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Isao Karube - One of the best experts on this subject based on the ideXlab platform.

  • An automatic flow-injection analysis system for determining phosphate ion in river water using pyruvate oxidase G (from Aerococcus viridans).
    Talanta, 1999
    Co-Authors: Hideaki Nakamura, Kazunori Ikebukuro, Yoshiko Arikawa, Hiroko Tanaka, Mami Hasegawa, Yuzo Masuda, Yoko Nomura, Isao Karube
    Abstract:

    An automated flow-injection system combining a pyruvate oxidase reaction and chemiluminescence for the detection of phosphate ion in river water has been developed. In this research, we used pyruvate oxidase G (PyrOxG), from Aerococcus viridans, immobilizing it on N-hydroxysuccinicacidimido beads without a cross-linker. In this sensor system, which was constructed as a trial system of desktop type, the temperature was precisely controlled. After the sensor system was optimized, a calibration curve was obtained with a detection limit of 96 nM phosphate ion, a range between 96 nM and 32 microM phosphate ion, and a relative standard deviation of 2.3% (n=5) at 25 degrees C. The sensitivity of this sensor was sufficient to determine the maximal permissible phosphate-ion concentration in the environmental waters of Japan (0.32 microM). In addition, the sensor could determine the calibration curves between 0.16 and 32 microM phosphate ion (five points, n=3; averaged correlation, r=1.00) for at least 2 weeks, demonstrating enough stability for practical use. Furthermore, we investigated the influence on the sensor response of dissolved substances in river water such as metal ions, heavy metal ions, inorganic ions, and organic compounds. Treatment with activated carbon could improve the response of the sensor when inhibited by dissolved substances in river water, except for manganese ion and uric acid. The sensor system could determine the concentrations of phosphate ion in various samples of river water from the Tone River. The results obtained by this sensor system and the modified molybdenum blue method were compared, and good correlation (r=0.94) was obtained.

  • an automatic flow injection analysis system for determining phosphate ion in river water using pyruvate oxidase g from Aerococcus viridans
    Talanta, 1999
    Co-Authors: Hideaki Nakamura, Kazunori Ikebukuro, Yoshiko Arikawa, Hiroko Tanaka, Mami Hasegawa, Yuzo Masuda, Yoko Nomura, Isao Karube
    Abstract:

    An automated flow-injection system combining a pyruvate oxidase reaction and chemiluminescence for the detection of phosphate ion in river water has been developed. In this research, we used pyruvate oxidase G (PyrOxG), from Aerococcus viridans, immobilizing it on N-hydroxysuccinicacidimido beads without a cross-linker. In this sensor system, which was constructed as a trial system of desktop type, the temperature was precisely controlled. After the sensor system was optimized, a calibration curve was obtained with a detection limit of 96 nM phosphate ion, a range between 96 nM and 32 μM phosphate ion, and a relative standard deviation of 2.3% (n = 5) at 25°C. The sensitivity of this sensor was sufficient to determine the maximal permissible phosphate-ion concentration in the environmental waters of Japan (0.32 μM). In addition, the sensor could determine the calibration curves between 0.16 and 32 μM phosphate ion (five points, n = 3; averaged correlation, r = 1.00) for at least 2 weeks, demonstrating enough stability for practical use. Furthermore, we investigated the influence on the sensor response of dissolved substances in river water such as metal ions, heavy metal ions, inorganic ions, and organic compounds. Treatment with activated carbon could improve the response of the sensor when inhibited by dissolved substances in river water, except for manganese ion and uric acid. The sensor system could determine the concentrations of phosphate ion in various samples of river water from the Tone River. The results obtained by this sensor system and the modified molybdenum blue method were compared, and good correlation (r = 0.94) was obtained.

Spencer J Greenwood - One of the best experts on this subject based on the ideXlab platform.

  • Aerococcus viridans var homari the presence of capsule and the relationship to virulence in american lobster homarus americanus
    Journal of Invertebrate Pathology, 2016
    Co-Authors: Fraser K Clark, Dorota W Wadowska, Spencer J Greenwood
    Abstract:

    The relationship between virulence and encapsulation of Aerococcus viridans var. homari was evaluated by growing virulent (Rabin's) and avirulent (ATCC 10400) strains under varying culture conditions, and during challenge trials. Changes in capsule thickness were monitored using a modified lysine-ruthenium red (LRR) fixation method and transmission electron microscopy. The virulent Rabin's strain possessed a prominent capsule of 0.252 μm±0.061 μm that was diminished by in vitro growth conditions to 0.206 μm±0.076 μm. The ATCC 10400 strain capsule thickness decreased from 0.157 μm±0.043 μm to 0.117 μm±0.043 μm after 10 in vitro passages. The virulent Rabin's strain capsule was significantly thicker than the avirulent ATCC 10400 strain under all growth conditions. Rabin's strain, regardless of pre-challenge growth conditions or dose (high dose 10(7) or low dose 10(2)), was able to kill lobsters in 7 days at 15°C. ATCC 10400 strain, regardless of pre-challenge growth conditions, killed lobster only at high doses (10(7)) with varying median time to death of ∼15 days, while at low doses (10(2)) all lobsters survived and no bacteria were present after 42 days. This work demonstrates the importance of the thickness of the A. viridans capsule to virulence in the American lobster.

  • differential expression of american lobster homarus americanus immune related genes during infection of Aerococcus viridans var homari the causative agent of gaffkemia
    Journal of Invertebrate Pathology, 2013
    Co-Authors: Fraser K Clark, Adam R Acorn, Spencer J Greenwood
    Abstract:

    This is the first transcriptomic study focusing on immunity in the commercially valuable American lobster (Homarus americanus). We have conducted an in vivo infection trial using the Gram-positive bacterium Aerococcus viridans var. homari to determine how H. americanus responds to this naturally occurring lethal-pathogen. A novel H. americanus microarray was used to measure the transcriptomic changes occurring in over 14,000 genes in the lobster hepatopancreas. Hundreds of new immune genes and isoforms were identified and measured for the first time in this species, and our findings highlight 148 genes of interest involved in H. americanus pathogen response. We verified our microarray results using RT-qPCR on three anti-lipopolysaccharide (ALFHa-1, ALFHa-2, ALFHa-4), a thioredoxin, acute phase serum amyloid protein A, hexokinase and two trypsin genes. RT-qPCR and microarray findings show close agreement and highlight the significant increase in gene expression in many lobster immune genes during A. viridans infection. Differential expression of the ALFHa isoforms may indicate that the H. americanus immune response can be tailored to the class of pathogen causing disease.

  • genetic characterization of the lobster pathogen Aerococcus viridans var homari by 16s rrna gene sequence and rapd
    Diseases of Aquatic Organisms, 2005
    Co-Authors: Spencer J Greenwood, Ian R Keith, Beatrice M Despres, Richard J Cawthorn
    Abstract:

    A combination of 16S rRNA sequencing and random amplified polymorphic DNA (RAPD) analysis was used to evaluate the genetic diversity within Aerococcus viridans var. homari, the causative agent of gaffkemia in lobsters. A collection of 7 A. viridans var. homari strains and 2 avirulent A. viridans-like cocci isolated from homarid lobsters harvested from different regions on the Atlantic Coast of North America were analyzed. The isolates are separated geographically and temporally between the years 1947 and 2000. Sequencing of 16S rRNA genes confirmed the inclusion of all 9 isolates in the monophyletic A. viridans clade (99.8 to 100% similarity). RAPD analysis revealed that the 9 A. viridans var. homari isolates could be separated into 2 distinct subtypes. Subtype 1 included the 7 pathogenic lobster isolates and constituted a homogeneous group regardless of their geographical, temporal or virulence differences. Subtype 2 contained the 2 avirulent A. viridans-like cocci that had distinct RAPD patterns and clustered separately with the non-marine A. viridans. RAPD analysis represented a useful method for determining molecular subtyping for the intraspecific classification and epidemiological investigations of A. viridans var. homari.

Sergio A Streitenberger - One of the best experts on this subject based on the ideXlab platform.

  • A study on the kinetic mechanism of apoenzyme reconstitution from Aerococcus viridans lactate oxidase.
    Journal of enzyme inhibition and medicinal chemistry, 2003
    Co-Authors: Sergio A Streitenberger, Jose Aniceto Lopez-mas, Álvaro Sánchez-ferrer, Francisco García-carmona
    Abstract:

    The preparation of a reconstitutable apoprotein is widely recognized as an important tool for studying the interactions between protein and coenzyme and also for characterizing the coenzyme-binding site of the protein. Here is described the kinetic analysis of the reconstitution of Aerococcus viridans lactate oxidase apoenzyme with FMN and FAD in the presence of substrate. The reconstitution was followed by measuring the increase in catalytic capacity with time. Lactate oxidase activity was easily removed by obtaining its apoenzyme in an acidic saturated ammonium sulphate solution. When the apoenzyme was reconstituted by the addition of FMN or FAD, a marked lag period was observed, after which the system reached a steady state (linear rate). To explain the binding mechanism of the cofactors to the apoenzyme, a kinetic model is proposed, in which the constants, k3 and k-3, representing the interaction of apoenzyme with cofactor are considered slow and responsible for the lag in the expression of activity. The affinity of apoenzyme was 51-fold higher for FMN than FAD.

  • Use of dye affinity chromatography for the purification of Aerococcus viridans lactate oxidase.
    Biotechnology progress, 2002
    Co-Authors: Sergio A Streitenberger, Jose Aniceto Lopez-mas, Álvaro Sánchez-ferrer, Francisco García-carmona
    Abstract:

    Lactate oxidase was purified from Aerococcus viridans (A. viridans) by dye affinity chromatography and FPLC ion exchange chromatography. The lactate oxidase could be purified by comparatively simple procedures, the purification achieved from a crude extract of A. viridans was 41-fold with a specific activity of 143 units/(mg of protein). The purified enzyme was a L-lactate oxidase, which catalyses the conversion of L-lactate in the presence of molecular oxygen to pyruvate and H(2)O(2). This purified lactate oxidase showed an apparent molecular mass of 48,200 in SDS-PAGE and the native molecular weight, as estimated by FPLC gel filtration, was 187,300. This molecular weight indicates that lactate oxidase exists in tetrameric form after gel filtration. To differing degrees, all the triazine dyes tested were inhibitors of lactate oxidase, solutions of free triazine dyes showing an inhibition mechanism which was both time- and pH-dependent.

  • microencapsulation of Aerococcus viridans with catalase and its application for the synthesis of dihydroxyacetone phosphate
    Applied Microbiology and Biotechnology, 2002
    Co-Authors: Sergio A Streitenberger, M J Villaverde, Alvaro Sanchezferrer, Francisco Garciacarmona
    Abstract:

    Dihydroxyacetone phosphate is essential for the synthesis of polyhydroxylated compounds used as components or precursors of active pharmaceutical substances, such as antibiotics or glycosidase inhibitors. Dihydroxyacetone phosphate was produced by enzymatic oxidation of L-α-glycerophosphate in the presence of glycerophosphate oxidase or Aerococcus viridans coimmobilized with a hydrogen peroxide-decomposing enzyme. The microencapsulation of A. viridans with catalase in sodium alginate showed a conversion of 98.5%; the conversion percentage remained constant in all five runs. Liquid chromatography of the product revealed that the product peak corresponded to that of the dihydroxyacetone phosphate internal standard. This indicated a high degree of product purity.

  • non linear slow binding inhibition of Aerococcus viridans lactate oxidase by cibacron blue 3ga
    Journal of Enzyme Inhibition, 2001
    Co-Authors: Sergio A Streitenberger, Alvaro Sanchezferrer, Jose Aniceto Lopezmas, Francisco Garciacarmona
    Abstract:

    Lactate oxidase (LOD) was purified from cells of Aerococcus viridans by phase partitioning in Triton X-114 (TX-114), ammonium sulphate fractionation and FPLC ion exchange chromatography. The purification achieved from a crude extract of A. viridans was 32-fold with a 60% recovery of activity. The isolated enzyme was a true FMN-containing LOD in tetrameric form with a subunit molecular weight of 48,000. The KM for L-lactate was 175 microM, a 6-fold less value than described in the literature. Among the inhibitors tested, Cibacron Blue 3GA showed the lowest Ki. At low concentrations, Cibacron Blue 3GA behaved as a dye-, pH- and time-dependent inhibitor. A Dixon plot of the steady-state rate showed the time-dependent inhibition to be non-linear, contrary to that described for other slow-binding inhibitors. A model to explain this phenomenon was proposed. The model implies the binding of Cibacron Blue 3GA to the isomerised form of the initial enzyme-inhibition complex (E'I).

  • highly efficient Aerococcus viridans l α glycerophosphate oxidase production in the presence of h2o2 decomposing agent purification and kinetic characterization
    Applied Microbiology and Biotechnology, 2001
    Co-Authors: Sergio A Streitenberger, Alvaro Sanchezferrer, Jose Aniceto Lopezmas, Francisco Garciacarmona
    Abstract:

    Glycerophosphate oxidase was purified from Aerococcus viridans cells by phase partitioning in Triton X-114, ammonium sulfate fractionation, FPLC ion-exchange chromatography and FPLC hydrophobic-interaction chromatography. The purification achieved from a crude extract of A. viridans was 38-fold with a 32% recovery of activity. Under the growth conditions used, A. viridans strain CECT 978 proved to be an excellent glycerophosphate-oxidase producer, with enzyme production 2,800-fold greater than that described in the literature for the same microorganism. The culture medium used in the present work is that commonly used for cultivation of this microorganism, except that an H2O2-decomposing enzyme was added. The addition of catalase to the growth medium had a clear effect on the growth rate. Furthermore, methylglyoxal, a metabolite that is formed enzymatically from triose phosphates, was found to be an inactivator of glycerophosphate oxidase activity.

Hideaki Nakamura - One of the best experts on this subject based on the ideXlab platform.

  • An automatic flow-injection analysis system for determining phosphate ion in river water using pyruvate oxidase G (from Aerococcus viridans).
    Talanta, 1999
    Co-Authors: Hideaki Nakamura, Kazunori Ikebukuro, Yoshiko Arikawa, Hiroko Tanaka, Mami Hasegawa, Yuzo Masuda, Yoko Nomura, Isao Karube
    Abstract:

    An automated flow-injection system combining a pyruvate oxidase reaction and chemiluminescence for the detection of phosphate ion in river water has been developed. In this research, we used pyruvate oxidase G (PyrOxG), from Aerococcus viridans, immobilizing it on N-hydroxysuccinicacidimido beads without a cross-linker. In this sensor system, which was constructed as a trial system of desktop type, the temperature was precisely controlled. After the sensor system was optimized, a calibration curve was obtained with a detection limit of 96 nM phosphate ion, a range between 96 nM and 32 microM phosphate ion, and a relative standard deviation of 2.3% (n=5) at 25 degrees C. The sensitivity of this sensor was sufficient to determine the maximal permissible phosphate-ion concentration in the environmental waters of Japan (0.32 microM). In addition, the sensor could determine the calibration curves between 0.16 and 32 microM phosphate ion (five points, n=3; averaged correlation, r=1.00) for at least 2 weeks, demonstrating enough stability for practical use. Furthermore, we investigated the influence on the sensor response of dissolved substances in river water such as metal ions, heavy metal ions, inorganic ions, and organic compounds. Treatment with activated carbon could improve the response of the sensor when inhibited by dissolved substances in river water, except for manganese ion and uric acid. The sensor system could determine the concentrations of phosphate ion in various samples of river water from the Tone River. The results obtained by this sensor system and the modified molybdenum blue method were compared, and good correlation (r=0.94) was obtained.

  • an automatic flow injection analysis system for determining phosphate ion in river water using pyruvate oxidase g from Aerococcus viridans
    Talanta, 1999
    Co-Authors: Hideaki Nakamura, Kazunori Ikebukuro, Yoshiko Arikawa, Hiroko Tanaka, Mami Hasegawa, Yuzo Masuda, Yoko Nomura, Isao Karube
    Abstract:

    An automated flow-injection system combining a pyruvate oxidase reaction and chemiluminescence for the detection of phosphate ion in river water has been developed. In this research, we used pyruvate oxidase G (PyrOxG), from Aerococcus viridans, immobilizing it on N-hydroxysuccinicacidimido beads without a cross-linker. In this sensor system, which was constructed as a trial system of desktop type, the temperature was precisely controlled. After the sensor system was optimized, a calibration curve was obtained with a detection limit of 96 nM phosphate ion, a range between 96 nM and 32 μM phosphate ion, and a relative standard deviation of 2.3% (n = 5) at 25°C. The sensitivity of this sensor was sufficient to determine the maximal permissible phosphate-ion concentration in the environmental waters of Japan (0.32 μM). In addition, the sensor could determine the calibration curves between 0.16 and 32 μM phosphate ion (five points, n = 3; averaged correlation, r = 1.00) for at least 2 weeks, demonstrating enough stability for practical use. Furthermore, we investigated the influence on the sensor response of dissolved substances in river water such as metal ions, heavy metal ions, inorganic ions, and organic compounds. Treatment with activated carbon could improve the response of the sensor when inhibited by dissolved substances in river water, except for manganese ion and uric acid. The sensor system could determine the concentrations of phosphate ion in various samples of river water from the Tone River. The results obtained by this sensor system and the modified molybdenum blue method were compared, and good correlation (r = 0.94) was obtained.

B. Horney - One of the best experts on this subject based on the ideXlab platform.

  • response of american lobsters homarus americanus to infection with a field isolate of Aerococcus viridans var homari gaffkemia survival and haematology
    Diseases of Aquatic Organisms, 2004
    Co-Authors: A L Battison, Richard J Cawthorn, B. Horney
    Abstract:

    American lobsters Homarus americanus were inoculated with a field isolate of the Gram-positive bacterium Aerococcus viridans var. homari, causative agent of gaffkemia, at 1 x 10(6), 1 x 10(4) or 1 x 10(2) colony forming units (CFU) kg(-1) or with sterile 3% NaCl and maintained at 10 or 15 degrees C until they died or were euthanised. Progression of disease in individual animals was monitored daily by total haemocyte count (THC) and haemolymph culture. Post-mortem examinations were performed on all lobsters. Effects of both ambient temperature and infective dose on survival time were observed. Marked bacteraemia occurred in all mortalities. Haemocytopenia (THC < 10 x 10(9) cells l(-1)) preceded death in most, but not all, mortalities.

  • Classification of Homarus americanus hemocytes and the use of differential hemocyte counts in lobsters infected with Aerococcus viridans var. homari (Gaffkemia)
    Journal of Invertebrate Pathology, 2003
    Co-Authors: Andrea L. Battison, Richard Cawthorn, B. Horney
    Abstract:

    Abstract Hemocytes of the American lobster ( Homarus americanus H. Milne Edwards) were classified after examination of Wright–Giemsa stained cytocentrifuge preparations by brightfield light microscopy. Eleven hemocyte types were identified using morphologic criteria. The classification system was then used to monitor changes in the differential hemocyte count (DHC) of lobsters infected with the Gram positive coccus Aerococcus viridans var. homari , etiologic agent of gaffkemia. The appearance of less mature hemocytes in the DHCs of lobsters in the late stages of infection was similar to the ‘left shift’ of vertebrate inflammation. Results from this study suggest that DHCs can be used to assess and characterize inflammation in H. americanus and possibly other crustaceans.