bluuebunny/crossref_metadata_2025 huggingface dataset:
Differences between Surface Antigenic Determinants of Polar Monotrichous Flagella of Vibrio parahaemolyticus and of Related Species
Authors: Shinoda Sumio , Senoh Toshinobu , Asano Kazuyo , Nakahara Noriko , Ono Bun‐Ichiro
Published: November 2013

Polar monotrichous flagella (M‐flagella) of Vibrio parahaemolyticus have antigens in common with those of various species of Vibrio including V. cholerae and V. anguillarum, and of Beneckea, revealed by gel diffusion tests with flagellin monomers. However, antiserum against M‐flagellin of V. parahaemolyticus did not agglutinate cells of V. cholerae and V. anguillarum, although it did agglutinate cells of V. parahaemolyticus. Agglutination tests after absorption of the antiserum with purified M‐flagellar filaments or flagellin monomers and H‐agglutination inhibition tests demonstrated that there are two different antigenic determinants in M‐flagella as in lateral flagella. One is on the surface of the M‐flagella (surface antigenic determinant, SA) and disappears or is buried in dissociated monomers. The other is inside the flagella (internal antigenic determinant, IA) and is exposed when the flagella are dissociated to flagellin monomers. SA of V. parahaemolyticus is different from those of V. cholerae and V. anguillarum, whereas the three species have a common IA.
Studies on Structural Proteins of Chikungunya Virus
Authors: Konishi Eiji , Hotta Susumu
Published: November 2013

Chikungunya virus (CHIKV) was purified and subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in discontinuous buffer systems. Three bands were revealed by staining with Coomassie blue; two of them (E1 and E2) were associated with the membrane, and one (C) with the core. Their molecular weights were estimated to be 56,000 (E1), 52,000 (E2), and 36,000 (C), irrespective of the concentration of acrylamide in the gels. The molar ratios of Ei, E2, and C were almost equal when the sample buffer was Tris‐HCl, whereas they were different when phosphate buffer was used.
Concanavalin A Agglutinability of Some Enveloped RNA Viruses Modified by Host Cell Transformation
Authors: Miki Takashi
Published: November 2013

The dependence on concanavalin A (Con A) concentration of agglutinability of some enveloped RNA viruses grown in transformed cells was compared with that of those grown in nontransformed cells. The avian oncoviruses were purified by centrifuging to equilibrium in a combination equilibrium: viscosity‐gradient of potassium tartrate and glycerol after conventional isopycnic sucrose density gradient centrifugation. Avian oncoviruses were more agglutinable with Con A when grown in transformed cells than when grown in nontransformed cells. Vesicular stomatitis virus grown in transformed cells was also more agglutinable than the virus grown in nontransformed cells. These results agree with the concept that the envelopes are modified by host cell transformation and that, therefore, viruses grown in transformed cells are expected to be more agglutinable with Con A than those grown in nontransformed cell.
Isolation of Nontoxigenic Variants Associated with Enhanced Sporulation and Alteration in the Cell Wall from Clostridium botulinum Type A 190L by Treatment with Detergents
Authors: Takumi Kenji , Kinouchi Takemi , Kawata Tomio
Published: November 2013

Nontoxigenic variants were isolated from Clostridium botulinum type A strain 190L after treatment with detergents such as deoxycholate, sodium dodecyl sulfate, Tween 80 and Brij‐58. Deoxycholate was most effective for obtaining the variants. The variants exhibited a markedly increased frequency of sporulation compared with the oligosporogenic parent strain. The cell wall of the parent strain was composed of an outer layer and an inner layer, whereas that of the variants lost the outer layer. After treatment with mitomycin C the parent strain was subjected to lysis and produced bacteriophages with a hexagonal head and a contractible tail, while the nontoxigenic variants did not yield bacteriophages or phage‐like structures. There appears to be a close relationship among the toxigenic and sporogenic properties, formation of the outer cell wall layer and lysogeny.
Ultrastructure of a Hexagonal Array in Exosporium of a Highly Sporogenic Mutant of Clostridium botulinum Type A Revealed by Electron Microscopy Using Optical Diffraction and Filtration
Authors: Masuda Kuniyoshi , Kawata Tomio , Takumi Kenji , Kinouchi Takemi
Published: November 2013

The ultrastructure of a hexagonal array in the exosporium from spores of a highly sporogenic mutant of Clostridium botulinum type A strain 190L was studied by electron microscopy of negatively stained exosporium fragments using optical diffraction and filtration. The exosporium was composed of three or more lamellae showing an equilateral, hexagonal periodicity. Images of the single exosporium layer from which the noise had been filtered optically revealed that the hexagonally arranged, morphological unit of the exosporium was composed of three globular subunits about 2.1 nm in diameter which were arranged at the vertices of an equilateral triangle with sides of about 2.4 nm. The morphological units were arranged with a spacing of about 4.5 nm. The adjacent globular subunits appeared to be interconnected by delicate linkers.
Regulation of the In Vitro Secondary Cell‐Mediated Cytotoxic Response against Syngeneic FBL‐3 Leukemia by Macrophages
Authors: Igarashi Tsuyoshi , Teranishi Tsuyoshi , Rodrigues Dennis , Yamamura Yuichi
Published: November 2013

Depletion of macrophages from immune spleen cells by treatment with carbonyl iron and magnet or by in vivo treatment with carrageenan enhanced the in vitro secondary cell‐mediated cytotoxic response against a syngeneic Friend virus‐induced leukemia, FBL‐3 cells of C57BL/6 mice. However, further depletion of macrophages by passing the carbonyl iron‐treated immune spleen cells through a nylon wool column abrogated the cytotoxic response. The addition of splenic macrophage‐enriched preparations from either FBL‐3‐immune or normal mice suppressed the cytotoxic response of immune spleen cells treated with carbonyl iron and magnet. This suppressive effect of splenic macrophages presented a marked contrast with the enhancing effect of normal peritoneal macrophages on the same cell‐mediated cytotoxic response, indicating regulation of the generation of killer T cells against a syngeneic tumor by functionally distinct macrophages. The suppressed cell‐mediated cytotoxic response against FBL‐3 cells by immune spleen cells was augmented by the addition of indomethacin to the culture medium, and this augmentation with indomethacin was greatly decreased by depletion of phagocytic cells from the immune spleen by treatment with carbonyl iron and magnet. The mechanisms of regulation of the cell‐mediated cytotoxic response with soluble factors released from macrophages are discussed.
Immunopharmacological Approach to Forssman Shock
Authors: Nagai Hiroichi , Kurimoto Yoshiyuki , Koda Akihide
Published: November 2013

Forssman shock (FS) following the intravenous injection of antisheep erythrocyte antibody into guinea pigs resulted in fatal systemic shock with marked decrease in CH50 values of complement, leucocyte, and platelet counts, and a prolongation of blood coagulation time. In addition, there was an increase in lactate dehydrogenase activity, and decreases in both esterase activity and fibrinogen levels were noted. F(ab′)2 of antisheep erythrocyte IgG antibody was not capable of eliciting FS. Cobra venom factor showed a fairly potent inhibition of FS. Leukopenia induced by cytosine arabinoside given intraperitoneally for 5 days had no effect on FS. Colchicine, which decreased the leucocyte count, did not inhibit fatal systemic shock. Administration of heparin or trasyrol did not prevent FS. The present findings demonstrate that FS is inhibited by anticomplementary agents but not by drugs which affect leucocyte and platelet counts, the coagulation system or serum proteases.
Formation of Mononuclear Phagocyte (Macrophage) Colonies by Mouse Spleen Cells in Liquid Culture
Authors: Yokochi Takashi , Nakashima Izumi , Nagase Fumihiko , Ohta Michio , Kato Nobuo
Published: November 2013

When spleen cells of the adult mouse were tested for the formation of mononuclear phagocyte (macrophage) colonies by the liquid culture technique with an incubation period of 7–8 days, about 100 macrophage colonies were produced from 1 × 106 cells. The number of macrophage colonies appearing after 2 days of incubation was small, but thereafter increased progressively up to at least 8 days. In the later stages of incubation (after day 6) large colonies consisting of more than 100 cells appeared. Macrophage colonies in the early stages consisted almost solely of macrophages. On day 6 significant numbers of small round mononuclear cells with no detectable phagocytic activity were seen in the center of large colonies, and by day 8 marked crowding of these cells had occurred. The peripheral region of the large colonies consisted mainly of macrophages and the intermediate region of middle‐sized round or slightly stretched cells with weak phagocytic activity. Approximately two‐thirds of the colony‐forming cells still remained after glass‐adherent cells were removed from the spleen cells by passing over a glass‐bead column. In cultures of glass‐nonadherent cells macrophage colonies were not generated in the early stage. The number of colony‐forming cells did not change significantly even after actively phagocytic cells were rigorously removed from the spleen cells. In addition, no macrophage colonies were generated in cultures of spleen cells treated with mitomycin C.
Sugar Composition of O‐Antigenic Lipopolysaccharides Isolated from Vibrio parahaemolyticus
Authors: Hisatsune Kazuhito , Kiuye Akira , Kondo Seiichi
Published: November 2013

Vibrio parahaemolyticus, a causative bacterium of food poisoning unique for its particular primary association with sea products, is now divided serologically into 11 or 12 O‐forms based on agglutination and agglutinin‐absorption tests. We determined the sugar composition of the somatic O‐antigens, i.e., lipopolysaccharides (LPS), of representative strains of each O‐form. Of particular interest is the absence of evidence for the presence of 2‐keto‐3‐deoxy‐octonic acid (KDO), a regular sugar component of gram‐negative bacterial LPS, in any LPS examined, with the exception of 06. Furthermore, 07 and 012 LPS contained a KDO‐like compound that is, however, not identical with KDO. Glucose, glucosamine, and L‐glycero‐D‐mannoheptose were found as common sugar constituents. Three unidentified amino sugars, designated here as P1, P2, and P3, were found. Various combinations of each of these unidentified amino sugars, and of galactose, fucose, arabinose, D‐glycero‐D‐mannoheptose, galactosamine, KDO, and the KDO‐like substance were detected in accordance with the O‐form of LPS. On the basis of the sugar composition, LPS of the 12 O‐forms of V. parahaemolyticus can be classified into nine chemotypes, because 03, 05, and 011 LPS belong to the same chemotype and 07 and 012 to another chemotype.
Numerical Changes in T Cell Subsets (Tγ and Tμ) in Leprosy Patients
Authors: Izumi Shinzo , Sugiyama Kazuko , Matsumoto Yoshiko , Nagai Takashi
Published: November 2013

Eighty‐six leprosy patients (49 active lepromatous, 24 inactive lepromatous, 7 borderline, and 6 tuberculoid) and nine healthy controls were examined for numerical changes in T cell subsets (Tγ and Tμ), and complement levels in peripheral blood to determine the roles of T cell subsets and complement in the etiology of leprosy.

The percentage and number of Tγ and Tμ cells showed no significant differences among the different clinical groups, but 4 out of 49 active lepromatous, 3 out of 24 inactive lepromatous and 3 out of 7 borderline cases showed a high percentage of Tγ cells.

Serum concentrations of C4, C3c, and C3 activator, an important factor in the alternative pathway of complement activation, were not significantly different among the groups. However, C3 activator and C3c concentrations were significantly high in active lepromatous patients complicated by an immune complex disease called “erythema nodosum leprosum” (ENL) compared with ENL‐free active lepromatous leprosy.
Studies on the Cross‐Resistance of Mycobacterium tuberculosis, Strain H37Rv, to Aminoglycoside‐ and Peptide‐Antibiotics
Authors: Tsukamura Michio , Mizuno Shoji
Published: November 2013

Various phenotypes of the resistance to aminoglycoside‐ and peptide‐antibiotics of Mycobacterium tuberculosis strain H37Rv were produced by single‐ and/or two‐step selection of the parent strain. Mutants obtained by single‐step selection with antibiotics were classified into ten phenotypes; one of single resistance, two of triple resistance, three of quadruple resistance, and four of sextuple resistance. There were two kinds of sextuple resistance (high resistance to enviomycin, viomycin, capreomycin, kanamycin, lividomycin, and paromomycin). One was isolated from the parent strain by single‐step selection and could be eliminated by mutation to isoniazid resistance, the other was obtained by two‐step selections and was not eliminated by mutation to isoniazid resistance. Interaction between mutation to streptomycin resistance and mutation to quadruple resistance (4R phenotype) was observed. Streptomycin resistance interfered with the formation of the 4R phenotype and produced a different phenotype, KR instead of the 4R phenotype. The existence of mutation of the 4R phenotype did not usually interfere with mutation to streptomycin resistance, but a small portion of the mutants with the 4R phenotype were altered in their phenotype from 4R to KR after addition of the mutation to streptomycin resistance. This effect of the mutation to streptomycin resistance was not observed in mutants which already had a mutation to klR phenotype (mutation to low concentrations of kanamycin only).
Purification and Some Properties of β‐Lactamases from Proteus rettgeri and Proteus inconstans
Authors: Ohya Satoshi , Fujii‐Kuriyama Yoshiaki , Yamamoto Mitsuyo , Sugawara Shinichi
Published: November 2013

Two β‐lactamases were isolated from strains of Proteus species and purified, one from a strain of P. rettgeri and the other from a strain of P. inconstans. Each enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis. Molecular weights of P. rettgeri and P. inconstans enzymes were found to be 42,000 and 43,000, and their isoelectric points pH 8.7 and 8.6, respectively. The two enzymes presented typical cephalosporinase profiles. Cefmetazole (CS‐1170) and cefoxitin, both cephamycin antibiotics, not only resisted hydrolysis by both of the enzymes, but also inhibited their activities competitively. Rabbit antiserum against purified P. rettgeri enzyme inhibited the activity of both purified and crude enzyme preparations from other strains of P. rettgeri so far tested. None of the β‐lactamases produced by other species of Proteus including P. inconstans was inhibited by the antiserum, thus showing that the purified cephalosporinase was of the species‐specific type. The enzymological properties of the preparations were compared with those of β‐lactamases derived from other gram‐negative enteric bacteria.
A Mouse Model for the Pathogenesis and Postexposure Prophylaxis of Rabies
Authors: Mifune Kumato , Shichijo Akehisa , Marino Yoshihiro , Takeughi Eriko , Yamada Akira , Sakamoto Kuniaki
Published: November 2013

A mouse model for the study of postexposure prophylaxis of rabies was established. Mice injected intramuscularly with a street strain of rabies virus were significantly protected from death by five daily 0.2‐ml doses of inactivated rabies vaccine of chick embryo cell culture origin initiated immediately or 3 hr after infection. In these mice, a large amount of circulating interferon was induced as early as 1 hr after the first dose of vaccine and lasted until at least 12 hr but no such amount of interferon was induced by additional doses of vaccine. Serum antibody was first detected in the mice on day 6. It was noted that some of the surviving mice manifested an ataxia or paralysis of the legs. Increasing mortality rates were shown in mice treated with decreasing doses of the vaccine. Passive protection tests using concentrated IgG and IgM antibodies with equivalent neutralization titers showed that IgG antibody gave total protection when given 24 hr before the infection, while it was almost totally ineffective in reducing the mortality when given 2 days or more after infection. IgM antibody did not protect the mice even when given 24 hr before infection. These results suggest that interferon production is more important than antibody production in the initial stages of protection by postexposure vaccination. However, the mechanisms of postexposure prophylaxis in this model could not be explained only by the interferon produced by the vaccine and the possible contributions of additional mechanisms were suggested.
Enhanced Production of Virus‐Inhibiting Factor (Interferon) in Human Diploid Cells by Ultraviolet Irradiation and Temperature Shift‐Down after Stimulation with Newcastle Disease Virus
Authors: Maehara Nobutoshi , Komatsu Haruko , Shimoda Kazunobu , Marino Satoshi , Nagano Yasuiti , Matumoto Minoru
Published: November 2013

The production of the virus‐inhibiting factor or interferon (IF) was highest in cells incubated at 37 C after inoculation with Newcastle disease (ND) virus and decreased as the incubation temperature was lowered. Shift‐down of incubation temperature to 32 C or 34 C after incubation at 37 C for 4–7 hr enhanced IF production in cell cultures stimulated with ND virus, as compared with cultures incubated continuously at 37 C. Shift‐down to 32 C after incubation at 37 C for 6 hr. was optimal for this enhancement of IF yield. Enhanced IF production was also observed in cell cultures irradiated by ultraviolet light 4–7 hr after stimulation with ND virus.
Effects of Dextran Sulfate 500 on Protective Responses to Sublethal Trichomonas foetus Infection in Mice
Authors: Oka Yoshikazu , Hayashi Hiromi , Ishikawa Fujiro , Osaki Humio
Published: November 2013

In order to analyze the host‐parasite interactions in experimental trichomoniasis, the growth of Trichomonas foetus in the peritoneal cavity and changes in the peritoneal exudate cells were followed in mice treated with dextran sulfate 500 (DS 500), a known macrophage‐toxic agent. Light microscopic observation showed that DS 500 treatment induced degeneration of peritoneal macrophages within about 48 hr after the treatment and the damaged macrophages did not phagocytize the parasites, whereas peritoneal neutrophils and lymphocytes were not affected by the drug. In the DS 500‐treated mice, growth of parasites in the peritoneal cavity was accelerated and a high susceptibility of the mice to T. foetus infection was observed. These results indicate that macrophages play the most important role among the peritoneal exudate cells in resistance to T. foetus infection, especially during the early stage of infection.
Polyclonal Antibody Production in Murine Spleen Cells Induced by Staphylococcus
Authors: Nakano Masayasu , Toyoda Hideko , Tanabe Masao J. , Matsumoto Takao , Masuda Shogo
Published: November 2013

Polyclonal plaque‐forming cell (PFC) responses in murine spleen cells induced by Staphylococcus aureus and S. epidermidis were studied. Injection of Balb/c mice with S. aureus strain 248βH resulted in the generation of anti‐trinitrophenyl (TNP) and anti‐sheep red blood cell PFC in their spleens. Cultures of Balb/c spleen cells in the presence of S. aureus 248βH, Cowan I, or a protein A‐deficient mutant yielded many anti‐TNP PFC. The larger the number of organisms that were added to the cultures, the better was the PFC response. Both living and killed organisms, were capable of inducing the response, but an excess of living 248βH organisms in the cultures abrogated the response. All of the organisms (12 strains of S. aureus and 11 strains of S. epidermidis) freshly isolated from patients had the ability to induce the polyclonal PFC response in cell cultures.

These organisms stimulated cultured C3H/HeJ mouse spleen cells, which were unresponsive to bacterial lipopolysaccharide (LPS). Cultured cells from the spleens of athymic nu/nu mice also responded to these organisms, and the number of PFC in nu/nu cell cultures was always greater than that in nu/+ cells prepared from a haired litter mate. Moreover, the responses of nu/nu spleen cell cultures to which nylon wool column‐filtered splenic nu/+ T cells were added were lower than expected. These findings suggest that the polyclonal PFC response to staphylococci is thymus independent, but that the magnitude of the response is regulated by mature T cells. Cultures of macrophage‐depleted spleen cells responded to the organisms to an extent similar to that of the control.

The 248βH organisms were less capable of stimulating spleen cells of 2‐week‐old mice (i.e., early maturing B cells) than LPS. However, spleen cells from adult (7‐week‐old) and aged (9‐month‐old) mice responded well to both the organisms and LPS. Previous sensitization with the organisms in vivo did not affect any polyclonal responses of spleen cells in vitro to either the organisms or LPS. The role of staphylococcal protein A in the polyclonal PFC response to staphylococci is discussed.
Adherence of Yersinia enterocolitica to Mammalian Epithelial Cell Lines
Authors: Okamoto Keinosuke , Inoue Takashi , Ighikawa Hidetaka , Kawamoto Yasuko , Hara Susumu , Miyama Akio
Published: November 2013

Yersinia enterocolitica RIMD 2501003 grown at 25 C avidly adhered to various kinds of cultured epithelial cell lines (HeLa, FL, Y‐1 adrenal, human intestine, human conjunctiva) but the bacteria grown at 37 C did not adhere. This phenomenon paralleled the temperature‐dependent motility of the bacteria. To clarify the adherence mechanism, we obtained two kinds of mutants, an immobile mutant and a nonadherent mutant, by treatment with A‐methyl‐A‐nitro‐A‐nitrosoguanidine. The immobile mutant did not move on soft agar but retained the capacity to adhere to cultured epithelial cells when grown at 25 C. The nonadherent mutant did not adhere to cultured epithelial cells but retained the ability to move on soft agar when grown at 25 C.

When the bacteria were killed by heat, ultraviolet light irradiation or formaldehyde they lost their capacity to adhere to the cultured epithelial cells.

Antiserum against Y. enterocolitica RIMD 2501003 grown at 25 C was absorbed with the bacteria grown at 37 C, with the bacteria grown at 25 C, with the nonadherent mutant grown at 25 C and with the bacteria killed by various means. Only the antiserum absorbed with bacteria grown at 37 C inhibited the adherence of bacteria.

These data indicate that motility does not correlate with adherence of Y. enterocolitica. It appears that the adherence factor involves both a temperature‐dependent surface factor and a factor synthesized de novo during the interaction of susceptible cells with the bacteria.
Mode of Subacute Sclerosing Panencephalitis (SSPE) Virus Infection in Tissue Culture Cells
Authors: Ohughi Masanobu , Ohuchi Reiko , Homma Morio
Published: November 2013

Cell‐free infectious viruses were successfully recovered by the aid of freezing and thawing from cultures infected with the Kitaken‐1 and Biken strains of subacute sclerosing panencephalitis (SSPE) virus. Our results including those in a previous report which dealt with the Niigata‐1 strain of SSPE virus show that cell‐free viruses can be detected from all of the SSPE virus‐carrying cultures established in Japan. It was also found that cell‐free infectious viruses can be recovered efficiently by dispersing the virus‐carrying cultures with EDTA. The inclusion of trypsin in the EDTA solution, however, caused a poor recovery of the infectious viruses. Infection of cells with the cell‐free viruses readily established the virus‐carrying cultures that have characteristics comparable to those of their original cultures. The culture infected with the Kitaken‐1 strain produced infectious viruses in about ten times the amount of the other two infected cultures. The buoyant densities of the cell‐free infectious viruses were almost the same among the three strains, the values being 1.120 to 1.132, but significantly less than that of 1.164 of measles virus. The low density can be ascribed to one of the characteristics of these SSPE viruses.
Mitogenic Activity of Cytoplasmic Membranes Isolated from L‐Forms of Staphylococcus aureus
Authors: Takada Haruhiko , Hirachi Yoshiyuki , Hashizume Hideo , Kotani Shozo
Published: November 2013

Cytoplasmic membranes of L‐forms of Staphylococcus aureus exerted a strong mitogenic effect on splenocytes of athymic nude mice as well as normal mice, while a cytoplasmic fraction of the same bacteria did not show definite mitogenicity. The mitogenic principle(s) of the membrane fraction was resistant to treatment with trypsin and was heat stable (at 100 C for 10 min). The active principle(s) in the insoluble residue of the membrane fraction digested with trypsin was not extracted with cold acetone, but could be solubilized by extraction with a cold chloroform‐methanol mixture (2:1, v/v). The mitogenic principle(s) in the extract was fractionated by silicic acid column chromatography. Among five fractions separated by chromatography, fractions eluted with chloroform‐methanol mixtures (1:1 and 1:20, v/v) were found to be strongly mitogenic.

The cytoplasmic membranes of the L‐forms also exerted a definite mitogenic effect on guinea pig splenocytes, but not on the thymocytes.
Drug Resistance and R Plasmids in Escherichia coli Strains Isolated from Imported Pet Birds
Authors: Nakamura Masayuki , Fukazawa Mitsuo , Yoshimura Haruo , Koeda Tetsuo
Published: November 2013

Drug resistance in Escherichia coli strains isolated from pet birds (mynahs, macaws, finches, common bengals, parrots, and flamingos) imported into Japan from 10 foreign countries in 1977 and 1978 was investigated. Of the 309 strains isolated from 127 pet birds in the Animal Quarantine Service, 232 (75.1%) were drug resistant. Furthermore, strains resistant to oxytetracycline hydrochloride, dihydrostreptomycin, and sulfadimethoxine were relatively common. Resistance patterns varied from single to sextuple resistance, and 148 (63.8%) of the resistant strains had conjugative R plasmids. These results suggest that the high incidence of drug resistance and R plasmids in E. coli strains isolated from these pet birds may be a reflection of the prophylactic use of antibiotics for the prevention of diseases which increasingly occur with importation of the birds. Furthermore, the results suggest that the birds may be potential reservoirs of drug‐resistant E. coli for families who raise and have intimate contact with such birds.
Restriction Endonuclease Cleavage Maps of the Ampicillin Transposons Tn2601 and Tn2602
Authors: Yamamoto Tomoko , Yamagata Somay , Hashimoto Yohko , Yamagishi Saburo
Published: November 2013

This paper reports the cleavage maps of ampicillin transposons Tn2601 and Tn2602, for restriction endonucleases BamHI, PvuII, AvaI, HincII, and HaeII. Both of the transposons are very similar to the well‐known ampicillin transposon Tn3 in size, endonuclease cleavage sites, and possession of a short inverted repeat sequence at both ends. A slight difference in the cleavage pattern among these three transposons was observed in the region around the BamHI site which was assumed to be a part of the repressor gene for transposition.
Effect of Lysozyme on Mycobacteria
Authors: Kanetsuna Fuminori
Published: November 2013

The effect of lysozyme on the growth of several strains of mycobacteria was examined at pH 5.0–7.0 in Dubos medium containing various concentrations of lysozyme (100–2,000 μg/ml). Mycobacterium smegmatis and M. phlei were susceptible to lysozyme at pH 5.0–7.0. The effect of lysozyme was marked between pH 6.0 and 7.0 and the colony counts were reduced to approximately 0.1–10% after incubation with 100 μg of lysozyme per ml for 48 hr. At pH 5.0, 10–40% of the organisms survived treatment with 1,000 μg of lysozyme per ml for 48 hr. M. bovis strain BCG, M. tuberculosis, and M. fortuitum appeared to be more resistant to lysozyme than M. smegmatis and M. phlei.

M. smegmatis and M. phlei did not contain detectable amounts of poly‐L‐glutamic acid, although the susceptibility of the mycobacteria to lysozyme did not correlate with the amounts of the polymer in the cell walls.

The role of lysozyme in animal infections with so‐called saprophytic mycobacteria is discussed.
Estimation of Type‐Specific Neutralizing Antibody to Herpes Simplex Virus Type 2 in Uterine Cervical Cancer Patients by a New Absorption Method
Authors: Kawana Takashi , Yoshino Kamesaburo
Published: November 2013

A simple method of estimating type‐specific neutralizing antibody to type 2 herpes simplex virus (HSV‐2) was devised with the use of the microneutralization system. Serially diluted serum was mixed in the well with a constant amount of type 1 virus (HSV‐1), and after 3 days' incubation at 37 C, the plate was irradiated with ultraviolet light. The absorbing HSV‐1 consisted of culture fluid plus an extract of infected Vero cells not especially concentrated. The well then received indicator HSV‐1 or HSV‐2, and after being left at 37 C for 1 hr a suspension of dispersed Vero cells was dropped into the wells, following our standard neutralization procedure. Preliminary tests with rabbit antisera showed that even a low level of HSV‐2 antibody was detected by this method, unless an exceptionally high titer of HSV‐1 antibody originally coexisted with the HSV‐2 antibody. Sera from acutely infected persons testified to the specificity of the antibody so detected. It was revealed by means of the new technique that the rate of HSV‐2 antibody was significantly higher in uterine cervical cancer patients than in control women. There was no correlation between the clinical stage of cervical cancer and the presence of HSV‐2 antibody.
Low Responsiveness of Human Neonatal Lymphocytes to a B‐Cell Mitogen, Staphylococcus aureus Cowan I (SpA CoI)
Authors: Kasahara Tadashi , Harada Hirotomo , Shioiri‐Nakano Kohei , Itoh Yoshihisa , Kawai Tadashi , Hida Keita
Published: November 2013

Responses of neonatal and adult lymphocytes to various mitogens were studied. Lymphocytes from umbilical cord blood (UCB) responded well to both phytohemagglutinin and concanavalin A, and also to pokeweed mitogen and Staphylococcus aureus Protein A. The responses of UCB lymphocytes to these mitogens were not significantly lower than those of adult peripheral blood lymphocytes (PBL). In contrast, UCB lymphocytes showed only a minimal response to killed Staphylococcus aureus Cowan I (SpA CoI), a potent B‐cell mitogen for human PBL, although the proportion of B cells in UCB was not less than that in PBL. The low level of response of lymphocytes from UCB to SpA CoI was not ascribed to differences in dose response or kinetics. Purified B cells from UCB were not stimulated by SpA CoI either, suggesting that the low responsiveness was not due to the suppressive effect of T cells or macrophages, but to some intrinsic defect in B cells in UCB. These results suggest that the B cells in neonates may be more immature than the T cells.
Cell Growth and Antimicrobial Activity of Mouse Peritoneal Macrophages in Response to Glucocorticoids, Choleragen and Lipopolysaccharide
Authors: Nozawa Ryushi T. , Yanaki Nobuko , Yokota Takeshi
Published: November 2013

Normal, thioglycollate‐stimulated and BCG‐activated mouse peritoneal macrophages were cultivated in vitro with the conditioned medium of mouse L‐929 cells. The thioglycollate‐ and BCG‐macrophages rapidly proliferated, whereas normal macrophages grew more slowly. A clear morphological difference between the three types of macrophages in the culture was observed. Glucocorticoids inhibited the growth of the macrophages at pharmacological concentrations. Other steroids, progesterone, diethylstilbesterol and testosterone in that order, had a far lower growth‐inhibiting effect. Macrophages cultured with 10‐6 M dexamethasone had a reduced antimicrobial effect on Candida parapsilosis compared with that of the untreated cells. Choleragen had the same effect on the macrophages as glucocorticoids. The toxin inhibited growth at a concentration as low as 10 pg/ml and cells treated with 1 ng of choleragen per ml had decreased antifungal activity. Similarly, Escherichia coli lipopolysaccharide at 10 ng/ml inhibited the growth of thioglycollate‐macrophages. However, macrophages incubated with the lipopolysaccharide had enhanced anticandida activity. Thus, the immunosuppressors glucocorticoid and choleragen inhibited both the increase in the number of macrophages and the microbicidal activity of the phagocytes. Lipopolysaccharide, an immunostimulant, stimulated macrophage activity, but was toxic for cell growth.
Macrophage Activation by Muramyl Dipeptide (MDP) without Lymphocyte Participation
Authors: Nagao Shigeki , Miki Tomohiro , Tanaka Atsushi
Published: November 2013

An increase in the numbers of spread macrophages caused by macrophage stimulants was found to be a very sensitive measure for macrophage activation. Muramyl dipeptide (MDP), bacterial lipopolysaccharide (LPS), and lymphokines were found to activate macrophages dose‐dependently as measured by this parameter. Macrophage activation by MDP was strictly dependent on its adjuvant‐active stereochemically specific structures. Macrophage activation by MDP and LPS occurred without lymphocyte participation. It is suggested that LPS also activates macrophages via lymphocytes.
Mutual Relationship between Antibiotics and Resting Spores of Bacillus subtilis: Binding of Cyclic Polypeptide and Aminoglycoside Antibiotics to Spores and Their Inhibitory Effect on Outgrowth and Vegetative Growth
Authors: Tochikubo Kunio , Hayakawa Yoshiaki , Kojima Kiyohide
Published: November 2013

Not only cyclic polypeptide antibiotics such as polymyxin B, colistin and gramicidin S but also aminoglycoside antibiotics such as streptomycin, kanamycin, gentamicin and kanamycin derivatives combined with the resting spores of Bacillus subtilis and inhibited outgrowth or vegetative growth after germination. All the antibiotics other than gramicidin S were released from the resting spores and their inhibitory action was reversed by the addition of Ca2+ and Fe3+. As the above antibiotics have free amino (or guanidine) groups in common, it was assumed that such groups play an important role in binding of the antibiotics to the resting spores. Moreover, it was shown that protamine and poly‐l‐lysine were also bound to the resting spores and were released from them by Ca2+. On the other hand, free carboxyl groups had been demonstrated in the outermost surface of the resting spores in a previous study. Thus, we assume that the mode of binding of the antibiotics to the resting spores may be due to the formation of reinforced ionic bonds between amino (or guanidine) groups in the antibiotics and carboxyl groups on the spore surface.
A New Candidate Type of Human Enterovirus (Drilon Strain) Isolated in the Philippines
Authors: Nakao Jose C. , Hara Minoru , Tagaya Isamu , Beran George W. , Macasaet Francisco F.
Published: November 2013

A cytopathogenic agent was isolated in monkey kidney (MK) cell cultures from the stool specimen of a 3‐month‐old Filipina hospitalized with lower respiratory disease. The agent was designated the Drilon strain. It was characterized as an enterovirus on the basis of electron microscopic morphology, nucleic acid type (RNA), resistance to ether and acid (pH 3.0) treatments, stabilization by molar MgCl2 against heat inactivation, and buoyant density in CsCl. The strain caused mild febrile illness in experimentally inoculated cynomolgus monkeys, but not in suckling mice. In addition to its effect in primary MK cells, the virus was cytopathogenic in primary and secondary human amnion or embryonic lung cell cultures and in WI‐38 or HEp‐2 cell lines, but not in primary bovine kidney, primary porcine kidney, primary embryonic mouse or primary embryonic chick cell cultures. The Drilon strain was not neutralized by reference antisera against the known enterovirus serotypes, and the antiserum prepared with the Drilon strain did not neutralize any of the recognized prototype enterovirus strains. Although the patient's sera were not available, antibodies against the Drilon strain were prevalent in normal Filipinos and Indonesians, but not in Japanese people. The Drilon strain fulfilled the criteria of human enterovirus and is considered a candidate for designation as a new type.
Experimental Myocarditis Induced in Mice by Infection with Influenza A2 Virus
Authors: Sakamoto Masahiro , Suzuki Fujio , Arai Sumio , Takishima Tamotsu , Ishida Nakao
Published: November 2013

A mouse model was established for the study of acute myocarditis that occurs during influenza infection. Challenge with more than 10 LD50 of mouse‐adapted influenza A2 virus (H2N2) induced myocarditis macroscopically discernible as white, irregularly shaped lesions which were shown by histological examination to consist of necrotic myofibers surrounded by infiltrating mononuclear inflammatory cells. After challenge with 10 LD50 of the virus, macroscopic myocarditis was found to advance in a progressive manner up to the 7th day, while the virus titer in the heart reached its peak on the 2nd day and began to decrease on the 5th day of infection. However, development of myocarditis was significantly suppressed in mice which were irradiated with 400 R of X‐rays before infection. In addition, myocarditis did not develop in congenitally athymic nude mice.

These data indicate that myocarditis was not brought about by viral action directly, but that it was mediated by some function of the host against viral in‐vasion, which was abolished by X‐irradiation. The data also suggest that T cells played a key role in the development of myocarditis.
Interferon‐Inducing Activity of an Immuno‐therapeutic Anticancer Agent, SSM, Prepared from Mycobacterium tuberculosis Strain Aoyama B
Authors: Hayashi Yoshiro , Ebina Takusaburo , Suzuki Fujio , Ishida Nakao
Published: November 2013

The interferon‐inducing capacity of arabinomannan‐lipid preparation (SSM) extracted from Mycobacterium tuberculosis Aoyama B in both BCG‐sensitized and unsensitized mice was studied in comparison with that of purified protein derivative (PPD) prepared from the same tubercle bacillus. Although it is known that PPD cannot stimulate interferon production in BCG‐unsensitized mice, interferon activity was found in sera of both groups of mice after intravenous injection of SSM at a dose of 5 mg/kg. The maximum titer was detected 5 hr after injection. The interferon induced by SSM in both groups of mice shared certain physicochemical properties with the immune interferon induced by PPD in BCG‐sensitized mice. In BCG‐unsensitized mice, interferon induction by SSM was markedly inhibited by pretreatment with trypan blue and carrageenan, whereas it was not depressed in BCG‐sensitized mice given the same treatment or when interferon was induced by PPD. In addition, induction of interferon in BCG‐sensitized mice by SSM and PPD and in unsensitized mice by SSM was completely abrogated by pretreatment with hydrocortisone acetate and whole‐body x‐irradiation (700 R). These results suggest that in BCG‐unsensitized mice macrophages, in addition to X‐ray or hydrocortisone‐sensitive cells, may be required for interferon induction by SSM.
A Possible Role of R Plasmids in Bacterial Permeability for β‐Lactam Antibiotics
Authors: Ikeuchi Toru , Osada Yasuaki
Published: November 2013

Four strains of clinical isolates of Serratia marcescens (13039, 13090, 13093, 14093) harboring R plasmids were highly resistant to ampicillin (ABPC) and cephaloridine (CER). With elimination of R plasmids from these strains by acriflavine treatment, ABPC‐resistance levels of these strains were markedly reduced. Reduction of CER‐resistance levels was also demonstrated in strains 13039 and 13093, but not in strains 13090 and 14093. The permeability of the former strains for CER was also decreased, but not in the latter strains. At the same time, β‐lactamase activity of these strains also almost completely disappeared when the R plasmids were eliminated. By broth matings with these strains, the recipient strains of S. marcescens 13031 (rif), Escherichia coli K‐12 (rif), and E. coli 15046 (rif) all acquired a high permeability barrier against CER with inheritance of the R plasmids from strains 13039 and 13093, but not from strains 13090 and 14093. The transconjugant of strain 13031 that inherited R plasmid 13093 was resistant not only to CER but also to cefazolin, cephalothin, and cephalexin. Its permeability to these antibiotics was significantly lower than that of the original strain. This fact suggests the possibility that the R plasmid from strain 13093 may be involved not only in production of β‐lactamases, but also in regulation of bacterial permeability for cephalosporins.
Clinical Studies on Cell‐Mediated Immunity in Gestational Trophoblastic Disease: Nonspecific Cellular Immunocompetence in Patients with Hydatid Mole and Trophoblastic Neoplasia
Authors: Sasaki Toshio , Fujisaki Syunichi
Published: November 2013

The nonspecific cell‐mediated immunocompetence of 51 patients with gestational trophoblastic disease (GTD), including 16 patients with hydatid mole (HM), 24 with nonmetastatic trophoblastic neoplasia (NTN) and 15 with metastatic TN (MTN), was studied with the use of both in vitro and in vivo parameters of cell‐mediated immunity (CMI) such as lymphocyte blastogenic response to phytohemagglutinin (PHA), subpopulation constitution, and delayed cutaneous hypersensitivity responses to 2,4‐dinitrochlorobenzene (DNCB) and to purified protein derivative of tuberculin (PPD).

The pretreatment cell‐mediated immune status of patients with HM developing no malignant sequelae was shown to be essentially similar, in terms of both in vitro PHA and in vivo DNCB reactivities, to that of normal women and of patients with benign gynecological diseases. In patients with TN, however, there was a significant depression in the blastogenic lymphocyte response to PHA before evacuation of the mole, which was persistently demonstrated after uterine evacuation and more marked throughout the course of disease in patients with MTN than in those with NTN, with a tendency to return to normal in remission. Moreover, patients with TN had a significant depletion of T lymphocytes as determined by rosette‐forming cell procedures before treatment, which was most evident in patients with MTN. Plasma from the MTN patients was also shown to have an inhibitory effect on PHA responsiveness of lymphocytes from normal women.

There was an increased incidence of impaired reactivity to DNCB in patients with TN (higher in MTN than in NTN), compared with HM and benign diseases, while no such difference in incidence was observed in response to PPD.

On the basis of these findings, a preliminary characterization of altered immunocompetence in patients with TN and its mechanism are discussed.
Induction of Transformed Phenotypes in Sheep Fibroblasts by Culture Fluids from Cells Persistently Infected with Bovine Leukemia Virus
Authors: Onuma Misao , Watarai Shinobu , Suneya Masahiko , Mikmi Takeshi , Izawa Hisao
Published: November 2013

FLK cells are fetal lamb kidney cells persistently infected with bovine leukemia virus (BLV). 3178 cells, originating from calf‐form bovine lymphosarcoma, also showed persistent production of BLV and alteration of cell morphology after treatment with 5'‐iodo‐2'‐deoxyuridine. In the present paper, the first in vitro transformation of sheep fibroblasts by inoculation with BLV materials from these two cell lines is described. In a few passages after inoculation with these viral materials, morphological alteration occurred. The morphologically altered cells were grown as stable cultures and showed such transformed phenotypes as growth in soft agar medium, increased uptake of 2‐deoxy‐d‐glucose and tumorigenicity in athymic nude mice. This result, together with our previous observation of simultaneous induction of BLV expression and morphological alteration in 3178 cells, suggests the presence of some transforming capacity in these BLV materials similar to that in, for example, murine or avian acute leukemia viruses. The possible acquisition of such capacity during the prolonged passage is discussed.
Proliferative Response of Bone Marrow Cells to Concanavalin A Does Not Require Ia Antigen‐Positive Cells
Authors: Tanabe Masao J. , Nakano Masayasu
Published: November 2013

The in vitro proliferative response of murine bone marrow cells to concanavalin A (Con A) and the effect of anti‐Ia serum on the response were studied. The incorporation of [3H]thymidine into cells prepared from the bone marrow of C3H/He, ATL, ATH, and C57BL/6 mice increased in the presence of certain doses of Con A. The bone marrow cells of athymic nude mice were also capable of responding to Con A, but cells prepared from the spleens of such mice were not. The addition of anti‐Ia serum to the cultures of bone marrow cells did not affect the responses of these cells to Con A, though their proliferative response to bacterial lipopolysaccharide was greatly reduced in the presence of the serum. Moreover, pretreatment of the bone marrow cells with anti‐Ia serum or anti‐Thy. 1.2 serum and rabbit complement did not abolish the ability of these cells to respond to Con A. These results indicate that there are some Ia negative and Thy. 1.2 negative cell populations in the marrow capable of responding to Con A. Furthermore, the effect of anti‐Ia serum on the Con A‐induced proliferative response of the spleen cells which had been obtained from γ‐irradiated and syngeneic bone marrow cell‐reconstituted mice was examined. The ability of these cells to respond to Con A increased gradually week by week after the reconstitution. The suppressive effect of anti‐Ia serum on the response of these cells gradually became much more pronounced after the reconstitution.
The Influence of Immunization with Live or Killed Staphylococcus aureus Vaccines on the Early Development of Opsonizing and Bactericidal Factors in Lymph and Blood of Sheep
Authors: Kennedy John W. , Watson Dennis L.
Published: November 2013

In order to obtain sensitive measurements on the synthesis of opsonins following immunization with live or killed S. aureus vaccines, lymph was collected from the efferent popliteal lymphatic duct of sheep during the early phase of the immune response. Lymph and blood serum were assayed for opsonizing capacity using 3H‐labeled S. aureus.

Within 1 hr after vaccination there was a rapid, transitory decrease in uptake by neutrophils of bacteria opsonized with lymph from sheep given the killed vaccine (Group 2). These results were in contrast to the relatively constant uptake rates of bacteria opsonized with lymph from sheep given the live vaccine (Group 1) and non‐vaccinated controls (Group 3) at this time. At 72, 96, and 120 hr post‐injection mean uptake values for bacteria opsonized with lymph from either vaccinated group were significantly greater than comparable values for controls.

Mean uptakes for organisms opsonized with blood serum from Group 1 at 72 and 96 hr post‐injection were significantly greater than comparable values for the control group. The percentage of viable neutrophil‐associated bacteria decreased when lymph collected from animals in Group 2 in the first hour post‐injection was used to opsonize the organisms.

Percentages of viable, neutrophil‐associated S. aureus for assays in which blood serum was used to opsonize remained relatively constant at around 45% for Groups 2 and 3. In contrast, however, values of viable neutrophil‐associated bacteria for Group 1 decreased during the 120 hr after immunization.