bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractThe 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

AbstractInbred 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

AbstractWe 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

AbstractA 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

AbstractTwo 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

AbstractThe 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

AbstractThe 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

AbstractStaphylococcus 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

AbstractEnteropathogenic 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

AbstractEndothelial 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

AbstractThe 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

AbstractIn 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

AbstractIn 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

AbstractThree 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

AbstractArcanobacterium (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

AbstractMacrophages (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

AbstractLipopolysaccharides 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.