bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative Analysis of Cereulide, an Emetic Toxin of Bacillus cereus, by Using Rat Liver Mitochondria
Authors: Kawamura‐Sato Kumiko , Hirama Yumi , Agata Norio , Ito Hideo , Torii Kenzo , Takeno Akira , Hasegawa Tadao , Shimomura Yoshiharu , Ohta Michio
Published: November 2013

An emetic toxin cereulide, produced by Bacillus cereus, causes emetic food poisonings, but a method for quantitative measurement of cereulide has not been well established. A current detection method is a bioassay method using the HEp‐2 cell vacuolation test, but it was unable to measure an accurate concentration. We established a quantitative assay for cereulide based on its mitochondrial respiratory uncoupling activity. The oxygen consumption in a reaction medium containing rat liver mitochondria was rapid in the presence of cereulide. Thus uncoupling effect of cereulide on mitochondrial respiration was similar to those of uncouplers 2,4‐dinitrophenol (DNP), carbonylcyanide m‐chlorophenylhydrazone (CCCP), and valinomycin. This method gave constant results over a wide range of cereulide concentrations, ranging from 0.05 to 100 μg/ml. The minimum cereulide concentration to detect uncoupled oxygen consumption was 50 ng/ml and increased dose‐dependently to the maximum level. Semi‐log relationship between the oxygen consumption rate and the cereulide concentraiton enables this method to quantify cereulide. The results of this method were highly reproducible as compared with the HEp‐2 cell vacuolation test and were in good agreement with those of the HEp‐2 cell vacuolation test. The enterotoxin of B. cereus or Staphylococcus aureus did not show any effect on the oxygen consumption, indicating this method is specific for the identification of cereulide as a causative agent of emetic food poisonings.
A Comparison of Mycolic Acid Analysis for Nontuberculous Mycobacteria Identification by Thin‐Layer Chromatography and Molecular Methods
Authors: Leite Clarisse Queico Fujimura , Rocha Adalgiza da Silva , de Andrade Leite Sergio Roberto , Ferreira Rosa Maria Carvalho , Suffys Philip N. , de Souza Fonseca Leila , Saad Maria Helena Féres
Published: November 2013

The development of fast, inexpensive, and reliable tests to identify nontuberculous mycobacteria (NTM) is needed. Studies have indicated that the conventional identification procedures, including biochemical assays, are imprecise. This study evaluated a proposed alternative identification method in which 83 NTM isolates, previously identified by conventional biochemical testing and in‐house M. avium IS1245‐PCR amplification, were submitted to the following tests: thin‐layer chromatography (TLC) of mycolic acids and PCR‐restriction enzyme analysis of hsp65 (PRA). High‐performance liquid chromatography (HPLC) analysis of mycolic acids and Southern blot analysis for M. avium IS1245 were performed on the strains that evidenced discrepancies on either of the above tests. Sixty‐eight out of 83 (82%) isolates were concordantly identified by the presence of IS1245 and PRA and by TLC mycolic acid analysis. Discrepant results were found between the phenotypic and molecular tests in 12/83 (14.4%) isolates. Most of these strains were isolated from non‐sterile body sites and were most probably colonizing in the host tissue. While TLC patterns suggested the presence of polymycobacterial infection in 3/83 (3.6%) cultures, this was the case in only one HPLC‐tested culture and in none of those tested by PRA. The results of this study indicated that, as a phenotypic identification procedure, TLC mycolic acid determination could be considered a relatively simple and cost‐effective method for routine screening of NTM isolates in mycobacteriology laboratory practice with a potential for use in developing countries. Further positive evidence was that this method demonstrated general agreement on MAC and M. simiae identification, including in the mixed cultures that predominated in the isolates of the disseminated infections in the AIDS patients under study. In view of the fact that the same treatment regimen is recommended for infections caused by these two species, TLC mycolic acid analysis may be a useful identification tool wherever molecular methods are unaffordable.
Characterization of Effector Cells with Anti‐Candida Activity Obtained from Murine Bone Marrow Cells Cultured in the Presence of rhG‐CSF: Comparison between Normal and CY‐Treated Mice
Authors: Morofuji Shinichiro , Abe Shigeru , Okinaga Kota
Published: November 2013

Bone marrow cells (BMC) obtained from normal and cyclophosphamide (CY)‐treated mice were cultured in the presence of recombinant human granulocyte‐colony stimulating factor (rhG‐CSF) and their effector cell activities inhibiting growth of C. albicans were examined. When BMC from CY‐treated mice were preincubated with 0.05 ng/ml of rhG‐CSF, effector cells with enhanced anti‐C. albicans activity were recovered in the adherent cell population, whereas anti‐C. albicans activity of BMC from normal mice was found in the non‐adherent cell population. During culture without the presence of rhG‐CSF, non‐adherent BMC, seemingly granulocytes, from normal mice showed apoptotic change, but addition of rhG‐CSF clearly inhibited this change. On the other hand, when BMC from CY‐treated mice were cultured with rhG‐CSF, adherent cells as the main effector had the appearance of monocytes. These differences between the effectors with anti‐C. albicans activity obtained from normal and CY‐treated mice are discussed.
Analysis of Polymorphism of 18S rRNA Gene in Wuchereria bancrofti Microfilariae
Authors: Bhandari Yashodhar , Dabir Pankaj , Nandhakumar Krithika , Dayananda Kannayakanahalli Maheshwarappa , Shouche Yogesh Shripad , Reddy Maryada Venkata Rami
Published: November 2013

The polymorphism of the 18S rRNA gene in Wuchereria bancrofti microfilariae (mf) collected from three different zones in India was analyzed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). The RFLPs of the amplified products obtained after digestion with restriction enzymes SspI, MspI and HhaI showed no difference in the banding patterns among the mf isolates from different endemic zones. Further the sequencing of PCR products did not show any difference in the nucleotide sequence either. The phylogenetic analysis of the sequences of W. bancrofti mf isolates from different endemic zones has shown branching with the earlier reported sequences of W. bancrofti and its close relative Brugia malayi.
Lactobacillus acidophilus Expressing Recombinant K99 Adhesive Fimbriae Has an Inhibitory Effect on Adhesion of Enterotoxigenic Escherichia coli
Authors: Chu Hyuk , Kang Seungha , Ha Seckho , Cho Kwangkeun , Park Sung‐moo , Han Kyung‐ho , Kang Sang Kee , Lee HongGu , Han Seung Hyun , Yun Cheol H. , Choi Yunjaie
Published: November 2013

The most common enteric colibacillosis in neonatal and newborns is caused by enterotoxigenic Escherichia coli (ETEC). Colonization of ETEC in the small intestine is associated with adhesions using fimbriae, which is known as a specific adhesion factor and provides highly specific means for anchoring and prerequisite for an infectious agent. In the present study we have engineered Lactobacillus acidophilus to produce recombinant K99 fimbriae, which is used for the colonization to the intestine of pigs. The expression of K99 fimbrial protein was confirmed using SDS‐PAGE, immunoblot and agglutination analyses. To evaluate a function of the K99 fimbrial protein, inhibition and competition tests were performed on pre‐screened intestinal brush border from pigs. The tests showed that recombinant L. acidophilus, not control L. acidophilus, had a significant inhibitory effect to and competition against K99+ E. coli in a dose dependent manner. In conclusion, we demonstrated that recombinant K99 fimbriae producing L. acidophilus was able to prevent E. coli binding to intestinal brush border.
Effect of Orally Administered Non‐Viable Lactobacillus Cells on Murine Humoral Immune Responses
Authors: He Fang , Morita Hirotsugu , Kubota Akira , Ouwehand Arthur C. , Hosoda Masataka , Hiramatsu Masaru , Kurisaki Jun‐ichi
Published: November 2013

BALB/c mice were immunized intraperitoneally with the food antigen ovalbumin (OVA) while they were fed with Lactobacillus GG heated killed cell preparation. The oral administration of Lactobacillus GG did not appear to modify the antigen‐augmented serum IgE in the tested mice but significantly augmented serum OVA specific IgG in the tested mice fed with a diet containing 0.1% Lactobacillus GG as the non‐viable cell preparation (P<0.05). The fecal OVA specific IgA of the tested mice fed with non‐viable Lactobacillus GG cells was also significantly elevated (P<0.05) compared to those from OVA immunized mice. The spleen cells of mice fed with non‐viable Lactobacillus GG cells secreted more IL‐6 (P<0.01). These results suggest that the non‐viable Lactobacillus GG can augment the systemic and mucosal immune responses in a host animal favoring secretory IgA but not IgE in an adjuvant‐like manner.
Gene Cloning and Properties of the RND‐Type Multidrug Efflux Pumps MexPQ‐OpmE and MexMN‐OprM from Pseudomonas aeruginosa
Authors: Mima Takehiko , Sekiya Hiroshi , Mizushima Tohru , Kuroda Teruo , Tsuchiya Tomofusa
Published: November 2013

We cloned two operons for putative RND‐type multidrug efflux pumps from Pseudomonas aeruginosa by a PCR method. We designated the genes in one operon mexPQ(‐opmE) and in another operon mexMN. Introduction of the mexPQ‐opmE into drug hypersensitive cells resulted in elevated MICs of macrolides, fluoroquinolones and some other drugs. Introduction of the mexMN into the hypersensitive cells possessing oprM, but not into cells not possessing oprM, resulted in elevated MICs of chloramphenicol and thiamphenicol. Thus, we conclude that MexPQ‐OpmE and MexMN‐OprM are functional multidrug efflux pumps when expressed in P. aeruginosa.
Mycobacterial RNase E‐Associated Proteins
Authors: Kovacs Lorand , Csanadi Agnes , Megyeri Klara , Kaberdin Vladimir R. , Miczak Andras
Published: November 2013

RNase E and its complex with other proteins (‘degradosome’) play an important role in RNA processing and decay in Escherichia coli and in many other bacteria. To identify the proteins which can potentially interact with this enzyme in mycobacteria, Mycobacterium tuberculosis H37Rv RNase E was cloned and expressed as a 6HisFLAG‐tagged fusion protein. Analysis of the mycobacterial RNase E overexpressed and purified from M. bovis BCG revealed the presence of GroEL and two other copurified proteins, products of the Mb1721 (inorganic polyphosphate/ATP‐NAD kinase) and Mb0825c (acetyltransferase) genes. Identical copies of these two genes can be found in M. tuberculosis H37Rv.
Inhibitory Effect of RNAi on Japanese Encephalitis Virus Replication In Vitro and In Vivo
Authors: Murakami Manabu , Ota Takahide , Nukuzuma Soichi , Takegami Tsutomu
Published: November 2013

Flaviviruses include many insect‐mediated small viruses and still cause serious problems in the world. In humans, JEV can cause acute meningioencephalomyelitis, resulting in fatality rates of 5 to 40%. RNA‐interference (RNAi) as an antiviral mechanism was originally discovered in plants and then found in the specific suppression of gene expression of other organisms such as Caenorhabditis elegans, Drosophila and vertebrates. As JEV is an RNA virus, RNAi could be a reasonable approach for therapeutic purposes to use against Japanese encephalitis. In this study, we examined the effect of RNAi on JEV replication. Viral reproduction in Vero cells was decreased to 7.2% and 39.0% of control by the transfection of small interference RNAs, JCR and JN3R at 250 NM, respectively. Under the transfection of 5 μg/ml pJRi which produces stem‐loop RNAi, viral reproduction was decreased to about 10% of control. Western blot analysis indicated that RNAi inhibited the translation level. We used pJRi in the animal experiment. After the inoculation of viruses at 5× 103 PFU, pJRi at 1.0 and 5.0 μg/g was injected into mice i.p. JEV‐infected control mice (n=5) died within 15 days. pJRi (1.0 or 5.0 μg/g)‐medicated mice survived 40 or 80% at 15 days. The data clearly indicate that pJRi has highly potent inhibitory activity against JEV replication in vivo. The results in vivo and in vitro provide evidence that JEV replication was efficiently inhibited by RNAi and RNAis could be used as an antiviral drug against JEV infection.
Susceptibility Loci to Coronary Arteritis in Animal Model of Kawasaki Disease Induced with Candida albicans‐Derived Substances
Authors: Oharaseki Toshiaki , Kameoka Yosuke , Kura Fumiaki , Persad Amanda S. , Suzuki Kazuo , Naoe Shiro
Published: November 2013

We have established an animal model of coronary arteritis which is histopathologically similar to that observed in cases of Kawasaki disease (KD), is a well‐known childhood vasculitis syndrome. Coronary arteritis in this mouse model has been induced by intraperitoneal injection of Candida albicans‐derived substances (CADS). Arteritis varied by mouse strain with the highest incidence by 71.1% (27/38) found in C3H/HeN mice, but absent in CBA/JN mice (0%, 0/27), suggesting association of genomic background to develop the disease. The present study aims to elucidate the susceptibility loci associated with coronary arteritis by using this animal model. The association of the onset of arteritis with polymorphic microsatellite markers between the two strains was examined using one hundred and fifteen of N1 backcross progeny [(CBA×C3H)F1×C3H]. Based on our analysis, arteritis‐susceptibility loci with suggestive linkage were mapped on D1Mit171 and D1Mit245 (map position 20.2 cM) on chromosome 1 (P=0.0019). These loci include several kinds of inflammatory cytokine receptors, such as interleukin 1 receptor and tumor necrosis factor receptor. We also found the cytokine response against CADS, levels of inflammatory cytokines interleukin‐1β, tumor necrosis factor‐α, and interleukin‐6 in sera increased within 24 hr after CADS injection. Our results may indicate based on genomics that ligand‐receptor interaction between these inflammatory cytokines and the receptors of these cytokines may affect the onset of arteritis.
Genotyping of Norovirus Strains Detected in Outbreaks between April 2002 and March 2003 in Osaka City, Japan
Authors: Seto Yoshiyuki , Iritani Nobuhiro , Kubo Hideyuki , Kaida Atsushi , Murakami Tsukasa , Haruki Kosuke , Nishio Osamu , Ayata Minoru , Ogura Hisashi
Published: November 2013

Noroviruses (NVs) are the major cause of food‐ and waterborne nonbacterial gastroenteritis in Japan. Between April 2002 and March 2003, a total of 111 fecal specimens from 40 outbreaks of acute nonbacterial gastroenteritis in Osaka City, Japan were subject to NV detection. Seventy‐two samples (64.9%) from 31 outbreaks (77.5%) were NV positive by a real time reverse transcription (RT)‐PCR assay. To further determine the genotype of individual NV strains, we sequenced the capsid N‐terminal/shell (N/S) domain of some representative strains from each outbreak. The 51 NV strains detected in this study were segregated into 15 genotypes (6 in genogroup I and 9 in genogroup II), and GII/5 genotype NV was a dominant outbreak genotype.
Duplex Shuttle PCR for Differential Diagnosis of Budgerigar Fledgling Disease and Psittacine Beak and Feather Disease
Authors: Ogawa Hirohito , Yamaguchi Tsuyoshi , Fukushi Hideto
Published: November 2013

Two common viral diseases in psittacine birds including budgerigar fledgling disease (BFD), generally called avian polyomavirus (APV) infection, and psittacine beak and feather disease (PBFD) have similar clinical manifestations characterized by feather disorders. A duplex shuttle PCR was developed for detection of APV and PBFD virus (PBFDV). Two pairs of oligonucleotide primers were designed to amplify a 298‐bp fragment of the t/T antigen region of APV genome and a 495‐bp fragment of the capsid protein region encoded by open reading frame (ORF) C1 of PBFDV genome, respectively. In the present study, APV and PBFDV were detected simultaneously in one tube by duplex shuttle PCR using these two pairs of primers. The detection limits were 2 viral copies of APV and 3 viral copies of PBFDV. In the clinical application, we detected 16 APV‐positive, 15 PBFDV‐positive, and 3 mixed infected samples in 39 samples examined. Sequences of the amplified products were read. The t/T antigen region was conserved in the APV‐positive samples as expected. ORF C1 of PBFDV genome showed diversity. Phylogenic analysis indicated that PBFDV ORF C1 consisted of 6 clusters which were related to subfamilies of psittacine birds. Our duplex shuttle PCR could be a useful method for differential diagnosis and molecular epidemiology of BFD and PBFD.
Quantitative Microarray‐Based DNA‐DNA Hybridization Assay for Measuring Genetic Distances among Bacterial Species and Its Application to the Identification of Family Enterobacteriaceae
Authors: Amano Makoto , Ohkusu Kiyofumi , Kusaba Koji , Ikeda Hironori , Nagasawa Zenzo , Aoki Yosuke , Kawamura Yoshiaki , Kobatake Shinzo , Tanaka Takumi , Matsuura Shuuji , Ezaki Takayuki
Published: November 2013

Quantitative DNA‐DNA hybridization to measure the genetic distances among bacterial species is indispensable for taxonomical determination. In the current studies, we developed a method to determine bacterial DNA relatedness on a glass microarray. Reference DNAs representing a total 93 species of Enterobacteriaceae were arrayed on a glass microplate, and signal intensities were measured after 2 hr of hybridization with Cy3‐labeled bacterial DNAs. All immobilized DNAs from members of the family Enterobacteriaceae were identified by this method except for DNAs from Yersinia pseudotuberculosis and Y. pestis. These results suggest that quantitative microarray hybridization could be an alternative to conventional DNA‐DNA hybridization for measuring chromosome relatedness among bacterial species.
Nonstructural Proteins 4A and 4B of Hepatitis C Virus Transactivate the Interleukin 8 Promoter
Authors: Kadoya Hiroyasu , Nagano‐Fujii Motoko , Deng Lin , Nakazono Naoki , Hotta Hak
Published: November 2013

Interleukin 8 (IL‐8) is induced in many cell types by various stimuli including virus infection. It was reported that nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) was involved in induction of IL‐8 expression at both mRNA and protein levels in cultured human cells. In this study, we aimed to determine whether or not another HCV protein(s) transactivates the IL‐8 gene expression, by means of an IL‐8 promoter‐driven luciferase reporter assay and measurement of endogenous IL‐8 mRNA and secreted IL‐8 protein levels. We observed that NS4B, and NS4A to a lesser extent, significantly transactivated the IL‐8 promoter, which resulted in enhanced production of IL‐8 protein. Also, the IL‐8 expression was augmented in Huh‐7 cells harboring an HCV subgenomic RNA replicon, compared with the control cells. Deletion mutational analysis of the IL‐8 promoter revealed the possible involvement of the transcription factor AP‐1 in both NS4A‐ and NS4B‐mediated IL‐8 gene activation. In addition, the IL‐8 gene activation by NS4B, but not that by NS4A, was likely to involve NF‐κB and/or NFIL‐6. The degree of the transactivation by NS4B and NS4A varied with different human cell lines, with HeLa cells showing the strongest activation followed by Huh‐7 cells, and with HepG2 cells exhibiting a marginal level of activation. Taken together, our present results suggest the possibility that NS4B and NS4A play an important role in inducing the IL‐8 gene expression under certain cellular conditions, which might be one of the strategies to establish persistent HCV infection.
The Time‐Dependent Serial Gene Response to Zeocin Treatment Involves Caspase‐Dependent Apoptosis in HeLa Cells
Authors: Hwang Jooyeon , Kim Young‐Youl , Huh Sungjin , Shim Junghee , Park Chan , Kimm Kuchan , Choi Dong Kug , Park Tae‐Kyu , Kim Soonhag
Published: November 2013

Zeocin, a member of the bleomycin/phleomycin family of antibiotics, is known to bind DNA and to induce apoptosis in cervical cancer cells, but the mechanism underlying this apoptotic response is poorly understood. The present study was undertaken to elucidate time‐dependent serial transcript patterns in the HeLa cervical carcinoma cell line, following treatment with Zeocin. The HeLa cell proliferation rate was found to gradually decrease following Zeocin exposure, in a time‐ and dose‐dependent manner. RNA transcript level measurements, for time‐dependent serial gene expression profiling, were determined at 0, 6, 12, 18 and 24 hr using a 0.5 k apoptosis functional microarray chip. Further statistical analysis, using a significance test at a 95% confidence level, for transcripts with a greater than 2‐fold change on the array chips, identified 49 up‐regulated and 57 down‐regulated genes. Our gene expression profile data indicate that Zeocin treatment induces an initial release of cytochrome c, the down‐regulation of Bcl‐XL, ENDOG, DAXX and MDM2, and the up‐regulation of CASP and BID. This suggests that a p53‐independent mitochondrial caspase cascade pathway is primarily involved in Zeocin‐induced apoptosis. Such caspase‐dependent cytotoxic activity also implies that this cell death pathway occurs via the caspase 8 and BID genes. However, disruption of either FAS or TNFR1 signaling did not interfere with the Zeocin induced apoptotic response in our experimental system. We hypothesize that Zeocin could be active against cervical cancer cell resistance to conventional chemotherapy and postulate that Zeocin is a novel candidate for the development of new chemotherapeutic treatments of gynecological cancers.
Effect of Cytokine‐Encoding Plasmid Delivery on Immune Response to Japanese Encephalitis Virus DNA Vaccine in Mice
Authors: Bharati Kaushik , Appaiahgari Mohan Babu , Vrati Sudhanshu
Published: November 2013

We have previously shown that immunization of mice with plasmid pMEa synthesizing Japanese encephalitis virus (JEV) envelope protein induced anti‐JEV humoral and cellular immune responses. We now show that intra‐muscular co‐administration of mice with pMEa and pGM‐CSF, encoding murine granulocyte‐macrophage colony‐stimulating factor or pIL‐2, encoding murine interleukin‐2 given 4 days after pMEa, augmented anti‐JEV antibody titers. This did not enhance the level of protection in immunized mice against JEV. However, intra‐dermal co‐administration of pMEa and pGM‐CSF in mice using the gene gun, enhanced anti‐JEV antibody titers resulting in an increased level of protection in mice against lethal JEV challenge.
Characterization of Bacterium Isolated from the Sediment at Coastal Area of Omura Bay in Japan and Several Biological Activities of Pigment Produced by This Isolate
Authors: Nakashima Takuji , Kurachi Maki , Kato Yoko , Yamaguchi Kenichi , Oda Tatsuya
Published: November 2013

Recently we discovered a bacterial strain (MS‐02–063) that produces large amounts of red pigment from coastal area of Nagasaki Prefecture, Japan. Comparative 16S rDNA gene sequencing analysis revealed that strain MS‐02–063 was phylogenetically closely related to γ‐proteobacterium Hahella sp. MBIC 3957 that produces prodigiosin. However, some physiological and biochemical differences between strain MS‐02–063 and Hahella sp. MBIC 3957 were observed. The red pigment (RP‐063) produced by this isolate was highly purified from the culture supernatant. It was speculated that RP‐063 might be prodigiosin‐like pigment in physical properties and biological activities such as antibacterial and cytotoxic activity. Antibacterial activity of RP‐063 was examined by an agar dilution method. The results indicated that RP‐063 showed antibacterial activity for specific for pathogenic gram‐positive bacteria such as Staphylococcus aureus. The potency of antibacterial activity against S. aureus was nearly equal to those of tetracycline. Moreover, RP‐063 showed inhibition of the superoxide generation by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐stimulated mouse macrophage RAW 264.7 cell line. Prodigiosin members have a wide variety of biological properties, including anticancer and antimalarial, etc. Especially, potent immunosuppressive properties have been reported for prodigiosin members with the mechanism of action different from that of the other well known immunosuppressors in atopic dermatitis therapy such as cyclosporin A, FK506 and rapamycin. It is suggested that RP‐063 may be able to arrest the inflammation caused by superantigens secreted from S. aureus, which colonized skin on atopic dermatitis as well as suppression of activated lymphocyte proliferation and superoxide generation from leucocytes.
Human IgM Monoclonal Antibodies Reactive with HIV‐1‐Infected Cells Generated Using a Trans‐Chromosome Mouse
Authors: Okada Noriko , Yin Shuping , Asai Suzuka , Kimbara Noriaki , Dohi Natsuki , Hosokawa Masato , Wu Xiaoshan , Okada Hidechika
Published: November 2013

The trans‐chromosome (TC) mouse that we used harbors human chromosomes 2, 14 and/or 22, and has undergone knock‐out of its endogeneous genes coding for μ‐ and κ‐chains of immunoglobulin. One of these TC mice was immunized with HIV‐1‐infected U937 cells, and spleen cells from the immunized animal were fused with the mouse myeloma cell line to generate hybridoma cells. We selected hybridomas that produce human IgM antibodies (Abs) reactive with HIV‐1‐infected MOLT4 cells but not with uninfected MOLT4 cells. Two hybridoma cell lines were established termed 9F11 and 2G9. Although 0.4 μg/ml of 9F11 was able to induce complement‐mediated cytolysis of the infected cells in the presence of fresh human serum, 2G9 could not. There was no difference between the two monoclonal Abs in the base sequences of cDNAs coding for the constant regions of μ‐ and κ‐chains. Therefore, we speculate that the ability to activate complement on homologous cell membranes might reflect the structural presentation of antigenic molecules, which could facilitate the binding of an IgM Ab to multiple binding sites resulting in escape from restriction by species‐specific inhibitors of complement such as DAF (CD55) and CD59. On the other hand, 2G9 induced apoptosis of HIV‐1‐infected cells, including latently infected OM10.1 cells, although the Ag for 2G9 remains to be identified. Since both of the Abs had reduced reactivity toward HIV‐1‐infected MOLT4 cells following cultivation in the presence of tunicamycin, the responsible antigens would involve a sugar moiety.
Simultaneous Multicolor Detection System of the Single‐Molecular Microbial Antigen with Total Internal Reflection Fluorescence Microscopy
Authors: Hoshino Akiyoshi , Fujioka Kouki , Manabe Noriyoshi , Yamaya Shun‐ichi , Goto Yoji , Yasuhara Masato , Yamamoto Kenji
Published: November 2013

Immunological diagnostic methods have been widely performed and showed high performance in molecular and cellular biology, molecular imaging, and medical diagnostics. We have developed novel methods for the fluorescent labeling of several antibodies coupled with fluorescent nanocrystal QDs. In this study we demonstrated that two bacterial toxins, diphtheria toxin and tetanus toxin, were detected simultaneously in the same view field of a cover slip by using directly QD‐conjugated antibodies. We have succeeded in detecting bacterial toxins by counting luminescent spots on the evanescent field with using primary antibody conjugated to QDs. In addition, each bacterial toxin in the mixture can be separately detected by single excitation laser with emission band pass filters, and simultaneously in situ pathogen quantification was performed by calculating the luminescent density on the surface of the cover slip. Our results demonstrate that total internal reflection fluorescence microscopy (TIRFM) enables us to distinguish each antigen from mixed samples and can simultaneously quantitate multiple antigens by QD‐conjugated antibodies. Bioconjugated QDs could have great potentialities for in practical biomedical applications to develop various high‐sensitivity detection systems.
Interleukin‐8 Secretion by Epithelial Cells Infected with Diffusely Adherent Escherichia coli Possessing Afa Adhesin‐Coding Genes
Authors: Arikawa Kentaro , Meraz Ismail Mustafa , Nishikawa Yoshikazu , Ogasawara Jun , Hase Atsushi
Published: November 2013

Escherichia coli that adhere sparsely to human epithelial (HEp‐2) cells are known as diffusely adherent E. coli (DAEC) and considered potentially diarrheagenic. The role of the afimbrial adhesive sheath (Afa)—identified originally as a uropathogenic factor—in diffuse adhesion is now understood. However, the role of DAEC in diarrheal disease remains controversial. Recently, ability to induce interleukin‐8 (IL‐8) secretion from intestinal epithelial cells has been suggested as one of the properties of enterovirulent bacteria. In this study, we examined whether DAEC strains possessing Afa genes induced IL‐8 in cultures of human carcinoma epithelial cells (e.g., HEp‐2, Caco‐2, and T84). Nineteen afa‐positive DAEC strains were examined for their ability to induce IL‐8 secretion, and only 7 strains (37%; 7/19) induced IL‐8 as much as enteroaggregative E. coli did. No marked differences in adhesion were observed between high and low inducers. Diffusive adhesiveness itself is unlikely to be sufficient to induce IL‐8. All high inducers were motile and others were nonmotile. Additional stimulation by flagella may be required to cause high levels of chemokine induction. Motility or presence of flagella can be an important criterion to predict DAEC diarrheagenicity at clinical laboratories.
Molecular Identification of Ehrlichia Species and ‘Candidatus Neoehrlichia mikurensis’ from Ticks and Wild Rodents in Shizuoka and Nagano Prefectures, Japan
Authors: Naitou Hirotaka , Kawaguchi Daizoh , Nishimura Yuusaku , Inayoshi Megumi , Kawamori Fumihiko , Masuzawa Toshiyuki , Hiroi Midori , Kurashige Hideaki , Kawabata Hiroki , Fujita Hiromi , Ohashi Norio
Published: November 2013

A total of 293 ticks and 111 wild rodents that were collected in Shizuoka and Nagano Prefectures, Japan, were examined for infection of Ehrlichia species and ‘Candidatus Neoehrlichia mikurensis.’ The 16S rDNA or the omp‐1 gene of these bacterial DNAs were detected from the spleens of tick‐inoculated mice (5 positive/total 29 mice) or from the spleens of wild rodents (25 positive/total 111 rodents) by PCR amplification. Sequencing of the 16S rDNA revealed Ehrlichia spp. from the 5 tick‐inoculated mice and 8 wild rodents, and ‘Candidatus N. mikurensis’ from 17 wild rodents. The data suggest the presence of additional genetic variants, and potential vectors and/or reservoirs for these bacteria in central Japan.
Proteomic Surveillance of Autoantigens in Relapsing Polychondritis
Authors: Tanaka Yasuhiko , Nakamura Manabu , Matsui Toshihiro , Iizuka Nobuko , Kondo Hirobumi , Tohma Shigeto , Masuko Kayo , Yudoh Kazuo , Nakamura Hiroshi , Nishioka Kusuki , Koizuka Izumi , Kato Tomohiro
Published: November 2013

Relapsing polychondritis (RP) is a systemic inflammatory disease, in which autoimmunity to cartilage‐related components is thought to be involved in its pathogenesis. However, the autoimmune profile in RP has not been studied fully. We therefore investigated autoantibodies/autoantigens in RP comprehensively, by 2‐dimensional electrophoresis (2DE), subsequent western blotting (WB) and mass spectrometry, using cell‐extracted proteins as the antigen source. As a result, we detected 15 autoantigens on 2DE‐WB, and further identified five of them. On average, one RP serum recognized approximately 8 out of the 15 autoantigens. Frequencies of the autoantibodies to the 5 identified antigens of tubulin alpha ubiquitous/6, vimentin, alpha enolase, calreticulin, and colligin‐1/‐2 were 91%, 46%, 36%, 82%, and 36%, respectively. ELISA using recombinant proteins for them revealed that frequencies of the autoantibodies to tubulin alpha ubiquitous, vimentin, alpha enolase, calreticulin, and colligin‐1 were 36%, 64%, 46%, 27%, and 18%, respectively. Our data demonstrated that the autoimmune reaction was not restricted to cartilage‐related components, rather a variety of autoimmune responses occurred in patients with RP, which may be involved in the pathophysiology of RP. In addition, the proteomic approach using cell‐extracted proteins would be a powerful way to investigate autoantigens.
Competitive Study of Monoclonal Antibodies against the HIV‐1 Gp41 Core Structure
Authors: Usami Osamu , Xiao Peng , Ling Hong , Hattori Toshio
Published: November 2013

Monoclonal antibodies (MAbs) 50.69, 98.6, and T26 bind specifically to the core structure of the human immunodeficiency virus type 1 (HIV‐1) envelope transmembrane glycoprotein (gp41). To clarify the specificity of the anti‐core structure MAbs, we performed competitive assays using the MAbs to the H9 human T cell line infected with the IIIB strain of HIV‐1 (H9/IIIB). Bound MAb 50.69 inhibited MAb 98.6 binding unidirectionally. The reason for the unidirectional cross competition between MAbs 50.69 and 98.6 is not clear, but these results help to define the antigenic structure of gp41 on the surface of infected cells.
A Novel Mouse Model for MPO‐ANCA‐Associated Glomerulonephritis
Authors: Yumura Wako , Itabashi Mitsuyo , Ishida‐Okawara Akiko , Tomizawa Kazuo , Yamashita Junji , Kaneshiro Yoshiaki , Nihei Hiroshi , Suzuki Kazuo
Published: November 2013

We established a novel model mouse for myeloperoxidase anti‐neutrophil cytoplasmic antibody (MPO‐ANCA)‐associated glomerulonephritis with crescentic formation, which was induced by administering bovine serum albumin (BSA). Neutrophil infiltration into the renal glomeruli began at 8 weeks and crescent formation was observed from 10 weeks after the first BSA injection. Platelet and neutrophil counts significantly increased, and proteinuria was observed from 5 weeks. MPO‐ANCA increased slightly at 4 and markedly at 9 weeks, and the TNF‐α level increased at 11 weeks. Glomerular neutrophil infiltration was correlated with MPO‐ANCA levels. In addition, proteinuria also significantly correlated with MPO‐ANCA levels. Finally, renal crescent formation was associated with an increase of MPO‐ANCA levels and neutrophil infiltration into glomeruli. The glomerular immune deposition of IgG and C3 was observed. These findings indicate that BSA induces neutrophil activation of peripheral blood followed by the elevation of MPO‐ANCA, resulting in the development of crescentic glomerulonephritis in mice.
A Viral Non‐Coding Region Determining Neuropathogenicity of Murine Leukemia Virus A8 Is Responsible for Envelope Protein Expression in the Rat Brain
Authors: Takase‐Yoden Sayaka , Wada Misaho , Watanabe Rihito
Published: November 2013

Friend murine leukemia virus clone A8 causes spongiform neurodegeneration in the rat brain. A 0.3‐kb fragment containing the R‐U5–5′ leader sequence of A8 is required in addition to the A8‐env gene to induce spongiosis. The A8‐env gene is a primary determinant of neuropathogenicity. Comparative studies of the neuropathogenic virus R7f, which carries the 0.3‐kb fragment of A8 and A8‐env on the background of the non‐neuropathogenic clone 57, and the non‐neuropathogenic virus Rec5, which carries A8‐env on the background of 57, showed that the 0.3‐kb fragment of A8 was responsible for increasing the ratio of Env/Gag expression in the brain, but not in the spleen.
Virulence‐Defective Strains of Salmonella enterica Serovar Typhi as Candidates for Education at Level 2 Facilities
Authors: Huang Xinxiang , Xu Huaxi , Sha Mohammad Monir , Zhao Licheng , Ohkusu Kiyofumi , Kawamura Yoshiaki , Ezaki Takayuki
Published: November 2013

The use of biosafety level 3 pathogens is an essential element of education and training at medical schools. We previously reported on invasion‐defective strains of Salmonella enterica serovar Typhi, GTC 3P408 (ΔinvA, ΔsipB) and GTC 3P409 (ΔinvA, ΔsipB, and ΔviaB), as candidates for use in educational programs. Vi negative strains of S. enterica serovar Typhi became extremely sensitive to complement attack but showed increased invasiveness. Therefore, this study was conducted to construct two virulence‐defective strains, GTC 3P460 (ΔinvA, ΔsipB, and ΔrpoS) and GTC 3P461 (ΔinvA, ΔsipB, ΔviaB, and ΔrpoS), of S. enterica serovar Typhi by deleting rpoS from the GTC 3P409 and GTC 3P408 strains. Stress tests demonstrated that GTC 3P460 and GTC 3P461 are sensitive to conditions of starvation, acid stress and oxidative stress. These results suggest that these virulence‐defective strains have difficulty surviving in the gastric environment and in macrophages, characteristics that make them ideal candidates for education at level 2 facilities. Colony morphology and conventional biochemical features of these strains are identical to the parent strain S. enterica serovar Typhi GIFU 10007.
Determination of Antifungal Activities in Serum Samples from Mice Treated with Different Antifungal Drugs Allows Detection of an Active Metabolite of Itraconazole
Authors: Maki Katsuyuki , Watabe Etsuko , Iguchi Yumi , Nakamura Hideko , Tomishima Masaki , Ohki Hidenori , Yamada Akira , Matsumoto Satoru , Ikeda Fumiaki , Tawara Shuichi , Mutoh Seitaro
Published: November 2013

To establish an in vitro method of predicting in vivo efficacy of antifungal drugs against Candida albicans and Aspergillus fumigatus, the antifungal activities of fluconazole, itraconazole, and amphotericin B were determined in mouse serum. The minimum inhibitory concentration (MIC) of each drug was measured using mouse serum as a diluent. For C. albicans, the assay endpoint of azoles was defined as inhibition of mycelial extension (mMIC) and for A. fumigatus, as no growth (MIC). The MICs of amphotericin B for both pathogens were defined as the MIC at which no mycelial growth occurred. Serum MIC or mMIC determinations were then used to estimate the concentration of the drugs in serum of mice treated with antifungal drugs by multiplying the antifungal titer of the serum samples by the serum (m)MIC. The serum drug concentrations were also determined by HPLC. The serum concentrations estimated microbiologically showed good agreement with those determined by HPLC, except for itraconazole. Analysis of the serum samples from itraconazole‐treated mice by a sensitive bioautography revealed the presence of additional spots, not seen in control samples of itraconazole. The bioautography assay demonstrated that the additional material detected in serum from mice treated with itraconazole was an active metabolite of itraconazole. The data showed that the apparent reduction in the itraconazole serum concentration as determined by HPLC was the result of the formation of an active metabolite, and that the use of a microbiological method to measure serum concentrations of drugs can provide a method for prediction of in vivo efficacy of antifungal drugs.
Immunodominant Epitope in the C‐Terminus of a Variable Major Protein in Borrelia duttonii, an Agent of Tick‐Borne Relapsing Fever
Authors: Tabuchi Norihiko , Tomoda Koichiro , Kawaguchi Hiroshi , Iwamoto Hiroyuki , Fukunaga Masahito
Published: November 2013

Borrelia duttonii strain Ly was isolated from a child with tick‐borne relapsing fever in Tanzania. B. duttonii produces variable major proteins (Vmps), which undergo antigenic variation. We previously reported transcription of the vmpP gene, which is one of the Vmp genes in strain Ly, detected in vitro cultivation. In the current study, we purified the recombinant non‐lipidated VmpP protein by affinity chromatography and produced VmpP polyclonal antibodies. Antigenicity of VmpP was examined by Western immunoblot analysis and peptide‐based enzyme‐linked immunosorbent assays. Antigenic epitopes were shown to comprise five regions interspersed within the VmpP primary amino acid sequence. Synthetic peptides spanning residues of three of five regions, 232–237 (LASIVD), 280–285 (AGGIAL), and 350–355 (KAADQQ), reacted strongly with the VmpP‐specific antibody and these residues were identified as epitopes. In particular, the C‐terminal domain (KAADQQ) of this protein was immunoreactive. Further research based on our results will promote the development of a recombinant vaccine for B. duttonii infection.
Gingipains Inactivate a Cell Surface Ligand on Porphyromonas gingivalis That Induces TLR2‐ and TLR4‐Independent Signaling
Authors: Kishimoto Mami , Yoshimura Atsutoshi , Naito Mariko , Okamoto Kuniaki , Yamamoto Kenji , Golenbock Douglas T. , Hara Yoshitaka , Nakayama Koji
Published: November 2013

Arginine‐specific gingipain and lysine‐specific gingipain are two major cysteine proteinases produced by Porphyromonas gingivalis. To clarify the role of gingipains in the interaction between P. gingivalis and the innate immune system, CHO reporter cells expressing TLR2 or TLR4 were stimulated with wild‐type or gingipain‐deficient P. gingivalis cells and activation of nuclear factor‐κB in these cells was examined. While CHO/CD14 cells and 7.19 cells, an MD‐2‐defective mutant derived from CHO/CD14 cells, failed to respond to wild‐type P. gingivalis, they responded to gingipain‐deficient P. gingivalis. On the other hand, CHO/CD14/TLR2 cells responded to both wild‐type and gingipain‐deficient P. gingivalis. These results suggested that gingipains have no effects on TLR2‐dependent signaling from P. gingivalis but have inhibitory effects on TLR2‐ and TLR4‐independent signaling in CHO cells. Indeed, the activity of gingipain‐deficient P. gingivalis to induce the activation of 7.19 cells was diminished after treatment of the bacterial cells with gingipains. We next partially purified bacterial cell components activating 7.19 cells from gingipain‐deficient P. gingivalis. The activity of the partially purified components was diminished by treatment with heat or gingipains. It is also noteworthy that anti‐CD14 mAb inhibited the activation of 7.19 cells induced by the partially purified components. These results indicated that the components of P. gingivalis that were able to induce TLR2‐ and TLR4‐independent signaling were inactivated by gingipains before being recognized by CD14. The inactivation of the components would be helpful for P. gingivalis to escape from the innate immune system.
Characterization of Biological Activities of Brucella melitensis Lipopolysaccharide
Authors: Tumurkhuu Gantsetseg , Koide Naoki , Takahashi Kazuko , Hassan Ferdaus , Islam Shamima , Ito Hiroyasu , Mori Isamu , Yoshida Tomoaki , Yokochi Takashi
Published: November 2013

Biological activities of lipopolysaccharide (LPS) from Brucella melitensis 16M were characterized in comparison with LPS from Escherichia coli O55. LPS extracted from B. melitensis was smooth type by electrophoretic analysis with silver staining. The endotoxin‐specific Limulus activity of B. melitensis LPS was lower than that of E. coli LPS. There was no significant production of tumor necrosis factor‐α and nitric oxide in RAW 264.7 macrophage cells stimulated with B. melitensis LPS, although E. coli LPS definitely induced their production. On the other hand, B. melitensis LPS exhibited a higher anti‐complement activity than E. coli LPS. B. melitensis LPS as well as E. coli LPS exhibited a strong adjuvant action on antibody response to bovine serum. The characteristic biological activities of B. melitensis are discussed.
Enhancement of Mice Susceptibility to Infection with Listeria monocytogenes by the Treatment of Morphine
Authors: Asakura Hiroshi , Kawamoto Keiko , Igimi Shizunobu , Yamamoto Shigeki , Makino Sou‐ichi
Published: November 2013

The effect of morphine on the susceptibility of BALB/c mice to diarrheagenic Escherichia coli, Shigella flexneri, Listeria monocytogenes, Salmonella Enteritidis, Yersinia enterocolitica, was examined via the intraperitoneal inoculation. Morphine treatment increased the susceptibility to S. Enteritidis and L. monocytogenes, resulting in bacteremia and central nervous system (CNS) invasion (for L. monocytogenes), while the infection with other bacteria did not show the systemic dissemination in the morphine‐treated mice. Notably, L. monocytogenes infection caused 100% mortality with a mean survival time (MST) of 1.3 days in morphine‐treated mice, but untreated mice did not die. The present data suggested that individuals using heroin or treated with morphine derivatives might be at high risk for listeriosis, especially those who are immunocompromised. Recent increasing consumption of morphine may propose the necessity for further epidemiological surveillance on infectious diseases.
Quantitative Analysis of Cutaneous Malassezia in Atopic Dermatitis Patients Using Real‐Time PCR
Authors: Sugita Takashi , Tajima Mami , Tsubuku Hisae , Tsuboi Ryoji , Nishikawa Akemi
Published: November 2013

We quantified the cutaneous Malassezia in patients with atopic dermatitis using a real‐time PCR assay. Seven to 12 times more Malassezia colonized the head and neck compared to the trunk or limbs, and the species M. globosa and M. restricta accounted for approximately 80% of all Malassezia colonization at any body site.
A Slow Spread of Adenovirus Type 7 Infection after Its Re‐Emergence in Yamagata, Japan, in 1995
Authors: Mizuta Katsumi , Abiko Chieko , Aoki Yoko , Murata Toshio , Katsushima Noriko , Sakamoto Michiyo , Itagaki Tsutomu , Hoshina Hitoshi , Ootani Katsumi
Published: November 2013

We have continued the epidemiological study on adenovirus type 7 (Ad7), which re‐emerged in 1995 in Yamagata, Japan. Between 1999 and 2004, we isolated only four strains from 10,778 throat swab specimens among children with acute respiratory infections. A serological survey of 303 specimens revealed the antibody‐positive rate against Ad7 to be 0–7.4% in children under 10 years of age in 2005, although it was 3.3–16.7% in 1997 and 0% in 1993. Our results suggest that a re‐emergence does not always provoke a sudden major outbreak, even if the antibody‐positive rate against Ad7 is low in the local community.
Cholera Toxin Modulates the Systemic Immune Responses against Vibrio cholerae Surface Antigens after Repeated Inoculations
Authors: Fernandez‐Miyakawa Mariano E. , Brero Maria Luisa , Mateo Nancy A.
Published: November 2013

The immunomodulating properties of a low cholera toxin (CT) dose over the systemic antibody response against Vibrio cholerae antigens after a comparatively extensive period of time were evaluated. Groups of 10 mice were injected intraperitoneally three times at 0, 30 and 86 days with 500 μl of buffer or 108 viable recombinant V. cholerae bacteria (lacking cholera toxin A subunit) with or without 100 ng of CT. Sera were obtained from inoculated mice at 0, 14, 28, 37, 58, 80, 93, 114, 236 and 356 days after the first injection. Vibriocidal activity and IgM and IgG anti‐lipopolysaccharide (LPS) or outer membrane protein (OMP) antibodies levels were estimated by ELISA in sera of inoculated mice. Anti‐LPS IgG subclasses were measured 2 weeks after each immunization by ELISA. Treatment of mice with CT markedly influenced the immune response to LPS but not against OMP of V. cholerae. Simultaneous intraperitoneal administration of CT with V. cholerae resulted in marked enhancement of both IgM anti‐LPS and vibriocidal titers which subsisted for a relatively extensive period of time after repeated antigen administration. No differences were observed in IgM and IgG anti‐OMP titers after extended periods of time between CT and control treatments. A similar pattern of IgG anti‐LPS subclasses was observed in the serum samples analyzed. These results suggest that long term CT administration modulates the IgM anti‐V. cholerae LPS response and the serum vibriocidal activity.
Antimicrobial Activities of Cinnamyl Rifamycin Derivatives, T‐9 and T‐11, against Mycobacterium tuberculosis and Mycobacterium avium Complex (MAC) with Special Reference to the Activities against Intracellular MAC
Authors: Sato Katsumasa , Shimizu Toshiaki , Dimova Velitchka , Tomioka Haruaki
Published: November 2013

The mycobacterial activities of cinamyl rifamycin derivatives, T‐9 and T‐11, especially against extracellular and intracellular Mycobacterium avium complex residing within macrophages and type II pneumocytes were compared with those of other rifamycins. The activities of test rifamycins were found to be in the order rifalazil, rifabutin, T‐9, T‐11, and rifampicin.