bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevalence of Drug‐Resistant Human Immunodeficiency Virus Type 1 in Therapy‐Naive Patients and Usefulness of Genotype Testing
Authors: Ibe Shiro , Hotta Naoe , Takeo Uta , Tawada Yukio , Mamiya Naoto , Yamanaka Katsuo , Utsumi Makoto , Kaneda Tsuguhiro
Published: November 2013

In the present study, we performed genotypic drug‐resistance testing in 116 therapy‐naive human immunodeficiency virus type 1 (HIV‐1)‐infected patients between 1999 and 2002 at Nagoya National Hospital, Japan. The prevalence of drug‐resistant HIV‐1 with one or more major mutations significantly increased from 5.3% (4/75) in 1999–2001 to 17.1% (7/41) in 2002 (P=0.05), suggesting the spread of drug‐resistant HIV‐1. We identified a patient who possessed a protease (PR) inhibitor‐resistant HIV‐1 with a major mutation consisting of L90M before the initiation of therapy. The patient was administered zidovudine, lamivudine, and efavirenz as highly active antiretroviral therapy (HAART), as PR inhibitors were excluded based on the result of the drug‐resistance testing. The treatment succeeded in strongly suppressing the proliferation of drug‐resistant HIV‐1 and concomitantly increased CD4 cell counts. Thus, we conclude that drug‐resistance testing prior to the initiation of therapy is important for therapy‐naive patients to devise the optimum therapy regimen for each individual.
Inhibition of the Protease Activity of Influenza Virus RNA Polymerase PA Subunit by Viral Matrix Protein
Authors: Hara Koyu , Shiota Mayumi , Kido Hiroshi , Watanabe Ken , Nagata Kyosuke , Toyoda Tetsuya
Published: November 2013

Influenza virus PA is a subunit of RNA‐dependent RNA polymerase. We demonstrated that PA has a unique chymotrypsin‐like serine protease activity with Ser624 as an active site. To obtain further insight into the role of the protease activity of PA in viral proliferation, we examined the interaction between PA and matrix protein (M1). Both M1 purified from virion and hexa‐histidine‐tagged M1 expressed in Escherichia coli bound to PA. Hexa‐histidine‐tagged M1 pulled down PA. The interaction of PA with M1 was sensitive to ionic strength, suggesting that the interaction is formed by electrostatic force. Using Suc‐Leu‐Leu‐Val‐Tyr‐MCA, a specific substrate for PA protease, M1 was demonstrated to inhibit the amidolytic activity of PA, whereas M1 did not inhibit that of chymotrypsin or trypsin at all. These results suggest that M1 binds to and inhibits the amidolytic activity of PA.
Prevalence of Inherited Myeloperoxidase Deficiency in Japan
Authors: Nunoi Hiroyuki , Kohi Fumikazu , Kajiwara Hideki , Suzuki Kazuo
Published: November 2013

The microbicidal activity of the myeloperoxidase (MPO)‐hydrogen peroxide‐halide system has been implicated as the most efficient, oxygen‐dependent antimicrobial component of neutrophil host defense. Unexpectedly, individuals with MPO deficiency suffer few clinical consequences. In order to understand better the clinical impact of MPO deficiency, we surveyed several clinical hematology laboratories in Japan to assess the prevalence of MPO deficiency in the general population. MPO activity was determined by flow cytometry using the Technicon H series of automated systems. We identified 26 cases of complete MPO deficiency, prevalence 1 in 57,135, and 129 cases of partial deficiency, prevalence 1 in 17,501. The distribution of complete and partial deficiencies differed among the laboratories studied.
Cloning and Expression of Bartonella henselae sucB Gene Encoding an Immunogenic Dihydrolipoamide Succinyltransferase Homologous Protein
Authors: Kabeya Hidenori , Maruyama Soichi , Hirano Kouji , Mikami Takeshi
Published: November 2013

Immunoscreening of a ZAP genomic library of Bartonella henselae strain Houston‐1 expressed in Escherichia coli resulted in the isolation of a clone containing 3.5 kb BamHI genomic DNA fragment. This 3.5 kb DNA fragment was found to contain a sequence of a gene encoding a protein with significant homology to the dihydrolipoamide succinyltransferase of Brucella melitensis (sucB). Subsequent cloning and DNA sequence analysis revealed that the deduced amino acid sequence from the cloned gene showed 66.5% identity to SucB protein of B. melitensis, and 43.4 and 47.2% identities to those of Coxiella burnetii and E. coli, respectively. The gene was expressed as a His‐Nus A‐tagged fusion protein. The recombinant SucB protein (rSucB) was shown to be an immunoreactive protein of about 115 kDa by Western blot analysis with sera from B. henselae‐immunized mice. Therefore the rSucB may be a candidate antigen for a specific serological diagnosis of B. henselae infection.
Phylogenetic Characterization of Orientia tsutsugamushi Isolated in Taiwan According to the Sequence Homologies of 56‐kDa Type‐Specific Antigen Genes
Authors: Qiang Yu , Tamura Akira , Urakami Hiroshi , Makisaka Yukie , Koyama Shoichiro , Fukuhara Masahiro , Kadosaka Teruki
Published: November 2013

Fourteen strains of Orientia tsutsugamushi isolated in Taiwan were characterized by sequencing 56‐kDa type‐specific antigen genes and patterns of restriction fragment length polymorphism (RFLP) predicted by a computer program. The strains showed high varieties in sequence homologies and were classified to 10 types by predicted patterns of RFLP. Furthermore, all Taiwan strains were not identical in typing with strains analyzed previously. These results suggest that there are various types of O. tsutsugamushi in Taiwan that are different from those distributed in other countries.
The Inhibitory Effect of Essential Oils on Herpes Simplex Virus Type‐1 Replication In Vitro
Authors: Minami Masato , Kita Masakazu , Nakaya Takaaki , Yamamoto Toshiro , Kuriyama Hiroko , Imanishi Jiro
Published: November 2013

The antiviral effect of 12 essential oils on herpes simplex virus type‐1 (HSV‐1) replication was examined in vitro. The replication ability of HSV‐1 was suppressed by incubation of HSV‐1 with 1% essential oils at 4 C for 24 hr. Especially, lemongrass completely inhibited the viral replication even at a concentration of 0.1%, and its antiviral activity was dependent on the concentrations of the essential oil. When Vero cells were treated with the essential oil before or after viral adsorption, no antiviral activity was found, which suggests that the antiviral activity of essential oils including lemongrass may be due to the direct interaction with virions.
Sensitive and Rapid Quantitative Detection of Anthrax Spores Isolated from Soil Samples by Real‐Time PCR
Authors: Ryu Chunsun , Lee Kyunghee , Yoo Cheonkwon , Seong Won Keun , Oh Hee‐Bok
Published: November 2013

Quantitative analysis of anthrax spores from environmental samples is essential for accurate detection and risk assessment since Bacillus anthracis spores have been shown to be one of the most effective biological weapons. Using TaqMan real‐time PCR, specific primers and probes were designed for the identification of pathogenic B. anthracis strains from pag gene and cap gene on two plasmids, pXO1 and pXO2, as well as a sap gene encoded on the S‐layer. To select the appropriate lysis method of anthrax spore from environmental samples, several heat treatments and germination methods were evaluated with multiplex‐PCR. Among them, heat treatment of samples suspended with sucrose plus non‐ionic detergent was considered an effective spore disruption method because it detected up to 105 spores/g soil by multiplex‐PCR. Serial dilutions of B. anthracis DNA and spore were detected up to a level of 0.1 ng/μl and 10 spores/ml, respectively, at the correlation coefficient of 0.99 by real‐time PCR. Quantitative analysis of anthrax spore could be obtained from the comparison between CT value and serial dilutions of soil sample at the correlation coefficient of 0.99. Additionally, spores added to soil samples were detected up to 104 spores/g soil within 3 hr by real‐time PCR. As a consequence, we established a rapid and accurate detection system for environmental anthrax spores using real‐time PCR, avoiding time and labor‐consuming preparation steps such as enrichment culturing and DNA preparation.
Differential Expression and Signaling of IFN‐γ in the Conjunctiva between Lewis and Brown Norway Rats
Authors: Fukushima Atsuki , Ozaki Akemi , Fukata Kazuyo , Ueno Hisayuki
Published: November 2013

Genetic background determines the histological features of experimental immune‐mediated blepharoconjunctivitis (EC) in rats, which is a model for human allergic conjunctivitis (AC). A great number of lymphocytes predominate in EC of Lewis rats, while less lymphocytes and more eosinophils are present in that of Brown Norway (BN) rats. Although this difference could be attributed to their systemic Th1/Th2 dominancy, it remains unclear whether some regulatory mechanisms may exist in the inflammatory site in the conjunctiva. Here, we aim to investigate this hypothesis by comparing the expression levels of inflammatory mediators in the conjunctiva between the two strains. EC was induced in Lewis and BN rats by transfer of ovalbumin (OVA)‐specific CD4+ T‐cell lines followed by eye drops of OVA as antigen challenge, and then was clinically and histologically evaluated. Reverse‐transcription (RT)‐PCR was performed to compare the expressions of cytokines and cytokine receptors (Rs) in conjunctivas of both strains of rats either with or without EC. To confirm the biological significance of interferon (IFN)‐γ R expression, phosphorylation of signal transducers and activators of transcription (STAT)‐1 was examined in the conjunctivas, followed by subconjunctival injection of IFN‐γ. BN T cells contained interleukin (IL)‐4 and IFN‐γ, while Lewis T cells expressed no IL‐4. Transfer of those cells induced more severe EC in Lewis rats. RT‐PCR using naive conjunctivas detected more IL‐4, IFN‐γ, and IFN‐γR β‐chain RNA expression in BN rats. After the EC induction, BN rats expressed significantly higher amounts of IFN‐γR β‐chain, and upregulation of interferon regulatory factor (IRF)‐1 was observed. Phosphorylation of STAT‐1 was more remarkable in BN rats. The findings demonstrate differential expression of IFN‐γR and signaling through IFN‐γ in the conjunctiva between the two strains. This may be due to differences in histopathological character between the two strains.
Distribution of the usp Gene in Uropathogenic Escherichia coli Isolated from Companion Animals and Correlation with Serotypes and Size‐Variations of the Pathogenicity Island
Authors: Kurazono Hisao , Nakano Masayuki , Yamamoto Shingo , Ogawa Osamu , Yuri Kazuyo , Nakata Katsuhisa , Kimura Miyuki , Makino Sou‐ichi , Nair G. Balakrish
Published: November 2013

Uncomplicated urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) is a serious problem not only among humans but also in companion animals such as dogs and cats. The uropathogenic specific protein gene (usp) is preferentially distributed in UPEC isolates from dogs and cats compared with the distribution of usp in E. coli strains from feces of healthy dogs and cats and this pattern of distribution resembles that observed in human UPEC strains. The UPEC strains from companion animals share common O serotypes like O1, O2, O4, O6, O16, O18, O22, O25 and O75 as those reported for human UPEC. The size variation of the pathogenicity island that includes usp in UPEC from dogs and cats was almost similar to those seen in human UPEC. We propose that dogs and cats are the alternative reservoirs for UPEC strains that are associated with human UTI.
Phylogenetic Analysis of Spotted Fever Group Rickettsiae Based on gltA, 17‐kDa, and rOmpA Genes Amplified by Nested PCR from Ticks in Japan
Authors: Ishikura Mitsuhiro , Ando Shuji , Shinagawa Yasuhiro , Matsuura Kumiko , Hasegawa Sumiyo , Nakayama Takashi , Fujita Hiromi , Watanabe Mamoru
Published: November 2013

In order to understand the natural situation of rickettsiae in the ticks in Japan, the rickettsial genes, gltA gene, rOmpA gene, and 17‐kDa gene, were amplified from the ticks by nested PCR. The prevalences of rickettsial gltA genes among Haemaphysalis formosensis, H. longicornis, H. megaspinosa, Ixodes ovatus, H. flava, H. kitaokai, and I. persulcatus were 62, 57, 24, 24, 19, 13, and 10%, respectively; 26% (186/722) being the average. The gltA genes amplified from the ticks were classified into 9 genotypes (I to IX) by the difference in nucleotide sequences. Genotype I was detected from 7 species of ticks. Genotype II mainly was detected from H. longicornis and H. formosensis. Genotypes III and VII mainly were detected from H. flava and I. ovatus. The polarization in the distribution of genotypes among regions where the ticks were collected was not clear. Based on the phylogenetic analysis of the three genes presented here, genotypes I, III, and IV (detected from H. formosensis, H. hystricia, and I. ovatus) are genetically close with each other, but rickettsiae of the same property still have not been isolated from ticks anywhere in the world. These genotypes should be considered as new species among SFG rickettsiae. Genotype II was identical with strain FUJ‐98, genetically close to R. japonica which has been isolated from ticks in China. Genotype V was identical with R. felis and strain California 2 isolated from the cat flea. This is the first report on the detection of R. felis from ticks. Genotype VI detected from Ixodes sp. did not seem to belong to genus Rickettsia. Based on the previous antigenic data and the phylogenetic analysis presented here, Genotype VII should be considered a variant of R. helvetica and genotype VIII detected from I. ovatus and I. persulcatus were identical with R. helvetica. Genotype IX detected from I. nipponensis was genetically close to the strains IRS3, IRS4, and IrR/Munich isolated from I. ricinus in Slovakia and German.
Mycobacterial Infection in MyD88‐Deficient Mice
Authors: Sugawara Isamu , Yamada Hiroyuki , Mizuno Satoru , Takeda Kiyoshi , Akira Shizuo
Published: November 2013

MyD88 is an adaptor protein that plays a major role in TLR/IL‐1 receptor family signaling. To understand the role of MyD88 in the development of murine tuberculosis in vivo, MyD88 knockout (KO) mice aerially were infected with Mycobacterium tuberculosis. Infected MyD88 mice were not highly susceptible to M. tuberculosis infection, but they developed granulomatous pulmonary lesions with neutrophil infiltration which were larger than those in wild‐type (WT) mice (P<0.01). The pulmonary tissue levels of mRNA for iNOS and IL‐18 were slightly lower, but levels of mRNA for IL‐1β, IL‐2, IL‐4, IL‐6, IL‐10, IFN‐γ, and TGF‐β were higher in MyD88 KO mice. IFN‐γ, TNF‐α, IL‐1β, and IL‐12 also were high in the sera of MyD88 KO mice. There were no statistically significant differences in the expression of TNF‐α, IL‐12, and ICAM‐1 mRNA between MyD88 KO and WT mice. Thus, MyD88 deficiency did not influence the development of murine tuberculosis. NF‐κB activity was similar in the alveolar macrophages from the lung tissues of MyD88 KO and WT mice. Also, there may be a TLR2‐specific, MyD88‐independent IL‐1 receptor/TLR‐mediated pathway to activate NF‐κB in the host defense against mycobacterial infection.
Molecular Epidemiology of Bordetella pertussis Isolated in Taiwan, 1992–1997
Authors: Lee Yeong‐Sheng , Yang Chiou‐Ying , Lu Cheng‐Hsiung , Tseng Yi‐Hsiung
Published: November 2013

In Taiwan, the number of pertussis cases including various types of infection has been increasing in recent years, especially in 1997. Since 71% of the reported cases concentrated in the densely populated Taipei metropolitan area, concerns have been raised that a highly contagious strain of Bordetella pertussis might have appeared in Taipei. In this study, 114 strains of B. pertussis including those isolated in 1992–1996 (n = 53) and 1997 (n=61) were subjected to pulsed‐field gel electrophoresis (PFGE) of the XbaI digests from their chromosomes. Based on the band patterns, they were divided into 21 subtypes, P1 to P21. The strains isolated in 1997 consist of 17 subtypes including 9 new subtypes which did not appear in the previous years, indicating that the outbreaks in 1997 were not caused by a sole specific virulent strain. Dendrogram analysis indicated that the 21 subtypes can be grouped into five clusters, with the first four subtypes possessing 60 to 95% relatedness to one another, whereas relatedness between cluster 5 (containing P21 only) and the other clusters is less than 50%. Notably, all the subtypes except P12 and P21 appeared at least once in Taipei and the majority of the strains (54%) belong to two clusters, 3 and 4. These results suggest that highly dense population may facilitate spread and accelerate genetic divergence of this pathogen. This is the first report on pertussis molecular epidemiology in Taiwan.
Effects of High Amylose Maize Starch and Clostridium butyricum on Metabolism in Colonic Microbiota and Formation of Azoxymethane‐Induced Aberrant Crypt Foci in the Rat Colon
Authors: Nakanishi Shuusuke , Kataoka Keiko , Kuwahara Tomomi , Ohnishi Yoshinari
Published: November 2013

High amylose maize starch (HAS) is not digested in the small intestine and most of it reaches the large intestine. In the large intestine, HAS is fermented by intestinal bacteria, resulting in production of short‐chain fatty acids (SCFA), particularly butyrate. Clostridium butyricum can utilize HAS and produce butyrate and acetate. It has been proposed that butyrate inhibits carcinogenesis in the colon. In this study, we examined the inhibitory effects of HAS and C. butyricum strain MIYAIRI588 (CBM588) on azoxymethane‐induced aberrant crypt foci (ACF) formation in rats. In the group of rats administered only CBM588 spores, the concentration of butyrate in the cecum increased, but there was no decrease in the number of ACF. In the group of rats fed an HAS diet, a decrease in the number of ACF was observed, and in the group of rats administered HAS and CBM588, the number of ACF decreased significantly. In these two groups, the concentrations of acetate and propionate in intestinal contents significantly increased, but the concentration of butyrate did not change. It was found that the β‐glucuronidase activity level of colonic contents decreased significantly in the two groups of rats fed HAS. This study showed that HAS and CBM588 changed the metabolism of colonic microbiota and decreased the level of β‐glucuronidase activity, phenomena that may play a role in the inhibition of ACF formation in the rat colon.
Serum Thymic Factor Prevents LPS‐Induced Pancreatic Cell Damage in Mice via Up‐Regulation of Bcl‐2 Expression in Pancreas
Authors: Saitoh Noriko , Awaya Akira , Sakudo Akikazu , SungWook Seo , Saeki Keiichi , Matsumoto Yoshitsugu , Onodera Takashi
Published: November 2013

The protective effect of synthetic serum thymic factor (FTS) nonapeptide on lipopolysaccharide (LPS)‐induced pancreatic cell damage in 10‐week‐old BALB/c male mice was investigated. Mice were divided into three groups. Group I was treated with LPS (10 μg/head; i.p.) (LPS‐treated mice). Group II was administered with FTS (50 μg/head; i.p.) 24 hr before treatment with LPS and complemented immediately before LPS injection with FTS (50 μg/head; i.p.) (FTS‐administered mice). Group III was only treated with the same volume of saline (control mice). Treatment of LPS in vivo resulted in the destruction of pancreatic acinar cells. In those cells, many apoptotic cells were detected by immunohistochemistry using an anti‐single stranded DNA antibody. Immunohistochemistry and reverse transcription‐polymerase chain reaction (RT‐PCR) revealed that LPS treatment also caused low or a lack of insulin expression in pancreatic islets. In contrast, morphological change was not seen and apoptotic cell death was suppressed in pancreatic cells of FTS‐administered mice. Moreover, insulin expression was normal in those mice. FTS administration enhanced expression of Bcl‐2 mRNA levels in pancreatic tissues and IL‐6 mRNA levels in splenocytes significantly compared with those of LPS treatment at 3 hr after LPS injection. These findings suggest that FTS prevents LPS‐induced cell damage via enhancing Bcl‐2 expression in the pancreas and systemic IL‐6 production.
Evaluation of Three Different Forward Primers by Terminal Restriction Fragment Length Polymorphism Analysis for Determination of Fecal Bifidobacterium Spp. in Healthy Subjects
Authors: Hayashi Hidenori , Sakamoto Mitsuo , Benno Yoshimi
Published: November 2013

The 27F forward primer is frequently used in 16S rRNA gene libraries and T‐RFLP analysis. However, Bifidobacterium spp. were barely detected with this primer in human fecal samples. In this study, fecal microbiota were analyzed using the T‐RFLP method with three different forward primers (27F, 35F, and 529F) in conjunction with one reverse primer (1492R). T‐RFLP analysis of fecal microbiota using 35F and 529F detected higher proportions of the terminal restriction fragment (T‐RF) corresponding to Bifidobacterium spp. than that using 27F. 27F is in imperfect agreement while 35F and 529F are in good concordance with the 16S rRNA gene sequences of Bifidobacterium spp., and the latter primers allowed for the detection of T‐RFs of Bifidobacterium spp. in fecal samples from five healthy subjects. The T‐RFs presumed to be Bifidobacterium spp. were cloned and sequenced, and found to match the 16S rRNA gene sequences of Bifidobacterium spp. Among the five fecal samples, two samples with low frequencies of T‐RFs of Bifidobacterium spp. were detected using these forward primers. This probably reflects a low prevalence of Bifidobacterium spp. in these two samples. Our study emphasizes the importance of selecting a suitable forward primer for detection of Bifidobacterium spp.
Differential Susceptibility of Cells Expressing Allogeneic MHC or Viral Antigen to Killing by Antigen‐Specific CTL
Authors: Lee Koutetsu , Takenaka Hiroshi , Yoneda Yukio , Goto Toshiyuki , Sano Kouichi , Nakanishi Mahito , Eguchi Akiko , Okada Masashi , Tashiro Junko , Sakurai Kanji , Kubota Takahiro , Yoshida Ryotaro
Published: November 2013

CD8+ cytotoxic T lymphocytes (CTLs) generated by immunization with allogeneic cells or viral infection are able to lyse allogeneic or virally infected in vitro cells (e.g., lymphoma and mastocytoma). In contrast, it is reported that CD8+ T cells are not essential for allograft rejection (e.g., heart and skin), and that clearance of influenza or the Sendai virus from virus‐infected respiratory epithelium is normal or only slightly delayed after a primary viral challenge of CD8‐knockout mice. To address this controversy, we generated H‐2d‐specific CD8+ CTLs by a mixed lymphocyte culture and examined the susceptibility of a panel of H‐2d cells to CTL lysis. KLN205 squamous cell carcinoma, Meth A fibrosarcoma, and BALB/c skin components were found to be resistant to CTL‐mediated lysis. This resistance did not appear to be related to a reduced expression of MHC class I molecules, and all these cells could block the recognition of H‐2d targets by CTLs in cold target inhibition assays. We extended our observation by persistently infecting the same panel of cell lines with defective‐interfering Sendai virus particles. The Meth A and KLN205 lines infected with a variant Sendai virus were resistant to lysis by Sendai virus‐specific CTLs. The Sendai virus‐infected Meth A and KLN205 lines were able to block the lysis of Sendai virus‐infected targets by CTLs in cold target inhibition assays. Taken together, these results suggest that not all in vivo tissues may be sensitive to CTL lysis.
Mechanisms of Action of Corilagin and Tellimagrandin I That Remarkably Potentiate the Activity of β‐Lactams against Methicillin‐Resistant Staphylococcus aureus
Authors: Shiota Sumiko , Shimizu Masato , Sugiyama Jun'ichi , Morita Yuji , Mizushima Tohru , Tsuchiya Tomofusa
Published: November 2013

Corilagin and tellimagrandin I are polyphenols isolated from the extract of Arctostaphylos uvaursi and Rosa canina L. (rose red), respectively. We have reported that corilagin and tellimagrandin I remarkably reduced the minimum inhibitory concentration (MIC) of β‐lactams in methicillin‐resistant Staphylococcus aureus (MRSA). In this study, we investigated the effect of corilagin and tellimagrandin I on the penicillin binding protein 2′ (2a) (PBP2′ (PBP2a)) which mainly confers the resistance to β‐lactam antibiotics in MRSA. These compounds when added to the culture medium were found to decrease production of the PBP2′ (PBP2a) slightly. Using BOCILLIN FL, a fluorescent‐labeled benzyl penicillin, we found that PBP2′ (PBP2a) in MRSA cells that were grown in medium containing corilagin or tellimagrandin I almost completely lost the ability to bind BOCILLIN FL. The binding activity of PBP2 and PBP3 were also reduced to some extent by these compounds. These results indicate that inactivation of PBPs, especially of PBP2′ (PBP2a), by corilagin or tellimagrandin I is the major reason for the remarkable reduction in the resistance level of β‐lactams in MRSA. Corilagin or tellimagrandin I suppressed the activity of β‐lactamase to some extent.
Identification of the Coxiella Sp. Detected from Haemaphysalis longicornis Ticks in Korea
Authors: Lee Jung‐Hee , Park Hyo‐Soon , Jang Won‐Jong , Koh Seong‐Eun , Park Tae‐Kyu , Kang Shin‐Seok , Kim Bum‐Joon , Kook Yoon‐Hoh , Park Kyung‐Hee , Lee Seung‐Hyun
Published: November 2013

Two Haemaphysalis longicornis ticks were found positive in PCR assay of com‐1 gene to detect Coxiella burnetii DNA from 100 ticks. The nucleotide sequences of com‐1 and 16S rRNA gene were determined from 2 ticks and compared with those of other C. burnetii strains. The results suggest that H. longicornis harbor Coxiella sp. bacteria in Korea. Furthermore, icd, cbhE′, and cbbE′ genes are C. burnetii specific genes whereas com‐1 gene is Coxiella genus specific gene. This study gives the first documentation to prove the existence of Coxiella sp. in tick collected in Korea.
Preparation of Virosomes Coated with the Vesicular Stomatitis Virus Glycoprotein as Efficient Gene Transfer Vehicles for Animal Cells
Authors: Shoji Jun'ichi , Tanihara Yuko , Uchiyama Tsuneo , Kawai Akihiko
Published: November 2013

The vesicular stomatitis virus (VSV) glycoprotein (G) was used to prepare virosomes as a model vehicle of gene transfer to animal cells, for which viral envelope functions (receptor recognition and binding and the pH‐dependent membrane‐fusion) were expected to work. Plasmid DNA (pEGFP‐N1; Clontech) was first encapsulated into liposomes by a method of repeated freezing and thawing of the mixture of DNA and lipids (phosphatidylcholine, phosphatidylserine and cholesterol mixed at a molar ratio of 5:1:4). Then, particle size of the liposomes was stepwise reduced to 200 nm or less in diameter by successive filtrations through a series of plastic filters of various pore sizes (10 μm, 2 μm, 0.65 μm, and then 0.45 μm). Assembly of the VSV G protein‐coated liposomes (VSV G‐virosomes) was performed by mixing the DNA‐encapsulated liposome suspensions with the purified VSV G proteins at pH 5.5, followed by ultracentrifugation in a discontinuous sucrose gradient. The highest gene‐transducing activity was detected in a single band formed between 20% and 45% sucrose layers. Negatively stained electron microscopic images showed that the band contained spherical particles of various sizes, ranging from 40 to 140 nm in diameter, that were covered with viral spike projections. The VSV G‐virosomes displayed a roughly similar level of gene‐transducing activity to that mediated by cationic liposomes (e.g., Lipofectamine), which was blocked either by pretreatment with anti‐VSV G antiserum or by addition of 20 MM NH4Cl to transfected cultures. From these results, we assume that the virosome‐mediated gene‐transduction was first achieved by using the whole functions of VSV G protein, and can also be used for further studies of the protein.
Human Cytomegalovirus (HCMV) Infection in Osteosarcoma Cell Line Suppresses GM‐CSF Production by Induction of TGF‐β
Authors: Kwon Ye Jin , Kim Dae‐Joong , Kim Jung Heon , Park Chung‐Gyu , Cha Chang‐Yong , Hwang Eung‐Soo
Published: November 2013

This study was performed to elucidate the possible mechanism of the disturbance of hemopoiesis by HCMV infection. Saos‐2 cells constitutively express mRNA of GM‐CSF, and its expression was profoundly decreased by HCMV infection, which required full replication of the virus and was mediated by soluble factors released from the HCMV‐infected Saos‐2 cells. TGF‐β1 production was statistically and significantly increased from one day after HCMV infection. Expression and production of GM‐CSF in Saos‐2 cells were restored when a culture supernatant of HCMV‐infected Saos‐2 cells was reacted with neutralizing anti‐TGF‐β antibody. Conclusively, HCMV inhibits GM‐CSF expression in Saos‐2 cells partly by the increased production of TGF‐β1.
Immunomagnetic Capture RT‐PCR for Detection of Norovirus from Foods Implicated in a Foodborne Outbreak
Authors: Kobayashi Shinichi , Natori Katsuro , Takeda Naokazu , Sakae Kenji
Published: November 2013

In February 2001, an outbreak of acute gastroenteritis due to Norovirus (NV) occurred among employees of 11 companies in Aichi Prefecture. The illness was strongly associated with eating a delivered box‐lunch. The use of magnetic beads coated with the antibody to the baculovirus‐expressed recombinant capsid proteins of the Chiba virus (rCV) facilitated capture of NV from the food items implicated in the outbreak. Following immunomagnetic capture, NV bound to the beads was detected by reverse transcription‐polymerase chain reaction (RT‐PCR). Of the nine food items tested, two were positive for a genogroup 1 NV. Sequence analysis of RT‐PCR products indicated that the nucleotide sequences of NV strains from foods were almost identical to those of NV strains detected in stool samples of ill patients. As the immunocapture RT‐PCR method is simple and easy to perform, this technique should be useful for the detection of NV from outbreak‐implicated foods.
Genetic Typing of Feline Rabies Virus Isolated in Greater Bangkok, Thailand
Authors: Kasempimolporn Songsri , Saengseesom Wachiraporn , Tirawatnapong Thaweesak , Puempumpanich Sununta , Sitprija Visith
Published: November 2013

To study the molecular epidemiology of rabies virus that is prevalent among cats in greater Bangkok, Thailand, a total of 17 rabies virus isolates from cats were characterized and compared with 120 rabies virus isolates from dogs. Analyses were performed on the genetic polymorphism in the rabies virus nucleoprotein (N) gene. Rabies virus N gene of isolates was amplified by reverse transcription‐polymerase chain reaction. The diversity of N gene was revealed by the restriction fragment length polymorphism (RFLP) method. The rabies virus isolates from cats could be classified into 5 types, designated as Dd I‐Hf I, Dd II‐Hf II, Dd III‐Hf I, Dd IV‐Hf I, and Dd IV‐Hf III. Type Dd I‐Hf I was encountered more frequently than the others. It was apparent that no less than five rabies virus types presented in the areas of Bangkok. Moreover, all five RFLP patterns were typical of those which had been observed in dogs. Our findings suggest that there had been viral transmission between the dogs and the cats.
Cytotoxicity and Enterotoxicity of the Thermostable Direct Hemolysin‐Deletion Mutants of Vibrio parahaemolyticus
Authors: Park Kwon‐Sam , Ono Takahiro , Rokuda Mitsuhiro , Jang Myoung‐Ho , Iida Tetsuya , Honda Takeshi
Published: November 2013

The thermostable direct hemolysin (TDH) has been proposed to be a major virulence factor of Vibrio parahaemolyticus. We have recently completed the genome sequence of a TDH‐producing V. parahaemolyticus strain, RIMD2210633. In this study, we constructed tdh‐deletion mutants from the sequenced strain by homologous recombination and analyzed their phenotypes. Although the deletion of both copies of tdh completely abolished the hemolytic activity of the wild‐type strain, the deletion did not affect the cytotoxicity to HeLa cells. Enterotoxicity, assayed by the rabbit ileal loop test, was lowered by tdh deletion, but the mutant still showed partial fluid accumulation in rabbit intestine. These results indicate that the cytotoxicity and enterotoxicity of TDH‐producing V. parahaemolyticus are not explained by TDH alone, and suggest that an unknown virulence factor(s) could be involved in these pathogenic activities.
Modulation of the Immune System by Listeria monocytogenes‐Mediated Gene Transfer into Mammalian Cells
Authors: Shen Hua , Kanoh Makoto , Liu Fengzhi , Maruyama Saho , Asano Yoshihiro
Published: November 2013

In this study, we established a method for Listeria monocytogenes (Lm)‐mediated gene transfer into mammalian cells to manipulate the immune response of the host during infection by pathogens. We used the Lm‐mediated gene transfer method in an in vivo study to manipulate host immune responses against Leishmania major (L. major)‐infection. The injection of Lm modulated the susceptible host into a resistant state against L. major‐infection. A more efficient protective effect was obtained with the injection of IL‐12‐cDNA containing Lm, and the protective effect was stronger than that of the resistant strain. The protective mechanism of Lm‐injection against L. major‐infection observed here appeared to be a result of the activation of the local immune system by the Lm‐mediated gene transfer method. The present study is the first demonstration that a gene introduced into a host by Lm works to modulate the murine host immune response against infections in vivo. Since this system strongly induces Th1 responses and suppresses Th2 responses in infected hosts, the system can be used for controlling infectious diseases and for protection against allergic responses in the future.
A Major Li+ Extrusion System NhaB of Pseudomonas aeruginosa: Comparison with the Major Na+ Extrusion System NhaP
Authors: Kuroda Teruo , Fujita Naoko , Utsugi Junko , Kuroda Masayuki , Mizushima Tohru , Tsuchiya Tomofusa
Published: November 2013

A gene encoding a Li+ extrusion system was cloned from the chromosomal DNA of Pseudomonas aeruginosa and expressed in Escherichia coli cells. The gene enabled growth of E. coli KNabc cells, which were unable to grow in the presence of 10 MM LiCl or 0.1 M NaCl because of the lack of major Na+(Li+)/H+ antiporters. We detected Li+/H+ and Na+/H+ antiport activities in membrane vesicles prepared from E. coli KNabc cells that harbored a plasmid carrying the cloned gene. Activity of this antiporter was pH‐dependent with an optimal pH activity between pH 7.5 and 8.5. These properties indicate that this antiporter is different from NhaP, an Na+/H+ antiporter from P. aeruginosa that we reported previously, and that is rather specific to Na+ but it cannot extrude Li+ effectively. The gene was sequenced and an open reading frame (ORF) was identified. The amino acid sequence deduced from the ORF showed homology (about 60% identity and 90% similarity) with that of the NhaB Na+/H+ antiporters of E. coli and Vibrio parahemolyticus. Thus, we designated the antiporter as NhaB of P. aeruginosa. E. coli KNabc carrying the nhaB gene from P. aeruginosa was able to grow in the presence of 10 to 50 MM LiCl, although KNabc carrying nhaP was unable to grow in these conditions. The antiport activity of NhaB from P. aeruginosa was produced in E. coli and showed apparent Km values for Li+ and Na+ of 2.0 MM and 1.3 MM, respectively. The antiport activity was inhibited by amiloride with a Ki value for Li+ and Na+ of 0.03 MM and 0.04 MM, respectively.
Antibacterial Activity of Hydrolyzable Tannins Derived from Medicinal Plants against Helicobacter pylori
Authors: Funatogawa Keiji , Hayashi Shunji , Shimomura Hirofumi , Yoshida Takashi , Hatano Tsutomu , Ito Hideyuki , Hirai Yoshikazu
Published: November 2013

Helicobacter pylori is a major etiological agent in gastroduodenal disorders. In this study, we isolated 36 polyphenols and 4 terpenoids from medicinal plants, and investigated their antibacterial activity against H. pylori in vitro. All hydrolyzable tannins tested demonstrated promising antibacterial activity against H. pylori. Monomeric hydrolyzable tannins revealed especially strong activity. Other compounds demonstrated minimal antibacterial activity with a few exceptions. A monomeric hydrolyzable tannin, Tellimagrandin I demonstrated time‐ and dose‐dependent bactericidal activity against H. pylori in vitro. On the other hand, hydrolyzable tannins did not affect the viability of MKN‐28 cells derived from human gastric epithelium. Hydrolyzable tannins, therefore, have potential as new and safe therapeutic regimens against H. pylori infection. Furthermore, we investigated effects of hydrolyzable tannins on lipid bilayer membranes. All the hydrolyzable tannins tested demonstrated dose‐dependent membrane‐damaging activity. However, it remains to be elucidated whether their membrane‐damaging activity directly contributes to their antibacterial action.
Infection of Different Cell Lines of Neural Origin with Subacute Sclerosing Panencephalitis (SSPE) Virus
Authors: Ishida Hiroshi , Ayata Minoru , Shingai Masashi , Matsunaga Isamu , Seto Yoshiyuki , Katayama Yuko , Iritani Nobuhiro , Seya Tsukasa , Yanagi Yusuke , Matsuoka Osamu , Yamano Tsunekazu , Ogura Hisashi
Published: November 2013

Measles virus is the causative agent of subacute sclerosing panencephalitis (SSPE). The viruses isolated from brain cells of patients with SSPE (called SSPE viruses) are defective in cell‐free virus production in vitro. To investigate the cell tropism of three strains of SSPE virus (Osaka‐1, Osaka‐2, Osaka‐3), SSPE virus‐infected cell cultures were treated with cytochalasin D to prepare virus‐like particles (CD‐VLPs). All CD‐VLPs formed syncytia after infection in CHO cells expressing CD150 but not in those expressing CD46. In addition, an antibody to CD46 did not block the infection of Vero cells by SSPE CD‐VLPs. The results were consistent with our previous suggestion that one or more unidentified receptors might be involved in the entry process. Infection with the CD‐VLPs from three SSPE strains was further examined in different human cell lines, including those of neural origin, and was found to induce syncytia in epithelial cells (HeLa and 293T) as well as neuroblastoma cells (IMR‐32 and SK‐N‐SH) with varying efficiency. SSPE CD‐VLPs also infected glioblastoma cells (A172) and astrocytoma cells (U‐251) but syncytial formation was rarely induced. These epithelial and neural cell lines were not permissive for the replication of wild‐type MV. Together with our previous observations, these results suggest that the cell entry receptor is the major factor determining the cell tropism of SSPE viruses. Further studies are necessary to identify other viral and/or cellular factors that might be involved in the replication of SSPE virus in specific neural cells and in the brain.
Identification of β‐Tubulin Isoform V as an Autoantigen in Allergic Rhinitis by a Proteomic Approach
Authors: Nakamura Manabu , Tsutsumi Kouichiro , Ooka Seido , Sekine Taichi , Koizuka Izumi , Nishioka Kusuki , Kato Tomohiro
Published: November 2013

Autoantibodies to IgE and p2‐adrenergic receptor have been reported in patients with allergic rhinitis. To investigate whether autoimmunity in allergic rhinitis is directed to such limited molecules or directed to a wide range of self proteins, we here attempted to survey autoantigens/autoantibodies comprehensively, using proteomics. Specifically, we separated proteins extracted from peripheral blood mononuclear cells by 2‐dimensional electrophoresis and then detected autoantigens by subsequent western blotting with sera from patients with allergic rhinitis. As a result, we detected multiple autoantigens, some of which were further identified by mass fingerprinting. Next, we confirmed antigenicity of one of the identified autoantigens, β‐tubulin isoform V (β‐tubV), using a recombinant protein and then measured prevalence of the anti‐β‐tubV autoantibodies. As a result, 52% of the tested patients with allergic rhinitis were found to possess anti‐β‐tubV autoantibodies. Our study indicates that autoimmunity is a common phenomena and β‐tubV is one of the major autoantigens in allergic rhinitis.
A Quorum‐Sensing Autoinducer Enhances the mexAB‐oprM Efflux‐Pump Expression without the MexR‐Mediated Regulation in Pseudomonas aeruginosa
Authors: Sawada Isao , Maseda Hideaki , Nakae Taiji , Uchiyama Hiroo , Nomura Nobuhiko
Published: November 2013

We have previously described that the quorum‐sensing autoinducer, N‐butyryl‐L‐homoserine lactone (C4‐HSL) enhances mexAB‐oprM expression, and this C4‐HSL‐mediated enhancement of mexAB‐oprM expression was repressed by MexT, a positive regulator of the mexEF‐oprN operon. In this study, we investigated the interaction between C4‐HSL and mexR by using a knockout mutant. It was indicated that the C4‐HSL‐mediated enhancement of mexAB‐oprM expression occurred without MexR‐mediated regulation. Furthermore, it was observed that the C4‐HSL‐mediated enhancement of mexAB‐oprM expression without the MexR‐mediated regulation was repressed by MexT.
Comparison of Five PCR Assays for Detecting Chlamydophila pneumoniae DNA
Authors: Fukano Hiroshi
Published: November 2013

We compared five different polymerase chain reaction (PCR) assays for the detection of Chlamydophila pneumoniae DNA using highly purified elementary bodies (EBs) and peripheral blood mononuclear cells (PBMCs) from healthy blood donors. The primers were as follows; two targeting the 16S rRNA gene, one targeting the ompA gene, one targeting the Pst‐I gene, and one targeting the 53 kDa outer membrane protein gene. The 16S rRNA touchdown enzyme time release (TETR) PCR, the ompA nested PCR and the 53 kDa nested PCR were the most sensitive assays and could detect one or more EB per assay. These three PCRs also had the same reproducibility, but the minimal amount of C. pneumoniae that could be reproducibly detected (10 of 10 testing positive) was 20 EBs. In a sample of specimens from healthy blood donors, we found 5 of 77 (6.5%) PBMCs specimens to have C. pneumoniae DNA according to the nested ompA PCR. Specimens with the 16S rRNA TETR and 53 kDa nested assays were found to have C. pneumoniae DNA 7 of 77 (9.1%) and 18 of 77 (23.4%) specimens, respectively. The other two assays failed to detect even a single positive. However, the detection rate decreased with repeated testing of the same samples. Our newly designed 53 kDa nested PCR may be as useful as the other four recommended PCR assays and may be a more useful assay for the detection of C. pneumoniae DNA from PBMCs.
Application of In Vitro Mutagenesis to Identify the Gene Responsible for Cold Agglutination Phenotype of Streptococcus mutans
Authors: Sato Yutaka , Okamoto Kazuko , Kagami Akiyoshi , Yamamoto Yasuhito , Ohta Kazumasa , Igarashi Takeshi , Kizaki Harutoshi
Published: November 2013

A previously unidentified protein with an apparent molecular mass of 120 kDa was detected in some Streptococcus mutans strains including the natural isolate strain Z1. This protein was likely involved in the cold‐agglutination of the strain, since a correlation between this phenotype and expression of the 120 kDa protein was found. We have applied random mutagenesis by in vitro transposition with the Himar1 minitransposon and isolated three cold‐agglutination‐negative mutants of this strain from approximately 2,000 mutants screened. A 2.5 kb chromosomal fragment flanking the minitransposon in one of the three mutants was amplified by PCR‐based chromosome walking and the minitransposon insertion in the other two mutants occurred also within the same region. Nucleotide sequencing of the region revealed a 1617 nt open reading frame specifying a putative protein of 538 amino acid residues with a calculated molecular weight of 57,192. The deduced eight amino acid sequence following a putative signal sequence completely coincided with the N‐terminal octapeptide sequence of the 120 kDa protein determined by the Edman degradation. Therefore, the 1617 nt gene unexpectedly encoded the 120 kDa protein from S. mutans. Interestingly, this gene encoded a collagen adhesin homologue. In vitro mutagenesis using the Himar1 minitransposon was successfully applied to S. mutans.
New World Relapsing Fever Borrelia Found in Ornithodoros porcinus Ticks in Central Tanzania
Authors: Mitani Harumi , Talbert Alison , Fukunaga Masahito
Published: November 2013

Ticks were collected from 8 houses in Mvumi Mission village, near Dodoma, Tanzania. All ticks were examined for Borrelia infestation by flagellin gene‐based nested polymerase chain reaction. All houses were highly infested with ticks, and all ticks collected were of the Ornithodoros porcinus species. Fifty‐one out of 120 ticks were infected with spirochetes, and a flagellin gene sequence comparison showed that most of the spirochetes belonged to Borrelia duttonii, which is the causative agent of tick‐borne relapsing fever in East Africa. The rest of the spirochetes were quite different from B. duttonii and instead resembled the New World tick‐borne relapsing fever borreliae. Phylogenetic analysis using 16S ribosomal RNA gene sequences also supported the interpretation that the spirochete was a Borrelia species distinct from previously described members of the genus.
Distribution of Virulence‐Associated Genes in Vibrio mimicus Isolates from Clinical and Environmental Origins
Authors: Shinoda Sumio , Nakagawa Tomoko , Shi Lei , Bi Kewei , Kanoh Yasuko , Tomochika Ken‐ichi , Miyoshi Shin‐ichi , Shimada Toshio
Published: November 2013

Distribution of virulence‐associated genes in Vibrio mimicus was studied including the toxin genes ctxA, tdh, st and vmh and the genes necessary for regulation of toxin production, toxR, toxS, toxT, tcpA and tcpP. Approximately half of clinical V. mimicus isolates possessed one or more genes encoding V. cholerae enterotoxic factors such as ctxA, tdh and st. All of the clinical and environmental isolates possessed vmh encoding V. mimicus hemolysin (VMH). The ctxA encoding cholera toxin was detected in only 2 strains, 5% of the clinical isolates. Furthermore, there were very few strains possessing tcpP and toxT needed for the expression of ctxA. These results may suggest that VMH is a more important pathogenic factor than well recognized toxins such as cholera toxin (CT) in V. mimicus infection.
A New Yeast, Malassezia yamatoensis, Isolated from a Patient with Seborrheic Dermatitis, and Its Distribution in Patients and Healthy Subjects
Authors: Sugita Takashi , Tajima Mami , Takashima Masako , Amaya Misato , Saito Masuyoshi , Tsuboi Ryoji , Nishikawa Akemi
Published: November 2013

Over the last few years, new Malassezia species have been found regularly in Japanese subjects. We isolated another new Malassezia species from a Japanese patient with seborrheic dermatitis (SD), and named it M. yamatoensis. In its physiological characteristics and the utilization of Tween by M. yamatoensis is similar to that of M. furfur and M. dermatis. It is distinguished by its growth temperature. To examine the distribution of the microorganism in the skin of patients with SD and atopic dermatitis (AD), and healthy subjects, we applied transparent dressings to the skin, and detected M. yamatoensis DNA using a non‐culture‐based method that consisted of nested PCR with specific primers. M. yamatoensis DNA was detected from 3 of 31 SD patients (9.7%), 5 of 36 AD patients (13.9%), and 1 of 22 healthy subjects (4.6%). Therefore, M. yamatoensis is a rare member of the cutaneous microflora.