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Articles by Mercedes Pardo in JoVE
Other articles by Mercedes Pardo on PubMed
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PST1 and ECM33 Encode Two Yeast Cell Surface GPI Proteins Important for Cell Wall Integrity
Microbiology (Reading, England).
Dec, 2004 |
Pubmed ID: 15583168 Pst1p was previously identified as a protein secreted by yeast regenerating protoplasts, which suggests a role in cell wall construction. ECM33 encodes a protein homologous to Pst1p, and both of them display typical features of GPI-anchored proteins and a characteristic receptor L-domain. Pst1p and Ecm33p are both localized to the cell surface, Pst1p being at the cell membrane and possibly also in the periplasmic space. Here, the characterization of pst1Delta, ecm33Delta and pst1Delta ecm33Delta mutants is described. Deletion of ECM33 leads to a weakened cell wall, and this defect is further aggravated by simultaneous deletion of PST1. As a result, the ecm33Delta mutant displays increased levels of activated Slt2p, the MAP kinase of the cell integrity pathway, and relies on a functional Slt2-mediated cell integrity pathway to ensure viability. Analyses of model glycosylated proteins show glycosylation defects in the ecm33Delta mutant. Ecm33p is also important for proper cell wall ultrastructure organization and, furthermore, for the correct assembly of the mannoprotein outer layer of the cell wall. Pst1p seems to act in the compensatory mechanism activated upon cell wall damage and, in these conditions, may partially substitute for Ecm33p.
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Genetic and Proteomic Evidences Support the Localization of Yeast Enolase in the Cell Surface
Proteomics.
Apr, 2006 |
Pubmed ID: 16544286 Although enolase, other glycolytic enzymes, and a variety of cytoplasmic proteins lacking an N-terminal secretion signal have been widely described as located at the cell surface in yeast and in mammalian cells, their presence in this external location is still controversial. Here, we report that different experimental approaches (genetics, cellular biology and proteomics) show that yeast enolase can reach the cell surface and describe the protein regions involved in its cell surface targeting. Hybrid enolase truncates, fused at their C terminus with the yeast internal invertase or green fluorescent protein (GFP) as reporter proteins, proved that the 169 N-terminal amino acids are sufficient to target the protein to the cell surface. Furthermore, the enolase-GFP fusion co-localized with a plasma membrane marker. Enolase was also identified among membrane proteins obtained by a purification protocol that includes sodium carbonate to prevent cytoplasmic contamination. These proteins were analyzed by SDS-PAGE, trypsin digestion and LC-MS/MS for peptide identification. Elongation factors, mitochondrial membrane proteins and a mannosyltransferase involved in cell wall mannan biosynthesis were also identified in this fraction.
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1,4-Bis(alkylamino)benzo[g]phthalazines Able to Form Dinuclear Complexes of Cu(II) Which As Free Ligands Behave As SOD Inhibitors and Show Efficient in Vitro Activity Against Trypanosoma Cruzi
Bioorganic & Medicinal Chemistry.
Mar, 2007 |
Pubmed ID: 17222558 The synthesis of a new series of 1,4-bis(alkylamino)benzo[g]phthalazines 1-3 is reported, and their ability to form dinuclear complexes with Cu(II) assayed. The geometry of the complexes is dependent on the nature of the electron-donor sites at the sidechains. Compounds 1 and 2, that contain sp3 or sp2 nitrogens at the end of the alkylamino groups, originate monopodal dinuclear complexes which seem to include endogenous OH bridges, and the sidechains seem to actively participate in complexation. However, the substitution of nitrogen by oxygen in 3 leads to a tripodal dinuclear complex in which the sidechains are not involved. The in vitro antiparasitic activity on Trypanosoma cruzi epimastigotes and amastigotes and the SOD activity inhibition have been evaluated for compounds 1-3, and, as expected, 1 and 2 show in all cases relevant results, whereas 3 is always the less active among the three substrates tested.
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[The Halo Sign in Computed Tomography Images: Differential Diagnosis and Correlation with Pathology Findings]
Archivos De Bronconeumologia.
Jul, 2008 |
Pubmed ID: 18727892 The halo sign is a circular area of ground-glass attenuation that is seen around pulmonary nodules at computed tomography (CT). Although the sign is most often an indication of pulmonary hemorrhage, it may also accompany other lesions associated with different disease processes. Examples are hemorrhagic nodules of infectious origin (mucormycosis, candidiasis, tuberculosis, viral pneumonia, and invasive aspergillosis--the last being the most common cause of the CT halo sign); hemorrhagic nodules of noninfectious origin (Wegener granulomatosis, Kaposi sarcoma, and hemorrhagic metastases); tumor cell infiltration (bronchioloalveolar carcinoma, lymphoma, and metastasis with intra-alveolar tumor growth); and nonhemorrhagic lesions (sarcoidosis and organizing pneumonia). Diagnosis must therefore be based on careful consideration of all the CT chest findings within the context of the patient's clinical state. The aim of this review was to describe and illustrate different disease processes that appear as a halo sign on CT scans, to analyze the value of this diagnostic tool, and to assess its correlation with pathology findings.
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Assignment of Protein Interactions from Affinity Purification/mass Spectrometry Data
Journal of Proteome Research.
Mar, 2012 |
Pubmed ID: 22283744 The combination of affinity purification with mass spectrometry analysis has become the method of choice for protein complex characterization. With the improved performance of mass spectrometry technology, the sensitivity of the analyses is increasing, probing deeper into molecular interactions and yielding longer lists of proteins. These identify not only core complex subunits but also the more inaccessible proteins that interact weakly or transiently. Alongside them, contaminant proteins, which are often abundant proteins in the cell, tend to be recovered in affinity experiments because they bind nonspecifically and with low affinity to matrix, tag, and/or antibody. The challenge now lies in discriminating nonspecific binders from true interactors, particularly at the low level and in a larger scale. This review aims to summarize the variety of methods that have been used to distinguish contaminants from specific interactions in the past few years, ranging from manual elimination using heuristic rules to more sophisticated probabilistic scoring approaches. We aim to give awareness on the processing that takes place before an interaction list is reported and on the different types of list curation approaches suited to the different experiments.
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Nuclear Receptor Binding Protein 1 Regulates Intestinal Progenitor Cell Homeostasis and Tumour Formation
The EMBO Journal.
May, 2012 |
Pubmed ID: 22510880 Genetic screens in simple model organisms have identified many of the key components of the conserved signal transduction pathways that are oncogenic when misregulated. Here, we identify H37N21.1 as a gene that regulates vulval induction in let-60(n1046gf), a strain with a gain-of-function mutation in the Caenorhabditis elegans Ras orthologue, and show that somatic deletion of Nrbp1, the mouse orthologue of this gene, results in an intestinal progenitor cell phenotype that leads to profound changes in the proliferation and differentiation of all intestinal cell lineages. We show that Nrbp1 interacts with key components of the ubiquitination machinery and that loss of Nrbp1 in the intestine results in the accumulation of Sall4, a key mediator of stem cell fate, and of Tsc22d2. We also reveal that somatic loss of Nrbp1 results in tumourigenesis, with haematological and intestinal tumours predominating, and that nuclear receptor binding protein 1 (NRBP1) is downregulated in a range of human tumours, where low expression correlates with a poor prognosis. Thus NRBP1 is a conserved regulator of cell fate, that plays an important role in tumour suppression.
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Characterization of Two Distinct Nucleosome Remodeling and Deacetylase (NuRD) Complex Assemblies in Embryonic Stem Cells
Molecular & Cellular Proteomics : MCP.
Mar, 2016 |
Pubmed ID: 26714524 Pluripotency and self-renewal, the defining properties of embryonic stem cells, are brought about by transcriptional programs involving an intricate network of transcription factors and chromatin remodeling complexes. The Nucleosome Remodeling and Deacetylase (NuRD) complex plays a crucial and dynamic role in the regulation of stemness and differentiation. Several NuRD-associated factors have been reported but how they are organized has not been investigated in detail. Here, we have combined affinity purification and blue native polyacrylamide gel electrophoresis followed by protein identification by mass spectrometry and protein correlation profiling to characterize the topology of the NuRD complex. Our data show that in mouse embryonic stem cells the NuRD complex is present as two distinct assemblies of differing topology with different binding partners. Cell cycle regulator Cdk2ap1 and transcription factor Sall4 associate only with the higher mass NuRD assembly. We further establish that only isoform Sall4a, and not Sall4b, associates with NuRD. By contrast, Suz12, a component of the PRC2 Polycomb repressor complex, associates with the lower mass entity. In addition, we identify and validate a novel NuRD-associated protein, Wdr5, a regulatory subunit of the MLL histone methyltransferase complex, which associates with both NuRD entities. Bioinformatic analyses of published target gene sets of these chromatin binding proteins are in agreement with these structural observations. In summary, this study provides an interesting insight into mechanistic aspects of NuRD function in stem cell biology. The relevance of our work has broader implications because of the ubiquitous nature of the NuRD complex. The strategy described here can be more broadly applicable to investigate the topology of the multiple complexes an individual protein can participate in.
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