bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of N‐Acetylchito‐Oligosaccharides on Activation of Phagocytes
Authors: Suzuki Ko , Tokoro Akio , Okawa Yoshio , Suzuki Shigeo , Suzuki Masuko
Published: November 2013

Four N‐acetylchito‐oligosaccharides, from tetra‐N‐acetylchitotetraose (NACOS‐4) to hepta‐N‐acetylchitoheptaose (NACOS‐7), were found to increase the number of peritoneal exudate cells (PEC) in male BALB/c mice after 3 hr intraperitoneal administration of 50 mg/kg of each oligosaccharide. The number of attracted cells, consisting largely of polymorphonuclear leukocytes (PMN), was proportional to the molecular weights of the administered oligosaccharides, except for NACOS‐7 which displayed the same activity as NACOS‐6. In an in vitro chemotaxis assay using normal mouse leukocytes, it was found that NACOS‐6 displayed stronger effects than muramyl dipeptide. The PEC from NACOS‐6 treated mice showed a higher active oxygen‐generating activity. PMN from normal mouse peripheral blood were also shown to have enhanced active oxygen‐generating activity in vitro. PEC from NACOS‐6 treated mice were shown to possess strong candidacidal activity in vitro.
Establishment of an Assay System for the Detection of Translated Materials of T Cell‐Replacing Factor mRNA in Xenopus Oocytes
Authors: Tominaga Akira , Matsumoto Mitsuhiro , Takahashi Takeo , Harada Nobuyuki , Takatsu Kiyoshi
Published: November 2013

We established an assay system for detecting T cell‐replacing factor (TRF) activity of translated materials in Xenopus oocytes of poly (A)‐positive mRNA extracted from a T cell hybrid cell line, B151K12 (B151) which constitutively produces TRF. Since it was difficult to detect TRF activity of the translated products of B151‐mRNA, partly because of low TRF activities, we developed the following two systems. First, RNA was prepared from B151 cells stimulated with phorbol myristate acetate and calcium ionophore A23187 because such stimulations augmented TRF production by approximately three to five‐fold. Second, interleukin 2 (IL‐2, 125 U/ml) was added to the culture of BCL1 cells to detect a small amount of TRF‐active materials since IL‐2 synergizes with a suboptimal dose of TRF to induce IgM secretion in TRF‐responding BCL1 cells (chronic B cell leukemic cells). Here we describe TRF activity of translation products of B151‐mRNA in Xenopus oocytes. B151‐TRF mRNA was detected in the fractions sedimented between 15 and 18S by analysis using sucrose density gradient centrifugation.
Induction of Human Interferon‐γ mRNA Analyzed by Cytoplasmic Dot Hybridization
Authors: Nagashima Hajime , Fukunaga Rikiro , Matsuyama Masako , Sokawa Yoshihiro
Published: November 2013

The cytoplasmic dot hybridization method was improved by eliminating non‐specific false signals. The non‐specific binding to a probe disappeared almost completely when the hybridized nitrocellulose sheet was treated with pronase. Using this improved method we demonstrated that reagents such as 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), concanavalin A (Con A) and a calcium ionophore (A23187) could induce the expression of IFN‐γ gene of a human T‐lymphoblastoid cell line, TCL‐Fuj 2M, where TPA functioned synergistically with Con A and A23187. However, when the cells were cultured with the ionophore for more than one day the amount of IFN‐γ mRNA was markedly reduced to a low level.
MDBK Nuclear Factor‐Binding Site of Various Serotypes of Adenovirus DNA
Authors: Endo Seiichi , Shinagawa Morikazu , Sato Gihei , Padmanabhan Radha
Published: November 2013

A fraction with the ability to bind the terminal fragment of equine adenovirus (EAd) DNA was prepared from MDBK cell nuclei. The fraction (MDBK nuclear factor) bound to the terminal fragment of all human and animal adenovirus DNAs examined except avian adenovirus EDS‐76. However, the terminal fragments of two animal adenoviruses, EAd and bovine adenovirus type 3 (BAd3), showed higher affinity for the nuclear factor than the others. The MDBK nuclear factor‐binding site determined by footprinting analysis was the sequence located between nucleotides 22 and 46 in EAd, between 36 and 53 in canine adenovirus type 2, and between 20 and 46 in BAd3, counting from the terminus. The respective binding site contained a sequence resembling the consensus sequence. The binding site of Ad4 DNA was not within the inverted terminal repetition, but was located at least 550 base pairs apart from the terminus.
Use of Protein A in the Serum‐in‐Agar Diffusion Method in Immune Electron Microscopy for Detection of Virus Particles in Cell Culture
Authors: Furui Sataro
Published: November 2013

A modified technique using protein A in the serum‐in‐agar (SIA) method for immune electron microscopy (IEM) was presented. Grids coated with staphylococcal protein A were floated on samples mounted on agar containing 2% antiserum and incubated at 37 C, for 60 min. After washing and staining, the grids were observed in an electron microscope. The effects of protein A on virus detection were evaluated using poliovirus and bovine rotavirus infected cell culture fluids. The results showed that the technique using protein A (PA‐SIA) had at least 10‐fold higher sensitivity for virus detection than the original SIA. The optimal concentration of protein A was 1 to 10 μg/ml for coating the grids to trap virus particles. The PA‐SIA method was also compared with immunosorbent electron microscopy (ISEM). The former showed higher or at least the same sensitivity and some advantages in detecting antigen‐antibody reaction than the latter method. These results indicate that our PA‐SIA method may be superior to other IEM techniques presented previously for the detection and identification of viruses.
Human Interferon‐γ (IFN‐γ) Containing Cells Bear Various Surface Phenotypic Markers
Authors: Sugawara Isamu , Kitagawa Hiroshi , Inagaki Hirofumi , De Ley Mark , Fukuda Akio
Published: November 2013

We explored the population of Interferon‐γ (IFN‐γ) containing cells in order to clarify their cell surface phenotypic markers. Here we define γ‐IFN containing cells as γ‐IFN plaque forming cells (PFC). By this method, it was found that IFN‐γ containing cells consist of two cell fractions, i.e., OKT3+, OKT4+, and OKT8‐ cells and OKM1+ cells. Effective IFN‐γ production seems to require participation of plastic‐adherent cells (presumably monocytes), while the addition of cyclosporin A (CyA) almost completely blocked generation of human IFN‐γ. To characterize Con A‐stimulated IFN‐γ containing cells, we performed two‐color flow cytometry using FACS IV. Most of the IFN‐γ containing cells have surface phenotypic markers for Leu3, Leu8, Leu15, HLA‐DR, and IL‐2 receptors, but most lack markers for Leu2 and Leu7. Interestingly, most of Leu3+ and IL‐2 receptor+ cells belong to the dimly illuminating cell fractions of the IFN‐γ containing cell population. Our results indicate that IFN‐γ containing cells are heterogeneous with respect to surface phenotypic markers but the predominant IFN‐γ containing cell type is the helper T cell (OKT4+). Lastly, OK432, glycyrrhizin, and CCA (lobenzarit disodium) increase the number of IFN‐γ containing cells and are thought to be immunomodulators.
Conjugal Acquisition and Stable Maintenance of Ent Plasmids in Nontoxigenic Wild‐Type Strains of Escherichia coli
Authors: Danbara Hirofumi , Arita Hiroshi , Baba Hisae , Yoshikawa Masanosuke
Published: November 2013

In spite of the ability of the genetic determinants for enterotoxin production to be conjugally transferred, mobilized or transposed, enterotoxigenic Escherichia coli (ETEC) isolated from diarrheal patients is restricted to certain serotypes. Four conjugative enterotoxigenic plasmids (Ent plasmids) encoding either a heat‐labile enterotoxin or a heat‐stable enterotoxin or both and belonging to one of three incompatibility groups IncFI, IncHl, or IncX, were examined for their transferability to and stability in 157 nonenterotoxigenic Escherichia coli strains belonging to various serotypes and 89 clinical isolates nonenterotoxigenic but belonging to those serotypes in which ETEC from diarrheal patients are usually found. The serotypes of the strains to which Ent plasmids were efficiently transferred and in which they were maintained stably were not always the serotypes in which ETEC had usually been found and vice versa. The frequencies of transfer of four Ent and two R plasmids to each of the 157 recipients were correlated with each other, indicating that the frequency of transfer of the plasmid is not determined by a resident plasmid, if there is one, but by a recipient factor which commonly affects transferability to all donors. These results have led to the conclusion that the reason why only certain serotypes are found among ETEC isolated from diarrheal patients is not the ability of these strains specifically and preferentially to acquire and maintain the Ent plasmids.
A Monoclonal Antibody with Broad Reactivity to Human Interferon‐α Subtypes Useful for Purification of Leukocyte‐Derived Interferon
Authors: Tsukui Kazuo , Uchida Shigeharu , Tokunaga Eiichi , Kawade Yoshimi
Published: November 2013

A monoclonal antibody to human interferon‐α, termed HT‐1 antibody, with a broad reactivity to various subtypes of interferon‐α was prepared. It bound and neutralized all of the four subtypes of E. coli‐derived human recombinant interferon‐α (aL1, aL2, aL4, and aL6) tested; it also neutralized human natural leukocyte interferon but only partially. Human interferon‐β and ‐γ were not bound. The antibody conjugated to Sepharose beads retained over 90% of human leukocyte interferon induced by Sendai virus. The bound interferon was recovered by acid elution in good yields and in almost pure form (specific activity was about 2 ′ 108 international units/mg protein). The purified interferon showed, in SDS‐polyacrylamide gel electrophoresis, an activity profile with major peaks in a mol. wt. range of 17,000–22,000, which completely agreed with the profile shown by polyclonal antibody‐purified interferon. Such purified leukocyte interferon‐α preparations containing most of the naturally occurring subtypes can be useful for clinical and other purposes.
Mechanism of Differences in Pathogenicity between Two Variants of a Laboratory Strain of Herpes Simplex Virus Type 1
Authors: Yamada Masao , Arao Yujiro , Uno Fumio , Nii Shiro
Published: November 2013

The mechanisms responsible for the difference in neurovirulence to inbred mice between two variants of the Miyama strain of herpes simplex virus type 1 (HSV‐1) were studied. After intraperitoneal (i.p.) inoculation, the +GC (LPV) variant reached the spinal cord and the brain, and caused death. Conversely, the ‐GCr variant lacked the ability to gain access to the central nervous system (CNS) after the same route of infection and failed to kill susceptible mice. The initial virus growth after i.p. inoculation, as indicated by the number of infective centers (ICs) produced by the peritoneal exudate cells (PECs), was compared between these two variants. The virulent +GC (LPV) strain induced much more ICs than the attenuated ‐GCr variant. When the attenuated variant was preinoculated i.p. 24 hr before the challenge inoculation with the virulent variant by the same route, the production of ICs by the pathogenic variant was highly inhibited, and growth of this variant did not occur in the CNS. Thus, mice were protected from lethal infection by the virulent variant by preinoculation with the attenuated one. Moreover, the ability of mice to resist i.p. infection by HSV‐1 was shown to be age‐dependent.
Role of Thymus–Derived Lymphocytes in Acquired Immunity to Salmonellosis in Mice
Authors: Chander Ramesh , Sainis Krishna Balaji , Lewis Norman Francis
Published: November 2013

The relative role of thymus‐derived (T‐) lymphocytes and bone marrow‐derived (B‐) cells in acquired immunity to salmonellosis was examined in mice. The results demonstrate that the protective capacity of the donor immunized mice could be passively transferred to the recipient mice by spleen cells but not with peritoneal exudate cells or sera. A high cell number of spleen cells (2 × 108/mouse) were required before passive transfer of immunity could be obtained. Of the T‐lymphocytes and B‐cell populations of spleen cells, T‐cells from immune mice were effective in conferring protection to the recipient mice.
Characterization and Mobilization of Nonconjugative Plasmids Encoding Resistance to Streptomycin and Sulfanilamide
Authors: Araki Yasuhisa , Inoue Matsuhisa , Hashimoto Hajime
Published: November 2013

Most of nonconjugative streptomycin (Sm)‐ and sulfanilamide (Su)‐resistance of clinical isolates belonging to various species of Enterobacteriaceae and Pseudomonas were encoded by an Inc Q plasmid, molecular size of which was 5.5 Md. The SmSu plasmids were efficiently mobilized by Inc P plasmids between E. coli strains. Inc I group and Inc F group plasmids could mobilize the Inc Q plasmids at lower efficiencies. The Inc Q plasmid was also mobilized to various species of Enterobacteriaceae at high frequencies without accompanying the conjugative Inc P plasmid; as a result, most of the SmSu‐resistant transconjugants were nontransferable. The above results may explain the wide distribution of nonconjugative SmSu strains among clinical isolates.
Effect of Various Sodium Taurocholate Preparations on the Recovery of Clostridium difficile Spores
Authors: Kamiya Shigeru , Yamakawa Kiyotaka , Ogura Hisashi , Nakamura Shinichi
Published: November 2013

The effect of four sodium taurocholate preparations, which are easily available in Japan, on recovery of Clostridium difficile spores was examined. All preparations, except for one, enabled the recovery of nearly all spores counted microscopically. Moreover, by using 69 toxigenic and 34 nontoxigenic C. difficile strains, the relationship between the recovery of spores in the medium with sodium taurocholate and toxigenicity of C. difficile was analyzed. It was noted that the number of strains with recovery rate of more than 70% was greater in toxigenic strains than in nontoxigenic strains, suggesting a more abundant recovery of toxigenic C. difficile strains in the presence of sodium taurocholate.
Enzymological Characteristics of Avian Influenza A Virus Neuraminidase
Authors: Kiyotani Katsuhiro , Takei Naomi , Senoo Masato , Takao Shinichi , Otsuki Koichi , Tsubokura Misao , Yoshida Tetsuya
Published: November 2013

Neuraminidases of 18 strains of avian influenza A virus were examined by both colorimetric and fluorometric assays using fetuin and 4‐methylumbelliferyl‐N‐Ac‐α‐d‐neuraminide as substrates, respectively, to compare them with those of human influenza A and B viruses. The ratios of the neuraminidase activity of avian influenza virus measured by the colorimetric assay method to that measured by the fluorometric assay were distributed in the range of 2.4–20.3. The enzyme of avian influenza virus showed calcium‐ion dependence in both assay methods. These results suggest that neuraminidase of avian influenza A virus is varies greatly from one strain to another in substrate specificity as compared with those of human influenza A and B viruses, and that some strains of avian influenza A virus have a neuraminidase with unique enzymological characteristics different from that of human influenza A virus as well as that of influenza B virus.
A Novel Enzyme Immunoassay Commonly Applied for Ten Strains of Pyricularia oryzae
Authors: Kitagawa Tsunehiro , Ohtani Wataru , Tanimori Hideaki , Kimura Sadao , Mogi Shizuo
Published: November 2013

Antiserum against a strain of the rice blast fungus Pyricularia oryzae was elicited in rabbits immunized with its cell fragments emulsified with incomplete Freund's adjuvant. The fragments were also used as solid‐phase antigens. A highly sensitive, competitive type enzyme‐linked immunosorbent assay for P. oryzae was developed by using these two preparations as the immune reagents together with the use of β‐d‐galactosidase‐labeled anti‐rabbit IgG as the tracer. Cross‐reactivity of nine different strains of P. oryzae were measured by the assay. Sensitivity and accuracy of the assay was improved by choosing the cell fragments of the least cross‐reactive strain as the solid‐phase antigen. The improved method was successfully applied for sensitive and accurate assay of all ten strains of P. oryzae with the common measuring range between 1 and 100 ng per tube. Other species of microorganisms had little reactivity in this immunoassay indicating that the assay is specific to P. oryzae group microorganisms.
Temperature‐Sensitive Growth Mutants as Live Vaccines against Experimental Murine Salmonellosis
Authors: Ohta Michio , Kido Nobuo , Fujii Yasuaki , Arakawa Yoshichika , Komatsu Takayuki , Kato Nobuo
Published: November 2013

Temperature‐sensitive growth mutants of Salmonella enteritidis, NUB 209 and NUB 323, characterized as protein synthesis mutants, were unable to proliferate at 37 C and lost the parent's virulence. In mice, these mutants conferred high levels of protection as live vaccines. Although the vaccination effect of NUB 323 was not so good as that of NUB 209, NUB 323 was preferred as a safer live vaccine because this mutant was completely avirulent and no back mutation appeared.
Comparative Antigenicity of Spore Coat Proteins from Bacillus Species Using Antibody to Spore Coat Proteins of Bacillus megaterium
Authors: El‐Belbasi Hussein I. , Imagawa Masayoshi , Oku Yuichi , Nishihara Tsutomu , Kondo Masaomi
Published: November 2013

Spore coat proteins obtained by extraction with sodium dodecylsulfate/dithiothreitol from six Bacillus spores were compared by immunoblot analysis using antibodies to spore coat proteins from two strains of B. megaterium. Although the extract from spores of each strain had heterogenous proteins with various molecular weights, there were some bands which cross‐reacted with specific antibodies from B. megaterium spores. Specific antibody to 48K protein from B. megaterium ATCC 12872 cross‐reacted with 17K protein from B. megaterium ATCC 19213, 13K protein from B. cereus and 50K protein from B. subtilis 60015 and B. subtilis NRRL B558. Also, specific antibody to 22K protein from the same strain cross‐reacted with 22K and 17K proteins from B. megaterium ATCC 19213 and 13K protein from B. cereus T. Specific antibody to 17K protein from B. megaterium ATCC 19213 reacted with 22K and 19K proteins in addition to 17K protein of own strain, and it was cross‐reactive with 16K protein from B. megaterium ATCC 12872, 19K and 27K proteins from B. thiaminolyticus, 13K protein from B. cereus.
The Efficacy of Biological Response Modifiers against Murine Cytomegalovirus Infection in Normal and Immunodeficient Mice
Authors: Okada Masanobu , Minamishima Yoichi
Published: November 2013

The host‐mediated antiviral effect of two biological response modifiers (BRM), OK‐432 and PS‐K, against murine cytomegalovirus (MCMV) was evaluated in normal and immunologically deficient mice of the same litters. In normal littermate mice, BALB/c (nu/+) or C57BL/6 (bg/+), the BRM‐induced resistance against MCMV infection was evidenced by increase in fifty percent lethal doses, decrease in titers of viruses replicated in the target organs and augmentation of natural killer (NK) cell activity of the spleen cells. In T cell‐deficient, athymic nude mice, BALB/c (nu/nu), the protective effect was manifested by prolongation of the survival, decrease in the virus titers, and increase in the NK‐cell activity, but without decrease in mortality. In NK cell‐deficient, beige mutant mice, C57BL/6 (bg/bg), the BRM‐induced protection was nullified or minimized, and there was little difference in those parameters between BRM‐treated and untreated mice. However, with higher doses of OK‐432, but not PS‐K, or with sublethal doses of MCMV, the NK cell activity was slightly augmented in the beige mutant mice. Thus both NK cell and T cell activity are essential for mice to overcome acute MCMV infection and it is likely that the protective effect of BRM manifests itself fully, at least in immunologically intact mice.
Functional Defects of Culture‐Grown Bone Marrow‐Derived Macrophages from Autoimmune MRL/MpJ‐1pr/1pr Mice
Authors: Nakanishi Atsushi , Ashiba Takashi , Imai Yasuyuki , Osawa Toshiaki
Published: November 2013

To investigate the primary defects and development of macrophages in MRL/MpJ‐1pr/1pr (MRL/1) mice, we used a pure population of macrophages derived from bone marrow precursor cells cultured in the presence of L‐cell conditioned medium (LCM) as a source of colony stimulating factor. Bone marrow‐derived macrophages (BMMφ) from MRL/1 mice had lower antigen presenting activity as detected by the induction of antigen‐specific T cell proliferation, than age‐ and sex‐matched control mice (CBA/J). Cell surface antigens (Ia and Mac‐1) were determined quantitatively by a cell sorter as markers of macrophage differentiation. The BMMφ from MRL/1 contained a much smaller number of Ia antigen‐positive macrophages than those from normal mice. Treatment of BMMφ with an Ia‐inducing of factor (IFN‐γ) markedly increased the expression of Ia antigens. This increase was significantly greater in BMMφ from MRL/1 mice than in BMMφ from control mice. Expression of Mac‐1 antigen was not different in BMMφ from the two strains. The Fc‐mediated phagocytosis of IgG‐coated sheep red blood cells was decreased in BMMφ from MRL/1 mice compared with those from control mice. The function of nonspecific phagocytosis as measured by latex‐bead incorporation was also impaired in MRL/1 mice. The functional defects of MRL/1 BMMφ found in these experiments are not secondary defects acquired under the influence of environmental signals during development, but are derived from the primary abnormalities which already exist in myeloid stem cells.
Muramyl Dipeptide Induced Augmentation of the Proliferative Response of Thymocytes to Phytohemagglutinin
Authors: Aida Yoshitomi , Aono Masao , Onoue Kaoru
Published: November 2013

N‐Acetylmuramyl‐l‐alanyl‐d‐isoglutamine (MDP) augmented the proliferative response of thymocytes to phytohemagglutinin (PHA). The augmenting effect of MDP disappeared by passage of glass‐nonadherent thymocytes through Sephadex G‐10 (G‐10) column or by removal of low density cells by the Ficoll‐Conray gradient centrifugation. The diminished augmenting effects of MDP on the proliferative response of glass‐nonadherent‐G‐10 nonadherent thymocytes was restored by the addition of the G‐10 adherent cells. When G‐10 adherent cell fraction was extensively depleted of macrophages by glass adherence and EA‐rosetting, it was found that neither the macrophage‐depleted G‐10 adherent cell fraction nor the macrophage fraction supported by itself the proliferative response of G‐10 nonadherent thymocytes. However, addition of macrophage fraction together with the macrophage‐depleted G‐10 adherent cells did support the proliferation of G‐10 nonadherent thymocytes. It was further shown that peritoneal exudate macrophages could be substituted for thymic macrophage fraction. These results suggested that both the G‐10 adherent‐glass nonadherent cells and macrophages were essential for the MDP‐induced augmentation of the proliferative response of thymocytes to PHA and these cells exerted different accessory roles in this response.
Characterization of Mouse‐Human Hybridoma as a Useful Fusion Partner for the Establishment of Mouse‐Human‐Human Hybridoma Secreting Anti‐Tetanus Toxoid Human Monoclonal Antibody of IgM or IgG Class
Authors: Hirata Yuko , Sugawara Isamu
Published: November 2013

We succeeded in establishing a mouse‐human (M‐H) heterohybridoma clone which provides parental cells useful for human monoclonal antibody (hMoAb) production. Electron micrographs show that the M‐H hybridoma cells retain characteristics of murine origin with regard to chromatin patterns, small granules, granular endoplasmic reticulum and Golgi apparatus. The human DNA incorporated into the M‐H hybridoma is estimated to be about 1% of the total human chromosomal DNA. Mouse‐human‐human (M‐H‐H) hybridomas obtained by hybridization of the M‐H hybridoma cells with Epstein‐Barr virus(EBV)‐transfected human B cells secrete immunoglobulin (Ig) in amounts comparable to those of murine hybridomas. Also the M‐H‐H hybridomas grow in nude mice and are capable of producing ascites containing large quantities of Ig. The Ig class switching takes place in the M‐H‐H hybridomas at a much higher frequency than in the original EBV transformant and the M‐H hybridoma. Cells secreting specific monoclonal antibody of different Ig classes could be separated and concentrated by the use of fluorescence activated cell sorter (FACS).
Heterogeneity in Immunologic Functions of Rat Alveolar Macrophages—Their Accessory Cell Function and IL‐1 Production
Authors: Oghiso Yoichi
Published: November 2013

Alveolar macrophages (AM) from normal rats were separated into 4 different density fractions by centrifugation on a discontinuous Percoll gradient. These fractionated (I–IV) AM, as well as unfractionated (UF) AM, were then tested for their capacities to regulate mitogen‐induced T cell proliferation. Concanavalin A (Con A)‐induced response of nylon wool‐passed non‐adherent splenic T lymphocytes was suppressed by addition of UF or higher density (III and IV) AM, while an intermediate density (II) AM fraction could enhance T cell response in a dose‐dependent manner. Similar effects of UF or fractionated (I–IV) AM on T cell responses were noted when the cultures were exposed in vitro to inert, non‐fibrogenic titanium dioxide (TiO2) particles. On the contrary, T cell response was sustained by addition of UF or higher density (III and IV) AM, and was also more prominently enhanced by an intermediate density (II) AM after the in vitro exposures to fibrogenic dust particles, like silica and asbestos. Higher interleukin 1 (IL‐1) activity was detected from these silica‐ or asbestos‐exposed cultures of UF and fractionated (II, III, and IV) AM. The IL‐1 activity was also highly detectable from the cultures of an intermediate density (II) AM fraction when cultures were unexposed or exposed in vitro to TiO2 particles. The Ia antigen expression on the surface of UF or fractionated (II, III, and IV) AM was elevated in the Con A‐pulsed co‐cultures, but not significantly different whether or not they were exposed in vitro to dust particles. These results may indicate the presence of heterogeneity in accessory cell functions and IL‐1 production among rat AM.
The Bactericidal Mechanisms of Polymorphonuclear Leukocytes against Bacteroides fragilis: Significance of the Oxygen‐Dependent System
Authors: Nishimura Toshiro , Sasada Masataka , Kubo Akemi , Kakita Tokio , Moriguchi Toshihiro , Uchino Haruto
Published: November 2013

The mechanism by which polymorphonuclear leukocytes (PMNs) kill ingested Bacteroides fragilis was examined using PMNs from patients with chronic granulomatous disease (CGD) which is an inherited disease characterized by the defect of their PMNs in oxygen‐radical generation. The phagocytosis of B. fragilis by PMNs from CGD patients was comparable to that by normal PMNs. Although CGD cells killed B. fragilis to some extent, they did so less effectively than the normal PMNS. B. fragilis was killed by a xanthine oxidase system that generates oxygen radicals. When PMNs were incubated with opsonized B. fragilis, B. fragilis triggered the release of O2‐ and H2O2 from normal PMNs. Thus, normal PMNs appear to kill B. fragilis by both oxygen‐dependent and oxygen‐independent mechanisms.
Characterization of Mesosomes of Micrococcus luteus: Isolation and Properties of Mesosomal Ribosomes, and Localization of Penicillin‐Binding Proteins in Mesosomal Membranes
Authors: Nakasone Noboru , Masuda Kuniyoshi , Kawata Tomio
Published: November 2013

Mesosomes were isolated and purified from Micrococcus luteus under hypertonic conditions throughout preparation processes. The purified mesosomal preparation was composed of closed tubules and vesicles. Electron‐dense ribosome‐like particles were observed within the isolated mesosomal vesicles by electron microscopy. The ribosome‐like particles were isolated from the purified mesosomes by a procedure involving solubilization of the membranes with detergents followed by centrifugation on a linear density gradient of sucrose. The isolated particles have a sedimentation coefficient of 70S in the presence of 10 mm Mg2+, when Mg2+ concentration was lowered to 0.1 mm, the particles were dissociated into two sub‐particles of 30S and 50S. The 70S particles had the same appearance as cytoplasmic 70S ribosome particles upon observations of negatively stained preparations. These findings indicate that mesosomal tubules contain ribosomes. The isolated mesosomal ribosomes had the ability for protein synthesis when polyuridylic acid‐directed polyphenylalanine synthesis was assayed. The sensitivity of mesosomal ribosomes to inhibitors, chloramphenicol and streptomycin, for protein synthesis was significantly lower than that of both cytoplasmic and cytoplasmic membrane‐bound ribosomes. In addition, three penicillin‐binding proteins were detected in the mesosomal membranes. One of these was localized predominantly in the mesosomal membranes and the other two were distributed almost equally in both mesosomal and cytoplasmic membranes.
Immunoreactivity of Cytomegalovirus‐Induced Fc Receptors
Authors: Mackowiak Philip A. , Marling‐Cason Margaret
Published: November 2013

The present studies were undertaken to characterize the affinity of CMV‐induced Fc receptors for each of the subclasses of human IgG and to define the specific region of the IgG Fc fragment interacting with such receptors. To do this, we infected confluent human embryonic lung (HEL) cell monolayers with CMV (strain AD169) and then used a double radiolabel assay to measure adherence of antibody‐coated E. coli 06 to such monolayers. Preincubating monolayers with each of the 4 subclasses of human IgG (but not IgA, IgM, or human or bovine albumin) abrogated the enhancing effect of CMV infection on adherence of antibody‐coated E. coli 06 to HEL monolayers. Pepsin‐derived, purified Fc fragments of human IgG had a similar abrogative effect. Preincubating these with staphylococcal protein A did not reduce their capacity to interfere with binding of antibody‐coated E. coli to CMV‐induced Fc receptors. These observations establish a broad range of immunoreactivity for CMV‐induced Fc receptors, that encompasses all 4 subclasses of human IgG. They also provide indirect evidence that the reaction site of CMV‐induced Fc receptors is in the CH2 domain of the Fc fragment.
Evaluation of the Experimental Pathogenicity of Some Cryptococcus Species in Normal and Cyclophosphamide‐Immunodepressed Mice
Authors: De Bernardis Flavia , Palliola Ennio , Lorenzini Rodolfo , Antonucci Giovanni
Published: November 2013

The pathogenic potential of distinct Cryptococcus species has been evaluated in mice rendered leukopenic by one or two injections of the potent immunosuppressive drug cyclophosphamide (Cy). Pathogenicity assessment included enumeration of viable cryptococcal cells in animal organs and histopathological observations. It was found that putatively non‐pathogenic species of Cryptococcus, in particular C. cereanus and C. albidus, showed significant lethality for Cy‐treated mice. In Cy‐immunodepressed mice, challenged with the infectious cryptococcal cells two days after pharmacological treatment, a significant decrease of LD50 (equivalent to at least one order of magnitude) was observed for all Cryptococcus species. However, the pathogenicity enhancement due to Cy immunodepression was greater with C. neoformans. In all cases, brain and kidney were the most invaded tissues as also evidenced by histopathological examination, which showed the typical cystic lesion. All the observations made point to the conclusion that the pathogenic potential, for the immunomodulated host, of Cryptococci other than C. neoformans is significant being quantitatively and not qualitatively different from that of C. neoformans, as evidenced by a similar organotropism and similar type of histological lesions in the target organs (brain and kidney).
Human Macrophage‐Activating Factors for Cytotoxicity
Authors: Higuchi Masahiro , Sugimoto Masamichi , Kobayashi Yoshiro , Osawa Toshiaki
Published: November 2013

Human T cell hybridoma, H3‐E9–6, that produces macrophage activating factors for cytotoxicity (MAF‐C) was prepared by somatic fusion of phytohemagglutinin‐activated peripheral blood lymphocytes with emetine/actinomycin D‐treated cloned human acute lymphocytic leukemia cells (CEM 11). The activities of the following were assayed: (1) macrophage‐activating factor for cytotoxicity of monocytes (MAF‐C 1 day), (2) macrophage‐activating factor for cytotoxicity of monocyte‐derived macrophages (MAF‐C 6 day), (3) macrophage‐activating factor for cytotoxicity of murine macrophages (MAF‐Cm), (4) macrophage‐activating factor for glucose consumption (MAF‐G), (5) macrophage‐activating factor for O2‐ formation (MAF‐O). The culture supernatant of H3‐E9–6 showed MAF‐C 1 day‐MAF‐C 6 day, MAF‐Cm, and MAF‐G activities. The MAF‐Cm activity was considerably enhanced by the addition of murine recombinant interferon gamma (rIFN‐γ). The MAF‐C 1 day activity in the H3‐E‐9–6 sup was not decreased by heat treatment (56 C, 30 min), by pH 2 treatment or by the addition of monoclonal anti‐human IFN‐γ antibody or polymyxin B. These data suggest that MAF‐C in H3‐E9–6 sup is distinct from human IFN‐γ or lipopolysaccharide (LPS).
Human Macrophage‐Activating Factors for Cytotoxicity
Authors: Higuchi Masahiro , Sugimoto Masamichi , Kobayashi Yoshiro , Osawa Toshiaki
Published: November 2013

Two different factors (MAF‐C I and MAF‐C II) were obtained by anion exchange chromatography of the culture supernatant of a human T‐cell hybridoma, H3‐E9–6, which produces macrophage‐activating factors for cytotoxicity (MAF‐C). These 2 factors induced the cytotoxicity of monocytes synergistically as a priming signal (MAF‐C I) and a triggering signal (MAF‐C II), respectively. On gel filtration on a column of Superose 12, MAF‐C II was eluted mainly at the void volume, whereas MAF‐C I was eluted in the fractions corresponding to approximate molecular weights of 30–300 K. On the other hand, gel filtration in the presence of sodium deoxycholate revealed that MAF‐C II has an approximate molecular weight of 40,000, but MAF‐C I was unstable under these conditions. When the activity for mouse macrophages (MAF‐Cm activity) was tested, the MAF‐C II fraction showed high MAF‐Cm activity in the presence of murine recombinant interferon gamma (rIFN‐γ), but the MAF‐C I fraction did not show MAF‐Cm activity even in the presence of lipopolysaccharide (LPS). These results suggest that MAF‐C I (priming lymphokine) has species specificity but MAF‐C II (triggering lymphokine) does not.
Temperature‐Modulated Physiological Characteristics of Candida albicans
Authors: Hazen Kevin C. , Hazen Beth W.
Published: November 2013

Despite numerous investigations on candidiasis, definitive conclusions concerning virulence factors are few because of oftentimes confusing and contradictory results. By utilizing various physiologic tests, which include germ tube induction, inhibition of germination by a morphogenic autoregulatory substance, enzyme production, susceptibility to exogenous chemicals, and cell surface hydrophobicity, we demonstrated that such variability is due, in part, to the environmental conditions in which cells were grown in preparation for analysis. Room‐temperature grown cells were generally less sensitive to environmental perturbation and germinated more uniformly than cells grown at 37 C. The implication of these results in relation to pathogenic studies and the epidemiology of candidiasis is suggested.
The Primary Immune Response of the Green Toad (Bufo viridis) to Challenge with Crithidia fasciculata
Authors: Al‐Yaman Fadwa , Ingram George A.
Published: November 2013

The primary immune response of the green toad (Bufo viridis) following immunization with Crithidia fasciculata choanomastigotes was studied. Lysins, agglutinins, and antibodies detectable by enzyme‐linked immunosorbent assay (ELISA) were first detected in the sera of immunized animals one week after injection. The antibody titers increased to significant levels (P<0.01) and maximum values were reached seven weeks post‐immunization. The stimulated immunoglobulins were antigen‐specific, partially heat‐labile, sensitive to the reducing agent dithiothreitol, possessed precipitin activity, effectively fixed complement and exhibited an electrophoretic mobility similar to the γ‐globulins of human serum. On this basis, it is probable that the antibody produced during the primary response in green toads is high molecular weight IgM. Increases in serum lysozyme levels paralleled the rise of antibody titers. Overall, the lysozyme concentration increased two‐fold compared to the appropriate controls. This is the first report of the immune response in amphibians to experimental injection with protozoan parasites and the use of the ELISA technique to detect antibodies in amphibian sera.
Hydrolysis of Phosphatidylinositol 4, 5‐Bisphosphate and Increase in Cytosolic Free Ca2+‐Concentration Induced in a Human T Cell Leukemia Line, JURKAT, by Monoclonal Antibodies against the T3 Complex
Authors: Sasaki Terukatsu , Takei Takashi , Hasegawa‐Sasaki Hiroko
Published: November 2013

The monoclonal antibodies against the T3 complex on human T lymphocytes, anti‐Leu‐4, OKT3, and T3, induced an accumulation of inositol phosphates in a human T cell leukemia line, JURKAT, in the presence of LiCl. The monoclonal antibodies also induced an increase in the cytosolic free Ca2+‐concentration ([Ca2+]i) in JURKAT. The accumulation of inositol phosphates and the increase in [Ca2+]i were specifically induced by the monoclonal antibodies against the T3 complex. Other monoclonal antibodies against differentiation antigens on human T lymphocytes were not active in inducing these responses in JURKAT. Stimulation of JURKAT by anti‐Leu‐4 induced a rapid and immediate decrease in phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P2] and an increase in the 32P‐labeling of phosphatidic acid, which occurred after a short lag period. An analysis of inositol phosphates formed in the anti‐Leu‐4‐stimulated JURKAT indicated the formation of inositol trisphosphate. These results strongly suggested that the T3 complex or T3/antigen receptor (Ti) complex functions as a receptor which transduces antigen signal, presented by either antigen‐presenting cells or target cells, into the hydrolysis of PtdIns(4,5)P2. Fetal bovine serum at a dose of 1–20 μl/ml induced a marked and transient [Ca2+]i increase in JURKAT immedately after addition. However, the level of formation of inositol phosphates was very small in cells stimulated by fetal bovine serum. Fetal bovine serum induced an immediate increase in the 32P‐labeling of phosphatidic acid in JURKAT. These and other results suggested that serum increased [Ca2+]i in JURKAT by a mechanism different from that for the anti‐Leu‐4‐induced [Ca2+]i response.
Two Groups of Mycobacterium avium Complex Strains Determined According to the Susceptibility to Rifampicin and Ansamycin
Authors: Tsukamura Michio
Published: November 2013

Mycobacterium avium complex strains previously not exposed to any antituberculosis agents could be divided into two groups according to their susceptibility to rifampicin and ansamycin; one group susceptible to 80 μg/ml rifampicin and to 1.25 μg/ml ansamycin, and another resistant to these concentrations. In each group, the ratio of the minimal inhibitory concentration of ansamycin against that of rifampicin was greatly different depending on the strain. This naturally occurring resistance to rifampicin and ansamycin was frequently correlated to naturally occurring resistance to ethambutol, kanamycin, enviomycin, kitasamycin, and minocycline, but not correlated to that to isoniazid and sulfadimethoxine. Ansamycin was more active than rifampicin against M. bovis, M. kansasii, M. marinum, M. xenopi, and M. haemophilum.
A Comparative Study of Alteration in Lymphocyte Subsets among Varicella, Hand‐Foot‐and‐Mouth Disease, Scarlet Fever, Measles, and Kawasaki Disease
Authors: Yanase Yoshio , Tango Toshiro , Okumura Ko , Tada Tomio , Kawasaki Tomisaku
Published: November 2013

Changes in the lymphocyte subsets of 13 patients with varicella, 5 with hand‐foot‐and‐mouth disease, 4 with scarlet fever, 10 with measles and 20 with Kawasaki disease were examined by immunofluorescent flow cytometric analysis using monoclonal antibodies against lymphocyte cell surface antigens. The results were compared with those of age‐matched normal controls. A significant increase in the percentage of Leu‐2a positive (Leu‐2a+) cells was shown during the early convalescence of varicella, scarlet fever and measles. A significant decrease in the percentage of Leu‐3a+ cells during the acute phase was common to all the diseases examined, and a significant decrease of Leu‐4+ cells was observed except in measles. As a result, a significant decrease in the Leu‐3a+/Leu‐2a+ ratio was common to all the diseases examined during the acute and/or early convalescent phases. Leu‐M3+ cells increased significantly in varicella, scarlet fever, and Kawasaki disease. HLA‐DR+ cells increased significantly in varicella and Kawasaki disease. No significant changes in the proportions of Leu‐7+, Leu‐10+, and 2H7+ cells were found throughout the course of all the diseases examined.
Fluorometric Enzyme‐Linked Immunosorbent Assay for the Measurement of IgE Antibody to Mite Dermatophagoides farinae
Authors: Sakaguchi Masahiro , Inouye Sakae
Published: November 2013

We have developed a fluorometric enzyme‐linked immunosorbent assay for measuring IgE antibody to Dermatophagoides farinae. Polystyrene microplates were coated with proteins extracted from the mites. The IgE antibody which attached to the solid‐phase antigen was detected by anti‐IgE antibody conjugated with β‐galactosidase. Four‐methylumbelliferyl‐β‐D‐galactoside was used as the enzyme substrate and the fluorescence intensity of the reaction product was measured. The antibody levels determined by this method well correlated with those determined by the radioallergosorbent test (RAST). This method is simpler and less expensive to carry out than the RAST when dealing with a large number of serum specimens for seroepidemiological studies of asthma and nasal allergy.
Overproduction of Gene Products by the Super‐High‐Copy‐Number Plasmid pNR333
Authors: Mochizuki Akihiko , Horiuchi Sankichi , Goto Nobuichi , Nakaya Rintaro
Published: November 2013

The plasmid pNR333 is a kanamycin‐resistant, deletion derivative of pNR113 with an extremely high copy number in Escherichia coli and in Proteus mirabilis. In order to determine the usefulness of pNR333 as a replication gene of vector, the genes encoding chloramphenicol acetyltransferase (CAT) and β‐galactosidase (β‐gal) were cloned individually into both pNR333 and other low‐copy‐number plasmids. The expression of the cloned genes was compared by measuring the specific activity of each enzyme and the amounts of the proteins produced. A hybrid plasmid pNR333‐cat expressed 53 times as much activity of CAT as the low‐copy plasmid S‐a which had a copy number of four. The lacZ gene cloned in pNR333 produced 17 times as much β‐gal as in the low‐copy‐number plasmid pNR1150. These results suggest that pNR333 is a useful vector plasmid for producing a large amount of polypeptides in E. coli hosts.
Quantitative Evaluation of Colonizing Ability of Vibrio cholerae O1
Authors: Nakasone Noboru , Iwanaga Masaaki
Published: November 2013

A new method to evaluate the adhesive ability of Vibrio cholerae O1 was proposed. Broth cultured V. cholerae O1 and a piece of formalin‐fixed rabbit intestinal wall were incubated together in KRT buffer and the number of adhered organisms was counted under a scanning electron microscope. This method was much less laborious than other methods that have been used so far, and most significantly, constant results were obtained in repeated experiments. The adhesive properties of toxigenic V. cholerae O1 evaluated by this method correlated well with its observed experimental pathogenicity.
Antigen‐Specific T Cell Cluster Formation on Antigen‐Pulsed Macrophage Monolayers in Mice
Authors: Saizawa Mitsuyoshi , Mabuchi Ayako , Yokomuro Kozo , Kimura Yoshitami
Published: November 2013

We describe the quantitative measurement of antigen‐specific clusters formed by antigen‐pulsed macrophages and immunized T cells in mice. We have found the peripheral blood T cells show very little non‐specific adhesion to macrophages in mice. By using this population of lymphocytes in the peripheral blood as the source of immunized T cells, we could quantitate antigen‐specific cluster formation. On OVA‐pulsed monolayers of peritoneal exudate macrophages from normal BALB/c mice, syngeneic peripheral blood T cells from donors immunized with the same antigen develop 20–40 clusters per 1,000 macrophages, whereas the same T cells on non‐pulsed monolayers develop only 0–5 cluster‐like accumulations of cells. On antigen‐pulsed monolayers of macrophages from allogeneic (C57BL/6 or A/J) mice, clusters are developed only in the negative range (0–5/1,000 macrophages). Considering the observation by Braendstrup et al, these data seem to suggest that histocompatibility between macrophages and T cells is required to develop antigen‐specific T cell clusters on antigen‐pulsed macrophage monolayers, and that the genetic restriction of immune responsiveness may be directly expressed in this initial form of cellular interaction between antigen‐bearing macrophages and specific T cells.
Characterization of Rat T Cell Subset Antigen by Monoclonal Antibody
Authors: Yamaki Toshiaki , Uede Toshimitsu , Sugawara Yukihiro , Wada Takuro , Yamaguchi Akira , Kokai Yasuo , Kikuchi Kokichi
Published: November 2013

6B2‐B8 T cell hybridoma cells were used to immunize mice, and immune spleen cells were fused with NS/1 myeloma cells. One clone, designated RTH‐7, reacted with 89.5% of rat thymocytes, 30.2% of rat spleen cells, and 42.3% of rat lymph node cells. The RTH‐7 reacted with a subset of rat T cells but not with B cells. Double staining analysis demonstrated that RTH‐7 stained a rat T cell subset distinct from R1‐10B5‐positive cells that were known to be equivalent to mouse Lyt‐2. It was revealed that RTH‐7 and W3/25 recognize different antigenic epitopes on the same molecule. The RTH‐7 as well as W3/25 substantially inhibited the production of interleukin 2 by cells in mixed lymphocyte reaction and the lymphocyte proliferation induced by mixed lymphocyte reaction. The RTH‐7 inhibited the lymphocyte proliferation induced by Con A whereas W3/25 failed to do so. The RTH‐7 defined antigen has a molecular weight of 53,000 under reducing condition and 47,000 under nonreducing condition. The RTH‐7 defined antigen showed a wide range of heterogeneity in pI (6.2–8.8). The associated molecule of approximate molecular weight of 27,000 was occasionally detected with the RTH‐7 defined antigen in 6B2‐B8 T cell hybridoma cells as well as peripheral T cells but not in thymocytes. Thus, RTH‐7 detects a cell surface antigen of a functional T cell subset of rat origin.
Antigenic Determinants on Fimbriae of Serratia marcescens US5 Analyzed Using Monoclonal Antibodies
Authors: Jingushi Seiya , Mitsuyama Masao , Moriya Tetsuhiro , Kawabata Shunichiro , Iwanaga Sadaaki , Amako Kazunobu
Published: November 2013

The antigenic sites on small thin fimbriae of Serratia marcescens strain US5 were investigated using immunoelectron microscopy and monoclonal antibodies (MAbs). Negative staining of the fimbriae after treatment with MAbs showed a regularly spaced arrangement of the antibody molecules. When the subunit peptide was subjected to immunoblotting using the MAbs, a single band with a molecular weight of approximately 19kD was evident. This binding of the MAbs to the subunit peptide was completely abrogated after treatment with 2‐mercaptoethanol, thereby suggesting the important role of disulfide linkage in the maintenance of the conformation of the antigenic site reacted with MAbs. Amino acid analysis of the subunit peptide revealed two cysteine residues, and cysteine residues were absent in the N‐terminal portion.