bluuebunny/crossref_metadata_2025 huggingface dataset:
Immune Response of Mice Infected with Recombinant Vaccinia Viruses Carrying the HIV gag Gene
Authors: Sugata Fumihiko , Aoki Naoto , Shioda Tatsuo , Hayashi Takuma , Shimada Kaoru , Mitamura Keiji , Shibuta Hiroshi
Published: November 2013

We examined mouse immune response to 4 kinds of recombinant vaccinia viruses carrying the HIV gag gene, including vac‐gag/pol, which produces HIV‐like particles with processed gag proteins; vac‐gag, which also produces HIV‐like particles but with unprocessed gag protein; and vac‐gag‐pol‐fuse and vac‐es‐gag/pol, neither of which produces such particles but releases reverse transcriptase and gag protein, respectively, from infected cells. Although infection of mice with recombinant vaccinia viruses induced production of the anti‐p24 antibody in all mice, vac‐gag/pol and vac‐es‐pol induced higher production than the other two recombinants. Increase in [3H]thymidine uptake by splenic lymphocytes following p24 antigen stimulation was most evident in mice infected with vac‐gag/pol. Thus, the highest immune reaction, both humoral and cellular, was elicited by vac‐gag/pol, indicating that among those tested, this recombinant vaccinia virus is the best candidate for a vaccine that induces anti‐HIV gag immunity.
Japanese Encephalitis Virus Fusion Protein with Protein A Expressed in Escherichia coli Confers Protective Immunity in Mice
Authors: Srivastava Ashok Kumar , Morita Kouichi , Matsuo Sachiko , Tanaka Mariko , Igarashi Akira
Published: November 2013

A complementary DNA (cDNA) that codes C‐terminal, one‐third of envelope glycoprotein (E) and N‐terminal 65 amino acids of NS1 protein of Japanese encephalitis (JE) virus was inserted into Escherichia coli expression vector pRIT2T. The inserted gene was expressed as a fusion protein with protein A, and the expressed protein was intraperitoneally injected into mice. The immunized mice produced anti‐JE antibodies measured by the hemagglutination‐inhibition and neutralization tests as well as ELISA and were protected from the lethal challenge of JE virus by intraperitoneal inoculation.
Detection and Quantitative Assessment of a Vibrio cholerae O1 Species in Several Foods by a Novel Enzyme Immunoassay
Authors: Kitagawa Tsunehiro , Tsutida Yoshiko , Murakami Ritsuko , Tanimori Hideaki , Hu Jian‐guo , Utsunomiya Akiyoshi , Naito Tatsuro
Published: November 2013

A selected antibody enzyme immunoassay (SAEIA) for the general detection of Vibrio cholerae O1 species has been developed using the immunological reagents of a rabbit antiserum specific for V. cholerae O1 classical Inaba 569B and immobilized cell fragments of V. cholerae O1 E1 Tor 85P6, and β‐D‐galactosidase‐labeled goat anti‐rabbit immunoglobulin G as tracer. The SAEIA was specific for V. cholerae O1 species and showed low cross‐reaction values to other microorganism species tested including Vibrio parahaemolyticus. The detection limit of the SAEIA was 4,500 cells per assay for all the 13 strains of V. cholerae O1 examined. Quantitative comparison on the growth of the E1 Tor 85P4 in several foods cultured for 24 hr were studied using the SAEIA. Preceding the experiments, little inhibition of every food homogenate for the measurement of the SAEIA was first demonstrated and then the homogenate was directly used for an assay sample. The interaction of the growth of Escherichia coli to that of V. cholerae O1 in a food was also found to be little under the mixed culturing of both bacteria using the SAEIA.
Hepatitis E Virus: cDNA Cloning and Expression
Authors: Uchida Toshikazu , Suzuki Koyu , Hayashi Nakanobu , Iida Fusae , Hara Tomijiro , Oo San San , Wang Chung‐kwe , Shikata Toshio , Ichikawa Munetaka , Rikihisa Tetsuji , Mizuno Kyosuke , Win Khin Maung
Published: November 2013

Viral hepatitis E is endemic, frequently provoking epidemic outbreaks in many developing countries. We have attempted to clone the viral genome and to develop an antibody assay system. A lambda gt11 cDNA library was constructed from the bile juice containing putative causative viruses and was immunoscreened by the antisera obtained from patients and monkeys infected with hepatitis E. Three virus‐specific clones were isolated and were revealed to overlap one another in sequence, with 1,459 nucleotides in total length. These clones direct the synthesis of polypeptides probably having common immunological epitope(s). Immunoplaque assay revealed the occurrence of antibodies against this epitope in the sera from experimental monkeys with the convalescent phase and from patients of Myanmar, Nepal and India. The data indicate that the cDNA fragments are useful for immunodiagnosis of hepatitis E.
A Latex Agglutination Test for the Detection of Mycoplasma pneumoniae in Respiratory Exudates: A Comparative Study with a Commercially Available DNA‐Probe Test
Authors: Masayoshi Toyohisa , Hirai Yoshikazu , Kanemasa Yasuhiro
Published: November 2013

We prepared polyclonal antibody specific to Mycoplasma pneumoniae. Using this antibody, we developed a latex agglutination test (LAT) for detecting the organism in respiratory exudates as rapid diagnosis of M. pneumoniae infection. Further, LAT was compared with DNA‐probe test (DP) which was the only commercially available test for the rapid detection of the organism. In LAT, both M. pneumoniae and M. genitalium give positive agglutination, but the titer of M. genitalium was significantly lower than that of M. pneumoniae. The detection limit of LAT was 2 × 105 CFU/ml and that of DP was 5 × 104 CFU/ml in vitro. It was considered that target molecules in LAT were accumulated in the pharyngeal portion of the patients, because of their long half‐life at 37 C. However, ribosomal RNA which was target molecule in DP was destroyed at 37 C much sooner, and the accumulation could not be expected. Actually, positive rate in LAT was higher than that in DP among clinical specimens in which M. pneumoniae was detected by culture method. The procedure of LAT is much easier and more rapid than that of DP in which radioactive isotope is required. LAT could be the choice of test for rapid diagnosis of M. pneumoniae infection.
Partial Characterization of Coliphage WPK and a Comparison with Coliphage T3
Authors: Loeffelholz Lisa , Maier Siegfried
Published: November 2013

Coliphage WPK was originally isolated from sewage in Kiel, Germany, because its plaque diameter continued to expand for days. Electron microscopy revealed an isometric capsid with dimensions of 54 nm between opposite apices, and a short, noncontractile tail 16 nm long, placing phage WPK into morphogroup C1. The nucleic acid of phage WPK was linear double stranded DNA. The host ranges of phages WPK and T3 were identical. Of ten E. coli strains tested for host range, two were resistant and of eighteen other Enterobacteriaceae only four were susceptible. Seven gram‐negative species which are not members of the Enterobacteriaceae were refractory. However, there were differences in plaque morphology and plaque expansion between the two phages. Phage T3 plaques expanded for at least seven days on E. coli B only, while phage WPK plaques expanded for at least seven days on four strains of E. coli. The buoyant density of WPK, determined by isopycnic density gradient centrifugation in CsCl, was 1.508 g/ml which was significantly different than that of T3 at 1.493 g/ml (P<0.05). Phage‐encoded proteins were examined for each phage using [35S]methionine incorporation, SDS‐PAGE, and autoradiography. Of thirty proteins identified in phage WPK and twenty‐eight in' phage T3, only fourteen were of the same size in both. We concluded that phage WPK was distinct, but related to T3.
Molecular Analysis of Virus‐Producing and Non‐Producing Clones Derived from a Defective SSPE Virus Yamagata‐1 Strain
Authors: Haga Takeshi , Komase Katsuhiro , Yoshikawa Yasuhiro , Yamanouchi Kazuya
Published: November 2013

Two virus clones were isolated from a defective SSPE virus, the Yamagata‐1 strain, and designated as the YA and YF clones. The YA clone‐infected cells produced neither cell‐free virus nor cell‐associated virus, whereas the YF clone‐infected cells produced both cell‐associated and cell‐free virus. No difference of epitopes on structural proteins was observed between these two clones. Both clones had hemadsorption activity. Quantitation of structural protein by Western dot blots showed relatively a lower amount of M protein in the YA‐infected cells than that in the YF‐infected cells. The ratio, P plus M dicistronic/M monocistronic mRNA, in the YA‐infected cells was about twice that in the YF‐infected cells. Sequence analysis of cDNA corresponding to P plus M dicistronic mRNA revealed that the deduced M protein of the YF virus was smaller than that of the YA virus by five amino acids from the carboxy terminal. These results suggest that abundant production of P plus M dicistronic mRNA is responsible for the reduced amount of M protein in the non‐productive YA clone.
Depressed CD4/CD8 Ratio in TPHA‐Negative Patients with Syphilis
Authors: Kanda Tsugiyasu , Shinohara Hiroshi , Suzuki Tadashi , Murata Kazuhiko
Published: November 2013

The Treponema pallidum hemagglutination assay (TPHA) is a widely used method for screening syphilis. We report our experience with six elderly (age 72.4±8.3 years) patients with syphilis, whose TPHA was negative. Their cardiolipin (RPR) and absorbed fluorescence treponemal tests (FTA‐ABS) were positive. TPHA‐negative patients with syphilis were compared with TPHA‐positive syphilitics by immunological analysis. We found that both the numbers and the percentage of CD4 cells in TPHA‐negative syphilitics were significantly lower than those in TPHA‐positive syphilitics (722±142 vs. 1,064± 141/mm3, P<0.01: 35.1±6.9 vs. 48.4±6.4%, P<0.01) and that the ratio of CD4 to CD8 was also lower in TPHA‐negative syphilitics compared with TPHA‐positive syphilitics (1.08±0.46 vs. 2.24± 1.07, P<0.05). These data suggest that the TPHA is insufficient for excluding elderly syphilitics because of immunological impairment seen in aged patients.
Augmentation of Host Resistance to Candida albicans Infection in Ascites Tumor‐Bearing Mice
Authors: Okawa Yoshio , Murata Yoshiko , Kobayashi Makiko , Suzuki Masuko , Suzuki Shigeo
Published: November 2013

We found that the number of peripheral blood polymorphonuclear leukocytes (PMN) dramatically increased in both sarcoma 180 (S‐180) and MM‐46 mammary carcinoma (MM‐46) ascites tumor‐bearing mice, and mice required a remarkable resistance to Candida albicans infection via intravenous route. When the resistance was determined by number of cells of C. albicans in the kidney, a significant decrease in the number of fungal cells was observed in the kidneys of infected ascites tumor‐bearing mice. An increase of active oxygen levels of PMN from ascites tumor‐bearing mice was observed, suggesting that this factor is important in developing of resistance in ascites tumor‐bearing mice. Additionally, a culture supernatant of tumor cells co‐cultivated with bone‐marrow cells in vitro increased the number of granulocytes and macrophages differentiated from the bone‐marrow cells.
Differentiation between Mycobacterium avium and Mycobacterium intracellulare by Thin‐Layer Chromatography of Lipid Fraction after Incubation with [35S] Methionine
Authors: Tsukamura Michio
Published: November 2013

Mycobacterium avium and Mycobacterium intracellular are differentiated from each other by thin‐layer chromatography of lipid fraction extracted after incubation with [35S]methionine. The former contained a petroleum ether‐soluble sulfolipid and the latter did not.
Existence of Phosphoenolpyruvate: Carbohydrate Phosphotransferase Systems in Lactobacillus fermentum, an Obligate Heterofermenter
Authors: Nagasaki Hiroshi , Ito Kazuyo , Matsuzaki Shigenobu , Tanaka Shuji
Published: November 2013

The presence or absence of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) in obligately heterofermentative group III lactobacilli including Lactobacillus brevis (3 strains), L. buchneri (2 strains) and L. fermentum (3 strains) was surveyed systematically for a series of sugars utilizable by these organisms. Contrary to common expectation, PTSs were found in two strains of L. fermentum: sucrose‐PTS in one strain; sucrose‐ and mannose‐PTSs in the other. All these activities were found to be constitutive.
Ultrastructural Study on the Adherence of Branhamella catarrhalis to Oropharyngeal Epithelial Cell
Authors: Ahmed Kamruddin , Rikitomi Naoto , Nagatake Tsuyoshi , Matsumoto Keizo
Published: November 2013

In the present study, it was observed that Branhamella catarrhalis adhere to the microplicae of the oropharyngeal epithelial cells. Both long and short microplicae patterns are present on the surface of oropharyngeal epithelial cells and the adherence ability of fimbriated Branhamella catarrhalis also varies according to the microplicae pattern. It was found that Branhamella catarrhalis attached more to one surface of the epithelial cell than to the other, suggesting that the presence of receptors are more on one surface than on the other. Branhamella catarrhalis did not attach to the mucus layer but directly to the epithelial cell surface. Ruthenium red staining specimen showed that Branhamella catarrhalis attached to a granular ruthenium red positive layer on the microplicae and also to a ruthenium red positive component, external to the unit membrane of the epithelial cell membrane.
Immunoglobulin G Fc Fragment‐Binding Proteins in Mycoplasma salivarium Cells
Authors: Sawa Yoshihiko , Watanabe Tsuguo , Shibata Ken‐ichiro
Published: November 2013

Cell proteins of Mycoplasma salivarium were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, transferred to membranes, then examined for reactivity with human IgG molecules, the Fc fragment of human IgG, and concanavalin A (ConA). Multiple protein bands bound IgG, and most of them also bound ConA. One (corresponding to a molecular mass of 90 kDa) of the IgG‐ and ConA‐binding bands intensely interacted with the Fc fragment of IgG. The reactivity of proteins eluted from the band with the Fc fragment, tested by dot‐blotting and ELISA, was inhibited (90%) by pre‐incubation with IgG and to a lesser extent (50%), with IgM. Thus, M. salivarium contained a cellular protein with a molecular mass of 90 kDa, that bound the Fc fragment of human IgG.
Focus Formation by the Human Immunodeficiency Virus (HIV) in the Immobilized MT‐4 Cell Culture and Its Application to the Evaluation of Anti‐HIV Agents
Authors: Nakane Masayuki , Kawai Akihiko
Published: November 2013

Immunofluorescence studies were performed on the infection of monolayer cultures of immobilized MT‐4 cells with human immunodeficiency virus type 1 (HIV‐1). By using the anti‐viral p24 monoclonal antibody, we could observe formation of foci of p24 antigen‐positive cells within 3 to 4 days when the infection was initiated with a relatively small amount of the virus. Frequency of the focus formation was in proportion to the dose of input virus (ranging from 0.001 to 0.1 PFU/cell), which allowed us to apply this phenomenon to the assay of anti‐HIV agents as well as to the estimation of relative infectivity of the virus stocks. When antiviral agents were added to the infected cultures, number of foci as well as the size of each focus was reduced in a concentration‐dependent manner. The dose required for reducing the number of foci by 50% was calculated to be 6 ng/ml and 8 ng/ml for tunicamycin (TM) and azidothymidine (AZT), respectively. These values are comparable to those obtained by other current assay methods. In addition, focus reduction assay is also useful in searching for such antiviral agents that would inhibit or block the early step of viral replication cycle.
An Enzyme‐Linked Immunosorbent Assay Using a Chaotropic Agent (Sodium Thiocyanate) for Serotype Specific Reaction between Crude Dengue Viral Antigen and Anti‐Dengue Mouse Antibody
Authors: Nawa Masaru
Published: November 2013

An enzyme‐linked immunosorbent assay (ELISA) has been developed to detect serotype specific reaction between crude dengue viral antigen and anti‐dengue mouse hyperimmunized antibody under the stringent condition in the presence of a Chaotropic agent, sodium thiocyanate (NaSCN), in the reaction mixture of antigen and antibody. Rapidly sedimenting hemagglutinin (RHA) derived from type 2 dengue virus‐infected mosquito cell culture fluid reacted to the antibody for both type 2 and type 3 dengue viruses in the ELISA. In contrast, its reactivity was reduced after the addition of NaSCN in the ELISA. Soluble complement‐fixing antigen (SCF) derived from type 2 dengue virus‐infected mosquito cell culture fluid reacted serotype specifically to anti‐dengue type 2 antibody, and was relatively stable for the NaSCN treatment in the ELISA. Anti‐type 2 RHA mouse antibody reacted to both type 1 and type 2 dengue viral antigens and its reactivity was reduced after the addition of NaSCN in the ELISA. Anti‐type 2 SCF antibody reacted serotype specifically to type 2 dengue viral antigen with and without NaSCN in the ELISA.
Antibody Response to Oral Streptococci in Behçet's Disease
Authors: Yokota Kenji , Hayashi Syunji , Fujii Nobuhiro , Yoshikawa Kouji , Kotake Satoshi , Isogai Emiko , Ohno Shigeaki , Araki Yoshio , Oguma Keiji
Published: November 2013

The serum antibody titers against oral streptococci were studied by enzyme‐linked immunosorbent assay (ELISA) both in patients with Behçet's disease (BD) and control groups. The patients with BD showed significantly higher antibody titers to S. sanguis strains 113‐20, 114‐23, and 118‐1 which were isolated from patients with BD, in comparison with control groups. Also, the reactions of hightitered sera to the crude cell wall and soluble (or membrane) fractions of the 113‐20 strain were observed by western blot test. The sera of the patients with BD demonstrated strong bands of approximately 36 kDa, 82 kDa, and 87 kDa in the crude cell wall fractions, and many bands of 80 kDa to 150 kDa in the membrane fractions, indicating that these proteins are the ones leading the high antibody titers to this bacterium in the sera of patients with BD.
A Prospective Study on Correlation between the Decrease in Anti‐P17 Antibody Level and Progression to AIDS in Asymptomatic Carriers of HIV
Authors: Choudhury Ahmed Murtaza , Yamada Osamu , Wakamiya Nobutaka , Kurimura Takashi
Published: November 2013

As the majority of human immunodeficiency virus (HIV) carriers are in asymptomatic stage for a long period of time, it is important to investigate the factors or surrogate markers for conversion from asymptomatic to symptomatic stage. Our study is designed to evaluate the relationship among virus isolation rate, anti‐p17 antibody status and progression to AIDS. We studied anti‐p17 antibody status along with virus isolation in 56 asymptomatic carriers and 46 AIDS cases. Progression to AIDS was markedly associated with high rate of virus isolation and loss of anti‐p17 antibody. In order to know the meaning of loss of anti‐p17 antibody during the clinical course, 15 anti‐p17 antibody positive and 16 anti‐p17 antibody negative cases were followed up prospectively for the development of AIDS. None of the anti‐p17 antibody positive cases developed AIDS while 6 out of 16 anti‐p17 negative cases developed AIDS during observation period (P<0.05). Progression to AIDS was associated with loss of anti‐p17 antibody. Identification of cases losing anti‐p17 antibody in peripheral blood during asymptomatic period may help high‐risk group who are in need of chemoprophylaxis. Moreover, study of anti‐p17 antibody may be helpful in designing vaccine in future if it works as a neutralizing antibody to HIV in vivo.
Effects of Gamma‐Interferon and FK506 on Resting B Cell Proliferation of New Zealand Black/White F1 Mice
Authors: Minoda Masahide , Ohno Motoki , Tomioka Yoshiki , Hamada Kinya , Yamazoe Yasushi , Higashikawa Mitsuhiro , Sugishima Hitoshi , Higashitani Sumihiko , Funauchi Masanori , Horiuchi Atsushi
Published: November 2013

We examined the effects of gamma‐interferon (γ‐IFN) and the new immunosuppressant FK506 on resting B cell proliferation of New Zealand black/white F1 hybrid (B/W F1) mice, an animal model of human systemic lupus erythematosus (SLE). γ‐IFN and FK506 inhibited in a dose‐dependent manner both B cell proliferation and autoantibody production of resting B cells respectively. There was a synergistic interaction between γ‐IFN and FK506 in their inhibition and they did not exhibit cell cytotoxicity. This in vitro synergism of γ‐IFN and FK506 may have clinical application in that low doses of γ‐IFN and FK506 combinations may be effective to correct polyclonal B cell activation of patients with SLE.
Involvement of Calcium in Germination of Coat‐Modified Spores of Bacillus cereus T
Authors: Shibata Hirofumi , Miyoshi Shigekatsu , Osato Tomohiko , Tani Isamu , Hashimoto Tadayo
Published: November 2013

The effect of calcium on germination of coat‐modified Bacillus cereus T spores was investigated. Coat‐modified spores produced either by chemical extraction (SDS‐DTT‐treated spores) or by mutagenesis (10LD mutant spores) were unable to germinate in response to inosine. While SDS‐DTT‐treated spores could germinate slowly in the presence of l‐alanine, 10LD mutant spores could not germinate at all. The lost or reduced germinability of coat‐modified spores was restored when exogenous Ca2+ was supplemented to the germination media. The calcium requirement of coat‐modified spores for germination was fairly specific. The simultaneous presence of germinant with Ca2+ was also required for germination of coat‐modified spores. The optimal recovery of germinability was observed in the presence of 1.0 mM of calcium acetate. The calcium requirement itself was remarkably diminished under the condition in which l‐alanine and a certain purine nucleoside analog, adenosine or inosine, coexisted. The lost or diminished germinability observed in SDS‐DTT‐treated spores or 10LD mutant spores may be attributed to the loss of calcium associated with the spore integuments.
IgG Subclass Alterations in Adult Asthma
Authors: Outschoorn Ingrid M. , Natta Clayton L.
Published: November 2013

Immunoglobulin levels were measured in serum samples of 12 black adult non‐smoking asthmatic patients, 11 females and 1 male, and compared with 15 age‐, sex‐matched normal controls. Their total IgG, IgA and IgM levels were within the normal range. However, on quantitation of subclasses, IgG1 levels were significantly above normal, while IgG2 and IgG3 levels were significantly lower than those of controls. No significant differences were found between the two groups when IgG4 levels were compared. These studies as well as those of others suggest that immunoglobulin administration, particularly of individual subclasses, might prove to be a beneficial addition in the management of this condition.
DNA from Bacteria, but Not from Vertebrates, Induces Interferons, Activates Natural Killer Cells and Inhibits Tumor Growth
Authors: Yamamoto Saburo , Yamamoto Toshiko , Shimada Shizuo , Kuramoto Etsuro , Yano Osamu , Kataoka Tetsuro , Tokunaga Tohru
Published: November 2013

The nucleic acid fraction from cells of 6 species of bacterium and 2 kinds of vertebrate, calf and salmon, was extracted and purified by the same procedures as described previously. When the spleen cells from BALB/c mice were incubated with the nucleic acid fraction from either of the bacteria, natural killer (NK) activity of the cells was remarkably elevated and the cells produced factors to activate macrophages and to inhibit viral growth. It was shown that the factor to activate macrophages was interferon (IFN)‐gamma and that to inhibit viral growth was IFN‐alpha/beta. On the other hand, the nucleic acid fraction from either of the vertebrate cells did not show such activities. Pretreatment of the bacterial nucleic acid fraction with DNase, but not with RNase, abrogated completely the biological activities. The activities of the bacterial nucleic acid were not influenced by the presence of polymyxin B, an inhibitor of lipopolysaccharide (LPS), and the spleen cells from not only BALB/c mice but also LPS‐insensitive C3H/HeJ mice were activated, indicating that the activities of the fraction were not ascribed to LPS contaminated possibly into the fraction, but to DNA itself. Intralesional injection with the bacterial DNA fraction caused regression of mouse IMC tumors, but the injection with the vertebrate DNA fraction did not. These findings prompted us to examine the biological activities of DNA samples from a variety of animals and plants, which were provided from other laboratories or purchased from manufacturers. All of the DNA samples from cells of 5 kinds of bacterium, 2 of virus and 4 of invertebrate augmented NK activity and induced IFN, more or less, in mouse spleen calls, while the DNA from 10 kinds of vertebrate, including 3 of fish and 5 of mammal, showed no such activities. The DNA from 2 species of plants, were also inactive. Possible mechanisms to explain the different biological activities of DNA from different cell sources were discussed based on our previous finding that the particular palindromic sequences with a G‐C motif (s) are required for induction of IFNs and activation of NK cells with synthetic 30‐mer oligonucleotides.
Seroepidemiological Survey for Antibody to Borrelia burgdorferi in Cows
Authors: Isogai Hiroshi , Isogai Emiko , Masuzawa Toshiyuki , Yanagihara Yasutake , Matsubara Mitsunori , Shimanuki Masamichi , Seta Toshiyuki , Fukai Kenji , Kurosawa Nobumichi , Enokidani Masafumi , Nakamura Toshio , Tajima Motoshi , Takahashi Kiyoshi , Takahashi Koichi , Fujii Nobuhiro
Published: November 2013

Antibody to Borrelia burgdorferi was examined in 380 healthy and 38 clinical cases of cows from Hokkaido and Shizuoka in Japan. In healthy animals, IgG and IgM antibody to B. burgdorferi HO14 strain were found in 44 cows (14.6%) and 24 cows (8.0%) from Hokkaido. In contrast, antibody‐positive case was not observed except for only 1 case which was IgM positive (1/79: 1.3%) in cows from Shizuoka. Mean antibody levels of healthy animals in Hokkaido and Shizuoka were 0.651 and 0.263 (IgG antibody to HO14 strain), 0.642 and 0.169 (IgG to HP3 strain), 0.613 and 0.367 (IgM to HO14 strain) and 0.582 and 0.286 (IgM to HP3 strain). The differences of the antibody levels between cows from Hokkaido and Shizuoka were significant. Seasonal difference was found in seropositive cows from Hokkaido. The rate of seropositive cows was high in summer (23.4% in June and 11.8% in July) but low in winter (0% in January and February). The pattern was discussed to be associated with activation of ticks. One of 4 cows with arthritis showed significantly higher IgG antibody level than that of healthy cows and cows with some disease, although the serum was collected from Shizuoka where antibody‐positive animals for B. burgdorferi were rare among healthy cows. This high IgG antibody may suggest that the arthritis of such cows was caused by infection with B. burgdorferi. Two of 7 cows with unclassified abortion showed positive antibody reaction in Hokkaido. These cases, however, may not be related to the B. burgdorferi infection because the positive rate was similar to those of healthy cows in the same season.
Nucleotide Sequence Analysis of the Class G Tetracycline Resistance Determinant from Vibrio anguillarum
Authors: Zhao Jiang , Aoki Takashi
Published: November 2013

The nucleotide sequence of the class G tetracycline resistance determinant previously isolated from Vibrio anguillarum has been determined. Two open reading frames of divergent polarity were identified. A resistance gene (tet A) encodes a protein of 393 amino acid residues (deduced molecular mass of 40.9 kDa), and a repressor gene (tet R) encodes a protein consisting of 210 amino acids with a calculated molecular mass of 23.6 kDa. Based on the deduced amino acid sequences, the proteins of tet A(G) and tet R(G) are about 60% homologous with those of RP1/Tn1721 (class A) and pSC101/pBR322 (class C), and about 50% homologous with Tn10 (class B). The relationship of the tet (G) sequence to five known tetracycline resistance determinants (class A to E) is discussed.
Impaired NK Response of Cancer Patients to IFN‐α but Not to IL‐2: Correlation with Serum Immunosuppressive Acidic Protein (IAP) and Role of Suppressor Macrophage
Authors: Aso Hisashi , Tamura Keiji , Yoshie Osamu , Nakamura Takehiko , Kikuchi Syuu , Ishida Nakao
Published: November 2013

In vitro NK responses of cancer patients (N=21) to rIFN‐αA and rIL‐2 were examined. The serum concentration of IAP (immunosuppressive acidic protein) was determined in parallel. Five out of seven patients whose serum IAP contents were within the normal range (270 μg/ml to 470 μg/ml), had their NK activities significantly augmented by rIFN‐αA and rIL‐2. On the other hand, NK cells from ten out of fifteen patients whose serum IAP concentrations were 650 μg/ml or more, were not activated by rIFN‐αA. NK cells of these fifteen patients yet were capable of responding to rIL‐2. NK cells from cancer patients, however, became responsive to rIFN‐αA by either removal of adherent cells or treatment with indomethacin. Therefore, macrophages in PBMC of cancer patients with high serum IAP levels seem to selectively suppress NK response to rIFN‐αA by an indomethacin‐sensitive mechanisms. It was further shown that PGE2 was not the mediator of this suppression.
Sensitivity of Polymerase Chain Reaction Assay for Rickettsia tsutsugamushi in Patients' Blood Samples
Authors: Murai Koichi , Tachibana Nobuyoshi , Okayama Akihiko , Shishime Eiichi , Tsuda Kazunori , Oshikawa Tatsumi
Published: November 2013

We developed a nested polymerase chain reaction (PCR) method to detect Rickettsia tsutsugamushi (R. tsutsugamushi) DNA and determined its sensitivity. Primers were selected from the DNA sequence of the 58‐kDa group‐specific antigen gene of the Karp strain. The target sequence of rickettsial DNA was detectable as the band corresponding to 88 bp in 1.0 μg of the DNA extracted from BS‐C‐1 cells infected with R. tsutsugamushi. Rickettsia‐specific bands were observed not only for the homologous Karp strain, but also for four heterologous strains: two other reference strains (Gilliam and Kato) and two prototype strains prevalent in Miyazaki district (Irie and Hirano). The minimum copy number detectable by this method was estimated to be five rickettsiae. All of nine peripheral blood mononuclear cell samples from patients with tsutsugamushi disease who were seen 2‐11 days after disease onset tested positive for rickettsial DNA. The PCR assay method presented here could be a specific diagnostic tool for tsutsugamushi disease, especially in its early acute stage.
Enteric Adenovirus Type 41 Isolates: Cloning, Physical Maps and Diversity in Restriction Enzyme Cleavage Pattern
Authors: Yamashita Yumi , Hotsubo Tomoyuki , Nakata Shuji , Yamaguchi Taihei , Sanekata Takeshi , Chiba Shuzo , Homma Morio , Fujinaga Kei
Published: November 2013

Adenovirus (Ad) type 40 and 41 DNAs were directly extracted from stool specimens of children with gastroenteritis. Two new strains of Ad41, Sanekata and Ehime strain, were cloned and their restriction maps were constructed. The left terminal end of the cloned Ad41 genome, EcoRI‐E fragment of the Sanekata strain and EcoRI‐F fragment of the Ehime strain, had transforming ability in rat 3Y1 cells. Only one of the 35 isolates of Ad40 tested showed a different restriction profile, while three different restriction profiles were found in DNAs from Ad41 isolates.
Polymorphism of Crystals of Salmonella minnesota Re and Ra Lipopolysaccharides
Authors: Kato Nobuo , Ohta Michio , Kido Nobuo , Arakawa Yoshichika , Sugiyama Tsuyoshi , Naito Setsuko , Ito Hideo
Published: November 2013

Salmonella minnesota Re and Ra lipopolysaccharides (LPSs) formed three‐dimensional crystals when they were precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and incubated in 70% ethanol containing 250 mM MgCl2 at 4 C. Besides typical shapes of crystals, hexagonal plates and solid columns, which were already reported (J. Bacteriol. 172: 1516–1528 (1990)), the LPSs thus treated formed crystals possessing various shapes such as square or rectangular plate, lozenge plate, discoid, and truncated hexangular pyramid forms. Electron diffraction patterns from all these crystals except square or rectangular plate crystals obtained by electron irradiation from the direction perpendicular to the basal plane were essentially the same as those from hexagonal plate crystals, indicating that they consist of hexagonal lattices with the lattice constant of 4.62 Å. From these results as well as the results of electron microscopic observations of these crystals, it was concluded that all these crystals except square or rectangular plate crystals are composed of hexagonal plate sheets as the basic structural units. Square or rectangular crystals were assumed to correspond to the {1011} planes of solid hexagonal column crystals.
Enhancement by Recombinant Human Interleukin 2 of Host Resistance to Toxoplasma gondii Infection in Pregnant Mice
Authors: Shirahata Toshikazu , Muroya Naoyoshi , Ohta Chikako , Goto Hitoshi , Nakane Akio
Published: November 2013

The lymphokine production by pregnant mice infected with a lethal dose of Toxoplasma gondii was evaluated in comparison with that by virgin mice infected with a sublethal dose of this protozoan parasite. Splenocytes taken from mice before and on the day after infection produced considerable amounts of IL‐2 in response to concanavalin A (Con A) stimulation, but the titers rapidly declined in both pregnant and virgin mice as infection progressed. A trace amount or undetectable level of IL‐2 was produced by splenocytes from acutely infected mice when stimulated with Toxoplasma lysate antigen (TLA). In contrast to the kinetics of IL‐2 production, the levels of IFN‐γ produced by splenocytes cultured with Con A or TLA increased steadily in the later stage of infection in both pregnant and virgin mice. Thus, the response to Con A or TLA of splenocytes to produce IL‐2 and IFN‐γ differed strikingly in acute toxoplasmosis in mice. The administration of rHuIL‐2 resulted in a significant decrease in the mortality of pregnant mice infected with a lethal dose of Toxoplasma. The combination of rHuIL‐2 and rMuIFN‐γ increased the survival rate slightly but not significantly compared with pregnant mice receiving either rHuIL‐2 or rMuIFN‐γ. Moreover, exogenously administered rHuIL‐2 enhanced the production of both IFN‐α and IFN‐γ in the bloodstreams of pregnant mice, in accordance with the decreased mortality. These results indicate that IL‐2 may play a significant role in modulating the host defense against Toxoplasma infection in pregnant mice.
Neutrophil Response to Nontypable Haemophilus influenzae in Respiratory Infections
Authors: Ahmed Kamruddin , Ichinose Akitoyo , Dai Tran Cong , Takahashi Atsushi , Utsunomiya Yoshiaki , Kawakami Kenji , Nagatake Tsuyoshi , Matsumoto Keizo
Published: November 2013

Sputa from patients with respiratory infections by nontypable Haemophilus influenzae (H. influenzae) were investigated by electron microscopy. The cell wall of H. influenzae appeared wavy and nonwavy. In the cell wall the peptidoglycan layer was ill‐defined. These patients had adequate IgG response in the serum against H. influenzae. However neither capsule nor fimbriae were found. Different stages of phagocytosis and destruction of the bacteria by polymorphonuclear neutrophils (PMN) were observed. PMNs were also found to phagocytose the debris. Evidences were found that the debris is formed mainly by the destruction of polymorphonuclear neutrophil. Extracellular lysosomes were also observed, which may have a role in destruction of both bacteria and host tissue. It was concluded that nontypable H. influenzae are nonfimbriated and noncapsulated during infection. Debris are the end product of PMN destruction, and phagocytosis of debris by PMNs has a role in the pathogenesis of chronic respiratory diseases.
Characterization of Borrelia Species Isolated from Ixodid Ticks, Ixodes ovatus
Authors: Takahashi Yukie , Sohnaka Masako , Nakao Minoru , Miyamoto Kenji , Fukunaga Masahito
Published: November 2013

Thirty‐two Borrelia isolates were obtained from the adult stage of ixodid ticks, Ixodes ovatus, collected in various localities in Japan. Borrelial isolates were cultivated and analyzed by polyacrylamide gel electrophoresis, with monoclonal antibodies, by pulsed field gel electrophoresis, and by genomic Southern hybridization. All borrelial isolates showed similar protein profiles and monoclonal antibody reactivities, while plasmid profiles were rather diverse. Genomic hybridization using rRNA gene probes demonstrated the genetic similarities of those isolates. We found no significant differences among the borrelial isolates tested, and the restriction fragment length polymorphism patterns of I. ovatus isolates were quite distinct from those of borrelial strains associated with Lyme disease. Therefore, the isolates of Borrelia obtained from I. ovatus were thought to fall into different genospecies.
Production of Monoclonal Anti‐Idiotype Antibodies Which Mimic an M‐Like Protein of Streptococcus equi
Authors: Jean‐Francois M. J. Bernadette , Poskitt David C. , MacDonald Lisa M. , Turnbull Sally J. , Yasmeen Dilruba
Published: November 2013

Rat monoclonal anti‐idiotype antibodies (mAb2) were raised against two mouse monoclonal antibodies (mAb1), 1D10 and 2A6, with specificity for the M‐like protein of Streptococcus equi. The capacity of the mAb2 to inhibit the binding between the corresponding mouse mAbl against which the mAb2 were raised and the M‐like protein was investigated in an inhibition EIA. One of the ten mAb2 examined, namely 5D1 (anti‐mAb1 1D10), was able to inhibit this binding. The mAb2 5D1 bound to the mAb1 1D10 in such a way as to completely inhibit the subsequent binding of the M‐like protein antigen to the paratope of the mAb1 1D10. The mAb2 5D1 is likely to represent a true image of the M‐like protein antigen and may thus be described as an Ab2β anti‐idiotype antibody.
The Structure of Mycoplasma pneumoniae as Determined by the Freeze‐Substitution Technique
Authors: Amako Kazunobu , Takade Akemi , Takada Tohoru , Watanabe Tsuguo , Nakamura Masahiro
Published: November 2013

The ultrastructure of Mycoplasma pneumoniae FH was examined by a mild fixation method, the freeze‐substitution technique, for thin‐section transmission electron microscopy and the following new findings were obtained. In the cytoplasm, no nuclear region could be clearly identified. The cytoplasm was filled with many ribosome‐like particles, fine fibers and electron‐dense particles. The electron‐dense particles appeared to be similar to the particles found in the nucleoid region of Pseudomonas aeruginosa and might therefore possibly be a kind of DNA binding protein. The cell surface was completely enveloped with a thin opaque layer. The presence of this surface layer prevented any direct contact of the cell surface with that of the two M. pneumoniae cells.
Purification and Characterization of a Protein Cryoprotective for Vibrio cholerae Extracted from the Prawn Shell Surface
Authors: Faming Dong , Shimodori Shoichi , Moriya Tetsuhiro , Iwanaga Sadaaki , Amako Kazunobu
Published: November 2013

A substance cryoprotective for Vibrio cholerae on the prawn shell surface was purified by ammonium sulfate precipitation and gel filtration. It was a protein of 81 kDa and called cryoprotective protein (CPP). The cryoprotective activity of this protein for V. cholerae was sensitive to heat at 100 C and trypsin treatment. In the presence of Mg ion the protein can bind to the bacterial cell surface. V. cholerae can adhere to the shell surface of the prawn. The number of adhered bacteria was reduced by treating the shell with anti‐CPP serum, heat or by trypsin. The presence of Mg ion promoted the adherence. These results suggest that the CPP could serve as an adherence site for V. cholerae on the shell surface.
Comparison between Enterotoxigenic Escherichia coli Strains Expressing “F42,” F41 and K99 Colonization Factors
Authors: Sperandio Vanessa , Dias da Silveira Wanderley
Published: November 2013

Two enterotoxigenic Escherichia coli (ETEC) strains (coded 567/7 and 103) isolated from piglets with neonatal diarrhea were described as producers of a new adhesin (F42). With the use of molecular biology and immunology techniques such as DNA hybridization with probes for F41 and K99 genes and Western‐blotting of the superficial proteins of these strains and standard E. coli strains carrying genes for F41 and K99 adhesins, it was demonstrated that this new adhesin either shares extensive genetic and immunological determinants with F41 adhesin or they are the same fimbriae.
Organization of Ribosomal RNA Genes in Borrelia burgdorferi sensu lato Isolated from Ixodes ovatus in Japan
Authors: Zhang Yanhua , Takahashi Yukie , Fukunaga Masahito
Published: November 2013

Borrelia burgdorferi sensu lato was obtained from adult ixodid ticks, Ixodes ovatus, collected in Nagano, Japan, and was named NT112. The genomic DNA was digested with enzymes, electrophoresed, blotted and hybridized with rRNA gene probes obtained from B. burgdorferi sensu stricto B31. The results showed that the borrelial chromosome contains a single rrs (16S rRNA gene) sequence and two copies of rrl/rrf (23S/5S rRNA genes) sequences. The rrl/rrf genes were tandemly repeated at intervals of 3.2 kb and were located separately from the rrs gene on the genome. Our findings indicate that the organization of rRNA genes in Borrelia from I. ovatus ticks is identical to that of B. burgdorferi sensu stricto.
Simple Purification Method for a Vibrio vulnificus Hemolysin by a Hydrophobic Column Chromatography in the Presence of a Detergent
Authors: Oh Eun‐gyoung , Tamanoi Yumi , Toyoda Atsuko , Usui Kaori , Miyoshi Shin‐ichi , Chang Dong‐suck , Shinoda Sumio
Published: November 2013

A Vibrio vulnificus hemolysin (VVH) was purified by two steps of hydrophobic column chromatography on Phenyl‐Sepharose HP. The first chromatography was carried out at pH 6.0. In this pH condition, VVH efficiently bound to the column, but the hemolysin fraction eluted was accompanied with colored substance(s). To eliminate this colored substance, the second chromatography was carried out at pH 9.8 in the presence of 1% 3‐[(3‐cholamidopropyl) dimethylammonio]‐1‐propanesulfonate (CHAPS), a zwitterionic detergent. Homogeneity of the hemolysin thus obtained was shown by polyacrylamide gel electrophoresis. The specific activity increased 33, 600 times and the yield was 35%. The method is simple and useful to supply enough VVH for study of the role of the hemolysin in the infection by V. vulnificus or on the mechanism of action of the hemolysin.
Detection of Verocytotoxin from Stool and Serological Testing of Patients with Diarrhea Caused by Escherichia coli O157: H 7
Authors: Yamada Sumio , Matsushita Shigeru , Kai Akemi , Sasaki Michiko , Tsuji Atsutoshi , Kanemitsu Takefumi , Yamashita Naoya , Anzai Eiko , Kudoh Yasuo
Published: November 2013

The detecion of verocytotoxin (VT) in stool and measurement of antibodies against VT and three antigens (unheated‐antigen, LPS, and flagellin) of Escherichia coli O157: H7 in the serum of patients with diarrhea were examined. Five of 14 inpatients during an outbreak had fecal VT2 in stool taken within 5 days of onset to hospitalization. Among these 5, 3 of them also had fecal VT‐producing E. coli (VTEC) serotype O157: H7, whereas the other 2 did not. In the passive hemagglutination (PHA) test with formalinized sheep red blood cells sensitized with theee VTEC O157: H7 antigens, 49 (74.2%) of 66 outbreak patients and 3 of 3 sporadic cases had antibodies against both or one of unheated‐antigen and LPS of E. coli O157, but none had antibody against flagellin. In addition, anti‐VT2 antibody was demonstrated in serum samples from 15 (94%) of 16 inpatients and 2 (4%) of 50 outpatients in an outbreak by a VT‐enzyme‐linked immunosorbent assay (VT‐ELISA). These results showed that serological assay particularly for antibodies against VT and unheated‐antigen or LPS of VTEC O157 may provide a useful tool for diagnosis of infection with VTEC O157.
A Chemotaxonomic Study of Vibrio fluvialis Based on the Sugar Composition of the Polysaccharide Portion of the Lipopolysaccharides
Authors: Iguchi Takehiro , Kondo Seiichi , Hisatsune Kazuhito
Published: November 2013

A chemotaxonomic study was carried out with a new serotyping scheme comprising 35 O‐antigen groups of Vibrio fluvialis on the basis of the sugar composition of the polysaccharide portion of their lipopolysaccharide (LPS). A previously developed rapid method of preparing samples for compositional sugar analysis was employed. The 35 O‐antigen groups were divided into 21 chemotypes. It is noted that a rarely occurring component sugar of gram‐negative bacterial LPS, D‐glycero‐D‐manno‐heptose, and two kinds of uronic acids, i.e., galacturonic acid of a weakly bound type and glucuronic acid of a strongly bound type, were found in common in all the 21 chemotypes. A characteristic sugar component of gram‐negative bacterial LPS, 2‐keto‐3‐deoxyoctonate (KDO), was not detectable in any of the 21 chemotypes. Instead, three kinds of “KDO‐like substances” were found, one in each of three chemotypes (III, XI and XVII). They were strongly positive in Weissbach's periodate‐thiobarbituric acid test for KDO, but definitely not identical to it in high‐voltage paper electrophoresis (HVPE); the substance present in chemotype XI was indicated by HVPE to be 3‐deoxy‐D‐threo‐hexulosonic acid which is a sugar constituent of Vibrio parahaemolyticus O7 and O12 LPS.
Cytotoxicity Induced by Monoclonal Antibody to α‐Fetoprotein Coadministered with Spleen Cells of Nude Mice
Authors: Masuda Kazuyoshi , Nagata Shunji , Hirano Koichiro , Takagishi Yasushi , Hirai Hidematsu
Published: November 2013

This paper describes an attempt to effectively induce antibody‐dependent cell‐mediated cytotoxicity (ADCC) in nude mice. A monoclonal antibody against α‐fetoprotein, 80G, coadministered with spleen cells from other nude mice bearing HuH‐7N (xenograft of human hepatoma cell line, HuH‐7) significantly suppressed the growth of HuH‐7N as compared to treatment with 80G alone. 80G with spleen cells from normal nude mice also had some suppressive effect. In contrast, no effect was observed with each spleen cells alone as well as 80G alone. These results suggest that further supply of effector cells could enhance ADCC activity in nude mice.
An Evaluation of Serodiagnostic Tests in Patients with Candidemia: Beta‐Glucan, Mannan, Candida Antigen by Cand‐Tec and D‐Arabinitol
Authors: Kohno Shigeru , Mitsutake Kotaro , Maesaki Shigefumi , Yasuoka Akira , Miyazaki Takashige , Kaku Mitsuo , Koga Hironobu , Hara Kohei
Published: November 2013

The serodiagnostic tests, beta‐glucan, mannan, candida antigen by Cand‐Tec, and D‐arabinitol were evaluated in 10 patients with candidemia, 14 patients with suspected fungemia, and 10 healthy persons. By blood culture or lysis centrifugation, C. albicans was isolated from 5 patients, C. parapsilosis from 4, and C. tropicalis from 1 patient; no organisms were isolated from the 14 patients with suspected fungemia or the 10 healthy subjects. Beta‐glucan was measured by the difference between two chromogenic limulus tests (Endotoxin test‐D® and Endospecy®), which was more than 60 pg/ml in 7 of 9 (78%) candidemic patients and 1 of 12 (8%) patients with suspected fungemia. Mannan was positive in 6 of 10 (60%) candidemic patients and 1 of 13 (8%) patients with suspected fungemia. Both antigens were very sensitive and highly specific for candidemia. However, the Cand‐Tec assay was less specific, because titers of more than 4 were observed in 5 of 14 (34%) patients with suspected fungemia. D‐Arabinitol was the least sensitive, because a D‐arabinitol/creatinine ratio greater than 2.0 μmol/mg was observed in only 2 of 7 (29%) candidemic patients. The titers of serodiagnostic tests decreased after successful treatment with an anti‐fungal agent. Our results show that the combined use of the assays in necessary for accurate serological diagnosis of candidemia.