bluuebunny/crossref_metadata_2025 huggingface dataset:
Further Characterization of a CD99‐Related 21‐kDa Transmembrane Protein (VAP21) Expressed in Syrian Hamster Cells and Its Possible Involvement in Vesicular Stomatitis Virus Production
Authors: Tochikura Tadafumi S. , Xiao Sa , Ego Takeshi , Sagara Junji , Kawai Akihiko
Published: November 2013

The VAP21, a CD99‐related 21‐kDa transmembrane protein, was first detected in the enveloped virions that were grown in a Syrian hamster‐derived cell line, BHK‐21 (Sagara et al., 1997; Yamamoto et al., 1999). We further tried to elucidate the nature and properties of VAP21. The VAP21 was detected in various organs of the Syrian hamster as well as in the Syrian hamster‐derived cell lines (BHK‐21 and HmLu‐1). We could not detect the VAP21 antigen in other cell lines derived from other animal species we examined, including a Chinese hamster (CHO‐K1), mouse (neuroblastoma C1300, clone NA), dog (MDCK), monkey (COS‐7), and human (HeLa, HepG2). We tried to introduce the VAP21 gene into VAP21‐negative cell lines using a tetracycline‐regulated gene expression system. All of our trials, however, resulted in failure to establish stably positive inducible cell lines. To the contrary, we could easily establish the VAP21‐overexpressing cell lines from the Syrian hamster cell lines, which were successfully grown and maintained without any loss of VAP21 expression even under the induced culture conditions. In such VAP21‐overexpressing cells, production of the vesicular stomatitis virus (VSV) was increased several‐fold, while suppression of the VAP21 expression resulted in reducing the VSV yields. From these results, we conclude that the VAP21 is a physiologically active cell membrane component of some animal species including the Syrian hamster, and might positively be involved in the VSV replication.
Prevalence of Infection and Genotypes of GBV‐C/HGV among Homosexual Men
Authors: Hattori Junko , Ibe Shiro , Nagai Hiromi , Wada Kaoru , Morishita Takayuki , Sato Katsuhiko , Utsumi Makoto , Kaneda Tsuguhiro
Published: November 2013

Since the discovery of GB virus‐C (GBV‐C) and hepatitis G virus (HGV), many studies have been performed. These viruses are now known to be parenterally, as well as sexually transmitted. A phylogenetic analysis also revealed that GBV‐C has five major genotypes: type 1 predominates in West Africa, type 2 in Europe and the United States, type 3 in parts of Asia, type 4 in Southeast Asia, and type 5 in South Africa. Despite the number of reports so far, there have been few large‐scale surveys of homosexual men to determine the prevalence of the GBV‐C/HGV infections. We examined the levels of GBV‐C/HGV viremia in 297 homosexual men who attended the Nagoya Lesbian and Gay Revolution held in Nagoya, Japan. Reverse transcription‐polymerase chain reaction (RT‐PCR)/nested PCR of the GBV‐C/HGV 5′‐non‐coding region (NCR), and base sequence analyses showed that the infection rate was 12.5%, and genotypes in this population were classified into type 2 (32%) and type 3 (68%). None were classified as types 1, 4, or 5 in this study. Our results indicate that the GBV‐C/HGV type 2 seen mainly in Europe and the US is spreading widely in Japan, especially in the Nagoya district.
Ability of CD8+ T Cell Anti‐Feline Immunodeficiency Virus Activity Correlated with Peripheral CD4+ T Cell Counts and Plasma Viremia
Authors: Hohdatsu Tsutomu , Yamazaki Ayanori , Yamada Mitsunobu , Kusuhara Hajime , Kaneshima Takashi , Koyama Hiroyuki
Published: November 2013

In the host defense mechanism against feline immunodeficiency virus (FIV) infection, CD8+ T cells specifically attack virus‐infected cells and suppress the replication of the virus in a non‐cytolytic manner by secreting soluble factors. In this study, we measured CD8+ T cell anti‐FIV activity in 30 FIV‐infected cats. We investigated its relationship with the number of peripheral blood lymphocytes, particularly the CD4+ T cell and CD8+ T cell counts, and the relationship between anti‐FIV activity and the number of T cells of CD8α+βlo and CD8α+β− phenotypes. A clearly significant correlation was observed between anti‐FIV activity and the number of CD4+ T cells. A weaker anti‐FIV activity was associated with a greater decrease in the number of CD4+ T cells. However, there was no significant correlation between anti‐FIV activity and the number of B or CD8+ T cells. Compared with SPF cats, FIV‐infected cats had significantly higher CD8α+βlo T cell and CD8α+β− T cell counts, but, no significant correlation was observed between these cell counts and anti‐FIV activity. This anti‐FIV activity significantly correlated with plasma viremia, which was detected in cats with a weak anti‐FIV activity. These results suggest that the anti‐FIV activity of CD8+ T cells plays an important role in plasma viremia and the maintenance of CD4+ T cells in the body. It is unlikely that CD8α+βlo or CD8α+β− T cells appearing after FIV infection represent a phenotype of CD8+ cells with anti‐FIV activity.
Effects of Quorum Sensing on flaA Transcription and Autoagglutination in Campylobacter jejuni
Authors: Jeon Byeonghwa , Itoh Kikuji , Misawa Naoaki , Ryu Sangryeol
Published: November 2013

Some bacteria can communicate with other species of bacteria by means of autoinducer‐2 (AI‐2)‐mediated quorum sensing. In this study, we demonstrated that AI‐2‐mediated quorum sensing influences the transcription of flaA, the major flagellin gene in Campylobacter jejuni. A null mutation of luxS in C. jejuni strain 81116 reduced flaA transcription (approximately 43% that of the wild‐type) and induced a reduction in motility. However, the luxS mutant had the same level of total flagellin protein as the wild‐type. Transmission electron microscopy showed that the flagellar structure was preserved in the luxS mutant. The agglutination capability was reduced in the mutant strain, implying that quorum sensing might be involved in the formation of surface structures of C. jejuni. These observations suggest that AI‐2‐mediated quorum sensing may play a role in regulation of motility and surface properties in C. jejuni.
Two Different Scrapie Prions Isolated in Japanese Sheep Flocks
Authors: Hirogari Yasuhiro , Kubo Masanori , Kimura Kumiko M. , Haritani Makoto , Yokoyama Takashi
Published: November 2013

Two different scrapie prion strains with different characteristics were obtained from two sheep naturally infected with scrapie in Japan. In mice transmission, one (Tsukuba‐1) showed shorter incubation periods (133±2 days) than the other (Tsukuba‐2) (288±5 days). Spongiform changes and accumulation of an abnormal isoform of prion protein (PrPSc) were observed throughout the brain in Tsukuba‐1 inoculated mice, while the lesions and the PrPSc accumulation were localized in the brain stem of mice with Tsukuba‐2. Western blot analysis showed that there was no strain‐specific glycoform of PrPSc within these two strains. A super‐infection experiment revealed that neither strain interfered with the other's propagation.
TCRδ Gene Rearrangements Revealed by Fine Structure of the Recombination Junction in Mice
Authors: Kanari Yasuyoshi , Muto Masahiro , Yamagishi Hideo
Published: November 2013

The standard products of V(D)J recombination of immunoglobulin and T cell receptor genes are two kinds of DNA junction, a coding joint and a signal joint. TCRδ V‐D and D‐D signal joints in adult mouse thymocytes were sequenced following PCR amplification. We observed differential nucleotide insertions at the Vδ‐Dδ signal joints, depending on the Vδ and Dδ gene usage in the developmental stage. Nucleotide insertions at the Vδ‐Dδ1 signal joints were less frequent for the Vδ4,5 genes preferentially utilized in adult thymocytes than for the Vδ3,6 genes, infrequently rearranged to Dδ1. In addition to standard signal joints, unexpectedly, novel nonstandard products, “replacement joints” of Dδ1 substituted downstream by the recombination signal sequence of Vδ were also found. However, no Dδ2‐associated replacement joints other than Vδ5 were found. The other replacement joints of Dδ1‐Dδ2 recombination were also observed. The mutation in TCRβ gene affected the frequency of nucleotide insertions at the Vδ‐Dδ signal joints and inhibited the formation of replacement joint. Recombination mechanism generating the replacement joint and the possible role of TCRβ in up‐regulation of TCRδ gene rearrangements are discussed.
Bacterial Artificial Chromosome Library of Finegoldia magna ATCC 29328 for Genetic Mapping and Comparative Genomics
Authors: Goto Takatsugu , Todo Kozo , Miyamoto Kazuaki , Akimoto Shigeru
Published: November 2013

We constructed a bacterial artificial chromosome (BAC) library of Finegoldia magna ATCC 29328 DNA to facilitate further genome analysis of F. magna. The BAC library contained 385 clones with an average insert size of 55 kb, representing a 10.1‐fold genomic coverage. Repeated DNA hybridization using primer sets designed on the basis of BAC‐end sequences yielded nine contigs covering 95% of the chromosome and two contigs covering 98% of the plasmid. The contigs were localized on the physical map of F. magna ATCC 29328 DNA. A total of 121 BAC‐end sequences revealed 103 unique genes, which had not been previously reported for F. magna. The homolog ORF of albumin‐binding protein (urPAB), one of the known virulence factors from F. magna, was sequenced and localized on the physical map. Homology analysis of 121 BAC‐end sequences revealed that F. magna is most closely related to clostridia, particularly Clostridium tetani. This close relationship is consistent with the recent classification of peptostreptococci based on 16S rRNA sequence analysis. The BAC library constructed here will be useful for the whole genome sequencing project and other postgenomic applications.
In Vivo Safety Assessment of Two Bifidobacterium longum Strains
Authors: Mäkeläinen Henna , Tahvonen Raija , Salminen Seppo , Ouwehand Arthur C.
Published: November 2013

Bifidobacteria are important members of the intestinal microbiota and are considered to contribute to maintaining health. However, the level of bifidobacteria colonising the intestine of elderly subjects tends to be lower than in younger adults. Therefore, two Bifidobacterium longum strains, isolated from healthy elderly, were chosen for supplementation of the endogenous Bifidobacterium microbiota in the elderly. Bifidobacteria are generally regarded safe for human consumption. However, since the strains are intended for consumption by the elderly, whom may be more prone to disease, it is important to ascertain their safety. For this purpose, the strains were given to healthy adult volunteers. No side effects were reported and no undesirable changes observed in the immune parameters measured. Based on this study it appears that the two strains are well tolerated by human subjects and there are no reservations about their food use.
The Basidiomycetous Yeasts Cryptococcus diffluens and C. liquefaciens Colonize the Skin of Patients with Atopic Dermatitis
Authors: Sugita Takashi , Saito Masuyoshi , Ito Tomonobu , Kato Yukihiko , Tsuboi Ryoji , Takeuchi Shohei , Nishikawa Akemi
Published: November 2013

Our previous research showed that lipophilic yeasts, Malassezia species, colonize the skin of patients with atopic dermatitis (AD) at a high frequency. In this study, we found that two basidiomycetous yeasts, Cryptococcus diffluens and C. liquefaciens, colonize the skin significantly more frequently in AD patients than in healthy subjects. Transparent dressings were applied to the skin of 36 AD patients and 30 healthy subjects and then transferred onto Sabouraud dextrose agar. Colonies recovered from the medium were identified by DNA sequence analysis of internal transcribed spacer regions and the D1/D2 26S rRNA gene. C. diffluens and C. liquefaciens were isolated from 42% (15/36) and 33% (12/36) of AD patients and from 20% (6/30) and 20% (6/30) of healthy subjects, respectively. In addition, fungal DNA was extracted directly from the dressings and amplified in a specific nested PCR assay. C. diffluens and C. liquefaciens DNA were detected in dressings from 97% (35/36) and 86% (31/36) of the AD patients and 47% (14/30) and 37% (11/30) of the healthy subjects, respectively. These findings show that Malassezia spp. are not the only yeasts that colonize the skin of AD patients; Cryptococcus spp. also are present in a high proportion of patients. The role of these microorganisms in AD is as yet unknown, but the current findings, in combination with previous results, indicate that C. diffluens, C. liquefaciens, M. globosa, and M. restricta together colonize the skin surface of AD patients at a high frequency.
Human Cytomegalovirus (HCMV) IE1 Plays Role in Resistance to Apoptosis with Etoposide in Cancer Cell Line by Cdk2 Accumulation
Authors: Kim Jinhee , Kwon Ye Jin , Park Eun‐Suk , Sung Bohyun , Kim Jung Heon , Park Chung‐Gyu , Hwang Eung‐Soo , Cha Chang‐Yong
Published: November 2013

Human cytomegalovirus (HCMV) has many strategies to survive the attack of the host. HCMV infection of host cells induces cellular activation and disturbance of the cell cycle. It is possible that HCMV modulates the behavior of certain cancer cells that are susceptible to HCMV infection. This study was performed to identify the possible mechanism of resistance to apoptotic stimuli in some cancer cell lines by HCMV infection. HCMV‐infected cancer cells showed resistance to apoptosis induced by the topoisomerase II inhibitor etoposide. UMG1–2, which constitutively expresses HCMV immediate‐early protein‐1 (IE1), had resistance to apoptosis induced by etoposide as compared with the parental cell line U373MG. Measurement of caspases activity with fluorogenic substrates in etoposide‐treated U373MG and UMG1–2 cells and the direct activation of caspase‐3 with peptides containing arginine‐glycine‐aspartate in U373MG and UMG1–2 cells revealed that the inhibition level of apoptosis by HCMV IE1 would be upstream of caspase‐3 in the caspase cascade pathway. Cellular expression of Cdk2 was increased in UMG1–2 after etoposide treatment while the expression of E2F‐1 in UMG1–2 was decreased as compared with that in U373MG. The Cdk2 inhibitor, roscovitine, decreased the resistance to apoptosis on etoposide‐treated UMG1–2. These results suggest that aberrant HCMV infection confers resistance to anticancer drugs on some cancer cells and protects cells from apoptosis, possibly due to the deregulation of cyclin‐dependent kinase by HCMV immediate‐early protein.
The Human‐Specific Action of Intermedilysin, a Homolog of Streptolysin O, Is Dictated by Domain 4 of the Protein
Authors: Nagamune Hideaki , Ohkura Kazuto , Sukeno Akiko , Cowan Graeme , Mitchell Timothy J. , Ito Wataru , Ohnishi Ooki , Hattori Kanako , Yamato Miki , Hirota Katsuhiko , Miyake Yoichiro , Maeda Takuya , Kourai Hiroki
Published: November 2013

Intermedilysin is a pore‐forming cytolysin belonging to the streptolysin O gene family known as the ‘Cholesterol‐binding/dependent cytolysins’ and is unique within the family in that it is highly human‐specific. This specificity suggests interaction with a component of human cells other than cholesterol, the proposed receptor for the other toxins of the gene family. Indeed, intermedilysin showed no significant degree of affinity to free or liposome‐embedded cholesterol. Characterization of intermedilysin undecapeptide mutants revealed that this lack of affinity to cholesterol was a result of the substitutions of intermedilysin in this region. Absorption assays with erythrocyte membranes from various animals, competitive inhibition with domain 4 of intermedilysin and liposome‐binding assays of streptolysin O and intermedilysin indicated that cell membrane binding is the human‐specific step of intermedilysin action, that the host cell membrane‐binding site is located within domain 4 in common with other members of the family and that the receptor for this toxin is not cholesterol. The species specificity of undecapeptide mutants of intermedilysin and streptolysin O and chimeric mutants between intermedilysin and streptolysin O, and intermedilysin and pneumolysin indicated that domain 4 of intermedilysin determines the human‐specific action step and the cell‐binding site of domain 4 lies within the 56 amino acids of the C‐terminal, excluding the undecapeptide region.
Role of Follicular Dendritic Cells in the Early HIV‐1 Infection: In vitro Model without Specific Antibody
Authors: Taruishi Midori , Terashima Kazuo , Dewan Md. Zahidunnabi , Yamamoto Norio , Ikeda Satoshi , Kobayashi Daisuke , Eishi Yoshinobu , Yamazaki Momoko , Furusaka Tohru , Sugimoto Masahiro , Ishii Masanori , Kitamura Ken , Yamamoto Naoki
Published: November 2013

About 90% of HIV‐1 RNA in the lymph nodes is reported to localize in follicular dendritic cells‐network (FDC‐NW) as early as several days after infection and as much as that in the late stage. But the mechanism remains to be fully understood. To elucidate the role of follicular dendritic cells (FDC) in the early stage of HIV‐1 infection, FDC‐like cell strains (FDCLC) were established and they were characterized in the co‐culture system with T cells for their effect on HIV‐1 trapping and replication in p24 immunoassay, immunohistochemistry as well as confocal and electronmicroscopy. Established FDCLC were positive for CNA‐42, S‐100α and intercellular desmosome‐like junctions. L‐SIGN and DC‐SIGN were also detected in FDCLC. Alu‐HIV‐1 PCR analysis showed no HIV‐1 integration in FDCLC. FDCLC trapped HIV‐1 and transferred them to uninfected MOLT‐4 T cells (MOLT‐4) efficiently in the absence of specific antibody. FDCLC also accelerated HIV‐1 replication in HIV‐1‐pre‐exposed MOLT‐4. These unique FDCLC effects were explained, at least partly, by the fact that FDCLC up‐regulated CD4 expression in MOLT‐4 and helped T cells escape from apoptosis in the co‐culture. These data suggest that FDC/FDCLC engage not only in trapping but also in active expansion of HIV‐1 in the absence of specific antibody.
Susceptibility to Killer T Cells of Gastric Cancer Cells Enhanced by Mitomycin‐C Involves Induction of ATBF1 and Activation of p21 (Waf1/Cip1) Promoter
Authors: Miura Yutaka , Kataoka Hiromi , Joh Takashi , Tada Toyohiro , Asai Kiyofumi , Nakanishi Makoto , Okada Noriko , Okada Hidechika
Published: November 2013

Alpha‐fetoprotein (AFP) expression is observed in embryonic tissues and, the expression of this protein is absent in normal adult tissues. The re‐elevation of serum AFP strongly suggests generation of a malignant tumor in an adult. We demonstrated here that AFP‐producing gastric cancer (AFP‐gastric cancer) could be treated by a combination therapy with a low dose of Mitomycin‐C (MMC) and lymphokine‐activated killer T (LAK‐T) cells. Treatment with MMC of AFP‐gastric cancer cells enhanced their susceptibility to LAK‐T cells and induced ATBF1 gene expression. We revealed here a novel signal pathway for regulation of the cell cycle of AFP‐gastric cancer cells through ATBF1, which enhances the promoter activity of the p21 (Waf1/Cip1) gene. Immunoprecipitation revealed the direct interaction between ATBF1 and p53. Overexpressed ATBF1 stimulated p21 (Waf1/Cip1) promoter activity up to 4‐fold compared with basal activity. The expression level of ATBF1 mRNA was doubled by MMC (0.05 μg/ml) treatment. The MMC treatment and ATBF1 overexpression synergistically activated the p21 (Waf1/Cip1) promoter activity in a dose‐dependent manner up to 7‐fold compared with basal activity.
The Genomic Heterogeneity among Mycobacterium terrae Complex Displayed by Sequencing of 16S rRNA and hsp65 Genes
Authors: Lee Chang Kyu , Gi Hyun Mi , Cho Yunjung , Kim Young Kee , Lee Kap No , Song Ki‐Joon , Song Jin‐Won , Park Kwang Sook , Park Eun Mi , Lee Hyeyoung , Bai Gill‐Han
Published: November 2013

The species identification within Mycobacterium terrae complex has been known to be very difficult. In this study, the genomic diversity of M. terrae complex with eighteen clinical isolates, which were initially identified as M. terrae complex by phenotypic method, was investigated, including that of three type strains (M. terrae, M. nonchromogenicum, and M. triviale). 16S rRNA and 65‐kDa heat shock protein (hsp65) gene sequences of mycobacteria were determined and aligned with eleven other references for the comparison using similarity search against the GenBank and Ribosomal Database Project II (RDP) databases. 16S rRNA and hsp65 genes of M. terrae complex showed genomic heterogeneity. Amongst the eighteen clinical isolates, nine were identified as M. nonchromogenicum, eight as M. terrae, one as M. mucogenicum with the molecular characteristic of rapid growth. M. nonchromogenicum could be subdivided into three subgroups, while M. terrae could be subdivided into two subgroups using a 5 bp criterion (>1% difference). Seven isolates in two subgroups of M. nonchromogenicum were Mycobacterium sp. strain MCRO 6, which was closely related to M. nonchromogenicum. The hsp65 gene could not differentiate one M. nonchromogenicum from M. avium or one M. terrae from M. intracellulare. The nucleotide sequence analysis of 16S rRNA and hsp65 genes was shown to be useful in identifying the M. terrae complex, but hsp65 was less discriminating than 16S rRNA.
Bacterial mRNA Purification by Magnetic Capture‐Hybridization Method
Authors: Pang Xin , Zhou Dongsheng , Song Yajun , Pei Decui , Wang Jin , Guo Zhaobiao , Yang Ruifu
Published: November 2013

A magnetic capture‐hybridization method was developed for purification of bacterial messenger RNA (mRNA) from total RNA by removing 5S, 16S and 23S ribosomal RNAs (rRNA). The quality of mRNA was evaluated by A260nm/A280nm value, denatured gel electrophoresis and RT‐PCR. The results showed that highly purified and intact mRNA was obtained by this method. The magnetic capture‐hybridization is a rapid and simple method for bacterial mRNA purification and has promising potential for improving studies using bacterial mRNA.
Augmenting Effect of Antibiotics on Endotoxin Activity May Cause a Safety Problem
Authors: Yamamoto Akihiko , Sakai Takeo , Ochiai Masaki , Kamachi Kazunari , Kataoka Michiyo , Toyoizumi Hiromi , Arakawa Yoshichika , Horiuchi Yoshinobu
Published: November 2013

Enhancing/interfering effect of antibiotics on endotoxin was evaluated using the endotoxin test and the cell line assay in 28SC cells that has a responsiveness consistent with that of human peripheral blood. When a total of 21 products of seven different kinds of antibiotics were tested, none showed any significant effect on the endotoxin test at its therapeutic dose. However, aminoglycosides showed a significant augmenting effect on IL‐6 induction of endotoxin in 28SC cells. Detailed examination of the augmenting effect was made on spectinomycin in the in vitro cell line assay and also in the lethal endotoxin challenge assay in D‐galactosamine‐treated mice. Spectinomycin also enhanced the endotoxin lethality in D‐galactosamine‐treated mice. A kinetic analysis in endotoxin‐sensitized 28SC cells revealed that the augmentation takes place as quickly as 10 min after spectinomycin treatment. Accordingly, a special caution concerning the augmenting effect was assumed necessary for the safety control of antibiotic products as well as for selecting antibiotics for the therapeutic use.
Effects of Artificial Codon Changes in the Movement Protein Gene on Adaptation of a Hybrid Bromovirus to Cowpea
Authors: Sasaki Nobumitsu , Kaido Masanori , Okuno Tetsuro , Mise Kazuyuki
Published: November 2013

A hybrid Cowpea chlorotic mottle virus (CCMV) with the movement protein (MP) gene replaced with that of the closely related Brome mosaic virus cannot infect cowpea systemically. Twenty‐nine spontaneous mutants from the hybrid CCMV capable of systemic infection in cowpea appeared through biased codon changes that resulted in Lys or Arg at five specific positions in the MP gene. In this study, we report that systemic infection of cowpea with the hybrid CCMV can be achieved by artificial codon changes that do not result in Lys or Arg. We discuss mechanisms that restrict the occurrence of cowpeaadapted mutants in nature.
Human Herpesvirus 6 Immediate‐Early 2 Protein Interacts with Heterogeneous Ribonucleoprotein K and Casein Kinase 2
Authors: Shimada Kazuya , Kondo Kazuhiro , Yamanishi Koichi
Published: November 2013

Human herpesvirus 6 (HHV‐6) immediate‐early (IE) 2 protein (IE2) may play important but incompletely defined roles during infection. We used yeast two‐hybrid screening to detect proteins interacting with HHV‐6 IE2, and found heterogeneous nuclear ribonucleoprotein K (hnRNP K) and the beta subunit of casein kinase 2 (CK2β) specifically interacted with HHV‐6 IE2. The interactions were confirmed by GST pull‐down assay, coimmunoprecipitation, and colocalization studies. These findings indicate that the HHV‐6 IE2 protein interacts with hnRNP K and CK2, and these interactions may affect viral and cellular RNA transcription and translation in viral replication.
Phenotypic Stability of Mature Dendritic Cells Tuned by TLR or CD40 to Control the Efficiency of Cytotoxic T Cell Priming
Authors: Nakamura Ichiro , Kajino Kiichi , Bamba Hiromichi , Itoh Fumito , Takikita Mikiko , Ogasawara Kazumasa
Published: November 2013

It is generally accepted that after stimulation immature DCs turn into mature DCs, which present exogenous antigens together with their MHC class I molecules and then activate the antigen‐specific CTLs. Although both TLR and CD40 stimulation appeared to provide the same effects on DC maturation, CD40‐dependent CTL activation is much more potent than CTL activation through LPS stimulation. Despite their different outcomes, the factors that lead mature DCs to different functions remain largely undefined. In this study, we defined the transient maturation and subsequent deactivation of DCs by TLR stimuli, including those by LPS and CpG‐ODN. In contrast, CD40 stimulation induced stable mature DCs that elicited sufficient CTL proliferation. The deactivated DCs, which we defined as “expired DCs,” were phenotypically similar to immature DCs, except for their phenotype stability, MHC class I expression level and IL‐10 production. Moreover, the functions of expired DCs were comparable to those of immature DCs in terms of CTL induction and tolerogenicity. These results may provide an explanation for the role of CD40 stimulation in antigen‐specific CTL induction.
Over‐Expression of the Testis‐Specific Gene TSGA10 in Cancers and Its Immunogenicity
Authors: Tanaka Ryo , Ono Toshiro , Sato Shuichiro , Nakada Tetsuya , Koizumi Fumihito , Hasegawa Kosei , Nakagawa Kazuhiko , Okumura Hideo , Yamashita Toshiharu , Ohtsuka Mikio , Asagoe Kenji , Yamasaki Osamu , Noguchi Yuji , Iwatsuki Keiji , Nakayama Eiichi
Published: November 2013

The TSGA10 gene was originally isolated in normal testis by differential mRNA display. TSGA10 is located on chromosome 2q11.2 and consists of 19 exons extending over 3 kb. TSGA10 mRNA expression was investigated in normal and malignant tissues using quantitative real‐time RT‐PCR. It was predominantly expressed in the testis in adult normal tissues. In malignant tissues, TSGA10 was over‐expressed in 4 of 20 hepatocellular carcinomas (HCC), 1 of 20 colon cancers, 7 of 20 ovarian cancers, 3 of 20 prostate cancers, 1 of 21 malignant melanomas, and 8 of 21 bladder cancers. Serological analysis revealed that 3 out of 346 patients with various types of cancer possessed antibody against recombinant TSGA10 protein. They included 2 patients with hepatocellular carcinoma and a patient with malignant melanoma.
Adsorption and Infectivity of Human Immunodeficiency Virus Type 1 Are Modified by the Fluidity of the Plasma Membrane for Multiple‐Site Binding
Authors: Harada Shinji , Akaike Takaaki , Yusa Keisuke , Maeda Yosuke
Published: November 2013

Based on the assumption that fluidity of the plasma membrane and viral envelope is necessary for recruiting additional receptors and ligands to the initial attachment site for “multiple‐site binding,” we determined the effect of increased temperature on viral infectivity. Infection of human immunodeficiency virus type 1 (HIV‐1) and a pseudotyped luciferase‐expressing chimeric virus using MAGI and GHOST/CXCR4 cells showed that in 1 hr of viral adsorption the extent of virus infection and the amount of tightly adsorbed viruses depended on temperature; and that membrane fluidity increased according to increased temperature. Augmented infection was observed as post‐attachment enhancement (PAE) when cells were washed and incubated at 40 C for 1 hr after viral adsorption. PAE was completely inhibited by 1 μM of anti‐CXCR4 peptide T140, and addition of T140 at 20 min resulted in a gradual loss of inhibition of PAE, indicating the need for a 30 to 40 min timelag to ensure tight multiple‐site binding. These data suggest that the accumulation of gp120 and receptor complex (multiple‐site binding) was needed to complete the infection. Treatments of cells with 0.05% Tween 20 or 2 μg/ml of anti‐HLA‐II antibody resulted in increases or decreases, respectively, of attached viruses and the infectivity. As well, Tween 20 and anti‐HLA‐II antibody enhanced and suppressed the fluidity of the plasma membrane, respectively. Amounts of adsorbed viruses and degrees of viral infectivity correlated with the intensity of fluidity of the plasma membrane, probably due to the formation of multiple‐site binding.
Heterogeneity of Envelope Molecules Shown by Different Sensitivities to Anti‐V3 Neutralizing Antibody and CXCR4 Antagonist Regulates the Formation of Multiple‐Site Binding of HIV‐1
Authors: Harada Shinji , Yusa Keisuke , Maeda Yosuke
Published: November 2013

Increased temperature enhances the infectivity of human immunodeficiency virus type 1 (HIV‐1), and this enhancement is inhibited by anti‐CXCR4 peptide T140, implying that multiple‐site binding is required to proceed to infection. Here, we tested whether the augmented infectivity induced by increased temperature could account for the heterogeneity of envelope molecules in the effectiveness of anti‐V3 neutralization and anti‐CXCR4 blocking. Pseudoviruses with the X4 envelope which were infectious at room temperature (RT) were more resistant to both anti‐V3 neutralizing antibody 0.5β and T140 than viruses infectious at 37 C and 40 C. Viruses infectious to cells treated with T140 were also resistant to 0.5β. Based on the hypothesis that the HIV‐1 viruses were carrying heterogeneity of functional and nonfunctional gp120 and required the formation of sufficient multiple‐site binding of functional gp120 with receptors to proceed to infection, viruses with many functional gp120 which were infectious at RT and infectious to cells with reduced numbers of CXCR4 by T140 treatment were resistant to 0.5β. Although viruses with many functional gp120 are a minority (less than 5%) of the infectious HIV‐1 fraction, they are regarded as able to escape from neutralizing antibodies and coreceptor antagonists.
Phenotypic Switching and β‐N‐Acetylhexosaminidase Activity of the Pathogenic Yeast Trichosporon asahii
Authors: Ichikawa Tomoe , Sugita Takashi , Wang Li , Yokoyama Koji , Nishimura Kazuko , Nishikawa Akemi
Published: November 2013

The pathogenic yeast Trichosporon asahii is the major causative agent of deep‐seated trichosporonosis in immunocompromised patients. Although infection by this microorganism is becoming increasingly frequent, information related to its pathogenicity and virulence factors is still limited. Therefore, we investigated phenotypic switching in colony morphology, and the production of extracellular enzymes as a virulence factor. Sixty‐one clinical isolates of T. asahii produced four different morphological types on Sabouraud dextrose agar (SDA): 69% WF (white farinose), 18% WP (white pustular), 10% Y (yellowish white), and 3% WC (white cerebriform). Strains of the three major types (WF, WP, and Y) produced two to five colony types when cultured on SDA at 37 C. The frequency of switching between colony types was 10–2 to 10–4, as in Candida albicans and Cryptococcus neoformans. Notably, most of the colonies switched to the smooth (S) type irreversibly, at frequencies of 10–2 to 10–3. No secreted aspartic proteinase or phospholipase activity was detected in T. asahii, while β‐N‐acetylhexosaminidase activity, which catalyzes the hydrolysis of terminal nonreducing N‐acetyl‐D‐hexosamine residues in N‐acetyl‐β‐D‐hexosaminides, was found. Furthermore, enzymatic activity of the S type was significantly greater than that of the parent type in all strains. No other clinically relevant Trichosporon species (T mucoides, T inkin, and T ovoides) produced this enzyme. These results provide basal information for understanding the pathogenic potential of T asahii.
Introduction of 65 kDa Antigen of Mycobacterium tuberculosis to Cancer Cells Enhances Anti‐Tumor Effect of BCG Therapy
Authors: Hara Isao , Sato Noriyuki , Miyake Hideaki , Muramaki Mototsugu , Hikosaka Satoko , Kamidono Sadao
Published: November 2013

Bacillus Calmette Guerin (BCG) immunotherapy has anti‐tumorigenic effects against bladder cancer. To improve the efficacy of BCG therapy, we introduced the gene encoding the 65 kDa heat shock protein (hsp) of Mycobacterium tuberculosis into a mouse malignant melanoma cell line (B16). An expression vector harboring the 65 kDa antigen gene was transfected into B16 using Lipofectamine, then expression of the antigen was confirmed by RT‐PCR and Western blotting. Several cell lines expressing 65 kDa antigen were established (B16/65kDa). We also established a control cell line transfected with the vector alone (B16/con). All cell lines (B16, B16/con, B16/65kDa) were injected intraperitoneally into syngeneic mice with or without BCG prior immunization and the development of tumor ascites was examined. To analyze the mechanism of the anti‐tumor effect, CD4 T cells or CD8 T cells were depleted in vivo by administering the corresponding monoclonal antibody. B16/65kDa expressed the 65 kDa hsp of M. tuberculosis. The tumor growth of B16/65kDa was slightly retarded in naive mice, but significantly inhibited by BCG. The anti‐tumor effect was totally abrogated in mice deficient in CD4 T cells, suggesting that CD4 T cells are involved in this process. The 65 kDa hsp of M. tuberculosis was expressed after gene transduction in a malignant melanoma cell line and significantly enhanced the anti‐tumor effect of BCG immunotherapy. CD4 T cells play an important role in this anti‐tumor effect.
Complex Formation of CD23/Surface Immunoglobulin and CD23/CD81/MHC Class II on an EBV‐Transformed Human B Cell Line and Inferable Role of Tetraspanin
Authors: Kijimoto‐Ochiai Shigeko , Noguchi Atsutaka , Ohnishi Toshiyuki , Araki Yoshio
Published: November 2013

CD23, a low‐affinity IgE receptor, is a type II transmembrane protein having a C‐type lectin domain and it associates noncovalently with MHC class II on B cells. The results of our immunoprecipitation analysis suggest that CD23 co‐exists with at least two additional molecules, surface immunoglobulin (sIg) and CD81 (and/or CD9), on the cell surface of L‐KT9 cells (an Epstein‐Barr virus (EBV)‐transformed human B cell line). When both CD23 and sIg molecules were stimulated simultaneously by the corresponding antibodies, a large increase in CD81 in the immunoprecipitation was observed as compared with the case of stimulation by only one antibody. Simultaneous stimulation by anti‐CD23 and anti‐Ig may mimic the situation of B cells stimulated by an antigen/IgE complex. In addition, a large increase in MHC class II in the immunoprecipitation was also observed by cross‐linking of CD23 with anti‐CD23 and its second antibody as compared with the case of stimulation by anti‐CD23 alone. The cross‐linking of CD23 with anti‐CD23 and its antibody may mimic the situation of B cells stimulated by an IgE/antigen/IgE complex. Therefore, the complex formation among CD23, sIg, MHC class II, and CD81 on the cell surface of L‐KT9 cells by the antigen/IgE or IgE/antigen/IgE complex is most likely to be closely related to B cell regulatory events by signaling through sIg or MHC class II. Tetraspanins such as CD81 and CD9 are thought to be involved in the formation and the preservation of various different membrane complexes consisting of several functional proteins.
IL‐7‐Dependent Homeostatic Proliferation in the Presence of a Large Number of T Cells in Gld Mice
Authors: Aranami Toshimasa , Iclozan Cristina , Iwabuchi Kazuya , Onoé Kazunori
Published: November 2013

In gld mice, CD4 and 8‐double‐negative (DN) T cells as well as naive and memory‐phenotype T cells accumulate in the peripheral lymphoid organs. Although Fas ligand (L) defect accounts for the progressive accumulation of abnormal DN T cells, the existence of other mechanisms which may be involved in the defective homeostasis in gld mice has been unclear. In this study, we analyze T‐cell homeostasis in gld mice using adoptive transfer systems. It was shown that a gld, but not C57BL/6 (B6), environment led to augmented proliferation of B6 T cells transferred without up‐regulation of CD69. Thus, the augmented T‐cell proliferation seemed to result from mal‐homeostatic proliferation even in the presence of a large number of recipient T cells. T cells from lpr mice showed no significant proliferation in the B6 environment, suggesting that the absence of Fas‐Fas L interaction was not responsible for the mal‐homeostatic proliferation. Although similar levels of IL‐7 mRNA were detected in gld and B6 spleens, the intensity of CD127 and the proportion of CD127+ cells in the T cells were significantly lower in gld mice than in B6 mice, suggesting that IL‐7 excess in a gld environment is responsible for the abnormal proliferation of transferred T cells. The administration of anti‐CD127 antibody inhibited the proliferation of transferred lymphocytes. Thus, IL‐7‐dependent proliferation seems to be involved in the abnormal proliferation of lymphocytes in gld recipients.
Reliability of the Detection of Meningococcal γ‐Glutamyl Transpeptidase as an Identification Marker for Neisseria meningitidis
Authors: Takahashi Hideyuki , Kuroki Toshiro , Watanabe Yuko , Dejsirilert Surang , Saengsuk Leelaowadee , Yamai Shiro , Watanabe Haruo
Published: November 2013

Detection of γ‐glutamyl transpeptidase (GGT; ggt) activity is one of the useful methods for a specific identification of Neisseria meningitidis. However, we previously happened to isolate a ggt‐deficient N. meningitidis strain (NIID113) from a healthy carrier. In this study, in order to re‐examine the reliability of the marker, we again investigated the GGT activity of 245 N. meningitidis human isolates and identified two other GGT‐defective N. meningitidis isolates besides NIID113. The isolation frequency (1.2%) of ggt mutants among human isolates strongly confirmed the 98.8% reliability of GGT activity as the identification marker for N. meningitidis.
Effect of Silver‐Carrying Photocatalyst “Hikari‐Gintech” on Mycobacterial Growth In Vitro
Authors: Matsui Yoshifumi , Otomo Koji , Ishida Shinichiro , Yanagihara Kazuo , Kawanobe Yasutaka , Kida Shoji , Taruoka Eiichi , Sugawara Isamu
Published: November 2013

The antimycobacterial activity of “Hikari‐Gintech” powder, which has photocatalytic activity, was examined in vitro. Both powder dissolved in liquid and Hikari‐Gintech‐coated cloths showed strong antimycobacterial activity against Mycobacterium tuberculosis H37Rv, M. bovis BCG Pasteur, multi‐drug‐resistant M. tuberculosis (a clinical isolate) and M. avium. Hikari‐Gintech powder appeared to affect mycobacterial cell wall metabolism rather than mycobacterial DNA because no damage to mycobacterial DNA was detected after spraying with Hikari‐Gintech solution.
Antibody Reactive to a Hepatitis C Virus (HCV)‐Derived Peptide Capable of Inducing HLA‐A2 Restricted Cytotoxic T Lymphocytes Is Detectable in a Majority of HCV‐Infected Individuals without HLA‐A2 Restriction
Authors: Takao Yukari , Yamada Akira , Yutani Shigeru , Sata Michio , Itoh Kyogo
Published: November 2013

Hepatitis C virus (HCV) is a single‐strand RNA virus. Approximately 170 million people around the world are persistently infected and are at risk of liver cirrhosis or cancer. There is an urgent need to develop both therapeutic and diagnostic modalities of HCV. One approach to achieve these goals would be to determine highly immunodominant HCV peptides which are recognized by both cellular and humoral immunities. This study reports one such peptide, HCV‐core protein at positions 35–44, having HLA‐A2 binding motifs. IgG specific to this CTL‐epitope peptide is consistently detectable in a majority of the patients with HCV infection regardless of the different HLA types, different disease conditions, and different HCV‐genotypes tested. The sequence LPRR at positions 37–40 is considered to be the fine epitope recognized by the IgG. These results may provide new insights for the development of both therapeutic and diagnostic modalities of HCV at lower costs.
Ultrastructure of a Japanese Rickettsial Strain Genetically Identified as Rickettsia helvetica Which Was Originally Found in Europe
Authors: Yano Yasuhiro , Fujita Hiromi , Takada Nobuhiro
Published: November 2013

A rickettsial strain IO‐1 has been isolated from a tick, Ixodes ovatus, in Japan and genetically identified as Rickettsia helvetica, a member of the spotted fever group rickettsiae. Ultrastructural observations were made on the microorganism. The ultrastructure of R. helvetica IO‐1 appeared to be generally the same as that previously shown for other rickettsiae of the spotted fever and typhus groups. The rickettsiae were primarily found free in the cytoplasm of L929 cultured cells. Occasionally, the rickettsiae may also invade the host cell nucleus; however, the frequency of the nuclear localization was very low.
Autolysis of Porphyromonas gingivalis Is Accompanied by an Increase in Several Periodontal Pathogenic Factors in the Supernatant
Authors: Kamaguchi Arihide , Nakano Miyo , Shoji Mikio , Nakamura Reiko , Sagane Yoshimasa , Okamoto Masaaki , Watanabe Toshihiro , Ohyama Tohru , Ohta Michio , Nakayama Koji
Published: November 2013

Porphyromonas gingivalis autolyzes in the culture media. To examine in more detail the molecular components of the autolysate, two‐dimensional gel electrophoresis was performed. Many protein spots varied both in number and volume. One of these spots included Arg‐gingipain (Rgp) as determined by N‐terminal amino acid sequencing. Corresponding to the increase in spot volume, Rgp activity also increased during autolysis. The results of this study suggested that Rgp and other proteins in the P. gingivalis autolysate may be involved with the prolongation of periodontal disease, even after the death of P. gingivalis cells.
A Plasmid Encoding Japanese Encephalitis Virus PrM and E Proteins Elicits Protective Immunity in Suckling Mice
Authors: Wu Yushui , Zhang Fuquan , Ma Wenyu , Song Jianhua , Huang Qingsheng , Zhang Hongyi
Published: November 2013

A plasmid encoding Japanese encephalitis virus (JEV) prM and E proteins was constructed, and its efficacy as a candidate vaccine against JEV was evaluated in suckling mice. Groups of 10 BALB/c mice (5–7 days old) were immunized twice via muscular injection with this DNA vaccine, an empty vector or PBS at an interval of 3 weeks, and were challenged with a lethal dose of JEV 3 weeks after the second inoculation. Both cellular and humoral immune responses were examined before the challenge. Two animals from each group were sacrificed to detect the JEV‐specific cytotoxic T lymphocyte activity. JEV‐specific lactate dehydrogenase release in the DNA vaccine, empty vector and PBS groups was 37.5%, 18% and 8.5% respectively. JEV‐specific antibody was detected in 8 of 10 animals in DNA vaccine group with a geometrical mean titer of 1:28.3. The pooled serum from the same group also showed a neutralizing activity. Six of 8 mice in the DNA vaccine group survived the challenge, with a protection rate of 75%, but all the mice died in the two control groups. These results show that this JEV prM and E DNA vaccine is immunogenic and protective against JEV infection in the mouse model.
The Role of IFN‐γ and Toll‐Like Receptors in Nephropathy Induced by Toxoplasma gondii Infection
Authors: Kudo Masamichi , Aosai Fumie , Mun Hye‐Seong , Norose Kazumi , Akira Shizuo , Iwakura Yoichiro , Yano Akihiko
Published: November 2013

The pathologic links between Toxoplasma gondii infections and renal diseases have not yet been established. Gamma interferon (IFN‐γ) and Toll‐like receptors (TLRs) are involved in the host defense mechanism against T. gondii infection. The role of IFN‐γ and TLRs in renal function of T. gondii‐infected mice was studied using wild type (WT), TLR2‐deficient and TLR4‐deficient mice perorally infected with cysts of an avirulent cyst‐forming Fukaya strain of T. gondii. T. gondii was abundant in kidneys in IFN‐γ KO (GKO) mice as determined by a quantitative competitive‐polymerase chain reaction (QC‐PCR). But, T. gondii was not detected in kidneys in WT, TLR2‐deficient and TLR4‐deficient mice. Interestingly, renal function of TLR2‐deficient and TLR4‐deficient mice was damaged as evaluated by serum creatinine, serum blood urea nitrogen (BUN), and urine albumin/creatinine ratio (ACR), whereas renal function of GKO and WT mice was not damaged. Histopathology of TLR2‐deficient mice exhibited glomerular and extracellular matrix swelling with advancing glomerular tissue proliferation, thickened Bowman's capsules and vacuolization of tubules. Renal immunofluorescence study of T. gondii‐infected TLR2‐deficient mice displayed positive staining of the glomerular basement membrane, mesangial areas and peritubular capillaries. The damage of kidney from TLR4‐deficient mice was less severe compared to TLR2‐deficient mice, and histopathological damage of kidney was not observed in WT and GKO mice. These results indicate that TLR2, but not IFN‐γ, plays a role in the protection of the renal function against T. gondii infection.
Amino‐Acid Residues Involved in the Expression of the Activity of Escherichia coli TolC
Authors: Yamanaka Hiroyasu , Morisada Naoyuki , Miyano Masaya , Tsuge Hideaki , Shinoda Sumio , Takahashi Eizo , Okamoto Keinosuke
Published: November 2013

The Escherichia coli TolC, composed of 471 amino‐acid residues, functions as a channel tunnel in the transport of various molecules across the outer membrane. We found previously that Leu‐412, the 60th amino‐acid residue from the carboxy terminal end, was crucial to the transport activity of TolC. Leu‐412 is located in a domain which protrudes from the main body of TolC into the periplasm. Subsequent study indicated that the hydrophobicity generated by Leu‐412 played an important role in the activity of TolC (H. Yamanaka, T. Nomura, N. Morisada, S. Shinoda, and K. Okamoto, Microb. Pathog. 33: 81–89, 2002). We predicted that other hydrophobic amino‐acid residues around Leu‐412 were also involved in the expression of the activity of TolC. To test this possibility, we substituted several hydrophobic residues around Leu‐412, (Leu‐3, Val‐6, Leu‐212, Leu‐213, Leu‐223, and Leu‐224), with serine and examined the activity of these mutant TolCs. The result showed that Leu‐3 is involved in the activity of TolC, but the other residues are not. The involvement of Leu‐3 was confirmed by the residue deletion experiment. A subsequent point‐mutational analysis of the residue showed that a hydrophobic side chain is required at position 3 for TolC to express its activity. As the distance between the α‐carbons of Leu‐3 and Leu‐412 is just 7.45 Å, hydrophobic interaction between the two leucine residues might be involved in the activity of TolC.
Consideration of Cysteine Protease Activity for Serological M‐Typing of Clinical Streptococcus pyogenes Isolates
Authors: Morita Masatomo , Ikebe Tadayoshi , Watanabe Haruo
Published: November 2013

Clinical isolates of Streptococcus pyogenes were classified by serological typing of their surface M protein. Non‐M typeable strains with the emm1 gene were characterized as the degradation of M protein caused by overproduction of the extracellular cysteine protease, SpeB. These events are dependent on the growth phase. M protein produced prior to expression of SpeB is degraded in the stationary phase when the active form of SpeB is detected. The proteolytic degradation of M protein should be considered for precise M typing analysis.