Nobuyuki Hamada

Kurume University, Куруме, Fukuoka, Japan

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Publications (38)80.58 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The influenza RNP, which is formed from PB1, PB2, PA, NP subunits, and vRNA, is autonomously replicated and transcribed in the infected cell. The simplest method to inhibit RNP activity is to impair the formation of the RNP. Thereupon we confirmed whether the peptides/fragments mimicking one of RNP components can interfere with their formation. During the process of this inhibitory study we found interesting suppression of protein expression of the RNP components by the N-terminal fragment of PA subunit. Especially, we found two residues (D108 and K134) on the fragment that were critical for the suppression. Furthermore, we determined the combination of three amino acids (P28, M86 and E100) on the fragment that are important for the strong suppression, and identified the minimum essential region (residues from 1 to 188) of the PA subunit that allowed its suppression. Our findings indicate that the N-terminal fragment of PA subunit may become one of candidates for an effective inhibitor of influenza RNP activity. Copyright © 2015 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
    Journal of Infection and Chemotherapy 01/2015; 21(4). DOI:10.1016/j.jiac.2014.12.007 · 1.38 Impact Factor
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    ABSTRACT: A patient, an adult Japanese traveler who had just returned from Thailand, had developed dengue hemorrhagic fever (DHF). A primary infection of dengue virus (DENV) was confirmed, in particular, DENV serotype 2 (DENV-2) via the detection of the virus genome, a significant increase in its specific neutralizing antibody and the isolation of DENV-2. DHF is often observed following a secondary infection from another serotype of dengue virus, particularly in children, but this case was a primary infection of DENV. Japan is a non-endemic country of dengue disease. Instead, only Japanese encephalitis (JE) is known to be an endemic flavivirus family. In this study, IgG antibody against Japanese encephalitis virus (JEV) was detected. JEV belongs to the family of dengue virus and prevails in Japan, particularly in Kyushu. Among many risk factors for the occurrence of DHF, a plausible candidate could be a cross-reactive antibody-dependent enhancement (ADE) mechanism by JEV antibody. This indicates that most Japanese travelers, who live in non-endemic areas of dengue, particularly in Kyushu, should pay attention to the occurrence of DHF.
    Tropical Medicine and Health 01/2015; 43(2). DOI:10.2149/tmh.2014-34
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    ABSTRACT: Influenza A virus has a RNA-dependent RNA polymerase (RdRp) that is composed of three subunits (PB1, PB2 and PA subunit), which assemble with nucleoproteins (NP) and a viral RNA (vRNA) to form a RNP complex in the host nucleus. Recently, we demonstrated that the combination of influenza ribonucleoprotein (RNP) components is important for both its assembly and activity. Therefore, we questioned whether the inhibition of the RNP combination via an incompatible component in the RNP complex could become a methodology for an anti-influenza drug. We found that a H5N1 PB2 subunit efficiently inhibits H1N1 RNP assembly and activity. Moreover, we determined the domains and important amino acids on the N-terminus of the PB2 subunit that are required for a strong inhibitory effect. The NP binding site of the PB2 subunit is important for the inhibition of RNP activity by another strain. A plaque assay also confirmed that a fragment of the PB2 subunit could inhibit viral replication. Our results suggest that the N-terminal fragment of a PB2 subunit becomes an inhibitor that targets influenza RNP activity that is different from that targeted by current drugs such as M2 and NA inhibitors.
    PLoS ONE 12/2014; 9(12):e114502. DOI:10.1371/journal.pone.0114502 · 3.53 Impact Factor
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    Dataset: RMCR83
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    ABSTRACT: A 64-year-old male patient was admitted with respiratory failure, although chest X-rays revealed only mild bronchiolitis. Streptococcus pneumonia, which usually presents as massive lobular pneumonia, was isolated from sputum, however, pan-pathogen screening using a next-generation sequencer also detected human metapneumovirus genome fragments.
    03/2014; 12. DOI:10.1016/j.rmcr.2013.12.007
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    ABSTRACT: The influenza virus RNA polymerase, composed of the PB1, PB2, and PA subunits, has a potential role in influencing genetic reassortment. Recent studies on the reassortment of human H3N2 strains suggest that the co-incorporation of PB2 and PA from the same H3N2 strain appears to be important for efficient virus replication; however, the underlying mechanism remains unclear. Here, we reconstituted reassortant ribonucleoprotein (RNP) complexes and demonstrated that the RNP activity was severely impaired when the PA subunit of H3N2 strain A/NT/60/1968 (NT PA) was introduced into H1N1 or H5N1 polymerase. The NT PA did not affect the correct assembly of the polymerase trimeric complex, but it significantly reduced replication-initiation activity when provided with a vRNA promoter and severely impaired the accumulation of RNP, which led to the loss of RNP activity. Mutational analysis demonstrated that PA residues184N and 383N were the major determinants of the inhibitory effect of NT PA and 184N/383N sequences were unique to human H3N2 strains. Significantly, NT PB2 specifically relieved the inhibitory effect of NT PA, and the PB2 residue 627K played a key role. Our results suggest that PB2 from the same H3N2 strain might be required for overcoming the inhibitory effect of H3N2 PA in the genetic reassortment of influenza virus.
    Journal of General Virology 08/2013; DOI:10.1099/vir.0.053959-0 · 3.53 Impact Factor
  • International Journal of Antimicrobial Agents 06/2013; 42:S133. DOI:10.1016/S0924-8579(13)70529-6 · 4.26 Impact Factor
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    ABSTRACT: SUMMARY Using a newly developed rapid test, an outbreak of human metapneumovirus (HMPV) infection in a long-term care facility was detected within only 2 days after the onset of symptoms in a putative index case. The outbreak was almost under control within 8 days mainly by zoning patients, with the exception of two cases of HMPV that were diagnosed 16 and 17 days after the onset of the outbreak. According to an immunological diagnosis as well as the rapid test, it was eventually proven that 18 patients had HMPV infections. We suspected that even asymptomatic residents, who had not been completely separated from the facility population, were a source of infection. That suggested that all asymptomatic residents should be tested and that the separation of the infected patients should be absolute, if an outbreak of HMPV infection is suspected in such a facility.
    Epidemiology and Infection 05/2013; DOI:10.1017/S0950268813001246 · 2.49 Impact Factor
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    ABSTRACT: We performed a community-based laboratory diagnosis of pandemic influenza A (H1N1) 2009 with the RT-PCR technique using originally constructed primers. Of 30 patients who were suspected to be infected with the influenza virus from May 2009 until January 2010, the A (H1N1) 2009 virus was detected in 13 patients (43.3%). Three cases were immunologically confirmed to be infected with the A (H1N1) 2009 virus, because significant increases in the HI titer were observed in the convalescent sera. We also measured the antibody titers to the A (H1N1) 2009 virus in 13 healthy individuals with the HI assay using originally isolated virus. In most cases, the HI antibody titers were less than 10, except two cases with titers of 40 and 20. Our inspection system organized in the early phase of the A (H1N1) 2009 pandemic contributed to disease control in an outpatient clinic and a hospital in a small city. The process which we used to construct the system would be a good reference for a treatment protocol in the case of a future literal pandemic.
    Kansenshogaku zasshi. The Journal of the Japanese Association for Infectious Diseases 05/2013; 87(3):368-74. DOI:10.11150/kansenshogakuzasshi.87.368
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    ABSTRACT: The oseltamivir-resistant pandemic influenza virus A (2009 H1N1) with H275Y mutation in neuraminidase (NA) has been sporadically reported, and its wide spread remains a potential threat. Here we detected the uneven distribution of H275Y mutant virus in a patient who received a 21-day long-term administration of oseltamivir. Intrahost variation of the virus showed that the H275Y mutant virus was the predominant population in both nasopharynx and right lung, whereas the oseltamivir-sensitive virus comprised half the population in the left lung. By constructing minimum spanning trees, it is proposed that the H275Y mutant might be generated primarily in the nasopharynx, then spread to the right and left lungs.
    Journal of Infection and Chemotherapy 06/2012; 18(6). DOI:10.1007/s10156-012-0429-0 · 1.38 Impact Factor
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    ABSTRACT: Genetic reassortment plays a critical role in the generation of pandemic strains of influenza virus. The influenza virus RNA polymerase, composed of PB1, PB2 and PA subunits, has been suggested to influence the efficiency of genetic reassortment. However, the role of the RNA polymerase in the genetic reassortment is not well understood. Here, we reconstituted reassortant ribonucleoprotein (RNP) complexes, and demonstrated that the PB2 subunit of A/HongKong/156/1997 (H5N1) [HK PB2] dramatically reduced the synthesis of mRNA, cRNA and vRNA when introduced into the polymerase of other influenza strains of H1N1 or H3N2. The HK PB2 had no significant effect on the assembly of the polymerase trimeric complex, or on promoter binding activity or replication initiation activity in vitro. However, the HK PB2 was found to remarkably impair the accumulation of RNP. This impaired accumulation and activity of RNP was fully restored when four amino acids at position 108, 508, 524 and 627 of the HK PB2 were mutated. Overall, we suggest that the PB2 subunit of influenza polymerase might play an important role for the replication of reassortant ribonucleoprotein complexes.
    PLoS ONE 04/2012; 7(2):e32634. DOI:10.1371/journal.pone.0032634 · 3.53 Impact Factor
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    ABSTRACT: The etiological agents associated with community-acquired pneumonia (CAP) in Thailand have been studied extensively in bacterial pathogens, but not in viral pathogens. To clarify the association of viral pathogens with CAP, we conducted a comprehensive study of viral and bacterial pathogens in patients with CAP. We enrolled 119 hospitalized patients with CAP in Nakornping Hospital, Chiang Mai, Thailand between 2006 and 2008. The severity of pneumonia was classified and the risk factors for death were estimated. Bacterial and fungal pathogens were determined from specimens taken from blood and sputum, and viral pathogens were identified from nasopharyngeal specimens by RT-PCR using primers specific for 7 respiratory viruses. Overall, 29 patients were HIV-infected and 90 patients were non-HIV-infected. The microbial pathogens most commonly isolated among HIV-infected patients were: 4 Klebsiella pneumoniae, 4 Mycobacterium tuberculosis and 3 Haemophilus influenzae. Among non-HIV infected patients, predominant microbial pathogens were: 6 Pseudomonas aeruginosa, 5 Haemophilus influenzae and 4 Klebsiella pneumoniae. As for viral pathogens for CAP, influenza virus was identified from 2 HIV-infected patients and 5 non-HIV infected patients. In addition, human rhinovirus (HRV) and respiratory syncytial virus (RSV) were identified from 2 patients each among non-HIV-infected patients. Our study demonstrates that the most common viral agent was influenza virus (5%), followed by HRV (2%) and RSV (2%) among CAP patients in northern Thailand. The underlying chronic obstructive pulmonary disease (COPD) seems to be correlated with the severity of illness.
    Internal Medicine 01/2011; 50(9):991-8. DOI:10.2169/internalmedicine.50.4738 · 0.97 Impact Factor
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    ABSTRACT: Influenza A virus can infect a variety of different hosts and therefore has to adapt to different host temperatures for its efficient viral replication. Influenza virus codes for an RNA polymerase of 3 subunits: PB1, PB2 and PA. It is well known that the PB2 subunit is involved in temperature sensitivity, such as cold adaptation. On the other hand the role of the PA subunit in thermal sensitivity is still poorly understood. To test which polymerase subunit(s) were involved in thermal stress we reconstituted artificial hybrids of influenza RNA polymerase in ribonucleoprotein (RNP) complexes and measured steady-state levels of mRNA, cRNA and vRNA at different temperatures. The PA subunit was involved in modulating RNP activity under thermal stress. Residue 114 of the PA subunit was an important determinant of this activity. These findings suggested that influenza A virus may acquire an RNA polymerase adapted to different body temperatures of the host by reassortment of the RNA polymerase genes.
    PLoS ONE 12/2010; 5(12):e15140. DOI:10.1371/journal.pone.0015140 · 3.53 Impact Factor
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    ABSTRACT: An outbreak of acute keratoconjunctivitis involving 27 patients occurred in the Department of Ophthalmology, Kurume University Hospital. Adenoviral DNA was detected in four inpatients, one outpatient and one healthcare worker. Sequence-based typing of adenoviral DNA indicated serotype 3 from one inpatient, the rest being serotype 37. At a later stage of the outbreak adenoviral DNA types 37 and/or 3 were also detected from almost all environmental instruments and commonly used eye drops, despite thorough disinfection of the environment and enforcement of various infection control measures. The detection rate of adenoviral DNA in environmental swabs was 81%. A further second disinfection of the environment reduced the detection rate of adenoviral DNA to 38%. The outbreak ceased after closing the ophthalmology ward and outpatient consulting room, accompanied by enhanced cleaning of environmental instruments and the introduction of disposable eye drops for individual patients.
    Journal of Hospital Infection 04/2008; 68(3):262-8. DOI:10.1016/j.jhin.2007.12.012 · 2.78 Impact Factor
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    ABSTRACT: We focused on the relationship between variation in the IRES of hepatitis C virus (HCV) genotype 1b and clinical outcome, since the internal ribosome entry site (IRES) has a comparatively low heterogeneity and it might be easy to find unique substitutions. Patients infected with HCV were selected using strict criteria, and unique mutations in the IRES were extracted by the subtraction of common mutations. We found that most mutations accumulated in domain III (dIII) of IRES in sustained virological responders (SVRs) and non-SVRs before therapy. However, these mutations were exclusively observed in domain II (dII) in non-SVR at 2 weeks after the start of therapy.
    Archives of Virology 02/2008; 153(8):1575-9. DOI:10.1007/s00705-008-0143-5 · 2.28 Impact Factor
  • Journal of Hepatology 01/2008; 48. DOI:10.1016/S0168-8278(08)60854-5 · 10.40 Impact Factor
  • Jun Iwahashi, Nobuyuki Hamada, Hiroshi Watanabe
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    ABSTRACT: Reticulon (RTN) proteins are localized to the endoplasmic reticulum (ER), and are related to intracellular membrane trafficking, apoptosis, inhibiting axonal regeneration, and Alzheimer's disease. The RTN proteins are produced without an N-terminal signal peptide. Their C-terminal domain contains two long hydrophobic segments. We analyzed the ER localization signal of human RTN1-A. Mutant proteins lacking the first (39 residues) or second (36 residues) hydrophobic segment showed ER localization. On the other hand, the mutant lacking both hydrophobic segments was cytosolic. Enhanced green fluorescent protein (EGFP) tagged with the first or second hydrophobic segment of RTN1-A was localized to the ER. These results suggest that each hydrophobic segment determines the ER localization. In addition, EGFP tagged with the truncated form of the first hydrophobic segment exhibited the localization to the Golgi rather than the ER. This suggests that the length of the hydrophobic segment contributes to the ER retention of RTN1-A.
    Biochemical and Biophysical Research Communications 05/2007; 355(2):508-12. DOI:10.1016/j.bbrc.2007.02.001 · 2.28 Impact Factor
  • Journal of the American Geriatrics Society 02/2006; 54(1):177-80. DOI:10.1111/j.1532-5415.2005.00575_10.x · 4.22 Impact Factor
  • J Iwahashi, N Hamada
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    ABSTRACT: Human reticulon family gene 1 (RTN1) is expressed predominantly in neuroendocrine tissues, and produces three proteins termed RTN1-A, RTN1-B, and RTN1-C. Yeast two-hybrid screening indicated that RTN1-A and RTN1-B interacted with AP50, a component of the AP-2 adaptor complex involved in endocytosis. In contrast, RTN1-C did not interact with AP50. Three RTN1 proteins contain the same C-terminal domain. In addition, RTN1-A and RTN1-B share N-terminal 168 amino acid region, suggesting that the 168 amino acid region might play a role in regulating the endocytic process. Although no apparent morphological change of the endocytic organelles was observed, the association of AP50 with the internalized clathrin-coated vesicles was moderately affected when CV-1 cells were directed to express stably RTN1-A or RTN1-B.
    Cellular and molecular biology 02/2003; 49 Online Pub:OL467-71. · 0.69 Impact Factor
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    ABSTRACT: Hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp). Sequences in the 3' termini of both the plus and minus strand of HCV genomic RNA harbor the activity of a replication origin and a transcription promoter. There are unique stem-loop structures in both termini of the viral RNA. We found that the complementary strand of the internal ribosome-binding site (IRES) showed strong template activity in vitro. The complementary strand RNA of the HCV genome works as a template for mRNA and viral genomic RNA. We analyzed the promoter/origin structure of the complementary sequence of IRES and found that the first and second stem-loops worked as negative and positive elements in RNA synthesis, respectively. The complementary strand of the second stem-loop of IRES was an important element also for binding to HCV RdRp.
    Journal of Biological Chemistry 09/2002; 277(32):28700-5. DOI:10.1074/jbc.M201251200 · 4.60 Impact Factor