bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Variation in the VP4 and NSP4 Genes of Human Rotavirus Serotype 3 (G3 Type) Isolated in China and Japan
Authors: Cao Xiu‐ru , Akihara Shiho , Fang Zhao‐yin , Nakagomi Osamu , Ushijima Hiroshi
Published: November 2013

Sequence analyses of the VP4 and NSP4 genes were performed on twenty human isolates of serotype G3 rotavirus obtained from China and Japan. One isolate from China, CHW17, possessed P[4] genotype VP4 and KUN group NSP4 genes which are associated with G2. One isolate (02/92) from Japan, which was shown to have a wider spacing between RNA segments 10 and 11 by RNA polyacrylamide gel electrophoretic analysis like AU‐1, possessed P[9] genotype VP4 and AU‐1 group NSP4 genes. The other isolates had P[8] genotype VP4 and Wa group NSP4 genes. While the nucleotide sequence conservation among the G3 VP7 genes was more than 79% (Wen et al, Arch. Virol., 1997, 142: 1481 1489), the conservation of VP4 and NSP4 genes in the same genotypes or groups was more than 85%.
High Molecular Weight Dextran Sulfate Increases the Activity of NF‐κB‐Regulated Promoter in Monocyte‐Derived Macrophages
Authors: Jagodzinski Pawel P. , Trzeciak Wiesław H.
Published: November 2013

It is known that sulfated polysaccharides can mimic the action of common T‐cell mitogens. To investigate the molecular basis of the mitogenic effect of high molecular weight dextran sulfate (HMDS), monocyte‐derived macrophages were transfected with recombinant plasmid containing chloramphenicol acetyl transferase (CAT) reporter gene under the control of the HIV‐1 long terminal repeat (LTR) promoter, which is regulated by transcription factor NF‐κB. We observed that HMDS, similar to bacterial lipopolysaccharide (LPS), increases the expression of CAT reporter gene suggesting increased activity of NF‐κB. The activation of NF‐κB correlated with the increased expression of B7.1 molecules. It was postulated that this NF‐κB‐regulated promoter might play a role in the activation of the accessory cells as well as the rate of replication of HIV‐1 in monocyte‐derived macrophages.
A Novel Negative Regulator Molecule, Cho‐1, Is Involved in the Cytotoxicity by Human Natural Killer Cells but Not in Cytotoxic T Lymphocytes
Authors: Akazawa Takashi , Hirai Itaru , Hirohashi Yoshihiko , Kamiguchi Kenjiro , Sahara Hiroeki , Torigoe Toshihiko , Nagasawa Shigeharu , Tamura Yasuaki , Sato Noriyuki
Published: November 2013

We previously reported the cytotoxic negative regulatory molecule, Cho‐1, that was expressed on the cell surface of rat fetal fibroblast cells in the cytotoxicity by natural killer (NK) cells. This molecule was IFN‐γ‐inducible, but appeared to be different from MHC class I. It was expressed on NK‐resistant cells but not on NK‐sensitive murine target cells such as YAC‐1. In this paper, first we determined whether Cho‐1 could also act as the negative regulatory molecule in a human NK‐resistant HEPM line. Our data strongly suggested that Cho‐1 could act as such a negative regulatory molecule in human NK cytotoxicity. The immunoprecipitates made with HEPM cell lysate and anti‐MHC class I monoclonal antibody (mAb) did not react against anti‐Cho‐1 mAb, indicating that Cho‐1 was different from MHC class I. Second, an assessment was made as to whether or not this molecule is involved in the cytotoxicity of CD8 (+) cytotoxic T lymphocytes (CTL) against human autologous tumor cells. The data indicated that although this cell surface molecule was expressed on certain tumor lines, it was not involved in the cytotoxic mechanism of CTL. Thus, Cho‐1 appeared to be the novel regulatory molecule in the NK cytotoxic mechanism.
Disappearance of Glyceraldehyde 3‐Phosphate Dehydrogenase from Erythrocyte Membrane by Hemolysis with Thermostable Direct Hemolysin of Vibrio parahaemolyticus or Vibrio cholerae El Tor Hemolysin
Authors: Zhang Dongyan , Honda Takeshi
Published: November 2013

It is believed that the thermostable direct hemolysin (TDH) of Vibrio parahaemolyticus and El Tor hemolysin (ETH) of Vibrio cholerae damage erythrocytes and other cells by acting as pore‐forming toxins. In this study, we found that a protein band with a molecular weight of 37,000 daltons specifically disappeared from erythrocyte membrane after hemolysis by TDH and ETH, but not after hypotonic hemolysis. The 37 kDa band was identified as glyceraldehyde 3‐phosphate dehydrogenase (G3PD), a glycolytic enzyme, based on N‐terminal 14 amino acid sequencing. The role of G3PD in hemolysis is discussed.
In Vitro Assessment of a Chemically Synthesized Shiga Toxin Receptor Analog Attached to Chromosorb P (Synsorb Pk) as a Specific Absorbing Agent of Shiga Toxin 1 and 2
Authors: Takeda Tae , Yoshino Ken‐ichi , Adachi Eriko , Sato Yuko , Yamagata Kyoko
Published: November 2013

A synthetic analog of Shiga toxin (Stx) receptor (Synsorb Pk) was quantitatively assessed to determine whether it can protect human renal adenocarcinoma cells (ACHN cells) from the cytotoxicity of Stx1 and Stx2 by coincubation experiments. Coincubation of 100 and 20 ng of Stx1 and Stx2 with 50 mg of Synsorb Pk for 1 hr at 37 C in 1 ml of Eagle's Minimum Essential Medium supplemented with 1% (v/v) nonessential amino acid and 10% (v/v) fetal calf serum protected 50% of the cells from the cytotoxic effect. Chromosorb P, an inert matrix control, did not absorb the Stxs at all. Heat‐treatment (boiled for 10 min) to Synsorb Pk caused a 50% decrease in Stx2‐binding activity, but did not effect the Stx1 binding. Further, Stxs bound to Synsorb Pk could be demonstrated. When 20 mg of Synsorb Pk was coincubated for 30 min at 37 C in 1 ml of phosphate‐buffered saline with 1 and 10 ng or more of Stx1 or Stx2, respectively, the toxins could be detected on the surface when the bound toxins on Synsorb Pk were used as the solid phase in enzyme immunoassay. The amount of 100 ng/ml of both Stx1 and Stx2 appeared to saturate 20 mg/ml of Synsorb Pk after coincubating for 30 min at 37 C. While assessing the Stxs' binding activity to Synsorb Pk, it was demonstrated that Stx1 had a higher affinity to Pk trisaccharide than Stx2. These observations provide useful information on the effectiveness of Synsorb Pk to trap and eliminate free Stxs produced in the gut of patients infected by Stx‐producing Escherichia coli, and to prevent the progression of hemorrhagic colitis to hemolytic uremic syndrome.
PCR‐RFLP Analysis of Cytomegalovirus Infections Associated with Bone Marrow Transplantation in Japanese Children
Authors: Kawasaki Satoshi , Oshitani Hitoshi , Suzuki Hiroshi , Arakawa Masaaki , Mizuta Katsumi , Imaizumi Masue , Tsuchiya Shigeru , Konno Tasuke
Published: November 2013

In order to investigate the longitudinal molecular epidemiology of cytomegalovirus (CMV) infections associated with bone marrow transplantation (BMT) in Japanese children, we analyzed 36 CMV strains from 11 cases. Three regions (DNA polymerase, glycoprotein H, and immediate‐early regions) of CMV DNA were amplified by polymerase chain reaction (PCR), and amplified products were each digested with two restriction enzymes, followed by electrophoresis. These restriction fragment length polymorphism (RFLP) analyses allowed the differentiation of 36 strains into 13 genotypes. Each patient excreted his or her own CMV with distinct genotype over the study period of up to one year. CMVs of two different genotypes were recovered during a one‐month study from one recipient, who received a peripheral blood stem cell transplantation. Although the majority of patients and donors were CMV‐seropositive before BMT, multiple CMV infections might not be common and the reactivation of latently infected CMV might be prominent in Japanese children receiving transplants.
Significance of Anaerobic Preincubation of Helicobacter pylori tor Measuring Metronidazole Susceptibility by the Etest
Authors: Chida‐Sakata Noriko , Baba Masaru , Inagawa Hiroko , Wada Akihito , Tanaka Tatsuro , Hoshihara Yoshio , Takemoto Tadayoshi
Published: November 2013

The Etest is widely used for measuring the susceptibility of Helicobacter pylori to metronidazole. By using 55 H. pylori isolates from 55 patients and a standard H. pylori strain, NCTC11637, we compared metronidazole susceptibility results obtained from the Etest with or without anaerobic preincubation to those obtained from the agar dilution method. Mueller Hinton agar plates supplemented with 5% horse blood were used for both methods. For the Etest, plates were incubated for 72 hr at 35 C under microaerophilic conditions after 0‐, 4‐ or 24‐hr periods of anaerobic preincubation. For the agar dilution method, the plates were incubated at the same microaerophilic conditions as those for the Etest. Without anaerobic preincubation for the Etest, 39 of the 56 (70%) H. pylori isolates were categorized as resistant to metronidazole (minimal inhibitory concentration >8 mg/liter), whereas only one of the 56 (1.8%) isolates was resistant according to the agar dilution method. The resistant and susceptible agreement rate was 32%. Four‐hour anaerobic preincubation did not alter the readings of the Etest significantly. However, when the Etest was performed with 24‐hr anaerobic preincubation, the number of isolates categorized as resistant was reduced to six (11%), improving the agreement rate to 91%. For measuring the metronidazole susceptibility of H. pylori by the Etest, 24‐hr anaerobic preincubation is necessary to agree with the results obtained by the agar dilution test.
Human Immunodeficiency Virus Type 1 Vpr Modifies Cell Proliferation via Multiple Pathways
Authors: Yamaguchi Tomoyuki , Watanabe Nobumoto , Nakauchi Hiromitsu , Koito Atsushi
Published: November 2013

Vpr, one of the accessory molecules of HIV‐1, has been demonstrated to arrest the cell cycle at the G2 phase. This Vpr‐mediated cell cycle arrest is implicated to have an important role in the viral life cycle. In the present study, we quantitate the extent of Vpr‐mediated cell cycle arrest with the use of a bicistronic vector consisting of a vpr gene and a green fluorescence protein sequence. Using this system, we examined the effect of several Vprs on cell cycle progression and growth of cells from different species quantitatively. We found that Vpr from the T‐cell line‐adapted HIV‐1SF2 strain (Vpr2) could not significantly induce G2 arrest in HeLa cells but was able to induce it in 293T cells. However, strong inhibition of cell proliferation in HeLa cells as well as in 293T cells was observed by Vpr2. This ability of Vpr2 to inhibit cell proliferation without G2 arrest was also observed when expressed in monkey cell line. Analyses of chimeric Vprs revealed that this species‐non‐specific growth inhibitory activity of Vpr was not mediated solely by the C‐terminal region of Vpr. These results indicated that the growth inhibitory activity of Vpr is independent of its G2 arresting activity. In addition, the species‐non‐specific nature of this activity suggests that Vpr has a novel mechanism to retard cell proliferation by influencing basic cellular functions.
Association of a Cellular 21‐kDa Transmembrane Protein (VAP21) with Enveloped Viruses
Authors: Yamamoto Kentaro , Tochikura Tadafumi S. , Xiao Sa , Sakurai Akira , Kawai Akihiko
Published: November 2013

We reported previously that the rabies virions contained a 21‐kDa cellular transmembrane protein (referred to as VAP21) as a minor component (Sagara, J. et al, Microbiol. Immunol. 41(12): 947‐955, 1997). In this study, we further examined the possible interactions of VAP21 with other enveloped viruses, including the vesicular stomatitis virus (VSV; negative‐stranded RNA virus), Sindbis virus (positive‐stranded RNA virus) and herpes simplex virus type 1 (HSV‐1; double‐stranded DNA virus). An immunoblot analysis demonstrated that all of these enveloped viruses contained VAP21 in the virion as a minor component. Immunoprecipitation studies suggested that VAP21 was associated with certain viral proteins in the cell, such as the matrix (M) protein of VSV, a capsid protein of Sindbis virus, and at least a capsid protein (VP5) of HSV‐1. The association was disrupted by treatment with 0.5% sodium dodecyl sulfate, but resistant to the treatment with 1% NP‐40 plus 1% deoxycholate. These results suggest that: 1) VAP21 is not primarily associated with the viral transmembrane glycoprotein but rather with the internal viral protein, and, 2) this association would cause the efficient incorporation of VAP21 into the virion.
Identification of Copper‐Zinc Superoxide Dismutase Gene from Enteroaggregative Escherichia coli
Authors: Kumar Rakesh , Sidhu Maninder Kaur , Ganguly Nirmal Kumar , Chakraborti Anuradha
Published: November 2013

We describe here the identification of sodC gene from enteroaggregative Escherichia coli (EAggEC). A 294 bp gene‐specific fragment was amplified from the organism by DNA as well as RT‐PCR using primers from bacterial sodC sequences. The metal co‐factor present in the protein was confirmed by running samples in native gels and inhibiting with 2 mM potassium cyanide. However, the nonpathogenic E. coli possesses the gene but does not express it. Thus, the presence of copper‐zinc superoxide dismutase encoded by sodC was demonstrated for the first time in EAggEC, which means it could be a novel candidate for a virulence marker.
Potentiation of Immune Response against the RESA Peptides of Plasmodium falciparum by Incorporating a Universal T‐Cell Epitope (CS. T3) and an Immunomodulator (Polytuftsin), and Delivery through Liposomes
Authors: Kumar Pawan , Biswas Sukla , Rao Donthamsetty Nageswara
Published: November 2013

Synthetic peptides representing repeat sequences of ring‐infected erythrocyte surface antigen (RESA) of Plasmodium falciparum have shown poor immunogenicity and protection. In this study, the RESA peptides [(EENVEHDA)2 and (DDEHVEEPTVA)2] were chemically linked to a universal T‐cell determinant, CS.T3, derived from the CS protein of P. falciparum. Polytuftsin (TKPR)40, a polymer of naturally occurring immunomodulator “tuftsin,” was physically mixed with these conjugates. These preparations in alum and liposomes were immunized in four inbred strains of mice with different genetic backgrounds to study the humoral response. In the case of liposome‐entrapped preparations, a 10 μg dose of antigen showed the optimum antibody response. Mice immunized with liposome containing RESA peptide(s)‐CS.T3 conjugate along with polytuftsin showed the highest antibody levels in all the strains, whereas the RESA peptide(S) alone, adsorbed on alum or entrapped in liposomes, showed either poor or moderate antibody levels. The antibodies raised against liposome‐entrapped preparations in both high‐responder strain (SJL/J H‐2s) and low‐responder strain (FVB/J H‐2q) showed 24‐fold lower Kd values as compared to the alum adsorbed preparations, suggestive of high affinity antibodies. All the antigen preparations predominantly induced IgG2a and IgG2b isotype response, suggesting that the T‐helper response involved is of the CD4+ Th1 type. The in vitro merozoite reinvasion inhibition assay showed 5092% inhibition with sera raised against different antigen formulations. The highest percentage inhibition was observed with the RESA peptide‐CS.T3 conjugate containing polytuftsin in liposomes. Thus, the incorporation of peptide antigens inside liposomes not only reduced the antigen dose by 5‐fold but also elicited a high titre with high affinity antibodies and the inhibition of merozoites to RBC in vitro. Therefore, we conclude that the incorporation of these synthetic constructs in liposomes could be a useful strategy for the development of a subunit immunogen against malaria.
Isolation of Feline Parvovirus from Peripheral Blood Mononuclear Cells of Cats in Northern Vietnam
Authors: Miyazawa Takayuki , Ikeda Yasuhiro , Nakamura Kazuya , Naito Risako , Mochizuki Masami , Tohya Yukinobu , Vu Dat , Mikami Takeshi , Takahashi Eiji
Published: November 2013

Feline parvovirus (FPV) was isolated rather frequently from the peripheral blood mononuclear cells (PBMCs) of cats in northern Vietnam by coculturing with MYA‐1 cells (an interleukin‐2‐dependent feline T lymphoblastoid cell line) or Crandell feline kidney (CRFK) cells (a feline renal cell line). Efficiency of virus isolation was higher in MYA‐1 cells than in CRFK cells. Interestingly, among the 17 cats from which FPV was isolated, 9 cats were positive for virus neutralizing (VN) antibody against FPV, indicating that FPV infected PBMCs and was not eliminated from PBMCs even in the presence of VN antibodies in the cats.
Differences in Genomic Macrorestriction Patterns of Arabinose‐Positive (Burkholderia thailandensis) and Arabinose‐Negative Burkholderia pseudomallei
Authors: Chaiyaroj Sansanee C. , Kotrnon Kanokorn , Koonpaew Surapong , Anantagool Narisara , White Nick J. , Sirisinha Stitaya
Published: November 2013

We reported previously two biochemically and antigenically distinct biotypes of Burkholderia pseudomallei. These two distinct biotypes could be distinguished by their ability to assimilate l‐arabinose. Some B. pseudomallei isolated from soil samples could utilize this substrate (Ara+), whereas the other soil isolates and all clinical isolates could not (Ara). Only the Ara− isolates were virulent in animals and reacted with monoclonal antibody directed at the surface envelope, most likely the exopolysaccharide component. In the present study, pulsed‐field gel electrophoresis was employed for karyotyping of these previously identified B. pseudomallei strains. We demonstrate here that the DNA macrorestriction pattern allows the differentiation between B. pseudomallei, which can assimilate l‐arabinose, and the proposed B. thailandensis, which cannot do so. Bacterial strains from 80 melioidosis patients and 33 soil samples were examined by genomic DNA digestion with NcoI. Two major reproducible restriction patterns were observed. All clinical (Ara) isolates and 9 Ara soil isolates exhibited macrorestriction pattern I (MPI), while 24 soil isolates (Ara+) from central and northeastern Thailand displayed macrorestriction pattern II (MPII). The study here demonstrated pulsed‐field gel electrophoresis to be a useful tool in epidemiological investigation possibly distinguishing virulent B. pseudomallei from avirulent B. thailandensis or even identifying closely related species of Burkholderia.
Phylogenetic Analysis of Saccharolytic Oral Treponemes Isolated from Human Subgingival Plaque
Authors: Sakamoto Mitsuo , Koseki Takeyoshi , Umeda Makoto , Ishikawa Isao , Benno Yoshimi , Nakase Takashi
Published: November 2013

A total of 74 strains of oral treponemes, which were isolated from subgingival plaque samples from patients with periodontitis, were taxonomically studied on the basis of biochemical characteristics, DNA‐DNA hybridization, and 16S rRNA gene sequences. These organisms fermented carbohydrates and required rumen fluid or short‐chain volatile fatty acids for growth. The isolates were divided into seven subgroups based on their biochemical characteristics. The levels of DNA relatedness among the representative strains of each subgroup and Treponema socranskii (including three subspecies) were greater than 78%, while the levels of DNA relatedness among these strains and other Treponema species, including T. denticola and “T. vincentii”, were less than 15%. DNA‐DNA hybridization indicated that all subgroups belonged to T. socranskii. This result correlated well with the cluster on the phylogenetic trees based on 16S rRNA sequences.
Phenotypic and Genotypic Homogeneity of the Strains of Rickettsia japonica Isolated from Patients with Oriental Spotted Fever
Authors: Uchiyama Tsuneo
Published: November 2013

Nine pathogenic strains of Rickettsia japonica isolated from patients with Oriental spotted fever were compared phenotypically and genotypically. Constitution and antigenicity of the proteins demonstrated to be the same among strains. Polymerase chain reaction (PCR) amplification of the two major outer membrane protein genes (ompA and ompB) and an intracellular spotted fever group‐common antigen protein gene (rps120) produced the same sizes of products for all strains. Restriction fragment length polymorphism of the PCR products showed the same pattern among strains with each endonuclease. Thus, these strains belong to a single type, the same as the type strain YH (=ATCC VR‐1363).
Detection of Clostridium difficile Toxin A by Reversed Passive Latex Agglutination
Authors: Toma Claudia , Nakamura Shinichi , Kamiya Shigeru , Nakasone Noboru , Iwanaga Masaaki
Published: November 2013

A reversed passive latex agglutination (RPLA) assay for detecting Clostridium difficile toxin A is presented. Purified monoclonal antibody (mAb 37B5) was used for latex sensitization. The culture supernatants of 93 strains of C. difficile were tested by RPLA assay and the results compared with those of a commercially available latex agglutination test, PCR and cytotoxin assay with Vero cells. There was agreement between RPLA, cytotoxicity and PCR assays, but 29 strains were positive in the RPLA assay while 35 were positive in the cytotoxicity test and PCR using primer pair NK3‐NK2 directed to the nonrepeating portion of the C. difficile toxin A gene. The 6 cytotoxic but RPLA‐negative strains were demonstrated to be toxin A‐negative/toxin B‐positive strains in the PCR assay by using primer pair NK11‐NK9 directed to the repeating portion of the C. difficile toxin A gene. There were no cross‐reactions with culture supernatants of the other clostridial strains except for two strains of C. sordelli that produced hemorrhagic toxin (which is immunologically related to C. difficile toxin A).
Production and Partial Characterization of Antibody to Cord Factor (Trehalose 6,6′‐Dimycolate) in Mice
Authors: Fujiwara Nagatoshi , Oka Shiro , Ide Michio , Kashima Kazutoshi , Honda Takeshi , Yano Ikuya
Published: November 2013

Antibody production against the trehalose 6,6′‐dimycolate (TDM, cord factor) of Rhodococcus ruber, a non‐pathogenic species of the Actinomycetales group, was investigated in mice by repeated intraperitoneal injection of TDM in water‐in‐oil‐in‐water micelles without carrier protein. The antigenic TDM was isolated and purified chromatographically from the chloroform‐methanol extractable lipids of R. ruber. The hydrophobic moiety of this TDM was composed of two molecules of monoenoic or dienoic α‐mycolic acids with a carbon chain length ranging from C44 to C48 centering at C46. To detect the antibody, an enzyme‐linked immunosorbent assay (ELISA) system was employed using plastic plates coated with TDM. The antibody reacted against the TDM of R. ruber. The antibody was reactive in similar fashion against glycosyl monomycolates differing in the carbohydrate moiety, such as that of glucose mycolate (GM) and mannose mycolate (MM), obtained from R. ruber. Moreover, the antibody reacted against mycolic acid methyl ester itself when it was used as the antigen in ELISA, and trehalose did not absorb the antibody to TDM or inhibit the reaction. These results indicate that the epitope of TDM recognized by the antibody is mycolic acid, an extremely hydrophobic part of the molecule. Next, we prepared monoclonal anti‐TDM antibody (moAb) in mice myeloma cells to examine its biological activities and the role of humoral immunity in mycobacterial infection. MoAb reacted against the TDM, glycosyl mycolate, and mycolic acid methyl ester in ELISA in the same manner as our polyclonal antibody did. The administration of moAb suppressed granuloma formation in the lungs, spleen, and liver induced by TDM and inhibited the production of interleukin‐1 (IL‐1) and chemotactic factor, which is reported to precede granuloma formation.
Production of Murine Collagen‐Induced Arthritis Using Klebsiella pneumoniae O3 Lipopolysaccharide as a Potent Immunological Adjuvant
Authors: Takahashi Kazuko , Kato Yutaka , Sugiyama Tsuyoshi , Koide Naoki , Kawai Makoto , Fukada Masako , Yoshida Tomoaki , Yokochi Takashi
Published: November 2013

Collagen‐induced arthritis (CIA) was produced in mice with non H‐2q and H‐2r haplotypes by repeated immunization of porcine type‐II collagen (CII) together with Klebsiella O3 lipopolysaccharide (KO3 LPS) as an immunological adjuvant. Histological changes that appeared in joints of repeatedly immunized mice were characterized by destruction of normal joint structure, synovial hyperplasia with proliferation of synovial cells, and infiltration of inflammatory cells. No such lesions were produced in mice receiving repeated injections of CII alone or KO3 LPS alone. Development of the humoral antibody and the delayed‐type hypersensitivity to CII was exclusively found in mice immunized with the mixture of CII and KO3 LPS. It was therefore suggested that arthritis lesions induced by repeated immunization with the mixture of CII and KO3 LPS might be caused by an autoimmune mechanism, and that the experimental model might be useful for characterization of human rheumatoid arthritis (RA).
Chitin Synthase 2 Gene Sequence of Malassezia Species
Authors: Kano Rui , Aizawa Tomohiro , Nakamura Yuka , Watanabe Shinichi , Hasegawa Atsuhiko
Published: November 2013

Nucleotide sequences of the chitin synthase 2 (CHS2) gene of seven species, Malassezia furfur, M. globosa, M. obtusa, M. pachydermatis, M. restricta, M. slooffiae and M. sympodialis, were analyzed for their phylogenetic relationship. About 620‐bp genomic DNA fragments of the CHS2 gene were amplified from these Malassezia species by polymerase chain reaction (PCR) and sequenced. The CHS2 nucleotide sequences of these Malassezia species showed more than 95% similarity between the species. A phylogenetic analysis of the nucleotide sequences of CHS2 gene fragments of seven Malassezia species revealed that the species were genetically distinct from each other.
Development of Restriction Fragment‐Length Polymorphism Method to Differentiate Five Subtypes of Feline Immunodeficiency Virus
Authors: Kurosawa Kyoko , Ikeda Yasuhiro , Miyazawa Takayuki , Izumiya Yoshihiro , Nishimura Yorihiro , Nakamura Kazuya , Sato Eiji , Mikami Takeshi , Kai Chieko , Takahashi Eiji
Published: November 2013

Feline immunodeficiency virus (FIV) isolates have been classified into five subtypes (A to E) based on the sequences of the env variable V3 to V5 region. In this study, we sequenced a partial gag region of 4 and 3 isolates belonging to subtypes C and E, respectively. Phylogenetic analysis revealed that the branching pattern based on the region was similar to that based on the env V3 to V5 region. Here, we propose a protocol to differentiate five subtypes by polymerase chain reaction amplifying 329 bp within the region followed by restriction fragment‐length polymorphism analysis using four restriction enzymes.
Characterization of an Outer Membrane Protein Gene, pgmA, and Its Gene Product from Porphyromonas gingivalis
Authors: Hongo Hirohisa , Osano Etsuo , Ozeki Masami , Onoe Takaya , Watanabe Koji , Honda Okahito , Tani Hiroshi , Nakamura Hiroshi , Yoshimura Fuminobu
Published: November 2013

A gene upstream from fimA, the gene encoding fimbrilin, on the chromosome of Porphyromonas gingivalis was sequenced and shown to be the gene encoding an outer membrane protein in this organism based on homology and biochemical analyses. Therefore, the gene (formerly ORF5) was designated pgmA, the P. gingivalis outer membrane protein A gene. The gene product, PgmA, was sensitive to protease, and was detected as a 60‐kDa protein from wild‐type strains with trichloroacetic acid treatment, which was carried out to destroy intrinsic proteases, and from protease‐deficient mutants without this treatment prior to electrophoresis. PgmA was indeed present in the membrane fraction. Its nature was determined to be that of outer membrane proteins in gram‐negative bacteria based on attempts at differential extraction of inner membrane proteins with detergents. No evidence has been found thus far from functional analyses that this protein is related to fimbrial morphogenesis and functions or to serum resistance of this organism.
A Clostridium perfringens hem Gene Cluster Contains a cysGB Homologue That Is Involved in Cobalamin Biosynthesis
Authors: Koyama Michio , Katayama Seiichi , Kaji Masato , Taniguchi Yuki , Matsushita Osamu , Minami Junzaburo , Morita Shushi , Okabe Akinobu
Published: November 2013

The hem gene cluster, which consists of hemA, cysGB, hemC, hemD, hemB, and hemL genes, and encodes enzymes involved in the biosynthetic pathway from glutamyl‐tRNA to uroporphyrinogen III, has been identified by the cloning and sequencing of two overlapping DNA fragments from Clostridium perfringens NCTC8237. The deduced amino acid sequence of the N‐terminal region of C. perfringens HemD is homologous to those reported for the C‐terminal region of Salmonella typhimurium CysG and Clostridium josui HemD. C, perfringens CysGB is a predicted 220‐residue protein which shows homology to the N‐terminal region of S. typhimurium CysG. Disruption of the cysGB gene in C. perfringens strain 13 by homologous recombination reduced cobalamin (vitamin B12) levels by a factor of 200. When grown in vitamin B12‐deficient medium, the mutant strain showed a four‐fold increase in its doubling time compared with that of the wild‐type strain, and this effect was counteracted by supplementing the medium with vitamin B12. These results suggest that C. perfringens CysGB is involved in the chelation of cobalt to precorrin II as suggested for the CysGB domain of S. typhimurium CysG, enabling the synthesis of cobalamin.
Decreased Prevalence of Orientia tsutsugamushi in Trombiculid Mites and Wild Rodents in the Primorye Region, Far East Russia
Authors: Urakami Hiroshi , Tamura Akira , Tarasevich Irina V. , Kadosaka Teruki , Shubin Felix N.
Published: November 2013

The isolation of Orientia tsutsugamushi was attempted from 249 rodents and approximately 14,000 trombiculid mites captured in the Primorye region, Far East Russia in 1993 and 1994, where high infection rates were recorded in both rodents and mites in the 1960s. However, no rickettsia was isolated from the samples. Low antibody titers against O. tsutsugamushi were detected in 7.1% of the rodents. These results indicate that the prevalence of O. tsutsugamushi in the Primorye region has decreased considerably in the past 30 years.
Sequence Analysis of the Gene Encoding a Spotted Fever Group‐Specific Intracytoplasmic Protein PS120 of Rickettsia japonica
Authors: Uchiyama Tsuneo
Published: November 2013

The 3,438‐nucleotide (nt) sequence containing a 3,054‐nt open reading frame of the gene (rps120) encoding an antigenic, intracytoplasmic, spotted fever group‐specific and heat‐stable 120‐kilodalton protein (PS120) of Rickettsia japonica was determined. The nt and deduced 1,018 amino‐acid (aa) sequences were compared to those of R. conorii since only those of this species had been determined among SFG rickettsiae. The homologies of these sequences between R. japonica and R. conorii were considerably high at 97 and 95%, respectively. These high homologies were comparable to those of β‐peptides encoded by the ompB genes among SFG rickettsiae. It was also found that the genome of R. prowazekii contained a nt sequence with 68% homology to that of the rps120 gene of R. japonica.
Complete Nucleotide Sequence and the Genome Organization of Tobacco Leaf Curl Geminivirus from Japan
Authors: Shimizu Sachiko , Ikegami Masato
Published: November 2013

The genomic DNA of tobacco leaf curl geminivirus (TLCV) from tomato plants with leaf curl disease in Japan has been sequenced. The single circular DNA molecule comprises 2,761 nucleotides. TLCV DNA contains six open reading frames (ORFs) capable of encoding proteins with a molecular weight greater than 10 K. In total nucleotide sequence comparisons with other geminiviruses, TLCV was most closely related to tomato leaf curl virus from Taiwan (TwToLCV) (76% identity), tomato leaf curl virus from Bangalore (ToLCV‐Ba) (74%) and agerantum yellow vein virus (AYVV) (74%), all possessing a monopartite genome. The significant but relatively low sequence similarity in the genomic DNA between TLCV and other geminiviruses suggests it is a distinct geminivirus in genus Begomovirus.
Genetic Diversity and the Absence of Regional Differences of Borrelia garinii as Demonstrated by ospA and ospB Gene Sequence Analysis
Authors: Yabuki Mihe , Nakao Minoru , Fukunaga Masahito
Published: November 2013

Unfed adult Ixodes persulcatus ticks were collected from four locations of Nagano and Hokkaido in Japan. Infected Borrelia garinii were investigated by PCR‐RFLP of the ospA and ospB gene sequences. The primer set amplified an approximately 1.6‐kb DNA fragment (0.7‐kb in some strains), and BsrI, BstYI, or NlaIII digestion of the product resulted in six distinctively different PCR‐RFLP groups and two independent borrelial strains. The representatives in each PCR‐RFLP group and individuals from the borrelial strains were sequenced, and their deduced amino acid sequences were aligned. A neighbor‐joining phylogenetic analysis showed that the B. garinii OspA or OspB sequences were each divided into three major clusters including isolates from both the Nagano and Hokkaido locations. There was no local difference in OspA/B sequences between Nagano and Hokkaido. The osp gene of Borrelia burgdorferi sensu lato is highly heterogeneous, and this was also confirmed by our sequence analysis. Some strains of the different PCR‐RFLP groups had closely related OspA sequences, while the OspB sequences of these strains were quite different. These findings suggested intraspecies gene exchange and recombination events between the two genes in B. garinii.
Complement Receptor Type 3 Plays an Important Role in Development of Protective Immunity to Primary and Secondary Corynebacterium pseudotuberculosis Infection in Mice
Authors: Lan Dinh Thi Bich , Makino Sou‐Ichi , Shirahata Toshikazu , Yamada Manabu , Nakane Akio
Published: November 2013

The present study was performed to investigate the role of CR3, the type 3 complement receptor, in host defense against primary and secondary Corynebacterium (C.) pseudotuberculosis infection in mice. Treatment of mice with 5C6, an anti‐CR3 monoclonal antibody (mAb), resulted in unrestricted multiplication of bacteria in the organs and dramatically increased mortalities of the infected mice. Histological examinations showed the inflammation, degeneration and necrosis of organs and revealed that the infection‐enhancing effect of 5C6 mAb was associated with the failure of mice to focus mononuclear phagocytes at sites of bacterial multiplication. These results suggest that CR3 plays an important role in host defense against primary as well as secondary C. pseudotuberculosis infection in mice.
Simple Preparation of Parapoxvirus Genome DNA for Endonuclease Analysis
Authors: Inoshima Yasuo , Morooka Akira , Murakami Kenji , Sentsui Hiroshi
Published: November 2013

We compared five methods for improved extraction of very‐large parapoxvirus DNA from infected cells: (i) alkaline‐lysis procedure followed by phenol extraction; (ii) modified Hirt procedure, which was a neutral lysis procedure followed by phenol extraction; (iii) Hirt procedure; (iv) method used for extraction of vaccinia virus DNA; and (v) standard procedure using virus purification with an ultracentrifuge and protease‐sodium dodecyl sulfate‐phenol treatment. The alkaline‐lysis procedure was more rapid, inexpensive and simpler than the other methods. Moreover, with this method it is not necessary to prepare any special facilities, reagents and kits. Although the extracted DNA was still crude, we could reproducibly prepare viral DNA from 2 × 106 infected cells in less than 2 hr and it could be readily digested by restriction endonuclease. This method will aid rapid genetic classification of parapoxvirus.
CPR‐Total (TAFI and Activated TAFI) Levels in Plasma/Serum of Hemophiliacs
Authors: Guo Xiaoyan , Okada Noriko , Okada Hidechika
Published: November 2013

Arginine carboxypeptidase (CPR) is a single‐chain plasma protein generated during coagulation from a precursor (proCPR). proCPR is the same molecule as thrombin activable fibrinolysis inhibitor (TAFI), which retards fibrin clot lysis in vitro and most likely modulates fibrinolysis in vivo. In this study, the amount of CPR‐total, which includes proCPR (TAFI) and CPR (activated TAFI), in hemophiliac patients was evaluated using a newly developed enzyme linked immunosorbent assay (ELISA). The amount of CPR‐total in plasma or serum of most of the hemophiliac patients was in the range of healthy individuals. There was no significant difference in hemophiliac patients with or without HIV‐1 infection. However, two out of the 74 hemophiliac patients showed a significantly high level. The upregulation of CPR‐total might contribute to compensate for inefficient coagulation in some hemophiliac individuals.
Molecular Characterization and Functional Analysis of a secA Gene Homolog in Actinobacillus actinomycetemcomitans
Authors: Novak Karen F. , Nonnemacher Michael R. , Pourhamidi Jaleh
Published: November 2013

Results of Southern blot analyses and polymerase chain reaction revealed that the Gram‐negative pathogen, Actinobacillus actinomycetemcomitans, harbored DNA homologous to the secA gene of Escherichia coli. In E. coli, the secA gene product is essential for translocation of proteins across the inner membrane via the Sec system. This A. actinomycetemcomitans secA homolog was cloned and its nucleotide sequence determined. Amino acid sequence analysis of the cloned gene revealed significant homology to the SecA proteins of Haemophilus influenzae, E. coli, Caulobacter crescentus and Bacillus subtilis. Although the cloned gene did not complement a temperature sensitive mutation in the E. coli secA gene, strains harboring the cloned gene did produce a protein that cross‐reacted with anti‐SecA antibody. In addition, the cloned gene did restore sensitivity to sodium azide in an E. coli azideR mutant. These data support the hypothesis that A. actinomycetemcomitans may use a system similar to the Sec system of E. coli to transport proteins across the cytoplasmic membrane, but suggest that the A. actinomycetemcomitans gene product may require genera‐specific Sec proteins to complement some Sec mutations in E. coli.
Analysis of Vibrio mimicus Clinical Strains by Arbitrarily Primed Polymerase Chain Reaction
Authors: Bi Kewei , Shi Lei , Maehara Yoko , Miyoshi Shin‐Ichi , Tomochika Ken‐Ichi , Shinoda Sumio
Published: November 2013

A total of 51 Vibrio mimicus clinical strains from different geographic locations were examined by arbitrarily primed polymerase chain reaction (AP‐PCR). The primer VMH‐3 divided them into 28 groups, although 18 groups consisted of a single strain at present. All groups had a common 1.0‐kb amplification fragment. Most of the groups consisted of strains from same region, although two exceptional groups showed a few amplification fragments including strains from different regions. AP‐PCR groups were not consistently associated with serogroups. AP‐PCR is thought to be a valuable and easy method for the epidemiological study of V. mimicus.
Phylogenetic Analysis of the Small Hydrophobic (SH) Gene of Mumps Virus in Korea: Identification of a New Genotype
Authors: Kim Sang Hyun , Song Ki‐joon , Shin Young Kyoo , Kim Jee Hee , Choi Soon Mi , Park Kwang Sook , Baek Luck Ju , Lee Yong Ju , Song Jin‐won
Published: November 2013

Viral RNAs extracted from fifteen mumps virus isolated from throat swab, saliva, blood, urine or CSF during mumps epidemics between 1997–1998 in Korea were amplified by reverse transcriptase‐polymerase chain reaction (RT‐PCR) and compared by nucleotide sequencing of the small hydrophobic (SH) gene. The deduced amino acid sequences of the SH gene were aligned with the published sequences of mumps virus isolated in different geographic areas. A comparison of the SH gene of mumps viruses in Korea indicated 96.2–100% and 91.2–100% similarity at the nucleotide and amino acid levels, respectively. Phylogenetic analysis, using the neighbor‐joining method, showed that Korean mumps virus strains formed a genetically distinct monophyletic group from previously reported genotypes based on the 315‐bp length nucleotide and 57 deduced amino acid sequences of the SH gene, and possibly be designated as a new genotype (I).
Disintegration of Mg2+‐Induced Hexagonal Assembly of an R‐Form Lipopolysaccharide from Klebsiella pneumoniae by Treatment with CaCl2
Authors: Ito Hideo , Naito Setsuko , Kato Nobuo
Published: November 2013

R‐form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN‐111 (O3—: K1—), which was precipitated by the addition of 2 volumes of ethanol containing 10 mm MgCl2 for the purification process, ultrastructurally exhibited membrane pieces consisting of an ordered hexagonal lattice structure with a lattice constant of 14 to 15 nm. When the R‐form LPS was suspended in 50 mm tris (hydroxymethyl) aminomethane buffer (at pH 8.5) containing 1 mm or higher concentrations of CaCl2 and kept at 4 C for 10 hr, the ordered hexagonal lattice structure of the R‐form LPS was disintegrated and changed to an irregular rough, mesh‐like structure. By treatment with CaCl2, the content of Mg in the LPS was markedly decreased, and conversely, the content of Ca was increased to a level depending upon the concentration of CaCl2. Results indicate that the addition of CaCl2 to suspensions of the Mg‐bound R‐form LPS result in a tighter binding of Ca2 to the R‐form LPS and the release of Mg2 from the R‐form LPS, and as a consequence, destroys the Mg2+‐induced ordered hexagonal lattice structure of the R‐form LPS.
Chemical and Serological Properties of Lipopolysaccharides from Vibrio parahaemolyticus O‐Untypeable Strains Isolated from Patients
Authors: Hashii Noritaka , Kondo Seiichi , Iguchi Takehiro , Nishibuchi Mitsuaki , Hisatsune Kazuhito
Published: November 2013

Chemical and serological studies have been carried out on the O‐antigenic lipopolysaccharides (LPS) of six strains, U‐6443, W‐90144, X‐3972, AD‐7999, 90A‐6611 and KX‐V212, of Vibrio parahaemolyticus isolated from patients. The O‐serotypes of these strains have not been identified because they were not agglutinated by any diagnostic antisera against known O‐serotype strains. A compositional sugar analysis of their LPS revealed that out of the six O‐untypeable (OUT) strains, U‐6443, W‐90144 and AD‐7999 strains belonged to chemotype II (chemotype of O2), 90A‐6611 and KX‐V212 strains to chemotype III (chemotype of O3, O5, O11 and O13) and X‐3972 strain to chemotype IV (chemotype of O4). A structural analysis of LPS isolated from KX‐V212 revealed that the inner core region of the LPS consisted of only one mole of 2‐keto‐3‐deoxy‐d‐manno‐octonic acid, which carried a phosphate group at position C4 and the outer core at position C5. In passive hemolysis tests performed by using LPS as the antigen to sensitize sheep red blood cells (SRBC), and diagnostic antisera (O1 to O11) or anti‐whole‐cell rabbit antisera raised against O12, O13 and the six OUT strains, strong cross‐reactivity was observed among LPS derived from the strains belonging to chemotype II (U‐6443, W‐90144, AD‐7999 and O2). Strong cross‐reactivity was also observed between X‐3972 (chemotype IV) and O4 LPS. In contrast, LPS from two of the strains belonging to chemotype III (90A‐6611 and KX‐V212) did not react with any of the antisera raised against known O‐serotypes. Cross‐absorption tests showed that the O‐antigens of U‐6443, W‐90144 and AD‐7999 were identical to that of O2, and the O‐antigen of X‐3972 to that of O4. On the other hand, after the absorption of antisera raised against 90A‐6611 and KX‐V212 with O2 cells, the hemolytic activities against SRBC sensitized with homologous LPS were still retained at a high titer, whereas the hemolytic activities against SRBC sensitized with LPS from other O‐serotype strains were completely eliminated. A cross‐absorption test revealed that the O‐antigens of these two strains were identical to each other. Thus, it was demonstrated that the O‐serotype of OUT strains 90A‐6611 and KX‐V212 was not involved in the known O‐serotypes; rather it represented a novel serotype which has not hitherto been reported.
Acyclovir Resistance in Herpes Simplex Virus Isolates from Keratitis Cases: An Analysis from a Developing Country
Authors: Pramod Naranatt Padmanabhan , Thyagarajan Sadras Panchatcharam , Mohan Keta Venkata Krishna , Anandakannan Kanagasabai
Published: November 2013

Seven herpes simplex type‐1 (HSV‐1) isolates from herpes simplex keratitis (HSK) cases clinically resistant to acyclovir (ACV) were analyzed for the mechanism of ACV resistance in them. The purpose of the study was to focus the attention of ophthalmologists on the frequency of occurrence of ACV resistance in HSK and to characterize such a phenomenon. We employed in‐vitro plaque reduction assay, thymidine kinase assay, polymerase chain reaction, single‐strand confirmation polymorphism analysis and sequencing to detect any mutation(s) in thymidine kinase gene in this analytical study. Four of the seven HSV‐1 isolates proved ACV resistant by plaque reduction assay and three of them showed reduced thymidine kinase activity. Altered mobility pattern indicative of mutation within 335 base pair PCR product bracketing the suggested homopolymer mutational hotspot (7 Guanosine) was detected in 2 of these 3 isolates. DNA sequencing showed a deletion at nucleotide position 336 from the tk gene transcription start in both the isolates. This mutation has generated the first TGA stop codon 27 nucleotides downstream in the tk open reading frame. Our study also suggests the need of clinical/molecular surveillance of ACV resistance in HSV types in a given geographic location for better management of HSV infections.
Cloning and Sequencing of the Gene Encoding the Candidate HtrA of Rickettsia typhi
Authors: Kim Jong‐hyun , Hahn Myong‐joon
Published: November 2013

We screened a phage library of Rickettsia typhi with a polyclonal antiserum to clone genes which encode immunogenic proteins of R. typhi. Among several clones obtained, one clone codes for a 466‐amino‐acid protein similar to the heat‐shock protein, HtrA. The deduced rickettsial HtrA contains a putative signal peptide sequence at the N‐terminus, a serine protease‐like domain, and two PDZ domains. The recombinant protein of rickettsial HtrA reacted with sera from patients with murine typhus and tsutsugamushi disease. We suggest that this gene and its recombinant protein would be valuable for the immunologic diagnosis of rickettsial diseases.
Protective Immune Responses Induced by a Non‐Pathogenic Simian/Human Immunodeficiency Virus (SHIV) against a Challenge of a Pathogenic SHIV in Monkeys
Authors: Yoshino Naoto , Ami Yasushi , Someya Kenji , Ando Shuji , Shinohara Katsuaki , Tashiro Fumio , Lu Yichen , Honda Mitsuo
Published: November 2013

A simian/human immunodeficiency virus (SHIV)‐NM3n containing the human nef, but not the monkey nef, and vpr genes of SIV was inoculated into two cynomolgus monkeys, resulting in systemic infection with a minimum level of transient virus load. In order to study the nature of immune responses associated with the prevention of a pathogenic SHIV, the SHIV‐NM3n‐inoculated monkeys and three naive monkeys were intravenously challenged with a pathogenic SHIV containing the envelope gene of HIV‐1 89.6. After the heterologous virus challenge, all of the SHIV‐NM3n‐inoculated animals completely avoided the loss of CD4+ T lymphocytes in PBMC as well as lymphoid tissues compared to pathogenic SHIV‐injected control animals. The inhibition of CD4+ cell depletion was associated with maintaining the proliferative response of helper T‐cells against SIV p27 in the previously nonpathogenic virus‐inoculated animals following the pathogenic virus challenge. Furthermore, the decline of CD28+ cells, the increase in CD95+ cells, and the enhancement of in vitro apoptosis in PBMC were inhibited in the non‐pathogenic virus‐inoculated animals. These results suggest that nonpathogenic SHIV‐NM3n infection induces the protection of monkeys from heterologous pathogenic viruses that may be associated with blocking the change in immune responses and the cell loss induced by a pathogenic virus.