bluuebunny/crossref_metadata_2025 huggingface dataset:
A Unique Mutation of Glycoprotein Gene of the Attenuated RC‐HL Strain of Rabies Virus, a Seed Virus Used for Production of Animal Vaccine in Japan
Authors: Ito Hiroshi , Minamoto Nobuyuki , Watanabe Tomiyoshi , Goto Hideo , Rong Luo Ting , Sugiyama Makoto , Kinjo Toshio , Mannen Kazuaki , Mifune Kumato , Konobe Takeo , Yoshida Iwao , Takamizawa Akihisa
Published: November 2013

Although the RC‐HL strain of rabies virus is avirulent in adult mice, the amino acid at position 333 of its G protein is arginine, which is thought to be necessary for virulence in adult mice upon intracerebral inoculation of the virus. This result suggests that besides arginine at position 333, some other positions of G protein might also be involved in determining the virulence of rabies virus.
Detection of Human Immunodeficiency Virus‐1 Nucleic Acid on Inactivated Filter Paper Disks by Polymerase Chain Reaction and Microtiter Plate Assay
Authors: Kunisada Takao , Ando Shuji , Saito Kunihiro , Eshita Yuki , Röder Wolfgang , Kruse Michael , Müller Werner E. G. , Ushijima Hiroshi
Published: November 2013

Human immunodeficiency virus type 1 (HIV‐1) in cultured cells, peripheral blood samples and sera were adsorbed on filter paper disks and inactivated by heat or ethanol. Two procedures, the polymerase chain reaction (PCR) and microtiter plate assay (HMPA) were used to detect the nucleic acid. The sensitivity after different heat treatments with nested PCR for HIV‐1 DNA (or nested reverse transcription‐PCR for HIV‐1 RNA) was identical regardless of whether the samples were examined immediately or one month later. Inactivation by ethanol treatment resulted in a slight loss of sensitivity. The HMPA proved to be as reliable and specific as the conventional PCR technique. We conclude that the heat‐treated filter paper disk assay is suitable for identifying HIV nucleic acid in clinical samples sent to the laboratory from a distance, e.g. in an envelope.
Binding of Host‐Associated Treponeme Proteins to Collagens and Laminin: A Possible Mechanism of Spirochetal Adherence to Host Tissues
Authors: Umemoto Toshihiko , Namikawa Isamu
Published: November 2013

The polypeptides of seven strains of human treponemes were investigated by immunoblot analysis for their binding to the human placental collagens and laminin. Of the treponemal polypeptides, eleven polypeptides, 45‐kDa, 49‐kDa, and 62‐kDa polypeptides from T. pallidum ATCC 27087, a 48‐kDa polypeptide from T. phagedenis biotype Reiter, 51‐kDa and 53‐kDa polypeptides from T. vincentii ATCC 35580, 30‐kDa, 53‐kDa and 63‐kDa polypeptides from T. socranskii subsp. buccale ATCC 35534, a 52‐kDa polypeptide from T. denticola ATCC 35405, and a 53‐kDa polypeptide from T. denticola ATCC 33520 possessed an ability to bind to the laminin, type I, III, IV, or V collagen. An intermediate‐sized human oral isolate strain G7201 did not possess any laminin‐ or collagen‐binding polypeptides. Immunoelectron microscopy using intact treponemal cells with a single collagen‐binding polypeptide and the corresponding antisera demonstrated that the 51‐kDa and 53‐kDa polypeptides from T. vincentii, the 53‐kDa polypeptide from T. socranskii subsp. buccale ATCC 35534 and the 52‐kDa polypeptide from T. denticola ATCC 35405, were outer envelope proteins.
Localization of Apoptosis (Programmed Cell Death) in Mice by Administration of Lipopolysaccharide
Authors: Zhang Xian‐min , Morikawa Akiko , Takahashi Kazuko , Jiang Guo‐zhi , Kato Yutaka , Sugiyama Tuyoshi , Kawai Makoto , Fukada Masako , Yokochi Takashi
Published: November 2013

Localization of apoptotic cells by administration of lipopolysaccharide into mice was studied by using the in situ specific labeling of fragmented DNA. This method clearly stained the nuclei of thymocytes at the cortex of the thymus. The nuclei of cells in the bone marrow and in the spleen were also positively stained. It was suggested that the cortex in the thymus is where the LPS‐induced programmed cell death occurs.
Detection and Typing of Human Rotavirus in Reference to Repeated Acute Gastroenteritis in Infants
Authors: Imamura Yoshihiro , Hamada Nobuyuki , Nagai Takayuki , Shingu Masahisa
Published: November 2013

Stool specimens from infants who visited a clinic because of acute gastroenteritis were tested for the presence of human rotavirus. Among the samples obtained were specimens taken from seven patients who had visited the clinic at two different times. In six of these seven children, human rotavirus (HRV) was detected in only one of the specimens taken (i.e. during only one of the two visits). One patient was shown to have excreted HRV twice; in both cases the HRV was serotyped to be type 1. The present results indicate that the symptomatic reinfection of HRV was not a widely occurring phenomenon in the group of infants tested.
Augmentation of Host Resistance against Bacterial Infection by Treatment with Leustroducsin B, a New CSF Inducer
Authors: Kohama Takafumi , Katayama Toshiaki , Inukai Masatoshi , Maeda Hiroaki , Shiraishi Akio
Published: November 2013

We tested the in vivo activity of leustroducsin B (LSN B), a new colony‐stimulating factor (CSF) inducer isolated from the culture broth of Streptomyces platensis, with mice infected with Escherichia coli. Treatment with LSN B augmented the host resistance to lethal infection of E. coli at doses between 0.1 mg/kg and 1 mg/kg. Serum interleukin‐6 (IL‐6) levels were found to increase after this treatment, and superoxide anion generation of neutrophils was enhanced in vivo, suggesting that LSN B augmented the host resistance at least in part by inducing IL‐6, which subsequently enhanced the bactericidal activity of the neutrophils.
Enterotoxigenicity, Hemolytic Activity and Antibiotic Resistance of Aeromonas Spp. Isolated from Freshwater Prawn Marketed in Dhaka, Bangladesh
Authors: Rahim Zeaur , Aziz Khwaja Muhammed Sultanul
Published: November 2013

Aeromonas spp. were isolated from gills, swimmerets, eggs, stomachs and ventral muscles of freshwater prawns (Macrobrachium malcolmsonii) available in the local fish market of Dhaka, Bangladesh. The density of Aeromonas spp. on these different body parts of the prawn samples ranged from 1.1±0.2 × 104 to 1.5 ± 0.16 × 107 cfu per gram. The viable counts of aeromonads, fecal coliforms (FC) and Escherichia coli gradually increased in prawn samples when stored at 4 C. At −20 C, the viable counts gradually decreased and became zero on the 12th day of storage. The isolation of A. sobria (56%) was more frequent than that of A. hydrophila (31%) and A. caviae (13%). In the rabbit ileal loop (RIL) model, fluid accumulation induced by live cultures and cell‐free culture filtrates of 11 strains ranged from 0.5 to 1.5 and 0.5 to 1.7 ml/cm of gut, respectively. Of 11 enterotoxigenic strains, 7 were A. sobria and 4 were A. hydrophila. Enterotoxigenicity correlated with hemolytic activity on blood agar. All enterotoxigenic strains were uniformly sensitive to chloramphenicol and gentamicin and resistant to novobiocin and vancomycin. Isolation of enterotoxigenic and antibiotic‐resistant Aeromonas from these prawn samples indicates possible public health problems for their handlers as well for raw prawn consumers.
In Vivo‐Like Antigenic Surface Properties of Staphylococcus aureus from Bovine Mastitis Induced upon Growth in Milk Whey
Authors: Mamo Wubshet , Fröman Gunnar
Published: November 2013

Antigenic surface properties of Staphylococcus aureus strains grown in milk whey were compared with TSB‐grown bacteria using immuno‐gold electron microscopy. It is shown that colloidal gold (CG) particles coated with polyclonal antibody raised against Staphylococcus aureus surface antigen expressed in vivo bound to the surface of S. aureus strain F1440 grown in milk whey, but not to homologous bacteria grown in TSB. S. aureus strains grown in milk whey agglutinated in the presence of the polyclonal antibody, whereas the corresponding bacteria grown in TSB did not agglutinate. Immuno‐gold particles did not bind to milk whey‐grown bacteria treated with periodate. Periodate‐treated milk whey‐grown bacteria did not agglutinate in the presence of the polyclonal antibody, whereas periodate treatment had no effect on TSB‐grown bacteria.
A Novel Assay System for Anti‐Human Immunodeficiency Virus Type 1 (HIV‐1) Activity Using a Subclone of a Monocytic Cell Line, U937
Authors: Miyano‐Kurosaki Naoko , Nakashima Hideki , Ichiyama Koji , Inazawa Kazuhiko , Tabata Hidenori , Tanabe Hideyuki , Ohnishi Kiyotaka , Mizusawa Hiroshi , Ohshiro Yukako , Yamamoto Naoki
Published: November 2013

A subclonal cl.1–14 cell was established from a monocytic cell line U937 by a limiting dilution method. The anti‐HIV‐1 activity of some antiviral compounds was evaluated in HIV‐1‐infected cl.1–14 cells. The results demonstrated that although AZT was a potent inhibitor of HIV‐1 replication in cl.1–14 cells, its 50% effective concentration (EC50) values was 80 times higher than that in HIV‐1 infected MT‐4 cells; the EC50 of AZT was 0.16 μM and 0.002 μM in cl.1–14 and MT‐4 cells, respectively. In contrast, the anti‐HIV‐1 activity of ddA, ddI and ddC in cl.1–14 cells was comparable to that in MT‐4 cells. The antiviral activity of nevirapine, dextran sulfate, curdlan sulfate and T22 did not differ significantly between the cl. 1–14 and MT‐4 cells. The antiviral activity of several compounds in the HIV‐1‐infected cl.1–14 cells was similar to that in the HIV‐1jr‐fl‐infected human peripheral macrophages. Our results suggest that cl.1–14 cell cultures are very useful for estimating antiviral activity and more advantageous than the use of peripheral blood macrophages.
Lipofection of Synthetic Oligodeoxyribonucleotide Having a Palindromic Sequence of AACGTT to Murine Splenocytes Enhances Interferon Production and Natural Killer Activity
Authors: Yamamoto Toshiko , Yamamoto Saburo , Kataoka Tetsuro , Tokunaga Tohru
Published: November 2013

A synthetic 22‐mer oligodeoxyribonucleotide having an AACGTT palindrome, AAC‐22, induced interferon (IFN) production and augmented the natural killer (NK) activity in murine splenocytes, whereas its analogue, ACC‐22, having an ACCGGT palindrome, did not. The binding of AAC‐22 to splenocytes was not different from that of ACC‐22. Lipofection of AAC‐22 to splenocytes remarkably enhanced IFN production and NK cell activity, whereas that of ACC‐22 caused little enhancement. These results strongly suggest that the prerequisite for IFN production is not the binding of AAC‐22 to the cell surface receptors, but its penetration into the spleen cells.
Alteration of Immune Responses of Rabbits Infected with Bovine Immunodeficiency‐Like Virus
Authors: Hirai Nobuaki , Xuan Shiwei , Ochiai Kenji , Onuma Misao
Published: November 2013

Nine 3‐month‐old rabbits were inoculated with bovine immunodeficiency‐like virus (BIV) to study the pathogenesis of BIV and alteration of the immune responses in experimentally infected rabbits. BIV proviral DNA and anti‐BIV antibodies were detected from all rabbits inoculated with BIV‐infected bovine embryo spleen (BESP) cells. Rabbits inoculated with spleen cells of the BIV‐infected rabbit also converted to proviral DNA‐positive and BIV‐antibody‐positive. The blastogenic responses to concanavalin A of peripheral blood mononuclear cells prepared from BIV‐infected rabbits were not significantly different from those from uninfected controls at 2 and 4 months post‐inoculation (PI). The humoral immune responses against bovine serum albumin (BSA) were depressed in two of four BIV‐infected rabbits at 1 to 3 months PI. The antibody responses against sheep red blood cells (SRBCs) were significantly depressed in all BIV‐infected rabbits at 2 to 4 months PI. BIV was rescued by cocultivation of spleen cells of infected rabbits with BESP cells. Distinct development of lymphoid follicle was observed in lymph nodes and spleens of uninfected rabbits which received BSA and SRBCs. In contrast, moderate lymphoid cell depletion was observed in BIV‐infected rabbits which received the same immunogens.
Mechanism for Macrophage Activation against Corynebacterium parvum—Participation of T Cells and Its Lymphokines
Authors: Mori Hiroshi , Mihara Masahiko , Uesugi Yuki , Nagai Hiroichi , Koda Akihide
Published: November 2013

It is well known that Corynebacterium parvum activates macrophages to produce tumor necrosis factor (TNF). It is suspected that the activation of macrophages by C. parvum requires T‐cell participation. The purpose of this study was to confirm that T cells participate in the activation of macrophages by C. parvum. TNF production in vitro from the spleen cells of BALB/c‐ + / + mice was abrogated completely by the pre‐treatment of spleen cells with anti‐Ia antiserum and complement, indicating that Ia+ cells are the source of TNF. TNF production was not elicited at all in BALB/c‐nu/nu mice. However, there was an increase in the number of Ia+ cells as well as an increase in the weight of spleen and liver. Supernatant from a culture of spleen cells stimulated with phytohemagglutinin‐P (a PHA‐induced lymphokine) made it possible for BALB/c‐nu/nu mice to produce TNF, associated with an induction of Lyt‐1+ cells and Lyt‐2+ cells. However, treatment with the lymphokine did not augment the increases of Ia+ cells or liver and spleen weights. These results suggest that increasing the number of Ia+ cells is not sufficient to bring about TNF production; Ia+ cells must also be stimulated by T cells or T‐cell lymphokines in order to produce TNF. These results suggest that T cells play an essential role in the activation of Ia+ cells against C. parvum.
Epitope Analysis Using Anti‐Oligosaccharide (G4) Monoclonal Antibody
Authors: Hirata Akio , Komoda Makiko , Itoh Wataru , Tabata Kengo , Sugawara Isamu
Published: November 2013

A murine monoclonal antibody recognizing (1→6)‐β‐d‐glucopyranosyl laminaritriose (G4) was prepared by immunizing BALB/c mice with G4‐bovine serum albumin conjugate and fusing the splenocytes with mouse myeloma cells. The monoclonal antibody (IgM) provoked by the cloned cells showed low reactivity with schizophyllan, an antitumor polysaccharide, but notable reactivity with some low‐molecular‐weight schizophyllans. This antibody was useful for determination of the epitope of several polysaccharides. The extent of reactivity of this monoclonal antibody was related only to the molecular weight of schizophyllan.
A Gene Encoding a Cytochrome c Oxidase‐Like Protein Is Located Closely to the Cytochrome c‐553 Gene in the Anaerobic Bacterium, Desulfovibrio vulgaris (Miyazaki F)
Authors: Kitamura Masaya , Mizugai Kazuya , Taniguchi Masahiro , Akutsu Hideo , Kumagai Izumi , Nakaya Tadao
Published: November 2013

The gene encoding cytochrome c‐553 from Desulfovibrio vulgaris (Miyazaki F) was cloned using a synthetic oligodeoxyribonucleotide probe. The nucleotide sequence indicated that cytochrome c‐553 was synthesized as a precursor protein with an NH2‐terminal signal sequence of 23 residues. In the cloned DNA fragment, there are three other open reading frames whose products have 191, 157, 541 amino acid residues, respectively. The putative ORF‐4 product is highly homologous with the cytochrome c oxidase subunit I from various organisms.
Increased Expression of Adhesion Molecules (CD54, CD29 and CD44) on Fibroblasts Infected with Cytomegalovirus
Authors: Ito Masahiro , Watanabe Masahiro , Ihara Toshiaki , Kamiya Hitoshi , Sakurai Minoru
Published: November 2013

The expression of ICAM‐1 (CD54), β1 integrin (CD29), and CD44 on cytomegalovirus (CMV)‐infected human embryonic fibroblasts (HEF) was analyzed by flow cytometry. The expression of these adhesion molecules increased significantly on CMV‐infected HEF, on days 2 and 5 after inoculation, compared to uninfected HEF. However, the expression of these adhesion molecules decreased on herpes simplex virus (HSV)‐1 and varicella‐zoster virus (VZV)‐infected HEF. Increased expression was not observed on HEF treated either with inactivated CMV or with supernatant fluid of CMV‐infected cells. The addition of anti‐cytokine (TNF‐α, IL‐1β, or IFN‐γ) antibodies had no effect on the increase of these adhesion molecules. This suggests that the increase in CD54, CD29, and CD44 on CMV‐infected cells requires active virus replication and was not mediated by a soluble factor released from CMV‐infected cells. Changes in adhesion molecules on CMV‐infected fibroblasts may contribute to inflammation induced by CMV infection.
Characterization of a Cloned pR72H Probe for Vibrio parahaemolyticus Detection and Development of a Nonisotopic Colony Hybridization Assay
Authors: Lee Chia‐yin , Chen Chien‐hsien , Chou Yi‐wen
Published: November 2013

Vibrio parahaemolyticus is a halophilic bacterium often found in shellfish and is an important causative agent of food poisoning in Taiwan. A rapid and efficient detection method is required to identify this foodborne pathogen. A 0.76‐Kb HindIII DNA fragment was cloned from the chromosomal DNA of V. parahaemolyticus strain no. 93, designated as pR72H fragment, was used as a polynucleotide probe. It was labeled with digoxigenin‐11‐dUTP (DIG) by the random primer‐labeling method. The sensitivity and specificity of the digoxigenin‐labeled 0.76‐Kb DNA probe was determined by colony hybridization assay. Under stringent hybridization conditions, 122 of 124 isolates of V. parahaemolyticus showed positive hybridization reaction with DIG‐0.76‐Kb DNA probe; the negative strains were attributed to slow growth. The DIG‐0.76‐Kb probe did not hybridize with 86 isolates of other vibrios and a number of other enterics as well as nonenteric microorganisms. The sensitivity and specificity of this DIG probe are 98% and 100%, respectively. This nonisotopic colony hybridization assay can be very useful for routine monitoring of V. parahaemolyticus in the food industry, environmental analysis and clinical laboratories.
Lethal and Dermonecrotic Activities of Clostridium perfringens Iota Toxin: Biological Activities Induced by Cooperation of Two Nonlinked Components
Authors: Sakurai Jun , Kobayashi Keiko
Published: November 2013

The effect of separate injections of two components of Clostridium perfringens iota toxin, designated Ia and Ib components, on the biological activities of the toxin was investigated. The intravenous injection of one component within 120 min after the injection of the other component killed mice. The activity of iota toxin was abolished by anti‐Ia or anti‐Ib antiserum. On the other hand, when Ib component was intravenously administered to mice given anti‐Ia antiserum within 120 min after the intravenous injection of Ia component, the lethal activity was completely neutralized, but when Ia component was injected into mice that were given anti‐Ib antiserum over 5 min after the injection of Ib component, the activity was not neutralized. The separate injections of Ia and Ib components in skin of guinea pigs indicated dermonecrosis at the injection site of Ib components, but not at the site of Ia components. Furthermore, when one component was intradermally injected in guinea pigs and then the other intraperitoneally, the dermonecrotic activity of the toxin was observed at the intradermal injection site of Ib component, but not at that of Ia component. From the data, it appears that the lethal and dermonecrotic activities of iota toxin are initiated by the binding of Ib component to specific sites on tissues.
Isolation of Influenza A and B Viruses in HeLa Cells
Authors: Yamanaka Takaya , Shirasawa Hiroshi , Yoshizawa Shigekatsu
Published: November 2013

The HeLa cell line which is one of the most popular cell lines was shown to be suitable for isolation of types A (H3N2) and B influenza viruses from throat washings of patients. Sixty‐nine and 67 out of 147 throat washings taken from patients during the period from January to April, 1994, were positive for influenza A virus in HeLa cells and MDCK cells, respectively. Seven out of 10 throat washings taken between January and March, 1993, were positive for influenza B virus in MDCK. Of these 7, 4 were also positive for HeLa cells.
Prevalence of Antibodies to Borrelia Spirochetes among Wild Small Rodents in Central and Western Japan
Authors: Ishiguro Fubito , Takada Nobuhiro , Yano Yasuhiro , Harada Masashi , Masuzawa Toshiyuki , Iida Hidetada
Published: November 2013

Field rodents serve as a reservoir for Lyme disease spirochetes. To evaluate the antibody responses of rodents against different Borrelia species in relation to fauna of vector ticks feeding on them, we examined 272 sera of wild rodents, Apodemus speciosus, A. argenteus, and Eothenomys smithii, obtained in 27 locations in central and western Japan from 1981 to 1994. As to prevalences by rodent species using immunoperoxidase test, A. speciosus, A. argenteus and E. smithii showed 29.4%, 11.6% and 30.8% reactivity to Borrelia japonica, 10.7%, 7.2% and 3.8% to B. afzelii, 0.6%, 1.4% and 0% to B. garinii, and 14.7%, 7.2% and 11.5% to an unknown Borrelia species designated as It type, respectively. Each antibody to B. japonica, B. afzelii and B. sp. It type was detected widely both in central and western Japan, but the antibody to B. garinii was scarcely detectable in any area and rodent species examined. Apodemus mice in high mountain altitudes tend to have antibody to B. afzelii or B. japonica, and those in lower altitudes tend to have B. japonica or B. sp. It type. All 13 Apodemus mice from which B. japonica or B. sp. It type were isolated showed higher titers of antibodies to each homologous Borrelia species. The present results indicate that these antibody prevalences among rodents may be associated with dominant Ixodes ovatus and sporadic I. persulcatus on the mainland of Japan, and that Apodemus mice may not be an efficient reservoir for B. garinii. Such a serosurvey is a useful measure to evaluate the natural distribution of the pathogen.
Salt‐Induced L‐Forms of Staphylococcus aureus
Authors: Yabu Kunihiko
Published: November 2013

When growing cultures of a salt‐sensitive strain of Staphylococcus aureus were inoculated on nutrient agar containing 0.8 m NaCl and 0.5% bovine serum albumin, typical colonies of L‐form developed extensively after 2 days of incubation at 30 C. Incubation of growing cultures with lipoteichoic acid, sodium polyanethole sulfonate and subtilisin resulted in inhibition of L‐form induction.
Characterization of Pseudomonas paucimobilis FP2001 Which Forms Flagella Depending upon the Presence of Rhamnose in Liquid Medium
Authors: Miake Fumio , Murata Kunihiko , Kuroiwa Ataru , Kumamoto Toshihiko , Kuroda Shinji , Terasawa Tadashi , Tone Hiroshi , Watanabe Kenji
Published: November 2013

A bacterial strain FP2001 isolated from the exudate of land reclaimed for municipal waste was identified as Pseudomonas paucimobilis. Cells of strain FP2001 were mobile by means of polar monotrichous flagellum, only when rhamnose was added as a carbon source in the liquid medium. The replacement of rhamnose by arabinose, galactose, glucose or xylose did not lead to the formation of flagella.
Overproduction, Purification, and Diagnostic Use of the Recombinant HIV‐1 Gag Proteins, the Precursor Protein p55 and the Processed Products p17, p24, and p15
Authors: Saito Atsushi , Morimoto Motoko , Ohara Takeshi , Takamizawa Akihisa , Nakata Atsuo , Shinagawa Hideo
Published: November 2013

HIV‐1 Gag protein precursor p55, and its processed products, p17, p24, and p15 were overproduced in Escherichia coli and purified to near homogeneity. To study the antigenic properties and the potentiality as the diagnostic and prognostic reagents, varying amounts of the purified Gag proteins were dotted onto the polyvinylidene difluoride membrane and reacted with 40 sera of HIV‐1‐infected individuals (35 AC, 1 ARC, and 4 AIDS patients) and 10 sera of normal healthy donors. p55 reacted with 40 (100%) sera of HIV‐1 carriers, while p17, p24, and p15 reacted with 37 (92.5%), 35 (87.5%) and 34 (85%) of the 40 sera of HIV‐1 carriers, respectively. On the whole, the reaction of p55 was especially strong and that of p15 was the weakest. p55 showed the strongest reaction among the four Gag proteins with all specimens, and it showed a positive reaction with a carrier serum with which none of the processed Gag proteins showed a positive reaction. Therefore, p55 is the most useful antigen among the four Gag proteins for detection of the Gag antibodies and may even be one of the most useful antigens for the diagnosis of HIV‐1 infection.
Interference between Neisseria meningitidis and PC‐KLH Induced Anti‐Phosphorylcholine PFC Responses in NZB/W Autoimmune Mice
Authors: Seoane Rafael , Eiras Adolfo , Quireza Otilia , Cancio Esperanza , Aguilera Antonio , Puentes Eugenia
Published: November 2013

In this work, we demonstrate that the simultaneous injection of PC‐KLH and Neisseria meningitidis‐derived antigens [NMB or PC‐(NMB)HI] induced in old NZB/W mice defective responses as does PC‐KLH challenge. On the other hand, the simultaneous injection of both immunogenic preparations of N. meningitidis evoked responses similar to those shown by old mice challenged with NMB alone. Alteration in PC‐specific PFC responses also affected hapten‐free inhibition profiles and their heterogeneities. The increase in PC50s of anti‐phosphorylcholine PFC responses and their heterogeneities induced by certain antigens with aging is correlated with a decrease in T15 idiotype expression, suggesting that after the T15 dominant clone disappears no other clone takes control of the anti‐PC response. These results suggest that the mechanism(s) involved in the regulation of T15 marker expression play an important role in the inability of old NZB/W mice to mount good anti‐PC responses and suggest that regulatory mechanisms induced by PC‐KLH dominate those elicited by NMB.
Analysis of the NH2‐Terminal 87th Amino Acid of Escherichia coli GyrA in Quinolone‐Resistance
Authors: Yonezawa Minoru , Takahata Masahiro , Banzawa Naoko , Matsubara Nobuyuki , Watanabe Yasuo , Narita Hirokazu
Published: November 2013

The functional contributions of amino acid residue Asp87 of Escherichia coli gyrase A protein (GyrA) was analyzed by site‐directed mutagenesis. We generated a series of mutants, in which Asp87 of GyrA was changed to Ala, Val, Phe, Asn, Ser, and Lys. By genetic analysis of gyrA genes in a gyrA temperature‐sensitive (Ts) background, it was shown that all these mutations caused the quinolone‐resistance. These results indicate that the 87th amino acid of E. coli GyrA must have negative charge in expressing the phenotype of quinolone sensitivity. These findings also suggest that the carboxyl group of Asp87 may interact with quinolone drugs.
Nucleotide Sequence of the Gene Encoding β‐Isopropylmalate Dehydrogenase (leuB) from Bacteroides fragilis
Authors: Sarker Mahfuzur R. , Akimoto Shigeru , Ugai Hideyo , Kuwahara Tomomi , Ohnishi Yoshinari
Published: November 2013

The complete nucleotide sequence of the gene (leuB) coding for β‐isopropylmaiate dehydrogenase of Bacteroides fragilis was determined. An open reading frame of 1,061 nucleotides was detected that could encode a polypeptide of 353 amino acid residues with a calculated molecular mass of 39,179 Da. The deduced amino acid sequence of the β‐isopropylmalate dehydrogenase from B. fragilis showed substantial sequence similarity with the β‐isopropylmalate dehydrogenases from other bacteria.
Distribution of C22‐, C24‐ and C26‐α‐Unit‐Containing Mycolic Acid Homologues in Mycobacteria
Authors: Kaneda Kenji , Imaizumi Sadao , Yano Ikuya
Published: November 2013

There are three mycolic acid homologues with C22‐, C24‐ and C26‐α‐units in Mycobacterium. In order to reveal the composition and distribution of these homologues in each subclass and molecular species of mycolic acids and to compare them with the composition of constitutive non‐polar fatty acids (free and bound forms), we have separated non‐polar fatty acids and each subclass of mycolic acids from 21 mycobacterial species by thin‐layer chromatography, and analyzed non‐polar fatty acid methyl esters by gas chromatography (GC) and the cleavage products of methyl mycolate by pyrolysis GC. We further performed mass chromatographic analysis of trimethylsilyl (TMS) ether derivatives of mycolic acid methyl esters by monitoring [B‐29]+ ions (loss of CHO from the α‐branched‐chain structure of mycolic acids) of m/z 426, 454 and 482 which are attributed to C22‐, C24‐ and C26‐α‐units of TMS ether derivatives of methyl mycolates, respectively, (Kaneda, K. et al, J. Clin. Microbiol. 24: 1060‐1070, 1986). By pyrolysis GC, C22:0, C24:0 and C26:0 fatty acid methyl esters generated by the C2‐C3 cleavage of C22‐, C24‐ and C26‐α‐unit‐containing mycolic acid methyl esters, respectively, were detected. Their proportion was almost the same among subclasses of mycolic acids in every Mycobacterium and also similar to the proportion of constitutive non‐polar C22:0, C24:0 and C26:0 fatty acids. By mass chromatography, the composition and distribution of C22‐ and C24‐α‐unit‐containing homologues were revealed to be similar between α‐ and α'‐mycolic acids in every Mycobacterium. We further analyzed in detail M. vaccae and demonstrated that the mass chromatogram of C22‐α‐unit‐containing homologue was analogous in shape to that of the C24‐α‐unit‐containing one, with the latter mass chromatogram being up‐shifted from the former by two carbon numbers, in every subclass of α‐, α'‐, keto and dicarboxy mycolic acids. The present study suggests that the compositions of three homologues of both mycolic acids and constitutive non‐polar fatty acids, which are characteristic to each mycobacterial species, may reflect the proportion of the amount of free C22:0, C24:0 and C26:0 fatty acids synthesized in the cell. It is further demonstrated that intermolecular condensation of two fatty acids which become α‐ and β‐units of mycolic acids will occur independently of the carbon chain length or kinds of polar moieties of fatty acids.
Seasonal Distribution of Adenoviruses, Enteroviruses and Reoviruses in Urban River Water
Authors: Tani Naoto , Dohi Yoshiko , Kurumatani Norio , Yonemasu Kunio
Published: November 2013

In the 63‐month period from January 1988 to March 1993, monthly levels of adenoviruses, enteroviruses (coxsackie B, polio, echo) and reoviruses in the urban river water in Nara Prefecture, Japan were in the range 0‐25, 0‐190 and 0‐325, plaque forming units per liter (PFU/liter), and the average levels were 2.4, 40.6 and 56.2 PFU/liter, respectively. The peak reovirus level was found in winter during the cold weather months (Nov. to Mar.). The peak enterovirus level was found in summer (May to Sept.) but continued to be found in autumn‐winter (Oct. to Jan.) from 1991 to 1993. The levels of adenoviruses were low throughout all 5 years, as compared to those of reoviruses and enteroviruses. Polioviruses were isolated following the administration of vaccine. Although a changing pattern of serotype prevalence was seen with the coxsackie B viruses and echoviruses from 1988 to 1993, this is not so for polioviruses, which remained almost unchanged for the five‐year period. Adenoviruses were isolated throughout all five years, though in small numbers. Reoviruses were isolated most frequently throughout five years.
Down‐Regulation of T‐Cell Proliferation in Response to Soluble Anti‐CD3 Antibodies through Development of Redirected Cytolytic Activity Eliminating Costimulatory Cells
Authors: Lwin Tint , Nakashima Izumi , Nagase Fumihiko
Published: November 2013

CD4+ T‐depleted spleen cells (CD8+ T cells) activated by anti‐CD3 antibodies (aCD3) suppressed proliferation of CD8+ T‐depleted spleen cells (CD4+ T cells) and fresh normal T cells in response to aCD3. Antigen‐nonspecific cytolytic activity was induced in splenic CD8+ T cells by stimulation with aCD3 and showed the peak level on day 3, whereas cytolytic activity induced in CD4+ T cells was weak. Intact Ig but not F(ab')2 of aCD3 induced and mediated cytolytic activity. Correspondingly, the cytolytic activity induced by aCD3 was directed against target cells bearing Ig‐binding Fc‐receptor activity and cytolysis was inhibited by the addition of free Ig into the assay system. We showed that aCD3‐activated T cells carried a high level of aCD3 on their surface at the time after the peak proliferation when they attained high cytolytic activity. This raised the possibility that the anti‐CD3‐induced aCD3‐redirected cytolytic activity eliminated Fc‐receptor‐bearing costimulatory cells in the culture for down‐regulation of the T‐cell proliferation. This view was supported by partial restoration of anti‐CD3‐induced low responsiveness of CD8+ T cells by the addition of fresh costimulatory cells. These results suggested a new pathway of down‐regulation of T‐cell proliferation by aCD3‐activated cytolytic CD8+ T cells.
Auxiliary Method for Clonal Identification of Staphylococcus aureus by Protein Band Pattern of Released Proteins on SDS‐Polyacrylamide Gel
Authors: Seki Keiko , Sakurada Junji , Murai Miyo , Usui Akemi , Seong Hee Kyong , Jitsukawa Hiroko , Masuda Shogo
Published: November 2013

A supportive method for clonal identification of Staphylococcus aureus strains was devised. Culture supernatant obtained by cellophane surface culture was subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) without performing any concentration procedure prior to electrophoresis. The combined use of cellophane surface culture and SDS‐PAGE was convenient for determining whether the strains belonged to the same clone or not when conducted in conjunction with other tests for bacteriological characterization.
Inhibition by Bacterial Lipopolysaccharide of Spontaneous and TNF‐α‐Induced Human Neutrophil Apoptosis In Vitro
Authors: Hachiya Osamu , Takeda Yuji , Miyata Hiroaki , Watanabe Hiroshi , Yamashita Takao , Sendo Fujiro
Published: November 2013

In the previous paper (Takeda et al, Int. Immunol., 5, 691‐694, 1993), we demonstrated that tumor necrosis factor‐α (TNF‐α) promptly accelerates apoptosis of human neutrophils in vitro. In order to determine the role of neutrophil apoptosis in defending against bacterial infection, we studied the effect of bacterial lipopolysaccharide (LPS) on this process. LPS inhibited spontaneous and TNF‐α‐induced human neutrophil apoptosis in vitro, as determined by 1) light and electron microscopy, 2) flow cytometry, and 3) agarose gel electrophoresis of DNA. Low concentrations of cycloheximide, a protein synthesis inhibitor, which alone did not affect neutrophil apoptosis, were able to reduce spontaneous apoptosis inhibition by LPS, suggesting the involvement of newly synthesized protein in this phenomenon.
Subcutaneous Growth of Staphylococcus aureus Concomitantly Inoculated with Ehrlich Ascites Tumor Cells
Authors: Murai Miyo , Seki Keiko , Sakurada Junji , Usui Akemi , Masuda Shogo
Published: November 2013

Intratumoral growth of Staphylococcus aureus Cowan I‐derived AP332 was examined by subcutaneous inoculation of cocci in doses ranging from 18 to 1.8 × 105 CFU with Ehrlich ascites tumor cells. Inoculation of 18 CFU AP332 resulted in staphylococcal growth in one of five mice, and the proportion of mice established intratumoral infection increased with the initial inocula. Six other strains of S. aureus also grew in the tumor tissue, and none of the three strains of coagulase‐negative staphylococci grew at all. Ethanol‐killed tumor cells did not promote staphylococcal growth as vigorously as the live tumor cells, especially when the initial inoculum of AP332 was smaller than 104 CFU.
Identification of Influenza C Virus Phosphoproteins
Authors: Nishimura Hidekazu , Sugawara Kanetsu , Gao Peng , Muraki Yasushi , Hongo Seiji , Kitame Fumio , Nakamura Kiyoto
Published: November 2013

The HMV‐II cells infected with influenza C virus were labeled with inorganic [32P]phosphate to identify phosphorylated proteins. Analysis by radioimmunoprecipitation with antiviral serum or monoclonal antibodies revealed that three major structural proteins of the virus, hemagglutinin‐esterase (HE), nucleoprotein (NP), and matrix protein (M1) are all phosphorylated in both infected cells and virions. It was also observed that, in the presence of trypsin (10 μg/ml), the unphosphorylated form of the HE glycoprotein was cleaved efficiently whereas the phosphorylated form was not, raising the possibility that phosphorylation of HE may influence its susceptibility to degradation by proteolytic enzymes.
Sequence Analysis of Flanking Regions of the pfoA Gene of Clostridium perfringens: β‐Galactosidase Gene (pbg) Is Located in the 3′‐Flanking Region
Authors: Shimizu Tohru , Kobayashi Takumi , Ba‐Thein William , Ohtani Kaori , Hayashi Hideo
Published: November 2013

The 3′‐flanking region of the perfringolysin O (theta‐toxin) gene (pfoA) of Clostridium perfringens was analyzed by chromosome walking. A total of 5,363 bp of the downstream region of the pfoA gene was sequenced and four open reading frames were found. ORF54 and ORF80 were found to be homologous to genes coding for membrane‐bound transporter proteins of other bacteria and the β‐galactosidase gene (bgaB) of Bacillus stearothermophilus, respectively. ORF80 was named the pbg gene. Clones which showed β‐galactosidase activities were selected from a λFIXII genomic library of C. perfringens by blue plaque screening using X‐Gal as a substrate. Four clones whose plaques showed blue appearances were obtained. Two of the four clones hybridized with the pbg probe but the others did not, indicating that there are two distinct β‐galactosidase genes in C. perfringens. The pbg gene was subcloned into pBR322 and was successfully expressed in Escherichia coli, suggesting that the pbg gene codes for a β‐galactosidase of C. perfringens.
Identification of the Sites for Suppressor Mutations on the Hemagglutinin Molecule to Temperature‐Sensitive Phenotype of the Influenza Virus
Authors: Tong Ning , Nakajima Katsuhisa , Nakajima Setsuko
Published: November 2013

A temperature‐sensitive (ts) mutant of the influenza virus A/WSN/ 33 strain, ts‐134, possessed a defect in intracellular transport at the nonpermissive temperature and marked thermolability of hemagglutinin (HA) activity at 51 C. These were caused by a change at amino acid residue 157 from tyrosine to histidine in the HA protein. We isolated 37 spontaneous revertant clones from ts‐134 at the nonpermissive temperature and determined their HA sequences. The deduced amino acid sequences demonstrated that one was a true revertant and the others were revertants with suppressor mutations, each of which had an additional amino acid change besides those of ts‐134. The changed amino acids were located at 14 positions on the HA molecule, and eight of them were found in multiple revertants. These were located in five to six distinct regions on the three‐dimensional structure of the HA molecule. However, the heat stability of HAs in the revertants was recovered differently depending on the sites of the changed amino acids. The kinetics of transport of the HA protein in the revertants were slightly delayed compared to the wild‐type both at permissive and nonpermissive temperatures.
Detection of Astroviruses from Stool Samples in Japan Using Reverse Transcription and Polymerase Chain Reaction Amplification
Authors: Saito Kunihiro , Ushijima Hiroshi , Nishio Osamu , Oseto Mituaki , Motohiro Hiroshi , Ueda Yuichi , Takagi Michio , Nakaya Shigekazu , Ando Tamie , Glass Rodger , Zaiman Kohji
Published: November 2013

We developed a reverse transcription and polymerase chain reaction (RT‐PCR) method for detecting astrovirus serotypes 1, 2, 3, 5, 6 and 7 (but not serotype 4). Furthermore, we developed the specific primers for detecting serotypes 1 and 2, the most predominant serotypes in the world. Sensitivity of the first PCR with serotype common primers was about 10 times higher than that of enzyme immunoassay with monoclonal antibody (EIA‐MAb). Sensitivity of the second PCR with the serotype‐specific primers was even higher. The RT‐PCR method was useful for detecting astroviruses from clinical samples, especially serotypes 1 and 2.
Genetic Variation in VP7 Gene of Human Rotavirus Serotype 2 (G2 Type) Isolated in Japan, China, and Pakistan
Authors: Wen Leying , Ushijima Hiroshi , Kakizawa Junko , Fang Zhao‐Yin , Nishio Osamu , Morikawa Shigeru , Motohiro Takashi
Published: November 2013

Sequence analysis of the gene encoding the major neutralization glycoprotein (VP7) was performed on sixteen human isolates of serotype 2 of rotavirus in Japan, China, and Pakistan and their genetic variations were examined. Comparative studies of their nucleotide and deduced amino acid sequences between the sixteen isolates and the HU5 strain revealed an overall homology of more than 94%. A higher degree of homology in nucleotides was observed among the sixteen isolates than between HU5 and the isolates. A total of thirteen amino acid residues frequently converted to another amino acid. Out of the thirteen, five amino acid residues belonging to the major neutralizing epitope regions (C, E, and F in this communication) converted frequently. From the amino acid sequences three subtypes, subtype 1, subtype 2, and intermediate, were suggested to be classified as previously reported for serotype 1 (Xin et al, Virology, 1993, 197: 813‐816).
Major Enteropathogenic Bacteria Isolated from Diarrheal Patients in Bolivia: A Hospital‐Based Study
Authors: Utsunomiya Akiyoshi , Elío‐Calvo Daniel , Reyes Armando Alberto Benitez , Castro Ernesto Sanzetenea , Rodríguez Enrique , Tress Carolina , de Corzo Jenny I. Zamora , Hannover Erika , Kai Akemi , Tamura Kazumichi , Higa Naomi
Published: November 2013

A total of 1,234 fecal samples from diarrhea cases were examined for etiological bacterial agents at medical facilities in La Paz and Sucre, Bolivia. Eighty strains of Shigella spp., 39 strains of Salmonella spp., 29 strains of Vibrio cholerae, and 222 strains of enteropathogenic Escherichia coli (139 EPEC, 55 ETEC, 29 EIEC, and 1 EHEC) were isolated. With regard to the serovars of Shigella, S. flexneri 2a, 3a, and 1b were predominant. In the case of Salmonella, S. enteritidis was the most common, followed by S. typhi, S. poona, and S. paratyphi B. Out of 29 cholera strains, 25 belonged to biovar El Tor, serovar Ogawa while the remaining 4 were serovar Inaba. Among 55 strains of ETEC serotypes, 5 showed ST producers but none showed LT producers. Likewise, among 55 strains of enterohemorrhagic serotypes, only one strain (O157:H7) produced verocytotoxin (VT 2). The results of drug sensitivity tests revealed the predominance of Shigella, EPEC, and ETEC strains resistant to aminobenzil‐penicillin (ABPC) and trimethoprim. Since diarrheal patients in Bolivia are treated mainly with ABPC or sulfamethoxazole/trimethoprim (SXT) and rarely with gentamicin, kanamycin, or other drugs, it is possible that ABPC‐ and SXT‐resistant strains will increase and persist in the near future.
Sequence Analysis of the Streptococcus mutans Ingbritt dexA Gene Encoding Extracellular Dextranase
Authors: Igarashi Takeshi , Yamamoto Ayako , Goto Nobuichi
Published: November 2013

The complete nucleotide sequence (3,747 bp) of the dextranase gene (dexA) and flanking regions of the chromosome of Streptococcus mutans Ingbritt (serotype c) were determined. The open reading frame for dexA was 2,550 bp, ending with a stop codon TGA. A putative ribosome‐binding site, promoter preceding the start codon, and potential stem‐loop structure were identified. The presumed dextranase protein (DexA) consisting of 850 amino acids was estimated to have a molecular size of 94,536 Da and a pI of 4.79. The nucleotide sequence and the deduced amino acid sequences of S. mutans dexA exhibited homologies of 57.8% and 47.0%, respectively, to those of Streptococcus sobrinus dex. The homologous region of dex of S. sobrinus was in the N‐terminal half. The C terminus of DexA consisted of a hexapeptide LPQTGD, followed by 7 charged amino acids, 21 amino acids with a strongly hydrophobic character, and a charged hexapeptide tail, which have been reported as a common structure of C termini of not only the surface‐associated proteins of Gram‐positive cocci but also the extracellular enzymes such as β‐fructosidase of S. mutans and dextranase of S. sobrinus. The DexA protein had no significant homology with the glucosyltransferases, the glucan‐binding protein, or the dextranase inhibitor of mutans streptococci.
Expression of 18.6/CD23 Antigen on Human Lymphoid Progenitor Cell Lines and Phorbol 12‐Myristate 13‐Acetate (PMA)‐Induced Microglia‐Shaped Cells
Authors: Okumura Akiou , Kishi Hiroyuki , Tagoh Hiromi , Minowada Jun , Muraguchi Atsushi
Published: November 2013

The nature of lymphoid progenitors and factor(s) determining commitment to either the T‐ or B‐lymphocyte pathway are poorly understood in the human system. In this study, we generated a monoclonal antibody (MoAb), 18.6, that recognizes a cell surface antigen on a human lymphoid progenitor cell line (FL4.4). MoAb 18.6 reacted with lymphoid progenitor lines, B lymphoid cell lines, and myelomonocytic cell lines. It did not react with any T cell or erythroid leukemic cell lines. Two color FACS analyses of normal lymphoid tissues showed that MoAb 18.6 reacted with a majority of CD20+ mature B cells and a minority of CD64+ monocytes. Molecules of 3 different sizes with MW of 34, 45, and 68 Kd were precipitated with MoAb 18.6 from the lymphoid progenitor cell line. The 18.6 antigen was not expressed on a fetal liver‐derived lymphoid progenitor‐like cell line, FL1.4, which has the capacity to differentiate into microglia‐shaped cells upon PMA‐stimulation. Stimulation of FL1.4 cells with PMA induced expression of the 18.6 antigen within 24 hr and the microglia‐shaped cells stained positively with MoAb 18.6. Finally, cloning of a cDNA that encoded the 18.6 antigen revealed that the 18.6 antigen is identical to the CD23 antigen. Taken together, these data suggest that the 18.6/CD23 antigen is expressed on lymphoid precursors at a very early stage of differentiation.
Prevention of Vascular Graft Infection by Sisomicin Incorporated into Fibrin Glue
Authors: Yamamura Keiko , Sakurai Tsunehisa , Yano Kohji , Osada Takashi , Nabeshima Toshitaka
Published: November 2013

To examine the efficacy of sisomicin (SISO) incorporated into fibrin glue (FG) for the prevention of graft infection in animal models, the susceptibility to infection of Dacron grafts (control) and SISO‐FG Dacron grafts following the inoculation of Staphylococcus aureus or S. epidermidis was compared. The results showed that SISO‐FG Dacron grafts displayed resistance to graft infection.
Correlation of the Polysaccharide Antigens of Francisella tularensis with Virulence in Experimental Mice
Authors: Fujita Hiromi , Sato Tadashi , Watanabe Yuriko , Ohara Yoshiro , Homma Morio
Published: November 2013

Francisella tularensis gives rise to two distinct colony types, acriflavine agglutination test‐positive (acf+) and ‐negative (acf−) colonies. The acf+ variants were exclusively low virulent in mice, while the acf− variants were shown to be either high or low virulent. Three fractions, phosphate‐buffered saline‐extractable without heating, with heating at 60 C, and with heating at 100 C, were obtained from cultures of both the acf+ and acf− variants on agar media, and the polysaccharide antigens in those fractions were quantitated. All of the highly virulent acf− variants possessed a large amount of the polysaccharide antigen in the fraction extractable with heating at 60 C. This antigen was not, however, detected in any of the acf+ variants and one low‐virulent acf− variant. It was also detected in a very low amount in some other acf− variants with low virulence. The amount of this polysaccharide antigen was therefore shown to be correlated with bacterial virulence in mice.