bluuebunny/crossref_metadata_2025 huggingface dataset:
Application of RT‐PCR Designed from the Sequence of the Local SRSV Strain to the Screening in Viral Gastroenteritis Outbreaks
Authors: Saito Hiroyuki , Saito Shioko , Kamada Kazuko , Harata Seizaburo , Sato Hiroyasu , Morita Morihiro , Miyajima Yoshimichi
Published: November 2013

The Yuri strain of small round structured virus (SRSV) was cloned from a fecal specimen in which virus particles were observed by electron microscopy. The most common RT‐PCR protocol in Japan, however, using 35/36 and NV81/NV82/SM82 nested primer pairs, could not detect the SRSV genome in this specimen. Nucleotide and amino acid sequence analysis revealed that the Yuri strain is genetically close to the genotype II of SRSV. A novel procedure using primer sets designed from the nucleotide sequence of the Yuri strain was applied to the screening of 119 stool samples obtained from subjects with sporadic diarrhea and 46 samples obtained during seven foodborne gastroenteritis outbreaks. Using this novel procedure, PCR bands were detected in 44% and 52% of the samples, respectively. These detection rates were approximately twice those obtained with the 35/36 and NV81/NV82/SM82 nested primers. In particular, more than 40% of positive samples could be detected by using only the Yuri primer sets. Furthermore, three improvements were made in the RNA preparation, cDNA synthesis, and amplification steps to save materials and time. The background, or extra bands, in the amplification reaction resulting from DNA in the fecal specimens was completely removed by DNase I treatment just before cDNA synthesis. Random nonamers were used as universal primers in the reverse transcription. No difference in sensitivity or specificity was noted in the final results when either random nonamers or specific primers were used. The use of a pre‐amplification step under low stringent conditions before standard amplification under highly stringent conditions compensated for any mismatched bases in the primers with respect to the target sequences. Thus our novel procedure using Yuri primer sets may be useful for the screening of SRSV in the recent SRSV outbreaks in Japan.
Participation of NK1.1+ T Cells in the Rejection of lpr αβT Cells When Bone Marrow Cells of Ipr Mice Are Transplanted into B6 Mice
Authors: Tsukahara Akihiro , Kawamura Hiroki , Iiai Tsuneo , Moroda Tetsuya , Suzuki Susumu , Tada Takashi , Minagawa Masahiro , Musha Nobuyuki , Hatakeyama Katsuyoshi , Abo Toru
Published: November 2013

When C57BL/6 (B6) mice were irradiated (9 Gy) and received bone marrow (BM) cells of B6‐lpr/lpr mouse origin (i.e., lpr→B6), all mice died within 6 days. In the irradiated B6 mice, radioresistant CD3− IL‐2Rβ+ NK cells and IL‐2Rβ CD3int cells (i.e., CD3int cells of extrathymic origin) remained, especially in the liver. There were two subsets, NK1.1+ and NK1.1−, among the IL‐2Rβ+ CD3int cells. However, the NK1.1+ subset (i.e., NK1.1+ T cells) was much more radioresistant, and the majority of CD3int cells belonged to this subset in irradiated mice. The expansion of lymphocytes from injected BM cells did not occur in the irradiated B6 mice. However, such expansion did take place in irradiated B6‐lpr/lpr mice injected with both BM cells of B6‐lpr/lpr and B6 origin. As a result, the mice subjected to BM cells survived. Irradiated B6 mice were treated in vivo with anti‐NK1.1 mAb or anti‐asialoGM1 antibody to eliminate NK cells alone or both NK cells and NK1.1+ T cells. When irradiated B6 mice were pretreated with anti‐NK1.1 mAb, the mice could survive. These results suggest that intact NK1.1+ T cells of extrathymic origin may recognize abnormal BM cells with the lpr gene and inhibit the expansion of lymphocytes, including abnormal double‐negative CD4−8− cells, in B6‐lpr/lpr mice. To inhibit the expansion of lymphocytes, mechanisms other than Fas ligand/Fas molecules on extrathymic T cells may be responsible.
Plasmid Maintenance Renders Bacteria More Susceptible to Heat Stress
Authors: Sato Takako , Kuramitsu Howard
Published: November 2013

The presence of shuttle plasmid pTS749 in the oral bacterium Streptococcus mutans resulted in growth inhibition under heat stress conditions. This effect was dependent upon the growth stage of the inoculum used to initiate growth. Likewise, the introduction of plasmids containing distinct ori sequences into Escherichia coli also resulted in growth inhibition at elevated temperatures and could be correlated with the plasmid levels within the cells. These results suggest that these plasmids in bacteria render the cells to be more susceptible to heat stress conditions.
Studies on Rabies Virus RNA Polymerase: 1. cDNA Cloning of the Catalytic Subunit (L Protein) of Avirulent HEP‐Flury Strain and Its Expression in Animal Cells
Authors: Morimoto Kinjiro , Akamine Takayuki , Takamatsu Fumihiko , Kawai Akihiko
Published: November 2013

To investigate the RNA polymerase of rabies virus, we cloned a cDNA of the catalytic subunit (called L protein because of its large molecular size) of the HEP‐Flury strain, an avirulent strain obtained by high frequencies of serial embryonated hen egg passages. Nucleotide sequencing showed that the cDNA encodes a long polypeptide of 2,127 amino acids (Mr. 242,938). A comparison of the deduced amino acid sequence with that of other strains (PV and SAD B19) indicated that the sequence was highly conserved, except for several amino acid substitutions which were accumulated in some limited regions. A fragment of the cDNA was used for expression in Escherichia coli (E. coli) to prepare the L antigen for raising the antibodies in rabbits. Immunoprecipitation studies with the rabbit antiserum showed that the polypeptides produced in the L cDNA‐transfected COS‐7 cells displayed almost the same electrophoretic mobility as that of authentic L protein. Immunofluorescence studies indicated that both L and P (another subunit of RNA polymerase) proteins displayed colocalized distribution with the nucleocapsid antigen (N) in the cytoplasmic inclusion bodies, where envelope proteins (G and M) were absent. On the other hand, expression of the L protein alone did not cause inclusion body‐like granular distribution, suggesting that the inclusion body‐like accumulation depends on certain interaction(s) with other viral gene products, probably with the ribonucleoproteins comprising the inclusion bodies.
Effect of IgA1 Protease on the Ability of Secretory IgA1 Antibodies to Inhibit the Adherence of Streptococcus mutans
Authors: Tyler Beth M. , Cole Michael F.
Published: November 2013

Secretory IgA (SIgA) is the principal immunoglobulin isotype present in the mucosal secretions of humans. SIgA is thought to play a major role in host defense at these surfaces by inhibiting the colonization of potentially pathogenic microorganisms. A number of bacteria that are mucosal pathogens of humans produce a protease that specifically cleaves the IgA1 subclass of humans and great apes at the hinge region to produce Fab and Fc fragments. In order to study the effect of IgA1 protease on the ability of SIgA1 antibodies to inhibit bacterial adherence, an in vitro assay that quantifies the adsorption of radiolabeled Streptococcus mutans to hydroxyapatite (HA) beads was employed. High titer S. mutans‐specific SIgA1 and SIgA2 antibodies were induced in chimpanzee milk for use in the assay. Fabα1 fragments had significantly reduced ability to inhibit adherence of S. mutans to saliva‐coated HA compared to intact SIgA1 or SIgA2 anti‐S. mutans antibodies. These data support the potential importance of IgA1 proteases as an ecological determinant in the oral cavity and their role as a determinant of pathogenesis of pathogenic bacteria whose portal of entry is the mucosal surface.
Heterogeneity in Expression of Lipopolysaccharide and Major Outer‐Membrane Proteins by Strains of Escherichia coli O157 with Different H‐Serotypyes
Authors: Fujimoto Shuji , Meno Yuko , Horikawa Kazumi
Published: November 2013

A total of 11 strains of Escherichia coli (E. coli) belonging to serogroup O157 were examined for the expression of long‐chain lipopolysaccharide (LPS) and major outer‐membrane proteins (OMPs) by means of SDS‐PAGE. The strains belonged to either one of four different flagellar (H) types or did not express flagella. Four of the eleven strains carried genes encoding Shiga‐like toxins (SLTs). All the strains exhibited one of four LPS profiles, designated A, B, C or D. Electron microscopic analysis with the freeze‐substitution technique demonstrated the differences in the cell surface structures of strains with each LPS profile. Strains with LPS profile A, B or C had layers of thin fibers 10, 20 and 20 nm long, respectively, on the outer membrane but strains with LPS profile D had no such structure. An analysis of the OMPs showed that all the strains had one of four OMP profiles, designated I, II, III or IV. Both LPS and OMP profiles were dependent on H‐serotypes, and the combination pattern of LPS and OMP profiles of the strains was unique for each H‐serotype. These data support the existence of heterogeneous groups of O157 strains.
Determination of Serotypes of Astroviruses by Reverse Transcription‐Polymerase Chain Reaction and Homologies of the Types by the Sequencing of Japanese Isolates
Authors: Matsui Mihoko , Ushijima Hiroshi , Hachiya Masahiko , Kakizawa Junko , Wen Leying , Oseto Mitsuaki , Morooka Keiichi , Kurtz John B.
Published: November 2013

Human standard astroviruses, serotypes 1 to 7, and 35 Japanese isolates were typed by reverse transcription and polymerase chain reaction (RT‐PCR) with serotype‐specific primers for the first time. The results were identical with those obtained by enzyme immunoassay with serotype‐specific polyclonal antibodies, a method which has already been reported. RT‐PCR with serotype‐specific primers is useful for epidemiological studies of astroviruses where serotype‐specific polyclonal antibodies are not available. Two parts of the capsid region, N terminus and C terminus, were sequenced. Serotypes differed in those regions. The N terminus differed less than the C terminus between serotypes. Both the N terminus and C terminus were similar intraserotypically with the exception of serotype‐4 isolates which could be divided into A and B subgroups on the basis of their C terminus sequences, which were not known previously.
The Functional Role of B7 Molecules on the Induction of Thymocyte Activation and Apoptosis
Authors: Aoki Naoko , Inobe Manabu , Murakami Masaaki , Abe Ryo , Lizuka Hajime
Published: November 2013

To evaluate the role of B7 on thymocyte activation and apoptosis, we took advantage of TCR transgenic mice in which the majority of thymocytes express a uniform TCR that is specific for ovalbumin. We also prepared Chinese hamster ovary (CHO) cells expressing B7 and appropriate class II molecules. We found that the apoptosis of double‐positive thymocytes by TCR‐mediated signaling, which presumably represents negative selection, requires a costimulatory signal provided by B7‐1 or B7‐2. The requirement of B7‐1 costimulation for the apoptosis of thymocytes does not change in either low or high antigenic peptide loading. We also demonstrated that two signals through TCR and CD28 augmented the proliferation of thymocytes, and the requirement of CD28‐mediated signal by B7‐1 or B7‐2 for thymocyte proliferation became less evident when high doses of antigenic peptide were loaded, indicating that the intensity of TCR‐mediated signal determines the requirement of B7‐mediated second signal for thymocyte proliferation.
Different Effects of Fibronectin on the Phagocytosis of Staphylococcus aureus and Coagulase‐Negative Staphylococci by Murine Peritoneal Macrophages
Authors: Shinji Hitomi , Sakurada Junji , Seki Keiko , Murai Miyo , Masuda Shogo
Published: November 2013

Fibronectin is known to be an important factor in colonization by Staphylococcus aureus of host tissues as well as other extracellular matrix proteins such as collagen and laminin. We investigated the effect of fibronectin on the phagocytosis of the S. aureus Cowan I strain by macrophages and of coagulase‐negative staphylococci (CNS) strains for comparison. Fibronectin‐reduced serum in place of normal serum lowered the phagocytic activity of the macrophages on the Cowan I strain. Purified fibronectin enhanced the phagocytic activity of the strain in a dose‐dependent manner. On the other hand, fibronectin did not show any opsonic effect on the ingestion of CNS strains, though the binding of fibronectin occurred equally well in CNS strains and the Cowan I strain. Fibronectin‐binding protein (FnBP), the specific fibronectin receptor on the surface on S. aureus, was detected in both the Cowan I strain and CNS strains. Polymerase chain reaction confirmed that not only the Cowan I strain, but also CNS strains possessed the FnBP gene. These results indicate that fibronectin shows an opsonic effect on the S. aureus, Cowan I strain but not on CNS strains, and suggest that the binding of fibronectin to FnBP is not sufficient for efficient phagocytosis of the staphylococci strains by macrophages.
Methods for Rapid Cloning and Detection for Sequencing of Cloned Inverse PCR‐Generated DNA Fragments Adjacent to Known Sequences in Bacterial Chromosomes
Authors: Pham Hong‐Son , Kiuchi Akio , Tabuchi Kiyoshi
Published: November 2013

Since the invention of PCR, many adaptation techniques have been developed for sequencing DNA fragments flanking known sequences. Of them, inverse PCR is a matter of interest because of the simplicity of its principle. However, the protocols for inverse PCR introduced so far consist of some time‐consuming procedures, and with them, we cannot “walk” chromosomes too far since the number of suitable restriction enzymes is limited. Our experiments led to confirming simpler technical approaches applicable to the case of bacterial chromosomes, that is, designing two end‐specific “contextual” sequences with which we can quickly detect the desired clones of targeted DNA fragments by simply analyzing PCR products, employing “the minimum value of the desired fragments” as a “discriminating minimum” value to decrease contaminant DNA fragments, and creating a new tandem of two cleaved end fragments of a known sequence (“reordering”) for PCR amplification in combination with cloning of the inverse PCR‐generated DNA. With the improvements, we could both simplify the procedures and broaden the capacity of the inverse PCR in “walking” chromosomes.
Fimbria‐Mediated Coaggregation between Human Oral Anaerobes Treponema medium and Porphyromonas gingivalis
Authors: Umemoto Toshihiko , Yoshimura Fuminobu , Kureshiro Hidetoshi , Hayashi Jun‐Ichiro , Noguchi Toshihide , Ogawa Tomohiko
Published: November 2013

Bacterial binding phenomena among different bacterial genera or species play an important role in bacterial colonization in a mixed microbiota such as in the human oral cavity. The coaggregation reaction between two gram‐negative anaerobes, Treponema medium and Porphyromonas gingivalis, was characterized using fimbria‐deficient mutants of P. gingivalis and specific antisera against purified fimbriae and bacterial whole cells. T. medium ATCC 700273 strongly coaggregated with fimbriate P. gingivalis strains ATCC 33277 and 381, but not with afimbriate strains including transposon‐induced fimbria‐deficient mutants and KDP98 as a fimA‐disrupted mutant of P. gingivalis ATCC 33277. In the P. gingivalis‐T. medium coaggregation assay, the presence of rabbit antiserum against the purified fimbriae or the whole cells of P. gingivalis ATCC 33277 produced different “aggregates” consisting predominantly of P. gingivalis cells with few spirochetes, but both preimmune serum and the antiserum against the afimbriate KDP98 cells did not inhibit the coaggregation reaction. Heated P. gingivalis cells lost their ability to bind both heated and unheated T. medium cells. This T. medium‐P. gingivalis coaggregation reaction was inhibited by a cysteine proteinase inhibitor, leupeptin, and also by arginine and lysine, but not by EDTA or sugars including lactose. A binding assay on nitrocellulose membranes and immunoelectron microscopy demonstrated that a heat‐stable 37 kDa surface protein on the T. medium cell attached to the P. gingivalis fimbriae.
Sequence Analysis of the Gene Encoding the Major Outer Membrane Protein rOmp B of Rickettsia japonica
Authors: Uchiyama Tsuneo
Published: November 2013

The complete nucleotide sequence of the gene encoding rOmp B (ompB), one of the two major outer membrane antigen proteins, of Rickettsia japonica was determined and compared to those of the other spotted fever group and typhus group rickettsiae. Open reading frame of the ompB gene of R. japonica consisted of 4,968 nucleotides coding for a putative precursor protein with 1,656 amino acids (aa) in which the N‐terminal 1,363 aa and the C‐terminal 293 aa encode 135‐kilodalton rOmp B and 32‐kilodalton β‐peptide, respectively. Putative promoter and terminator sequences for transcription were present in the upstream region of the ATG start codon and downstream of the TAA stop codon. Overall sequences of the ompB genes were well conserved beyond the group, especially in the β‐peptide regions.
Stable Marker on Flagellin Gene Sequences Related to Arabinose Non‐Assimilating Pathogenic Burkholderia pseudomallei
Authors: Wajanarogana Sumet , Sonthayanon Piengchan , Wuthiekanun Vanaporn , Panyim Sakol , Simpson Andrew J.H. , Tungpradabkul Sumalee
Published: November 2013

Using PCR‐based isolation and sequence analysis of the flagellin gene from two distinct biotypes of Burkholderia pseudomallei, a 15‐bp deletion was found within the variable domain of the gene in isolates capable of assimilating arabinose (Ara+). This finding led to the development of a PCR‐based method in order to differentiate and identify pathogenic B. pseudomallei for epidemiological study. A pair of specific primers was designed covering the 15‐bp deletion region at the variable domain. PCR‐amplification products of 176 and 191 bp in size were detected from 41 Ara+ isolates and 39 Ara— isolates of B. pseudomallei, respectively. Moreover, flagellin gene fragments of other bacterial species tested in this study were not amplified using these primers. The results suggest that the flagellin gene sequences of both B. pseudomallei biotypes in this region are stable and distinct. This method can be applied and useful for the epidemiological study of B. pseudomallei.
Cloning and Sequencing of the Central Region of the Flagellin Gene from the Gram‐Positive Bacterium Clostridium tyrobutyricum ATCC 25755
Authors: Arnold Françoise , Bédouet Laurent , Batina Pierre , Robreau Georges
Published: November 2013

The purpose of this study was to sequence the central part of the coding region of the Clostridium tyrobutyricum flagellin gene to improve the immunoenzymatic counting of cells after milk filtration. The coding region was amplified by PCR, and the amplified products were cloned. A DNA sequence analysis of positive clones gave us 1,131 nucleotides with a partial calculated flagellin molecular mass of 40,143 Da. The flagellar filament protein sequence exhibited high levels of homology to sequences of flagellin protein from other bacteria in both N‐ and C‐terminal parts, but little homology in the central domain. A PCR‐restriction fragment length polymorphism analysis of amplified C. tyrobutyricum flagellin gene products confirmed the variability of the central domain. The flagellin mRNA was determined to be 1.1 kb in size, which suggests a monocistronic mRNA. Furthermore, the deduced protein flagellin contains eleven potential N‐glycosylation sites and one sequence rich in serine, which could be modified by O‐glycosylation.
Experimental Murine Model for Autoimmune Enterocolitis Using Klebsiella pneumoniae O3 Lipopolysaccharide as a Potent Immunological Adjuvant
Authors: Paeng Noriko , Morikawa Akiko , Kato Yutaka , Sugiyama Tsuyoshi , Koide Naoki , Yoshida Tomoaki , Yokochi Takashi
Published: November 2013

An experimental model for autoimmune enterocolitis was produced in mice by repeated immunization of homologous colon extract together with Klebsiella O3 lipopolysaccharide (KO3 LPS) as an immunological adjuvant. Histological changes in the intestinal lesions were characterized by infiltration with polymorphonuclear leukocytes in the lamina propria, muscularis mucosae and submucosa of repeatedly immunized mice. No such intestinal lesions were produced in mice receiving injections of colon extract alone or KO3 LPS alone. Development of the autoantibody and delayed‐type hypersensitivity against colon extract were found in mice immunized with the mixture of colon extract and KO3 LPS. Distinct positive staining was detected specifically on the columnar epithelium of villi. Sera from hyperimmunized mice defined organ‐specific antigens present in the intestine. Therefore, it was suggested that the intestinal lesions might be caused by an autoimmune mechanism.
Antisense Macrophage Migration Inhibitory Factor (MIF) Prevents Anti‐IgM Mediated Growth Arrest and Apoptosis of a Murine B Cell Line by Regulating Cell Cycle Progression
Authors: Takahashi Akio , Iwabuchi Kazuya , Suzuki Masaki , Ogasawara Kazumasa , Nishihira Jun , Onoé Kazunori
Published: November 2013

Macrophage migration inhibitory factor (MIF) is involved in the generation of cell‐mediated immune responses. Recently it has been reported that MIF also plays a role in cell proliferation and differentiation. In the present study, using a B‐cell line, WEHI‐231, and its stable MIF‐antisense transfectant, WaM2, as a representative transfectant, we investigated the mechanism underlying regulation of the cell growth by MIF. WaM2 cells produced less MIF than vector control or parental WEHI‐231 cells. Reduced and increased proportions were seen in G1 and S‐phase cells, respectively, in WaM2 as compared with WEHI‐231. Growth arrest and apoptosis after stimulation via surface Ig (sIg) were less prominent in WaM2 cells than those in WEHI‐231. However, the addition of recombinant rat MIF did not reverse the inhibition of the growth arrest and apoptosis induced in WaM2 by cross‐linking sIg. Almost the same amount of p27kipI expression was detected in WaM2 cells as those in WEHI‐231 and vector control cells. Cross‐linking of sIg elevated the p27kipI level equally in these cells irrespective of the MIF‐antisense expression. Taken together, it seems that MIF plays a role in inducing apoptosis in B cells upon IgM cross‐linking by regulating the cell cycle via a novel intracellular pathway.
Prolonged Incubation Period of Salmonellosis in an Outbreak of Salmonella enteritidis Infection
Authors: Nagai Kazushige , Mori Toshihiko , Tsuda Shinichiro , Izumiya Hidemasa , Terajima Jun , Watanabe Haruo
Published: November 2013

An outbreak of Salmonella enteritidis infections occurred in Otaru, Japan, in September 1997. A total of 143 cases of salmonellosis were reported to the local Public Health Center. In this outbreak, one case had a 214‐hr incubation period. We investigated 5 isolates including this case by phage typing and pulsedfield gel electrophoresis (PFGE) to determine the genetic heterogeneity of S. enteritidis. Five isolates were phage typed as reacted, but did not conform (RDNC) with identical reaction patterns and had quite similar PFGE patterns. Thus, the prolonged incubation period may not be attributed to genetic heterogeneity of the organism but rather to other factors.
Difference in Response of NK Cell Activity in Newborns and Adults to IL‐2, IL‐12 and IL‐15
Authors: Hoshina Takayuki , Kida Kazunori , Ito Masahiro
Published: November 2013

The killing activity of cord blood mononuclear cells (cMNC) against cytomegalovirus (CMV)‐uninfected and ‐infected fibroblasts was comparable to that of adult peripheral blood mononuclear cells (aPBMC). The killing activity of cMNC against K562 cells was significantly lower compared with that of aPBMC. Treatment of cMNC and aPBMC with interleukin‐2 (IL‐2), IL‐12 or IL‐15 significantly enhanced killing activity against K562 cells and CMV‐uninfected and ‐infected cells. By comparison of cMNC with aPBMC, killing activity against the K562 cells of cMNC was augmented to the level of aPBMC when cultured with IL‐2, IL‐12 or IL‐15. The killing activity of cMNC against CMV‐uninfected and ‐infected fibroblasts did not increase to the level of adult PBMC by treatment with IL‐2, IL‐12 or IL‐15. These data suggest that cord blood contains a functionally different NK cell subpopulation than that among adult NK cells.
Genotypic and Phenotypic Analysis of Bromovirus Adaptive Mutants Derived from a Single Plant
Authors: Fujita Yasunari , Mise Kazuyuki , Furusawa Iwao
Published: November 2013

Eight adaptive mutant clones have been made from the total RNA extracted from uninoculated upper leaves of a single cowpea plant exhibiting systemic infection after inoculation with a hybrid cowpea chlorotic mottle bromovirus (CCMV) with the 3a movement protein gene of CCMV replaced by that of cowpea‐nonadapted brome mosaic bromovirus (BMV). Sequence and mutational analyses of these clones showed genotypic and phenotypic diversity of the cloned virus population, but all examined clones had the adaptive mutation, A to C at position 776 within the BMV 3a gene, required for the systemic infection of cowpea. The data support the quasispecies model for RNA virus population, and suggest that the maintenance of the adaptive mutation may be due to powerful selection pressure in an infection process.
Effective Inhibition of Candida albicans Growth by the Combination of Murine Peritoneal Neutrophils and Activated Macrophages
Authors: Tansho Shigeru , Abe Shigeru , Tansho Tohru , Yamaguchi Hideyo
Published: November 2013

The effect of leukocytes on the anti‐Candida activity of neutrophils was examined. Murine neutrophils which were purified from casein‐induced peritoneal cells inhibited the mycelial growth of Candida albicans. This anti‐Candida activity of neutrophils was augmented by the addition of spleen cells prepared from mice pretreated with bacterial lipopolysaccharide 3 hr before, but not from non‐treated mice. The population in the spleen cells, which enhanced the anti‐Candida activity of neutrophils, was plastic‐plate adherent, nylon‐fiber columns adherent and anti‐Mac‐1 antigen‐positive. These immunological profiles suggested that the enhancing cells are classified to splenic macrophages. Peritoneal‐exudated macrophages from mice treated with lipopolysaccharide also augmented the anti‐Candida activity of neutrophils. These results suggest that the anti‐Candida activity of neutrophils may be upregulated by activated macrophages.
Recruitment of Apoptotic Cysteine Proteases (Caspases) in Influenza Virus‐Induced Cell Death
Authors: Takizawa Takenori , Tatematsu Chizuru , Ohashi Kayoko , Nakanishi Yoshinobu
Published: November 2013

Influenza virus infection induces apoptosis in cultured cells with an augmented expression of Fas (APO‐1/CD95). Caspases, a family of cysteine proteases structurally related to interleukin‐1‐β‐eonverting enzyme (ICE), play crucial roles in apoptosis induced by various stimuli, including Fas. However, activation of the caspase‐cascade seems to be different in various pathways of apoptotic stimuli. We therefore examined the involvement of caspases in influenza virus‐induced apoptosis using caspase inhibitors. We found that z‐VAD‐fmk and z‐IETD‐fmk effectively inhibited virus‐induced apoptosis, whereas Ac‐DEVD‐CHO and Ac‐YVAD‐CHO showed partial and little effect on virus‐induced cell death, respectively. Consistently, caspase‐3‐like activity, but not caspase‐1‐like activity, was increased in the virus‐infected cells. The transfection of plasmids encoding viral inhibitors of caspase (v‐FLIP or crmA) into HeLa cells inhibited apoptosis by virus infection. The peptide inhibitors of caspases used in this study did not inhibit viral replication. We conclude that influenza virus infection activates some caspases, and that this activation may be downstream of viral replication.
The Distribution of HIV‐1 Subtypes in Fukuoka, Japan
Authors: Chijiwa Katsumi , Ishibashi Tetsuya , Kashiwagi Seizaburo , Mori Ryoichi
Published: November 2013

To determine the incidence of human immunodeficiency virus type‐1 (HIV‐1) subtypes in Fukuoka, Japan, viruses from 41 HIV‐1 infected individuals were subtyped. Subtyping by V3‐loop enzyme‐linked immunosorbent assay (ELISA) showed 31 of the 41 subjects as subtype B (MN type), one as subtype A, one as subtype C, and eight untypable. The subject infected with subtype C was identified as a foreigner; the subtype A subject was Japanese. A phylogenetic analysis of nucleic acid sequences from the env C2‐V3 region was also conducted. Genetic subtyping was successful for 25 samples: 23 samples were determined as subtype B, one subtype A and one subtype E. One of the individuals infected with subtype B, as well as the subtype A and subtype E subjects, were not Japanese. This study indicated that subtype B (USA and European type) is still dominant among HIV‐1 infections in Fukuoka. Further, no Japanese were subtype E positive, which is increasing in the Kanto region. It is notable, however, that subtype A and subtype C infections, which are rare in Japan, were found in Fukuoka, located far from the metropolitan area of Tokyo.
Proinflammatory Cytokines Secreted by Monocytes of Filarial Patients
Authors: Raman Uma , Eswaran Devarajan , Narayanan Rangarajan Badri , Jayaraman Kunthala , Kaliraj Perumal
Published: November 2013

The levels of interleukin 1, granulocyte macrophage colony stimulating factor (GM‐CSF) and tumour necrosis factor (TNF)‐α secreted by the monocytes of filarial patients, such as asymptomatic microfilaremics (MF), chronic pathology (CP), and normal individuals, residing in a Wuchereria bancrofti endemic area (EN) in response to whole Brugia malayi antigen (BmA) and Setaria digitata (Sd‐cuticular) and a recombinant filarial antigen (pRJ51) were studied. Stimulation of peripheral blood adherent cells with whole parasite antigen showed marked increase in IL‐1 levels in MF as compared to CP or EN. The recombinant antigen stimulation, however, resulted in similar levels of IL‐1 in MF and CP. In contrast, stimulation of peripheral blood adherent cells with whole parasite antigen produced high levels of GM‐CSF and TNF‐α in CP as opposed to MF or EN. Recombinant antigen stimulation, however, produced high levels of GM‐CSF in EN as compared to MF or CP, while no significant change in the release of TNF‐α was observed in these patients. These results suggest that monocytes from filarial patients exhibit functional activity similar to that observed by the monocytes of endemic normals (control group).
Molecular Cloning and Characterization of the oprQ Gene Coding for Outer Membrane Protein OprE3 of Pseudomonas aeruginosa
Authors: Okamoto Kiyomi , Gotoh Naomasa , Tsujimoto Hideto , Yamada Hiroshi , Yoshihara Eisaku , Nakae Taiji , Nishino Takeshi
Published: November 2013

We cloned and characterized the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa PAO1. The oprQ gene was composed of 1,275 base pairs including a sequence encoding for the signal sequence and a mature protein with a Mr of 44,602. Computer‐aided alignment and hydropathy analyses of the predicted amino acid sequences suggested that OprE3 is a transmembrane protein homologous to outer membrane proteins of P. aeruginosa such as OprD2 (OprD) porin and OprE1 (OprE) porin. Susceptibility to several antibiotics of the strains lacking or overproducing OprE3 was indistinguishable from that of the wild‐type strain, suggesting that OprE3 is unlikely involved in the diffusion of carbapenems and other β‐lactam antibiotics.
Induction of Neutralizing Antibody against Human Cytomegalovirus (HCMV) with DNA‐Mediated Immunization of HCMV Glycoprotein B in Mice
Authors: Hwang Eung‐soo , Kwon Kyung‐bae , Park Jae‐won , Kim Dae‐joong , Park Chung‐gyu , Cha Chang‐yong
Published: November 2013

Immunization was accomplished by inoculating pcGB containing human cytomegalovirus (HCMV) glycoprotein B (gB) gene into BALB/c mice intramuscularly. IgM antibody was detected in all the immunized group. IgG antibody was also found in all the tested mice with a mean peak antibody titer of 1:262 in three‐times immunized groups. IgG antibody appeared at 2 weeks postinoculation, raised peak levels at 7 weeks postinoculation and persisted over 6 months. Neutralizing antibody was developed, and the percent reduction of input infectivity in 1:100 diluted sera was 74.5% in three‐times immunized groups. This study suggested that DNA vaccine using the gene encoding HCMV gB is a candidate method for developing immunity to HCMV.
Role of Superoxide Anion in the Fungicidal Activity of Murine Peritoneal Exudate Macrophages against Penicillium marneffei
Authors: Kudeken Norifumi , Kawakami Kazuyoshi , Saito Atsushi
Published: November 2013

Penicillium marneffei is an important opportunistic fungal pathogen. The mechanisms of host defense against P. marneffei are not fully understood. In the present study, we, for the first time, investigated the role of superoxide anion (O2−) in the killing of two forms of P. marneffei, yeast cells and conidia, and the role of this killing mediator in the fungicidal activity of IFN‐γ‐stimulated murine peritoneal macrophages. P. marneffei yeast cells were susceptible to the killing effect of activated macrophages and chemically generated O2−, while conidia were not. These results suggested that O2− played some role in the fungicidal activity of macrophages. However, an oxygen radical scavenger, superoxide dismutase (SOD), did not suppress, but rather enhanced the fungicidal activity of IFN‐γ‐stimulated macrophages against P. marneffei yeast cells. This inconsistency was explained by the release of insufficient concentrations of O2− by activated macrophages as compared with the amount of O2− necessary for the killing of yeast cells, which was predicted in a chemical generating system. On the other hand, SOD enhanced the production of nitric oxide (NO) by IFN‐γ‐activated macrophages, and their increased fungicidal activity was significantly inhibited by NG‐monomethyl‐l‐arginine (l‐NMMA), a competitive inhibitor of NO synthase. Our results suggested that O2− does not function as the killing mediator of macrophages against P. marneffei, but rather plays an important role in the regulation of the NO‐mediated killing system by suppressing NO production.
Genetic Variation in the 5′ End and NS5B Regions of Classical Swine Fever Virus Genome among Japanese Isolates
Authors: Harasawa Ryô , Giangaspero Massimo
Published: November 2013

Sixteen clinical strains of classical swine fever virus (CSFV) isolated in Japan were subjected to analyses of nucleotide sequence variations in the 5′ end and NS5B regions of the genome. These isolates were divided into three genovars, CSFV‐1, CSFV‐2 and CSFV‐3, based on palindromic nucleotide substitutions at the three variable loci in the 5′ untranslated region (UTR). Phylogenetic trees constructed from nucleotide sequences in the 5′‐UTR and NS5B gene indicated that the CSFV strains were divided into three clusters, I, II and III. CSFV strains included in clusters I, II and III were identical to those in the CSFV‐1, CSFV‐2 and CSFV‐3 genovars, respectively.
A Filamentous Phage of Vibrio parahaemolyticus O3:K6 Isolated in Laos
Authors: Nakasone Noboru , Ikema Masahide , Higa Naomi , Yamashiro Tetsu , Iwanaga Masaaki
Published: November 2013

A filamentous phage, ‘lvpf5’, of Vibrio parahaemolyticus O3:K6 strain LVP5 was isolated and characterized. The host range was not restricted to serotype O3:K6, but 7 of 99 V. parahaemolyticus strains with a variety of serotypes were susceptible to the phage. The phage was inactivated by heating at 80 C for 10 min and by treating with chloroform. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of the phage exhibited a 3.8 kDa protein. The amino‐terminal amino acid sequence of the coat protein was determined as AEGGAADPFEAIDLLGVATL. The phage genome consisted of a single‐stranded DNA molecule. The activity of the phages was inhibited by anti‐Na2 pili antibody.
Phylogenic and Phenotypic Evidence for the Transfer of Eubacterium fossor to the Genus Atopobium as Atopobium fossor Comb. Nov.
Authors: Kageyama Akiko , Benno Yoshimi , Nakase Takashi
Published: November 2013

The 16S rRNA primary structure of Eubacterium fossor was determined by sequencing in vitro amplified rDNA. Sequence comparisons indicated that E. fossor has a specific phylogenetic association with the Atopobium species and is far from E. limosum, the type species of the genus Eubacterium. Phenotypic characters of E. fossor resemble those of the genus Atopobium. Therefore, we propose that E. fossor should be transferred to the genus Atopobium as Atopobium fossor comb. nov.
Are Autoantibodies against Lewis Antigens Involved in the Pathogenesis of Helicobacter pylori‐lnduced Peptic Ulcers?
Authors: Kamiya Kenji , Arisawa Tomiyasu , Goto Hidemi , Shibayama Keigo , Horii Toshinobu , Hayakawa Tetsuo , Ohta Michio
Published: November 2013

We examined whether anti‐Lewis x (Lex) and y (Ley) autoantibodies affect the pathogenesis of Helicobacter pylori‐induced peptic ulcers. Of 11 patients with peptic ulcers, 10 patients had both anti‐Lex and ‐Ley immunoglobulin G (IgG) antibodies, and 1 patient had only anti‐Lex antibody. After successful eradication, we measured the serum titer of anti‐Lex and ‐Ley antibodies. Six patients had a reduction of the titers of anti‐Lex and/or ‐Ley antibodies, whereas no notable changes were detected in 5 patients in the follow‐up. This result suggests that anti‐Lex and ‐Ley autoantibodies had no critical role in the development of H. pylori‐induced peptic ulcer.
Conservation of Putative Promoter Sequences Located Upstream of Chlamydial Major Sigma Factor Gene, sigA among Chlamydia Spp.
Authors: Ochiai Yoshitsugu , Fukushi Hideto , Cai Yan , Yamaguchi Tsuyoshi , Hirai Katsuya
Published: November 2013

A highly conserved 40‐nucleotide sequence was identified. Two completely conserved sequences, TAGATT and TAAACT, separated by 17 nucleotides resemble the consensus sequence recognized by the Escherichia coli major sigma factor and sequence found in other chlamydial promoters. In addition, the adenine‐rich sequence present in many chlamydial promoters was also conserved upstream of the putative − 35 element. These findings suggest that the conserved sequence may play a role in the regulatory function at the transcriptional level. Multiple ATG codons were found at the 5′‐terminal region of the chlamydial sigA ORFs except for Chlamydia pneumoniae, although the putative Shine‐Dargarno sequence was absent.
Efficient Propagation of Human Herpesvirus 6B in a T‐Cell Line Derived from a Patient with Adult T‐Cell Leukemia/Lymphoma
Authors: Zhou Chang Fang , Abe Kenji , Wang Ping , Ojima Tomoko , Yamamoto Kohtaro
Published: November 2013

The efficient propagation of the OK strain of the B variant of human herpesvirus 6 (HHV‐6B) was demonstrated in a line of T cells, TaY, established from the peripheral blood lymphocytes of a patient with adult T‐cell leukemia/lymphoma (ATL). Growth of TaY cells depends on the presence of IL‐2 and the cells harbor HTLV‐I genomes. A severe cytopathic effect (CPE) was observed in many HHV‐6B(OK)‐infected TaY cells one week after infection. The release of virus from HHV‐6B(OK)‐infected TaY cells [TaY(OK)] was first detected after three days and increased rapidly for up to seven days after infection, as demonstrated by PCR. The titer of HHV‐6B(OK) in the supernatant was comparable to the value of 103.5 TCID50/ml obtained with PHA‐activated cord blood lymphocytes (CBL) that had been infected with HHV‐6B(OK). The replication of the virus was shown to depend to a considerable extent on cell viability. Electron microscopy revealed many herpesvirus‐type capsid‐ and enveloped‐viruses in the nuclei and cytoplasm of degenerated cells in TaY(OK) cultures. The U1102 strain of HHV‐6A and the Z29 strain of HHV‐6B also infected TaY cells productively, as detected by PCR and an immunofluorescence test. These results suggest that the activation of CD4+ T lymphocytes with mitogens such as PHA or IL‐2 and the expression of some cellular gene or the HTLV‐I gene might be essential for efficient propagation of HHV‐6B. TaY cells should play an important role in future investigations of cell‐virus interactions and genetic variations or cell tropism of HHV‐6 isolates since no cell line that shows propagation of both HHV‐6A and HHV‐6B has been reported to date.
The Role of Intrahepatic γδ‐T Cells for Liver Injury Induced by Salmonella Infection in Mouse
Authors: Ishigami Masatoshi , Nishimura Hitoshi , Yoshioka Kentaro , Kakumu Shinichi , Yoshikai Yasunobu
Published: November 2013

Liver injury was induced after infection with Salmonella choleraesuis 31N‐1. In T‐cell receptor‐δ knockout mice, serum alanine transferase level was significantly decreased in comparison with normal control mice after Salmonella infection. On the contrary, in vivo administration of anti‐γδ T‐cell receptor monoclonal antibody (UC7‐13D5) to stimulate γδ‐T cells in infected mice significantly increased serum alanine transferase level but decreased bacterial growth compared with infected mice given control antibody (UC8; hamster IgG). These data suggest that γδ‐T cells have effector activities not only for protection but also for liver injury during Salmonella infection.
Role of Toxoplasma gondii HSP70 and Toxoplasma gondii HSP30/bag1 in Antibody Formation and Prophylactic Immunity in Mice Experimentally Infected with Toxoplasma gondii
Authors: Mun Hye‐seong , Aosai Fumie , Yano Akihiko
Published: November 2013

Production of antibodies against Toxoplasma gondii (T. gondii)‐derived stress proteins, T. gondii HSP70 (T.g.HSP70) and T.g.HSP30/bag1, in C57BL/6 and BALB/c mice perorally infected with cysts of the avirulent Fukaya strain of T. gondii was analyzed. Production of anti‐T.g.HSP70 IgG antibodies was transient, whereas production of anti‐T.g.HSP30/bag1 IgG antibodies persisted after infection in both C57BL/6 and BALB/c mice. C57BL/6 mice, a susceptible strain, predominantly produced IgG antibodies specific for T.g.HSP70, whereas BALB/c mice, a resistant strain, predominantly produced IgG antibodies specific for T.g.HSP30/bag1, after T. gondii infection. Immunization with rT.g.HSP30/bag1 enhanced, whereas immunization with rT.g.HSP70 reduced host protective immunity against T. gondii infection with a cyst‐forming avirulent strain, Fukaya, and a virulent strain, RH.
Cloning, Sequencing, and Functional Expression in Escherichia coli of Chaperonin (groESL) Genes from Vibrio cholerae
Authors: Mizunoe Yoshimitsu , Wai Sun Nyunt , Umene Kenichi , Kokubo Takayuki , Kawabata Shun‐ichiro , Yoshida Shin‐ichi
Published: November 2013

Using a series of oligonucleotides synthesized on the basis of conserved nucleotide motifs in heat‐shock genes, the groESL heat‐shock operon from a Vibrio cholerae TSI‐4 strain has been cloned and sequenced, revealing that the presence of two open reading frames (ORFs) of 291 nucleotides and 1,632 nucleotides separated by 54 nucleotides. The first ORF encoded a polypeptide of 97 amino acids, GroES homologue, and the second ORF encoded a polypeptide of 544 amino acids, GroEL homologue. A comparison of the deduced amino acid sequences revealed that the primary structures of the V. cholerae GroES and GroEL proteins showed significant homology with those of the GroES and GroEL proteins of other bacteria. Complementation experiments were performed using Escherichia coli groE mutants which have the temperature‐sensitive growth phenotype. The results showed that the groES and groEL from V. cholerae were expressed in E. coli, and groE mutants harboring V. cholerae groESL genes regained growth ability at high temperature. The evolutionary analysis indicates a closer relationship between V. cholerae chaperonins and those of the Haemophilus and Yersinia species.
Detection of Mycoplasma fermentans in Saliva Sampled from Infants, Preschool and School Children, Adolescents and Adults by a Polymerase Chain Reaction‐Based Assay
Authors: Shibata Ken‐ichiro , Kaga Masayuki , Kudo Masaki , Dong Li , Hasebe Akira , Domon Hikouji , Sato Yoshiaki , Oguchi Haruhisa , Watanabe Tsuguo
Published: November 2013

Attempts were made to detect Mycoplasma fermentans in saliva sampled from 201 subjects (108 males and 93 females) aged from 4 months to 59 years by a polymerase chain reaction‐based assay. M. fermentans was detected in saliva from 110 (54.7%) of 201 subjects, and 10 (28.6%) of 35 subjects aged from 4 months to 3 years. Of ten positive subjects, three were aged from 16 to 23 months and five were from 26 to 31 months. The incidence tended to increase with age up to the teens. The incidence was significantly greater in teenagers than in subjects aged from 7 to 12 years, but there was no significant difference in the incidence between the group of teenagers and each of the groups of subjects older than the teenagers. Thus, it was suggested that M. fermentans colonized the mouth at the age of about 16 months up to the age of 19 years.
The Production of Nitric Oxide and Tumor Necrosis Factor by Murine Macrophages Infected with Mycobacterial Strains Differing by Hemolytic Activity
Authors: Rudnicka Wiesława , Brzychcy Marzena , Klink Magdalena , Lopez Arley Gomez , Fonteyne Pierre Alain , Rüsch‐Gerdes Sabine , Rózalska Barbara
Published: November 2013

In this study, we compared the secretion of nitric oxide (NO) and tumor necrosis factor (TNF‐α) by murine macrophages infected in vitro with hemolytic or unhemolytic mycobacteria isolates. We observed that unhemolytic mycobacteria induced more intensive NO production by macrophages and were more susceptible to bactericidal effect of mononuclear phagocytes than hemolytic mycobacterial strains. In contrast, the high‐virulence hemolytic isolates induced significantly stronger TNF‐α production by infected macrophages than the low‐virulence unhemolytic bacilli.
Arginine Carboxypeptidase (CPR) in Human Plasma Determined with Sandwich ELISA
Authors: Guo Xiaoyan , Morioka Akihiro , Kaneko Yoko , Okada Noriko , Obata Kyoko , Nomura Tomoyuki , Campbell William , Okada Hidechika
Published: November 2013

There are two types of carboxypeptidases present in human blood, carboxypeptidase N (CPN) and arginine carboxypeptidase (CPR). CPR is generated during coagulation from a precursor (proCPR) which can be converted to the active form by trypsin in vitro. Since it is difficult to distinguish the two types of carboxypeptidases in human blood by the measurement of enzyme activity, we established a quantitative sandwich ELISA by which CPR can be quantitated. The amount of CPR in plasma, fresh serum and heated serum were essentially the same. Therefore the ELISA assay does not distinguish proCPR, activated CPR and inactivated CPR. With the ELISA method, CPR was quantitated in plasma from fifty patients with rheumatoid arthritis and eleven patients with severe hepatitis as well as healthy individuals. The amount of CPR in plasma obtained from patients with rheumatoid arthritis was not found to be lower than that of normal subjects. Furthermore, the patients who suffered severe hepatitis and had very low levels of CPR‐total were fatal. This suggests that a decrease of CPR level might be a good indication of a patient's prognosis to death by hepatitis.
The Characterization of Shiga Toxin‐Non‐Producing Escherichia coli Serotype O157:H7 Isolated from Carcasses of Cattle at a Slaughter House
Authors: Itoh Yoshihiro , Hayashi Naoko , Katoh Mikio , Yamamoto Atuko , Hayashi Seizou , Maeda Shigeki , Ezaki Takayuki
Published: November 2013

A bacteriological investigation of Shiga toxin (Stx)‐producing Escherichia coli (STEC) 0157:H7 was performed on 298 carcasses of cattle at slaughter houses between July 1996 and January 1997 in Gifu Prefecture, Japan. As a result, four Stx‐non‐producing Escherichia coli O157:H7 strains were isolated from two slaughtered carcasses of cattle. The purpose of this study was to examine the characterization of isolates. Isolates possessed the E. coli attaching and effacing gene (eaeA), and hemolysin gene (hlyA), and harbored 3.0‐MDa and 60‐MDa plasmids. The Xba I pulsed‐field gel electrophoresis (PFGE) pattern showed three similar patterns. Consequently, a closely related genotype of Stx‐non‐producing E. coli O157:H7 may widely exist in cattle.