bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and Sequencing of the Vibrio parahaemolyticus fur Gene
Authors: Yamamoto Shigeo , Funahashi Tatsuya , Ikai Hisato , Shinoda Sumio
Published: November 2013

A ferric uptake regulatory gene (fur) was cloned from Vibrio parahaemolyticus WP1 by a polymerase chain reaction‐based technique followed by functional complementation of a fur mutation in Escherichia coli. A sequence analysis showed that, at the amino acid level, the V. parahaemolyticus Fur protein is 81% identical with the Fur protein from E. coli and over 90% identical with those of the Vibrio species.
Morphological Features of a Filamentous Phage of Vibrio cholerae O139 Bengal
Authors: Shimodori Shoichi , Lida Kenichiro , Kojima Fumiko , Takade Akemi , Ehara Masahiko , Amako Kazunobu
Published: November 2013

A filamentous phage was isolated from carrier strain AI‐1841 of Vibrio cholerae O139 Bengal and thus was termed fs phage. The phage was measured to be approximately 1 μm in length and 6 nm in width. One end of the phage was slightly tapered and had a fibrous appendage. The plaques developed on strain AI‐4450 of V. cholerae O139 were small and turbid. The phage grew in strain AI‐4450 and reached a size of 108 to 109 pfu/ml at 5 hr after infection without inducing any lysis of the host bacteria. The group of phages attached on rod‐shaped materials like fimbriae of this bacteria, with their fibrous appendages at the pointed end, were often found in the phage‐infected culture. The anti‐fimbrial serum effectively inhibited the infection of fs phage to the host strain AI‐4450. We thus concluded that the phage can be adsorbed on fimbriae with a fibrous appendage on the pointed end of the phage filament.
Interaction of Drugs for Eradication Therapy against Antibiotic‐Resistant Strains of Helicobacter pylori
Authors: Gotoh Akira , Kawakami Yoshiyuki , Akamatsu Taiji , Katsuyama Tsutomu
Published: November 2013

To clarify the interactions of drugs for combination therapy of Helicobacter pylori infection, especially due to antibiotic‐resistant strains, we have evaluated the in vitro effect of combining different drugs. Using a modified time‐kill assay, we tested the effect of combining 2 drugs from 4 agents; amoxicillin (AMPC), clarithromycin (CAM), metronidazole (MTZ) and lansoprazole (a proton pump inhibitor). The H. pylori in the study consisted of 4 strains sensitive to the all drugs, 2 strains resistant only to CAM, 2 strains resistant only to MTZ, and 2 strains resistant to both CAM and MTZ. From the 6 different drug combinations, synergism was observed for 5 of the combinations, among which the combination of AMPC and CAM revealed such effects most frequently. However, all of the strains which showed synergism were sensitive to both of the drugs. In the case of the strains resistant to CAM and/or MTZ, no synergism was demonstrated in any of the combinations including CAM and/or MTZ. When a strain was resistant to one drug from a combination, no synergism was detected. Thus, the administration of a drug to which the strains are resistant may have no advantage in the eradication therapy of H. pylori. For a more effective and safer therapy, susceptibility testing should be performed before treatment.
Detection of EBV RNA (EBER‐1 and EBER‐2) in Malaria Lymph Nodes by In Situ Hybridization
Authors: Facer Christine , Khan George
Published: November 2013

Acute Plasmodium falciparum malaria in African children allows expansion of latent Epstein‐Barr virus infection, leading to colonization of lymph nodes by virus‐infected lymphoblasts in 60% of cases as demonstrated by in situ hybridization for the detection of EBER‐1 and EBER‐2 RNA. This probably arises against a background of malaria‐induced immunosuppression to EBV and concurrent lymphoid activation. The relevance of the results to the pathogenesis of African endemic Burkitt's lymphoma is discussed.
Shotgun Cloning and Characterization of the Thymidylate Synthase‐Encoding Gene from Mycobacterium bovis BCG
Authors: Matsumoto Sohkichi , Yukitake Hideharu , Ohara Naoya , Dairi Tohru , Kanbara Hiroji , Yamada Takeshi
Published: November 2013

The shotgun cloning of a Mycobacterium bovis BCG (BCG) genome into pBluescript SK (+) successfully yielded a 0.9 kbp fragment, confirming the ability of Escherichia coli thyA mutant MH2702 to grow in a thymine‐depleted medium. This DNA fragment contained a gene homologous to the thymidylate synthase (TS)‐encoding genes (thyA) of other organisms. An inverted repeat sequence and open reading frame (ORF) were observed at the upstream region of the thyA. A computer analysis revealed that the protein encoded by this ORF possessed a structure unique for a DNA binding protein.
Biochemical and Immunological Analyses of the Flagellin of Clostridium tyrobutyricum ATCC 25755
Authors: Arnold Françoise , Bédouet Laurent , Batina Pierre , Robreau Georges , Talbot Françoise , Lécher Pierre , Malcoste Roger
Published: November 2013

The monoclonal antibody 21E7‐B12 (IgG3) can be used in a direct method of Clostridium tyrobutyricum detection based on an immunoenzymatic assay. Immunoelectron microscopy demonstrated that the 21E7‐B12 antibody recognized the surface‐exposed epitopes on the flagellar filaments of C. tyrobutyricum. After flagellar extraction, the purified flagellin showed an apparent molecular mass of 46 kDa with an isoelectric point of 3.6. Sugar staining, mild periodate oxidation and é‐elimination experiments showed that the flagellin was glycosylated and that the 21E7‐B12 epitope was located in the sugar moiety. Amino acid composition showed that the flagellar filament protein contained a high percentage of serine and threonine, while proline was absent. The first 23 residues of the N‐terminal were determined and sequence homology with other flagellins was found.
Follow‐Up Study of Hypervariable Region Sequences of the Hepatitis C Virus (HCV) Genome in an Infant with Delayed Anti‐HCV Antibody Responses
Authors: Katayama Yuko , Tajiri Hitoshi , Tada Kanae , Okada Shintaro , Tong Wen‐yan , Ishido Satoshi , Hotta Hak
Published: November 2013

An infant born prematurely and infected with hepatitis C virus (HCV) one month after birth was followed for 4.5 years. The patient did not produce detectable anti‐HCV antibodies until two years after the onset of hepatitis. Before seroconversion, a single clone of HCV, as determined by quasispecies of the hypervariable region (HVR) of the HCV genome, was almost exclusively found in the serum. After seroconversion, however, another distinct lineage of HCV clones replaced it within half a year. As HCV infection persisted further in the presence of anti‐HCV antibodies, many derivatives of both sequence lineages emerged to exhibit the typical quasispecies feature of HVR sequences. Neither seroconversion nor the changes in HVR sequences influenced the serum aminotransferase titers.
Partial Analysis of the Flagellar Antigenic Determinant Recognized by a Monoclonal Antibody to Clostridium tyrobutyricum
Authors: Bédouet Laurent , Arnold Francoise , Robreau Georges , Batina Pierre , Talbot Francoise , Malcoste Roger
Published: November 2013

In order to count Clostridium tyrobutyricum spores in milk after membrane filtration, murine 21E7‐B12 monoclonal antibody was produced. Elution of the monoclonal antibody from this antigen, the flagellar filament protein, by carbohydrate ligands was used to study the epitope structure. A competitive elution of an anti‐dextran monoclonal antibody by carbohydrate ligands served as a control in order to validate the immunological tool applied to flagellin epitope study. The carbohydrate moiety of flagellin contained D‐glucose and N‐acetyl‐glucosamine in a molar ratio of 11:1 as determined by gas‐liquid chromatography and 2 low‐abundancy unidentified compounds. In ELISA, D‐glucose and N‐acetyl‐glucosamine did not dissociate the antibody‐flagellin complex contrary to maltose, maltotriose, maltotetraose and maltopentaose. The efficiency of elution increased from the dimer to the pentamer and became nil for maltohexaose and maltoheptaose. The fact that the hexamer and heptamer could not react with the 21E7‐B12 monoclonal antibody could be explained by a drastic conformational change. The overall stretched maltopentaose switch to a helical‐shaped maltoheptaose which could not fit the 21E7‐B12 monoclonal antibody antigen‐combining site. Thus, flagellin epitope may contain α(1→4) linked glucose residues plus either N‐actyl‐glucosamine or an unidentified compound that maintain it in an extended shape.
Liver‐Associated Natural Killer Activity in Cirrhotic Rats
Authors: Shirachi Miki , Sata Michio , Miyajima Ichiro , Tanaka Masatoshi , Tanikawa Kyuichi
Published: November 2013

An impaired host defense mechanism is well known in patients with liver cirrhosis (LC). Using a sinusoidal lavage method, lymphocytes were obtained from LC rats that were administered thioacetamide, and natural killer (NK) activity was measured by 5lCr‐release assay. The NK cell count was measured by flow cytometric analysis using monoclonal antibody (Mab) 3.2.3 and/or CD 3‐8+ as markers for NK cells, and by immunohistochemical staining using Mab 3.2.3. Furthermore, interferon (IFN) α was administered to LC rats and the subsequent changes in hepatic NK activity and NK cell count were observed. In the large granular lymphocyte (LGL)‐rich fraction (Fr.1, LGLs: 60‐90%), the NK activity was significantly lower in the LC rats (40.0±3.8%) compared to that in the control rats (48.4±4.3%) (P<0.005). In addition, the number of NK cells in the liver tissues of the LC rats was significantly lower compared to that in the liver tissues of the control rats by morphometric analysis (P<0.05). For LC rats, NK activity of the Fr.1 24 hr after IFNα administration (5×104 IU / 100 g body weight) increased significantly (P<0.005). Hepatic NK activity and NK cell count were reduced in the LC rats, and recovered following IFNα administration. The results obtained in this study may give clues to better understanding the impaired host defense mechanism in LC patients.
Construction of the Chimeric Reverse Transcriptase of Simian Immunodeficiency Virus Sensitive to Nonnucleoside Reverse Transcriptase Inhibitor
Authors: Isaka Yoshitaka , Sato Akihiko , Kawauchi Shinobu , Suyama Akemi , Miki Shigeru , Hayami Masanori , Fujiwara Tamio
Published: November 2013

A number of structurally diverse compounds have been shown to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase (RT). The compounds can be grouped into two broad classes; nucleoside analogs and nonnucleoside RT inhibitors. The nonnucleoside RT inhibitors are quite specific for HIV‐1 RT but not human immunodeficiency virus type 2 (HIV‐2) and simian immunodeficiency virus (SIV) RT. We have investigated the property of SIV/HIV‐1 chimeric viruses in which portions of SIVMAC RT were exchanged with the corresponding domain of HIV‐1 RT; amino acids 176‐190, 176‐383 and 176‐495 of HIV‐1 RT. The chimeric virus, which was substituted amino acids 176‐190 of RT, had detectable RT activity, and this chimeric RT was sensitive to three nonnucleoside RT inhibitors [nevirapine, HEPT derivative (E‐EBU‐dM) and TIBO derivative (R82913)]. To further study this chimeric virus, we purified the chimeric RT enzyme expressed in Escherichia coli and determined its kinetic properties; the Km and Vmax values, and the Ki value of HEPT derivative calculated for the DNA polymerase activity. This study reveals that amino acids 176‐190 of SIVMAC RT were important for the enzymatic activity and the SIV/HIV‐1 chimeric RT, which had amino acids 176‐190 of HIV‐1, was sensitive to the nonnucleoside RT inhibitor.
Filamentous Phage fs1 of Vibrio cholerae O139
Authors: Nakasone Noboru , Honma Yasuko , Toma Claudia , Yamashiro Tetsu , Iwanaga Masaaki
Published: November 2013

Filamentous phage, fs1, was obtained from Vibrio cholerae O139. The lysogenized strains produced a large amount of fs1 phage in the culture supernatant. This phage was previously reported as novel fimbriae of that organism. The genome of the phage was a 6.5 kb single‐stranded DNA. The capsid of fs1 consists of a small molecule peptide (about 2.5 kDa).
Human Immunodeficiency Virus Type 1 (HIV‐1) Infection and Transcytosis Activity of a HIV‐1 Susceptible Clone from HeLa Cell
Authors: Morizono Kouki , Harada Shinji
Published: November 2013

In order to clarify the transmission process of human immunodeficiency virus type 1 (HIV‐1) through the epithelial cell barrier, HeLa cells susceptible and non‐susceptible to HIV‐1 were cloned and designated as P6 HeLa and N7 HeLa cells, respectively. P6 HeLa cells could be infected with the LAI strain of HIV‐1 and mediated HIV‐1 transcytosis. In contrast, N7 HeLa cells exhibited neither HIV‐1 infection nor transcytosis. CD4 and galactosylceramide as the receptors for HIV‐1 were not detected on P6 HeLa cells, although an anti‐CD4 monoclonal antibody (mAb) blocked HIV‐1 infection. Since HIV‐1‐infected P6 HeLa cells exhibited no fusion and survived, we speculated that the P6 HeLa cells expressed molecules other than CD4 which facilitated HIV‐1 infection. Two mAbs (A‐14 ITK and C57 a9‐9) which inhibited the HIV‐1 infection of P6 HeLa cells were generated. Each mAb recognized distinct molecule(s) as shown by Western blotting. Transcytosis by the P6 HeLa cells was inhibited by C57 a9‐9 but not by A‐14 ITK or anti‐CD4 mAb. Both infection and transcytosis may be responsible for HIV‐1 transmission through epithelial cells in a complex manner. Although infection and transcytosis occurred via different mechanisms, the molecule(s) recognized by C57 a9‐9 mAb may be associated with both processes.
Evidence for a Dual Strategy in the Expression of Cauliflower Mosaic Virus Open Reading Frames I and IV
Authors: Kobayashi Kappei , Tsuge Seiji , Nakayashiki Hitoshi , Mise Kazuyuki , Furusawa Iwao
Published: November 2013

Studies have indicated that cauliflower mosaic virus (CaMV) gene expression is mediated by the translation of polycistronic 35S pregenomic RNA, but the involvement of some minor subgenomic RNA species is also suspected. We examined the involvement of the 35S promoter in the expression of CaMV open reading frames (ORFs) I and IV using both 35S RNA‐driven and promoter‐less ORF I‐ and ORF IV‐β‐glucuronidase (GUS) fusion constructs. In addition to the 35S promoter‐dependent expression of both ORF I‐ and IV‐GUS fusions, we detected the 35S promoter‐independent expression of both fusion genes via subgenomic mRNAs, which were detected by Northern blotting in the protoplasts transfected with the 35S promoter‐driven constructs as well as in those transfected with the promoter‐less constructs. These results suggest the involvement of subgenomic RNAs in the expression of CaMV ORFs I and IV, and the operation of a dual strategy in the expression of two viral genes.
Epitope Specificity of a Monoclonal Antibody Generated against the Dissociated CFA/I Fimbriae of Enterotoxigenic Escherichia coli
Authors: De Luna Maria Das Graças , Rudin Anna , Vinhas Solange Alves , de Almeida Darcy Fontoura , de Souza Ferreira Luis Carlos
Published: November 2013

A monoclonal antibody (MAb 84) raised against the dissociated CFA/I fimbriae of enterotoxigenic Escherichia coli was characterized with regard to antigen binding and epitope specificity. Enzyme‐linked immunosorbent assay (ELISA) showed that MAb 84 had higher affinity to CFA/I subunits than to intact CFA/I fimbriae and recognized a Salmonella flagellin carrying an insert corresponding to amino acids 32 to 45 of the CFA/I subunit. Fine epitope mapping based on the Pepscan technique showed that the peptide 39TFESY43, derived from the sequence of the mature CFA/I subunit, was specifically recognized by MAb 84. The 39TFESY43 sequence is probably not accessible on the surface of the native CFA/I fimbriae since MAb 84 did not bind to intact fimbriae as evaluated in inhibition ELISA tests. Moreover, MAb 84 did not agglutinate fimbriated ETEC cells nor inhibit CFA/I‐mediated hemagglutination or the adhesion to Caco‐2 cells.
Measurement of Arginine Carboxypeptidase‐Generating Activity of Adult Plasma
Authors: Watanabe Motoi , Ishikawa Yasushige , Campbell William , Okada Hidechika
Published: November 2013

Arginine carboxypeptidase (CPR) is a novel carboxypeptidase which was first described by Campbell and Okada. CPR is generated from a stable precursor of CPR (proCPR) during coagulation or under other circumstances and is promptly inactivated at 37 C. Therefore, it is not easy to determine CPR in blood samples. Since proCPR can be separated from the other basic carboxypeptidase (carboxypeptidase N; CPN) by passing plasma through DEAE gel, we have established a method to determine the amount of proCPR after converting it to active CPR by trypsin treatment. We first separated the proCPR from CPN using a filter cup tube (FC tube) packed with DEAE Sephadex, and measured activity after conversion of the enzyme to its active form using trypsin. With this method, no significant decrease in proCPR was noted in the plasma of patients including those with rheumatoid arthritis (RA), although CPR activity in fresh sera has been reported to be decreased. This discrepancy suggests that proCPR is not depleted in most patient sera, but that the level of activity of the enzyme which converts proCPR into active CPR may be compromised in RA patients.
Regulation of Promoter and Intron Enhancer Activity in Immunoglobulin Heavy‐Chain Genes during B‐Cell Differentiation
Authors: Naito Akihiro , Suzuki Yasuhiko , Azuma Takachika
Published: November 2013

Chloramphenicol acetyltransferase (CAT) transgenic mice, in which the transgene is regulated by the VH promoter and heavy‐chain intron enhancer (Eμ), were examined to investigate the variation of activity of these cis‐acting elements during the late stage of B‐cell development. CAT enzyme activity decreased when resting B cells were stimulated through B‐cell receptors (BCR) with goat anti‐mouse IgM antibodies in vitro. On the other hand, when these B cells were stimulated by lipopolysaccharide (LPS) in vitro, they showed enhanced CAT activity, accompanied by an increase in the number of CD43+ B220+ cells (pro‐plasma and plasma cells). In addition, the CAT activities in CD43+ B220+ and PNAhi B220+ cells from immunized mice were higher than those in CD43− B220+ and PNAlo B220+ cells, respectively. These results suggest that the activity of Eμ in the context of VH promoter was transiently down‐regulated by stimulation through the BCR but enhanced at the pro‐plasma and plasma stages.
Occurrence of Coagulase Serotype among Staphylococcus aureus Strains Isolated from Healthy Individuals —Special Reference to Correlation with Size of Protein‐A Gene—
Authors: Sekr Keiko , Sakurada Junji , Seong Hee Kyong , Murai Miyo , Tachi Hidemi , Ishii Hiroko , Masuda Shogo
Published: November 2013

One‐hundred‐and‐nineteen strains of Staphylococcus aureus isolated from healthy individuals for 3 years between 1991 and 1993 were subjected to an investigation on the producibility of proteins including protein A, coagulase, enterotoxins and toxic‐shock syndrome toxin‐1. Especially, protein A was the center of our interest. Among these strains, 69, 43, 3 and 1 strains were found to have the protein‐A gene containing 5, 4, 3 and 2 IgG‐binding domains, respectively. On the other hand, only one strain was devoid of the protein‐A gene. There were some differences in the profile of the coagulase serotype between the group with 4 IgG‐binding domains and that with 5 IgG‐binding domains. Differences in the profile of toxin production were also observed between the two groups.
Differentiation of Chlamydia Species by Combined Use of Polymerase Chain Reaction and Restriction Endonuclease Analysis
Authors: Yoshida Hiroshi , Kishi Yuichiro , Shiga Sadashi , Hagiwara Toshikatsu
Published: November 2013

To differentiate Chlamydia spp., a primer pair designed to generate a genus‐specific region of the major outer membrane protein (MOMP) gene was used in a PCR to amplify a single DNA fragment of 245‐259 bp. In the PCR, the expected single DNA fragment was amplified from strains of Chlamydia trachomatis, C. psittaci, C. pneumoniae and C. pecorum, respectively. By restriction endonuclease analysis with AluI and PvuII, the amplified products exhibited four distinct patterns, corresponding to the four species. It is, therefore, concluded that one‐step PCR followed by restriction endonuclease analysis as described in this study could be a valuable method for the detection and differentiation of Chlamydia species.
Induction of Cross‐Reactivity in an Endogenous Viral Peptide Non‐Reactive to FBL‐3 Tumor‐Specific Helper T‐Cell Clones
Authors: Uenishi Hirohide , Iwanami Norimasa , Yamagishi Hideo , Nakatani Takako , Kawasaki Toshisuke , Tamamura Hirokazu , Fujii Nobutaka , Kuribayashi Kagemasa
Published: November 2013

We previously reported a helper T‐cell (Th) epitope (peptide i) which corresponded to the sequence ranging from positions 462 to 479 from the N‐terminus of the Friend‐murine leukemia virus (F‐MuLV) envelope protein (env462‐479). Homologous sequences exist in both Moloney‐murine leukemia (M‐MuLV env452‐469) and endogenous AKV (AKV env453‐470) viruses, which differ from F‐MuLV env462‐479 in 5 and 7 amino acids, respectively. However, peptide i‐specific Th clones did not respond to either of the corresponding exogenous or endogenous peptides. One amino acid substitution in M‐MuLV env452‐469 (Asn to Tyr at position 465: N465Y) and three amino acids in AKV env453‐470 (H460S, A466Y and Y468H) endowed both peptides with the reactivity to one of the Th clones, F5‐5, almost to the same degree as peptide i. However, the other Th clones responded differently to each of the modified endogenous peptides substituted by one to three amino acids. The cells responsive to the cross‐reactive peptides occupied only a minor portion, if any, of the bulk cultured lymph node cells from peptide i‐immune mice, and in particular, no significant response to the modified endogenous peptides was observed in repeated experiments. The exchange of at least 3 residues was necessary for the endogenous peptide to acquire sufficient cross‐reactivity to two of the three Th clones. However, it was noticeable that a single substitution of alanine by tyrosine at the dominant T‐cell receptor (TCR) contact position of the peptide ie generated a weak but significant cross‐reactivity to one of the three Th clones in this study. Thus, peptides of endogenous retroviral origin that would be modified by mutational events might become ‘non‐self’ and prime Th cells leading to auto‐antibody production and resulting in autoimmune disease.
Utilization of Proteinase K‐Treated Cells as Lipopolysaccharide Antigens for the Serodiagnosis of Helicobacter pylori Infections
Authors: Amano Ken‐ichi , Yokota Shin‐ichi , Ishioka Tomonori , Hayashi Shunji , Kubota Toru , Fujii Nobuhiro
Published: November 2013

We have evaluated the use of proteinase K (PK)‐treated cells isolated from Helicobacter pylori as lipopolysaccharide (LPS) antigens in an immunoblot assay and an enzyme‐linked immunosorbent assay (ELISA) for the serodiagnosis of H. pylori infection. The sera from patients with chronic gastritis, gastric ulcer, duodenal ulcer or gastric cancer, and from healthy adults with or without H. pylori infection were assayed with three commercial serodiagnostic kits (HM‐CAP, Helico‐G, and G.A.P. II) and novel methods relying on the use of PK‐treated cells. The PK‐treated cells used in these assays were selected on the basis of their possibility to possess a common epitope in the O‐polysaccharides of H. pylori, which is known to be highly immunogenic in humans. Of the sera from these patients, 71‐94% were positive with the commercial kits, 97% with immunoblot assay, and 90% with ELISA. On the other hand, of the healthy adults infected with H. pylori, 72‐97% were positive with the commercial kits, 86% with immunoblot assay, and 72% with ELISA. PK‐treated cells that did not contain the common epitope were unsuitable as an antigen for immunoblot assay or ELISA. Furthermore, the reactivity of these sera reacted specifically with H. pylori PK‐treated cells but not with LPSs from other gram‐negative bacteria, such as Campylobacter, Proteus, Bordetella, and Salmonella. These results demonstrate that the serological assays relying on the use of H. pylori PK‐treated cells possessing a highly antigenic epitope are potentially useful as a serodiagnostic test for H. pylori infection.
Induction of Apoptosis and Tyrosine Phosphorylation of Cellular Proteins in T Cells and Non‐T Cells by Stimulation with Concanavalin A
Authors: Nagase Fumihiko , Abo Tomoko , Hiramatsu Kumiko , Suzuki Shin‐ichi , Du Jun , Nakashima Izumi
Published: November 2013

A high concentration (30 μg/ml or more) of Con A caused the death of not only thymocytes but also splenic cells of BALB/c mice, whereas a moderate concentration (3 μg/ml) of Con A induced proliferation of these cells. A high concentration of Con A also induced the death of splenic cells of athymic BALB/c‐nu/nu mice and the bone marrow cells of BALB/c mice which mainly consist of non‐T cells. However, any concentration (1‐30 μg/ml) of Con A failed to induce the proliferation of these cells. Specific binding of tetrameric Con A to mannose‐containing receptors was required for the induction of cell death. DNA fragmentation was observed by both laser flow cytometry and electrophoresis in Con A‐stimulated T cells and non‐T cells. This indicated that the mechanism of induction of apoptosis with Con A is not necessarily TCR‐dependent. Con A induced tyrosine phosphorylation of a number of proteins in various types of cells. Interestingly, phosphorylation of the 40 kDa protein developed only in the thymocytes and spleen cells that contain T cells, whereas phosphorylation of the 80 and 120 kDa proteins appeared in both T cells and non‐T cells. These results suggested that the Con A‐induced apoptosis of T cells and non‐T cells involves different but possibly mutually related protein tyrosine phosphorylation‐linked signals.
Rapid Analysis of Allelic Variants of the Sheep PrP Gene by Oligonucleotide Probes
Authors: Ishiguro Naotaka , Shinagawa Morikazu , Onoe Sadao , Yamanouchi Kazunori , Saito Toshiro
Published: November 2013

A rapid method to determine the allelic variants of the sheep PrP gene was developed. DNA samples from 128 Suffolk sheep (39 rams and 89 ewes) were screened by using polymerase chain reactions and dot‐blot hybridization with 32P‐labeled nine allele‐specific oligonucleotide probes corresponding to the polymorphic PrP codons 112, 136, 154 and 171. Three allelic variants of the PrP gene, PrPMARQ, PrPTARQ and PrPMARR, were found in the flocks. Among those variants, nearly half of the ewes had alleles of the 171‐Arg variant that is closely associated with resistance to natural scrapie. Assessments of allelic mutations of the PrP gene may help to select the scrapie‐resistant progenitors in the flocks.
Regulation of GM‐CSF‐Induced Dendritic Cell Development by TGF‐β1 and Co‐Developing Macrophages
Authors: Yamaguchi Yasunori
Published: November 2013

Using a culture system of bone marrow progenitor cells with GM‐CSF and TGF‐β1, a study was performed to analyze the effect of TGF‐β1 on the development of dendritic cells (DC) and to elucidate the regulatory role of macrophages co‐developing with dendritic cells. The results demonstrate that DC generated in the presence of TGF‐β1 were immature with respect to the expression of CD86, nonspecific esterase activity and cell shape. Such inhibitory effects of TGF‐β1 were dependent on FcR+ macrophages, which were depleted by panning. TGF‐β1 did not appear to inhibit the commitment of progenitor cells to the DC lineage. In addition, TGF‐β1 also acted directly on the intermediate stage of DC to prevent their over‐maturation, which results in a preferential decrease in MHC class II, but not in CD86, in the presence of TNF‐α. FcR+ suppressive macrophages were also shown to facilitate DC maturation when stimulated via FcR‐mediated signals even in the presence of TGF‐β1. These results indicate that TGF‐β1 indirectly and directly regulate the development of DC and that co‐developing macrophages have a regulatory role in DC maturation.
Developmental Regulation by Cytokines of Bone Marrow‐Derived Dendritic Cells and Epidermal Langerhans Cells
Authors: Yamaguchi Yasunori
Published: November 2013

Dendritic cells (DC) are specialized antigen‐presenting cells involved in T cell‐mediated immune responses. Differentiation and functional maturation of the DC are now known to be regulated by various cytokines, including TGF‐β1. The experiments of this study examined the effect of other cytokines, such as IL‐4, IL‐10 and IL‐6, on the differentiation and maturation of bone marrow (BM)‐derived DC (BM‐DC) and epidermal Langerhans cells (LC). When IL‐6 or IL‐10 was added to cultures of BM cells in the presence of GM‐CSF, both cytokines, as in the case of TGF‐β1, suppressed the maturation of DC in terms of the expression of adhesion and costimulatory molecules and T cell‐stimulating activity. In contrast, IL‐4 was not suppressive but rather supportive for the differentiation of DC. However, these suppressive cytokines hardly counteracted the maturation‐inducing activity of TNF‐α when added to cultures of immature DC. In addition, they appeared to block the overmaturation of DC, which is characterized by a loss of MHC class II molecules. Regarding LC maturation in epidermal cell cultures, IL‐6 and IL‐10 were inhibitory for the expression of CD86 and CD80 in a dose‐dependent fashion. Unlike BM‐DC, LC maturation was slightly enhanced by TGF‐β1. The protein antigen‐presentation by LC to Th1 clone was not affected by IL‐6, but slightly reduced by IL‐10. These results suggest that each cytokine contributes to regulate the differentiation and maturation of DC at a different developmental stage.
Rapid Serodiagnosis of Mycobacterium avium‐intracellulare Complex Infection by ELISA with Cord Factor (Trehalose 6, 6′‐Dimycolate), and Serotyping Using the Glycopeptidolipid Antigen
Authors: Enomoto Kyoko , Oka Shiro , Fujiwara Nagatoshi , Okamoto Takashi , Okuda Yoshinari , Maekura Ryoji , Kuroki Tetsuo , Yano Ikuya
Published: November 2013

Mycobacterium avium‐intracellulare complex (MAC) is one of the most important opportunistic pathogens, particularly in patients with acquired immunodeficiency syndrome (AIDS). The aim of this study was to determine whether an enzyme‐linked immunosorbent assay (ELISA) using trehalose 6,6′‐dimycolate (TDM) as an antigen can be used for the rapid serodiagnosis of MAC infection. We also identified MAC serotypes by ELISA using serotype‐specific glycopeptidolipid (GPL) antigen. To confirm our findings, the thin‐layer chromatographic (TLC) behavior of serotype‐specific GPL of the strains isolated from MAC‐infected patients was also tested. Forty patients infected with MAC and 30 healthy controls were tested. Thirty‐two of the 40 MAC‐infected patients had higher titers of serum antibodies against MAC TDM than against MTB TDM, while all 30 healthy control sera were unreactive to MAC TDM and MTB TDM. Results of the GPL ELISA indicated that 20 of the 40 MAC‐infected patients' sera were reactive against serotype 4 GPL, 3 against serotype 8 GPL, and 1 against serotype 16 GPL. A TLC analysis of the GPL of the 40 MAC isolates showed that 16 strains were of serotype 4, 5 of serotype 8, and 2 of serotype 16. Results of the GPL ELISA were in good accord with those of the TLC analysis for most patients. Our findings suggest that ELISA using TDM is useful for rapid serodiagnosis of MAC infection, and that complementary ELISA testing using serotype‐specific GPL gives additional detailed information concerning MAC serotypes.
Studies on the Rabies Virus RNA Polymerase: 2. Possible Relationships between the Two Forms of the Non‐Catalytic Subunit (P Protein)
Authors: Takamatsu Fumihiko , Asakawa Naoki , Morimoto Kinjiro , Takeuchs Kenji , Eriguchi Yoshiro , Toriumi Harufusa , Kawai Akihiko
Published: November 2013

We investigated the relationship between the two forms of rabies virus P protein, a non‐catalytic subunit of rabies virus RNA polymerase. The two displayed different electrophoretic mobilities as 37‐ and 40‐kDa polypeptides, hence termed as p37 and p40, respectively. Double labeling experiments with [3H]leucine and [32P]orthophosphate demonstrated that p40 was much more phosphorylated than p37. Treatment of the virion proteins with alkaline phosphatase eliminated only p40, and not 37‐kDa polypeptide. The p37 was a major product of the P gene, and was accumulated in the infected cell and incorporated into the virion. On the other hand, p40 was apparently detected only in the virion, and little detected in the cells. Treatment of infected cells with okadaic acid, however, resulted in significant accumulation of p40 in the cell, suggesting that p40 was continuously produced in the cell but dephosphorylated quickly. We detected both 37‐ and 40‐kDa products in P cDNA‐transfected animal cells, while only a 37‐kDa product was produced in Escherichia coli. Incubation of 37‐kDa products from E. coli with the lysates of animal cells in vitro resulted in the production of a 40‐kDa product, which was also shown to be suppressed by the heparin. From these results, it is suggested that p40 is produced by the hyperphosphorylation of a 37‐kDa polypeptide, which depends on certain heparin‐sensitive cellular enzyme(s) and occurs even in the absence of the other viral gene products, and that p40 is reverted quickly to p37 in the infected cells, probably being dependent on some virus‐induced factor(s).
Detection and Genetical Characterization of Shiga Toxin‐Producing Escherichia coli from Wild Deer
Authors: Asakura Hiroshi , Makino Sou‐ichi , Shirahata Toshikazu , Tsukamoto Teizo , Kurazono Hisao , Ikeda Tetsuya , Takeshi Kouichi
Published: November 2013

Shiga toxin (Stx)‐producing Escherichia coli (STEC) strains isolated from wild deer in Japan were examined. A total of 43 fecal samples were collected 4 times from 4 different sites around Obihiro City, Hokkaido, Japan, in June and July 1997. Seven STEC strains were isolated by PCR screening, all of them were confirmed by ELISA and Vero cell cytotoxicity assay to be producing only active Stx type 2 (Stx2). Moreover, they seemed to carry the hemolysin and eaeA genes of STEC O157:H7, and some isolates harbored large plasmids which were similar to the 90‐kilobase virulence plasmid of STEC O157:H7. Based on their plasmid profiles, antibiotic resistance patterns, PCR‐based DNA fingerprinting data obtained by using random amplified polymorphic DNA (RAPD) and the stx2 gene sequences, all isolates were divergent from each other except for 3 isolates from the first and second samplings. A DNA sequence analysis of representative isolates revealed that deer originating STEC strains were closely related to each other, but not to the Stx2‐producing STEC strains isolated from a mass outbreak in Obihiro at the same time. A phylogenic analysis of the deduced Stx2 amino acid sequences demonstrated that three distinct clusters existed in the deer originating STEC strains and that the Stx of deer originating STEC was closely associated with that originating from humans, but not those of STEC originating from other animals. These results suggest that STEC contamination of deer carcasses should be considered as a potential source of human infection and adequate sanitary inspection of meat for human consumption is also essential for wild animals.
Identification and Semiquantitation of Mycobacterium avium Using a Competitive PCR Method
Authors: Hashimoto Toru , Suzuki Katsuhiro
Published: November 2013

A competitive PCR method with standard DNA (MIMIC) was developed for the rapid detection and semiquantitation of Mycobacterium avium (M. avium) using primers specific for the alpha antigen sequence of the bacteria. DNA from both M. avium and Mycobacterium marinum was amplified by polymerase chain reaction (PCR), but only M. avium could be detected by subsequent blotting confirmation with a probe specific for the bacteria. With the PCR and subsequent dot blot hybridization, as little as 10 fg of the M. avium DNA could be detected, equivalent to about 2 cells of the mycobacteria. In addition, we could distinguish 105 CFUs of M. avium from 104 CFUs or less by competitive PCR using a MIMIC. The present competitive PCR test enabled rapid identification and semiquantitation of M. avium, and could be used clinically to monitor disease severity and response to treatment of human M. avium disease.
Specific Microbial Colonizations in the Periodontal Sites of HIV‐infected Subjects
Authors: Chattin Blanca Rosa , Ishihara Kazuyuki , Okuda Katsuji , Hirai Yoshito , Ishikawa Tatsuya
Published: November 2013

The purpose of this study was to examine colonization by specific organisms at periodontal sites in HIV‐seropositive [HIV(+)] subjects. A total of 67 HIV(+) and 32 HIV(‐) subjects were investigated. The specific pathogens included black‐pigmented anaerobic rods (BPAR), Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Candida albicans and mycoplasma species. P. gingivalis was present in the HIV(+) subjects more frequently than in the HIV(‐) periodontitis patients (P < 0.01). The cell numbers of BPAR and P. gingivalis and percentages as the total of CFUs on blood agar cultured in an anaerobic chamber were statistically higher in periodontal pocket samples from HIV(+) than from HIV(‐). A. actinomycetemcomitans was also detected at a high rate (41.8%) in HIV(+) patients. The average cell numbers of C. albicans were higher in samples from the HIV(+) group (P < 0.05). The detection rate of mycoplasma species in the HIV(+) patients was significantly lower than that in HIV(‐) subjects (P < 0.05), and most isolated mycoplasma strains were Mycoplasma salivarium.
Siderophore Production of Vibrio parahaemolyticus Strains from Different Sources
Authors: Yamamoto Shigeo , Okujo Noriyuki , Miyoshi Shin‐Ichi , Shinoda Sumio , Narimatsu Shizuo
Published: November 2013

Vibrio parahaemolyticus strains isolated from different sources were assayed for their ability to produce a siderophore, vibrioferrin, under iron‐limited growth conditions. The mean value } standard error of mean μM vibrioferrin in spent culture supernatant/optical density at 660 nm) was 832.3 } 66.9 for clinical isolates (n 44), which was significantly higher (P 0.01) than those for food isolates (461.0 } 66.5; n 37) and coastal isolates (378.8 } 37.2; n 26). This suggests that greater productivity of vibrioferrin by clinical isolates may be associated with a selective advantage for survival and proliferation under conditions of iron‐limitation such as in the intestine.
Partial Purification and Characterization of the Active Entity Responsible for Inducing Interleukin‐6 Production by Human Gingival Fibroblasts from Mycoplasma salivarium Cells
Authors: Hasebe Akira , Shibata Ken‐Ichiro , Domon Hikouji , Dong Li , Watanabe Tsuguo
Published: November 2013

The active entity responsible for inducing interleukin‐6 production by human gingival fibroblasts was partially purified by ion‐exchange chromatography from the water‐soluble fraction of Mycoplasma salivarium cells. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of the final preparation revealed one densely stained band with a molecular weight of 20.6 kilodaltons and two faint bands with molecular weights of 40.5 and 82.5 kilodaltons. The specific activity of the final preparation was 34‐fold higher than that of the starting water‐soluble fraction. The interleukin‐6‐inducing activity was destroyed by proteinase K and reduced 70% by lipoprotein lipase and heat treatment, but was not affected by deoxyri‐bonuclease I or endoglucosidase D. The final preparation induced small amounts of tumor necrosis factore‐ α and interleukin‐1β in a myelomonocytic cell line, THP‐1 cells, but did not induce interleukin‐6. The ability of Escherichia coli lipopolysaccharide to stimulate human gingival fibroblasts to release interleukin‐6 was dependent upon the presence of serum in the assay medium, but that of the final preparation from M. salivarium was not. Thus, we partially purified the protein(s) from M. salivarium which were capable of stimulating human gingival fibroblasts to release interleukin‐6 by a mechanism different from that of E. coli lipopolysaccharide.
Adenovirus Type 5 E1A Immortalizes Primary Rat Cells Expressing Wild‐Type p53
Authors: Yamashita Toshiharu , Tonoki Hidefumi , Nakata Daichi , Yamano Shigeru , Segawa Kaoru , Moriuchi Tetsuya
Published: November 2013

Adenovirus (Ad) E1A induces apoptosis in cells expressing wild‐type p53, and stable transformation by Ad E1A requires the co‐introduction of an anti‐apoptotic gene such as Ad E1B 19K. Thus, cells immortalized by Ad E1A alone might have lost functional p53. In order to analyze the p53 in rat cells expressing Ad E1A, we established rat cell lines by transfecting primary rat embryo fibroblast (REF) and baby rat kidney (BRK) cells with cloned Ad5 E1A. By using a yeast functional assay, we analyzed p53 in six primary REF and three BRK cell lines immortalized by Ad5 E1A as well as five spontaneously immortalized rat cell lines (REF52, NRK, WFB, Rat‐1 and 3Y1). The yeast functional assay revealed that all of the spontaneously and Ad5 E1A‐immortalized rat cell lines except for 3Y1 expressed wild‐type p53. All of the Ad5 E1A‐immortalized rat cell lines contained p53 detectable by immunoprecipitation. Recombinant adenovirus expressing rat p53 cloned from a REF cell line immortalized by Ad5 E1A, as well as that expressing murine wild‐type p53, induced apoptosis in p53‐null cells in collaboration with E1A. Thus, it is suggested that the mutation of p53 appears to be not frequent in the spontaneous immortalization of primary rat cells, and that the functional loss of wild‐type p53 is not a prerequisite of E1A‐mediated immortalization.
The hsp Operons Are Repressed by the hrc37 of the hsp70 Operon in Staphylococcus aureus
Authors: Kuroda Makoto , Kobayashi Daisuke , Honda Kyoko , Hayashi Hideo , Ohta Toshiko
Published: November 2013

The heat‐shock proteins are coded for the polycistronic operons hsp70 and hsp60 in Staphylococcus aureus. The hsp70 operon is comprised of five genes, hrc37, hsp20, hsp70, hsp40 and orf35, and the hsp60 is comprised of two genes, hsp10 and hsp60. The hsp70 operon transcribed five different sizes of mRNA from three promoters: P1, the most active promoter, transcribed 6.0 and 3.6 kb mRNAs; P2 transcribed a single 1.8 kb mRNA; and P3 transcribed 4.2 and 2.4 kb mRNAs. The hsp60 operon transcribed a single 2.1 kb transcript from only one promoter, P1. Both operons had a common structure of inverted repeat element (CIRCE, Controlling Inverted Repeat of Chaperon Expression) at the promoter region. All of the transcripts were heat (46 C) inducible. One of the unidentified genes, hrc37, was characterized. The disruptant of the hrc37 in the hsp70 operon enhanced the transcription of both operons at 37 C (derepression). Complementation of the disruptant with the cloned hrc37 plasmid recovered the repression of the transcription of both operons at 37 C. The product of hrc37, Hrc37, was found to bind to the CIRCE element. These findings indicated that Hrc37 from the hsp70 operon repressed the transcription of both the hsp70 and hsp60 operons by binding to the CIRCE element located at the promoter region.
The Hemagglutinating Action of Vibrio vulnificus Metalloprotease
Authors: Miyoshi Shin‐ichi , Kawata Koji , Tomochika Ken‐ichi , Shinoda Sumio
Published: November 2013

Vibrio vulnificus protease (VVP), a 45‐kDa zinc metalloprotease, consists of two functional domains: an N‐terminal 35‐kDa polypeptide having endoproteinase activity, and a C‐terminal 10‐kDa polypeptide that mediates the binding of VVP to the erythrocyte membrane. Therefore, VVP, but not its N‐terminal endoproteinase domain alone, has agglutinating activity to rabbit erythrocytes. When a single zinc atom in the catalytic center was substituted by treatment with CuCl2 or NiCl2, proteolytic and hemagglutinating activities were reduced by Ni substitution but not by Cu substitution. Cu‐treated 35‐kDa polypeptide showed sufficient affinity of the catalytic center and weak binding ability to the erythrocyte membrane, but the Ni‐treated polypeptide did not. These results suggest that the binding of endoproteinase domain to membrane is also necessary for hemagglutination.
Expression of Lymphotoxin Gene Inserted into Theiler's Murine Encephalomyelitis Virus
Authors: Obuchi Masatsugu , Odagiri Takato , Lizuka Hideaki , Ohara Yoshiro
Published: November 2013

Theiler's murine encephalomyelitis virus (TMEV) belongs to the Picornaviridae genus. DA subgroup strains of this virus induce early, non‐fatal polioencephalomyelitis followed by demyelination in the spinal cord, with virus persistence. We investigated the use of DA strain as a vector for the introduction of a foreign gene into the central nervous system. Human lymphotoxin (LT) gene was inserted in the L region, the most upstream part of the polyprotein coding region of DA genome. Expression of LT was demonstrated by an immunoblot and an enzyme‐linked immunosorbent assay on BHK‐21 cells that were infected with the recombinant virus. In addition, the expressed LT showed cytotoxicity against L‐929 cells.
Protective Immunity Induced by Vaccination with SAG1 Gene‐Transfected Cells against Toxoplasma gondii‐Infect ion in Mice
Authors: Aosai Fumie , Mun Hye‐seong , Norose Kazumi , Chen Mei , Hata Hidekazu , Kobayashi Masashi , Kiuchi Masahiro , Stauss Hans J. , Yano Akihiko
Published: November 2013

To develop a vaccine by augmenting the protective cellular immunity against Toxoplasma gondii (T. gondii)‐infection, T. gondii SAG1 gene‐transfectants were established by using RMA.S (H‐2b), a murine transporter associated with the antigen processing (TAP) molecule‐deficient lymphoma line, as a host antigen‐presenting cell (APC). Immunization of C57BL/6 mice with the SAG1‐transfected RMA.S induced CD8+ cytotoxic T lymphocytes (CTL) specific for not only SAG1‐transfected RMA.S but also T. gondii‐infected RMA.S, and elicited protective responses to infection with a virulent T. gondii strain, RH.
The Effects of S‐Carboxymethylcysteine and N‐Acetylcysteine on the Adherence of Moraxella catarrhalis to Human Pharyngeal Epithelial Cells
Authors: Zheng Can Hong , Ahmed Kamruddin , Rikitomi Naoto , Martinez Glenda , Nagatake Tsuyoshi
Published: November 2013

We investigated the effects of two mucoregulating drugs, S‐carboxymethyleysteine (S‐CMC) and N‐acetylcysteine (NAC), on the attachment of Moraxella catarrhalis (M. catarrhalis) to pharyngeal epithelial cells. The attachment of M. catarrhalis decreased (3357%) significantly (P<0.01) in a dose‐dependent manner in cells treated with mucoregulating drugs as compared to the control. There was a significant (P< 0.01) decrease (35 45%) in the attachment of M. catarrhalis to pharyngeal cells after oral administration of S‐CMC. By electron microscopic observation, it was found that there was a fine, granular, electron‐dense, ruthenium red‐positive layer on the surface of pharyngeal epithelial cells; this layer was absent on cell surfaces treated with mucoregulating drugs. Possibly, this layer contained the portion of M. catarrhalis receptor which is responsible for the attachment of this bacteria to pharyngeal epithelial cells. From the above results, it may be concluded that one of the mechanisms of mucoregulating drugs to decrease the episode of respiratory infections in patients with chronic respiratory diseases is by inhibiting the attachment of bacteria to the upper respiratory tract.
Genetic Relationships among Mycoplasmas Based on the 16S‐23S rRNA Spacer Sequence
Authors: Harasawa Ryô
Published: November 2013

The nucleotide sequences of the spacer regions between the 16S and 23S rRNA genes of 20 Mycoplasma species were determined following amplification by PCR. Although the spacer regions lacked spacer tRNA genes, they contained the box B and box A sequences in this order from the 5′ terminus. The sequence alignment indicated that the 20 species were divided into four clusters, the M. pneumoniae, M. hominis, M. hyorhinis and M. fermentans clusters, and a single floating species, M. hyopneumoniae.