bluuebunny/crossref_metadata_2025 huggingface dataset:
Modification of Autolysis by Synthetic Peptides Derived from the Presumptive Binding Domain of Staphylococcus aureus Autolysin
Authors: Takano Mayumi , Oshida Tadahiro , Yasojima Akira , Yamada Masaki , Okagaki Chieko , Sugai Motoyuki , Suginaka Hidekazu , Matsushita Tadahiro
Published: November 2013

The autolytic cell wall hydrolase of Staphylococcus aureus, Atl, contains three highly cationic repeats in the central region of the amino acid sequence, and the repeats are presumed to have the role of binding the enzyme to some components on the cell surface. To explain the possible function of the repeats, we synthesized a number of 10‐ to 30‐mer oligopeptides based on the Atl amino acid sequence (Thr432–Lys610) containing repeat 1, and examined their effects on the autolysis of S. aureus cells. When the peptides were added to a cell suspension of S. aureus under low ionic strength conditions, five peptides, A10, A11, A14, A16 and B9, showed immediate increases in optical density (OD) of the cell suspension accompanied by decreases in viable cell counts. After the immediate increases, the ODs for A10 and A14 changed little in the first 2 hr. In contrast, the ODs for A11 and A16 decreased rapidly. When peptide A10 was added to suspensions of heat‐killed whole cells, crude cell walls and a crude peptidoglycan preparation, their ODs were increased approximately 2‐fold. In contrast, the OD was not increased when the peptide was added to a suspension of pure peptidoglycan from which anionic polymers had been removed. Light microscopic and transmission electron microscopic study showed that A10 and A14 inhibited autolysis and that A11 and A16 induced autolysis earlier than the control. These results suggest strongly that the peptides adsorb to and precipitate on the anionic cell surface polymers such as teichoic acid and lipoteichoic acid via ionic interaction. The effects of peptides on the autolysis may be the results of the modification of S. aureus autolysin activities. These peptides, especially the 10‐mer peptide B9 (PGTKLYTVPW) that represents the C‐terminal half of A10 and N‐terminal half of A11, may be important segments for Atl to bind to the cell surface.
Mumps Virus Can Suppress the Effective Augmentation of HPC‐Induced Apoptosis by IFN‐γ through Disruption of IFN Signaling in U937 Cells
Authors: Hariya Yasushi , Yokosawa Noriko , Yonekura Noriyuki , Kohama Gen‐iku , Fujii Nobuhiro
Published: November 2013

Cells of the human promonocytic cell line U937 were found to be sensitive to hexadeeylphosphocholine (HPC), which is a potential anticancer drug. Induction of apoptosis was found in U937 cells after treatment with HPC for 24 to 48 hr. The apoptosis in U937 cells exposed to HPC was increased significantly in the presence of interferon‐gamma (IFN‐γ). The augmentation of HPC‐induced apoptosis by IFN‐γ is repressed in cells (U937‐MP) persistently infected with mumps virus. A persistently infected cell line, U937‐MP, showed poor induction of signal transducers and activators of transcription‐1α (STAT‐1α), STAT‐2, p48 and IFN‐regulatory factor‐1 (IRF‐1), which are closely correlated with interferon‐α (IFN‐α) and IFN‐γ signaling pathways. Expression of MHC class‐I or class‐II was augmented by IFN‐α or IFN‐γ in U937 cells, but not in persistently infected cells. Therefore, it is suggested that the IFN‐γ signaling pathway plays an important role in the augmentation of HPC‐induced apoptosis. Mumps virus can suppress the IFN‐γ signaling pathway and subsequent development of apoptosis.
Hepatitis C Virus NS5B RNA Replicase Specifically Binds Ribosomes
Authors: Tanaka Torahiko , Sugiyama Kazuo , Ikeda Masanori , Naganuma Atsushi , Nozaki Akito , Saito Masaki , Shimotohno Kunitada , Kato Nobuyuki
Published: November 2013

Hepatitis C virus (HCV) non‐structural protein 5B (NS5B) is an RNA replicase. We expressed full‐length NS5B (591 amino acid residues) in Escherichia coli as a fusion protein with maltose binding protein (MBP‐NS5B). MBP‐NS5B was recovered in the soluble fraction after centrifugation at 40,000 × g and affinity‐purified with amylose resin. The purified MBP‐NS5B had a high‐level of poly (A), oligo (U)‐dependent UMP incorporation with a Km of 2 μm for UTP. Surprisingly, the enzymatically active MBP‐NS5B was sedimented by ultracentrifugation at 160,000 × g. The pellet contained 16S and 23S ribosomal RNAs, suggesting that ribosomes were associated with MBP‐NS5B. Ribosomes and MBP‐NS5B were subsequently co‐purified on amylose resin. Deletion study revealed that either the N‐terminal (amino acid residues 1 107) or the C‐terminal (amino acid residues 498–591) region of NS5B were sufficient for this association with ribosomes. We further found that NS5B also bound with human ribosomes. Our results implicate a novel mechanism of coupling between replication and translation of the viral genome in the life cycle of HCV.
Genotyping of Pneumocystis carinii f. sp. hominis Isolates in Japan Based on Nucleotide Sequence Variations in Internal Transcribed Spacer Regions of rRNA Genes
Authors: Hosoya Noriaki , Takahashi Takashi , Wada Miki , Endo Tokiomi , Nakamura Tetsuya , Sakashita Hiroyuki , Kimura Kyoko , Ohnishi Kenji , Nakamura Yoshikazu , Mizuochi Tsuguo , Iwamoto Aikichi
Published: November 2013

Genotyping of Pneumocystis carinii (Pc) isolated from 24 bronchoalveolar lavage (BAL) fluid specimens in Japan was examined based on nucleotide sequence variations in internal transcribed spacer regions 1 and 2 (ITS1 and ITS2, respectively) of rRNA genes. We found 11 ITS1 genotypes including 2 novel ones and 11 ITS2 genotypes including 3 new ones. Combining the ITS1 and ITS2 genotypes resulted in 30 ITS genotypes, of which 10 are newly described in this report. Two or more genotypes in ITS regions in a specimen were observed in 16 of 24 patients. Our results will be of help for the epidemiological investigation of Pc infection.
Detection of Microsporum canis in the Skin Scrapings and Hairs of Dogs with Dermatophytosis Based on Sequences of the Chitin Synthase 1 Gene
Authors: Kano Rui , Nakamura Yuka , Watanabe Shinichi , Hasegawa Atsuhiko
Published: November 2013

In the present study, to confirm Microsporum canis infection rapidly, we detected the chitin synthase gene 1 (CHS1) gene of M. canis in the hair and skin samples of four dogs with dermatophytosis. Amplification of the DNAs in the four samples with CHS1 primers yielded fragments of about 620‐bp. Nucleotide sequence analysis of the CHS1 gene fragments from samples and a reference strain of M. canis gene showed more than 99% similarity. The method presented in this study can rapidly detect the DNA of M. canis in skin scrapings, and we anticipate that it will be a useful microbiological tool for the diagnosis of M. canis infections in animals and humans.
Firm Adherence of Staphylococcus aureus and Staphylococcus epidermidis to Human Hair and Effect of Detergent Treatment
Authors: Mase Kiyomi , Hasegawa Tadao , Horii Toshinobu , Hatakeyama Kazuto , Kawano Yasushi , Yamashino Takafumi , Ohta Michio
Published: November 2013

Staphylococcus aureus and S. epidermidis are common pathogens in hospitals, and care should be taken not to disseminate these organisms among patients. We have focused on human hair as a source of bacterial contamination. We treated hair with culture solutions of S. aureus and S. epidermidis, and then performed scanning electron microscopy. Bacteria were detected on the surface of the cuticles of the hair, and the attached bacteria were not completely removed even by repeated washing with detergents. These results suggested that hair could be a source of bacterial contamination and indicated the importance of decontamination of hair.
Identification of Amino Acids of Influenza Virus HA Responsible for Resistance to a Fusion Inhibitor, Stachyflin
Authors: Yoshimoto Jun , Kakui Mayumi , Iwasaki Hiroko , Sugimoto Hirohiko , Fujiwara Tamio , Hattori Naohiko
Published: November 2013

We have recently described a novel hemagglutinin (HA) conformational change inhibitor of human influenza virus, Stachyflin (Yoshimoto et al, Arch. Virol., 144, 1–14, 1999). Stachyflin‐resistant variants of human influenza A/WSN/33 (H1N1) virus were isolated in vitro and the nucleotide sequences of their HA genes were determined. The relation of amino acid substitutions and Stachyflin resistance was analyzed with in vitro membrane fusion between HA‐expressing cells and octadecylrhodamine (R18)‐labelled chick erythrocytes (RBC). The amino acid substitutions, lysine to arginine at position 51 or lysine to glutamic acid at position 121 of the HA2 subunit of the HA protein was enough to confer a Stachyflin‐resistant phenotype of HA protein. The molecular mechanism of anti‐HA conformational change activity of Stachyflin is discussed.
Expression of Recombinant Capsid Proteins of Chitta Virus, a Genogroup II Norwalk Virus, and Development of an ELISA to Detect the Viral Antigen
Authors: Kobayashi Shinichi , Sakae Kenji , Suzuki Yasumoto , Ishiko Hiroaki , Kamata Kunio , Suzuki Kenji , Natori Katsuro , Miyamura Tatsuo , Takeda Naokazu
Published: November 2013

The second open reading frame (ORF2) gene of the Chitta virus (CHV) was cloned to construct a recombinant baculovirus. The CHV ORF2 is predicted to encode a capsid protein of 535 amino acids (aa). CHV showed a high aa identity in the capsid region with genogroup II Norwalk virus (NV) (65–85%), but a low aa identity with genogroup I NV (44–46%). Phylogenetic analysis of the ORF2 gene demonstrated that CHV is genetically closely related to the Hawaii virus included in genogroup II NV. The recombinant capsid protein of CHV (rCHV) self‐assembled to form empty virus‐like particles (VLPs) when expressed in insect cells with the recombinant baculovirus. An enzyme‐linked immunosorbent assay (ELISA) based on antisera to rCHV was developed to detect CHV antigen in stools. The antigen ELISA appeared to be highly specific to both rCHV and CHV‐like strains. In addition, combined use of antigen ELISAs using antibodies against two antigenically distinct recombinant VLPs, the recombinant Chiba virus (rCV) and recombinant Seto virus (rSEV), enabled us to determine the genetic as well as antigenic relationship among these three viruses.
Determination of the Protective Effects of Neutralizing Anti‐Hepatitis B Virus (HBV) Immunoglobulins by Epitope Mapping with Recombinant HBV Surface‐Antigen Proteins
Authors: Park Jung‐hyun , Cho Eun‐wie , Lee Yun‐jung , Shin Song Yub , Kim Kil Lyong
Published: November 2013

Anti‐hepatitis B virus (HBV) surface‐antigen immunoglobulins prepared from human sera are clinical reagents which have been approved for prophylactic treatment in HBV‐exposed persons. The passive immunoprophylaxis with immunoglobulins is meant to cross‐link viral particles, which are then further cleared by the host's own immune system. While antibodies specific for both anti‐S‐ and anti‐preS proteins have been proved to serve as effective anti‐viral agents, so far the fine antigen specificity of clinical immunoglobulin preparations has not been determined. Using recombinant proteins covering the hepatitis B surface antigen, in the present study, the specificity of a commercially available immunoglobulin preparation was determined and immunodominant epitopes were mapped. Here, it is shown that the major reactivity of anti‐HBV immunoglobulins is directed against the S‐protein, and that no reactivity to the preS2 but a weak binding activity to the preS1 region was detectable. The antigen reactivity within the preS1 region was biased to the C‐terminal region, which indicates the presence of a putative B‐cell epitope. The evaluation of the antigen specificity and determination of novel protective epitopes will provide valuable information for the further development and improvement of prophylactic HBV immunoglobulins.
First Isolation of Stephanoascus ciferrii from a Cat
Authors: Kano Rui , Makimura Koichi , Kushida Toshiaki , Nomura Megumi , Yamaguchi Hideo , Hasegawa Atsuhiko
Published: November 2013

The present study deals with the first isolation of Stephanoascus ciferrii from a cat. A 2‐year‐old female Persian cat weighing 2.25 kg was referred to an animal hospital with a chief complaint of otitis externa of the left ear. Microscopic examination of specimen from the left ear disclosed yeast cells. The colony of the clinical isolate was cream‐colored, rough, raised and wrinkled. The microscopic examination of the clinical isolate revealed abundant branched and septated mycelia with small ramified chains of oval blastoconidia, variable in size, and arranged alongside the hyphae. Amplification of the isolate DNA with LSU rDNA primers yielded a fragment of about 570 bp, whose nucleotide sequence of the isolate showed 100% similarity to that of Stephanoascus ciferrii in the GenBank database. Therefore, the isolate was identified as Stephanoascus ciferrii, confirming the result of mycological examination by molecular analysis.
Molecular Cloning and Partial Characterization of Rat Procarboxypeptidase R and Carboxypeptidase N
Authors: Kato Tomomi , Akatsu Hiroyasu , Sato Tomoo , Matsuo Seiichi , Yamamoto Takayuki , Campbell William , Hotta Nigishi , Okada Noriko , Okada Hidechika
Published: November 2013

Carboxypeptidase R (EC 3.4.17.20) (CPR) and carboxypeptidase N (EC 3.4.17.3) (CPN) cleave carboxy‐terminal arginine or lysine residues from biologically active peptides such as kinins or anaphylatoxins in the circulation thereby regulating their activities. Although CPN is present in a stable active form in plasma, CPR is generated from proCPR, a plasma zymogen, by proteolytic enzymes such as thrombin, thrombin‐thrombomodulin complex and plasmin. We have isolated rat proCPR and CPN cDNA clones which can induce enzymatic activities in culture supernatants of the transfected cells. mRNA of proCPR was detected only in rat liver by Northern hybridization and showed hepatocyte‐specific expression. Expression of proCPR mRNA was enhanced following LPS injection, indicating that proCPR production is increased under inflammatory conditions.
Upregulated Expression of Iba1 Molecules in the Central Nervous System of Mice in Response to Neurovirulent Influenza A Virus Infection
Authors: Mori Isamu , Imai Yoshinori , Kohsaka Shinichi , Kimura Yoshinobu
Published: November 2013

The present study deals with the expression of Ibal molecules, a novel EF‐hand Ca2+‐binding protein, in the brain after stereotaxic introduction of the neurovirulent WSN strain of influenza A virus into the olfactory bulb of C57BL/6 mice. The virus selectively targeted the paraventricular and anterior olfactory nuclei. Infected neurons appeared as early as at day 3 post infection and degenerated and vanished by day 12. The Ibal molecule was normally expressed in resting microglia. The overexpression of the Ibal in microglial cells was detected at day 3 post infection, culminating at day 7 with a morphological activation. Ibal‐immunopositive macrophages outnumbered microglia in the paraventricular and anterior olfactory nuclei, where the infected neurons had degenerated. Macrophages totally disappeared by day 12, and the Ibal‐expression in microglia was reduced to a normal level by day 35. Lack of perforin predisposed the mice to long‐term virus infection of the brain, leading to the prolonged Ibal‐overexpression. These results show that the Ibal is upregulated in the mouse brain in response to influenza virus infection and may play significant roles in the regulation of some immunological and pathophysiological functions of microglia during virus infection.
Lack of Evidence of an Association between the Carriage of Virulence Plasmid and the Bacteremia of Salmonella typhimurium in Humans
Authors: Chiu Cheng‐hsun , Chu Chishih , Ou Jonathan T.
Published: November 2013

The involvement of the virulence plasmid (pSTV) of Salmonella typhimurium in human salmonellosis was examined. Most of the 224 clinical strains isolated from the blood (53) and nonblood samples (171) contained a 90 kb or larger plasmid, most of which were pSTV. The rates of pSTV carriage in the isolates showed no statistically significant difference between those derived from the blood and those from other sources (87% vs. 83%; χ2=0.49, 0.1 < P < 0.9), suggesting that pSTV may not play a critical role in promoting S. typhimurium bacteremia in humans. Nine strains with representative plasmid profiles were tested for the mouse virulence. The result revealed that these clinical isolates contained all three virulent types known: the avirulent, the highly virulent when a pSTV was present, and the moderately virulent regardless of the presence or absence of pSTV. This indicated that mouse virulence of S. typhimurium did not correlate their virulence in humans. Clinical data showed that most patients with primary bacteremia had underlying immunosuppressive diseases, whereas only a few patients with secondary bacteremia had preexisting diseases (87% vs. 13%; χ2 = 22.73, P < 0.005). It is suggested that the contribution of pSTV to S. typhimurium bacteremia in humans is likely to be limited, and both the host factor and the microbial virulence determinants on the chromosome are more important than virulence plasmid in predisposing patients to bacteremia.
Localization of Major, High Molecular Weight Proteins in Bacteroides forsythus
Authors: Higuchi Naoya , Murakami Yukitaka , Moriguchi Keiichi , Ohno Norikazu , Nakamura Hiroshi , Yoshimura Fuminobu
Published: November 2013

Bacteroides forsythus produces species‐specific major proteins with high molecular weights of 270 and 230‐kDa (270K and 230K). A specific antibody raised against 270K was used for Western blot analysis and immunoelectron microscopy. Western blot analysis showed that the 270K and 230K proteins were immunologically similar. Immunogold labeling of ultrathin‐sectioned bacterial cells and biochemical fractionation revealed that these proteins were localized at the outermost cell surface, not in the cytoplasm. These results suggest that major proteins ubiquitous to this species may form the S‐layer.
Th1‐Th2 Cytokine Kinetics in the Bronchoalveolar Lavage Fluid of Mice Infected with Cryptococcus neoformans of Different Virulences
Authors: Abe Koh , Kadota Jun‐ichi , Ishimatsu Yuji , Iwashita Tetsuji , Tomono Kazunori , Kawakami Kazuyoshi , Kohno Shigeru
Published: November 2013

Th1 immune response plays an important role in protection against infection with Cryptococcus neoformans in mice. We investigated the effect of virulence of C. neoformans on cytokine production in the lung of a mouse model of pulmonary cryptococcosis. BALB/c mice were inoculated intratracheally with a high or low virulence strain of C. neoformans, followed by serial measurements of Th1 and Th2 cytokine concentrations in the bronchoalveolar lavage (BAL) fluid using appropriate enzyme‐linked immunosorbent assay kits. The number of colony‐forming units (CFU) increased with time, and all mice infected with the highly virulent strain were dead at 28 days after inoculation. In contrast, the number of microorganisms diminished with time in the mice infected with the low virulence strain during the 4‐week study. The numbers of neutrophils and lymphocytes in the BAL fluid paralleled those of CFU. High neutrophil counts were observed in the BAL fluid of mice infected with the highly virulent strain, while lymphocyte counts were increased only in the later part of the study in mice infected with the high and low virulence strains. The concentrations of Th2 cytokine, interleukin (IL)‐4 were significantly higher in mice infected with the highly virulent strain at days 14 and 21 of infection, whereas the level of Th1 cytokine, interferon‐gamma, was significantly higher in the latter strain at days 7 and 14. Our results suggest that strain‐specific difference in the organism's ability to induce (or evade) the host immune system contributes to the outcome of infection.
Emergence of New Influenza A Viruses Which Carry an Escape Mutation of the HLA‐B27‐Restricted CTL Epitope of NP in Japan
Authors: Kashiwagi Takahito , Hamada Nobuyuki , Iwahashi Jun , Hara Koyu , Ueda Tatsuo , Noguchi Hidetaro , Toyoda Tetsuya
Published: November 2013

Influenza A viruses (H3N2) isolated in 1998 in Nagasaki, Japan, carried a mutation (384R→G) in one of the anchor amino acids of the HLA‐B27‐restricted cytotoxic T lymphocyte (CTL) epitope of NP (383–391). Phylogenetic analysis revealed that these viruses have been isolated only in Japan to date and belong to the unique lineages.
Control of Immunologically Crossreactive Leptospiral Infection by Administration of Lipopolysaccharides from a Nonpathogenic Strain of Leptospira biflexa
Authors: Matsuo Kouki , Isogai Emiko , Araki Yoshio
Published: November 2013

In our previous paper (Matsuo, K., Isogai, E., and Araki, Y., Carbohydr. Res., 328: 517–524, 2000), antigenic polysaccharides obtained from the lipopolysaccharide (LPS) fraction of a nonpathogenic leptospira, Leptospira biflexa patoc Patoc I, are shown to be broadly crossreactable with most rabbit antisera elicited by immunization with various pathogenic leptospires. The result led us to test a protective effect of the same LPS in a hamster model system by heterologously challenging with a pathogenic leptospira, L. interrogans manilae UP‐MMG. Firstly, a similarity in the antigenic epitopes of L. biflexa and L. interrogans was confirmed by the following assays. In the microscopic agglutination test (MAT), a hamster antiserum elicited by immunization with the L. biflexa‐LPS preparation was shown to agglutinate cells of L. interrogans. Contrarily, in the enzyme‐linked immunosorbent assay (ELISA), the L. biflexa‐LPS preparation was shown to crossreact with a hamster antiserum elicited by immunization with whole cells of L. interrogans. These results suggest that the same or closely related antigens may be present on the cell surfaces of both L. biflexa patoc Patoc I and L. interrogans manilae UP‐MMG. Furthermore, in a protective assay, the prior administration of a L. biflexa‐LPS preparation resulted in raising a protective response in hamsters against challenge by L. interrogans without any side effect. The protective effect was strongly dependent on the dose amounts and/or administration times of L. biflexa‐LPS. Thus, L. biflexa‐LPS preparations can use as a potent vaccine against leptospirosis caused by various leptospires.
Effect of Bicozamycin on the Eradication of Shiga Toxin‐Producing Escherichia coli in Calves
Authors: Misawa Naoaki , Sueyoshi Masuo , Uemura Ryoko , Kakemizu Yoshihiro , Kawashima Kumiko , Nagatomo Hiroshi , Kondo Fusao , Murakami Takayuki , Takahashi Yoshihiro
Published: November 2013

Fifty‐nine calves, aged 11 days to 9 months, from three farms breeding Japanese Black beef cattle in Miyazaki Prefecture, Japan, were examined for the presence of Shiga toxin‐producing Escherichia coli (STEC). A high prevalence of STEC was detected among calves, with 45 (76.3%) animals carrying STEC including different serogroups (O26, O74, O111, O114, O119, O127, O153, O157, and ONT) and toxin types. The number of STEC in the feces was estimated by a combined method involving enumeration of colony‐forming units by a plate‐most‐probable‐number (plate‐MPN) technique and polymerase chain reaction for the detection of Shiga toxin genes. Fecal shedding ranged from 101 to 10s MPN/g feces. To evaluate the safety and efficacy of bicozamycin (BCM: previously named as bicyclomycin) in eradicating STEC, 30 calves carrying STEC with or without diarrhea were examined. Fifteen calves were treated orally with BCM (10 mg/kg/day) once daily for 5 days, and the other 15 were untreated. Twenty‐four hours after the last dose, fecal specimens were collected from both groups to compare the number of coliforms and STEC with those before treatment. BCM‐treated animals had a significantly lower number of coliforms and STEC compared to the untreated calves. The STEC eradication rate was 86.6% (13/15) in the BCM‐treated group, compared to 0% (0/15) in the control group. The corresponding cure rates for diarrhea were 87.5 (7/8) and 0% (0/3), respectively. No adverse reactions were observed in the calves during treatment. It is concluded that BCM is an effective agent for the eradication of STEC in calves with or without diarrhea.
Apoptosis Observed in Murine Peritoneal Macrophages Treated with Interferon γ through Staphylococcal Enterotoxin‐Dependent Cell‐Mediated Cytotoxicity
Authors: Sakurada Junji , Tajima Akiko , Shinji Hitomi , Seki Keiko , Masuda Shogo
Published: November 2013

The concept of superantigens is well‐known and widely accepted. In this brief communication, we analyze the behaviour of antigen‐presenting cells after T‐cell activation by staphylococcal enterotoxin B, a representative superantigen. We tried to activate murine T cells by inflammatory mouse peritoneal macrophage in the presence of staphylococcal enterotoxin B, but no T‐cell activation was observed. We, therefore, analyzed surface‐specific antigens of the macrophages. They expressed insufficient amounts of MHC class II, CD80 and CD86 molecules on the surface of the cells. On the contrary, increased amounts of MHC class II and CD86 molecules on the cell surfaces were observed after incubation with interferon γ. Interferon γ‐primed macrophages were found to be competent to activate T cells in the presence of staphylococcal enterotoxin B. To our surprise, these macrophages underwent apoptosis in parallel with T‐cell activation.
Size Conversion of a Linear Plasmid in the Relapsing Fever Agent Borrelia duttonii
Authors: Takahashi Yukie , Cutler Sally J. , Fukunaga Masahito
Published: November 2013

Borreliae have genomes composed of both linear and circular replicons. We have characterized the organization of linear DNA molecules from the Borrelia duttonii strain Ly. It contains a linear one megabase chromosome and 12 linear plasmids of 11 to 200 kb in size. A variant of the strain obtained after successive in vitro cultivation in BSKII medium had a 69 kb molecule instead of the 44 kb linear plasmid. No detectable differences in the growth rates and cellular structures were found. Southern hybridization using the vsp33 gene sequence from Borrelia hermsii as a probe showed that both plasmids (69 and 44 kb molecules) contained a similar part of the sequence. The spirochetes of the parental strain cause erythrocytes to aggregate in mice blood, but the variant did not form such aggregates and seemed to have lost its infectivity in mice. Size conversion of the linear plasmid may be associated with the host‐parasite relationship in mammals.
The Significance of Cytomegalovirus Infection over the Clinical Course of Adult T‐Cell Leukemia/Lymphoma
Authors: Fujiwara Hiroshi , Eizuru Yoshito , Matsumoto Tadashi , Kukita Toshimasa , Imaizumi Rie , Kawada Hideaki , Ohtsubo Hideo , Matsushita Kakushi , Arima Naomi , Tei Chuwa
Published: November 2013

The significance of cytomegalovirus (CMV) infections developed over the clinical course of adult T‐cell leukemia/lymphoma (ATLL) were evaluated in relation to the patient survival rate, ATL activity and immunocompetent cells. ATLL patients with CMV infections on admission exhibited a poor survival rate, while patients with CMV infections at any time after admission survived longer than those not infected with this virus. ATLL patients who exhibited a numbers of CMV infection on admission showed higher ATL activity and had lower numbers of CD8‐positive and CD56‐positive cells than those who developed CMV infections at any time after admission. Therefore, it appears likely that patients with CMV infections on admission were in an immunosuppressive state due to aggressive ATL activity.
PCR Detection of Capnocytophaga Species in Dental Plaque Samples from Children Aged 2 to 12 Years
Authors: Hayashi Fumiko , Okada Mitsugi , Zhong Xiaobo , Miura Kazuo
Published: November 2013

The purpose of this study was to detect the presence of Capnocytophaga sputigena, C. ochracea, and C. gingivalis in plaque samples from the toothbrushes of 122 children, using a polymerase chain reaction (PCR) method. The subjects were 25, 85, and 12 children with healthy gingiva, gingivitis, and periodontitis, respectively, ranging in age from 2–12 years old. Plaque samples were collected from all erupted tooth sites using a sterile toothbrush. The mean amount of DNA recovered from the samples was approximately 19.3 μg, which was deemed sufficient for performing a PCR‐based survey. C. sputigena prevalence in healthy, gingivitis, and periodontitis subjects was 48.0%, 36.5% and 25.0%, respectively, that for C. ochracea was 100%, 89.4%, and 50.0%, respectively, and that for C. gingivalis was 96.0%, 84.7%, and 75.0%, respectively. The lowest age of positive subjects was approximately 2 years. Our results showed that C. sputigena was moderately prevalent, whereas C. ochracea and C. gingivalis were commonly detected in the oral cavities of the tested children, suggesting that all of these species become established in the early years.
Relatedness between the Coagulase Gene 3′‐End Region and Coagulase Serotypes among Staphylococcus aureus Strains
Authors: Kanemitsu Keiji , Yamamoto Hiroyuki , Takemura Hiromu , Kaku Mitsuo , Shimada Jingoro
Published: November 2013

The 3′‐end region of the coagulase gene from 22 strains of Staphylococcus aureus including 10 standard serotype strains was sequenced, and five subgroups with 48 tandem repeating units were distinguished among the tested strains. Phylogenetic analysis of the 3′‐end region of the coagulase gene indicated that strains belonging to the same serotype were clustered in the same branch. A phylogenetic tree of the deduced amino acid sequences revealed that the C‐terminal region might not be responsible for the epitope of the coagulase protein.
Intraperitoneal Immune Cell Responses to Eubacterium saphenum in Mice
Authors: Salam Mohammad Abdus , Sato Michiko , Hoshino Etsuro
Published: November 2013

Oral asaccharolytic Eubacterium saphenum, which are newly isolated Gram‐positive rods and one of the predominant microorganisms in human periodontal pockets, were injected intraperitoneally in mice to elucidate their pathogenicity in periodontal diseases. Infiltrating immune cells in the peritoneal exudate were quantitated and intracellular T cell (CD4+/CD8+/γδ+) production of cytokines IL‐4 and IFN‐γ which are related to cellular and humoral immunity, respectively, was determined. Neutrophils appeared first in peritoneal exudates, followed by macrophages and lymphocytes, after the injection of either E. saphenum or Porphyromonas gingivalis. Intracellular IL‐4+ and IFN‐γ+ γδ T cells were detected in the exudates after the injection of E. saphenum (4.6±0.8% and 10.1±1.4%, respectively) and P. gingivalis (5.3±1.6% and 10.1±2.1%, respectively). The intracellular production of IL‐4/IFN‐γ in CD4+/CD8+ T cells was rather low indicating that the main response was from γδ T cells which initiated the immune reactions in mouse peritoneal cavities after injection of E. saphenum or P. gingivalis. Serum IgG and IgM levels were elevated in animals injected with E. saphenum and similarly with P. gingivalis. The present study showed that with slight differences, similar modes of cell response and cytokine and Ig production were observed after intraperitoneal injection of both E. saphenum and P. gingivalis, indicating that E. saphenum may play just as important a role in periodontal diseases as P. gingivalis.
A Comparison of Complete Genome Sequences of the Attenuated RC‐HL Strain of Rabies Virus Used for Production of Animal Vaccine in Japan, and the Parental Nishigahara Strain
Authors: Ito Naoto , Kakemizu Misako , Ito Kumi A. , Yamamoto Atsuko , Yoshida Yoshio , Sugiyama Makoto , Minamoto Nobuyuki
Published: November 2013

In order to establish the molecular basis of the pathogenicity of the attenuated RC‐HL strain of rabies virus used for the production of animal vaccine in Japan, the complete genome sequence of this strain was determined and compared with that of the parental Nishigahara strain which is virulent for adult mice. The viral genome of both strains was composed of 11,926 nucleotides. The nucleotide sequences of the two genomes showed a high homology of 98.9%. The homology of the G gene was lower than those of N, P, M and L genes at both nucleotide and deduced amino acid levels, and the percentage of radical amino acid substitutions on the G protein was the highest among the five proteins. These findings raise the possibility that the structure of the G protein is the most variable among the five proteins of the two strains. Furthermore, we found two clusters of amino acid substitutions on the G and L proteins. The relevance of these clusters to the difference in the pathogenicity between the two strains is discussed.
Hemadsorption Expressed by Cloned H Genes from Subacute Sclerosing Panencephalitis (SSPE) Viruses and Their Possible Progenitor Measles Viruses Isolated in Osaka, Japan
Authors: Furukawa Kyoko , Ayata Minoru , Kimura Masatsugu , Seto Toshiyuki , Matsunaga Isamu , Murata Ryosuke , Yamano Tsunekazu , Ogura Hisashi
Published: November 2013

Most subacute sclerosing panencephalitis (SSPE) viruses, including our Osaka‐1, −2, and −3 strains isolated in Osaka, have shown negative hemadsorption (HAD) by African green monkey red blood cells. This property has been thought to be characteristic of SSPE virus as compared to the positive reaction of the standard Edmonston strain of measles virus (MV). However, this assumption has become quite obscure because MV mutates frequently at the genetic level during its multiplication and also because recent field strains isolated by lymphoblastoid cell lines have shown negative HAD. To investigate the above issue, the nucleotide sequences of the hemagglutinin (H) genes from SSPE virus Osaka‐1, −2, or −3 strains were compared to those of various MV field strains isolated in Osaka by Vero cells. The H gene sequences of three SSPE strains were relatively conserved without such biased hypermutation as had been observed in the matrix (M) gene of three SSPE strains. However, this analysis of the H gene sequence of the SSPE viruses enabled us to deduce possible progenitor MVs, which are in agreement with the deduction from the M gene analysis we reported previously. The HAD of Vero cells transfected with the cloned H cDNAs from the SSPE strains and their progenitors suggested that negative HAD of the SSPE viruses has been maintained as one of original properties of the progenitor MVs rather than having been acquired as an altered one during long‐term persistent infection in the brains of patients with SSPE.
Cytokine Responses to Recombinant Cholera Toxin B Subunit Produced by Bacillus brevis as a Mucosal Adjuvant
Authors: Maeyama Jun‐ichi , Isaka Masanori , Yasuda Yoko , Matano Keiko , Kozuka Satoshi , Taniguchi Tooru , Ohkuma Kunio , Tochikubo Kunio , Goto Norihisa
Published: November 2013

We attempted to clarify the mechanism of the mucosal adjuvanticity of recombinant cholera toxin B subunit (rCTB), which is inherently uncontaminated with the holotoxin produced by Bacillus brevis and has a powerful mucosal adjuvant activity, on cytokine responses compared with that of cholera toxin (CT). rCTB had no ability to stimulate cyclic AMP formation in mouse peritoneal macrophages (Mφ). Cytokine production by non‐immunized Mφ cultured with rCTB or CT and by the spleen cells of mice co‐immunized intranasally with ovalbumin (OVA) and rCTB or CT was examined. rCTB alone did not induce interleukin (IL)‐1α/β or IL‐6 production by Mφ, but combination of rCTB with lipopolysaccharide (LPS) enhanced both IL‐1α/β production. Conversely, CT plus LPS suppressed IL‐1α/β production more than LPS alone. Both rCTB and CT suppressed IL‐12 secretion induced by interferon γ (IFN γ) plus LPS. IL‐2, IL‐4, IL‐5, and IL‐10 were secreted by mouse spleen cells restimulated with OVA after intranasal co‐administration of OVA together with rCTB, and in response to CT, the same cytokines were secreted. The different effect of rCTB on Mφ from that of CT may mean a difference between the mechanisms of rCTB and CT during the early stage of an immune response.
Identification and Characterization of the sodA Genes Encoding Manganese Superoxide Dismutases in Vibrio parahaemolyticus, Vibrio mimicus, and Vibrio vulnificus
Authors: Kimoto Ryoko , Funahashi Tatsuya , Yamamoto Noriko , Miyoshi Shin‐ichi , Narimatsu Shizuo , Yamamoto Shigeo
Published: November 2013

Sequencing of Fur titration assay‐positive clones obtained from genomic DNA libraries of Vibrio parahaemolyticus, V. mimicus and V. vulnificus revealed open reading frames encoding proteins of 202, 205 and 202 amino acid residues, respectively. Each open reading frame was preceded by a predicted Fur box which overlaps a likely promoter with similarity to the −10 and −35 consensus sequence of Escherichia coli. The deduced amino acid sequences shared considerable homology with bacterial Mn‐containing superoxide dismutases (MnSODs). Consistent with this, these Vibrio strains produced proteins with SOD activity resistant to inhibition by H2O2 and KCN only when grown under iron‐limiting conditions. Primer extension analysis of the total RNA from these vibrios revealed iron‐repressible expression of the genes. Furthermore, when grown under iron‐limiting conditions, E. coli carrying a plasmid with each cloned gene over‐expressed protein with the same electrophoretic mobility and insensitivity of SOD activity to H2O2 and KCN. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis followed by N‐terminal amino acid sequencing revealed that proteins (MnSODs) having N‐terminal amino acid sequences consistent with those deduced from the corresponding genes were present in cell lysates of the vibrios grown under these iron‐limited conditions. These results demonstrate that the genes cloned in this study are sodA homologs encoding MnSODs, whose expression is regulated by the iron status of the growth medium. PCR using a primer set based on the V. parahaemolyticus sodA sequence revealed the presence of homologous genes in certain other Vibrio species.
Plasminogen Activator Production in a Rat Model of Pneumocystis carinii Pneumonia
Authors: Angelici Elena , Contini Carlo , Spezzano Massimiliano , Romani Roberto , Carfagna Paolo , Serra Pietro , Canipari Rita
Published: November 2013

Several studies have indicated that the serine protease urokinase‐plasminogen‐activator (uPA) is an important factor in host defense against pulmonary pathogens. To gain a better insight into the role of uPA in Pneumocystis carinii (P. carinii) pneumonia (PCP), we evaluated PA production in alveolar macrophages (AMs) obtained from rats with steroid‐induced PCP. Treatment with cortisone acetate favored PCP in 91% of rats. In the bronchoalveolar lavage (BAL) samples of immunosuppressed rats both with and without PCP, we observed a decrease in uPA activity as well as a decrease in cell number. Urokinase‐PA production by AMs was reduced in rats treated with cortisone alone. However, an increase in cell‐associated uPA was observed in rats with PCP. This increase appears to be produced in response to P. carinii infection. In fact, when AMs obtained from untreated healthy or immunosuppressed uninfected rats were challenged with P. carinii, a significant increase in PA activity in cell lysates was observed, though a lower response was obtained in cortisone‐treated animals. Our results suggest that healthy AMs respond to the presence of P. carinii with an increase in uPA production and that this response in immunodepressed rat‐AMs is partially impaired.
Enterocolitis Caused by Methicillin‐Resistant Staphylococcus aureus: Molecular Characterization of Respiratory and Digestive Tract Isolates
Authors: Watanabe Hiroshi , Masaki Hironori , Asoh Norichika , Watanabe Kiwao , Oishi Kazunori , Kobayashi Shinobu , Sato Akiyoshi , Nagatake Tsuyoshi
Published: November 2013

We investigated the mechanism of outbreak of enterocolitis caused by methicillin‐resistant Staphylococcus aureus (MRSA). Five epidemiological markers [coagulase type, enterotoxin type, toxic shock syndrome toxin‐1 (TSST‐1) production, β‐lactamase production and pulsed‐field gel electrophoresis (PFGE)] of 45 strains of MRSA isolated simultaneously from the respiratory tract (nasal cavity and/or pharynx and/or sputum) and stool (plus one sample of gastric juice) in 13 patients (8 males and 5 females, mean age, 77.1 years) were compared retrospectively. Forty‐four of the 45 isolates of MRSA were positive for enterotoxin C and TSST‐1 production, and the remaining isolate was positive for enterotoxin A and negative for TSST‐1 production. All isolates were coagulase type II, and 27 showed β‐lactamase production. The patterns of coagulase type, enterotoxin type, TSST‐1 and β‐lactamase production of MRSA isolated from the respiratory tract were similar to those of MRSA isolated from the intestine in 12 of 13 patients. Molecular typing by PFGE demonstrated that the pattern of respiratory tract isolates was identical to those of stool isolates in 9 (69.2%), similar in 3 (23.1%), and different in 1 (7.7%). The data suggested that enterocolitis might be caused by the MRSA colonized in the respiratory tract and incorporated into the digestive tracts. Therefore, we propose that early eradication of MRSA in the respiratory tract is important for protection of patients against the development of enterocolitis, particularly in susceptible patients, e.g., immunocompromised or pre‐operated patients with digestive diseases, especially malignant disease.
DNA Substrates Influence the Recombination Efficiency Mediated by FLP Recombinase Expressed in Mammalian Cells
Authors: Nakano Masakazu , Ishimura Masakazu , Chiba Joe , Kanegae Yumi , Saito Izumu
Published: November 2013

The FLP recombinase derived from Saccharomyces cerevisiae mediates precise site‐specific recombination between a pair of FLP recognition targets (FRTs). Like the Cre/loxP system derived from bacteriophage P1, the FLP/FRT system has recently been applied to gene regulation systems using an FLP‐expressing recombinant adenovirus (rAd) (Nakano et al, Nucleic Acids Res. 29: e40, 2001). In an attempt to improve the FLP/FRT system by altering its DNA substrates, we compared the recombination efficiency among different substrates by a quantitative in vitro assay using FLP expressed in mammalian cells. Unexpectedly, we found that one linearized DNA substrate showed 4‐ to > 20‐fold lower recombination efficiency than other substrates, which phenomenon has not been observed in the Cre/loxP system. The quantitative in vitro assay using truncated DNA substrates suggested that the recombination efficiency seemed to be influenced not only by the linearized position of the substrate, but also by the length between a pair of FRTs. Such substrate preference of FLP expressed in mammalian cells should probably be noted when designing versatile applications of the FLP/FRT system as a gene regulation system in mammalian systems. Fortunately, however, we demonstrated that no substrate preference was observed when using a particular substrate (pCAFNF5) and the preference was reduced when using a certain pair of mutant FRTs (f72), which will also be a promising tool for simultaneous gene regulation in combination with wild‐type FRT.
Bacterial Arylsulfate Sulfotransferase as a Reporter System
Authors: Yun Hee‐Jeong , Kwon Ae‐Ran , Choi Eung‐Chil
Published: November 2013

To investigate whether the arylsulfate sulfotransferase (ASST) is suitable as a reporter system for monitoring gene expression, a reporter vector carrying the fragments of the astA coding region without the promoter region was constructed and designated as pSY815. As a test of the ASST reporter system's suitability, the regulatory regions of ermC and lacZ were inserted upstream of the coding region of the reporter gene to generate pSY815‐EC and pSY815‐LZ, respectively. In the absence of the inserted regulatory regions, the plasmids displayed very low background activities in Bacillus subtilis and Escherichia coli. The ASST activity under the control of the ermC regulatory region was increased 4.4‐fold in B. subtilis when induced by 0.1 μgml–1 of erythromycin. These results were consistent with a lacZ reporter gene assay of the ermC regulatory region. Furthermore, we confirmed that the lacZ promoter in E. coli was strongly induced to a 17.9‐fold increase by 0.05 mM of isopropyl‐β‐D‐thiogalactopyranoside (IPTG) in this reporter system. These results indicate that the ASST is a suitable reporter system. The lack of endogenous activity, the simple detection of enzyme activity in the living cell, the commercially available non‐toxic substrates, and the high sensitivity make ASST a useful genetic reporter system for monitoring gene expression and understanding gene regulation.
Menadione‐Catalyzed Luminol Chemiluminescent Assay for the Viability of Escherichia coli ATCC 25922
Authors: Yamashoji Shiro , Takeda Mitsue
Published: November 2013

Escherichia coli ATCC 25922 produced O2− in the presence of menadione, and O2−‐dependent luminol chemiluminescence intensity was proportional to colony‐forming unit (CFU) in the exponential phase. CFU was determined by using a 96‐well plate at a range of 3 × 103 to 8 × 107 CFU/well (0.1 ml) after a 10‐min incubation with menadione, followed by chemiluminescent assay for 5 s. After a 4‐hr incubation of E. coli (105 CFU/0.1 ml) with menadione and an antimicrobial agent inhibiting the synthesis of peptidoglycan, protein, and DNA, the inhibitory concentration (IC) of the antimicrobial agent determined by menadione‐catalyzed luminol chemiluminescent assay was in good agreement with minimal inhibitory concentration (MIC) of the NCCLS (National Committee for Clinical Laboratory Standard) method requiring 18 hr. Menadione‐catalyzed luminol chemiluminescent assay is expected to be useful for the rapid determination of cell viability under the conditions of various cell growths and stresses.
Role of Interleukin (IL)‐1 Type 1 Receptor in Mycobacterial Infection
Authors: Sugawara Isamu , Yamada Hiroyuki , Hua Shucheng , Mizuno Satoru
Published: November 2013

It is important to gain a better understanding of IL‐1‐mediated signaling events in mycobacterial infection. In order to clarify the role of IL‐1 receptor type 1 (IL‐1 R1) in IL‐1 R1, knockout (KO) mice were infected with either Mycobacterium tuberculosis H37Rv or Kurono strain by the respiratory route, and their ability to control mycobacterial growth, pulmonary granuloma formation, and cytokine mRNA expression was investigated. IL‐1 R1 KO mice developed significantly larger (P < 0.01) granulomatous lesions with neutrophil infiltration in their lungs than wild‐type mice did after infection with the M. tuberculosis Kurono strain. The number of mycobacterial colonies in lungs and spleen increased from five weeks post‐infection. Interferon‐γ production by spleen cells was low in IL‐1 R1 KO mice. It is concluded that the IL‐1 R1 is essential for IL‐1‐mediated signaling events in mycobacterial infection.
Staphylococcus aureus Requires Increased Level of Ca2+ or Mn2+ to Grow Normally in a High‐NaCl/Low‐Mg2+ Medium
Authors: Ochiai Toshiro
Published: November 2013

Mg2+‐availability in Staphylococcus aureus cells decreased significantly with increasing NaCl concentration in growth media. Cells grew in a NaCl‐free, Chelex resin‐treated complex medium only if the medium was supplemented with 50 μM MgCl2, while, growth was limited when the medium was further supplemented with 1.0 M NaCl. Cells grown in such a high‐NaCl/low‐Mg2+ medium exhibited the morphologic abnormality of larger than normal cells. Both sufficient growth and normal cell morphology were restored by increasing Mg2+ concentration in a high‐NaCl medium, or by supplementation with either CaCl2 or MnSO4 in a high‐NaCl/low‐Mg2+ medium. Supplementing with BaCl2, SrCl2 or FeSO4, however, had no effect. These results indicate that Ca2+ and Mn2+ might play some essential role in the growth of Staphylococcus aureus in a high‐NaCl/low‐Mg2+ environment.