bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of the dnaJ Gene for the Detection and Identification of All Legionella pneumophila Serogroups and Description of the Primers Used to Detect 16S rDNA Gene Sequences of Major Members of the Genus Legionella
Authors: Liu Hongsheng , Li Ying , Huang Xinxiang , Kawamura Yoshiaki , Ezaki Takayuki
Published: November 2013

We sequenced about 930 bp of the dnaJ gene from 15 Legionella pneumophila serogroups and some other members of the genus Legionella. As L. pneumophila 16S rDNA sequences could not discriminate between all subspecies and serogroups, we assessed the use of dnaJ gene sequences to differentiate between Legionella subspecies as well as between L. pneumophila serogroups. A phylogenetic analysis revealed that dnaJ gene sequences were more variable between the L. pneumophila serogroups than mip gene and 16S rDNA sequences. By studying 61 strains from 41 species of the genus Legionella, as well as other genera, we established a PCR method that could amplify 285 bp of dnaJ gene from all L. pneumophila serogroups. This primer set was more sensitive than mip gene primers and was able to detect 0.25 ng of purified DNA. We also describe the 16S rDNA primers that were used to detect most Legionella genus members.
Biofilm Formation by a Fimbriae‐Deficient Mutant of Actinobacillus actinomycetemcomitans
Authors: Inoue Tetsuyoshi , Shingaki Ryuji , Sogawa Norio , Sogawa Chiharu Aoki , Asaumi Jun‐ichi , Kokeguchi Susumu , Fukui Kazuhiro
Published: November 2013

Actinobacillus actinomycetemcomitans strain 310‐TR produces fimbriae and forms a tight biofilm in broth cultures, without turbid growth. The fimbriae‐deficient mutant 310‐DF, constructed in this study, was grown as a relatively fragile biofilm at the bottom of a culture vessel. Scanning electron microscopy revealed that on glass coverslips, 310‐TR formed tight and spherical microcolonies, while 310‐DF produced looser ones. These findings suggest that fimbriae are not essential for the surface‐adherent growth but are required for enhancing cell‐to‐surface and cell‐to‐cell interactions to stabilize the biofilm. Treatment of the 310‐DF biofilm with either sodium metaperiodate or DNase resulted in significant desorption of cells from glass surfaces, indicating that both carbohydrate residues and DNA molecules present on the cell surface are also involved in the biofilm formation.
Characterization and Localization of Fluorescent Pseudomonas Cold Shock Protein(s) by Monospecific Polyclonal Antibodies
Authors: Khan Mahejibin , Bajpai Virendra Kumar , Anasari Shaheen Akhtar , Kumar Anil , Goel Reeta
Published: November 2013

Cold shock protein (CSP) from Pseudomonas fluorescens MTCC 103 and cold resistant protein (CRP) from its mutant CRPF8 of 14 and 35 kd, respectively were purified to homogeneity by HPLC. Polyclonal antibodies were raised against these proteins and the expression level was checked at different temperatures, i.e., 4, 10, 20, 30 and 37 C. Furthermore, morphological changes in P. fluorescens MTCC 103 and its mutant (CRPF8) were analyzed by transmission electron microscopy (TEM). Localization of CSP and CRP documented with immunoelectron microscopy, using colloidal gold particles conjugated with secondary antibodies being the probe were used. Nevertheless, the results of cytosolic localization of CSP and CRP were evident. Furthermore, the expression of CSP and CRP increased with decrease in temperature and the cell wall thickness of the mutant exhibited 2‐fold increase, thus facilitating low temperature survival.
Characterization of a Slow‐Migrating Component of the Rabies Virus Matrix Protein Strongly Associated with the Viral Glycoprotein
Authors: Nakahara Tomomi , Toriumi Harufusa , Irie Takashi , Takahashi Takuo , Ameyama Satoshi , Mizukoshi Masami , Kawai Akihiko
Published: November 2013

We investigated multiple forms of rabies virus matrix (M) protein. Under non‐reducing electrophoretic conditions, we detected, in addition to major bands of monomer forms (23‐ and 24‐kDa) of M protein, an M antigen‐positive slow‐migrating minor band (about 54 kDa) in both the virion and infected cells. Relative contents of the 54‐kDa and monomer components in the virion were about 20–30% and 70–80% of the whole M protein, respectively, while the content of the 54‐kDa component was smaller (about 10–20% of the total M protein) in the cell than in the virion. The 54‐kDa components could be extracted from the infected cells with sodium deoxycholate, but they were quite resistant to extraction with 1% non‐ionic detergents by which most monomer components were solubilized. The 54‐kDa component was precipitated more efficiently than the monomer by a monoclonal antibody (mAb; #3‐9‐16), which recognized a linear epitope located at the N‐terminal of the M protein. The mAb #3‐9‐16 coprecipitated the viral glycoprotein (G), which was demonstrated to be due to strong association between the G and 54‐kDa component of the M protein. Monomers and the 54‐kDa polypeptide migrated to the same isoelectric point (pI) in two‐dimensional (2‐D) gel electrophoresis, implicating that the 54‐kDa component was composed of component(s) of the same pI as that of the M protein monomers. From these results, we conclude that the M antigen‐positive 54‐kDa polypeptide is a homodimer of M protein, taking an N‐terminal‐exposed conformation, and is strongly associated with the viral glycoprotein. Possible association with a membrane microdomain of the cell will be discussed.
Identification and Characterization of Class 1 Integron Resistance Gene Cassettes among Salmonella Strains Isolated from Healthy Humans in China
Authors: Zhang Hongmei , Shi Lei , Li Lin , Guo Siyuan , Zhang Ximei , Yamasaki Shinji , Miyoshi Shin‐ichi , Shinoda Sumio
Published: November 2013

Twenty‐three strains of Salmonella spp. isolated from healthy humans in Guangdong, China, were examined for their susceptibility to ten common antibiotics and the presence of antibiotic resistance integrons. All the strains were resistant to at least one antibiotic, and 4 strains were positive for the intI1 gene. Polymerase chain reaction using in‐F and in‐B primers showed the existence of amplicons of 1,009 bp in two, 1,664 bp in one, and 1,009 bp and 1,664 bp in one of the intI1‐positive isolates, respectively. Sequence analysis revealed that the 1,009‐bp amplicon harbored gene cassette aadA2, conferring resistance to spectinomycin, and the 1,664‐bp amplicon harbored genes aadA5 and dfr17, conferring resistance to spectinomycin, streptomycin and trimethoprim. Meanwhile the experiments of plasmid conjugation and Southern hybridization with intI1 as the DNA probe indicated that all the integrons found in these strains were chromosomal. Because the strains carrying class 1 integrons were isolated from healthy humans, it suggests the need for all‐round surveillance of the antibiotic resistance of pathogens.
Role of Tumor Necrosis Factor‐Alpha and Interferon‐Gamma in Helicobacter pylori Infection
Authors: Yamamoto Toshiro , Kita Masakazu , Ohno Tomoyuki , Iwakura Yoichiro , Sekikawa Kenji , Imanishi Jiro
Published: November 2013

Immune responses to Helicobacter pylori infection play important roles in gastroduodenal diseases. The contributions of tumor necrosis factor‐α (TNF‐α) and interferon‐7 (IFN‐γ) to the induction of gastric inflammation and to the protection from H. pylori infection were investigated using TNF‐α gene‐knockout (TNF‐α−/−) mice and IFN‐γ gene‐knockout (IFN‐γ−/−) mice. We first examined the colonizing ability of H. pylori strain CPY2052 in the stomach of C57BL/6 wild‐type and knockout mice. The number of H. pylori colonized in the stomach of IFN‐γ−/− and TNF‐α−/− mice was higher than that of wild‐type mice. These findings suggest that TNF‐α and IFN‐γ may play a protective role in H pylori infection. Furthermore, we examined the contribution of TNF‐α and IFN‐γ to gastric inflammation. The CPY2052‐infected TNF‐α−/− mice showed a moderate infiltration of mononuclear cells in the lamina propria and erosions in the gastric epithelium as did wild‐type mice, whereas the CPY2052‐infected IFN‐γ−/− mice showed no inflammatory findings even 6 months after infection. These results demonstrate that IFN‐γ may play an important role in gastric inflammation induced by H. pylori infection, whereas TNF‐α may not participate in the development of inflammatory response.
Application of Monoclonal Antibody, Specific for Intracellular Orientia tsutsugamushi, to Immunofluorescent Antibody Test for Determining Antibiotic Susceptibility
Authors: Kim Mee‐Kyung , Odgerel Zagga , Kim Mi‐Jeong , Chung Moon‐Hyun , Lim Byung‐Uk , Kang Jae‐Seung
Published: November 2013

The simple quantification of viable intracellular bacteria is important for the study of an obligate intracellular bacterium, Orientia tsutsugamushi. We applied a novel monoclonal antibody (M686–13) —specific for intracellular Orientia— to an immunofluorescent antibody (IFA) test for determining antibiotic susceptibility of O. tsutsugamushi. M686–13 did not react with Orientia that was inhibited by doxycycline, although bacterial particles still remained in the cells. This preferential staining of proliferating bacteria made the IFA test rapid and precise. Using this method, we could successfully measure the minimal inhibitory concentration (MIC) of a Korean strain of O. tsutsugamushi to doxycycline and clindamycin. This method may be used in other procedures to evaluate the growth of Orientia.
In Vitro Susceptibility to Tetracycline and Fluoroquinolones of Japanese Isolates of Coxiella burnetii
Authors: Andoh Masako , Naganawa Takashi , Yamaguchi Tsuyoshi , Fukushi Hideto , Hirai Katsuya
Published: November 2013

Coxiella burnetii is the agent of the worldwide zoonosis, Q fever. The in vitro susceptibility to tetracycline and fluoroquinolones of Japanese isolates of C. burnetii was evaluated for the first time. The MICs against Japanese isolates were almost the same as the MICs against the foreign reference isolates. The results suggest that the common antibiotics therapy for Q fever used in other countries is also effective for Japanese Q fever patients.
The Anthracycline Antitumor Agents Doxorubicin and Daunorubicin Reduce the Activity of Candida albicans Phospholipase B
Authors: Arai Rika , Sugita Takashi , Nishikawa Akemi
Published: November 2013

A pathogenic yeast, Candida albicans, causes life‐threatening infection in immunocompromised patients. Inhibiting the production or release of phospholipase B by C. albicans should reduce direct host cell damage, and inhibit the release of eicosanoids from cells of this microorganism. Of the antitumor agents tested, doxorubicin and daunorubicin inhibited the activity of phospholipase B, and prostaglandin production by C. albicans. These two agents have the potential to inhibit the activity of C. albicans phospholipase B, although the inhibitory concentrations exceeded the clinical dose.
Effects of Tacrolimus (FK506) on Encephalomyocarditic Virus‐Induced Diabetes in Mice
Authors: Yama Sachiko , Nishioka Wakako , Hirokami Yumiko , Setoguchi Ruka , Takeyama Natsumi , Saeki Keiichi , Matsumoto Yoshitsugu , Hayashi Toshiharu , Doi Kunio , Onodera Takashi
Published: November 2013

The effects of tacrolimus on insulin‐dependent diabetes mellitus (IDDM) induced by the D‐variant of encephalomyocarditis virus (D‐EMCV) have been investigated. Male BALB/c mice were treated with tacrolimus before viral inoculation, and then were inoculated with 10 plaque forming units (PFU) of D‐EMCV. The mice continued to be treated with tacrolimus until the animals were sacrificed. D‐EMCV‐infected mice, which were treated with saline as controls, showed abnormal glucose tolerance test (GTT) values, whereas all infected mice with tacrolimus pretreatment were normal on 7 days‐post inoculation (DPI). Histological observations revealed that non‐treated tacrolimus D‐EMCV‐infected mice and which developed diabetes showed severe insulitis in their islets of Langerhans. On the other hand, D‐EMCV‐infected mice treated with tacrolimus were normal. In D‐EMCV‐infected mice, viruses in the pancreata were detected at the same level regardless of treatment with tacrolimus or saline. Expressions of TNF‐α and IFN‐γ mRNA in spleens of tacrolimus‐treated D‐EMCV‐infected mice were lower than that of non‐treated tacrolimus D‐EMCV‐infected mice on 7 DPI. The results suggest that tacrolimus suppresses expressions of TNF‐α and IFN‐γ mRNAs to prevent the onset of D‐EMCV‐induced IDDM.
Detection of Antigen‐Specific T Cells in Experimental Immune‐Mediated Blepharoconjunctivitis in DO11.10 T Cell Receptor Transgenic Mice
Authors: Ozaki Akemi , Ishida Waka , Fukata Kazuyo , Fukushima Atsuki , Ueno Hisayuki
Published: November 2013

Antigen (Ag)‐specific T cells are thought to play a key role in pathogenesis of chronic allergic conjunctivitis (AC) such as atopic keratoconjunctivitis (AKC) and vernal keratoconjunctivitis (VKC). In order to investigate the trafficking of Ag‐specific T cells in experimental immune‐mediated blepharoconjunctivitis (EC), we established a novel AC model in DO11.10 T cell receptor (TcR) transgenic (Tg) mice. DO11.10 TcR‐Tg mice were challenged with eye drops of whole OVA protein, OVA peptide 1–15, 321–335, or 323–339. Their eyes were histologically examined. Conventional proliferation assay was performed against each Ag. Phenotypes of infiltrating cells and kinetics of Ag‐specific T cells were investigated by immunohistochemistry. Adoptive transfer of CD4+ Ag‐specific T cells from DO11.10 TcR‐Tg to WT mice was performed. The distribution of KJ1–26+ cells was investigated in recipient mice. The challenge of OVA peptide 323–339 induced infiltration of inflammatory cells in conjunctivae in a dose dependent manner, accompanied by the proliferative responses of splenocytes. Immunohistochemical analysis revealed Ag‐specific/non‐Ag‐specific T cells, macrophages, and eosinophils in conjunctivae. Infiltration of Ag‐specific T cells increased 24 hr later. Transfer of CD4+ cells from DO11.10 TcR‐Tg to WT mice induced EC depending on the number of transferred cells. Ag‐specific T cells were detected in the conjunctivae and spleens of recipient mice, though its numbers were significantly smaller compared to DO11.10 TcR‐Tg mice. The challenge of OVA peptide 323–339 induced EC in DO11.10 TcR‐Tg mice without prior sensitization. The response was mediated by CD4+ Ag‐specific T cells. The trafficking of Ag‐specific T cells in EC was clearly visualized.
New Lactococcus Strain with Immunomodulatory Activity: Enhancement of Th1‐Type Immune Response
Authors: Kimoto Hiromi , Mizumachi Koko , Okamoto Takashi , Kurisaki Jun‐ichi
Published: November 2013

Few studies exist dealing with the probiotic activity of lactococci, which are commonly used as starter bacteria in the manufacture of many kinds of fermented dairy products. Fifteen strains of the genus Lactococcus were examined for their probiotic activities, such as immunomodulatory effects. Six strains induced the production of cytokines (IL‐12, IL‐6, and TNF‐α) in macrophage‐like cell line J774.1, and the highest induction was observed with Lactococcus lactis subsp. lactis G50. The cytokine induction in the J774.1 cell line was almost entirely sustained after heat‐killing of the strain. Spleen cells from BALB/c mice fed G50 culture produced more IL‐12 and IFN‐γ and slightly less IL‐4 and IL‐6 than the control (i.e., without strain G50), indicating that strain G50 can enhance Th1‐type immune response in vivo. The effect of the oral administration of strain G50 on antibody response in mice was also investigated. Mice were immunized with ovomucoid (OVM), a potent egg allergen, and the antibody level in the serum was then determined. The total IgE antibody level in the group treated with strain G50 was significantly lower than that of the control. The response of OVM‐specific IgG1 and IgE antibodies tended to be low in the group that was administered strain G50, compared with the response of the control group. These results suggest that strain G50 has an ability to suppress the Th2 response. Thus, Lactococcus lactis subsp. lactis G50 is a potential probiotic strain for the suppression of hypersensitive reactions caused by the Th2 response.
Nucleolin and the Packaging Signal, ψ, Promote the Budding of Human Immunodeficiency Virus Type‐1 (HIV‐1)
Authors: Ueno Tomonori , Tokunaga Kenzo , Sawa Hirofumi , Maeda Masae , Chiba Joe , Kojima Asato , Hasegawa Hideki , Shoya Yuko , Sata Tetsutaro , Kurata Takeshi , Takahashi Hidehiro
Published: November 2013

Gag proteins of human immunodeficiency virus type 1 (HIV‐1) play a pivotal role in the budding of the virion, in which the zinc finger motifs of the gag proteins recognize the packaging signal of genomic RNA. Nucleolin, an RNA‐binding protein, is identified as a cellular protein that binds to murine leukemia virus (MuLV) gag proteins and regulates the viral budding, suggesting that HIV‐1 gag proteins, the packaging signal, ψ and nucleolin affect the budding of HIV‐1. Here we report that nucleolin enhances the release of HIV‐1 virions which contain ψ. Furthermore, nucleolin and gag proteins form a complex incorporated into virions, and nucleolin promotes the infectivity of HIV‐1. Our results suggest that an empty particle which contains neither nucleolin nor the genomic RNA is eliminated during the budding process, and this mechanism is beneficial for escape from the host immune response against HIV‐1.
Comparison of Immunological Responses to the Various Types of Circumsporozoite Proteins of Plasmodium vivax in Malaria Patients of Korea
Authors: Suh In Bum , Lee Kee‐Hoyung , Kim Young Ree , Woo Sang Kyu , Kang Heui Yeon , Won Yoo Duck , An Seong Soo A. , Cho Min , Cho Duck , Lim Chae Seung
Published: November 2013

We compared the seroreactivities against four synthetic peptide antigens (VK210, VK247, Korean type 1, and type 2) and a full length recombinant circumsporozoite protein (CSP) antigen of Plasmodium vivax (P. vivax) in samples of sixty‐three tertian malaria patients in Korea. Among the various CSP antigens, the full‐length recombinant CSP showed the highest reactivity in malaria‐exposed groups (85.7%, 54/63). No significant difference was found in the percentage of malaria patients with antibodies among four peptides examined, except a full‐length recombinant CSP. Absorbance values from the peptide‐based ELISA showed high correlations (r>0.9, P<0.05) at significant values. Five sera without the immunoaffinity against peptides were reactive towards the full‐length recombinant CSP in ELISA. Sera, which were not reactive to a full length recombinant CSP antigen, were not recognized by any of peptide‐based ELISA. These data suggested that peptide structures included in Korean isolates, GNGAGGQAA, and VK247 peptides had immune reactivity and recognition epitopes. Among the antigens, GNGAGGQAA was less recognized by patients exposed to Korean strains of P. vivax in comparison to the VK210 structures.
WT1 Peptide‐Based Immunotherapy for Patients with Lung Cancer: Report of Two Cases
Authors: Tsuboi Akihiro , Oka Yoshihiro , Osaki Tadashi , Kumagai Toru , Tachibana Isao , Hayashi Seiji , Murakami Masaki , Nakajima Hiroko , Elisseeva Olga A. , Fei Wu , Masuda Tomoki , Yasukawa Masaki , Oji Yusuke , Kawakami Manabu , Hosen Naoki , Ikegame Kazuhiro , Yoshihara Satoshi , Udaka Keiko , Nakatsuka Shin‐ichi , Aozasa Katsuyuki , Kawase Ichiro , Sugiyama Haruo
Published: November 2013

The Wilms' tumor gene WT1 is overexpressed in various types of solid tumors, including lung and breast cancer and WT1 protein is a tumor antigen for these malignancies. In phase I clinical trials of WT1 peptide‐based cancer immunotherapy, two patients with advanced lung cancer were intradermally injected with 0.3 mg of an HLA‐A*2402‐restricted, 9‐mer WT1 peptide emulsified with Montanide ISA51 adjuvant. Consecutive WT1 vaccination at 2‐week intervals resulted in a reduction in tumor markers such as chorio‐embryonic antigen (CEA) and sialyl Lewis (x) (SLX) and by a transient decrease in tumor size. No adverse effects except for local erythema at the injection sites of WT1 vaccine were observed. These results provided us with the first clinical evidence demonstrating that WT1 peptide‐based immunotherapy should be a promising treatment for patients with lung cancer.
Evaluation of the Effects of Sulfamethoxazole on Toxoplasma gondii Loads and Stage Conversion in IFN‐γ Knockout Mice Using QC‐PCR
Authors: Belal Usama S. , Norose Kazumi , Aosai Fumie , Mun Hye‐Seong , Ahmed Azza K. , Chen Mei , Mohamed Rabie M. , Piao Lian‐Xun , Iwakura Yoichiro , Yano Akihiko
Published: November 2013

Toxoplasma gondii abundance with or without sulfamethoxazole treatment was evaluated by quantitative competitive polymerase chain reaction (QC‐PCR) assay in various organs of IFN‐γ knockout BALB/c (B/c) mice after peroral infection with the cyst‐forming Fukaya strain. T. gondii infection was observed in the brain, skin, tongue, heart, and skeletal muscle of the mice treated with sulfamethoxazole, although the parasite was not observed during the treatment in the mesenteric lymph node, spleen, small intestine or kidney. After discontinuing the therapy, T. gondii reappeared within five days in all organs. Reverse transcriptase (RT)‐PCR showed that sulfamethoxazole treatment accelerated the stage conversion of T. gondii from tachyzoites into bradyzoites in the brain, lung, and heart. In contrast, after discontinuing sulfamethoxazole treatment, T. gondii underwent stage conversion from bradyzoites into tachyzoites in these organs. These results indicate that we successfully established an animal model for evaluating chemotherapy regimens in immunocompromised hosts infected with T. gondii.
Infection with Campylobacter jejuni Induces Tyrosine‐Phosphorylated Proteins into INT‐407 Cells
Authors: Biswas Debabrata , Niwa Hidekazu , Itoh Kikuji
Published: November 2013

The mechanisms used by Campylobacter jejuni to induce internalization into host intestinal epithelial cells have not been defined. In this study, we obtained evidence that exposure of INT‐407 cells to protein kinase inhibitors results in decreased invasion of these cells by C. jejuni in a dose dependent manner. Preincubation of INT‐407 cells in the presence of staurosporine, tyrphostin 46 and genistein decreased invasion of these cells by C. jejuni significantly. Moreover, C. jejuni infection of INT‐407 cells induced tyrosine phosphorylation of several Triton X‐100 soluble proteins with approximate molecular weights of 170, 145, 90, 60 and 55 kDa that were absent or reduced in the presence of genistein in cells after 1 hr of pretreatment. These data suggest that tyrosine protein kinase‐linked pathways strongly regulate the internalization of C. jejuni into intestinal epithelial cells.
Identification of Signature Genes for Rapid and Specific Characterization of Yersinia pestis
Authors: Zhou Dongsheng , Han Yanping , Dai Erhei , Pei Decui , Song Yajun , Zhai Junhui , Du Zongmin , Wang Jin , Guo Zhaobiao , Yang Ruifu
Published: November 2013

Polymerase chain reaction (PCR) amplification of DNA‐based unique markers, the signature sequences, is ideal for rapid detection and identification of pathogens. We described the discovery of twenty‐eight signature genes of Yersinia pestis by DNA microarray‐based comparative genome hybridization in conjunction with PCR validation. Three pairs of Y. pestis‐specific primers designed from signature genes were demonstrated to have the expected specificity to this target bacterium, without cross‐reaction with the closely related Y. pseudotuberculosis or a large collection of genomic DNAs from other organisms.
Effects of Recombinant Cholera Toxin B Subunit (rCTB) on Cellular Immune Responses: Enhancement of Delayed‐Type Hypersensitivity Following Intranasal Co‐Administration of Mycobacterium bovis‐BCG with rCTB
Authors: Maeyama Jun‐ichi , Isaka Masanori , Yasuda Yoko , Matano Keiko , Morokuma Kazunori , Ohkuma Kunio , Tochikubo Kunio , Yamamoto Saburo , Goto Norihisa
Published: November 2013

Recombinant cholera toxin B subunit (rCTB) is a safe and potent mucosal adjuvant. To gain insight into the mechanism underlying the adjuvant effect of rCTB, the effects of rCTB on cell‐mediated immune responses of mice and guinea pigs were examined after intranasal administration of Mycobacterium bovis‐bacillus Calmette‐Guérin (BCG) with and without rCTB. Delayed‐type hypersensitivity, for skin reactions in guinea pigs and for footpad swelling reactions in mice, to purified protein derivative (PPD) were enhanced by intranasal co‐administration of BCG and rCTB, as compared to giving BCG alone to these animals. Moreover, tumor necrosis factor (TNF)‐α and interferon (IFN)‐γ production of spleen cells and antigen specific spleen cell proliferation, stimulated with PPD, were enhanced in the presence of rCTB. These results strongly suggest that rCTB enhances cellular as well as humoral immune responses.
Zymographic Characterization of Bacteriolytic Enzymes Produced by Oral Streptococci
Authors: Yoshimura Goh , Komatsuzawa Hitoshi , Kajimura Junko , Fujiwara Tamaki , Ohara Masaru , Kozai Katsuyuki , Sugai Motoyuki
Published: November 2013

Zymographic analysis was performed to know the bacteriolytic enzyme profiles of 4% SDS extracts of oral streptococci, Streptococcus mutans, S. sobrinus, S. sanguis, S. mitis and S. salivarius. We investigated the five strains in each species and found that the profile was very similar among strains of the same species except for S. salivarius (the profile was classified into two types). On the other hand, the profile was considerably different among species. Two major bacteriolytic enzymes of S. mutans showing molecular mass of 80 and 100 kDa were found using SDS‐boiled S. mutans or S. sobrinus cells as substrate. These bacteriolytic activities were less apparent in the gel containing S. mitis or S. salivarius, and also not detectable in the gel containing S. sanguis. S. sobrinus extract showed only one bacteriolytic band (78 kDa) as strong activity using S. sobrinus cells as substrate. S. sanguis extract showed no bacteriolytic bands using any streptococcal cells. Extracts of either S. mitis or S. salivarius showed weak activity by using respective strains as substrate.
Epidemiological Characterization of Salmonella Typhimurium DT104 Prevalent among Food‐Producing Animals in the Japanese Veterinary Antimicrobial Resistance Monitoring Program (1999–2001)
Authors: Esaki Hidetake , Morioka Ayako , Kojima Akemi , Ishihara Kanako , Asai Tetsuo , Tamura Yutaka , Izumiya Hidemasa , Terajima Jyun , Watanabe Haruo , Takahashi Toshio
Published: November 2013

In the course of nationwide investigation on epidemiological characteristics in Salmonella Typhimurium isolates from food‐producing animals in Japan between 1999 and 2001, fifty‐seven isolates of S. Typhimurium DT104 and 104B obtained from cattle and swine at farm level in Japan between 1999 and 2001 were classified with pulsotype and antimicrobial resistance type. Most of the isolates were resistant to five or more antimicrobials and were genotyped into four groups. The present nationwide investigation shows that at least 11 types of S. Typhimurium related to DT104 are prevalent among food‐producing animals across the country.
Detection of the Impairment of CD80 Expression on Circulating Monocytes in HIV‐Infected Thai Children
Authors: Ubol Sukathida , Aiamkitsumrit Benjamaz , Nitayaphan Sorachai , Sirinavin Sayomporn , Kitthawee Sangvorn
Published: November 2013

The mechanism of progressive anergic response in HIV‐infected children has yet to be adequately described. One possibility is inappropriate delivery of an essential second signal for T‐cell activation due to the inappropriate presentation of co‐stimulatory molecules. To determine whether the ligand for the secondary signal is impaired in pediatric AIDS, we compared the level of CD80 expression by circulating monocytes in HIV‐infected and ‐noninfected children (15 mild/asymptomatic, 13 symptomatic and 12 HIV seronegative children). By two‐color flow cytometry analysis, there was no statistically significant difference in the percentage of monocytes expressing CD80 among the groups (i.e., 63.2±15.8, 60.9±12.7, 61.04±10.9 for uninfected children, mild‐asymptomatic children and symptomatic children, respectively). However, both infected groups showed statistically significant lower levels of CD80 expression, with mean fluorescent intensities of 40.9±15.9 and 38.8±10.7 compared to 57.05±16.3 for the uninfected control group. Our data demonstrated a correlation between HIV infection and impairment of CD80 by circulating monocytes. Whether the impairment on CD80 expression contributes to destruction of the immunological network in HIV‐infected children requires further investigation.
Microbiota Composition of the Intestinal Mucosa: Association with Fecal Microbiota?
Authors: Ouwehand Arthur C. , Salminen Seppo , Arvola Taina , Ruuska Tarja , Isolauri Erika
Published: November 2013

The fecal and mucosal microbiota of infants with rectal bleeding and the fecal microbiota of healthy age‐matched controls were investigated by fluorescent in situ hybridization. Bifidobacteria were the main genus in both the feces and mucosa. The other genera tested, Bacteroides, Clostridium, Escherichia coli and lactobacilli/enterococci, represented only minor constituents. No differences in fecal microbiota were observed between patients and controls. In the patients, however, four times greater numbers of bifidobacteria were observed in the feces when compared to the mucosa. Notwithstanding this difference, a strong positive correlation prevailed for bifidobacteria in feces and mucosal samples. The genera assessed accounted for 16% of total bacterial counts on mucosal samples and for 47% of total bacterial counts in feces. This indicates that the unidentified part of the microbiota, especially on the mucosa, deserves more attention.
Epidemiological and Serological Survey of Brucellosis in Mongolia by ELISA Using Sarcosine Extracts
Authors: Erdenebaatar Janchivdorj , Bayarsaikhan Balgan , Yondondorj Agchbazar , Watarai Masahisa , Shirahata Toshikazu , Jargalsaikhan Enkhtuya , Kawamoto Keiko , Makino Sou‐ichi
Published: November 2013

Brucellosis is an important zoonosis, and serological surveillance is essential to its control. However, cross‐reactions of attenuated live cells of Brucella abortus strain S‐19 and B. melitensis strain Rev‐1 with Yersinia enterocolitica O9 or vaccinated animal sera interfere with accurate serological diagnosis by the Rose Bengal test (RBT). Therefore, we used ELISA with sarcosine extracts from the virulent B. abortus strain 544 to eliminate false‐positives among RBT positive‐sera. A total of 697 serum samples were collected in Mongolia from humans and animals in 23 nomadic herds. The herds were classified into three groups as brucellosis‐endemic (BE), brucellosis‐suspected (BS), or Brucella‐vaccinated (BV). The number of 295 animals (43.0%) was positive by RBT, but 206 (69.8%) of these were positive according to ELISA; therefore, 30.2% of the RBT‐positive sera were found to be false positives. The false positive samples for RTB represent 4.1%, 27.4%, and 68.2% of the animals from the BE, BS, and BV herds, respectively. In addition, 32% of RBT‐positive human sera were also false positives. Thus, our ELISA would be more specific than RTB and useful for epidemiological surveillance for brucellosis.
Suppression of Hepatitis C Virus Replicon by RNA Interference Directed against the NS3 and NS5B Regions of the Viral Genome
Authors: Takigawa Yuki , Nagano‐Fujii Motoko , Deng Lin , Hidajat Rachmat , Tanaka Motofumi , Mizuta Hiroyuki , Hotta Hak
Published: November 2013

RNA interference (RNAi) is a phenomenon in which small interfering RNA (siRNA), an RNA duplex 21 to 23 nucleotides (nt) long, or short hairpin RNA (shRNA) resembling siRNA, mediates degradation of the target RNA molecule in a sequence‐specific manner. RNAi is now expected to be a useful therapeutic strategy for hepatitis C virus (HCV) infection. In the present study we compared the efficacy of a number of shRNAs directed against different target regions of the HCV genome, such as 5′‐untranslated region (5′UTR) (nt 286 to 304), Core (nt 371 to 389), NS3–1 (nt 2052 to 2060), NS3–2 (nt 2104 to 2122), and NS5B (nt 7326 to 7344), all of which except for NS5B are conserved among most, if not all, HCV subtype 1b (HCV‐1b) isolates in Japan. We utilized two methods to express shRNAs, one utilizing an expression plasmid (pAVU6+27) and the other utilizing a recombinant lentivirus harboring the pAVU6+27‐derived expression cassette. Although 5′UTR has been considered to be the most suitable region for therapeutic siRNA and/or shRNA because of its extremely high degree of sequence conservation, we observed only a faint suppression of an HCV subgenomic replicon by shRNA against 5′UTR. In both plasmid‐ and lentivirus‐mediated expression systems, shRNAs against NS3–1 and NS5B suppressed most efficiently the replication of the HCV replicon without suppressing host cellular gene expression. Synthetic siRNA against NS3–1 also inhibited replication of the HCV replicon in a dose‐dependent manner. Taken together, the present results imply the possibility that the recombinant lentivirus expressing shRNA against NS3–1 would be a useful tool to inhibit HCV‐1b infection.
A Highly Effective Method for Removing Suspended Poliovirus from Water Using a Positively‐Charged Carbon Felt Electrode
Authors: Kondo Yasuhito , Morita Yukio , Yamada Atsushi , Kimura Hirokazu
Published: November 2013

We developed an effective system to eliminate poliovirus from modified tap water using a positively‐charged carbon felt electrode. The zeta potential of polioviruses was measured using laser microscopic electrophoresis. Poliovirus adsorption to the electrode was examined by indirect immunofluorescence. The tissue culture infective dose (TCID) of poliovirus type 2 (Sabin strain) was determined using human rhabdomyosarcoma cells (RD cells). Poliovirus VP2 gene copy numbers were assessed by reverse transcription followed by a quantitative real‐time polymerase chain reaction. The mean zeta potential of the viruses was −20 mV. Relatively large numbers of polioviruses (103 or 4 × 103 TCID50/0.1 ml) could be removed by adsorption to the electrode, drastically decreasing TCID and copy numbers of poliovirus genome in the water. Virus elimination was dependent on electric current and time. Thus, the positively‐charged carbon felt electrode effectively adsorbed polioviruses. The system may prove applicable to the elimination of certain viruses from water.
PCR‐Based Identification of Pandemic Group Vibrio parahaemolyticus with a Novel Group‐Specific Primer Pair
Authors: Okura Masatoshi , Osawa Ro , Iguchi Atsushi , Takagi Michihiro , Arakawa Eiji , Terajima Jun , Watanabe Haruo
Published: November 2013

A PCR‐based assay to identify pandemic group Vibrio parahaemolyticus has been developed. The assay employs an oligonucleotide primer pair derived from the group‐specific sequence of an arbitrarily primed‐PCR fragment, which is located in the genome encoding a “hypothetical protein,” approximately 80% homologous to the Mn2+ and Fe2+ transporter of the NRAMP family of V. vulnificus. The assay distinguished the pandemic group from other V. parahaemolyticus strains by yielding a 235‐bp specific amplicon, and can be a useful diagnostic tool for identification of pandemic group strains.
Piceatannol Prevents Lipopolysaccharide (LPS)‐Induced Nitric Oxide (NO) Production and Nuclear Factor (NF)‐κB Activation by Inhibiting IκB Kinase (IKK)
Authors: Islam Shamima , Hassan Ferdaus , Mu Mya Mya , Ito Hiroyasu , Koide Naoki , Mori Isamu , Yoshida Tomoaki , Yokochi Takashi
Published: November 2013

The effect of piceatannol on lipopolysaccharide (LPS)‐induced nitric oxide (NO) production was examined. Piceatannol significantly inhibited NO production in LPS‐stimulated RAW 264.7 cells. The inhibition was due to the reduced expression of an inducible isoform of NO synthase (iNOS). The inhibitory effect of piceatannol was mediated by down‐regulation of LPS‐induced nuclear factor (NF)‐κB activation, but not by its cytotoxic action. Piceatannol inhibited IκB kinase (IKK)‐α and β phosphorylation, and subsequently IKB‐α phosphorylation in LPS‐stimulated RAW 264.7 cells. On the other hand, piceatannol did not affect activation of mitogen‐activated protein (MAP) kinases including extracellular signal regulated kinase 1/2 (Erk1/2), p38 and stress‐activated protein kinase/c‐Jun NH2‐terminal kinase (SAPK/JNK). Piceatannol inhibited the phosphorylation of Akt and Raf‐1 molecules, which regulated the activation of IKK‐α and β phosphorylation. The detailed mechanism of the inhibition of LPS‐induced NO production by piceatannol is discussed.
Comparative Evaluation of Standard Dilution Method and Commercial Kit for Frozen Plate Antifungal Susceptibility Testing of Yeasts Using 200 Clinical Isolates
Authors: Makimura Koichi , Suzuki Takako , Tamura Takashi , Ikedo Masanari , Hanazawa Ryo , Takahashi Yuko , Yamada Yohko , Uchida Katsuhisa , Yamaguchi Hideyo
Published: November 2013

A comparative evaluation of standard microdilution methods and a commercial kit for frozen plate antifungal susceptibility testing of yeasts was performed using amphotericin B, flucytosine, fluconazole, miconazole, and itraconazole on 200 yeast isolates. The isolates included 100 strains of Candida albicans, eight of C. tropicalis, twelve of C. parapsilosis, eight of C. glabrata, five of Cryptococcus neoformans, thirteen of Trichosporon asahii, and 54 other strains of seven other species of ascomycotic yeasts. Microdilution testing was performed according to the standard method for antifungal susceptibility testing published by the Japanese Society for Medical Mycology (JSMM), which are a modification of the method developed by the National Committee for Clinical Laboratory Standards (NCCLS) M27‐P. The commercial kit was prepared according to the manufacturer's instructions. The degree of agreement within ± 1 dilution for 200 clinical isolates against five antifungal agents was excellent with values for amphotericin B, flucytosine, fluconazole, miconazole, and itraconazole of 100%, 99.0%, 97.5%, 97.0%, and 97.0%, respectively. Overall, the frozen plate antifungal susceptibility testing kit provided convenient and reproducible results comparable to those obtained with the JSMM standard method.
Delayed HIV‐1 Infection of CD4+ T Lymphocytes from Therapy‐Naïve Patients Demonstrated by Quantification of HIV‐1 DNA Copy Numbers
Authors: Wada Kaoru , Nagai Hiromi , Hagiwara Tomoko , Ibe Shiro , Utsumi Makoto , Kaneda Tsuguhiro
Published: November 2013

Measuring the amount of HIV‐1 DNA in infected cells is important to estimate the size of the viral reservoir in patients. However, the clinical impact of the intracellular viral DNA level remains unclear. The present study examines the clinical significance of the HIV‐1 DNA level in peripheral CD4+ T lymphocytes from 21 therapy‐naïve patients. HIV‐1 DNA levels in purified peripheral CD4+ T lymphocytes were measured by the real‐time PCR method using the Roche LightCycler system that can detect 200 copies/106 cells. We detected intracellular HIV‐1 DNA in 15 (71.4%) of 21 patients at levels ranging from 270 to 98,120 copies/106 CD4+ cells, with a median of 2,220 copies/106 cells. We also found HIV‐1 DNA that was below the detection limit in the remaining 6 patients, although 8,800–150,000 copies/ml of HIV‐1 RNA were detected in plasma. Circular HIV‐1 DNA was not detected in 5 of 6 cases, suggesting that reverse transcription in CD4+ T lymphocytes of these cases was not active. Thus, delayed HIV‐1 infection of CD4+ T lymphocytes was demonstrated in these patients. The level of HIV‐1 DNA in peripheral CD4+ T lymphocytes indicates the clinical status of therapy‐naïve patients.
Study on Immunological Status of Chinese HIV‐Infected Individuals
Authors: Shang Hong , Jiang Yongjun , Zhang Zining , Han Xiaoxu , Zhang Min , Wang Yanan , Liu Jing
Published: November 2013

HIV‐1 infection is characterized by a gradual decline of immune function, and the immune dysfunction is widely regarded as one of the most important determinants of disease progression. The present study was performed to analyze in more detail the immunological status of HIV‐infected people in China. T cell counts, activation of T cells, HIV‐1 specific CTL and plasma levels of cytokines were determined with flow cytometry, IFN‐γ Elispot or ELISA techniques. The HIV viral load was negatively correlated with CD4+, CD8+ T cell counts (r = –0.654, P<0.001; r = –0.228, P<0.05); the breadth and magnitude of HIV‐1 specific CTL responses against HIV‐1 Gag peptides was related to disease progression; the activation of CD8+ T cells was significantly higher than that in HIV‐negative controls; the level of plasma IL‐12 was much lower and the plasma IFN‐γ, IL‐10 and IL‐6 were much higher in HIV‐infected persons than in HIV‐negative controls (P<0.05). Study on immunological status in HIV‐infected Chinese is very important in predicting the disease progression and providing information for HAART therapy in China.
Novel Evidence Suggesting Clostridium difficile Is Present in Human Gut Microbiota More Frequently than Previously Suspected
Authors: Iizuka Masahiro , Konno Shiho , Itou Hiroaki , Chihara Junichi , Toyoshima Itaru , Horie Yasuo , Sasaki Kenji , Sato Akiko , Shindo Kenichi , Watanabe Sumio
Published: November 2013

Prevalence rate of Clostridium difficile in healthy human adults is believed to be very low. Our RT‐PCR system using glass powder, which can eliminate PCR inhibitors, detected C. difficile toxin B mRNA in 16 of 30 fecal samples (53.3%) from healthy human adults. In contrast, we failed to detect toxin B in the same fecal samples by PCR using DNA templates extracted with phenol‐chloroform. Our results suggest that PCR inhibitors in feces carried through phenol‐chloroform extraction procedure might suppress the sensitivity of PCR and that C. difficile is actually present in human gut microbiota more frequently than previously suspected.
Effects of Two Probiotic Lactobacillus Strains on Jejunal and Cecal Microbiota of Broiler Chicken under Acute Heat Stress Condition as Revealed by Molecular Analysis of 16S rRNA Genes
Authors: Lan Pham Thi Ngoc , Sakamoto Mitsuo , Benno Yoshimi
Published: November 2013

We examined the effects of probiotic Lactobacillus strains of Lactobacillus agilis JCM 1048 and Lactobacillus salivarius subsp. salicinius JCM 1230 on jejunal and cecal microbiota of broiler chicken under heat stress condition using terminal restriction fragment length polymorphism (T‐RFLP) analysis. The jejunal bacterial community was limited to a few bacterial groups, mostly Lactobacillus spp. A relatively abundant and higher prevalence of Lactobacillus spp. were observed in the jejunal and cecal microbiota of the probiotic chickens compared with those of the control chickens under heat stress condition. In general, the probiotic strains did not significantly affect the abundance of L. agilis and L. salivarius in chicken intestine but clearly contributed to increasing their prevalence in the probiotic chickens. The probiotic Lactobacillus strains enriched the diversity of Lactobacillus flora in chicken jejunum and cecum by increasing the abundance and prevalence of Lactobacillus spp. inhabiting the intestine. The richness of Lactobacillus species tended to be similar among the jejunal and cecal microbiota. The bacterial community of cecum was complex and age‐dependent. The major components of the cecal microbiota were clostridia and lactobacilli. The Clostridium subcluster XIVa was the most predominant group in chicken cecum. Probiotic Lactobacillus strains restored the microbial balance and maintained the natural stability of indigenous bacterial microbiota following heat stress‐induced changes.
Comparison of Japanese Isolates of Coxiella burnetii by PCR‐RFLP and Sequence Analysis
Authors: Andoh Masako , Nagaoka Hiromi , Yamaguchi Tsuyoshi , Fukushi Hideto , Hirai Katsuya
Published: November 2013

The genetic variation of Japanese isolates of Coxiella burnetii, the agent of Q fever, was found for the first time. Forty‐nine out of 72 isolates had the chronic pattern of the isocitrate hydrogenase gene. Sequence analysis revealed that the isolates have a specific nucleotide sequence. The putative amino acid sequence was the same as that of chronic reference strains. These results suggest the variation of C. burnetii isolates in Japan.
B1‐B Cells Are the Main Antigen Presenting Cells in CpG‐ODN‐Stimulated Peritoneal Exudate Cells
Authors: Bamba Hiromichi , Ishigaki Hirohito , Ishida Hideaki , Kajino Kiichi , Fujiyama Yoshihide , Ogasawara Kazumasa
Published: November 2013

Peritoneal exudate cells (PEC) have long been used as antigen presenting cells (APC), because they have been considered to contain mainly macrophages. However, it is still unclear specifically which cells of the peritoneal exudate function as APC. Herein, we focused on macrophages and B1‐B cells of the PEC and examined their APC function and cytokine production. B1‐B cells purified from PEC functioned effectively as APC after CpG‐stimulation and mainly produced IL‐10. In contrast, macrophages purified from PEC were not able to present incorporated antigens to T cells, despite the production of IL‐12 and expression of co‐stimulatory molecules after CpG stimulation. These results suggest that previously held ideas regarding the functions of the mixture of cells in the PEC need to re‐evaluated. In summary, the antigen presenting function of PEC was mainly attributed to B1‐B cells and immunoenhancing cytokine production was dominantly derived from peritoneal macrophages.
Seroepidemiology of Spotted Fever Group and Typhus Group Rickettsioses in Humans, South Korea
Authors: Jang Won‐Jong , Choi Yeon‐Joo , Kim Jong‐Hyun , Jung Kwang‐Don , Ryu Ji‐Sun , Lee Seung‐Hyun , Yoo Cheon‐Kwon , Paik Hyung‐Suk , Choi Myung‐Sik , Park Kyung‐Hee , Kim Ik‐Sang
Published: November 2013

The prevalence of spotted fever group (SFG) and typhus group (TG) rickettsioses was investigated in 3,362 sera by immunofluorescence assay. The serum samples were obtained from patients with acute febrile episodes in South Korea from December 1992 to November 1993. The number of polyvalent positive sera against SFG rickettsial agents at the level of 1:40 dilution was 269 (8%) in Rickettsia sibirica, 482 (14.34%) in R. conorii, and 546 (16.24%) in R. akari. Many of the positive sera contained immunoglobulin (Ig) M antibodies rather than IgG antibodies. These results strongly suggest that SFG rickettsioses are prevalent in Korea. For TG rickettsial agents, the number of positive sera was 1,096 (32.60%) in R. typhi and 951 (28.29%) in R. prowazekii. Only a few epidemic typhus positive sera contained IgM antibodies. The result suggests that recent and/or primary infections of epidemic typhus were very rare in Korea during the said period. Among seven patients who had high titers (1:5,120) of IgG antibody to R. prowazekii, six were over 50 years old. The result suggests that Brill‐Zinsser disease was prevalent in Korea.