bluuebunny/crossref_metadata_2025 huggingface dataset:
Interferon Gamma Response to Combinations 38 kDa/CFP‐10, 38 kDa/MPT‐64, ESAT‐6/MPT‐64 and ESAT‐6/CFP‐10, Each Related to a Single Recombinant Protein of Mycobacterium tuberculosis in Individuals from Tuberculosis Endemic Areas
Authors: Tavares Ricardo C. Oliveira , Salgado Jorge , Moreira Valéria Barbosa , Ferreira Mônica Antonia S. , Mello Fernanda C. Queiroz , Leung Janaína W. , Fonseca Leila de Souza , Spallek Ralf , Singh Mahaviir , Saad Maria Helena F.
Published: November 2013

Several antigens of Mycobacterium tuberculosis have been identified and specificity to one or multiple antigens could determine the distinction between protective and pathogenic host reaction. Therefore T cell immune response to combinations 38 kDa/CFP‐10, 38 kDa/MPT‐64, ESAT‐6/MPT‐64 and ESAT‐6/CFP‐10 (each related to a single protein of Mycobacterium tuberculosis) in individuals from tuberculosis endemic areas have been examined. ELISA was used to detect IFN‐γ production in PBMC priming with single proteins and combinations in a panel of 105 individuals: 38 tuberculosis patients (6 untreated and 32 treated) and 67 healthy controls with tuberculin skin test positive or negative (TST). Brazilian TB patients highly recognized ESAT‐6 (66%), but combinations improved response in the following order: ESAT‐6/MPT‐64 (89%) > ESAT‐6/CFP‐10 (73%) > 38 kDa/CFP‐10 (70%), the last combination showing the highest specificity (TST+=42% and TST–=83%). Average IFN‐γ production in TB patients was significantly higher for 38 kDa/CFP‐10 (P=0.012) and 38 kDa/MPT‐64 (P<0.035), when compared to single antigens. None of the combinations was able to discriminate TB patients from TST+ controls; however, 38 kDa/CFP‐10 displayed a borderline significance (P=0.053). Similar to the ESAT‐6/CFP‐10 combination, IFN‐γ response to 38 kDa/CFP‐10 showed an increased tendency in treated patients, although not significant (P=0.16). We demonstrated for the first time that 38 kDa/CFP‐10 had prediction sensitivity for TB patients similar to the ESAT‐6/CFP‐10 combination and also significant response improvement related to the single proteins with more selective reactivity among TST‐positive individuals, which could be of potential interest for diagnostic evaluation for tuberculosis infection.
Increased Inhibitory Capacity of an Anti‐C5a Complementary Peptide Following Acetylation of N‐terminal Alanine
Authors: Okada Noriko , Asai Suzuka , Hotta Aya , Miura Noriko , Ohno Naohito , Farkas Imre , Hau Lewis , Okada Hidechika
Published: November 2013

Amino acids 37 to 53 (RAARISLGPRCIKAFTE) of C5a anaphylatoxin form an essential region for C5a function. To target this sequence, we generated a complementary peptide (ASGAPAPGPAGPLRPMF) designated PepA which has a potent inhibitory effect on C5a activity. By introducing an acetyl group at the N‐terminal alanine of PepA, an acetylated form was generated which was designated AcPepA. The acetylation resulted in increased inhibition of C5a stimulation of neutrophils as determined by Ca influx. Furthermore, AcPepA partially inhibited the lethal shock induced in mice by intravenous administration of Candida albicans water‐soluble mannoprotein‐β‐glucan complex. In addition, local skin inflammation in rats caused by an anti‐Crry monoclonal antibody was suppressed when AcPepA and the antibody were injected together, while PepA had little inhibitory capacity. The potent inhibitory capacity of AcPepA was also confirmed by a skin reaction of guinea pigs inoculated with recombinant human C5a together with AcPepA.
C Terminal Half Fragment (50 kDa) of Heavy Chain Components of Clostridium botulinum Type C and D Neurotoxins Can Be Used as an Effective Vaccine
Authors: Lee Jae‐Chul , Hwang Hyun‐Jung , Sakaguchi Yoshihiko , Yamamoto Yumiko , Arimitsu Hideyuki , Tsuji Takao , Watanabe Toshihiro , Ohyama Tohru , Tsuchiya Tomofusa , Oguma Keiji
Published: November 2013

Recombinant whole heavy chains (H, 100 kDa) and their N‐terminal (Hn, 50 kDa) and C‐terminal (Hc, 50 kDa) half fragments of Clostridium botulinum type C and D neurotoxins were expressed as glutathione S‐transferase (GST) fusion proteins in Escherichia coli. GST eliminated‐preparations of H (10 μg), Hn (5 μg), Hc (5 μg), or a mixture of Hn (5 μg) and Hc (5 μg) of types C and D were mixed with an equal volume of adjuvant, and then were twice injected into mice subcutaneously. After immunization, the mice were challenged with up to 106 the minimum lethal doses (MLD)/0.5 ml of C or D toxin, the type of which was same as that of the immunogens. All of the mice immunized with antigens except for Hn survived against 105 to 106 MLD/0.5 ml of the toxins, but the mice immunized with Hn were killed by 100 MLD/0.5 ml. The mice immunized with a mixture of C‐Hc and D‐Hc, each 5 μg, also showed a high level of resistance against both C and D toxins. Antibody levels immunized with GST fused‐ or GST eliminated‐preparation were quite similar. These results indicate that recombinant GST‐fused Hc can be used as a safe and effective vaccine for type C and D botulism in animals. It also became clear that one time inoculation with a large amount of C‐Hc or D‐Hc, 100 μg, is useful for vaccine trials in mice.
Serum Withdrawal‐Induced Apoptosis in ZrchI Prion Protein (PrP) Gene‐Deficient Neuronal Cell Line Is Suppressed by PrP, Independent of Doppel
Authors: Nishimura Takuya , Sakudo Akikazu , Hashiyama Yoriko , Yachi Akiko , Saeki Keiichi , Matsumoto Yoshitsugu , Ogawa Masaharu , Sakaguchi Suehiro , Itohara Shigeyoshi , Onodera Takashi
Published: November 2013

Previous studies have shown that cellular prion protein (PrPC) plays anti‐apoptotic and anti‐oxidative role against cell death induced by serum‐deprivation (SDP) in an immortalized prion protein gene‐deficient neuronal cell line derived from Rikn prion protein (PrP) gene‐deficient (Prnp–/–) mice, which ectopically produce excess Doppel (Dpl) (PrP‐like glycoprotein). To investigate whether PrPC inhibits apoptotic neuronal cell death without Dpl, an immortalized cell line was established from the brain of ZrchI Prnp–/– mice, which do not show ectopic expression of Dpl. The results using a ZrchI neuronal Prnp–/– cell line (NpL2) showed that PrPC potently inhibited SDP‐induced apoptotic cell death. Furthermore, PrPC expression enhanced the superoxide dismutase (SOD) activity in NpL2 cells. These results indicate that Dpl production did not affect anti‐apoptotic and anti‐oxidative functions of PrP, suggesting that PrPC may be directly correlated with protection against oxidative stress.
Biofilm Acts as a Microenvironment for Plankton‐Associated Vibrio cholerae in the Aquatic Environment of Bangladesh
Authors: Islam Mohammad Sirajul , Jahid Mohammad Iqbal Kabir , Rahman Mohammad Majibur , Rahman Mohammed Ziaur , Islam Mohammad Shafiqul , Kabir Mohammad Shahidul , Sack David Allen , Schoolnik Gary K.
Published: November 2013

The role of biofilm as a microenvironment of plankton‐associated Vibrio cholerae was investigated using plexiglass as a bait A total of 72 biofilm samples were tested using culture, direct fluorescent antibody (DFA) and molecular techniques following standard procedures. Culturable V. cholerae (smooth and rugose variants) were isolated from 33% of the samples. V. cholerae O1 were detected by FA technique throughout the year except April and June. All V. cholerae O1 isolates were positive for tcpA, ctxA and ace genes while V. cholerae non‐O1, non‐O139 isolates lacked these genes. V. cholerae O1 (both Inaba and Ogawa) strains had identical ribotype pattern (R1), but V. cholerae non‐O1, non‐O139 had different ribotype patterns. All V. cholerae O1 strains were resistant to vibrio‐static compound (O/129). All V. cholerae O1 except one were resistant to trimethoprime‐sulphamethoxazole, streptomycin, nalidixic acid and furazolidone but sensitive to ciprofloxacin, and tetracycline. This study indicates that plexiglass can act as a bait to form biofilm, a microenvironment that provides shelter for plankton containing V. cholerae in the aquatic environment of Bangladesh.
Distinct Function of Pseudomonas aeruginosa Type IV Pili Disclosed in the Bacterial Pass‐Through of Membrane Filter with Smaller Pore Sizes
Authors: Hasegawa Hiroyuki , Tanikawa Taichiro , Nozawa Takashi , Nakazawa Kentaro , Nakagawa Yoji , Matsuyama Tohey
Published: November 2013

Membrane filter pass‐through ability of Pseudomonas aeruginosa was analyzed with isogenic mutants. A flagellum‐deficient fliC mutant required two‐times longer time (12 hr) to pass through a 0.45‐μm pore size filter. With 0.3‐ and 0.22‐μm filters, however, the fliC mutant showed no remarkable disability. Meanwhile a pilA mutant defective in twitching motility failed to pass through the 0.22‐μm filter. Complementation of the mutant with pilA gene on a plasmid restored the twitching motility and the 0.22‐μm filter pass‐through activity. Thus, the distinctive role of P. aeruginosa type IV pili in infiltration into finer reticulate structures was indicated.
A Novel In Vitro Pharmacokinetic/Pharmacodynamic Model Based on Two‐Compartment Open Model Used to Simulate Serum Drug Concentration‐Time Profiles
Authors: Tomita Tsutomu , Ohara‐Nemoto Yuko , Moriyama Hitomi , Ozawa Ayako , Takeda Yoshifumi , Kikuchi Ken
Published: November 2013

An in vitro pharmacokinetic/pharmacodynamic perfusion model that simulates a two‐compartment open model of serum drug concentration‐time profiles following intravenous bolus injection and infusion was developed and mathematically described. In the present apparatus model, flow was kept in a one‐way mode to avoid liquid traffic, and the washout effect seen in dilution models was overcome by embedding the tested bacteria in low melting point agarose gel. The validity of the equations and the reproducibility of the apparatus model were ascertained by simulating the concentration‐time profiles of cefazolin and fosfomycin by substitution of their pharmacokinetic parameters obtained from humans for the equations. An empirical regimen 1X(q24h) of 1 g with cefazolin administered by intravenous infusion effectively killed a Staphylococcus aureus strain. The same regimen with fosfomycin produced a marked kill‐curve with a fosfomycin‐susceptible enterohaemorrhagic Escherichia coli O157:H7, whereas considerable regrowth was observed with a resistant strain. These results indicated that the present model was able to provide a convenient and reliable method for evaluating the efficacy of antimicrobial agents administered by intravenous infusion.
Extensive Accumulation of Influenza Virus NS1 Protein in the Nuclei Causes Effective Viral Growth in Vero Cells
Authors: Ozaki Hiroichi , Kida Hiroshi
Published: November 2013

We previously showed that modified A/Puerto Rico/8/34 (PR8) influenza master strain had improved viral rescue and growth properties in African green monkey kidney (Vero) cell line by introducing NS gene of Vero‐adapted A/England/1/53 (vaEng53). In the present study, it was found that the NS1 protein derived from vaEng53 was extensively accumulated in the nuclei than that of PR8. This accumulation was caused by 7 amino acid differences in C‐terminal region of NS1 protein. These results suggest that specific accumulation of NS1 protein may contribute to efficient viral replication in Vero cells.
Chlamydia pneumoniae Respiratory Infections in Taiwan
Authors: Hsu Min‐Chih , Lin Tsun‐Mei , Huang Chung‐Ter , Liao Mei‐Hui , Li Shu‐Ying
Published: November 2013

To understand the epidemiology of Chlamydia pneumoniae acute infections in Taiwan, we collected 116 paired and 244 single sera from patients suspected of C. pneumoniae infection and conducted microimmunofluorescence test. Eighty‐three patients (83/360, 23%) met the diagnostic criteria of current C. pneumoniae infection. The C. pneumoniae infections were significantly higher in men than in women (P≤0.0001) and were most frequent in the group of 40–49 year‐olds, and the people older than 70 years old. C. pneumoniae infection often occurred in the late autumn lasting to the cold winter and in the transition period between the spring and summer.
Neutralizing Activity of Polyvalent Gb3, Gb2 and Galacto‐Trehalose Models against Shiga Toxins
Authors: Neri Paola , Nagano Saori Itoh , Yokoyama Shin‐ichiro , Dohi Hirofumi , Kobayashi Kazukiyo , Miura Tsuyoshi , Inazu Toshiyuki , Sugiyama Tsuyoshi , Nishida Yoshihiro , Mori Hiroshi
Published: November 2013

Shiga toxin (Stx) is one of the most critical factors in the development of hemolytic uremic syndrome and other systemic complications following enterohemorrhagic Escherichia coli (EHEC) infection. Substances neutralizing Stx by interfering with toxin‐receptor binding have been explored as therapeutic candidates for EHEC infection. In this study, we examined globotriaosyl (Gb3), galabiosyl (Gb2) and galacto‐trehalose, each of which was synthetically conjugated with a polyacrylamide backbone, for Stx‐neutralizing activity. Galacto‐trehalose was designed as a Gb2 mimicking, unnatural Stx‐ligand that was expected to show tolerance to enzymatic degradation in vivo. Galacto‐trehalose copolymer showed neutralizing activity against Stx‐1 but not Stx‐2 in a HeLa cell cytotoxicity assay. It was thought that galacto‐trehalose copolymer could be a lead compound for the treatment of Stx‐mediated diseases, although it requires modification to show neutralizing activity to Stx‐2. The Gb3 copolymer with high sugar unit density showed stronger neutralizing activity against Stx‐2 than those with lower density. However, the density‐dependency of the neutralizing activity was less obvious against Stx‐1. Intravenous administration of the Gb3 copolymer prevented death in mice lethally infected with Stx‐1‐ and Stx‐2‐producing E. coli O157:H7. Thus, we demonstrated that the artificial Gb3 copolymer could neutralize Stx‐1 and the more clinically relevant Stx‐2 in vitro and effectively inhibit Stx toxicity in vivo.
Failure of Multinucleated Giant Cell Formation in K562 Cells Infected with Newcastle Disease Virus and Human Parainfluenza Type 2 Virus
Authors: Yamakawa Izumi , Tsurudome Masato , Kawano Mitsuo , Nishio Machiko , Komada Hiroshi , Ito Morihiro , Uji Yukitaka , Ito Yasuhiko
Published: November 2013

When K562 cells were infected with Newcastle disease virus (NDV) or human parainfluenza type 2 virus (hPIV‐2), polykaryocyte formation could not be detected. Failure of multinucleated giant cell formation in K562 cells infected with either NDV or hPIV‐2 is due to disturbance of the viral envelope‐cell fusion step or to defect in the cell‐cell fusion step, respectively. Especially, NDV completely replicated in K562 cells, and the hemagglutinin‐neuraminidase and fusion proteins expressed on the cell surface of NDV‐infected K562 cell were fully functional for fusion inducing activity. Therefore, the cell membranes of K562 cells are considered to be resistant to virus‐induced cell fusion. Membrane fusion is regulated by many host factors including membrane fluidity, cytoskeletal systems, and fusion regulatory proteins system. An unknown regulatory mechanism of virus‐induced cell fusion may function on the cell surface of K562 cells.
Genetic Characterization of Thermal Tolerance in Enterobacter sakazakii
Authors: Asakura Hiroshi , Morita‐Ishihara Tomoko , Yamamoto Shigeki , Igimi Shizunobu
Published: November 2013

Enterobacter sakazakii is an opportunistic pathogen that causes meningitis and necrotizing enterocolitis in neonates. Here we characterized the thermal tolerance of E. sakazakii isolates obtained from powdered infant formula and other food products in Japan. Isolates were categorized into three classes according to their thermal tolerance, and differential gene expression analysis showed that the heat‐resistant clones expressed a higher level of infB (which encodes a translation initiation factor), than did the heat‐sensitive isolates. Gene expression and DNA polymorphism analyses suggested that this gene target might be useful to unequivocally detect and identify heat‐resistant clones, permitting epidemiological surveillance for this pathogen.
Rhamnolipid‐Dependent Spreading Growth of Pseudomonas aeruginosa on a High‐Agar Medium: Marked Enhancement under CO2‐Rich Anaerobic Conditions
Authors: Nozawa Takashi , Tanikawa Taichiro , Hasegawa Hiroyuki , Takahashi Chihiro , Ando Yumi , Matsushita Mitsugu , Nakagawa Yoji , Matsuyama Tohey
Published: November 2013

Anaerobiosis of Pseudomonas aeruginosa in infected organs is now gaining attention as a unique physiological feature. After anaerobic cultivation of P. aeruginosa wild type strain PAO1 T, we noticed an unexpectedly expanding colony on a 1.5% agar medium. The basic factors involved in this spreading growth were investigated by growing the PAO1 T strain and its isogenic mutants on a Davis high‐agar minimal synthetic medium under various experimental conditions. The most promotive environment for this spreading growth was an O2‐depleted 8% CO2 condition. From mutational analysis of this spreading growth, flagella and type IV pili were shown to be ancillary factors for this bacterial activity. On the other hand, a rhamnolipid‐deficient rhlA mutant TR failed to exhibit spreading growth on a high‐agar medium. Complementation of the gene defect of the mutant TR with a plasmid carrying the rhlAB operon resulted in the restoration of the spreading growth. In addition, an external supply of rhamnolipid or other surfactants (surfactin from Bacillus subtilis or artificial product Tween 80) also restored the spreading growth of the mutant TR. Such activity of surfactants on bacterial spreading on a hard‐agar medium was unique to P. aeruginosa under CO2‐rich anaerobic conditions.
Evaluation of Repetitive Extragenic Palindromic‐PCR for Discrimination of Fecal Escherichia coli from Humans, and Different Domestic‐ and Wild‐Animals
Authors: Mohapatra Bidyut R. , Broersma Klaas , Nordin Rick , Mazumder Asit
Published: November 2013

The objective of this study was to investigate the potential of repetitive extragenic palindromic anchored polymerase chain reaction (rep‐PCR) in differentiating fecal Escherichia coli isolates of human, domestic‐ and wild‐animal origin that might be used as a molecular tool to identify the possible source(s) of fecal pollution of source water. A total of 625 fecal E. coli isolates of human, 3 domestic‐ (cow, dog and horse) and 7 wild‐animal (black bear, coyote, elk, marmot, mule deer, raccoon and wolf) species were characterized by rep‐PCR DNA fingerprinting technique coupled with BOX A1R primer and discriminant analysis. Discriminant analysis of rep‐PCR DNA fingerprints of fecal E. coli isolates from 11 host sources revealed an average rate of correct classification of 79.89%, and 84.6%, 83.8%, 83.3%, 82.5%, 81.6%, 80.8%, 79.8%, 79.3%, 77.4%, 73.2% and 63.6% of elk, human, marmot, mule deer, cow, coyote, raccoon, horse, dog, wolf and black bear fecal E. coli isolates were assigned to the correct host source. These results suggest that rep‐PCR DNA fingerprinting procedures can be used as a source tracking tool for detection of human‐ as well as animal‐derived fecal contamination of water.
Methicillin Resistance in Staphylococcus aureus and Coagulase‐Negative Staphylococci: Epidemiological and Molecular Aspects
Authors: Martins André , de Lourdes R.S. Cunha Maria
Published: November 2013

Infections caused by the genus Staphylococcus are of great importance for human health. Staphylococcus species are divided into coagulase‐positive staphylococci, represented by S. aureus, a pathogen that can cause infections of the skin and other organs in immunocompetent patients, and coagulase‐negative staphylococci (CNS) which comprise different species normally involved in infectious processes in immunocompromised patients or patients using catheters. Oxacillin has been one of the main drugs used for the treatment of staphylococcal infections; however, a large number of S. aureus and CNS isolates of nosocomial origin are resistant to this drug. Methicillin resistance is encoded by the mecA gene which is inserted in the SCCmec cassette. This cassette is a mobile genetic element consisting of five different types and several subtypes. Oxacillin‐resistant strains are detected by phenotypic and genotypic methods. Epidemiologically, methicillin‐resistant S. aureus strains can be divided into five large pandemic clones, called Brazilian, Hungarian, Iberian, New York/Japan and Pediatric. The objective of the present review was to discuss aspects of resistance, epidemiology, genetics and detection of oxacillin resistance in Staphylococcus spp., since these microorganisms are increasingly more frequent in Brazil.
Proteomic Characterization of Enterohemorrhagic Escherichia coli O157:H7 in the Oxidation‐Induced Viable but Non‐Culturable State
Authors: Asakura Hiroshi , Panutdaporn Nantika , Kawamoto Keiko , Igimi Shizunobu , Yamamoto Shigeki , Makino Sou‐ichi
Published: November 2013

Enterohemorrhagic Escherichia coli (EHEC) O157 strain F2, a food isolate of an outbreak, is resistant to oxidative stress, but has increased stress‐sensitivity after passage through mice. The stress‐sensitive variant of F2 (designated MP37) has decreased culturability, but retains membrane integrity under stress conditions, indicating that the cells enter a viable but non‐culturable (VBNC) state. Proteomic analyses revealed that MP37 in the VBNC state had decreased levels of some oxidation‐responsive factors (AhpCF, AceF), but it markedly increased levels of outer membrane protein W (OmpW). Because F2 expressed higher levels of some ribosome‐associated proteins (RaiA, S6, Bcp) than MP37, the effect of animal passage on the induction of the VBNC state in the EHEC O157 cells might be due to ribosomal activity.
Development of an Immunochromatographic Kit for Rapid Diagnosis of H5 Avian Influenza Virus Infection
Authors: Tsuda Yoshimi , Sakoda Yoshihiro , Sakabe Saori , Mochizuki Tsuyoshi , Namba Yasuharu , Kida Hiroshi
Published: November 2013

Highly pathogenic avian influenza (HPAI) caused by the H5N1 subtype has given rise to serious damage in poultry industries in Asia. The virus has expanded its geographical range to Europe and Africa, posing a great risk to human health as well. For the control of avian influenza, a rapid diagnosis by detecting the causative virus and identifying its subtype is essential. In the present study, a rapid diagnosis kit combining immunochromatography with enzyme immunoassay which detects the H5 HA antigen of influenza A virus was developed using newly established anti‐H5 HA monoclonal antibodies. The present kit specifically detected all of the H5 influenza viruses tested, and did not react with the other HA subtypes. H5 HA antigens were detected from swabs and tissue homogenates of chickens infected with HPAI virus strain A/chicken/Yamaguchi/7/04 (H5N1) from 2 days post inoculation. The kit showed enough sensitivity and specificity for the rapid diagnosis of HPAI.
Detection and Phylogenetic Analysis of Norovirus inCorbicula flumineain a Freshwater River in Japan
Authors: Saitoh Mika , Kimura Hirokazu , Kozawa Kunihisa , Nishio Osamu , Shoji Akira
Published: November 2013

To study the molecular epidemiology of noroviruses (NoVs) in bivalves residing in freshwater rivers, we detected, quantified and phylogenetically analyzed the NoV genome in purified concentrates obtained from the gills and digestive diverticula ofCorbicula flumineain a freshwater river in Gunma Prefecture, Japan. We detected the NoV genome in 35 of the 58C. flumineasamples. Based on our phylogenetic analysis, the NoV genome detected in the samples was classified into 4 genotypes (GI/1, GI/2, GI/3 and GI/4) in genogroup I and 5 genotypes (GII/3, GII/4, GII/5, GII/8 and GII/12) in genogroup II. The phylogenetic tree showed wide genetic diversity among the genogroups. In addition, more than 104copies of the NoV genome were detected in 2 of 35 samples. These results suggest that the freshwater bivalveC. flumineais a reservoir for NoVs, similar to seawater bivalves such as oysters.
Biological and Genetic Classification of Canine Intestinal Lactic Acid Bacteria and Bifidobacteria
Authors: Kim So‐Young , Adachi Yoshikazu
Published: November 2013

To investigate the distribution of lactic acid bacteria (LAB) inhabiting canine intestines, a total of 374 gram‐positive LAB and bifidobacteria (BF) isolated from large intestinal contents in 36 dogs were classified and identified by phenotypic and genetic analyses. Based on cell morphological sizes, these isolates were divided into seven biotypes containing the genera Lactobacillus, Bifidobacterium, Enterococcus, and Streptococcus. The LAB and BF isolates were classified into 38 chemotypes based on SDS‐PAGE protein profile analysis of whole cells. Furthermore, partial 16S rDNA sequencing analysis demonstrated the presence of 24 bacterial species in the 38 chemotypes from 36 dogs. The identified species consisted of ten species belonging to the genus Lactobacillus (78.8%), seven species to the genus Bifidobacterium (6.8%), five species to the genus Enterococcus (11.6%), one species of Streptococcus bovis (2.0%), and one species of Pediococcus acidilactici (0.8%). In particular, the most predominant species in canine intestines were L. reuteri, L. animalis, and L. johnsonii and were found in the high frequency of occurrence of 77.8, 80.6, and 86.1%, respectively. Besides these, Enterococcus faecalis, Bifidobacterium animalis subsp. lactis, Pediococcus acidilactici, and Streptococcus bovis were also isolated in the present study. The sequences of the isolates also showed high levels of similarity to those of the reference strains registered previously in the DDBJ and the similarity was above 97.2%. Their partial 16S rRNA genes were registered in the DDBJ.
Induction of Preferential Chemotaxis of Unstimulated B‐Lymphocytes by 2‐Arachidonoylglycerol in Immunized Mice
Authors: Tanikawa Takashi , Kurohane Kohta , Imai Yasuyuki
Published: November 2013

2‐Arachidonoylglycerol (2‐AG) is an endogenous ligand for cannabinoid receptors. There are two types of cannabinoid receptors, CB1 and CB2. We investigated the chemotactic activity of 2‐AG using mouse lymphocytes because cells in the immune system are known to express CB2. Spleen cell migration toward 2‐AG was observed, which was completely inhibited by SR144528, a CB2‐specific antagonist 2‐AG has been reported to induce a preferential B cell chemotaxis. We examined whether there is any difference in responsiveness during the activation of B cells. When spleen cells from immunized mice were tested, naive B cells but not germinal center B cells (GL7‐positive) were increased in the fraction attracted by 2‐AG. Furthermore, when Peyer's patch lymphocytes were tested after oral administration of cholera toxin, the number of IgA– B cells was increased in the fraction attracted by 2‐AG. These results suggested that 2‐AG preferentially attracts unstimulated naive B cells rather than activated and/or class‐switched B cells. This property may influence the structure of B cell compartments in secondary lymphoid tissues.
Correlation between Substitutions in Penicillin‐Binding Protein 1 and Amoxicillin Resistance in Helicobacter pylori
Authors: Rimbara Emiko , Noguchi Norihisa , Kawai Takashi , Sasatsu Masanori
Published: November 2013

The correlation between the substitutions of penicillin‐binding protein 1 (PBP1) and amoxicillin resistance was studied for the determination of the substitutions in PBP1 which confer amoxicillin resistance in Helicobacter pylori. By the comparison of the amino acid sequences of PBP1 in the amoxicillin‐resistant (n=3), low‐susceptible (n=3), and susceptible (n = 13) H. pylori isolates, the substitution Asn562→Tyr, which is adjacent to KTG motif (555–557), was common and specific to amoxicillin‐resistant H. pylori. Additionally, all amoxicillin‐resistant isolates had multiple substitutions such as Ser414→Arg in the transpeptidase region of PBP1 of H. pylori. Furthermore all transformants obtained by the natural transformation using the pbp1 genes of amoxicillin‐resistant H. pylori isolates had multiple substitutions including Asn562→Tyr. These results suggest that multiple amino acid substitutions in the transpeptidase region of PBP1 are closely related to amoxicillin resistance in H. pylori.
Evaluation of the Levels of Specific IgE against Cryptococcus diffluens and Cryptococcus liquefaciens in Patients with Atopic Dermatitis
Authors: Kato Hiroshi , Sugita Takashi , Ishibashi Yoshio , Nishikawa Akemi
Published: November 2013

Cryptococcus diffluens and Cryptococcus liquefaciens, 2 basidiomycetous yeasts, frequently colonize the skin of patients with atopic dermatitis (AD). In this study, we investigated the presence of specific IgE antibodies against C. diffluens and C. liquefaciens in the sera of AD patients by using an enzyme immunoassay. Of the 122 AD serum samples tested, 43 (35.2%) and 50 (41.0%) were positive for specific IgE antibodies against C. diffluens and C. liquefaciens, respectively. The levels of specific IgE against the C diffluens antigen and that against the C. liquefaciens antigen were strongly correlated (r = 0.96). In contrast, no remarkable correlation was observed between the levels of specific IgE against the 2 Cryptococcus species and that of specific IgE against Malassezia restricta. Competitive enzyme‐linked immunosorbent assay (ELISA) inhibition tests revealed that C. diffluens and C. liquefaciens shared common antigens. This finding was consistent with the IgE immunoblotting data which demonstrated that several IgE‐binding proteins with molecular masses of 77, 54, and 30 kDa were recognized in both C. diffluens and C. liquefaciens antigens. These results suggest that fungal components from C. diffluens and C. liquefaciens may act as allergens and play a role in the pathogenesis of AD.
Salt in Surroundings Influences the Production of Serine Protease into Milieu by Aeromonas sobria
Authors: Khan Rasel , Takahashi Eizo , Ramamurthy Thandavarayan , Takeda Yoshifumi , Okamoto Keinosuke
Published: November 2013

Previously we have shown that the open reading frame 2 protein (ORF2 protein), which is encoded at the 3′ end of serine protease of Aeromonas sobria (ASP), functions as a chaperone protein in periplasm in the production of ASP. Both proteins, ASP and ORF2 protein, associate in periplasm and ORF2 protein helps ASP to take an active form. ASP which is dissociated from ORF2 protein emerges in milieu. In this study, we examined the effect of sodium chloride (NaCl) in medium on ASP production by A. sobria. The ASP activity of culture supernatant was extremely decreased when A. sobria was cultured in medium containing 3.0% NaCl (concentration almost equivalent to sea water salinity). Our analysis showed that the transcription of asp by A. sobria is not inhibited by NaCl in medium and that A. sobria synthesizes and releases ASP in milieu even under the condition of 3.0% NaCl. However, these ASPs in milieu formed complex as with ORF2 proteins. This indicates that the maturation pathway of ASP is disturbed in A. sobria cultured in medium containing 3.0% NaCl. It is likely that ASP does not associate with ORF2 protein in the correct form in periplasam when A. sobria is cultured in medium containing 3.0% NaCl, though both proteins, ASP and ORF2 protein, make complexes and emerge outside of the cell. This idea suggests that the chaperone system of ASP possesses the ability to sense NaCl in surroundings and regulates the production of active ASP.
Mechanism of Inhibition of DNA Gyrase by ES‐1273, a Novel DNA Gyrase Inhibitor
Authors: Oyamada Yoshihiro , Yamagishi Jun‐ichi , Kihara Takahiro , Yoshida Hiroaki , Wachi Masaaki , Ito Hideaki
Published: November 2013

We investigated the mode of action of ES‐1273, a novel DNA gyrase inhibitor obtained by optimization of ES‐0615, which was found by screening our chemical library using anucleate cell blue assay. ES‐1273 exhibited the same antibacterial activity against S. aureus strains with amino acid change(s) conferring quinolone‐ and coumarin‐resistance as that against a susceptible strain. In addition, ES‐1273 inhibited DNA gyrase supercoiling activity, but not ATPase activity of the GyrB subunit of DNA gyrase. Moreover, ES‐1273 did not induce cleavable complex. These findings demonstrate that the mechanism by which ES‐1273 inhibits DNA gyrase is different from that of the quinolones or the coumarins. Preincubation of DNA gyrase and substrate DNA prevented inhibition of DNA gyrase supercoiling activity by ES‐1273. ES‐1273 antagonized quinolone‐induced cleavage. In electrophoretic mobility shift assay, no band representing DNA gyrase‐DNA complex was observed in the presence of ES‐1273. Taken together, these results indicate that ES‐1273 prevents DNA from binding to DNA gyrase. Furthermore, our results from surface plasmon resonance experiments strongly suggest that ES‐1273 interacts with DNA. Therefore, the interaction between ES‐1273 and DNA prevents DNA from binding to DNA gyrase, resulting in inhibition of DNA gyrase supercoiling. Interestingly, we also found that ES‐1273 inhibits topoisomerase IV and human topoisomerase IIoc, but not human topoisomerase I. These findings indicate that ES‐1273 is a type II topoisomerase specific inhibitor.
Novel Functions of Herbal Medicines in Dendritic Cells: Role of Amomi Semen in Tumor Immunity
Authors: Fukui Hajime , Mitsui Seika , Harima Nobue , Nose Mitsuhiko , Tsujimura Kunio , Mizukami Hajime , Morita Akimichi
Published: November 2013

Dendritic cells (DCs) have a major role in regulating immune responses, including tumor immunity and peripheral tolerance. In the present study, we identified novel functions of herbal medicines in DCs by screening 99 herbal medicines, most of which are among the 210 Chinese medicines approved by the Ministry of Health, Labour, and Welfare, Japan. Ethanol extracts were prepared, and a murine epidermal‐derived Langerhans cell line, XS106, was used to screen the 99 extracts by analyzing major histocompatibility complex (MHC) class II expression. Amomi Semen (amomum seed), Polyporus (polyporus sclerotium), and Plantaginis Semen (plantago seed) potently activated XS106 and were selected for further analysis. The effects of these extracts on bone marrow‐derived DCs (BM‐DCs) generated in vitro were then analyzed using surface phenotype (MHC class II, CD80, and CD86) and interleukin (IL)‐12p70 production as indicators. BM‐DCs treated with Amomi Semen extract exhibited activated phenotypes and secreted IL‐12p70. The activation level was similar to that induced by lipopolysaccharides. Finally, an E.G7‐OVA tumor model (E.L4‐OVA transfectant) was used to examine the anti‐tumor effects of Amomi Semen extract. Vaccination of mice with a subcutaneous injection of BM‐DCs treated with Amomi Semen extract and OVA peptide significantly inhibited the growth of tumor cells and prolonged survival time compared to controls. Furthermore, therapeutic effects were observed on established tumors. The inhibition rates for both the prophylactic and therapeutic protocols were comparable to those of lipopolysaccharides. These results indicate that Amomi Semen extract potently activate DCs and is potentially useful for DC vaccination.
Direct Comparison of the Pharmacodynamics of Four Antifungal Drugs in a Mouse Model of Disseminated Candidiasis Using Microbiological Assays of Serum Drug Concentrations
Authors: Maki Katsuyuki , Holmes Ann R. , Watabe Etsuko , Iguchi Yumi , Matsumoto Satoru , Ikeda Fumiaki , Tawara Shuichi , Mutoh Seitaro
Published: November 2013

The aim of this study was to compare the pharmacodynamics of the azole antifungal drugs fluconazole, itraconazole and ketoconazole, and the polyene antifungal amphotericin B, in a mouse model of disseminated Candida albicans infection. In order to directly compare effective serum concentrations of these antifungals, drug concentrations were assayed microbiologically by measuring inhibition of C. albicans mycelial growth (mMIC) in a mouse serum‐based assay (serum antifungal titer). Efficacy in the mouse infection model was determined using an organ‐based (kidney burden) endpoint. For all four drugs, the serum antifungal titers, 8 hr after administration of single doses of drugs at a range of drug concentrations, correlated closely with C. albicans kidney fungal burden in the mouse model. The results showed that determining serum antifungal titer may be used to accurately represent kidney fungal burden in a mouse model of disseminated candidiasis and allowed direct comparison of the pharmacodynamics of differing classes of antifungal drugs.
Molecular Cloning and Characterization of All RND‐Type Efflux Transporters in Vibrio cholerae Non‐O1
Authors: Rahman M. Mushfequr , Matsuo Taira , Ogawa Wakano , Koterasawa Motohiro , Kuroda Teruo , Tsuchiya Tomofusa
Published: November 2013

Resistance Nodulation cell Division (RND) efflux transporters are thought to be involved in mediating multidrug resistance in Gram‐negative bacteria, including Vibrio cholerae non‐O1. There are six operons for putative RND‐type efflux transporters present in the chromosome of V. cholerae O1 including two operons, vexAB and vexCD, which had already been identified. All of the six operons were cloned from V. cholerae non‐O1, NCTC4716 by the PCR method, introduced, and expressed in cells of drug hypersusceptible Escherichia coli KAM33 (AacrAB, AydhE). Only vexEF conferred elevated minimum inhibitory concentrations (MICs) of some antimicrobial agents in the E. coli cells. However, VexEF did not confer increased MIC of any drug tested in tolC‐deficient E. coli KAM43 cells. On the other hand, when E. coli KAM43 was transformed with vexAB, vexCD or vexEF together with tolCVc of V. cholerae NCTC4716, we observed elevated MICs of various antimicrobial agents. Among them, E. coli KAM43 expressing both VexEF and TolCVc showed much higher MICs and much broader substrate specificity than the other two. We also observed ethidium efflux activity via VexEF‐TolCVc, and the activity required Na+. Thus, VexEF‐TolCVc is either a Na+‐activated or a Na+‐coupled transporter. To our knowledge, this is the first report on the requirement of Na+ for an RND‐type efflux transporter.
Mode of Infection of Hokkaido Virus (Genus Hantavirus) among Grey Red‐Backed Voles, Myodes rufocanus, in Hokkaido, Japan
Authors: Daud Nur Hardy Abu , Kariwa Hiroaki , Tanikawa Yoich , Nakamura Ichiro , Seto Takahiro , Miyashita Daisuke , Yoshii Kentaro , Nakauchi Mina , Yoshimatsu Kumiko , Arikawa Jiro , Takashima Ikuo
Published: November 2013

Hokkaido virus (HOKV) is a member of the genus Hantavirus, in the family Bunyaviridae. To investigate HOKV infection in the host Myodes rufocanus, the grey red‐backed vole, 199 animals were captured at Tobetsu (October 2004 and July 2005) and Nakagawa (October 2004) in Hokkaido, Japan, for detection of antibody, antigen, and viral RNA. In the surveys in Tobetsu (2004) and Nakagawa (2004), seropositive animals were detected at a frequency of 6.0% (5/84) and 10.4% (5/48), respectively. No seropositive animals were detected in Tobetsu in 2005. Seroprevalence in males in Tobetsu and Nakagawa in 2004 was 25% (1/4) and 45.5% (5/11), respectively, which was higher than in females, at 5.0% (4/80) and 0% (0/37), respectively (P<0.01). These results suggest that male animals play an important role in the maintenance of HOKV in M. rufocanus. Two females were seronegative but viral RNA‐positive, indicating that these animals had acute infections before antibody was produced. Another five infected animals in Nakagawa were all male and had high levels of antibodies and viral RNA, suggesting that they had persistent infections. Viral RNA copies in organs of infected animals in Nakagawa were quantified by real‐time polymerase chain reaction. Two acutely infected animals had ≥10 times the number of RNA copies in their lungs compared to those of persistently infected animals. In most cases, lungs or spleen had the highest RNA copy number, regardless of infection status.
Evasion of Legionella pneumophila from the Bactericidal System by Reactive Oxygen Species (ROS) in Macrophages
Authors: Harada Toshihiko , Miyake Masaki , Imai Yasuyuki
Published: November 2013

We demonstrated that the production of reactive oxygen species (ROS) by U937 macrophage‐like cells was suppressed upon infection with a wild type Legionella pneumophila strain, whereas such suppression was not observed in the case of infection with intracellular growth‐deficient mutants. This was supported not only by measuring ROS released into the supernatants of cell cultures by chemiluminescence assaying but also by detecting intracellular ROS with a fluorescent probe, 2‐[6‐(4′‐amino)phenoxy‐3H‐xanthen‐3‐on‐9‐yl]benzoic acid (APF), under a confocal laser scanning microscope. Furthermore, more than 60% of the phagosomes containing intracellular growth‐deficient mutants were colocalized with p47phox, which is the cytosolic subunit of NADPH oxidase, consistently throughout the observation period in an early stage of bacterial infection. In contrast, the colocalization of p47phox was suppressed after infection with the wild type strain. These results suggest that the interference with ROS production by U937 cells infected with wild type L. pneumophila is due to a failure of NADPH oxidase activation through inhibition of p47phox recruitment to phagosomes harboring bacteria. The results also highlighted the difference in the nature of phagosomes between ones harboring the wild type and ones the intracellular growth‐deficient strains.
Comparison of Epitope Structures of H3HAs through Protein Modeling of Influenza A Virus Hemagglutinin: Mechanism for Selection of Antigenic Variants in the Presence of a Monoclonal Antibody
Authors: Nakajima Setsuko , Nakajima Katsuhisa , Nobusawa Eri , Zhao Jin , Tanaka Shigenori , Fukuzawa Kaori
Published: November 2013

Starting with nine plaques of influenza A/Kamata/14/91(H3N2) virus, we selected mutants in the presence of monoclonal antibody 203 (mAb203). In total, amino acid substitutions were found at nine positions (77, 80, 131, 135, 141, 142, 143, 144 and 146), which localized in the antigenic site A of the hemagglutinin (HA). The escape mutants differed in the extent to which they had lost binding to mAb203. HA protein with substitutions of some amino acid residues created by site‐directed mutagenesis in the escape mutants retained the ability to bind to mAb203. Changes in the amino acid character affecting charge or hydrophobicity accounted for the binding capacity to the antibody of the HA with most of the substitutions in the escape mutants and binding‐positive mutants. However, the effect of some amino acid substitutions remained unexplained. A three‐dimensional model of the 1991 HA was constructed and used to analyze substituted amino acids in these mutants for the accessible surface hydrophobic and hydrophilic characters. One amino acid substitution in an escape mutant and another amino acid substitution in a binding‐positive mutant seemed to be explained by the changes noted on this model.
Impact of a 4q25 Genetic Variant in Atrial Flutter and on the Risk of Atrial Fibrillation After Cavotricuspid Isthmus Ablation
Authors: ROBERTS JASON D. , HSU JONATHAN C. , AOUIZERAT BRADLEY E. , PULLINGER CLIVE R. , MALLOY MARY J. , KANE JOHN P. , OLGIN JEFFREY E. , MARCUS GREGORY M.
Published: November 2013

Role of a 4q25 Genetic Variant in Atrial Flutter

Background

The prediction of atrial fibrillation (AF) following catheter ablation of atrial flutter (Afl) would be helpful to facilitate targeted arrhythmia monitoring and anti‐coagulation strategies. A single nucleotide polymorphism, rs2200733, is strongly associated with AF. We sought to characterize the association between rs2200733 and prevalent Afl and to determine if the variant could predict AF after cavotricuspid isthmus ablation.

Methods and Results

We performed a genetic association study of 295 patients with Afl and/or AF and 469 controls using multivariable logistic regression. The variant was then assessed as a predictor of incident AF after cavotricuspid isthmus ablation in 87 consecutive typical Afl patients with Cox proportional hazards models. The rs2200733 rare allele was associated with an adjusted 2.06‐fold increased odds of isolated Afl (95% CI: 1.13–3.76, P = 0.019) and an adjusted 2.79‐fold increased odds of a combined phenotype of AF and Afl (95% CI: 1.81–4.28, P < 0.001). Following catheter ablation for Afl, carrier status of rs2200733 failed to predict an increased risk of AF either among all subjects (adjusted HR: 0.94; 95% CI: 0.58–1.53, P = 0.806) or among those with isolated Afl (adjusted HR: 1.29; 95% CI: 0.51–3.26, P = 0.585).

Conclusions

Our study demonstrates that Afl, whether occurring in isolation or along with AF, is associated with the rs2200733 AF risk allele. Genetic carrier status of rs2200733 failed to predict an increased risk of incident or recurrent AF following catheter ablation for Afl. These findings suggest that the causal mechanism associated with rs2200733 is germane to both AF and Afl.
Impact of Structural Heart Disease on the Acute Complication Rate in Atrial Fibrillation Ablation: Results from the German Ablation Registry
Authors: HOFFMANN BORIS A. , KUCK KARL‐HEINZ , ANDRESEN DIETRICH , SPITZER STEFAN G. , HOFFMANN ELLEN , SCHUMACHER BURGHARD , ECKARDT LARS , BRACHMANN JOHANNES , BECKER RÜDIGER , STEVEN DANIEL , ROSTOCK THOMAS , JÜNGER CLAUS , SENGES JOCHEN , WILLEMS STEPHAN
Published: November 2013

Acute Complication Rate in AF Ablation

Introduction

Catheter ablation (CA) has emerged as a widespread therapeutic option in the treatment of atrial fibrillation (AF). Currently, no safety data with regard to the impact of the underlying structural heart diseases (SHD) are available. We sought to assess the risk for acute and long‐term complications during CA of AF in relation to underlying SHD.

Methods and Results

We included 6,211 patients in a prospective registry undergoing CA of AF in 41 nationwide centers. All patients were divided into 4 groups according to the underlying heart disease: No SHD (69.4%), hypertensive heart disease (HHD) (12.0%), coronary artery disease (CAD) (15.1%), and cardiomyopathy (CM) (3.6%). In univariate analysis, patients with HHD had an overall complication rate of 7.28%, whereas patients without an SHD had a significantly lower rate of 6.01% (P < 0.01). Multivariate analysis revealed that HHD (adjusted odds ratio [OR]: 1.97 [95% confidence interval (CI): 1.02–3.83], P = 0.0442) and age (years; OR: 1.04 [95% CI: 1.01–1.07], P = 0.0155) were independent predictors of severe, nonfatal complications and death. Other SHD including CAD (OR: 1.48 (0.73–3.00), P = 0.2797) and CM (OR: 2.37 [0.70–7.99], P = 0.1630) failed to reach statistical significance. Male sex was protective (OR: 0.47 [95% CI: 0.27–0.81], P = 0.0062).

Conclusion

In general, CA of AF has a low number of severe complications. In our prospective registry HHD emerged as an independent predictor of severe, nonfatal complications during AF ablation but other SHD including CAD and CM did not. The influence of HHD on the complication rate should be considered in patient selection.