RESUMEN
Here we describe a proteomic analysis of Escherichia coli in which 3,199 protein forms were detected, and of those 2,160 were annotated and assigned to the cytosol, periplasm, inner membrane, and outer membrane by biochemical fractionation followed by two-dimensional gel electrophoresis and tandem mass spectrometry. Represented within this inventory were unique and modified forms corresponding to 575 different ORFs that included 151 proteins whose existence had been predicted from hypothetical ORFs, 76 proteins of completely unknown function, and 222 proteins currently without location assignments in the Swiss-Prot Database. Of the 575 unique proteins identified, 42% were found to exist in multiple forms. Using DIGE, we also examined the relative changes in protein expression when cells were grown in the presence and absence of amino acids. A total of 23 different proteins were identified whose abundance changed significantly between the two conditions. Most of these changes were found to be associated with proteins involved in carbon and amino acid metabolism, transport, and chemotaxis. Detailed information related to all 2,160 protein forms (protein and gene names, accession numbers, subcellular locations, relative abundances, sequence coverage, molecular masses, and isoelectric points) can be obtained upon request in either tabular form or as interactive gel images.
Asunto(s)
Aminoácidos/deficiencia , Proteínas Bacterianas/química , Escherichia coli/química , Escherichia coli/crecimiento & desarrollo , Genoma Bacteriano , Proteínas Bacterianas/aislamiento & purificación , Bases de Datos de Proteínas , Electroforesis en Gel Bidimensional/métodos , Sistemas de Lectura Abierta , Mapeo Peptídico/métodos , Análisis de Secuencia/métodosRESUMEN
To gain insight into the cell envelope of Escherichia coli grown under aerobic and anaerobic conditions, lipoproteins were examined by using functional genomics. The mRNA expression levels of each of these genes under three growth conditions--aerobic, anaerobic, and anaerobic with nitrate--were examined by using both Affymetrix GeneChip E. coli antisense genome arrays and real-time PCR (RT-PCR). Many genes showed significant changes in expression level. The RT-PCR results were in very good agreement with the microarray data. The results of this study represent the first insights into the possible roles of unknown lipoprotein genes and broaden our understanding of the composition of the cell envelope under different environmental conditions. Additionally, these data serve as a test set for the refinement of high-throughput bioinformatic and global gene expression methods.
Asunto(s)
Escherichia coli/genética , Genoma Bacteriano , Lipoproteínas/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Mitogen-activated protein kinases (MAPKs), including extracellular-responsive kinase (ERK) and p38 MAPK, are activated by stresses associated with hypothermia-rewarming and ischemia-reperfusion. Their activation in heart is associated with beneficial (preconditioning) and adverse effects (apoptosis and impaired contractility). This study determined whether ERK and p38 MAPK activities are altered by hypothermic ischemia and normothermic reperfusion and the consequences of their inhibition on recovery of myocardial function. METHODS: Left ventricular work (L x min(-1) x mm Hg) was assessed during normothermic perfusion (30 min) of isolated rat hearts that were either freshly excised or previously subjected to hypothermic storage (8 hr, 3 degrees C) and rewarming (10 min, 37 degrees C) before normothermic reperfusion (30 min). Phospho-specific immunoblot analysis of p38 MAPK was performed in hearts and various cultured cells. RESULTS: Compared with fresh hearts, hearts subjected to hypothermia and rewarming demonstrated impaired left ventricular work (1.96+/-0.53, n=12 vs. 8.37+/-0.46, n=4, <0.05) during reperfusion. The ERK inhibitor, PD98059 (20 microM), present during storage and rewarming, caused modest improvement (3.66+/-0.75, n=9, <0.05). The p38 MAPK inhibitor, SB202190 (10 microM), when present during reperfusion, improved recovery (to 6.12+/-0.75, n=6, <0.05); it was ineffective if present only during rewarming (1.52+/-0.88, n=4). In rat2 fibroblasts, hypothermia and rewarming activated p38 MAPK and its downstream kinase MAPK-activated protein kinase 2, but not c-Jun N-terminal kinase/stress-activated protein kinase. CONCLUSIONS: Myocardial p38 MAPK and MAPK-activated protein kinase 2 are stimulated by hypothermia, ischemia, and rewarming and are detrimental to recovery of mechanical function of hearts subjected to prolonged hypothermic storage. Inhibition of p38 MAPK may be useful in protocols to improve the recovery of mechanical function of cold-stored hearts.
Asunto(s)
Flavonoides/farmacología , Hipotermia Inducida , Imidazoles/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Isquemia Miocárdica/fisiopatología , Piridinas/farmacología , Función Ventricular Izquierda/efectos de los fármacos , Animales , Activación Enzimática , Masculino , Fosforilación , Ratas , Ratas Sprague-Dawley , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
To investigate the role of Raf-1 in v-Ha-ras transformation, we have isolated and characterized a number of Raf-1 mutants that display increased transforming activity in Rat2 fibroblasts. A dipeptide deletion (Delta144-145) in the cysteine-rich domain (CRD) of conserved region (CR) 1 increased the interaction between Raf-1 and v-Ha-ras effector loop mutants in the yeast two-hybrid system, supporting the proposal that the CRD serves as a secondary ras-binding domain. Many activating mutations were located in CR2. Two representative CR2 mutants (Delta250-258 and S257L) displayed increased interaction with v-Ha-ras effector loop mutants and with mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) 1 in the two-hybrid system. One novel mutation in CR3 was recovered; G361S affected the third glycine of the GXGXXG protein kinase motif involved in ATP binding. Expression of G361S Raf-1 in Rat2 fibroblasts activated MEK and ERK. The CR1, CR2, and CR3 activating mutations, when combined in cis, cooperated in transforming Rat2 fibroblasts. Conversely, Raf-1 transforming activity was decreased when the S257L or G361S mutation was combined in cis with the R89E substitution, which disrupts ras-Raf interaction. This mutant analysis provides additional information about the distinct functions of individual Raf-1 regions and documents a novel genetic mechanism for activating an oncogenic kinase.