bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationship between β‐Lactamase Activity and Resistance to β‐Lactam Antibiotics in Mycobacterium smegmatis
Authors: Yabu Kunihiko , Kaneda Shunko , Oghiai Toshiro
Published: November 2013

Penicillin‐susceptible mutants and β‐lactamase‐negative mutants were isolated from Mycobacterium smegmatis after nitrosoguanidine mutagenesis. Both the mutants were found to be susceptible to low levels of penicillin and cephalosporins by twofold dilution testing. Clavulanic acid reduced the minimal inhibitory concentrations of β‐lactamase‐labile β‐lactams for the penicillin‐susceptible mutants and the parent strain, but had no effect on the susceptibility of the β‐lactamase‐negative mutants. Comparison of the β‐lactamase activities found in these mutants and the parent strain indicated that there was a rough correlation between the β‐lactamase level in these organisms and their susceptibility to β‐lactams.
Enhancement of Influenza Virus Hemolysis by Physical and Serological Treatments
Authors: Watanabe Yasuo , Tashiro Masato , Kitame Fumio , Homma Morio
Published: November 2013

The mode of hemolysis by influenza A virus was compared with that of Sendai virus. The WSN strain of influenza virus grown in either eggs or MDCK cells expressed hardly any hemolytic activity by itself. Treatment of the MDCK cell‐grown WSN virus with sonication or freezing and thawing moderately enhanced the hemolytic activity, but the maximum level attainable was considerably lower than that of Sendai virus. A high level of hemolytic activity comparable to that of Sendai virus was obtained only after treatment of the virus with antibody and complement. An electron microscopic study revealed that non‐ or low‐hemolytic WSN virions were not permeable to uranyl acetate stain in contrast with the hemolytic virions obtained after treatment with antibody and complement, indicating that the hemolytic virions had sustained some injury to their envelopes. These phenomena were comparable to those found with Sendai virus, showing that damage to the envelope is also responsible for the hemolysis of influenza virus. The influenza viruses, however, remained spherical after every treatment and the stain did not penetrate into the core of the virion. These observations suggest that the envelope of influenza virus is more rigid than that of Sendai virus but that the hemolytic process of influenza virus is nevertheless mediated through envelope‐membrane fusion as in the case of Sendai virus (Y.K. Shimizu et al 1976. Virology 71: 48–60).
Epidemiological Studies on the Background of the Endemic Occurrence of Tsutsugamushi Disease in Toyama Prefecture
Authors: Ishikura Mitsuhiro , Watanabe Mamoru , Morita Osayuki , Uetake Hisao
Published: November 2013

In order to clarify the epidemiological background of the endemic occurrence of tsutsugamushi disease in Toyama Prefecture, Japan, since 1978, comparative surveys have been carried out between endemic and nonendemic areas. Rickettsia tsutsugamushi (Rt) was isolated at a rate of about 36% (158/439) from field rodents in the endemic area while it was not isolated from any of 280 in nonendemic areas. In all of six stations in the endemic area, a significantly high proportion of rodents were found to be Rt carriers. However, no Rt was isolated from rodents captured from July to September. The organism was isolated from rodents captured in the other months, especially in a high proportion in November when infestation of rodents with Leptotrombidium pallidum was at its peak. When the rodents were examined by indirect immunofluorescence staining, the rate of anti‐Rt antibody‐positive animals was about 55% (157/287) and about 17% (62/368) in endemic and nonendemic areas, respectively. Larvae of mites collected from the rodents were found to belong to four genera and 11 species. Among them L. pallidum was the only mite that had been known to be a vector of Rt. L. pallidum was found most frequently and in abundance from rodents in the endemic area, whereas it was present in very small numbers in rodents in nonendemic areas. The infestation of rodents with L. pallidum showed a seasonal variation, i.e. two peaks per year, in spring and autumn, and the number of mites detected was markedly greater in November than in spring. Rt was isolated from L. pallidum on rodents captured in the endemic area.
Distribution of the Level of Antibody to AIDS‐Associated Virus (LAV) in Sera from AIDS or AIDS Related Complex and Japanese Hemophiliacs Infected with AIDS‐Associated Virus
Authors: Tsuchie Hideaki , Kawatani Toshio , Nakayama Erika , Matsui Toshio , Kurimura Takashi , Hinuma Yorio
Published: November 2013

The level of antibody to AIDS‐associated virus (LAV) in sera from patients with AIDS or AIDS related diseases (AIDS related complex; ARC) and Japanese hemophiliacs was studied using indirect immunofluorescence. Titer of anti‐LAV antibody in sera from 89 patients with AIDS or ARC ranged between 10 and 40,960 (median: 1,280) and that of 83 Japanese hemophiliacs ranged from 20 to 20,480 (median: 640). The distribution of the level of anti‐LAV antibody in hemophiliacs was similar to that of patients with AIDS or ARC, and no correlation between the titer of antibody and the presence of symptoms of AIDS was observed. Our results suggested that hemophiliacs in Japan might have been infected with live LAV rather than been immunized with inactivated LAV by injection of factor VIII or IX concentrate, and more hemophiliacs in Japan might show symptoms of AIDS in the future as in the United States.
Synthesis and Deposition of Spore Coat Proteins during Sporulation of Bacillus megaterium
Authors: Imagawa Masayoshi , Oku Yuichi , El‐Belbasi Hussein I. , Teraoka Mie , Nishihara Tsutomu , Kondo Masaomi
Published: November 2013

Rabbit (anti‐spore coat protein) IgG was prepared by immunization with coat proteins extracted with sodium dodecyl sulfate and dithiothreitol from isolated spore coats of Bacillus megaterium ATCC 12872. Coat proteins were detected from 3 hr after the end of exponential growth (t3) in the mother cell cytoplasmic fiaction by sandwich enzyme immunoassay using this antibody. The proteins in the forespore coat protein fraction increased from t3 and reached a plateau at t10. Immunoblot analysis for the coat proteins in sporulating cells revealed the sequential synthesis of various proteins in the mother cell cytoplasmic fraction and simultaneous deposition of the same proteins as in the forespore coat fraction. These results suggest that turnover of precursor proteins of the spore coat is very rapid if precursor proteins are produced and they are proteolytically processed to produce mature proteins. Specific antibody to the 48,000‐dalton protein, which is a major protein, did not cross‐react with any other major (36,000, 22,000, 19,500, and 17,500‐dalton) proteins. Specific antibody to the 22,000‐dalton protein did not cross‐react with the 48,000, 36,000, 19,500, 17,500, and 16,000‐dalton proteins, but did cross‐react with the 44,000, 25,000, and 12,000‐dalton proteins.
The Role of NK Cell Activity in Age‐Dependent Resistance of Mice to Murine Cytomegalovirus Infection
Authors: Hayashi Kozaburo , Eizuru Yoshito , Sato Shoichiro , Minamishima Yoichi
Published: November 2013

The resistance of mice to cell culture passaged murine cytomegalovirus (CC‐MCMV) infection developed with age. In parallel with this finding, augmentation of the splenic NK cell activity in older mice was always higher than that of younger mice. The splenic NK cell activity reached the maximum level at 6 day post infection (PI) in 2–4‐week‐old mice while in 6–8‐week‐old mice it peaked at 4 days PI. When the dose of CC‐MCMV was increased, the NK cell activity was potentiated accordingly. However, it was decreased on the infection with increased doses of the salivary gland passaged MCMV (SG‐MCMV).

NK cells augmented by MCMV infection actually inhibited in vitro replication of MCMV when they were added to mouse embryonic fibroblast (MEF) monolayers infected with CC‐MCMV. Splenic and peritoneal macrophages inhibited in vitro replication of MCMV, but their activities were less potent than those of NK cells.
Active Substance of Histoplasma capsulatum that Inhibits Blastogenic Response of Lymphocytes
Authors: Yamamoto Yoshimasa , Jingu Shigeji , Iwata Kazuo
Published: November 2013

A substance inhibiting blast transformation of murine spleen lymphocytes stimulated with various mitogens, such as LPS, PHA, and PWM, was obtained from yeast‐form cells of Histoplasma capsulatum. This active substance was partially purified from the cell‐free extract by DEAE‐Sepharose CL‐6B column chromatography. As a result of this partial purification, the inhibitory activity was 1.26 μg/ml in terms of ID50. Materials from H. capsulatum also inhibited blast transformation of murine spleen lymphocytes stimulated with the antigen PPD as well as mitogens LPS, PHA, and PWM. However, the con A‐induced proliferative response was only slightly affected. A similar result was observed for the MLR. These inhibitory activities were abolished by heating at 70 C for 30 min. These results suggest that the heat‐labile active substance produced by H. capsulatum might directly affect the lymphocytes, leading to inhibition of their blast transformation.
Stability of the Hexagonal Lattice Structure Formed by an R‐Form Lipopolysaccharide of Klebsiella: Decrease in the Stability by Electrodialysis and Recovery by Addition of the Magnesium
Authors: Kato Nobuo , Ohta Michio , Kido Nobuo , Ito Hideo , Naito Setsuko , Kuno Tsuneharu
Published: November 2013

The R‐form lipopolysaccharide (LPS) from Klebsiella strain LEN‐111 (O3‐:K1‐) forms a hexagonal lattice structure with a lattice constant of 14 to 15 nm when it is precipitated by addition of two volumes of 10 mM MgCl2‐ethanol. When the LPS was suspended in various buffers (50 mM) at pH 2 to 12 for 24 hr at 4 C, at pH 2 and 3 pits of the hexagonal lattice structure markedly disappeared, at pH 4 to 8.5 the lattice structure was stable, and at pH 9 to 12 it tended to loosen somewhat. The LPS from which cations were removed by electrodialysis retained the ability of hexagonal assembly, although the lattice constant of the hexagonal lattice of the electrodialyzed LPS was large. The lattice structure of the electrodialyzed LPS was much more labile than that of the non‐electrodialyzed LPS at alkaline pH levels and the former was completely disintegrated into ribbon‐like structures when the LPS was suspended in 50 mM Tris buffer at pH 7.7 or higher. However, the electrodialyzed LPS formed a hexagonal lattice structure in Tris buffer at pH 8.5 containing 0.1 to 100 mM MgCl2. The lattice constants of the hexagonal lattice formed by the electrodialyzed LPS at 10 or 100 mM MgCl2 were very similar to that of the lattice of the non‐electrodialyzed LPS. From these results it is concluded that the lability of the hexagonal lattice structure of the electrodialyzed LPS at alkaline conditions is due to removal of Mg2+ by electrodialysis.
Suppressive Effect of Ultraviolet‐B‐Irradiation of Epidermal Cells on the Induction of Contact Sensitivity
Authors: Tsuchida Tetsuo , Takemura Masahiko , Qian Ja‐Hua , Izumi Yutaka , Mizuochi Toshiaki , Maekawa Ryuji , Fujiwara Hiromi , Hamaoka Toshiyuki
Published: November 2013

Contact sensitivity to trinitrophenyl (TNP) hapten was induced by subcutaneous (s.c.) administration of TNP‐modified syngeneic spleen cells or epidermal cells (EC) (TNP‐EC). Intraperitoneal (i.p.) inoculation of TNP‐EC resulted in a comparable response, whereas i.p. administration of TNP‐spleen cells or TNP‐modified‐ultraviolet (UV)‐preirradiated EC (TNP‐UV‐EC) failed to induce TNP‐contact sensitivity responses. The present study investigates the effect of UV‐irradiation on the potential of EC for inducing the contact sensitivity response. Exposure of BALB/c mouse EC in vitro to 1600 J/m2 of UV‐B before they were modified with TNP had no discernible effect on the Ia‐positivity and viability of EC. Coexistence of TNP‐UV‐EC had no inhibitory effect upon the contact sensitivity response induced by TNP‐EC via the i.p. route. The absence of suppressor cell generation was substantiated by the adoptive transfer of spleen cells from mice administered TNP‐UV‐EC i.p. to normal syngeneic mice. The effect of interleukin 1 (IL‐1) or epidermal cell‐derived thymocyte‐activating factor (ETAF) in restoring the ability of TNP‐UV‐EC to induce contact sensitivity was examined. IL‐1 or ETAF administered along with TNP‐spleen cells i.p. induced a potent contact sensitivity response, whereas the same preparations of IL‐1 or ETAF were unable to restore the contact sensitivity induction by TNP‐UV‐EC. The results are discussed in the context of UV‐induced cell surface changes of the Langerhans cell population.
Biochemical Differentiation between Enterotoxigenic Heat‐Sensitive and Heat‐Resistant Clostridium perfringens Strains
Authors: Tórtora João C.O. , Pinheiro Márcia S. , Pitard Maria C.
Published: November 2013

Some biochemical characteristics of 37 enterotoxigenic Clostridium perfringens strains isolated from human feces, ground beef, and soil samples by heat‐selection methods and of two NCTC strains were studied. Two different biochemical patterns closely related to the heat resistance of the strains were found. The strains placed into group 1 were trehalose, inositol, and sorbitol negative and synthesized heat‐resistant spores, while those placed into group 2 were trehalose and inositol positive and synthesized heat‐sensitive spores. Sorbitol fermentation was variable among the strains of this last group. The strains of group 1 were more cellobiose, melibiose, and salicin fermentative than those of group 2. Only the strains placed into group 2 synthesized toxins of sufficient levels for typing. In spite of having been isolated by mild heat treatment of the specimens, two strains showed the same biochemical and toxigenic characteristics of the strains of group 1. The heating of these two strains did not modify their characteristics. We conclude that enterotoxigenic C. perfringens strains showing the two different toxigenic and biochemical patterns are present in the human gut, ground beef, and, probably, in soil. These strains may be differentiated on the basis of their capacity to produce acid from trehalose, inositol, and sorbitol, heat resistance of the spores and grade of toxigenicity. The heat‐selection methods used for isolation of C. perfringens strains from different sources exerted a selection of strains from one or another group, but had no influence on their toxigenic and biochemical properties.
Protection of Mice against Herpes Simplex Virus Infection by a Lactobacillus casei Preparation (LC 9018) in Combination with Inactivated Viral Antigen
Authors: Watanabe Takashi , Saito Hajime
Published: November 2013

Heat‐killed Lactobacillus casei YIT 9018 (LC 9018) cells enhanced the resistance to herpes simplex virus type 1 (HSV‐1) in adult mice, but not significantly. The protection of mice against HSV‐1 infection and the production of neutralizing antibodies were significantly enhanced by the administration of LC 9018 in combination with inactivated HSV‐1 antigen. The optimal enhancement of resistance was seen in mice 14 days after the simultaneous administration of these substances. The resistance to HSV‐1 infection in mice could be transferred with peritoneal exudate cells from syngeneic mice previously treated with LC 9018 alone and LC 9018 in combination with inactivated HSV‐1 antigen or with thioglycollate broth, whereas the transfer of peritoneal exudate cells induced by thioglycollate broth alone and of spleen cells induced by LC 9018 in combination with thioglycollate broth or by thioglycollate broth alone was not effective. These results suggest that mouse peritoneal macrophages induced by the administration of LC 9018 in combination with inactivated HSV‐1 antigen may play an important role in host defense mechanisms against HSV‐1 infection.
The Production of a Cytotoxic Factor by Mouse Peritoneal Macrophages and Macrophage Hybridomas Treated with Various Stimulating Agents
Authors: Takeda Yasuhisa , Higuchi Masahiro , Sugimoto Masamichi , Shimoda Olina , Woo Hee Jong , Shimada Shigetoshi , Osawa Toshiaki
Published: November 2013

Murine peritoneal macrophages elicited with a streptococcal preparation, OK‐432, produced as much of a cytotoxic factor after stimulation with lipopolysaccharide (LPS) as BCG‐elicited macrophages did. Proteose peptone‐elicited macrophages produced a very small amount, if any, of the factor, and resident peritoneal macrophages did not release it at all even after LPS‐stimulation. A newly established macrophage hybridoma, D/O‐3.3, produced the factor after LPS‐stimulation, but another hybridoma, D/O‐3.2, did not. Experiments using these peritoneal macrophages and macrophage hybridomas demonstrated that macrophages can be divided into three subpopulations with regard to stages of activation for production of the cytotoxic factor. The first is fully activated macrophages which produce the factor after stimulation with LPS or MAF‐C alone, the second is partially activated macrophages which produce the factor only after stimulation with a combination of recombinant interferon‐γ (rlFN‐γ) and LPS or rIFN‐γ and macrophage activating factor for cytotoxicity (MAF‐C), and the third is nonactivated macrophages which cannot produce the factor at all.
Specific Binding Sites for Monomeric and Aggregated β2‐Microglobulin on Surface of Groups A, B, C, and G Streptococci
Authors: Chhatwal Gursharan S. , Dutra Iveraldo S. , Blobel Hans
Published: November 2013

Human β2‐microglobulin (β2‐m) was isolated from urine samples of patients with tubular dysfunctions and aggregated with glutaraldehyde. Four aggregates with molecular weights of 800,000, 480,000, 260,000, and 60,000 were separated by filtration on Sephacryl S‐300. The aggregates and monomeric β2‐m (11,800 MW) were subsequently labeled with 125I and tested for binding to streptococci. Group A streptococci bound only aggregated β2‐m with a mean binding of 44.5%. Most of the group G streptococci, on the other hand, bound only monomeric β2‐m with a mean binding of 58%. Among group B streptococci the serotypes with protein antigens interacted mainly with monomeric β2‐m and those without protein antigens preferentially with aggregated β2‐m. Nontypable group B streptococcal serotypes did not bind monomeric or aggegated β2‐m. Of the streptococci belonging to group C, S. equisimilis reacted with monomeric β2‐m and S. dysgalactiae with aggregated β2‐m. S. equi did not interact with monomeric β2‐m or aggregated β2‐m. Bindings of monomeric β2‐m and aggregated β2‐m were saturable and could be inhibited by the respective unlabeled forms of β2‐m. Fibrinogen, fibronectin, α2‐macroglobulin, haptoglobin, or immunoglobulin G did not inhibit the binding of either form of β2‐m. The binding sites for monomeric β2‐m were more susceptible to trypsin than those for aggregated β2‐m. Treatment of streptococci with pronase destroyed their binding activities for monomeric and aggregated β2‐m. Both monomeric β2‐m and aggregated β2‐m binding sites were sensitive to heat. The Scatchard plots of monomeric β2‐m and aggregated β2‐m were linear with Kd of 1.29 × 10−9 M and >1.9 × 10−9 M respectively. The number of binding sites per bacterium were estimated to be 81,000 for monomeric β2‐m and 1,210 for aggregated β2‐m.
Antigen Presentation by Human Antigen‐Presenting Cells to Antigen‐Specific Xenogeneic Murine T Cells
Authors: Yabu Kouji , Yano Akihiko
Published: November 2013

Successful antigen presentation by xenogeneic human antigen‐presenting cells (APC) to stimulate the proliferation of antigen‐specific, keyhole limpet hemocyanin (KLH)‐specific, ovelbumin (OVA)‐specific, and purified protein derivative of Mycobacterium tuberculosis (PPD)‐specific murine T cells was observed. Evidence indicating a direct cell interaction between antigen‐specific murine T cells and xenogeneic human APC was given by experiments using antigen‐specific murine T cell clones. The OVA‐specific B10.S(9R) T cell line (9‐0‐A1) and PPD‐specific B10.A(4R) T cell line (4‐P‐1) were stimulated by both xenogeneic human APC and murine APC from syngeneic or I‐A compatible strains, while the PPD‐specific human T cell line (Y‐P‐5) was stimulated by autologous human APC but not by murine APC. Anti‐HLA‐DR monoclonal antibodies (MoAb) blocked the xenogeneic human APC‐antigen‐specific murine T cell clone interaction. Thus, human xenogeneic APC can stimulate antigen‐specific murine T cells through HLA‐DR molecules in the same manner as syngeneic murine APC do through Ia molecules coded for by the I region of the H‐2 complex, while murine APC failed to present antigen to stimulate human antigen‐specific T cells.
Effects of Lactobacillus casei on Pseudomonas aeruginosa Infection in Normal and Dexamethasone‐Treated Mice
Authors: Saito Hajime , Watanabe Takashi , Horikawa Yoshiya
Published: November 2013

A single intraperitoneal injection of Lactobacillus casei YIT 0003 into normal or dexamethasone‐treated mice led to nonspecific resistance against intraperitoneal challenge with lethal doses of Pseudomonas aeruginosa PAO 3047. The enhanced resistance was retained for 14 days (P<0.05) after injection with living L. casei. In contrast, the statistically significant duration of the enhanced resistance in mice treated intraperitoneally with living L. acidophilus YIT 0075 was only 5 days. The in vivo killing activity of peritoneal exudate cells (PECs) against P. aeruginosa 5 and 7 days after intraperitoneal injection of living L. casei was significantly higher than in the case of PECs elicited by L. acidophilus. In the case of intravenous injection of heat‐killed L. casei before intraperitoneal challenge with P. aeruginosa, there were no survivors in the late period after administration of L. casei. A high correlation existed between the patterns of in vivo killing of P. aeruginosa by PECs and survival rate of mice injected intravenously with heat‐killed L. casei. The reduced in vivo killing activity of PECs from dexamethasone‐treated mice against P. aeruginosa infection was also augmented by the intraperitoneal injection of heat‐killed L. casei. These results indicate that L. casei possesses a resistance‐enhancing capacity against P. aeruginosa infection in vivo. Differences in the duration of the enhanced resistance caused by L. casei and by L. acidophilus may be due to differences in chemical composition and/or physicochemical properties of the cell walls of the two kinds of bacteria.
Characterization of an Echovirus Type 30 Variant Isolated from Patients with Aseptic Meningitis
Authors: Matsumoto Kazuo , Yokochi Takashi , Matsuda Susumu , Itoi Yoshiyuki , Kimura Yoshinobu
Published: November 2013

An outbreak of echovirus type 30 infection associated with aseptic meningitis occurred among newborn babies in a hospital neonatal room at Fukui City in 1983. The isolated virus was identified as an antigenic variant of echovirus type 30 by cross‐neutralization tests with antisera against the prototype Bastianni strain and the present isolate. Western blot analysis demonstrated that the antigenic determinants responsible for virus neutralization, some of which were type specific and others strain specific, were present on the capsid protein VP1. The current strain was more thermoresistant than the prototype, suggesting some alterations in virus structural proteins.
New Monoclonal Antibodies That Define Multiple Epitopes and a Human‐Specific Marker on the Interleukin 2 Receptor Molecules of Primates
Authors: Tanaka Yuetsu , Inoi Takeshi , Tozawa Hideki , Sugamura Kazuo , Hinuma Yorio
Published: November 2013

Twelve hybridoma cell lines producing monoclonal antibodies to the human interleukin 2 (IL‐2) receptor (IL‐2R) molecule were prepared. These antibodies were characterized by competitive antibody‐binding assay and sequential immunoprecipitation assay with four known monoclonal antibodies to the human IL‐2R molecule. The twelve new monoclonal antibodies were divided among the four known antibody types, the HIEI‐, H‐A26‐, H‐31‐, and anti‐Tac‐type, and an additional new type, the H‐48‐type. The H‐48 antibody did not compete with any other antibodies in the competitive binding assay. The binding of 125I‐IL‐2 to MT‐2 cells and the IL‐2‐dependent growth of normal activated T‐cells were both strongly inhibited by all the H‐31‐ and anti‐Tac‐type antibodies, and partially or slightly inhibited by HIEI‐ and H‐A26‐type antibodies, but were not inhibited by the H‐48 antibody. Thus, the same type of monoclonal antibodies had a similar effect on the function of IL‐2R. These results suggest that epitopes for the same type of antibodies could be single identical epitopes or epitopes closely associated with each other. On the other hand, these antibodies also reacted variously with a panel of various human and simian lymphoid cell lines immortalized with human T‐cell leukemia virus type‐I (HTLV‐I): the H‐45 antibody reacted only with the human cell lines, the H‐Cl and H‐44 and H‐47 antibodies reacted with human and ape cell lines, and the other antibodies reacted with cell lines of humans, apes and Old and New World monkeys. These differences in the reactivity of the antibodies with the primate cell lines suggest that the antigenic structure of the IL‐2R molecule changed during evolutionary divergence of the primates.
Mechanisms of Plasmid‐Mediated Antibiotic Resistances in Vibrio parahaemolytcus
Authors: Hasegawa Hiroko , Hamashima Hajime , Arai Taketoshi
Published: November 2013

The mechanisms of drug resistance of clinical isolate, Vibrio (V.) parahaemolyticus ST550, resistant to chloramphenicol (CP), aminoglycoside antibiotics (AGs) and β‐lactam antibiotics were investigated. The mechanisms of resistance to CP, AGs and β‐lactam antibiotics were dependent on chloramphenicol acetyltransferase (CAT), aminoglycoside‐3″‐adenylyltransferase AAD(3″) and aminoglycosides‐3′‐phosphotransferase APH(3′) and TEM type penicillinase, respectively.
Number of Hits Necessary for Complement‐Mediated Hemolysis
Authors: Takeda Junji , Kozono Haruo , Takata Yuko , Hong Kyongsu , Kinoshita Taroh , Sayama Koji , Tanaka Etsuko , Inoue Kozo
Published: November 2013

The number of hits necessary for the C8 and C9 steps of immune hemolysis was reexamined with a previously unemployed experimental design, in which various numbers of EAC1–7, excess of the supplementary component and a constant amount of the component tested were incubated in a constant volume (Inoue et al. 1976. Infect. Immun. 13: 337). Our results were consistent with previous findings; the steps of guinea pig C8 and C9, and human C8 each followed a one‐hit mechanism, while that of human C9 showed a multi‐hit response. When lysis of sensitized erythrocytes (EA) by normal human serum was analysed in a similar way, one‐hit curves were obtained. This result, taken together with the above results, suggests that immune hemolysis occurs by a single lesion including a single C8 and multiple C9 in the case of human complement and that normal human serum contains sufficient excess of C9. On the other hand, when C9‐deficient human serum was used for lysis of EA, multiple‐hit curves were obtained. The mechanism of lysis by C5b‐8 may differ from that by C5b‐9.
Survey of Modifying Enzymes and Plasmids in Amikacin‐Resistant Serratia marcescens
Authors: Katoh‐Kanno Rieko , Kimura Mitsuko , Ikeda Tatsuo , Kimura Sadao
Published: November 2013

Forty amikacin‐resistant strains of Serratia marcescens isolated from four different hospitals (A, B, C, and D) were examined for modifying enzymes and plasmids. Twenty‐one of the isolates produced acetyltransferase that modified amikacin. Eighteen of the 21 acetyltransferase‐bearing isolates were from different inpatients in hospital A and the other three were from hospital C. Amikacin resistance was mediated by conjugative plasmid of 24 megadaltons in 15 of the 18 acetyltransferase‐bearing isolates of hospital A and by nonconjugative plasmids, derivatives of the 24‐megadalton plasmids, in the remaining three isolates of the same hospital. The 24‐megadalton plasmid determined aminoglycoside acetyltransferase (6′) IV. This plasmid‐borne enzyme conferred amikacin resistance on S. marcescens but not on Escherichia coli K12. The frequency of transfer of the 24‐megadalton plasmid from the S. marcescens isolate to E. coli K12 by conjugation was approximately 10‐7 (transconjugants/donors) and was 0.1% of that between E. coli strains. In acetyltransferase‐bearing isolates from hospital C, the enzyme was mediated by a nonconjugative plasmid in one case and could not be associated with a plasmid in the remaining two cases. Neither enzymes nor plasmids could be associated with amikacin resistance of the isolates of the other two hospitals.
A Novel Rat Monoclonal Antibody Directed against a Population of Activated Mouse Peritoneal Macrophages
Authors: Someya Akira
Published: November 2013

A hybridoma clone secreting rat monoclonal antibody (MAB) designated as 3F3.5F and which reacted with a population of activated tumoricidal mouse peritoneal macrophage (MΦ) was produced by the fusion of mouse myeloma cells with rat spleen cells immunized against adherent BCG‐activated mouse peritoneal exudate cells (adherent BCG‐PEC). The antibody was cytotoxic and of the rat IgM class. The specific reactivity of the antibody with mouse primary cells and cell lines was examined by complement‐dependent cytotoxicity and indirect immunofluorescence flow cytometry analysis. The antibody was found to bind to about 40% of the adherent BCG‐PEC activated in vivo and elicited peritoneal macrophages activated in vitro by lymphokine and lipopolysaccharide (LPS), to about 35% of polymorphonuclear neutrophils (PMN) 15 hr after intraperitoneal injection of BCG, to about 30% of bone marrow cells from BCG‐infected mice, to about 10% of P815 mastocytoma cells and to thioglycollate‐induced PEC to some degree. It did not bind to other cells tested including BCG‐induced peritoneal lymphocytes, non‐tumoricidal PEC, thymocytes, spleen cells, resting bone marrow cells from normal mice, lymphomas, myelomas, fibroblasts, or macrophage‐cell lines. Pretreatment of adherent BCG‐PEC with MAB 3F3.5F and rabbit complement caused a considerable decrease in tumor cytotoxicity toward P815 cells, but the same pretreatment of non‐adherent BCG‐PEC had no inhibitory effect on natural killer activity for YAC‐1 cells.
Age‐Specific Prevalence of Hepatitis B e Antigen (HBeAg) and Antibody to HBeAg (Anti‐HBe) among Asymptomatic Hepatitis B Surface Antigen (HBsAg) Carriers in Okinawa and Kyushu, Japan
Authors: Kashiwagi Seizaburo , Hayashi Jun , Nomura Hideyuki , Kajiyama Wataru , Ikematsu Hideyuki , Shingu Toshimitsu , Hayashida Kazuo , Kaji Masaro
Published: November 2013

A total of 1,741 asymptomatic hepatitis B surface antigen (HBsAg) carriers in two areas (Okinawa and Kyushu) in Japan were surveyed for the presence of hepatitis B e antigen (HBeAg) and the corresponding antibody (anti‐HBe) to determine the age‐specific prevalence of these markers and the mean age of carriers with HBeAg. Prevalence of HBeAg was significantly higher in Kyushu (36.4% of 755 carriers) than in Okinawa (20.0% of 986 carriers) (P<0.001). The mean age of carriers with HBeAg was 25.5 years in Kyushu and 16.1 years in Okinawa, suggesting that HBeAg converted to anti‐HBe earlier in Okinawa than in Kyushu. In contrast, the prevalence of anti‐HBe was significantly higher in Okinawa (74.6% of 986) than in Kyushu (56.3% of 755) (P<0.001). The prevalence of HBeAg decreased with age up to 40–49 years of age and then increased in both areas. Prevalence of anti‐HBe was inversely related to the prevalence of HBeAg in both areas. These data suggest that HBeAg and anti‐HBe are chronological markers of chronic hepatitis B virus infection and that the duration of HBeAg persistence can be different in different area, even in the same country.
Plasmid‐Like Properties of the Four Virulence‐Associated Factors of Yersinia pestis
Authors: Tsukano Hiroko , Wake Akira , Sakakibara Yoshimasa
Published: November 2013

The virulence‐associated factors of Yersinia pestis, which determine the abilities to produce pesticin I (Pst+), capsular fraction I antigen (Fra+), V and W antigen complex (Vwa+) and a cell‐surface component for adsorption of exogenous pigments (Pgm+), were independently eliminated by cultivation of the cells in the presence of acridine orange, ethidium bromide or sodium dodecyl sulfate at a subinhibitory concentration. A virulent Y. pestis strain, Yreka, harbored at least five extrachromosomal DNA molecules of different sizes. In these molecules, a novel 13‐megadalton DNA which was cured concomitantly with the elimination of the Fra factor was found, in addition to the known species of 7 and 44 megadaltons which were lost with the conversions to Pst‐ and Vwa‐, respectively. Although the conversion to Pgm‐ could not be correlated with the lack of any proper extrachromosomal DNA, the factor was transmitted to Pgm‐ cells with the aid of self‐conjugative RP4 plasmid. The cells acquiring the Pgm factor regained virulence for mice.
Structural Changes in the Nucleoid of Bacillus subtilis at Low Temperature
Authors: Amako Kazunobu , Takade Akemi , Umeda Akiko
Published: November 2013

The external shape of the nucleoid of Bacillus subtilis strain w23 was examined with a new electron microscopic technique, the rapid freezing and substitution fixation method. The nucleoid of the log and stationary phase cells was recognized as an area devoid of ribosomes and widely dispersed in the cytoplasm, which was different from that observed in OsO4‐fixed cells. If the bacteria were exposed to low temperatures (0 to 10 C), the nucleoid showed a highly concentrated shape in the middle of the cytoplasm. These structural changes were observed only when the bacteria were maintained in a high‐salt buffer. The results are discussed in relation to the membrane fluidity at low temperature.
Stimulation of Macrophages and Antitumor Activity of Radiodetoxified Endotoxin
Authors: Nerkar Devdas Punjaji , Bandekar Jayant Ramchandra
Published: November 2013

Lipopolysaccharide of Salmonella typhimurium was irradiated with gamma radiation at 10, 15, and 30 kGy doses. A dose of 30 kGy significantly detoxified the LPS (180 times). Mice were injected intraperitoneally with the radiodetoxified LPS, and it was found that it stimulated peritoneal macrophages as was evident from the enhancement of their acid hydrolases and cellular RNA content. Both LPS and radiodetoxified LPS exhibited antitumor activity against S180 cells in Swiss mice. Treatment with 20 μg/mouse of either LPS or 30 kGy LPS gave maximum survival of the mice (90%). These mice were found to resist the challenge of S180 cells (1 × 106).
Metabolic Activation of 1–Nitropyrene and 1, 6–Dinitropyrene by Nitroreductases from Bacteroides fragilis and Distribution of Nitroreductase Activity in Rats
Authors: Kinouchi Takemi , Ohnishi Yoshinari
Published: November 2013

Nitrated polycyclic aromatic compounds, 1–nitropyrene (1–NP) and 1,6‐dinitropyrene (1,6–diNP), are environmental mutagens and carcinogens. Nitroreductases purified from an anaerobic bacterium, Bacteroides fragilis, catalyzed the metabolic activation of these compounds to produce DNA‐ and tRNA‐bound adducts in vitro. Formation of the adducts was inhibited by p‐chloromercuribenzoic acid, which is an inhibitor of nitroreductases from B. fragilis. The enzyme and coenzyme (NADPH) were essential for the adduct formation. These results suggest that nitroreduction is a necessary step in the metabolic activation of nitropyrenes. 1–NP bound specifically to poly(G) and poly(dG), and 1,6–diNP bound to poly(G), poly(dG), and poly(X). The other purine polynucleotides were weak acceptors. However, the reactive products of nitropyrenes formed by nitroreductases could not bind to pyrimidine polynucleotides. Enzymatic hydrolysis of 1–NP‐bound DNA and subsequent analysis by high‐performance liquid chromatography showed one major and two minor adducts in the hydrolysate. The peak of the major adduct corresponded to that of N‐(deoxyguanosin‐8–yl)‐1‐aminopyrene, which is the same as an adduct formed by xanthine oxidase, a mammalian nitroreductase. Nitroreductase activity in the various organs and intestinal contents of Sprague‐Dawley rats was assayed in the presence of NADPH or NADH under nitrogen gas. Nitroreductase activity was widely distributed in the organs of the rats; in particular, that of the liver and of the small intestine was relatively high, but that of the respiratory organs such as lung and alveolar macrophages was very low. Intestinal contents had high nitroreductase activity, which was proportional to the number of bacteria, especially anaerobic bacteria, in the intestine. These results suggest that the nitroreductase activity of the normal bacterial flora is very high in rats and that the intestinal bacteria play a major role in the metabolism of nitropyrenes in vivo.
Effect of Mannitol and Glucose on the Distribution and Trypsin Susceptibility of Protein A of Staphylococcus aureus
Authors: Yamada Sakuo , Matsumoto Akira
Published: November 2013

The biological activity and morphological distribution of protein A on the cell surface were studied in a medium containing an excess of either mannitol or glucose, which suppressed protein A production of Staphylococcus aureus, Cowan I strain. Preculture of the organisms in the presence of a sugar suppressed the expression of protein A, resulting in a decrease in the number of cells bound with anti‐ferritin rabbit IgG molecules, which specifically indicate protein A distribution. The distribution pattern of protein A on the cell surface changed with glucose but not with mannitol. The two‐layered ferritin distribution on the organism grown in the control medium was altered into a heavily labeled, thick and rough layer with glucose, suggesting the induction of a conformational change in the polypeptide chain forming protein A. This was positively supported by the increase in trypsin susceptibility of protein A.
Culture Conditions for Stimulating Cholera Toxin Production by Vibrio cholerae O1 El Tor
Authors: Iwanaga Masaaki , Yamamoto Koichiro , Higa Naomi , Ichinose Yoshio , Nakasone Noboru , Tanabe Masao
Published: November 2013

A method that stimulates cholera toxin (CT) production by Vibrio cholerae O1 biotype El Tor (El Tor vibrios) to the level of several micrograms per ml in the culture fluid was established. Such a large amount of CT was obtained by the following method: El Tor vibrios were cultured in AKI medium (1.5% Bacto peptone, 0.4% yeast extract‐Difco, 0.5% NaCl, 0.3% NaHCO3) at 37 C for 4 hr in a stationary test tube and then for 16 hr in a shaken flask, with inoculum sizes of 105 to 107/ml. With this method, 35 strains out of 60 examined produced 2 to 16 μg/ml of CT as determined by the reversed passive latex agglutination test (RPLA). Thirty‐three randomly selected strains out of the 60 produced reasonable amounts of rabbit skin vascular permeability factor, reflecting the amount of CT titrated with RPLA.
Appearance of Uridine 5′‐Diphospho‐N‐Acetylglucosamine‐4‐Epimerase during Sporulation of Bacillus megaterium
Authors: Nishikawa Jun‐Ichi , Iwawaki Hiromi , Takubo Yoshihiro , Nishihara Tsutomu , Kondo Masaomi
Published: November 2013

In a biosynthetic study of the spore coat of Bacillus megaterium ATCC 12872 spore with galactosamine phosphate as a major component of the outer coat, high‐performance liquid chromatography (HPLC) and enzyme immunoassay were applied for the measurement of UDP‐N‐acetylglucosamine‐4‐epimerase [EC 5.1.3.7] activity and the enzyme protein concentration, respectively. The new HPLC system using an ion‐pair (or anion‐exchange) column allowed us to determine successfully the enzyme activity and its application, proving that the specific activity of the enzyme in the cells increased at the later stage of sporulation. This increase in activity was parallel to the induction of enzyme protein synthesis, which was detected by sandwich enzyme immunoassay using antiserum to the purified enzyme. These results suggested that the regulation of this enzyme is at the genetic level and it plays an important role in the outer coat synthesis in the later sporulation stage of B. megaterium.
Cytopathogenic Effect of Salmonella typhi GIFU 10007 on M cells of Murine Ileal Peyer's Patches in Ligated Ileal Loops: An Ultrastructural Study
Authors: Kohbata Shunro , Yokoyama Hideaki , Yabuuchi Eiko
Published: November 2013

An electron microscopic study revealed that, within 30 min after inoculation into the ligated ileal loop of anesthetized mice. cells of Salmonella typhi GIFU 10007 adhered to the M cell surface of Peyer's patch lymphoid follicle epithelium, and induced almost complete destruction of M cells. The M cell cytoplasms were pinched off and extruded from the epithelial lining into the luminal space together with the lymphoid cells primarily enfolded into the corresponding M cells. When two or more M cells were destroyed, a large defect in the epithelial lining was apparent, and a number of bacteria appeared near the basal lamina of the epithelial lining. These findings suggest, as far as anesthetized murine ileal loops and strain 10007 are concerned, that ileal M cells are the target cell at an early stage of S. typhi infection and the infection may further progress to deeper tissues and to the general circulation.
Characterization of an Intergroup Serological Mutant from Group II RNA Phage GA
Authors: Harigai Harumi , Furuse Kohsuke , Inokuchi Yoshio , Hirashima Akikazu
Published: November 2013

Starting from the group II RNA phage GA which has an amber mutation in the maturation protein cistron, a spontaneous mutant of group II phage GA, whose serological and electrophoretic properties became similar to those of group I phage MS2, was isolated and analyzed. The mutant has now become sensitive to anti‐MS2 serum and resistant to anti‐GA serum. Analysis of the nucleotide sequence of the coat protein gene revealed that G↔A transition was the main change. The deduced amino acid sequence showed that five amino acids were substituted in the mutant, and three of the five became identical to MS2, resulting in increased molecular weight of the coat protein. However, it did not complement MS2. These results suggested that the serological change from group II phage GA type to group I phage MS2 type is induced spontaneously at high frequency by minor nucleotide changes in coat protein gene, and confirmed the previous results at the RNA level that MS2 and GA were related although the closeness between them seems somewhat remoter than that of groups III and IV (18, Inokuchi et al, unpublished data for the nucleotide sequence of group IV phage SP).
Two Monoclonal Antibodies Distinguish between Human Recombinant Interferon‐α5s Produced by Escherichia coli and by Mouse Cells
Authors: Tsukui Kazuo , Fukunaga Rikiro , Nagata Shigekazu , Ito Keizo
Published: November 2013

Two monoclonal antibodies against human IFN‐α‐one against natural leukocyte IFN‐α and the other against recombinant human IFN‐α 2 produced in E. coli—were prepared, and designated as HT‐1, and 104‐5‐f, respectively. These monoclonal antibodies were used to examine the antigenicities of recombinant human IFN‐α5s produced by E. coli and by mouse cells. The HT‐1 antibody could bind and neutralize recombinant human IFN‐α5 synthesized in mouse cells, but not recombinant human IFN‐α5 synthesized in E. coli. On the other hand, the 104‐5‐f antibody could bind and neutralize recombinant human IFN‐α5 synthesized in E. coli but not recombinant human IFN‐α5 synthesized in mouse cells. Then these monoclonal antibodies or sheep polyclonal antibody against human IFN‐α were used to immunoprecipitate the radioactively labeled recombinant human IFN‐α5 synthesized either in E. coli or mouse cells, and analysed on polyacrylamide gel electrophoresis in the presence of NaDodSO4. The labeled recombinant human IFN‐α5 produced by mouse cells could be immunoprecipitated with the HT‐1 monoclonal antibody or sheep anti‐(human IFN‐α) polyclonal antibody but not with the 104‐5‐f monoclonal antibody and showed a band of Mr. 17,500 on polyacrylamide gel electrophoresis in the presence of NaDodSO4. On the other hand, the labeled recombinant human IFN‐α5 produced by E. coli could be immunoprecipitated with the 104‐5‐f monoclonal antibody but not with the HT‐1 monoclonal antibody and showed a band of similar Mr. on polyacrylamide gel electrophoresis. These results indicate that the two different preparations of recombinant human IFN‐α5, one produced by mouse cells and the other in E. coli, have different epitopes on their molecules which are supposed to have the same primary amino acid sequence. Although the reason why these different epitopes are expressed on the two molecules is unclear, the results agree with our previous observation that recombinant human IFN‐α5 produced by mouse cells but not by E. coli has a slight cross reactivity on mouse cells [Fukunaga, Sokawa, and Nagata, 1984. Proc. Natl. Acad. Sci. U.S.A. 81: 5086–5096]. And these results may suggest the different tertiary structure between recombinant human IFN‐α5 produced in E. coli and mammalian cells.
A Study on Location of Synthetic Site Which Mainly Synthesizes and Delivers Fifth Component of Complement System In Vivo
Authors: Geng Li , Iwabuchi Kazuya , Sakai Shunnosuke , Ogasawara Masahiro , Fujita Masaki , Noguchi Munechika , Good Robert A. , Morikawa Kazuo , Onoé Kazunori
Published: November 2013

Tissue sites for synthesis of the fifth component of complement (C5) in vivo have been investigated by using allogeneic bone marrow chimeras and bone marrow chimeras which were transplanted in addition with hepatocytes. Our prior studies have demonstrated that bone marrow chimeras which had been prepared by transplanting marrow cells from C5‐sufficient donor mice into irradiated C5‐deficient recipients lacked detectable levels of C5 in the sera. However, when such potentially C5‐deficient [C5(+)→C5(−)] chimeras were introduced into their spleens by means of injections of fully dispersed single cell suspensions of hepatocytes isolated from the C5‐sufficient donor strain, they accepted the transplantation of hepatocytes for prolonged periods and developed a measurable amount of C5 in the sera. These results indicate that C5 protein in sera is not synthesized in significant amount by cells that are descendants of bone marrow cells but rather that this complement component is synthesized and delivered to the blood in vivo by somatic cells including liver cells that are not derivatives of the bone marrow.
Rabbit Autoantibodies to Actin Induced by Immunization with Modified Homologous Actins
Authors: Nishioka Mikio , Aibiki Toshiyuki , Shirai Mutsunori , Terada Soichiro , Kagawa Hiroyuki , Watanabe Seishiro
Published: November 2013

Actin is a major antigen involved in the reaction of smooth muscle antibody positive sera from patients with chronic active hepatitis. In the present study, actin extracted from rabbit skeletal muscle was denatured by sodium dodecyl sulfate and was immunized into the rabbit, a homologous animal for actin. The rabbits, thus immunized, produced antibodies reactive with actins of homologous and heterologous animals. In addition, the antibodies showed reactivity with autologous actin. It indicates that the denatured homologous actin is capable of terminating immunological tolerance to actin and induces formation of autoantibody to rabbit actin. This phenomenon may be implicated in the occurrence of anti‐actin antibody in sera from patients with chronic liver disease and several other diseases.