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Master

Affiliation (Master)

  • International Institute for Zoonosis Control　Division of Infection and Immunity

Affiliation (Master)

  • International Institute for Zoonosis Control　Division of Infection and Immunity

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  • Name (Japanese)

    Higashi

  • Name (Kana)

    Hideaki

  • Name

    200901099560352225

Achievement

Research Interests

  • Anthrax   細菌   ヘリコバクターピロリ   感染症   癌   

Research Areas

  • Life sciences / Molecular biology

Awards

  • 2003 日本癌学会奨励賞
  • 2002 消化管細胞機能研究会 特別賞
  • 2002 日本ヘリコバクター学会 上原H. pylori 最優秀賞

Published Papers

  • Taona Sinyawa, Misheck Shawa, Geoffrey M. Muuka, Fusya Goma, Paul Fandamu, Joseph Yamweka Chizimu, Cynthia Sipho Khumalo, Malala Mulavu, Masuzyo Ngoma, Herman Moses Chambaro, Harvey Kakoma Kamboyi, Masahiro Kajihara, Hirofumi Sawa, Yasuhiko Suzuki, Hideaki Higashi, Geoffrey Mainda, Musso Munyeme, John Bwalya Muma, Christian Owusu Nyantakyi, Beverly Egyir, Bernard Mudenda Hang’ombe
    Antibiotics 13 (5) 467 - 467 2024/05/20 
    Antimicrobial resistance (AMR) among Escherichia coli from food animals is a rising problem, and heavy antimicrobial use in poultry is a contributing factor. In Zambia, studies linking poultry-associated AMR and antibiotic use (AMU) are rare. This study aimed to investigate commercial and medium-/small-scale poultry farmers’ usage of antimicrobials based on a questionnaire survey in ten districts of Zambia. In addition, the study characterized extended-spectrum β-lactamase (ESBL)-producing E. coli isolates obtained from poultry in the same districts. Data regarding knowledge and usage of antimicrobials were collected from commercial and medium-/small-scale poultry farmers using a pre-tested structured questionnaire. At the same time, cloacal samples were collected and analyzed. One hundred and fifty E. coli isolates were tested for antimicrobial susceptibility using eight antibiotic classes. The isolates were further screened for ESBL production by streaking them on cefotaxime (CTX)-supplemented MacConkey agar, then subjecting them to sequencing on a NextSeq. The questionnaire survey showed that more medium-/small-scale than commercial poultry farmers used antimicrobials (OR = 7.70, 95% CI = 2.88–20.61) but less prescriptions (OR = 0.02, 95% CI = 0.00–0.08). Susceptibility testing revealed that resistance was highest to ampicillin (128/148, 86.5%) and tetracycline (101/136, 74.3%) and that the prevalence of multidrug resistance (MDR) (28/30, 93.3%) was high. Whole-genome sequencing (WGS) of eight (8/30, 26.7%) isolates with CTX Minimum Inhibitory Concentration (MIC) ≥ 4 µg/mL revealed the presence of ESBL-encoding genes blaCTX-M-14, blaCTX-M-55, and blaTEM. WGS also detected other AMR genes for quinolones, aminoglycosides, phenicols, tetracycline, macrolides, and folate-pathway antagonists. Altogether, the questionnaire survey results showed a higher proportion of AMU and lower prescription usage among medium-/small-scale farmers. In addition, our results emphasize the circulation of ESBL-producing E. coli strains with associated MDR. It is critical to educate farmers about AMR risks and to encourage responsible usage of antimicrobials. Furthermore, there is a need to strengthen regulations limiting access to antimicrobials. Finally, there is a need to establish a one health system to guide public health response.
  • Chikwanda Chileshe, Misheck Shawa, Nelson Phiri, Joseph Ndebe, Cynthia Sipho Khumalo, Chie Nakajima, Masahiro Kajihara, Hideaki Higashi, Hirofumi Sawa, Yasuhiko Suzuki, Walter Muleya, Bernard Mudenda Hang'ombe
    Antibiotics (Basel, Switzerland) 13 (3) 2024/03/15 
    Poultry products in Zambia form an integral part of the human diet in many households, as they are cheap and easy to produce. The burden of poultry diseases has, however, remained a major challenge. Growing consumer demand for poultry products in Zambia has resulted in non-prudent antimicrobial use on farms, intending to prevent and treat poultry diseases for growth optimisation and maximising profits. This cross-sectional study aimed to identify the different types of bacteria causing diseases in chickens in Lusaka and to detect the extended-spectrum lactamase (ESBL)-encoding genes. We collected 215 samples from 91 diseased chickens at three post-mortem facilities and screened them for Gram-negative bacteria. Of these samples, 103 tested positive for various clinically relevant Enterobacteriaceae, including Enterobacter (43/103, 41.7%), Escherichia coli (20/103, 19.4%), Salmonella (10/103, 9.7%), and Shigella (8/103, 7.8%). Other isolated bacteria included Yersinia, Morganella, Proteus, and Klebsiella, which accounted for 21.4%. E. coli, Enterobacter, Salmonella, and Shigella were subjected to antimicrobial susceptibility testing. The results revealed that E. coli, Enterobacter, and Shigella were highly resistant to tetracycline, ampicillin, amoxicillin, and trimethoprim-sulfamethoxazole, while Salmonella showed complete susceptibility to all tested antibiotics. The observed resistance patterns correlated with antimicrobial usage estimated from sales data from a large-scale wholesale and retail company. Six (6/14, 42.9%) E. coli isolates tested positive for blaCTX-M, whilst eight (8/14, 57.1%) Enterobacter samples tested positive for blaTEM. Interestingly, four (4/6, 66.7%) of the E. coli isolates carrying blaCTX-M-positive strains were also positive for blaTEM. Sanger sequencing of the PCR products revealed that five (5/6, 83.3%) of the abovementioned isolates possessed the blaCTX-M-15 allele. The results suggest the presence of potentially pathogenic ESBL-producing Enterobacteriaceae in poultry, threatening public health.
  • Misheck Shawa, Atmika Paudel, Herman Chambaro, Harvey Kamboyi, Ruth Nakazwe, Luke Alutuli, Tuvshinzaya Zorigt, Taona Sinyawa, Mulemba Samutela, Joseph Chizimu, Manyando Simbotwe, Kyoko Hayashida, Naganori Nao, Masahiro Kajihara, Yoshikazu Furuta, Yasuhiko Suzuki, Hirofumi Sawa, Bernard Hang'ombe, Hideaki Higashi
    PloS one 19 (4) e0302053  2024 
    Increased antimicrobial resistance (AMR) among bacteria underscores the need to strengthen AMR surveillance and promote data-based prescribing. To evaluate trends and associations between antimicrobial usage (AMU) and AMR, we explored a dataset of 34,672 bacterial isolates collected between 2015 and 2020 from clinical samples at the University Teaching Hospital (UTH) in Lusaka, Zambia. The most frequently isolated species were Escherichia coli (4,986/34,672; 14.4%), Staphylococcus aureus (3,941/34,672; 11.4%), and Klebsiella pneumoniae (3,796/34,672; 10.9%). Of the 16 drugs (eight classes) tested, only amikacin and imipenem showed good (> 50%) antimicrobial activity against both E. coli and K. pneumoniae, while nitrofurantoin was effective only in E. coli. Furthermore, 38.8% (1,934/4,980) of E. coli and 52.4% (2,079/3,791) of K. pneumoniae isolates displayed multidrug resistance (MDR) patterns on antimicrobial susceptibility tests. Among S. aureus isolates, 44.6% (973/2,181) were classified as methicillin-resistant (MRSA). Notably, all the MRSA exhibited MDR patterns. The annual hospital AMR rates varied over time, while there was a weak positive relationship (r = 0.38, 95% CI = 0.11-0.60) between the monthly use of third-generation cephalosporins (3GCs) and 3GC resistance among Enterobacterales. Overall, the results revealed high AMR rates that fluctuated over time, with a weak positive relationship between 3GC use and resistance. To our knowledge, this is the first report to evaluate the association between AMU and AMR in Zambia. Our results highlight the need to strengthen antimicrobial stewardship programs and optimize AMU in hospital settings.
  • Saicheng Zhang, Yuki Funahashi, Satoho Tanaka, Torahiko Okubo, Jeewan Thapa, Shinji Nakamura, Hideaki Higashi, Hiroyuki Yamaguchi
    Microbes and Infection 25 (5) 105097 - 105097 1286-4579 2023/06
  • Hirofumi Uchiyama, Daisuke Muramatsu, Hideaki Higashi, Hiroshi Kida, Atsushi Iwai
    PloS one 18 (4) e0284343  2023 
    Chondroitin sulfate (CS) is a glycosaminoglycan, and CS derived from various animal species is used in drugs and food supplements to alleviate arthralgia. The CS is a high molecular weight compound, and hydrolysis of CS by intestinal microbiota is thought to be required for absorption in mammalians. Chondroitin sulfate oligosaccharides (Oligo-CS) are produced by hydrolysis with subcritical water from CS isolated from a species of skate, Raja pulchra for the improvement of bioavailability. The present study conducted in vitro experiments using murine cell lines, to compare the biological activities of Oligo-CS and high molecular weight CS composed with the similar disaccharide isomer units of D-glucuronic acid and N-acetyl-D-glucosamine (CS-C). The results show that Oligo-CS inhibits osteoclast differentiation of RAW264 cells significantly at lower concentrations than in CS. The cell viability of a myoblast cell line, C2C12 cells, was increased when the cells were grown in a differentiated medium for myotubes with Oligo-CS, where there were no effects on the cell viability in CS. These results suggest that in vitro Oligo-CS exhibits stronger bioactivity than high-molecular weight CS.
  • Daisuke Muramatsu, Hirofumi Uchiyama, Hideaki Higashi, Hiroshi Kida, Atsushi Iwai
    PloS one 18 (5) e0286255  2023 
    Betanin is a red pigment of red beetroot (Beta vulgaris L.), providing the beneficial effects to maintain human health. Betanin is involved in the characteristic red color of red beetroot, and used as an edible dye. Betanin is known to be a highly unstable pigment, and water solutions of betanin are nearly fully degraded after heating at 99°C for 60 min in the experimental conditions of this study. The present study investigated the effects of red beetroot juice (RBJ) and betanin on immune cells, and found that stimulation with RBJ and betanin induces interleukin (IL)-1β, IL-8, and IL-10 mRNA in a human monocyte derived cell line, THP-1 cells. This mRNA induction after stimulation with RBJ and betanin was not significantly changed after heat treatment when attempting to induce degradation of the betanin. Following these results, the effects of heat degradation of betanin on the inhibition of lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW264 cells and the antioxidant capacity were investigated. The results showed that the inhibition activity of RBJ and betanin with the LPS induced NO production is not altered after heat degradation of betanin. In addition, the results of FRAP (ferric reducing antioxidant power) and DPPH (1,1-Diphenyl-2-picrylhydrazyl) assays indicate that a not inconsiderable degree of the antioxidant capacity of RBJ and betanin remained after heat degradation of betanin. These results suggest that it is important to consider the effects of degradation products of betanin in the evaluation of the beneficial effects of red beetroot on health.
  • Ryoya Tsujikawa, Jeewan Thapa, Torahiko Okubo, Shinji Nakamura, Saicheng Zhang, Yoshikazu Furuta, Hideaki Higashi, Hiroyuki Yamaguchi
    Current Microbiology 79 (9) 0343-8651 2022/09
  • Jeewan Thapa, Gen Yoshiiri, Koki Ito, Torahiko Okubo, Shinji Nakamura, Yoshikazu Furuta, Hideaki Higashi, Hiroyuki Yamaguchi
    Frontiers in Cellular and Infection Microbiology 12 2022/05/26 
    Chlamydia trachomatis (Ct) is an intracellular energy-parasitic bacterium that requires ATP derived from infected cells for its growth. Meanwhile, depending on the O2 concentration, the host cells change their mode of ATP production between oxidative phosphorylation in mitochondria (Mt) and glycolysis; this change depends on signaling via reactive oxygen species (ROS) produced by NADPH oxidases (NOXs) as well as Mt. It has been proposed that Ct correspondingly switches its source of acquisition of ATP between host-cell Mt and glycolysis, but this has not been verified experimentally. In the present study, we assessed the roles of host-cell NOXs and Mt in the intracellular growth of CtL2 (L2 434/Bu) under normoxia (21% O2) and hypoxia (2% O2) by using several inhibitors of NOXs (or the downstream molecule) and Mt-dysfunctional (Mtd) HEp-2 cells. Under normoxia, diphenyleneiodonium, an inhibitor of ROS diffusion, abolished the growth of CtL2 and other Chlamydiae (CtD and C. pneumoniae). Both ML171 (a pan-NOX inhibitor) and GLX351322 (a NOX4-specific inhibitor) impaired the growth of CtL2 under normoxia, but not hypoxia. NOX4-knockdown cells diminished the bacterial growth. SB203580, an inhibitor of the NOX4-downstream molecule p38MAPK, also inhibited the growth of CtL2 under normoxia but not hypoxia. Furthermore, CtL2 failed to grow in Mtd cells under normoxia, but no effect was observed under hypoxia. We conclude that under normoxia, Ct requires functional Mt in its host cells as an ATP source, and that this process requires NOX4/p38MAPK signaling in the host cells. In contrast to hypoxia, crosstalk between NOX4 and Mt via p38MAPK may be crucial for the growth of Ct under normoxia.
  • Yoshikazu Furuta, Mai Tsujinouchi, Misheck Shawa, Tuvshinzaya Zorigt, Yuya Miyajima, Atmika Paudel, Satowa Suzuki, Hideaki Higashi
    Microbiology resource announcements 11 (4) e0120321  2022/04/21 
    Bacillus cereus is mainly associated with foodborne illness but sometimes causes nosocomial infections. We previously reported that B. cereus strains of a specific sequence type, ST1420, were associated with nosocomial infection. Here, we determined the complete genome sequences of B. cereus strains isolated from nosocomial infection cases in Japanese hospitals.
  • Misheck Shawa, Yoshikazu Furuta, Atmika Paudel, O'Brian Kabunda, Evans Mulenga, Maron Mubanga, Harvey Kamboyi, Tuvshinzaya Zorigt, Herman Chambaro, Manyando Simbotwe, Bernard Hang'ombe, Hideaki Higashi
    FEMS microbiology letters 2022/01/14 
    Multidrug-resistant (MDR) Escherichia coli in food animals such as chickens is an emerging public health concern in Zambia. Additionally, the country's high demand for poultry products necessitates further investigation into the link between poultry and human MDR E. coli. Twenty cefotaxime-resistant E. coli isolates collected from poultry in Lusaka, Zambia, were screened for multidrug resistance and sequenced on MiSeq and MinION platforms. Genomes were assembled de novo and compared to 36 previously reported cefotaxime-resistant E. coli isolates from inpatients at the University Teaching Hospital, Lusaka. All (20/20, 100%) poultry isolates exhibited resistance to ampicillin, chloramphenicol, and doxycycline. Phylogenetic analysis and hierarchical clustering showed a high degree of genetic relatedness between E. coli O17:H18-ST69 from poultry and humans. The E. coli O17:H18-ST69 clone accounted for 4/20 (20%) poultry- and 9/36 (25%) human-associated isolates that shared two plasmids harboring 14 antimicrobial resistance (AMR) genes. However, comparison analysis showed that the isolates also had other AMR plasmids distinct for each niche. Our results suggested clonal transmission of MDR E. coli between poultry and humans, with the potential acquisition of niche-specific AMR plasmids. Thus, the control of MDR E. coli requires a One Health approach involving both human and animal health sectors.
  • Misheck Shawa, Yoshikazu Furuta, Gillan Mulenga, Maron Mubanga, Evans Mulenga, Tuvshinzaya Zorigt, Christone Kaile, Manyando Simbotwe, Atmika Paudel, Bernard Hang’ombe, Hideaki Higashi
    Antimicrobial Resistance & Infection Control 10 (1) 79 - 79 2021/12 [Refereed]
     
    Abstract Background The epidemiology of extended-spectrum β-lactamases (ESBLs) has undergone dramatic changes, with CTX-M-type enzymes prevailing over other types. blaCTX-M genes, encoding CTX-M-type ESBLs, are usually found on plasmids, but chromosomal location is becoming common. Given that blaCTX-M-harboring strains often exhibit multidrug resistance (MDR), it is important to investigate the association between chromosomally integrated blaCTX-M and the presence of additional antimicrobial resistance (AMR) genes, and to identify other relevant genetic elements. Methods A total of 46 clinical isolates of cefotaxime-resistant Enterobacteriaceae (1 Enterobacter cloacae, 9 Klebsiella pneumoniae, and 36 Escherichia coli) from Zambia were subjected to whole-genome sequencing (WGS) using MiSeq and MinION. By reconstructing nearly complete genomes, blaCTX-M genes were categorized as either chromosomal or plasmid-borne. Results WGS-based genotyping identified 58 AMR genes, including four blaCTX-M alleles (i.e., blaCTX-M-14, blaCTX-M-15, blaCTX-M-27, and blaCTX-M-55). Hierarchical clustering using selected phenotypic and genotypic characteristics suggested clonal dissemination of blaCTX-M genes. Out of 45 blaCTX-M gene-carrying strains, 7 harbored the gene in their chromosome. In one E. cloacae and three E. coli strains, chromosomal blaCTX-M-15 was located on insertions longer than 10 kb. These insertions were bounded by ISEcp1 at one end, exhibited a high degree of nucleotide sequence homology with previously reported plasmids, and carried multiple AMR genes that corresponded with phenotypic AMR profiles. Conclusion Our study revealed the co-occurrence of ISEcp1-blaCTX-M-15 and multiple AMR genes on chromosomal insertions in E. cloacae and E. coli, suggesting that ISEcp1 may be responsible for the transposition of diverse AMR genes from plasmids to chromosomes. Stable retention of such insertions in chromosomes may facilitate the successful propagation of MDR clones among these Enterobacteriaceae species.
  • Tuvshinzaya Zorigt, Satoshi Ito, Norikazu Isoda, Yoshikazu Furuta, Misheck Shawa, Natsagdorj Norov, Baasansuren Lkham, Jargalsaikhan Enkhtuya, Hideaki Higashi
    PLOS ONE 16 (11) e0260299 - e0260299 2021/11/19 
    Anthrax is a worldwide zoonotic disease. Anthrax has long been a public health and socio-economic issue in Mongolia. Presently, there is no spatial information on carcass burial sites as a potential hazard of future anthrax outbreaks and possible risk factors associated with anthrax occurrences in Mongolia. Here, we analyze retrospective data (1986–2015) on the disposal sites of livestock carcasses to describe historical spatio-temporal patterns of livestock anthrax in Khuvsgul Province, which showed the highest anthrax incidence rate in Mongolia. From the results of spatial mean and standard deviational ellipse analyses, we found that the anthrax spatial distribution in livestock did not change over the study period, indicating a localized source of exposure. The multi-distance spatial cluster analysis showed that carcass sites distributed in the study area are clustered. Using kernel density estimation analysis on carcass sites, we identified two anthrax hotspots in low-lying areas around the south and north regions. Notably, this study disclosed a new hotspot in the northern part that emerged in the last decade of the 30-year study period. The highest proportion of cases was recorded in cattle, whose prevalence per area was highest in six districts (i.e., Murun, Chandmani-Undur, Khatgal, Ikh-Uul, Tosontsengel, and Tsagaan-Uul), suggesting that vaccination should prioritize cattle in these districts. Furthermore, size of outbreaks was influenced by the annual summer mean air temperature of Khuvsgul Province, probably by affecting the permafrost freeze-thawing activity.
  • Tuvshinzaya Zorigt, Yoshikazu Furuta, Manyando Simbotwe, Akihiro Ochi, Mai Tsujinouchi, Misheck Shawa, Tomoko Shimizu, Norikazu Isoda, Jargalsaikhan Enkhtuya, Hideaki Higashi
    PLOS ONE 16 (10) e0258317 - e0258317 2021/10/11 [Refereed]
     
    Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacterium Bacillus anthracis. Detecting naturally acquired antibodies against anthrax sublethal exposure in animals is essential for anthrax surveillance and effective control measures. Serological assays based on protective antigen (PA) of B. anthracis are mainly used for anthrax surveillance and vaccine evaluation. Although the assay is reliable, it is challenging to distinguish the naturally acquired antibodies from vaccine-induced immunity in animals because PA is cross-reactive to both antibodies. Although additional data on the vaccination history of animals could bypass this problem, such data are not readily accessible in many cases. In this study, we established a new enzyme-linked immunosorbent assay (ELISA) specific to antibodies against capsule biosynthesis protein CapA antigen of B. anthracis, which is non-cross-reactive to vaccine-induced antibodies in horses. Using in silico analyses, we screened coding sequences encoded on pXO2 plasmid, which is absent in the veterinary vaccine strain Sterne 34F2 but present in virulent strains of B. anthracis. Among the 8 selected antigen candidates, capsule biosynthesis protein CapA (GBAA_RS28240) and peptide ABC transporter substrate-binding protein (GBAA_RS28340) were detected by antibodies in infected horse sera. Of these, CapA has not yet been identified as immunoreactive in other studies to the best of our knowledge. Considering the protein solubility and specificity of B. anthracis, we prepared the C-terminus region of CapA, named CapA322, and developed CapA322-ELISA based on a horse model. Comparative analysis of the CapA322-ELISA and PAD1-ELISA (ELISA uses domain one of the PA) showed that CapA322-ELISA could detect anti-CapA antibodies in sera from infected horses but was non-reactive to sera from vaccinated horses. The CapA322-ELISA could contribute to the anthrax surveillance in endemic areas, and two immunoreactive proteins identified in this study could be additives to the improvement of current or future vaccine development.
  • Yoshikazu Furuta, Cheng Cheng, Tuvshinzaya Zorigt, Atmika Paudel, Shun Izumi, Mai Tsujinouchi, Tomoko Shimizu, Wim G. Meijer, Hideaki Higashi
    mSystems 6 (4) 2021/08/31 [Refereed]
     
    Bacillus anthracis is the Gram-positive bacterial species that causes anthrax. Anthrax is still prevalent in countries mainly in Asia and Africa, where it causes economic damage and remains a public health issue.
  • Mulemba Tillika Samutela, Geoffrey Kwenda, Edgar Simulundu, Panji Nkhoma, Hideaki Higashi, Andrew Frey, Matthew Bates, Bernard M. Hang'ombe
    International Journal of Infectious Diseases 109 38 - 49 1201-9712 2021/08 [Refereed]
  • Chiho Kaneko, Michihito Sasaki, Ryosuke Omori, Ryo Nakao, Chikako Kataoka-Nakamura, Ladslav Moonga, Joseph Ndebe, Walter Muleya, Edgar Simulundu, Bernard M. Hang’ombe, George Dautu, Masahiro Kajihara, Akina Mori-Kajihara, Yongjin Qiu, Naoto Ito, Herman M. Chambaro, Chihiro Sugimoto, Hideaki Higashi, Ayato Takada, Hirofumi Sawa, Aaron S. Mweene, Norikazu Isoda
    Pathogens 10 (6) 738 - 738 2021/06/11 [Refereed]
     
    Rabies remains endemic in Zambia. Despite conducting canine vaccinations in Lusaka district, the vaccination coverage and actual seropositivity in the dog population in Lusaka district are rarely evaluated. This study estimated the seropositivity-based immunization coverage in the owned dog population in Lusaka district using the expanded program on immunization cluster survey method. The time-series trend of neutralizing antibodies against rabies in vaccinated dogs was also evaluated. Of 366 dogs in 200 dog-owning households in Lusaka district, blood samples were collected successfully from 251 dogs. In the sampled dogs, 42.2% (106/251) had an antibody titer ≥0.5 IU/mL. When the 115 dogs whose blood was not collected were assumed to be seronegative, the minimum immunization coverage in Lusaka district’s owned dog population was estimated at 29.0% (95% confidence interval: 22.4–35.5). It was also found that a single vaccination with certified vaccines is capable of inducing protective levels of antibodies. In contrast, higher antibody titers were observed in multiple-vaccinated dogs than in single-vaccinated dogs, coupled with the observation of a decline in antibody titer over time. These results suggest the importance of continuous booster immunization to maintain herd immunity and provide useful information to plan mass vaccination against rabies in Zambia.
  • Chiho Kaneko, Ryosuke Omori, Michihito Sasaki, Chikako Kataoka-Nakamura, Edgar Simulundu, Walter Muleya, Ladslav Moonga, Joseph Ndebe, Bernard M. Hang’ombe, George Dautu, Yongjin Qiu, Ryo Nakao, Masahiro Kajihara, Akina Mori-Kajihara, Herman M. Chambaro, Hideaki Higashi, Chihiro Sugimoto, Hirofumi Sawa, Aaron S. Mweene, Ayato Takada, Norikazu Isoda
    PLOS Neglected Tropical Diseases 15 (4) e0009222 - e0009222 2021/04/28 [Refereed]
     
    Background An estimated 75% or more of the human rabies cases in Africa occur in rural settings, which underscores the importance of rabies control in these areas. Understanding dog demographics can help design strategies for rabies control and plan and conduct canine mass vaccination campaigns effectively in African countries. Methodology/Principal findings A cross-sectional survey was conducted to investigate domestic dog demographics in Kalambabakali, in the rural Mazabuka District of Zambia. The population of ownerless dogs and the total achievable vaccination coverage among the total dog population was estimated using the capture-recapture-based Bayesian model by conducting a canine mass vaccination campaign. This study revealed that 29% of the domestic dog population was under one year old, and 57.7% of those were under three months old and thus were not eligible for the canine rabies vaccination in Zambia. The population growth was estimated at 15% per annum based on the cross-sectional household survey. The population of ownerless dogs was estimated to be small, with an ownerless-to-owned-dog ratio of 0.01–0.06 in the target zones. The achieved overall vaccination coverage from the first mass vaccination was estimated 19.8–51.6%. This low coverage was principally attributed to the owners’ lack of information, unavailability, and dog-handling difficulties. The follow-up mass vaccination campaign achieved an overall coverage of 54.8–76.2%. Conclusions/Significance This paper indicates the potential for controlling canine rabies through mass vaccination in rural Zambia. Rabies education and responsible dog ownership are required to achieve high and sustainable vaccination coverage. Our findings also propose including puppies below three months old in the target population for rabies vaccination and emphasize that securing an annual enforcement of canine mass vaccination that reaches 70% coverage in the dog population is necessary to maintain protective herd immunity.
  • Miho Okude, Junji Matsuo, Tomohiro Yamazaki, Kentaro Saito, Yoshikazu Furuta, Shinji Nakamura, Jeewan Thapa, Torahiko Okubo, Hideaki Higashi, Hiroyuki Yamaguchi
    Microbiology and Immunology 65 (3) 115 - 124 0385-5600 2021/03 [Refereed]
     
    We previously isolated a symbiotic environmental amoeba, harboring an environmental chlamydia, Neochlamydia S13. Interestingly, this bacterium failed to survive outside of host cells and was immediately digested inside other amoebae, indicating bacterial distribution via cytokinesis. This may provide a model for understanding organelle development and chlamydial pathogenesis and evolution; therefore, we assessed our hypothesis of Neochlamydia S13 distribution via cytokinesis by comparative analysis with other environmental Chlamydiae (Protochlamydia R18 and Parachlamydia Bn9 ). Dual staining with 4',6-diamidino-2-phenylindole and phalloidin revealed that the progeny of Neochlamydia S13 and Protochlamydia R18 existed in both daughter cells with a contractile ring on the verge of separation. However, in contrast to other environmental Chlamydiae, little Neochlamydia S13 16S ribosomal DNA was amplified from the culture supernatant. Interestingly, Neochlamydia S13 failed to infect aposymbiotic amoebae, indicating an intimate interaction with the host cells. Furthermore, its infectious rates in cultures expanded from a single amoeba were always maintained at 100%, indicating distribution via cytokinesis. We concluded that unlike other environmental Chlamydiae, Neochlamydia S13 has a unique ability to divide its progeny only via host amoebal cytokinesis. This may be a suitable model to elucidate the mechanism of cell organelle distribution and of chlamydial pathogenesis and evolution.
  • Atmika Paudel, Yoshikazu Furuta, Hideaki Higashi
    Virulence 12 (1) 2285 - 2295 2150-5594 2021/01/01 [Refereed]
  • Kosuke Okuya, Nao Eguchi, Rashid Manzoor, Reiko Yoshida, Shinji Saito, Tadaki Suzuki, Michihito Sasaki, Takeshi Saito, Yurie Kida, Akina Mori-Kajihara, Hiroko Miyamoto, Osamu Ichii, Masahiro Kajihara, Hideaki Higashi, Ayato Takada
    Viruses 12 (7) 780 - 780 2020/07/20 [Refereed]
     
    The influenza A virus (IAV) matrix-2 (M2) protein is an antigenically conserved viral envelope protein that plays an important role in virus budding together with another envelope protein, hemagglutinin (HA). An M2-specific mouse monoclonal IgG antibody, rM2ss23, which binds to the ectodomain of the M2 protein, has been shown to be a non-neutralizing antibody, but inhibits plaque formation of IAV strains. In this study, we generated chimeric rM2ss23 (ch-rM2ss23) IgG and IgA antibodies with the same variable region and compared their antiviral activities. Using gel chromatography, ch-rM2ss23 IgA were divided into three antibody subsets: monomeric IgA (m-IgA), dimeric IgA (d-IgA), and trimeric and tetrameric IgA (t/q-IgA). We found that t/q-IgA had a significantly higher capacity to reduce the plaque size of IAVs than IgG and m-IgA, most likely due to the decreased number of progeny virus particles produced from infected cells. Interestingly, HA-M2 colocalization was remarkably reduced on the infected cell surface in the presence of ch-rM2ss23 antibodies. These results indicate that anti-M2 polymeric IgA restricts IAV budding more efficiently than IgG and suggest a role of anti-M2 IgA in cross-protective immunity to IAVs.
  • Yongjin Qiu, Masahiro Kajihara, Ryo Nakao, Evans Mulenga, Hayato Harima, Bernard Mudenda Hang’ombe, Yoshiki Eto, Katendi Changula, Daniel Mwizabi, Hirofumi Sawa, Hideaki Higashi, Aaron Mweene, Ayato Takada, Martin Simuunza, Chihiro Sugimoto
    Pathogens 9 (6) 469 - 469 2020/06/13 [Refereed][Not invited]
     
    Bat-associated bartonellae, including Bartonella mayotimonensis and Candidatus Bartonella rousetti, were recently identified as emerging and potential zoonotic agents, respectively. However, there is no report of bat-associated bartonellae in Zambia. Thus, we aimed to isolate and characterize Bartonella spp. from bats and bat flies captured in Zambia by culturing and PCR. Overall, Bartonella spp. were isolated from six out of 36 bats (16.7%), while Bartonella DNA was detected in nine out of 19 bat flies (47.3%). Subsequent characterization using a sequence of five different genes revealed that three isolates obtained from Egyptian fruit bats (Rousettus aegyptiacus) were Ca. B. rousetti. The isolates obtained from insectivorous bats (Macronycteris vittatus) were divided into two previously unclassified bat-associated bartonellae. A phylogenetic analysis of the six genotypes of Bartonella gltA sequences from nine pathogen-positive bat flies revealed that three genotypes belonged to the same clades as bat-associated bartonellae, including Ca. B. rousetti. The other three genotypes represented arthropod-associated bartonellae, which have previously been isolated only from ectoparasites. We demonstrated that Ca. B. rousetti is maintained between bats (R. aegyptiacus) and bat flies in Zambia. Continuous surveillance of Bartonella spp. in bats and serological surveys in humans in Africa are warranted to evaluate the public health importance of bat-associated bartonellae.
  • Kosuke Okuya, Reiko Yoshida, Rashid Manzoor, Shinji Saito, Tadaki Suzuki, Michihito Sasaki, Takeshi Saito, Yurie Kida, Akina Mori-Kajihara, Tatsunari Kondoh, Masahiro Sato, Masahiro Kajihara, Hiroko Miyamoto, Osamu Ichii, Hideaki Higashi, Ayato Takada
    Journal of Virology 94 (12) 0022-538X 2020/06 [Refereed][Not invited]
     
    Mucosal immunity represented by pSIgA plays important roles in protection from IAV infection. Furthermore, IAV HA-specific pSIgA antibodies are thought to contribute to cross-protective immunity against multiple IAV subtypes. However, the mechanisms by which pSIgA exerts such versatile antiviral activity are not fully understood. In this study, we generated broadly cross-reactive recombinant IgG and pSIgA having the same antigen-recognition site and compared their antiviral activities in vitro . These recombinant antibodies did not show “classical” neutralizing activity, whereas pSIgA, but not IgG, significantly inhibited the production of progeny virus particles from infected cells. Plaque formation was also significantly reduced by pSIgA, but not IgG. These effects were seen in infection with IAVs of several different HA subtypes. Based on our findings, we propose an antibody-mediated host defense mechanism by which mucosal immunity may contribute to broad cross-protection from IAVs of multiple HA subtypes, including viruses with pandemic potential.
  • Takashi Ohnishi, Katsuhisa Yamada, Koji Iwasaki, Takeru Tsujimoto, Hideaki Higashi, Taichi Kimura, Norimasa Iwasaki, Hideki Sudo
    Scientific Reports 9 (1) 19324 - 19324 2019/12 [Refereed][Not invited]
     
    AbstractApproximately 40% of people under 30 and over 90% of people 55 or older suffer from moderate-to-severe levels of degenerative intervertebral disc (IVD) disease in their lumbar spines. Surgical treatments are sometimes effective; however, the treatment of back pain related to IVD degeneration is still a challenge; therefore, new treatments are necessary. Apoptosis may be important in IVD degeneration because suppressing cell apoptosis inside the IVD inhibits degeneration. Caspase-3, the primary effector of apoptosis, may be a key treatment target. We analyzed caspase-3’s role in two different types of IVD degeneration using caspase-3 knockout (Casp-3 KO) mice. Casp-3 KO delayed IVD degeneration in the injury-induced model but accelerated it in the age-induced model. Our results suggest that this is due to different pathological mechanisms of these two types of IVD degeneration. Apoptosis was suppressed in the IVD cells of Casp-3 KO mice, but cellular senescence was enhanced. This would explain why the Casp-3 KO was effective against injury-induced, but not age-related, IVD degeneration. Our results suggest that short-term caspase-3 inhibition could be used to treat injury-induced IVD degeneration.
  • Reiko Akamatsu, Masato Suzuki, Keiji Okinaka, Teppei Sasahara, Kunikazu Yamane, Satowa Suzuki, Daisuke Fujikura, Yoshikazu Furuta, Naomi Ohnishi, Minoru Esaki, Keigo Shibayama, Hideaki Higashi
    Emerging infectious diseases 25 (5) 883 - 890 2019/05 [Refereed][Not invited]
     
    Bacillus cereus is associated with foodborne illnesses characterized by vomiting and diarrhea. Although some B. cereus strains that cause severe extraintestinal infections and nosocomial infections are recognized as serious public health threats in healthcare settings, the genetic backgrounds of B. cereus strains causing such infections remain unknown. By conducting pulsed-field gel electrophoresis and multilocus sequence typing, we found that a novel sequence type (ST), newly registered as ST1420, was the dominant ST isolated from the cases of nosocomial infections that occurred in 3 locations in Japan in 2006, 2013, and 2016. Phylogenetic analysis showed that ST1420 strains belonged to the Cereus III lineage, which is much closer to the Anthracis lineage than to other Cereus lineages. Our results suggest that ST1420 is a prevalent ST in B. cereus strains that have caused recent nosocomial infections in Japan.
  • Kochi Toyomane, Yoshikazu Furuta, Daisuke Fujikura, Hideaki Higashi
    PeerJ 7 e6718 - e6718 2167-8359 2019/04/12 [Refereed]
     
    The anthrax toxin is a virulence factor produced by the bacteriumBacillus anthracis. Transcription of anthrax toxin genes is controlled by the transcription factor AtxA. Thus, AtxA is thought to be a key factor for the pathogenicity ofB. anthracis. Despite its important role inB. anthracisinfection, the molecular mechanism by which AtxA controls expression of anthrax toxin remains unclear. This study aimed to characterize the molecular mechanism of AtxA-mediated regulation of protective antigen (PA), a component of anthrax toxin encoded by thepagAgene. First, the interaction between the upstream region ofpagAand AtxA was evaluated in vivo by constructing a transcriptional fusion of the upstream region with an auxotrophic marker. The results showed that (i) the upstream region ofpagAsuppressed transcription of the downstream gene and (ii) AtxA recovered suppressed transcription. Second, in vitro analysis using a gel mobility shift assay was performed to evaluate binding specificity of the AtxA–DNA interaction. The result showed sequence-independent binding of AtxA to DNA. Taken together, our findings suggest that the expression of PA was suppressed by the upstream region ofpagAand that an interaction of AtxA and the upstream region releases the suppression.
  • Geographic distribution of cattle anthrax in Western Zambia
    Simbotwe M, Mulenga E, Furuta Y, Muuka GM, Hang’ombe BM, Higashi H
    JJVR 67 (2) 195 - 202 2019/02 [Refereed]
  • Yoshikazu Furuta, Hayato Harima, Emiko Ito, Fumito Maruyama, Naomi Ohnishi, Ken Osaki, Hirohito Ogawa, David Squarre, Bernard Mudenda Hang'ombe, Hideaki Higashi
    mSystems 3 (5) 2018/10/30 [Refereed][Not invited]
     
    Anthrax is caused by Bacillus anthracis , an endospore-forming soil bacterium. The genetic diversity of B. anthracis is known to be low compared with that of Bacillus species. In this study, we performed whole-genome sequencing of Zambian isolates of B. anthracis to understand the genetic diversity between closely related strains. Comparison of genomic sequences revealed that closely related strains were separated into three groups based on single nucleotide polymorphisms distributed throughout the genome. A large genomic deletion was detected in the region containing a bacitracin resistance gene cluster flanked by rRNA operons, resulting in the loss of bacitracin resistance. The structure of the deleted region, which was also conserved among species of the Bacillus cereus group, has the potential for both deletion and amplification and thus might be enabling the species to flexibly control the level of bacitracin resistance for adaptive evolution.
  • Manyando Simbotwe, Daisuke Fujikura, Miyuki Ohnuma, Ryosuke Omori, Yoshikazu Furuta, Geoffrey Munkombwe Muuka, Bernard Mudenda Hang’ombe, Hideaki Higashi
    PLOS ONE 13 (10) e0205986 - e0205986 1932-6203 2018/10/18 [Refereed]
     
    In Zambia, anthrax outbreaks among cattle are reported on nearly an annual basis. Presently, there is a lack of serological assays and information to develop an anthrax management and control strategy. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) based on recombinant protective antigen domain 1 (rPA-D1) of Bacillus anthracis was developed and used to detect anti-PA antibodies in cattle in Zambia. An antigen coating of 10 ng/well and a serum dilution of 1: 100 were determined to be the optimal rPA-D1 ELISA titration conditions. The intra-and inter-assay% coefficients of variation were less than 10% and 15%, respectively. The rPA-D1 ELISA could detect seroconversion in the cattle 1 month after anthrax vaccination. In a cross-sectional study conducted in the Western Province, Zambia, 187 serum samples from 8 herds of cattle were screened for anti-PA antibodies using the rPA-D1 ELISA. The seropositive rate of the serum samples was 8%, and the mean anti-PA antibody was 0.358 ELISA units. Additionally, we screened 131 cattle serum samples from Lusaka, which is a nonendemic area, and found no significant association between the antibody levels and sampling area (endemic versus nonendemic area). Conversely, significant differences were observed between the anti-PA antibody levels and herds, anti-PA antibody levels and vaccination status and anti-PA antibody levels and vaccination timing. Collectively, these findings suggest that the rPA-D1 ELISA is a useful tool for the detection of anti-PA antibodies in cattle in Zambia. The low proportion of seropositive sera indicates that there is inadequate cattle vaccination in the Western Province and, in addition to other epidemiological factors, this may precipitate the anthrax outbreak recurrence.
  • Daisuke Fujikura, Daisuke Muramatsu, Kochi Toyomane, Satoko Chiba, Takuji Daito, Atsushi Iwai, Takahisa Kouwaki, Masaaki Okamoto, Hideaki Higashi, Hiroshi Kida, Hiroyuki Oshiumi
    The Journal of Biochemistry 163 (1) 31 - 38 0021-924X 2018/01/01 [Refereed]
     
    Several microbial molecules with pathogen-associated molecular patterns stimulate host innate immune responses. The innate immune system plays a crucial role in activating acquired immune response via cytokine production and antigen presentation. Previous studies have shown that Aureobasidium pullulans-cultured fluid (AP-CF), which contains beta-glucan, exhibits adjuvant activity and renders mice resistance to influenza A virus infection; however, the underlying mechanism remains elusive. In this study, we investigated the innate immune response to AP-CF. We found that intraperitoneal administration of AP-CF increased the serum level of IL-18 and the number of splenic IFN-gamma producing CD4(+) cells during influenza A virus infection. The adjuvant effect of AP-CF was distinct from that of alum, which is known to have the ability to stimulate a Th2 immune response. In addition, AP-CF injection barely increased the number of peritoneal neutrophils and inflammatory macrophages, whereas alum injection markedly increased the number of neutrophils and inflammatory macrophages, suggesting that AP-CF is a weak inducer of inflammation compared to alum. AP-CF induced IL-18 production by DC2.4 cells, a dendritic cell line, and by peritoneal exudate cells that include peritoneal macrophages. Collectively, our findings indicate that AP-CF is an adjuvant that promotes the Th1 response during influenza A virus infection.
  • Hirohito Ogawa, Masahiro Kajihara, Naganori Nao, Asako Shigeno, Daisuke Fujikura, Bernard Hang’ombe, Aaron Mweene, Alisheke Mutemwa, David Squarre, Masao Yamada, Hideaki Higashi, Hirofumi Sawa, Ayato Takada
    Viruses 9 (12) 371 - 371 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.
  • Daisuke Fujikura, Masahiro Ikesue, Tsutomu Endo, Satoko Chiba, Hideaki Higashi, Toshimitsu Uede
    Nature Communications 8 (1) 13957 - 13957 2017/04 [Refereed]
     
    Expansion of autoreactive follicular helper T (Tfh) cells is tightly restricted to prevent induction of autoantibody-dependent immunological diseases, such as systemic lupus erythematosus (SLE). Here we show expression of an orphan immune regulator, death receptor 6 (DR6/TNFRSF21), on a population of Tfh cells that are highly expanded in lupus-like disease progression in mice. Genome-wide screening reveals an interaction between syndecan-1 and DR6 resulting in immunosuppressive functions. Importantly, syndecan-1 is expressed specifically on autoreactive germinal centre (GC) B cells that are critical for maintenance of Tfh cells. Syndecan-1 expression level on GC B cells is associated with Tfh cell expansion and disease progression in lupus-prone mouse strains. In addition, Tfh cell suppression by DR6-specific monoclonal antibody delays disease progression in lupus-prone mice. These findings suggest that the DR6/syndecan-1 axis regulates aberrant GC reactions and could be a therapeutic target for autoimmune diseases such as SLE.
  • Daisuke FUJIKURA, Kochi TOYOMANE, Kozue KAMIYA, Memi MUTOH, Etsuko MIFUNE, Miyuki OHNUMA, Hideaki HIGASHI
    Journal of Veterinary Medical Science 78 (8) 1311 - 1317 0916-7250 2016 [Refereed]
     
    Several animal models have shown that anthrax toxin (ATX) elicits a cytotoxic effect on host cells through anthrax toxin receptor (ANTXR) function. In this study, compared with mouse cells, cells obtained from humans exhibited low sensitivity to ATX-mediated cytotoxicity, and the sensitivity was not correlated with expression levels of ANTXRs. ATX treatment also induced a cytotoxic effect in other cultured human cells, human embryonic kidney (HEK) 293 cells, that express ANTXRs at undetectable levels. Furthermore, ectopic expression of ANTXRs in HEK293 cells did not affect the sensitivity to ATX treatment. These findings suggest that there is an ANTXR-independent cytotoxic mechanism in human cells.
  • Kameoka S, Kameyama T, Hayashi T, Sato S, Ohnishi N, Hayashi T, Murata-Kamiya N, Higashi H, Hatakeyama M, Takaoka A
    Biomedical research (Tokyo, Japan) 37 (1) 21 - 27 0388-6107 2016 [Refereed][Not invited]
     
    More than 50% of people in the world are infected with Helicobacter pylori (H. pylori), which induces various gastric diseases. Especially, epidemiological studies have shown that H. pylori infection is a major risk factor for gastric cancer. It has been reported that the levels of interleukin (IL)-1β are upregulated in gastric tissues of patients with H. pylori infection. In this study, we investigated the induction mechanism of IL-1β during H. pylori infection. We found that IL-1βmRNA and protein were induced in phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells after H. pylori infection. This IL-1β production was inhibited by a caspase-1 inhibitor and a ROS inhibitor. Furthermore, K(+) efflux and Ca(2+) signaling were also involved in this process. These data suggest that NOD-like receptor (NLR) family, pyrin domain containing 3 (NLRP3) and its complex, known as NLRP3 inflammasome, are involved in IL-1β production during H. pylori infection because it is reported that NLRP3 inflammasome is activated by ROS, K(+) efflux and/or Ca(2+) signaling. These findings may provide therapeutic strategy for the control of gastric cancer in H. pylori-infected patients.
  • Ogawa H, Ohnuma M, Squarre D, Mweene AS, Ezaki T, Fujikura D, Ohnishi N, Thomas Y, Hang'ombe BM, Higashi H
    The Journal of veterinary medical science 77 (8) 993 - 995 0916-7250 2015/08 [Refereed][Not invited]
     
    To follow-up anthrax in Zambia since the outbreak in 2011, we have collected samples from the environment and the carcasses of anthrax-suspected animals, and have tried to isolate Bacillus anthracis. In the process of identification of B. anthracis, we collected two isolates, of which colonies were similar to B. anthracis; however, from the results of identification using the molecular-based methods, two isolates were genetically related to the highly pathogenic B. cereus, of which clinical manifestation is severe and fatal (e.g., pneumonia). In this study, we showed the existence of bacteria suspected to be highly pathogenic B. cereus in Zambia, indicating the possibility of an outbreak caused by highly pathogenic B. cereus.
  • Yabe J, Hamambulu P, Simulundu E, Ogawa H, Kajihara M, Mori-Kajihara A, Changula-Chitanga K, Mwase M, Mweemba-Muwowo M, Chambaro HM, Mataa L, Hang'ombe B, Namangala B, Fandamu P, Sawa H, Takada A, Higashi H, Mweene AS
    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.
  • Ogawa H, Fujikura D, Ohnuma M, Ohnishi N, Hang'ombe BM, Mimuro H, Ezaki T, Mweene AS, Higashi H
    PloS one 10 (3) e0122004  1932-6203 2015 [Refereed][Not invited]
     
    Anthrax is an important zoonotic disease worldwide that is caused by Bacillus anthracis, a spore-forming pathogenic bacterium. A rapid and sensitive method to detect B. anthracis is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify B. anthracis by using previously reported molecular-based methods because of the emergence of B. cereus, which causes severe extra-intestinal infection, as well as the human pathogenic B. thuringiensis, both of which are genetically related to B. anthracis. The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a B. anthracis multiplex PCR that can screen for the presence of B. anthracis virulent plasmids and differentiate B. anthracis and its genetically related strains from other B. cereus group species. Six sets of primers targeting a chromosome of B. anthracis and B. anthracis-like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of B. anthracis DNA per PCR reaction and was sensitive to B. anthracis. The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to B. anthracis, which were B. cereus strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated B. anthracis and its genetically related strains from other B. cereus group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting B. anthracis.
  • Research Activities of Hokudai Center for Zoonosis Control in Zambia
    Higashi H, Kida, H
    2014/10 [Refereed]
  • Yamada K, Sudo H, Iwasaki K, Sasaki N, Higashi H, Kameda Y, Ito M, Takahata M, Abumi K, Minami A, Iwasaki N
    The American journal of pathology 184 (3) 753 - 764 0002-9440 2014/03 [Refereed][Not invited]
     
    Intervertebral disk (IVD) degeneration causes debilitating low back pain in much of the worldwide population. No efficient treatment exists because of an unclear pathogenesis. One characteristic event early in such degeneration is the apoptosis of nucleus pulposus (NP) cells embedded in IVDs. Excessive biomechanical loading may also be a major etiology of IVD degeneration. The present study used in vitro and in vivo models of compressive loading to elucidate the underlying mechanism of IVD degeneration. In addition, we investigated whether the inhibition of apoptosis is a potential clinical therapeutic strategy for the treatment of IVD degeneration induced by biomechanical stress. A TUNEL assay showed that NP cell-agarose three-dimensional composite cultures subjected to uniaxial, unconfined, static, compressive loading exhibited a time-dependent increase in apoptosis. Western blot analysis revealed the up-regulation of several extracellular matrix-degrading enzymes and down-regulation of tissue inhibitor of metalloproteinase 1. These responses to compressive loading were all significantly inhibited by caspase 3 siRNA. In the in vivo model of compressive loading-induced IVD degeneration, a single local injection of caspase 3 siRNA significantly inhibited IVD degeneration by magnetic resonance imaging, histological findings, IHC, and TUNEL assay. The present study suggests that caspase 3 siRNA attenuates overload-induced IVD degeneration by inhibiting NP cell apoptosis and the expression of matrix-degrading enzymes.
  • Ohnishi N, Maruyama F, Ogawa H, Kachi H, Yamada S, Fujikura D, Nakagawa I, Hang'ombe MB, Thomas Y, Mweene AS, Higashi H
    Genome announcements 2 (2) 2014/03 [Refereed][Not invited]
     
    In August 2011, an anthrax outbreak occurred among Hippopotamus amphibius hippopotamuses and humans in Zambia. Here, we report the draft genome sequence of the Bacillus anthracis outbreak strain CZC5, isolated from tissues of H. amphibius hippopotamuses that had died in the outbreak area.
  • Iwasaki K, Sudo H, Yamada K, Higashi H, Ohnishi T, Tsujimoto T, Iwasaki N
    PloS one 9 (10) e109851  2014 [Refereed][Not invited]
     
    BACKGROUND: Analgesic discography (discoblock) can be used to diagnose or treat discogenic low back pain by injecting a small amount of local anesthetics. However, recent in vitro studies have revealed cytotoxic effects of local anesthetics on intervertebral disc (IVD) cells. Here we aimed to investigate the deteriorative effects of lidocaine and bupivacaine on rabbit IVDs using an organotypic culture model and an in vivo long-term follow-up model. METHODS: For the organotypic culture model, rabbit IVDs were harvested and cultured for 3 or 7 days after intradiscal injection of local anesthetics (1% lidocaine or 0.5% bupivacaine). Nucleus pulposus (NP) cell death was measured using confocal microscopy. Histological and TUNEL assays were performed. For in vivo study, each local anesthetic was injected into rabbit lumbar IVDs under a fluoroscope. Six or 12 months after the injection, each IVD was prepared for magnetic resonance imaging (MRI) and histological analysis. RESULTS: In the organotypic culture model, both anesthetic agents induced time-dependent NP cell death; when compared with injected saline solution, significant effects were detected within 7 days. Compared with the saline group, TUNEL-positive NP cells were significantly increased in the bupivacaine group. In the in vivo study, MRI analysis did not show any significant difference. Histological analysis revealed that IVD degeneration occurred to a significantly level in the saline- and local anesthetics-injected groups compared with the untreated control or puncture-only groups. However, there was no significant difference between the saline and anesthetic agents groups. CONCLUSIONS/SIGNIFICANCE: In the in vivo model using healthy IVDs, there was no strong evidence to suggest that discoblock with local anesthetics has the potential of inducing IVD degeneration other than the initial mechanical damage of the pressurized injection. Further studies should be performed to investigate the deteriorative effects of the local injection of analgesic agents on degenerated IVDs.
  • Hideki Sudo, Katsuhisa Yamada, Koji Iwasaki, Hideaki Higashi, Manabu Ito, Akio Minami, Norimasa Iwasaki
    PLOS ONE 8 (3) e58806  1932-6203 2013/03 [Refereed][Not invited]
     
    Background: Intervertebral disc degeneration is a significant cause of degenerative spinal diseases. Nucleus pulposus (NP) cells reportedly fail to survive in large degenerated discs with limited nutrient availability. Therefore, understanding the regulatory mechanism of the molecular response of NP cells to nutrient deprivation may reveal a new strategy to treat disc degeneration. This study aimed to identify genes related to nutrient deprivation in NP cells on a global scale in an experimental nutrient deprivation model. Methodology/Principal Findings: Rat NP cells were subjected to serum starvation. Global gene expression was profiled by microarray analysis. Confirmation of the selected genes was obtained by real-time polymerase chain reaction array analysis. Western blotting was used to confirm the expression of selected genes. Functional interactions between p21(Cip1) and caspase 3 were examined. Finally, flow cytometric analyses of NP cells were performed. Microarray analysis revealed 2922 differentially expressed probe sets with >= 1.5-fold changes in expression. Serum starvation of NP cells significantly affected the expression of several genes involved in DNA damage checkpoints of the cell cycle, including Atm, Brca1, Cdc25, Gadd45, Hus1, Ppm1D, Rad 9, Tp53, and Cyclin D1. Both p27(Kip1) and p53 protein expression was upregulated in serum-starved cells. p21(Cip1) expression remained in NP cells transfected with short interfering RNA targeting caspase 3 (caspase 3 siRNA). Both G1 arrest and apoptosis induced by serum starvation were inhibited in cells transfected with caspase 3 siRNA. Conclusions/Significance: Nutrient deprivation in NP cells results in the activation of a signaling response including DNA damage checkpoint genes regulating the cell cycle. These results provide novel possibilities to improve the success of intervertebral disc regenerative techniques.
  • 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.
  • Takeru Hayashi, Miki Senda, Hiroko Morohashi, Hideaki Higashi, Masafumi Horio, Yui Kashiba, Lisa Nagase, Daisuke Sasaya, Tomohiro Shimizu, Nagarajan Venugopalan, Hiroyuki Kumeta, Nobuo N. Noda, Fuyuhiko Inagaki, Toshiya Senda, Masanori Hatakeyama
    CELL HOST & MICROBE 12 (1) 20 - 33 1931-3128 2012/07 [Refereed][Not invited]
     
    The Helicobacter pylori type IV secretion effector CagA is a major bacterial virulence determinant and critical for gastric carcinogenesis. Upon delivery into gastric epithelial cells, CagA localizes to the inner face of the plasma membrane, where it acts as a pathogenic scaffold/hub that promiscuously recruits host proteins to potentiate oncogenic signaling. We find that CagA comprises a structured N-terminal region and an intrinsically disordered C-terminal region that directs versatile protein interactions. X-ray crystallographic analysis of the N-terminal CagA fragment (residues 1-876) revealed that the region has a structure comprised of three discrete domains. Domain I constitutes a mobile CagA N terminus, while Domain II tethers CagA to the plasma membrane by interacting with membrane phosphatidylserine. Domain III interacts intramolecularly with the intrinsically disordered C-terminal region, and this interaction potentiates the pathogenic scaffold/hub function of CagA. The present work provides a tertiary-structural basis for the pathophysiological/oncogenic action of H. pylori CagA.
  • Naoko Murata-Kamiya, Kenji Kikuchi, Takeru Hayashi, Hideaki Higashi, Masanori Hatakeyama
    CELL HOST & MICROBE 7 (5) 399 - 411 1931-3128 2010/05 [Refereed][Not invited]
     
    When delivered into gastric epithelial cells via type IV secretion, Helicobacter pylori CagA perturbs host cell signaling and thereby promotes gastric carcinogenesis. However, the mechanisms of CagA delivery, localization, and action remain poorly understood. We show that direct contact of H. pylori with epithelial cells induces externalization of the inner leaflet enriched host phospholipid, phosphatidylserine, to the outer leaflet of the host plasma membrane. CagA, which is exposed on the bacterial surface via type IV secretion, interacts with the externalized phosphatidylserine to initiate its entry into cells. CagA delivery also requires energy-dependent host cell processes distinct from known endocytic pathways. Within polarized epithelial cells, CagA is tethered to the inner leaflet of the plasma membrane through interaction with phosphatidylserine and binds the polarity-regulating host kinase PAR1/MARK to induce junctional and polarity defects. Thus, host membrane phosphatidylserine plays a key role in the delivery, localization, and pathophysiological action of CagA.
  • Acacia Lamb, Xiao-Dong Yang, Ying-Hung N. Tsang, Jiang-Dong Li, Hideaki Higashi, Masanori Hatakeyama, Richard M. Peek, Steven R. Blanke, Lin-Feng Chen
    EMBO REPORTS 10 (11) 1242 - 1249 1469-221X 2009/11 [Refereed][Not invited]
     
    Helicobacter pylori-initiated chronic gastritis is characterized by the cag pathogenicity island-dependent upregulation of proinflammatory cytokines, which is largely mediated by the transcription factor nuclear factor (NF)-kappa B. However, the cag pathogenicity island-encoded proteins and cellular signalling molecules that are involved in H. pylori-induced NF-kappa B activation and inflammatory response remain unclear. Here, we show that H. pylori virulence factor CagA and host protein transforming growth factor-beta-activated kinase 1 (TAK1) are essential for H. pylori-induced activation of NF-kappa B. CagA physically associates with TAK1 and enhances its activity and TAK1-induced NF-kappa B activation through the tumour necrosis factor receptor-associated factor 6-mediated, Lys 63-linked ubiquitination of TAK1. These findings show that polyubiquitination of TAK1 regulates the activation of NF-kappa B, which in turn is used by H. pylori CagA for the H. pylori-induced inflammatory response.
  • Huai-Sheng Lu, Yasuhiro Saito, Mayumi Umeda, Naoko Murata-Kamiya, Hong-Mei Zhang, Hideaki Higashi, Masanori Hatakeyama
    CANCER SCIENCE 99 (10) 2004 - 2011 1347-9032 2008/10 [Refereed][Not invited]
     
    Helicobacter pylori (H. pylori) cagA-positive strains are associated with gastritis, peptic ulcerations, and gastric adenocarcinoma. Upon delivery into gastric epithelial cells, the cagA-encoded CagA protein specifically binds and aberrantly activates SHP-2 oncoprotein in a manner that is dependent on CagA tyrosine phosphorylation. CagA-deregulated SHP-2 then elicits aberrant Erk activation while causing an elongated cell shape known as the hummingbird phenotype. In polarized epithelial cells, CagA also binds to PAR1b/MARK2 and inhibits the PAR1b kinase activity, thereby disrupting tight junctions and epithelial cell polarity independent of CagA tyrosine phosphorylation. We show here that the CagA-multimerization (CM) sequence that mediates interaction of CagA with PAR1b is not only essential for the CagA-triggered junctional defects but also plays an important role in induction of the hummingbird phenotype by potentiating CagA-SHP-2 complex formation. We also show that the CM sequence of CagA isolated from East Asian H. pylori (referred to as the E-CM sequence) binds PAR1b more strongly than that of CagA isolated from Western H. pylori (referred to as the W-CM sequence). Within Western CagA species, the ability to bind PAR1b is proportional to the number of W-CM sequences. Furthermore, the level of PAR1b-binding activity of CagA correlates with the magnitude of junctional defects and the degree of hummingbird phenotype induction. Our findings reveal that structural diversity in the CM sequence is an important determinant for the degree of virulence of CagA, a bacterial oncoprotein that is associated with gastric carcinogenesis. (Cancer Sci 2008; 99: 2004-2011)
  • D. Miyamoto, M. Miyamoto, A. Takahashi, Y. Yomogita, H. Higashi, S. Kondo, M. Hatakeyama
    ONCOGENE 27 (25) 3508 - 3515 0950-9232 2008/06 [Refereed][Not invited]
     
    SHP-2 protein tyrosine phosphatase plays an important role in activation of the RAS-dependent signaling. Gain-of-function mutations in the PTPN11 gene, which encodes SHP-2, have been found in the leukemia-prone developmental disorder Noonan syndrome as well as sporadic childhood leukemias, indicating that SHP-2 is a bona. de human oncoprotein. However, the role of SHP-2 mutations in non-hematological malignancies remains obscure. Here, we screened for PTPN11 mutations in primary solid tumors and identified a 1520C > A mutation that causes threonine-507 to lysine (T507K) substitution in the phosphatase domain of SHP-2 in a case of hepatocellular carcinoma. T507K SHP-2 exhibited altered substrate specificity with slightly elevated basal phosphatase activity. Upon expression in NIH3T3 cells, T507K SHP-2 induced transformed foci, which was not observed with wild type, Noonan-specific or leukemia-specific SHP-2. Furthermore, NIH3T3 cells transformed by T507K SHP-2 showed anchorage-independent growth and developed tumors in nude mice. These results indicate that quantitative and/or qualitative alteration in phosphatase activity determines the transforming potential as well as target cell/tissue spectrum of individual SHP-2 mutants as oncoproteins. Although rare in solid tumors, the identified T507K SHP-2 represents a distinct class of SHP-2 mutants with oncogenic RAS-like transforming activity, which could contribute to the development of solid tumors.
  • Yo Kurashima, Naoko Murata-Kamiya, Kenji Kikuchi, Hideaki Higashi, Takeshi Azuma, Satoshi Kondo, Masanori Hatakeyama
    INTERNATIONAL JOURNAL OF CANCER 122 (4) 823 - 831 0020-7136 2008/02 [Refereed][Not invited]
     
    Infection with Helicobacter pylori cagA-positive strains causes gastritis and peptic ulceration and is associated with gastric adenocarcinoma. The cagA gene product CagA is delivered into gastric epithelial cells, where it undergoes tyrosine phosphorylation by Src family kinases at the C-terminal EPIYA-repeat region. Tyrosine-phosphorylated CagA specifically binds and activates SHP-2 tyrosine phosphatase, causing cell morphological transformation known as the hummingbird phenotype. CagA also destabilizes the E-cadherin/beta-catenin complex to elicit aberrant activation of the beta-catenin signal that underlies intestinal metaplasia. Here we show that translocalization of membranous beta-catenin and subsequent activation of the beta-catenin signal by CagA requires the EPIYA-repeat region, which is characterized by structural variation between CagA of H. pylori isolated in Western countries (Western CagA) and that of East Asian H. pylori isolates (East Asian CagA), but is independent of CagA tyrosine phosphorylation. Detailed analysis using a series of Western and East Asian CagA mutants revealed that deregulation of beta-catenin requires residues 10091086 and residues 908-1012 of ABCCC Western CagA and ABD East Asian CagA, respectively, and is mediated by the 16-aminoacid CagA multimerization sequence that is conserved between the 2 geographically distinct H. pylori CagA species. Our results indicate that aberrant activation of the P-catenin signal, which promotes precancerous intestinal metaplasia, is an inherent and fundamental CagA activity that is independent of the structural polymorphism of CagA. (C) 2007 Wiley-Liss, Inc.
  • 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.
  • N. Murata-Kamiya, Y. Kurashima, Y. Teishikata, Y. Yamahashi, Y. Saito, H. Higashi, H. Aburatani, T. Akiyama, R. M. Peek, T. Azuma, M. Hatakeyama
    ONCOGENE 26 (32) 4617 - 4626 0950-9232 2007/07 [Refereed][Not invited]
     
    Infection with Helicobacter pylori cagA-positive strains is associated with gastric adenocarcinoma. Intestinal metaplasia is a precancerous lesion of the stomach characterized by transdifferentiation of the gastric mucosa to an intestinal phenotype. The H. pylori cagA gene product, CagA, is delivered into gastric epithelial cells, where it undergoes tyrosine phosphorylation by Src family kinases. Tyrosine-phosphorylated CagA specifically binds to and activates SHP-2 phosphatase, thereby inducing cell-morphological transformation. We report here that CagA physically interacts with E-cadherin independently of CagA tyrosine phosphorylation. The CagA/E-cadherin interaction impairs the complex formation between E-cadherin and beta-catenin, causing cytoplasmic and nuclear accumulation of beta-catenin. CagA-deregulated beta-catenin then transactivates beta-catenin-dependent genes such as cdx1, which encodes intestinal specific CDX1 transcription factor. In addition to b-catenin signal, CagA also transactivates p21(WAF1/Cip1), again, in a phosphorylation-independent manner. Consequently, CagA induces aberrant expression of an intestinal-differentiation marker, goblet-cell mucin MUC2, in gastric epithelial cells that have been arrested in G1 by p21(WAF1/Cip1). These results indicate that perturbation of the E-cadherin/beta-catenin complex by H. pylori CagA plays an important role in the development of intestinal metaplasia, a premalignant transdifferentiation of gastric epithelial cells from which intestinal-type gastric adenocarcinoma arises.
  • Iraj Saadat, Hideaki Higashi, Chikashi Obuse, Mayumi Umeda, Naoko Murata-Kamiya, Yasuhiro Saito, Huaisheng Lu, Naomi Ohnishi, Takeshi Azuma, Atsushi Suzuki, Shigeo Ohno, Masanori Hatakeyama
    NATURE 447 (7142) 330 - U8 0028-0836 2007/05 [Refereed][Not invited]
     
    Helicobacter pylori cagA-positive strains are associated with gastritis, ulcerations and gastric adenocarcinoma(1). CagA is delivered into gastric epithelial cells(2) and, on tyrosine phosphorylation, specifically binds and activates the SHP2 oncoprotein(3-7), thereby inducing the formation of an elongated cell shape known as the 'hummingbird' phenotype(2,3). In polarized epithelial cells, CagA also disrupts the tight junction and causes loss of apical basolateral polarity(8,9). We show here that H. pylori CagA specifically interacts with PAR1/MARK kinase, which has an essential role in epithelial cell polarity(10,11). Association of CagA inhibits PAR1 kinase activity and prevents atypical protein kinase C ( aPKC)-mediated PAR1 phosphorylation, which dissociates PAR1 from the membrane(12,13), collectively causing junctional and polarity defects. Because of the multimeric nature of PAR1 (ref. 14), PAR1 also promotes CagA multimerization, which stabilizes the CagA - SHP2 interaction(15). Furthermore, induction of the hummingbird phenotype by CagA-activated SHP2 requires simultaneous inhibition of PAR1 kinase activity by CagA. Thus, the CagA - PAR1 interaction not only elicits the junctional and polarity defects but also promotes the morphogenetic activity of CagA. Our findings revealed that PAR1 is a key target of H. pylori CagA in the disorganization of gastric epithelial architecture underlying mucosal damage, inflammation and carcinogenesis.
  • Shumei Ren, Hideaki Higashi, Huaisheng Lu, Takeshi Azuma, Masanori Hatakeyama
    JOURNAL OF BIOLOGICAL CHEMISTRY 281 (43) 32344 - 32352 0021-9258 2006/10 [Refereed][Not invited]
     
    Helicobacter pylori cagA-positive strains are associated with gastric adenocarcinoma. The cagA gene product CagA is delivered into gastric epithelial cells where it localizes to the plasma membrane and undergoes tyrosine phosphorylation at the EPIYA-repeat region, which contains the EPIYA-A segment, EPIYA-B segment, and Western CagA-specific EPIYA-C or East Asian CagA-specific EPIYA-D segment. In host cells, CagA specifically binds to and deregulates SHP-2 phosphatase via the tyrosine-phosphorylated EPIYA-C or EPIYA-D segment, thereby inducing an elongated cell shape known as the hummingbird phenotype. In this study, we found that CagA multimerizes in cells in a manner independent of its tyrosine phosphorylation. Using a series of CagA mutants, we identified a conserved amino acid sequence motif (FPLXRXXXVXDL-SKVG), which mediates CagA multimerization, within the EPIYA-C segment as well as in a sequence that located immediately downstream of the EPIYA-C or EPIYA-D segment. We also found that a phosphorylation-resistant CagA, which multimerizes but cannot bind SHP-2, inhibits the wild-type CagA-SHP-2 complex formation and abolishes induction of the hummingbird phenotype. Thus, SHP-2 binds to a preformed and tyrosine-phosphorylated CagA multimer via its two Src homology 2 domains. These results, in turn, indicate that CagA multimerization is a prerequisite for CagA-SHP-2 interaction and subsequent deregulation of SHP-2. The present work raises the possibility that inhibition of CagA multimerization abolishes pathophysiological activities of CagA that promote gastric carcinogenesis.
  • M Naito, T Yamazaki, R Tsutsumi, H Higashi, K Onoe, S Yamazaki, T Azuma, M Hatakeyama
    GASTROENTEROLOGY 130 (4) 1181 - 1190 0016-5085 2006/04 [Refereed][Not invited]
     
    Background & Aims: Helicobacter pylori CagA-positive strain is associated with gastric adenocarcinoma. CagA is delivered into gastric epithelial cells, where it undergoes tyrosine phosphorylation at the EPIYA sites by Src family kinases (SFKs). Owing to homologous recombination within the 3'-region of the cagA gene, 4 distinct EPIYA sites, each of which is defined by surrounding sequences, are variably assembled in both number and order among CagA proteins from different clinical H pylori isolates. Tyrosine-phosphorylated CagA specifically binds and deregulates SHP-2 via the Western CagA-specific EPIYA-C or East Asian CagA-specific EPIYA-D site, and C-terminal Src kinase (Csk) via the EPIYA-A or EPIYA-B site. Here we investigated the influence of EPIYA-repeat polymorphism on the CagA activity. Methods: A series of EPIYA-repeat variants of CagA were expressed in AGS gastric epithelial cells and the ability of individual CagA to bind SHP-2 or Csk was determined by the sequential immunoprecipitation and immunoblotting method. Results: CagA proteins carrying multiple EPIYA-C or EPIYA-D sites bound and deregulated SHP-2 more strongly than those having a single EPIYA-C or EPIYA-D. Furthermore, the ability of CagA to bind Csk was correlated with the number of EPIYA-A and EPIYA-B sites. Because Csk inhibits SFK, CagA with greater Csk-binding activity more strongly inhibited Src-dependent CagA phosphorylation and more effectively attenuated induction of cell elongation caused by CagA-SHP-2 interaction. Conclusions: EFIYA-repeat polymorphism of CagA greatly influences the magnitude and duration of phosphorylation-dependent CagA activity, which may determine the potential of individual CagA as a bacterial virulence factor that directs gastric carcinogenesis.
  • R Tsutsumi, A Takahashi, T Azuma, H Higashi, M Hatakeyama
    MOLECULAR AND CELLULAR BIOLOGY 26 (1) 261 - 276 0270-7306 2006/01 [Refereed][Not invited]
     
    Infection with cagA-positive Helicobacter pylori (H. pylori) is associated with atrophic gastritis, peptic ulcer, and gastric adenocarcinoma. The cagA gene product CagA is translocated from H. pylori into gastric epithelial cells and undergoes tyrosine phosphorylation by Src family kinases (SFKs). Tyrosine-phosphorylated CagA binds and activates SHP-2 phosphatase and the C-terminal Src kinase (Csk) while inducing an elongated cell shape termed the "hummingbird phenotype." Here we show that CagA reduces the level of focal adhesion kinase (FAK) tyrosine phosphorylation in gastric epithelial cells. The decrease in phosphorylated FAK is due to SHP-2-mediated dephosphorylation of FAK at the activating phosphorylation sites, not due to Csk-dependent inhibition of SFKs, which phosphorylate FAK. Coexpression of constitutively active FAK with CagA inhibits induction of the hummingbird phenotype, whereas expression of dominant-negative FAK elicits an elongated cell shape characteristic of the hummingbird phenotype. These results indicate that inhibition of FAK by SHP-2 plays a crucial role in the morphogenetic activity of CagA. Impaired cell adhesion and increased motility by CagA may be involved in the development of gastric lesions associated with cagA-positive H. pylori infection.
  • N Nakano, K Urasawa, Y Takagi, T Saito, S Kaneta, S Ishikawa, H Higashi, H Tsutsui, M Hatakeyama, A Kitabatake
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 338 (3) 1661 - 1667 0006-291X 2005/12 [Refereed][Not invited]
     
    Immature vascular smooth muscle cells (VSMCs) proliferate responding to extrinsic mitogens and accumulate in neointima after arterial injuries. Cell proliferation is positively regulated by cyclin/cyclin-dependent kinase (CDK) complex and negatively controlled by CDK inhibitors; CKIs Such as p27(kip1) and p57(kip2). In this Study, embryonic rat thoracic aorta VSMCs; A10 were G0/G1 arrested by serum starvation, re-stimulated with serum, and harvested every four hours. Both CKIs co-expressed in quiescent VSMCs and rapidly diminished by stimulation. Protein level of p27(kip1) was regulated by both transcription and post-transcription, but that of p57(kip2) was mainly by post-transcription. Supplemental overexpression of p57(kip2) inhibited the activations of G1 cyclin/CDKs and subsequent hyperphosphorylations of all three retinoblastoma pocket proteins as well as G1/S transition of cell cycle. Our findings suggest that the downregulations of not only p27(kip1), but also p57(kip2) responding to mitogenic stimulation, play key roles in the cell cycle progression of VSMCs. (c) 2005 Elsevier Inc. All rights reserved.
  • M Hatakeyama, H Higashi
    CANCER SCIENCE 96 (12) 835 - 843 1347-9032 2005/12 [Refereed][Not invited]
     
    Infection with cagA-positive Helicobacter pylori is associated with the development of gastric adenocarcinoma. The cagA gene product CagA is injected directly from the bacterium into the bacterium-attached gastric epithelial cells via the type-IV secretion system. Upon membrane localization and subsequent tyrosine phosphorylation by Src family kinases, CagA functions as a scaffolding adaptor and interacts with a number of host proteins that regulate cell growth, cell motility and cell polarity in both CagA phosphorylation-dependent and phosphorylation-independent manners. Of special interest is the interaction of CagA with the SHP-2 tyrosine phosphatase, gain-of-function mutations that of which have recently been found in a variety of human malignancies. The CagA-SHP-2 interaction is entirely dependent on CagA tyrosine phosphorylation and, through the complex formation, SHP-2 is catalytically activated and induces morphological transformation with elevated cell motility. Intriguingly, structural diversity of the tyrosine phosphorylation sites of CagA accounts for the differential activity of individual CagA to bind and activate SHP-2. Deregulation of SHP-2 and other intracellular signaling molecules by H. pylori CagA may predispose cells to accumulate multiple genetic and epigenetic changes involved in gastric carcinogenesis. Furthermore, the differential potential of individual CagA to disturb cellular functions indicates that H. pylori strains carrying biologically more active CagA are more virulent than those with less active CagA and are more closely associated with gastric carcinoma.
  • S Yamazaki, A Yamakawa, T Okuda, M Ohtani, H Suto, Y Ito, Y Yamazaki, Y Keida, H Higashi, M Hatakeyama, T Azuma
    JOURNAL OF CLINICAL MICROBIOLOGY 43 (8) 3906 - 3916 0095-1137 2005/08 [Refereed][Not invited]
     
    Colonization of the stomach mucosa by Helicobacter pylori is a major cause of acute and chronic gastric pathologies in humans. Several H. pylori virulence genes that may play a role in its pathogenicity have been identified. The most important determinants are vacA and cagA in the cag pathogenicity island (cagPAI) genes. In the present study, to consider the association of molecular genetics between vacA and the cagPAI regarding clinical outcome, we selected H. pylori strains with various genotypes of vacA in Japan and sequenced full-length vacA, cagA, and cagE genes. Sequencing of vacA and cagA genes revealed variable size, whereas the cagE gene was well conserved among strains. Each of the phylogenetic trees based on the deduced amino acid sequences of VacA, CagA, and CagE indicated that all three proteins were divided into two major groups, a Western group and an East Asian group, and the distributions of isolates exhibited similar patterns among the three proteins. The strains with s2 and s1a/m1a vacA genotypes and the Western-type 3' region cagA genotype were classified into the Western group, and the strains with the s1c/m1b vacA genotype and the East Asian-type 3' cagA genotype were included in the East Asian group. In addition, the prevalence of infection with the Western group strain was significantly higher in patients with peptic ulcer (90.0%, 9/10) than in patients with chronic gastritis (22.7%, 5/22) (x(2) = 12.64, P = 0.00057). These data suggest that the molecular genetics of vacA and cagPAI are associated and that the Western group with vacA and cagPAI genes is associated with peptic ulcer disease.
  • K Yokoyama, H Higashi, S Ishikawa, Y Fujii, S Kondo, H Kato, T Azuma, A Wada, T Hirayama, H Aburatani, M Hatakeyama
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102 (27) 9661 - 9666 0027-8424 2005/07 [Refereed][Not invited]
     
    Chronic infection with cagA-positive Helicobacter pylori is associated with the development of atrophic gastritis, peptic ulcers, and gastric adenocarcinoma. The cagA gene product CagA is injected into gastric epithelial cells, where it undergoes tyrosine phosphorylation by Src family kinases. Translocated CagA disturbs cellular functions by physically interacting with and deregulating intracellular signaling transducers through both tyrosine phosphorylation-dependent and -independent mechanisms. To gain further insights into the pathophysiological activities of CagA in gastric epithelial cells, we executed a genome-wide screening of CagA-responsive genes by using DNA microarray and identified nuclear factor of activated T cells (NFAT) transcription factors whose binding sites were overrepresented in the promoter regions of CagA-activated genes. Results of reporter assays confirmed that CagA was capable of activating NFAT in a manner independent of CagA phosphorylation. Expression of CagA in gastric epithelial cells provoked translocation of NFATc3, a member of the NFAT family, from the cytoplasm to the nucleus and activated an NFAT-regulated gene, p21(WAF1/CiP1). CagA-mediated NFAT activation was abolished by inhibiting calcineurin or phospholipase C gamma activity. Furthermore, treatment of cells with H. pylori VacA (vacuolating toxin), which inhibits NFAT activity in T lymphocytes, counteracted the ability of CagA to activate NFAT in gastric epithelial cells. These findings indicate that the two major H. pylori virulence factors inversely control NFAT activity. Considering the pleiotropic roles of NFAT in cell growth and differentiation, deregulation of NFAT, either positively or negatively, depending on the relative exposure of cells to CagA and VacA, may contribute to the various disease outcomes caused by H. pylori infection.
  • S Yamazaki, S Kato, N Matsukura, M Ohtani, Y Ito, H Suto, Y Yamazaki, A Yamakawa, S Tokudome, H Higashi, M Hatakeyama, T Azuma
    FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY 44 (3) 261 - 268 0928-8244 2005/06 [Refereed][Not invited]
     
    The CagA protein is one of the virulence factors of Helicobacter pylori, and two major subtypes of CagA have been observed, the Western and East Asian type. CagA is injected from the bacteria into gastric epithelial cells, undergoes tyrosine phosphorylation, and binds to Src homology 2 domain-containing protein-tyrosine phosphatase SHP-2. The East Asian type CagA binds to SHP-2 more strongly than the Western type CagA. Here, we tried to distinguish the CagA type by highly sensitive real-time PCR with the objective of establishing a system to detect H. pylori and CagA subtypes from gastric biopsies. We designed primers and probe sets for Western or East Asian-cagA at Western-specific or East Asian-specific sequence regions, respectively, and H. pylori 16S rRNA. We could detect the H. pylori 16S rRNA gene, Western and East Asian-cagA gene from DNA of gastric biopsies. The sensitivity and specificity for H. pylori infection was 100% in this system. In Thai patients, 87.8% (36/41) were cagA-positive; 26.8% (11/41) were Western-cagA positive and 53.7% (22/41) were East Asian-cagA positive, while 7.3% (3/41) reacted with both types of cagA. These results suggest that this real-time PCR system provides a highly sensitive assessment of CagA type as a new diagnostic tool for the pathogenicity of H. pylori infection. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
  • H Higashi, K Yokoyama, Y Fujii, S Ren, H Yuasa, Saadat, I, NM Kamiya, T Azuma, M Hatakeyama
    JOURNAL OF BIOLOGICAL CHEMISTRY 280 (24) 23130 - 23137 0021-9258 2005/06 [Refereed][Not invited]
     
    Helicobacter pylori contributes to the development of peptic ulcers and atrophic gastritis. Furthermore, H. pylori strains carrying the cagA gene are more virulent than cagA-negative strains and are associated with the development of gastric adenocarcinoma. The cagA gene product, CagA, is translocated into gastric epithelial cells and localizes to the inner surface of the plasma membrane, in which it undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motif. Tyrosine-phosphorylated CagA specifically binds to and activates Src homology 2-containing protein-tyrosine phosphatase-2 (SHP-2) at the membrane, thereby inducing an elongated cell shape termed the hummingbird phenotype. Accordingly, membrane tethering of CagA is an essential prerequisite for the pathogenic activity of CagA. We show here that membrane association of CagA requires the EPIYA-containing region but is independent of EPIYA tyrosine phosphorylation. We further show that specific deletion of the EPIYA motif abolishes the ability of CagA to associate with the membrane. Conversely, reintroduction of an EPIYA sequence into a CagA mutant that lacks the EPIYA-containing region restores membrane association of CagA. Thus, the presence of a single EPIYA motif is necessary for the membrane localization of CagA. Our results indicate that the EPIYA motif has a dual function in membrane association and tyrosine phosphorylation, both of which are critically involved in the activity of CagA to deregulate intracellular signaling, and suggest that the EPIYA motif is a crucial therapeutic target of cagA-positive H. pylori infection.
  • lac repressorを利用した誘導的RNA干渉による細胞内タンパク質の可逆的発現抑制
    堤 良平, 樋口 恵, 東 秀明, 畠山 昌則
    生化学 (公社)日本生化学会 76 (9) 1238 - 1238 0037-1017 2004/09
  • H Ozawa, S Ashizawa, M Naito, M Yanagihara, N Ohnishi, T Maeda, Y Matsuda, Y Jo, H Higashi, A Kakita, M Hatakeyama
    ONCOGENE 23 (39) 6590 - 6602 0950-9232 2004/08 [Refereed][Not invited]
     
    The eukaryotic cell cycle is regulated by sequential activation and inactivation of cyclin-cyclin-dependent kinase (Cdk) complexes. In this work, we screened human cDNAs that can rescue yeast Saccharomyces cerevisiae from lethality caused by ectopic expression of human cyclin E and isolated a cDNA encoding ESXR1, a paired-like homeodomain-containing protein with a unique C-terminal proline-rich repeat region. In adult tissues, ESXR1 is primarily expressed in the testis. We demonstrate that ESXR1 prevents degradation of ubiquitinated cyclins in human cells. Accordingly, elevation of ESXR1 level results in accumulation of cyclin A and cyclin B1 and thereby provokes M-phase arrest. In human cells, the 65-kDa full-length ESXR1 protein is capable of proteolytically processing into N-terminal 45-kDa and C-terminal 20-kDa fragments. The C-terminal fragment, containing a proline-rich repeat region, is localized to the cytoplasm and displays the ability to inhibit cyclin degradation. In contrast, the N-terminal fragment, containing a paired-like homeodomain, is localized exclusively in the nucleus, suggesting that it plays a role in transcription. Our results indicate that proteolytic processing of ESXR1 plays a role in concerted regulation of the cell cycle and transcription in human cells.
  • T Azuma, M Ohtani, Y Yamazaki, H Higashi, M Hatakeyama
    GASTROENTEROLOGY 126 (7) 1926 - 1927 0016-5085 2004/06 [Refereed][Not invited]
  • T Azuma, A Yamakawa, S Yamazaki, M Ohtani, Y Ito, A Muramatsu, H Suto, Y Yamazaki, Y Keida, H Higashi, M Hatakeyama
    JOURNAL OF CLINICAL MICROBIOLOGY 42 (6) 2508 - 2517 0095-1137 2004/06 [Refereed][Not invited]
     
    The severity of Helicobacter pylori-related disease is correlated with the presence of a cag pathogenicity island (PAI). Genetic diversity within the cag PAI may have a modifying effect on the pathogenic potential of the infecting strain. We analyzed the complete cag PAI sequences of 11 representative Japanese strains according to their vacA genotypes and clinical effects and examined the relationship between the diversity of the cag PAI and clinical features. The cag PAI genes were divided into two major groups, a Western and a Japanese group, by phylogenetic analysis based on the entire cag PAI sequences. The predominant Japanese strains formed a Japanese cluster which was different from the cluster formed by Western strains. The diversity of the cag PAI was associated with the vacA and cagA genotypes. All strains with the s1c vacA genotype were in the Japanese cluster. In addition, all strains with the East Asian-type cagA genotype were also in the Japanese cluster. Patients infected with the Japanese-cluster strain had high-grade gastric mucosal atrophy. These results suggest that a distinct diversity of the cag PAI of H. pylori is present among Japanese strains and that this diversity may be involved in the development of atrophic gastritis and may increase the risk for gastric cancer.
  • M Higuchi, R Tsutsumi, H Higashi, M Hatakeyama
    CANCER SCIENCE 95 (5) 442 - 447 1347-9032 2004/05 [Refereed][Not invited]
     
    RNA interference (RNAi) is a newly described biological phenomenon mediated by small interfering RNA (siRNA) that targets mRNA for degradation by cellular enzymes and has become a powerful method for studying gene functions in mammalian systems. The development of systems for inducing siRNA expression should enable examination of acute loss-of-function phenotypes in a cell of interest without the need to consider lethality or epigenetic adaptation of cells. We describe in this report an inducible siRNA expression system made by combined utilization of the RNA polymerase III-dependent promoter H1 and the bacterial lac repressor. Using this system, we established AGS gastric epithelial cells in which expression of SHP-2, a cellular tyrosine phosphatase known to specifically bind the Helicobacter pylori virulence factor CagA, is conditionally and reversibly silenced by the lactose analog isopropyl-l-thio-beta-D-galactopyranoside (IPTG). Upon expression in AGS cells, CagA provoked a morphological transformation, termed the hummingbird phenotype, which is associated with CagA virulence. This morphogenetic activity of CagA was totally abolished when SHP-2 expression was silenced by inducible siRNA expression in AGS cells. Our results indicate that SHP-2 is a critical downstream effector of H. pylori CagA. The conditional gene silencing system described here should become a powerful tool for investigating the roles of cancer-related genes through a reversed genetic approach.
  • H Higashi, A Nakaya, R Tsutsumi, K Yokoyama, Y Fujii, S Ishikawa, M Higuchi, A Takahashi, Y Kurashima, Y Teishikata, S Tanaka, T Azuma, M Hatakeyama
    JOURNAL OF BIOLOGICAL CHEMISTRY 279 (17) 17205 - 17216 0021-9258 2004/04 [Refereed][Not invited]
     
    The CagA protein of Helicobacter pylori, which is injected from the bacteria into bacteria-attached gastric epithelial cells, is associated with gastric carcinoma. CagA is tyrosine-phosphorylated by Src family kinases, binds the SH2 domain-containing SHP-2 phosphatase in a tyrosine phosphorylation-dependent manner, and deregulates its enzymatic activity. We established AGS human gastric epithelial cells that inducibly express wildtype or a phosphorylation-resistant CagA, in which tyrosine residues constituting the EPIYA motifs were substituted with alanines. Upon induction, wild-type CagA, but not the mutant CagA, elicited strong elongation of cell shape, termed the "hummingbird" phenotype. Time-lapse video microscopic analysis revealed that the CagA-expressing cells exhibited a marked increase in cell motility with successive rounds of elongation-contraction processes. Inhibition of CagA phosphorylation by an Src kinase inhibitor, PP2, or knockdown of SHP-2 expression by small interference RNA ( siRNA) abolished the CagA-mediated hummingbird phenotype. The morphogenetic activity of CagA also required Erk MAPK but was independent of Ras or Grb2. In AGS cells, CagA prolonged duration of Erk activation in response to serum stimulation. Conversely, inhibition of SHP-2 expression by siRNA abolished the sustained Erk activation. Thus, SHP-2 acts as a positive regulator of Erk activity in AGS cells. These results indicate that SHP-2 is involved in the Ras-independent modification of Erk signals that is necessary for the morphogenetic activity of CagA. Our work therefore suggests a key role of SHP-2 in the pathological activity of H. pylori virulence factor CagA.
  • T Azuma, S Yamazaki, A Yamakawa, M Ohtani, A Muramatsu, H Suto, Y Ito, M Dojo, Y Yamazaki, M Kuriyama, Y Keida, H Higashi, M Hatakeyama
    JOURNAL OF INFECTIOUS DISEASES 189 (5) 820 - 827 0022-1899 2004/03 [Refereed][Not invited]
     
    We investigated the relationship between the diversity of Helicobacter pylori CagA protein and clinical outcome. The cagA gene was sequenced in 115 clinical isolates. The binding affinity of CagA to Src homology 2 domain containing tyrosine phosphatase (SHP-2) was examined by in vitro infection. Two major CagA subtypes were observed - the East Asian and the Western type. The grades of inflammation, activity of gastritis, and atrophy were significantly higher in patients with gastritis infected with the East Asian CagA-positive strain than in patients with gastritis infected with cagA-negative or Western CagA-positive strains. All strains isolated from patients with gastric cancer were East Asian CagA positive. East Asian CagA exhibited stronger SHP-2-binding activity than did Western CagA. These findings suggest that infection with East Asian CagA - positive H. pylori is associated with atrophic gastritis and gastric cancer and that persistent active inflammation induced by the East Asian CagA- positive strain may play a role in the pathogenesis of disease.
  • W Zhou, S Yamazaki, A Yamakawa, M Ohtani, Y Ito, Y Keida, H Higashi, M Hatakeyama, JM Si, T Azuma
    FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY 40 (1) 81 - 87 0928-8244 2004/01 [Refereed][Not invited]
     
    It has been reported that Helicobacter pylori infection with the type I strain, which expresses the VacA and CagA antigens, is associated with duodenal ulcer. We examined the diversity of vacA and cagA genes in 143 isolates obtained from patients with duodenal ulcer or chronic gastritis in East Asia (two different areas of Japan, Fukui and Okinawa, and also in Hangzhou, China) by polymerase chain reaction (PCR) and sequence analysis. Diversities of cagA and vacA genes were detected in East Asia. The prevalence of cagA-positive H. pylori was significantly different between Fukui and Okinawa (P = 0.0032). The prevalence of Western type CagA was significantly higher in Okinawa than in Fukui (P < 0.0001). However, there was no significant association between the genotype of cagA and clinical outcome. In Japan, the predominant vacA genotype was slc/mlb. In contrast, in Hangzhou, the predominant vacA genotype was s1c/m2, and they were all East Asian CagA-positive. These findings suggest that a distinct distribution of the vacA and cagA genotypes is present in East Asia, regardless of clinical outcome. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
  • S Umehara, H Higashi, N Ohnishi, M Asaka, M Hatakeyama
    ONCOGENE 22 (51) 8337 - 8342 0950-9232 2003/11 [Refereed][Not invited]
     
    Helicobacter pylori (H. pylori) is a causative agent of gastrointestinal diseases such as atrophic gastritis and gastroduodenal ulcer. Infection of cagA-positive H. pylori is also associated with gastric carcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The cagA gene product CagA is directly injected into the bacteria-attached host cells via the bacterial type IV secretion system. The translocated CagA deregulates intracellular signaling pathways and thereby initiates pathogenesis. In this work, we examined the biological effects of CagA on B cells, from which MALT lymphoma arises. Ectopic expression of CagA in interleukin 3-dependent B cells inhibited cell proliferation by suppressing the JAK-STAT signaling. CagA was also capable of preventing hydroxyurea-induced B-cell apoptosis through inhibiting p53 accumulation. In contrast to the effects of CagA in gastric epithelial cells, the observed CagA activities in B cells were independent of its tyrosine phosphorylation. Our results indicate that CagA possesses both phosphorylation-dependent and -independent activities in mammalian cells and that biological impacts of CagA depend on cell-type context. As a result of B-cell growth inhibition, CagA may diminish anti-H. pylori immune responses. Furthermore, CagA may play a role in the development of MALT lymphoma by impairing p53-dependent apoptosis.
  • T. Azuma, S. Yamazaki, A. Yamakawa, Y. Ito, M. Ohtani, M. Dojo, Y. Yamazaki, H. Higashi, M. Hatakeyama
    Alimentary Pharmacology and Therapeutics, Supplement 18 (1) 39 - 44 0953-0673 2003/07 [Refereed][Not invited]
     
    Background: The CagA protein of Helicobacter pylori is directly injected from the bacteria into cells via the bacterial type IV secretion system and undergoes tyrosine phosphorylation in the gastric epithelial cells. Translocated CagA forms a physical complex with the SRC homology 2 domain (SH2)-containing tyrosine phosphatase SHP-2, which plays an important role in mitogenic signal transduction in the host cells. Aim: We examined the effect of eradication therapy on the signal transduction pathway of gastric epithelial cells induced by the CagA protein of H. pylori. Methods: Gastric biopsy samples were obtained from 20 H. pylori-positive atrophic gastritis patients before, and 3 months after, H. pylori infection eradication therapy, and subjected to immunoblot analysis to detect tyrosine phosphorylated CagA protein and CagA co-immunoprecipitated endogenous SHP-2. Results: Tyrosine phosphorylated CagA protein and CagA co-immunoprecipitated endogenous SHP-2 were detected in the gastric mucosa from H. pylori-positive atrophic gastritis patients. All H. pylori strains from these patients were cagA-positive type I strains. After curing H. pylori infection, the tyrosine phosphorylated CagA protein and CagA co-immunoprecipitated endogenous SHP-2 disappeared from the gastric mucosa. Conclusion: The cure of infection reduces the stimulated signal transduction of gastric epithelial cells by the translocated CagA protein of H. pylori, and may confer a beneficial effect on the reduction of cancer risk.
  • T Azuma, S Yamazaki, A Yamakawa, Y Ito, M Ohtani, M Dojo, Y Yamazaki, H Higashi, M Hatakeyama
    ALIMENTARY PHARMACOLOGY & THERAPEUTICS 18 39 - 44 0269-2813 2003/07 [Refereed][Not invited]
     
    Background: The CagA protein of Helicobacter pylori is directly injected from the bacteria into cells via the bacterial type IV secretion system and undergoes tyrosine phosphorylation in the gastric epithelial cells. Translocated CagA forms a physical complex with the SRC homology 2 domain (SH2)-containing tyrosine phosphatase SHP-2, which plays an important role in mitogenic signal transduction in the host cells. Aim: We examined the effect of eradication therapy on the signal transduction pathway of gastric epithelial cells induced by the CagA protein of H, pylori. Methods: Gastric biopsy samples were obtained from 20 H. pylori-positive atrophic gastritis patients before, and 3 months after. H. pylori infection eradication therapy, and subjected to immunoblot analysis to detect tyrosine phosphorylated CagA protein and CagA co-immunoprecipitated endogenous SHP-2. Results: Tyrosine phosphorylated CagA protein and CagA co-immunoprecipitated endogenous SHP-2 were detected in the gastric mucosa from H. pylori-positive atrophic gastritis patients. All H. pylori strains from these patients were cagA-positive type I strains. After curing H. pylori infection, the tyrosine phosphorylated CagA protein and CagA co-immunoprecipitated endogenous SHP-2 disappeared from the gastric mucosa. Conclusion: The cure of infection reduces the stimulated signal transduction of gastric epithelial cells by the translocated CagA protein of H. pylori, and may confer a beneficial effect on the reduction of cancer risk.
  • T Takebayashi, H Higashi, H Sudo, H Ozawa, E Suzuki, O Shirado, H Katoh, M Hatakeyama
    JOURNAL OF BIOLOGICAL CHEMISTRY 278 (17) 14897 - 14905 0021-9258 2003/04 [Refereed][Not invited]
     
    The retinoblastoma protein (pRB) and its homologues, p107 and p130, prevent cell cycle progression from G(0)/G(1) to S phase by forming complexes with E2F transcription factors. Upon phosphorylation by G, cyclin-cyclin-dependent kinase (Cdk) complexes such as cyclin D1-Cdk4/6 and cyclin E-Cdk2, they lose the ability to bind E2F, and cells are thereby allowed to progress into S phase. Functional loss of one or more of the pRB family members, as a result of genetic mutation or deregulated phosphorylation, is considered to be an essential prerequisite for cellular transformation. In this study, we found that pRB family proteins have the ability to stimulate cyclin D1 transcription by activation of the NF-kappaB transcription factor. The cyclin D1-inducing activity of pRB is abolished by adenovirus E1A oncoprotein but not by the deletion of the A-box, the B-box, or the C-terminal region of the pocket, indicating that multiple pocket sequences are independently involved in cyclin D1 activation. Intriguingly, tumor-derived pRB pocket mutants retain the cyclin D1-inducing activity. Our results reveal a novel role of pRB family proteins as potential activators of NF-kappaB and inducers of G(1) cyclin. Certain pRB pocket mutants may give rise to a cellular situation in which deregulated E2F and cyclin D1 cooperatively promote abnormal cell proliferation.
  • R Tsutsumi, H Higashi, M Higuchi, M Okada, M Hatakeyama
    JOURNAL OF BIOLOGICAL CHEMISTRY 278 (6) 3664 - 3670 0021-9258 2003/02 [Refereed][Not invited]
     
    diseases ranging from gastritis to cancer. The CagA protein is the product of the cagA gene carried among virulent H. pylori strains and is associated with severe disease outcomes, most notably gastric carcinoma. CagA is injected from the attached H. pylori into gastric epithelial cells and undergoes tyrosine phosphorylation. The phosphorylated CagA binds and activates SHP-2 phosphatase and thereby induces a growth factor-like morphological change termed the "humming-bird phenotype." In this work, we demonstrate that CagA is also capable of interacting with C-terminal Src kinase (Csk). As is the case with SHP-2, Csk selectively binds tyrosine-phosphorylated CagA via its SH2 domain. Upon complex formation, CagA stimulates Csk, which in turn inactivates the Src family of protein-tyrosine kinases. Because Src family kinases are responsible for CagA phosphorylation, an essential prerequisite of CagA.SHP-2 complex formation and subsequent induction of the hummingbird phenotype, our results indicate that CagA-Csk interaction down-regulates CagA.SHP-2 signaling by both competitively inhibiting CagA.SHP-2 complex formation and reducing levels of CagA phosphorylation. We further demonstrate that CagA.SHP-2 signaling eventually induces apoptosis in AGS cells. Our results thus indicate that CagA-Csk interaction prevents excess cell damage caused by deregulated activation of SHP-2. Attenuation of CagA activity by Csk may enable cagA-positive H. pylori to persistently infect the human stomach for decades while avoiding excess CagA toxicity to the host.
  • S Yamazaki, A Yamakawa, Y Ito, M Ohtani, H Higashi, M Hatakeyama, T Azuma
    JOURNAL OF INFECTIOUS DISEASES 187 (2) 334 - 337 0022-1899 2003/01 [Refereed][Not invited]
     
    Recent experiments have indicated that CagA of Helicobacter pylori is injected into epithelial cells via the type IV secretion system and undergoes tyrosine phosphorylation in cells and that translocated CagA binds the SRC homology 2 domain-containing tyrosine phosphatase (SHP-2). We investigated these phenomena in in vivo human gastric mucosa. Tyrosine-phosphorylated CagA and CagA-coimmunoprecipitated SHP-2 were detected in gastric mucosa from H. pylori-positive patients with atrophic gastritis and in noncancerous tissues from H. pylori-positive patients with early gastric cancer. In contrast, CagA was not detected in gastric mucosa with either intestinal metaplasia or cancer. Our results provide the first evidence that CagA is translocated into the gastric epithelial cells, receives tyrosine phosphorylation, and binds SHP-2 in in vivo human gastric mucosa. Deregulation of SHP-2 by CagA may play a role in the acquisition of a cellular-transformed phenotype at a relatively early stage of multistep gastric carcinogenesis.
  • H Higashi, R Tsutsumi, A Fujita, S Yamazaki, M Asaka, T Azuma, M Hatakeyama
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 99 (22) 14428 - 14433 0027-8424 2002/10 [Refereed][Not invited]
     
    Helicobacter pylori is a causative agent of gastritis and peptic ulcer. cagA(+) H. pylori strains are more virulent than cagA(-) strains and are associated with gastric carcinoma. The cagA gene product, CagA, is injected by the bacterium into gastric epithelial cells and subsequently undergoes tyrosine phosphorylation. The phosphorylated CagA specifically binds SHP-2 phosphatase, activates the phosphatase activity, and thereby induces morphological transformation of cells. CagA proteins of most Western H. pylori isolates have a 34-amino acid sequence that variably repeats among different strains. Here, we show that the repeat sequence contains a tyrosine phosphorylation site. CagA proteins having more repeats were found to undergo greater tyrosine phosphorylation, to exhibit increased SHP-2 binding, and to induce greater morphological changes. In contrast, predominant CagA proteins specified by H. pylori strains isolated in East Asia, where gastric carcinoma is prevalent, had a distinct tyrosine phosphorylation sequence at the region corresponding to the repeat sequence of Western CagA. This East Asian-specific sequence conferred stronger SHP-2 binding and morphologically transforming activities to Western CagA. Finally, a critical amino acid residue that determines SHP-2 binding activity among different CagA proteins was identified. Our results indicate that the potential of individual CagA to perturb host-cell functions is determined by the degree of SHP-2 binding activity, which depends in turn on the number and sequences of tyrosine phosphorylation sites. The presence of distinctly structured CagA proteins in Western and East Asian H. pylori isolates may underlie the strikingly different incidences of gastric carcinoma in these two geographic areas.
  • M Yamada, T Kondo, S Ashizawa, T Takebayashi, H Higashi, M Hatakeyama
    CYTOKINE 17 (2) 91 - 97 1043-4666 2002/01 [Refereed][Not invited]
     
    Interleukin 3 (IL-3)-dependent proliferation of haematopoietic cells is specifically inhibited by p130, a member of the pRB-family proteins. p130 interacts with the cell-cycle regulatory E2F transcription factors, notably E2F-4 and E2F-5, and affects promoters containing E2F-binding sites through two distinct mechanisms. First, upon complex formation with E2F, it blocks transcriptional activation by E2F. Second, the formed p130-E2F complex binds to E2F sites and actively represses transcription by inhibiting the activity of surrounding enhancer elements on the promoter. To pursue the relative contributions of each mechanism in the p130-mediated inhibition of IL-3-dependent cell proliferation, we employed a dominant-negative DP-1, which suppresses both E2F-dependent transactivation and the formation of active transcriptional repressors. Ectopic expression of the dominant negative DP-1 in the IL-3-dependent BaF3 lymphoid cells gave rise to an inhibition of cell proliferation, which was concomitantly associated with a decrease in levels of cyclin E, an indispensable molecule for G1 to S-phase cell-cycle progression. Our results indicate that blocking E2F-dependent transactivation, but not the formation of p130-E2F transcriptional repressor complexes, is responsible for the inhibition of IL-3-dependent cell growth by p130. (C) 2002 Elsevier Science Ltd.
  • H Higashi, R Tsutsumi, S Muto, T Sugiyama, T Azuma, M Asaka, M Hatakeyama
    SCIENCE 295 (5555) 683 - 686 0036-8075 2002/01 [Refereed][Not invited]
     
    Helicobacter pylori CagA protein is associated with severe gastritis and gastric carcinoma. CagA is injected from the attached Helicobacter pylori into host cells and undergoes tyrosine phosphorylation. Wild-type but not phosphorylation-resistant CagA induced a growth factor-like response in gastric epithelial cells. Furthermore, CagA formed a physical complex with the SRC homology 2 domain (SH2)-containing tyrosine phosphatase SHP-2 in a phosphorylation-dependent manner and stimulated the phosphatase activity. Disruption of the CagA-SHP-2 complex abolished the CagA-dependent cellular response. Conversely, the CagA effect on cells was reproduced by constitutively active SHP-2. Thus, upon translocation, CagA perturbs cellular functions by deregulating SHP-2.
  • T Kondo, H Higashi, H Nishizawa, S Ishikawa, S Ashizawa, M Yamada, Z Makita, T Koike, M Hatakeyama
    JOURNAL OF BIOLOGICAL CHEMISTRY 276 (20) 17559 - 17567 0021-9258 2001/05 [Refereed][Not invited]
     
    PRE family pocket proteins consisting of pRB, p107, and p130 are thought to act as a set of growth regulators that inhibit the cell cycle transition from G(1) to S phases by virtue of their interaction with E2F transcription factors. When cells are committed to progressing through the cell cycle at the late G(1) restriction point, they are hyperphosphorylated by G(1) cyclin-cyclin-dependent kinase and are functionally inactivated. Consistent with such a G(1) regulatory role, pRB and p130 are abundantly expressed in quiescent cells. In contrast, p107 is present at low levels in the hypophosphorylated form in quiescent cells. As cells progress toward late G(1) to S phases, the levels of p107 increase, and the majority become hyperphosphorylated, suggesting a possible role of p107 in post-G(1) cell cycle regulation. In this study, we have demonstrated that a nonphosphorylatable and thus constitutively active p107 has the potential to inhibit S phase progression. The levels of the phosphorylation-resistant p107 required for the S phase inhibition are significantly less than those of endogenous p107. We further show herein that the exposure of cells to the DNA-damaging agent, cisplatin, provokes S phase arrest, which is concomitantly associated with the accumulation of hypophosphorylated p107. Furthermore, the S phase inhibitory response to cisplatin is augmented by the ectopic expression of wild type p107, although it is diminished by the adenovirus E1A oncoprotein, which counteracts the pocket protein functions. Because p107 is a major pRB family protein expressed in S phase cells, our results indicate that p107 participates in an inhibition of cell cycle progression in response to DNA damage in S phase cells.
  • S Ashizawa, H Nishizawa, M Yamada, H Higashi, T Kondo, H Ozawa, A Kakita, M Hatakeyama
    JOURNAL OF BIOLOGICAL CHEMISTRY 276 (14) 11362 - 11370 0021-9258 2001/04 [Refereed][Not invited]
     
    The activity of the retinoblastoma protein pRB is regulated by phosphorylation that is mediated by G(1) cyclin-associated cyclin-dependent kinases (CDKs). Since the pRB-related pocket proteins p107 and p130 share general structures and biological functions with pRB, their activity is also considered to be regulated by phosphorylation. In this work, we generated phosphorylation-resistant p107 and p130 molecules by replacing potential cyclin CDK phosphorylation sites with non-phosphorylatable alanine residues. These phosphorylation-resistant mutants retained the ability to bind E2F and cyclin. Upon introduction into p16(INK4a)-deficient U2-OS osteosarcoma cells, in which cyclin D-CDK4/6 is dysregulated, the phosphorylation-resistant mutants, but not wild-type p107 or p130, were capable of inhibiting cell proliferation. Furthermore, when ectopically expressed in pRB-deficient SAOS-8 osteosarcoma cells, the wild-type as well as the phosphorylation-resistant pRB family proteins were capable of inducing large flat cells. The flat cell-inducing activity of the wild-type proteins, but not that of the phosphorylation-resistant mutants; was:abolished by coexpressing cyclin E. Our results indicate that the elevated cyclin D- or cyclin E-associated kinase leads to systemic inactivation of the pRB family proteins and suggest that dysregulation of the pRB kinase provokes an aberrant cell cycle in a broader range of cell types than those induced by genetic inactivation of the RB gene.
  • R Mizuguchi, S Noto, M Yamada, S Ashizawa, H Higashi, M Hatakeyama
    JAPANESE JOURNAL OF CANCER RESEARCH 91 (5) 527 - 533 0910-5050 2000/05 [Refereed][Not invited]
     
    Cytokines exert their activities in cell growth and differentiation by binding specific cell membrane receptors, Janus kinases (JAKs) are cytoplasmic protein tyrosine kinases that physically interact with intracellular domains of the cytokine receptors and they play crucial roles in transducing signals triggered by the cytokine-receptor interaction. We have previously shown that conditional activation of JAK through membrane-proximal dimerization confers cytokine-independence on interleukin-3 (IL-3)-dependent Ba/F3 lymphoid cells and that the cytokine-independent proliferation is completely inhibited by dominant negative Ras. In this work, we demonstrate that ectopic expression of a dominant negative form of Stat5, a major signal transducer and activator of transcription (STAT) expressed in Ba/F3 cells, also inhibits JAK-triggered mitogenesis. In contrast, overexpression of constitutively active Ras or conditional activation of Stat5 by chemical dimerization fails to confer cytokine-independence. However, concomitant activation of ectopic Ras and Stat5 molecules in Ba/F3 cells suffices for cell proliferation in the absence of IL-3, Our results indicate that Ras and STAT are essential and sufficient components of JAK-triggered mitogenesis. Our findings further indicate that the cytokine signal bifurcates into Ras and STAT pathways following JAK activation.
  • T Aso, K Yamazaki, K Amimoto, A Kuroiwa, H Higashi, Y Matsuda, S Kitajima, M Hatakeyama
    JOURNAL OF BIOLOGICAL CHEMISTRY 275 (9) 6546 - 6552 0021-9258 2000/03 [Refereed][Not invited]
     
    The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II by suppressing the transient pausing of the polymerase at many sites along the DNA template. Elongin is composed of a transcriptionally active A subunit and two small regulatory B and C subunits, the latter of which bind stably to each other to form a binary complex that interacts with Elongin A and strongly induces its transcriptional activity. To further understand the roles of Elongin in transcriptional regulation, Re attempted to identify Elongin-related proteins. sere, we report on the cloning, expression, and characterization of human Elongin A2, a novel transcription elongation factor that exhibited 47% identity and 61% similarity to Elongin A. Biochemical studies have shown that Elongin A2 stimulates the rate of transcription elongation by RNA polymerase II and is capable of forming a stable complex with Elongin BC, However, in contrast to Elongin A, its transcriptional activity is not activated by Elongin BC. Northern blot analysis revealed that Elongin A2 mRNA was specifically expressed in the testis, suggesting that Elongin A2 may regulate the transcription of testis-specific genes.
  • A Ohtoshi, T Maeda, H Higashi, S Ashizawa, M Hatakeyama
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 268 (2) 530 - 534 0006-291X 2000/02 [Refereed][Not invited]
     
    The initiation of anaphase and exit from mitosis depend on the activation of the anaphase-promoting complex/cyclosome (APC/C), a multicomponent, ubiquitin-protein ligase, The WD-repeat protein called p55(CDC)(Cdc20) directly binds to and activates APC/C. By using yeast two-hybrid screening, we found that cyclin A, a critical cell cycle regulator in the S and G2/M phases, specifically interacts with p55(CDC). Ectopically expressed p55(CDC) and cyclin A form a stable protein complex in mammalian cells. The p55(CDC)-cyclin A interaction occurs through the region containing the WD repeats of p55(CDC) and the region between the destruction box and the cyclin box of cyclin A. In addition to the physical interaction, p55(CDC) is phosphorylated by cyclin A-associated kinase. These findings suggest that the function of p55(CDC) is mediated or regulated by its complex formation with cyclin A. (C) 2000 Academic Press.
  • A Ohtoshi, T Maeda, H Higashi, S Ashizawa, M Yamada, M Hatakeyama
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 267 (3) 947 - 952 0006-291X 2000/01 [Refereed][Not invited]
     
    Cyclin A is indispensable for S phase cell cycle progression and is suggested to be a crucial target of cell adhesion signals. In this study, we demonstrate that beta 3-endonexin, a molecule known to associate with the integrin beta 3 cytoplasmic domain, specifically binds cyclin A. Deletion of the amino-terminal 52-amino-acid residues including the cyclin-binding RxL motif abolishes the ability of beta 3-endonexin to interact with cyclin A. In an in vitro kinase assay, beta 3-endonexin inhibits pRB kinase activity associated with cyclin A-Cdk2 while leaving its histone H1 kinase activity unaffected. Coexpression of beta 3-endonexin in yeast cells overcomes growth suppression caused by an activation of cyclin A-associated kinase. Our results indicate that beta 3-endonexin is a novel cyclin A-binding molecule that regulates cyclin A-associated pRB kinase activity. (C) 2000 Academic Press.
  • A Mori, H Higashi, Y Hoshikawa, M Imamura, M Asaka, M Hatakeyama
    ONCOGENE 18 (46) 6209 - 6221 0950-9232 1999/11 [Refereed][Not invited]
     
    The retinoblastoma protein (pRB) and the related pocket proteins, p107 and p130, play crucial roles in mammalian cell cycle control. Recent studies indicate that these pocket proteins are also involved in cellular differentiation processes. We demonstrate in this work that the pRB-related p130 selectively accumulates during the in vitro differentiation of the myeloid progenitor cell, 32Dc13, into granulocyte in response to granulocyte-colony stimulating factor (G-CSF). This G-CSF-dependent granulocytic differentiation is blocked by the adenovirus E1A oncoprotein, which binds to and inactivates the pRB family of pocket proteins including p130. Furthermore, enforced overexpression of p130 but not PRE inhibits the myeloid cell proliferation that is concomitantly associated with granulocytic differentiation morphologically characterized by nuclear segmentation. However, simple G1-cell cycle al rest induced by cytokine deprivation or ectopic overexpression of the p27 cyclin-dependent kinase inhibitor, or inhibition of E2F activities by dominant negative DP-1 is not sufficient to trigger granulocytic differentiation. The differentiation-promoting activity of p130 in myeloid cells requires both the pocket domain and the spacer domain. Our results indicate that the pRB-related p130 plays a critical role in myeloid cell differentiation and suggest that coupling of cell cycle exit with the cellular differentiation program may be specifically achieved by p130.
  • S Hatakeyama, M Kitagawa, K Nakayama, M Shirane, M Matsumoto, K Hattori, H Higashi, H Nakano, K Okumura, K Onoe, RA Good, K Nakayama
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 96 (7) 3859 - 3863 0027-8424 1999/03 [Refereed][Not invited]
     
    Activation of the transcription factor nuclear factor kappa B (NF-kappa B) is controlled by proteolysis of its inhibitory subunit (I kappa B) via the ubiquitin-proteasome pathway, Signal-induced phosphorylation of I kappa B alpha by a large multisubunit complex containing I kappa B kinases is a prerequisite for ubiquitination. Here, we show that FWD1 (a mouse homologue of Slimb/beta TrCP), a member of the F-box/WD40-repeat proteins, is associated specifically with I kappa B alpha only when I kappa B alpha is phosphorylated, The introduction of FWD1 into cells significantly promotes ubiquitination and degradation of I kappa B alpha in concert with I kappa B kinases, resulting in nuclear translocation of NF-kappa B. In addition, FWD1 strikingly evoked the ubiquitination of I kappa B alpha in the ira vitro system. In contrast, a dominant-negative form of FWD1 inhibits the ubiquitination, leading to stabilization of I kappa B alpha. These results suggest that the substrate-specific degradation of I kappa B alpha is mediated by a Skp1/Cull 1/F-box protein (SCF) FWD1 ubiquitin-ligase complex and that FWD1 serves as an intracellular receptor for phosphorylated I kappa B alpha. Skp1/Cullin/F-box protein FWD1 might play a critical role in transcriptional regulation of NF-kappa B through control of I kappa B protein stability.
  • T Terano, T Tanaka, Y Tamura, M Kitagawa, H Higashi, Y Saito, A Hirai
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 254 (2) 502 - 506 0006-291X 1999/01 [Refereed][Not invited]
     
    Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the form of triacylglycerol (TG) were dose dependently incorporated into phospholipid fraction of vascular smooth muscle cells (VSMC) and suppressed the proliferation of VSMC. Flow cytometric analysis demonstrated both EPA and DHA inhibited G(1)/S progression. EPA and DHA inhibited the phosphorylation of Cdk2 protein and Cdk2 kinase activity without altering the amount of cyclin E and p27(kip1) proteins and cyclin dependent kinase activating kinase activity by growth stimulation. This mechanisms remained to be clarified but this is the first report of a novel mechanisms of inhibition of DNA synthesis by EPA and DHA. (C) 1999 Academic Press.
  • S. Hatakeyama, M. Kitagawa, K. Nakayama, M. Shirane, M. Matsumoto, K. Hattori, H. Higashi, H. Nakano, K. Okumura, K. Onoe, R. A. Good, K. I. Nakayama
    Proceedings of the National Academy of Sciences of the United States of America 96 6571  0027-8424 1999/01/01 [Not refereed][Not invited]
  • T Tanaka, Tatsuno, I, Y Noguchi, D Uchida, T Oeda, S Narumiya, T Yasuda, H Higashi, M Kitagawa, K Nakayama, Y Saito, A Hirai
    JOURNAL OF BIOLOGICAL CHEMISTRY 273 (41) 26772 - 26778 0021-9258 1998/10 [Refereed][Not invited]
     
    The role of the mevalonate cascade in the control of cell cycle progression in astrocytes has been investigated. Serum stimulation of rat astrocytes in primary culture induces the expression of cyclin E followed by the activation of cyclin-dependent kinase 2 (Cdk2) during G(1)/S transition. The expression of p27(hip1) cyclin D1, and the activities of Cdk4 and Cdk-activating kinase (CAB), composed of Cdk7 and cyclin H, mere not affected. Serum did, however, stimulate the expression of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA at mid-G(1) phase. Moreover, an inhibitor of HMG-CoA reductase, pravastatin, reduced cyclin E expression and Cdk2 activation and caused G(1) arrest in the astro cytes. In contrast, mevalonate and its metabolite, geranylgeranylpyrophosphate (GGPP) but not farnesylpyrophosphate (FPP), reversed the inhibitory effects of pravastatin on cyclin E expression and Cdk2 activation and allowed G(1)/S transition. Rho small GTPase(s) were geranylgeranylated and translocated to membranes in the presence of GGPP during G(1)/S transition. The effect of GGPP on cyclin E expression was abolished by botulinum C3 exoenzyme, which specifically inactivates Rho. These data indicate that geranylgeranylated Rho small GTPase(s) are essential for the induction of cyclin E expression, Cdk2 activation, and G(1)/S transition in rat astrocytes.
  • SuzukiTakahashi, I, H Higashi, E Yoshida, S Nishimura, M Kitagawa
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 234 (2) 386 - 392 0006-291X 1997/05 [Refereed][Not invited]
     
    p16(INK4a), a protein that inhibits cyclin-dependent kinase 4 (Cdk4) and Cdk6, is deficient in many human cancers and in established lines of tumor cells. It has been reported that transfection with cDNA for p16(INK4a) inhibits the growth of cell lines that express retinoblastoma protein (pRB). However, it is unclear whether the introduction of cDNA for p16(INK4a) affects the growth of cells that express p16(INK4a) protein. Moreover, the effects of other cell-cycle regulators on the inhibition of cell growth by p16(INK4a) remain unknown. In this study, cDNA for p16(INK4a) was used to transfect human cell lines that had various status of expression of RE pathway-related proteins, such as members of the RE family proteins and Cdk-inhibitory proteins. We found that status of p107, p130, p15(INK4b), p18(INK4c) p21(Cip1), p27(Kip1) cyclin D1, and Cdk4 were not correlated with the growth-inhibitory activity of exogenous p16(INK4a). BY contrast, transfection with cDNA for p16(INK4a) had a significant effect on the growth of cells depended on the status not only of pRB but also of p16(INK4a). Although exogenous p16(INK4a) inhibited the growth of cells that expressed pRB but did not express p16(INK4a) (pRB(+)/p16(-) cells), it had little affect on either pRB(-)/p16(+) cells or pRB(-)/p16(+) cells. Moreover, transfection with cDNA for p16(INK4a) also inhibited the activity of the E2 promoter of the dehydrofolate reductase gene in the same manner that depended on the absence of p16(INK4a), as well as on the presence of pRB. These results suggest that deregulation of the RE pathway by p16(INK4a) deficiency plays a very important role in the proliferation of cells that lack p16(INK4a) protein. (C) 1997 Academic Press.
  • H Higashi, SuzukiTakahashi, I, E Yoshida, S Nishimura, M Kitagawa
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 231 (3) 743 - 750 0006-291X 1997/02 [Refereed][Not invited]
     
    p16(INK4a) is a inhibitory protein of Cyclin-dependent kinase 4 (Cdk4). p16 negatively regulates the cell cycle progression from G1 to S phase. Functional pie is absent from many human cancers, as well as from many established lines of tumor cells. However, it is not clear whether expression of p16 in p16-deficient tumor cells can suppress their anchorage-independent growth. Therefore, we introduced a cDNA for p16(INK4a) into the human glioblastoma cell line T98G, which lacks a gene for p16(INK4a). We isolated several clones that stably expressed various amounts of pie protein. The doubling time of the various clones was generally prolonged. Clones with high-level expression of p16 protein had characteristics of restricted growth, such as contact inhibition, while the parental T98G cells had no such characteristics. Furthermore, the efficiency of colony formation in soft agar was dramatically decreased in the case of cells that expressed exogenous pie. Our observations suggest that the expression of pie protein restricts the unbounded growth and the anchorage-independent growth of tumor cells. (C) 1997 Academic Press.
  • Y Taya, HK Jung, M Ikeda, K Tamai, H Higashi, M Kitagawa
    GENOMIC INSTABILITY AND IMMORTALITY IN CANCER 8 229 - 231 1997 [Refereed][Not invited]
  • M Kitagawa, H Higashi, HK Jung, SuzukiTakahashi, I, M Ikeda, K Tamai, J Kato, K Segawa, E Yoshida, S Nishimura, Y Taya
    EMBO JOURNAL 15 (24) 7060 - 7069 0261-4189 1996/12 [Refereed][Not invited]
     
    Cyclin D-Cdk4/6 and cyclin A/E-Cdk2 are suggested to be involved in phosphorylation of the retinoblastoma protein (pRB) during the G(1)/S transition of the cell cycle. However, it is unclear why several Cdks are needed and how they are different from one another. We found that the consensus amino acid sequence for phosphorylation by cyclin D1-Cdk4 is different from S/T-P-X-K/R, which is the consensus sequence for phosphorylation by cyclin A/E-Cdk2 using various synthetic peptides as substrates. Cyclin D1-Cdk4 efficiently phosphorylated the G1 peptide, RPPTLS(780)PIP- that contained a part of the sequence of pRB, while cyclins E-Cdk2 and A-Cdk2 did not. To determine the phosphorylation state of pRB in vitro and in vivo, we raised the specific antibody against phospho-Ser780 in pRB. We confirmed that cyclin D1-Cdk4, but not cyclin E-Cdk2, phosphorylated Ser780 in recombinant pRB. The Ser78O in pRB was phosphorylated in the GI phase in a cell cycle-dependent manner, Furthermore, we found that pRB phosphorylated at Ser780 cannot bind to E2F-1 in vivo. Our data show that cyclin D1-Cdk4 and cyclin, A/E Cdk2 phosphorylate different sites of pRB in vivo.
  • H Higashi, SuzukiTakahashi, I, S Saitoh, K Segawa, Y Taya, A Okuyama, S Nishimura, M Kitagawa
    EUROPEAN JOURNAL OF BIOCHEMISTRY 237 (2) 460 - 467 0014-2956 1996/04 [Refereed][Not invited]
     
    Cyclin-dependent kinases (Cdks) form complexes with cyclins, and as a consequence they generally express kinase activities. One of these Cdks, Cdk2, is known to bind with cyclins A and E, and plays an important role in the progression of the cell cycle via phosphorylation of target proteins such as the product of the retinoblastoma tumor-suppressor gene (pRB). It has been suggested that Cdk2 bound with cyclin D1 and Cdk2-cyclin-D1 complex show neither H1 histone nor pRB kinase activity. However, it is not clear whether Cdk2-cyclin-D1 has unknown targets and why Cdk2 is not activated by binding with cyclin D1. We investigated these questions using Cdk, cyclin and Cdk-cyclin complexes produced in a baculovirus expression system. Cdk2 formed a complex with cyclin D1 in this system. After extensive purification, Cdk2 was still bound to cyclin D. The Cdk2-cyclin-D1 complex did not phosphorylate any tested substrates, such as H1 histone, pRB, SV40 large T antigen, p53, E2F-1 or a preparation of nuclear proteins from HeLa cells; in contrast, Cdk2-cyclin-E and Cdk2-cyclin-A phosphorylated these proteins. Moreover, the Cdk2-cyclin-D1 complex was not activated by incubation with Cdk4 or cyclin E. Thus, Cdk2 and cyclin D1 formed a stable complex that was not activated. In order to determine why Cdk2-cyclin-D1 lacks kinase activity, we investigated the phosphorylation of Cdk2. Under-shifted Cdk2, the active form of Cdk2, was not detected in the Cdk2-cyclin-D1 complex in the baculovirus system. In human WI-38 cells, cyclin D1 began to form a complex with Cdk2 as well as with Cdk4 from the mid-G1 phase of the cell cycle. The Cdk2 bound to cyclin D1 in human cells was also the inactive form that was slowly migrated. Moreover, we found that Cdk2 bound to cyclin D1 was not phosphorylated by Cdk7-cyclin-H, while Cdk2 bound to cyclin E, as well as free Cdk2, was phosphorylated by Cdk7-cyclin-H. Additionally, Cdk2 phosphorylated by Cdk7-cyclin-H did not bind to cyclin D1. These results strongly suggest that Cdk2 forms a stable complex with cyclin D1 but is not activated because the Cdk2 molecule in the complex is not phosphorylated by Cdk7-cyclin-H and the phosphorylated Cdk2, an active form, does not bind to cyclin D1.
  • H HIGASHI, SUZUKITAKAHASHI, I, Y TAYA, K SEGAWA, S NISHIMURA, M KITAGAWA
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 216 (2) 520 - 525 0006-291X 1995/11 [Refereed][Not invited]
     
    Cyclin-dependent kinase 2 (Cdk(2)), when bound to either cyclin A or cyclin E, recognizes the Ser/Thr-Pro-X-basic amino acid (motif A) as a phosphorylation site. In this study, we designed several peptides based on motif A and examined the substrate specificity of Cdk2-cyclin A and Cdk2-cyclin E using these peptides, Peptides containing a proline residue in the sequence Pro-X-Thr-Pro-X-basic amino acid (motif B) had higher affinity for both Cdk2 complexes than peptides; containing motif A. Furthermore, differences in substrate affinity between the two Cdk2 complexes were caused by a proline residue adjacent to or three positions before the threonine residue. Similarly, the presence of different basic amino acids in motif B also had different effects on affinity for each complex. We demonstrate the possibility that the substrate specificity of Cdk2 bound to cyclin might be regulated by the species of cyclin. (C) 1995 Academic Press, Inc.
  • SUZUKITAKAHASHI, I, M KITAGAWA, M SAIJO, H HIGASHI, H OGINO, H MATSUMOTO, Y TAYA, S NISHIMURA, A OKUYAMA
    ONCOGENE 10 (9) 1691 - 1698 0950-9232 1995/05 [Refereed][Not invited]
     
    It has been postulated that the product (pRB) of the retinoblastoma gene dissociates from the E2F-pRB complex upon phosphorylation by cyclin-dependent kinase(s) (cdk). However, there is no direct evidence for the regulation of formation of the E2F-pRB complex via phosphorylation by purified cdk, Therefore,we investigated the regulation of formation of this complex by phosphorylation using pRB and purified cyclin A-cdk2, cyclin E-cdk2 or cyclin D1-cdk4. Purified pRB was incubated with nuclear extracts prepared from pRB-defective cells and then subjected to gel mobility shift assays, We confirmed that unphosphorylated pRB associated with various types of E2F but pRB has been phosphorylated by cyclin A-cdk2 did not. We found that E2F-pRB complexes were disrupted as a consequence of phosphorylation by cyclin A-cdk2, and the levels of the free forms of E2Fs increased. We also found that not only the E2F-pRB complexes but also the E2F-p107 complexes were disrupted upon phosphorylation by cyclin A-cdk2. Furthermore, E2F-pRB complexes were disrupted through phosphorylation by cyclin D1-cdk4 and cyclin E-cdk2, as web as by cyclin A-cdk2. These results clearly demonstrate that the phosphorylation of pRB and p107 by cdks regulates the formation of complexes between E2F and pRB or p107.
  • S KOTANI, T ENDO, M KITAGAWA, H HIGASHI, T ONAYA
    ONCOGENE 10 (4) 663 - 669 0950-9232 1995/02 [Refereed][Not invited]
     
    Cyclin-dependent kinase 2 (Cdk2) controls the transition from the G1 to the S phase in the mammalian cell cycle. We found by immunoblotting that anti-Cdk2 antibodies recognize three Cdk2 proteins (of 33, 34 and 39 kDa) in FRTL-5 and FRTL-Tc cells (malignantly transformed FRTL cells). Although 33 kDa protein is a phosphorylated form of 34 kDa protein previously reported, the nature of 39 kDa protein is unknown. In order to determine the nature of this protein, we screened a FRTL-5 cDNA library. Two cDNA clones of the rat homologue (rat Cdk2-alpha and -beta) of human Cdk2 were isolated. The open reading frame of rat Cdk2-alpha cDNA encoded a protein with 428 amino acids and has a high degree of conservation with human Cdk2. The calculated molecular weight of Cdk2-alpha protein is 33892 Da. The rat Cdk2-beta cDNA was identical to Cdk2-alpha cDNA except that it had extra 144 bp; this coincided with insertion of 48 amino acids into Cdk2-alpha protein between Met 196 and Val 197. The calculated molecular weight of Cdk2-beta protein is 39087 Da. Northern blot analysis indicated that the sizes of rat Cdk2-alpha and -beta mRNAs are approximately 2.5 kb and 3.0 kb, respectively. Partial proteolytic mapping showed that Cdk2-beta gene product is 39 kDa Cdk2 in the immunoblotting. We also found that Cdk2-beta protein binds to cyclin A and suc1 proteins. During G1-S phase in FRTL-Tc cells, Cdk2-alpha protein level is constant, but is gradually phosphorylated. In contrast, the level of Cdk2-beta protein increases through the S phase and decreases at the early G2 phase. These results suggest that a variant form of Cdk2 protein might be required for entry into the S phase of the cell cycle in FRTL-Tc cells.
  • M KITAGAWA, H HIGASHI, SUZUKITAKAHASHI, I, K SEGAWA, SK HANKS, Y TAYA, S NISHIMURA, A OKUYAMA
    ONCOGENE 10 (2) 229 - 236 0950-9232 1995/01 [Refereed][Not invited]
     
    Transcription factor E2F-1 has a putative consensus sequence for phosphorylation by cyclin dependent kinase (Ser-Pro-X-Lys/Arg). Therefore, we studied the phosphorylation of E2F-1 in vivo and in vitro and its biological functions. E2F-1 was prepared by immunoprecipitation with anti-E2F-1 antibody from IMR32 lysates and was effectively phosphorylated by human cyclin A-cdk2 which was expressed in insect cells using baculovirus system. GST-EZF-1 was phosphorylated by cyclin A-cdk2 more efficiently than by cyclin E-cdk2. Cyclin D1-cdk4 phosphorylated PRE but scarcely phosphorylated GST-E2F-1 or H1 histone. The 60 kd protein precipitated with anti-E2F-1 antibody was phosphorylated in vivo. Phospho-peptide mapping indicated that its cleavage profile was identical with that of E2F-1 phosphorylated by cyclin A-cdk2 in vitro. This 60 kd protein, which is likely to be E2F-1, was not phosphorylated during the GO and early G1 phase, Phosphorylation of E2F-1 began from the S phase while phosphorylation of pRB started nearly at G1/S. The in vivo phosphorylation of E2F-1 was inhibited by butyrolactone I, a cyclin-dependent kinase inhibitor (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). The binding of E2F-1 to E2 promoter was found to be reduced by phosphorylation of E2F-1 by cyclin A-cdk2, suggesting that phosphorylation of E2F-1 may induce shut off of gene expression at the transcriptional level. These results suggest that E2F-1 is phosphorylated by cyclin A-cdkZ in the S phase in vivo as well as in vitro and that its phosphorylation by cyclin A-cdkZ may modulate its activity.
  • M KITAGAWA, H HIGASHI, IS TAKAHASHI, T OKABE, H OGINO, Y TAYA, S NISHIMURA, A OKUYAMA
    ONCOGENE 9 (9) 2549 - 2557 0950-9232 1994/09 [Refereed][Not invited]
     
    Butyrolactone I is a selective inhibitor of the cyclin-dependent kinase (cdk) family. It inhibits both cdk2 and cdc2 kinase, but scarcely affects C-kinase, A-kinase, casein kinases, MAP kinase or EGF receptor-tyrosine kinase (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). We studied the effects of butyrolactone I on the cell cycle as well as on phosphorylation of retinoblastoma protein (pRB). Butyrolactone I inhibited phosphorylation of pRB catalyzed by cyclin A-cdk2 produced by baculovirus in vitro. Furthermore, it inhibited phosphorylation of pRB and cell cycle progression from G1 to S phase in WI38 cell cultures. WI38 cells arrested at the G0 phase by serum starvation progressed in the cell cycle after serum stimulation. pRB was phosphorylated after 10 h serum stimulation. Incorporation of [H-3]thymidine into the cells began to increase after 16 h serum stimulation. These processes were inhibited by butyrolactone I. Flow cytometric analysis showed that exposure to butyrolactone I inhibited progression of the cell cycle from G1 to S phase. These data suggested that initiation of DNA synthesis was inhibited by butyrolactone I and that the cell cycle was arrested in the G1 phase. Butyrolactone I also inhibited H1 histone phosphorylation in human WI38 cells and their G2/M progression, tsFT210 cells, a temperature-sensitive cdc2 mutant cell line, were synchronized at G2/M at a nonpermissive temperature, butyrolactone I inhibited the cell cycle progression of these cells at G2/M at the permissive temperature. Thus butyrolactone I, a cyclin-dependent kinase family inhibitor, which prevented the phosphorylations of the cell cycle-regulating proteins pRB and H1 histone, inhibited the cell cycle at G1/S and G2/M, respectively. These results suggest that the phosphorylations of pRB and H1 histone may play crucial roles in G1/S and G2/M progression, respectively, although it is possible that phosphorylations of other proteins by cdks are involved in G1/S and G2/M progression.
  • M KITAGAWA, T OKABE, H OGINO, H MATSUMOTO, SUZUKITAKAHASHI, I, T KOKUBO, H HIGASHI, S SAITOH, Y TAYA, H YASUDA, Y OHBA, S NISHIMURA, N TANAKA, A OKUYAMA
    ONCOGENE 8 (9) 2425 - 2432 0950-9232 1993/09 [Refereed][Not invited]
     
    We screened cdc2 kinase inhibitors from cultured mediums of micro organisms using purified mouse cyclin B-cdc2 kinase and a specific substrate peptide for cdc2 kinase. A selective inhibitor of cdc2 kinase was isolated from the cultured medium of Aspergillus species F-25799, and identified as butyrolactone I. Butyrolactone I inhibited cdc2 and cdk2 kinases but it had little effect on mitogen-activated protein kinase, protein kinase C, cyclic-AMP dependent kinase, casein kinase II, casein kinase I or epidermal growth factor-receptor tyrosine kinase. Its inhibitory effect was found to be due to competition with ATP. Butyrolactone I selectively inhibited the H1 histone phosphorylation in nuclear extracts. It also inhibited the phosphorylation of the product of retinoblastoma susceptibility gene in nuclear extracts and intact cells. Thus butyrolactone I should be very useful for elucidating the function of cdc2 and cdk2 kinases in cell cycle regulation.
  • T YOSHIMOTO, H HIGASHI, A KANATANI, XS LIN, H NAGAI, H OYAMA, K KURAZONO, D TSURU
    JOURNAL OF BACTERIOLOGY 173 (7) 2173 - 2179 0021-9193 1991/04 [Refereed][Not invited]
     
    The 7-alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) gene from Escherichia coli HB101 was cloned and expressed in E. coli DH1. The hybrid plasmid pSD1, with a 2.8-kbp insert of chromosomal DNA at the BamHI site of pBR322, was subcloned into pUC19 to construct plasmid pSD3. The entire nucleotide sequence of an inserted PstI-BamHI fragment of plasmid pSD3 was determined by the dideoxy chain-termination method. Within this sequence, the mature enzyme protein-encoding sequence was found to start at a GTG initiation codon and to comprise 765 bp, as judged by comparison with the protein sequence. The deduced amino acid sequence of the enzyme indicated that the molecular weight is 26,778. The transformant of E. coli DH1 harboring pSD3 with a 1.8-kbp fragment showed about 200-fold-higher enzyme activity than the host. The enzyme was purified by a single chromatography step on DEAE-Toyopearl and obtained as crystals, with an activity yield of 39%. The purified enzyme was homogeneous, as judged by sodium dodecyl sulfate gel electrophoresis. The enzyme was most active at pH 8.5 and stable between pH 8 and 9. The enzyme was NAD+ dependent and had a pI of 4.3. The molecular mass was estimated to be 120 kDa by the gel filtration method and 28 kDa by electrophoresis, indicating that the enzyme exists in a tetrameric form.
  • T YOSHIMOTO, H OYAMA, T TAKESHITA, H HIGASHI, SL XU, D TSURU
    JOURNAL OF FERMENTATION AND BIOENGINEERING 70 (6) 370 - 375 0922-338X 1990 [Refereed][Not invited]
     
    The neutral protease gene of Bacillus subtilis var. amylosacchariticus was isolated and the nucleotide sequence encoding the signal and pro-peptides followed by the mature protein region was established. Putative -35 and -10 sequences, TTGAGT and TAATAT, were observed as the consensus sequence for the promoter recognized by the sigma-55RNA polymerase of B. subtilis, and the ribosome binding site, whose sequence was AAAGGGGG, was complementary to the binding sequence of B. subtilis 16S rRNA except for one base. The nucleotide sequence of the gene was highly homologous to that of the neutral protease gene from B. subtilis IA72, particularly in the mature protein coding region: only two nucleotide discrepancies out of 900 nucleotides were found, which resulted in no amino acid replacement. However, sequences in pre-pro and putative terminator regions were significantly different between the neutral protease genes of these two strains of B. subtilis. In B. subtilis var. amylosacchariticus, a stem-loop structure with a calculated free energy of -46.0 kcal/mol, which was much lower than that of B. subtilis IA72, was postulated at the 3' terminal region.

MISC

  • 小川寛人, 小川寛人, 小川寛人, 梶原将大, 直亨則, 丸山隼輝, 上野恵介, 石井秋宏, 石井秋宏, 藤倉大輔, HANG’OMBE Bernard, AARON Mweene, 山田雅夫, 東秀明, 東秀明, 澤洋文, 澤洋文, 高田礼人, 高田礼人  日本獣医学会学術集会講演要旨集  159th-  376  2016/08/30  [Not refereed][Not invited]
  • M Hatakeyama, K Yokoyama, H Higashi, S Ishikawa, T Azuma, T Hirayama, H Aburatani  HELICOBACTER  10-  (5)  471  -471  2005/10  [Not refereed][Not invited]
  • 【H.pylori感染の基礎と臨床】 H.pyloriの胃粘膜障害機序 宿主側から
    堤 良平, 東 秀明, 畠山 昌則  Surgery Frontier  11-  (2)  122  -125  2004/06  [Not refereed][Not invited]
  • 東 秀明, 畠山 昌則  細胞工学  23-  (5)  551  -555  2004/05
  • M Higuchi, R Tsutsumi, H Higashi, M Hatakeyama  CANCER SCIENCE  95-  (5)  442  -447  2004/05  [Not refereed][Not invited]
     
    RNA interference (RNAi) is a newly described biological phenomenon mediated by small interfering RNA (siRNA) that targets mRNA for degradation by cellular enzymes and has become a powerful method for studying gene functions in mammalian systems. The development of systems for inducing siRNA expression should enable examination of acute loss-of-function phenotypes in a cell of interest without the need to consider lethality or epigenetic adaptation of cells. We describe in this report an inducible siRNA expression system made by combined utilization of the RNA polymerase III-dependent promoter H1 and the bacterial lac repressor. Using this system, we established AGS gastric epithelial cells in which expression of SHP-2, a cellular tyrosine phosphatase known to specifically bind the Helicobacter pylori virulence factor CagA, is conditionally and reversibly silenced by the lactose analog isopropyl-l-thio-beta-D-galactopyranoside (IPTG). Upon expression in AGS cells, CagA provoked a morphological transformation, termed the hummingbird phenotype, which is associated with CagA virulence. This morphogenetic activity of CagA was totally abolished when SHP-2 expression was silenced by inducible siRNA expression in AGS cells. Our results indicate that SHP-2 is a critical downstream effector of H. pylori CagA. The conditional gene silencing system described here should become a powerful tool for investigating the roles of cancer-related genes through a reversed genetic approach.
  • S Umehara, H Higashi, N Ohnishi, M Asaka, M Hatakeyama  ONCOGENE  22-  (51)  8337  -8342  2003/11  [Not refereed][Not invited]
     
    Helicobacter pylori (H. pylori) is a causative agent of gastrointestinal diseases such as atrophic gastritis and gastroduodenal ulcer. Infection of cagA-positive H. pylori is also associated with gastric carcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The cagA gene product CagA is directly injected into the bacteria-attached host cells via the bacterial type IV secretion system. The translocated CagA deregulates intracellular signaling pathways and thereby initiates pathogenesis. In this work, we examined the biological effects of CagA on B cells, from which MALT lymphoma arises. Ectopic expression of CagA in interleukin 3-dependent B cells inhibited cell proliferation by suppressing the JAK-STAT signaling. CagA was also capable of preventing hydroxyurea-induced B-cell apoptosis through inhibiting p53 accumulation. In contrast to the effects of CagA in gastric epithelial cells, the observed CagA activities in B cells were independent of its tyrosine phosphorylation. Our results indicate that CagA possesses both phosphorylation-dependent and -independent activities in mammalian cells and that biological impacts of CagA depend on cell-type context. As a result of B-cell growth inhibition, CagA may diminish anti-H. pylori immune responses. Furthermore, CagA may play a role in the development of MALT lymphoma by impairing p53-dependent apoptosis.
  • 図解 Helicobacter pylori遺伝子の細胞内導入
    堤 良平, 東 秀明, 畠山 昌則  Helicobacter Research  7-  (3)  204  -208  2003/06  [Not refereed][Not invited]
  • T Takebayashi, H Higashi, H Sudo, H Ozawa, E Suzuki, O Shirado, H Katoh, M Hatakeyama  JOURNAL OF BIOLOGICAL CHEMISTRY  278-  (17)  14897  -14905  2003/04  [Not refereed][Not invited]
     
    The retinoblastoma protein (pRB) and its homologues, p107 and p130, prevent cell cycle progression from G(0)/G(1) to S phase by forming complexes with E2F transcription factors. Upon phosphorylation by G, cyclin-cyclin-dependent kinase (Cdk) complexes such as cyclin D1-Cdk4/6 and cyclin E-Cdk2, they lose the ability to bind E2F, and cells are thereby allowed to progress into S phase. Functional loss of one or more of the pRB family members, as a result of genetic mutation or deregulated phosphorylation, is considered to be an essential prerequisite for cellular transformation. In this study, we found that pRB family proteins have the ability to stimulate cyclin D1 transcription by activation of the NF-kappaB transcription factor. The cyclin D1-inducing activity of pRB is abolished by adenovirus E1A oncoprotein but not by the deletion of the A-box, the B-box, or the C-terminal region of the pocket, indicating that multiple pocket sequences are independently involved in cyclin D1 activation. Intriguingly, tumor-derived pRB pocket mutants retain the cyclin D1-inducing activity. Our results reveal a novel role of pRB family proteins as potential activators of NF-kappaB and inducers of G(1) cyclin. Certain pRB pocket mutants may give rise to a cellular situation in which deregulated E2F and cyclin D1 cooperatively promote abnormal cell proliferation.
  • 消化管の分子生物学 Helicobacter pylori CagA蛋白と細胞内シグナル伝達系の相互作用
    堤 良平, 東 秀明, 畠山 昌則  G.I.Research  11-  (1)  70  -73  2003/02  [Not refereed][Not invited]
  • R Tsutsumi, H Higashi, M Higuchi, M Okada, M Hatakeyama  JOURNAL OF BIOLOGICAL CHEMISTRY  278-  (6)  3664  -3670  2003/02  [Not refereed][Not invited]
     
    diseases ranging from gastritis to cancer. The CagA protein is the product of the cagA gene carried among virulent H. pylori strains and is associated with severe disease outcomes, most notably gastric carcinoma. CagA is injected from the attached H. pylori into gastric epithelial cells and undergoes tyrosine phosphorylation. The phosphorylated CagA binds and activates SHP-2 phosphatase and thereby induces a growth factor-like morphological change termed the "humming-bird phenotype." In this work, we demonstrate that CagA is also capable of interacting with C-terminal Src kinase (Csk). As is the case with SHP-2, Csk selectively binds tyrosine-phosphorylated CagA via its SH2 domain. Upon complex formation, CagA stimulates Csk, which in turn inactivates the Src family of protein-tyrosine kinases. Because Src family kinases are responsible for CagA phosphorylation, an essential prerequisite of CagA.SHP-2 complex formation and subsequent induction of the hummingbird phenotype, our results indicate that CagA-Csk interaction down-regulates CagA.SHP-2 signaling by both competitively inhibiting CagA.SHP-2 complex formation and reducing levels of CagA phosphorylation. We further demonstrate that CagA.SHP-2 signaling eventually induces apoptosis in AGS cells. Our results thus indicate that CagA-Csk interaction prevents excess cell damage caused by deregulated activation of SHP-2. Attenuation of CagA activity by Csk may enable cagA-positive H. pylori to persistently infect the human stomach for decades while avoiding excess CagA toxicity to the host.
  • S Yamazaki, A Yamakawa, Y Ito, M Ohtani, H Higashi, M Hatakeyama, T Azuma  GUT  51-  A13  -A13  2002/09  [Not refereed][Not invited]
  • 細菌感染症発症機序における宿主因子 ヘリコバクター・ピロリ菌の細胞内標的分子と胃癌
    畠山 昌則, 東 秀明, 堤 良平, 武藤 修一  日本細菌学雑誌  57-  (1)  79  -79  2002/02  [Not refereed][Not invited]
  • M Yamada, T Kondo, S Ashizawa, T Takebayashi, H Higashi, M Hatakeyama  CYTOKINE  17-  (2)  91  -97  2002/01  [Not refereed][Not invited]
     
    Interleukin 3 (IL-3)-dependent proliferation of haematopoietic cells is specifically inhibited by p130, a member of the pRB-family proteins. p130 interacts with the cell-cycle regulatory E2F transcription factors, notably E2F-4 and E2F-5, and affects promoters containing E2F-binding sites through two distinct mechanisms. First, upon complex formation with E2F, it blocks transcriptional activation by E2F. Second, the formed p130-E2F complex binds to E2F sites and actively represses transcription by inhibiting the activity of surrounding enhancer elements on the promoter. To pursue the relative contributions of each mechanism in the p130-mediated inhibition of IL-3-dependent cell proliferation, we employed a dominant-negative DP-1, which suppresses both E2F-dependent transactivation and the formation of active transcriptional repressors. Ectopic expression of the dominant negative DP-1 in the IL-3-dependent BaF3 lymphoid cells gave rise to an inhibition of cell proliferation, which was concomitantly associated with a decrease in levels of cyclin E, an indispensable molecule for G1 to S-phase cell-cycle progression. Our results indicate that blocking E2F-dependent transactivation, but not the formation of p130-E2F transcriptional repressor complexes, is responsible for the inhibition of IL-3-dependent cell growth by p130. (C) 2002 Elsevier Science Ltd.
  • pRB相同分子p107はDNA損傷に反応してS期の進行を抑制する
    近藤 琢磨, 東 秀明, 山田 雅文, 石川 晋, 芦澤 敏, 畠山 昌則  日本癌学会総会記事  60回-  101  -101  2001/09  [Not refereed][Not invited]
  • リンパ系細胞増殖制御におけるE2F転写因子の機能的役割
    山田 雅文, 近藤 琢磨, 芦澤 敏, 東 秀明, 畠山 昌則  日本癌学会総会記事  60回-  100  -100  2001/09  [Not refereed][Not invited]
  • T Kondo, H Higashi, H Nishizawa, S Ishikawa, S Ashizawa, M Yamada, Z Makita, T Koike, M Hatakeyama  JOURNAL OF BIOLOGICAL CHEMISTRY  276-  (20)  17559  -17567  2001/05  [Not refereed][Not invited]
     
    PRE family pocket proteins consisting of pRB, p107, and p130 are thought to act as a set of growth regulators that inhibit the cell cycle transition from G(1) to S phases by virtue of their interaction with E2F transcription factors. When cells are committed to progressing through the cell cycle at the late G(1) restriction point, they are hyperphosphorylated by G(1) cyclin-cyclin-dependent kinase and are functionally inactivated. Consistent with such a G(1) regulatory role, pRB and p130 are abundantly expressed in quiescent cells. In contrast, p107 is present at low levels in the hypophosphorylated form in quiescent cells. As cells progress toward late G(1) to S phases, the levels of p107 increase, and the majority become hyperphosphorylated, suggesting a possible role of p107 in post-G(1) cell cycle regulation. In this study, we have demonstrated that a nonphosphorylatable and thus constitutively active p107 has the potential to inhibit S phase progression. The levels of the phosphorylation-resistant p107 required for the S phase inhibition are significantly less than those of endogenous p107. We further show herein that the exposure of cells to the DNA-damaging agent, cisplatin, provokes S phase arrest, which is concomitantly associated with the accumulation of hypophosphorylated p107. Furthermore, the S phase inhibitory response to cisplatin is augmented by the ectopic expression of wild type p107, although it is diminished by the adenovirus E1A oncoprotein, which counteracts the pocket protein functions. Because p107 is a major pRB family protein expressed in S phase cells, our results indicate that p107 participates in an inhibition of cell cycle progression in response to DNA damage in S phase cells.
  • S Ashizawa, H Nishizawa, M Yamada, H Higashi, T Kondo, H Ozawa, A Kakita, M Hatakeyama  JOURNAL OF BIOLOGICAL CHEMISTRY  276-  (14)  11362  -11370  2001/04  [Not refereed][Not invited]
     
    The activity of the retinoblastoma protein pRB is regulated by phosphorylation that is mediated by G(1) cyclin-associated cyclin-dependent kinases (CDKs). Since the pRB-related pocket proteins p107 and p130 share general structures and biological functions with pRB, their activity is also considered to be regulated by phosphorylation. In this work, we generated phosphorylation-resistant p107 and p130 molecules by replacing potential cyclin CDK phosphorylation sites with non-phosphorylatable alanine residues. These phosphorylation-resistant mutants retained the ability to bind E2F and cyclin. Upon introduction into p16(INK4a)-deficient U2-OS osteosarcoma cells, in which cyclin D-CDK4/6 is dysregulated, the phosphorylation-resistant mutants, but not wild-type p107 or p130, were capable of inhibiting cell proliferation. Furthermore, when ectopically expressed in pRB-deficient SAOS-8 osteosarcoma cells, the wild-type as well as the phosphorylation-resistant pRB family proteins were capable of inducing large flat cells. The flat cell-inducing activity of the wild-type proteins, but not that of the phosphorylation-resistant mutants; was:abolished by coexpressing cyclin E. Our results indicate that the elevated cyclin D- or cyclin E-associated kinase leads to systemic inactivation of the pRB family proteins and suggest that dysregulation of the pRB kinase provokes an aberrant cell cycle in a broader range of cell types than those induced by genetic inactivation of the RB gene.
  • Ohtoshi A., Maeda T., Higashi H., Ashizawa S., Yamada ′ M., Hatakeyama M.  Collected papers from Institute for Genetic Medicine Hokkaido University  2-  290  -294  2001
  • Ohtoshi A., Maeda T., Higashi H., Ashizawa S., Yamada M., ′ Hatakeyama M.  Collected papers from Institute for Genetic Medicine Hokkaido University  2-  284  -289  2001
  • 細胞周期制御におけるpRB相同分子・p107の役割
    近藤 琢磨, 山田 雅文, 石川 晋, 芦澤 敏, 東 秀明, 畠山 昌則  日本癌学会総会記事  59回-  328  -328  2000/09  [Not refereed][Not invited]
  • リン酸化によるRBファミリータンパクの包括的機能制御
    芦澤 敏, 山田 雅文, 東 秀明, 近藤 琢磨, 畠山 昌則  日本癌学会総会記事  59回-  294  -294  2000/09  [Not refereed][Not invited]
  • A Ohtoshi, T Maeda, H Higashi, S Ashizawa, M Hatakeyama  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS  268-  (2)  530  -534  2000/02  [Not refereed][Not invited]
     
    The initiation of anaphase and exit from mitosis depend on the activation of the anaphase-promoting complex/cyclosome (APC/C), a multicomponent, ubiquitin-protein ligase, The WD-repeat protein called p55(CDC)(Cdc20) directly binds to and activates APC/C. By using yeast two-hybrid screening, we found that cyclin A, a critical cell cycle regulator in the S and G2/M phases, specifically interacts with p55(CDC). Ectopically expressed p55(CDC) and cyclin A form a stable protein complex in mammalian cells. The p55(CDC)-cyclin A interaction occurs through the region containing the WD repeats of p55(CDC) and the region between the destruction box and the cyclin box of cyclin A. In addition to the physical interaction, p55(CDC) is phosphorylated by cyclin A-associated kinase. These findings suggest that the function of p55(CDC) is mediated or regulated by its complex formation with cyclin A. (C) 2000 Academic Press.
  • A Mori, H Higashi, Y Hoshikawa, M Imamura, M Asaka, M Hatakeyama  BLOOD  94-  (10)  269A  -269A  1999/11  [Not refereed][Not invited]
  • A Mori, H Higashi, Y Hoshikawa, M Imamura, M Asaka, M Hatakeyama  ONCOGENE  18-  (46)  6209  -6221  1999/11  [Not refereed][Not invited]
     
    The retinoblastoma protein (pRB) and the related pocket proteins, p107 and p130, play crucial roles in mammalian cell cycle control. Recent studies indicate that these pocket proteins are also involved in cellular differentiation processes. We demonstrate in this work that the pRB-related p130 selectively accumulates during the in vitro differentiation of the myeloid progenitor cell, 32Dc13, into granulocyte in response to granulocyte-colony stimulating factor (G-CSF). This G-CSF-dependent granulocytic differentiation is blocked by the adenovirus E1A oncoprotein, which binds to and inactivates the pRB family of pocket proteins including p130. Furthermore, enforced overexpression of p130 but not PRE inhibits the myeloid cell proliferation that is concomitantly associated with granulocytic differentiation morphologically characterized by nuclear segmentation. However, simple G1-cell cycle al rest induced by cytokine deprivation or ectopic overexpression of the p27 cyclin-dependent kinase inhibitor, or inhibition of E2F activities by dominant negative DP-1 is not sufficient to trigger granulocytic differentiation. The differentiation-promoting activity of p130 in myeloid cells requires both the pocket domain and the spacer domain. Our results indicate that the pRB-related p130 plays a critical role in myeloid cell differentiation and suggest that coupling of cell cycle exit with the cellular differentiation program may be specifically achieved by p130.
  • S Hatakeyama, M Kitagawa, K Nakayama, M Shirane, M Matsumoto, K Hattori, H Higashi, H Nakano, K Okumura, K Onoe, RA Good, K Nakayama  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA  96-  (11)  6571  -6571  1999/05  [Not refereed][Not invited]
  • Hatakeyama S., Kitagawa M., Nakayama K., Shirane M., ′ Matsumoto M., Hattori K., Higashi H., Nakano H., ′ Okumura K., Ono_ K., Good R.A., Nakayama K.  Collected papers from Institute for Genetic Medicine Hokkaido University  1-  271  -275  1999
  • T Tanaka, Tatsuno, I, Y Noguchi, D Uchida, T Oeda, S Narumiya, T Yasuda, H Higashi, M Kitagawa, K Nakayama, Y Saito, A Hirai  JOURNAL OF BIOLOGICAL CHEMISTRY  273-  (41)  26772  -26778  1998/10  [Not refereed][Not invited]
     
    The role of the mevalonate cascade in the control of cell cycle progression in astrocytes has been investigated. Serum stimulation of rat astrocytes in primary culture induces the expression of cyclin E followed by the activation of cyclin-dependent kinase 2 (Cdk2) during G(1)/S transition. The expression of p27(hip1) cyclin D1, and the activities of Cdk4 and Cdk-activating kinase (CAB), composed of Cdk7 and cyclin H, mere not affected. Serum did, however, stimulate the expression of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA at mid-G(1) phase. Moreover, an inhibitor of HMG-CoA reductase, pravastatin, reduced cyclin E expression and Cdk2 activation and caused G(1) arrest in the astro cytes. In contrast, mevalonate and its metabolite, geranylgeranylpyrophosphate (GGPP) but not farnesylpyrophosphate (FPP), reversed the inhibitory effects of pravastatin on cyclin E expression and Cdk2 activation and allowed G(1)/S transition. Rho small GTPase(s) were geranylgeranylated and translocated to membranes in the presence of GGPP during G(1)/S transition. The effect of GGPP on cyclin E expression was abolished by botulinum C3 exoenzyme, which specifically inactivates Rho. These data indicate that geranylgeranylated Rho small GTPase(s) are essential for the induction of cyclin E expression, Cdk2 activation, and G(1)/S transition in rat astrocytes.
  • SuzukiTakahashi, I, H Higashi, E Yoshida, S Nishimura, M Kitagawa  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS  234-  (2)  386  -392  1997/05  [Not refereed][Not invited]
     
    p16(INK4a), a protein that inhibits cyclin-dependent kinase 4 (Cdk4) and Cdk6, is deficient in many human cancers and in established lines of tumor cells. It has been reported that transfection with cDNA for p16(INK4a) inhibits the growth of cell lines that express retinoblastoma protein (pRB). However, it is unclear whether the introduction of cDNA for p16(INK4a) affects the growth of cells that express p16(INK4a) protein. Moreover, the effects of other cell-cycle regulators on the inhibition of cell growth by p16(INK4a) remain unknown. In this study, cDNA for p16(INK4a) was used to transfect human cell lines that had various status of expression of RE pathway-related proteins, such as members of the RE family proteins and Cdk-inhibitory proteins. We found that status of p107, p130, p15(INK4b), p18(INK4c) p21(Cip1), p27(Kip1) cyclin D1, and Cdk4 were not correlated with the growth-inhibitory activity of exogenous p16(INK4a). BY contrast, transfection with cDNA for p16(INK4a) had a significant effect on the growth of cells depended on the status not only of pRB but also of p16(INK4a). Although exogenous p16(INK4a) inhibited the growth of cells that expressed pRB but did not express p16(INK4a) (pRB(+)/p16(-) cells), it had little affect on either pRB(-)/p16(+) cells or pRB(-)/p16(+) cells. Moreover, transfection with cDNA for p16(INK4a) also inhibited the activity of the E2 promoter of the dehydrofolate reductase gene in the same manner that depended on the absence of p16(INK4a), as well as on the presence of pRB. These results suggest that deregulation of the RE pathway by p16(INK4a) deficiency plays a very important role in the proliferation of cells that lack p16(INK4a) protein. (C) 1997 Academic Press.
  • H Higashi, SuzukiTakahashi, I, E Yoshida, S Nishimura, M Kitagawa  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS  231-  (3)  743  -750  1997/02  [Not refereed][Not invited]
     
    p16(INK4a) is a inhibitory protein of Cyclin-dependent kinase 4 (Cdk4). p16 negatively regulates the cell cycle progression from G1 to S phase. Functional pie is absent from many human cancers, as well as from many established lines of tumor cells. However, it is not clear whether expression of p16 in p16-deficient tumor cells can suppress their anchorage-independent growth. Therefore, we introduced a cDNA for p16(INK4a) into the human glioblastoma cell line T98G, which lacks a gene for p16(INK4a). We isolated several clones that stably expressed various amounts of pie protein. The doubling time of the various clones was generally prolonged. Clones with high-level expression of p16 protein had characteristics of restricted growth, such as contact inhibition, while the parental T98G cells had no such characteristics. Furthermore, the efficiency of colony formation in soft agar was dramatically decreased in the case of cells that expressed exogenous pie. Our observations suggest that the expression of pie protein restricts the unbounded growth and the anchorage-independent growth of tumor cells. (C) 1997 Academic Press.
  • Y Taya, K Hitomi, HK Jung, M Ikeda, K Tamai, H Higashi, M Kitagawa  EUROPEAN JOURNAL OF CELL BIOLOGY  72-  118  -118  1997  [Not refereed][Not invited]
  • M Kitagawa, H Higashi, HK Jung, SuzukiTakahashi, I, M Ikeda, K Tamai, J Kato, K Segawa, E Yoshida, S Nishimura, Y Taya  EMBO JOURNAL  15-  (24)  7060  -7069  1996/12  [Not refereed][Not invited]
     
    Cyclin D-Cdk4/6 and cyclin A/E-Cdk2 are suggested to be involved in phosphorylation of the retinoblastoma protein (pRB) during the G(1)/S transition of the cell cycle. However, it is unclear why several Cdks are needed and how they are different from one another. We found that the consensus amino acid sequence for phosphorylation by cyclin D1-Cdk4 is different from S/T-P-X-K/R, which is the consensus sequence for phosphorylation by cyclin A/E-Cdk2 using various synthetic peptides as substrates. Cyclin D1-Cdk4 efficiently phosphorylated the G1 peptide, RPPTLS(780)PIP- that contained a part of the sequence of pRB, while cyclins E-Cdk2 and A-Cdk2 did not. To determine the phosphorylation state of pRB in vitro and in vivo, we raised the specific antibody against phospho-Ser780 in pRB. We confirmed that cyclin D1-Cdk4, but not cyclin E-Cdk2, phosphorylated Ser780 in recombinant pRB. The Ser78O in pRB was phosphorylated in the GI phase in a cell cycle-dependent manner, Furthermore, we found that pRB phosphorylated at Ser780 cannot bind to E2F-1 in vivo. Our data show that cyclin D1-Cdk4 and cyclin, A/E Cdk2 phosphorylate different sites of pRB in vivo.
  • M Kitagawa, H Higashi, HK Jung, SuzukiTakahashi, I, M Ikeda, K Tamai, J Kato, K Segawa, E Yoshida, S Nishimura, Y Taya  EMBO JOURNAL  15-  (24)  7060  -7069  1996/12  [Not refereed][Not invited]
     
    Cyclin D-Cdk4/6 and cyclin A/E-Cdk2 are suggested to be involved in phosphorylation of the retinoblastoma protein (pRB) during the G(1)/S transition of the cell cycle. However, it is unclear why several Cdks are needed and how they are different from one another. We found that the consensus amino acid sequence for phosphorylation by cyclin D1-Cdk4 is different from S/T-P-X-K/R, which is the consensus sequence for phosphorylation by cyclin A/E-Cdk2 using various synthetic peptides as substrates. Cyclin D1-Cdk4 efficiently phosphorylated the G1 peptide, RPPTLS(780)PIP- that contained a part of the sequence of pRB, while cyclins E-Cdk2 and A-Cdk2 did not. To determine the phosphorylation state of pRB in vitro and in vivo, we raised the specific antibody against phospho-Ser780 in pRB. We confirmed that cyclin D1-Cdk4, but not cyclin E-Cdk2, phosphorylated Ser780 in recombinant pRB. The Ser78O in pRB was phosphorylated in the GI phase in a cell cycle-dependent manner, Furthermore, we found that pRB phosphorylated at Ser780 cannot bind to E2F-1 in vivo. Our data show that cyclin D1-Cdk4 and cyclin, A/E Cdk2 phosphorylate different sites of pRB in vivo.
  • H Higashi, SuzukiTakahashi, I, S Saitoh, K Segawa, Y Taya, A Okuyama, S Nishimura, M Kitagawa  EUROPEAN JOURNAL OF BIOCHEMISTRY  237-  (2)  460  -467  1996/04  [Not refereed][Not invited]
     
    Cyclin-dependent kinases (Cdks) form complexes with cyclins, and as a consequence they generally express kinase activities. One of these Cdks, Cdk2, is known to bind with cyclins A and E, and plays an important role in the progression of the cell cycle via phosphorylation of target proteins such as the product of the retinoblastoma tumor-suppressor gene (pRB). It has been suggested that Cdk2 bound with cyclin D1 and Cdk2-cyclin-D1 complex show neither H1 histone nor pRB kinase activity. However, it is not clear whether Cdk2-cyclin-D1 has unknown targets and why Cdk2 is not activated by binding with cyclin D1. We investigated these questions using Cdk, cyclin and Cdk-cyclin complexes produced in a baculovirus expression system. Cdk2 formed a complex with cyclin D1 in this system. After extensive purification, Cdk2 was still bound to cyclin D. The Cdk2-cyclin-D1 complex did not phosphorylate any tested substrates, such as H1 histone, pRB, SV40 large T antigen, p53, E2F-1 or a preparation of nuclear proteins from HeLa cells; in contrast, Cdk2-cyclin-E and Cdk2-cyclin-A phosphorylated these proteins. Moreover, the Cdk2-cyclin-D1 complex was not activated by incubation with Cdk4 or cyclin E. Thus, Cdk2 and cyclin D1 formed a stable complex that was not activated. In order to determine why Cdk2-cyclin-D1 lacks kinase activity, we investigated the phosphorylation of Cdk2. Under-shifted Cdk2, the active form of Cdk2, was not detected in the Cdk2-cyclin-D1 complex in the baculovirus system. In human WI-38 cells, cyclin D1 began to form a complex with Cdk2 as well as with Cdk4 from the mid-G1 phase of the cell cycle. The Cdk2 bound to cyclin D1 in human cells was also the inactive form that was slowly migrated. Moreover, we found that Cdk2 bound to cyclin D1 was not phosphorylated by Cdk7-cyclin-H, while Cdk2 bound to cyclin E, as well as free Cdk2, was phosphorylated by Cdk7-cyclin-H. Additionally, Cdk2 phosphorylated by Cdk7-cyclin-H did not bind to cyclin D1. These results strongly suggest that Cdk2 forms a stable complex with cyclin D1 but is not activated because the Cdk2 molecule in the complex is not phosphorylated by Cdk7-cyclin-H and the phosphorylated Cdk2, an active form, does not bind to cyclin D1.
  • H HIGASHI, SUZUKITAKAHASHI, I, Y TAYA, K SEGAWA, S NISHIMURA, M KITAGAWA  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS  216-  (2)  520  -525  1995/11  [Not refereed][Not invited]
     
    Cyclin-dependent kinase 2 (Cdk(2)), when bound to either cyclin A or cyclin E, recognizes the Ser/Thr-Pro-X-basic amino acid (motif A) as a phosphorylation site. In this study, we designed several peptides based on motif A and examined the substrate specificity of Cdk2-cyclin A and Cdk2-cyclin E using these peptides, Peptides containing a proline residue in the sequence Pro-X-Thr-Pro-X-basic amino acid (motif B) had higher affinity for both Cdk2 complexes than peptides; containing motif A. Furthermore, differences in substrate affinity between the two Cdk2 complexes were caused by a proline residue adjacent to or three positions before the threonine residue. Similarly, the presence of different basic amino acids in motif B also had different effects on affinity for each complex. We demonstrate the possibility that the substrate specificity of Cdk2 bound to cyclin might be regulated by the species of cyclin. (C) 1995 Academic Press, Inc.
  • SUZUKITAKAHASHI, I, M KITAGAWA, M SAIJO, H HIGASHI, H OGINO, H MATSUMOTO, Y TAYA, S NISHIMURA, A OKUYAMA  ONCOGENE  10-  (9)  1691  -1698  1995/05  [Not refereed][Not invited]
     
    It has been postulated that the product (pRB) of the retinoblastoma gene dissociates from the E2F-pRB complex upon phosphorylation by cyclin-dependent kinase(s) (cdk). However, there is no direct evidence for the regulation of formation of the E2F-pRB complex via phosphorylation by purified cdk, Therefore,we investigated the regulation of formation of this complex by phosphorylation using pRB and purified cyclin A-cdk2, cyclin E-cdk2 or cyclin D1-cdk4. Purified pRB was incubated with nuclear extracts prepared from pRB-defective cells and then subjected to gel mobility shift assays, We confirmed that unphosphorylated pRB associated with various types of E2F but pRB has been phosphorylated by cyclin A-cdk2 did not. We found that E2F-pRB complexes were disrupted as a consequence of phosphorylation by cyclin A-cdk2, and the levels of the free forms of E2Fs increased. We also found that not only the E2F-pRB complexes but also the E2F-p107 complexes were disrupted upon phosphorylation by cyclin A-cdk2. Furthermore, E2F-pRB complexes were disrupted through phosphorylation by cyclin D1-cdk4 and cyclin E-cdk2, as web as by cyclin A-cdk2. These results clearly demonstrate that the phosphorylation of pRB and p107 by cdks regulates the formation of complexes between E2F and pRB or p107.
  • S KOTANI, T ENDO, M KITAGAWA, H HIGASHI, T ONAYA  ONCOGENE  10-  (4)  663  -669  1995/02  [Not refereed][Not invited]
     
    Cyclin-dependent kinase 2 (Cdk2) controls the transition from the G1 to the S phase in the mammalian cell cycle. We found by immunoblotting that anti-Cdk2 antibodies recognize three Cdk2 proteins (of 33, 34 and 39 kDa) in FRTL-5 and FRTL-Tc cells (malignantly transformed FRTL cells). Although 33 kDa protein is a phosphorylated form of 34 kDa protein previously reported, the nature of 39 kDa protein is unknown. In order to determine the nature of this protein, we screened a FRTL-5 cDNA library. Two cDNA clones of the rat homologue (rat Cdk2-alpha and -beta) of human Cdk2 were isolated. The open reading frame of rat Cdk2-alpha cDNA encoded a protein with 428 amino acids and has a high degree of conservation with human Cdk2. The calculated molecular weight of Cdk2-alpha protein is 33892 Da. The rat Cdk2-beta cDNA was identical to Cdk2-alpha cDNA except that it had extra 144 bp; this coincided with insertion of 48 amino acids into Cdk2-alpha protein between Met 196 and Val 197. The calculated molecular weight of Cdk2-beta protein is 39087 Da. Northern blot analysis indicated that the sizes of rat Cdk2-alpha and -beta mRNAs are approximately 2.5 kb and 3.0 kb, respectively. Partial proteolytic mapping showed that Cdk2-beta gene product is 39 kDa Cdk2 in the immunoblotting. We also found that Cdk2-beta protein binds to cyclin A and suc1 proteins. During G1-S phase in FRTL-Tc cells, Cdk2-alpha protein level is constant, but is gradually phosphorylated. In contrast, the level of Cdk2-beta protein increases through the S phase and decreases at the early G2 phase. These results suggest that a variant form of Cdk2 protein might be required for entry into the S phase of the cell cycle in FRTL-Tc cells.
  • M KITAGAWA, H HIGASHI, SUZUKITAKAHASHI, I, K SEGAWA, SK HANKS, Y TAYA, S NISHIMURA, A OKUYAMA  ONCOGENE  10-  (2)  229  -236  1995/01  [Not refereed][Not invited]
     
    Transcription factor E2F-1 has a putative consensus sequence for phosphorylation by cyclin dependent kinase (Ser-Pro-X-Lys/Arg). Therefore, we studied the phosphorylation of E2F-1 in vivo and in vitro and its biological functions. E2F-1 was prepared by immunoprecipitation with anti-E2F-1 antibody from IMR32 lysates and was effectively phosphorylated by human cyclin A-cdk2 which was expressed in insect cells using baculovirus system. GST-EZF-1 was phosphorylated by cyclin A-cdk2 more efficiently than by cyclin E-cdk2. Cyclin D1-cdk4 phosphorylated PRE but scarcely phosphorylated GST-E2F-1 or H1 histone. The 60 kd protein precipitated with anti-E2F-1 antibody was phosphorylated in vivo. Phospho-peptide mapping indicated that its cleavage profile was identical with that of E2F-1 phosphorylated by cyclin A-cdk2 in vitro. This 60 kd protein, which is likely to be E2F-1, was not phosphorylated during the GO and early G1 phase, Phosphorylation of E2F-1 began from the S phase while phosphorylation of pRB started nearly at G1/S. The in vivo phosphorylation of E2F-1 was inhibited by butyrolactone I, a cyclin-dependent kinase inhibitor (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). The binding of E2F-1 to E2 promoter was found to be reduced by phosphorylation of E2F-1 by cyclin A-cdk2, suggesting that phosphorylation of E2F-1 may induce shut off of gene expression at the transcriptional level. These results suggest that E2F-1 is phosphorylated by cyclin A-cdkZ in the S phase in vivo as well as in vitro and that its phosphorylation by cyclin A-cdkZ may modulate its activity.
  • M KITAGAWA, H HIGASHI, IS TAKAHASHI, T OKABE, H OGINO, Y TAYA, S NISHIMURA, A OKUYAMA  ONCOGENE  9-  (9)  2549  -2557  1994/09  [Not refereed][Not invited]
     
    Butyrolactone I is a selective inhibitor of the cyclin-dependent kinase (cdk) family. It inhibits both cdk2 and cdc2 kinase, but scarcely affects C-kinase, A-kinase, casein kinases, MAP kinase or EGF receptor-tyrosine kinase (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). We studied the effects of butyrolactone I on the cell cycle as well as on phosphorylation of retinoblastoma protein (pRB). Butyrolactone I inhibited phosphorylation of pRB catalyzed by cyclin A-cdk2 produced by baculovirus in vitro. Furthermore, it inhibited phosphorylation of pRB and cell cycle progression from G1 to S phase in WI38 cell cultures. WI38 cells arrested at the G0 phase by serum starvation progressed in the cell cycle after serum stimulation. pRB was phosphorylated after 10 h serum stimulation. Incorporation of [H-3]thymidine into the cells began to increase after 16 h serum stimulation. These processes were inhibited by butyrolactone I. Flow cytometric analysis showed that exposure to butyrolactone I inhibited progression of the cell cycle from G1 to S phase. These data suggested that initiation of DNA synthesis was inhibited by butyrolactone I and that the cell cycle was arrested in the G1 phase. Butyrolactone I also inhibited H1 histone phosphorylation in human WI38 cells and their G2/M progression, tsFT210 cells, a temperature-sensitive cdc2 mutant cell line, were synchronized at G2/M at a nonpermissive temperature, butyrolactone I inhibited the cell cycle progression of these cells at G2/M at the permissive temperature. Thus butyrolactone I, a cyclin-dependent kinase family inhibitor, which prevented the phosphorylations of the cell cycle-regulating proteins pRB and H1 histone, inhibited the cell cycle at G1/S and G2/M, respectively. These results suggest that the phosphorylations of pRB and H1 histone may play crucial roles in G1/S and G2/M progression, respectively, although it is possible that phosphorylations of other proteins by cdks are involved in G1/S and G2/M progression.
  • M KITAGAWA, H HIGASHI, IS TAKAHASHI, T OKABE, H OGINO, Y TAYA, S NISHIMURA, A OKUYAMA  ONCOGENE  9-  (9)  2549  -2557  1994/09  [Not refereed][Not invited]
     
    Butyrolactone I is a selective inhibitor of the cyclin-dependent kinase (cdk) family. It inhibits both cdk2 and cdc2 kinase, but scarcely affects C-kinase, A-kinase, casein kinases, MAP kinase or EGF receptor-tyrosine kinase (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). We studied the effects of butyrolactone I on the cell cycle as well as on phosphorylation of retinoblastoma protein (pRB). Butyrolactone I inhibited phosphorylation of pRB catalyzed by cyclin A-cdk2 produced by baculovirus in vitro. Furthermore, it inhibited phosphorylation of pRB and cell cycle progression from G1 to S phase in WI38 cell cultures. WI38 cells arrested at the G0 phase by serum starvation progressed in the cell cycle after serum stimulation. pRB was phosphorylated after 10 h serum stimulation. Incorporation of [H-3]thymidine into the cells began to increase after 16 h serum stimulation. These processes were inhibited by butyrolactone I. Flow cytometric analysis showed that exposure to butyrolactone I inhibited progression of the cell cycle from G1 to S phase. These data suggested that initiation of DNA synthesis was inhibited by butyrolactone I and that the cell cycle was arrested in the G1 phase. Butyrolactone I also inhibited H1 histone phosphorylation in human WI38 cells and their G2/M progression, tsFT210 cells, a temperature-sensitive cdc2 mutant cell line, were synchronized at G2/M at a nonpermissive temperature, butyrolactone I inhibited the cell cycle progression of these cells at G2/M at the permissive temperature. Thus butyrolactone I, a cyclin-dependent kinase family inhibitor, which prevented the phosphorylations of the cell cycle-regulating proteins pRB and H1 histone, inhibited the cell cycle at G1/S and G2/M, respectively. These results suggest that the phosphorylations of pRB and H1 histone may play crucial roles in G1/S and G2/M progression, respectively, although it is possible that phosphorylations of other proteins by cdks are involved in G1/S and G2/M progression.
  • M KITAGAWA, T OKABE, H OGINO, H MATSUMOTO, SUZUKITAKAHASHI, I, T KOKUBO, H HIGASHI, S SAITOH, Y TAYA, H YASUDA, Y OHBA, S NISHIMURA, N TANAKA, A OKUYAMA  ONCOGENE  8-  (9)  2425  -2432  1993/09  [Not refereed][Not invited]
     
    We screened cdc2 kinase inhibitors from cultured mediums of micro organisms using purified mouse cyclin B-cdc2 kinase and a specific substrate peptide for cdc2 kinase. A selective inhibitor of cdc2 kinase was isolated from the cultured medium of Aspergillus species F-25799, and identified as butyrolactone I. Butyrolactone I inhibited cdc2 and cdk2 kinases but it had little effect on mitogen-activated protein kinase, protein kinase C, cyclic-AMP dependent kinase, casein kinase II, casein kinase I or epidermal growth factor-receptor tyrosine kinase. Its inhibitory effect was found to be due to competition with ATP. Butyrolactone I selectively inhibited the H1 histone phosphorylation in nuclear extracts. It also inhibited the phosphorylation of the product of retinoblastoma susceptibility gene in nuclear extracts and intact cells. Thus butyrolactone I should be very useful for elucidating the function of cdc2 and cdk2 kinases in cell cycle regulation.
  • M KITAGAWA, T OKABE, H OGINO, H MATSUMOTO, SUZUKITAKAHASHI, I, T KOKUBO, H HIGASHI, S SAITOH, Y TAYA, H YASUDA, Y OHBA, S NISHIMURA, N TANAKA, A OKUYAMA  ONCOGENE  8-  (9)  2425  -2432  1993/09  [Not refereed][Not invited]
     
    We screened cdc2 kinase inhibitors from cultured mediums of micro organisms using purified mouse cyclin B-cdc2 kinase and a specific substrate peptide for cdc2 kinase. A selective inhibitor of cdc2 kinase was isolated from the cultured medium of Aspergillus species F-25799, and identified as butyrolactone I. Butyrolactone I inhibited cdc2 and cdk2 kinases but it had little effect on mitogen-activated protein kinase, protein kinase C, cyclic-AMP dependent kinase, casein kinase II, casein kinase I or epidermal growth factor-receptor tyrosine kinase. Its inhibitory effect was found to be due to competition with ATP. Butyrolactone I selectively inhibited the H1 histone phosphorylation in nuclear extracts. It also inhibited the phosphorylation of the product of retinoblastoma susceptibility gene in nuclear extracts and intact cells. Thus butyrolactone I should be very useful for elucidating the function of cdc2 and cdk2 kinases in cell cycle regulation.
  • T YOSHIMOTO, H HIGASHI, A KANATANI, XS LIN, H NAGAI, H OYAMA, K KURAZONO, D TSURU  JOURNAL OF BACTERIOLOGY  173-  (7)  2173  -2179  1991/04  [Not refereed][Not invited]
     
    The 7-alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) gene from Escherichia coli HB101 was cloned and expressed in E. coli DH1. The hybrid plasmid pSD1, with a 2.8-kbp insert of chromosomal DNA at the BamHI site of pBR322, was subcloned into pUC19 to construct plasmid pSD3. The entire nucleotide sequence of an inserted PstI-BamHI fragment of plasmid pSD3 was determined by the dideoxy chain-termination method. Within this sequence, the mature enzyme protein-encoding sequence was found to start at a GTG initiation codon and to comprise 765 bp, as judged by comparison with the protein sequence. The deduced amino acid sequence of the enzyme indicated that the molecular weight is 26,778. The transformant of E. coli DH1 harboring pSD3 with a 1.8-kbp fragment showed about 200-fold-higher enzyme activity than the host. The enzyme was purified by a single chromatography step on DEAE-Toyopearl and obtained as crystals, with an activity yield of 39%. The purified enzyme was homogeneous, as judged by sodium dodecyl sulfate gel electrophoresis. The enzyme was most active at pH 8.5 and stable between pH 8 and 9. The enzyme was NAD+ dependent and had a pI of 4.3. The molecular mass was estimated to be 120 kDa by the gel filtration method and 28 kDa by electrophoresis, indicating that the enzyme exists in a tetrameric form.
  • T YOSHIMOTO, H HIGASHI, A KANATANI, XS LIN, H NAGAI, H OYAMA, K KURAZONO, D TSURU  JOURNAL OF BACTERIOLOGY  173-  (7)  2173  -2179  1991/04  [Not refereed][Not invited]
     
    The 7-alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) gene from Escherichia coli HB101 was cloned and expressed in E. coli DH1. The hybrid plasmid pSD1, with a 2.8-kbp insert of chromosomal DNA at the BamHI site of pBR322, was subcloned into pUC19 to construct plasmid pSD3. The entire nucleotide sequence of an inserted PstI-BamHI fragment of plasmid pSD3 was determined by the dideoxy chain-termination method. Within this sequence, the mature enzyme protein-encoding sequence was found to start at a GTG initiation codon and to comprise 765 bp, as judged by comparison with the protein sequence. The deduced amino acid sequence of the enzyme indicated that the molecular weight is 26,778. The transformant of E. coli DH1 harboring pSD3 with a 1.8-kbp fragment showed about 200-fold-higher enzyme activity than the host. The enzyme was purified by a single chromatography step on DEAE-Toyopearl and obtained as crystals, with an activity yield of 39%. The purified enzyme was homogeneous, as judged by sodium dodecyl sulfate gel electrophoresis. The enzyme was most active at pH 8.5 and stable between pH 8 and 9. The enzyme was NAD+ dependent and had a pI of 4.3. The molecular mass was estimated to be 120 kDa by the gel filtration method and 28 kDa by electrophoresis, indicating that the enzyme exists in a tetrameric form.
  • Tadashi Yoshimoto, Hiroshi Oyama, Tomoaki Takeshita, Hideaki Higashi, Sheng-lin Xu, Daisuke Tsuru  Journal of Fermentation and Bioengineering  70-  (6)  370  -375  1990  [Not refereed][Not invited]
     
    The neutral protease gene of Bacillus subtilis var. amylosacchariticus was isolated and the nucleotide sequence encoding the signal and pro-peptides followed by the mature protein region was established. Putative -35 and -10 sequences, TTGAGT and TAATAT, were observed as the consensus sequence for the promoter recognized by the σ55RNA polymerase of B. subtilis, and the ribosome binding site, whose sequence was AAAGGGGG, was complementary to the binding sequence of B. subtilis 16S rRNA except for one base. The nucleotide sequence of the gene was highly homologous to that of the neutral protease gene from B. subtilis IA72, particularly in the mature protein coding region: only two nucleotide discrepancies out of 900 nucleotides were found, which resulted in no amino acid replacement. However, sequences in pre-pro and putative terminator regions were significantly different between the neutral protease genes of these two strains of B. subtilis. In B. subtilis var. amylosacchariticus, a stem-loop structure with a calculated free energy of -46.0 kcal/mol, which was much lower than that of B. subtilis IA72, was postulated at the 3′ terminal region. © 1990.
  • Tadashi Yoshimoto, Hiroshi Oyama, Tomoaki Takeshita, Hideaki Higashi, Sheng-lin Xu, Daisuke Tsuru  Journal of Fermentation and Bioengineering  70-  (6)  370  -375  1990  [Not refereed][Not invited]
     
    The neutral protease gene of Bacillus subtilis var. amylosacchariticus was isolated and the nucleotide sequence encoding the signal and pro-peptides followed by the mature protein region was established. Putative -35 and -10 sequences, TTGAGT and TAATAT, were observed as the consensus sequence for the promoter recognized by the σ55RNA polymerase of B. subtilis, and the ribosome binding site, whose sequence was AAAGGGGG, was complementary to the binding sequence of B. subtilis 16S rRNA except for one base. The nucleotide sequence of the gene was highly homologous to that of the neutral protease gene from B. subtilis IA72, particularly in the mature protein coding region: only two nucleotide discrepancies out of 900 nucleotides were found, which resulted in no amino acid replacement. However, sequences in pre-pro and putative terminator regions were significantly different between the neutral protease genes of these two strains of B. subtilis. In B. subtilis var. amylosacchariticus, a stem-loop structure with a calculated free energy of -46.0 kcal/mol, which was much lower than that of B. subtilis IA72, was postulated at the 3′ terminal region. © 1990.

Books etc

  • Recent Advances in Gastrointestinal Carcinogenesis
    Higashi H (Joint workHelicobacter pylori CagA Deregulates the Cell Signaling.)
    Transworld Research Network 2006

Association Memberships

  • JAPANESE SOCIETY FOR BACTERIOLOGY   日本ヘリコバクター学会   日本癌学会   

Research Projects

  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2018/06 -2023/03 
    Author : 東 秀明
     
    炭疽の予防法として、無莢膜弱毒炭疽菌の芽胞懸濁液が動物用炭疽生ワクチンとして世界で広く利用されている。このワクチンは、動物では炭疽予防に一定の効果を示す一方、ヒトに対しては重篤な副作用を示す。一部の国では、炭疽菌毒素の一つProtective antigen(PA)を利用した無細胞炭疽ワクチンをヒトに使用しているが毒性を有することから実用性に乏しい。本研究では、新規ヒト炭疽ワクチン開発を目的として、炭疽菌毒素PAの多量体構造情報を基にin silico 分子計算手法を用い、ワクチン抗原候補分子を人工分子として設計した。これまでに、組換え体タンパク質として大腸菌内に産生させた人工分子を高度精製し、約2 mgの精製人工分子を調製した。さらに精製分子をin vitroで2量体化させた多量体化人工分子を用い、メルボルン大学においてマウス免疫試験を実施した。マウスを用いた免疫試験で得られた血清において、人工分子は全長PAとほぼ同等の抗原性を示し、かつ同等以上の毒素中和活性を示すことが示された。免疫原性の強さに関しては全長PAと大きな差はみられなかったが、人工分子においてはPA毒性発現に関わる細胞膜結合領域が欠失していることから、現行のワクチンで問題となっている毒性問題を解決できる抗原であると予想された。免疫効果を高めるため、メルボルン大学で開発されたアジュバントが人工分子抗原のワクチン効果に与える影響を検討する計画だが、COVID-19感染拡大により実施を見合わせている。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2017/04 -2020/03 
    Author : Higashi Hideaki
     
    To elucidate the transmission system of zoonotic pathogens in Zambia, we focused on anthrax spores in river water and attempted to reveal the waterborne transmission of the pathogen. In the Luangwa River basin, where anthrax outbreaks have been occurring repeatedly, river water samples were collected by the anthrax spore enrichment method developed in this study. By using PCR and next-generation sequencing, the presence of anthrax in the river water was demonstrated. Additionally, we also found the presence of enteroviruses in the river water. Furthermore, environmental information such as river water depth, temperature, turbidity and flow rate were measured at the sampling sites, and a model of pathogen diffusion by river water was constructed.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2016/04 -2019/03 
    Author : Sudo Hideki
     
    The current surgical procedure of choice for lumbar intervertebral disc (IVD) herniation is discectomy. However, defects within IVD produced upon discectomy may impair tissue healing and predispose patients to subsequent IVD degeneration. This study aimed to investigate whether the use of an acellular bioresorbable ultra-purified alginate (UPAL) gel implantation system is safe and effective as a reparative therapeutic strategy after lumbar discectomy. The UPAL gel was biocompatible with human IVD cells and promoted extracellular matrix production after discectomy, demonstrating sufficient biomechanical characteristics without material protrusion. The present results indicate the safety and efficacy of UPAL gels in a large animal model and suggest that these gels represent a novel therapeutic strategy after discectomy in cases of lumbar IVD herniation.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2015/04 -2019/03 
    Author : Suzuki Satowa, Kobayashi Ayako
     
    Bacillus cereus is ubiquitous in environment, but several nosocomial outbreaks of B. cereus blood stream infections including fatal cases were reported in Japan. Analysis of JANIS data showed diverse isolation rate of Bacillus spp. among hospitals in Japan. Isolation rate of B. cereus had clear seasonality which peaks in summer time but not in other Bacillus spp. Increase of isolation rate of total Bacillus spp. may reflect contamination of hospital environment by B. cereus. This was supported by species identification of all Bacillus spp. isolates from clinical samples of three hospitals. Molecular analysis of B. cereus isolates from nosocomial outbreaks revealed that unique sequence type (ST) 1420 was dominant. The ST1420 belonged to the Cereus III lineage which is close to the Anthracis lineages. Species identification and molecular analysis of Bacillus spp. in case of summer increase may useful for risk assessment to prevent future nosocomial B. cereus infection.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2012/04 -2015/03 
    Author : HIGASHI Hideaki
     
    As infection with intestinal residential bacteria depends on the host immune system and induces tumor formation, bacterial infection was predicted to be a potential risk factor of carcinogenesis, as is the case for oncoviral infection. The aim of this study was to reveal the effect of bacterial infection on cell proliferation through the functional analysis of Tarp molecules in Chlamydia trachomatis. We created a humanized C. trachomatis tarp gene and successfully constructed a system for ectopic Tarp expression in animal cells, which has been considered difficult thus far. We also studied an approach to create genetically modified animals using the humanized genes.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2011/04 -2014/03 
    Author : HIGASHI Hideaki
     
    Helicobacter pylori CagA protein is injected into gastric epithelial cells, where it interacts with cellular proteins such as SHP-2 and PAR1. CagA perturbs intracellular machineries involved in the regulation of cell growth and cell polarity through its C-terminal region containing Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs. To elucidate molecular basis for the pathogenic activity of CagA, we sought to determine three-dimensional structure of CagA. From results of proteolysis experiment, we found that CagA consists of the possible N-terminal and C-terminal structural domains. The CagA C-terminal fragment largely consists of an unfolded structure, although the fragment possesses an ability to bind with target molecules. Furthermore, at results of X-ray crystallographic analysis, the CagA N-terminal fragment indicate that the region has a structure comprised of three domains.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2009 -2010 
    Author : 東 秀明
     
    全長CagAのプロテアーゼ限定分解などの解析結果から、CagAはN末端側およびC末端側それぞれにドメイン構造を有し、個々のドメインを構成するフラグメントは分子内で相互作用を示すことをこれまでに明らかにしてきた。そこで、CagA分子内相互作用に関わる構造基盤の解明を目的として、CagA分子内相互作用に分子機構に関する解析を行った。 1)In vitro再構成実験の結果から、CagAのN末側領域(アミノ酸1-876)におけるアミノ酸配列554-617及び708-821の双方が、CagAC末側領域(アミノ酸877-1186)との分子内相互作用に必要であった。一方、CagAC末側領域配列を基に合成されたペプチドによる競合阻害実験から、CagAアミノ酸配列977-1077に相当するペプチドの存在により、CagAN末側及びCagAC末側フラグメント間の結合が抑制された。アミノ酸配列977-1077領域における一連の欠失変異体を用いた解析から、アミノ酸配列1027-1038に相当する11個のアミノ酸が、CagAC末側領域の分子内相互作用に関わる責任部位であることが明らかとなった。 2)アミノ酸配列998-1038を欠失させた変異型CagAを胃上皮AGS細胞に発現させたところ、野生型CagAに較べ細胞形態変化を示した細胞数及び細胞の伸長率が大きく減少した。この時、変異型CagAのチロシンリン酸化レベルは野生型CagAと差は見られなかったが、リン酸化型CagAの細胞内標的分子であるSHP-2との複合体形成量が低下していた。 これらのことから、CagAにおける分子内相互作用は構造的にSHP-2複合体形成に深く関与し、SHP-2の異常活性化を必要とするCagAの癌タンパク質としての機能発現に深く関わっているものと推察される。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2008 -2010 
    Author : HIGASHI Hideaki
     
    Helicobacter pylori CagA protein is injected into gastric epithelial cells, where it interacts with cellular proteins such as SHP-2 and Par1 through its C-terminal region containing Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs. Consequently, CagA perturbs intracellular machineries involved in the regulation of cell growth and cell polarity. To elucidate molecular basis for the pathophysiological activity of CagA, we sought to determine three-dimensional structure of CagA. We performed a limited proteolysis experiment of CagA and identified a specific cleavage site, which could represent a linker region that connects the possible N-terminal and C-terminal structural domains of CagA. Intriguingly, the C-terminal proteolytic fragment of CagA was bound to the remaining N-terminal proteolytic fragment. Furthermore, we found that N-terminal fragment enhances CagA biological activity that induces cell morphological changes in depending on the C-terminal region of CagA. Our finding indicates that an intramolecular interaction between the N-terminal and C-terminal domains plays an important role in the structural integrity of the biologically active CagA protein.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2008 -2009 
    Author : 東 秀明
     
    (1)全長CagAのプロテアーゼ限定分解解析の結果から、CagAはN末端側およびC末端側それぞれにドメイン構造を有しており、CagAのC末端側フラグメントは^1H NMR及びCDスペクトル解析から高次構造を形成しない内因性不規則構造タンパク質であることが示唆されていた。そこで、CagAと標的分子間の分子相互作用機構を解明するため、PAR1との結合に十分なCagA C末側34アミノ酸残基の同定を行い、^<15>N標識ペプチドを用いたPAR1共存下HSQCスペクトル解析を行った。その結果、PAR1結合に関わるCagAのアミノ酸残基による化学シフトの観測に成功した。 (2)CagA分子内相互作用に関わる構造基盤の解明を目的として、CagA分子内相互作用に関わる結合責任領域の同定をおこなった。CagAのN末側領域(アミノ酸1-876)に対する一連の欠失変異体を用いた実験から、CagA C末側領域(アミノ酸877-1186)との分子内相互作用にはN末側アミノ酸配列554-617及び708-821の双方が必要であることが明らかとなった。一方、CagA C末側領域配列を基に合成されたペプチドによる競合阻害実験から、CagAアミノ酸977-1077に相当するペプチドの存在により、CagA N末側及びCagA C末側フラグメント間の結合が抑制された。これらのことから、CagAはN末側領域における2つの領域が近接した立体構造をとり、この部位に対してアミノ酸977-1077で構成されるC末側領域が分子内相互作用すると推察された。また、CagAのC末側フラグメントを単独発現させた場合に比べ、N末側及びC末側フラグメントを共発現させた胃上皮AGS細胞において、より顕著な細胞形態変化が確認されるとともに形態変化を示した細胞数の増加が見られた。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2007 -2008 
    Author : 東 秀明
     
    (1) 昨年度、ヘリコバクターピロリ菌病原因子CagA全長タンパク質(約140kDa)を異所性に可溶性タンパク質として大量発現させることに成功し、組換えCagAのタンパク質を用いた構造解析結果から、CagAは二つのドメインで構成され、CagA生物活性に重要なC末端領域においては高次構造の存在が示唆される一方で、自由度の高い不規則構造も併せて存在していることを報告した。マイクロインジェクション法を用いて、組換えC末端フラグメントを胃上皮細胞へ直接注入し、組換えタンパク質の生物活性を細胞形態変化誘導活性を指標に検討した。その結果、不規則構造から構成されているC末端フラグメントは、全長CagAタンパク質と同様に細胞形態変化を誘導し生物活性を有していることが示唆された。CagAは生物活性に重要なEPIYA領域周辺を不定形構造として持つ内因性不規則構造タンパク質であることが示唆された。NMR解析を用いたC末端フラグメントとCagAの標的分子であるPAR1分子間相互作用の解析を進め、CagA分子内のPAR1相互作用に関わるアミノ酸残基の同定を進めている。一方、CagA分子のN末端フラグメントを用いた結晶化スクリーニングを行ったところ、複数の沈殿化剤によりN末端フラグメントのタンパク質結晶を得ることに成功した。セレノシステイン置換体等の調製を進め、得られたタンパク質結晶の構造解析を現在進めている。 CagA分子におけるPAR1結合責任部位の詳細を解析した。その結果、CagAは分子内の多量体化モチーフ依存的にPAR1と複合体を形成し、菌株間で見られるモチーフ内のアミノ酸置換に依存してPAR1結合能が大きく変化することが明らかとなった。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2006 -2007 
    Author : 東 秀明
     
    1)全長CagA精製タンパク質を用いた結晶化スクリーニングの結果、一つの試料において再現性良く微小結晶の形成が確認されたが、X線構造解析に適した大型結晶は得られなかった。精製CagAのCDスペクトル解析を行ったところ、二次構造予測解析で得られた高次構造含有量に較べ測定値は低く、また、高次構造形成能をinsilico解析した結果CagA分子内に内因性不規則構造が存在することが示唆された。全長CagA単分子ではX線構造解析に適した結晶形成は困難であると予想された。そこで、CagAの標的分子PAR1との共結晶化を計画し、現在共結晶化スクリーニングを行う段階こ至った。一方プロテアーゼ分解により(CagAのドメイン構造解析を行い、CagAは二つのドメインで構成されている可能性が推察された。EPIYA領域を含むC末端側ドメインのNMR及びCD解析を進めたところ、高次構造の存在が示唆された一方で、不規則構造の存在も併せて観測された。CagAは主要な生物活性に重要なEPIYA領域周辺を不定形構造として持つ内因性不規則構造タンパク質であることが推察された。 2)EPIYA領域に相互作用を示す細胞内タンパク質を質量分析法により同定した。その結果、CagAは細胞極性制御因子PAR1とEPIYA配列近傍の分子構造に依存して複合体を形成し、PAR1キナーゼ活性の抑制を通して細胞極性形成を崩壊させることが明らかとなった。このCagA分子活性に依存し、CagA陽性ピロリ菌は胃上皮組織構造を破壊し胃粘膜病変を発症させていることが推察された。 3)ピロリ菌cagA遺伝子を導入したトランスジェニックマウスを樹立し、個体レベルにおけるCagAの病変発症への関与を検討した。その結果、cagAマウスにおいて胃上皮細胞の過増殖にともなう胃粘膜肥厚ならびに過形成性ポリープ、さらには胃ならびに小腸における癌の発症を認めた。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2005 -2006 
    Author : HIGASHI Hideaki
     
    1.Membrane association of CagA is important for the pathogenic action of CagA in gastric epithelial cells and requires the EPIYA motif but is independent of EPIYA tyrosine phosphorylation. 2.The activated SHP-2 with CagA directly dephosphorylates phospho-tyrosine residues of FAK, which are the activating phosphorylation sites. Accordingly, FAK activation is abolished in cells, and the inhibition of FAK by SHP-2 plays a crucial role in the morphogenetic activity of CagA. 3.By screening of CagA-responsive genes using DNA microarray, we identified NFAT. CagA induced nuclear translocation and transcriptional activity of NFAT depending on calcineurin/PLC_γ pathway. Furthermore, H. pylori VacA toxin counteracted the ability of CagA to activate NFAT. 4.Using a series of EPIYA-repeat variants of CagA that represent reported variations in clinical isolates of H. pylori, we show that EPIYA-repeat polymorphism, 'which is due to differences of both number and order of EPIYA segment, influences the phosphorylation-dependent CagA biological activity. 5.We identified CagA multimerization motif that is composed of 16 amino acid residues and mediates an intermolecular interaction between CagA molecules. The CagA multimerization is necessary to express CagA biological activity 'that relates with cell morphogenetic activity and stimulation of cell motility.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2004 -2005 
    Author : HIGASHI Hideaki
     
    1. Membrane association of CagA is important for the pathogenic action of CagA in gastric epithelial cells and requires the EPIYA motif but is independent of EPIYA tyrosine phosphorylation. 2. The activated SHP-2 with CagA directly dephosphorylates phospho-tyrosine residues of FAK, which are the activating phosphorylation sites. Accordingly, FAK activation is abolished in cells, and the inhibition of FAK by SHP-2 plays a crucial role in the morphogenetic activity of CagA. 3. Induction of cell morphological change with CagA is due to the Ras-independent modification of Erk signals. 4. By screening of CagA-responsive genes using DNA microarray, we identified NFAT. CagA induced nuclear translocation and transcriptional activity of NFAT depending on calcineurin/PLCg pathway. Furthermore, H. pylori VacA toxin counteracted the ability of CagA to activate NFAT. 5. Using a series of EPIYA-repeat variants of CagA that represent reported variations in clinical isolates of H. pylori, we show that EPIYA-repeat polymorphism, which is due to differences of both number and order of EPIYA segment, influences the phosphorylation-dependent CagA biological activity. 6. We identified CagA multimerization motif that is composed of 16 amino acid residues and mediates an intermolecular interaction between CagA molecules. The CagA multimerization is necessary to express CagA biological activity that relates with cell morphogenetic activity and stimulation of cell motility.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2003 -2004 
    Author : HIGASHI Hideaki
     
    1.We established AGS human gastric epithelial cells that conditionally express CagA by employing Tet-On system. The cells expressing CagA exhibited the growth factor-like morphological change as in the case of cagA-positive H.pylori infection. Using the cells, we investigated the CagA effect on Ras/MAPK pathway. As results, we revealed that the morphological change induced by CagA was dependent on MAPK activity but not on Ras activity. On the other hand, we observed that tyrosine phosphorylation levels of tyrosine kinase FAK decreased in CagA-expressing cells. SHP-2 directly dephosphorylates FAK phosphorylation sites which associate with FAK activation, and thereby inactivates FAK. From these results, we clarified that CagA disrupt intracellular signaling by in activation of FAK through SHP-2 activation as well as activation of Ras-independent MAPK cascade. 2.We analyzed changes in gene expression caused by ectopic expression of the cagA gene in gastric epithelial cells using a DNA microarray, and accordingly found multiple cagA-responsive genes. Analyzing putative promoter sequences of the cagA-responsive genes, binding sites for particular transcription factors were significantly over-represented in the promoter regions of CagA-activated genes. Furthermore, we found that CagA is capable of activating gene transcription which is dependent on a specific transcriptional factor in the cells. It is suggested that CagA biological activity causes dysregulation of intracellular gene-expression profile. 3.We constructed cagA transgenic mouse to investigate CagA biological activity and the mechanism of CagA mediated-development of gastric cancer in vivo. We have established several transgenic lines which show expression of cagA gene.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2003 -2004 
    Author : 畠山 昌則, 東 秀明
     
    (1)ヘリコバクター・ピロリ菌CagAと胃発癌機構 胃上皮細胞内に侵入したピロリ菌CagAはチロシンリン酸化を受けた後、SHP-2ならびにCskと特異的に結合しそれらの酵素活性を脱制御することを明らかにした。脱制御されたSHP-2はErk-MAPキナーゼの持続的活性化を引き起こし、細胞の形態変化・運動能亢進・細胞増殖異常を誘導した。SHP-2との結合に関わるEPIYAモチーフ周辺のアミノ酸配列は東アジア諸国で単離される東アジア型CagAならびに欧米諸国で単離される欧米型CagAとの間で著しく異なる。この分子多型を反映し、東アジア型CagAは欧米型CagAに比べて有意に強いSHP-2結合活性ならびに細胞形態変化誘導活性を示した。 (2)pRB癌抑制分子ファミリーの機能ならびに制御 pRB, p107およびp130からなるpRBファミリーの細胞増殖抑制活性が、cyclin Dならびにcyclin E依存的なリン酸化により包括的に不活化されることを明らかにした。また、S期におけるDNA損傷依存的細胞周期停止にp107が関与することを見い出した。さらに、新たなpRBファミリーの生物活性としてサイクリンD1誘導を見い出した。 (3)ESXR1の単離と機能解析 新規ヒトpaired-likeホメオ蛋白としてESXR1を単離した。ESXR1は蛋白分解によりホメオドメインを含む45KDaのN末断片ならびに20KDaのC末断片に分断される。C末断片は細胞質においてプロテアソーム依存的なM期サイクリン分解を阻害し、細胞周期をM期に停止させる。一方、N末断片は核内に移行後、転写抑制因子として機能することが明らかとなった。このESXR1-N末断片の標的遺伝子としてK-ras遺伝子を同定した。ESXR1-N末断片はK-ras遺伝子発現を抑えることにより、K-ras変異を有するヒト癌の増殖を特異的かつ強力に抑制した。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2003 -2003 
    Author : 東 秀明
     
    RBファミリータンパク質は、ポケット活性依存的にE2F転写活性を抑制し細胞周期の進行を阻止する。その増殖抑制は、サイクリン依存性キナーゼ(CDK)によるリン酸化で解除される。一方、pRBは自身の機能を不活化するサイクリンD1遺伝子の転写量を増加させることに加えて、機能的なRB遺伝子を持たない細胞ではサイクリンD1の発現量が低下していることが知られている。本研究では、RBファミリー従来の増殖抑制能とは異なる、細胞増殖亢進につながるRBファミリー依存的なサイクリンD1発現誘導機構の解明を目標とした。機能的なRB遺伝子を持たない細胞において、RBファミリーp107及びp130を異所性発現させることで、pRBと同様にサイクリンD1の発現量が増加することを見出した。さらに、RBファミリーによるサイクリンD1転写活性化に関与する転写因子の同定を試みたところ、NFκB依存的な転写を介して転写活性化が引き起こされていた。一方、pRBにおけるサイクリンD1発現誘導に関わる責任領域の同定を行ったところ、pRBのポケットAおよびB領域、またはC端領域それぞれが個別にサイクリンD1発現誘導能をもつことが明らかとなった。また、癌細胞由来のE2F結合能を失った変異型pRBによってもサイクリンD1の誘導が認められた。以上のことよりRBファミリーは、ポケット活性依存的な増殖抑制能とは区別されるサイクリンD1発現誘導活性を有しており、RB遺伝子ポケット領域の変異は増殖抑制活性の不活化に止まらず、細胞の異常増殖をさらに亢進させる可能性が示唆された。また、RBの機能が遺伝子欠失またはアミノ酸置換という異なる様式で不活性化された場合、その細胞の増殖抑制能に与える影響は大きく異なる可能性が示唆された。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2003 -2003 
    Author : 東 秀明
     
    Helicobacter pylori CagAタンパク質は菌体内で合成後、感染後宿主細胞内に注入され分子内に存在する複数のチロシン残基がリン酸化を受ける。CagAの細胞内標的分子であるSHP-2は、Rasの活性化に関わる分子として考えられている。そこで、CagAにより修飾されるSHP-2介在性シグナル伝達系をより詳細に検討するため、Tet-On systemを用い野生型CagAおよびリン酸化を受けるチロシン残基をアラニン残基に置換したリン酸化耐性CagAの発現制御が可能なAGSヒト胃上皮細胞株を樹立した。野生型CagAを誘導発現させた細胞株では、CagA陽性H.pylori感染時に観察される特徴的な細胞形態変化hummingbird表現型を示した。hummingbird細胞をタイムラプスビデオで経時的な観察を行ったところ、hummingbird表現型を示した細胞は細胞運動性が亢進していることが明らかとなった。次に、CagAがチロシンリン酸化依存的にRas/MAPK系に与える影響を検討した。その結果、CagAの細胞形態変化誘導能は、優性陰性型Ras分子の異所性発現では全く影響を受けず、またRasの活性調節因子であるGrb2の優性陰性型分子を発現させた細胞においても同様の結果であった。一方、MAPKK阻害剤により細胞内Erk活性化を阻害したところ、CagAによる細胞形態変化誘導が抑制された。また、SHP-2特異的siRNAを用いてSHP-2の発現を抑制した細胞においてもCagA依存的な細胞形態変化誘導が阻害された。これらのことより、CagAはSHP-2の活性化とRas非依存的なMAPKカスケードの活性化を介して、細胞形態変化の誘導のみならず細胞運動能を亢進していることが明らかとなった。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2002 -2002 
    Author : 畠山 昌則, 東 秀明
     
    ヘリコバクター・ピロリ菌由来cagA遺伝子を胃上皮細胞に一過性導入・発現させる系を樹立した。発現されたCagAタンパク質は細胞内で細胞膜近傍に局在し、Srcファミリーキナーゼによりチロシンリン酸化を受けた。CagAはチロシンリン酸化依存的にSHP-2チロシンホスファターゼのSH2ドメインと特異的に結合し、この結合を介してSHP-2のホスファターゼ活性を増強した。また、CagA-SHP-2相互作用の結果、胃上皮細胞には細胞質の著しい進展で特徴付けられる形態的変化(hummingbird表現型)が誘導された。CagAのチロシンリン酸化に関わるEPIYAモチーフは単離される菌株ごとにその数ならびに周辺アミノ酸組成が変動する。欧米諸国で単離されるピロリ菌由来CagAのSHP-2結合活性ならびにhummingbird表現型誘導活性はEPIYAモチーフの数に比例した。一方、胃癌の多発する日本、韓国など東アジア諸国で単離されるピロリ菌CagAは、欧米型CagAと周辺アミノ酸を異にするEPIYAモチーフを有し、この東アジア特異的EPIYAモチーフは欧米型EPIYAモチーフに比べはるかに強いSHP-2結合能ならびにhummingbird表現型誘導活性を示した。CagAとチロシンリン酸化特異的に結合する第二の細胞性蛋白としてCskを同定した。CskはCagAとの結合により活性化され、Srcを抑制することによりCagAのリン酸化レベルを低下させた。CagAによるSHP-2の構成的活性化は胃上皮細胞のapoptosisを誘導することから、CagA-Csk相互作用はCagAの細胞毒性を現弱させるフィードバック制御に関与するものと推察された。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2002 -2002 
    Author : 畠山 昌則, 東 健, 東 秀明
     
    cagA陽性ヘリコバクター・ピロリ菌(ピロリ菌)は高度の萎縮性胃炎を惹起し、胃癌との強い関連が示されている。CagA遺伝子産物であるCagAタンパク質はピロリ菌の保有する注射針様のIV型注入装置を介して菌体内から胃上皮細胞に直接注入され、さらに細胞内でSrcファミリーキナーゼによりチロシンリン酸化を受ける。ピロリ菌由来cagA遺伝子を胃上皮細胞内に導入・異所性発現させることにより、CagAがHGFなど増殖因子刺激時と同様の胃上皮細胞形態変化を引き起こすことを見い出した。一方、CagAのチロシンリン酸化部位となるEPIYAモチーフを構成するチロシン残基をアラニンに置換したリン酸化抵抗性CagAは全く増殖因子様活性を示さず、またこのCagA活性はSrcインヒビターであるPP2添加により著しく抑制された。すなわち、CagAはチロシンリン酸化により活性化されるものと結論ずけられた。次に、リン酸化CagAと特異的に相互作用する胃上皮細胞内分子を検索し、SH2ドメインを有するチロシンフォスファターゼSHP-2を同定した。リン酸化されたCagAはSHP-2のSH2ドメインにチロシンリン酸化部位特異的に結合し、その結果SHP-2のフォスファターゼ活性は著しく増強された。CagAとSHP-2の細胞内複合体形成は高度にstoichiometricであり、SHP-2が過剰な状態では細胞内に発現されたCagAの80%以上がSHP-2と結合した。胃上皮細胞に対するCagAの増殖因子様活性は、細胞内でのCagA-SHP-2相互作用を阻害することにより抑制された。一方、細胞膜直下でSHP-2を構成的に活性化させることにより、CagA発現と同様の細胞形態変化が誘導された。CagAによる細胞増殖シグナル伝達系ならびに細胞運動制御系の持続的脱制御は、cagA陽性ピロリ菌の慢性感染を基盤とする胃上皮細胞の多段階発癌プロセスに重要な役割を担うことが推察される。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2001 -2002 
    Author : 東 秀明
     
    Helicobacter pylori CagAタンパク質は菌体内で合成後、感染後宿主細胞内に注入され分子内に存在する複数のチロシン残基がリン酸化を受ける。申請者は細胞内におけるCagA分子の機能ならびにCagA分子が宿主細胞に与える影響を検証し、CagAタンパク質固有の性質を解明することを目的として研究を遂行した。その結果、胃上皮細胞にCagAを異所性発現させる系を用いCagA細胞内標的分子としてチロシンホスファターゼSHP-2を同定した。CagA-SHP-2複合体の形成にはCagAのチロシン残基のリン酸化が必須であることを示し、加えてCagA-SHP-2複合体の形成により、SHP-2のフォスファターゼ活性は著しく増強していることを明らかにした。また遺伝子導入細胞は、CagA陽性H. pylori感染時に観察される細胞形態変化(hummingbird phenotype)を、SHP-2活性化依存的に示すことを見い出した。そのような細胞形態の変化は増殖因子で細胞を刺激した時にも出現することから、細胞内へのCagAタンパク質の侵入はSHP-2との相互作用を通して増殖刺激と同様の影響を細胞にもたらしていることが明らかとなった。また、胃癌の高い発症率を示す東アジアで単離されたH. pylori株が保有するCagAが、他の地域で単離されたH. pylori株由来のCagAと構造的に異なり、SHP-21に対して強い親和性を示すことを明らかにした。そして、この事象が東アジア型CagAのみに保存される一つのアミノ酸残基に起因することを明らかにした。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2001 -2001 
    Author : 畠山 昌則, 東 秀明
     
    本研究は、増殖・分化・形態といったある特定の細胞形質発現を担う遺伝子群を(1)細胞への効率良いcDNA発現ライブラリーの導入;(2)目的とする細胞形質を獲得した細胞の選択・選別;(3)部位特異的DNAリコンビナーゼによるcDNAの染色体からの切り出し・回収;(4)回収されたcDNAの再導入による細胞の再形質転換;から構成される過程を繰り返し、目的の形質発現に関わる遺伝子群を包括的に単離するシステムを樹立することを目的とする。本研究では既に、酵母由来のKw DNAリコンビナーゼを異所性に誘導発現するIL-3依存性細胞株BaF/Kwを樹立するとともに、3'LTR内にKwリコンビナーゼの認識配列を持つMarXレトロウイルスベクターを用いcDNA発現ライブラリーを作成している。本年度は、MarX-lacZレトロウイルスをBaF/Kwに感染させた後、Kwリコンビナーゼを誘導発現させることにより染色体DNAからのlacZ遺伝子切り出しに成功した。また、レトロウイルスcDNA発現ライブラリーをBaF/Kw細胞に感染後IL-3非存在下に培養し、自律的増殖能を獲得した細胞プールを複数樹立した。Kwリコンビナーゼの誘導発現により、自律増殖能を獲得した細胞プールからのレトロウイルスベクター由来cDNA回収を進めている。一方、本システムをより一般化するため、HIV由来TATトランスダクションドメインを融合したKwリコンビナーゼを作成した。この融合蛋白がTATドメイン依存的に細胞膜ならびに核膜を自由に通過する能力を有することを確認した。組み換えTAT-Kw融合蛋白を,Kwリコンビナーゼ認識配列を有するテスト遺伝子に作用させたところ、TAT-Kwは機能的リコンビナーゼとして機能した。現在、TAT-Kwによる染色体からの遺伝子切り出しのための至適条件を検討している。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2000 -2001 
    Author : 畠山 昌則, 東 秀明
     
    ゲノム情報が急速に蓄積されつつある現在、その情報が本来有する生物活性の迅速な同定ならびにゲノム多型情報に対応する生物活性定量法の開発は重要な意義をもつ。本研究は、特定の細胞形質発現をつかさどる遺伝子群を (1)細胞への効率良いcDNA発現ライブラリーの導入 (2)目的とする細胞形質を獲得した細胞の選択・選別 (3)部位特異的リコンビナーゼによるcDNAの染色体からの切り出し・回収 (4)回収されたcDNAの再導入による細胞の再形質転換 から構成される過程を繰り返すことにより、迅速かつ包括的に濃縮・単離する系を樹立することを目的とした。 まず、テトラサイクリンならびにIPTGの二重制御を受ける誘導発現プロモーター(TcIPプロモーター)の下流に酵母由来の部位特異的リコンビナーゼであるKwリコンビナーゼ遺伝子を接続し、インターロイキン3(IL-3)依存性リンパ系細胞株BaF3に安定的に導入した。得られたトランスフェクタントから、IPTG添加によりKwリコンビナーゼを誘導発現する安定変異細胞株BaF/Kwを樹立した。次に、BaF3細胞株から調整したpolyA(+)RNAを用いてcDNAライブラリーを作製し、レトロウイルスベクター(MarX)に組み込んだ。MarXベクターは3′LTR内にKwリコンビナーゼの認識配列が挿入されており、プロウイルスとして宿主ゲノム内に組み込まれたcDNAはKwリコンビナーゼによりゲノムから選択的に切り出される。同ベクターにBaF3由来cDNAを挿入し、約130万個の独立cDNAから構成される発現ライブラリーを作製した。今後は、BaF/Kw細胞へのウイルス感染によるcDNAライブラリーの導入ならびにIL-3非依存性細胞選択の後、リコンビナーゼ発現依存的にIL-3非依存性増殖能が喪失するcDNAの選択を行う。
  • Regulation of cell cycle

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