bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vitro Selection of Variants of Herpes Simplex Virus Type 1 Which Differ in Cytopathic Changes
Authors: Padilla Jorge , Yamada Masao , Takahashi Yasushi , Tsukazaki Takashi , Nakamura Jun , Yoshida Mariko , Uno Fumio , Arao Yujiro , Nii Shiro
Published: November 2013

To analyze the mechanisms for in vitro emergence of the syncytial variants of herpes simplex virus type 1 (HSV‐1), several cell lines were infected with a mixture of equal amounts of two HSV‐1 variants, one syncytial and the other non‐syncytial, and changes in their relative abundance were monitored during passage. With a combination of two variants of the Miyama strain of HSV‐1, the syncytial variant became dominant during passage in Vero, RK‐13 and FL cells. On the other hand, the ratios of the two variants remained around 1:1 during the passage in HEp‐2, MGC and HEL cells. In another set of variants of the SKO strain of HSV‐1, the outcomes were different from those of the Miyama strain in the FL, MGC and HEp‐2 cells. The ratios of the two variants remained around 1:1 during passage in FL cells, while the non‐syncytial variant became dominant during passage in MGC and HEp‐2 cells. In addition, we examined the effects of a complement and interferon‐β (IFN‐β) on the outcome of the selection. As a result, the complement slowed the selection of a syncytial variant, whereas IFN‐β facilitated it.
Thiadiazole Derivatives: Highly Potent and Selective Inhibitors of Human Immunodeficiency Virus Type 1 (HIV‐1) Replications In Vitro
Authors: Fujiwara Masatoshi , Ijichi Katsushi , Hanasaki Yasuaki , Ide Teruhiko , Katsuura Kimio , Takayama Hiromitsu , Aimi Norio , Shigeta Shiro , Konno Kenji , Yokota Tomoyuki , Baba Masanori
Published: November 2013

We have recently reported that thiadiazole (TDA) derivatives are highly potent inhibitors of human immunodeficiency virus type 1 (HIV‐1) replication. These compounds belong to the family of nonnucleoside reverse transcriptase inhibitors (NNRTIs). In an attempt to develop more effective and pharmacologically favorable compounds, novel TDA derivatives have been synthesized and examined for their anti‐HIV‐1 activity in vitro. Among them, RD4‐2217 was found to be the most potent inhibitor of HIV‐1 replication. It inhibited replication of the HTLV‐IIIB strain in MT‐4 cells at a concentration of 6 nM. RD4‐2217 was also inhibitory to clinical isolates and zidovudine‐resistant mutants of HIV‐1. The combination of RD4‐2217 with zidovudine or the protease inhibitor A‐75925 synergistically inhibited HIV‐1 replication. Studies on the emergence of drug‐resistant mutants revealed that, although much higher concentrations (1‐10 μM) were required, RD4‐2217 completely suppressed the breakthrough of HIV‐1 in the supernatants during long‐term culturing of infected cells. Furthermore, RD4‐2217 at low concentrations (10 or 100 nM), in combination with zidovudine, also completely inhibited viral breakthrough. In addition, RD4‐2217 had lower lipophilicity and improved protein binding as compared to its congener RD4‐2024 and loviride. These results suggest that RD4‐2217, one of the TDA derivatives, is worth pursuing as a candidate drug for the treatment of HIV‐1 infections.
Breast Milk Is Not a Significant Source for Early Epstein‐Barr Virus or Human Herpesvirus 6 Infection in Infants: A Seroepidemiologic Study in 2 Endemic Areas of Human T‐Cell Lymphotropic Virus Type I in Japan
Authors: Kusuhara Koichi , Takabayashi Akira , Ueda Kohji , Hidaka Yasufumi , Minamishima Ikuko , Take Hiromichi , Fujioka Katsuyoshi , Imai Shosuke , Osato Toyoro
Published: November 2013

In order to evaluate the possibility of Epstein‐Barr virus (EBV) and human herpesvirus 6 (HHV‐6) transmission via breast milk, a total of 331 serum specimens collected from bottle‐fed and breast‐fed children and their mothers, in 2 endemic areas of human T‐cell lymphotropic virus type I (HTLV‐I) in Japan, were assayed for antibodies to EBV and HHV‐6. The seroprevalences of EBV and HHV‐6 were over 95% both in the mothers of bottle‐fed children and in those of breast‐fed children. The seroprevalence of EBV at 12–23 months of age was 54.5% (36/66) and 55.8% (24/43) in breast‐fed children and bottle‐fed children, respectively. The seroprevalence of HHV‐6 at 12–23 months of age was 90.9% (60/66) and 93.0% (40/43) in breast‐fed children and bottle‐fed children, respectively. No difference was observed between the seroprevalences of EBV and HHV‐6 in breast‐fed and bottle‐fed children at 12–23 months of age. Our seroepidemiologic data indicate that breast milk is not a significant source of early EBV or HHV‐6 infection in infancy.
Further Clonal Expansion of T Cells upon Rechallenge of Superantigen Staphylococcal Enterotoxin A
Authors: Aoki Yoshiyasu , Yoshikai Yasunobu
Published: November 2013

Superantigens are known to induce clonal anergy and/or deletion in reactive T cells peripherally. This study was undertaken to investigate the T‐cell status early after exposure to staphylococcal enterotoxin A (SEA) in vivo and in vitro. At the peak of clonal expansion following the administration of 5 μg SEA (i.e., 2 days after the injection), C57BL/6 mice were rechallenged with the same dose of SEA in vivo. The secondary stimulation augmented clonal expansion of the T cells bearing Vβ3 and Vβ11 in both CD4+ and CD8+ populations. In vitro restimulation of the spleen cells taken from the SEA‐primed mice also induced further expansion of the Vβ3+ T cells during 2 days of culturing, whereas without restimulation, a marked reduction of Vβ3+ T cells occurred. The spleen cells from the SEA‐primed mice were hyper‐reactive to in vitro restimulation with SEA as measured by 3H‐TdR uptake on day 1 of culturing, but augmented proliferation leveled off thereafter. By day 3, the values of 3H‐TdR uptake were less than 20% of those of the controls in which spleen cells from native mice were stimulated with SEA in vitro. These results suggest that T cells exposed to SEA in vivo are still capable of proliferating upon SEA rechallenge, but subsequently, the proliferation starts to wane.
Analysis of Cytokine Producing Activity of Intestinal Intraepithelial T Cells from TCR β‐Chain and 5‐Chain Mutant Mice
Authors: Kohyama Masako , Hachimura Satoshi , Nanno Masanobu , Ishikawa Hiromichi , Kaminogawa Shuichi
Published: November 2013

Intestinal intraepithelial T cells (IELs) expressing either 78 TCR or αβ TCR have been proposed to play an important role in the regulation of intestinal epithelia by producing cytokines that directly influence the adjoining intestinal epithelial cell (IEC) functions. To illuminate this issue, we utilized TCR mutant mice to obtain γδ IELs, αβ IELs and mixed γδ and αβ IELs from corresponding αβ T‐cell‐deficient (β−/‐), γδ T‐cell‐deficient (δ−/‐) and wild‐type (WT) littermate mice. The production of IFN‐γ by these IELs as well as the mRNA for IFN‐γ, TGF‐α, TGF‐β1, TNF‐α and TNF‐β in these IELs, in conjunction with the effect of produced cytokines on the expression of class II MHC molecules by the in vitro cell line IEC‐6, were investigated. IFN‐γ and TGF‐α specific mRNA were detectable in all freshly isolated γδ, αβ and WT IELs. In addition to the IFN‐γ and TGF‐α mRNA, αβ and WT IELs that had been activated in culture plates coated with anti‐CD3 mAb contained mRNA for TGF‐β1 and TNF‐β proteins. In the cultured γδ IELs, however, the signals for IFN‐γ and TGF‐α transcripts were weak, and mRNA for the latter two cytokines was almost undetectable. Supernatants from in vitro culturing of αβ and WT IELs but not γδ IELs induced class II MHC gene expression in IEC‐6, whereas, in the presence of anti‐IFN‐γ mAb, the same culture supernatants failed to do so. In fact, the concentration of IFN‐γ in supernatants from αβ and WT IEL cultures was ten‐ to twentyfold higher than that in the supernatant from the γδ IEL culture. Finally, TGF‐α specific mRNA was not detectable in the γδ and αβ IELs even after in vitro activation. These results indicate that αβ IELs are superior to γδ IELs in the ability to produce IFN‐γ, TGF‐β1, TGF‐α and TNF‐β through TCR crosslinking primary in vitro stimulation.
Experimental Helicobacter pylori Infection in Association with Other Bacteria
Authors: Isogai Hiroshi , Isogai Emiko , Hayashi Shunji , Kimura Koichi , Kubota Tom , Fujii Nobuhiro , Oguma Keiji
Published: November 2013

We performed surgical treatment on normal ddY mice before Helicobacter pylori inoculation. The treatment was expected to obstruct bacterial flow out of the stomach and increase the chance of bacterial attachment to the gastric epithelium in mice. The bacterial challenge induced inflammation in the stomach. H. pylori was recovered from the stomach throughout the observation period. Lactobacilli and streptococci tended to relate to the increase in number of H. pylori recovered. Pretreatment with atropine was considered to confuse the gastric flora and affect the number of H. pylori recovered. These results suggested that a certain amount of time is necessary for H. pylori to contact with the gastric epithelium and that the composition of flora is important for the establishment of H. pylori infection.
Moraxella (Branhamella) catarrhalis Adherence to Human Bronchial and Oropharyngeal Cells: The Role of Adherence in Lower Respiratory Tract Infections
Authors: Rikitomi Naoto , Ahmed Kamruddin , Nagatake Tsuyoshi
Published: November 2013

To study the role of Moraxella (subgenus Branhamella) catarrhalis (B. catarrhalis) adherence to airway cells in lower respiratory tract infections, the in vitro attachments of B. catarrhalis to upper airway (oropharyngeal) and lower airway (bronchial) epithelial cells were compared. The adherence of 4 strains (1 nonfimbriated and 3 fimbriated) of B. catarrhalis to respiratory tract epithelial cells collected from 11 patients with chronic pulmonary disease (CPD) and 11 healthy individuals was evaluated. Both the fimbriated and nonfimbriated strains showed increased attachment to oropharyngeal cells in the CPD patients (mean ± SEM; 25.0 ± 3.2/cell; P < 0.01) when compared to the control subjects (12.1 ± 1.1/cell). On the average, the attachment to bronchial cells was 6.1 to 13.6 times greater per surface area (bacteria/μ2) than the attachment to oropharyngeal cells. The fimbriated strains tended to adhere in higher numbers to bronchial cells (19.0 ± 1.8/cell) than the nonfimbriated strain (8.7 ± 1.2/cell), although there was no difference between the CPD and control groups. In conclusion, the attachment of B. catarrhalis to oropharyngeal cells may be an enhancing factor for colonization in the upper respiratory tract in patients with CPD, and elevated adherence of the bacteria to bronchial cells may suggest pathogenic importance when mucociliary function is impaired.
Lambda‐Toxin of Clostridium perfringens Activates the Precursor of Epsilon‐Toxin by Releasing Its N‐ and C‐Terminal Peptides
Authors: Minami Junzaburo , Katayama Seiichi , Matsushita Osamu , Matsushita Chieko , Okabe Akinobu
Published: November 2013

The effect of γ‐toxin, a thermolysin‐like metalloprotease of Clostridium perfringens, on the inactive ε‐prototoxin produced by the same organism was examined. When the purified ε‐prototoxin was incubated with the purified γ‐toxin at 37 C for 2 hr, the 32.5‐kDa ε‐prototoxin was processed into a 30.5‐kDa polypeptide, as determined by SDS‐polyacrylamide gel electrophoresis. A mouse lethality test showed that the treatment activated the prototoxin: the 50% lethal doses (LD50) of the prototoxin with and without γ‐toxin treatment were 110 and 70,000 ng/kg of body weight, respectively. The lethal activity of the prototoxin activated by γ‐toxin was comparable to that with trypsin plus chymotrypsin and higher than that with trypsin alone: LD50 of the prototoxin treated with trypsin and trypsin plus chymotrypsin were 320 and 65 ng/kg of body weight, respectively. The ε‐toxin gene was cloned and sequenced. Determination of the N‐terminal amino acid sequence of each activated ε‐prototoxin revealed that γ‐toxin cleaved between the 10th and 11th amino acid residues from the N‐terminus of the prototoxin, while trypsin and trypsin plus chymotrypsin cleaved between the 13th and 14th amino acid residues. The molecular weight of each activated ε‐prototoxin was also determined by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. The C‐terminus deduced from the molecular weight is located at the 23rd or 30th amino acid residue from the C‐terminus of the prototoxin, suggesting that removal of not only N‐terminal but also C‐terminal peptide is responsible for activation of the prototoxin.
Endogenous Tumor Necrosis Factor (TNF) α Mediates Neutrophil Accumulation at the Mid‐Phase of a Murine Model of Pseudomonas aeruginosa Pneumonia
Authors: Sonoda Fuminari , Oishi Kazunori , Iwagaki Akitaka , Matsumoto Keizo
Published: November 2013

To determine the role of endogenous tumor necrosis factor (TNF) α on neutrophil influx into the lungs in acute Pseudomonas aeruginosa pneumonia, we evaluated TNF α activity, inflammatory cell response and neutrophil chemotactic activity in the bronchoalveolar lavage fluids (BALFs) of P. aeruginosa‐infected mice. In the case of fatal pneumonia, the TNF α activity in the BALFs appeared within 3 hr, peaked at 6–12 hr and attenuated within 24 hr after intratracheal challenging, while no TNF α activity was detected in the plasma. The elevation of TNF α activity in the BALFs was closely associated with neutrophil accumulation. Mirroring the TNF α activity response and the influx of neutrophils into the murine airway, the number of neutrophils in the BALFs increased within 3 hr, peaked at 6–12 hr and remained elevated up to 24 hr after challenging. Neutralization of the TNF α activity in the BALFs with anti‐murine TNF antiserum decreased the level of neutrophil migration by BALF 45.0–49.7% at 6 hr and 49.3–54.2% at 12 hr, while the neutralizing antiserum had no effect on the level of neutrophil migration by BALFs at 3 and 24 hr. Furthermore, the intravenous administration of anti‐murine TNF antiserum 2 hr before challenging significantly inhibited neutrophil migration into the lungs of mice with sublethal pneumonia (P < 0.05; compared with mice receiving pre‐immune serum). These data suggest that intra‐alveolar TNF α plays an important role in causing lung neutrophil accumulation at the mid‐phase of murine P. aeruginosa pneumonia.
Intestinal Distribution and Intraluminal Localization of Orally Administered Clostridium butyricum in Rats
Authors: Sato Rumiko , Tanaka Mamoru
Published: November 2013

Clostridium butyricum has been used as a probiotic in animals and humans for years, however, its fate in the intestine has not been clarified yet. We investigated the intestinal fate of C. butyricum using a selective medium and a monoclonal antibody after orally administering C. butyricum spores to rats. The number of C. butyricum, both viable and dead cells, in the intestinal contents were counted by enzyme‐linked immunosorbent assay (ELISA) at various times after a single oral administration. The total viable number of C. butyricum was counted using a selective medium, and viable resting spores were selectively detected by treating the samples with ethanol. To investigate the intraluminal localization of the C. butyricum cells, frozen intestinal tracts were imprinted onto slides and stained with immunogold‐silver. Total viable spores exceeded the number of viable resting spores by more than 10‐fold from the proximal to middle of the small intestine 30 min after administration. Vegetative cells of C. butyricum were first detected in the distal small intestine after 2 hr, and vegetative growth was observed from the cecum to the colon 5 hr after administration. Dead vegetative cells were detected 9 hr after administration, and C. butyricum cells were not detected in the intestine after 3 days. The C. butyricum cells in the intestinal imprints were stained specifically by immunogold‐silver staining, and proliferative cells were observed in the cecum after 3 hr. These results suggest that the administered C. butyricum germinated in the upper small intestine, grew mainly from the distal small intestine to the colon and were excreted from the rat intestine within 3 days.
A Long‐Term Survey of Methicillin‐Resistant Staphylococcus aureus in the Oral Cavity of Children
Authors: Suzuki Junji , Komatsuzawa Hitoshi , Sugai Motoyuki , Suzuki Takako , Kozai Katsuyuki , Miyake Yoichiro , Suginaka Hidekazu , Nagasaka Nobuo
Published: November 2013

Methicillin‐resistant Staphylococcus aureus (MRSA), an indigenous bacteria in healthy people, often causes nosocomial infections. If the host human becomes compromised, MRSA can cause a serious infection. The long‐term colonization of MRSA increases this risk. The purpose of this study was to demonstrate the incidence of S. aureus and MRSA colonization in the oral cavities of healthy children, and to examine the stability of identical strains of MRSA over a long‐term period. Fourteen children were examined in two stages (first stage: 1987–88, second stage: 1992–93). Five of the 14 children were negative for S. aureus in both stages, seven children were positive in both stages and two children were positive in only the second stage. The children who were colonized with S. aureus in the first stage always harbored the bacteria in the second stage. Of the seven children that were positive for S. aureus in both stages, three persisted in carrying MRSA. We compared two MRSA strains isolated from the same children in both stages by coagulase typing, antibiogram typing and DNA fingerprinting. In two children, the strains showed the same coagulase types, similar antibiograms and similar DNA fragment profiles. These data strongly suggest that identical strains of MRSA persisted in the oral cavities for more than five years, and that the oral cavity can serve as a reservoir for MRSA with the potential to cause nosocomial infections.
Different Susceptibility of Three Clinically Isolated Strains of Cryptococcus neoformans to the Fungicidal Effects of Reactive Nitrogen and Oxygen Intermediates: Possible Relationships with Virulence
Authors: Xie Qifeng , Kawakami Kazuyoshi , Kudeken Norifumi , Zhang Tiantuo , Qureshi Mahboob Hossain , Saito Atsushi
Published: November 2013

We investigated the susceptibility of three clinically isolated strains of Cryptococcus neoformans with different virulences to reactive nitrogen and oxygen intermediates (RNI and ROI, respectively), representing two important mediators of macrophage microbicidal activity. All mice infected with the highly virulent strain of C. neoformans, YC‐11, died within 3 to 6 weeks because of rapid multiplication of the organism in the lungs and dissemination to the brain. In contrast, a weakly virulent strain, YC‐13, was almost completely eradicated from the lungs and did not disseminate to the brain, leading to survival of all infected animals during the period of observation (15 weeks). The virulence of the third strain, YC‐5, was intermediate between the other two strains. To examine the susceptibility of C. neoformans to the fungicidal effect of nitric oxide (NO) and superoxide anions (O2‐), the organisms were exposed to these oxidants, which were chemically generated in a cell‐free system. Interestingly, the number of live YC‐13 yeast cells was markedly reduced after exposure to NO and O2−. In contrast, YC‐11 was almost completely resistant to the killing effect of these oxidants. YC‐5 showed an intermediate susceptibility. Our results demonstrate that the resistance of C. neoformans to the fungicidal effects of RNI and ROI is related to virulence, and suggest that the resistance to nitrogen‐ and oxygen‐derived oxidants may be one of the factors to determine the outcome of infection with C. neoformans.
Fc Receptor‐Mediated Phagocytosis, Superoxide Production and Calcium Signaling of β2Integrin‐Deficient Bovine Neutrophils
Authors: Nagahata Hajime , Sawada Chikari , Higuchi Hidetoshi , Teraoka Hiroki , Yamaguchi Mamoru
Published: November 2013

Fc receptor for immunoglobulin G‐mediated phagocytosis, superoxide production and intracellular calcium ([Ca2+]i) signaling of complement receptor type 3 (CR3)‐deficient neutrophils from a heifer with leukocyte adhesion deficiency (BLAD) were compared to those of control heifers. The mean phagocytic activity of IgG‐coated yeasts and aggregated bovine IgG (Agg‐IgG)‐induced superoxide production of CR3‐deficient neutrophils were 10% and 77.9%, respectively, of those of control neutrophils. The [Ca2+]isignals in CR3‐deficient neutrophils stimulated with Agg‐IgG or concanavalin A were different with mean peak [Ca2+]iconcentrations of 78% and 41.9%, respectively, of those of control neutrophils. These findings suggest that Fc receptor‐mediated neutrophil functions are closely dependent on the presence of CR3 (CD11b/CD18) on the neutrophil cell surfaces.
Participation of CD14 in the Phagocytosis of Smooth‐Type Salmonella typhimurium by the Macrophage‐Like Cell Line, J774.1
Authors: Onozuka Kazuyasu , Kirikae Teruo , Kirikae Fumiko , Suda Yasuo , Kusumoto Shoichi , Yamamoto Shunsuke , Shimamura Tadakatsu , Nakano Masayasu
Published: November 2013

The role of CD14 in the phagocytosis and killing of microorganisms was investigated using macrophage‐like cell lines, CD14‐positive J774.1 cells and CD14‐negative mutant J7.DEF3 cells derived from J744.1 cells. The cells were infected with Salmonella typhimurium organisms of the smooth (S)‐form LT2, mutant rough (R)‐form TV148 or Staphylococcus aureus 248βH. At 30 or 180 min incubation, the cells were washed and disrupted. Colony‐forming units (CFUs) liberated from the disrupted cells were determined by quantitative cultivation, and the phagocytic index and killing rate were calculated. Both the phagocytic index and killing rate of J774.1 cells against LT2 organisms were greater than those of J7.DEF.3 cells. However, the index and rate of J774.1 cells against TV148 and 248βH organisms were similar to those of the J7.DEF.3 cells. The phagocytosis of LT2 organisms by J774.1 cells was partially inhibited by S‐form LPS (S‐LPS) and anti‐CD14 antibody, but not by R‐chemotype LPS (R‐LPS). These results suggest that CD14 participates in the phagocytosis of S‐form Salmonella.
Epidemiological Study on Infectious Diarrheal Diseases in Children in a Coastal Rural Area of Kenya
Authors: Saidi Suleiman M. , Lijima Yoshio , Sang William K. , Mwangudza Anderson K. , Oundo Joseph O. , Taga Kenichiro , Aihara Masanori , Nagayama Kenichi , Yamamoto Hiroyuki , Waiyaki Peter G. , Honda Takeshi
Published: November 2013

Diarrheal diseases are major causes of morbidity and mortality among children in developing countries. We have analyzed the causative agents of diarrhea in children under five years of age who resided in rural environments but attended a hospital in Malindi, a coastal town in Kenya. Bacterial diarrhea was found in 239 (27.7%) of 862 patients with diarrhea. Diarrheagenic Escherichia coli, including enteropathogenic, enterotoxigenic, and enterohaemorrhagic strains, was isolated from 119 (13.8%) patients, followed by Salmonella spp. (63 cases, 7.3%) and Shigella spp. (56 cases, 6.5%). Intestinal parasites were found in 109 (12.6%) of the patients. Entamoeba histolytica and Giardia lamblia were found in 67 (7.8%) and 42 (4.9%) of the cases, respectively. Rotavirus was found in 69 (16.1%) of 428 cases, a part of the 862 cases. Significant differences in age distribution were seen in diarrheal cases due to Campylobacter spp., G. lamblia, and rotavirus. No significant seasonal incidence of specific pathogens was found, but the number of diarrheal patients was significantly correlated to rainfall. Drinking water was contaminated with bacteria at concentrations ranging from 103 to 106 CFU/ml in 98% of the households and by coliform bacteria at concentrations of 102 to 105 CFU/ml in 72% of the households. These results suggest that the main routes of infection may be contaminated drinking water and fecal‐oral transmission of enteric pathogens. Consequently, we propose that the enhancement of hygienic practice through health education is a feasible control measure of diarrhea in the study area.
Characteristics of IgA Anti‐HIV Antibodies in Plasma from Patients with HIV Infection
Authors: Matsuda Shunji , Noda Masahird , Toyota Akie , Tokumoto Shizuyo , Miyata Masahiko
Published: November 2013

The concentration of total IgA and the specificity and molecular size of IgA anti‐human immunodeficiency virus (HIV) type‐1 antibodies in plasma obtained from individuals at different stages of HIV infection were analyzed. The concentration of total IgA in the plasma was not decreased even in the late stage of HIV infection, in contrast with those of total IgG and IgM. The IgA anti‐HIV antibodies differed to the IgG anti‐HIV antibodies in their specificity as determined by Western blotting. The IgA antibodies mainly bind to Env glycoproteins. The IgA anti‐HIV antibodies in plasma were detected between IgG and IgM by gel filtration, suggesting the presence of polymeric IgA anti‐HIV antibodies. These results indicate that the production of non‐specific IgA in plasma is enhanced by unknown mechanisms in every stages of HIV infection, and suggest that IgA anti‐HIV antibodies in plasma which are possibly polymeric and have unique specificity may play an important role in HIV infection.
Hyaluronidase Activity in Human Pus from Which Streptococcus intermedius Was Isolated
Authors: Takao Ayuko , Nagashima Hiroyuki , Usui Hiroyuki , Sasaki Fumihiko , Maeda Nobuko , Ishibashi Katsunori , Fujita Hiroshi
Published: November 2013

Hyaluronidase (HAase) activity was detected in both a human pus sample and the culture supernatant of the only bacterial isolate from the pus, Streptococcus intermedius, using a zymographic technique. The optimum pH range for HAase activity was similar for both samples. Although the bands showing the strongest HAase activity of these samples differed from each other with respect to molecular size, both samples were equally inhibited by an antiserum raised against HAase of S. intermedius. These results suggest that S. intermedius may produce HAase in vivo as well as in vitro, and that this enzyme and/or its fragments may play an important role in host tissue degradation.
Intracytoplasmic Localization of Antigenic Heat‐Stable 120‐ to 130‐Kilodalton Proteins (PS120) Common to Spotted Fever Group Rickettsiae Demonstrated by Immunoelectron Microscopy
Authors: Uchiyama Tsuneo
Published: November 2013

Immunoelectron microscopy demonstrated antigenic heat‐stable 120‐ to 130‐kilodalton proteins (PS120) of spotted fever group (SFG) rickettsiae with antiserum against recombinant PS120 of Rickettsia japonica. In the case of R. japonica, a major part of the protein was shown to be localized outside the electron‐lucent nucleoid‐like region in the cytoplasm of the organisms. The other SFG rickettsiae represented a similar localization of the PS120 antigens cross‐reactive to that of R. japonica. On the other hand, a typhus group rickettsia demonstrated no antigens cross‐reactive to the PS120 of SFG rickettsiae.
Genotypic Analysis of the 5′‐Untranslated Region of a Pestivirus Strain Isolated from Human Leucocytes
Authors: Giangaspero Massimo , Harasawa Ryô , Verhulst Antoine
Published: November 2013

The 5′‐untranslated genomic region of the pestivirus strain Europa, originated in human leucocytes and previously identified as bovine diarrhea virus (BVDV), was amplified by reverse transcription‐PCR and sequenced. Analyses based on primary nucleotide sequence homology and on secondary palindromic sequence structures characteristic to genotypes revealed that this human isolate should be assigned to a novel genotype of pestivirus, type Ic. This newly emerged genotype was related to, but distinguishable from the three known BVDV genotypes, Ia, Ib and II. Three other bovine field isolates of BVDV originated from Germany were also found to belong to this new genotype Ic. Within pestivirus genotype Ic strains, the overall nucleotide sequence homology was 95–96%, and 88–92%, 88–90% and 77–79% with the other BVDV genotypes Ia, Ib and II, respectively. With the strains from border disease virus (genotype III) and hog cholera virus (genotype IV), homologies were less than 75%.
Differentiation of Coxiella burnetii by Sequence Analysis of the Gene (com1) Encoding a 27‐kDa Outer Membrane Protein
Authors: Zhang Guo Quan , To Ho , Yamaguchi Tsuyoshi , Fukushi Hideto , Hirai Katsuya
Published: November 2013

The gene (com1) encoding a 27‐kDa outer membrane protein in 21 strains of Coxiella burnetii from a variety of clinical and geographical sources was sequenced for strain differentiation. The com1 gene was highly conserved among all the strains tested but there were several differences in nucleotide and deduced amino acid sequences. Based on the com1 gene‐specific nucleotides and deduced amino acids, the 21 strains were divided into four groups. Group 1 contained 14 strains originating from ticks, cattle and human cases of acute Q fever. Groups 2 and 3 included 2 and 3 strains, respectively, originating from human cases of chronic Q fever. Group 4 contained 2 strains originating from a human case of acute Q fever and a goat with abortion. The results indicated that the strains originating from ticks, cattle and human cases of acute Q fever differed at the molecular level from those of human chronic Q fever. This study suggests that a sequence analysis of the com1 gene can be used for strain differentiation of C. burnetii.
Antigenic Characteristics of Polypeptides of Coxiella burnetii Isolates
Authors: To Ho , Hotta Akitoyo , Zhang Guo Quan , Van Nguyen Sa , Ogawa Motohiko , Yamaguchi Tsuyoshi , Fukushi Hideto , Amano Ken‐ichi , Hirai Katsuya
Published: November 2013

Eighteen Coxiella burnetii strains from a variety of clinical and geographical sources were screened for antigenic variation of polypeptides by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) coupled with Coomassie brilliant blue (CBB) staining or immunoblotting. These polypeptide profiles showed the greatest variability in the region from 33 to 8.1 kDa. Such differences in the antigenicity of the polypeptides were also recognized by immunoblotting with 15 various mouse anti‐C. burnetii antisera. In addition, we detected a polypeptide at about 28 kDa which was immunodominant in strains from human cases of acute Q fever, milk and ticks but not immunogenic in strains from human cases of chronic Q fever. These findings suggest that this polypeptide is a marker to distinguish between acute and chronic strains.
Enhancement of Human Immunodeficiency Virus Type 1 (HIV‐1) Infection via Increased Membrane Fluidity by a Cationic Polymer
Authors: Owada Takashi , Miyashita Yuko , Motomura Tadahiro , Onishi Makoto , Yamashita Shuzo , Yamamoto Naoki
Published: November 2013

Cationic polymers are known to have potent activity against bacteria, but their effects on viral activity have been little studied. We investigated the effect of one such polymer, polyethyleneimine (PEI), on HIV‐1 infection. Although virus‐cell binding was significantly inhibited by PEI, HIV‐1 infection in human T‐cell lines such as MT‐4 and MOLT‐4 was accelerated conversely when the drug treatment was carried out, after the virus had attached to the cells or PEI was simultaneously added to the virus and cell culture system. This paradoxical effect of PEI on HIV‐1 infection was examined using HIV‐1 chronically infected cells (MOLT‐4/HIV‐1). Dissociation of the glycoprotein gp120 (as revealed by exposure of transmembrane protein gp41) from MOLT‐4/HIV‐1 cells and the resultant fusion of these cells was shown to be induced by the addition of PEI. Accordingly, it was suggested that the binding inhibition of HIV‐1 to CD4‐positive cells by PEI was due to the shedding of gp120 from HIV‐1 particles, and this PEI rather promoted membrane fusion between the virus and cells leading to the enhancement of HIV‐1 infection. Similarly, dissociation of gp120 from MOLT‐4/HIV‐1 was also induced by sCD4. The effect of these reagents on changes in membrane fluidity was evaluated by polarization (p) measurements, and it was observed that the acceleration of membrane fluidity occurred only in the PEI system. Therefore, it is likely that PEI accelerates HIV‐1 infection by facilitating virus entry into the host cells through an increase in membrane fluidity.
Evaluation of a Recombinant 27‐kDa Outer Membrane Protein of Coxiella burnetii as an Immunodiagnostic Reagent
Authors: Zhang Guo Quan , Hotta Akitoyo , Ho To , Yamaguchi Tsuyoshi , Fukushi Hideto , Hirai Katsuya
Published: November 2013

The 27‐kDa outer membrane protein from eight strains of Coxiella burnetii was expressed in the pET‐21c protein expression system. Two fusion proteins with molecular masses of 30 and 32 kDa were evident in all eight of the recombinants by SDS‐PAGE and immunoblotting. A protein having an approximate size of 30 kDa was purified from the Escherichia coli lysates by one‐step affinity purification. The utility of the purified recombinant protein in ELISA was also evaluated by testing its reactivity with human sera and comparing this reactivity with that of Nine Mile phase II antigen. All of the 40 IF‐positive serum samples were ELISA‐positive for both the Nine Mile phase II and recombinant antigens, and negative serum controls were negative for both antigens. These results suggest that ELISA with the 27‐kDa recombinant antigen is a sensitive and specific method for detecting anti‐C. burnetii antibodies in human sera.
Combined Use of Bacteriophage Typing and Pulsed‐Field Gel Electrophoresis in the Epidemiological Analysis of Japanese Isolates of Enterohemorrhagic Escherichia coli O157:H7
Authors: Izumiya Hidemasa , Masuda Takashi , Ahmed Rafiq , Khakhria Rasik , Wada Akihito , Terajima Jun , Itoh Ken‐ichiro , Johnson Wendy M. , Konuma Hirotaka , Shinagawa Kunihiro , Tamura Kazumichi , Watanabe Haruo
Published: November 2013

A total of 236 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates in Japan were investigated by bacteriophage typing, and the results were compared with those of pulsed‐field gel electrophoresis (PFGE). Seven phage types (PTs) were observed in 71 isolates which were derived from 22 outbreaks. All of the isolates from ten outbreaks in the Kinki region (midwestern part of Japan) in July‐August 1996 were grouped into the same PFGE type (IIa) and PT 32, while among total isolates, there were such varieties as PFGE type IIa containing five phage types and PT32 containing two PFGE types. These results suggest that the ten outbreaks should be considered to be a single outbreak, and show that the combined use of bacteriophage typing and PFGE enhances reliability in epidemiological surveys.
Incidence of Prevotella intermedia and Prevotella nigrescens in Periodontal Health and Disease
Authors: Maeda Nobuko , Okamoto Masaaki , Kondo Kenichiro , Ishikawa Hiroyuki , Osada Reiko , Tsurumoto Akihisa , Fujita Hiroshi
Published: November 2013

The incidence of black‐pigmented rods (BPRs), especially Prevotella intermedia and Prevotella nigrescens, in periodontal health and disease were examined. Furthermore, the degradative enzyme activities of P. intermedia were compared among the strains from periodontal health and disease. Microbiological specimens were collected from subgingival crevice or periodontal pocket by paper point. The BPRs were found in 71.1% of periodontally healthy subjects (n = 45), and in 47.1% of healthy sites (n = 34) and 87.8% of active sites (n = 41) among periodontally diseased patients. Porphyromonas gingivalis was detected only in active sites of periodontally diseased patients (17.8% of 180 strains). P. intermedia was the predominant BPR in both healthy and active sites (37.3 and 41.7%, respectively) of the patients. However, P. nigrescens was the predominant BPR (70.5% of 173 strains) in periodontally healthy subjects. The enzyme activities of esterase, esterase‐lipase, acid‐phosphatase and α‐fucosidase of P. intermedia strains isolated from active sites in patients were significantly higher (P < 0.05) than those of healthy subjects. The results suggest that P. intermedia might increase the activity of degradative enzymes under a certain condition and support the progression of periodontitis.
Immune Control of Chlamydial Growth in the Human Epithelial Cell Line RT4 Involves Multiple Mechanisms That Include Nitric Oxide Induction, Tryptophan Catabolism and Iron Deprivation
Authors: Igietseme Joseph U. , Ananaba Godwin A. , Candal Debra H. , Lyn Deborah , Black Carolyn M.
Published: November 2013

The antimicrobial activity of T cell‐derived cytokines, especially interferon (IFN)‐γ, against intracellular pathogens, such as Chlamydia trachomatis, involves the induction of 3 major biochemical processes: tryptophan catabolism, nitric oxide (NO) induction and intracellular iron (Fe) deprivation. Since the epithelial cell is the natural target of chlamydial infection, the presence of these antimicrobial systems in the cell would suggest that they may be involved in T cell control of intracellular multiplication of Chlamydia. However, the controversy over whether these 3 antimicrobial processes are present in both mice and humans has precluded the assessment of the relative contribution of each of the 3 mechanisms to chlamydial inhibition in the same epithelial cell from either mice or humans. In the present study, we identified a Chlamydia‐susceptible human epithelial cell line, RT4, that possesses the 3 antimicrobial systems, and we examined the role of nitric oxide (NO) induction, and deprivation of tryptophan or Fe in cytokine‐induced inhibition of chlamydiae. It was found that the 3 antimicrobial systems contributed to cytokine‐mediated inhibition of the intracellular growth of Chlamydia. NO induction accounted for ~20% of the growth inhibition; tryptophan catabolism contributed approximately 30%; iron deprivation was least effective; but the combination of the 3 systems accounted for greater than 60% of the inhibition observed. These results indicate that immune control of chlamydial growth in human epithelial cells may involve multiple mechanisms that include NO induction, tryptophan catabolism and Fe deprivation.
Prevalence of Human Melioidosis on Hainan Island in China
Authors: Yang Song , Tong Shide , Mo Chengjin , Jiang Zhongjun , Yang Sun , Ma Yuhai , Lu Zhenzi
Published: November 2013

Eight human cases of melioidosis were diagnosed at Hainan People's Hospital over a period of one year. Four of the cases were of septicemia form, while the rest were of chronic form with abscesses in different organs. All the isolates were intrinsically resistant to cefazolin, cefuroxime, and gentamicin, while being rather sensitive to ampicillin/sulbactam, ticarcillin/clavulanic acid, and imipenem. A serological survey of exotoxin antibodies at different farms showed that Xinglong was a farm seriously devastated by Burkholderia pseudomallei, while the mountainous farm of Licai had the lowest prevalence (P < 0.01). From the results of serological survey and melioidosis case distribution, it could be clearly seen that melioidosis predominantly exists in coastal plain regions around this island, where the altitude above sea‐level is below 100 m, the annual rainfall is up to 2,300 mm and a rather warm climate in coldest months of December and January.
Purification and Characterization of Enterotoxin Produced by Aeromonas sobria
Authors: Fujii Yoshio , Nomura Tomohiko , Kanzawa Hiromi , Kameyama Michio , Yamanaka Hiroyasu , Akita Masahiko , Setsu Kojun , Okamoto Keinosuke
Published: November 2013

We purified the toxin of Aeromonas sobria capable of inducing a positive response in the mouse intestinal loop assay. The purified toxin showed a positive response not only in the loop assay but also in a hemolytic assay. Subsequently, we cloned the toxin gene and demonstrated that the product of this gene possessed both hemolytic and enterotoxic activities. These results showed that the enterotoxin of A. sobria possesses hemolytic activity. Nucleotide sequence determination of the toxin gene and amino acid sequence analysis of the purified toxin revealed that it is synthesized as a precursor composed of 488 amino acid residues, and that the 24 amino‐terminal amino acid residues of the precursor is removed in the mature toxin. As antiserum against the purified toxin neutralized the fluid accumulation induced by living cells not only of A. sobria but also of A. hydrophila, this and antigenically related toxin(s) are thought to play an essential role in the induction of diarrhea by these organisms. The toxin‐injured Chinese hamster ovary (CHO) cells induced the release of intracellular lactose dehydrogenase (LDH). The release of LDH from CHO cells and the lysis of erythrocytes by the toxin were repressed by the addition of dextran to the reaction solution, indicating that the toxin forms pores in the membranes and that the cells were injured by the osmotic gradient developed due to pore formation. However, the histopathological examination of intestinal cells exposed to the toxin showed that it caused fluid accumulation in the mouse intestinal loop without causing cellular damage.
Heterogeneity of Phenotypic and Genotypic Traits Including Organic‐Acid Resistance in Escherichia coli O157 Isolates
Authors: Horii Toshinobu , Barua Soumitra , Kimura Taku , Kasugai Shin , Sato Kumiko , Shibayama Keigo , Ichiyama Satoshi , Ohta Michio
Published: November 2013

We undertook an epidemiologic study for the sensitivity of both Shiga‐like toxin (Slt)‐producing Escherichia coli (STEC) O157 and non‐STEC O157 strains isolated from different patients with diarrhea to hydrochloric acid (HCl) and organic acids such as acetate, propionate, butyrate and lactate, and other pathogenic factors. The E. coli O157 isolates examined showed a wide variety of organic‐acid susceptibility patterns. E. coli O157 isolates resistant to HCl or acetate were found more frequently than those resistant to other organic acids. These isolates also showed diverse pathogenicity patterns for the presence of the virulence genes, antibiotic susceptibility and plasmid profile.
Identification of a Novel DNA‐Binding Protein from Mycobacterium bovis Bacillus Calmette‐Guérin
Authors: Matsumoto Sohkichi , Yukitake Hideharu , Furugen Makoto , Matsuo Takemitsu , Mineta Takao , Yamada Takeshi
Published: November 2013

A novel DNA‐binding protein expressed (8–10% in total protein) in Mycobacterium bovis bacillus Calmette‐Guérin was observed. This protein was designated mycobacterial DNA‐binding protein 1 (MDP1). MDP1 recognized bases, sugar moieties, phosphate‐backbone on DNA and preferentially bound to DNA guanine and cytosine. In the gel retardation assay, MDP1 preferentially bound to closed circular plasmid DNA than open circular and linear form plasmid DNA and also bound to RNA. MDP1 formed a highly polymerized structure and localized not only in the nucleoid but also at the 50S ribosomal subunits and cell surface. MDP1 was conserved in Mycobacterium thus far examined and the expression was enhanced in stationary growth phases. These results will provide a reasonable basis for further study of the function of MDP1 in living mycobacteria.
A 63 kDa Tumor Necrosis Factor Inhibitor Released from a Human Monocytic Leukemia Cell Line, THP‐1
Authors: Aso Hisashi , Yoshie Osamu , Tomioka Yoshihisa , Rose Michael T. , Mizugaki Michinao
Published: November 2013

A human monocytic cell line, THP‐1‐S, was cultured in a serum‐free medium. The effect of the culture supernatant of THP‐1‐S on the cytotoxicity of rTNF‐α to three kinds of cell lines and the binding of rTNF to its receptor were tested. The supernatant inhibited the cytotoxicity of rTNF‐α when tested by the neutral red uptake method. In addition, the supernatant blocked the binding of 125I‐rTNF‐α to its receptor. Furthermore, following precipitation with PEG we detected complexes between rTNF‐α and the inhibitory factor which formed during incubation with the culture supernatant from THP‐1‐S cells. However, the supernatant did not bind to or down‐regulate the receptor for TNF‐α on the cell surface of L‐M‐2d6 cells. This factor eluted with an apparent molecular mass of 63,000 Da by gel filtration and did not react with antibodies against p55 and p75 TNF receptors. These data suggest that human monocytic cells are capable of releasing an inhibitory factor against rTNF‐α in serum‐free culture conditions.
Intrafamilial Distribution of Mutans Streptococci in Japanese Families and Possibility of Father‐to‐Child Transmission
Authors: Kozai Katsuyuki , Nakayama Ryusuke , Tedjosasongko Udijanto , Kuwahara Satsuki , Suzuki Junji , Okada Mitsugi , Nagasaka Nobuo
Published: November 2013

The purpose of this study was to investigate the intrafamilial distribution of mutans streptococci in Japanese families using chromosomal DNA fingerprinting with three endonucleases; EcoRI, HindIII and HaeIII. The analysis of 1,908 isolates cultured from the dental plaque of 76 subjects from 20 families (20 married couples and 36 of their children) resulted in the identification of 144 genotypes containing 114 strains of Streptococcus mutans (serotype c, 66.7%; e, 12.5%) and 30 strains of S. sobrinus (d, 13.2%; g, 7.6%). A mean of 1.89 genotypes (from one to four) was harbored in individual subjects, and a mean of 4.10 genotypes from two to seven was harbored in individual families. Among the 70 genotypes found in the children, 36 (51.4%) were in agreement with their mothers and 22 (31.4%) were in agreement with their fathers. The other genotypes (18.6%) did not correspond with the parents. Homologous strains between parents were found in only two couples. This result showed that fathers or others as well as mothers can be sources of transmission. Further, the serotype d, e and g strains showed significantly higher probabilities of transmission than serotype c.
Diffusely Adhering Escherichia coli (DAEC) Strains of Fecal Origin Rarely Express F1845 Adhesin
Authors: Campos Leila Carvalho , Vieira Mônica Aparecida Midolli , Trabulsi Luiz Rachid , Da Silva Luzinete Alves , Monteiro‐Neto Valério , Gomes Tânia Aparecida Tardelli
Published: November 2013

A total of 398 diffusely adhering Escherichia coli (DAEC) strains of fecal origin were analyzed for the presence of sequences homologous to the structural subunit gene (daaE) of the F1845 fimbria. For that purpose, a DNA fragment homologous to daaE, obtained by PCR, was used as a probe in colony hybridization assays. Only two strains carried daaE and expressed F1845, suggesting that this fimbria is rare among DAEC strains.
Antibacterial Activities of New Synthetic Divalent Cation Chelators
Authors: Ashoori Mandana , Ohta Michio , Ohsuka Shinji , Shibayama Keigo , Horii Toshinobu , Ueda Minoru , Kurosaki Hiromasa
Published: November 2013

A series of new synthetic ligand compounds which chelate divalent cations was examined for the antibacterial activities of the compounds. Only 2 of 14 synthetic chelators, 9‐trans‐anthryl‐1, 4, 8, 11‐tetraaza‐tetradecane (No. 6) and bis(2‐pyridyl)methylamine (No. 13) showed antibacterial activities, whereas none of the diamines, hydrophilic triamines nor tetramines showed antibacterial activities. Chelators No. 6 and No. 13 inhibited the growth of both Gram‐negative and ‐positive bacteria at doses of 25–200 μg/ml, comparable to those of common antibiotics such as polymixin B, fosfomycin and macrolides. Ethylenedi‐aminetetraacetate (EDTA) potentiated these antibacterial activities, whereas an inhibitory effect of Mg2+ on the MICs of these chelators was observed. Moreover, these chelators enhanced the leakage of periplasmic β‐lactamase. It is therefore suggested that chelators No. 6 and No. 13 disrupt both the membranes and cytoplasmic functions of bacteria, resulting in cell death.