bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic Variation in 16S‐23S rDNA Internal Transcribed Spacer Regions and the Possible Use of This Genetic Variation for Molecular Diagnosis of Bacteroides Species
Authors: Kuwahara Tomomi , Norimatsu Izumi , Nakayama Haruyuki , Akimoto Shigeru , Kataoka Keiko , Arimochi Hideki , Ohnishi Yoshinari 
Published: November 2013

AbstractThe structural variation in 16S‐23S rDNA internal transcribed spacer regions (ITS) among Bacteroides species was assessed by PCR amplification and sequencing analysis, and its possible use for molecular diagnosis of these species was evaluated. Ninety strains of the genus Bacteroides, including the species B. distasonis, B. eggerthii, B. fragilis, B. ovatus, B. thetaiotaomicron, B. uniformis and B. vulgatus, produced one to three ITS amplification products with sizes ranging from 615 to 810 bp. Some Bacteroides strains could be differentiated at species level on the basis of ITS amplification patterns and restriction fragment length polymorphism (RFLP) analysis using a four‐nucleotide‐recognizing enzyme, Msp I. The results of sequence analysis of ITS amplification products revealed genes for Ile‐tRNA and Ala‐tRNA in all strains tested. The nucleotide sequence, except for that in tRNA‐coding regions, was highly variable and characteristic for each species, but a common sequence among B. fragilis, B. thetaiotaomicron and B. ovatus was observed. A digoxigenin‐labeled oligonucleotide probe (named FOT1), which was designed from this conserved sequence, specifically hybridized to the ITS amplification products from B. fragilis, B. thetaiotaomicron and B. ovatus. These results suggest that the ITS region is a useful target for the development of rapid and accurate techniques for identification of Bacteroides species.
Effect of Sealing and Tween 80 on the Antifungal Susceptibility Testing of Essential Oils
Authors: Inouye Shigeharu , Tsuruoka Tsutomu , Uchida Katsuhisa , Yamaguchi Hideyo 
Published: November 2013

AbstractConcentrations of essential oils showing high volatility decreased substantially in broth and agar media when incubated under open conditions. The decrease in the half life was from 0.7 to 38 hr in broth medium at 27 C. When evaporation was prevented by sealing, MIC values against Aspergillus fumigatus, Candida albicans and Trichophyton mentagrophytes by broth or agar dilution assay were lowered two to eight‐fold, as compared with those obtained under open conditions. Addition of Tween 80 caused a rise of the MICs against A. fumigatus by two to four‐fold in broth dilution assay, but little affected the MICs in agar dilution assay.
Cluster Analysis of Human and Animal Pathogenic Microsporum Species and Their Teleomorphic States, Arthroderma Species, Based on the DNA Sequences of Nuclear Ribosomal Internal Transcribed Spacer 1
Authors: Makimura Koichi , Tamura Yoshiko , Murakami Arisa , Kano Rui , Nakamura Yuka , Hasegawa Atsuhiko , Uchida Katsuhisa , Yamaguchi Hideyo 
Published: November 2013

AbstractWe performed a cluster analysis of human and animal pathogenic Microsporum species and their teleomorphic states, Arthroderma species, including A. otoe‐related species (M. canis, M. audouinii, M. distortum, M. equinum, M. langeronii, and M. ferrugineum) and M. gypseum complex (A. fulvum, A. gypseum, and A. incurvatum) using DNA sequences of nuclear ribosomal internal transcribed spacer 1 (ITS1). The dendrogram showed the members of A. otae‐related species to be monophyletic and to construct an extremely closely related cluster with a long horizontal branch. This ITS1‐homologous group of A. otae was organized in 6 unique genotypes, while sequences of the members of the ITS1‐homologous group of M. gypseum complex are more diverse. This ITS1‐based database of Microsporum species and their teleomorphic states will provide a useful and reliable species identification system: it is time‐saving (takes two to three days), accurate and applicable even to strains with atypical morphological features or in a non‐culturable state.
Flow Cytometric Evaluation of Antiviral Agents against Human Herpesvirus 6
Authors: Amjad Muhammad , Gillespie Michael A. , Carlson Robert M. , Karim M. Reza‐ul 
Published: November 2013

AbstractAntiviral activities of acyclovir (9‐[(2‐hydroxyethoxy) methyl] guanine, ACV), penciclovir (9‐[4‐hydroxy‐3‐(hydroxymethyl) butyl] guanine, PCV), ganciclovir ([9‐(1,3‐dihydroxy‐2‐propoxy) methyl] guanine, GCV), and foscarnet (phosphonoformic acid, PFA) were determined against Human Herpesvirus 6 (HHV‐6) by flow cytometric technique. The technique is based on the detection of gp116 antigen expression in virus infected cells. Susceptibility was defined in terms of drug concentration which reduced the number of cells expressing HHV‐6 gp116 antigen with a mean fluorescent intensity (MFI) by 50% as compared to virus infected untreated cells. GCV was found to be most effective against HHV‐6 followed by PFA, PCV and ACV. For HHV‐6A, the mean 50% inhibitory concentrations (IC50) of GCV and PFA were found to be 3.4 μm and 34.7 μm respectively, whereas the IC50 of ACV and PCV were found to be 53.7 μm and 37.9 μm respectively. For HHV‐6B, the IC50 of GCV and PFA were found to be 5.7 μm and 71.4 μm respectively, whereas the IC50 of ACV and PCV were found to be 119.0 μm and 77.8 μm respectively. Flow cytometry is a valuable technique for the evaluation of antiviral compounds against viruses including HHV‐6.
Cross‐Linking of β2 Integrins Caused Diminished Responses of Neutrophils to Priming Agents Like Lipopolysaccharide or Tumor Necrosis Factor‐α: Possible Involvement of Tyrosine Kinase Syk
Authors: Kusumoto Keisuke , Aida Yoshitomi , Maeda Katsumasa , Pabst Michael J. 
Published: November 2013

AbstractNeutrophils up‐regulate β2 integrins like CD11b/CD18 in response to lipopolysaccharide (LPS). Up‐regulation of β2 integrins causes neutrophils to adhere to surfaces, and to release superoxide anion (O2–). When neutrophils are exposed to LPS plus plasma under conditions not favorable for adherence (absence of Mg2+), the cells do not spontaneously release O2–, but instead they are primed for enhanced release of O2– after subsequent triggering by fMLP. In the presence of Mg2+, neutrophils adhere in response to LPS but fMLP‐triggered O2– release by LPS‐primed neutrophils is diminished. To understand why adherence interferes with the response of neutrophils to N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP), β2 integrins were cross‐linked by mouse monoclonal antibodies that had been immobilized by surface‐bound anti‐mouse antibody. When unprimed neutrophils were trapped on the surface by these cross‐linked monoclonal antibodies, O2– release was triggered, and priming by LPS for fMLP‐triggered O2– release was diminished, indicating that this cross‐linking of β2 integrins mimicked adherence. Alkaline phosphatase is up‐regulated by LPS or tumor necrosis factor‐α, and this response was also diminished by the cross‐linking antibodies. The diminished alkaline phosphatase up‐regulation was reversed by genistein, a general inhibitor of tyrosine kinases, and by piceatannol, an inhibitor for Syk kinase. Piceatannol also inhibited the phosphorylation of Syk caused by cross‐linking of β2 integrins. These results suggested that adherence‐induced triggering and Syk kinase activation might be responsible for the diminished response of LPS‐primed neutrophils to fMLP when neutrophils were adherent.
Rapid Detection of Human Fecal Eubacterium Species and Related Genera by Nested PCR Method
Authors: Kageyama Akiko , Benno Yoshimi 
Published: November 2013

AbstractPCR procedures based on 16S rDNA gene sequence specific for seven Eubacterium spp. and Eggerthella lenta that predominate in the human intestinal tract were developed, and used for direct detection of these species in seven human feces samples. Three species of Eggerthella lenta, Eubacterium rectale, and Eubacterium eligens were detected from seven fecal samples. Eubacterium biforme was detected from six samples. It was reported that E. rectale, E. eligens, and E. biforme were difficult to detect by traditional culture method, but the nested PCR method is available for the detection of these species. This result shows that the nested PCR method utilizing a universal primer pair, followed by amplification with species‐specific primers, would allow rapid detection of Eubacterium species in human feces.
Much Higher Risk of Premalignant and Malignant Cervical Diseases in Younger Women Positive for HPV16 than in Older Women Positive for HPV16
Authors: Tanaka Hidenori , Karube Akihiko , Tanaka Toshinobu , Nakagomi Osamu 
Published: November 2013

AbstractHuman papillomavirus (HPV), particularly HPV16, is strongly associated with premalignant lesions of the uterine cervix and cervical cancer. However, HPV infection is a common sexually transmitted disease and only a few women develop cervical cancer. Although the presence of HPV and abnormal cytology are independent risk factors for cervical diseases, implementing both tests on every woman is argued not to be recommended mainly in terms of cost‐effectiveness. During a 20‐month period between October, 1994, and May, 1996, cervical swabs from 207 women who were referred for colposcopy because of cervical dyskaryosis (Papanicolaou class IIIa or higher) were examined by PCR for the presence of HPV16. When these women were divided into two groups; i.e., group A consisting of women who were 44 years old or younger (n = 111), and group B consisting of women who were 45 years old or older (n = 96), the risk of having premalignant and malignant cervical diseases upon infection with HPV16 was approximately 8 times higher in group A than in group B. Thus, we conclude that HPV‐testing should be implemented on every young woman with an abnormal Papanicolaou smear test.
Mechanism for Maintenance of Dominant T Helper 1 Immune Responses in Lewis Rats
Authors: Sakamoto Shuji , Fukushima Atsuki , Ozaki Akemi , Ueno Hisayuki , Kamakura Maki , Taniguchi Taketoshi 
Published: November 2013

AbstractLewis rats are prone to T helper (Th) 1 immune responses, whereas Brown Norway (BN) rats are susceptible to Th2 immune responses. Yet, the precise mechanism of induction of the different outcome between these two strains remained elusive. We investigated the expression levels of some cytokines, their receptors and accessory molecules responsible for the polarization of antigen‐specific immune response into a predominant Th1 or Th2 profile in Lewis and BN rats. Lymph node (LN) cells collected from rats immunized with short ragweed (RW) were used directly or after stimulation in vitro with RW for 3 days. Expression of cytokines, their receptors and accessory molecules in these LN cells were tested by reverse transcriptase‐PCR. Culture supernatant was used for ELISA to detect IL‐12 protein. We observed clear differences between these strains in the expression of IL‐12p40, which was high in LN cells of Lewis rats even before stimulation in vitro. In addition, a higher amount of IL‐12 was present in the culture supernatant in Lewis rats. Upregulation of the expression of IL‐12 receptor β1, β2, IFN‐γ receptor α and β genes were more prominent in Lewis rats rather than BN rats. Furthermore, attenuated expression of CD40 and CD40 ligand by stimulation in vitro was noted only in BN rats. Changes in expression of these molecules by stimulation as well as higher basal level of IL‐12p40 might have led to the activation of Th1 cells in Lewis rats.
Resistance to Antibiotics in Injured Coliforms Isolated from Drinking Water
Authors: Cordoba María Alejandra , Roccia Irene Laura , De Luca Maria Marta , Pezzani Betina Cecilia , Basualdo Juan Angel 
Published: November 2013

AbstractWe studied the antibiotic sensitivity of injured coliforms isolated from drinking water of La Plata, Argentina. The antibiotic sensitivity test by the agar diffusion method were proved in: Klebsiella oxytoca (14 strains), Enterobacter aerogenes (4 strains) and Enterobacter cloacae genomic group 3 (14 strains). We found that while these impaired total coliforms were sensitive to piperacillin‐tazobactam (TAZ), netilmicin (NTL), ofloxacin (OFLX), and norfloxacin (NFLX) (100%), they had resistant to aminopenicillin‐sulbactam (AMS) and nitrofurantoin (NIT) (100%). The resistance to antibiotics demonstrated in these strains would point to the need to promote a rational and judicious use of antimicrobial agents while at the same time implementing a program of active vigilance aimed at ensuring the highest quality of drinking water throughout the system.
Negative Incidence of Lyme Disease‐Related Borrelia Spp. in Alishan, Taiwan
Authors: Masuzawa Toshiyuki , Pan Ming‐jeng , Yu Qin , Kadosaka Teruki , Imai Yasuyuki , Yanagihara Yasutake 
Published: November 2013

AbstractTo investigate the prevalence of Lyme disease‐related Borrelia species, wild rodents were captured around Yushan National Park and Alishan Forest Recreation Area Park in Taiwan 2,000 to 3,000 meters above sea level. Borrelia was not isolated from 67 small mammals of 7 species. Sera from rodents showed no positive reactivity against whole cell antigens of B. garinii, B. afzelii or B. valaisiana by ELISA. These results suggested that Lyme disease is not endemic to the Alishan area.
Molecular Heterogeneity of Heat‐Stable Toxin‐Producing and Non‐Producing Escherichia coli O25 Strains Isolated in Japan
Authors: Kaneko Aki , Fujino Tomoko , Sugawara Chizu , Kagami Yutaka , Miyadai Hidenori , Kirikae Teruo , Kawahara Kazuyoshi 
Published: November 2013

AbstractEscherichia coli O25 strains that produce heat‐stable toxin (ST) have been recently isolated in Japan, and epidemiological study of this type of enterotoxigenic E. coli is required. In this study the heterogeneity of 16 ST‐producing and non‐producing strains of E. coli O25 was investigated. All eight ST‐producing strains were shown to have STIb gene, and seven of them had similar profiles of plasmids, ladder‐banding of LPS in SDS‐polyacrylamide gel electrophoresis, and chromosomal DNA digestions in pulsed‐field gel electrophoresis (PFGE). In contrast, ST‐non‐producing strains were more heterogeneous in all parameters examined. PFGE of the digested chromosomal DNA with several restriction enzymes was proved to be an effective procedure to compare the closely related strains of E. coli O25.
Helicobacter pylori DNA in Drinking Water in Japan
Authors: Horiuchi Takao , Ohkusa Toshifumi , Watanabe Mamoru , Kobayashi Daisuke , Miwa Hiroto , Eishi Yoshinobu 
Published: November 2013

AbstractHelicobacter pylori has been detected in drinking water in Peru and Sweden, suggesting the possibility of water‐borne transmission. To date there have been few reports of H. pylori being detected in water; one was of the ureA gene of H. pylori in wells and springs in rural Japan. We examined water sampled in or near urban areas of Japan for H. pylori DNA by three assays using the polymerase chain reaction (PCR). Near Tokyo, samples were obtained: 10 of tap water, 6 of well water, 10 of river water, and 10 of sea water. Samples were filtered with membranes with 0.05‐ or 0.22‐μm pores, which bacterial cells are caught by. Bacterial nucleic acids were extracted and purified and the PCR was done to amplify adhesin specific for H. pylori and the ureA gene, if present. Real‐time PCR that measured the yield in terms of fluorescence was done with primers for 16S rRNA. None of the samples of tap, river, or sea water contained adhesin, ureA or 16S rRNA. None of the 6 samples of well water contained adhesin or ureA, but 2 of the 6 samples contained 16S rRNA. Some of the users of the well had had H. pylori infection in the past H. pylori DNA was detected in well water and the users had been infected, so water‐borne transmission via well water may occur even in towns in Japan.
Morphological Variety of Intracellular Microcolonies of Legionella Species in Vero Cells
Authors: Ogawa Midori , Takade Akemi , Miyamoto Hiroshi , Taniguchi Hatsumi , Yoshida Shin‐ichi 
Published: November 2013

AbstractIntracellular microcolonies of six Legionella species growing in Vero cells showed distinctly varied morphologies. The varieties were observed by light microscopy of Gimenez‐stained, Legionella‐infected Vero cells and by electron microscopy (EM). Legionella pneumophila Philadelphia‐1 formed needle‐shaped crystal‐like microcolonies. Legionella bozemanii WIGA formed microcolonies like wool balls containing filamentous cells. In EM, these organisms proliferated in endosomes, which were adjacent to swollen rough endoplasmic reticula. Legionella oakridgensis OR‐10 showed serpentine chains. Many mitochondria were observed around the microcolonies. Legionella jordanis BL‐540 formed spherical moss‐like microcolonies which were or were not surrounded by endoplasmic membranes. Legionella feeleii WO‐44C spread throughout the cytoplasm without making clusters. Legionella dumoffii Tex‐KL made big clusters that spread in the cytoplasm, a portion of which was outside the endosome membranes. These different morphologies imply diversity in modes of intracellular multiplication of Legionella spp.
Effect of Anticoagulants in Colorimetric Assay for Basic Carboxypeptidases
Authors: Komura Hidefumi , Obata Kyoko , Campbell William , Yumoto Miho , Shimomura Yasuyo , Katsuya Hirotada , Okada Noriko , Okada Hidechika 
Published: November 2013

AbstractCarboxypeptidases (CP) in plasma and sera serve as regulators of anaphylatoxins such as C3a and C5a. The activity of CP can be measured by determining hippuric acid after cleavage of the small synthetic substrate hippuryl‐l‐arginine. Although a colorimetric assay is convenient for determining hippuric acid generated by CP, we noticed that some anticoagulants, such as citrate, interfere with the color development of the reagents used. EDTA and heparin provide an appropriate value. EGTA used as anticoagulant also provides an appropriate value. Therefore, concentration of citrate in samples should be controlled to be constant for background subtraction.
Engineering of a Sagiyama Alphavirus RNA‐Based Transient Expression Vector
Authors: Yamaguchi Yuka , Shirako Yukio 
Published: November 2013

AbstractSagiyama virus (SAGV), a strain of Getah virus in the genus Alphavirus in the family Togaviridae, has a broad host range in vertebrates and invertebrates but is not pathogenic for humans. We engineered the SAGV genome as an efficient transient expression vector using the full‐length infectious cDNA clone pSAG2 as the background. A green fluorescent protein (GFP) gene was used as a reporter gene and expressed from a subgenomic mRNA. When the GFP gene was placed downstream of the intact capsid protein gene or an internally deleted capsid protein gene encoding the N‐terminal 9 amino acids and C‐terminal 149 amino acids, autoproteolysis occurred efficiently at the boundary site to release GFP from the N‐terminally‐fused capsid‐protease domain. GFP was also expressed efficiently without the 5′‐terminal region of the capsid protein gene, suggesting that SAGV capsid protein gene does not have a translation enhancer sequence. To provide structural proteins for pseudovirion formation, a nonviable mutant construct, pSAG2.3L, which contains a Gly‐to‐Leu substitution at the − 2 position of the nsP3/4 cleavage site, was used as a helper. GFP was expressed up to 50 μg from 1 × 106 BHK21 cells after inoculation of pseudovirions. The C6/36 mosquito cell was also a suitable host for a large scale expression of GFP using pseudovirions. In addition to high‐level transient expression, safeness of SAGV should give an advantage over other alphavirus expression vectors.
Inactivation of C3a and C5a Octapeptides by Carboxypeptidase R and Carboxypeptidase N
Authors: Campbell William D. , Lazoura Eliada , Okada Noriko , Okada Hidechika 
Published: November 2013

AbstractPro‐carboxypeptidase R (proCPR), also known as thrombin‐activatable fibrinolysis inhibitor (TAFI), precursor of carboxypeptidase U and plasma carboxypeptidase B is present in plasma and following activation by thrombin/thrombomodulin and/or plasmin can remove arginine from the carboxyterminal of C3a and C5a. We have shown that this enzyme can remove terminal arginine from the C5a octapeptide much more efficiently than the classical anaphylatoxin inactivator, carboxypeptidase N (CPN). Since we have previously demonstrated that proCPR is significantly upregulated in the inflammatory state, this enzyme would appear to significantly contribute to the inactivation of C5a, the most potent of the complement derived anaphylatoxins.
Two‐Dimensional Analysis of Exoproteins of Methicillin‐Resistant Staphylococcus aureus (MRSA) for Possible Epidemiological Applications
Authors: Nakano Miyo , Kawano Yasushi , Kawagishi Mika , Hasegawa Tadao , Iinuma Yoshitsugu , Ohta Michio 
Published: November 2013

AbstractWe applied two‐dimensional gel electrophoresis (2‐DE) to the total exoproteins secreted from pathogenic MRSA strains and identified major protein spots by N‐terminal amino acid sequence analysis. In approximately 300 to 500 spots visualized on each gel, various exoproteins and cell‐associated proteins were identified and their sites on the gels confirmed for construction of a reference map. Major exotoxins such as enterotoxins SEA, SEB, and SEC3, toxic shock syndrome toxin‐1 (TSST‐1), and hemolysins were distributed in the region of pI 6.8 to 8.1 and MW 21 to 35 kDa. Although the differences between calculated and observed values of pI and MW were relatively small in each exoprotein, those of several proteins including alpha‐hemolysin and SEB were considerably deviated from the positions of the expected values. Some exoproteins were detected as multiple spots. These included beta‐hemolysin, enterotoxins SEA, SEB, and SEC3, glutamic acid‐specific endopeptidase, glycerophosphoryl diester phosphodiesterase and triacylglycerol lipase. The multiple spots of these exoproteins may be generated by the action of own proteases. Certain similarities of 2‐DE patterns among strains belonging to the same coagulase types were observed. On the basis of 2‐DE image analysis, coagulase type II strains secreted somewhat larger amounts of SEB and SEC3 as well as TSST‐1 than the strains belonging to other coagulase types. Taken together, 2‐DE analysis of exoproteins is applicable to epidemiological studies for MRSA, as compared with pulsed field gel electrophoresis of restricted chromosomal DNA.
Cross‐Reactivity and Sensitivity of Two Legionella Urinary Antigen Kits, Biotest EIA and Binax NOW, to Extracted Antigens from Various Serogroups of L. pneumophila and Other Legionella Species
Authors: Okada Chikako , Kura Fumiaki , Wada Akihito , Inagawa Hiroko , Lee Gang‐hong , Matsushita Hiroshi 
Published: November 2013

AbstractLegionella antigen detection kits for diagnosing legionellosis from urine have become widely used, but basic information about reactivity of the kits to non‐serogroup (SG) 1 L. pneumophila and other Legionella species remains incomplete. We evaluated Biotest EIA and the most recently developed Binax NOW by using in‐vitro extracted antigens of 22 L. pneumophila SG 1 to 15 strains and of 27 other Legionella species. Both kits showed excellent sensitivity to L. pneumophila SG 1 antigens, but reacted to different sets of non‐SG 1 L. pneumophila with different sensitivity. No cross‐reactivity was observed to Legionella species other than L. pneumophila.