bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of Mucosal and Serum Antibodies Specific for the Capsular Polysaccharide of Haemophilus influenzae Type b by Enzyme‐Linked Immunosorbent Assay
Authors: Harada Teruhiko , Sakakura Yasuo , Shimura Keishiro
Published: November 2013

A sensitive enzyme‐linked immunosorbent assay (ELISA) with rough‐surfaced glass beads used as the solid phase was developed for detection of IgG, IgM, and IgA antibodies to Haemophilus influenzae type b capsular polysaccharide (HITB‐CP). A successful method for indirect coating of glass bead surfaces with HITB‐CP, and parameters affecting the specificity and sensitivity of the assay are described. This ELISA system proved to be 100 times as sensitive as the standard indirect fluorescent‐antibody assay. The assay was applied to the measurement of antibodies to HITB‐CP in serum and nasal secretions and proved to be a useful tool in the evaluation of immunological response to HITB infection.
Formation of Varicella‐Zoster Virus Antigens in Infected Vero Cells
Authors: Takayama Michiko , Oya Akira
Published: November 2013

The formation of varicella‐zoster (V‐Z) virus‐associated antigens was studied in V‐Z virus‐infected Vero cells by means of indirect immunofluorescence. Early antigen (EA) was first detected inside V‐Z virus‐infected Vero cells 4 to 6 hr after infection, whereas surface membrane antigen (SMA) was expressed on the outer surface of infected cells 2 to 3 hr later than EA, and intranuclear late antigen (LA) was detected several hours later than SMA antigen. EA expression was not inhibited by cytosine arabinoside (Ara‐C) treatment, whereas LA formation was completely blocked by Ara‐C. The presence of two components of SMA early SMA (ESMA) and late SMA (LSMA), was suggested by this difference in susceptibility to Ara‐C. The formation of all viral antigens, EA, SMA, and LA, was blocked by inhibitors of RNA and protein synthesis.
Mycoplasma pulmonis Arthritis in Mice: Microbiological and Immunological Features
Authors: Kono Masako , Yoshioka Morimasa , Imai Miki , Hirose Shunichi
Published: November 2013

Mycoplasma pulmonis m53 was inoculated intraarticularly in the bilateral hind footpads and bilateral knee joints of BALB/c mice. Mycoplasmas were recovered from the affected joints over 20 weeks accompanying acute or subacute inflammation. Intensive deposition of immunoglobulins, a complement (C3) and mycoplasma cell antigens occurred in synovial and adjacent connective tissues. The histopathologically intact kidneys, brain, and lungs showed deposition of IgG and the complement on the endothelial cells of blood vessels. An IgG‐rheumatoid factor like substance (RFLS) was detected in the serum of the mice by an enzyme‐linked immunoabsorbent assay. Persistence of mycoplasma cells and immune complexes in the articular tissues might cause the prolongation of inflammatory responses in murine mycoplasmal arthritis.
Collapse of Cortex Expansion during Germination of Bacillus megaterium Spores
Authors: Nakatani Yoshihiro , Tanida Ikuko , Koshikawa Tomihiko , Imagawa Masayoshi , Nishihara Tsutomu , Kondo Masaomi
Published: November 2013

When spores of Bacillus megaterium ATCC 12872 were incubated with CdCl2, they germinated without decomposition of the cortex. Moreover, the volume ratio of cortex to protoplast‐plus‐cortex, C/(P + C), of the CdCl2‐germinated spores was reduced. Incubation of isolated cortex with the divalent compounds Cd2+, Ca2+, and Mg2+ reduced the gel volume to about 1/5 but incubation with a nonionic compounds, glucose, did not. The spores with reduced C/(P+C) were observed in the early period of glucose‐induced germination. The time required for a 50% change in cortex morphology to occur was 2.5 min, which corresponds well with the time for 50% loss of heat resistance. This time was shorter than that necessary for release of peptidoglycan fragments and hydrolysis of cortex glycan chains. These data indicate that cortex hydrolysis is not related to the initiation of germination. 50% of the dipicolinic acid, calcium and magnesium were released at 3.4, 4.0, and 2.4 min, respectively. These results suggest that collapse of cortex expansion by the interaction of cortex with dipicolinic acid and cations released from the core, or exogenous ionic germinants is an important step in the initiation of germination.
Formation of a Hexagonal Lattice Structure by an R‐Form Lipopolysaccharide of Klebsiella: Relationship between Lattice Formation and Uniform Salt Forms
Authors: Kato Nobuo , Ohta Michio , Kido Nobuo , Ito Hideo , Naito Setsuko , Kuno Tsuneharu
Published: November 2013

Various uniform salt forms of an R‐form lipopolysaccharide (LPS) extracted from Klebsiella strain LEN‐111 (O3‐:K1‐) were prepared and their ultra‐structure was examined. The LPS, which was extracted by the phenol‐water method, freed from contamination with RNA by treatment with RNase, and precipitated by addition of two volumes of 10 mM MgCl2‐ethanol, was used as the original preparation for uniform salt forms. The original LPS preparation formed a hexagonal lattice structure with a lattice constant of 14.9±0.2 nm. The LPS after electrodialysis retained the ability to form a hexagonal lattice structure, although its lattice constant was large (18.7±0.5 nm) and the lattice structure of the electro‐dialyzed LPS was labile at pH 8.0 in contrast to that of the original LPS preparation. The magnesium salt form of the LPS formed essentially the same ordered hexagonal lattice structure (lattice constant of 15.0±0.2 nm) as that of the original LPS preparation. The calcium and ammonium salt forms formed a hexagonal lattice structure, but the lattice constants of the calcium and ammonium salt forms were larger (18.6±0.6nm and 19.3±0.4nm, respectively) than that of the magnesium salt form. The sodium and potassium salt forms consisted of freely branching ribbon‐like structures with an average width of 13 nm and an average thickness of 9 nm. The triethylamine salt form consisted principally of short rods (10 nm × 9–13 nm).
Comparison of Murine B Cell Clonal Expansions by Synthetic Lipid A and Muramyl Dipeptide Analogs
Authors: Saito‐Taki Tatsuo , Nakano Masayasu , Kiso Makoto , Hasegawa Akira
Published: November 2013

Direct stimulations of murine B lymphocytes with synthetic lipid A analogs and synthetic muramyl dipeptide (MDP) derivatives were studied using a limiting dilution assay system. Synthetic lipid A analogs, GLA‐27 and GLA‐40, when conjugated with bovine serum albumin (BSA) had the ability to induce B cell clonal expansion of a single B cell from the spleen or bone‐marrow. Their activities were almost the same as those of naturally obtained lipid A, but were lower than that of bacterial lipopolysaccharide (LPS). Addition of dextran sulfate (DXS) enhanced the effect of lipid A analogs. In contrast, synthetic MDP and its derivatives, although they had many biological and immunological activities in experimental animals, could not stimulate a single B cell to induce clonal expansion regardless of the presence or absence of DXS. These results suggested that lipid A analogs can directly cause the proliferation of B cells, but MDPs can not.
The Development of Complement Activating Ability as an Age Related Factor in Murine Brains
Authors: Tanaka Morihisa , Cyong Jong‐Choi
Published: November 2013

We recently reported on the ability of the myelin fraction of the murine brain to activate the complement system through the classical pathway, which might be important in the induction of secondary inflammation in various pathological conditions where brain tissue has been exposed to the complement. The present study was undertaken to investigate the relationship between the appearance of complement activity in the mouse brain and the synthesis of myelin in ICR mice up to ninety days of age.

Here, we show that anti‐complementary activity in the murine brain is closely related to murine brain weight and that its activity seems to be dependent on the amount of myelin in the murine brain. Myelin was isolated from brains of equal weight taken from both two‐day‐old and ninety‐day‐old mice, and we found that ninety‐day‐old myelin consumed a much greater amount of complement (C) than two‐day‐old myelin. However, for equal concentrations of myelin, almost an equal amount of C was consumed by the myelin of the two‐day‐old mice and by that of the ninety‐day‐old mice. It was suggested that the difference of anti‐complementary activity was caused by the myelin contents of the murine brains, but the possibility of maturation of myelin was not excluded. The mechanism involved in the anti‐complementary activity of the myelin was found to be related to the consumption of complement, mainly via the classical pathway but also less activity via the alternative pathway.
Serological Analysis of Serogroup Icterohaemorrhagiae Using Monoclonal Antibodies
Authors: Kobayashi Yuzuru , Tamai Tomonori , Sada Eiji
Published: November 2013

Eighteen serovars (19 strains) of serogroup Icterohaemorrhagiae were serologically analyzed using 18 monoclonal antibodies against serovar copenhageni Shiromizu, M20 and serovar icterohaemorrhagiae RGA strains. The reaction patterns of the serovars against these monoclonal antibodies were different. According to these results, we divided the serovars, except for serovar tonkini, into the following three subgroups:

Subgroup 1 reacted to many monoclonal antibodies including serovars icterohaemorrhagiae, copenhageni, hualien, monymusk, mankarso, and budapest.

Subgroup 2 fell between subgroups 1 and 3 including serovars dakota, naam, bogvere, birkini, smithi, ndambari, gem, ndahambukuje and mwogolo.

Subgroup 3 reacted to only a few monoclonal antibodies: serovars weaveri and sarmin.

Serovar tonkini did not react to any of the monoclonal antibodies used. There is a possibility that serovar tonkini does not belong to serogroup Icterohaemorrhagiae. Further studies on the serological reactions of each strain revealed that it was impossible to distinguish the RGA strain from the serovar hualien LT11–31 strain, indicating that they may be identical. It was also observed that serovar copenhageni and monymusk seemed to be closely related. Serovars birkini and smithi, and serovars ndambari and gem were alike in their serological reactivities.

Among the 18 monoclonal antibodies, RGAMA‐1 was a unique antibody which reacted only to serovar icterohaemorrhagiae and serovar hualien, indicating that it must be the serovar icterohaemorrhagiae specific antibody. On the other hand, SHIRMA‐2, 5, 6 reacted to all the serovars except for serovars weaveri, sarmin, and tonkini. These antibodies exhibited a broad reaction spectrum.
Reversion to Bacillary Forms of Serratia marcescens Spheroplasts Induced by Carbenicillin
Authors: Furutani Akio , Tada Yoshifumi , Yamaguchi Junji
Published: November 2013

Bacterial cells of Serratia marcescens were easily induced to form spheroplasts in liquid medium by the addition of carbenicillin. The spheroplasts were unable to divide, but they were able to revert to the bacillary forms in liquid medium not containing carbenicillin. Four phases of the reversion sequence could be differentiated by scanning electron microscopy. (1) After 3 hr of incubation in carbenicillin‐free medium, some projections arose out of the spheroplasts, and grew and elongated. (2) Their elongation resulted in a morphological change in the spheroplasts from spherical bodies to long irregular bacillary forms. (3) Further incubation caused several constricted areas in the bacillary form. (4) The long bacillary forms split along the constricted areas to become the parent bacillary forms of S. marcescens. When the long bacillary form that developed during the reversion was retreated with carbenicillin, it was immediately induced to become a spheroplast again.
Plasmid Profiles of Neisseria gonorrhoeae in Peninsular Malaysia
Authors: Kalaimathee K.K. , Koh Chong‐Lek , Ngeow Yun‐Fong
Published: November 2013

The plasmid profiles of 160 strains of Neisseria gonorrhoeae isolated in Peninsular Malaysia, comprising 80 penicillinase‐producing (PPNG) and 80 non‐penicillinase‐producing (non‐PPNG) isolates, were determined. The 80 PPNG isolates were divided into two plasmid groups. All of them harbored two common plasmid species, a 4.4 megadalton (Md) R plasmid previously associated with β‐lactamase production in PPNG strains from the Far East and a 2.6 Md multicopy plasmid of unknown function. In addition to these two plasmids, 60 (75%) PPNG isolates also carried a large 24.5 Md conjugative plasmid.

In contrast, the 80 non‐PPNG strains were divided into three plasmid groups. All of them possessed the 2.6 Md cryptic plasmid, and 35 (44%) isolates also harbored the 24.5 Md transfer plasmid. Besides these two plasmids, one non‐PPNG isolate carried an additional 7.8 Md cryptic plasmid.
Chemical Characterization of Capsular Polysaccharide from Cryptococcus neoformans Serotype A‐D
Authors: Ikeda Reiko , Nishikawa Akemi , Shinoda Takako , Fukazawa Yoshimura
Published: November 2013

During a study of serotyping of Cryptococcus neoformans, we found that the type strain of C. neoformans (CBS 132) was serotype A‐D. This strain agglutinated with both factor 7 serum (specific for serotype A) and factor 8 serum (specific for serotype D) in our serotyping system. Therefore, we investigated the chemical structure of the antigenic capsular polysaccharide of this strain. The soluble capsular polysaccharide was obtained from the culture supernatant fluid by precipitation with ethanol. Column chromatography of the polysaccharide on DEAE‐cellulose yielded three fractions (F‐1 to F‐3). The major antigenic activity was found in the F‐3 fraction. The results obtained by methylation analysis, controlled Smith degradation‐methylation analysis, partial acid hydrolysis, and other structural studies of F‐3 polysaccharide indicated that the polysaccharide contains mannose, xylose, and glucuronic acid at a ratio of 7:2:2, and has a backbone of α(1–3)‐linked D‐mannopyranoside residues with a single branch of β(1–2)‐xylose and glucuronic acid. The ratio of mannose residues with or without a branch in the F‐3 polysaccharide was 4:3 and its molecular weight calculated from the average of the degree of polymerization was 46,500 daltons. These results indicate that the chemical structure of the capsular polysaccharide of serotype A‐D is very similar to those from serotypes A and D, suggesting that small differences in the molar ratio and pattern of linkage of monosaccharides in the branch of the polysaccharides of the three serotypes may be responsible for their different specificities.
IgM Induction in Murine B Hybridomas with la‐Restricted T Cell Clones: A Monoclonal Model for T and B Cell Interactions in Antibody Response
Authors: Yoshikubo Takashi , Kimoto Masao , Kishimoto Tadamitsu
Published: November 2013

The mechanism of MHC‐restricted T and B cell interactions in antibody response was studied with IgM‐inducible B hybridomas and antigen‐specific helper T cell clones. B hybridomas were prepared by fusion between splenic B cells from (CBA/N (H‐2k) × BALB/c (H‐2d)) F1 (NBF1) male mice and a B lymphoma cell line, M12.4.5. A B hybridoma clone, 1M70, which expressed I‐Ad but not I‐Ak determinants was chosen in the present study. IgM secretion was induced in 1M70 when it was cocultured with a “resting” KLH‐specific and H‐2d restricted helper T cell clone in the presence of KLH. A “resting” KLH‐specific and H‐2k restricted T cell clone did not induce IgM secretion in 1M70 even in the presence of KLH. However, when these KLH‐specific T cell clones were activated by KLH and appropriate antigen presenting cells, both H‐2d and H‐2k restricted T cell clones induced IgM secretion in 1M70 even in the absence of KLH. A monoclonal anti‐I‐Ad antibody inhibited IgM secretion induced by a “resting” H‐2d restricted T cell clone, but not by an “activated” T cell clone. These results indicated that T cell clones recognized antigens in the context of la molecules on B hybridomas in a MHC‐restricted manner and were activated to produce B cell stimulatory factors which in turn acted on B hybridomas in a non‐MHC‐restricted manner and induced differentiation of B hybridomas into IgM secreting cells.
One I Region Restriction Determinant Can Associate with Multiple Antigenic Epitopes
Authors: Kimoto Masao , Fathman C. Garrison
Published: November 2013

Studies presented in this paper show that T cell clones recognizing different epitopes of multideterminant antigens can be restricted by the same I‐A molecule. These data further support the concept that a single I‐A restriction site can present more than one antigenic epitope. This concept was supported by data on the proliferation of T cell clones reactive with either poly(L‐Glu60, L‐Ala30, L‐Tyr10)n (GAT) or poly(Tyr, Glu)‐poly D,L‐Ala–poly Lys [(T.G)‐A–L] which recognized different epitopes on these multideterminant antigens. Two clones recognizing different epitopes on the same multideterminant antigen can be blocked by the same monoclonal anti‐I‐A antibody. Additionally, the mutation in the Abm12 chain utilized in [B6.C‐H‐2bm12(bm12) × B10.A(4R)]F1 mice can affect the restriction determinant of clones recognizing different antigenic epitopes. These results suggest that in the strictest sense, the determinant selection theory is not tenable and would support the concept that T cell specificity is controlled by the T cell repertoire.
Impairment of T Cell‐Mediated Immunity to Listeria monocytogenes in Pregnant Mice
Authors: Sano Masatoshi , Mitsuyama Masao , Watanabe Yoshitsugu , Nomoto Kikuo
Published: November 2013

In order to study pregnancy‐induced changes in cell‐mediated immunity to Listeria monocytogenes, acquired resistance and T cell functions in pregnant mice were compared with those in nonpregnant mice after immunization with viable listerial cells. Impaired generation of acquired resistance was evident in pregnant mice from the impaired elimination of bacteria and poor survival after secondary challenge. Delayed footpad reactivity to listerial antigen was also lower in the pregnant mice. When immune spleen cells were examined for their ability to produce macrophage activating factor in vitro, culture supernatants from pregnant‐mouse spleen cells with listerial antigen showed far less ability to render macrophages cytostatic for P815 mastocytoma cells. To elucidate further the impairment of listeria‐immune T cell generation in pregnant mice, a local transfer experiment was carried out. When a given number of immune spleen cells was transferred locally into the footpads of naive mice, both delayed footpad reaction and local protection were much lower in the pregnant mice. This local transferability of the reactions was abrogated after treatment of cells with anti‐Thy 1 antibody plus complement. These findings indicate that pregnancy impairs the generation of specific T cells capable of contributing to acquired resistance to L. monocytogenes. Possible mechanisms for this impairment and the relationship to macrophage functions are discussed.
Composition of Major Outer Membrane Proteins of Vibrio vulnificus Isolates: Effect of Different Growth Media and Iron Deficiency
Authors: Koga Tetsuro , Kawata Tomio
Published: November 2013

The outer membrane proteins of Vibrio vulnificus including isolates from humans, seawater and an asari clam were examined by sodium dodecyl sulfate (SDS)‐Polyacrylamide gel electrophoresis. A major outer membrane protein with an apparent molecular weight of 48,000 (48K protein) was common to all the strains grown in 3% NaCl‐nutrient broth; however this 48K protein was not produced in any of the strains grown in chemically defined medium. Other major outer membrane proteins with molecular weights ranging from 33,000 to 40,000 varied in number, relative amount and molecular weight depending on the strain. One to three new outer membrane proteins with molecular weights ranging from 74,000 to 85,000 were produced in the cells grown in iron‐deficient medium. The 48K protein and one or two major proteins with molecular weights ranging from 35,000 to 37,000 in the cells grown in 3% NaCl‐nutrient broth were not solubilized by 2% SDS at 60 C for 30 min and were resistant to trypsin, indicating that they are porins. On the other hand, in cells grown in chemically defined medium, one or two major outer membrane proteins with molecular weights ranging from 33,000 to 40,000 might be porins.
Immunochemical Similarity of Synthetic α‐(1→3)‐Branched D‐Glucans to Dextran B1375
Authors: Torii Mitsuo , Yamazoe Mari , Ogawa Setsuko , Watabe Kazuhito , Koshikawa Tomihiko , Uryu Toshiyuki , Schuerch Conrad
Published: November 2013

Anti‐dextran B1375 antibodies were raised in rabbits by injecting formalin‐killed Leuconostoc mesenteroides strain NRRL B1375, and the anti‐dextran serum was used to examine native dextran B1375, and synthetic linear and four α‐(1→3)‐branched α‐(1→6)‐d‐glucopyranans for similarities. The antiserum reacted with the homologous dextran B1375 and also with all the synthetic linear and branched glucans. Precipitation and precipitation‐inhibition studies indicated that the antiserum contained at least three groups of antibodies with different specificities, the first specific for linear α‐(1→6)‐D‐glucopyranan structure, the second specific for α‐D‐glycopyranosyl‐(1→3)‐branching and the last specific for another, unknown structure present in the dextran B1375 molecule. Two samples of the synthetic branched glucans were shown to be immunochemically the most similar to natural dextran B1375 by inhibition experiments.
Laser Raman Spectroscopy of Lyophilized Bacterial Spores
Authors: Shibata Hirofumi , Yamashita Shinsuke , Ohe Mayumi , Tani Isamu
Published: November 2013

Laser‐excited Raman spectra were examined in lyophilized spores of Bacillus cereus. In a comparison of the spectrum of the dormant spore with that of the germinated spore, we found several Raman bands which occurred in the former but not in the latter. Among these Raman bands, the 1,573, 1,395, 1,017, 822, and 662 cm−1 bands were assigned to the vibrational frequencies of calcium dipicolinate (CaDPA). No Raman bands and peaks due to dipicolinic acid (H2DPA) were observed. This Raman evidence indicates that CaDPA is the predominant DPA species in this spore.

We also proposed a tentative assignment for other vibrational frequencies due to several components of the spore.
Induction of Antibodies against Newcastle Disease Virus with Syngeneic Anti‐Idiotype Antibodies in Mice
Authors: Tanaka Masayuki , Sasaki Norimasa , Seto Akira
Published: November 2013

Anti‐idiotype antibodies were induced by injecting BALB/c mice with syngeneic antibody against the hemagglutinin of Newcastle disease virus (NDV). These anti‐idiotype antibodies were purified and injected into syngeneic mice. Anti‐anti‐idiotype sera thus prepared contained antibodies against the hemagglutinin of NDV. This NDV‐mouse experimental system might provide a good experimental model for investigation of basic problems of idiotype vaccine.
Isolation, and Structural and Chemical Characterization of Outer Sheath Carrying a Polygonal Array from Treponema phagedenis Biotype Reiter
Authors: Masuda Kuniyoshi , Kawata Tomio
Published: November 2013

An outer sheath was isolated from Treponema phagedenis biotype Reiter by our previously developed method (Masuda, K., and Kawata, T. 1982. J. Bacteriol. 150: 1405–1413). The isolated outer sheath was observed as a triple‐layered, closed vesicle carrying a polygonal array by electron microscopy. The outer sheath was mainly composed of protein (41.0%), phospholipid (38.7%), and carbohydrate (11.0%). Sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of the isolated outer sheath in the presence of 2‐mercaptoethanol (EtSH) gave one major protein band with an apparent molecular weight of about 69,000 and several minor protein bands. On the other hand, in the absence of EtSH, the major protein band disappeared but two new protein bands at positions of molecular weights of about 65,000 and 72,000 appeared. The SDS‐PAGE profiles of the minor protein bands did not change with or without EtSH. Sodium deoxycholate (DOC)‐solubilized materials from the isolated outer sheath were reassembled into thin membranous sheets carrying a roughly polygonal array upon removal of DOC by dialysis against Tris‐HCl buffer in the absence of Mg2+.
Increased Resistance to Salmonella Infection of Hypoferremic Mice Fed a Low‐Protein Diet
Authors: Gauthier Yves , Isoard Pierre
Published: November 2013

BALB/c and CBA/CA mice fed a protein‐deficient diet developed a plasma hypoferremia corresponding to a 30 percent lowering of serum iron concentration. This hypoferremia persisted as long as the diet was maintained. Hypoferremic CBA/CA mice had increased resistance to Salmonella typhimurium C5 infection, as shown by the reduced lethal activity and the decreased growth of the bacteria in the spleen and in the peritoneal exudate of the deficient animals. This induced resistance was abolished after injection of iron or Desferal into the restricted animals. Such resistance was not observed with BALB/c mice fed a protein‐deficient diet, in spite of the plasma hypoferremia. The growth of S. typhimurium C5 in the spleen and in the peritoneal exudate of these animals did not differ from the growth observed in control animals fed a protein‐sufficient diet. This study suggests that hypoferremia induced by a protein‐deficient diet is probably involved in the enhancement of resistance of CBA/CA mice to Salmonella infection, and that the phenomenon is host‐strain dependent.
Study of the Biological Activities of Toxic Shock Syndrome Toxin‐1
Authors: Uchiyama Takehiko , Kamagata Yusuke , Wakai Mariko , Yoshioka Morimasa , Fujikawa Hiroshi , Igarashi Hideo
Published: November 2013

The mitogenic and interleukin 2 (IL 2) production‐inducing effects of toxic shock syndrome toxin‐1 (TSST‐1) on murine lymphocytes were investigated. TSST‐1, an exotoxin produced by Staphylococcus aureus recovered from patients with toxic shock syndrome (TSS), is thought to be a causative agent of the syndrome.

TSST‐1 was mitogenic for splenic T cells and peanut agglutinin (PNA)‐negative thymocytes, but not for T cell‐depleted spleen cells, PNA‐positive thymocytes or IL 2‐dependent CTLL 2‐cells. A factor mitogenic for CTCC‐2 cells with a molecular weight of 30–35 kdaltons was obtained by stimulating spleen cells with TSST‐1 and it was absorbed by CTLL‐2 cells, indicating that the factor is IL 2. For substantial amounts of IL 2 to be produced, 10 ng or more of TSST‐1 per ml and 48 hr or more of incubation were required. Removal of T cells abrogated the IL 2 production by spleen cells. T cells obtained by the nylon wool column method alone produced IL 2 on TSST‐1 stimulation in the presence of either macrophages or a macrophage lysate containing interleukin 1. However, T cells obtained by a combination of the nylon wool column method and anti‐Ia antibody treatment produced IL 2 in the presence of macrophages but not of the macrophage lysate, indicating that IL 2 production by TSST‐1‐stimulated T cells is absolutely dependent on the presence of accessory cells.
A Probable New Adhesive Factor (F42) Produced by Enterotoxigenic Escherichia coli Isolated from Pigs
Authors: Yano Tomomasa , Leite Domingos Da Silva , De Camargo Irineu José Barsanti , De Castro Antonio Fernando Pestana
Published: November 2013

Three enterotoxigenic Escherichia coli (ETEC) strains (coded 62, 104, and 567/7) isolated from piglets with neonatal diarrhea produced only a thermostable enterotoxin. Although these strains showed mannose‐resistant microhemagglutination (MRMH), the responsible factor was serologically different from the known hemagglutinating colonization factors from porcine strains (K88, K99, and F41). Bacterial cells from these strains adhered to HeLa cells and pig brush borders. Electron microscope studies revealed the presence of fimbria‐like structures on bacterial cells grown at 37 C but not on cells grown at 18 C. The antiserum prepared from partially purified fimbrial antigen (provisionally called F42) inhibited chicken erythrocyte MRMH caused by these strains as well as adherence of strain 567/7 to HeLa cells and to pig brush borders. These data taken together suggest the existence of a new hemagglutinating adhesin that is different from those so far described for porcine ETEC.
Budding Process and Fine Structure of Lymphadenopathy‐associated Virus (LAV)
Authors: Tsuchie Hideaki , Katsumoto Tetsuo , Hattori Naohiko , Kawatani Toshio , Kurimura Takashi , Hinuma Yorio
Published: November 2013

The budding process and fine structure of lymphadenopathy‐associated virus (LAV), were studied by indirect immunofluorescence (IF) and electron microscopy (EM). By IF, LAV antigen was seen to be distributed focally within infected CCRF‐CEM cells. Consistent with this finding, electron micrographs showed that LAV particles occurred in a focally aggregated state in a restricted area of the surface of the infected cells. LAV particles possessed bar‐shaped, dense and central or eccentric cores. In addition, two or more cores were occasionally observed in one virus particle, or the cores were sometimes absent when thin sections were examined. The envelope of the virus particles had an irregular structure, although LAV particles were approximately spherical.
Actinobacillus (Haemophilus) actinomycetemcomitans in Periodontal Disease
Authors: Ohta Hiroyuki , Kokegughi Susumu , Fukui Kazuhiro , Kato Keijiro
Published: November 2013

Actinobacillus (Haemophilus) actinomycetemcomitans is a facultatively anaerobic, gram‐negative coccobacillus which is a possible etiological agent in juvenile periodontitis (JP). In this study, bacterial flora, especially the occurrence of A. actinomycetemcomitans, in the periodontal pockets of one juvenile with gingivitis (G), one JP patients, five rapidly progressive periodontitis (RP) patients and one adult periodontitis(AP) patient, and one adult with healthy periodontium was investigated using a blood agar medium and a selective medium for A. actinomycetemcomitans. Eight hundred and sixty‐five bacteria were isolated from the periodontal pockets, examined for their gram‐stain, cell morphologies, relations to O2 and CO2 and calatase reaction, and divided into 21 groups on the basis of these characters. Among the isolates, 604 isolates were further characterized biochemically and identified. A. actinomycetemcomitans was found as 0.2% of the flora of a site in the JP patient, as 9% of the flora of a site in the G patient, and as 19% and 1%, respectively, of the flora of a site in the two RP patients. However, the organism was not detected in another lesion site of the JP patient. In our JP and RP patients, Fusobacterium, Wolinella, Streptococcus, and obligately anaerobic, gram‐positive cocci were frequently found at high levels. The bacterial flora of the G and AP patients were more heterogenous and included Bacteriodes at relatively high proportions. These results indicate that A. actinomycetemcomitans is not always associated with JP but occurred in some patients with RP and G.
Pathogenic Significance of Acinetobacter calcoaceticus: Analysis of Experimental Infection in Mice
Authors: Obana Yoshiki
Published: November 2013

The phenomenon that mixed infection with certain species of bacteria and Acinetobacter calcoaceticus is more virulent than single infection was analyzed experimentally. In mixed infections with A. calcoaceticus paired with either Escherichia coli, Serratia marcescens, or Pseudomonas aeruginosa, the virulence of the latter three organisms was markedly increased over that of single infections only by slime‐producing strains of A. calcoaceticus. Of the 100 strains of A. calcoaceticus tested, 14 had slime‐producing ability. There was scarcely any difference in the chemical components of the slimes of the two strains tested, but the components of the slime of P. aeruginosa were different from those of these strains. The slime of these two strains exhibited lethal activity in mice, but no correlation was found between the amount of slime produced and the virulence. The slime enhanced the virulence of E. coli, S. marcescens, and P. aeruginosa when it was inoculated along with their viable cells. Furthermore, the slime exhibited potent cell‐impairing activity against mouse neutrophils both in vitro and in vivo. This activity was considered to be mainly responsible for the enhancement of virulence in mixed infections.
Staining of O‐Specific Polysaccharide Chains of Lipopolysaccharides with Ruthenium Red
Authors: Kuno Tsuneharu , Naito Setsuko , Ohta Michio , Kido Nobuo , Ito Hideo , Kato Nobuo
Published: November 2013

S‐form lipopolysaccharides (LPS) from Klebsiella strain LEN‐1 (O3: K1 —) and from Salmonella minnesota strain 1114 were positively stained with ruthenium red, whereas R‐form LPS from Klebsiella strain LEN‐111 (O3— : K1 —) and Ra, Rb1, RcP+, Rd1P‐, and Re LPS from the respective mutant strains of S. minnesota were not or only faintly stained by such treatment. From these results it was concluded that ruthenium red stains the O‐specific polysaccharide chains of LPS. The appearance of stained preparations of S‐form LPS suggested that the material responsible for this positive staining corresponded to the surface projections which were seen by the negative staining technique as attached to the ribbon‐like structures and spherules of the LPS.
Modification of Susceptibility to Klebsiella pneumoniae during Murine Cytomegalovirus Infection
Authors: Leung Wai Chi Teresa , Hashimoto Kazuo
Published: November 2013

The effect of murine cytomegalovirus (MCMV) infection on susceptibility to bacterial infection was studied in mice by a combination of intraperitoneal (ip) inoculation of a sublethal dose of MCMV with subsequent ip challenge of 2 × 103 cfu of a strain of Klebsiella pneumoniae (KP). When given alone, KP produced a mortality of 30–40%. Mortality was increased when KP was given 1 to 7 days after MCMV injection with the peak increase at the 4th to 5th day when 100% mortality occurred. Virus levels in various organs of mice infected with MCMV alone, or superinfected with KP did not differ. Bacterial counts on the other hand, showed that increased mortality in mixed MCMV and KP infected mice was due to an uncontrolled growth of bacteria at the site of primary lodgment, i.e., the peritoneum, and severe systemic infection. Neutrophil response to growth of KP during the first 3 days of bacterial infection was defective in MCMV infected mice. While the initial clearance of KP from the blood was more efficient in MCMV infected mice, probably due to activated reticuloendothelial function, it did not protect the mice against KP infection. Using the in vivo model, it was shown that poor neutrophil response and possibly other defective neutrophil functions were responsible for increased mortality to KP infection in MCMV infected mice.
Lipid Metabolism in Endotoxic Rats
Authors: Kawakami Masanobu , Murase Toshio , Itakura Hiroshige , Yamada Nobuhiro , Ohsawa Nakaaki , Takaku Fumimaro
Published: November 2013

Studies were conducted to investigate the effect of E. coli endotoxin administration on hepatic triglyceride lipase (H‐TGL) activity in rats, since H‐TGL activity is known to behave differently from lipoprotein lipase (LPL) activity in various situations. Plasma triglyceride and free fatty acid concentrations were markedly elevated in animals after injection of endotoxin. Cholesterol and phospholipids were also increased significantly. Lipoprotein analysis by ultracentrifugation showed that the most pronounced increase of lipoproteins was in the VLDL and IDL fractions. Triglyceride lipase activities in post‐heparin plasma were markedly decreased. A selective assay for H‐TGL activity using a specific antibody revealed that this enzyme as well as LPL is significantly decreased (26% of control) in endotoxic animals. Thus, the increase of VLDL and IDL appears to result from the decrease of both of LPL and H‐TGL.
Numerical Classification of Rapidly Growing Nonphotochromogenic Mycobacteria
Authors: Tsukamura Michio , Ichiyama Satoshi
Published: November 2013

Numerical analysis of 211 strains of rapidly growing, nonphotochromogenic mycobacteria was carried out by using 116 characters. New species reported after 1981 and not yet compared with known species by numerical classification were included. At the level of 90% of the matching coefficient, the following species could be differentiated from each other and were shown as distinct clusters: Mycobacterium pulveris, M. moriokaense, M. chitae, M. diernhoferi, M. porcinum, M. fortuitum, M. chelonae subspecies chelonae, M. chelonae subspecies abscessus, M. smegmatis, M. agri, M. fallax, M. parafortuitum, M. fortuitum subspecies acetamidolyticum. Four taxa, M. fortuitum, M. porcinum, M. chelonae subsp. chelonae, and M. chelonae subsp. abscessus, were regarded as distinct. However, these were combined into one large cluster at a level of 90% of the matching coefficient and, therefore, were regarded as forming one series or complex. M. fortuitum subsp. acetamidolyticum was reported as a subspecies of M. fortuitum, but this was differentiated clearly from the other species including M. fortuitum.
Antigens Associated with Various Cytotoxic Activities of Murine Peripheral Macrophages
Authors: Okutomi Takafumi , Satoh Motonobu , Oshima Haruyuki , Yamazaki Masatoshi
Published: November 2013

BCG‐ or glucan‐elicited murine peripheral macrophages released a cytotoxin in the presence of loach egg lectin, whereas proteose peptone‐, glycogen‐, or thioglycollate‐elicited or resident macrophages did not. The macrophages that released cytotoxin coincided with those that showed lectin‐dependent macrophage‐mediated cytolysis (LDMC) in response to loach egg lectin. The cytotoxin released by BCG‐elicited macrophages in response to loach egg lectin had a molecular weight of 55 K daltons. The macrophages that released cytotoxin and other cytotoxic macrophages such as those that showed LDMC‐ and antibody‐dependent macrophage‐mediated cytolysis (ADMC) were examined by using several antibodies to surface antigens of macrophages. The results showed that murine peripheral macrophages could be divided into three types. Resident macrophages (Type I) which had common macrophage antigens (Mac‐1 and B12) showed only LDMC in response to wheat germ agglutinin. Some elicited macrophages (Type II) were asialo GM1‐positive and showed both ADMC and LDMC in response to wheat germ agglutinin. Activated macrophages (Type III) showed LDMC in response to loach egg lectin and cytotoxin‐release, but had no antigen detectable with monoclonal anti‐macrophage antibody (C14). These three types of macrophages were clearly distinguished diagrammatically by their roof‐shaped, rocket‐shaped and irregular‐shaped profiles of activities and antigens. These data suggest that several selected surface antigens of macrophages are associated with distinct cytotoxic stages of peripheral macrophages.
In Vitro Hexagonal Assembly of Lipopolysaccharide of Escherichia coli K‐12
Authors: Kato Nobuo , Ohta Michio , Kido Nobuo , Ito Hideo , Naito Setsuko , Kuno Tsuneharu
Published: November 2013

We examined Escherichia coli K‐12 lipopolysaccharide (LPS), which is known to be an R‐form LPS, for its ability to form a hexagonal lattice structure in vitro. The LPS from E. coli K‐12 strain JE1011 did not form a hexagonal lattice structure when it was precipitated by addition of two volumes of 10 mm MgCl2‐ethanol, but it did form such a structure when it was electrodialyzed and then converted to the magnesium or calcium salt form. The lattice constant of the magnesium salt form was 15.2±0.3 nm and that of the calcium salt form 18.5±0.3 nm. Since prior treatment of the LPS with proteinase K in the presence of sodium dodecyl sulfate did not affect its capability of hexagonal assembly, the lattice formation by the LPS does not require the presence of proteins.
Evaluation of the Efficacy of Split‐Product Trivalent A(H1N1), A(H3N2), and B Influenza Vaccines
Authors: Ochiai Hiroshi , Shibata Mitsuo , Kamimura Katsura , Niwayama Seihachiro
Published: November 2013

A total of 1,995 primary school children (1,464 vaccinees and 531 nonvaccinees) were studied to evaluate the protective efficacy of Tween‐ether split trivalent A(H1N1), A(H3N2), and B influenza vaccines by comparison of the incidence of confirmed infection in two groups during 1980 to 1984. During the study period, epidemics caused by antigenically different influenza viruses, that is A(H1N1) epidemics in 1981 and 1984, a B epidemic in 1982 and an A(H3N2) epidemic in 1983, were experienced, and at the same time strains changed by antigenic drift were frequently isolated. In these epidemics, 61% to 87% of the children reported respiratory illnesses and 18% to 48% of the illnesses were influenza confirmed by seroconversion. Throughout these four epidemics, the incidence of confirmed infection among the vaccinees (7.8% to 33.8%) was 6.5% to 34.8% lower than that among the nonvaccinees (35.4% to 51.6%), demonstrating that the vaccine was effective (χ2=76.34, P<0.001). However, this effectiveness was not seen in an epidemic in one of the entrant schools in 1984, possibly caused by a strain with intense antigenic drift. On the basis of data on incidence of various symptoms, duration of fever and the number of days of absence from class, it was considered that clinical symptoms in the vaccinees were milder than those in the nonvaccinees. When the titers of hemagglutination‐inhibiting (HAI) antibody against the vaccine strains were measured, the protective level of HAI antibody giving ≦50% incidence of infection was 1:64, but it increased to 1:256 in the 1984 epidemic, reflecting the high rate of isolates with intense antigenic drift.
Microimmunofluorescence Using Terasaki Plates and Direct Plate Freezing Method—Rapid and Reliable Screening System of Hybridomas
Authors: Nakagawa Shoko , Tsuji Yoshiyuki , Nishiura Haruhiko , Isojima Shinzo
Published: November 2013

Microimmunofluorescence using Terasaki plates and a direct plate freezing method were combined for effective screening of hybridoma supernatants. The microplates, in which the fused cells (myeloma and spleen cells) were cultured and hybridoma colonies were growing, were frozen after harvest of supernatants and saved at −80 C for several weeks without affecting antibody production ability of hybridomas. Microimmunofluorescence was performed in Terasaki plates on which target cells were attached by poly‐l‐lysine and glutaraldehyde or by short time culture of the cells in Terasaki plates. The direct plate freezing method prevented initial hybridoma cells from changes or disappearance of antibody productions during screening of hybridoma supernatants; the microimmunofluorescence staining method permits fast and detailed estimation of specificity of antibodies of hybridomas by saving time and minimal consumption of supernatant for checking. The combination of these two methods is a powerful tool for obtaining desired monoclonal antibodies.
Invasiveness of Salmonella typhi Strains in HeLa S3 Monolayer Cells
Authors: Yabuuchi Eiko , Ikedo Masanari , Ezaki Takayuki
Published: November 2013

The internalization and intracellular multiplication, i.e., the invasiveness, of Salmonella typhi strains recently isolated from typhoid fever patients were confirmed in HeLa cell monolayers. When stained with Giemsa solution, intracellular bacteria were 0.6 × 1.2 μm in size and stained purple, whereas extracellular bacteria associated or not with the HeLa cell surface were 1.0 × 3.0 μm and stained deep blue. Strain GIFU 10007 was internalized into 23% of the HeLa cells within 10 min after inoculation. About 90% of the HeLa cells were infected after 24 hr incubation in kanamycin (KM)‐containing medium. Intracellular multiplication of the challenge organism was verified by a large number of intracellular bacteria after 24 hr incubation in KM‐containing medium by both light‐microscopy of the Giemsa stained preparation and viable counts of intracellular bacteria. The viable counts of strain 10007 showed an increase of more than 40‐fold within 24 hr after inoculation, whereas in the four other less or non‐infective strains, recovery of viable bacteria was poor or nil. Strains which were highly invasive usually failed to show strong adhesion. The contribution of Vi antigen to the internalization of challenge organisms was not proved. Infective strains, when killed by formalin were still adhesive, but were not internalized. The same strains, when killed by boiling, were neither adhesive nor internalized. From these findings it was concluded that the internalization and multiplication of infective S. typhi strains in cultured HeLa cells should be regarded as an invasion rather than phagocytosis by host cells, and such invasiveness could be an indicator to estimate the virulence of S. typhi strains.
Effect of Bordetella Heat‐Labile Toxin on Perfused Lung Preparations of Guinea Pigs
Authors: Endoh Masahiko , Nagai Masaaki , Nakase Yasukiyo
Published: November 2013

In vitro effects of the Bordetella HLT on the isolated perfused lung and some other tissue preparations from guinea pigs were examined. When HLT (30 to 300 MNDs/ml) was administered, an increase of the perfusion pressure was induced in the perfused lungs, indicating vasoconstriction. When 100 or 300 MNDs/ml of HLT was given, the pressure increase appeared after a lag period of 3.5 to 4 min, reached a maximum within 8 to 13 min, and then slowly decreased by 60 to 80% 25 min after exposure. In calcium‐free medium, the pressure increase dur to HLT did not occur, but these HLT‐treated lungs manifested an increase without any lag period immediately after the calcium‐free medium was replaced by normal medium containing calcium. No difference in the response of the perfused lungs to histamine was observed before and after exposure to HLT. The arterial strip did not respond to HLT, but after predigestion with a collagenase and elastase solution the contractive response to 100 MNDs/ml of HLT appeared with a lag period of 1 min. HLT had no effect on the pharmacological responses of the isolated atria, deferent canal or intestinal preparations, or on the ciliary movement of cultured tracheal rings.
Effect of Streptococcus parvulus and Peptostreptococcus magnus on Cytotoxin Levels of Clostridium difficile in Anaerobic Continuous Flow Culture
Authors: Yamamoto Takako , Takahashi Yasuyuki , Aiba Yuji , Ohnishi Nobuhiko , Ozawa Atsushi
Published: November 2013

An anaerobic continuous flow (CF) culture method was used in order to study the effect of Peptostreptococcus magnus and Streptococcus parvulus, anaerobic gram‐positive cocci which are members of intestinal bacterial flora, on growth and cytotoxin‐activity of Clostridium difficile. The growth‐ and the cytotoxin activity‐patterns of C. difficile in an established CF culture of P. magnus were similar to those of C. difficile alone. On the other hand, in the mixed culture system of C. difficile and S. parvulus, the cytotoxin levels were significantly lower as compared with C. difficile alone in spite of the fact that no differences existed between growth of C. difficile in mixed and single culture systems. The culture filtrate of P. magnus did not influence the growth and cytotoxin production of C. difficile, nor did that of S. parvulus have any effect on growth of C. difficile in static culture. The cytotoxin activity of C. difficile was, however, suppressed by the culture filtrate of S. parvulus. Furthermore, when P. magnus or S. parvulus was statically cultured in a medium containing cytotoxic culture filtrate of C. difficile, the toxin in the medium was not inactivated.