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1.
Mol Cell Proteomics ; 12(11): 3148-59, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23897580

RESUMO

Pre-eclampsia (PE) is a serious complication of pregnancy with potentially life threatening consequences for both mother and baby. Presently there is no test with the required performance to predict which healthy first-time mothers will go on to develop PE. The high specificity, sensitivity, and multiplexed nature of selected reaction monitoring holds great potential as a tool for the verification and validation of putative candidate biomarkersfor disease states. Realization of this potential involves establishing a high throughput, cost effective, reproducible sample preparation workflow. We have developed a semi-automated HPLC-based sample preparation workflow before a label-free selected reaction monitoring approach. This workflow has been applied to the search for novel predictive biomarkers for PE. To discover novel candidate biomarkers for PE, we used isobaric tagging to identify several potential biomarker proteins in plasma obtained at 15 weeks gestation from nulliparous women who later developed PE compared with pregnant women who remained healthy. Such a study generates a number of "candidate" biomarkers that require further testing in larger patient cohorts. As proof-of-principle, two of these proteins were taken forward for verification in a 100 women (58 PE, 42 controls) using label-free SRM. We obtained reproducible protein quantitation across the 100 samples and demonstrated significant changes in protein levels, even with as little as 20% change in protein concentration. The SRM data correlated with a commercial ELISA, suggesting that this is a robust workflow suitable for rapid, affordable, label-free verification of which candidate biomarkers should be taken forward for thorough investigation. A subset of pregnancy-specific glycoproteins (PSGs) had value as novel predictive markers for PE.


Assuntos
Glicoproteínas/sangue , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/diagnóstico , Proteínas da Gravidez/sangue , Proteômica/métodos , Adolescente , Adulto , Sequência de Aminoácidos , Automação , Biomarcadores/sangue , Cromatografia Líquida de Alta Pressão , Estudos de Coortes , Diagnóstico Precoce , Ensaio de Imunoadsorção Enzimática , Feminino , Glicoproteínas/genética , Humanos , Dados de Sequência Molecular , Gravidez , Proteínas da Gravidez/genética , Segundo Trimestre da Gravidez/sangue , Estudos Prospectivos , Espectrometria de Massas em Tandem , Fluxo de Trabalho , Adulto Jovem , beta-Tromboglobulina/análise , beta-Tromboglobulina/genética
3.
Cell Adh Migr ; 2(3): 161-6, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19262118

RESUMO

Desmosomes are intercellular junctions responsible for strong cell-cell adhesion in epithelia and cardiac muscle. Numerous studies have shown that the other major type of epithelial cell adhesion, the adherens junction, is destabilized by src-induced tyrosine phosphorylation of two of its principal components, E-cadherin and beta-catenin. Here we show that treatment of epithelial cells with the potent tyrosine phosphatase inhibitor sodium pervanadate causes tyrosine phosphorylation of the major desmosomal components desmoglein 2 and plakoglobin in both the non-ionic detergent soluble and insoluble cell fractions and, surprisingly, stabilizes desmosomal adhesion, inducing the hyper-adhesive form normally found in tissues and confluent cell sheets. Taken together with the few other studies on desmosomes these results suggest that the effects of tyrosine phosphorylation on desmosomal adhesion are complex.


Assuntos
Desmossomos/efeitos dos fármacos , Vanadatos/farmacologia , Animais , Linhagem Celular , Desmossomos/metabolismo , Cães , Fosfotirosina/metabolismo
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