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Objective To investigate the role of protein phosphorylation in Pseudomonas aeruginosa (P.aeruginosa) strains in response to stress triggered by mouse macrophages.Methods The strong cation exchange-immobilized metal affinity chromatography (SCX-IMAC) was performed to enrich phosphopeptides.The nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS) was carried out to identify and analyze phosphoproteome.Results Fourteen phosphopeptides from twelve proteins were identified within thirty-one phosphorylation sites on serine,threonine and tyrosine residues.Fifty percent of these phosphorylated proteins were membrane proteins,indicating that their phosphorylation modification was more critical for bacteria in response to the stress.In terms of biological process of Gene Ontology,these identified proteins were involved in stress response,iron transport,anaerobic respiration,response to hydrogen peroxide and signal transduction by phosphorylation,etc.Conclusion These phosphorylated proteins in P.aeruginosa strains are necessary for signal transduction and their response to harsh environment within the macrophages,such as iron limitation,hypoxia and oxidative stress.This study provides evidence for further investigation on virulence and pathogenesis of P.aeruginosa.
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Objective To study NonO protein expression changes in murine erythroleukemia ( MEL ) cell differentiation induced by sodium butyrate .Methods Benzidine staining was used to test sodium butyrate-induced erythroid differentiation of MEL cells .We detected NonO protein expression changes in MEL cell differentiation induced by sodium butyrate and NonO protein localization in MEL cells by Western blotting and immunocytochemistry .Furthermore , we applied PCR technique to detect NonO RNA expression in differentiation process .Results We found that NonO protein was upregulated at gene and protein levels in the erythroid differentiation process of MEL cells induced by sodium butyrate and located in cytoplasm and nucleus in MEL cells .Conclusion These results show that NonO protein is closely related with MEL cell differentiation induced by sodium butyrate , which may provide important clues for further study of the mechanism of leukemia .
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Objective To explore the differentially expressed proteins during erythroid differentiation .Methods The murine erythroleukemia ( MEL) cell were treated by DMSO , and the comparative proteomic was systematically analyzed and identified on different differentiating time points .ratio of cell differentiation and viability were detected by benzidine staining, MTT assay and Ter119 immunofluorescence.Using two-dimensional gel electrophoresis combined with mass spectrometry technology and bioinformatics analysis , we conducted a comparative proteomic analysis on MEL cells during the process of induced differentiation to screen and identify differential proteins .Results The MEL cells induced by 1.2%DMSO for 0 hour, 6hours, 12hours, 24hours, 36hours, 48hours, 72 hours, 96 hours, 120 hours were collected for proteomic analysis, by two-dimensional gel electrophoresis combined with mass spectrometry .A total of 87 kinds of proteins were successfully identified .MEL cells exposed to DMSO at a final concentration of 1.2% for 120 hours reached the highest differentiation rate of 67%.MTT assay showed that 1.0%, 1.2%, 1.4% DMSO had no inhibiting effect on cell vitality.Conclusion DMSO may induce MEL cells to differentiate and have no inhibiting effect on cell vitality .The 87 kinds of differentially expressed proteins from two-dimentional gel electrophoresis may be divided into twelve categories ;the most three parts are 41%enzyme protein, 15%structural protein and 13%regulatory protein.
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Objective To explore the effect of mouse proliferation-associated protein 2G4 (p38-2G4) high-expression on the proliferation and erythriod differentiation of murine erythroleukemia ( MEL ) cells.Methods To establish the recombinant lentivirus vector p 38-2G4-pLJM1, the p38-2G4-pLJM1 was cotransfected into HEK293T cells to obtain lentivirus with pCMV-VSV-G and pCMV-dR8.2.Lentivirus were infected into MEL cells to establish the stably p 38-2G4 high-expressed MEL cells.Western blotting was used to analyse the high-expression efficiency.MTT assay and benzidine staining were applied to detect the cell viability and hemoglobin synthesis of the stable cell line in presence /absence of inducers.Results Western blotting showed that the p38-2G4 high-expression stable cell strain had a higher expression of p38-2G4 as compared to the control group ( MEL) ( P 0.05), but the hemoglobin synthesis had been reduced as compared to the control group (P<0.05).Conclusion p38-2G4 high-expression does not affect the cell viability of MEL cells , but inhibits the erythriod differentiation of MEL cells in three independent experiments .
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Proteomic analysis of core needle biopsy (CNB) sample from patient populations is critical to our understanding of human disease,but has been hindered by its particular small size.Here,we present a method for the proteomic analysis of CNB sample based on the two dimensional electrophoresis.Proteins were extracted directly from 3 rat liver CNB specimens and a human prostate CNB sample.respectively.24 cm Immobiline DryStrip (pH 3-10NL) and 12.5% SDS-PAGE were introduced to separate the proteins.Interesting spots were analyzed by MALDI TOF/TOF mass spectrometry after tryptic digestion.With this method,consistent electrophoretic patterns of more than 2 500 protein spots were reproducibly obtained after silver staining,from rat liver CNB specimens.Qualitatively and quantitatively reproducible results also yield when the method was applied to a human prostate CNB sample.57 stochastically selected protein spots were analyzed by MALDI TOF/TOF moss spectrometry.and were identified with high confidence including faint ones.This simple and reproducible approach raises the opportunity of defining key molecular events of human disease pathologies.