bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Identification of Members of the Genus Corynebacterium at Genus and Species Levels with 16S rDNA‐Targeted Probes
Authors: Hou Xiao‐gang , Kawamura Yoshiaki , Sultana Ferdousi , Hirose Kenji , Miyake Masaki , Otsuka Yoshihito , Misawa Shigeki , Oguri Toyoko , Yamamoto Hiroyuki , Ezaki Takayuki
Published: November 2013

16S rRNA gene‐targeted probes were designed for the identification of corynebacteria at the genus and species levels. The genus‐specific probe hybridized all clinically important members of the genus Corynebacterium and could distinguish them from other coryneform bacteria and phylogenetically related high G + C% gram‐positive bacteria, including Actinomyces, Rhodococcus, Gordona, Nocardia, Streptomyces, Brevibacterium and Mycobacterium. The species‐specific probes for C. jeikeium and C. diphtheriae could differentiate these two species from other members of this genus. The probes were used to select corynebacteria among gram‐positive clinical isolates which had been tentatively identified as corynebacteria by biochemical tests. We screened 59 strains with the genus‐specific probe; 51 strains hybridized to the genus‐specific probe, 8 did not. Of the 51 strains that hybridized to the genus‐specific probe, 1 hybridized to the C. diphtheriae species probe and 13 hybridized to the C. jeikeium species probe. The 8 strains that did not hybridize to the genus probe were further characterized by analyzing cell wall diaminopimelic acid and partial 16S rRNA sequencing. The results indicated that these strains were distributed in the genera Arthrobacter and Brevibacterium.
Amplification of a Full‐Length Borna Disease Virus (BDV) cDNA from Total RNA of Cells Persistently Infected with BDV
Authors: Shoya Yuko , Kobayashi Takeshi , Koda Toshiaki , Lai Patrick K. , Tanaka Hidetoshi , Koyama Tsukasa , Ikuta Kazuyoshi , Kakinuma Mitsuaki , Kishi Masahiko
Published: November 2013

We have developed a novel reverse transcriptase‐polymerase chain reaction (RT‐PCR) to amplify the full‐length 8.9 kilobase (kbp) cDNA of the Borna disease virus (BDV) RNA genome from the total cellular RNA of MDCK cells persistently infected with BDV (MDCK/BDV). Antigenomic BDV cDNA was reverse transcribed using a 53‐mer oligonucleotide primer, corresponding to the 5′‐terminus of a putative 3′‐leader sequence of the BDV RNA genome, for 2 hr at 42 C followed by 30 min at 55 C. PCR was performed in the presence of this 53‐mer antigenomic primer and a 25‐mer primer, corresponding to the 3′‐terminus of the BDV antigenomic cDNA, by use of an rTth DNA polymerase with proof‐reading activity. The amplified full‐length BDV cDNA was detected in as little as 20 ng of total cellular RNA of MDCK/BDV. This RT‐PCR method should be a useful technique to study the molecular quasispecies of BDV.
Cellular Immune Responses to Cell Wall Peptidoglycan Associated Protein Antigens in Tuberculosis Patients and Healthy Subjects
Authors: Dhiman Neelam , Verma Indu , Khuller Gopal Krishan
Published: November 2013

We have isolated cell wall peptidoglycan associated proteins (CW‐Pr) of Mycobacterium tuberculosis H37Ra by chemical treatment with trifluoromethanesulfonic acid:anisole (2:1), which further resolved into 71, 60 and 45 kDa proteins on SDS‐PAGE. A study was carried out to investigate the immunoreactivity of these proteins with blood samples from 4 categories, including 15 tuberculous patients (TB), 5 tuberculous patients on ATT (TBT), 10 PPD non‐reactive healthy controls (HPPD−) and 11 PPD reactive healthy controls (HPPD+). Comparing the proliferative responses to cell wall protein antigens, it was observed that the 71 kDa protein gave maximum stimulation with PBMCs from the TB and HPPD+ groups. The adherent PBMCs from the TB group also demonstrated enhanced phagocytosis, particularly in the presence of 71 and 45 kDa proteins, and the phagocytic index was significantly higher (P < 0.05) than the TBT group. However, PBMCs from of the groups recognized the 60 kDa cell wall antigen. Our results suggest that the 71 kDa protein from the cell wall of M. tuberculosis is highly immunogenic.
Sequence Analysis of the Influenza Virus Strain A/Shearwater/Australia/1/72 (H6N5)
Authors: Ward Alister C. , Harley Vincent R.
Published: November 2013

Analysis of the NS and M genes of the archetype H6N5 influenza virus strain A/shearwater/Australia/1/72 shows it to be a typical example of the avian host reservoir, containing Old World/Eurasian internal proteins with divergent surface proteins, which is a potential source of new pandemic strains.
Effects of Chronic Isoproterenol Treatment or Submandibular and Sublingual Ablation on Microflora of Mouse Tooth Surfaces
Authors: Maeda Nobuko , Okamoto Masaaki , Nakagawa Yoichi , Fujita Hiroshi , Humphreys‐Beher Michael G.
Published: November 2013

BALB/cA mice were examined for the effects of chronic isoproterenol treatment or submandibular‐sublingual gland ablation on the natural patterns of oral bacterial colonization on tooth surfaces. Indigenous microflora on the tooth surfaces of BALB/cA mice was relatively simple. The predominant bacterial groups were Enterobacteriaceae (45.9%), enterococci (29.4%) and staphylococci (15.7%). Isoproterenol, which resulted in the induced synthesis of proline‐rich proteins, caused a decrease in the total cultivable bacteria on the tooth surfaces. The proportion of Enterobacteriaceae in the isoproterenol‐treated mice decreased, although the proportion of other bacterial groups increased. Salivary gland ablation, which caused the loss of mucins in saliva, showed essentially the same number of total bacteria as the control. Salivary gland ablation resulted in a decrease in the proportion of Enterobacteriaceae, while the proportion of Gram‐positive rods and staphylococci increased.
Heat Shock Cognate Protein 71‐Associated Peptides Function as an Epitope for Toxoplasma gondii‐Specific CD4+ CTL
Authors: Yang Tian‐hui , Aosai Fumie , Norose Kazumi , Mun Hye‐seong , Yano Akihiko
Published: November 2013

HLA‐DR‐restricted CD4+ cytotoxic T‐lymphocyte (CTL) lines specific for Toxoplasma gondii (T. gondii)‐infected melanoma cells have been established from peripheral blood lymphocytes (PBLs) of a patient with chronic toxoplasmosis. The role of heat shock cognate protein (HSC) 71 in antigen (Ag) processing and presentation of T. gondii‐infected melanoma cells to these CD4+ CTL lines was investigated. A human melanoma cell line (P36) pulsed with T. gondii‐infected P36 cell‐derived HSC71 was lysed by a T. gondii‐specific CD4+ CTL line (Tx‐HSC‐1). The Tx‐HSC‐1 also killed T. gondii‐infected P36 cells. The lytic activity of Tx‐HSC‐1 against P36 cells pulsed with T. gondii‐infected P36 cell‐derived HSC71 was inhibited by monoclonal antibodies (mAbs) against HSC71. Anti‐human leukocyte antigen (HLA)‐DR mAb also partially blocked the lytic activity, whereas anti‐HLA‐A,B,C mAb did not block the lytic activity. In addition, a flow cytometric analysis with these specific mAbs against HSC71 showed HSC71 to be expressed on the cell surface of T. gondii‐infected P36 cells as well as uninfected P36 cells. These data indicate that HSC71 molecules are expressed on human melanoma cell line P36, and that HSC71 may play a potential role in Ag presentation and processing of T. gondii‐infected P36 cells to CD4+ CTL.
Humoral Immune Response in Yersinia enterocolitica O:5 Induced Arthritis in Hamsters
Authors: Di Genaro María Silvia , Escudero María Esther , Velázquez Lidia Del Carmen , Muñoz Estela , Aguilera Claudia , Juárez Américo , Scardapane Luis , de Guzmán Ana María Stefanini
Published: November 2013

Yersinia enterocolitica can cause extraintestinal sequelae such as reactive arthritis. The immunopathogenic mechanisms of this disease have not been completely clarified. Autoimmunity and persistent immune responses against bacterial antigens have been related to Yersinia‐induced arthritis. The arthritogenic capacity of Y. enterocolitica O:5 and the kinetics of the development of autoantibodies and Yersinia antigen‐specific antibodies were studied in hamsters. The results indicated that Y. enterocolitica O:5 was arthritogenic in the animal model studied. The animals developed septic arthritis on day 2 post‐infection (p.i.) and reactive arthritis on day 65 p.i. An important IgG response to types I and II collagen and the persistence of antibodies against lipopolysaccharide and bacterial cellular extract were observed. By immunoblotting, it was obtained that IgG reacted against a large number of bacterial antigens, the strongest being the responses against 88, 76, 63 and 36‐33 kDa peptides. From the results obtained, it can be concluded that serovar O:5 was experimentally arthritogenic, and that both autoimmune mechanisms and Yersinia‐specific antibodies participated in the development of Yersinia‐induced reactive arthritis in the animal model studied.
Combined Use of Ribotyping, PFGE Typing and IS431 Typing in the Discrimination of Nosocomial Strains of Methicillin‐Resistant Staphylococcus aureus
Authors: Yoshida Tatsumi , Kondo Noriko , Hanifah Yasmin Abu , Hiramatsu Keiichi
Published: November 2013

We have previously reported the phenotypic characterization of methicillin‐resistant Staphylococcus aureus (MRSA) clinical strains isolated in Malaya University Hospital in the period 1987 to 1989 using antibiogram, coagulase typing, plasmid profiles, and phage typing. Here, we report the analysis of the same strains with three genotyping methods; ribotyping, pulsed‐field gel electrophoresis (PFGE) typing, and IS431 typing (a restriction enzyme fragment length polymorphism analysis using an IS431 probe). Ribotyping could discriminate 46 clinical MRSA strains into 5 ribotypes, PFGE typing into 22 types, and IS431 typing into 15 types. Since the differences of the three genotyping patterns from strain to strain were quite independent from one another, the combined use of the three genotyping methods could discriminate 46 strains into 39 genotypes. Thus, the powerful discriminatory ability of the combination was demonstrated.
Amplification of rfbE and fliC Genes by Polymerase Chain Reaction for Identification and Detection of Salmonella Serovar Enteritidis, Dublin and Gallinarum‐Pullorum
Authors: Itoh Yoshihiro , Hirose Kenji , Miyake Masaki , Khan Abdul Q. , Hashimoto Yasuhiro , Ezaki Takayuki
Published: November 2013

Polymerase chain reaction (PCR) primers for O9 antigen (rfbE) and phase 1 flagellin antigen (fliC) were designed for the rapid identification and detection of Salmonella serovar Enteritidis and Dublin. The rfbE primer pairs selectively amplified the rfbE region of group O9 Salmonella serovars. The fliC primer pairs amplified the DNAs of g,m and g,p‐type flagellar antigen; Salmonella serovar Enteritidis, Dublin, and Essen. However, DNA from flagellar‐negative Salmonella serovar Gallinarum‐Pullorum was also amplified. The sensitivity of PCR primer pairs was 10 CFU/assay by boiled DNA preparation and 102 CFU/assay by proteinase K‐treated DNA preparation.
Serologic Evidence That Streptococcal Superantigens Are Not Involved in the Pathogenesis of Kawasaki Disease
Authors: Morita Atsushi , Imada Yoshio , Igarashi Hisanaga , Yutsudo Takashi
Published: November 2013

Kawasaki disease (KD) is an acute multisystem vasculitis of unknown etiology and is associated with marked activation of T cells and monocyte macrophages, leading to the assumption that superantigens are involved in its pathogenesis. To determine if an association exists between streptococcal superantigens and KD, we examined serum antibody responses to superantigens in sera from 50 paired acute and convalescent KD patients using purified recombinant streptococcal superantigens, such as SPEA, SPEC, SSA and MF. We found a very low frequency of detection of anti‐superantigen antibodies by ELISA and no marked IgG seroconversion to each superantigen, indicating the absence of a serological relationship between toxin‐producing streptococcal infection and the onset of KD.
Growth Inhibition of Helicobacter pylori by Monoclonal Antibody to Heat‐Shock Protein 60
Authors: Yamaguchi Hiroyuki , Osaki Takako , Taguchi Haruhiko , Hanawa Tomoko , Yamamoto Tomoko , Fukuda Minoru , Kawakami Hayato , Hirano Hiroshi , Kamiya Shigeru
Published: November 2013

The H20mAb recognizing the 60‐kilodalton protein, which existed in the outer membrane and was induced by heat shock at 42 C, was established. The molecule recognized with the mAb was a heat‐shock protein 60 (HSP60) of Helicobacter pylori. To understand the role of HSP60 on the cell surface of H. pylori, whether or not H20mAb affects the growth of H. pylori was investigated. When bacteria were cultured with H20mAb, growth was markedly inhibited after 24 hr, although an initial 5 hr‐incubation with the mAb induced no significant inhibition of H. pylori growth. The 24‐ and 48 hr growth of the bacteria after washing to remove the mAb at 5 hr was also inhibited though the inhibitory effect was not strong. In electron microscopical analysis, the spots with high electron density in the cytoplasm of the bacteria treated with H20mAb were increased, depending on the length of incubation time from 5 to 24 hr. After 24 hr treatment with H20mAb, bacterial destruction was also observed, indicating bactericidal activity by H20mAb. These results suggest that the HSP60 on the cell surface of H. pylori might have an essential role in the growth of the bacteria.
Adherence of Human Oral Spirochetes by Collagen‐Binding Proteins
Authors: Umemoto Toshihiko , Li Mingyu , Namikawa Isamu
Published: November 2013

The ability of spirochetes to adhere to collagens was compared among three species of human oral treponemes. Immunoblot analysis demonstrated that type I‐, IV‐, and V‐collagen‐binding polypeptides (CBPs) were detected in the heated and unheated preparations from both Treponema denticola ATCC 33520 and T. socranskii subsp. buccale ATCC 35534. Few CBPs, however, were detected in the heated and unheated preparations from a recently characterized isolate, T. medium strain G7201. Immunoelectron microscopy using rabbit antisera against the CBPs from the unheated preparations demonstrated that four CBPs, a 27 kDa type V‐CBP of T. denticola ATCC 33520, a 95 kDa type IV‐CBP and a 110 kDa type I‐CBP of T. socranskii subsp. buccale ATCC 35534, and a 95 kDa type IV‐CBP of T. medium strain G7201, were located on the outer envelopes of the individual cells. The adherence of T. denticola to the collagen‐coated surfaces was significantly greater than that of T. medium, suggesting that the CBPs on the oral spirochetal cells play an important role in their adherence to collagen‐rich connective tissues of the host.
Application of Equine Infectious Anemia Virus Core Proteins Produced in a Baculovirus Expression System to Serological Diagnosis
Authors: Kong Xian‐gang , Pang Hai , Sugiura Takeo , Sentsui Hiroshi , Onodera Takashi , Matsumoto Yoshitsugu , Akashi Hiroomi
Published: November 2013

Equine infectious anemia virus (EIAV) core proteins were obtained from a baculovirus expression system. Recombinant baculoviruses (rBVs) highly expressed the Gag precursor and p26 antigens in an rBV‐infected Sf21 cell culture supernatant. Enzyme‐linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) were conducted using the expressed proteins to detect antibodies from experimentally infected horses. The expressed antigens showed low background levels, high specificity and sensitivity in ELISA and AGID. The results of the serological tests using the expressed antigens were identical to those using a manufactured trial antigen. rBVs containing gag and p26 genes were found to express high quality and large quantities of Gag and p26 antigens, respectively. The antigens were quite useful for detecting anti‐EIAV antibodies from virus‐infected horses.
Immunomodulatory Activities of Oat β‐Glucan In Vitro and In Vivo
Authors: Estrada Alberto , Yun Cheol‐heui , Van Kessel Andrew , Li Bing , Hauta Shirley , Laarveld Bernard
Published: November 2013

Previous studies have shown that β‐glucans extracted from yeast or fungi potentiate immune responses. In the present study, the immunomodulatory activities of β‐(1→3, 1→4)‐glucan, derived from oats, were investigated. The ability of oat β‐glucan (OβG) to stimulate IL‐1 and TNF‐α release from murine peritoneal macrophages and the murine macrophage cell line P338D1, was assessed. In vitro stimulation of macrophages with OβG resulted in the production of IL‐1 in a dose and time‐dependent manner, whereas only small amounts of TNF‐α could be detected in the culture supernatants. OβG also induced the production of IL‐2, IFN‐γ and IL‐4 secretion in a dose‐dependent manner in cultured spleen cells. The intraperitoneal administration of OβG in mice resulted in the accumulation of leucocytes, predominantly macrophages, in the peritoneal cavity. Furthermore, OβG was tested for its ability to enhance non‐specific resistance to a bacterial challenge in mice. Survival of mice challenged with Staphylococcus aureus was enhanced by a single intraperitoneal administration of 500 μg of OβG 3 days prior to bacterial challenge. In conclusion, these studies demonstrated that OβG possesses immunomodulatory activities capable of stimulating immune functions both in vitro and in vivo.
Immunobiology of Lipopolysaccharide (LPS) and LPS‐Derived Immunoconjugates Vaccinate Mice against Salmonella typhimurium
Authors: Tiwari Ram Parkash , Gupta Winney , Rishi Praveen
Published: November 2013

The immunobiology of lipopolysaccharide (LPS) of Salmonella typhimurium LT2–71 was studied in its native, modified and conjugated states using mice as the experimental model. An alkali‐treated detoxified fraction of LPS (D‐LPS) was found to be not only non‐toxic but also equally immunogenic, like LPS. In addition D‐LPS alone or conjugated with enterotoxin or hemolysin was also non‐pyrogenic and non‐indurogenic. The immunoprophylactic activity of D‐LPS conjugates to a 100 ID50 challenge dose of S. typhimurium was also higher than that of detoxified LPS or native LPS.
Structural and Immunochemical Studies of Two Cross‐Reactive Proteus mirabilis O‐Antigens, O6 and O23, Containing β1→3‐Linked 2‐Acetamido‐2‐Deoxy‐d‐Glucopyranose Residues
Authors: Cedzyñski Maciej , Świerzko Anna St , Zi´łkowski Andrzej , Różalski Antoni , Paramonov Nikolay A. , Vinogradov Evgeny V. , Knirel Yuriy A. , Kaca Wiesław
Published: November 2013

A marked serological cross‐reactivity was observed by ELISA and a precipitation test between anti‐Proteus mirabilis O23 serum and the lipopolysaccharide as well as the O‐specific polysaccharide from the Proteus mirabilis strain belonging to serogroup O6. The structures of the O‐specific polysaccharides were elucidated using chemical and NMR spectroscopic analyses, and the only common component, 2‐acetamido‐2‐deoxy‐β‐d‐glucopyranose (β‐d‐GlcNAc), was revealed, which was suggested to be responsible for the cross‐reactivity observed. Both anti‐O23 and anti‐O6 sera were shown to react with 1, 3‐linked β‐d‐GlcNAc‐containing O‐antigen from Salmonella enterica ssp. arizonae O59 also. The lack of reactivity of Smith‐degraded P. mirabilis O6 O‐specific polysaccharide with homologous antiserum indicated the crucial role of α‐d‐glucuronic acid in specific antibody binding.
Response of Blood Platelets to Proteus mirabilis Lipopolysaccharide
Authors: Wachowicz Barbara , Saluk Joanna , Kaca Wiesław
Published: November 2013

The effects of the lipopolysaccharide (LPS) of Proteus mirabilis on the production of thiobarbituric acid reactive substances (TBARS) and the generation of superoxide radicals (O2−) by pig blood platelets were studied in vitro. The effect of LPS on TBARS formation in platelets was dependent on the concentration of endotoxin. LPS at concentrations above 0.1 μg/108 platelets caused the production of TBARS concomitant with the generation of superoxide radicals. The responses of platelets to LPS suggest that endotoxin, like thrombin (a strong platelets agonist), stimulates an enzymatic cascade of platelet arachidonate via cyclooxygenase and produces thromboxane A2 (TXA2) concomitant with malonyldialdehyde (MDA).
In Vitro Susceptibility of Chlamydia pecorum to Macrolides, Tetracyclines, Quinolones and β‐Lactam
Authors: Pudjiatmoko  , Fukushi Hideto , Ochiai Yoshitsugu , Yamaguchi Tsuyoshi , Hirai Katsuya
Published: November 2013

The in vitro susceptibility of Chlamydia pecorum to two macrolides (clarithromycin and erythromycin), two tetracyclines (doxycycline and minocycline), two quinolones (ofloxacin and ciprofloxacin) and one β‐lactam (ampicillin) was determined. The MICs were 0.004 to 0.008 μg/ml for clarithromycin, 0.008 to 0.031 μg/ml for doxycycline and minocycline, 0.063 to 0.125 μg/ml for erythromycin, 0.25 to 0.5 μg/ml for ofloxacin and 0.25 to 1.0 μg/ml for ciprofloxacin. The MIC for ampicillin was greater than 1,024 μg/ml. The results show clarithromycin and doxycycline are the two most effective drugs against C. pecorum.
Accumulation Kinetics of Viral Gene Products in Cauliflower Mosaic Virus‐Infected Turnip Protoplasts
Authors: Kobayashi Kappei , Nakayashiki Hitoshi , Tsuge Seiji , Mise Kazuyuki , Furusawa Iwao
Published: November 2013

The expression of cauliflower mosaic virus (CaMV) genes was studied in a turnip protoplast system. Six CaMV‐encoded gene products were detected in infected turnip protoplasts by means of Western blotting. The infected turnip protoplasts showed different patterns of protein accumulation; e.g. an open reading frame (ORF) I‐encoded movement protein, an ORF V‐encoded reverse transcriptase and an ORF VI‐encoded posttranscriptional transactivator representing the early accumulated proteins, an ORF II‐encoded aphid transmission factor and an ORF IV‐encoded coat protein the late accumulated proteins and an ORF III‐encoded DNA binding protein the intermediate protein. The results suggest that the expression of CaMV genes is differentially regulated.
Infectivity and Immunogenicity of SIVmac/HIV‐1 Chimeric Viruses (SHIVs) with Deletions in Two or Three Genes (vpr, nef and vpx)
Authors: Igarashi Tatsuhiko , Kuwata Takeo , Yamamoto Hiroshi , Moriyama Hiroyuki , Ui Masahiro , Miyazaki Yasuyuki , Hayami Masanori
Published: November 2013

Two SHIVs with two or three genes deleted (SHIV‐drn and SHIV‐dxrn) were constructed. The inoculation of monkeys with SHIV‐drn resulted in short‐term viremia, but inoculation with SHIV‐dxrn did not. At 68 weeks post‐inoculation, the monkeys were reinoculated with a 100‐fold higher dose of each SHIV, but none showed viremia. Killer cell activities against HTV‐1 Env were detected in the SHIV‐drn‐ and SHIV‐dxrn‐inoculated monkeys. Cross‐reactive killer activity against HIV‐1 Gag and SIVmac Gag was observed in one monkey. Antibodies were not detected in the SHIV‐dxrn‐inoculated monkeys, but the SHIV‐drn‐inoculated monkeys showed an anamnestic antibody reaction. These data indicate that SHIV‐drn is infectious to and immuno‐inducible in macaques but SHIV‐dxrn is not.
Protective Effect of Japanese Green Tea Extract on Gnotobiotic Mice Infected with an Escherichia coli O157:H7 Strain
Authors: Isogai Emiko , Isogai Hiroshi , Takeshi Koichi , Nishikawa Takeshi
Published: November 2013

We examined the effect of Japanese green tea extract (JGTE) on enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection in a gnotobiotic mouse model. Gnotobiotic mice inoculated with an EHEC strain developed neurologic and systemic symptoms, usually culminating in death. In contrast, none of mice receiving dietary JGTE showed clinical signs or death. This report describes the effect of JGTE, which includes the inhibition of bacterial growth in vivo. The Shiga‐like toxin (SLT) level in the feces of the JGTE diet group was significantly lower than that of the control group.
Cloning and Characterization of the Genomic RNA Sequence of the Mumps Virus Strain Associated with a High Incidence of Aseptic Meningitis
Authors: Saito Hiroyuki , Takahashi Yoshihiro , Harata Seizaburo , Tanaka Keiko , Sato Hiroyasu , Suto Tsunehisa , Yamada Akio , Yamazaki Shudo , Morita Morihiro
Published: November 2013

cDNA clones of the mumps virus wild‐type strain, associated with a high incidence of aseptic meningitis (ODATE‐1 strain), were isolated and analyzed from genomic nucleotide position 22 to 8520 containing the NP, P, M., F, SH and HN protein coding region. The ODATE‐1 strain exhibited a RFLP profile identical to that of the Urabe vaccine strain in spite of the fact that the virus was isolated from non‐vaccinated cases. However, a comparison of nucleotide and amino acid sequences among the ODATE‐1 strain, Urabe strain and Miyahara strain revealed that the ODATE‐1 strain was not related to the Urabe strain.
Surface Characteristics of Gram‐Negative and Gram‐Positive Bacteria in an Atomic Force Microscope Image
Authors: Umeda Akiko , Saito Mitsumasa , Amako Kazunobu
Published: November 2013

Bacterial images can be obtained rather easily with an atomic‐force microscope (AFM) in the magnification range of 5,000 to 30,000 times without any pretreatment of the specimens for such observations as chemical fixation, dehydration or staining. The bacterial shapes or the presence of flagella can be clearly recognized in these magnification ranges. In addition, we were also able to distinguish between Gramnegative and Gram‐positive bacteria based on the specific wavy surface appearance of the former. AFM could thus be a useful tool for the identification of bacteria in the resolution range between electron and light microscopy.
Resistance to Oxyimino β‐Lactams Due to a Mutation of Chromosomal β‐Lactamase in Citrobacter freundii
Authors: Haruta Shin , Nukaga Michiyoshi , Taniguchi Kazuo , Sawai Tetsuo
Published: November 2013

The duplicative mutation of an Ala‐Val‐Arg sequence at positions 208 to 210 in the loop structure of Enterobacter cloacae class C β‐lactamase caused substrate specificity extension to oxyimino β‐lactam antibiotics and this chromosomal mutation provided bacterial cells with high resistance to the β‐lactams (M. Nukaga et al, 1995, J. Biol. Chem. 270, 5729‐5735). In order to confirm the universality of this phenomenon among other class C β‐lactamases, the duplicative mutation was applied to a class C β‐lactamase of Citrobacter freundii, which has 74% homology to the E. cloacae β‐lactamase amino acid sequence. The counterpart sequence to the Ala‐Val‐Arg of the E. cloacae enzyme in C. freundii β‐lactamase was identified to be Pro‐Val‐His. A Pro‐Val‐His sequence was inserted just after the native Pro‐Val‐His sequence at positions 208 to 210 in the C. freundii β‐lactamase. The resulting mutant of C. freundii β‐lactamase obtained a striking characteristic that we expected, showing substrate specificity extension to oxyimino β‐lactams. Nearly the same result was obtained with the insertion of an Ala‐Val‐Arg sequence after the native Pro‐Val‐His sequence. These results indicate that structural modification of this locus commonly induces modification of the substrate specificity to unfavorable substrates for many chromosomal class C β‐lactamases produced by Gram‐negative bacteria.
Increased Resistance to Quinolones in Campylobacter jejuni: A Genetic Analysis of gyrA Gene Mutations in Quinolone‐Resistant Clinical Isolates
Authors: Ruiz Joaquim , Goñi Pilar , Marco Francesc , Gallardo Francesc , Mirelis Beatriz , De Anta Teresa Jimenez , Vila Jordi
Published: November 2013

Campylobacter jejuni is a frequent cause of enteritis and sometimes it requires antimicrobial therapy. We have studied the evolution of resistance to nine antibiotics from 1990 to 1994 and investigated how frequently gyrA mutations are involved in the acquisition of quinolone resistance. The percentage of chloramphenicol‐, clindamycin‐, tertracycline‐ and amoxicillin plus clavulanic acid‐resistant strains has remained practically unchanged and erythromycin and gentamicin resistance has decreased, whereas the percentage of ampicillin‐, nalidixic acid‐ or ciprofloxacin‐resistant strains has almost doubled in the follow‐up period, from 56 to 76% for ampicillin‐ and from 47.5 to 88% for quinolone‐resistant strains. This study clearly shows that a mutation in Thr‐86 to Ile or Lys is a frequent mechanism associated with the acquisition of a high level of resistance to quinolones in clinical isolates of C. jejuni.
A New Approach to AIDS Research and Prevention: The Use of Gene‐Mutated HIV‐1/SIV Chimeric Viruses for Anti‐HIV‐1 Live‐Attenuated Vaccines
Authors: Haga Takeshi , Kuwata Takeo , Ui Masahiro , Igarashi Tatsuhiko , Miyazaki Yasuyuki , Hayami Masanori
Published: November 2013

The lack of a suitable animal model is a major obstacle to developing anti‐HIV‐1 vaccines. We successfully generated an SIVmac/HIV‐1 chimeric virus (SHIV) (designated as NM‐3rN) that contains the HIV‐1 env gene and is infectious to macaque monkeys. Challenging the vaccinated macaque monkeys with NM‐3rN, we developed an evaluation system for anti‐HIV‐1 Env‐targeted vaccines. For the purpose of making the vaccine, a series of gene‐mutated SHIVs were constructed. The monkeys vaccinated with these SHIVs had long‐term anti‐virus immunities without manifesting the disease, and became resistant to a challenge inoculation with NM‐3rN. The sera from a monkey showed that, after the vaccination, the neutralizing antibodies not only against the parental HIV‐1 but also against an antigenically different HIV‐1 were raised. In vivo experiments confirmed that the vaccinated monkeys were protected from the challenge inoculum of an antigenically different SHIV‐MN. Vaccination of monkeys with the attenuated SHIVs showed that further gene‐deletion of the SHIV resulted in less immunogenicity. Nevertheless, the attenuated SHIVs had a vaccine effect against the challenge inoculation. In addition to specific immunities including neutralizing antibodies and cytotoxic T cells, a more complicated immune mechanism induced by live vaccine appears to play a role in this protection. Our data suggest that the live vaccine can induce strong and wide‐range immunity against HIV‐1. These SHIVs should contribute to understanding the pathogenicity of AIDS and to the development of future anti‐HIV‐1 live vaccines for humans.
Culture Supernatants of Lactobacillus acidophilus and Bifidobacterium adolescentis Repress Ileal Ulcer Formation in Rats Treated with a Nonsteroidal Antiinflammatory Drug by Suppressing Unbalanced Growth of Aerobic Bacteria and Lipid Peroxidation
Authors: Kinouchi Takemi , Kataoka Keiko , Bing Shan Ruo , Nakayama Haruyuki , Uejima Motoo , Shimono Kazuyuki , Kuwahara Tomomi , Akimoto Shigeru , Hiraoka Isao , Ohnishi Yoshinari
Published: November 2013

A nonsteroidal antiinflammatory drug, 5‐bromo‐2‐(4‐fluorophenyl)‐3‐(4‐methylsulfonylphenyl) thiophene (BFMeT), induced ileal ulcers in rats after oral administration, while no ulcers were observed after subcutaneous injection. The ileal ulcer formation in BFMeT‐treated rats was examined to correlate the administration of cultures of Lactobacillus acidophilus or Bifidobacterium adolescentis with intestinal bacteria in the ileal contents and lipid peroxidation of the small intestinal mucosa. Ileal ulcers were observed in more than 85% of the rats treated with BFMeT at a dose of 1,000 mg/kg when they were given tap water as drinking water. The incidence of ulcer formation was repressed by giving culture supernatants of L. acidophilus or B. adolescentis as drinking water, but not by giving the cell suspension as drinking water. Gram staining of the ileal contents of normal rats revealed that 97% of the stained bacteria were Gram‐positive rods and only 1.5% were Gram‐negative rods. The percentage of Gram‐negative rods 72 hr after BFMeT administration was 49.8% and increased over 30‐fold in BFMeT‐treated rats. However, the percentage of Gram‐negative rods was 9.7% or 16%, respectively, in rats taking culture supernatants of L. acidophilus or B. adolescentis. In addition, thiobarbituric acid‐reactive substances in the ileal mucosa increased significantly in the rats given tap water for 72 hr after BFMeT treatment, but not in rats given the culture supernatants of L. acidophilus or B. adolescentis. Since BFMeT induced an unbalanced intestinal microflora, the effect of antibiotic treatment on ulcer formation in rats was examined. The magnitude of the ulcer formation in the antibiotic‐treated rats was, in decreasing order, metronidazole >none > kanamycin > a mixture (bacitracin, neomycin and streptomycin). These results suggest that the intestinal microflora plays an important role in ulcer formation and that a metabolite(s) of L. acidophilus and B. adolescentis inhibits ileal ulcer formation by repressing changes in the intestinal microflora and lipid peroxidation in BFMeT‐treated rats.
Immunomodulatory Effects of Oat β‐Glucan Administered Intragastrically or Parenterally on Mice Infected with Eimeria vermiformis
Authors: Yuri Cheol‐heui , Estrada Alberto , Van Kessel Andrew , Gajadhar Alvin , Redmond Mark , Laarveld Bernard
Published: November 2013

The immunostimulatory effect of intragastrically or parenterally administered β‐(1→3; 1→4) glucan, extracted from oats (OβG), on disease resistance to Eimeria vermiformis was studied in C57BL/6 mice. Multiple administrations of OβG by intragastric or subcutaneous routes reduced fecal oocyst shedding compared to the non‐treated control group. The administration of OβG by subcutaneous route resulted in higher levels of total serum immunoglobulins and antigen (sporozoite and merozoite)‐specific immunoglobulins as compared with the non‐treated group. To evaluate the effect of a single subcutaneous dose, groups of mice were treated with OβG 2 days before E. vermiformis infection, at the time of infection and at 2 or 6 days after infection. From day 11 post‐infection the oocyst discharge was significantly diminished (P< 0.05‐0.01) in the OβG‐treated groups, except in those treated 6 days after infection, as compared to the non‐treated control group. The proliferative responses to E. vermiformis sporozoite antigen of lymphocytes isolated from the spleen were significantly increased (P < 0.05) when OβG was administered 2 days before or at the time of E. vermiformis infection. Lymphocyte proliferative responses to merozoite antigen were not influenced by treatment. In conclusion, OβG appeared to up‐regulate immune mechanisms and provide enhanced resistance against eimerian coccidiosis in mice.
Transient Dissociation between Infectious HIV‐1 Titer and Viral RNA during the Early Phase of AZT Treatment
Authors: Zöllner Bernhard , Stellbrink Hans‐Jürgen , Feucht Heinz‐hubert , Schröter Matthias , Baumgartner Eva‐maria , Laufs Rainer
Published: November 2013

The short‐term kinetics of infectious HIV titers, HIV copy numbers and p24‐antigen during the first 28 days of AZT monotherapy were evaluated. In three of four patients, infectious HIV was culturable and infectious titers rose 2‐ and 4‐fold compared to baseline values. This increase was neither associated with mutations conferring resistance to AZT nor a switch from NSI to SI phenotypes. Two patients showed an increase of plasma infectivity associated with a reduction of HIV copies and p24‐antigen. We conclude that transient dissociations of plasma infectivity and HIV copy numbers occur during early AZT monotherapy.
Molecular Composition of the 16S Toxin Produced by a Clostridium botulinum Type D Strain, 1873
Authors: Nakajima Hiroshi , Inoue Kaoru , Ikeda Tetsuya , Fujinaga Yukako , Sunagawa Hiroyuki , Takeshi Kouichi , Ohyama Tohru , Watanabe Toshihiro , Inoue Katsuhiro , Oguma Keiji
Published: November 2013

The 16S toxin was purified from a Clostridium botulinum type D strain 1873 (D‐1873). Furthermore, the entire nucleotide sequences of the genes coding for the 16S toxin were determined. It became clear that the purified D‐1873 16S toxin consists of neurotoxin, nontoxic nonhemagglutinin (NTNH), and hemagglutinin (HA), and that HA consists of four subcomponents, HA1, HA2, HA3a, and HA3b, the same as type D strain CB16 (D‐CB16) 16S toxin. The nucleotide sequences of the nontoxic components of these two strains were also found to be identical except for several bases. However, the culture supernatant and the purified 16S toxin of D‐1873 showed little HA activity, unlike D‐CB16, though the fractions successively eluted after the D‐1873 16S toxin peak from an SP‐Toyopearl 650S column showed a low level of HA activity. The main difference between D‐1873 and D‐CB16 HA molecules was the mobility of the HA1 on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Therefore it was presumed that the loss of HA activity of D‐1873 16S toxin might be caused by the differences of processing HA after the translation.
Expression Analysis of the Autolysin Gene (atl) of Staphylococcus aureus
Authors: Oshida Tadahiro , Takano Mayumi , Sugai Motoyuki , Suginaka Hidekazu , Matsushita Tadahiro
Published: November 2013

The bifunctional autolysin gene (atl) of Staphylococcus aureus was transcribed into a 4.1‐kb transcript. The transcription initiation site was located at an adenine residue 33‐nt upstream from the putative atl start codon. Analysis using a promoter‐reporter plasmid showed that promoter activity increased during the exponential growth phase. The Tn551 insertion site of the autolysis‐deficient mutant S. aureus RUSAL2 was located in the putative catalytic region of the glucosaminidase domain.
Detection and Long‐Term Existence of Shiga Toxin (Stx)‐Producing Escherichia coli in Sheep
Authors: Asakura Hiroshi , Makino Sou‐ichi , Shirahata Toshikazu , Tsukamoto Teizo , Kurazono Hisao , Ikeda Tetsuya , Takeshi Kouichi
Published: November 2013

The isolation and characterization of Shiga‐like toxin (Stx)‐producing Escherichia coli (STEC) from sheep are described. The distribution of stx genes in E. coli isolates was detected by PCR. When brain heart infusion (BHI) broth and novobiocin supplemented m‐EC broth (N‐mEC) were used as enrichment culture for the isolation of STEC, N‐mEC, compared to BHI, showed clearly lower efficiency. Finally, 5 STEC isolates from 4 sheep were isolated and characterized by biochemical and genetical analysis. All of them were confirmed by ELISA and Vero cell cytotoxicity assay for the production of Stx. Moreover, some strains carried hemolysin and eaeA genes and harbored large plasmids. Based on their plasmid profiles, antibiotic patterns and PCR‐based DNA fingerprinting analysis using random amplified polymorphic DNA (RAPD), all isolates were different from each other. Three of the isolates were identified to belong to serogroups O2, O153 and O165, respectively, and the STEC strains belonging to these serogroups had been isolated from STEC outbreaks in humans. Four months after the first isolation in July 1997, STEC from sheep #1 was isolated again. A new isolate, HI‐11, was identified as STEC O2: Hnt. Simultaneously, 2 STEC, which were genetically and phenotypically different from each other, were isolated from the same sheep at intervals of 4 months. These results demonstrate that sheep may be an important animal for studying human STEC infections, and that further epidemiological surveys on STEC are necessary.
Attachment of Nontypable Haemophilus influenzae to Human Pharyngeal Epithelial Cells Mediated by a Ganglioside Receptor
Authors: Kawakami Kenji , Ahmed Kamruddin , Utsunomiya Yoshiaki , Rikitomi Naoto , Hori Akihiro , Oishi Kazunori , Nagatake Tsuyoshi
Published: November 2013

Nontypable Haemophilus influenzae (NTHI) is one of the major pathogens of human respiratory infections and has the ability to attach to pharyngeal epithelial cells. We characterized the epithelial cell receptor to which NTHI bind. Neuraminidase pretreatment of pharyngeal epithelial cells resulted in a significant decrease in NTHI attachment, suggesting sialic acid as an important component of the receptor. The attachment was not decreased in NTHI pretreated with 1,000 μg/ml of fucose, N‐acetyl‐neuraminic acid, N‐acetyl‐glucosamine, N‐acetyl‐galactosamine, acetyl‐salicylic acid and colominic acid. Only treatment with gangliosides D1a, D1b and D2 at a concentration of 12.5 μg/ml significantly decreased the attachment. On the other hand, treatment with gangliosides M1, M2, M3, D3, T1b and asialoganglioside M1 did not decrease the attachment of NTHI. Only ganglioside D2 inhibited the attachment significantly at a concentration of 12.5 ng/ml. Other isolates of NTHI showed a decrease in attachment after treatment with ganglioside D2. Treatment of cells with anti‐human GD2 monoclonal antibody also decreased the attachment of NTHI in a dose‐dependent manner. This study indicates that sialic acid glycoconjugate, GD2, is one of the receptors of NTHI on human pharyngeal epithelial cells.
Genomic Analysis of the Viral Population in Genital Secretions Early after Infection of Simian Immunodeficiency Viruses in Macaque Monkeys
Authors: Enose Yoshimi , Ibuki Kentaro , Shimada Toshihide , Ui Masahiro , Hayami Masanori
Published: November 2013

To clarify the change in the viral population during passage from the vaginal cavity to blood circulation and vice versa, we examined the viral clones detected in cells in vaginal washes (VWCs) early after inoculation and after systemic infection with polyclonal SIV. In two intravaginally inoculated monkeys, the viral clones found in VWCs at 18 days p.i. were shown to be some of those contained in the inoculum, whereas the viral population in the peripheral blood mononuclear cells (PBMCs) was a monotype. This gradual decrease of viral clones suggested the possible existence of two barriers, one at the genital tract and the other between the genital tract and the blood. Later, at one month p.i., the viral clones in VWCs became rather restricted, whereas those in PBMCs diverged from a single clone to several clones. This suggested that different mechanisms affect the viral populations in PBMCs and VWCs. In order to examine how the viral population was affected by passage from the blood to the vaginal cavity, a monkey was intravenously inoculated and the viral clones in VWCs were analyzed at 14 days p.i., at a time of the heterogeneous population in PBMCs. The viral population in VWCs was found to be a single clone and this clone was a minor type in PBMCs, suggesting that the major clone in PBMCs was not always secreted to the vaginal cavity.
Induced Expression and Localization to Nuclear‐Inclusion Bodies of hsp70 in Varicella‐Zoster Virus‐Infected Human Diploid Fibroblasts
Authors: Ohgitani Eriko , Kobayashi Kappei , Takeshita Kazushige , Imanishi Jiro
Published: November 2013

The expression and subcellular localization of cellular heat‐shock protein hsp70 were examined in varicella‐zoster virus (VZV)‐infected human diploid fibroblasts. Infection with VZV elevated the steady‐state levels of hsp70 mRNA by 24 hr post‐infection (hpi). Western blotting analysis revealed an increase in accumulation of hsp70 from 24 hpi. Subcellular localization of the hsp70 in VZV‐infected cells was examined by indirect immunofluorescence. In most VZV‐infected cells, hsp70 was localized to inclusion bodies induced in the cell nucleus by infection with VZV. In some cells, however, the remaining parts of the cell nucleus and the cytoplasm were also stained with anti‐hsp70 antibody. These results indicate that infection with VZV induces the expression of hsp70 and its localization to VZV‐specific inclusion bodies, which suggests the involvement of hsp70 in molecular events within inclusion bodies.
The Matrix Gene Expression of Subacute Sclerosing Panencephalitis (SSPE) Virus (Osaka‐1 Strain): A Comparison of Two Sibling Viruses Isolated from Different Lobes of an SSPE Brain
Authors: Ayata Minoru , Hayashi Kaoru , Seto Toshiyuki , Murata Ryosuke , Ogura Hisashi
Published: November 2013

The Fr/V and Oc/V sibling viruses of the Osaka‐1 strain of the subacute sclerosing panencephalitis (SSPE) virus were defective in cell‐free virus production. By radioimmunoprecipitation assay, the matrix (M) protein was not detected in cells persistently infected with the Osaka‐1 strain. This undetectable expression was consistent with the selective reduction of antibody response to the M protein in the patient from whom the Osaka‐1 strain was isolated. The sequence of the M gene, however, predicted that the protein could be synthesized because the translational start and stop codons for the protein were not altered. Northern blot hybridization demonstrated the selective defect of the monocistronic mRNAs for the M protein and the phosphoprotein (P) together with the dominant presence of the P‐M bicistronic mRNA. This absence of the M mRNA was further confirmed by primer extension analysis. Therefore, the undetectable expression of the M protein in the infected cells was proved to be caused by a transcriptional defect. The two sibling viruses, isolated from remote portions of an SSPE brain, were indistinguishable in their viral characters, including the M gene sequences, which indicates the possibility of clonal expansion of the strain in the brain.
Intranasal Immunization with Yersinia enterocolitica O:8 Cellular Extract Protects against Local Challenge Infection
Authors: Genaro María Silvia Di , Escudero María Esther , Muñoz Estela , Aguilera Claudia , Scardapane Luis , De Guzmán Ana María Stefanini
Published: November 2013

Yersinia enterocolitica is enteropathogenic for humans and rodents. Immune protection from oral and respiratory pathogens may be most effectively elicited following intranasal (i.n.) vaccination. An experimental murine intranasal challenge model was used to evaluate the immunogenicity of a Y. enterocolitica O:8 cellular extract (CE) in mucosa. This antigenic preparation has demonstrated to induce protection by subcutaneous immunization. Mice were immunized intranasally with two doses of CE. Immunized and nonimmunized animals were challenged with 5×106 colony‐forming units (CFU) by nasal infection. Antibodies in serum and bronchoalveolar lavage (b.a.l.) fluid were assessed before and 48 hr after challenge. The CFU were determined by analysis of lung homogenate samples. The CE immunization induced significant b.a.l.‐specific IgA and IgG, and serum‐specific IgG, IgA and IgM. Histopathological studies 24 and 48 hr postchallenge demonstrated that immunization protected against progressive lesions resulting from Y. enterocolitica invasion of the pulmonary mucosa. The CFU in the lungs showed that CE immunization led to significant clearance as compared to the bacterial level in nonimmunized controls. From the results obtained, it can be concluded that CE can induce local and systemic immunity and protect against nasal infection.
Cytotoxicity of Vibrio vulnificus Cytolysin on Rat Peritoneal Mast Cells
Authors: Kim Jong‐suk , Chae Mee‐ree , Chang Keun , Park Kwang‐hyun , Rho Hye‐won , Park Byung‐hyun , Park Jin‐woo , Kim Hyung‐rho
Published: November 2013

Histamine has been thought to be a permeability enhancing factor in Vibrio vulnificus infection. The injection of living bacteria or purified V. vulnificus cytolysin (VVC) can cause lethality in mice by inducing hemoconcentration and increased vascular permeability. In the present study, we tried to identify whether histamine release causes the increased vascular permeability that is responsible for the lethal effect of VVC. Treatment of rat peritoneal mast cells with high concentrations of VVC caused the release of whole cellular histamine and lactate dehydrogenase (LDH). At concentrations less than 10 HU/ml, histamine and LDH were not released whereas preloaded 2‐deoxy‐d‐glucose was rapidly effluxed with the concomitant decrease in cellular ATP. VVC‐treated mast cells were refractory to the stimulation of histamine secretion by Compound 48/80 but remained fully responsive to Ca2+ plus GTP‐γ‐S. These results indicate that histamine can be released from mast cells only when the concentration of VVC is high enough to cause the lysis of cells. At low concentrations, VVC does not induce the release of stored histamine from damaged cells. The intravenous injection of 80 HU purified VVC to rats, which can produce the calculated blood concentration of about 3 HU/ml, caused a marked increase in pulmonary vascular permeability, hemoconcentration and death. However, no increase in blood histamine level was detected. This level of VVC in rat blood was enough to cause severe hemoconcentration and lethality but might not be enough to cause cytolysis of the mast cells and resulting histamine release.