Jun Iwahashi

MSF, a novel cytoplasmic chaperone which functions in precursor targeting to mitochondria.

Mitochondrial import stimulation factor (MSF) unfolds wheat germ lysate synthesized aggregated mitochondrial precursor proteins and stimulates their mitochondrial import in an ATP dependent manner. Here we analysed the function of MSF mainly by utilizing chemically pure adrenodoxin precursor (pAd). MSF bound to the unfolded pAd and prevented it from losing import competence and also restored the import competence of the aggregated pAd dependent on ATP hydrolysis. The import incompetent aggregated mitochondrial precursors induced the ATPase activity of MSF and the activity was strongly inhibited by isolated mitochondrial outer membrane (OM) but not by trypsin treated outer membrane (tOM). The precursor induced ATPase activity of N‐ethylmaleimide (NEM)‐treated MSF was not inhibited by OM. In this context, the MSF‐precursor complex specifically bound to OM and binding was abolished both by the treatment of OM with trypsin and by the treatment of MSF with NEM. These results show that MSF is a novel cytoplasmic chaperone protein with a mitochondrial precursor‐targeting function.

Influenza virus RNA polymerase PA subunit is a novel serine protease with Ser624 at the active site

Influenza virus RNA polymerase is a multifunctional enzyme that catalyses both transcription and replication of the RNA genome. The function of the influenza virus RNA polymerase PA subunit in viral replication is poorly understood, although the enzyme is known to be required for cRNA → vRNA synthesis. The protease related activity of PA has been discussed ever since protease‐inducing activity was demonstrated in transfection experiments.

Analysis of the Functional Domain of the Rat Liver Mitochondrial Import Receptor Tom20

Tom20 is an outer mitochondrial membrane protein and functions as a component of the import receptor complex for the cytoplasmically synthesized mitochondrial precursor proteins. It consists of the N-terminal membrane-anchor segment, the tetratricopeptide repeat (TPR) motif, a charged amino acids-rich linker segment between the membrane anchor and the TPR motif, and the C-terminal acidic amino acid cluster. To assess the functional significance of these segments in mammalian Tom20, we cloned rat Tom20 and expressed mutant rat Tom20 proteins in Δtom20 yeast cells and examined their ability to complement the defects of respiration-driven growth and mitochondrial protein import. Tom20N69, a mutant consisting of the membrane anchor and the linker segments, was targeted to mitochondria and complemented the growth and import defects as efficiently as wild-type Tom20, whereas a mutant lacking the linker segment did not. In vitro protein import into mitochondria isolated from the complemented yeast cells revealed that the precursor targeted to yeast Tom70 was efficiently imported into the mitochondria via rat Tom20N69. Thus the linker segment is essential for the function of rat Tom20, whereas the TPR motif and the C-terminal acidic amino acids are not.

Caenorhabditis elegans reticulon interacts with RME-1 during embryogenesis

Reticulon (RTN) family proteins are localized in the endoplasmic reticulum (ER). At least four different RTN genes have been identified in mammals, but in most cases, the functions of the encoded proteins except mammalian RTN4-A and RTN4-B are unknown. Each RTN gene produces 1-3 proteins by different promoters and alternative splicing. In Caenorhabditis elegans, there is a single gene (rtn gene) encoding three reticulon proteins, nRTN-A, B, and C. mRNA of nRTN-C is expressed in germ cells and embryos. However, nRTN-C protein is only expressed during embryogenesis and rapidly disappears after hatch. By yeast two-hybrid screening, two clones encoding the same C-terminal region of RME-1, a protein functioning in the endocytic recycling, were isolated. These findings suggest that nRTN-C functions in the endocytic pathway during embryogenesis.

Nosocomial outbreak of epidemic keratoconjunctivitis accompanying environmental contamination with adenoviruses.

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.

Inhibition of influenza virus replication in cultured cells by RNA‐cleaving DNA enzyme

Influenza virus replication has been effectively inhibited by antisense phosphothioate oligonucleotides targeting the AUG initiation codon of PB2 mRNA. We designed RNA‐cleaving DNA enzymes from 10‐23 catalytic motif to target PB2‐AUG initiation codon and measured their RNA‐cleaving activity in vitro. Although the RNA‐cleaving activity was not optimal under physiological conditions, DNA enzymes inhibited viral replication in cultured cells more effectively than antisense phosphothioate oligonucleotides. Our data indicated that DNA enzymes could be useful for the control of viral infection.

Promoter/Origin Structure of the Complementary Strand of Hepatitis C Virus Genome

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.

Isolation of Amantadine-Resistant Influenza A Viruses (H3N2) from Patients following Administration of Amantadine in Japan

ABSTRACT In Japan, the use of amantadine for treatment of influenza A virus infection was not accepted until November 1998, although it was widely used for treatment of Parkinsonism. Since then, we have monitored the emergence of amantadine-resistant viruses and isolated two viruses from patients on long-term treatment with amantadine.

Antibody-Scanning and Epitope-Tagging Methods Molecular Mapping of Proteins Using Antibodies

Because synthetic short peptides bearing critical binding residues, can chemically mimic the folded antigenic determinants on proteins, short synthetic peptides can generate antibodies that react with cognate sequences in intact folded proteins. According to this mimotope theory, we produced site-specific antibodies by immunization with short peptides which overlapped each other and covered the entire protein, and used them for domain mapping of influenza virus RNA polymerase (antibody-scanning method). We also used a tagged-epitope and its monoclonal antibodies for topology mapping of clathrin light chains in clathrin triskelions by electron microscopy. Both methods using specific epitopes in combination with their antibodies enable us to determine the domains of interesting proteins systematically without the need to generate monoclonal antibodies or mutant proteins.

Post-Travel Consultations in a Regional Hub City Hospital, Japan

OBJECTIVE To clarify the characteristics of post-travel consultation services in Japan, particularly in the provinces, we analyzed our post-travel patients in the travel clinic of Kurume University Hospital located in Kurume City (a regional hub City in southwestern Japan). METHODS Sixty post-travel patients visited our clinic between April 2008 and October 2014 and participated in this study: 55 were Japanese and five were foreign. We summarized and compared the characteristics of the patients after dividing the Japanese participants into long-term travelers (>14 days) and short-term travelers (≤14 days). The foreign travelers were described in a separate analysis. RESULTS Of the 55 Japanese travelers, the mean age (± standard deviation) was 37.3 ± 16.3 years, and 36 patients (65%) were men. Southeast Asia was the major destination (30/55, 55%), and business was stated as the major reason for travel (16/55, 29%). Post-exposure rabies prophylaxis (16/55, 29%) was the most common purpose for the consultations. There were 34 participants (62%) who were classified as short-term travelers. Fewer of the short-term travelers stated receiving pre-travel consultations compared with long-term travelers (11% vs. 79%, p=0.0002). The five foreign travelers included one dengue fever patient and two malaria patients. CONCLUSION Most post-travel Japanese patients visited our clinic were short-term travelers who had not received any pre-travel consultation. One of the most common complaints, post-exposure rabies prophylaxis, could have been avoided to some extent by appropriate pre-travel consultations. The results of this study suggest that pre-travel consultations should therefore be encouraged for both long- and short-term travelers.