bluuebunny/crossref_metadata_2025 huggingface dataset:
Morphological Properties of a Human Intestinal Spirochete First Isolated from a Patient with Diarrhea in Japan
Authors: Tasu Chollada , Nakamura Shin‐ichi , Tazawa Hideki , Hara Hiromichi , Adachi Yoshikazu
Published: November 2013

A human intestinal spirochete isolated from a rectal biopsy specimen was morphologically characterized. The isolate was comma‐shaped, 3–6 μm in length, 0.2 μm in diameter and had tapered ends. The surface layer, external to the outer envelope, was amorphous. Four string‐like periplasmic flagella with a diameter of 20 nm were presented at each end of the SDS‐treated cells. Thin sections of the bacterial cell revealed a high‐density cytoplasmic membrane and flagella in the periplasmic space between the cytoplasmic membrane and the outer envelope. Three segments of equal length were observed in some of the cells, while other cells were bi‐segmented and more frequently observed.
Induction of Protective Immunity by Primed B‐1 Cells in Toxoplasma gondii‐Infected B Cell‐Deficient Mice
Authors: Chen Mei , Mun Hye‐Seong , Piao Lian‐Xun , Aosai Fumie , Norose Kazumi , Mohamed Rabie M. , Belal Usama S. , Fang Hao , Ahmed Azza K. , Kang Hyun‐Kyu , Matsuzaki Goro , Kitamura Daisuke , Yano Akihiko
Published: November 2013

We examined the role of B‐1 cells in protection against Toxoplasma gondii infection using B cell‐deficient mice (μMT mice). We found that primed but not naïve B‐1 cells from wild‐type C57BL/6 mice protected B cell‐deficient recipients from challenge infection. All μMT mice transferred with primed B‐1 cells survived more than 5 months after T. gondii infection, whereas 100% of μMT mice transferred with naïve B‐1 cells succumbed by 18 days after infection. Additionally, high expression of both T help (Th) 1‐ and Th2‐type cytokines and a high level of nitric oxide production were observed in T. gondii‐infected μMT mice transferred with primed B‐1 cells. Thus, it was clearly demonstrated that B‐1 cells play an important role in host protection against T. gondii infection in μMT mice.
Moenomycin‐Resistance Is Associated with Vancomycin‐Intermediate Susceptibility in Staphylococcus aureus
Authors: Nishi Hiromi , Komatsuzawa Hitoshi , Yamada Sakuo , Fujiwara Tamaki , Ohara Masaru , Ohta Kouji , Sugiyama Masaru , Ishikawa Takenori , Sugai Motoyuki
Published: November 2013

We have previously isolated a vancomycin‐intermediate susceptibility mutant from methicillin‐resistant Staphylococcus aureus (MRSA) strain COL, and demonstrated the increased glycan‐chain length and the decreased moenomycin‐susceptibility. To further investigate the relationship between the resistance to vancomycin and to moenomycin, we isolated moenomycin‐resistant mutants (4–16 fold higher compared to the parent) from 5 MRSA and 2 methicillin‐sensitive S. aureus (MSSA) strains. The MRSA mutants showed a decreased susceptibility to vancomycin (2–4 fold), teicoplanin (2–4 fold) and an increased susceptibility to methicillin (2–8 fold). MSSA strains also showed similar results with those of MRSA strains except that there was no alteration of methicillin susceptibility. Among the mutants, three mutants including two MRSA mutants and one MSSA mutant were analyzed by electron microscopy, and they showed thickened cell walls compared to those of the parents. The glycan‐chain length of the peptidoglycan of the mutant was shown to be slightly longer than that of the parent, but the muropeptide profile was very similar. The expression levels of all PBPs were similar to those of the parent. Furthermore, the nucleotide sequences of sgtA, sgtB and pbp2 in the mutant were identical to those of the parent. These results indicate that the moenomycin‐resistance is closely associated with vancomycin‐intermediate susceptibility in S. aureus.
Serological Identification of Endothelial Antigens Predominantly Recognized in Kawasaki Disease Patients by Recombinant Expression Cloning
Authors: Kaneko Miho , Ono Toshiro , Matsubara Tomoyo , Yamamoto Yukiyo , Ikeda Hitoshi , Yoshiki Takashi , Furukawa Susumu , Nakayama Eiichi
Published: November 2013

We showed IgG immune response against endothelial antigens in sera obtained from convalescent Kawasaki disease (KD) patients after recovery from the disease during the follow‐up period using serological analysis of recombinant cDNA expression library (SEREX) methodology. We identified 46 antigens represented by 69 clones by immunoscreening of a cDNA expression library from tumor necrosis factor‐α (TNFα) treated human umbilical vein endothelial cells (HUVEC) with sera from 4 KD patients. They included ubiquitin pathway proteins, transcriptional factors, signal transduction molecules, metabolic enzymes, cytoskeletal proteins, an adhesion molecule, and a cell cycle protein. By serological survey using phage plaque assay, sera from 5 non‐KD patients were rarely reactive with the antigens. Among the antigens, tropomyosin was most frequently isolated (18 of 69 clones). Seventeen of the 18 clones were identified using KD3 serum. The second most frequently isolated antigen was also a cytoskeletal protein, called T‐plastin (3 of 69 clones).
On the Cyto‐Toxicity Caused by Quantum Dots
Authors: Shiohara Amane , Hoshino Akiyoshi , Hanaki Ken‐ichi , Suzuki Kazuo , Yamamoto Kenji
Published: November 2013

Quantum dots (QDs) such as CdSe QDs have been introduced as new fluorophores. The QDs conjugated with antibody are starting to be widely used for immunostaining. However there is still not sufficient analysis of the toxicity of QDs in the literature. Therefore we evaluated the cell damage caused by the quantum dots for biological applications. We performed cell viability assay to determine the difference in cell damage depending on the sizes and colors of mercapto‐undecanoic acid (MUA) QDs and the cell types. The results showed that the cell viability decreased with increasing concentration of MUA‐QDs. But in the case of Vero cell (African green monkey's kidney cell) with red fluorescence QD (QD640), the cell damage was less than for the others. Furthermore through the flow cytometry assay we found that this cell damage caused by MUA‐QD turned out to be cell death after 4‐6‐hr incubation. From the two assays described above, we found that there is a range of concentration of MUA‐QDs where the cell viability decreased without cell death occurring and thus we conclude that attention should be given when MUA‐QDs are applied to living organisms even in low concentrations.
Molecular Characterization of Dextranase from Streptococcus rattus
Authors: Igarashi Takeshi , Morisaki Hirobumi , Goto Nobuichi
Published: November 2013

The complete nucleotide sequence of the dextranase gene of Streptococcus rattus ATCC19645 was determined. An open reading frame of the dextranase gene was 2,760 bp long and encoded a dextranase protein consisting of 920 amino acids with a molecular weight of 100,163 Da and an isoelectric point of 4.67. The S. rattus dextranase purified from recombinant Escherichia coli cells showed dextran‐hydrolyzing activity with optimal pH (5.0) and temperature (40 C) similar to those of dextranases from Streptococcus mutans and Streptococcus sobrinus. The deduced amino acid sequence of the S. rattus dextranase revealed that the dextranase molecule consists of two variable regions and a conserved region. The variable regions contained an N‐terminal signal peptide and a C‐terminal cell wall sorting signal; the conserved region contained two functional domains, catalytic and dextran‐binding sites. This structural feature of the S. rattus dextranase is quite similar to that of other cariogenic species such as S. mutans, S. sobrinus, and Streptococcus downei.
Down‐Regulation of gp96 by Orientia tsutsugamushi
Authors: Cho Nam‐Hyuk , Choi Cheol‐Young , Seong Seung‐Yong
Published: November 2013

gp96 plays a central role in innate as well as acquired immunity, maturation and chemotaxis of dendritic cells, Ab production, and cross‐priming, and is a peptide acceptor in endoplasmic reticulum and an accessory to peptide loading of MHC class I molecules. The remarkable conservation of essential immunological properties of gp96 suggests their important roles during the evolution of the immune system. Considering their importance in immunity, immune evasion mechanisms of pathogens by modulating gp96 expression have been speculated. By differential display PCR, we observed that obligate intracellular bacteria, Orientia tsutsugamushi, inhibit gp96 expression of a macrophage cell line, J774A.1. Not only gp96 transcripts but also protein was lower than for null‐infected cells. The down‐regulation was also consistent in an endothelial cell line, HMEC‐1, and in murine peritoneal cells. These data support the idea that gp96 may be one of the target molecules for the immune evasion by intracellular bacteria.
Virulence Properties of Rough and Smooth Strains of Vibrio cholerae O1
Authors: Islam Mohammad Sirajul , Ahsan Sunjukta , Khan Sirajul Islam , Ahmed Qazi Shafi , Rashid Mohammad Harunur , Islam Khan M. Nasirul , Sack Richard Bradley
Published: November 2013

A comparative study was carried out to see the differences in pathogenicity of rough and smooth strains. A total of 10 strains including 5 each of rough and smooth strains of Vibrio cholerae O1 were tested and found positive for toxin production by enzyme‐linked immunosorbent assay (ELISA) in Richardson's and AKI media. All the smooth and rough strains, except one, showed a titre of 1:10 and 1:100 in Richardson's and AKI media, respectively. Both types of strains produced enterotoxin in rabbit ileal loop (RIL). The differences in multiplication abilities of smooth and rough strains in RIL were statistically significant (P<0.05). However, these differences in multiplying abilities did not influence the adherence potential or enterotoxin production as there was no significant difference (P>0.05) between these properties. This study demonstrated that the rough strains are equally pathogenic and as important as smooth strains.
Comparison among Fecal Secondary Bile Acid Levels, Fecal Microbiota and Clostridium scindens Cell Numbers in Japanese
Authors: Kitahara Maki , Sakata Shinji , Sakamoto Mitsuo , Benno Yoshimi
Published: November 2013

Bile acid 7α‐dehydroxylation by intestinal bacteria, which converts cholic acid and chenodeoxycholic acid to deoxycholic acid (DCA) and lithocholic acid (LCA), respectively, is an important function in the human intestine. Clostridium scindens is one of the most important bacterial species for bile acid 7α‐dehydroxylation because C. scindens has high levels of bile acid 7α‐dehydroxylating activity. We quantified C. scindens and secondary bile acids, DCA and LCA, in fecal samples from 40 healthy Japanese and investigated their correlation. Moreover, we used terminal restriction fragment length polymorphism (T‐RFLP) analysis to investigate the effect of fecal microbiota on secondary bile acid levels. There was no correlation between C. scindens and secondary bile acid in fecal samples. On the other hand, T‐RFLP analysis demonstrated that fecal microbiota associated with high levels of DCA were different from those associated with low levels of DCA, and furthermore that fecal microbiota in the elderly (over 72 years) were significantly different from those in younger adults (under 55 years). These results suggest that intestinal microbiota have a stronger effect on DCA level than does the number of C. scindens cells.
Identification and Characterization of Two Contiguous Operons Required for Aerobactin Transport and Biosynthesis in Vibrio mimicus
Authors: Moon Yong‐Hwa , Tanabe Tomotaka , Funahashi Tatsuya , Shiuchi Kei‐ichi , Nakao Hiroshi , Yamamoto Shigeo
Published: November 2013

In response to iron deprivation, Vibrio mimicus produces aerobactin as a major siderophore. Application of the Fur titration assay to a V. mimicus genomic DNA library followed by further cloning of the surrounding regions led to the identification of two adjacent, iron‐regulated operons. One contains three genes encoding homologs of the Escherichia coli FhuCDB and the other, five genes encoding homologs of the E. coli IucABCD IutA. Construction of the V. mimicus polar disruptants in the respective operons allowed us to confirm their functions. The genetic arrangement of the aerobactin‐mediated iron acquisition system in V. mimicus is unique in that the aerobactin operon (iucABCD iutA) is contiguous to the operon (matCDB) encoding components of an ATP‐binding cassette transport system for ferric aerobactin. This is the first report demonstrating that aerobactin transport and biosynthesis genes are present in a species outside the family Enterobacteriaceae.
Association of Rabies Virus Nominal Phosphoprotein (P) with Viral Nucleocapsid (NC) Is Enhanced by Phosphorylation of the Viral Nucleoprotein (N)
Authors: Toriumi Harufusa , Kawai Akihiko
Published: November 2013

We investigated possible role(s) of N protein phosphorylation in the rabies virus replication process. A large amount of P proteins are associated with the viral nucleocapsid (NC) in the infected cell, the amount which was greatly decreased by phosphatase‐treatment of the isolated NC, indicating that the phosphate group of N and/or P proteins is essential for their stable association with the NC. Immunoprecipitation studies were performed on the coexpressed normal N or phosphorylation deficient N(S389A) and P proteins, demonstrating that the P protein associated with phosphorylation‐deficient NC‐like structures was much less in amount than that associated with the wild type NC. Similar results were also obtained with a mutant P protein, PΔN19, which lacked the N‐terminal 19 amino acids and was capable of binding to the NC‐like structures but incapable of forming the RNA‐free N‐P complexes. Immunoprecipitation studies with mAb #402‐13 further suggested that the NC‐specific linear 402–13 epitope was exposed even on the P proteins which were associated with the phosphorylation‐deficient NC‐like structures, but such association was very weak as demonstrated by greatly decreased amounts of coprecipitated NC‐like structures. From these results, we assume that the phosphorylation of N protein enhances the association between the 402–13 epitope‐positive P protein and the NC probably by stabilizing such P‐NC binding.
Isolation and Phylogenetic Analysis of Arcobacter Spp. in Ground Chicken Meat and Environmental Water in Japan and Thailand
Authors: Morita Yukio , Maruyama Soichi , Kabeya Hidenori , Boonmar Sumalee , Nimsuphan Burin , Nagai Akira , Kozawa Kunihisa , Nakajima Takashi , Mikami Takeshi , Kimura Hirokazu
Published: November 2013

Prevalence of Arcobacter spp. in chicken meat samples and environmental water samples in Japan and Thailand was investigated. Arcobacter was isolated from 48% of chicken meat samples (20/41) and 23% of river water samples (4/17) from Japan, and 100% of chicken meat samples (10/10) and 100% of canal water samples (7/7) from Thailand. A. butzleri was among the species isolated from all positive samples. About 10% genetic diversity was seen in the rpoB‐rpoC in Arcobacters, and phylogenetic trees were divided into two clusters. In both countries, the results suggested that chicken and environmental water were highly contaminated with a genetically diverse population of Arcobacter.
Fluconazole‐Resistant Pathogens Candida inconspicua and C. norvegensis: DNA Sequence Diversity of the rRNA Intergenic Spacer Region, Antifungal Drug Susceptibility, and Extracellular Enzyme Production
Authors: Sugita Takashi , Takeo Kanji , Ohkusu Misako , Virtudazo Eric , Takashima Masako , Asako Emi , Ohshima Fumiko , Harada Satoshi , Yanaka Chikako , Nishikawa Akemi , Majoros Laszlo , Sipiczki Matthias
Published: November 2013

The opportunistic fungal pathogens Candida inconspicua and C. norvegensis are very rarely isolated from patients and are resistant to fluconazole. We collected 38 strains of the two microorganisms isolated from Europe and Japan, and compared the polymorphism of the rRNA intergenic spacer (IGS) and internal transcribed spacer (ITS) regions, antifungal drug susceptibility, and extracellular enzyme production as a potential virulence factor. While the IGS sequences of C. norvegensis were not very divergent (more than 96.7% sequence similarity among the strains), those of C. inconspicua showed remarkable diversity, and were divided into four genotypes with three subtypes. In the ITS region, no variation was found in either species. Since the sequence similarity of the two species is approximately 70% at the ITS region, they are closely related phylogenetically. Fluconazole resistance was reconfirmed for the two microorganisms but they were susceptible to micafungin and amphotericin B. No strain of either species secreted aspartyl proteinase or phospholipase B. These results provide basal information for accurate identification, which is of benefit to global molecular epidemiological studies and facilitates our understanding of the medical mycological characteristics of C. inconspicua and C. norvegensis.
Effects of Endocytosis Inhibitory Drugs on Rubella Virus Entry into VeroE6 Cells
Authors: Kee Sun‐Ho , Cho Eun‐Jin , Song Jin‐Won , Park Kwang Sook , Baek Luck Ju , Song Ki‐Joon
Published: November 2013

It has been suggested that infectious entry of rubella virus (RV) is conducted by receptor mediated endocytosis. To explore the cellular entry mechanism of RV, inhibitory effects of drugs affecting various endocytic pathways on RV entry into VeroE6 cells were analyzed. Results showed that RV infectious entry into VeroE6 cells is mediated by clathrin‐dependent endocytosis and not by caveolae‐mediated endocytosis. Moreover, chemical inhibition of macropinocytosis such as treatments of amiloride, actin and microtubule‐disrupting drug significantly reduced RV infection. Considering that macropinocytosis is inducible endocytosis by cellular stimulations, clathrin‐mediated endocytosis is likely to be a major route of RV infectious entry.
Growth and Virulence Alterations of Equine Herpesvirus 1 by Insertion of a Green Fluorescent Protein Gene in the Intergenic Region between ORFs 62 and 63
Authors: Ibrahim El Sayed Moustafa , Pagmajav Ochir , Yamaguchi Tsuyoshi , Matsumura Tomio , Fukushi Hideto
Published: November 2013

Nucleotide sequences of the intergenic region between ORF 62 and ORF 63 of equine herpesvirus 1 (EHV‐1) isolates were analyzed. The sequences of this region consisted of variable and conserved domains among EHV‐1 isolates. An EHV‐1 mutant, Ab4‐GFP, was constructed by inserting a green fluorescent protein (GFP) expression cassette flanked by loxP at both ends into the intergenic region between ORF 62 and ORF 63. Another mutant, Ab4‐loxP, which contains one loxP site, was constructed by excision of the GFP cassette from the Ab4‐GFP virus genome by cre enzyme. The recombinant Ab4‐GFP formed smaller plaques than the wild type in MDBK cells. Virus production also decreased for Ab4‐GFP in multi‐step growth analyses. Virulence of Ab4‐GFP in both mice and hamsters was weaker than that of the wild type. Ab4‐loxP exhibited properties similar to those of the wild type. These results suggest that the intergenic region between ORF 62 and ORF 63 plays various roles in the virus growth.
Stimulation of Phagocytosis of Influenza Virus‐Infected Cells through Surface Desialylation of Macrophages by Viral Neuraminidase
Authors: Watanabe Yuichi , Shiratsuchi Akiko , Shimizu Kazufumi , Takizawa Takenori , Nakanishi Yoshinobu
Published: November 2013

Cells infected with influenza A virus undergo apoptosis and become susceptible to phosphatidylserine‐mediated phagocytosis by macrophages. This study was undertaken to elucidate the mechanism underlying our previous finding that the activity of viral neuraminidase (NA) is required for efficient phagocytosis. Treatment of macrophages, not influenza virus‐infected cells, with Arthrobacter ureafaciens NA or virus‐infected cells expressing viral NA augmented the level of phagocytosis of virus‐infected cells but not of latex beads or cells undergoing Fas‐induced apoptosis. Oligosaccharides, including sialyllactose, bound to influenza virus‐infected cells and inhibited phagocytosis by macrophages. These results indicate that surface desialylation of macrophages by influenza virus NA modulates the mode of association between macrophages and target virus‐infected cells and stimulates phosphatidylserine‐mediated phagocytosis.
Yersinia enterocolitica Serovar O:8 Infection in Breeding Monkeys in Japan
Authors: Iwata Taketoshi , Une Yumi , Okatani Alexandre Tomomitsu , Kaneko Sei‐ichi , Namai Satoshi , Yoshida Shin‐ichiro , Horisaka Tomoko , Horikita Tetsuya , Nakadai Aya , Hayashidani Hideki
Published: November 2013

In the period from December 2002 to January 2003, 5 of 50 squirrel monkeys (Saimiri sciureus) housed at a Zoological Garden in the Kanto region of Japan died following a few days' history of diarrhea. After this outbreak had ended in the squirrel monkeys, 1 of 2 dark‐handed gibbons (Hylobates agilis) died in April of 2003, showing similar clinical signs. We examined the organs of 3 of the dead squirrel monkeys and of the dark‐handed gibbon, and Yersinia enterocolitica serovar O:8, which is the most pathogenic serovar of Y. enterocolitica, was isolated. In order to determine the source and the transmission route of infection, 98 fecal samples (45 from squirrel monkeys, 20 from other monkeys of 18 different species, and 33 from black rats captured around the monkey houses) and 7 water samples were collected in the Zoological Garden, and were examined for the prevalence of Y. enterocolitica serovar O:8. Serovar O:8 was isolated from 21 of 65 monkeys (32.3%) and 5 of 33 (15.2%) black rats (Rattus rattus). Furthermore, we examined the 30 isolates using molecular typing methods, pulsed field gel electrophoresis (PFGE), ribotyping using the RiboPrinter system, and restriction endonuclease analysis of virulence plasmid DNA (REAP), and compared the isolates in this outbreak with Japanese O:8 isolates previously identified. Genotyping showed that almost all the isolates were identical, and the genotype of the isolates was highly similar to that from wild rodents captured in Niigata Prefecture. This is the first report of fatal cases of Y. enterocolitica serovar O:8 infection in monkeys anywhere in the world.
Sequence Analysis of the Hypervariable Regions of the 56 kDa Immunodominant Protein Genes of Orientia tsutsugamushi Strains in Malaysia
Authors: Tay Sun Tee , Rohani Yasin Mohd , Ho Tze Ming , Shamala Devi
Published: November 2013

The DNA sequences encompassing two hypervariable regions, VD II and III of the 56 kDa immunodominant protein gene of 21 Malaysian strains of Orientia tsutsugamushi were determined. Two strains demonstrated a 100% DNA homology with the Gilliam prototype strain, and one with TH1817 strain and TA678 strain respectively. High percentages of DNA similarity (95–99%) were observed with Karp (4 strains), Gilliam (2 strains), TH1817 (4 strains), TC586 (3 strains) and TA763 (1 strain). The remaining strains demonstrated the highest DNA similarity with TA763 (1 strain, 89%), TA678 (1 strain, 86%) and TA686 (1 strain, 87%). Our study provides additional evidence on the existence and the genetic heterogeneity of TA strains of the Southeast Asia and their closely related strains in Malaysia.
Mutations in the gyrA and parC Genes in Ciprofloxacin‐Resistant Clinical Isolates of Acinetobacter baumannii in Korea
Authors: Lee Jeom Kyu , Lee Yeong Seon , Park Yong Keun , Kim Bong Su
Published: November 2013

Mutation with Ser‐83→Leu in gyrA gene was associated with the principal mutation for ciprofloxacin resistance in clinical isolates of Acinetobacter baumannii. Double mutation, Ser‐83→Leu in gyrA gene and Ser‐80→Leu in parC gene, was the most frequently detected among ciprofloxacin‐resistant isolates. A novel mutation with Ser‐80→Trp in parC gene, in addition to mutation in gyrA gene, was associated with a high‐level ciprofloxacin resistance. These results suggested that the presence of an additional mutation in the parC gene contributed to a higher‐level of ciprofloxacin resistance than a single mutation in the gyrA gene (geometric mean MICs of ciprofloxacin, 44.1 versus 16 μg/ml, P<0.05).
Molecular Subtyping for Escherichia coli O157:H7 Isolated in Taiwan
Authors: Tsai Tsung‐Yu , Luo Wei‐Chen , Wu Fang‐Tzy , Pan Tzu‐Ming
Published: November 2013

Enterohaemorrhagic Escherichia coli O157:H7 is an important pathogen these days. Outbreaks of its infection have been reported all over the world, in Australia, Canada, Japan, the United States, south Africa, and various countries in Europe. In the summer of 2001, the first clinical infection by E. coli O157:H7 was identified in Taiwan. In this study, the standard procedures for molecular subtyping were applied to several strains collected in Taiwan as well as from elsewhere. The two molecular subtyping methods we used are pulsed‐field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP). The isolates from the U.S.A., Canada, Japan, and Taiwan each showed a unique molecular fingerprinting pattern. The environmental strains isolated in Taiwan showed closer relationships with each other, and their similarity was in the range of 75–85%. The first clinical strain isolated in Taiwan in 2001 was similar to the strains from North America but not closely related to the Taiwanese environmental strains. Our surveys showed that some local E. coli O157:H7 strains did exist in Taiwan, but there had been only one official case report of the infection by local E. coli O157:H7. The eating habits of the people and the geographic distribution of the pathogen are considered crucial risk factors in Taiwan. The establishment of a database of our own and joining the global network database are important tasks if we want to control such agricultural and food‐borne pathogens, and reduce the number of victims and amount sufferings, as well as the economic losses due to the infection.
Vaccine Efficacy of Recombinant GAPDH of Edwardsiella tarda against Edwardsiellosis
Authors: Liu Ying , Oshima Syun‐ichirou , Kurohara Kenrou , Ohnishi Kouhei , Kawai Kenji
Published: November 2013

Thirty‐seven kilodalton glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) of Edwardsiella tarda was suggested to be an effective vaccine candidate against E. tarda infection in previous research. For developing a vaccine, obtaining GAPDH in large quantities is necessary. In this study, we determined the complete nucleotide sequence of the gene that encodes GAPDH of E. tarda, and overexpressed the GAPDH of E. tarda by using the Escherichia coli expression system. We immunized Japanese flounder with recombinant GAPDH (rGAPDH) and evaluated its vaccine efficacy. Our results showed that rGAPDH effectively protected Japanese flounder from experimental E. tarda infection, and will contribute to the development of a vaccine against E. tarda.
Detection and Identification of Human Papillomavirus Types Isolated from Korean Patients with Flat Warts
Authors: Yoo Hoon , Won Sung‐Sik , Choi Hyun‐Chul , Yoon Taek‐Joon , Ye Sang‐Kyu , Park Tae‐Kyu , Lee Kwang‐Ho
Published: November 2013

Flat warts, also called verruca planna (VP) or juvenile warts, are benign epithelial proliferations of the skin caused by infection with human papillomaviruses (HPV). Several HPV types are known to be associated with flat warts, and particularly HPV type 3 and 10 have been most frequently reported in other countries. In this study, for the detection and typing of human papillomavirus isolated from Korean patients with flat warts, polymerase chain reaction (PCR) and restriction endonuclease digestion were carried out with a set of restriction endonucleases, using the cloned HPV DNA and DNA from clinical specimens. A unique digestion pattern for HPV type 3 and 10, a form of miniature fingerprinting, enabled us to identify HPV type from the amplified fragments. A total of thirty clinical samples, as either frozen tissue or paraffin‐embedded tissue, were investigated to verify the type. All the clinical samples except one were confirmed to be type 3, one of the most frequently observed types in flat warts, and one sample was neither type 3 nor type 10. Further investigation of the unidentified sample by DNA sequencing and sequence alignment with other known HPV types revealed that the sample was a variant of HPV type 94, one of the EV‐related HPVs, with the closest evolutionary distance to the HPV type 10 among the known flat wart‐associated HPV types.
The Recombinant Immunogen with High‐Density Epitopes of ELDKWA and ELDEWA Induced Antibodies Recognizing Both Epitopes on HIV‐1 gp41
Authors: Wang Zuguang , Liu Zuqiang , Cheng Xiwen , Chen Ying‐Hua
Published: November 2013

The high mutation rate of HIV‐1 (human immunodeficiency virus‐1) is a major obstacle to developing an effective vaccine. The mutation of ELDKWA‐ (aa669–674) to ELDEWA‐epitope on HIV‐1 gp41 caused the immune escape from neutralization by potent anti‐HIV‐1 human monoclonal antibody (mAb) 2F5. In this study, we suggested and evaluated a multi‐epitope vaccine as a new strategy to develop HIV‐1 vaccines. A glutathione S‐transferase (GST) fusion protein (GST‐K8E8) containing 8 copies of ELDKWA‐ and mutated ELDEWA‐epitopes was constructed and used to immunize mice or rabbits. Analysis of the antisera (rAS3) induced by GST‐K8E8 suggested that multi‐epitope vaccine immunogen could raise antibodies in mice and rabbits against either the original ELDKWA‐epitope or the mutated ELDEWA‐epitope that resulted in immune escape. Briefly, ELDKWA‐epitope‐specific antibodies, directly purified from rAS3 by ELDKWA‐epitope‐peptide affinity chromatography, recognized either original gp41 protein (ELDKWA, rgp41K) or mutated gp41 (ELDEWA, rgp41E) in immunoblotting assay; in contrast, the existing ELDKWA‐epitope antibodies recognized only rgp41K but not rgp41E, which were purified by ELDKWA‐epitope‐peptide affinity chromatography from rAS3 that were firstly completely pre‐absorbed by ELDEWA‐epitope‐peptide affinity beads. And the same results were also observed when detecting the ELDEWA‐epitope‐specific antibodies in rAS3 by a means similar to the above. All the data presented here demonstrated that a high density multi‐epitope vaccine could be an interesting strategy against HIV‐1 mutation.
Rapid PrPSc Detection in Lymphoid Tissue and Application to Scrapie Surveillance of Fallen Stock in Japan: Variable PrPSc Accumulation in Palatal Tonsil in Natural Scrapie
Authors: Shimada Kimi , Hayashi Hiroko K. , Ookubo Yuka , Iwamaru Yoshifumi , Imamura Morikazu , Takata Masuhiro , Schmerr Mary Jo , Shinagawa Morikazu , Yokoyama Takashi
Published: November 2013

Rapid western blot (WB) procedure for an abnormal isoform of prion protein (PrPSc) detection in lymphoid tissues was established and has been applied to the surveillance of fallen stock. In this program, brain and palatal tonsil were examined by WB and three cases of sheep scrapie were detected. While one clinically scrapie‐infected sheep harbored PrPSc in the brain and palatal tonsil, the two sheep in the pre‐clinical stage harbored PrPSc in the brain, but not in the palatal tonsil. This study shows that PrPSc accumulation in palatal tonsil is variable in natural scrapie, even among genetically susceptible sheep.
Further Studies on the Mechanism of Rabies Virus Neutralization by a Viral Glycoprotein‐Specific Monoclonal Antibody, #1‐46‐12
Authors: Irie Takashi , Kawai Akihiko
Published: November 2013

We previously reported that a conformational epitope‐specific monoclonal antibody (mAb; #1‐46‐12) neutralized the rabies virus by binding only a small number (less than 20) of the antibody molecules per virion, while a linear epitope‐specific mAb (#7‐1‐9) required more than 250 IgG molecules for the neutralization. We also isolated both the epitope‐negative (R‐31) and ‐positive (R‐61) escape mutants that resisted mAb #1‐46‐12. Co‐infection studies with wild type (wt) and R‐61 mutant have shown that although the infectivity of R‐61 mutant was not affected by the binding of about 300 IgG molecules per virion, incorporation of a small number of wt G protein into the R‐61 virion resulted in dramatic loss of the resistance. In this study, we further investigated properties of the mutant G proteins. The R‐61 G protein lost reactivity to the mAb when solubilized, even keeping a trimer form, suggesting that membrane‐anchorage is essential for the maintenance of its epitope‐positive conformation. On the other hand, incorporation of wt G proteins into the R‐31 virions did not affect their resistance to the mAb very much. Although we have not so far found the presumed conformational changes induced by the mAb‐binding, we think that these results are not inconsistent with our previously proposed novel model (referred to as a domino effect model) for the virus neutralization by mAb #1‐46‐12 other than a classical spike‐blocking model, which implicates successive spreading of the postulated antibody‐induced conformational changes of G protein to the neighboring spikes until abolishing the host cell‐binding ability of the virion.
A Novel B‐2 Suppressor Cell Regulating Susceptibility/Resistance of Mice to Toxoplasma gondii Infection
Authors: Mun Hye‐Seong , Aosai Fumie , Fang Hao , Piao Lian‐Xun , Winn Tin , Norose Kazumi , Yano Akihiko
Published: November 2013

Irradiation treatment enhanced resistance of C57BL/6, but not BALB/c against Toxoplasma gondii infection. Six Gy‐irradiated (IR) C57BL/6 recipients of B‐2 cells from T. gondii‐infected C57BL/6 died after infection. B‐2 suppressor cells from infected C57BL/6 enhanced production of IL‐4 and IL‐10 in peritoneal exudate cells (PECs), and down‐regulated NO release in peritoneal macrophages after infection. On the other hand, B‐2 suppressor cells were not detected in a strain, BALB/c, resistant against infection. These data indicated that irradiation‐sensitive B‐2 cells regulated susceptibility/resistance in mice against T. gondii infection.
Rapid Detection and Differentiation of the Major Mycoplasma Contaminants in Cell Cultures Using Real‐Time PCR with SYBR Green I and Melting Curve Analysis
Authors: Harasawa Ryô , Mizusawa Hiroshi , Fujii Masashi , Yamamoto Junko , Mukai Hiroyuki , Uemori Takashi , Asada Kiyozo , Kato Ikunoshin
Published: November 2013

A quantitative real‐time polymerase chain reaction (PCR) procedure followed by melting curve analysis, using the green fluorescence dye SYBR Green I, was developed for rapid detection and differentiation of mycoplasma contaminants in cell cultures. This method showed that the detection of the target sequence was linear over a range from 104 to 10 colony‐forming units (CFU) of the mycoplasma cells. Analysis of the melting temperature of the PCR products allowed differentiation of the major mycoplasma contaminants. These results demonstrate that the protocol described in the present study can decrease the time to obtain reproducible results by simultaneous detection and differentiation of the Mycoplasma species contaminating cell cultures.
Identification of an HLA‐A24‐Restricted OY‐TES‐1 Epitope Recognized by Cytotoxic T‐Cells
Authors: Okumura Hideo , Noguchi Yuji , Uenaka Akiko , Aji Toshiki , Ono Toshiro , Nakagawa Kazuhiko , Aoe Motoi , Shimizu Nobuyoshi , Nakayama Eiichi
Published: November 2013

OY‐TES‐1 was identified as a human homologue of the mouse, guinea pig, and pig proacrosin binding protein sp32 precursor. Differential expression levels of OY‐TES‐1 mRNA between testis and other normal tissues, and its expression in cancers indicated that OY‐TES‐1 would be classified as a cancer/testis antigen and considered to be a candidate of target antigen for cancer immunotherapy. In this study, we showed identification of HLA‐A24‐binding OY‐TES‐1 peptide, TES401–409 (KTPFVSPLL) recognized by CD8 T‐cells. Purified CD8 T‐cells from healthy donors stimulated in vitro with the peptide‐pulsed autologous DC and PBMC produced IFN7 in response to the peptide‐pulsed PBMC and showed cytotoxicity against the peptide‐pulsed autologous EBV‐B specifically. Furthermore, cytotoxicity was also observed against an OY‐TES‐1 mRNA‐expressing tumor line, LK79. The retention time of the fraction in HPLC of the acid eluate from LK79 cells that showed positive sensitization against autologous EBV‐B cells in recognition by CD8 CTL was the same as that of the fraction of the TES401–409 peptide itself, suggesting that the TES401–409 was a naturally processed peptide on LK79.
Characterization of M Gene‐Deficient Rabies Virus with Advantages of Effective Immunization and Safety as a Vaccine Strain
Authors: Ito Naoto , Sugiyama Makoto , Yamada Kentaro , Shimizu Kenta , Takayama‐Ito Mutsuyo , Hosokawa Junji , Minamoto Nobuyuki
Published: November 2013

Matrix (M) protein of rabies virus is known to play an important role in assembly and budding of the progeny virus. We generated an M gene‐deficient rabies virus, RC‐HLAM, using a reverse genetics system of rabies virus RC‐HL strain to develop a novel type of vaccine. RC‐HLAM infection was confined within a single cell in mouse neuroblastoma cells. This deficient virus failed to generate the progeny virus in the cells. In contrast, RC‐HLAM propagated in BHK cells inductively expressing M protein. Suckling and adult mice inoculated intracerebrally with the parental RC‐HL strain showed lethal infection and transient body weight loss, respectively, whereas both suckling and adult mice inoculated with RC‐HLAM showed no symptoms. The neutralizing antibody against rabies virus was successfully induced by intramuscular immunization with 105 focus‐forming units of RC‐HLAM but not UV‐inactivated RC‐HL. Intranasal immunization with RC‐HLAM resulted in almost the same antibody titer to rabies virus as that in the case of immunization with live RC‐HL strain. These findings indicate that RC‐HLAM is a candidate for a novel rabies vaccine that is safer and more effective than are current vaccines.
Contribution of Anaphylatoxins to Allergic Inflammation in Human Lungs
Authors: Abe Masayoshi , Hama Hiroshi , Shirakusa Takayuki , Iwasaki Akinori , Ono Nobuhumi , Kimura Nobuhiro , Hugli Tony E. , Okada Noriko , Katsuragi Takeshi , Okada Hidechika
Published: November 2013

The contribution of complement activation to allergic asthma remains controversial. In order to elucidate the role played by the complement split products, anaphylatoxins C3a and C5a, we evaluated their effects on production of cysteinyl‐leukotrienes (cysLTs) by human lung fragments following an anaphylactic reaction. The lung tissues obtained from two patients with lung cancer showed C5aR‐, C5L2R‐, and C3aR‐mRNA expression. When the chopped lung fragments passively sensitized with human IgE were incubated with anti‐human IgE antibody, a significant amount of cysLTs was generated in comparison with the control (without anti‐IgE antibody). The co‐addition of human C5a at doses of 0.1 to 10 ng/ml to the anti‐IgE antibody potentiated cysLT production. The response was bell‐shaped in distribution, significant, and peaked at a C5a concentration of 1 ng/ml. The co‐addition of human C3a up to 1,000 ng/ml seemed to increase cysLT production, but not to any significant extent. A novel C5a receptor complementary peptide, acetylated peptide A, dose‐dependently inhibited cysLT production by the human lung fragments following the anaphylactic reaction in the presence of 1 ng/ml C5a. However, this peptide did not inhibit cysLT production in the presence of 100 ng/ml C3a. It is suggested that the anaphylatoxin C5a potentiates cysLT production in human lung tissues and contributes to allergic inflammation in disorders such as asthma, thus acetylated peptide A may be useful for suppressing allergic inflammation in the lungs.
Evaluation of the Lon‐Deficient Salmonella Strain as an Oral Vaccine Candidate
Authors: Kodama Chie , Eguchi Masahiro , Sekiya Yukie , Yamamoto Tomoko , Kikuchi Yuji , Matsui Hidenori
Published: November 2013

We evaluated the efficacy of CS2022 (the Lon protease‐deficient mutant strain of Salmonella enterica serovar Typhimurium) as a candidate live oral vaccine strain against subsequent oral challenge with a virulent strain administered to BALB/c and C57BL/6 mice. CS2022 persistently resided in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum of both strains of mice after a single oral inoculation with 1 × 108 colony‐forming units. Finally, CS2022 almost disappeared from each tissue sample by week 12 in BALB/c mice, whereas CS2022 still resided in each tissue type at week 12 after inoculation of C57BL/6 mice. A significant increase in the serovar Typhimurium lipopolysaccharide‐specific secretory immunoglobulin A (s‐IgA), as measured for one of the mucosal immune responses, was detected in bile and intestinal samples of both strains of immunized mice at week 4 after immunization. In addition, the expression of gamma interferon mRNA in the spleens of both strains of immunized mice, especially those of C57BL/6 mice, was significantly increased at week 4 after immunization and was boosted during the following 5 days after the challenge was administered to the mice. Furthermore, peritoneal macrophages isolated from immunized mice at week 4 after immunization exhibited an increase in intracellular killing activity against both virulent and avirulent Salmonella. The present results suggested that salmonellae‐specific s‐IgA on the mucosal surfaces induced by immunization with CS2022 generally prevented mice from succumbing to an oral challenge with a virulent strain. Simultaneously, CS2022 promoted the protective immunity associated with macrophages in both strains of mice.
Evaluation of the ESPLINE® INFLUENZA A&B‐N Kit for the Diagnosis of Avian and Swine Influenza
Authors: Bai Gui‐Rong , Sakoda Yoshihiro , Mweene Aaron Simanyengwe , Kishida Noriko , Yamada Takashi , Minakawa Hidetaka , Kida Hiroshi
Published: November 2013

The ESPLINE® INFLUENZA A&B‐N kit was evaluated for its applicability to the rapid diagnosis of influenza in chickens and pigs. The kit specifically detected viral antigens in tracheal swabs and tissue homogenates of the trachea, liver, spleen, and colon of chickens inoculated with a highly pathogenic avian influenza virus strain, A/chicken/Yamaguchi/7/04 (H5N1), at 48 hr post‐inoculation (p.i.) as well as in the tracheal and cloacal swabs and tissue homogenates of dead chickens. For those infected with a low pathogenic strain, A/chicken/aq‐Y‐55/01 (H9N2), antigens were detected only in the samples from tracheal swabs and organs 1–4 days p.i. The kit also detected viral antigens in the nasal swabs of miniature pigs infected with swine and avian influenza viruses. The kit was found to be sensitive and specific enough for the rapid diagnosis of infections of influenza A virus in chickens and pigs.
Epidemiology of Typhoid Carriers among Blood Donors and Patients with Biliary, Gastrointestinal and Other Related Diseases
Authors: Vaishnavi Chetana , Kochhar Rakesh , Singh Gurpreet , Kumar Sudarshan , Singh Satnam , Singh Kartar
Published: November 2013

Enteric fever due to Salmonella Typhi is a major public health problem. Typhoid carriers have high titres of Vi agglutinins in their sera. We worked out the baseline data for Vi agglutinins from 705 healthy blood donors (controls) by ELISA and compared it with 446 patients with biliary, gastrointestinal and other related diseases (cases). The samples were divided into five groups based on the disease condition of the patients from whom they were collected. Group A (n = 196) consisted of patients with stones in the gall bladder/common bile duct and Group B (n=27) with gall bladder carcinoma. Group C (n=33) comprised patients with carcinoma of the pancreas/ampulla, obstructive jaundice and/or cholangiocarcinoma. Group D (n = 112) had patients with acute/chronic pancreatitis, abdominal pain, intestinal obstruction, peritonitis, carcinoma oesophagus, chronic diarrhoea, gastrointestinal bleeding and dyspepsia. Group E (n=78) included patients with miscellaneous diseases. The mean absorbance value obtained for healthy subjects +3 standard deviations was taken as the cut‐off value for a positive typhoid carrier. In Group A, 10.2% samples were positive; in Group B, 7.4%; in Group C, 12.0%; in Group D, 9.8% and in Group E, 9.0%. There was a highly significant (P<0.001) increase in the presence of Vi agglutinins in the cases compared to the controls. High prevalence of typhoid carriers occurs in patients with biliary, gastrointestinal and other related diseases. Vi serology employing highly purified Vi antigen offers a practical and cost‐effective way of screening for S. Typhi carriers.
Natural Recombinant between Equine Herpesviruses 1 and 4 in the ICP4 Gene
Authors: Pagamjav Ochir , Sakata Tohru , Matsumura Tomio , Yamaguchi Tsuyoshi , Fukushi Hideto
Published: November 2013

Equine herpesvirus 1 (EHV‐1) is a pathogen causing rhinopneumonia in young horses, abortion in mares, and myeloencephalitis in adult horses. Two types, EHV‐1 P and EHV‐1 B, have recently been dominant among 16 electropherotypes. EHV‐1 P and EHV‐1 B viruses were compared by long and accurate polymerase chain reaction (LA‐PCR) and restriction fragment length polymorphism (RFLP) analysis. Differences in restriction sites were found to be focused in ORF64, which encodes the infected cell protein 4 (ICP4), and downstream of the ICP4 gene. The 3′‐end and downstream of ICP4 gene of EHV‐1 B were found to be replaced by the corresponding region of EHV‐4, indicating that EHV‐1 B is a naturally occurring recombinant virus between progenitors of EHV‐1 P and EHV‐4. This is the first report showing a natural interspecies recombinant in alphaherpesviruses.
Diversity of Borrelia burgdorferi Sensu Lato in Russian Far East
Authors: Mediannikov Oleg Y. , Ivanov Leonid , Zdanovskaya Nina , Vorobyova Rimma , Sidelnikov Yuri , Fournier Pierre‐Edouard , Tarasevich Irina , Raoult Didier
Published: November 2013

Thirty strains of Borrelia burgdorferi sensu lato have been isolated from Ixodes persulcatus ticks and from skin lesions of Lyme disease patients in the Russian Far East from 1997 to 2003. We amplified full‐length outer surface protein A (ospA) gene of all strains. BLAST search and following phylogenetic analysis showed that strains form four well‐defined groups. Four strains belong to Borrelia afzelii species. Other strains distributed into tree major groups, identified as Borrelia garinii. Indeed, based on the ospA gene comparison, phylogenetic relationship of these groups among each other does not differ from relationship among other previously defined groups inside B. burgdorferi sensu lato genogroup, such as B. afzelii or Borrelia bissettii. Further investigations of genetic and serologic properties of the strains belonging to those groups are required in order to clarify their taxonomic status.
Molecular Basis for Adaptation of a Chimeric Dengue Type‐4/Japanese Encephalitis Virus to Vero Cells
Authors: Tang Wei‐Feng , Eshita Yuki , Tadano Masayuki , Morita Kouichi , Makino Yoshihiro
Published: November 2013

The premembrane and envelope (E) genes of a full‐length cDNA clone of the dengue type‐4 (DEN4) virus 814669 strain were replaced with those of the Japanese encephalitis (JE) virus JaOH0566 strain. The in vitro‐synthesized RNA transcripts prepared from chimeric cDNA were used to transfect mosquito C6/36 cells. A viable chimeric virus (designated DEN4/JE) was recovered. Unexpectedly, DEN4/JE exhibited restricted growth in Vero cells. After a serial passage in Vero cells, the Vero‐adapted chimeras were obtained (two clones, designated Strain I and Strain II, respectively). The entire genomes of DEN4/JE, Strain I, and Strain II were sequenced and compared. There were multiple mutations, but amino acid substitutions occurred only in E and nonstructural (NS) protein NS4B. Our findings in this study indicate that the 5′ nontranslated region, E, and NS4B may be involved in Vero cell adaptation in this chimeric system.