bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of thecolHGene EncodingClostridium histolyticumCollagenase inBacillus subtilisand Its Application to Enzyme Purification
Authors: Jung Chang‐Min , Matsushita Osamu , Katayama Seiichi , Minami Junzaburo , Ohhira Lichiro , Okabe Akinobu
Published: November 2013

ThecolHgene encoding 116‐kDa collagenase ofClostridium histolyticum(cColH) was cloned into anEscherichia coli‐Bacillus subtilisshuttle vector to develop a method for purification of recombinant collagenase (rColH). When plasmid pJCM310 containing thecolHgene was introduced intoB. subtilisDB104 and the transformant was grown in LB broth at 37 C, stability of the plasmid was not maintained. However, stability was partly improved by growing the transformant in a modified LB broth containing 0.5Msodium succinate with gentle shaking at 35 C. When the transformant was grown to an optical density of 0.4 at 600 nm in this medium, pJCM310 was stable and rColH was produced in sufficient amounts. rColH was purified to homogeneity by ammonium sulfate precipitation, gel filtration and ion‐exchange chromatography. The yield of rColH from an 800‐ml culture was 0.53 mg and its specific activity was estimated to be 1,210 U per mg of protein. The purified rColH was capable of degrading native type‐I collagen fibril from bovine achilles tendon, as was demonstrated by zymography. A comparison of the N‐terminal amino acid sequence between cColH and rColH revealed that rColH has 10 extra N‐terminal amino acid residues. However, the peptide mapping of rColH with V8 protease was virtually identical to that of cColH. Furthermore, the molecular mass of rColH was estimated to be 112,999 Da by mass spectrometry, coinciding with the value of 112,977 Da, which was predicted from the nucleotide sequence of thecolHgene. Therefore, the recombinantB. subtilisculture is capable of serving as a useful source for enzyme purification.
Effects of Granulocyte‐Macrophage Colony Stimulating Factor (GM‐CSF) on Biomaterial‐Associated Staphylococcal Infection in Mice
Authors: Rózalska Barbara , Ljungh Asa , Paziak‐Domańska Beata , Rudnicka Wieslawa
Published: November 2013

Staphylococcal infections are a major complication in the usage of biomaterials. Different modifications of polymers have been made to reduce the incidence of such infections. We studied the effects of modifying heparinized polyethylene (H‐PE) with mouse recombinant granulocyte‐macrophage stimulating factor (rGM‐CSF). The elimination of staphylococci (Staphylococcus aureus, S. epidermidis) from the peritoneum of mice implanted with rGM‐CSF‐coated H‐PE was slightly more effective than the elimination of the bacteria from the peritoneum of animals implanted with uncoated H‐PE. Most interestingly, the number of staphylococci present in the biofilms covering rGM‐CSF‐coated implants were significantly lower than the number of bacteria detected on the surface of H‐PE not coated with rGM‐CSF. In vitro, rGM‐CSF restored the anti‐bacterial potency of the phagocytes, which had been reduced by surface contact with H‐PE. The results suggest that modification of biomaterials with rGM‐CSF could be one way of preventing staphylococcal infections; especially in neutropenic disorders, which constitute the highest risk factor for foreign body‐associated infections.
The Thermostable Direct Hemolysin Gene (tdh) of Vibrio hollisae Is Dissimilar in Prevalence to and Phylogenetically Distant from the tdh Genes of Other Vibrios: Implications in the Horizontal Transfer of the tdh Gene
Authors: Nishibuchi Mitsuaki , Janda J. Michael , Ezaki Takayuki
Published: November 2013

Vibrio hollisae strains isolated recently from patients in various locations were examined for the presence of the thermostable direct hemolysin gene (tdh) using nucleic acid hybridization and polymerase chain reaction assays. The results were consistent with the previous finding that all strains of V. hollisae carry the tdh gene. In contrast, the tdh gene has been detected in a minority of strains for other Vibrio species (V. parahaemolyticus, V. cholerae non‐O1, and V. mimicus). Detailed phylogenetic analysis showed that the tdh genes of the non‐V. hollisae species were very closely related to each other and that the tdh gene of V. hollisae was distantly related to the tdh genes of the non‐V. hollisae species. These results and the proposed insertion sequence‐mediated tdh transfer mechanism suggest that the tdh gene may have been maintained stably in V. hollisae and that the tdh genes of the non‐V. hollisae species may have been involved in recent horizontal transfer.
Cloning and Sequencing of the Spore Germination Gene of Bacillus megaterium ATCC 12872: Similarities to the NaH‐Antiporter Gene of Enterococcus hirae
Authors: Tani Katsuji , Watanabe Takuya , Matsuda Hideyasu , Nasu Masao , Kondo Masaomi
Published: November 2013

The germination mutant TM‐31 of Bacillus megaterium ATCC 12872, was isolated by transposon Tn917 insertional mutagenesis. Glucose, L‐proline, L‐leucine and KNO3 germinated TM‐31 poorly. The DNA in the region of the Tn917 insertion was cloned, and its nucleotide sequence determined. One major open reading frame was present on the cloned DNA. The hydrophobic protein encoded is presumably membrane‐associated. A homology search revealed that the gene encoded in the region of the Tn917 insertion is homologous to napA of Enterococcus hirae. napA codes for the NaH‐antiporter. It is hypothesized that transport of cations must play an important role in spore germination in B. megaterium ATCC 12872.
Prophylactic Effect of Enterococcus faecalis FK‐23 Preparation on Experimental Candidiasis in Mice
Authors: Satonaka Katsuhito , Ohashi Kazutomo , Nohmi Takao , Yamamoto Tetsuro , Abe Shigeru , Uchida Katsuhisa , Yamaguchi Hideyo
Published: November 2013

The prophylactic effects of heat‐killed cells of Enterococcus faecalis FK‐23 (FK‐23 preparation) on experimental candidiasis were investigated in normal and leukopenic mice. In cyclophosphamide‐induced leukopenic mice, oral or intraperitoneal administration of the FK‐23 preparation at a daily dose of 1.25 or 5 mg/mouse for 3 consecutive days prior to Candida albicans infection significantly prolonged survival periods of the infected mice, and decreased viable counts of C. albicans recovered from their kidneys. In normal mice, the FK‐23 preparation administered at dosages ranging from 0.63 to 10 mg/mouse/day for 3 consecutive days was ineffective, while in leukopenic mice, the FK‐23 administered orally caused a facilitated recovery in the number of white blood cells including neutrophils. Furthermore, intraperitoneal administration of the FK‐23 preparation into mice augmented the anti‐Candida activity of immunocompromised peritoneal exudate cells obtained from the animals. These results suggested the potential usefulness of the FK‐23 preparation as a prophylactic agent for the management of patients with opportunistic fungal infections.
Susceptibility of Helicobacter pylori Isolates against Agents Commonly Administered for Eradication Therapy and the Efficacy of Chemotherapy
Authors: Gotoh Akira , Kawakami Yoshiyuki , Akahane Takayuki , Akamatsu Taiji , Shimizu Toshiki , Kiyosawa Kendo , Katsuyama Tsutomu
Published: November 2013

Although chemotherapy has been documented to be effective in the treatment of Helicobacter pylori‐associated gastritis and gastroduodenal ulcers, some cases are known to have been unsuccessful in the attempt to eradicate this species. In this study, we examined the relation between the susceptibility of H. pylori isolates and the efficacy of chemotherapy. We utilized the modified agar plate dilution method to determine the minimum inhibitory concentrations (MICs) of 63 H. pylori strains isolated before treatment with several drugs routinely used during eradication chemotherapy. Among the drugs tested, amoxicillin (AMPC) and clarithromycin (CAM) demonstrated high degrees of activity with MIC90, 0.39 and 0.2 μg/ml, respectively. No highly resistant strain against AMPC was detected among the strains examined, while for CAM and metronidazole (MTZ), 9.5% and 7.9% of the strains, respectively, were resistant before treatment. It should be noted that all of the MICs of the strains from patients with successful therapy were lower than those from patients with unsuccessful therapy. These findings indicate that susceptibility tests should be carried out prior to the commencement of drug administration in order to provide safer and more effective chemotherapy.
Detachment Activity but Not Cytotoxicity Induced in a T‐Cell Clone Following Antigen Presentation in the Presence of Thymic Epithelial Cells
Authors: Kaneda Rika , Kasai Michiyuki , Itoh Yasushi , Iwabuchi Kazuya , Onoé Kazunori
Published: November 2013

The selective induction of effector functions of a T‐cell clone (DB14), specific to pigeon cytochrome c 43–58 (p 43–58) and restricted to I‐Ab, was analyzed using a professional antigen‐presenting cell, B hybridoma (Th 2.58), and various non‐professional antigen‐presenting cells (APC), L cells transfected with I‐Ab (I‐Ab L cells), a medullary thymic epithelial cell line (m‐TEC) and a cortical thymic epithelial cell line (c‐TEC). The m‐TEC and c‐TEC expressed I‐Ab upon induction with interferon γ (IFN‐γ). When stimulated with p 43–58 in the presence of I‐Ab L cells as well as Th 2.58 cells, the DB14 cells showed marked proliferation and, after 18 hr of culturing, exhibited significant cytotoxicity against the APC. By contrast, in the presence of m, c‐TEC, the DB14 cells showed neither proliferation nor cytotoxicity against these TEC but exhibited considerable detachment activity towards them. Furthermore, DB14 cells became expressed activation markers (CD69 or CD44) following stimulation with p 43–58 plus m‐TEC or c‐TEC. The addition of rIL‐2 to the culture of DB14 cells, p 43–58 and m‐TEC or c‐TEC, restored the proliferative responses. However, it was shown that anergy was not involved in the negligible proliferative responses of DB14 cells after stimulation with p 43–58 plus m, c‐TEC. The present findings indicate that differences in APC functions are present among non‐professional APC and suggest that the selective induction of T‐cell functions can be achieved using the appropriate non‐professional APC. The characteristic activation of T cells by TEC may be related to their functional roles in situ.
Antibiotic Treatment in Mice Infected with Japanese Borrelia garinii: Efficacy of Ceftriaxone for Eradicating the Infection Induced by Ixodes persulcatus Tick Bites
Authors: Konishi Tatsuya , Nakao Minoru
Published: November 2013

We compared the efficacy of three antibiotics (ceftriaxone, erythromycin and clarithromycin) against Borrelia garinii infection in mice. The nymphal ticks of Ixodes persulcatus infected with the strain JEM6 of Japanese B. garinii were allowed to feed on female C3H mice. The mice were treated with each of the antibiotics for 5 consecutive days 1, 3, or 7 weeks after tick detachment. The doses of antibiotics per day were as follows: 5 mg intraperitoneal injection of ceftriaxone, 2 mg intraperitoneal injection of erythromycin and 1 mg peroral administration of clarithromycin. The infection status in treated mice was monitored by culturing their earlobes, hearts and urinary bladders in BSK II medium. Ceftriaxone eliminated borreliae completely; however, a recurrence of infection was observed in mice treated with erythromycin and clarithromycin.
Prevalence and Characteristics of Enteropathogenic Escherichia coli with the eae Gene in Diarrhoeic Rabbits
Authors: Blanco Jesús E. , Blanco Miguel , Blanco Jorge , Mora Azucena , Balaguer Luis , Cuervo Luis , Balsalobre Carlos , Muñoa Francisco
Published: November 2013

A field study was carried out with the objective of investigating the prevalence of enteropathogenic Escherichia coli (EPEC) with the eae gene in diarrhoeic rabbits. EPEC eae+ were isolated from 60 (74%) of 81 diarrhoeic rabbits sampled in 30 industrial fattening farms localized in the four provinces of Galicia (northwestern Spain). Attaching and effacing lesions were found in 44 of 50 animals processed for histology. The 111 E. coli strains identified belonged to 19 different O serogroups and 13 biotypes. However, 53 (48%) of the strains belonged to serogroup O103 and 36 (32%) showed the serobiotype O103:B14. The eae gene was significantly more frequent (100%; 47 of 47) among the highly pathogenic rhamnose‐negative strains of serobiotypes O103:B6 and O103:B14 than among the E. coli strains belonging to other serobiotypes (36%; 23 of 64) (P < 0.001). In this first report about the prevalence of EPEC with the eae gene in rabbits, we conclude that the class of E. coli strains observed is a common cause of diarrhoea in Galician rabbit farms, and that highly pathogenic rhamnose‐negative strains of serotype O103:K‐:H2 and biotype B14 are specially predominant.
Accelerated Progression of a Murine Retrovirus‐Induced Immunodeficiency Syndrome In Fas Mutant C57BL/6/lpr/lpr Mice
Authors: Hiromatsu Kenji , Usami Jun , Aoki Yoshiyasu , Makino Masahiko , Yoshikai Yasunobu
Published: November 2013

We reported previously that CD4+ T cells and B cells in mice with retrovirus‐induced murine acquired immunodeficiency syndrome (MAIDS) caused by LP‐BM5 murine leukemia virus (MuLV) mixtures increased the expression of Fas antigen (Fas) during progression of the disease. However, the contribution of the Fas/Fas ligand (Fas L) system to the pathogenesis of MAIDS remained unknown. Here, we examined the susceptibility of C57BL/6 (B6) lpr/lpr mice, which has been reported to be defective for the expression of Fas, to MAIDS. We found that the Thy 1.2− CD4 T cells and IgK dull B220+ cells, which are characteristic of MAIDS, increased after the inoculation of LP‐BM5 MuLV in B6 lpr/lpr mice. B22+ TCR αβ T cells, unique to lupus prone mice, also increased in the B6 lpr/lpr mice after infection. CD4+ B220+ TCR αβ T cells increased profoundly among the B220+ TCR αβ T cells from LP‐BM5 MuLV‐infected B6 lpr/lpr mice, while the B220+ TCR αβ T cells observed in non‐infected B6 lpr/lpr mice were largely of the CD4− CD8− phenotype. A DNA PCR analysis of the LP‐BM5 MuLV‐infected B6 lpr/lpr mice revealed the genome integration of defective LP‐BM5 virus, further confirming that MAIDS is inducible to B6 lpr/lpr mice. LP‐BM5 MuLV‐infected lpr/lpr mice died within 3 months, while MAIDS‐infected B6 +/+ mice usually died within 5 to 6 months, and B6 lpr/lpr mice not infected with LP‐BM5 MuLV lived more than 6 months. Taken together, these results suggest that MAIDS is inducible independently with functional Fas expression and the possibility of accelerated progression of murine AIDS and lpr‐associated autoimmune disease in B6 lpr/lpr mice infected with LP‐BM5 MuLV.
Antigenic and Genetic Variations among Japanese Encephalitis Virus Strains Belonging to Genotype 1
Authors: Ali Ayub , Igarashi Akira
Published: November 2013

Hyperimmune antisera against four Japanese encephalitis (JE) virus strains, ThCMAr4492 and ThCMAr6793 from Thailand and Nakayama and JaGAr01 from Japan, were used to analyze the antigenic relationships among 12 Thai strains belonging to genotype 1, and two Japanese strains and one Chinese strain belonging to genotype 3. The antiserum for ThCMAr6793 significantly neutralized nine of the 12 Thai strains, none of which was significantly neutralized by antisera for the Nakayama and JaGAr01 strains. The antiserum for ThCMAr4492 neutralized only its homologous strain; therefore, ThCMAr4492 was antigenically different from all other strains. Two Thai strains (Subin and KE‐093/83) were significantly less neutralized by all four of the antisera tested. In the deduced amino‐acid sequence of the E protein, the 12 Thai strains revealed 100 to 98.2% identity among them and 90.0 to 98.8% identity with the published strains, respectively. Among significant amino‐acid substitutions, three residues at positions E‐222, E‐327 and E‐366 were shared by all of the Thai strains, whereas residues at E‐89, E‐123, E‐131, E‐178, E‐293, E‐351 and E‐373 seemed to be strain‐specific. The amino acids at positions E‐178, E‐327, E‐351, E‐373 and E‐366 are found either in the peptides with functional T‐helper cell epitopes or in the ectodomain of the E protein of other flaviviruses. These amino acids may therefore be responsible for determining the antigenic heterogeneity of these strains.
Recombinant Interleukin‐1 Promotes Leishmaniasis in Susceptible Mice
Authors: Ajdary Soheila , Hosseini Mohammad Hossein , Alimohammadian Mohammad Hossein
Published: November 2013

Interleukin‐1 exacerbates leishmanial lesions in Leishmania major‐infected BALB/c mice. Indomethacin can modulate the effect of IL‐1, so at least part of the IL‐1 effect on disease progression is due to the induction of prostaglandin synthesis.
Synovial Mononuclear Cells Consist with T Cells Which Produce High Levels of Tumor Necrosis Factor α
Authors: Akatsuka Hidenobu , Okubo Mitsuo , Ishida Hironori , Chiba Katsumi , Imanishi Kenichi , Uchiyama Takehiko , Yamamoto Kazuhiko , Kasukawa Reiji , Nishimaki Tomoe
Published: November 2013

To determine whether synovial mononuclear cells include a population of tumor necrosis factor α‐produeing T cells, we measured tumor necrosis α levels in culture supernatants of synovial mononuclear cells by ELISA and analyzed tumor necrosis α mRNA‐positive cell frequencies. There were no significant differences in the spontaneous levels of TNF α between synovial mononuclear cells and peripheral mononuclear cells. The frequency of tumor necrosis factor α mRNA‐positive cells in synovial mononuclear cells was higher than that of peripheral mononuclear cells. When stimulated with a superantigen, mononuclear cells from the synovial fluid of rheumatoid arthritis patients showed higher levels of tumor necrosis factor α production (1,035 ± 817 pg/ml) than did mononuclear cells from their peripheral blood (236 ± 180 pg/ml). In addition, we observed that a few T cell clones were resistant to superantigenic restimulation in vitro. We conclude that when these types of T cells persist in the synovium, they play a role in the development of rheumatoid arthritis via a mechanism involving tumor necrosis factor α production.
Antibodies Are Generated during Infection to Coxiella burnetii Macrophage Infectivity Potentiator Protein (Cb‐Mip)
Authors: Seshu J. , McLvor Keith L. , Mallavia Louis P.
Published: November 2013

Antisera from rabbits immunized with formalin inactivated Coxiella burnetii isolates associated with either acute (Nine Mile, phase I or phase II) or chronic (Priscilla) Q fever showed reactivity to a C. burnetii macrophage infectivity potentiator protein (Cb‐Mip) cloned in Escherichia coli. Further, antisera generated in BALB/c mice after infection with live Nine Mile phase I or Priscilla isolates also showed reactivity to Cb‐Mip by immunoblot analysis. In addition, human serum from an individual with previous serological and clinical evidence of Q fever showed reactivity to Cb‐Mip. This study indicates that Cb‐Mip is immunogenic in both experimental and natural infections, and is the first report on the presence of antibodies to Mip/Mip‐like proteins of intracellular bacteria in human sera. Cb‐Mip may serve as a potential target antigen for developing recombinant vaccines or diagnostic assays for Q fever.
Serological Indicators of Helicobacter pylori Infection in Adult Dyspeptic Patients and Healthy Blood Donors
Authors: Rechciński Tomasz , Chmiela Magdalena , Matecka‐Panas Ewa , Płaneta‐Małsecka Izabela , Rudnicka Wiesława
Published: November 2013

The levels of IgM, IgG and IgA antibodies reacting with two Helicobacter pylori antigens (glycine acid extract (GE) and a recombinant CagA protein) were determined in the sera from adult dyspeptic patients, positive (H.p.(+)) or negative (H.p.(‐)) for H. pylori urease/culture, and from healthy blood donors. All sera were also examined against GE by Western blot (immunoblot) technique. Similar levels of anti‐GE IgG were detected in the sera from all H.p.(+) and almost all H.p.(‐) patients and from over 40% of the healthy volunteers. In contrast, higher levels of anti‐GE IgA were found in the sera from patients than that from healthy subjects, although such antibodies were not detected in the sera from 30% of the H.p.(+) patients. In general, our results suggest that a combination of ELISA and immunoblot may be more sensitive in the detection of H. pylori infection in dyspeptic patients than the examination of biopsy specimens by culturing or histology.
Ultrastructural Alterations of Candida albicans Induced by a New Imidazole Antimycotic Omoconazole Nitrate
Authors: Nishiyama Yayoi , Itoyama Toshio , Yamaguchi Hideyo
Published: November 2013

The antifungal effects of an imidazole‐antimyeotic omoconazole nitrate (OMZ) on the morphology and ultrastructure of Candida albicans yeast cells were studied using scanning and transmission electron microscopy. The treatment of growing Candida cultures with fungistatic doses (0.4 to 4 μg/ml) of OMZ produced the formation of a chain or cluster of cells. Thickening of the cell wall and accumulation of electron‐dense vesicles in the wall were clearly observed. Development of Golgi‐like complex membranous structures in the cytoplasm was the most prominent finding. The cytological alteration induced by exposure to a higher concentration (40 μg/ml) of the drug was characterized by disruption of the intracytoplasmic organelles. Our results confirm the strong antifungal activity of OMZ against fungal cells.
Unification of the Genera Serpulina and Brachyspira, and Proposals of Brachyspira hyodysenteriae Comb. Nov., Brachyspira innocens Comb. Nov. and Brachyspira pilosicoli Comb. Nov.
Authors: Ochiai Sachiyo , Adachi Yoshikazu , Mori Katsumi
Published: November 2013

The phylogenetic positions of Serpulina hyodysenteriae, Serpulina innocens, Serpulina pilosicoli and Brachyspira aalborgi were studied. Complete 16S ribosomal DNA sequences of these three species and B. aalborgi revealed that their 16S rDNA sequences were related more than 96.0%. The mol% guanine plus cytosine (G + C) of B. aalborgi DNA was 27.1, and was similar to those of the 3 members of the genus Serpulina. The homologous rates using 32P‐labeled B. aalborgi chromosome DNA in DNA‐DNA reassociation tests were 22.0% to S. hyodysenteriae, 19.1% to S. innocens and 17.2% to S. pilosicoli. Therefore, we propose to transfer the three species of the genus Serpulina to the genus Brachyspira. Descriptions of Brachyspira hyodysenteriae comb. nov., Brachyspira innocens comb. nov. and Brachyspira pilosicoli comb. nov., and an emended description of B. aalborgi are given. Phenotypic characteristics of the 4 members of the genus Brachyspira were also studied. They fermented fructose, galactose, glucose, lactose, maltose, man‐nose, raffinose and trehalose; however, B. aalborgi did not ferment raffinose. All of them hydrolyzed esculin but did not produce indole except for B. hyodysenteriae. The protein profile of B. aalborgi was different from those of the four strains of B. hyodysenteriae, B. innocens and B. pilosicoli, but the heavy bands with molecular sizes of 49.4 and 52.3 kDa of B. aalborgi were quite similar to those of B. innocens in the points of quantity and molecular size. In immunoblotting tests, B. aalborgi reacted well with anti‐B. innocens and B. pilosicoli sera, but reacted weakly with anti‐B. hyodysenteriae serum. Only one heavy band and several faint bands were revealed by the reaction between B. aalborgi and anti‐B. hyodysenteriae serum, and the heavy band was common among these strains.
Prevalence of Genotypes of Orientia tsutsugamushi in Patients with Scrub Typhus in Miyazaki Prefecture
Authors: Horinouchi Hisato , Murai Koichi , Okayama Akihiko , Nagatomo Yasuhiro , Tachibana Nobuyoshi , Tsubouchi Hirohito
Published: November 2013

The genotypes of Orientia tsutsugamushi in patients with scrub typhus in Miyazaki Prefecture were examined by polymerase chain reaction (PCR) and the restriction fragment length polymorphism method. Specific patterns for genotypes Irie, Hirano, Tazume and Yoshimura were detected in 26, 6, 5 and 2 of 39 DNA samples obtained from peripheral blood mononuclear cells, respectively. DNA sequences of the PCR products from the Tazume strain were genetically very close to the Hirano strain and the Yoshimura strain was also very close to the Karp strain. Furthermore, the DNA sequences from the Irie and Tazume strains were completely homologous to the reported sequences of the Kawasaki and Kuroki strains, respectively.
Production of Monoclonal Antibody Discriminating Serological Difference in Escherichia coli O9 and O9a Polysaccharides
Authors: Kido Nobuo , Morooka Naoki , Paeng Noriko , Ohtani Taizo , Kobayashi Hidemitsu , Shibata Nobuyuki , Okawa Yoshio , Suzuki Shigeo , Sugiyama Tsuyoshi , Yokochi Takashi
Published: November 2013

A monoclonal antibody (mAb) with a unique antigenic specificity against Escherichia coli O9 was produced. The O9a mAb was reactive with a part of the strains in E. coli O9. The O9a mAb did not react with LPS from the E. coli O9 test strain Bi316‐42. The distribution of the antigen defined by the O9a mAb in E. coli O9 was consistent with that of E. coli O9a present in E. coli O9 strains. The chemical structure of the repeating unit of the O‐specific polysaccharide detected by the mAb was demonstrated to be a mannotetraose by two‐dimensional nuclear magnetic resonance spectroscopy. It was confirmed that the mAb recognized E. coli O9a serotype in E. coli O9 serotype strains, suggesting that E. coli O9a serotype might be a dominant strain in E. coli O9.
Effect of Arginine on Toxin Production by Clostridium difficile in Defined Medium
Authors: Karasawa Tadahiro , Maegawa Tsuneo , Nojiri Takashi , Yamakawa Kiyotaka , Nakamura Shinichi
Published: November 2013

Twenty strains of Clostridium difficile were examined for the effect of arginine on toxin production in a defined medium. In three strains, the production of toxins A and B was greatly enhanced in the absence of arginine. These strains showed distinctively poorer growth in the absence of arginine in comparison with the remaining 17 strains, indicating that the presence of arginine is required for good growth among the three strains. From the present results, test strains were divided into two groups: a group in which arginine insufficiency caused distinctly poor growth and enhanced toxin production, and another group in which there was neither distinctly poor growth nor enhanced toxin production. The phenomenon is discussed in relation to the biosynthesis and catabolism of arginine.
Stability of C3 Convertase in the Rat Classical Complement Pathway
Authors: Naito Akihiro , Okada Hidechika
Published: November 2013

It has been reported that rat serum complement causes efficient hemolysis of antibody‐sensitized sheep erythrocytes (EA) at 20 C but not at 37 C. In connection with this, we demonstrated that C3 convertase of rat complement was significantly unstable at 37 C using purified components of rat complement.
Involvement of Ca2+ ‐Calmodulin‐Dependent Protein Kinase II in the Intestinal Secretory Action of Escherichia coli Heat‐Stable Enterotoxin II
Authors: Fujii Yoshio , Nomura Tomohiko , Yamanaka Hiroyasu , Okamoto Keinosuke
Published: November 2013

Treating the mouse intestine with the calmodulin antagonist W‐7 and KN‐93, an inhibitor of Ca2+ ‐calmodulin‐dependent protein kinase II (CaMK II), reduced the sensitivity of the host to the action of Escherichia coli heat‐stable enterotoxin II (STII). CaMK II activity in mouse intestinal cells increased after exposure to STII. These results indicate that CaMK II is involved in the mechanism of action of STII.
Genetic Analysis of the Capsid Region of Human Astrovirus Serotype 3 Isolated in Japan
Authors: Kakizawa Junko , Ushijima Hiroshi , Wen Leying , Ikeda Yusei , Oseto Mitsuaki
Published: November 2013

The total capsid region of astrovirus serotype 3 was analyzed using five isolates including four from Japan and one from the United Kingdom. The nucleic acid and deduced amino acid sequences in the region were homologous (over 97%). However, the sequences of serotype 3 were different from those of serotypes 1, 2, 4, 5, 6 and 8, especially in the C terminus.
Deletion in the Genes Encoding Outer Surface Proteins OspA and OspB of Borrelia garinii Isolated from Patients in Japan
Authors: Wang Jianhui , Masuzawa Toshiyuki , Li Muqing , Yanagihara Yasutake
Published: November 2013

We detected the expression of outer surface proteins OspA and OspB, and characterized the genes encoding the two Osps of eight Borrelia garinii isolates from patients in Japan. Six of the eight strains shared a common antigenic epitope in their OspA and/or OspB proteins to monoclonal antibody P3134 against OspB, and were identified to have a conserved carboxyl terminus on their ospA and ospB genes by Southern blot hybridization. One strain, JEM4, did not express OspB protein, which was due to lack of the ospB gene. Gene cloning and sequencing analysis revealed that it had only one osp open reading frame with 819 nucleotides, which was similar to the ospA gene. The deletion of the ospB gene could be explained by a homologous recombination based on the common C‐terminal sequences on the ospAB operon.
Role of Curdlan Sulfate in the Binding of HIV‐1 gp120 to CD4 Molecules and the Production of gp120‐Mediated TNF‐α
Authors: Takeda‐Hirokawa Nanako , Neoh Lian‐pin , Akimoto Hiroaki , Kaneko Hiroshi , Hishikawa Takashi , Sekigawa Iwao , Hashimoto Hiroshi , Hirose Shun‐ichi , Murakami Tsutomu , Yamamoto Naoki , Mimura Tohru , Kaneko Yutaro
Published: November 2013

To clarify the mechanism by which curdlan sulfate (CRDS) inhibits human immunodeficiency virus (HIV)‐1 infection, we examined its influence on the binding of gp120 to CD4 molecules on T cells and macrophages, as well as on the production of TNF‐α by gp120‐stimulated macrophages (which promotes HIV‐1 replication). CRDS treatment of cells not only inhibited the binding of HIV‐1 gp120 to CD4+ cells, but also inhibited TNF‐α production induced by gp120. Inhibition of HIV‐1 infection by CRDS may be related to these two actions.
Modification of Delivery System Enhances MHC Nonrestricted Immunogenicity of V3 Loop Region of HIV‐1 gp120
Authors: Ahluwalia Amrita , Gokulan Kuppan , Nath Indira , Rao D. Nageswara
Published: November 2013

A successful peptide vaccine for AIDS is desired to elicit T‐helper and cytotoxic T lymphocyte responses besides neutralizing antibodies. The V3 loop peptide of HIV‐1 has been shown to contain the principal neutralizing domain, one of the most immunodominant regions, having both B‐cell and T‐cell determinants. In this study, the tip of the V3 loop region was mutated from GPGR to GPGQ based on the sequence of Indian isolates (CKRKIHIGPGQAFYT). To further enhance the immunogenicity of this epitope, two delivery systems of immune stimulating complexes (ISCOMs) and liposomes were used to incorporate the peptide. Mice of differing haplotypes, H‐2b, H‐2d, H‐2k and H‐2S, showed no MHC restriction when immunized with these formulations. The IgG levels as assessed by ELISA were found to be significantly higher (P<0.05 to P< 0.001) for even five‐fold lower doses of the peptide in ISCOMs and liposomes as compared to the conventional alum‐based preparation. The major subtype elicited was IgG2a/IgG2b, suggestive of a Th1‐like response for all the formulations. Thus, it would appear that the same peptide incorporated in ISCOMs and liposomes selects a Th1 response and may therefore be important not only for neutralization but also for virus clearance.
Cloning and Sequence Analysis of Another Shiga‐Like Toxin IIe Variant Gene (slt‐IIera) from an Escherichia coli R107 Strain Isolated from Rabbit
Authors: Kim Sang‐hyun , Cha In‐ho , Kim Kyoung‐sook , Kim Yong‐hwan , Lee Young‐choon
Published: November 2013

An Escherichia coli R107 strain (O26 serotype) producing a Shiga‐like toxin IIe variant (SLT‐IIera) was isolated from the mesenteric lymph node of a freshly dead rabbit carcass. The entire structural gene for this SLT‐IIera was cloned from chromosomal DNA by PCR using primers based on previously published slt‐IIe sequences. Nucleotide sequence analysis indicated that the slt‐llera gene was very similar to slt‐IIe (formerly called slt‐IIy) from E. coli strains S1191 and 412; five and one nucleotide changes were detected in A and B subunits, respectively, which resulted in changes in amino acid sequences of the corresponding subunits by three and one residues. Recombinant SLT‐IIera and SLT‐IIe produced using an E. coli host‐vector system showed similar cytotoxicity, suggesting that the variations in the structural gene of SLT‐IIera have no significant effect on cytotoxic level.
Direct Detection by PCR of Escherichia coli O157 and Enteropathogens in Patients with Bloody Diarrhea
Authors: Takeshi Kouichi , Ikeda Tetsuya , Kubo Akiko , Fujinaga Yukako , Makino Souichi , Oguma Keiji , Isogai Emiko , Yoshida Shin‐ichi , Sunagawa Hiroyuki , Ohyama Tohru , Kimura Hiroo
Published: November 2013

Direct detection of Escherichia coli O157 and foodborne pathogens associated with bloody diarrhea were achieved using polymerase chain reaction (PCR) after the preparation of DNA from stool specimens using the microspin technique. PCR was compared with cultivation and toxin production tests with respect to the efficiency of detection of each pathogen; E. coli O157, Vibrio parahaemolyticus, Salmonella serovar Enteritidis and Campylobacter jejuni. Detection of some or all of the above pathogens in clinical stool specimens was achieved using PCR. The minimum number of cells required for the detection of the above pathogens by PCR was 101 CFUs/0.5 g of stool sample. PCR was completed within 6 hr. The above pathogens were also detected in cultivation and toxin production tests. Partial purification of the template DNA using the microspin technique was essential for the elimination of PCR inhibitors from the DNA samples. This PCR method is an accurate, easy‐to‐read screening method for the detection of Shiga‐like toxin producing E. coli O157 and enteropathogens associated with bloody diarrhea in stool specimens.
Prevalence of Herpes Simplex Virus Type 1‐ and 2‐ Specific Antibodies among the Acute, Recurrent, and Provoked Types of Female Genital Herpes
Authors: Hashido Madoka , Lee Francis K. , Nahmias Andre J. , Kawana Takashi
Published: November 2013

Sixty‐eight sera from the acute, recurrent, and provoked types of female genital herpes were compared for the seroprevalence of herpes simplex virus (HSV) types 1 and 2 by immunodot assay using HSV glycoprotein G. In the HSV‐1‐isolated patients, no HSV‐2 antibodies were detected, whereas in the HSV‐2‐isolated patients, HSV‐1 seroprevalence was 9% for the acute type, 89% for the provoked type (P< 0.005), and 55% for the recurrent type (P<0.05). The natural history of female genital herpes and the possible protective role of pre‐existing antibodies in preventing the acquisition or clinical manifestation of a subsequent HSV infection are discussed.
Sho‐Saiko‐To, a Traditional Kampo Medicine, Enhances the Anti‐HIV‐1 Activity of Lamivudine (3TC) In Vitro
Authors: Piras Giovanna , Makino Masahiko , Baba Masanori
Published: November 2013

Sho‐saiko‐to (SST), a traditional Kampo medicine, has been examined for its inhibitory effect on human immunodeficiency virus type 1 (HIV‐1) replication in peripheral blood mononuclear cells (PBMCs). SST alone moderately inhibited HIV‐1 replication at a concentration of 25 μg/ml. When SST was combined with zidovudine (AZT), lamivudine (3TC) or AZT plus 3TC, SST enhanced the anti‐HIV‐1 activity of 3TC. In contrast, SST slightly enhanced the anti‐HIV‐1 activity of AZT plus 3TC but did not enhance the activity of AZT alone. These results suggest that the combination of SST and 3TC has potential as a chemother‐apeutic modality of HIV‐1 infection.
Immune Response to Neutralizing Epitope on Human Cytomegalovirus Glycoprotein B in Japanese: Correlation of Serologic Response with HLA‐Type
Authors: Wada Kaoru , Mizuno Shinichi , Ohta Hirotoshi , Nishiyama Yukihiro
Published: November 2013

Antigenic domain 1 (AD‐1), located between amino acids 608 and 625 of human cytomegalovirus (CMV) gB protein, is the major domain recognized by neutralizing antibodies. Amino acids 552 to 630 are essential for the binding of neutralizing antibodies. We developed an enzyme‐linked immunosorbent assay (ELISA) to detect antibodies against a fusion protein containing amino acid residues 549 to 644 of the gB polypeptide and maltose binding protein (MBP). Of 180 seropositive samples, 106 (58.9%) showed positive immuno‐reactivity against the fusion protein. None of the seronegative samples reacted with the fusion protein. Among 57 seropositive individuals typed for HLA, subjects with HLA‐DR9 had a higher positive rate against the fusion protein (13/14=92.9%) than those without HLA‐DR9 (25/43=58.1%). In addition, subjects with HLA‐DR15 had a lower positive rate against the fusion protein (7/16=43.3%) than those without HLA‐DR15 (31/41 = 75.6%). Mean OD values of HLA‐DR15‐positive individuals were significantly lower than those of HLA‐DR15‐negative individuals. Thus, among CMV‐infected individuals, HLA‐DR9 may be associated with responders for neutralizing antibodies and HLA‐DR15 may be associated with non/low‐responders.
Molecular Epidemiology of Vibrio cholerae O1 Isolated from Sporadic Cholera Cases in Okinawa, Japan
Authors: Iwanaga Masaaki , Honma Yasuko , Enami Mika
Published: November 2013

In July 1994, 6 cholera cases due to Vibrio cholerae O1 El Tor Ogawa sporadically appeared in Okinawa. All 6 patients had no history of traveling abroad. In the period of this cholera outbreak, a strain of V. cholerae O1 El Tor Ogawa was detected from an imported fish at the Naha port quarantine station. The isolates were characterized to clarify whether or not, they belonged to a common clone. Phenotypes were identical except that one strain revealed cured Celebes and the others were original Celebes in kappa phage typing. The restriction fragment patterns of DNA of the isolates hybridized with an enzyme‐labeled oligonucleotide probe for cholera toxin gene (ctx) were identical. Randomly amplified polymorphic DNA of the isolates were identical when a primer was used, but 2 patterns were seen when another primer was used. Pulsed‐field gel electrophoresis of the chromosomal DNA digested with NotI restriction enzyme showed 3 patterns. The DNA fragment pattern of the strain isolated from the imported fish was different from the clinical isolates. These results suggested that there was no epidemiological relation among the strains of V. cholerae O1 isolated during this period.
Evaluation of Enzyme‐Labeled Oligonucleotide Probes to Identify Enterohaemorrhagic Escherichia coli
Authors: Yoh Myonsun , Takagi Kazuhiro , Eda Junji , Ohtomo Masato , Takarada Yutaka , Shibata Shuji , Honda Takeshi
Published: November 2013

Alkaline phosphatase‐conjugated oligonucleotide probes were developed to detect the gene coding for Vero toxin 1 (VT1) and Vero toxin 2 (VT2). Using these probes, 3 hr was enough to detect VT genes when suspicious colonies of enterohaemorrhagic Escherichia coli (EHEC) were obtained on an agar plate. The results of a hybridization test with 144 isolates of EHEC O157 and one isolate of Shigella dysenteriae Type 1 agreed exactly with the immunological detection, reversed passive latex agglutination (RPLA) test, of VTs in their culture supernatants. The sensitivity levels of these probes for the detection of VT genes were 100%. The specificity of these probes were also tested with a total of 1,002 strains of Escherichia coli other than EHEC and 8 strains of Shigella sp. other than Shigella dysenteriae Type 1; the results showed 100% specificity.
A Thymic Hormone Protects Mice from Enteropathy during Acute Graft‐versus‐Host Disease
Authors: Inagaki‐Ohara Kyoko , Sakai Tetsu , Koya Goyo , Awaya Akira , Yoshikai Yasunobu
Published: November 2013

We have previously reported that a nonapeptide thymic hormone, facteur thymique serique (FTS), is involved in the differentiation and activation of intestinal intraepithelial lymphocytes (i‐IEL) in mice. In this study, we examined the effect of FTS treatment on enteropathy in a murine model for acute graft‐vs.‐host disease (GVHD) induced by injection of parental C57BL/6 splenocytes into unirradiated (C57BL/6XDBA/2) F1 hybrids. FTS treatment significantly protected mice from developing acute GVHD as assessed by mortality rate, splenomegaly and enteropathy. The infiltration of donor‐derived TCRαβ i‐IEL bearing CD8αβ was significantly inhibited in the small intestine of FTS‐treated mice, and the frequencies of apoptosis of crypt cells in the intestinal mucosa were decreased in these mice during acute GVHD. These results suggest that FTS treatment contributes to protection against enteropathy of acute GVHD. Thus, FTS may provide a useful approach to control acute GVHD after blood transfusion or bone marrow transplantation.
Molecular Typing of Staphylococcus epidermidis and Other CNS with Repetitive Element Sequence‐Based PCR
Authors: Hu Xiaohong , Totake Kyoko
Published: November 2013

The chromosomal distribution of the repetitive DNA sequence found in Mycoplasma pneumoniae (REP‐MP2) provides an ideal target for detecting DNA fragment patterns specific to individual Staphylococcus epidermidis and S. haemolyticus strains. A REP‐MP2 sequence‐based PCR (rep‐PCR) was developed and applied to CNS isolates. We identified a 450 bp genomic DNA fragment which was common and specific to S. epidermidis isolates and not found in other CNS. In addition, S. epidermidis isolates showed several bands that could be grouped into 14 different fragment patterns. Similarly, S. haemolyticus isolates were classified into 10 groups. Significant correlations between the typing patterns of S. epidermidis and resistance to oxacillin (P<0.05), gentamicin (P<0.01), erythromycin (P<0.02), and sulfamethoxazole‐trimethoprim (P<0.001) were found. The rep‐PCR method is a rapid and reproducible discriminatory means for molecular typing of S. epidermidis and other CNS.
Production of TNFα and IL‐6 by Activated Whole Blood from HIV‐1 Infected Patients Detected by a One‐Stage Procedure: Relationship with the Phenotype of HIV‐1 Isolates
Authors: Benyoucef Samira , Hober Didier , Shen Lu , Ajana Faïza , De Groote Donat , Bocket‐Mouton Laurence , Gérard Yann , Lion Georges , Vilain Virginie , Wattré Pierre
Published: November 2013

Diluted whole blood (WB) culturing may be the most appropriate milieu in which to study cytokine production in vitro. We tested TNFα and IL‐6 production using small volumes of WB (25 μl) from HIV‐1 positive patients with a one‐step procedure that combines WB stimulation with LPS, PHA and cytokine measurement. We studied 49 patients without secondary infection or at distance of secondary infection staged according to the 1993 classification of the CDC and 12 healthy seronegative subjects. Heparinized blood from 5 control subjects had been collected sequentially during a period of 5 months. The individual variations of TNFα and IL‐6 production were limited for all these individuals. In 1 out of 20 CDC group A patients, 6 out of 17 CDC group B patients and 3 out of 12 CDC group C patients, we obtained higher values of TNFα than the mean + 2 S.D. of the control group. In 3 out of 20 CDC group A patients, 1 out of 17 CDC group B patients without AIDS and 5 out of 12 CDC group C patients, the TNFα values were lower than the mean −2 S.D. of the control group. Low IL‐6 values were obtained in 1 out of 20 CDC group A patients and 1 out of 17 CDC group B patients and 3 out of 12 CDC group C patients. There was no correlation between TNFα production in vitro and plasma level of TNFα. We found no correlation between the levels of cytokines and monocyte count or between the levels of cytokines and CD4 T‐cell count in peripheral blood. Our data point out a disarray in TNFα and IL‐6 production by WB from HIV‐1 infected patients. The relationship between the disarray of cytokine production and cytopathogenicity of HIV‐1 isolates in the P4 cell line was investigated in this study. We found a correlation between the high level of TNFα produced by WB and the phenotype of HIV‐1 isolates isolated from patients. The one‐stage procedure used in this work is of potential value to investigate the activation status of cells for monitoring HIV‐1 positive individuals and predicting HIV‐1 phenotype.
Bioactive Fragment of Human IL‐1β [163‐171] Modulates the Immune Response to Synthetic Peptides of HIV
Authors: Gokulan Kuppan , Rao D. Nageswara
Published: November 2013

The activation of T helper cells specific for viral antigens is critical for antibody production and the generation of cytotoxic T cells during retroviral infection. In this study, we examined the effect of linking HIV peptides with a bioactive fragment of human interleukin‐1β (IL‐1β) (163–171) on the induction of immune response to the peptides. A panel of highly purified synthetic peptides representing defined regions of gp41, Gag and gp120 were used as antigens. Mouse spleen cells primed with the peptide conjugates produced greater proliferation on in vitro stimulation than spleen cells primed with peptide alone. In addition, antibody production as assessed by ELISA was observed after immunization with conjugated peptides but not with peptide alone, indicating B‐cell activation. We also found that a high level of IgG2a antibody production correlated with a high level of IFN‐γ production. These findings favor the notion that IL‐1β plays an important role in immune responses. These observations support the formulation and design of synthetic vaccines against HIV using synthetic HIV peptides conjugated with immunomodulators. Such an approach may provide an effective vaccination against other infectious agents.
Bactericidal Activity of a Fermented Hot‐Water Extract from Stevia rebaudiana Bertoni towards Enterohemorrhagic Escherichia coli O157:H7 and Other Food‐Borne Pathogenic Bacteria
Authors: Tomita Toshio , Sato Naohiko , Arai Tomio , Shiraishi Hiroyuki , Sato Minoru , Takeuchi Masaaki , Kamio Yoshiyuki
Published: November 2013

A fermented aqueous extract from Stevia rebaudiana Bertoni showed strong bactericidal activity towards a wide range of food‐borne pathogenic bacteria including enterohemorrhagic Escherichia coli O157:H7. The colony‐forming ability of the food‐borne pathogenic bacteria tested so far was reduced to < 10−7 when exposed to ⩾40% (v/v) solutions of the fermented extract at 37 C for 2 hr. Secretion of verocytotoxin 1 and 2 by enterohemorrhagic E. coli was also diminished by fermented extract at a concentration of ⩾10% (v/v). In contrast, the fermented extract did not significantly kill Bifidobacteria or Lactobacilli. The active principle(s) of the fermented Stevia extract were bactericidal under acidic conditions.
Adhesive Property of Toxin‐Coregulated Pilus of Vibrio cholerae O1
Authors: Tamamoto Tohru , Nakashima Kazutoshi , Nakasone Noboru , Honma Yasuko , Higa Naomi , Yamashiro Tetsu
Published: November 2013

The adhesive property of toxin‐coregulated pilus (TCP) to the human intestine (jejunum), and whether or not TCP mediates the adhesion of Vibrio cholerae 395 organisms to the intestinal epithelium were investigated using visually proving methods. The purified TCP did not agglutinate human erythrocytes nor adhere to the surface of human intestinal epithelium. V. cholerae 395 adhered to the epithelium, but the adhesion was not inhibited by blocking the pili with the Fab fraction of anti‐TCP IgG. The organisms adhered to the intestine treated with purified TCP in advance, as well as to the intact intestine. These findings suggest that TCP is not involved in the adhesion of these organisms to the intestinal epithelium.
Detection of Coxiella burnetii from Dust in a Barn Housing Dairy Cattle
Authors: Yanase Tsuyoshi , Muramatsu Yasukazu , Inouye Iwao , Okabayashi Tamaki , Ueno Hiroshi , Morita Chiharu
Published: November 2013

We attempted to detect Coxiella burnetii in dust samples collected from a barn housing dairy cattle by the polymerase chain reaction (PCR) method. Ten dust samples (five from ventilation fans and five from crossbeams) were collected from two areas in a barn on a farm near Sapporo, Hokkaido. C. burnetii was detected in 5 of the 10 dust samples. It was believed that aerial contamination by C. burnetii occurred in the barn.