Transcriptoma e proteoma em sangue periférico na busca de novos marcadores de doenças cardiovasculares / Transcriptomic and proteomic of peripheral blood as approaches to biomarkers cardiovascular discovers

INTRODUÇÃO: A principal manifestação clínica da doença aterosclerótica é o infarto agudo do miocárdio, caracterizada como emergência médica, que necessita de diagnóstico correto, rápido, preciso e terapia eficaz. Os estudos de transcriptoma e proteoma possibilitam obter informações que nos permite compreender de forma mais abrangente a evolução fisiopatológica das doenças, sendo as cardiovasculares particularmente favorecidas por terem etiologia multifatorial e sem dúvida multigênica, portanto a utilização destas ferramentas num modelo de doença aguda pode auxiliar, de forma singular, na obtenção de novas informações, tais como novos marcadores precoces de injúria. OBJETIVO: Identificar novos biomarcadores de doenças cardiovasculares através da análise do perfil de expressão de RNAm de células do sangue periférico e proteínas plasmáticas de pacientes com SCA. CASUÍSTICA E MÉTODOS: É um estudo caso-controle de pacientes com SCA recrutados no Pronto-Socorro do Instituto Dante Pazzanese de Cardiologia. Foram recrutados 84 indivíduos com Síndrome Coronariana Aguda (SCA), 47 indivíduos sem doença cardiovascular (grupo controle), de ambos os sexos com idade entre 30 a 65 anos, atendidos no Instituto Dante Pazzanese do Estado de São Paulo - Brasil. A avaliação de expressão gênica global de 10 pacientes e 6 controles (pareados) durante as primeiras 48 h após o IAM foi realizada através dos microarranjos de DNA (sistema Affymetrix) e a análise de plasma por separação em sistema de microarranjos (ProteinChip®), seguida da identificação e quantificação por espectrometria de massa, em sistema SELDI-TOF/MS. Os resultados de microarranjo de DNA foram validados tecnicamente (a partir das mesmas amostras processadas por microarranjo de DNA) e, posteriormente validados biologicamente (a partir das outras amostras não processadas por microarranjo de DNA) através da PCR em tempo real. RESULTADOS: Foram observados ao total 599 genes diferentemente expressos nas primeiras 48 h...

BACKGROUND: The main clinical manifestation of atherosclerosis is the acute myocardial infarction (AMI), which is a medical emergency that requires prompt diagnosis and efficient therapy. The transcriptomic and proteomic approaches are both powerful tools for the study of AMI and may be instrumental to identify news biomarkers involved in the inflammatory and apoptotic process of cardiovascular diseases. OBJECTIVE: The aim of this study is to determine the gene expression of RNAm and protein in blood cells following an AMI to identify new biomarkers. PATIENTS AND METHODS: For this study eighty four patients with acute coronary syndrome (ACS) and forty seven control individuals were selected among patients of the Instituto Dante Pazzanese, São Paulo state, Brazil. A global gene expression profile by GeneChip® Exon 1.0 ST Array (Affymetrix) and proteomic plasma profile by ProteinChip® Biomarker System and SELDI-TOF/MS were evaluated for ten patients from the ACS group and six from the control group. These patients were followed up for the first 48 h following the AMI. The genes differently expressed by microarray analysis were submitted to technical (same casuistic) and biologic (new casuistic) validation by PCR real time. RESULTS: 599 genes were differentially expressed at the first 48 h after AMI. Thirty-three genes were selected and submitted to the technical validation, and 20 were subjected to biological validation by real time PCR afterwards. The validated ones were: ALOX15, Areg, BCL2A1, BCL2L1, CA1, COX7B, ECDHC3, KCNE1, IL18R1, IRS2, MYL4, MMP9 and TREML4. At proteomic analysis, 479 peaks of plasma proteins differentially expressed were identified. 16 peaks were considered as high potentially biomarkers. Their molecular weight was between 6386.5 Da and 17807.7 Da. CONCLUSION: Results from this study were able to identify changes in gene and protein profiles in the plasma, and suggest new markers for evaluation of acute coronary syndrome and...

Cycle modulation of insulin-like growth factor-binding protein 1 in human endometrium

Endometrium is one of the fastest growing human tissues. Sex hormones, estrogen and progesterone, in interaction with several growth factors, control its growth and differentiation. Insulin-like growth factor 1 (IGF-1) interacts with cell surface receptors and also with specific soluble binding proteins. IGF-binding proteins (IGF-BP) have been shown to modulate IGF-1 action. Of six known isoforms, IGF-BP-1 has been characterized as a marker produced by endometrial stromal cells in the late secretory phase and in the decidua. In the current study, IGF-1-BP concentration and affinity in the proliferative and secretory phase of the menstrual cycle were measured. Endometrial samples were from patients of reproductive age with regular menstrual cycles and taking no steroid hormones. Cytosolic fractions were prepared and binding of 125I-labeled IGF-1 performed. Cross-linking reaction products were analyzed by SDS-polyacrylamide gel electrophoresis (7.5 percent) followed by autoradiography. 125I-IGF-1 affinity to cytosolic proteins was not statistically different between the proliferative and secretory endometrium. An approximately 35-kDa binding protein was identified when 125I-IGF-1 was cross-linked to cytosol proteins. Secretory endometrium had significantly more IGF-1-BP when compared to proliferative endometrium. The specificity of the cross-linking process was evaluated by the addition of 100 nM unlabeled IGF-1 or insulin. Unlabeled IGF-1 totally abolished the radioactivity from the band, indicating specific binding. Insulin had no apparent effect on the intensity of the labeled band. These results suggest that IGF-BP could modulate the action of IGF-1 throughout the menstrual cycle. It would be interesting to study this binding protein in other pathologic conditions of the endometrium such as adenocarcinomas and hyperplasia

Affinity labels as probes for the study of the nucleotide binding site of enzymes

Affinity labeling has proved to be a very useful tool for searching important amino acid residues located in active or allosteric sites of enzymes. In this article, the general principles and specific examples of the use of affinity labeling are discussed

Photoaffinity labeling and photoaffinity crosslinking of enzymes

Photoaffinity labeling is a special type of chemical modification, where the label is activated by the action of light. This article presents the general principles and limitations of this technique, its application to the study of Micrococcus luteus ATPase and the use of photoaffinity crosslinking to probe the structure of this enzyme

Potential infectivity of blood from HBsAg asymptomatic carriers due to the presence of HBV-DNA and comparison with other markers of HBV infection

Em 356 soros HBsAg positivos de portadores assintomáticos, titulados por hemaglutinaçäo passiva reversa, foi analisada a presença de HBV-DNA, HBeAg e IgM anti-HBc, da seguinte forma: as amostras foram divididas em três classes de acordo com o título de HBsAg e IgM anti-HBc e foi verificada a distribuiçäo de HBV-DNA e HBsAg nestas classes. Em amostras com títulos elevados de HBsAg, 65% das amostras tiveram um ou ambos os marcadors HBV-DNA e HBsAg e as mesmas foram encontradas em apenas 19% das amostras com baixo título de HBsAg. Do total de 356 amostras, 121 foram positivas par aIgM anti-HBc (33.9%). Destas, 38.9% de HBV-DNA e 47.9% de HBeAG foram observadas, ao passo que em amostras com ausência de IGM anti-HBc, 18.3% e 16.6% foram respectivamente encontrados. A concordância entre os três marcadores foi encontrada em amostras com alto título de HBsAg, entretanto HBV-DNA foi também detectado em grupo de baixo título de HBsAg na ausência de IgM anti-HBc ou em níveis baixos, mostrando o potencial de infectividade mesmo em amostras de HBsAg com valores próximos ao limite da positividade

Photoaffinity labeling of extracellular and intracellular androgen binding proteins

Se describe un método de marcación por fotoafinidad de la SHBG sérica humana y de conejo en un precipitado de sulfato de amonio utilizando delta6-testosterona 3H como ligando. La precipitación disminuye la contaminación con albúmina y concentra al mismo tiempo la globulina ligadora de hormonas sexuales. La marcación por fotoafinidad se realizó mediante un flash electrónico. El esteroide libre y el no unido covalentemente se adsorbieron mediante un tratamiento prolongado con una solución de carbón dextrán. La cromatografía en columna de Sephadex G-200 mostró un único pico radioactivo unido covalentemente con el volumen de elución de la SHBG. Este preparado es útil para ser utilizado en estudios de metabolización de la SHBG in vivo. Con algunas modificaciones, el método fue aplicado a la marcación por fotoafinidad del receptor citosólico de andrógenos de próstata utilizando al R 1881 como ligando. El receptor de andrógenos de citosol de próstata también fue precipitado con sulfato de amonio. Luego de marca, irradiar y calentar a 50- C el R 1881-3H no unido covalentemente fue removido mediante el tratamiento con carbón dextrán. En presencia de fotólisis previa al tratamiento con calor, la cromatografía en microcolumnas de Sephadex G-25 mostró un pico radiactivo que eluyó con el volumen de elución de las macromoléculas, el cual desaparecía en las muestras no fotolizadas previamente; sin embargo el receptor fotomarcado tuvo un coeficiente de sedimentación, luego de ultracentrifugación en gradientes lineares de sucrosa, diferente del receptor no irradiado, sugiriendo que la fotólisis generó a algún cambio en la configuración del complejo