bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Genetic Determinants That Regulate Tumorigenicity of Friend Murine Leukemia Virus in Rats
Authors: Takase‐Yoden Sayaka , Watanabe Rihito
Published: November 2013

A neuropathogenic variant of Friend murine leukemia virus (FrMLV), clone A8, has been shown to cause thymoma and infiltration of leukemic cells to organs at 7–8 weeks post‐infection in rats with a more rapid progression than clone 57. We have previously reported that the determinant for induction of aggressive leukemia in rats is located in the ClaI‐AatII fragment containing the long terminal repeat (LTR) and the 5′ half of the 5′ leader sequence of A8 virus. Further studies of chimeric viruses restricted the determinant for the induction of thymoma to only the 0.6‐kb ClaI‐KpnI fragment of A8. This fragment contains a 0.1 kb region of the 3′ terminus of the env gene, the intergenic region, the U3, and the 5′ half of the R region in the LTR. Major differences in the fragment between A8 and 57 viruses were found in the U3 region, especially in the enhancer motifs. These results indicate that the enhancer region of A8‐LTR contributes to the manifestation of thymoma with rapid progression in rats.
Effect of Environmental Conditions on Proteins Secreted by Enterohemorrhagic Escherichia coli O26:H11
Authors: Yoh Myonsun , Bi Zhenqing , Matsuyama Junko , Nagayama Kenichi , Honda Takeshi
Published: November 2013

Infections due to Shiga toxin‐producing Escherichia coli (STEC) are responsible for severe diarrheal diseases in humans, and these bacteria have recently emerged as a leading cause of renal failure and encephalitis in children and the aged. In this study, we examined the environment‐dependent production of proteins secreted from a strain of STEC O26:H11 by trichloroacetic acid precipitation, SDS‐PAGE, Western blotting and N‐terminal amino acid sequence analysis. Growth of bacteria in essential minimum medium (M9) led to the detection of secreted proteins of 104, 80, 40, 37 and 25 kDa (P104, P80, P40, P37 and P25, respectively). When grown in serum‐free MEM, only P104, P40, P37 and P25 were observed in supernatant fluids. Growth of the bacteria in Luria‐Bertani broth (LB) enhanced the expression of P104, but the productions of the other proteins were remarkably reduced. CO2 increased the secretion of P80 and P37, but reduced the production of P104. N‐terminal amino acid sequencing revealed that P104 was EspP of STEC, which was homologous to EspC of enteropathogenic Escherichia coli (EPEC), and both proteins belong to a subclass of the IgA protease family. P80, which was identified as EspE of STEC, was homologous to Tir of EPEC. P40, P37 and P25 were found to be highly homologous to the similarly sized EspD, EspB and EspA proteins, previously detected in culture supernatants of EPEC. Those proteins are thought to be STEC virulence factors. Sera were obtained from two patients, one with colitis and another with hemolytic uremic syndrome (HUS), caused by STEC O157:H7, to study immune response to secreted proteins. Our results suggested that Tir caused immune response following STEC disease.
Transposon‐Induced Norfloxacin‐Sensitive Mutants of Bacteroides thetaiotaomicron
Authors: Ueda Ohmi , Yoshimura Fuminobu
Published: November 2013

To elucidate the mechanism of norfloxacin (a fluoroquinolone) resistance of Bacteroides thetaiotaomicron, a member of the B. fragilis group, we isolated transposon‐induced mutants sensitive to this agent using Tn4351. Four norfloxacin‐sensitive mutants showed reduced levels of resistance, at least, to ethidium bromide. Cloning and sequencing of three chromosomal fragments adjacent to Tn4351 from the mutants revealed that two partial open reading frames (orfs) were disrupted by a transposon. Amino acid sequences of partial orf products had strong homologies to those of Escherichia coli RecB and B. ovatus transketolase. Two mutants carried a recB homolog inserted by Tn4351 together with R751 (cointegration) and by itself (simple transposition) at the amino‐ and carboxyl‐terminal portions, respectively. Since mutations in recB produce E. coli cells sensitive to DNA‐damaging treatments by quinolones, it is concluded that decreases of the minimum inhibitory concentrations (MICs) of the agents for B. thetaiotaomicron resulted from disruption of the recB homolog. Another mutant carried a transketolase gene inserted by Tn4351. There is no reasonable explanation why disruption of the transketolase gene caused a decrease of the MIC of norfloxacin for this organism, although Streptococcus pneumoniae RecP related to DNA recombination was reported to be transketolase.
Isolation of Escherichia coli O157:H7 from Dung Beetles Catharsius molossus
Authors: Xu Jianguo , Liu Qiyong , Jing Huaiqi , Pang Bo , Yang Jinchuan , Zhao Guangfa , Li Hongwei
Published: November 2013

In an epidemiological survey, Escherichia coli O157:H7 was isolated from the intestine 4 of 113 dung beetle Catharsius molossus captured below ground at Tongshan County, Jiangsu Province of China. In parallel, 10 strains of E. coli O157:H7 were isolated from fecal samples of 383 diarrhea patients from the same region. Most importantly, using pulsed field gel electrophoresis (PFGE) of chromosomal DNA restriction fragments and PCR method, we found that the PFGE pattern and virulence genes of beetle isolates were identical to those of the human isolates, such as Shiga‐toxins (stx) and enterohemorrhagic Escherichia coli hemolysin A (EHEC‐hlyA). Therefore, dung beetle might acquire pathogenic E. coli O157:H7 through contact with feces of domestic animals.
Individuals Exhibiting Conspicuous Nevi (Lentigo Simplex) Are Resistant to Allergic Rhinitis/Conjunctivitis (Pollinosis), but Those Who Do Not Show Increased Susceptibility to Pollinosis
Authors: Awaya Akira , Watanabe Ken‐ichi , Kato Shinsuke
Published: November 2013

We investigated 115 individuals in Tokyo from two groups of persons with conspicuous nevi on the head, neck, and arms, etc. (nevus‐positive group) and persons who do not exhibit such nevi (nevi‐negative group) interviewing them to determine whether they suffered from allergic rhinitis/conjunctivitis (pollinosis) and examining them for the presence and extent of nevi. There was a distinct correlation: only 5 persons among 85 individuals of the nevus‐positive group suffered from pollinosis, and all 30 people in the nevus‐negative group had pollinosis. Thus, individuals exhibiting conspicuous nevi are resistant to pollinosis, but those who do not show increased susceptibility to pollinosis.
Screening Method for Salmonella enterica Serovar Typhi and Serovar Paratyphi A with Reduced Susceptibility to Fluoroquinolones by PCR‐Restriction Fragment Length Polymorphism
Authors: Hirose Kenji , Tamura Kazumichi , Watanabe Haruo
Published: November 2013

Salmonella enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones (MICs of ciprofloxacin, 0.25 to 2 μg/ml) have a mutation at codon either Ser‐83 or Asp‐87 of gyrA gene. A screening method by PCR‐restriction fragment length polymorphism (PCR‐RFLP) was designed to screen the mutations at codon Ser‐83 and Asp‐87 of the gyrA gene of S. enterica serovar Typhi and serovar Paratyphi A clinical isolates. This method successfully screened the gyrA mutations of S. enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones.
Degradation Process of Mycobacterium leprae Cells in Infected Tissue Examined by the Freeze‐Substitution Method in Electron Microscopy
Authors: Amako Kazunobu , Takade Akemi , Umeda Akiko , Matsuoka Masanori , Yoshida Shin‐ichi , Nakamura Masahiro
Published: November 2013

Mycobacterium leprae cells (strain Thai‐53) harvested from infected mouse foot pads were examined by electron microscopy using the freeze‐substitution technique. The population of M. leprae cells from the infected tissue consisted of a large number of degraded cells and a few normal cells. These thin sectioned cell profiles could be categorized into four groups depending on the alteration of the membrane structures, and the degradation process is considered to occur in stages, namely from stages 1 to 3. These are the normal cells with an asymmetrical membrane, a seemingly normal cell but with a symmetrical membrane (stage 1), a cell possessing contracted and highly concentrated cytoplasm with a membrane (stage 2), and a cell that has lost its membrane (stage 3). The peptidoglycan layer was found to remain intact in these cell groups.
Disruption of the Human Pathogenic Yeast Candida albicans Catalase Gene Decreases Survival in Mouse‐Model Infection and Elevates Susceptibility to Higher Temperature and to Detergents
Authors: Nakagawa Yoshiyuki , Kanbe Toshio , Mizuguchi Ikuyo
Published: November 2013

Catalase‐deficient strains of the human pathogenic yeast Candida albicans were constructed using the URA‐blaster method. The disruptant was viable and grew normally in an ordinary culture condition, but became extremely sensitive to treatment with hydrogen peroxide. No catalase activity was observed in a catalase (CCT)‐gene‐disrupted strain, 1F5‐4‐1, suggesting that there were no other catalase or catalase‐like enzymes in this yeast. The disruptant was shown to be sensitive to higher temperature and to low concentrations of SDS, NP‐40, or Triton X‐100. After a wild‐type CCT gene was reintroduced into the disruptant, catalase activity was restored and the strain became moderately sensitive to treatment with hydrogen peroxide. However, neither the temperature sensitivity nor the susceptibility to SDS observed in the disruptant was restored in the CCT‐reintroduced strain. A model infection experiment using wild‐type and dCCT strains showed that the disruptants disappeared more rapidly than the wild‐type strain in mouse liver, lung, and spleen. These results suggest that the catalase plays a significant role in survival in the host immune system and thus leads this organism to establish infection in the host.
Chimeric Human/Murine Monoclonal IgM Antibodies to HIV‐1 Nef Antigen Expressed on Chronically Infected Cells
Authors: Kawai Masahiro , He Lianying , Kawamura Takeshi , Omoto Shinya , Fujii Yoichi R. , Okada Noriko
Published: November 2013

Human IgM antibody (Ab) to gangliosides induced cytolysis of HIV‐1‐infected cells by homologous human complement. We expected that any human IgM Ab reactive with HIV‐1 infected cells could cause complement‐mediated cytolysis. The trans‐chromosome mouse (TC mouse) contains human chromosomes harboring genes responsible for immunoglobulin production. Spleen cells from TC mice immunized with recombinant Nef were fused with mouse myeloma cells to generate hybridomas, and we selected those that produced human μ‐chain‐positive Abs reactive with Nef fixed on an ELISA plate. However, the L‐chain of the monoclonal Abs (mAbs) were murine λ in type and were chimeric, and we could not succeed in obtaining mAb with human μ‐ and human κ‐chains. The chimeric mAbs reacted with the HIV‐1 infected cells as seen with flow cytometric analysis, and the surface expression of Nef was also detectable on chronically infected OM10.1 cells which had no detectable gp120. However, although the reaction of the chimeric IgM mAb with HIV‐1‐infected MOLT4 cells induced C3 deposition on cell surfaces on incubation with fresh human serum, the cells remained unlysed, as determined by 51Cr release assay. The amount of Nef antigen on the cells might not have been high enough to overcome the function of HRF20 (CD59) that restricts formation of membrane attack complexes of homologous complement. However, combination of anti‐Nef IgM mAb with other IgM mAbs reactive with the surface of HIV‐1‐infected cells may induce a synergistic effect in complement mediated cytolysis.
The Urease Gene Cluster of Vibrio parahaemolyticus Does Not Influence the Expression of the Thermostable Direct Hemolysin (TDH) Gene or the TDH‐Related Hemolysin Gene
Authors: Nakaguchi Yoshitsugu , Okuda Jun , Iida Tetsuya , Nishibuchi Mitsuaki
Published: November 2013

In order to investigate why the thermostable direct hemolysin (TDH) and the TDH‐related hemolysin (TRH) of Vibrio parahaemolyticus are produced at low levels from urease‐positive strains, the effect of the functional urease gene cluster of V. parahaemolyticus on the expression of the tdh and trh genes was examined. Transcriptional lacZ fusions with the tdh1, tdh2, trh1 and trh2 genes representing variants of the tdh and trh genes were integrated into the chromosome of an Escherichia coli strain and a urease‐negative V. parahaemolyticus strain. The plasmid‐borne urease gene cluster introduced and expressed in these constructs did not affect expression of any of the fusion genes. The amount of TDH produced from a Kanagawa phenomenon‐positive V. parahaemolyticus did not change by introduction of the urease gene cluster either. It was concluded therefore that the urease gene cluster is not involved in the regulation of tdh and trh expression.
An Effective Sporicidal Reagent against Bacillus subtilis Spores
Authors: Kida Nori , Mochizuki Yasushi , Taguchi Fumiaki
Published: November 2013

We developed a reagent which showed significant sporicidal activity against Bacillus subtilis spores. This reagent was composed of ethylenediaminetetraacetic acid, disodium salt (EDTA‐2Na), ferric chloride hexahydrate (FeCl3 · 6H2O) and ethanol (tentatively designated as the ethanol reagent). The ethanol reagent showed pH‐ and temperature‐dependent sporicidal activity. At pH 0.3, its activity was almost the same as that of 0.05% sodium hypochlorite at 20 C and was higher at 37 C than at 20 C. The activity of the ethanol reagent was similar both with and without 10% serum. The ethanol reagent might be applicable for disinfecting Bacillus spores.
Preferential Detection of Pro‐Carboxypeptidase R by Enzyme‐Linked Immunosorbent Assay
Authors: Tani Sayuri , Akatsu Hiroyasu , Ishikawa Yasushige , Okada Noriko , Okada Hidechika
Published: November 2013

We generated two monoclonal antibodies (mAbs), 2A16 and 10G1, against pro‐carboxypeptidase R (proCPR), also known as thrombin activatable fibrinolysis inhibitor (TAFI). By use of these mAbs, we developed a sandwich enzyme‐linked immunosorbent assay (ELISA) system to detect proCPR. Since the amount of the antigen detectable by the ELISA was essentially the same in fresh plasma and serum incubated at 37 C for 1 hr, we concluded that the ELISA system detected not only proCPR, but also inactivated CPR generated from proCPR. However, an appreciable amount of proCPR remained unactivated in serum. For extensive activation of proCPR in plasma, thrombin and thrombomodulin complexes (T‐TM) can be used together with CaCl2. Following extensive conversion of proCPR to CPR by T‐TM and CaCl2, converting plasma to serum (T‐TM serum), antigenicity became undetectable by ELISA. Further analysis revealed that 2A16 reacts only with proCPR although 10G1 reacts with proCPR, active CPR and inactivated CPR. Therefore, we concluded that the ELISA system preferentially detects proCPR and not CPR. Our sandwich ELISA system utilizing 2A16 and 10G1 provides a suitable method for detecting proCPR and can be used to determine levels of proCPR in plasma samples from patients.
Morphology of the Type Strain of Bacillus anthracis EY 3169T=ATCC 14578T Grown Either Aerobically or Anaerobically on Agar Plates —Observation by Light and Laser Microscopes—
Authors: Yabuuchi Eiko , Koseki Masayuki
Published: November 2013

Growth characteristics including cell‐arrangement of the type strain of Bacillus anthracis EY 3169T=ATCC 14578T grown on agar plates in level 3 laboratory were observed by both light and laser microscopes. Small daughter colonies appeared on parent colonies grown on 5% sheep blood or chocolate agar plates after 12 days incubation at room temperature. Daughter colonies, stained by Wirtz‐Conklin method, were composed with vegetative cells and spores. Growth of daughter colonies might be supported by the debris of cells in the parent colony. Colonies grown under anaerobic conditions were flat with smooth edges, and the cells neither formed chains of any length, nor produced any spores after 25 days incubation at room temperature. It was thought that spores of B. anthracis were produced at the terminal stage of individual cell life instead of under unfavorable conditions for the organism. Air is needed for spore formation and cell‐chain formation. More nutrients, probably amino acids, are needed for anaerobic growth rather than aerobic.
Hepatitis C Virus Nonstructural Protein NS3 Binds to Sm‐D1, a Small Nuclear Ribonucleoprotein Associated with Autoimmune Disease
Authors: Iwai Atsushi , Hasumura Yasushi , Nojima Takayuki , Takegami Tsutomu
Published: November 2013

Hepatitis C virus (HCV) causes a persistent infection, chronic hepatitis, and leads to hepatocellular carcinoma. Nonstructural protein 3 (NS3) of HCV possesses protease, nucleoside triphosphatase, and helicase activities. Using the yeast two hybrid assay, we identified Sm‐D1, a host protein that binds to NS3. Sm‐D1 is a component of small nuclear ribonucleoprotein (snRNP) complexes which are associated with autoimmune disease. Sm‐D1 has Gly‐Arg (GR) repeats at the C terminus, which contains dimethylarginine modified by post‐translational modification and may constitute an immunoreactive determinant. Deletion mutants revealed that the C‐terminal region of Sm‐D1 containing the GR repeats was the binding region for NS3, and the expression feature of Sm‐D1 was affected by co‐expression of NS3. Immunostaining assay demonstrated that NS3 was also present in the nucleus of cells overexpressing Sm‐D1, although it was usually found in cytoplasm. The localization of NS3 could change following interaction with Sm‐D1 and affect the function of Sm‐D1.
Improvements of PCR‐Based Identification Targeting the DNA Topoisomerase II Gene to Determine Major Species of the Opportunistic Fungi Candida and Aspergillus fumigatus
Authors: Kanbe Toshio , Arishima Takuro , Horii Toshinobu , Kikuchi Akihiko
Published: November 2013

For the simple and rapid detection/identification of major pathogenic fungal species such as Candida albicans, C. tropicalis, C. parapsilosis, C. glabrata and Aspergillus fumigatus, common primers for these species and specific primers for each species, designed on the basis on the genomic nucleotide sequences of the DNA topoisomerase II genes, were prepared and tested for their specificities in PCR amplifications. Twelve specific primers were pooled and designated PsVI. Genomic DNAs were amplified by the common primer pair, and followed by PCR amplification using PsVI. Using PsVI, six unique DNA fragments, all of which corresponded to a Candida or A. fumigatus species, were specifically and acceptably amplified from each template DNA even in the presence of other DNAs. Similarly, the results of identification of clinical samples based on the PCR amplification coincided with those of conventional identification techniques. The sensitivities of the direct PCR and the nested PCR using PsVI were found to be 1,000 and 50 yeast cells, respectively.
Herpes Simplex Virus UL14 Protein Blocks Apoptosis
Authors: Yamauchi Yohei , Daikoku Tohru , Goshima Fumi , Nishiyama Yukihiro
Published: November 2013

We report that an HSV‐2 UL14 protein expressing cell line (14/HEp‐2) was more resistant to apoptosis induced by osmotic shock and certain drugs than its parental cell line. Furthermore, HSV‐1 UL14 protein deletion virus (UL14D) showed weaker inhibition of apoptosis compared to the rescued virus UL14R. The protein' s anti‐apoptotic function may derive from its heat shock protein‐like properties.
Depolymerization of Actin Filament by Cytochalasin E Induces Interleukin‐8 Production and Up‐Regulates CD54 in the HeLa Epithelial Cell Line
Authors: Ikewaki Nobunao , Yamada Akira , Inoko Hidetoshi
Published: November 2013

We previously reported that the depolymerization of actin filament by cytochalasin E enhances low affinity Fcε receptor II (CD23) expression on the human monocyte‐like cell line, U937 (J. Clin. Immunol. 20: 235, 2000). In this study, we found that cytochalasin E strongly induces interleukin‐8 through an epithelial cell line, HeLa, in dose‐ and time‐dependent manners as assessed by enzyme‐linked immunoassay and reverse transcription‐polymerase chain reaction techniques. In addition, interleukin‐8 production in the HeLa cells cultured with cytochalasin E was blocked in the presence of protein kinase C inhibitors, Go6976 and H‐7. On the other hand, it was found that CD54 (intercellular adhesion molecule‐1; ICAM‐1) expression on the HeLa cells and the secretion of soluble CD54 were significantly up‐regulated after culturing with cytochalasin E, and that these up‐regulations of CD54 were also suppressed by Go6976. Taken together, these findings indicate that cytochalasin E activates protein kinase C under the depolymerization of actin filament, leading to the induction of interleukin‐8 production and the up‐regulation of CD54 in HeLa cells.
Genetic and Immunological Analysis of a Novel Variant of Shiga Toxin 1 from Bovine Escherichia coli Strains and Development of Bead‐ELISA to Detect the Variant Toxin
Authors: Ohmura‐Hoshino Mari , Ho Soon‐Tae , Kurazono Hisao , Igarashi Kazuei , Yamasaki Shinji , Takeda Yoshifumi
Published: November 2013

A novel variant of Shiga toxin 1 (Stx1) was identified from bovine Escherichia coli strains. The stx1 variant genes designated as stx1v51 and stx1v52 were cloned and sequenced. The two variant genes differed each other by 2 bp, but the deduced amino acid sequences of the two Stx1 variant toxins were the same and had 94% and 92% homology to that of prototype A and B subunits of Stx1, respectively. The variant toxin designated as Stx1v52 was purified to homogeneity. Although inhibition of protein synthesis in vitro by purified Stx1v52 was almost equal to that of purified Stx1, Vero cell cytotoxicity and mouse lethality of Stx1v52 were several folds lower than those of prototype Stx1. In Ouchterlony double gel diffusion test, the precipitin line between Stx1v52 and Stx1 formed a spur against anti‐Stx1 serum but was fused against anti‐Stx1v52 serum. Stx1v52 and Stx1v52‐specific‐bead‐ELISA was developed, and both Stx1 and Stx1v52 could be detected with high sensitivity using Stx1v52 conjugate. However, Stx1v52 but not Stx1 could be detected with Stx1v52‐specific bead‐ELISA.
Electron Microscopic Observations of Orientia tsutsugamushi in Salivary Gland Cells of Naturally Infected Leptotrombidium pallidum Larvae during Feeding
Authors: Kadosaka Teruki , Kimura Eisaku
Published: November 2013

We performed a detailed electron microscopic observation on the escaping process of Orientia tsutsugamushi from the salivary gland cells of naturally infected trombiculid larvae into the acinar lumen of the gland during feeding on mice. In unfed larvae, many O. tsutsugamushi were intermingled with secretory granules in the cytoplasm of the salivary gland cell. O. tsutsugamushi was neither found in the acinar lumen nor observed escaping from the apical surface of the gland cell. In contrast, in the larvae fed on mice, many O. tsutsugamushi were observable in the acinar lumen. They were enveloped with the host glandular cell membrane. In salivary gland cells, secretory granules changed the distribution and accumulated in the apical region. In such cells, the majority of O. tsutsugamushi were found at the base of the cell. Some O. tsutsugamushi were pushing the glandular cell membrane outward in various degrees, showing different stages of escape. These findings suggest that larval feeding induced O. tsutsugamushi escape from salivary gland cells, that the escape was by budding, during which O. tsutsugamushi were enveloped in the host cell membrane, and that O. tsutsugamushi would be injected into the mouse skin as a mixture with mite saliva. The study also revealed the presence of many small vesicles that had the same cell wall structure as O. tsutsugamushi in the cytoplasm of the salivary gland cell. Most of them seemed to be products from degenerated Orientia.
A Novel Type of Two‐Component Regulatory System Affecting Gingipains in Porphyromonas gingivalis
Authors: Hasegawa Yoshiaki , Nishiyama So‐ichiro , Nishikawa Kiyoshi , Kadowaki Tomoko , Yamamoto Kenji , Noguchi Toshihide , Yoshimura Fuminobu
Published: November 2013

We surveyed the Porphyromonas gingivalis W83 genome database for homologues of FimS, the first two‐component sensor histidine kinase, which could possibly control virulence factors. Including fimS, we found six putative sensor kinase genes in the genome. The gene encoding one of the homologues was cloned from a P. gingivalis plasmid library, sequenced, and analyzed using its mutants. Two gene‐disruption mutants were created in strain ATCC 33277 by introducing a drug cassette into the gene. The mutants formed nonpigmented colonies, indicating that they might be defective in proteinase production, a characteristic of this organism. Proteinase activities, measured as arginine‐ and lysine‐specific (Rgp and Kgp gingipains, respectively) activities, of the mutants were almost half those of the parent strain. Unlike the parent and wild‐type strains, most of the gingipain activities were detected in the culture supernatant, not in cells, of the mutants. Abnormal production of gingipains was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analyses. These results strongly suggest that this newly‐discovered two‐component sensor kinase is involved in maturation and proper localization of gingipains to the outer membrane through an unknown mechanism. The gene encoding the sensor histidine kinase was designated gppX, which represents regulation (X) of gingipains and black pigmentation in P. gingivalis.
Quantities of Interleukin‐12p40 in Mature CD8α Negative Dendritic Cells Correlate with Strength of TCR Signal and Determine Th Cell Development
Authors: Fujimoto Noriki , Ishida Hideaki , Nakamura Ichiro , Ogasawara Kazumasa , Itoh Yasushi
Published: November 2013

Strength of T cell antigen receptor (TCR) signaling drives the development of Th1 and Th2 subsets from naive T helper precursors. The quantity of interleukin‐12 (IL‐12) from antigen presenting cells (APC) is also profoundly involved in Th development. TCR signal strength and IL‐12 production from dendritic cells (DCs) are linked by CD40 ligand (CD40L) expression on activated T cells. CD40L on the activated T cells interacts with CD40 on DC, resulting in induction of IL‐12 from DCs. However, the subsets of DC in spleen that produce the IL‐12 have not been clearly identified. Purification of DC subsets itself may provide maturation signals to immature DCs. Thus, we used non‐purified mouse spleen cells to analyze IL‐12 producing cells, near to steady states, during the interaction of naive T cells either with or without agonist. Mature CD86highCD8α−DCs in spleen mainly produced IL‐12p40 after stimulation of high dose agonist. The ratio of CD40L positive T cells and IL‐12p40 secreting CD86highDCs correlated with the concentration of agonist and Th1 development. However, anti‐IL‐12 did not completely inhibit the Th1 development. Altogether, strength of TCR signaling directs Th cell development by regulating CD40L expression on T cells which determines production of IL‐12p40 from CD86highCD8α−DC via CD40.
Need to Differentiate Lethal Toxin‐Producing Strains of Burkholderia gladioli, Which Cause Severe Food Poisoning: Description of B. gladioli Pathovar cocovenenans and an Emended Description of B. gladioli
Authors: Jiao Zhenquan , Kawamura Yoshiaki , Mishima Noriko , Yang Ruifu , Li Na , Liu Xiumei , Ezaki Takayuki
Published: November 2013

Burkholderia cocovenenans produces a lethal toxin (Bongkrekic acid) that leads to high fatality in food poisoning cases. However, B. cocovenenans was combined in Burkholderia gladioli in 1999. B. gladioli was originally described as a phytopathogenic bacteria that sometimes causes pneumonia in humans and that acts as an opportunistic pathogen. We thought that it was clinically dangerous to describe these two species without considering their pathogenicities. From our data of 16S rRNA sequence analysis, DNA‐DNA hybridization, and fatty acid analysis, we could confirm that B. cocovenenans and B. gladioli should be categorized as a single species. However the species really weaved lethal toxin‐producing strains with non‐lethal strains. To emphasize that B. gladioli contains two different pathogens, we describe a new pathovar, B. gladioli pathovar cocovenenans, for the lethal toxin‐producing strains. We provide characteristics that differentiate this lethal toxin‐producing pathovar from other phytopathogenic pathovars within B. gladioli, together with an emended description of B. gladioli.
A Multi‐Prefectural Outbreak of Shigella sonnei Infections Associated with Eating Oysters in Japan
Authors: Terajima Jun , Tamura Kazumichi , Hirose Kenji , Izumiya Hidemasa , Miyahara Michiko , Konuma Hirotaka , Watanabe Haruo
Published: November 2013

Among roughly one thousand incidents of shigellosis annually in Japan, approximately 70% of the cases are estimated to be associated with overseas travel. However, at the end of 2001, reports of domestically acquired Shigella sonnei infections suddenly increased. We report here the first multi‐prefectural outbreak of Shigella sonnei infections linked to the consumption of imported oysters in Japan at the end of 2001. Isolates of S. sonnei from patients epidemiologically linked to eating contaminated oysters and from the imported oysters themselves showed an indistinguishable pulsed‐field gel electrophoresis pattern and drug resistance pattern.
Contribution of a Single Hydrogen Bond between βHis81 of MHC Class II I‐Ek and the Bound Peptide to the pH‐Dependent Thermal Stability
Authors: Saito Keigo , Oda Masayuki , Sarai Akinori , Azuma Takachika , Kozono Haruo
Published: November 2013

To determine the energetic contribution of the hydrogen bond between βHis81 of the major histocompatibility complex class II (MHC II) molecule, I‐Ek, and the bound hemoglobin peptide (Hb), we analyzed the thermal stability of the hydrogen bond‐disrupted mutant, I‐Ek‐Hb βH81Y, in which the βHis81 residue was replaced with Tyr, by differential scanning calorimetry. The thermal stability of the I‐Ek‐Hb βH81Y mutant was lower than that of the I‐Ek‐Hb wild‐type, mainly due to the decreased enthalpy change. The difference in the denaturation temperature of the I‐Ek‐Hb βH81Y mutant as compared with that of the I‐Ek‐Hb wild‐type at pH 5.5 was only slightly smaller than that at pH 7.4, in agreement with the increased stability at an acidic pH, a unique characteristic of MHC II. Thus, the hydrogen bond contributed by βHis81 is critical for peptide binding, and is independent of pH, which can alter the hydrophilicity of the His residue.
Toll‐Like Receptor 3: A Link between Toll‐Like Receptor, Interferon and Viruses
Authors: Matsumoto Misako , Funami Kenji , Oshiumi Hiroyuki , Seya Tsukasa
Published: November 2013

Production of type I interferon (IFN‐α/β) by virus‐infected cells is the central event in their antiviral immune responses. In mammalian cells, IFN‐α/β gene transcription is induced through distinct signaling pathways by viral infection or by treatment with double‐stranded (ds) RNA, which is an intermediate of virus replication. Toll‐like receptor 3 (TLR3) was found to recognize dsRNA and transmit signals to activate NF‐κB and the IFN‐β promoter. Recent identification of the TLR3‐adaptor protein and its downstream signaling molecules, which are involved in IFN‐α/β production, revealed a novel IFN‐inducing pathway for an anti‐viral immune response. Here, we summarize the current knowledge of TLR3‐mediated immune responses.
Nocardia testaceus Sp. Nov. and Nocardia senatus Sp. Nov., Isolated from Patients in Japan
Authors: Kageyama Akiko , Yazawa Katsukiyo , Nishimura Kazuko , Mikami Yuzuru
Published: November 2013

Two actinomycete strains isolated from sputum between 1999 and 2001 in Japan were provisionally assigned to the genus Nocardia based on morphological criteria. These isolates were further studied in order to determine their specific taxonomic status. Detailed chemotaxonomic characterization and 16S rDNA gene sequence analysis of these isolates also confirmed that they belong to the genus Nocardia. The 16S rDNA sequence data of the two strains showed that they are most similar to that of Nocardia carnea and Nocardia flavorosea. However, DNA‐DNA relatedness data showed that the two strains could be distinguished from N. carnea and N. flavorosea and therefore represented two new species within the genus Nocardia. The designation of the two isolated strains are Nocardia testaceus for IFM 0937T (=JCM 12235T, DSM 44765T) and Nocardia senatus for IFM 10088T (=JCM 12236T, DSM 44766T).
Expression of Equine Interleukin‐18 by Baculovirus Expression System and Its Biologic Activity
Authors: Wu Donglai , Murakami Kenji , Liu Nihong , Konishi Misako , Muneta Yoshihiro , Inumaru Shigeki , Kokuho Takehiro , Sentsui Hiroshi
Published: November 2013

The equine interleukin‐18 (IL‐18) cDNA that contains the coding sequence was cloned and a recombinant baculovirus, named AcEIL‐18, was constructed. The recombinant protein of the equine IL‐18 was expressed by AcEIL‐18 and its expression was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and Western blotting. Insect cells infected with AcEIL‐18 secreted a precursor IL‐18 with 24 kilo dalton (kDa) into the culture supernatant. Western blot analysis showed that mature equine IL‐18 about 18 kDa was also confirmed without co‐expression of caspase‐1. Culture supernatant from AcEIL‐18 infected cells showed a synergistic effect with recombinant human lnterleukin‐12 for induction of interferon‐γ gene expression in equine peripheral mononuclear cells, indicating that the recombinant equine IL‐18 expressed in this study also has biological activity without any treatment.
Development and Evaluation of a Rapid Flow‐Through Immuno Filtration Test Using Recombinant Filarial Antigen for Diagnosis of Brugian and Bancroftian Filariasis
Authors: Baskar Lakshmi Kanthan Vijaya , Srikanth Tokuluva Ramalingam , Suba Salahudeen , Mody Himansu Chirag , Desai Pradeep Kumar , Kaliraj Perumal
Published: November 2013

There is an imperative need to develop a rapid antibody test that can be used for diagnosis of clinical cases in travelers and expatriates, primary surveillance in areas of unknown endemicity, detection of early infection in childhood and for monitoring chemotherapeutic programs. A rapid‐format, simple and qualitative flow through immuno filtration test has been developed for the identification of total IgG antibodies to recombinant filarial antigen WbSXP‐1. This test system employs colloidal gold‐protein A reagent as the antibody capture reagent. The sensitivity and specificity of the test was evaluated in a total of 1,230 serum samples. The sensitivity of the test was found to be 90.8% with brugian (n=70) and 91.4% with bancroftian (n = 140) microfilaraemic subjects. The test showed minimum reactivity (4/10) with Loa loa microfilaria (MF) positive sera and no reactivity (0/20) with Onchocerca MF positive sera. This rapid diagnosis is found to be non‐reactive with individuals having other parasitic diseases including schistosomiasis (n = 10), soil‐transmitted helminthiases (n=34) and protozoan infections (n=33) indicating the potential of this test as a prospective method of diagnosis for both brugian and bancroftian lymphatic filariasis. Stability kinetics was studied at different temperatures and different time periods. The rapid flow‐through immuno filtration test is advantageous since it can be stored at room temperature, is user friendly and is particularly applicable in the field as an initial screening method, for epidemiological monitoring of filarial infections in bancroftian and brugian endemic regions of the world.
Analysis of Major Virulence Factors in Porphyromonas gingivalis under Various Culture Temperatures Using Specific Antibodies
Authors: Murakami Yukitaka , Masuda Takashi , Imai Masashi , Iwami Jun , Nakamura Hiroshi , Noguchi Toshihide , Yoshimura Fuminobu
Published: November 2013

Porphyromonas gingivalis is implicated in the occurrence of adult periodontitis. We have previously identified major outer membrane proteins from P. gingivalis, which include representative virulence factors such as gingipains, a 75 kDa major protein, RagA, RagB, and putative porin. Fimbriae, another important virulence factor, exist on the cell surface. In this study, we identified major supernatant proteins. They were fimbrilin, the 75 kDa major protein, gingipains and their adhesin domains. Microscopic examination showed that supernatant proteins formed vesicle‐like and fimbrial structures. To learn more about the character of this bacterium, we examined effects of growth temperature on localization and expression of these virulence factors. In general, localization of major virulence factors did not change at the various growth temperatures used. Most of the 75 kDa major protein, RagA, RagB, and putative porin were found in the envelope fraction, not in cell‐free culture supernatant. Gingipains were found in both the envelope fraction and supernatant. More than 80% of fimbriae were associated with cells, less than 20% migrated to the supernatant. Most fimbriae existed in the whole cell lysate, although there was a small amount in the envelope fraction. When the growth temperature was increased, expression of fimbriae, gingipains, the 75 kDa major protein, RagA, and RagB decreased. However, temperature had almost no effect on expression of putative porin. The tendency for expression of major virulence factors to decrease at higher temperatures may enable P. gingivalis to survive under hostile conditions.
The Participation of Nitric Oxide in Peritoneal Exudate Cell Cytotoxicity of Mice by Fusobacterium nucleatum
Authors: Kato Chihomi , Mikami Masato , Saito Kazuko
Published: November 2013

Previously we reported that mice infected recurrently with live Fusobacterium nucleatum (Fn) synthesize a significant amount of NO between 12 hr and 24 hr after Fn injection. Fn is a gram‐negative rod periodontal pathogen. NO could not be induced by heat‐killed Fn or in untreated mice. This NO, derived from the iNOS after infection of live Fn, was not involved in the Fn reduction because Fn clearance occurs within 6 hr. We investigated in this study whether this NO was involved in cytotoxicity in peritoneal exudate cells (PEC) in vivo. The mice were divided into two groups: those treated with live Fn (immune) and those left untreated (normal). PEC number, NO production, detection of apoptosis or death cells, and lactate dehydrogenase (LDH) release activity after injection of live Fn were compared in these groups. In the immune group, the increase of the total cell numbers caused by an increase in neutrophils, a significant NO production only after injection of live Fn at 24 hr and identification of iNOS positive macrophages were confirmed. The apoptotic rate was very low and did not increase at 24 hr in vivo. Therefore, apoptosis was seldom relevant to the NO. In the immune group, LDH activity was remarkable high at 24 hr, and dead cells and macrophages phagocytizing cell fragments increased at the same time. Pretreatment of L‐NMMA, an inhibitor of iNOS, suppressed LDH activity and cell death. Therefore, the NO derived from the iNOS is involved in the cytotoxicity. These results suggest that NO may contribute to the inflammatory response during Fn infection in periodontitis.
Acetate‐Mediated Production of Orotic Acid by ura3 Mutants of Candida albicans
Authors: Nezu Takashi , Shimokawa Osamu
Published: November 2013

Candida albicans ura3 mutants were found to produce large amounts of orotic acid when the growth medium was supplemented with sodium acetate. Experiments with 13C‐labeled acetate showed that the acetate served as a precursor of orotic acid. This system of acetate‐mediated production of orotic acid is similar to other documented microbial producers in yield but unique for its acetate requirement.
Genotype Analysis of Malassezia restricta as the Major Cutaneous Flora in Patients with Atopic Dermatitis and Healthy Subjects
Authors: Sugita Takashi , Tajima Mami , Amaya Misato , Tsuboi Ryoji , Nishikawa Akemi
Published: November 2013

Lipophilic yeasts of the genus Malassezia colonize the skin surface of humans and are an exacerbating factor in atopic dermatitis (AD). Two species, M. restricta and M. globosa are major cutaneous microflora in both AD patients and healthy subjects. We compared the DNA sequences of the intergenic spacer (IGS) region, located between the 26S and 5S rRNA genes of M. restricta colonizing the skin surfaces of 13 AD patients and 12 healthy subjects, and of three CBS stock strains as references. The IGS 1 sequences were divided into two major groups, corresponding to AD patients and healthy subjects. These findings suggest that a specific genotype of M. restricta plays a significant role in AD, although M. restricta commonly colonizes both AD patients and healthy subjects.
Variation of Toxigenic Vibrio cholerae O1 in the Aquatic Environment of Bangladesh and Its Correlation with the Clinical Strains
Authors: Islam Mohammad Sirajul , Talukder Kaisar Ali , Khan Nurul Huda , Mahmud Zahid Hayat , Rahman Mohammed Ziaur , Nair Gopinath Balakrish , Siddique Abul K.M. , Yunus Mohammad , Sack David Allen , Sack Richard Bradley , Huq Anwar , Colwell Rita R.
Published: November 2013

The diversity of toxigenic V. cholerae O1 in the aquatic environment of Bangladesh is not known. A total of 18 environmental and 18 clinical strains of toxigenic V. cholerae O1 were isolated simultaneously from four different geographical areas and tested for variation by the pulsed‐field gel electrophoresis method. Environmental strains showed diversified profiles and one of the profiles was common to some environmental strains and most clinical strains. It appears that one clone has an advantage over others to cause disease. These findings suggest that the study of the molecular ecology of V. cholerae O1 in relation to its environmental reservoir is important in identifying virulent strains that cause disease.
Rapid Identification of Borrelia burgdorferi Sensu Lato by PCR‐Restriction Fragment Length Polymorphism Analysis of groEL Gene
Authors: Park Hyo‐Soon , Lee Jung‐Hee , Jeong Eun‐Ju , Park Tae‐Kyu , Jang Won‐Jong , Park Kyung‐Hee , Kim Bum‐Joon , Kook Yoon‐Hoh , Lee Seung‐Hyun
Published: November 2013

Thirty‐one reference strains and 23 Korean isolates of the genus Borrelia were identified through the PCR‐RFLP analysis using the groEL gene. This will be useful for the rapid differentiation of B. burgdorferi sensu lato and complements one of the 5S–23S intergenic spacers.
Nocardia aobensis Sp. Nov., Isolated from Patients in Japan
Authors: Kageyama Akiko , Suzuki Shin‐ichi , Yazawa Katsukiyo , Nishimura Kazuko , Kroppenstedt Reiner M. , Mikami Yuzuru
Published: November 2013

Five clinical isolates, strains IFM 0137, 0372T, 0496, 0556, and 0952, were provisionally assigned to the genus Nocardia based on morphological criteria. Nearly complete 16S rDNA sequences were determined for these strains. These data showed that they are most similar to that of Nocardia africana, Nocardia cerradoensis and Nocardia veterana. However, DNA‐DNA relatedness data showed that the five strains were of a single species and were distinguishable from N. africana, N. cerradoensis and N. veterana. Therefore, these strains represent a new species within the genus Nocardia. The designation of these five strains is Nocardia aobensis sp. nov. The type strain is IFM 0372T (=NBRC 100429T=JCM 12352T=DSM 44805T).
Further Studies on the Hyperphosphorylated Form (p40) of the Rabies Virus Nominal Phosphoprotein (P)
Authors: Toriumi Harufusa , Eriguchi Yoshiro , Takamatsu Fumihiko , Kawai Akihiko
Published: November 2013

We investigated possible mechanisms involved in production of a hyperphosphorylated form (p40) of rabies virus P protein, to which two dimensional (2‐D) gel electrophoresis was applied. The P gene products produced in Escherichia coli cells could be detected as a single spot of unphosphorylated 37‐kDa form (termed as p37–0) in a 2‐D gel. The 37‐kDa proteins in the virus‐infected cells are composed of some phosphorylated forms, including a major p37–1 and more phosphorylated minor forms (e.g., p37–2, p37–3, etc.), but little p37–0 is detected (Eriguchi et al., 2002). When the E. coli‐produced P protein analogues were incubated with BHK‐21 cell lysates, heparin‐sensitive phosphorylation occurred as described previously (Takamatsu et al., 1998), giving an additional 40‐kDa spot. However, such a p40‐like derivative displayed a little more basic pI value than that of the authentic p40 produced in the infected cells; hence, the former was termed p40–0 (pI=4.78), while the latter, p40–1 (pI=4.73). In contrast, p40 produced in the P cDNA‐transfected animal cell was detected at the p40–1 position. In addition, staurosporine did not affect the p40–1 production in virus‐infected nor the P cDNA‐transfected animal cells, while the agent reduced production of hyperphosphorylated forms of p37, resulting in accumulation of p37–1, but not of p37–0. These results suggest that, although p37–0 may become a substrate for the heparin‐sensitive protein kinase (PK) in vitro, only p37–1 is a substrate for p40 production catalyzed by heparin‐sensitive PK in animal cells, and staurosporine‐sensitive PK is involved in the production of more phosphorylated forms of p37, but not in p37–1 production from p37–0.
Characterization of Mechanisms of Fluconazole Resistance in a Candida albicans Isolate from a Japanese Patient with Chronic Mucocutaneous Candidiasis
Authors: Kamai Yasuki , Maebashi Kazunori , Kudoh Michinari , Makimura Koichi , Naka Wataru , Uchida Katsuhisa , Yamaguchi Hideyo
Published: November 2013

We examined the mechanisms of fluconazole resistance in a fluconazole‐resistant Candida albicans isolate from a Japanese patient with chronic mucocutaneous candidiasis. It was demonstrated that the highly resistant phenotype of this strain was associated with combined mechanisms of the energy‐dependent reduced intracellular accumulation of fluconazole, presumably due to the increased expression of the ATP‐binding cassette efflux pump CDR gene(s), and the reduced affinity of the target enzyme, Erg11p, to fluconazole. In particular, the reduced affinity of Erg11p was considered to contribute largely to the fluconazole resistance in the TIMM3209 strain. Biochemical studies indicated that the Erg11p from the TIMM3209 strain showed reduced susceptibility both to fluconazole and itraconazole of cell‐free ergosterol biosynthesis, and cytochrome P‐450 also showed reduced affinity to fluconazole in the carbon monoxide‐cytochrome P‐450 complex formation assay. We identified two amino acid substitutions, Y132H and G448V, in Erg11p from the TIMM3209 strain. We found that the cytochrome P‐450 from the TIMM3209 strain decayed during incubation at 37 C without fluconazole although it is unknown whether or not the phenomenon is linked to the resistant phenotype. These mutations are thought to confer the above‐mentioned characteristics to Erg11p.