RESUMEN
The room-temperature liquid salt, ethylammonium nitrate (EAN), has been used to enhance the recovery of denatured-reduced hen egg white lysozyme (HEWL). Our results show that EAN has the ability to prevent aggregation of the denatured protein. The use of EAN as a refolding additive is advantageous because the renaturation is a one-step process. When HEWL was denatured reduced using routine procedures and renatured using EAN as an additive, HEWL was found to regain 75% of its activity. When HEWL was denatured and reduced in neat EAN, dilution resulted in over 90% recovery of active protein. An important aspect of this process is that renaturation of HEWL occurs at concentrations of 1.6 mg/mL, whereas other renaturation processes occur at significantly lower protein concentrations. Additionally, the refolded-active protein can be separated from the molten salt by simple desalting methods. Although the use of a low-temperature molten salt in protein renaturation is unconventional, the power of this approach lies in its simplicity and utility.
Asunto(s)
Muramidasa/química , Renaturación de Proteína , Compuestos de Amonio Cuaternario , Animales , Rastreo Diferencial de Calorimetría , Pollos , Femenino , Técnicas In Vitro , Indicadores y Reactivos , Cinética , Modelos Moleculares , Muramidasa/metabolismo , Oxidación-Reducción , Desnaturalización Proteica , Estructura Secundaria de ProteínaRESUMEN
Ethylammonium nitrate (EAN) is a liquid organic salt that has many potential applications in protein chemistry. Because this solvent has hydrophobic and ionic character as well as the ability to hydrogen bond, it is especially well suited for broad use in protein crystallography. For example, EAN may be used as an additive, a detergent, a precipitating agent or to deliver ligands into protein crystals. A discussion of the crystallization of lysozyme using EAN as a precipitating agent is given here.
Asunto(s)
Cristalización , Indicadores y Reactivos , Proteínas/aislamiento & purificación , Animales , Precipitación Química , Muramidasa/aislamiento & purificación , Nitratos , Compuestos de Amonio CuaternarioRESUMEN
OBJECTIVE: To study the interaction of interleukin-1alpha (IL-1alpha) and oncostatin M (OSM) in promoting cartilage collagen destruction. METHODS: Bovine, porcine, and human cartilage and human chondrocytes were studied in culture. The levels of collagenase (matrix metalloproteinase 1 [MMP-1]) and tissue inhibitor of metalloproteinases 1 (TIMP-1) were measured by bioassay and enzyme-linked immunosorbent assay (ELISA). The levels of OSM in rheumatoid synovial fluid were measured by ELISA. RESULTS: When combined with OSM, IL-1alpha, IL-1beta, and tumor necrosis factor alpha released proteoglycan and collagen from cartilage. OSM was the only member of the IL-6 family to have this effect. Human tendon also responded to IL-1alpha and OSM. OSM increased the production of MMP-1 and TIMP-1 but when combined with IL-1alpha, synergistically promoted MMP-1 production in human chondrocytes and synovial fibroblasts. High levels of OSM were found in human rheumatoid synovial fluids, and confocal microscopy showed that OSM was produced by macrophages in rheumatoid synovial tissue. CONCLUSION: These results highlight an important new mechanism by which there is irreversible loss of collagen from cartilage.
Asunto(s)
Artritis Reumatoide/metabolismo , Colágeno/metabolismo , Tejido Conectivo/química , Inhibidores de Crecimiento/fisiología , Péptidos/fisiología , Animales , Northern Blotting , Western Blotting , Cartílago Articular/efectos de los fármacos , Cartílago Articular/enzimología , Cartílago Articular/metabolismo , Bovinos , Condrocitos/efectos de los fármacos , Colagenasas/metabolismo , Citocinas/metabolismo , Humanos , Interleucina-1/farmacología , Interleucina-6/farmacología , Articulación de la Rodilla/química , Microscopía Confocal , Oncostatina M , Osteoartritis/metabolismo , Fenotipo , Porcinos , Líquido Sinovial/química , Membrana Sinovial/química , Membrana Sinovial/metabolismo , Tendones/efectos de los fármacosRESUMEN
The causes and outcome of cardiopulmonary arrests were studied in a paediatric hospital over a 12 month period. Forty five resuscitation attempts were made involving 41 children and one adult. Twenty eight (68%) of the children were under 1 year of age and 10 (24%) were neonates. Twenty one (47%) arrests were primarily respiratory and 11 (24%) primarily cardiac in origin. Eighty two per cent of the respiratory arrests had an initially successful outcome, compared with 36% of the cardiac arrests. Overall 70% of cardiopulmonary resuscitation attempts were initially successful. There were no survivors from resuscitation attempts longer than 30 minutes. At 12 months after cardiopulmonary resuscitation 15 (37%) of the children were still alive. The 11 children who had been neurologically normal before the arrest showed no evidence of neurological damage after successful cardiopulmonary resuscitation.