bluuebunny/crossref_metadata_2025 huggingface dataset:
Extracellular Antigens of Actinobacillus actinomycetemcomitans Y4 in Severe Alveolar Bone Loss Patients Studied by Two‐Dimensional Electrophoresis and Western Blots
Authors: Tani Yoshiko , Tani Masahiko , Yamada Takeshi , Kato Ihachi
Published: November 2013

The human immune response to extracellular substances (ES) from Actinobacillus actinomycetemcomitans Y4 were analyzed. Twenty‐nine periodontal patients with generalized severe alveolar bone loss and 13 healthy volunteers were examined for serum IgG antibody titers against ES. Among the patients, 17 had higher antibody titers than healthy individuals (high‐titer patients) but the remainder (low‐titer patients) did not. Two‐dimensional electrophoresis (2DE) and Western blots demonstrated that two proteins (40 and 37 kDa) and three smeared substances reacted with IgGs from high‐titer patients, but not with IgGs from healthy volunteers. The low‐molecular‐mass smear at the acid side reacted with over 90% of all patients. This smear reacted with anti‐A. actinomycetemcomitans lipopolysaccharide (LPS) monoclonal antibody which recognized LPS from each A. actinomycetemcomitans serotype. The high molecular mass smear at the acid side might be serotype‐specific O‐antigens of LPS. Another high molecular mass smear which located from the alkaline to the neutral side reacted with anti‐serotype‐b‐specific capsular polysaccharide monoclonal antibody. Moreover, the 40‐ and 37‐kDa proteins reacted with this anti‐capsular polysaccharide antibody. This results suggested that 40‐and 37‐kDa proteins which reacted with 100% or 88% of high‐titer patients might be glycoproteins linked with capsular polysaccharide.
Acute and Chronic Experimental Trypanosoma cruzi Infection in the Rat. Response to Systemic Treatment with Recombinant Rat Interferon‐Gamma
Authors: Revelli Silvia , Davila Hector , Ferro Maria E. , Romero‐Piffiguer Marta , Musso Orlando , Valenti Jose , Bernabo Jorge , Falcoff Ernesto , Wietzerbin Jeanne , Bottasso Oscar
Published: November 2013

We examined the effects of recombinant rat inteferon‐gamma (IFN‐γ) injections on the parasitologic, serologic, immunologic and histopathologic features of acute and chronic experimental Trypanosoma cruzi (T. cruzi) infections in “l” rats. Upon infection at weaning, two rat groups were allocated to receive a 20‐day cycle of IFN‐γ injections, 20,000 IU/rat each, which started at 1, and 7 days post‐infection (pi). Treatment with IFN‐γ, initiated at either 1 or 7 days pi, resulted in comparatively lower peak parasitemias (P<0.02) but in similar levels of anti‐T. cruzi circulating antibodies and serum IFN‐γ activities. The latter appeared significantly increased during acute infection whereas biologically active tumor necrosis factor was virtually undetectable in serum from infected rats regardless of whether they had been given IFN‐γ or not. The prevalence of chronic focal myocarditis in IFN‐γ‐treated infected rats showed no differences with respect to the one recorded in control‐infected counterparts. The inverse CD4/CD8 ratio of spleen and lymph node T cells that usually accompanies chronic infection was reversed by IFN‐γ. Mononuclear cells carrying class III‐A and I‐E molecules, that were found to have increased at both compartments, appeared also modified upon IFN‐γ treatment with an overincrease of I‐A‐positive cells, and a normalization of I‐E‐bearing cells.
Electron Microscopic Examination of Factors Influencing the Expression of Filamentous Surface Structures on Clinical and Environmental Isolates of Aeromonas veronii biotype sobria
Authors: Kirov Sylvia M. , Jacobs Irene , Hayward Laura J. , Hapin Rosario H.
Published: November 2013

Strains of Aeromonas veronii biotype sobria isolated from clinical and environmental sources were examined for their expression of surface structures under a variety of culture conditions. When grown on solid media at 37 C, more than 95% of bacteria from the majority of strains isolated from human diarrheal feces and chicken carcasses were non‐piliated or expressed only a few pili of long, flexible morphology per cell. Strains isolated from water or other foods were much more likely to express pili. Heavily piliated strains (all sources) possessed pili of several morphological types, including long, flexible pili of varying widths and rigid pili of varying lengths. Expression of pili was favored by growth at temperatures ca. 20 C and below and growth in liquid medium. Most fecal strains expressed some pili under these conditions. In addition, other surface structures (fibrillar aggregates, fibrillar networks, bundle‐forming pili) were seen on some strains from most sources. These were also seen most frequently when bacteria were grown in liquid media at temperatures ca. 20 C and below. Pili expression was not dramatically influenced by growth under anaerobic conditions, or in iron‐depleted media, or by combinations of the above conditions. The role of the above surface structures in Aeromonas pathogenicity remains to be elucidated.
Sheep Red Blood Cell Instillation at Palatine Tonsil Effectively Induces Specific IgA Class Antibody in Saliva in Rabbits
Authors: Fukuizumi Takaki , Inoue Hiromasa , Anzai Yuichi , Tsujisawa Toshiyuki , Uchiyama Choji
Published: November 2013

We have shown that the palatine tonsil effectively incorporates exogenous foreign substances instilled at its surface. It is not clear whether antigen‐specific IgA can be induced by the instillation. Sheep red blood cells (SRBC) were instilled at the palatine tonsil every three days as the antigen, and the agglutination titer of specific IgA in saliva was examined. Nasal or intragastric administration, which have been shown to induce specific antibody in saliva, were done as control experiments. Anti‐SRBC antibody in saliva from the tonsillar instillation group was detected in the second week, and the agglutination titer reached a maximum in the 6th week after the instillation. The maximum titers in the tonsillar instillation group and nasal administration group were 16 (P<0.01, n=7) and 4 times (P<0.01, n = 7) higher, respectively, than that in the intragastric administration group. In the tonsillar instillation group, the number of specific antibody‐producing cells per 105 lymphocytes was the highest in the parotid glands compared with the lymphoid tissues such as the retropharyngeal lymph nodes, nasal mucosa, mesenteric lymph nodes, Peyer's patches, cervical lymph nodes, palatine tonsil and spleen. In the nasal administration group, the number of lymphocytes was the highest in the nasal mucosa. The results indicate that tonsillar instillation was more effective than nasal administration in inducing specific IgA in saliva.
A Flow Cytometric Assay Reveals a Suppression of Phagocytosis by Rabbit Defensin NP‐3A in Mouse Peritoneal Macrophages
Authors: Ichinose Mitsuyuki , Sawada Masashi
Published: November 2013

Phagocytosis of fluorescein isothiocyanate (FITC)‐labeled polystyrene microparticles by peritonea] macrophages from thioglycollate‐elicited mice was examined by means of flow cytometry (FCM). This assay revealed that rabbit defensin NP‐3A suppressed the phagocytosis in a dose‐dependent manner. The present results suggest that NP‐3A released from neutrophils is one of the mediators which modulates the activity of macrophages in response to infection.
Distribution of Virulence Factors in Escherichia coli Isolated from Urine of Cystitis Patients
Authors: Yamamoto Shingo , Tsukamoto Teizo , Terai Akito , Kurazono Hisao , Takeda Yoshifumi , Yoshida Osamu
Published: November 2013

The distribution of 7 urovirulence factors, such as type 1 pilus (pil), pilus associated with pyelonephritis (pap), S fimbriae (sfa), afimbrial adhesin I (afaI), hemolysin (hly), aerobactin (aer) and cytotoxic necrotizing factor 1 (cnf1) was examined by a DNA colony hybridization test among 194 Escherichia coli strains isolated from the urine of cystitis patients and in 80 strains isolated from the stool specimens of healthy adults. All virulence factors examined, except pil, were significantly more frequently detected among the cystitis isolates than among the fecal isolates. When individual virulence factors were analyzed against the others, an association was discernible which was not apparent when all 7 virulence factors were considered collectively. There was an apparent correlation between the genotypes and serotypes of the E. coli strains from the cystitis patients. From the data presented, it was proposed that genetic detection of virulence factors would be useful for rapid diagnosis of cystitis, especially in patients without severe pyuria or bacteriuria.
Isolation of Cytopathic Small Round Virus (Aichi Virus) from Pakistani Children and Japanese Travelers from Southeast Asia
Authors: Yamashita Teruo , Sakae Kenji , Kobayashi Shinichi , Ishihara Yuichi , Miyake Takashi , Mubina Agboatwalla , Isomura Shin
Published: November 2013

Aichi virus was isolated in Vero cells from 5 (2.3%) of 222 Pakistani children with gastroenteritis but none was found in 91 healthy children. Aichi virus was also isolated from 5 (0.7%) of 722 Japanese travelers returned from tours to Southeast Asian countries and complained of gastrointestinal symptoms at the quarantine station of Nagoya International Airport in Japan. Of 5 Japanese travelers, 3 were returning from Indonesia, and 2 from Thailand or Malaysia. These results indicate that Aichi virus or a similar agent is endemic in Southeast Asian countries and is a cause of gastrointestinal symptoms in children in these areas or in Japanese travelers who visit there.
The Release of Outer Membrane Vesicles from the Strains of Enterotoxigenic Escherichia coli
Authors: Wai Sun Nyunt , Takade Akemi , Amako Kazunobu
Published: November 2013

The clinically isolated heat labile enterotoxin (LT)‐producing strains of Escherichia coli can be separated into two groups, namely spontaneous LT‐releasing strain and non‐spontaneous LT‐releasing strain, based on their phenotypes of spontaneous release of LT into the culture medium. The phenotype of spontaneous LT release was observed to correlate closely with the phenotype of the release of numerous small vesicles into the culture medium. Both morphological and biological examinations of the vesicle showed that the vesicle was released from the outer membrane. It can, therefore, be assumed that LT may be released from the cell at the time the vesicles form.
Detection of Methicillin‐Resistant Staphylococcus aureus (MRSA) with Antibodies against Synthetic Peptides Derived from Penicillin‐Binding Protein 2′
Authors: Sekiguchi Kiyoshi , Saito Michihiro , Yajima Ryuichi
Published: November 2013

Ten kinds of peptides (21 to 32 amino acids in length) were synthesized based on the reported amino acid sequences of the penicillin‐binding protein 2′ (PBP2′) of methicillin‐resistant Staphylococcus aureus (MRSA). Antibodies against these synthetic peptides (SPs) were generated by immunizing rabbits. The antibodies raised against all the peptides except for one reacted to PBP2′ of MRSA and to SPs used for immunization but not to any other protein of MRSA or methicillin‐susceptible S. aureus (MSSA) tested by ELISA and Western blotting. A sandwich immunoradiometric assay (IRMA) for the detection of PBP2′ was developed using these antibodies. The method could detect PBP2′ extracted from as few as 3 × 104 cells of a clinical MRSA isolate, and a good correlation between cell number and signal radio‐count was observed. IRMA was positive for all 51 methicillin‐resistant staphylococci isolated from patients, and was negative for all the 28 methicillin‐susceptible ones and 19 strains of other bacterial species. IRMA could be a simple and reliable method for MRSA detection in the clinical bacterial laboratory.
Segmented Filamentous Bacteria Are Indigenous Intestinal Bacteria That Activate Intraepithelial Lymphocytes and Induce MHC Class II Molecules and Fucosyl Asialo GM1 Glycolipids on the Small Intestinal Epithelial Cells in the Ex‐Germ‐Free Mouse
Authors: Umesaki Yoshinori , Okada Yasushi , Matsumoto Satoshi , Imaoka Akemi , Setoyama Hiromi
Published: November 2013

In ex‐germ‐free mice conventionalized by association with fecal microorganisms, the induction of major histocompatibility complex class II molecules and fucosylation of asialo GM1 glycolipid occur in the small intestinal epithelial cells (IEC). The intestinal intraepithelial lymphocytes (IEL), especially αβ T‐cell receptor‐bearing ones, also remarkably expand and show cytolytic activity. In this study, we investigated the immunological and physiological characteristics of the small intestine induced by a kind of indigenous bacteria of the small intestine, segmented filamentous bacteria (SFB), among chloroform‐resistant intestinal bacteria. Monoassociation of SFB with germ‐free mice was confirmed by the determination of the base sequences of polymerase chain reaction products of 16S rRNA genes of the fecal bacteria of these mice and in situ hybridization using fluorescein‐labeled probes based on them. SFB increased the number of αβTCR‐bearing IEL and induced Thy‐1 expression and cytolytic activity of IEL. The induction of MHC class II molecules and fucosyl asialo GM1 glycolipids and the increases in the mitotic activity and the ratio of the number of columnar cells to those of goblet cells also occurred in the small intestinal epithelial cells on monoassociation of these bacteria. SFB are important indigenous bacteria for the development of the mucosal architecture and immune system in the small intestine, at least in mice.
Inactivation of Lactobacillus Bacteriophage PL‐1 by Microwave Irradiation
Authors: Kakita Yukari , Kashige Nobuhiro , Murata Kunihiko , Kuroiwa Ataru , Funatsu Michiko , Watanabe Kenji
Published: November 2013

The effect of microwave irradiation on the survival of bacteriophage PL‐1, which is specific for Lactobacillus casei, was studied using a commercial 2,450 MHz microwave oven. The phages were inactivated by microwave irradiation according to almost first‐order reaction kinetics. The rate of phage inactivation was not affected by the difference in the continuous or intermittent irradiation, nor by the concentrations of phages used, but was affected by the volume of phage suspensions, which prevented the loss of generated heat. Microwave irradiation of phage suspensions produced a number of ghost phages with empty heads, but fragmentation of the tail was hardly noticed. The breakage of phage genome DNA was primarily caused by the heat generated by microwave irradiation, whereas the phage DNA was not affected by the same temperature achieved by heat from outside. Thus we concluded that the phage‐inactivating effect of microwave irradiation was mainly attributed to a thermal microwave effect, which was much stronger than a simple thermal exposure.
Humoral Immunity to Commensal Oral Bacteria: Quantitation, Specificity and Avidity of Serum IgG and IgM Antibodies Reactive with Actinobacillus actinomycetemcomitans in Children
Authors: Cole Michael F. , Fitzsimmons Sean P. , Sheridan Michael J. , Xu Yong
Published: November 2013

The levels, specificity and avidities of serum IgM and IgG antibodies reactive with Actinobacillus actinomycetemcomitans (Aa) serotypes a, b and c were determined in periodontally healthy (PH) children and compared with subjects with localized juvenile periodontitis (LJP). All PH children exhibited IgM and IgG Aa‐reactive antibodies whether or not Aa was detected subgingivally but the antibodies were not specific for Aa. In contrast, LJP sera contained high concentrations of IgM and IgG antibodies reactive with Aa that were largely specific for this bacterium. IgM and IgG antibodies in both PH and LJP subjects were of low avidity. With one exception, the avidities of IgG anti‐Aa antibodies were significantly greater than those of IgM antibodies in both PH and LJP subjects. However, although the LJP subjects had as much as 115‐fold more Aa‐reactive IgG antibody than did the PH subjects the avidities of their IgG antibodies were no greater than those of the PH group. The induction by the host of low‐avidity antibodies, that are ineffective in immune elimination, may be a reason why commensal bacteria persist at mucosal surfaces and why persons with LJP fail to eliminate Aa from their periodontal pockets.
Alteration of Bacteriolytic Enzyme Profile of Staphylococcus aureus during Growth
Authors: Komatsuzawa Hitoshi , Sugai Motoyuki , Nakashima Seiji , Suginaka Hidekazu
Published: November 2013

Profiles of cell‐associated bacteriolytic activities and those in the culture supernatant of Staphylococcus aureus FDA209P at various stages of growth were analyzed using sodium dodecyl sulfate‐polyacrylamide gels containing Micrococcus luteus or S. aureus. In the logarithmic growth phase, the cell‐associated bacteriolytic activities extracted with Triton X‐100 contained a number of bacteriolytic proteins, the profiles of which were similar to those we reported elsewhere (Sugai, M., Akiyama, T., Komatsuzawa, H., Miyake, Y., and Suginaka, H.(1990) J. Bacteriol., 172, 6494‐6498). The proteins include P1, P2, P7, P9, PX, P13, P18 and other minor components. At the stationary growth phase, the bacteriolytic band‐profile of the Triton X‐100 extract changed dramatically. P1, P7 and P9 disappeared, and the other minor bands had markedly decreased band intensities. On the other hand, P2, PX, P13, and P18 retained their band intensities during the stationary growth phase. The band intensities of P7, P13, PX, and P18 increased in the supernatant during the logarithmic growth phase. These results indicated that the bacteriolytic band‐profile changes during growth.
Antiviral Activity of Trichothecene Mycotoxins (Deoxynivalenol, Fusarenon‐X, and Nivalenol) against Herpes Simplex Virus Types 1 and 2
Authors: Tani Naoto , Dohi Yoshiko , Onji Yoshiki , Yonemasu Kunio
Published: November 2013

The effect of trichothecene mycotoxins, deoxynivalenol (DON), fusarenon‐X (FX) and nivalenol (NIV), on plaque formation of herpes simplex virus types 1 and 2 (HSV‐1 and HSV‐2) in HEp‐2 cells was examined. The 50% effective concentrations (EC50) of DON, FX, and NIV for HSV‐1 plaque formation were 160, 56, and 120 ng/ml, respectively. Those for HSV‐2 plaque formation were 94, 26, and 50 ng/ml, respectively. These three mycotoxins showed about 2‐fold higher selectivity to HSV‐2 than to HSV‐1. Plaque formation of HSV‐1 was not inhibited with trichothecenes at concentrations completely inhibiting plaque formation when cells were treated during virus adsorption period or 15 hr before infection. These results indicate that trichothecenes affect replication of HSV‐1 after virus adsorption, but not before or during virus adsorption to the host cells.
Suppression of Cytokine Production in T Helper Type 2 Cells by Nitric Oxide in Comparison with T Helper Type 1 Cells
Authors: Nukaya Ikuei , Takagi Kuniaki , Kawabe Takefumi , Suketa Yasunobu
Published: November 2013

We examined the effect of nitric oxide (NO) on cytokine production in T helper (Th) cell subsets, using murine splenic CD4+ T cells and two types of Th clones. Interferon‐gamma‐treated murine peritoneal exudate cells (IFN‐PEC) suppressed DNA synthesis to 60% of the control level in CD4+ T cells stimulated with the anti‐CD3 monoclonal antibody. The production of IL‐2 and IL‐4 in the CD4+ T cells decreased to 63.2% and 9.2%, respectively, of the control value by co‐culture with IFN‐PEC. The addition of NG‐monomethyl‐L‐arginine (L‐NMMA) partially recovered the suppression of DNA synthesis. In the presence of indomethacin, the suppression of DNA synthesis was partially inhibited and the reduction in the cytokine production caused by IFN‐PEC was partially recovered. The simultaneous addition of NG‐monomethyl‐L‐arginine (L‐NMMA) and indomethacin completely inhibited not only the suppression of DNA synthesis but also the reduction in the cytokine production caused by IFN‐PEC. Moreover, DNA synthesis in the Th2 clone was suppressed to a greater extent than that in the Th1 clone by co‐culture with IFN‐PEC. This suppression in the Th1 clone was inhibited by the addition of L‐NMMA, whereas the DNA synthesis in the Th2 clone was not recovered by L‐NMMA. In addition, sodium nitroprusside (SNP) suppressed IL‐4 production in the Th2 clone but had no effect on IL‐2 production in the Th1 clone. In the experiment of the co‐culture with IFN‐PEC, the inhibitory‐effect of NO on T cell activation was not clarified by the influence of prostaglandins. However, in conclusion, cytokine production in Th2 cells may be more susceptible to NO than that in Th1 cells.
Growth Conditions Influence Expression of Cell Surface Hydrophobicity of Staphylococci and Other Wound Infection Pathogens
Authors: Ljungh Åsa , Wadström Torkel
Published: November 2013

The initial adhesion of microbes to tissue and solid surfaces can be mediated by hydrophobic interaction. Expression of microbial cell surface hydrophobicity (CSH) is influenced by growth conditions, and often best expressed after growth under nutrient‐poor conditions, or “starvation.” In the present study, the CSH of 133 strains of Enterobacteriaceae, Staphylococcus aureus, coagulase‐negative staphylococci, Enterococcus faecalis, group A streptococcus, Pseudomonas aeruginosa, Clostridium perfringens, Bacteroides fragilis, Peptococcus magnus, and of 8 Candida albicans strains was measured by the salt aggregation test after growth on hematin agar in a 5% CO2 atmosphere, or under anaerobiosis. Cells of all but 8 strains expressed pronounced or moderate CSH, i.e., they aggregated in 0.01‐2 M ammonium sulfate. When the agar surface was covered by human serum (diluted 1:5) to mimic growth conditions in a wound, 94 strains expressed higher CSH, and 44 strains the same CSH as after growth without serum. The CSH of 12 strains of different species was measured after growth on blood, hematin and PDM agar, with or without serum, and in an aerobic or a 5% CO2 atmosphere. The highest CSH was expressed after growth in 5% CO2 with serum, and the lowest growth after on blood agar in aerobic atmosphere. Identical results were obtained with native and heat‐inactivated (56 C, 20 min) serum. The reduced surface tension obtained in 5% CO2, as well as yet unidentified serum factors, promotes expression of CSH.
Class‐Specific Antibody Response in Acyclovir‐Treated and Adenine Arabinoside‐Treated Patients with Primary Genital Herpes Simplex Virus Infection
Authors: Kawana Takashi , Hashido Madoka , Koizumi Yoshio
Published: November 2013

Herpes simplex virus (HSV) class‐specific antibody responses after primary genital herpes infection were evaluated with an enzyme‐linked immunosorbent assay (ELISA) in 16 patients treated with acyclovir (ACV), given orally, and 17 patients treated with adenine arabinoside (Ara‐A), given topically. ACV significantly suppressed the levels of IgM, IgA, and IgG. In the ACV‐treated patients, IgM and IgG were not detected in 4 of the 16 and in 1 of the 16 patients, respectively. We must take into account this suppressive effect of ACV on antibody responses, especially on the IgM response, when serodiagnosis of HSV infection is made.
Cross‐Reactivity of Rickettsia japonica and Rickettsia typhi Demonstrated by Immunofluorescence and Western Immunoblotting
Authors: Uchiyama Tsuneo , Zhao Licheng , Yan Yansheng , Uchida Takahiro
Published: November 2013

Cross‐reactivity between Rickettsia japonica and R. typhi was observed by immunofluorescence tests using sera from patients with Oriental spotted fever (OSF), from whom the causative agent was isolated and identified as R. japonica. Western immunoblotting with these sera revealed that only the 120‐kilodalton surface polypeptide, i.e., rickettsial outer membrane protein (rOmp) B, has a common antigenicity with the 105‐kilodalton surface polypeptide of R. typhi. In some cases, antibodies specifically reactive with R. typhi were detected in acute‐phase sera followed by a significant rise in titers, possibly because of an anamnestic response to a previous infection with an R. typhi‐like agent; the sera retained reactivity to R. typhi even after absorption by a homologous strain. A lipopolysaccharide (LPS)‐like antigen of R. typhi was found to be reactive with some sera of OSF patients. The ladder bands on Western immunoblot of rickettsial organisms were confirmed to be polysaccharide in nature, which was demonstrated by comparing them with the pattern of silver‐stained gel of proteinase K‐treated rickettsial specimens after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.
Purification and Characterization of a Fibrinogen‐Degrading Protease in Bacteroides fragilis Strain YCH46
Authors: Chen Yong , Kinouchi Takemi , Kataoka Keiko , Akimoto Shigeru , Ohnishi Yoshinari
Published: November 2013

A novel fibrinogenolytic protease was purified from Bacteroides fragilis strain YCH46. The protease was extracted from cells by ultrasonic treatment and was purified 425‐fold with a recovery of 2.1% by sequential procedures using azocasein as a substrate. The purified protease showed a single band on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis with an estimated molecular weight of 100 kDa, which was consistent with the value obtained by gel filtration, indicating a monomeric native structure. Its optimal pH, Km, and Vmax for azocasein were 7.5, 0.2%, and 286 U/min/mg, respectively. The protease activity was completely inhibited by addition of 1 mM Hg2+, Cu2+, Zn2+, diisopropyl fluorophosphate, N‐ethylmaleimide or p‐chloromercuribenzoate but not by the inhibitors of metalloprotease or aspartic protease, suggesting that the enzyme is a serine‐thiol‐like protease. The protease hydrolyzed azocasein, casein, fibrinogen, gelatin, and azocoll, but not bovine serum albumin, ovalbumin, fibrin, fibronectin, immunoglobulins, transferrin, hemoglobin or types I, III, and IV collagen. The enzyme also hydrolyzed the chromogenic substrates alanyl‐alanine p‐nitroanilide, L‐valyl‐alanine p‐nitroanilide, alanyl‐alanyl‐valyl‐alanine p‐nitroanilide, and glycyl‐proline p‐nitroanilide, but was inert toward L‐alanine p‐nitroanilide, alanyl‐alanyl‐alanine p‐nitroanilide, and N‐α‐benzoyl‐DL‐arginine p‐nitroanilide. The protease completely hydrolyzed the α‐chain of fibrinogen at 37 C within 10 hr and at the same time the time required for clotting of protease‐treated fibrinogen by thrombin was prolonged. The fibrinogenolytic activity of a crude extract of B. fragilis was stronger than that of other species of the Bacteroides fragilis group tested: B. ovatus, B. distasonis, B. eggerthii, B. uniformis, and B. thetaiotaomicron. These results suggest that the fibrinogenolytic protease is an important biological factor in Bacteroides infection.
Demonstration and Genotyping of Pestivirus RNA from Mammalian Cell Lines
Authors: Harasawa Ryô , Mizusawa Hiroshi
Published: November 2013

We examined 20 cell lines of various animal origins for the presence of pestivirus contamination by the reverse transcription‐polymerase chain reaction (RT‐PCR), and found 15 (75%) cell lines were positive. The RT‐PCR products of the 5′ untranslated region (UTR) of pestivirus genome were sequenced and subjected to genotyping. Stem‐loop structures at three variable regions in the 5′ UTR render genotyping of the contaminated pestiviruses. Bovine cell lines tested were all contaminated with genotypes I, II, or III of bovine diarrhea virus (BVDV). Cell lines of canine, feline, and primate origin were contaminated with genotype II of BVDV. Cell line Ch1Es of caprine origin was contaminated with border disease virus (BDV).
Extraction and Characterization of the Smooth‐Type Lipopolysaccharide from Fusobacterium nucleatum JCM 8532 and Its Biological Activities
Authors: Onoue Sakura , Niwa Motō , Isshiki Yasunori , Kawahara Kazuyoshi
Published: November 2013

The lipopolysaccharide (LPS) of Fusobacterium nucleatum JCM 8532 was isolated by hot‐phenol water extraction. Most of the LPS was extracted in the phenolic phase and shown to be the smooth‐type, whereas the aqueous phase contained mainly rough‐type LPS. The chemical composition of the LPS was similar to that reported in other studies, but D‐quinovosamine, which may be a major component of O‐antigenic polysaccharide, and 3‐deoxy‐D‐manno‐2‐octulosonic acid (Kdo) were detected for the first time by gas chromatography‐mass spectrometry. The biological activities of smooth‐type LPS, including limulus activity, lethal toxicity, pyrogenicity, and B lymphocyte mitogenicity, were comparable to those of enterobacterial LPS. Smooth‐type LPS inhibited the cell growth and DNA synthesis of adult and fetal human gingival fibroblasts in a dose‐dependent manner, suggesting that LPS may play a role in the occurrence of human gingivitis.
Typing of Verotoxins by DNA Colony Hybridization with Poly‐ and Oligonucleotide Probes, a Bead‐Enzyme‐Linked Immunosorbent Assay, and Polymerase Chain Reaction
Authors: Yamasaki Shinji , Lin Zaw , Shirai Hiromasa , Terai Akito , Oku Yuichi , Ito Hideaki , Ohmura Mari , Karasawa Tadahiro , Tsukamoto Teizo , Kurazono Hisao , Takeda Yoshifumi
Published: November 2013

To identify the type of Verotoxins (VT) produced by Verocytotoxin‐producing Escherichia coli (VTEC), a sensitive bead‐enzyme‐linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo‐ and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.
Transient Expression of CD20 Antigen (Pan B Cell Marker) in Activated Lymph Node T Cells
Authors: Murayama Yuichi , Mukai Ryozaburo , Sata Tetsutaro , Matsunaga Satoko , Noguchi Atsuo , Yoshikawa Yasuhiro
Published: November 2013

In contrast to the case of peripheral T cells, the surface expression of CD20 antigen and the expression of CD20 mRNA in monkey lymph node (LN) T cells underwent a noticeable increase when they were cultured with mitogen and interleukin‐2 (IL‐2). To confirm in vivo regulation of CD20 expression during the activation of LN T cells, we examined LNs derived from monkeys experimentally inoculated with simian immunodeficiency virus (SIV). Significant expression of CD20 antigen was detected in the T cells of the LNs at the stage of lymphadenopathy. These findings suggest that lymphocyte activation in the LNs induced expression of the CD20 molecule in some T cells.
Role of Intestinal Bacteria in Ileal Ulcer Formation in Rats Treated with a Nonsteroidal Antiinflammatory Drug
Authors: Uejima Motoo , Kinouchi Takemi , Kataoka Keiko , Hiraoka Isao , Ohnishi Yoshinari
Published: November 2013

The role of intestinal bacteria in induction and repression of ulcer formation in the ileum of rats treated with one of the nonsteroidal antiinflammatory drugs (NSAIDs), 5‐bromo‐2‐(4‐fluorophenyl)‐3‐(4‐methylsulfonylphenyl) thiophene (BFMeT), was examined in this study. BFMeT was administered by intragastric gavage once at doses of 500‐1,500 mg/kg of body weight to Wistar rats treated with and without antibiotics (bacitracin, neomycin, streptomycin), germ‐free rats and gnotobiotic rats, and 72 hr later their gastrointestinal tracts were examined for ulcer formation. A single oral administration of BFMeT induced ileal ulcers in specific pathogen‐free rats. However, the rats given antibiotics to reduce the intestinal bacteria had no ulcers. BFMeT‐treated germ‐free rats and gnotobiotic rats mono‐associated with Bifidobacterium adolescentis or Lactobacillus acidophilus also had no intestinal ulcers. However, the drug induced ileal ulcers in gnotobiotic rats mono‐associated with Eubacterium limosum or Escherichia coli. An overnight culture of B. adolescentis or L. acidophilus or yogurt containing Bifidobacterium breve and Streptococcus thermophilus, when given as drinking water, inhibited ulcer formation in the ileum of rats treated with BFMeT. Gram staining of the ileal contents of normal rats revealed that 97.4% of the stained microorganisms were Gram‐positive rods and only 1.2% were Gram‐negative rods. In the group of rats with ulcers induced by BFMeT, the Gram‐positive rods decreased by 56.4% and the Gram‐negative rods including Escherichia coli, Klebsiella, Proteus and Bacteroides increased by 37.3%. However, in the group of rats administered the Bifidobacterium culture, the Lactobacillus culture or yogurt, the percentages of the Gram‐negative rods were decreased. Although Lactobacillus was a major bacterium in the ileum of normal rats, the Gram‐negative facultatively anaerobic rods E. coli, Klebsiella and Proteus were increased in the ulcerated ileum of rats treated with BFMeT, suggesting that these bacteria are associated with ulcer formation in rats treated with NSAIDs, and that Lactobacillus and Bifidobacterium inhibit it by repressing the growth of ulcer‐inducing bacteria.
Expression of Virulence‐Associated Antigens of Rhodococcus equi Is Regulated by Temperature and pH
Authors: Takai Shinji , Fukunaga Nariaki , Kamisawa Kei , Imai Yumiko , Sasaki Yukako , Tsubaki Shiro
Published: November 2013

We recently reported that there are two different virulence‐associated antigens correlated with virulence levels in Rhodococcus equi isolates from AIDS patients: virulent R. equi that kills mice with 106 cells expresses 15‐ to 17‐kDa antigens and intermediately virulent R. equi that kills mice with 107 cells expresses a 20‐kDa antigen. Environmental parameters were evaluated for their effects on the expression of these virulence‐associated antigens in virulent R. equi strains by immunoblotting using monoclonal antibodies in this study. Expression of these two virulence‐associated antigens of R. equi was regulated by pH and temperature; the antigens were produced maximally when the isolates were grown at 38 C and pH 6.5, but were not produced when grown at 38 C and pH 8, nor at temperatures below 30 C. The 20‐kDa antigen was found to be located on the cell surface, as were the 15‐ to 17‐kDa antigens, and showed susceptibility to proteolysis by trypsin. These results indicate that expression of the virulence‐associated antigens of R. equi is dependent on the environmental conditions.
Demonstration of Antigenic and Genotypic Variation in Orientia tsutsugamushi Which Were Isolated in Japan, and Their Classification into Type and Subtype
Authors: Ohashi Norio , Koyama Yoko , Urakami Hiroshi , Fukuhara Masahiro , Tamura Akira , Kawamori Fumihiko , Yamamoto Seigo , Kasuya Shiro , Yoshimura Kentaro
Published: November 2013

A total of 40 strains of Orientia tsutsugamushi (34 isolates from patients and trombiculid mites in Japan, and 6 prototype strains of antigenic variants) were examined for classification based on the reactivities with type‐specific monoclonal antibodies in indirect immunofluorescence tests, and on the restriction fragment length polymorphism of a polymerase chain reaction (PCR)‐amplified 56‐kilodalton type‐specific antigenic protein gene. By these methods, several antigenic and genotypic variants were found among the strains, and these variants were classified into types and further into subtypes. These results suggest that there are many variants in O. tsutsugamushi, and the methods used here seem to be useful for the systematic classification of the numerous variants. A strain which may be a new type distinguishable from those identified previously was also found in this study. Furthermore, variety in the degree of pathogenicity in mice related to type and/or subtype classification were observed.
Characterization of Actinobacillus actinomycetemcomitans Hemolysin
Authors: Kimizuka Ryuta , Miura Tadashi , Okuda Katsuji
Published: November 2013

Twenty out of 33 Actinobacillus actinomycetemcomitans strains formed hemolytic colonies on horse blood agar plates under anaerobic conditions. The hemolytic activity found in A. actinomycetemcomitans strain 137HE was examined. This activity was detected in the late exponential to early stationary phases of growth. Human erythrocytes were the most susceptible, followed by rabbit, sheep, horse and swine red blood cells. The majority of activity was detected in the cell‐associated vesicle fraction. Zwitterionic detergent 3‐[(3‐cholamidopropyl)‐dimethyl‐ammonio]‐1‐propanesulfonate (CHAPS) extract from whole cells was semipurified by ammonium sulfate precipitation, preparative isoelectric focusing (IEF) and gel‐filtration chromatography to yield a major band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) with a molecular mass of 12 kDa. Heating at 80 C for 30 min and treatment with proteinase K or trypsin resulted in complete disappearance of the hemolytic activity. Sulphydryl reagents enhanced activity and small amounts of cholesterol inhibited it. In summary, we demonstrated the presence of hemolysin in A. actinomycetemcomitans, and examined and characterized it.
Electron Microscopy of the Major Outer Membrane Protein of Campylobacter jejuni
Authors: Amako Kazunobu , Wai Sun Nyunt , Umeda Akiko , Shigematsu Mika , Takade Akemi
Published: November 2013

The surfaces of the disrupted‐cell surfaces of the Campylobacter jejuni strains FUM158432 and M1 were examined using the negative‐staining technique and electron microscopy. The surfaces of the whole cells and the outer membranes were covered with small dark dots which, in some areas, were arranged in hexagonal patterns. The hexagonal arrangement was more clearly seen in extracted outer membrane. The size of each structure was measured based on a center‐to‐center distance with the adjacent structure, and was determined to be 9.9±0.9 nm. A profile of the proteins in the outer membrane by SDS‐PAGE, performed in 0.1% SDS and at 100 C, showed 42 kDa proteins to comprise the major outer membrane protein of this bacterium. Digestion of the outer membrane materials with proteinase reduced this protein band in the SDS‐PAGE, and the amount of dark dots on the electron micrograph indicated the structure to be the major outer membrane protein (porin) of this bacterium. The power spectrogram of a computer‐assisted Fourier transformation of the hexagonally arranged porin proteins suggests that the porin has a trimeric structure rather than a monomeric one.
Seroepidemiology of Feline Chlamydiosis by Microimmunofluorescence Assay with Multiple Strains as Antigens
Authors: Pudjiatmoko  , Fukushi Hideto , Ochiai Yoshitsugu , Yamaguchi Tsuyoshi , Hirai Katsuya
Published: November 2013

The prevalence of anti‐chlamydia antibodies was examined in 232 cat sera collected in 1985 and from 1993 to 1995 from laboratories and veterinary hospitals located in 11 prefectures of Japan. The antibodies were determined by an indirect microimmunofluorescence test using six strains of feline Chlamydia: one strain each of avian‐ and guinea pig‐derived C. psittaci and one strain each of C. pecorum, C. pneumoniae and C. trachomatis. Positive rates of IgG antibodies to chlamydiae were 34.4% in 1985 and 16.5–21.4% from 1993 to 1995. Positive rates of IgM antibodies to chlamydiae were 8.2% in 1985 and 6.6–14.3% from 1993 to 1995. Variations in antibody reactivity to the different feline strains were observed. The results suggest the wide prevalence of chlamydial infection in cats in Japan, and antigenic diversity in the feline strains of C. psittaci.
Detection and Identification of Yersinia pestis by Polymerase Chain Reaction (PCR) Using Multiplex Primers
Authors: Tsukano Hiroko , Itoh Ken‐Ichiro , Suzuki Sosuke , Watanabe Haruo
Published: November 2013

A PCR method for detection of Yersinia pestis‐virulence determinants by the use of multiplex primers was developed. Four pairs of oligonucleotide primers were designed from each gene of three kinds of virulent plasmids and a chromosomal DNA; 60‐Md plasmid‐located gene (caf1) encoding Y. pestis‐specific capsular antigen fraction 1, a Y. pestis‐specific region of a yopM gene encoded on 42‐Md virulent plasmid, a plasminogen activator gene (pla) encoded on Y. pestis‐specific 7‐Md plasmid and an invasin protein gene (inv) encoded on chromosomal DNA. This multiplex‐primer system was specific for the detection of Y. pestis among pathogenic Yersinia species and other enterobacteriaceae having antigens common to Y. pestis. Since this method is simple and safe, it will be useful to identify and confirm Y. pestis in cases of emergency and for the surveillance of epidemics.
Migration of Salmonella typhi through Intestinal Epithelial Monolayers: An In Vitro Study
Authors: Kops Sandra Kraeuter , Lowe Daniel K. , Bement William M. , West A. Brian
Published: November 2013

This study characterizes the transmigration of enteroinvasive Salmonella typhi in vitro, using a human intestinal epithelial cell line as a model of small intestinal epithelium. C2BBe cells, a subclone of CACO‐2 with a highly differentiated enterocytic phenotype, were grown to maturity on Transwell filters. S. typhi Ty2 and the vaccine strain, Ty21a, the S. typhi mutant X7344 and parent strain SB130, and S. typhimurium 5771 in logarithmic phase were introduced to the upper chamber of the filter units. Numbers of bacteria in the lower chamber, TER and permeability of the monolayer to mannitol were measured over time. Monolayers were examined by light, electron and confocal microscopy to determine the pathway of bacterial transmigration, and intracellular bacteria were estimated by gentamicin assay. Epithelial cell injury was quantified by light microscopy. S. typhi transmigrated earlier and in larger numbers than S. typhimurium, inducing marked changes in electrical resistance and permeability. Unlike S. typhimurium, S. typhi selected epithelial cells in small number and caused their death and extrusion from the monolayers leaving holes through which S. typhi transmigrated. Ty2 consistently transmigrated in larger numbers and with more injury to monolayers than Ty21a. S. typhi crosses the monolayers of C2BBe cells by a paracellular route in contrast to the transcellular pathway described for other Salmonellae. This may be related to the unique pathophysiology of S. typhi infection and the restricted host specificity of this pathogen. In these assays the vaccine strain, Ty21a, is slightly less invasive than its parent, though more invasive than S. typhimurium.
Cooperative Anti‐Candida Effects of Lactoferrin or Its Peptides in Combination with Azole Antifungal Agents
Authors: Wakabayashi Hiroyuki , Abe Shigeru , Okutomi Takafumi , Tansho Shigeru , Kawase Kouzou , Yamaguchi Hideyo
Published: November 2013

The effects of lactoferrin (LF), an antimicrobial protein secreted in body fluids, and its peptides in combination with azole antifungal agents were investigated by the micro‐broth‐dilution method in a study of Candida albicans. In the case of LF, its pepsin hydrolysate (LFhyd) or the LF‐derived antimicrobial peptide Lactoferricin® B (LF‐B), the concentrations required to inhibit the growth of Candida decreased in the presence of relatively low concentrations of clotrimazole (CTZ). The minimum inhibitory concentration (MIC) of all azole antifungal agents tested was reduced by 1/41/16 in the presence of a sub‐MIC level of each of these LF‐related substances. Polyene and fluoropyrimidine antifungal agents did not show such a combined effect with these LF‐related substances. The anti‐Candida activity of LF or LF‐B in combination with CTZ was shown to be synergistic by checkerboard analysis. These results indicate that LF‐related substances function cooperatively with azole antifungal agents against C. albicans.
Production, Binding Characteristics and Protective Immunity of Monoclonal Antibody to Pneumococcal Type‐9V Conjugate
Authors: Lee Chi‐Jen , Karpas Arthur , Wang Theresa R. , Kosaka Tadashi , Koizumi Kaio
Published: November 2013

A monoclonal antibody (MAb) to pneumococcal type‐9V polysaccharide (PS) was produced using PS conjugated to inactivated pneumolysin as the immunogen. The MAb to 9V PS was of the IgG1 subclass. The antigen‐antibody reaction increased rapidly at low concentrations and reached a plateau at 10 μg PS/ml as measured by nephelometry of the group 9 PS against 9V MAb binding. In contrast, the binding of group 9 PS against rabbit 9V antiserum (AS) increased proportionally and continued to increase up to the highest concentration of PS tested (20 μg PS/ml). The 9V MAb reacted with all group 9 PSs (9A, 9L, 9N and 9V) by immunodiffusion. In the homologous 9V Ag‐MAb reaction, there were marked differences in the inhibition of binding by the cross‐reactive 9L PS (19.2% inhibition) and the 9N PS (0.2%). In contrast, inhibition of the homologous 9V Ag‐rabbit AS binding by cross‐reactive 9L and 9N PSs ranged from 57.8 to 62.7%. Removal of the O‐acetyl group from 9V PS by alkali hydrolysis resulted in decreased binding with rabbit 9V AS. However, the binding reaction with 9V MAb was less affected by the loss of O‐acetyl content. The 9V MAb was both opsonic and passively protected young mice against challenge with type‐9V pneumococci.
An Epidemiological Study of Salmonella enteritidis by Pulsed‐Field Gel Electrophoresis (PFGE): Several PFGE Patterns Observed in Isolates from a Food Poisoning Outbreak
Authors: Murase Toshiyuki , Nakamura Akiko , Matsushima Akiyoshi , Yamai Shiro
Published: November 2013

An epidemiological analysis of Salmonella enteritidis from a food poisoning was done using pulsed‐field gel electrophoresis (PFGE) of BlnI‐ or XbaI‐digested fragments of chromosomal DNA of isolates. S. enteritidis isolates obtained from 19 patients had identical PFGE patterns. Therefore, a strain giving the same pattern was considered to be the causative agent of this outbreak. In addition, four isolates that had different BlnI‐digested PFGE patterns were obtained from three patients, suggesting that the observed variations in PFGE patterns might occur as the result of some point mutations of chromosomal DNA during growth or from the existence of several S. enteritidis strains from various sources. Subsequent PFGE analysis of continuously subcultured strains supported the former possibility. These observations indicate that PFGE analysis on multiple numbers of colonies from each patient are necessary for the epidemiologic investigation of S. enteritidis.
Differential Expression of a Mycobacterium tuberculosis Protein Recognized by a Mouse Monoclonal Antibody
Authors: Cummings Patrick J. , Rowland Sharon S. , Hooper Nancy E. , Schwalbe Richard S.
Published: November 2013

Murine monoclonal antibodies were produced against Mycobacterium tuberculosis (Mtb) using standard hybridoma procedures. By a whole cell enzyme‐linked immunosorbent assay (ELISA), one monoclonal antibody (mAb), HB28, demonstrated high level specific reactivity to Mtb. Western blot analysis demonstrated reactivity to a single 65 kDa Mtb protein in the cell wall extract and culture filtrate. HB28 mAb appears to be recognizing a 65 kDa Mtb protein that is over‐expressed by Mtb but not other species under certain culture conditions. Differential expression and detection of this protein by HB28 mAb may have potential for diagnostic applications.
Rapid Increase of Pneumococcal Resistance to β‐Lactam and Other Antibiotics in Isolates from the Respiratory Tract (Nagasaki, Japan: 1975–1994)
Authors: Rikitomi Naoto , Sow Papa Salif , Watanabe Kiwao , Nunez Duilio S. , Martinez Glenda , Nagatake Tsuyoshi
Published: November 2013

The susceptibility of 101 pneumococcal isolates from the respiratory tract during 1991–1994 was examined and compared with the susceptibility of isolates over the period of 1975–1990. A rapid increase of resistance was seen not only to penicillin but also other antimicrobial agents. During 1991–1994, 38% of all the isolates were resistant to penicillin. The rates of resistance during this period were 16–23% for three newer cephalosporins, 18% for imipenem, 69% for tetracycline, 31% for erythromycin, 20% for chloramphenicol and 9% for clindamycin. The use of antibiotics within one month prior to pneumococcal isolation was correlated with penicillin resistance (P < 0.05). Serotyping of the isolates by antiserum revealed differences in predominant types between penicillin‐resistant (19F, 23F, 4) and ‐susceptible isolates (15, 4, 11A). Our data suggests that anti‐pneumococcal antibiotics should be carefully chosen on the basis of susceptibility tests.