bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of B‐L Antigens on Reticular Epithelial Cells of the Bursa of Fabricius
Authors: Hoshi Sumio , Nunoya Tetsuo , Ueda Susumu
Published: November 2013

We have established two monoclonal antibodies against B‐L antigens (chicken Ia‐like antigens). The specificity of the antibodies for B‐L antigens was determined by two criteria, the cellular expression and the molecular structure of antigens with which they reacted. They reacted with antigens expressed on bursacytes, Con A‐blast thymocytes, macrophages, and MDCC MSB1, but not with thymocytes and erythrocytes. In molecular basis, they recognized 64,000 dalton glycoprotein consisting of two polypeptides, 35,000 and 32,000 dalton, which bound non‐covalently. To investigate the distribution of B‐L antigens on non‐lymphoid cells of the bursa of Fabricius, which were thought to play important roles in the differentiation of B cells, anti‐B‐L antigen and anti‐chicken immunoglobulin (Ig) monoclonal antibodies were used. B‐L antigen‐positive cells were detected in both cortical and medullary areas, whereas Ig‐positive lymphoid cells were confined to the medullary areas of normal chicken bursal follicles. In the bursal follicles of cyclophosphamide (CY)‐treated chickens, lymphoid cells were depleted but epithelial cells remained intact. And B‐L antigen‐positive but Ig‐negative cells were easily detected in the medullary areas of almost all follicles. These cells were identified to be reticular epithelial cells (REp cells) from the result of their keratin expression.
The Structure of Marek's Disease Virus DNA: Amplification of Repeat Sequence in IRs and TRs
Authors: Hayashi Masanobu , Jessip John , Fukuchi Kunihiko , Smith Mary , Tanaka Akiko , Nonoyama Meihan
Published: November 2013

The structure of Marek's disease virus (MDV) DNA was investigated by using Southern blot hybridization analysis. A heterogeneous region was observed in the inverted repeats region, IRs and TRs, as well as in the TRL and IRL. The results of DNA sequencing of the heterogeneous region showed that the heterogeneity of IRs and TRs was due to amplification of a 178‐bp repeat sequence. Amplification of IRs and TRs was found in viral DNA from both pathogenic and nonpathogenic strains. The structure of DNA from the latent MDV genome present in established lymphoblastic cells was also determined. Amplification of the 132‐bp repeat sequence in IRL and TRL was not found in latent MDV DNA of established lymphoblastic cells, whereas amplification of the 178‐bp repeat sequence in IRs and TRs was found in the same DNA.
Tolerance Induction of Alloreactivity by Portal Venous Inoculation with Allogeneic Cells Followed by the Injection of Cyclophosphamide
Authors: Kokudo Shuhei , Sato Soichiro , Qian Jia‐hua , Wada Kentaroh , Ikegami Ryoichi , Hamaoka Toshiyuki , Fujiwara Hiromi
Published: November 2013

BALB/c mice receiving allogeneic C3H/He or C57BL/6 spleen cells via portal venous (p.v.) route or a single administration of cyclophosphamide (Cy) were capable of rejecting the respective allogeneic C3H/He‐ or C57BL/6‐derived tumor cells. In contrast, the combined treatment of p.v. inoculation with allogeneic lymphocytes and Cy administration abrogated the capability of rejecting allogeneic tumor cells. Such abrogation of alloreactivity was alloantigen‐specific and associated with the suppression of potentials to generate delayed‐type hypersensitivity (DTH) and cytotoxic T lymphocyte (CTL) responses to alloantigens. This was further substantiated by the inhibition of molecular mechanisms underlying anti‐allo‐DTH and ‐CTL responses. Thus, the above combined treatment led to the decreased production of lymphokines such as macrophage‐activating factor (MAF) and interleukin 2 (IL2) following the stimulation with the relevant alloantigens. These results demonstrate that p.v. inoculation of allogeneic cells followed by a single administration of Cy results in the effective elimination of alloreactivity as verified by the suppression of cellular and molecular mechanisms of alloreactive responses.
Some Applications of the Surface‐Spreading Technique to Virological Studies
Authors: Yazaki Kazumori , Sano Toshihiko , Ikegami Masato , Urushibara Toshiyuki
Published: November 2013

By application of the surface‐spreading technique, virus particles in infected cells and viremia serum, and precipitates in agar plates of the double immunodiffusion technique of Ouchterlony could easily and clearly be visualized without any purification process.
Two‐Dimensional Flow Cytometric Analysis of T Cell Subsets Maintained in IL‐2 Medium after Autologous Mixed Leukocyte Reaction Activation
Authors: Goto Makoto , Miyamoto Terumasa , Nishioka Kusuki
Published: November 2013

Human T cells are stimulated with an autologous mixed leukocyte reaction (AMLR) and can be propagated in interleukin‐2. Staining of the cultured cells with the combination of two monoclonal antibodies was evaluated by two‐dimensional flow cytometry at weekly intervals. AMLR activation resulted in an initial preservation of the CD4+ (helper/inducer T) subset predominance over the CD8+ (suppressor/cytotoxic T) cells, noted on normal circulating blood lymphocytes. However, during culture in interleukin‐2, there was a progressive increase in the percentages of CD8+ Leu 15− cytotoxic T, CD4+ Leu 8− helper T, and CD3+ HLA‐DR+ activated T cells, and a concomitant decrease in those of CD4+ Leu 8+ suppressor inducer T and CD8+ Leu 15+ suppressor T cells if the responder sheep red blood cell (SRBC)‐rosetting T cells were made up by tris ammonium chloride, but not by hypotonic shock treatment to lyse SRBC. The significant difference between hypotonic shock‐T cells and ammonium chloride‐T cells in the phenotypic changes of T cell subsets after long‐term culture in an interleukin‐2 medium may suggest a regulatory role of the ammonium chloride‐sensitive T cells in the AMLR.
Relationship between Mycobacterial Species and Their Carotenoid Pigments
Authors: Ichiyama Satoshi , Shimokata Kaoru , Tsukamura Michio
Published: November 2013

A study of the relationship between mycobacterial species and their carotenoid pigments was carried out. According to the carotenoid pigments contained, the mycobacterial species tested were divided into four groups: the first group ofMycobacterium kansasiiandM. marinum, which formed principally onlyβ‐carotene; the second group ofM. gordonae, M. scrofulaceum, M. szulgai, M. xenopi, M. flavescens, M. phlei, M. rhodesiae, M. neoaurum, andM. aichiense, which formedβ‐carotene and a zeaxanthin‐like substance; the third group ofM. aurumandM. obuense, which formedβ‐carotene and an eschscholtzxanthin‐like substance; and the fourth group ofM. chubuenseandM. tokaiense, which formedβ‐carotene and zeaxanthin‐ and eschscholtzxanthin‐like substances. The common carotenoid pigment throughout the genusMycobacteriumwasβ‐carotene and the hypophasic carotenoids differed according to the species.
Formation of a Hexagonal Lattice Structure by an R‐Form Lipopolysaccharide of Klebsiella: Effect of Various Divalent Cations on the Lattice Formation
Authors: Kato Nobuo , Ohta Michio , Kido Nobuo , Ito Hideo , Naito Setsuko
Published: November 2013

The R‐form lipopolysaccharide from Klebsiella pneumoniae strain LEN‐111 (O3‐: Kl‐), from which cationic material had been removed by electrodialysis, was previously shown to form a hexagonal lattice structure with the lattice constant of 14 to 15 nm when suspended in 50 mm tris(hydroxymethyl)aminomethane buffer at pH 8.5 containing 10 mm Mg2+. Under this experimental condition, effects of other divalent metal cations on the hexagonal assembly of the electrodialyzed LPS were compared with that of Mg2+. The Zn2+, Hg2+, Cu2+, and Ni2+ could produce essentially the same hexagonal lattice structure with the lattice constant of 14.5 to 15.0 nm as that formed with Mg2+. The Cd2+, Co2+, and Fe2+ produced the hexagonal lattice structure with the lattice constant of 15.5 to 16.0 nm, and Ba2+, Sr2+, and Ca2+ produced that with the lattice constant of 18 to 19 nm. In addition, the hexagonal lattice structures formed with the latter three cations were less orderly than those formed with the other cations. When the higher concentrations of Ba2+, Sr2+, and Ca2+ were used, the lattice constants were not shortened. The length of lattice constants of the hexagonal lattice structures formed with the divalent cations did not relate to the quantity of the cations bound to the LPS. Among the divalent cations tested, Hg2+ was bound to the LPS in the smallest amount (its atomic ratio to P, 0.07), and Zn2+ and Fe2+ were bound in very large amounts (their atomic ratios to P, 2.94 and 8.28, respectively).
T Cell‐Mediated Cytotoxicity against Dengue‐Infected Target Cells
Authors: Pang Tikki , Devi Shamala , Blanden Robert V. , Lam Sai Kit
Published: November 2013

A cytotoxic T lymphocyte (CTL) response to dengue virus‐infected target cells is described. Effector cells were generated in an in vitro secondary culture and appeared to be T cells possessing both the Lyt 1.1 and Lyt 2.1 surface antigens. A stronger CTL response was noted with the H‐2k haplotype compared to H‐2d, and H‐2 compatibility was required between CTL and target cells. CTL generated showed some cross‐reactivity with target cells infected with Japanese encephalitis virus (JEV), another flavivirus, but not with target cells infected with an alphavirus, Sindbis. The significance and importance of these findings are discussed.
Purification and Some Properties of Cytotoxin Produced by Clostridium difficile
Authors: Katoh Takahiro , Honda Takeshi , Miwatani Toshio
Published: November 2013

The cytotoxin produced by Clostridium difficile was highly purified by using ammonium sulfate fractionation and successive column chromatographies of DEAE‐Sephadex A‐25, hydroxyapatite, Bio‐Gel A‐0.5m, Phenyl‐Sepharose CL‐4B, and Mono Q. The purified cytotoxin gave a single band on conventional and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the presence of dithiothreitol. Its molecular weight was estimated to be 260,000 and 50,000 by gel filtration and sodium dodecyl sulfate‐polyacrylamide slab gel electrophoresis in the presence of dithiothreitol, respectively. Thus it was supposed that the toxin consists of 5 subunits having molecular weight of approximately 50,000. It had an isoelectric point of 6.6. The toxin was heat‐labile (60 C for 10 min) and inactivated by treatment with trypsin and pronase, or at pH below 4 or over 10. The minimum cytotoxic dose of the cytotoxin against Chinese hamster ovary cells was 3 ng. It was also demonstrated that the toxin is antigenically different from enterotoxin of C. difficile.
Activities and Properties of Putrescine‐Biosynthetic Enzymes in Vibrio parahaemolyticus
Authors: Yamamoto Shigeo , Nakao Hiroshi , Yamasaki Keiko , Takashina Kimiko , Suemoto Yasuo , Shinoda Sumio
Published: November 2013

The biosynthetic pathways for putrescine (Put) in Vibrio parahaemolyticus were delineated by measuring activities of the enzymes which would be involved in its biosynthesis. Experiments with labeled arginine and ornithine revealed that both of these amino acids were converted into Put by intact cells. The activities of three enzymes, arginine decarboxylase (ADC), ornithine decarboxylase (ODC), and agmatine ureohydrolase (AUH), were detected in cell extracts. ADC and ODC of V. parahaemolyticus were similar in the following properties to the corresponding enzymes of Escherichia coli: 1) both decarboxylases showed a pH optimum at 8.25 and required pyridoxal phosphate and dithiothreitol for full activity; 2) while ODC was considerably activated by GTP, ADC was only slightly; 3) both decarboxylases were inhibited by polyamines; 4) ADC was inhibited by difluoromethylarginine, a potent inhibitor of bacterial ADC. However, in contrast to the corresponding enzymes of E. coli, the V. parahaemolyticus ADC showed no requirement for Mg2+, and the AUH was active over a wide pH range of 8.5–9.5 with a maximum at pH 9.0. Furthermore, in all 6 strains tested, the activity of ADC was obviously high compared with that of ODC, and AUH was present with a relatively high activity. Cultivation of these strains at a suboptimal NaCl concentration (0.5%) resulted in a pronounced increase in both ADC and AUH activities. These observations suggest that the important pathway for Put biosynthesis in V. parahaemolyticus is the decarboxylation of arginine by ADC and the subsequent hydrolysis of its product, agmatine, by AUH.
Effect of C‐Reactive Protein on Peritoneal Macrophages
Authors: Miyagawa Nobuyuki , Okamoto Yasuyuki , Miyagawa Sachiko
Published: November 2013

The effect of human C‐reactive protein (CRP) isolated and purified from pooled patients' sera on macrophage function, especially on macrophage migration, was studied. Peritoneal exudate cells (PEC) from guinea pigs were used for macrophage migration inhibition (MMI) test of capillary method. Migration of either PEC or adherent purified macrophages exposed to CRP were inhibited dose‐dependently. These findings indicate that CRP inhibits macrophage migration directly, not via activation of lymphocytes contained in PEC. As control, we examined the effect of normal human serum, anti C‐polysaccharide antibodies isolated from patients' sera, and free endotoxin at the dose contaminated in CRP preparation on macrophage migration and found that none of them were effective. The effect of CRP on MMI of sensitized PEC exposed to antigen was also studied. Large amounts of CRP inhibited MMI induced by antigen, indicating the possibility that CRP may act on macrophages competitively with migration inhibitory factor (MIF) and may modulate MMI. CRP possesses MIF‐like activity and may play a functional role at the site of tissue injury by causing the accumulation of macrophages.
Effect of C‐Reactive Protein on Peritoneal Macrophages
Authors: Miyagawa Nobuyuki , Okamoto Yasuyuki , Nakano Hiroshi
Published: November 2013

The effect of human C‐reactive protein (CRP) on macrophage function was studied in an assay of superoxide anion (O2−) production. Peritoneal exudate macrophages (PEM) of guinea pigs exposed in vitro to various doses of CRP for 72 hr resulted in the development of O2− production dose‐dependently, measured by increases in superoxide dismutase‐inhibitable nitro blue tetrazolium reduction. The O2−‐producing activity of PEM cultured without CRP, used as a control, decreased markedly in proportion to incubation time. The O2− production by PEM exposed to CRP for 18 hr when control PEM were still high in O2− production, was decreased by larger doses of CRP, while PEM cultured without CRP for 72 hr, when O2− production by control PEM was very low, followed by incubation with CRP for another 18 hr, produced O2− CRP‐dose‐dependently as in the case of that observed after 72‐hr incubation with CRP. These results indicate that CRP is capable of activating macrophages and acts on macrophage function as a modulator. CRP possesses migration inhibitory factor (MIF)‐like activity (as reported in the preceding paper) and also macrophage‐activating factor (MAF)‐like activity, indicating that CRP may play a functional role at the site of inflammation and tissue damage by accumulating and activating macrophages.
Contractile Action of Heat‐Labile Toxin of Bordetella parapertussis on Aortic Smooth Muscles of Pigs
Authors: Endoh Masahiko , Nagai Masaaki , Nakase Yasukiyo
Published: November 2013

Using both vascular smooth muscle strips (VSMS) and cultured cells (VSMC) from aortas of pigs, the contractile action of Bordetella heat‐labile toxin (HLT) purified from B. parapertussis was studied in an attempt to elucidate the mechanisms of its action. HLT induced contractions in VSMC in parallel with the increase of Ca2+‐influx. The HLT‐induced Ca2+‐influx and contraction were not influenced by verapamil or diltiazem, though a certain extension of the lag period was seen. The contractile action of HLT on VSMS and VSMC was not influenced either by diltiazem or quinacrine; that on VSMC was not influenced by prednisolone, indomethacin, aspirin, CV‐3988, FPL‐55712, ruthenium red, or TEAC. On VSMS, prednisolone caused the extension of lag period following HLT exposure. The action of HLT on VSMS was inhibited by TMB‐8, whereas that on VSMC was not though the extension of lag period was seen. The HLT‐induced contraction in both VSMS and VSMC was completely inhibited by H‐7. The contraction in VSMS, but not in VSMC, was inhibited by H‐8. HLT did not induce specific activation of the protein kinases in VSMC. The addition of cGMP or cAMP brought about relaxation in the HLT‐exposed VSMS contracting in maximum. HLT caused a significant increase in permeability of VSMC membrane to trypan blue, accompanied with contraction. Both HLT‐induced contraction and increase in permeability were inhibited by dextran of M.W. 8,000, but not of M.W. 5,000. These results suggested that HLT acted on vascular smooth muscle cells by damaging the membrane permeability, but not by disturbing the known cascades or systems for physiological contractions, resulting in the increase in Ca2+‐influx and then contractions.
Electron Microscopic Observations of the Embryo Leptotrombidium (Leptotrombidium) pallidum Naturally Infected with Rickettsia tsutsugamushi
Authors: Urakami Hiroshi , Takahashi Mamoru , Tamura Akira , Hori Eitaro
Published: November 2013

Embryos of Leptotrombidium (Leptotrombidium) pallidum mites naturally infected with Rickettsia tsutsugamushi were examined by electron microscopy. Rickettsiae were not found in eggs just after oviposition, but were easily detected in cells at the various parts of the embryos just before hatching, indicating that the rickettsiae are surely vertically transmitted from infected adult mites to the larvae through embryos, and the rickettsiae may multiply in situ during the developing process of the embryo.
Isolation and Characterization of Outermost Layer Deficient Mutant Spores of Bacillus megaterium
Authors: Takubo Yoshihiro , Atarashi Miyuki , Nishihara Tsutomu , Kondo Masaomi
Published: November 2013

Outermost layer deficient mutant spores of Bacillus megaterium ATCC 12872 were isolated by Urografin density gradient centrifugation after mutagenesis with ethyl methanesulfonate. Although the composition of the cortex peptidoglycan was the same as that of the parent spores, three major proteins (48, 36, and 22 K daltons) were missing, suggesting that these proteins are components of the outermost layer. All mutant spores were also found to have very hydrophobic surface by ‘salt aggregation test,’ which would facilitate selection of such mutants.
Enhancement of Antigen‐Presenting Function of Dendritic Cells with Culture Supernatants of Mouse Peritoneal Macrophages Stimulated with Certain Particulate Substances
Authors: Miyazaki Hiroshi , Ito Akira , Osawa Toshiaki
Published: November 2013

The production from murine resident peritoneal macrophages (Mφ) of a soluble factor, which was capable of enhancing the antigen‐presenting (AP) function of dendritic cells (DC), was examined. The supernatants of peritoneal Mφ (Mφ sup) were prepared by culturing peritoneal Mφ with particles, i.e., zymosan A, latex, and sheep red blood cells (SRBC), or antigen‐antibody (Ag‐Ab) complexes such as keyhole limpet hemocyanin (KLH)‐anti‐KLH, ovalbumin (OVA)‐anti‐OVA, and SRBC‐anti‐SRBC complexes. When exposed to Mφ sup during antigen pulsing DC induced a marked antigen‐specific T cell proliferation, relative to DC treated with the supernatants from Mφ cultured without stimuli (control sup). On the other hand, Mφ sup‐treated splenic Mφ stimulated antigen‐specific T cell activation to almost the same extent as did splenic Mφ treated with control sup. These results indicated that peritoneal Mφ elaborated a soluble factor which preferentially enhanced the AP capacity of DC when stimulated with particles or Ag‐Ab complexes. Analytical gel filtration of Mφ sup revealed that the factor had an apparent molecular weight of 27,000 daltons which was distinct from interleukin 1.
Suppression of HBsAg Production in PLC/PRF/5 Human Hepatoma Cell Line by Interferons
Authors: Yamashita Yumi , Koike Kaori , Takaoki Muneo , Matsuda Shinobu
Published: November 2013

Recombinant human interferon alpha 2a as well as natural human interferons α and β significantly suppressed the production of hepatitis B surface antigen by PLC/PRF/5 cells (which have been established from a human primary hepatocellular carcinoma and proven to carry the hepatitis B virus DNA) and inhibited proliferation of these cells in vitro. However, the production of α‐fetoprotein by PLC/PRF/5 cells was less significantly affected by any of the interferons. These results suggest that these interferons not only suppress cellular proliferation but also selectively inhibit the action of the HBV gene which is persistently present in these cells.
Study on rRNA Genes in Mycobacterium smegmatis
Authors: Suzuki Yasuhiko , Yamada Takeshi
Published: November 2013

The number of rRNA genes in Mycobacterium smegmatis was examined by hybridization of BamHI and SalI digests of chromosomal DNA with 3′‐end‐labeled 5S, 16S, 23S rRNA and tRNA. Each RNA probe gave two hybridization bands. The PstI fragments of 6.6 kilobases were cloned to pBR322. The cloned DNA was characterized by restriction endonuclease mapping, DNA‐RNA hybridization, and the R‐loop technique.
Reversion of Neurovirulence and in vitro Phenotype of Neural Cell‐Adapted Canine Distemper Virus after Passage in Non‐Neural Cells
Authors: Morikawa Yuko , Yoshikawa Yasuhiro , Yamanouchi Kazuya
Published: November 2013

The stability of neurovirulence and in vitro phenotypes of canine distemper viruses adapted to neural cells was examined. Neurovirulence was estimated by the morbidity, mortality, and histopathological changes in the central nervous system of mice. After a single passage of the adapted viruses in Vero cells in which the unadapted virus had been passed, the neurovirulence of glioblastoma‐adapted and oligodendroglioma‐adapted viruses reverted completely to that of the unadapted virus. However, the neurovirulence of a neuroblastoma‐adapted virus reverted partially. In vitro phenotypes such as the two‐dimensional electrophoretic patterns of viral proteins and the cross‐neutralization patterns also reverted to those of the unadapted virus. However, plaque sizes remained similar to those of the viruses adapted to neural cells.
A Possible Correlation between Histological Changes in Regional Subcutaneous Tissue Induced by Bacterial Lipopolysaccharides and Their Adjuvant Activities
Authors: Yokochi Takashi , Inoue Yoshiko , Miyadai Toshiaki , Kimura Yoshinobu , Ito Hidenobu , Kato Nobuo
Published: November 2013

Previously it was demonstrated that Klebsiella pneumoniae O3 lipopolysaccharide (KO3 LPS) exhibited much stronger adjuvant action on antibody response to subcutaneously (s.c.) injected sheep red blood cells or deaggregated bovine serum albumin than did other kinds of LPS, the R‐form LPS lacking the O‐specific polysaccharide chain of KO3 LPS (R‐LPS), and the lipid A fractionated from KO3 LPS. We compared histological changes in the regional subcutaneous tissues of mice injected subcutaneously (s.c.) with KO3 LPS, the lipid A, and R‐LPS. At the early stage after injection, KO3 LPS induced the infiltration of a large number of inflammatory cells, mainly polymorphonuclear leukocytes (PMN), at the site of injection. Neither R‐LPS nor the lipid A induced the accumulation of PMN so much as KO3 LPS did. When injected s.c. with LPS from Escherichia coli O111 (EO111 LPS) and O55 (EO55 LPS), and Salmonella enteritidis (Sent LPS), the appearance of PMN at the regional site was much less than KO3 LPS. KO3 LPS could accumulate more 51Cr‐labeled leukocytes at the injection site than EO111 LPS and Sent LPS. Administration of acetylsalicylic acid, which can inhibit leukocyte migration in inflammatory lesions, suppressed its adjuvant action. It was therefore suggested that the strong adjuvant action of KO3 LPS in s.c. injection might be dependent on its potent capability of accumulating PMN at the regional subcutaneous tissue. Furthermore, at the late stage after injection, the formation of several lymphoid follicles at the regional site was seen only in mice injected with KO3 LPS. It might be also related to the strong adjuvant action of KO3 LPS.
Toxicity of Microcystis aeruginosa K‐139 Strain
Authors: Nakano Masayasu , Nakano Yasunobu , Saito‐Taki Tatsuo , Mori Naoyoshi , Kojima Mizu , Ohtake Akio , Shirai Makoto
Published: November 2013

Toxicity of the cells of a newly established axenic Microcystis aeruginosa K‐139 strain to mice was studied. LD50 of the cells harvested in the mid‐log phase was 7.3 mg/kg. The organs of acute dead mice were examined histopathologically. The blood congestion and necrosis of the parenchymal cells around the central veins in the liver were observed, but other organs seemed to be normal. The liver damage was confirmed by the tests of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) activities in the sera of the mice after the injection with the K‐139 cells. Furthermore, the K‐139 cells were capable of inducing interleukin 1 (IL‐1) production by peritoneal macrophages in vitro.
Identification of Species and Capsular Types of Klebsiella Clinical Isolates, with Special Reference to Klebsiella planticola
Authors: Mori Masashi , Ohta Michio , Agata Norio , Kido Nobuo , Arakawa Yoshichika , Ito Hideo , Komatsu Takayuki , Kato Nobuo
Published: November 2013

In the 77 reference strains for Klebsiella K types, there are 17 strains (22.1%) of Klebsiella planticola, 6 strains (7.8%) of Klebsiella oxytoca, 1 strain (1.3%) of Klebsiella terrigena, and 53 strains (68.8%) of Klebsiella pneumoniae. The species K. planticola, which was originally isolated from botanical and aquatic environments and hence thus named, was also identified at high incidence (81 strains, 18.5%) among the 439 recent clinical isolates of Klebsiella species. Among these K. planticola strains of hospital origin, 52 (64%) were isolated from sputum, 17 (21%) from urine, and the remaining 12 (15%) from other sources. The capsular types of these isolates were determined by the gel precipitation reaction. Seventy of 81 K. planticola isolates (86.4%) were typable by antisera to Klebsiella reference strains for K types and the K types of the clinical isolates distributed to 35 kinds of K types. The proportion of typable strains among clinical isolates of K. planticola was very similar to those in K. pneumoniae (87.5%) and K. oxytoca (86.0%).
In Situ Infiltration of Natural Killer‐Like Cells Induced by Intradermal Injection of the Nucleic Acid Fraction from BCG
Authors: Kuramoto Etsuro , Toizumi Shin‐Ichi , Shimada Shizuo , Tokunaga Tohru
Published: November 2013

Intradermal injection of MY‐1, a nucleic acid fraction extracted from Mycobacterium bovis strain BCG, induced in situ infiltration of mononuclear cells, most of which were asialo GM1 (GA1)‐positive as determined by immunofluorescence microscopy. The infiltration occurred with as little as 1 μg of MY‐1 and lasted for a week. Double immunofluorescence microscopy revealed that the infiltrating GA1‐positive cells were all positive for Ly‐5, and partially positive for Thy‐1.2, but negative for Mac‐1, Ia, μ‐chain, Lyt‐1, Lyt‐2, L3T4, and Fc receptor II. They contained neither peroxidase nor nonspecific esterase. The infiltrating cells thus markedly resembled natural killer (NK) cells in their cytochemical characteristics and surface markers. DNase and RNase destroyed the GA1‐positive cell‐inducing activity of MY‐1. These results indicate that the nucleic acid components of MY‐1 are responsible for this effect.
Isolation of a Coxiella burnetii Strain That Has Low Virulence for Mice from a Patient with Acute Q Fever
Authors: Oda Hiroshi , Yoshiie Kiyotaka
Published: November 2013

Coxiella burnetii was isolated from a patient with Q fever. It could not be determined whether this was an imported case or an indigenous one. Identification of the isolate was made by electron microscopic morphology and the indirect fluorescent antibody test with convalescent‐phase serum from a Q fever patient having a known titer of antibody to C. burnetii. The isolated strain, named TK‐1, caused no symptoms in ddY and BALB/c mice except when the mice were treated with cyclophosphamide.
Pili of Vibrio cholerae O1 Biotype El Tor: A Comparative Study on Adhesive and Non‐Adhesive Strains
Authors: Iwanaga Masaaki , Nakasone Noboru , Ehara Masahiko
Published: November 2013

Pili were found on the cell surface of non‐adhesive Vibrio cholerae O1 Biotype El Tor as well as the adhesive strain. Purified pili of the adhesive and non‐adhesive strains were morphologically, electrophoretically, and immunologically, indistinguishable from each other. The molecular weights of both pilin (subunit protein of the pilus) were about 16,000 daltons as determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. These 16 kDa pili are different from the pilus colonization factor, which is a 20.5 kDa protein, reported by Taylor et al. The 16 kDa pili of Vibrio cholerae O1 Biotype El Tor have hemagglutinating activity, but may have no role in colonization, because non‐adhesive strains also have such pili.
Penicillin Amidohydrolases in Fungal Autolysis
Authors: Alfonso Carlos , Cribeiro Lourdes , Reyes Fuensanta
Published: November 2013

The production of penicillin G and penicillin V amidohydrolases or acylases (E.C.3.5.1.11) was studied during the autolysis of filamentous fungi in a mineral medium, and in the same medium with phenoxyacetic acid as inducer. In all the studied fungi, enzymes showing penicillin G and penicillin V amidohydrolase activities were found. Generally, an increase of these activities during fungal autolysis was observed. The presence of phenoxyacetic acid in the medium did not increase these activities. The activities found in the culture fluids were generally higher than that found in the mycelial extracts. Under these conditions, β‐lactamases (penicillinases) were not found. The fungi Alternaria alternata, Fusarium culmorum, Penicillium oxalicum, and the species Penicillium 222 were chosen to study penicillin G and penicillin V acylases. The enzymes were precipitated with tannic acid from the culture fluid of their autolyzed cultures. Some kinetic constants of these activities were determined.
A Simple Method for Observation of Phagocytosis on Bacterial Thin‐Layer
Authors: Seki Keiko , Murai Miyo , Sakurada Junji , Shirahige Akira , Kobayashi Noriyuki , Hwang Soo‐myung , Masuda Shogo
Published: November 2013

A simple method was developed to visually present the phagocytic activity of leukocytes by using adherent Staphylococcus aureus cells and blood applied on a plastic dish. When heparinized blood was applied on thin‐layer of heat‐killed S. aureus cells on the plastic dish, plaques due to the phagocytic activity of leukocytes were observed with a microscope under a low magnification. Fewer and smaller plaques were observed when plasma‐deprived rather than whole blood was used. Some analyses were made in respect to the fundamental conditions required for optimal results. This method was considered to be useful for conveniently evaluating the serum opsonin activities and phagocytic function of leukocytes in various kinds of diseases.
Antigenic Variation among Sendai Virus Strains Detected by Monoclonal Antibodies
Authors: Yamaguchi Ryoji , Iwai Hiroshi , Ueda Katsumoto
Published: November 2013

Thirteen strains of Sendai virus isolated from various sources in the 1950′s and after 1976 were compared for their reactivities with monoclonal antibodies prepared against the prototype strain MN of Sendai virus. Results revealed that while the 5 strains isolated in the 1950′s reacted with all the monoclonal antibodies as the prototype strain did, the 2 strains isolated in 1976 and 1978 did not react with an F‐specific monoclonal antibody, and the other 6 strains isolated after 1978 lacked reactivity with an HN‐specific monoclonal antibody.
Single‐Serum Diagnosis of Recent Rubella Infection with the Use of Hemagglutination Inhibition Test and Enzyme‐Linked Immunosorbent Assays
Authors: Katow Shigetaka , Sugiura Akira , Janejai Noppavan
Published: November 2013

Single‐serum diagnosis of recent rubella infection was attempted with the use of hemagglutination inhibition (HI) test and two commercially available enzyme‐linked immunosorbent assays (ELISAs). The period during which IgM antibody was detectable by IgM capture ELISA was 4 to 30 days after the onset of rash. Rubella IgG ELISA values relative to HI titer were lower in the sera from the patients with recent infection than in the sera from the subjects with remote infection. IgM ELISA and the combined use of IgG ELISA and HI test are two useful methods of single‐serum diagnosis of recent rubella infection.
Simple Method for Quantitation of Cell‐Bound Protein A on Staphylococcus aureus Cells by Means of Hemagglutination with Sheep Erythrocytes Differentially Sensitized with Rabbit Antibody and Its Clinical Application
Authors: Hwang Soo Myung , Seki Keiko , Murai Miyo , Sakurada Junji , Nishihara Shoko , Maeda Toshiro , Masuda Shogo
Published: November 2013

A simple method for quantitation of cell‐bound protein A (SpA) on organisms of Staphylococcus aureus was successfully devised by using hemagglutination between staphylococcal organisms and a series of sheep erythrocyte suspensions sensitized with different amounts of anti sheep erythrocyte rabbit antiserum. The validity of the principle and the reproducibility of the method presented here were precisely analyzed and the details of the method are presented. The hemagglutination was quantitatively inhibited both by normal rabbit serum and by soluble SpA. Using the method presented here, 376 strains of S. aureus freshly isolated from clinical materials were subjected to SpA quantitation in cell‐bound form. According to the results, there was an unexpected distribution profile in the amounts of cell‐bound SpA among the clinical isolates, which showed a two‐peak pattern. A possible usefulness of the method presented here in clinical investigations is briefly discussed also.
Annual Change in Immunoglobulin G and M Antibody Levels to Toxoplasma gondii in Human Sera
Authors: Konishi Eiji
Published: November 2013

Sera collected from a Japanese population over 4 years at yearly intervals were examined for antibodies to Toxoplasma gondii by enzyme‐linked immunosorbent assay (ELISA). Distributions of immunoglobulin (Ig) G antibody levels for 6 age groups consistently showed two antibody groups with constant mean ELISA values. Quantitative analysis of annual changes in IgG antibody levels for 1,366 pairs indicated increase of ≥0.1 in 28 samples and decrease of ≥0.1 in 58 samples, with the remaining 1,280 samples showing changes of <0.1. Mean ELISA values of samples with increase and decrease were 0.479 and 0.833, respectively, and the difference of 0.354 was similar to 0.412, that was the sum total of mean annual changes in samples with increase (0.228) and decrease (0.184). Increase of ≥0.1 from the initial IgG antibody level of <0.1, which was considered as a serological evidence for newly acquired infection, was observed in 3 samples, but no increase was demonstrated in IgM antibody level. The changing pattern of IgG and IgM antibody levels in people during the initial and chronic phases of asymptomatic infections is discussed.
Prevalence of Antibody to Human Herpesvirus 6 in Women and Children
Authors: Balachandra Kruavon , Ayuthaya Panasda Isarangkul Na , Auwanit Wattana , Jayavasu Chruindee , Okuno Toshiomi , Yamanishi Koichi , Takahashi Michiaki
Published: November 2013

The antibody prevalence to human herpesvirus 6 (HHV‐6) was compared between pregnant women and control women of similar ages in Thailand. No significant difference was detected in the antibody positive rate and antibody titers between both groups. The antibody titers in sera collected from pregnant women at 1st and 3rd trimester remained unchanged. Next, the antibody prevalence in infants were examined and the positive rate decreased until 3 months and started to increase from 6 months after birth. The present results suggest that the reactivation of HHV‐6 might not occur during pregnancy and this virus infects infants postnatally.
Different Correlations of Drug Susceptibilities to Colonial Morphology in Mycobacterium avium Complex Strains
Authors: Tsukamura Michio , Mizuno Shoji , Miyama Akio
Published: November 2013

In Mycobacterium avium and Mycobacterium intracellulare complex strains isolated from patients who were not treated previously by any antituberculosis drugs or from fowls, the colonial morphology, smooth, domed, opaque (SmD) or smooth, flat, transparent (SmT) colonial forms, significantly correlated with susceptibilities to rifampicin, minocycline, streptomycin, kanamycin, enviomycin, ethambutol, and sulfadimethoxine, whereas it did not correlate with susceptibilities to isoniazid, cycloserine, and ethionamide. Strains with the SmT colonial morphology were more resistant to the former seven drugs than strains with the SmD colonial morphology. Since the susceptibilities to antituberculosis drugs with large molecules correlated with the colonial morphology, it has been suggested that a permeability barrier that allows passage of small molecules but prevents passage of large molecules exists in the strains with the SmT colonial morphology.
Altered Immune Responsiveness in Patients with Chronic Glomerulonephritis
Authors: Nakauchi Hiromitsu , Tango Toshiro , Umezawa Yoshimi , Ohno Iwao , Okumura Ko
Published: November 2013

In order to detect abnormalities in humoral immunity and to determine immunogenetic traits underlying chronic glomerulonephritis, sera from 260 patients who had chronic glomerulonephritis and who were undergoing hemodialysis were tested for naturally occurring antibodies against mycoplasma and 22 different viruses. Among the 23 microorganisms tested, antibody titers were significantly lower against 12, higher against 3, and no different against 8 when compared with titers of 43 normal subjects. The data were analyzed further by plotting each in a 23‐dimensional space according to their standardized antibody titers. Multivariate cluster analysis by the Ward's method revealed 3 large clusters differing from each other in natural antibody titers, and one of the clusters included 74% of the normal controls, while two other distinct clusters comprised the majority of the patients. The level of BUN, creatinine, and duration of hemodialysis treatment did not differ significantly among patients in these three different clusters. Our study suggests that patients with chronic glomerulonephritis being treated by hemodialysis have altered levels of naturally occurring antibodies to microorganisms. This alteration is not caused by just the uremic state or hemodialysis but immunogenetic regulation may also play a part.
Isolation of the Saprophytic Strain of MC‐3 and Participation of the Cell Surface Structure in Predation
Authors: Takubo Yoshihiro , Nishimura Yoshie , Zytoon Eiman M. , Nishihara Tsutomu , Kondo Masaomi
Published: November 2013

From a predatory bacterium, MC‐3, a mutant strain which lost predation ability was isolated by chance selection. Biological properties of the mutant were the same as the parent except only saprophytic property. Properties of the parent and the mutant strains of MC‐3, such as bacteriolytic activity of the culture supernatant, digestion of peptidoglycan of the host bacteria, and growth by utilizing the host cells or their cytoplasmic substances, suggested that cell surface structure of the host cell plays an important role in predation and host specificity.
Specificity of Salmonella Porin as an Eliciting Antigen for Cell‐Mediated Immunity (CMI) Reaction in Murine Salmonellosis
Authors: Matsui Katsuhiko , Arai Toshihiko
Published: November 2013

The specificities of Salmonella porin on elicitations of delayed‐type hypersensitivity (DTH) reaction and interleukin‐2 (IL‐2) production in BALB/c mice immunized with Salmonella typhimurium were examined. Only porin from S. typhimurium was capable of eliciting significant levels of DTH and IL‐2 production in S. typhimurium‐immunized mice, whereas no significant DTH and IL‐2 production were induced by porin from Salmonella enteritidis or Escherichia coli. Our observations suggested that Salmonella porin was a serovar‐specific antigen for the elicitation of cell‐mediated immunity (CMI) in salmonellosis.
Granuloma‐Forming Activity and Antitumor Activity of Newly Isolated Mycoloyl Glycolipid from Rhodococcus terrae 70012 (Rt. GM‐2)
Authors: Natsuhara Yayoi , Yoshinaga Junji , Shogaki Takeshi , Sumi‐Nishikawa Yukie , Kurano Satoko , Kato Yoshiko , Kaneda Kenji , Oka Shiro , Yano Ikuya
Published: November 2013

A newly isolated mycoloyl glycolipid (Rt. GM‐2) from Rhodococcus terrae 70012 was identified and the granulomagenic and antitumor activities were studied as compared with trehalose‐6,6′‐dimycolate (cord factor) also from R. terrae (Rt. TDM). The alkaline hydrolysis products of Rt. GM‐2 contained trehalose, methyl‐α‐mycolate and a less‐polar ester than the usual methyl‐α‐mycolate, possibly β‐keto mycolate (1: 1: 1, by mol. ratios). On the other hand, analysis of alditol acetate obtained after the mild permethylation, NaBH4 reduction, and acetylation showed the occurrence of 2,3,4‐tri‐O‐methyl‐6‐O‐acetylglucitol. Therefore, the original glycolipid (Rt. GM‐2) was identified tentatively as 6‐O‐α‐mycoloyl 6′‐O‐β‐ketomy‐coloyl trehalose. Intravenous injection of Rt. GM‐2 in the form of water‐in‐oil‐in‐water emulsion caused prominent granulomas in lungs and spleen of ICR and BALB/c mice. The granulomagenic effects were as strong as those caused by Rt. TDM. The lung and spleen weights reached peaks one week after an injection of Rt. GM‐2 in mice and then gradually decreased. Multiple intravenous injections of Rt. GM‐2 and Rt. TDM showed antitumor activity against subcutaneously implanted Sarcoma‐180, and caused prominent granulomatous changes and growth suppression of mice.
Pili of Aeromonas hydrophila: Purification, Characterization, and Biological Role
Authors: Honma Yasuko , Nakasone Noboru
Published: November 2013

Aeromonas hydrophila (Ae6) has 2 morphologically distinctive kinds of pili. One appeared rigid, channeled, and straight with a diameter of 9 nm (Ae6‐R pili). The other looked flexible, wavy, and having helical structure with a diameter of 7 nm (Ae6‐W pili). Ae6‐R pili were purified and characterized. The pili consisted of a subunit protein with a molecular weight of 18 kDa as estimated by SDS‐PAGE, and contained 42.3% hydrophobic amino acids and one cysteine residue. The pilus was solubilized to 18 kDa subunit protein by 2‐mercaptoethanol, dithiothreitol, hydrochloric acid, or heating at 120 C for 5 min. The organism Ae6 was strongly adhesive to rabbit intestines as well as human intestines, and agglutinated erythrocytes. Anti‐pili antibody (Fab fraction) did not block the adhesion. Purified Ae6‐R pili did not adhere to the intestine or to the erythrocytes. However, the anti‐pili Fab inhibited pellicle formation of the organisms cultured in broth, and also inhibited salt agglutination with ammonium sulfate. From these results, Ae6‐R pili are not likely a colonization factor but probably play a role in the autoaggregation of the organisms.
Comparative Evaluation on Mouse Nasal Immunogenicity of Arylmethane‐, Xanthene‐, Quinone‐Imine‐, and Acridine‐Dye‐Inactivated Sendai Virus Vaccines
Authors: Miyamae Takeo
Published: November 2013

Twenty‐seven kinds of organic dye‐inactivated Sendai virus vaccines were prepared by treatment in dark at 23 C for 2 months or more, and selected with the high HA titers as a guide. Their nasal immunogenicities were examined in mice by contact infection and immunofluorescent method, and the relative merits of the dye‐inactivants were determined. The strongest protection was elicited with acriflavine‐. auramine O‐, eosin Y‐, neutral red‐, night blue‐, patent blue V‐, thymol blue‐. uranin‐, and xylene cyanol FF‐treated vaccines. Middling protective efficacy was induced by use of erio green B‐, malachite green‐, methyl green‐, proflavine‐, pyronin B‐, and thionin‐inactivated vaccines. Dye‐inactivated vaccines that resulted in the weakest protection were Bindschedler's green‐, bromothymol blue‐, erythrosin B‐, ethyl violet‐, gallein‐, light green SF yellowish‐, methyl violet‐, new methylene blue N‐, phenol red‐, rhodamine 6G‐, spirit blue‐ and victoria blue B‐treated ones. Serum HI titers developed by nasal vaccination were variable, and rose still more in most vaccinated groups postexposure. Elicitation of the most effective nasal immunogenicity in dye‐inactivated vaccines appeared to depend on selective modification of capsid protein or ribose in viral core with dyes possessing definite functions, despite the different molecular structures.
Drug Susceptibilities of Isolates of Varicella‐Zoster Virus in a Clinical Study of Oral Brovavir
Authors: Machida Haruhiko , Nishitani Makiko
Published: November 2013

Susceptibilities to brovavir [l‐β‐D‐arabinofuranosyl‐E‐5‐(2‐bromovinyl)‐uracil] and acyclovir of clinical isolates of varicella‐zoster virus obtained from 58 patients with herpes zoster, included in a clinical trial of oral brovavir, were tested by a plaque reduction method. All 101 isolates were significantly susceptible to brovavir; 50% effective dose of brovavir for these isolates ranged between 0.6‐4.0 ng/ ml (average: 1.29 ng/ml). Brovavir was about 3,000 times more potent than acyclovir against these isolates. No marked change in the susceptibility of isolates from these patients during treatment with brovavir was observed.
Rapid Small‐Scale Preparation Method of Cell Surface Polysaccharides
Authors: Sugiyama Tsuyoshi , Kido Nobuo , Arakawa Yoshichika , Mori Masashi , Naito Setsuko , Ohta Michio , Kato Nobuo
Published: November 2013

A rapid small‐scale method of extraction of lipopolysaccharide (LPS) and capsular polysaccharides was developed for the purpose of identification of chemotypes of LPS and serotypes of capsular antigens. Cell surface polysaccharides were prepared within less than 2 hr from 1.5 ml of broth or suspension of colonies cultured overnight. The preparations were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis for LPS, and by double diffusiori gel precipitation (Ouchterlony) test and blotting to nitrocellulose membrane for capsular polysaccharide. The analyses with the preparations obtained by the method could provide adequate results capable of identifying chemotypes of LPS and serotypes of capsular antigens.
Effect of Tween 80 on the Growth of Mycobacterium avium Complex
Authors: Masaki Shunichiro , Sugimori Giichi , Okamoto Atuko , Imose Jun , Hayashi Yoshiyuki
Published: November 2013

The effect of Tween 80 on the growth of Mycobacterium avium complex (MAC) in liquid culture condition was investigated. Observation of the colony‐forming units (CFU) and the morphology of MAC with transmission and scanning electron microscopy showed that Tween 80 at 0.05% in the medium (ca. 0.5 mg/ml) had bacteriostatic action and caused cell elongation. Tween 80 at 0.5% or more in the medium (ca. 5 mg/ml) reduced the quantity of MAC glycolipids and also led to false positive or positive results in biochemical tests for mycobacterial identification using nitrate reductase, urease, or arylsulfatase. To determine whether or not surfactants could reduce the MAC permeability barrier, the minimal inhibitory concentration (MIC) of antituberculosis drugs on MAC was determined in liquid medium with or without several kinds of surfactants including Tween 80. Five surfactants including Tween 80 increased the activity of antituberculosis drugs to MAC. These findings suggest that Tween 80 acts directly on the cell wall of MAC.