bluuebunny/crossref_metadata_2025 huggingface dataset:
Ia Restriction Specificity of KLH‐Specific T Cells from Allogeneic Bone Marrow Chimeras Is Influenced by Histocompatibility at the H‐2 and Minor Histocompatibility Loci
Authors: Ogasawara Kazumasa , Fukushi Noriko , Mishima Machiko , Good Robert A. , Onoé Kazunori
Published: November 2013

Ia restriction specificity involved in T cell proliferative responses to keyhole limpet hemocyanin (KLH) has been analyzed using a variety of allogeneic bone marrow chimeras. The chimeric mice were prepared by reconstituting irradiated AKR, SJL, B10.BR and B10.A(4R) mice with bone marrow cells from B10 mice. When such chimeric mice had first been primed with KLH in complete Freund's adjuvant (CFA), T cells from H‐2 incompatible fully allogeneic chimeras showed significantly higher responses to KLH in the presence of antigen‐presenting cells (APC) of donor strain (B10) than APC of recipient strain. However, in H‐2 subregion compatible chimeras, [B10→B10.A(4R)], which were matched at the H‐2D locus and at minor histocompatible loci, the T cells could mount vigorous responses to KLH with antigen‐presenting cells (APC) of either donor or recipient type. The same results were obtained as well with chimeras that had been thymectomized after full reconstitution of lymphoid tissues by donor‐derived cells. A considerable proportion of KLH‐specific T cell hybridomas established from [B10→B10.A(4R)] chimeras exhibited both I‐Ab and I‐Ak restriction specificities. The present findings indicate that the bias to donor Ia type of antigen specific T cells is determined by donor‐derived APC present in the extrathymic environment but that cross‐reactivity to the recipient Ia is influenced to some degree by histocompatibility between donor and recipient mice, even though the histocompatible H‐2D locus and minor histocompatibility loci seem not to be directly involved in the I‐A restricted responses studied herein.
The Nucleotide and Deduced Amino Acid Sequences of EcoRI Fragment Containing the 5′‐Terminal Region of Clostridium botulinum Type E Toxin Gene Cloned from Mashike, Iwanai and Otaru Strains
Authors: Fujii Nobuhiro , Kimura Kouichi , Murakami Tadayuki , Indoh Tomokazu , Yashiki Teruo , Tsuzuki Kayo , Yokosawa Noriko , Oguma Keiji
Published: November 2013

Chromosomal DNAs were extracted from toxigenic three Clostridium botulinum type E strains isolated from food‐borne botulism. After digestion by EcoRI, the fragments were cloned into Escherichia coli by using bacteriophage lambda gtl 1 and screened with monoclonal antibody recognizing the light chain component of botulinum type E toxin. The fragments (about 1 kbp size) cloned from each strain were recloned into a plasmid vector pUC118. The E. coli cells transformed with the recombinant plasmids produced 33 kDa protein with or without IPTG (isopropyl‐β‐D‐thiogalactopyranoside) which reacted with the monoclonal antibody. The nucleotide sequences of the cloned EcoRI fragments from the three type E strains were identical and contain the 5′‐terminal region of the type E toxin gene. It was also found that there exist several highly homologous nucleotide sequences among the botulinum types A, C and E, and tetanus toxin genes in both translated and untranslated regions.
In vitro and in vivo Transferrable β‐Lactam Resistance Due to a New Plasmid‐Mediated Oxyiminocephalosporinase from a Clinical Isolate of Proteus mirabilis
Authors: Watanabe Yuji , Yokota Takeshi , Higashi Yasuyuki , Wakai Yoshimi , Mine Yasuhiro
Published: November 2013

A new plasmid‐mediated β‐lactamase (FPM‐1) with an isoelectric point of 7.2 and a molecular weight of 26,000 was found in a cefuroxime‐resistant clinical isolate of Proteus mirabilis strain 6003. FPM‐1 can be classified as a type I oxyiminocephalosporinase on the basis of its substrate specificity and inhibition pattern by clavulanic acid etc., and it conferred resistance on both the strain and transconjugants against most oxyme‐type cephalosporins as well as the older ones but not against cefamycins and a few exceptional oxyme‐type cephalosporins such as ceftizoxime, ceftazidime and cefixime. In a murine systemic infection model, only these FPM‐1‐stable drugs exhibited protective activity against the FPM‐1‐producing P. mirabilis 6003 similar to that against a nonproducing derivative strain. The FPM‐1‐mediated cefuroxime resistance in P. mirabilis 6003 was transferred to co‐infected Escherichia coli 7004 at frequencies between 3.8 × 10−3 and 4.0 × 10−2 in a murine ascending urinary tract infection model. In the same infection model due to the FPM‐1‐producing E. coli transconjugant, FPM‐1‐stable cefixime was significantly more effective than FPM‐1‐labile cefteram pivoxil, although both drugs had similar therapeutic effect against its FPM‐1‐nonproducing counterpart strain.
Detection of Bacillus cereus Flagellar Antigen by Enzyme‐Linked Immunosorbent Assay (ELISA)
Authors: Murakami Tomonori , Hiraoka Kenji , Mikami Takeshi , Matsumoto Tatsuji , Katagiri Susumu , Suzuki Masuko
Published: November 2013

A serological typing scheme of Bacillus cereus has been developed by immunochemical analyses of flagellar antigen using an agglutination method. Enzyme‐linked immunosorbent assay (ELISA) for the classification of flagellar serotype of Bacillus cereus had greater sensitivity, 10–500 times, than that of agglutination method. The specificity of flagellar antigen and antibody was determined by immunogold electron microscopy and ELISA inhibition assay. Application of ELISA is useful for the detection of the small amounts and many kinds of antigen‐antibody reactions.
Macrophage Migration Inhibitory Factor (MIF) Produced by a Human T Cell Hybridoma Clone
Authors: Hirose Shin‐ichiro , Ooki Shinji , Higuchi Masahiro , Osawa Toshiaki
Published: November 2013

A human T cell hybridoma clone, F5, producing high levels of macrophage migration inhibitory factor (MIF) was established by the emetine‐actinomycin D selection method. This clone produced two species of MIF which were separated on a Phenyl Sepharose column. We purified MIF‐2 (the more hydrophobic species of the two) to homogeneity from the conditioned medium of stimulated F5 cells by a series of steps that included hydrophobic chromatography, ion‐exchange chromatography, Ricinus communis lectin affinity chromatography, and high‐performance liquid chromatography on anion exchange and reverse‐phase columns. Purified MIF was digested with endoproteinase Lys‐C and Asp‐N. The amino acid sequences of the generated peptides were determined. No sequence similarity with any other protein was found. The molecular weight of MIF‐2 was estimated to be 45 kDa from sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis of immunoprecipitates with anti‐peptide antibodies. These results show that F5MIF‐2 is a novel cytokine.
Genetic and Molecular Analysis of Spontaneous Respiratory Deficient (res−) Mutants of Escherichia coli K‐12
Authors: Lewis Leslie A. , Li Koibong , Gousse Angelo , Pereira Faria , Pacheco Nancy , Pierre Stella , Kodaman Pinar , Lawson Steve
Published: November 2013

Respiratory deficient (res−) mutants of E. coli are slow growing micro‐colonial, anaerobic, catalase and benzidine negative strains whose broad phenotypic alteration may result from pleiotropic mutations in genes of the hemin biosynthetic pathway. They are easily recovered from platings of sensitive cells on concentrations of gentamicin higher than the minimal inhibitory concentration. These mutants show a dramatic change in their biochemical diagnostic profile resulting primarily from deficiencies in the active transport mechanisms of the cell. Using well‐marked F− and Hfr strains, 157 mutants were analyzed from 3 different parent strains; all but 2 resulted from mutations in 3 loci of the hemin biosynthetic pathway. Of these a marked skew to hemB− mutations was seen, with more than 80% mapping there. The possibility that this hot spot resulted from transpositional activity was tested by Southern hybridization of EcoRI digests of the chromosomal DNA, using as a probe, a 2.8‐kb fragment containing the hemB gene. The WT and other hemB+ control strains contained a 14.6‐kb fragment. Of 18 hemB strains tested, 14 showed deletion and insertion mutations which fell into four classes based on the variation in the size of the fragment or on the absence of hybridization. The latter resulted from complete deletion of the hemB gene. An increase in fragment size from 1.5‐kb to 3.4‐kb was observed in some of the strains.
Distribution of Plesiomonas shigelloides in Various Components of Pond Ecosystems in Dhaka, Bangladesh
Authors: Islam Md. Sirajul , Alam Md. Jahangir , Khan Sirajul Islam
Published: November 2013

Plesiomonas shigelloides is considered to be a waterborne agent of human gastroenteritis. An ecological study was carried out in five ponds in Dhaka city over a period of one year to elucidate the distribution and seasonality of this organism in various components of pond ecosystems. Samples were collected from hydrophytes, water, phytoplankton and sediment every 15 days over 12 months and cultured for P. shigelloides. P. shigelloides was isolated from a total of 120 samples including 25 (20.8%), 16 (13.3%), 22 (18.3%) and 35 (29.2%) of hydrophytes, water, phytoplankton and sediment samples, respectively. Distinct seasonal patterns of isolation of P. shigelloides were observed in the four components with two distinct peaks. The highest peaks were observed in hydrophytes and water samples in May and in phytoplankton and sediment in November. P. shigelloides was isolated from all components from all ponds during the study period. These results suggest that P. shigelloides is an autochthonous member in the freshwater pond ecosystems in Dhaka, Bangladesh.
Adaptational Changes of Fatty Acid Composition and the Physical State of Membrane Lipids Following the Change of Growth Temperature in Yersinia enterocolitica
Authors: Nagamachi Eiko , Shibuya Sei‐ichiro , Hirai Yoshikazu , Matsushita Osamu , Tomochika Ken‐ichi , Kanemasa Yasuhiro
Published: November 2013

Yersinia enterocolitica is capable of growing in a broad range of temperatures from 4 to 45 C. How this organism alters its membrane lipids in response to the change of growth temperature is very interesting. The fatty acids of membrane lipids of cells cultured at 5, 15, 25 and 37 C were analyzed and the physical states of these membrane lipids were characterized. The major phospholipids of this bacterium were phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, lysophosphatidylglycerol and lysophosphatidylethanolamine. No significant difference in phospholipid composition in response to culture temperatures was observed. It was reported in our previous paper that the major fatty acids of membrane phospholipids of Y. enterocolitica were C15:0, C16:0, C16:1, cyclopropane C17:0 and C18:0, Some differences in the fatty acid composition were, however, observed with the change of culture temperature. When the culture temperature was raised, the saturated and cyclopropane fatty acids substantially increased and the unsaturated ones decreased. A reverse phenomenon was observed when culture temperature was lowered. From the viewpoints of membrane physical state, adaptational changes were analyzed using a nylon microcapsule method. Phase transition in membrane lipids of cells grown at each culture temperature took place in the range of about 5 C below and about 10 C above the culture temperature. It is, therefore, considered that Y. enterocolitica maintains its membrane rigidity and fluidity in response to growth temperature by changing the membrane fatty acid composition.
Immunological Properties of Borrelia burgdorferi Isolated from the Ixodes ovatus in Shizuoka, Japan
Authors: Masuzawa Toshiyuki , Okada Yutaka , Beppu Yoshihito , Oku Tomozou , Kawamori Fumihiko , Yanagihara Yasutake
Published: November 2013

Three strains of spirochetes (IKA1 to 3) were isolated from the midgut of Ixodes ovatus collected in the Ikawa region of the northern part of Shizuoka, Japan. These isolates had eight flagella, and their size and other morphological features were similar to Borrelia burgdorferi. They showed similar motility and reacted with monoclonal antibody (MAb) H9724 against borrelial flagella and with MAb H5332 against the outer surface protein A. These strains showed similar SDS‐PAGE profiles to that of B. burgdorferi strain B31 and P/Bi isolated in the U.S.A. and Europe, respectively. Immunoblot with Lyme disease patient serum showed positive reactions with the flagella (41 Kilodalton, kDa), protein C (20 to 22 kDa), and outer surface protein A (29 kDa) of the isolates. Immunological properties, morphological characteristics, and epidemiological features revealed that these isolates were B. burgdorferi.
Detection and Discrimination of Mycoplasma pneumoniae and Mycoplasma genitalium by the in vitro DNA Amplification
Authors: Sasaki Yuko , Shintani Miharu , Shimada Toru , Watanabe Haruo , Sasaki Tsuguo
Published: November 2013

By using the primers designed on the bases of the sequences of the 16S rRNA genes of Mycoplasma pneumoniae and Mycoplasma genitalium, respectively, specific and sensitive in vitro DNA amplification assay system for the detection and discrimination of these two mycoplasmas was established. The detection limit of the assay was 100 cells for M. pneumoniae and 1,000 cells for M. genitalium. Neither other human mycoplasmas nor oral bacteria existing in human saliva showed any cross‐reactions with these primers.
Some Improved Versions of Methods for the Indirect Detection of Staphylococcal Protein A Gene Expressed in Escherichia coli
Authors: Sakurada Junji , Sumi Yoshiko
Published: November 2013

Several versions of methods for the indirect detection of expression of staphylococcal protein A gene (spa) in Escherichia coli (E. coli) were devised by making use of biological properties of staphylococcal protein A (SpA). i) Hemagglutination of sheep red blood cells (SRBC) sensitized with anti‐SRBC‐antibodies using heat‐treated spa‐transformed E. coli organisms; Native spa‐transformed E. coli organisms did not agglutinate the sensitized SRBC. The heat‐treatment (60 C, 4 hr) of the transformants, however, caused positive hemagglutination like SpA‐positive Staphylococcus aureus (S. aureus) organisms. ii) Halo formation around colonies on agar plates containing normal dog serum, which is originally used for the detection of SpA of S. aureus. A mutant strain NMJ was isolated, which showed formation of the halo of precipitate due to interaction between immunoglobulin and SpA. iii) A new version of immunodetection; After lysis of the transformants grown on a nitrocellulose membrane by alkali, SpA could be directly detected by immuno‐detection procedures after inactivation of endogenous peroxidase in bacteria by phenylhydrazine and hydrogen peroxide.
Correlation between Increased Susceptibility to Primary Toxoplasma gondii Infection and Depressed Production of Gamma Interferon in Pregnant Mice
Authors: Shirahata Toshikazu , Muroya Naoyoshi , Ohta Chikako , Goto Hitoshi , Nakane Akio
Published: November 2013

To explore a possible mechanism of pregnancy‐associated suppression of T cell‐mediated immunity to Toxoplasma gondii, acquired resistance and gamma interferone (IFN‐γ) production in pregnant mice were compared with those in virgin mice after infection with the S‐273 strain of this protozoan parasite. The 50% lethal dose of this strain was less than 200 tachyzoites for pregnant mice and 2,800 organisms for virgin controls. Toxoplasma‐induced production of both IFN‐α and IFN‐γ in the bloodstream of pregnant mice was significantly depressed as compared with that in virgin controls. The administration of recombinant murine IFN‐γ (rMuIFN‐γ) resulted in a significant decrease of mortality and parasitic growth in the organs of pregnant mice infected with a lethal dose of S‐273 strain tachyzoites. Thus, the impairment of T cell‐mediated immune responses was evident in pregnant mice from the impaired IFN‐γ‐generating capacity and poor survival rate after primary infection with Toxoplasma. When mice with chronic Toxoplasma infection were injected with specific antigen, the resultant production of IFN‐γ was also significantly suppressed during pregnancy. However, there was no direct correlation between the serum levels of IFN‐γ and susceptibility to reinfection, since the mortality rate of chronically infected pregnant mice after the challenge with the high virulent RH strain was not significantly higher than that of virgin controls.
A Broad‐Host‐Range Vibriophage, KVP40, Isolated from Sea Water
Authors: Matsuzaki Shigenobu , Tanaka Shuji , Koga Tetsuro , Kawata Tomio
Published: November 2013

A broad‐host‐range vibriophage, KVP40, was isolated from sea water by using Vibrio parahaemolyticus 1010 (EB101) as the indicator host. The host range of KVP40 extended over at least 8 Vibrio and 1 Photobacterium species. KVP40 was a large tailed phage containing double‐stranded DNA and belonged to Ackermann's morphotype A2. KVP40 DNA was cleaved by 11 different type II restriction endo‐nucleases including EcoRl and Hindlll, but not by 17 other enzymes including BamHI, KpnI and SalI.1
A Unique Monoclonal Antibody That Recognizes Mature p17 of HIV‐1 but Not Its Precursor
Authors: Saitoh Atsushi , Tanaka Naoto , Nakata Atsuo , Ikuta Kazuyoshi , Shinagawa Hideo
Published: November 2013

The entire and partial gag regions of human immunodeficiency virus type 1 (HIV‐1) were overproduced in Escherichia coli and used for epitope mapping of antibodies against p17. We found that a mouse monoclonal antibody to p17, V17 recognizes the mature p17 but not the unprocessed Gag proteins containing the entire p17 moiety. Further analysis revealed that V17 recognizes the C‐terminal 12‐amino‐acid region of p17 having free C‐terminus. This monoclonal antibody may be useful for monitoring the maturation of virus particles.
Separate Isolation of Clostridium difficile Spores and Vegetative Cells from the Feces of Newborn Infants
Authors: Miyazaki Shuichi , Matsunaga Toshiyuki , Kawasaki Kenji , Kobayashi Intetsu , Tada Hiroshi , Yamaguchi Keizo , Goto Sachiko
Published: November 2013

A modified taurocholate‐cefoxitin‐cycloserine‐fructose agar medium, pH 5.5, on which vegetative cells alone could grow, was newly devised for separate isolation of Clostridium difficile vegetative cells and spores from feces. The ratio of C. difficile‐positive feces from healthy newborn infants younger than 10 days of the age was 30.8%, and 93.3% of feces from healthy infants older than 20 days were positive for C. difficile. C. difficile spores alone were detected in twenty‐one samples (75%) of C. difficile‐positive Twenty‐eight specimens. Only 10.7% (3/28) C. difficile vegetative cells alone were detected. C. difficile spores alone were detected in one of nine healthy adults. These collective results offer potential explanations for high frequent isolations of C. difficile from newborn infants without occurrence of pseudomembranous colitis.
Molecular Cloning and Nucleotide Sequencing of a Novel Aminoglycoside 6′‐N‐Acetyltransferase Gene from an R‐Plasmid of Salmonella typhimurium S24 Isolated in Taiwan
Authors: Peng Chien‐fang , Chang Shui‐feng
Published: November 2013

A conjugative aminoglycoside resistance plasmid pST2 has been isolated from Escherichia coli K‐12 14R525, which was mated with a clinical isolate of Salmonella typhimurium S24. A novel resistance gene of aminoglycoside 6′‐N‐acetyltransferase[AAC(6′)] was cloned from plasmid pST2 on a 1,393 kilobase (kb) of Sphl‐SalI fragment into vector pACYC184 and pUC18. This novel A AC (6′) gene in plasmid pST2 acetylated kanamycin, amikacin, dibekacin, tobramycin, gentamicin, netilmicin, and sisomicin. The complete nucleotide sequence of the novel AAC(6′) gene and its neighboring sequences were also determined. Minicell experiments detected only one protein of 24.7 kilodaltons (kDa) translated from an open reading frame of the 618 base pairs (bp) gene.
Serological Cross‐Reaction between Yersinia enterocolitica O9 and Non‐O1 Vibrio cholerae Bio‐Serogroup Hakata and Antigenic Analysis of Their Relationship by Their Lipopolysaccharides
Authors: Isshiki Yasunori , Haishima Yuji , Kondo Seiichi , Shimada Toshio , Hisatsune Kazuhito
Published: November 2013

Cross‐agglutination and cross‐agglutinin absorption experiments were carried out on non‐O1 Vibrio cholerae bio‐serogroup Hakata (Hakata) and Yersinia enterocolitica O9 (O9). It was shown that the O‐antigen of Hakata was closely related to that of O9 in an a, b‐a, c type of relationship. The antigenic relationship between the O‐antigens of the two bacteria was analyzed by passive hemolysis (PH) and passive hemolysis inhibition (PHI) tests by using their lipopolysaccharides (LPS) as antigen for sensitizing sheep red blood cells (SRBC) and, in the case of the latter, as an inhibitor in a PH system consisting of LPS‐coated SRBC, guinea‐pig complement and anti‐Hakata or O9 antiserum, both unabsorbed and absorbed with the heterologous Hakata or O9 antigen. In the PH experiment, unabsorbed anti‐Hakata antiserum had hemolytic titers of 126,100 and 2,600 against Hakata‐ and O9‐LPS‐coated SRBC, respectively, and anti‐O9 antiserum had hemolytic titers of 19,400 and 38,800, respectively, against these SRBC. The PH experiment showed that anti‐O9 antiserum contains a hemolysin reacting with the heterologous Hakata antigen at a high titer (19,400), while anti‐Hakata antiserum contains a hemolysin reacting with the heterologous O9 antigen at a significant titer (2,600). The former was completely removed from anti‐O9 antiserum with the Hakata antigen and the latter from anti‐Hakata antiserum with the O9 antigen. Thus, serological cross‐reactivity was demonstrated between the Hakata and O9 strains.
Type 1 Pili Enhance the Invasion of Salmonella braenderup and Salmonella typhimurium to HeLa Cells
Authors: Horiuchi Sankichi , Inagaki Yoshio , Okamura Noboru , Nakaya Rintaro , Yamamoto Naoki
Published: November 2013

The relationship between type 1 pili‐associated adhesion and invasion to HeLa cells by Salmonella braenderup and S. typhimurium was studied. When the clinical isolates of these strains were grown in L‐broth, they showed both type 1 pili formation and mannose‐sensitive adhesion to HeLa cells. On the other hand, the type 1 pili‐defective mutants, which were obtained either by repeated subcultures on L‐agar plates or by the transposon Tn1‐insertion mutagenesis of the S. braenderup and S. typhimurium strains, concomitantly lost mannose‐sensitive adhesion to HeLa cells. When the HeLa cells were incubated with Salmonella, the type 1 piliated strains invaded the HeLa cells with much higher infection rate than did the type 1 pili‐defective strains. The invasion of type 1 piliated strains to HeLa cells was markedly inhibited in the presence of d‐mannose. The infectivity of the strain, which lost type 1 pili but still had mannose‐resistant adhesion, was slightly higher than that of the strains defective in both mannose‐sensitive and mannose‐resistant adhesion. These results suggested that type 1 pili have a role in enhancing the invasion of S. braenderup and S. typhimurium to HeLa cells.
Seroepidemiology of Spotted Fever Group Rickettsiae in Small Field Rodents in Japan
Authors: Ishikura Mitsuhiro , Watanabe Mamoru , Nakayama Takashi , Matsuura Kumiko , Morita Osayuki , Uchida Takahiro
Published: November 2013

To clarify the geographic distribution of spotted fever group (SFG) rickettsiae in Japan, small field rodents captured in endemic and nonendemic areas were screened for anti‐SFG rickettsia antibodies by the immunofluorescence test. Among total 716 specimens tested, 73–75% of rodents were antibody‐positive against Rickettsia japonica and/or Rickettsia montana, showing different degree of antibody‐positive proportions among each species of the rodents. Interestingly, these profiles were not different in the rodents from each endemic and nonendemic areas, indicating that the SFG rickettsiae are prevailing in the wider areas where patients have not been found yet.
Cloning and Nucleotide Sequence Analysis of a Chloramphenicol Acetyltransferase Gene from Vibrio anguillarum
Authors: Zhao Jiang , Aoki Takashi
Published: November 2013

The chloramphenicol resistant gene (cat) encoding chloramphenicol acetyltransferase (CAT) in a transferable R plasmid (pJA7324) isolated from the fish pathogen Vibrio anguillarum strain PT24 was cloned into the plasmid vector pUC19. The nucleotide sequence analysis of 1,348 base pair DNA identified an open reading frame encoding a protein of 216 amino acid residues with a calculated molecular mass of 25,471 daltons. The predicted amino acid sequences for this cat gene are 37–69% homologous with other CAT proteins of both Gram‐negative and ‐positive bacteria. Colony hybridization performed with a PvuII‐BamHI fragment including this cat gene as a probe, revealed that the same or similar chloramphenicol resistance genes existed among V. anguillarum isolates.
Comparison of Borrelia burgdorferi Isolated from Humans and Ixodid Ticks in Hokkaido, Japan
Authors: Nakao Minoru , Miyamoto Kenji , Kawagishi Naoko , Hashimoto Yoshio , Iizuka Hajime
Published: November 2013

Four spirochetal isolates (JEM1 to JEM4) were obtained from cutaneous lesions of patients manifesting erythema chronicum migrans in Hokkaido, Japan. In the protein profiles by SDS‐PAGE and the reactivities with monoclonal antibodies (H5332 and H9724) by immunoblotting, all the human isolates were identical with the tick isolates from Ixodes persulcatus. These data indicate that I. persulcatus is an important vector of Lyme disease for humans in Japan.
Proposal of Burkholderia gen. nov. and Transfer of Seven Species of the Genus Pseudomonas Homology Group II to the New Genus, with the Type Species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov.
Authors: Yabuuchi Eiko , Kosako Yoshimasa , Oyaizu Hiroshi , Yano Ikuya , Hotta Hisako , Hashimoto Yasuhiro , Ezaki Takayuki , Arakawa Michio
Published: November 2013

Based on the 16S rRNA sequences, DNA‐DNA homology values, cellular lipid and fatty acid composition, and phenotypic characteristics, a new genus Burkholderia is proposed for the RNA homology group II of genus Pseudomonas. Seven species in this group were transfered to the new genus. Thus seven new combinations, Burkholderia cepacia (Palleroni and Holmes 1981), Burkholderia mallei (Zopf 1885), Burkholderia pseudomallei (Whitmore 1913), Burkholderia caryophylli (Burkholder 1942), Burkholderia gladioli (Severini 1913), Burkholderia pickettii (Ralston et al 1973) and Burkholderia solanacearum (Smith 1896) were proposed.
Antiviral Activity of 1‐β‐D‐Arabinofuranosyl‐E‐5‐(2‐Bromovinyl)Uracil against Thymidine Kinase Negative Strains of Varicella‐Zoster Virus
Authors: Kawai Hideki , Yoshida Itsuro , Suzutani Tatsuo
Published: November 2013

Mechanism of antiviral activity of 1‐β‐d‐arabinofuranosyl‐E‐5‐(2‐bromovinyl)uracil (BV‐araU) against the YSR strain of varicella‐zoster virus (VZV), which is a mutant derived from the wild YS strain and is completely deficient in viral thymidine kinase (TK), was searched in comparison with antiviral activity of other thymidine analogues, guanosine analogue and thymidylate synthase (TS) inhibitor in human embryo lung fibroblast cells.

Thymidine analogues, such as BV‐araU, 5‐iododeoxyuridine (IUDR), 1‐β‐d‐arabinofuranosylthymine (araT), and guanosine analogue, such as 9‐(2‐hydroxyethoxymethyl)guanine (ACV), showed higher antiviral activity to the YS strain than to the YSR strain. Though, BV‐araU also had the antiviral activity of a microgram level against the YSR strain. In contrast to these results, TS inhibitor, 5‐fluorodeoxyuridine (FUDR), had higher antiviral activity to the YSR strain than to the YS strain. Highly synergistic antiviral activities of FUDR to the YS strain and the YSR strain were observed in combination with IUDR, araT, or ACV. However, weakly synergistic or additive inhibition to the YSR strain was shown in combination of BV‐araU and FUDR, in spite of highly synergistic effect of this combination to the YS strain. The viral and cellular TS activity was partially inhibited by BV‐araU monophosphate, but not by BV‐araU. These results indicate that BV‐araU is converted into BV‐araU monophosphate by cellular TK, and the inhibition of TS activity by BV‐araU monophosphate in the YSR strain‐infected cells results in the suppression of viral replication.
Serological Reactivity of Sera from Scrub Typhus Patients against Weil‐Felix Test Antigens
Authors: Amano Ken‐ichi , Suzuki Nagao , Fujita Miki , Nakamura Yoko , Suto Tsunehisa
Published: November 2013

Sera from 17 patients of scrub typhus in the acute and convalescent phases were tested by indirect immunoperoxidase test, Weil‐Felix (WF) test, enzyme‐linked immunosorbent assay (ELISA), and immunoblotting. In the comparison of antibody titers between acute‐ and convalescent‐phase sera, we recognized a parallelism of increment between the titers in WF test and titers of immunoglobulin M (IgM) in ELISA against Proteus mirabilis strain OXK‐whole cells and OXK‐lipopolysaccharides (Proteus OXK‐LPS). Furthermore, IgM antibodies from almost all of WF test‐positive sera recognized LPS from Proteus OXK in immunoblotting. Based on these results, it was concluded that IgM antibody rather than IgG may participate in WF test, and that Proteus OXK‐LPS may have one of antigenic epitopes common to the components of R. tsutsugamushi.
Localization and Functions of Japanese Encephalitis Virus Nonstructural Proteins NS3 and NS5 for Viral RNA Synthesis in the Infected Cells
Authors: Edward Zulkarnain , Takegami Tsutomu
Published: November 2013

Recently it has been reported that Japanese encephalitis virus (JEV)‐specific RNAs can be synthesized in vitro in the subcellular fraction including outer‐nuclear membrane (Takegami and Hotta, 1989). The results of Western blot analysis and indirect immunofluorescence test using two kinds of monospecific antisera against JEV nonstructural proteins NS3 and NS5 showed that NS3 and NS5 were membrane‐associated proteins and formed the complex at the perinuclear site in the infected cells. Both antisera against NS3 and NS5 inhibited in vitro RNA synthesis. These results suggest that NS5 and NS3 play important role(s) in flavivirus RNA replication.
Extrathymic Pathways of T‐Cell Differentiation: A Primitive and Fundamental Immune System
Authors: Abo Toru
Published: November 2013

In addition to an intrathymic pathway of T‐cell differentiation, extrathymic pathways of T‐cell differentiation have recently been demonstrated to occur in multiple sites in mice. Such sites include the sinusoids of the liver, the intraepithelial region of the intestine, and the omentum of the peritoneal cavity. Although these extrathymic pathways are minimal at a young age, they become predominant with aging. Extrathymically differentiated T cells display many properties distinct from those of regular T cells of thymic origin. For instance, they consist of a considerably large proportion of γδT cells as well as αβT cells, contain double‐negative CD4–CD8– cells and self‐reactive oligoclones, constitutively express the II‐2 receptor β‐chain, and have an αα homodimer of CD8 if they carry it. Cumulative evidence reveals that the extrathymic pathways comprise a primitive and fundamental immune system in the body and play a pivotal role in immune reactions under conditions of aging, bacterial infections, malignancies, autoimmune diseases, and pregnancy.
Enhancement and Neutralization of Feline Infectious Peritonitis Virus Infection in Feline Macrophages by Neutralizing Monoclonal Antibodies Recognizing Different Epitopes
Authors: Hohdatsu Tsutomu , Yamada Hitomi , Ishizuka Yuzuru , Koyama Hiroyuki
Published: November 2013

The interaction between the enhancing and neutralizing activities of three monoclonal antibodies (MAbs) (5‐6‐2, 6‐4‐2 and 7‐4‐1) to the spike protein of feline infectious peritonitis virus (FIPV) strain 79‐1146 was determined using feline macrophages. At a high MAb concentration, all of the three MAbs completely inhibited the FIPV infection at 37 C. However, two of them (6‐4‐2 and 7‐4‐1) enhanced FIPV infection when either the MAb concentration or reaction temperature was lowered. These MAbs also exerted an immediate infectivity‐enhancing activity for up to 10 min of reaction and by 20 min, neutralizing activities were observed. Only MAb 5‐6‐2 consistently showed neutralizing activity regardless of the reaction conditions. Competition with sera from cats experimentally infected with FIPV strain 79‐1146 or feline enteric coronavirus strain 79‐1683 showed that the two epitopes recognized by MAb 5‐6‐2 and MAb 6‐4‐2, respectively, are also recognized by the natural host.
Production of Slime Polysaccharides by Shigella dysenteriae Type 1
Authors: Qadri Firdausi , Haque Md. Azizul , Hossain Anwar , Albert Manuel John
Published: November 2013

Electron microscopy of ruthenium red‐stained ultrathin section of strains of Shigella dysenteriae type 1 grown in the Casamino Acids‐yeast extract broth medium showed the presence of an extracellular slime layer. The slime appeared as a dense sheath covering bacteria. The presence of slime promoted hemagglutinating activity of the bacteria. The slime polysaccharide (SPS) isolated from the cell‐free culture supernatant or the bacterial surface was less than 162,000 daltons in size and immunochemically similar. The SPS showed cross‐reaction with lipopolysaccharide (LPS) antigen in immunological tests; however, it also appeared to be different from LPS since it did not contain 2‐keto‐3‐deoxyoctonate, a core sugar of LPS. A different pattern of separation from LPS was also observed by silver staining of SDS‐polyacrylamide gels. From these data it appeared that either LPS and SPS are contaminated with each other or that SPS is the polysaccharide portion of LPS.
Resistance to Pseudorabies Virus with Enhanced Interferon Production and Natural Killer Cell Activity in Mice Treated with Serum Thymic Factor
Authors: Onodera Takashi , Yoshihara Kazuhiro , Suzuki Toshihito , Tsuda Tomoyuki , Ikeda Toshio , Awaya Akira , Kobayashi Hisashi , Yukawa Masayosi
Published: November 2013

Susceptibility to pseudorabies virus (PRV) infection in mice, which were continuously depleted of natural killer (NK) cell activity by injection of anti‐asialo GM1, was examined. Effects of serum thymic factor (FTS) on susceptibility of mice to PRV were also investigated. In mice with depleted NK cell activity, the mortality of PRV‐infected mice was markedly high, whereas that of FTS‐pretreated mice was significantly lower than the controls. Reduced susceptibility to PRV was demonstrated in mice treated with anti‐asialo GM1 antisera before the PRV infection. Such a reduced susceptibility was not observed in mice inoculated with the antisera on day 1 post‐infection (PI). To analyze the FTS‐induced resistance to PRV infection, NK cell activity, macrophage activity, and interferon (IFN) productions were studied. Interferon production and NK cell activity were enhanced in the FTS‐pretreated mice, suggesting that interferon may play an important role in this FTS‐induced resistance to PRV infection.
Transfer of Protection to Murine Typhoid Conferred by L‐Form Salmonella typhimurium in Dependence of Cooperation between L Form‐Adopted Macrophages and L Form‐Induced Lyt‐2+ T Cells
Authors: Nishikawa Fumiko , Kita Eiji , Matsui Norio , Kashiba Shuzo
Published: November 2013

The effector cells responsible for protection to Salmonella typhimurium in C3H/HeJ mice, conferred by L‐form S. typhimurium, were determined by cell transfer test. Nonfractionated spleen cells from 6‐week immune mice but not from 24‐week immune animals transferred anti‐S. typhimurium immunity. Treatment with anti‐macrophage antiserum and complement most effectively abolished protective capacity in 6‐week immune cells, while anti‐T cell monoclonal antibody plus complement reduced it to a lesser extent. However, adoptive protection was achieved only by transfer of immune macrophages along with Lyt‐2+ T cells selected from 6‐week immune spleen cells. These Lyt‐2+ T cells were cytotoxic to Kupffer cells from C3H/HeJ mice which had been infected 48 hr previously and from the mice which had been immunized 1 week previously, but not to the cells from 6‐week immune mice and from normal animals. Moreover, protective capacity in immune macrophages seemed to be correlated to the degree of colonization by the L forms, and the inability to transfer immunity of 24‐week immune spleen cells may be due to the decrease in the L form‐colonization. These results suggest that cooperation between the L form‐colonized macrophages and L form‐induced cytotoxic Lyt‐2+ T cells contributes to anti‐S. typhimurium immunity, and might imply the immunological difference between the 6‐week immune phagocytes and the cells at an early stage of infection or immunization.
Escherichia coli Heat‐Labile Enterotoxin Binds to Glycosylated Proteins with Lactose by Amino Carbonyl Reaction
Authors: Shida Kan , Takamizawa Kotaro , Nagaoka Masato , Tsuji Takao , Osawa Toshiaki
Published: November 2013

The binding of Escherichia coli heat‐labile enterotoxin (LT) type I to glycosylated proteins with lactose (Galβ1‐4Glc) by amino carbonyl reaction was studied by the Western blot assay and by the microtiter well binding assay. LT bound to a lactose‐α‐lactalbumin amino carbonyl product (Lac‐LA), whereas cholera toxin did not. The binding ability of Lac‐LA was abolished by β‐galactosidase treatment, indicating that the terminal galactose is essential for the binding of LT. The binding of LT to Lac‐LA was inhibited by galactose and lactose, and most effectively inhibited by lactulose (Galβ1‐4Fru), which is a structural analog of the Amadori rearrangement product of the amino carbonyl reaction between lactose and an ε‐amino group of a lysine residue (lactuloselysine). The results suggest that LT recognizes the portion of lactuloselysine in Lac‐LA. LT also bound to a melibiose (Galα1‐6Glc)‐α‐lactalbumin amino carbonyl product (Mel‐LA), but the binding ability of Mel‐LA was weaker than that of Lac‐LA, suggesting that the β1‐4 linked terminal galactose is dispensable but preferable for the binding. Furthermore, LT bound to the amino carbonyl products of lactose with β‐lactoglobulin, caseins, bovine serum albumin, and ovalbumin. These results indicate that LT binds to the amino carbonyl products between proteins and sugars containing the terminal galactose, such as lactose.
Morphological Effects, Rate of Incorporation, and the Enzymatic Action of Botulinum ADP‐Ribosyltransferase, Known as C3 Exoenzyme, on Human Neuroblastoma GOTO Cells
Authors: Kamata Yoichi , Nishiki Tei‐ichi , Matsumura Kunihiko , Hiroi Toyoko , Kozaki Shunji
Published: November 2013

The susceptibility of various lines of cultured cells to botulinum ADP‐ribosyltransferase, known as C3 exoenzyme, was examined. Human neuroblastoma GOTO cells were most sensitive. The C3 exoenzyme caused a change in cell shape that involved extension of neurites. The exoenzyme evoked the outgrowth of neurites from chick ganglion as effectively as nerve growth factor, suggesting that C3 exoenzyme possesses neurotropic activity. Experiments with125I‐labeled enzyme revealed that C3 exoenzyme was rapidly incorporated into cells but the number of incorporated enzyme molecules was small. Once C3 exoenzyme had been incorporated, ADP‐ribosylation of the substrate (Rho protein) in GOTO cells occurred immediately and rapidly reached a maximum level. However, some of Rho proteins remained unmodified even after induction of the change in morphology. These findings suggest that ADP‐ribosylation by C3 exoenzyme is directly associated with the differentiation of GOTO cells but that other events may also participate in this process.
Linear and Conformation‐Dependent Antigenic Sites on the Nucleoprotein of Rabies Virus
Authors: Minamoto Nobuyuki , Tanaka Harumi , Hishida Miyuki , Goto Hideo , Ito Hiroshi , Naruse Shinji , Yamamoto Keiko , Sugiyama Makoto , Kinjo Toshio , Mannen Kazuaki , Mifune Kumato
Published: November 2013

A set of 29 monoclonal antibodies (MAbs) specific for the rabies virus nucleoprotein (N protein) was prepared and used to analyze the topography of antigenic sites. At least four partially overlapping antigenic sites were delineated on the N protein of rabies virus by competitive binding assays. Indirect immunofluorescent antibody tests using MAbs with a series of rabies and rabies‐related viruses showed that epitopes shared by various fixed and street strains of rabies virus were mainly localized at antigenic sites II and III, while epitopes representing the genus‐specific antigen of Lyssavirus were widely presented at sites I, III and IV. All but one of seven MAbs specific for antigenic sites I, IV and bridge site (I and II) reacted with the antigen that had been denatured by sodium dodecyl sulfate or 2‐mercaptoethanol, as well as with the denatured N protein in Western blotting assays. However, none of the MAbs against antigenic sites II and III reacted with the denatured antigen. These data indicate that antigenic sites I and IV, and sites II and III on the N protein of rabies virus are composed of linear and conformation‐dependent epitopes, respectively.
The Non‐Random Pattern of Insertion of IS2 into the hemB Gene of Escherichia coli
Authors: Lewis Leslie A. , Gopaul Shamila , Marsh Camile
Published: November 2013

The hemB gene of Escherichia coli has been identified as a hot spot for the insertion of the transposable element IS2. The insertional specificity of IS2 is still unclear. This study reports on the attempt to sequence a statistically significant number of insertions in hemB, in order to determine whether there might be a basis for future studies to determine a molecular basis of IS2 insertional specificity. The results indicate that IS2 inserts in a non‐random manner into a 240 bp segment at the 5′ end of the gene (region I). Twenty‐one of 24 insertions occurred in region I. Three insertions have been identified in the two middle 250 bp segments of the 975 bp gene, and none in the 3′ terminal segment. A seventeen bp sequence showing 88.2% identity with a segment of IS2, 221 bp from the 3′ terminus has been identified in region I. Four instances of repeated insertion between the same pair of nucleotides have been observed at four different sites.
Polymerase Chain Reaction Analysis of Borrelia Species Isolated in Japan
Authors: Kawabata Hiroki , Tashibu Hiroyuki , Yamada Kazuto , Masuzawa Toshiyuki , Yanagihara Yasutake
Published: November 2013

Primer reactivities of 25 Borrelia burgdorferi sensu lato isolates from the ticks, Ixodes persulcatus and I. ovatus, in Japan and 10 isolates in Europe and North America were investigated. The methods used in this study were the polymerase chain reaction (PCR) on the flagellin structural gene (fla), the outer surface protein A gene (osp A) and the outer surface protein B gene (osp B), and the restriction fragment length polymorphism (RFLP) analysis of PCR products from osp A and osp B, The flagellin PCR primer set reacted with all the Borrelia strains tested. Four genospecies, B. burgdorferi sensu stricto, B. garinii, B. afzelii and B. japonica, were differentiated by PCR using osp A and osp B primers combined with RFLP analysis. Some Japanese isolates from I. persulcatus were identified as B. garinii or B. afzelii. The other isolates from I. persulcatus did not fit in any of the 4 genospecies.

These results suggested that Japanese isolates from I. persulcatus are highly heterogeneous in their osp A and osp B structures. Furthermore, PCR primers targeting fla are applicable to the gene diagnosis for Lyme disease in Japan and osp A and osp B primers can be used to classify B. burgdorferi sensu lato isolates into genospecies by PCR and RFLP analyses.
Comparison of the Virulence of Methicillin‐Resistant and Methicillin‐Sensitive Staphylococcus aureus
Authors: Mizobuchi Sadao , Minami Junzaburo , Jin Fu , Matsushita Osamu , Okabe Akinobu
Published: November 2013

The virulence of methicillin‐resistant Staphylococcus aureus (MRSA) was compared with that of methicillin‐sensitive S. aureus (MSSA), using 13 MRSA and 7 MSSA strains isolated from clinical specimens. The infectivity and lethality of the two groups were examined as to the inoculum required to infect 50% of guinea pigs (ID50) and to kill 50% of mice (LD50), respectively. The mean ID50 [log10 colony forming units (CFU)] for MRSA strains was 7.1 ± 0.60 standard deviation, which was 1.5 higher than that for MSSA strains (P < 0.001). The mean LD50 (log10 CFU) for MRSA strains was 9.0 ± 0.42, being 1.1 higher than that for MSSA strains (P = 0.001). Pretreatment of mice with cyclophosphamide decreased the mean LD50 for MRSA strains more than that for MSSA strains, resulting in the difference in the mean LD50 being insignificant (P = 0.502). These results indicate that MRSA is less virulent than MSSA in normal hosts, but that they are equally virulent in immunocompromised hosts. The growth of MRSA strains was much slower than that of MSSA strains in the lag phase, although their growth rates were almost the same in the exponential growth phase, suggesting that the difference in virulence between them may be at least partly due to such a difference in growth.
The Role of Tumor Necrosis Factor in Host Defense against Scrub Typhus Rickettsiae. I. Inhibition of Growth of Rickettsia tsutsugamushi, Karp Strain, in Cultured Murine Embryonic Cells and Macrophages by Recombinant Tumor Necrosis Factor‐Alpha
Authors: Geng Peigin , Jerrells Thomas R.
Published: November 2013

Recombinant murine tumor necrosis factor‐alpha (TNF‐α) inhibited intracellular growth of Rickettsia tsutsugamushi, Karp strain, in the mouse embryo cell line C3H/10T1/2 clone 8 at doses of 100 to 10 U/ml. The growth inhibitory effect of TNF‐α was also evident when peritoneal exudate macrophages or bone marrow‐derived macrophages were used as the host cell for rickettsial growth. Interferon‐gamma (IFN‐γ), at doses up to 1,000 U/ml, did not affect the growth of this strain of rickettsiae in the mouse embryo cell line but, as expected, profoundly inhibited rickettsial growth in peritoneal exudate macrophages and bone marrow‐derived macrophages. The effect of TNF‐α on rickettsial growth in the mouse embryo cell line was not reproducibly enhanced by IFN‐γ. Treatment of the cell line with TNF‐α delayed rickettsial cytopathic effects, but the rickettsiae ultimately grew to high numbers in the cells and caused cell death. These findings show that, at least in our system, R. tsutsugamushi is resistant to IFN‐γ‐mediated antirickettsial effects in cells other than macrophages. The results of this study support the suggestion that TNF‐α may inhibit rickettsial growth in cells other than macrophages.
Target Cells of Cytotoxic T Lymphocytes Directed to the Individual Structural Proteins of Rabies Virus
Authors: Fujii Hiroyuki , Mannen Kazuaki , Takita‐Sonoda Yoshiko , Hirai Kazuhiro , Cruz‐Abrenica M.S.E. , Kawano Yasushi , Nishizono Akira , Mifune Kumato
Published: November 2013

Target cells of cytotoxic T lymphocytes (CTL) directed to the individual structural proteins (except for the large polymerase (L) protein) of rabies virus were established by expressing only the respective protein in murine neuroblastoma (NA) and murine macrophage (J774‐1) cell lines. Mice infected with the ERA strain of rabies virus developed CTL responses to all of these rabies virus proteins. The cytotoxic activity was abrogated by pretreatment of the effector cells with anti‐CD8 monoclonal antibody (MAb) and complement but not with anti‐CD4 MAb. Cell lysis by CTL was blocked in the presence of anti‐major histocompatibility complex (MHC) class 1 antibodies in J774‐1 cell lines. Rabies virus‐infected cells express these proteins at the surface, which can be recognized and lysed by the respective CTL. Mice immunized with β‐propiolactone‐inactivated virus induced a CTL response against glycoprotein but not against internal viral components. This assay system might be useful for further analysis of the possible contribution of these proteins in the cell‐mediated immune protection against rabies.