bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Determination of Infection Source of a Sporadic Legionella Pneumonia Case Associated with a Hot Spring Bath
Authors: Miyamoto Hiroshi , Jitsurong Siroj , Shiota Ryoko , Maruta Koji , Yoshida Shin‐Ichi , Yabuuchi Eiko
Published: November 2013

To determine the infection source of a sporadic Legionella pneumonia case associated with a hot spring bath, we used five molecular methods, including repetitive element polymerase chain reaction (rep‐PCR), arbitrarily primed PCR (AP‐PCR), ribotyping, restriction endonuclease analysis (REA), and macrorestriction endonuclease analysis (MREA) by pulsed‐field gel electrophoresis. L. pneumophila serogroup (SG) 3 strain EY 3702, isolated from an intratracheal specimen of a 71‐year‐old Japanese female who developed pneumonia after nearly drowning in a hot spring spa bath, produced rep‐PCR and AP‐PCR fingerprints identical to those of L. pneumophila SG 3 strains EY 3768 and EY 3769 isolated from the bath water. Four epidemiologically unrelated L. pneumophila SG 3 strains showed different rep‐PCR or AP‐PCR fingerprints from those of the three EY strains (EY 3702, 3768, and 3769). The three EY strains were also genotypically indistinguishable by ribotyping with EcoRI and PstI, by REA with EcoBI or HindIII, and by MREA with NotI. Based on these results, we identified the bath water of the hot spring spa as the source of infection of this patient, even though the viable number of the organisms in the bath water was low (3 CFU/100 ml) when determined 27 days after her nearly drowning.
Detection of Flaviviruses by Reverse Transcriptase‐Polymerase Chain Reaction with the Universal Primer Set
Authors: Meiyu Fang , Huosheng Chen , Cuihua Chen , Xiaodong Tian , Lianhua Jiang , Yifei Peng , Weijun Chen , Huiyu Guo
Published: November 2013

Using a universal primer set designed to match the sequence of the NS1 gene of flaviviruses, the virus RNA of dengue (DEN), Japanese encephalitis (JEV), powassan and langat of Flaviviridae were successfully amplified by polymerase chain reaction (PCR) via cDNA; and with different internal primers, the serotypes of the dengue viruses were identified. Of the 78 clinically diagnosed dengue fever patients, 18 patients were positive for DEN 1, 48 patients for DEN 2 and 8 patients concurrently infected with DEN 4. Of the 52 patients admitted with Japanese encephalitis (JE), 45 were determined to be JEV infections. By nested PCR, we completed the identification of flaviviruses within 2 days. The results show that seven primers have a potential value for rapid clinical diagnosis of flavivirus infections.
Advantage of Dimeric Peptide Antigens in Serodiagnosis of HIV‐1 Infection
Authors: Gokulan Kuppan , Tripathi Srikant Prasad , Rao D. Nageshwara
Published: November 2013

The reactivity of antibodies with dimeric and monomelic peptide antigens was compared by ELISA. A panel of highly purified synthetic peptides of HIV‐1 representing defined regions, 598–609 and 524 533 (fusion domain) of gp41 and 306–320 of gpl20, were used as antigens in the ELISA. These peptides were selected and synthesized taking into account the level of sequence conservation of various strains and hydrophilicity. The analysis included sera from 52 HIV‐1 infected individuals and 53 HIV‐1 negative controls. Both peptides from gp41 were found to be particularly immunoreactive with sera from HIV‐1 infected individuals. The frequency of reactivity to the selected peptide from gp120 (V3 loop) in infected individuals was 82%. An interesting observation was that the dimeric peptide antigens had a detection rate more than 4‐fold higher than the monomeric antigens. We found that lower levels of antibodies could be detected with dimeric antigens. The peptides reacted with few sera other than HIV‐1 positive sera. These results implicate the potential dimeric peptide antigens to be utilized in the serodiagnosis of HIV‐1 infection.
Identification of a Phosphatase‐Sensitive Epitope of Rabies Virus Nucleoprotein Which Is Recognized by a Monoclonal Antibody 5‐2‐26
Authors: Anzai Jun , Takamatsu Fumihiko , Takeuchi Kenji , Kohno Takashi , Morimoto Kinjiro , Goto Hideo , Minamoto Nobuyuki , Kawai Akihiko
Published: November 2013

We have investigated a phosphatase‐sensitive sequential epitope of the nucleoprotein (N), one of the phosphoproteins of rabies virus, which is recognized by the monoclonal antibody (MAb) #5‐2‐26. The epitope was shared in common by all of the rabies virus strains we tested, including the HEP, ERA, CVS and Japanese strains (Nishigahara and Komatsukawa). Thin layer chromatography of the acid hydrolyzates of 32P‐labeled N protein showed that the protein contained phosphoserine and phospho‐threonine at a molar ratio of about 4 to 1, while no phosphotyrosine was detected. Immunoprecipitation studies with several deletion mutants of the N protein showed that the epitope is located in a region spanning from amino acid 344 to 415. If the phosphatase‐sensitive epitope is located at or near the phosphoamino acid, the location of the latter could be narrowed further to a region from amino acid 354 to 389 by comparing the amino‐acid sequences among the viral strains. To examine this assumption, point mutation was introduced by amino‐acid substitution with alanine at either of five potential phosphorylation sites (i.e., positions 354, 375, 377, 386 and 389) in the 354–389 region. Among those, only one substitution, at position 389, greatly affected the antigenicity. Substitution of serine‐389 by threonine also reduced the antigenicity. These results strongly suggest that serine‐389 is a phosphorylation site and essential for constructing or stabilizing the antigenic structure for MAb 5‐2‐26.
Double‐Negative BV8S3 T Cells Expanded in MRL lpr/lpr Lymph Nodes Conserve TCRBJ Gene Usage of Single‐Positive BV8S3 T Cells
Authors: Fan Jiong , Nanki Toshihiro , Miyasaka Nobuyuki , Kohsaka Hitoshi
Published: November 2013

Enlarged lymph nodes of mice with lpr mutation consist predominantly of CD4−CD8− (double‐negative: DN) T cells. Among them, TCRBV8S3 (Vβ 8.3) T cells are overrepresented as compared to those in single‐positive (SP) T cells. To address the question of whether the expansion of oligoclonal T cells is responsible for the increase in TCRBV8S3 cells, we examined the TCRBJ gene repertoires of BV8S3 DN and SP T cells from multiple MRL lpr/lpr mice. The BJ repertoires of BV3 (Vβ3), BV8S1 (Vβ8.1) and BV8S2 (Vβ8.2) were studied for comparison with those of BV8S3 T cells. The employed method, which was based on a PCR‐ELISA technique, was newly developed and allowed us to make a precise quantitation of TCRBJ gene usage of the multiple lymphocyte samples. The results showed that there were no biases of the BJ gene usage by BV8S3 DN T cells as well as other BV T cells. Furthermore, the BJ gene usage of CD4 and CD8 BV8S3 T cells was conserved by the DN T cells. It is suggested that the BV8S3 DN T cells were not expanded by specific antigens. The expansion may result from aberrant regulation specific to the BV8S3‐expressing T cells.
Role of Platelet‐Actlvatlng‐Factor (PAF) on Cellular Responses after Stimulation with Leptospire Lipopolysaccharide
Authors: Isogai Emiko , Hirose Kimiharu , Kimura Koichi , Hayashi Shunji , Kubota Toru , Fujii Nobuhiro , Isogai Hiroshi
Published: November 2013

Leptospire lipopolysaccharide (LPS) stimulated the adherence of polymorphonuclear neutrophils (PMNs) to human umbilical vein endothelial cells (HUVEC). Enhanced PMN adherence in response to leptospire LPS can be mediated by platelet‐activator‐factor (PAF), because a PAF antagonist reduced adherence. Leptospire LPS also induced the adherence platelets or U937. The second experiment involved leptospire LPS elicited platelet aggregation in a PMN‐platelet mixture, because leptospire LPS stimulated human PMN but not the human platelets. The platelet response was observed only in the mixture system and was inhibited by a PAF antagonist. PAF could be an important pathogenic factor in human leptospirosis.
Polymerase Chain Reaction Test for Differentiation of Five Toxin Types of Clostridium perfringens
Authors: Yamagishi Takayoshi , Sugitani Kayo , Tanishima Kiyoo , Nakamura Shinichi
Published: November 2013

In order to avoid the use of experimental animals, the polymerase chain reaction (PCR) method was applied to differentiate Clostridium perfringens into five toxin types. Twenty‐two out of 23 strains tested produced the toxin(s) corresponding to the toxin gene(s) identified by PCR, and vice versa. Consequently, the gene typing was consistent with conventional typing by animal tests. Twenty‐five strains were identified as types different from original ones by the PCR method as well as a toxin neutralization test. These findings suggest that the PCR method, which is easy and timesaving, is applicable to identify the toxin types of C. perfringens as an alternative to animal tests, and that beta‐, epsilon‐ and iota‐toxin genes might be lost by long‐term preservation. The reasons why the strains lost the genes are discussed.
Unique Expression of HRF20 (CD59) in Human Nervous Tissue
Authors: Akatsu Hiroyasu , Yamada Tatsuo , Okada Noriko , Yamamoto Takayuki , Yamashina Manabu , Okada Hidechika
Published: November 2013

Damage to autologous tissue by complement is limited by several widely distributed membrane‐associated glycoproteins which restrict the action of the complement in homologous species. These include decay accelerating factor (DAF), membrane cofactor protein (MCP) and 20 kDa homologous restriction factor (HRF20,CD59). Using immunohistochemical techniques, we examined the localization of these proteins in the centra] nervous system (CNS) and peripheral nervous system (PNS) using non‐neurological human nervous tissue since some complement components have been demonstrated to be synthesized in the CNS. There was no evidence of parenchymal staining by anti‐DAF or anti‐MCP antibodies in either type of tissue except for the staining of the endothelium in capillaries. On the other hand, anti‐HRF20 antibody clearly stained myelinated axons in the CNS as well as Schwann cells in the PNS. In addition, we detected positive staining by anti‐DAF antibody in the PNS of a Paroxysmal nocturnal hemoglobinuria (PNH) patient who is genetically deficient in HRF20.
Overproduction of Campylobacter Ferritin in Escherichia coli and Induction of Paracrystalline Inclusion by Ferrous Compound
Authors: Wai Sun Nyunt , Nakayama Koji , Takade Akemi , Amako Kazunobu
Published: November 2013

The ferritin gene (cft) of Campylobacter jejuni was overexpressed in cells of Escherichia coli using a T7 RNA polymerase expression system. Many round particles which were the same size as the ferritin particles purified from C. jejuni were observed in the lysate of the cft‐overexpressed E. coli cells. Since most of them were devoid of a central electron dense core consisting of ferric irons, the Campylobacter ferritins overproduced in E. coli seemed to be apoferritin. When large amounts of ferrous iron (supplied as FeSO4) were added to culture medium, the cft‐overexpressed cells formed large inclusion bodies of paracrystalline arrays comprised of ferritin particles with central electron dense cores. The addition of ferric irons did not produce paracrystalline inclusion.
Subcellular Localization of the Major Autolysin, ATL and Its Processed Proteins in Staphylococcus aureus
Authors: Komatsuzawa Hitoshi , Sugai Motoyuki , Nakashima Seiji , Yamada Sakuo , Matsumoto Akira , Oshida Tadahiro , Suginaka Hidekazu
Published: November 2013

The Staphylococcus aureus autolysin gene, atl, encodes a unique 138‐kDa protein (ATL) with amidase and glucosaminidase domains. ATL has been suggested to undergo proteolytic processing to generate two extracellular peptidoglycan hydrolases, 51‐kDa endo‐β‐N‐acetylglucosaminidase (51‐kDa GL) and 62‐kDa N‐acetylmuramyl‐L‐alanine amidase (62‐kDa AM). To investigate cell‐associated bacteriolytic enzymes for atl gene products, proteins were extracted from the cells as follows. The cells were exposed to 3 M LiCl followed by 4% SDS. Thereafter, the cells were disrupted and again extracted with 4% SDS. Whole SDS‐stable cell‐associated bacteriolytic proteins were extracted without disrupting the cells. Exposure to 3 M LiCl released major 138‐, 115‐, 85‐, 62‐ and 51‐kDa bacteriolytic proteins, and subsequent 4% SDS extraction released major 138‐ and 115‐kDa bacteriolytic proteins. These bacteriolytic proteins were missing in extracts of atl mutant RUSAL2 (S. aureus RN450 atl:: Tn551). Immunoblotting studies suggest that these are all atl gene products: the 138‐kDa protein is an ATL with a cleaved signal sequence; the 115‐and 85‐kDa proteins are intermediates; and the 51‐ and 62‐kDa proteins are cell‐associated 51‐kDa GL and 62‐kDa AM, respectively. The trypsin susceptibility of these proteins suggests that they are located outside the cell membrane. Differences in extractability and immunoelectron microscopic studies suggest that atl gene products are associated with cells in two different ways, LiCl extractable and non extractable. We suggest that the 138‐kDa ATL undergoes processing through intermediate proteins (115‐ and 85‐kDa proteins) to mature as the active cell cluster‐dispersing enzymes 51‐kDa GL and 62‐kDa AM on the cell surface.
Pathogenicity of Glycoprotein C‐Deficient Herpes Simplex Virus 1 Strain TN‐1 Which Encodes Truncated Glycoprotein C
Authors: Minagawa Hiroko , Yoshida Ying Liu Tetsuhiko , Hidaka Yasufumi , Toh Yasushi , Mori Ryoichi
Published: November 2013

A clinical isolate of herpes simplex virus 1 (TN‐1) from a stromal keratitis patient was found to be defective in the glycoprotein C (gC) gene (UL44), thus resulting in the production of truncated gC upon infection. To study the pathogenetic role of truncated gC, we prepared a recombinant LTN‐8 derived from TN‐1 with deletions of the 1.5 kilobase pairs of the gC gene including the initiation codon. A penetration assay revealed LTN‐8 to be less efficient in its penetration ability than TN‐1, the laboratory strain KOS and RTN‐1‐20‐3, a recombinant derived from TN‐1 with the KOS gC gene. The penetration of LTN‐8 was facilitated by the addition of TN‐1‐infected culture medium. TN‐1 virus preparations had no hemagglutinating activity. However, the animals infected with TN‐1 did develop hemagglutination inhibition (HI) antibodies. The LTN‐8‐infected animals did not develop HI antibodies. The pathogenicity in BALB/c mice following either corneal, intraperitoneal or intracerebral inoculation did not significantly differ among TN‐1, RTN‐1‐20‐3 or LTN‐8. Our results indicate that truncated gC was sufficient for the induction of HI antibodies and was also able to facilitate penetration in vitro. Although truncated gC might be a virulence factor acting as a decoy, both truncated gC and intact gC had little effect on the outcome following intracerebral, intraperitoneal or corneal inoculation.
MHC Class I Presentation of an Exogenous Polypeptide Antigen Encoded by the Murine AIDS Defective Virus
Authors: Yee Sung‐tae , Okada Yoshiaki , Ogasawara Kazumasa , Omura Satoshi , Takatsuki Akira , Kakiuchi Terutaka , Muno Daisaku , Kominami Eiki , Mizuochi Toshiaki
Published: November 2013

Peptides derived from endogenous proteins are presented by MHC class I molecules, whereas those derived from exogenous proteins are presented by MHC class II molecules. This strict segregation has been reconsidered in recent reports in which exogenous antigens are shown to be presented by MHC class I molecules in the phagocytic pathway. In this report, the presentation pathway of an exogenously added highly antigenic polypeptide encoded by the murine AIDS (MAIDS) defective virus gag p12 gene is investigated. A 25‐mer polypeptide (P12–25) encoded within the gag p12 region of the MAIDS defective virus was found to be effective in stimulating unprimed B6 (H‐2b) CD8+ T cells in vitro. The presentation of P12–25 is sensitive to cytochalasin B and D, brefeldin A and gelonin, a ribosome‐inactivating protein synthesis inhibitor, but less sensitive or resistant to lactacystin, a highly specific inhibitor of the proteasome. Interestingly, CA‐074, a selective inhibitor of cathepsin B, inhibited presentation of the polypeptide, indicating its involvement in the degradation of the P12–25 polypeptide. In fact, when P12–25 was digested with purified cathepsin B in vitro, a highly antigenic 11‐mer peptide containing the class I (H‐2Db)‐binding motif was obtained. Our results favor the phagosome/macropinosome‐to‐cytosol‐to‐endoplasmic reticulum (ER)‐to‐cell surface pathway for exogenous antigens presented by MHC class I molecules. These findings may be relevant to exploiting peptide vaccines that specifically elicit CD8+ T cell immunity in vivo.
Partial Sequences of Large Subunit Ribosomal DNA of a New Yeast Species, Trichosporon domesticum and Related Species
Authors: Sugita Takashi , Makimura Koichi , Nishikawa Akemi , Uchida Katsuhisa , Yamaguchi Hideyo , Shinoda Takako
Published: November 2013

We determined the partial sequences of large subunit rDNA of a new yeast species, Trichosporon domesticum, which was isolated from the house of a summer‐type hypersensitivity pneumonitis patient. Phylogenetically, T. domesticum was positioned in the taxonomic group containing T. montevideense and T. brassicae, which indicated an identical serotype. A phylogenetic relationship among all species of the genus Trichosporon which belong to the basidiomycetous yeast is clarified.
cDNA Cloning and Characterization of Rat C5a Anaphylatoxin Receptor
Authors: Akatsu Hiroyasu , Miwa Takashi , Sakurada Chikai , Fukuoka Yoshihiro , Ember Julia A. , Yamamoto Takayuki , Hugli Tony E. , Okada Hidechika
Published: November 2013

Activation of the complement cascade plays an esssential role in the early stages of inflammation. C5a and its receptor are particularly active in anaphylaxis. To determine the pathological roles played by C5a and C5a receptor (C5aR) in rats, we cloned C5aR cDNA and analyzed distribution of its mRNA in various organs including lung from an LPS‐stimulated rat. Furthermore, we generated a polyclonal antiserum which specifically recognizes rat C5aR, as confirmed by its specific interaction with cells transfected with rat C5aR cDNA.
Cyclophosphamide‐Induced Bacterial Translocation in Escherichia coli C25‐Monoassociated Specific Pathogen‐Free Mice
Authors: Nakayama Masaharu , Itoh Kikuji , Takahashi Eiji
Published: November 2013

The kinetics of bacterial translocation (BTL) from the intestine to the mesenteric lymph nodes (MLN) and the number of peripheral white blood cells (WBC), macrophages in Peyer's patches (PP) and M‐cells on the surface of cecal PP after cyclophosphamide (CY) injection were examined in penicillin‐G and streptomycin sulfate decontaminated and Escherichia coli C25‐monoassociated specific pathogen‐free mice. WBC were counted to confirm the immunological state of the mice. Until 8 days after CY injection, the number of WBC, bacteria in MLN and macrophages in PP decreased, but then significantly increased on day 14. The levels again decreased to the control levels on day 16. Although the number of M‐cells decreased up to day 8, it did not return to the control level on day 16. These results indicate that BTL is stimulated in an immunopotentiated state after CY injection, and this phenomenon may be closely related to the number of macrophages in the blood and PP.
Genetic Diversity of Borrelia burgdorferi Sensu Lato Isolated in Far Eastern Russia
Authors: Masuzawa Toshiyuki , Iwaki Atsue , Sato Yukita , Miyamoto Kenji , Korenberg Edward I. , Yanagihara Yasutake
Published: November 2013

One‐hundred and fifty‐seven Borrelia isolated from adult ticks, Ixodes persulcatus, and wild rodents, Clethrionomys rufocanus and Apodemus peninsulae, in the far eastern part of Russia were characterized and identified by restriction fragment length polymorphism (RFLP) of the 5S‐23S rRNA intergenic spacer. Some isolates showed unique RFLP patterns and were determined as Borrelia garinii on the basis of a sequence analysis of the intergenic spacer amplicon and reactivity with species‐specific monoclonal antibodies (MAbs). 86.5 and 12.7% of the tick isolates, and 74.2 and 12.9% of the rodent isolates were determined as Borrelia garinii and Borrelia afzelii, respectively, but no Borrelia burgdorferi sensu stricto was detected. This finding is similar to the results obtained from Borrelia surveys of I. persulcatus and wild rodents in Hokkaido, Japan.
Genetic Control of Immune Responses to a Synthetic Fimbrial Antigen of Actinobacillus actinomycetemcomitans
Authors: Honma Kiyonobu , Ishii Norihisa , Kato Tetsuo , Ishihara Kazuyuki , Okuda Kenji , Okuda Katsuji
Published: November 2013

The incidence of infection by Actinobacillus actinomycetemcomitans, one of the important pathogens in human periodontal diseases, has been reported to be associated with racial background and genetic factors. We attempted to determine the genetic regulation of immune responses to A. actinomycetemcomitans fimbriae, an attachment factor, using various inbred strains of mice. For this purpose, we synthesized an oligopeptide antigen using the amino acid sequence of the fimbriae and conjugated this antigen to branched lysine polymer resin beads. After immunization with the synthetic A. actinomycetemcomitans fimbrial antigen, serum antibody levels and the delayed‐type hypersensitivity (DTH) reaction to the antigen were measured by enzyme‐linked immunosorbent assay (ELISA) and footpad swelling responses, respectively. The strains of mice found to be high‐IgG responders to the antigen were B10.HTT, B10.RIII, B10A (5R) and B10.S (9R). These results indicate that mice with Eβs: Eαk, Eβr: Eαr and Eβb: Eαk respond strongly to the synthetic peptide. All of the high‐IgG responders showed a high DTH response. A cell transfer experiment confirmed that CD4 T cells mediated with a DTH response to the synthetic peptide. Thus, the results of this study demonstrate that the immune responses to A. actinomycetemcomitans fimbriae are genetically controlled.
Genomic Diversity in the pfoA Region of Theta‐Toxin‐Deficient Strains of Clostridium perfringens
Authors: Ba‐Thein William , Inui Satoko , Shimizu Tohru , Swe Tint , Banu Sayera , Ohtani Kaori , Oe Misari , Sakurai Naomi , Nakamura Shinichi , Hayashi Hideo
Published: November 2013

The genomic structure of the pfoA‐colA region in six theta‐toxin‐deficient strains of Clostridium perfringens was examined by Southern hybridization using the pfoR, pfoA, pbg, arcABDC and colA genes, encoding regulator for pfoA, theta‐toxin, beta‐galactosidase, arginine metabolism enzymes and kappa‐toxin, respectively, as gene probes. It is suggested that the productivity of theta‐toxin in these strains is diverse because of the multiple genetic backgrounds including single deletion of pfoA, large deletion of the pfoA‐colA region and the putative point mutations.
Interferon‐α and ‐γ Differentially Reduce Rapid Immature T‐Cell Death by Contact with HIV‐1 Carrier Cell Clones In Vitro
Authors: Kinzl Petra , Otani Takeshi , Benz Roland , Minowada Jun
Published: November 2013

The non‐antigen specific rapid cytotoxic (CT) death of immature TdT+CD4+CD8+ T cells due to contact with HIV‐1 carrier T‐cell clones we have found recently is a novel phenomenon. The effects of interferons (IFN) on this CT reaction were studied in vitro. Treatment of the HIV‐1 carrier clones, referred to as “effectors,” with IFN‐α but not IFN‐γ, or of the susceptible immature TdT+CD4+CD8+ T cells, referred to as “targets,” with IFN‐γ but not IFN‐α, for 24 hr prior to CT testing was found to reduce the CT reaction. Simultaneously, a down‐regulated CD8 expression and an up‐regulated antigen expression of both major histocompatibility antigen complex class I (MHC‐I) and HIV‐1 gp120/gp160 in the IFN‐α treated effector (gp120+CD8+ HPB‐ALL/HIV), and/or simultaneously up‐regulated antigen expression of both CD8 and MHC‐I in the IFN‐γ treated target (CD4+CD8+ HPB‐ALL) were found to be associated with reduced CT reaction. However, altered antigen expression in the IFN‐γ treated effectors or IFN‐α treated targets did not affect the ultimate degree of CT reaction. This study thus suggests a possible therapeutic efficacy of IFN by reducing the direct elimination of the T‐cell precursors in HIV‐1 infection.
Negative Finding in Cross‐Protective Activity of JapaneseBorreliaIsolates against Infection with Three Species of Lyme DiseaseBorreliain Outbred Mice
Authors: Masuzawa Toshiyuki , Kurita Toru , Yanagihara Yasutake
Published: November 2013

Outer surface protein A (OspA) is the most promising candidate for a component vaccine against Lyme disease caused byBorrelia burgdorferisensu lato. Active cross‐protection using a whole‐cell vaccine prepared from strains belonging various OspA serotypes observed in Japan and worldwide was examined. No cross‐protection was obtained by heterologous OspA‐serotype vaccines. Since OspA is a highly variable protein expressed byBorrelia, this suggests that immunologically different OspA serotypes need to be combined for the development of an effective vaccine in Japan.
Cloning and Characterisation of the aroA and aroD Genes of Shigella dysenteriae Type 1
Authors: Walker John C. , Verma Naresh K.
Published: November 2013

The aroA and aroD genes from Shigella dysenteriae type 1, encoding 5‐enolpyruvylshikimate 3‐phosphate synthase and 3‐dehydroquinase, respectively, were cloned by polymerase chain reaction (PCR). Their nucleotide sequences were determined and predicted to code for 46 kDa and 27.5 kDa proteins, respectively. Protein expressed from these genes using the minicell system, corresponded to the size of the predicted protein products. The cloned genes were shown to be functional by complementation of Escherichia coli aroA and aroD− mutants. The predicted amino acid sequences of the cloned aroA (427 amino acids) and aroD (252 amino acids) genes of S. dysenteriae type 1 were found to be highly homologous to the corresponding genes in other bacterial species, indicating the high conservation of these housekeeping genes. The use of the cloned aroA and aroD genes in the development of a vaccine strain against S. dysenteriae is discussed.
IgA Protease Produced by Streptococcus sanguis and Antibody Production against IgA Protease in Patients with Behçet's Disease
Authors: Yokota Kenji , Oguma Keiji
Published: November 2013

The production of IgA protease in twelve strains of Streptococcus sanguis isolated from patients with Behçet's disease (BD) was examined. Protease activity was detected in 10 out of 12 strains. The protease was purified from one representative strain, S. sanguis 113–20, by employing Rotofor and DEAE‐Sephacel chromatography. The molecular mass of the purified protease was approximately 100 kDa, and it cleaved the proline‐threonine site of the IgA. Both IgG and IgA titers against the cells (113–20) and the purified IgA protease in the sera of BD patients and healthy controls, 36 each, were assayed. The IgG titers against the cells and protease were not significant in the BD patients or controls, but the IgA titers against the cells and protease in the BD patients were significantly higher than those of the controls. These data indicate that the BD patients are infected with IgA protease‐producing S. sanguis strains, which cause an increase of IgA titer against these organisms and IgA protease antigen. Since the organisms can proliferate in BD patients for a long period of time (years), it seems that IgA antibodies cannot effectively eliminate the organisms.
Mechanisms of Allografted Tumor Rejection: The Roles of T Cells in Allograft Rejection Mediated by a Type of Bone Marrow‐Derived Macrophage
Authors: Ushio‐Umeda Yumiko , Yoshida Ryotaro
Published: November 2013

Allografted tumor rejection does not occur in the absence of T cells, but the main effector cells responsible for the rejection are allograft‐induced macrophages (AIM). We examined the roles of T cells in the AIM‐mediated rejection of Meth A (H‐2) tumor cells from C57BL/6 (H‐2b) mice. Irradiation of C57BL/6 mice abrogated both the induction of AIM and the allograft rejection. Reconstitution of the irradiated mice with F1 (C57BL/6 X C3H/He: H‐2b/k) bone marrow cells led to the appearance of H‐2b/k haplo‐type of AIM exclusively in the rejection site and to allograft rejection, indicating that radiosensitive cells prerequisite for both the induction of AIM and allograft rejection were bone marrow‐derived cells, and that the progenitors of AIM existed in the bone marrow cells to be activated into AIM in the rejection site. To understand the role of T cells in the induction of AIM, we used adult‐thymectomized, X‐irradiated C57BL/6 mice reconstituted with F1 bone marrow (ATXBM). The ATXBM mice could neither induce AIM nor reject allogeneic Meth A cells, whereas adoptive transfer of F1 lymph node T cells to the ATXBM mice restored not only the induction of AIM but also rejection of the allograft. Among the lymph node T cells, CD4+, but not CD8+, cells were found to be essential for the activation of AIM progenitors to AIM; and CD8+ T cells were further required for rejection, at least in part, to enhance the number of AIM in the rejection site.
The Inhibitory Effect of Staphylococcus epidermidis Slime on the Phagocytosis of Murine Peritoneal Macrophages Is Interferon‐Independent
Authors: Shiau Ai‐li , Wu Chao‐liang
Published: November 2013

The extracellular slime produced by Staphylococcus epidermidis has been shown to interfere with several human neutrophil functions in vitro, such as chemotaxis, degranulation and phagocytosis. Slime production has been suggested as a useful marker for clinically significant infections with coagulase‐negative Staphylococcus. Since the main role of macrophages in defense mechanisms is phagocytosis, the effect of slime on the phagocytic activity of macrophages was investigated. The phagocytic activity of murine peritoneal macrophages treated with slime in vitro decreased in a dose‐dependent fashion. A similar decrease was also observed in macrophages isolated from mice that had previously received intraperitoneal injection of slime. To investigate whether interferon also plays a role in this process, mice were treated with interferon or an interferon inducer, polyinosinic‐polycytidylic acid (poly I:C), together with slime before macrophage isolation. The slime‐suppressed phagocytic activity of macrophages was partially relieved by both agents, and the recovery effect of poly I:C in slime‐suppressed phagocytosis of macrophages in vivo might be attributed to the increased interferon level in peritoneal fluid and sera. However, when slime was given to poly I:C‐pretreated mice, the phagocytic activity remained suppressed. Thus, it appears that slime is able to suppress the phagocytic activity of macrophages regardless of the state of macrophage activation by poly I:C. The results suggest that the inhibition of phagocytosis by S. epidermidis slime may be independent from the activation of interferon.
Development of Reference Procedures for Broth Microdilution Antifungal Susceptibility Testing of Yeasts with Standardized Endpoint Determination
Authors: Makimura Koichi , Sudo Takako , Kudo Michinari , Uchida Katsuhisa , Yamaguchi Hideyo
Published: November 2013

Standard guidelines for the broth microdilution antifungal susceptibility testing of amphotericin B, flucytosine, fluconazole, miconazole and itraconazole are reported. These are a modification of the method developed by the National Committee for Clinical Laboratory Standards (NCCLS) on the following two points: standardization of the means of endpoint determination and the inclusion of miconazole and itraconazole in the testing. MIC was determined to be when the positive control had a turbidity of 0.2 at the 630 nm wavelength. The endpoint was 80% inhibition for azoles and 100% inhibition for other drugs. The method provided good reproducibility, and a wide range of MIC distribution was observed in all antifungal agents except amphotericin B.
Elevated Serum Levels of the Soluble Form of gp130, the IL‐6 Signal Transducer, in HTLV‐1 Infection and No Involvement of Alternative Splicing for Its Generation
Authors: Zhou Yongwei , Horiuchi Sankichi , Yamamoto Mikio , Yamamoto Naoki
Published: November 2013

By using an ELISA, increased levels of the soluble form (sgp130) of gp130, the IL‐6 signal transducer, were detected in the sera of various HTLV‐1‐associated conditions (HC, ATL, HAM) as compared to normal healthy individuals. Sgp130 levels seemed to be correlated with disease severity. The 94 KD of sgp130 was specifically precipitated in the sera of HTLV‐1‐infected patients as revealed by Western blot analysis. A reverse transcriptase (RT)‐polymerase chain reaction (PCR) method was used to detect the message for transmembrane (TM) lacking gp130 in mRNA isolated from peripheral blood mononuclear cells (PBMCs) of patients infected with or without HTLV‐1 and those of various hematopoietic cell lines. Two PCR products, 648 and 507 bp were observed in the PBMCs from HTLV‐1‐infected patients. But the 507 bp PCR product was not detected in the PBMCs from normal healthy individuals and HTLV‐1‐positive cell lines although the 648 bp product was equally expressed. A nucleotide sequence analysis of the 507 bp fragment showed deletion of the 141 bp at the region spanning from nucleotide 1702 (G) to 1842 (T) of the 648 bp product that matched completely with a conventional gp130 molecule. This deleted region was located upstream of the transmembrane (TM) domain, but not within the TM region itself. However, no frame shift was observed. These results indicate that the generation of sgp130 may not be due to an alternative splicing mechanism.
Infectivity and Arthritis Induction of Borrelia japonica on SCID Mice and Immune Competent Mice: Possible Role of Galactosylceramide Binding Activity on Initiation of Infection
Authors: Kaneda Kazuhide , Masuzawa Toshiyuki , Simon Markus M. , Isogai Emiko , Isogai Hiroshi , Yasugami Kitsuzo , Suzuki Takashi , Suzuki Yasuo , Yanagihara Yasutake
Published: November 2013

We investigated the relationship between the binding activity to galactosylceramide (GalCer) and the arthritis induction activity of Borrelia japonica. The B. japonica strains maintained the ability to induce arthritis in inbred C3H/HeN and immunodeficient SCID mice, but the ability was lower than that of Borrelia burgdorferi sensu stricto virulent strain 297. Histopathological changes were restricted to the joints, and a marked effusion of polymorphonuclear neutrophils into the joint space was found. The binding activity of B. japonica strains to GalCer was lower than that of the virulent strain 297 but higher than that of the high‐passage strain 297. The lower infectivity and virulence of B. japonica may explain its lower binding ability to GalCer.
Kinetical Analysis of Tumor Cell Death‐Inducing Mechanism by Polymorphonuclear Leukocyte‐Derived Calprotectin: Involvement of Protein Synthesis and Generation of Reactive Oxygen Species in Target Cells
Authors: Mikami Masaaki , Yamazaki Masatoshi , Yui Satoru
Published: November 2013

We have previously shown that calprotectin, the most abundant cytosolic protein existing in polymorphonuclear leukocytes (PMNs), induces apoptotic cell death in various tumor cells, suggesting that calprotectin is an effector molecule against tumor cells in PMNs. To explore the cell death‐inducing mechanism of the factor, we examined the involvement of target protein synthesis and generation of reactive oxygen species (ROS) in the reaction. Calprotectin induced cell death in MM46 mouse mammary carcinoma cells after a 14‐16 hr lag time. When the factor was removed from the medium up to about 12 hr after culturing, the effect was diminished. The induction of cell death by calprotectin was markedly inhibited by the presence of the RNA synthesis inhibitor actinomycin D or the protein synthesis inhibitor cycloheximide. However, the addition of these inhibitors after 12 hr of culturing was unable to inhibit the reaction. Up to 12 hr of culturing, the net protein synthesis of MM46 cells was augmented by the presence of calprotectin, but thereafter was impaired. The induction of cell death was also inhibited by the antioxidative reagents N‐acetyl‐l‐cysteine (NAC) or propyl gallate. The addition of NAC even 15 hr later significantly attenuated the calprotectin effect. Flow cytometry analysis showed that calprotectin began to increase the ROS content in MM46 cells after 8‐12 hr of culturing, and that the increase was abrogated by the antioxidants. Thus, protein synthesis and ROS generation may be essential elements in the early or later phases of the cell death‐inducing reaction of calprotectin, respectively.
Zymographic Characterization of Staphylococcus aureus Cell Wall
Authors: Ohta Kouji , Komatsuzawa Hitoshi , Sugai Motoyuki , Suginaka Hidekazu
Published: November 2013

Susceptibilities of several preparations of Staphylococcus aureus cells to various peptidoglycan hydrolases with known bond specificity were analyzed by zymography. The substrates were intact S. aureus cells, cells boiled in the presence of SDS and cells treated with trichloroacetic acid after treatment with boiling SDS solution (TCA‐cells). Twofold dilutions of lysostaphin (LS), lysozyme (LZ), S. aureus 51 kDa glucosaminidase (GL) or S. aureus 62 kDa amidase (AM) were electrophoresed, and the minimal enzyme dose showing a visible bacteriolytic band was defined as MBD (minimal bacteriolytic dose). Under the same experimental conditions, this method gave reproducible results. As the substrate for zymogram, TCA‐cells were the most sensitive to LS, LZ and AM, whereas the three substrate were equally sensitive to GL. A zymographic analysis of methicillin‐resistant S. aureus treated with methicillin together with previous studies suggest that this method can be used for the preliminary characterization of S. aureus cell wall peptidoglycan.
Molecular Characterization of Low‐Molecular‐Weight Component Protein, Flp, in Actinobacillus actinomycetemcomitans Fimbriae
Authors: Inoue Tetsuyoshi , Tanimoto Ichiro , Ohta Hiroyuki , Kato Keijiro , Murayama Yoji , Fukui Kazuhiro
Published: November 2013

Fimbriae preparation from Actinobacillus actinomycetemcomitans was found to contain an abundant low‐molecular‐weight protein (termed Flp) with an apparent molecular mass of approximately 6.5 kDa, in addition to a small amount of 54‐kDa protein. Immunogold electron microscopy localized the Flp protein at the bacterial fimbriae but not at the cell surface. The DNA fragment including the flp gene was cloned from A. actinomycetemcomitans 304‐a and its nucleotide sequence was determined. An open reading frame of the flp gene was composed of 225 bp encoding a protein of 75 amino acids. Comparison of the translated amino acid sequence with the sequence of native Flp determined by Edman degradation indicated that the N‐terminal part of 26 amino acids is leader peptide. The N‐terminal sequence of mature Flp exhibited some similarity to type‐IV pilin. Furthermore, the processing site of premature Flp is also similar to that of type‐IV prepilin, and a gene encoding a protein homologous to type‐IV prepilin‐like protein leader peptidase was found downstream of the flp gene. These findings indicate that Flp is the major component protein of A. actinomycetemcomitans fimbriae.
Epidemiologic Study of Shigella sonnei from Sequential Outbreaks and Sporadic Cases Using Different Typing Techniques
Authors: Matsumoto Masakado , Suzuki Yasumoto , Saito Makoto , Ishikawa Naohisa , Ohta Michio
Published: November 2013

We noted that eight outbreaks of Shigella sonnei from an unknown source occurred sequentially in Aichi Prefecture, Japan, between October 1992‐June 1993. For comparative purposes we analyzed 53 outbreak‐related isolates of Shigella sonnei using different subtyping methods and studied the epidemiology of the outbreaks. It appeared from our study that DNA‐based techniques such as plasmid typing and pulsed‐field gel electrophoresis (PFGE) were more useful tools for subtyping Shigella sonnei than colicin typing and the antimicrobial susceptibility test. Moreover, according to PFGE analysis, four genetically related isolates of Shigella sonnei were responsible for the eight sequential outbreaks. To further investigate the epidemiology of outbreaks, 58 sporadic isolates of Shigella sonnei from overseas travelers with shigellosis during the same period were also examined. We found that some sporadic isolates from travelers in Asia were genetically related to those of the outbreak‐related isolates, indicating that genetically related isolates prevailed in Asia during this period, probably because of the extensive movement of people or food.
Studies of Escherichia coli Cultured on RainbowTM Agar O157 with Particular Reference to Enterohaemorrhagic Escherichia coli (EHEC)
Authors: Bettelheim Karl A.
Published: November 2013

RainbowTM Agar O157 is designed for the rapid isolation and identification of enterohaemorrhagic Escherichia coli (EHEC), particularly O157, characterised by black colonies. Five‐hundred‐eighty‐five E. coli strains, including O157, O111 and O113 serogroups from many sources were examined on RainbowTM Agar O157. EHEC O157 could readily be isolated and recognized uniquely by typical black colonies. Some other EHEC also stand out as blue‐black, whereas O113 and some other EHEC strains were mauve, red or pink and indistinguishable from SLT‐negative strains of E. coli.
Diagnostic Value of the Amplicor PCR Assay for Initial Diagnosis and Assessment of Treatment Response for Pulmonary Tuberculosis
Authors: Iinuma Yoshitsugu , Ichiyama Satoshi , Yamori Sadaaki , Oohama Jinya , Takagi Norio , Hasegawa Yoshinori , Shimokata Kaoru , Nakashima Nobuo
Published: November 2013

We evaluated the Amplicor PCR assay as an initial diagnostic tool on the basis of clinical diagnosis, and assessed this assay as a follow‐up test for patients with pulmonary tuberculosis during chemotherapy. Of the 208 specimens from 155 patients who were bacteriologically and/or clinically diagnosed with active tuberculosis before chemotherapy, 144 were Amplicor PCR‐positive (sensitivity, 69.2%), which was equal to the results of culturing. Among 89 specimens which showed positive results by smear and culturing, the Amplicor PCR assay detected 87 (97.8%), whereas among 55 specimens which showed smear‐negative but culture‐positive results, the Amplicor PCR assay detected 46 (83.6%)(P = 0.003). No false positive results were found in the two systems (specificity, 100%, 120/120). The Amplicor PCR assay was also evaluated as a follow‐up test using 926 specimens from 207 patients receiving active tuberculosis chemotherapy. Among 433 specimens which showed Amplicor‐PCR positive, 222 (51.3%) were culture‐negative. On the other hand, among 233 culture‐positive specimens, only 12 (5.2%) were Amplicor PCR‐negative. Therefore, this assay is useful for the rapid diagnosis of tuberculosis. The duration of Amplicor PCR‐positive after culture‐negative conversion was significantly associated with the presence of cavitary lesion, smear‐positive specimens before treatment, and smear‐positive specimens with negative cultures during chemotherapy.
The 21‐kDa Polypeptide (VAP21) in the Rabies Virion Is a CD99‐Related Host Cell Protein
Authors: Sagara Junji , Tochikura Tadafumi S. , Tanaka Hajime , Baba Yoshihiro , Tsukita Shoichiro , Tsukita Sachiko , Kawai Akihiko
Published: November 2013

In our monoclonal antibody (MAb) stocks prepared against the BHK‐21 cell antigens, two (#11875 and 28276) recognized a 21‐kDa polypeptide (referred to as VAP21) which is efficiently incorporated into the rabies virion. By using these MAbs, we isolated the cDNA clones that encoded a polypeptide of 144 amino acids from our BHK‐21 cell cDNA library. Based on the following evidence, the cDNA was assumed to encode a full‐length sequence of VAP21 antigen: i) expression of the cDNA in animal cells resulted in the production of a polypeptide recognized by the two MAbs, and its electrophoretic mobility was the same as that of authentic VAP21 antigen; and ii) immunization with the products from the cDNA‐transformed E. coli cells raised specific antibodies in rabbits that recognized a 21‐kDa polypeptide in the virion. From the deduced amino acid sequence, it is suggested that the VAP21 antigen has a molecular structure of type‐I transmembrane protein containing characteristic proline‐rich and glycine‐rich regions in its ectodomain. Homology searches resulted in finding homologous sequences (totally about 40% homology) in the human MIC2 gene product (CD99; 32‐kDa) of T lymphocytes. These results suggest that the VAP21 antigen in the rabies virion is a cellular CD99‐related transmembrane protein.
Cellular Reaction to Mycobacterium avium Complex (MAC) Clinical Isolates Differing in Hemolytic Activity and Virulence for C57BL/6 Mice
Authors: Brzychcy Marzena , Zwolska Zofia , Andrzejczyk Zofia , Rudnicka Wiesława
Published: November 2013

In this study we showed that Mycobacterium avium complex (MAC) clinical isolates differed by the expression of hemolytic activity. Two hemolytic MAC strains were less susceptible to the mycobactericidal effect of murine macrophages than two unhemolytic MAC isolates. In vivo, hemolytic MAC bacilli survived in the spleens of infected mice for a longer time than unhemolytic MAC strains. This suggested a role of hemolysins in the virulence of MAC strains. There was no difference in the cytotoxicity of T cells from mice immunized with M. bovis BCG towards macrophages infected in vitro with MAC strains expressing or not expressing hemolytic activity.
Morphological Alterations of Saccharomyces cerevisiae Induced by Benanomicin A, an Antifungal Antibiotic with Mannan Affinity
Authors: Watanabe Maki , Nishiyama Yayoi , Inouye Shigeharu , Yamaguchi Hideyo , Kondo Shinichi , Takeuchi Tomio
Published: November 2013

The effects of benanomicin A, a mannose‐binding antifungal antibiotic, on yeast cells of Saccharomyces cerevisiae were studied by electron microscopy. Cytological studies using vital stain with methylene blue demonstrated that benanomicin A at 20 and 80 μg/ml killed buds in preference to parent cells. In confirmation, examination by TEM revealed that benanomicin A at 80 μg/ml damaged buds more severely than parent cells. The major effect on the ultrastructure was characterized by severe damage to the cell membrane. In addition, it caused expansion and vacuolation of the endoplasmic reticulum (ER), and partial fragmentation and disappearance of nuclear membranes. The membrane‐disruptive activity of benanomicin A may be closely associated with its membrane affinity.
Serological Differentiation between HCV Subtypes 1a and 1b by a Recombinant Immunoblot Assay
Authors: Schröter Matthias , Feucht Heinz‐hubert , Zöllner Bernhard , Laufs Rainer
Published: November 2013

Until now, no serological assay has been available for the differentiation of HCV subtypes. Since there is evidence that the subtypes differently influence the clinical course of HCV infection and the outcome of interferon therapy, we established a strip immunoblot assay (NS‐4 IBA) with recombinant HCV proteins of the nonstructural 4 (NS‐4) region propagated in Escherichia coli. Using this NS‐4 IBA, we were able to distinguish HCV subtypes la and 1b, which are the most prevalent subtypes in Europe and the U.S.A. The results of the serotyping assay were compared with those obtained by nucleotide sequencing from the NS‐5 region. Concordant results were observed to match 94.9% (n = 100) by the NS‐4 IBA and nucleotide sequencing. Discrepant results were obtained in only 5.1% (n = 6). These data indicate that HCV subtypes can be serologically distinguished, providing the possibility for easier identification of infection with different KCV subtypes.