bluuebunny/crossref_metadata_2025 huggingface dataset:
Fate of Legionella pneumophila in Macrophages of C57BL/6 Chronic Granulomatous Disease Mice
Authors: Saito Mitsumasa , Kajiwara Hideko , Miyamoto Hiroshi , Yoshida Shin‐ichi
Published: November 2013

We compared the intracellular survival and growth of Legionella pneumophila Philadelphia‐1 in peritoneal macrophages obtained from A/J, C57BL/6, and X‐linked chronic granulomatous disease (CGD) mice produced from C57BL/6 strain. The initial killing was observed in A/J and C57BL/6 macrophages at 2,4 and 6 hr after in vitro phagocytosis, but not in the CGD macrophages. Thereafter, there was a 10‐fold increase of CFU in A/J macrophages. The bacteria, however, did not proliferate in C57BL/6 and CGD macrophages at 24 or 48 hr after in vitro phagocytosis. These results suggest that effector molecules for the initial killing are a superoxide anion and its metabolites, and Lgn1 gene product inhibits the intracellular growth of L. pneumophila independently of NADPH oxidase.
Identification of a H+/Glucose and Galactose Symporter Gene glt from Xanthomonas oryzae pv. oryzae
Authors: Tsuge Seiji , Furutani Ayako , Kubo Yasuyuki , Horino Osamu
Published: November 2013

We identified a glucose and galactose transporter gene from the plant‐pathogenic bacterium Xanthomonas oryzae pv. oryzae. Sequence analysis indicated that the gene, named glt, encoded a polypeptide of 592 amino acid residues and the product was significantly homologous with members of the Na+/glucose cotransporter (SGLT) family from mammalian and bacterial origin, especially with vSGLT from Vibrio parahaemolyticus (50% identity). GLT functioned as a glucose and galactose transporter in an Escherichia coli mutant deficient in glucose and galactose transport activity. A protonophore inhibited the transport activity, suggesting that GLT is a H+‐coupled glucose/galactose symporter.
Immunomodulatory Effect of Gold Sodium Thiomalate on Murine Acquired Immunodeficiency Syndrome
Authors: Yamaguchi Koushi , Ushijima Hiroshi , Hisano Michi , Inoue Yousuke , Shimamura Tadakatsu , Hirano Takao , Müller Werner E.G.
Published: November 2013

Induction of IL‐2 production and increased expression of CD25 were observed in C57BL/10 mice after weekly treatment with gold sodium thiomalate (GST). LP‐BM5 murine leukemia virus (MuLV) infected mice treated with GST survived longer, had less cervical lymph node swelling, lower spleen weight, and fewer abnormalities in the expression of the cell surface markers, CD4, CD8a and CD45R/B220 on spleen cells than those that were not treated with GST. Thus, GST treatment may be beneficial through a decrease in disease progression via IL‐2 induction in MuLV infected mice. This may have application in human immunodeficiency virus‐infected individuals.
Development of a New Medium Useful for the Recovery of Dermatophytes from Clinical Specimens by Minimizing the Carryover Effect of Antifungal Agents
Authors: Nakashima Takuji , Nozawa Akira , Ito Takao , Majima Toshiro , Yamaguchi Hideyo
Published: November 2013

Two surface‐active compounds, egg lecithin and polysorbate 80, usually used as the deactivators of various preservatives were tested whether they also counteract either or all of the three major topical antifungal drugs, bifonazole (BFZ), lanoconazole (LCZ) and terbinafine (TBF). Both egg lecithin and polysorbate 80, when added to culture media up to final concentrations of 1.0 and 0.7%, respectively, antagonized the anti‐dermatophytic activity of the three drugs in a concentration‐dependent manner. A greater extent of antagonistic action was exerted when the two deactivators combined at their maximal levels tested were added; MIC's of BFZ were increased more than 30‐fold and those of LCZ and TBF more than 200‐fold compared with the values obtained in the absence of the deactivators. Using the agar medium supplemented with the combined deactivators, culture studies were carried out with skin tissues specimens taken from guinea pigs whose feet were infected with dermatophytes and subsequently treated with 1% topical preparations of the three antifungal drugs. The experimental data from this animal study demonstrated that the combined deactivators‐supplemented medium yielded increased numbers of fungi compared with the basal medium. It looks, therefore, likely that the fungal recovery on the former medium more correctly reflects to actual fungal burden in the infected lesions than the latter. All these results suggest that the combined deactivators‐supplemented medium is more useful for mycological evaluation of therapeutic efficacy of imidazole and allylamine drugs against dermatophytoses in both preclinical and clinical studies.
Oral Delivery of Antigens in Liposomes with Some Lipid Compositions Modulates Oral Tolerance to the Antigens
Authors: Masuda Kazuyoshi , Horie Kazutoshi , Suzuki Ryuji , Yoshikawa Takayoshi , Hirano Koichiro
Published: November 2013

Several liposomes containing ovalbumin (OVA), a model antigen, with different lipid compositions were prepared in order to evaluate their ability to induce oral tolerance. Oral administration of these liposomal OVAs induced suppression of the proliferative responses of popliteal lymph node cells from the treated mice to OVA, suggesting that these treated mice were tolerized. The efficiency of the induction of oral tolerance was affected by the liposome composition. OVA entrapment in these liposomes could modulate the tolerizing dose of OVA itself. These results suggest that some liposomes can be suitable antigen‐delivery systems for modulated and/or effective induction of oral tolerance.
Elastase from Activated Human Neutrophils Activates Procarboxypeptidase R
Authors: Kawamura Takeshi , Okada Noriko , Okada Hidechika
Published: November 2013

Carboxypeptidase R (EC 3.4.17.20; CPR) is an unstable basic carboxypeptidase found in fresh serum in addition to carboxypeptidase N (CPN) which is a stable enzyme. CPR in fresh serum is generated from its zymogen (proCPR) during coagulation by trypsin‐like enzymes such as thrombin and thrombin/thrombomodulin complexes. Since removal of the C‐terminal arginine abrogates the anaphylatoxin activity of C3a and C5a, CPR and CPN are regarded as anaphylatoxin inactivators. We report here that the culture supernatant of activated human neutrophils converts proCPR to CPR. Addition of an elastase specific inhibitor, N‐methoxysuccinyl‐Ala‐Ala‐Pro‐Val‐chloromethyl ketone (MSAAPVCK) to the supernatant of stimulated neutrophils completely inhibited activation of proCPR. On the other hand, a thrombin specific inhibitor, p‐Nitrophenyl‐p'‐amidinophenyl‐methanesulfonate hydrochloride (pNP‐pAPMS) inhibited only 16% of proCPR activation by the neutrophil supernatant. Furthermore, purified elastase converted proCPR to CPR. Therefore, elastase can activate proCPR directly, or indirectly through activation of some proteases, which have been contaminating in reagents. Release of CPR generating enzymes from neutrophils should play an important role in regulation of excess inflammation.
Characterization of Spotted Fever Group Rickettsiae Detected in Dogs and Ticks in Okinawa, Japan
Authors: Satoh Hiroshi , Motoi Yurie , Camer Gerry A. , Inokuma Hisashi , Izawa Masako , Kiyuuna Tsuyoshi , Kumazawa Norichika , Muramatsu Yasukazu , Ueno Hiroshi , Morita Chiharu
Published: November 2013

Spotted fever group (SFG) rickettsial DNAs were detected in 2.4% of 340 canine blood samples and a pool of 84 tick pool samples (229 ticks) collected in Okinawa, Japan by PCR using a citrate synthase and an SFG rickettsial 190‐kDa surface antigen gene primer pair. The sequences of both genes from canine blood and tick samples showed high levels of similarity with those of Rickettsia japonica and several SFG rickettsiae (R. aeschlimannii, R. massiliae, R. rhipicephali and Bar‐29 strain). Phylogenesis of canine blood and tick samples was closely related to that of reference SFG rickettsiae. Serological evidence of SFG rickettsial infection in dogs and humans in Okinawa, where no clinical human cases have been reported, has been obtained. In this study, genetical characterization of SFG rickettsia in Okinawa was investigated phylogenetically.
Adhesion of Lactic Acid Bacteria to Caco‐2 Cells and Their Effect on Cytokine Secretion
Authors: Morita Hirotsugu , He Fang , Fuse Tetsuo , Ouwehand Arthur C. , Hashimoto Hideo , Hosoda Masataka , Mizumachi Koko , Kurisaki Jun‐ichi
Published: November 2013

Cytokines secreted by human enterocytes play a critical role in mucosal and systemic immunity. Intestinal microorganisms can influence this secretion. In the present study, 30 strains of lactic acid bacteria were characterized for their adhesion to Caco‐2 cells and their potential to stimulate proinflammatory cytokine secretion by this cell line. The bacteria adhered in a strain‐dependent manner to Caco‐2 cells. Contact with lactobacilli did not result in the production of IL‐6 or IL‐8. A slight IL‐6 and IL‐8 production by a Caco‐2 cell was detected after exposure to 8 of the tested Bifidobacterium strains. No correlation was found between adhesion and cytokine induction among the bacteria tested. This indicates that lactic acid bacteria, even those with strong adhesive properties, are not very likely to trigger an inflammatory response in human enterocytes.
Hemagglutinating Factor (HAF) Associated with Adhesiveness in Enteroinvasive Escherichia coli (EIEC)
Authors: Simi Silvia , Pelosi‐Teixeira E. , Yamada Áaureo T. , Joazeiro Paulo P. , Catani Cleide F. , Yano Tomomasa
Published: November 2013

A cell‐associated mannose‐resistant hemagglutinating factor (HAF) was extracted from enteroinvasive Escherichia coli (EIEC) serotype O124:H by sonication. Ultrastructural analysis of EIEC and immunocytochemical assays with the cell‐free HAF and EIEC bacterial cells on HeLa cells, suggested that the HAF is a non‐fimbrial putative adhesive factor that mediates in vivo adherence of EIEC to human epithelial cells.
Genetic Analysis of Rabies Virus Isolates in the Philippines
Authors: Nishizono Akira , Mannen Kazuaki , Elio‐Villa Luningning P. , Tanaka Seiichi , Li Kang‐sheng , Mifune Kumato , Arca Benito F. , Cabanban Arturo , Martinez Benjamin , Rodriguez Arlito , Atienza Victor C. , Camba Rolando , Resontoc Nilo
Published: November 2013

To determine the genetic characteristics of the rabies virus in the Philippines, 59 rabies virus isolates were obtained from domestic rabid dogs and their partial nucleotide sequences of nucleoprotein (N) gene were compared. Based on comparison with reported sequences, phylogenetic analysis revealed that all isolates from the Philippines had close genetic relations and formed two subgroups. The Philippines isolates belonged to a different lineage from other Asian isolates but were closer to them than to terrestrial isolates and laboratory strains. Several specific nucleotide and amino acid substitutions were observed among the Philippines isolates. Our results suggest that rabies viruses in the Philippines might have a characteristic evolution.
Studies on the Escape Mutants of Rabies Virus Which Are Resistant to Neutralization by a Highly Conserved Conformational Epitope‐Specific Monoclonal Antibody #1‐46‐12
Authors: Irie Takashi , Matsuda Yukihiro , Honda Yoshikazu , Morimoto Kinjiro , Kawai Akihiko
Published: November 2013

We investigated a virus‐neutralizing conformational epitope of the rabies virus glycoprotein (G) that is recognized by an anti‐G monoclonal antibody (mAb; #1‐46‐12) and shared by most of the laboratory strains of the virus. To investigate the epitope structure, we isolated escape mutants from the HEP‐Flury virus (wild‐type; wt) after repeated passages in culture in the presence of the mAb. Immunofluorescence studies indicated that the mutants could be classified into two groups; the Group I lacked the epitope, while Group II preserved the epitope. The latter was dominant under the passage conditions, since Group I disappeared during the continuous passages. G proteins showed different electrophoretic mobilities; G protein of Group I migrated at the same rate as wt G protein, while that of Group II migrated at a slower rate, which was shown to be due to acquisition of an additional oligosaccharide side chain. Nucleotide sequencing of the G gene strongly suggested that amino acid substitutions at Thr‐36 by Pro and Ser‐39 by Thr of the G protein are responsible for the escape mutations of Groups I and II, respectively. The latter is a unique mutation of the rabies virus that allows the G protein to be glycosylated additionally at Asn‐37, a potential glycosylation site that is not glycosylated in the parent virus, in preserving the epitope‐positive conformation. These results suggest that to keep the 1‐46‐12 epitope structure is of greater survival advantage for the virus to escape the neutralization than to destroy it, which could be achieved by acquiring an additional oligosaccharide chain at Asn‐37.
Isolation and Genetic Characterization of a Novel Filamentous Bacteriophage, a Deleted Form of Phage f237, from a Pandemic Vibrio parahaemolyticus O4:K68 Strain
Authors: Chang Bin , Miyamoto Hiroshi , Taniguchi Hatsumi , Yoshida Shin‐ichi
Published: November 2013

We isolated a filamentous bacteriophage, VfO4K68, from the pandemic Vibrio parahaemolyticus strain belonging to O4:K68 serovar. The VfO4K68 DNA lacked a 1,893‐bp fragment present in that of the distinctive region of f237, a filamentous phage isolated from a pandemic O3:K6 strain (Nasu, H. et al., J. Clin. Microbiol., 38, 2156–2161, 2000). The deletion resulted in the formation of a novel open reading frame (ORF) that possesses homology to the ORF 27 of ETA phage and staphylococcal enterotoxin E (SEE) of Staphylococcus aureus. VfO4K68 was able to infect the recipient O3:K6 serovar strains. These results suggest that VfO4K68 might act as a genetic transmitter and play some roles in the pandemic V. parahaemolyticus infection.
Menadione‐Catalyzed Luminol Chemiluminescent Assay for Viability of Mycobacterium bovis
Authors: Yamashoji Shiro
Published: November 2013

Stable luminol chemiluminescence was observed 10 min after the addition of menadione to a suspension of Mycobacterium bovis homogenized in Middlebrook 7H9 broth base including OADC enrichment. The chemiluminescence intensity was proportional to the absorbance of the bacterial suspension at 600 nm in a range of 0.005 to 0.15. Luminol chemiluminescence disappeared after 10 min incubation of M. bovis at over 60% of ethanol or 4 days of cultivation of M. bovis in the presence of 40 μg/ml of streptomycin. The bacterium showing the disappearance of chemiluminescence could not grow after being washed, suggesting that the inhibition concentration of the antimicrobials can be estimated on the basis of the disappearance of chemiluminescence. Menadione‐catalyzed luminol chemiluminescent assay was rapid and sensitive in comparison to turbidimetry, tetrazolium (WST‐8) reduction assay, and the assay using the Mycobacteria growth indicator tube (MGIT).
Identification of Staphylococcus xylosus Isolated from C57BL/6J‐Nos2tm1Lau Mice with Dermatitis
Authors: Won Young Suk , Kwon Hyo Jung , Oh Goo Taeg , Kim Bang Hyun , Lee Chul Ho , Park Yong Ho , Hyun Byung Hwa , Choi Yang Kyu
Published: November 2013

Coagulase negative staphylococci (CNS) are significant pathogens, particularly in medical device related infections and in immunocompromised patients. Five CNS strains were isolated from 5 NOS2 knockout mice with dermatitis. Histologically, granulomatous dermatitis was found in the skin around the ear with epidermal ulceration. Dermal lesions included pustules, necrosis, and accumulations of neutrophils and macrophages. Isolates of the bacterial strains were identified to be Staphylococcus xylosus by the API STAPH kit and 16S–23S intergenic transcribed spacer‐PCR. These results demonstrate the potential of this organism to be an opportunistic pathogen in immunocompromised mice.
Characterization of Borrelia afzelii Isolated from Ixodes nipponensis and Apodemus agrarius in Chungju, Korea, by PCR‐RFLP Analyses of ospC Gene and rrf (5S)‐rrl(23S) Intergenic Spacer
Authors: Lee Seung‐hyun , Jung Kwang‐don , Lee Jung‐hee , Kim Sang‐choin , Kim Jong‐hyun , Jang Won‐jong , Park Kyung‐hee
Published: November 2013

Nine Borrelia afzelii strains, which had been isolated from two vectors, Ixodes nipponensis and Apodemus agrarius in Chungju, Korea, were characterized by PCR‐RFLP analyses of ospC genes and rrf (5S)‐rrl (23S) intergenic spacer. DraI restriction patterns of Chungju strains were identical to those of B. afzelii VS461. But MseI restriction patterns of rrf(5S)‐rrl (23S) intergenic spacer genes of KK2, KM4, KK5 differed from those of previously reported B. burgdorferi sensu lato strains. Nine Chungju strains were classified with four distinct ospC RFLP patterns, which differed from the eight ospC RFLP patterns (A‐1 to A‐8) of previously reported B. afzelii. Moreover, five additional restriction patterns were deduced from published ospC sequences of reference strains. These results suggest that Chungju strains are very heterogeneous.
Human Immunodeficiency Virus Type 1 Subtype C Exhibits Higher Transactivation Activity of Tat than Subtypes B and E
Authors: Kurosu Takeshi , Mukai Tetsu , Komoto Satoshi , Ibrahim Madiha S. , Li Yong‐gang , Kobayashi Takeshi , Tsuji Shoutaro , Ikuta Kazuyoshi
Published: November 2013

Although human immunodeficiency virus type 1 (HIV‐1) subtypes C and E are expanding faster and seem to be of greater global significance than HIV‐1 subtype B, there is only little information about Tat activity of such non‐B subtypes. Here, we showed evidence that subtype C Tat exhibits higher transcriptional activity from the HIV‐1 long‐terminal repeat (LTR) in a human T‐cell line, compared with subtypes B and E. This higher activity of subtype C Tat was not due to the LTR, but to the Tat sequence variability. We examined three candidate regions with sequence for the higher activity of subtype C Tat, such as the cysteine‐rich motif, the basic domain, and the 2nd exon. The results showed that the variation in subtype C Tat at two amino acid residues, Ser57 and Glu63 in stead of Arg57 and Gln63 in subtypes B and E, within and close to the basic domain were involved in the higher activity of subtype C Tat. This variation did not affect its nuclear localization activity. Thus, there may be a significant advantage for the high Tat activity on subtype C replication.
Niche‐Specific Association of Aeromonas Ribotypes from Human and Environmental Origin
Authors: Bauab Tais Maria , Levy Carlos Emílio , Rodrigues Josias , Falcão Deise Pasetto
Published: November 2013

A total of 88 Aeromonas isolates from distinct locations and sources (39 from extraintestinal infections, 31 from diarrhoeic, ten from non‐diarrhoeic faeces, all human, and eight from fresh water) were subjected to phenospecies identification, serotyping, ribotyping and detection of some virulence markers. The strains belonged to four different phenospecies marked by 19 O serogroups and 38 ribotypes. No strong correlation between these parameters was found, and no group, as defined by the typing methods, could be characterized with a particular set of virulence markers. There was a clear association of ribotypes with the source of the strains. Cluster analysis allowed the identification of a complex of ribotypes belonging to distinct but related sources, including clinical and environmental isolates. These results suggest that ribotyping may be an epidemiological tool suitable for the study of Aeromonas infections.
The Reduction of Fusobacterium nucleatum in Mice Is Irrelevant to the Nitric Oxide Induced by iNOS
Authors: Kato Chihomi , Mikami Masato , Suzuki Anri , Saito Kazuko
Published: November 2013

Previously we reported that mice infected recurrently with live Fusobacterim nucleatum (Fn) synthesize a significant amount of NO between 12 hr and 24 hr after the Fn injection. We now investigated whether the NO has the capability of killing Fn, a gram‐negative rod periodontal pathogen. The mice were divided into three groups: treated with live bacteria (LB), treated with heat‐killed bacteria (HKB) and untreated: normal (N). The Fn reduction, NO production and cell number after Fn injection were then compared in these mice. In the LB group, no Fn was detected at 6 hr, whereas it was still detected in the HKB and N groups at 24 hr as assessed by both colony counts and PCR assays. A significant amount of NO was synthesized in the LB group at 24 hr after the Fn injection. Fn is not killed by SNAP‐generated NO in vitro. An increase in the total cell number was accompanied by an increase of the neutrophil numbers in the LB group. Intracellular O2– generation (including ONOO–) was visualized using dihydrorhodamine (DHR)‐123. The peak of O2– generation by PEC was shown to be at 3 hr in all 3 groups. The number of O2– positive cells in the LB group at 3 hr was remarkably high, and most of them were likely to be neutrophils. The Fn reduction would be performed cooperatively via oxygen dependent and oxygen independent mechanisms. Thus reactive oxygen species (ROS) included in the oxygen dependent mechanism appear to be important for Fn reduction. However the significant amounts of NO derived from the iNOS synthesized in the LB group between 12 hr and 24 hr after injection of LFn were not involved in the Fn reduction.
Lactoferrin Feeding Augments Peritoneal Macrophage Activities in Mice Intraperitoneally Injected with Inactivated Candida albicans
Authors: Wakabayashi Hiroyuki , Takakura Natsuko , Teraguchi Susumu , Tamura Yoshitaka
Published: November 2013

Oral administration of lactoferrin (LF), an innate‐defense protein present in exocrine secretions such as milk and in neutrophils, is reported to improve host‐protection against infections with microorganisms including pathogenic fungi, possibly due to an immunomodulatory effect. This study aimed to evaluate the effect of bovine LF feeding on peritoneal macrophage activities in mice intraperitoneally injected with inactivated Candida albicans. Time course analysis during the 14 days following Candida‐priming revealed that LF administration slightly increased the number of peritoneal exudate cells, and significantly enhanced the production of superoxide anion (O2–) and nitric oxide (NO) by peritoneal macrophages at day 7. LF administration facilitated NO production and Candida hyphal‐growth inhibition by macrophages derived from Candida‐primed mice but not non‐primed mice, suggesting that the action of LF is dependent on the immune status of the host. LF administration altered the kinetics of cytokines in the peritoneal lavage fluid of Candida‐primed mice. Enhancement of cytokine levels by LF was observed for IL‐12 at day 5 and IFN‐7 at day 9, but not for TNF‐α or IL‐10. In conclusion, LF feeding augmented the activities of macrophages in a manner dependent on Candida‐priming and these effects may be related to enhanced cytokine levels.
Proliferation of Intracellular Structure Corresponding to Reduced Affinity of Fluconazole for Cytochrome P‐450 in Two Low‐Susceptibility Strains of Candida albicans Isolated from a Japanese AIDS Patient
Authors: Maebashi Kazunori , Kudoh Michinari , Nishiyama Yayoi , Makimura Koichi , Kamai Yasuki , Uchida Katsuhisa , Yamaguchi Hideyo
Published: November 2013

Three Candida albicans isolates, TIMM 3164, 3165 and 3166 with reduced fluconazole susceptibility, were isolated from two Japanese AIDS patients. We earlier reported that a reduced intracellular accumulation of fluconazole in these isolates played an important role in the resistance mechanism of fluconazole, but we did not exclude the involvement of other factors. We here examined characteristics related to cytochrome P‐450 (CYP), especially sterol 14α‐demethylase encoded by the ERG11 gene which is the target molecule for fluconazole. In TIMM 3164 and 3165, the ergosterol synthesis by cell‐free extracts was somewhat less susceptible to fluconazole, due to a decrease in fluconazole affinity for CYP. The nucleotide substitutions in the ERG11 gene were identified to result in three amino acid changes of K143R, E266D and V488I in TIMM 3164, and of E266D, V404L and V488I in TIMM 3165. These amino acid substitutions might contribute to the decreased affinity for CYP in both isolates. However, a single amino acid change, E266D, observed in TIMM 3166 was unrelated to the decreased affinity for CYP. The most prominent finding on the ultrastructure of TIMM 3164 and 3165 was the development of mesh membrane structures of the endoplasmic reticula, which is a location related to sterol synthesis. This phenomenon was not observed in the cells of TIMM 3166 or the susceptible control strains of ATCC 90028 and 10231. In addition to the reduced intracellular accumulation, the decreased affinity of fluconazole for CYP in TIMM 3164 and 3165 is assumed to be associated with the fluconazole‐resistance phenotype.
Relationship between Canine Mucosal and Serum Immunoglobulin A (IgA) Concentrations: Serum IgA Does Not Assess Duodenal Secretory IgA
Authors: Rinkinen Minna , Teppo Anna‐maija , Harmoinen Jaana , Westermarck Elias
Published: November 2013

A double‐sandwich enzyme immunoassay method was developed for determination of serum immunoglobulin A (S‐IgA) and mucosal secretory immunoglobulin A (sIgA) in duodenal brush samples obtained via endoscopy and the relationship between enteric mucosal sIgA, salivary sIgA and S‐IgA in dogs was examined. Twenty healthy dogs underwent routine endoscopy. A brush sample from the duodenal mucosa was obtained and washed in PBS, with a serum sample being taken concurrently. A saliva sample was collected from twelve of these dogs. S‐IgA and sIgA with total protein concentrations in the duodenal washings and saliva samples were determined. A significant negative correlation (r = −0.64, P = 0.0059) was found between duodenal sIgA/protein ratios and S‐IgA concentrations. Saliva sIgA/protein ratios did not correlate with sIgA/protein ratios of duodenal samples. The method described here allows for direct assessment of duodenal IgA; therefore indirect measures based on serum IgA or salivary IgA can be avoided. In addition, these indirect measures appear to be poor indicators of duodenal sIgA competence in dogs.
The Effect of Lactobacillus rhamnosus on Enterohemorrhagic Escherichia coli Infection of Human Intestinal Cells In Vitro
Authors: Hirano Jyunko , Yoshida Tomoaki , Sugiyama Tsuyoshi , Koide Naoki , Mori Isamu , Yokochi Takashi
Published: November 2013

There are many examples of probiotic effects of various lactic bacteria on enteropathogens. In this study, Lactobacillus strains (L. rhamnosus, L. gasseri, L. casei and L. plantarum) were tested in an in vitro model of enterohemorrhagic Escherichia coli (EHEC) infection of a human colon epithelial cell line, C2BBe1. While the adhesion and colonization of EHEC was not affected by any of the lactobacillus strains tested, the internalization of EHEC into the cell line was markedly suppressed by L. rhamnosus, though not by others. Concerning the possible mechanisms, the viabilities of EHEC and host cell were not affected by the presence of L. rhamnosus. Simple competitions at certain receptors were unlikely because the suppressive effect on EHEC internalization was strictly dependent on viable L. rhamnosus and could not be observed with the conditioned medium or killed L. rhamnosus. The fact that L. rhamnosus showed outstanding potential for adhering to the colon epithelial cell line, compared with other strains, suggested that an avid interaction between L. rhamnosus and the host cell might be modulating intra‐cellular events responsible for the internalization of EHEC.
Cloning and Characterization of the fur Gene from Moraxella bovis
Authors: Kakuda Tsutomu , Oishi Daiki , Tsubaki Shiro , Takai Shinji
Published: November 2013

A homologue of the ferric uptake regulator gene (fur) was isolated from Moraxella bovis by degenerate polymerase chain reaction and cloning. Fur protein of M. bovis exhibited 72.1% amino acid identity with Acinetobacter calcoaceticus Fur. Western blot analysis showed a decrease of Fur expression in response to sufficient‐iron conditions compared with deficient‐iron conditions. An electrophoretic mobility‐shift assay indicated that Fur protein binds to DNA fragments containing a putative Fur‐box derived from the upstream region of the M. bovis fur gene. Fur of M. bovis may regulate the expression of iron transport systems in response to iron limitation in the environment.
The Influence of Ovine MHC Class IIDRB1Alleles on Immune Response in Bovine Leukemia Virus Infection
Authors: Konnai Satoru , Takeshima Shin‐nosuke , Tajima Shigeru , Yin Shan Ai , Okada Kosuke , Onuma Misao , Aida Yoko
Published: November 2013

We have reported previously that the alleles of the ovine leukocyte antigen (OLA)‐DRB1gene that encode the Arg‐Lys (RK) motif and the Ser‐Arg (SR) motif at positions β70/71of the OLA‐DRβ1 domain are associated with resistance and susceptibility, respectively, to development of bovine leukemia virus (BLV)‐induced ovine lymphoma. Here, to investigate the different immune response in sheep that carried alleles associated with resistance and susceptible for 30 weeks after infection with BLV, we selected sheep that had the RK/RK or SR/SR genotype among the 52 sheep analyzed by polymerase chain reaction‐restriction fragment length polymorphism and DNA sequencing of PCR product for theOLA‐DRB1exon 2 and infected them with BLV. Although the number of BLV‐infected cells and virus titer had been maintaining low levels throughout the experimental period, the sheep with the RK/RK genotype could induce expansion of CD5−B‐cells and rapid production of neutralizing antibody in the early phase of infection. The level of incorporation of [3H]thymidine by peripheral blood mononuclear cells from the sheep with RK/RK genotype gave a strong response to BLV virion antigen and synthetic antigenic peptides that corresponded to T‐helper epitope of the BLV envelope glycoprotein gp51. In contrast, the sheep with SR/SR genotype showed a strong response to BLV virion antigen and synthetic antigenic peptides that corresponded to T‐cytotoxic and B‐cell epitopes. In such cases, the animals with the RK/RK strongly expressed IFN‐7, the animals with SR/SR genotype strongly expressed IL‐2. To determine the proliferating cells, we tried a blocking assay with monoclonal antibodies such as anti‐CD4, ‐CD8 and ‐DR molecule. We found that these proliferating cells were MHC‐restricted CD4+T‐cells.
Comparison of a Multiplex‐PCR Assay with Mycolic Acids Analysis and Conventional Methods for the Identification of Mycobacteria
Authors: Tanaka Ioshie Ibara , Anno Ivone Shizuko , de Andrade Leite Sergio Roberto , Cooksey Robert C. , Leite Clarice Queico Fujimura
Published: November 2013

A fast, sensitive and cost‐effective multiplex‐PCR assay for Mycobacterium tuberculosis complex (MTC) and Mycobacterium avium (M. avium) identification for routine diagnosis was evaluated. A total of 158 isolates of mycobacteria from 448 clinical specimens from patients with symptoms of mycobacterial disease were analyzed. By conventional biochemical methods 151 isolates were identified as M. tuberculosis, five as M. avium and two as Mycobacterium chelonae (M. chelonae). Mycolic acid patterns confirmed these results. Multiplex‐PCR detected only IS6110 in isolates identified as MTC, and IS1245 was found only in the M. avium isolates. The method applied to isolates from two patients, identified by conventional methods and mycolic acid analysis, one as M. avium and other as M. chelonae, resulted positive for IS6110, suggesting co‐infection with M. tuberculosis. These patients were successfully submitted to tuberculosis treatment. The multiplex‐PCR method may offer expeditious identification of MTC and M. avium, which may minimize risks for active transmission of these organisms and provide useful treatment information.
Analysis of the Envelope Proteins of Heat‐Shocked Vibrio parahaemolyticus Cells by Immunoblotting and Biotin‐Labeling Methods
Authors: Wong Hin‐chung , Chen Yu‐Chih
Published: November 2013

Vibrio parahaemolyticus, a common enteropathogen in tropical and subtropical coastal regions, exhibits significant adaptive acid tolerance response and heat‐shock response, and the envelope proteins induced by stresses are suggested to be associated with virulence. This work examined the heat‐shock proteins located in the envelope of V. parahaemolyticus by two rapid methods; namely, the immunoblotting and biotin‐labeling methods. The bacterial cells were cultured at 25 C and heat shocked at 37 or 42 C for 1 or 2 hr. The cells were first lysed, then proteins were separated by gel electrophoresis and probed with antiserum raised against heat‐shocked cells. Next, the heat‐shocked cells were examined by labeling with water soluble sulfo‐NHS‐LC‐biotin. Proteins of 33, 61, 66, 71, 78, 92 and 101 kDa were induced, while 55, 86, 102, 120 and 160 kDa proteins were markedly enhanced in the envelope of the heat‐shocked V. parahaemolyticus cells. The biotin tagged envelope proteins were purified using a monomeric avidin column, and the N‐terminal sequence was determined and compared with other high identity protein sequences. The sequence results suggest that Vph1 (55 kDa), Vph2 (46 kDa) and Vph3 (42 kDa) are de novo synthesized heat‐shock proteins located in the envelope of this pathogen, and the functions of these proteins in stress protection and virulence have yet to be determined.
Mycobacterial Infection in TLR2 and TLR6 Knockout Mice
Authors: Sugawara Isamu , Yamada Hiroyuki , Li Chuanyou , Mizuno Satoru , Takeuchi Osamu , Akira Shizuo
Published: November 2013

To investigate the role of TLR in the development of murine tuberculosis in vivo, TLR2 and TLR6 knockout (KO) mice were infected with Mycobacterium tuberculosis by placing them in the exposure chamber of an airborne infection apparatus. Both TLR2 and TLR6 KO mice survived until sacrifice at 12 weeks after infection. Infected TLR2 KO mice developed granulomatous pulmonary lesions with neutrophil infiltration, which were slightly larger in size than those in wild‐type mice. Pulmonary levels of the mRNAs for inducible nitric oxide synthase (iNOS), TNF‐α, TGF‐β, IL‐1β, and IL‐2 were significantly lower, but levels of the mRNAs for IL‐4 and IL‐6 were higher, than in wild‐type (WT) mice. No significant difference was recognized in cytokine mRNA expression between TLR2 KO and WT mice at 12 weeks after infection. DNA binding by NF‐κB was low in TLR2 KO mice. On the other hand, TLR6 KO mice were not different from WT mice in terms of pulmonary histopathology, mRNA expression and CFU assay. Therefore, TLR2 does not play an essential role in the pathogenesis of murine tuberculosis, although it is important for defense against mycobacterial infection.
Further Characterization of the Rabies Virus Glycoproteins Produced by Virus‐Infected and G cDNA‐Transfected Cells Using a Monoclonal Antibody, #1‐30‐44, Which Recognizes an Acid‐Sensitive Epitope
Authors: Kankanamge Pushpa Jenette , Irie Takashi , Shoji Jun'ichi , Tochikura Tadafumi S. , Kawai Akihiko
Published: November 2013

Expression of rabies virus glycoprotein (G) by G cDNA‐transfected mammalian cells resulted in the production of only a fusion‐negative form. Low pH‐dependent fusion activity, however, was seen when the expression was done under control of the T7 promoter with the help of recombinant vaccinia virus (RVV‐T7) that provided T7 RNA polymerase. Fusion‐inactive G proteins were transported to the cell surface as being detected by a conformational epitope‐specific monoclonal antibody (mAb; #1‐46‐12). The fusion‐inactive G proteins were recognized by most of our 13 conformation‐specific mAbs, except for one mAb, #1‐30‐44, that recognized the low pH‐sensitive conformational epitope. When the G gene expression was done with the help of RVV‐T7, although most G proteins remained in the epitope‐negative form, a small fraction of G gene products were 1‐30‐44 epitope‐positive, and cell fusion activity could be seen when cells were exposed to low pH conditions. From these results, we conclude that acquisition of low pH‐dependent fusion activity is closely related to structural maturation of the G protein to form the low pH‐sensitive 1‐30‐44 epitope. Such maturation seems to be dependent on certain rabies virus‐induced cellular conditions or functions, which might also be provided in part by the vaccinia virus infection. We further assume that expression of G cDNA alone mostly results in the production of mis‐folded and/or differently folded forms of G protein, and only a small fraction is correctly folded even under RVV‐T7‐mediated expression conditions.
T‐Cell Receptor Variable γ Chain Gene Expression in the Interaction between Rat γδ‐Type T Cells and Heat‐Shock Protein 70‐Like Molecule
Authors: Ichinohe Takashi , Ichimiya Shingo , Kishi Akihiko , Tamura Yasuaki , Kondo Nobuhiko , Ueda Gosei , Torigoe Toshihiko , Yamaguchi Akira , Hiratsuka Hiroyoshi , Hirai Itaru , Kohama Genn‐iku , Sato Noriyuki
Published: November 2013

We previously reported that rat T‐cell receptor (TCR) Vδ6 of T‐cell hybridomas was preferentially involved in recognition of the cell surface‐expressed 70 kDa rat heat‐shock cognate (hsc70, a constitutively expressed member of the hsp 70 family) protein‐like molecule (#067 molecule). In the present study, we analyzed usage of the TCR Vγ family of #067‐restricted T‐cell hybridomas. Our data indicated that most of these hybridomas expressed transcripts of TCR Vγ1 and/or Vγ2. However, some of the Vγ2 transcripts were out‐of‐frame, suggesting that the TCR Vγ1 family may be important for the recognition of #067‐defined molecules. TCR Vγ1 transcripts were detected in not only #067‐restricted T‐cell hybridomas, but #067‐non restricted ones as well. However, V‐J nucleotide sequences of #067‐restricted and #067‐non restricted T‐cell hybridomas suggested that #067‐restricted T‐cell hybridomas showed limited insertion of nucleotide stretch as compared with #067‐non restricted ones. In terms of amino acids, only one amino acid was added in #067‐restricted T‐cell hybridomas, whereas two or three amino acids were added in #067‐non restricted ones. These data suggest that the heterodimer of the TCR relatively short stretch form of Vγ1 molecule and TCR Vδ6 may participate in recognition of the #067 molecule.
A Rare Appearance of Influenza A(H1N2) as a Reassortant in a Community Such as Yamagata Where A(H1N1) and A(H3N2) Co‐Circulate
Authors: Mizuta Katsumi , Katsushima Noriko , Ito Sueshi , Sanjoh Kanako , Murata Toshio , Abiko Chieko , Murayama Shoko
Published: November 2013

To find a new influenza subtype A(H1N2), 383 isolates identified as H1 by hemagglutination inhibition test between the 1998–1999 and 2001–2002 seasons in Yamagata, Japan, were screened by reverse transcription polymerase chain reaction. As a result, 3 strains from the 1999–2000 season were identified as possibly being A(H1N2). Although several of their clones were found to be A(H1N2), A(H1N1) and A(H3N2), we could not confirm the origin of the A(H1N2) clones without the original specimens. These results suggest that a reassortment to produce A(H1N2) does not readily occur even when A(H1N1) and A(H3N2) co‐circulate in a community such as Yamagata.
Pathogenicity of Toxoplasma gondii through B‐2 Cell‐Mediated Downregulation of Host Defense Responses
Authors: Mun Hye‐Seong , Aosai Fumie , Chen Mei , Piao Lian‐Xun , Norose Kazumi , Iwakura Yoichiro , Yano Akihiko
Published: November 2013

IFN‐γ is the primary mediator of anti‐parasite effector mechanisms against Toxoplasma gondii. After intraperitoneal infection with the Fukaya strain of T. gondii, unirradiated IFN‐γ knock‐out (GKO) mice transferred with wild type (WT) CD8+ effector T cells from infected mice failed to induce the production of IFN‐γ and died, whereas irradiated (IR) GKO mice transferred with WT CD8+ T cells induced IFN‐γ production and survived more than 6 months. IR GKO mice transferred with WT CD8+ T cells together with GKO B‐2 cells died 8 days after infection, whereas those transferred with WT CD8+ T cells together with B‐1a or T cells survived. B‐2 cells of infected GKO mice activated CD11b+ cells for IL‐4 production, and down‐regulated NO release, STAT1 phosphorylation, and interferon regulatory factor‐1 expression in the peritoneal exudates cells of IR GKO mice transferred with WT CD8+ T cells together with GKO B‐2 cells after infection. Thus, B‐2 cells in T. gondii‐infected mice act as suppressor cells in the host defense of infected mice.
Characterization of Epstein‐Barr Virus (EBV)‐Positive NK Cells Isolated from Hydroa Vacciniforme‐Like Eruptions
Authors: Demachi Ayako , Nagata Hiroshi , Morio Tomohiro , Oyoshi Michiko K. , Zhang Yu , Tabata Nobuko , Kimura Nobuhiro , Shimizu Norio , Yamamoto Kohtaro
Published: November 2013

Recently, the involvement of Epstein‐Barr virus (EBV) in hydroa vacciniforme (HV)‐like eruptions has been suggested. To elucidate the role of EBV in this disease, we isolated EBV‐infected cell clones from peripheral blood mononuclear cells (PBMC) and the skin lesions of a patient with HV‐like eruptions; cells isolated from PBMC were designated SNK‐12, and those from the eruption SNK‐11. Both cells expressed CD16, CD56, and HLA‐DR and had germline configurations of the T‐cell receptor and the immunoglobulin genes, indicating that the cell clones were of NK cell lineage. The analysis of EBV terminal repeats indicated that the cells were monoclonal, had identical clonality, and originated from EBV‐positive cells in the PBMC and eruption. Both clones expressed EBNA‐1, but not EBNA‐2. Although LMP‐1 was weakly detected in SNK‐11, no LMP‐1 was detected in SNK‐12. Interestingly, EBV‐infected cells required less IL‐2 for in vitro growth in the later phase of this disease and this appeared to correlate with the expression of LMP‐1, suggesting that the proliferative capacity of the EBV‐positive NK cells increased during the time course of the disease, and LMP‐1 expression might be responsible for that. This is the first report of the isolation of EBV‐infected cells from the skin lesions of HV‐like eruptions and strongly suggests that the HV‐like eruption in the patient was caused by clonal NK cells with latent EBV infection.
The Role of Non‐Chromosomal Histones in the Host Defense System
Authors: Kanai Yoshiyuki
Published: November 2013

Histone H1 is located at the inter‐nucleosome and more correctly at both ends of the double‐stranded DNA that protrude from the nucleosome unit. It has long been recognized to be localized only inside the nuclei as a constituent for packaging nucleosome into chromatin. Thus, it could be hardly believed that detatched or solubilized histone H1 plays the role of a host defense molecule. Given the old reports on histone‐like basic proteins that show bacteriostatic functions, I herein chose some recent related articles and tried review them. Recent advances in research on the cell death mechanism makes it possible to understand that programmed cell death, (i.e. apoptosis) could serve as a good source of soluble histones. Some forms of them are highly probable to be bacteriostatic.
A Quantitative In Vitro Assay Method for Detecting Biological Activity of Endotoxin Using Prostaglandin E2 Induction in Rabbit Peripheral Blood
Authors: Ochiai Masaki , Kataoka Michiyo , Toyoizumi Hiromi , Yamamoto Akihiko , Kamachi Kazunari , Horiuchi Yoshinobu
Published: November 2013

The pyrogen test and the endotoxin test (the LAL test) have been playing crucial roles in detecting endotoxin in parenteral drugs. The current test methods, however, have disadvantages such as requiring a large number of animals or an inadequacy in evaluation of in vivo endotoxin activity. We attempted to establish a new assay method that can overcome the shortcomings of the current methods. We standardized the in vitro assay method by the use of prostaglandin E2 (PGE2) induction from peripheral blood of rabbits for detecting endotoxin activity. A linear dose‐response regression was attained from approximately 0.15 to 5 endotoxin units/ml of Japanese national reference standard endotoxin by the in vitro assay. The assay showed a fine correlation with the pyrogen test but not with the LAL test, when endotoxins from various bacterial sources were tested. The in vitro assay was also shown to have the capability of detecting a synergistic effect of endotoxin and parenteral drugs. The in vitro PGE2 induction test using rabbit blood was, therefore, suggested to be the appropriate test method for guaranteeing the same level of safety of parenteral drugs as the pyrogen test does.
Antigenic Analysis of Nonstructural Protein (NSP) 4 of Group A Avian Rotavirus Strain PO‐13
Authors: Borgan Mohammed Ali , Mori Yoshio , Ito Naoto , Sugiyama Makoto , Minamoto Nobuyuki
Published: November 2013

In order to analyze the antigenic structure of nonstructural protein (NSP) 4 of group A avian rotavirus strain PO‐13, 25 monoclonal antibodies (MAbs) against NSP4 expressed in Escherichia coli were produced. All MAbs reacted with NSP4 on Western blotting, indicating that they recognized sequential epitopes. To determine the antigenic sites (ASs) recognized by the produced MAbs, seven truncated NSP4s were expressed in E. coli. Western blotting analysis showed that there are at least four major ASs on PO‐13 NSP4, designated as AS I located in amino acids (aa) 151 to 169, AS II (aa 136 to 150), AS III (aa 112 to 133) and AS IV (aa 1 to 24). Two MAbs reacted exclusively with AS III encompassing the region that has been reported to be an enterotoxin domain. MAbs against ASs II, III and IV reacted with all avian rotaviruses tested by indirect immunofluorescent antibody assays. MAbs against AS I reacted with turkey strains, Ty‐1 and Ty‐3, but not with a chicken strain, Ch‐1. Nine of 11 MAbs against AS II cross‐reacted with NSP4 of mammalian rotavirus strains with different NSP4 genotypes. These results suggest that AS II on NSP4 is widely conserved among a variety of rotaviruses.
SATB1 Makes a Complex with p300 and Represses gp91phox Promoter Activity
Authors: Fujii Yoshito , Kumatori Atsushi , Nakamura Michio
Published: November 2013

The expression of gp91phox, the key component of the phagocyte NADPH oxidase, is regulated by various factors binding to its proximal promoter. Two nuclear matrix attachment region (MAR)‐binding proteins, special AT‐rich binding protein 1 (SATB1) and CCAAT displacement protein (CDP), have been reported as rare examples of gp91phox gene repressors. However, their individual roles and interactions with other factors in the promoter have not been elucidated in detail. We have focused on these two repressive proteins recognizing the bp −115 to bp −106 segment of the gene and obtained the following results: 1. SATB1 makes a complex, mainly with p300, regardless of the presence of DNA. 2. SATB1/p300 complex binding to the 5′ upstream AT‐rich region in the bp −115 to bp −106 segment represses the gp91phox promoter activity, and the repressed activity is partially released by CDP binding to the CCAAT element directly downstream of the AT‐rich region. Our findings imply a novel role for p300 in SATB1‐associated global transcription regulation.