RESUMO
Since the function of the spinal cord depends on the proteins found there, better defing the normal Spinal Cord Proteome is an important and challenging task. Although brain and cerebrospinal fluid samples from patients with different central nervous system (CNS) disorders have been studied, a thorough examination of specific spinal cord proteins and the changes induced by injury or associated to conditions such as neurodegeneration, spasticity and neuropathies has yet to be performed. In the present study, we aimed to describe total protein content in the spinal cord of healthy rats, employing different proteomics tools. Accordingly, we have developed a fast, easy, and reproducible sequential protocol for protein extraction from rat spinal cords. We employed conventional two dimensional electrophoresis (2DE) in different pH ranges (eg. 4-7, 3-11 NL) combined with identification by mass spectrometry (MALDI-TOF/TOF), as well as first dimension protein separation combined with Liquid Chromatography Mass Spectrometry/Mass Spectrometry (LC-MS/MS) to maximise the benefits of this technology. The value of these techniques is demonstrated here by the identification of several proteins known to be associated with neuroglial structures, neurotransmission, cell survival and nerve growth in the central nervous system. Furthermore this study identified many spinal proteins that have not previously been described in the literature and which may play an important role as either sensitive biomarkers of dysfunction or of recovery after Spinal Cord Injury.
RESUMO
Objetivo: Analizar si existe alguna asociación entre la resistencia a aspirina (RA) y la presencia de polimorfismos genéticos de un único nucleótido (SNPs) en el gen de la COX-1, así como su relación con modificaciones en la expresión de proteínas plasmáticas en pacientes con enfermedad isquémica estable y tratamiento continuado de aspirina. Materiales y métodos: Analizamos el proteoma plasmático de 19 pacientes sensibles, 19 resistentes. RA se definió mediante el sistema PFA-100. Se realizó electroforesis bidimensional (IPG 17cm, pH(4-7), geles SDS-PAGE 10%) y tinción con plata. Se analizaron cambios en tres SNPs (A-842G, C22T y C50T) en 50 pacientes sensibles, 33 resistentes y 83 controles mediante PCR a tiempo real. Resultados: La expresión de cuatro isoformas de alfa1-antitripsina estaba aumentada en los pacientes resistentes. No encontramos diferencias en la expresión de ceruloplasmina, precursor de haptoglobina, apolipoproteína AI y precursor de albúmina entre ambos tipos de pacientes. Ningún paciente presentó cambios en el SNP A-842G. La frecuencia de cambio en C22T y C50T fue relativamente baja con respecto a la población total. Conclusiones: No encontramos asociación entre la presencia de polimorfismos en el gen de la COX-1 y la peor respuesta a la aspirina. Los cambios en observados alfa1-antitripsina podrían estar relacionados con un diferente estado inflamatorio entre ambos tipos de pacientes (AU)
Aim: To evaluate the existence of a possible association between Aspirin resistance (AR), COX-1 single-nucleotide polymorphisms (SNPs) and the modifications in the plasma proteome of clinically stable coronary patients. Materials and methods: AR was defined according to the PFA-100 assay. AR-sensitive and AR-resistant patients had been taken aspirin for the last 9 months. The proteomic study (19 AR-sensitive, 19 AR-resistant) was performed using IPG strips (17cm, pH 4-7), SDS-PAGE gels (10%) and silver staining. We study three SNPs (A- 842G, C22T y C50T) in 50 AR-sensitive patients, 33 AR-resistant and 83 controls using a real-time PCR. Results: The expression of four alpha1-antitripsin isoforms was increased in the aspirin-resistant patients. No differences were found in the expression of ceruloplasmin, haptoglobin-precursor, apolipoprotein-AI and albumin- precursor between both groups of patients. The A-842G SNP was undetectable in all subjects. The remaining two SNPs (C22T y C50T) showed a low frequency with respect the global population. Conclusions: The low SNPs frequencies were unlikely to explain the difference in aspirin responsiveness between both groups of patients. The changes in alpha1-antitripsin could be linked with a different inflammatory state in these patients (AU)
