bluuebunny/crossref_metadata_2025 huggingface dataset:
An Application of Microplate Hybridization Assay for the Confirmation and Probe Typing of “Norwalk‐Like Viruses”
Authors: Fukuda Shinji , Takao Shinichi , Shimazu Yukie , Miyazaki Kazuo
Published: November 2013

“Norwalk‐like virus” (NLV) genomes are generally detected by using reverse transcription‐PCR and confirmed by blot hybridization and nucleotide sequencing because of their fastidious nature. In the present study, the confirmation and typing of NLV genomes were carried out using a streptavidin‐biotin binding technique and microplate hybridization assay with digoxigenin labeled probes. Eight probe typing sets (G1A, G1B, G2A, G2B, G2C, G2D, G2E, and G2F) formatted from 6 newly designed probes and 8 probes reported elsewhere were used for hybridization. The correlation between probe typing and nucleotide sequencing was found and our 8 probe sets were useful for the typing of NLVs.
Saccharomyces cerevisiae‐ and Candida albicans‐Derived Mannan Induced Production of Tumor Necrosis Factor Alpha by Human Monocytes in a CD14‐ and Toll‐Like Receptor 4‐Dependent Manner
Authors: Tada Hiroyuki , Nemoto Eiji , Shimauchi Hidetoshi , Watanabe Toshihiko , Mikami Takeshi , Matsumoto Tatsuji , Ohno Naohito , Tamura Hiroshi , Shibata Ken‐ichiro , Akashi Sachiko , Miyake Kensuke , Sugawara Shunji , Takada Haruhiko
Published: November 2013

The cytokine‐inducing activities of fungal polysaccharides were examined in human monocytes in culture, with special reference to CD14 and Toll‐like receptors (TLRs). Tumor necrosis factor alpha (TNF‐α) production by monocytes was markedly induced in a dose‐dependent manner upon stimulation with cell walls from Candida albicans and mannan from Saccharomyces cerevisiae and C. albicans, although relatively high concentrations (10 to 100 μg/ml) of stimulants were required for activation as compared with the reference lipopolysaccharide (LPS) (1 to 10 ng/ml). The yeast form C. albicans and its mannan and cell wall fractions exhibited higher TNF‐α production than respective preparations from the hyphal form. Only slight TNF‐α production was induced by the S. cerevisiae glucan. The TNF‐α production triggered by reference LPS and purified fungal mannans required the presence of LPS‐binding protein (LBP), and these responses were inhibited by anti‐CD14 and anti‐TLR4 antibodies, but not by anti‐TLR2 antibody. In contrast to the activity of LPS, the activity of purified S. cerevisiae mannan was not inhibited by polymyxin B. These findings suggested that the mannan‐LBP complex is recognized by CD14 on monocytes and that signaling through TLR4 leads to the production of proinflammatory cytokines in a manner similar to that induced by LPS.
Immune Responses against Allogeneic and Syngeneic Tumors in Aged C57BL/6 Mice
Authors: Win Sanda , Uenaka Akiko , Nakayama Eiichi
Published: November 2013

Aged C57BL/6 (B6) mice could reject allogeneic BALB/c RL male 1 tumor as efficiently as young B6 mice. However, in vitro analysis showed impaired generation of cytotoxic T cell response in aged B6 mice against allogeneic tumor. The reaction could be augmented by the addition of recombinant interleukin‐2 (rIL‐2). Enzyme‐linked immunospots (ELISPOT) produced by CD8+ T cells purified from spleen cells showed no reduction in aged mice. The findings suggested that the number of CD8+ T cells capable of reacting against allogeneic H‐2 antigens was similar in young and aged B6 mice. Low cytotoxic T lymphocyte (CTL) responsiveness in aged B6 mice appeared to have resulted from low responsiveness of CD4+ T cells producing IL‐2. Although CTL generation was apparently impaired, strong multiple antigenicity of allogeneic tumor evoked a rejection response in aged B6 mice. On the other hand, no rejection response was observed against syngeneic EL4 tumor in aged B6 mice even after depletion of CD4CD25+ immunoregulatory cells. Depletion of CD4+CD25+ cells caused rejection of EL4 tumor in young B6 mice. The findings suggested that aged B6 mice were incapable of inducing effector cells against weak tumor antigens. Only marginal CTL response and small number of ELISPOTs were generated in young but not aged B6 mice against EL4. Addition of rIL‐2 to the culture augmented EL4 killing and ELISPOTs in spleen cells from young and aged B6 mice.
A Novel Method Using Micropig Stratum Corneum In Vitro for the Evaluation of Anti‐Trichophyton mentagrophytes Activity
Authors: Nakashima Takuji , Nozawa Akira , Majima Toshiro
Published: November 2013

Antifungal susceptibility testing under conditions close to clinical status is expected to provide more helpful information than that obtained by a conventional microdilution method. For this purpose, we developed a novel method to evaluate anti‐Trichophyton mentagrophytes activity of antifungal agents in vitro by using disks of micropig stratum corneum epidermis (SCE). Basal agar medium containing K2HPO4, MgSO4, CaCl2 and three kinds of antibiotics. Bifonazole (BFZ), lanoconazole (LCZ) or terbinafine (TBF) was added to the basal agar medium to give serially doubling dilutions ranging from 0.0006 to 10 μg/ml. Five‐hundred‐μl portions of the agar media thus prepared were solidified in wells of flat‐bottomed plates. SCE disks (6 mm in diameter) were placed on surfaces of the agar medium and 104 conidia of T. mentagrophytes were inoculated on each SCE disk There was very good correlation between the initial concentration of the antifungal agents added to the basal agar medium (μg/ml) and the concentration of the agents impregnated into the SCE disks (μg/g) (r2>0.99). The minimum inhibitory concentration (MIC) values of BFZ, LCZ and TBF were respectively 26‐, 10‐ and 78‐times higher than those measured by the standard microdilution method. From the correlation between the concentration of the agents in the basal medium and that in the SCE disks, the above MIC values corresponded to the concentrations in SCE disks (μg/g), 832.95 for BFZ, 1.42 for LCZ and 8.87 for TBF. This novel method of antidermatophytic susceptibility testing using SCE would be useful as an in vitro screening of proper antimycotics for topical treatment of dermatophytosis.
A Limulus Amoebocyte Lysate Activating Activity (LAL Activity) That Lacks Biological Activities of Endotoxin Found in Biological Products
Authors: Ochiai Masaki , Tamura Hiroshi , Yamamoto Akihiko , Aizawa Maki , Kataoka Michiyo , Toyoizumi Hiromi , Horiuchi Yoshinobu
Published: November 2013

Pyrogenic substances in influenza HA (IHA) vaccine have been controlled by the pyrogen test or the mouse body weight decreasing toxicity (BWD) test. We examined the possibility of replacing the animal tests with the endotoxin test. Commercial IHA vaccines were found to show considerable levels of LAL activity ranging from 0.2 to 160 EU/ml. However, a batch of the vaccine having even 100 EU/ml of LAL activity showed neither pyrogenicity in rabbits nor tumor necrosis factor alpha (TNF‐α) induction in RAW264.7 cells. The LAL activity of IHA vaccine was abolished by a monoclonal antibody that recognizes LPS‐binding epitope of LAL factor C. The activity of IHA vaccine showed different physicochemical properties from those of LAL activity of endotoxin. LAL activity of endotoxin is known to be sensitive to polymyxin B treatment and was found to be resistant to polyoxyethylene 10 cetyl ether (Brij56) treatment. On the contrary, the LAL activity of IHA vaccine was shown to be resistant to polymyxin B but sensitive to Brij56 treatment. The difference in sensitivity of the two LAL activities to polymyxin B and Brij56 might suggest the possibility of their discriminative measurements.
Genetic Background Determines Sensitivity to the Inhibitory Function of NO on T Cell Proliferation and the Amounts of NO Production Mediated through IFN‐γ
Authors: Ozaki Akemi , Fukushima Atsuki , Fukata Kazuyo , Sakamoto Shuji , Taniguchi Taketoshi , Hashiguch Masaaki , Ueno Hisayuki
Published: November 2013

Nitric oxide (NO) inhibits T cell proliferation. We demonstrate that the action of NO on T cell proliferation is different for Lewis and Brown Norway (BN) rats. Splenocytes from Lewis rats consistently showed higher proliferation against concanavalin A than splenocytes from BN rats did. In contrast, NO production was higher in BN rats than in Lewis rats. A depletion of adherent cells increased proliferation in BN rats to a level similar to that in Lewis rats. Thus NO produced by adherent splenocytes could be considered to inhibit proliferation. The addition of NG‐monomethyl‐l‐arginine, a potent inhibitor of NO production, increased proliferation in Lewis rats, but much less so in BN rats. Similar results were obtained by the addition of anti‐interferon (IFN)‐γ. It is surprising that, low doses of sodium nitroprusside, an NO donor, increased proliferation in BN rats but not in Lewis rats. To investigate the mechanism of differential NO production between the two strains, splenocytes were stimulated with IFN‐γ. The early signaling event evaluated by the phosphorylation of Stat‐1 was similar in both strains, whereas inducible NO synthase (iNOS) mRNA expression seemed more sustained in BN rats. Thus the differential production of NO might be related to the differential transcriptional regulation of iNOS. Altogether, genetic background might be involved in sensitivity to the inhibitory function of NO for T cell proliferation and NO production.
Viral Load in Epstein‐Barr Virus‐Associated Hemophagocytic Syndrome
Authors: Kimura Hiroshi , Hoshino Yo , Hara Shinya , Nishikawa Kazuo , Sako Masahiro , Hirayama Masahiro , Komada Yoshihiro , Morishima Tsuneo
Published: November 2013

The viral load in peripheral blood from patients with Epstein‐Barr virus (EBV)‐associated hemophagocytic syndrome was measured by real‐time quantitative PCR and compared with that in infectious mononucleosis. Patients with EBV‐associated hemophagocytic syndrome generally had larger viral burdens, although it was difficult to differentiate EBV‐associated hemophagocytic syndrome from infectious mononucleosis simply by viral load. The difference in viral load seemed to be clearer in peripheral blood mononuclear cells than in plasma.
A Structurally Variant Form of the 2B4 Antigen Is Expressed on the Cell Surface of Mouse Mast Cells
Authors: Kubota Koichi
Published: November 2013

2B4 (CD244) is a 66‐kDa CD2 family protein expressed on natural killer (NK) cells. Mouse NK cells express two isoforms of 2B4, termed 2B4L and 2B4S, whose molecular masses are 42 kDa and 36 kDa, respectively. In this study, we biochemically characterize the 2B4 antigen that was newly found on mouse bone marrow‐derived mast cells (BMMC). Anti‐2B4 mAb immunoprecipitated glycoproteins with a molecular mass of 60 kDa from BMMC. Removal of N‐linked sugars from the antigen by N‐glycosidase F treatment yielded two protein backbones of 35 kDa and 25 kDa, indicating that BMMC express the 2B4S isoform, but not 2B4L. Nucleotide sequence analyses confirmed that BMMC transcribe 2B4S mRNA. The preferential expression of the 2B4S isoform and the detection of an additional 25‐kDa glycoprotein on BMMC indicate that differences in the structure of 2B4 antigen exist between BMMC and NK cells.
Development of an Immunofluorescence Method for the Detection of Antibodies to Ebola Virus Subtype Reston by the Use of Recombinant Nucleoprotein‐Expressing HeLa Cells
Authors: Ikegami Tetsuro , Saijo Masayuki , Niikura Masahiro , Miranda Mary E.G. , Calaor Alan B. , Hernandez Marvin , Manalo Daria L. , Kurane Ichiro , Yoshikawa Yasuhiro , Morikawa Shigeru
Published: November 2013

An indirect immunofluorescent assay (IFA) to detect Ebola virus subtype Reston (EBO‐R) antibodies was developed by the use of a HeLa cell line stably expressing EBO‐R nucleoprotein (NP). This IFA has a high specificity for the detection of EBO‐R IgG antibodies in both hyperimmune rabbit sera and monkey sera collected during an EBO‐R outbreak in the Philippines in 1996. Furthermore, this IFA showed a higher sensitivity for the detection of EBO‐R antibodies than did the IFA using HeLa cells expressing the NP of Ebola virus subtype Zaire. These results suggest that this new IFA is useful for seroepidemiological studies of EBO‐R infection among monkeys.
Antimicrobial Activity and Stability to Proteolysis of Small Linear Cationic Peptides with D‐Amino Acid Substitutions
Authors: Hamamoto Kumiko , Kida Yutaka , Zhang Ye , Shimizu Takashi , Kuwano Koichi
Published: November 2013

Antimicrobial peptides contribute to innate host defense against a number of bacteria and fungal pathogens. Some of antimicrobial synthetic peptides were systemically administered in vivo; however, effective protection has so far not been obtained because the effective dose of peptides in vivo seems to be very high, often close to the toxic level against the host. Alternatively, peptides administered in vivo may be degraded by certain proteases present in serum. In this study, D‐amino acids were substituted for the L‐amino acids of antimicrobial peptides to circumvent these problems. Initially a peptide (L‐peptide) rich in five arginine residues and consisting of an 11‐amino acid peptide (residues 32–42) of human granulysin was synthesized. Subsequently, the L‐amino acids of the 11‐amino acid peptide were replaced partially (D‐peptide) or wholly (AD‐peptide) with D‐amino acids. Activity and stability to proteolysis, in particular, in the serum of antimicrobial peptides with D‐amino acid substitutions were examined. Peptides with D‐amino acid substitutions were found to lyse bacteria as efficiently as their all‐L‐amino acid parent, L‐peptide. In addition, the peptide composed of L‐amino acids was susceptible to trypsin, whereas peptides containing D‐amino acid substitutions were highly stable to trypsin treatment. Similarly, the peptide consisting of L‐amino acids alone was also susceptible to fetal calf serum (FCS), however, protease inhibitors restored the lowered antimicrobial activity of the FCS‐incubated peptide. Thus, D‐amino acid substitutions can make antimicrobial peptides resistant to proteolysis, suggesting that the antimicrobial peptides consisting of D‐amino acids are potential candidates for clinical therapeutic use.
Molecular Fingerprinting of Isoniazid‐Resistant Mycobacterium tuberculosis Isolates from Chest Diseases Hospital in Kuwait
Authors: Mokaddas Eiman , Ahmad Suhail , Abal Adnan T.
Published: November 2013

Touchdown double‐repetitive‐element‐PCR (DRE‐PCR) was carried out for typing 38 consecutive isoniazid‐resistant Mycobacterium tuberculosis strains isolated at Chest Diseases Hospital, Kuwait, during 1998–2000. The polymorphism at codon 463 in the katG gene was also determined and correlated with genotypic relationships among the isolates. The isolates exhibited 21 distinct patterns in DRE‐PCR. Nearly half of the isolates (18 of 38) exhibited unique patterns. Majority of isolates (16 of 20) yielding multiple DNA fragments in DRE‐PCR were unique strains while most of the isolates (16 of 18) yielding a single DNA fragment in DRE‐PCR clustered together. The prevalence of L463 in the katG gene was much higher in isolates from Middle Eastern (mostly Kuwaiti) patients than is reported for this ethnic group. The data indicate the possibility of some strains of South Asian/Southeast Asian origin spreading among local populations.
Monoclonal Antibody to Shiga Toxin 1, Which Blocks Receptor Binding and Neutralizes Cytotoxicity
Authors: Nakao Hiroshi , Kataoka Chiwa , Kiyokawa Nobutaka , Fujimoto Junichiro , Yamasaki Shinji , Takeda Tae
Published: November 2013

A monoclonal antibody, 5–5B, which neutralizes Shiga toxin 1 (Stx1) cytotoxicity of Escherichia coli, was constructed. An epitope analysis indicated that Asn55 in Stx1 B subunit was an important residue. This result and our previous results using an anti‐Stx2 monoclonal antibody indicate that the region around the cysteine residue of the disulfide bond might be important for the neutralization of Stx cytotoxicity, making it a potential vaccination candidate.
Double‐Stranded nef RNA Interferes with Human Immunodeficiency Virus Type 1 Replication
Authors: Yamamoto Takuya , Omoto Shinya , Mizuguchi Masaaki , Mizukami Hajime , Okuyama Harumi , Okada Noriko , Saksena Nitin K. , Brisibe Ebiamadon A. , Otake Kaori , Fujii Yoichi R.
Published: November 2013

RNA interference (RNAi) has been reported to be post‐transcriptional gene silencing (PTGS) by approximately 500 nucleotide‐(nt)‐long double‐stranded (ds) RNA that specifically targets homologous sequences of messenger RNA. In this report, we describe inhibition of HIV‐1 transcription by synthetic dsRNAs constructed with mutated nef genes (nef dsRNAs) derived from long‐term non‐progressors (LTNPs) using cotransfection of the target gene‐expressing plasmid and dsRNA. The effects of nef dsRNAs were examined with luciferase (Luc) reporter which is combined with the HIV‐1 (SF2) LTR in persistently HIV‐1‐infected T cell and macrophage cell lines. At 48 hr, a defective nef dsRNA (556 nt) suppressed Luc activity more potently than did SF2 full‐length nef dsRNA (744 nt), suggesting that approximately 500 nt‐long nef dsRNA could interfere with the HIV‐1 transcription.
Human Immunodeficiency Virus Type 1 Neutralization by a Single Molecule of V3‐Targeted Antibody
Authors: Harada Shinji
Published: November 2013

Human immunodeficiency virus type‐1 (HIV‐1) infection generally provokes antibody responses to the viral envelope glycoprotein. Two major regions of gp120, the third variable (V3) domain and the CD4‐binding site, have been identified as neutralization targets. The precise mechanism of HIV‐1 neutralization by antibodies against the V3 domain is still unknown. It is shown that by kinetic neutralization studies, one molecule of V3‐targeted monoclonal antibody (0.5P) is enough to neutralize one virion. This antibody, which neutralized more than 99% of the virus, inhibited the binding of the virus to cells by 42%. HIV‐1 pseudotyped with G glycoprotein from vesicular stomatitis virus was also neutralized by 0.5β, suggesting that the antibody did not inhibit the viral attachment but caused some alteration in the envelope. These results indicate that the antibody plays an additional role on steric change of the envelope involved in inhibition of viral entry.
Distribution of Aeromonas hydrophila Serogroups in Different Clinical Samples and the Development of Polyclonal Antibodies for Rapid Identification of the Genus Aeromonas by Direct Agglutination
Authors: Korbsrisate Sunee , Dumnin Suwat , Chawengkirttikul Rungrawan , Gherunpong Vanchit , Eampokalap Boonchuay , Gongviseisoog Charles , Janyapoon Kritsana , Lertpocasombat Kancharee , Shimada Toshio
Published: November 2013

We characterized a collection of 256 Aeromonas hydrophila strains isolated from blood, discharge and stool for their serogroup designation. Of these, 2.3% were untypable and 15.2% were rough strains. Among the typable strains, about 50% comprised serogroups O:11, O:16, O:18, O:34 and O:83. To develop rapid differentiation of Aeromonas from other oxidase‐positive bacteria, antisera against Aeromonas were produced to establish a direct, genus‐specific, agglutination test. It was found that among 105 isolates of Aeromonas, 102 showed positive results with the agglutination test. The calculated sensitivity and specificity were 97.1% and 90.7%, respectively.
Antimicrobial Susceptibilities of Shiga Toxin‐Producing Escherichia coli Isolates from Pigs with Edema Disease in Japan
Authors: Uemura Ryoko , Sueyoshi Masuo , Nagayoshi Miki , Nagatomo Hiroshi
Published: November 2013

Fifty‐seven Shiga toxin‐producing Escherichia coli (STEC) strains isolated from pigs with edema disease (ED) from 1997 to 2001 in Japan were examined for antimicrobial susceptibilities. The susceptibilities were compared with those of E. coli ATCC 23546 isolated from pig with ED in the 1950's. Consequently, the isolated STECs showed high susceptibility to peptides and bicozamycin in a way similar to the reference strain. On the other hand, the STECs showed low susceptibility to beta‐lactams, tetracyclines, novobiocin, fosfomycin, trimethoprim, and old quinolones. It became clear that the susceptibilities of the isolated STECs had diminished in regard to antimicrobials.
Efficient Adeno‐Associated Virus‐Mediated Gene Expression in Human Placenta‐Derived Mesenchymal Cells
Authors: Zhang Xiaohong , Nakaoka Takashi , Nishishita Toshihide , Watanabe Nobukazu , Igura Koichi , Shinomiya Ken‐ichi , Takahashi Tsuneo A. , Yamashita Naohide
Published: November 2013

Mesenchymal cells from various sources are pluripotent and are attractive sources for cell transplantation. In this study, we analyzed recombinant adeno‐associated virus (rAAV)‐mediated gene expression in human placenta‐derived mesenchymal cells (hPDMCs), which reside in placental villi. After transduction of AV‐CAG‐EGFP, a rAAV expressing enhanced green fluorescence protein (EGFP), hPDMCs showed much higher level of EGFP expression than human umbilical vein endothelial cells or rat aortic smooth muscle cells. The number of EGFP‐positive hPDMCs infected by AV‐CAG‐EGFP alone did not increase significantly by coinfection of adenovirus, which enhanced expression level of the rAAV vector. Moreover, flow cytometric analysis showed discrete positive fraction of EGFP‐expressing hPDMCs, which is about 15–20% of the cells infected with AV‐CAG‐EGFP. Therefore, some cell population in hPDMCs might be highly susceptible to rAAV‐mediated gene transduction. In addition, stable EGFP expressions were observed in about 1% of hPDMCs infected with AV‐CAG‐EGFP at 4 weeks post‐infection. Collectively, hPDMCs have characters favorable for rAAV‐mediated gene expression.
Characterization of the F Gene of Contemporary Mumps Virus Strains Isolated in Japan
Authors: Uchida Kazue , Shinohara Michiyo , Shimada Shin‐ichi , Segawa Yukari , Kimura Kazuhiro , Hoshino Yoji
Published: November 2013

Mumps virus (MuV) strains isolated in Saitama Prefecture, Japan, from 1997 to 2001, were examined by analyzing the SH and the F gene nucleotide sequences. The results of the SH gene analysis showed that only genotype G was found in 2001 as well as in 2000, and that genotype J, which we proposed as a new genotype in a previous study, was from a different lineage than the genotype J described by Tecle et al. (J. Gen. Virol. 82, 2675–2680). We therefore, propose to rename the genotype as K to avoid confusion. Then, the F gene of genotypes G, H, and K strains were analyzed together with previously reported strains in this study. The results of phylogenetic analysis of the F gene nucleotide sequences showed that these strains formed a cluster as described by the SH gene analysis. Alignment of the F amino acid sequences showed that the F protein was well conserved among strains of different genotypes with a few amino acid differences. These results provide better information for the characterization of contemporary MuV strains in Japan.
High Molecular Weight Vimentin Complex Is Formed after Proteolytic Digestion of Vimentin by Caspase‐3: Detection by Sera of Patients with Interstitial Pneumonia
Authors: Fujita Jiro , Bandoh Shuji , Yang Yu , Wu Fei , Ohtsuki Yuji , Yoshinouchi Takeo , Ishida Toshihiko
Published: November 2013

In a previous study, we demonstrated anti‐vimentin antibodies in sera of patients with interstitial pneumonia. We hypothesized that antibodies in sera might detect vimentin fragments formed during the process of apoptosis. To prove this, recombinant human vimentin was digested by recombinant human caspase‐3 or caspase‐8. Then, Western blotting using several commercially available antibodies against human vimentin or patients' sera which had anti‐vimentin autoantibodies, was performed. As a result, after recombinant human vimentin was digested by caspase‐3 or caspase‐8, several vimentin fragments were formed and detected by 2 kinds of monoclonal anti‐vimentin antibodies (clone 3B4 and clone V9) as well as by polyclonal sheep anti‐human vimentin antibody. It was demonstrated that high molecular weight vimentin was formed after the digestion of vimentin by caspase‐3, which was only detected by patients' sera. The high molecular weight vimentin was not formed after digestion of vimentin by caspase‐8. Our present results show that high molecular weight vimentin was formed after the digestion of vimentin by caspase‐3. In addition, it is suggested that this high molecular weight vimentin acted as an autoantigen to form anti‐vimentin autoantibody in vivo.
Role of Microfilaments and Microtubules in the Invasion of INT‐407 Cells by Campylobacter jejuni
Authors: Biswas Debabrata , Itoh Kikuji , Sasakawa Chihiro
Published: November 2013

The internalization mechanisms triggered by Campylobacter jejuni were studied by invasion assays conducted with different inhibitors that act on the cytoskeleton structure of eukaryotic cells. The depolymerization of microfilaments by cytochalasin‐D and that of microtubules by colchicines and nocodazole inhibited the uptake of C. jejuni into INT‐407 cells in a dose‐dependent manner. The inhibitory effect of microfilament depolymerization on C. jejuni internalization was more pronounced than that of microtubule depolymerization. By immunofluorescence microscopic observations, it was demonstrated that both microfilaments and microtubules were localized in INT‐407 cells after C. jejuni infection. These data suggest that the internalization mechanism triggered by C. jejuni is associated with the combined effect of microfilaments and microtubules of host cells.
DNA Base Alignment and Taxonomic Study of Genus Malassezia Based upon Partial Sequences of Mitochondrial Large Subunit Ribosomal RNA Gene
Authors: Yamada Yohko , Makimura Koichi , Ueda Kumiko , Nishiyama Yayoi , Uchida Katsuhisa , Yamaguchi Hideyo , Osumi Masako
Published: November 2013

The sequences of the large subunit of mitochondrial ribosomal RNA (LsmtrRNA) gene of Malassezia species were analysed. The sequences of the seven species of Malassezia are well separated in each species. Therefore the LsmtrRNA gene is thought to be one of the gene targets for species identification in the genus Malassezia. The dendrogram obtained from this gene supports the previous study of Malassezia species based upon the chromosomal genes. This is the first report of taxonomic analysis of Malassezia species based upon the mitochondrial gene.
Possible Relationship of PFGE Patterns of Moraxella catarrhalis between Hospital‐ and Community‐Acquired Respiratory Infections in a Community Hospital
Authors: Masaki Hironori , Asoh Norichika , Kawazoe Kaneo , Watanabe Kiwao , Onizuka Tomoko , Shimogama Seiji , Yamaryo Takeshi , Watanabe Hiroshi , Oishi Kazunori , Nagatake Tsuyoshi
Published: November 2013

We describe a prospective study of molecular analysis of Moraxella catarrhalis isolated from a community hospital. Our study was designed to investigate the possible relationship of pulsed‐field gel electrophoresis (PFGE) patterns of M. catarrhalis between hospital‐ and community‐acquired respiratory infections. A nosocomial outbreak of M. catarrhalis was observed between September 2000 and September 2001. During the study period, 40 strains of M. catarrhalis were isolated from a total of 32 patients with respiratory infections (26 strains from 18 inpatients, and 14 strains from 14 outpatients). We compared the PFGE patterns in 40 strains of M. catarrhalis isolated from the respiratory tract of the study patients. The genomic types of M. catarrhalis were classified into three PFGE patterns (A, B, and C). Interestingly, the nosocomial outbreak of M. catarrhalis included two patterns (A and B). Of the three patterns, two patterns (A and B) were found in both inpatients and outpatients. More interestingly, two subtypes of pattern B (B1 and B4) were simultaneously found in both inpatients and outpatients. Our results indicated that PFGE with SmaI chromosomal digestion is a suitable technique to establish the inter‐strain genetic relatedness of M. catarrhalis, and suggested that the outbreak of M. catarrhalis occasionally included miscellaneous PFGE patterns. The results also showed that PFGE patterns of M. catarrhalis isolates were similar between hospital‐ and community‐acquired respiratory infections. Analysis of the subtypes suggested that there might be some association between hospital‐ and community‐acquired respiratory infections caused by M. catarrhalis.
Hepatitis C Virus Core Protein Selectively Inhibits Synthesis and Accumulation of p21/Waf1 and Certain Nuclear Proteins
Authors: Oka Kiyomasa , Nagano‐Fujii Motoko , Yoshida Isao , Hidajat Rachmat , Deng Lin , Akutsu Masato , Hotta Hak
Published: November 2013

By using a vaccinia virus‐T7 expression system, possible effects of hepatitis C virus (HCV) core protein on synthesis and accumulation of host cellular proteins transiently expressed in cultured cells were analyzed. Immunoblot and immunofluorescence analyses revealed that synthesis and accumulation of certain nuclear proteins, such as p21/Waf1, p53, proliferating cell nuclear antigen and c‐Fos, were strongly inhibited by HCV core protein. On the other hand, synthesis and accumulation of cytoplasmic proteins, such as 2′‐5′‐oligoadenylate synthetase (2′‐5′‐OAS), RNase L and MEK1, were barely affected by HCV core protein. Northern blot analysis showed that the degrees of mRNA expression for those proteins did not differ between HCV core protein‐expressing cells and the control, suggesting that the inhibition occurred at the post‐transcription level. Pulse‐labeling analysis suggested that HCV core protein strongly inhibited synthesis of p21/Waf1 at the translation level. Once being accumulated in the nucleus, p21/Waf1 stability was not significantly affected by HCV core protein. Mutants of HCV core protein C‐terminally deleted by 18 or 41 amino acids (aa), which were localized almost exclusively in the nucleus, lost their ability to inhibit synthesis/accumulation of p21/Waf1 whereas another mutant C‐terminally deleted by 8 aa still maintained the same properties (subcellular localization and the inhibitory effect) as the full‐length HCV core protein of 191 aa. Taken together, our present results suggest that expression of HCV core protein in the cytoplasm selectively inhibits synthesis of p21/Waf1 and some other nuclear proteins at the translation level.
Discrimination of West Nile Virus and Japanese Encephalitis Virus Strains Using RT‐PCR RFLP Analysis
Authors: Shirato Kazuya , Mizutani Tetsuya , Kariwa Hiroaki , Takashima Ikuo
Published: November 2013

West Nile (WN) virus is a mosquito‐borne flavivirus that induces lethal encephalitis in humans and horses. Since an outbreak of WN encephalitis in humans and horses occurred in New York City in late August 1999, the possibility exists that WN virus will invade regions that have close links with the United States, such as Japan. We developed a genetic diagnostic method that discriminates between strains of WN virus and Japanese encephalitis (JE) virus. The method involves RT‐PCR restriction fragment length polymorphism (RFLP) analysis with a RT‐PCR primer set, a nested PCR primer set, and a restriction enzyme. We detected WN and JE viruses in experimentally infected animal brain, spleen, and serum samples. Our method is useful in distinguishing WN viruses from the endemic background of JE viruses, and in discriminating the highly virulent WN strain, which was isolated in New York in 1999, from other WN virus strains.
The First Isolation of Ustilaginomycetous Anamorphic Yeasts, Pseudozyma Species, from Patients' Blood and a Description of Two New Species: P. parantarctica and P. thailandica
Authors: Sugita Takashi , Takashima Masako , Poonwan Natteewan , Mekha Nanthawan , Malaithao Kaewjai , Thungmuthasawat Benjaporn , Prasarn Soem , Luangsook Pakeenee , Kudo Toshiaki
Published: November 2013

The genus Pseudozyma is ustilaginomycetous anamorphic yeasts, and are mainly isolated from plants. We isolated three Pseudozyma strains from the blood of patients in Thailand. While one isolate was identified as P. antarctica by rDNA sequence analysis, the other two were considered to be new species and were named P. parantarctica and P. thailandica. The three isolates proved to be resistant to 5‐flucytosine, and P. thailandica was also resistant to fluconazole and itraconazole. As far as we know, this is the first isolation of Pseudozyma strains from humans. The two new species are described.
Coenzyme Q1‐Catalyzed Luminol Chemiluminescent Assay for Rapid Antimicrobial Susceptibility Testing of Mycobacterium bovis
Authors: Yamashoji Shiro
Published: November 2013

Coenzyme Q1 is herein proposed as the best catalyst among coenzymes Q and vitamins K for quinone‐catalyzed luminol chemiluminescent assays applied to rapid determination of viability or rapid antimicrobial susceptibility tests of Mycobacterium bovis. Luminol chemiluminescence intensity (LCI) was determined 10 min after the incubation of M. bovis with coenzyme Q1, and was proportional to CFU (colony‐forming unit)/ml in the range of 9,000 to 2,250,000. LCI depended on the the production of the superoxide anion (O2−) rather than H2O2 during a 10‐min incubation of M. bovis with coenzyme Q1, as superoxide dismutase reduced LCI more effectively than catalase. The minimal inhibitory concentrations (MICs) of 10 kinds of antituberculous agents estimated on the basis of decrease in LCI after one or two days' cultivation were in good agreement with MICs determined by turbidity analysis, which requires upwards of 1 week to complete.
The Role of Glucan‐Binding Proteins in the Cariogenicity of Streptococcus mutans
Authors: Matsumura Mieko , Izumi Tomohiro , Matsumoto Michiyo , Tsuji Masato , Fujiwara Taku , Ooshima Takashi
Published: November 2013

Streptococcus mutans produces glucan‐binding proteins (Gbps), which appear to contribute to the virulence of S. mutans. GbpA and GbpC genes were inactivated by the insertion of antibiotic‐resistant genes into each gbp gene of S. mutans MT8148 to generate Gbp‐defective mutants. Sucrose dependent adherences of the GbpA‐ and GbpC‐defective mutants were found to be significantly lower than those of their parent strains MT8148. Caries inducing activity of the mutants in rats was significantly lower than that of strain MT8148R (streptomycin‐resistant strain of MT8148). These results suggest that GbpA and GbpC participate in cellular adherence to tooth surfaces and contribute to the cariogenicity of S. mutans.
Correlation between the Avidity Maturation of Anti‐HSP70 IgG Autoantibody and Recombination Activating Gene Expressions in Peripheral Lymphoid Tissues of Toxoplasma gondii‐Infected Mice
Authors: Chen Mei , Aosai Fumie , Mun Hye‐Seong , Norose Kazumi , Piao Lian Xun , Yano Akihiko
Published: November 2013

The avidity maturation of anti‐TgHSP70 IgG antibody produced by B‐2 cells of BALB/c mice (a resistant strain) and that of anti‐mHSP70 IgG autoantibody produced by B‐1 cells of C57BL/6 mice (B6; a susceptible strain) was observed after Toxoplasma gondii infection. Recombination‐activating genes (RAGs) were predominantly expressed in B‐1 cells from peritoneal exudate cells (PECs) of T. gondii‐infected B6 mice, while RAGs were expressed in B‐2 cells from PECs of BALB/c mice. These results suggest that the involvement of RAG gene activations in the peripheral lymphoid tissues in the avidity maturation of anti‐TgHSP70 IgG antibody and anti‐mHSP70 IgG autoantibody in T. gondii‐infected mice.
Effects of Melanin upon Susceptibility of Cryptococcus to Antifungals
Authors: Ikeda Reiko , Sugita Takashi , Jacobson Eric S. , Shinoda Takako
Published: November 2013

Melanin is a recognized virulence factor in Cryptococcus neoformans; several pathogenetic mechanisms have been suggested. We studied melanin as an antifungal resistance factor. The growth of laccase‐active strains of C. neoformans and C. albidus in L‐DOPA resulted in the production of black pigment. The formal minimal inhibitory concentrations (MICs) of amphotericin B and fluconazole were not changed by melanization. However, when we examined those wells which contained inhibited cells, we found live cells only in wells containing melanized C. neoformans. In contrast, melanization did not protect C. albidus from killing by amphotericin B. In an amphotericin B time‐kill study of C. neoformans, significantly more melanized cells than non‐melanized survived for the first few hours. Fluorescence microscopy and flow cytometry analyses showed that fewer melanized cells were stained with the fluorescent dye MitoRed. Incubation of MitoRed (the model) or amphotericin B with melanin extracted from C. neoformans decreased the free concentrations of these substances. Fluconazole, in contrast, was not removed from solution by melanin. This suggests that neoformans cryptococcal melanin deposited amphotericin B in the cell wall binds, reducing its effective concentrations.
Cell‐Mediated Fas‐Based Lysis of Dendritic Cells Which Are Apparently Resistant to Anti‐Fas Antibody
Authors: Yokota Aya , Oikawa Atsuhiko , Matsuda Chieko , Shinohara Nobukata , Eshima Koji
Published: November 2013

In this report, the controversy concerning the sensitivity of dendritic cells (DCs) to Fas‐dependent induction of apoptosis was examined using murine DCs. Although DCs could not be lysed when exposed to an anti‐Fas antibody, Jo2, the observed resistance turned out to reflect their lack of the expression of FcγR necessary for crosslinking the antibody, rather than their intrinsic resistance. Thus, at least a fraction of DCs was sensitive to Jo2 in the presence of FcγR‐expressing by‐standers. Consistently, a significant fraction of DCs was sensitive to Fas‐dependent lysis mediated by T cells including the antigen‐specific killing by CD4+ T cells. Both immature (class II MHClow) and mature (class II MHChigh) DCs were sensitive to the Fas‐based induction of apoptosis.
Probable Laboratory Contamination of Clinical Specimens with Leptospira meyeri
Authors: Koizumi Nobuo , Kawabata Hiroki , Watanabe Haruo
Published: November 2013

Leptospira meyeri were isolated from the blood or the pleural effusion cultures of four patients at a commercial clinical laboratory. All isolates were identical in their 16S rDNA sequences and NotI or SfiI restriction profiles of the chromosome DNA on pulsed‐field gel electrophoresis. However, intraperitoneal inoculation of the isolate (NIID1) failed to kill hamsters and none of the patients' sera reacted with L. meyeri strains isolated, indicating leptospiral contamination occurred during laboratory investigation.
3‐Methylcholanthrene Activates Human Immunodeficiency Virus Type 1 Replication via Aryl Hydrocarbon Receptor
Authors: Ohata Hirokazu , Tetsuka Toshifumi , Hayashi Hidetoshi , Onozaki Kikuo , Okamoto Takashi
Published: November 2013

We found that 3‐methylcholanthrene (3‐MC) could induce the reactivation of human immunodeficiency virus type 1 (HIV‐1) replication in OM 10.1 cell, promyelocytic cell line latently infected with HIV‐1. Transient luciferase expression experiments have revealed no particular transcription factors that are responsible for the effect of 3‐MC in inducing HIV‐1 gene expression as HIV‐1 LTR mutants lacking various upstream transcriptional activators similarly responded to 3‐MC. In addition, there was no effect of 3‐MC on the DNA binding activity of nuclear factor‐kappa B (NF‐κB) that was previously reported to be crucial for the effect of 2, 3, 7, 8‐tetrachlorodibenzo‐p‐dioxin (TCDD), a chemical homologue of 3‐MC. However, overexpression of wild type aryl hydrocarbon receptor (AhR), a nuclear receptor of polycyclic aromatic hydrocarbons (PAHs) such as 3‐MC, augmented the effect of 3‐MC in the induction of gene expression from HIV‐1 LTR. Moreover, a dominant negative mutant of AhR dramatically reduced the 3‐MC‐mediated activation of HIV‐1 LTR. These findings suggest that 3‐MC stimulates HIV‐1 transcription by interacting with general transcription factors. Our observations indicate that chronic exposure of the HIV‐1 infected individuals to PAHs may be contributable to the clinical development of acquired immunodeficiency syndrome (AIDS) among the individuals infected with HIV.
Mapping of the Low pH‐Sensitive Conformational Epitope of Rabies Virus Glycoprotein Recognized by a Monoclonal Antibody #1‐30‐44
Authors: Kankanamge Pushpa Jenette , Irie Takashi , Mannen Kazuaki , Tochikura Tadafumi S. , Kawai Akihiko
Published: November 2013

Monoclonal antibody (mAb) #1‐30‐44 recognized an acid‐sensitive conformational epitope of rabies virus glycoprotein (G). The antigenicity of G protein exposed on the cell surface was lost when the infected cells were exposed to pH 5.8. By comparing the deduced amino acid sequence of G protein between the HEP‐Flury strain and the epitope‐negative CVS strain as well as the mAb‐resistant escape mutants, two distant sites that contained Lys‐202 and Asn‐336 were shown to be involved in the epitope formation. Lys‐202 is located in the so‐called neurotoxin‐like sequence, while Asn‐336 is included in antigenic site III and is very near the amino acid at position 333, which is known to affect greatly the neuropathogenicity of rabies virus when changed. Consistent with this finding, antigenicity of a neurovirulent revertant of the HEP‐Flury strain, in which Gln‐333 of G protein was replaced by Arg, was also affected as shown by its greatly decreased reactivity with mAb #1‐30‐44 compared to that of the original avirulent HEP virus. Based on these results, we hypothesize that the neurotoxin‐like domain and some amino acids in antigenic site III come into contact with each other to form a conformational epitope for mAb #1‐30‐44, and such a configuration would be lost when exposed to acidic conditions to perform a certain low pH‐dependent function of G protein.
Distribution of Human Rotaviruses, Especially G9 Strains, in Japan from 1996 to 2000
Authors: Zhou Yumei , Li Lei , Okitsu Shoko , Maneekarn Niwat , Ushijima Hiroshi
Published: November 2013

A 4‐year (1996–2000) survey of rotavirus infection involving 2,218 diarrheal fecal specimens of children collected from five regions of Japan was conducted. A total of 642 (28.9%) specimens were found to be rotavirus positive. A changed prevalence pattern of rotavirus G serotype was found with an increase of G9 and G2 and a decrease of G1, although G1 remained the prevailing serotype. Serotype G9 was unexpectedly determined to be the prevailing serotype in Sapporo (62.5%) and Tokyo (52.9%) in 1998–1999, and in Saga (78.4%) in 1999–2000. G9 strains isolated from 1998–1999 belonged to the P[8]‐NSP4‐Wa‐group with long RNA pattern, while, G9 strains isolated from 1999–2000 belonged to three groups, the P[8]‐NSP4‐Wa‐group with long RNA pattern, the P[4]‐NSP4‐KUN‐group with short RNA pattern and a mixed‐type group (P[4]/P[8]‐NSP4‐KUN/Wa‐group with long RNA pattern). Both sequence and immunological analysis of VP7 revealed that the G9 strains from 1999–2000 were much more closely related to the G9 strains isolated worldwide in the 1990s, including G9 strains found in Thailand in 1997. However, the G9 strains from 1998–1999 were distinct from these and more closely related to the G9 prototype strains F45, AU32 and WI61 discovered in Japan and the US in the 1980s. Thus the G9 strains isolated in 1998–1999 had progenitors common to the G9 prototype strains, while the strains isolated in 1999–2000 did not directly evolve from them but were related to global G9 strains that have emerged in recent years. These data supported our previous report that G9 rotavirus might exist as two or more subtypes with diverse RNA patterns, P‐genotype and NSP4 genogroup combinations (Y.M. Zhou et al., J. Med. Virol. 65: 619–628, 2001) and suggested that G9 rotavirus prevalent in Japan during two successive years belonged to different subtypes. The nucleotide sequences presented in this paper were submitted to DDBJ, EMBL and GenBank nucleotide sequence databases. The accession numbers are: 00‐Ad2863VP7 (AB091746), 00‐OS2986VP7 (AB091747), 00‐SG2509VP7 (AB091748), 00‐SG2518VP7 (AB091749), 00‐SG2541 (AB091750), 00‐SG2864 (AB091751), 00‐SP2737VP7 (AB091752), 99‐SP1542VP7 (AB091753), 99‐SP1904VP7 (AB091754), 99‐TK2082VP7 (AB091755) and 99‐TK2091VP7 (AB091756).
The Size of cagA Based on Repeat Sequence Has the Responsibility of the Location of Helicobacter pylori in the Gastric Mucus and the Degree of Gastric Mucosal Inflammation
Authors: Karita Mikio , Matsumoto Satoru , Kamei Toshiaki
Published: November 2013

The aim of this study was to examine whether there is a relationship between cagA size of Japanese Helicobacter pylori strains and the location of these strains in the mucous layer, the degree of gastric inflammation and acid survival. Upper gastrointestinal endoscopies were done to 144 patients with dyspeptic symptom with informed consent, sera, biopsy specimens and H. pylori strains were obtained, and gastric histology and susceptibility to pH 3 of the strains were evaluated. To determine cagA size of Japanese strains using PCR, cagA of strain CPY3401 was sequenced. 74 H. pylori samples (72 cagA +) were obtained from the body and 56 samples (56 cagA +) obtained from the antrum. cagA size of 72 H. pylori strains from the body was mainly classified into 3 groups (short (48), middle (8), long (9), and others (7)) by PCR and all of that of 56 strains from the antrum except 2 was short. The size of cagA of isolated strains from the body is associated with enhanced gastritis, acid survival, and the location in the mucus. The long size cagA of which strain is acid sensitive, may be a strong selective pressure on strain that colonizes close to the host, which enhanced gastritis.
Genetic Characterization of Rabies Field Isolates from Thailand
Authors: Susetya Heru , Sugiyama Makoto , Inagaki Akio , Ito Naoto , Oraveerakul Kanisak , Traiwanatham Nimit , Minamoto Nobuyuki
Published: November 2013

We sequenced 512 nucleotides in two variable regions of the N gene of 23 rabies isolates from the northeastern part of Thailand by direct sequencing of PCR‐amplified products. The sequencing data revealed two new lineages in these rabies isolates. Based on the results of this study together with the findings of our earlier study, the rabies isolates in Thailand were divided into two genogroups, designated as T1 and T2, which were predominantly localized in the northern and northeastern areas, respectively. Each of these two genogroups consisted of four lineages. There was a correlation between these eight lineages and the geographical origins of the isolates. Two lineages belonging to the T2 genogroup from the northeastern area of Thailand were newly identified in this study. The isolates in both genogroups were also prevalent in the central area of Thailand. Each lineage in the T1 and T2 genogroups was found independently in dogs in the upper and lower southern areas of Thailand, respectively. These genetic data and the historical background suggest that rabies viruses belonging to the T2 genogroups were prevalent many years ago in the central and northeastern areas of Thailand and were later transferred to the lower southern parts of Thailand.
Gamma Interferon‐Induced Nitric Oxide Production in Mouse CD5+ B1‐Like Cell Line and Its Association with Apoptotic Cell Death
Authors: Koide Naoki , Sugiyama Tsuyoshi , Mu Mya Mya , Mori Isamu , Yoshida Tomoaki , Hamano Teruaki , Yokochi Takashi
Published: November 2013

The in vitro effect of gamma interferon (IFN‐γ) on nitric oxide (NO) production in a mouse CD5+ B1‐like cell line, TH2.52, was studied. The TH2.52 cell line is the hybridoma line between mouse B lymphoma line and mouse splenic B cells and expresses a series of B1 markers. IFN‐γ induced a marked NO production in TH2.52 cells through the expression of an inducible type of NO synthase (iNOS). IFN‐γ‐induced NO production was triggered by the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT) pathway since it was inhibited by AG490, a JAK2 inhibitor. The growth of TH2.52 cells significantly was inhibited in the presence of IFN‐γ. A significant number of cells underwent apoptotic cell death, accompanied by the DNA fragmentation, annexin V binding, and caspase 3 activation. N(G)‐monomethyl‐L‐arginine, an iNOS inhibitor, prevented IFN‐γ‐induced cell death. Therefore, IFN‐γ‐induced NO production was possible in causing cell death in TH2.52 cells. Further, IFN‐γ‐induced NO production and cell death significantly were prevented by interleukin‐4, a representative Th2 cytokine. The immunological significance of IFN‐γ‐induced NO production in a mouse B1‐like cell line is discussed.