Lysyl oxidase-like protein-2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane.

Sprouting angiogenesis is associated with extensive extracellular matrix (ECM) remodeling. The molecular mechanisms involved in building the vascular microenvironment and its impact on capillary formation remain elusive. We therefore performed a proteomic analysis of ECM from endothelial cells maintained in hypoxia, a major stimulator of angiogenesis.

Organellar protein complexes of Caco-2 human cells analyzed by two-dimensional blue native/SDS-PAGE and mass spectrometry.

The complexome is essential for a better understanding of protein functions. In order to study protein complexes, an approach allowing the extraction and the analysis in native conditions is needed. Two-dimensional blue native/SDS-PAGE (2D BN/SDS-PAGE) technology is thus an interesting and powerful approach for this purpose.

Electrospray tandem mass spectrometry of alendronate analogues: fingerprints for characterization of new potential prodrugs.

1-hydroxymethylene-1,1-bisphosphonic acids (HMBPs) are important drugs for the treatment of a variety of bone diseases. Since these compounds have no chromophore, their detection is challenging and mass spectrometry (MS) appears to be an appropriate sensitive tool. Our work deals with the analysis by electrospray ionization tandem mass spectrometry (ESI-MSn) of the well-known nitrogen-containing HMBP alendronate and of three analogues, considered as potential prodrugs.

Fragmentation patterns of new esterified and unesterified aromatic 1-hydroxymethylene-1, 1-bisphosphonic acids by ESI-MSn.

1-Hydroxymethylene-1,1-bisphosphonic acids (HMBPs) are compounds that have interesting pharmacological applications. Unfortunately few studies exist on their analyses by mass spectrometry (MS). In this work, we have analyzed new aromatic HMBPs and their prodrugs with electrospray tandem mass spectrometry (ESI-MS(n)).

HPLC-chip-mass spectrometry for protein signature identifications.

This work investigates the use of an HPLC-chip microfluidic device interfaced to an IT mass spectrometer to search for biomarker signatures. To that end, the identification of autoantigens is chosen as a model. It not only constitutes a proof of concept model but also the growing interest in autoantibodies and autoantigens as new markers of diseases provides a practical application at the same time.

Usefulness of autoantigens depletion to detect autoantibody signatures by multiple affinity protein profiling.

Patients with cancer produce specific autoantibodies against protein antigens present in limited amount among a large background of immunoglobulins (Igs), nonrelevant as biomarkers, including natural antibodies. Multiple affinity protein profiling (MAPPing) that combines 2-D immunoaffinity chromatography, enzymatic digestion of the isolated proteins, and identification by MS/MS, may facilitate the identification of these so far unknown patient antibodies. The first immunoaffinity chromatography is crucial, as it is used for selectively removing proteins (autoantigens) recognized by natural antibodies.

Proteomic Analysis of the MCF7 Breast Cancer Cell Line.

Background: The MCF7 breast cancer cell line is a cellular model for breast cancer studies and marker discovery. Therefore, a better knowledge of its proteome is a prerequisite for a more efficient use of this model.

Materials And Methods: Proteins expressed during the exponential growth phase of MCF7 cells were analyzed and mapped using two-dimensional gel electrophoresis and mass spectrometry.

Usefulness of an integrated microfluidic device (HPLC-Chip-MS) to enhance confidence in protein identification by proteomics.

Nanoflow liquid chromatography/mass spectrometry (nanoLC/MS) has become a current tool in proteomics applications increasingly used in the search for new biomarkers. A new integrated microfluidic device (HPLC-Chip), coupled to ion trap mass spectrometry (ITMS), appears as an innovative and robust tool for improving the identifications commonly performed by nanoLC/MS/MS. We tested this device for the identification of proteins obtained from two-dimensional gel electrophoresis or chromatography.

Thiophilic adsorption revisited.

Specific and efficient selection of serum immunoglobulins, but not other proteins, on T-gel remains difficult. T-gel capacity was determined for different activation conditions and serum loadings. Mass spectrometry analysis was used to identify the proteins found in the flow-through and in the eluted fractions.

A proteomic approach to investigate potential biomarkers directed against membrane-associated breast cancer proteins.

The identification of specific protein markers for breast cancer would provide the basis for early diagnosis. Particularly, membrane and membrane-associated proteins are rich in targets for antibodies that may constitute suitable biomarkers of carcinogenesis. However, membrane proteins separation using 2-DE remains difficult.

Proteomics in hematologic malignancies.

Basic science research in hematology has been determining the functions of gene products using classical approaches that typically involve studying one or a few genes at a time. Proteomics, defined as the study of protein properties on a large scale, provides tools to globally analyze malignant hematologic cells. A major challenge in cancer therapy is the identification of drugs that kill tumor cells while preserving normal cells.

An efficient proteomics-based approach for the screening of autoantibodies.

This study presents an improved method for the complete transfer of proteins separated by two-dimensional gel electrophoresis to a membrane, specifically designed for the screening and identification of antigens recognized by autoantibodies in patients with breast cancer (BCP) and healthy volunteers. This paper reports the evaluation of this technique using proteins from MCF7 as a source of antigens following 2-DE separation. The appropriate quantity of protein to be loaded on gels (150 microg) has been determined, the aim being a complete and reproducible recovery of all separated proteins onto the polyvinylidene fluoride membrane (2D-blot) after a semi-dry electrotransfer.

Proteome analysis in the study of lymphoma cells.

This review provides an overview on recent studies in the field of proteome analysis of lymphoma cells, and highlights the potentials of such studies for a better knowledge of drug effects at the molecular level. After giving general information on the field of proteome analysis of lymphoma cells, some characteristics of the strategies used during this analysis are pointed out, such as cell extraction strategies and affinity captures. Therefore, the issue of proteome analysis of lymphoma cells content will be covered with respect to those protein extracts that can be prepared in saline solutions, such as cytoplasm proteins, or that are associated with the cell membranes.

Purification of human galectin-1 produced in high-cell density cultures of recombinant Escherichia coli: a comparison with classic shake flask cultivation.

The aim of the present work was to develop a highly productive and simplified process for active human galectin-1 (Gal1) production. Gal1 is a beta-galactoside binding lectin that differentially affects biological and cellular functions such as immune surveillance and apoptosis. These effects have attracted the attention of researchers in cell biology, biochemistry and immunology.

[Proteomic analysis of proteins involved in the renal phenotype in renovascular hypertension].

Renovascular hypertension is characterised by stenosis of the renal artery and high plasma renin levels due to the recruitment of renin-producing cells along the afferent arterioles. This increase in myoepithelioid cells is mainly a result of the differentiation of existing smooth muscle cells with acquisition of a secretory phenotype. To understand the molecular mechanisms involved in this recruitment, we used the model of renovascular hypertension known as the two-kidney, one-clip model in the Lewis rat.

Troponin T as a marker of differentiation revealed by proteomic analysis in renal arterioles.

Renovascular hypertension is characterized by stenosis of the renal artery and high plasma renin levels. The renal phenotype is characterized by high levels of renin in the hypoperfused kidney due to the recruitment of renin-producing cells along the afferent arterioles. This increase in myoepithelioïd cells is due mainly to the differentiation of existing smooth muscle cells with acquisition of a secretory phenotype.

Proteomic analysis of a lymphoma-derived cell line (DG75) following treatment with a demethylating drug: modification of membrane-associated proteins.

5'-azacytidine (AZC) is a potent DNA demethylating agent used clinically for treatment of patients with malignant hemopathies. We have previously shown that AZC induces a halt in cell growth and a decrease of cell activity, without affecting cell viability. We have also shown using proteomics, that 35 polypeptides were differentially expressed in a cytoplasmic fraction.

Subproteomics analysis of phosphorylated proteins: application to the study of B-lymphoblasts from a patient with Scott syndrome.

Proteomics based approaches, which examine the expressed proteins of a tissue or cell type, complement the genome initiatives and are increasingly used to address biomedical questions. Proteins are the main functional output, and post-translational modifications such as phosphorylation are very important in determining protein function. To address this question, we developed a method for specific immunoprecipitation using anti-phosphotyrosine antibodies.

Proteomic map and database of lymphoblastoid proteins.

Advances in genomics have led to the accumulation of an unprecedented amount of data, giving rise to a new field in biochemistry, proteomics. We used a combination of two dimensional gel electrophoresis, analysis and annotation using third-generation software, and mass spectrometry to establish the proteome maps of lymphoblastoid B-cells, a prerequisite for analysis of drug effects and lymphocyte cell diseases. About 1200 protein spots were detected and characterised in terms of their isoelectric point, molecular mass and expression.

Receptor tyrosine phosphatase, CD45 binds galectin-1 but does not mediate its apoptotic signal in T cell lines.

Galectin-1 (Gal-1) is an endogenous mammalian S-type lectin with highly pleiotropic effect on different tissues. The viability of the lymphoid cells is reduced by gal-1 by triggering apoptosis, however, the mechanism of the gal-1 induced apoptosis is still under investigation. The receptor tyrosine phosphatase, CD45, a heavily glycosylated cell surface molecule binds to gal-1 with high affinity, however, its contribution to the gal-1 induced apoptosis is still controversial.

Effect of 5-azacytidine and galectin-1 on growth and differentiation of the human b lymphoma cell line bl36.

BACKGROUND: 5-AzaCytidine (AzaC) is a DNA demethylating drugs that has been shown to inhibit cell growth and to induce apoptosis in certain cancer cells. Induced expression of the galectin1 (Gal1) protein, a galactoside-binding protein distributed widely in immune cells, has been described in cultured hepatoma-derived cells treated with AzaC and this event may have a role in the effect of the drug. According to this hypothesis, we investigated the effect of AzaC and Gal1 on human lymphoid B cells phenotype.