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Jean-charles Dalphin - One of the best experts on this subject based on the ideXlab platform.
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Microbial exposure to dairy farmers' dwellings and COPD occurrence.
International Journal of Environmental Health Research, 2018Co-Authors: Coralie Barrera, Jean-charles Dalphin, Laurence Millon, Bruno Degano, Steffi Rocchi, Thibaud Soumagne, Lucie Laurent, Anne-pauline Bellanger, Jean-jacques Laplante, G. RebouxAbstract:Dairy farming is a risk factor for chronic obstructive pulmonary disease (COPD). The aim was to determine predictive markers either in blood samples or in dwelling dust samples by comparing COPD and healthy controls with or without farming activity. Dust was collected and analyzed by real-time quantitative PCR. ELISA and DELFIA® were performed to assay the level of specific IgG and IgE of 10 targeted microorganisms. The dwelling exposure of farmers was higher than in the non-farmers (Especially Eurotium amstelodami and Lichtheimia corymbifera). The IgG response against Wallemia sebi and Saccharopolyspora rectivirgula was more often higher in the farmers than the non-farmers. However, exposure and sensitization to the microorganisms tested cannot explain the occurrence of COPD in the dairy farmers' population. COPD development is probably caused by multiple factors associated with exposure over a period of several years.
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Immunoreactive proteins of Saccharopolyspora rectivirgula for farmer's lung serodiagnosis.
PROTEOMICS - Clinical Applications, 2014Co-Authors: Coralie Barrera, Jean-charles Dalphin, Bénédicte Rognon, Laurence Millon, Manfredo Quadroni, Sandrine Roussel, Isabelle Court-fortune, Denis Caillaud, Stephane Jouneau, Jean-marc FellrathAbstract:Saccharopolyspora rectivirgula is the principal cause of farmer's lung disease (FLD). Serodiagnosis is based on immunoprecipitation techniques or enzyme immunoassays with homemade crude antigens and is not standardized. We aimed to produce specific recombinant antigens for the development of a standardized ELISA. We recruited 41 patients and 43 healthy exposed controls from five university hospital pneumology departments in France and Switzerland. S. rectivirgula proteins were extracted, separated by 2D electrophoresis, and subjected to Western blotting, with sera from FLD patients or controls. FLD-specific proteins were identified by MS and were produced as recombinant antigens. The diagnostic performance of ELISA tests using the recombinant antigens was assessed with all the sera from FLD patients and controls. We identified 25 FLD-specific proteins, some of which play important roles in transport, nutrition, or virulence. We produced 17 of these proteins as recombinant antigens and assessed their suitability for inclusion in the ELISA test. A combination of three of these proteins (SR1FA, SR17, and SR22) proved remarkably effective at discriminating between patients and controls, with a sensitivity of 83% and a specificity of 77%. The recombinant antigens produced in this study constitute a major step toward the improvement of diagnostic performance and the standardization of FLD serodiagnosis.
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Hypoxaemia during pregnancy: pulmonary arteriovenous dilatation as a likely cause.
European Respiratory Review, 2014Co-Authors: Matthieu Veil-picard, Jean-charles Dalphin, Julie Cattin, Romain Chopard, François Schiele, Didier Riethmuller, Bruno DeganoAbstract:Saccharopolyspora rectivirgula is the main cause of farmer's lung disease. The development of recombinant antigens to standardize the serodiagnosis of the disease requires knowledge of the S. rectivirgula genome. We sequenced the genome of an environmental strain, S. rectivirgula DSM 43113. A total of 3,221 proteins were found to be encoded in a short 3.9-Mb genome.
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Immunoreactive proteins of Saccharopolyspora rectivirgula for farmer's lung serodiagnosis.
PROTEOMICS - Clinical Applications, 2014Co-Authors: Coralie Barrera, Jean-charles Dalphin, Bénédicte Rognon, Laurence Millon, Manfredo Quadroni, Sandrine Roussel, Isabelle Court-fortune, Denis Caillaud, Stephane Jouneau, Jean-marc FellrathAbstract:PURPOSE: Saccharopolyspora rectivirgula is the principal cause of farmer's lung disease (FLD). Serodiagnosis is based on immunoprecipitation techniques or enzyme immunoassays with homemade crude antigens and is not standardized. We aimed to produce specific recombinant antigens for the development of a standardized ELISA. EXPERIMENTAL DESIGN: We recruited 41 patients and 43 healthy exposed controls from five university hospital pneumology departments in France and Switzerland. S. rectivirgula proteins were extracted, separated by 2D electrophoresis, and subjected to Western blotting, with sera from FLD patients or controls. FLD-specific proteins were identified by MS and were produced as recombinant antigens. The diagnostic performance of ELISA tests using the recombinant antigens was assessed with all the sera from FLD patients and controls. RESULTS: We identified 25 FLD-specific proteins, some of which play important roles in transport, nutrition, or virulence. We produced 17 of these proteins as recombinant antigens and assessed their suitability for inclusion in the ELISA test. A combination of three of these proteins (SR1FA, SR17, and SR22) proved remarkably effective at discriminating between patients and controls, with a sensitivity of 83% and a specificity of 77%. CONCLUSIONS AND CLINICAL RELEVANCE: The recombinant antigens produced in this study constitute a major step toward the improvement of diagnostic performance and the standardization of FLD serodiagnosis.
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Human monocyte-derived dendritic cells exposed to microorganisms involved in hypersensitivity pneumonitis induce a Th1-polarized immune response.
Clinical and Vaccine Immunology, 2013Co-Authors: Anne-pauline Bellanger, Jean-charles Dalphin, G. Reboux, Jean-rené Pallandre, Christophe Borg, Loeffert Sophie, Laurence MillonAbstract:Hypersensitivity pneumonitis (HP) is an immunoallergic disease characterized by a prominent interstitial infiltrate composed predominantly of lymphocytes secreting inflammatory cytokines. Dendritic cells (DCs) are known to play a pivotal role in the lymphocytic response. However, their cross talk with microorganisms that cause HP has yet to be elucidated. This study aimed to investigate the initial interactions between human monocyte-derived DCs (MoDCs) and four microorganisms that are different in nature (Saccharopolyspora rectivirgula [actinomycetes], Mycobacterium immunogenum [mycobacteria], and Wallemia sebi and Eurotium amstelodami [filamentous fungi]) and are involved in HP. Our objectives were to determine the cross talk between MoDCs and HP-causative agents and to determine whether the resulting immune response varied according to the microbial extract tested. The phenotypic activation of MoDCs was measured by the increased expression of costimulatory molecules and levels of cytokines in supernatants. The functional activation of MoDCs was measured by the ability of MoDCs to induce lymphocytic proliferation and differentiation in a mixed lymphocytic reaction (MLR). E. amstelodami-exposed (EA) MoDCs expressed higher percentages of costimulatory molecules than did W. sebi-exposed (WS), S. rectivirgula-exposed (SR), or M. immunogenum-exposed (MI) MoDCs (P < 0.05, Wilcoxon signedrank test). EA-MoDCs, WS-MoDCs, SR-MoDCs, and MI-MoDCs induced CD4 T cell proliferation and a Th1-polarized immune response. The present study provides evidence that, although differences were initially observed between MoDCs exposed to filamentous fungi and MoDCs exposed to bacteria, a Th1 response was ultimately promoted by DCs regardless of the microbial extract tested.
E. Israël-assayag - One of the best experts on this subject based on the ideXlab platform.
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Viral infection enhances the response to Saccharopolyspora rectivirgula in mice prechallenged with this farmer's lung antigen
Lung, 1996Co-Authors: Yvon Cormier, N. Samson, E. Israël-assayagAbstract:Sendai viral infection enhances mice lung response to Saccharopolyspora rectivirgula (SR). The mechanisms of this viral enhancement remain unclear. The present study was done to verify if the viral infection was required and if the presence of the viral infection needed to be given simultaneously with the SR antigen for the enhanced response to occur. In a first experiment groups of C57BL/6 mice were instilled concomitantly with SR and live or inactivated Sendai virus. In a second experiment the viral infection in the appropriate group preceded the SR challenges by 4 weeks. As reported previously SR induced a cellular inflammatory response. This effect of SR was enhanced by the viral infection but not by inactivated virus particles. Total lavage cells 3 weeks after the virus inoculation in the appropriate groups were: saline = 69 ± 15 × 10^3; SR = 678 ± 104 × 10^3; Sendai = 277 ± 61 × 10^3; inactivated Sendai = 73 ± 23 × 10^3; SR + Sendai = 1232 ± 232 × 10^3; SR + inactivated Sendai = 515 ± 54 × 10^3. In the second experiment, where the infection preceded the SR challenge, no enhancement was observed. We conclude that Sendai virus enhances mice lung response to SR by an infectious process when both SR and the virus are present simultaneously.
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PC: PS liposomes induce a recruitment of neutrophils and the release of TNFa in the lungs of mice sensitized with Saccharopolyspora rectivirgula
European Journal of Clinical Investigation, 1995Co-Authors: F Bellemare, E. Israël-assayag, Y CormierAbstract:The aim of this study was to verify the effect of nasally instilled liposomes (L) or dexamethasone-containing L (Ldexa) on normal or inflamed lung tissue. Three groups of mice were studied. Group I was given saline instillations for 3 weeks prior to the instillation with liposomes. In groups II and III lung inflammation was induced by repeated instillations of Saccharopolyspora rectivirgula before the instillation of liposomes (group II) or liposomes containing dexamethasone (group III). Animals from all groups were killed at regular time intervals for up to 48 h after the instillation of liposomes. The total cell count in bronchoalveolar lavage fluid did not differ significantly between groups I and II. However, in group III it decreased rapidly from 6.2 to 2.8 x 10(5) cells mL-1 within 2 h. Differential counts did not change in group I, but in group II a transient neutrophilia was observed 180 min after the instillation of liposomes. In group III, the instillation of dexamethasone-containing liposomes depleted all neutrophils and lymphocytes after 4 h. No TNF alpha was found in samples of lavage fluid from any of the groups at time 0. In group I, liposomes induced the production of 0.03 ng mL-1 of TNF alpha in the 1 h sample only. In group II, TNF alpha peaked to 1 ng mL-1 at 1 h and had decreased to 0.35 ng mL-1 by 3 h. In group III, TNF alpha peaked at 1 h, but only reached a level of 0.1 ng mL-1.(ABSTRACT TRUNCATED AT 250 WORDS)
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Long-term viral enhancement of lung response to Saccharopolyspora rectivirgula
The American review of respiratory disease, 1994Co-Authors: Yvon Cormier, G. M. Tremblay, M. Fournier, E. Israël-assayagAbstract:The current study was done to look at the long-term enhancing effect of a single viral infection on repeated Saccharopolyspora rectivirgula (SR) antigenic challenges in mice and at inflammatory cytokines in this enhancement. Four groups of C57BI/6 mice were studied: Group 1 received intranasal instillations of saline, 3 days per week; Group 2, intranasal instillations of saline plus one intranasal instillation of 80 hemagglutination units (HAU) of Sendai virus after 3 wk of saline; Group 3, instillations of SR, 3 days per week; and Group 4, instillations of SR, 3 days per week, plus one instillation of Sendai virus after 3 wk of SR. Bronchoalveolar lavages (BAL) were performed 15 and 30 wk after the virus inoculation in the appropriate groups
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Long-term viral enhancement of lung response to Saccharopolyspora rectivirgula.
American Journal of Respiratory and Critical Care Medicine, 1994Co-Authors: Yvon Cormier, G. M. Tremblay, M. Fournier, E. Israël-assayagAbstract:The current study was done to look at the long-term enhancing effect of a single viral infection on repeated Saccharopolyspora rectivirgula (SR) antigenic challenges in mice and at inflammatory cytokines in this enhancement. Four groups of C57BI/6 mice were studied: Group 1 received intranasal instillations of saline, 3 days per week; Group 2, intranasal instillations of saline plus one intranasal instillation of 80 hemagglutination units (HAU) of Sendai virus after 3 wk of saline; Group 3, instillations of SR, 3 days per week; and Group 4, instillations of SR, 3 days per week, plus one instillation of Sendai virus after 3 wk of SR. Bronchoalveolar lavages (BAL) were performed 15 and 30 wk after the virus inoculation in the appropriate groups. Each time, a two- to threefold increase in BAL cell counts was obtained from virus-infected animals challenged with SR compared with animals that received the SR alone. Animals infected with virus only showed values similar to those of control animals. A higher perc...
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Long-term viral enhancement of lung response to Saccharopolyspora rectivirgula.
American journal of respiratory and critical care medicine, 1994Co-Authors: Yvon Cormier, G. M. Tremblay, M. Fournier, E. Israël-assayagAbstract:The current study was done to look at the long-term enhancing effect of a single viral infection on repeated Saccharopolyspora rectivirgula (SR) antigenic challenges in mice and at inflammatory cytokines in this enhancement. Four groups of C57BI/6 mice were studied: Group 1 received intranasal instillations of saline, 3 days per week; Group 2, intranasal instillations of saline plus one intranasal instillation of 80 hemagglutination units (HAU) of Sendai virus after 3 wk of saline; Group 3, instillations of SR, 3 days per week; and Group 4, instillations of SR, 3 days per week, plus one instillation of Sendai virus after 3 wk of SR. Bronchoalveolar lavages (BAL) were performed 15 and 30 wk after the virus inoculation in the appropriate groups. Each time, a two- to threefold increase in BAL cell counts was obtained from virus-infected animals challenged with SR compared with animals that received the SR alone. Animals infected with virus only showed values similar to those of control animals. A higher percentage of large foamy multinucleated cells were found in the BAL from the SR+Sendai group than the SR alone group (7.92 +/- 0.730% compared with 1.8 +/- 0.296%). These cells were not seen in the other groups. BAL levels of TNF-alpha and IL-1 alpha at 15 wk were much higher in SR+Sendai-treated (1,439 +/- 268 and 96 +/- 9 pg/ml, respectively) than SR alone-treated animals (361 +/- 100 and 23 +/- 4 pg/ml). BAL fluid of control animals and Sendai alone animals contained 64 +/- 24 and 65 +/- 15 pg/ml of TNF-alpha, but no IL-1 alpha was detected.(ABSTRACT TRUNCATED AT 250 WORDS)
Yvon Cormier - One of the best experts on this subject based on the ideXlab platform.
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Hypersensitivity Pneumonitis Caused by Fungi
Proceedings of the American Thoracic Society, 2010Co-Authors: Moisés Selman, Yves Lacasse, Annie Pardo, Yvon CormierAbstract:Hypersensitivity pneumonitis (HP) is a complex syndrome caused by an exaggerated immune response to the inhalation of a large variety of organic particles. The most frequent antigens that cause HP worldwide are bird proteins (pigeon breeders' disease) and bacteria (Saccharopolyspora rectivirgula). However, fungi are also implicated in many cases, including occupational and nonoccupational outbreaks. The clinical course of the disease is highly variable and its diagnosis clinically challenging since no specific test or biomarker allows a consistent diagnosis. Therefore, a combination of symptoms, bronchoalveolar lavage findings, chest imaging, lab tests, and often biopsies are needed for an accurate diagnosis. Regardless of the cause or the responsible environment, the histopathology is similar and usually consists of a granulomatous interstitial bronchiolocentric pneumonitis characterized by the presence of poorly formed granulomas and a prominent interstitial infiltrate composed of lymphocytes, plasma ce...
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Mature CD11c+ cells are enhanced in hypersensitivity pneumonitis
European Respiratory Journal, 2009Co-Authors: M. Girard, Evelyne Israël-assayag, Yvon CormierAbstract:The present study verified the hypothesis that enhanced maturation of antigen-presenting CD11c + cells could explain the viral-induced exacerbated immune response to Saccharopolyspora rectivirgula (SR), the main antigen responsible for farmer’s lung, a classic form of hypersensitivity pneumonitis (HP). Four groups of mice were studied: group 1 received intranasal instillations of saline; group 2 received instillations of SR for 12 weeks; group 3 received instillations of saline and a single infection with Sendai virus on week 3; and group 4 received instillations of SR for 12 weeks with a single administration of Sendai virus on week 3. On week 13, mice were sacrificed and bronchoalveolar lavage was performed. Lungs were harvested, digested with enzymes, and CD11c + cells were analysed in flow cytometry with anti-CD11c, anti-CD86 and anti-major histocompatibility complex class II markers. Immunofluorescence studies were also performed with the same cell surface markers. Both flow cytometry and immunofluorescence results demonstrate that mature CD11c + cells are significantly enhanced in SR-challenged mice simultaneously infected with Sendai virus, compared with other groups. These CD11c + cells persist in the lung for 9 weeks after the virus infection. Maturation of CD11c + cells could explain, at least in part, the virus-induced increased immune response to SR antigens in this model of HP, but mechanisms have still to be elucidated.
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Inhibitory effect of nicotine on experimental hypersensitivity pneumonitis in vivo and in vitro.
American Journal of Respiratory and Critical Care Medicine, 2004Co-Authors: Marie-renée Blanchet, Evelyne Israël-assayag, Yvon CormierAbstract:The incidence of hypersensitivity pneumonitis (HP) is lower in smokers than in nonsmokers. Because nicotine is immunosuppressive, we hypothesized that it could have a protective effect on HP induction in vivo. HP was induced in mice that were treated with nicotine either intraperitoneally (IP) (0.5 to 2.0 mg/kg/day) or intranasally (IN) (0.025 to 2.0 mg/kg/day). Both IP- and IN-treated animals had fewer bronchoalveolar lavage total cells and lymphocytes and a decreased lung tissue inflammation. IFN-γ but not interleukin-10 mRNA expression was reduced in lung tissue of 2.0-mg/kg IN-treated animals. To test the effect of nicotine on alveolar macrophages, AMJ2-C11 cells were treated with nicotine and stimulated with lipopolysaccharide or Saccharopolyspora rectivirgula, a causative agent of HP. Nicotine reduced tumor necrosis factor release and tumor necrosis factor, interleukin-10, and IFN-γ mRNA expression after stimulation and decreased CD80 expression by 55% in lipopolysaccharide-stimulated cells and by 4...
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Saccharopolyspora rectivirgula from Quebec dairy barns: application of simplified criteria for the identification of an agent responsible for farmer's lung disease
Journal of Medical Microbiology, 1999Co-Authors: Caroline Duchaine, Anne Mériaux, Gilles Brochu, Kathryn Bernard, Yvon CormierAbstract:Saccharopolyspora rectivirgula (Micropolyspora faeni) is one of the major agents responsible for farmer's lung disease, a form of hypersensitivity pneumonitis. It is frequently isolated from the air of contaminated barns. The identification of this actinomycete is difficult because most of its phenotypic characteristics are variable and classical tests are not easy to perform on actinomycetes. Fatty acid analysis is very useful for the identification of these strains, but is not available except in some research or reference laboratories. Morphological (microscopic and macroscopic observations), physiological and biochemical tests (growth properties; macromolecules degraded; citrate utilisation and acid production from carbohydrates; resistance to antibiotics, lysozyme and heat), cell wall and fatty acid analyses and IgG analyses with serum from patients with farmer's lung were performed on 12 environmental isolates presumed to be S. rectivirgula and two control strains of S. rectivirgula. From this, a simple and rapid scheme for the identification of this actinomycete is proposed: optimal growth temperature (55°C); colony appearance based on morphology (filamentous) and colour (beige to orange-brown); microscopic morphology (chains of spores on both aerial and substrate mycelium); growth on NaCl 10%; cell-wall analysis (type IV); and the verification of antibody response with serum from a patient with farmer's lung. This last criterion is important to confirm the immunogenicity of the strains identified as S. rectivirgula. This scheme provides an accurate and efficient way of identifying S. rectivirgula strains and evaluating exposure to this bacterium. The study shows the limited value and the lack of reproducibility of some classical biochemical tests.
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Airborne microflora in Quebec dairy farms: lack of effect of bacterial hay preservatives.
American Industrial Hygiene Association Journal, 1999Co-Authors: Caroline Duchaine, Anne Mériaux, Gilles Brochu, Yvon CormierAbstract:Pediococcus pentosaceus is a lactic-acid producing bacterium inoculated in hay to prevent hay deterioration. This study sought to verify the effect of this treatment on the barn microenvironment. Air samples were obtained from 19 barns using bacterial hay treatment and from 18 control barns with six-stage Andersen samplers and all-glass impingers. Appropriate culture media were used for the recovery and identification of microorganisms. Endotoxins were measured with chromogenic Limulus amoebocyte lysate assay. Median values (respectively for treated and untreated hay barns) were: 5.28 × 105 and 3.84 × 105 colony-forming units (CFU)/m3 for total bacteria; 3.18 × 106 and 4.5 × 106 CFU/m3 for molds; 1.36 × 103 and 1.74 × 103 endotoxin units/m3 for endotoxin levels; and 1.03 × 103 and 3.00 × 103 CFU/m3 for Saccharopolyspora rectivirgula. No viable P. pentosaceus were recovered. The presence of S. rectivirgula, the causative agent for farmer's lung, was not influenced by the hay treatment. Since no significant...
Teruo Amachi - One of the best experts on this subject based on the ideXlab platform.
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Divalent metal ion requirements of a thermostable multimetal beta-galactosidase from Saccharopolyspora rectivirgula.
The Journal of biological chemistry, 1994Co-Authors: M Harada, Toru Nakayama, Masahiro Nakao, Yuji Shibano, M Inohara, A. Kakudo, Teruo AmachiAbstract:Abstract To understand the roles of metal ions on the catalytic properties and thermostability of the thermostable beta-galactosidase of Saccharopolyspora rectivirgula, a thermophilic actinomycete, we have investigated the binding kinetics and requirements of divalent metal ions by equilibrium dialysis, titration, and metal ion buffer techniques. We found that the monomeric multimetal enzyme (M(r) 136,977) had eight specific binding sites for divalent metal ions. These sites were classified as follows: a very tight (class I) site for Ca2+, three tight (class II) sites consisting of two Ca(2+)-specific sites (class IICa) and one Mn(2+)-specific site (class IIMn; Kd for Mn2+, 2.0 nM), and four loose (class III) sites for Mn2+ (Kd, 1.2 microM) and Mg2+ (Kd, 2 microM). Removal of metal ions bound to class II and III sites of the holoenzyme (Ca3Mn5 species; relative Vmax (Vrel), 100%) by a chelating resin at 4 degrees C yielded a less thermostable Ca1 species (Vrel, 1.7%) with a class I Ca2+ ion, removal of which by a chelating resin at 50 degrees C caused a complete irreversible inactivation of the enzyme. Titration studies revealed that stoichiometric binding of Mn2+ to a class IIMn site of the Ca1 species caused a 33-fold activation whereas binding of Ca2+ to class IICa sites had no effect on enzyme activity. Ca1 species could be also activated 8-fold by heating at 60 degrees C for 20 min, suggesting that the catalytically important class II Mn2+ plays important roles in maintaining the native structure essential for activity. Occupation of class III sites by Mg2+ or Mn2+ was of physiological importance to attain sufficient thermostability by which this extracellular beta-galactosidase remained active for a prolonged time at elevated temperatures as was observed during growth of S. rectivirgula.
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Purification and characterization of a thermostable β-galactosidase with high transgalactosylation activity from Saccharopolyspora rectivirgula
Applied Microbiology and Biotechnology, 1994Co-Authors: Masahiro Nakao, Toru Nakayama, Yuji Shibano, Masami Harada, Yukiko Kodama, Teruo AmachiAbstract:We purified an extracellular thermostable β-galactosidase of Saccharopolyspora rectivirgula strain V2-2, a thermophilic actinomycete, to homogeneity and characterized it to be a monomeric enzyme with a relative molecular mass of 145 000 and s °_20,w of 7.1 s . In addition to the hydrolytic activity of 1- O -substituted β- d -galactopyranosides such as lactose [a Michaelis constant K _m=0.75 m m and molecular activity ( k _cat)= 63.1 s^−1 at pH 7.2 and 55° C] and p -nitrophenyl β- d -galactopyranoside ( K _m=0.04 m m k _cat= 55.8 s^−1), the enzyme had a high transgalactosylation activity. The enzyme reacted with 1.75 m lactose at 70°C and pH 7.0 for 22 h to yield oligosaccharides in a maximum yield (other than lactose) of 41% (w/w). A general structure for the major transgalactosylic products could be expressed as (Gal)_c-Glc, where n is 1, 2, 3, and 4 with a glucose at a reducing terminal. These oligosaccharides could selectively promote the growth of the genus Bifidobacterium found in human intestines. S. rectivirgula β-galactosidase was stable at pH 7.2 up to 60°C (for 4 h in the presence of 10 μ m MnCl_2) or 70°C (for 22 h in the presence of 1.75 m lactose and 10 μ m MnCl_2). Thus the enzyme is applicable to an immobilized enzyme system at high temperatures (60°C
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purification and characterization of a thermostable β galactosidase with high transgalactosylation activity from Saccharopolyspora rectivirgula
Applied Microbiology and Biotechnology, 1994Co-Authors: Masahiro Nakao, Toru Nakayama, Yuji Shibano, Masami Harada, Yukiko Kodama, Teruo AmachiAbstract:We purified an extracellular thermostable β-galactosidase of Saccharopolyspora rectivirgula strain V2-2, a thermophilic actinomycete, to homogeneity and characterized it to be a monomeric enzyme with a relative molecular mass of 145 000 and s°20,w of 7.1 s. In addition to the hydrolytic activity of 1-O-substituted β-d-galactopyranosides such as lactose [a Michaelis constant Km=0.75 mm and molecular activity (kcat)= 63.1 s−1 at pH 7.2 and 55° C] and p-nitrophenyl β-d-galactopyranoside (Km=0.04 mmkcat= 55.8 s−1), the enzyme had a high transgalactosylation activity. The enzyme reacted with 1.75 m lactose at 70°C and pH 7.0 for 22 h to yield oligosaccharides in a maximum yield (other than lactose) of 41% (w/w). A general structure for the major transgalactosylic products could be expressed as (Gal)c-Glc, where n is 1, 2, 3, and 4 with a glucose at a reducing terminal. These oligosaccharides could selectively promote the growth of the genus Bifidobacterium found in human intestines. S. rectivirgula β-galactosidase was stable at pH 7.2 up to 60°C (for 4 h in the presence of 10 μm MnCl2) or 70°C (for 22 h in the presence of 1.75 m lactose and 10 μm MnCl2). Thus the enzyme is applicable to an immobilized enzyme system at high temperatures (60°C <) for efficient production of the oligosaccharides from lactose.
Masahiro Nakao - One of the best experts on this subject based on the ideXlab platform.
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Unique primary structure of a thermostable multimetal β-galactosidase from Saccharopolyspora rectivirgula
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1998Co-Authors: Misa Inohara-ochiai, Toru Nakayama, Masahiro Nakao, Tsuyoshi Fujita, Takashi Ueda, Toshihiko Ashikari, Tokuzo Nishino, Yuji ShibanoAbstract:The gene of the monomeric multimetal β-galactosidase of Saccharopolyspora rectivirgula was cloned and sequenced. Although the enzyme could be assigned as a member of β-galactosidases belonging to the glycosyl hydrolase family 2, it has unusual structural features for β-galactosidase of this family; it contained a unique sequence which consists of approximately 200 amino acid residues with no similarity to known proteins. This 200-residue sequence exists as if it is inserted into a sequence homologous to the active-site domain of the Escherichia coli lacZ enzyme.
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Divalent metal ion requirements of a thermostable multimetal beta-galactosidase from Saccharopolyspora rectivirgula.
The Journal of biological chemistry, 1994Co-Authors: M Harada, Toru Nakayama, Masahiro Nakao, Yuji Shibano, M Inohara, A. Kakudo, Teruo AmachiAbstract:Abstract To understand the roles of metal ions on the catalytic properties and thermostability of the thermostable beta-galactosidase of Saccharopolyspora rectivirgula, a thermophilic actinomycete, we have investigated the binding kinetics and requirements of divalent metal ions by equilibrium dialysis, titration, and metal ion buffer techniques. We found that the monomeric multimetal enzyme (M(r) 136,977) had eight specific binding sites for divalent metal ions. These sites were classified as follows: a very tight (class I) site for Ca2+, three tight (class II) sites consisting of two Ca(2+)-specific sites (class IICa) and one Mn(2+)-specific site (class IIMn; Kd for Mn2+, 2.0 nM), and four loose (class III) sites for Mn2+ (Kd, 1.2 microM) and Mg2+ (Kd, 2 microM). Removal of metal ions bound to class II and III sites of the holoenzyme (Ca3Mn5 species; relative Vmax (Vrel), 100%) by a chelating resin at 4 degrees C yielded a less thermostable Ca1 species (Vrel, 1.7%) with a class I Ca2+ ion, removal of which by a chelating resin at 50 degrees C caused a complete irreversible inactivation of the enzyme. Titration studies revealed that stoichiometric binding of Mn2+ to a class IIMn site of the Ca1 species caused a 33-fold activation whereas binding of Ca2+ to class IICa sites had no effect on enzyme activity. Ca1 species could be also activated 8-fold by heating at 60 degrees C for 20 min, suggesting that the catalytically important class II Mn2+ plays important roles in maintaining the native structure essential for activity. Occupation of class III sites by Mg2+ or Mn2+ was of physiological importance to attain sufficient thermostability by which this extracellular beta-galactosidase remained active for a prolonged time at elevated temperatures as was observed during growth of S. rectivirgula.
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Purification and characterization of a thermostable β-galactosidase with high transgalactosylation activity from Saccharopolyspora rectivirgula
Applied Microbiology and Biotechnology, 1994Co-Authors: Masahiro Nakao, Toru Nakayama, Yuji Shibano, Masami Harada, Yukiko Kodama, Teruo AmachiAbstract:We purified an extracellular thermostable β-galactosidase of Saccharopolyspora rectivirgula strain V2-2, a thermophilic actinomycete, to homogeneity and characterized it to be a monomeric enzyme with a relative molecular mass of 145 000 and s °_20,w of 7.1 s . In addition to the hydrolytic activity of 1- O -substituted β- d -galactopyranosides such as lactose [a Michaelis constant K _m=0.75 m m and molecular activity ( k _cat)= 63.1 s^−1 at pH 7.2 and 55° C] and p -nitrophenyl β- d -galactopyranoside ( K _m=0.04 m m k _cat= 55.8 s^−1), the enzyme had a high transgalactosylation activity. The enzyme reacted with 1.75 m lactose at 70°C and pH 7.0 for 22 h to yield oligosaccharides in a maximum yield (other than lactose) of 41% (w/w). A general structure for the major transgalactosylic products could be expressed as (Gal)_c-Glc, where n is 1, 2, 3, and 4 with a glucose at a reducing terminal. These oligosaccharides could selectively promote the growth of the genus Bifidobacterium found in human intestines. S. rectivirgula β-galactosidase was stable at pH 7.2 up to 60°C (for 4 h in the presence of 10 μ m MnCl_2) or 70°C (for 22 h in the presence of 1.75 m lactose and 10 μ m MnCl_2). Thus the enzyme is applicable to an immobilized enzyme system at high temperatures (60°C
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purification and characterization of a thermostable β galactosidase with high transgalactosylation activity from Saccharopolyspora rectivirgula
Applied Microbiology and Biotechnology, 1994Co-Authors: Masahiro Nakao, Toru Nakayama, Yuji Shibano, Masami Harada, Yukiko Kodama, Teruo AmachiAbstract:We purified an extracellular thermostable β-galactosidase of Saccharopolyspora rectivirgula strain V2-2, a thermophilic actinomycete, to homogeneity and characterized it to be a monomeric enzyme with a relative molecular mass of 145 000 and s°20,w of 7.1 s. In addition to the hydrolytic activity of 1-O-substituted β-d-galactopyranosides such as lactose [a Michaelis constant Km=0.75 mm and molecular activity (kcat)= 63.1 s−1 at pH 7.2 and 55° C] and p-nitrophenyl β-d-galactopyranoside (Km=0.04 mmkcat= 55.8 s−1), the enzyme had a high transgalactosylation activity. The enzyme reacted with 1.75 m lactose at 70°C and pH 7.0 for 22 h to yield oligosaccharides in a maximum yield (other than lactose) of 41% (w/w). A general structure for the major transgalactosylic products could be expressed as (Gal)c-Glc, where n is 1, 2, 3, and 4 with a glucose at a reducing terminal. These oligosaccharides could selectively promote the growth of the genus Bifidobacterium found in human intestines. S. rectivirgula β-galactosidase was stable at pH 7.2 up to 60°C (for 4 h in the presence of 10 μm MnCl2) or 70°C (for 22 h in the presence of 1.75 m lactose and 10 μm MnCl2). Thus the enzyme is applicable to an immobilized enzyme system at high temperatures (60°C <) for efficient production of the oligosaccharides from lactose.
