bluuebunny/crossref_metadata_2025 huggingface dataset:
Antimicrobial Susceptibility of Clostridium difficile from Different Sources
Authors: Nakamura Shinichi , Nakashio Satoshi , Mikawa Masato , Yamakawa Kiyotaka , Okumura Seiichi , Nishida Shoki
Published: November 2013

A total of 79 Clostridium difficile strains from healthy young and elderly adults, elderly patients without gastrointestinal disease, elderly patients receiving antibiotics without gastrointestinal complications, and elderly patients with antibiotic‐associated diarrhea or pseudomembranous colitis were tested for their susceptibilities to 24 antimicrobial agents. All of the 79 strains were inhibited by low concentrations of rifampicin, metronidazole, fusidic acid, vancomycin, ampicillin, and penicillin G. The strains were highly resistant to aminoglycosides, trimethoprim, sulfamethoxazole, nalidixic acid, and cycloserine and often resistant to neomycin, cefoxitin, and cefalexin. Wide variations in the susceptibility of C. difficile strains to erythromycin, clindamycin, lincomycin, chloramphenicol, and tetracycline were found. Strains resistant to erythromycin, clindamycin, and lincomycin were more frequently found among strains isolated from elderly adults than those isolated from young adults, with particularly high frequency among strains isolated from elderly patients receiving antibiotics. None of the 23 strains isolated from healthy young adults was resistant to chloramphenicol. All of the 14 strains resistant to erythromycin, clindamycin, lincomycin, and chloramphenicol were sensitive to tetracycline and all of the 15 strains resistant to erythromycin, clindamycin, lincomycin, and tetracycline were sensitive to chloramphenicol. Only one out of 19 tetracycline‐resistant strains was highly toxigenic, whereas 42 (70%) of 60 sensitive strains were highly toxigenic.
Group H Streptococcal Bacteriocins Having No Relation to Bacterial Transformation
Authors: Ito Takako
Published: November 2013

The culture filtrate of group H streptococcus strain Challis produced a competence factor (CF) for bacterial transformation as well as a bactericidal factor(s) against Wicky cells. Strain 36658, in the same streptococcal group, also produced the bactericidal factor(s) but not CF. The effect of the Challis bacteriocin was limited to strains Wicky and 58, whereas the 36658 bacteriocin affected 67% of 49 strains tested. Strain 58, one of the indicator strains, was affected by the bactericidal activity of these bacteriocins but not by CF activity, and failed to transform. No relationship between the bacteriocin‐producing strains and indicator strains was observed. Both Challis and 36658 bacteriocin activities decreased markedly either when the bacteriocins were heated at 50 C for 30 min or with the addition of a protein synthesis inhibitor, but showed different sensitivities to trypsin, papain and lipase. The bacteriocins were of at least protein nature and their molecular weight was roughly estimated as 100,000 daltons by membrane filtration experiments. The 36658 bacteriocin is a new type of streptocin previously not reported. The possible absence of bacteriophage or phage‐like particles in the preparations is discussed.
Purification and Characterization of Spirosomes in Lactobacillus brevis
Authors: Ueki Yukihide , Masuda Kuniyoshi , Kawata Tornio
Published: November 2013

Spirosomes, very fine spiral particles, were isolated from a protoplastlysate of Lactobacillus brevis ATCC 8287 by differential centrifugation and purified further by potassium tartrate density gradient centrifugation. The purified spirosome preparation showed a maximum peak around 275 nm on the ultraviolet absorption spectrum and it consisted of about 94.5% protein. The buoyant density in CsCl of the spirosomes was 1.320 g/cm3. The spirosomes were composed mainly of a single protein (spirosin) with an apparent molecular weight of about 95,000 as determined by sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis. The protein of the spirosomes was found to be composed predominantly of neutral amino acids accompanied by approximately equal amounts of acidic and basic amino acids. The spirosomes showed one antigenic determinant in the immunodiffusion test. The spirosomes were readily degraded by the action of proteolytic enzymes and lost their antigenicity, but they were not affected by treatment with either deoxyribonuclease or ribonuclease. The spiral structure of the spirosome was also found to be disintegrated by treatment with 1 m guanidine hydrochloride, 4 m urea or 0.1% SDS, but not by the action of deoxycholate, non‐ionic detergents or mercaptoethanol, as observed in the electron microscope.
Regulatory Mechanism of Delayed‐Type Hypersensitivity in Mice: IV. Effect of Suppressor T Cells on the Development of Memory T Cells Involved in Accelerated Generation of DTH‐Effector Cells In Vitro
Authors: Tamura Shin‐Ichi , Kojima Asato
Published: November 2013

The effect of suppressor T cells (Ts) on the induction and the subsequent development of memory T cells for delayed‐type hypersensitivity (DTH) was examined. The memory cells were induced in the spleens of mice primed previously with a low dose of reduced and alkylated ovalbumin (RA‐OA), and they generated DTH‐effector T cells (DTH‐Te) in a significantly accelerated fashion when cultured with OA in vitro. Ts were obtained from the spleens of mice which received OA‐coupled spleen cells i.v. 4 days previously, and they inhibited antigen‐specifically the induction of DTH responses in the recipient mice sensitized with alum‐absorbed OA only when transferred within 5 weeks before sensitization. The spleen cells from mice given Ts together with the priming antigen 7 weeks before culture failed to generate DTH‐Te in an accelerated manner on restimulation with OA in vitro. The memory cells from primed mice also did not cause accelerated generation of DTH‐Te, when cultured with Ts in the presence of OA in vitro. These results indicate that both the induction of the memory cells by priming with antigen in vivo and the subsequent development of memory cells to DTH‐Te by restimulation in vitro are inhibited independently by Ts. This corresponded well with the effect of Ts on the development of DTH‐memory in vivo.
Regulatory Mechanism of Delayed‐Type Hypersensitivity in Mice: V. Augmentor Cells for DTH Responses
Authors: Kojima Asato , Tamura Shin‐Ichi , Egashira Yasuyuki
Published: November 2013

Mice primed with 1 μg of reduced and alkylated ovalbumin (RA‐OA) developed not only long‐lived memory cells for delayed‐type hypersensitivity (DTH), capable of differentiating into DTH‐effector T cells (DTH‐Te) against ovalbumin (OA) when restimulated in vitro with OA, but also spleen cells capable of augmenting recipients' DTH responses to OA when transferred into cyclophosphamide (CY)‐pretreated mice. The augmenting activity in spleen cells, upon transfer, was found 7 days, but not 21 or 91 days, after priming with RA‐OA, although memory DTH‐Te were present throughout the period of observation. The loss of augmenting activity after day 7 of priming was not due to the presence of suppressor cells; spleen cells taken 21 days after priming failed to suppress, upon transfer, the augmenting activity in 7‐day‐primed spleen cells as well as induction and expression of DTH responses to OA. When 7‐day‐primed spleen cells were fractionated on a discontinuous bovine serum albumin density gradient, the augmenting activity was found only in the medium‐density‐cell layer, although memory DTH‐Te were separated in the high‐density layer. Augmentation of DTH‐Te generation could also be demonstrated in vitro when 7‐day‐primed spleen cells, but not 21‐day‐primed spleen cells, were added to cultures of spleen cells from CY‐pretreated mice. These results indicate that, in the 7‐day‐primed spleen, there is an augmentor cell population which is different from memory DTH‐Te and interacts with CY‐resistant unprimed cells to facilitate DTH‐Te generation.
Chemical and Biological Properties of Lipopolysaccharide from a Marine Bacterium, Photobacterium phosphoreum PJ‐1
Authors: Kuwae Toyoyasu , Sasaki Takeji , Kurata Munetsugu
Published: November 2013

The chemical and biological properties of the lipopolysaccharide (LPS) isolated from a marine bacterium, Photobacterium phosphoreum PJ‐1, were studied.

This LPS consists of 40.6% carbohydrate, 27.3% fatty acid, 0.2% 2‐keto‐3‐deoxyoctonate (KDO) and other components. One characteristic of this LPS is its small amount of KDO, the basic component of the usual LPS. Electrophoresis in sodium dodecylsulfate polyacrylamide gel revealed at least two staining bands for carbohydrates. These bands were continuous and broad, and showed rapid electrophoretic mobility which corresponded closely to the fastest moving band of LPS from Salmonella typhimurium.

This LPS preparation had adjuvant activity, lethality for ddY mice, and the ability to gel Limulus amebocyte lysate, and the strength of these activities corresponded closely to those of LPS preparations from Escherichia coli 0111:B4 and S. typhimurium. In the test for lethality of the LPS for ddY mice, the lethal action appeared in two phases depending on the dose used for intravenous (i.v.) injection : the early lethal action appeared within 30 min after injection of 250 μg or less, and the late lethal action occurred gradually after 16 hr at doses of 500 μg or more. The total (both phases) LD50 of this LPS (i.v.) for ddY mice was 265 μg per mouse and in only the late phase it was 500 μg.

These results show that in spite of structual differences in regard to KDO content, LPS from P. phosphoreum PJ‐1 has some biological properties similar to those of LPS from E. coli 0111:B4 and S. typhimurium but it shows no immunological cross‐reaction with other LPS.
Sugar Composition of Lipopolysaccharides of Family Vibrionaceae
Authors: Hisatsune Kazuhito , Kondo Seiichi , Igughi Takehiro , Machida Masaaki , Asou Shinobu , Inaguma Makoto , Yamamoto Fumihiro
Published: November 2013

A comparative study was carried out on the sugar composition of lipopolysaccharides (LPS) isolated from representative strains of members of the family Vibrionaceae including all of the constituting genera, i.e., Vibrio, Aeromonas, Photobacterium, Plesiomonas, and Lucibacterium. More than 100 strains were examined. It was found that, with the exception of Vibrio parahaemolyticus 06, 2‐keto‐3‐deoxyoctonate (KDO), known generally as a component sugar in the core region of usual gram‐negative bacterial LPS, is virtually absent from LPS of the Vibrionaceae strains so far examined. Furthermore, mannose was also lacking in LPS of Vibrionaceae strains with the exception of only one strain, A. anaerogenes (ATCC 15467). Instead, some KDO‐like substances were found in LPS from Vibrio (“Beneckea”) nereida (ATCC 25917) and Plesiomonas shigelloides including the type strain (ATCC 14029), the same as those found in LPS from V. parahaemolyticus O7 and O12, and three strains of V. alginolyticus. These substances were strongly positive in the periodate‐thiobarbituric acid test, yielding a color with maximum absorption at 549 nm. The spectra were identical to that of KDO, whereas substances differed from KDO at least in behavior in high‐voltage paper electrophoresis and thin‐layer chromatography.

A particularly interesting feature from the chemotaxonomical point of view was found in the sugar composition of LPS isolated from V. cholerae. Fructose was present exclusively in LPS of V. cholerae (both O1 and non‐O1 groups and classical and eltor biotypes) with the exception of one strain of Photobacterium phosphoreum (NCMB 844). In addition, a pair of rarely occurring amino sugars, perosamine and quinovosamine, was found in LPS from O1 group V. cholerae regardless of either the biotype (classical or eltor) or the serotype (Inaba or Ogawa), whereas this pair was not present in non‐O1 group V. cholerae (the so‐called NAG vibrios). This feature was confirmed with LPS from more than 30 additional strains of O1 group V. cholerae isolated from patients.

The virtual absence of KDO in LPS of the family Vibrionaceae was demonstrated for the first time in this study. These results are compatible with the interpretation that the absence of KDO in LPS can be used as one of the taxonomical characteristics of Vibrionaceae in addition to (G+C) content, DNA (or RNA) homology, and numerical analysis data.
An Epidemiological Study of Serratia marcescens Infections by Bacteriocin Typing
Authors: Nasu Masaru , Goto Jun , Goto Ikuo
Published: November 2013

Bacteriocin sensitivity typing according to the method of Traub (Appl. Microbiol. 1971. 21: 837–840) was carried out on 226 clinical isolates of Serratia marcescens obtained from inpatients at Nagasaki University Hospital during the period from January 1976 to December 1978. The isolates were divided into 16 different bacteriocin types, mainly 26, 4, and 9. The distribution of the types suggests that Serratia marcescens infections may be caused by cross infection. Reproducibility of bacteriocin typing and the relationship between serotypes (O‐antigen) and bacteriocin types are discussed in regard to the application of this method to the study of nosocomial infections.
Delayed‐Type Hypersensitivity to Hen Egg‐White Lysozyme: I. The Surface Phenotypes of Suppressor T Cells Induced by Intravenous Injection of Lysozyme‐Modified Spleen Cells, and Their Moleculor Interactions
Authors: Okuda Kenji , Ishii Norihisa , Ikezawa Zenroh , Takahashi Toyozoh , Arai Katsuji , Tadokoro Ichiro
Published: November 2013

Suppressor T cells (Ts) induced by lysozyme‐modified syngeneic lymphocytes were characterized. Hen egg‐white lysozyme (HEL)‐specific delayed‐type hypersensitivity (DTH) was suppressed when HEL‐induced Ts were transferred into naive mice. These HEL‐induced Ts had surface markers of both Thy‐1 antigen, and I‐J gene products. The suppression of HEL‐specific DTH was greatly increased, when these Ts had been enriched with HEL‐coated petri dishes.

Isolated anti‐HEL antibodies from BIO.BR or A/Sn mice were inoculated into rabbits to induce anti‐cross‐reactive idiotype (CRI) antibodies. The rabbit antisera were extensively absorbed with normal B10.BR or A/Sn immunoglobulins (Igs) and MOPC 104E ascites Igs to render them idiotype (Id) specific. Using these anti‐CRI antibodies, we observed that these Ts possessed Id receptors on their cell surface. Results of both fluorescence techniques and cytotoxicity tests revealed that about 10% of the enriched T cells containing these Ts were Id positive. Moreover, these enriched T cells were substantially killed by anti‐I‐J antiserum plus complement. However, this killing was completely blocked by HEL antigen. These results suggest that both Id receptors and I‐J gene products might be forming the same molecular complexes or might coexist in the vicinity of the molecule.
Mitogenic Effect of the Mannans from Saccharomyces cerevisiae on Mouse Spleen Lymphocytes
Authors: Mikami Takeshi , Nagase Toshihiko , Matsumoto Tatsuji , Suzuki Masuko , Suzuki Shigeo , Kumano Nobuko
Published: November 2013

The DNA synthetic activities of mannans isolated from two Saccharomyces cerevisiae strains were examined in vitro using spleen cells obtained from normal or nude BALB/c strain mice. A highly branched mannan isolated from the S. cerevisiae wild type strain induced a greater increase in mitogenic activity than those displayed by the mannan of the S. cerevisiae X2180–1A‐5 mutant strain which possessed fewer branching moieties. Acid‐hydrolyzed wild type strain mannan with two‐thirds of the molecular weight of the parent intact mannan showed weak mitogenicity.

Increases in the DNA synthetic activities of nude and normal spleen cells were almost the same as that of wild type strain mannan, while nylon wool column‐passed spleen cells obtained from both normal and nude mice did not show mitogenicity with this mannan. The results indicated that the mitogenic activity was responsible for the highly branched structure of the wild type strain mannan, and that this mannan is a B‐cell mitogen.
Effect of Clindamycin on Cytotoxin Production by Clostridium difficile
Authors: Nakamura Shinichi , Mikawa Masato , Tanabe Naomi , Yamakawa Kiyotaka , Nishida Shoki
Published: November 2013

A total of 80 strains of Clostridium difficile, 33 toxigenic and 11 nontoxigenic clindamycin (CLDM)‐sensitive (MIC less than 12.5 μg/ml), and 23 toxigenic and 13 nontoxigenic CLDM‐resistant (MIC 200 to 6,400 μg/ml) were tested for cytotoxin production in the presence of CLDM. None of the 24 nontoxigenic strains produced cytotoxin regardless of the presence of CLDM and only six out of the 56 toxigenic strains showed 16‐ to 64‐fold higher levels of cytotoxic activity in the presence of CLDM at the concentrations of 1/2 to 1/32 of the MIC than in the absence of CLDM; all of the six strains were CLDM sensitive. Further studies revealed that addition of CLDM to the culture caused enhanced cytotoxin synthesis, and that the maximum production of cytotoxin was obtained when CLDM was added to the medium at the time of inoculation or of the ensuing early logarithmic phase. Also, the influence of other antibiotics on the effect of CLDM was examined. Addition of metronidazole, vancomycin, chloramphenicol, cephaloridine, or penicillin, which induced cytotoxin to medium containing CLDM did not increase the effect of CLDM any further. Addition of CLDM to medium containing tetracycline, which inhibited cytotoxin production, induced cytotoxin production but not fully.
Interferon Production Potentials of Various Human Lymphoblastoid Cell Lines
Authors: Matsuyama Masako , Sairenji Takeshi , Yonemura Kensuke , Hinuma Yorio
Published: November 2013

A number of human lymphoblastoid cells were examined concerning their ability to produce spontaneously liberated and virus‐induced interferon (IFN). It was found that, in addition to B cells, various T and nonT‐nonB lymphoblastoid cells responded well to Sendai virus infection to form IFN, the characterization of which has been recently reported (20). One B lymphoblastoid cell line from an infectious mononucleosis (IM) patient produced a large amount of IFN‐α and might become an alternative source of IFN production.

Among 68 cell lines examined, 35 cell lines liberated 10 U/ml or more of IFN spontaneously in culture fluid. The presence of Epstein‐Barr virus (EBV) genome or its activation appears to have no correlation with the spontaneous liberation of IFN. Spontaneously produced IFN from three cell lines was characterized as IFN‐α. Comparatively higher amounts of IFN were produced in cells from IM patients than those from Burkitt's lymphoma cases or healthy adults. Spontaneously produced IFN was detected more easily in cells transformed by EBV alone than in those transformed by EBV and a tumor promoter, TPA.
Gaschromatography of Constitutive Fatty Acids in Mycobacterium leprae
Authors: Kusaka Takashi , Izumi Shinzo
Published: November 2013

A constitutive saturated and monounsaturated fatty acid pattern of Mycobacterium leprae, isolated from the liver of a nine‐banded armadillo with experimental leprosy, was analyzed gaschromatographically and compared with that of cultured M. lepraemurium, M. avium, M. bovis, strain BCG and M. smegmatis. In comparing the fatty acid pattern thus obtained and the known structure of mycolic acids in these mycobacteria, an experiential rule that each species of mycobacteria has a relatively high content of normal (straight‐chained) saturated fatty acid having two more carbons than those of the α‐branch in this species' mycolic acids, coincided well for all mycobacteria tested. In particular, M. leprae was found to contain a relatively high content of behenic acid (n‐C22:0) and the carbon‐number of the α‐branch in this species' mycolic acids is 20 as we previously reported. These data suggested the possibility of simple detection of M. leprae by gaschromatography, and results sustaining this possibility were obtained.
Characterization of Small Plaque Mutants of Mouse Hepatitis Virus, JHM Strain
Authors: Makino Shinji , Taguchi Fumihiro , Hayami Masanori , Fujiwara Kosaku
Published: November 2013

Two small plaque mutants designated as 1a and 2c were isolated from DBT cells persistently infected with the JHM strain of mouse hepatitis virus. Unlike the wild type JHM, these two mutant viruses grew more slowly with no prominent cell fusion. The buoyant densities of the mutants were slightly lower and 2c was revealed to have fewer peplomers than JHM by electron microscopy. The purified JHM contained five polypeptides with molecular weights (M.W.) of 260,000, 105,000 (GP105), 65,000, 60,000 (P60), and 23,000 (GP23). In addition to two polypeptides, P60 and GP23, which were common to JHM and the mutants, 1a was found to contain three other specific polypeptides with M.W. of 180,000 (GP160), 110,000, and 95,000 (GP95), while 2c had GP180, GP105, GP95, and one with a M.W. of 175,000. All of these polypeptides were shown to be glycosylated except for P60. After bromelain treatment, all these viruses lost the peplomers and contained P60 and another new 18,000 dalton polypeptide.
Drug Resistance and R Plasmids in Escherichia coli Strains Isolated from Broilers
Authors: Kanai Hisashi , Hashimoto Hajime , Mitsuhashi Susumu
Published: November 2013

In 1978, 1,021 Escherichia coli strains were isolated from 105 field broilers (F) and 1,058 strains from 106 broilers in a zootechnical experiment station (Z), and their drug‐resistance patterns and the presence of conjugative R plasmids were compared. The resistance markers examined were tetracycline (TC), chloramphenicol (CM), streptomycin (SM), sulfonamides (SA), kanamycin (KM), and ampicillin (APC). The populations of individuals that excreted resistant strains were 100% in F and 58% in Z. Frequencies of isolation of drug‐resistant strains among the total isolates were 93% in F and 36% in Z, indicating that the resistant strains are a rather high proportion of the intestinal flora in F but are slightly less prevalent in Z. The resistance pattern to (TC.SM.SA.KM) was seen at the highest frequency in both groups. Conjugative R plasmids were demonstrated more frequently in field broilers (F). The results reflect the wide use of antibiotics in the livestock industry, resulting in the appearance of drug‐resistant strains mostly due to the presence of R plasmids.
Two Active Forms of Valyl‐tRNA Synthetase from Bacillus subtilis
Authors: Ohyama Kunio , Kaneko Ichiro , Yamakawa Toshio , Ohkuma Seiichi
Published: November 2013

Two enzymatically active forms, E‐I and E‐II, of valyl‐tRNA synthetase [EC 6.1.1.9] from cells at various stages in the life cycle of Bacillus subtilis 168 LTT, germinated cells, vegetative cells (t−0.5), sporulating cells (t0, t1, t3, and t4), forespores and mature spores, were analyzed by hydroxyapatite column chromatography. The E‐II activity was detected in the main fraction of valyl‐tRNA synthetase during the life cycle of B. subtilis 168 LTT. The high activity of E‐II at t0 decreased rapidly in the stationary and sporulating phases. On the other hand, the E‐I activity increased in the early sporulating stage and was about twofold higher at t3 than at t0. After t3, this activity also decreased rapidly and was not detected in forespores and mature spores. The relative amount of E‐I at t0 was 3.4% of the total valyl‐tRNA synthetase activity eluted from the hydroxyapatite column, 12.9% at t1 and 29.2% at t3, but it was less than 10% at t4 and in germinated cells. The alteration in E‐I and E‐II activities was also observed in cells of B. subtilis NIG 1121 (spo+), W23 and 168W, but not in any asporogenous mutant strain studied. These results show that the alteration in the valyl‐tRNA synthetase activity appears only during the early stages of sporulation and is closely related to the sporulation of B. subtilis.
Mode of Protection of Mice against Herpes Simplex Virus Type 2 Infection by Propionibacterium
Authors: Ohashi Masami , Amagai Takashi , Ushijima Tsutomu , Imanishi Jiro , Kishida Tsunataro , Ozaki Yoshikatsu
Published: November 2013

We compared various strains of Propionibacterium with regard to protection of young adult mice against lethal infection with herpes simplex virus type 2 (HSV‐2). Propionibacterium acnes, P. granulosum, and P. avidum were protective, while P. acidi‐propionici and P. lymphophilum were ineffective. The protective effect proved to be in the cell wall fraction. Attempts were made to elucidate possible mechanisms of the protection using both effective and ineffective strains. The results strongly suggest that induction of interferon rather than activation of macrophages and natural killer cells by Propionibacterium pretreatment plays a crucial role, directly or indirectly, in protection against infection by herpes simplex virus. Propionibacterium only moderately protected newborn mice against HSV‐2 infection.
New β‐Lactamase‐Resistant Cephem Treatment of Guinea Pigs Infected with Legionella pneumophila
Authors: Toro Julio , Sawatari Katsuhiko , Kanda Tetsuro , Saito Atsushi , Hara Kohei
Published: November 2013

The in vivo antimicrobial effect of seven new β‐lactamase‐resistant cephems (cefotaxime, latamoxef, ceftazidime, ceftriaxone, cefotiam, cefbuperazone, and MT‐141) on Legionella pneumophila (strain 81–066, serogroup IV) in guinea pigs was compared with that of erythromycin. As the minimal LD100 within one week was about 4.0 × 109 CFU/ml by intraperitoneal injection of the strain, the animals were inoculated with 2.0 ml of twofold dilutions of a suspension of this bacterium. The animals developed purulent peritonitis and systemic involvement demonstrated by the development of periangitis, pneumonia and pleuritis in the lungs. Three different doses of antibiotics were administered intraperitoneally immediately after the rectal temperature reached more than 40 C. Erythromycin had a significant therapeutic effect but none of the new cephems tested death of the infected guinea pigs.
Purification and Characterization of β‐Glucuronidase Inhibitor from Mycobacterium tuberculosis
Authors: Kiyotani Katsuhiro , Tasaka Hiromichi , Matsuo Yoshiyasu
Published: November 2013

Factors inhibitory to β‐glucuronidase were found in the culture filtrate and in a bacillary extract of Mycobacterium tuberculosis H37Rv grown for 6 weeks on Sauton medium. The inhibitors were purified by ammonium sulfate fractionation, treatment with n‐butanol and streptomycin, and chromatography on DEAE‐Sepharose CL‐6B. Two inhibitors were obtained from the culture filtrate. The molecular weights were estimated to be 25,500 and 15,500 by gel filtration on a Sephadex G‐75 column. Three inhibitors were purified from the bacillary extract, two of which were similar to those from the culture filtrate. The molecular weight of the third inhibitor was 21,000. However, the molecular weight of all the denatured inhibitors was 8,600 in the presence of sodium dodecyl sulfate.

The inhibitors contained extremely high amounts of glutamic and aspartic acids and had a highly acidic isoelectric point of pH 2.5. The inhibitors acted noncompetitively against β‐glucuronidase of guinea pig origin at an optimal pH 4.5. β‐Glucuronidases from human peripheral leukocytes and beef liver were partially sensitive to the inhibitors; all the other enzymes tested for sensitivity were unaffected by the inhibitors.
Muramyl Peptides as Possible Endogenous Immunopharmacological Mediators
Authors: Chedid Louis
Published: November 2013

Synthetic N‐acetylmuramyl‐L‐alanyl‐d‐isoglutamine, also called MDP for muramyl dipeptide, is a copy of a fragment of bacterial peptidoglycan. Soon after the recognition of MDP as being the minimal subunit responsible for the activity of Freund's complete adjuvant, a great number of derivatives were synthesized. Because of their very low molecular weight it was hoped that they could retain selectively certain of the numerous effects produced by complex bacterial agents. Evidence was gathered showing MDP's direct effect on lymphocytes and on macrophages. The ensuing studies reviewed that MDP and several of its derivatives have marked immunopharmacological and neuropharmacological activities. Thus, besides being adjuvants, they are capable of producing hyperthermia by acting directly on thermoregulation centers or by inducing in vivo and in vitro endogenous pyrogens (EP). More recently, Krueger et al have shown that slow‐wave sleep (SWS) factor was a muramyl peptide of a molecular weight close to 1,000 daltons. They have also shown that MDP and several of its synthetic analogs had a somnogenic activity.

It has previously been hypothesized that several of the immunological activities of the muramyl peptides could be due to biological mimicry with endogenous products. Recent observations argue in favor of the presence of an MDP bacterial structure in mammalian mediators which increase slow‐wave sleep and/or produce fever. The implications of these findings will be discussed.
Induction of L‐Phase Variant from Protoplast of Staphylococcus aureus
Authors: Makino Toshikazu
Published: November 2013

Protoplasts of Staphylococcus aureus 209P and Cowan 1 were induced by treatment with lysostaphin. These protoplasts were sensitive to detergent, a low concentration of sodium chloride and low temperature.

Almost all protoplast cells spread on CLYS agar medium (casein hydrolysate, yeast extract, Na‐lactate, and NaCl) formed typical L‐form colonies. Horse serum (0.25%) and Mg2+ (109 mm) are essential factors for formation of the L‐form colonies of 209P. In the case of Cowan 1, Mg2+ was not required.

The active factor(s) in horse serum was heat‐resistant and protein in nature.
Incomplete Growth of Varicella‐Zoster Virus in Human Monocytes
Authors: Baba Masanori , Shigeta Shiro
Published: November 2013

Infection of human peripheral blood monocytes by varicella‐zoster virus (VZV) was investigated. When freshly isolated monocytes of young adult volunteers were infected with cell‐free VZV and examined by indirect immunofluorescence, specific antigens appeared at 8 hr and the number of antigen‐positive cells reached the maximum between 24 and 48 hr postinfection. The proportion of antigen‐positive cells to total cells was similar to that of the permissive control (HeLa cells), while very few infectious centers (IC) of monocytes were formed in comparison with the infected control cells. Monocytes isolated from infants and old persons formed a larger number of IC than those of young adults. Electron microscopic study of VZV‐infected monocytes from three young adult volunteers demonstrated that the proportion of VZV particle‐positive cells to total cells was similar to that of antigen‐positive cells, and most of the particles seen in the nuclei were low in density and lacked a central core. These results suggest that the growth of VZV in human adult monocytes is incomplete. This restriction of VZV growth by monocytes may play an important role in defense against VZV infection.
Serogrouping of Rhodococcus equi
Authors: Nakazawa Muneo , Kubo Masanori , Sugimoto Chihiro , Isayama Yasuro
Published: November 2013

The serological relationships among 27 isolates of Rhodococcus equi selected from a total of 1,195 isolates were investigated by cross‐agglutination and absorption tests. The presence of capsular material was demonstrated in all the 27 isolates by electron microscopic observation. Antisera were prepared by employing formalized antigen of each isolate. In the cross‐agglutination test with formalized antigen, 13 antisera reacted with homologous antigens alone, but the remaining 14 antisera reacted not only with homologous antigens but also with one to four heterologous antigens. When these 14 antisera possessing heterologous agglutinins were absorbed with each of the cross‐reacting antigens, 14 specific antisera were obtained. The cross‐agglutination test with these 27 antisera proved the 27 strains examined to be serologically distinct from one another. These strains were designated serogroups 1 to 27. Thus the same number of grouping antisera were prepared.

The distribution of each serogroup among the 1,195 isolates and 15 reference strains was investigated by the slide agglutination test. All the strains were found to be groupable. Most of them belonged to serogroups 1 to 4, 7 to 9, 11, 14, and 15. Of the serogroups designated, 4, 16, 2, 12, 21, 1, and 9 were identical with Prescott's serovars 1, 2, 3, 4, 5, 6, and 7, respectively.
Relationship between Luminous Fish and Symbiosis
Authors: Kuwae Toyoyasu , Andoh Motohiro , Fukasawa Shigeki , Kurata Munetsugu
Published: November 2013

In order to investigate the relationship between host and symbiosis in the luminous marine fish, Physiculus japonicus, the bacterial lipopolysaccharides (LPS) of symbiotic luminous bacteria were compared serologically and electrophoretically.

Five symbiotic luminous bacteria (PJ strains) were separately isolated from five individuals of this fish species caught at three points, off the coasts of Chiba, Nakaminato, and Oharai. LPS preparations were made from these bacteria by Westphal's phenol‐water method and highly purified by repeated ultracentrifugation. These LPSs contained little or no 2‐keto‐3‐deoxyoctonate and had powerful mitogenic activity.

In sodium dodecylsulfate polyacrylamide gel electrophoresis, these PJ‐1 to −5 LPSs were separated by their electrophoretic patterns into three groups; the first group included PJ‐1 and PJ‐4, the second group PJ‐2 and PJ‐3, and the third group PJ‐5 alone. The results agreed with those of the double immunodiffusion test; precipitin lines completely coalesced within each group but not with other groups.

In immunoelectrophoresis, one precipitin line was observed between anti PJ‐2 LPS serum and PJ‐5 LPS but the electrophoretic mobility of PJ‐5 LPS was clearly different from that of the PJ‐2 LPS group. Furthermore, in a 50% inhibition test with PJ‐2 LPS by the passive hemolysis system, the doses of PJ‐2 LPS, PJ‐3 LPS, and PJ‐5 LPS required for 50% inhibition (ID50) in this system were 0.25, 0.25, and 21.6 μg/ml for each alkali‐treated LPS, respectively, and the ID50's of both PJ‐1 LPS and PJ‐4 LPS were above 1,000 μg/ml. These results indicate that PJ‐5 LPS has an antigenic determinant partially in common with LPS from the PJ‐2 group but not with LPS from the PJ‐1 group and that the symbiotic luminous bacterium PJ‐5 is more closely related to the PJ‐2 group than to the PJ‐1 group.

These results show that the species Physiculus japonicus is symbiotically associated with at least three immunologically different strains of luminous marine bacteria in its specialized light organ.
Dose‐Dependent Induction of Immunologic Enhancement and Suppression after Oral Administration of Antigen
Authors: Yasui Hisako , Ohwaki Makoto
Published: November 2013

The effects of feeding various quantities of a particulate antigen, sheep red blood cells (SRBC), on plaque‐forming cells (PFC) in the spleen were determined. Mice were given various numbers of SRBC orally daily for 14 days, then injected with SRBC intravenously. Splenic IgA PFC responses to SRBC were enhanced in the mice fed 5 × 108 SRBC and splenic IgG PFC responses to SRBC were depressed in the mice fed 5 × 109 SRBC.

Adoptive transfer experiments showed that enhancement of splenic IgA PFC responses and suppression of splenic IgG PFC responses were induced by the T‐cell rich fraction from Peyer's patches (PP) and the spleen in 5 × 108 SRBC‐ and 5 × 109 SRBC‐fed mice, respectively. Kinetic studies revealed that IgA helper cells or IgG suppressor cells appeared in PP 2 days after oral administration and 4 days after it in the spleen.
Endogenous Inhibitors of Factor B
Authors: Moriyama Takeshi , Kawamoto Yasuko , Ichikawa Hidetaka , Okamoto Keinosuke , Inoue Takashi , Kobayashi Tatsuhiko , Miyama Akio
Published: November 2013

Proteins which were able to bind noncovalently with mouse factor B were found in cells that are nonsecretors of factor B such as mouse‐established monocytic cells and L cells but not in peritoneal resident macrophages. These proteins were isolated from lysates of L cells and separated into four distinct proteins by preparative SDS‐polyacrylamide gel electrophoresis, with molecular weights of 25K, 28K, 33K, and 35K. The individual proteins formed a complex with purified mouse factor B at a molecular ratio of 1: 1 and inhibited its hemolytic activity. Proteins 25K and 28K inhibited the hemolytic activity of an activated form of factor B combined with cobra venom factor as well as that of the native form. These inhibitors did not affect the hemolytic activity of the second component of complement in mouse serum. The inhibitory activity of the 25K protein was partially inhibited by antiserum raised against it in rabbits.
A Monoclonal Antibody Defining Human B Cell Differentiation Antigen (HLB‐1 Antigen)
Authors: Kasai Kiyoshi , Koshiba Hirofumi , Ishii Yoshifumi , Kikuchi Koukichi
Published: November 2013

A new monoclonal antibody specific for human B cell differentiation antigen (HLB‐1) is produced by a hybridoma established by fusion of splenocytes of mice immunized with the Epstein‐Barr virus (EBV)‐transformed peripheral B cell line, RPMI‐8057. This monoclonal, antibody designated anti‐HLB‐1 monoclonal antibody (anti‐HLB‐1), reacted with surface immunoglobulin (sIg)‐positive B cells of normal peripheral blood and lymphoid tissues and sIg‐positive leukemic cells. The cells of T cell leukemia, non‐T non‐B acute lymphoblastic leukemia (ALL) and nonlymphoid leukemia were HLB‐1 negative. These data were further confirmed by studying a panel of cultured human hematopoietic cell lines. Anti‐HLB‐1 reacted with B cell lines derived from pre‐B, Burkitt's lymphoma, B cell type ALL and EBV‐transformed peripheral B cells. Anti‐HLB‐1 was reactive with only one of three human myeloma cell lines, and with none of the T cell, myeloid and non‐T non‐B ALL cell lines. This newly defined HLB‐1 antigen is different from other conventional human B cell markers such as sIg, Ia antigens, and receptors for the Fc portion of Ig and complement C3.
Immunogenic Dialyzable Factor Derived from a Ribosomal Fraction of Salmonella typhimurium
Authors: Kita Eiji , Tsukao Noriaki , Kitajima Shin , Takahashi Shiro , Kashiba Shuzo
Published: November 2013

An immunogenic dialyzable factor was obtained by dialysis of the freeze‐thawed ribosomal fraction derived from a smooth virulent strain (LT2) of Salmonella typhimurium. Ion exchange chromatography of the dialyzable factor on Dowex 1‐X2 (Cl– form) demonstrated the presence of four peaks and the fourth peak eluted with 0.4 m NaCl in 0.005 n HCl was found to be necessary for protection. This effective peak was not obtained by chromatography of nonprotective dialyzable factors such as an RNase digest. Dowex chromatography of the dialyzable factors isolated from rough mutants of strain LT2 revealed that the dialyzable factor of strain SL1004 whose live vaccine is capable of inducing protective immunity contained fairly large amounts of peak IV. DEAE‐cellulose for two‐dimensional thin layer chromatography was used to identify the composition of the dialyzable factor and peak IV. Eight spots were located under ultraviolet light and seven spots were characterized by their absorption ratios. In peak IV, four nucleotides were located and identified by comparison with a map of the original dialyzable factor. The data show that the effective components of the dialyzable factor are mixed nucleotides and may be unique to ribonucleic acids of strains of S. typhimurium in which live vaccines are capable of affording mouse protection.
In Vitro Induction of Altered Response of Rat Atria to Acetylcholine with Histamine‐Sensitizing Factor of Bordetella pertussis
Authors: Endoh Masahiko , Nakase Yasukiyo
Published: November 2013

Purified histamine‐sensitizing factor (HSF) of Bordetella pertussis induced in vitro an alteration in the pharmacologic response of rat atria to acetylcholine. Spontaneously beating atrial preparations isolated from rats were exposed to HSF at a concentration of 50 ng/ml at 37 C for 30 min to 4 hr and washed with Krebs‐Ringer solution, and then tested at 1‐hr intervals up to 28 hr during incubation for their responses to epinephrine and acetylcholine. At 13 hr after exposure to HSF, irrespective of the exposure period, the HSF‐treated atria, in which positive‐inotropic action of epinephrine was manifested, depressed the negative‐inotropic response to acetylcholine. The activity of HSF was neutralized by anti‐HSF serum only in the first 3 hr of exposure. Only 1.8% of the added 125I‐labeled HSF bound “specifically” to one pair of atria for the manifestation of the altered response.
Chemical and Biological Properties of Lipopolysaccharides from Symbiotic Luminous Bacteria from Several Luminous Marine Animals
Authors: Kuwae Toyoyasu , Kurata Munetsugu
Published: November 2013

The chemical and biological properties of lipopolysaccharides (LPS) in five strains of symbiotic luminous bacteria isolated from four species of luminous marine fishes, Coelorhynchus kishinouyei (CK‐1), Chlorophthalmus albatrossis (CA‐1), Ventrifossa garmani (VG‐1), and Acropoma japonicum (AJ‐1b), as well as from a luminous squid, Doryteuthis kensaki (DK‐1) were examined.

The LPS isolated from these symbiotic luminous bacteria were characterized by the absence of 2‐keto‐3‐deoxyoctonate, known to be a basic component of the usual gram‐negative bacterial LPS.

All LPS from these symbiotic luminous bacteria upon electrophoresis in sodium dodecylsulfate polyacrylamide gel exhibited one or two clear main bands with high mobility, and one or two obscure minor bands with low mobility when stained with periodate‐Schiff reagent. LPS from CA‐1 and VG‐1 exhibited similar electrophoretic patterns, whereas the electrophoretic patterns of the LPS from CK‐1, AJ‐1b, and DK‐1 were easily distinguishable from each other.

All these LPS also had similarly potent and diverse biological activities in regard to their adjuvanticity, immunosuppression, polyclonal effect, B‐cell mitogenicity, and activation of the phagocytic function of macrophages.
A Study of the Taxonomy of the Mycobacterium nonchromogenicum Complex and Report of Six Cases of Lung Infection Due to Mycobacterium nonchromogenicum
Authors: Tsukamura Michio , Kita Nobuhiko , Otsuka Wataru , Shimoide Hisao
Published: November 2013

In numerical classification, four species of the Mycobacterium nonchromogenicum complex, Mycobacterium nonchromogenicum, M. terrae, M. novum, and M. triviale, formed one cluster. These four species appeared to be reduced to one species, Mycobacterium nonchromogenicum. Furthermore, relationships between the species were numerically analyzed by using the hypothetical median organism pattern. The results showed that the M. nonchromogenicum complex can be divided into two subgroups: M. nonchromogenicum and the other three. These two subgroups were differentiated from each other by scores based on two or more positive reactions in the following three characteristics: resistance to bleomycin (5 μg/ml); heat‐stable acid phosphatase activity; nicotinamidase or pyrazinamidase activity or both activities. M. nonchromogenicum gave two or three positive reactions among these three, and M. terrae, M. novum, and M. triviale gave two or three negative reactions.

Three cases of lung infection due to M. nonchromogenicum, as well as three other cases of probable lung infection due to M. nonchromogenicum, were observed in this study. Only one organism isolated from one doubtful case was M. terrae. Up to now, M. nonchromogenicum was considered a nonpathogen. It was shown, however, that this organism causes lung infection in humans.
Penetration of Rickettsia tsutsugamushi into Cultured Mouse Fibroblasts (L Cells): An Electron Microscopic Observation
Authors: Urakami Hiroshi , Tsuruhara Takashi , Tamura Akira
Published: November 2013

The mechanism of penetration of purified Rickettsia tsutsugamushi (Gilliam strain) into cultured mouse fibroblasts (L cells) was examined by electron microscopy. After 10–40 min of infection, rickettsiae in the process of being phagocytized were often seen on the cell surface. These were restricted to the rickettsiae which seemed to be intact in morphology, while heavily plasmolyzed ones were never phagocytized. Additionally, rickettsiae were taken up individually into a phagosome, and phagocytosis of several rickettsiae together was rarely observed, except in the case of heat‐inactivated microorganisms. In the cells, phagosomes whose membranes enclosed rickettsiae either tightly or loosely were seen. Rickettsiae in the loose phagosomes often showed signs of plasmolysis and were rarely released into the cell cytoplasm. Partial disintegration of phagosomal membranes and the escape of rickettsiae from the phagosomes were seen only in tight phagosomes. Large phagosomes containing a clump of several rickettsiae were observed occasionally, in which case the microorganisms were deformed and seemed to be denatured. From the above observations and the frequency of appearance of these different penetration stages in the specimens 10, 20, and 40 min after infection, it was concluded that the rickettsiae enter initially into a tight phagosome by phagocytosis and are then released into the cell cytoplasm by disruption of the phagosomal membrane. No other mechanisms of penetration were found. On the other hand, rickettsiae inactivated by trypsin did not attach to host cells. Inactivation by heat or UV irradiation resulted in reduction of phagocytosis, and rickettsiae treated with rifamycin could penetrate into the host cell cytoplasm to the same extent as in the case of infection with intact rickettsiae.
Rapid Purification of Extracted Bacterial Lipopolysaccharides by Continuous Free‐Flow Electrophoresis
Authors: Kuwae Toyoyasu , Kurata Munetsugu
Published: November 2013

The use of continuous free‐flow electrophoresis for the purification of extracted lipopolysaccharides (LPSs) was investigated. Commercial (nucleic acid contaminated) LPS preparations, isolated by the hot phenol‐water method of Westphal from Salmonella typhimurium and Escherichia coli 0111: B4, were analyzed. Continuous free‐flow electrophoresis for purification of crude LPSs proved to be a rapid and useful means for the continuous purification of large amounts of LPS (more than 45 mg crude LPS per hr) and it showed good reproducibility and pure LPS. The electrophoretic profile of both crude LPSs obtained by continuous free‐flow electrophoresis showed two distinct, sharp peaks; one representing the nucleic acid fraction and the other the LPS fraction. Under the continuous free‐flow electrophoresis conditions employed, nucleic acid in the crude LPSs possessed low electrophoretic mobility, whereas LPS migration was negligible. Thus for both preparations pure LPS (no detectable nucleic acid) was obtained. Electrophoretic profiles of these purified LPSs on sodium dodecylsulfate‐polyacrylamide gel electrophoresis were similar in both cases to those of crude LPS and of LPS purified by repeated ultracentrifugation. By immunological analysis using double immunodiffusion and immunoelectrophoresis, it was found that two components of crude E. coli 0111: B4 LPS were eliminated by continuous free‐flow electrophoresis, but each component of purified E. coli 0111: B4 LPS was immunologically identical to the corresponding component in its crude LPS. In S. typhimurium LPS, none of its components were influenced by continuous free‐flow electrophoresis but not by ultracentrifugation. In spite of these results, both purified LPSs possessed stronger mitogenic activity than each crude LPS. These results indicated that continuous free‐flow electrophoresis is a useful means of purifying extracted crude LPS.