCV agents.
  • H Hasegawa, M Tatsumi, K Ogawa-Goto, H Takahashi, A Kojima, T Iwasaki, T Kurata, T Sata, T Takeuchi, N Sheehy, H Sawa, K Nagashima, WW Hall
    AIDS RESEARCH AND HUMAN RETROVIRUSES 18 (17) 1253 - 1260 0889-2229 2002/11 [Refereed][Not invited]
     
    To investigate the relationship between the fusogenic properties of HTLV-II and the processing of the envelope precursor glycoprotein gp63, recombinant cowpox virus expressing this protein was used to infect a range of cell lines derived from different species. Syncytium formation and gp63 processing were observed in all cells with the exception of LoVo cells, which are known to have a dysfunctional form of the endoprotease, furin. Furin has been shown to be necessary for the processing of a number of viral envelope glycoproteins, and gp63 contains a consensus sequence (305)Arg-Arg-Arg-Arg, which is a furin substrate motif. Pulse-chase studies demonstrated gp63 processing in Vero but not in LoVo cells. In addition it could be shown that expression of recombinant furin restored the processing of gp63 to gp46 in LoVo cells, and this resulted in syncytium formation. Our findings suggest that furin plays a pivotal role in cleavage of the HTLV-II envelope gp63, which in turn is a prerequisite for the fusogenic properties of the virus.
  • K Matsumoto, H Sawa, M Sato, Y Orba, K Nagashima, H Ariga
    ACTA NEUROPATHOLOGICA 104 (5) 448 - 454 0001-6322 2002/11 [Refereed][Not invited]
     
    Tenascin-X (TNX) is an extracellular matrix protein that is highly expressed in the peripheral nervous system as well as muscular tissues, especially the heart and skeletal muscle. However, the expression manner and the physiological role of TNX in the peripheral nervous system have not been fully investigated. In this study, we elucidated its distribution in adult mouse sciatic nerves by immunohistochemical staining. TNX was found to be localized in the perineurium and the endoneurium of sciatic nerve fibers. To examine the physiological role of TNX, we investigated sciatic nerves of TNX-deficient mice that are viable and fertile and have no obvious deficits in general performance. The thickness of myelin sheaths and the size of the individual axons in these mice appeared normal. The ultrastructure of the sciatic nerves of TNX-deficient mice were similar to those of wild-type mice. Thus, the lack of a discernible phenotype in the sciatic nerves of TNX-deficient mice suggests that TNX has either a redundant or a very subtle function in the macromolecular organization in the peripheral nerve.
  • H Takai, K Naka, Y Okada, M Watanabe, N Harada, S Saito, CW Anderson, E Appella, M Nakanishi, H Suzuki, K Nagashima, H Sawa, K Ikeda, N Motoyama
    EMBO JOURNAL 21 (19) 5195 - 5205 0261-4189 2002/10 [Refereed][Not invited]
     
    The mammalian Chk2 kinase is thought to mediate ATM-dependent signaling in response to DNA damage. The physiological role of mammalian Chk2 has now been investigated by the generation of Chk2-deficient mice. Although Chk2(-/-) mice appeared normal, they were resistant to ionizing radiation (IR) as a result of the preservation of splenic lymphocytes. Thymocytes and neurons of the developing brain were also resistant to IR-induced apoptosis. The IR-induced G(1)/S cell cycle checkpoint, but not the G(2)/M or S phase checkpoints, was impaired in embryonic fibroblasts derived from Chk2(-/-) mice. IR-induced stabilization of p53 in Chk2(-/-) cells was 50-70% of that in wild-type cells. Caffeine further reduced p53 accumulation, suggesting the existence of an ATM/ ATR-dependent but Chk2-independent pathway for p53 stabilization. In spite of p53 protein stabilization and phosphorylation of Ser23, p53-dependent transcriptional induction of target genes, such as p21 and Noxa, was not observed in Chk2(-/-) cells. Our results show that Chk2 plays a critical role in p53 function in response to IR by regulating its transcriptional activity as well as its stability.
  • N Makita, M Horie, T Nakamura, T Ai, K Sasaki, H Yokoi, M Sakurai, Sakuma, I, H Otani, H Sawa, A Kitabatake
    CIRCULATION 106 (10) 1269 - 1274 0009-7322 2002/09 [Refereed][Not invited]
     
    Background-Subclinical mutations in genes associated with the congenital long-QT syndromes (LQTS) have been suggested as a risk factor for drug-induced LQTS and accompanying life-threatening arrhythmias. Recent studies have identified genetic variants of the cardiac K+ channel genes predisposing affected individuals to acquired LQTS. We have identified a novel Na+ channel mutation in an individual who exhibited drug-induced LQTS. Methods and Results-An elderly Japanese woman with documented QT prolongation and torsade de pointes during treatment with the prokinetic drug cisapride underwent mutational analysis of LQTS-related genes. A novel missense mutation (L1825P) was identified within the C-terminus region of the cardiac Na+ channel (SCN5A). The L1825P channel heterologously expressed in tsA-201 cells showed Na+ cur-rent with slow decay and a prominent tetrodotoxin-sensitive noninactivating component, similar to the gain-of-function phenotype most commonly observed for SCN5A-associated congenital LQTS (LQT3). In addition, L1825P exhibited loss of function Na+ channel features characteristic of Brugada syndrome. Peak Na+ current density observed in cells expressing L1825P was significantly diminished, and the voltage dependence of activation and inactivation was shifted toward more positive and negative potentials, respectively. Conclusions-This study demonstrates that subclinical mutations in the LQTS-related gene SCN5A may predispose certain individuals to drug-induced cardiac arrhythmias.
  • H Takahashi, H Sawa, H Hasegawa, T Sata, WW Hall, T Kurata
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 297 (3) 593 - 599 0006-291X 2002/09 [Refereed][Not invited]
     
    Cellular topoisomerase I has been reported to be present in retroviral particles and to enhance viral cDNA synthesis; however, the mechanisms involved remain unknown. In the present study, it has been demonstrated that human topoisomerase I combines with a stem-loop RNA and that the bound topoisomerase I can be dissociated from RNA substrates in the presence of ATP. In addition, in vitro cleaved synthetic RNA bound by topoisomerase I is subsequently religated when the topoisomerase I is dissociated by ATP. A mechanism is proposed in which human topoisomerase I is carried into virions and regulates the repair of genomic RNA by its ligation activity. (C) 2002 Elsevier Science (USA). All rights reserved.
  • Y Okada, H Sawa, S Endo, Y Orba, T Umemura, H Nishihara, AC Stan, S Tanaka, H Takahashi, K Nagashima
    ACTA NEUROPATHOLOGICA 104 (2) 130 - 136 0001-6322 2002/08 [Refereed][Not invited]
     
    To examine the function of JC virus (JCV) agnoprotein, we examined the brains of cases of progressive multifocal leukoencephalopathy (PML), which is caused by JCV infection, using a newly generated antibody. The antibody reacted with 8 kDa protein specific for JCV agnoprotein by Western blotting. In vitro analyses showed that JCV capsid protein VP1 and large T antigen (T-Ag) were localized in the nuclei, but that agnoprotein was mainly detected in the cytoplasm of JCV-infected cells with an occasional nuclear staining. In the PML brain, an immunoreactive signal for agnoprotein was distributed in the perinuclear areas and cytoplasmic processes with occasional punctate staining in demyelinating lesions as well as adjacent myelinated areas. Agnoprotein presented mostly in the infected oligodendrocytes and partly in the astrocytes. Using double immunostaining, agnoprotein was seen to be expressed in the cytoplasmic processes of the cells, the nuclei of which were labeled with VP1 and T-Ag, where virus particles existed. Thus, JCV agnoprotein was mostly expressed in the infected oligodendrocytes and mainly localized in the cytoplasmic processes apart from virus particles in the demyelinated lesions.
  • H Nishihara, M Maeda, M Tsuda, Y Makino, H Sawa, K Nagashima, S Tanaka
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 296 (3) 716 - 720 0006-291X 2002/08 [Refereed][Not invited]
     
    DOCK2, a CDM family protein exclusively found in hematopoietic cells, has been shown to play a role in lymphocyte migration by the regulation of actin cytoskeleton. Although DOCK2 has been shown to induce the activation of Rac1, the regulatory mechanism of Rac2, which is a hematopoietic cell-specific small GTPase, is still unknown. In this study, we examined the role of DOCK2 in the activation of Rac2 in hematopoietic cells. DOCK2 was found to associate with the zeta subunit of the CD3 complex of T cell receptors in Jurkat cells and to activate forced expressed Rac2 in 293T cells. In addition, the stable expression of DOCK2 in Jurkat cells exhibited the elevated activity of endogenous Rac2. Furthermore, the transcriptional activity of interleukin-2 (IL-2) was enhanced in DOCK2-expressing Jurkat cells and the dominant negative form of Rac2 suppressed its elevated IL-2 promoter activity. These results suggest that DOCK2 mediates TCR-dependent activation of Rac2, leading to the regulation of IL-2 promoter activity in T cells. (C) 2002 Elsevier Science (USA). All rights reserved.
  • H Nishihara, S Tanaka, M Tsuda, S Oikawa, M Maeda, M Shimizu, H Shinomiya, A Tanigami, H Sawa, K Nagashima
    CANCER LETTERS 180 (1) 55 - 61 0304-3835 2002/06 [Refereed][Not invited]
     
    Crk is a signaling adaptor protein which is mostly composed of SH2 and SH3 domains. and has been shown to play a pivotal role in cell proliferation. differentiation, and migration. Because Crk was originally isolated as an avian sarcoma virus CT10 encoding oncoprotein v-Crk. we examined a potential role for c-Crk in the carcinogenesis of human cancers. First, to analyze gene mutations of c-Crk, we isolated a human bacterial artificial chromosome clone containing Crk genome and exon/intron structures, However, polymerase chain reaction-single strand conformation polymorphism methods failed to show any genomic mutations in the Crk exon which could be related to carcinogenesis. Second, immunohistochemical analysis of c-Crk-II demonstrated that the levels of c-Crk-II were significantly elevated in most of the tumors, particularly in the colon and lung cancers. Furthermore, immunoblot analysis using human lung cancer cell lines revealed that the expression levels of c-Crk-II were correlated to growth rates of L cells. The elevated expression levels of c-Crk-II might be related to the development of human cancers. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.
  • H Takahashi, H Sawa, H Hasegawa, Y Shoya, T Sata, WW Hall, K Nagashima, T Kurata
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 294 (2) 509 - 517 0006-291X 2002/06 [Refereed][Not invited]
     
    Replication of human immunodeficiency virus type 1 (HIV-1) is regulated at reverse transcription. Cellular topoisomerase I has been reported to be carried into HIV-1 virions and enhance cDNA synthesis in vitro, suggesting that topoisomerase I expressed in virus producer cells regulates reverse transcription. Here, by employing both indicator cell assay and endogenous reverse transcription (ERT) assay, we show that topoisomerase I and adenosine triphosphate (ATP) enhanced cDNA synthesis of HIV-1. In addition, topoisomerase I mutants, R488A and K532A, lacking enzymatic activity, attenuated the efficiency of cDNA synthesis and resulted in inhibition of the infectivity of HIV-1, suggesting that the activity of topoisomerase I lacking in these mutants is indispensable for the cDNA synthesis in the HIV-1 replication process. Furthermore, ATP could dissociate topoisomerase I from the topoisomerase I-RNA complex and enhance cDNA synthesis in vitro. These findings suggest that cellular topoisomerase I and ATP play a pivotal role in the synthesis of cDNA of HIV-1. (C) 2002 Elsevier Science (USA). All rights reserved.
  • H Takahashi, H Sawa, H Hasegawa, T Sata, WW Hall, K Nagashima, T Kurata
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 293 (3) 1084 - 1091 0006-291X 2002/05 [Refereed][Not invited]
     
    A human immunodeficiency virus type 1 (HIV-1) particle contains approximately 1200 molecules of gag proteins and two copies of a 9.2-kb genomic RNA which has been reported to be dimerized and rapidly cleaved and to form a complex with a nucleocapsid protein, p7 (NCp7), during viral budding. These suggest that the cleavage can be reconstituted with gag proteins in vitro. Here we show that the p15(gag) coding region of viral RNA is fragmented in viral particles and that in vitro-synthesized RNA transcripts of HIV-1 undergo cleavage which is activated by NCp7 and other factors. Single-stranded oligoribonucleotides were cleaved between C and A or U and A, leaving 2',3'-cyclic phosphate and 5'-hydroxyl termini. These findings might explain the rapid degradation of genomic RNAs in HIV-1 particles. (C) 2002 Elsevier Science (USA). All rights reserved.
  • Y Kobayashi, M Watanabe, Y Okada, H Sawa, H Takai, M Nakanishi, Y Kawase, H Suzuki, K Nagashima, K Ikeda, N Motoyama
    MOLECULAR AND CELLULAR BIOLOGY 22 (8) 2769 - 2776 0270-7306 2002/04 [Refereed][Not invited]
     
    A growing number of DNA polymerases have been identified, although their physiological function and relation to human disease remain mostly unknown. DNA polymerase lambda (Pol lambda; also known as Pol beta2) has recently been identified as a member of the X fancily of DNA polymerases and shares 32% amino acid sequence identity with DNA Pot R within the polymerase domain. With the use of homologous recombination, we generated Pol lambda(-/-) mice. Pol lambda(-/-) mice develop hydrocephalus with marked dilation of the lateral ventricles and exhibit a high rate of mortality after birth, although embryonic development appears normal. Pol lambda(-/-) mice also show situs inversus totalis and chronic suppurative sinusitis. The surviving male, but not female, Pol lambda(-/-) mice are sterile as a result of spermatozoal immobility. Microinjection of sperm from male Pol lambda(-/-) mice into oocytes gives rise to normal offspring, suggesting that the meiotic process is not impaired. Ultrastructural analysis reveals that inner dynein arms of cilia from both the ependymal cell layer and respiratory epithelium are defective, which may underlie the pathogenesis of hydrocephalus, situs inversus totalis, chronic sinusitis, and male infertility. Sensitivity of Pol lambda(-/-) cells to various kinds of DNA damage is indistinguishable from that of Pol lambda(+/+) cells. Collectively, Pol lambda(-/-) mice may provide a useful model for clarifying the pathogenesis of immotile cilia syndrome.
  • T Okamoto, S Tanaka, AC Stan, T Koike, M Kase, Z Makita, H Sawa, K Nagashima
    MICROVASCULAR RESEARCH 63 (2) 186 - 195 0026-2862 2002/03 [Refereed][Not invited]
     
    Advanced glycation end products (AGEs) have been thought to participate in diabetic microangiopathy. However, the effects of AGEs on angiogenesis have so far been mainly examined either in vitro or by using cultured cells. In the present study, we have analyzed whether AGES induce angiogenesis in vivo by using the chorioallantoic membrane (CAM) assay. The CAM assay was carried out in embryonated hen eggs to determine the effects of AGEs. Following generation of AGEs based on bovine serum albumin (BSA), either AGE-BSA or nonglycated BSA was administered to the CAM and their effects on angiogenesis were assessed, together with an inhibitory effect of an anti-AGE antibody against AGE-BSA-induced angiogenesis. The histological features of AGE-induced vascular lumens were examined by immunohistochemical analysis for Factor VIII and smooth muscle a-actin. AGE-BSA induced angiogenesis in CAM in a dose- and time-dependent manner. AGE-induced angiogenesis on CAM was neutralized by the anti-AGE antibody. Inummohistochemical analysis demonstrated that AGE-induced vascular lumens were devoid of pericytes. Our data demonstrated that AGES are an angiogenetic factor and that our system of AGE-induced abnormal vessels in CAMs is useful in further investigations of the mechanism of diabetic retinal angiogenesis and can also be used to provide a therapeutic model for diabetic angiopathy. (C) 2002 Elsevier Science (USA).
  • Signaling adaptor protein v-Crk activates Rho and regulates cell motility in 3Y1 rat fibroblast cell line.
    Tsuda M, Tanaka S, Sawa H, Hanafusa H, Nagashima K
    Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research 3 13 (3) 131 - 139 1044-9523 2002/03 [Refereed][Not invited]
  • [Molecular neuropathology of JC virus].
    Orba Y, Sawa H, Nagashima K
    No to shinkei = Brain and nerve 2 54 (2) 101 - 109 0006-8969 2002/02 [Refereed][Not invited]
  • Tsuda, M., Tanaka, S., Sawa, H., Hanafusa, H., Nagashima: Signalling adaptor protein v-Crk activates Rho and regulates cell motility in 3Y1 rat fibroblast cell line. Cell Growth Diff 13: 131-139, 2002*
    2002 [Not refereed][Not invited]
  • Takahashi H, Sawa H, Hasegawa H, Sata T, Hall WW, Kurata T: Binding and dissociation of human topoisomerase I with hair-pin loop RNAs: implications for the regulation of HIV-1 replication. Biochem Biophys Res Commun 297: 593-599, 2002*
    2002 [Not refereed][Not invited]
  • Nishihara H, Tanaka S, Tsuda M., Oikawa S, Maeda M, Shimizu M, Shimoyama H, Tanigami A, Sawa H, Nagashima K: Molecular and immunohistochemical analysis of adaptor protein Crk in human cancers. Cancer Lett 180, 55-61, 2002*
    2002 [Not refereed][Not invited]
  • Takahashi H, Sawa H, Hasegawa H, Sata T, Hall WW, Nagashima K, Kurata T: Reconstitution of cleavage of human immunodeficiency virus type-1 (HIV-1) RNAs. Biochem Biophys Res Commun 293: 1084-1091, 2002*
    2002 [Not refereed][Not invited]
  • Matsumoto K, Sawa H, Sato M, Orba Y, Nagashima K, Ariga H: Distribution of extracellular matrix tenascin-X in sciatic nerves. Acta Neuropathol 104: 448-454, 2002*
    2002 [Not refereed][Not invited]
  • Okada Y, Sawa H, Endo S, Orba Y, Umemura T, Nishihara H, Stan AC, Tanaka S, Nagashima K. Expression of JC virus (JCV) agnoprotein in progressive multifocal leukoencephalopathy (PML) brain. Acta Neuropathol 104: 130-136, 2002*
    2002 [Not refereed][Not invited]
  • Makita N, Horie M, Nakamura T, Ai T, Otani H, Sawa H, Kitabatake A: Drug-induced long-QT syndrome associated with a subclinical SCN5A mutation. Circulation 106: 1269-1274, 2002*
    2002 [Not refereed][Not invited]
  • Hasegawa H, Tatsumi M, Ogawa-Goto K, Takahashi H, Iwasaki T, Kurata T, Sata T, Takeuchi T, Sheehy N, Sawa H, Nagashima K, Hall WW: Processing of the HTLV-II envelope precursor glycoprotein, gp63 by furin is essential for cell fusion activity. AIDS Res・・・
    2002 [Not refereed][Not invited]
     
    Hasegawa H, Tatsumi M, Ogawa-Goto K, Takahashi H, Iwasaki T, Kurata T, Sata T, Takeuchi T, Sheehy N, Sawa H, Nagashima K, Hall WW: Processing of the HTLV-II envelope precursor glycoprotein, gp63 by furin is essential for cell fusion activity. AIDS Res Hum Retroviruses 18: 1253-1260, 2002*
  • Nishihara H, Maeda M, Oda A, Tsuda M, Sawa H, Tanaka S, and Nagashima K: DOCK2 associates with CrkL and regulates Rac1 in hematopoietic cells. Blood 100: 3968-3974, 2002*
    2002 [Not refereed][Not invited]
  • Yoshida H, Okada Y, Kinoshita N, Hara H, Sasaki M, Sawa H, Nagashima K, Mak TW, Motoyama N: Differential requirement for Apaf1 and Bcl-XL in the regulation of programmed cell death during development. Cell Death Differ 9: 1273-1276, 2002*
    2002 [Not refereed][Not invited]
  • Nishihara H, Maeda M, Tsuda M, Makino Y, Sawa H, Nagashima K, Tanaka S: DOCK2 mediates T cell receptor-induced activation of Rac2 and IL-2 transcription. Biochem Biophys Res Commun 296: 716-720, 2002*
    2002 [Not refereed][Not invited]
  • Takai H, Naka K, Okada Y, Watanabe M, Harada N, Saito S, Anderson CW, Appella E, Nakanishi M, Suzuki H, Nagashima K, Sawa H, Ikeda K, Motoyama N: Chk2-deficient mice exhibit increased resistance to ionizing radiation and defective p53-mediated transcri・・・
    2002 [Not refereed][Not invited]
     
    Takai H, Naka K, Okada Y, Watanabe M, Harada N, Saito S, Anderson CW, Appella E, Nakanishi M, Suzuki H, Nagashima K, Sawa H, Ikeda K, Motoyama N: Chk2-deficient mice exhibit increased resistance to ionizing radiation and defective p53-mediated transcription. EMBO J 21: 5195-5205, 2002*
  • Komagome R, Sawa H*, Suzuki T, Suzuki Y, Tanaka S, Atwood WJ, Nagashima K: Oligosaccharides as receptors for JC virus. J Virol 76: 12992-13000, 2002 (* corresponding author)*
    2002 [Not refereed][Not invited]
  • Takahashi H, Sawa H, Hasegawa H, Shoya Y, Sata T, Hall WW, Nagashima K, Kurata T: Topoisomerase I and ATP activate cDNA synthesis of human immunodeficiency virus type-1 (HIV-1). Biochem Biophys Res Commun 294: 509-517, 2002
    2002 [Not refereed][Not invited]
  • Hasegawa H, Tatsumi M, Ogawa-Goto K, Takahashi H, Iwasaki T, Kurata T, Sata T, Takeuchi T, Sheehy N, Sawa H, Nagashima K, Hall WW: "Processing of the HTLV-II envelope precursor glycoprotein, gp63 by furin is essential for cell fusion activity." AIDS R・・・
    2002 [Not refereed][Not invited]
     
    Hasegawa H, Tatsumi M, Ogawa-Goto K, Takahashi H, Iwasaki T, Kurata T, Sata T, Takeuchi T, Sheehy N, Sawa H, Nagashima K, Hall WW: "Processing of the HTLV-II envelope precursor glycoprotein, gp63 by furin is essential for cell fusion activity." AIDS Res Hum Retroviruses 18: 1253-1260, 2002*
  • Nishihara H, Tanaka S, Tsuda M., Oikawa S, Maeda M, Shimizu M, Shimoyama H, Tanigami A, Sawa H, Nagashima K: "Molecular and immunohistochemical analysis of adaptor protein Crk in human cancers." Cancer Lett 180, 55-61, 2002*
    2002 [Not refereed][Not invited]
  • Matsumoto K, Sawa H, Sato M, Orba Y, Nagashima K, Ariga H: "Distribution of extracellular matrix tenascin-X in sciatic nerves." Acta Neuropathol 104: 448-454, 2002*
    2002 [Not refereed][Not invited]
  • Tsuda, M., Tanaka, S., Sawa, H., Hanafusa, H., and Nagashima: "Signalling adaptor protein v-Crk activates Rho and regulates cell motility in 3Y1 rat fibroblast cell line." Cell Growth Diff 13: 131-139, 2002*
    2002 [Not refereed][Not invited]
  • Takahashi H, Sawa H, Hasegawa H, Sata T, Hall WW, Nagashima K, Kurata T: "Reconstitution of cleavage of human immunodeficiency virus type-1 (HIV-1) RNAs." Biochem Biophys Res Commun 293: 1084-1091, 2002*
    2002 [Not refereed][Not invited]
  • Yoshida H, Okada Y, Kinoshita N, Hara H, Sasaki M, Sawa H, Nagashima K, Mak TW, Motoyama N: "Differential requirement for Apaf1 and Bcl-XL in the regulation of programmed cell death during development." Cell Death Differ 9: 1273-1276, 2002*
    2002 [Not refereed][Not invited]
  • Takahashi H, Sawa H, Hasegawa H, Sata T, Hall WW, Kurata T: "Binding and dissociation of human topoisomerase I with hair-pin loop RNAs: implications for the regulation of HIV-1 replication." Biochem Biophys Res Commun 297: 593-599, 2002*
    2002 [Not refereed][Not invited]
  • Komagome R, Sawa H*, Suzuki T, Suzuki Y, Tanaka S, Atwood WJ, Nagashima K: "Oligosaccharides as receptors for JC virus." J Virol 76: 12992-13000, 2002 (* corresponding author)*
    2002 [Not refereed][Not invited]
  • Nishihara H, Maeda M, Oda A, Tsuda M, Sawa H, Tanaka S, and Nagashima K: "DOCK2 associates with CrkL and regulates Rac1 in hematopoietic cells." Blood 100: 3968-3974, 2002*
    2002 [Not refereed][Not invited]
  • Takai H, Naka K, Okada Y, Watanabe M, Harada N, Saito S, Anderson CW, Appella E, Nakanishi M, Suzuki H, Nagashima K, Sawa H, Ikeda K, Motoyama N: "Chk2-deficient mice exhibit increased resistance to ionizing radiation and defective p53-mediated transcr・・・
    2002 [Not refereed][Not invited]
     
    Takai H, Naka K, Okada Y, Watanabe M, Harada N, Saito S, Anderson CW, Appella E, Nakanishi M, Suzuki H, Nagashima K, Sawa H, Ikeda K, Motoyama N: "Chk2-deficient mice exhibit increased resistance to ionizing radiation and defective p53-mediated transcription." EMBO J 21: 5195-5205, 2002*
  • Nishihara H, Maeda M, Tsuda M, Makino Y, Sawa H, Nagashima K, Tanaka S: "DOCK2 mediates T cell receptor-induced activation of Rac2 and IL-2 transcription." Biochem Biophys Res Commun 296: 716-720, 2002*
    2002 [Not refereed][Not invited]
  • Okada Y, Sawa H, Endo S, Orba Y, Umemura T, Nishihara H, Stan AC, Tanaka S, Nagashima K. "Expression of JC virus (JCV) agnoprotein in progressive multifocal leukoencephalopathy (PML) brain." Acta Neuropathol 104: 130-136, 2002*
    2002 [Not refereed][Not invited]
  • Xie Z, Koyama T, Suzuki J, Fujii Y, Togashi H, Sawa H, Nagashima K
    Jpn Heart J 42 (6) 759 - 770 0021-4868 2001 [Refereed][Not invited]
  • Angiotensin-converting enzyme inhibition attenuates hypofibrinolysis and reduces cardiac perivascular fibrinolysis in genetically obese diabetic mice.
    Zaman AK, Fujii S, Sawa H, Goto D, Ishimori N, Watano K, Kaneko T, Furumoto T, Sugawara T, Sakuma I, Kitabatake A, Sobel BE
    Circulation 103 3123 - 3128 2001 [Refereed][Not invited]
     
    Zaman AK, Fujii S, Sawa H, Goto D, Ishimori N, Watano K, Kaneko T, Furumoto T, Sugawara T, Sakuma I, Kitabatake A, Sobel BE: "Angiotensin-converting enzyme inhibition attenuates hypofibrinolysis and reduces cardiac perivascular fibrinolysis in genetically obese diabetic mice." Circulation 103: 3123-3128, 2001*
  • Okada Y, Endo S, Takahashi H, Sawa H, Umemura T, Nagashima K: "Distribution and function of JCV agnoprotein." J Neurovirol 7: 302-306, 2001
    2001 [Not refereed][Not invited]
  • Suzuki S, Sawa H*, Komagome R, Orba Y, Yamada M, Okada Y, Ishida Y, Nishihara H, Tanaka S, Nagashima K: "Broad distribution of the JC virus receptor contrasts with a marked cellular restriction of virus replication." Virology 286: 100-112, 2001 (* cor・・・
    2001 [Not refereed][Not invited]
     
    Suzuki S, Sawa H*, Komagome R, Orba Y, Yamada M, Okada Y, Ishida Y, Nishihara H, Tanaka S, Nagashima K: "Broad distribution of the JC virus receptor contrasts with a marked cellular restriction of virus replication." Virology 286: 100-112, 2001 (* corresponding author)*
  • Shirane M, Sawa H, Kobayashi Y, Nakano T, Shinkai Y, Nagashima K, and Negishi I. "Deficiency of phospholipase C-1 impares renal development and hematopoiesis." Development 128: 5173-5180, 2001*
    2001 [Not refereed][Not invited]
  • Okamoto T, Tanaka S, Stan AC., Koike T, Kase, Makita Z, Sawa H, Ngashima K: "Advanced glycation end products induce angiogenesis in vivo." Microvascular Research 63: 186-195, 2002*
    2001 [Not refereed][Not invited]
  • Tanaka S, Katano H, Tsukamoto K, Jin M, Oikawa S, Nishihara H, Sawa H, Sawada K, Shimizu M, Sata T, Fujioka Y, Nagashima K. "HHV8-negative primary effusion lymphoma of the peritoneal cavity presenting with a distinct immunohistochemical phenotype." Pa・・・
    2001 [Not refereed][Not invited]
     
    Tanaka S, Katano H, Tsukamoto K, Jin M, Oikawa S, Nishihara H, Sawa H, Sawada K, Shimizu M, Sata T, Fujioka Y, Nagashima K. "HHV8-negative primary effusion lymphoma of the peritoneal cavity presenting with a distinct immunohistochemical phenotype." Pathol Int 51: 293-300, 2001*
  • Ohwatari R, Iwabuchi K, Iwabuchi C, Morohashi T, Sawa H, Hioki K, Kobayash i K, Fukuda S, Inuyama Y, and Onoe K. "Developmental and functional analyses of CD8+ NK1.1+ T cells in the class I restricted TCR transgenic mice." Cell Immunol. 213: 24-33, 2001*
    2001 [Not refereed][Not invited]
  • 2001 [Not refereed][Not invited]
     
    Nagai M, Tanaka S, Tsuda M, Endo S, Kato H, Sonobe H, Minami A, Hiraga H, Nishihara H, Sawa H, Nagashima K: "Analysis of transforming activity of human synovial sarcoma-associated chimeric protein SYT-SSX1 bound to chromatin remodeling factor hBRM/hSNF2alpha." Proc Natl Acad Sci USA 98, 3843-3848, 2001*
  • Nagashima T, Mori M, Fujimoto M, Nunomura M, Sakurai Y, Okada Y, Itoh T, Sawa H, Stan AC, Nagashima K: "Adult T-cell lymphoma involving the leptomeninges associated with a spinal cord schwannoma." Neuropathology 21, 229-235, 2001
    2001 [Not refereed][Not invited]
  • Furuta Y, Ohtani F, Sawa H, Fukuda S, Inuyama Y: "Quantitation of varicella-zoster virus DNA in patients with Ramsay Hunt syndrome and Zoster Sine Herpete." J Clin Microbiol 39: 2856-2859, 2001.*
    2001 [Not refereed][Not invited]
  • Kamimura E, UenoY, Tanaka S, Sawa H, Yoshioka M, Ueno K, Ishikawa R, Inoue T, Li X, Koyama T, Nagashima K: "New rat model for attention deficit hyperactive disorder (ADHD)." Comp Med 51: 245-251, 2001*
    2001 [Not refereed][Not invited]
  • Ohnishi J, Ohnishi E, Hirano W, Nakane D, Matsui H, Kimura A, Sawa H, Nakayama K, Shibuya H, Nagashima K, Takahashi T: Cloning and characterization of a rat ortholog of MMP-23 (matrix metalloproteinase-23), a unique type of membrane-anchored matrix met・・・
    2001 [Not refereed][Not invited]
     
    Ohnishi J, Ohnishi E, Hirano W, Nakane D, Matsui H, Kimura A, Sawa H, Nakayama K, Shibuya H, Nagashima K, Takahashi T: Cloning and characterization of a rat ortholog of MMP-23 (matrix metalloproteinase-23), a unique type of membrane-anchored matrix metalloproteinase and conditioned switching of its expression during the ovarian follicular development. Mol Endocrinol 15, 747-764, 2001*
  • Hayashi H, Endo S, Suzuki S, Tanaka S, Sawa H, Ozaki Y, Sawamura Y, Nagashima K: "JC virus large T protein transforms rodent cells but is not involved in human medulloblastoma." Neuropathology 21: 129-137, 2001*
    2001 [Not refereed][Not invited]
  • Xie Z, Koyama T, Suzuki J, Fujii Y, Togashi H, Sawa H, Nagashima K: Coronary reperfusion following ischemia. Different expression of Bcl-2 and Bax proteins, and cardiomyocyte apoptosis. Jpn Heart J 42: 759-770, 2001*
    2001 [Not refereed][Not invited]
  • Sato-Matsumura KC, Matsumura T, Nakamura H, Sawa H, Nagashima K, Koizumi H: Membranous expression of annexin I is enhanced by calcium and TPA in cultured human keratinocytes. Arch Dermatol Res 292, 496-499, 2000*
    2000 [Not refereed][Not invited]
  • Shintaku M, Matsumoto R, Sawa H, Nagashima K: Infection with JC virus and possible dysplastic ganglion-like transformation of the cerebral cortical neurons in a case of progressive multifocal leukoencephalopathy. J Neuropathol Exp Neurol 59, 921-929, ・・・
    2000 [Not refereed][Not invited]
     
    Shintaku M, Matsumoto R, Sawa H, Nagashima K: Infection with JC virus and possible dysplastic ganglion-like transformation of the cerebral cortical neurons in a case of progressive multifocal leukoencephalopathy. J Neuropathol Exp Neurol 59, 921-929, 2000*
  • Hiroi Y, Chen R, Sawa H, Hosoda T, Kudoh S, Kobayashi Y, Aburatani H, Nagashima K, Nagai R, Yazaki Y, Medof ME, Komuro I: Cloning of murine glycosyl phosphatidylinositol anchor attachment protein, GPAA1. Am J Physiol Cell Physiol 279, C205-C212, 2000*
    2000 [Not refereed][Not invited]
  • Okada Y, Sawa H, Tanaka S, Takada A, Suzuki S, Hasegawa H, Umemura T, Fujisawa J-i, Tanaka Y, Hall WW, Nagashima K
    J Biol Chem 275 (22) 17016 - 17023 0021-9258 2000 [Refereed][Not invited]
     
    Okada Y, Sawa H*, Tanaka S, Takada A, Suzuki S, Hasegawa H, Umemura T, Fujisawa J-i, Tanaka Y, Hall WW, Nagashima1 K: Transcriptional activation of JC virus (JCV) by human T-lymphotropic virus type I (HTLV-I) tax protein in human neuronal cell lines. J Biol Chem, 275, 17016-17023, 2000 (* corresponding author)*
  • Xie Z, Koyama T, Abe K, Fujii Y, Sawa H, Nagashima K: Upregulation of p53 protein in rat heart subjected to a transient occlusion of the coronary artery followed by reperfusion. Jpn J Physiol 50: 159-162, 2000*
    2000 [Not refereed][Not invited]
  • Suzuki G, Sawa H, Kobayashi Y, Nakata Y, Nakagawa Ki, Uzawa A, Sakiyama H, Kakinuma S, Iwabuchi K, Nagashima K: Pertussis toxin-sensitive signal controls the trafficking of thymocytes across corticomedullary junction in the thymus. J Immunol 162: 5981・・・
    1999 [Not refereed][Not invited]
     
    Suzuki G, Sawa H, Kobayashi Y, Nakata Y, Nakagawa Ki, Uzawa A, Sakiyama H, Kakinuma S, Iwabuchi K, Nagashima K: Pertussis toxin-sensitive signal controls the trafficking of thymocytes across corticomedullary junction in the thymus. J Immunol 162: 5981-5985, 1999*
  • Ohba Y, Suzuki H, Hiraga H, Ito T, Sawa H, Nagai M, Satoh S, Iwaki H, Nagashima K. Melanotic peritoneal sarcomatosis originating from clear cell sarcoma. Pathol Int 49: 653-657, 1999*
    1999 [Not refereed][Not invited]
  • Hiraga H, Nojima T, Abe S, Sawa H, Yamashiro K, Yamawaki S, Kaneda K, Nagashima K: Diagnosis of synovial sarcoma with the reverse transcriptase-polymerase chain reaction: analyses of 84 soft tissue and bone tumors. Diagn Mol Pathol 7: 102-110, (1998)*
    1998 [Not refereed][Not invited]
  • Sasaki H, Kojima H, Yabe I, Tashiro K, Hamada T, Sawa H, Hiraga H, Nagashima K: Neuropathological and molecular studies of spinocerebellar ataxia type 6 (SCA6). Acta Neuropathology (Berl) 95: 199-204, 1998*
    1998 [Not refereed][Not invited]
  • Nordt TK, Sawa H, Fujii S, Bode C, Sobel BE: Augmentation of arterial endothelial cell expression of the plasminogen activator inhibitor type-1 (PAI-1) gene by proinsulin and insulin vivo. J Mol Cell Cardiol 30: 1535-1543, 1998*
    1998 [Not refereed][Not invited]
  • Kobayashi Y, Sawa H, Akagi H, Itakura C, Fujioka Y, Nagashima K: Distributional pattern of apoptotic cells in rat cerebellar vermis experimentally induced by methylmercury intoxication. Neuropathology 18: 33-37, 1998*
    1998 [Not refereed][Not invited]
  • Kobayashi Y, Sawa H, Watanabe M, Furuoka H, Matsui T, Nagashima K: Calbindin D immunoreactivity and chronic lesions of rat cerebella in methylmercury chrolide intoxication. Neuropathology 18: 402-407, 1998*
    1998 [Not refereed][Not invited]
  • Nordt TK, Sawa H, Fujii S, Bode C, Sobel BE: Augmentation of arterial endothelial cell expression of the plasminogen activator inhibitor type-1 (PAI-1) gene by proinsulin and insulin in vivo. J Mol Cell Cardiol 30: 1535-1543, (1998)*
    1998 [Not refereed][Not invited]
  • Nordt TK, Sawa H, Fujii S, Sobel BE: Hyperinsulinemia increases plasma activity of PAI-1 in vivo independently of an acute phase reaction. Fibrinolysis Proteolysis 11 : 51-54, 1997
    1997 [Not refereed][Not invited]
  • Takahashi HR, Sawa H, Takada A, Kitabatake A, Nagashima K: Expression of beta-amyloid precursor protein mRNAs in the vascular smooth muscle cell of human brain. Neuropathology 17: 11-14, 1997*
    1997 [Not refereed][Not invited]
  • Shinohara K, Shinohara T, Mochizuki N, Mochizuki Y, Sawa H, Kohya T, Fujita M, Fujioka Y, Kitabatake A, Nagashima K: Expression of vascular endothelial growth factor associated with human myocardial infarction. Heart Vessels 11: 113-122, 1996*
    1996 [Not refereed][Not invited]
  • Takahashi HR, Sawa H, Kuroda S, Saito H, Fujita M, Fujioka Y, Fukatsu R, Nagashima K: Pathologic processes leading to cerebral hemorrhage in amyloid angiopathy. Neuropathology 16: 99-105, 1996*
    1996 [Not refereed][Not invited]
  • Okada H, Kawaguchi H, Kudo T, Sawa H, Okamoto H, Watanabe S, Urasawa K, Murakami T, Kitabatake A: Alteration of extracellular matrix in dilated cardiomyopathic hamster heart. Mol Cell Biochem 156: 9-15, 1996*
    1996 [Not refereed][Not invited]
  • Sawa H, Lundgren CH, Brown SL, Fujii S: Dependence of human vascular cell surface proteolysis on expression of the urokinase receptor. J Thromb Thromboly 3: 331-336, 1996*
    1996 [Not refereed][Not invited]
  • EXPRESSION OF INTERCELLULAR AND VASCULAR CELL-ADHESION MOLECULES AND CLASS-II MAJOR HISTOCOMPATIBILITY ANTIGENS IN HUMAN LUNGS - LACK OF INFLUENCE BY CONDITIONS OF ORGAN PRESERVATION
    S HASEGAWA, JH RITTER, GA PATTERSON, DM OCKNER, H SAWA, T MOHANAKUMAR, JD COOPER, MR WICK
    JOURNAL OF HEART AND LUNG TRANSPLANTATION 14 (5) 897 - 905 1053-2498 1995/09 [Refereed][Not invited]
     
    Background: The expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and class II major histocompatibility complex antigens was studied in control lung tissue and preserved human donor lungs. The three controls were represented by wedge biopsy specimens taken from non-neoplastic lung surrounding bronchogenic carcinomas. Methods: Nine lungs were harvested from six brain-dead donors, flushed with Euro-Collins solution or low potassium-dextran-glucose solution, and stored at 1 degrees or 10 degrees C. Samples of the latter organs were taken at the time of surgical harvest (baseline) and after 2, 12, 24, and 48 hours of preservation time. Immunostains with monoclonal antibodies against intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and class II major histocompatibility complex molecules were performed on all samples, and the relative presence of these determinants was evaluated. Results: In both the controls and preserved lungs, intercellular adhesion molecule-1 expression was intense in the septal capillary endothelium and alveolar pneumocytes, but essentially absent in bronchial epithelium. Vascular cell adhesion molecule-1 was moderately to strongly labeled in the endothelia of large and small blood vessels of all types, and it was not seen in other cell types. Class II major histocompatibility complex antigens were variably observed in pulmonary epithelial cells, but they were not expressed by endothelia. There appeared to be no significant difference in the immunohistologic density of intercellular adhesion molecule-1 or vascular cell adhesion molecule-1 immunostaining in allografts at the specified time points of preservation; this conclusion was confirmed by Western blot analysis. Similar findings pertained to staining results for human leukocyte DR antigens. There was likewise no significant difference in the expression of the three analytes when donor lungs perfused with Euro-Collins solution versus low potassium-dextran-glucose solution were compared; this was also true of organs preserved at 1 degrees C versus 10 degrees C. Conclusions: These results suggest that, in the immediate postharvest period, modulations in the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, or class II major histocompatibility complex antigens in pulmonary allografts are not attributable to the influences of preservation conditions.
  • Mochizuki EY, Mochizuki N, Sawa H, Takada A, Okamoto H, Kawaguchi H, Nagashima K, Kitabatake A: Expression of renin and angiotensin converting enzyme in human hearts. Heart Vessels 10: 285-293, 1995*
    1995 [Not refereed][Not invited]
  • Nordt TK, Sawa H, Fujii S, Sobel BE: Induction of plasminogen activator inhibitor type-1 (PAI-1) by proinsulin and insulin in vivo. Circulation 91: 764-770, 1995*
    1995 [Not refereed][Not invited]
  • Motoyama N, Wang F, Roth KA, Sawa H, Nakayama Ki, Nakayama K, Negishi I, Senju S, Zhang Q, Fujii S, Loh DY: Massiva cell death of post-mitotic immature lymphocytes in Bcl-x-deficient mice. Science 267: 1506-1510, 1995*
    1995 [Not refereed][Not invited]
  • Nakayama K, Nakayama K-i, Negishi I, Kuida K, Sawa H, Loh DY: Targeted disruption of Bcl-2 alpha beta in mice: occurrence of gray hair,polycystic kidney disease, and lymphocytopenia. Proc Natl Acad Sci U S A 91: 3700-3704, 1994*
    1994 [Not refereed][Not invited]
  • Lundgren CL, Sawa H, Sobel BE, Fujii S: Modulation of expression of monocyte/macropharge plasminogen activator activity and its implications for attenuation of vasculopathy. Circulation 90: 1927-1934, 1994*
    1994 [Not refereed][Not invited]
  • Sawa H, Kawaguchi H, Mochizuki N, Endo Y, Kudo T, Tokuchi F, Fujioka Y, Nagashima K, Kitabatake A: Distribution of angiotensinogen in diseased human hearts. Mol Cell Biochem 132: 15-23, 1994*
    1994 [Not refereed][Not invited]
  • Sawa H, Lundgren C, Sobel BE, Fujii S: Increased intramural expression of plasminogen activator inhibitor type-1 after balloon injury: A potential progenitor of restenosis. J Am Coll Cardiol 24: 1742-1748, 1994*
    1994 [Not refereed][Not invited]
  • Sawa H, Sobel BE, Fujii S: Inhibition of type-1 plasminogen activator inhibitor production by antisense oligonucleotides in human vascular endothelial and smooth muscle cells. J Biol Chem 269: 14149-14152, 1994*
    1994 [Not refereed][Not invited]
  • Fujii S, Sawa H, Sobel BE: Inhibition of endothelial cell expression of plasminogen activator inhibitor type-1 by gemfibrozil. Thromb Haemost 70: 642-647, 1993*
    1993 [Not refereed][Not invited]
  • Sawa H, Sobel BE, Fujii S: Potentiation by hypercholesterolemia of the induction of aortic intramural synthesis of plasminogen activator inhibitor type-1 by endothelial injury. Circ Res 73: 671-680, 1993*
    1993 [Not refereed][Not invited]
  • Fujii S, Sawa H, Sobel BE: Induction of endothelial cell expression of the plasminogen activator inhibitor type-1 gene by thrombosis in vivo. Circulation 86: 2000-2010, 1992
    1992 [Not refereed][Not invited]
  • Furuta Y, Takasu T, Asai T, Shinohara T, Sawa H, Nagashima K, Inuyama Y: Detection of human papillomavirus DNA in carcinomas of the nasal cavities and paranasal sinuses by polymerase chain reaction. Cancer 69: 353-357, 1992
    1992 [Not refereed][Not invited]
  • Sawa H, Fujii S, Sobel BE: Augmented arterial wall expression of type-1 plasminogen activator inhibitor induced by thrombosis. Arterioscler Thromb 12: 1507-1515, 1992
    1992 [Not refereed][Not invited]
  • Kawaguchi H, Shoki M, Sano H, Kudo T, Sawa H, Mochizuki N, Okamoto H, Endo Y, Kitabatake A: Polyphosphoinositide metabolism in hypertrophic rat heart. J Mol Cell Cardiol 24: 1003-1010, 1992*
    1992 [Not refereed][Not invited]
  • Sawa H, Tokuchi F, Mochizuki N, Endo Y, Furuta Y, Shinohara T, Takada A, Kawaguchi H, Yasuda H, Nagashima K: Expression of the angiotensinogen gene and localization of its protein in the human heart. Circulation 86: 138-146, 1992
    1992 [Not refereed][Not invited]
  • Shoki M, Kawaguchi H, Okamoto H, Sano H, Sawa H, Kudo T, Hirao N, Sakata Y, Yasuda H: Phosphatidylinositol and inositolphosphatide metabolism in hypertrophied rat heart. Jpn Circ J 56:142-147, 1992*
    1992 [Not refereed][Not invited]
  • Kawaguchi H, Shoki M, Sano H, Kudo T, Sawa H, Okamoto H, Sakata Y, Yasuda H: The studies of cell damaging and cell growth factors which induce cardiomyopathy. Jpn Circ J 56: 1037-1044, 1992*
    1992 [Not refereed][Not invited]
  • Kawaguchi H, Sawa H, Yasuda H: Effect of endothelin on angiotensin converting enzyme activity in cultured pulmonary artery endothelial cells. J Hypertens 9: 171-174, 1991*
    1991 [Not refereed][Not invited]
  • Kawaguchi H, Shoki M, Sano H, Kudo T, Sawa H, Okamoto H, Sakata Y, Yasuda H: Phospholipid metabolism in cardiomyopathic hamster heart cells. Circ Res 69: 1015-1021, 1991*
    1991 [Not refereed][Not invited]
  • Mochizuki N, Sawa H, Yasuda H, Shinohara T, Nagashima K, Yamaji N, Ohnuma N, Hall WW: Distribution of atrial natriuretic peptide in the conduction system and ventricular muscles of the human heart. Virchows Arch A Pathol Anat Histopathol 418: 9-16, 1991*
    1991 [Not refereed][Not invited]
  • Kawaguchi H, Sawa H, Yasuda H: Mechanism of increased angiotensin converting enzyme activity stimulated by platelet-activating factor. Biochim Biophys Acta 1052: 503-508, 1990*
    1990 [Not refereed][Not invited]
  • Kawaguchi H, Sawa H, Iizuka K, Yasuda H: Platelet-activating factor stimulates angiotensin converting enzyme activity. J Hypertens 8: 173-177, 1990*
    1990 [Not refereed][Not invited]
  • Kawaguchi H, Sawa H, Yasuda H: Endothelin stimulates angiotensin I to angiotensin II conversion in cultured pulmonary artery endothelial cells. J Mol Cell Cardiol 22: 893-842, 1990*
    1990 [Not refereed][Not invited]
  • Kawaguchi H, Sawa H, Yasuda H: Effect of atrial natriuretic factor on angiotensin converting enzyme. J Hypertens 8: 749-753, 1990*
    1990 [Not refereed][Not invited]
  • Kawaguchi H, Sawa H, Yasuda H: Effect of atrial natriuretic factor on angiotensin converting enzyme. J Mol Cell Cardiol 21: 959-961, 1989*
    1989 [Not refereed][Not invited]

Books etc

  • プリオン病と遅発性ウイルス感染症
    金原出版 2010
  • Molecular Biology of Tumor Virus Gene Products.
    Research Signpost 2009
  • DNA viruses, Methods and Protocols.
    Humana Press 2005
  • Understanding of Minamata disease. Methylmercury poisoning in Minamata and Niigata, Japan
    Japan Public Health Association 2001
  • Endokrine und zellbiologische aspekte der arteriosklerose 10
    Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung 1996
  • Arteriosklerose zerebraler gefäß 9
    Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung 1995

MISC

  • Rinsho Shinkeigaku  51-  (11)  1051  -1057  2011  [Not refereed][Not invited]
  • Case of highly active anti-retroviral therapy-induced immune reconstitution inflammatory syndrome in AIDS-related progressive multifocal leukoencephalopathy.
    Rinsho Shinkeigaku  65-  1495  -1500  2007  [Not refereed][Not invited]
  • Etiological agent and pathogenicity mechanism of PML.
    Nippon Rinsho  65-  1495  -1500  2007  [Not refereed][Not invited]
  • Recent research on the JC virus.
    Brain Nerve  59-  101  -108  2007  [Not refereed][Not invited]

Industrial Property Rights

  • JCウイルスのVP-1に対するsiRNA、およびそれを含有してなる医薬組成物
    特許第4672654号
  • JCウイルスagnoを対象としたPMLの治療
    特許第4840792号

Awards & Honors

  • 2009 日本神経病理学会賞

Educational Activities

Teaching Experience

  • 感染症学特別研究Ⅰ
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • Microbiology
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 医学部
    キーワード : 細菌、ウイルス、グラム陽性菌、グラム陰性菌、抗酸性菌、感染症、ワクチン、プリオン、寄生虫、原虫、マイコプラズマ、クラミジア、スピロヘータ、真菌
  • 感染症学特別演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • Zoonotic Science
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 人獣共通感染症、新興・再興感染症、疫学、自然宿主、媒介動物、感染環、ウイルス、細菌、原虫、寄生虫 
  • 研究倫理演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 人獣共通感染症対策専門特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 感染症学特別研究ⅡA
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 感染症学特別研究ⅡB
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 海外インターンシップA
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 海外インターンシップB
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 獣医科学・感染症学基礎科目 微生物学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 国際感染症学コア科目Ⅰ 人獣共通感染症制御学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 国際感染症学コア科目ⅡB 感染病理学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 獣医科学特別研究
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 海外インターンシップ
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 国内インターンシップ
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 人獣共通感染症対策専門特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学特論演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学基礎科目B 動物実験倫理特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学基礎科目B 獣医微生物学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学基礎科目A 人獣共通感染症制御学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 研究倫理演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • インターンシップ
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 獣医科学基礎科目 微生物学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学院

Campus Position History

  • 2016年4月1日 
    2018年3月31日 
    人獣共通感染症リサーチセンター副センター長
  • 2018年4月1日 
    2020年3月31日 
    人獣共通感染症リサーチセンター副センター長

Position History

  • 2016年4月1日 
    2018年3月31日 
    人獣共通感染症リサーチセンター副センター長
  • 2018年4月1日 
    2020年3月31日 
    人獣共通感染症リサーチセンター副センター長


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