bluuebunny/crossref_metadata_2025 huggingface dataset:
Canine Ehrlichiosis Caused Simultaneously by Ehrlichia canis and Ehrlichia platys
Authors: Hua Pan , Yuhai Ma , Shide Tong , Yang Sun , Bohai Wen , Xiangrui Cheng
Published: November 2013

To identify the causative agent of canine ehrlichiosis that has occurred in the suburbs of Guangzhou, China, since 1998, the 16S rRNA gene was amplified and sequenced. Two sequences of 1,482 and 1,483 base pairs were obtained and named as Gzh981 and Gzh982, respectively. The level of similarity of these two was 91.50%, and Gzh981 closely resembled the 16S rRNA gene of Ehrlichia canis, whereas Gzh982 resembled Ehrlichia platys. We therefore conclude that E. canis and E. platys together caused recent outbreaks of canine ehrlichiosis in China.
Glycyrrhizin Inhibits the Lytic Pathway of Complement — Possible Mechanism of Its Anti‐Inflammatory Effect on Liver Cells in Viral Hepatitis —
Authors: Fujisawa Yumiko , Sakamoto Motoko , Matsushita Misao , Fujita Teizo , Nishioka Kusuya
Published: November 2013

Glycyrrhizin, an aqueous extract of licorice root, has anti‐inflammatory activity and has been used for the treatment of chronic viral hepatitis. In the present study we describe the mechanism by which glycyrrhizin inhibits complement. Glycyrrhizin inhibited the cytolytic activity of complement via the activation of both the classical and alternative pathways, while it had no effect on immune adherence, suggesting that it blocks C5 or a later stage of the complement cascade. Further analysis revealed that glycyrrhizin inhibits the lytic pathway in which the membrane attack complex (MAC) is formed. This mechanism suggests that glycyrrhizin may prevent tissue injury caused by MAC not only in chronic hepatitis but in many autoimmune and inflammatory diseases.
Bifidobacterium lactis Meile et al. 1997 Is a Subjective Synonym of Bifidobacterium animalis (Mitsuoka 1969) Scardovi and Trovatelli 1974
Authors: Cai Yimin , Matsumoto Mitsuharu , Benno Yoshimi
Published: November 2013

Bifidobacterium lactis JCM 10602T (T = type strain) and Bifidobacterium animalis JCM 1190T were found to be phenotypically similar. These strains were subjected to investigation of their genetic relationships. The 16S rRNA sequence of B. animalis JCM 1190T was aligned with that of other Bifidobacterium species. B. animalis and B. lactis were the most closely related species in the phylogenetic tree and showed a high similarity in sequences (98.8%). The levels of DNA‐DNA hybridization between the type strains of B. lactis and B. animalis ranged from 85.5 to 92.3%, showing that they represent a single species. It is proposed that B. lactis should be considered as a junior subjective synonym of B. animalis.
Characteristics of DNA‐Binding Activity of Human Cytomegalovirus ppUL44
Authors: Hwang Eung‐soo , Kim Jinhee , Jong Hyun‐soon , Park Jae‐won , Park Chung‐gyu , Cha Chang‐yong
Published: November 2013

Human cytomegalovirus (HCMV)‐specific monoclonal antibody, SCMVM34, recognizes the early antigen encoded by UL44 of HCMV. This antigen is confined to the nucleus of HCMV‐infected cells. This study was performed to characterize the DNA‐binding activity of the protein encoded by UL44 of HCMV. The nuclear and cytoskeletal fraction of HCMV‐infected cells was subjected to 0.4 m NaCl extraction, DEAE‐Sephacel ion exchange chromatography, DNA‐cellulose chromatography and SDS‐PAGE analysis with monitoring of the reactive protein using SCMVM34 monoclonal antibody. The molecular weights of the resultant proteins were found to be 34, 40 and 52 kDa. The internal peptide fragments were isolated by tryptic digestion and reverse‐phase HPLC. The internal amino acid sequence analysis of the peptides from the HPLC profile revealed that the antigen recognized by SCMVM34 monoclonal antibody was ppUL44. The reactive antigen began to be eluted from 250 mm NaCl (Tris‐HCl pH 7.4) in DNA cellulose. The 34 kDa protein seems to bind to DEAE more tightly than the 52 kDa protein. The surface charge of 34 kDa might be more basic. Conclusively, the antigen recognized by SCMVM34 was the protein encoded by HCMV UL44, which was localized in the nuclei after HCMV infection, and was the DNA‐binding protein with the characteristic that the surface charge of the molecule was more basic, as the molecular weights of the protein were decreased.
Comparison of the Evolution of Recent and Late Phase of Old Influenza A (H1N1) Viruses
Authors: Nakajima Setsuko , Nishikawa Fumio , Nakajima Katsuhisa
Published: November 2013

A comparison of the evolutionary tree of new influenza A (H1N1) viruses to that of old H1N1 viruses which disappeared in 1957 was performed. The evolutionary trees of the hemagglutinin (HA) molecule based on amino acid sequences of the HA1 polypeptide were constructed with old and new H1N1 viruses isolated from 1947 to 1957 and 1986 to 2000, respectively. The evolutionary history of recent H1N1 viruses was similar to that of old H1N1 viruses just before the disappearance in two respects. Firstly, both viruses did not originate from the viruses of the previous H1N1 epidemic season but originated from the viruses branched off at the same point on the mainstream stem as the viruses of two H1N1 epidemic seasons earlier. Secondly, recent H1N1 viruses mainly circulating in Japan have a deletion at amino acid residue 134, located close to residue 131, which was deleted in old H1N1 viruses at the time of the disappearance. However, different from the evolutionary history of old H1N1 viruses, in the 1999/2000 H1N1 epidemic season, the H1N1 viruses which were located on the same lineage as the previous epidemic viruses were also isolated sporadically in Japan.
Characterization of Monoclonal Antibodies Generated against Norwalk virus Gil Capsid Protein Expressed in Escherichia coli
Authors: Yoda Tomoko , Terano Yoshitake , Suzuki Yasuhiko , Yamazaki Kenji , Oishi Isao , Utagawa Etuko , Shimada Akira , Matsuura Shiro , Nakajima Masaharu , Shibata Tadayoshi
Published: November 2013

The Norwalk virus (NV) causes outbreaks of acute non‐bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. The capsid protein of NV36 (genogroup II, Mexico virus type) was expressed in an Escherichia coli system and ten monoclonal antibodies (MAbs) were generated against it. The reactivity of these MAbs was characterized using enzyme‐linked immunosorbent assay (ELISA) and Western blot (WB) analysis towards 20 overlapping fragments of the NV36 capsid protein expressed in E. coli. All of the MAbs recognized sequential (continuous) epitopes on the three antigenic regions. Six of the 10 MAbs recognized fragment 2 (equivalent residues 31–70), three MAbs recognized fragment 13 (residues 361–403) and one MAb recognized fragment 7 (residues 181–220), suggesting that the N‐terminal domain (residues 1–220) may contain more antigenic epitopes than the C‐terminal domain (residues 210–548). Furthermore, two MAbs (1B4 and 1F6) reacted in WB with three purified NV strains (genogroup II) derived from patients' stool samples. It was also found that genogroup I recombinant NV96–908 (genogroup I, KY89 type) could be detected as sensitively as recombinant NV36 (genogroup II) by ELISA with a set of the MAbs produced here.
Giraffe Strain of Pestivirus: Its Taxonomic Status Based on the 5′‐Untranslated Region
Authors: Harasawa Ryô , Giangaspero Massimo , Ibata Georgina , Paton David J.
Published: November 2013

The 5′‐untranslated region (5′‐UTR) of the ‘Giraffe’ strain of pestivirus was sequenced for comparison with those of other pestiviruses from cattle, sheep, goats, and swine. A phylogenetic tree constructed with these strains suggested that the ‘Giraffe’ strain was allocated to a new taxon. This observation was also confirmed by a newly proposed method based on palindromic nucleotide substitutions (PNS) at the three variable regions in the 5′‐UTR. Other reported pestivirus strains isolated from deer were assigned as bovine viral disease virus (BVDV)‐1 according to the PNS as well as phylogenetic analysis, suggesting that BVDV‐1 strains can cross‐infect deer as well as cattle, sheep, goats, and swine, and that wild deer may serve as a reservoir of BVDV‐1. We also identified the genovar of a deer isolate, SH9/11, as BVDV‐1c by the PNS method.
Comparative Study of Staphylococcus aureus Isolated from Lesional and Non‐Lesional Skin of Atopic Dermatitis Patients
Authors: Matsui Katsuhiko , Nishikawa Akemi , Suto Hajime , Tsuboi Ryoji , Ogawa Hideoki
Published: November 2013

The skin of patients with atopic dermatitis (AD) is often colonized by Staphylococcus aureus, and superantigenic exotoxins produced by the organism are thought to be an important precipitating factor of AD. However, there are few reports comparing the characteristics of S. aureus isolated from the lesional and non‐lesional skin of identical AD patients. In this study, therefore, we examined whether the presence of superantigen‐producing S. aureus correlates with the formation of eczematous lesion of AD patients. The detection rate of S. aureus on the lesional skin of AD patients was higher than on the non‐lesional skin of AD patients. Furthermore, the bacterial cell count of S. aureus on the lesional skin of AD patients was also significantly higher than that of the non‐lesional skin of AD patients. However, there was no significant difference between the detection rate of superantigenic exotoxin‐producing S. aureus on the lesional and non‐lesional skin of AD patients. These results suggest that the number of S. aureus present is more important in the formation of eczematous lesion of AD patients than the presence of superantigenic exotoxin‐producing S. aureus strains per se.
Effect of Sulbactam on Anti‐Pseudomonal Activity of β‐Lactam Antibiotics in Cells Producing Various Levels of the MexAB‐OprM Efflux Pump and β‐Lactamase
Authors: Nakae Taiji , Saito Kohjiro , Nakajima Akira
Published: November 2013

The β‐lactamase inhibitor, sulbactam, was tested for β‐lactamase inhibitory activity in Pseudomonas aeruginosa cells producing various levels of both the MexAB‐OprM efflux pump and β‐lactamase. We found that sulbactam lowered the MICs of cefoperazone and piperacillin by inhibiting the β‐lactamase 8‐fold in the cell producing a constitutively high level of AmpC‐type β‐lactamase and a wild‐type level of MexAB‐OprM pump compared with that without sulbactam. The MICs of cefoperazone and piperacillin in the cell producing a constitutively high level of both the efflux pump and β‐lactamase under the presence of sulbactam were 8 and 4 times, respectively, lower than that without sulbactam. The MICs of sulbactam in the cell producing a constitutively high and a wild‐type level of the efflux pump were 16‐ and 8‐fold higher, respectively, than that in the mutant lacking the efflux pump. We concluded that sulbactam exerts potent β‐lactamase inhibitory activity in the cell producing a high level of efflux pump, in spite of the fact that sulbactam serves as a substrate of the MexAB‐OprM pump. Increasing amounts of sulbactam over the weight of β‐lactams further strengthen the effect of β‐lactam antibiotics.
Characterization and Identification of Borrelia Isolates as Borrelia valaisiana in Taiwan and Kinmen Islands
Authors: Masuzawa Toshiyuki , Pan Ming‐jeng , Kadosaka Teruki , Kudeken Midori , Takada Nobuhiro , Yano Yasuhiro , Imai Yasuyuki , Yanagihara Yasutake
Published: November 2013

Eleven pure cultures of Borrelia from 3 species of wild rodents (Apodemus agrarius, Mus formosanus, Rattus losea) captured in Taichung, located in the center of Taiwan island, and on Kinmen Island were characterized. Five isolates showed restriction fragment length polymorphism (RFLP) patterns of 5S‐23S rRNA gene intergenic spacer sequences identical to those of strains 5MT and 10MT, identified as Borrelia valaisiana, which were isolated in the southern tip of South Korea. Although the remaining six isolates showed novel RFLP patterns, these isolates showed more similarity to members of B. valaisiana from Korea, Japan and Europe based on 16S rRNA gene and flagellin gene sequences. This led us to speculate that transmission and proliferation of this type of borrelia occurred between Taiwan and the southern part of South Korea.
IL‐12, IFN‐γ, and TNF‐α Released from Mononuclear Cells Inhibit the Spread of Varicella‐Zoster Virus at an Early Stage of Varicella
Authors: Torigoe Sadayoshi , Ihara Toshiaki , Kamiya Hitoshi
Published: November 2013

The activity of mononuclear cells to inhibit plaque formation of varicella‐zoster virus (VZV) was investigated by an in vitro infectious center assay. Peripheral blood mononuclear cells (PBMC) inhibited VZV plaque formation by co‐cultivation with VZV‐infected fibroblasts. As compared to mononuclear cells from normal individuals, mononuclear cells from umbilical cord blood and from patients receiving corticosteroids showed a significant decrease in the ability to inhibit viral replication. This ability was significantly increased for mononuclear cells collected during the acute phase of varicella. PBMC obtained from patients in the acute phase of varicella produced significantly higher amounts of interferon (IFN)‐γ, tumor necrosis factor (TNF)‐α, and interleukin (IL)‐12 in the supernatant compared with those of healthy individuals. These data suggest that the cytokines have an important role in the inhibition of the spread of VZV at an early stage of varicella. Th1 type adaptive immunity might play a major role in VZV infection.
High‐Dose Corticosteroid Administration Induces Increase of Serum Macrophage Migration Inhibitory Factor in Patients with Vogt‐Koyanagi‐Harada's Disease
Authors: Kitaichi Nobuyoshi , Kotake Satoshi , Mizue Yuka , Sasamoto Yoichi , Goda Chiho , Iwabuchi Kazuya , Onoé Kazunori , Matsuda Hidehiko , Nishihira Jun
Published: November 2013

To investigate the influence of corticosteroid administration on the serum level of macrophage migration inhibitory factor (MIF), sera obtained from 9 patients with Vogt‐Koyanagi‐Harada's disease who had been treated with high‐dose corticosteroid were analyzed. The serum MIF levels of most patients were prominently increased on day 7 and/or day 14 after corticosteroid treatment. No TNF‐α was detected in the sera. The average serum MIF level of nine patients at the highest stages after corticosteroid administration was significantly higher than that before the corticosteroid treatment. It seems that MIF is a unique cytokine and acts together with corticosteroid to regulate inflammation and immunity.
First Detection ofEhrlichia platysin Dogs and Ticks in Okinawa, Japan
Authors: Motoi Yurie , Satoh Hiroshi , Inokuma Hisashi , Kiyuuna Tsuyoshi , Muramatsu Yasukazu , Ueno Hiroshi , Morita Çhiharu
Published: November 2013

We investigatedEhrlichia. platysinfection of dogs and ticks in Okinawa, Japan. UsingE. platysspecific primers, E. platys and HE3‐R, PCR‐positive results were obtained with 32.0% (64/200) of blood samples of dogs and 3.8% (3/77) of ticks. The nucleotide sequences of the amplified DNA fragment from the dogs and the ticks infesting them were identical, and the sequence corresponded to that of theE. platysGzh981 strain. We concluded that there is a cyclic maintenance ofE. platysbetween dogs and ticks in Okinawa.
Elimination of Latently HIV‐1‐Infected Cells by Lymphoblasts Armed with Bifunctional Antibody
Authors: Yin Shuping , Okada Noriko , Okada Hidechika
Published: November 2013

The Fab′ fragment of a monoclonal antibody (mAb) to CD3 and the F(ab′)2 fragment of a mAb to human immunodeficiency virus 1 (HIV‐1) gp41 were combined to generate a bifunctional antibody (BFA). The mAb to gp41 (IV1‐4G6) has previously been shown to react with a number of HIV‐1 strains and T‐lymphoblastoid cells (TLBC) armed with the BFA (BFA‐TLBC) effectively inhibited HIV‐1 in primarily cultured lymphoblasts infected with the clinically isolated virus which was reactive to the mAb. Although BFA‐TLBC could not cause cytolysis of 51Cr‐labeled latently infected cells (OM‐10.1) in 6 hr incubation, cocultivation of OM‐10.1 cells with BFA‐TLBC for 3 days or more eliminated the latently infected cells making the cells susceptible to BFA‐TLBC. Therefore, BFA‐TLBC may be beneficial for HIV‐infected patients in eradicating latently infected cells which can not be eliminated even with highly active antiretroviral therapy (HAART).
Rapid Detection and Quantification of Five Periodontopathic Bacteria by Real‐Time PCR
Authors: Sakamoto Mitsuo , Takeuchi Yasuo , Umeda Makoto , Ishikawa Isao , Benno Yoshimi
Published: November 2013

The quantity of periodontopathic bacteria in plaque samples is an important determinant for understanding the etiologic role of bacteria. The real‐time PCR method was used to detect and quantify periodontopathic bacteria, such as Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Porphyromonas gingivalis, Treponema denticola, and Treponema socranskii, in saliva and subgingival plaque samples. There was good agreement between the results of conventional PCR and real‐time PCR methods. Using the LightCycler™ system we were able to determine the amount of periodontopathic bacteria within an hour. The real‐time PCR method was linear for samples containing from 103 to more than 108 cells (r2=0.999). The application of the real‐time PCR method should be useful in the rapid detection and quantification of periodontopathic bacteria in clinical samples.
Cross Reaction of Recombinant Equine Infectious Anemia Virus Antigen to Heterologous Strains and Application for Serological Survey among Horses in the Field
Authors: Sentsui Hiroshi , Inoshima Yasuo , Murakami Kenji , Akashi Hiroomi , Purevtseren Byamba , Pagmajav Ochir , Sugiura Takeo
Published: November 2013

Cross reactivity of equine infectious anemia virus (EIAV) antigen prepared using a recombinant baculovirus containing the p26 gene of strain P337‐V70 was examined by the agar gel immunodiffusion (AGID) test and enzyme‐linked immunosorbent assay (ELISA). Serum samples serially collected from 13 horses experimentally infected with six different EIAV strains (two or three horses per strain) were subjected to the test Positive reactions were observed in the AGID test and ELISA before or soon after the first feverish period and continued persistently in most of the horses. The results with recombinant antigens were essentially the same as those with the virion antigen prepared from horse cell cultures both in the AGID test and ELISA. The reactivities of the antigens were further compared using serum samples collected from horses in 1999 in certain districts of Mongolia where equine infectious anemia has been prevalent, and from horses in Japan in 1973 when EIA had not been eliminated completely from Japanese horses. These results were completely concurrent. Generally, recombinant antigens have high specificity but low cross reactivity to heterologous strains. However, the present study showed that the recombinant EIAV p26 antigen has cross reactivity to the heterologous strain and is useful for diagnosis of EIA in the field.
Role of Furin in Delivery of a CTL Epitope of an Anthrax Toxin‐Fusion Protein
Authors: Zhang Ye , Kida Yutaka , Kuwano Koichi , Misumi Yoshio , Ikehara Yukio , Arai Sumio
Published: November 2013

Anthrax toxin lethal factor (LF) in combination with anthrax toxin protective antigen (PA) was endocytosed and translocated to the cytosol of mammalian cells. Residues 1–255 of anthrax toxin lethal factor (LFn) was fused to a cytotoxic T lymphocyte (CTL) epitope of an influenza virus. For processing the toxins, PA must be cleaved into a 63‐kDa fragment (PA63) by furin, which is a subtilisin‐like processing endoprotease expressed by many eukaryotic cells. To test the ability of cells treated with the LFn fusion protein plus PA to deliver the epitope, CTL assay was performed. Two types of cell lines were identified, one was able to deliver CTL epitope while the other failed to efficiently deliver the epitope. To further elucidate the differences between these cells, the role of furin in these cells was examined. Disruption of the furin gene reduced its ability to deliver the CTL epitope. Furin expression in cells capable of efficiently delivering CTL epitope was quantitatively higher than in cells unable to deliver the epitope. The results suggest that furin plays a critical role in delivery of the CTL epitope of LFn fusion protein.
Use of Antiserum‐Coated Latex Particles for Serotyping Streptococcus pneumoniae
Authors: Arai Satoru , Konda Toshifumi , Wada Akihito , Matsunaga Yasuko , Okabe Nobuhiko , Watanabe Haruo , Inouye Sakae
Published: November 2013

The Quellung reaction provides a standard means for serotyping Streptococcus pneumoniae, but it requires microscopic examination with skillful technique. We have developed an improved agglutination method with anti‐rabbit IgG‐coated latex particles, which are sensitized with pooled antisera for serotyping/serogrouping S. pneumoniae. Our method is as specific and sensitive as the Quellung test, and much easier to perform.
First Records of Tick‐Borne Pathogens, Borrelia, and Spotted Fever Group Rickettsiae in Okinawajima Island, Japan
Authors: Takada Nobuhiro , Fujita Hiromi , Yano Yasuhiro , Ishiguro Fubito , Iwasaki Hiromichi , Masuzawa Toshiyuki
Published: November 2013

In early April 2000, tick‐borne pathogens were surveyed in the northern area of Okinawajima Island, Okinawa Prefecture, which is the southernmost area of Japan. Borrelia valaisiana, a Lyme disease spirochete, was isolated from a field mouse Mus calori, and unidentified rickettsiae of the spotted fever group were isolated from all stages of Amblyomma testudinarium. These are the first reports of these pathogens on Okinawajima Island.
Stenotrophomonas maltophilia Interaction with Human Epithelial Respiratory Cells In Vitro
Authors: de Abreu Vidipó Luciana , de Andrade Marques Elizabeth , Puchelle Edith , Plotkowski Maria‐Cristina
Published: November 2013

Bacteria of Stenotrophomonas maltophilia have been isolated with increasing frequency from the airways of cystic fibrosis (CF) patients, usually following P. aeruginosa infections, but their adherence to human epithelial respiratory cells has never been investigated. In this study, various S. maltophilia strains were seen to adhere to epithelial respiratory cells in vitro, mainly along intercellular junctions. Bacteria could also enter into host cells, as determined by the gentamicin exclusion assay and transmission electron microscopy. Cells co‐incubated with P. aeruginosa and S. maltophilia exhibited a significantly decreased adherence of these latter bacteria. No decrease in S. maltophilia adherence was observed when co‐infection was carried out with heat‐killed P. aeruginosa or when respiratory cells were first incubated with P. aeruginosa, before incubation with S. maltophilia. Our data suggest that P. aeruginosa infections do not account for the increased prevalence of S. maltophilia in CF patient airways, that thermolabile products from P. aeruginosa can control the adherence of S. maltophilia to respiratory cells and also that these two bacteria do not compete for cell receptors.
Characteristics of Macrolide‐Resistant Mycoplasma pneumoniae Strains Isolated from Patients and Induced with Erythromycin In Vitro
Authors: Okazaki Norio , Narita Mitsuo , Yamada Satoshi , Izumikawa Kinichi , Umetsu Masao , Kenri Tsuyoshi , Sasaki Yuko , Arakawa Yoshichika , Sasaki Tsuguo
Published: November 2013

Some patients with Mycoplasma pneumoniae infection are clinically resistant to antibiotics such as erythromycin, clarithromycin, or clindamycin. We isolated M. pneumoniae from such patients and found that one of three isolates showed a point mutation in the 23S rRNA gene. Furthermore, 141 EM‐sensitive clinical isolates of M. pneumoniae were cultured in broth medium containing 100 μg/ml of erythromycin (EM). Among 11 EM‐resistant strains that grew in the medium, point mutations in the 23S rRNA were found in 3 strains at A2063G, 5 strains at A2064G and 3 strains at A2064C. The relationship between the point mutation pattern of these EM‐resistant strains and their resistance phenotypes to several macrolide antibiotics was investigated.
The First Large‐Scale Nucleic Acid Amplification Testing (NAT) of Donated Blood Using Multiplex Reagent for Simultaneous Detection of HBV, HCV, and HIV‐1 and Significance of NAT for HBV
Authors: Ohnuma Hitoshi ,   Japanese Red Cross NAT Screening Research Group, Tanaka Takeshi , Yoshikawa Akira , Murokawa Hiroyuki , Minegishi Kiyoshi , Yamanaka Retsuji , Lizuka Hisao Yugi , Miyamoto Masaki , Satoh Shuhei , Nakahira Seiji , Tomono Tsugikazu , Murozuka Takashi , Takeda Yoshio , Doi Yasushi , Mine Hideko , Yokoyama Shigeki , Hirose Toru , Nishioka Kusuya
Published: November 2013

The first nationwide nucleic acid amplification testing (NAT) for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV‐1) of voluntarily donated blood after serological pre‐screening and before release of cellular components and plasma for fractionation was implemented by the Japanese Red Cross Blood Transfusion Services. From February 1, 2000 to April 30, 2001, specimens from 6,805,010 units of serologically negative donation were screened in minipools of 50 samples within 24 hr after blood donation by NAT using multiplex HBV/HCV/HIV‐1 reagent for blood transfusion including short shelf‐life platelets. Among them, 112 HBV DNA‐positives, 25 HCV RNA positives and 4 HIV‐1 RNA positives were screened out and we could prevent transfusion of these NAT positive units. Subtypes/genotypes of HBV DNA, adr/C, adw/A, adw/B, adw/C, ayr/C and ayw/D were found and adr/C was predominant. A total of 61.6% of them (69/112) were negative by overnight EIA. Sixth three of HBV NAT‐positive samples carried virus loads less than 104 copies/mL and 92.1% of them (58/63) were negative by overnight EIA. The virus growth curves of HBV in 6 cases obtained by retrospective and prospective follow‐up study showed exponential straight lines in the early stage of serological window periods and the log times of HBV growth (10 fold increase) in serological window period were between 4.6 and 7.6 days. NAT screening with highly sensitive reagents in pool of specimens is useful to exclude blood units with low level of HBV and HBV mutants from blood transfusion.
Inhibition of p21/Waf1/Cip1/Sdi1 Expression by Hepatitis C Virus Core Protein
Authors: Yoshida Isao , Oka Kiyomasa , Hidajat Rachmat , Nagano‐Fujii Motoko , Ishido Satoshi , Hotta Hak
Published: November 2013

The possibility of interaction between hepatitis C virus (HCV) core protein and the cell cycle regulator protein p21/Waf1/Cip1/Sdi1 (p21/Waf1) in cultured cells was analyzed. Although colocalization of HCV core protein and p21/Waf1 was not clearly observed, p21/Waf1 expression was much weaker in HCV core protein‐expressing cells than in the control. A Northern blot analysis showed nearly the same level of p21/Waf1 mRNA in both cells, suggesting that HCV core protein inhibited p21/Waf1 expression post‐transcriptionally. The degradation patterns of p21/Waf1 did not differ significantly in HCV core protein‐expressing cells and in the control, suggesting that the stability of p21/Waf1, once it was accumulated in the cell, was not significantly affected by HCV core protein. But this does not necessarily exclude the possibility that synthesis, maturation, and nuclear transport of p21/Waf1 is impaired, or that the degradation of newly synthesized, improperly processed p21/Waf1 is promoted by HCV core protein. The decrease in p21/Waf1 accumulation was partially inhibited by proteasome inhibitors and a calpain inhibitor in both HCV core protein‐expressing cells and the control. In vitro kinase assay revealed that a p21/Waf1‐mediated inhibition of cyclin‐dependent kinase 2 activity was partially negated by HCV core protein. Taken together, the present results suggest that HCV core protein inhibits p21/Waf1 expression post‐transcriptionally and impairs the function of p21/Waf1 in the cell.
Changes in the Cell Structure of Flavobacterium psychrophilum with Length of Culture
Authors: Kondo Motoki , Kawai Kenji , Yagyu Ken‐ichi , Nakayama Kouichi , Kurohara Kenrou , Oshima Syun‐ichirou
Published: November 2013

Flavobacterium psychrophilum, the pathogen of bacterial cold‐water disease, causes serious problems in ayu Plecoglossus altivelis culture. This study investigated the effect of the culture period of F. psychrophilum and on the structure of its cells. From the SDS‐PAGE of total proteins of cellular components, much difference was found between the 36 hr culture and the 48 and 72 hr cultures. A SEM observation of the cells showed many fragments, especially on the cell surface of the 36 hr culture. These fragments consisted of an outer membrane, seen by TEM observation, and may contain substances causing the virulence. Specific proteins observed by the SDS‐PAGE and fragments in the 36 hr culture may be related to the virulence of F. psychrophilum.
Bacterial Population in Russian Space Station “Mir”
Authors: Kawamura Yoshiaki , Li Ying , Liu Hongsheng , Huang Xinxiang , Li Zhiyu , Ezaki Takayuki
Published: November 2013

We had the opportunity to investigate the bacterial population in air samples, condensation water, and inner wall swabs from the Russian space station Mir. From the first and second air samples during the mission, 29 and 7 bacterial colonies were collected, respectively. The values were equivalent to 16.8 and 4.0 cfu/100 liter air, respectively. Condensation water was collected from three different sites. The total viable bacterial counts were 2.1 × 106, 5.2 × 102, and 3.0 × 101 cfu/ml. The phylogenetic position of each isolate was determined by total 16S rDNA sequencing. Bacteria from air samples were mainly Gram‐positive (35/36 colonies), and staphylococci occupied dominant specifically (23/36 colonies). On the other hand, Gram‐negative bacteria were mainly isolated from condensation water samples. Most strains were thought to be opportunistic pathogens or environmental bacteria (such as those that inhabit soil, water, or air) found on earth. However, 6 of 23 isolates were suspected to be new species according to phylogenetic analysis and quantitative DNA‐DNA hybridization data. The isolation of the other levels 3 and 2 bacteria, using specific selective media, was unsuccessful because all samples were heavily contaminated with fungi. To overcome this situation, PCR methods were applied to survey most levels 3 and 2 pathogenic bacteria in the condensation water samples. Up to 380 different primers for bacterial pathogens were used in this study. Only Mycobacterium avium 16S DNA sequences, however, could be amplified from the three water samples. The average bacteria count was estimated to be about 104 organisms/ml water.
Correlation between Human Herpesvirus 6 and 7 Infections after Living Related Liver Transplantation
Authors: Ihira Masaru , Yoshikawa Tetsushi , Suzuki Kyoko , Ohashi Masahiro , Suga Sadao , Asonuma Katsuhiro , Tanaka Koichi , Asano Yoshizo
Published: November 2013

Human herpesvirus 6 (HHV‐6) and human herpesvirus 7 (HHV‐7) are closely related to each other. Interaction between the two viruses at the time of primary HHV‐7 infection is suggested by in vivo and in vitro studies. However, interaction between the two viruses in organ transplant recipients has not been analyzed. We analyzed serially collected plasma samples obtained from 40 living related liver transplant recipients by serological assay (indirect immunofluorescence assay, IFA) and polymerase chain reaction (PCR). Significant increase or seroconversion of HHV‐6 IgG and HHV‐7 IgG antibody titers were observed in 45% and 58% of recipients respectively. Positive rate of IgM HHV‐6 antibody increased up to 35% at 4 weeks after transplantation. However, no remarkable peak in the positive rate of HHV‐7 IgM antibody was demonstrated. HHV‐6 and HHV‐7 DNA were detected in plasma in 15 (38%) and 16 (40%) of the 40 recipients respectively. HHV‐6 DNA was detected in 10 (26%) of the 38 recipients at 2 weeks after transplantation. The positive rate of the virus genome in plasma gradually decreased after that time. HHV‐7 DNA was detected in 5 (14%) of the 37 recipients at 2 weeks after transplantation; no obvious peak in the positive rate of HHV‐7 DNA was demonstrated. Antibody responses involving both HHV‐6 and HHV‐7, including either a significant increase in IgG antibody titers or positive identification of IgM antibody were observed in 17 (43%) of the 40 recipients. Thirteen out of the 17 recipients demonstrated concurrent antibody response against both viruses. HHV‐7 antibody response preceded the HHV‐6 antibody response in 2 of the remaining 4 recipients, whereas the opposite was true in the other 2 recipients. Both HHV‐6 and HHV‐7 DNA were detected in 7 (18%) of the 40 recipients. In 4 of those 7 recipients, DNA from both viruses was concurrently detected, 3 of whom had HHV‐7 DNA repeatedly detected after first detection of the virus DNA. The detection of HHV‐7 DNA preceded the detection of HHV‐6 DNA in 2 recipients, whereas HHV‐6 DNA appeared first in 1 recipient.
Investigation of Varicella‐Zoster Virus DNA in Lymphocyte Subpopulations by Quantitative PCR Assay
Authors: Ito Yoshinori , Kimura Hiroshi , Hara Shinya , Kido Shinji , Ozaki Takao , Nishiyama Yukihiro , Morishima Tsuneo
Published: November 2013

To investigate the nature of viremia during the acute phase of varicella, we studied the viral load in nine otherwise healthy children with varicella. Plasma and peripheral blood mononuclear cells (PBMC) were obtained, then PBMC were divided into CD4+T, CD8+T, and B lymphocytes and monocyte/macrophage fractions. The viral DNA in each component was quantified using a real‐time quantitative polymerase chain reaction assay. Varicella‐zoster virus (VZV) DNA was detected in plasma, PBMC and all subpopulations. The amount of viral DNA was similar in each PBMC subpopulation, suggesting that each lymphocyte fraction and monocytes carry similar amounts of VZV DNA during viremia.
Proteolytic Cleavage of Staphylococcal Exoproteins Analyzed by Two‐Dimensional Gel Electrophoresis
Authors: Kawano Yasushi , Kawagishi Mika , Nakano Miyo , Mase Kiyomi , Yamashino Takafumi , Hasegawa Tadao , Ohta Michio
Published: November 2013

Extracellular proteases of Staphylococcus aureus are emerging as potential virulence factors that are relevant to the pathogenicity of staphylococcal infections. These proteases may also be involved in the proteolytic cleavage of other exoproteins released from this organism. To define the target exoproteins and their sites of cleavage by proteases, high‐resolution two‐dimensional polyacrylamide gel electrophoresis followed by N‐terminal amino acid sequencing of exoprotein spots was performed. Two to three hundred exoprotein spots were detected at the early‐stationary phase of cultures of S. aureus NCTC8325, and then at the late‐stationary stage most of these high molecular protein spots became invisible due to further proteolytic degradation. As the result of N‐terminal analysis, lipase, triacylglycerol lipase, orf619 protein and orf388 protein were detected as multiple spots at the early‐stationary phase. We found that these exoproteins were cleaved at 3, 7, 4 and 4 different sites, respectively, by proteases. According to the M.W. and pI of each peptide spot obtained from the gel and their matches with calculated values in addition to their N‐terminal sequences, we showed that the positions of putative peptides resulted from proteolytic cleavage of these proteins.
Burkholderia pseudomallei Interferes with Inducible Nitric Oxide Synthase (iNOS) Production: A Possible Mechanism of Evading Macrophage Killing
Authors: Utaisincharoen Pongsak , Tangthawornchaikul Nattaya , Kespichayawattana Wanapa , Chaisuriya Predawan , Sirisinha Stitaya
Published: November 2013

Burkholderia pseudomallei is a causative agent of melioidosis, a life threatening disease which affects humans and animals in tropical and subtropical areas. This bacterium is known to survive and multiply inside cells such as macrophages. The mechanism of host defense against this bacterium is still unknown. In this study, we demonstrated that B. pseudomallei exhibited unique macrophage activation activity compared with Escherichia coli and Salmonella typhi. The mouse macrophage cell line (RAW 264.7) infected with B. pseudomallei at MOI of 0.1:1, 1:1 and 10:1 did not express a detectable level of inducible nitric oxide synthase (iNOS). Moreover, the B. pseudomallei infected cells released TNF‐α only when they were infected with high MOI (10:1). Unlike the cells infected with B. pseudomallei, the cells infected with E. coli, and S. typhi expressed iNOS even at MOI of 0.1:1. These infected cells also released a significantly higher level of TNF‐α at the low MOI ratio. The cells that were preactivated with IFN‐γ prior to being infected with B. pseudomallei exhibited an enhanced production of iNOS and TNF‐α release. The increased macrophage activation activity in the presence of IFN‐γ also correlated with the restriction of the intracellular bacteria survival. Moreover, IFN‐γ also prevented cell fusion and multinucleated cell formation induced by B. pseudomallei, a phenomenon recently described by our group. Altogether, these results indicate that internalization of B. pseudomallei failed to trigger substantial macrophage activation, a phenomenon which could prolong their survival inside the phagocytic cells and facilitate a direct cell to cell spreading of B. pseudomallei to neighboring cells.
Identification of Shiga Toxin‐Producing Escherichia coli Possessing Insertionally Inactivated Shiga Toxin Gene
Authors: Okitsu Tadayuki , Kusumoto Masahiro , Suzuki Rieko , Sata Shin , Nishiya Yoshiaki , Kawamura Yoshihisa , Yamai Shiro
Published: November 2013

We have investigated the Shiga toxin genes of Shiga toxin‐producing Escherichia coli (STEC) strains, using polymerase chain reaction (PCR) amplifying the full lengths of these genes. As a result, we found the Shiga toxin 2 gene which was insertionally inactivated by an insertion sequence (IS). This IS element was identical to IS1203v which has been also found in inactivated Shiga toxin 2 genes, and was inserted at the same site as in the previous paper. On the other hand, both Shiga toxin 2 genes were different (98.3% identity). These suggested that IS1203v independently inserted into each Shiga toxin 2 genes, and STEC strains possessing the insertionally inactivated Shiga toxin genes are most likely to have a wide distribution. Amplification of the full length of the Shiga toxin gene is one of the effective methods to detect the gene no matter where the IS element is included, i.e., the insertion can be reflected in the size of amplicon.
Region of Heat‐Stable Enterotoxin II of Escherichia coli Involved in Translocation across the Outer Membrane
Authors: Okamoto Keinosuke , Yamanaka Hiroyasu , Takeji Miho , Fujii Yoshio
Published: November 2013

Heat‐stable enterotoxin II of Escherichia coli (STII) is synthesized as a precursor form consisting of pre‐ and mature regions. The pre‐region is cleaved off from the mature region during translocation across the inner membrane, and the mature region emerges in the periplasm. The mature region, composed of 48 amino acid residues, is processed in the periplasm by DsbA to form an intramolecular disulfide bond between Cys‐10 and Cys‐48 and between Cys‐21 and Cys‐36. STII formed with these disulfide bonds is efficiently secreted out of the cell through the secretory system, including TolC. However, it remains unknown which regions of STII are involved in interaction with TolC. In this study, we mutated the STII gene and examined the secretion of these STIIs into the culture supernatant. A deletion of the part covering from amino acid residue 37 to the carboxy terminal end did not markedly reduce the efficiency of secretion of STII into the culture supernatant. On the other hand, the efficiency of secretion of the peptide covering from the amino terminal end to position 18 to the culture supernatant was significantly low. These observations indicated that the central region of STII from amino acid residue 19 to that at position 36 is involved in the secretion of STII into the milieu. The experiment using a dsbA‐deficient strain of E. coli showed that the disulfide bond between Cys‐21 and Cys‐36 by DsbA is necessary for STII to adapt to the structure that can cross the outer membrane.
Effector Mechanism and Clinical Response of BAK (BRM‐Activated Killer) Immuno‐Cell Therapy for Maintaining Satisfactory QOL of Advanced Cancer Patients Utilizing CD56‐Positive NIE (Neuro‐Immune‐Endocrine) Cells
Authors: Ebina Takusaburo , Ogama Naoko , Shimanuki Hiroko , Kubota Tomoka , Isono Noriko
Published: November 2013

A new type of immuno‐cell therapy called BRM‐activated killer (BAK) therapy using non‐MHC‐restricted lymphocytes, CD56‐positive cells, was devised. Peripheral blood lymphocytes were selected by immobilization with anti‐CD3 monoclonal antibody and cultured for 2 weeks in the presence of IL‐2. Thereafter, they were reactivated by 1,000 U/ml of IFN‐α for 15 min. Twenty‐six outpatients with cancer whose performance status were over 80% on Karnofsky scale were selected for this study. About 6 × 109 BAK cells were returned by intravenous drip infusion, at one month intervals at an outpatient clinic to each of 20 advanced cancer patients in whom many metastatic lesions were found postoperatively, and to 6 patients with no postoperatively detectable metastases. The proportion of CD56‐positive cells increased from 20% to 50% with culture. CD56‐positive cells have strong cytotoxic activity and produced 20 ng/109 cells of β‐endorphin, an intracerebral hormone. During the course of BAK therapy, we adopted the Face scale as a QOL indicator. The QOL of all patients remained satisfactory or improved. β‐Endorphin is thought to make patients fell well and maintains good QOL because of its potent analgesic, sedative activity. From that facts that CD56 is a neural cell adhesion molecule and a member of the Ig superfamily, and that the CD56‐positive cell produces β‐endorphin, we concluded that the CD56‐positive cell is a multifunctional, integrated NIE (neuro‐immune‐endocrine) cell. Administration of BAK cells allowed all 20 advanced cancer patients with metastases to survive for over one year. All 6 patients receiving the same therapy for prevention of postoperative metastasis have been recurrence‐free for one to five years.
Gene Analysis of Vibrio cholerae NAGV14 Pilus and Its Distribution
Authors: Kuroki Hiromi , Toma Claudia , Nakasone Noboru , Yamashiro Tetsu , Iwanaga Masaaki
Published: November 2013

Adhesive pilus of Vibrio cholerae O34, strain NAGV14, was genetically analyzed. The deduced amino acid (aa) sequence of the major pilin structural gene (VcfA) was 67% homologous to the MshA pilin in the N‐terminal region, but no homology was found in the C‐terminal region which contained the antigenic epitopes. Upstream and downstream flanking regions examined were highly homologous to mshB and mshC of the MSHA (mannose‐sensitive hemagglutinin) gene locus. A short leader sequence and a pair of cysteines near the C‐terminus which are the characteristics of type 4a pilus family were found. The major pilin structural gene of NAGV14 was compared to that of a strain V10 producing non‐adhesive pili. The deduced aa sequences showed 60% homology, and the distance between two cysteines in the C‐terminal region was different. A total of 177 V. cholerae strains were investigated for the presence of a type 4 pilus gene locus by PCR, and 95% were positive. The major pilin gene of NAGV14 was detected in 4 of 93 V. cholerae non‐O1, non‐O139 strains tested, but none of the V. cholerae O1 and O139 (72 and 12 strains, respectively). Our result suggested that a type 4 pilus gene locus similar to the MSHA gene locus is widely distributed among V. cholerae strains. We proposed naming this type 4 pilus gene locus the VCF (for V. cholerae flexible pili) gene locus.
3′‐Fluorine‐Substitution in 3‐Fluorocarbocyclic Oxetanocin A Augments the Drug's Inhibition of HHV‐6B Propagation in Chronically Infected Cultures
Authors: Wang Ping , Abe Kenji , Ojima Tomoko , Ohyashiki Junko H. , Satoh Hitoshi , Maruyama Tokumi , Nagata Hiroshi , Tanaka Hiroshi , Yamamoto Kohtaro
Published: November 2013

An infection of TaY cells, which originated from an adult T‐cell leukemia, with an HHV‐6B OK isolate resulted in a chronically infected culture, termed TaY(OK). Cell cloning analysis revealed that the TaY(OK) culture consisted of a mixture of cells permissive and refractory to the infection, and that the permissive cells were continuously produced from the refractory cell population. Since the chronically infected culture has been maintained for over 2 years without the addition of uninfected TaY cells, we used it for an evaluation of the antiviral potency of nucleoside analogs, especially carbocyclic oxetanocins (COXTs). MTT (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide) assays showed a lack of toxicity of ganciclovir (GCV), COXTs, and their derivatives, to TaY(OK) cells at 1 μm. Therefore we compared the antiviral potencies of these drugs at 1 μm by monitoring the viral loads produced during a 1‐day period during the course of the drug treatment. Among the drugs tested, 3′‐fluorocarbocyclic oxetanocin A (3′‐C.OXT‐A) was the most effective for inhibiting the virus production, and at concentrations ranging from 0.5 μm to 10 μm, the inhibition of the viral production was dose‐dependent. A comparison of the chemical structures of the derivatives with that of C.OXT‐A, which is the parental molecule, suggested that the 3′‐fluorine‐modification might account for the higher anti‐HHV‐6 activity and lower cytotoxicity.
A Sandwich ELISA for Detection of Soluble GPI‐80, a Glycosylphosphatidyl‐Inositol (GPI)‐Anchored Protein on Human Leukocytes Involved in Regulation of Neutrophil Adherence and Migration—Its Release from Activated Neutrophils and Presence in Synovial Fluid of Rheumatoid Arthritis Patients—
Authors: Huang Jibiao , Takeda Yuji , Watanabe Tadashi , Sendo Fujiro
Published: November 2013

GPI‐80, mainly distributed on human neutrophils and monocytes, is a novel glycosylphosphatidylinositol (GPI)‐anchored protein regulating neutrophil adherence and migration. We prepared a polyclonal antibody specific to GPI‐80 by immunizing rabbits with the purified GPI‐80 and established a sandwich ELISA for detecting soluble GPI‐80. We found that soluble GPI‐80 was released from fMLP activated neutrophils and was present at high concentrations in synovial fluids but not sera of rheumatoid arthritis patients, suggesting that GPI‐80 may play a role in inflammatory diseases. The detection of soluble GPI‐80 may help us to understand its physiological and pathological functions.
Regulation of Fas‐Mediated Apoptosis by NF‐κB Activity in Human Hepatocyte Derived Cell Lines
Authors: Marusawa Hiroyuki , Hijikata Makoto , Watashi Koichi , Chiba Tsutomu , Shimotohno Kunitada
Published: November 2013

Nuclear Factor‐κB (NF‐κB) is known to be one of the key regulators of genes involved in survival signaling. The purpose of this study is to investigate the role of NF‐κB activity in signaling events related to cell survival in hepatocytes, which has been supposed to be one of the most sensitive organs against Fas‐induced cytotoxicity. The functions of NF‐κB activity on Fas‐mediated apoptosis in different human cell lines were investigated by a magnetic concentration system for cells with exogenous protein production. We demonstrated that the activation of NF‐κB was triggered by anti‐Fas treatment in hepatocyte derived cell lines, HepG2 and Huh‐7 cells. Overexpression of kinase‐inactive NF‐κB‐inducing kinase (NIK) and IκB kinase (IKK) inhibited the activation of NF‐κB introduced by anti‐Fas treatment in these cells. Notably, inactivation of NF‐κB by the production of IκB‐α protein made these cells more susceptible to apoptosis induced by Fas stimulation. In contrast, transient expression of NIK showed a suppressive effect on Fas‐mediated apoptosis in the same cell lines. These findings suggest the involvement of NF‐κB in suppression of Fas‐mediated apoptosis in human hepatocyte derived cell lines, in which concomitant activation of this transcriptional factor was observed through the stimulation of Fas signaling.