RESUMO
PURPOSE: In clinical conditions trauma is associated with high mortality and morbidity. Neutrophils play a key role in the development of multiple organ failure after trauma EXPERIMENTAL DESIGN: To have a detailed understanding of the neutrophil activation at primary stages after trauma, neutrophils are isolated from control and surgical trauma rats in this study. Extracted proteins are analyzed using nano liquid chromatography coupled with tandem mass spectrometry. RESULTS: A total of 2924 rat neutrophil proteins are identified in our analysis, of which 393 are found differentially regulated between control and trauma groups. By using functional pathways analysis of the 190 proteins up-regulated in surgical trauma, we found proteins related to transcription initiation and protein biosynthesis. On the other hand, among the 203 proteins down-regulated in surgical trauma we found enrichment for proteins of the immune response, proteasome degradation and actin cytoskeleton. Overall, enzyme prediction analysis revealed that regulated enzymes are directly involved in neutrophil apoptosis, directional migration and chemotaxis. Our observations are then confirmed by in silico protein-protein interaction analysis. CONCLUSIONS AND CLINICAL RELEVANCE: Collectively, our results reveal that neutrophils drastically regulate their biochemical pathways after the early stages of surgical trauma, showing lower activity. This implies higher susceptibility of the trauma patients to infection and bystander tissues damage.
Assuntos
Enzimas/metabolismo , Neutrófilos/metabolismo , Proteoma/análise , Proteômica , Animais , Apoptose , Cromatografia Líquida de Alta Pressão , Regulação para Baixo , Interações Hidrofóbicas e Hidrofílicas , Isomerases/análise , Masculino , Neutrófilos/imunologia , Oxirredutases/análise , Mapas de Interação de Proteínas , Ratos , Ratos Wistar , Transdução de Sinais , Espectrometria de Massas em Tandem , Regulação para Cima , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologia , Ferimentos e Lesões/cirurgiaRESUMO
We have developed a novel method for the continuous assay of protein disulfide oxidoreductase activity using as substrate bovine pancreas insulin in which both N-terminal amino groups are chemically modified with fluorescein isothiocyanate. The reduction of intercatenary disulfide bonds of di-fluoresceinthiocarbamyl-insulin with dithiothreitol initially lowers but subsequently enhances the emission intensity. In this biphasic kinetics, the rate of increase is sensitive enough for the estimation of Escherichia coli thioredoxin concentrations from 5 nM (0.06 microgram/ml) to 500 nM (6 micrograms/ml). Neither changes of pH over a range of 6.2 to 8.4 nor neutral salts (K+, Mg2+, and Ca2+) at concentrations lower than 100 mM affect this simple reaction system. Moreover, the fluorometric method is functional for measuring the reductive capacity of Brassica napus protein disulfide isomerase. Hence, a highly reproducible and accurate one-state assay for protein disulfide oxidoreductase activity not only greatly improves the sensitivity compared to the commonly used turbidimetric assay but also represents a reliable alternative to assays based on accessory enzymes or radiolabeled substrates.
Assuntos
Fluorometria , Insulina/química , Isomerases/análise , Proteína Dissulfeto Redutase (Glutationa)/análise , Tiorredoxinas/análise , Estudos de Viabilidade , Insulina/análogos & derivados , Nefelometria e Turbidimetria , Isomerases de Dissulfetos de Proteínas , Reprodutibilidade dos Testes , Espectrometria de FluorescênciaRESUMO
Fourteen population samples of Drosophila pavani were obtained from a number of localities in Chile. The populations sampled were dispersed over 7 degrees of latitude and 1800 meters of elevation, and were drawn at three different times. Sixteen electrophoretic loci were assayed for each population; eight of the loci were analyzed statistically for geographic variation; the other eight were essentially monomorphic. For all eight variable loci, variation in allelic frequencies among populations was highly significant. In all cases, a significant portion of the variation among populations was associated with variation in gross environmental variables (latitude, elevation, month of collection). The implications of the evidence were discussed, and the authors concluded that there was suggestive evidence for selection.