Calmodulin Regulates Intracellular Trafficking of Epidermal Growth Factor Receptor and the MAPK Signaling Pathway

The epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase receptor family involved in signal transduction and the regulation of cellular proliferation and differentiation. It is also a calmodulin-binding protein. To examine the role of calmodulin in the regulation of EGFR, the effect of calmodulin antagonist, W-13, on the intracellular trafficking of EGFR and the MAPK signaling pathway was analyzed. W-13 did not alter the internalization of EGFR but inhibited its recycling and degradation, thus causing the accumulation of EGF and EGFR in enlarged early endosomal structures. In addition, we demonstrated that W-13 stimulated the tyrosine phosphorylation of EGFR and consequent recruitment of Shc adaptor protein with EGFR, presumably through inhibition of the calmodulin-dependent protein kinase II (CaM kinase II). W-13¿mediated EGFR phosphorylation was blocked by metalloprotease inhibitor, BB94, indicating a possible involvement of shedding in this process. However, MAPK activity was decreased by W-13; dissection of this signaling pathway showed that W-13 specifically interferes with Raf-1 activity. These data are consistent with the regulation of EGFR by calmodulin at several steps of the receptor signaling and trafficking pathways.

Estudi del procés de neurodegeneració

Les anomenades malalties neurodegeneratives tenen una simptomatologia i unes manifestacions clíniques molt diferents entre elles. No obstant, totes elles convergeixen en el mateix procés final, la neurodegeneració, que es manifestarà en diferents localitzacions o tipus cel·lulars del sistema nerviós. Nosaltres, plantegem la hipòtesi de que els processos moleculars i cel·lulars subjacents a la neurodegeneració són comuns per totes elles. Després de dur a terme un procés de selecció, es decideix treballar amb la malaltia de Parkinson, la d’Alzheimer, l’Esclerosi lateral amiotròfica i l’esclerosi múltiple. Hem pogut determinar que hi ha set processos moleculars o cel·lulars que estan associats al procés de neurodegeneració i que són comuns a totes elles. Havent-les estudiat per separat s’observa que el procés de neurodegeneració consisteix en una fallada en cadena de diferents sistemes moleculars i cel·lulars que tenen com a punt d’origen l’estrès oxidatiu. A aquest estrès s’hi pot arribar de diferents maneres. Una d’elles és l’exposició excessiva a certs metalls, que provoca la pèrdua dels sistemes antioxidants cel·lulars. Degut a això, els mitocondris reben un impacte oxidatiu massa gran i comencen a fallar. El fet que aquest orgànul actuï com a tampó del calci intracel·lular en provoca la seva desregulació, alterant d’aquesta manera el senyal nerviós. En resposta a l’estrès oxidatiu i tèrmic que genera la disfunció mitocondrial, s’activen les Proteïnes de Xoc Tèrmic (HSP) que actuant de citocines i presentadores d’antígens, inicien la resposta immunològica contra les cèl·lules danyades. Paral·lelament, s’observa un increment de la permeabilitat de la barrera hematoencefàlica degut a la pèrdua de les adhesions cel·lulars estretes per l’alta presència d’espècies reactives. Com a conseqüència de l’afebliment o el trencament de la barrera hematoencefàlica, es pot produir una entrada al SNC de diferents substàncies neurotòxiques i de cèl·lules del sistema immunitàri que, en condicions normals tenen l’accés restringit. Juntament amb aquestes cèl·lules immunològiques, també s’activen les cèl·lules del sistema immunitari innat residents al cervell, la micròglia, i totes elles secreten citocines proinflamatòries que contribueixen al procés de neurodegeneració. Nosaltres presentem els mecanismes pels quals aquesta inflamació, lluny d’atenuar-se, es cronifica per l’acció de certs bucles de retroalimentació positiva. Les diferents peculiaritats de cada malaltia contribueixen en aquest procés de diferents maneres, com és el cas dels pèptids β-amilides en la malaltia d’Alzheimer, l’α-sinucleina en el Parkinson, la superòxid dismutasa (SOD) en l’esclerosi lateral amiotròfica, o l’infiltració de leucòcits al cervell degut a la resposta autoimmune de l’esclerosi múltiple.Deixant de banda aquestes diferències, si el procés és comú entre totes elles, l’estudi a fons d’aquest procés hauria de poder permetre identificar dianes tarapèutiques que siguin comunes per les quatre malalties.

Differential effects of polyphenols and alcohol of red wine on the expression of adhesion molecules and inflammatory cytokines related to atherosclerosis: a randomized clinical trial

Background: Few clinical studies have focused on the alcoholindependent cardiovascular effects of the phenolic compounds of red wine (RW). Objective: We aimed to evaluate the effects of ethanol and phenolic compounds of RW on the expression of inflammatory biomarkers related to atherosclerosis in subjects at high risk of cardiovascular disease. Design: Sixty-seven high-risk, male volunteers were included in a randomized, crossover consumption trial. After a washout period, all subjects received RW (30 g alcohol/d), the equivalent amount of dealcoholized red wine (DRW), or gin (30 g alcohol/d) for 4 wk. Before and after each intervention period, 7 cellular and 18 serum inflammatory biomarkers were evaluated. Results: Alcohol increased IL-10 and decreased macrophage-derived chemokine concentrations, whereas the phenolic compounds of RW decreased serum concentrations of intercellular adhesion molecule- 1, E-selectin, and IL-6 and inhibited the expression of lymphocyte function-associated antigen 1 in T lymphocytes and macrophage-1 receptor, Sialil-Lewis X, and C-C chemokine receptor type 2 expression in monocytes. Both ethanol and phenolic compounds of RW downregulated serum concentrations of CD40 antigen, CD40 ligand, IL-16, monocyte chemotactic protein-1, and vascular cell adhesion molecule-1. Conclusion: The results suggest that the phenolic content of RW may modulate leukocyte adhesion molecules, whereas both ethanol and polyphenols of RW may modulate soluble inflammatory mediators in high-risk patients. The trial was registered in the International Standard Randomized Controlled Trial Number Register at http://www. isrctn.org/ as ISRCTN88720134

Differential effects of polyphenols and alcohol of red wine on the expression of adhesion molecules and inflammatory cytokines related to atherosclerosis: a randomized clinical trial

Background: Few clinical studies have focused on the alcoholindependent cardiovascular effects of the phenolic compounds of red wine (RW). Objective: We aimed to evaluate the effects of ethanol and phenolic compounds of RW on the expression of inflammatory biomarkers related to atherosclerosis in subjects at high risk of cardiovascular disease. Design: Sixty-seven high-risk, male volunteers were included in a randomized, crossover consumption trial. After a washout period, all subjects received RW (30 g alcohol/d), the equivalent amount of dealcoholized red wine (DRW), or gin (30 g alcohol/d) for 4 wk. Before and after each intervention period, 7 cellular and 18 serum inflammatory biomarkers were evaluated. Results: Alcohol increased IL-10 and decreased macrophage-derived chemokine concentrations, whereas the phenolic compounds of RW decreased serum concentrations of intercellular adhesion molecule- 1, E-selectin, and IL-6 and inhibited the expression of lymphocyte function-associated antigen 1 in T lymphocytes and macrophage-1 receptor, Sialil-Lewis X, and C-C chemokine receptor type 2 expression in monocytes. Both ethanol and phenolic compounds of RW downregulated serum concentrations of CD40 antigen, CD40 ligand, IL-16, monocyte chemotactic protein-1, and vascular cell adhesion molecule-1. Conclusion: The results suggest that the phenolic content of RW may modulate leukocyte adhesion molecules, whereas both ethanol and polyphenols of RW may modulate soluble inflammatory mediators in high-risk patients. The trial was registered in the International Standard Randomized Controlled Trial Number Register at http://www. isrctn.org/ as ISRCTN88720134

Lysine-based surfactants in nanovesicle formulations: the role of cationic charge position and hydrophobicity in in vitro cytotoxicity and intracellular delivery

Understanding nanomaterial interactions within cells is of increasing importance for assessing their toxicity and cellular transport. Here, we developed nanovesicles containing bioactive cationic lysine-based amphiphiles, and assessed whether these cationic compounds increase the likelihood of intracellular delivery and modulate toxicity. We found different cytotoxic responses among the formulations, depending on surfactant, cell line and endpoint assayed. The induction of mitochondrial dysfunction, oxidative stress and apoptosis were the general mechanisms underlying cytotoxicity. Fluorescence microscopy analysis demonstrated that nanovesicles were internalized by HeLa cells, and evidenced that their ability to release endocytosed materials into cell cytoplasm depends on the structural parameters of amphiphiles. The cationic charge position and hydrophobicity of surfactants determine the nanovesicle interactions within the cell and, thus, the resulting toxicity and intracellular behavior after cell uptake of the nanomaterial. The insights into some toxicity mechanisms of these new nanomaterials contribute to reducing the uncertainty surrounding their potential health hazards.