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1.
Methods Mol Biol ; 878: 175-83, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22674133

RESUMO

The use of biosensors has become a standard method to characterize biomolecular interactions. Data obtained from biosensor studies are widely used to evaluate drug candidates, particularly in relation to their binding properties towards a selected target. The importance of measuring such interactions in a biologically relevant environment has become the new challenge for the biosensor technologies. In this chapter we describe a method for studying interactions between proteins and targets at the surface of intact cells using a quartz crystal microbalance (QCM) biosensor.


Assuntos
Biomarcadores Tumorais/metabolismo , Técnicas Biossensoriais/métodos , Técnicas de Microbalança de Cristal de Quartzo/métodos , Linhagem Celular Tumoral , Humanos , Metástase Neoplásica , Ligação Proteica
2.
Biosens Bioelectron ; 35(1): 200-205, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22410484

RESUMO

A novel approach to the study of molecular interactions on the surface of mammalian cells using a QCM biosensor was developed. For this study, an epidermoid carcinoma cell line (A-431) and a breast adenocarcinoma cell line (MDA-MB-468) were immobilized onto polystyrene-coated quartz crystals. The binding and dissociation between the lectin Con A and the cells as well as the inhibition of the binding by monosaccharides were monitored in real time and provided an insight into the complex avidic recognition of cell glycoconjugates. The real-time lectin screening of a range of lectins, including Con A, DBA, PNA and UEA-I, enabled the accurate study of the glycosylation changes between cells, such as changes associated with cancer progression and development. Furthermore, the kinetic parameters of the interaction of Con A with MDA-MB-468 cells were studied. This application provides investigators in the field of glycobiology with a novel tool to study cell surface glycosylation and may also have impacts on drug discovery.


Assuntos
Carboidratos/análise , Técnicas de Microbalança de Cristal de Quartzo/métodos , Ligação Competitiva , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/metabolismo , Células Imobilizadas , Sistemas Computacionais , Concanavalina A/metabolismo , Feminino , Glicoconjugados/análise , Glicômica/métodos , Glicosilação , Humanos , Cinética
3.
Analyst ; 136(18): 3777-82, 2011 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-21789315

RESUMO

Collagen, the major component of extracellular matrix (ECM) and the most abundant protein in the human body, is implicated in the development of atherosclerosis. Collagen types I and III were immobilized on fused-silica capillary to investigate their shape, size and structure by atomic force microscopy (AFM). For comparison, collagen was also immobilized on a mica surface. Our studies demonstrated that not only does the adsorption pattern on the substrate vary with the type of collagen, but also the substrate material plays an important role in the fibril formation process. Decorin, which promotes the binding of low-density lipoprotein (LDL) particles with collagen, was investigated for its effect on the fibrillogenesis. On both substrate materials, addition of decorin clearly reduced the fibril diameter of collagen surfaces. Moreover, a quartz crystal microbalance (QCM)-based biosensor approach was applied to clarify and evaluate the affinity of different collagen coatings immobilized on a silicon dioxide sensor chip toward apolipoprotein B-100, the major protein of LDL. The results confirmed the importance of collagen type and their fibrillogenesis on the binding of the positive residues of apolipoprotein B-100 on negatively charged collagen surfaces.


Assuntos
Apolipoproteína B-100/metabolismo , Colágeno Tipo III/metabolismo , Colágeno Tipo I/metabolismo , Decorina/metabolismo , Microscopia de Força Atômica , Técnicas de Microbalança de Cristal de Quartzo , Colágeno Tipo I/química , Colágeno Tipo III/química , Decorina/química , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Ligação Proteica , Dióxido de Silício/química , Propriedades de Superfície
4.
Anal Bioanal Chem ; 396(4): 1373-80, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20041230

RESUMO

Atherosclerosis has received wide attention as a primary cause of premature death in developed countries. The retention of low-density lipoprotein (LDL) particles in the intima, the inner layer of the capillaries, has been imputed as the main cause of the development of atherosclerotic plaques. The entrapment of LDL is mainly due to the specific interaction between the lysine-rich site on apolipoprotein B-100 (apoB-100), a major apolipoprotein of LDL, and extracellular matrix (ECM) components such as collagen, proteoglycans, and glycosaminoglycans (GAGs). Although valuable techniques already exist for studies on apoB-100 and ECM interactions, there is continued need for miniaturized tools that can complement the tools already available and even provide totally new data. This work explores the applicability of the quartz crystal microbalance (QCM) for interaction studies between apoB-100 peptide fragments and various components of the ECM. Two positive peptide fragments, PP and PP(2), and two components of the ECM, collagen I and a selected GAG, chondroitin 6-sulfate (C6S), were immobilized on polystyrene and carboxyl sensor chips. C6S was injected as analyte for PP- and PP(2)-coated surfaces, while PP was the analyte for collagen I and C(6)S surfaces. The estimated dissociation constant (K(D)) indicates that the interactions occur via the positive residues, lysine and arginine, of apoB-100. The continuous-flow QCM system employed in this study is shown to be an excellent tool for the elucidation of interactions between these types of biomolecules.


Assuntos
Apolipoproteína B-100/metabolismo , Matriz Extracelular/metabolismo , Quartzo , Pesos e Medidas/instrumentação , Apolipoproteína B-100/química , Arginina/química , Matriz Extracelular/química , Lisina/química , Ligação Proteica
5.
Proteomics ; 7(22): 4082-9, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17994629

RESUMO

The lectin from Helix pomatia (HPA) binds to adenocarcinomas with a metastatic phenotype but the glycoconjugates of cancer cells that bind to the lectin have yet to be characterized in detail. We used a model of metastatic (HT29) and nonmetastatic (SW480) human colorectal cancer cells and a proteomic approach to identify HPA binding glycoproteins. Cell membrane proteins purified by HPA affinity chromatography, were separated by 2-DE and analyzed by MS. Competitive inhibition experiments with N-acetylgalactosamine, N-acetylglucosamine, and sialic acid confirmed that HPA binding was via a glycan-mediated interaction. Western blot analysis showed that HPA binds to proteins not recognized by an antibody against blood group A epitope. The proteomic study showed the main HPA binding partners include integrin alphav/alpha6 and annexin A2/A4. These proteins were found complexed with microfilament proteins alpha and beta tubulin, actin, and cytokeratins 8 and 18. HPA also bound to Hsp70, Hsp90, TRAP-1, and tumor rejection factor 1. This study revealed that the prognostic utility of HPA lies in its ability to bind simultaneously to many glycoproteins involved in cell migration and signaling, in addition, the proteins recognized by HPA are glycosylated with structures distinct from the blood group A epitope.


Assuntos
Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/secundário , Lectinas/análise , Proteômica , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Humanos , Lectinas/efeitos dos fármacos , Lectinas/imunologia , Proteínas de Membrana/imunologia , Microscopia Confocal , N-Acetilgalactosaminiltransferases/farmacologia , N-Acetilglucosaminiltransferases/farmacologia , Ácido N-Acetilneuramínico/farmacologia , Prognóstico , Ligação Proteica , Sensibilidade e Especificidade , Relação Estrutura-Atividade , Células Tumorais Cultivadas
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