bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on the Conditions Required for Structural and Functional Maturation of Rabies Virus Glycoprotein (G) in G cDNA‐Transfected Cells
Authors: Sakai Mai , Kankanamge Pushpa J. , Shoji Jun'ichi , Kawata Sanae , Tochikura Tadafumi S. , Kawai Akihiko
Published: November 2013

When the rabies virus G cDNA was expressed with the help of T7 RNA polymerase provided by a recombinant vaccinia virus (RVV‐T7), functional G proteins were produced in terms of their ability to induce low pH‐dependent syncytium formation and the formation of conformational epitopes, including the acid‐sensitive epitope recognized by mAb #1‐30‐44. Such an ability and the 1‐30‐44 epitope formation, however, were not associated with the G gene products when G cDNA was expressed without the help of RVV‐T7 using a tetracycline‐regulated expression vector (pTet‐G), although they were normally transported to the surface of established G protein‐producing BHK‐21 (G‐BHK) cells. But, when the G‐BHK cells were treated with 2.5 MM sodium butyrate (NaB) after the removal of tetracycline, we could observe not only a much increased frequency of G protein‐producing cells, but also the greatly enhanced maturation of the protein. Another short acylate, sodium propionate (NaP), similarly induced increased G protein synthesis at a concentration of 2.5 MM as NaB; however, such proteins were mostly not endowed with the fusion activity nor the 1‐30‐44 epitope, while NaP at a higher concentration as 5.0 MM did induce similarly the increased production and enhanced maturation of G protein, including the 1‐30‐44 epitope formation. From these results, we conclude that functional maturation of G protein to acquire fusogenic activity is correlated with 1‐30‐44 epitope formation, and 2.5 MM NaB not only stimulates G protein production, but also provides such cellular conditions as are required for the structural and functional maturation of the protein.
Pi30 DNA Probe May Be Useful for the Identification of Prevotella intermedia at the Species or Strain Level
Authors: Shin Yong Kook , Jeong Seung‐U , Yoo So Young , Kim Mi‐Kwang , Kim Hwa‐Sook , Kim Byung‐Ock , Kim Do Kyung , Hwang Ho‐Keel , Kook Joong‐Ki
Published: November 2013

Recently, we introduced a new method for the rapid screening of bacterial species‐ or subspecies‐specific DNA probes, named the “inverted dot blot hybridization screening method.” This method has subsequently been then applied to develop species‐ or strain‐specific DNA probes for Prevotella intermedia and Prevotella nigrescens. In a previous study, the inverted dot blot hybridization data showed that a probe, Pi30, was specific for P. intermedia. In this study, the DNA probe Pi30 was evaluated by Southern blot analysis to determine if it could distinguish P. intermedia from P. nigrescens. The data showed that the probe Pi30 reacted with the genomic DNAs from the reference strains and clinical isolates of both P. intermedia and P. nigrescens, but the size of the signal bands was different. In addition, the probe Pi30 reacted with a 1.4 kbp fragment from the genomic DNAs digested with PstI of the P. intermedia strains but not with any fragments of P. nigrescens strains. The result indicates that the probe Pi30 could be useful for the identification of P. intermedia by restriction fragment length polymorphism (RFLP) at the species or strain level.
Isolation of Mini‐Tn5Km2 Insertion Mutants of Salmonella enterica Serovar Oranienburg Sensitive to NaCl‐Induced Osmotic Stress
Authors: Asakura Hiroshi , Panutdaporn Nantika , Kawamoto Keiko , Igimi Shizunobu , Yamamoto Shigeki , Makino Sou‐ichi
Published: November 2013

We previously reported that the viability of Salmonella Oranienburg strains under NaCl stress was variable and depended on the strain's origin; food strains were resistant and patient strains sensitive to NaCl. Therefore, we mutagenized a food strain with a mini‐Tn5Km2 transposon. Of 2,400 mutants screened, 15 NaCl‐sensitive mutants were isolated, and 7 genes associated with NaCl‐sensitivity were identified. The intact genes complemented their own food‐strain mutants, but not patient‐strain mutants, suggesting that the difference in NaCl‐sensitivity between food and patient S. Oranienburg strains might not arise from a single gene mutation, but from change in multiple osmoregulatory mechanisms in Salmonella.
Detection of Tannerella forsythia and/or Prevotella intermedia Might Be Useful for Microbial Predictive Markers for the Outcome of Initial Periodontal Treatment in Koreans
Authors: Kook Joong‐Ki , Sakamoto Tomonori , Nishi Kazuya , Kim Mi‐Kwang , Seong Jin‐Hyo , Son Young Nam , Kim Dong‐Kie
Published: November 2013

A proportion of diseased sites in periodontal disease do not respond to the initial treatment, which might be due in part to the presence of specific microbial pathogens. The aim of this study was to clarify the value of microbial screening for predicting the outcome of periodontal treatment in Koreans using a polymerase chain reaction (PCR). This study enrolled 32 adults with periodontal disease. Microbial and clinical examinations were performed at the baseline and after the initial treatment (professional toothbrushing, scaling, and root planing). Subgingival plaque samples were taken from four sites in each subject (total 128 samples). PCR was used to detect the four putative pathogenic bacteria. There was an improvement in the average of each clinical measurement after the initial treatment. However, approximately half of the sites exhibiting bleeding upon probing (BOP) at the baseline still exhibited bleeding after treatment. There was a close association between the presence of BOP and the presence of Tannerella forsythia (formerly Bacteroides forsythus) and/or Prevotella intermedia. Furthermore, the sites harboring both T. forsythia and P. intermedia at the baseline had a poorer response to treatment than the sites where these two species were not detected. Therefore, microbial screening for T. forsythia and P. intermedia might be useful for predicting the treatment outcome in Koreans.
Induction of Inducible Nitric Oxide (NO) Synthase mRNA and NO Production in Macrophages Infected with Influenza A/PR/8 Virus and Stimulated with Its Ether‐Split Product
Authors: Imanishi Nobuko , Andoh Tsugunobu , Sakai Shinya , Satoh Miyuki , Katada Yuko , Ueda Kyouka , Terasawa Katsutoshi , Ochiai Hiroshi
Published: November 2013

We investigated the inductive activity of infective influenza A/PR/8/34 (PR8) virus and its ether‐split product (ESP) on the expression of inducible nitric oxide (NO) synthase (iNOS) and NO production in RAW264.7 (RAW) cells, a murine macrophage (MΦ) cell line, and thioglycolate‐elicited peritoneal MΦ (TPM). In both cells, PR8 virus infection induced iNOS mRNA between 4 hr and 24 hr, attaining a peak value at 12 hr. In correlation with induction of iNOS mRNA, NO amounts increased significantly from 12 to 24 hr. Moreover, this study demonstrated that ESP with the same hemagglutination titer as PR8 virus could induce iNOS mRNA and NO production, although the inductive activity of ESP was weaker than that of PR8 virus. Considering the dual role (beneficial and detrimental roles) of NO on certain inflammatory disorders and virus infections, the inductive activity of influenza virus on the iNOS‐mediated NO production independent of its infectivity might contribute to a modification of influenza virus infection.
Further Studies on the Soluble Form (Gs) of Rabies Virus Glycoprotein (G): Molecular Structure of Gs Protein and Possible Mechanism of the Shedding
Authors: Thirapanmethee Krit , Ootaki Naohiro , Sakai Mai , Lien Chah Keng , Kawai Akihiko
Published: November 2013

In this study, we investigated the antigenic structures and maturation of some C‐terminal‐deficient derivatives of rabies virus glycoprotein (G). The Gs protein, a soluble form of G protein shed from infected cells, displayed antigenicity to most of our conformational epitope‐specific anti‐G mAbs, but took the 1‐30‐44 epitope‐deficient conformation (termed GC form). (The 1‐30‐44 epitope was acid‐sensitive and dependent on two separate regions, the Lys‐202‐containing and Asn‐336‐containing regions; Kankanamge et al., Microbiol. Immunol., 47: 507–519). Intact G proteins took the 1‐30‐44 epitope‐positive form (referred to as GB form) on the cell surface, but not inside the cell. A deletion mutant G(1–429) (termed GATC), lacking the transmembrane (TM) and cytoplasmic domains, was shown to be accumulated in the rough endoplasmic reticulum (rER) with BiP and did not seem to be shed. Another C‐terminal‐deficient mutant G(1–462) (termed CT1) was deprived of the whole cytoplasmic domain except for a basic amino acid left at the C‐terminus, but was transported to the cell surface, where it showed pH‐dependent cell fusion activity and almost full antigenicity to most of the anti‐G mAbs with the exception of very weak antigenicity to mAb #1‐30‐44. No Gs protein could be detected in the CT1‐producing cultures. Based on these results, we think that the cytoplasmic domain was not necessary for the G protein to be transported to the cell surface, but was necessary to keep its 1‐30‐44 epitope‐positive GB conformation. Gs proteins might have lost the C‐terminal regions during the maturation process after being exported from the rER.
Structural Difference Recognized by a Monoclonal Antibody #404‐11 between the Rabies Virus Nucleocapsid (NC) Produced in Virus Infected Cells and the NC‐Like Structures Produced in the Nucleoprotein (N) cDNA‐Transfected Cells
Authors: Toriumi Harufusa , Kawai Akihiko
Published: November 2013

We investigated structural changes in the rabies virus (HEP‐Flury strain) nucleocapsid (NC) during the virus replication, for which we used two anti‐nucleoprotein (N) monoclonal antibodies (mAbs), #404‐11 (specific for a conformation‐dependently exposed linear epitope) and #1‐7‐11 (specific for a conformational epitope which is exposed after the nucleocapsid formation). Both mAbs recognized the N protein of the viral NC, but not of the RNA‐free N‐P complex. The 1‐7‐11 and 404–11 epitopes could be mapped to the N‐terminal and the C‐terminal regions of N protein, respectively. Immunoprecipitation studies demonstrated that treatment of the NC either with the alkaline phosphatase or sodium deoxycholate (DOC) resulted in dissociation of most P proteins from the NC and in the reduced reactivity to mAb #404‐11, but not to mAb #1‐7‐11. NC‐like structures produced in the N cDNA‐transfected cells displayed strong reactivity to mAb #1‐7‐11; however, reactivity to mAb #404‐11 was very weak. And, co‐expression with viral phosphoprotein (P) resulted in little increase in reactivity to mAb #404‐11 of the NC‐like structures, while the reactivity was significantly increased by cotransfection with P and the viral minigenome whose 3′‐ and 5′‐end structures were derived from the viral genome. From these results, we assume that, although the 404–11 epitope is a linear one, the epitope‐containing region is exposed only when N proteins encapsidate properly the viral RNA in collaboration with the P protein. Further, exposure of the 404–11 epitope region might be function‐related, and be regulated by association and dissociation of the P protein.
Organization of the CTX Prophage in Environmental Isolates of Vibrio mimicus
Authors: Islam Mohammad Sirajul , Rahman Mohammed Ziaur , Khan Sirajul Islam , Mahmud Zahid Hayat , Ramamurthy Thandavarayan , Nair Gopinath Balakrish , Sack Richard Bradley , Sack David Allen
Published: November 2013

The organization of the CTX prophage in environmental strains of Vibrio mimicus was investigated. Sixteen hundred non‐sucrose fermenting vibrios were examined for ctx gene by hybridization. Out of 1,600 isolates, 6 V. mimicus isolates contained ctxA gene. The organization of CTX prophage was determined by RFLP using ctxA probe. The CTX prophage integrated at a single site in V. mimicus genome which was present as a single copy flanked by at least a single RS element. Ribotype pattern revealed that a particular clone of V. mimicus acquired the CTXφ in the aquatic environment. This study demonstrated that V. mimicus could act as a reservoir of CTXφ in the aquatic environment.
Effects of Type II Alveolar Epithelial Cells on T Cell Mitogenic Responses to Concanavalin A and Purified Protein Derivatives
Authors: Sato Katsumasa , Shimizu Toshiaki , Sano Chiaki , Tomioka Haruaki
Published: November 2013

To investigate the role of type II alveolar epithelial cells during the T cell‐dependent host immune response to Mycobacterium tuberculosis (MTB), effects of MTB‐infected A‐549 human type II alveolar epithelial cells (A‐549 cells) on T cell mitogenesis in response to concanavalin A (Con A) and purified protein derivatives (PPD) were studied. Human peripheral blood mononuclear cells (PBMCs) were co‐cultivated with uninfected or MTB‐infected A‐549 cells and Con A‐ and PPD‐induced T cell mitogeneses were examined, and the following findings were obtained. T cell mitogenesis was inhibited by uninfected as well as MTB‐infected A‐549 cells, even when a dual‐chamber culture system was used to prevent direct cell contact between PBMCs and A‐549 cells. Therefore, it appears that A‐549 cells suppress T cell mitogenesis by producing some unknown humoral suppressor factors.
In Silico Analysis of ORF1ab in Coronavirus HKU1 Genome Reveals a Unique Putative Cleavage Site of Coronavirus HKU1 3C‐Like Protease
Authors: Woo Patrick C.Y. , Huang Yi , Lau Susanna K.P. , Tsoi Hoi‐wah , Yuen Kwok‐yung
Published: November 2013

Recently we have described the discovery and complete genome sequence of a novel coronavirus associated with pneumonia, coronavirus HKU1 (CoV‐HKU1). In this study, a detailed in silico analysis of the ORF1ab, encoding the 7,182‐amino acid replicase polyprotein in the CoV‐HKU1 genome showed that the replicase polyprotein of CoV‐HKU1 is cleaved by its papain‐like proteases and 3C‐like protease (3CLpro) into 16 polypeptides homologous to the corresponding polypeptides in other coronaviruses. Surprisingly, analysis of the putative cleavage sites of the 3CLpro revealed a unique putative cleavage site. In all known coronaviruses, the P1 positions at the cleavage sites of the 3CLpro are occupied by glutamine. This is also observed in CoV‐HKU1, except for one site at the junction between nsp10 (helicase) and nsp11 (member of exonuclease family), where the P1 position is occupied by histidine. This amino acid substitution is due to a single nucleotide mutation in the CoV‐HKU1 genome, CAG/A to CAT. This probably represents a novel cleavage site because the same mutation was consistently observed in CoV‐HKU1 sequences from multiple specimens of different patients; the P2 and P1′‐P12′ positions of this cleavage site are consistent between CoV‐HKU1 and other coronaviruses; and as the helicase is one of the most conserved proteins in coronaviruses, cleavage between nsp10 and nsp11 should be an essential step for the generation of the mature functional helicase. Experiments, including purification and C‐terminal amino acid sequencing of the CoV‐HKU1 helicase and trans‐cleavage assays of the CoV‐HKU1 3CLpro will confirm the presence of this novel cleavage site.
Legionella pneumophila Serogroup 1 Isolates from Cooling Towers in Japan Form a Distinct Genetic Cluster
Authors: Amemura‐Maekawa Junko , Kura Fumiaki , Chang Bin , Watanabe Haruo
Published: November 2013

Thirty‐one epidemiologically unrelated Legionella pneumophila serogroup 1 isolates (10 from cooling towers, 10 from public spas and/or hot spring baths, and 11 from patients) were analyzed by pulsed‐field gel electrophoresis (PFGE) and sequence‐based typing (SBT) using 6 loci, flaA, pilE, asd, mip, mompS, and proA. The results of PFGE and SBT analysis indicated that all 10 isolates from cooling towers clustered into a unique type, which was distinct from strains of other environmental sources.
Increased Expression of c‐myc Is Associated with Thymoma in Rats Infected with Murine Leukemia Virus A8
Authors: Hirano Tomoko , Watanabe Rihito , Takase‐Yoden Sayaka
Published: November 2013

Infection of rats with Friend murine leukemia virus (Fr‐MLV) clone A8 causes thymoma in all the animals within 7 weeks. The rapid induction of thymoma is associated with a unique enhancer structure in the U3 region of the A8‐LTR. Our Southern blot analyses showed that the thymomas were oligo clonal. The A8‐induced thymomas showed 3‐ to 11‐fold overexpression of c‐myc mRNA. These results suggest that provirus insertion into particular positions of the host genome is correlated with tumorigenesis after A8 infection and that up‐regulation of c‐myc plays an important role in the induction of thymoma.
Production of Autoantibodies Associated with Polyclonal Activation in Yersinia enterocolitica O:8‐Infected Mice
Authors: Ramos Orivaldo Pereira , Silva Eloisa Elena Cangiani , Falcão Deise Pasetto , de Medeiros Beatriz Maria Machado
Published: November 2013

Polyclonal lymphocyte stimulation is one of the immunomodulatory mechanisms induced by arthritogenic pathogens. In this study we examined the polyclonal activation potential of a virulent strain of Y. enterocolitica serotype O:8 (WA 2707+) and its plasmidless isogenic pair (WA 2707−). SPF Swiss mice were infected intragastrically and spleen cells were obtained on days 7, 14, 21, 28, 35 and 42 after infection. The number of cells secreting nonspecific immunoglobulins of IgG, IgM and IgA isotypes was determined by the ELISPOT technique. The presence of serum‐specific antibodies was investigated by ELISA and the presence of autoantibodies by dot‐blot assay. Although the patterns of infection of the two bacterial strains were almost the same, only the animals infected with the virulent strain presented clinical anomalies. Neither arthritic nor inflammatory signs were observed in the joints of the infected animals. The greatest activation observed was that of the nonspecific IgM‐secreting cells, and their peak of secretion occurred between the 28th and the 42nd day after infection, for both strains of Y. enterocolitica O:8. Only the animals infected with the virulent strain (WA 2707+) produced IgG‐specific antibodies in the serum, from the 28th day after infection. The serum of animals infected with either strain showed reactivity to all the autologous constituents tested, mainly on the 28th and 42nd day after infection. It was concluded that infection of mice with either the virulent strain of Y. enterocolitica O:8 or with its plasmidless isogenic pair resulted in the polyclonal activation of the splenic B lymphocytes including some autoreactive clones.
Quantification of Aquabirnaviruses Isolated from Different Host Species by Real‐Time RT‐PCR
Authors: Hirayama Takeshi , Imajoh Masayuki , Oshima Syun‐ichirou
Published: November 2013

Real‐time reverse transcription (RT)‐PCR was developed to detect and quantify successfully aquatic birnaviruses (ABVs) from fish tissue and seawater samples. This method detected marine birnavirus (MABV) RNA in several samples, and the detection was specific for ABVs. Monitoring the MABV strain Y‐6 RNA quantification by real‐time RT‐PCR showed replication kinetics of MABV after experimental infection in vitro. We found the quantity of ABVs isolated from different host species by using combined virus absorption in cell culture and real‐time RT‐PCR. Although all specimens showed no symptoms of viral infection, ABVs were detected regardless of host species. In conclusion, real‐time RT‐PCR was shown to be a sensitive and reliable tool to detect and quantify ABVs in cultured fish. This method is useful procedure to show details of horizontal or vertical infections by ABVs in the breeding water of aquatic animals.
Practical Range of Effective Dose for Cre Recombinase‐Expressing Recombinant Adenovirus without Cell Toxicity in Mammalian Cells
Authors: Baba Yasuko , Nakano Masakazu , Yamada Yoshitsugu , Saito Izumu , Kanegae Yumi
Published: November 2013

The site‐specific recombinase Cre is valuable for regulation of gene expression not only in vitro but also in vivo. We previously reported that replication‐deficient recombinant adenovirus (rAd) expressing Cre can mediate efficient and strict regulation in 100% of cultured cells. Recently, the constitutive‐expression of Cre using retrovirus or lentivirus vector reportedly inhibited cell‐growth, but the effect of transient Cre expression have not yet been examined. Here we showed that an excess amount of Cre produced from Cre‐expressing rAd caused a deleterious effect in cells even when Cre was transiently expressed. We used three rAds carrying promoters with different activities: the SV40 early promoter (AxSVENCre), the SRα promoter (AxSRCre) and the CAG promoter (AxCANCre). Cell toxicity was clearly caused by Cre itself and was distinguishable from that caused by rAd virions when the cytopathic effects of these rAds were compared with that of a control virus lacking the Cre expression unit. Cre toxicity was strongly correlated with the expression level of Cre. Importantly, AxSRCre and AxCANCre gave a 60‐fold range of effective MOIs (“effective range”) sufficient for gene activation without causing cell toxicity from either the rAd particles or Cre itself, while AxSVENCre failed to give such a range because the expression level of Cre was too low. When Cre was tagged with a nuclear localization signal (NLS), not only its activity but also Cre toxicity was increased fourfold, and the effective range was unchanged. Therefore, AxSRNCre might be more useful to control cell toxicity from the rAd virions than AxSRCre. Cre‐induced cell toxicity can be avoided by pre‐examining the “effective range” using the purpose cell lines before starting experiments utilizing the experiment of Cre‐expressing rAd.
Application of Real‐Time PCR for Quantitative Detection of Clostridium botulinum Type A Toxin Gene in Food
Authors: Yoon So‐Yeon , Chung Gyung Tae , Kang Do‐Hyun , Ryu Chunsun , Yoo Cheon‐Kwon , Seong Won‐Keun
Published: November 2013

The TaqMan real‐time PCR method for the quantitative detection of C. botulinum type A was developed based on sequence‐specific hybridization probes. The validity of this assay was verified by using 10 genera of 20 strains, including reference strains of C. botulinum types A, B, C, D, E and F. The detection limit of this assay was evaluated on C. botulinum type A, using a 10‐fold dilution series of DNA and spores. The DNA and spores were detected up to level of 0.1 ng/ml and 102 spores/ml, respectively. Spore spiked food sample preparation prior to the real‐time PCR was performed by two methods, heat treatment and GuSCN. The detection limits after heat treatment showed 102 spores/ml for spiked sausage slurry, and 103 spores/ml for spiked canned corn slurry, while detection limits after GuSCN precipitation showed 102 spores/ml in both sausage and canned corn. Therefore the real‐time PCR assay after GuSCN precipitation is useful for the quantification of C. botulinum type A because it showed identical CT values in both pure spore solutions and food slurries. We suggest that quantitative analysis of C. botulinum type A by TaqMan real‐time PCR can be a rapid and accurate assessment method for botulinal risk in food samples.
Highly Pathogenic H5N1 Influenza Virus Causes Coagulopathy in Chickens
Authors: Muramoto Yukiko , Ozaki Hiroichi , Takada Ayato , Park Chun‐Ho , Sunden Yuji , Umemura Takashi , Kawaoka Yoshihiro , Matsuda Haruo , Kida Hiroshi
Published: November 2013

Severe hemorrhage at multiple organs is frequently observed in chickens infected with highly pathogenic avian influenza (HPAI) A viruses. In this study we examined whether HPAI virus infection leads to coagulation disorder in chickens. Pathological examinations showed that the fibrin thrombi were formed in arterioles at the lung, associated with the viral antigens in endothelial cells of chickens infected intravenously with HPAI virus. Hematological analyses of peripheral blood collected from the chickens revealed that coagulopathy was initiated at early stage of infection when viral antigens were detected only in the endothelial cells and monocytes/macrophages. Furthermore, gene expression of the tissue factor, the main initiator of blood coagulation, was upregulated in the spleen, lung, and brain of HPAI virus‐infected chickens. These results suggest that dysfunction of endothelial cells and monocytes/macrophages upon HPAI virus infection may induce hemostasis abnormalities represented by the excessive blood coagulation and consumptive coagulopathy in chickens.
Correlation between the Anti‐Virus‐Induced Cytopathic Effect Activity of Interferon‐α Subtypes and Induction of MxA Protein In Vitro
Authors: Schanen Cécile , Chieux Vincent , Lobert Pierre‐Emmanuel , Harvey Jeanne , Hober Didier
Published: November 2013

There are several interferon‐α (IFN‐α) subtypes. Mechanism of disparity in biological effects among members of IFN‐α subtypes remains unexplained. Biological activity of IFN‐α is mediated in part by induction of intracellular antiviral proteins. We studied whether differences in biologic effects of IFN‐α subtypes may rely on their antiviral protein inducing effect. Intracellular induction of MxA protein and anti‐virus‐induced cytopathic effect (CPE) activity of 11 IFN‐α subtypes in human amnion WISH cells have been studied. MxA protein quantitation in cell lysates was performed by immunochemiluminescence assay and anti‐virus‐induced CPE activity was assessed by protection against vesicular stomatitis virus (VSV)‐induced CPE. Range of MxA values was high when cells were treated with 10 and 100 IU/ml of each IFN‐α subtype. Levels of MxA correlated with anti‐VSV‐induced CPE obtained with 10 IU/ml IFN‐α subtype. Together our data show a disparity in MxA‐inducing activity of IFN‐α subtypes and suggest that differences in anti‐VSV‐induced CPE of IFN‐α subtypes in WISH cells can be related to their different ability to induce MxA.
Multigenic Relation to the Attenuation of Rabies Virus
Authors: Yamada Kentaro , Ito Naoto , Takayama‐Ito Mutsuyo , Sugiyama Makoto , Minamoto Nobuyuki
Published: November 2013

Rabies virus Nishigahara strain causes lethal infection in adult mice after intracerebral inoculation. On the other hand, the RC‐HL strain, derived from the Nishigahara strain, does not cause lethal infection in adult mice. We previously demonstrated that a chimeric virus, R(G), with the open reading frame of the G gene (G‐ORF) from the Nishigahara strain in the background of the RC‐HL genome, is virulent. Reversely, in order to demonstrate that the G gene of the RC‐HL strain is related to the attenuated phenotype, we established a reverse genetics system of the Nishigahara strain and generated a chimeric virus, Ni(G), with the G‐ORF from RC‐HL in the background of the Nishigahara genome. Contrary to our prediction, Ni(G) killed adult mice after intracerebral inoculation with neuropathic symptoms like those of Nishigahara strain infection. Therefore, the G‐ORF of the RC‐HL strain is not the sole determinant of the attenuated phenotype. In additional investigation, we examined other genes, including N, P, M and L genes, and generated chimeric viruses exhaustively. We found that chimeric viruses with a single gene from the RC‐HL were not attenuated and that chimeric viruses with the G‐ORF and at least one other ORF from the RC‐HL were attenuated. In conclusion, attenuation from the Nishigahara to RC‐HL strain is multigenic.
Organic Solvent‐Selective Domain of the Resistance‐Nodulation‐Division‐Type Xenobiotic‐Antibiotic Transporters of Pseudomonas aeruginosa
Authors: Li Xiaohong , Eda Shima , Nakaeu Taiji
Published: November 2013

The hydrophobic substrate‐selective domain of the resistance‐nodulation‐division‐type xenobiotic transporter of Pseudomonas aeruginosa was assigned based on the different organic‐solvent selectivities of MexB and MexY. The MexB‐MexY hybrid protein consisting of two large periplasmic domains of MexB and the transmembrane domains of MexY showed MexB‐type organic solvent selectivity. The results imply that the resistance‐nodulation‐division‐type xenobiotic transporters recognize hydrophobic substrates such as organic solvents by their periplasmic domains and expel them to the external milieu. This is an elegant way to protect the cytoplasmic membrane from membrane‐deteriorating agents.
Analysis of Six New Genes Encoding Lysis Proteins and Coat Proteins in Escherichia coli Group A RNA Phages
Authors: Nishihara Tohru , Fujisaki Shingo , Nishimura Yukinobu , Minami Yoshiko , Yubisui Toshitsugu
Published: November 2013

Group A RNA phages consist of four genes—maturation protein, coat protein, lysis protein and replicase genes. We analyzed six plasmids containing lysis protein genes and coat protein genes of Escherichia coli group A RNA phages and compared their amino acid sequences with the known proteins of E. coli (group A), Pseudomonas aeruginosa (PP7) RNA phages and Rg‐lysis protein from Qβ phage. The size of lysis proteins was different by the groups but the coat proteins were almost the same size among phages. The phylogenetic analysis shows that the sub‐groups A‐I and A‐II of E. coli RNA phages were clearly dispersed into two clusters.
A Novel Receptor on Allograft (H‐2d)‐Induced Macrophage (H‐2b) toward an Allogeneic Major Histocompatibility Complex Class I Molecule, H‐2Dd, in Mice
Authors: Tashiro‐Yamaji Junko , Einaga‐Naito Kuniko , Kubota Takahiro , Yoshida Ryotaro
Published: November 2013

The generation of knockout mice demonstrated that noncytotoxic CD4+, but not cytotoxic CD8+, T cells were essential for the rejection of skin or organ allografts. Earlier we reported that allograft‐induced macrophages (AIM) in mice lysed allografts with H‐2 haplotype specificity, implying screening of grafts by AIM. Here, we isolated a cDNA clone encoding a novel receptor on AIM (H‐2Db) for an allogeneic major histocompatibility complex (MHC) class I molecule, H‐2Dd, by using H‐2Dd tetramer and a monoclonal antibody (mAb; R15) specific for AIM. The cDNA (1,181‐bp) encoded a 342‐amino acid polypeptide with a calculated molecular mass of 45 kDa and was found to be expressed on AIM, but not on resident macrophages or other cells, infiltrating into the rejection site. HEK293T cells transfected with this cDNA reacted with R15 mAb and H‐2Dd, but not H‐2Ld, H‐2Kd, H‐2Db, H‐2Kb, H‐2Dk, or H‐2Kk, molecules; and the H‐2Dd binding was suppressed by the addition of R15 or anti‐H‐2Dd mAb. AIM yielded a specific saturation isotherm in the presence of increasing concentrations of H‐2Dd, but not H‐2Db or H‐2Dk, molecules. The dissociation constant of AIM toward H‐2Dd tetramers was 1.9×10–9 M; and the binding was completely inhibited by the addition of R15 or anti‐H‐2Dd mAb. These results reveal that a novel receptor for an allogeneic H‐2Dd molecule was induced on effector macrophages responsible for allograft (H‐2d) rejection in H‐2b mice.
Diagnostic Surface Expression of SWAP‐70 on HIV‐1 Infected T Cells
Authors: Kimbara Noriaki , Dohi Natsuki , Miyamoto Mizuyu , Asai Suzuka , Okada Hidechika , Okada Noriko
Published: November 2013

Following immunization with HIV‐1 infected cells, a hybridoma cell line termed 9F11 was established from the P3U‐1 myeloma line fused with lymphocytes from a trans‐chromosome (TC) mouse, that harbors human chromosomes containing immunoglobulin genes. The 9F11 human IgM monoclonal antibody (9F11 Ab) reacts with HIV‐1 infected MOLT4 cells but not with uninfected MOLT4 cells, and causes immune cytolysis with homologous human complement at a concentration as low as 0.4 μg/ml. This Ab was used to perform immunoscreening of a cDNA expression library derived from HIV‐1 infected cells. All positive cDNA clones contained SWAP‐70 cDNA. SWAP‐70 RNA and protein expression are much stronger in HIV‐1 infected cells. SWAP‐70 was also detected on the surface of HIV‐1 infected cells by flow cytometric analysis. The monocyte cell line U937 cells expresses SWAP‐70 on its cell surface regardless of whether it was infected with HIV‐1. Furthermore, among PBMCs surface expression of SWAP‐70 was detected on CD21+, CD56+ and CD14+ cells. Although CD3+ cells scarcely express SWAP‐70 on their surface, once activated, they become positive. SWAP‐70 may therefore serve as a marker for T cell differentiation as well as for HIV‐1 infection.
Functional Analysis of spoT, relA and dksA Genes on Quinolone Tolerance in Pseudomonas aeruginosa under Nongrowing Condition
Authors: Viducic Darija , Ono Tsuneko , Murakami Keiji , Susilowati Heni , Kayama Shizuo , Hirota Katsuhiko , Miyake Yoichiro
Published: November 2013

To assess the contribution of ppGpp in antibiotic tolerance to quinolone in Pseudomonas aeruginosa, knockout mutants of the genes involved or linked with the stringent response, such as relA, spoT and dksA, were constructed and investigated for their antibiotic susceptibility to quinolones. The survival of the dksA and spoT mutants in the presence of 8 μg/ml of ofloxacin and 1 μg/ml of ciprofloxacin were shown to be approximately 20–180 and 10–40 times respectively, higher than the same for the wild type strain. The intracellular levels of ppGpp determined with high performance liquid chromatography (HPLC) demonstrated that spoT and dksA mutants possess higher basal levels of ppGpp. The data suggest that elevated basal levels of ppGpp may be responsible for rendering these mutants tolerant to quinolones and expand the importance of ppGpp as an antimicrobial target in P. aeruginosa.
Molecular Typing of Pasteurella pneumotropica Isolated from Rodents by Amplified 16S Ribosomal DNA Restriction Analysis and Pulsed‐Field Gel Electrophoresis
Authors: Sasaki Hiraku , Kawamoto Eiichi , Okiyama Emi , Ueshiba Hidehiro , Mikazuki Katsumi , Amao Hiromi , Sawada Takuo
Published: November 2013

A total of 52 isolates of Pasteurella pneumotropica obtained from rodents were examined for their genetic heterogeneity. On the basis of DNA restriction analysis, including amplified 16S ribosomal DNA restriction analysis (ARDRA) and pulsed‐field gel electrophoresis (PFGE), differences were identified among the isolates. ARDRA typing with HaeIII revealed 4 different banding patterns of the P. pneumotropica isolates. Eighty‐two percent of the 23 isolates identified as a‐1 were derived from mice, whereas all the isolates identified as a‐3 were derived from rats. Most of the isolates, which showed hemolytic activity on blood agar, obtained from mice and rats, were identified as a‐2 and a‐4, respectively. By restriction analysis of genomic DNA, ApaI and NotI digestion differentiated 9 variants and an undiscriminating group. However, no close relation with regard to the phenotypic characteristics was observed among the variants. The isolates identified as a‐2 and a‐4 could not be distinguished by PFGE analysis. DNA restriction analysis revealed that the genetic diversity of the P. pneumotropica isolates was more complex than the phenotypic characteristics among the species, and that at least the P. pneumotropica isolates were clearly differentiated into 4 groups by ARDRA typing with HaeIII.
Quorum Sensing System Lactones Do Not Increase Invasiveness of a MexAB‐OprM Efflux Mutant but Do Play a Partial Role in Pseudomonas aeruginosa Invasion
Authors: Kondo Akira , Hirakata Yoichi , Gotoh Naomasa , Fukushima Kazuko , Yanagihara Katsunori , Ohno Hideaki , Higashiyama Yasuhito , Miyazaki Yoshitugu , Nishide Kiyoharu , Node Manabu , Yamada Yasuaki , Kohno Shigeru , Kamihira Shimeru
Published: November 2013

We studied the quorum sensing (QS) system and the related homoserine lactones (HSLs) observing Pseudomonas aeruginosa invasion using the epithelial cell monolayer penetration assay model. Compared to the PAO1 wild‐type, the QS mutants, ΔlasI and ΔrhlI, were compromised in their capacity to invade. The decreased invasiveness of ΔrhlI was restored by adding 100 μM exogenous C4‐HSL. However, the decreased invasiveness of an efflux mutant, AmexAB‐oprM, was not restored in the presence of exogenous HSLs. The QS system partially plays a role in P. aeruginosa invasion; however, C4‐HSL and 3‐O‐C12‐HSL are not the essential determinants for invasiveness for P. aeruginosa.
First Record of Leptospira borgpetersenii Isolation in the Amami Islands, Japan
Authors: Kawabata Hiroki , Sakakibara Seiji , Imai Yasuyuki , Masuzawa Toshiyuki , Fujita Hiromi , Tsurumi Miyako , Sato Fumio , Takano Ai , Nogami Sadao , Kaneda Kazuhide , Watanabe Haruo
Published: November 2013

In 2003, a Leptospira survey was performed on Yoroshima Island of the Amami Islands located in the southwestern part of Japan. Seven Leptospira strains were isolated from the field rat Rattus rattus, which were identified as L. borgpetersenii by flaB sequencing, 16S rDNA sequencing and gyrB sequencing, and serovar Javanica was determined by a microscopic agglutination test. NotI‐long restriction fragment analysis indicated that these isolates were genetically indistinguishable from an isolate from the Okinawa Islands. The present results suggest that L. borgpetersenii is migrating into the Amami Islands in Japan.
Two Different Conformations of Rabies Virus Glycoprotein Taken under Neutral pH Conditions
Authors: Irie Takashi , Kankanamge Pushpa Jenette , Kawai Akihiko
Published: November 2013

We previously reported that the rabies virus glycoprotein (G) takes either of two different conformations (referred to as B and C forms) under neutral pH conditions, that could be differentiated by their reactivity to a monoclonal antibody (mAb), #1–30–44, that recognizes the acid‐sensitive conformational epitope, and the formation taken is dependent on two separate regions containing Lys‐202 and Asn‐336 of the protein (Kankanamge et al., Microbiol. Immunol., 47, 507–519, 2003). Semi‐quantitative antibody‐binding assays demonstrated that only one‐third to one‐fourth of mature G proteins on the cell surface were taking the 1–30–44 epitope‐positive B form even at pH 7.4. The ratio of B to C varied, depending on the environmental pH, but did not decrease to zero even at pH 5.8–6.2, preserving a certain content (about 15–20%) of B form. Immunoprecipitation studies demonstrated that a portion of G proteins were intimately associated with a dimer form of matrix (M) protein in terms of resistance to treatment with a mixture of 1% deoxycholate and 1% Nonidet P‐40, and seemed to preserve the B form even at lower pHs. Similar results were also obtained with the virion‐associated G proteins, including the intimate association of a portion of the G proteins with the M protein dimer. From these results, we assume that a certain portion of the rabies virion‐associated G proteins are associated with a dimer form of M protein, keeping the 1–30–44 epitope‐positive B conformation under various pH conditions, which might possibly assure the virion's recognition of host cell receptor molecules in the body.
β‐Glucan Derived from Zymosan Acts as an Adjuvant for Collagen‐Induced Arthritis
Authors: Hida Shunsuke , Nagi‐Miura Noriko , Adachi Yoshiyuki , Ohno Naohito
Published: November 2013

Collagen‐induced arthritis (CIA) is an experimental model of rheumatoid arthritis (RA) and has helped researchers to analyze the pathogenesis of inflammatory joint disease. In classical CIA, Freund's complete adjuvant (FCA), which contains heat‐killed Mycobacterium tuberculosis, is used as an adjuvant. In our previous study, we reported that particles of β‐glucan, OX‐CA, derived from Candida albicans, acted as a proper adjuvant in the CIA model. In this study, to establish pure β‐glucan as an adjuvant for CIA, we tested a commercially available preparation of Zymosan A (ZYM) and modified its products. β‐Glucan fractions of ZYM were prepared by oxidation with various concentrations of NaClO. The oxidized ZYM (OX‐ZYM) was mainly composed of β‐glucan. In this study, we examined its effect as an adjuvant for CIA. DBA/1 mice injected with CII and OX‐CA developed arthritis 7–10 days after receiving booster injections; the OX‐ZYM fractions induced arthritis with the same time course. 0.01% OX‐ZYM (oxidized with a 0.01% NaClO solution) caused arthritis faster than 0.1% OX‐ZYM or 0.5% OX‐ZYM. In conclusion, β‐glucan derived from ZYM by brief oxidation with NaClO is a suitable adjuvant for a CIA model with anti‐CII antibody production.
Deletion Polymorphisms in the Promoter Region of Fcγ Receptor IIB Is Not Associated with Antigen‐Specific IgG2a and IgG2b Antibody Responses in NC/Nga Mice
Authors: Sakai Tohru , Kogiso Mari , Mitsuya Kaori , Komatsu Tatsushi , Yamamoto Shigeru
Published: November 2013

Fcγ receptor (R) IIB, a low‐affinity FcR for IgG, inhibits B cell Ag R (BCR)‐mediated activation when these two receptors are cross‐linked by Ag and IgG‐containing immune complexs (ICs). We found deletion polymorphisms in the promoter region of fcgr2b in NC/Nga mice, a model for human atopic dermatitis. NC/Nga mice produced significantly higher levels of ovalbumin (OVA)‐specific IgG, IgG2a and IgG2b than did BALB/c mice. Analysis of (BALB/c×NC/Nga)F1×BALB/c or (BALB/c×NC/Nga) F1×NC/Nga backcross mice revealed that deletion polymorphisms of fcgr2b in NC/Nga mice does not directly regulate hyper OVA‐specific IgG2a and IgG2b Ab responses.
Clostridium glycyrrhizinilyticum Sp. Nov., a Glycyrrhizin‐Hydrolysing Bacterium Isolated from Human Faeces
Authors: Sakuma Keita , Kitahara Maki , Kibe Ryoko , Sakamoto Mitsuo , Benno Yoshimi
Published: November 2013

Screening of faecal bacteria for glycyrrhetic acid (GA) production by hydrolysing of glycyrrhizin (GL) resulted in the isolation of two strains, designated ZM35T and ZM38. Strains ZM35T and ZM38 were Gram‐positive, obligate anaerobic, non‐spore‐forming and rod‐shaped bacteria. Analysis of the 16S rRNA gene sequences indicated that strains ZM35T and ZM38 belonged to cluster XIVa of the genus Clostridium. The 16S rRNA gene sequences of strains ZM35T and ZM38 were identical. Strain ZM35T exhibited approximately 94% to 95% identity with the validly described species, Clostridium oroticum (94.5%), Eubacterium contortum (93.8%), Ruminococcus gnavus (94.5%) and R. torques (95.1%). In an experiment of DNA‐DNA hybridization, it was confirmed that strains ZM35T and ZM38 were the same species. The guanine‐plus‐cytosine (G+C) content of strain ZM35T is 45.7 mol%. Based on the phylogenetic and phenotypic findings, we propose that strains ZM35T and ZM38 be assigned to a novel species named Clostridium glycyrrhizinilyticum. The type strain is ZM35T (=JCM 13368T=DSM 17593T).
Full‐Length Sequence Analysis of Subacute Sclerosing Panencephalitis (SSPE) Virus, a Mutant of Measles Virus, Isolated from Brain Tissues of a Patient Shortly after Onset of SSPE
Authors: Hotta Hak , Nihei Kenji , Abe Yu‐ichi , Kato Seiichi , Jiang Da‐Peng , Nagano‐Fujii Motoko , Sada Kiyonao
Published: November 2013

Subacute sclerosing panencephalitis (SSPE) virus, a measles virus (MeV) mutant, was isolated from brain tissues of a patient shortly after the clinical onset, and the entire viral genome was sequenced. The virus, named SSPE‐Kobe‐1, formed syncytia on B95a and Vero/SLAM cells without producing cell‐free infectious virus particles, which is characteristic of SSPE virus. Phylogenetic analysis classified SSPE‐Kobe‐1 into genotype D3. When compared with an MeV field isolate of the same genotype (Ich‐B strain), SSPE‐Kobe‐1 exhibited mutation rates of 0.8–1.6% at the nucleotide level in each of the protein‐coding regions of the viral genome. It is noteworthy that the mutation rate of the M gene (1.2%) of SSPE‐Kobe‐1 was considerably lower than for other SSPE virus strains reported so far, but that the majority of the mutations (75%) were the uridine‐to‐cytidine biased hypermutation characteristic of the SSPE virus M gene. At the amino acid level, the viral proteins, such as N, P., C, V, M., F, H and L proteins, had point‐mutations on 3, 7, 1, 4, 3, 9, 8 and 14 residues, respectively, compared with the Ich‐B strain. In addition, the F and H proteins had mutated C‐termini due to single‐point mutations near or at the stop codons. Two of the three mutations in the M protein were Leu‐to‐Pro mutations, which are likely to affect the conformation and, therefore, the function of the protein. Because of the relatively small number of mutations, SSPE‐Kobe‐1 would be a useful tool to study genetic evolution of SSPE virus.
Immunostimulating Factor Isolated from Actinobacillus actinomycetemcomitans Stimulates Monocytes and Inflammatory Macrophages
Authors: Jo Wol‐Soon , Yee Sung‐Tae , Yoon Sik , Nam Byung‐Huouk , Do Eunju , Jung Beom‐Seok , Jeong Soo‐Jin , Hong Sook‐Hee , Yoo Young‐Hyun , Kang Chi‐Dug , Lim Young‐Jin , Jeong Min‐Ho , Lee Jae‐Dong
Published: November 2013

Immunostimulating factor (ISTF) isolated from Actinobacillus actinomycetemcomitans which has been described previously, is distinct from lipopolysaccharide and induces proliferation of B cells. This study was undertaken to investigate whether ISTF might enhance the stimulation of other immune cells. Immunohistochemically, ISTF exhibited a profound stimulating effect on macrophages and dendritic cells as well as B cells in the spleen of BALB/c mice. ISTF was also recognized for its capacity to induce direct activation of mouse macrophages to produce IL‐6, TNF‐α, and NO and MHC class II expression. Therefore, it is postulated that ISTF stimulates macrophages and possibly other cells to produce a wide variety of proinflammatory mediators, which may be involved in the chronicity and tissue destruction of periodontal disease.
Possible Mechanism of Adenovirus Generation from a Cloned Viral Genome Tagged with Nucleotides at Its Ends
Authors: Fukuda Hiromitsu , Terashima Miho , Koshikawa Michiko , Kanegae Yumi , Saito Izumu
Published: November 2013

The entire cloned human adenovirus type 5 (Ad5) genome is known to be able to generate infectious virus after transfection into 293 cells when the both ends of the genome are exposed by digestion with appropriate restriction enzymes. However, when one or both ends of the genome are tagged with nucleotides and are not intact, whether the tagged end of the viral genome was remained tagged or corrected to be intact during the generation of viral clones has been unclear and, if such oligonucleotide removal occurs, how does the virus remove these tagged sequences and thereby restore its proper structure? Here, we show in our semi‐quantitative study that the generation efficiency of virus clones decreases depending on the length of nucleotide tags at the both ends and that both the oligonucleotide tags were precisely removed during virus generation with restoration of the proper terminal sequences. Interestingly the viral genome of which one end was tagged, while the other was attached about 12‐kb sequences, did generate intact viral clones at a reduced but significant efficiency. From these results, we here propose a possible mechanism whereby the terminal‐protein‐deoxycytidine complex enters from the enzyme‐cleaved end and reaches deoxyguanine at the initiating position of DNA synthesis in vivo. A replication origin at one end, embedded deeply in double‐stranded DNA, can be activated by two cycles of one‐directional full‐length DNA synthesis initiated by the other exposed replication origin about 30 kilobases away. We also describe new cassette cosmids which can use not only PacI but also BstBI for construction of an adenovirus vector, without reducing construction efficiency.