bluuebunny/crossref_metadata_2025 huggingface dataset:
Abscess Forming Ability of Streptococcus milleri Group: Synergistic Effect with Fusobacterium nucleatum
Authors: Nagashima Hiroyuki , Takao Ayuko , Maeda Nobuko
Published: November 2013

The abscess forming abilities of “Streptococcus milleri” strains (Streptococcus constellatus, Streptococcus anginosus, and Streptococcus intermedius) isolated from dentoalveolar abscesses and the synergistic effect of Fusobacterium nucleatum co‐inoculated with the isolates were examined on a mouse subcutaneous abscess model. Five days after inoculation, all S. milleri strains formed abscesses, which showed less pathological spread to surrounding connective tissues than those formed by Staphylococcus aureus 209P strain and were similar to those by F. nucleatum ATCC25586. When each S. milleri strain and F. nucleatum were co‐inoculated, abscess sizes and each bacterial number recovered from abscesses increased in comparison to those treated by bacterial mono‐inoculation of each S. milleri strain or F. nucleatum alone. The strongest synergistic effect was observed in the combination of S. constellatus and F. nucleatum. In a time course experiment with this combination, the recovery of S. constellatus subsequently decreased after the decrement of F. nucleatum, and it appeared that the association with F. nucleatum maintained the bacterial number of S. constellatus in the abscess. The cell‐free supernatant of F. nucleatum had a tendency to increase the abscess size caused by S. constellatus in this model. When S. constellatus was cultured with F. nucleatum culture supernatant in vitro, growth enhancement in the early phase was observed. Furthermore, the phagocytic killing of S. constellatus by human polymorphonuclear leukocytes (PMNs) was significantly suppressed and the PMN membranes appeared to be injured by addition of the F. nucleatum culture supernatant. These results suggest that the pathogenicity of S. milleri strains in odontogenic infections may be enhanced by the co‐existence of F. nucleatum.
Antigenic Relationship among the Eight Prototype and New Serotype Strains of Orientia tsutsugamushi Revealed by Monoclonal Antibodies
Authors: Kang Jae‐seung , Chang Woo‐hyun
Published: November 2013

Orientia tsutsugamushi, the etiologic agent of tsutsugamushi disease, exhibits great antigenic variation. Three classical strains (Karp, Gilliam, and Kato) and new antigenic types from Thailand (TA686, TA678, TA716, TA763, and TH1817) have been used as prototype strains of O. tsutsugamushi in many studies. In this study, monoclonal antibodies to the five Thailand strains were produced, and their reactivity against prototype strains and newly identified isolates from Korea and Japan was tested. With a panel of these monoclonal antibodies, we could analyze the antigenic relationship among various strains of O. tsutsugamushi from Thailand, Japan, and Korea. Twelve strains of the O. tsutsugamushi tested showed various reactivities to monoclonal antibodies, and no distinct pattern of reactivity was found according to their location of isolation. Although the Boryong and Kuroki strains were similar in reactivities to most monoclonal antibodies, several monoclonal antibodies could differentiate the two strains. These results indicate that the immunofluorescence antibody test using monoclonal antibodies used in this study is valuable for analyzing the antigenic relationship and classification of O. tsutsugamushi.
Intracellular Behavior of Rabies Virus Matrix Protein (M) Is Determined by the Viral Glycoprotein (G)
Authors: Nakahara Koichiro , Ohnuma Hiroki , Sugita Shigeo , Yasuoka Kayou , Nakahara Tomomi , Tochikura Tadafumi S. , Kawai Akihiko
Published: November 2013

To investigate the nature and intracellular behavior of the matrix (M) protein of an avirulent strain (HEP‐Flury) of rabies virus, we cloned and sequenced the cDNA of the protein. Using expression vectors pZIP‐NeoSV(X)1 and pCDM8, the cDNA was transfected to animal cells (BHK‐21 and COS‐7) with or without coexpression of viral glycoprotein (G). When M protein alone was expressed in the cells, it displayed homogeneous distribution in the whole cell including the nucleus. In contrast, coexpression with G protein resulted in the abolishment of nuclear distribution of M antigen, and both of the antigens displayed a colocalized distribution in the cell, especially at the cellular membrane as seen in the virus‐infected cells, while the distribution of G antigen was not affected by coexpressed M antigen. Immunoprecipitation studies revealed that M protein was coprecipitated with G protein by anti‐G antibody, and vice versa, although cross‐linking with dithiobis(succinimidyl propionate) was necessary for coprecipitation because of their easier dissociation in the presence of sodium deoxycholate. These results suggest that M protein intimately associates with G protein, which may affect or regulate the behavior (e.g., intracellular localization) of M protein. Studies with deletion mutants of M protein indicate that an internal region around the amino acids from 115 to 151 is essential for the M protein to preserve its binding ability to G protein.
TTG as the Initiation Codon of Salmonella slyA, a Gene Required for Survival within Macrophages
Authors: Kawakami Takatoshi , Kaneko Aki , Okada Nobuhiko , Imajoh‐Ohmi Shinobu , Nonaka Takeshi , Matsui Hidenori , Kawahara Kazuyoshi , Danbara Hirofumi
Published: November 2013

The slyA gene, which has been implicated in the virulence of Salmonella serovar Typhimurium and its survival in macrophages, is widely distributed among different Salmonella serovars. In this study, we cloned and sequenced the translational initiation region of the slyA gene from nine different serovars and found sequence differences in the previously proposed ATG initiation codon but not in a TTG triplet, another putative initiation codon in the slyA gene. Therefore, we determined the actual translational initiation site of the slyA gene by analyzing slyA genes with defined mutation in either the ATG or TTG sequences in an in vitro translation assay and a quantitative hemolytic assay in Escherichia coli. The replacement of TTG by TTC in the slyA gene significantly reduced both the amount of protein synthesized and the hemolytic activity of a transformed strain of E. coli, while replacement of ATG by ATC had no effect in these assays. In addition, the amino acid sequence analysis of the His‐tagged SlyA protein showed that it was identical with the amino acid sequence deduced from the 5′ end of the slyA gene with a TTG initiation codon. Our results suggest that TTG serves as the translational initiation codon for the slyA gene of Salmonella.
Overproduction of Gamma Interferon in B/Jas Inbred Rabbits with Herpes Simplex Virus Encephalitis
Authors: Yuasa Takeshi , Isono Takahiro , Tooyama Ikuo , Seto Akira
Published: November 2013

Inbred rabbits of the B/Jas strain are highly susceptible to herpes simplex virus type‐1 (HSV‐1) encephalitis, developing seizures of encephalitis after intravenous injection of the KOS strain of the virus. Anti‐viral interferon activity became detectable in the serum just prior to or at the onset of seizures, its level being lower in the serum than in the cerebrospinal fluid. The activity was of gamma interferon, as suggested by the acid instability and the inability of Mx protein induction. An immunohistochemical analysis of the brain tissues of encephalitic rabbits showed that MHC class I antigen was expressed on the microglia cells of inflamed lesions but not on these cells in uninflamed areas. These findings were discussed in correlation with the pathogenesis of herpetic encephalitis in the inbred rabbits.
Molecular Epidemiological Study of a Mass Outbreak Caused by Enteropathogenic Escherichia coli O157:H45
Authors: Makino Sou‐ichi , Asakura Hiroshi , Shirahata Toshikazu , Ikeda Tetsuya , Takeshi Koichi , Arai Keisuke , Nagasawa Motohiro , Abe Tamotsu , Sadamoto Terukazu
Published: November 2013

We made a molecular analysis of O157:H45 Escherichia coli isolated from a mass outbreak that occurred in Obihiro City. Using DNA analysis, we confirmed this infection case as a mass outbreak. Although the isolates expressed O157 antigen, they did not produce Vero toxin. We concluded they were enteropathogenic E. coli (EPEC) because they had a bfp gene and an EAF plasmid, and further they exhibited local adherence to HEp‐2 cells. We believe this is the first report of a mass outbreak by O157 EPEC, and we suggest that PCR using eae‐ and bfp‐specific primers and HEp‐2 adherence assay are useful to identify EPEC.
Protective Efficacy and Immunogenicity of Vi‐Porin Conjugate against Salmonella typhi
Authors: Singh Mantej , Ganguly Nirmal Kumar , Kumar Lata , Vohra Harpreet
Published: November 2013

A conjugate vaccine against Salmonella typhi was prepared by covalently binding capsular polysaccharide (Vi) with porin, both isolated from S. typhi. First, Vi and porins were extracted. The Vi was purified from S. typhi Ty2. The purified Vi conformed to the requirements of the World Health Organization. Porins were purified from S. typhi 0901. The Vi was bound to the porins by a heterobifunctional cross‐linking reagent, N‐succinimidyl‐3‐(2‐pyridyl dithio)‐propionate (SPDP). After preparing the Vi‐porin conjugate, its protective ability and immunogenicity were studied in mice following systemic immunization. The results showed that the conjugate is 6.5‐fold more protective than Vi alone against S. typhi. The mice immunized with conjugate elicited higher anti‐Vi antibody (IgG) levels (P<0.01) than the mice immunized with Vi alone. Anti‐porin antibodies were also induced by the conjugate. To study the mucosal immune responses, secretory IgA (sIgA) in the intestinal fluid was measured. Conjugate‐immunized mice showed the induction of sIgA as compared to Vi alone. The results showed that when Vi is bound to porins, both isolated from same organism, the resultant conjugate induced both systemic and mucosal immune responses and provided better protection against S. typhi than Vi alone.
Correlation of T‐Cell Response and Lymphokine Profile with RESA Peptides of Plasmodium falciparum Containing a Universal T‐Cell Epitope and an Immunopotentiator, Polytuftsin
Authors: Kumar Pawan , Rao Donthamsetty Nageswara
Published: November 2013

Two RESA repeat sequences, (EENVEHDA)2 and (DDEHVEEPTVA)2, were chemically linked to a universal T‐cell epitope, CS.T3 and polytuftsin, and a natural immunopotentiator, was physically mixed with these conjugates. The immunogens were studied for in vitro antigen‐induced T‐cell proliferation, and cytokine levels were measured in the culture supernatants. The RESA peptide(s)‐CS.T3 conjugate containing polytuftsin showed the highest stimulation index (SI) as compared to the RESA peptide‐CS.T3 conjugates or RESA peptides alone. Spleen cells from mice primed with either RESA peptide(s)‐CS.T3 conjugate or RESA peptide‐CS.T3 conjugate containing polytuftsin, when pulsed in vitro with the respective RESA peptide, showed a higher proliferation index as compared to spleen cells primed and pulsed in vitro with the respective RESA peptides. This observation has an important relevance during natural reinfection for boosting the immune response. The culture supernatants from the cells primed and pulsed in vitro with RESA peptide‐CS.T3 conjugate and RESA peptide‐CS.T3 conjugate containing polytuftsin showed higher IL‐2 and IFN‐γ levels as compared to the RESA peptides alone. Very low IL‐4 levels were detected with the above formulations. The cytokine profile is suggestive of a CD4+ TH1 type of immune response, which is ideal for the killing of intracellular pathogens like the malarial parasite.
A Single Cell Analysis of TCR AV24AJ18+ DN T Cells
Authors: Keino Hiroshi , Matsumoto Isao , Okada Seiji , Kurokawa Manae , Kato Tomohiro , Tokuhisa Takeshi , Usui Masahiko , Taniguchi Masaru , Nishioka Kusuki , Sumida Takayuki
Published: November 2013

The T‐cell receptor (TCR) BV gene of human TCR AV24+ double‐negative (DN) T cells, a novel subset of natural killer (NK) T cells, was investigated by single‐cell sorting and single‐cell polymerase chain reaction (PCR) methods. Seven of eleven TCR AV24+ DN T‐cell clones utilized TCR BV8, three BY9, and one BV6. Six of seven TCR AV24/BV8+ DN T‐cell clones had identical TCR β and α chains, indicating that they were the same clone. All three TCR AV24/BV9+ DN T‐cell clones also demonstrated the same amino acids in the CDR3 region. These findings strongly suggest that the usage of TCR β and α chains on TCR AV24+ DN T cells is extremely restricted, supporting the notion that these cells recognize highly limited T‐cell epitopes on antigens. All TCR AV24+ clones expressed the NKR‐P1A mRNA, and so were true NK T cells. IL‐2 and IL‐4 mRNAs were detected in all clones, suggesting that the majority of these cells were Th0‐type T cells. Six clones overexpressed Fas‐ligand (Fas‐L) mRNA and Fas antigen was detected on all clones at the mRNA level. In conclusion, TCR AV24+ DN T cells might recognize restricted T‐cell epitopes on antigens and function as Th0‐type T cells, inducer cells to Th1‐ or Th2‐type T cells (regulatory T cells), and as Fas‐L‐positive cytolytic T cells.
mRNA Expression of Complement Components and Regulators in Rat Arterial Smooth Muscle Cells
Authors: Li Wenxin , Tada Toyohiro , Miwa Takashi , Okada Noriko , Ito Jin‐ichi , Okada Hidechika , Tateyama Hisashi , Eimoto Tadaaki
Published: November 2013

The presence of C5b‐9 complexes, some complement regulators, and abundant cytokines in atherosclerotic lesions has been reported. However, it is unclear whether these complement‐associated proteins are produced by vascular smooth muscle cells (SMCs) and how they are influenced by the cytokines. In the present study, we demonstrated, by the reverse transcription‐polymerase chain reaction method, the mRNA expression of complement components (C3, C4, and C5) and membrane regulators (decay‐accelerating factor, membrane cofactor protein, Crry, and CD59) in cultured SMCs derived from the rat carotid artery. The expression of C9 mRNA was also induced upon stimulation by interferon‐gamma (IFN‐γ), tumor necrosis factor‐alpha (TNF‐α) and/or lipopolysaccharide (LPS). Northern blot analysis showed that the mRNA expression of C3, C4, DAF and Crry was up‐regulated, but that of CD59 was down‐regulated by IFN‐7, TNF‐α and/or LPS alone or by synergy. The increase of C3 mRNA by TNF‐α or LPS and that of C4 mRNA by IFN‐γ was induced in a dose‐dependent manner. The results indicate that the arterial SMCs of rat have the ability to produce complement components and regulators, which is affected by cytokines and/or LPS. Since atherosclerosis is characterized by the intimal proliferation of SMCs, the complement system including its regulators may be involved in the pathogenesis of the disease.
Apoptosis Observed in BALB/3T3 Cells Having Ingested Staphylococcus aureus
Authors: Murai Miyo , Sakurada Junji , Seki Keiko , Shinji Hitomi , Hirota Yasuhisa , Masuda Shogo
Published: November 2013

Staphylococcus aureus was previously shown to be internalized by murine fibroblast. We examined the intracellular events of S. aureus ingested by BALB/3T3 cells. After uptake of strains A191 and A151, isolates from atopic lesion, and a laboratory strain, Cowan I, for 1 hr, BALB/3T3 cells were incubated with 1.25 μ/ml lysostaphin. Laddering of the DNA in multiples of approximately 180 bp occurred within 4 hr following bacterial addition in BALB/3T3 cells infected with A191 and within 18 hr in BALB/3T3 cells infected with A151: histochemical staining by the terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labeling method revealed that the rate of the fragmentation of nucleic DNA in Cowan I‐infected BALB/3T3 cells at 21 hr following bacterial addition was 0.52 ± 0.25%, significantly higher than that in the control cells. Transmission electron micrographs of BALB/3T3 cells at 4 hr following A191 addition showed that the apoptotic features, including electron‐dense nucleus and plasma membrane blebbing, occurred in some cells in which many staphylococci escaped the endosome and went on to cell division. At the same time, A151 organisms enclosed with endosome membrane were static in the intact BALB/3T3 cells. The significant increase of A191 was confirmed by counting intracellular live bacteria during 2‐ to 6‐hr incubation. These results suggest that internalized S. aureus escapes the endosome, multiplies and induces apoptosis in the fibroblast cell.
Development of an Enzyme‐Labeled Oligonucleotide Probe for Detecting the Escherichia coli Attaching and Effacing A Gene
Authors: Bi Zhenqiang , Nagayama Kenichi , Akeda Yukihiro , Cantareli Vlademir , Kodama Toshio , Takarada Yutaka , Shibata Shuji , Honda Takeshi
Published: November 2013

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) can produce attaching and effacing (AE) lesions on intestinal epithelium in vitro and in vivo. A gene necessary to cause the AE lesion has been identified and designated Escherichia coli attaching and effacing A (eaeA) gene. In this study, an alkaline phosphatase (ALP)‐conjugated oligonucleotide probe for the eaeA gene was developed and used to detect the eaeA gene among 163 strains of classical EPEC and 25 strains of EHEC O157. The prevalence rates of eaeA gene in the strains of classical EPEC and EHEC O157 were 51.5 and 100%, respectively. The eaeA‐positive rate (60.0%) in strains of class I EPEC serogroups (026, 055, 086, O111, O119, O125, O126, O127, O128ab, and O142) was significantly higher than that (22.9%) in strains of the class II EPEC serogroups (O18, O44, O114) (P<0.01). A total of 109 eaeA‐positive classical EPEC and EHEC O157 were positive for fluorescent actin staining (FAS) assay, whereas 79 eaeA‐negative classical EPEC were negative. Both the sensitivity and specificity of the eaeA probe versus the FAS assay positivity were 100%. Thus, use of the ALP‐conjugated oligonucleotide probe for the eaeA gene would be specific and reliable in identifying the adherence capability of EPEC and EHEC.
Apoptosis of Endothelial Cell Line ECV304 Persistently Infected with Orientia tsutsugamushi
Authors: Kim Mee‐kyung , Kee Sun‐ho , Cho Kyung‐A , Chung Moon‐hyun , Lim Byung‐uk , Chang Woo‐hyun , Kang Jae‐seung
Published: November 2013

Endothelial cells are major targets of Orientia tsutsugamushi. To examine the consequences of the infection of endothelial cells with O. tsutsugamushi, we used human endothelial cell line ECV304. Persistent infection was established and infected cultures could be maintained for over seven months without the addition of normal cells. The heavily infected cells became round and floated in the culture medium, harboring large numbers of organisms inside them. Some of the infected ECV304 cells showed features of apoptotic cells, as determined by the terminal deoxytransferase‐mediated dUTP nick end‐labeling reaction and DNA fragmentation. We also found that O. tsutsugamushi increased transcription of the mRNAs of proinflammatory cytokines such as IL‐6 and IL‐8. These results show the first evidence of in vitro‐persistent infection by O. tsutsugamushi, which may be related to in vivo persistence reported previously.
Evaluation of AFLP, A High‐Resolution DNA Fingerprinting Method, as a Tool for Molecular Subtyping of Enterohemorrhagic Escherichia coli O157:H7 Isolates
Authors: Lyoda Sunao , Wada Akihito , Weller Jeniffer , Flood Susan J. A. , Schreiber Edgar , Tucker Bill , Watanabe Haruo
Published: November 2013

The amplified fragment‐length polymorphism (AFLPTM) technique is based on the selective PCR amplification of restriction fragments. We investigated the utility of AFLP in the molecular subtyping of enterohemorrhagic Escherichia coli serotype O157:H7 isolates. We analyzed a total of 46 isolates of E. coli O157:H7 along with other serotypes, O26:H11, O114:H19 and O119:NT. Isolates of E. coli O157:H7 derived from the same outbreak showed an identical AFLP‐banding pattern and were subtyped into the same group, giving results almost consistent with those of a pulsed‐field gel electrophoresis (PFGE) study, while other serotypes showed clearly different patterns from those of E. coli O157:H7. These results suggest that the AFLP technique has potential as an alternative tool for the molecular epidemiology of E. coli O157:H7.
Limited Stress Response in Streptococcus pneumoniae
Authors: Choi In‐hwa , Shim Jai‐heon , Kim Seung‐whan , Kim Su‐nam , Pyo Suhk‐neung , Rhee Dong‐kwon
Published: November 2013

In Streptococcus pneumoniae, heat shock induces the synthesis of 65‐, 73‐, and 84‐kDa proteins, and ethanol shock induces a 104‐kDa protein. In this study, the 65‐, 84‐, and 104‐kDa proteins were identified as members of the GroEL, ClpL and alcohol dehydrogenase families, respectively, and the general properties of the stress response of S. pneumoniae to several other stresses were characterized. However, several stresses which are known to induce stress responses in Escherichia coli and Bacillus subtilis failed to induce any high molecular weight heat‐shock proteins (HSPs) such as GroEL and DnaK homologues. A minor temperature shift from 30 to 37 C triggered induction of the homologues of DnaK and GroEL of E. coli. These features may provide a foundation for evaluating the role of heat‐shock proteins relative to the physiology and pathogenesis of pneumococcus.
Chemokine Receptor‐Usage of Clinical HIV‐1 Isolates Obtained from Patients with HIV‐1 Infection in Late Clinical Stages Using PHA‐Blast
Authors: Matsuda Shunji , Miyata Masahiko
Published: November 2013

In the present sudy, chemokine receptor‐usage of primary HIV‐1 isolates was examined using U87‐CD4 cells expressing chemokine receptors CCR3, CCR5 and CXCR4. HIV‐1 was isolated from the peripheral blood mononuclear cells (PBMC) and/or plasma of eight HIV‐1‐infected individuals in late CDC‐II and CDC‐IV clinical stages using PHA‐blast prepared from the PBMC of healthy blood donors. The primary HIV‐1 isolates from patients in late CDC‐II stage rarely infected monocyte‐derived macrophages in the present study, whereas most isolates from patients in the CDC‐IV stage infected the macrophages. In the experiments using U87‐CD4 cells expressing chemokine receptors, the isolates from patients in the late CDC‐II stage infected U87‐CD4 cells expressing CXCR4, but not U87‐CD4 cells expressing CCR5. In contrast, most isolates from patients in the CDC‐IV stage infected both U87‐CD4 cells expressing CXCR4 or CCR5. The isolates which infected both U87‐CD4 cells were supposed to contain dual tropic HIV‐1 or a mixture of CXCR4‐tropic and CCR5‐tropic HIV‐1s. Analysis of the deduced amino acid sequence of the V3 region in proviral env gene showed that the V3 region in U87‐CD4 cells infected with CXCR4‐tropic HIV‐1 isolates was largely different from that in the cells infected with CCR5‐tropic isolates, but were highly similar to that in cells infected with dual tropic isolates. These results suggest that PHA‐blasts may preferentially support the replication of the CXCR4‐tropic and dual tropic HIV‐1s, and that CXCR4‐tropic and dual tropic HIV‐1s are also present in peripheral blood from patients in the late stage of the asymptomatic phase.
Isolation of Orientia tsutsugamushi from Patients in Shikoku and Finding of a Strain Which Grows Preferentially at Low Temperatures
Authors: Tamura Akira , Makisaka Yukie , Enatsu Teruyuki , Urakami Hiroshi , Okubo Koji , Fukuhara Masahiro , Mahara Fumihiko
Published: November 2013

Three strains of Orientia tsutsugamushi were isolated from patients in Anan City, Tokushima Prefecture. The strains were identified as Karp type by analyses of reactivities with type‐specific monoclonal antibodies. One strain, Okazaki, was isolated in L cells cultivated at 31 C, but not in cells at 36 C or in mice. This strain showed better growth at 31 C than 36 C. This is the first report of an O. tsutsugamushi strain which grows preferentially at low temperatures.
Genetic and Biochemical Properties of a Hemolysin (Pyolysin) Produced by a Swine Isolate of Arcanobacterium (Actinomyces) pyogenes
Authors: Ikegami Mitsuhiro , Hashimoto Nozomu , Kaidoh Toshio , Sekizaki Tsutomu , Takeuchi Shotaro
Published: November 2013

Arcanobacterium (Actinomyces) pyogenes, a causative agent of various pyogenic diseases in domestic animals, produces a hemolysin which is thought to be an important virulence factor. This hemolysin was purified from the culture supernatant of A. pyogenes swine isolate. The purified hemolysin showed a single band with a molecular mass of 56 kDa on SDS‐polyacrylamide gel electrophoresis, and its isoelectric point was 9.2. The activity of this hemolysin was not enhanced by the addition of L‐cysteine or sodium thioglycolate, but it was inhibited by cholesterol. The gene encoding the hemolysin was cloned, sequenced and expressed in Escherichia coli by means of ZAP Express vector. Analysis by SDS‐polyacrylamide gel electrophoresis with immunoblotting showed that the molecular weight of the hemolysin expressed in E. coli is the same as that of the hemolysin purified from A. pyogenes. Nucleotide sequence analysis revealed an open reading frame of 1,605 bp encoding a 534 amino acid protein of 57,989 Da. The nucleotide sequence of the hemolysin gene from A. pyogenes swine isolate differed only slightly (97.6% identity) from the sequence of plo gene from A. pyogenes strain BBR1 reported by Billington et al (J. Bacteriol. 179: 6100–6106, 1997). The cysteine residue existed in the undecapeptide region of the hemolysin, which is highly conserved in thiol‐activated cytolysins (cholesterol‐binding cytolysins), and is replaced with alanine. Therefore, the hemolysin of A. pyogenes seems to be a novel member of the thiol‐activated cytolysin family.
Leukocyte Integrin‐Dependent and Antibody‐Independent Cytotoxicity of Macrophage against Allografts
Authors: Yoshida Ryotaro , Oku Tohru , Takikawa Osamu , Einaga‐Naito Kuniko , Yoneda Yukio , Hirota Ryuichiro , Kubota Takahiro
Published: November 2013

Macrophages (Mφs), but not T cells, infiltrating into the rejection site of either i.p. allografted Meth A (H‐2) fibrosarcoma cells in C57BL/6 (B6) (H‐2b) mice or BALB/c (H‐2d) skin onto B6 mice are cytotoxic against allografts with H‐2d specificity. To determine the mechanisms of specific killing of allografts by allograft‐induced Mφ (AIM), we raised ≈5,000 rat monoclonal antibodies (mAbs) against AIM and selected three of them (R1–73, R2–40 and R1–34), each of which inhibited cytotoxic activity against allografts in a dose‐dependent manner. The antigens recognized by R1–73, R2–40 and R1–34 mAbs were defined by immunoprecipitation and Western blot analyses as CD11a, CD18 and CD11b, respectively; and the allografts expressed CD54, a ligand of CD11a or CD11b, suggesting leukocyte integrin‐dependent killing. Although Ab‐dependent cellular cytotoxicity has been recognized as a mechanism of specific killing by Mφs, the infiltration of AIM into the rejection site of allografts far (≈6 days) preceded the appearance of serum IgG Ab specific for the allograft. AIM exhibiting full cytotoxic activity against allografts was also induced in the transplantation site of Fcγ receptor knockout [(B6×129) F1] mice as well as B10.D2 (H‐2 compatible with allograft) and B6‐xid (X‐linked immunodeficiency with B cell‐specific defect) strains of mice. In the latter two strains of mice, the levels of serum IgG Ab to the allograft were negligible. Moreover, the cytotoxic activity of AIM against allografts was not affected by pretreatment of the cells with anti‐mouse IgG serum, suggesting Ab‐independent cytotoxicity.
Efficient Induction of Peptide‐Specific Cytotoxic T Lymphocytes by LPS‐Activated Spleen Cells
Authors: Kakugawa Kiyokazu , Udaka Keiko , Nakashima Kosuke , Inaba Kayo , Oka Yoshihiro , Sugiyama Haruo , Tamamura Hirokazu , Yamagishi Hideo
Published: November 2013

Lipopolysaccharides of Gram‐negative bacteria are potent activators of B cells, dendritic cells and monocytes/macrophages. We have investigated the use of LPS‐activated spleen cells as antigen‐presenting cells to induce CD8+ cytotoxic T lymphocytes in vivo that are reactive to MHC class I binding peptides. Compared with resting spleen cells, CTL induction was more efficient and less variable for different peptides with LPS‐activated spleen cells. Cytotoxic responses were specific for the immunized peptides and contained high affinity CD8+ T cells. The removal of dendritic cells and monocytes/macrophages by Sephadex G10 column did not show profound effects on CTL induction, indicating that B‐cell blasts were largely responsible. This easily accessible method should facilitate the screening of MHC class I binding peptides to determine whether or not the host's T‐cell repertoire contains reactive T cells.
Seroepidemiological Investigation of Feline Chlamydiosis in Cats and Humans in Japan
Authors: Yan Cai , Fukushi Hideto , Matsudate Hiroki , Ishihara Kanako , Yasuda Keiko , Kitagawa Hitoshi , Yamaguchi Tsuyoshi , Hirai Katsuya
Published: November 2013

The prevalence of chlamydia antibodies in Japan was investigated in 215 cat sera, consisting of 88 sera of stray cats and 127 sera of pet cats, and 2,184 human sera, taken from 2,003 general persons and 181 small animal clinic veterinarians, by microimmunofluorescence (MIF) testing withChlamydia psittaciFe/Pn1 of feline origin and Prk/6BC of avian origin as antigens. The prevalence rates of anti‐Fe/Pn1 antibodies were 45.5% in stray cats, 17.3% in pet cats, 1.7% in general persons and 8.8% in small animal clinic veterinarians. The prevalence rates of anti‐Prk/6BC antibodies were 51.1% in stray cats, 15.0% in pet cats, 3.1% in general persons and 5.0% in small animal clinic veterinarians. These results suggested that feline chlamydia infection is widely spread in cats especially in stray cats in Japan, and suggested that feline chlamydiosis could be transmitted to people who are in close contact with infected cats.
An Exfoliative Toxin A‐Converting Phage Isolated from Staphylococcus aureus Strain ZM
Authors: Yoshizawa Yukio , Sakurada Junji , Sakurai Susumu , Machida Katsuhiko , Kondo Isamu , Masuda Shogo
Published: November 2013

Exfoliative toxin A (ETA) causes staphylococcal scalded‐skin syndrome in children. The gene for ETA was believed to be coded by the chromosomal DNA. We isolated temperate phages from an ETA‐producing strain, ZM, using a restriction minus strain, 1039, as an indicator. One of the prophages, designated φ‐ZM‐1 mediated lysogenic conversion of ETA. The polymerase chain reaction assay of the eta gene revealed that phage φ‐ZM‐1 carries the structural gene for ETA.
A Simple Screening Method for Detecting Bindings between Oligopeptides and HLA‐DR Molecules on Filter Papers: Possible Application for Mapping of Putative Helper T‐Cell Epitopes on MSP1 of Plasmodium falciparum
Authors: Fu Jun , Hato Mariko , Igarashi Karen , Suzuki Takashi , Matsuoka Hiroyuki , Ishii Akira , Leafasia Judson L. , Chinzei Yasuo , Ohta Nobuo
Published: November 2013

Binding capacities of synthetic peptides to HLA‐DR molecules were tested on filter papers to identify putative helper T‐cell epitopes on a malarial protein. The antigen tested was the merozoite surface glycoprotein 1 (MSP1) of Plasmodium falciparum, a vaccine candidate targeting the asexual erythrocytic stage. Bindings between synthetic oligopeptides and HLA‐DR molecules were tested. Such bindings were not non‐specific, and a known helper T‐cell epitope peptide showed positive binding to the restricting HLA‐DR molecule. By using this screening system, we observed the unequal distribution of HLA‐DR‐binding peptides in 10 out of 17 MSP1 blocks tested. Block #6 of MSP1 seemed to show the highest frequency in the positive binding; on the other hand, blocks #1 and #17, both of which were thought to be vaccine candidate regions, contained fewer HLA‐DR binding peptides. This was not inconsistent with the results that block #17 was less stimulatory to peripheral T cells than block #6. The peptides with positive binding to HLA‐DR showed actual epitope activities when we tested peptide‐driven proliferation of human bulk T‐cell lines, and association between the two parameters was statistically significant (P < 0.001). For more detailed information for vaccine development, peptides with both IgG‐ and HLA‐DR binding activities were mapped in block #17 of MSP1. Together with these results, we demonstrate that our simple screening system seems to provide essential information for vaccine development through uncovering locations of putative epitopes for human helper T cells.
Preventive Effect of Ninjin‐to (Ren‐Shen‐Tang), a Kampo (Japanese Traditional) Formulation, on Spontaneous Autoimmune Diabetes in Non‐Obese Diabetic (NOD) Mice
Authors: Kobayashi Takao , Song Qing‐hua , Hong Tie , Kitamura Hajime , Cyong Jong‐choi
Published: November 2013

We previously found that ingestion of an extract of Ninjin‐to (NJT; Ren‐Shen‐Tang) suppressed the development of autoimmune diabetes in C57BL/KsJ mice induced by multiple low doses of streptozotocin. To verify this effects on spontaneous autoimmune diabetes, the effects of NJT on NOD mice were investigated in the present study. NJT, provided in drinking water (0.25%, 450 mg/kg/day) from 6 weeks of age, significantly prevented the incidence of spontaneous diabetes in female NOD mice at 30 weeks of age (2/10) compared with that of the controls (7/10), with no effects on body growth or food intake. Even in non‐diabetic mice, the blood glucose levels of the NOD controls gradually increased with age, while such increase in NJT‐treated mice was significantly suppressed by preventing any deficiency of glucose tolerance. NJT also significantly suppressed the progression of insulitis, which causes insulin deficiency and diabetes. It is well known that NOD mice develop insulitis and diabetes because of their Th1‐dominant autoimmune response. IFN‐γ production from splenic T lymphocytes stimulated with anti‐CD3 monoclonal antibodies was increased, whereas IL‐4 production was decreased in NOD controls compared to age‐ and sex‐matched normal ICR mice. NJT‐treatment reduced these deviations of cytokine production in NOD mice. These data all suggest that NJT can prevent spontaneous insulitis and diabetes by the modification of deviated cytokine production in NOD mice.
Cell Surface‐Associated Enolase in Actinobacillus actinomycetemcomitans
Authors: Hara Hiroaki , Ohta Hiroyuki , Inoue Tetsuyoshi , Ohashi Toshio , Takashiba Shogo , Murayama Yoji , Fukui Kazuhiro
Published: November 2013

Cell surface‐associated materials of Actinobacillus actinomycetemcomitans were extracted by a short incubation of the cell suspension in a Tris‐buffered saline in the presence and absence of a restriction enzyme, EcoRI. The supernatants (which we termed EcoRI extract and surface extract, respectively) contained a number of extracellularly released proteins. Of these proteins, four major proteins were identified by N‐terminal sequencing to be the 34 and 39 kDa outer membrane proteins, the GroEL‐like protein, and a 47 kDa protein homologous to Haemophilus influenzae enolase. Enolase activity was found in the extracts and its relative amount of activity in the EcoRI extract from a culture of the mid‐exponential growth phase was estimated as 5.7% of total enzyme activity. In contrast, the relative amount of activity of another cytosolic enzyme, lactate dehydrogenase, was extremely low in the extracts and also in the culture supernatant. These results suggest the external localization of enolase in this bacterium.
Hexagonal Assembly of the Magnesium Salt of an R‐Form Lipopolysaccharide from Klebsiella pneumoniae: Its Lowered Stability Compared with Original Non‐Electrodialyzed Preparation
Authors: Ito Hideo , Naito Setsuko , Kato Nobuo
Published: November 2013

The magnesium salt of R‐form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN‐111 (O3— :K1—) that was prepared after the removal of cationic materials by electrodialysis formed essentially the same ordered hexagonal lattice structure with a lattice constant of 14 to 15 nm as the original non‐electrodialyzed preparation of the R‐form LPS. When the magnesium salt was suspended in 50 mm glycine buffer or Tris buffer at pH 1.4 to 9.5 and kept at 4 C for 24 hr, its content of Mg was markedly decreased, and its hexagonal lattice structure was changed to a swollen hexagonal lattice structure with extended lattice constants at pH 1.4 and to a loose mesh‐like structure at pH 3.0 or higher. In the original non‐electrodialyzed preparation of the R‐form LPS, the release of Mg and disintegration of the hexagonal lattice structure did not occur by suspending in buffers at pH 1.4 to 8.5 at 4 C for 24 hr, but occurred only at pH 9.0 or higher. The results suggest that organic cations that can be removed by electrodialysis play some part in tight binding to Mg2+ and in stabilizing the ordered hexagonal assembly of the R‐form LPS.
Evaluation of Nine Sets of PCR Primers in the RNA Dependent RNA Polymerase Region for Detection and Differentiation of Members of the Family Caliciviridae, Norwalk Virus and Sapporo Virus
Authors: Honma Shinjiro , Nakata Shuji , Kinoshita‐Numata Kazuko , Kogawa Keiko , Chiba Shunzo
Published: November 2013

Norwalk virus and Sapporo virus were approved as type species of the genus “Norwalk‐like viruses” and the genus “Sapporo‐like viruses,” respectively, in the family Caliciviridae. A total of 116 stool specimens containing Norwalk virus (NV) or Sapporo virus (SV) were tested by RT‐PCR and Southern hybridization to evaluate nine sets of PCR primers and seven internal oligonucleotide probes in the RNA dependent RNA polymerase region of NV and SV for detection and differentiation of viruses in the NV and SV. Fifty‐five stool samples were collected from 11 outbreaks of NV and/or SV gastroenteritis in an infant home, where residents were infants under 2 years of age, in Sapporo, Japan. Sixty specimens were obtained in Sapporo from sporadic cases in children, mainly under 6 years of age, of acute gastroenteritis due to small round structured viruses detected by EM. There is no single primer pair to detect all NV and SV, and at least three primer pairs, G1 set, G2 set and Sapp35/Sapp36, are required to detect viruses in the NV and SV clades. Many NV and SV strains were successfully classified into one of the NV/genogroup I, NV/genogroup II and SV by single‐round RT‐PCR and Southern hybridization. The new detection method for SV reported in this study combined with those for NV previously reported may elucidate the importance of Norwalk virus and Sapporo virus as a cause of viral gastroenteritis in all age groups in the world.
Interleukin‐8 Secretion of Human Epithelial and Monocytic Cell Lines Induced by Middle Ear Pathogens
Authors: Kita Hideaki , Himi Tetsuo , Fujii Nobuhiro , Ylikoski Jukka
Published: November 2013

Otitis media with effusion (OME) is one of the most common diseases in children. Alloiococcus otitidis, a new Gram‐positive bacterial species, was isolated from the middle ear fluid of children with OME; however, the pathogenic role of this bacteria is yet unknown. In this study, the ability of cultured epithelial cell lines (Hep‐2 and Hela) and monocytic cell lines (THP‐1 and U 937) to secrete chemokine interleukin‐8 (IL‐8) in response to the A. otitidis organism and three bacterial organisms mainly detected from middle ear fluid in OME, and bacterial cell components was investigated. When stimulated with four viable bacterial cells, epithelial cells and monocytes secreted IL‐8 in a time‐dependent manner. The monocytes produced significantly higher levels of IL‐8 than the epithelial cells. Compared with that by viable bacterial cells, IL‐8 secretion by stimulated epithelial cells and monocytes was reduced when the bacteria were heated and treated with glutaraldehyde. With bacterial stimulations, cell treatment of interferon‐gamma caused monocytes to increase the induction of IL‐8 production, however, the induction of monocyte differentiation caused monocytes to reduce the induction of IL‐8 production. Furthermore, epithelial cells and monocytes stimulated by four viable bacterial organisms physically separated from cultured cells reduced the induction of IL‐8 compared with directly stimulated cells, and monocytes stimulated with soluble extracts prepared from A. otitidis organisms produced IL‐8 in a dose‐dependent manner. These results suggest that part of the IL‐8 stimulation of the A. otitidis organism may exist in a diffusable factor released by the bacteria or soluble components of the bacteria itself.
Genetic Analysis of the ycgJ‐metB‐cysK‐ygaG Operon Negatively Regulated by the VirR/VirS System in Clostridium perfringens
Authors: Ohtani Kaori , Takamura Hisao , Yaguchi Harumi , Hayashi Hideo , Shimizu Tohru
Published: November 2013

The 5′‐flanking region of the metB‐cysK‐ygaG operon, whose expression is negatively regulated by the VirR/VirS system in C. perfringens, was analyzed. The region contained the ycgJ, mscL, and colA genes encoding a hypothetical protein, a large conductance mechanosensitive channel protein, and kappa‐toxin (collagenase), respectively. Northern analysis revealed that the ycgJ gene was transcribed as a 4.9‐kb operon together with the metB‐cysK‐ygaG genes and that this operon was negatively regulated by the VirR/VirS system. It is indicated that the pfoA (theta‐toxin or perfringolysin O), colA, and ycgJ‐metB‐cysK‐ygaG genes that belong to the VirR/VirS regulon are situated very close together in a 26.5‐kb region of the chromosome, but do not form a pathogenic island.
Expression of Fimbriae and Hemagglutination Activity in Shigella boydii
Authors: Utsunomiya Akiyoshi , Nakamura Michio , Hamamoto Akihiro
Published: November 2013

This report describes the presence of type 1 fimbriae on Shigella boydii 5 which agglutinate guinea pig erythrocytes and feature mannose‐sensitive adherence. Morphologically, the fimbriae were thin, rigid cylinders 2–5 μm in length and 3–5 nm in diameter, and the organella retained axial holes. This is the first study to have revealed the existence of type 1 fimbriae on S. boydii.
Combined Effect of Rebamipide and Ecabet Sodium on Helicobacter pylori Adhesion to Gastric Epithelial Cells
Authors: Hayashi Shunji , Sugiyama Toshiro , Yokota Kenji , Isogai Hiroshi , Isogai Emiko , Shimomura Hirofumi , Oguma Keiji , Asaka Masahiro , Hirai Yoshikazu
Published: November 2013

Helicobacter pylori is a major etiological agent in gastroduodenal disorders. The adhesion of H. pylori to gastric epithelial cells is the initial step of H. pylori infection. Inhibition of H. pylori adhesion is thus a therapeutic target in the prevention of H. pylori infection. We have reported that rebamipide and ecabet sodium, mucoprotective antiulcer agents, independently inhibit H. pylori adhesion. However, the antiadhesion activity of each antiulcer agent was incomplete. Experiments were performed to evaluate the combined effect of rebamipide and ecabet sodium on H. pylori adhesion to gastric epithelial cells. MKN‐28 and MKN‐45 cells, derived from human gastric carcinomas, were used as target cells. Twelve clinical isolates of H. pylori were used in this study. We evaluated the effects of rebamipide and ecabet sodium, individually and in combination, on H. pylori adhesion to target cells quantitatively using our previously established enzyme‐linked immunosorbent assay. Rebamipide and ecabet sodium each partially inhibited H. pylori adhesion. In contrast, adhesion was almost completely inhibited by pretreating target cells and H. pylori with the combination of rebamipide and ecabet sodium. Our studies suggest that the synergistic antiadhesion activity of rebamipide and ecabet sodium is greater than that of each antiulcer agent alone.
Role of Tryptophan‐1 in Hemolytic and Phospholipase C Activities of Clostridium perfringens Alpha‐Toxin
Authors: Nagahama Masahiro , Mukai Masakazu , Ochi Sadayuki , Sakurai Jun
Published: November 2013

Replacement of the Trp‐1 in Clostridium perfringens alpha‐toxin with tyrosine caused no effect on hemolytic and phospholipase C (PLC) activities or on binding to the zinc ion, but that of the residue with alanine, glycine and histidine led to drastic decreases in these activities and a significant reduction in binding to the zinc ion. The hemolytic and PLC activities of W1H and W1A were significantly increased by the preincubation of these variant toxins with zinc ions, but the preincubation of WIG with the metal ion caused little effect on these activities. Gly‐Ile‐alpha‐toxin, which contained an additional Gly‐Ile linked to the N‐terminal amino acid of alpha‐toxin, did not show hemolytic activity, but showed about 6% PLC activity of the wild‐type toxin. A mutant toxin, which contained an additional Gly‐Ile linked to the N‐terminus of a protein lacking 4 N‐terminal residues of alpha‐toxin, showed about 1 and 6% hemolytic and PLC activities of the wild‐type toxin, respectively. Incubation of the mutant toxin with zinc ions caused a significant increase in PLC activity. These observations suggested that Trp‐1 is not essential for toxin activity, but plays a role in binding to zinc ions.
The Mechanism of Actinomycin D‐Mediated Increase of Borna Disease Virus (BDV) RNA in Cells Persistently Infected by BDV
Authors: Mizutani Tetsuya , Inagaki Hisae , Tada Mitsuhiro , Hayasaka Daisuke , Murphy Michael , Fujiwara Toshiyoshi , Hamada Jun‐ichi , Kariwa Hiroaki , Takashima Ikuo
Published: November 2013

The transcriptional mechanism of Borna disease virus (BDV) has been poorly understood. We have analyzed transcription of the virus upon various stimuli in Madin‐Darby canine kidney cells which were persistently infected by BDV (MDCK/BDV). Treatment with actinomycin D (ActD) increased the level of BDV RNA, shifting the size of RNA from 1.9 kb to 2.3 kb beginning 5 hr after the treatment. To understand the mechanism of this unique modulation of BDV RNA, we conducted several experiments. The RNA increase occurred at the stage in which synthesis of cellular intrinsic mRNA was intact, suggesting BDV does not compete with cellular transcriptional machinery for intrinsic RNA polymerase II. The BDV transcription was also enhanced by cycloheximide treatment, indicating that newly synthesized viral or cellular proteins are not necessary for viral transcription. However, a shift in the RNA size was not observed for cycloheximide‐induced BDV RNA. The increase in viral transcription persisted during the cellular apoptotic process consequent to p53 gene accumulation beginning 1 hr after ActD treatment. Caspase inhibitors Z‐VAD and DEVD‐CHO repressed the apoptotic process but failed to block the increase in BDV transcription. In addition, adenovirus‐mediated transduction of wild‐type p53 did not alter the BDV transcription, indicating that the increase in BDV transcription was independent of the p53‐mediated apoptotic process. Other various stimuli that evoke cellular signal transductions failed to alter BDV transcription. Agents inhibitory to topoisomerase except adriamycin failed to enhance BDV transcription, indicating that the increase in BDV transcription is not mediated by an inhibitory action to the topoisomerase II of ActD. Adriamycin showed an increase and size‐shift of BDV RNA similar to ActD. These results suggest that intercalation of the viral genome itself with ActD is related to the stabilization of viral RNA and alteration of RNA size rather than secondary host cell changes.
Antiviral Substance from Silkworm Faeces: Characterization of Its Antiviral Activity
Authors: Hiraki Akihiro , Hirayama Etsuko , Kim Jeman
Published: November 2013

The antiviral activity of a substance (L4–1) purified from silkworm faeces was examined in an HVJ (Sendai virus)—LLC‐MK2 cell system. Its antiviral effect depended on the period of light irradiation and was inhibited by sodium sulfite and anaerobic conditions. These results indicate that the antiviral activity of L4–1 is associated with active oxygen species produced from the substance. SDS‐polyacrylamide gel electrophoretic analysis showed that viral proteins were damaged by this substance under light irradiation. The results suggest that the antiviral activity is due to damage to viral protein(s) caused by active oxygen species produced from L4–1.