bluuebunny/crossref_metadata_2025 huggingface dataset:
Appearance of Enterotoxigenic Escherichia coli in Piglets with Diarrhea in Connection with Feed Changes
Authors: Shimizu Mikio , Terashima Toyoaki
Published: November 2013

Twelve weaned piglets (3‐week‐old) were divided into three groups according to the time of feed change and observed for diarrhea during the time they were 3 to 8 weeks of age. A total of 553 strains of Escherichia coli were isolated from rectal fecal samples and examined for heat‐labile (LT) and heat‐stable (ST) enterotoxins, pilus antigens (K88, K99, and 987P), hemolysin (Hly), raffinose utilization (Raf) and drug resistance. Enterotoxigenic and/or hemolytic E. coli strains appeared in the rectal feces of 5‐ to 6‐week‐old piglets with diarrhea in connection with feed change and changing temperatures. Most of the isolates showed multiple drug resistance to sulfonamides (Sa), streptomycin (Sm), chloramphenicol (Cp), tetracycline (Tc), kanamycin (Km), spectinomycin (Sp), ampicillin, and/or mercury. Enterotoxigenic E. coli isolates represented four phenotypes: K88+.LT+.Hly+.Raf+.(SaSmCpTcKmSp) (12 strains), K99+.ST+.Raf+.(TcKm) (7 strains), ST+.Raf+.(TcKm) (7 strains), and ST.+(SaSmKm) (25 strains). The drug resistance determinants were transferable concurrently and some of them mobilized the determinants for K88, LT, Hly, and Raf to an E. coli C strain.
Dengue Virus Multiplication in Cultures of Mouse Peritoneal Macrophages: Effects of Macrophage Activators
Authors: Hotta Hak , Hotta Susumu
Published: November 2013

Dengue‐2 virus multiplied in cultures of methylcellulose‐induced peritoneal macrophages of BALB/c mice. The in vitro‐cultivated macrophages from dengue‐1 virus‐immune mice produced larger amounts of dengue‐2 virus than did those from nonimmune controls. The effect of macrophage activators was examined by using nonimmune macrophages. Enhanced virus production was demonstrated in cultures of macrophages pretreated with phytohemagglutinin (PHA) or bacterial lipopolysaccharide (LPS). The number of virus‐infected cells in the pretreated cultures was estimated to be about 0.01% or less of the total macrophages. Continuous treatment of macrophages with PHA before and after virus inoculation brought about the most marked enhancement of dengue‐2 virus multiplication. On the other hand, treatment with concanavalin A or pokeweed mitogen showed little effect on the multiplication of the same virus. Treatment with carrageenan, a specific macrophage blocking agent, markedly suppressed dengue‐2 virus production in both dengue‐1 virus‐immune macrophages and LPS‐treated macrophages. The indirect fluorescent‐antibody (FA) technique revealed dengue‐2 viral antigen in the cytoplasm of infected macrophages, and the FA‐positive macrophages were more numerous in PHA‐treated cultures than in untreated controls. The results obtained are discussed in relation to a possible role of activated monocytes/macrophages in the pathogenesis of dengue hemorrhagic fever.
The Effect of Oxidation‐Reduction Potential on Spore Germination, Outgrowth, and Vegetative Growth of Clostridium tetani, Clostridium butyricum, and Bacillus subtilis
Authors: Hachisuka Yoetsu , Suzuki Ikuo , Morikawa Keisuke , Maeda Shigeaki
Published: November 2013

Spores of Clostridium tetani germinated in liver broth with +580 mV as the starting Eh value, and those of Clostridium butyricum germinated in liver broth with an initial Eh of +400 mV regardless of the presence or absence of a liquid paraffin covering.

Spores of Bacillus subtilis germinated in liver broth with −100 mV as the starting Eh value.

Also, it was found that there are two ranges of starting Eh values for germination and vegetative growth of Cl. tetani, Cl. butyricum, and B. subtilis. In the first range these spores germinated and grew, but in the second range they only germinated and then died without outgrowth.
Appearance of a Cell Surface Antigen Associated with the Activation of Peritoneal Macrophages in Mice
Authors: Akagawa Kiyoko S. , Tokunaga Tohru
Published: November 2013

A relatively large population of murine peritoneal exudate macrophages induced with viable BCG or heat‐killed Corynebacterium parvum was stained by the antiserum prepared against purified gangliotetraosyl ceramide (asialo GM1), while only a small population of peritoneal resident macrophages or peritoneal exudate macrophages induced with proteose peptone was stained. The cytotoxicity assay of those macrophages with anti‐asialo GM1 plus complement supported these results. Peritoneal macrophages induced with BCG or C. parvum showed strong cytotoxicity for EL4 cells in vitro, while resident or peptone‐induced peritoneal macrophages showed no cytotoxicity. BCG‐ or C. parvum‐induced peritoneal cells contained both NK cells and cytotoxic macrophages, and either in vivo or in vitro pretreatment of the cells with anti‐asialo GM1 and complement abolished the activities of both types of cells. Peptone‐induced peritoneal macrophages incubated with lymphokines (LK) or lipopolysaccharide (LPS) were cytotoxic for EL4 cells and contained an increased number of cells stained by anti‐asialo GM1. The cytotoxicity of these in vitro activated macrophages was reduced by treatment with anti‐asialo GM1 plus complement. When peptone‐induced peritoneal macrophages were incubated with LK, the number of cells stained by anti‐Ia antiserum increased, but the number did not increase when the macrophages were incubated with LPS. Pretreatment of peptone‐induced macrophages with anti‐asialo GM1 plus complement did not affect the ability of the macrophages to be activated by LK. These results taken together strongly suggest that the antigen (s) reactive with anti‐asialo GM1 is expressed on the cell surface of cytotoxic peritoneal macrophages in mice.
Hapten‐Specific T Cell Response to Azobenzenearsonate‐N‐Acetyl‐L‐Tyrosine (ABA‐tyr) in Lewis Rats: II. Induction and Suppression of ABA‐Specific Helper Cell Activity for Antibody Production
Authors: Miyagawa Nobuyuki , Lawn Chieh Yuan , Leskowitz Sidney
Published: November 2013

Immunization of Lewis rats with azobenzenearsonate‐N‐acetyl‐l‐tyrosine (ABA‐tyr) in complete Freund's adjuvant (CFA), produces a hapten‐specific helper T cell response measured by an increase in plaque forming cells (PFC) against a different hapten. The response seen is primarily direct (IgM) PFC unless B cells are primed by injection of trinitrophenylated keyhole limpet hemocyanin (TNP‐KLH) prior to immunization with ABA‐tyr. The response requires both ABA and TNP to be on the same carrier molecule which can be as diverse as bovine serum albumin (BSA), poly l‐glutamine‐lysine‐tyrosine (l‐GLT); however, a d‐amino acid polypeptide does not work. The in vitro demonstration of such help was successful only with peritoneal exudate lymphocytes, not spleen or lymph node cells. Repeated pretreatment of rats by intraperitoneal injection of ABA‐tyr in incomplete Freund's adjuvant (IFA) induced an unresponsiveness for helper activity to subsequent immunization with the same antigen in CFA. Passive transfer of lymphoid cells from spleens and lymph nodes from rats pretreated with ABA‐tyr in IFA followed by boosting with ABA‐tyr in CFA induced unresponsiveness to subsequent induction of hapten‐specific help.
Biotransformation of 1‐Nitropyrene in Intestinal Anaerobic Bacteria
Authors: Kinouchi Takemi , Manabe Yoshiki , Wakisaka Kazumi , Ohnishi Yoshinari
Published: November 2013

Mutagenic nitroaromatic compounds have recently been found in photocopies, urban atmosphere, automobile exhaust and wastewater. 1‐Nitropyrene (1‐NP) is readily formed when pyrene, ubiquitous in the environment, is exposed to nitrogen dioxide in the urban atmosphere or in automobile exhaust, and is highly mutagenic, inducing 449 his+ revertants/plate/nmol from Salmonella typhimurium strain TA98 in the absence of S9 fraction in the Salmonella‐microsome test. It is possible to swallow sputum or some food containing 1‐NP and it would come into contact with the normal bacterial flora. We determined the 1‐NP nitroreductase activity in environmental and laboratory bacterial strains. We found that the mutagenicity of 1‐NP mixed with the feces of a healthy man or a culture of anaerobic bacteria was decreased. The product proved to be 1‐aminopyrene (1‐AP), based on its fluorescence spectrum, its mass spectrum, and its characteristic thin layer chromatographic and high performance liquid chromatographic patterns. The 1‐NP nitroreductase activity of aerobic bacteria was low, but crude extracts from the anaerobic bacteria, i.e., Bacteroides fragilis, B. thetaiotaomicron, B. vulgatus, Fusobacterium mortiferum, F. nucleatum, Clostridium perfringens, C. sporogenes, Bifidobacterium adolescentis, B. bifidum, Eubacterium lentum, E. limosum, and Peptostreptococcus anaerobius, all easily converted 1‐NP to 1‐AP and proportionally decreased the mutagenic activity of 1‐NP.
Production by Cultured Spleen Cells of Inflammatory Substances and Other Lymphokines that Mediate Delayed‐Type Hypersensitivity in Mice
Authors: Tamura Shin‐Ichi , Tsuru Sumiaki , Chiba Joe , Kojima Asato
Published: November 2013

In vitro cultivation of normal mouse spleen cells with human serum albumin generated effector cells that mediate the delayed‐type hypersensitivity (DTH) reaction. The cultured cells, when incubated in a serum‐free medium for a further 24 hr, released substances (FPRF) which caused a footpad inflammatory reaction at a maximum of 6 hr after injection into normal syngeneic or allogeneic strains of mice, as well as macrophage migration inhibition factor (MIF) and macrophage activating factor (MAF). The DTH‐effector cells in the culture were fractionated in the low density layers by discontinuous bovine serum albumin density gradient centrifugation. The effector cells in the low density layers were further enriched in the Lyt 1 subpopulation of T cells when fractionated on a fluorescence activated cell sorter. Cells capable of producing the inflammatory substances (FPRF), MIF and MAF were also enriched in the same fraction containing DTH‐effector cells. These results suggest that low density, Lyt 1‐positive T cells mediating the DTH reaction produce FPRF as well as MIF and MAF.
Antigenic Relationship between Downy and Granular Forms of Trichophyton mentagrophytes and Trichophyton rubrum
Authors: Shukla Narmada Prasada , Agarwal Ganga Prasada
Published: November 2013

One strain each of granular and downy varieties of Trichophyton mentagrophytes and two strains each of granular and floccose colonial types of Trichophyton rubrum were used for preparation of antigens. The antigens were analyzed by double diffusion against homologous and heterologous antisera raised in rabbits. A total of 17 distinct antigens were recognized, seven of which were common to the two dermatophytes. Granular and downy varieties of these Trichophyton species produced one additional line of nonidentity. Cross reactions failed to establish any relationship between similar morphologic forms of different dermatophytes. One isolate of T. rubrum, which was originally the floccose culture type but changed into the granular form after artificial infection, showed an antigenic relationship to the floccose culture.
Numerical Classification of 280 Strains of Slowly Growing Mycobacteria
Authors: Tsukamura Michio
Published: November 2013

Numerical classification of 280 strains of slowly growing mycobacteria was carried out by testing each strain for 76 characters. The following fourteen clusters were observed: 1. M. tuberculosis, M. bovis, M. africanum, and M. microti; 2. M. haemophilum; 3. M. ulcerans; 4. M. xenopi; 5. M. kansasii; 6. M. szulgai; 7. M. gordonae; 8. M. gastri; 9. M. avium, M. intracellulare, M. scrofulaceum, and M. asiaticum; 10. M. marinum; 11. M. simiae; 12. M. nonchromogenicum, “M. novum,” M. terrae, and M. triviale; 13. M. malmoense; 14. M. shimoidei.

The clusters composed of M. tuberculosis, M. bovis, M. africanum, and M. microti, of M. avium, M. intracellulare, M. scrofulaceum, and M. asiaticum, and of M. nonchromogenicum, “M. novum,” M. terrae, and M. triviale appeared to be reduced to a single species each. The names having priority for each species should be M. tuberculosis, M. avium, and M. nonchromogenicum, respectively. However, the clusters may, in practice, be called the M. tuberculosis series (complex), the M. avium series (complex), and the M. nonchromogenicum series (complex). The type species of these series are M. tuberculosis, M. avium, and M. nonchromogenicum, respectively. These series were characterized in this study.
Effect of Phorbol Myristate Acetate, a Tumor‐Promoting Agent, on the Growth of Mycobacterium lepraemurium in the Mouse Footpad
Authors: Tomioka Haruaki , Saito Hajime
Published: November 2013

Phorbol myristate acetate (PMA), a potent inflammatory agent with tumor‐promoting activity, was examined for its effect on the growth of Mycobacterium lepraemurium (MLM) in the left hind footpad of mice. When the animals were infected with 104 MLM and received multiple injections of 3 μg of PMA in the infection site weekly during the first 2 months and biweekly thereafter, the growth of the bacilli was markedly enhanced. PMA injection in the infection site resulted in severe footpad swelling accompanied by inflammatory signs such as redness, edema, induration, and sometimes ulcer. Acetic acid, as potent an inflammatory and hyperplastic agent as PMA but without any appreciable tumor‐promoting action, did not stimulate MLM growth when it was injected biweekly in the site of infection with MLM at a dose of 30 μmol per injection. When mice were infected with 108 MLM, proper elimination of bacilli from the infection site was observed during the first 3 months. In this case, multiple injections of PMA in the infection site resulted in the enhancement of the elimination of MLM by host defense mechanisms, although PMA caused as severe inflammation as that observed when MLM infection was produced with a small inoculum (104 MLM). In both cases, dexamethasone was synergistic with, but indomethacin and L‐1‐tosylamide‐2‐phenyl‐ethylchloromethyl ketone were antagonistic to, the effect of PMA.
Cytotoxin Production by Clostridium sordellii Strains
Authors: Nakamura Shinichi , Tanabe Naomi , Yamakawa Kiyotaka , Nishida Shoki
Published: November 2013

A total of 55 strains of Clostridium sordellii, 21 lethal toxin‐positive and 34 lethal toxin‐negative, were tested for cytotoxin production in brain heart infusion medium supplemented with 0.2% Na2HPO4 (m‐BHI) and cooked‐meat‐glucose (CMG) medium using baby hamster kidney (BHK‐21/WI‐2) cells as indicator cells. The m‐BHI medium was preferred to CMG medium and 24 hr of incubation was sufficient for cytotoxin production. Nineteen of the 21 toxigenic strains were also cytotoxigenic, and the strength of the cytotoxigenicity was approximately parallel with that of the lethal toxigenicity. Clostridium difficile antitoxin neutralized C. sordellii cytotoxin and also C. sordellii antitoxin neutralized C. difficile cytotoxin.
Physicochemical Characterization of Tween 80‐Hydrolyzing Esterases Produced by Rapidly Growing Mycobacteria
Authors: Tomioka Haruaki , Yoneyama Toshimi , Asano Kyoko , Watanabe Takashi , Saito Hajime
Published: November 2013

Tween 80‐hydrolyzing esterases produced by various species of rapidly growing mycobacteria were partially purified from sonicated cell lysates by diethylaminoethyl (DEAE) cellulose and subsequent Sephadex G‐150 column chromatographies. The amount of the esterase produced per gram of bacterial cells varied markedly with each species. Mycobacterium smegmatis, M. chelonei, and M. phlei were high producers and M. chitae and M. diernhoferi were low producers of Tween‐hydrolyzing esterase. The resistance of each mycobacterial strain to oleic acid correlated well with their esterase‐producing ability. All the esterases studied were adsorbed on DEAE cellulose in 50 mM Tris‐HCl buffer (pH 7.5), indicating that they are acidic proteins. Esterases of M. smegmatis, M. chitae, M. fortuitum, and M. phlei were eluted from DEAE at high concentrations (0.11–0.18 m) of ammonium sulfate, while those of M. parafortuitum and M. diernhoferi were eluted at lower concentrations (0.05–0.08 m). With Sephadex G‐150 gel filtration, all esterases were shown to have similar molecular weights (36,000 to 58,000). On the basis of heat‐stability and trypsin‐ or chymotrypsin‐sensitivity, these esterases were divided into three groups: one was heat‐stable and protease‐sensitive (M. smegmatis and M. fotuitum), another was heat‐labile and protease‐resistant (M. chelonei), and the other was the intermediate of the above two groups (M. diernhoferi).
Effect of Maltose on Glucan Synthesis by Glucosyltransferases of Streptococcus mutans
Authors: Fukui Kazuhiro , Moriyama Takafumi
Published: November 2013

The effects of added maltose on the activities of a preparation of crude glucosyltransferases (GTases) and purified dextransucrase (DS) were investigated to elucidate the inhibition mechanism of maltose on the synthesis of water‐insoluble glucan (ISG) in Streptococcus mutans HS‐6. Tri‐ and tetra‐saccharides produced by crude GTases from sucrose in the presence of maltose were identified as panose (4‐α‐isomaltosylglucose) and 4‐α‐isomaltotriosylglucose which were responsible for the activity of DS involved in crude GTases. Kinetic studies on crude GTases in the presence of maltose showed similar results to those of DS except that the synthesis of ISG in the crude GTases was inhibited. Comparative studies of soluble products of crude GTases and DS in the presence of maltose were performed employing gel filtration on Sephadex G‐15. The existence of oligosaccharides above hexasaccharide was revealed as the products of DS but not of crude GTases. These findings were interpreted in terms of the previously proposed mechanism of ISG sythesis by S. mutans, i.e., ISG should be synthesized from the preformed soluble glucan. It was indicated that oligosaccharides above hexasaccharide are utilized for ISG synthesis in the crude GTases system. From these results, the inhibitory mechanism of added maltose on ISG synthesis by crude GTases is considered as follows: DS synthesizes a series of 4‐α‐isomaltodextrinylglucose from sucrose and maltose, and the increase of added maltose results in the decrease of oligosaccharides responsible for synthesis of ISG.
A Family Study of IgG Subclasses in Sickle Cell Anemia
Authors: Outschoorn Ingrid M. , Natta Clayton
Published: November 2013

Three siblings with sickle cell anemia were studied immunologically and hematologically. Their patterns of Protein A‐Sepharose chromatography distribution showed considerable heterogeneity, particularly with respect to the IgG2 and IgG3 subclasses, even though their hematological make up was similar. An attempt was made to correlate their IgG2: IgG1 subclass ratios with their clinical history of recurrent bacterial infections, as well as a possible compensatory IgG3 heterogeneity.
A Selective Medium for Isolation and Presumptive Identification of the Bacteroides fragilis Group
Authors: Ushijima Tsutomu , Takahashi Megumi , Tatewaki Kenichi , Ozaki Yoshikatsu
Published: November 2013

A new selective medium, Bacteroides fragilis ammonium‐sulfate gentamicin (BFAG) agar, for isolation and presumptive identification of the Bacteroides fragilis group is presented in this paper. This semisynthetic medium includes 0.2 g of ammonium sulfate, 0.7 g of lactose, 10 mg of gentamicin, 0.1 mg of aminobenzylpenicillin, 60 units of bacitracin, 20 mg of sodium cholate and 1 mg of sodium azide per 100 ml of medium. Stock cultures of the B. fragilis group grew well on this medium. None of the other 126 gram‐positive or negative strains belonging to 40 aerobic or 45 anaerobic species tested grew on this medium. Three of the seven specimens in the clinical trials yielded colonies of only the B. fragilis group on BFAG agar plates. Also BFAG agar plates inoculated with human feces and contents of the alimentary tract (stomach, small intestine, cecum and colon) of mice gave rise to colonies of only the B. fragilis group. The high selectivity and good plating efficiency of BFAG agar enabled us to isolate the B. fragilis group rapidly from various clinical specimens.
Antigenic and Biochemical Characterization of Poliovirus Type 1 Isolates of Non‐Vaccine Origin
Authors: Hara Minora , Hagiwara Akio , Yoneyama Tetsuo , Saito Yoshiko , Shimojo Hiroto
Published: November 2013

During the past 15 years, five poliovirus type 1 strains with non‐vaccinelike antigenicity have been isolated in Japan. Of these isolates, two were from paralytic poliomyelitis patients not associated with the use of Sabin vaccine, and three were apparently introduced from abroad. All the isolates could be readily distinguished from the corresponding Sabin type 1 vaccine strain by oligonucleotide mapping of the viral RNA and by polyacrylamide gel electrophoresis of the viral proteins. The oligonucleotide map of the virulent Mahoney strain which has non‐vaccine‐like antigenicity was very similar to the map of Sabin type 1 strain. These data indicate that none of the isolates were derived from Sabin type 1 vaccine or its parental Mahoney strain. In addition, some isolates had close antigenic relationship with one another. It is probable that all these strains were introduced from foreign lands where wild poliovirus strains are prevalent.
Isolation and Characterization of the Serotype g Carbohydrate Moiety from an Enzyme Lysate of Streptococcus mutans 6715 Cell Walls
Authors: Hamada Shigeyuki , Torii Mitsuo , Okahashi Nobuo , Kotani Shozo , Kato Keijiro , Kawata Shigeo , Yokogawa Kanae , Michalek Suzanne M. , McGhee Jerry R.
Published: November 2013

The serotype‐specific carbohydrate moiety of Streptococcus mutans was isolated by mild degradation of purified cell walls with a cell‐wall lytic enzyme. Cell walls of serotype g S. mutans strain 6715 were digested with M1 enzyme, an endo‐N‐acetylmuramidase purified from culture supernatants of Streptomyces globisporus strain 1829. The enzyme lysate of the cell walls was applied to a CM Sephadex C‐25 column to remove the M1 enzyme from the cell wall lysate and then subjected to Sephadex G‐100 column chromatography. Carbohydrate antigens with serotype g specificity, designated M1g, and a peptidoglycan—polysaccharide complex lacking serotype specificity (M1PG) were separated. Purified serotype g antigen was also obtained by autoclaving the S. mutans 6715 whole cells in saline at 120 C for 30 min. The extract was applied to a DEAE Sephadex A‐25 column to remove nucleic acids and teichoic acids. The unbound peak fraction was concentrated and re‐chromatographed on a Bio‐Gel P‐100 column. The void volume fraction contained serotype g carbohydrate and was designated RRg antigen. M1g and RRg antigens formed a band of identity with anti‐serotype g serum by immunodiffusion. These antigens were composed mainly of galactose, glucose, and rhamnose at an approximate weight ratio of 8 : 4 : 1, while constituent sugars of M1PG consisted of rhamnose and glucose, with no detectable galactose. M1g also contained peptidoglycan residues other than threonine, an interpeptide bridge component of the native cell wall peptidoglycan. Marked inhibition of the quantitative precipitin reaction between M1g and anti‐serotype g serum was obtained with melibiose and galactose, which suggests that the immunodeterminant of the serotype g carbohydrate is an α‐linked galactose‐glucose terminal linkage.
Chemical and Ultrastructural Differences in Endotoxic Glycolipids from Salmonella Minnesota Re Mutant Extracted with Various Solvent Systems
Authors: Amano Ken‐Ichi , Fukushi Kazue
Published: November 2013

Endotoxic glycolipids (ReGl) extracted from the whole cells (WC) and cell walls of heptose‐less Re mutant of Salmonella minnesota with hot phenol‐water (PW), phenol‐chloroform‐petroleum ether (PCP), and chloroform‐methanol (CM) were analyzed chemically and examined with an electron microscope. ReGl‐PW‐(WC) contained mannose and proteins as contaminants, ReGl‐PCP(WC) consisted of an excess amount of amino compound (cadaverine), and ReGl‐CM(WC) consisted of proteins and cadaverine, in addition to the ReGl constituents.

The ultrastructure of ReGl‐PW(WC) resembled onions when stained with uranyl formate, and was spherical when stained with uranyl acetate, sodium phosphotungstate and ammonium molybdate, whereas those of ReGl‐PCP(WC) and ReGl‐CM(WC) were shaped like ribbons. However, the shadowed ultrastructures of ReGl‐PCP(WC) and ReGl‐CM(WC) showed small pieces of flat and wide sheets, respectively. ReGl‐PCP(WC) re‐extracted by the PW method was found to be converted into the onion‐like structure which was similar to that of ReGl‐PW(WC), while an intermediate form (fingerprint‐like) was observed after re‐extraction of ReGl‐PW(WC) with PCP. It was strongly suggested that the ultrastructural arrangement of ReGl was dependent on the solvent systems used for extraction.
Electron Microscopic Studies of Endotoxins Treated with Alkaline and Acid Reagents
Authors: Amano Ken‐Ichi , Fukushi Kazue
Published: November 2013

Endotoxins extracted from Salmonella minnesota wild strain and R mutants (Ra to Re) were treated with two different alkaline reagents. Treatment with diluted sodium hydroxide, which caused partial removal of O‐ester linked fatty acids, changed the ultrastructures of endotoxins from an onion‐like structure to monolayer particles (approximately 100 Å in diameter) except for endotoxic glycolipids from Rd2 and Re mutants which showed mixed ultrastructures of untreated and treated endotoxins. Treatment with alkaline hydroxylamine, which caused liberation of all O‐ester linked fatty acids, changed the ultrastructures of all endotoxins to monolayer particles. The results suggested that the ultrastructures of alkaline‐treated endotoxins were dependent on the degree of their hydrophobicity. On the other hand, the micrographs of acid treated endotoxins did not show a constant structure because of the high hydrophobicity.
Isolation and Characterization of a Filamentous Phage, Vf33, Specific for Vibrio parahaemolyticus
Authors: Taniguchi Hatsumi , Sato Kiyoshi , Ogawa Midori , Udou Takezo , Mizuguchi Yasuo
Published: November 2013

Phage Vf33, a filamentous phage about 1,400 nm long and 7 nm wide, specific for Vibrio parahaemolyticus, was isolated and characterized. The buoyant density of Vf33 in CsCl was 1.292 g/cm3. As with other filamentous phages, the lytic activity of Vf33 was resistant to heating below 80 C and to treatment with diethylether, acetone or methanol but sensitive to chloroform. The nucleic acid of this phage is single‐stranded circular DNA 8.4 kb in size. The viral genome was converted to a double‐stranded replicative form in the host cell. Among the strains tested, only V. parahaemolyticus strains possessing K38 antigen was sensitive to the phage.
Immunochemical Characteristics of Streptococcus mutans Serotype h Carbohydrate Antigen
Authors: Okahashi Nobuo , Nishida Yasukuni , Koga Toshihiko , Hamada Shigeyuki
Published: November 2013

Serotype A carbohydrate antigen was prepared from cell walls of Streptococcus mutans strain MFe28 of monkey origin. The h antigen was extracted from the cell walls with 5% trichloroacetic acid at 4 C, and purified by DEAE‐Sephadex A‐25 ion exchange chromatography followed by Sephacryl S‐300 gel filtration. The purified antigen was composed of galactose (75%), glucose (16%), and rhamnose (3%). Although the antiserum against whole cells of S. mutans MFe28 gave a strong cross reaction with serotype d S. mutans, serotype h‐specific antiserum could be obtained by adequate adsorption. The precipitin reaction and hapten inhibition test using serotype h‐specific antiserum showed that galactose, glucose, and their derivative sugars were markedly potent inhibitors. It was concluded that the serotype h antigen is immunologically distinguishable from the known serotypes of S. mutans, although it is closely related to serotype d antigen of S. mutans.
Immunoglobulin Classes of Anti‐Sendai Virus Antibody Detected by ELISA in Infected Nude Mouse Serum
Authors: Iwai Hiroshi , Yamaguchi Ryoji , Otsuka Yuko , Ueda Katsumoto , Saito Muneo
Published: November 2013

Sendai virus‐infected nude mouse sera obtained on the seventh day after infection or later, in which anti‐Sendai virus antibodies were undetectable by hemagglutination‐inhibition and neutralization tests, were found to be reactive with the virus antigen by ELISA using horseradish peroxidase‐conjugated anti‐mouse IgG rabbit IgG. The reactivity was blocked by rabbit anti‐Sendai virus antiserum and was not observed against influenza virus which served as a control antigen. Anti‐Sendai virus antibody activity of fractions from Sephadex G‐200 gel filtration was detected in the IgM fraction when anti‐mouse μ chain‐specific antiserum was used and in both IgG and IgM fractions when heavy and light chain‐specific anti‐mouse IgG serum was employed in ELISA. ELISA of the fractions from protein A‐Sepharose affinity chromatography of Sendai virus‐infected nude mouse sera showed that the eluates at pH 6.0 and pH 3.5 contained IgG1 and IgG2b anti‐Sendai virus antibodies, respectively, and that the eluate at pH 4.5 contained both IgG2a and IgG3 antibodies.
Sugar Synthesis in Leptospira
Authors: Yanagihara Yasutake , Kobayashi Susumu , Mifuchi Ichiji
Published: November 2013

The presence and some properties of the key enzymes of the glyoxylate cycle, isocitrate lyase (threo‐Ds‐isocitrate glyoxylate‐lyase, EC 4.1.3.1) and malate synthase (L‐malate glyoxylate‐lyase (CoA‐acetylating) EC 4.1.3.2), were investigated in Leptospira biflexa.

Isocitrate lyase activity was found for the first time in the organism. The enzyme was induced by ethanol but not by acetate. The optimum pH was 6.8. The activity was inhibited by phosphoenolpyruvate, a specific inhibitor of isocitrate lyase.

The optimum pH of malate synthase of L. biflexa was about 8.5. The Km value for glyoxylate was 3.0 × 10−3 M and the activity was inhibited by glycolate, the inhibitor.

The results strongly suggested the presence of a glyoxylate cycle in Leptospira. The possibility that the glyoxylate cycle plays an essential role in the synthesis of sugars, amino acids and other cellular components as an anaplerotic pathway of the tricarboxylic acid cycle in Leptospira was discussed.
Chemical Compositions of Cell Walls and Polysaccharide Fractions of Spirochetes
Authors: Yanagihara Yasutake , Kamisango Kei‐Ichi , Yasuda Seiji , Kobayashi Susumu , Mifuchi Ichiji , Azuma Ichiro , Yamamura Yuichi , Johnson Russell C.
Published: November 2013

Cellular polysaccharide fractions of various representative members of genera of the family Spirochaetaceae were obtained by the ammonium hydroxide extraction method. The sugar composition of the polysaccharide preparations was complex and many kinds of sugars such as rhamnose, fucose, ribose, xylose, mannose, galactose, and glucose were detected in all of the spirochetes tested. Of particular interest was the presence of 4‐O‐methylmannose as a constituent polysaccharide in members of the genus Leptospira. This sugar was not detected in the polysaccharides of Spirochaeta, Borrelia, and Treponema.

The chemical compositions of cell wall fractions were also examined. 4‐O‐Methylmannose was detected in the cell wall polysaccharides of the genus Leptospira but not in cell walls prepared from the Spirochaeta, Borrelia, and Treponema. The diaminopimelic acid present in cell wall peptidoglycans of the genus Leptospira was meso‐diaminopimelic acid (A2pm). The molar ratios of alanine, glutamic acid, A2pm, glycine, muramic acid, and glucosamine in leptospiral cell walls were found to be approximately 2: 1: 1: 1: 1: 1. In contrast to the Leptospira, the peptidoglycans of genera Spirochaeta, Borrelia, and Treponema contained ornithine (Orn) but not A2pm. Since 4‐O‐methylmannose and A2pm were found in the cell wall fractions of genus Leptospira but not in Spirochaeta, Borrelia, or Treponema, it was suggested that the chemical compositions of the cell wall might become an important criterion for the chemotaxonomy of Spirochaetales.
Phospholipases of Leptospira
Authors: Yanagihara Yasutake , Taniyama Tadayoshi , Misaki Hideo , Suzuki Yasuo , Matsumoto Makoto , Mifuchi Ichiji
Published: November 2013

The hydrolysis of phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and trioleoylglycerol by Leptospira biflexa strain Urawa was studied in vitro.

Phospholipase A1 was identified by the formation of 32P‐ and 14C‐labeled lyso‐derivatives from 32P‐phosphatidylcholine, 32P‐phosphatidylethanolamine, or 1‐acyl‐2‐[1‐14C]oleoyl‐sn‐glycero‐3‐phosphorylcholine. Phospholipase A1 activity was independent of lipase in the microorganism since 14C‐labeled trioleoylglycerol was scarcely attacked under the same conditions in which the phospholipids were hydrolyzed.

Lysophospholipase activity was also demonstrated using 32P‐ and non‐labeled lysophosphatidylcholine.

The activity of phospholipase A1 was found in a broad range of pH but no optimal pH was determined. The pH optimum of lysophospholipase was 8.0. Both enzymes were labile to heat.

Phospholipase C activity, however, could not be detected because no radioactive di‐ and monoacylglycerol was found in the experiment with 1‐acyl‐2‐[1‐14C]‐oleoyl‐sn‐glycero‐3‐phosphorylcholine as the substrate.

It was inferred that phosphatidylethanolamine, which was the major component of phospholipids in leptospirae, was hydrolyzed serially by phospholipase A (A1 and/or A2?) and lysophospholipase to glycerophosphorylethanolamine via 2‐acyl‐type‐lyso‐derivative as one metabolic pathway of the substrate.
Slow Development of Measles Virus (Edmonston Strain) Infection in the Brain of Nude Mice
Authors: Ohuchi Reiko , Ohuchi Masanobu , Mifune Kumato
Published: November 2013

The Edmonston strain of measles virus caused neurologic disease in athymic nude mice by intracerebral inoculation. The incubation periods of the disease, however, were extremely long, ranging from 59 to 140 days when the mice were inoculated with 104 plaque forming units (PFU) of the virus. The Edmonston strain was highly infectious in the nude mouse brain since virus infection was established even with 1 PFU of the virus. Virus titers in the brains of infected mice increased with the time of incubation. These results indicate that the extremely long incubation period of the disease is ascribed to very slow development of virus infection in the mouse brain. On the other hand, the incubation periods of the Biken strain of SSPE virus were very short (generally within 2 weeks) even with inoculations of 1 PFU of the virus. However, the extent of the dissemination of infection in brains was not significantly different between the two viruses as examined by immunofluorescent staining.
Differentiation of Mycobacterial Species by Investigation of Petroleum Ether‐Soluble Sulfolipids Using Thin‐Layer Chromatography after Incubation with [35S]Sulfate
Authors: Tsukamura Michio , Mizuno Shoji , Toyama Haruo
Published: November 2013

The method of detecting petroleum ether‐soluble sulfolipids by thin‐layer chromatography after incubation with [35S]sulfate is useful for differentiation between mycobacterial species. Rapidly growing mycobacteria, including two subspecies of Mycobacterium chelonei, were differentiated by this method. Most species of slowly growing mycobacteria were characterized by the pattern of distribution of radioactive sulfolipids in the thin‐layer chromatograms. Mycobacterium nonchromogenicum was clearly differentiated from Mycobacterium terrae and Mycobacterium triviale by the presence of a sulfolipid. The latter two did not contain any significant amount of sulfolipids. Mycobacterium avium contained a large amount of sulfolipids, whereas Mycobacterium scrofulaceum did not contain any detectable sulfolipid. Rapidly growing, scotochromogenic mycobacteria, except for Mycobacterium flavescens, did not contain any remarkable amounts of sulfolipids.
A New Monoclonal Antibody Recognizing an Antigen of Human Lymphocytes Similar or Identical to Tac Antigen
Authors: Tanaka Yuetsu , Tozawa Hideki , Koyanagi Yoshio , Yamamoto Naoki , Hinuma Yorio
Published: November 2013

A new mouse monoclonal antibody (HIEI, IgG1 type) that reacts with a cell surface glycoprotein of human lymphocytes was isolated. Membrane immunofluorescence assay showed that HIEI, like the anti‐Tac monoclonal antibody, reacted preferentially with activated normal human T‐cells and adult T‐cell leukemia (ATL) virus (ATLV)‐carrying human T‐ and B‐cell lines. However, an interesting difference between HIEI and anti‐Tac antibody was that HIEI did not react with ATLV‐transformed simian cell lines or those cultured with interleukin‐2 (IL‐2), whereas the anti‐Tac antibody did. The immunoprecipitation assay showed that both HIEI and anti‐Tac antibody precipitated a glycoprotein with a molecular weight of 60,000 daltons (gp60) from activated normal T‐cells and ATLV‐positive T‐ and B‐cells, and also gp53 from MT‐2 and MT‐2‐related T‐cell lines transformed with ATLV in vitro by the MT‐2 cocultivation method. HIEI inhibited the Independent proliferation of normal T‐cells, but its inhibitory effect was much weaker than that of the anti‐Tac antibody. The anti‐Tac antibody interfered with the binding of HIEI to target cells, but HIEI did not block binding of the anti‐Tac antibody to the cells. These observations indicate that HIEI antibody recognizes a new antigenic determinant of the human Tac antigen.
Isotype Specificity and Heterogeneity of Fcγ‐Receptors on Guinea Pig Splenic B and T Lymphocytes
Authors: Yamashita Toshiyuki , Koyama Jiro
Published: November 2013

The isotype specificity of Fc‐receptors for IgG (Fcγ‐Rs) on normal guinea pig splenic B and T cells was determined by a rosette assay using sheep erythrocytes sensitized with either homologous IgG1 or IgG2 anti‐sheep erythrocyte antibody [EA(IgG1) or EA(IgG2)]. Approximately 70% of the lymphocytes in the highly purified B‐cell fraction could form rosettes with EA(IgG2), and 55% of the cells with EA(IgG1). Inhibition experiments with soluble complexes of IgG1 or IgG2 antibody with ovalbumin demonstrated that approximately 20% of the EA(IgG2) rosette‐forming B cells bore the Fcγ‐R monospecific for IgG2, whereas 80% of the cells had two distinct Fcγ‐Rs simultaneously; one monospecific for IgG2 and the other bispecific for IgG1 and IgG2. The existence of a B cell bearing the Fcγ‐R monospecific for IgG1 was not definitively demonstrated in the B‐cell fraction. In the T cell‐enriched fraction, approximately 40% of the cells could form rosettes with EA(IgG2). The EA(IgG1)rosette‐forming cells, however, comprised only 6% of the total cells, indicating that most of the EA(IgG2) rosette‐forming T cells bear essentially the Fcγ‐R monspecific for IgG2 alone.

The results obtained revealed that guinea pig splenic lymphocytes bear two distinct Fcγ‐Rs, which are not equally distributed on the B‐ and T‐cell populations and also on their respective subsets.
Serological Cross‐Reactivities of Murine and Human Class II Antigen Determined by Murine Xeno Anti‐Human Class II Antibody
Authors: Aosai Fumie , Yui Katsuyuki , Shigematsu Hidekazu , Yano Akihiko
Published: November 2013

Murine anti‐human class II antibodies were shown to cross‐react with polymorphic determinants of murine class II antigens. The cross‐reacting antibodies were raised in B10.S(9R) mice by immunizing with human nylon wool adherent cells (Ad cells) from peripheral blood leukocytes. The B10.S(9R) anti‐human Ad cell antiserum bound to the molecules consisting of two chains with molecular weights of 35K and 28K dimers which were purified with a lentil‐lectin column. The B10.S (9R) anti‐human class II antiserum was also revealed to contain two distinct cross‐reacting antibodies with polymorphic determinants of murine class II antigens coded for by the I‐A subregion of the H‐2. One is specific for a determinant of class II molecules coded for by I‐Ab,d,q, and the other seems to be specific for class II molecules coded for by I‐Aa,k,r.
Demonstration of Protective Antigen Carried by Flagella of Clostridium chauvoei
Authors: Tamura Yutaka , Minamoto Nobuyuki , Tanaka Shozo
Published: November 2013

The protective antigen present on the flagella of Clostridium chauvoei was studied by the mouse protection test. A partially purified flagella preparation (PPF) showed protective antigenicity after two intraperitoneal injections of 2 μg as protein, while the protective antigenicity of nonflagellated mutants (NFM) was 100‐fold less than that of the flagellated parent strain. Although the protective effect of antisera against the whole cells and PPF, in terms of ED50 values, was mostly lost after absorption with the parent strain, that of antisera after absorption with NFMs showed no appreciable loss. These results suggest that the flagella of Cl. chauvoei play some role in inducing protective immunity in mice.
Drug Resistance and R Plasmids in Vibrio anguillarum Isolated in Cultured Ayu (Plecoglossus altivelis)
Authors: Aoki Takashi , Kitao Tadatoshi , Watanabe Satoshi , Takeshita Sunao
Published: November 2013

Two hundred twenty‐six strains of Vibrio anguillarum collected from cultured ayu (Plecoglossus altivelis) between 1978 and 1980 were studied for their sensitivities to 10 chemotherapeutics. In order to determine whether the drug‐resistant strains possessed transferable R plasmids, they were conjugated with Escherichia coli. Almost all the strains isolated during the 3 years showed resistance to nalidixic acid (NA) and/or furazolidone (NF). NA and NF resistance were not transferred to Escherichia coli from any of the strains. Chloramphenicol‐resistant strains were isolated in every year and almost all of them carried transferable R plasmids. Only one strain with tetracycline resistance was found among the strains tested. Strains resistant to sulfonamides, streptomycin, ampicillin (ABP), and trimethoprim (TMP) increased rapidly in 1980, and a large number of them carried transferable R plasmids.

Transferable R plasmids encoded with resistance to ABP and TMP were detected for the first time in V. anguillarum strains. The R plasmids detected in the strains isolated in 1980 were classified into incompatibility groups E, A, and an untypable group. The R plasmid DNAs were cleaved by EcoRI to yield 11 to 13 fragments. The estimated molecular weights of the R plasmids from the five strains ranged from 97 to 104 M daltons.
Properties and Origin of Filamentous Appendages on Spores of Bacillus cereus
Authors: Kozuka Satoshi , Tochikubo Kunio
Published: November 2013

Some physical, chemical, and immunological properties of filamentous appendages and the exosporium on the spores of Bacillus cereus were examined for the purpose of elucidating the origin of filamentous appendages. The main components of both filamentous appendages and the exosporium were protein and their amino acid compositions were similar in point of a high content of glycine, alanine, threonine, valine, and acidic amino acids and a low content of basic and sulphur‐containing amino acids. Treatment with 1 N NaOH at 50 C solubilized the isolated appendages completely and the isolated exosporia partially. In both preparations the solubilized proteins consisted of highly acidic monomeric subunits with molecular weights between 2,000 and 5,000. Treatment of the spores with 2% 2‐mercaptoethanol at 37 C resulted in the isolation of long filamentous appendages without segmentation. When the spores were treated with 10% 2‐mercaptoethanol, there was partial destruction of the exosporium as well as detachment of the filamentous appendages. There was a common antigenic component in the exosporium and the tips of the filamentous appendages. Five strains of B. cereus having a common appendage antigen also had a common exosporium antigen, whereas six other strains had neither a common appendage antigen nor a common exosporium antigen. From these facts it was concluded that the filamentous appendages arose from the exosporium.
Taxonomic Studies on the Mycobacterium tuberculosis Series
Authors: Tsukamura Michio , Mizuno Shoji , Toyama Haruo
Published: November 2013

Numerical classification of slowly growing mycobacteria, including 159 strains received as Mycobacterium tuberculosis, M. bovis, M. africanum, and M. microti, was carried out using 88 characters, and the following results were obtained. 1) All 159 strains received as M. tuberculosis, M. bovis, M. africanum, and M. microti formed one cluster, and no clear‐cut differentiation among these four species was achieved. These four species should be reduced to one species, M. tuberculosis. 2) Within the cluster, two subclusters appeared, with a number of strains located outside the subclusters. One subcluster was composed, except for two strains, of only M. tuberculosis strains, and another of M. bovis and M. africanum strains only. The subclusters were regarded as subspecies tuberculosis and subspecies bovis, respectively. M. africanum was regarded as a synonym of M. bovis, i.e., a niacin‐positive variety of M. bovis (subsp. bovis). 3) An intermediate subcluster was observed outside the M. tuberculosis and M. bovis subclusters. This may be regarded as intermediate between the two subspecies, tuberculosis and bovis. 4) In the last two decades, the characteristics of M. tuberculosis strains isolated from patients seemed to be approaching those of M. bovis strains. The recently isolated strains showed stronger arylsulfatase activity and lower resistance to thiophene‐2‐carboxylic acid hydrazide than the strains isolated 16 to 19 years ago.
Reverse Effect of Gram‐Positive Bacteria vs. Gram‐Negative Bacteria on Adjuvant‐Induced Arthritis in Germfree Rats
Authors: Kohashi Osamu , Kohashi Yukiko , Takahashi Tokutaro , Ozawa Atsushi , Shigematsu Nobuaki
Published: November 2013

Germfree (GF) F344 rats developed severe adjuvant‐induced arthritis with a 100% incidence after a single intradermal injection of heat‐killed Mycobacterium bovis (BCG). Specific pathogene‐free (SPF) rats developed less severe arthritis with a lower incidence. The rats colonized with Escherichia coli or Bacteroides developed mild disease comparable to that in SPF rats. The rats colonized with Bifidobacterium, Propionibacterium acnes, Lactobacillus casei, L. fermentum, L. murini, and L. acidophilus developed more severe disease than that in GF rats. Furthermore, the rats colonized with a mixture of E. coli and the above lactobacilli developed very mild disease similar to that in SPF rats. These results suggest that (1) gram‐negative bacteria, such as E. coli and Bacteroides, may suppress the disease, possibly through their lipopolysaccharides, and may be responsible for the lower susceptibility of SPF rats; (2) gram‐positive bacteria, such as Bifidobacterium, P. acnes, and lactobacilli, may enhance the disease, possibly through their peptidoglycans; and (3) E. coli may play a dominant role in modulating the development of adjuvant‐induced arthritis.