bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Differences of Dyes in Inducing Respiratory Immunogenicity by Azo‐, Thiazole‐, Quinoline‐, Reactive‐, and Naphthol‐Dye‐Inactivated Sendai Viruses
Authors: Miyamae Takeo
Published: November 2013

The prophylactic effects by mouse nasal inoculation of 31 kinds of organic dye‐inactivated Sendai viruses were investigated by contact exposure experiment that used mouse nasally infected with 105,8 EID50, immunofluorescent examination of the entire respiratory tract, and check of rise of serum HI titer postexposure. The relative merits of the dye‐structures for inciting nasal immunogenicity were determined. Of 14 azo‐dye‐inactivated vaccines, only azo blue‐ and amido black 10B treated ones brought about nearly complete protection, while the other 5 dye‐groups provided partial protection and the remaining 7 dye‐groups the least protection. Of 6 thiazole dye‐vaccines, primuline‐, thioflavine S‐, and thioflavine T‐vaccines induced complete or almost complete protection, and the others moderate or the least protection. With 3 quinolinevaccines, both pinacyanol‐ and quinaldine red‐inactivated ones provided complete protection, but not with quinoline blue‐vaccine. Of 4 reactive dye‐vaccines, both cibacron brilliant yellow 3G‐P‐and reactive blue 4‐treated ones brought about nearly complete protection, but the remaining 2 vaccines induced regional infection. Nevertheless, all 4 naphthol group (AS, AS‐BS, AS‐BI, and AS‐MX)‐treated vaccines yielded complete or almost complete protection. The thirteen most effective vaccine groups suppressed marked rise of high or low serum HI titers developed through nasal vaccination postexposure. In short, specified dyestuffs having a great affinity for cellulosic fibers, evidently incited mucosal immunogenicity, probably by major union of the dyes to viral core ribose.
In Vitro Cultivation of Borrelia duttonii on Cultures of SflEp Cells
Authors: Konishi Hisanori , Morshed Muhammad Golam , Akitomi Hisaji , Nakazawa Teruko
Published: November 2013

Borrelia duttonii strain 406 K, a causative agent of relapsing fever, could not be cultivated in vitro in currently available media for borreliae. We have developed an in vitro cultivation system by using SflEp cell cultures. The average increases of the number of borreliae, when inoculated with 1.0 × 105 organisms per ml from infected mice, were 23‐fold and 150‐fold in the primary culture and the 3rd subculture, respectively. Even a single borrelia could propagate in this cultivation system. This system will be useful for immunological and physiological studies on uncultivable Borrelia strains.
Mutations in the rfbT Gene Are Responsible for the Ogawa to Inaba Serotype Conversion in Vibrio cholerae O1
Authors: Ito Teruyo , Hiramatsu Keiichi , Ohshita Yoshihiro , Yokota Takeshi
Published: November 2013

In cultures of Vibrio cholerae strains of Ogawa serotype, variant strains which had undergone serotype conversion from Ogawa to Inaba were identified. The rfbT genes cloned from the parent strains were found to produce a 31‐kDa protein in the maxicell system, and to cause serotype conversion when introduced into E. coli cells expressing Inaba serotype specificity. On the other hand, rfbT genes cloned from the variant strains neither produced the 31‐kDa protein nor caused serotype conversion. Nucleotide sequence of these rfbT genes as well as those of two clinical Vibrio cholerae strains of Inaba serotype revealed that mutations causing premature termination of their rfbT genes were invariably present in strains expressing Inaba serotype specificity. The result strongly suggested that genetic alteration of the rfbT gene is responsible for serotype conversion of Vibrio cholerae O1.
Detection of Dengue Virus Antigens in Cultured Cells by Using Protein A‐Gold‐Silver Staining (pAgs) Method
Authors: Chen Wei‐June , Chen Su‐Lih , Fang Ay‐Huey , Wang Ming‐Tsan
Published: November 2013

A protein A‐gold‐silver (pAgs) staining was developed to detect dengue virus antigens in cultured cells. The method can be carried out in either newly‐subcultured or monolayered cells. Dengue virus‐inoculated C6/36 clone of Aedes albopictus cells and human endothelial cells appeared brown‐yellowish color on the peripheral membrane of the infected cells. In many cases, the infected C6/36 cells appeared darker than that of the infected endothelial cells. The positive results from the inoculated C6/36 cells usually appeared as early as 2 days post‐inoculation for types 1, 2, and 4 of dengue viruses and 3 days for the dengue 3 virus. The same batch of specimens detected by direct immunofluorescence antibody test (DFA) showed positive 4 days post‐inoculation for the types 2, 3, and 4 of dengue viruses and 6 days for the dengue 1 virus. The result also showed that all pAgs‐positive specimens were also DFA‐positive, but not vice versa. It suggested that pAgs is not only sensitive but also specific for dengue virus detection from inoculated cultured cells.
Yersinia pseudotubetculosis in Italy. Attempted Recovery from 37,666 Samples
Authors: Chiesa Claudio , Pacifico Lucia , Nanni Fulvio , Renzi Anna Maddalena , Ravagnan Giampietro
Published: November 2013

From 1981 to 1991, 37,666 human, animal, food and environmental samples were cultured for Yersinia pseudotuberculosis using direct plating methods and/or cold enhancement techniques. Despite an intensive surveillance and adequate culture methods, Y. pseudotuberculosis was isolated from stools of 0.05% (5/9,720) of patients with acute enteritis, and alimentary tracts of 0.1% (10/6,849) of apparently healthy animals. No Y. pseudotuberculosis strains were recovered from stools of 4,726 healthy controls nor from the appendices (656), mesenteric lymph nodes (84), and stools (421) of 656 patients operated for suspected appendicitis. Of the 10,842 food and 4,368 environmental samples, none yielded positive cultures for Y. pseudotuberculosis.
Gene Transfer of a Part of a β‐Lactamase Gene?
Authors: Ogawara Hiroshi , Kuma Kei‐Ichi , Miyata Takashi
Published: November 2013

β‐Lactamase is an enzyme which catalyzes the hydrolysis of the β‐lactam ring of penicillins and cephalosporins. By similarity analysis of amino acid sequences in a database, the amino acid sequence deduced from the nucleotide sequence of the upstream region of cytochrome c oxidase subunit II from Paracoccus denitrificans was found to have an unusually high score of homology to that of a portion of β‐lactamases from Gram‐negative bacteria. Furthermore, the nucleotide sequences corresponding only to this region had a very high score of similarity among them. The phylogenetic tree constructed on the basis of the amino acid sequences was in accord with that constituted on the 5S rRNA's. Moreover, the molar G + C contents and the codon usage were similar to those in their respective bacteria. It is suggested, therefore, that the nucleotide sequence in P. denitrificans was positioned by a transfer of a part of a β‐lactamase gene formed as a result of gene duplication or it was formed by a deletion of the essential region of the β‐lactamase gene, although no β‐lactamase gene has been yet detected in P. denitrificans.
Differentiation of Rickettsia japonica by Restriction Endonuclease Fragment Length Polymorphism Using Products of Polymerase Chain Reaction Amplification with Rickettsia rickettsii 190‐Kilodalton Surface Antigen Gene Primers
Authors: Yan Yansheng , Uchiyama Tsuneo , Uchida Takahiro
Published: November 2013

Restriction fragment length polymorphism of polymerase chain reaction (PCR) amplification products differentiated Rickettsia japonica, a causative agent of Oriental spotted fever, from other spotted fever group (SFG) rickettsiae. Primer pair Rr190. 70p and Rr190. 602n of R. rickettsii 190‐kDa antigen gene sequence primed genomic DNAs obtained from R. japonica, type strain YH and strains NT, NK, YKI, and TKN. The products were cleaved by PstI but not by AfaI restriction endonuclease. The PstI digestion pattern of PCR‐products amplified from all strains of R. japonica was identical and easily differentiated from that of other SFG rickettsiae. The present study demonstrated a genotypic difference between R. japonica and other pathogenic SFG rickettsiae.
An Occurrence of Diarrheal Cases Associated with Group C Rotavirus in Adults
Authors: Oishi Isao , Yamazaki Kenji , Minekawa Yoshiichi
Published: November 2013

Six of the 23 college students who joined a group trip in February of 1991 developed acute nonbacterial gastroenteritis with severe diarrhea. The causal agent was identified as group C rotaviruses by electron microscopy (EM), immune‐EM (IEM) and the molecular examinations including polyacrylamide gel electrophoresis (PAGE) and polymerase chain reaction (PCR) on virus particles detected in the extract of watery fecal specimens of the patients. The patients positive for virus isolation showed significant increase in IEM antibody to the isolated virus in their paired sera. These findings suggest that the group C rotavirus is an important etiological agent of diarrhea and may also cause serious food‐borne diarrheal disease in adults.
Detection of Caries‐Inducing Microorganisms in Hyposalivated Rats without Infection of Mutans Streptococci
Authors: Ooshima Takashi , Yoshida Toshihiko , Hamada Shigeyuki
Published: November 2013

Rampant dental caries was induced in hyposalivated rats fed a high sucrose diet without infection of mutans streptococci, in which increased numbers of lactobacilli and S. aureus were demonstrated in the oral flora. Administration of either penicillin or piperacillin, effective against all isolates of lactobacilli, markedly inhibited the caries induction in these rats, while severe dental caries was induced in hyposalivated rats given vancomycin that is inhibitory against S. aureus. These results suggested that certain lactobacilli might induce dental caries in hyposalivated rats fed a sucrose diet. Three strains of Lactobacillus species isolated from the hyposalivated rats were made resistant to erythromycin. The caries‐inducing activity of these erythromycin‐resistant lactobacilli was studied in hyposalivated rats giving erythromycin in the drinking water at a concentration of 500 μg/ml. After a 61‐day experimental period, severe dental caries was induced in hyposalivated rats infected with L. fermentum TY1R. On the other hand, low caries incidence was found in hyposalivated rats infected with either L. acidophilus TY7R or L. plantarum TY3R. These results indicate that L. fermentum may be one of causative agents of dental caries in hyposalivated rats fed a sucrose diet.
High‐Molecular‐Weight Hyaluronic Acids Inhibit Chemotaxis and Phagocytosis but Not Lysosomal Enzyme Release Induced by Receptor‐Mediated Stimulations in Guinea Pig Phagocytes
Authors: Tamoto Koichi , Nochi Hiromi , Tada Masahito , Shimada Sachiyo , Mori Yoki , Kataoka Syuichi , Suzuki Yoshihiro , Nakamura Tohoru
Published: November 2013

The effects of high‐molecular‐weight (HMW) hyaluronic acids (HAs) of 1.9 × 106 Da, 8 × 105 Da and 3 × 105 Da on the receptor‐mediated functions of guinea pig peritoneal phagocytes were studied. HMW‐HAs of 1.9 × 106 Da (HA190) and 8 × 105 Da (HA80) effectively inhibited the chemotactic activity of polymorphonuclear leukocytes (PMNs) for formyl‐Met‐Leu‐Phe (fMLP). The degree of inhibition was dose‐dependent and the concentrations of HA190 and HA80 required for 50% inhibition were 0.5–1.5 mg/ml and 1.5–2.5 mg/ml, respectively. HMW‐HA of 3 × 105 Da (HA30) hardly affected the chemotaxis within a concentration range of 0.5–5.0 mg/ml. The phagocytic activities of PMNs and macrophages (Møs) for serum‐opsonized zymosan (SOZ) and polystyrene latex particles were also inhibited by these HAs in a dose‐ and molecular‐weight‐dependent manner and HA190 was again the most inhibitory. By contrast, the release of lysosomal enzyme from Møs stimulated with SOZ was not significantly affected by HMW‐HAs at any concentration used. Furthermore, the binding of [3H]fMLP with PMNs and the rosette formation of Møs with SOZ were not influenced by the presence of HMW‐HAs. These findings suggested that the binding of HMW‐HAs to the HA receptors on PMNs and Møs might produce certain intracellular signals which would be responsible for the suppression of the chemotaxis and the phagocytosis but not for the release of lysosomal enzyme. For the generation of such signals, higher‐molecular‐weight HMW‐HAs would be more effective than lower one.
Effect of Escherichia coli Lipopolysaccharide on Bacteroides fragilis Abscess Formation and Mortality in Mice
Authors: Tanaka Yasunori , Jotwani Ravi , Watanabe Kunitomo , Tanaka Kaori , Kato Naoki , Ueno Kazue
Published: November 2013

To study the mechanism of synergism between Bacteroides fragilis and Escherichia coli, the effect of sublethal dose of E. coli lipopolysaccharide (LPS) (25μg/mouse) was checked on B. fragilis abscess formation. LPS was administered prior or after inoculum injection. No significant difference in the abscess size was observed at necropsy on day 6. However, all the groups receiving LPS showed higher incidence of recovery of additional intestinal bacteria (23.5–45.5%) from the abscess pus. When LPS was given 4 hr prior to inoculum administration, 83–100% mortality was observed. Detailed investigation showed autoclaved cecal contents alone could also cause similar mortality. Studies with stimulation of endogenous cytokines by E. coli LPS demonstrated induction of all of them within 3 hr in the blood stream with TNF‐α demonstrating peak at 1 hr, IL‐1α and IL‐6 at 4 hr and IFN‐γ between 6–9 hr with moderately high levels at 4 hr. This E. coli LPS‐triggered cytokine cascade possibly gets further stimulated by injection of autoclaved cecal contents containing high concentration of endotoxins (1.6 × 105 EU/ml) contributed by dead bacteria and lead to the mortality of animals.
Purification and Characterization of an Acid Phosphatase from Mycoplasma fermentans
Authors: Noda Mamoru , Shibata Ken‐ichiro , Sawa Yoshihiko , Shimokoube Hirokata , Watanabe Tsuguo
Published: November 2013

An acid phosphatase associated with the cell membranes of Mycoplasma fermentans was released from the membranes with Triton X‐100, then purified by ion‐exchange chromatography on DEAE‐Sephacel and CM‐Sepharose, followed by affinity chromatography on Con A‐Sepharose. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of the purified enzyme revealed a single band with a molecular mass of 31.2 kilodaltons. The enzyme activity toward p‐nitrophenyl phosphate was enhanced remarkably by Cu2+, Co2+ and Mg2+, but the activity was not inhibited by EDTA. The enzyme dephosphorylated O‐phospho‐l‐tyrosine as well as p‐nitrophenyl phosphate, but not O‐phospho‐l‐threonine, O‐phospho‐l‐serine, glucose‐1‐phosphate, phosphoryl choline and adenosine triphosphate. The level of the O‐phospho‐l‐tyrosine phosphatase activity was the highest in Mycoplasma faucium and the second highest in Mycoplasma fermentans of all tested human mycoplasmas.
Effect of BV‐araU and Acyclovir on Varicella‐Zoster Virus Replication with Various Length and Timing of Drug Exposure
Authors: Machida Haruhiko , Nishitani Makiko , Ashida Noriyuki
Published: November 2013

We studied antiviral effects of 1‐β‐d‐arabinofuranosyl‐5‐[(E)‐2‐bromovinyl]uracil (BV‐araU) and acyclovir against varicella‐zoster virus (VZV) multiplication varying the length or timing of drug exposure. First, residual anti‐VZV effect of drugs, exposed to cells for various periods followed by incubation in drug‐free medium, was determined by the plaque inhibition assay. None of the drugs showed activity when removed within 24 hr of incubation. Weakened efficacy of BV‐araU was seen in 2 days of treatment. When it was removed after 3 or 4 days, the ED50 was as low as that for cultures in which the drug was not removed. Still, plaque inhibition was not complete even at high concentrations. Acyclovir inhibited plaque formation only by 50% or less in 2 days of treatment. It gave a much higher ED50 in 3 days of treatment than that observed without drug removal. In the experiments, in which BV‐araU was added to VZV‐infected cells 1 day after infection, BV‐araU immediately suppressed increase in the number of infective centers at a concentration of 0.001 μg/ml, and reduced it at concentrations of 0.01 μg/ml or higher. The reduction of infective centers was seen with a dose‐dependent manner when added 2 or 3 days after infection. BV‐araU stimulated the decrease in the number of infective centers when added 4 days after infection. This inhibitory effect of acyclovir was very weak. Microscopic observations supported the above results. BV‐araU was still much superior to acyclovir in the anti‐VZV effect when the length and timing of drug exposure were varied.
Protective Capacity of L‐Form Salmonella typhimurium against Murine Typhoid in C3H/HeJ Mice
Authors: Nishikawa Fumiko , Kita Eiji , Yamada Hideki , Nakano Akira , Kashiba Shuzo
Published: November 2013

L forms of Salmonella typhimurium LT2 conferred strong protection to a lethal challenge with its parental bacterium on innately hypersusceptible C3H/HeJ mice, and its minimal protective dose was approximately 150 L‐forming units. Although L‐form S. typhimurium was avirulent for C3H/HeJ mice, it multiplied slowly in both the liver and spleen with the maximal growth 2–3 weeks after immunization and thereafter it persisted in the liver until 24 weeks. Protective immunity began to work between 4 and 6 weeks after immunization, and it remained active as long as the L forms colonized the liver (until 24 weeks after immunization). Vaccination with the L form induced a population of T cells responding to L‐form whole‐cell lysate (WCL), while delayed‐type hypersensitivity (DTH) to the extract of S. typhimurium was induced after the establishment of solid immunity. Moreover, neither T‐cell responses nor DTH to heat‐killed S. typhimurium was generated. In addition, antibody responses were elicited to WCL but not to heat‐killed S. typhimurium. These results indicate that protection conferred by the L forms is attributable to the persistent colonization of the L forms rather than the presence of DTH, and also that Salmonella cytoplasmic antigens are involved in induction of immunological responses by vaccination with the L forms.
Cytoplasmic Membrane‐Associated Protein (CAP) Isolated from Streptococcus pyogenes: As a New Bacterial Superantigen
Authors: Sato Hideki , Itoh Tetsuro , Rikiishi Hidemi , Kumagai Katsuo
Published: November 2013

A protein isolated from the cytoplasmic membranes of Streptococcus pyogenes (cytoplasmic membrane‐associated protein, CAP) stimulated human T cells in vitro to induce their mitogenic response. This CAP‐induced T cell proliferation required the presence of nylon‐adherent accessory cells (AC) of either autologous or allogeneic origin in the reaction mixtures. In addition, the reaction was inhibited by monoclonal antibodies (mAbs) against major histocompatibility complex (MHC) class II molecules, HLA‐DR and ‐DQ, but not ‐DP. Human lymphoid cell lines positive for HLA‐DR but not those lacking it were also effective as AC for the reaction. A binding test using fluorescein‐labeled protein revealed that CAP bound to the adherent monocytes and HLA‐DR+ but not to ‐DR– lymphoid cell lines. The proliferative response of T cells to CAP was, however, not inhibited by the addition of the lysosomotrophic agent NH4Cl to the reaction mixtures. These results suggest that the presentation of CAP by AC to human T cells is mediated through binding of the protein to the MHC class II molecules but without being processed in the AC. The proliferative response of T cells was also found to be inhibited by addition of anti‐CD2, ‐CD3 or ‐T cell receptor (TcR) mAbs. A major population responding to CAP was CD3+4+8– T cells. CAP also appears to stimulate T cells bearing Vβ8 sequences much more selectively than T cells bearing other Vβs. These results indicate that this streptococcal membrane protein, CAP, may be a new protein belonging to a group of bacterial superantigens.
Greater Amount of HCV‐RNA in Tears Compared to Blood
Authors: Feucht Heinz‐hubert , Polywka Susanne , Zöllner Bernhard , Laufs Rainer
Published: November 2013

Plasma, tear fluid and swabs from eye, nose and pharynx of 33 patients were examined for presence of hepatitis C virus (HCV) RNA by polymerase chain reaction (PCR). All samples from plasma, tear fluid and eyeswabs were found to show a positive reaction in HCV‐RNA PCR. Remarkably, we regularly found greater amounts of amplification products in tear fluid and eyeswabs compared to plasma using the same conditions for sample preparation.
Monoclonal Antibodies to Spirosin of Yersinia enterocolitica and Analysis of the Localization of Spirosome by Use of Them
Authors: Yamato Masayuki , Takahashi Yuko , Tomotake Hiroyuki , Ota Fusao , Hirota Katsuhiko , Yamaguchi Kazuo
Published: November 2013

Two hybridomas producing monoclonal antibodies (MAbs) were prepared by fusing myeloma cells (Sp2/0‐Ag14) with mouse spleen cells immunized with purified spirosin from Yersinia enterocolitica SYT‐11–72 (YE72). The antibodies produced by them were designated MAbs‐S5 and S27. They were IgG2a and IgG1, respectively, both with χ light chains. MAbs‐S5 and S27 reacted specifically with spirosin from YE72. On Western blotting after limited proteolysis with Staphylococcus aureus V8 protease, YE72 spirosin revealed peptide fragments of 35 and 37 kDa reacting markedly with MAb‐S5, which suggested the presence of an antigenic determinant on these fragments. By cellular fractionation of YE72 and subsequent EIA and Western blot analysis, spirosome was shown to be present in the cytoplasm of YE72.
Unique Properties of a Cytotoxic CD4+CD8+ Intraepithelial T‐Cell Line Established from the Mouse Intestinal Epithelium
Authors: Sasahara Takeshi , Tamauchi Hidekazu , Ikewaki Nobunao , Kubota Koichi
Published: November 2013

Growth factor‐dependent gut intraepithelial lymphocyte (IEL) cell lines were established from a long‐term in vitro culture of BALB/c IEL with syngeneic irradiated spleen cells in the presence of concanavalin A‐stimulated spleen supernatant fluids. The cell lines were preferentially consisted of very limited thymoindependent subsets of IEL; i.e., Thy‐1+ CD5–TCRαβ+ CD4+CD8 α+β– (double‐positive; DP) IEL and Thy‐1+ CD5– TCRαβ+ CD4–CD8α+β– (CD8 single‐positive; CD8 SP) IEL. The CD8 SP IEL cell line had cytotoxic activities and was triggered to proliferate by T‐cell receptor (TCR)‐directed stimuli. The DP IEL cell line expressed high levels of the CD3‐TCRαβ, exhibited cytotoxic activity in redirected lysis assays, and had perforin in the cytoplasm, indicating the functional maturity of this cell line. However, the DP IEL cell line did not proliferate in response to TCRαβ‐directed stimuli, which indicated that TCRαβ‐mediated signalling was able to initiate cytotoxic function but not to induce proliferation of the DP IEL cell line. Although both cell lines were shown to have functional competence, they expressed J11d antigen which marks immaturity in thymocyte differentiation pathways. These results indicate that the established thymoindependent DP and CD8 SP IEL cell lines have unique properties distinct from DP thymocytes and CD8 SP peripheral T cells. Together with a recent report on freshly isolated DP IEL (10), the unique properties of the DP IEL cell line seems to support the notion that DP IEL may undergo a unique maturation process in the gut microenvironment.
Characterization of Effector Cells against B16 Melanoma in Mice Inoculated with Allogeneic Spleen Cells
Authors: Nishi Yasuaki , Hosokawa Tomohide , Aoike Akira , Han Di , Takehara Hiroshi , Kawai Keiichi , Kamahora Takeshi
Published: November 2013

Inbred C57BL/6 (B6) mice which had received an inoculation of allogeneic spleen cells showed remarkable antitumor activity against syngeneic tumor challenge with B16 melanoma cells 3 days after the allogeneic cell inoculation. This antitumor activity was not specific to the inoculated alloantigen, since the challenging B16 cells are syngeneic to B6 mice and since it was induced by BALB/c spleen cells as well as C3H/He spleen cells. The antitumor activity was sensitive to an in vivo treatment with anti‐asialo GM1 (AGM1) antiserum or anti‐Thy.1 monoclonal antibody (mAb) just before the tumor challenge and was resistant to an in vivo treatment with anti‐CD8 (Ly. 2) mAb. These results suggest that AGM1+Thy.1+CD8– activated natural killer (NK) cells were generated by alloantigen inoculation and took an important part in the antitumor effect of the alloantigen inoculation.
Two G3 Feline Rotavirus Strains Lacking Cross‐Neutralization Reactions Represent Distinct Subtypes of Serotype G3
Authors: Mochizuki Masami , Nakagomi Osamu
Published: November 2013

Two feline rotavirus strains, FRV‐1 and FRV64, that have been shown to lack cross‐neutralization reactions despite the sharing of serotype G3 were examined by plaque‐reduction neutralization assays in relation to other G3 strains originating from cats, dogs, humans and monkeys. While FRV‐1 and human G3 strains constituted one subtype (G3A), FRV64, canine strains and simian strains constituted another subtype (G3B).
Molecular Cloning of the Wild‐Type and Mutant thyA Gene from Shigella flexneri Y
Authors: Nur‐E‐Kamal Mohammed Shah Alam , Mamun Abu Amar Md Al , Ahmed Zia Uddin
Published: November 2013

The thyA gene which codes for thymidylate synthase has been cloned and sequenced from the wild‐type Shigella flexneri Y strain SH4 and a thyA mutant TSF21 after amplifying the gene by polymerase chain reaction (PCR). The nucleotide sequence revealed 98% homology to the E. coli K‐12 thyA gene. The sequence of the wild‐type thyA gene of Shigella flexneri Y was identical with that of the thyA mutant except that the residue T at position 345 was replaced by residue A in the thyA mutant. This change would cause a predicted amino acid substitution of leucine at position 44 in the polypeptide product of the wild type by glutamine in the mutant. Thus, Leu44 may be critical in enzymatic activity of the thyA gene product thymidylate synthase.
Herpes Simplex Virus‐Induced Expression of 70 kDa Heat Shock Protein (HSP70) Requires Early Protein Synthesis But Not Viral DNA Replication
Authors: Kobayashi Kappei , Ohgitani Eriko , Tanaka Yasuyuki , Kita Masakazu , Imanishi Jiro
Published: November 2013

The major 70 kDa heat shock protein (HSP70), which is scarcely expressed in unstressed rodent cells, was apparently induced by infection with herpes simplex virus (HSV). Infection with HSV types 1 and 2 elevated HSP70 mRNA levels within 4 hr post‐infection. HSP70 synthesis and accumulation increased in HSV‐infected cells. Irradiation of HSV with UV‐light abolished the ability to induce HSP70 mRNA. Inhibitors of viral DNA synthesis did not affect the induction of HSP70 in infected cells. Protein synthesis within 2 hr after infection was necessary for HSP70 induction.
Amniotic Fluid Enhances Allogeneic Cytotoxic T Cell Responses, Whereas It Suppresses Mitogen‐Stimulated Lymphocyte Proliferation
Authors: Parvin Mahbuba , Isobe Ken‐ichi , Zohreh Heidari , Goto Setsuko , Nakashima Izumi , Tomoda Yutaka
Published: November 2013

Mouse amniotic fluid has been shown to suppress T lymphocyte proliferation and suggested to be important in regulating immunity during pregnancy. In allogeneic pregnancy, cytotoxic T cells in pregnant lymphocytes against paternal transplantation antigen are impaired. We examined the effect of amniotic fluid to the alloreactive CTL responses. Although the amniotic fluid suppressed Con A or LPS stimulated lymphocyte proliferation as previously reported, the amniotic fluid taken from syngeneic C57BL/6 pregnant mice or allogeneic C57BL/6 × BALB/c pregnant mice enhanced the anti‐H‐2d or anti‐H‐2k CTL responses dose‐dependently. We speculate that amniotic fluid contains not only immunosuppressive factors but also immunoenhancing factors which upregulate the allogeneic CTL responses.
Purification and Characterization of Type 1 Fimbriae of Salmonella typhi
Authors: Muscas Patrizia , Rossolini Gian Maria , Chiesurin Alessandra , Santucci Annalisa , Satta Giuseppe
Published: November 2013

Type 1 fimbriae have been purified from a Salmonella typhi strain of clinical origin. Purified fimbriae retained their ability to bind to erythrocytes in a mannose‐inhibitable fashion and, in doing so, behaved preferentially as a monovalent adhesin. SDS‐PAGE analysis of the fimbrial preparation showed the presence of a 20‐kDa major polypeptide component (fimbrillin) and of additional larger polypeptides present in smaller amounts. The amino‐terminal sequence of fimbrillin was determined and turned out to be very similar but not identical to that of type 1 fimbrillins of other Salmonella serovars. A Western blot analysis of the purified fimbrial preparation using an antiserum raised against native fimbriae suggested that fimbrial proteins did not carry any major sequential epitope and that, in native fimbriae, conformational epitopes, possibly generated between different subunits, might provide for the major immunogenic epitopes. Analysis of different S. typhi clinical isolates using the anti‐fimbrial antiserum showed an overall immunological similarity of these structures within this serovar.
Opsonic Activity of Sera and Blister Fluid from Severely Burned Patients Evaluated by a Chemiluminescence Method
Authors: Ono Yasuo , Kunii Otohiko , Suzuki Hiromasa , Ikeda Hirohito , Kobayashi Kunio , Kanegasaki Shiro
Published: November 2013

Burn wound sepsis is the most common and severe complication in the patients with severe burn. To know the systemic and local defect in immunity of burned patients, we measured the luminol‐enhanced chemiluminescence (CL) response of normal polymorphonuclear leukocytes (PMNs) upon exposure to zymosan particles, bacteria or Candida albicans that were opsonized with any of patient's serum, blister fluid of burn wound or pooled normal serum (blood type AB). Sera from patients exhibited lower opsonic activities than those of pooled normal serum in the early postburn days. The levels of serum immunoglobulins, complement components and plasma fibronectin were found to correlate well with opsonin‐index (OI), which was determined based on the CL response data obtained during the course of infusion therapy with fresh frozen plasma. Furthermore, patient's blister fluid showed much lower opsonic activity against bacteria such as Pseudomonas aeruginosa than patient's own serum. These results indicate that blister fluid is also not effective to opsonize bacteria because of the marked depression of the levels of immunoglobulins and complement components. Destruction of the skin barrier by thermal injury and impairment of systemic or local humoral immunity may predispose these patients to burn wound sepsis.
In Vitro Antibiotic Susceptibilities of Borrelia Isolates from Erythema Migrans Lesion of Lyme Disease Patients in Japan
Authors: Masuzawa Toshiyuki , Yamada Kazuto , Kawabata Hiroki , Yanagihara Yasutake
Published: November 2013

Antibiotic susceptibilities of twelve borrelial isolates from skin of patients with erythema migrans (EM) and ticks (Ixodes persulcatus and I. ovatus) in Japan were examined by in vitro microdilution MIC method and macrodilution MBC method. Nine EM isolates and 3 tick isolates were susceptible to amoxicillin, erythromycin, and minocycline. MICs for Japanese isolates were 0.038–0.30 μg/ml, < 0.012 μg/ml, and < 0.012–0.05 μg/ml, respectively. MBCs were as follows: 0.038–0.88 μg/ml, < 0.012–0.10 μg/ml, and <0.025–0.78 μg/ml, respectively. These antibiotics could be recommended for treatment of patients in early stage of Lyme disease in Japan.
Heat Shock Protein Produced by Helicobacter pylori
Authors: Yokota Kenji , Hirai Yoshikazu , Haque Mahmudul , Hayashi Shyunji , Isogai Hiroshi , Sugiyama Toshiro , Nagamachi Eiko , Tsukada Yutaka , Fujii Nobuhiro , Oguma Keiji
Published: November 2013

The cells of Helicobacter pylori were suspended in the medium containing35S‐methionine. After a heat shock of the cells at 42 C for 5, 10, and 30 min, the production of proteins was analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and autoradiography. Out of many proteins produced by the cells, only 66 kDa protein production was dramatically increased by heat treatment. The N‐terminal amino acid sequence of 66 kDa protein was quite similar to that of 62 kDa and 54 kDa proteins previously suggested as heat shock protein (HSP) of H. pylori based on the reaction with polyclonal and monoclonal antibodies against HSP 60 family proteins produced by other bacteria. Therefore, it was concluded that H. pylori produces the 66 kDa protein as its major heat shock protein which belongs to HSP 60 family.
Lidocaine Hydrochloride and Acetylsalicylate Kill Bacteria by Disrupting the Bacterial Membrane Potential in Different Ways
Authors: Ohsuka Shinji , Ohta Michio , Masuda Koji , Arakawa Yoshichika , Kaneda Toshio , Kato Nobuo
Published: November 2013

Lidocaine hydrochloride (LH), a local anesthetic, and acetylsalicylate (AcSAL), show antibacterial activity for both gram‐negative and gram‐positive bacteria. Kinetic studies indicated that antibacterial activity of LH was different from that of AcSAL. A subinhibitory concentration of LH and AcSAL enhanced the sensitivity of Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa to novobiocin and nalidixic acid. The synergistic effect of AcSAL with novobiocin and nalidixic acid was higher than that of LH. The effect of both drugs on the membrane potential of inner membrane was also studied using inverted membrane vesicles of bacteria. Both LH and AcSAL depolarized the membrane potential after the vesicles were energized with nicotinamide adenine dinucleotide. However, unlike AcSAL, pre‐treatment of vesicles with LH had no effect on the generation of membrane potential. These results suggest that depolarization of the cytoplasmic membrane, preceded by the permeabilization of the outer membrane for gram‐negative bacteria, is associated with antibacterial activity of LH and AcSAL. The difference in actions of LH and AcSAL was discussed.
Specific Detection of a Verotoxin 2 Variant, VT2vp1, by a Bead‐Enzyme‐Linked Immunosorbent Assay
Authors: Cao Chunyu , Yamasaki Shinji , Lin Zaw , Kurazono Hisao , Takeda Yoshifumi
Published: November 2013

A bead‐enzyme‐linked immunosorbent assay to specifically detect a Verotoxin 2 variant, VT2vpl, was developed. The sensitivity of the bead‐ELISA was 200 pg/ml of the purified VT2vpl and it did not react with 20 ng/ml of the purified VT2. The specificity of the bead‐ELISA was examined with 107 strains of Verocytotoxin‐producing Escherichia coli that include VT1‐, VT2‐, VT2vha‐, VT2vhb‐ and VT2vp1‐producing E. coli, and only VT2vp1‐producing E. coli that were confirmed by VT2vp1‐specific polymerase chain reaction gave positive results. It was noted that all 58 VT2vp1‐producing E. coli strains were from pigs, but not from cows and humans.
Existence of a Novel Hemagglutinin Having No Protease Activity in Vibrio mimicus
Authors: Alam Munirul , Miyoshi Shin‐ichi , Maruo Ikuyo , Ogawa Chiemi , Shinoda Sumio
Published: November 2013

The protease elaborated by Vibrio mimicus is known to possess hemagglutinating ability to chicken erythrocytes, the well‐known HA/protease. A non‐protease hemagglutinin (HA) with strong agglutinating ability towards rabbit erythrocytes was obtained from 32 hr culture supernatant of a pathogenic environmental strain of V. mimicus. This HA (V. mimicus HA: VMHA) appeared stable at relatively higher temperature and agglutinated the erythrocytes from rabbit, guinea pig and mouse but not the erythrocytes from chicken, bovine, horse and sheep. Simple sugars, metal ions and chelating agents failed to inhibit the activity of VMHA. The activity of VMHA was found to be sensitive to digestion by proteolytic enzymes including HA/protease. These results provide evidence for the existence of novel HA other than HA/protease in V. mimicus.
Detection and Characterization of Antibodies to Bacterial Heat‐Shock Protein 60 in Sera of Patients with Primary Biliary Cirrhosis
Authors: Yamaguchi Hiroyuki , Miura Hitoshi , Ohsumi Kazuoki , Ishimi Norikuni , Taguchi Haruhiko , Ishiyama Narihiro , Shiraishi Yasumori , Yamamoto Tomoko , Ogata Sachio
Published: November 2013

The enzyme‐linked immunosorbent assay (ELISA) with bacterial heat‐shock protein 60 (HSP60) purified from Yersinia enterocolitica (Ye) revealed that the antibodies directed against YeHSP60 existed in sera of patients with primary biliary cirrhosis (PBC). To characterize the epitope specificity of the antibodies in patients, the epitope mapping of HSP60 by means of the antibodies was performed. The results have suggested that the epitope recognized with anti‐HSP60 antibodies in PBC relates to the amino acid sequence of YeHSP60 molecule as follows: DLGQA‐KRVVINKDTTIIIDGVGDEAAIQGRLAQIRQQIEEATSDYDKEK.
Genotyping of Staphylococcus epidermidis by Small‐Fragment Restriction Endonuclease Analysis and Pulsed‐Field Gel Electrophoresis of Genomic Restriction Fragments
Authors: Haertl Rolf , Bandlow Gert
Published: November 2013

Small‐fragment restriction endonuclease analysis (SF‐REA) was established as a typing tool for Staphylococcus epidermidis. A total of 60 isolates comprising 48 epidemiologically nonrelated strains and 12 putatively linked isolates from 7 patients in 2 wards were analyzed. Nonrelated isolates were characterized by unique fingerprints when DNA was cleaved with EcoRI or ClaI, electrophoretically separated in a polyacrylamide gel, and silver stained. Three blood culture isolates from one patient in an intensive care unit, 4 isolates obtained from a child over a span of 2 weeks, and 5 isolates from 5 newborns in the same ward were grouped into 3 DNA pattern types, indicating identity of sequential isolates from 2 patients and nosocomial transmission of one Staphylococcus epidermidis strain between 5 babies. Results from pulsed‐field gel electrophoresis of SmaI and SacII DNA digests and conventional marker systems such as antibiogram and plasmid profile were in accordance with these interpretations, whereas slight variation was observed in the biotypes of several strains. From the results of this study, we conclude that SF‐REA is a precise and efficient method for the genotypic characterization of Staphylococcus epidermidis strains that can be used as a rapid and reliable typing tool.
Pig Intestinal Membrane‐Bound Receptor (Guanylyl Cyclase) for Heat‐Stable Enterotoxin: cDNA Cloning, Functional Expression, and Characterization
Authors: Wada Akihiro , Hirayama Toshiya , Kitao Saori , Fujisawa Jun‐ichi , Hidaka Yuji , Shimonishi Yasutsugu
Published: November 2013

A cDNA encoding the receptor protein for a heat‐stable enterotoxin (STa) produced by enterotoxigenic Escherichia coli was cloned from intestinal epithelial cells of a 10‐week‐old pig. The cDNA had an open reading frame of 3,219 base pairs and coded for a protein with 1,073 amino acid residues. The mature protein consisted of 1,050 amino acid residues with a molecular mass of ca. 121 kDa and was 87% and 82% identical with the human and rat protein, respectively. The CHO cell line overexpressing the pig recombinant STa receptor specifically bound to a photoaffinity‐labeled analog of STa and showed marked elevation of the cellular content of cGMP in response to STa.
Stimulation of Macrophage Oxygen Free Radical Production and Lymphocyte Blastogenic Response by Immunization with Porins
Authors: Tabaraie Bahman , Sharma Bal Krishan , Sharma Praveen Rishi nee , Sehgal Rakesh , Ganguly Nirmal Kumar
Published: November 2013

Porins were prepared from smooth strain of Salmonella typhi 0–901 and chemotype rough mutant of S. typhimurium Ra‐30. Porins could significantly stimulate the immune systems of mice. Immunization of mice with the porins provoked synthesis of anti‐porin antibodies. Macrophages from the immunized mice showed increased capacity to generate oxygen free radicals, and lymphocytes from these mice showed proliferative response to the porins. Thus porins may play a role in providing protection from salmonellosis by stimulating the antibody production and increasing the capacity of macrophages to generate oxygen free radicals along with stimulation of lymphocytes.
Analysis of Major Surface Polypeptides of Rickettsia japonica
Authors: Uchiyama Tsuneo , Uchida Takahiro , Walker David H.
Published: November 2013

Major surface polypeptides of Rickettsia japonica migrated to the position of 120, 135, and 145 kDa on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, when the organisms were solubilized at room temperature. Two major bands at the position of 135 and 185 kDa were seen, when the organisms were solubilized by heating before electrophoresis. Heat‐denaturation of the 120‐ and 145‐kDa polypeptides in excised gel bands changed their mobility and caused them to migrate to 135‐ and 185‐kDa positions, respectively. Two polypeptides at the 120‐kDa position were demonstrated: one is a major heat‐modifiable polypeptide and the other a minor heat‐stable. Peptide mapping was performed to determine the identity between native and denatured polypeptides.
Molecular Comparison of Dengue Type 1 Mochizuki Strain Virus and Other Selected Viruses Concerning Nucleotide and Amino Acid Sequences of Genomic RNA: A Consideration of Viral Epidemiology and Variation
Authors: Zulkarnaini Edward , Hotta Susumu , Takegami Tsutomu
Published: November 2013

Dengue‐1 (D1) Mochizuki strain was examined for its nucleotide and amino acid sequences of genomic RNA and the data obtained were compared with those of other selected virus strains reported previously. Genomic regions corresponding to C, preM and M proteins were the major subjects of study. Parts of E protein were additionally examined. Among the D1 viruses investigated, the Mochizuki virus which was isolated in 1943 in Japan was shown to be close to Philippine 836‐1 strain isolated in 1984 and Nauru Island strain isolated in 1974 at the respective places, in contrast with Thai AHF 82‐80 strain isolated in 1980 and Caribbean CV1636/77 strain isolated in 1977. At the same time, a difference was noted between the Mochizuki and Philippine/Nauru strains at the cleavage site of preM/M junction: Mochizuki possessed RRGKR/S sequence whereas the Philippine/Nauru had RRDKR/S. The glycosylation site in preM and hydrophobic regions at the carboxyl termini of M and E were well conserved. Significances of the data are discussed in connection with viral epidemiology and variation.
Detection of DNA of Causative Agent of Spotted Fever Group Rickettsiosis in Japan from the Patient's Blood Sample by Polymerase Chain Reaction
Authors: Tange Yoshiki , Matsumoto Mitsuru , Okada Takanori , Hasegawa Hitoshi , Kobayashi Yuzuru
Published: November 2013

The polymerase chain reaction (PCR) was applied for the etiological diagnosis of spotted fever group (SFG) rickettsiosis in Japan. Nucleotide primers derived from the 17‐kDa antigen gene of Rickettsia rickettsii primed a rickettsia‐specific 246‐base‐pair product for all of the Katayama, Abe, Misaka and Kojima strains, which we had isolated previously. Moreover, we were able to detect the same product by PCR amplification from the peripheral blood of a patient in the acute stage of the illness. The PCR method is considered to be useful for rapid etiological diagnosis of SFG rickettsiosis in Japan.
Comparison of Cytokine Induction by Lipopolysaccharide of Bacteroides fragilis with Salmonella typhimurium in Mice
Authors: Jotwani Ravi , Tanaka Yasunori , Watanabe Kunitomo , Tanaka‐Bandoh Kaori , Kato Naoki , Ueno Kazue
Published: November 2013

Comparison of cytokine stimulation by lipopolysaccharide (LPS) of Bacteroides fragilis and Salmonella typhimurium was done to study the early events occurring in vivo. Mice injected intraperitoneally with either LPS demonstrated endogenous production of all the cytokines studied (tumor necrosis factor‐alpha, interferon‐gamma and interleukin‐6) within 6 hr in the bloodstream. However induction of all the cytokines by B. fragilis LPS (50 μg/mouse) was much weaker compared with S. typhimurium LPS (50 μg/mouse). Even a dose of S. typhimurium LPS 40 times smaller (1.2 μg/mouse) induced cytokines more strongly compared with B. fragilis LPS. Thus, a weak biological response to B. fragilis LPS as evidenced by chick embryo lethality, limulus lysate gelation, LD50 for mice and rabbit pyrogenicity could be due to weak induction of bioactive mediators by LPS.
Determination of the Agretopic Residues of a Peptide Co‐Restricted to Different Class II Isotypes, I‐Au and I‐Eu, and Its Application for Preparation of a Synthetic Peptide Vaccine against Influenza Virus A/Aichi/2/68
Authors: Takami Kimitaka , Ogasawara Kazumasa , Itoh Yasushi , Kajino Kiichi , Naruse Hirohito , Onoé Kazunori
Published: November 2013

We have defined that residues 46 and 54 on a synthetic peptide composed of residues 43–58 of pigeon cytochrome c (p43–58) work as agretopes (sites bound to an MHC molecule) in I‐Ab mice. Substitution of amino acid residues on these positions altered the peptide to bind with the other MHC molecules. Furthermore, by substituting the agretopic residues with a variety of amino acids, we could determine the class II binding motif for each MHC molecule. In the present study, immunogenicity of a peptide, 46R50V54A, carrying valine (V) at epitopic (site bound to TCR) position 50, arginine (R) and alanine (A) at agretopic positions 46 and 54 of the p43–58, respectively has been analyzed in B10.PL (H‐2u) mice. We found that this peptide bound to two different class II isotypes, I‐Au and I‐Eu. Arginine at position 46 or alanine at position 54 of the 46R50V54A was shown to be critical for binding to I‐Au or I‐Eu, respectively. Further, on the basis of this class II binding motif we could prepare potent peptide vaccines against influenza A/Aichi/2/68 virus in B10. PL mice.
CD8+ T Lymphocytes Are the Major Cell Population Involved in the Early Gamma Interferon Response and Resistance to Acute Primary Toxoplasma gondii Infection in Mice
Authors: Shirahata Toshikazu , Yamashita Takumi , Ohta Chikako , Goto Hitoshi , Nakane Akio
Published: November 2013

Gamma interferon (IFN‐γ) is known to be a major mediator influencing host defense against Toxoplasma (T.) gondii. To evaluate lymphocyte populations involved in this cytokine‐mediated early resistance to T. gondii, the effects of in vivo administration of monoclonal antibodies (MAbs) against T‐cell subsets and anti‐asialo GM1 antibody on the course of infection and IFN‐γ response were investigated in mice infected acutely with this parasitic protozoan. A single injection of anti‐CD8 MAb on day −1 or day 4 severely exacerbated the infection, in accordance with a marked suppression of endogenous IFN‐γ production. Moreover, the administration of anti‐IFN‐γ MAb on day 0 but not later than day 4 resulted in a total abrogation of resistance to T. gondii, suggesting that endogenous IFN‐γ produced during the first several days of infection is critical for the generation of antitoxoplasmal resistance in mice. In contrast, no significant increase in mortality was observed when injected with either anti‐CD4 MAb or anti‐asialo GM1 antibody on day − 1, while these antibodies reduced significantly the ability of mice to produce IFN‐γ. Indeed, simultaneous depletion of CD4+ and CD8+ cells had no greater suppressive effect on host defense and endogenous IFN‐γ production than depletion of CD8+ cells alone. Together, these results suggest that CD8+ T cells play a central role for resolution of acute toxoplasmosis by participating in endogenous IFN‐γ production. The possible role of early produced IFN‐γ in the development of protective immune response to T. gondii is also discussed.