bluuebunny/crossref_metadata_2025 huggingface dataset:
Sugar Synthesis in Leptospira
Authors: Yanagihara Yasutake , Kobayashi Susumu , Mifuchi Ichiji
Published: November 2013

The presence of glucosephosphate isomerase, one of the key enzymes in carbohydrate metabolism, was confirmed for the first time in the cell‐free extract of Leptospira biflexa.

The glucosephosphate isomerase of L. biflexa was heat‐labile and its optimum pH was about 8.5. The enzyme showed an optimal temperature of about 45 C but was more stable at 30 C. Km value of the enzyme was 5.6 × 10−3M. The activity of the enzyme was inhibited by the inhibitor, 6‐phosphogluconate.

From this study, the presence of a metabolic pathway, the phosphogluconate pathway, other than non‐oxidative pentose phosphate pathway presented by Baseman and Cox was suggested.
Rapid Isolation of Endogenous Inhibitor of Factor B
Authors: Moriyama Takeshi , Kawamoto Yasuko , Kobayashi Tatsuhiko , Inoue Takashi , Okamoto Keinosuke , Miyama Akio
Published: November 2013

An inhibitor of mouse factor B was purified from lysate of L cells by a human factor B‐affinity column. The purified inhibitor was found to be homogeneous with a molecular weight of about 25,000 and identical to the 25K protein isolated from L cell‐lysate by the coprecipitation method with anti mouse factor B. Hemolytic titration of the activity and PAGE‐analysis of the inhibitor‐factor B complex showed that 1 mol of inhibitor reacted with 1 mol of mouse factor B and inactivated its hemolytic activity.
Antibody‐Dependent Cellular Cytotoxicity and Natural Killer Cytotoxicity of Peritoneal Cells from Nude Mice to Herpes Simplex Virus‐Infected Cells
Authors: Kohl Steve , Tang Jy‐Ping , Loo Lian Sim
Published: November 2013

Nude BALB/c mice (athymic) were more susceptible to fatal herpes simplex virus (HSV) than normal BALB/c mice (P=0.002). The peritoneal cells of nude mice mediated levels of antibody‐dependent cellular cytotoxicity (ADCC) of equal or greater magnitude than cells from normal BALB/c, heterozygote nu/+, or C57BL/6 mice. Unstimulated natural killer cytotoxicity of peritoneal cells from nude mice was higher (P<0.05) than that mediated by cells from C57BL/6 mice. Nude mice failed to make anti‐HSV ADCC antibody 6 to 14 days post HSV inoculation, at times when nu/+, BALB/c, and C57BL/6 mice produced antibody. Passive reconstitution of nude mice with high titer intraperitoneal anti‐HSV immune globulin provided circulating anti‐HSV ADCC antibody and significant protection against lethal HSV infection.
Biological Activities of Pertussigen from Bordetella pertussis Strains of Various Agglutinogen Types
Authors: Watanabe Mitsuru
Published: November 2013

Pertussigen prepared from Bordetella pertussis strains of various agglutinogen types was found to have similar biological activities in mice, and to give precipitin lines of identity in agar diffusion tests. The doses required to induce histamine hypersensitivity ranged from 0.9 to 9.5 ng, and to induce leukocytosis from 124 to 190 ng. When pertussigen was detoxified by treatment with glutaraldehyde, it protected mice from intracerebral challenge with B, pertussis at doses of from 12.7 to 24.4 fig. The results showed that all smooth strains of B. pertussis produced pertussigen that was biologically and serologically similar, and that it was produced independently of fimbrial hemagglutinin.
Calcium Behavior in Endotoxin‐Poisoned Mice: Especially Calcium Accumulation in Mitochondria
Authors: Sakaguchi Shuhei , Abe Hiroharu , Sakaguchi Osamu
Published: November 2013

A possible role of intracellular Ca2+ and participation of calmodulin in cellular metabolism in endotoxin‐poisoned mice were investigated. The levels of calcium in liver cytosol and liver mitochondria fractions in poisoned mice were markedly higher 18–48 hr after endotoxin injection than in the control mice. On the other hand, the levels of serum calcium in the poisoned mice were about 20% lower at 18 hr than in the controls. The serum calcium levels in mice injected with 50 and 100 μg of endotoxin showed no dose‐response effect, but a dose‐response effect was observed at a dose of 200–400 μg. The serum Ca2+ levels in endotoxin‐tolerant mice were similar to those in the control mice. The levels in mice injected with glucocorticoid‐antagonizing factor mice were about 14% lower at 3 hr than in the controls. The mice fed a vitamin D3‐ and calcium‐free diet showed a higher mortality rate in the early stage (12–18 hr) of endotoxication than that of the mice fed a normal diet.

The lipid peroxide levels and Ca2+‐ATPase activity in the liver mitochondria fraction in endotoxin‐poisoned mice showed a higher level than those of the control mice. There was little or no difference in the levels of serum glucose between the mice injected with calmodulin antagonist (trifluoperazine, TFP) plus endotoxin and those given endotoxin alone. However, the liver glycogen levels in TFP plus endotoxin‐treated mice were markedly higher than that in mice given endotoxin alone. Furthermore, calcium antagonist (verapamil) plus endotoxin‐treated mice had about a 40% higher survival rate after 72 hr than those given endotoxin alone.

The findings suggest that there is a possibility of participation of the Ca2+‐calmodulin system in carbohydrate metabolic disorders during endotoxemia and that the changes in intracellular Ca2+ may result in various metabolic disorders.
Cells Participating in Immunologic Memory Induced with RNA from the Spleens of Immunized Mice
Authors: Kodama Kazue , Yoshida Toshiharu , Kurashige Satonori , Mitsuhashi Susumu
Published: November 2013

Ribonucleic acid (immune RNA, iRNA) extracted from the spleens of mice immunized with heterologous red blood cells induced antigen‐specific immunologic memory. The type of cells (T cells, B cells) which participate in immunologic memory induced with iRNA was investigated. Immune RNA‐primed T cells cooperated with normal, iRNA‐primed or antigen‐primed B cells and induced a high IgM response. Immune RNA‐primed B cells did not cooperate with normal T cells, but did with iRNA‐ or antigen‐primed T cells. The activities of iRNA‐primed T and B cells were antigen‐specific.
Suppressor Macrophages in Lymphocyte Proliferation of Cynomolgus Monkeys
Authors: Tatsumi Masashi
Published: November 2013

The present study demonstrated the presence of cells belonging to monocyte/macrophage lineage which suppressed mitogen‐induced blastogenesis of peripheral blood lymphocytes in cynomolgus monkeys. Depletion of adherent or phagocytic cells from peripheral mononuclear cells caused a substantial increase in the blastogenic response of cynomolgus monkey lymphocytes whereas the same treatment led to marked reduction rather than enhancement in human lymphocyte blastogenesis. Addition of thioglycollate‐elicited peritoneal exudate adherent cells as macrophages suppressed the blastogenic response of nonadherent lymphocytes in a dose‐dependent manner. The suppressive effect was observed not only in autologous but also in allogeneic macrophages to the responder lymphocytes. Treatment of macrophages with silica, carrageenan or freezing‐thawing reduced their suppressive effect but there was no reduction with mitomycin C or indomethacin. No suppressive activity was detected in the cell‐free supernatant of macrophages cultured in the presence or absence of mitogens for up to 4 days. From these findings, it appeared that monocyte/macrophage lineage might be responsible for the observed suppressive effect on mitogen‐induced blastogenesis of cynomolgus monkey lymphocytes.
Studies on the Enteropathogenic Mechanism of Non‐O 1 Vibrio cholerae Isolated from the Environment and Fish in Toyama Prefecture
Authors: Gyobu Yotaku , Kodama Hirohide , Uetake Hisao , Katsuda Shogo
Published: November 2013

Enteropathogenic mechanisms of non‐O 1 Vibrio cholerae were investigated using strains from the environment and those from fish in Toyama Prefecture.

None of the 93 non‐O 1 V. cholerae strains produced a detectable level of cholera‐toxin‐like‐enterotoxin (CT‐like‐enterotoxin) in Syncase medium, while 23 strains showed a distinct fluid accumulation in the rabbit ileal loop test (RIL). These RIL‐positive strains neither produced CT‐like‐enterotoxin in vitro in the other four kinds of media which are considered suitable for CT production, nor in vivo in the ligated ileal loop. Approximately one‐third of RIL‐positive strains produced a fluid accumulating factor (FAF) which was not neutralized with anti‐CT serum. FAF of a representative strain (Strain 79–9‐2) was inactivated by heating at 100 C for 10 min, and has a molecular weight within the range of 50,000 to 100,000 daltons. Most accumulated fluids in RIL after inoculation with whole cultures of RIL‐positive strains contained both hemolytic and cytotoxic principles.

Desquamation of epithelial cells, inflammatory edema, neutrophile infiltration, loss of goblet cells and frequent hemorrhages were observed in sections of ligated ileal loop inoculated with whole cultures or concentrated culture filtrates of CT‐like‐enterotoxin‐negative but RIL‐positive strains. In contrast, neither desquamation of epithelial cells nor hemorrhage was observed in sections after inoculation with those of a CT‐like‐enterotoxin positive strain (Strain E 8498).

These results indicated that most RIL‐positive non‐O 1 V. cholerae strains from the environment and fish isolated in Toyama Prefecture produce little CT‐like‐enterotoxin, but some of them produce FAF with cytotoxic activities.
Development of a New Cell System for the Infectivity Assay of Dengue Viruses: Plaque Formation and Virus Growth of Prototype and Wild‐Type Dengue Virus Strains in a Newly Established Cell Line, GK
Authors: Nawa Masaru
Published: November 2013

A newly established cell line, GK, derived from the kidney tissue of Mongolian gerbils, produced plaques by infection of prototype and wild‐type dengue virus strains. Both prototype and wild strains of type 2 virus grew in GK cells and formed plaques at 35.5 C and at 31 C, while types 1, 3, and 4 wild strains grew and formed plaques only at 31 C. In GK cells, plaque formation and the growth of dengue viruses depended on the high (35.5 C) and low (31 C) incubation temperatures.

Virus yields in GK cells of all the 14 dengue virus strains tested, including four prototype and ten wild‐type viruses, were 5 to 1,000‐times lower than those in C6/36 cells. After five serial passages in GK cells, types 2, 3, and 4 prototype viruses and type 2 wild strain increased virus yields, and one strain of prototype virus and one strain of wild‐type virus decreased mouse neurovirulence.
Age of Appearance of IgG, IgM, and IgE Antibodies Specific for Loa loa in Gabonese Children
Authors: Goussard Bernard , Ivanoff Bernard , Frost Eric , Garin Yves , Bourderiou Christiane
Published: November 2013

IgG, IgM, and IgE antibodies against the filaria Loa loa were measured in umbilical cord blood and in blood from young Gabonese children by an ELISA technique using a homologous metabolic antigen. For children in eight consecutive age groups and adults the percentage of the population positive for each of the antibody classes was determined. The number of children with maternal IgG decreased until one year of age when new synthesis began to become apparent. IgM antibodies were detected only after six months, probably indicating an early infancy as opposed to a fetal infection. The percentage of individuals positive for IgM or IgE reached a peak between two and three years old, followed by a slight decline. Over half of the individuals over one year of age had IgM antibody against L. loa, indicating long‐term synthesis of this class of immunoglobulin in many people. In the first two years of life, IgE antibodies were usually accompanied by L. loa‐specific IgM. This specific IgE did not appear to trigger the synthesis of nonspecific IgE. By the age of two, 95% of the population had some antibodies against L. loa and by five the percentage of individuals positive for each antibody class had reached adult levels.
Immunosuppressive Activities of Peritoneal and Splenic Macrophages in Murine Leprosy: Effect on Lymphocyte Transformation and Tumor Growth
Authors: Ha David K.K. , Lawton John W.M. , Gardner Ian D.
Published: November 2013

The ability of peritoneal macrophages (PM) and splenic macrophages (SM) to suppress tumor growth and lymphocyte transformation in vitro was studied in infected mice with Mycobacterium lepraemurium (MLM). Both PM and SM of leprous mice showed cytostatic activity against tumor cells in vitro. However, such cells showed significantly less cytostatic activity on a per cell basis than highly activated macrophages obtained from Corynebaclerium parvum‐immunized mice. Furthermore, this cytostatic activity declined as the infection progressed.

Mitogen‐induced transformation of splenic lymphocytes was also suppressed in the presence of adherent PM and SM from leprous mice. PM from leprous mice showed significantly less activity than PM from C. parvum‐immunized mice in terms of suppression of lymphocyte transformation. Moreover, PM from leprous mice treated with C. parvum or sodium thioglycollate broth demonstrated significantly less ability to suppress lymphocyte transformation than did PM from similarly treated normal mice or untreated leprous mice. These findings demonstrated that MLM infection stimulates the mononuclear phagocyte system but does not activated it to the extent that it confers enhanced resistance to MLM on the host.
Requirement of Anionic Groups for the Mitogenicity of a Fungal Mitogen, Vesiculogen
Authors: Ohno Naohito , Suzuki Iwao , Miyazaki Toshio , Yadomae Toshiro
Published: November 2013

Active site(s) of a B‐cell mitogen, vesiculogen, was investigated by means of chemical and enzymic modifications. Vesiculogen is a non‐dialyzable fraction of a hot water extract from a fungus, Peziza vesiculosa (Yadomae et al, Microbiol. Immunol. 23, 997 (1979)), and was composed of protein (~60%), carbohydrate (~30%), and a small amount of amino sugar, uronic acid, phosphate, and lipid. The mitogenicity was not affected by periodate oxidation, N‐acylation, defatting treatment, destruction of the three dimentional structure, protease digestion, nuclease digestion, N‐bromosuccinimide treatment, and β‐mercaptoethanol treatment. However, the mitogenicity was decreased by a modification of carboxyl groups, such as 1‐ethyl‐3(3‐dimethylamino propyl)‐carbodiimide or dicyclohexylcarbodiimide treatment. Vesiculogen contains a large amount of acidic amino acids. These results suggest that the mitogenicity of vesiculogen is due to the presence of anionic groups, such as aspartic acid and glutamic acid, and that the mitogenic substance is a novel, heat stable polyanionic B‐cell activator obtained from Ascomycotina.
Constitutive Production of Phagocytosis Inducing Factor(s) in a Monocyte/Macrophage Lineage Cell Line (THP‐1) by Retrovirus‐Transformed Human T Cell Lines
Authors: Hinuma Shuji , Sugamura Kazuo , Tsukamoto Kyozo , Hinuma Yorio
Published: November 2013

Four human T cell lines, MT‐2, TCL‐Kan, TCL‐As 2, and TCL‐Haz, established from normal leukocytes by cocultivation with adult T‐cell leukemia (ATL) virus (ATLV)‐producing cells, produced constitutively phagocytosis inducing factor(s) (PIF) that induced phagocytosis in a human monocytic cell line, THP‐1. These cell lines expressed ATLV‐associated antigens (ATLA) as well as numerous virus particles, whereas the other twelve leukocyte cell lines tested, including T cell lines, B cell lines, and non‐T and non‐B cell lines, did not produce detectable amounts of the factor(s) in the culture supernatants. PIF was produced in the absence of serum and was not related to either ATLV‐particles or viral structural proteins. Its activity was stable at 56 C for 30 min, but labile at 80 C for 30 min and at pH 2 for 20 hr. MT‐2 and TCL‐Kan produced large amounts of the factor(s) in the culture supernatants but little interferon‐γ (IFN‐γ) or colony stimulating factor (CSF) activity was detected; furthermore, the activity was not neutralized by rabbit anti‐IFN‐γ sera. These observations suggest that some ATLV‐transformed Tcell lines produce PIF that is different from IFN‐γ and CSF.
Tumoricidal Adsorption of Staphylococcus aureus Organisms on Ehrlich Ascites Tumor Cells Sensitized with Rabbit Antibody
Authors: Masuda Shogo , Seki Keiko , Nishihara Shoko
Published: November 2013

A tumoricidal effect was observed when protein A‐bearing Staphylococcus aureus organisms were adsorbed on Ehrlich ascites tumor cells previously sensitized with antiserum from a rabbit immunized with Ehrlich ascites tumor cells. Electron micrographs showed that staphylococci were firmly attached to the tumor cells, which might explain how effectively the attached cocci killed the tumor cells. The tumoricidal effect was confirmed not only by an in vitro experiment but also by an in vivo one. The possible applications of the tumoricidal adsorption as an indicator for staphylococcal virulence or for selective anti‐tumor action was discussed.
Lethal Effect of Neutral Mannan Fraction of Bakers' Yeast in Mice
Authors: Nagase Toshihiko , Mikami Takeshi , Suzuki Shigeo , Schuerch Conrad , Suzuki Masuko
Published: November 2013

A simple polysaccharide, the neutral mannan from Saccharomyces cerevisiae wild type strain (WNM) was found to kill ddY strain mice by intravenous administration, showing a LD50 value of 12.2 mg/kg. On the other hand, the acidic mannan fraction from the same yeast containing phosphate (WAM025), and chemically phosphorylated WNM (WNM‐P) were practically non‐toxic. Concerning the relationship between chemical structure and lethal effect of these mannans, it was demonstrated that a mannan possessing a highly branched structure exhibited stronger lethality than those with less branched structures. Against C3H/HeJ strain mice with no responsiveness to lipopolysaccharide, the LD50 value of WNM was as high as 75 mg/kg. Pretreatment with 500 mg/kg of D‐mannose, N‐acetyl‐D‐glucosamine, D‐galactose, and L‐fucose prevented mice from the lethal effects of WNM. However, WNM (LD100) did not show any lethal effect in mice for 2 to 12 hr after treatment with dexamethasone, an anti‐inflammatory steroid.
Characterization of a Monoclonal Antibody Specific for Lipoteichoic Acid from Various Gram‐Positive Bacteria
Authors: Hamada Shigeyuki , Furuta Takushi , Okahashi Nobuo , Nisizawa Tosiki , Yamamoto Toshiko , Chiba Joe
Published: November 2013

A hybrid cell line, 3G6, producing monoclonal antibody (mAb) against the polyglycerophosphate (PGP) backbone of lipoteichoic acids has been derived by the polyethylene glycol‐induced fusion of mouse myeloma cells and spleen cells from mice immunized with partially purified glucosyltransferase from culture supernatant of Streptococcus mutans strain 6715. Immunodiffusion tests and ELISA revealed that the antibody reacted with purified PGP from group A Streptococcus pyogenes strain Sv as well as crude phenol‐water and saline extracts of various gram‐positive bacteria except for a few species such as biotype B S. sanguis, Micrococcus sp., and Actinomyces viscosus. Whole cells of serotype b S. mutans and Staphylococcus epidermidis were agglutinated upon addition of 3G6 mAb, while those of most other species were not significantly affected by this procedure. A hapten inhibition study showed that glycerophosphate was only a potent inhibitor of passive hemagglutination reactions between LTA coated sheep erythrocytes and 3G6 mAb.
Antigenic Analysis of Flaviviruses with Monoclonal Antibodies against Negishi Virus
Authors: Furuta Itsuko , Takashima Ikuo , Arikawa Jiro , Hashimoto Nobuo
Published: November 2013

Twelve monoclonal antibodies against Negishi virus were obtained and characterized by hemagglutination inhibition (HI) and neutralization (NT) tests using five flaviviruses isolated in the pan‐Pacific region. The reaction pattern of the antibodies showed that Negishi virus was most closely related to Langat virus, followed by 3‐Arch, JE and Apoi viruses in that order. Hemagglutinating (HA) antigen of the virus had distinct HI relating sites which were Negishi virus specific, tick borne encephalitis (TBE) virus complex specific and flavivirus cross‐reactive. Monoclonal anti‐Negishi antibodies cross‐reactive to Japanese encephalitis (JE) virus in the HI test had neutralizing activity to JE virus but no activity to homologous Negishi virus.
Properties of TAS‐1D3, a Tuberculin‐Active Substance from BCG, in Regard to Delayed Hypersensitivity
Authors: Yano Osamu , Toizumi Shin‐Ichi , Sudo Tadashi
Published: November 2013

The properties of TAS‐1D3, a tuberculin‐active substance purified from the cell extract of Mycobacterium bovis BCG, were studied in vivo and in vitro. In the delayed hypersensitivity skin reaction, TAS‐1D3 showed far more potent activity than tuberculin purified protein derivative (PPD) in guinea pigs sensitized with BCG vaccine. This was consistently observed from 6 to 24 weeks after sensitization. The histological findings of the skin reaction to TAS‐1D3 were similar to those of the reaction to PPD. Moreover, TAS‐1D3 induced well both thymidine incorporation and the production of migration inhibitory factor (MIF) by the spleen cells from guinea pigs sensitized with BCG vaccine. In contrast, TAS‐1D3 showed weaker activity than PPD in guinea pigs sensitized with either heat‐killed M. tuberculosis Aoyama B or heat‐killed M. tuberculosis H37Ra, and it weakly stimulated the spleen cells from animals sensitized with M. tuberculosis Aoyama B to incorporate thymidine and to produce MIF.
Effect of Muramyl Dipeptide Analog on Salmonella enteritidis Infection in Beige Mice with Chediak‐Higashi Syndrome
Authors: Onozuka Kazuyasu , Saito‐Taki Tatsuo , Nakano Masayasu
Published: November 2013

Beige mutant (bg/bg) mice with Chediak‐Higashi syndrome (CHS) were much more sensitive to virulent Salmonella enteritidis No. 11 strain than parental C57 BL/6 (+/+) or heterozygous (bg/ +) mice, and they had weaker bactericidal activity against the organisms. Muramyl dipeptide (MDP) and Nα‐(N‐acetyl‐muramyl‐L‐alanyl‐D‐isoglutamyl)‐Nε‐stearoyl‐L‐lysine [MDP‐Lys(L18)], a synthetic derivative of MDP, failed to confer any protection against the infection, but the MDPs showed some ability to stimulate the bactericidal activity in the peritoneal cavities and spleens of these mice. The bactericidal effect of MDP‐Lys(L18) was dose‐dependent, and the greatest effect was seen when it had been injected 24 hr before the infection. Multiple injections of MDP were much more beneficial than a single injection.

Previous injection of N2, O2′‐dibutyryl guanosine 3′: 5′‐cyclic monophosphate (DB‐cGMP) improved the impaired bactericidal capacity in beige mice, but the simultaneous injection of N6, O2′‐dibutyryl adenosine 3′: 5′‐cyclic monophosphate (DB‐cAMP) with DB‐cGMP abolished the effect of DB‐cGMP. The augmentation of bactericidal capacity by MDP‐Lys(L18) was not affected by the injection of either DB‐cGMP or DB‐cAMP, suggesting that the effect of the MDPs was not related directly to cyclic nucleotide regulation in beige mice.
The Presence of Anti‐Sheep Red Blood Cell Heterophile Antibodies and Their Characteristics in Murine Schistosomiasis japonica
Authors: Asahi Hiroko , Kawabata Masato , Sendo Fujiro , Naiki Masaharu , Hosaka Yukio , Kobayakawa Takatoshi
Published: November 2013

Sensitive methods of an enzyme‐linked immunosorbent assay (ELISA) using red blood cells (RBC) have been developed and were applied to the detection of anti‐sheep red blood cell (SRBC) heterophile antibodies (Ab) present in sera of Schistosoma japonicum (SJ)‐infected mice. The indirect hemagglutination test (IHA) was used for the purpose as well. By these methods a significant increase in the heterophile Ab levels was demonstrated in the mice particularly after 6–10 weeks of infection. The heterophile Ab in SJ‐infected mice were predominantly immunoglobulins resistant to 2‐mercaptoethanol treatment and had temperature‐independent reactivity. In an attempt to investigate the immunological specificity of the heterophile Ab, various absorption tests were performed; Davidsohn's differential absorption test revealed that the heterophile Ab were distinct from Forssman antibody, Paul‐Bunnell antibody and heterophile agglutinins known to appear in serum sickness. The heterophile Ab were absorbed only with SRBC and goat red blood cells, not with other species of RBC such as human O Rh‐, O Rh+, A Rh+, B Rh+, mouse, ox, chicken, horse, rabbit, guinea pig, and rat RBC, or syngeneic tissue homogenates including brain, lung, liver, spleen, kidney, muscle, and thymus. This heterophile antibody response is not a consequence of a specific immune response directed to the antigens of SJ parasites, since absorption of the heterophile Ab with SJ adult worms or an egg preparation did not reduce the heterophile Ab level.
Immunochemical Properties of Mannan‐Protein Complex Isolated from Viable Cells of Saccharomyces cerevisiae 4484‐24D‐1 Mutant Strain by the Action of Zymolyase
Authors: Shibata Nobuyuki , Mizugami Kunihito , Suzuki Shigeo
Published: November 2013

Viable cells of Saccharomyces cerevisiae 4484‐24D‐1 mutant strain were treated with an Arthrobacter sp. β‐1,3‐glucanase, Zymolyase‐60,000, in the presence of a serine protease inhibitor, phenylmethylsulfonyl fluoride. Fractionation of the solubilized materials with Cetavlon (cetyltrimethylammonium bromide) yielded a purified mannan‐protein complex, which had a molecular weight of ca. 150,000, approximately three times higher than that of the mannan isolated from the same cells by the hot‐water extraction method at 135 C. The amino acid composition of the mannan‐protein complex was found to be very similar to that of the mannan‐protein complexes of S. cerevisiae X2180‐1A wild and S. cerevisiae X2180‐1A‐5 mutant strains, indicating the presence of large amounts of serine and threonine. It was unexpected that the antibody‐precipitating activity of this complex against the homologous anti‐whole cell serum was about twice as great as that of the mannan isolated by hot‐water extraction. Treatment of this complex with 100 mM NaOH, hot water at 135 C, and pronase, respectively, gave degradation products having the same molecular weight and antibody‐precipitating activity as those of the hot‐water extracted mannan, allowing the assumption that the protein moiety participated in a large part of this activity.
Experimental Infection in Newborn Mice and Rats by Hemorrhagic Fever with Renal Syndrome (HFRS) Virus
Authors: Yamanouchi Takahisa , Domae Kayoko , Tanishita Osamu , Takahashi Yoshiyuki , Yamanishi Koichi , Takahashi Michiaki , Kurata Takeshi
Published: November 2013

Newborn mice and rats were inoculated intracerebrally (ic) or intraperitoneally (ip) with Hantaan virus (76–118 strain) or HFRS‐related virus (B‐1 strain). The mortality and the influence on the increase of body weight in newborn mice were higher in the groups infected with the 76–118 strain than in the groups infected with the B‐1 strain, while the B‐1 strain was more virulent in rats than the 76–118 strain. Virus isolation from rats inoculated with either strain was attempted 7 and 11 weeks after inoculation. Virus could be isolated from various organs of rats infected with the B‐1 strain, while it was recovered from only the brain and lungs of rats infected with the 76–118 strain. Viral antigen was readily detected in various organs of rats infected with the B‐1 strain, but the amount and distribution of antigens were less in rats infected with the 76–118 strain. Our results suggest that the virulence of HFRS‐related virus is variable, depending on the species of infected animals as well as on the. virus strains. The virus also persists in the injected animals with high titers of antibodies for at least 11 weeks.
Development of DTH Skin Reaction to HBsAg in Individuals Immunized with HB Vaccine
Authors: Kashiwagi Seizaburo , Nagafuchi Seiho , Inoue Takatoshi , Hayashi Shin , Takeshita Shigemori , Imayama Shuhei
Published: November 2013

To examine the temporal relationship between the DTH skin reaction to HBsAg and antibody response to HBsAg, serial skin tests for HBsAg were carried out on seven individuals immunized with HB vaccine.

All cases developed both a skin reaction and antibody response. In many cases, the DTH skin reaction appeared four weeks before the antibody response. These data suggest that DTH skin reaction may be a more sensitive method than humoral antibody assessment for monitoring the success of vaccination.
Significance of Filter Mating in Integrative Incompatibility Test for Plasmid Classification
Authors: Yoshida Takaharu , Takahashi Isamu , Tubahara Hikokichi , Sasakawa Chihiro , Yoshikawa Masanosuke
Published: November 2013

For classification of plasmids in epidemiological studies, an integrative incompatibility test using liquid mating was developed by Sasakawa et al (Plasmid 3: 116–127, 1980). This test was designed to compare the relative mating frequency of a donor carrying a test plasmid with that of recA recipients carrying various integrated plasmids. To improve the accuracy of this method by increasing transfer frequency of a test plasmid, filter mating was introduced.

A transfer frequency 10 to 30,000 times higher than that achieved by liquid mating was attained by filter mating. The degree of increase varied among the incompatibility groups and the majority of members belonging to the same incompatibility group exhibited a similar degree of increase.

Standard plasmids were classified correctly with the integrative incompatibility test using filter mating. Out of 26 naturally occurring plasmids of poor transferability in liquid mating, all of domestic animal origin, 25 were correctly classified as IncH with the integrative incompatibility test using filter mating. Moreover, the method is capable of subdividing IncH plasmids directly into IncH1 and IncH2, because IncH2, but not IncH1, plasmids showed incompatibility with the integrated plasmid, R478, of the IncH2 group.
Biological Activities of Lipopolysaccharide Fractionated by Preparative Acrylamide Gel Electrophoresis
Authors: Ohta Michio , Rothmann Joseph , Kovats Enikö , Pham Phuc Hong , Nowotny Alois
Published: November 2013

Lipopolysaccharide from a smooth strain of Salmonella minnesota was fractionated into two major fractions and one intermediate fraction by using sodium dodecylsulfate‐polyacrylamide gel electrophoresis. On the basis of the study by Hitchcock and Brown, it was deduced that the top fraction was mainly long O‐side chain LPS and the bottom fraction was O‐side chain‐less LPS. The middle fraction was a mixture of both short O‐side chain LPS and O‐side chain‐less LPS. The antigenic properties and biological activities were not altered in this fractionation procedure. Comparison of the biological activities of the top fraction with those of the bottom fraction revealed that the bottom fraction had higher activity in polyclonal B‐cell activation and spleen‐swelling effect and that there was no significant difference in adjuvant activity, ability to render macrophages cytotoxic, induction of colony‐stimulating factor and the ability to induce the Shwartzmann reaction. It was suggested that O‐side chain makes no contribution to the latter biological activities including adjuvant activity of S. minnesota LPS.
A Focus Assay Method for Japanese Encephalitis Virus Using Complement and Anti‐Virus Serum
Authors: Kimura‐Kuroda Junko , Yasui Kotaro
Published: November 2013

A sensitive, quantitative, short‐time, and reproducible focus assay for Japanese encephalitis (JE) virus is described. After 2 or 3 days of incubation, the infected cells were treated with anti‐JE virus serum and complement, and subsequently stained with trypan blue; then clear foci were produced. This method made it easy to titrate the infectivities not only of all seven JE virus strains tested but also of West Nile (WN), Murray Valley encephalitis (MVE), and St. Louis encephalitis (SLE) viruses using hyperimmune anti‐JE virus serum for the latter. Moreover, even cell lines which hardly formed plaques by the agar overlay method easily produced foci within 2 or 3 days by this method.
Studies on Toxin of Aspergillus fumigatus
Authors: Ebina Keiichi , Ichinowatari Shyunya , Yokota Katsushi
Published: November 2013

The fashion of binding of Asp‐hemolysin to human erythrocytes and the isolation of Asp‐hemolysin‐binding proteins from erythrocyte membranes were investigated by the immunocytochemical technique and affinity chromatography. Asp‐hemolysin bound best at a pH range from 5 to 7. The erythrocytes treated with Asp‐hemolysin showed diffuse, ring‐like or cap‐like staining by the peroxidase‐labeled antibody method under the light microscope. The distribution of Asp‐hemolysin on the erythrocyte surface was clearly observed as patches or caps in the scanning electron microscope. The erythrocyte ghosts were extracted with 1% sodium deoxycholate‐0.1 M Tris‐HCl buffer (pH 7.5) containing 0.2 M NaCl and 1 mM EDTA, and the extract was chromatographed on an affinity column consisting of Asp‐hemolysin attached to activated thiol‐Sepharose 4B. Four proteins present in the membrane extract were retained by activated thiol‐Sepharose 4B and eluted with 50 mM cysteine as toxin‐membrane components. Sodium dodecyl sulfate Polyacrylamide gel electrophoresis indicated that the polypeptides correspond to band 2.1, one protein of the 2 region, band 3 and band 7 in the Steck nomenclature system.
Germination‐Initiation and Inhibitory Activities of L‐ and D‐Alanine Analogues for Bacillus subtilis Spores
Authors: Yasuda Yoko , Tochikubo Kunio
Published: November 2013

The ability of 33 compounds of L‐alanine analogues over a wide range of concentrations to initiate germination of Bacillus subtilis spores was determined, and the inhibitory activity against L‐alanine‐initiated germination was determined for the above compounds and 22 of their D‐, DL‐isomers. Nineteen L‐isomers were able to initiate the germination. The maximum germination rate and the apparent binding affinity of the germinant were obtained from concentration‐germination response curves. Not only D‐isomers but also L‐isomers of many alanine analogues showed inhibitory action on L‐alanine‐initiated germination. The apparent binding affinity of an inhibitor was calculated by Schild's method. D‐Alanine, D‐serine, glycine, D‐2‐amino‐n‐butyric acid, D‐cysteine, D‐norvaline, and D‐threonine were competitive inhibitors for the L‐alanine action. Analysis of the relation between the structure of the side chain of L‐, D‐alanine analogues and their apparent affinity suggested that there are separate binding portions, which differ in size and electrostatic nature, for germination and for inhibition on the receptor. Certain L‐alanine analogues had a dualistic property of initiating germination at low concentrations and inhibitory activity at higher concentrations, i.e., autoinhibition. The autoinhibitory phenomenon might be explained by the above postulation of the presence of separate binding portions for germination and for inhibition.
Decrease in Respiration Activity Related to Prodigiosin Synthesis in Serratia marcescens
Authors: Kobayashi Nobuharu , Ichikawa Yoichi
Published: November 2013

Variation in the cell respiration rate of pigmented and nonpigmented strains of Serratia marcescens was exhibited. The respiration rate of a pigmented strain decreased earlier than that of nonpigmented strains in the late exponential or early stationary phase. However when prodigiosin synthesis was not induced by exchange of carbon sources in the medium, the decrease in the respiration rate of the pigmented strain was the same as that of nonpigmented strains. Measurement of the oxygen consumption rate in the sonicated cell membrane by adding NADH solution showed that the rate in the pigmented strain was lower than that in nonpigmented strains. Furthermore, the cell membrane of prodigiosin‐induced organisms was more sensitive to respiration inhibitors than that of pigment‐noninduced organisms of the pigmented strain. These results showed that the respiration activity was decreased by prodigiosin synthesis in S. marcescens.
Nucleotide Sequence of the Replication Region of Plasmid R401 and Its Incompatibility Function
Authors: Tabughi Akira
Published: November 2013

The plasmids R401 and Rtsl belong to the same incompatibility group, IncT. The nucleotide sequence of the basic replicon of R401 consisting of 1,857 base pairs was determined and compared with that of mini‐Rtsl previously reported. The mini‐R401 was found to be composed of two clusters of direct repeated sequences flanking a large open reading frame that could encode a 33,000 Mr protein (RepA protein) consisting of 288 amino acids. This structure of mini‐R401 is quite similar to that of mini‐Rtsl. Furthermore, the nucleotide sequence of mini‐R401 is identical to that of mini‐Rtsl except for eleven nucleotides; three are located near the carboxyl terminus portion of the RepA coding region (repA) and four are in the repeated sequences (incI) located downstream from repA. Incompatibility study showed that mini‐R401 plasmid coexisted stably with the cloned incI repeats of mini‐Rtsl, suggesting that mini‐R401 RepA protein binds to incI repeats of mini‐Rtsl less efficiently than does mini‐Rtsl RepA protein.
A Staphylococcal Coagglutination Test for Detecting and Serogrouping Legionella pneumophila
Authors: Yoh Myonsun , Honda Takeshi , Miwatani Toshio , Uemura Takaaki , Kuwata Hiroyuki , Fukai Konosuke
Published: November 2013

For detection of Legionella pneumophila and determination of its serogroup, the modified staphylococcal coagglutination test was studied in detail. Cross‐reactions in serum‐agglutination tests were observed among serogroups, but immunoglobulin‐coated staphylococcal cells could detect L. pneumophila with high sensitivity (a cell concentration with an absorbance of 0.008 at 660 nm could be detected) and serogroups could be determined without cross‐reactions. Moreover the coexistence of other bacteria did not affect the results of the test. These results suggest that the staphylococcal coagglutination test is useful for detection and identification of Legionella, especially in environmental samples, and for serogrouping of isolates of L. pneumophila from clinical specimens.
Comparison of the Efficacy of Egg Yolk Extract and Horse Serum for Growth of Mycoplasma pneumoniae
Authors: Sasaki Tsuguo , Shintani Miharu , Kihara Kojo
Published: November 2013

The usefulness of chicken egg yolk extract as a substitute for horse serum in culture media for Mycoplasma pneumoniae was investigated. As a growth‐supporting factor in the growth medium for M. pneumoniae and some other mycoplasmal species, the primary isolation medium for M. pneumoniae, and the metabolism‐inhibition test medium for diagnosis of M. pneumoniae infection, egg yolk extract may be an excellent substitute for horse serum. The particular superiority of egg yolk extract to horse serum is that egg yolk extract, unlike horse serum, did not show any inhibitory effect on the growth of M. pneumoniae.
The Suitability of Tórtora's Medium for the Production of Enterotoxin in Clostridium perfringens Strains
Authors: de Oliveira TÓRtora JoãO Carlos , Costa Gobert Araujo
Published: November 2013

Examination of 200 samples from soil and the same number of samples from healthy human feces yielded 49 (24.5%) and 105 (52.5%) strains of heat‐resistant Clostridium perfringens respectively. Fourteen (7.0%) strains isolated from soil and 37 (18.5%) from feces synthesized enterotoxin, as demonstrated by Tórtora's method, at sufficient levels to permit its detection by mouse lethality, microslide double gel diffusion or counterimmunoelectrophoresis tests. By using the Duncan‐Strong (DS) method, only four (2%) enterotoxigenic strains from soil and 14 (7.0%) from feces were obtained. The supernatant fluid from two enterotoxigenic‐negative strains grown in DS medium gave a false‐positive reaction when they were injected intravenously into mice. Tórtora's medium was preferable because a larger number of isolated strains produced spores and enterotoxin to permit their recognition as enterotoxigenic strains.
Characterization of Scrapie Agent Isolated from Sheep in Japan
Authors: Shinagawa Morikazu , Takahashi Kaname , Sasaki Satoshi , Doi Satoshi , Goto Hitoshi , Sato Gihei
Published: November 2013

A pathogenic agent isolated in mice from the brain of a sheep affected by scrapie‐like disease was characterized. The incubation period of the disease in the primary transmission from the sheep to mice was longer than in the secondary and the tertiary transmission in the same strain of mice. Progressive dilution of the inoculum caused prolongation of the incubation period. The infectivity of the agent in a 10% brain homogenate persisted, but decreased about 103 to 104 times after heating at 100 C for 30 min. Histological changes in the diseased mouse brains consisted of vacuolation of the nerve cells and spongiform degeneration in the gray matter of the central nervous system. Fine rod‐shaped granulae with a length of 3 to 5 nm were observed within the swollen neuropil, axon, and perivascular astrocytic process. No serum antibodies against available mouse viruses, parainfluenza type 1 virus, lymphocytic choriomeningitis virus, and mouse reovirus type 3, were detected in any mice used in the experiments. These findings demonstrate that the disease of the sheep was the first case of scrapie in Japan.
Effect of Aculeacin A, a Wall‐Active Antibiotic, on Synthesis of the Yeast Cell Wall
Authors: Yamaguchi Hideyo , Hiratani Tamio , Baba Misuzu , Osumi Masako
Published: November 2013

A wall‐active, amphophilic antibiotic aculeacin A significantly but incompletely inhibited in vitro the activity of β‐(1,3)glucan synthase prepared from highly susceptible yeasts Saccharomyces cerevisiae and Candida albicans. In contrast, comparable cell‐free preparations from S. cerevisiae active in chitin synthase or mannan synthase were insensitive to the antibiotic, suggesting selectivity of its action in synthesis of the yeast cell wall.

An electron microscopic study of the effects of aculeacin A at 0.31 μg/ml, the optimally active concentration, on osmotically stabilized C. albicans cells revealed morphological alterations in both cell walls and cell membranes. Deformation in contour and derangement of the layered structure of the cell wall were prominent. In addition, massive fibrous material of β‐glucan‐like microfibrils was occasionally extruded from the cell surface. Accompanying this effect on the cytology of the cell wall, ultrastructural and functional impairment of the cell membrane was demonstrated by transmission and freeze‐fracture electron microscopic techniques.

These data suggest that aculeacin A affects synthesis of the yeast cell wall through not only selective blockage of β‐(1,3)glucan synthase, as a result of a primary interaction with the cell membrane, but also inhibition of the fabrication of β‐glucan or other wall components into well‐organized cell walls.
Detection of Antibody to M Protein of Measles Virus in Patients with Subacute Sclerosing Panencephalitis: A Comparative Study on Immunoprecipitation
Authors: Ohara Yoshiro , Tashiro Masato , Takase Sadao , Homma Morio
Published: November 2013

Consistent results have not been obtained yet on the presence of antibody to the M protein of measles virus in the sera of patients with subacute sclerosing panencephalitis (SSPE). We performed a comparative study on various immunoprecipitation systems which appeared in the literature and found that the difference in the composition of the solubilizing buffer produced a large variety of results on the immunoprecipitation. [35S]Methionine‐labeled Vero cells infected with the Edmonston strain of measles virus were solubilized by 10 different buffers and reacted with hyperimmune rabbit serum to whole virus, monospecific antisera to H, NP, and M proteins of the virus, normal adults' sera, and the sera from 16 SSPE patients. The immune complex was absorbed by protein A and both solubilization and precipitation rates were compared with each viral protein. Although viral proteins were solubilized by all buffers, the solubilization rate varied considerably. M protein was solubilized and was not coprecipitated nonspecifically with any of the other viral proteins. Purified protein A conjugated to Sepharose was preferable to Staphylococcus aureus for absorption of the immune complex since the latter absorbed both viral and host proteins nonspecifically. The precipitation rates of the viral proteins also varied according to the buffers. Better solubilization of the viral proteins seemed to reduce their rate of precipitation for which the presence of SDS may be responsible, and the presence of the protease inhibitors may also affect the results of immunoprecipitation. Detection of M protein in the immunoprecipitates was largely influenced by the kind of buffer used: some buffers could detect it clearly, but others could not defect it at all. Among the solubilizing buffers tested, Saleh's buffer (Virology 93: 369–376 (1979)), which contains 0.5% DOC and 0.5% Triton X‐100, was most reliable for detection of the anti‐M antibody in the rabbit serum, because it showed a high solubilization and high precipitation rates of viral proteins without nonspecific absorption by protein A or coprecipitation of M proteins with any of the other proteins. Using this buffer, we could definitely detect M proteins in the immunoprecipitates from the sera of all six healthy adults and 15 out of 16 patients with SSPE. It was found, however, that the amount of M proteins in SSPE patients was lower than that in healthy adults and varied considerably.
Mononuclear Phagocyte Colony Formation of Human Spleen Cells in Liquid Culture
Authors: Kojima Takashi , Ito Yohichi , Hasegawa Yasuhisa , Kamei Hideo , Kondo Tatsuhei , Nakashima Izumi , Kato Nobuo
Published: November 2013

We found that mononuclear phagocytes formed a distinct number of clusters and colonies on the bottom of a culture dish 7 days later but granulocytes did not, when a large number of human spleen cells were cultured in liquid medium. In all gastric cancer bearers and patients with portal hypertension operated on, however, colony formation was restricted to spleen cells from patients with advanced gastric cancer and from a group of patients with portal hypertension. These spleen cells formed mononuclear phagocyte colonies without the help of exogeneous colony stimulating factor (CSF). We further demonstrated that the colony‐forming cells were glass non‐adherent and nylon wool adherent, and that spontaneous colony formation required cooperation between the colony‐forming cells and colony‐stimulating cells adherent to a plastic surface.
Production of Macrophage Activating Factor by Human Leukemic T Cell Lines
Authors: Imai Katsuyuki , Suzuki Yukio , Harada Takayuki , Morikawa Shigeru , Tanaka Atsushi
Published: November 2013

Human leukemic T cell lines were tested for their ability to produce a macrophage activating factor. When mouse peritoneal macrophages were cultured for 48 hr in the presence of culture supernatants from cell lines HPB‐ALL, CCRF‐CEM, or MOLT‐4, glucose oxidation via the hexose monophosphate pathway was enhanced by five to seven fold. Culture supernatants from cell line HPB‐MLT stimulated the oxidation to a lesser extent. However, cell line CCRF‐HSB‐2 was essentially inactive as a producer. The active supernatants also stimulated the release of hydrogen peroxide from macrophages, whereas the inactive one did not. Since treatment of the cell lines with 12‐o‐tetradecanoyl phorbol acetate or phytohemagglutinin had little effect on the production of the factor except HPB‐ALL, the cell lines seemed to secrete the factor constitutively. The stimulatory effect was dose‐dependent and evident at a concentration as low as a 1/80 dilution. The factor was resistant to heat treatment at 100 C for 20 min, nondialysable and sensitive to protease digestion. The activating factor could be partially purified by anion exchange and gel filtration chromatographies.