bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Pathogenic Constituents of Cryptococcus neoformans Strains
Authors: Kagaya Keiko , Yamada Toshihiko , Miyakawa Yozo , Fukazawa Yoshimura , Saito Shū
Published: November 2013

We examined seven strains, comprising five serotypes, of Cryptococcus neoformans to determine what constituents of the organisms are responsible for pathogenicity and virulence in BALB/c mice. C. neoformans strains were divided into three virulence classes by survival rates after intravenous inoculation of 1 × 105 or 1 × 107 viable cells, and virulence was found not to be correlated with serotype or capsular size. C. neoformans cells resisted phagocytosis in different degrees in the presence of normal serum. Sensitivity of the C. neoformans strains to singlet oxygen ranged from resistance to susceptibility. Histological examination revealed that a weakly encapsulated virulent strain induced inflammatory responses with granuloma formation in the liver, lung, and kidney in addition to formation of cystic foci in the brain. In contrast, although the heavily encapsulated virulent strain produced granulomatous lesions in the liver, this strain preferably produced mucinous cystic foci in the lung, kidney, and brain. Correlation between virulence, and biological, histopathological and physiological evidence suggests that C. neoformans strains are endowed with the implicated multiple pathogenic constituents in various degrees and proportions. The following are suggested as the most important pathogenic constituents: (i) a polysaccharide capsule responsible for resistance to phagocytosis and formation of cystic foci; (ii) a cell surface structure for responsible for resistance to intra‐ or extracellular killing and induction of the granulomatous lesion; (iii) a growth rate suitable for interacting with phagocytic elimination.
Studies of Latent Cytomegalovirus Infection: The Macrophage as a Virus‐Harboring Cell
Authors: Hayashi Kozaburo , Saze Koichi , Ughida Yukio
Published: November 2013

During chronic infection of mice with mouse cytomegalovirus (MCMV), the virus was isolated from various tissues by cocultivation with allogeneic mouse embryonic fibroblasts (MEF). Infectious virus was recovered from over 15% of the pancreases, salivary glands, kidneys, lacrimal glands, and spleens. When activated macrophages were obtained by intraperitoneal injection of peptone into mice infected 3 months earlier, they harbored MCMV.

Macrophages or lymphocytes were infected with MCMV in vitro and injected into normal mice intravenously. The peritoneal cavities of these mice were then stimulated by peptone injection 3 months after the transfer, and peritoneal or splenic macrophages and lymphocytes were cocultured with allogeneic MEF. MCMV was recovered from the peritoneal and splenic macrophages and not from the lymphocytes.
Experimental Infection of Young Broiler Chicks with Treponema hyodysenteriae
Authors: Adachi Yoshikazu , Sueyoshi Masuo , Miyagawa Eiichi , Minato Hajime , Shoya Shigemi
Published: November 2013

In young broiler chicks which were inoculated with 108 cells of Treponema hyodysenteriae within 24 hr after hatching, numerous treponemes were observed by scanning electron microscopy on the surface of the cecal mucosa 7 and 14 days after the inoculation. However, in the groups inoculated with 107 cells, treponemes were not observed on the cecal mucosa 14 days after the inoculation, and the isolation rate from the cecal contents was lower than that from cecal contents of chicks inoculated with 108 cells.

While the cecal mucosa of noninfected chicks had a smooth surface, that of the chicks infected with treponemes was generally roughened and the epithelium was eroded. Numerous treponemes were also observed within the eroded epithelium.
Immunogenicity and Protective Effect of Hemolysis Mutants of Mycoplasma pneumoniae
Authors: Yayoshi Masumi , Araake Minako , Hayatsu Eizo , Takezawa Toshiyuki , Yoshioka Morimasa
Published: November 2013

Two attenuated strains of Mycoplasma pneumoniae, P24‐S1 and P24‐S11, were tested for their ability as a live vaccine to confer on hamsters immune resistance against challenge infection with a virulent strain of M. pneumoniae, FH‐P24. Fifty percent protection was obtained by vaccination with the P24‐S1 strain administered once or twice. In contrast, only 10% of the animals were protected by the P24‐S11 vaccine even when it was given three times. Vaccination with the P24‐S1 strain resulted in higher humoral and cellular immune responses than the P24‐S11 did. These results suggest that the P24‐S1 strain has the primary qualities a vaccine which may be used for protection against human mycoplasmal infection.
Formation of a Hexagonal Lattice Structure by an R‐Form Lipopolysaccharide of Klebsiella: Comparative Study of Preparations Extracted by the Phenol‐Water and the Phenol‐Chloroform‐Petroleum Ether Methods
Authors: Kato Nobuo , Ohta Michio , Kido Nobuo , Ito Hideo , Naito Setsuko , Kuno Tsuneharu
Published: November 2013

An R‐form lipopolysaccharide (LPS) extracted from Klebsiella strain LEN‐111 (O3‐:K1‐) by the phenol‐chloroform‐petroleum ether method was compared with that extracted by the phenol‐water method in the ability to form a hexagonal assembly. The LPS which was extracted by the phenol‐water method and dialyzed against tap water to remove phenol showed ribbon‐like structures, and it formed a hexagonal lattice structure with a lattice constant of 14.5 ± 0.3 nm when it was precipitated by addition of two volumes of 10 mM MgCl2‐ethanol. The LPS which was extracted by the phenol‐chloroform‐petroleum ether method and lyophilized consisted of ribbon‐like structures and their fragments and it often formed small pieces of a hexagonal lattice, although the LPS before lyophilization did not form such a lattice. When the LPS extracted by the phenol‐chloroform‐petroleum ether method was precipitated by addition of two volumes of 10 mM MgCl2‐ethanol, it formed essentially the same hexagonal lattice structure as that formed by the LPS extracted by the phenol‐water method. From these results it is concluded that the ability of the LPS to form a hexagonal lattice structure does not depend upon the method of its extraction from bacterial cells.
Studies on the Antigenic Structure of Japanese Encephalitis Virus Using Monoclonal Antibodies
Authors: Kobayashi Yuzuru , Hasegawa Hitoshi , Yamauchi Tetsuzo
Published: November 2013

The antigenic relationships among 11 strains of Japanese encephalitis (JE) virus were analyzed by using monoclonal antibodies (NARMA) against the Nakayama‐RFVL strain in hemagglutination‐inhibition (HI) and neutralization (Nt) tests. Of the 14 JE virus‐specific HI antibodies, all except NARMA 5 showed Nt reactivity with the homologous strain. The HI and Nt titers of these antibodies were not parallel. The 14 antibodies included the following characteristic antibodies: NARMA 3 is a species‐specific antibody with HI and Nt reactivities against JE virus, NARMA 13 is a species‐specific HI antibody, NARMA 6 is a Nakayama strain‐specific antibody with HI and Nt reactivities, and NARMA 5 is a Nakayama strain‐specific HI antibody. The 11 strains of JE virus were divided into four major antigenic groups. However, slight antigenic differences were found among some strains of the same group. Furthermore, competitive binding assays were performed to determine the distribution of antigenic determinants by enzyme‐linked immunosorbent assay. The results suggest the existence of at least five HI sites on the JE virus virion, and indicate that the JE species‐specific HI site and the flavivirus genus‐specific HI site are topologically distinct.
Suppression of Phorbol Myristate Acetate‐Triggering of Macrophage H2O2 Release by Sarcoma 180 Originating Factor
Authors: Tomioka Haruaki , Saito Hajime
Published: November 2013

A high molecular weight proteinaceous factor in the cell extract of sarcoma 180 (S‐180) was found to inhibit phorbol myristate acetate (PMA)‐triggering of macrophage H2O2 release. This factor (S‐180 factor) was stable at 56 C for 1 hr and resistant to ultraviolet‐irradiation. The S‐180 factor inhibited the specific binding of PMA to macrophages and this was accompanied by a parallel reduction of PMA‐triggered H2O2 release. S‐180 factor preferentially depressed macrophage H2O2 release in response to phorbol diesters including PMA, 4β‐phorbol 12β,13α‐diacetate, 4β‐phorbol 12β,13α‐didecanoate, 4β‐phorbol 12β,13α‐dibenzoate, and 4‐o‐methyl‐PMA rather than the H2O2 release triggered by wheat germ aggulutinin or by phagocytosis of latex particles. The S‐180 factor failed to affect the PMA‐elicited macrophage cell spreading and macrophage phagocytic activity against latex beads with or without PMA‐mediated stimulation. A similar inhibitory factor was found in the extracts of some other murine tumor cells (Ehrlich carcinoma and thymic leukemia) and normal cells (liver, spleen, and peritoneal exudate cells).
Effect of Mutacin Administration on Streptococcus mutans‐Induced Dental Caries in Rats
Authors: Ooshima Takashi , Yasufuku Yoshiaki , Izumitani Akira , Sumi Norio , Iwanami Takefumi
Published: November 2013

A bacteriocin from serotype c Streptococcus mutans strain C3603 was examined for its inhibitory effect on experimental dental caries in rats infected with S. mutans MT8148R (serotype c). Significant reduction in the incidence of dental caries was found only when bacteriocin was incorporated both in the drinking water and in the diet at a high concentration. However, caries reduction was not as great as expected and the addition of bacteriocin to drinking water alone had no effect on the recovery of S. mutans, plaque deposition or caries incidence. The bacteriocin activity must have been reduced in the oral cavity of rats, and the reasons were examined. Bacteriocin‐resistant mutants were not detected and the bacteriocin was not inactivated by saliva.

Whereas the bacteriocin did not kill the S. mutans cells grown in a sucrose‐containing medium, it completely killed the cells grown in a sucrose‐free medium.
Solubilization and Partial Properties of Receptor Substance for Bacteriophage α2 Induced from Clostridium botulinum Type A 190L
Authors: Takumi Kenji , Takeoka Aya , Kinouchi Takemi , Kawata Tomio
Published: November 2013

Bacteriophage α2, one of the two inducible phages from Clostridium botulinum type A 190L, had a latent period of 55 min and an average burst size of 75 in C. botulinum type A Hall used as the host bacterium. The phage particles were adsorbed on the cell walls extracted with hot trichloroacetic acid (TCA‐walls). The receptor substance for the phage was solubilized from the TCA‐walls with Achromopeptidase and fractionated by gel filtration on Sephadex G‐150. The fraction having the highest level of receptor activity for the phage contained large amounts of muramic acid and glucosamine. Both authentic muramic acid and glucosamine significantly inactivated the phage, whereas glucose, galactose, L‐and D‐alanine, diaminopimeric acid, or D‐glutamic acid did not exhibit similar activity. There results strongly suggest that the receptor site for phage α2 is closely associated with glycan moieties of the cell wall peptidoglycan.
An Assay for the Receptor‐Destroying Activity of Influenza C Virus
Authors: Sugawara Kanetsu , Kitame Fumio , Homma Morio , Nakamura Kiyoto
Published: November 2013

We have developed a convenient method for assaying the receptor‐destroying enzyme (RDE) activity of influenza C virus. This method measures the ability of the RDE to destroy the hemagglutination‐inhibition activity of a potent inhibitor present in rat serum. Some physico‐chemical properties of the RDE of influenza C virus were investigated by using this method. The temperature optimum for maximal activity of this enzyme was found to be 45 C to 53 C. There was little difference in thermostability between the RDE and hemagglutinating activities of influenza C virus. When influenza C virions were treated with various concentrations of trypsin, the RDE activity decreased in parallel with the decrease in the amount of residual gp88 glycoprotein, suggesting association of RDE with this glycoprotein.
Determination of the T‐Cell Subset Producing γ‐Interferon in Tuberculous Pleural Effusion
Authors: Shimokata Kaoru , Kishimoto Hirotsugu , Takagi Eishi , Tsunekawa Hiroshi
Published: November 2013

The percentage of cells of different T‐cell subsets and their functions in tuberculous pleural effusion were investigated. The percentage of OKT4‐positive cells was 65 ± 2% (mean ± SEM, n=8) and that of OKT8‐positive cells was 19 ± 3% (n=8). Pleural T lymphocytes of patients with tuberculous pleurisy responded well to stimulation with purified protein derivative of tuberculin, and deoxyribonucleic acid synthesis was observed along with gamma interferon (IFN‐γ) production. When pleural T lymphocytes of patients with tuberculous pleurisy were treated with OKT4 monoclonal antibody and complement, a significant decrease in IFN‐γ production was observed in all eight patients (P<0.005), whereas no definite decrease in IFN‐γ production was found after treatment with OKT8 monoclonal antibody and complement. These results suggest that at least the OKT4+/OKT8− T‐cell subset is responsible for the antigen‐specific IFN‐γ production in pleural T lymphocytes of patients with tuberculous pleurisy.
Comparison of the Fecal Microflora in Rural Japanese and Urban Canadians
Authors: Benno Yoshimi , Suzuki Kunio , Suzuki Kunihiko , Narisawa Kimiko , Bruce W. Robert , Mitsuoka Tomotari
Published: November 2013

The fecal microflora of nine rural healthy Japanese and eight urban healthy Canadians was examined. The two populations ate typical Japanese and western diets, respectively. The numbers of eubacteria (P<0.01), bifidobacteria (P<0.05), bacilli (P<0.01), lactobacilli and veillonellae and the frequency of occurrence of bifidobacteria were higher in the Japanese than in the Canadians. Higher numbers of bacteroides and lecithinase‐negative Clostridia were found in the Canadians. Twenty‐three genera and over 75 species or biovars were isolated from the feces of Japanese and 18 genera and over 66 species or biovars from the Canadians. The numbers of Bacteroides vulgatus (P<0.05), Clostridium coccides (P<0.001), and C. tertium (P<0.05) and the incidence of B. uniformis (P<0.01), C. innocuum (P<0.05), and Bacillus spp. (P<0.01) were significantly lower in the Japanese than in the Canadians. In contrast, the numbers of Eubacterium aerofaciens (P<0.001), and the incidence of Bifidobacterium adolescentis biovar b (P<0.01) and Bacillus subtilis (P<0.01) were significantly higher in the Japanese than in the Canadians. These findings suggest that significant reductions in anaerobic grampositive bacilli and increased numbers of bacteroides and Clostridia in the feces were induced by the intake of a western diet.
Production and Characterization of Monoclonal Strain‐Specific Antibodies against Prototype Strains of Rickettsia tsutsugamushi
Authors: Murata Michisato , Yoshida Yoshiya , Osono Michiyo , Ohashi Norio , Oyanagi Miho , Urakami Hiroshi , Tamura Akira , Nogami Sadao , Tanaka Hiroshi , Kawamura Akiyoshi
Published: November 2013

We have developed 18 hybridoma cell lines which secrete murine monoclonal strain‐specific antibodies to prototype strains of Rickettsia tsutsugamushi: nine anti‐Gilliam, four anti‐Karp and five anti‐Kato antibodies. All the monoclonal antibodies reacted only with their homologous strains in direct and indirect immunofluorescence (IF), or indirect immunoperoxidase (IP) test. By IF and IP tests with the monoclonal antibodies, 22 strains of R. tsutsugamushi, which were newly isolated from mites, field rodents and patients with Tsutsugamushi disease (scrub typhus) in Japan, were all clearly identified as either Gilliam or Karp type. Analysis by polyacrylamide gel electrophoresis and immunoblotting techniques revealed that the monoclonal antibodies recognized primarily the polypeptides of an apparent molecular weight of 54 to 56 kilodaltons of the homologous rickettsial surface. The monoclonal antibodies produced in the present study should enhance the serotyping and further analytical investigation of the rickettsial antigens since they recognize the strain‐ or type‐specific polypeptides and do not show any cross‐reaction among strains.
Transformation by Extracellular DNA Produced by Pseudomonas aeruginosa
Authors: Muto Yumiko , Goto Sachiko
Published: November 2013

Most Pseudomonas aeruginosa strains are capable of producing extracellular DNA. Very closely linked chromosomal markers (leu+ and trp+) were co‐transferred to P. aeruginosa PAO1819 (leu9001, trp9008) by the extracellular DNA produced by P. aeruginosa strains IFO3445 and PAO1 at a frequency of 10‐7 to 10‐8. Treatment of the extracellular DNA with DNase, heating at 95 C or sonication completely destroyed its transforming ability. The R plasmid in the extracellular DNA produced by P. aeruginosa IFO3445 (RP4) or PAO2142 (RLb679) could be transferred to Escherichia coli ML4901 or P. aeruginosa PAO1819. The resultant transformants showed identical resistance patterns in the respective donors, and the sizes of the DNAs of RLb679 and RP4 isolated from the transformants were the same as those in the respective donors. These results demonstrate that the extracellular DNA contains both chromosomal DNA and plasmid DNA, and that it exhibits transforming ability. This implies that transformation by the extracellular DNA produced by P. aeruginosa may occur in nature and this seems to be of clinical importance in view of the spread of R plasmids among pathogens.
Purification and Characterization of Heat‐Labile Toxin from Bordetella bronchiseptica
Authors: Endoh Masahiko , Amitani Makoto , Nakase Yasukiyo
Published: November 2013

The heat‐labile toxin (HLT) of Bordetella bronchiseptica was purified successively from sonic extracts of phase I organisms grown in Stainer‐Scholte medium, by partition in hydrophobic interaction, sucrose density gradient centrifugation, gel filtration through Sepharose 4B and 6B, isoelectric precipitation and isoelectric focusing. The purified HLT was homogeneous by disc Polyacrylamide gel electrophoresis and the gel diffusion‐test, and free of detectable hemagglutinin and endotoxin activity. A 386‐fold purification over the crude extract was obtained at a yield of about 28%, and a minimum dose of 0.9 ng was dermonecrotizing with a lesion 5 mm in diameter in guinea pigs and induced splenoatrophy. The mouse LD50 was 200 ng (intraperitoneal) or 70 ng (intravenous). The HLT was found to be a simple protein with an isoelectric point of pi 6.9. It has a molecular weight of 102,000 estimated by Sepharose 6B gel filtration and was found to consist of two different types of polypeptide by SDS‐polyacrylamide gel electrophoresis, their molecular weights being 30,000 and 20,000. Amino acid analysis showed 15 common amino acid residues, and methionine, cysteine and tryptophan were undetectable. The HLT crystallized by methylpentanediol showed a block form. The HLT was inactivated at 56 C when heated for 10 min, and at above pH 9 and below pH 4.
Degranulation and Histamine Release from Murine Mast Cells Sensitized with Dengue Virus‐Immune Sera
Authors: Sanchez Luis Fernando , Hotta Hak , Hotta Susumu , Homma Morio
Published: November 2013

Mice were immunized with dengue type 2 virus (DEN 2) under a schedule favoring the production of IgE antibody. The antibody obtained could sensitize peritoneal resident mast cells both in vitro and in vivo so that the sensitized cells were degranulated and released histamine on challenge with the DEN 2 antigen. It was also demonstrated that the antibody was cytophilic and heat‐labile. The above observations suggest that the present experimental system can be used to detect anti‐DEN 2 IgE antibody in mice.
Evaluation of the Efficacy of Split‐Product Trivalent A(H1N1), A(H3N2), and B Influenza Vaccines
Authors: Ochiai Hiroshi , Shibata Mitsuo , Kamimura Katsura , Niwayama Seihachiro
Published: November 2013

The reactogenicity and immunogenicity of Tween‐ether split trivalent A(H1N1), A (H3N2), and B influenza vaccine in primary school children aged seven to 12 years, and the persistence of antibodies following two doses of vaccine were studied during 1980–1984. Adverse reactions were infrequent, and, even when reported, were chiefly local ones, mild in nature and of short duration. Most of the reactions were less frequent after the second dose than after the first dose. Most of the systemic reactions occurred during the intervaccination period with almost equal frequency, indicating that careful consideration is required to judge whether they were induced by vaccination or not. This vaccine had induced adequate hemagglutination inhibiting (HAI) antibody because the geometric mean titers (GMTs) of the vaccinees were two‐to eightfold higher than those of the nonvaccinees to any of the vaccine antigens following two doses of vaccine. In general, the responses to A(H3N2) virus were the best among the vaccine antigens through the four vaccination seasons, but there was a tendency to show a poorer response to the same type (or subtype) of virus antigen as the causative one during a protracted epidemic. The antibodies induced by either vaccination or natural infection were shown to persist for less than a year, supporting the recommendation for annual vaccination.
Experimental Pulmonary Cavity Formation by Mycobacterial Components and Synthetic Adjuvants
Authors: Yamamura Yoshihiro , Maeda Hideo , Ogawa Yasaka , Hashimoto Taikichi
Published: November 2013

An investigation was undertaken to determine the components of mycobacteria responsible for pulmonary cavity formation in tuberculosis. Rabbits received an intrapulmonary injection through the chest wall, of mycobacterial protein, II‐p, mixed with either mycobacterial lipids, synthetic adjuvants or Nocardia cell wall skeleton as adjuvant. Six weeks later, they were killed and the lung lesions were examined. Cavities and necrosis were produced by the injection of II‐p mixed with cord factor, Nocardia cell wall skeleton or N‐acetylmuramyl dipeptide conjugated with long‐chain branched fatty acids. Cavities were not produced by either the injection of II‐p together with phospholipid, N‐acetylmuramyl dipeptide (MDP), MDP‐derivatives having no long‐chain branched fatty acid, or by the injection of individual components of the mixtures. The results suggest that in tuberculosis a mycobacterial component with a long‐chain branched fatty acid such as mycolic acid plays an important role in pulmonary cavity formation by enhancing the antigenicity of mycobacterial protein and helping it induce cell‐mediated immunity at the site of the lesion. Passive transfer with sera from rabbits highly sensitized with tubercle bacilli failed to enhance cavity formation in the recipient animals.
Enhanced Interleukin 1 Production by Alveolar Macrophages and Increase in Ia‐Positive Lung Cells in Silica‐Exposed Rats
Authors: Oghiso Yoichi , Kubota Yoshihisa
Published: November 2013

Inhalation exposure to silica dust enhanced interleukin 1 (IL‐1) production by alveolar macrophages (AM), which is attributable to an increase in Ia‐positive lung cells. While the proportion of Ia‐positive cells in lavaged bronchoalveolar cells (BAC) was much lower (0–3%) in unexposed control rats, about a third of the rats that inhaled silica showed higher proportions (8.0–18.5%); these were designated “Ia‐high” exposed animals. The number of total cells, Ia‐positive cells and lymphocytes in BAC was significantly increased (P<0.05, P<0.001, and P<0.001, respectively) in these “Ia‐high” exposed animals, compared to the control animals. Adherent AM populations obtained from BAC preparations also contained significantly higher (P<0.001) proportions of Ia‐positive cells in the “Ia‐high” exposed animals. When these adherent AM cultures were stimulated with lipopolysaccharide, IL‐1 activity of the culture supernatants was enhanced and was significantly higher (P<0.001) in the “Ia‐high” exposed rats, compared to the control animals. These results indicate that silica‐exposure can induce populational changes in lung cells and also activation of AM associated with the increase in Ia‐positive cells.
Effects of Disodium Ethane‐1‐Hydroxy‐1,1‐Diphosphonate (EHDP) on Interleukin 1 Production by Macrophages
Authors: Aida Yoshitomi , Toda Yoshiko , Shimakoshi Yoshikazu , Yamada Kuniko , Aono Masao
Published: November 2013

The effects of disodium ethane‐1‐hydroxy‐1,1‐diphosphonate (EHDP) on the in vitro functions of guinea pig macrophages were studied. A high dose (1 mg/ml) of EHDP inhibited interleukin 1 (IL 1) production by oil‐induced peritoneal macrophages stimulated with muramyl dipeptide (MDP), lipopolysaccharide (LPS), phorbol myristic acetate (PMA), heat‐aggregated IgG2 or calcium ionophore A23187. On the other hand, low doses (<0.125 mg/ml) of EHDP augmented the MDP induced IL 1 production by macrophages. This biphasic effect was also observed when macrophages were exposed to EHDP at 37 C for 24 hr and then stimulated with IL 1 inducers. Superoxide anion generation induced by formyl peptide or PMA was not affected by preincubation of the macrophages with doses of EHDP up to 1 mg/ml. Adherence and spreading of macrophages was inhibited by EHDP in a dose dependent manner without affecting cell viability. These results demonstrated that EHDP acted on macrophages directly and modulated IL 1 production in vitro.
Structure of a Defective Provirus of Bovine Leukemia Virus
Authors: Ogawa Yasuki , Sagata Noriyuki , Tsuzuku‐Kawamura Junko , Koyama Hiroyuki , Onuma Misao , Izawa Hisao , Ikawa Yoji
Published: November 2013

Defective proviruses of bovine leukemia virus (BLV) in the genomes of infected cells were investigated by using Southern blotting hybridization analysis with various portions of a cloned BLV DNA as probes. When nine independent tumors of enzootic bovine leukosis with a single proviral copy per cell were examined, a single defective provirus of BLV was found in one tumor and also in a bovine B cell line derived from this tumor. Hybridization analysis of this defective provirus revealed that it underwent deletion between the pol and env genes and contained no major deletion in the other regions.
A Microplate Method for Isolation of Viruses from Infants and Children with Acute Respiratory Infections
Authors: Numazaki Yoshio , Oshima Takeko , Ohmi Akira , Tanaka Akira , Oizumi Yoshihumi , Komatsu Shigeo , Takagi Tokuro , Karahashi Minoru , Ishida Nakao
Published: November 2013

Between December 1984 and December 1986, a microplate technique was adopted for isolation of viruses from infants and children with acute respiratory infections. By using two kinds of tissue culture microplates, i.e., the HHVM plate, containing human embryonic fibroblast (HEF), HEp‐2, Vero and MDCK cells, and the MK plate which contains secondary monkey kidney cells, 1,080 field viruses were isolated from 1,061 (24.9%) out of 4,254 throat swabs. Of these 1,080 isolates, 1,003 (92.9%) were recovered in the HHVM plates and the remaining 77 (7.1%) in the MK plates. With the HHVM plate, influenza A and B viruses were cultivated in MDCK, RS virus in HEp‐2, parainfluenza and mumps viruses in Vero, adenoviruses in both HEF and HEp‐2, polioviruses in HEF, HEp‐2 and Vero, coxsackie B viruses in both HEp‐2 and Vero, rhino and echo viruses in HEF, herpes simplex virus in both HEF and HEp‐2, and cytomegalovirus in HEF, although MK were more sensitive than Vero to parainfluenza and coxsackie B viruses. There was no difference in the rate of isolation of viruses between the microplate and ordinary tube methods. Cross contamination in the microplates was negligible for routine work.
Distribution of the Antibody to Influenza C Virus in Dogs and Pigs in Yamagata Prefecture, Japan
Authors: Ohwada Kazuo , Kitame Fumio , Sugawara Kanetsu , Nishimura Hidekazu , Homma Morio , Nakamura Kiyoto
Published: November 2013

The distribution of the antibody to influenza C virus in the dogs and pigs in Yamagata prefecture, Japan was investigated by using three different serological methods: hemagglutination‐inhibition (HI), radioimmunoprecipitation (RIP), and immunoblotting. The antibody against influenza C virus glycoprotein (gp88) was detected in 5 out of 112 sera collected from mongrel dogs, three by RIP test and two by any of the three methods, suggesting that the virus can cause natural infection in dogs. Significant levels of HI activity were found in 58 out of 269 sera collected from domestic pigs, but none of them showed positive reaction in the more sensitive method, RIP, which suggests that the inhibitors against the hemagglutination by influenza C virus rather than the antibody to gp88 are responsible for the observed HI activity. It appears, therefore, that at least in the Yamagata area, pigs do not play significant roles in the spread of influenza C virus in humans.
Differential Expression of the Major Histocompatibility Antigen Complex (MHC) on a Series of Burkitt's Lymphoma Lines
Authors: Yokochi Takashi , Inoue Yoshiko , Iwata Hitoshi , Miyadai Toshiaki , Kimura Yoshinobu
Published: November 2013

We compared the expressions of class I and class II major histocompatibility antigen complex (MHC) on the surface of Jijoye and P3HR‐1 cells of Burkitt's lymphoma sublines. Jijoye cells had a large amount of class I and class II MHC antigens, whereas these antigens were less expressed on P3HR‐1 cells. On a subline of P3HR‐1 K cells the expression of class I antigen markedly diminished and class II antigen was undetectable. On the other hand, Jijoye, P3HR‐1, and P3HR‐1 K cell lines were confirmed to be Epstein‐Barr virus (EBV) nonproducer, low producer, and high producer, respectively. The chemical activation of EBV genome by treating P3HR‐1 cells with 12‐O‐tetradecanyl phorbol‐13 acetate (TPA) and n‐butyrate resulted in inhibition of the expression of class I and II antigens, while the addition of retinoic acid, an inhibitor of virus replication, blocked the decrease in the MHC antigen expression. These findings suggested that there might be an inverse correlation between the virus production and the expression of class I and II MHC antigens.
Purification and Characterization of Vibrio vulnificus Protease
Authors: Miyoshi Noriko , Shimizu Chie , Miyoshi Shin‐Ichi , Shinoda Sumio
Published: November 2013

A protease was purified from a strain of Vibrio vulnificus isolated from the blood of a septicemic human. The vibrio was cultured in bacto peptone‐yeast extract medium, and the protease was purified by a purification procedure including ultrafiltration of the culture supernatant with an Amicon YM 5 membrane, diethylaminoethyl‐Sephacel column chromatography, Sephacryl S‐200 column chromatography and fast protein liquid chromatography on Mono Q column. The protease preparation revealed homogeneity on polyacrylamide gel electrophoresis and about 30,000‐fold purification was achieved, with a yield of about 30%. The isoelectric point of the purified V. vulnificus protease was about 5.80 and its molecular weight was ca. 45,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH of the protease activity was 8.0. The V. vulnificus protease was inhibited by a metalloprotease inhibitor and zinc ion and/or ferrous ion were essential for its enzyme activity. No cysteine residue was detected in the V. vulnificus protease. The protease had caseinolytic, elastolytic and collagenolytic activities.
The Mode of Entry of Herpes Simplex Virus Type 1 into Vero Cells
Authors: Koyama A. Hajime , Uchida Takahiro
Published: November 2013

The mode of entry of herpes simplex virus type 1 (HSV‐1) into Vero cells was investigated quantitatively with biological techniques. The entry of virus occurred rapidly when the virus‐adsorbed cells were incubated at 37 C. The kinetics of virus entry was found to be similar to that of the process of uncoating, indicating that the uncoating of HSV‐1 occurs simultaneously with the entry of virus into the cell. Experiments with ammonium chloride revealed that acidity in endosomes is not necessary for the entry or uncoating of HSV‐1, in contrast with the cases of enveloped RNA viruses. In addition, endocytosis of the virus seems to be one of the processes of entry for HSV‐1. However, the kinetics of endocytosis showed that the cell‐bound virus is endocytosed gradually and suggested that the endocytosis of HSV‐1 does not lead the virus to an uncoating process. These results are most consistent with a mechanism of entry for HSV‐1 involving fusion of the viral envelope with the plasma membrane of the host cell.
Incidence and Some Characteristics of Fimbriae FY and 31A of Escherichia coli Isolates from Calves with Diarrhea in Japan
Authors: Shimizu Mikio , Sakano Tetsuya , Yamamoto Junya , Kitajima Katsuyoshi
Published: November 2013

Escherichia coli isolates from calves with diarrhea (1 day to 8 weeks old, 140 individuals) were surveyed for the three immunologically distinct fimbrial adhesins FY, 31A, and K99. Of a total of 1,370 strains isolated, 96 (7.0%), 34 (2.5%), 75 (5.5%), and 13 (0.9%) were identified as FY+, 31A+, FY+·31A+, and K99+, respectively. The K99+ strains also manifested heat‐stable enterotoxin production (ST+), while FY+, 31A+, and FY+·31A+ strains were ST‐. Expression of FY and 31A was repressed at lower temperatures or poor aeration. The FY+ and 31A+ E. coli showed mannose‐resistant hemagglutinating activity with bovine erythrocytes. Electron microscopy revealed that FY is a gently curled fimbria with a mean diameter of 4.2 nm, and 31A is a fimbria with a mean diameter of 5.1 nm. The molecular mass of protein subunits was found to be approximately 20 kilodaltons (Kd) and 19 Kd for FY and 31A, respectively. Lethal diarrhea of neonatal calves was induced by challenge with the combination of a K99+·ST+ E. coli strain and either a 31A+ E. coli strain or a 31A+ E. coli strain plus an FY+ E. coli strain under the experimental conditions in which lethal diarrhea was not induced by challenge with a K99+·ST+ E. coli strain alone.
Cross‐Reactive Antibodies to Mycoplasmas Found in Human Sera by the Enzyme‐Linked Immunosorbent Assay (ELISA)
Authors: Sasaki Tsuguo , Bonissol Christiane , Stoiljkovic Bob
Published: November 2013

Antibodies against Mycoplasma pneumoniae in patients' sera with M. pneumoniae infection were measured by the complement fixation (CF) test and enzyme‐linked immunosorbent assay (ELISA). Many patients' sera cross‐reacted with heterologous mycoplasmal ELISA antigens such as M. hominis, M. hyorhinis, M. orale, M. pulmonis and M. salivarium. The sera with high CF (CF≥40) titers gave significantly higher ELISA values to M. hyorhinis (P<0.001) and M. pulmonis (P<0.001), which are not parasitic for humans, than those with low CF (CF<20) titer. Human normal immunoglobulin G (human normal IgG) containing 98% or more IgG, prepared from pooled plasma of at least 500 normal human donors, showed ELISA reactions with all mycoplasmal strains used. The nonspecific adsorption of human normal IgG on the surface of plate wells and on medium components which might contaminate mycoplasmal ELISA antigens could be disregarded. These results suggest that cross‐reactive antibodies to mycoplasmas exist in human sera, and they affect the results of ELISA for serodiagnosis of M. pneumoniae infection.
Activation of the Human Complement Cascade by Bacterial Cell Walls, Peptidoglycans, Water‐Soluble Peptidoglycan Components, and Synthetic Muramylpeptides—Studies on Active Components and Structural Requirements
Authors: Kawasaki Akinori , Takada Haruhiko , Kotani Shozo , Inai Shinya , Nagaki Kazuyoshi , Matsumoto Misako , Yokogawa Kanae , Kawata Shigeo , Kusumoto Shoichi , Shiba Tetsuo
Published: November 2013

Cell walls isolated from 29 strains of 24 gram‐positive bacterial species, whose peptidoglycans belong to the group A type of Schleifer and Kandler's classification, with one exception (Arthrobacter sp.), were shown to activate the complement cascade in pooled fresh human serum mainly through the alternative pathway and partly through the classical one. The complement‐activating effect of cell walls (5 species) possessing group B type peptidoglycan, except those of Corynebacterium insidiosum, was weaker than that of the walls with group A type peptidoglycan. Preparations of peptidoglycan isolated from cell walls of Staphylococcus aureus, Streptococcus pyogenes, and Lactobacillus plantarum also activated the alternative pathway of the complement cascade, but less effectively than the respective parent cell walls. A water‐soluble “polymer” of peptidoglycan subunits (SEPS), which was prepared from Staphylococcus epidermidis peptidoglycans by treatment with a cross‐bridge degrading endopeptidase, retained most of the complement‐activating ability of the parent cell walls. A peptidoglycan “monomer,” SEPS‐M, which was obtained by hydrolysis of the glycan chain of SEPS with endo‐N‐acetylmuramidase to disaccharide units did not activate complement. In conformity with this finding, neither synthetic N‐acetylmuramyl‐l‐alanyl‐d‐isoglutamine (MDP) nor MDP‐l‐Lys‐d‐Ala activated the complement cascade. Among several lipophilic derivatives of MDP, 6‐O‐(3‐hydroxy‐3‐docosylhexacosanoyl)‐MDP‐l‐Lys‐d‐Ala (BH48‐MDP‐l‐Lys‐d‐Ala) and 6‐O‐(2‐tetradecylhexadecanoyl)‐MDP (B30‐MDP) were shown to activate complement through the alternative as well as the classical pathway and exclusively through the classical pathway, respectively. The finding that a d‐isoasparagine analog of B30‐MDP caused the same effect as the parent molecule strongly suggests that the activation of complement by B30‐MDP is different from that caused by cell wall peptidoglycans and a water‐soluble “polymer” of peptidoglycan subunits.
Isolation of Campylobacter pyloridis from Human Gastric Mucosa and Characterization of the Isolates
Authors: Itoh Takeshi , Yanagawa Yoshitoki , Shingaki Masao , Takahashi Masaki , Kai Akemi , Ohashi Makoto , Hamana Gennichi
Published: November 2013

Biopsy specimens of human gastric mucosa of patients with gastric complaints and subjected to endoscopic examination were cultured microaerobically, and Campylobacter pyloridis was detected in 46 out of 80 cases (57.5%). The organism was found in 13 out of 22 patients with gastritis, 11 out of 16 with gastric ulcer scar, 7 out of 16 with gastric ulcer, 3 out of 9 with gastric polyp, 4 out of 5 with gastric carcinoma, 2 out of 2 with esophagus carcinoma, and 6 out of 9 with other gastric diseases. The isolates were identified as C. pyloridis, demonstrating its characteristic features such as positive for oxidase and catalase, negative for reduction of nitrite and nitrate, positive for urease, no growth at 25 C, growth at 37 C, not tolerant to 1% glycine, and resistant to nalidixic acid. Positive alkaline phosphatase activity was considered as an additional feature characteristic for the strains of C. pyloridis. The major cellular fatty acids were tetradecanoic acid and 19‐carbon‐cyclopropane acid. This pattern is unique among Campylobacter species. The survival of the organism for a longer period than 60 min at pH 2.5 indicates its significant resistance to acidic environment.
Effect of Aculeacin A on Reverting Protoplasts of Candida albicans
Authors: Yamaguchi Hideyo , Hiratani Tamio , Baba Misuzu , Osumi Masako
Published: November 2013

Protoplasts of Candida albicans were prepared by digestion with Zymolyase and the effect of aculeacin A, a wall‐active antibiotic, on the synthesis of microfibrillar structures by the protoplasts incubated for 5 hr in an osmotically stabilized medium was studied using several electron microscopical techniques. Chemical analyses of the protoplasts before and after reversion with or without the antibiotic were also performed. Aculeacin A not only inhibited synthesis of microfibrils mainly composed of alkali‐insoluble β‐glucan, but also their assembly to form thicker bundles. The antibiotic appeared to have no effect on other wall components constituting the surface structure of reverting protoplasts. These data confirmed our previous postulation that aculeacin A is a specific and potent inhibitor of β‐glucan synthesis as well as biogenesis of cell walls including β‐glucans in susceptible yeasts.
Chemical Properties of Lipopolysaccharide‐Like Substance (LLS) Extracted from Leptospira interrogans Serovar canicola Strain Moulton
Authors: Shimizu Tadayori , Matsusaka Eiichiro , Nagakura Naoki , Takayanagi Kazuhisa , Masuzawa Toshiyuki , Iwamoto Yoshihisa , Morita Tamotsu , Mifuchi Ichiji , Yanagihara Yasutake
Published: November 2013

The aqueous layer was isolated from Leptospira interrogans serovar canicola strain Moulton by the hot phenol‐water method. After ultracentrifugation, the precipitate was designated as lipopolysaccharide‐like substance (LLS) fraction and the chemical composition was compared with that of bacterial LPS. The LLS fraction consists of 35.2% carbohydrate, 3.8% amino sugar, 36.4% lipid, 15.2% protein, and 0.3% phosphorus. Neutral sugars were detected as rhamnose, arabinose, xylose, 4‐O‐methylmannose, mannose, galactose, and a small amount of erythrose, fucose and glucose by gas‐liquid chromatography (GLC), but 2‐keto‐3‐deoxyoctonic acid was not detected in the LLS by thiobarbituric acid test and high voltage paper electrophoresis. Fatty acids detected by GLC were decanoic acid (C10: 0), dodecanoic acid (C12: 0), dodecenoic acid (C12: 1), tridecenoic acid (C13: 1), tetradecanoic acid (C14: 0), hexadecanoic acid (C16: 0), hexadecenoic acid (C16: 1), and octadecenoic acid (C18: 1). With SDS‐polyacrylamide gel electrophoresis, bacterial LPS showed many orderly bands, while the banding pattern of the leptospiral LLS was very simple. These findings demonstrate that the physicochemical properties and chemical composition of LLS fraction from Leptospira are different from those of LPS extracted from gram‐negative bacteria such as Enterobacteriaceae, and suggesting that Leptospira has no typical LPS.
Biological Activities of Lipopolysaccharide‐Like Substance (LLS) Extracted from Leptospira interrogans Serovar canicola Strain Moulton
Authors: Shimizu Tadayori , Matsusaka Eiichiro , Takayanagi Kazuhisa , Masuzawa Toshiyuki , Iwamoto Yoshihisa , Morita Tamotsu , Mifuchi Ichiji , Yanagihara Yasutake
Published: November 2013

The biological activities of lipopolysaccharide‐like substance (LLS) extracted from Leptospira interrogans serovar canicola strain Moulton by the hot phenol‐water method were studied in mice. The addition of 12.5 μg/ml or more of LLS fraction increased the incorporation of [3H] thymidine into in vitro cultured spleen cells of C57BL/6 mice, while the activity of the LLS fraction was about 20 times weaker than that of Salmonella typhimurium lipopolysaccharide (LPS). Pretreatment of murine spleen cells with rabbit anti‐mouse thymocyte antiserum did not diminish the mitogenic activity of leptospiral LLS, and the LLS could not increase the incorporation of [3H] thymidine into thymocytes, suggesting that LLS acts on a B‐lymphocyte population of lymphocytes. When sheep erythrocytes and LLS fraction were injected intraperitoneally into BALB/c mice, LLS exhibited an enhancing effect on antibody response in vivo. However, lethal toxicity of the LLS fraction was about 500 times lower than that of LPS in C57BL/6 mice loaded with galactosamine. No antitumor activity of leptospiral LLS (250–1,000 μg/mouse) against the ascites form of Ehrlich carcinoma in ddY mice was observed. The biological activities of the LLS fraction from the organism were weaker than those of gram‐negative bacterial LPS, suggesting that Leptospira possesses no typical LPS.
Identification of Re Lipopolysaccharide‐Binding Protein on Murine Erythrocyte Membrane
Authors: Kirikae Teruo , Inada Katsuya , Hirata Michimasa , Yoshida Masao , Kondo Seiichi , Hisatsune Kazuhisa
Published: November 2013

Our recent studies have suggested that bacterial lipopolysaccharide (LPS) attaches to Pronase‐sensitive proteins on the murine erythrocyte membrane. In the present study, in order to identify the LPS‐binding protein on the murine erythrocyte membrane, a unique method to detect LPS‐binding protein on a nitrocellulose membrane was developed. Murine erythrocyte membrane proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, then transferred electrophoretically onto a nitrocellulose membrane. The membrane was incubated with LPS of Salmonella minnesota R595 (Re LPS) in phosphate‐buffered saline (PBS), after the remaining sites were blocked with gelatin in PBS. We were able to obtain a non‐background stain by adding the nonionic detergent octylglucoside at the low concentration of 0.1% to the Re LPS solution. The Re LPS bound to the protein on the nitrocellulose membrane was exposed to affinity purified anti‐Re LPS antibodies (IgG) and then to alkaline phosphatase‐conjugated anti‐IgG. The alkaline phosphatase was detected on the membrane by an enzymatic reaction. This method demonstrated that Re LPS was bound to an erythrocyte protein of 96 kDa. Treatment of erythrocytes with Pronase led to disappearance of the Re LPS‐binding protein on the erythrocyte membrane. There was no difference between LPS‐responder and LPS‐nonresponder murine erythrocyte membranes in amount and molecular weight of the Re LPS‐binding protein.
“Transformation” of Human Endothelial Cells by SV40 Virions
Authors: Ide Hideyuki , Minamishima Yoichi , Eizuru Yoshito , Okada Masanobu , Sakihama Kuniharu , Katsuki Taketo
Published: November 2013

Human endothelial cells derived from the umbilical vein were transformed with SV40 virions. A cell line subcultured for over 60 serial passages was characterized in comparison with its untransformed counterpart which was culturable for less than five passages.

The SV40‐transformed human endothelial cells, designated SV‐HUVEC, were positive not only for tumor (T) antigen specific to the SV40‐transformed cell, but also for two markers of endothelial cells, Factor VIII‐related antigen and a receptor for Ulex europaeus agglutinin I. By transformation the growth potential of the human endothelial cells was increased and their serum requirement was decreased. The SV40‐transformed endothelial cells were, however, unable to form colonies in soft agar or to form tumors in athymic nude mice, although a small nodule was produced at the site of inoculation. Subcultivation of these cells up to the 62nd passage eventually resulted in crisis and loss of further cell division. Thus, the human endothelial cells were transformed by SV40 while retaining certain normal functions but without showing tumorigenicity.
Homologous Human Macrophage Hybridomas That Produce a Novel Cytotoxic Factor in Their Culture Supernatants
Authors: Woo Hee Jong , Shimoda Olina , Imai Yasuyuki , Osawa Toshiaki
Published: November 2013

Homologous human macrophage hybridoma cell lines were obtained by somatic cell fusion between peripheral blood monocyte‐derived macrophages and a subclone of the myelomonocytic cell line, U937‐F9. The hybridoma cell lines grown in vitro for more than a year were confirmed by manifestations of phagocytosis, adherence, nonspecific esterase, acid phosphatase, chromosome numbers and other cell surface antigens. Cell surface antigens on hybridomas were detected by flow cytometry analysis with monoclonal antibodies. With interclonal differences, a typical phenotype of hybridoma cells was CDw14+, OKM5+, Mac‐1+ (equivalent to OKM1 and Mol), OKT9+, HLA‐DR− and CD20+. After stimulation with lipopolysaccharide and calcium ionophore A23187, culture supernatants of clones c18A and c29A showed cytotoxic activity against human melanoma A375 Met‐Mix and other cell lines which were resistant to the tumor necrosis factor, lymphotoxin and interleukin 1. This cytotoxic factor was found to be distinct from the tumor necrosis factor, lymphotoxin and interleukin 1 using the anti‐tumor necrosis factor, anti‐lymphotoxin and anti‐interleukin 1 antisera.