bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and Characterization of a Major Antigenic Component of Malassezia globosa to IgE Antibodies in Sera of Patients with Atopic Dermatitis
Authors: Koyama Tomoki , Kanbe Toshio , Ishiguro Ayako , Kikuchi Akihiko , Tomita Yasushi
Published: November 2013

Three major components of Malassezia globosa were isolated from 2‐ME extracts of this fungus by ion‐exchange column chromatography and are referred to as Malg46a, Malg46b and Malg67, respectively. IgE antibodies to these components in the sera of patients with AD were detected by immunoblots. In Western blot, IgE antibodies to Malg46b were most frequently detected in the sera of AD patients. Dot blot with the Malg46b‐containing fraction immunologically reacted with 69% of the sera of the patients, and with 83% of the sera of the patients who were positive for IgE antibodies to the 2‐ME extract of M. globosa in the Western blot. The intensities generated for each dot correlated well with the total intensities generated for the 2‐ME extract of M. globosa in the Western blot (r=0.763). In the lectin blot, Con A reacted with both Malg46a and Malg46b but not with Malg67. The polyclonal antibody to Malg46b reacted strongly only with the 2‐ME extract of M. globosa and reacted slightly with M. restricta. In conclusion, a glycoprotein, Malg46b of M. globosa, is dominantly expressed in this fungus and is a possible major antigen for IgE antibodies in patients with AD.
Effect of the Sialyl Lewis X (SLe) Moiety on Splenic Accumulation of SLex‐Carboxymethylpullulan Conjugate
Authors: Horie Kazutoshi , Sakagami Masahiro , Kuramochi Kentaro , Ito Teruomi , Hamana Hiroshi
Published: November 2013

Sialyl Lewis X (SLex), an E‐selectin ligand, was conjugated with carboxymethylpullulan (CMPul) and the disposition characteristics of this conjugate after intravenous administration were investigated using mice with ear edema. The concentration of 3H‐labeled SLex‐CMPul in the spleen was significantly high. When CMPul was modified with a saccharide unable to bind to E‐selectin, this splenic accumulation was not observed. The uptake of radiolabeled SLex‐CMPul by the spleen was completely inhibited by a 100‐fold molar of cold SLe‐CMPul but not by a sialyl N‐acetyllactosamine‐CMPul conjugate (SLN‐CMPul). Microautoradiography analyses revealed that SLex‐CMPul accumulated in the marginal zone of the spleen.
Dithiothreitol Enhances Listeria monocytogenes Mediated Cell Cytotoxicity
Authors: Westbrook David G. , Bhunia Arun K.
Published: November 2013

The hybridoma Ped‐2E9 based cytotoxicity assay was developed to distinguish virulent from avirulent Listeria species in 6 hr. The cytotoxicity effect on Ped‐2E9 was reported to be primarily due the cytolytic action of listeriolysin O (LLO), produced by L. monocytogenes. In this study, the effect of a reducing agent, dithiothreitol (DTT, 0–2 mm) that is known to activate LLO was investigated to make the Ped‐2E9 based cytotoxicity assay an even more sensitive and rapid. Also, we examined the effect of fetal bovine serum (FBS, 0–50%), a common ingredient of tissue culture media on cytotoxicity. A DTT concentration of 0.5 mm gave an optimum cytotoxicity effect, which could be measured by both alkaline phosphatase (AP) and lactate dehydrogenase (LDH) assays in just 1.5–2 hr. FBS, at levels between 10 to 50%, significantly inhibited Listeria‐mediated cytotoxicity. Concentrated culture filtrates from L. monocytogenes or LLO producing recombinant L. innocua (prfA+hlyA+) strain also caused cytotoxicity effects, which were observed by scanning electron microscopy or a cytotoxicity assay in 2–3 hr. Interestingly, addition of DTT to culture filtrates produced 100% cell cytotoxicity in just 15 min. This indicated that LLO activity, which is responsible for Ped‐2E9 cytotoxicity, was augmented several folds with the addition of a reducing agent. Examination of Listeria isolates belonging to different serogroups from clinical sources or naturally contaminated meat products with DTT gave cytotoxicity results in 2 hr, which were comparable to the 5‐hr assay analyzed concurrently without DTT. These results indicated that DTT, which activated the LLO, could be used in the cytotoxicity assay to enhance Listeria‐mediated Ped‐2E9 cell cytotoxicity. This knowledge will greatly assist us to develop a user‐friendly rapid assay to screen cytopathogenic properties of Listeria species.
Blockade ofSalmonella enteritidisPassage across the Basolateral Barriers of Human Intestinal Epithelial Cells by Specific Antibody
Authors: Sugita‐Konishi Yoshiko , Ogawa Michinaga , Arai Soichi , Kumagai Susumu , Igimi Shizunobu , Shimizu Makoto
Published: November 2013

Antibodies specific toSalmonella enteritidis(S.E.) were obtained from immunized egg yolk, and their protective effects against S.E. were studied by using monolayer‐cultured human intestinal epithelial cells, Caco‐2 and T84. TheSalmonellaadherence and entry to the cells were partially inhibited by the antibodies. The antibodies inhibited the decrease in transepithelial electrical resistance (TEER) of the intestinal epithelial monolayers and IL‐8 secretion of the cells induced by S.E. invasion. Also, the antibodies blocked the penetration of bacteria through the cell layer although they did not inhibit the growth of bacteria in the cells. Confocal microscopic photographs revealed the bacteria in the infected monolayer cells were bound to antibodies. These results indicate that anti‐S.E. antibodies may protect the cells from destruction induced by S.E. invasion in intestinal epithelial cells in addition to the partial inhibition of adhesion and invasion of S.E. at the cell surface. Passive antibodies against invasive bacteria would be useful to prevent the migration of S.E. to blood not only at the cell surface but also inside of intestinal epithelial cells.
Differential Level in Co‐Down‐Modulation of CD4 and CXCR4 Primed by HIV‐1 gp120 in Response to Phorbol Ester, PMA, among HIV‐1 Isolates
Authors: Tahara‐Hanaoka Satoko , Ushijima Yuki , Tarui Hiroshi , Wada Masayuki , Hara Toshio , Imanishi Shigeo , Yamaguchi Tomoyuki , Hattori Toshio , Nakauchi Hiromitsu , Koito Atsushi
Published: November 2013

HIV‐1 enters cells through interacting with cell surface molecules such as CD4 and chemokine receptors. We generated recombinant soluble gp120s derived from T‐cell line‐tropic (T‐tropic) and macrophage‐tropic (M‐tropic) HIV‐1 strains using a baculovirus expression system and investigated the association of CD4‐gp120 complex with the chemokine receptor and/or other surface molecule(s). For monitoring the co‐down‐modulations of the CD4‐gp120 complex, a cytoplasmic domain deletion mutant (tailless CD4), which is not capable of undergoing down‐modulation by itself in response to phorbol ester PMA, was used. Our studies revealed both cell‐type and HIV‐1 strain‐specific differences. We found that T‐tropic gp120s were capable of priming co‐down‐modulation with tailless CD4 by interacting with CXCR4, whereas M‐tropic SF162 gp120 could not after PMA treatment even in the presence of CCR5. Among the T‐tropic HIV‐1 envelopes, IIIB gp120 was the most potent. Furthermore, the ability of gp120 to prime the PMA induced co‐down‐modulation of tailless CD4 appeared to be dependent on the concentration of the principal coreceptor CXCR4. Nevertheless, the observation that IIIB gp120 strongly primed tailless CD4 co‐down‐modulation on human osteosarcoma HOS cells that express undetectable levels of surface CXCR4 raised the possibility that membrane component(s) other than those recently identified can be involved in down‐modulation of the CD4/gp120 complexes.
Development of a Chromosome‐Plasmid Balanced Lethal System for Lactobacillus acidophilus with thyA Gene as Selective Marker
Authors: Fu Xiaoli , Xu Jian‐guo
Published: November 2013

A chromosome‐plasmid balanced lethal gene delivery system for Lactobacillus acidophilus based on the thyA gene was developed. The selected L. acidophilus DOM La strain carries a mutated thyA gene and has an obligate requirement for thymidine. This strain can be used as a host for the constructed shuttle vector pFXL03, lacking antibiotic‐resistant markers but having the wild‐type thyA gene from L. casei which complements the thyA chromosomal mutation. The vector also contains the replicon region from plasmid pUC19 and that of the Lactococcus plasmid pWV01, which allows the transfer between Escherichia coli, L. casei and L. acidophilus. Eight unique restriction sites (i.e., PstI, HindIII, SphI, SafI, AccI, XbaI, KpnI and SacI) are available for cloning. After 40‐time transfers in modified MRS medium, no plasmid loss was observed. The vector pFXL03 is potentially useful as a food‐grade vaccine delivery system for L. acidophilus.
Heterogeneous Post‐Translational Modification of Actinobacillus actinomycetemcomitans Fimbrillin
Authors: Inoue Tetsuyoshi , Ohta Hiroyuki , Tanimoto Ichiro , Shingaki Ryuji , Fukui Kazuhiro
Published: November 2013

Fresh isolates of Actinobacillus actinomycetemcomitans produce bundle‐forming fimbriae. The exact molecular mass of A. actinomycetemcomitans fimbrillin, a structural subunit of fimbriae, was determined by liquid chromatography‐electrospray ionization mass spectrometry. Three major molecular species with 6,226.0, 6,366.0, and 6,513.0 Da were detected in a purified fimbrial fraction from the strain 310‐a. These molecular masses were significantly higher than the molecular weight (5,118 Da) calculated from nucleotide sequence data of the fimbrillin gene, flp, suggesting that the fimbrial peptides were post‐translationally modified. Modification of the fimbrial peptides was also suggested by an N‐terminal amino acid sequence analysis of fimbrillin peptic fragments, with the modified amino acids being due to seven serine or asparagine residues located in the C‐terminal region. A periodate oxidation/biotin‐hydrazide labeling assay of fimbrillin suggested that it might be glycosylated.
Nucleotide Sequencing and Transcriptional Analysis of Two Tandem Genes Encoding Glucosyltransferase (Water‐Soluble‐Glucan Synthetase) in Streptococcus cricetus HS‐6
Authors: Inoue Miho , Inoue Tetsuyoshi , Miyagi Atsushi , Tanimoto Ichiro , Shingaki Ryuji , Ohta Hiroyuki , Fukui Kazuhiro
Published: November 2013

Two tandem genes encoding glucosyltransferase synthesizing water‐soluble glucan (GTF‐S) were cloned from the lambda gene library of Streptococcus cricetus HS‐6 (serotype a) using anti‐GTF‐S antibody, and the nucleotide sequences were analyzed. The two genes (ORF1 and ORF2) were identified as streptococcal glucosyltransferases based on the following evidence: [1] the deduced amino acid sequences of their products have an active site for catalytic action and C‐terminal repeated units for dextran binding, and [2] a homology search revealed that the ORF1 and ORF2 products are homologous to the GtfS protein (77.4%) of S. downei Mfe28 and GtfT protein (83.8%) of S. sobrinus OMZ176, respectively, which are both known to have GTF‐S activity. Therefore, ORF1 and ORF2 might be designated gtfS and gtfT of S. cricetus, respectively. A Northern blotting and RNase protection assay suggested that the gtfS and gtfT of S. cricetus are transcribed as a single bicistronic mRNA as well as separate monocistronic mRNAs. Primer extension analysis indicated multiple transcriptional start points for each gene.
Adherence Protects the Binding Sites of Helicobacter pylori Urease from Acid‐Induced Damage
Authors: Icatlo Faustino C. , Yokoyama Hideaki , Kuroki Masahiko , Kobayashi Chizu , Goshima Hideo , Ikemori Yutaka , Kodama Yoshikatsu
Published: November 2013

Colonization by Helicobacter pylori partly depends on acid‐dependent adherence by urease to gastric mucin. To further verify the relevance of urease adherence to colonization, the influence of acidity on the binding sites of H. pylori urease was investigated. When enzyme‐based in vitro ligand capture assays were used, the effect of acidity on the binding site of H. pylori urease was determined against a backdrop medium consisting of acidic buffers simulating the luminal side of gastric mucus. A high degree of stability was exhibited by adherent urease, suggesting a pivotal role by the denatured enzyme in the persistence of the bacterium within the acidified compartment of gastric mucus.
Metal Content and Biochemical Analyses of a Recombinant Collagenase PrtV from Vibrio parahaemolyticus
Authors: Yu Mei‐shiuan , Yap Mee‐ngan , Lee Chia‐yin
Published: November 2013

PrtV is an extracellular metalloprotease of Vibrio parahaemolyticus and regarded as a collagenase. Inductively coupled plasma‐optical emission spectrometry analysis indicated that the recombinant PrtV contains 1 mol of zinc per mol of the native enzyme. On the basis of a kinetic study using 2‐furanacryloyl‐Leu‐Gly‐Pro‐Ala (FALGPA, the specific substrate for bacterial collagenase) as a substrate, it was suggested that metal ions may play a significant role in the binding and catalytic steps of the substrate. PrtV hydrolyzed type I, II, III, and IV collagens; however, it did not hydrolyze type V. In addition, the hydrolysis of native proteins and synthetic substrates revealed that PrtV possesses higher activity toward collagen and collagenlike sequences. The result of the thermal stability study indicated that PrtV was thermostable up to 40 C; at 50 C, stability gradually decreased. In addition, PrtV showed higher storage stability at −20 and 4 C, respectively, than at 25 C. Compared with collagenases from Clostridium histolyticum and Vibrio alginolyticus, PrtV was immunologically different and had no significant effect on the growth of CHO, HeLa, and Vero cells. Taken together, the results of the studies described in this paper advance our knowledge concerning the metal content and biochemical properties of PrtV.
Functional Analysis of the Type III Secretion System in Enteropathogenic Escherichia coli O157: H45
Authors: Abe Akio , Nagano Hideki
Published: November 2013

A mass outbreak of Escherichia coli O157:H45 was first reported in Japan in 1998. This pathogen was classified as an enteropathogenic E. coli (EPEC) O157 because it was characterized by the Shiga toxin gene (stx)‐negative and bundle‐forming pilus (bfp) gene‐positive genotypes. In this study, we investigated the type III secretion system in EPEC O157. Although no type III secreted proteins, Esps (E. coli‐secreted proteins), in EPEC O157:H45 were detectable in culture supernatant, secreted proteins were induced by the introduction of an EPEC plasmid‐encoded regulator, per. In further contrast to EHEC O157:H7, EPEC O157:H45 triggered the accumulation of tyrosine phosphorylated proteins beneath the adherent bacteria. These results suggest that regulation of the type III secretion apparatus and host signal transduction events between E. coli O157:H45 and O157:H7 are completely different.
Molecular Analysis of the folC Gene of Pseudomonas aeruginosa
Authors: Murata Takahiro , Bognar Andrew L. , Hayashi Tetsuya , Ohnishi Makoto , Nakayama Keisuke , Terawaki Yoshiro
Published: November 2013

We have cloned the Pseudomonas aeruginosa folC gene coding for folylpolyglutamate synthetase‐dihydrofolate synthetase, which was located between the trpF and purF loci, and determined the nucleotide sequence of the folC gene and its flanking region. The deduced amino acid sequence of P. aeruginosa FolC was highly homologous to that of Escherichia coli FolC. The cloned gene complemented E. coli folC mutations and was found to encode both folylpolyglutamate synthetase and dihydrofolate synthetase activities. The gene organization around the folC gene in P. aeruginosa was completely conserved with that in E. coli; the accD gene was located upstream of the folC gene, and dedD, cvpA and purF genes followed the folC gene in this order. The gene arrangement and the result of the promoter activity assay suggested that the P. aeruginosa accD and folC genes were co‐transcribed.
Decline in the HIV‐1 Isolation Rate in Japan: A 12‐Year Observation
Authors: Nakasone Tadashi , Takamatsu Junki , Watanabe Kuhomi , Naganawa Satoshi , Someya Kenji , Yoshino Naoto , Kaizu Masahiko , Ohsu Takeaki , Takizawa Mari , Izumi Yasuyuki , Kawahara Mamoru , Hara Takashi , Fujimura Yoshihiro , Yamada Kaneo , Nagai Yoshiyuki , Yamazaki Shudo , Honda Mitsuo
Published: November 2013

Since 1988, we have isolated HIV‐1 from 614 HIV‐1‐infected persons (total sample = 2,785) in Japan. During the past 12 years, we have found a decline in the HIV‐1 isolation rate in Japan, with two identifiable turning points, 1991–1992 and 1996–1997. The two turning points correspond to shifts in anti‐HIV‐1 therapy. These findings suggest that HIV‐1 in Japan is currently biologically well controlled, probably due to anti‐HIV‐1 therapy. On the other hand, this decline is inconsistent with the recent increase of genetic drug‐resistant HIV‐1 in Japan. Further studies are needed to clarify mechanisms that might explain the discrepancy.
Characterization of a Novel Vibrio parahaemolyticus Phage, KVP241, and Its Relatives Frequently Isolated from Seawater
Authors: Matsuzaki Shigenobu , Inoue Tetsuyoshi , Tanaka Shuji , Koga Tetsuro , Kuroda Masayuki , Kimura Susumu , Imai Shosuke
Published: November 2013

A vibriophage, KVP241, and six of its relatives were isolated independently from seawater using Vibrio parahaemolyticus as the host. All of the phages had the same morphology (a hexagonal head and a tail with a contractile sheath) and the same host range (specific for some V. parahaemolyticus strains). DNA‐DNA hybridization experiments elucidated that their genomes are highly homologous to each other. Analyses of amino acid sequences of putative major capsid proteins indicated that KVP241 may be weakly related to T4‐type phages having a more elongated head.
IL‐4 Is Required for Defense against Mycobacterial Infection
Authors: Sugawara Isamu , Yamada Hiroyuki , Mizuno Satoru , Iwakura Yoichiro
Published: November 2013

Although the involvement of T helper (Th1) cells is central to protection against intracellular bacteria, including Mycobacterium tuberculosis, the involvement of Th2 cells, characterized by potent interleukin (IL)‐4 secretion in mycobacterial infection is still unclear. In order to clarify the role of IL‐4 in murine tuberculosis, IL‐4‐deficient mutant mice, IL‐4 knockout (IL‐4 KO) mice, were utilized. The mice were infected with H37Rv, Kurono or BCG Pasteur via an airborne infection route by placing them in the exposure chamber of a Middlebrook airborne infection apparatus. Their capacity to control mycobacterial growth, granuloma formation, cytokine secretion, and nitric oxide (NO) production were examined. These mice developed large granulomas, but not necrotic lesions in the lungs, liver or spleen (P < 0.05). This was consistent with a significant increase in lung colony‐forming units (CFU). Compared with levels in wild‐type mice, upon stimulation with mycobacteria, splenic IL‐10 levels were low and IL‐6 levels were intermediate, but interferon (IFN)‐γ and IL‐12 levels were significantly higher. IL‐18 levels were within the normal range. The level of NO production by alveolar macrophages of the IL‐4 KO mice was similar to that of the wild‐type mice. Granulomatous lesion development by IL‐4 KO mice was inhibited significantly by treatment with exogenous recombinant IL‐4. These findings were not specific to the IL‐4 KO mice used. Our data show that IL‐4 may play a protective role in defense against mycobacteria, although IFN‐γ and TNF‐α play major roles in it. Our data do not rule out an IFN‐γ‐independent function of IL‐4 in controlling tuberculosis.
Salmonella typhimurium Induces Apoptosis in Human Monocyte‐Derived Macrophages
Authors: Zhou Xin , Mantis Nicholas , Zhang Xiao‐ru , Potoka Douglas A. , Watkins Simon C. , Ford Henri R.
Published: November 2013

Salmonella species represent a leading cause of gastroenteritis worldwide. More recently, they have been proposed as putative vaccine delivery vehicles in humans. Oral infection with Salmonella leads to invasion of the intestinal epithelial barrier and subsequent interaction with mucosal macrophages. In this study, we investigated the fate of Salmonella typhimurium‐infected human macrophages differentiated from blood monocytes by GM‐CSF. Wild type S. typhimurium strain SL1344 induced macrophage surface blebbing and caused the release of host cytoplasmic lactate dehydrogenase beginning 30 min post‐infection. Three hours later more than 80% of the macrophages in the culture were killed. In contrast, during the same period, macrophages infected with the non‐invasive S. typhimurium strain BJ66 remained viable. Chromatin fragmentation is a hallmark of cells undergoing apoptosis. Using TUNEL analysis, we observed chromatin fragmentation in macrophages infected with SL1344 but not in BJ66 infected cells. Consistent with this observation, we found that pretreatment of human macrophages with an inhibitor of caspase‐3, a member of the pro‐apoptotic enzyme family shown to be involved in S. typhimurium‐induced killing of mouse macrophages, reduced SL1344‐mediated cytotoxicity by 40%. Our study provides the first evidence that invasive S. typhimurium induces apoptosis in human macrophages that were differentiated from blood monocytes by GM‐CSF, and that cell death is a caspase‐dependent phenomenon.
Circulating Soluble CD4 Directly Prevents Host Resistance and Delayed‐Type Hypersensitivity Response to Cryptococcus neoformans in Mice
Authors: Kawakami Kazuyoshi , Koguchi Yoshinobu , Qureshi Mahboob Hossain , Yara Satomi , Kinjo Yuki , Miyazato Akiko , Nishizawa Akio , Nariuchi Hideo , Saito Atsushi
Published: November 2013

In the present study, we examined the effect of soluble CD4 (sCD4) on host resistance and delayed‐type hypersensitivity (DTH) response to Cryptococcus neoformans using a novel mutant mouse that exhibits a defect in the expression of membrane‐bound CD4 but secretes high levels of sCD4 in the serum. In these mice, host resistance to this pathogen was impaired as indicated by an increased number of live pathogens in the lung. To elucidate the mechanism of immunodeficiency, three different sets of experiments were conducted. First, administration of anti‐CD4 mAb restored the attenuated host defense. Second, in CD4 gene‐disrupted (CD4KO) mice, host resistance was not attenuated compared to control mice. Third, implantation of sCD4 gene‐transfected myeloma cells rendered the CD4KO mice susceptible to this infection, while similar treatment with mock‐transfected cells did not show such an effect. These results indicated that immunodeficiency in the mutant mice was attributed to the circulating sCD4 rather than to the lack of CD4+ T cells. In addition, DTH response to C. neoformans evaluated by footpad swelling was reduced in the mutant mice compared to that in the control, and the reduced response was restored by the administration of anti‐CD4 mAb. Finally, serum levels of IFN‐γ, IL‐12 and IL‐18 in the mutant mice were significantly reduced, while there was no difference in Th2 cytokines, such as IL‐4 and IL‐10. Considered collectively, our results demonstrated that sCD4 could directly prevent host resistance and DTH response to C. neoformans through interference with the production of Th1‐type cytokines.
Molecular Cloning of Rat NK Target Structure —The Possibility of CD44 Involvement in NK Cell‐Mediated Lysis
Authors: Kanki Kazushige , Torigoe Toshihiko , Hirai Itaru , Sahara Hiroeki , Kamiguchi Kenjiro , Tamura Yasuaki , Yagihashi Atsuhito , Sato Noriyuki
Published: November 2013

The nature of target molecules of natural killer (NK) cell‐mediated lysis remains to be elucidated. As we previously reported, mAb 109 recognizes one of the tumor‐associated antigens, designated as 109 antigen (Ag), expressed on the cell surface of rat fibrosarcomas W31 and W14, which are transformants of WFB (rat fetal fibroblast cell line) with H‐ras oncogene. 109Ag was thought to be a target structure of NK cells since mAb 109 inhibited NK cell‐mediated lysis against W31 and W14. Here, we demonstrate by molecular cloning that 109Ag is identical to rat CD44. Immunoprecipitation and immunoblotting studies also showed that mAb 109 and anti‐rat CD44 mAb OX‐50 recognize the same protein of W31 cell lysates with an 86 kDa molecular size. CD44 was suggested to be a target structure of NK cell‐mediated lysis; however, rat CD44 cDNA transfection alone into CD44 null cell lines did not result in up‐regulation of target cell susceptibility to NK cell‐mediated lysis. Our results therefore indicated that CD44 may play a crucial role as one of the target structures in our rat fibrosarcoma system though the cell surface expression of CD44 alone does not affect NK susceptibility of the target cells.
A Simple and Rapid Method for Purifying Staphylococcal Exfoliative Toxin A
Authors: Yamazaki Taisuke , Sakurai Susumu , Matsuo Naruto , Machida Katsuhiko , Kohno Minoru
Published: November 2013

A rapid and efficient method of purification of staphylococcal exfoliative toxin A, the causative agent of staphylococcal scalded skin syndrome (SSSS), has been developed. It is based on ammonium sulfate precipitation of the culture supernatant and hydrophobic interaction chromatography on Phenyl‐Sepharose CL‐4B. This procedure results in 87‐fold purification of this toxin, which appears as a single band in sodium dodecyl sulfate‐polyacrylamide gels.
A Role for Lipid Rafts in Immune Cell Signaling
Authors: Katagiri Yohko U. , Kiyokawa Nobutaka , Fujimoto Junichiro
Published: November 2013

Cross‐linking of surface receptors in hematopoietic cells results in the enrichment of these receptors in the rafts along with other downstream signaling molecules. A possible explanation how signal is transduced through the plasma membrane has arisen from the concept of raft. From the study of cellular responses in the plasma membrane which enrich members of the Src‐family tyrosine kinase, rafts can function as centers of signal transduction by forming patches. Under physiological conditions, these elements synergize to transduce successfully a signal at the plasma membrane. Rafts are suggested to be important in controlling appropriate protein interactions in hematopoietic cells, and aggregation of rafts following receptor ligation may be a general mechanism for promoting immune cell signaling.
Effect of Constitutively Expressed phoP Gene on the Localization of Salmonella typhimurium within Mac‐1 Positive Phagocytes
Authors: Matsui Hidenori , Eguchi Masahiro , Kikuchi Yuji
Published: November 2013

Intracellular localization of the wild‐type (Spv+), the phoP‐constitutively expressed strain (PhoPc), and the spv‐deleted strain (Spv–) of Salmonella typhimurium was examined by the use of confocal laser scanning microscopy analysis of immunostained sections of mouse spleens after oral or subcutaneous inoculation. Only 40% of salmonellae of both the PhoPc and the Spv– strains were detected intracellularly within Mac‐1 positive cells at day five after oral or day four after subcutaneous inoculation. In contrast, over 85% of salmonellae of the Spv+ strain were detected inside Mac‐1 positive cells. In both inoculation trials, the splenic colony‐forming unit values for the PhoPc and Spv– strains were significantly lower than the corresponding value for the Spv+ strain. These findings suggest that the constitutively expressed phoP gene of S. typhimurium attenuated virulence by limiting intracellular proliferation within mouse spleen phagocytes, and that the lack of spv genes had the same effect.
A Nested PCR Assay to Detect DNA in Sera for the Diagnosis of Deep‐Seated Trichosporonosis
Authors: Sugita Takashi , Nakajima Masamitsu , Ikeda Reiko , Niki Yoshihito , Matsushima Toshiharu , Shinoda Takako
Published: November 2013

Deep‐seated trichosporonosis caused by Trichosporon asahii has a high mortality rate and a very poor prognosis. New species‐specific oligonucleotide primers for T. asahii were developed from a sequence analysis of rRNA genes that included the internal transcribed spacer regions. A nested PCR assay with specific primers was used to examine 11 serum samples from 7 patients, who were diagnosed with deep‐seated trichosporonosis histologically at autopsy. In addition, Trichosporon cell wall polysaccharide (PS) was detected by a latex agglutination (LA) test. Of 11 samples, seven had a positive LA test, and T. asahii DNA was also detected with the nested PCR assay. Of the four samples in which PS antigen was not detected, the nested PCR of two samples was positive. Our new nested PCR assay may be used as an adjunct to conventional methods for diagnosing T. asahii infection.
Chemotaxis of Oral Treponemes toward Sera and Albumin of Rabbit
Authors: Umemoto Toshihiko , Jinno Takayashi , Taiji Yasuhiro , Ogawa Tomohiko
Published: November 2013

The motility and chemotaxis of human oral spirochetes Treponema denticola ATCC 35404, T. medium ATCC 700293, and T. vincentii ATCC 35580 were examined by a capillary assay method. Of five sera three human oral treponemes were dominantly chemoattractant to the rabbit serum. The checkerboard analysis of chemotaxis toward rabbit serum clearly showed that the motile T. denticola cells swam toward the culture media containing higher concentrations of the rabbit serum. T. denticola chemotaxis to the rabbit serum was clearly reduced by heating serum, and rabbit albumin contributed by 60 to 70% to its chemotaxis to the rabbit serum. Western blotting analysis demonstrated that these treponemes possessed rabbit albumin‐binding polypeptides with approximate molecular sizes of 65 kDa and 70 kDa. Immunoelectron microscopy demonstrated that a 65 kDa rabbit albumin‐binding polypeptide was located on the outer envelopes, suggesting that the rabbit albumin‐binding polypeptide is responsible for chemotaxis toward rabbit serum.
Detection of Virulence Associated Genes in Clinical Strains of Vibrio mimicus
Authors: Bi Kewei , Miyoshi Shin‐ichi , Tomochika Ken‐ichi , Shinoda Sumio
Published: November 2013

A total of 42 clinical strains of Vibrio mimicus were examined for the presence of virulence associated genes toxR, toxS, toxT, tcpP, ctx and tcpA by PCR assay. Almost all strains were shown to have the toxR gene, while the toxS gene was found in 27 strains. On the other hand, five strains possessed both toxT and tcpP genes, but others had neither. Only two strains were positive for amplification of the ctx gene, whereas no PCR product with tcpA primers was detected. The results indicate the incomplete copies of virulence cascade in V. mimicus strains. The pathogenesis and epidemic potential of this species is also discussed.
Molecular Evolution of Human Echovirus 9 Isolated from Patients with Aseptic Meningitis in Northern Kyushu during the Summer of 1997
Authors: Hara Koyu , Kashiwagi Takahito , Ohtsu Yasushi , Masunaga Kenji , Akasu‐Tsuji Yuko , Tsumura Naoki , Kato Hirohisa , Iwahashi Jun , Hamada Nobuyuki , Toyoda Michiko , Toyoda Tetsuya
Published: November 2013

An epidemic of aseptic meningitis caused by human echovirus 9 (E‐9) occurred in the summer of 1997 in northern Kyushu, Japan. Sequences of genome position 2504–3358, which encoded a part of VP1, of the nine isolated viruses were determined. An RGD motif and B‐C loop were found in all. They were almost identical and closely related to the virulent strain Barty.
Characterization of Two Genes Encoding Ferritin‐Like Protein in Actinobacillus actinomycetemcomitans
Authors: Hirosue Masaru , Kokeguchi Susumu , Maeda Hiroshi , Nishimura Fusanori , Takashiba Shogo , Murayama Yoji
Published: November 2013

Two genes encoding ferritin‐like protein, designated afnA and afnB, were identified in the upstream region of actX on the Actinobacillus actinomycetemcomitans chromosomal DNA. The actX has been reported to be a regulatory gene homologous to the Escherichia coli fnr, which controls the growth and virulence of A. actinomycetemcomitans under anaerobic conditions. The afnB located 340 bp‐upstream from the actX, and the afnA located just 15 bp‐upstream from afnB. The afnA and afnB encoded 161 and 165 amino acid residues, respectively, which were similar to ferritin‐like proteins of other microorganisms. Western immunoblotting using rabbit antiserum against E. coli ferritin showed these two proteins, which are reactive with the serum with 19‐kDa molecular masses, are produced from A. actinomycetemcomitans. The N‐terminal amino acid sequences of the two proteins were consequent with those deduced from afnA and afnB. Northern hybridization revealed that the afnA and afnB constituted a bicistronic operon and the accumulation of afnA and afnB mRNA was upregulated under aerobic conditions. These findings suggested that the operon was regulated by the presence of oxygen. The two ferritin‐like proteins may have important roles in the adaptation of A. actinomycetemcomitans to oxidative environmental changes.
Diversity among Strains of Cryptococcus neoformans var. gattii as Revealed by a Sequence Analysis of Multiple Genes and a Chemotype Analysis of Capsular Polysaccharide
Authors: Sugita Takashi , Ikeda Reiko , Shinoda Takako
Published: November 2013

We demonstrated the diversity of Cryptococcus neoformans var. gattii strains by a sequence analysis of multiple genes: (i) the intergenic spacer (IGS) 1 and 2 regions of the rRNA gene; (ii) the internal transcribed spacer (ITS) region, including 5.8S of the rRNA gene; (iii) TOP1 (topoisomerase); and (iv) CAP59. In these studies, we compared C. neoformans var. gattii with varieties grubii, and neoformans of C. neoformans. Phylogenetic analysis indicated that both C. neoformans var. grubii and C. neoformans var. neoformans are monophyletic, but C. neoformans var. gattii showed polyphyletic. C. neoformans var. gattii can be divided into three phylogenetic groups, I, II, and III, with high bootstrap support Phylogenetic group I contains serotype B and C strains, and groups II and in include serotype B strains. Because the serotype B strains of C. neoformans var. gattii exhibited more genetic divergence, the serological characteristics and chemotypes of their capsular polysaccharide were further investigated. No remarkable difference among the serotype B strains was found in the reactivities to factor serum 5, which is specific for serotype B. The NMR spectra of the capsular polysaccharide from serotype B strains could be divided into three characteristic patterns, but the chemical shifts were very similar. These results suggested that the serotype B strain of C. neoformans var. gattii has more genetic diversity than the serotype C strain of C. neoformans var. gattii or the varieties grubii and neoformans of C. neoformans, but there was no correlation between genotype and chemotype.
Common Cell‐Surface Antigens Functioning in Self‐Recognition Reactions by Both Somatic Cells and Gametes in the Solitary Ascidian Halocynthia roretzi
Authors: Arai Makoto , Suzuki‐Koike Miho , Ohtake Shin‐Ichi , Ohba Hiroyoshi , Tanaka Kunio , Chiba Joe
Published: November 2013

The “contact reaction” is an extremely rapid allogeneic cytotoxic reaction (ACR) mediated by hemocytes in the solitary ascidian Halocynthia roretzi. It has been proposed that regulation of the alloreactivity of hemocytes may be involved in preference for fertilization or self‐sterility in this species. To identify the receptors and target ligands involved both in self‐recognition by somatic cells and self‐discrimination by gametes, we produced monoclonal antibodies (mAbs) that inhibit the ACR mediated by hemocytes and tested their effects on fertilization. Six different mAbs that inhibit the ACR were prepared and categorized into three groups. Although all three mAbs seemed to have the same ability to inhibit the ACR, almost constant and statistically significant inhibition (CRB1.1) and infrequent but significant inhibition (CRB2.1, and CRB3.1) of the ACR were observed in the same pairs of animals. Pretreatment of the unfertilized eggs with CRB1.1, CRB2.1, and CRB3.1, resulted in the constant and statistically significant inhibition, infrequent but significant inhibition, and no inhibition, respectively, of fertilization. Antigens recognized by CRB1.1 (CRB1.1 antigens) were detected on the cell surface of all types of hemocytes and on the vitelline coat and follicle cells of unfertilized eggs. CRB2.1 and CRB3.1 antigens were detected on the surface of certain types of hemocytes and follicle cells, but not on the vitelline coat. CRB mAbs were directed against different epitopes in the N‐linked glycan on glycoproteins. These common carbohydrate antigens on somatic cells and gametes may function in some recognition processes in ACR and fertilization in H. roretzi.
Mechanism of Inhibitory Effect of NCC164 on Replication of Human Immunodeficiency Virus
Authors: Nakamura Mariko , Terada Masaki , Matsuzaki Katsuhiro , Yoshida Kiyotsugu , Ohno Tsuneya
Published: November 2013

The effect of a novel anti‐HIV (human immunodeficiency virus) compound, designated NCC164, has been studied with HIV infected cultures. The agent exhibited concentration‐dependent inhibition of HIV replication in primary infected cultures of H9 cell line and PBMCs. Substantial inhibition of viral replication was observed at concentration of NCC164 that showed little cytotoxicity. The ratio of IC50 values for the MTT (3–4,5 dimethylthiazol‐2,5 diphenyl tetrazolium bromide) to RT (reverse transcriptase) assays means the selectivity index was more than 100. In attempting to define the inhibitory mechanism of NCC164, we investigated its effect on each step of HIV replication. This agent was highly effective against HIV replication regardless of the addition period during early stages of infection (adsorption to integration) but did not inhibit reverse transcriptase activity directly. The agent efficiently blocked virus maturation without side effect and the number of progeny produced by NCC164‐treated cells was markedly reduced.
PCR Detection Method of Clostridium scindens and C. hiranonis in Human Fecal Samples
Authors: Kitahara Maki , Sakamoto Mitsuo , Benno Yoshimi
Published: November 2013

Clostridium scindens and C. hiranonis have high bile acid 7α‐dehydroxylating activity. Direct PCR and nested PCR with the specific primers for C. scindens and C. hiranonis were developed and tested for the detection of them in human fecal samples. Nested PCR detected C. scindens in all samples, but direct PCR detected it in 27 of 34 samples. Regarding C. hiranonis, nested PCR detected it in only 18 of 34 samples although nested PCR has a high level of sensitivity. Nested PCR was a specific and sensitive method for the detection of C. scindens and C. hiranonis in fecal samples.
Characterization of the Lactobacillus casei Group and the Lactobacillus acidophilus Group by Automated Ribotyping
Authors: Ryu Chun Sun , Czajka John W. , Sakamoto Mitsuo , Benno Yoshimi
Published: November 2013

A total of 91 type and reference strains of the Lactobacillus casei group and the L. acidophilus group were characterized by the automated ribotyping device Riboprinter® microbial characterization system. The L. casei group was divided into five (C1–C5) genotypes by ribotyping. Among them, the strain of L. casei ATCC 334 was clustered to the same genotype group as most of L. paracasei strains and L. casei JCM 1134T generated a riboprint pattern that was different from the type strain of L. zeae. These results supported the designation of L. casei ATCC 334 as the neotype strain, but were not consistent with the reclassification of L. casei JCM 1134T as L. zeae. The L. acidophilus group was also divided into 14 (A1–A11, B1–B3) genotypes by ribotyping. L. acidophilus, L. amylovorus, L. crispatus and L. gallinarum generated ribotype patterns that were distinct from the patterns produced by L. gasseri and L. johnsonii. This result confirmed previous data that the L. acidophilus group divided to two major clusters. Five strains of L. acidophilus and two strains of L. gasseri were correctly reidentified by ribotyping. Most strains belonging to the L. casei group and the L. acidophilus group were discriminated at the species level by automated ribotyping. Thus this RiboPrinter® system yields rapid, accurate and reproducible genetic information for the identification of many strains.
Structural Characterization of the O‐Antigenic Polysaccharide Chain of Porphyromonas circumdentaria NCTC 12469
Authors: Matsuo Kouki , Isogai Emiko , Araki Yoshio
Published: November 2013

Structural studies were carried out on an O‐antigenic polysaccharide moiety derived from Porphyromonas circumdentaria NCTC 12469, a reference strain of Porphyromonas species. The polysaccharide chain was composed of d‐glucose, d‐galactose, N‐acetyl‐d‐glucosamine, and N‐acetyl‐d‐galactosamine in a molar ratio of 1:2:1:1. On the basis of results from 1H‐ and 13C‐NMR spectroscopic analyses including COSY, TOCSY, and HMQC experiments together with results of Smith degradation, methylation analysis, and partial acid hydrolysis, it is concluded that the polysaccharide chain has a pentasaccharide repeating unit of →6)‐β‐d‐Glcp‐(1→6)‐β‐d‐Galp‐(1→6)‐β‐d‐Galp‐(1→3)‐β‐d‐GlcpNAc‐(1→3)‐β‐d‐GalpNAc‐(1→. The immunoreaction between P. circumdentaria LPS and the corresponding antiserum was strongly inhibited by the pentasaccharide fragment (Glc‐Gal‐Gal‐GlcNAc‐GalNAc) isolated from partial acid hydrolysis of the above polysaccharide, suggestive of O‐antigen specific antibodies in the used antiserum.
Comparative Sequences of Two Type 1 Dengue Virus Strains Possessing Different Growth Characteristics In Vitro
Authors: Ishak Hasanuddin , Takegami Tsutomu , Kamimura Kiyoshi , Funada Hisashi
Published: November 2013

The complete genome sequences of two dengue‐1 virus strains having different growth characteristics (Mochizuki and A88) were compared with other published strains. The sequence analysis indicated several unique amino acid changes throughout the coding region of Mochizuki strain, mostly in envelope (E) protein. A unique amino acid, Ile‐69 for Mochizuki strain at E protein resulted in the loss of an Asn‐67‐linked glycosylation site. A Thr substitution for Ala‐114 at C protein and amino acid changes found in E, non‐structural NS3, NS4a, and NS5 proteins were unique for A88 strain. These substitutions might be correlated to their different growth characteristics in vitro.
Stabilization of Cauliflower Mosaic Virus P3 Tetramer by Covalent Linkage
Authors: Tsuge Seiji , Okuno Tetsuro , Furusawa Iwao , Kubo Yasuyuki , Horino Osamu
Published: November 2013

Cauliflower mosaic virus (CaMV) open reading frame (ORF) III encodes a 15 kDa protein (P3) that is indispensable for viral infectivity. Although P3 has been shown to be a prerequisite for CaMV aphid transmission, its role in viral replication remains unknown. We previously showed that P3 forms a tetramer in planta and that P3 tetramer co‐sediments with viral coat protein on sucrose gradient centrifugation, suggesting that a tetramer may be the functional form of P3. We presumed that disulfide bonds were involved in tetramer formation because 1) the tetramer was detected by Western blotting after electrophoresis under non‐reducing conditions, and 2) the cysteine‐X‐cysteine motif is well conserved in CaMV P3 and P3 homologues among Caulimoviruses. Therefore we mutated either or both of the cysteine residues of CaMV P3. The mutant viruses were infectious and accumulated to a similar extent as the wild‐type. An analysis of mutant proteins confirmed that the wild‐type P3 molecules in the tetramer are covalently bound with one another through disulfide bonds. It was also suggested that mutant proteins are less stable than wild‐type protein in planta. Furthermore, sedimentation study suggested that the disulfide bonds are involved in stable association of P3 with CaMV virions or virion‐like particles, or both. The mutant viruses could be transmitted by aphids. These results suggested that the covalent bonds in P3 tetramer are dispensable for biological activity of P3 under experimental situations and may have some biological significance in natural infection in the field.
Cytokine Kinetics in the Plasma of Monkeys Infected with Pathogenic and Nonpathogenic Simian and Human Immunodeficiency Chimeric Viruses at an Early Stage of Infection
Authors: Kwofie Theophilus B. , Haga Takeshi , Iida Tohko , Hayami Masanori , Miura Tomoyuki
Published: November 2013

To evaluate the pattern of cytokines as a result of pathogenic and nonpathogenic SHIV infections in monkeys, we analyzed the cytokines interleukin (IL)‐2, IL‐4, IL‐10, IL‐12, and interferon (IFN)‐γ in the plasma of 8 monkeys infected with either pathogenic 89.6P or nonpathogenic NM‐3rN chimeric viruses. The cytokine kinetics in the 89.6P‐infected monkeys was characterized by increases of IL‐2, IL‐10, and to some extent IFN‐γ and a decrease of IL‐12. Although that of NM‐3rN‐infected monkeys was characterized by an increase of IFN‐γ, and a transient decrease of IL‐12. IL‐4 was not detected in any of the monkeys. The results, therefore, showed a mixture of Th‐1 and Th‐2 cytokine profiles implying these cytokines are not clear enough to use as an index of the pathogenicity of the viruses at an early stage of infection.
Epidemiological Survey of Orientia tsutsugamushi Distribution in Field Rodents in Saitama Prefecture, Japan, and Discovery of a New Type
Authors: Tamura Akira , Yamamoto Narishige , Koyama Shoichiro , Makisaka Yukie , Takahashi Masakazu , Urabe Ken‐ichi , Takaoka Masatoshi , Nakazawa Kiyoaki , Urakami Hiroshi , Fukuhara Masahiro
Published: November 2013

There are various antigenic variants of Orientia tsutsugamushi which are distinguished by immunological and molecular genetic methods targeted at the antigenic diversity of 56‐kDa type‐specific antigen proteins. The present study was performed to analyze 15 strains successfully isolated from rodents in Saitama Prefecture, Japan, by 56‐kDa gene sequence homologies, reactivities with type‐specific monoclonal antibodies and polymerase chain reaction (PCR) using type‐specific primer‐pairs. We demonstrated the presence of a new type of O. tsutsugamushi among the isolates. This new type, designated as the Saitama type, was located in the branch of Karp type in the phylogenetic tree based on 56‐kDa gene sequences, but distant from the known Karp types, such as Karp, JP‐1 and JP‐2, showing less than 90% homology. Strains of this type could not be distinguished by immunological methods from Karp type strains, but a new primer‐pair for PCR which specifically amplifies the DNA of this new type strain was designed. This primer‐pair may serve to find this strain type in future studies.
Growth Inhibition of Cancer Cells by Co‐Transfection of Diphtheria Toxin A‐Chain Gene Plasmid with Bovine Leukemia Virus‐Tax Expression Vector
Authors: Tana  , Watarai Shinobu , Aida Yoko , Tajima Shigeru , Kakidani Hitoshi , Onuma Misao , Kodama Hiroshi
Published: November 2013

We constructed a plasmid containing bovine leukemia virus (BLV)‐tax gene driven by SRα promoter, designated as pME‐BLVtax, to activate the promoter of the long terminal repeat (LTR) of BLV in various tumor cells. Activation of the promoter of BLV‐LTR by pME‐BLVtax was confirmed by luciferase assay. When the cells, such as COS‐1, C8, and KU‐1, were transfected with a plasmid pBLV‐LUC1, which contained the luciferase gene under the control of BLV‐LTR, and pME‐BLVtax, luciferase was expressed in these cells, whereas no luciferase gene expression was observed when only pBLV‐LUC1 was introduced into the cells. Activation of the BLV‐LTR promoter was regulated by pME‐BLVtax and 0.5 μg of pME‐BLVtax was sufficient for the expression of the gene under the control of BLV‐LTR. Furthermore, pME‐BLVtax was used to direct the cell expression of the gene for diphtheria toxin A‐chain under the control of BLV‐LTR (pLTR‐DT) to various tumor cell lines, KU‐1, C8, COS‐1, BL2M3, and HeLa cells. The transfection was carried out with cationic liposomes. In this experiment, co‐transfection of pLTR‐DT with pME‐BLVtax exerted selective growth inhibitory effects on the tumor cell lines. Moreover, three co‐introductions of pLTR‐DT with pME‐BLVtax into the cell lines resulted in significant inhibition of the cell growth. This result suggests that the delivery of the pLTR‐DT and pME‐BLVtax genes into tumor cells by the use of cationic liposomes may be potentially useful as a novel approach for the treatment of tumor cells.