3 research outputs found
Purification and characterization of lipopolysaccharide from Eikenella corrodens 23834 and Porphyromonas gingivalis W83
La purificaci贸n de lipopolisac谩ridos (LPS) o endotoxinas y su caracterizaci贸n es un aspecto esencial para estudios que buscan aclarar el papel de estas biomol茅culas de bacterias Gram negativas presentes en la cavidad oral y su relaci贸n con enfermedades locales periodontales y sist茅micas. Este estudio implementa una metodolog铆a para la extracci贸n, purificaci贸n y caracterizaci贸n de LPS a partir de bacteria completa de Eikenella corrodens 23834 y Porphyromonas gingivalis W83, utilizando t茅cnicas previamente descritas. La extracci贸n cruda de LPS se realiz贸 con fenol-agua caliente; la purificaci贸n se realiz贸 con tratamiento enzim谩tico con nucleasas y proteasa, seguido de cromatograf铆a de exclusi贸n por tama帽o (Sephacryl S-200 HR) con deoxicolato de sodio como fase m贸vil. La caracterizaci贸n de los extractos purificados se realiz贸 por barrido espectrofotom茅trico, pruebas bioqu铆micas de electroforesis SDS-PAGE, ensayo Purpald y la prueba cromog茅nica de LAL. Como control para la identificaci贸n y caracterizaci贸n de los extractos purificados se utilizaron LPS comerciales de Escherichia coli, Salmonella typhimurium, Rodobacter sphaeroides y Porphyromonas gingivalis. La metodolog铆a implementada permiti贸 la obtenci贸n de LPS de elevada pureza con la identificaci贸n de KDO o heptosas, un quimiotipo de LPS-S (liso) para E. corrodens y LPS-SR (semi-rugoso) para P. gingivalis W83. Ambos LPS purificados mostraron capacidad endot贸xica a bajas concentraciones. La metodolog铆a implementada en este estudio para la purificaci贸n y caracterizaci贸n de LPS a partir de bacteria completa fue eficiente al compararla con los LPS comerciales.Purification of lipopolysaccharide (LPS) or endotoxins and its characterization is an important aspect for studies aimed at clarify the role of these biomolecules from Gram negative bacteria present in the oral cavity and its relationship with periodontal and systemic diseases. This study describes an extraction, purification and characterization method of LPS from Eikenella corrodens 23834 and Porphyromonas gingivalis W83. LPS extraction was performed by using hot phenol-water; the purification was done with nuclease and protease enzymatic treatment, followed by size-exclusion chromatography (Sephacryl S-200 HR) with sodium deoxycholate as mobile phase. The characterization of the purified extracts was performed by spectrophotometric scanning, SDS-PAGE biochemical tests, Purpald assay and chromogenic LAL test. As control, commercial LPS from Escherichia coli, Salmonella typhimurium, P. gingivalis, and Rodobacter sphaeroides were used. The methodology mentioned above had allowed obtaining high purity LPS by identifying KDO or heptoses, a chemotype S-LPS (smooth) to E. corrodens; SR-LPS (semi-rough) for P. gingivalis W83. Both purified LPS showed endotoxic capacity at low valoraconcentrations. The methodology used in this study for purification and characterization of LPS from the whole bacteria was efficient when it was compared with commercial LPS.Departamento Administrativo de Ciencia, Tecnolog铆a e Innovaci贸n [CO] Colciencias1308-519-28960Inducci贸n de disfunci贸n endotelial in vitro por lipopolisacarido de bacterias periodontopaticas e inhibici贸n de la inflamaci贸n por resolvina (rvd1) y estatina (rosuvastatina)n
Purificaci贸n y caracterizaci贸n de lipopolisac谩ridos de Eikenella corrodens 23834 y Porphyromonas gingivalis W83
Purification of lipopolysaccharide (LPS) or endotoxins and its characterization is an important aspect for studies aimed at clarifying the role of these biomolecules from Gram negative bacteria present in the oral cavity and its relationship with periodontal and systemic diseases. This study describes an extraction, purification and characterization method of LPS from Eikenella corrodens 23834 and Porphyromonas gingivalis W83. LPS extraction was performed using hot phenol-water; the purification was done with nuclease and protease enzymatic treatment, followed by size-exclusion chromatography (Sephacryl S-200 HR) with sodium deoxycholate as mobile phase. The characterization of the purified extracts was performed by spectrophotometric scanning, SDS-PAGE biochemical tests, Purpald assay and chromogenic LAL test. As control, commercial LPS from Escherichia coli, Salmonella typhimurium, P. gingivalis, and Rodobacter sphaeroides were used. The methodology mentioned above had allowed obtaining high purity LPS by identifying KDO or heptoses, a chemotype S-LPS (smooth) to E. corrodens; SR-LPS (semi-rough) for P. gingivalis W83. Both purified LPS showed endotoxic capacity at low concentrations. The methodology used in this study for purification and characterization of LPS from the whole bacteria was efficient when compared with commercial LPS.La purificaci贸n de lipopolisac谩ridos (LPS) o endotoxinas y su caracterizaci贸n es un aspecto esencial para estudios que buscan aclarar el papel de estas biomol茅culas de bacterias Gram negativas presentes en la cavidad oral y su relaci贸n con enfermedades locales periodontales y sist茅micas. Este estudio implementa una metodolog铆a para la extracci贸n, purificaci贸n y caracterizaci贸n de LPS a partir de bacteria completa de Eikenella corrodens 23834 y Porphyromonas gingivalis W83,聽 utilizando t茅cnicas previamente descritas. La extracci贸n cruda de LPS se realiz贸 con fenol-agua caliente; la purificaci贸n se realiz贸 con tratamiento enzim谩tico con nucleasas y proteasa, seguido de cromatograf铆a de exclusi贸n por tama帽o (Sephacryl S-200 HR) con deoxicolato de sodio como fase m贸vil. La caracterizaci贸n de los extractos purificados se realiz贸 por barrido espectrofotom茅trico, pruebas bioqu铆micas de electroforesis SDS-PAGE, ensayo Purpald y la prueba cromog茅nica de LAL. Como control para la identificaci贸n y caracterizaci贸n de los extractos purificados se utilizaron LPS comerciales de Escherichia coli, Salmonella typhimurium, Rodobacter sphaeroides y Porphyromonas gingivalis. La metodolog铆a implementada permiti贸 la obtenci贸n de LPS de elevada pureza con la identificaci贸n de KDO o heptosas, un quimiotipo de LPS-S (liso) para E. corrodens y LPS-SR (semi-rugoso) para P. gingivalis W83. Ambos LPS purificados mostraron capacidad endot贸xica a bajas concentraciones. La metodolog铆a implementada en este estudio para la purificaci贸n y caracterizaci贸n de LPS a partir de bacteria completa聽 fue eficiente al compararla con los LPS comerciales
Purificaci贸n y caracterizaci贸n de lipopolisac谩ridos de eikenella corrodens 23834 y porphyromonas gingivalis w83
T铆tulo corto: Metodolog铆a para el aislamiento e identificaci贸n聽 de LPS de periodonto-pat贸genosT铆tulo en ingl茅s: Purification and characterization of lipopolysaccharide from Eikenella corrodens 23834 and Porphyromonas gingivalis W83Resumen:聽La purificaci贸n de lipopolisac谩ridos (LPS) o endotoxinas y su caracterizaci贸n es un aspecto esencial para estudios que buscan aclarar el papel de estas biomol茅culas de bacterias Gram negativas presentes en la cavidad oral y su relaci贸n con enfermedades locales periodontales y sist茅micas. Este estudio implementa una metodolog铆a para la extracci贸n, purificaci贸n y caracterizaci贸n de LPS a partir de bacteria completa de Eikenella corrodens 23834 y Porphyromonas gingivalis W83,聽 utilizando t茅cnicas previamente descritas. La extracci贸n cruda de LPS se realiz贸 con fenol-agua caliente; la purificaci贸n se realiz贸 con tratamiento enzim谩tico con nucleasas y proteasa, seguido de cromatograf铆a de exclusi贸n por tama帽o (Sephacryl S-200 HR) con deoxicolato de sodio como fase m贸vil. La caracterizaci贸n de los extractos purificados se realiz贸 por barrido espectrofotom茅trico, pruebas bioqu铆micas de electroforesis SDS-PAGE, ensayo Purpald y la prueba cromog茅nica de LAL. Como control para la identificaci贸n y caracterizaci贸n de los extractos purificados se utilizaron LPS comerciales de Escherichia coli, Salmonella typhimurium, Rodobacter sphaeroides y Porphyromonas gingivalis. La metodolog铆a implementada permiti贸 la obtenci贸n de LPS de elevada pureza con la identificaci贸n de KDO o heptosas, un quimiotipo de LPS-S (liso) para E. corrodens y LPS-SR (semi-rugoso) para P. gingivalis W83. Ambos LPS purificados mostraron capacidad endot贸xica a bajas concentraciones. La metodolog铆a implementada en este estudio para la purificaci贸n y caracterizaci贸n de LPS a partir de bacteria completa聽 fue eficiente al compararla con los LPS comerciales.Palabras clave: endotoxinas, cromatograf铆a, 谩cido 2-ceto-3-desoxioctulos贸nico (KDO), test LAL, periodontitis.Abstract:聽Purification of lipopolysaccharide (LPS) or endotoxins and its characterization is an important aspect for studies aimed at clarify the role of these biomolecules from Gram negative bacteria present in the oral cavity and its relationship with periodontal and systemic diseases. This study describes an extraction, purification and characterization method of LPS from Eikenella corrodens 23834 and Porphyromonas gingivalis W83. LPS extraction was performed by using hot phenol-water; the purification was done with nuclease and protease enzymatic treatment, followed by size-exclusion chromatography (Sephacryl S-200 HR) with sodium deoxycholate as mobile phase. The characterization of the purified extracts was performed by spectrophotometric scanning, SDS-PAGE biochemical tests, Purpald assay and chromogenic LAL test. As control, commercial LPS from Escherichia coli, Salmonella typhimurium, P. gingivalis, and Rodobacter sphaeroides were used. The methodology mentioned above had allowed obtaining high purity LPS by identifying KDO or heptoses, a chemotype S-LPS (smooth) to E. corrodens; SR-LPS (semi-rough) for P. gingivalis W83. Both purified LPS showed endotoxic capacity at low concentrations. The methodology used in this study for purification and characterization of LPS from the whole bacteria was efficient when it was compared with commercial LPS.Key words: endotoxin, 2-keto-3-deoxyoctonate (KDO), Chromatography, Limulus test (LAL), periodontitis