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M Vidal

Publications and source records attributed to M Vidal.

At least 73 records · Page 4Linked to original sources
PubMed2000

Inhibitors of Ras signal transduction as antitumor agents.

Anarchic cell proliferation, observed in some leukemia and in breast and ovarian cancers, has been related to dysfunctioning of cytoplasmic or receptor tyrosine kinase activities coupled to p21 Ras. The growth factor receptor-bound protein 2 (Grb2) adaptor when complexed with Sos (Son of sevenless), the exchange factor of Ras, conveys the signal induced by tyrosine kinase-activated receptor to Ras by recruiting Sos to the membrane, allowing activation of Ras. This review shows how it is possible to stop the Ras-deregulated signaling pathway to obtain potential antitumor agents. Grb2 protein is comprised of one SH2 surrounded by two SH3 domains and interacts by means of its Src homology (SH2) domain with phosphotyrosine residues of target proteins such as the epidermal growth factor (EGF) receptor or the Shc adaptor. By means of its SH3 domains, Grb2 recognizes proline-rich sequences of Sos, leading to Ras activation. Inhibitors of SH2 and SH3 domains were designed with the aim of interrupting Grb2 recognition. On the one hand, using structural data and molecular modeling, peptide dimers or "peptidimers", made up of two proline-rich sequences from Sos linked by an optimized spacer, were developed. On the other, using the structure of the Grb2 SH2 domain complexed with a phosphotyrosine (pTyr)-containing peptide and molecular modeling studies, a series of N-protected tripeptides containing two phosphotyrosine or mimetic residues, with one pTyr sterically constrained, were devised. These compounds show very high affinities for Grb2 in vitro. They have been targeted into cells showing selective antiproliferative activity on tumor cells. These results suggest that inhibiting SH2 or SH3 domains of signaling proteins might provide antitumor agents.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Yeast two-hybrid systems and protein interaction mapping projects for yeast and worm.

The availability of complete genome sequences necessitates the development of standardized functional assays to analyse the tens of thousands of predicted gene products in high-throughput experimental settings. Such approaches are collectively referred to as 'functional genomics'. One approach to investigate the properties of a proteome of interest is by systematic analysis of protein-protein interactions. So far, the yeast two-hybrid system is the most commonly used method for large-scale, high-throughput identification of potential protein-protein interactions. Here, we discuss several technical features of variants of the two-hybrid systems in light of data recently obtained from different protein interaction mapping projects for the budding yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans.

Animals↗
PubMed2000

Inhibition of the ras-dependent mitogenic pathway by phosphopeptide prodrugs with antiproliferative properties.

Phosphopeptide prodrugs bearing two S-acyl-2-thioethyl (SATE) biolabile phosphate protections were developed. They are capable to inhibit the Shc/Grb2 interaction and MAP kinases (ERK1 and ERK2) phosphorylation in cellular assay. The S-acetyl-2-thioethyl (MeSATE) analogue showed an IC50 of 1 microM in the inhibition of the colony formation of tumor cell line NIH3T3/HER2.

3T3 Cells↗
PubMed2000

Protein interaction mapping in C. elegans using proteins involved in vulval development.

Protein interaction mapping using large-scale two-hybrid analysis has been proposed as a way to functionally annotate large numbers of uncharacterized proteins predicted by complete genome sequences. This approach was examined in Caenorhabditis elegans, starting with 27 proteins involved in vulval development. The resulting map reveals both known and new potential interactions and provides a functional annotation for approximately 100 uncharacterized gene products. A protein interaction mapping project is now feasible for C. elegans on a genome-wide scale and should contribute to the understanding of molecular mechanisms in this organism and in human diseases.

Animals↗
PubMed2000

Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth.

Cyclin-dependent kinase 5 (Cdk5) is a small serine/threonine kinase that plays a pivotal role during development of the CNS. Cables, a novel protein, interacts with Cdk5 in brain lysates. Cables also binds to and is a substrate of the c-Abl tyrosine kinase. Active c-Abl kinase leads to Cdk5 tyrosine phosphorylation, and this phosphorylation is enhanced by Cables. Phosphorylation of Cdk5 by c-Abl occurs on tyrosine 15 (Y15), which is stimulatory for p35/Cdk5 kinase activity. Expression of antisense Cables in primary cortical neurons inhibited neurite outgrowth. Furthermore, expression of active Abl resulted in lengthening of neurites. The data provide evidence for a Cables-mediated interplay between the Cdk5 and c-Abl signaling pathways in the developing nervous system.

Amino Acid Sequence↗
PubMed2000

Optical biosensing of nitrite ions using cytochrome cd1 nitrite reductase encapsulated in a sol-gel matrix.

Nitrite is an important human health and environmental analyte. As such, the European Union (EU) has imposed a limit for nitrite in potable water of 0.1 mg l-1 (2.18 microM). In order to develop an optical biosensing system for the determination of nitrite ions in environmental waters, cytochrome cd1 nitrite reductase has been extracted and purified from the bacterium Paracoccus pantotrophus. The protein has been spectroscopically characterised in solution and important kinetic parameters of nitrite reduction of the cytochrome cd1 enzyme, i.e., Km, Vmax and kcat have been determined. The influence of pH on the activity of the cytochrome cd1 has been investigated and the results suggest that this enzyme can be used for the determination of nitrite in the pH range 6-9. Biosensing experiments with the cytochrome cd1 in solution suggested that the decrease in intensity of the absorption band associated with the d1 haem (which is the nitrite binding site), at 460 nm, with increasing nitrite concentrations would enable the measurement of this analyte with the optimum limit of detection. The cytochrome cd1 has been encapsulated in a bulk sol-gel monolith with no structural changes observed and retention of enzymatic activity. The detection of nitrite ions in the range 0.075-1.250 microM was achieved, with a limit of detection of 0.075 microM. In order to increase the speed of response, a sol-gel sandwich thin film structure was formulated with the cytochrome cd1. This structure enabled the determination of nitrite concentrations within ca. 5 min. The sol-gel sandwich entrapped cytochrome cd1 enzyme was found to be stable for several months when the films were stored at 4 degrees C.

Biosensing Techniques↗
PubMed2000

Exosomes released during reticulocyte maturation bind to fibronectin via integrin alpha4beta1.

Exosomes are vesicles formed in the endosomal compartment and released in the extracellular medium during reticulocyte maturation into erythrocytes. They have a clearing function because of their enrichment with some proteins known to decrease or disappear from the cell surface during maturation, e.g. acetylcholinesterase and transferrin receptor. We show here that integrin alpha4beta1, present on the surface of erythroid precursors but absent from the mature red cell membrane, is at least partly cleared from the reticulocyte plasma membrane by the exosomal pathway. Using flow cytometry, we found that the alpha4 subunit disappears from the reticulocyte surface during in vitro maturation. Two different monoclonal antibodies (B-5G10 and HP 2/1) were used to demonstrate the presence of the alpha4 chain on the exosome surface. Moreover, membrane acetylcholinesterase and lumenal peroxidase-like (i.e. hemoglobin) enzymatic activities were assayed to demonstrate exosome binding to plates coated with increasing fibronectin (FN) concentrations. This interaction was dependent on divalent cations (MnCl2 > MgCl2 > CaCl2). Similarly, vesicles bound to plates coated with the chymotryptic 40 K fragment (FN-40) containing the heparin-binding region of FN. This binding was inhibited by exosome preincubation with fibronectin CS1 peptide and with a monoclonal antibody (HP 2/1) against the integrin alpha4-chain, confirming an alpha4beta1-induced interaction. The importance of the exosome clearance function is highlighted here, since the presence of VLA-4 on reticulocytes often leads to blood circulation complications in some diseases. Moreover, the presence of alpha4beta1 on the exosome surface, by allowing binding to endothelial cells through vascular cell adhesion molecule 1 (VCAM-1), might confer another physiological function to the secreted vesicles.

Animals↗
PubMed2000

Loss- and gain-of-function mutations show a polycomb group function for Ring1A in mice.

The products of the Polycomb group (PcG) of genes act as transcriptional repressors involved in the maintenance of homeotic gene expression patterns throughout development, from flies to mice. Biochemical and molecular evidence suggests that the mouse Ring1A gene is a member of the PcG of genes. However, genetic evidence is needed to establish PcG function for Ring1A, since contrary to all other murine PcG genes, there is no known Drosophila PcG gene encoding a homolog of the Ring1A protein. To study Ring1A function we have generated a mouse line lacking Ring1A and mouse lines overexpressing Ring1A. Both Ring1A(-/-)and Ring1A(+/-) mice show anterior transformations and other abnormalities of the axial skeleton, which indicates an unusual sensitivity of axial skeleton patterning to Ring1A gene dosage. Ectopic expression of Ring1A also results in dose-dependent anterior transformations of vertebral identity, many of which, interestingly, are shared by Ring1A(-/-) mice. In contrast, the alterations of Hox gene expression observed in both type of mutant mice are subtle and involve a reduced number of Hox genes. Taken together, these results provide genetic evidence for a PcG function of the mouse Ring1A gene.

Animals↗
PubMed2000

The Methodus medendi innovation in Giorgio Baglivi's work.

The need to overcome the critical situation in the medical art of his time and to establish a unifying foundation for medicine urged Giorgio Baglivi to write his De praxi medica. His innovative methodus medendi was achieved by reinstating Hippocrates and adopting Francis Bacon's scientific method. A comparison with T. Sydenham's work evidences further aspects of Baglivi's complex method, in which skepticism towards theoretical medicine is overcome and mechanist theories are embraced. In his solidistic work of physiopathology, De fibra motrice et morbosa, Baglivi goes deeper into the matter, applying to medical knowledge the new scientific concepts of Galilei, Bellini, Borelli, Torricelli, Mayow and Boyle.

Biology↗
PubMed1999

Prediction of the impact of the Aznalcóllar toxic spill on the trace element contamination of agricultural soils.

The interaction of several trace elements (Cd, Zn, Cu, Pb, As, Bi, and Tl) was studied by leaching experiments in agricultural soils affected by the Aznalcóllar toxic spill. The spill led to contamination by acid waste waters and sludge deposition. The levels of contamination recorded after the sludge was removed from soils showed that highly contaminated areas remained. A comparison of soils directly affected by sludge deposition and acid waste waters with soils contaminated only by acid waste waters demonstrated that Zn/As and Cd/As ratios were good indicators of the two contributions to the contamination. Soil samples were characterised and grouped according to their texture and carbonate content. The response of elements to single extractions with CaCl2 0.01 mol l-1, CaCl2 1 mol l-1, CH3COOH 0.43 mol l-1, and EDTA 0.05 mol l-1 enabled us to estimate their mobility in the soils. Cd and Zn were found to be the most mobile elements. Cu showed an intermediate mobility, especially in an acidic medium. Pb, As, Bi and Tl were found to be non-mobile elements. A comparison of referent, low and highly contaminated samples showed that the presence of sludge had an effect on desorption yields, in part due to the short-term after the contamination. Calculations of a relative scale of long-term mobility, between soils and trace elements, provided further conclusions derived from the use of single extractions.

Accidents, Occupational↗
PubMed1999

Identification of a family of human F-box proteins.

F-box proteins are an expanding family of eukaryotic proteins characterized by an approximately 40 aminoacid motif, the F box (so named because cyclin F was one of the first proteins in which this motif was identified) [1]. Some F-box proteins have been shown to be critical for the controlled degradation of cellular regulatory proteins [2] [3]. In fact, F-box proteins are one of the four subunits of ubiquitin protein ligases called SCFs. The other three subunits are the Skp1 protein; one of the cullin proteins (Cul1 in metazoans and Cdc53 or Cul A in the yeast Saccharomyces cerevisiae); and the recently identified Roc1 protein (also called Rbx1 or Hrt1). SCF ligases bring ubiquitin conjugating enzymes (either Ubc3 or Ubc4) to substrates that are specifically recruited by the different F-box proteins. The need for high substrate specificity and the large number of known F-box proteins in yeast and worms [2] [4] suggest the existence of a large family of mammalian F-box proteins. Using Skp1 as a bait in a yeast two-hybrid screen and by searching DNA databases, we identified a family of 26 human F-box proteins, 25 of which were novel. Some of these proteins contained WD-40 domains or leucine-rich repeats; others contained either different protein-protein interaction modules or no recognizable motifs. We have named the F-box proteins that contain WD-40 domains Fbws, those containing leucine-rich repeats, Fbls, and the remaining ones Fbxs. We have further characterized representative members of these three classes of F-box proteins.

Amino Acid Motifs↗
PubMed1999

Small peptides containing phosphotyrosine and adjacent alphaMe-phosphotyrosine or its mimetics as highly potent inhibitors of Grb2 SH2 domain.

A series of small peptides with the sequence mAZ-pTyr-Xaa-Asn-NH(2), where Xaa denotes alpha-methylphosphotyrosine or its carboxylic mimetics, were synthesized as inhibitors of the Grb2 SH2 domain. Peptide 3 with (alpha-Me)pTyr as Xaa has the highest affinity for Grb2 (K(d) = 3 +/- 1 nM) and exhibits to date the best inhibitory activity (IC(50) = 11 +/- 1 nM) to displace PSpYVNVQN-Grb2 interaction in an ELISA test. The lower affinities of peptides with (alpha-Me)Tyr, (alpha-Me)Phe(4-CO(2)H), or (alpha-Me)Phe(4-CH(2)CO(2)H) as Xaa demonstrate the importance of a double charged phosphate group at the pY+1 position. Molecular modeling showed additional hydrogen bond interactions provided by the (alpha-Me)pTyr residue with the Grb2 SH2 domain. These results thus show that the effect of hydrophobic pY+1 residues, initially put forth to increased the binding affinities, can be further enhanced by a (-Me)pTyr residue which has both hydrophobic and hydrophilic properties.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Molecular and cellular analysis of Grb2 SH3 domain mutants: interaction with Sos and dynamin.

Quantitative analysis of Grb2/dynamin interaction through plasmon resonance analysis (BIAcore) using Grb2 mutants showed that the high affinity measured between Grb2 and dynamin is essentially mediated by the N-SH3 domain of Grb2. In order to study the interactions between Grb2 and either dynamin or Sos in more detail, Grb2 N-SH3 domains containing different mutations have been analysed. Two mutations were located on the hydrophobic platform binding proline-rich peptides (Y7V and P49L) and one (E40T) located in a region that we had previously shown to be essential for Grb2/dynamin interactions. Through NMR analysis, we have clearly demonstrated that the structure of the P49L mutant is not folded, while the other E40T and Y7V mutants adopt folded structures that are quite similar to that described for the reference domain. Nevertheless, these point mutations were shown to alter the overall stability of these domains by inducing an equilibrium between a folded and an unfolded form. The complex formed between the peptide VPPPVPPRRR, derived from Sos, and the E40T mutant was shown to have the same 3D structure as that described for the wild-type SH3 domain. However, the VPPPVPPRRR peptide adopts a slightly different orientation when it is complexed with the Y7V mutant. Finally, the affinity of the proline-rich peptide GPPPQVPSRPNR, derived from dynamin, for the Grb2 N-SH3 domain was too low to be analyzed by NMR. Thus, the interaction between either Sos or dynamin and the SH3 mutants were tested on a cellular homogenate by means of a far-Western blot analysis. In these conditions, the P49L mutant was shown to be devoid of affinity for Sos as well as for dynamin. The Y7V SH3 mutant displayed a decrease of affinity for both Sos and dynamin, while the E40T mutant exhibited a decrease of affinity only for dynamin. These results support the existence of two binding sites between dynamin and the Grb2 N-SH3 domain.

Adaptor Proteins, Signal Transducing↗
PubMed1999

RYBP, a new repressor protein that interacts with components of the mammalian Polycomb complex, and with the transcription factor YY1.

The products of the Polycomb group (PcG) of genes are necessary for the maintenance of transcriptional repression of a number of important developmental genes, including the homeotic genes. A two-hybrid screen was used to search for putative new members of the PcG of genes in mammals. We have identified a new Zn finger protein, RYBP, which interacts directly with both Ring1 proteins (Ring1A and Ring1B) and with M33, two mutually interacting sets of proteins of the mammalian Polycomb complex. Ring1 binds RYBP and M33 through the same C-terminal domain, whereas the RYBP-M33 interaction takes place through an M33 domain not involved in Ring1 binding. RYBP also interacts directly with YY1, a transcription factor partially related to the product of the Drosophila pleiohomeotic gene. In addition, we show here that RYBP acts as a transcriptional repressor in transiently transfected cells. Finally, RYBP shows a dynamic expression pattern during embryogenesis which initially overlaps partially that of Ring1A in the central nervous system, and later becomes ubiquitous. Taken together, these data suggest that RYBP may play a relevant role in PcG function in mammals.

Amino Acid Sequence↗
PubMed1999

Yeast forward and reverse 'n'-hybrid systems.

Since its original description almost 10 years ago, the yeast two-hybrid system has been used extensively to identify protein-protein interactions from many different organisms. Simultaneously, a number of 'variations on a theme' based on the original concept have been described. In one set of variations, systems were developed to detect other macromolecular interactions: DNA-protein (one-hybrid), RNA-protein (RNA-based three-hybrid) and small molecule-protein interactions (ligand-based three-hybrid). These different versions are collectively referred to here as 'n-hybrid systems'. In another set of variations, the original configuration of the two-hybrid fusion proteins was modified to expand the range of possible protein-protein interactions that could be analyzed. For example, systems were developed to detect trimeric interactions, ligand-receptor interactions or interactions that require particular post-translational modifications. Finally, the original concept was turned upside down and 'reverse n-hybrid systems' were developed to identify mutations, peptides or small molecules that dissociate macromolecular interactions. These reagents can be used to validate, in the relevant biological systems, the potential interactions identified with the 'forward n-hybrid systems'. The powerful genetic selections of the forward and reverse n-hybrid systems are proving useful in proteomic projects aimed at generating macromolecular interaction maps.

Animals↗