bluuebunny/crossref_metadata_2025 huggingface dataset:
A Rapid Bioluminescent Enzyme Immunoassay (BLEIA) for the Detection of Shiga Toxin Types 1 and 2
Authors: Yamazaki Makoto , Sato Sachihiro , Gondaira Fumio , Sugiyama Jun‐ichi
Published: November 2013

In recent years, Escherichia coli O157: H7 has emerged as a global public health concern. Among the more important virulence characteristics of this strain is its ability to produce one or more Shiga toxins (Stx). Traditional culture‐based methods for assay of enteric toxins in foods and clinical samples are relatively slow and results can be ambiguous. In this work, we established a toxin‐detection system based on bioluminescent enzyme immunoassay (BLEIA) using a simple and inexpensive device. The system could detect both Shiga toxin types 1 and 2 individually within 150 min with a detection limit for each toxin at 5 pg/ml. In our study of previously characterized Shigatoxigenic and all non‐Shigatoxigenic E. coli and other bacterial species, we found all Shigatoxigenic strains to be positive and non‐Shigatoxigenic E. coli and other bacterial species to be negative. This assay was also used to detect Stxs in milk and supernatant fluids from minced chicken and beef. For clinical stool samples we noted a tendency for the system to give unexpectedly high background level. Our results suggest the feasibility of using BLEIA methodology for the simple, rapid and sensitive detection of toxins from culture supernatant, various foods and clinical samples.
Paramyxovirus Accessory Proteins as Interferon Antagonists
Authors: Gotoh Bin , Komatsu Takayuki , Takeuchi Kenji , Yokoo Junko
Published: November 2013

A new role of the Paramyxovirus accessory proteins has been uncovered. The P gene of the subfamily Paramyxovirinae encodes accessory proteins including the V and/or C protein by means of pseudotemplated nucleotide addition (RNA editing) or by overlapping open reading frame. The Respirovirus (Sendai virus and human parainfluenza virus (hPIV)3) and Rubulavirus (simian virus (SV)5, SV41, mumps virus and hPIV2) circumvent the interferon (IFN) response by inhibiting IFN signaling. The responsible genes were mapped to the C gene for SeV and the V gene for rubulaviruses. On the other hand, wild type measles viruses isolated from clinical specimens suppress production of IFN, although responsible viral factors remain to be identified. Both human and bovine respiratory syncytial viruses (RSVs) counteract the antiviral effect of IFN with inhibiting neither IFN signaling nor IFN production. Bovine RSV NS1 and NS2 proteins cooperatively antagonize the antiviral effect of IFN. Studies on the molecular mechanism by which viruses circumvent the host IFN response will not only illustrate co‐evolution of virus strategies of immune evasion but also provide basic information useful for engineering novel antiviral drugs as well as recombinant live vaccine.
Fungal Flora on Board the Mir‐Space Station, Identification by Morphological Features and Ribosomal DNA Sequences
Authors: Makimura Koichi , Hanazawa Ryo , Takatori Kosuke , Tamura Yoshiko , Fujisaki Ryuichi , Nishiyama Yayoi , Abe Shigeru , Uchida Katsuhisa , Kawamura Yoshiaki , Ezaki Takayuki , Yamaguchi Hideyo
Published: November 2013

This report is on the morphological and molecular biological identification, using 18S‐ and ITS1‐rDNA sequences, of the “space fungi” isolated on board the Russian Mir‐Space Station as the major constituents of the fungal flora. The six fungal strains were isolated from air by using an air sampler or from condensation. Strains were identified as Penicillium chrysogenum, Aspergillus versicolor, or Penicillium sp. by both methods. The species of space fungi were common saprophytic fungi in our living environment, potential pathogens, and allergens. This study concluded that the environment on board the space station Mir allows the growth of potentially pathogenic fungi as true in residential areas on the earth. Therefore, to prevent infection or other health disorders caused by these fungi, easy and reliable methods should be established to survey the fungal flora in a space station.
Prevention of Bacterial Diarrhea by Pasteurization of Drinking Water in Kenya
Authors: Iijima Yoshio , Karama Mohamed , Oundo Joseph O. , Honda Takeshi
Published: November 2013

Diarrheal disease is one of the major causes of morbidity and mortality in developing countries. Drinking water is a primary transmission route of infectious diarrheagenic bacteria in a rural area of Kenya (Microbiol. Immunol. 41: 773–778, 1997). We tried to prevent diarrhea at villages with approximately 1,500 households in Kenya by pasteurizing drinking water. A durable simple thermoindicator which changes color at 70 C was used as an indicator of pasteurization. The number of households in which drinking water was coliform bacteria‐free increased from 10.7% to 43.1% after adoption of a pasteurization practice. Consequently, the incidence of severe diarrhea among people drinking pasteurized water was significantly lower than in people taking raw water (odds ratio = 0.55, P = 0.0016). The reduction ratio of the incidence after pasteurization was nearly equivalent with that after the adoption of a boiling method. Employment of women leaders as fieldworkers and demonstration of bacterial colony disappearance on agar plates by pasteurization also affected reduction of the diarrheal incidence.
Reconstitution and Purification of Cytolethal Distending Toxin of Actinobacillus actinomycetemcomitans
Authors: Saiki Keitarou , Konishi Kiyoshi , Gomi Tomoharu , Nishihara Tatsuji , Yoshikawa Masanosuke
Published: November 2013

Cytolethal distending toxin (CDT) has been found in various pathogenic bacterial species and causes a cell distending and a G2 arrest against eukaryotic cells. All the cdtABC genes, which encode CDT, are known to be required for the CDT activities although the CDT holotoxin structure has not been elucidated. We cloned the cdtABC genes of Actinobacillus actinomycetemcomitans and constructed an Escherichia coli expression system for them. We found that crude extracts from six deletion mutants (ΔcdtA, ΔcdtB, ΔcdtC, ΔcdtBC, ΔcdtAC, and ΔcdtAB) of recombinant E. coli, which showed very weak or no detectable CDT activities, restored the CDT activities when pre‐mixing and pre‐incubation of them were performed in combinations to contain all the CdtA, CdtB, and CdtC proteins. These results indicate that all the Cdt proteins are required for the CDT activities. We also found that the chimera CdtB protein, CdtB‐intein‐CBD (chitin binding domain) like CdtB protein itself assembled with CdtA and CdtC. The reconstituted CDT containing the chimera CdtB protein was specifically extracted by chitin beads and the only CDT portion was isolated from the chitin beads by a cleavage reaction of the intein. The purified reconstituted‐CDT was found to consist of CdtA, CdtB, and CdtC proteins, and showed appreciable CDT activities, indicating that the CDT holotoxin structure is the CdtABC complex. To our knowledge, this is the first report succeeded in complete purification of an active CDT and may offer useful tools for elucidation of the toxic mechanism of CDT.
Immunogenicity of a Recombinant MVA and a DNA Vaccine for Japanese Encephalitis Virus in Swine
Authors: Nam Jae‐hwan , Chae Soo‐lim , Cho Hae‐wol
Published: November 2013

We previously reported that mice immunized with recombinant modified vaccinia virus Ankara (MVA) encoding Japanese encephalitis virus (JEV) prM and E genes were completely protected against JEV challenge (Nam, J.H., Wyatt, L.S., Chae, S.L., Cho, H.W., Park, Y.K., Moss, B. Vaccine 1999, 17: 261–268). In this study, we examined the immunogenicity in swine of this recombinant MVA (vJH9) or a DNA vaccine (pcJH‐1) expressing the same JEV genes. Although the booster effect in mice with a combination of vJH9, pcJH‐1 and inactivated JEV commercial vaccine was not apparent by measuring JEV antibodies, the recombinant MVA vaccine (vJH9) and the DNA vaccine (pcJH‐1) efficiently produced neutralizing antibodies in swine and 2 doses of each showed a booster effect in mice and swine. Therefore, both vJH9 and pcJH‐1 are good candidates for a second generation JEV vaccine.
Antigenic Relatedness between Burkholderia pseudomallei and Burkholderia mallei
Authors: Anuntagool Narisara , Sirisinha Stitaya
Published: November 2013

Burkholderia pseudomallei and Burkholderia mallei are causative agents of distinct diseases, namely, melioidosis and glanders, respectively. The two species are very closely related, based on DNA‐DNA homology, base sequence of the 16S rRNA, and phenotypic characteristics. Based on the use of polyclonal antisera, B. pseudomallei and B. mallei are also found to be antigenically closely related to one another. We previously reported the production of monoclonal antibodies (MAbs) against B. pseudomallei antigens; one group was specific for the 200‐kDa exopolysaccharide present on the surface of all B. pseudomallei isolates, and the other was specific for the lipopolysaccharide (LPS) structure present on more than 95% of the B. pseudomallei tested. In the present study, we showed that the MAbs against 200‐kDa antigen of B. pseudomallei cross‐reacted with a component present also in some B. mallei isolates (3/6), but the positive immunoblot reaction was noted below the 200‐kDa position. On the other hand, none of the six B. mallei isolates reacted with the MAb specific for B. pseudomallei LPS. It was of interest to observe that only the 3 exopolysaccharide‐positive B. mallei isolates reacted with a commercial MAb against B. mallei LPS. The data presented suggest that B. mallei can be classified antigenically into two types based on their reactivities with different MAbs, i.e., the presence or absence of exopolysaccharide and the types of lipopolysaccharide. The heterogeneity of the LPS from these two closely related organisms is most likely related to the differences in its O‐polysaccharide side chain.
Heat Stability of Carboxypeptidase R of Experimental Animals
Authors: Komura Hidefumi , Shimomura Yasuyo , Yumoto Miho , Katsuya Hirotada , Okada Noriko , Okada Hidechika
Published: November 2013

Carboxypeptidase N (CPN) and carboxypeptidase R (CPR) are present in fresh serum, and cleave C‐terminal arginine or lysine residues from bioactive peptides such as anaphylatoxins and kinins resulting in regulation of peptide activity. Although CPN is present in the active form in plasma, CPR is generated from proCPR by trypsin‐like enzymes such as thrombin. CPR regulates not only inflammatory peptides but also restricts fibrinolysis. To elucidate the complex role of CPN and CPR in vivo, studies in animal models will be essential. CPR of guinea pig, rat and rabbit decayed at 37 C rapidly as in the case of human CPR. However, at 25 C, CPR of those species decayed to some extent, although human serum CPR did not decay within 60 min. In the presence of thrombin inhibitor, CPR in the sera of animals tested decayed more rapidly than CPR in serum without thrombin inhibitor suggesting that additional generation of CPR may have been prevented during decay evaluation. However, human serum CPR decayed more rapidly in the absence of thrombin inhibitor indicating that thrombin may accelerate the decay in human serum.
An Outbreak of Neonatal Toxic Shock Syndrome‐Like Exanthematous Disease (NTED) Caused by Methicillin‐Resistant Staphylococcus aureus (MRSA) in a Neonatal Intensive Care Unit
Authors: Nakano Miyo , Miyazawa Hirofumi , Kawano Yasushi , Kawagishi Mika , Torii Keizo , Hasegawa Tadao , Iinuma Yoshitsugu , Ohta Michio
Published: November 2013

Neonatal toxic shock syndrome‐like exanthematous disease (NTED) is a new entity of methicillin‐resistant Staphylococcus aureus (MRSA) infection. Most of NTED cases reported previously in the literature were sporadic ones. In the present report, we describe an outbreak of NTED that occurred in a neonatal intensive care unit (NICU) between April, 1999 and April, 2000 in Japan. All MRSA strains isolated from 14 patients (6 NTED, 2 infections and 6 colonizations) in this outbreak belonged to the group of coagulase II and produced toxic shock syndrome toxin 1 (TSST‐1). Of these, 14 strains produced staphylococcal enterotoxin C (SEC). No other superantigenic toxins were produced by these strains. The pulsed field gel electrophoresis (PFGE) patterns of genomic DNA digested with SmaI were indistinguishable each other due to no band shifting in all of the 13 strains except for strain O‐21 and M56. Strain M56 was different from the dominant type in the positions of only 2 bands, whereas the pattern of strain O‐21 had no similarity with the other pattern, suggesting that this outbreak was associated with the spread of a unique MRSA strain in the NICU. Two‐dimensional electrophoresis (2‐DE) analysis of exoproteins revealed that the patterns of these 14 strains were very indistinguishable to each other, and that these strains produced very large amounts of TSST‐1 and SEC3 subtype superantigens, as measured with computer‐assisted image analysis of the intensity of 2‐DE spots. The 2‐DE gel of O‐21 showed the different pattern from the others. These results as well as the profiles of toxin production also supported the conclusion drawn from PFGE analysis. Based on these results, the involvement of TSST‐1 and SEC3 in the pathogenesis of NTED is discussed.
Rapid and Sensitive Detection Method of a Bacterium by Using a GFP Reporter Phage
Authors: Funatsu Takashi , Taniyama Tadayoshi , Tajima Takashi , Tadakuma Hisashi , Namiki Hideo
Published: November 2013

A rapid, sensitive, and convenient method for detecting a specific bacterium was developed by using a GFP phage. Here we describe a model system that utilizes the temperate Escherichia coli‐restricted bacteriophage lambda, which was genetically modified to express a reporter gene for GFP to identify the colon bacillus E. coli in the specimen. E. coli infected with GFP phage was detected by GFP fluorescence after 4–6 hr of incubation. The results show that a few bacteria in a specimen can be detected under fluorescence microscopy equipped with a sensitive cooled CCD camera. When E. coli and Mycobacterium smegmatis were mixed in a solution containing GFP phage, only E. coli was infected, indicating the specificity of this method. The method has the following advantages: 1) Bacteria from biological samples need not be purified unless they contain fluorescent impurities; 2) The infection of GFP phage to bacteria is specific; 3) The fluorescence of GFP within infected bacteria enables highly sensitive detection; 4) Exogenous substrates and cofactors are not required for fluorescence. Therefore this method is suitable for any phage‐bacterium system when bacteria‐specific phages are available.
Evaluation of Therapeutic Efficacy of Adjuvant Helicobacter pylori Whole Cell Sonicate in Mice with Chronic H. pylori Infection
Authors: Maeda Kosaku , Yamashiro Tetsu , Minoura Takanori , Fujioka Toshio , Nasu Masaru , Nishizono Akira
Published: November 2013

Successful prophylactic administration of Helicobacter pylori whole cell sonicate (WCS) plus complete Freund's adjuvant (CFA) or aluminum hydroxide (ALM) against subsequent H. pylori infection was reported recently. Here we tested the effect of WCS plus TiterMax® Gold (TMX) or ALM in mice with chronic H. pylori infection. Mice with chronic (18 weeks) H. pylori infection were injected intraperitoneally with H. pylori (Sydney strain) WCS plus ALM or TMX once weekly for three times. The number of colonizing H. pylori in the stomach, IgG1 and IgG2a levels, and local inflammatory status were determined after therapeutic immunization. H. pylori specific IgG1, but not IgG2a, was significantly induced in mice immunized with H. pylori WCS plus TMX or ALM. Immunization did not result in reduction of bacterial count or recruiting inflammatory cells to the stomach. Adjuvant H. pylori WCS resulted in induction of CD4+ Th2 cell‐mediated immunity although it did not reduce bacterial density in mice with chronic H. pylori infection. Our results implied that CD4+ Th1 cell‐mediated immunity, rather than Th2 cell dominant immunity, might play a role in reducing the number of bacteria in chronic H. pylori infection.
A Very Late Activating Antigen‐α4 (CD49d) Monoclonal Antibody, BU49 Induces Phosphorylation of a cAMP Response Element‐Binding Protein (CREB), Resulting in Induction of Homotypic Cell Aggregation and Enhancement of Interleukin‐8 (IL‐8) Production
Authors: Ikewaki Nobunao , Inoko Hidetoshi
Published: November 2013

A very late activating antigen‐α4 (CD49d) monoclonal antibody (mAb), BU49 was found to induce phosphorylation of a cAMP response element‐binding protein (CREB) in the human monocyte‐like cell line, U937. This phosphorylation of CREB was completely inhibited by a protein kinase A (PKA) inhibitor H‐89 with the optimum concentration (completely inhibits PKA). Furthermore, BU49 strongly and rapidly (within 5 hr) induced homotypic cell aggregation in the U937 cells accompanied by CREB phosphorylation. This cell aggregation was also completely inhibited by the addition of H‐89. Interestingly, both of two mAbs (mAb13 and 4B4) recognizing different epitopes on the CD29 (β1 integrin) completely inhibited this aggregation at the late phase (18 to 24 hr) but not at the early phase (5 hr) after cultured with BU49. On the other hand, BU49 significantly enhanced interleukin‐8 (IL‐8) production from the U937 cells into the culture supernatant. In addition, this IL‐8 production was significantly blocked in the presence of H‐89 with the optimum concentration. However, a CD29 mAb which inhibits homotypic cell aggregation could not block this IL‐8 production. Taken together, these findings indicate that BU49 induces CREB phosphorylation mainly mediated by PKA, which finally results in the induction of homotypic cell aggregation and the enhancement of IL‐8 production. Furthermore, these findings also indicate that the enhancement of IL‐8 production from the U937 cells induced by BU49 partially depends on CREB phosphorylation mainly mediated by PKA.
Role of Internalization in the Pathogenicity of Shiga Toxin‐Producing Escherichia coli Infection in a Gnotobiotic Murine Model
Authors: Aiba Yuji , Ishikawa Hiroki , Shimizu Keiko , Noda Satoshi , Kitada Yukie , Sasaki Masafumi , Koga Yasuhiro
Published: November 2013

We investigated the role of bacterial internalization in the killing caused by Shiga toxin‐producing Escherichia coli (STEC) infection using a gnotobiotic murine model. A high number of internalized STEC was found in the colonic epithelial cells of STEC‐infected mice by both an ex vivo assay and transmission electron microscopy. Most of these mice were killed within 10 days after infection. However, the implantation of lactic acid bacteria in such mice before infection markedly decreased the number of internalized STECs and also completely protected these hosts from killing by a STEC infection. The inhibition of such internalization by immunoglobulin also prevented the hosts from being killed. The Shiga toxin levels in these hosts indicated an inhibition of the penetration of Shiga toxins produced in the colon to the underlying tissue. These results suggested that the internalization plays an important role in the pathogenicity caused by STEC infection in a gnotobiotic murine model.
Lethal Factor to Mice Produced by Escherichia coli Isolated from Chickens with Swollen Head Syndrome
Authors: Salvadori Marcia Regina , Chudzinski‐Tavassi Ana Marisa , Baccaro Maria Regina , Ferreira Claudete Serrano Astolfi , Ferreira Antonio José Piantino , Prado‐Franceschi Júlia , Yano Tomomasa
Published: November 2013

A lethal toxin similar to Bacillus cererus lethal toxin was detected in the culture supernatants of Escherichia coli isolated from chickens with swollen head syndrome. The lethal activity was heat‐labile, protease‐sensitive and killed mice within 10 min. The light microscopy of the histopathological studies revealed that the principal organ affected by this toxin was the lung but the liver and kidneys also showed lesions. The relevance of this lethal activity from E. coli remains to be determined.
Identification of the Pathogenic Aspergillus Species by Nested PCR Using a Mixture of Specific Primers to DNA Topoisomerase II Gene
Authors: Kanbe Toshio , Yamaki Kenichi , Kikuchi Akihiko
Published: November 2013

For PCR‐based identification of Aspergillus species, a common primer of the DNA topoisomerase II genes of Candida, Aspergillus and Penicillium, and species‐specific primers of the genomic sequences of DNA topoisomerase II of A. fumigatus, A. niger, A. flavus (A. oryzae), A. nidulans and A. terreus were tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by a common primer set, followed by a second PCR with a primer mix consisting of 5 species‐specific primer pairs for each Aspergillus species. By using the common primer pair, a DNA fragment of approximately 1,200 bp was amplified from the Aspergillus and Penicillium genomic DNAs. Using each species‐specific primer pair, unique sizes of PCR products were amplified, all of which corresponded to a species of Aspergillus even in the presence of DNAs of several fungal species. The sensitivity of A. fumigatus to the nested PCR was found to be 100 fg of DNA in the reaction mixture. In the nested PCR obtained by using the primer mix (PsIV), the specific DNA fragment of A. fumigatus was amplified from clinical specimens. These results suggest that this nested PCR method is rapid, simple and available as a tool for identification of pathogenic Aspergillus to a species level.
Construction and In Vitro Properties of Chimeric Simian and Human Immunodeficiency Virus with the Human TNF‐Alpha Gene
Authors: Haga Takeshi , Shimizu Yuya , Okoba Masashi , Kumabe Shino , Goto Yoshitaka , Shinjo Toshiharu , Ichimura Hiroshi , Kuwata Takeo , Hayami Masanori , Miura Tomoyuki
Published: November 2013

Tumor necrosis factor‐alpha (TNF‐α) has been reported to be involved in the development and progression of acquired immunodeficiency syndrome (AIDS). To study the role of this cytokine in AIDS pathogenesis, we constructed a chimeric simian and human immunodeficiency virus (SHIV) having the human TNF‐α gene (SHIV‐TNF) and characterized its properties in vitro. SHIV‐TNF replicated both in M8166, a human T cell line, and in monkey peripheral blood mononuclear cells (PBMCs). Along with SHIV‐TNF replication, TNF‐α was detected in the culture supernatant by ELISA. The maximum expression level of TNF‐α reached 120 ng/ml in M8166 cells, and 2.5 ng/ml in monkey PBMCs. The expressed TNF was biologically active, as shown by a cytotoxic assay using TNF‐sensitive L929 mouse fibroblasts. This activity was detected at least until 10 passages of SHIV‐TNF (74 days after the initial infection). In monkey PBMCs, SHIV‐TNF replicated much better than the parental SHIV‐NI. Flow cytometric analysis showed that the death of monkey PBMCs infected with SHIV‐TNF was severer than that caused by the parental SHIV‐NI. These results suggest that SHIV‐TNF would be useful for inducing the disease in a monkey model, which may contribute to a better understanding of the role of TNF‐α in AIDS etiology.
Serum Antibodies of Periodontitis Patients Compared to the Lipopolysaccharides of Porphyromonas gingivalis and Fusobacterium nucleatum
Authors: Onoue Sakura , Imai Toshio , Kumada Hidefumi , Umemoto Toshio , Kaca Wieslaw , Isshiki Yasunori , Kaneko Aki , Kawahara Kazuyoshi
Published: November 2013

Serum antibody titers against the lipopolysaccharides (LPSs) of Porphyromonas gingivalis and Fusobacterium nucleatum were compared between 9 periodontitis patients and 24 healthy persons. The IgG titers against the LPSs of P. gingivalis ATCC 33277T and W50 were clearly higher in the patients than in the healthy persons. However, IgM titers against the LPSs of P. gingivalis strains were relatively low, and no significant difference was observed between the patients and healthy persons. On the other hand, IgG and IgM titers against the LPS of Fusobacterium nucleatum JCM 8532T in some patients were significantly higher than those in the healthy persons, although the difference in IgG titers was not large compared to that of the LPS of P. gingivalis. These results suggest that the antibody measurement of patients' sera against the LPS of periodontal bacteria can be applied for the diagnosis of periodontitis.
Tyrosine Phosphorylation of a Novel 100‐kDa Protein Coupled to CD28 in Resting Human T Cells Is Enhanced by a Signal through TCR/CD3 Complex
Authors: Matsumoto Aki , Dobashi Hiroaki , Ohnishi Hiroaki , Tanaka Terukazu , Kubota Yoshitsugu , Kitanaka Akira , Ishida Hiroshi , Tokuda Michiaki , Waki Masato , Kubo Akihito , Ishida Toshihiko
Published: November 2013

For T cell activation, two signals are required, i.e., a T cell receptor (TCR)/CD3‐mediated main signal and a CD28‐mediated costimulatory signal. CD28 binds to its ligand (CD80 or CD86) and transduces the most important costimulatory signal. The cytoplasmic domain of the CD28 molecule, composed of 41 amino acids, does not contain any intrinsic enzyme activity. The cytoplasmic domain of CD28 is remarkably conserved among species and is associated with a number of signaling molecules that affect the main signal. We report here that a tyrosine phosphorylated 100‐kDa protein (pp100) was coupled to the CD28 cytoplasmic domain in Jurkat and human peripheral T cells. The pp100 was distinguished from other CD28 associated molecules such as Vav, STAT5, PI 3‐kinase, Valosin‐containing protein (VCP), Nucleolin, Gab2 (Grb2‐associated binding protein 2), and STAT6. The tyrosine phosphorylation of pp100 coprecipitated with CD28 was enhanced by CD3 stimulation by the specific antibody, tyrosine phosphatase inhibitor and PKC activator. Tyrosine phosphorylation of pp100 was attenuated by the prior addition of PKC inhibitor. These findings indicate that pp100 is a novel tyrosine phosphorylated protein coupled to CD28 under continuous control of tyrosine phosphatases and might play a role in T cell activation augmented by a TCR/CD3‐mediated main signal.
High Molecular Weight Factor in FCS Inhibits Helicobacter pylori VacA‐Binding to Its Receptor, RPTPβ, on AZ‐521
Authors: Kimura Takahiro , Wada Akihiro , Nakayama Masaaki , Ogushi Ken‐ichi , Nishi Yoshito , De Guzman Blanquita B. , Moss Joel , Hirayama Toshiya
Published: November 2013

VacA, a secretory product of Helicobacter pylori, binds to its cell surface receptor, receptor tyrosine phosphatase (RPTP) β, leading to cytoplasmic vacuolization of gastric epithelial AZ‐521 cells. VacA binding to the cell surface and VacA‐dependent vacuolization were inhibited by cell culture media containing fetal calf serum (FCS). The high molecular weight fraction of FCS isolated by Superose 12 gel filtration chromatography inhibited VacA binding, whereas only weak effects were observed with other fractions. These data show that the high molecular weight fraction of FCS inhibits VacA action though its ability to block toxin binding to its receptor, RPTPβ, on AZ‐521 cells.
Gene Cloning and Characterization of VcrM, a Na+‐Coupled Multidrug Efflux Pump, from Vibrio cholerae Non‐O1
Authors: Huda Md. Nazmul , Chen Jing , Morita Yuji , Kuroda Teruo , Mizushima Tohru , Tsuchiya Tomofusa
Published: November 2013

We cloned a DNA fragment responsible for drug resistance from chromosome of Vibrio cholerae non‐O1. Nucleotide sequence analysis of this fragment revealed the presence of a single open reading frame encoding a protein consisting of 445 amino acid residues. We designated the gene as vcrM. Hydropathy analysis of the deduced amino acid sequence of VcrM suggests the presence of 12 trans‐membrane segments. A dendrogram showed that VcrM is a member of the DinF‐subfamily within the MATE family of multidrug efflux pumps. Expression of the cloned vcrM gene in drug‐hypersensitive Escherichia coli KAM32 cells made them resistant to acriflavine, 4′, 6‐diamidino‐2‐phenylindole, Hoechst 33342, rhodamine 6G, tetraphenylphosphonium chloride (TPPCl) and ethidium bromide. Efflux of acriflavine due to VcrM was dependent on Na+ or Li+. Moreover, Na+ efflux was observed with VcrM when TPPCl was added to Na+‐loaded cells. Therefore, we conclude that VcrM is a Na+/drug antiporter‐type multidrug efflux pump.
A Novel Murine Model of Oral Candidiasis with Local Symptoms Characteristic of Oral Thrush
Authors: Takakura Natsuko , Sato Yuichi , Ishibashi Hiroko , Oshima Haruyuki , Uchida Katsuhisa , Yamaguchi Hideyo , Abe Shigeru
Published: November 2013

A conventional and easy method to establish a murine oral candidiasis model, which has not only a stable yeast population in the oral cavity but also symptoms characteristic of oral thrush, was developed by using a sedative agent. Mice were immunosuppressed with prednisolone and were given tetracycline hydrochloride. They were orally infected with 106 viable cells of Candida albicans by means of a cotton swab and enough chlorpromazine chloride had been injected to keep them in a sedative state about for 3 hr after inoculation. From day 3 to day 7 post inoculation, 105–106 colony forming units of Candida were recovered from the oral cavity of each mouse and whitish, curd‐like patches were observed on most parts of tongue. Microscopically, germ tubes had appeared on the tongue surface. This model would be a useful experimental oral candidiasis for investigating the pathogenesis of C. albicans oral infection and the efficacy of various antifungal agents microbiologically and symptomatically.
Genotype and Subtype Analyses of Hepatitis B Virus (HBV) and Possible Co‐Infection of HBV and Hepatitis C Virus (HCV) or Hepatitis D Virus (HDV) in Blood Donors, Patients with Chronic Liver Disease and Patients on Hemodialysis in Surabaya, Indonesia
Authors: Lusida Maria I. , Surayah  , Sakugawa Hiroshi , Nagano‐Fujii Motoko , Soetjipto  , Mulyanto  , Handajani Retno , Boediwarsono  , Setiawan Poernomo B. , Nidom Chairul A. , Ohgimoto Shinji , Hotta Hak
Published: November 2013

Four subtypes (adw, adr, ayw, and ayr) and eight genotypes (A to H) of the hepatitis B virus (HBV) have been identified. They appear to be associated with particular geographic distribution, ethnicity, and possibly clinical outcomes. In this study, hepatitis B surface antigen (HBsAg) subtyping and HBV genotyping were carried out on sera obtained from HBsAg‐positive HBV carriers, including healthy blood donors; patients with acute hepatitis, chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma; and patients on hemodialysis all located in Surabaya, Indonesia. We report here that all HBV isolates tested in Surabaya belonged to genotype B, with more than 90% of them being classified into subtype adw. Our results also revealed that prevalence of hepatitis C virus (HCV) co‐infection among HBV carriers in Surabaya was approximately 10% for healthy blood donors and patients with chronic liver disease, and approximately 60% for patients on maintenance hemodialysis. Interestingly, HBsAg titers were lower in HBV carriers with HCV co‐infection than in those without HCV co‐infection. We also found that prevalence of hepatitis D virus (HDV) co‐infection was <0.5% among HBV carriers in Surabaya.
Molecular Cloning and Characterization of the HmrM Multidrug Efflux Pump from Haemophilus influenzae Rd
Authors: Xu Xing‐Jue , Su Xian‐Zhong , Morita Yuji , Kuroda Teruo , Mizushima Tohru , Tsuchiya Tomofusa
Published: November 2013

We cloned a gene responsible for norfloxacin resistance from the chromosomal DNA of Haemophilus influenzae Rd, and designated the gene as hmrM. HmrM showed sequence similarity with NorM of Vibrio parahaemolyticus and YdhE of Escherichia coli and others that belong to the MATE family multidrug efflux pumps. The recombinant plasmid carrying the hmrM gene conferred elevated resistance not only to norfloxacin but also to acriflavine, 4′,6‐diamidino‐2‐phenylindole, doxorubicin, ethidium bromide, tetraphenylphosphonium chloride, Hoechst 33342, daunomycin, berberine, and sodium deoxycholate in Escherichia coli KAM32, a drug‐hypersensitive strain. We observed an Na+‐dependent efflux of ethidium and an ethidium‐induced efflux of Na+ in E. coli KAM32 cells harboring the plasmid carrying the hmrM gene. These results indicate that HmrM is an Na+/drug antiporter‐type multidrug efflux pump. A difference in substrate preference was observed between HmrM, NorM, and YdhE.
Homologous and Heterologous Antibody Responses to Lipopolysaccharide after Enterohemorrhagic Escherichia coli Infection
Authors: Tsutsumi Reiko , Ichinohe Naoko , Shimooki Osamu , Obata Fumiko , Takahashi Kiomi , Inada Katsuya , Sasaki Mika , Sato Shigehiro , Chida Shoichi
Published: November 2013

To evaluate antibody responses against lipopolysaccharide (LPS: O157, O26, and O111) in enterohemorrhagic Escherichia coli (EHEC) infection, sera of 24 schoolchildren associated with the Morioka outbreak in 1997 and of 74 sporadic patients suspected of having EHEC infection were examined. Using a positive standard serum, quantitative evaluation of LPS antibodies by an enzyme‐linked immunosorbent assay (ELISA) was established. High levels of specific IgM and IgA antibodies against homologous E. coli LPS were present in the acute period and are characteristic of EHEC. This could be used for the serological diagnosis of EHEC infection, except for early infants and the elderly. In addition to the specific homologous response, multiple antibody responses against different serotypes other than those isolated were demonstrated in many cases by qualitative analysis using Western blotting.
CD72 Stimulation Modulates Anti‐IgM Induced Apoptotic Signaling through the Pathway of NF‐κB, c‐Myc and p27Kip1
Authors: Fujiwara Naruyoshi , Fusaki Noemi , Hozumi Nobumichi
Published: November 2013

Engagement of mIgM induces G1 arrest and apoptosis in immature B cells. The biochemical mechanism(s) regulating the cell death process are poorly understood. Cross‐linking of CD72 (a B cell co‐receptor) with anti‐CD72 antibody was shown to protect B cells from apoptosis. We investigated the molecular mechanism involved in apoptosis preventing signaling mediated by CD72 ligation using a derivative (WEHIδ) of the WEHI231 cell line which is representative of immature B cells. Apoptotic WEHIδ cells following cross‐linking of mIgM demonstrate a dramatic loss of c‐Myc protein after transient up‐regulation. In contrast, pre‐ligation of CD72 was able to sustain c‐Myc expression after transient up‐regulation. Cross‐linking of mIgM of WEHIδ cells causes accumulation of the Cdk inhibitor, p27Kip1. CD72 pre‐ligation was shown to inhibit the accumulation of p27Kip1 protein. Moreover, NF‐κB activity was not suppressed in WEHIδ cells after mIgM cross‐linking when the cells were pre‐treated with anti‐CD72 antibody. These results strongly suggest that the apoptosis preventing signal evoked by CD72 ligation is delivered through the pathway of NF‐κB, c‐Myc, p27Kip1 and cyclin.
First Isolation of Bartonella henselae Type I from a Cat‐Scratch Disease Patient in Japan and Its Molecular Analysis
Authors: Maruyama Soichi , Izumikawa Koichi , Miyashita Mayumi , Kabeya Hidenori , Mikami Takeshi , Yamanouchi Hirotsugu , Sasaki Eisuke , Yoshida Hiroshi , Izumikawa Kinichi
Published: November 2013

We isolated Bartonella henselae from an inguinal lymph node of a 36‐year‐old male patient with cat‐scratch disease. The patient had many areas of erythema on his body, swelling of the left inguinal lymph nodes with pain and slight fever. The diagnosis was made on the basis of polymerase chain reaction for B. henselae DNA from the lymph node biopsies and blood sample, and isolation of the organism, histology of the lymph node and serology with an indirect immunofluorescent antibody test. We also analyzed the genome profiles for five strains of 90 isolates from the lymph node by pulsed‐field gel electrophoresis after NotI endonuclease digestion. We found two different genomic profiles. These results suggest that the patient had been either co‐infected or re‐infected with two genetically different strains of B. henselae.
Emergence and Serovar Transition of Vibrio parahaemolyticus Pandemic Strains Isolated during a Diarrhea Outbreak in Vietnam between 1997 and 1999
Authors: Chowdhury Ashrafuzzaman , Ishibashi Masanori , Thiem Vu Dinh , Tuyet Dinh Thi Ngoc , Van Tung Tran , Chien Bui Trong , von Seidlein Lorenz , Canh Do Gia , Clemens John , Trach Dang Duc , Nishibuchi Mitsuaki
Published: November 2013

We characterized 523 Vibrio parahaemolyticus strains isolated during a survey of diarrhea patients in Khanh Hoa province, Vietnam between 1997 and 1999. Forty‐nine percent of the strains were judged to belong to the pandemic strains that emerged around 1996 and spread to many countries. These strains were positive in the GS‐PCR assay and carried the tdh gene. The ORF8 of the f237 phage genome, a possible marker of the pandemic clone, was absent in 10% of these strains. Eleven O:K serovars were detected among the pandemic strains and the strains representing all 11 serovars of pandemic strains were shown to be closely related regardless of the ORF8 genotype using arbitrarily primed PCR and pulsed field gel electrophoresis analyses. It was clear that a transition of major serovars occurred among the pandemic strains represented by the emergence of O3:K6 in 1997, O4:K68 in 1998, and O1:K25 in 1998 and 1999.
Characterization of a Shiga‐Toxin 1‐Resistant Stock of Vero Cells
Authors: Sekino Takaomi , Kiyokawa Nobutaka , Taguchi Tomoko , Takenouchi Hisami , Matsui Jun , Tang Wei‐Ran , Suzuki Toyo , Nakajima Hideki , Saito Masahiro , Ohmi Kazuhiro , Katagiri Yohko U. , Okita Hajime , Nakao Hiroshi , Takeda Tae , Fujimoto Junichiro
Published: November 2013

Shiga toxins (Stxs, also referred to as verotoxins) were first described as a novel cytotoxic activity against Vero cells. In this study, we report the characterization of an Stx1‐resistant (R‐) stock of Vero cells. (1) When the susceptibility of R‐Vero cells to Stx1 cytotoxicity was compared to that of Stx1‐sensitive (S‐) Vero cells by methylthiazolyldiphenyl‐tetrazolium bromide (MTT) assay, cell viability after 48‐hr exposure to 10 pg/ml of Stx1 was greater than 80% and less than 15%, respectively. (2) Although both a binding assay of fluorescence‐labeled Stx1 and lipid analysis indicated considerable expression of Gb3Cer, a functional receptor for Stxs, in both Vero cells, anti‐Gb3Cer monoclonal antibodies capable of binding to S‐Vero cells failed to effectively label R‐Vero cells, suggesting a conformational difference in the Gb3Cer expressed on R‐Vero cells. (3) The lipid analysis also showed that the R‐Vero cells contained significant amounts of Gb4Cer. In addition, introduction of exogenous Gb4Cer into S‐Vero cells slightly inhibited Stx1 cytotoxicity, suggesting some correlation between glycosphingolipid composition and Stx1 resistance. (4) Both butyrate treatment and serum depression eliminated the Stx1 resistance of R‐Vero cells. (5) The results of the analysis by confocal microscopy suggest a difference in intracellular transport of Stx1 between R‐Vero and S‐Vero cells. Further study of R‐Vero cells may provide a model of Stx1 resistance via distinct intracellular transport of Stx1.
Comparison of Real‐Time and Nested PCR Assays for Detection of Herpes Simplex Virus DNA
Authors: Kawada Jun‐ichi , Kimura Hiroshi , Ito Yoshinori , Hoshino Yo , Tanaka‐Kitajima Naoko , Ando Yoshihiro , Futamura Masahide , Morishima Tsuneo
Published: November 2013

We performed a real‐time PCR assay to detect herpes simplex virus (HSV) DNA, and compared it prospectively with a nested PCR assay in 164 clinical samples (109 cerebrospinal fluid and 55 sera) from patients suspected of having neonatal HSV infection or HSV encephalitis. In 25 of 164 samples, HSV DNA was detected by the nested PCR assay. All samples positive for HSV DNA in the nested PCR assay were also positive in the real‐time PCR assay, and all but two samples negative for HSV DNA in the nested assay were negative in the real‐time assay. The real‐time PCR assay thus had a sensitivity of 100% and a specificity of 99%, when compared with the nested assay. Sequential assays in a case of disseminated HSV showed that a decrease in HSV DNA paralleled clinical improvement. Quantification of HSV DNA by real‐time PCR was useful for diagnosing and monitoring patients with HSV encephalitis and neonatal HSV infection.
Characterization of Ehrlichia Species from Ixodes ovatus Ticks at the Foot of Mt. Fuji, Japan
Authors: Inayoshi Megumi , Naitou Hirotaka , Kawamori Fumihiko , Masuzawa Toshiyuki , Ohashi Norio
Published: November 2013

A total of 390 adult ticks (288 Ixodes ovatus and 102 I. persulcatus) collected at the foot of Mt. Fuji and two near cities in Shizuoka prefecture, Japan, were examined for Ehrlichia infection by isolation with laboratory mice from whole tick tissues. Ehrlichial DNAs were detected from the spleens of mice inoculated with tissues from I. ovatus, but not I. persulcatus. The prevalence of ehrlichiae in the ticks was estimated to be ca. 3%. The 16S rDNA analysis revealed that the sequences of 8 ehrlichial isolates (termed “Shizuoka” isolates) obtained were identical, and they were very similar, but not identical, to those of two Ehrlichia species strain variants recently isolated in Japan, followed by Ehrlichia chaffeensis in the US. Analysis of parts of the omp‐1 multigene family specific for monocytic ehrlichiosis agents showed that the Shizuoka isolates were distinct from other ehrlichial organisms. The Shizuoka isolates caused death in immunocompetent laboratory mice, suggesting that they are highly pathogenic in mice. The data show that the Shizuoka isolates are likely to be a new strain variant of Ehrlichia species in Japan. Further characterization and surveillance will be required in Japan due to the presence of these human ehrlichiosis agent‐like organisms.
The Throat Flora and Its Mitogenic Activity in Patients with Kawasaki Disease
Authors: Horita Norihisa , Yokota Shin‐ichi , Fuse Shigeto , Takamuro Motoki , Tomita Hideshi , Sato Kiyoshi , Fujii Nobuhiro , Tsutsumi Hiroyuki
Published: November 2013

The etiology of Kawasaki disease (KD) remains unknown, although some infectious organism has been suggested as the cause. Recent studies suggest that some bacterial toxins with superantigen activity are involved in its pathogenesis, but no specific bacterial toxin has yet been identified. Throat swabs for bacterial culture were obtained from 21 patients with KD and 20 with other febrile illnesses as controls. Mitogenic activity in culture supernatants obtained from individual bacterial strains was measured by lymphocyte proliferation assay. Sixty‐one bacterial strains were isolated from KD patients, and 62 strains from control patients. There was no apparent difference in bacterial species in the throat flora between KD patients and febrile controls. Moreover, total and individual mitogenic activity of strains from KD patients was no greater than that of strains from febrile controls. The bacterial superantigen activity of throat flora may not play a major role in the pathogenesis of KD.
Roles of Toxoplasma gondii‐Derived Heat Shock Protein 70 in Host Defense against T. gondii Infection
Authors: Ahmed Azza Kamal , Mun Hye‐Seong , Aosai Fumie , Piao Lian‐Xun , Fang Hao , Norose Kazumi , Yano Akihiko
Published: November 2013

C57BL/6 mice receiving intraperitoneal injection of Toxoplasma gondii‐derived heat shock protein 70 (T.g. HSP70) on day 3 post T. gondii infection succumbed by day 9 post infection, while vector protein‐injected control mice survived more than 6 months. The deteriorating effect of T.g.HSP70 on host immune responses was dose‐dependent. By T.g. HSP70 injection, T. gondii loads increased in various organs of T. gondii‐infected mice. Th2 cytokines such as IL‐4 and IL‐10 were continuously produced from spleen and peritoneal exudate cells of T. gondii‐infected mice by injection of T.g. HSP70. Furthermore, nitric oxide production from peritoneal macrophages in T. gondii‐infected mice was reduced by T.g. HSP70.
Role of p38 Mitogen‐Activated Protein Kinase (MAPK) for Vacuole Formation in Lipopolysaccharide (LPS)‐Stimulated Macrophages
Authors: Hassan Ferdaus , Islam Shamima , Koide Naoki , Mu Mya Mya , Ito Hiroyasu , Mori Isamu , Yoshida Tomoaki , Yokochi Takashi
Published: November 2013

The role of p38 mitogen‐activated protein kinase (MAPK) on vacuole formation in lipopolysaccharide (LPS)‐stimulated RAW 264.7 cells was examined. LPS definitely induced the formation of vacuoles in RAW 264.7 cells and SB202190 as a p38 specific inhibitor also induced slight vacuole formation. The simultaneous treatment with LPS and SB202190 induced many more vacuoles in RAW 264.7 cells than the treatment with LPS or SB202190 alone, and the vacuoles were extraordinarily large in size. On the other hand, an inactive inhibitor of p38 MAPK did not augment LPS‐induced vacuole formation. Further, the inhibitors of other MAPKs and nuclear factor (NF)‐κB pathways did not affect it. The extraordinarily large vacuoles in RAW 264.7 cells treated with LPS and SB202190 were possibly formed via fusion of small vacuoles. However, SB202190 did not augment vacuole formation in CpG DNA or interferon (IFN)‐γ‐stimulated RAW 264.7 cells. The role of p38 MAPK in the vacuole formation in LPS‐stimulated macrophages is discussed.
The Larval Specific Lymphatic Filarial ALT‐2: Induction of Protection Using Protein or DNA Vaccination
Authors: Ramachandran Sabarinathan , Kumar Mishra Pankaj , Rami Reddy Maryada Venkata , Chinnaiah Harinath Basker , Nutman Thomas , Kaliraj Perumal , McCarthy James
Published: November 2013

Genes from the infective stage of lymphatic filarial parasites expressed at the time of host invasion have been identified as potential vaccine candidates. By screening an L3 cDNA library with sera from uninfected longstanding residents of an area endemic for onchocerciasis, so‐called “endemic normals” (EN), we have cloned and characterized one such gene termed the abundant larval transcript two (ALT‐2). The stage specificity of ALT‐2 gene transcription and protein synthesis was confirmed by PCR using gene‐specific primers, and by western blot analysis of protein extracts from various stages of the parasite life cycle using specific antisera. Significant differences in antibody response to the recombinant ALT‐2 were observed in endemic populations with differing clinical manifestations of lymphatic filariasis with an antibody response present in sera from 18 of 25 (72%) EN subjects compared to 9 of 25 (36%) with subclinical microfilaracmia (MF) and 14 of 25 (52%) of those with chronic lymphatic obstruction (CP) (P=0.01 for comparison of EN to CP or to MF). This differential responsiveness suggests that the protective immunity postulated to account for their uninfected status might be associated with a response to this protein. When the utility of ALT‐2 as a vaccine candidate was tested in a murine model using either recombinant protein or a DNA vaccine construct, statistically significant protection was observed when compared to a control filarial gene product expressed across all stages of the parasite lifecycle (SXP‐1; P=0.02 for protein and P=0.01 for the DNA vaccine) or compared to adjuvant alone. This level of protection indicates that this vaccine is a promising candidate for further development.
Release of Shiga Toxin by Membrane Vesicles in Shigella dysenteriae Serotype 1 Strains and In Vitro Effects of Antimicrobials on Toxin Production and Release
Authors: Dutta Shanta , Iida Ken‐ichiro , Takade Akemi , Meno Yuko , Nair Gopinath Balakrish , Yoshida Shin‐ichi
Published: November 2013

Effects of various antimicrobials on in vitro Shiga toxin production and release by Shigella dysenteriae serotype 1 was investigated in this study with particular reference to the role of outer membrane vesicles in toxin release by the organism. Five antimicrobials, namely nalidixic acid, ciprofloxacin, norfloxacin, fosfomycin and mitomycin C, were chosen for the study and the toxin titre was measured by the reverse passive latex agglutination (RPLA) method using an available kit. Only mitomycin C was found to induce production of Shiga toxin in the bacteria and its release by outer membrane vesicles. The highest titre of toxin was obtained in vesicle fraction suggesting that the vesicles play an important role in the release of Shiga toxin from periplasmic space by the organism.