bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification of Bacillus subtilis Spore Coat Protein by Electrophoretic Elution Procedure and Determination of NH2‐Terminal Amino Acid Sequences
Authors: Abe Akiko , Ogawa Setsuko , Kohno Takeyuki , Watabe Kazuhito
Published: November 2013

Spore coat protein of Bacillus subtilis was purified by electrophoretic elution procedure. Solubilized coat protein components were separated on SDS‐PAGE and the desired protein was recovered from the gel pieces under the optimal condition examined. Two purified polypeptides with molecular weights of about 40 kDa were obtained; each of them was in very closed size on SDS‐PAGE, both retaining antigenic activity against anti‐spore coat protein serum on immunoblot analysis. The N‐terminal 23 and 30 amino acid sequences of them were determined, and they were not identical to each other and also not homologous in the sequences of coat proteins previously reported.
Murine Endothelial Cell Line Cells, F‐2: Interaction with Leukocytes and Cytokines Production
Authors: Maruyama Hidenori , Toda Ken‐ichi , Uno Kazuko , Miyake Kensuke , Matsushima Koji , Yamamoto Kazuo , Mori Kazuhiro J. , Masuda Tohru
Published: November 2013

Flowcytometry demonstrated that murine endothelial cell line F‐2 expresses MHC class I antigen, FcR II, Mac‐1 and vascular cell adhesion molecule‐1 (VCAM‐1), but not intercellular adhesion molecule‐1 (ICAM‐1) and class II antigen. However, co‐culturing with TNF‐α for 24 hr resulted in the increased expression of ICAM‐1, and the decreased expression of VCAM‐1. IL‐1α and IFN‐γ exerted this regulatory effect on VCAM‐1 but not on ICAM‐1. T (Con A blast) and B (LPS blast) cells adhered to F‐2 cells at almost equal levels, and the adhesion was enhanced 20 to 50% when the cells were precultured with TNF‐α for 24 hr. The inhibition assay using either (anti‐ICAM‐1 + anti‐LFA‐1, lymphocyte function‐associated antigen‐1) or (anti‐VCAM‐1 + anti‐VLA‐4, very late antigen‐4) mAbs demonstrated that the ICAM‐1 system was utilized more preferentially by T than B blasts when F‐2 cells were stimulated with TNF‐α, and the VCAM‐1 system was vice versa under the unstimulated and stimulated conditions. Granulocytes also adhered to F‐2 cells, but no mAbs could inhibit the adhesion. Although F‐2 cells produced a considerable amount of IL‐6, GM‐CSF and neutrophil chemotactic activity, a 24 hr incubation with TNF‐α resulted in an increase of 12 fold in IL‐6 and 3 fold in neutrophil chemotactic activity production.
Preliminary Observations on Ultrastructure of Borreliae in Tissues of Ixodes persulcatus
Authors: Yano Yasuhiro , Ishiguro Fubito , Takada Nobuhiro
Published: November 2013

We observed Lyme borrelia by electron microscopy in the tissues of the ticks, Ixodes persulcatus, which were indicated positive for borreliae by BSK cultures of their internal organs. Borreliae (0.25 μm in diameter) were found only in the lumen of the midgut. They were closely associated with the microvilli on the midgut epithelium but never penetrated into the epithelial cells. Ultrastructural features common to Lyme borreliae., i.e., the three‐layered membranes surrounding the cytoplasm and orientation of the flagella insertions, were obviously confirmed. The present results are useful to understand tick tissue‐borrelia interface.
Immunological Properties of TtT/M‐87 Cell Line Established from Murine Pituitary Tumor‐Associated Macrophages
Authors: Mitsuishi Toshimi , Akuzawa Yuki , Sato Shinobu , Rui Jin , Kodama Kazue , Inoue Kinji , Kurashige Satonori
Published: November 2013

TtT/M‐87 cell is a macrophage cell line established from thyrotropic pituitary tumor tissues in mouse. In this paper, we report the immunological properties of M‐87 cells as a model of tumor‐associated macrophage. Contrasting with resident peritoneal macrophages, M‐87 cells constitutively secreted small but significant amounts of TNF‐α and IL‐1α, which were detectable in both biological assays (cytotoxic activity for L929 and co‐mitogenic activity for Con A‐induced T cell proliferation, respectively) and ELISA, and produced larger amounts of these cytokines upon stimulation with LPS. They expressed MHC class II molecules on their cell surface without stimulation by IFN‐γ. The accessory or antigen‐presenting cell activity in antibody‐producing response of spleen lymphocytes to sheep red blood cells was shown to be much higher in M‐87 cells than normal peritoneal macrophages. In addition, when normal spleen lymphocytes were cultured with allogeneic tumor cells, such as EL‐4 and S‐180, in the presence of M‐87 cells, lymphocytes reactive to stimulator cells were activated to manifest inhibitory effect on the tumor cell growth and also to manifest specific cytotoxic effect on the allogeneic tumor cells. These results show that M‐87 cells derived from tumor‐associated tissue are activated macrophages and that they are inhibitory to tumor cell growth and augmentative in the induction of T‐cell‐mediated immune responses.
Small, Round‐Structured Viruses (SRSVs) Associated with Acute Gastroenteritis Outbreaks in Gifu, Japan
Authors: Kawamoto Hiroyoshi , Hasegawa Sumiyo , Sawatari Seiko , Miwa Chieko , Morita Osayuki , Hosokawa Takehiko , Tanaka Hiroshi
Published: November 2013

Two outbreaks of non‐bacterial gastroenteritis occurred in Gifu prefecture in January 1989 and in January 1991. Both outbreaks were closely related to the consumption of raw oysters, and showed similar clinical features. Small, round‐structured virus particles were found in patient stools in both outbreaks by electron microscopy. The role of these particles as the causative agents of the outbreaks were strongly suggested by immune electron microscopy and/or western‐blotting immunoassay. When compared with SRSV‐9 (Tokyo/SRSV/86–510) reported previously (Hayashi et al, J. Clin. Microbiol., 27: 1728–1733, 1989), it was found that these viral particles were antigenically similar to SRSV‐9, and had a major structural protein of 63 kilodaltons (kDa). Further, the prevalence of this agent in Gifu area was examined by western blot antibody assay using 67 serum samples collected from the inhabitants in 1991. The results indicated the circulation of the same or antigenically similar agent in this area.
Pili of Vibrio cholerae Widely Distributed in Serogroup O1 Strains
Authors: Iwanaga Masaaki , Nakasone Noboru , Yamashiro Tetsu , Higa Naomi
Published: November 2013

The distribution of Vibrio cholerae O1 pili consisting of 16 kDa subunit protein (16K‐pili) was examined by Western blotting, using 211 strains from various origins and specific anti‐16K‐pili sera. The 16 kDa protein was detected in all 211 strains. The pili were purified from 3 El Tor and 3 classical strains, and characterized by hemagglutination and inhibition tests. All purified pili were hemagglutinative. However, the hemagglutinating activity of classical pili disappeared after exposure to 5 M urea and the agglutination induced by the classical pili was inhibited by D‐mannose, alpha‐methylmannoside, D‐glucose and N‐acetylglucosamine. On the contrary, El Tor pili were resistant to these sugars and urea.
Applications of Polymerase Chain Reaction for Identification of Dengue Viruses Isolated from Patient Sera
Authors: Maneekarn Niwat , Morita Kouichi , Tanaka Mariko , Igarashi Akira , Usawattanakul Wipawee , Sirisanthana Virat , Innis Bruce L. , Sittisombut Nopporn , Nisalak Ananda , Nimmanitya Suchitra
Published: November 2013

A simple and sensitive procedure of reverse transcriptase polymerase chain reaction (RT‐PCR) was developed previously such that all 4 serotypes of dengue viruses could be detected and their serotypes identified simultaneously in a single‐step procedure. In this study we compared the RT‐PCR with a conventional immunoperoxidase (PAP) staining method for the identification of dengue viruses currently isolated from patient sera. Sixty‐six sera taken from dengue hemorrhagic fever (DHF) patients were subjected to virus isolation by inoculating onto C6/36 cell cultures. Screening for the presence of dengue viruses in culture fluids was done after 7 days of incubation by PAP staining using hyperimmune rabbit anti‐dengue virus antibody as the primary reagent. Dengue viruses in positive cultures were further identified for their serotypes by PAP using type‐specific monoclonal antibodies (MAb) and by RT‐PCR. Thirty‐two out of the 66 serum specimens tested (48.5%) were positive for dengue viruses. Of these, 5 were type 1 (DEN‐1), 25 were type 2 (DEN‐2) and 2 contained both DEN‐1 and DEN‐2. All cultures that were positive by PAP method were also positive by RT‐PCR and vice versa. Thus, the results obtained by RT‐PCR were in good agreement with those by PAP. It is important to point out that while all 5 DEN‐1 isolates reacted readily with the MAb 1F1, only 2 of them could be identified by the MAb 15F3. Our data suggest that antigenic variation among DEN‐1 isolates occur frequently and this should be taken into consideration in the selection of appropriate type‐specific MAb for serotyping of dengue viruses. We also demonstrated dual infection of DEN‐1 and DEN‐2 in two patients' sera by RT‐PCR.
Fibronectin‐Binding Proteins of a Human Oral Spirochete Treponema denticola
Authors: Umemoto Toshihiko , Nakatani Yoshihiro , Nakamura Yoshinori , Namikawa Isamu
Published: November 2013

Major polypeptides from a human oral spirochete Treponema denticola ATCC 33520 were examined to demonstrate their ability to bind to human plasma fibronectin by immunoblot analysis. Of three main polypeptides separated on sodium dodecyl sulfate polyacrylamide gels 53,000‐daltons (53‐kDa) and 72‐kDa surface antigenic proteins and a 38‐kDa axial flagellar protein showed the ability to bind to fibronectin, suggesting that fibronectin on host cells can mediate cytoadherence of T. denticola by its binding to the surface proteins or the exposed 38‐kDa axial flageller protein.
Evidence for Interference by Immune Complexes in the Serodiagnosis of Cryptococcosis
Authors: Sadamoto Shinya , Ikeda Reiko , Nishikawa Akemi , Shinoda Takako
Published: November 2013

The latex agglutination test was used to compare cryptococcal antigen titers before and after protease treatment in 19 patients with pulmonary cryptococcosis. Antigen was detected by the LA test in 13 of 33 serum samples before protease treatment, and in an additional 13 samples following treatment. Of 26 antigen‐positive serum samples, 22 (84.6%) showed an increased antigen titer after protease treatment. Using the cell slide agglutination test, antibody was detected in 3 of 19 cases. In one of these 3, antigen could only be detected after protease treatment. Soluble immune complex was prepared in vitro using anti‐C. neoformans rabbit antiserum and polysaccharide antigen of C. neoformans, and the effects of immune complexes on the LA test were examined. In this experimental model, soluble immune complexes seemed to be observed in antibody excess region, because the antigen titers were increased after the protease treatment. We concluded that C. neoformans capsular polysaccharide antigen and anti‐C. neoformans antibody formed soluble immune complexes in patients' sera, which interfered with antigen detection by the latex agglutination test without protease treatment.
Antigenic Behaviors of Two Axial Flagellar Proteins Detected in Treponema denticola
Authors: Umemoto Toshihiko , Wadood Abdul , Nakamura Yoshinori , Nakatani Yoshihiro , Namikawa Isamu
Published: November 2013

Two polypeptide antigens with molecular sizes of 34,000 daltons (34 kDa) and 38 kDa were separated from heated cells of a human clinical treponeme strain G7201 and Treponema denticola ATCC 35404, respectively. The rabbit polyclonal antisera against these antigens were produced and examined for their immunological reactions with the two heated antigens or intact spirochetal cells. Immunoblot analysis showed that the 34‐kDa protein was also detected in T. denticola ATCC 35404 and ATCC 33520, and the 38‐kDa protein was detected only in the two ATCC strains. Immunoelectron microscopy using the two rabbit antisera and protein A‐gold complexes demonstrated that the 38‐kDa protein antigen was present on the axial flagella of two T. denticola strains, and that the 34‐kDa protein was located in the axial flagella of the G7201 cell, but neither in axial flagella nor on outer envelopes of the two ATCC strains cells, suggesting that the native 34‐kDa axial flagellar protein of the G7201 strain may be different from that of T. denticola in terms of immunological reactivity.
Sub‐Minimum Inhibitory Concentrations of Ceftibuten Reduce Adherence of Escherichia coli to Human Cells and Induces Formation of Long Filaments
Authors: Braga Pier Carlo , Piatti Gioia
Published: November 2013

The minimal inhibitory concentrations (MICs) of an antibiotic are present for only a certain period of time, after which they become sub‐inhibitory concentrations (sub‐MICs). These sub‐MICs are still active because they can interfere with the mechanism of bacterial adhesion, which is the first step in the sequence of events leading to infection. The purpose of the present study was to investigate the effects of sub‐MICs of ceftibuten, a new third‐generation cephalosporin, on the adhesion of Escherichia coli (E. coli) to human buccal cells. The degree of inhibition was maximal at 1/2 MIC and then gradually returned toward to the control values at 1/128 the MIC. The differences were statistically significant from 1/2 to 1/32 MIC. Since the MIC was 0.5 μg/ml, concentrations from 0.25 to 0.015 μg/ml significantly reduce bacterial adhesion. Ceftibuten also caused marked elongation of E. coli. These findings could help to explain the efficacy showed by ceftibuten in the treatment of respiratory and urinary tract infections when administered once daily.
Role of Calcium in Biphasic Germination of Bacillus cereus T Spores Sensitized to Lysozyme
Authors: Shibata Hirofumi , Adachi Sawako , Hirose Yoko , Ike Mika , Tani Isamu , Hashimoto Tadayo
Published: November 2013

Biphasic germination induced by inosine in the presence of Ca2+ was examined using Bacillus cereus T spores treated with sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) at pH 10. The first phase of the germination was stimulated by Ca2+ in the concentration‐dependent manner, showing the optimal concentration at 0.5‐1.0 mM. The second phase appeared to be insensitive to the cation. The optimal temperatures for the first and the second phase were 25 C and 40 C, respectively; the optimal pHs for the two phases were 7‐9 and around 7.5, respectively. Heat resistance and dipicolinic acid of the SDS‐DTT‐treated spores were lost mostly during the first phase. A Ca2+‐specific chelator, glycoletherdiamine‐N,N,N',N'‐tetraacetic acid (GEDTA), inhibited the first phase evoked by Ca2+, while it had no inhibitory effect on the second phase. In contrast, the divalent cations examined, except Mg2+ and Sr2+, affected not only the first phase but also the second phase. The order of inhibitory effect on the first phase was Hg2+ > Zn2+ > Ba2+, Co2+, Cu2+ > Mn2+; on the second phase, it was Hg2+ > Cu2+ > Zn2+ > Co2+ > Mn2+ > Ba2+.
Variation in Field Isolates of Measles Virus during an 8‐Year Period in Japan
Authors: Sakata Hiroko , Kobune Fumio , Sato Takeshi A. , Tanabayashi Kiyoshi , Yamada Akio , Sugiura Akira
Published: November 2013

Field isolates of measles virus (MV) during an 8‐year period in four areas of Japan, i.e., Osaka, Nagoya, Tokyo and Akita, were classified into three types in regard to the electrophoretic mobility of the hemagglutinin (HA) proteins: S type with small (78 K) HA, M type with intermediate (80 K) HA and L type with large (82K) HA. The type of field isolates was closely related with the geographical location and the year of virus isolation. The S type strain was isolated only in an outbreak from 1983 to 1984, whereas the M and L type strains were isolated between 1983 and 1990. The HA genes of the M and L type strains of MV were found to have a nucleotide substitution which introduces a new potential glycosylation site. In addition, the matrix proteins of all field strains isolated after 1977 showed slower electrophoretic mobility of 42 K than 39 K of the Edmonston and Toyoshima strains. These results indicate that MV strains of different HA types existed concomitantly and that major populations of MV currently circulating in Japan are changing from those prevalent in 1983‐1984.
Comparison of the Virulence for Mice of Mycobacterium avium and Mycobacterium intracellulare Identified by DNA Probe Test
Authors: Tomioka Haruaki , Saito Hajime , Sato Katsumasa , Dawson David J.
Published: November 2013

The virulence of various serovars of Mycobacterium avium and M. intracellulare identified by DNA probe test was compared with each other. We found species‐ and serovar‐dependencies of M. avium complex (MAC) virulence to mice in terms of mortality, incidence of lung lesions and bacterial load in the visceral organs, as follows. First, human‐ or environment‐derived M. intracellulare was more virulent for mice, as compared to M. avium isolated from patients or environmental sources. Second, the virulence of MAC isolates belonging to serovars 1, 8, 9 (M. avium), 14 and 16 (M. intracellulare) is in the order of serovars 16 > 14 > 8 > 1 > 9. These aspects were different from those for MAC virulence to human and bird, swine and cattle.
A Sensitive Serodiagnosis of Hepatitis C Virus (HCV) Infection with Two Non‐Fused Peptides: Comparison of Antibody Responses Detected with a Newly Developed Assay and a Commercial Second‐Generation Test
Authors: Sato Atsushi , Ida Nobuo , Ishikawa Mari , Tanahashi Kazuhiro , Nakamura Haruji , Sho Yukihiko , Arima Terukatsu , Kunitomo Tetsunosuke
Published: November 2013

An enzyme‐linked immunosorbent assay (ELISA) was developed for the detection of anti‐HCV antibody. We assayed for antibodies against either oligopeptide (S29‐1) deduced from the nucleocapsid gene or the product of nonstructural region (NS3) synthesized in a recombinant Escherichia coli (S4). To reduce false‐positive results induced by non‐specific binding of antibodies with a carrier protein and to increase the sensitivity of an immunoassay, non‐fused S4 peptide was prepared by the recombinant DNA technique and site‐specific proteolysis (by factor Xa). In 71 non‐A, non‐B hepatitis patients with chronic liver disease, 70 (98.5%) were positive by S29‐1/S4 ELISA as well as by a second‐generation test (Abbott II). On the other hand, of 40 serum samples from blood donors, in which anti‐N14 (core) and C100‐3 antibodies were not detected but hepatitis C virus (HCV) RNA was detectable by polymerase chain reaction (PCR), 24 (60%) were positive by S29‐1/S4 ELISA, whereas only 18 (45%) were diagnosed by Abbott II. In addition, based on results in a small group of 92 blood donors, detection of anti‐S29‐1/S4 antibody correlated well with HCV viremia as confirmed by PCR. These results indicated that the preparation of non‐fused protein (S4) by recombinant DNA technique and a combination of S29‐1 and S4 as immobilized antigens in an ELISA provide a sensitive and specific diagnosis for HCV infection with good correlation with the presence of viral RNA as confirmed by PCR.
Ecological Studies on Reovirus Pollution of Rivers in Toyama Prefecture. II. Molecular Epidemiological Study of Reoviruses Isolated from River Water
Authors: Matsuura Kumiko , Ishikura Mitsuhiro , Nakayama Takashi , Hasegawa Sumiyo , Morita Osayuki , Katori Kouji , Uetake Hisao
Published: November 2013

In order to clarify the source of reovirus pollution in river water, comparative surveys have been carried out between reovirus isolates from river water and those from sewage, human or animal, by making use of the analysis of genomic RNA‐migration pattern of reovirus in polyacrylamide gel electrophoresis (electropherotype). The strains of reovirus serotype 1 and 2 isolated from river water were classified into 3 and 9 electropherotypes, respectively, and 8 out of these 12 types were also found among strains isolated from sewage or human. When the monthly distribution of the river isolates classified by electropherotypes was compared with that of the sewage isolates, there were cases in which strains of the same electropherotype were simultaneously isolated from both sources. The electropherotypes of 3 isolates from pig and field rodents were different from those of the other isolates. The electropherotype of an oyster isolate coincided with that of some of the isolates from humans and river water. These results indicate that the major sources of reoviruses polluting river water may be the human excretion.
Cholera Toxin Inhibits Lethal Hit Stage of Natural Killer Cell‐Mediated Cytotoxicity
Authors: Watanabe Masami , Kiyohara Tatsuo , Murata Ken‐Ichirou , Yodoi Junji , Itakura Hideyo
Published: November 2013

Cytolytic process which was affected by cholera toxin (CT) resulting in the loss of natural killer (NK) cell activity was analyzed. Conjugate formation assay, membrane phospholipid methylation assay and serine esterase (granzyme A) release assay were used to determine the stage of the CT‐induced inhibition of NK cell‐mediated cytotoxicity. A human NK cell line YT cell‐mediated cytotoxicity was completely abolished by CT pretreatment or addition of CT to the assay system. The conjugate formation assay revealed that the binding between YT cells and target cells was not affected by CT. The defined triggering stage which is coupled with membrane phospholipid methylation was not affected by CT treatment, either. On the other hand, the lethal hit stage which is represented by serine esterase (SE) release was completely inhibited by CT treatment of YT cells. Therefore, CT inhibits the stage after binding and triggering—i.e., lethal hit stage of NK cell‐mediated cytotoxicity. The results also suggest that there exists a CT‐sensitive negative cytotoxic signal transduction pathway as well as usual positive signal transduction pathway and these pathways might cross talk each other in the NK cell cytotoxic process.
Chemical Analysis of Lipopolysaccharides of Shigella sonnei Form II Strains Expressed by Cloned Form I Antigen Genes
Authors: Okamura Noboru , Chida Toshio , Kinoshita Masao , Yoshida Yoko , Kondo Seiichi , Hisatsune Kazuhito
Published: November 2013

A compositional sugar analysis was carried out on lipopolysaccharide (LPS) from Shigella sonnei form II in which a plasmid with cloned form I antigen genes had been introduced. The recipient form II strains contained galactose, glucose, heptose, glucosamine, and 2‐keto‐3‐deoxyoctonic acid (KDO) (2: 3: 1: 2: 2) in its LPS, while the transformant form I LPS contained, besides these sugars, N‐acetyl‐L‐altrosaminouronic acid as an additional sugar constituent, which is known to be one of the antigenic determinants of form I antigen.
Passive Hemagglutination Assays for the Detection of Antibodies to Herpes Viruses
Authors: Kino Yoichiro , Minamishima Yoichi
Published: November 2013

A simple and effective method for the detection of antibodies to herpes simplex virus (HSV), human cytomegalovirus (HCMV) and varicella‐zoster virus (VZV), has been established using the passive hemagglutination assay (PHA) in combination with viral specific glycoproteins. The results obtained with the PHA were compared with those from neutralization (NT) and complement fixation (CF) tests. The PHA test for each of the herpes viruses appears to compare favorably with the other assays tested. The specificity and sensitivity of HSV PHA to NT were 100%, whereas the specificity and sensitivity of HSV CF test to NT were 98% and 100%, respectively. For HCMV, the specificity and sensitivity of PHA to NT and PHA to CF were 100%. Similarly, the specificity and sensitivity of VZV PHA to NT were 100%. Because of the low sensitivity of the VZV CF, the sensitivity of CF to NT was 83%. Furthermore, the range of antibody titers and their absolute levels obtained in the PHAs were significantly greater than those in the NT and CF tests.
Cloning and Sequencing of Two New Verotoxin 2 Variant Genes of Escherichia coli Isolated from Cases of Human and Bovine Diarrhea
Authors: Lin Zaw , Yamasaki Shinji , Kurazono Hisao , Ohmura Mari , Karasawa Tadahiro , Inoue Takahiro , Sakamoto Seisuke , Suganami Takahiro , Takeoka Tomohiro , Taniguchi Yoshihiro , Takeda Yoshifumi
Published: November 2013

We cloned and sequenced two new Verotoxin 2 (VT2) variant genes: one from an Escherichia coli strain from a case of bovine diarrhea and the other from an E. coli strain from a patient with diarrhea. The nucleotide and amino acid sequences of these two genes were highly homologous with, but distinct from those of the VT2, VT2vha, VT2vhb, SLT‐IIv (VT2vp1) and SLT‐IIva (VT2vp2) genes. Their nucleotide sequences were much more closely homologous to that of VT2vh than to that of VT2vp. Search for these two new genes in other Verocytotoxin‐producing E. coli strains resulted in the isolation of 2 strains carrying one of the new VT2 variant genes, one strain from Tokyo and the other from Canada.
Antigenic Characterization of Chlamydia pneumoniae Isolated in Hiroshima, Japan
Authors: Kanamoto Yasuo , Iijima Yoshio , Miyashita Naoyuki , Matsumoto Akira , Sakano Takashi
Published: November 2013

The morphology and antigenic property of elementary bodies (EBs) of new Chlamydia pneumoniae YK‐41 strain isolated in Hiroshima, Japan, were compared with those of C. pneumoniae strains TW‐183 and AR‐39, C. trachomatis L2/434/Bu strain and C. psittaci Cal 10 and Budgerigar‐1 strains by SDS‐PAGE and immunoblotting techniques. In spite of a clear difference in EB morphology between the YK‐41 and the other C. pneumoniae strains used, protein profile of the YK‐41 strain in SDS‐PAGE was similar to that of the other strains. However, some quantitative difference in 200 and 98 kDa peptides and a faint difference in SDS‐PAGE pattern was also observed in the molecular masses from 42 to 50 kDa. Immunoblot analysis with the patient serum at the convalescent stage revealed the presence of genus‐specific and species‐specific antigens in YK‐41 EBs: i. e., the major outer membrane protein and 73 kDa peptides were genus‐specific and the peptides of 43, 46, 53, 60 and 98 kDa appeared to be C. pneumoniae‐specific.
Characterization of Hemolytic and Antifungal Substance, Cepalycin, from Pseudomonas cepacia
Authors: Abe Mitsuko , Nakazawa Teruko
Published: November 2013

Hemolytic and antifungal substances, cepalycin I and cepalycin II, have been isolated from Pseudomonas cepacia JN106. A large amount of cepalycins were produced by growing the cells on 1% glycerin‐nutrient agar medium covered with a cellophane membrane. The cell‐washed supernatant was applied to an Amberlite XAD2 column, and cepalycins were eluted with 70% ethanol containing 1mM HCl. Cepalycins were separated by reverse phase HPLC in two fractions which were designated as cepalycin I and cepalycin II. The two cepalycins have indistinguishable UV absorption spectra but have different levels of hemolytic activity relative to the UV absorption. From the inhibition of hemolytic activity of cepalycin by sterols, both cepalycins were suggested to interact with cholesterol in erythrocyte membrane. Such an interaction may contribute to their hemolytic and antifungal activities.
Electrophoretic Karyotypes of Candida Yeasts Recurrently Isolated from Single Patients
Authors: Doi Matsuko , Mizuguchi Ikuyo , Homma Michio , Tanaka Kenji
Published: November 2013

Candida strains were isolated repeatedly from single patients during recurrent episodes of Candida infection in a hospital, and their electrophoretic karyotypes were analyzed by pulsed‐field gel electrophoresis using CHEF system. When only C. albicans (in 6 patients) or C. glabrata (in 1 patient) were recurrently isolated, their karyotypes from each patient were almost identical to one another, suggesting that they carried single type of the yeast. When multiple species were recovered from single patients (6 cases), the karyotypes of the most frequently recovered yeast species were almost identical with respect to each patient. The electrophoretic karyotype analysis has been proved to be useful for epidemiological studies because the method can tell not only the species identification but also the differences among the strains of the same species.
Helicobacter pylori: A Fickle Germ
Authors: Cellini Luigina , Allocati Nerino , Di Campli Emanuela , Dainelli Benedetto
Published: November 2013

The morphologic changes from bacillary to coccoid forms of Helicobacter pylori were studied. These form changes were analyzed by bacterial growth in Brucella broth plus 2% fetal calf serum. The coccoid forms were observed at five days of incubation and a rapid decrease of CFU/ml was recorded. At two weeks of microaerophilic incubation, all coccoid forms observed were not culturable in vitro. The coccoid morphology was observed earlier when the culture of H. pylori was incubated in aerobic conditions and with subinhibitory concentrations of omeprazole and roxithromycin. To evaluate the possibility of resistance of coccal forms, before plating, the cultures were heated to 80 C for 10 min and sonicated. In the absence of these treatments the cultures did not show growth in vitro. The proteic patterns of the same strains of two different morphologies were studied revealing significant differences.
The Transposon‐Like Structure of IS26‐Tetracycllne, and Kanamycin Resistance Determinant Derived from Transferable R Plasmid of Fish Pathogen, Pasteurella piscicida
Authors: Kim Eun‐heui , Aoki Takashi
Published: November 2013

Tetracycline (pp‐tet), and kanamycin (pp‐kan) resistance genes were cloned from a transferable R plasmid of fish pathogen Pasteurella piscicida, and complete nucleotide sequences were determined. The pp‐tet was a class D Tet determinant constructed with the tetA resistance gene of 1,182 bp encoding a protein with a deduced molecular mass of 41 kDa and the tetR repressor gene of 654 bp encoding a product of 24 kDa. The pp‐tet was highly homologous to the tet(D) of plasmid RA1 isolated from Aeromonas hydrophila with two nucleotide differences in the tetR, and of plasmid pIP173 from Salmonella ordonez with two nucleotide differences in the tetA. The pp‐kan contained 813 bp encoding a 31 kDa protein of 271 amino acids, and was classified into type aph‐Ic. It was identical to the aphA7 in the IAB operon of pBWH77, in which was originally found an isolate of Klebsiella pneumoniae, in its nucleotide sequences and hybrid promoter construction. The genes were connected by an insertion sequence IS 26 of 820 bp, and were flanked by repeated copies in direct orientation at the 3′ flanking region of the pp‐tetA and in inverted orientation at the 3′ flanking region of the pp‐kan. The genetic elements are organized like a complex transposon by close linkage of the IS 26 and the pp‐tet and ‐kan.
Evaluation of the Sensitivity and Specificity of Polymerase Chain Reaction Test Kit, AMPLICOR Chlamydia trachomatis
Authors: Miyashita Naoyuki , Iijima Yoshio , Matsumoto Akira
Published: November 2013

The sensitivity and specificity of the polymerase chain reaction (PCR) test kit, AMPLICOR Chlamydia trachomatis, were examined by the use of purified elementary bodies (EBs), cells having inclusions containing reticulate bodies alone and 20 clinical isolates. The numbers of EB and inclusion of C. trachomatis at the detection limit were determined to be approximately 2 to 4 EBs and one inclusion per assay, respectively. No reaction occurred for C. psittaci and C. pneumoniae. All clinical isolates were positively reacted in the PCR assay.
Epidemiological and Ecological Studies of Japanese Encephalitis in Okinawa, Subtropical Area in Japan. II. Prevalence of Japanese Encephalitis Antibody in Residents in Okinawa, Miyako and Ishigaki Islands
Authors: Tadano Masayuki , Kanemura Kazumi , Arakaki Sakae , Makino Yoshihiro , Fukunaga Toshihiko , Hokama Tetsuo , Kyan Takeyoshi , Makiya Hiroshi , Taira Youichi , Ohama Nagateru , Iju Choyu , Sakurai Kaori
Published: November 2013

During 1989 to 1990, human sera were collected by age groups in Okinawa (the northern, central and southern areas), Miyako and Ishigaki islands and examined for the neutralization (N) antibodies to two strains, Nakayama (vaccine strain) and C307 (Okinawan strain), of Japanese encephalitis (JE) virus. In Okinawa island, the N antibody positive rate to C307 was higher than that to Nakayama, while in Miyako and Ishigaki islands, the positive rate to Nakayama was higher than that to C307, suggesting that JE virus transmission rate was higher in Okinawa than in Miyako and Ishigaki islands. In Okinawa Prefecture, JE vaccine had not been administered to most of residents over 31 years of age at the time of serum collection. In residents over 31 years old, the positive rate to C307 was highest in the north of Okinawa (83.3%) and was lowest in Miyako (26.3%), with the second lowest in Ishigaki (33.3%). The distribution of N antibody titers to C307 gave hyperbolic patterns in the 0–5 age groups in Miyako and Ishigaki, and also in the 31–40, 41–50 age groups in Miyako and the 41–50 age group in Ishigaki, suggesting low rates of natural infection in these 4–5 decades in both islands. In residents of ages subjected to JE vaccine, a characteristic pattern was obtained, in which the curves to Nakayama shifted to higher titers than those to C307, suggesting that the first antigenic stimulation was caused by vaccine, not by natural infection of JE virus.
A Function of 40 kDa Outer Membrane Protein in Serratia marcescens
Authors: Tada Yoshifumi , Yamaguchi Junji
Published: November 2013

The function of one of the outer membrane proteins of Serratia marcescens was investigated. S. marcescens with an abundant 40 kDa outer membrane protein was induced to form spheroplast at a high rate in an isotonic medium in the presence of calcium, although the spheroplasts were generally fragile in the isotonic environment. The degree of spheroplast induction was correlated to the amount of the 40 kDa protein present in the membrane. In the 40 kDa proteinless mutant strains, the spheroplast induction rate was remarkably decreased. Autoradiography of the outer membrane revealed the presence of a calcium‐binding protein as a radioactive band whose position coincided with the 40 kDa protein. These results suggest that the 40 kDa protein has an important role in maintaining the structural integrity of the cell wall against osmotic shock.
Effective Induction of Human NK Cells with OK‐432 and Further Augmentation of Their Cytolytic Function by rIL‐2
Authors: Hu Zhao‐liang , Kubota Eiro , Imamura Hideo , Katano Mitsuo , Katsuki Takeshi
Published: November 2013

Low concentrations of exogenously added recombinant interleukin 2 (rIL‐2) were able to augment OK‐432‐induced natural killer (NK) cell activity. This kind of augmenting effect depended on the dose of rIL‐2 and manifested itself only in PBMC stimulated with OK‐432 (OK‐MC) followed by rIL‐2; augmentation did not happen in the reverse order. The existence of CD16+/CD25+ (IL‐2 receptor positive; IL‐2R+) and CD57+/CD25+ double positive cells which possess NK cell surface markers in OK‐MC markedly increased in a long‐term culture (12 days). A strong positive correlation was observed between the IL‐2‐dependent augmentation of NK activity and the quantitative changes in cell populations that possessed NK cell phenotypes. Treatment of the day‐12‐OK‐MC with monoclonal anti‐CD56 antibody plus complement could almost completely abrogate the augmented NK cytotoxicity. Furthermore, this augmenting effect was detectable within 4 hr after addition of rIL‐2 at single cell level, suggesting that the effect did not require NK cell's DNA synthesis. Thus it was suggested that OK‐432 could promote and upregulate the expression of IL‐2 receptor (CD25) on CD56+ NK cell populations. Moreover, it was considered that the interaction of low concentration rIL‐2 with IL‐2 receptors on OK‐432‐activated NK cells could augment their lytic function.
Pili of a Vibrio cholerae O139
Authors: Nakasone Noboru , Yamashiro Tetsu , Albert M. John , Iwanaga Masaaki
Published: November 2013

The pili of a strain of Vibrio cholerae O139 were purified and characterized. They were morphologically, electrophoretically and immunologically indistinguishable from the pili with 16 kDa subunit protein of V. cholerae O1. All 22 strains of V. cholerae O139 examined possessed the pili. The pili were different in hemagglutination inhibition pattern from V. cholerae O1 16K pili.
Purification and Characterization of Membrane Protein (90 kDa) from Mycoplasma salivarium, Which Binds Immunoglobulin (Ig) G Fc Fragment
Authors: Sawa Yoshihiko , Shibata Ken‐ichiro , Noda Mamoru , Watanabe Tsuguo
Published: November 2013

A 90 kDa protein of Mycoplasma salivarium was released from cell membranes of the organism with Triton X‐100 and purified by ion‐exchange chromatography and chromatofocusing. The protein was eluted at pH 5.5 by chromatofocusing. The protein was shown to react with the Fc fragments of IgG from human and nine different animal species and did not distinguish between IgG from different species, while protein A, tested for comparative purposes, displayed a strong specificity for human and swine IgG. Furthermore, the protein reacted with antigen specific goat IgG (specific for gamma chains of human IgG), sheep red blood cells (SRBC) sensitized with rabbit antiserum to SRBC, that is, the Fc part of rabbit IgG, and concanavalin A as well. These findings may suggest that the protein is a lectin which binds the carbohydrate moiety of the Fc part of IgG.
Experimental Induction of Lyme Arthritis in Outbred Mice
Authors: Masuzawa Toshiyuki , Kurita Toru , Kawabata Hiroki , Suzuki Harumi , Yanagihara Yasutake
Published: November 2013

Outbred ddY mice inoculated with live cells ofBorrelia burgdorferistrain 297 into hind footpad displayed swelling of the footpad at days 7 to 11 after inoculation. Marked neutrophilic infiltration was observed in the subcutaneous tissue and the part of bone tissue which was partially destroyed, and synovial layer of articular capsule was thickened and protruded into the joint space in the histopathological examination of footpad inoculated with liveBorreliacells. The inflammation peaked at day 7 andB. burgdorferiwas cultured from bladder and heart of the mice at day 14 after inoculation. The mice inoculated with heat‐inactivated cells at 56 C for 30 min did not show any significant histopathological change. In this mice model, nontreated littermates were not infected in contact with infected littermates for 14 days of experimental period. The outbred ddY mice model is useful for evaluating the effectiveness of vaccination against Lyme disease.
“Silent” Hepatitis B Virus Mutants Are Responsible for Non‐A, Non‐B, Non‐C, Non‐D, Non‐E Hepatitis
Authors: Uchida Toshikazu , Shimojima Masakazu , Gotoh Kenichirou , Shikata Toshio , Tanaka Eiji , Kiyosawa Kendo
Published: November 2013

Since the recent introduction of diagnostic kits for hepatitis C and E, some cases of nonA, non‐B, non‐C, non‐D, non‐E hepatitis (so‐called hepatitis F) have been revealed. We attempted to demonstrate that so‐called hepatitis F is caused by hepatitis B virus (HBV) variants. Polymerase chain reaction (PCR) was used to amplify serum HBV DNAs from 20 patients with acute hepatitis and 20 patients with chronic hepatitis who had been diagnosed as having so‐called hepatitis F on the basis of conventional serological markers. The PCR technique successfully amplified HBV DNAs in 18 (90%) cases of acute hepatitis and 17 (85%) cases of chronic hepatitis. Sequencing of HBV DNAs of six patients (acute 3, chronic 3) revealed equally a T‐to‐C mutation of DR2 and an 8‐nucleotide deletion of the 3′‐terminus of the X gene coding region, giving rise to the generation of a C‐terminally truncated X protein and probable damage to the enhancer II/core promoter elements. These mutations of the X gene coding region may lead to suppression of replication and expression of HBV DNAs. Thus virtually all cases of so‐called hepatitis F appear to be caused by “silent” HBV mutants, at least in Japan.
Detection of Antibody‐Coated Bacteria in Expectorated Sputum for Diagnosis of Lower Respiratory Infections
Authors: Matsumoto Tetsuya , Kaku Mitsuo , Tateda Kazuhiro , Furuya Nobuhiko , Hirakata Yoichi , Yamaguchi Keizo
Published: November 2013

We evaluated antibody‐coated bacteria (ACB) in expectorated sputum to discriminate contaminating or colonizing organisms from true pathogens. We examined 60 expectorated sputum samples from 51 patients with lower respiratory infections (chronic obstructive pulmonary disease 25, pneumonia 20, purulent tracheobronchitis 6). All samples were examined with quantitative culture and immunofluorescent demonstration of ACB. From the results of quantitative culture, we divided specimens into pathogen‐isolated and pathogen‐free samples. Among pathogen‐isolated samples, in which we isolated accepted pathogenic organisms at ≥ 107 colony‐forming units per ml, 16 of 23 samples were ACB‐positive (69.5%). In contrast, among pathogen‐free samples, in which we isolated accepted pathogens at < 107 colony forming units per ml or only upper respiratory flora, only 3 of 37 samples were ACB‐positive (8.1%). The ACB‐positive rate was significantly higher in pathogen‐isolated than in pathogen‐free samples (P < 0.001). Consequently, detecting ACB in expectorated sputum shows good potential as another criterion for distinguishing contaminating or colonizing organisms from true pathogens.
Helicobacter pylori Induces Inflammation in Mouse Urinary Bladder and Pelvis
Authors: Isogai Hiroshi , Isogai Emiko , Kimura Koichi , Fujii Nobuhiro , Yokota Kenji , Oguma Keiji
Published: November 2013

Helicobacter pylori was transurethrally inoculated into the mouse urinary tract. The organism established infection and induced inflammation in the urinary bladder and pelvis. During the infection, urinary pH was elevated, probably due to the production of NH3 by bacterial urease. H. pylori was recovered from the urinary bladder, kidney and urine of the infected mice. Histopathologically, severe neutrophil infiltration was observed in the mucosal layer of both organs. H. pylori was detected on the surface of the epithelial cells. These results indicate that low pH and bacterial flora were not essential factors in establishing the mucosal infection with H. pylori. This experimental system is useful to investigate the pathogenicity of H. pylori in mucosal organs.
The Rice Culture Filtrate of Bacillus cereus Isolated from Emetic‐Type Food Poisoning Causes Mitochondrial Swelling in a HEp‐2 Cell
Authors: Sakurai Naomi , Koike Kazuko A. , Irie Yuji , Hayashi Hideo
Published: November 2013

Rice culture filtrates of Bacillus cereus SA‐50, an emetic‐type strain, produced a toxin which caused cytoplasmic vacuole formation in HEp‐2 and HeLa cells. Electron microscopic observation revealed that the apparent vacuoles in HEp‐2 cells seen under a light microscope were actually swollen mitochondria. The oxygen consumption of HEp‐2 cells was accelerated by the addition of the rice culture filtrate as was measured with a polarographic oxymeter; a respiratory control ratio was 1.0 for control cells, while 1.4 for ones with the filtrates. The culture filtrates showed a similar effect on the isolated mouse liver mitochondria; respiratory control ratios for the mitochondria with and without the filtrates were 3.6 and 1.0, respectively. The affecting manner of the culture filtrates on the oxygen consumption of mitochondria was similar to that of 2,4‐dinitrophenol, suggesting that the culture filtrate contains a toxin acting as an uncoupler of oxidative phosphorylation in mitochondria. It is likely that the culture filtrates containing the emetic toxin of B. cereus causes mitochondrial swelling with a close relationship to the uncoupling of the oxidative phosphorylation of mitochondria.
Regulation of Iron on Bacterial Growth and Production of Thermostable Direct Hemolysin by Vibrio parahaemolyticus in Intraperitoneal Infected Mice
Authors: Wong Hin‐chung , Lee Yeong‐sheng
Published: November 2013

Pathogenesis of Vibrio parahaemolyticus is not clearly understood. Effects of iron on the bacterial proliferation and production of thermostable direct hemolysin (TDH) in intraperitoneal infected mice were studied. Injection of bacterial culture in the presence of ferric ammonium citrate (100 μg/ml) significantly enhanced the lethality for mice, and simultaneously activated bacterial proliferation in vivo. The iron‐limited cultures showed better proliferation than those iron‐rich cultures in response to the addition of supplementary iron source. Production of TDH by the hemolytic strains ST550 and D62 was higher in the iron‐limited cultures than the iron‐rich cultures. Production of TDH by both the iron‐limited or iron‐rich cultures was inhibited by the addition of iron. In conclusion, the virulence enhancement effect of iron in V. parahaemolyticus was probably by activating bacterial proliferation in vivo and not by stimulating the production of TDH. V. parahaemolyticus precultured in iron‐limited condition may be more adaptable to in vivo environment.
Cross‐Reactivity and Neutralizing Ability of Monoclonal Antibodies against Microcystins
Authors: Saito Shinji , Nakano Yasunobu , Kushida Katsutoshi , Shirai Makoto , Harada Ken‐ichi , Nakano Masayasu
Published: November 2013

Monoclonal antibodies (MAbs) against the microcystin‐leucine‐arginine variant (MCYST‐LR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. The specificity of the MAbs and their ability to neutralize the toxin were investigated by an indirect enzyme‐linked immunosorbent assay (ELISA) and by a neutralizing test in mice, respectively. All MAbs reacted with MCYST‐LR and also with the microcystin‐arginine‐arginine variant (MCYST‐RR), 3, 7‐didesmethylmicrocystin (MCYST‐3, 7‐dDMLR) and 7‐desmethylmicrocystin (MCYST‐7‐DMLR). Furthermore, the antibodies reacted with cell‐extracts of toxic and non‐toxic M. aeruginosa strains. The MAbs can apparently recognize the common configuration, but not the variant‐specific structure, in the microcystin molecules. The non‐toxic strains apparently contain some substance(s) related antigenically to microcystin. The in vivo toxin‐neutralizing ability of MAbs was minimal.
Construction of Mutant Genes for a Non‐Toxic Verotoxin 2 Variant (VT2vp1) of Escherichia coli and Characterization of Purified Mutant Toxins
Authors: Cao Chunyu , Kurazono Hisao , Yamasaki Shinji , Kashiwagi Keiko , Igarashi Kazuei , Takeda Yoshifumi
Published: November 2013

The gene encoding a Verotoxin 2 variant, VTvp1, was mutated by oligonucleotide‐directed site‐specific mutagenesis. Among 6 mutant toxins encoded by the mutated genes, E167Q‐R170L (glutamic acid at position 167 and arginine at position 170 from N‐terminus of the A subunit were replaced by glutamine and leucine, respectively) was found to have markedly decreased activities; inhibition of protein synthesis, Vero cell cytotoxicity and mouse lethality of the purified E167Q‐R170L were 1/1,900, 1/125,000 and 1/2,000, respectively, of those of the purified wild‐type VT2vp1. Since the antigenic property of the E167Q‐R170L was demonstrated to be similar to that of the wild‐type VT2vp1 by Ouchterlony double gel diffusion test and by neutralization test of Vero cell cytotoxicity of the VT2vp1, a possibility to use the mutant VT2vp1, E167Q‐R170L, as a toxoid is discussed.
Coaggregation between Porphyromonas gingivalis and Mutans Streptococci
Authors: Kamaguchi Arihide , Baba Hisae , Hoshi Masaaki , Inomata Koshiro
Published: November 2013

Coaggregation occurred between Porphyromonas gingivalis and mutans streptococci. The coaggregation was completely inhibited by l‐arginine, Nα‐p‐tosyl‐l‐lysine chloromethyl ketone (TLCK), and a trypsin inhibitor, and weakly inhibited by l‐lysine, N‐ethylmaleimide, lysozyme, and human whole saliva. The results of heat and proteinase K treatment suggested that a heat‐labile proteinaceous substance of P. gingivalis and a heat‐stable substance of mutans streptococci may play a role in the coaggregation. Mutans streptococci also aggregated in the presence of the heat‐labile factor in the supernatant of P. gingivalis. The aggregation was also inhibited by l‐arginine, TLCK, and a trypsin inhibitor.