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1.
Age (Dordr) ; 35(4): 1091-104, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22661299

RESUMO

Calorie restriction (CR) remains the most robust metabolic intervention to extend lifespan and improve healthspan in several species. Using global and targeted mass spectrometry-based metabolomics approaches, here we show that chronic CR prevents age-related changes in specific metabolic signatures. Global metabolomic analysis using ultra-performance liquid chromatography-tandem mass spectrometry detected more than 7,000 metabolites in sera from ad-libitum-fed young, aged, and aged C57BL/6 mice maintained on 40 % CR. Multivariate statistical analysis of mass spectrometry data revealed a clear separation among the young, aged, and aged-CR mice demonstrating the potential of this approach for producing reliable metabolic profiles that discriminate based on age and diet. We have identified 168 discriminating features with high statistical significance (p ≤ 0.001) and validated and quantified three of these metabolites using targeted metabolite analysis. Calorie restriction prevented the age-related alteration in specific metabolites, namely lysophosphatidylcholines (16:1 and 18:4), sphingomyelin (d18:1/12:0), tetracosahexaenoic acid, and 7α-dihydroxy-4-cholesten-3-one, in the serum. Pathway analysis revealed that CR impacted the age-related changes in metabolic byproducts of lipid metabolism, fatty acid metabolism, and bile acid biosynthesis. Our data suggest that metabolomics approach has the potential to elucidate the metabolic mechanism of CR's potential anti-aging effects in larger-scale investigations.


Assuntos
Envelhecimento/sangue , Restrição Calórica , Metabolismo dos Lipídeos/fisiologia , Metaboloma/fisiologia , Animais , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Feminino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL
2.
Analyst ; 137(14): 3227-9, 2012 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-22575916

RESUMO

Here we report a novel method for the manipulation and concentration of Aß amyloid fibrils, implicated in Alzheimer's disease, using DC insulating gradient dielectrophoresis (DC-iGDEP). Fibril enrichment was found to be ∼400%. Simulations suggest that capture of the full range of amyloid protein aggregates is possible with optimized device design.


Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/isolamento & purificação , Condutividade Elétrica , Eletroforese/métodos , Multimerização Proteica , Impedância Elétrica , Estrutura Secundária de Proteína
3.
Anal Biochem ; 425(2): 104-12, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-22446499

RESUMO

The separation and detection of individual amyloid beta (Aß) aggregates by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was demonstrated. Samples were prepared with either Aß (1-40) or Aß (1-42) peptides and were characterized by CE with ultraviolet (UV) absorbance detection and transmission electron microscopy (TEM). Using thioflavin T (ThT) in the electrophoresis buffer, electrophoresis of aggregate-containing samples (5.0-s injection) produced up to several hundred narrow (< 20 ms FWHM [full width at half maximum]) fluorescence peaks. Injection of Aß (1-40) monomer samples resulted in no additional peaks compared with controls. The CE-LIF results were validated by bulk ThT fluorescence measurements for the same samples. The potential of laser-induced fluorescence anisotropy (LIFA) with CE to characterize individual Aß aggregates also was investigated.


Assuntos
Peptídeos beta-Amiloides/análise , Eletroforese Capilar , Lasers , Fragmentos de Peptídeos/análise , Espectrofotometria Ultravioleta , Peptídeos beta-Amiloides/isolamento & purificação , Benzotiazóis , Microscopia Eletrônica de Transmissão e Varredura , Fragmentos de Peptídeos/isolamento & purificação , Tiazóis/química
4.
J Chromatogr B Analyt Technol Biomed Life Sci ; 879(9-10): 627-32, 2011 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-21334989

RESUMO

A method based on capillary electrophoresis (CE) with UV absorbance detection is presented to characterize synthetic amyloid beta (Aß) peptide preparations at different aggregation states. Aggregation of Aß (1-40) and Aß (1-42) is closely linked to Alzheimer's disease (AD), and studying how Aß peptides self-assemble to form aggregates is the focus of intense research. Developing methods capable of identifying, characterizing and quantifying a wide range of Aß species from monomers to fully formed fibrils is critical for AD research and is a major analytical challenge. Monomer and fibril samples of Aß (1-40) and Aß (1-42) were prepared and characterized for this study. The monomer-equivalent concentration for each sample was determined by HPLC-UV, and aggregate formation was confirmed and characterized by transmission electron microscopy. The same samples were studied using CE with UV absorbance detection. Analysis by mass spectrometry of collected CE fractions was used to confirm the presence of Aß for some CE-UV peaks. The CE-UV method reported here clearly indicates that monomeric and aggregated Aß were electrophoretically separated, and substantial differences in the electrophoretic profiles between samples of Aß (1-40) and Aß (1-42) were observed. This CE-UV method can differentiate between Aß monomer, oligomeric intermediates, and mature fibrils.


Assuntos
Peptídeos beta-Amiloides/análise , Eletroforese Capilar/métodos , Fragmentos de Peptídeos/análise , Peptídeos beta-Amiloides/química , Humanos , Espectrometria de Massas , Fragmentos de Peptídeos/química , Multimerização Proteica , Subunidades Proteicas , Espectrofotometria Ultravioleta
5.
J Proteome Res ; 10(4): 1519-27, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21261302

RESUMO

Adipose tissue contains a heterogeneous population of mature adipocytes, endothelial cells, immune cells, pericytes, and preadipocytic stromal/stem cells. To date, a majority of proteomic analyses have focused on intact adipose tissue or isolated adipose stromal/stem cells in vitro. In this study, human subcutaneous adipose tissue from multiple depots (arm and abdomen) obtained from female donors was separated into populations of stromal vascular fraction cells and mature adipocytes. Out of 960 features detected by 2-D gel electrophoresis, a total of 200 features displayed a 2-fold up- or down-regulation relative to each cell population. The protein identity of 136 features was determined. Immunoblot analyses comparing SVF relative to adipocytes confirmed that carbonic anhydrase II was up-regulated in both adipose depots while catalase was up-regulated in the arm only. Bioinformatic analyses of the data set determined that cytoskeletal, glycogenic, glycolytic, lipid metabolic, and oxidative stress related pathways were highly represented as differentially regulated between the mature adipocytes and stromal vascular fraction cells. These findings extend previous reports in the literature with respect to the adipose tissue proteome and the consequences of adipogenesis. The proteins identified may have value as biomarkers for monitoring the physiology and pathology of cell populations within subcutaneous adipose depots.


Assuntos
Adipócitos/química , Eletroforese em Gel Bidimensional/métodos , Células Endoteliais/química , Proteoma/análise , Células Estromais/química , Gordura Subcutânea/citologia , Adulto , Anidrase Carbônica II/metabolismo , Diferenciação Celular , Células Cultivadas , Biologia Computacional/métodos , Células Endoteliais/citologia , Feminino , Humanos , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Fenótipo , Células-Tronco/química , Células-Tronco/citologia , Células Estromais/citologia
6.
Methods Mol Biol ; 702: 163-90, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21082402

RESUMO

Proteomics refers to the analysis of expression, localization, functions, posttranslational modifications, and interactions of proteins expressed by a genome at a specific condition and at a specific time. Mass spectrometry (MS)-based proteomic methods have emerged as a key technology for unbiased systematic and high-throughput identification and quantification of complex protein mixtures. These methods have the potential to reveal unknown and novel changes in protein interactions and assemblies that regulate cellular and physiological processes. Both gel-based (one-dimensional [1D] gel electrophoresis, two-dimensional [2D] polyacrylamide gel electrophoresis, 2D difference in-gel electrophoresis [DIGE]) and gel-free (liquid chromatography [LC], capillary electrophoresis) approaches have been developed and utilized in a variety of combinations to separate proteins prior to mass spectrometric analysis. Detailed protocols for global proteomic analysis from adipose-derived stem cells (ASCs) using two central strategies, 2D-DIGE-MS and 2D-LC-MS, are presented here.


Assuntos
Proteômica/métodos , Sequência de Aminoácidos , Bioensaio , Fracionamento Celular , Precipitação Química , Cromatografia por Troca Iônica , Cromatografia Líquida , Cromatografia de Fase Reversa , Eletroforese em Gel Bidimensional , Humanos , Focalização Isoelétrica , Marcação por Isótopo , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas/química , Proteínas/isolamento & purificação , Coloração e Rotulagem
7.
Phytother Res ; 24(9): 1278-84, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20175108

RESUMO

An ethanolic extract of Artemisia dracunculus L. (PMI 5011) has been observed to decrease glucose and insulin levels in animal models and enhance cellular signaling in cultured cells. To determine the mechanism of action of PMI-5011, we have measured changes in protein expression in human primary skeletal muscle culture (HSMC) from subjects with Type 2 diabetes. After obtaining skeletal muscle biopsies, HSMCs were initiated, grown to confluence, and exposed to 10 microg/mL PMI 5011 overnight. Two-dimensional difference in-gel electrophoresis was used to separate proteins, and liquid chromatography mass spectrometry was used to identify differentially regulated proteins. Additionally, real-time polymerase chain reaction (PCR) was used to confirm candidate proteins identified. These data demonstrate that a well characterized botanical extract of Artemisia dracunculus L. significantly modulates proteins involved in regulating inflammatory pathways, particularly the NFkappaB complex system.


Assuntos
Artemisia , Diabetes Mellitus Tipo 2/metabolismo , Hipoglicemiantes/farmacologia , Insulina/metabolismo , Músculo Esquelético/metabolismo , Obesidade/metabolismo , Extratos Vegetais/farmacologia , Células Cultivadas , Humanos , Mediadores da Inflamação/metabolismo , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/efeitos dos fármacos , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase , Proteômica/métodos , Transdução de Sinais/efeitos dos fármacos
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