bluuebunny/crossref_metadata_2025 huggingface dataset:
Anti‐Fibrin Antibody Binding in Valvular Vegetations and Kidney Lesions during Experimental Endocarditis
Authors: Yokota Mitsumasa , Basi David L. , Herzberg Mark C. , Meyer Maurice W.
Published: November 2013

In Streptococcus sanguinis (sanguis) induced experimental endocarditis, we sought evidence that the development of aortic valvular vegetation depends on the availability of fibrin. Endocarditis was induced in New Zealand white rabbits by catheter placement into the left ventricle and inoculation of the bacteria. Fibrin was localized in the developing vegetation with 99mTechnetium (Tc)‐labeled anti‐fibrin antibody one or three days later. When rabbit anti‐fibrin antibody was given intravenously on day 1, the mass of aortic valvular vegetation was significantly reduced at day 3; infusion of non‐specific rabbit IgG showed no effect. The 99mTc‐labeled anti‐fibrin antibody also labeled kidneys that showed macroscopic subcapsular hemorrhage. To learn if the deposition of fibrin in the kidneys was a consequence of endocarditis required a comparison of farm‐bred and specific pathogen‐free rabbits before and after the induction of endocarditis. Before induction, the kidneys of farm‐bred rabbits were labeled, but specific pathogen‐free rabbits were free of labeling and signs of macroscopic hemorrhage. After 3 days of endocarditis, kidneys of 10 of 14 specific pathogen‐free rabbits labeled with 99mTc‐labeled anti‐fibrin antibody and showed hemorrhage. Kidney lesions were suggested to be a frequent sequellae of S. sanguinis infective endocarditis. For the first time, fibrin was shown to be required for the continued development of aortic valvular vegetations.
Mycobacterium leprae and Leprosy: A Compendium
Authors: Sasaki Shin , Takeshita Fumihiko , Okuda Kenji , Ishii Norihisa
Published: November 2013

Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which was discovered by G.H.A. Hansen in 1873. M. leprae is an exceptional bacterium because of its long generation time and no growth in artificial media. Entire sequencing of the bacterial genome revealed numerous pseudogenes (inactive reading frames with functional counterparts in M. tuberculosis) which might be responsible for the very limited metabolic activity of M. leprae. The clinical demonstration of the disease is determined by the quality of host immune response. Th1‐type immune response helps to kill the bacteria, but hosts are encroached upon when Th2‐type response is predominant. The bacteria have affinity to the peripheral nerves and are likely to cause neuropathy. M. leprae/laminin‐α2 complexes bind to α/β dystroglycan complexes expressed on the Schwann cell surface. WHO recommends a chemotherapy protocol [multidrug therapy (MDT)] which effectively controls the disease and contributes to the global elimination program. Leprosy has been stigmatized throughout history, and recent topics regarding the disease in Japan are also discussed.
Immunological Properties of Trehalose Dimycolate (Cord Factor) and Other Mycotic Acid‐Containing Glycolipids–‐A Review
Authors: Ryll Roland , Kumazawa Yoshio , Yano Ikuya
Published: November 2013

Mycolic acids are characteristic fatty acids of Mycobacteria and are responsible for the wax‐like consistence of these microorganisms. Decades of research revealed that mycolic acid‐containing glycolipids, in particular trehalose‐6,6′‐dimycolate (TDM, cord factor) as their best‐studied representative, exert a number of immunomodifying effects. They are able to stimulate innate, early adaptive and both humoral and cellular adaptive immunity. Most functions can be associated with their ability to induce a wide range of chemokines (MCP‐1, MIP‐1α, IL‐8) and cytokines (e.g., EL‐12, IFN‐γ, TNF‐α, IL‐4, IL‐6, IL‐10). This review tries to link well‐known properties of mycolic acid‐containing glycolipids, e.g., stimulation of cellular and humoral immunity, granuloma formation and anti‐tumor activity, with recent findings in molecular immunology and to give an outlook on potential practical applications.
Crossreactivity of SLE Autoantibodies with 70 kDa Heat Shock Proteins of Mycobacterium tuberculosis
Authors: Tasneem Subia , Islam Najmul , Ali Rashid
Published: November 2013

Heat shock proteins (hsp) may be involved in the initiation and perpetuation of autoimmune diseases. In order to investigate the possible role of hsp and other intracellular proteins of Mycobacterium tuberculosis in the autoantibody production in SLE, the immuno‐crossreactivity of SLE autoantibodies with Mycobacterium tuberculosis sonic extract and hsp‐70 kDa was investigated. These proteins showed significant binding with Protein A‐Sepharose isolated SLE IgG. Western blotting of hsp‐70 with SLE IgG showed strong recognition, suggesting possible involvement of hsp and other intracellular proteins of Mycobacterium tuberculosis in the autoantibody induction in SLE.
Adhesion of Bifidobacterium Spp. to Human Intestinal Mucus
Authors: He Fang , Ouwehand Arthur C. , Hashimoto Hideo , Isolauri Erika , Benno Yoshimi , Salminen Seppo
Published: November 2013

Twenty‐four Bifidobacterium strains were examined for their ability to bind to immobilized human and bovine intestinal mucus glycoproteins. Each of the tested bacteria exhibited its characteristic adhesion to human and bovine fecal mucus. No significant differences were found among the taxonomic species. Among the tested bacteria, B. adolescentis, B. angulation, B. bifidum, B. breve, B. catenulatum, B. infantis, B. longum and B. pseudocatenulatum adhered to human fecal mucus better than bovine fecal mucus, while the binding of B. animalis and B. lactis was not preferential. These results suggest that the mucosal adhesive properties of bifidobacteria may be a strain dependent feature, and the mucosal binding of the human bifidobacteria may be more host specific.
Intraspecies Diversity of Cryptococcus albidus Isolated from Humans as Revealed by Sequences of the Internal Transcribed Spacer Regions
Authors: Sugita Takashi , Takashima Masako , Ikeda Reiko , Nakase Takashi , Shinoda Takako
Published: November 2013

The basidiomycetous yeast, Cryptococcus albidus, shows intraspecies diversity, but it is rarely isolated from immunocompromised patients. Nineteen strains of C. albidus, including nine clinical isolates, were re‐classified by sequences of their rRNA internal transcribed spacer (ITS) regions. The nine clinical isolates were genetically diverse and included both C. albidus and C. diffluens. One clinical isolate, recovered from the blood of an AIDS patient, represented a new species. Only small differences were found in the biochemical and serological characteristics of C. albidus and C. diffluens. All isolates were sensitive to amphotericin B, but several isolates were resistant to fluconazole and itraconazole. C. albidus heterogeneity should be taken into consideration when identifying clinical isolates.
Menadione‐Catalyzed O2– Production by Escherichia coli Cells: Application of Rapid Chemiluminescent Assay to Antimicrobial Susceptibility Testing
Authors: Yamashoji Shiro , Manome Isao , Ikedo Masanari
Published: November 2013

This study proposes a novel chemiluminescent assay of bacterial activity. Luminol chemiluminescence (LC) was amplified on addition of menadione to Escherichia coli suspension, and it was effectively inhibited by addition of superoxide dismutase rather than catalase. This fact suggests that H2O2 produced from O2– by superoxide dismutase is decomposed by catalase of E. coli. NAD(P)H:menadione reductase activities in periplasm and cytosol corresponded to the amplification of menadione‐catalyzed LC, and outer and cytoplasmic membranes were only slightly involved in the LC. The total activity and Vmax of NAD(P)H:menadione reductase in the cytoplasm were greater than those in the periplasm. A transient increase in menadione‐catalyzed LC was observed in the exponential phase and the LC decreased in the stationary phase during growth of E. coll Menadione‐catalyzed LC was sensitive to antibiotic action. A decrease in menadione‐catalyzed LC by the impairment of membrane functions and by the inhibition of protein synthesis was observed at 5 min and 3 hr, respectively. These findings suggest the possibility that menadione‐catalyzed luminol chemiluminescent assay is applicable to rapid antimicrobial assay because LC is sensitive to the change in growth and cytotoxic events caused by antimicrobial agents.
flaB‐Polymerase Chain Reaction (flaB‐PCR) and Its Restriction Fragment Length Polymorphism (RFLP) Analysis Are an Efficient Tool for Detection and Identification of Leptospira Spp.
Authors: Kawabata Hiroki , Dancel Louella A. , Villanueva Sharon Y.A.M. , Yanagihara Yasutake , Koizumi Nobuo , Watanabe Haruo
Published: November 2013

For establishment of a rapid‐identification method of Leptospira species, aflaB gene of Leptospira was investigated and the following results were obtained. 1) HaeIII‐ or HindIII‐restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) products (793 bp) of flaB gene was effectual for the classification of species of Leptospira. 2) Twenty cells of Leptospira in 1 ml of coagulated blood and 100 cells of Leptospira in 1 ml of anti‐coagulated blood could be detected by flaB‐PCR. These results suggested that PCR‐RFLP based on the flaB gene was an efficient tool for rapid detection and identification of species of infected Leptospira from clinical specimens.
Effects of Yersinia enterocolitica O:3 Derivatives on B Lymphocyte Activation In Vivo
Authors: de Moraes Costa Crespo Adriana , Falcão Deise Pasetto , de Araújo Paulo Maria Ferreira , de Medeiros Beatriz Maria Machado
Published: November 2013

The potential sequelae of intestinal infection with Yersinia enterocolitica include reactive arthritis, erythema nodosum, Reiter's syndrome and other autoimmune diseases. The role of the immune response in the pathogenesis of these diseases has not been fully defined, but autoimmune manifestations may be a consequence of the increase in autoantibodies as a result of polyclonal B‐cell activation induced by Yersinia. We investigated the effects of Y. enterocolitica O:3 derivatives on B lymphocyte activation in vivo. Groups of five specific pathogen free (SPF) Swiss mice were inoculated with bacterial cell extract, Yersinia outermembrane proteins (Yops) or lipopolysaccharide (LPS) obtained from Y. enterocolitica O:3 and their immunoglobulin‐secreting spleen cells were detected by isotype‐specific protein A plaque assay. The presence of specific anti‐Yersinia antibodies and autoantibodies was determined in mouse sera by ELISA. In all experiments a marked increase in the number of secretory cells of different isotypes was observed as early as the third day after inoculation. IgG and IgM anti‐Yersinia antibodies were detected in the sera of all inoculated mice, and autoantibodies against myosin in the sera of those inoculated with bacterial cell extract. The sera from animals stimulated with LPS reacted with myelin, actin and laminin, while the sera from mice inoculated with Yops reacted with myelin, thyroglobulin and cardiolipin. These results suggest that SPF Swiss mice inoculated with any one of the Y. enterocolitica derivatives tested exhibited polyclonal activation of B lymphocytes as a result of stimulation by various bacterial components and not only LPS stimulation.
Suppressed Proliferative Response of Spleen T Cells from Metallothionein Null Mice
Authors: Mita Masaharu , Imura Nobumasa , Kumazawa Yoshio , Himeno Seiichiro
Published: November 2013

To investigate the role of metal‐binding protein, metallothionein (MT), in lymphocyte activation, the mitogen‐induced proliferation of freshly isolated spleen cells was compared among MT‐I, II null, and control 129/Sv mice. Spleen cells from MT null mice exhibited a markedly reduced proliferation compared with control cells when stimulated by concanavalin A or anti‐CD3(∈) mAb, but not by lipopolysaccharide, indicating that only the response of T cells to mitogens was suppressed in MT null mice. Flow cytometric analysis of unstimulated spleen cells demonstrated no significant difference in the relative percentages of either B220+ and CD3+ cells or CD4+ and CD8+ cells between the two strains of mice. The production of interleukin (IL)‐2 by MT null spleen cells after the stimulation by anti‐CD3(∈) mAb was lower than that of control spleen cells, especially within 24 hr after the stimulation. The addition of IL‐2 recovered the proliferation of MT null spleen cells to the control level. The reduced proliferative response to mitogenic stimulation of MT null T cells was confirmed by using purified splenic T cells. These results suggest that the MT expressed at basal level in the splenocytes plays an important role in T cell mitogen‐induced proliferative response, probably by positively regulating the production of IL‐2.
Promoted Cell Death of Cells Expressing Human MxA by Influenza Virus Infection
Authors: Mibayashi Masaki , Nakade Koji , Nagata Kyosuke
Published: November 2013

Interferon‐inducible MxA protein plays a crucial role in cellular protection from RNA virus infection, although the protection mechanism is not completely clarified. Here, we examined effects of MxA on either uninfected or influenza virus A/PR/8/34‐infected cells. Viral protein synthesis was reduced in cells expressing MxA. Under serum‐starved conditions, not only viral but also cellular protein synthesis was reduced by expression of MxA. Of interest is that MxA promoted cell death induced by apoptotic stimuli as well as influenza virus infection. These results lead to a possibility that MxA suppresses multiplication of influenza virus by affecting cellular functions including the apoptotic pathway.
The 44‐kb Linear Plasmid Molecule in the Relapsing Fever Agent Borrelia duttonii Strain Ly Serve as a Preservation of vmp Genes
Authors: Tabuchi Norihiko , Mitani Harumi , Seino Satoshi , Fukunaga Masahito
Published: November 2013

Borrelia duttonii strain Ly, a causative agent of relapsing fever, contains a linear one megabase chromosome and 12 linear plasmid molecules. Here we report that the sequence of the 44‐kb linear plasmid of strain Ly is found to contain variable major protein (vmp) genes for antigenic variation of relapsing fever borreliae. The determined sequence is of 44,010 bp except for both ends of the molecule. Of 39 open reading frames (ORFs) found in the sequence, 21 ORFs (named vmpA to U) showed moderate similarities with vmp genes for Borrelia hermsii. However, most of the vmp homologues are apparently nonfunctional because of their frameshifts within the sequence and/or absence of promoter and ribosome‐binding signals upstream of their genes. RT‐PCR experiments using the specific primer for each vmp gene revealed that vmpE, one of the vmp genes, was expressed at the location of the 44‐kb plasmid molecule. The result suggests that the plasmid molecule may play a role in the preservation of the serotype switching of vmp genes in a mammalian host.
Anti‐CD11b Monoclonal Antibody Suppresses Brain Dissemination of Cryptococcus neoformans in Mice
Authors: Kawakami Kazuyoshi , Koguchi Yoshinobu , Qureshi Mahboob Hossain , Zhang Tiantuo , Kinjo Yuki , Yara Satomi , Uezu Kaori , Shibuya Kazutoshi , Naoe Shiro , Saito Atsushi
Published: November 2013

To elucidate the role of the β2 integrin family of adhesion molecules in the disseminated infection of Cryptococcus neoformans from the lung to the central nervous system, we examined the effects of monoclonal antibodies (mAbs) against CD11a, CD11b, CD11c and CD18 on the number of live microorganisms in both the lung and brain of mice three weeks after intratracheal infection. Administration of anti‐CD11b mAb partially, but reproducibly, reduced the fungal loads in the brain in three independent experiments, while the lung loads were not affected. In addition, the same treatment significantly decreased the number of live microorganisms in the blood. In sharp contrast, the brain loads one week after intravenous injection with C. neoformans were not affected by treatment with anti‐CD11b mAb. Finally, administration of mAb against other adhesion molecules (CD11a, CD11c or CD18) failed to affect the fungal loads in the brain as well as in the lung three weeks after intratracheal instillation, except for anti‐CD18 mAb which rather increased the brain loads. Our results suggested that CD11b might be involved at least in part in the process of fungal dissemination from lung to brain, although the significance of other β2 integrin family adhesion molecules remains to be substantiated.
Purification of a Serine Protease of Vibrio parahaemolyticus and Its Characterization
Authors: Ishihara Masami , Kawanishi Ayako , Watanabe Hirofumi , Tomochika Ken‐ichi , Miyoshi Shin‐ichi , Shinoda Sumio
Published: November 2013

A 50 kDa protease designated as VPP1 was purified from the culture supernatant of a clinical strain of Vibrio parahaemolyticus by ammonium sulfate fractionation, Sephacryl S‐200 HR gel filtration and Fractogel EMD TMAE 650 ion‐exchange chromatography. VPP1 was inhibited by EDTA, EGTA and serine protease inhibitors, suggesting that it is a calcium‐dependent serine protease. N‐terminal amino acid sequence of VPP1 was quite similar to that of V. metschnikovii protease and antibody against VPP1 inhibited the activity of V. metschnikovii protease, suggesting the similarity of the two proteases. It was demonstrated that VPP1 or its related protease widely distribute in not only V. parahaemolyticus but also V. alginolyticus.
Development of Antigen Detection ELISA for the Diagnosis of Brugian and Bancroftian Filariasis Using Antibodies to Recombinant Filarial Antigens Bm‐SXP‐1 and Wb‐SXP‐1
Authors: Lalitha Pattabhiraman , Eswaran Devarajan , Gnanasekar Muniratnam , Rao Kakuturu Venkata Nagaraja , Narayanan Rangarajan Badri , Scott Alan , Nutman Thomas , Kaliraj Perumal
Published: November 2013

Antibodies specific to recombinant filarial antigens Wb‐SXP‐1 and Bm‐SXP‐1 have been used to develop a sandwich ELISA for the detection of circulating filarial antigen (CFA) in sera from patients with lymphatic filariasis caused by Wuchereria bancrofti of Brugia malayi. In patients with W. bancrofti infections, a high proportion of microfilaria (mf) positive (MF) and low proportions of patients with chronic pathology (CP) and endemic normals (EN) showed the presence of CFA. Similarly in patients with brugian infections a high proportion of mf positive individuals contained CFA while none of the patients with chronic pathology or endemic normals showed the presence of CFA. Sera from patients with other parasitic infections (OPI) like O. volvulus, Loa loa, Ascaris lumbricoides and from individuals residing in areas non‐endemic to filariasis did not exhibit any reactivity. This assay shows promise for the detection of microfilaremic infections in lymphatic filariasis and its usefulness as a diagnostic tool especially in B. malayi infections, needs to be further evaluated.
Design of Species‐Specific Primers to Identify 13 Species of Clostridium Harbored in Human Intestinal Tracts
Authors: Kikuchi Eisaku , Miyamoto Yukiko , Narushima Seiko , Itoh Kikuji
Published: November 2013

The genus Clostridium is dominant in human intestinal tracts and plays an important role in human health. We designed species‐specific primers to identify 13 species of Clostridium (C. perfringens, C. paraputrificum, C. bifermentans, C. difficile, C. clostridiiforme, C. nexile, C. sphenoides, C. indolis, C. ramosum, C. cocleatum, C. butyricum, C. sordellii, and C. innocuum) easily and rapidly. The PCR annealing temperature was set at a uniform 60 C for application to all strains at the same time. To confirm the specificities of these primers, 85 intestinal bacteria in total, including type strains, reference strains, and isolates were used. Ten primers (including those for C. perfringens to C. cocleatum) indicated high specificities. Although there were some cross‐reactions with the other three primers, the target species were distinguishable from other bacteria by the different sizes of PCR products.
emm Typing of Group A Streptococcus Clinical Isolates: Identification of Dominant Types for Throat and Skin Isolates
Authors: Tanaka Daisuke , Gyobu Yotaku , Kodama Hirohide , Isobe Junko , Hosorogi Shiho , Hiramoto Yuko , Karasawa Tadahiro , Nakamura Shinichi
Published: November 2013

T and emm types were determined for group A streptococci isolated from patients with various infections during 1990–1999 in Toyama Prefecture, Japan. Out of 906 isolates, 872 isolates were divided into 20 T serotypes, and 34 isoltes were T nontypeable (TNT). T12, T1, and T4 were dominant among 699 throat isolates; on the other hand, T11, T28, TB3264, and TNT were dominant among 80 skin isolates. The emm types of 190 isolates were determined following specific PCR amplification and sequencing of the products. Twenty T serotypes were divided into 34 T type/emm type combinations. Thirty‐four TNT isolates were divided into 14 emm types, in which emm58 was the most common (38%). Among 82 throat isolates randomly selected, predominant T types T12, T1, and T4 isolates were of the respective same numbers in emm type. T11/emm89, T28/emm28, TB3264/emm 13w, and TNT/emm58 were predominant among 80 skin isolates. emm‐type distribution observed in the present study was that usually reported in the western world. To our knowledge, 3 T/emm is a novel combination. These results show that emm typing allows the characterization of group A streptococci from various sources.
Antibacterial Effect of Kampo Herbal Formulation Hochu‐Ekki‐To (Bu‐Zhong‐Yi‐Qi‐Tang) on Helicobacter pylori Infection in Mice
Authors: Yan Xiaoqun , Kita Masakazu , Minami Masato , Yamamoto Toshiro , Kuriyama Hiroko , Ohno Tomoyuki , Iwakura Yoichiro , Imanishi Jiro
Published: November 2013

Because Helicobacter pylori (H. pylori) infection is a major cause of gastroduodenal diseases in humans, the eradication of H. pylori using antibiotics is very effective for the treatment of gastroduodenal diseases. However, it has recently been reported that resistance to these antibiotics is developing. In the present study, the antibacterial effect of a Kampo (traditional Japanese medicine) herbal formulation, Hochu‐ekki‐to (HET; Formula repletionis animalis et supletionis medii), against H. pylori was examined in vitro and in vivo. HET inhibited the growth of antibiotic‐resistant strains of H. pylori as well as antibiotic‐sensitive strains at a dose of 2.5 mg/ml in vitro. When 1,000 mg/kg of HET was administered orally to C57BL/6 mice for 7 days before or after inoculation with H. pylori, H. pylori in the stomach was significantly reduced in the HET‐pre‐treatment group compared with the control group. Furthermore, HET in combination with antibiotics completely eradicated the bacteria in mice. The expression of interferon (IFN)‐γ was induced in the gastric mucosa of the mice pre‐treated with HET. There were no significant differences between the colonization of H. pylori in the control and HET treatment groups in IFN‐γ gene‐deficient mice. These results suggest that the antibacterial effect of HET may be partly due to IFN‐γ induction, and that HET may be clinically useful for treatment of H. pylori infection.
Comparison of Four Microbial Enzymes in Clostridia and Bacteroides Isolated from Human Feces
Authors: Nakamura Joe , Kubota Yoshihiko , Miyaoka Masaaki , Saitoh Toshihiko , Mizuno Fumio , Benno Yoshimi
Published: November 2013

The activities of four microbial enzymes (azoreductase, nitroreductase, β‐glucuronidase, and β‐glucosidase) in major anaerobic members of human fecal microflora were quantified and the influence of the host factors on expression of these microbial enzyme activities was also investigated. Clostridium paraputrificum and C. clostridiiforme showed much higher activities than other fecal anaerobes tested. Nitroreductase activity in C. paraputrificum isolated from fecal specimens of patients with colon cancer was significantly (P<0.05) higher than that in the clostridia isolated from healthy subjects and the subjects given high beef diets. However, the activities of some microorganisms tested showed marked differences in each strain.
Effects of Recombinant Cholera Toxin B Subunit on IL‐1β Production by Macrophages In Vitro
Authors: Maeyama Jun‐ichi , Isaka Masanori , Yasuda Yoko , Matano Keiko , Taniguchi Tooru , Morokuma Kazunori , Ohkuma Kunio , Tochikubo Kunio , Goto Norihisa
Published: November 2013

Recombinant cholera toxin B subunit (rCTB) is a safe and potent mucosal adjuvant. As a clue to the mechanism of the adjuvant effect of rCTB, the profile of cytokines secreted in vitro by the mouse peritoneal macrophage (MΦ) treated with rCTB was examined. IL‐1β secretion, intracellular production, and expression of its mRNA of LPS‐stimulated MΦ was greatly enhanced by treatment with rCTB. IL‐1β production in response to other microbial stimulators, such as Pansorbin, Sansorbin, insoluble peptidoglycan, and Taxol, was also potentiated by rCTB. MΦ pretreated with rCTB before 24 hr could maintain the ability to produce a high level of IL‐1β, suggesting that this ability may be involved in the adjuvant activity of rCTB on MΦ stimulation. The possibility of close association between rCTB and signal transduction of a Toll‐like receptor family in MΦ is discussed.
Purification, Cloning and Characterization of Variant LukE‐LukD with Strong Leukocidal Activity of Staphylococcal Bi‐Component Leukotoxin Family
Authors: Morinaga Naoko , Kaihou Yuriko , Noda Masatoshi
Published: November 2013

Staphylococcus aureus produces bi‐component leukotoxins composed of non‐associated soluble proteins, S and F. Neither S nor F component alone is cytotoxic, but components together are active. These include Panton‐Valentine leukocidin (PVL), γ‐hemolysin, LukE‐LukD and others. Purification of leukotoxin from Staphylococcus aureus V8 strain (ATCC 27733) which does not have PVL genes, identified an F component with 100% identical to that of PVL in the first twenty‐five N‐terminal amino acids. Molecular cloning of this toxin obtained 2,595 nucleotides sequences containing two novel open reading frames for S and F. Deduced amino acid sequences of the S and F were respectively 91 and 94% identical to those of LukE and LukD. These were named variant of LukE‐LukD (LukEv‐LukDv). The activity of the recombinant LukEv‐LukDv to rabbit leukocytes was similar to that of recombinant PVL. LukEv‐LukDv was hemolytic to rabbit red blood cells although the activity was only 8% of γ‐hemolysin, but PVL was not. These activities were quite different from the LukE‐LukD which was reported no hemolytic and poorly cytotoxic to leukocytes compared to PVL. The lukEv‐lukDv was found in 87% of clinical isolates of Staphylococcus aureus but lukE‐lukD was not detected. These data demonstrate the existence of variant LukE‐LukD in V8 strain (ATCC 27733).
Epstein‐Barr Virus (EBV) Nuclear Antigen Leader Protein (EBNA‐LP) Forms Complexes with a Cellular Anti‐Apoptosis Protein Bcl‐2 or Its EBV Counterpart BHRF1 through HS1‐Associated Protein X‐1
Authors: Matsuda Go , Nakajima Kaori , Kawaguchi Yasushi , Yamanashi Yuji , Hirai Kanji
Published: November 2013

Epstein‐Barr virus (EBV) nuclear antigen leader protein (EBNA‐LP) plays a critical role in EBV‐induced transformation. An earlier report (Y. Kawaguchi et al., J. Virol. 74: 10104–10111, 2000) showed that EBNA‐LP interacts with a cellular protein HS1‐associated protein X‐1 (HAX‐1). The predicted amino acid sequence of HAX‐1 exhibits similarity to that of another cellular protein Nip3 which has been shown to interact with cellular and viral anti‐apoptotic proteins such as Bcl‐2 and BHRF1, an EBV homolog of Bcl‐2. Here we investigated whether HAX‐1, like Nip3, interacts with Bcl‐2 proteins and report the following. (i) A purified chimeric protein consisting of gluthathione S‐transferase (GST) fused to BHRF1 (GST‐BHRF1) or Bcl‐2 (GST‐Bcl‐2) specifically pulled down HAX‐1 transiently expressed in COS‐7 cells. (ii) GST‐BHRF1 or GST‐Bcl‐2 was not able to pull down EBNA‐LP transiently expressed in COS‐7 cells, whereas each of the GST fusion proteins formed complexes with EBNA‐LP in the presence of HAX‐1. These results indicated that EBNA‐LP interacts with the viral and cellular Bcl‐2 proteins through HAX‐1, suggesting that EBNA‐LP possesses a potential function in the regulation of apoptosis in EBV‐infected cells.
Adhesion and Colonization of Enterohemorrhagic Escherichia coli O157:H7 in Cecum of Mice
Authors: Nagano Keiji , Taguchi Kazuki , Hara Takumi , Yokoyama Shin‐ichiro , Kawada Kenji , Mori Hiroshi
Published: November 2013

Infectious diseases due to enterohemorrhagic Escherichia coli (EHEC) are characterized by diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. The adherence of EHEC on intestinal epithelial cells is a first step for developing these diseases. In the present study, we examined whether EHEC O157: H7 adhere to intestinal epithelial cells of mice and cause F‐actin accumulation in the epithelial cells following the intragastric inoculation of the pathogen. Fecal shedding of the EHEC O157:H7 strain was observed in ICR mice up to 3 weeks. Fecal shedding periods of the type III secretion system‐related gene (espA and sepL) deletion mutants were clearly shorter than that of the wild‐type EHEC O157:H7 strain. The EHEC O157:H7 colonies were found on the epithelial surfaces of the ceca in association with F‐actin accumulation beneath the attached bacteria.
Terminal Restriction Fragment Length Polymorphism Analysis for Human Fecal Microbiota and Its Application for Analysis of Complex Bifidobacterial Communities
Authors: Sakamoto Mitsuo , Hayashi Hidenori , Benno Yoshimi
Published: November 2013

Terminal restriction fragment length polymorphism (T‐RFLP) analysis was used to characterize and compare human fecal microbiota among individuals. T‐RFLP patterns of fecal 16S ribosomal DNA (rDNA) PCR products from three adults revealed host‐specific bacterial communities and were in good agreement with those reported in our previous study. In addition, we applied T‐RFLP analysis for the analysis of complex bifidobacterial communities in human fecal samples. The developed method based on Bifidobacterium genus‐specific PCR and T‐RFLP could identify more than one bifidobacterial species. T‐RFLP patterns of Bifidobacterium genus‐specific PCR products from the fecal samples were host‐specific as well as those of fecal 16S rDNA PCR products. These results were confirmed by PCR‐denaturing gradient gel electrophoresis (DGGE) with primers specific for the genus Bifidobacterium and Bifidobacterium species‐ and group‐specific PCR. Our study demonstrates that T‐RFLP analysis is useful for assessment of the diversity of the human fecal microbiota and rapid comparison of the community structure among individuals, and that the applied method is useful for rapid and sensitive analysis of bifidobacterial community.
The Suppressive Effect of Bifidobacteria on Bacteroides vulgatus, a Putative Pathogenic Microbe in Inflammatory Bowel Disease
Authors: Shiba Tadahiko , Aiba Yuji , Ishikawa Hiroki , Ushiyama Atsushi , Takagi Atsushi , Mine Tetsuya , Koga Yasuhiro
Published: November 2013

Bacteroides, a predominant commensal bacteria in the gut, are thought to be responsible for the development of inflammatory bowel disease (IBD). In the present study, we examined whether or not bifidobacteria suppress B. vulgatus, a representative pathogenic Bacteroides species, in both the coculture system and the gnotobiotic murine model. As a result, Bifidobacterium infantis 1222 highly inhibited the growth of B. vulgatus in the coculture and also significantly suppressed the systemic antibody response raised by B. vulgatus colonizing the gut in gnotobiotic mice. Colonization of the mice by B. vulgatus increased the number of Peyer's patch (PP) cells bearing PNA (peanut agglutinin)+/anti‐κ+ phenotype, which represents plasma cell‐like B cells. Moreover, treatment of those B. vulgatus‐implanted mice with B. infantis 1222 abrogated such increase in the number of PNA+/anti‐κ+ cells. These results thus suggested that B. infantis 1222 protected the gut epithelial layer including the PP from being invaded by Bacteroides, thereby suppressing the systemic antibody response raised by Bacteroides.
Proinflammatory Cytokines in Bovine Colostrum Potentiate the Mitogenic Response of Peripheral Blood Mononuclear Cells from Newborn Calves through IL‐2 and CD25 Expression
Authors: Yamanaka Hitoki , Hagiwara Katsuro , Kirisawa Rikio , Iwai Hiroshi
Published: November 2013

Bovine colostrum contains high concentrations of cytokines, and colostral cytokines are considered to be an important factor in stimulation of maturation of the immune system in newborns. In this study, 5 proinflammatory cytokines (IL‐1β, IL‐6, TNF‐α, IFN‐γ and IL‐1 receptor antagonist, IL‐1ra) present in colostrum were tested for their potential to enhance mitogenic response and to elicit expression of IL‐2 mRNA and CD25 in peripheral blood mononuclear cells (PBMC) from newborn calves before being fed colostrum. PBMC were pretreated with each recombinant bovine cytokine for 2 hr before stimulation with concanavalin A (ConA). Pretreatment of PBMC from newborn calves with IL‐1β, TNF‐α or IFN‐γ significantly enhanced the ConA response, whereas IL‐1ra inhibited the response. The degree of enhancement or inhibition of mitogenic response by these cytokines was more pronounced in PBMC from newborn calves than in those from adult cows. Although IL‐2 mRNA expression in ConA‐stimulated PBMC from newborn calves was weaker than that in those from adult cows of ConA‐stimulated controls, the expression levels became comparable after pretreatment with IL‐1β, TNF‐α or IFN‐γ. The CD25 expression in PBMC from newborn calves was also enhanced by pretreatment with IL‐1β, TNF‐α and IFN‐γ. These results suggest that pretreatment of neonatal PBMC with IL‐1β, TNF‐α or IFN‐γ promotes mitogenic response to ConA through up‐regulating the production of IL‐2 and the expression of the mature IL‐2 receptor.
Structure and Biological Activities of β‐Glucans from Yeast and Mycelial Forms of Candida albicans
Authors: Miura Noriko N. , Adachi Yoshiyuki , Yadomae Toshiro , Tamura Hiroshi , Tanaka Shigenori , Ohno Naohito
Published: November 2013

We have achieved the extraction of cell wall β‐glucan from the mycelial form of Candida albicans (C. albicans) IFO 0579 (M‐CSBG) by using acetic acid, sodium hypochlorite (NaClO), and dimethylsulfoxide (DMSO) treatments. The yield of M‐CSBG was significantly lower (7.5% from dried mycelial cells) than that of the yeast form from C. albicans IFO 1385 (Y‐CSBG, 25.9% from dried yeast cells). The properties of M‐CSBG were similar to those of Y‐CSBG in terms of nuclear magnetic resonance (NMR) spectra and limulus reactivity. Molecular weight (Mw) of M‐CSBG was slightly higher than that of Y‐CSBG. Both Y‐CSBG and M‐CSBG induced the production of comparable amounts of macrophage inflammatory protein‐2 (MIP‐2), a chemotactic factor, from mouse peritoneal exudate cells (PEC) in vitro. These findings suggest that the structure and properties of CSBG from yeast and mycelial cells are similar to each other.
Modulation of Procarboxypeptidase R (ProCPR) Activation by Complementary Peptides to Thrombomodulin
Authors: Shimomura Yasuyo , Kawamura Takeshi , Komura Hidefumi , Campbell William , Okada Noriko , Okada Hidechika
Published: November 2013

We designed complementary peptides (C‐peptides) using a novel computer program (MIMETIC), which generates a series of peptides designed to interact with a target peptide sequence. Carboxypeptidase R (CPR) is an unstable basic carboxypeptidase found in fresh serum in addition to carboxypeptidase N (CPN) which is stable. CPR is generated from its precursor form (proCPR) by trypsinlike enzymes, and its activation is mediated by thrombin generated in the coagulation cascade. The efficiency of activation is enhanced approximately 1,200‐fold when thrombin (T) is bound to thrombomodulin (TM). We attempted to generate C‐peptides which recognize the T‐binding site within TM assuming that some of these might interfere with the generation of T and TM complexes (T‐TM). Among three peptides designed, two inhibited the enhancement in activation of proCPR by T in the presence of TM. One of the peptides at 16 μM reduced the activation of proCPR to the level obtained by T alone.
Identification of the Spotted Fever Group Rickettsiae Detected from Haemaphysalis longicornis in Korea
Authors: Lee Jung‐Hee , Park Hyo‐Soon , Jung Kwang‐Don , Jang Won‐Jong , Koh Seong‐Eun , Kang Shin‐Seok , Lee In‐Yong , Lee Won‐Ja , Kim Bum‐Joon , Kook Yoon‐Hoh , Park Kyung‐Hee , Lee Seung‐Hyun
Published: November 2013

Seven Haemaphysalis ticks were found positive in PCR assay of gltA gene to detect the spotted fever group (SFG) rickettsiae DNA from 100 ticks. The nucleotide sequence of 16S rRNA gene was determined from 5 ticks and compared to those of other Rickettsia strains. The nucleotide sequence from 4 ticks showed high homologies (99.7 to 100%) with that of R. japonica YH, and that from 1 tick (tick no. 48) was identical with that of R. rickettsii R, suggesting that SFG rickettsiae exists in Korea. This is the first documentation of SFG rickettsiae in Korea.
Chemistry and Biology of Angiitis Inducer, Candida albicans Water‐Soluble Mannoprotein‐β‐Glucan Complex (CAWS)
Authors: Ohno Naohito
Published: November 2013

Deep mycoses have been clearly demonstrated to release β‐glucans into the blood. Structure of the β‐glucan was, at least in part, suggested to be a mannoprotein β‐glucan complex (CAWS) as assessed by biochemical and immunochemical analyses of the extracellular macromolecular fraction of Candida albicans. Half clearance time of i.v. administered CAWS was about 30 min in mice. In addition to the reactivity with limulus G‐test, CAWS was found to exhibit various biological activities, such as cytokine synthesis by leukocyte, platelet aggregation, lethal toxicity, enhancement of side effect of indomethacin, induction of coronary arteritis in mice, and so on. In this review, the chemical properties and biological activities of CAWS are discussed.
Molecular Analysis of Fecal Microbiota in Elderly Individuals Using 16S rDNA Library and T‐RFLP
Authors: Hayashi Hidenori , Sakamoto Mitsuo , Kitahara Maki , Benno Yoshimi
Published: November 2013

Fecal microbiota in six elderly individuals were characterized by the 16S rDNA libraries and terminal restriction fragment length polymorphism (T‐RFLP) analysis. Random clones of 16S rRNA gene sequences were isolated after PCR amplification with universal primer sets from total genomic DNA extracted from feces of three elderly individuals. These clones were partially sequenced (about 500 bp). T‐RFLP analysis was performed using 16S rDNA amplified from six subjects. The lengths of the terminal restriction fragment (T‐RF) were analyzed after digestion by HhaI and MspI. Among 240 clones obtained, approximately 46% belonged to 27 known species. About 54% of the other clones were 56 novel “phylotypes” (at least 98% homology of clone sequence). These libraries included 83 species or phylotypes. In addition, about 13% (30 phylotypes) of these phylotypes were newly discovered in these libraries. A large number of species that are not yet known exist in the feces of elderly individuals. 16S rDNA libraries and T‐RFLP analysis revealed that the majority of bacteria were Bacteroides and relatives, Clostridium rRNA cluster IV, IX, Clostridium rRNA subcluster XIVa, and “Gammaproteobacteria”. The proportion of Clostridium rRNA subcluster XIVa was lower than in healthy adults. In addition, although Ruminococcus obeum and its closely related phylotypes were detected in high frequency in healthy young subjects, hardly any were detected in our elderly individuals. “Gammaproteobacteria” were detected at high frequency.
Improved Recovery of Rabies Virus from Cloned cDNA Using a Vaccinia Virus‐Free Reverse Genetics System
Authors: Ito Naoto , Takayama‐Ito Mutsuyo , Yamada Kentaro , Hosokawa Junji , Sugiyama Makoto , Minamoto Nobuyuki
Published: November 2013

To improve efficiency of recovery of rabies virus from cloned cDNA, we established a BHK cell clone that stably expresses T7 RNA polymerase, which we named BHK/T7–9. We also constructed new helper plasmids for expression of nucleoprotein and RNA polymerase of the RC‐HL strain using the pTM1 plasmid vector, which makes the T7 RNA polymerase‐transcripts from the plasmid cap‐independent for translation. After co‐transfection of these helper plasmids and the previously constructed full‐length genome plasmid of the RC‐HL strain to BHK/T7–9 cells, recombinant rabies virus was efficiently recovered from the cloned cDNA.
Monoclonal Antibody #3‐9‐16 Recognizes One of the Two Isoforms of Rabies Virus Matrix Protein That Exposes Its N‐Terminus on the Virion Surface
Authors: Ameyama Satoshi , Toriumi Harufusa , Takahashi Takuo , Shimura Yuji , Nakahara Tomomi , Honda Yoshikazu , Mifune Kumato , Uchiyama Tsuneo , Kawai Akihiko
Published: November 2013

We investigated behaviors of the rabies virus matrix (M) protein using a monoclonal antibody (mAb), #3‐9‐16, that recognized a linear epitope located at the N‐terminus of the protein. Based on the reactivity with this mAb, M proteins could be divided into at least two isoforms; an ordinary major form (Mα) whose 3‐9‐16 epitope is hidden, and an N‐terminal‐exposed epitope‐positive form (Mβ). The Mβ protein accounted for about 25–30% of the total M proteins in the virion, while its content in the cell ranged from 10 to 15% of total M protein. Fluorescent antibody (FA) staining showed that the Mβ antigen distributed in the Golgi area where the colocalized viral glycoprotein antigen was also detected. Mβ antigen was shown to be exposed on the surface of infected cells by both immunoprecipitation and FA staining with the mAb, whereby the cells might have become sensitive to the mAb‐dependent complement‐mediated cytolysis. Similarly, the Mβ antigen was shown to be exposed on the virion surface, and the infectivity of the virus was destroyed by the mAb in the presence of a complement. Together with these results, we think that the M protein molecule takes either of two conformations, one (Mβ) of which exposes the 3‐9‐16 epitope located in the N‐terminal region of the M protein, that are also exposed on the surface of the virion and infected cells, whereby it might play a certain important role(s) in the virus replication process differently from the other form (Mα), probably through its intimate association with the Golgi area and/or the cell membrane.
A Silkworm Larvae Plasma Test for Detecting Peptidoglycan in Cerebrospinal Fluid Is Useful for the Diagnosis of Bacterial Meningitis
Authors: Inada Katsuya , Takahashi Kiyomi , Ichinohe Sadato , Suda Hidetoshi , Tsuchiya Masakazu , Takahashi Junkichi , Matsuura Shuji , Kasai Takeshi , Yoshida Masao , Endo Shigeatsu , Sato Shigehiro
Published: November 2013

The silkworm larvae plasma (SLP) test has been established based on a cascade reaction triggered by either peptidoglycan or (1, 3)‐β‐D‐glucan to produce melanin. We applied this test to the diagnosis of bacterial meningitis. Cerebrospinal fluid (CSF) obtained from patients with bacterial meningitis due to gram‐positive bacteria, gram‐negative bacteria, or fungi, showed positive reactions to the test. In contrast, CSF from patients with viral meningitis or noninfectious illnesses gave negative reactions. Therefore, this test seems to be useful for diagnosis of bacterial and fungal meningitis. When this test was used together with two types of limulus tests, an endotoxin‐specific test, and a conventional test, meningitis was further characterized as gram‐positive, gram‐negative or fungal meningitis. The SLP test requires a computerized instrument for quantitative colorimetric measurement. A qualitative alternative of this test also can be accomplished by visually observing the darkening color. Thus, this method can be applied for simple and rapid diagnosis of meningitis.
Two Different Mechanisms Are Involved in the Extremely High‐Level Benzalkonium Chloride Resistance of a Pseudomonas fluorescens Strain
Authors: Nagai Katsuhiro , Murata Takahiro , Ohta Shin , Zenda Hiroshi , Ohnishi Makoto , Hayashi Tetsuya
Published: November 2013

A Pseudomonas fluorescens strain, PFRB, which we previously isolated as a contaminant in a batch of benzalkonium chloride (BAC) stock solution, exhibits high‐level resistance, not only to BAC, but also to other cationic surfactants belonging to disinfectants classified as quaternary ammonium compounds (QACs). In this study, we analyzed the resistance mechanism of the strain to BAC and other disinfectants. We obtained results suggesting that two different mechanisms, reduced adsorption of BAC to the cell surface and an energy‐dependent mechanism which is most probably an efflux system, were implicated in the high‐level resistance to BAC. Reduced adsorption of BAC is likely due to the decreased negative cell surface charge of the strain. The putative efflux system seems to be unique in that it excretes only a certain range of cationic membrane‐acting disinfectants belonging to QACs.
Peptidase Activity of Dipeptidyl Aminopeptidase IV Produced by Porphyromonas gingivalis Is Important but Not Sufficient for Virulence
Authors: Kumagai Yumi , Yajima Ayako , Konishi Kiyoshi
Published: November 2013

Porphyromonas gingivalis is a pathogen associated with adult periodontitis, which is a chronic inflammatory disease characterized by breakdown of the periodontal tissue. Dipeptidyl aminopeptidase IV (DPPIV) produced by P. gingivalis has been considered to be a potential virulence factor based on the finding that the virulence was reduced by disruption of the gene (dpp) coding for DPPIV. In the present study, we constructed a shuttle vector that is mobilized from Escherichia coli to P. gingivalis and is maintained stably in both bacteria, and we showed that the virulence was restored by introducing the cloned wild‐type dpp gene into the null mutant of P. gingivalis using our vector system. To assess the implications of the peptidase activity in the virulence, mutant DPPIV with the catalytic Ser mutagenized to Ala (DPPSA) was produced. The P. gingivalis strain expressing DPPSA exhibited an intermediate virulence between the strain expressing wild‐type DPPIV and the strain harboring a vector. From these results, it is suggested that peptidase activity is very important but not sufficient for virulence.
Rate of Helicobacter pylori Infection in Children and Clonality of Taiwan Strains
Authors: Chu Chi shih , Yu Yu‐Jen , Kong Man Shan , Ou Jonathan T.
Published: November 2013

The infection rate of Helicobacter pylori in children from <1 to 17 years old was investigated. Three techniques, namely culture, CLO test, and PCR, were employed to check the presence or absence of the organism in the antrum of the stomach. Several PCR positives without viable cultures were observed in babies of less than one year old. On the other hand, only two viable cultures were obtained from toddlers of less than two years old. The percentage of positive cultures steadily increased from 8% (3 of 42 cases) in the 0–4 years old age group to 32% (32 of 99 cases) in the 13–17 years old age group. A steady increase also was observed in the result of the CLO test. In PCR, the percentage of positives was greatly higher than that seen with the culture or CLO test. The rate of PCR positives also showed an increase with age but of a much slower rate. The overall infection rate in 295 children was 22% (64 of 295 cases) positive with culture and 76% (225 of 295 cases) with PCR, in contrast to 85% (40 of 49 cases) and 92% (43 of 47 cases), respectively, in adults. The urease activity of the H. pylori derived from children was much lower than that derived from adults (P<0.001). Taken together, these results suggest that a child might be repeatedly infected and some infecting strains eventually might obtain a steady infection, perhaps by a strain of higher virulence such as higher urease activity. The base variations in the nucleotide sequences did not correlate to the varied urease activities or to the age of the child. The sequences, however, indicated that there were two types of strains. The strains in Taiwan appeared to be derived from the French type strain and not the English type strain. The amino acid sequences of the ureA and the phylogenetic relationship of the 29 strains indicated that the strains in Taiwan are rapidly evolving into a unique clone.