bluuebunny/crossref_metadata_2025 huggingface dataset:
Interferon and Cytotoxic Factor (Cytotoxin) Released in the Blood of Mice Infected with Mycobacterium bovis BCG
Authors: Kato Nobuo , Nakashima Izumi , Ohta Michio , Nagase Fumihiko , Yokochi Takashi , Naito Setsuko
Published: November 2013

The time course of development and decline of the ability of BCG‐infected mice to produce interferon in the serum in response to the intravenous injection of purified protein derivative of tuberculin (PPD) was very similar to that of their systemic hypersensitivity to PPD. A cytotoxic factor (cytotoxin) was produced in parallel with interferon in the serum of BCG‐infected mice after stimulation with PPD. The duration of the period in which cytotoxin‐production responsiveness to PPD was definitely detectable was much shorter than that for interferon‐production responsiveness although the periods for the maximum production of interferon and cytotoxin coincided. The kinetics of release of interferon in the serum of BCG‐infected mice after stimulation with PPD did not parallel that of release of cytotoxin.

The four kinds of activities, interferons and cytotoxins induced by PPD and lipopolysaccharide (LPS) in the serum of BCG‐infected mice, were compared for their stability to heating at 56 C and to treatment at pH 2. The kinetics of inactivation of these four activities differed significantly, when the serum was either heated at 56 C or treated at pH 2. Interferon produced in response to LPS could be neutralized by anti‐L cell(NDV) interferon rabbit serum as easily as L cell (NDV) interferon, 16 times as much antiserum was required to neutralize the same amount of interferon in response to PPD, but cytotoxins induced by PPD and LPS were not neutralized at all by the antiserum. From these findings it is thought likely that interferons and cytotoxins induced by PPD and LPS in the serum of BCG‐infected mice are different substances, although the antigenic relationship between cytotoxins induced by PPD and LPS remains unknown.
Studies on the Neutralization of Herpes Simplex Virus
Authors: Yoshino Kamesaburo , Urayama Mari , Abe Kazuko
Published: November 2013

Late IgG and IgM from a rabbit immunized with herpes virus were tested for ordinary neutralizing (N) antibody, complement‐requiring neutralizing (CRN) antibody and in addition CRN antibody detectable by overnight sensitization at 0 C (s‐CRN antibody). Heat stability tests showed that IgG s‐CRN antibody was slightly less resistant to heating at 70 C than were N and CRN antibodies, whereas all three activities of IgM were quickly degraded at this temperature. Dose‐response curves with varying amounts of complement (C) or anti‐antibody revealed a marked difference between IgG s‐CRN and IgM s‐CRN antibodies. While 1‐hr sensitization at 37 C was insufficient to detect IgG s‐CRN antibody, it had the same effect as overnight sensitization at 0 C for IgM s‐CRN antibody. When sensitization at 0 C was prolonged to 3 days, unexpectedly high endpoints exceeding 1:10,000 were obtained even with IgM. Consequently, enhancement by C was several hundred‐fold with IgM in contrast to 5‐ to 10‐fold enhancement of IgG s‐CRN antibody, which was similar to that after overnight sensitization. Also IgM obviously required more C than did IgG. These results suggest that IgM of late serum is slower reacting and more C‐dependent than IgG s‐CRN antibody. Tests with early rabbit sera indicated that the s‐CRN antibody detectable by 3‐day sensitization reaches a high level before the appearance of N antibody.
Production and Properties of Immune Interferon from Spleen Cell Cultures of Toxoplasma‐Infected Mice
Authors: Shirahata Toshikazu , Shimizu Kiheiji
Published: November 2013

In response to antigenic stimulation, spleen cells from Toxoplasma‐infected mice produce a factor showing inhibitory activity against vesicular stomatitis virus infection in L cell cultures. When BALB/C and ICR mice were inoculated intraperitoneally with the low‐virulent S‐273 strain of T. gondii, such activity was first detected in 4 and 7 days and reached maximum levels at 10 and 14 days respectively, and retained these levels for at least three weeks. However, BALB/C mice, which are considerably more sensitive to Toxoplasma infection than ICR mice, produced significantly smaller amounts of interferon (IF) after challenge with the high virulent strain.

The IF produced in this system possessed certain known properties of immune (type II) IF and was not neutralized by rabbit antiserum against mouse type I IF. The immune IF preparation also inhibited multiplication of Toxoplasma within nonphagocytic L cells in an IF‐like fashion, whereas Newcastle disease virus‐induced (type I) IF had no effect on this parasite. The antiviral and anti‐Toxoplasma activity in immune IF preparations could not be distinguished solely on the bases of their molecular weight and isoelectric point. The experiments with anti‐θ serum plus complement and with nylon wool column effluent cells strongly suggest that immune IF was produced by T lymphocytes and required the assistance of macrophages.
B Cell Tolerance
Authors: Uchiyama Takehiko
Published: November 2013

The mechanisms of B cell tolerance were studied in an attempt to learn whether B cells rendered tolerant are present in the immune system in a potentially responsive form. The author tested the in vitro anti‐trinitrophenyl (TNP) antibody‐forming cell (anti‐TNP AFC) response to TNP‐immunogens and polyclonal B cell activators (PBA) of spleen cells taken from mice injected with a tolerogen, TNP‐carboxymethylcellulose (TNP‐CMC). Spleen cells from mice injected 5 days previously with 10 μg of TNP‐CMC did not respond to TNP‐sheep red blood cells (TNP‐SRBC), T‐dependent (TD) antigen or TNP‐Ficoll, T‐independent (TI) antigen. However, the same spleen cells responded to PBA, lipopolysaccharide (LPS) of Salmonella enteritidis and purified protein derivative (PPD) of BCG. The results indicate that B cells specific for TNP are present in a potentially responsive form. Spleen cells from mice injected with 500 μg of TNP‐CMC did not respond to either TNP‐immunogens or PBA. The state of unresponsiveness to PBA lasted for 12 days after the tolerogen injection. Responsiveness to PBA reappeared within the short period of 2 days, whereas unresponsiveness to TNP‐immunogens lasted much longer. Unresponsiveness to PBA was relieved considerably by treating tolerant spleen cells with the proteolytic enzyme trypsin before in vitro stimulation. These results indicate that B cells rendered refractory are present in the immune system in a potentially responsive form.
Formation of Mononuclear Phagocyte (Macrophage) Colonies by Mouse Spleen Cells in Liquid Culture
Authors: Yokochi Takashi , Nakashima Izumi , Ohta Michio , Hasegawa Takaaki , Fujii Yasuaki , Kato Nobuo
Published: November 2013

The effect of the capsular polysaccharide of Klebsiella pneumoniae type 1 Kasuya strain (CPS‐K) on the formation of macrophage colonies in cultures of mouse spleen cells was investigated by the liquid culture technique during an incubation period of 7–8 days. CPS‐K markedly inhibited further generation of macrophage colonies when added at any time after the beginning of culture, whereas it showed no destructive effect on macrophage colonies which were already formed before its addition. When CPS‐K was present throughout the incubation period, such a low concentration as 0.05 μg/ml significantly inhibited colony formation, and the intensity of its inhibitory effect depended on its dose in the range of 0.005–50 μg/ml. The inhibitory effect persisted even if CPS‐K was washed out after spleen cells were kept in contact with 20 μg of CPS‐K per ml at 37 C for 6 hr. It was found that the inhibitory effect of CPS‐K on colony formation was not mediated through its action on T cells, B cells or macrophages, and that it was not due to the generation of suppressor cells capable of inhibiting colony formation. It is concluded therefore that CPS‐K directly inhibits the proliferation of macrophage colony‐forming cells. The active substance responsible for the inhibitory effect of CPS‐K on colony formation is the neutral polysaccharide fraction of CPS‐K.
Effect of Monovalent Cations on the Aggregation of Tobacco Mosaic Virus by Chondroitin Sulfate
Authors: Sano Yoh , Inoue Hideo
Published: November 2013

The mechanism of precipitation of tobacco mosaic virus by chondroitin sulfate in the presence of various monovalent cations was investigated kinetically by means of turbidimetry. The virus solution became turbid on the addition of chondroitin sulfate, and led to separation of the virus as a crystalline phase. In the presence of monovalent cations the degree of precipitation of the virus by chondroitin was reduced with the increase in monovalent cation concentration. The order of the reduction was Li+<Na+<K+, which is reversely analogous to the lyotropic series. The least hydrated cation, K+, reduced the degree of precipitation of tobacco mosaic virus the most because the radius of the sphere equivalent to chondroitin sulfate was diminished most strongly by K+ ion. The effect of the monovalent cations on the precipitation supports our assumption that the virus precipitation results from incompatibility and mutual spatial exclusion between the virus and chondroitin. The maximum turbidities, the initial slopes, and the aggregation half‐time were measured by varying the monovalent cation concentrations. The former two parameters decreased with the increase in cation concentration, whereas the aggregation half‐time increased. A lag time was present and the effect of the cations increased in the order K+<Na+<Li+.
On the Immunogenicity of Ribosomes and Ribosomal Proteins Isolated from Klebsiella pneumoniae and Streptococcus pneumoniae
Authors: Robert Daniel , Michel Serge , Ivanoff Bernard , Cozzone Alain J. , Fontanges Robert
Published: November 2013

The two pathogenic species Streptococcus pneumoniae and Klebsiella pneumoniae were used to analyze the immunogenic role of proteins in ribosomal preparations. The protective activity of ribosomes prepared from either strain and further purified by washing with high‐salt concentrations, followed or not by sucrose gradient separation of the particles, was identical to that of crude unwashed ribosomes. Similarly, no substantial alteration of the level of protection was observed after treatment with the antibiotic puromycin. Therefore, the immunizing efficacy of ribosomes does not appear to be due either to the nonribosomal proteins adsorbed at the surface of organelles or to the growing polypeptide chain. It seems rather to be attributable to the structural ribosomal proteins themselves, which were indeed shown to induce alone a significant level of protection.
Studies on Thin‐Layer Chromatography after Incubation with [35S]Methionine as an Aid to Identification of Mycobacteria
Authors: Tsukamura Michio , Mizuno Shoji
Published: November 2013

Thin‐layer chromatography (TLC) of ethyl ether‐ethanol extracts of mycobacteria obtained after incubation with [35S] methionine is useful for differentiation among mycobacterial species, as the distribution of radioactive spots in TLC shows a characteristic pattern except for a few species, including M. intracellulare and M. gordonae. Some supplementary studies have been carried out in the present investigation and the following results have been obtained.

Pretreatment of bacterial cells with trichloroacetic acid (TCA) is required to remove radioactive sulfur‐containing amino acids.

The radioactive spots that appear in TLC of the ethyl ether‐ethanol extract are divided into the following groups: (1) Petroleum ether‐soluble fraction (the spot at Rf value 0.95); (2) acetone‐soluble (petroleum ether‐insoluble) fraction (the spots at Rf values 0.50, 0.30, and 0.13–0.20); (3) ethanol‐soluble (petroleum ether‐ and acetone‐insoluble) fraction (the spot at Rf value 0.10); (4) petroleum ether‐, acetone‐, and ethanol‐insoluble fraction (the spot at Rf value 0.00). The spot which appears in the region of Rf values 0.13–0.20 (it may appear as two spots) in the TLC of ethyl ether‐ethanol extracts is composed of two compounds, one soluble in acetone and the other insoluble in acetone but soluble in ethanol.

Strains of the M. avium‐M. intracellulare complex have been divided into three subgroups by the pattern of radioactive spots B, C, and D, which show Rf values of 0.13–0.20, 0.30, and 0.50, respectively, in the TLC of ethyl ether‐ethanol extracts. The first group shows none of these spots, and strains of the serotypes 1, 2, and 8 belong in this group. The second group shows spot B, and strains of the serotypes 4, 18, and 20 belong in this group. The third group shows spots B, C, and D, and strains of the serotype 16 belong in it.
Transformation of Bile Acids by Mixed Microbial Cultures from Human Feces and Bile Acid Transforming Activities of Isolated Bacterial Strains
Authors: Hirano Seiju , Masuda Noriyuki , Oda Hiroshi , Imamura Teisuke
Published: November 2013

Microbial transformation of cholic acid and chenodeoxycholic acid by anaerobic mixed cultures of human fecal microorganisms was investigated, and the results were examined in relation to the bile acid transforming activities of 75 bacterial strains isolated from the same fecal cultures. The reactions involved in the mixed cultures were dehydrogenation and dehydroxylation of the 7α‐hydroxy group in both primary bile acids and epimerization of the 3α‐hydroxy group in all metabolic bile acids. Extensive epimerization of the 7α‐hydroxy group of chenodeoxycholic acid yielding ursodeoxycholic acid was also demonstrated by certain fecal samples. 7α‐Dehydrogenase activity was widespread among the fecal isolates (88% of 16 facultative anaerobes and 51% of 59 obligate anaerobes), and 7α‐dehydroxylase activity was revealed in one of the isolates, an unidentified gram‐positive nonsporeforming anaerobic bacterium. 3α‐Epimerization was effected by seven strains assigned to Eubacterium lentum, which were also active for 3α‐ and 7α‐dehydrogenations. No microorganism accounting for 7α‐epimerization was recovered among the isolates. Splitting of conjugated bile acid was demonstrated by the majority of obligate anaerobes but the activity was rare among facultative anaerobes.
Mitogenic Effect of Cytoplasmic Membranes and a Cytoplasmic Fraction of Staphylococcus aureus L‐Forms on Human Peripheral Blood Lymphocytes
Authors: Takada Haruhiko , Hirachi Yoshiyuki , Hashizume Hideo , Kotani Shozo
Published: November 2013

The cytoplasmic membranes and a cytoplasmic fraction of Staphylococcus aureus L‐forms increased the incorporation of [3H] thymidine by human lymphocytes in the presence of fetal bovine serum. Both fractions stimulated cord blood lymphocytes as well as adult peripheral lymphocytes, suggesting the possibility that the observed effect was not due to an antigen‐specific reaction, but to an immunologically nonspecific action.

The membrane mitogen(s) was resistant to trypsin, although it was partially solubilized by trypsin treatment. The mitogen (s) could not be extracted with a chloroform‐methanol mixture (2:1, v/v), although the chloroform‐methanol soluble fraction was strongly mitogenic to murine splenocytes.

Human serum which was added to the assay system in place of fetal bovine serum definitely suppressed the mitogenic effect of both cytoplasmic membranes and the cytoplasmic fraction, especially the latter.
Immunopotentiating Activity of Whole Cells and Mini‐Cells of Salmonella typhimurium
Authors: Kurashige Satonori , Yoshida Toshiharu , Kodama Kazue , Mitsuhashi Susumu
Published: November 2013

Salmonella typhimurium strain 9 produces mini‐cells during cell proliferation. Mini‐cells are viable but cannot proliferate since they do not contain chromosomal DNA. Effects of whole cells and mini‐cells of S. typhimurium on the immune responses were investigated, with the following results. Phagocytosis of peritoneal macrophages was enhanced by in vivo stimulation of both whole cells and mini‐cells. Cellular immunity against L1210 cells (mouse leukemia cells) and Sarcoma 180 cells was also enhanced by both whole cells and mini‐cells. Mini‐cells slightly stimulated in vitro blast cell transformation of normal mouse lymphocytes. Whole cells of S. typhimurium induced antibody‐forming cells to produce IgG of higher affinity but mini‐cells did not. Mini‐cells were not directly cytotoxic for normal lymphocytes or L1210 cells.
Properties of the Biotin Transport System in Bifidobacterium breve N4
Authors: Yazawa Kouhei , Nakamura Hiroshi , Shibamura Seiichi , Tamura Zenzo
Published: November 2013

Binding of biotin to resting cells of Bifidobacterium breve N4, which grew in a biotin‐deficient medium, was independent of pH from 1 to 9 and of temperature below 50 C. It was not inhibited by metabolic inhibitors including sulfhydryl reagents, but it was inhibited by treatment with 80% ethanol or 5% trichloroacetic acid. It was also competitively inhibited by biotin‐sulfone, but not by tetrahydrothiophene nor dethiobiotin. The binding constant was calculated to be 3.3 × 108m−1.

The amount of biotin unextractable with hot water, representing part of the transported biotin, increased gradually for 20 min, this increase was inhibited by NaF, hydroxylamine and low temperature.

14C‐biotin on the cells was displaced by cold biotin and biotin‐sulfone; the displacement was not inhibited by metabolic inhibitors, but it was dependent on temperature.

A few minutes after binding, the biotin was released to the medium. The release was dependent on pH and temperature, was affected by energy sources and was inhibited by metabolic inhibitors, e.g. NaF, p‐chloromercuribenzoic acid and hydroxylamine. It could be stopped at any time by cooling to 0 C or by adding NaF, and the amount of accumulated biotin did not increase under those conditions. These results suggest that the binding sites on the cell surface decreased in number or in their binding affinity for biotin through an energy‐dependent process.
Mutual Relationship between Antibiotics and Resting Spores of Bacillus subtilis: Morphological Changes and Macromolecular Synthesis after Germination of Spores Treated with Cyclic Polypeptide and Aminoglycoside Antibiotics
Authors: Hayakawa Yoshiaki , Tochikubo Kunio , Kozuka Satoshi
Published: November 2013

Morphological changes and synthesis of DNA, RNA, protein, and cell wall were investigated during germination of resting spores of Bacillus subtilis exposed transiently to the cyclic polypeptide antibiotics, polymyxin B and gramicidin S, and the aminoglycoside antibiotics, streptomycin, kanamycin, and gentamicin. Normal germinated spores showed breaks of the spore coat, a diminution in size and a fibrillar appearance of the cortex, a swelling core, a cell wall as thick as that of vegetative cells, some mesosomes and DNA fibrils. On the other hand, no breaks of the spore coat, a spore core with a slight swelling and irregular form, a thin cell wall, no demonstration of the nuclear material and no granularity in the cytoplasm were characteristic of the germinated spores derived from polymyxin B‐ and gramicidin S‐treated resting spores. With gramicidin S‐treated germinated spores a few vacuoles were formed in the cytoplasm. Both polymyxin B‐ and gramicidin S‐treated germinated spores showed little or no synthesis of DNA, RNA, and protein. The vegetative cells derived from streptomycin‐treated resting spores demonstrated several finely granular regions in the cytoplasm and a disorder of the fibrillar nucleoid, and their autolysis occurred early. Their DNA and RNA synthesis was normal, whereas protein synthesis was low. In spite of no occurrence of cell division and very low protein synthesis, the most striking characteristics of the outgrowing cells derived from kanamycin‐treated resting spores were a markedly thickened cell wall and a continuous incorporation of labeled d‐alanine suggesting cell wall synthesis; RNA synthesis was slightly lower and DNA synthesis was almost normal. The outgrowing cells from gentamicin‐treated resting spores also revealed relatively thick cell walls and a very slight incorporation of labeled d‐alanine. Their DNA and RNA synthesis was fairly low and protein synthesis was almost completely inhibited. These results coincide with the growth curves of individual antibiotic‐treated resting spores.
Production of Interferon by an SV40‐Transformed Macrophage Line, BB‐W‐531–2
Authors: Takayama Hisao , Tanigawa Takahiko , Takagi Atsushi
Published: November 2013

A simian virus 40‐transformed mouse macrophage line, BB‐W‐531–2, was examined for its ability to produce interferon. BB‐W‐531–2 cells showed a phenotypic change between the macrophage and the nonmacrophage states. A viral inhibitor (interferon) was produced by the cells during the phenotypic change from the nonmacrophage to the macrophage state. Cells having macrophage properties were well capable of producing interferon when they were stimulated with ultraviolet‐inactivated vaccinia virus, lipopolysaccharide, a streptococcal preparation (OK‐432) or polyinosinate polycytidylate. In contrast, cells that had lost their macrophage properties did not produce interferon even when they were given the same treatments as the cells having macrophage properties. The results suggest that the ability of BB‐W‐531–2 cells to produce interferon is associated with the expression of several macrophage properties.
Idiotypic Analysis of Antibodies to Hen Egg‐White Lysozyme (HEL): III. Further Studies on the Idiotypic Cross‐Reactivity among Anti‐HEL Antibodies in Mice
Authors: Sakato Nobuo , Semma Masanori , Fujio Hajime , Amano Tsunehisa
Published: November 2013

An isolated antibody preparation directed to the native hen egg‐white lysozyme (HEL) from a single A/J mouse (#a‐11), termed IdHELa‐11, was inoculated into rabbits to produce anti‐idiotypic sera (anti‐Id). The antisera were extensively absorbed with normal A/J Ig to render them idiotype specific. Radioimmunoassay utilizing 125IdHELa‐11 and the anti‐Id sera (R103 and R104) was performed to examine the idiotypic cross‐reactivity of the humoral immune response to HEL in various mouse strains and other animal species. Idiotypes shared by IdHELa‐11 were detected in the sera of five mouse strains tested, but not in any of the examined sera of other animal species such as rats, goats, guinea pigs, or sheep, indicating the occurrence of species‐specific cross‐reactive idiotypes (CRI) of antibodies to HEL. Our experiments also suggested the presence of intrastrain CRI. The present experiments, along with our earlier results (15), suggest that idiotypic cross‐reactivity among murine antibodies to HEL appears to be weak. Thus when R103 and R104 were the anti‐Id sera used, the frequency of occurrence of CRI shared by IdHELa‐11 in 5 μg of anti‐HEL antibody in strain A mice was 74 ng and 111 ng; in other strains it was 25–44 ng and 60–98 ng, respectively.
Titration of Bactericidal Activity against Smooth and Rough Strains of Salmonella which Are Resistant in Isotonic Medium
Authors: Ihara Izumi , Kawakami Masaya
Published: November 2013

Bactericidal factors in antisera against various chemotype strains of Salmonella were assayed by means of the spot method. Certain smooth and rough strains were resistant to the killing effect of immune mouse sera when the activity was assayed in an isotonic salt medium and guinea pig complement was used. However, the sensitivity was found to be increased in various degrees by assaying it in a medium containing hypotonic concentrations of NaCl or tris(hydroxymethyl)‐aminomethane‐HCl (Tris‐HCl). Keeping the resistant bacteria in hypotonic solutions before or after treatment with complement increased the titer of antiserum, indicating that the hypotonic solution increases the sensitivity of bacterial cells to the antiserum and/or complement. The optimal salt concentration for the assay of serum‐sensitive and ‐resistant smooth strains was 0.02 M NaCl. With Ra through Rd chemotype strains, 0.1 m NaCl before complement treatment and 0.05 m NaCl after the treatment was the best. Isotonic medium was necessary for the titration of the Re chemotype strain. Specificity of the killing effect which was assayed by the hypotonic spot method was shown by adsorption studies on the immune sera. Use of C4‐deficient guinea pig complement resulted in low titers against certain strains of bacteria and high titers were restored by the addition of the C4 component of complement. These results indicate that serologically specific bactericidal factors including antibody can be assayed by the spot method using hypotonic NaCl or Tris‐HCl.
Studies on the Bacterial Spore Coat: IX. The Role of Surface Charge in Germination of Bacillus megaterium Spores
Authors: Nishihara Tsutomu , Yoshimoto Ikuko , Kondo Masaomi
Published: November 2013

The surface charge of Bacillus megaterium QM B1551 spores was estimated to be negative, ‐0.2 and ‐0.4 μeq/mg by colloidal titration using glycol chitosan (GCh) and methylglycol chitosan (MGCh), respectively, as positive colloids. MGCh, which reacts with all of the negatively charged groups including carboxylate, inhibited the second stage of the germination to result in semirefractile spores, but GCh, which reacts only with strong acidic groups such as phosphate, did not. The spores produced in a medium with limited phosphate had coats with low phosphate content and carried less negative charge, and they were induced to germinate with 0.4 mm KNO3, which is one‐tenth of the minimum concentration required for the germination of the control spores. A similar increase in germinability was observed in spores incubated with calcium acetate.

These results suggest that the role of the surface charge in germination is as follows. Strong acidic groups (such as phosphate) in the coat may block the action of ionic germinants and act as a barrier against the initiation of ionic germination. Positively charged compounds (such as calcium) may compensate for this blocking effect. Weak acidic groups (such as carboxylate) may be involved in the later stage of germination.
Effect of α‐Tocopherol on Endotoxicosis
Authors: Sakaguchi Osamu , Kanda Norio , Sakaguchi Shuhei , Hsu Cheng‐Chin , Abe Hiroharu
Published: November 2013

The effect of α‐tocopherol in endotoxicosis was studied. The α‐tocopherol level significantly decreased in mouse liver 18 hr after endotoxin administration, thereafter tending to increase to approach the normal range. In endotoxin‐tolerant mouse liver, the lipid peroxide level was reduced to less than half of that in nontolerant animals following endotoxin challenge. The liver lipid peroxide level and serum lactate dehydrogenase or acid phosphatase leakage were studied in mice fed a vitamin E‐deficient (ED) diet and a vitamin E‐supplemented (ES) diet for 40 days. ED mouse liver exhibited a higher formation of lipid peroxide after endotoxin was given while there was a markedly lower level in ES mouse liver. There was significantly more serum lactate dehydrogenase or acid phosphatase leakage in ED mice than in ES mice after endotoxin administration. There was about a 25% decrease in liver superoxide dismutase (SOD) activity in endotoxin‐poisoned mice fed both the normal and the ED diets, while the activity was at a higher level in ES‐fed mice. These results suggest that α‐tocopherol may be helpful in preventing membrane instability in endotoxin poisoning.
Effect of Treatment of Soil with Keratinaceous Material on Soil Fungi
Authors: Moubasher Abdel‐Aal H. , Abdel‐Fattah Hassan M. , Maghazy Soad M.
Published: November 2013

In the early periods of soil storage, the total number of fungi almost regularly and significantly increased with the concentration of keratinaceous material (ground buffalo hooves) between 1% and 10%. A concentration of 20% was stimulative after 7 days and became toxic after 15 days and remained so through the end of the experiment. With increase in the storage period, the beneficial effect of all concentrations was lost and in the case of high concentrations (5–20%) toxic ammonia was produced.

The occurrence of Scopulariopsis brevicaulis, Cephalosporium acremonium, Chrysos‐porium tropicum, C. indicum, and C. keratinophilum increased markedly, especially at the higher concentrations of the keratinaceous material and after long periods of incubation.

With increase in the concentration of keratinaceous material, the rate of evolution of ammonia from the soil sharply increased during the second week after treatment and fell off sharply during the third and fourth weeks.

In soil stored at 35 C, the rate of decline in total fungi after longer periods of incubation was sharper at 35 C than at 25 C. Also the amount of evolved ammonia during the first and the second weeks was almost always higher at 35 C than at 25 C.

Fourteen species were tested for keratinolytic activity. Three of them were highly keratinolytic and five moderately so.
An L‐Form of Staphylococcus aureus Adapted to a Brain Heart Infusion Medium without Osmotic Stabilizers
Authors: Takahashi Toyozoh , Tadokoro Ichiro , Adaghi Shoji
Published: November 2013

An L‐form derived from halotolerant Staphylococcus aureus Tasaki was adapted to growth in a brain heart infusion medium without any supplemental osmotically protective solutes (360 mOsm/kg). This L‐form had no chemically detectable peptidoglycan residues on its surface. Electron microscopic observations confirmed morphologically the absence of the structures and also of other osmotically protective polymers within or exterior to the cytoplasmic membrane. The osmotic stability and susceptibility to bacitracin, d‐cycloserine, and vancomycin of the L‐form adapted to growth in 360 mOsm osmotically unprotective medium was higher than that of the L‐form grown in 1,950 mOsm supplemented with 4.5% NaCl. The adapted L‐form tended to be more sensitive to almost all of the antibiotics examined, other than the inhibitors for cell wall‐synthesis, than the original L‐form strain requiring osmotic protection for growth. Chemical analysis of the membrane of the adapted L‐form indicated 16.3% total lipids and 20.6% proteins by dry weight of the membrane, and it contained larger amounts of lipid phosphorus (20.0 μ/mg).
Essential Role of T Cells in the Postexposure Prophylaxis of Rabies in Mice
Authors: Mifune Kumato , Takeuchi Eriko , Napiorkowski Patricia A. , Yamada Akira , Sakamoto Kuniaki
Published: November 2013

Athymic nude mice injected intramuscularly with a street strain of rabies virus were not protected against rabies by postexposure administration of beta‐propiolactone‐inactivated rabies vaccine. In contrast, their normal littermates were completely protected from death by the same vaccination regimens. Nude mice did not produce IgG antibody as a result of the vaccine during the test period of 15 days, whereas normal littermates produced IgG antibody from day 5 after vaccination. However, passive immunization with antirabies hyperimmune mouse ascites showed that antibody was completely ineffective in protecting either nude mice or their normal littermates against rabies when given later than 2 days after infection. No significant difference in the induction of circulating interferon by the vaccination was noted in these mice. Passive transfer of immune spleen cells to nude mice immediately after infection resulted in 30 to 37.5% protection of the mice. Passively transferred spleen cells did not produce detectable amounts of neutralizing antibody in the recipient mice except on day 2 after the transfer, when a low level of antibody was detected. These observations demonstrate the essential role of T cells in the postexposure prophylaxis of rabies in mice. The mechanisms of the failure of postexposure vaccination in nude mice are discussed.
Mitogenicity and Adjuvanticity of a Marine Bacterium, Vibrio anguillarum, in Mice
Authors: Shimizu Tadayori , Nitta Masamichi , Itoh Yoshiaki , Yanagihara Yasutake , Mifuchi Ichiji
Published: November 2013

The effects of whole cells of three different O serotypes of Vibrio anguillarum on the murine immune response were studied. The addition of different doses (1–100/ig/ml) of V. anguillarum cells, as well as Salmonella typhimurium lipopolysaccharide, markedly increased the incorporation of [3H] thymidine into in vitro cultured spleen cells of C57BL/6 mice. All three serotype strains of V. anguillarum were able to induce the mitogenic effect at 10 μg/ml and 100 μg/ml, but serotype I strains were more potent than the others. Since pretreatment of spleen cells with rabbit anti‐mouse thymocyte antiserum did not affect the mitogenic activity of V. anguillarum, Vibrio cells may be a B‐lymphocyte mitogen.

When sheep or horse erythrocytes and Vibrio cells were injected intraperitoneally into ddY mice, Vibrio cells exhibited an enhancing effect on antibody response in vivo, regardless of the different serotypes. Vibrio cells, when injected intraperitoneally into mice before the antigen, markedly suppressed the antibody response. Several days after the injection of Vibrio cells, these mice showed an enhanced carbon clearance activity. Acid phosphatase activity in their peritoneal cells was also augmented, suggesting that Vibrio cells activated macrophages in the mice.
Preparation of Stable Target Cells for Anti‐Herpes Simplex Virus Cytotoxic T Lymphocytes
Authors: Sonoda Shunro , Hitsumoto Yasuo , Utsumi Sayaka , Tarami Shunsai , Oseto Mitsuaki , Minamishima Yoichi
Published: November 2013

Using an avirulent strain of herpes simplex virus (HSV), SKa, and a methylcholanthrene induced sarcoma cell line, Meth A cells, we have developed a reliable target cell system for detection of cell‐mediated cytotoxicity directed against HSV‐infected cells. SKa‐infection in Meth A produced no progeny virus but induced HSV‐specific surface antigens as revealed by radioimmunoassay using 125I‐labeled HSV antibody. Spontaneous release of 51Cr from the SKa‐infected Meth A cells was no more than that from uninfected control cells but a strong spontaneous 51Cr release was produced in Meth A cells infected with KOS, a virulent strain which produced a progeny virus in Meth A and was lytic for the cells. When used as a target, SKa‐infected Meth A cells could detect HSV‐specific cytotoxicity by spleen and lymph node lymphocytes of mice immunized with SKa and KOS. This system also detected effector cytotoxic lymphocytes stimulated in vitro by mixed cultures of immune spleen cells and KOS‐infected Meth A cells. Thus, the system should be valuable in studies of cell‐mediated cytotoxicity directed against HSV‐infected cells.
Effect of Immune Heterozygous Spleen Cell Transfer on Resistance to Mouse Hepatitis Virus Infection in Nude Mice
Authors: Kai Chieko , Tamura Teruko , Fujiwara Kôsaku
Published: November 2013

The role of cell mediated immune response to mouse hepatitis virus (MHV) infection in mice was studied by transferring spleen cells from immune heterozygous littermates (nu/+). A suppressive effect on viral growth was seen in infected nude (nu/nu) mice, whereas immune nu/+ serum transfer had no effect. The protective effect of immune nu/+ spleen cells was significantly reduced by treatment with anti‐theta serum plus complement but not with anti‐Ig serum. In infected nu/nu mice which received transfers of immune nu/+ cells, neutralizing antibody appeared although the titer was not high enough to protect nu/nu mice from fatal infection. Histopathologically, lymphocyte infiltration in hepatic lesions was evident in infected nu/nu mice with nu/+ cell transfer, while it was slight without nu/+ cell transfer.
A Micro‐Neutralization System for Detection of s‐CRN Antibody to Herpes Simplex Virus
Authors: Yoshino Kamesaburo , Abe Kazuko
Published: November 2013

It was learned that the ordinary micro‐neutralization system with herpes simplex virus (HSV) gave a composite result of the initial neutralization and the effect of antibody on subsequent growth of unneutralized virus. In the case of slow‐reacting complement‐requiring neutralizing (s‐CRN) antibody, which was detected by incubating virus‐serum mixtures at 4 C for 3 days before addition of complement, the titer obtained was lower than expected from the result of the plaque reduction test. This was thought ascribable to its low ability to prevent viral breakthrough caused by growth of unneutralized virus. This was overcome by adding an appropriate amount of hyperimmune antibody at 3 hr after addition of cells. The endpoint of s‐CRN antibody so determined was but slightly lower than that obtained by the plaque reduction test. Early (1‐week) rabbit sera, which were negative in the ordinary micro‐neutralization test, titered 1:2,560 to 1:5,120 when tested by this method. When the 3‐day sensitization in the cold was substituted by 5‐hr incubation at 37 C, the titer obtained was 2 to 4‐fold lower; in this case, however, the whole process could be finished within 3 days. Also, s‐CRN antibody reactive with type 2 HSV in homologous and heterologous sera could be detected by the same method using type 1 hyperimmune serum as the additional antibody.
Chemotaxis of Kupffer Cells Isolated from Rodent Liver
Authors: Katano Mitsuo , Miyazaki Kohji , Aso Masakazu , Torisu Motomichi
Published: November 2013

Kupffer cells (KC) were isolated from the liver of guinea pigs, rats, and mice using enzymatic digestion with collagenase, followed by differential centrifugation and plastic adherence. Purity of the isolated KC was 96.0±2.2, 97.2±2.1, and 96.0±2.3 per cent in guinea pigs, rats, and mice respectively. These isolated KC were tested for migratory response to bacterial factor, which is one of the representative chemotactic factors for inflammatory macrophages, using a modified Boyden chamber technique. KC from the three animal species similarly migrated to the bacterial factor. The migratory response of the KC to the bacterial factor is due to Chemotaxis but not chemokinesis. These results show the possibility that KC may recognize a chemoattractant and directionally migrate to it.
Sensitivity in Detection of Monoclonal Immunoglobulins by Thin‐Layer Isoelectric Focusing and Certain Electrophoreses in Laboratory Use
Authors: Kamiya‐Harada Yasuko , Sato Yoshiko , Otani Hideki , Kawakami Masaya
Published: November 2013

By applying sera containing various myeloma proteins to thin‐layer isoelectric focusing at various pH ranges, we found that the best range of pH gradient for the detection of the band group specific for monoclonal immunoglobulin was from 5 to 8. The lowest concentration of monoclonal immunoglobulin detected by this method was approximately 0.1 mg/ml which was 8 to 32 times lower than the concentration detectable by cellulose acetate membrane electrophoresis or immunoelectrophoresis in routine laboratory use. Monoclonal protein concentration from 0.1 to 20 mg/ml was determined quantitatively. Blind tests on sera resulted in a disagreement between the concentration of monoclonal immunoglobulin assumed from the results of cellulose acetate membrane electrophoresis and immunoelectrophoresis and that determined by isoelectric focusing. These results suggest that isoelectric focusing is useful for the surveillance of monoclonal immunoglobulinemia provided that the technique and equipment are improved for laboratory tests.
T Cell Growth Factor from Human Splenic Cell Cultures: Conditions for Its Production, and Its Utilization for Maintenance of Cytotoxic T Cell Lines
Authors: Tanaka Yuetsu , Sugamura Kazuo , Hinuma Yorio
Published: November 2013

We prepared the T cell growth factor (TCGF) from human spleen cell cultures stimulated with phytohemagglutinin (PHA). Various cell culture conditions and agents supporting the active TCGF production of the spleen cells were examined. The highest TCGF activity was obtained in the supernatants under the conditions that 2 × 106/ml spleen cells were stimulated with PHA for 48 hr. Production of TCGF from spleen cells depended markedly on their individual sources. Addition of indomethacin to the culture or irradiation of the responding spleen cells increased TCGF activity in the supernatant of the culture. Further, addition of irradiated cells of an Epstein‐Barr virus (EBV)‐transformed lymphoblastoid cell line (LCL) to spleen cell cultures stimulated with PHA greatly enhanced TCGF production.

Human splenic TCGF facilitated the establishment of human cytotoxic T cell (Tc) lines specific for EBV‐transformed LCL cells when those Tc line cells were stimulated periodically with irradiated autologous LCL cells but not with the other two types (K‐562 or Molt‐4) of cells. Allogeneic LCL stimulators allowed the Tc line cells to proliferate. However, Tc line cells cocultured once with allogeneic LCL stimulators no longer exhibited EBV‐specificity in their cytotoxicities.
Comparative Activity of N‐Formimidoyl Thienamycin with Third Generation Cephalosporins and Ureido Penicillins against Multiple Resistant Serratia marcescens
Authors: Miller Marcia A. , Lefrock Jack L. , Vercler Mary J.
Published: November 2013

Twenty multiple resistant clinical isolates were tested with N‐formimidoyl thienamycin, moxalactam, cefotaxime, cefoperazone, and the three ureidopenicillins: azlocillin, mezlocillin, and piperacillin. A concentration of < 0.97 μg/ml inhibited 100% of organisms for N‐f‐thienamycin and cefotaxime, 90% for moxalactam, and 60% for cefoperazone. An increase in inoculum from 103 to 106 cells reduced activity fourfold for 95% of isolates with cefoperazone, 70% with N‐formimidoyl thienamycin, 65% for cefotaxime, but only 15% for moxalactam. For ureidopenicillins, 85% of strains tested had MIC's ≤ 15.6 μg/ml. An inoculum effect was observed in only 35–50%. At 103, the cidal concentration was the same or twofold greater than the inhibitory level for N‐f‐thienamycin and cephalosporins in 70% of strains tested and 65% for penicillins. With 106, the 70% value remained for N‐f‐thienamycin but was reduced to 45% for cefotaxime and 25% for moxalactam; 85% demonstrated > eightfold differences with cefoperazone. Single step high‐level resistance was observed to moxalactam (20%). Carbenicillin resistant strains were cross‐resistant to the ureidopenicillins. N‐f‐thienamycin and cefotaxime appeared comparable, although important differences between morphological alteration and metabolism may influence their therapeutic effectiveness.
Sensitivity to Ultraviolet Light of Tobacco Mosaic Virus Modified by Cetyltrimethylammonium Bromide
Authors: Abe Tetsuya , Watanabe Motoo , Sai Toshiyuki , Asano Taisuke , Kiho Yukio
Published: November 2013

Cetyltrimethylammonium bromide (CTAB) modified tobacco mosaic virus (TMV) virions so that the intrinsic fluorescence changed, viral infectivity decreased, sensitivity to RNase or UV irradiation increased, and coat protein subunits were released by the addition of Triton X‐100. The change in fluorescence emission at 320 nm shifted to 340 nm was observed at 100 μg of CTAB per ml. This represents a change in the tryptophan environment inside the virion. At a lower concentration of CTAB, intersubunit contact was weakened, resulting in the release of coat protein subunits and an increase in RNase sensitivity. The release of coat protein took place gradually and two relatively stable intermediates were observed. Increase in UV sensitivity was observed at a lower concentration of CTAB and formation of pyrimidine hydrate was involved in this inactivation. The nature of the minor structural change leading to UV inactivation is discussed.
Role of T Cells in the Mitogen‐Induced Proliferation and Polyclonal Antibody Response of Murine B Cells
Authors: Ishikawa Hiromichi , Saito Kazuhisa
Published: November 2013

The effect of thymus‐derived lymphocytes (T cells) on the responsiveness of bone marrow‐derived lymphocytes (B cells) to bacterial lipopolysaccharide (LPS) was determined in in vitro experiments. Radiation resistant splenic T cells obtained from euthymic nu/+ mice increased the number of proliferating cells in the cultures of splenic B cells from athymic nu/nu mice even in a nonstimulated state. The radiation resistant T cells augmented significantly the responsiveness of B cells to LPS, as determined by an increase in proliferating cells and polyclonally induced anti‐sheep erythrocyte (SRBC) IgM hemolysin plaque‐forming cells (PFC). Addition of the T cells to B cell cultures not only augmented the responsiveness of B cells to suboptimal doses of LPS but also enabled B cells to respond to supraoptimal doses of LPS. As is well documented, the radiation resistant T cells were unable to induce the generation of anti‐SRBC PFC in B cell cultures, unless the cultures were simultaneously stimulated with SRBC.

Colcemid, a specific inhibitor of cell mitosis, blocked almost completely the exponential generation of anti‐SRBC PFC in B cell cultures responding to SRBC with the aid of radiation resistant T cells. In contrast, colcemid did not affect the exponential generation of anti‐SRBC PFC of a polyclonal nature in B cell cultures responding to LPS, either in the presence or absence of radiation resistant T cells.
Cloning of a 1.1‐kb Fragment Including srnB+ Gene in the F Plasmid and Isolation of an srnB Mutant
Authors: Ohnishi Yoshinari , Ono Tsuneko , Ito Masaki , Akimoto Shigeru
Published: November 2013

The srnB+ gene, promoting stable RNA degradation at 42 C in the presence of rifampin, was cloned by using pBR322 as a vector; it was located on a 1.1‐kilobase (kb) EcoRI/BamHI fragment between 1.4 and 2.5 kb of the F plasmid. The region between 93.3 and 4.0 kb of the F plasmid was physically mapped by using restriction endonucleases EcoRI, HindIII, BamHI, PstI, and SmaI, with reference to a standard HindIII site in IS3. An srnB1 mutant was isolated from a chimeric plasmid, pOY54, after treatment of its DNA with hydroxylamine. The srnB1 allele on the F fragment of the mutant plasmid was recessive to the wild‐type allele. Thermal elevation of cell cultures to 39 C was high enough to promote RNA degradation in strain YS12 carrying plasmid pOY54.
Grouping of Adenoviruses and Identification of Restriction Endonuclease Cleavage Patterns of Adenovirus DNAs Using Infected Cell DNA: Simple and Practical Methods
Authors: Suzuki Nobutaka , Ueno Tetsuji , Yamashita Toshiharu , Fujinaga Kei
Published: November 2013

Simple and practical methods for grouping of adenoviruses and for identification of restriction endonuclease cleavage patterns of viral DNA were established by using infected cell DNA. DNA homology groupings of adenoviruses could be examined by spot hybridization, and restriction endonuclease cleavage patterns of viral DNAs could be obtained by Southern blot hybridization, by using infected cell DNA. The method was very sensitive and allowed the identification of the cleavage pattern of viral DNA of the inoculum by means of cell DNA extracted from infected cells with undetectable cytopathic effect (CPE). In ethidium bromide‐stained gels without Southern blot hybridization, the restriction endonuclease cleavage pattern of viral DNA could be detected precisely in spite of background staining due to cellular DNA. The preparation of infected cell DNA used in these procedures was technically much easier than that of viral DNA. These methods require only a small number of infected cells and allow many isolates to be investigated with ease.