bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemical and Ultrastructural Comparison of Endotoxins Extracted from Salmonella minnesota Wild Type and R Mutants
Authors: Amano Ken‐Ichi , Fukushi Kazue
Published: November 2013

Endotoxic lipopolysaccharide and glycolipids (RGl) extracted from Salmonella minnesota wild type and R mutant cells (chemotypes Ra, Rb, Re, Rd1, and Rd2), respectively, with hot phenol‐water (PW) and phenol‐chloroform‐petroleum ether (PCP) were analyzed chemically and electron microscopically. All RGl extracted with PW (RGl‐PW) contained excess amounts of phosphate, O‐ester linked fatty acids and neutral sugars, while all RGl extracted with PCP (RGl‐PCP) contained excess amounts of free amino groups and fatty acids, in addition to the RGl constituents. Polyamine (cadaverine), phosphoethanolamine, and an unidentified amino compound were contained in RGl‐PCP as free amino groups.

When stained with uranyl formate, the ultrastructure of RGl‐PW showed a spherical form (onion‐like form), whereas the micrographs of RGl‐PCP showed a filamentous structure, regardless of strain differences. On the other hand, the micrographs of RGl‐PW represented spherical and doughnut‐shaped forms, and the micrographs of RGl‐PCP showed filamentous or stick forms, when stained with uranyl acetate. Thus, it is suggested that the ultrastructures of RGl were dominated by the solvent systems used for extraction, and not by the strains used here.
Occurrence of Galactosamine‐6‐Phosphate as a Constituent of Bacillus megaterium Spore Coat
Authors: Nasu Masao , Ogaki Junji , Nishihara Tsutomu , Nakanishi Tsutomu , Ichikawa Tomio , Kondo Masaomi
Published: November 2013

Galactosamine‐6‐phosphate was identified as a component of the coat of the Bacillus megaterium QM B1551 spore. It was one of the main constituents of the outermost layer of the spore coat, but it was absent from the other integuments including the cortex. These findings suggest that galactosamine‐6‐phosphate comprises the phosphorus‐containing skeleton structure of the spore coat.
Augmentation of Mouse Natural Killer Cell Activity by Lactobacillus casei and Its Surface Antigens
Authors: Kato Ikuo , Yokokura Teruo , Mutai Masahiko
Published: November 2013

Lactobacillus casei YIT 9018 (LC 9018) augmented the natural killer (NK) cell activity of spleen cells from inbred BALB/c mice injected intravenously with LC 9018 or intraperitoneally with polyinosinate‐polycytidylate. Augmentation of this activity by LC 9018 was also observed in male C3H/He, CBA/N, and C57BL/6 mice. The spleen cells exhibited no cytolytic activity against P815, a cell line insensitive to NK cells. The cytolytic activity of the spleen cells increased 2 days after the injection of 250 μg of LC 9018/mouse, peaked on day 3, and gradually declined thereafter. The increase caused by LC 9018 was also observed in normal and Meth A‐bearing mice.

In vitro treatment with anti‐asialo GM1 antibody plus complement completely‐abrogated the LC 9018‐augmented murine NK cell activity. The NK activity on the 3rd day after LC 9018 injection was reduced by in vitro treatment with anti‐Thy 1.2 monoclonal antibody plus complement to half of that observed when treatment was with complement alone. This suggests that there were two populations of NK cells in the spleen cell suspension derived from LC 9018‐treated mice. One population was asialo GM1‐positive and Thy 1‐negative, the other was asialo GM1‐positive and Thy 1‐positive.
Two Distinct Cytotoxic T Lymphocyte Subpopulations in Patients with Vogt‐Koyanagi‐Harada Disease that Recognize Human Melanoma Cells
Authors: Maezawa Nobuyoshi , Yano Akihiko
Published: November 2013

The functional properties of cytotoxic lymphocytes from patients with Vogt‐Koyanagi‐Harada disease (VKH) specific for human melanoma cells (P‐36 melanoma cell line established from a patient with malignant melanoma) were investigated by using monoclonal antibodies specific for human T cell subsets. Peripheral blood lymphocytes (PBL) from patients with VKH showed significant cytotoxic activity against the P‐36 (SK‐MEL‐28) human melanoma cell line, but not against a human cervical carcinoma of the uterus cell line (HeLa‐S3 cell line) or against a mouse melanoma cell line (B‐16 cell line) originating from a C57BL/6 strain mouse or against the EL‐4 mouse lymphoma cell line from a C57BL/6 mouse. The cytotoxic activity of the patients' PBL against the P‐36 melanoma cell line was markedly reduced by pretreatment of the PBL with monoclonal anti‐human Leu‐1 antibody plus rabbit complement, but it was reduced to much less extent by pretreatment with either monoclonal anti‐human Leu‐2a or Leu‐3a antibody plus rabbit complement. The specific cytotoxic activity of the patients' PBL against the P‐36 human melanoma cell line is, therefore, mediated by T cells bearing Leu‐1+ Leu‐2a+ or Leu‐1+ Leu‐3a+ antigens. Furthermore, the cytotoxic activity was shown to be blocked not only by anti‐Leu‐2a antibody specific to human cytotoxic/suppressor T cells but also unexpectedly by anti‐Leu‐3a antibody which has previously been considered to be specific to human inducer/helper T cells. The results of this study suggest that at least two distinct subpopulations of cytotoxic T cells specific for P‐36 human melanoma cells are present in the peripheral blood of VKH patients. These cytotoxic T cells have different surface antigens, Leu‐2a and Leu‐3a.
Characterization of Inhibitor to Leptospiral Hemolysin Present in Bovine Serum
Authors: Kojima Tsuyoshi , Yanagihara Yasutake , Mifuchi Ichiji
Published: November 2013

Hemolysis by leptospiral hemolysin was strongly inhibited by bovine serum. The inhibitory activity was observed in the chloroform‐methanol‐soluble fraction of bovine serum. The inhibitor was eluted in a complex lipid fraction and was separated into two fractions (Fr. I and II) by silicic acid column chromatography. Fractions I and II inhibited approximately 75% and 95%, respectively, of hemolysis by leptospiral hemolysin. Fraction I was identified as phosphatidylethanolamine (PdE) by silica gel thin‐layer chromatography (TLC). Two kinds of phospholipids (PLs) were detected in Fr. II by TLC. One was resistant to alkaline treatment and was identified as sphingomyelin (Spm), and the other was sensitive to such treatment and was identified as phosphatidylcholine (PdC). PLs, such as Spm, PdC, phosphatidylglycerol, PdE, phosphatidylserine and cardiolipin, inhibited hemolysis by leptospiral hemolysin, but phosphatidylinositol did not show any inhibitory activity. PLs lacking the amino group in the polar backbone of the molecules were more effective.

From experiments using erythrocytes of various kinds of animals, it was revealed that the hemolytic sensitivity of mammalian erythrocytes to leptospiral hemolysin depended on the Spm content in the erythrocyte membrane. On the other hand, phospholipase C (PLase C) activity with Spm and PdC as substrates was detected in the culture supernatant of Leptospira. Therefore, leptospiral hemolysin was presumed to be PLase C, perhaps sphingomyelinase. The inhibitors of leptospiral hemolysin present in bovine serum were identified as PLs. PLs in bovine serum were suggested to function as inhibitors of the interaction between leptospiral hemolysin and the surface of the erythrocyte membrane.
Characterization and Pathogenicity of Hemolysis Mutants of Mycoplasma pneumoniae
Authors: Yayoshi Masumi , Araake Minako , Hayatsu Eizo , Kawakubo Yasuaki , Yoshioka Morimasa
Published: November 2013

Hemolysis mutants were produced by treating Mycoplasma pneumoniae FH‐P24 strain with N‐methyl‐N‐nitro‐nitrosoguanidine and were classified into three different groups. The first group of mutants, strains P24‐L1, L2, and L11, showed wide and clear hemolytic zones. Their attachment ability to erythrocytes of various animals and to hamster lung cells were the same as those of the parent strain. The second group, strain P24‐S1, showed non‐hemolysis and non‐hemadsorption, but retained the attachment ability to lung cells, although not to erythrocytes. The third group, strain P24‐S11, was non‐hemolytic, had completely lost the attaching ability, and did not proliferate in vivo. Strains in the first group produced significant microscopic pneumonic lesions in hamsters while strain P24‐S1 produced milder lung lesions. Strain P24‐S11 did not cause any lung lesions, and organisms were not recovered from the lungs of hamsters. The attachment of M. pneumoniae to respiratory epithelium as a cause of infection and the existence of a relationship between the hemolytic abilities of the organisms and histopathogenicity in the hamster lung tissue were further supported by the present data.

It was also shown that the use of hemolysis mutants is useful for the elucidation of pathogenesis in mycoplasmal infections.
Studies on the Toxin of Aspergillus fumigatus
Authors: Yokota Katsushi , Kamaguchi Arihide , Sakaguchi Osamu
Published: November 2013

The site of hemolytic activity of a toxin isolated from Aspergillus fumigatus designated Asp‐hemolysin was determined by photooxidation techniques. The hemolytic activity of this toxin was strongly inhibited by photooxidation with methylene blue, rose bengal, riboflavin, or eosin G as a sensitizer, whereas crystal violet, hematoxylin, naphthol yellow S, bromothymol blue, methyl orange, and cresol red had no effect. pH dependence of the inactivation with methylene blue was observed in the narrow range of pH values from 7.0 to 8.0, like that of the inactivation with rose bengal or riboflavin. The histidine, cysteine, methionine, tryptophan, and tyrosine content of methylene blue‐photooxidized Asp‐hemolysin was significantly decreased, while other amino acids were not affected. The hemolytic activity of the toxin was lost more slowly than the histidine residue, being maintained at about 50% even at the time when the histidine residue was completely lost after 30 min. Photooxidation of Asp‐hemolysin in the presence of rose bengal also caused a decrease in histidine, methionine, and threonine content. These findings suggest that residues of cysteine, methionine, threonine, tryptophan, and/or tyrosine but not histidine may play an important role through stereostructure in the manifestation of the hemolytic activity of Asp‐hemolysin.
“Original Antigenic Sin” Phenomenon in Experimental Flavivirus Infections of Guinea Pigs: Studies by Enzyme‐Linked Immunosorbent Assay
Authors: Inouye Sakae , Matsuno Shigeo , Tsurukubo Yoshito
Published: November 2013

Guinea pigs were sequentially infected with two closely related flavivituses (Japanese encephalitis and West Nile viruses), and their antibody responses were studied both by enzyme‐linked immunosorbent assay (ELISA) and hemagglutination‐inhibition (HI). The results by ELISA always showed “original antigenic sin” responses: antibody activity of sera after the second infection was higher to the first infecting virus than to the second infecting virus. However, the results obtained by HI were variable.
Pyrogenicity of Yeast Mannans in Rabbits
Authors: Nagase Toshihiko , Mikami Takeshi , Suzuki Shigeo , Suzuki Masuko
Published: November 2013

A few yeast mannans free from protein and phosphorus showed pyrogenic activity in rabbits although the extent of this activity was considerably lower than that of the bacterial lipopolysaccharides (LPS). The pyrogenic activity was not abolished by treatment with sodium deoxycholate. This result showed that the mannans themselves participated in the pyrogenicity, excluding any possibility of LPS contamination in the mannans. Concerning the relationship between chemical structure and pyrogenicity of these mannans, it was demonstrated that a mannan possessing a highly branched structure exhibited stronger pyrogenicity than that of a less branched one.
Biological and Biochemical Characterization of Macrophage Activating Factor (MAF) in Murine Lymphocytes: Physicochemical Similarity of MAF to Gamma Interferon (IFN‐γ)
Authors: Fukazawa Yoshimura , Kagaya Keiko , Miura Hiroshi , Shinoda Takako , Natori Katsuro , Yamazaki Shudo
Published: November 2013

During the course of an investigation designed to separate macrophage activating factor (MAF) activity from interferon (IFN) antiviral activity in the lymphokine‐rich fraction (LKF) produced by stimulation of murine splenic cells with concanavalin A (Con A), we found molecular evidence for the similarity of the two activities. MAF activity was expressed as the rate of inhibition of intracellular growth of Salmonella typhimurium in macrophages based on the linear correlation between relative MAF activity and LKF concentration. The antiviral substance in LKF was identified as IFN‐γ based on the observation that its activity was inactivated at pH 2 and neutralized with anti‐mouse IFN‐γ serum but not with anti‐mouse IFN‐α/β serum. MAF and IFN antiviral activities displayed identical sensitivity to pH 2 and temperature. Further, neither activity was affected by β‐mercapto‐ethanol, but both were inactivated by guanidine hydrochloride and by sodium dodecyl sulfate, suggesting that the structures related to conformation of the protein of the two molecules may be similar. In affinity chromatography of the LKF on a Con A‐Sepharose column, MAF and IFN activities were found in both the nonadsorbing (F I) and adsorbing (F II) fractions. However, the rates of F II of MAF and IFN activities increased proportionally when the sample was applied on a column of higher capacity, suggesting that the molecular structure of the mannose‐containing glycosyl moiety of the two molecules may also be similar. Moreover, the intact or modified form of MAF and IFN activities of different LKF preparations showed a strong correlation, indicating that the production and denaturation of MAF activity were proportional to those of IFN antiviral activity. The results of this study provide strong evidence that MAF and IFN antiviral activities may reside in virtually the same molecular species.
Some Biochemical and Functional Characteristics of Macrophages Activated by Tetrahymena pyriformis
Authors: Makioka Asao , Kobayashi Akio
Published: November 2013

Phagocytosis, enzyme activities and capacity to release hydrogen peroxide (H2O2) and superoxide anion (O2−) of peritoneal macrophages from mice inoculated with Tetrahymena pyriformis, a free‐living ciliate, were examined in comparison with resident and BCG‐activated macrophages. Fc receptor‐mediated phagocytosis of sheep erythrocytes was markedly increased in Tetrahymena‐activated macrophages to the same level as that seen in BCG‐activated ones. Tetrahymena‐activated macrophages showed an increased level of acid phosphatase (lysosomal enzyme) and a reduced level of alkaline phosphodiesterase I (plasma membrane ectoenzyme) as compared with resident macrophages. Similar changes in the activities of the two enzymes were also observed in BCG‐activated macrophages. Both Tetrahymena‐ and BCG‐activated macrophages exhibited more enhanced capacity to release H2O2 and O2− than resident macrophages when stimulated with phorbol myristate acetate. In the macrophages from mice inoculated with varying doses of Tetrahymena, a significant correlation was observed between the increased capacity of H2O2 and O2− release as observed in the present study, and the enhanced toxoplasmacidal activity as observed in a previous study, in a dose‐dependent fashion.
Polysaccharides with Sulfate Groups Are Human T Cell Mitogens and Murine Polyclonal B Cell Activators (PBAs)
Authors: Sugawara Isamu , Ishizaka Shigeaki
Published: November 2013

In our previous paper, we reported that various types of carrageenan, dextran sulfate and fucoidan, which are sulfated homopolysaccharides with high molecular weights, were human T cell mitogens and murine polyclonal B cell activators (PBAs) and that heparin, a sulfated heteropolysaccharide, was a very weak human mitogen and mouse PBA.

Here we used cellulose sulfate (Mr 7–9 × 103), dextran sulfate with two different low molecular weights (Mr 5 × 103 and 8 × 103), two different condroitin sulfates (Mr 3.5 × 104), polyvinyl sulfate and polygalacturonic acid to investigate mitogenic activities of polysaccharides in detail.

The following results were obtained. 1. Low‐molecular‐weight sulfated homopolysaccharides, dextran sulfate and cellulose sulfate, were very weak or not human T cell mitogens. However, they were better murine PBAs. 2. Sulfated hetero‐polysaccharides, chondroitin 4‐sulfate and chondroitin 6‐sulfate, hardly induced mitogenic changes in human T cells and mouse B cells, even though the molecular weight of these substances was more than 1 × 104. 3. There were no other polymers examined so far which activated both human T cells and murine B cells.

The relationship among molecular size, sulfate groups and lymphocyte activation is discussed in detail.
Ultrastructures of Leuco‐Adsorption on Monolayers Infected with Influenza Virus
Authors: Saburi Yasuo , Uno Fumio , Akatsuka Kazuya , Uno Naomichi , Kumon Hiromi , Takahashi Toyozoh , Okuda Kenji
Published: November 2013

Leuco‐adsorption occurring in influenza virus infected‐cell cultures was studied morphologically to clarify the mechanisms of adsorption of leukocytes. Among the various types of chicken leukocytes studied, such as lymphocytes, monocytes, granulocytes, and thrombocytes, all were found to adhere to the virus‐infected cells. The adsorption seems to occur through at least two processes, one is mediated by microvilli (microvillus‐attachment), and the other is direct adherence of both cells (cell‐to‐cell‐attachment). In the former, the leukocytes are bound to the microvilli protruding from the infected MDCK cells and in the latter both cell membranes attach directly. In the cell‐to‐cell‐attachment, there was an electron‐lucent gap of about 12 nm in width in the intermembranous space of the junctional regions. This region was similar morphologically to the gap junction. As a result of leuco‐adsorption no cytolytic effects occurred in the MDCK cells under the experimental conditions.
Processing of Glycoprotein gB Related to Neutralization of Marek's Disease Virus and Herpesvirus of Turkeys
Authors: Ikuta Kazuyoshi , Ueda Shigeharu , Kato Shiro , Hirai Kanji
Published: November 2013

The glycoprotein gB related to neutralization of Marek's disease virus (MDV) and herpesvirus of turkeys (HVT) is composed of several glycosylated polypeptides, which were immunoprecipitated with monoclonal antibodies and rabbit antiserum cross‐reactive to MDV‐gB and HVT‐gB, and analyzed by SDS‐polyacrylamide gel electrophoresis. The present pulse‐chase experiments showed that the precursor forms of MDV‐ and HVT‐gB were glycoproteins with molecular weights of 110K to 115K (gp 115/110) and 115K (gp115), respectively. These precursor forms were processed to smaller gB's (gp63 and gp50 for MDV; gp62, gp52, and gp48 for HVT), at least in part by sialylation. The proteins synthesized in the presence of tunicamycin were two polypeptides of 88K and 83K in MDV‐infected cells and a 90K polypeptide in HVT‐infected cells, indicating the presence of unglycosylated precursor forms of MDV‐ and HVT‐gB. Differences between virulent and avirulent MDV's and between HVT's with and without protective activity against Marek's disease were observed in the processed forms of MDV‐ and HVT‐gB, especially at the processing step of sialylation.
The Intestinal Microflora of Infants: Composition of Fecal Flora in Breast‐Fed and Bottle‐Fed Infants
Authors: Benno Yoshimi , Sawada Ken , Mitsuoka Tomotari
Published: November 2013

The fecal flora of 35 breast‐fed and 35 bottle‐fed babies was determined. Bifidobacteria were the predominant fecal bacteria in both groups. Conversely, the counts of most of the other bacteria, such as bacteroides, eubacteria, peptococci, veillonella, clostridia, enterobacteria, streptococci, and bacilli in the bottle‐fed group were significantly higher than those in the breast‐fed group. The frequencies of occurrence of lecithinase positive Clostridia, clostridia‐others, pseudomonas and bacilli in the bottle‐fed group were significantly higher than those in the breast‐fed group. Twenty‐one genera and 103 species or biovars of microorganisms were isolated from the feces of the breast‐fed group and 20 genera and 97 species or biovars from the bottle‐fed group. The organism that showed the highest number and the highest frequency of occurrence in both groups was Bifidobacterium breve. Bifidobacterium infantis, which was formerly the most prevalent Bifidobacterium species in baby feces, was never isolated in this study. Further, the counts and incidences of Clostridium paraputrificum, C. perfringens, and Bacillus subtilis, the counts of C. clostridiiforme, Bacteroides vulgatus, Veillonella parvula, Lactobacillus acidophilus, Escherichia coli, Streptococcus bovis, S. faecalis, and S. faecium and the incidences of C. difficile, C. tertium, and Pseudomonas aeruginosa in the bottle‐fed infants were significantly higher than those in the breast‐fed infants.
Participation of Macrophages in Enhanced In Vitro Immune Interferon (IFNγ) Production with Mouse Spleen Cells
Authors: Yamaguchi Takahiro , Kuroda Yoko , Yokoyama Takahiko , Saito Motoo , Ebina Takusaburo , Hoshino Fumihiko , Ishida Nakao
Published: November 2013

IFNγ production in cultures of spleen cells obtained from mice sensitized with TH69, a live Streptococcus faecalis preparation, was examined to determine how macrophages participate. It was demonstrated that sensitized spleen macrophages participated in enhanced IFNγ production by T cells at an early stage (0–6 hr) of incubation, and that this production is mainly dependent on Ia‐bearing macrophages. In the reconstitution experiments where different combinations of spleen macrophages and T cells obtained from mice sensitized with TH69, OK‐432, and BCG were used, T cells required that the identity between the sensitizing organisms in vivo and the stimulating organisms in vitro be the same for enhanced IFNγ production while macrophages did not. Macrophage‐mediated production of IFNγ appears to be genetically restricted because IFNγ was only produced in cultures where the H‐2 region of macrophages and T cells matched.

Further examination revealed that for macrophages to participate in enhanced IFNγ production, first contact between cycloheximide‐treated macrophages and T cells was required. Second, enhanced IFNγ production occurred when culture supernatants of macrophages obtained from sensitized spleen cells were added to T cells. However, the addition of culture supernatant obtained from sensitized peritoneal macrophages resulted in inhibition of IFNγ production.

These results clearly showed the crucial role of macrophages in enhanced IFNγ production by spleen T cells in vitro.
Disappearance of Terminal Deoxynucleotidyltransferase in Human Malignant T‐Lymphoblasts Treated with a Human Alpha Interferon Preparation
Authors: Takano Saeko , Nakamura Hiromu , Mita Haruhisa , Nagano Yasuiti
Published: November 2013

Human T‐lymphoblastoid cell lines RPMI 8402, MOLT‐3, and CCRF‐CEM were treated with interferon (IFN) to determine if the treatment would result in the disappearance of cellular terminaldeoxynucleotidyltransferase (TdT), a possible differentiation marker for T‐lymphocytes. Incubation of RPMI 8402 cells in the presence of IFN preparation caused a decrease in the number of TdT‐positive cells and in TdT activity of the cell extract. The inhibition of cell multiplication was dose dependent. The anticellular effect of IFN preparation was cytostatic, not cytocidal. The IFN preparation modified neither the TdT content nor proliferation of MOLT‐3 and CCRF‐CEM cell lines. The effects of IFN preparation thus varied with the cell line.
Partial Characterization of Thymocyte‐Activating Factor Derived from MDP‐Stimulated Guinea Pig Fibroblasts
Authors: Iribe Hideaki , Koga Toshitaka
Published: November 2013

The activity of fibroblast‐derived thymocyte activating factor (FTAF) of the guinea pig was measured, and the factor was partially characterized. The FTAF activity was heat labile, and destroyed by treatment with trypsin, chymotrypsin, and Streptomyces griseus protease, suggesting the protein nature of FTAF. FTAF bound to DEAE‐Sepharose CL‐6B in Tris‐HCl buffer at pH 8.0, and was eluted with 0.1–0.2 M NaCl. FTAF was absorbed with Blue Sepharose CL‐6B. The factor bound to a hydroxylapatite column in 10 mM phosphate buffer and was eluted in two major fractions, one fraction with 40 mM phosphate buffer, the other with 70–110 mM phosphate buffer. Finally, FTAF did not have as much effect on the proliferation of lymph node T cells as T‐cell‐activating monokines which exhibited marked stimulating effects on both T lymphocytes and thymocytes.
Composition of O‐Antigenic Lipopolysaccharides from Enterobacter cloacae
Authors: White Pippa J. , Gaston Michael A. , Wilkinson Stephen G.
Published: November 2013

Analyses have been carried out on lipopolysaccharides (LPS) from 14 strains of Enterobacter cloacae representing different O serotypes. All of the products appeared to have a composition and architecture typical of enterobacterial LPS, but points of interest include the absence of phosphate residues from the core oligosaccharide, the presence of both L‐glycero‐D‐mannoheptose and D‐glycero‐D‐mannoheptose (ratio usually about 4: 1), and the presence in lipid A of small amounts of fatty acids with odd numbers of carbon atoms (mainly C13) in addition to tetradecanoic acid and 3‐hydroxytetradecanoic acid. Monosaccharides identified as components of polymeric fractions from the LPS were glucose, galactose, mannose, rhamnose, glucosamine, galactosamine, fucosamine, and galacturonic acid. Most polymeric fractions also probably contained an O‐acetyl substituent. Closely similar chemotypes found for the polymeric fractions from the LPS of cross‐reacting serotypes support the view that these fractions contain the O‐antigenic determinants and represent the side chains of the LPS.
Effect of Glucose on the Interaction of Hydrophobic Compounds with the Alanine Receptor Field of Bacillus subtilis Spores during Initiation of Germination
Authors: Yasuda Yoko , Tochikubo Kunio
Published: November 2013

Glucose interfered with the inhibitory action of hydrophobic compounds, such as n‐octanol, diphenylamine and 2‐tert‐butylphenol, during L‐alanine‐initiated germination of Bacillus subtilis spores. The action of glucose on the action of the hydrophobic compounds was not competitive, and the binding affinity of glucose was not essentially affected by the hydrophobic compounds, indicating the presence of separate binding sites for glucose and the hydrophobic compounds. The binding affinity of D‐alanine, a competitive inhibitor of L‐alanine, was not affected by the hydrophobic compounds, indicating separate binding sites for D‐alanine and the hydrophobic compounds. A possible arrangement of the binding sites for glucose and for the hydrophobic compounds in relation to those for L‐ and D‐alanine on the spores is discussed.
Preparation of Complement Fixation Antigen of Chlamydia psittaci Grown in Tissue Culture by Treatment with β‐Propiolactone
Authors: Chiba Nobuyuki , Takashima Ikuo , Arikawa Jiro , Hashimoto Nobuo
Published: November 2013

A new method of preparing a chlamydial complement fixation (CF) antigen by treatment with β‐propiolactone (BPL) is presented. Chlamydia psittaci strains Pigeon‐1041 and Budgerigar‐No. 1, and Chlamydia trachomatis strain L2/434/BU, propagated in L‐929 cell monolayers, were inactivated with BPL. This BPL‐treated antigen was useful for detecting CF antibodies in both human and pigeon sera, and it did not cause false‐positive reactions, as are sometimes observed between some human sera and phenol‐treated antigen derived from eggs. When this CF antigen was treated with potassium periodate and tested for reactivity with mouse immune ascitic fluid, it was found that the antigen contained type‐ or strain specificity as well as genus specificity. Immunization with the BPL‐treated antigen elicited type‐ or strain‐specific neutralizing antibody.
Relationships among Broad‐Spectrum and Narrow‐Spectrum Mannose‐Resistant Fimbrial Hemagglutinins in Different Yersinia Species
Authors: Old David C. , Adegbola Richard A.
Published: November 2013

When 117 strains of Yersinia were grown in serial broth culture at 22 or 30 C, 44 (38%) formed a broad‐spectrum, mannose‐resistant hemagglutinin (MR/Y‐HA) associated with thick (8 nm) channelled (MR/Y) fimbriae; eight other strains (7%) formed a narrow‐spectrum, mannose‐resistant hemagglutinin (MR/K‐like HA) associated with thin (4 nm) non‐channelled (type 3‐like) fimbriae. The distribution of these two fimbrial hemagglutinins in different Yersinia species is discussed and their properties are compared with those of other reported mannose‐resistant hemagglutinins. The thick fimbriae of four Yersinia species were antigenically similar as judged by immunoelectronmicroscopy. Likewise, the thin fimbriae of three Yersinia species were antigenically similar, though different from the thick fimbriae.
Constitutive Production and Characterization of Interferon‐Gamma in a Human T‐Lymphoblastoid Cell Line Transformed by a Human Retrovirus
Authors: Matsuyama Masako , Sugamura Kazuo , Hinuma Yorio
Published: November 2013

A human T‐lymphoblastoid cell line, TCL‐Fuj, constitutively produced a large amount of human gamma interferon (IFN) in culture fluids and has sustained stable IFN production for more than two years. When cells were incubated in RPMI‐1640 medium with 10% fetal calf serum for three days, IFN activity was detectable at a cell density of 6 × 104 cells/ml, whereas 2,000–16,000 units of IFN per ml were produced at 5–10 × 105 cells/ml. IFN production was also detected even in serum‐free medium and as early as 2 hr after cultivation in fresh medium. IFN was inhibited by treatment of cells with either actinomycin D or cycloheximide, indicating the requirement of IFN‐mRNA and protein for de novo synthesis. The molecular weight of the IFN was 45,000–60,000 as determined by Sephacryl S200 gel filtration. Two activity peaks corresponding to molecular weights of 22,000 and 39,000 were obtained by SDS‐polyacrylamide gel electrophoresis. Analysis by isoelectric focusing revealed charge heterogeneity with four species at pIs of 6.0, 7.1, 8.6, and 9.3. Conventional IFN‐gamma inducers, concanavalin A and 12‐O‐tetradecanoyl‐phorbol‐13‐acetate, further enhanced the production of IFN in this cell line.
Antigenic Relationship between Candida parapsilosis and Candida albicans Serotype B
Authors: Funayama Masahiro , Nishikawa Akemi , Shinoda Takako , Suzuki Motofumi , Fukazawa Yoshimura
Published: November 2013

We examined the antigenic relationship between Candida parapsilosis and C. albicans serotype B with respect to antigenic factors 13 and 13b, specific for the former species and common to both species, respectively. Acetolysis of C. albicans serotype B cell‐wall mannan gave six oligosaccharides. Their chemical structure was determined by 1H‐nuclear magnetic resonance (NMR) spectroscopy, methylation analysis, and partial acid hydrolysis. The structure of the hexasaccharide derived from C. albicans serotype B mannan was α‐D‐Manp‐(1–2)‐α‐D‐Manp‐(1–3)‐α‐D‐Manp‐(1–2)‐α‐D‐Manp‐(1–2)‐α‐D‐Manp‐(1–2)‐D‐Man (M6) which is identical to that from C. parapsilosis mannan. Inhibition of two precipitin reaction systems (anti‐C. albicans serotype B serum and anti‐C. parapsilosis serum to the respective homologous mannan), by oligosaccharides from homologous and heterologous mannans indicated that M6 from either C. albicans serotype B or C. parapsilosis was the most effective inhibitor. Moreover inhibition of the agglutination reaction between factor serum containing anti‐factors 13 and 13b and C. albicans serotype B or C. parapsilosis cells by oligosaccharides from both mannans also indicated that the M6s were the most effective inhibitors. These results suggest that the M6s derived from the two species are identical in their chemical structure, although the structures of the whole mannans of the two species are not identical as demonstrated by gel diffusion precipitation patterns, and that M6s may be involved in the specificities of antigenic factors 13 and 13b. The amount of M6 is larger in C. parapsilosis cell‐wall mannan, suggesting that high repeating frequency of M6 fragment may induce the antibody specific for C. parapsilosis.
Panning Separation for Monoclonal Antibody‐Specific T‐Cell Subsets: Effects of Enzymes and Metabolic Inhibitors
Authors: Goto Makoto , Bluestein Harry G. , Zvaifler Nathan J.
Published: November 2013

The mechanisms of “panning,” a simple positive cell separation technique, were examined. Using monoclonal antibodies to “pan” for T cells (T101+, T4+, and T8+), we obtained enriched populations with 90% purity and viability from unfractionated human peripheral blood lymphocytes (PBL). We found that the “panning” method reflects an active process. It is sodium azide inhibitable and independent of the divalent cation concentration. Effective panning does not require capping, patch formation, or DNA synthesis. The cell yields are unaffected by mitomycin‐C or microtubule and microfilament blocking reagents such as concanavalin A, colchicine, and cytochalasin B. This economical technique provides large numbers of functionally intact monoclonal antibody‐specific cells within a relatively short time for further functional and biochemical characterization.
Cytotoxicity and Calcium‐Dependent Antigen of Yersinia
Authors: Okamoto Keinosuke , Kobayashi Tatsuhiko , Shinoda Sumio , Inoue Takashi , Yukitake Jun , Shimizu Kiyoji , Kawamoto Yasuko , Moriyama Takeshi , Miyama Akio
Published: November 2013

The relationship between invasiveness and calcium dependency was examined in various strains of Yersinia enterocolitica and Y. pseudotuberculosis by using established cell lines. Infection with calcium‐dependent bacteria resulted in the formation of microvilli and the adherence of bacteria on the cell surface, and the adherent bacteria were ingested 1.5 hr after infection. Morphological changes in the cells became visible 2 to 3 hr after infection, and intracellular multiplication of the ingested bacteria was noted. When the cells were incubated with bacteria at 37 C for 1.5 hr and then at 25 C, however, the morphological changes in the infected cells were not observed. No isogenic strains that had lost calcium dependency for growth at 37 C were able to elicit the morphological changes in the cells, though they possessed the ability to adhere to and penetrate the cells.

The antigen(s) supposedly related to cytotoxicity of the calcium‐dependent Yersinia was sought by using antibodies prepared against calcium‐dependent bacteria and then absorbed with calcium‐independent bacteria and with calcium‐independent bacterial cytosol. Double diffusion tests between the antisera and bacterial cytosol extracts revealed the presence of an antigen which was a cytoplasmic substance common to all calcium‐dependent but not calcium‐independent strains of Y. enterocolitica and Y. pseudotuberculosis.
Effect of E‐64, Thiol Protease Inhibitor, on the Secondary Anti‐SRBC Response In Vitro
Authors: Amamoto Toshiro , Okazaki Tadayasu , Komurasaki Toshi , Hanada Kazunori , Omura Sadafumi
Published: November 2013

E‐64, L‐trans‐epoxysuccinyl‐leucylamido (4‐guanidino) butane, a specific inhibitor of thiol proteases originally isolated from the culture of a fungus, was examined in connection with the immune responses to the splenocytes of mice.

In cultures of C3H/He mouse splenocytes, E‐64 and its analogues showed mitogenic activity, and some of them enhanced the lymphocyte blast transformation induced by a suboptimal concentration of concanavalin A. E‐64 caused a significant suppressive effect on the secondary anti‐SRBC responses when 7‐ or 14‐day‐primed BDF1 mouse splenocytes were cultured with SRBC, while it induced no effect on cultured splenocytes either from mice treated with cyclophosphamide, from mice sensitized with dinitrophenyl‐Ficoll. The results with E‐64 and its close analogues revealed that their effects on the immune response roughly correlated with their inhibitory activity against thiol protease.

These results suggest that a thiol protease might be involved in the process of secondary immune response in mouse splenocytes.
Isolation and Characterization of Canine Distemper Virus‐Specific RNA
Authors: Hirayama Norio , Senda Megumi , Yamamoto Hisashi , Yoshikawa Yasuhiro , Yamanouchi Kazuya
Published: November 2013

Ten species of virus‐specific RNA were detected in Vero cells infected with the FXNO strain of canine distemper virus (CDV). The largest RNA was the genome‐sized RNA and the nine smaller species were polyadenylated RNAs. Similar results were obtained for nine other strains of CDV. The molecular weights of these ten RNAs were determined to be 4.61 × 106, 2.46 × 106, 1.52 × 106, 1.32 × 106, 1.19 × 106, 1.07 × 106, 0.77 × 106, 0.65 × 106, 0.58 × 106. and 0.48 × 106. By in vitro translation of the polyadenylated RNAs in a rabbit reticulocyte lysate system, three different proteins which probably correspond to H, NP, and M were synthesized from the fraction containing RNAs 7, 8, 9, and 10.
Induction of Interferon and Activation of NK Cells and Macrophages in Mice by Oral Administration of Ge‐132, an Organic Germanium Compound
Authors: Aso Hisashi , Suzuki Fujio , Yamagughi Takahiro , Hayashi Yoshiro , Ebina Takusaburo , Ishida Nakao
Published: November 2013

After oral administration of an organic germanium compound, Ge‐132 (300 mg/kg), a significant level of interferon (IFN) activity was detected in the sera of mice at 20 hr and it reached a maximum of 320 U/ml at 24 hr. This IFN activity was lost after heat‐ or acid‐treatment, suggesting that the induced IFN is of ‐nature. The molecular weight of this IFN was estimated to be 50,000 daltons by gel filtration. The NK activity of spleen cells was increased 24 hr after the oral administration of Ge‐132, and cytotoxic macrophages were induced in the peritoneal cavity by 48 hr. In the mice receiving an intraperitoneal (ip) injection of trypan blue or carrageenan 2 days before oral administration of Ge‐132, neither induction of IFN nor augmentation of NK activity occurred, and X‐ray irradiation of mice also rendered the mice incapable of producing IFN, all indicating that both macrophages and lymphocytes are required for this IFN induction. Both NK and cytotoxic macrophages appeared 18 hr after ip administration of the induced IFN with a titer as low as 20 U/ml. These facts suggest that both the augmentation of NK activity and activation of macrophages in mice after oral administration of Ge‐132 are mediated by the induced IFN.
Isolation of a New Drug‐Resistance Plasmid from a Strain of Vibrio parahaemolyticus
Authors: Arm Taketoshi , Hamashima Hajime , Hasegawa Hiroko
Published: November 2013

A new R plasmid, pSA55, with a molecular weight of 112 mcgadaltons (Md), was isolated from a strain of Vibrio parahaemolyticus with multiple drug resistance. The pSA55 plasmid conferred on its host resistance to chloramphenicol, tetracycline, streptomycin, kanamycin, ampicillin, trimethoprim and 2,4‐diamino‐6,7‐diisopropyl pteridine, and belongs to incompatibility group C. The plasmid was transferable to Escherichia coli, V. parahaemolyticus, V. alginolyticus and NAG bivrio at a frequency of 10−3~‐7, and was stably inherited by the transconjugants of these species. The conjugal transfer of pSA55 plasmid was significantly affected by the growth culture phase. The resistance pattern and resistance levels of transconjugants were the same as those of the donor strain. We did not observe fluctuations in minimal inhibitory concentrations with transfer, unlike the case of V. cholerae. The relationship between the pSA55 plasmid and the Kanagawa phenomenon was not clarified in the present study.
Bactericidal Effect of Fatty Acids on Mycobacteria, with Particular Reference to the Suggested Mechanism of Intracellular Killing
Authors: Kanetsuna Fuminori
Published: November 2013

The effect of fatty acids on Mycobacterium smegmatis was examined in vitro at pH 5.0 to 7.0 to determine the role of fatty acids in the intracellular killing of mycobacteria. Unsaturated fatty acids showed strong bactericidal activity in low concentrations (0.005 to 0.02 mM), whereas saturated fatty acids, except for lauric and myristic acids, were not very effective even at a concentration of 0.2 mM. Addition of a saturated fatty acid (palmitic or stearic acid) to an unsaturated fatty acid (oleic or linoleic acid) did not strongly interfere with the bactericidal effect of the unsaturated fatty acid at pH 5.0 and 6.0. Ca2+ (3.0 mM), Mg2+ (1.0 mM), and γ‐globulin (0.4%) showed weak reversal effects on the bactericidal activity of unsaturated fatty acids at pH 5.0 and 6.0. Serum albumin and serum showed strong reversal effects. The concentrations of each fatty acid in a mixture (molar ratio, 1: 1: 1: 1) of oleic, linoleic, palmitic, and stearic acids required for the killing of M. smegmatis in the presence of 2% serum (bovine, rabbit, or human) were 0.05 to 0.10 mM at pH 5.0 and 6.0 and 0.05 to 0.20 mM at pH 7.0, depending on the serum used.

The susceptibilities of M. kansasii, M. bovis strain BCG, and M. tuberculosis to the mixture of the four fatty acids in the presence of 2% bovine serum were similar to that of M. smegmatis, although M. fortuitum was more resistant.
Immunomodulating Activities of Staphylococcal Enterotoxins
Authors: Kawaguchi‐Nagata Kumiko , Okamura Haruki , Shoji Ko , Kanagawa Hisami , Semma Masanori , Shinagawa Kunihiro
Published: November 2013

The immunomodulating effects of staphylococcal enterotoxins on in vivo immune responses in C57BL/6 mice were examined. Of the five serological types A (SEA), B, C, D, and E (SEE), only SEA and SEE markedly suppressed the antibody response to sheep red blood cells (SRBC) when injected 1 day before or on the day of immunization with SRBC.

Further study of SEA revealed that it did not affect the antibody response to a thymus‐independent antigen, salmonella flagella, but did affect the T‐cell‐mediated immune response. Contact sensitivity to dinitrofluorobenzene (DNFB) was suppressed when SEA was injected before sensitization or before challenge with DNFB, indicating that SEA affected both the afferent and efferent phases of DNFB contact sensitivity. As the suppression of DNFB contact sensitivity could be transferred by anti‐Thy‐1.2 antibody‐sensitive spleen cells of SEA injected donors into normal or DNFB‐sensitized recipients, the suppression was thought to be an active one. However, SEA could augment the DNFB contact sensitivity when injected on the third day after sensitization with DNFB.

These results indicate that the immunomodulating effects of SEA can be mediated by the T‐cell function.
Electrophoretic Patterns of Extracellular Deoxyribonuclease (DNase) and Their Correlation with T‐Type in Group A Streptococci
Authors: Miyakawa Yozo , Yamada Toshihiko , Shitara Masatsugu , Fukazawa Yoshimura
Published: November 2013

Molecular heterogeneity of the extracellular deoxyribonuclease (DNase) in group A streptococci was demonstrated in 42 clinical isolates. Although Polyacrylamide gel electrophoretic patterns of the extracellular DNase of all the isolates were heterogeneous, they could be divided into five main patterns with respect to the presence or absence of three DNase components including DNase B. By comparing the electrophoretic patterns of DNase in all the isolates with their T‐types, we found that the patterns were quite characteristic for their T‐types, especially in the prevalent T‐types 12 and 1, and that the isolates of T‐types 12 and 1 produced DNase B as their major extracellular DNase. Relative DNase B activity in the total extracellular DNase activity of group A, B, and G isolates was determined by the rapid method of neutralization with anti‐DNase B antibody. The results showed neutralization of DNase activity in all the isolates of group A streptococci, largely corresponding to their T‐types, but not of the isolates of groups B and G. These results indicate that the electrophoretic patterns of the extracellular DNase of group A streptococci are closely correlated with their T‐types, suggesting the physicochemical taxonomic value of these properties.
Adjuvant Activity of Klebsiella O3 Lipopolysaccharide: Inhibition of the Adjuvant Activity by Concanavalin A
Authors: Kato Nobuo , Kido Nobuo , Ohta Michio , Naito Setsuko
Published: November 2013

Klebsiella O3 lipopolysaccharide (KO3 LPS) was found to exhibit extraordinarily strong adjuvant activity in augmenting antibody responses and delayed‐type hypersensitivity (DTH) to protein antigens in mice. The O‐specific polysaccharide chain of KO3 LPS consists of α‐mannoside. We investigated the effect of concanavalin A (Con A) or succinyl Con A, which is known to bind to α‐mannoside, on the adjuvant activity of KO3 LPS in augmenting DTH to ovalbumin. When KO3 LPS was mixed with Con A prior to injection, the strong adjuvant activity of KO3 LPS in augmenting DTH was inhibited and the degree of inhibition depended upon the dose of Con A. An equal amount of Con A elicited nearly complete inhibition of the adjuvant activity of KO3 LPS, Con A at 1/10 the amount of LPS elicited partial inhibition, and Con A at 1/100 the amount of LPS showed no inhibition. An equal amount of succinyl Con A, which induced less marked aggregation of KO3 LPS than Con A, elicited inhibition of the adjuvant activity of KO3 LPS to an extent similar to that by Con A. On the other hand, Con A or succinyl Con A bound to KO3 LPS did not impair in any way the lethal toxicity of KO3 LPS for mice which is known to be due to the lipid A moiety. From these findings it is concluded that the strong adjuvant activity of KO3 LPS does not solely depend upon the lipid A moiety but the O‐specific polysaccharide moiety plays an important role in expression of the adjuvant activity.
The Intestinal Microflora of Infants: Fecal Flora of Infants with Vitamin K Deficiency
Authors: Benno Yoshimi , Sawada Ken , Mitsuoka Tomotari
Published: November 2013

The fecal flora of 10 infants with vitamin K deficiency (VKD) and 10 healthy infants was examined. All the infants were breast‐fed. In the infants with VKD, the total counts (P<0.01) and the numbers of bifidobacteria (P< 0.001) were lower than in the healthy infants, whereas bacteroides, veillonella and enterococci were present in greater numbers in the feces of infants with VKD. The incidence of the Bacteroides fragilis group was higher (P<0.05) in the infants with VKD than in the healthy infants. A significant reduction (P<0.05) in Bifidobacterium breve was shown in the infants with VKD. The data emphasize the abnormal flora in infants with VKD.
Hybridoma Antibodies Produced against Bromelain Derived Cores of Influenza Virus
Authors: Lecomte Jacqueline
Published: November 2013

Splenic lymphocytes from BALB/c mice immunized with “cores” of influenza virus, obtained after bromelain cleavage of the surface glycoprotein, were fused with the P3‐NS1/1‐Ag‐1 mouse cell line to yield hybridoma cultures. Among 20 stable cloned hybrid cells secreting monoclonal antibodies, one was specific for the nucleoprotein (NP), 11 were specific for the membrane (M) protein and eight were specific for the hemagglutinin (HA). These “cores” used as immunogen contained only the internal proteins of the influenza virus, namely the three polymerases, the NP and the M protein and no HA when examined by standard procedures of SDS‐PAGE, electron microscopy and hemagglutination activity. It thus appeared that a small amount of contaminating antigens can sensitize a sufficient number of mouse B cells to be selected as hybrid partners. These antibodies were provisionally assigned as anti‐carbohydrate attached to the HA.