bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of West Nile and Japanese Encephalitis Viral Genome Sequences in Cerebrospinal Fluid from Acute Encephalitis Cases in Karachi, Pakistan
Authors: Igarashi Akira , Tanaka Mariko , Morita Kouichi , Takasu Toshiaki , Ahmed Akhtar , Ahmed Arsalam , Akram D.S. , Waqar M. Anwar
Published: November 2013

Reverse transcriptase‐polymerase chain reaction (RT‐PCR) on 24 cerebrospinal fluid (CSF) specimens collected between February and August 1992 detected genome sequence of West Nile (WN) virus in 8 specimens and Japanese encephalitis (JE) virus in a single specimen. The results, combined with the data by IgM‐ELISA on CSF indicated that a significant proportion of acute encephalitis cases in Karachi, Pakistan, were caused by WN virus infection, while JE virus caused a small fraction.
Identification of Borrelia burgdorferi Isolated in Korea Using Outer Surface Protein A (OspA) Serotyping System
Authors: Kee Sunho , Hwang Kyu‐jam , Oh Hee‐bok , Park Kyung‐seok
Published: November 2013

Two characteristic strains (935T, 934U) of B. burgdorferi isolated from Ixodes persulcatus and a wild rodent (Apodemus agrarius) in Korea were selected and analyzed by an immunoblot method using the monoclonal antibodies directed to different epitopes of outer surface protein A (OspA). The reactive pattern of strain 934U with these monoclonal antibodies was identical to that of strains belonging to B. afzelii and that of strain 935T was different from other isolates. Monoclonal antibody (5TEE3) which is specific to strain 935T did not react with any other Western and Japanese isolates. So, it was suggested that there exist at least two groups of B. burgdorferi in Korea. One could be classified as B. afzelii and the other is a divergent group from three known species of B. burgdorferi sensu stricto, B. garinii and B. afzelii.
Isolation and Characterization of Bile Acid 7‐Dehydroxylating Bacteria from Human Feces
Authors: Takamine Fusae , Imamura Teisuke
Published: November 2013

Methods for isolation of fecal 7α‐dehydroxylating bacteria are presented. A total of 219 strains were isolated from feces of healthy humans, and their ability to 7‐dehydroxylate cholic, chenodeoxycholic, and ursodeoxycholic acids were examined. Of all the isolates, 14 strains were found to be capable of eliminating the hydroxy group at C‐7α and/or C‐7β. All the isolates were strictly anaerobic, Gram‐positive rods. Thirteen isolates were non‐sporeforming bacteria showing certain saccharolytic properties with the production of acid and gas from dextrose, and were catalase‐positive but indole‐, lecithinase‐, urease‐ and oxidase‐negative. Based on the data available at present, it was concluded that they could be regarded as members of the genus Eubacterium. One strain, however was identified as Clostridium sordellii. The isolated strains capable of 7α‐dehydroxylating cholic acid and chenodeoxycholic acid were also able to oxidize the hydroxy group at C‐7α. Nine strains (10, 12, 36S, M‐2, M‐17, M‐18, Y‐98, Y‐1112, and Y‐1113) of the 7α‐dehydroxylating bacteria were confirmed to have 7β‐dehydroxylation ability, but five strains (O‐51, O‐52, O‐71, O‐72, and Y‐67) could not transform ursodeoxycholic acid to lithocholic acid.
Natural Killer (NK)‐Like Cytotoxicity of Murine Intraepithelial Lymphocytes in the Small Intestine (iIEL) and the Effect of the Serine Proteases
Authors: Kato Yutaka , Yokochi Takashi , Maeda Kenji , Sasaki Keiko , Kawamoto Yasuko , Tsuji Takao , Miyama Akio
Published: November 2013

The natural killer (NK)‐like cytotoxicity of murine intraepithelial lymphocytes in the small intestine (iIEL) and the participation of serine proteases in it were investigated. We monitored the cytotoxicity of iIEL with a sensitive cytotoxic assay using laser flow cytometry. iIEL exhibited NK‐like cytotoxicity on YAC‐1 target cells. Benzamidine, a serine protease inhibitor, inhibited significantly both Na‐CBZ‐l‐lysine thiobenzyl ester (BLT)‐specific serine protease activity and iIEL‐mediated NK‐like cytotoxicity. These results suggest that BLT‐specific serine proteases may participate in NK‐like cytotoxicity of murine iIEL.
Metastasis‐Promoting Activity of a Novel Molecule, Ag 243‐5, Derived from Mycoplasma, and the Complete Nucleotide Sequence
Authors: Ushio Shimpei , Iwaki Kanso , Taniai Madoka , Ohta Tsunetaka , Fukuda Shigeharu , Sugimura Kazuhisa , Kurimoto Masashi
Published: November 2013

We found that mycoplasma‐infected cells have a higher ability to metastasize in vivo than non‐mycoplasma‐infected cells. To investigate this phenomenon, we obtained a monoclonal antibody, MAb 243‐5, by immunization with Mycoplasma arginini‐infected RPMI 4788 cells. This MAb recognized a mycoplasmal protein with an MW of 47 kDa and completely inhibited the experimental metastasis of M. arginini‐infected RPMI 4788 cells using a nude mouse model. Using this MAb, we purified a molecule called Ag 243‐5 and determined the N‐terminal amino acid sequence and clarified the entire nucleotide sequence of the Ag 243‐5 gene. PCR analysis showed the existence of a homologous gene in Mycoplasma hyorhinis. Four sequential injections of Ag 243‐5 (30 μg/shot) promoted the experimental metastasis of non‐mycoplasma‐infected RPMI 4788 cells more than 10‐fold using a nude mouse model. Ag 243‐5 also promoted the experimental metastasis of the non‐mycoplasma‐infected mouse colon cancer cell line colon 26. This metastasis‐promoting effect was neutralized by MAb 243‐5.
Anti‐Hepatitis A Virus Antibody Response Elicited in Mice by Different Forms of a Synthetic VP1 Peptide
Authors: Haro Isabel , Pinto Rosa M. , Gonzalez‐Dankaart Juan F. , Perez Jose A. , Reig Francisca , Bosch Albert
Published: November 2013

Peptide VP1 (11‐25) of the capsid of hepatitis A virus was synthesized by the Fmoc‐polyamide solid phase method, and administered to mice in different forms: (1) free, (2) encapsulated in multilamellar liposomes, (3) coupled to keyhole limpet hemocyanin (KHL), and (4) incorporated into a tetrameric branched lysine core. The highest anti‐VP1 peptide responses were generated by synthetic peptides entrapped into liposomes and coupled to KLH. No anti‐HAV response was generated with the free peptide, while all the other forms induced both anti‐HAV and HAV‐neutralizing antibodies. Maximum neutralization indices were observed in ascites from mice treated with liposome‐entrapped and KLH peptides.
Temperature Effects on Legionella pneumophila Killing by and Multiplication in Phagocytes of Guinea Pigs
Authors: Miyamoto Hiroshi , Ogawa Midori , Maruta Koji , Nikaido Yoshihiko , Yamamoto Chiyuki , Taniguchi Hatsumi , Yoshida Shin‐ichi
Published: November 2013

We examined the effects of temperature on the interaction between Legionella pneumophila and phagocytes of guinea pigs. The body temperatures of guinea pigs infected with a sublethal dose (1.2 × 104 CFU) or a lethal dose (1.0 × 105 CFU) of L. pneumophila elevated from 38.4±0.15 C to 40.2±0.42 C or 40.3 ± 0.62 C, respectively. The intracellular bacterial killing by and bacterial proliferation in the phagocytes were examined at 33, 37, 40, and 42 C, using in vitro culture systems of peritoneal macrophages or polymorphonuclear leukocytes (PMN) of guinea pigs. In all the macrophages incubated at different temperatures, significant intracellular bacterial killings were observed at 4 hr after in vitro phagocytosis. After 24 hr of incubation, there was about a 100‐fold increase of CFU and the number reached a maximum after 48 hr of incubation in the macrophages incubated at 42 C as well as 37 and 40 C, suggesting that macrophages support the intracellular bacterial growth in hyperthermia. In the PMN, L. pneumophila CFU 4 hr or 12 hr after the infection were significantly lower at 42 C than those at 37 C (P<0.05), indicating that the bactericidal capacity of PMN was enhanced at 42 C compared to 37 C. However, in all the PMN incubated at different temperatures, there were about 10‐fold increases of CFU 24 hr after the infection, suggesting that PMN as well as macrophages support intracellular bacterial growth in hyperthermia. The extracellular bacterial growth was examined at 33, 37, 40, and 42 C in buffered yeast extract (BYE) broth or RPMI 1640 medium containing 50% guinea pig serum as a permissive or non‐permissive liquid medium for the bacterial growth, respectively. Inhibition of bacterial growth in BYE broth at 42 C, and a decrease of CFU in RPMI 1640 medium containing 50% guinea pig serum at 42 C were observed. In conclusion, hyperthermia may be beneficial by restricting extracellular bacterial survival, but it exerts no beneficial effect on the restriction of intracellular bacterial growth in phagocytes, though PMN showed enhanced initial killing at 42 C. These results suggest that fever, or hyperthermia itself, may not largely contribute as a nonspecific host defense early in the course of legionellosis.
Evaluation of DNA Fingerprinting by PFGE as an Epidemiologic Tool for Salmonella Infections
Authors: Murase Toshiyuki , Okitsu Tadayuki , Suzuki Rieko , Morozumi Hirotoshi , Matsushima Akiyoshi , Nakamura Akiko , Yamai Shiro
Published: November 2013

To evaluate DNA fingerprinting as an epidemiologic tool, pulsed‐field gel electrophoresis (PFGE) was performed on isolates of Salmonella, including S. typhimurium, S. thompson, and S. enteritidis. Chromosomal DNA was digested with the restriction endonucleases Bln I and Xba I. The patterns of S. thompson and S. typhimurium isolates from various sources were different from one another. There was no correlation between the phage type and the digestion pattern of S. enteritidis isolates. Some strains belonging to one phage type were distinguished by their PFGE pattern in this study. These results suggest that the Bln I and Xba I digestion patterns of chromosomal DNA are useful for epidemiological analysis of an outbreak of Salmonella infection or food poisoning.
Vibrio trachuri Sp. Nov., a New Species Isolated from Diseased Japanese Horse Mackerel
Authors: Iwamoto Yoshihisa , Suzuki Yusaku , Kurita Akinobu , Watanabe Yoko , Shimizu Tadayori , Ohgami Hirohisa , Yanagihara Yasutake
Published: November 2013

A new species, Vibrio trachuri sp. nov., was isolated from the cultured Japanese horse mackerel (Trachurus japonicus). These Vibrio were Gram negative, motile rods and formed yellow colonies on BTB teepol and TCBS plate, turned TSI medium to yellow and was sensitive to 150 μM O/129 (2,4‐diamino‐6,7‐diisopropyl pteridine phosphate) like Listonella anguillarum which has been described as Vibrio anguillarum. However, the results of VP test and decarboxylation of lysine or dihydrolation of arginine suggested that these Vibrio are rather closely related to V. parahaemolyticus. DNA similarity determined by the microplate hybridization technique revealed that these Vibrio are genetically quite distant from Listonella anguillarum or V. parahaemolyticus and rather close to V. harveyi, although there was no Vibrio species which had more than 70% similarity value. From these results we propose to nominate Vibrio trachuri sp. nov. for this new Vibrio species.
Endotoxin Induces Severe Inflammatory Reactions with Necrosis at Sites Primed with Delayed‐Type Hypersensitivity Reactions in Guinea Pigs
Authors: Nagao Shigeki , Kawabata Yoshihiro , Nishikawa Takashige , Takada Haruhiko
Published: November 2013

Guinea pigs immunized with Freund's complete adjuvant received challenge injection of the purified protein derivative of Mycobacterium tuberculosis in the flanks and the corneas to prepare delayed‐type hypersensitivity (DTH) reactions. The animals were injected subcutaneously with lipopolysaccharide (LPS) or a synthetic lipid A (LA‐15‐PP). At the skin site primed with DTH reaction, increased swelling and hemorrhagic reaction followed by a definite necrotic reaction occurred. Severe corneal reactions were also observed in the animals. These findings indicate that bacterial endotoxin modulates DTH reactions and induces severe inflammatory reactions.
Targeting of Chrolamphenicol Acetyltransferase to Human Immunodeficiency Virus Particles via Vpr and Vpx
Authors: Sato Akihiko , Isaka Yoshitaka , Kodama Makoto , Yoshimoto Jun , Kawauchi Shinobu , Kuwata Takeo , Adachi Akio , Hayami Masanori , Yoshie Osamu , Fujiwara Tamio
Published: November 2013

Vpr and Vpx are the auxiliary proteins of human immunodeficiency viruses (HIVs) selectively incorporated into mature viral particles. We showed that the bacterial chloramphenicol acetyltransferase (CAT) fused to the N‐terminus of HIV‐1 Vpr, HIV‐2 Vpr, or HIV‐2 Vpx was incorporated into mature virions in a type‐selective manner. By using chimeric proteins between HIV‐1 Vpr and HIV‐2 Vpx, we found that the N‐terminal side of these proteins was mainly important for type‐selective virion incorporation. The C‐terminal arginine‐rich region of HIV‐1 Vpr was also found to transport CAT fusion proteins into virions but without any type selectivity. Furthermore, the corresponding regions of HIV‐2 Vpr and HIV‐2 Vpx had no such activity. This region of HIV‐1 Vpr may interact nonspecifically with viral genomic RNA. Collectively, Vpr and Vpx may provide a means to introduce foreign proteins and other molecules into HIV virions for therapeutic purposes.
The Structure and Characterization of the Surface Protein Layer of a Comamonas‐Like Organism
Authors: Wai Sun Nyunt , Takade Akemi , Fujimoto Shuji , Amako Kazunobu
Published: November 2013

The regular surface layer of a strain of a Comamonas‐like organism was examined by electron microscopy. The surface layer protein was easily extracted from the cell surface by a 2.5 M solution of lithium chloride. The protein subunit has a molecular size of 32,000 daltons, but usually forms a large aggregate of more than 1,200,000 daltons. In the extract it formed a regular array of p4 symmetry and was observed to be intimately associated with fragments of lipopolysaccharide. The size of a subunit determined by the negative staining method and the image processing method measured 5.2 × 6.4 nm (width and length), was arranged in a cobblestone‐like pattern, and was located in a lattice space measuring 13.0 nm square.
Actions of Vibrio vulnificus Metalloprotease on Human Plasma Proteinase‐Proteinase Inhibitor Systems: A Comparative Study of Native Protease with Its Derivative Modified by Polyethylene Glycol
Authors: Miyoshi Shin‐ichi , Narukawa Hitoshi , Tomochika Ken‐ichi , Shinoda Sumio
Published: November 2013

Vibrio vulnificus, an opportunistic human pathogen causing wound infection and septicemia, produces a metalloprotease (VVP) which is suspected to be a virulent determinant. The interactions of VVP, as well as its derivative (PEG1‐VVP) modified with polyethylene glycol, with a variety of human plasma proteins were investigated. We found that native VVP and its derivative were able to act directly on many biologically important human plasma proteins even in the presence of α‐macroglobulin, the sole plasma inhibitor of native VVP. The activities of both classical and alternative pathways of the complement cascade system were drastically abolished by incubation with either VVP. Furthermore, these proteases rapidly digested the Aα‐chain of human fibrinogen into fragment(s) with no clotting ability. Therefore both VVPs are thought to function as a fibrinogenolytic enzyme, causing delay of the coagulation reaction. VVP and PEG1‐VVP were also shown to destroy plasma proteinase inhibitors including α1‐proteinase inhibitor, a major inhibitor in human plasma. Because endogenous proteolytic enzymes and their inhibitors are indispensable in maintaining physiological homeostasis, these findings suggest that VVP (and PEG1‐VVP) may cause an imbalance of human plasma proteinase‐proteinase inhibitor systems, thus eliciting an immunocompromised state in the host and facilitating the development of a systemic V. vulnificus infection such as septicemia.
Cytokine‐Independent Proliferation of IL‐2‐Nonproducing CTL Clones in Association with High TCR‐Signal Transduction Responses
Authors: Nagase Fumihiko , Lwin Tint , Nakashima Izumi
Published: November 2013

Alloreactive CTL clone D2‐23 proliferated in response to antigenic cells without IL‐2 production. Among subclones of D2‐23, the F1 but not F2 clone proliferated in response to soluble aCD3 or PMA, although both clones proliferated in response to immobilized aCD3, antigenic cells or soluble aCD3 plus costimulatory cells. The difference in responsiveness between F1 and F2 was not caused by distinct expression of CD3 or Fc receptors. Cyclosporin A, which totally blocks IL‐2 production of Th1 cells, barely or only partially inhibited PMA‐ or aCD3‐induced proliferation of F1. F1 did not produce cytokines for proliferation of F2 in response to soluble aCD3. Tyrosine phosphorylation developed for various proteins of F1 and F2 at the levels apparently correlated to the extent of cell proliferation when the cells were stimulated with soluble aCD3 or PMA. The proliferative responsiveness of F1 and F2 to the described stimulators was maintained by stimulation with IL‐2 plus antigenic cells, or even IL‐2 alone, but was decreased during resting culture or by stimulation with immobilized aCD3. These results show evidence of a new TCR‐linked mechanism for CTL proliferation that is independent of costimulatory cell‐ or cytokine‐mediated signaling, but is originally prepared by prior stimulation with IL‐2.
Impaired Antigen Specific Responses and Enhanced Polyclonal Stimulation in Mice Infected with Ehrlichia muris
Authors: Kawahara Makoto , Suto Chiharu , Shibata Shinichiro , Futohashi Masataka , Rikihisa Yasuko
Published: November 2013

The immune status of BALB/c mice infected by intraperitoneal inoculation with Ehrlichia muris was examined. The level of E. muris infection in both peritoneal cavity and spleen was greatest at day 10 postinoculation (PI). Thereafter, the infection level was dramatically reduced while the organism persisted for up to 400 days PI. The greatest intraperitoneal infiltration of leukocytes, splenomegaly, and leukocytosis were observed on days 10, 15, and 20 PI, respectively. Infected mice developed marked hypergammaglobulinemia of IgG and IgM that peaked at day 20 PI; however, IgA plummeted at day 15 PI. Of IgG, G2a and G3 increased while G1 and G2b remained constant. Despite hypergammaglobulinemia, both IgG and IgM antibody titers against E. muris were very low throughout the 30‐day study. Antibody development and plaque‐forming cells against sheep red blood cells (SRBC) were abolished when the antigen was inoculated on day 10 PI. IgM antibody development against SRBC was more severely inhibited than IgG antibody development. However, when mice were immunized with SRBC prior to E. muris infection, antibody development against SRBC was not reduced. Delayed type hypersensitivity reaction to dinitrofluorobenzene was also maximally inhibited when the antigen was administered on day 10 PI. The IFN‐γ level in the blood was maximal at day 10 PI. These results indicate that although the vigorous polyclonal activation and protective IFN‐γ responses occurred by day 10 PI—which cleared most of the ehrlichial infection—antigen‐specific immune stimulation was impaired primarily at the level of antigen‐priming at peak parasitemia.
Sequence and Product Analyses of the Four Genes Downstream from the Fimbrilin Gene (fimA) of the Oral Anaerobe Porphyromonas gingivalis
Authors: Watanabe Koji , Onoe Takaya , Ozeki Masami , Shimizu Yasuaki , Sakayori Toshiko , Nakamura Hiroshi , Yoshimura Fuminobu
Published: November 2013

The downstream DNA region of the fimbrilin gene (fimA), which encodes the major subunit protein of Porphyromonas gingivalis fimbriae, was fully sequenced. Gene products, expressed from this region in Escherichia coli, were purified by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and their partial amino acid sequences were determined to verify open reading frames (ORFs) found in the region by DNA sequencing. Four ORFs, designated ORF1, ORF2, ORF3 and ORF4, were found in the 5.8‐kb PstI fragment downstream from fimA, which was previously cloned and partially characterized by Yoshimura, Takahashi, Hibi, Takasawa, Kato, and Dickinson (Infect. Immun. 61: 5181–5189, 1993). The direction of transcription of all the ORFs was the same as that of fimA. The 50 and 80 kDa encoded proteins, ORF2 and ORF3, respectively, have been reported to be minor components associated with fimbriae. The 15 and 19 kDa proteins, ORF1 and ORF4, respectively, have been expressed in E. coli but not identified in P. gingivalis. However, all the gene products of the ORFs, expressed in E. coli, appeared to contain intact signal peptides based on their N‐terminal amino acid sequences.
Virulence in Mice of Orientia tsutsugamushi Isolated from Patients in a New Endemic Area in Japan
Authors: Nagano Isao , Kasuya Shiro , Noda Nobuji , Yamashita Teruo
Published: November 2013

Four strains of Orientia tsutsugamushi (KN‐1, KN‐2, KN‐3 and GJ‐1) isolated from patients in an area of Gifu Prefecture, Japan, in which tsutsugamushi disease is newly endemic, were examined for their virulence in mice. Among these, KN‐1 (identified as Kawasaki type), GJ‐1 (identified as Kuroki type) and KN‐2 strains were found to be non‐lethal for BALB/c mice as well as CH3/HeJ mice, even with high doses (106 × being the 50% mouse infectious dose). On the other hand, the KN‐3 strain was found to be sufficiently virulent to kill BALB/c mice. Among the prototype strains (Gilliam, Karp and Kato), the Karp and Kato strains exhibited high virulence to mice, while the Gilliam strain killed only a susceptible strain of mouse. BALB/c mice infected with KN‐1 and KN‐2 strains showed significant splenomegaly and moderate ascites accumulation in the first week of infection, while these symptoms became prominent during the second week of infection using KN‐3, Karp and Kato strains. After infection with the GJ‐1 strain, these symptoms were not observed. Antibody responses induced by infections with highly virulent strains were lower than that with low or intermediate virulent strains.
Role of Cell Surface Glycosaminoglycans of Human T Cells in Human Immunodeficiency Virus Type‐1 (HIV‐1) Infection
Authors: Ohshiro Yukako , Murakami Tsutomu , Matsuda Kazuhiro , Nishioka Kiyoshi , Yoshida Keiichi , Yamamoto Naoki
Published: November 2013

To investigate the role of cell surface glycosaminoglycans (GAGs), including heparan sulfate (HS), on HIV‐1 infection in human T cells, HIV‐1 binding and infection were determined after treatment of T‐cell lines and CD4 + T cells from normal peripheral blood mononuclear cells (PBMC) with GAG‐degrading enzyme or a GAG metabolic sulfation inhibitor. Heparitinase I (hep I) and sodium chlorate prevented binding of HIV‐1/IIIB to MT‐4 cells as revealed by indirect immunofluorescence procedures, thereby inhibiting infection. Hep I was less effective in the binding inhibition of the macrophage‐tropic strain HIV‐1/SF162 than that of the T‐cell line‐tropic strain HIV‐1/IIIB. The binding of HIV‐1/SF162 was about 100‐fold less dependent on cell surface HS than HIV‐1/IIIB. Human HTLV‐I positive T‐cell lines expressed more HS than HTLV‐I negative T‐cell lines or normal CD4 + T cells when stained with anti‐HS mAbs against either native or heparitinase‐treated HS. With the exception of endo‐β‐galactosidase (endo‐β‐gal), GAG‐degrading enzymes, including hep I, chondroitinase ABC (chon ABC), chondroitinase AC II (chon AC II) and keratanase, did not prevent the binding of HIV‐1/IIIB to CD4+ T cells from normal PBMC. These results indicate that the cell surface HS of human T cells participates in HIV‐1 infection by facilitating HIV‐1/IIIB binding to MT‐4 cells. In particular, the sulfation of HS chains is critical. Since the expression of cell surface HS varies among T cells, which are not consistently sensitive to hep I treatment in HIV‐1 binding inhibition, other GAG‐like molecules may also be involved.
Induction of Tumor Necrosis Factor Alpha (TNFα) and Enhancement of HIV‐1 Replication in the J22HL60 Cell Line by Mycoplasma penetrans
Authors: Lyama Kaori , Ono Saori , Kuwano Koichi , Ohishi Masahiro , Shigematsu Hideki , Arai Sumio
Published: November 2013

Mycoplasma penetrans isolated from clinical specimens of AIDS patients showed potent activity in tumor necrosis factor alpha (TNFα) production in THP‐1, U937 and J22HL60 cell lines, and in the enhancement of HIV‐1 replication in a dormantly‐infected J22HL60 cell line as compared with the activities of other mycoplasmas. Both activities were found in the methanol layer but not in the chloroform layer of the membrane extracted by the Bligh‐Dyer method. TNFα production was observed in the peritoneal macrophages from both lipopolysaccharide‐responsive and ‐unresponsive mouse strains, and was not inhibited by polymyxin B. The induction of TNFα production and enhancement of HIV‐1 replication were strongly inhibited by Concanavalin A‐Sepharose. The inhibitory effect of Concanavalin A‐Sepharose was partially prevented by sugars in the order methyl‐α‐D‐mannopyranoside and methyl‐α‐D‐glucopyranoside but not methyl‐α‐D‐galactopyranoside. Anti‐human TNFα antibody, however, did not reduce the activity of the methanol layer to enhance HIV‐1 replication, suggesting that the methanol layer could enhance HIV‐1 replication directly. These results suggest that the carbohydrate derived from M. penetrans might be responsible for the progression of HIV‐1 infection.
Borrelia burgdorferi Sensu Lato in an Endemic Environment: Wild Sika Deer (Cervus nippon yesoensis) with Infected Ticks and Antibodies
Authors: Isogai Emiko , Isogai Hiroshi , Masuzawa Toshiyuki , Postic Daniele , Baranton Guy , Kamewaka Yuri , Kimura Koichi , Nishikawa Takeshi , Fujii Nobuhiro , Ishii Norihisa , Ohno Shigeaki , Yamaguti Noboru
Published: November 2013

Ticks and blood samples were collected from wild sika deer (Cervus nippon yesoensis) during a hunting season (August to October) of 1991 at a selected location in Hokkaido, Japan. Ixodes persulcatus (adult and nymph) and I. ovatus (adult) were the common ticks on sika deer. Spirochetes were detected in the midgut of the ticks by the indirect peroxidase‐conjugated antibody staining method and by dark‐field microscopy after cultivation. By the reactive pattern of monoclonal antibodies, isolates were considered to belong to Borrelia garinii or B. japonica. In an antibody test, the percentage of seropositive deer was 69.0%. Most of the adult sika deer were positive for antibodies to the spirochetes. There are significant age‐dependency in antibody level and seropositive rate. The surveillance of deer should be valuable in monitoring the transmission risk of B. burgdorferi sensu lato in nature.
Nucleotide Sequence of the SrRNA Gene and Phylogenetic Analysis of Trichomonas tenax
Authors: Fukura Katsuhisa , Yamamoto Ayako , Hashimoto Tetsuo , Goto Nobuichi
Published: November 2013

The small subunit ribosomal RNA (SrRNA) gene of Trichomonas tenax ATCC30207 was amplified by PCR and the 1.55‐kb product was cloned into plasmid vector pUC18. Four clones were isolated and sequenced. The insert DNAs were 1,552 bp long and their G+C contents were 48.1%; three of them had exactly the same DNA sequences and one had only one nucleotide change. A representative SrRNA sequence was analyzed and a phylogenetic tree was estimated by the neighbor‐joining (NJ) method. Among the protists examined, T. tenax was placed as the closest relative of Tritrichomonas foetus, as expected from the traditional taxonomy. The total homology between the two SrRNA sequences was 89.2%.
Reactivation of Herpes Simplex Virus Type 1 in Cells of Latently Infected Mouse Trigeminal Ganglia Cultured in Serum‐Free Medium
Authors: Kimura Susumu
Published: November 2013

Herpes simplex virus type 1 (HSV‐1) was reactivated more rapidly in cells of latently infected mouse trigeminal ganglia which were cultured in serum‐free medium (after 3.7 days of cultivation) than in those cultured in serum‐containing Dulbecco's modified Eagle's medium (after 8.5 days of cultivation). The concentration of calcium ion (Ca2+) in the medium affected HSV‐1 reactivation in ganglionic cultures, and 0.9 mM was the optimum concentration for the reactivation. Reactivation was delayed significantly in ganglia put into culture 4 months or more after infection compared with those cultured 1 month after infection.
Seroepidemiological Survey of Lymphocytic Choriomeningitis Virus in Wild House Mice in China with Particular Reference to Their Subspecies
Authors: Morita Chiharu , Tsuchiya Kimisachi , Ueno Hiroshi , Muramatsu Yasukazu , Kojimahara Akiko , Suzuki Hitoshi , Miyashita Nobumoto , Moriwaki Kazuo , Jin Mei‐Lei , Wu Xiang‐Lin , Wang Feng‐Shan
Published: November 2013

Serum samples from 337 wild house mice (Mus musculus) from 35 sites in China, collected in 1992 and 1993, were examined for antibodies against lymphocytic choriomeningitis virus (LCMV). Ten samples from eight sites were found to contain such antibodies. Six of the eight positive sites were located in the territory of M. m. gansuensis. One of the other two sites was located in the territory of M. m. castaneus in southern China and the other site was in a habitat of M. m. castaneus which had invaded into the western end of the territory of M. m. homourus. It seems likely that LCMV is distributed in the territories of M. m. gansuensis and M. m. castaneus in China. This is the first report of detection of these antibodies in wild house mice in China and specifically in the territories of M. m. gansuensis and M. m. castaneus.
Japanese Encephalitis Virus Infection in Vero Cells: The Involvement of Intracellular Acidic Vesicles in the Early Phase of Viral Infection Was Observed with the Treatment of a Specific Vacuolar Type H+‐ATPase Inhibitor, Bafilomycin A1
Authors: Nawa Masaru
Published: November 2013

The involvement of intracellular acidic vesicles in the early phase of Japanese encephalitis (JE) virus infection in Vero cells was observed by adding a specific vacuolar type H+ ‐ATPase (V‐ATPase) inhibitor (bafilomycin A1) in the cell culture medium. Studies with the detection of viral envelope (E) protein suggested that bafilomycin A1 inhibited virus infection in the cells. Subcellular distribution of incoming biotinylated virions and 3H‐uridine‐labeled viral RNA were observed in fractions of a Percoll density gradient. At 10 min of the chasing period, virions and viral RNA were found mainly in fractions with a mean density of 1.04 g/ml corresponding to the endosome both in the control and bafilomycin A1‐treated cells. At 60 min of the chasing period, the peak of biotin activity was detected in fractions with a mean density of 1.08 g/ml corresponding to the lysosome, whereas the peak of radioactivity did not run parallel with that of biotin and shifted to fractions with a mean density of 1.05 g/ml and higher than 1.084 g/ml, respectively. At 60 min of the chasing period in bafilomycin A1‐treated cells, the peak of biotin and radioactivity were still found mainly in the fraction with a density of 1.04 g/ml, representing the endosome. Subcellular fractionation by a Percoll density gradient revealed that bafilomycin A1 treatment resulted in the accumulation of virions in the endosome fraction and suggested the prevention of intracellular translocation of the virions which occurs during the early entry process of an infecting virus to the cells.
Biovar Diversity Is Reflected by Variations of Genes Encoding Urease of Ureaplasma urealyticum
Authors: Ruifu Yang , Minli Zhang , Zhaobiao Guo , Xiaohui Wang
Published: November 2013

Five oligonucleotide primers derived from the gene encoding urease of Ureaplasma urealyticum were designed to evaluate the relationship between the urease gene and biovar diversity of this organism. Five combinations of these primers were tested by PCR and the result revealed that there were variations in urease genes among different serovars of U. urealyticum. This result, in agreement with other PCRs based on other functionally unrelated (rRNA and MB antigen) genes, may reflect the phylogenetic relationship among organisms taxonomically classified as U. urealyticum.
Phylogenetic Positions and Assignment of Swine and Ovine Corynebacterial Isolates Based on the 16S rDNA Sequence
Authors: Takahashi Tatsufumi , Mori Yukari , Kobayashi Hideaki , Ochi Masaru , Kikuchi Naoya , Hiramune Takashi
Published: November 2013

The nucleotide sequences of the 16S ribosomal RNA gene (rDNA) of swine and ovine corynebacterial strains were determined. The sequences of the strains that identified as Corynebacterium pseudotuberculosis by their biochemical characteristics were homologous with each other. The phylogenetic position of C. pseudotuberculosis strains was closest to C. ulcerans and next closest to C. diphtheriae. The nucleotide sequence of another swine isolate, SC8, was similar to that of a recently proposed species, C. seminale, and a non‐validated species, “C. glucuronolyticum,” with about 0.01 to 0.02 evolutionary distances. Analysis of the predicted secondary structure of the 16S rRNA molecule agreed with the close phylogenetic relationships between C. pseudotuberculosis and C. ulcerans and between C. seminale and strain SC8.
Anti‐Chemotactic Activity of Capsular Polysaccharide of Cryptococcus neoformans In Vitro
Authors: Fujihara Hitomi , Kagaya Keiko , Fukazawa Yoshimura
Published: November 2013

In this study, we demonstrated the anti‐chemotaetic activity of the capsular polysaccharides (CPSs) isolated from each of the heavily (H)‐ and weakly (W)‐encapsulated strains of Cryptococcus neoformans in vitro. The capacity for activation of the alternative complement pathway (ACP) of cells of the two C. neoformans strains in fresh human sera was comparable to that of zymosan (insoluble control), whereas the capacity for generation of the chemotactic factor (CF) of the cells of the two strains in fresh murine sera was markedly lower in the order H‐ < W‐strain than that of zymosan. Conversely, the capacities for ACP activation and CF generation of the CPSs were extremely lower than those of lipopolysaccharide (LPS, soluble control). When zymosan‐activated murine serum was incubated with CPS, both CPSs inhibited CF activity dose dependently. When zymosan‐activated serum was incubated with heat‐killed cells of each strain of C. neoformans, H and W, the CF activity of the treated sera decreased significantly, suggesting that CPS per se did not affect the neutrophils directly, but CPS absorbed CF. On the other hand, both CPSs were shown to possess the O‐acetyl groups in their molecules by 1H‐nuclear magnetic resonance spectroscopy. The de‐O‐acetylation of both CPSs increased the capacity for ACP activation to a level similar to that of LPS, and the de‐O‐acetylated CPS of both strains exhibited a lower ability to inhibit CF than did native CPS. Collectively, these results suggest that the anti‐chemotactic activity of CPS accounts for its ability to absorb the CF which was mostly generated at the sites around the cell wall of whole cells via the ACP, thus suppressing the inflammatory response by preventing dispersal of CF to the extracellular space; and also that the O‐acetyl group is partly, if any, involved in the mechanism for incompetence in ACP activation as well as the inhibition of CF.
Protease‐Induced Multicell Formation in Staphylococcus haemolyticus
Authors: Yabu Kunihiko , Nishiyama Yayoi , Ochiai Toshiro
Published: November 2013

Multicellular cells were efficiently induced in Staphylococcus haemolyticus by the addition of protease to exponentially growing cultures at 30 C. Electron microscopy revealed the formation of tetrad‐shaped multicells that were septated but not separated from each other. Incubation of the multicells with extract from the cells grown without protease resulted in a fourfold increase in the number of colony‐forming units as compared with the untreated control. An electrophoretic analysis showed that protease caused a loss of cell wall‐lytic activity of the cell, which possibly led to the formation of multicells through cessation of cross wall separation.
The Host Response to Listeria monocytogenes Mutants Defective in Genes Encoding Phospholipases C (plcA, plcB) and Actin Assembly (actA)
Authors: Rudnicka Wiesława , Kaczmarek Małgorzata , Szeliga Jacek , Germann Tieno , Więckowska Marzena , Różalska Barbara
Published: November 2013

Several genes involved in the determination of Listeria monocytogenes pathogenesis have been identified. Among them, plcA gene encodes phosphatidylinositol‐specific phospholipase C (PI‐PLC), plcB gene encodes a broad‐range phospholipase C (PC‐PLC), and actA encodes a protein contributing to actin assembly in infected cells. The interaction of L. monocytogenes wild type (LO 28) strain and two derivative mutants, plcA− (BUG 206) and actA−/plcB− (LUT 12), with macrophages and T lymphocytes was investigated in a mouse model of listeriosis. Both mutants showed evidence of attenuation. The plcA− mutant, but not the plcB− mutant, expressed an increase in susceptibility to the anti‐listerial activity of macrophages. Both mutants showed a decreased ability to induce IL‐12 production by bone marrow macrophages when co‐stimulated with E. coli LPS or IFN‐γ. In vivo, L. monocytogenes plcA− mutant was found to be a more effective stimulator of T cells than the wild LO 28 strain.
A Structural Analysis of the Regularly Arranged Porin on the Outer Membrane of Campylobacter jejuni Based on Correlation Averaging
Authors: Amako Kazunobu , Baba Norio , Suzuki Nobuyuki , Wai Sun Nyunt , Umeda Akiko
Published: November 2013

A negatively stained electron micrograph of regularly arranged porin proteins of Campylobacter jejuni on the isolated outer membrane of bacteria was analyzed in detail by the correlation averaging method using a computer‐assisted program. The results showed that the porin of C. jejuni had a trimeric structure separated by about 10.4±0.15 nm. In addition, the pores in the trimers were also separated by about 4.3 ±0.1 nm.
Immunological Studies of a 21 kDa Cellular Component Efficiently Incorporated into Rabies Virion Grown in a BHK‐21 Cell Culture
Authors: Sagara Junji , Tochikura Tadafumi S. , Yamamoto Toshiko , Tsukita Shoichiro , Tsukita Sachiko , Kawai Akihiko
Published: November 2013

To investigate cellular components incorporated into the rabies virion, monoclonal antibodies (MAbs) were screened based on their reactivity with additional virion components. Two of the MAbs we prepared recognized a virion‐associated 21 kDa polypeptide (referred to as VAP21) from a BHK‐21 cell. Since the MAbs precipitated the rabies virion and trypsin digestion eliminated the VAP21 antigen from the virion but alkaline treatment (pH 11) did not, VAP21 seems to be anchored into the viral envelope and exposed on the virion surface. Although quantitative immunoblot analyses indicated an apparently increased concentration of VAP21 in the virion, the ratio of the content of VAP21 to that of viral glycoprotein (G) was several times decreased as compared to the ratio of those in the cell. These data suggest that sorting of VAP21 occurs during the viral budding process on the cell but that it might be inefficient, probably due to a more intimate association of VAP21 with the viral envelope proteins. This assumption seems to be consistent with the results of immunofluorescence studies; that is, VAP21 displayed colocalized distribution with viral envelope antigens in the cell. From these results, it is suggested that VAP21 closely associates with the viral envelope proteins in the cell, and this association might cause passive but relatively efficient incorporation of VAP21 into the virion.
Metabolic Fate of [3H]1‐β‐d‐Arabinofuranosyl‐5‐[(E)‐2‐bromovinyl]uracil in Herpes Simplex Virus Type 1‐Infected Cells
Authors: Ashida Noriyuki , Sakata Shinji , Machida Haruhiko
Published: November 2013

The metabolic fate of 1‐β‐d‐arabinofuranosyl‐5‐[(E)‐2‐bromovinyl]uracil (BV‐araU) in herpes simplex virus type 1‐infected cells was studied using tritium‐labeled BV‐araU. [3H]BV‐araU was selectively taken‐up by infected cells. Approximately 10% of the total uptake of [3H]BV‐araU was recovered from the acid‐insoluble fraction at any time post‐infection. Both cellular uptake of [3H]BV‐araU and its incorporation into the acid‐insoluble fraction increased with increasing incubation time through 8 hr post‐infection. Uptake of [3H]BV‐araU and its incorporation into the acid‐insoluble fraction also increased proportionally to the duration of exposure to [3H]BV‐araU. An alkaline sucrose gradient sedimentation analysis revealed that the radioactive DNA obtained from cells pulse‐labeled with [3H]BV‐araU were small DNA fragments which remained at the top following a chasing period in isotope‐free medium, whereas that pulse‐labeled with [3H]thymidine was chased to a fraction of high molecular weight DNA. Nuclease P1 digestion reduced 99% of the [3H]BV‐araU‐labeled DNA extracted from infected cells to a low molecular weight. Following digestion of [3H]BV‐araU‐labeled DNA with micrococcal nuclease and spleen exonuclease, all of the radioactivity was recovered as [3H]BV‐araU 3′‐monophosphate. Thus, BV‐araU strongly inhibits the elongation of viral DNA strands as demonstrated by the alkaline sucrose gradient sedimentation analysis, whereas at least a portion of the [3H]BV‐araU is incorporated inside viral DNA strands in infected cells.
Salmonella typhi Ty2 OmpC Porin Induces Bactericidal Activity on U937 Monocytes
Authors: Blanco Luz P. , Toro Cecilia S. , Romero Jaime M. , Santiviago Carlos A. , Mora Guido C.
Published: November 2013

The immunogenic effect of Salmonella typhi OmpC porin during typhoid fever in humans was evaluated in vitro. Peripheral blood mononuclear cells from 17 patients were challenged with outer membrane preparations from Escherichia coli UH302 and UH302/pSTP2K2 strains, both lacking E. coli OmpF and OmpC porins, although UH302/pSTP2K2 expressed a plasmid‐encoded S. typhi Ty2 OmpC. The mononuclear cell supernatants, immunized in vitro with OmpC antigen, derived from 10 out of 17 patients activated U937 bactericidal capacity. In contrast, the supernatants from the immunization with outer membrane preparation lacking S. typhi Ty2 OmpC induced a significantly reduced bactericidal capacity of U937 cells. This procedure should prove useful for in vitro characterization of cellular immunogens from exclusive human pathogens.
Sequence Analysis of the actA Gene of Listeria monocytogenes Isolated from Human
Authors: Moriishi Kohji , Terao Michinori , Koura Minako , Inoue Satoshi
Published: November 2013

The region encoding proline‐rich units of actA genes was amplified from 24 strains of Listeria monocytogenes using polymerase chain reaction (PCR). PCR products of 13 strains showed the expected size of 623 bp, whereas those of 11 strains showed a short size of 518 bp. The shortening of these PCR products resulted from the deletion of one proline‐rich unit. These results indicate that ActA proteins are divided into at least two different types which are unrelated to bacterial serotypes.
Effect of Acidic Fibroblast Growth Factor (aFGF) on Phagocytosis in Mouse Peritoneal Macrophages
Authors: Ichinose Mitsuyuki , Sawada Masashi , Sasaki Kazuo , Oomura Yutaka
Published: November 2013

The effects of acidic fibroblast growth factor (aFGF) on phagocytosis in peritoneal macrophages from thioglycollate‐elicited mice were examined using flow cytometry. aFGF enhanced phagocytosis of fluorescein isothiocyanate‐labeled latex particles in a dose‐dependent manner. Basic fibroblast growth factor (bFGF) also enhanced phagocytosis. This study suggests that aFGF can modulate an important activity of macrophages.
Intervening Sequences in 16S rRNA Genes of Campylobacter sp.: Diversity of Nucleotide Sequences and Uniformity of Location
Authors: Etoh Yumiko , Yamamoto Ayako , Goto Nobuichi
Published: November 2013

We found and sequenced intervening sequences (IVSs) in the PCR‐amplicons of 16S rRNA genes of 3 strains of Campylobacter rectus, 2 strains of C. curvus and 2 strains of C. sputorum. The lengths of the IVSs were 140 to 233 bp. The IVSs of C. rectus were identical and had a sequence homology of 55 to 79% against those of C. curvus and C. helveticus. The IVSs of C. sputorum were 97.9‐100% homologous but poorly homologous to the other IVSs. In spite of the diversities of the lengths and the nucleotide sequences, all of the IVSs were located at the same position in the 16S rRNA genes.
Requirement of Cauliflower Mosaic Virus Open Reading Frame VI Product for Viral Gene Expression and Multiplication in Turnip Protoplasts
Authors: Kobayashi Kappei , Tsuge Seiji , Nakayashiki Hitoshi , Mise Kazuyuki , Furusawa Iwao
Published: November 2013

Cauliflower mosaic virus (CaMV) open reading frame (ORF) VI product (P6) has been shown to be the major constituent of viral inclusion body, to function as a post‐transcriptional transactivator, and to be essential for infectivity on whole plants. Although these findings suggest that P6 has an important role in viral multiplication, it is unknown whether P6 is required for viral multiplication in a single cell. To address this question, we transfected turnip protoplasts with an ORF VI frame‐shift (4 bp deletion) mutant (pCaFS6) of an infectious CaMV DNA clone (pCa122). The mutant was uninfectious. Co‐transfection of plasmids expressing P6 complemented the mutant. Overexpression of P6 elevated the infection rate in co‐transfection experiments with either pCa122 or pCaFS6. This would have been achieved by elevating the level of pregenomic 35S RNA, a putative polycistronic mRNA for ORFs I, II, III, IV and V, and by enhancing the accumulation of these five viral gene products. When CaMV ORFs I, II, III, IV and V were expressed from monocistronic constructs in which each of the ORFs was placed just downstream of the 35S promoter, the accumulation of ORF III, IV and V products depended on the co‐expression of P6. The accumulation of ORF I and II products was not detected, even in the presence of P6. These results suggest that P6 is involved in the stabilization of other viral gene products as well as in the activation of viral gene expression, and thus, is a prerequisite for CaMV multiplication.
The First Isolation of Arthroderma benhamiae in Japan
Authors: Kano Rui , Nakamura Yuka , Yasuda Kazuo , Watari Toshihiro , Watanabe Shinichi , Takahashi Hisashi , Tsujimoto Hajime , Hasegawa Atsuhiko
Published: November 2013

A clinical isolate of Trichophyton mentagrophytes from rabbit was examined by polymerase chain reaction (PCR) analysis and a mating experiment. The species‐specific primers designed from the nucleotide sequences of the chitin synthase 1 (CHS1) gene in the teleomorph of Arthroderma benhamiae amplified a fragment from genomic DNA samples of A. benhamiae and the clinical isolate but not from those of A. simii and A. vanbreuseghemii. On the other hand, the species‐specific primers of A. simii and A. vanbreuseghemii did not amplify any fragment from the genomic DNA of the clinical isolates. When the isolate was respectively crossed with (+) or (‐) tester strains of A. benhamiae, A. simii and A. vanbreuseghemii, ascospores were produced in the crossing with the A. benhamiae (+) strain. Therefore, the isolate was identified to be A. benhamiae (‐), confirming the result of molecular analysis. This is the first report on the isolation of A. benhamiae in Japan.