bluuebunny/crossref_metadata_2025 huggingface dataset:
Engraftment of Tonsillar Mononuclear Cells in Human Skin/SCID Mouse Chimera —Validation of a Novel Xenogeneic Transplantation Model for Autoimmune Diseases—
Authors: Yamanaka Noboru , Yamamoto Yoshikazu , Kuki Kiyonori
Published: November 2013

Pustulosis palmaris et plantaris (PPP) has been considered as one of the typical tonsillar focal infections, based on the marked clinical improvement of the skin lesions after tonsillectomy. Despite the accumulation of data showing the clinical efficacy of tonsillectomy for this skin lesion, fundamental etiological and pathophysiological issues have yet to be addressed. One primary obstacle hindering investigators has been the lack of an appropriate animal model for this human skin disorder. In the early stage of PPP, it has been reported that lymphocytes, predominantly CD4+ T lymphocytes, infiltrate the palmar and plantar skins. However, the origin and mechanism of infiltration by these lymphocytes is not clear and there are very few reports on whether tonsillar mononuclear cells react directly with the skin. We have been intrigued by the ability to engraft human cells onto severe combined immunodeficiency (SCID) mice, together with the opportunity for long‐term graft survival and ability to adoptively transfer various human immunocompetent cells. In this review, we addressed the existing deficiencies in our understanding of the relationship between tonsils and PPP by using emerging transplantation technology involving SCID mice.
Mechanism of Selective Inhibition of Respiratory Syncytial Virus by a Benzodithiin Compound (RD3–0028)
Authors: Sudo Kenji , Konno Kenji , Watanabe Wataru , Shigeta Shiro , Yokota Tomoyuki
Published: November 2013

RD3–0028, a compound with a benzodithiin structure, was found to be a potent inhibitor of respiratory syncytial virus (RSV) replication. Its action is specific; no activity is seen against influenza A virus, measles virus, herpes simplex virus type 1 or 2, or human cytomegalovirus. A time‐dependent drug addition experiment indicated that the antiviral activity occurs in the late stage of the RSV replication cycle, since this compound completely inhibited syncytium formation even when added up to 16 hr after the infection of cell monolayers at an MOI of 3. RD3–0028 had no direct virucidal effect on RSV. Western blotting analysis showed that RD3–0028 significantly decreased the amount of RSV proteins released into the cell culture medium. Moreover, five independent isolates of the RSV long strain were selected for growth in RD3–0028 (5–20 μg/ml). These resistant viruses were more than 80‐fold less sensitive to RD3–0028 than the long strain. The F gene segment of each of these viruses was sequenced and in each case the mutant RNA segment contained at least one sequence alteration, converting asparagine 276 to tyrosine (F1 protein). These results suggest that RD3–0028 inhibits RSV replication by interfering with intracellular processing of the RSV fusion protein, or a step immediately thereafter, leading to loss of infectivity.
Immunohistochemical Distribution of Epithelioid Cell, Myofibroblast, and Transforming Growth Factor‐β1 in the Granuloma Caused by Mycobacterium avium intracellulare Complex Pulmonary Infection
Authors: Fujita Jiro , Ohtsuki Yuji , Suemitsu Ichizo , Yamadori Ichiro , Shigeto Eriko , Shiode Masahiko , Nishimura Kazutaka , Hirayama Takeshi , Matsushima Toshiharu , Ishida Toshihiko
Published: November 2013

The present study was designed to evaluate the distribution of epithelioid cells, myofibroblasts, and TGF‐β1 in the formation of granuloma caused by Mycobacterium avium intracellulare complex (MAC) lung infection. A retrospective study was performed for 9 cases of positive MAC culture in which lung resections were performed between January 1989 and August 1999. Resected lung specimens were evaluated histologically and immunohistochemically for CD68 (stain for monocytes and macrophages, and epithelioid cells) and α‐smooth muscle actin as well as vimentin (stain for myofibroblasts), and TGF‐β1 was performed. When granuloma was initially formed, no myofibroblasts were found, but as caseous necrosis appeared, the thin epithelioid cell layer was detected and the outer myofibroblast layer gradually became thick. In the cavitary wall, the layer of epithelioid cells and multinucleated giant cells surrounded necrosis, and was associated with the outer layer of myofibroblasts. In addition, the anti‐TGF‐β1 antibody stained the cytoplasm of epithelioid cells and multinucleated giant cells, preceding the advent of myofibroblasts. In summary, our present study evaluated distributions of epithelioid cells, myofibroblasts, and TGF‐β along with the morphogenesis of granuloma, and clearly demonstrated the immunohistochemical difference between granuloma with caseous necrosis and granulomas without caseous necrosis.
Antibacterial Properties of Antimicrobial‐Finished Textile Products
Authors: Takai Kenichi , Ohtsuka Tokuko , Senda Yoshiko , Nakao Miyuki , Yamamoto Kouichiro , Matsuoka Junji , Hirai Yoshikazu
Published: November 2013

The antibacterial properties of five kinds of antimicrobial‐finished textile products (AFTPs) were examined against Staphylococcus aureus, including methicillin‐resistant (MRSA) strains and Pseudomonas aeruginosa, under wet and dry conditions. Textile products containing Ag. Zn. ammonium Zeolite and chitosan were found to be effective against methicillin‐sensitive S. aureus (MSSA) for up to 6 hr of incubation under wet and dry conditions, and effective against MRSA for up to 24 hr of incubation only under wet conditions. Under dry conditions, however, all AFTPs were ineffective against one MRSA strain. When organic matter was added to the incubation mixture, textile products containing Ag. Zn. ammonium Zeolite and chitosan still showed antibacterial activities, but not as strongly. The results of this study suggested the following: (1) There are differences in antibacterial properties among commercially available AFTPs; (2) Determining effectiveness requires several hours of incubation; (3) Water content as an environmental factor can affect effectiveness; and (4) Some bacterial species and strains are not affected by AFTPs. The antibacterial properties of AFTPs in the clinical setting may be of limited value.
Antifungal Pharmacodynamic Characteristics of Amphotericin B against Trichosporon asahii, Using Time‐Kill Methodology
Authors: Toriumi Yoshimi , Sugita Takashi , Nakajima Masamitsu , Matsushima Toshiharu , Shinoda Takako
Published: November 2013

We determined the MIC of amphotericin B against 45 Trichosporon asahii isolates from various clinical and environmental sources, and used in vitro time‐kill methods to characterize the relationship between amphotericin B concentrations and MIC for four representative T. asahii isolates. Amphotericin B had concentration‐dependent antifungal activity. MICs ranged from 0.5 to 16 μg/ml, and most T. asahii isolates (76%, 34/45) were inhibited at safely achievable amphotericin B serum concentrations (≤ 2 μg/ml). However, 40% (18/45) of isolates were not killed at these concentrations (MFCs from 1.0 to 32 μg/ml). At concentrations ≥ 2 × MIC, amphotericin B exhibited fungicidal activity (< 99.9% reduction in CFU) over a 12‐hr time‐period; the maximal effect was achieved at ≥ 4 × MIC. Susceptibility testing confirmed the resistance of T. asahii to amphotericin B, and in vitro pharmacodynamic results also suggest that amphotericin B is not suitable therapy for T. asahii infection.
FimZ Binds the Salmonella typhimurium fimA Promoter Region and May Regulate Its Own Expression with FimY
Authors: Yeh Kuang‐sheng , Tinker Juliette K. , Clegg Steven
Published: November 2013

The FimZ protein, an activator of FimA production in Salmonella typhimurium, acts in conjunction with FimY to facilitate the expression of type 1 fimbriae. The predicted amino acid sequence of FimZ suggests that this protein may be a DNA‐binding protein related to BvgA, a sensory regulator of virulence gene expression in Bordetella pertussis. Purification of FimZ following overexpression of the protein by a strong inducible promoter and gel mobility shift assays confirm that FimZ is a 25‐kDa polypeptide that binds to the promoter region of fimA. The region of DNA protected from DNase I digestion by FimZ binding is located between 47 and 98 nucleotides upstream from the fimA transcription initiation site. This region possesses a pair of 7‐base pair tandem repeats, of which at least one is necessary for FimZ binding. One copy of the 7‐base pair sequence is also located in the fimZ promoter region. In addition, expression from a fimZ‐lacZ reporter construct confirms that FimZ plays a role in its own expression. Both FimZ and FimY are required for high‐level expression of FimZ, which suggests that these two fimbrial proteins are involved in regulating both FimA and FimZ.
Lactoferrin Stimulates AStaphylococcus aureusKilling Activity of Bovine Phagocytes in the Mammary Gland
Authors: Kai Kenzo , Komine Ken‐ichi , Komine Yumiko , Kuroishi Toshinobu , Kozutsumi Tomoyuki , Kobayashi Jin , Ohta Minoru , Kitamura Hajime , Kumagai Katsuo
Published: November 2013

Lactoferrin (Lf) may play a key role in the clearance of microorganisms from a host. To studyin vitrothe bactericidal mechanisms of Lf during nonlactating periods, we investigated whether the effects of Lf were influenced by bovine mammary gland secretory cells (MGSC) and fresh normal bovine serum (NBS) as a source of complement. Phagocytic killing tests demonstrated that a phagocytic mixture of unopsonizedStaphylococcus aureus (S. aureus)and MGSC in the presence of Lf reduced bacterial growth, compared with that of unopsonizedS. aureusand MGSC without Lf. The opsonization with Lf and fresh NBS together resulted in more than a 95% reduction in CFU. The activation of complement induced by Lf also resulted in increased deposition of C3 onS. aureus, and the phagocytic activity of MGSC was augmented by opsonization with Lf and fresh NBS. Inhibition of C3 deposition by Lf was not induced in the presence of Mg‐EGTA, but was induced by the addition of bovine Lf antiserum. These results strongly suggest that Lf induces the activation of complement in fresh NBS mainly through an alternative pathway. The results demonstrated a Lf‐dependent, antibody‐independent and complement‐mediated phagocytic killing ofS. aureus, and implied that Lf was synergistically capable of activating both the alternative pathway of the bovine complement cascade and phagocytosis by phagocytes.
Minimal Contribution of Vα14 Natural Killer T Cells to Th1 Response and Host Resistance against Mycobacterial Infection in Mice
Authors: Kawakami Kazuyoshi , Kinjo Yuki , Uezu Kaori , Yara Satomi , Miyagi Kazuya , Koguchi Yoshinobu , Nakayama Toshinori , Taniguchi Masaru , Saito Atsushi
Published: November 2013

We elucidated the contribution of Vα14 NKT cells to Th1 response and host resistance against mycobacterial infection. In Vα14 NKT cell‐deficient mice, host defense and DTH response to Mycobacterium bovis BCG were not different from wild‐type mice after pulmonary infection. There was no significant difference in the lung concentrations of IFN‐γ between the two strains of mice. In addition, host defense to systemic infection with M. tuberculosis was similar to that of M. bovis. Our results indicate that Vα14 NKT cells play only a marginal role, if any, in the Th1 response and host resistance to mycobacterial infection.
A Novel Genetic Algorithm for Designing Mimetic Peptides That Interfere with the Function of a Target Molecule
Authors: Campbell William , Kleiman Laurence , Baranyi Lajos , Li Zhou , Khorchid Ahmad , Fujita Emiko , Okada Noriko , Okada Hidechika
Published: November 2013

We designed a new computer program (MIMETIC), which generates a series of peptides for interaction with a target peptide sequence. The genetic algorithm employed ranks the sequences obtained from one generation to the next by “goodness of fit” to the target. MIMETIC designed recognition peptides to various regions of HIV‐1 reverse transcriptase. Among ten peptide candidates synthesized, three inhibited reverse transcription in vitro. TLMA2993 and PSTW1594 both targeted the connection domain of reverse transcriptase and ESLA2340 targeted the thumb domain.
Advance in Treatment Strategy and Immune Reconstruction against HIV‐1 Infection
Authors: Matsushita Shuzo , Kimura Tetsuya
Published: November 2013

HIV‐1 can be considered an infection of the immune system, resulting in progressive and ultimately profound immune suppression. The availability of highly active antiretroviral therapy (HAART) has resulted in dramatic changes in the disease course in persons fortunate enough to have access to these medications, but long‐term therapy is limited by the development of resistance as well as toxicities of the potent medication regimens. Emerging data indicate that individuals who have non‐progressive clinical course control HIV‐1 immunologically. This has bolstered hope that the immune response might be effectively augmented in persons with HIV infection. Recent data indicating that immediate treatment of acute infection leads to augmentation of antiviral immune responses have provided evidence that the immune system might be enhanced in certain situations. Therefore, investigation in the reconstitution of anti‐HIV immune response in patients under HAART should provide encouragement for continuing to explore methods to obtain meaningful and durable immune enhancement as an adjunct to HAART in HIV‐1 infection.
Inhibition by Apple Polyphenols of ADP‐Ribosyltransferase Activity of Cholera Toxin and Toxin‐Induced Fluid Accumulation in Mice
Authors: Saito Takao , Miyake Masami , Toba Masamichi , Okamatsu Hiroshi , Shimizu Seiichi , Noda Masatoshi
Published: November 2013

The effects of crude polyphenol extracted from immature apples on the enzymatic and biological activities of a cholera toxin (CT) were investigated. When the apple polyphenol extract (APE) was examined for properties to inhibit CT‐catalyzed ADP‐ribosylation of agmatine, it was found that APE inhibited it in a dose‐dependent manner. The concentration of APE to inhibit 50% of the enzymatic activity of CT (15 μg/ml) was approximately 8.7 μg/ml. The APE also diminished CT‐induced fluid accumulation in two diarrhea models for in vivo mice. In the ligated ileum loops, 25 μg of APE significantly inhibited fluid accumulation induced by 500 ng of CT. In a sealed mouse model, even when APE was administered orally 10 min after a toxin injection, fluid accumulation was significantly inhibited at a comparable dosage. Lineweaver‐Burk analysis demonstrated that APE had negative allosteric effects on CT‐catalyzed NAD: agmatine ADP‐ribosyltransferase. We fractionated the APE into four fractions using LH‐20 Sephadex resin. One of the fractions, FAP (fraction from apple polyphenol) 1, which contains non‐catechin polyphenols, did not significantly inhibit the CT‐catalyzed ADP‐ribosylation of agmatine. FAP2, which contains compounds with monomeric, dimeric, and trimeric catechins, inhibited the ADP‐ribosylation only partially, but significantly. FAP3 and FAP4, which consist of highly polymerized catechin compounds, strongly inhibited the ADP‐ribosylation, indicating that the polymerized structure of catechin is responsible for the inhibitory effect that resides in APE. The results suggest that polymerized catechin compounds in APE inhibit the biological and enzymatic activities of CT and can be used in a precautionary and therapeutic manner in the treatment of cholera patients.
Differential Expression and Polarized Secretion of CXC and CC Chemokines by Human Intestinal Epithelial Cancer Cell Lines in Response to Clostridium difficile Toxin A
Authors: Kim Jung Mogg , Kim Joo Sung , Jung Hyun Chae , Oh Yu‐kyoung , Song In Sung , Kim Chung Yong
Published: November 2013

Intestinal epithelial cells are the initial sites of host response to Clostridium difficile infection and can play a role in signaling the influx of inflammatory cells. To further explore this role, the regulated expression and polarized secretion of CXC and CC chemokines by human intestinal epithelial cells were investigated. An expression of the CXC chemokines, including IL‐8 and growth‐related oncogene (GRO)‐α, and the CC chemokine monocyte chemoattractant protein (MCP)‐1 from HT‐29 cells increased in the 1–6 hr following C. difficile toxin A stimulation, assessed by quantitative RT‐PCR. In contrast, the expression of neutrophil activating protein‐78 (ENA‐78) was delayed for 18 hr. The up‐regulated mRNA expression of chemokines was paralleled by the increase of protein levels. However, the expression of macrophage inflammatory protein (MIP)‐1α, RANTES (regulated on activation normal T cells expressed and secreted), and interferon‐γ‐inducible protein‐10 (IP‐10) was not changed in HT‐29 or Caco‐2 cells stimulated with toxin A. Upon stimulation of the polarized Caco‐2 epithelial cells in a transwell chamber with toxin A, CXC and CC chemokines were released predominantly into the basolateral compartment. Moreover, the addition of IFN‐γ and TNF‐α to toxin A stimulated Caco‐2 cells increased the basolateral release of CC chemokine MCP‐1. In contrast, IFN‐γ and TNF‐α had no effect on the expression of the CXC chemokines IL‐8 and GRO‐α. These results suggest that a CXC and CC chemokine expression from epithelial cells infected with C. difficile may be an important factor in the mucosal inflammatory response.
Phylogenetic Analysis of Cecal Microbiota in Chicken by the Use of 16S rDNA Clone Libraries
Authors: Lan Pham Thi Ngoc , Hayashi Hidenori , Sakamoto Mitsuo , Benno Yoshimi
Published: November 2013

The chicken cecum contains a great many bacteria, most of which are strict anaerobes. A strictly anaerobe culture‐based method was used in the present study, in conjunction with the 16S rDNA clone library, to elucidate bacterial diversity and the phylogenetic relationship of cecal microbiota in the chicken. A comparative 16S rDNA sequence analysis of cultivated strains and retrieved clones from cecal contents was performed. Approximately 90% of the bacterial cells detected by microscopy did not form colonies on a medium 10 in plate‐in‐bottle. The 19 isolated strains yielded 11 distinct rDNA sequences, 58% of which were classified as low G + C Gram‐positive bacteria, 26% were related to Bacteroides spp., and 16% were classified as Proteobacteria. Based on the sequence analysis of 164 clones, 24% were identified to belong to 8 known species and 76% were considered to be 65 novel phylotypes. Approximately 94% of cloned sequences were classified into low G + C Gram‐positive bacteria, 4% were related to Bacteroides spp., and 2% were classified into Proteobacteria. Clostridium subcluster XIVa (38%), Clostridium cluster IV (13%), Lactobacillus spp. (24%), and Bacteroides spp. (4%) were the major groups constituting the cecal microbiota in chicken, in which the Clostridium subcluster XIVa was the most phylogenetically diverse group in chicken cecum. The 16S rDNA sequences of Lactobacillus acidophilus, L. crispatus, L. salivarius, and L. reuteri were the most frequently found in the Lactobacillus group in chicken cecum.
High‐Level Fluoroquinolone Resistance in Pseudomonas aeruginosa Due to Interplay of the MexAB‐OprM Efflux Pump and the DNA Gyrase Mutation
Authors: Nakajima Akira , Sugimoto Yohko , Yoneyama Hiroshi , Nakae Taiji
Published: November 2013

Fluoroquinolone resistance in Pseudomonas aeruginosa is mainly attributable to the constitutive expression of the xenobiotic efflux pump and mutation in DNA gyrase or topoisomerase IV. We constructed cells with a double‐mutation in gyrA and mexR encoding DNA gyrase and repressor for the mexAB‐oprM operon, respectively. The mutant showed 1,024 times higher fluoroquinolone resistance than cells lacking the MexAB‐OprM. Cells with a single mutation in gyrA and producing a wild‐type level of the MexAB‐OprM efflux pump showed 128 times higher fluoroquinolone resistance than cells lacking the MexAB‐OprM. In contrast, a single mutation in gyrA or mexR caused only 4 and 64 times higher resistance, respectively. These findings manifested the interplay between the MexAB‐OprM efflux pump and the target mutation in fluoroquinolone resistance.
The First Isolation of Nocardia veterana from a Human Mycetoma
Authors: Kano Rui , Hattori Yousuke , Murakami Noboru , Mine Naoko , Kashima Masato , Kroppenstedt Reiner M. , Mizoguchi Masako , Hasegawa Atsuhiko
Published: November 2013

The clinical isolates from biopsy specimen human subcutaneous nodule developed orange‐colored and wrinkled colonies on Sabouraud's dextrose agar at 24 C for 2 weeks. The isolates were aerobic and gram‐positive. The bacteria were rod‐shaped to coccoid and 1 × 5 μm in size. The assimilation tests revealed that the clinical isolate was identical to a reference strain of Nocardia veterana. A nucleotide sequence analysis of the 16S ribosomal DNA from the isolate and a reference strain of N. veterana showed 99.8% similarity. All data are consistent with the conclusion that the isolate in this human case of mycetoma is N. veterana.
Additional Inhibitory Effect of Tea Extract on the Growth of Influenza A and B Viruses in MDCK Cells
Authors: Imanishi Nobuko , Tuji Yumiko , Katada Yuko , Maruhashi Miyuki , Konosu Satoko , Mantani Naoki , Terasawa Katutoshi , Ochiai Hiroshi
Published: November 2013

It has been previously reported that green‐tea extract (GTE) inhibits the growth of influenza virus by preventing its adsorption. In this study, we further investigated whether GTE exerts an additional inhibitory effect on the acidification of intracellular compartments such as endosomes and lysosomes (referred to as ELS) and thereby inhibits the growth of influenza A and B viruses in Madin‐Darby canine kidney cells. The vital fluorescence microscopic study showed that GTE inhibited acidification of ELS in a concentration‐dependent manner. Moreover, the growth of influenza A and B viruses was equally inhibited when the cells were treated with GTE within as early as 5 to 15 min after infection, depending on the virus strains. The fact that (—)epigallocatechin (EGC), one of major catechin molecules in GTE, exerts the inhibitory effects on the acidification of ELS and virus growth in a manner similar to that of GTE strongly suggests that EGC is one of the active components in the extract.
Expanding Distribution of Human Serotype G6 Rotaviruses in Australia
Authors: Diwakarla Shanti , Clark Ruth , Palombo Enzo A.
Published: November 2013

Serotype G6 rotaviruses are common pathogens of cattle but are rarely found in humans. In Australia, human G6 isolates have previously been detected in two major southern population centres. A new isolate, ASG6.02, was detected in central Australia (Alice Springs) in 1997. Comparison of the deduced amino acid sequence of the major neutralizing antigen, VP7, indicated that ASG6.02 was related to human G6 viruses isolated from children in Italy and Australia. Phylogenetic analysis supported the close relationship between ASG6.02 and other Australian isolates and indicated that G6 VP7 sequences generally clustered according to the species of origin (human, bovine or porcine). The VP4 type of ASG6.02 was determined as P‐type [14], in common with other isolates from Australia and Italy. The detection of ASG6.02 indicates that the distribution of this serotype is increasing in this country and may have implications for successful vaccine development.
Phylogenetic Analysis of the Human Gut Microbiota Using 16S rDNA Clone Libraries and Strictly Anaerobic Culture‐Based Methods
Authors: Hayashi Hidenori , Sakamoto Mitsuo , Benno Yoshimi
Published: November 2013

The human gut microbiota from three healthy subjects were compared by the use of a sequence analysis of 16S rDNA libraries and a culture‐based method. Direct counts ranged from 1.9 × 1011 to 4.0 × 1011 cells/g (wet weight), and plate counts totaled 6.6 × 1010 to 1.2 × 1011 CFU/g (wet weight). Sixty to seventy percent of the bacteria in the human intestinal tract cannot be cultured with currently available methods. The 16S rDNA libraries from three subjects were generated from total community DNA in the intestinal tract with universal primer sets. Randomly selected clones were partially sequenced. All purified colonies detected from the surface of the agar plate were used for a partial sequencing of 16S rDNA. On the basis of sequence similarities, the clones and colonies were classified into several clusters corresponding to the major phylum of the domain Bacteria. Among a total of 744 clones obtained, approximately 25% of them belonged to 31 known species. About 75% of the remaining clones were novel “phylotypes” (at least 98% similarity of clone sequence). The predominant intestinal microbial community consisted of 130 species or phylotypes according to the sequence data in this study. The 16S rDNA libraries and colonies included the Bacteroides group, Streptococcus group, Bifidobacterium group, and Clostridium rRNA clusters IV, IX, XIVa, and XVIII. Moreover, several previously uncharacterized and uncultured microorganisms were recognized in clone libraries and colonies. Our results also showed marked individual differences in the composition of intestinal microbiota.
Characterization of Parapoxviruses Circulating among Wild Japanese Serows (Capricornis crispus)
Authors: Inoshima Yasuo , Murakami Kenji , Wu Donglai , Sentsui Hiroshi
Published: November 2013

We antigenically and molecularly compared 5 parapoxvirus isolates and 7 viral DNA samples from clinical lesions of Japanese serows with 3 viruses from sheep and goats. All isolates from Japanese serows except one, Ishikawa‐S, reacted with six monoclonal antibodies to orf virus (ORFV). Restriction endonuclease analysis using amplified viral DNA showed the ORFV‐specific pattern in all samples except Ishikawa‐S, which showed a bovine papular stomatitis virus (BPSV)‐specific pattern. Partial nucleotide sequences of the envelope genes were determined and those of all samples from Japanese serows and sheep except Ishikawa‐S were completely identical and also had high identities with the goat virus. These findings suggest that parapoxvirus infection in Japanese serows might be mainly caused by ORFV and accidentally by BPSV. The envelope gene sequenced here seems to be conserved in Japanese ORFVs.
Molecular Characterization of an atl Null Mutant of Staphylococcus aureus
Authors: Takahashi Junko , Komatsuzawa Hitoshi , Yamada Sakuo , Nishida Tetsuya , Labischinski Harald , Fujiwara Tamaki , Ohara Masaru , Yamagishi Jun‐ichi , Sugai Motoyuki
Published: November 2013

atl is a gene encoding a bifunctional peptidoglycan hydrolase of Staphylococcus aureus. The gene product of atl is a 138 kDa protein that has an amidase domain and a glucosaminidase domain, and undergoes processing to generate two major peptidoglycan hydrolases, a 51 kDa glucosaminidase and a 62 kDa amidase in culture supernatant. An atl null mutant was isolated by allelic replacement and characterized. The mutant grew in clusters and sedimented when grown in broth culture. Analysis of peptidoglycan prepared from the wild type and the mutant revealed that there were no differences in muropeptide composition or in glycan chain length distribution. On the other hand, the atl mutation resulted in pleiotropic effects on cell surface nature. The mutant cells showed complete inhibition of metabolic turnover of cell wall peptidoglycan and revealed a rough outer cell wall surface. The mutation also decreased the amount of protein non‐covalently bound to the cell surface and altered the protein profile, but did not affect proteins covalently associated with the cell wall. Lysis of growing cells treated with otherwise lytic concentration of penicillin G was completely inhibited in the mutant, but that of non‐growing cells was not affected by the mutation. The atl mutation did not significantly affect the ability of S. aureus to provoke an acute infection when inoculated intraperitoneally in a mouse sepsis model. These results further support the supposition that atl gene products are involved in cell separation, cell wall turnover and penicillin‐induced lysis of the cells.
Serological Evidence of Transmission of Human Influenza A and B Viruses to Caspian Seals (Phoca caspica)
Authors: Ohishi Kazue , Ninomiya Ai , Kida Hiroshi , Park Chun‐ho , Maruyama Tadashi , Arai Takaomi , Katsumata Etsuko , Tobayama Teruo , Boltunov Andrei N. , Khuraskin Lev S. , Miyazaki Nobuyuki
Published: November 2013

Seroepidemiological surveillance of influenza in Caspian seals (Phoca caspica) was conducted. Antibodies to influenza A virus were detected in 54% (7/13), 57% (4/7), 40% (6/15) and 26% (11/42) of the serum samples collected in 1993, 1997, 1998 and 2000 by enzyme‐linked immunosorbent assay (ELISA). In an hemagglutination‐inhibition (HI) test using H1‐H15 reference influenza A viruses as antigens, more than half of the examined ELISA‐positive sera reacted with an H3N2 prototype strain A/Aichi/2/68. These sera were then examined by HI test with a series of naturally occurring antigenic variants of human H3N2 virus, and H3 viruses of swine, duck, and equine origin. The sera reacted strongly with the A/Bangkok/1/79 (H3N2) strain, which was prevalent in humans in 1979–1981. The present results indicate that human A/Bangkok/1/79‐like virus was transmitted to Caspian seals probably in the early 1980s, and was circulated in the population. Antibodies to influenza B virus were detected by ELISA in 14% (1/7) and 10% (4/42) serum samples collected from Caspian seals in 1997 and 2000, respectively. Our findings indicate that seal might be a reservoir of both influenza A and B viruses originated from humans.
Extracellular ATP Regulates Cell Death of Lymphocytes and Monocytes Induced by Membrane‐Bound Lipoproteins of Mycoplasma fermentans and Mycoplasma salivarium
Authors: Into Takeshi , Okada Kazutaka , Inoue Nobuo , Yasuda Motoaki , Shibata Ken‐ichiro
Published: November 2013

The cytotoxicities of lipoproteins of Mycoplasma fermentans and Mycoplasma salivarium to a lymphocytic cell line, MOLT‐4, and a monocytic cell line, HL‐60, was upregulated by ATP added extracellularly in a dose‐dependent manner. These lipoproteins induced ATP release and plasma membrane permeability increase in these cell lines. In addition, periodate‐oxidized ATP, an antagonist for P2X purinergic receptors, suppressed the cytotoxicity of the lipoproteins, suggesting the possibility that P2X receptors for ATP play crucial roles in the cytotoxicity. Activation of caspase‐3 induced by the lipoproteins, which was assessed by the cleavage of the synthetic substrate DEVD‐pNA and the endogenous substrate poly(ADP‐ribose) polymerase, was also upregulated and downregulated by extracellular ATP and periodate‐oxidized ATP, respectively. On the basis of these results, this study suggests that mycoplasmal lipoproteins induce the permeability increase in lymphocytes and monocytes, by which ATP is released, and the ATP regulates the cytotoxicities of the lipoproteins to the cells, possibly by interaction with ATP receptors such as P2X purinergic receptors.
Monitoring of Active HHV‐6 Infection in Bone Marrow Transplant Recipients by Real Time PCR; Comparison to Detection of Viral DNA in Plasma by Qualitative PCR
Authors: Ihira Masaru , Yoshikawa Tetsushi , Suzuki Kyoko , Ohashi Masahiro , Suga Sadao , Horibe Keizo , Tanaka Naoko , Kimura Hiroshi , Kojima Seiji , Kato Koji , Matsuyama Takaharu , Nishiyama Yukihiro , Asano Yoshizo
Published: November 2013

Twelve (46%) of the 26 patients had human herpesvirus 6 (HHV‐6) viremia after bone marrow transplant (BMT). All isolates were recovered from the samples obtained at 2 weeks after BMT. The sensitivity and the specificity of detection of viral DNA in plasma by qualitative polymerase chain reaction (PCR) for monitoring active virus replication were 92% and 97% respectively. Moreover, the positive (85%) and negative (99%) predictive values were also high. The patients with HHV‐6 viremia showed a clear peak in HHV‐6 DNA in peripheral blood mononuclear cells (PBMCs) at 2 weeks after BMT, which was measured by real time PCR. The virus DNA level in PBMCs between the two groups (patients with viremia and patients without viremia) was statistically different at 2 weeks after BMT (P = 0.033). In patients with HHV‐6 viremia, mean HHV‐6 DNA copy number was higher in the samples collected at 2 weeks after BMT than the samples collected at any other time period.
Phosphorylation of Cytokeratin 17 by Herpes Simplex Virus Type 2 US3 Protein Kinase
Authors: Murata Takayuki , Goshima Fumi , Nishizawa Yuji , Daikoku Tohru , Takakuwa Hiroki , Ohtsuka Kenzo , Yoshikawa Tetsushi , Nishiyama Yukihiro
Published: November 2013

We previously reported the establishment of an HEp2 cell line which expresses the US3 protein kinase (PK) of herpes simplex virus type 2 (HSV‐2) upon induction with IPTG. Here we report that expression, phosphorylation and ubiquitination of cytokeratin 17 (CK17) are enhanced in US3‐expressing HEp2 cells. In vitro kinase and co‐immunoprecipitation assays provided evidence that US3 PK directly phosphorylates CK17. Expression of US3 PK caused a significant decrease in filamentous staining of CK17, suggesting that phosphorylation of CK17 by US3 PK causes a disruption of intermediate filaments. Our observations suggest a role for US3 in the regulation of CKs and intermediate filaments in cells. Moreover, we found that infection of a keratinocyte‐derived cell line, A431, with a US3‐deficient virus, results in cytopathic effects that are morphologically distinct from those induced by wild‐type and revertant viruses, suggesting that US3 PK may be important for interaction between HSV‐2 and peripheral epithelial cells.
Interferon Regulatory Factor 1 in Mycobacterial Infection
Authors: Yamada Hiroyuki , Mizuno Satoru , Sugawara Isamu
Published: November 2013

In order to understand the role of IRF‐1 in the development of murine tuberculosis in vivo, IRF‐1 knockout mice were infected with Mycobacterium tuberculosis by placing them in the exposure chamber of an airborne infection apparatus. These knockout mice developed multifocal necrotic lesions in the lung, liver and spleen tissues and died of disseminated tuberculosis within 43 days of infection. Compared with the levels in wild‐type mice, the pulmonary inducible NO synthase (iNOS) mRNA expression level was significantly lower, but IL‐18 and IL‐6 mRNA levels were higher. There was no statistically significant difference in the expression of IFN‐γ and TNF‐α mRNA between the IRF‐1 knockout and wild‐type mice. IRF‐1 is indirectly responsible for iNOS mRNA expression and plays an important role in the pathogenesis of murine tuberculosis.
Extracellular Proteinase and Phospholipase Activity of Three Genotypic Strains of a Human Pathogenic Yeast, Candida albicans
Authors: Sugita Takashi , Kurosaka Shoko , Yajitate Maki , Sato Hitomi , Nishikawa Akemi
Published: November 2013

Strains of a human pathogenic yeast, Candida albicans, have (A) intronless, (B) intron‐containing, and (C) a mixture of intron‐containing and intronless 26S rRNA genes. To elucidate the significance of these three genotypes in pathogenesis, we measured two major virulence factors, extracellular proteinase and phospholipase activity, in 56 clinical isolates of C. albicans, and investigated the relationship between genotype and enzymatic activity. The genotype B strains had significantly higher proteinase and phospholipase activities than genotypes A or C. These results suggest that to understand the pathogenesis of C. albicans, the genotypes should be considered.
Development of a Mucosal Complex Vaccine against Oral Salmonella Infection in Mice
Authors: Harada Hiroko , Nishikawa Fumiko , Higashi Nobutaka , Kita Eiji
Published: November 2013

We examined the immunogenicity of a Salmonella enterica complex vaccine (CV), consisting of flagellin and polysome purified from serotype Typhimurium LT2. CV plus cholera toxin (CT), in three oral doses given at 7‐day intervals, conferred complete protection on C57BL/6 mice against lethal oral infection with a wild‐type strain. It elicited mucosal IgA>IgG2a>IgG1 and systemic IgG2a>IgG1>IgA antibodies to flagellin and polysome, and delayed footpad response (DFR) to both antigens. In Peyer's patches (PPs) and lamina propria (LP), IgA was produced under a Th1‐dominant environment; CD4T cells from produced interleukin (IL)‐2, interferon (IFN)‐γ, and IL‐10 by stimulation with salmonella extract. On the same protocol, flagellin plus CT induced flagellin‐specific mucosal and systemic IgA and IgG1 antibodies, CD4T cells producing IL‐10 and IFN‐γ in PPs and LP, and only minimal levels of flagellin‐specific DFR. Polysome plus CT induced polysome‐specific mucosal and systemic IgG2a in addition to IgG1 and IgA antibodies, CD4+T cells producing IFN‐γ and IL‐2 in PPs and LP, and polysome‐specific DFR. These two vaccines, however, conferred at most 50–60% survival rates. Our results suggest that polysomes in CV provide effective adjuvant activity for the induction of both mucosal and systemic Th1‐biased responses toward flagellin.
Selection of Human Immunodeficiency Virus Type 1 Variants with an Insertion Mutation in the p6gag and p6pol Genes under Highly Active Antiretroviral Therapy
Authors: Ibe Shiro , Shibata Naomi , Utsumi Makoto , Kaneda Tsuguhiro
Published: November 2013

We detected several types of human immunodeficiency virus type 1 (HIV‐1) variants with an insertion mutation in the p6gag and p6pol genes in eight of twenty‐two (36.4%) patients who possessed drug‐resistant viruses under highly active antiretroviral therapy (HAART). It was characteristic that a conserved proline‐rich motif “PTAPP” in the N‐terminus of p6gag protein was completely or partially duplicated in all cases. Five among the eight cases were retrospectively investigated in terms of the occurrence of dynamic change in the gag gene between the inserted and wild‐type HIV‐1 in the course of HAART. The longitudinal analysis revealed the following: 1) The inserted‐type viruses were selected over the wild‐type during HAART in three cases in which the both types coexisted in the beginning of the therapy. 2) In two cases in which the inserted‐type HIV‐1 alone was detected before the beginning of HAART, the inserted‐type HIV‐1 alone was continuously detected during the therapy. The inserted‐type HIV‐1 was also detected in four of thirty‐nine (10.3%) therapy‐naive patients. However, the frequency of inserted‐type HIV‐1 detection in the HAART‐receiving patients is significantly higher than that in the therapy‐naive patients (P=0.02). These results suggest that this type of insertion mutation is a polymorphism of the p6gag and p6pol genes, however, it consequently gave an advantage on proliferation and/or survival of the HIV‐1 variant under the presence of antiretroviral drugs.
Achievement of Complete Mycological Cure by Topical Antifungal Agent NND‐502 in Guinea Pig Model of Tinea Pedis
Authors: Uchida Katsuhisa , Tanaka Takashi , Yamaguchi Hideyo
Published: November 2013

We examined the therapeutic effect of a 1% cream preparation of NND‐502, a novel topical antifungal agent, in a guinea pig tinea pedis model produced by infecting the plantar skin of guinea pigs with Trichophyton mentagrophytes. Animals developing tinea pedis were divided into two groups: an untreated control group and a treated group. In the latter group, after confirming infection had been established, the infected animals were topically treated with the NND‐502 cream once daily for one week. The animals were reared in a clean environment free from exposure to exogenous dermatophytes. At one week (5 weeks post‐infection), 6 weeks (10 weeks post‐infection) and 16 weeks (20 weeks post‐infection) after completion of the treatment, plantar skin samples were taken from a certain number of both groups of animals. The results demonstrated that all of the animals in the untreated control group and none of those in the treated group were culture‐positive in this animal model of tinea pedis. The topical treatment with NND‐502 achieved a mycological cure. Thus NND‐502 can be considered a promising candidate as a new anti‐dermatophytic agent for topical use.
Seroprevalence ofBartonella henselae, Toxoplasma gondii, FIV and FeLV Infections in Domestic Cats in Japan
Authors: Maruyama Soichi , Kabeya Hidenori , Nakao Ruriko , Tanaka Shigeo , Sakai Takeo , Xuan Xuenan , Katsube Yasuji , Mikami Takeshi
Published: November 2013

Seroprevalence ofBartonella henselae, Toxoplasma gondii, feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infections was investigated in 1,447 domestic cats derived from the north (Hokkaido) to the south (Okinawa) prefectures in Japan. Of the cats investigated, 8.8% (128/1,447) were seropositive toB. henselae, 5.4% (78/1,447) toT. gondii, 9.8% (107/1,088) to FIV, and 2.9% (32/1,088) to FeLV, respectively. ForB. henselaeinfection, the positive rate varied from 11.5% in cats of 1 to <2 years old to 7.2% in those over 3 years old. Outdoor cats showed higher positive rate (14.5%) than that (7.0%) in indoor ones. The rate (13.5%) in flea‐infested cats was significantly higher than that (7.4%) in flea‐negative cats. The positive rates in southern and urban sites were more likely to be higher than those in northern and suburban sites, suggesting that warm and humid environments, density of cat population, and raising status, including hygienic condition and flea infestation in cats may correlate to higher seroprevalence ofB. henselaeinfection. ForT. gondii, FIV and FeLV infections, the seroprevalence also tended to be higher in outdoor, flea‐infested cats and advanced age groups. For FIV infection, the positive rates in male (14.3%) and outdoor cats (15.0%) were significantly higher than those in female (5.0%) and indoor cats (4.6%). On the other hand, no significant difference in seropositivities was observed in FeLV andT. gondiiinfections concerning to both genders and raising status.
Overcoming Tolerance in Hepatitis B Virus Transgenic Mice: A Possible Involvement of Regulatory T Cells
Authors: Roh Sujin , Kim Kilhyoun
Published: November 2013

The hepatitis B virus (HBV) transgenic mouse (Tg) 50–4 strain is immunologically tolerant to HBV antigens. Various vaccination strategies have been attempted but failed to break the tolerance in the mouse. Although the tolerance to HBV antigen is maintained, this mouse strain develops spontaneous liver disease beginning at the age of about 3 months. We attempted to induce immune responses to HBV surface antigen (HBsAg) in the Tg by immunization with recombinant vaccinia virus expressing HBsAg (vvHBV), and observed different immunological responsiveness between 2‐month‐old and 5‐month‐old Tg. In contrast to the unbreakable tolerance reported previously, we could induce both the cytotoxic T lymphocyte (CTL) and the antibody response against HBsAg by the vvHBV immunization. The cytokine expression pattern indicated that T helper 1 type immune response was induced. However, interestingly, these immune responses were observed only in the 5‐month‐old Tg, but not in the 2‐month‐old Tg. Furthermore, CD4+ T cells from 2‐month‐old mice, but not those from 5‐month‐old mice, inhibited CTL response to HBV antigen when adoptively transferred to C57BL/6. These results suggest the possible involvement of regulatory T cell function in the HBV Tg for maintaining tolerance. This study would contribute to a better understanding of immune status of the HBV Tg as a model of human chronic hepatitis and to the search for new therapeutic targets for chronic viral infections.
Induction of Apoptosis in Macrophage Cell Line, J774, by the Cell‐Free Supernatant from Pseudomonas aeruginosa
Authors: Zhang Jianling , Takayama Hisao , Matsuba Takashi , Jiang Ru , Tanaka Yoshinori
Published: November 2013

Pseudomonas aeruginosa is able to secrete many virulence factors that are cytotoxic towards eukaryotic cells. To investigate the effect of the bacterium on macrophages, we obtained cell‐free supernatants from P. aeruginosa (Pa) IID1117 (elastase‐positive and protease‐positive) and Pa IID1130 (elastase‐positive and protease‐negative). After 6 hr of incubation with the cell‐free supernatant from the Pa IID1117 strain, the viability of J774 macrophages was shown to be significantly reduced (47.5±11%), but not Pa IID1130 (96.4±1.6%) at a concentration of 10% (v/v) compared to control J774 macrophages without any supernatant (97.2±1.7%) by the detection of trypan blue dye exclusion. The death of cells was further demonstrated to be due to apoptosis characterized by chromatin condensation and apoptotic bodies by Hoechst 33258 staining, DNA fragmentation by agarose gel electrophoresis and terminal deoxynucleotidyl transferase‐mediated d‐UTP nick end labeling (TUNEL). An activated subunit was found to be released from procaspase‐3 in cell lysate. But in the presence of protease inhibitor, the apoptosis was completely blocked. The findings indicate that the Pa IID1117 strain is capable of inducing apoptosis in J774 macrophages. The apoptosis induced by the cell‐free supernatant from Pa IID1117 strain is suggested to be dependent on protease, but not elastase.
Effects of Tungstosilicate on Strains of Methicillin‐Resistant Staphylococcus aureus with Unique Resistant Mechanisms
Authors: Tajima Yutaka
Published: November 2013

In a previous study, it was found that polyoxotungstates such as undecatungstosilicate (SiW11) greatly sensitized strains of methicillin‐resistant Staphylococcus aureus (MRSA) to β‐lactams. In this report, the effects of SiW11 on several MRSA strains with unique resistant mechanisms were studied. SiW11 was still effective to MRSA mutants with higher β‐lactam resistance due to reduced cell‐lytic activity. Since the antimicrobial effect of TOC‐39 (a cephem antibiotic with strong affinity to penicillin‐binding protein (PBP) 2′) was not strongly enhanced in any case, it was confirmed that the sensitizing effect of SiW11 is due to reduced expression of PBP2′. However, the sensitizing effect of SiW11 was relatively weak in MRSA strains with lowered susceptibility to glycopeptide antibiotics. A certain resistant mechanism other than the mecA‐PBP2′ system worked in such a strain. Interestingly, an MRSA mutant with the Eagle‐type resistance was dramatically sensitized. This result suggests that SiW11 has another site of action besides reducing the expression of PBP2′.
Autologous Cancer Vaccine: A Novel Formulation
Authors: Ohno Tadao
Published: November 2013

A novel cancer vaccine consisting of fixed autologous cancer tissue‐fragments, microparticles encapsulating cytokines, and an adjuvant was developed. In a Phase I/IIa clinical trial, vaccination to patients after resection of hepatocellular carcinoma induced significantly longer time before the first recurrence than that in historical control patients operated in the same department (P<0.05). This formulation will be promising against recurrence of many types of human cancers.
Comparative Studies of the Cell Structures of Mycobacterium leprae and M. tuberculosis Using the Electron Microscopy Freeze‐Substitution Technique
Authors: Takade Akemi , Umeda Akiko , Matsuoka Masanori , Yoshida Shin‐ichi , Nakamura Masahiro , Amako Kazunobu
Published: November 2013

The cell envelope and cytoplasmic architecture of the Mycobacterium leprae Thai‐53 strain were examined using the freeze‐substitution technique of electron microscopy and compared with those of the M. tuberculosis H37Rv strain. Both strains had similarly multilayered envelope architectures composed of an electron‐translucent layer, a peptidoglycan layer and the plasma membrane, from outside to inside. A comparison of the structures of these two mycobacteria revealed that the M. leprae cell was smaller in size and had a thinner peptidoglycan layer than the M. tuberculosis cell. The cell widths measured on electron micrographs were 0.44 μm for M. tuberculosis and 0.38 μm for M. leprae. The peptidoglycan layer of M. leprae was 4–5 nm, while the corresponding layer of M. tuberculosis was 10–15 nm.