bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on Complement‐Fixation Reaction in Equine Infectious Anemia
Authors: Ueda Susumu , Nakamura Junji , Kuroda Sadao , Samejima Togo
Published: November 2013

The substances responsible for inhibiting complement‐fixation (CF) reaction of the late‐stage serum of an equine infectious anemia (EIA)‐infected horse were investigated. It was found that the IgG and IgG(T) classes in the late‐stage serum were responsible for the CF inhibition. IgA could not be detected in partially purified IgG(T) by an immunodiffusion test using rabbit anti‐human IgA serum. Other serum components could not be demonstrated in purified IgG by immunoelectrophoresis using rabbit anti‐horse serum. The IgG class simultaneously showed CF and CF‐inhibiting (CFI) activities, whereas the IgG(T) class showed only CFI activity. The IgG(T) class could exert CFI activity only when it had been reacted with the EIA antigen before addition of the reference CF serum and complement. In contrast, the IgG class converted the CF‐active reference serum into a non‐CF‐reactive one irrespective of whether it was simultaneously reacted with the EIA antigen and the reference CF serum, whether it was added to the reaction mixture of the EIA antigen and the reference CF serum, or whether it was sensitized with the EIA antigen before addition of the reference CF serum. Inhibitory activities of the IgG and IgG(T) classes seemed to be different from each other in their reaction pattern as far as tested under our experimental conditions. Their CFI activities seemed to be specific for EIA, being negative in CFI activity in reaction with other antigens.
Studies on the Establishment of Multi‐Drug‐Resistant Strain BIO‐4R of Streptococcus faecalis in the Intestinal Tract of Germ‐Free Mice
Authors: Hara Noriyoshi
Published: November 2013

Germ‐free ICR mice were mono‐ or dicontaminated with a multi‐drug‐resistant strain BIO‐4R of Streptococcus faecalis (BIO‐4R) and Escherichia coli 026 : K60 (E. coli) and administered aminobenzyl penicillin (ABPC). BIO‐4R was established in the intestinal tract at a level of 108 viable cells per gram of stool on the fourth day following oral inoculation and the BIO‐4R population was stably maintained thereafter. The drug resistance of BIO‐4R remained unchanged in the intestinal tract of gnotobiotes throughout the experiment. Highly resistant cells of E. coli were isolated from the feces of some dicontaminated mice after ABPC administration. However, it seems that the high resistance of these E. coli is not due to the transfer of resistance of BIO‐4R to E. coli. All animals given a large amount of BIO‐4R (108 cells) per os survived throughout the study period of two weeks without symptoms.
Lethal Effect of Fresh Sea Water on Vibrio parahaemolyticus and Isolation of Bdellovibrio Parasitic against the Organism
Authors: Miyamoto Saburo , Kuroda Koichi
Published: November 2013

Halophilic Bdellovibrio, which is parasitic and lytic to Vibrio parahaemolyticus, was isolated from fresh sea water in the winter. It had a lethal effect on V. parahaemolyticus. The optimum temperature for multiplication ranged from 25 C to 30 C and growth was not observed at 35 C. Plaque numbers of the isolate reached a maximum in 17 hr under conditions of shaking at 25 C in autoclaved sea water supplemented with V. parahaemolyticus cells, and were as high as ten times the number of host cells. With respect to the host‐suspended medium, the isolate multiplied in natural sea water ten times more than in Herbst's artificial sea water but did not grow in saline. V. parahaemolyticus, Vibrio alginolyticus and several species in the Vibrio genus were susceptible to the parasite on the basis of plaque formation but Escherichia coli and Staphylococcus aureus were not.
Studies of Mycobacterium lepraemurium in Cell Culture
Authors: Matsuo Yoshiyasu
Published: November 2013

A serially diluted bacterial suspension of the Kurume‐42 strain of Mycobacterium lepraemurium maintained for 1255 days in a mouse foot pad (MFP) cell culture was inoculated in mice subcutaneously. The ID50 value was estimated at more than 10.7 and less than 85 organisms, indicating that pathogenicity of the organism had been maintained well in a long‐term cell culture. The cells infected and maintained for a long period in the cell culture showed all the stages of cell mitosis. This suggests that the bacterial increase in cell cultures of M. lepraemurium is not only due to rephagocytosis of the bacilli released from the infected cells but also to a constant intracellular growth cycle of the bacilli accompanied by mitosis of the infected cells. In acid phosphatase activity, no appreciable differences were noted between the infected and uninfected cells as far as the present cell culture system was concerned. Most of the bacilli within the cells were ultrastructurally normal. Solid bacilli in phagosomes were surrounded by less electron‐dense clear zones.
Relationship between the Molecular Size of Poly I·Poly C and Its Biological Activity1
Authors: Machida Haruhiko , Kuninaka Akira , Yoshino Hiroshi
Published: November 2013

Seven polyinosinic·polycytidylic acid (poly I·poly C) preparations, ranging from 4.2 S to 21.2 S, prepared from various sizes of polyinosinate and polycytidylate, were examined for toxicity and interferon‐inducing activity in mice. The increase in size of poly I·poly C was accompanied by increases both in the maximal amount of interferon produced and in the length of persistence of a high level of interferon in plasma. Toxicity of poly I·poly C was proportional to the molecular size within the range of 8 S to 16 S. The amount of interferon induced by 1/5 LD50 of poly I·poly C depended on the size of the inducer, being increasingly lower with progressively smaller sizes. Next, activities of poly I·poly C in culture cells were examined. The resistance‐inducing activity of poly I·poly C in primary chick embryo cells (CEC) increased with the size of the inducer (4.2 S to 11.6 S), whereas the activity in L cells was not so markedly dependent upon its molecular size as in CEC. In the presence of calf serum during induction of resistance the activity was lowered. The activities of preparations with small molecular sizes were affected by calf serum more markedly than those of large molecular sizes. The interferon‐inducing activity in RK13 was not appreciably influenced by the size of poly I·poly C, especially in the presence of DEAE‐dextran, while the activity in L cells was markedly dependent upon the size of the inducer. These results suggest that the influence of the molecular size of poly I·poly C upon the resistance‐inducing and interferon‐inducing activities varies among different kinds of cells, and alters in the presence of serum or DEAE‐dextran.
Induction of Antiviral Activity In Vivo and In Vitro by Human Placenta Ribonucleic Acid Treated with Nitrous Acid
Authors: Arimura Hirofumi
Published: November 2013

Induction of antiviral activity and interferon by human placenta ribonucleic acid deaminated with sodium nitrite (NO2‐RNA) was studied in vitro and in vivo. (1) Viral multiplication in diploid cells from human kidney (HK cells) was depressed by pretreatment with NO2‐RNA, but not by pretreatment with the original placenta RNA. (2) NO2‐RNA showed an interferon‐inducing activity in rabbits and mice. (3) NO2‐RNA sedimenting in 18 S and 28 S regions showed a higher antiviral activity than that sedimenting in 4 S region.
Immunochemical Studies on Bacterial Blood Group Substances
Authors: Kishi Koichiro , Iseki Shoei
Published: November 2013

Blood group H‐active polysaccharide has been prepared from “smooth” strain Escherichia coli 2B‐V by Freeman's method. a‐Fucosidase derived from Bacillus fulminans caused the liberation of fucose from this polysaccharide, together with concomitant loss of blood group H activity. The results of quantitative microanalysis, borohydride reduction, the Morgan‐Elson reaction and enzymic hydrolysis with β‐galactosidase using isolated oligosaccharides obtained by partial acid hydrolysis indicated that the O‐specific side chain of the polysaccharide has a pentasaccharide unit which is β‐d‐Gal‐(1→3)‐d‐GalNAc‐(1→3)‐d‐GalNAc‐Fuc with a D‐glucose residue bound at some undetermined point on this structure. It was considered that terminal non‐reducing fucose of the polysaccharide was liberated by partial acid hydrolysis.
Separation of a Water‐Soluble Adjuvant (MAF3) from Delipidated Cells of Mycobacterium tuberculosis Strain Aoyama B by Hydrogenolysis and Gel Filtration
Authors: Kumazawa Yoshio , Shibusawa Atsuko , Suzuki Takakazu , Mizunoe Kimifusa
Published: November 2013

A water‐extract from hydrogenolyzed cells of Mycobacterium tuberculosis strain Aoyama B was separated into four portions (F‐1 to F‐4 fractions) by gel filtration with a Sephadex G‐100 column. The third peak (called MAF3) eluted from the column was the most adjuvant‐active fraction. The molecular weight determined by gel filtration was around 16 000 daltons. MAF3 consisted of heteropolymer(s) composed of approximately 76 to 79% neutral sugars (Ara, Gal, Man, and Glc) and 19% mucopeptide (MurN, GlcN, Glu, Ala, Dpm, Gly, Asp, Thr, Ser, Leu, Lys, Arg, His, Pro, Tyr, and Phe). The adjuvanticities of MAF3 and other fractions in water‐in‐oil emulsion were estimated by the enhancing effect on immune response to egg albumin (EA) in guinea pigs. MAF3 stimulated the production of humoral antibodies, particularly IgG2 antibody specific to the antigen, and induced delayed type hypersensitivity against EA in the skin and cornea of antigen‐primed guinea pigs. These adjuvanticities of MAF3 were similar to the characteristics of mycobacterial cell wall in Freund's complete adjuvant.
Inhibition by Thiopeptin of Bacterial Protein Synthesis
Authors: Liou Yei‐Fei , Kinoshita Tadatoshi , Tanaka Nobuo
Published: November 2013

Thiopeptin, a sulfur‐containing antibiotic, was found to inhibit protein synthesis in a bacterial ribosomal system. The pretreatment of ribosomal subunits with the antibiotic revealed that thiopeptin may act on the 50 S ribosomal subunit. The elongation of peptide chain on the ribosome is more profoundly blocked by the antibiotic than the initiation of protein synthesis. It was demonstrated that thiopeptin inhibits elongation factor (EF)‐Tu‐dependent GTP hydrolysis and binding of aminoacyl‐tRNA to the ribosome. The peptidyl transferase‐catalyzed puromycin reaction is not significantly affected by the antibiotic. Thiopeptin inhibits EF‐G‐associated GTPase reaction, and translocation of peptidyl‐tRNA and mRNA from the acceptor site to the donor site. Protein synthesis in ribosomal systems, obtained from rat liver and rabbit reticulocytes are insensitive to the antibiotic.
Tolerance and Other Biological Properties of Vibrio parahaemolyticus Endotoxins
Authors: Sochard M.R. , Colwell R.R.
Published: November 2013

Biological properties of endotoxins prepared from three strains of Vibrio parahaemolyticus were compared with reference to commercially prepared Salmonella typhi endotoxin. Endotoxin assays performed in rabbits included dermal Shwartzman reactivity, pyrogenicity, heat stability, and ability to induce tolerance as well as cross‐tolerance. Mice were used for endotoxin LD50 determinations. Results showed V. parahaemolyticus endotoxins were similar to that of S. typhi strain O901. Induction of tolerance to V. parahaemolyticus strain 11590 endotoxin resulted in complete cross‐tolerance to S. typhi endotoxin, and vice versa. Partial cross‐tolerance to S. typhi endotoxin was demonstrated with rabbits rendered tolerant to endotoxin from V. parahaemolyticus strains Sak‐3 and FC1011. Absorption spectra, nitrogen, phosphorus and carbohydrate analyses revealed additional similarities between endotoxins from V. parahaemolyticus and endotoxin from a member of the Enterobacteriaceae.
Inhibition of Rat Dental Caries by Dextranase from a Strain of Spicaria violacea
Authors: Hamada Shigeyuki , Ooshima Takashi , Masuda Norio , Mizuno Junn , Sobue Shizuo
Published: November 2013

Dextranase AD17 obtained from a culture liquor of a strain of Spicaria violacea was assessed for its ability to inhibit the development of dental caries in conventional Sprague‐Dawley rats which had been infected with one of the Streptococcus mutans strains, MT6R (serotype c), OMZ 176R (d), or MT‐703R (e). These experiments showed that caries was significantly inhibited when rats were given cariogenic diet # 2000 and drinking water containing AD17 at a concentration of 10 units/g, as compared to control rats not given dextranase. The inhibitory effects of AD17 were more prominent in smooth surface caries than in total caries. AD17 had a tendency to retard both the establishment of inoculated S. mutans and plaque deposition on tooth surfaces. However, S. mutans could be implanted in the rat oral cavity after repeated inoculation of the bacteria, even in the presence of AD17. These results suggest that the anticaries activity of AD17 is due to not only inhibition of adherence of S. mutans cells on tooth surfaces but also to physicochemical changes of dental plaque formed under the enzymatic action of AD17. Preliminary histopathological examination showed that AD17 had no significant toxicity in rats.
Enhanced Growth and Plaquing of Rabies Virus in Static Chick Embryo Cell Culture
Authors: Serine Noriyoshi , Yoshino Kamesaburo
Published: November 2013

The 7‐day egg passage line of HEP Flury strain of rabies virus was inoculated to primary chick embryo (CE) cells prepared in different ways to compare efficiencies of viral growth and plaquing. Special care to minimize cellular damage due to trypsin at the step of monodispersion and sowing a comparatively large number of cells for monolayer preparation were required for rabies plaquing, whereas such cares were not necessary for plaquing of vesicular stomatitis virus. Plaque number and size were increased by incorporation of a high concentration of thymidine into cell growth medium. Various other means to produce a static state of CE cells were tested, and a maximal plaquing efficiency was obtained when dishes receiving a massive number of dispersed cells in MEM plus 1% calf serum were incubated at 37 C for 1 day without any buffering for monolayer preparation and postinfection incubation was done at 32 C in a CO2‐incubator. Bottle cultures of CE cells prepared in a similar manner, when infected with HEP Flury virus, yielded a markedly higher titer of virus than CE cells prepared by our previous standard method.
Viral DNA Synthesis In Vitro with the Inclusions Isolated from Adenovirus 12‐Infected Cells
Authors: Ariga Hiroyoshi , Shimojo Hiroto , Yamaguchi Kazuyoshi
Published: November 2013

A fraction defined as the inclusions was isolated by banding in CsCl gradients from nuclei of adenovirus 12‐infected KB cells. When examined by electron microscopy, the isolated inclusions were relatively homogeneous, finely granular materials of moderate electron density, possibly representing the disintegrated type II or IV inclusions. The conditions of endogenous DNA synthesis in vitro with the inclusions were determined. The product of DNA synthesis in vitro with the inclusions was mainly viral and scarcely cellular, as revealed by DNA‐DNA hybridization and methylated albumin kieselgur column chromatography. However, viral DNA synthesized in vitro was smaller (18 S, 22 S) than viral DNA in virions (31 S, 34 S) in neutral and alkaline sucrose gradients. Effects of various treatment of the inclusions on the DNA‐synthesizing activity showed that phospholipase C inhibited the activity efficiently. The in vitro DNA synthesis was stimulated by addition of the cytoplasmic extract from adenovirus 12‐infected cells and not that from unifected cells. The analysis of the composition of the inclusions showed that the inclusions contained DNA, protein, phospholipid and a small amount of RNA and carbohydrate.
Properties of Virus Isolated from an Epidemic of Hand‐Foot‐and‐Mouth Disease in 1973 in the City of Matsue
Authors: Gobara Fumiko , Itagaki Asao , Ito Yoshihiro , Saito Koichi , Katsumoto Tetsuo , Kimura Genki
Published: November 2013

The virus strains isolated from clinical cases in an epidemic of hand‐foot‐and‐mouth disease in Matsue in 1973 were characterized and its properties were compared with those of the Coxsackievirus group A type 16 (CA 16) prototype strain. The virus isolated in 1973 was similar to CA16 prototype virus with respect to morphology in electron microscopy, resistance to ether and capability to replicate in medium containing fluorodeoxyuridine. Cross neutralization tests using guinea‐pig and horse antisera revealed that there was little or no detectable common antigen between the two viruses. The two viruses also differed in heat stability of virion infectivity: the 1973‐viruses were much more resistant to heat than the prototype virus. Under one‐step growth conditions in Vero cell cultures, growth rate and virus yield of the 1973‐viruses were lower than those of CA16, but this property was independent of incubation temperatures, pH of culture medium and other culture conditions. Several other differences in property between the 2 strains are also described. It is concluded that the epidemic in 1973 was caused by a virus whose properties differed greatly from those of the CA16 prototype.
Speculation and C2 Toxin Production by Clostridium botulinum Type C Strains Producing No C1 Toxin
Authors: Nakamura Shinichi , Serikawa Toshihiko , Yamakawa Kiyotaka , Nishida Shoki , Kozaki Shunji , Sakaguchi Genji
Published: November 2013

All of the 8 strains that were previously assumed to be nontoxigenic Clostridium botulinum type C were re‐examined for their toxigenicity and were demonstrated by trypsinization of the culture filtrates to produce C2 toxin under improved cultural conditions. One per cent glucose added to trypticase peptone medium enhanced C2 toxin production. The larger the spore population, the higher the C2 toxicity and when spore population was smaller than a level of 104/ml, no C2 toxicity was demonstrated. The C2 toxin was produced only during sporulation and not during vegetative growth.
Serotype‐Dependent Inhibition of Glucan Synthesis and Cell Adherence of Streptococcus mutans by Antibody against Glucosyltransferase of Serotype e S. mutans
Authors: Hamada Shigeyuki , Tai Stella , Slade Hutton D.
Published: November 2013

A crude glucosyltransferase (GTase) preparation was obtained from the culture supernatant of Streptococcus mutans strain MT703 (serotype e) by 50% ammonium sulphate precipitation. Antiserum specific against the GTase was prepared by immunizing rabbits intramuscularly with the GTase in Freund incomplete adjuvant, followed by GTase without adjuvant intravenously. Gamma globulin fractions of the antiserum and normal serum were partially purified by 1/3 saturated ammonium sulphate precipitation. The antibody strongly inhibited the GTase activity (>90%) of type c, e and f S. mutans, whereas the GTase of types a, d and g was not affected by the antibody. The GTase from type b S. mutans was slightly inhibited. The adherence of viable cells of type c, e, and f S. mutans to a glass surface due to synthesis of glucan by the cell‐associated GTase was also significantly inhibited by the antibody to the enzyme. These results suggest that type c, e, and f and types a, d, and g S. mutans can be separated into two major groups in terms of the immunological relationship of GTase.
Bacteriocinogeny and Antibiotic Resistance of Naturally Occurring Strains of Streptococcus mutans
Authors: Katayama Ario , Ishikawa Etsuko , Arai Toshihiko
Published: November 2013

We studied bacteriocinogenies and antibiotic resistances of naturally occurring strains of Streptococcus mutans isolated from different sources. It was found that naturally occurring strains from humans and rats were distinguished by their bacteriocin susceptibilities and production. We had a few incidences of tetracycline resistant strains only among those isolated from rats. It is quite interesting that none of the resistant strains we collected produced bacteriocins.
Investigation on an Intracellular Hemolytic Agent of Vibrio parahaemolyticus: Lipid Fraction of Dried Cells
Authors: Iida Atsushi , Takagi Mitsuzo
Published: November 2013

The dried cells of two strains of Vibrio parahaemolyticus were fractionated by extracting first with water and then with organic solvents. The hemolytic activity of the fractions was determined, and some of them were assayed for their effect in mice. The hemolytic agent present in the water‐insoluble fraction was extractable in organic solvents such as 70% aqueous ethanol, chloroform–methanol–water (1:2:0.8) and acetone. The extracts showed no toxic effect in mice after intraperitoneal inoculation. No hemolytic activity was observed in the remaining cell residue, which bore the toxicity.
Effects of Age and Starvation on the Gastrointestinal Microflora and the Heat Resistance of Fecal Bacteria in Rats
Authors: Morishita Yoshiyuki , Miyaki Komei
Published: November 2013

The microflora of the gastrointestinal tract in rats 1 day to 100 weeks old, of the cecal contents and wall in starved rats, and of heat‐treated feces of normal rats was determined by cultural examination. Streptococci, staphylococci, lactobacilli, actinobacilli, and coliforms colonized the tract during the 1st week of life. Bacteroidaceae, veillonellae, catenabacteria (composed of eubacteria and anaerobic lactobacilli), clostridia, bifidobacteria, anaerobic gram‐positive cocci, fusiform bacteria, curved rods, and spirochetes appeared when the rats were 2 to 4 weeks old. Yeasts were slower in colonizing the tract than any other organism. Dramatic changes occurred in the microflora of rats 2 to 4 weeks of age. There was a time lag between the changes in enterococcal and coliform populations. The enterococcal population was depressed over a period from 2 to 6 weeks of age. Bifidobacteria showed a larger population at 4 to 9 weeks than at any other age. The microflora of the stomach was the same as that of the small intestine, with some exceptions. It differed markedly from that of the cecum. The ratio of total aerobic count to anaerobic count gradually increased in the stomach, but decreased in the cecum, with advance in age. The microorganisms distributed in the tract could be divided roughly into 3 types. The population of each organism, except spirochetes, in the cecal wall was approximately 1/1,000 of that in the cecal contents. One of the 2 types of spirochetes was found only in the cecal wall and in a high incidence, forming a large population. In rats starved for 48 hr, coliforms, Proteus spp., anaerobic gram‐positive cocci, Clostridia, and some bacteroidaceae showed an increase in population in the cecum, but lactobacilli, veillonellae, and spirochetes decreased. The major organisms cultured from the heat‐treated feces were fusiform and curved bacteria, some members of Bacillus, minor anaerobic cocci, and straight rods.
Partial Purification, Physicochemical Characterization and Biological Function of a Rosette‐Decreasing Factor (RDF) Extracted from Guinea Pig Thymus
Authors: Mabuchi Ayako , Kanamori Sachio , Yokomuro Kozo , Kimura Yoshitami
Published: November 2013

A factor that decreases rosette formation between guinea pig T‐cells and rabbit red blood cells (RRBC) was extracted from the thymus of the guinea pig.

The active factor could be extracted from the spleen as well as the thymus, but not from the liver or kidney. The active factor of the thymic extract was found in the precipitates produced by 80% saturated ammonium sulfate and it was separated from the water‐soluble fraction of the precipitates. The molecular weight of the partially purified substance was estimated to range between 10,000 and 30,000 by filtration through a diaflow membrane.

From the studies on physicochemical characterization, it might be a heat‐resistant basic peptide probably bound to a ribonucleotide moiety. This factor reduced rosette formation between RRBC and guinea pig T‐cells, but did not reduce erythrocyte‐antibody‐complement rosette formation.

This factor also inhibited mitogen (concanavalin A, phytohemagglutinin‐P)‐induced DNA synthesis of guinea pig lymphocytes and antigen‐induced DNA synthesis of sensitized guinea pig lymphocytes.
Characterization of Multi‐Layered Membrane Generated by Rabies Virus
Authors: Tanabe Kiyokatsu , Matsumoto Seiichi
Published: November 2013

Electron microscopy revealed multi‐layered membranes within the cytoplasmic inclusion (accumulation of nucleocapsids) produced by rabies virus. When infected BHK cells were maintained at 31 C, an enhancement in production of these membranes occurred in approximately 60% of inclusion‐containing cells. Multi‐layered membranes were composed of an alternate array of two different layers; an electron‐dense, thin membrane and a less dense layer which was thicker. SDS‐polyacrylamide gel electrophoresis and immune electron microscopy of isolated multi‐layered membrane preparations demonstrated that the structures contained viral G and M2 polypeptides. Our observations suggest that these membranous structures are not a degenerative product of rabies virus infection but rather are related to the replication of viral envelope constituents, although they represent themselves to be an abortive form of viral assembly.
Heterogeneous RNA of the Prague Strain of Rous Sarcoma Virus Caused by Undiluted Passages
Authors: Nasu Masao , Yutsudo Masuo , Toyoshima Kumao
Published: November 2013

The size of genomic RNA in PR‐RSV A passaged in chick embryo fibroblasts (CEF) or quail embryo fibroblasts (QEF) was determined by gel electrophoresis. The results showed that 3 undiluted passages resulted in heterogeneity of RNA. The heterogeneity of the smaller incomplete RNAs in the virus stock was decreased by diluted passage or cloning, but RNA of the b subunit size and a subunit RNA of complete genome size were relatively stable.

These heterogeneous RNAs were characterized by hybridization analysis. The RNAs from 4 peaks hybridized with both cDNAtotal and cDNAsrc to appreciable extents, indicating that they were derived from viral RNA and that at least some of them contained the src sequence.

This finding and the failure to isolate a td mutant from the undiluted‐passaged virus stock or from some subclones that had a and b subunits of RNA indicate that the td virus was only a minor constituent of the incomplete virus population caused by undiluted passages.

Some viruses with incomplete RNA in the virus stock could produce foci with the aid of td B77 or RAV‐60. The emergence of rd viruses by undiluted passages was indicated.
“Tween Egg Medium” for Isolating Mycobacteria from Sputum Specimens
Authors: Tsukamura Michio , Toyama Haruo , Fukaya Yuji
Published: November 2013

‘Tween egg medium,’ in which Tween 80 was used as a carbon source, showed a higher rate of positive cultures than Ogawa egg medium in isolation of mycobacteria from sputum specimens.
Trypsin Action on the Growth of Sendai Virus in Tissue Culture Cells
Authors: Muramatsu Michiko , Homma Morio
Published: November 2013

A trypsin‐like protease which is responsible for activation of Sendai virus was found in the chorioallantoic fluid (CAF) of embryonated chicken eggs. Treatment of the inactive form of Sendai virus, grown in LLC‐MK2 cells, with CAF enhanced both hemolytic activity and infectivity for the cells. Soybean trypsin inhibitor restrained the enhancing activity of CAF. These results indicate that CAF contains a trypsin‐like protease which activates the inactive form of Sendai virus.

The activation was strongly inhibited by phenylmethylsulfonylfluoride, ethyl‐enediaminetetraacetate, antipain, and leupeptin but not by tosyllysylchloromethylketone, suggesting that the activating enzyme in CAF is a protease similar to but not identical with trypsin. The inactive form of the virion was produced in ovo when the seed virus was inoculated along with antipain or leupeptin.

In deembryonated chicken eggs in which CAF was substituted for a culture medium, multiple cycle growth occurred, but not when soybean trypsin inhibitor was present. These observations indicate that some activating enzyme, possibly the same one as found in CAF, was secreted from the chorioallantoic membrane.
Transformation of Mouse Peritoneal Macrophages and Bone Marrow Cells by Simian Virus 40
Authors: Takayama Hisao
Published: November 2013

A fraction of cultured mouse peritoneal macrophages and bone marrow cells acquired the ability to divide after infection by simian virus 40 (SV40). Two types of transformed lines were obtained. Most transformants isolated 40–60 days after infection did not display macrophage specific properties such as ingestion of opsonized red blood cells, possession of Fc receptors and complement receptors, and acid phosphatase activity throughout the whole culture phase. Cells of the transformed lines isolated by trypsin selection 2–6 months after infection displayed these properties when the cell density became high and cell growth was arrested. In the cells of the latter type of transformed lines, SV40 T‐antigen was intensely demonstrated by immunofluorescence in the growing phase, but weakly or not at all in the stationary phase. It is suggested that the reversion to the phenotype of the progenitor (expression of macrophage specific functions) depends on the physiological state of the culture; however, it is uncertain whether the development of the macrophage functions is directly related to the SV40 T‐antigen.
Intestinal Microflora and Bile Acids
Authors: Sakai Kosei , Marino Takashi , Kawai Yasuo , Mutai Masahiko
Published: November 2013

The population levels of intestinal microflora and bile acid composition in the digestive tract were examined in rats fed bile acids to determine the relationships between gastrointestinal microflora and the host. The population level of Bacteroides was increased in the ceca of rats fed cholic acid or deoxycholic acid. In the ileum, the concentration of conjugated bile acid in rats fed cholesterol, cholic acid, hyodeoxycholic acid or lithocholic acid was higher than that in control rats, and was very low in ceca and feces of all the rats. The concentration of total free bile acid was much higher in the ceca than in the ilea of rats fed hyodeoxycholic acid or lithocholic acid. Cholic acid and deoxycholic acid were found in the ilea, ceca and feces of the cholic acid‐fed rats. In the deoxycholic acid‐fed rats, cholic acid was localized in the ileum. 7‐Ketodeoxycholic acid was also found in the ceca of the cholic acid‐fed rats. 12‐Ketolithocholic acid was found in the feces of rats fed cholic acid or deoxycholic acid. 3‐Ketocholanic acid was found in some samples from the lithocholic acid‐fed rats. Therefore, some kinds of bile acids influence the population levels of gastrointestinal microflora and bile acid composition in the intestine.
Effects of a Bacteriocin from Mycobacterium smegmatis on BALB/3T3 and Simian Virus 40‐Transformed BALB/c Mouse Cells
Authors: Saito Hajime , Watanabe Takashi
Published: November 2013

The effects of a bacteriocin from Mycobacterium smegmatis ATCC 14468 on simian virus 40‐transformed BALB/c mouse cells (mKS‐A TU‐7 cells) and non‐transformed BALB/3T3 cells originating from the BALB/c mouse strain were studied. The percentage of nigrosin‐unstained (viable) cells in the bacteriocin‐treated mKS‐A TU‐7 cells decreased time‐dependently with an increase in the bacteriocin activity. There was a time‐dependent decrease in the bacteriocin activity after treatment with the cell membrane preparation from mKS‐A TU‐7 cells. There was no apparent effect of the bacteriocin on the viability of non‐transformed BALB/3T3 cells. Wheat germ agglutinin blocked the toxic effect of bacteriocin on mKS‐A TU‐7 cells. These results indicate that the higher sensitivity and binding capacity of the tumor cells to the bacteriocin is probably due to the presence of a larger amount of N‐acetyl‐glucosamine or closely related sugar residues with a high affinity for bacteriocin, as compared with normal cells. The bacteriocin produced morphological alterations and inhibition of synthesis of ribonucleic acid, deoxyribonucleic acid and protein in the transformed but not in the nontransformed cells.
Effect of Lactobacilli on Urinary Indican Excretion in Gnotobiotic Rats and in Man
Authors: Tohyama Kiyoshi , Kobayashi Yoichi , Kan Tatsuhiko , Yazawa Kazunaga , Terashima Tsuneo , Mutai Masahiko
Published: November 2013

The effect of Lactobacillus species on urinary indican excretion in two types of gnotobiotic rats (GBH‐9 and GB‐5) and in man was studied. L. salivarius, L. plantarum and L. casei colonized in the cecum and colon of both types of rats at levels of 107 to 108 per gram of contents following one‐dose oral administratron and caused a significant reduction in urinary indican excretion, whereas two strains of L. acidophilus which colonized at low levels (104 to 105/g) did not reduce indican excretion. Daily feeding of Lactobacillus concentrates caused a further significant reduction in indican excretion which was corrected by tryptophan intake, even in the case of L. acidophilus. There was a clear relationship between fecal lactobacillus counts and urinary indican. A significant reduction of indican secretion was obtained only when the fecal counts of lactobacilli exceeded 107.4/g in rats.

L. casei also had an effect in man, reducing urinary excretion of indican and p‐cresol. Reduction in indican excretion seems to be accompanied by decreasing fecal tryptophanase activity in rats and man. A negative correlation was also obtained between fecal resident lactobacilli and urinary indican in man (r = − 0.532, n = 28, p<0.01). Subjects with lactobacilli at a level of 106/g or more excreted less indican than those harboring the bacteria at levels below 105/g.
Complement Proteins and Macrophages
Authors: Miyama Akio , Kawamoto Yasuko , Ighikawa Hidetaka , Moriyama Takeshi , Okamoto Keinosuke , Takahashi Tetsuomi
Published: November 2013

A kinetic study of factor B secretion by particle‐ingesting macrophages showed that the ingestion of zymosan but not latex lead to an early suppression of factor B secretion followed by a significant enhancement. This dual response was proportional to the number of ingested zymosans. After a suppressive period of 24–48 hr, the ingested zymosan stimulated the synthesis of factor B. The response of acid phosphatase synthesis to ingested zymosan was the same as that of factor B, while the synthesis of lactic dehydrogenase proceeded without an early suppression. These results suggest that factor B should be considered an enzyme released into the lysosomes of macrophages.
Anticomplement Immunofluorescence for the Titration of Antibody to Varicella‐Zoster Virus
Authors: Shigeta Shiro , Baba Masanori , Ogata Masahiro , Iijima Susumu , Murai Chiaki
Published: November 2013

Anticomplement immunofluorescence (ACIF) was tested for its use for the titration of antibody against varicella‐zoster virus (VZV). ACIF antibody responses of patients with VZV infection were specific for VZV antigen and heterotypic responses to herpes simplex virus type‐1 and cytomegalovirus antigens were not observed. Comparative studies of ACIF, membrane immunofluorescence (MIF) and indirect immunofluorescence (IF), using acetone‐fixed antigen, were carried out with nonimmune sera and convalescent sera of patients who had recovered from varicella, herpes zoster and Rumsey Hunt disease. Nonspecific staining occurred with some nonimmune sera at a 1:4 dilution in the MIF and IF tests, after freezing and thawing of the serum, but not in the ACIF test. The antibody titers in convalescent sera agreed well in these three methods and the highest titer was obtained by MIF. The titers in ACIF and IF were similar but the ACIF antibody decreased earlier than the IF antibody during convalescence. On the other hand there was a discrepancy between the titers of ACIF and those of MIF and IF antibody in the sera of healthy adults, all sera with titers higher than 10 in the MIF and IF tests had titers below 10 in the ACIF test. The average titer of ACIF antibody declined to less than 10 with increasing age (13 to more than 20 years), whereas the MIF antibody increased during the same period of life.
Protein Synthesis in the Isolated Forespores from Sporulating Cells of Bacillus subtilis
Authors: Watabe Kazuhito , Iida Seiichi , Nakamura Keiko , Ichikawa Tomio , Kondo Masaomi
Published: November 2013

Developing forespores were isolated from Bacillus subtilis at different stages of sporulation and protein synthesis in the forespore compartment was examined. Pulse‐labeling experiments indicated that [14C]phenylalanine was continuously incorporated into the sporangium throughout sporulation, and at t5 (early stage V of sporulation) 58% of the radioactivity was located in the forespore compartment. Significantly high incorporation of [14C]phenylalanine was observed when the isolated forespores at t5 were incubated with the corresponding mother‐cell cytoplasmic fraction or an amino acid mixture. About 73% of the radioactivity incorporated into the isolated forespore at t5 was found in the cytoplasmic fraction and 26% in the membranous fraction. Analysis by sodium dodecyl sulfate‐gel electrophoresis showed that the 14C‐labeled cytoplasmic protein had a molecular weight of about 20,000, and that a protein having the same molecular weight was present in the t5 forespore as a slight protein band and also in the mature spore as a clear protein band. Gel electrophoresis also revealed that the 14C‐labeled membranous‐soluble protein (prepared by solubilization with detergents) had broad peaks with molecular weights of about 74,000, 33,000, 20,000, and 12,000.
Isolation of Antigenically Active Components from Leptospiral Serovar‐Specific Lipopoly‐saccharide Antigen by Alkaline Treatment
Authors: Tsuji Masayoshi , Kawaoka Yoshihiro , Naiki Masaharu , Yanagawa Ryo
Published: November 2013

The serovar‐specific main antigen (TM antigen) of Leptospira interrogans serovar canicola, which has lipopolysaccharide properties, was treated with 0.1 n sodium hydroxide. This treatment degraded the antigen into two major antigenic components, one of high and one of low molecular weight. The component with the lower molecular weight (approximately 4,000 daltons) consisted mainly of carbohydrates, having lost almost all of the fatty acid and protein components of the original antigen. Although the substance lacked immunoprecipitable activity, it continued to show serovar‐specific inhibitory potency in a radioimmunoassay system as well as in a microscopic immunoagglutination reaction of the organisms. The antigenic activity of the compound was also reduced by periodate oxidation as was that of the TM antigen. A component with the same chemical and physicochemical properties was also produced by alkaline treatment from a different serotype TM antigen (serovar kremastos Kyoto), but it showed no antigenic activity.
Spontaneously Induced Suppressor Cells In Vitro: Nonspecific Suppression of In Vitro Antibody Formation
Authors: Kodama Kazue , Kurashige Satonori , Mitsuhashi Susumu
Published: November 2013

Nonspecific suppressor cells were induced during in vitro culture of normal mouse spleen cells (SPC) using the Marbrook culture system. The suppressor cells inhibited both the primary and secondary antibody‐formation responses antigen nonspecifically in vitro, and both IgM‐ and IgG‐responses were inhibited. The supernatants from suppressive precultured cells were not suppressive. The suppressor cells also inhibited the response of allogeneic SPC beyond H‐2 compatibility. The induction of the suppressor cells did not require the presence of antigen but required fetal calf serum (FCS) or both FCS and 2‐mercaptoethanol (2‐ME). The suppressor cells were generated from the nylon‐wool adherent, radiation‐sensitive T cell population. On the other hand, the suppressor cells were nylon‐wool nonadherent, relatively radiation‐sensitive T cells.

Actively antibody‐producing cells were not affected by the suppressor cells. The suppressor cells inhibited the mitogenic responses of normal SPC to phytohemagglutinin‐P (PHA), bacterial lipopolysaccharide (LPS) and concanavalin A (Con A). The suppressor cells themselves inhibited the growth of EL4 cells (T‐cell leukemia of C57BL/6 mouse origin) and MOPCll cells (B cells, plasmacytoma of BALB/c mouse origin) even at a low effector‐to‐target cell ratio (E:T ratio = 1:1), but did not kill these tumor cells. These results indicate that the target cells of the suppressor cells are both T and B cells, and that the mechanism of action of the suppression is either inhibition of proliferation or inhibition of early events in the course of the immune response.
Expression of Herpes Simplex Virus Type‐Common Surface Antigens in Clonal Cells of an HSV Type 2‐Transformed Line: Effect of Adriamycin
Authors: Kako Daijiro , Ohnishi Yoshinari , Kimura Susumu
Published: November 2013

The expression of herpes simplex virus (HSV) type‐common surface antigens (CSA) in a representative cell clone (155–4‐03) of hamster cell line 155–4 transformed by HSV type 2 was enhanced by treatment with inhibitors of RNA synthesis [adriamycin (ADM) and daunomycin] but not with inhibitors of DNA synthesis (2‐iododeoxyuridine, bleomycin, mitomycin C and cytosine arabinoside), although all these drugs decreased the number of viable cells to a similar extent. ADM‐enhanced CSA expression in the clone was inhibited by puromycin and 2‐deoxy‐D‐glucose, suggesting that the enhanced expression required both protein synthesis and glycosylation. This enhanced expression was sensitive to protease inhibitors (antipain and p‐nitrophenyl‐p'‐guanidinobenzoate) and procaine, which is known to inhibit trypsin action and the organization of cell membrane‐associated cytoskeletal elements (microfilaments and microtubules). Furthermore, low concentrations of ADM (0.1 μg/ml) and actinomycin D (0.5 μg/ml) enhanced CSA expression additively, but the most effective concentrations of ADM (0.25 μg/ml) and actinomycin D (2 μg/ml) did not. These findings indicate that the two drugs enhance CSA expression in the clone by a common mechanism.
Analysis of the Inhibitory Effect of Canavanine on the Replication of Influenza RI/5+ Virus: II. Interaction of M Protein with the Plasma Membrane
Authors: Aoki Hiizu , Maeno Koichiro , Tsurumi Tatsuya , Takeura Shigeki , Shibata Motohiro , Hamaguchi Michinari , Nagai Yoshiyuki , Sugiura Yasuo
Published: November 2013

When influenza A/RI/5+ virus‐infected cells were incubated in medium to which 2 μg of canavanine (arginine analog) per ml had been added 4 hr after infection, all viral polypeptides were synthesized but the budding‐like process with the appearance of extracellular virus was completely inhibited. The plasma membrane isolated from these cells contained exclusively hemagglutinin (HA), and membrane (M) protein and nucleoprotein (NP) appeared to be associated with the nucleus, in contrast to untreated cells whose plasma membrane contained abundant HA, M protein, and NP. Disruption of canavanine‐treated cells by freeze‐thawing generated a number of hemagglutinating membranous vesicles or fragments containing exclusively HA. By isotope labeling it was found that the M protein synthesized in the presence of canavanine, together with HA and NP, is a canavanine‐substituted polypeptide. It is suggested that canavanine inhibits the formation of the mature envelope of influenza RI/5+, because of the inability of M protein to associate with the plasma membrane.
Purine Metabolic Enzymes in Lymphocytes: II. Adenosine Deaminase and Purine Nucleoside Phosphorylase Activities in the Immune Responses
Authors: Kurashige Satonori , Akuzawa Yuki , Yoshida Toshiharu , Teshima Chisato , Kodama Kazue , Mitsuhashi Susumu
Published: November 2013

ICR mice were immunized with sheep red blood cells (sRBC). Both adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in spleen lymphocytes increased faster than the serum antibody titer and reached a peak one week after the immunization. ADA activity increased significantly in T lymphocytes but not in B lymphocytes collected from the spleens of the immunized mice. A statistically significant increase in PNP activity was found in both T and B lymphocytes from the spleens of the immunized mice.

Spleen lymphocytes collected from ICR mice which had been immunized with mitomycin C‐treated sarcoma 180 (S180) cells one week earlier showed cytotoxic activity against viable S180 cells. Both ADA and PNP activities in spleen lymphocytes of S180‐immunized mice increased significantly, and both activities increased in T lymphocytes prepared from spleens of immunized mice. In contrast, an increase was found in PNP activity but not in ADA activity in B lymphocytes.

These results suggest that an increase in both ADA and PNP activities may be necessary for the T‐cell response in both humoral and cellular immune responses, and that an increase in PNP activity may be necessary for the B‐cell response.
Modulation of Immune Response by Bacterial Lipopolysaccharide (LPS): Roles of Macrophages and T Cells in In Vitro Adjuvant Effect of LPS on Antibody Response to T Cell‐Dependent and T Cell‐Independent Antigens
Authors: Uchiyama Takehiko
Published: November 2013

The roles of macrophages and T cells in the adjuvant effect of lipopolysaccharide (LPS) were studied. In vitro anti‐trinitrophenyl (anti‐TNP) antibody responses to TNP‐Ficoll and TNP‐keyhole limpet hemocyanine (TNP‐KLH) in spleen cells of C57BL/6 mice showed the most enhancement, when LPS was added to cultures at 1 μg/ml 48 hr after culture was started. The responses to these antigens were enhanced markedly by LPS in whole and macrophage‐depleted spleen cells. The enhancement was greater in the latter group than in the former. The adjuvant effect among whole, T cell‐depleted, macrophage‐depleted and both macrophage‐ and T cell‐depleted spleen cells was compared. The response to TNP‐Ficoll was enhanced markedly by LPS in all groups. The enhancement was greater in the latter two groups than in the first two groups. The response to TNP‐KLH was enhanced by LPS strongly in macrophage‐depleted spleen cells, moderately in whole and both macrophage‐ and T cell‐depleted spleen cells, and only slightly in T cell‐depleted spleen cells. Enhancement was restored to T cell‐depleted spleen cells by adding T cells. The response to TNP‐KLH of macrophage‐depleted spleen cells of LPS‐responsive C3H/HeN mice which was enhanced by LPS was suppressed by adding splenic macrophages of C3H/HeN mice, but not of LPS‐nonresponsive C3H/HeJ mice. The response to TNP‐KLH of macrophage‐depleted spleen cells of C3H/HeJ mice was not enhanced by LPS, irrespective of the addition of macrophages of C3H/HeN mice. The results indicate that B cells are activated directly by LPS, and T cells enhance and macrophages suppress the adjuvant effect of LPS.
Purification of Rickettsia tsutsugamushi by Percoll Density Gradient Centrifugation
Authors: Tamura Akira , Urakami Hiroshi , Tsuruhara Takashi
Published: November 2013

Purification of Rickettsia tsutsugamushi has been achieved by Percoll density gradient centrifugation. The microorganisms purified showed good retention of infectivity and intracellular morphology. Budding rickettsiae in the egressing stage and intracellular rickettsiae in the multiplying process were harvested separately and purified by this technique. In electron microscopic observations, the intracellular rickettsiae obtained were surrounded with double membrane‐layers of cell wall and cell membrane, and the budding rickettsiae were enveloped with an additional outermost membrane which may have originated from host cell membrane obtained in the budding process.