bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin‐4 Downregulates Interleukin‐6 Production by Human Alveolar Macrophages at Protein and mRNA Levels
Authors: Yanagawa Hiroaki , Sone Saburo , Sugihara Kohji , Tanaka Keiji , Ogura Takeshi
Published: November 2013

Effects of interleukin‐4 (IL‐4) on IL‐6 production by human alveolar macrophages (AM) obtained by bronchoalveolar lavage from healthy donors was examined at the protein and gene levels. IL‐6 production was quantitated by enzyme immunoassay (EIA) and bioassay using the IL‐6 dependent murine hybridoma cell line MH60.BSF2. Results showed that when activated with LPS, AM released significantly more biologically active IL‐6 than blood monocytes. Human rIL‐4 significantly suppressed IL‐6 production by AM and monocytes stimulated with LPS. Northern blot analysis revealed that IL‐4 reduced the expression of IL‐6 mRNA in LPS‐stimulated AM and monocytes. The inhibitory effect was most pronounced when IL‐4 was added with LPS or within the first 4 hr after LPS to AM or monocytes. The suppressive effect of IL‐4 was completely neutralized by pretreatment with anti‐IL‐4 antibody. IL‐4 also showed a suppressive effect on IL‐6 production by macrophages generated in vitro by maturation of blood monocytes with granulocyte‐macrophage colony stimulating factor (GM‐CSF). These observations suggest that IL‐4 may play a critical role in in situ regulation of immune responses through suppression of IL‐6 production.
Immunoelectron Microscopy of Chlamydia psittaci with Monoclonal Antibodies
Authors: Ando Shuji , Takashima Ikuo , Hashimoto Nobuo
Published: November 2013

An immunoelectron microscopic study was performed to determine the distribution of antigenic components on particles of Chlamydia psittaci and infected cells using a number of monoclonal antibodies (MAbs). Of three anti‐lipopolysaccharide (LPS) antibodies (4D5, A2 and 4G5), two antibodies (4D5 and A2) reacted with the surface of reticulate bodies (RBs) but not with that of elementary bodies (EBs). The other antibody (4G5) reacted with both EBs and RBs. Examination of infected cells in thin sections revealed that 4D5 and A2 combined with the membranes of both EBs and RBs. These results indicate that each LPS epitope localized at a different position in the chlamydial membrane. Most MAbs directed to protein antigens reacted on the surface of both EBs and RBs though 3E9 specific for the 90 kDa and 50 kDa protein components combined with RBs only.
Genetic and Biochemical Analysis of Ribosomal Proteins of Minocycline‐Susceptible and ‐Resistant Mycobacterium smegmatis
Authors: Yamada Takeshi , Mizuguchi Yasuo , Isono Setsuko , Isono Katsumi
Published: November 2013

A minocycline (MINO)‐resistant mutant was isolated from Mycobacterium smegmatis strain Rabinowitschi. Polypeptide synthesis in the cell‐free system prepared from the mutant was resistant to minocycline (MINO) because of alterated 30S ribosomal subunits. Upon two‐dimensional gel electrophoresis, two proteins of 30S subunit were found to be altered. MINO resistance phenotype was transferred by mating to the recipient strain P‐53. MINO resistance phenotype of a recombinant thus obtained was transferred by a different mating system to the recipient strain Jucho, once again. Ribosomal proteins of each of the donors, recipients and recombinants were analyzed and compared on 2‐dimensional (2D) electrophoresis. Approximately 50 ribosomal proteins were observed in 70S ribosomes. Some proteins were differently electrophoresed in different strains. The 30S ribosomal subunits contained at least 19 proteins and 50S ribosomal subunits contained at least 23 proteins. Some proteins were easily washed off during dissociation of subunits in sucrose gradients. At least one protein (designated F) in both subunits was observed at the same position. One protein designated C in 30S subunits could be co‐transferred to the recipient cells together with resistance phenotype at the frequency of 100% in the 30 recombinants examined so far. The other protein designated D in 30S subunits could be transferred at the frequency of 86–88%. Three other proteins in 50S subunits could be co‐transferred to the recipient strain at a lower frequency. Minocycline resistance, therefore, could be mapped close to genes encoding the structure of ribosomal proteins in M. smegmatis.
The Other Mediator for Adherence of Haemophilus influenzae Organisms without Involvement of Fimbriae
Authors: Miyazaki Shuichi , Matsunaga Toshiyuki , Kobayashi Intetsu , Yamaguchi Keizo , Goto Sachiko
Published: November 2013

The number of the nontypeable Haemophilus influenzae (HiN) organisms that adhered to the primary mouse fetal lung cells was significantly more than type b Haemophilus influenzae (Hib) organisms. The average number of HiN organisms adherent to host cells was 2,291/100 host cells (range, 1,654–3,182), but that of Hib was markedly reduced to 147/100 host cells (range, 102–238). In this case, P value was <0.05 by using a paired Student t‐test. The sonicated extract from HiN TMS11 organisms inhibited adherence of H. influenzae TMS11 organisms to monolayer at 76.3% and it inhibited adherence of Hib TMS24 organisms at 92.3%. This result indicates that a mediator existing on the surface of HiN organisms may be the same as that on type b organisms. The number of detected organisms in broncho and lung tissues 3 days after intranasal infection with HiN strains was significantly greater than that in infection with Hib strains. Therefore, in vitro adhesive capacity of H. influenzae organisms was correlative to infectivity by intranasal injection.
Cloning and Whole Nucleotide Sequence of the Gene for the Light Chain Component of Botulinum Type E Toxin from Clostridium butyricum Strain BL6340 and Clostridium botulinum Type E Strain Mashike
Authors: Fujii Nobuhiro , Kimura Kouichi , Yashiki Teruo , Tsuzuki Kayo , Moriishi Kohji , Yokosawa Noriko , Syuto Bunei , Oguma Keiji
Published: November 2013

Chromosomal DNAs were extracted from Clostridium butyricum strain BL6340 and Clostridium botulinum type E strain Mashike. The 6.0 Kbp fragment coding for the entire light chain (L) component and the N‐terminus of heavy chain (H) component of botulinum type E toxin was obtained from each extracted DNAs after digestion with HindIII. The entire nucleotide sequences for the light chain components of these cloned genes were determined, and the derived amino acid sequences were compared to each other, and with those of botulinum type A, Cl, D, and tetanus toxins reported previously. The cleavage site of L and H components of type E toxin was presumed to be Arg‐422. In a total of 422 amino acid residues of L component, 17 residues were different between butyricum and type E toxins, and all these differences were found within 200 residues of N‐terminus of L component. On the contrary, five regions showing highly homologous sequences were found in L components among these six toxins, and one more region between botulinum type E and tetanus toxins.
The Comparison of Cell‐Mediated Immunity Induced by Immunization with Porin, Viable Cells and Killed Cells of Salmonella typhimurium
Authors: Matsui Katsuhiko , Arai Toshihiko
Published: November 2013

A marked level of cell‐mediated immunity (CMI) to Salmonella typhimurium‐infection in mice, as determined by acquired resistance, delayed‐type hypersensitivity, interleukin‐2 production and interferon‐γ production, was induced by immunization with porin or viable cells but not with killed cells of S. typhimurium LT2. When the up‐regulation of immune system to each immunogen was studied by comparing increases of la‐bearing macrophages, the immunization with porin or viable cells, but not killed cells, could stimulate the immune system for more than 14 days. Interleukin‐1 (IL‐1) production of macrophages to each immunogen was also examined; the result showed that immunization with porin or viable cells could induce a notable level of IL‐1 production, while killed cells could not. However, when the abilities to induce these immune responses were compared between UV‐killed and heat‐killed cells, UV‐killed cells were superior to heat‐killed cells. These results suggested that the ineffectiveness of immunogen that lacked CMI‐inducing ability might be ascribed to the denaturation of antigen and the insufficient inductions of Ia‐bearing macrophages and IL‐1 production.
Generation of Helper T Cells That Recognize a Cross‐Reactive Idiotype through a Network Mechanism
Authors: Masaki Hideyuki , Irimajiri Kiyohiro
Published: November 2013

T cells that recognize the cross‐reactive idiotype expressed on the heavy (H) chain of M104E (IgM, λ1) were induced in BALB/c mice by immunization with Dextran B‐1355. T cells derived from mice immunized with 1 mg of Dextran B‐1355 showed a marked proliferative response against M104E, whereas T cells from mice immunized with Ficoll or lesser amounts of Dextran B‐1355 did not. BCLiId, which had an immunoglobulin isotype identical to M104E, did not induce proliferation of the T cells. These T cells also proliferated against J558 (IgA, λ1) which shared the cross‐reactive idiotype of the anti‐a (1—>3) glucosidic linkage antibody with M104E on the H chain. The T cells proliferated more efficiently against F(ab)2‐104E, Fab‐104E and H104E, the H chain of M104E, than against intact M104E. The T cell proliferative response against the idiotype on M104E or even H104E required macrophages as antigen‐presenting cells (APC) and the response was inhibited when APC were treated with NH4CI or chloroquine, inhibitors of antigen processing. Moreover, anti‐CD4 antibody or anti‐Ia antibody inhibited the proliferative response. These results indicated that anti‐idiotypic T cells of the helper type, which recognized a cross‐reactive idiotype associated with la molecules in processed form, could be induced physiologically through a network mechanism.
Lateral Flagella of Vibrios: Serological Classification and Genetical Similarity
Authors: Shinoda Sumio , Yakiyama Ichiro , Yasui Satoshi , Kim Young‐man , Ono Bun‐ichiro , Nakagami Satoru
Published: November 2013

Lateral (L‐) flagella‐having vibrios were classified into 13 H‐serogroups (flagellar antigen serogroups) by means of H‐agglutination test. Vibrio parahaemolyticus was classified into 3 serogroups, HL1 to 3. V. alginolyticus and V. harveyi were classified into 5 and 3 serogroups, respectively, but 2 of those were serogroups common to the both species. V. fluvialis and V. furnissii constituted a same serogroup, HL8. Cross‐reactivity between each serogroup was not observed in H‐agglutination test, although some cross‐reactivity was observed in gel diffusion test. Furthermore, similarity of DNA sequence of L‐flagellar structure gene was demonstrated by dot blot hybridization test with a DNA probe of HL2 L‐flagellar gene fragment. These results suggest conservation of DNA sequence of the L‐flagellar gene of vibrios.
Oligo‐2′,5′‐Adenylate Synthetase Activity in Cells Persistently Infected with Human T‐Lymphotropic Virus Type I (HTLV‐I)
Authors: Fujii Nobuhiro , Kwon Kil‐won , Yashiki Teruo , Kimura Kouichi , Isogai Emiko , Isogai Hiroshi , Sekiguchi Sadayoshi , Oguma Keiji
Published: November 2013

Spontaneous production of interferon‐gamma (IFN‐γ) was shown in several T‐lymphoblastoid cell lines persistently infected with human T‐lymphotropic virus (HTLV‐1). However, the produced IFN‐γ was not always associated with the induction of the antivirus state. The induction of oligo‐2′,5′‐adenylate synthetase (2‐5AS) by IFN was studied in five human T‐cell lines persistently infected with HTLV‐I (MT‐1, MT‐2, SMT‐1, HUT 102 and OKM‐2). Four cell lines are able to produce IFN‐γ spontaneously, while the OKM‐2 cell line is not. Poor induction of 2‐5AS was recognized in three (MT‐1, MT‐2 and SMT‐1) of the four cell lines producing IFN‐γ, though the poor induction was improved after long‐term cultivation of cells with IFN‐α. On the contrary, in the OKM‐2 cell line. significant activity of the enzyme was induced by IFN‐α. Induction of 2‐5AS was not correlated with cell growth inhibition, but with the antivirus state. Furthermore, an inverse relationship between IFN‐γ production and 2‐5AS induction was demonstrated in these cell lines with the exception of HUT 102 cells.
Induced CD25 Expression in a Human B‐Lymphoma Cell Line Transfected with the Epstein‐Barr Virus Nuclear Antigen 2 Gene
Authors: Harada Shizuko , Yanagi Kazuo
Published: November 2013

Two EBV‐negative human B‐lymphoma cell lines, BJAB and DG75, were transfected with an Epstein‐Barr virus (EBV) nuclear antigen 2 (EBNA‐2) gene, which plays a critical role in the EBV‐induced immortalization of primary B lymphocytes. Furthermore, DG75 cells were co‐transfected with the EBNA‐2 gene and a latent membrane protein (LMP) gene. Expression of eight surface antigens on the resultant EBNA‐2‐expressing cell clones was analyzed by flowcytometry. None of the EBNA‐2‐expressing cell clones derived from BJAB and DG75 showed a significant increase in the expression of cell surface marker CD23, of which enhancement by EBNA‐2 in a different EBV‐negative human B cell line, Louckes, was previously reported. Expression of CD25 (IL‐2R/Tac) on cell surface, however, was induced in two of six DG75‐derived cell clones. One of the two CD25‐induced cell clones was expressing EBNA‐2 only, and the other was co‐expressing EBNA‐2 and LMP. The results suggest that EBNA‐2 has a potential to up‐regulate CD25 independently of CD23 on human B cells.
Induction of Cytokines in Human Peripheral Blood Mononuclear Cells by Mycoplasmas
Authors: Kita Masakazu , Ohmoto Yasukazu , Hirai Yoshikatsu , Yamaguchi Nozomi , Imanishi Jiro
Published: November 2013

Various species of mycoplasmas were tested for their ability to induce cytokine production in human peripheral blood mononuclear cells (PBMC). Human PBMC were incubated with Mycoplasma pneumoniae, M. hyorhinis, M. arginini, M. salivarium, M. orale, M. gallisepticum or A. laidlawii for 48 hr, and the activities of interleukin‐1β (IL‐1β), IL‐2, IL‐4, IL‐6, tumor necrosis factor‐α (TNF‐α) and interferon (IFN) in the supernatants were determined by ELISA or bioassay. All mycoplasma species induced IL‐1β, IL‐6 and TNF‐α, although IL‐2 was induced only by M. pneumoniae. IFN was induced by 5 of the 7 species, and the IFN produced was antigenically confirmed to be mainly IFN‐α. On the other hand, mycoplasma‐stimulated cultures did not contain detectable amounts of IFN‐β and IL‐4 activities. Furthermore, the cytokines were induced by mycoplasmal contaminating cells in human PBMC as well as by mycoplasma alone. These results suggest that many kinds of cytokines induced by mycoplasma contamination in cell culture affect immunological experiments in vitro.
Prevalence of Clostridium botulinum Type E and Coexistence of C. botulinum Nonproteolytic Type B in the River Soil of Japan
Authors: Yamakawa Kiyotaka , Nakamura Shinichi
Published: November 2013

Soil samples from 98 sites in the whole systems of four rivers in Japan were examined for the presence of Clostridium botulinum. Type E organism was prevalently shown throughout the whole river systems including upper part; detection rates of type E toxin in soil culture ranged from 33 to 82%. This type was also detected in soil of adjacent mountainous district. Type B and C toxins were detected at 7% and 9% of the sites examined, respectively. C. botulinum type E and nonproteolytic type B strains were isolated from enrichment cultures of soil samples. These results suggest that the terrestrial origin of type E organism would be considered as one of the reasons for the high incidence of this organism in the sea areas, and prove that C. botulinum nonproteolytic type B exists in the soil of Japan.
Role of the Upstream Region Containing an Intrinsic DNA Curvature in the Negative Regulation of the Phospholipase C Gene of Clostridium perfringens
Authors: Toyonaga Tatsuo , Matsushita Osamu , Katayama Sei‐ichi , Minami Junzaburo , Okabe Akinobu
Published: November 2013

The phospholipase C (α‐toxin) gene (plc) of Clostridium perfringens was cloned into pUC19 and the effects of the upstream regions on expression of the plc gene were examined in Escherichia coli JM109. When the 0.7‐kb region just upstream of the putative — 35 site of the gene was deleted, production of phospholipase C increased approximately 10‐fold. Northern blot hybridization analysis of the plc transcript showed that the upstream region inhibited transcription from the plc promoter. Nucleotide sequencing of this upstream region revealed that there are three periodically repeated (dA)5–6 tracts between positions −66 and −40 of the plc gene. A fragment containing this sequence showed anomalously slow electrophoretic mobility at low temperature, indicating that the region immediately upstream of the plc promoter is a locus of sequence directed DNA‐bending. Nested deletions of the upstream region were created from its 5′ end by exonuclease III and the effects of deletions on the expression of the plc gene were examined. When the 77‐bp fragment containing the two (dA)5–6 tracts was deleted, phospholipase C production increased markedly. These results indicate that the intrinsic DNA curvature upstream of the plc promoter is involved in the negative regulation of the plc gene transcription.
Purification and Characterization of Secretory Proteinase of Candida albicans
Authors: Yamamoto Tetsuro , Nohara Kumiko , Uchida Katsuhisa , Yamaguchi Hideyo
Published: November 2013

Proteolytic activity of medically important yeasts was tested in both YCB‐BSA agar and medium. All of 134 strains of Candida albicans, 13 of 18 strains of Candida tropicalis and 11 of 18 strains of Candida parapsilosis had this activity, while none of 52 Candida glabrata strains or of 11 Cryptococcus neoformans strains tested had proteolytic activity. Strains of C. albicans fell into five groups based on the level and time‐course of in vitro proteinase productivity. Five strains randomly selected from each group were tested for pathogenicity in mice. The strain possessing the strongest pathogenicity was used to purify proteinase. The molecular weight of the proteinase was approximately 44,000 daltons and its isoelectric point was pH 4.2. Optimal pH of the proteinase was 3.2 and the enzyme was stable below pH 7.0 and lost its activity above pH 8.0 at 37 C in a 60‐min incubation. The 23 amino acid sequence of the proteinase N‐terminus was determined.
Arginine Deiminase Gene of an AIDS‐Associated Mycoplasma, Mycoplasma incognitus
Authors: Sugimura Kazuhisa , Ohno Takeshi , Kimura Yoshihito , Kimura Tatsuji , Azuma Ichiro
Published: November 2013

Recently, we have demonstrated by molecular cloning that a strong immunosuppressive factor derived from Mycoplasma arginini is arginine deiminase. We show here that mycoplasma species identified in many of AIDS patients also bear the arginine deiminase gene. The implication of the arginine deiminase gene detected in AIDS‐associated mycoplasma species is discussed.
A Phage in Bartonella bacilliformis
Authors: Umemori Eriko , Sasaki Yoshimichi , Amano Ken‐ichi , Amano Yasuji
Published: November 2013

Bacteriophage‐like particles were found in Bartonella bacilliformis culture. The particles consisted of head (icosahedral), 40 nm in diameter, and tail, 16 nm in length.
Studies on Novel Pili from Shigella flexneri
Authors: Utsunomiya Akiyoshi , Naito Tatsuro , Ehara Masahiko , Ichinose Yoshio , Hamamoto Akihiro
Published: November 2013

Pili were detected using electron microscopy in clinical isolates of Shigella flexneri which had been continuously subcultivated in liquid media. Morphologically, the pili appeared as thin, flexible, cylindrical structures of up to 2–5 μm in length and about 3–5 nm in diameter. Two strains showed mannose‐resistant (MR) hemagglutination to fresh fowl erythrocytes (type 4), and one to tannic acid‐treated horse erythrocyte (type 3) pili. These pili are novel and different from the mannose‐sensitive (MS) type 1 pili described by Duguid and Gillies.
Latent Infection of SCID Mice with Herpes Simplex Virus 1 and Lethal Cutaneous Lesions in Pregnancy
Authors: Moriyama Kosei , Mohri Shirou , Watanabe Takeshi , Mori Ryoichi
Published: November 2013

Some SCID mice survived primary infection with herpes simplex virus 1 without the development of peripheral lesions but established coculture‐positive ganglionic latency when a low dose of a wild‐type strain was inoculated intracutaneously. The latency was also evidenced by the development of the fatal zosteriform skin lesions and the isolation of the virus during pregnancy. We consider that the viral entry into neurons without successive replication, rather than the arrest of the lytic infection within the cells, is an important mechanism in the establishment of latency.
Molecular Analysis of Immunoglobulin Heavy Chain Genes Coding for Idiotypic and Anti‐Idiotypic Antibodies Involved in B‐B Cellular Interaction
Authors: Takahashi Seiichi , Matsuura Yasushi , Taniguchi Taketoshi , Tamura Hiroo , Bitoh Soji , Onishi Saburo , Yamamoto Yasutake , Yamamoto Hiroshi , Fujimoto Shigeyoshi
Published: November 2013

We recently reported that a unique B cell clone (B19‐1d), specific for a cross‐reactive idiotype (CRI) on MOPC104E myeloma protein (M104E), enhances Igh‐restricted CRI+ antibody production. In this paper, we report the nucleotide sequences of immunoglobulin heavy chain variable regions (VH) of both M104E and B19‐1d‐derived hybridoma (HB19) antibodies. The sequence data revealed that both belong to the J558 germ line VH gene subfamily. Strikingly, not only the VH region, but also the leader sequences of M104E and HB19 are very similar to each other at 88% (VH) and 91 % (leader) homology, but they use different D and J segments. The VH region sequence similarity is highest among the germ line VH gene sequences of the BALB/c J558 subfamily so far screened. Southern hybridization data, using 5′‐noncoding regions of either M104E or HB19 genomic VH gene clones as probes, revealed that both VH genes are conserved in the M104E CRI producer strains of mice. Moreover, these probes show the restriction length polymorphism pattern of mouse VH genes in various strains. That the HB19 VH gene locates to the 5′ upper arm of the M104E VH gene on the chromosome was suggested by Southern blot hybridization. Immunoglobulin VH gene restriction of idiotypic and antiidiotypic B‐B cellular interaction is discussed from a molecular point of view.
Monoclonal Antibodies against Human α‐Fetoprotein More Reactive to Cell‐Surface α‐Fetoprotein than to Free α‐Fetoprotein
Authors: Masuda Kazuyoshi , Nagata Shunji , Harada Shigenori , Hirano Koichiro , Takagishi Yasushi
Published: November 2013

This paper describes the finding of monoclonal antibody (MoAb) more reactive to cell‐surface α‐fetoprotein (AFP) than to free AFP by using a simple in vitro system. Twelve mouse MoAbs, ten IgG1, one IgG2a and one IgG2b, against human AFP from hepatocellular carcinoma were obtained by the cell fusion technique. Each hybridoma supernatant was assayed by enzyme‐linked immunosorbent assay (ELISA) to solid‐phase AFP. The assay results showed that two MoAbs, 67D and 80G, were most reactive to AFP. 80G had a higher affinity constant than 67D, while the both reactions were similarly difficult to inhibit by free AFP in ELISA. 67D and 80G reacted with AFP on the surface of ethanol‐fixed cells from the human hepatoma cell line HuH‐7 and this reaction was also difficult to inhibit by free AFP in Cell ELISA. Furthermore, Western blot analysis showed that 67D and 80G were more reactive to membrane‐bound AFP than other antibodies. These findings first suggest that there could be anti‐AFP MoAbs preferably binding to cell‐surface AFP rather than to serum AFP.
Temperature‐Sensitive Immunoglobulin A‐Binding and Dimerization of C‐Terminus‐Impaired Protein Arp4 Produced in Escherichia coli
Authors: Nakatani Tomoyuki , Higuchi Atsuko , Noguchi Hiroshi
Published: November 2013

A gene for protein Arp4, an IgA receptor protein derived from Streptococcus pyogenes AP4, was expressed in Escherichia coli. The product was demonstrated to be accumulated in a periplasmic space as a polypeptide with an apparent molecular weight of 40 kDa with the deleted C‐terminal membrane anchor portion of protein Arp4. This 40‐kDa peptide of the C‐terminus‐impaired recombinant protein Arp4 produced in E. coli, designated ir‐protein Arp4, was purified from a periplasmic fraction of transformants and its IgA‐binding activity was analyzed. The IgA binding of ir‐protein Arp4 was temperature‐sensitive, that is, ir‐protein Arp4 bound IgA at 4 and 25 C, but did not at 37 C. In addition, the dimerization of ir‐protein Arp4 was also temperature‐sensitive in parallel with temperature‐dependent binding activity, suggesting that the dimerization of ir‐protein Arp4 may be required for its active binding to IgA. In contrast, ir‐protein Arp4 immobilized on Sepharose 4B did bind to IgA even at 37 C as well as 4 and 25 C. The immobilized ir‐protein Arp4 might acquire the temperature‐resistant IgA binding activity in part through the formation of a stable dimerized ir‐protein Arp4 on the solid support.
Characterization of the Dextranase Purified from Streptococcus mutans Ingbritt
Authors: Igarashi Takeshi , Yamamoto Ayako , Goto Nobuichi
Published: November 2013

We purified dextranase from the culture supernatant of Streptococcus mutans Ingbritt by procedures including ammonium sulfate precipitation, ion‐exchange chromatography, and gel filtration. The molecular weight of the enzyme was estimated as 78 kDa by SDS‐PAGE. The enzyme degraded dextran at the optimum pH of 5.5, but not other glucans and fructans at all. Paper chromatographic analysis revealed that the enzyme cleaved dextran by an endo‐type mechanism. The enzyme was inhibited by Hg2+, Fe3+, Zn2+, and anionic detergents SDS and deoxycholic acid, but not inhibited by non‐ionic detergents Triton X‐100, Lubrol PX, Nonidet P‐40, and Tween 80. SDS‐blue dextran‐PAGE analysis of the culture supernatant revealed that the enzyme activity detected in the 96 kDa band shifted gradually to the 78 kDa band during handling the supernatant. This shift was inhibited by phenylmethylsulfonyl fluoride, suggesting that the shift of the molecular size is due to proteolytic degradation of the enzyme by serine protease.
Fimbriation, Hemagglutination and Adherence Properties of Fresh Clinical Isolates of Branhamella catarrhalis
Authors: Ahmed Kamruddin , Rikitomi Naoto , Matsumoto Keizo
Published: November 2013

This study investigated the fimbriation on 24 fresh clinical isolates of Branhamella catarrhalis by electron microscopy. All the strains were isolated from patients with respiratory infections. The Branhamella catarrhalis strains were classified into three groups according to the grade of fimbriation. Among these 24 strains the incidence of densely fimbriated, moderately fimbriated and sparsely fimbriated isolates were 12 (50%), 7 (29%) and 5 (21%), respectively. After five‐times serial subculture on Brain Heart Infusion agar, the average number of fimbriae per bacteria was decreased from 174 to 114 in the densely fimbriated strain and from 48 to 10 in the moderately fimbriated strain. Moreover, 20% of the population became non‐fimbriated in moderately fimbriated strain after the serial subculture. In strains with higher hemagglutination titer the number of fimbriae was significantly (P<0.04) more than in strains with lower hemagglutination titer.
Effects of Human Intravenous Immune Globulin on Diarrhea Caused by Shiga‐Like Toxin I and Shiga‐Like Toxin II in Infant Rabbits
Authors: Havens Peter L. , Dunne W. Michael , Burd Eileen M.
Published: November 2013

Shiga toxin and the related Shiga‐like toxins (SLT), produced by Escherichia coli, can cause hemorrhagic colitis and hemolytic uremic syndrome (HUS). Human intravenous immune globulin (HIVIg) blocks the cytotoxicity of some SLTs in vitro. To examine the ability of HIVIg to modify disease caused by Shiga‐like toxin I or Shiga‐like toxin II (SLT‐I or SLT‐II), we injected 3‐day‐old rabbits intraperitoneally with SLT‐containing cell‐free supernatants from Escherichia coli O157: H7. A subset of rabbits was treated with subcutaneous HIVIg. All rabbits given 104 CD50 of SLT‐1 developed severe diarrhea, and 5 died. When HIVIg 500 mg/kg was given in addition to SLT‐I, only 6 of 18 rabbits (33.3%) developed diarrhea (P < 0.0001), and 1 died. HIVIg 500 mg/kg or 1,000 mg/kg protected against diarrhea when given one hour prior to toxin. HIVIg 1,000 mg/kg was protective when administered one hour after toxin, but not at 6 or 24 hr. Seventeen of 18 rabbits given 106 CD50 of SLT‐II developed severe diarrhea, and 4 died. In contrast to SLT‐I‐associated disease, HIVIg had no effect on diarrhea in rabbits given SLT‐II. We conclude that HIVIg protects infant rabbits from diarrhea and death caused by by intrapertoneally administered SLT‐I, but does not affect the course of SLT‐II‐associated illness.
Characterization of an Amorphous and Soluble Hemagglutinin from Yersinia pseudotuberculosis
Authors: Sheng Yao Jian , Otsuki Koichi , Sanekata Takeshi , Tsubokura Misao , Koga Tetsuro , Tsuji Hideaki , Oka Tatsuzo , Takumi Kenji
Published: November 2013

Yersinia pseudotuberculosis which were screened out depending on auto‐agglutination and Ca2+ dependency, were examined for their production of hemagglutinin (HA), and its purification and characterization were performed. The HA with a broad reactivity with various mammalian erythrocytes was recovered from the culture supernatant of these strains grown at 37 C but not 25 C. HAs from two strains, R148R and T1040, were purified by salt precipitation, gel filtration and anion‐exchange chromatography by HPLC. Both purified HAs were cysteine‐deficient acidic protein with an apparent molecular weight in the range of 15,000 to 16,000. N‐terminal amino acid sequences of the first 25 residues were found to share 12% identity with that of afimbrial adhesin from enterotoxigenic Escherichia coli 2230. Immunoelectron microscopy and immunodiffusion test with polyclonal antiserum raised against the purified R148RHA demonstrated that the HA was associated with the amorphous aggregates which were detached from bacteria. These results suggest that the HA of Y. pseudotuberculosis belongs to a third type of HA produced by the yersinial species.
Detection of Herpes Simplex and Varicella‐Zoster Virus DNA by Field‐Inversion Gel Electrophoresis from Clinical Materials
Authors: Arao Yujiro , Yoshida Mariko , Sata Tetsutaro , Nakatsukasa Akihiko , Miyoshi Kaoru , Yamada Masao , Uno Fumio , Kurata Takeshi , Nii Shiro
Published: November 2013

A simple method using field‐inversion gel electrophoresis (FIGE) was applied to detect herpes simplex virus (HSV) and varicella‐zoster virus (VZV) genomes in clinical specimens. The whole genomes of these viruses could be detected in small vesicle tissues by the FIGE method regardless of their clinical stages of skin lesions. And the sensitivity of the FIGE method was equivalent to that of an immunofluorescent (IF) method. These data indicated usefulness of the FIGE method to detect the whole genomes of HSV and VZV in clinical specimens.
Suppression of Cell‐Mediated Immunity by Street Rabies Virus Infection
Authors: Hirai Kazuhiro , Kawano Hiroshi , Mifune Kumato , Fujii Hiroyuki , Nishizono Akira , Shichijo Akehisa , Mannen Kazuaki
Published: November 2013

An attempt to define a severe suppression of cell‐mediated immunity by street rabies virus infection was undertaken by using the mice lethally and peripherally infected with a street rabies virus (1088 strain). The cell‐mediated cytotoxic (CMC) activity of the spleen cells from those mice once slightly increased until day 4 after infection but declined rapidly thereafter until their death on days 10 to 12 after infection. In parallel with a decrease of CMC response of the spleen cells from 1088‐infected mice, proliferative response to Con A, IL‐2 activity in the culture supernatants of Con A‐induced proliferation, responsiveness to exogenously added IL‐2 and to Con A to express IL‐2R, of those cells became suppressed, and the marked decrease of the total number of spleen cells was observed. Selective depletion of CD4+ and CD8+ cells in the spleens, abnormalities of IL‐1 and E‐type prostaglandins (PGE2) production or production of inhibitory component able to block IL‐2 activity by spleen cells were not observed and these factors did not appear to be associated with the suppression of proliferative response to Con A. However, an apparent association of CD8+ cells in the suppression of differentiation of pre‐cytotoxic lymphocytes (CTL) into CTL was demonstrated in the co‐culture experiments of the spleen cells from 1088‐infected mice with spleen cells of mice infected with an attenuated rabies virus (ERA strain) which can induce higher levels of CMC response. There was no evidence of the productive replication of rabies virus in thymus and spleen of 1088‐infected mice. The relationship of these observations to current theories on virus‐induced immunosuppression was discussed.
Establishment of Stable Cell Lines Producing Anti‐Pseudomonas aeruginosa Monoclonal Antibodies and Their Protective Effects for the Infection in Mice
Authors: O'Oka Hisayoshi , Chonan Eiko , Mizutani Kazunori , Fukuda Tamotsu , Kuroiwa Yasuyuki , Ono Yasushi , Shigeta Shiro
Published: November 2013

Human‐human hybridomas producing monoclonal antibodies (MoAbs) specific for five major serotypes of Pseudomonas aeruginosa were developed by fusing P. aeruginosa primed and Epstein‐Barr virus‐transformed cells with human myeloma P109 cells using polyethyleneglycol. The MoAbs which were produced by the hybridomas were protective against lethal intraperitoneal (i.p.) challenge of P. aeruginosa (10 LD50) in mice. The 50% effective dose (ED50) values of MoAbs ranged from 0.5 to 10.2 μg/mouse and were 26 to 240 times more protective than a commercial human IgG preparation. MoAb administration to mice promoted bacterial clearance in peritoneal cavity, and prevented bacterial invasion into blood in the way of increasing both the number of bacteria trapped by a macrophage and the ratio of macrophages that trapped bacteria. MoAbs also showed protective effects against lethal infection of P. aeruginosa in the mice which were decreased in polymorphonuclear cells (PMN) by cyclophosphamide (CY). All MoAbs showed serotype‐specific binding to the clinical isolates of P. aeruginosa as well as to the immunized strains. The hybridoma cell lines maintained their capacity to produce MoAb continuously for more than 12 months and produced 10 to 60 μg MoAbs per 106 cells in 24 hr. It is practicable to use these cell lines for large‐scale production of anti‐P. aeruginosa MoAbs and such MoAbs must be useful for the therapeutics of patients with P. aeruginosa infection.
Priming Effect of Pseudomonal Leukocidin on Chemiluminescence Response of Rabbit Polymorphonuclear Leukocytes
Authors: Nishiya Hajime , Kunii Otohiko , Noda Masatoshi
Published: November 2013

To clarify effects of pseudomonal leukocidin (42.5 kd) on chemiluminescence (CL) production of polymorphonuclear leukocytes (PMNs), rabbit PMNs were stimulated by zymosan or phorbol myristate acetate (PMA) after pretreatment with the leukocidin, which by itself stimulated little chemiluminescence response. The extent of CL responses stimulated by zymosan or PMA was respectively 5.3‐ or 3.5‐fold greater in leukocidin (1.5 μg/ml)‐pretreated PMNs than in non‐pretreated ones. The priming effect of the leukocidin was greater than that of G‐CSF and related to some steps before NADPH oxidase activation. The increased CL productions might be related to tissue damages caused by pseudomonal infections in vivo.
Isolation of rfb Gene Clusters Directing the Synthesis of O Polysaccharides Consisting of Mannose Homopolymers and Serological Analysis of Lipopolysaccharides
Authors: Saeki Atsushi , Kido Nobuo , Sugiyama Tsuyoshi , Ohta Michio , Iwashita Toshihide , Uchiya Keiichi , Kato Nobuo
Published: November 2013

Four serotypes of two genera, Escherichia coli O8 and O9 and Klebsiella O3 and O5, produce the O polysaccharides consisting of mannose homopolymers. Previously we reported the isolation and expression of E. coli O9 rfb in E. coli K‐12 strains (Kido et al, J. Bacteriol., 171: 3629–3633, 1989). In this study, R' plasmids carrying his‐rfb region of the other three strains were isolated and expressed in E. coli K‐12 strain. Serological study of lipopolysaccharides (LPS) synthesized in E. coli K‐12 strain was carried out. His‐linked rfb genes from E. coli O9 and Klebsiella O3 directed the synthesis of O polysaccharides with the same antigenicity as those of the parental strains in E. coli K‐12 strain. On the other hand, rfb genes from E. coli O8 and Klebsiella O5 directed the synthesis of O polysaccharides which were antigenically not identical but partially common to those of the parental strains. A rough strain derived from E. coli O8 synthesized LPS which showed the identical antigenicity as the wild strain when the his‐rfb region of E. coli O8 was introduced. The results suggest that some genes located distantly from his are additionally required to complete the synthesis of O polysaccharides of E. coli O8 and Klebsiella O5.
Functional and Phenotypical Analysis of Subsets of Rat CD4+ T Cells
Authors: Nagai Yumiko , Inobe Manabu , Kikuchi Kokichi , Uede Toshimitsu
Published: November 2013

Rat CD4+ T cells were divided into two distinct subsets by a monoclonal antibody RTH‐1 recognizing a unique epitope on rat CD45R. Cellular distribution of OX‐22‐ and RTH‐1‐defined antigens was the same. However, OX‐22 and RTH‐1 recognized different epitopes that exist on rat CD45R. The expression of IL‐4 gene was detected only in RTH‐1low CD4+ T cell subset upon various stimulations. In contrast, the expression of IL‐2 and IFN‐γ gene varied depending upon the nature of stimuli. The increased cell surface expression of CD44 was detected in RTH‐1high CD4+ T cell subset. Conversely the increased expression of CD2 was detected in RTH‐1low CD4+ T cell subset. The expression of CD3 and LFA‐1 was not significantly different between RTH‐1high and RTH‐1low subsets.
Radioresistance of Intermediate TCR Cells and Their Localization in the Body of Mice Revealed by Irradiation
Authors: Kimura Motohiko , Watanabe Hisami , Ohtsuka Kazuo , Iiai Tsuneo , Tsuchida Masanori , Sato Shotaro , Abo Toru
Published: November 2013

Extrathymic generation of T cells in the liver and in the intestine was recently demonstrated. We investigated herein whether such T cells, especially those in the liver, are present in other organs of mice. This investigation is possible employing our recently introduced method with which even a minor proportion of extrathymic, intermediate TCR cells in organs other than the liver can be identified. Intermediate TCR cells expressed higher levels of IL‐2Rβ and LFA‐1 than bright TCR cells (i.e., T cells of thymic origin) as revealed by two‐color staining. Although intermediate TCR cells were present at a small proportion in the spleen and thymus, they predominated in these organs after irradiation (9 Gy) and bone marrow reconstitution, or after low dose irradiation (6 Gy). This was due to that intermediate TCR cells were relatively radioresistant, whereas bright TCR cells were radiosensitive. Microscopic observation and immunochemical staining showed that intermediate TCR cells in the spleen localized in the red pulp and those in the thymus localized in the medulla. These intermediate TCR cells displayed a large light scatter, similar to such cells in the liver. The present results suggest that intermediate TCR cells may proliferate at multiple sites in the body.
Host‐Specific Hemagglutination of Influenza A (H1N1) Virus
Authors: Morishita Takayuki , Kobayashi Shinichi , Miyake Takashi , Ishihara Yuichi , Nakajima Setsuko , Nakajima Katsuhisa
Published: November 2013

H1N1 strains of influenza A virus isolated during the influenza season of 1991–92 were divided into two groups according to the property of host‐specific hemagglutination. Group 1 viruses agglutinated human and chicken red blood cells. Group 2 viruses agglutinated human but not chicken red blood cells. The viruses of both groups, however, showed the same antigenic structure determined with ferret antisera. The virus clones which were plaque‐purified twice from a group 2 virus retained the characteristic of host‐specific hemagglutination after five successive passages in MDCK cells, indicating that this phenomenon is genetically determined. However, the amino acid, sequences of the hemagglutinin (HA) polypeptides deduced from the nucleotide sequences of the HA gene of the two groups did not show any differences between them. This suggests a difference in amino acids in some other polypeptide(s), which affects the host‐specific hemagglutination.
Antigenic Heterogeneity of Qa‐2 Antigens in C57BL/6
Authors: Park Eunkyue , Shinohara Nobukata , Arn J. Scott , Sharrow Susan O. , Waneck Gerry L. , Sacks David H.
Published: November 2013

The Qa‐2 antigens are class I‐like molecules encoded by genes mapped telomeric to the H‐2D region on chromosome 17 in the mouse. A panel of 8 new monoclonal anti‐Qa‐2 antibodies derived from a C3H.KBR anti‐C3H. SW immunization was studied. Immunoprecipitation of125I‐labeled C57BL/6 splenocyte antigens showed that all of these antibodies precipitated 40 kDa molecules which could be completely precleared by the monoclonal antibody 20‐8‐4, which had previously been shown to crossreact with Qa‐2. One of the monoclonal antibodies (1‐12‐1), however, was found not to completely preclear Qa‐2 antigens precipitable by the other 7 antibodies or by 20‐8‐4, suggesting the existence of at least two different species of Qa‐2 molecules. Cell lines transfected with Q7 or Q9 genes were reactive with all 9 antibodies and the Qa‐2 antigens expressed on surface membranes of these cells were completely precleared by both 20‐8‐4 and 1‐12‐1. Therefore, the observed heterogeneity of these molecules cannot be explained by an antigenic difference between the Q7 and Q9 gene products. 2D gel analyses showed identical pI spectra between Qa‐2 molecules precipitated with 20‐8‐4 and 1‐12‐1. In addition, all of the monoclonal antibodies reacted with labeled antigen preparations following treatment with Endo F or neuraminidase, indicating that carbohydrate moieties are probably not responsible for the antigenic difference between the two species of Qa‐2 antigen.
Binding of Streptavidin to Bacteria or Fungi and Its Applications in Detecting These Microbes
Authors: Matsuhisa Akio , Saito Yoshihiro , Ueyama Hiroshi , Yamamoto Makiko , Ohono Tsuneya
Published: November 2013

We have investigated the characteristics and utilities of streptavidin‐binding to gram‐negative and gram‐positive bacteria and Candida spp. The pre‐treatment of these microbes with chemical reagents such as CHCl3, NaOH, and Tween 20 have allowed colorimetric visualization under light microscopy or quantitation on nitrocellulose membranes, using streptavidin/biotinylated alkaline phosphatase conjugates. Analysis of this binding was confirmed by western blot. These binding reactions were due to the specific interaction of streptavidin with biotinylated proteins present in the microbes. Competition assays with free biotin or inhibition by an antibiotin antibody confirmed binding to these proteins. With knowledge of these strongly specific interactions, we attempted to reveal the biotinylated proteins within these microbes using clinical specimens. Using phagocyte‐smears from blood, urine, and ascites, these intracellular microbes were easily detected by light microscopy. One of the septic blood samples stained by our technique revealed semi‐digested microbial signals despite the absence of a signal with routine staining. This detection system, which combines streptavidin as a probe and biotinylated proteins as a microbial marker, is useful in staining for intracellular bacteria or fungi (e.g., microbial infections in phagocyte‐smears).
Granulocyte‐Macrophage Colony‐Stimulating Factor and Tumor Necrosis Factor Alpha in Patients with Human Immunodeficiency Virus (HIV) Type 1 Infection
Authors: Hober Didier , Ajana Faïza , Petit Marie‐Cécile , Sartiaux Claudine , Boniface Michel , Caillaux Michèle , Mouton Yves , Wattre Pierre , Maniez‐Montreuil Michèle
Published: November 2013

Variations in cytokine production in patients with human immunodeficiency virus (HIV) infection could be involved in the physiopathology and in the progression of the disease. Therefore we studied the level of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and tumor necrosis factor α (TNFα) produced in patients with HIV infection at stage II (asymptomatic seropositives) and stage IV (AIDS) of the CDC classification, by using an enzyme amplified sensitivity immunoassay. We measured the level of GM‐CSF and TNFα in supernatant of phytohemagglutinin‐activated peripheral blood mononuclear cells from patients and healthy individuals. In one out of 10 stage II patients and 4 out of 14 stage IV patients, we obtained higher levels of GM‐CSF than the mean + 2 S.D. of controls, but in 3 stage IV patients with very low CD4+ T lymphocyte counts (< 50/mm–3) compared to other patients, the GM‐CSF values were very low. High levels of TNFα were detected in 3 out of 10 stage II and 6 out of 11 stage IV patients. The high values of TNFα were associated with high values of GM‐CSF in stage II and in most of AIDS patients except those with very low CD4+ T cell counts, who produced low levels of GM‐CSF. Plasma levels of cytokines were evaluated in 10 stage II, 22 stage IV patients and 20 controls. Increased levels of GM‐CSF (more than 9 pg/ml) were observed in the plasma from 8 out of 10 stage II patients and 17 out of 22 stage IV patients. The tendency that increased levels of GM‐CSF were associated with increased levels of TNFα was observed in plasma from stage IV patients. We report a disarray of GM‐CSF production in patients with HIV infection that could be involved in clinical manifestations and progression of the disease.
Augmentation and Suppression of TNF Release from Macrophages by Inflammatory Polymorphonuclear Leukocytes
Authors: Yui Satoru , Sasaki Toshinori , Yamazaki Masatoshi
Published: November 2013

It is known that polymorphonuclear leukocytes (PMNs) emerge first in local inflammatory sites, and then they are followed and scavenged by macrophages. We focused on the effect of PMN on tumor necrosis factor (TNF) release activity of macrophages, which is viewed as a possible indicator of the status of macrophage activation. One day after macrophages were cultured with fresh, intact murine PMNs which were induced with sodium casein, the release of TNF triggered by lipopolysaccharide (LPS) was augmented by low concentrations of PMNs, but suppressed by their high concentrations. When the PMN samples were fractionated into soluble and insoluble fractions, the augmenting and suppressing activity was partitioned; the relatively high concentrations of soluble fraction showed the suppressive effect whereas the insoluble fraction in lower concentrations showed augmentation. The suppressive activity was stable at 100 C, but the filtrates of the soluble fraction with membranes having cut‐offs of 5,000 or 10,000 were not suppressive at all, suggesting the suppression is not due to low molecular compounds. It was also suggested that the suppressive effect for TNF release was not due to contaminating LPS or transforming growth factor‐β. Inflammatory processes may thus be positively and negatively controlled by a quantitative factor of initial PMN populations by regulating the TNF release activity of the subsequent macrophages.