bluuebunny/crossref_metadata_2025 huggingface dataset:
Tinctorial Properties of Spherical Bodies in Broth Cultures of Nocardia asteroides GUH‐2
Authors: Kohbata Shunro
Published: November 2013

In yeast extract‐supplemented brain heart infusion (BHI) broth cultures of Nocardia asteroides GUH‐2, many spherical bodies (SBs) were frequently seen nearby filamentous cells. They showed no Gram‐positivity when Gram stain was applied. When acridine orange stain was applied, many of them showed different green fluorescence from bright orange fluorescence of the filamentous nocardiae under ultraviolet light. Their acid‐fastness appeared to depend on the presence of paraffin. Using the polymerase chain reaction (PCR) method, 16S rRNA genes were detected in SB‐containing broth cultures inoculated with culture filtrates from broth cultures of the strain and identical to that of N. asteroides. These results suggest that SBs are cell wall‐defective (CWD) forms which result from the spontaneous mutation of N. asteroides GUH‐2.
The Use of Heterologous Promoters for Adeno‐Associated Virus (AAV) Protein Expression in AAV Vector Production
Authors: Ogasawara Yoji , Urabe Masashi , Ozawa Keiya
Published: November 2013

Although adeno‐associated virus (AAV) vectors are potentially useful gene transfer vehicles for gene therapy, the vector production system is currently at the developmental stage. We constructed AAV helper plasmids (Rep and Cap expression plasmids) by replacing a native AAV promoter, p5, with various heterologous promoters to examine whether the efficiency of AAV vector production was influenced by modulating the AAV protein expression pattern. The helper plasmids containing heterologous promoters (EF, CMV, SV40, B19p6, and CAG promoters, respectively) expressed Rep78/68 more efficiently than a conventional helper plasmid (pIM45), but the expression of Rep52/40 and Cap decreased, resulting in a significant reduction in AAV vector production. Furthermore, the efficiency of vector production never fully recovered even if the Cap proteins were supplied by an additional expression plasmid. A large amount of Rep78/68 and/or a reduced level of Rep52/40 may have deleterious effects on AAV vector production. The present findings will aid in the development of a more efficient AAV vector production system.
Antigenic and Functional Analyses of Glycoprotein of Rabies Virus Using Monoclonal Antibodies
Authors: Luo Ting Rong , Minamoto Nobuyuki , Hishida Miyuki , Yamamoto Keiko , Fujise Toru , Hiraga Shinya , Ito Naoto , Sugiyama Makoto , Kinjo Toshio
Published: November 2013

Thirty‐five monoclonal antibodies (MAbs) against glycoprotein (G protein) of the RC‐HL strain of the rabies virus have been established. Using these MAbs, two antigenic sites (I and II) were delineated on the G protein of the RC‐HL strain in a competitive binding assay. Of these, 34 MAbs recognized the epitopes on site IL Site II was further categorized into 10 subsites according to their patterns in a competitive binding assay. Each site II‐specific MAb showed 5 to 23 nonreciprocal competitions. The reactivities of 35 MAbs to rabies and rabies‐related viruses in an indirect immunofluorescent antibody test showed that six MAbs in group A binded to rabies and rabies‐related viruses and eight MAbs in group E reacted only with rabies viruses, considering that the former represent the genus‐specific of Lyssavirus and the latter are rabies virus‐specific. From biological assays, 28 of the 35 MAbs showed neutralization activity, 31 showed hemagglutination inhibition (HI) activity, and 18 showed immunolysis (IL) activity. The MAbs recognizing neutralization epitopes fell into at least three groups: those exhibiting both HI and IL activity, those showing only HI activity, and those showing neither HI nor IL activity. All IL epitopes overlap with HA epitopes. Five of the nine MAbs which reacted with the antigen treated by sodium dodecyl sulfate in ELISA were not reduced, or reduced only slightly, in the titer. None of the MAbs reacted with 2‐mercaptoethanol‐treated antigen. Only one MAb that recognized site I reacted with the denatured G protein in a Western blotting assay, indicating that its epitope is linear. These results suggest that almost all of the epitopes on the G protein of the rabies virus are conformation‐dependent and the G protein forms a complicated antigenic structure.
Protective Effect of Administration of Skim Milk on Exogenous and Endogenous Infection in Mice
Authors: Kobayashi Toshiya , Ohmori Toshrhiro , Yanai Minoru , Takeshita Yasuyoshi , Mitsuyama Masao
Published: November 2013

In order to minimize the denaturation of proteins in milk, normal cow's milk was pasteurized at 61 C for 20 min. The protective effects of the thus prepared skim milk (low‐heat skim milk) on exogenous and endogenous infection were examined as compared with conventional skim milk which was pasteurized at 121 C for 2 sec. The antibody titers to Listeria monocytogenes and Escherichia coli of low‐heat skim milk were almost equal to that of raw milk, while no antibody was detected in the conventional skim milk. When mice were given low‐heat skim milk or conventional skim milk, the incidence of the translocation of orally inoculated Listeria monocytogenes to the spleen was lower in the low‐heat skim milk group than that in the conventional skim milk group. The life span of 7 Gy X‐ray irradiated mice given low‐heat skim milk was significantly prolonged in comparison to that of mice given conventional skim milk. However, there were no differences in the number of bacteria in the feces or IgA production by Peyer's patch cells between the two groups. These results suggest that antibodies in low‐heat skim milk, which still have reactivity to exogenous or indigenous bacteria, may contribute to the protective effects against bacterial infection.
Discrimination among the Clinical Isolates of Candida albicans by Amplification of the Repetitive Sequences, alts
Authors: Doi Matsuko , Chibana Hiroji , Nakagawa Yoshiyuki , Tanaka Kenji
Published: November 2013

A primer pair, PB and BSH, which amplified alts, a portion of Candida albicans‐specific repetitive sequence, RPS, gave stable and reproducible fingerprint patterns of the strains by polymerase chain reaction (PCR). We applied this method to clinical isolates of C. albicans for strain discrimination. Using PCR fingerprint patterns, we could analyze the relatedness of C. albicans strains including those isolated from children with leukemia and their bedside parents. The results indicated that PCR analysis targeting an alt region gives rise to the same conclusion as the previous study obtained by SmaI RFLP analysis.
Multiplication of Legionella pneumophila in HeLa Cells in the Presence of Cytoskeleton and Metabolic Inhibitors
Authors: Goldoni Paola , Cattani Lorena , Carrara Stefania , Pastoris Maddalena Castellani , Sinibaldi Laura , Orsi Nicola
Published: November 2013

A study has been carried out on the action of cytoskeleton and metabolic inhibitors on intracellular multiplication in HeLa cells of a virulent strain of Legionella pneumophila serogroup 6. The effects of the substances were separately tested on both penetration and intracellular multiplication of L. pneumophila. Only cytochalasin A and 2‐deoxy‐d‐glucose (2dG) affected bacterial internalisation, whereas intracellular multiplication was inhibited by cytochalasins A, B, C, D and J (D being the most active) and by 2dG with a dose‐response effect. The action of 2dG was counteracted by 50 mM glucose. Experiments carried out with cytochalasin D and a rhodamine‐phalloidin conjugate showed the involvement of cytoskeletal elements in intracellular multiplication of Legionella; compounds acting on microtubules had no effect.
Relation between Reactivity to the NS‐4 Region Peptides of Hepatitis C Virus (HCV) and Clinical Features among Patients Infected with HCV Genotype 1b
Authors: Sakugawa Hiroshi , Nakasone Hiroki , Nakayoshi Tomofumi , Kawakami Yuko , Kinjo Fukunori , Saito Atsushi , Nakayoshi Tomokuni , Yamashiro Akihiro
Published: November 2013

Nearly all patients infected with hepatitis C virus (HCV) genotype 1b have reactivity to the core (c22‐3) or non‐structural (NS)‐3 region (c33c) protein in a second‐generation recombinant immunoblot assay (RIBA‐2). However, reactivities to the NS‐4 region antigens (5‐1‐1, c100‐3) vary among patients. To clarify whether differences in serological reactivities to the NS‐4 antigens are associated with the clinical features or response to interferon (IFN) therapy of patients infected with hepatitis C virus (HCV) genotype 1b, we clinically investigated 115 such patients. Positive reactions to 5‐1‐1 and c100‐3 were seen in 75.7 and 79.1%, respectively, of the patients. There were no differences between the patients with and those without antibodies to NS‐4 region antigens (5‐1‐1, c100‐3) with regard to age, duration of HCV infection, severity of liver disease and virus load. Fifty‐one of the patients were treated with recombinant IFN‐α, and 17 of the 51 patients showed sustained response to the therapy. The sustained response was more frequently seen in the patients positive for antibodies to both 5‐1‐1 and c100‐3 as compared with those negative for either or both antibodies (41.0% vs. 8.3%, P < 0.05).
Detection of HIV‐Gag p24‐Specific Antibodies in Sera and Saliva of HIV‐1‐Infected Adults and in Sera of Infants Born to HIV‐1‐Infected Mothers
Authors: Yasuda Sachiko , Iwasaki Masahiro , Oka Shin‐ichi , Naganawa Satoshi , Nakasone Tadashi , Honda Mitsuo , Sata Tetsutaro , Kojima Asato , Matsuda Syunji , Takemori Toshitada , Tsunetsugu‐Yokota Yasuko
Published: November 2013

Secretory immunoglobulin A (IgA) is known to play an important role in the mucosal defense against a variety of pathogens. Although the role of IgA antibodies during sexual transmission of HIV is not clear, HIV‐specific IgA antibodies have been detected in various mucosal secretions of HIV‐infected individuals. Using a monoclonal antibody against human IgA, we established an ELISA system to detect anti‐HIV p24 IgA antibodies in sera and saliva. We have analyzed the levels of anti‐HIV p24 IgG and IgA antibodies in sera and saliva of 107 and 119 adults, respectively, with HIV infection at different clinical stages, and in the sera of 13 infants born to HIV‐infected mothers. The level of anti‐HIV p24 IgA antibodies was lower in sera and higher in saliva as compared to that of anti‐HIV p24 IgG antibodies. Where the percentage of HIV‐specific serum antibody‐positive cases decreased with disease progression, that of saliva antibody‐positive cases increased in AIDS patients. Among the 13 infants born to HIV‐infected mothers, 7 infants were HIV‐p24‐specific serum IgA positive. These sera were negative for anti‐HIV p24 secretory IgA, suggesting that some infants develop their own immune responses against HIV infection. Thus, the detection of HIV‐specific IgA antibodies, especially in saliva, could be a simple and reliable test for the diagnosis of HIV infection.
Inhibitory Effect of Bacterial Attachment on Candidal Growth Due to Adherence with Mannose‐Sensitive Pili
Authors: Seki Keiko , Kobayashi Kiyoshi , Tomiya Yoshinori , Sakurada Junji , Murai Miyo , Usui Akemi , Masuda Shogo
Published: November 2013

A bacterial strain with affinity to Candida albicans was successfully obtained from a natural environment. An uncovered Petri dish containing a suspension of heat‐killed C. albicans cells was allowed to stand in a laboratory for several days. Some bacteria which had adhered to the candidal cells were tested for their ability to agglutinate the cells. A bacterial strain, designated later as CAB‐1, was found to agglutinate candidal cells through bridging by mannose‐sensitive pili. CAB‐1 showed similar bacteriological characteristics to those of Citrobacter freundii by ID test. The adherence of CAB‐1 to candidal cell was precisely presented by scanning electron microscopy. The inhibitory effect of CAB‐1 attachment to candidal cells on the growth of Candida was also preliminarily confirmed.
Systemic Dissemination by Intrarectal Infection with Listeria monocytogenes in Mice
Authors: Nishikawa Shinsuke , Hirasue Mayuko , Miura Tomisato , Yamada Kyogo , Sasaki Sanae , Nakane Akio
Published: November 2013

Orally ingested Listeria monocytogenes is known to penetrate into Peyer's patches (PP) and translocate to the spleen and liver. Herein, extraintestinal dissemination of the bacterium independent of PP was investigated. Dissemination of Listeriae to the spleen and liver was observed in intrarectally infected mice as well as in intragastrically infected animals in spite that no Listeriae were detected in the small intestines of mice infected intrarectally. Decreased numbers of intestinal intraepithelial lymphocytes (iIEL) and increased numbers of lymphocytes in the contents of the small and large intestines were observed after intragastric infection and in the large intestine after intrarectal infection, giving the assumption that the leakage of iIEL caused by injury of epithelial layers in intestines might occur during infection. These results suggest that L. monocytogenes might be able to disseminate through small and large intestines in part by a PP‐independent mechanism.
Pathogenicity of a Mutant Friend Spleen Focus‐Forming Virus Encoding an Env‐Like Membrane Glycoprotein (gp55) with Substitution by a Xenotropic Murine Leukemia Virus Env gp70 Sequence
Authors: Yugawa Takashi , Amanuma Hiroshi
Published: November 2013

Friend spleen focus‐forming virus (F‐SFFV) is a replication‐defective acutely leukemogenic mouse retrovirus and encodes an envelope protein (Env)‐like membrane glycoprotein (gp55) in its defective env gene, which is responsible for the early stage of the viral leukemogenesis. Gp55 is a modified Env protein and contains a polytropic mink cell focus‐inducing (MCF) murine leukemia virus (MuLV) Env gp70‐derived sequence in its amino‐terminal region. To evaluate the possibility that the presumed binding of gp55 to an MCF MuLV receptor protein has some role in leukemogenesis, we examined the biological activities of a mutant gp55 (XE gp55), which has a xenotropic MuLV Env gp70 amino‐terminal region. XE gp55 displayed almost the same biological activities as the wild‐type gp55, excluding the above possibility.
Detection of Verotoxin‐Producing Escherichia coli O157:H7 by Multiplex Polymerase Chain Reaction
Authors: Nagano Isao , Kunishima Miyuki , Itoh Yoshihiro , Wu Zhiliang , Takahashi Yuzo
Published: November 2013

We constructed primers for multiplex polymerase chain reaction (PCR) to detect verotoxin‐producing Escherichia coli (VTEC) O157:H7. The multiplex PCR primers were designed from the sequence of the flagellin structural gene of Escherichia coli flagellar type H7 (GenBank under accession number L07388), and from the sequence of the rfbE gene of Escherichia coli O157:H7 (GenBank under accession number S83460). In addition to these primers, we used a primer pair reported by Karch and Meyer (J. Clin, Microbiol. 27: 2751‐2757, 1989) to amplify various VT genes from VTEC. All of the examined specimens (18 isolates) of VT‐producing E. coli O157:H7 showed a positive result by the multiplex PCR test with the three sets of primers. The sensitivity of detection for VT‐producing E. coli O157:H7 was shown to be at least 3,000 cells per PCR tube.
Emendation of GenusAchromobacterandAchromobacter xylosoxidans(Yabuuchi and Yano) and Proposal ofAchromobacter ruhlandii(Packer and Vishniac) Comb. Nov.,Achromobacter piechaudii(Kiredjian et al.) Comb. Nov., andAchromobacter xylosoxidansSubsp.denitrificans(Rüger and Tan) Comb. Nov.
Authors: Yabuuchi Eiko , Kawamura Yoshiaki , Kosako Yoshimasa , Ezaki Takayuki
Published: November 2013

Based on the results of GC content determination and 16S rRNA sequence analysis among the type strains ofAchromobacter xylosoxidans, 4Alcaligenesspecies, 5Bordetellaspecies, and 12 species of 4 other genera, the separation of genusAchromobacterYabuuchi and Yano 1981, with the type speciesAchromobacter xylosoxidans, is confirmed.Alcaligenes ruhlandii(Packer and Vishniac) Aragno and Schlegel 1992 is a distinct species and not a senior synonym ofAchromobacter xylosoxidans. Alcaligenes ruhlandiiandAlcaligenes piechaudiiKiredjian et al 1986 are transferred to genusAchromobacter. Thus 2 new combinations,Achromobacter ruhlandii(Packer and Vishniac) andAchromobacter piechaudii(Kiredjian et al) are proposed; their type strains are ATCC 15749 and ATCC 43552, respectively.Alcaligenes denitrificansRüger and Tan 1983 is also transferred to genusAchromobacterand ranked down to the subspecies ofAchromobacter xylosoxidans. Thus a new subspecies name,Achromobacter xylosoxidanssubsp.denitrificans(Rüger and Tan) is proposed. The type strain of the subspecies is ATCC 15173. This proposal automatically creates type subspecies,Achromobacter xylosoxidanssubsp.xylosoxidans, with type strain ATCC 27061. An emended description of genusAchromobacterand of type speciesAchromobacter xylosoxidansare given.
First Report of Trichosporon ovoides Isolated from the Home of a Summer‐Type Hypersensitivity Pneumonitis Patient
Authors: Sugita Takashi , Nishikawa Akemi , Ikeda Reiko , Shinoda Takako , Sakashita Hiroyuki , Sakai Youko , Yoshizawa Yasuyuki
Published: November 2013

Trichosporon species have been known to cause summer‐type hypersensitivity pneumonitis (SHP). During the isolation of yeasts from an SHP patient's house, we recovered a strain belonging to the genus Trichosporon. Morphologically, the isolate produced rectangular arthroconidia when grown on corn meal agar. DNA‐DNA hybridization experiments identified the isolate as T. ovoides. A slide agglutination test using specific factor sera demonstrated that the serotype of the strain was type II. Previously, T. asahii, a serotype II species, was considered to be the major antigen of SHP, but it is possible that T. ovoides may also be responsible for SHP. This is the first report of T. ovoides isolated from an SHP patient's home environment.
Trimethoprim Resistance and Susceptibility Genes in Staphylococcus epidermidis
Authors: Totake Kyoko , Kobayashi Nobuharu , Odaka Takeshi
Published: November 2013

Genes encoding trimethoprim (TMP)‐resistant and ‐susceptible dihydrofolate reductases (DHFR) in Staphylococcus epidermidis isolated in Saitama Prefecture were compared with the TMP‐resistant DHFR gene of S. aureus, dfrA. The nucleotide sequences of TMPr and TMPS genes in five S. epidermidis isolates tested could be divided into three types: type 1, identical with the TMPr gene dfrA that had been found in S. aureus; type 3, identical with the TMPs gene dfrC in S. epidermidis; and type 2, having only two nucleotide substitutions to dfrC with no amino acid change. TMPr isolates carried either one of the type 2 or type 3 sequences in addition to the type 1 sequence. A Southern hybridization analysis revealed that, in TMPr S. epidermidis, the type 1 sequence was located on a 5.5 kb EcoRI‐EcoRV restriction fragment together with the sequence for the gentamicin (GM)‐resistant gene, while the type 2 or type 3 sequence was located on the 1.0 kb EcoRI‐EcoRV fragment. No plasmid‐carrying dfrA‐homologous sequence was detected in the S. epidermidis isolates we tested. These results suggest that the TMPr and GMr genes are closely linked and located on the chromosome in S. epidermidis isolated in Japan.
Chemical and Immunological Characterization of a Low Molecular Weight Outer Membrane Protein of Salmonella typhi
Authors: De Andrade Carlos Maurício , Ferreira Antônio Gomes Pinto , Da Silva Joana D'Arc Cardoso , Nascimento Hilton Jorge , Junior José Godinho Da Silva
Published: November 2013

A new immunogenic outer membrane protein, Omp‐28 (MW 28,000 and pI 4.6), was isolated from smooth Salmonella typhi cells by the use of an extracting medium containing 6 m urea, 1% deoxycholate and 5 mM EDTA. The purification of Omp‐28 was performed by gel filtration and fast ion exchange chromatography. This protein showed to be the prevalent component isolated by the latter methodology. Omp‐28 is formed by three identical subunits (MW 9,000), not linked by disulfide bonds. The partial N‐terminal amino acid sequence of Omp‐28 presented great homology with part of the sequence of an Escherichia coli protein found in a precursor whose sequence was predicted by c‐DNA. ELISA and Western blotting identified Omp‐28 as the major antigenic protein present in the outer membrane protein fraction, isolated by gel filtration. Antibodies against Omp‐28 were detected by ELISA in 43% of 28 sera from typhoid fever convalescent patients. The antisera from mice immunized with Omp‐28 and the highest positive typhoid fever convalescent serum gave a positive bactericidal test, killing 50% of Salmonella typhi cells in serum dilutions of 1/80 and 1/320, respectively. These results indicate the immunogenic importance of Omp‐28 isolated from Salmonella typhi outer membrane and strongly suggest it should be used in further studies of animal protection against the disease caused by this pathogenic bacteria.
Mechanism of Membrane Damage by Clostridium perfringens Alpha‐Toxin
Authors: Nagahama Masahiro , Michiue Kei , Mukai Masakazu , Ochi Sadayuki , Sakùrai Jun
Published: November 2013

The effect of Clostridium perfringens alpha‐toxin on liposomes prepared from phosphatidylcholine (PC) containing the fatty acyl residues of 18 carbon atoms was investigated. The toxin‐induced carboxyfluorescein (CF) leakage and phosphorylcholine release from multilamellar liposomes increased as the phase transition temperature of the phosphatidylcholines containing unsaturated fatty acyl residues decreased. However, there was no difference between the sensitivity of the different phosphatidylcholines solubilized by deoxycholate to the phospholipase C (PLC) activity of the toxin. However, the toxin did not hydrolyze solubilized distearoyl‐l‐α‐phosphatidylcholine (DSPC) or phosphatidylcholine containing saturated fatty acyl residue, and caused no effect on liposomes composed of DSPC. These results suggest that the activity of the toxin is closely related to the membrane fluidity and double bond in PC. The N‐terminal domain of alpha‐toxin (AT1‐246) and variant H148G did not induce CF leakage from liposomes composed of dioleoyl‐l‐α‐phosphatidylcholine (DOPC). H148G bound to the liposomes, but AT1‐246 did not. However, the C‐terminal domain (AT251‐370) conferred binding to liposomes and the membrane‐damaging activity on AT1‐246. These observations suggest that the membrane‐damaging action of alpha‐toxin is due to the binding of the C‐terminal domain of the toxin to the double bond in the PC in the bilayer and hydrolysis of the PC by the N‐terminal domain.
Single‐Step Reverse Transcription‐Polymerase Chain Reaction for the Detection of Hepatitis C Virus RNA
Authors: Mizutani Tetsuya , Ikeda Masanori , Saito Satoru , Sugiyama Kazuo , Shimotohno Kunitada , Kato Nobuyuki
Published: November 2013

We developed a novel single‐step reverse transcription‐polymerase chain reaction (RT‐PCR), which is equal in sensitivity and specificity to RT‐nested PCR, based on both reverse transcriptase and Taq DNA polymerase working efficiently under single buffer reaction conditions. Using in vitro synthesized hepatitis C virus (HCV) RNA, it was demonstrated that 10‐100 copies of HCV RNA could be detected with a set of primers that amplify a 144 base‐pair sequence unique to the 5′‐noncoding region of HCV RNA. Furthermore, this method was successfully performed on serum and liver biopsy specimens obtained from patients with chronic hepatitis C. In addition, HCV RNA from in vitro HCV‐infected MT‐2C cells, which supported HCV replication, was also detected by this method. The method is anticipated to improve the detection of small amounts of RNA, such as that of HCV, promoting both labor savings and the prevention of carry‐over contamination.
Lipopolysaccharide (LPS) Stimulates the Production of Tumor Necrosis Factor (TNF)‐α and Expression of Inducible Nitric Oxide Synthase (iNOS) by Osteoclasts (OCL) in Murine Bone Marrow Cell Culture
Authors: Kikkawa Ichiro , Saito Shinji , Tominaga Kaoru , Hoshino Yuichi , Ooi Yoshio , Nakano Masayasu
Published: November 2013

Osteoclasts (OCL) resorb bone. They are essential for the development of normal bones and the repair of impaired bones. The function of OCL is presumed to be supported by cytokines and other biological mediators, including tumor necrosis factor (TNF)‐α and nitric oxide (NO). Bacterial lipopolysaccharide (LPS) is a potent inducer of TNF‐α and inducible nitric oxide synthase (iNOS), which is the specific enzyme for synthesizing NO from L‐arginine. To obtain direct evidence on LPS‐induced TNF‐α production and iNOS expression by OCL, OCL‐enriched cultures were prepared by 7‐day cocultures of bone marrow cells of adult BALB/c mice and osteoblastic cells (OBs) derived from calvaria of newborn BALB/c mice, and the generation of TNF‐α and iNOS in OCL stimulated with LPS was examined immunocytochemically. When the cultured cells were stimulated with 100 ng/ml of LPS, OCL clearly showed TNF‐α and iNOS expression. Without LPS‐stimulation, no expression was observed. TNF activity in the culture supernatants of the OCL‐enriched cultures in the presence of LPS was also detected by cytotoxic assay that used TNF‐sensitive L929 cells. The dentin resorption activity of OCL was estimated by area and number of pits formed on dentin slices, which were covered by the OCL fraction and cultured in the presence or absence of LPS, sodium nitroprusside (SNP; a NO generating compound), NG‐monomethyl L‐arginine acetate (L‐NMMA; a competitive inhibitor of NO synthase (NOS)), or LPS plus L‐NMMA. Pit formation was obviously inhibited in the presence of SNP and slightly inhibited in the presence of L‐NMMA, but it was not affected in the presence of LPS or LPS plus L‐NMMA. These findings indicate that OCL produces TNF and expresses iNOS in response to LPS, but the LPS‐activation of OCL scarcely affects pit formation by them.
Phylogenetic Analysis of the Genus Bifidobacterium and Related Genera Based on 16S rDNA Sequences
Authors: Miyake Taeko , Watanabe Koichi , Watanabe Tsugio , Oyaizu Hiroshi
Published: November 2013

The 16S rRNA gene sequences were determined for type strains of 21 Bifidobacterium species. A phylogenetic tree was constructed using the determined sequences and sequences from DNA databases, which contain the sequences of 11 type strains of Bifidobacterium species and 11 strains of related genera. All species of the genus Bifidobacterium and Gardnerella vaginalis ATCC 14018 belonged to a cluster phylogenetically distinct from the other genera. The cluster was divided into two subclusters: subcluster 1 composed of most species of Bifidobacterium and G. vaginalis, and subcluster 2 consisting of two species, B. denticolens and B. inopinatum; both of which were isolated from human dental caries. In the genus Bifidobacterium, four groups of species are known to be moderately to highly related by DNA‐DNA hybridization. The four groups of species exhibited more than 99% similarity among their 16S rDNA sequences within each group. These results indicated that species with around 99% or more similarity in their 16S rDNA sequences should be confirmed for species identities.
Serological Studies of an Acid‐Labile O‐Polysaccharide of Proteus vulgaris OX19 Lipopolysaccharide Using Human and Rabbit Antibodies
Authors: Kaca Wieslaw , Swierzko Anna ST. , Ziolkowski Andrzej , Amano Ken‐ichi , Senchenkova Sof'Ya N. , Knirel Yuriy A.
Published: November 2013

In a Weil‐Felix test, sera from patients infected with Rickettsia sp. agglutinate Proteus OX types of bacteria and Proteus lipopolysaccharide (LPS) are responsible for the cross‐reaction. Data on the character of LPS of one of the OX group strains, Proteus vulgaris OX19, are contradictory, and it remained unclear whether it has an O‐polysaccharide (OPS) and is thus LPS of the smooth type (S) or not (rough‐type LPS). Our studies showed that P. vulgaris OX19 (strain PZH‐24) produces a smooth‐type LPS that contains a long‐chain OPS, but it undergoes depolymerization during mild acid hydrolysis conventionally used for LPS delipidation and loses the serological activity. An elucidation of the complete structure of OPS demonstrated the presence of a glycosyl phosphate linkage responsible for the acid‐lability of the polysaccharide chain. In ELISA, both IgM type antibodies in a Weil‐Felix test with human anti‐Rickettsia typhi sera and rabbit anti‐P. vulgaris OX19 antibodies reacted with OPS. Rabbit antibodies did not inhibit the cross‐reaction with human antibodies and thus bind to different epitopes.
Analysis of Genetic Relationships and Antimicrobial Susceptibility of Verotoxin‐Producing Escherichia coli Strains Isolated in Nagasaki Prefecture, Japan in 1996
Authors: Izumikawa Koichi , Hirakata Yoichi , Yamaguchi Toshiyuki , Yoshida Ryoji , Nakano Michiko , Matsuda Junichi , Mochida Chikako , Maesaki Shigefumi , Tomono Kazunori , Yamada Yasuaki , Tashiro Takayoshi , Kohno Shigeru , Kamihira Shimeru
Published: November 2013

A total of 19 Escherichia coli O157 isolates were obtained in Nagasaki Prefecture, in the southwestern part of Japan, between 1990 and 1996. Pulsed‐field gel electrophoresis (PFGE) and computer‐assisted analysis were applied to determine genetic relationships among these strains. Fragment patterns of the isolates in Nagasaki, as determined by PFGE, were compared with those of isolates in other areas where large outbreaks and sporadic cases of E. coli O157 infection occurred. Similarity values of all the strains isolated in Nagasaki Prefecture were over 0.65 except for E. coli O26. Some strains were identical to the strains isolated from the areas where large outbreaks occurred. All strains were susceptible to ampicillin, fosfomycin, minocycline, amikacin, ofloxacin and sulfamethoxazole‐trimethoprim.
Epidemiologic Typing of Methicillin‐Resistant Staphylococcus aureus in Neonate Intensive Care Units Using Pulsed‐Field Gel Electrophoresis
Authors: Saito Yumi , Seki Keiko , Ohara Tomoko , Shimauchi Chieko , Honma Yoko , Hayashi Mutsumu , Masuda Shogo , Nakano Masayasu
Published: November 2013

To elucidate the mode of dissemination of methicillin‐resistant Staphylococcus aureus (MRSA) in neonate intensive care units (NICUs), a total of 223 isolates from 3 separate hospitals were investigated between 1994 and 1996 by a DNA fingerprinting technique with pulsed‐field gel electrophoresis (PFGE). Exoprotein profiles of some strains were also examined using SDS‐polyacrylamide gel‐electrophoresis (SDS‐PAGE) and the assessment of enzyme/toxin production such as coagulase, enterotoxin and TSST‐1. Judging from the strain typing data from PFGE results and the epidemiological data, 2 different types of PFGE patterns (A and B) and their subtypes (A′, A″ and B′) were identified. The A type including A′ and A″ (comprising approximately 95% of the isolates) was markedly dominant. Only 5% of the isolates belonged to type B and subtype B′. On the other hand, MRSA isolated from adult patients admitted to the same hospital showed many different PFGE patterns. The results strongly suggested that some strain(s) with specific PFGE pattern(s) is prevalent in NICUs. Furthermore, isolates which expressed the same PFGE pattern did not always express the same SDS‐PAGE pattern. There were some isolates with different abilities to produce coagulase, enterotoxin C and toxic‐shock syndrome toxin (TSST)‐1, and the abilities had no relation with a particular type of PFGE pattern. Therefore, a combination of PFGE analysis and biochemical analyses of coagulase, enterotoxin C and TSST‐1 may provide us with more detailed information for the epidemiological study of MRSA in NICUs.
A Rapid Latex Agglutination Assay for the Detection of Penicillin‐Binding Protein 2′
Authors: Nakatomi Yasuo , Sugiyama Junichi
Published: November 2013

A simple and rapid slide latex agglutination assay was developed to detect penicillin‐binding protein 2′ (PBP2′) from isolates of staphylococi. PBP2′ present in the membranes of methicillin‐resistant Staphylococcus aureus (MRSA) or methicillin‐resistant coagulase negative staphylococci (MRCNS) was rapidly extracted by alkaline treatment and, by combining with a slide agglutination reaction using latex particles sensitized with monoclonal antibodies raised against it, PBP2′ could be detected from a single loopful of cells taken from agar plates not containing beta‐lactum antibiotics within 15 min. In a study of clinical isolates previously characterized as either MRSA or methicillin‐susceptible Staphylococcus aureus (MSSA) by antibiotic susceptibility testing, 231 specimens of 232 MRSA were PBP2′ positive by latex agglutination, and the 87 specimens of MSSA were all negative. One specimen identified as MRSA by susceptibility testing but PBP2′ negative by latex agglutination was confirmed as mecA gene negative by PCR. This simple and rapid slide latex reagent should be useful in clinical diagnostics.
Protective Effects of a Culture Supernatant of Lactobacillus acidophilus and Antioxidants on Ileal Ulcer Formation in Rats Treated with a Nonsteroidal Antiinflammatory Drug
Authors: Bing Shan Ruo , Kinouchi Takemi , Kataoka Keiko , Kuwahara Tomomi , Ohnishi Yoshinari
Published: November 2013

Ileal ulcers and thiobarbituric acid (TBA)‐reactive substances in the ileal mucosa were induced in rats treated with a nonsteroidal antiinflammatory drug, 5‐bromo‐2‐(4‐fluorophenyl)‐3‐(4‐methylsulfonylphenyl)thiophene (BFMeT), at a dose of 1,000 mg/kg administered with tap water as drinking water. However, the formation of ileal ulcers and TBA‐reactive substances in the ileal mucosa was repressed by giving the animals a culture supernatant of Lactobacillus acidophilus as drinking water. We measured the antioxidative activity of the culture supernatant and found that the supernatant inhibited the formation of t‐butyl hydroperoxide‐induced TBA‐reactive substances in erythrocyte membrane ghosts. Therefore, the effects of various known antioxidative compounds on the ileal ulcer formation induced by BFMeT were investigated. While α‐tocopherol, t‐butyl‐1,4‐hydroxyanisole and allopurinol did not repress ulcer formation after BFMeT treatment, ascorbic acid, dimethyl sulfoxide, glutathione and β‐carotene significantly inhibited formation. Among these compounds, ascorbic acid was the most effective. Accumulation of TBA‐reactive substances in the ileal mucosa after BFMeT treatment also decreased significantly in rats treated with ascorbic acid. In addition, the percentage of Gram‐negative rods in the ileal contents of rats treated with BFMeT and tap water was dramatically increased, but it was not increased in rats treated with BFMeT and these antioxidants. A positive correlation between the percentage of Gram‐negative rods and the number of ileal ulcers was also observed. These results suggest that lipid peroxidation mediated by oxygen radicals plays an important role in the induction of ileal ulcers by BFMeT in rats, and that lipopolysaccharide‐activated neutrophils probably produce highly reactive hypochlorous acid and hydrogen peroxide, which are inactivated by ascorbic acid and glutathione, respectively.
Anti‐Candida Activity of Cal protect in in Combination with Neutrophils or Lactoferrin
Authors: Okutomi Takafumi , Tanaka Takashi , Yui Satoru , Mikami Masaaki , Yamazaki Masatoshi , Abe Shigeru , Yamaguchi Hideyo
Published: November 2013

The effect of an anti‐microbial protein, calprotectin, in combination with neutrophils on the growth of Candida albicans was investigated. The growth inhibition of C. albicans by murine neutrophils was augmented by the addition of a low concentration of calprotectin prepared from rat peritoneal exudate cells. The concentrations of calprotectin causing 50% inhibition of growth of C. albicans in the absence or presence of neutrophils at an effector‐to‐target (E/T) ratio of 30 and 60 were estimated to be 0.45, 0.34 and 0.28 U/ml, respectively. The anti‐Candida activity of calprotectin was completely inhibited by 2 μM of zinc ion, while it only partially lowered the activity of the combination of calprotectin and neutrophils. Lactoferrin, which is an anti‐microbial protein released from neutrophils, strongly inhibited the growth of C. albicans in combination with calprotectin. These results suggest that calprotectin and lactoferrin released from neutrophils may cooperate to inhibit the growth of C. albicans at a local lesion of the infection where there is an accumulation of neutrophils.
Detection of Genes Encoding Cholera Toxin (CT), Zonula Occludens Toxin (ZOT), Accessory Cholera Enterotoxin (ACE) and Heat‐Stable Enterotoxin (ST) in Vibrio mimicus Clinical Strains
Authors: Shi Lei , Miyoshi Shin‐ichi , Hiura Mieko , Tomochika Ken‐ichi , Shimada Toshio , Shinoda Sumio
Published: November 2013

A total of 51 clinical strains of Vibrio mimicus were searched for the presence of virulence‐associated genes, like ctx, zot or ace genes which locate in “cholera virulence cassette,” and the st gene by polymerase chain reaction. Moreover, the pathological potential of each clinical strain was also examined by rabbit ileal loop (RIL). Three strains showed to have the ctx gene, of which only one strain was zot gene‐positive. Meanwhile, one other strain was zot+ but ctx−. All of these four strains were found to have the ace gene and to belong to serogroup O115. Nine strains showed to carry the st gene. However, none of these ST‐gene‐positive strains was indicated to contain the genes located in the “cholera virulence cassette.” It is of interest to note that all of the RIL‐positive and/or virulence gene‐positive strains were restricted to three serogroups, O20, O41 and O115. These results suggest a significant association between O antigens and enterotoxic activities in V. mimicus clinical strains, and clearly demonstrate multifactorial virulence potentials of this human pathogen.
New Evidence That the Tyr‐157 and Tyr‐159 Residues of Staphylococcal Exfoliative Toxin B Are Essential for Its Toxicity
Authors: Sakurai Susumu , Suzuki Hitoshi , Saito Shinobu , Konishi Yasuhiko , Machida Katsuhiko , Kohno Minoru
Published: November 2013

To determine the active site of exfoliative toxin B (sETB) of Staphylococcus aureus, the etb gene was cloned from an S. aureus SU strain obtained from a patient with impetigo. We prepared a frame shift mutant protein from a recombinant plasmid with a BglII linker inserted into the Tyr‐155 codon of the ETB gene (pETB/BglIIL). The recombinant mutant protein (ETB/BglIIL) obtained from Escherichia coli containing pETB/BglIIL showed no toxicity in neonatal mice and no agglutination activity. The 20‐kDa ETB/BglIIL contained 185 amino acid residues. Site‐directed mutagenesis was used to introduce mutations at either Tyr‐155, Tyr‐157, Tyr‐159, or Tyr‐162. Substitution of any of the Tyr residues decreased exfoliative activity compared with that of native sETB (4,000 EU/ml). Substitution of Tyr‐155 with a Phe (ETB/Y155) decreased activity 5‐fold (800 EU/ml). Substitution of Tyr‐157 with Leu (ETB/Y157) decreased activity 80‐fold (50 EU/ml) and decreased agglutination titer 5‐fold compared with that of native sETB (400,000). Substitution of Tyr‐159 with Leu (ETB/Y159)decreased activity 4‐fold (1,000 EU/ml). When both Tyr‐157 and Tyr‐159 were mutated (ETB/Y157‐159), both toxicity and antigenicity were completely lost. On an immunodiffusion test, ETB/Y157 showed a faint precipitation line, but ETB/BglIIL and ETB/Y157‐159 had no activity, showing that the Tyr‐157 and Tyr‐159 residues are essential for the toxicity and antigenicity of ETB.
Role of Endogenous Tumor Necrosis Factor Alpha and Gamma Interferon in Resistance to Corynebacterium pseudotuberculosis Infection in Mice
Authors: Lan Dinh Thi Bich , Taniguchi Shunji , Makino Sou‐ichi , Shirahata Toshikazu , Nakane Akio
Published: November 2013

The production and roles of endogenous tumor necrosis factor alpha (TNF‐α) and gamma interferon (IFN‐γ) in the infection of Corynebacterium (C.) pseudotuberculosis were investigated in mice. The maximum levels of TNF‐α and IFN‐γ were detected on day 4 after infection. The administration of anti‐TNF‐α monoclonal antibody (mAb) as well as anti‐IFN‐γ mAb increased bacterial proliferation in the organs, leading to the death of infected mice, but anti‐IFN‐γ mAb showed a less marked effect than anti‐TNF‐α mAb. The suppressive effect of anti‐TNF‐α and anti‐IFN‐γ mAbs on anticorynebacterial resistance was augmented by the simultaneous administration of these antibodies. Anti‐TNF‐α mAb was found to be highly effective when administered on day 0 and day 4, suggesting that TNF‐α produced during the early stage of infection is critical for the generation of resistance. Histologically, many microabscesses, severe follicular swelling and lymphocyte destruction were observed in mice treated with anti‐TNF‐α or anti‐IFN‐γ mAb. Injection of anti‐CD4 or anti‐CD8 mAb also resulted in significantly increased mortality and a marked suppression of IFN‐γ production, but had no effect on TNF‐α production. Carrageenan also showed a marked effect on the exacerbation of infection. Taken together, these results suggest that endogenously produced TNF‐α and IFN‐γ are both essential to the host defense against C. pseudotuberculosis infection and that these cytokines may have an additive effect.
The Gene Encoding the Prepilin Peptidase Involved in Biosynthesis of Pilus Colonization Factor Antigen III (CFA/III) of Human Enterotoxigenic Escherichia coli
Authors: Taniguchi Tooru , Yasuda Yoko , Tochikubo Kunio , Yamamoto Koichiro , Honda Takeshi
Published: November 2013

The assembly of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli requires the processing of CFA/III major pilin (CofA) by a peptidase, likely another type IV pilus formation system. Western blot analysis of CofA reveals that CofA is produced initially as a 26.5‐kDa preform pilin (prepilin) and then processed to 20.5‐kDa mature pilin by a prepilin peptidase. This processing is essential for exportation of the CofA from the cytoplasm to the periplasm. In this experiment, the structural gene, cofP, encoding CFA/III prepilin peptidase which cleavages at the Gly‐30‐Met‐31 junction of CofA was identified, and the nucleotide sequence of the gene was determined. cofP consists of 819 bp encoding a 273‐amino acid protein with a relative molecular mass of 30,533 Da. CofP is predicted to be localized in the inner membrane based on its hydropathy index. The amino acid sequence of CofP shows a high degree of homology with other prepilin peptidases which play a role in the assembly of type IV pili in several gram‐negative bacteria.
Absence of Cecal Secondary Bile Acids in Gnotobiotic Mice Associated with Two Human Intestinal Bacteria with the Ability to Dehydroxylate Bile Acids In Vitro
Authors: Narushima Seiko , Itoh Kikuji , Takamine Fusae , Uchida Kiyohisa
Published: November 2013

Germ‐free mice were orally inoculated with human intestinal 7α‐dehydroxylating bacterial strains to evaluate their ability to transform bile acids in vivo. Three weeks after inoculation of the bacteria, cecal bile acids were examined. Among free‐form bile acids, only β‐muricholic acid was detected in the cecal contents of gnotobiotic mice associated with Bacteroides distasonis strain K‐5. No secondary bile acid was observed in the cecal contents of any of the gnotobiotic mice associated with 7α‐dehydroxylating bacteria, Clostridium species strain TO‐931 or Eubacterium species strain 36S.
Facilitated Expansion of Pneumococcal Colonization from the Nose to the Lower Respiratory Tract in Mice Preinfected with Influenza Virus
Authors: Takase Hiroyuki , Nitanai Hironobu , Yamamura Eiko , Otani Tsuyoshi
Published: November 2013

A strain of Streptococcus pneumoniae, when inoculated intranasally in 2 μl of suspension into BALB/c mice preinfected with influenza virus, colonized first in the nose, and several days thereafter also colonized significantly in the trachea and lungs with purulent inflammation. Pneumoccocal colonization was also observed in the noses of normal mice after the same bacterial inoculation, but not apparently in the lower respiratory tract. These results suggest that pneumococcal infection may develop from the upper to the lower respiratory tract as a possible sequence preferentially in influenza virus‐infected subjects.
Growth Conditions of and Emetic Toxin Production by Bacillus cereus in a Defined Medium with Amino Acids
Authors: Agata Norio , Ohta Michio , Mori Masashi , Shibayama Keigo
Published: November 2013

The growth and emetic toxin (cereulide) production of Bacillus cereus strains in defined culture media were studied. We found that a fully synthetic medium (CADM) allowed the production of emetic toxin and the addition of glucose enhanced it. By subtracting each amino acid from CADM, we found that only three amino acids, valine, leucine and threonine, were essential for growth and toxin production by B. cereus. The addition of high levels (50 mM) of leucine, isoleucine and glutamic acid decreased the toxin production. Other amino acids had no effect at this concentration.
Comparison of Substrate Specificities against the Fusion Glycoprotein of Virulent Newcastle Disease Virus between a Chick Embryo Fibroblast Processing Protease and Mammalian Subtilisin‐Like Proteases
Authors: Fujii Yutaka , Sakaguchi Takemasa , Kiyotani Katsuhiro , Yoshida Tetsuya
Published: November 2013

The fusion (F) protein precursor of virulent Newcastle disease virus (NDV) strains has two pairs of basic amino acids at the cleavage site, and its intracellular cleavage activation occurs in a variety of cells; therefore, the viruses cause systemic infections in poultry. To explore the protease responsible for the cleavage in the natural host, we examined detailed substrate specificity of the enzyme in chick embryo fibroblasts (CEF) using a panel of the F protein mutants at the cleavage site expressed by vaccinia virus vectors, and compared the specificity with those of mammalian subtilisin‐like proteases such as furin, PC6 and PACE4 which are candidates for F protein processing enzymes. It was demonstrated in CEF cells that Arg residues at the −4, −2 and −1 positions upstream of the cleavage site were essential, and that at the −5 position was required for maximal cleavage. Phe at the +1 position was also important for efficient cleavage. On the other hand, furin and PC6 expressed by vaccinia virus vectors showed cleavage specificities against the F protein mutants consistent with that shown by the processing enzyme of CEF cells, but PACE4 hardly cleaved the F proteins including the wild type. These results indicate that the proteolytic processing enzymes of poultry for virulent NDV F proteins could be furin and/or PC6 but not PACE4. The significance of individual contribution of the three amino acids at the −5, −2 and +1 positions to cleavability was discussed in relation to the evolution of virulent and avirulent NDV strains.
Characterization of Serotypes and Outer Membrane Protein Profiles in Enteroaggregative Escherichia coli Strains
Authors: Suzart Sérgio , Gomes Tânia Aparecida Tardelli , Guth Beatriz Ernestina Cabilio
Published: November 2013

Twenty‐four Escherichia coli strains mainly isolated from children with diarrhea in São Paulo, and showing characteristics of enteroaggregative E. coli (EAEC), were characterized by serotyping and outer membrane protein (OMP) profiles. The relationship between these characteristics was evaluated, as well as the usefulness of OMP profiles in the clonal analysis of EAEC strains. All strains presented aggregative adherence to HeLa cells and were classified in two groups based on their interaction with the EAEC DNA probe. A diversity of serotypes and OMP profiles was observed in both groups studied. Although no significant correlation between serotypes and OMP profiles was observed, unique OMP profiles were identified in 80% of the probe‐positive strains which were distributed in only 4 OMP profiles. This result may indicate the presence of a few clones in the probe‐positive group. On the other hand, probe‐negative strains seem to constitute a more diverse group. In general, the observed heterogeneity in serotypes and OMP profiles described in the present study suggest a great genetic diversity in EAEC isolates of either the same or different serotypes and in strains presenting the same EAEC markers identified in our community.