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1.
PLoS One ; 14(6): e0217690, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31194764

RESUMO

INTRODUCTION: Long-term head-down bed rest (HDBR) results in musculoskeletal losses similar to those observed during long-term space flight. Agents such as testosterone, in addition to regular exercise, are effective countermeasures for reducing loss of skeletal muscle mass and function. OBJECTIVE: We investigated the skeletal muscle proteome of healthy men in response to long term HDBR alone (CON) and to HDBR with exercise (PEX) or exercise plus testosterone (TEX) countermeasures. METHOD: Biopsies were performed on the vastus lateralis before (pre) HDBR and on HDBR days 32 (mid) and 64 (post). Extracted proteins from these skeletal muscle biopsies were subjected to 2-dimensional gel electrophoresis (2DE), stained for phosphoproteins (Pro-Q Diamond dye) and total proteins (Sypro Ruby dye). Proteins showing significant fold differences (t-test p ≤ 0.05) in abundance or phosphorylation state at mid or post were identified by mass spectroscopy (MS). RESULTS: From a total of 932 protein spots, 130 spots were identified as potentially altered in terms of total protein or phosphoprotein levels due to HDBR and/or countermeasures, and 59 unique molecules emerged from MS analysis. Top canonical pathways identified through IPA included calcium signaling, actin cytoskeleton signaling, integrin linked kinase (ILK) signaling, and epithelial adherens junction signaling. Data from the pre-HDBR proteome supported the potential for predicting physiological post-HDBR responses such as the individual's potential for loss vs. maintenance of muscle mass and strength. CONCLUSIONS: HDBR resulted in alterations to skeletal muscle abundances and phosphorylation of several structural and metabolic proteins. Inclusion of exercise alone or in combination with testosterone treatment modulated the proteomic responses towards cellular reorganization and hypertrophy, respectively. Finally, the baseline proteome may aid in the development of personalized countermeasures to mitigate health risks in astronauts as related to loss of muscle mass and function.


Assuntos
Repouso em Cama/efeitos adversos , Decúbito Inclinado com Rebaixamento da Cabeça/efeitos adversos , Músculo Esquelético/fisiopatologia , Adulto , Atrofia/tratamento farmacológico , Exercício Físico/fisiologia , Terapia por Exercício/métodos , Decúbito Inclinado com Rebaixamento da Cabeça/fisiologia , Voluntários Saudáveis , Humanos , Masculino , Fenômenos Fisiológicos Musculoesqueléticos/efeitos dos fármacos , Proteômica/métodos , Músculo Quadríceps/metabolismo , Testosterona/uso terapêutico , Simulação de Ausência de Peso
2.
Alcohol Clin Exp Res ; 41(10): 1675-1685, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28792616

RESUMO

BACKGROUND: Chronic alcohol abuse, a major risk factor for such diseases as hepatitis and cirrhosis, impairs hepatic alcohol dehydrogenase (ADH; key ethanol [EtOH]-metabolizing enzyme). Therefore, differentially altered hepatic and plasma proteomes were identified in chronic EtOH feeding model of hepatic ADH-deficient (ADH- ) deer mice to understand the metabolic basis of alcoholic liver disease (ALD). METHODS: ADH- deer mice were fed 3.5 g% EtOH via Lieber-DeCarli liquid diet daily for 3 months and histology of the liver assessed. Liver and plasma proteins were separated by 2-dimensional gel electrophoresis. The proteins differentially expressed were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. RESULTS: Histology of the liver showed panlobular steatosis and infiltration of T lymphocytes. Using the criteria of ≥1.5 for fold change (p-value ≤0.05) with expectation value (E ≤10-3 ) and protein score (≥64), 18 proteins in the livers and 5 in the plasma of EtOH-fed mice were differentially expressed and identified. Prolyl 4-hydroxylase, cytochrome b-5, endo A cytokeratin, ATP synthase, heat-shock 70 kD proteins, enoyl CoA hydratase, stress-70 protein, peroxiredoxin 1, and ornithine carbamoyl transferase were up-regulated in the livers. However, carbonic anhydrase 3, mitochondrial ATP synthase, aldolase 2, actin γ, laminin receptor, and carbamoyl phosphate synthase were down-regulated. Contrary to the increased expression of creatine kinase M-type, a decreased expression of serine protease inhibitor A3A precursor, sulfated glycoprotein-2 (clusterin), and apolipoprotein E isoforms were found in the plasma of EtOH group. CONCLUSIONS: Chronic EtOH feeding in ADH- deer mice causes steatosis and infiltration of T lymphocytes in the livers along with increased expression of proteins involved in endoplasmic reticulum (ER) stress, fibrosis, fatty acid ß oxidation and biogenesis, and decreased expression of proteins involved in ATP synthesis, carbohydrate metabolism, in cell regulation and architecture. Reduced expression of various carrier proteins as found in the plasma of EtOH group has a biomarker potential.


Assuntos
Álcool Desidrogenase/deficiência , Etanol/toxicidade , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/patologia , Proteômica/métodos , Álcool Desidrogenase/genética , Animais , Etanol/administração & dosagem , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Hepatopatias Alcoólicas/genética , Masculino , Camundongos , Peromyscus
3.
J Proteome Res ; 16(8): 2663-2679, 2017 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-28679203

RESUMO

Activated eosinophils contribute to airway dysfunction and tissue remodeling in asthma and thus are considered to be important factors in asthma pathology. We report here comparative proteomic and phosphoproteomic changes upon activation of eosinophils using eight cytokines individually and in selected cytokine combinations in time-course reactions. Differential protein and phosphoprotein expressions were determined by mass spectrometry after 2-dimensional gel electrophoresis (2DGE) and by LC-MS/MS. We found that each cytokine-stimulation produced significantly different changes in the eosinophil proteome and phosphoproteome, with phosphoproteomic changes being more pronounced and having an earlier onset. Furthermore, we observed that IL-5, GM-CSF, and IL-3 showed the greatest change in protein expression and phosphorylation, and this expression differed markedly from those of the other five cytokines evaluated. Comprehensive univariate and multivariate statistical analyses were employed to evaluate the comparative results. We also monitored eosinophil activation using flow cytometry (FC) analysis of CD69. In agreement with our proteomic studies, FC indicated that IL-5, GM-CSF, and IL-3 were more effective than the other five cytokines studied in stimulating a cell surface CD69 increase indicative of eosinophil activation. Moreover, selected combinations of cytokines revealed proteomic patterns with many proteins in common with single cytokine expression patterns but also showed a greater effect of the two cytokines employed, indicating a more complex signaling pathway that was reflective of a more typical inflammatory pathology.


Assuntos
Citocinas/farmacologia , Eosinófilos/efeitos dos fármacos , Fosfoproteínas/análise , Proteínas/análise , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Asma/sangue , Células Cultivadas , Citocinas/metabolismo , Eletroforese em Gel Bidimensional , Eosinófilos/metabolismo , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/patologia , Interleucina-3/farmacologia , Interleucina-5/farmacologia , Lectinas Tipo C/análise , Masculino , Proteômica/métodos , Espectrometria de Massas em Tandem , Fatores de Tempo
4.
Pancreas ; 46(6): 806-812, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28609370

RESUMO

OBJECTIVES: The aim of this study was to identify differentially expressed proteins in the pancreatic tissue of hepatic alcohol dehydrogenase-deficient deer mice fed ethanol to understand metabolic basis and mechanism of alcoholic chronic pancreatitis. METHODS: Mice were fed liquid diet containing 3.5 g% ethanol daily for 3 months, and differentially expressed pancreatic proteins were identified by protein separation using 2-dimensional gel electrophoresis and identification by mass spectrometry. RESULTS: Nineteen differentially expressed proteins were identified by applying criteria established for protein identification in proteomics. An increased abundance was found for ribosome-binding protein 1, 60S ribosomal protein L31-like isoform 1, histone 4, calcium, and adenosine triphosphate (ATP) binding proteins and the proteins involved in antiapoptotic processes and endoplasmic reticulum function, stress, and/or homeostasis. Low abundance was found for endoA cytokeratin, 40S ribosomal protein SA, amylase 2b isoform precursor, serum albumin, and ATP synthase subunit ß and the proteins involved in cell motility, structure, and conformation. CONCLUSIONS: Chronic ethanol feeding in alcohol dehydrogenase-deficient deer mice differentially expresses pancreatic functional and structural proteins, which can be used to develop biomarker(s) of alcoholic chronic pancreatitis, particularly amylase 2b precursor, and 60 kDa heat shock protein and those involved in ATP synthesis and blood osmotic pressure.


Assuntos
Álcool Desidrogenase/deficiência , Consumo de Bebidas Alcoólicas , Etanol , Fígado/enzimologia , Pâncreas/metabolismo , Pancreatite Alcoólica/metabolismo , Proteínas/metabolismo , Álcool Desidrogenase/genética , Animais , Modelos Animais de Doenças , Genótipo , Masculino , Camundongos Knockout , Pancreatite Alcoólica/genética , Peromyscus , Fenótipo , Proteômica/métodos , Fatores de Tempo
5.
Adv Exp Med Biol ; 919: 443-462, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27975230

RESUMO

Properly performed, biomarker discovery can lead to effective candidates that can ultimately serve as predictors of disease, medical condition, define therapeutic parameters, and many other applications in medicine. Preferably, biomarkers comprise a panel of indicators, e.g. proteins and/or peptides that can be predictive or diagnostic of the medical condition of interest. Emphasis here is placed on "panel," as single candidates are rarely sufficient to provide the necessary sensitivity and specificity. To develop an effective panel that survives the development process described in Chap. 19 , proper experimental design and attention to important statistical parameters are critical to ensure success. Errors in discovery can lead to an inefficient use of expensive resources, as these may not be uncovered until the latter stages in biomarker development. Hence, accuracy, precision, and an estimate of the power of the proposed analyses are critical in the discovery of the panel of candidate biomarkers by proteomic methods, as is the selection of statistical approaches to refine and appropriately reduce the dataset for subsequent confirmatory assays.


Assuntos
Proteínas Sanguíneas/análise , Biologia Computacional/métodos , Mineração de Dados/métodos , Bases de Dados de Proteínas , Espectrometria de Massas/métodos , Proteoma , Proteômica/métodos , Algoritmos , Biomarcadores/sangue , Ensaios de Triagem em Larga Escala , Humanos , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Software
6.
Int J Proteomics ; 2016: 1384523, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27635260

RESUMO

Nitric oxide (NO) protects the heart against ischemic injury; however, NO- and superoxide-dependent S-nitrosylation (S-NO) of cysteines can affect function of target proteins and play a role in disease outcome. We employed 2D-GE with thiol-labeling FL-maleimide dye and MALDI-TOF MS/MS to capture the quantitative changes in abundance and S-NO proteome of HF patients (versus healthy controls, n = 30/group). We identified 93 differentially abundant (59-increased/34-decreased) and 111 S-NO-modified (63-increased/48-decreased) protein spots, respectively, in HF subjects (versus controls, fold-change | ≥1.5|, p ≤ 0.05). Ingenuity pathway analysis of proteome datasets suggested that the pathways involved in phagocytes' migration, free radical production, and cell death were activated and fatty acid metabolism was decreased in HF subjects. Multivariate adaptive regression splines modeling of datasets identified a panel of proteins that will provide >90% prediction success in classifying HF subjects. Proteomic profiling identified ATP-synthase, thrombospondin-1 (THBS1), and vinculin (VCL) as top differentially abundant and S-NO-modified proteins, and these proteins were verified by Western blotting and ELISA in different set of HF subjects. We conclude that differential abundance and S-NO modification of proteins serve as a mechanism in regulating cell viability and free radical production, and THBS1 and VCL evaluation will potentially be useful in the prediction of heart failure.

7.
Digestion ; 93(4): 288-99, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27198566

RESUMO

BACKGROUND AIMS: Esophageal eosinophilia (EE) can be caused by gastroesophageal reflux disease (GERD), proton-pump inhibitor-responsive EE (PPI-REE) or eosinophilic esophagitis (EoE). This study quantified protein expression and S-nitrosylation (SNO) post-translational modifications in EE to elucidate potential disease biomarkers. METHODS: Proximal and distal esophageal (DE) biopsy proteins in patients with EE and in controls were assayed for protein content and fluorescence-labeled with and without ascorbate treatment. Protein SNO was determined, and selected protein spots were identified by matrix-assisted laser desorption ionization time-of-flight/mass spectrometry. Western blot and ingenuity pathway analysis were performed. RESULTS: Ninety-one of 648 proteins showed differential expression. There were significantly altered levels of abundance for 11 proximal and 14 DE proteins. Hierarchal clustering revealed differential SNO in inflamed tissues, indicating reactive nitrogen/oxygen species involvement. Galectin-3 was upregulated in both proximal (p < 0.04) and distal (p < 0.004) esophageal EE biopsies compared to controls. In distal EE samples, galectin-3 was significantly S-nitrosylated (p < 0.004). Principal component analysis revealed sample group discrimination distally. CONCLUSION: Proteomic analysis in EE esophageal mucosa revealed a distinct abundance and nitrosylation profile, most prominently in distal biopsies. Galectin-3 was upregulated in expression and SNO, which may indicate its potential role in mucosal inflammation. These results call for more studies to be performed to investigate the role of galectin-3 in GERD, PPI-REE and EoE.


Assuntos
Eosinofilia/metabolismo , Esofagite Eosinofílica/metabolismo , Mucosa Esofágica/metabolismo , Galectina 3/metabolismo , Refluxo Gastroesofágico/metabolismo , Processamento de Proteína Pós-Traducional , Adolescente , Biomarcadores/metabolismo , Biópsia , Criança , Pré-Escolar , Eosinofilia/patologia , Esofagite Eosinofílica/tratamento farmacológico , Esofagite Eosinofílica/patologia , Mucosa Esofágica/patologia , Refluxo Gastroesofágico/tratamento farmacológico , Refluxo Gastroesofágico/patologia , Humanos , Óxido Nítrico/metabolismo , Nitrosação , Proteômica , Inibidores da Bomba de Prótons/uso terapêutico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem
8.
PLoS Negl Trop Dis ; 10(2): e0004490, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26919708

RESUMO

Trypanosoma cruzi (Tc) infection causes chagasic cardiomyopathy; however, why 30-40% of the patients develop clinical disease is not known. To discover the pathomechanisms in disease progression, we obtained the proteome signature of peripheral blood mononuclear cells (PBMCs) of normal healthy controls (N/H, n = 30) and subjects that were seropositive for Tc-specific antibodies, but were clinically asymptomatic (C/A, n = 25) or clinically symptomatic (C/S, n = 28) with cardiac involvement and left ventricular dysfunction. Protein samples were labeled with BODIPY FL-maleimide (dynamic range: > 4 orders of magnitude, detection limit: 5 f-mol) and resolved by two-dimensional gel electrophoresis (2D-GE). After normalizing the gel images, protein spots that exhibited differential abundance in any of the two groups were analyzed by mass spectrometry, and searched against UniProt human database for protein identification. We found 213 and 199 protein spots (fold change: |≥ 1.5|, p< 0.05) were differentially abundant in C/A and C/S individuals, respectively, with respect to N/H controls. Ingenuity Pathway Analysis (IPA) of PBMCs proteome dataset identified an increase in disorganization of cytoskeletal assembly and recruitment/activation and migration of immune cells in all chagasic subjects, though the invasion capacity of cells was decreased in C/S individuals. IPA predicted with high probability a decline in cell survival and free radical scavenging capacity in C/S (but not C/A) subjects. The MYC/SP1 transcription factors that regulate hypoxia and oxidative/inflammatory stress were predicted to be key targets in the context of control of Chagas disease severity. Further, MARS-modeling identified a panel of proteins that had >93% prediction success in classifying infected individuals with no disease and those with cardiac involvement and LV dysfunction. In conclusion, we have identified molecular pathways and a panel of proteins that could aid in detecting seropositive individuals at risk of developing cardiomyopathy.


Assuntos
Cardiomiopatia Chagásica/metabolismo , Leucócitos Mononucleares/química , Proteínas/química , Proteoma/química , Cardiomiopatia Chagásica/parasitologia , Doença Crônica , Eletroforese em Gel Bidimensional , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/parasitologia , Proteínas/metabolismo , Proteoma/metabolismo , Proteômica , Trypanosoma cruzi/fisiologia
9.
J Clin Virol ; 64: 97-106, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25728087

RESUMO

OBJECTIVES: Dengue virus (DENV) infection is a significant risk to over a third of the human population that causes a wide spectrum of illness, ranging from sub-clinical disease to intermediate syndrome of vascular complications called dengue fever complicated (DFC) and severe, dengue hemorrhagic fever (DHF). Methods for discriminating outcomes will impact clinical trials and understanding disease pathophysiology. STUDY DESIGN: We integrated a proteomics discovery pipeline with a heuristics approach to develop a molecular classifier to identify an intermediate phenotype of DENV-3 infectious outcome. RESULTS: 121 differentially expressed proteins were identified in plasma from DHF vs dengue fever (DF), and informative candidates were selected using nonparametric statistics. These were combined with markers that measure complement activation, acute phase response, cellular leak, granulocyte differentiation and viral load. From this, we applied quantitative proteomics to select a 15 member panel of proteins that accurately predicted DF, DHF, and DFC using a random forest classifier. The classifier primarily relied on acute phase (A2M), complement (CFD), platelet counts and cellular leak (TPM4) to produce an 86% accuracy of prediction with an area under the receiver operating curve of >0.9 for DHF and DFC vs DF. CONCLUSIONS: Integrating discovery and heuristic approaches to sample distinct pathophysiological processes is a powerful approach in infectious disease. Early detection of intermediate outcomes of DENV-3 will speed clinical trials evaluating vaccines or drug interventions.


Assuntos
Biomarcadores/análise , Vírus da Dengue/genética , Dengue/diagnóstico , Dengue Grave/diagnóstico , Reação de Fase Aguda , Adulto , Biomarcadores/sangue , Ativação do Complemento , Dengue/genética , Dengue/virologia , Diagnóstico Precoce , Feminino , Humanos , Masculino , Fenótipo , Contagem de Plaquetas , Proteômica , Curva ROC , Dengue Grave/genética , Dengue Grave/virologia , Tropomiosina/análise , Carga Viral , Adulto Jovem , alfa-Macroglobulinas/análise
10.
Clin Transl Sci ; 6(6): 463-8, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24330691

RESUMO

Reductions in skeletal muscle function occur during the course of healthy aging as well as with bed rest or diverse diseases such as cancer, muscular dystrophy, and heart failure. However, there are no accepted pharmacologic therapies to improve impaired skeletal muscle function. Nitric oxide may influence skeletal muscle function through effects on excitation-contraction coupling, myofibrillar function, perfusion, and metabolism. Here we show that augmentation of nitric oxide-cyclic guanosine monophosphate signaling by short-term daily administration of the phosphodiesterase 5 inhibitor sildenafil increases protein synthesis, alters protein expression and nitrosylation, and reduces fatigue in human skeletal muscle. These findings suggest that phosphodiesterase 5 inhibitors represent viable pharmacologic interventions to improve muscle function.


Assuntos
Contração Muscular/efeitos dos fármacos , Fadiga Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/uso terapêutico , Piperazinas/uso terapêutico , Biossíntese de Proteínas/efeitos dos fármacos , Sulfonas/uso terapêutico , Adulto , Idoso , GMP Cíclico/metabolismo , Método Duplo-Cego , Esquema de Medicação , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/enzimologia , Óxido Nítrico/metabolismo , Inibidores da Fosfodiesterase 5/administração & dosagem , Piperazinas/administração & dosagem , Purinas/administração & dosagem , Purinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Citrato de Sildenafila , Sulfonas/administração & dosagem , Texas , Fatores de Tempo , Resultado do Tratamento , Adulto Jovem
11.
Nat Commun ; 4: 1403, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23360994

RESUMO

Noradrenaline can modulate multiple cellular functions important for cancer progression; however, how this single extracellular signal regulates such a broad array of cellular processes is unknown. Here we identify Src as a key regulator of phosphoproteomic signalling networks activated in response to beta-adrenergic signalling in cancer cells. These results also identify a new mechanism of Src phosphorylation that mediates beta-adrenergic/PKA regulation of downstream networks, thereby enhancing tumour cell migration, invasion and growth. In human ovarian cancer samples, high tumoural noradrenaline levels were correlated with high pSrc(Y419) levels. Moreover, among cancer patients, the use of beta blockers was significantly associated with reduced cancer-related mortality. Collectively, these data provide a pivotal molecular target for disrupting neural signalling in the tumour microenvironment.


Assuntos
Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Receptores Adrenérgicos beta/metabolismo , Quinases da Família src/metabolismo , Antagonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/uso terapêutico , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Camundongos , Modelos Moleculares , Invasividade Neoplásica , Metástase Neoplásica , Norepinefrina/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/mortalidade , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Análise de Sobrevida , Tirosina/metabolismo , Quinases da Família src/química
12.
Toxicol Appl Pharmacol ; 266(3): 470-80, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23200777

RESUMO

Fatty liver is an early stage of alcoholic and nonalcoholic liver disease (ALD and NALD) that progresses to steatohepatitis and other irreversible conditions. In this study, we identified proteins that were differentially expressed in the livers of rats fed 5% ethanol in a Lieber-DeCarli diet daily for 1 and 3 months by discovery proteomics (two-dimensional gel electrophoresis and mass spectrometry) and non-parametric modeling (Multivariate Adaptive Regression Splines). Hepatic fatty infiltration was significantly higher in ethanol-fed animals as compared to controls, and more pronounced at 3 months of ethanol feeding. Discovery proteomics identified changes in the expression of proteins involved in alcohol, lipid, and amino acid metabolism after ethanol feeding. At 1 and 3 months, 12 and 15 different proteins were differentially expressed. Of the identified proteins, down regulation of alcohol dehydrogenase (-1.6) at 1 month and up regulation of aldehyde dehydrogenase (2.1) at 3 months could be a protective/adaptive mechanism against ethanol toxicity. In addition, betaine-homocysteine S-methyltransferase 2 a protein responsible for methionine metabolism and previously implicated in fatty liver development was significantly up regulated (1.4) at ethanol-induced fatty liver stage (1 month) while peroxiredoxin-1 was down regulated (-1.5) at late fatty liver stage (3 months). Nonparametric analysis of the protein spots yielded fewer proteins and narrowed the list of possible markers and identified d-dopachrome tautomerase (-1.7, at 3 months) as a possible marker for ethanol-induced early steatohepatitis. The observed differential regulation of proteins have potential to serve as biomarker signature for the detection of steatosis and its progression to steatohepatitis once validated in plasma/serum.


Assuntos
Fígado Gorduroso Alcoólico/metabolismo , Proteômica/métodos , Animais , Modelos Animais de Doenças , Eletroforese em Gel Bidimensional , Etanol/administração & dosagem , Etanol/toxicidade , Fígado Gorduroso Alcoólico/genética , Regulação da Expressão Gênica , Masculino , Ratos , Ratos Endogâmicos F344 , Análise de Regressão , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
13.
Planta ; 236(2): 623-33, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22481138

RESUMO

Although the hypersensitive reaction in foliar plant diseases has been extensively described, little is clear regarding plant defense strategies in vascular wilt diseases affecting numerous economically important crops and trees. We have examined global genetic responses to Verticillium wilt in tomato (Lycopersicon esculentum Mill.) plants differing in Ve1 resistance alleles. Unexpectedly, mRNA analyses in the susceptible plant (Ve1-) based on the microarrays revealed a very heroic but unsuccessful systemic response involving many known plant defense genes. In contrast, the response is surprisingly low in plants expressing the Ve1+ R-gene and successfully resisting the pathogen. Similarly, whole-cell protein analyses, based on 2D gel electrophoresis and mass spectrometry, demonstrate large systemic increases in a variety of known plant defense proteins in the stems of susceptible plants but only modest changes in the resistant plant. Taken together, the results indicate that the large systemic increases in plant defense proteins do not protect the susceptible plant. Indeed, since a number of the highly elevated proteins are known to participate in the plant hypersensitive response as well as natural senescence, the results suggest that some or all of the disease symptoms, including ultimate plant death, actually may be the result of this exaggerated plant response.


Assuntos
Interações Hospedeiro-Patógeno , Lycopersicon esculentum/metabolismo , Doenças das Plantas/imunologia , Proteínas de Plantas/metabolismo , Verticillium/fisiologia , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Lycopersicon esculentum/genética , Lycopersicon esculentum/imunologia , Lycopersicon esculentum/microbiologia , Espectrometria de Massas , Análise de Sequência com Séries de Oligonucleotídeos , Doenças das Plantas/microbiologia , Imunidade Vegetal , Folhas de Planta/genética , Folhas de Planta/imunologia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Caules de Planta/genética , Caules de Planta/imunologia , Caules de Planta/metabolismo , Caules de Planta/microbiologia , Proteômica , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA de Plantas/análise , RNA de Plantas/genética , Plântula/genética , Plântula/imunologia , Plântula/metabolismo , Plântula/microbiologia , Verticillium/imunologia
14.
Toxicol Appl Pharmacol ; 255(1): 103-12, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21708182

RESUMO

Aniline exposure is associated with toxicity to the spleen which is characterized by splenomegaly, hyperplasia, fibrosis, and a variety of sarcomas on chronic exposure in rats. However, mechanisms by which aniline elicits splenotoxic responses are not well understood. Earlier we have shown that aniline exposure leads to increased nitration of proteins in the spleen. However, nitrated proteins remain to be characterized. Therefore, in the current study using proteomic approaches, we focused on characterizing the nitrated proteins in the spleen of aniline-exposed rats. Aniline exposure led to increased tyrosine nitration of proteins, as determined by 2D Western blotting with anti-3-nitrotyrosine specific antibody, compared to the controls. The analyzed nitrated proteins were found in the molecular weight range of 27.7 to 123.6kDa. A total of 37 nitrated proteins were identified in aniline-treated and control spleens. Among them, 25 were found only in aniline-treated rats, 11 were present in both aniline-treated and control rats, while one was found in controls only. The nitrated proteins identified mainly represent skeletal proteins, chaperones, ferric iron transporter, enzymes, nucleic acids binding protein, and signaling and protein synthesis pathways. Furthermore, aniline exposure led to significantly increased iNOS mRNA and protein expression in the spleen, suggesting its role in increased reactive nitrogen species formation and contribution to increased nitrated proteins. The identified nitrated proteins provide a global map to further investigate alterations in their structural and functional properties, which will lead to a better understanding of the role of protein nitration in aniline-mediated splenic toxicity.


Assuntos
Compostos de Anilina/toxicidade , Proteínas/metabolismo , Proteômica , Baço/efeitos dos fármacos , Animais , Western Blotting , Masculino , Óxido Nítrico Sintase Tipo II/genética , Ratos , Ratos Sprague-Dawley , Baço/metabolismo , Estresse Fisiológico
15.
Artigo em Inglês | MEDLINE | ID: mdl-20706531

RESUMO

The arenaviruses include a number of important pathogens including Lassa virus and Junin virus. Presently, the only treatment is supportive care and the antiviral Ribavirin. In the event of an epidemic, patient triage may be required to more effectively manage resources; the development of prognostic biomarker signatures, correlating with disease severity, would allow rational triage. Using a pair of arenaviruses, which cause mild or severe disease, we analyzed extracts from infected cells using SELDI mass spectrometry to characterize potential biomarker profiles. EDGE analysis was used to analyze longitudinal expression differences. Extracts from infected guinea pigs revealed protein peaks which could discriminate between mild or severe infection, and between times post-infection. Tandem mass-spectrometry identified several peaks, including the transcriptional regulator prothymosin-alpha. Further investigation revealed differences in secretion of this peptide. These data show proof of concept that proteomic profiling of host markers could be used as prognostic markers of infectious disease.


Assuntos
Infecções por Arenaviridae/metabolismo , Vírus Pichinde/fisiologia , Precursores de Proteínas/biossíntese , Timosina/análogos & derivados , Análise de Variância , Animais , Infecções por Arenaviridae/virologia , Biomarcadores , Extratos Celulares/química , Linhagem Celular , Modelos Animais de Doenças , Cobaias , Camundongos , Peritônio/citologia , Precursores de Proteínas/metabolismo , Proteômica/métodos , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Timosina/biossíntese , Timosina/metabolismo
16.
J Virol ; 84(18): 9533-45, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20610706

RESUMO

The respiratory epithelium plays a central role in innate immunity by secreting networks of inflammatory mediators in response to respiratory syncytial virus (RSV) infection. Previous proteomic studies focusing on the host cellular response to RSV indicated the existence of a nuclear heat shock response and cytoplasmic depletion of antioxidant proteins in model type II-like airway epithelial cells. Here, we increased the depth of nuclear proteomic interrogation by using fluorescence difference labeling followed by liquid isoelectric focusing prefractionation/two-dimensional gel electrophoresis (2-DE) to identify an additional 41 proteins affected by RSV infection. Surprisingly, we found inducible oligomers and shifts in isoelectric points for peroxiredoxin 1 (Prdx-1), Prdx-3, and Prdx-4 isoforms without changes in their total abundance, indicating that Prdxs were being oxidized in response to RSV. To address the role of Prdx-1 and Prdx-4 in RSV infection, isoforms were selectively knocked down by small interfering RNA (siRNA) transfection. Cells lacking Prdx-1, Prdx-4, or both showed increased levels of reactive oxygen species formation and a higher level of protein carbonylation in response to RSV infection. Using a novel saturation fluorescence labeling 2-DE analysis, we showed that 15 unique proteins had enhanced oxidative modifications of at least >1.2-fold in the Prdx knockdowns in response to RSV, including annexin A2 and desmoplakin. Our results suggest that Prdx-1 and Prdx-4 are essential for preventing RSV-induced oxidative damage in a subset of nuclear intermediate filament and actin binding proteins in epithelial cells.


Assuntos
Cisteína/metabolismo , Proteínas do Citoesqueleto/metabolismo , Peroxirredoxinas/metabolismo , Vírus Sinciciais Respiratórios/patogenicidade , Linhagem Celular , Células Epiteliais/virologia , Técnicas de Silenciamento de Genes , Humanos , Proteínas Nucleares , Oxirredução , Peroxirredoxinas/genética , Proteoma/análise , Espécies Reativas de Oxigênio/análise
17.
Proteomics Clin Appl ; 3(10): 1151-1173, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-21048890

RESUMO

Eosinophils are granular leukocytes that have significant roles in many inflammatory and immunoregulatory responses, especially asthma and allergic diseases. We have undertaken a fairly comprehensive proteomic analysis of purified peripheral blood eosinophils from normal human donors primarily employing 2-dimensional gel electrophoresis with protein spot identification by matrix-assisted laser desorption/ionization mass spectrometry. Protein subfractionation methods employed included isoelectric focusing (Zoom(®) Fractionator) and subcellular fractionation using differential protein solubilization. We have identified 3,141 proteins which had Mascot expectation scores of 10(-3) or less. Of these 426 were unique and non-redundant of which 231 were novel proteins not previously reported to occur in eosinophils. Ingenuity Pathway Analysis showed that some 70% of the non-redundant proteins could be subdivided into categories that are clearly related to currently known eosinophil biological activities. Cytoskeletal and associated proteins predominated among the proteins identified. Extensive protein posttranslational modifications were evident, many of which have not been previously reported that reflected the dynamic character of the eosinophil. This dataset of eosinophilic proteins will prove valuable in comparative studies of disease versus normal states and for studies of gender differences and polymorphic variation among individuals.

18.
J Neurosci Methods ; 158(1): 1-12, 2006 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-16766040

RESUMO

Surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) is an analytical technology for proteomic analysis that combines chromatography and mass spectrometry. At present, this technology is most commonly being exploited for the simultaneous measurement of numerous proteins in serum, but has also been utilized in organ tissue, although rarely in the brain. We applied SELDI-TOF MS technology to study protein expression in the brain of rats withdrawn from repeated cocaine exposure. Our goals were to optimize sample preparation and ProteinChip Array protocols for brain tissue, to verify the reproducibility of SELDI-TOF mass spectra and to determine whether SELDI-TOF MS detects differentially expressed proteins in cocaine- versus saline-treated rats. Consequently, we have developed an optimal protocol and generated a reproducible spectral pattern with six dominant peaks in all test samples. We have detected two smaller peaks (m/z: 5179, 5030) that were significantly increased (p < 0.05) in cocaine-treated rats compared to saline-treated rats. In summary, the application of SELDI-TOF MS to the study of protein expression in a rat model of cocaine withdrawal is feasible and has the potential to generate new hypotheses.


Assuntos
Cocaína/efeitos adversos , Proteínas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Síndrome de Abstinência a Substâncias/etiologia , Síndrome de Abstinência a Substâncias/metabolismo , Animais , Comportamento Animal , Modelos Animais de Doenças , Masculino , Análise Serial de Proteínas/métodos , Ratos , Ratos Sprague-Dawley , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/metabolismo
19.
Biophys J ; 91(4): 1302-14, 2006 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-16751247

RESUMO

The electrostatic environments near the acetylcholine binding sites on the nicotinic acetylcholine receptor (nAChR) and acetylcholinesterase were measured by diffusion-enhanced fluorescence energy transfer (DEFET) to determine the influence of long-range electrostatic interactions on ligand binding kinetics and net binding energy. Changes in DEFET from variously charged Tb3+ -chelates revealed net potentials of -20 mV at the nAChR agonist sites and -14 mV at the entrance to the AChE active site, in physiological ionic strength conditions. The potential at the alphadelta-binding site of the nAChR was determined independently in the presence of d-tubocurarine to be -14 mV; the calculated potential at the alphagamma-site was approximately threefold stronger than at the alphadelta-site. By determining the local potential in increasing ionic strength, Debye-Hückel theory predicted that the potentials near the nAChR agonist binding sites are constituted by one to three charges in close proximity to the binding site. Examination of the binding kinetics of the fluorescent acetylcholine analog dansyl-C6-choline at ionic strengths from 12.5 to 400 mM revealed a twofold decrease in association rate. Debye-Hückel analysis of the kinetics revealed a similar charge distribution as seen by changes in the potentials. To determine whether the experimentally determined potentials are reflected by continuum electrostatics calculations, solutions to the nonlinear Poisson-Boltzmann equation were used to compute the potentials expected from DEFET measurements from high-resolution models of the nAChR and AChE. These calculations are in good agreement with the DEFET measurements for AChE and for the alphagamma-site of the nAChR. We conclude that long-range electrostatic interactions contribute -0.3 and -1 kcal/mol to the binding energy at the nAChR alphadelta- and alphagamma-sites due to an increase in association rates.


Assuntos
Acetilcolina/química , Acetilcolinesterase/química , Membrana Celular/química , Potenciais da Membrana , Modelos Químicos , Modelos Moleculares , Receptores Nicotínicos/química , Sítios de Ligação , Simulação por Computador , Transferência Ressonante de Energia de Fluorescência , Ligação Proteica , Eletricidade Estática
20.
Surgery ; 138(2): 382-90, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16153451

RESUMO

BACKGROUND: The stromal microenvironment influences many steps of tumor progression through the elaboration of signals from myofibroblasts. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway transduces signals initiated by growth factors and is involved in colonic epithelial proliferation. The purpose of this study was to determine (1) the influence of myofibroblasts on colon cancer cell proliferation and PI3K activity, and (2) the protein alterations associated with myofibroblasts derived from polyp versus normal margins. METHODS: Myofibroblasts were derived from polyps and corresponding normal mucosa. Myofibroblasts were cocultured with colon cancer cells HT29 stably transfected with green fluorescent protein and KM20 cells. Proliferation was quantitated by green fluorescent protein count and cytokeratin enzyme-linked immunosorbent assay. HT29 cells were incubated with conditioned medium from myofibroblasts, and the effect on proliferation and PI3K activity was determined by 5-bromo 2-deoxyuridine incorporation and Akt kinase assay, respectively. Protein profiles were obtained by SELDI-TOF MS analysis. RESULTS: In coculture experiments, all myofibroblasts significantly enhanced HT29 and KM20 cell proliferation. However, polyp myofibroblasts enhanced proliferation of the cancer cells to a greater extent than normal myofibroblasts. Conditioned medium from all myofibroblasts stimulated Akt kinase activity. SELDI-TOF MS profiles showed more than 40 protein peaks for each isolate. One protein was differentially expressed in polyps versus normal cells. CONCLUSIONS: Utilizing a novel proteomic approach, we identify distinct protein profiles in myofibroblasts of polyps compared with stromal cells of normal mucosa. Moreover, myofibroblasts can stimulate indirectly PI3K activity and enhance colon cancer cell proliferation. These findings suggest that targeted therapy to signaling pathways in myofibroblasts may be useful in colorectal cancer chemoprevention and possible treatment.


Assuntos
Pólipos Adenomatosos/patologia , Comunicação Celular/fisiologia , Colo/citologia , Neoplasias do Colo/patologia , Fibroblastos/citologia , Proteômica , Pólipos Adenomatosos/fisiopatologia , Divisão Celular/fisiologia , Técnicas de Cocultura , Neoplasias do Colo/fisiopatologia , Meios de Cultivo Condicionados/metabolismo , Fibroblastos/fisiologia , Células HT29/citologia , Humanos , Espectrometria de Massas , Fosfatidilinositol 3-Quinases/metabolismo
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