bluuebunny/crossref_metadata_2025 huggingface dataset:
Morphological Changes in Escherichia coli Cells Exposed to Low or High Concentrations of Hydrogen Peroxide
Authors: Brandi Giorgio , Fiorani Mara , Pierotti Cinzia , Albano Amedeo , Cattabeni Flaminio , Cantoni Orazio
Published: November 2013

Escherichia coli cells challenged with low or high concentrations of hydrogen peroxide are killed via two different mechanisms and respond with morphological changes which are also dependent on the extracellular concentration of the oxidant. Treatment with low concentrations (<2.5 mM) of H2O2 is followed by an extensive cell filamentation which is dependent on the level of H2O2 or the time of exposure. In particular, addition of 1.75 mM H2O2 results in a growth lag of approximately 90 min followed by partial increase in optical density, which was mainly due to the onset of the filamentous response. In fact, microscopic analysis of the samples obtained from cultures incubated with the oxidant for various time intervals has revealed that this change in morphology becomes apparent after 90 min of exposure to H2O2 and that the length of the filaments gradually increases following longer time intervals. Analysis of the ability of these cells to form colonies has indicated a loss in viability in the first 90 min of exposure followed by a gradual recovery in the number of cells capable of forming colonies. Measurement of lactate dehydrogenase in culture medium (as a marker for membrane damage) has revealed that a small amount of this enzyme was released from the cells at early times (< 150 min) but not after longer incubation periods (300 min). Cells exposed to high concentrations of H2O2 (>10 mM) do not filament and their loss of viability is associated with a marked reduction in cell volume. In fact, treatment with 17.5 mM H2O2 resulted in a time‐dependent decrease of the optical density, clonogenicity, and cellular volume. In addition, these effects were paralleled by a significant release in the culture medium of lactate dehydrogenase thus suggesting that the reduced cell volume may be dependent on membrane damage followed by loss of intracellular material. This hypothesis is supported by preliminary results obtained in electron microscopy studies. In conclusion, this study further demonstrates that the response of E. coli to hydrogen peroxide is highly dependent on the concentration of H2O2 and further stresses the point that low or high concentrations of the oxidant result in the production of different species leading to cell death via two different mechanisms and/or capable of specifically affecting the cell shape.
Experimental Group A Rotaviral Infection in Cynomolgus Monkeys Raised on Formula Diet
Authors: Leong Yap Kok , Awang Asri
Published: November 2013

Rotaviral infections in cynomolgus monkeys (Macaca fasicularis) were studied to ascertain its suitability as a model of infection and diarrhea caused by group A human rotaviruses. Formula‐fed monkeys were used as they could be observed closely. Experimental rotaviral infection of cynomolgus monkeys was age‐dependent; only young monkeys were readily infected. Formula‐fed newborns were readily infected with cell‐culture‐adapted human (WA) and simian (SA11) viruses and with a rotavirus from a human fecal specimen. However, diarrhea was detected only in very young animals. A number of rotaviral shedding patterns as a function of time were observed. Although there was no typical viral shedding pattern which represented exclusive association of viral infection with diarrhea, the initial level of viral excretion and the maximum level of viral shedding attained were much higher in animals with diarrhea. Seroconversion occurred in less than half of the inoculated animals. The presence of maternal rotaviral antibodies did not prevent infection or diarrhea.
Protection of Mice against Sendai Virus Pneumonia by Non‐Neutralizing Anti‐F Monoclonal Antibodies
Authors: Mochizuki Yasushi , De Ming Tan , Hayashi Takako , Itoh Masae , Hotta Hak , Homma Morio
Published: November 2013

Nine monoclonal antibodies (MAbs) directed to F protein of Sendai virus were obtained and characterized for their protective ability against Sendai virus infection in mice. None of the MAbs showed hemagglutination‐inhibition (HI), hemolysis‐inhibition (HLI), or neutralization (NT) activities in vitro when assayed by standard methods. Some of the MAbs, however, showed complement‐requiring NT (C‐NT) and complement‐requiring hemolysis (C‐HL) activities when assayed in the presence of complement. Passive immunization experiments revealed that the MAbs with higher C‐NT and C‐HL activities showed protective activity against Sendai virus pneumonia in mice, and that some MAbs with IgGl isotype having neither C‐NT nor C‐HL activity also showed the protective activity. Digestion of the MAbs with pepsin which split immunoglobulin molecules into F(ab')2 and Fc fragments greatly suppressed the protective activity. These results suggest that not only complement‐mediated immunological responses such as immune virolysis but also antibody‐dependent cellular cytotoxicity (ADCC) and/or immune phagocytosis, in which complement system is not necessarily involved. play an important role in the protection of mice from Sendai virus infection.
In vitro Effect of Actinomycin D on Human Neutrophil Function
Authors: Chang Feng‐Yee , Shaio Men‐Fang
Published: November 2013

The effect of actinomycin D (ACT‐D) on human neutrophil chemotaxis, chemiluminescence (CL), superoxide (O2‐) production, phagocytic uptake, and intracellular bacterial killing has been examined. The viability of the ACT‐D‐treated neutrophils was 9800 even at a concentration of 10 μg/ml for 4 hr. Using fMLP as the chemotactic factor, depressed chemotaxis was demonstrated following ACT‐D (1 10 μg/ml) pretreatment of neutrophils as compared with the non‐treated controls. Similar ACT‐D pretreatment produced the depressed responses in phorbol myristate acetate‐induced CL and superoxide production by neutrophils. Moreover, using heat‐inactivated human serum as an opsonin for Salmonella enteritidis (NCTC 6676), there was a significant difference in intracellular killing (P<0.01) but no difference in phagocytic uptake between ACT‐D‐treated and non‐treated neutrophils. These studies indicate that ACT‐D profoundly impairs both intracellular bacterial killing by human neutrophil through an effect on respiratory burst activity and directed cell migration of human neutrophils.
Pathogenesis of Vibrio parahaemolyticus: Intraperitoneal and Orogastric Challenge Experiments in Mice
Authors: Hoashi Kikuo , Ogata Kikuyo , Taniguchi Hatsumi , Yamashita Hiroto , Tsuji Koichi , Mizuguchi Yasuo , Ohtomo Nobuya
Published: November 2013

It has been noted that V. parahaemolyticus isolated from patients with food poisoning are almost always thermostable direct hemolysin (TDH)‐positive, whereas, isolates from foods or environmental sources are usually TDH‐negative. The virulence of V. parahaemolyticus in mice was examined by using intraperitoneal and orogastric challenge models, where the strains used were those isolated from patients and foods with food poisoning and included TDH‐positive and ‐negative. The LD50 in mice was estimated to be approximately 107 in the former and 108 in the latter challenge model. In the mice killed by challenge, either intraperitoneal or orogastric, the following pathological changes were almost always observed: swelling, redness and fluid accumulation in the small intestine, particularly the upper part of it. Histologically, congestion, edema, and vacuolation were observed in mucosal and submucosal tissues; furthermore, ulceration at tips of some villi was noticed. The lethality and pathological finding in mice due to V. parahaemolyticus were demonstrated similarly in both challenge models and irrespective of the TDH phenotype of the strain used. It will be suggested that the findings in challenged mice may be. attributed to enteropathogenic factor(s) other than TDH of V. par ahaemolyticus.
Demonstration of RNA N‐Glycosidase Activity of a Vero Toxin (VT2 Variant) Produced by Escherichia coli 091: H21 from a Patient with the Hemolytic Uremic Syndrome
Authors: Furutani Masayuki , Ito Kiyoshi , Oku Yuichi , Takeda Yoshifumi , Igarashi Kazuei
Published: November 2013

A new Vero toxin purified from Escherichia coli O91: H21 isolated from a patient with the hemolytic uremic syndrome (VT2vh) was shown to inhibit elongation factor 1‐dependent aminoacyl‐tRNA binding to ribosomes, resulting in inhibition of protein synthesis in rabbit reticulocytes. VT2vh, like Shiga toxin, VT1 and VT2, showed RNA N‐glycosidase activity and cleaved the N‐glycosidic bond of the adenosine residue at position 4324 in 28S ribosomal RNA.
Linkage of Ribosomal RNA Genes in Leptospira
Authors: Fukunaga Masahito , Masuzavva Toshiyuki , Okuzako Noriko , Mifuchi Ichiji , Yanagihara Yasutake
Published: November 2013

We determined the linkage of 16S, 23S, and 5S rRNA genes in several strains of Leptospira and Leptonema by DNA‐DNA hybridization. Almost all the hybridizations in all leptospires used in these experiments gave two radioactive bands and the results strongly suggest that the number of the 16S and the 23S rRNA genes in those strains is two, respectively. In contrast with the larger rRNAs, the number of 5S rRNA gene was different. In the strains of leptospires, L. biflexa, which were non‐parasitic, there are two genes for 5S rRNA, whereas only one gene for 5S rRNA is carried in L. interrogans, which were originally isolated as parasitic. Southern hybridization experiments suggest that those rRNA genes are interspersed on the leptospiral chromosome.
Antiviral Effects of 2′,5′ Oligoadenylates (2‐5As), and Related Compounds
Authors: Tominaga Akihiro , Saito Sakura , Kohno Seiya , Sakurai Katsukiyo , Hayakawa Yoshihiro , Noyori Ryoji
Published: November 2013

Dephosphorylated “core” of 2′,5′‐oligoadenylate (2‐5A) dimer (A2′P5′A), exogenously added to nonpermeabilized FL cells, inhibited the multiplication of Sindbis virus and vesicular stomatitis virus (VSV). The compound was shown to inhibit viral protein synthesis. The addition of A2′P5′A at the early stage of viral replication was more effective than that at the late stage. In contrast with the core, phosphorylated 2‐5A (P5′A2′P5′A and PPP5′A2′P5′A) and 2‐5A analogs containing cordycepin (3′‐deoxyadenosine) did not show such antiviral effects. The rate of uptake of [3H]ppp5′ A 2′P5′A into acid‐soluble and acid‐insoluble fractions, especially into the acid‐insoluble fraction, was faster than that of [3H]A2′P5′A. These results suggest that the difference of antiviral activity between A2′P5′A and PPP5′A2′P5′A does not result from the different rate of uptake by cells, but from the different rate of from acid‐soluble to acid‐insoluble fractions.
Synthesis and Localization of Japanese Encephalitis Virus RNAs in the Infected Cells
Authors: Takegami Tsutomu , Hotta Susumu
Published: November 2013

Synthesis and localization of virus‐specific RNA in cells infected with Japanese encephalitis virus (JEV) were examined. To prepare specific RNA probes, we constructed four kinds of plasmids which contained DNA fragments corresponding to JEV genomic RNA. Minus probes, JT18V and JT19III, transcribed by T7 RNA polymerase were able to recognize a negative strand of JEV‐specific RNA synthesized in cells as early as 6 hr postinfection (p.i.). In the experiments using a plus‐strand probe JT19V to hybridize the 3′ end of JEV‐RNA, not only full‐length 42S(+) RNA but also 10S(+) RNA were detected in the infected cells at 24 hr p.i. The positive‐strand 42S RNA was found in much greater abundance in the membrane fraction than in the supernatant fraction of the infected cells. In contrast, larger amounts of the negative‐strand RNAs existed in the supernatant fraction. It is suggested from the data that the JEV‐specific negative‐ and positive‐strand RNAs accumulate at different sites in the infected cells.
Antiviral Potencies of BV‐araU and Related Nucleoside Analogues against Varicella‐Zoster Virus in Different Cell Lines
Authors: Machida Haruhiko , Ijichi Katsushi , Ohta Arihito , Honda Mariko , Niimura Michihito
Published: November 2013

1‐β‐D‐Arabinofuranosyl‐E‐5‐(2‐bromovinyl)uracil (BV‐araU) and nine other antiherpesviral nucleoside analogues were compared for their potencies against four strains of varicella‐zoster virus (VZV) on three different cell lines: HEL cells, Vero cells, and MS cells established from a human malignant schwannoma. In contrast to the activity against herpes simplex virus type 1 previously reported, BV‐araU showed extremely marked antiviral activity against VZV even on Vero cells. ED50, 5000 plaque reduction dose, of BV‐araU for VZV was 0.20–3.1 and 0.14–0.63 ng/ml on Vero cells and on HEL cells, respectively. Potency of BV‐araU on MS cells was similar to that on these cell lines. There was not significant variation in anti‐VZV activities of other nucleoside analogues on these three different cell lines except a few combinations of VZV strain and test compound.
Antibodies to Borrelia burgdorferi in Dogs in Hokkaido
Authors: Isogai Emiko , Isogai Hiroshi , Sato Nanao , Yuzawa Mutsumi , Kawakami Mitsuaki
Published: November 2013

During 1985 to 1990, serum samples were obtained from 229 healthy dogs. The dogs lived in Hokkaido, known to be infested with ticks. An enzyme‐linked immunosorbent assay (ELISA) was used to detect IgG and IgM antibodies against Borrelia burgdorferi HO14 and HP3, which were isolated from Ixodes ovatus and I. persulcatus in the area. IgG antibody to B. burgdorferi HO14 was detected in 8.8% (1985). 16.4% (1987) and 18.5% (1990). IgM antibody to the bacteria was detected in 1.8% (1987) and 2.5% (1990). Antibodies to the strain HP3 of B. burgdorferi were also detected in the serum samples of dogs, but the percentage of seropositive sample to the strain HP3 was lower than that to the strain HO14. Statistical differences were not noticed between pet and street dogs. No antibody to B. burgdorferi was observed in 13 beagle dogs as experimental animal.
Seroepidemiology of Herpes Simplex Virus Type 1 in Yanji, Jilin, China
Authors: Li Yu‐Yu , Hidaka Yasufumi , Kino Yoichiro , Mori Ryoichi
Published: November 2013

Sera from 158 individuals in Yanji, Jilin, China, were tested for antibodies to herpes simplex virus type 1 (HSV‐1) by the passive hemagglutination method. Age‐specific incidence rates for antibodies to HSV‐1 were calculated. For sera from persons in the age group 10 years or less, the positive rate was 54% but in the age group higher than 10 years, it was more than 91% (P<0.01). In the part of China surveyed, primary HSV‐1 infection occurred in early generation before about age 10. In children, the positive rate in the Han race was significantly higher than that in the Korean race (P<0.05).
Platelet Aggregation Induced by Strains of Various Species of Coagulase‐Negative Staphylococci
Authors: Usui Yukio , Ohshima Yukio , Ichiman Yoshitoshi , Ohtomo Toshichika , Suganuma Masaru , Yoshida Kosaku
Published: November 2013

Major species of coagulase‐negative staphylococci (CNS) were tested for their ability to induce platelet aggregation in rabbit platelet‐rich plasma (PRP). Among 11 species of CNS tested, a majority of the strains of 10 species of CNS (S. epidermidis, S. simulans, S. capitis, S. hyicus, S. sciuri, S. cohnii, S. xylosus, S. hominis, S. haemolyticus, S. warneri) caused induction of the platelet aggregation and serotonin release, while S. saprophyticus did not show such activity. The addition of aspirin (10 mM) or quinacrine (1 mM) to PRP resulted in no remarkable effect on the platelet aggregation induced by these strains and it was shown that the platelet aggregation did not require arachidonate pathways. Complement system components were shown to be one of the plasma factors required for platelet aggregation by ten strains of each species of CNS. The bacterial substance participating in the platelet aggregation by ten species of CNS tested was indicated to be heat‐stable and trypsin‐resistant, while the activity of a strain of S. epidermidis was susceptible to trypsin.
Practical Methods for Chemical Inactivation of Creutzfeldt‐Jakob Disease Pathogen
Authors: Tateishi Jun , Tashima Takatoshi , Kitamoto Tetsuyuki
Published: November 2013

Chemical inactivation of pathogen of Creutzfeldt‐Jakob disease (CJD; was examined using the mouse‐adapted CJD strain. A high concentration of formic acid, guanidine compounds, trichloroacetate and phenol prevented CJD transmission. NaOH between 0.25 and 2 N lengthened the incubation periods. Sodium dodecyl sulfate (SDS) in a concentration between 1 and 3% did not alter incubation at room temperature but did completely block the transmission after boiling for 3 min in 300 SDS. This method is recommended for practical disinfection.
Lymphocyte Subpopulations and Cytokine Production in Paracoccidioidomycosis Patients
Authors: Bava A. Javier , Mistchenko Alicia S. , Palacios M. Fernanda , Estevez Maria E. , Tiraboschi Nora I. , Sen Luisa , Negroni Ricardo , Diez Roberto A.
Published: November 2013

Despite the postulated role of the immune system in the control of the infection by Paracoccidioides brasiliensis, only a few studies have addressed this point in patients. The determination of total lymphocytes and their subpopulations in 6 untreated patients with the chronic form of paracoccidiodomycosis showed that half of them were lymphopenic, because of low number of CD4+ T‐lymphocytes. All patients had low CD4/CD8 ratios. On the contrary, B‐lymphocytes were normal in all patients. An additional patient, studied on treatment with ketoconazole, had normal lymphocyte counts in all subpopulations, as did one of the patients previously studied at diagnosis when he received specific antimycotic treatment. The production of interferon and tumor necrosis factor, determined by bioassay in supernatants of mononuclear blood cells of the patients, induced by interleukin 2 in vitro was significantly lower than that of normal subjects. These results show that patients with paracoccidioidomycosis have a defect in blood lymphocyte subsets as well as in the ability to produce regulatory cytokines.
Accumulation of Phosphate‐Containing Granules in the Nucleoid Area of Pseudomonas aeruginosa
Authors: Takade Akemi , Umeda Akiko , Misumi Tsuyosi , Sawae Yoshirou , Amako Kazunobu
Published: November 2013

Many electron‐dense granules were found in the nucleoid area of Pseudomonas aeruginosa strain K by electron microscopy with the technique of the freeze‐substitution method. These granules contained phosphorus and calcium as determined by X‐ray microanalysis. The size and the numbers of the granules decreased when the bacteria was cultured in the medium from which phosphate‐containing compounds were depleted. From these observations we concluded that the granule was a phosphate‐containing granule and possibly a polyphosphate granule. The excellent preservation of the fine structures by the freeze‐substitution technique enables us to show very small polyphosphate granules in the nucleoid area of the bacterial cells which cannot be revealed by the conventional chemical fixation method. As we could not see the granules in other bacteria cultured in nutrient medium such as Serratia, Escherichia, Bacillus and Vibrios, the accumulation of the phosphate granules in Ps. aeruginosa might be a unique character of this bacteria and might be related to the growing capability of this bacteria in extremely low nutrient supply.
Protease Inhibitors Prevent the Development of Human Rotavirus‐Induced Diarrhea in Suckling Mice
Authors: Ebina Takusaburo , Tsukada Keiko
Published: November 2013

Oral inoculation of human rotavirus MO strain (serotype 3) into 5‐day‐old BALB/c mice caused gastroenteritis characterized by diarrhea (90% on the average, on day 2). Using this animal model, preventive effect of antiviral agents on the development of rotavirus‐induced diarrhea was examined. The infectivity of human rotavirus was enhanced by treatment with protease in vitro. A cysteine protease inhibitor, E‐64‐c, was given orally at 12 hr and 24 hr after MO infection. Oral administration of 0.3 mg of E‐64‐c decreased the diarrhea ratio to 17.5% on day 2 and to 10% on day 3. Oral administration of 0.15 mg of cysteine protease inhibitor, ovocystatin, completely prevented the diarrhea on day 2. Serine protease inhibitor, aprotinin (0.15 mgx2), also prevented the diarrhea on day 2 to 14.3%. These protease inhibitors were nontoxic in vitro and to suckling mice. The histopathological changes in the small intestine due to infection recovered 2 days after MO infection in mice treated with E‐64‐c and ovocystatin. These results suggest that protease inhibitors are protective agents for human rotavirus infection by inhibiting proteases required for viral replication.
Two Generalized Transducing Phages in Vibrio parahaemolyticus and Vibrio alginolyticus
Authors: Muramatsu Kouichi , Matsumoto Hideki
Published: November 2013

Two bacteriophages named φVP253 and φVP143 isolated after ultraviolet induction from lysogenic strains of Vibrio parahaemolyticus have been shown to be generalized transducing phages. So far, seven different auxotrophic markers of a V. parahaemolyticus strain could be transduced at the frequencies ranging from 2.2× 10−7 to 7.5×10−5 per infected cell at the m.o.i. of approximately 1.0. The phage φVP143, but not φVP253, lysed 20 of the 28 strains of V. alginolyticus and the occurrence of generalized transduction by this phage in this Vibrio species has been confirmed. Molecular size of the genomes of both phages were estimated to be approximately 48 kb as judged from electrophoretic mobilities of the DNAs digested with HindIII endonuclease. The results and similarity of the two phages in morphology and other properties suggest very close relatedness of the phages.
Paradoxical Effect of Tween 80 between the Susceptibility to Rifampicin and Streptomycin and the Susceptibility to Ethambutol and Sulfadimethoxine in the Mycobacterium avium‐Mycobacterium intracellulare Complex
Authors: Yamori Sadaaki , Tsukamura Michio
Published: November 2013

The susceptibility to rifampicin and streptomycin of the Mycobacterium avium‐Mycobacterium intracellulare complex was augmented by the addition of Tween 80 into 7H10 agar medium with OADC, whereas the susceptibility to ethambutol and sulfadimethoxine was either not changed or reduced by the addition of Tween 80. In 7H10 agar medium without OADC, however, susceptibilities to both rifampicin and sulfadimethoxine were reduced by the addition of Tween 80 to the medium. A number of hypotheses are made to explain these phenomena.
Detection of Cholera Toxin by a Highly Sensitive Bead‐Enzyme Linked Immunosorbent Assay
Authors: Uesaka Yoshihiko , Otsuka Yoko , Kashida Mitsuaki , Oku Yuichi , Horigome Kazuki , Nair G. Balakrish , Pal S. C. , Yamasaki Shinji , Takeda Yoshifumi
Published: November 2013

A bead‐enzyme linked immunosorbent assay (bead‐ELISA) for detection and quantification of cholera toxin (CT) in broth cultures of Vibrio cholerae O1 has been developed. Under optimal buffer and pH conditions the bead‐ELISA could consistently detect 40 pg/ml of CT. None of the ingredients of commonly used media for in vitro culture of V. cholerae O1 hindered the performance of the bead‐ELISA. Evaluation of the sensitivity and specificity of the bead‐ELISA against the commonly used reversed passive latex agglutination (RPLA) test for detection of CT was performed using a collection of 239 strains of V. cholerae O1 (including both biotypes and serotypes) which were examined by a gene probe encoding for the A1 subunit of CT. Although both the assays were highly specific, the bead‐ELISA was more sensitive than the RPLA. Quantification of CT by the bead‐ELISA revealed that the concentration of CT produced by the strains of V. cholerae O1 which were negative by the RPLA was lower than 1 ng/ml and therefore below the minimum detection ability of the RPLA. The bead‐ELISA is a simple, specific and highly sensitive assay for routine detection of CT and is recommended for routine use in clinical microbiology laboratories.
Studies on Osmotic Stability of Liposomes Prepared with Bacterial Membrane Lipids by Carboxyfluorescein Release
Authors: Nagamachi Eiko , Hirai Yoshikazu , Tomochika Ken‐ichi , Kanemasa Yasuhiro
Published: November 2013

The authors measured the osmotic stability of liposomes prepared with membrane lipids of bacteria, using the osmotic‐shock release of entrapped carboxyfluorescein as an indicator. The sub‐second physical changes of liposomes suspended in a solution of low osmotic pressure were examined by stopped flow spectrophotometry. The entrapped carboxyfluorescein was released when the liposomes burst on inflow of excess water. Liposomes prepared with the lipids of a stable Staphylococcus aureus L‐form strain were more resistant to low osmotic pressure than those prepared from the wild strain of S. aureus, and liposomes prepared from Mycoplasma orale were even more resistant. Cardiolipin enhanced the lipid membrane stability in S. aureus and cholesterol in M. orale. The stability of lipid membranes to low osmotic pressure could be precisely determined by the present method.
Interferon Induction in Murine Peritoneal Macrophage by Stimulation with Lactobacillus acidophilus
Authors: Kitazawa Haruki , Matsumura Kazuki , Itoh Takatoshi , Yamaguchi Takahiro
Published: November 2013

Induction of interferon for a kind of dairy lactic acid bacteria, Lactobacillus acidophilus (L. acidophilus), was investigated in murine peritoneal macrophage (MØ) cultures. Lactobacillus acidophilus JCM 1034, 1132T, 1229 and 2125 induced IFN (12–34 I.U./ml) in MØ cultures in vitro. Strain 1132T‐ and 2125‐induced IFNs were characterized as IFNα/β by treatment with anti‐IFNs serum. The results indicate that the inducing activity of IFNs may be one of the available biological parameters for designating the dairy products containing L. acidophilus as “physiologically functional foods.”
Lipopolysaccharide Composition and Virulence Properties of Clinical and Environmental Strains of Vibrio fluvialis and Vibrio mimicus
Authors: Rahman Mohammed Mahbubur , Qadri Firdausi , Albert Manuel John , Hossain Anwar , Mosihuzzaman M.
Published: November 2013

Vibrio mimicus strains W‐26768 (stool isolate) and N‐1301 (environmental isolate) and Vibrio fluvialis strains AA‐18239 (stool isolate) and M‐940 (environmental isolate) were studied for virulence properties and lipopolysaccharide composition. All four strains were hydrophobic, produced cytotoxin, adhered to HeLa cells and showed mannose‐sensitive agglutination of guinea pig erythrocyte. The strains were negative for enterotoxin production and were mostly susceptible to the common antibiotics. The environmental and clinical isolates of both species were anti‐genically unrelated to each other. Lipopolysaccharide antigen analysis showed that O‐antigen polysaccharides of two strains of V. fluvialis and two strains of V. mimicus differed with respect to the sugar components. Only LPS from V. mimicus W‐26768 showed the presence of an unusual sugar, 3,6‐dideoxy‐3‐acetamido‐hexose. The sugar compositions of these V. fluvialis and V. mimicus strains differed from those of previously reported Japanese isolates. These differences probably reflect differences in the serogroup of strains.
Bacteriostatic and Bactericidal Activity of Antituberculosis Drugs against Mycobacterium tuberculosis, Mycobacterium avium‐Mycobacterium intracellulare Complex and Mycobacterium kansasii in Different Growth Phases
Authors: Yamori Sadaaki , Ichiyama Satoshi , Shimokata Kaoru , Tsukamura Michio
Published: November 2013

Bacteriostatic and bactericidal activities of rifampicin, isoniazid, streptomycin, enviomycin and ethambutol against Mycobacterium tuberculosis, Mycobacterium avium—M. intracellulare complex and Mycobacterium kansasii were studied in different growth phases. Bacteriostatic activities of the drugs were similar in different growth phases, except isoniazid. M, tuberculosis was much less susceptible to isoniazid in the lag phase than in the log and the stationary phases. In contrast, bactericidal activity was influenced by the growth phase. M. tuberculosis was killed by isoniazid, streptomycin and rifampicin. The bactericidal activity of isoniazid was strongest. The bactericidal activity of isoniazid and streptomycin was most marked in the log phase. M. avium complex and M. kansasii resisted the bactericidal activity, but some strains of M. avium complex were killed by streptomycin and enviomycin, and the activities of these two drugs were most marked in the lag phase.
Proposal of a New Scheme for the Serological Typing of Enterococcus faecalis Strains
Authors: Maekawa Shizue , Yoshioka Migaku , Kumamoto Yoshiaki
Published: November 2013

No systematic study on serotyping of Enterococcus faecalis has been reported since 1964 when M.E. Sharpe conducted serotyping of group D streptococcus in U.K. So, we attempted to re‐evaluate serotyping of E. faecalis. For this purpose, we received 42 Sharpe's strains and first examined for their biochemical characteristics as E. faecalis. Only 9 of the 42 strains were identified as E. faecalis. We raised rabbit antisera against a large number of E. faecalis strains, including the 9 Sharpe's strains, 2 strains obtained from CDC in U.S.A. and 36 strains isolated from patients hospitalized in different cities of Japan. From the results of cross‐agglutination tests and absorption tests performed on these antisera using a large number of E. faecalis strains, we were able to classify 21 distinct serotype strains and to prepare 21 mono‐specific typing antisera by absorption of the antisera to the type strains with appropriate cross‐agglutinating strains. When 832 E. faecalis strains were serotyped with the 21 typing antisera, 638 strains (76.7%) were typable. Thus, we propose a provisional scheme of 21 distinct serovars in E. faecalis.
Phage Pattern and Antibiotic Resistance Pattern of Coagulase‐Negative Staphylococci Obtained from Immunocompromised Patients
Authors: Barcs István , Herendi Ágnes , Lipcsey András , Bognár Csaba , Hashimoto Hajime
Published: November 2013

A total of 152 coagulase‐negative staphylococcal strains were isolated from clinical samples of 14 patients hospitalized after bone‐marrow transplantation in a specialized hospital ward in Hungary, during an 18‐month period between 1987 and 1989. Two species, Staphylococcus epidermidis and Staphylococcus haemolyticus, predominated (each, 45%). Using Pulverer and co‐workers' phage set for typing, 68% of the isolates were typable; 16 phage patterns were observed. A characteristic long pattern with phages Ph10/Ph13/Ph15/U4/U15/U16/U20/U33/U46 appeared only in S. epidermidis, among 5 of 11 colonized patients (8.5% of all strains). Single lysis with phage Phl3 was observed in 7 of the 14 patients (49% of all strains), in species S. capitis, S. epidermidis, S. haemolyticus, S. hominis, and S. warneri. In S. haemolyticus, non‐typable strains predominated (66%); this character occurred only in 2% among other species. The strains colonizing the immunocompromised patients differed from each other in phage pattern, antibiotic resistance pattern, and/or slime production. No hospital infection was suggested. On the other hand, high incidence of two well‐definable phage patterns raises some relationship between phage receptors or some regulatory systems in phage multiplication and factors responsible for special colonization as common surface properties.
Pathogenicity of Bacteroides fragilis Group in Rat Intra‐Abdominal Abscesses
Authors: Jotwani Ravi , Gupta Usha , Watanabe Kunitomo , Ueno Kazue
Published: November 2013

Attempts were made to study the pathogenicity of some strains of Bacteroides fragilis group in the rat intra‐abdominal abscess model. Multiple intra‐abdominal abscesses were produced in 50 to 70% of animals when an inoculum containing 109 CFU/ml of any of the five species of Bacteroides fragilis group was injected. Rising homologous antibody titers determined by indirect fluorescent antibody test were observed till the 3rd week when tested last, indirectly confirming the multiplication of the organisms as also evident by viable count of bacteria in the abscesses. In some cases in addition to inoculated organisms some intestinal bacteria like Escherichia coli, Proteus mirabilis and Streptococcus spp. were also recovered from the abscess pus. Studies with the electron microscope showed presence of capsular polysaccharide only in Bacteroides fragilis and Bacteroides thetaiotaomicron. It was doubtful in Bacteroides distasonis and absent in Bacteroides ovatus and Bacteroides vulgatus, suggesting that virulence factor beside the capsular polysaccharide may be playing a role. Further studies are required to investigate the virulence factor responsible for the pathogenicity of noncapsulated species.
Mechanisms of Augmented Resistance of Cyclosporin A‐Treated Mice to Influenza Virus Infection by Trehalose‐6, 6′‐Dimycolate
Authors: Sazaki Katsuhiko , Yoshida Itsuro , Azuma Masanobu
Published: November 2013

Cyclosporin A (CsA), which is an immunosuppressive drug of helper T lymphocytes, diminished a resistance of mice to influenza virus infection. Mice inoculated intravenously with trehalose‐6,6′‐dimycolate (TDM, a glycolipid component of the cell wall of Mycobacterium) in an oil‐in‐water emulsion (TDM emulsion) recovered the resistance to influenza virus infection impaired by CsA. Number of antibody‐producing cells was markedly reduced in CsA‐ and/or TDM‐treated mice. Interferon production in lung of TDM‐treated mice was augmented; however, it was extremely reduced not only in CsA‐treated mice, but also in CsA‐ and TDM‐treated mice. Activities of natural killer cells of CsA‐ and/or TDM‐treated mice were not different from that of control mice. Numbers of lymphocytes in lung of TDM‐treated mice and CsA‐ and TDM‐treated mice were more predominantly increased than that of control mice. Analysis of lung lymphocytes by flow cytometry revealed no difference between the populations of L3T4+ T lymphocytes and Lyt‐2.2+ T lymphocytes in CsA‐ and/or TDM‐treated mice and the populations in control mice. However, the population of γδ T cell receptor positive (γδ TCR+) lymphocytes increased markedly in lung of TDM‐treated mice and also CsA‐ and TDM‐treated mice. In vitro experiments showed that macrophage cultures treated with TDM emulsion released a mediator(s) which activates T lymphocytes, but not B lymphocytes. These and our earlier results suggest that the recovered anti‐influenza virus resistance of CsA‐treated mice by treatment with TDM emulsion was caused by elicitation of macrophages with TDM, then activation of T lymphocytes, especially γδ TCR+ lymphocytes.
Serological Cross‐Reaction between Intact and Chemically Modified Lipopolysaccharides of O1 Vibrio cholerae Inaba and Non‐O1 V. cholerae Bio‐Serogroup Hakata
Authors: Isshiki Yasunori , Haishima Yuji , Kondo Seiichi , Hisatsune Kazuhito
Published: November 2013

Serological cross‐reaction of intact as well as chemically modified LPS from O1 Vibrio cholerae 569B (Inaba) with non‐O1 V. cholerae Hakata LPS, which contain α(1→2)‐linked N‐acetyl perosamine‐homopolymer constituting their O polysaccharide chain, was studied by passive hemolysis test by using their LPS as antigen for sensitizing sheep red blood cells (SRBC). The N‐deacylation of the α(1→2)‐linked linear 3‐deoxy‐tetronyl perosamine‐homopolymer constituting the O polysaccharide chain in 569B LPS resulted in virtual elimination of their serological reactivity with both homologous Inaba and heterologous Hakata antisera. Furthermore, when the resultant NH2 groups of the N‐deacylated perosamine‐homopolymers in 569B LPS were N‐acylated with acetyl, propionyl or butanoyl groups, they markedly recovered the serological reactivity to a marked extent, in particular, their pronounced cross‐serological reactivity with heterologous Hakata antiserum. These results are believed to be compatible with the interpretation that the Inaba antigen factor C possessed by the two bacteria studied is related to the common occurrence of the N‐acyl groups, regardless of what the acyl groups are, residing in the perosamine residues of the perosamine‐homopolymers constituting the O polysaccharide chain of their LPS.
cDNA Cloning of an Aspartic Proteinase Secreted by Candida albicans
Authors: Mukai Hiroyuki , Takeda Osamu , Asada Kiyozo , Kato Ikunoshin , Murayama Somay Yamagata , Yamaguchi Hideyo
Published: November 2013

cDNA of an aspartic proteinase secreted by Candida albicans No. 114 was isolated using the polymerase chain reaction (PCR). The primary structure of the enzyme was deduced from the nucleotide sequence of the cDNA and compared with the structures of Saccharomyces cerevisiae proteinase A and vacuolar aspartyl proteinase of C. albicans. The mature aspartic proteinase consisted of 341 amino acid residues, and was 17.6 and 15.3% identical with the proteinase A and the aspartyl proteinase, respectively. Two active aspartic acid sites and the amino acids near those sites were conserved in the aspartic proteinase. We also showed that there is another gene of aspartic proteinase than that of strain ATCC10231 reported by Hube et al (J. Med. Vet. Mycol. 29 (1991)) in the same C. albicans genome, both in that strain and in No. 114.
An Improved Method for the Serotyping of Free Coagulase from Staphylococcus aureus
Authors: Tajima Yutaka , Nagasawa Zenzo , Tanabe Ichiro , Yamada Hisashi , Kusaba Koji , Tadano Jutaro
Published: November 2013

The serotyping of free coagulase, one of the most reliable ways to identify strains of Staphylococcus aureus, and widely employed in Japan, has been improved by adding magnetite sand to the reaction mixture. Culture medium supernatant and a type‐specific antibody are mixed in a well of a microtiter plate, and plasma‐enriched bovine fibrinogen is treated with magnetite sand. The use of tranexamic acid and gum arabic in the reaction mixture also increases the sensitivity of the reaction. Finally, the plate is placed on a magnetic stirrer. If the type of the coagulase corresponds to that of the antibody, no clot formation will occur, and this is easily confirmed by the movement of the sand. Although the amount of reaction mixture required is much less than that for the conventional tube method, our new method is able to detect slight increases in viscosity of the reaction mixture due to fibrin formation even before complete clotting occurs, thus providing very high sensitivity. Clot formation can also be judged by observing a turbid mass of fibrin in the well (Hwang's method), but this approach is a little slower than our method involving immobilization of magnetite sand.
Cloning of the Genes That Control Formation of the Fimbrial Colonization Factor Antigen III (CFA/III) from an Enterotoxigenic Escherichia coli
Authors: Shinagawa Hideo , Taniguchi Tooru , Yamaguchi Osamu , Yamamoto Koichiro , Honda Takeshi
Published: November 2013

Enterotoxigenic Escherichia coli strain 260‐1 produces colonization factor antigen III and heat‐labile enterotoxin. A 55‐kb plasmid controlling the expression of the colonization factor antigen was isolated from this strain after it was labeled with ampicillin resistance transposon, Tn3. When this plasmid was introduced into E. coli K‐12 strains, it induced the formation of pili that were morphologically and immunologically identical to those on the surface of 260‐1 cells, as examined by electron microscopic observation and with the specific antiserum. The physical map of the plasmid was constructed, and the 17.4‐kb region was found to be responsible for the expression of the pili.
A Factor Found in Aged Tungstate Solution Enhanced the Antibacterial Effect of β‐Lactams on Methicillin‐Resistant Staphylococcus aureus
Authors: Tajima Yutaka , Nagasawa Zenzo , Tadano Jutaro
Published: November 2013

We have found a factor (Factor T) in aged mixtures of tungstate and phosphate which greatly enhances the antibacterial effects of β‐lactams on both inducible and constitutive methicillin‐resistant Staphylococcus aureus, but not on methicillin‐susceptible S. aureus. Factor T alone did not strongly inhibit bacterial growth. There was no synergism of Factor T with other classes of antibiotics, nor with other groups of bacteria, and it reduced the efficacy of aminoglycosides and tetracycline. Upon preparation of Factor T, acidifying and heating the mixture of tungstate and phosphate resulted in a high yield and reproducibility, and no enhancing effect was observed when other anions such as sulfate or molybdate were used instead. Factor T is heat‐ and acid‐stable but labile to alkalization, and is probably a complex of phosphate and tungstate.
Suppressive Effect of Liposomes Containing DNA Coding for Diphtheria Toxin A‐Chain on Cells Transformed with Bovine Leukemia Virus
Authors: Kakidani Hitoshi , Watarai Shinobu , Onuma Misao , Tomochika Ken‐ichi , Yasuda Tatsuji
Published: November 2013

A recombinant plasmid which contained a gene for diphtheria toxin A‐chain (DT‐A) under the control of the long terminal repeat (LTR) of bovine leukemia virus (BLV) (BLV‐LTR) was constructed to test a novel application of liposomes as antiviral agents. The promoter activity of BLV‐LTR was estimated by the chloramphenicol acetyltransferase (CAT) assay using a plasmid which contains the coding sequence of CAT under the control of BLV‐LTR (pBLVCAT). When BLV‐infected cells were transfected with pBLVCAT, CAT activity was detected. BLV‐uninfected cell lines, however, showed no detectable CAT activity. The plasmid DNA entrapped in liposomes was added to BLV‐infected cells in culture. Syncytium formation induced by BLV‐infected cells was effectively suppressed by the liposomes containing the gene for DT‐A under the control of BLV‐LTR. Conversely, liposomes containing the gene for DT‐A without a promoter showed no such effect. DT‐A gene‐containing liposomes with BLV‐LTR did not affect formation of syncytium induced by bovine immunodeficiency virus. These observations indicate that BLV‐infected cells were readily targeted on the level of gene expression. This strategy could be applied to the treatment of BLV‐induced B‐cell proliferation of cattle, and further to other viral/neoplastic diseases where specific gene expression is exerted.
Characterization of a Phenol Oxidase from Cryptococcus neoformans var. neoformans
Authors: Ikeda Reiko , Shinoda Takako , Morita Takashi , Jacobson Eric S.
Published: November 2013

In Cryptococcus neoformans, enzymic oxidation of various catechols leads to melanin, a proposed virulence factor. A phenol oxidase enzyme of Cryptococcus neoformans var. neoformans produced at 25 C has been purified from an ultracentrifugal supernatant of an extract of broken cells. Hydrophobic interaction chromatography followed by anion‐exchange column chromatography allowed purification of the phenol oxidase. The molecular weight of the enzyme estimated by gel filtration was about 80,000 and a dimeric species (Mw = 160,000) was suggested. The isoelectric point of the protein was approximately 4.1. An NH2‐terminal 31 amino acid sequence was determined using phenol oxidase electroblotted onto a PVDF membrane after nondenaturing gel electrophoresis. Upon searching the Peptide Institute (Osaka) data base, no proteins with high degrees of homology were found.
Genomic Analysis of Borrelia japonica Sp. Nov. Isolated from Ixodes ovatus in Japan
Authors: Kawabata Hiroki , Masuzawa Toshiyuki , Yanagihara Yasutake
Published: November 2013

Genetic characteristics of 12 Borrelia isolates from the tick, Ixodes ovatus, I. persulcatus, and the rodent, Apodemus speciosus ainu, in Japan were compared to members of the three genospecies of Borrelia burgdorferi sensu lato; B. burgdorferi sensu stricto, B. garinii and group VS461. The methods used in this study were the quantitative microplate DNA hybridization assay and restriction fragment length polymorphism (RFLP) analyses of the flagellin structural genes and the 16S rRNA genes. The six isolates from I. persulcatus and A. speciosus ainu were identified as genospecies B. garinii using RFLP analysis of the flagellin and 16S rRNA genes. In contrast, RFLP analysis of the six isolates from I. ovatus indicated that they were different from the three reported genospecies. DNA homology studies confirmed the RFLP results. The six isolates from I. ovatus had DNA homologies ranging from 85 to 99%, whereas DNA relatedness of the I. ovatus isolate with strains belonging to the three genospecies was 50 to 64%. These results suggest that the strains isolated from I. ovatus in Japan differ from the three genospecies and should be classified as a new genospecies of B. burgdorferi sensu lato. We propose that strains isolated from I. ovatus should be classified as B. japonica sp. nov.
Cytotoxic Effect of Hemolytic Culture Supernatant from Enterococcus faecalis on Mouse Polymorphonuclear Neutrophils and Macrophages
Authors: Miyazaki Shuichi , Ohno Akira , Kobayashi Intetsu , Uji Tatsuya , Yamaguchi Keizo , Goto Sachiko
Published: November 2013

We reconfirmed that the LD50S of hemolytic Enterococcus faecalis strains were significantly less than those of nonhemolytic E. faecalis strains in normal mice. Hemolysin produced by E. faecalis lysed human, horse, rabbit, and mouse erythrocytes, but not cow and sheep erythrocytes. Sphingomyelin comprises a part of the lipid composition of the erythrocyte membrane of all mammalian species tested. But phosphatidylcholine exists only in human, horse, rabbit, and mouse. These two lipids inhibited lysis of horse erythrocytes by hemolytic E. faecalis. Phosphatidylcholine is probably the binding component on the membrane of erythrocytes for E. faecalis hemolysin. The hemolytic culture supernatant lysed not only erythrocytes but also mouse polymorphonuclear neutrophils (PMNs) and macrophages.
Neurologic Abnormalities in Two Dogs Suspected Lyme Disease
Authors: Azuma Yuzo , Kawamura Katsuzi , Isogai Hiroshi , Isogai Emiko
Published: November 2013

A 2‐year‐old mongrel dog developed neurological signs following tick bite. These included astasia, persistent tonic convulsions and hyper‐reflexia. Both serum IgG and IgM antibody titers against Borrelia burgdorferi were positive in enzyme‐linked immunosorbent assay (ELISA). The neurological signs subsided after high‐dose penicillin and streptomycin treatment. A strain of spirochetes (P427a) was isolated from the midgut of Ixodes persulcatus feeding on the dog. Morphological characteristic, immunological property and protein profile revealed that the isolate was B. burgdorferi. Similarly, a 2‐year‐old Labrador retriever dog developed neurological signs after tick bite and showed a positive IgG antibody titer against B. burgdorferi. Antibiotic treatment was effective also in this case. These findings suggest that neurological symptoms shown in both dogs were caused by infection with B. burgdorferi.