bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of Opsonophagocytic Dysfunctions in Severely Burned Patients by Luminol‐Dependent Chemiluminescence
Authors: Ono Yasuo , Kunii Otohiko , Kobayashi Kunio , Kanegasaki Shiro
Published: November 2013

We compared the luminol‐dependent chemiluminescence (CL) response of peripheral blood from severely burned patients with that from normal controls to evaluate the primary defense level against bacterial infection in the patients. The CL was measured upon addition to diluted whole blood of a soluble stimulus, phorbol myristate acetate (PMA) or particulate stimuli such as bacteria or zymosan without special opsonization. In the early post‐burn days, the initial rate of whole blood CL induced with the particulate stimulus was much lower than that in the normal controls, whereas the rate was higher when PMA was used as a stimulus. The number of granulocytes in the patients' blood had increased and isolated polymorphonuclear leukocytes (PMNs) from the patients exhibited higher CL responses to the particulate or soluble stimulus as compared with those of normal controls. The results suggest that the PMNs in burn patients were activated and normally mobilized in the early post‐burn period but the opsonizing capacity in the blood decreased. In fact, the serum levels of complement, immunoglobulins and fibronectin were found to be lower in the blood from the patients than those from normal controls and a supplement of freshly frozen plasma of human immunoglobulin preparations restored the initial rate of the whole blood CL upon phagocytosis. The prognosis is still poor when severe infection occurs in the patients with decreased CL response of whole blood. Recombinant human granulocyte colony‐stimulating factor (rhG‐CSF) enhanced the CL response of PMNs from burn patients. The administration of rhG‐CSF may be useful for decreasing the morbidity of severe infection following burn injury in the near future.
Comparison of Detection Methods for Legionella Species in Environmental Water by Colony Isolation, Fluorescent Antibody Staining, and Polymerase Chain Reaction
Authors: Yamamoto Hiroyuki , Hashimoto Yasuhiro , Ezaki Takayuki
Published: November 2013

Three detection methods for Legionella species in water samples from cooling towers and a river were examined. Direct counting of bacteria stained with fluorescent antibody (FA) for L. pneumophila (serogroups 1 to 6) could detect the cell of 104 to 106 cell/100 ml in all 14 samples, while colony counting method detected 10 to 103 CFU/100 ml only in 8 samples from cooling towers. Polymerase chain reaction (PCR) assay with primers to amplify 16S ribosomal DNA sequence of most Legionella species (LEG primer) detected legionellae in 13 samples, while species‐specific primers for L. pneumophila detected the DNAs from 3 samples. In laboratory examination, LEG primers could amplify DNAs of 29 species of genus Legionella with high sensitivity, even from 1 cell of L. pneumophila GIFU 9134. The PCR assay with LEG primers was specific and sensitive methods to be satisfied the survey of legionellae. Thus, PCR assay is a suitable method to detect and monitor Legionella species in an environment.
Heterogeneity of HLA‐G Genes Identified by Polymerase Chain Reaction/Single Strand Conformational Polymorphism (PCR/SSCP)
Authors: Tamaki Junko , Arimura Yutaka , Koda Toshiaki , Fujimoto Seiichiro , Fujino Takafumi , Wakisaka Akemi , Kakinuma Mitsuaki
Published: November 2013

A genomic HLA‐G clone named 7.0E was isolated from a Japanese placenta. The deduced amino acid sequence of the 7.0E was identical to two HLA‐G genomic clones and two cDNA clones previously described. The DNA sequences of α1 and α2 domains of the HLA‐G gene from 5 cell lines also encoded the same amino acids. However, a 14 bp insertion, ATTTGTTCATGCCT, was present in the 3′ untranslated region of 7.0E compared with the originally described HLA‐G clone (HLA 6.0). Polymerase chain reaction (PCR)/single strand conformational polymorphism (SSCP) analysis of exon 8 allowed the HLA‐G gene to be classified into two alternative types, G6.0 and 7.0 E, those correlated to the absence or the presence of the 14 bp stretch. Each group had minor sequence variant(s), and the alleles of the 7.0E‐type were more heterogeneous than those of the G6.0‐ type. The 14 bp deletion is present only in the G6.0‐type of HLA‐G alleles among HLA class I genes. Thus it was suggested that G6.0 alleles were generated after diversification of the HLA‐G.
Immunogenicity of Vibrio cholerae O1 Fimbriae in Animal and Human Cholera
Authors: Ehara Masahiko , Ichinose Yoshio , Iwami Mamoru , Utsunomiya Akiyoshi , Shimodori Shoichi , Kangethe Stanley K. , Neves Bianca C. , Supawat Krongkaew , Nakamura Satoshi
Published: November 2013

Parenteral immunization with either formalin‐fixed whole cells of the fimbriate Bgd17 strain or purified fimbriae protected against Vibrio cholerae O1 infection in rabbits, independent of biotype and serotype. Parenteral immunization of adult rabbits with purified fimbriae prior to V. cholerae O1 challenge resulted in a reduction of 2 to 3 orders of magnitude in the number of bacteria recovered from the small intestines of immunized rabbits in comparison to non‐immunized controls. IgG and IgA antibodies against fimbrillin of V. cholerae O1 were detected in the convalescent sera of patients with cholera; however, little fimbrial antigen was detected in the commercially available cholera vaccines when examined by polyclonal and monoclonal antibodies against fimbriae. These data suggest that fimbrial hemagglutinin is a major adhesin of V. cholerae O1 and that parenteral immunization with fimbriae generates a specific immune response in the gut that may serve as one means of mitigating subsequent V. cholerae O1 gut infection.
Molecular Characterization of Human Rotavirus VP4 Genes by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Assay
Authors: Iizuka Masahiro , Chiba Mitsuro , Masamune Osamu , Gerna Giuseppe , Nakagomi Osamu
Published: November 2013

A restriction fragment length polymorphism (RFLP) assay was developed to examine the genetic variability and similarity of the VP4 genes of human rotaviruses. The VP4 genes of 14 human rotavirus strains, including VP4 serotype P1A strains (Wa, P, VA70), serotype P1B strain (DS‐1), serotype P2 strains (M37, 1076, McN, ST3) and serotype P3 strains (AU‐1, AU228, K8, PA151, PCP5, MZ58), and those of 2 feline strains (FRV‐1 and Cat2) were reverse‐transcribed and amplified by the polymerase chain reaction (PCR). The amplified VP4 cDNAs were then digested with a panel of restriction endonucleases (HindIII, NruI, HaeIII, and EcoRI), resulting in the identification of at least one enzyme with which digestion produced an RFLP profile specific for a particular P serotype. Of interest was the presence of two distinct RFLP patterns within the serotype P3 VP4 genes: one corresponding to the VP4 gene carried by the members of the AU‐1 genogroup and the other corresponding to the VP4 genes carried by naturally‐occurring reassortants between members of the AU‐1 and other genogroups.
Neutralization of Chlamydia psittaci with Monoclonal Antibodies
Authors: Ando Shuji , Takashima Ikuo , Hashimoto Nobuo
Published: November 2013

Neutralization of Chlamydia (C.) psittaci avian strain P‐1041 was examined in vitro using monoclonal antibodies (MAbs). Of the 10 MAbs used, 6 were found to exhibit neutralizing capability. These include 3 against major outer membrane protein (MOMP), 1 against lipopolysaccharide (LPS) and 2 against other protein molecules [90 kilodalton (kDa) and 90/50 kDa]. Most neutralizing MAbs were dependent on complement for efficient neutralization, while a strain‐specific MAb (2B5) against the 90 kDa protein displayed a different requirement for complement and neutralized the infectivity of the P‐1041 at high concentrations without complement. By competitive inhibition enzyme‐linked immunosorbent assay (competitive inhibition ELISA), all 3 neutralizing anti‐MOMP MAbs were demonstrated to recognize different epitopes found in very close proximity to each other on the outer membrane.
An Assay Method for Herpes Simplex Virus Type 1 Seroprevalence Survey—Detection of Antibody in Saliva by Avidin‐Biotin Complex Enzyme‐Linked Immunosorbent Assay (ABC‐ELISA)
Authors: Zhang Zhi‐qiang
Published: November 2013

To determine whether the avidin‐biotin complex enzyme‐linked immunosorbent assay (ABC‐E) is a potentially useful method for detection of herpes simplex virus type 1 (HSV‐1) antibody in saliva, paired serum and saliva samples from 129 healthy individuals aged 18 to 25 years were collected simultaneously and subjected to a neutralization test (NT) for neutralizing antibody and also to an indirect ELISA (IE) and ABC‐E for HSV‐1 specific IgG detection. Compared with the results of NT, the sensitivities of the IE and ABC‐E for serum were both 100% (45/45), and for saliva 82.2% (37/45) and 93.3% (42/45), respectively. The specificity of all these methods was 100% (84/84). With the same ABC‐E method, a significant correlation (r=0.66, P < 0.001) between the OD‐difference (d‐OD) values of positive serum and saliva samples was observed. Furthermore, the consistency of ABC‐E for salivary antibody detection was confirmed with the paired serum and saliva samples which were collected from four individuals followed up for eight months. It was clear that the ABC‐E method for saliva can be used in place of the NT and ABC‐E method for serum for seroprevalence studying of HSV‐1 infection.
Lymphocytic Proliferative Response to Outer‐Membrane Proteins Isolated from Salmonella
Authors: González César R. , Isibasi Armando , Ortiz‐Navarrete Vianney , Paniagua Jorge , García José A. , Blanco Francisco , Kumate Jesús
Published: November 2013

Porins isolated from Salmonella typhi have been demonstrated to protect against the challenge with this bacteria in mice. The mechanism has not been clarified, but could be associated with activation of both humoral and cellular immunity. In order to evaluate the induction of specific T cell responses, the lymphocytic proliferation to porins isolated from Salmonella typhimurium, Salmonella typhi and Escherichia coli was examined by 3H‐thymidine incorporation assay in mice immunized with three different antigens: acetone‐killed S. typhimurium, its porins, or outer‐membrane proteins (OMPs) isolated from S. typhi. Higher proliferative responses were observed in mice immunized with porins and OMPs compared with those which received the acetone‐killed bacteria. Although cross‐reactivity was observed between porins, they were not mitogenic. Moreover, porins were able to activate T lymphocytes isolated from mice immunized with S. typhi OMPs. These results suggest that T cell activation, through the release of lymphokines, may play a role in the induction of protective immunity with porins.
A Quantitative Microanalysis of Bacterial Endotoxin Using [3H]‐Labeled L‐Glycero‐D‐mannoheptitol as a Marker
Authors: Haishima Yuji , Tanamoto Ken‐ichi
Published: November 2013

Quantitative microanalysis of bacterial endotoxin was performed using [3H]‐labeled L‐glycero‐D‐mannoheptitol (LD‐Heptitol) as a marker. Several different amounts of authentic L‐glycero‐D‐mannoheptose (LD‐Heptose) were reduced with 20 μg of cold NaBH4 containing 2 μg of NaB3H4 (40 Ci/mmol) in 20 μ1 of 1 mM NaOH at 4 C for 48 hr. The product, [1‐3H]‐labeled LD‐Heptitol, has high specific activity, and was purified by HPLC and detected using a liquid‐scintillation counter. As little as 50 pg of LD‐Heptose was detectable, and the radioactivity increased dose‐dependently in the 100 pg to 80 ng range tested. More than 2 ng of Salmonella abortus equi endotoxin could be accurately determined by this method. It is possible to detect 50 pg of endotoxin by this method, if 100% hot material (NaB3H4) is used for [3H]‐labeling.
Two Distinct Clusterings of the VP8* Gene of Rotaviruses Possessing the AU‐1 Gene 4 Allele
Authors: Nakagomi Osamu , Isegawa Yuji , Ueda Sigeharu , Flores Jorge
Published: November 2013

A phylogenetic tree constructed by the unweighted pair group method with arithmetic means (UPGMA) for the VP8* gene from 13 human and two feline rotavirus strains possessing the AU‐1 gene 4 allele revealed that these strains could be classified into two clusters which did not correspond with the year of isolation or the host species from which they originated. Nine of 10 human strains and one feline strain isolated in Japan formed one cluster, whereas three human strains from Italy and one feline strain from Australia formed the other. Human strain K8 from Japan was distantly related to the Australo‐European clustering.
The Entry and Intracellular Multiplication of Francisella tularensis in Cultured Cells: Its Correlation with Virulence in Experimental Mice
Authors: Fujita Hiromi , Watanabe Yuriko , Sato Tadashi , Ohara Yoshiro , Homma Morio
Published: November 2013

Five acriflavine agglutination test‐positive (acf+) colonies and five negative (acf–) colonies were isolated from each of the four strains (Ebina, CMB2, N9, and Schu) of Francisella tularensis, and the correlation between the virulence in experimental mice and the entry and intracellular multiplication in cultured mouse fibroblast cells (L‐929 cells) was examined. All of the acf– colonies derived from the Ebina and CMB2 strains were highly virulent in mice, readily entering and growing well in the cells, while all of the acf‐ colonies from N9 and Schu strains were of low virulence and neither entered nor grew in the cells effectively. On the other hand, regardless of their parent strains, the acf+ colonies were low virulent and most of those colonies did neither enter nor grow in L‐929 cells. In addition, two acf‐ colonies, one from the N9 and the other from the Schu strain, gained virulence through several passages in mice, and in parallel, their entry and multiplication also improved. However, two acf+ colonies from the Ebina strain and one acf+ colony from the N9 strain showed a moderate degree of the entry and multiplication although they were all low virulent. The overall results indicate that the entry and multiplication in cells are important factors regulating the virulence of F. tularensis. The results also showed, however, that they were not sole factors to elucidate the virulence of the bacterium in mice.
Potent In Vitro Bactericidal Activity of Polymyxin B against Methicillin‐Resistant Staphylococcus aureus (MRSA)
Authors: Yoshida Tatsumi , Hiramatsu Keiichi
Published: November 2013

Eradication of methicillin‐resistant Staphylococcus aureus (MRSA) carried by inpatients or healthy hospital personnel by topical use of antibiotics is an important step for preventing outbreak of MRSA nosocomial infection. In the screening of the antibiotic best suited for this purpose, we have found that polymyxin B, a commonly used antibiotic for gram‐negative infection, had an unexpected strong cytokilling activity towards MRSA clinical strains, which was more potent than that of vancomycin or gentamicin. The data suggested that polymyxin B could be an antibiotic of choice in the treatment of topical carriage of or infection caused by MRSA.
Influence of a Small Number of Mature T Cells in Donor Bone Marrow Inocula on Reconstitution of Lymphoid Tissues and Negative Selection of a T Cell Repertoire in the Recipient
Authors: Arase‐Fukushi Noriko , Arase Hisashi , Wang Bingyan , Hirano Mari , Ogasawara Kazumasa , Good Robert A. , Onoé Kazunori
Published: November 2013

Allo‐chimerism and clonal elimination of self antigen (Ag) (Ia + Mls‐1a) reactive Vβ6+ T cells were analyzed and compared between allogeneic bone marrow (BM) chimeras reconstituted with BM cells which had been treated with anti‐Thy‐1 monoclonal antibody (mAb) plus complement (C) (T– chimeras) and BM chimeras which had been reconstituted with BM cells pretreated with anti‐Thy‐1 mAb alone (T+ chimeras). When lethally irradiated AKR (Mls‐1a) mice were reconstituted with BM cells from B10 or B10 H‐2 congenic mice, both T+ and T– chimeras were entirely free of signs of graft‐versus‐host reaction (GVHR). However, complete replacement of the AKR lymphoid tissues by donor BM cells was accomplished at an early stage in T+ chimeras but not in T– chimeras. On the other hand, clonal elimination of Vβ6+ T cells reactive to the recipient Ag (Mls‐1a) was abolished in T+ chimeras but successfully induced in T– chimeras. The Vβ6+ T cells not eliminated in T+ chimeras showed depressed responses against Mls‐1a antigens. The findings herein demonstrate that T cells which contaminate a BM inoculum survive in recipient mice after treatment with anti‐Thy‐1 mAb without C in vitro followed by BMT. The surviving T cells have been estimated to represent fewer than 0.5% of the BM cells inoculated. These cells appear to accelerate the full replacement of recipient lymphoid tissues by donor cells. Furthermore, the T cells which survive in the marrow inoculum influence eventually the development of a tolerant state in the T cell repertoire of the donor.
Quantitative Analysis of Polymyxin B Released from Polymyxin B‐Treated Dormant Spores of Bacillus subtilis and Relationship between Its Permeability and Inhibitory Effect on Outgrowth
Authors: Fujita‐Ichikawa Yuko , Tochikubo Kunio
Published: November 2013

Polymyxin B, one of the cyclic polypeptide antibiotics, binds to the coat of Bacillus subtilis dormant spores and inhibits them from growing after germination. When about 2.8 × 108 cells/ml of polymyxin B‐treated dormant spores were incubated in heart infusion broth, 3.6 μg/ml of polymyxin B were released into the liquid medium during germination. Incubation of the same concentration of polymyxin B‐treated ones in 100 mM CaCl2 solution released 4.0 μg/ml of the antibiotic. The effect of various concentrations of polymyxin B on germination, outgrowth and vegetative growth of the dormant spores was investigated; the results showed that concentrations of 4.0 μg/ml and higher of the antibiotic inhibited their outgrowth and vegetative growth after germination. Young vegetative cells were less sensitive to the antibiotic than germinated spores. In addition to these results, immunoelectron microscopy with colloidal gold particles indicated that polymyxin B permeated into the core of the germinated spores and inhibited them from outgrowing.
Antigen‐Specific Proliferative Response of Peritoneal Exudate Lymphocytes Primed with Antigen and Bacterial Lipopolysaccharide: The Roles of Ia+ Accessory Cells and IL‐2
Authors: Nitta Toshimasa , Imai Hiroaki , Ogasawara Yuko , Hirayama Nobukuni , Nakano Masayasu
Published: November 2013

In vitro antigen‐specific proliferation was investigated in a lymphocyte population that had been taken from the peritoneal exudate cells (PEC) of C3H/HeN mice (Iak) primed in vivo with both bacterial lipopolysaccharide (LPS) and horse red blood cells (HRBC) and had been purified by passage through a nylon fiber column (Nfc). The proliferative response of the Nfc‐passed lymphocytes primed with HRBC and LPS [T(HRBC + LPS) cells] depended on the dose of antigen in the cultures, and the response was higher than that of cells prepared from mice primed with HRBC alone [T(HRBC) cells]. No response was seen in the cells prepared from the LPS‐primed mice [T(LPS) cells] or normal mice [T(N) cells]. The response of the T(HRBC) cells was abolished by previous treatment of the cells with anti‐Iak antibody and complement (C), whereas the response of the T(HRBC + LPS) cells was retained after the same treatment, indicating that the Ia– T(HRBC + LPS) cells can proliferate in response to antigen in spite of Ia+ accessory cell‐depletion. Supernatants from the cultures of Ia– T(HRBC + LPS) cells in the presence of HRBC showed abundant IL‐2 activity, while those of Ia– T(HRBC) cells did not. The IL‐2 should be produced by the L3T4 cell population in T(HRBC + LPS) cells in response to antigen, since the previous treatment of the cells with anti‐L3T4 antibody and C abrogated the production. On the other hand, the Ia– T(HRBC + LPS) cells as well as the Ia– T(LPS) cells could respond to IL‐2 dose‐dependently when recombinant IL‐2 was added into the cultures, but the response of Ia– T(HRBC) cells to IL‐2 was very weak. The cell population responding to IL‐2 in the T(HRBC + LPS) cells as well as T(LPS) cells must be AsGM1‐positive or natural killer (NK) cells, since previous treatment of the cells with anti‐AsGM1 antibody and C abrogates the response. Together these results suggest that L3T4 lymphocytes capable of producing IL‐2 in response to HRBC antigen without Ia+ accessory cells are generated in the PEC of the mice after priming with LPS and antigen together, and the IL‐2 produced by the L3T4 lymphocytes induces the proliferation of the LPS‐primed AsGM1+ cells.
Characterization of Cell Growth‐Inhibitory Factor in Inflammatory Peritoneal Exudate Cells of Rats
Authors: Yui Satoru , Yang De , Mikami Masaaki , Yamazaki Masatoshi
Published: November 2013

We characterized the nature and reaction mode of the cell growth‐inhibitory factor (here designated CGIF) from rat peritoneal exudate cells (PEC). The soluble fraction separated from the lysate of Enterococcus faecalis‐induced 24 hr PEC completely inhibited Con A‐induced thymocyte mitogenesis. Gel filtration chromatography showed that CGIF has a molecular weight of approximately 23–25 kDa. Isoelectric focusing with Rotofor indicates that the factor has an isoelectronic point of 5.8–6.4. CGIF was inactivated by treatment at 70 C, for 30 min or by tryptic digestion, but the activity was not destroyed by the reduction with dithiothreitol. As well as thymocyte proliferation, CGIF completely suppressed 3H‐thymidine incorporation of splenocytes which were stimulated by either Con A or LPS, suggesting the factor is effective on both T and B cells. The acting point of the inhibitor appeared to be a later stage of the lymphocyte activation sequence, since it was still effective when added 28.5 hr after the addition of Con A. CGIF also reduced the viability of these cells when added with mitogens such as Con A or LPS. CGIF thus appears to be distinct from interleukin‐1 receptor antagonist or transforming growth factor‐β.
Diagnosis of Genital Herpes by Polymerase Chain Reaction Amplification
Authors: Goto Tetsuya , Yamaguchi Yuki , Hashido Madoka , Yoshikawa Hiroyuki , Kawana Takashi
Published: November 2013

A new polymerase chain reaction (PCR) method employing type‐specific primers and probes was applied to 114 clinical specimens obtained from 58 female patients with genital lesions or who had a history of genital herpes. Ten and 15 specimens, respectively, were positive for herpes simplex virus (HSV)‐1 and HSV‐2 by cell culture. All of 10 culture‐confirmed HSV‐1 cases and 11 of 15 (73%) culture‐confirmed HSV‐2 cases were identified by PCR. Although there were several cases with discrepancy between cell culture and PCR for HSV‐2, the results suggest that this PCR procedure could be applied to clinical specimens from the female genital tract.
Efficient Adsorption of Lysostaphin on Bacterial Cells of Lysostaphin‐Resistant Staphylococcus aureus Mutant
Authors: Sakurada Junji , Murai Miyo , Zhijun Li , Usui Akemi , Seki Keiko , Kobayashi Kiyoshi , Sumi Yoshiko , Jitsukawa Hiroko , Masuda Shogo
Published: November 2013

A simple and efficient method for the purification of staphylolytic endopeptidase (lysostaphin) contained in culture supernatant of Staphylococcus simulans biovar staphylolyticus strain by adsorption of the enzyme on bacterial cells of lysostaphin‐resistant S. aureus mutant was successfully devised. Lysostaphin was sufficiently adsorbed on the heat‐killed mutant cells derived from S. aureus Cowan I and efficiently eluted by 3 M KSCN. Enzyme preparation obtained by a single procedure of the affinity purification was pure enough for practical use. The yield of the enzyme was 25 mg from 1 liter culture and recovery rate was 64%.
Construction of a Slime Negative Transposon Mutant in Staphylococcus epidermidis Using the Enterococcus faecalis Transposon Tn917
Authors: Grüter Ludwig , Feucht Hubert , Mempel Martin , Laufs Rainer
Published: November 2013

The slime‐producing Staphylococcus epidermidis strain sensu strictu CNS23 was transformed by protoplast transformation with the plasmid pTV1 which carries transposon Tn917. Using this transposon mutagenesis system we obtained the Tn917‐inserted mutant CT512, which has lost the ability to produce slime. A single insertion of the trasposon Tn917 into the chromosome of CT512 could be detected by Southern hybridization. This mutant showed a significantly higher stability concerning its slime‐negative phenotype compared with spontaneous slime‐negative mutants of S. epidermidis strain CNS23. In slime‐ELISA no slime‐associated antigen could be detected in extracts of the transposon mutant. Compared to slime‐positive S. epidermidis strains, CT512 lacked in accumulative growth in microtiter tube test.
Suppression of ADCC by Immune Complexes Formed in Vitro in Mycobacterium leprae‐Infected Mice
Authors: Vaishnavi Chetana , Ganguly Nirmal Kumar , Kaur Surrinder , Kumar Bhushan
Published: November 2013

Antibody‐dependent cellular cytotoxicity (ADCC) was assessed in mice infected experimentally with Mycobacterium leprae and injected simultaneously with in vitro‐formed immune complexes (IC). Significant decrease in the ADCC function was observed in animals given IC at zero day (OdIC) and 3 months (3mIC) post inoculation with M. leprae, when ADCC activity was assessed at 3, 6 and 9 months period. From the data obtained we believe that ADCC is suppressed by IC formed in vitro.
Inhibitory Effect of 8‐(N, N‐Diethylamino)octyl‐3, 4, 5‐Trimethoxybenzoate Hydrochloride (TMB‐8) on Germination of Bacillus cereus T Spores
Authors: Shibata Hirofumi , Mimura Akiko , Mitani Hirotoshi , Tani Isamu , Hashimoto Tadayo
Published: November 2013

Effect of 8‐(N,N‐diethylamino)octyl‐3, 4, 5 ‐ trimethoxybenzoate hydrochloride (TMB‐8), a calcium antagonist, on germination of Bacillus cereus T spores induced by L‐alanine and inosine was investigated. TMB‐8 had no effect on the germination of heat‐activated spores, whereas it inhibited that of nonactivated spores. The TMB‐8 inhibitory effect was antagonized competitively by inosine, but not by L‐alanine. Addition of Ca2+ reversed the inhibitory effect of TMB‐8 in a dose‐related fashion. Based on the results, a role of inosine and a site(s) for inhibitory action of TMB‐8 in the process leading to the germination of nonactivated spores were discussed.
Protective Activity of Antisera against Isolates of Borrelia burgdorferi from Various Geographical Origins
Authors: Masuzawa Toshiyuki , Okada Yutaka , Yanagihara Yasutake
Published: November 2013

Antisera from rabbits immunized with two Japanese strains of Borrelia burgdorferi, HP3 an isolate from Ixodes persulcatus and HO14 an isolate from I. ovatus, or the European strain P/Bi isolated from human cerebrospinal fluid (CSF) did not passively protect hamsters from challenge with the infectious strain 297, a North American isolate from patient CSF. Antisera to strains 297 and B31, a North American isolate from I. dammini, however, provided protective effect to challenge with strain 297. Immune mice sera in the presence of homologous B. burgdorferi antigen induced the production of oxygen intermediates from mouse peritoneal exudate cells. Heterologous B. burgdorferi antigen had no effect. These results suggest that antigenic properties of Japanese strains are different from those of North American and European isolates.
Identification of a Human Cancer Related Organ‐Specific Neoantigen
Authors: Ilantzis Christian , Thomson David M.P. , Michaelidou Alice , Benchimol Sarita , Stanners Clifford P.
Published: November 2013

Human cancers express organ‐specific neoantigens (OSNs) which elicit specific cellular immune responses in the cancer patient, as demonstrated by leukocyte adherence inhibition (LAI), an in vitro immune response assay. A purified protein of MW 40,000 (p40) exhibiting OSN (colon specific) activity was cleaved into specific peptide fragments and their partial amino acid sequences determined. This information was used in the polymerase chain reaction (PCR) to obtain a 992 bp cDNA clone (PCR‐992) from a human colon adenocarcinoma cell line (LS‐180). By comparison of the predicted amino acid sequence of PCR‐992 with the known sequence of p40 peptides, PCR‐992 was shown to correspond to almost the entire coding region of p40. Nucleotide sequence analysis suggested that the protein was mycoplasmal in origin due to its high A+T content (76%) and the presence of five in frame TGA termination codons; at least two of the latter are actually read as tryptophan, a known feature of mycoplasma translation. We have confirmed this origin by direct isolation of a contaminating mycoplasma species from the LS‐180 cell line and demonstration that it could be hybridized with the PCR‐992 probe. Northern and PCR analysis of RNA preparations from the contaminated LS‐180 cell line showed that p40 was part of the high affinity transport system operon of Mycoplasma hyorhinis (Dudler et al, EMBO J., 7: 3963‐3970, 1988). Total protein lysates of Mycoplasma hyorhinis cultivated without animal cells could elicit positive LAI responses when incubated with cancer patient leukocytes but not with normal patient leukocytes. The organ‐specific nature of the response was, however, not observed indicating that host cell‐mycoplasmal interactions may play a role in determining the organ‐specific nature of p40 seen with the LAI. The significance of these findings will be discussed in the context of previous thinking regarding the origin of OSNs.
Collagen‐Induced Arthritis Suppressed with Monoclonal Anti‐Idiotypic Antibody
Authors: Tarutani Susumu
Published: November 2013

In foregoing work, we identified at least 5 distinct epitopes on human type II collagen (CII), using 8 murine monoclonal antibodies (mAb) against human CII, and suggested that a species‐nonspecific epitope on CII recognized by anti‐CII mAb termed 1‐5 is an arthritogenic epitope. We also found that antibody response against a selected epitope of human CII could be induced by immunization with rabbit anti‐idiotypic (Id) antibody against anti‐CII mAb. The author developed and characterized monoclonal anti‐Id antibodies against 1‐5 mAb recognizing a putative arthritogenic epitope. The author also investigated whether the anti‐Id mAb could regulate antibody response directed against a selected epitope recognized by 1‐5 mAb, and the induction of collagen‐induced arthritis in DBA/1J mice. DBA/1J mice intravenously preinjected with anti‐Id mAb to 1‐5, did not produce anti‐CII antibody expressing 1‐5 Id upon immunization with human CII. Furthermore, as the development of collagen‐induced arthritis (CIA) in DBA/1J mice pretreated with anti‐Id mAb to 1‐5 was significantly suppressed, anti‐Id mAb will be a useful tool for studying the regulation of antibody response to a selected epitope. This study lends support to our hypothesis that the 1‐5 epitope is an arthritogenic epitope.
Lipopolysaccharide Isolated from a New O‐Antigenic Form (O13) of Vibrio parahaemolyticus
Authors: Hisatsune Kazuhito , Iguchi Takehiro , Haishima Yuji , Tamura Norihiko , Kondo Seiichi
Published: November 2013

The chemical properties of a lipopolysaccharide (LPS) isolated from a new O‐antigenic form (O13) of Vibrio parahaemolyticus were investigated. The LPS contained glucose, galactose, L‐glycero‐D‐manno‐heptose and glucosamine. 2‐Keto‐3‐deoxy‐octonate (KDO) was not detected in the LPS by the periodate‐thiobarbituric acid test (Weissbach's reaction) under conventional hydrolysis conditions. Instead, phosphorylated KDO (X1 and X2) was found in its strong‐acid hydrolysate. This sugar composition was identical to that of V. parahaemolyticus O3, O5 and O11 LPS, indicating that, based on the sugar composition, O13 LPS belongs to Chemotype III to which O3, O5 and O11 belong. In addition, structural study demonstrated the presence of KDO 4‐phosphate in its inner‐core region.
Identification of Heat‐Stable Enterotoxin‐Producing Strains of Yersinia enterocolitica and Vibrio cholerae Non‐O1 by a Monoclonal Antibody‐Based Enzyme‐Linked Immunosorbent Assay
Authors: Nair G. Balakrish , Bhattacharya Sujit K. , Takeda Tae
Published: November 2013

Using a mouse monoclonal antibody (MAb) 2F raised against Vibrio cholerae non‐O1 heat‐stable enterotoxin (NAG‐ST) which also recognizes a shared epitope of Yersinia enterocolitica heat‐stable enterotoxin (Y‐ST), a competitive enzyme‐linked immunosorbent assay (ELISA) was developed for independent detection of NAG‐ST and Y‐ST. There was good concordance between the Y‐ST ELISA and the suckling mouse assay (SMA) for detection of Y‐ST from test strains of Y. enterocolitica, and the Y‐ST ELISA can effectively replace the SMA for routine detection of Y‐ST. On the contrary, evaluation of the NAG‐ST ELISA and the SMA using 139 strains of V. cholerae non‐O1 showed discordant results and this was attributed to the presence of the suckling mice active factor(s) such as El Tor hemolysin and to the production of low amounts of NAG‐ST. Concentration of culture supernatants of V. cholerae non‐O1 followed by heating at 100 C was essential to obtain reproducible results by both the NAG‐ST ELISA and the SMA. The ELISA developed in this study can be used for the identification of biologically active strains. While recently genetic methods such as polymerase chain reaction became available and were very reliable and simple techniques, the ELISA in this study has an advantage in detecting biologically toxic gene products of the strains. The genetic methods cannot differentiate silent STa genes which we often encounter in the case of Y. enterocolitica.
Functional Properties of Pro Region of Escherichia coli Heat‐Stable Enterotoxin
Authors: Yamanaka Hiroyasu , Fuke Yasunori , Hitotsubashi Shunji , Fujii Yoshio , Okamoto Keinosuke
Published: November 2013

Escherichia coli heat‐stable enterotoxin Ip (STp) is synthesized as the 72‐amino‐acid residue precursor consisting of three regions: pre region (amino acid residues 1 to 19), pro region (amino acid residues 20 to 54), and mature ST (mST) region (amino acid residues 55 to 72). We examined the role of the pro sequence of STp in enterotoxigenicity of a strain by deleting the gene fragment encoding amino acids 22 to 57. This deletion caused a remarkable reduction of its enterotoxic activity of culture supernatant. In order to analyze the sequence responsible for the function of the pro region, two additional deletion mutants were made. The deletion of the sequence covering amino acids 29 to 38, which is conserved in all sequences of ST reported, brought about a significant reduction of enterotoxic activity but the deletion of the non‐conserved sequence (amino acids 40 to 53) did not. This result shows that conserved sequence is mainly responsible for the function. Subsequently, to examine the mechanism of action of the pro region, plasmids carrying DNA sequences of hybrid proteins consisting of pre‐pro‐nuclease, pre‐mST‐nuclease, pre‐pro‐mST‐nuclease and pre‐pro‐nuclease‐mST were constructed. Amino acid sequence determination and SDS‐polyacrylamide gel analysis revealed that these fusion proteins were cleaved between pre sequence and pro sequence during secretion and the cleaved fusion proteins were accumulated in periplasmic space. But the amount of hybrid protein accumulated in the periplasmic space varied among the strains. That is, the amount of the pre‐pro‐nuclease gene product that accumulated in the periplasmic space was the highest of all fusion gene products. These results indicate that the existence of the mST region strongly interferes with the translocation of the gene product into the periplasmic space and that the pro region functions to guide the mST region into the periplasmic space.
Restriction Fragment Length Polymorphism Analysis of Epidemiologically Related Mycobacterium tuberculosis Isolates
Authors: Takahashi Mitsuyoshi , Kazumi Yuko , Fukasawa Yutaka , Hirano Kazue , Mori Toru , Dale Jeremy W. , Abe Chiyoji
Published: November 2013

Restriction fragment length polymorphism (RFLP) analysis of a large number of Japanese isolates of Mycobacterium tuberculosis, containing isolates from small outbreaks of M. tuberculosis infection, and clinical isolates of M. bovis BCG, was carried out using a DNA probe derived from the insertion sequence IS986. Clinical isolates of M. tuberculosis had a high degree of RFLP. The occurrences of the IS element varied from 1 to 19, the majority of isolates having 8 to 15 copies. Very similar fingerprints, however, were seen among strains isolated in the Kanto district. In particular, 3 strains were of the same pattern with or without an additional band. Similarity of the banding patterns of strains islated in the same district was observed in other areas. Six groups of strains, each group arising from a suspected common source of infection, were analyzed. Of these, 5 showed identical fingerprints within each group, but one showed different fingerprints. RFLP patterns of three strains isolated from individuals with lymphadenitis developed about two months after BCG vaccination, and one strain isolated from a bladder cancer patient with BCG instillation therapy were identical to those of BCG‐Tokyo which had been used for the vaccination and therapy. These results confirm that RFLP analysis using IS986 is a suitable tool for epidemiology of tuberculosis.
In Vitro Modification of Human Immunodeficiency Virus Type 1 (HIV‐1) Infectivity by the U937 Cells
Authors: El‐Farrash Mohamed A. , Masuda Takao , Kuroda Marcelo J. , Harada Shinji
Published: November 2013

The effect of host cell factors on infectivity of human immunodeficiency virus type 1 (HIV‐1) was studied by infecting a monoblastoid cell line (U937) or a T‐cell line (MOLT‐4) with a highly infective single clone of HIV‐1 and comparing the infectivity of the produced viruses to different cell lines. Chronically infected U937 cells consistently produced viruses with minimal infectivity. This phenotypic change was host‐dependent as the back‐passage of the U937‐produced low infective viruses into MOLT‐4 cells resulted in regaining their original high infectivity. Southern and Northern blot analyses of the HIV‐1 grown in U937 cells did not reveal any genomic difference between it and the virus grown it MOLT‐4 cells. The radioimmunoprecipitation analysis of viral proteins showed that the HIV‐1‐infected U937 cells had a different pattern of envelope glycoproteins and core proteins, which well correlated with the low infectivity of the produced viruses. This experimental system using MOLT‐4 and U937 cell lines would be useful to further explore host cell factor(s) which play an important role in the regulation of HIV‐1 infectivity.
Induction of Interleukin 2‐Responsiveness in Thymocytes of the Transgenic Mice Carrying lck‐Transgene
Authors: Moroi Yoichi , Koga Yasuhiro , Nakamura Kazuhiko , Ohtsu Masumi , Kimura Genki , Nomoto Kikuo
Published: November 2013

The role of lck gene in T cell proliferation and differentiation was investigated with transgenic mice carrying human lck cDNA whose expression was regulated by the promoter of mouse H‐2Kb and the enhancer element of mouse IgH. RNase protection assay revealed that the lck transgene was expressed in the thymus and spleen, whereas immunoblot analysis demonstrated that amounts of p56lck in freshly isolated lymphoid organs were almost equal between transgenic mice and negative littermates. Cell‐surface marker analyses of the thymocytes and peripheral lymphocytes revealed no remarkable difference between both groups. Notable finding is that the thymocytes from transgenic mice showed a significant proliferative response to the stimulation with IL‐2, but not the thymocytes from negative littermates. Further analysis revealed that CD4+8– single positive thymocytes proliferated in response to IL‐2. While surface expression levels of IL‐2Rα and IL‐2Rβ of these CD4+8– thymocytes from transgenic and control mice were almost equal before stimulation with IL‐2, the expression of IL‐2Rβ was induced only in transgenic thymocytes after stimulation with IL‐2. Immunoblot analysis demonstrated that the expression of p56lck of transgenic thymocytes was not down‐reguated at 4 hr after stimulaion with IL‐2, whereas p56lck of control ones were not detectable any more at 4 hr after stimulation with IL‐2. Moreover, in vitro kinase assay substantiated such unchanged expression of p56lck in the thymocytes from transgenic mice: the kinase activities of p56lck did not decrease in thymocytes from transgenic mice after stimulation with IL‐2, while kinase activities of control ones were significantly down‐regulated by stimulation of IL‐2. These results suggested that a significant proliferative response found in the thymocytes from lck‐transgenic mice after the stimulation with IL‐2 was caused by a constitutive expression of p56lck in these thymocytes even after the stimulation. Our findings, therefore, support a possibility that p56lck may play a role in the IL‐2R‐mediated signaling system in CD4+8– thymocytes.
Similarity in Nucleotide Sequence of the Gene Encoding Nontoxic Component of Botulinum Toxin Produced by Toxigenic Clostridium butyricum Strain BL6340 and Clostridium botulinum Type E Strain Mashike
Authors: Fujii Nobuhiro , Kimura Kouichi , Yokosawa Noriko , Oguma Keiji , Yashiki Teruo , Takeshi Kouichi , Ohyama Touru , Isogai Emiko , Isogai Hiroshi
Published: November 2013

The complete nucleotide and deduced amino acid sequence of the nontoxic component of botulinum type E progenitor toxin is determined in recombinant plasmid pU9BUH containing about 6.0 kb HindIII fragment obtained from chromosomal DNA of Clostridium butyricum strain BL6340. The open reading frame (ORF) of this nontoxic component gene is composed of 3,486 nucleotide bases (1,162 amino acid residues). The molecular weight calculated from deduced amino acid residues is estimated 13,6810.1. The present study revealed that 33 nucleotide bases of 3,486 are different in the nontoxic component gene between C.butyricum strain BL6340 and C. botulinum type E strain Mashike. This corresponds to the difference of 17 amino acid residues in these nontoxic component.
Studies on Hemolytic Action of a Hemolysin Produced by Vibrio mimicus
Authors: Shinoda Sumio , Ishida Kazuhiko , Oh Eun‐Gyoung , Sasahara Kazuhiro , Miyoshi Shin‐Ichi , Chowdhury Mohammad Afzalur Rahim , Yasuda Tatsuji
Published: November 2013

Some properties and mechanism of action of a hemolysin (VMH) produced by an enteropathogenic Vibrio mimicus strain was examined. VMH was heat‐labile and inhibited by addition of divalent cations, including Ca2+, Mg2+ and Mn2+. The hemolysis by VMH was inhibited by incubating with gangliosides, suggesting that the ganglioside was the binding site on the erythrocyte membrane for VMH. Existence of a galactose moiety on reducing end of the ganglioside molecule and a sialic acid on the galactose moiety was suggested to be important for the binding of VMH molecule. Colloid osmotic manner of the hemolysis by VMH was demonstrated.
Identification and Characterization of Heat‐Stable Enterotoxin II‐Producing Escherichia coli from Patients with Diarrhea
Authors: Okamoto Keinosuke , Fujii Yoshio , Akashi Noriko , Hitotsubashi Shunji , Kurazono Hisao , Karasawa Tadahiro , Takeda Yoshifumi
Published: November 2013

Stock strains of Eschericia coli isolated from patients with traveller's diarrhea were examined for production of heat‐stable enterotoxin II (STII). Of 400 strains examined, 3 were found to produce STII. The nucleotide sequence of the STII gene of these human strains was shown to be identical to that of porcine strains. Cultured cells of these strains induced fluid accumulation in ligated mouse intestinal loops and the activity was neutralized by anti‐STII antiserum. These results suggest that STII‐produciing enterotoxigenic E. coli can cause human diarrhea.
Establishment of a Feline T‐Lymphoblastoid Cell Line Susceptible to Feline Immunodeficiency Viruses
Authors: Fujimoto Satoshi , Masuda Mahito , Ono Etsuro , Kida Hiroshi , Shimizu Yukio
Published: November 2013

A feline T‐lymphoblastoid cell line susceptible to feline immunodeficiency viruses (FIV) was established and designated as Yu‐1 cells. Yu‐1 cells continued to grow over one year with more than 100 successive passages in the presence of human recombinant interleukin‐2. Surface antigens of Yu‐1 cells were feline Pan‐T+, CD4+, and CD8. Susceptibility of Yu‐1 cells to FIV strains were higher than that of the primary culture of the feline peripheral blood mononuclear cells, indicating that this cell line should be useful for isolation, propagation, and neutralization test of FIV.
Effect of Clostridium perfringens Epsilon Toxin on Rat Isolated Aorta
Authors: Nagahama Masahiro , Iida Hiroshi , Sakurai Jun
Published: November 2013

The effect of Clostridium perfringens epsilon toxin on rat isolated aorta was investigated. The toxin caused contraction of the isolated aorta in a dose‐dependent manner. The toxin induced no contraction of the isolated aorta in low‐Na medium and of the tissue stored at 4 C for 7 days. However, tetrodotoxin (TTX) had no effect on the toxin‐induced contraction. The toxin‐induced contraction was significantly inhibited by phentolamine and prazosine, but did not by atropine, mecamylamine, chlorpheniramine and methysergide. These data suggest that the toxin‐caused contraction is mediated through nervous system in rat isolated aorta.
The Nucleotide Sequence and Genome Structure of Mung Bean Yellow Mosaic Geminivirus
Authors: Morinaga Tsutou , Ikegami Masato , Miura Kin‐Ichirou
Published: November 2013

Complete nucleotide sequences of the infectious cloned DNA components (DNA 1 and DNA 2) of mung bean yellow mosaic virus (MYMV) were determined. MYMV DNA 1 and DNA 2 consists of 2,723 and 2,675 nucleotides respectively. DNA 1 and DNA 2 have little sequence similarity except for a region of approximately 200 bases which is almost identical in the two molecules. Analysis of open reading frames revealed nine potential coding regions for proteins of mol. wt. > 10,000, six in DNA 1 and three in DNA 2. The nucleotide sequence of MYMV DNA was compared with that of bean golden mosaic virus (BGMV), tomato golden mosaic virus (TGMV) and African cassava mosaic virus (ACMV). The 200‐base region common to the two DNAs of each virus had little sequence similarity, except for a highly conserved 33‐36 base sequence potentially capable of forming a stable hairpin structure. The potential coding regions in the MYMV DNAs had counterparts in the BGMV, TGMV and ACMV, suggesting an overall similarity in genome organization, except for absence of 1L3 in MYMV DNA 1. The most highly conserved ORFs, MYMV 1R1, BGMV 1R1, TGMV 1R1 and ACMV 1R1, are the putative genes for the coat proteins of MYMV, BGMV, TGMV and ACMV, respectively. MYMV 1L1 has also a high degree of sequence similarity with BGMV 1L1, TGMV 1L1 and ACMV 1L1.
Suppressive Activity of Splenic Macrophages Induced by Transparent and Opaque Colonial Variants of Mycobacterium avium Complex against Lymphoproliferative Response
Authors: Tomioka Haruaki , Saito Hajime
Published: November 2013

Splenic macrophages (Møs) induced by the two colonial variants of Mycobacterium avium complex, SmT variant (smooth, transparent, flat) and SmD variant (smooth, opaque, dome‐shaped), were studied for their suppressive activity against mitogenic response of splenic T cells to concanavalin A (ConA). The SmT variant is much more virulent than the SmD variant and causes more severe infection in host mice. Moreover, suppressed state of ConA mitogenic response of host spleen cells persisted much longer in SmT‐infected mice than in SmD‐infected ones. However, the suppressive activity of SmT‐induced Møs, induced around 2 weeks after infection, against lymphoproliferative response was significantly weaker than SmD‐induced Møs. In contrast, the former Møs showed somewhat higher active oxygen‐producing ability than the latter, as based on the chemiluminescence. These findings indicate that the immunosuppressive Møs transiently induced in the early phase of M. intracellulare infection hardly affect the host resistance to the organisms and, thus, play relatively minor role in the progression of the infection.