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Sérgio Simões

Publications and source records attributed to Sérgio Simões.

15 recordsLinked to original sources
PubMed2006

Compartmentalisation of Rho regulators directs cell invagination during tissue morphogenesis.

During development, small RhoGTPases control the precise cell shape changes and movements that underlie morphogenesis. Their activity must be tightly regulated in time and space, but little is known about how Rho regulators (RhoGEFs and RhoGAPs) perform this function in the embryo. Taking advantage of a new probe that allows the visualisation of small RhoGTPase activity in Drosophila, we present evidence that Rho1 is apically activated and essential for epithelial cell invagination, a common morphogenetic movement during embryogenesis. In the posterior spiracles of the fly embryo, this asymmetric activation is achieved by at least two mechanisms: the apical enrichment of Rho1; and the opposing distribution of Rho activators and inhibitors to distinct compartments of the cell membrane. At least two Rho1 activators, RhoGEF2 and RhoGEF64C are localised apically, whereas the Rho inhibitor RhoGAP Cv-c localises at the basolateral membrane. Furthermore, the mRNA of RhoGEF64C is also apically enriched, depending on signals present within its open reading frame, suggesting that apical transport of RhoGEF mRNA followed by local translation is a mechanism to spatially restrict Rho1 activity during epithelial cell invagination.

Animals↗
PubMed2006

Cellular uptake of S413-PV peptide occurs upon conformational changes induced by peptide-membrane interactions.

In face of accumulated reports demonstrating that uptake of some cell-penetrating peptides occurs through previously described endocytic pathways, or is a consequence of cell fixation artifacts, we conducted a systematic analysis on the mechanism responsible for the cellular uptake of the S4(13)-PV karyophilic cell-penetrating peptide. The results reviewed here show that the S4(13)-PV peptide is able to very efficiently accumulate inside live cells in a rapid, non-toxic and dose-dependent manner, through a mechanism distinct from endocytosis. Comparative analysis of peptide uptake by mutant cells lacking heparan sulfate proteoglycans demonstrates that, although not mandatory, their presence at cell surface facilitates the cellular uptake of the S4(13)-PV peptide. Furthermore, we demonstrate that upon interaction with lipid vesicles, the S4(13)-PV peptide undergoes significant conformational changes that are consistent with the formation of helical structures. Such conformational changes occur concomitantly with a penetration of the peptide into the lipid bilayer, strongly suggesting that the resulting helical structures are crucial for the non-endocytic cellular uptake of the S4(13)-PV peptide. Overall, our data support that, rather than endocytosis, the cellular uptake of the S4(13)-PV cell-penetrating peptide is a consequence of its direct translocation through cell membranes following conformational changes induced by peptide-membrane interactions.

Amino Acid Sequence↗
PubMed2006

Nuclear export of African swine fever virus p37 protein occurs through two distinct pathways and is mediated by three independent signals.

Nucleocytoplasmic shuttling activity of the African swine fever virus p37 protein, a major structural protein of this highly complex virus, has been recently reported. The systematic characterization of the nuclear export ability of this protein constituted the major purpose of the present study. We report that both the N- and C-terminal regions of p37 protein are actively exported from the nucleus to the cytoplasm of yeast and mammalian cells. Moreover, experiments using leptomycin B and small interfering RNAs targeting the CRM1 receptor have demonstrated that the export of p37 protein is mediated by both the CRM1-dependent and CRM1-independent nuclear export pathways. Two signals responsible for the CRM1-mediated nuclear export of p37 protein were identified at the N terminus of the protein, and an additional signal was identified at the C-terminal region, which mediates the CRM1-independent nuclear export. Interestingly, site-directed mutagenesis revealed that hydrophobic amino acids are critical to the function of these three nuclear export signals. Overall, our results demonstrate that two distinct pathways contribute to the strong nuclear export of full-length p37 protein, which is mediated by three independent nuclear export signals. The existence of overlapping nuclear export mechanisms, together with our observation that p37 protein is localized in the nucleus at early stages of infection and exclusively in the cytoplasm at later stages, suggests that the nuclear transport ability of this protein may be critical to the African swine fever virus replication cycle.

Active Transport, Cell Nucleus↗
PubMed2005

On the mechanisms of the internalization of S4(13)-PV cell-penetrating peptide.

Cell-penetrating peptides have been shown to translocate across eukaryotic cell membranes through a temperature-insensitive and energy-independent mechanism that does not involve membrane receptors or transporters. Although cell-penetrating peptides have been successfully used to mediate the intracellular delivery of a wide variety of molecules of pharmacological interest both in vitro and in vivo, the mechanisms by which cellular uptake occurs remain unclear. In the face of recent reports demonstrating that uptake of cell-penetrating peptides occurs through previously described endocytic pathways, or is a consequence of fixation artifacts, we conducted a critical re-evaluation of the mechanism responsible for the cellular uptake of the S4(13)-PV karyophilic cell-penetrating peptide. We report that the S4(13)-PV peptide is able to accumulate inside live cells very efficiently through a rapid, dose-dependent and non-toxic process, providing clear evidence that the cellular uptake of this peptide cannot be attributed to fixation artifacts. Comparative analysis of peptide uptake into mutant cells lacking heparan sulphate proteoglycans demonstrates that their presence at the cell surface facilitates the cellular uptake of the S4(13)-PV peptide, particularly at low peptide concentrations. Most importantly, our results clearly demonstrate that, in addition to endocytosis, which is only evident at low peptide concentrations, the efficient cellular uptake of the S4(13)-PV cell-penetrating peptide occurs mainly through an alternative, non-endocytic mechanism, most likely involving direct penetration across cell membranes.

Amino Acid Sequence↗
PubMed2005

Targeting of sterically stabilised pH-sensitive liposomes to human T-leukaemia cells.

The main aim of this work was to develop novel targeted sterically stabilised pH-sensitive liposomes tailored to promote efficient intracellular delivery of therapeutic molecules into human T-leukaemia cells. Our results indicate that the targeting moiety (thiolated transferrin) was successfully coupled to the distal reactive maleimide terminus of poly(ethylene glycol)-phospholipid conjugates incorporated in the liposomal bilayer. Results from atomic force microscopy studies, performed to characterise vesicle surface topology, indicated that, to a certain extent, thiolated transferrin has the ability to associate in a non-specific manner with the lipid membrane of pegylated liposomes. This is an issue not commonly reported in the literature but which is crucial to demonstrate the targeting proof of principle. Nevertheless, fluorimetric studies together with confocal microscopy clearly demonstrate that liposomes bearing covalently coupled transferrin associate more extensively to human T-leukaemia cells in vitro than non-targeted liposomes. Cell mechanistic studies indicate that targeted liposomes bind specifically to transferrin receptors and are internalised via receptor-dependent endocytotic pathway. In addition, the biophysical features exhibited by the developed liposomes, namely their ability to promote pH-triggered cytoplasmic delivery of loaded material, make them promising delivery systems for in vivo targeting of therapeutic molecules to tumours.

Cell Line, Tumor↗
PubMed2005

Cationic liposomes for gene delivery.

Cationic liposome-DNA complexes (lipoplexes) constitute a potentially viable alternative to viral vectors for the delivery of therapeutic genes. This review will focus on various parameters governing lipoplex biological activity, from their mode of formation to in vivo behaviour. Particular emphasis is given to the mechanism of interaction of lipoplexes with cells, in an attempt to dissect the different barriers that need to be surpassed for efficient gene expression to occur. Aspects related to new trends in the formulation of lipid-based gene delivery systems aiming at overcoming some of their limitations will be covered. Finally, examples illustrating the potential of cationic liposomes in clinical applications will be provided.

Animals↗
PubMed2004

On the formulation of pH-sensitive liposomes with long circulation times.

Strategies used to enhance liposome-mediated drug delivery in vivo include the enhancement of stability and circulation time in the bloodstream, targeting to specific tissues or cells, and facilitation of intracytoplasmic delivery. pH-sensitive liposomes have been developed to mediate the introduction of highly hydrophilic molecules or macromolecules into the cytoplasm. These liposomes destabilize under acidic conditions found in the endocytotic pathway, and usually contain phosphatidylethanolamine (PE) and titratable stabilizing amphiphiles. Formulations without PE have also been developed. Encapsulated compounds are thought to be transported into the cytoplasm through destabilization of or fusion with the endosome membrane. Incorporation of a low mole percentage of poly(ethylene glycol) (PEG)-conjugated lipids into pH-sensitive liposomes confers prolonged circulation times to these liposomes, which are otherwise cleared rapidly. While the incorporation of PEG-lipids reduces the pH-dependent release of encapsulated fluorescent markers in vitro, it does not hinder the cytoplasmic delivery of the markers per cell-associated liposome. This suggests that intracellular delivery is not dictated simply by the destabilization of the liposomes. Antibodies or ligands to cell surface receptors can be coupled to pH-sensitive or sterically stabilized pH-sensitive liposomes for targeting. pH-sensitive liposomes have been used to deliver anticancer drugs, antibiotics, antisense oligonucleotides, ribozymes, plasmids, proteins and peptides to cells in culture or in vivo.

Animals↗
PubMed2004

Improving lipoplex-mediated gene transfer into C6 glioma cells and primary neurons.

The development of methodologies for gene transfer into the central nervous system is crucial for gene therapy of neurological disorders. In this study, different cationic liposome formulations were used to transfer DNA into C6 glioma cells and primary hippocampal and cortical neurons by varying the nature of the helper lipid (DOPE, Chol) or a mixture of DOPE and cholesterol (Chol) associated to DOTAP. In addition, the effect of the lipid/DNA (+/-) charge ratio, the association of the ligand transferrin to the lipoplexes, and the stage of differentiation of the primary cells on the levels of transfection activity, transfection efficiency, and duration of gene expression were evaluated. Mechanistic studies were also performed to investigate the route of delivery of the complexes into neurons. Our results indicate that DOTAP:Chol (1:1 mol ratio) was the best formulation to transfer a reporter gene into C6 glioma cells, primary hippocampal neurons, and primary cortical neurons. The use of transferrin-associated lipoplexes resulted in a significant enhancement of transfection activity, as compared to plain lipoplexes, which can be partially attributed to the promotion of their internalization mediated by transferrin. While for hippocampal neurons the levels of luciferase gene expression are very low, for primary cortical neurons the levels of transgene expression are high and relatively stable, although only 4% of the cells has been transfected. The stage of cell differentiation revealed to be critical to the levels of gene expression. Consistent with previous findings on the mechanisms of cell internalization, the experiments with inhibitors of the endocytotic pathway clearly indicate that transferrin-associated lipoplexes are internalized into primary neurons by endocytosis. Promising results were obtained in terms of the levels and duration of gene expression, particularly in cortical neurons when transfected with the Tf-associated lipoplexes, this finding suggesting the usefulness of these lipid-based carriers to deliver genes within the CNS.

Animals↗
PubMed2003

Notch and lymphopoiesis: a view from the microenvironment.

The differentiation of B- and T-cells in primary lymphoid organs depends on, or is strongly influenced by, signals provided by stromal cells, extracellular matrix components as well as by direct contacts between differentiating lymphocytes and distinct environmental cells. Notch receptors and their ligands mediate intercellular contacts and are crucially important for the development of T- and B-cell lineages. Here we start by reviewing current knowledge on the expression patterns of Notch receptors and their ligands in primary lymphoid organs and the effects induced by their functional interactions. Then we shall attempt to discuss how those interactions may regulate not only lymphopoiesis per se but also morphogenesis and the functional compartmentalization of lymphopoietic organs during development.

Animals↗
PubMed2003

Cationic liposomes for gene delivery: novel cationic lipids and enhancement by proteins and peptides.

Cationic liposome-DNA complexes, also called "lipoplexes", constitute a potentially viable alternative to viral vectors for the delivery of therapeutic genes. Here we review the mechanisms of lipoplex-mediated gene delivery, the barriers to efficient gene expression, and novel cationic lipids used for transfection. We also describe methods for enhancing gene transfer via the use of proteins, including transferrin, albumin and asialofetuin, and synthetic peptides, including GALA and nuclear localization signal peptides. We underscore the importance of understanding the mechanisms of cytoplasmic and nuclear entry of DNA and its dissociation from lipoplexes. We emphasize that the in vitro transfection activity of new lipoplex constructs should be tested in the presence of high serum concentrations to emulate in vivo conditions.

Animals↗
PubMed2003

Technology evaluation: LErafAON, NeoPharm.

NeoPharm is developing liposome-encapsulated, c-Raf antisense oligodeoxynucleotides (LErafAON) for the potential treatment of various solid tumors, including those that have become resistant to radiation or chemotherapy. Phase I/II trials commenced in March 2001 and were ongoing as of June 2003.

Animals↗
PubMed2002

Paclitaxel-loaded PLGA nanoparticles: preparation, physicochemical characterization and in vitro anti-tumoral activity.

The main objective of this study was to develop a polymeric drug delivery system for paclitaxel, intended to be intravenously administered, capable of improving the therapeutic index of the drug and devoid of the adverse effects of Cremophor EL. To achieve this goal paclitaxel (Ptx)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (Ptx-PLGA-Nps) were prepared by the interfacial deposition method. The influence of different experimental parameters on the incorporation efficiency of paclitaxel in the nanoparticles was evaluated. Our results demonstrate that the incorporation efficiency of paclitaxel in nanoparticles was mostly affected by the method of preparation of the organic phase and also by the organic phase/aqueous phase ratio. Our data indicate that the methodology of preparation allowed the formation of spherical nanometric (<200 nm), homogeneous and negatively charged particles which are suitable for intravenous administration. The release behaviour of paclitaxel from the developed Nps exhibited a biphasic pattern characterised by an initial fast release during the first 24 h, followed by a slower and continuous release. The in vitro anti-tumoral activity of Ptx-PLGA-Nps developed in this work was assessed using a human small cell lung cancer cell line (NCI-H69 SCLC) and compared to the in vitro anti-tumoral activity of the commercial formulation Taxol. The influence of Cremophor EL on cell viability was also investigated. Exposure of NCI-H69 cells to 25 microg/ml Taxol resulted in a steep decrease in cell viability. Our results demonstrate that incorporation of Ptx in nanoparticles strongly enhances the cytotoxic effect of the drug as compared to Taxol, this effect being more relevant for prolonged incubation times.

Chemical Phenomena↗