bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Phosphocholine‐Containing Glycoglycerolipids Purified from Mycoplasma fermentans‐Infected Human Helper T‐Cell Culture as Components of M. fermentans
Authors: Matsuda Kazuhiro , Harasawa Ryo , Li Jin‐liang , Kasama Takeshi , Taki Takao , Handa Shizuo , Yamamoto Naoki
Published: November 2013

Previously, we have reported the occurrence of novel phosphocholine‐containing glycoglycerolipids (GGPLs: GGPL‐I and GGPL‐III) in human helper T‐cell culture (MT‐4 cell line) (Matsuda et al, Glycoconjugate J. 10: 340). However, the GGPLs disappeared from the MT‐4 cells after treatment with an antimycoplasma agent. This disappearance suggested the involvement of microorganisms in the GGPL expression. In this paper, we show that the novel lipids are components of Mycoplasma fermentans itself. The supernatant fluid of the antimycoplasma agent‐untreated MT‐4 cell culture produced mycoplasma‐like colonies on PPLO agar plates, and PCR and immunological methods revealed the presence of M. fermentans. GGPLs were expressed again in the treated MT‐4 cells after infection with the isolated M. fermentans. The isolated M. fermentans had glycoglycerolipids corresponding to GGPL‐I and GGPL‐III. Thin‐layer chromatography‐mass spectrometry and immunological analyses showed that these glycoglycerolipids which were derived from the isolated M. fermentans were identical with GGPL‐I and GGPL‐III previously obtained. This is the first report that shows mycoplasma has phosphocholine‐containing glycoglycerolipids.
Typing of Staphylococcus epidermidis Colonizing in Human Nares by Pulsed‐Field Gel Electrophoresis
Authors: Hu Lan , Umeda Akiko , Amako Kazunobu
Published: November 2013

From the nares of 11 healthy adults, 253 strains of coagulase negative staphylococcus were isolated and 88% of them were identified as Staphylococcus epidermidis using the API STAPH system. Chromosomal DNA fingerprinting of the isolated strains revealed that each person carried multiple types of S. epidermidis in his or her nares. The colonization of the strains was not stable; the types of the isolates changed in the first and the second examinations 5 months apart. The results contrasted with previous findings in which only one strain of S. aureus colonized persistently in the nares of healthy adults.
Heat Shock Proteins in the Human Periodontal Disease Process
Authors: Ando Tomohiro , Kato Tetsuo , Ishihara Kazuyuki , Ogiuchi Hideki , Okuda Katsuji
Published: November 2013

The production of HSP by periodontopathic Gram‐negative bacteria was examined by SDS‐PAGE, two dimensional gel electrophoresis, and Western blotting using monoclonal antibodies against HSPs. Strains of Actinobacillus actinomycetemcomitans, Eikenella corrodens, Fusobacterium nucleatum, Prevotella intermedia, Prevotella nigrescens, Prevotella melaninogenica, and Treponema socranskii species produced HSP which reacted with anti‐Yersinia enterocolitica HSP 60 and/or mycobacterial 65‐kDa HSP monoclonal antibodies. It was found that gingival homogenate samples from patients with adult periodontitis reacted with anti‐human HSP 60 and bovine brain HSP 70 monoclonal antibodies. Antibodies which reacted with bacterial HSP were also found in a serum sample from a periodontitis patient. The present study suggests that HSPs are implicated in the human periodontal disease process.
Seroepidemiological Survey ofBartonella (Rochalimaea) henselaein Domestic Cats in Japan
Authors: Ueno Hiroshi , Muramatsu Yasukazu , Chomel Bruno B. , Hohdatsu Tsutomu , Koyama Hiroyuki , Morita Chiharu
Published: November 2013

A total of 199 domestic cat serum samples from 3 geographical areas (northeastern, central and southwestern) of Japan collected between 1992 to 1994 were examined for serum antibody againstBartonella henselaeusing an immunofluorescent assay. The antibody prevalence was 15.1% (30/199). A significant difference in the prevalence ofB. henselaeantibody was observed between the northeastern area (6.3%:3/48) and the central area (22.0%: 13/59) in Japan. There was no significant difference between the average age of seropositive cats (4.39 ±3.26 years) and that of seronegative cats (4.03 ±3.84 years), and also between the frequency of seropositive male cats (16.5%: 15/91) and that of seropositive female cats (11.8%:9/76). This is the first report ofB. henselaeantibodies in cats in Japan.
Correlation between the Presence of Virulence‐Associated Genes as Determined by PCR and Actual Virulence to Mice in Various Strains of Listeria Spp.
Authors: Nishibori Takeaki , Cooray Karven , Xiong Huabao , Kawamura Ikuo , Fujita Masashi , Mitsuyama Masao
Published: November 2013

Five chromosomal genes, prfA, plcA, hlyA, mpl and plcB, are implicated in the virulence of Listeria monocytogenes and some of these genes have been used for the identification of bacteria by polymerase chain reaction (PCR). Using 6 strains of L. monocytogenes and 3 L. innocua strains, the relationship was examined between the presence of five virulence‐associated genes and actual virulence to mice in terms of 50% lethal dose (LD50), bacterial viability in the organ of infected mice and the intracellular growth in cultured macrophages. None of the five genes could be amplified by PCR in all the L. innocua strains and they were actually avirulent to mice. All L. monocytogenes strains were shown to be virulent and to have intact virulence‐associated genes except for the strain ATCC15313. This particular strain was revealed to be avirulent and defective in hlyA and plcA in PCR amplification. It was suggested that PCR detection of genes prfA, mpl, or plcB may not be sufficient to detect virulent strains of L. monocytogenes. It appeared that the ability to produce listeriolysin O (LLO), which is encoded by hlyA, was critical for the expression of virulence regardless of the amount of LLO produced.
Optimum Culture Conditions for Production of Exfoliative Toxin by Staphylococcus hyicus
Authors: Watanabe Takao , Tanabe Taishi , Sato Hisaaki , Fuse Yoko , Ueda Kengo , Nakano Katsushige , Saito Hiroshi , Maehara Nobutoshi
Published: November 2013

Optimum culture conditions for the production of exfoliative toxin by Staphylococcus hyicus (shET) were examined. High shET activity was obtained from the culture filtrate of HI and TY broth inoculated with S. hyicus. The pH in these two media ranged from 7 to 8.5 during bacterial culture, while the lowest pH in TS and BHI broth was less than 6. shET activity in the culture filtrate from TY broth inoculated with 107 CFU of S. hyicus per ml was higher than that in TY broth inoculated with 106 and 108 CFU of bacteria per ml. When shET activity in the culture filtrate was measured under various shaking conditions, the culture filtrate shaken at 75 oscillations per min had the highest shET activity of the five shaking conditions. shET activity of the culture filtrate of TY broth to which protease inhibitor had been added was the same as that of TY broth without inhibitor. shET activity in a shaking culture in an Erlenmeyer flask was also the same as that in sac culture and that in shaking culture using a shaking (Sakaguchi) flask. shET activity in TY broth supplemented with 100 mM glucose was significantly lower than that in TY broth without glucose. Based on the above results, the optimum culture conditions for the production of shET were as follows: inoculation of 3 × 109 CFU of S. hyicus strain P‐1 into 300 ml of TY broth in a 2,000‐ml Erlenmeyer flask, and incubation at 37 C with shaking at 75 oscillations per min. Then shET activity of the culture filtrate under appropriate culture conditions was measured after various incubation periods. shET activity was detected 6 hr after inoculation, reached the maximum (253 exfoliative unit/0.1 ml) at 16 hr and decreased between 20 and 48 hr. Thus, the optimum incubation period was determined to be 16 hr. Then the optimum concentration of ammonium sulfate for isolation of shET from the culture filtrate under appropriate culture conditions was examined. The greatest shET activity was obtained from the fraction salted out with 90% saturated ammonium sulfate. Thus, the optimum concentration of ammonium sulfate for the isolation of shET was determined to be 90% saturation.
Production of Tumor Necrosis Factor‐α in Granulocytopenic Mice with Pulmonary Candidiasis and Its Modification with Granulocyte Colony‐Stimulating Factor
Authors: Futenma Mitsuhiko , Kawakami Kazuyoshi , Saito Atsushi
Published: November 2013

In the present study, a lethal model of pulmonary candidiasis was established using granulocytopenic mice with cyclophosphamide. These mice started to die 1 day after infection and had all died within the next 48 hr. The counts of live C. albicans in the lung gradually increased with time, while the organisms were quickly eliminated in the normal mice. From the histology and measurements on bronchoalveolar lavage fluid (BALF), polymorphonuclear cells (PMN) response was almost zero up to 24 hr, and then a weak but significant response was observed at 48 hr, while a marked accumulation of PMN was detected from as early as 6 hr in normal mice. In contrast, macrophages had accumulated in BALF by 48 hr in granulocytopenic mice, but not in normal mice. Both in serum and BALF, a considerable level of tumor necrosis factor‐α (TNF‐α) was detected from 6 hr, peaking at 24 to 48 hr, while in normal mice the quantity was under the detection limit in serum and very low in BALF. The effects of administering granulocyte colony‐stimulating factor (G‐CSF) on these parameters were next examined. G‐CSF significantly prolonged the survival time of granulocytopenic mice, promoted the clearance of organisms through increasing the counts of PMN in the lung, and strongly inhibited the production of TNF‐α both in BALF and serum. These results suggest that this cytokine does not protect them, but plays some role in their death due to candidial infection in granulocytopenic mice.
Nucleotide Sequence Analysis of Recent Epidemic Strains of Enterovirus 70
Authors: Yamazaki Kenji , Oishi Isao , Minekawa Yoshiichi
Published: November 2013

Nucleotide sequences of the genome of enterovirus 70 (EV70) isolated in Osaka in 1993 were determined, and compared with those of the past epidemic strains. Nucleotide substitution rates in 332 bp, 174 bp, and 178 bp of the genes encoding viral protein (VP)1, VP2, or VP3 were 9.0, 7.5, and 5.6% between Kumoi‐2/93 and J670/71, respectively. Likewise, the putative amino acid substitution rates were 1.8, 0, and 0%. It seems that the epidemic strains of EV70 in Japan have been evolving at a constant high nucleotide substitution rate but almost all the substitutions were synonymous.
Characterization of Nontoxic‐Nonhemagglutinin Component of the Two Types of Progenitor Toxin (M and L) Produced by Clostridium botulinum Type D CB‐16
Authors: Ohyama Tohru , Watanabe Toshihiro , Fujinaga Yukako , Inoue Kaoru , Sunagawa Hiroyuki , Fujii Nobuhiro , Inoue Katsuhiro , Oguma Keiji
Published: November 2013

A 9.8‐kbp DNA fragment which contained a neurotoxin gene and its upstream region was cloned from Clostridium botulinum type D strain CB‐16. Nucleotide sequencing of the fragment revealed that genes encoding for hemagglutinin (HA) subcomponents and one for a nontoxic‐nonhemagglutinin (NTNH) component were located upstream of the neurotoxin gene. This strain produced two toxins of different molecular size (approximately 300 kDa and 500 kDa) which were designated as progenitor toxins (M and L toxins). The molecular size of the NTNH component of L toxin was approximately 130 kDa on SDS‐PAGE and its N‐terminal amino acid sequence was M‐D‐I‐N‐D‐D‐L‐N‐I‐N‐S‐P‐V‐D‐N‐K‐N‐V‐V‐I which agreed with that deduced from the nucleotide sequence. In contrast, the M toxin had a 115‐kDa NTNH component whose N‐terminal sequence was S‐T‐I‐P‐F‐P‐F‐G‐G‐Y‐R‐E‐T‐N‐Y‐I‐E, corresponding to the sequence from Ser141 of the deduced sequence. A 15‐kDa fragment, which was found to be associated with an M toxin preparation, possessed the same N‐terminal amino acid sequence as that of the 130‐kDa NTNH component. Furthermore, five major fragments generated by limited proteolysis with V8 protease were shown to have N‐terminal amino acid sequences identical to those deduced from the nucleotide sequence of 130‐kDa NTNH. These results indicate that the 130‐kDa NTNH of the L toxin is cleaved at a unique site, between Thr and Ser, leading to the 115‐kDa NTNH of the M toxin.
TCR‐Independent Induction of Low Responsiveness by Chemically Fixed Cells in Alloreactive CTL Clones and Its Prevention through Cognate Cell‐Cell Interaction
Authors: Lwin Tint , Nakashima Izumi , Nagase Fumihiko
Published: November 2013

We established BALB/c‐derived CD8+ CTL clones D2‐22 (Vβ 6+), D2‐23 (Vβ 8+) and D2‐24 (Vβ 8+) specific for B10.D2 minor H antigen. D2‐22 and D2‐23 proliferated without producing IL‐2 in response to X‐ray‐irradiated antigenic cells, Con A, aCD3, PMA and IL‐2. Paraformaldehyde‐fixed antigenic spleen cells neither induced proliferation in the presence of costimulatory cells nor inhibited responses to irradiated antigenic cells added simultaneously. Unlike the previously reported results with IL‐2‐producing CTL clones and Th1 clones, the fixed antigenic cells failed to induce antigen‐specific unresponsiveness in these IL‐2‐nonproducing CTL clones. Instead, the responsiveness of these clones to fresh stimulation was found to be reduced severely after 2 days in the culture added with either antigenic or syngeneic fixed cells. Induction of their antigen‐nonspecific low responsiveness by the fixed cells was prevented by adding irradiated syngeneic cells into the culture or even by increasing the concentration of responder D2‐23 cells. Close contact of D2‐23 and irradiated syngeneic cells was required to prevent the reduction of the responsiveness, although this cognate cell‐cell interaction could be replaced by exogenously added IL‐2 or PMA. Cytolytic and tumor cell growth inhibitory activities of D2‐23 were also reduced by incubation with the fixed cells, which was prevented by the addition of irradiated syngeneic cells. These findings showed the unique properties of IL‐2‐nonproducing CTL clones in signal requirements for maintaining normal responsiveness for proliferation and cytolytic activity.
Monoclonal Antibody Developed against a Hemolysin of Bacillus thuringiensis
Authors: Matsuyama Junko , Yamamoto Koichiro , Miwatani Toshio , Honda Takeshi
Published: November 2013

A total of five hybridoma cell lines that produced monoclonal antibodies (MAb) against a hemolysin (Bt‐hemolysin) produced by Bacillus thuringiensis were established and characterized. All of these monoclonal antibodies reacted similarly not only to Bt‐hemolysin but also to a hemolysin (Bc‐hemolysin) produced by B. cereus, suggesting that the two hemolysins are immunologically indistinguishable. The MAb developed in this study was also successfully applied for rapid and simple purification of both Bt‐ and Bc‐hemolysins by immunoaffinity column chromatography. The partial N‐terminal amino acid sequence of the purified hemolysins was determined to be Ile‐Glu‐Gln‐Thr.
Functional Activities of Antibodies Directed against Surface Lipoproteins of Borrelia hermsii
Authors: Sambri Vittorio , Marangoni Antonella , Massaria Francesca , Farencena Aldo , Placa Michelangelo La , Cevenini Roberto
Published: November 2013

Enriched preparations for mouse polyclonal immunoglobulin G (IgG) antibodies reactive with surface‐exposed epitopes (Ab‐SEE) of the 22‐kDa and 24‐kDa membrane lipoproteins of living Borrelia hermsii (HS 1 strain) cells were obtained by an antibody absorption technique using living spirochetes. In vitro, the antibody preparations both inhibited spirochetal growth and were borreliacidal in the presence of complement. The monovalent Fab antibody fragments, prepared from antibody‐enriched preparations, did not inhibit the growth of the bacteria, whereas they killed the bacteria in the presence of complement. The two‐dimension gel electrophoresis of B. hermsii cells showed that 3H‐labeled fatty acids incorporated into the 22‐kDa and 24‐kDa lipoproteins were resolved into one and three compact spots, respectively. The spots were recognized by the Ab‐SEE preparations reactive with the 22‐kDa and 24‐kDa proteins, by Western blotting.
Characterization of Streptococcus sanguis Isolated from Patients with Behçet's Disease
Authors: Yokota Kenji , Hayashi Shynji , Araki Yoshio , Isogai Emiko , Kotake Satoshi , Yoshikawa Kouji , Fujii Nobuhiro , Hirai Yoshikazu , Oguma Keiji
Published: November 2013

The DNA homology and cell wall sugar constituents of eight Streptococcus sanguis(‐like) strains, three isolated from the patients with Behçet's disease (BD114‐23, BD113‐20, BD118‐1), two from patients with Kawasaki disease (MCLS‐1, MCLS‐2), and three type and reference strains of ATCC (ATCC10556T: S. sanguis, ATCC10557: S. oralis, and ATCC10558T: S. gordonii) were analyzed. Strains BD114‐23 and BD118‐1 showed high DNA homology to ATCC10556T, and their cell wall constituents were identical. Conversely, BD113‐20, MCLS‐1, MCLS‐2, and ATCC10557 showed little DNA homology to ATCC10556T and ATCC10558T, but showed approximately 50 to 60% homology to each other. The cell wall constituents of BD113‐20, MCLS‐1, MCLS‐2, and ATCC10557, however, were somewhat different, indicating that some of the clinical isolates have different characters from those of the three ATCC strains.
Isolation, and Virulence Profiles, of Aeromonas hydrophila Implicated in an Outbreak of Food Poisoning in Sweden
Authors: Krovacek Karel , Dumontet Stefano , Eriksson Erik , Baloda Suraj B.
Published: November 2013

A case of food poisoning outbreak involving Aeromonas hydrophila is reported in this study. A group of 27 people consumed a typical Swedish food “landgång” which is a type of smörgåsbord containing shrimps with mayonnaise, liver paté, ham, sausage, and legume salad which was purchased from a food store. Twenty‐two of the 27 persons became ill within 20‐34 hr of consumption of the food and reported the symptoms ranging from severe acute diarrhea, abdominal pain, headache, fever and vomiting. One person also fainted. The symptoms lasted for a couple of days. Of the remaining 5 healthy persons who consumed the left‐over food the next day, 2 became ill with similar symptoms. The bacteriological examination of left‐over food samples resulted in the isolation of A. hydrophila from shrimps with mayonnaise, smoked sausage, liver paté and boiled ham. The total number of A. hydrophila in these foods were log 106 to log >107 organisms per gram of food sample. A. hydrophila was however, not isolated from legume/mayonnaise salad samples. All the food samples tested showed low numbers of other expected food contaminating organisms such as coliforms at 37 C and 44 C, fecal streptococci, Staphylococcus aureus, fungi and yeast etc., while Bacillus cereus, Clostridium perfringens and Salmonella spp. were not detected in the food samples. Investigations of the virulence profiles of the A. hydrophila isolates showed their capacity to produce β‐hemolysin, cytotoxins, cytotonic toxins, enterotoxins, and adhesion to and invasion of human intestinal (Henle 407) cells in culture.
The Measurement of Swimming Velocity of Vibrio cholerae and Pseudomonas aeruginosa Using the Video Tracking Method
Authors: Shigematsu Mika , Meno Yuko , Misumi Hiroyasu , Amako Kazunobu
Published: November 2013

The swimming velocities of two monotrichous flagellated bacteria were measured by a computer‐assisted video tracking method. Tracing the moving path of the individual bacterium revealed that the bacterial cell did not swim continuously in a straight direction, but frequently changed swimming direction and velocity. The average swimming velocities calculated from the 3‐sec path were 75.4 ±9.4 μm/sec in four strains of Vibrio cholerae and 513 ±8.4 μm/sec in five strains of Pseudomonas aeruginosa. These results suggest that V. cholerae swim faster than P. aeruginosa at 30 C in nutrient broth. This method is useful for a detailed analysis of bacterial movement and moving patterns in different environmental conditions.
Monomer of the B Subunit of Heat‐Labile Enterotoxin from Enterotoxigenic Escherichia coli Has Little Ability to Bind to GM1 Ganglioside Compared to Its Coligenoid
Authors: Tsuji Takao , Watanabe Katsumi , Miyama Akio
Published: November 2013

Coligenoid, composed of the B subunit of heat‐labile enterotoxin from enterotoxigenic Escherichia coli, was separated into monomers in the presence of 2% propionic acid containing 6 M urea (pH 3.8). Monomers equilibrated against 0.75% or 0.5% propionic acid containing 3 M urea (pH 3.8) did not reassemble into coligenoid. Complexes of GM1 ganglioside and coligenoid in these buffers were detected by SDS‐polyacrylamide gel electrophoresis, but those of the GM1 ganglioside and monomers were not. The binding ability of monomer to GM1 ganglioside in these buffers was about 1% of that of normal coligenoid by GM1‐enzyme‐linked immunosorbent assay. Moreover, monomers in these buffers reassembled into coligenoid by buffering against original TEAN buffer, and the binding ability of the resulting coligenoid to GM1 ganglioside was identical to that of native coligenoid. These data suggest that although coligenoid formation is important for the receptor binding of the B subunit, little binding ability to GM1 ganglioside remains in monomer of the B subunit.
Construction of a Combined NotI/SmaI Physical and Genetic Map of Moraxella (Branhamella) catarrhalis Strain ATCC25238
Authors: Furihata Kakuya , Sato Kaori , Matsumoto Hideki
Published: November 2013

Using pulsed field gel electrophoresis (PFGE) and Southern hybridization techniques, a physical map of Moraxella catarrhalis strain ATCC25238 was constructed to provide basic genetic knowledge of this bacterium that has attracted attention in recent years as a human pathogen. Restriction endonuclease NotI cut the genome into 10 fragments and SmaI into 9, and the molecular size of the genome was estimated to be 1,940 kilobases. Location of the 12 genes participating in the biosynthesis of purine, pyrimidine and nine kinds of amino acids were determined on the circular physical map of the strain.
Analysis of the Relationship between Cellular Thymidine Kinase Activity and Virulence of Thymidine Kinase‐Negative Herpes Simplex Virus Types 1 and 2
Authors: Suzutani Tatsuo , Koyano Shin , Takada Minoru , Yoshida Itsuro , Azuma Masanobu
Published: November 2013

The virulence of thymidine kinase‐negative herpes simplex virus type 1 (HSV‐1; VRTK− strain) and type 2 (HSV‐2; UWTK− strain) was studied in comparison with that of their parental strains (VR‐3 and UW‐268, respectively) in an encephalitis model of adult (4‐week‐old) and newborn (3‐day‐old) mice. Viral thymidine kinase (TK) activity was essential for the maximum expression of virulence of HSV‐1, because the 50% lethal dose (LD50) of VRTK− was 60 times higher than that of VR‐3 in the brains of newborn mice expressing high levels of cellular TK activity. However, the UWTK− strain showed the same virulence as the parental strain in newborn mice, despite the lack virulence in adults, suggesting that replication of the UWTK− strain was completely supported by cellular TK activity. This difference in the role of viral and cellular TKs for virus growth between HSV‐1 and HSV‐2 was confirmed with the one‐step growth of virus strains in L‐M and L‐M(TK−) cells.
Growth and Cytopathogenicity of Herpes Simplex Virus in a Macrophage Cell Line, RAW264: A Good Indicator of Intraperitoneal Pathogenicity
Authors: Jiang Yue‐Mei , Daikoku Tohru , Yamamoto Mitsuaki , Morishima Tsuneo , Nishiyama Yukihiro
Published: November 2013

Macrophages are known to play a critical role in host resistance to herpes simplex virus (HSV). In this study, we investigate the interaction between various HSV strains with different virulence and a murine macrophage cell line, RAW264. Highly attenuated strains replicated poorly in RAW264 cells and were cleared from the cultures. For the eleven viruses tested, there was good correlation between intraperitoneal pathogenicity for adult mice and replication in RAW264 cells. It was also shown that interferon α/β was involved in restricted replication of some strains.
Specific Deposition of Passively Transferred Monoclonal Antibodies against Herpes Simplex Virus Type 1 in Rat Brain Infected with the Virus
Authors: Tamada Koh‐ichi , Fujinaga Shigeki , Watanabe Rihito , Yamashita Ryoko , Takeuchi Yoshinao , Osano Mitsuru
Published: November 2013

The kinetics of human monoclonal antibody (anti‐gB) to herpes simplex virus type 1 (HSV‐1) were investigated after intravenous injection of anti‐gB into an HSV‐1 encephalitis animal model. Immunohistochemical study revealed specific deposition of passively tansferred anti‐gB in the hippocampus and thalamus of the infected rat brain, and it bound to the same neurons in which HSV‐1 antigen was positively stained. To examine the macroscopic distribution of anti‐gB in the infected brain, we undertook an 125I‐labeled anti‐gB injection study, and the same distribution of 125I‐labeled anti‐gB deposition was observed by brain semimicroautoradiography as in the immunohistochemical study. These results suggest that anti‐gB easily permeates the capillary wall and is deposited in the inflammatory site where HSV‐1‐specific antigen is detectable. The use of radioisotope‐labeled anti‐gB injection and external brain imaging could lead to a noninvasive diagnostic tool for the early detection of HSV‐1 antigen in cases of suspected HSV‐1 encephalitis.
Flow Cytometric Analysis Using Lipophilic Dye PKH‐2 for Adhesion of Vibrio cholerae to Intestine 407 Cells
Authors: Taguchi Haruhiko , Osaki Takako , Yamaguchi Hiroyuki , Kamiya Shigeru
Published: November 2013

A comparative study of indirect and direct flow cytometric analysis for adherence of Vibrio cholerae to Intestine 407 cells was performed. The direct flow cytometric analysis employed the lipophilic dye PKH‐2. It was concluded that direct flow cytometry using the lipophilic dye PKH‐2 is useful and convenient for analyzing bacterium‐host cell interactions, since it does not require any specific antibody as the first antibody.
Recognition of Nonstructural Protein Peptides by Cytotoxic T Lymphocytes Raised against Japanese Encephalitis Virus
Authors: Murali‐Krishna Kaja , Ramireddy Bommireddy , Ravi Vasanthapuram , Manjunath Ramanathapuram
Published: November 2013

We have previously reported that Lyt2+ cytotoxic T lymphocytes (CTL) can be raised against Japanese encephalitis virus (JEV) in BALB/c mice. In order to confirm the presence of H‐2Kd‐restricted CTL and to examine their cross‐recognition of West Nile virus (WNV), we tested the capacity of anti‐JEV CTL to lyse uninfected syngeneic target cells that were pulsed with synthetic peptides. The sequence of the synthetic peptides was predicted based upon the H‐2Kd binding consensus motif. We show here that preincubation of uninfected syngeneic targets (P388D1) with JEV NS1‐ and NS3‐derived peptides [NS1 (891‐899) and NS3 (1804‐1812)], but not with JEV NS5‐derived peptide [NS5 (3370‐3378)], partially sensitized them for lysis by polyclonal anti‐JEV CTL. These results indicate the CTL recognition of NS1‐ and NS3‐derived peptides of JEV.
Suppression of HIV‐1 Reverse Transcriptase Activity by Culture Supernatants of Mycoplasmas
Authors: Shang Hong , Miyakawa Yozo , Sasaki Tsuguo , Nakashima Hideki , Ito Masahiko
Published: November 2013

Coinfection with mycoplasmas has been shown to enhance cytopathic changes in T lymphocytes in culture brought about by human immunodeficiency virus type‐1 (HIV‐1), accelerate disease progression, and suppress reverse transcriptase (RT) activity simultaneously. We attempted to identify the components in culture supernatants of mycoplasmas which suppress RT activity. The marked inhibitory effect on RT by culture supernatants was dependent upon Mg2+. The culture supernatants exhibited the activities of DNase and RNase, which degraded the products and substrates in RT assay, respectively. Gel filtration studies revealed that two major protein peaks, peak 1 (MW 67‐100 kDa) and peak 2 (MW 10‐25 kDa), exhibited DNase and/or RNase activities, and that both peaks contained a significant degree of inhibitory activity on RT. These results indicate that suppression of RT activity by the culture supernatants of mycoplasmas is due to DNase and RNase activities in the culture supernatants. The results of the present investigation suggest that RT assay of certain biological materials that are contaminated with mycoplasmas must be conducted carefully.
Reduced Anti‐Pseudomonas aeruginosa Activity of a Cephalosporin, Cefodizime, in Rats Whose Neutrophils Were Selectively Depleted by a Monoclonal Antibody
Authors: Araki Akemi , Sendo Fujiro
Published: November 2013

In order to clarify the mechanisms of the in vivo antibacterial activity of a cephalosporin, cefodizime (CDZM), the effect of this antibiotic on Pseudomonas aeruginosa E7 infection was examined in rats whose neutrophils had been selectively depleted by monoclonal antibody RP‐3. CDZM was less effective in RP‐3‐treated rats than in untreated rats. However, treatment of rats with recombinant human granulocyte‐colony stimulating factor (rhG‐CSF) augmented the in vivo activity of this antibiotic. Furthermore, the in vivo antibacterial activity of two other cephalosporins, cefpimizole (CPIZ) and cefoperazone (CPZ), was bilaterally affected by a rise or fall in the neutrophil number, although to a lesser degree than was the case with CDZM. Taken together, neutrophils play an important role in the in vivo antibacterial activity of certain cephalosporins.
Electron Microscopic Observation of the Antibody‐Induced Capsular Swelling Phenomenon in Klebsiella pneumoniae
Authors: Meno Yuko , Amako Kazunobu
Published: November 2013

The capsular swelling phenomenon of Klebsiella pneumoniae strain 277 was examined morphologically using the electron microscopy techniques of freeze‐substitution. The capsules of strain 277 measured about 52 nm in thickness, and were composed of a number of fine fibers. After treating the bacteria with anti‐capsular serum, the size of the capsules increased to about twice the normal size and they lost their electron density. The capsular fibers that are tightly packed in normal cells became loose and thus the identification of the individual capsular fibers was difficult in the swollen capsules. Capsule swelling was induced by washing the cells with phosphate‐buffered saline. The removal of either divalent cations or some other materials might thus be important for maintaining the normal capsule structure. The mechanism of the swelling phenomenon was also discussed.
Sulfonamide Resistance Gene in a Transferable R Plasmid of Pasteurella piscicida
Authors: Kim Eun‐Heui , Aoki Takashi
Published: November 2013

The sulfonamide resistance (SAr) determinant was cloned from a transferable R plasmid of Pasteurella piscicida, pSP9351, and the sequence was determined. The resistance gene (pp‐sul) was localized to an approximately 1‐kb region that includes the PstI‐EcoRI site in the restriction map. An open reading frame coding a sul II‐type gene composed of 810 nucleotides was identified. A direct repeat sequence was shown in the 5′ flanking region of pp‐sul, and a plasmid recombinational event may have occurred during the construction of pSP9351. In the 3′ flanking region of the gene, a sequence homologous to the 5′ noncoding sequence of the trimethoprim resistance gene, dhfr IX was found.
Hepatitis C Virus Infection‐Associated Markers in Sera from Blood Donors in Surabaya, Indonesia
Authors: Darmadi Siswanto , Soetjipto  , Handajani Retno , Lusida Maria Inge , Soemarto  , Sakugawa Hiroshi , Ishido Satoshi , Hotta Hak
Published: November 2013

Among 2,233 sera obtained from volunteer blood donors, 259 (11.6%) showed elevated alanine aminotransferase (ALT) levels. A second‐generation enzyme‐linked immunosorbent assay (ELISA) revealed that 23 (8.9%) of the 259 sera were positive for antibodies against hepatitis C virus (HCV), whereas only 9 (1.4%) of 646 sera randomly collected from blood donors with normal ALT levels were positive (P<0.001). The overall prevalence of anti‐HCV antibodies among blood donors was estimated to be 2.3%. HCV RNA was detected in 19 (83%) of the 23 anti‐HCV‐positive sera with elevated ALT levels, and 8 (89%) of the 9 sera with normal ALT levels. Among the anti‐HCV‐positive sera, IgM anti‐HCV was detected in 5 (22%) of 23 sera with elevated ALT levels and in 2 (22%) of 9 sera with normal ALT levels. All of the IgM anti‐HCV‐positive sera were positive for HCV RNA, irrespective of ALT levels.
Biological Mimicry of the Bluetongue Virus Core Protein VP7 by Rabbit Anti‐Idiotype
Authors: Lin Min , Zhou En‐Min
Published: November 2013

A subpopulation of rabbit polyclonal anti‐idiotypic antibody (anti‐Id) was previously produced to a murine monoclonal antibody (mAb) (M1875) specific for the bluetongue virus core protein VP7. In this report, mimicry of VP7 by this anti‐Id (designated RAb2‐A) was functionally analyzed through immunization of Balb/c mice with RAb2‐A or purified VP7. Animals immunized with RAb2‐A were able to produce an M1875‐like Ab3 antibody response with idiotype and epitope specificity resembling that of M1875 without subsequent exposure to the nominal antigen. This conclusion was supported by experiments showing that the RAb2‐A‐induced Ab3 antibodies (i) reacted specifically with the immunizing anti‐Id; (ii) were capable of binding VP7; (iii) inhibited M1875 from binding to VP7; and (iv) inhibited M1875 from binding to RAb2‐A. Similarly, mice immunized with purified VP7 also produced antibodies that exhibited characteristics such as idiotype and epitope specificity in common with M1875. No antibody response to VP7 was detected in control groups of mice immunized with either normal rabbit IgG or BHK‐21 cell components. Therefore, it can be concluded that rabbit anti‐Id RAb2‐A mimics an M1875‐defined VP7 epitope sufficiently to function as a surrogate antigen for inducing an anti‐bluetongue virus response.
Involvement of Two Types of TNF Receptor in TNF‐α Induced Neutrophil Apoptosis
Authors: Gon Songi , Gatanaga Tetsuya , Sendo Fujiro
Published: November 2013

We previously demonstrated that tumor necrosis factor‐α (TNF‐α) induces rapid human neutrophil apoptosis. In this paper, we examined which of the TNF receptors, p55 kDa TNF receptor (55‐R) or p75 kDa TNF receptor (75‐R), or both are involved in this process using specific rabbit antisera. Antibodies to 55‐R (anti55‐R) or 75‐R (anti75‐R) alone did not induce neutrophil apoptosis. Further addition of cycloheximide and anti‐rabbit immunoglobulin to anti55‐R but not to anti75‐R accelerated apoptosis, although anti75‐R augmented the capacity of anti55‐R to do so. These results suggest that 55‐R is a prerequisite for TNF‐α induced neutrophil apoptosis.
Failure to Reject an Allografted Tumor after Elimination of Macrophages in Mice
Authors: Ushio Yumiko , Yamamoto Naoki , Sanchez‐Bueno Antonio , Yoshida Ryotaro
Published: November 2013

After an i.p. transplantation of an allogeneic tumor (Meth A) to C57BL/6 mice, a macrophage (MΦ)‐rich, non‐T, non‐NK cell population is induced as the major infiltrate and cytotoxic cells. We here evaluated the role of the MΦs in the rejection of allografted Meth A cells and characterized the MΦs in comparison with other well‐known MΦs. At all time intervals after transplantation, the highest cytotoxic activities against Meth A tumor were obtained with the MΦ‐rich population. In addition, the lymphocyte‐rich population had a significant but low cytotoxic activity, whereas two other population types, granulocytes and large granular cells, were inactive. When the MΦ‐rich or the T cell‐depleted MΦ‐rich population was i.p. transplanted simultaneously with Meth A cells into untreated C57BL/6 mice, the tumor cells were rejected without growth. After specific elimination of MΦs by in vivo application of dichloromethylene diphosphonate‐containing liposomes, the cytotoxic activity against Meth A cells was hardly induced at the transplantation site of Meth A cells and the allografted Meth A tumor continued to grow, indicating that a type of MΦ is the effector cell essential for the rejection. In contrast to other well‐known MΦs, the cytotoxic activity against Meth A cells was cell‐to‐cell contact dependent and soluble factor (e.g., NO and TNF‐α) independent. Moreover, the cytotoxic activity of the MΦs (H‐2b) against 51Cr‐labeled Meth A (H‐2d) cells was inhibited by the addition of unlabeled H‐2d, but not H‐2a, H‐2k or H‐2b, lymphoblasts as well as Meth A cells, implying the specific interaction of the MΦs with H‐2d cells.
Seroprevalence of Helicobacter pylori Infection in Patients with Connective Tissue Diseases
Authors: Showji Yumiko , Nozawa Ryushi , Sato Koji , Suzuki Harumi
Published: November 2013

To assess the possibility that Helicobacter pylori might be an etiologic agent, titers of anti‐H. pylori IgG in sera of patients with connective tissue diseases [rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), polymyositis or dermatomyositis (PM/DM), progressive systemic sclerosis (PSS), mixed connective tissue disease (MCTD) and Sjögren's syndrome (SjS)] were compared with those of non‐patient (healthy) volunteers and of patients with chronic pulmonary diseases (CPD) by ELISA using an extract of sonicated H. pylori as the antigen. Among patients with connective tissue diseases, those with SLE and RA had anti‐H. pylori titers as low as healthy volunteers. Patients with SjS had much higher average titers than patients with CPD (P<0.05). We previously reported that levels of myeloid calcium‐binding protein (MRP8 and MRP14) were elevated in the serum of patients with connective tissue diseases. No correlation was found between serum levels of anti‐H. pylori IgG and of MRP, a novel marker of inflammation. Furthermore, sera with high IgG titers were selected, and their reactivity with the H. pylori antigen were analyzed by Western blotting. H. pylori antigens with a variety of molecular masses were immunostained with sera from patients and from healthy volunteers, but a 16‐kDa antigen was only immunostained by reaction with the sera of patients with MCTD and SjS, although the number of test samples was small.
Cloning and Sequencing of the Klebsiella K‐36 astA Gene, Encoding an Arylsulfate Sulfotransferase
Authors: Baek Moon‐Chang , Kim Sook‐Kyung , Kim Dong‐Hyun , Kim Byong‐Kak , Choi Eung‐Chil
Published: November 2013

A gene‐encoding arylsulfate sulfotransferase (ASST) was cloned from a Klebsiella K‐36 genomic library. ASST tranfers a sulfate group from phenolic sulfate esters to a phenolic acceptor substrate. The gene, designated astA, was subcloned into vector pGEM3Zf(–) and sequenced. Recombinant clone‐harbouring astA was directly identified using a fluorescent product. The nucleotide sequencing revealed an open reading frame (ORF) of 2,082 bp encoding a protein of 694 amino acids with a secretory signal sequence. A protein of similar size was visualized after in vitro transcription and translation using a plasmid carrying the cloned 3.1‐kb fragment as a template. The N‐terminal amino acid sequence of the purified processed protein was found to be identical to that predicted from the gene sequence. When searching the database for astA nucleotide or its deduced amino acid sequence, no significant homology to any sequence was found.
High Levels of sTNFR p75 and TNFα in Dengue‐Infected Patients
Authors: Hober Didier , Delannoy Anne‐Sophie , Benyoucef Samira , De Groote Donat , Wattré Pierre
Published: November 2013

Soluble forms of the two molecular species of the cell surface TNF receptors (sTNFR p55 and sTNFR p75) can reduce the activity of TNFα but they may also enhance its function by stabilizing the active TNFα oligomer. Considering the pathophysiological importance of sTNFR p75 for the regulation of the bioavailability of TNFα in the body, we determined the serum levels of sTNFR p75 and TNFα in 45 children and 28 adults with laboratory‐confirmed dengue infection by using immunoassays. The serum samples were obtained from day 1 to day 15 after the onset of the disease during the 1989–1990 outbreak of dengue‐3 in Tahiti, French Polynesia. The patients were clinically classified as having dengue hemorrhagic fever (DHF) and graded according to the criteria of the World Health Organization (WHO) into four grades from less severe (grade I) to severe (grade IV). The sera of both children and adult patients of all severity grades contained higher levels of sTNFR p75 than the sera of control subjects. Although high levels of TNFα were also detected in children and adults among grade I, II, III and IV patients, we found no correlation between sTNFR p75 and TNFα. We observed in adults a moderate elevation of sTNFR p75 and TNFα in sera compared with that observed in children. The raised levels of immunoreactive sTNFR p75 and TNFα in all clinical groups of dengue‐infected patients strongly indicate activation of the TNFα system during dengue infection. The balance between sTNFR p75 and TNFα may be altered in dengue infection. Further investigations are needed to understand the role of sTNFR p75 and TNFα in the pathogenesis of DHF and to improve the management of dengue infection.
Characterization of the Growth Factor Activity of Amniotic Fluid on Cells from Hematopoietic and Lymphoid Organs of Different Life Stages
Authors: Heidari Zohreh , Isobe Ken‐Ichi , Goto Setsuko , Nakashima Izumi , Kiuchi Kazutoshi , Tomoda Yutaka
Published: November 2013

We investigated the proliferation‐promoting effects of murine amniotic fluid (MAF) on in vitro cultured cells originally obtained from murine hematopoietic and lymphoid organs at different life stages. MAF promoted proliferation of the fetal liver cells (FLC), newborn spleen cells and adult bone marrow cells. The proliferation‐promoting activity of MAF was extended to liver cells and spleen cells from mice younger than 2 weeks old. MAF did not, however, promote the proliferation of newborn or adult thymocytes, or of spleen cells, liver cells or peritoneal cells from 2‐week‐old or older mice. Rather, it partially inhibited the proliferation of spleen cells, thymocytes and peritoneal cells from 1‐year‐old mice. These results suggest that MAF contains growth factors for hematopoietic stem cells but not for either mature or immature T lymphocytes. Supporting this view, the MAF activity was partially neutralized by a polyclonal anti‐mouse stem cell factor (SCF) antibody. Moreover, the immunoblotting of MAF against anti‐mouse SCF antibody revealed a band at 30–32 kDa corresponding to the previously reported SCF. Interestingly, MAF was able to maintain FLC and adult bone marrow cells alive in culture for a relatively long time (2 weeks). The MAF activity was further shown to be partially and cell type‐dependently antagonized by TNF‐α and TGF‐β. These results provided evidence that MAF contains potentially multiple growth factors preferentially affecting the early stage of hematopoiesis, one of which is SCF.
Assessment of the Significance of Virulence Factors of Uropathogenic Escherichia coli in Experimental Urinary Tract Infection in Mice
Authors: Yamamoto Shingo , Nakata Katsuhisa , Yuri Kazuyo , Katae Hiromi , Terai Akito , Kurazono Hisao , Takeda Yoshifumi , Yoshida Osamu
Published: November 2013

Four Escherichia coli strains, isolated from cystitis patients, belonging to serotype O2:H− and possessing different combinations of urovirulence factors were examined in an experimental pyelonephritis mouse model to assess the relative importance of virulence factors in causation of urinary tract infections (UTI). The results suggest not only that the each virulence factor has a role in causation of UTI but also that the presence of P fimbriae and production of hemolysin significantly reduced the LD50 and ID50 of the strains in the mouse model. The results also demonstrate that the presence of additional virulence factors acts in an additive or synergetic fashion enhancing the cumulative impact of the strain.
A Modified PCR‐Based Method for Rapid Non‐Radioactive Detection of Clinically Important Pathogens
Authors: Gorelov Victor N. , Dumon Kristoffel , Barteneva Natalia S. , Röher Hans‐Dieter , Goretzki Peter E.
Published: November 2013

We have devised a sensitive and rapid method for the detection of several bacterial pathogens in clinical specimens using PCR. This method has been named Direct Labeling and Detection Procedure (DLDP) and is based on the direct incorporation of a nonradioactive digoxigenin label (DIG‐11‐dUTP) into a microbial species‐specific gene fragment during amplification. Following amplification, the resulting PCR products are cleansed of nonincorporated DIG‐11‐dUTP, spotted onto a nylon membrane, fixed by UV‐crosslinking and the labeled DNA is visualized by digoxigenin detection reagents. Using cultivated reference bacteria (Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa) we were able to demonstrate a rapid and sensitive detection of < 20 CFU of bacteria in human secretions (sputum, urine, mucous). The present study suggests that DLDP can be used as a reliable method for indication of bacteria in clinical or environmental specimens with the proviso that the selected corresponding oligonucleotide primers provide amplification of strong species‐specific genes.
Development of Functional Rat‐Derived T Cells in SCID Mice Engrafted with the Fetal Thymus of LEC Rats Which Are Defective in CD4+ T Cells
Authors: Chai Jian‐Guo , Sakai Tohru , Hisaeda Hajime , Nagasawa Hideyuki , Yasutomo Koji , Furukawa Atuko , Ishikawa Hiroyuki , Maekawa Yoichi , Uehara Hisanori , Izumi Keisuke , Matsumoto Kozo , Himeno Kunisuke
Published: November 2013

We reported that LEC rats are genetically deficient in the development of thymic CD4+8− cells and that this defect is caused by bone marrow (BM)‐derived stem cells. To determine which BM‐derived cells are responsible for the arrest of T‐cell development in LEC rats, fetal thymuses of LEC rats, or LEA rats which bear the same major histocompatibility complex (MHC) as LEC rats but are immunologically normal, were engrafted under the kidney capsule of severe combined immunodeficiency (SCID) mice (LEC‐TG and LEA‐TG mice, respectively). We then examined the differentiation of T cells and their immunological functions in the SCID mice. A large number of rat‐derived CD4+ T cells appeared in the peripheral blood, lymph nodes (LN) and spleens in LEC‐TG mice. Furthermore, the peripheral LN cells in LEC‐TG mice appeared to be functional. These cells produced IL‐2 upon Con A stimulation, whereas LN cells from LEC rats produced no IL‐2 in the same conditions. Thymopoiesis was observed at 3 weeks in LEC‐TG as well as LEA‐TG mice. The distribution of thymocyte subsets with respect to CD4 and CD8 expression in LEC‐TG mice closely resembled that of LEA rat thymus and that in LEA‐TG mice, suggesting that normal T‐cell differentiation occurred in LEC‐TG mice. The results indicated that BM‐derived progenitor T cells of LEC rats could differentiate to functional CD4+ T cells.
Serological Study of Bartonella henselae in Cat Scratch Disease in Japan
Authors: Yoshida Hiroshi , Kusaba Nobuhide , Omachi Kei , Miyazaki Norihisa , Yamawaki Makoto , Tsuji Yuichiro , Nakahara Keita , Sumino Michihiro , Noudomi Masanori , Shimokawa Yutaka , Tanikawa Kyuichi
Published: November 2013

It has become clear that Bartonella henselae is a common cause of cat scratch disease (CSD). The indirect fluorescence antibody (IFA) test for detection of IgG and IgM antibodies to B. henselae concerning CSD showed that 5 (50%) of 10 patients with CSD had a serum IgG antibody titer of 1:128 or more and that 2 (20%) patients had a serum IgM antibody titer of 1:20 or more. One of 7 asymptomatic members of patients' families (14%) had IgG antibody to B. henselae at a titer of 1:256. IgM antibody to B. henselae was not detected in sera from the patients' families. Both IgG and IgM antibodies to B. henselae were not detected in sera from the healthy control group. These data suggest that B. henselae may be a cause of CSD in Japan.