Facultat de Farmàcia
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Cocoa flavanol metabolites activate HNF-3 beta , Sp1, and NFY-mediated transcription of apolipoprotein AI in human cells

Carlota Oleaga, Carlos J. Ciudad, Maria Izquierdo-Pulido and Véronique Noé *

* Departament de Bioquímica i Biologia Molecular.

Abstract

Scope: To identify the mechanisms by which cocoa induces HDL levels and since apolipoprotein AI (ApoAI) is the major protein in HDLs, we analyzed, upon incubation with cocoa metabolites, ApoAI mRNA levels, its transcriptional regulation, and the levels of the transcription factors involved in this process.

Methods and results: Epicatechin and cocoa metabolites caused an increase in ApoAI expression in HepG2 cells. Electrophoretic mobility shift assays revealed the involvement of Sites A and B of the ApoAI promoter in the induction of ApoAI mRNA upon incubation with cocoa metabolites. Using supershift assays, we demonstrated the binding of HNF-3beta, HNF-4, ER-alpha, and RXR-alpha to Site A and the binding of HNF-3beta, NFY, and Sp1 to Site B. Luciferase assays performed with a construct containing Site B confirmed its role in the upregulation of ApoAI by cocoa metabolites. Incubation with 3-methyl-epicatechin led to an increase in HNF-3beta mRNA, HNF-3beta, ER-alpha, Sp1, and NFY protein levels and the activation of ApoAI transcriptionmediated by NFY, Sp1, and ER-alpha.

Conclusion: The activation of ApoAI transcription through Site B by cocoa flavanol metabolites is mainly mediated by an increase in HNF-3beta, with a significant contribution of Sp1 and NFY, as a mechanism for the protective role of these compounds in cardiovascular diseases.

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PI3 k/akt inhibition induces apoptosis through p38 activation in neurons  

Aurelio Vázquez de la Torre, Felix Junyent, Jaume Folch, Carme Pelegrí, Jordi Vilaplana, Carme Auladell, Carlos Beas-Zarate, Mercè Pallàs, Ester Verdaguer, Antoni Camins *

* Departament de Farmacologia i Química Terapèutica, Unitat de Farmacologia i Farmacognòsia.

Abstract

Accumulating evidence suggests that the PI3 K/AKT pathway is a pro-survival signalling system in neurons. Therefore, the inhibition of this pathway may be implicated in the degeneration of neurons in Parkinson's disease (PD), Alzheimer's disease (AD), and other neurological disorders. Here we study the participation of the mitogen-activated protein kinase (MAPK) pathway on apoptosis induced by PI3 K/AKT inhibition in cultured cerebellar granule cells (CGCs).

LY294002, a specific PI3 K/AKT inhibitor, selectively activated the p38 MAPK kinase pathway and enhanced c-Jun phosphorylation, but did not activate JNK. The pharmacological inhibitors SB203580 (p38 inhibitor) and SP600125 (a JNK inhibitor) protected primary cultures of rat CGCs from LY294002-induced apoptosis. Furthermore, both compounds decreased the phosphorylation of c-Jun and lowered mRNA levels of the pro-apoptotic gene dp5, a direct target of c-Jun. Taken together, our data demonstrate that PI3 K/AKT inhibition induces neuronal apoptosis, a process that is mediated by the activation of p38MAPK/c-Jun/dp5.

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Assessment of the safety of maslinic acid, a bioactive compound from Olea europaea L.  

Marta Sánchez-González, Glòria Lozano-Mena, M. Emília Juan, Andrés García-Granados and Joana M. Planas *

* Departament de Fisiologia.

Abstract

Scope: Maslinic acid, the main pentacyclic triterpene of the cuticle of Olea europaea L. fruit, has multiple beneficial effects on health, most notably antitumor and hypoglycemic properties. Notwithstanding the biological activities, there is a lack of knowledge about its safety. Therefore, the purpose of this study was to evaluate whether high doses of maslinic acid have harmful effects on Swiss CD-1 male mice.

Methods and results: The single oral administration of the pentacyclic triterpene at 1000 mg/kg to mice did not produce any signs of morbidity or mortality. The repeated daily oral administration of 50 mg/kg of maslinic acid for 28 days did not induce any sign of toxicity during the experimental period. Body weight did not differ between mice that received the triterpene and the control group. Similarly, hematological and biochemical variables were not affected by the treatment. Histopathologic examination of the organs revealed that there were no differences between the control and the treated mice.

Conclusion: Taken together the results obtained from the acute and the repeated intake of maslinic acid indicate that the compound does not exert any adverse effects on the variables tested in mice, thus suggesting a sufficient margin of safety for its putative use as a nutraceutical.

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Oleate prevents saturated-fatty-acid-induced ER stress, inflammation and insulin resistance in skeletal muscle cells through an AMPK-dependent mechanism 

L. Salvadó, T. Coll, A. M. Gómez-Foix, E. Salmerón, E. Barroso, X. Palomer, and M. Vázquez-Carrera *

* Departament de Farmacologia i Química Terapèutica, Unitat de Farmacologia i Farmacognòsia.

Abstract

Aims/hypothesis : Although the substitution of saturated fatty acids with oleate has been recommended in the management of type 2 diabetes mellitus, the mechanisms by which oleate improves insulin resistance in skeletal muscle cells are not completely known. Here, we examined whether oleate, through activation of AMP-activated protein kinase (AMPK), prevented palmitate-induced endoplasmic reticulum (ER) stress, which is involved in the link between lipid-induced inflammation and insulin resistance.

Methods: Studies were conducted in mouse C2C12 myotubes and in the human myogenic cell line LHCN-M2. To analyse the involvement of AMPK, activators and inhibitors of this kinase and overexpression of a dominant negative AMPK construct (K45R) were used.

Results: Palmitate increased the levels of ER stress markers, whereas oleate did not. In palmitate-exposed cells incubated with a lower concentration of oleate, the effects of palmitate were prevented. The induction of ER stress markers by palmitate was prevented by the presence of the AMPK activators AICAR and A-769662. Moreover, the ability of oleate to prevent palmitate-induced ER stress and inflammation (nuclear factor-kappa B [NF-?B] DNA-binding activity and expression and secretion of IL6) as well as insulin-stimulated Akt phosphorylation and 2-deoxyglucose uptake was reversed in the presence of the AMPK inhibitor compound C or by overexpression of a dominant negative AMPK construct. Finally, palmitate reduced phospho-AMPK levels, whereas this was not observed in oleate-exposed cells or in palmitate-exposed cells supplemented with oleate.

Conclusions/interpretation : Overall, these findings indicate that oleate prevents ER stress, inflammation and insulin resistance in palmitate-exposed skeletal muscle cells by activating AMPK.

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DNA delivery via cationic solid lipid nanoparticles (SLNs)  

Carolina Carrillo,* Noemí Sánchez-Hernández, Encarna García-Montoya, Pilar Pérez-Lozano, Josep M. Suñé-Negre, Josep R. Ticó, Carlos Suñé, Montserrat Miñarro

* Departament de Farmàcia i Tecnologia Farmacèutica, Unitat de Tecnologia Farmacèutica.

Abstract

In recent years the use of solid lipid nanoparticles (SLNs) as transport systems for the delivery of drugs and biomolecules has become particularly important. The use of cationic SLNs developed by the technique of microemulsion, which are complexed with DNA in order to study their application as non-viral vectors in gene therapy, is reported. The nanoparticles are characterized by scanning electron microscopy and transmission electron microscopy (SEM and TEM), atomic force microscopy (AFM) and differential scanning calorimetry (DSC). Furthermore, the process of lyophilization of the samples and their stability was studied. The nanoparticles obtained presented a particle size of 340 nm with a positive surface charge of 44 mV and the capability of forming lipoplexes with DNA plasmids was stated.

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First diastereoselective [3 + 2] cycloaddition reaction of diethyl isocyanomethylphosphonate and maleimides  

Carlos Arróniz, Juan Molina, Sonia Abás, Elies Molins, Josep M. Campanera, F. Javier Luque, and Carmen Escolano*

* Departament de Farmacologia i Química Terapèutica, Unitat de Química Orgànica.

Abstract

Bicyclic alpha-iminophosphonates were prepared via the first diastereoselective silver catalyzed [3 + 2] cycloaddition reaction of diethyl isocyanomethylphosphonate and diversely N-substituted maleimides. The reduction of the resulting imine by catalytic hydrogenation led to cyclic alpha-aminophosphonates, which are alpha-aminoester surrogates. The relative stereochemistry of the adducts was confirmed by X-ray crystallographic analysis of 4. The diastereoselectivity of the cycloaddition reaction was rationalised by theoretical studies.

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In vitro antitumor activity of methotrexate via pH-sensitive chitosan nanoparticles  

Daniele Rubert Nogueira, Lorena Tavano, Montserrat Mitjans, Lourdes Pérez, M. Rosa Infante, M. Pilar Vinardell*

* Departament de Fisiologia.

Abstract

Nanoparticles with pH-sensitive behavior may enhance the success of chemotherapy in many cancers by efficient intracellular drug delivery. Here, we investigated the effect of a bioactive surfactant with pH-sensitive properties on the antitumor activity and intracellular behavior of methotrexate-loaded chitosan nanoparticles (MTX-CS-NPs). NPs were prepared using a modified ionotropic complexation process, in which was included the surfactant derived from N-alpha,N-epsylon-dioctanoyl lysine with an inorganic lithium counterion. The pH-sensitive behavior of NPs allowed accelerated release of MTX in an acidic medium, as well as membrane-lytic pH-dependent activity, which facilitated the cytosolic delivery of endocytosed materials. Moreover, our results clearly proved that MTX-CS-NPs were more active against the tumor HeLa and MCF-7 cell lines than the free drug. The feasibilty of using NPs to target acidic tumor extracellular pH was also shown, as cytotoxicity against cancer cells was greater in a mildly acidic environment. Finally, the combined physicochemical and pH-sensitive properties of NPs generally allowed the entrapped drug to induce greater cell cycle arrest and apoptotic effects. Therefore, our overall results suggest that pH-sensitive MTX-CS-NPs could be potentially useful as a carrier system for tumor and intracellular drug delivery in cancer therapy.

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Primary prevention of cardiovascular disease with a mediterranean diet.

Ramón Estruch, Emilio Ros, Jordi Salas-Salvadó, Maria-Isabel Covas, Dolores Corella, Fernando Arós, Enrique Gómez-Gracia, Valentina Ruiz-Gutiérrez, Miquel Fiol, José Lapetra, Rosa Maria Lamuela-Raventos,* Lluís Serra-Majem, Xavier Pintó, Josep Basora, Miguel Angel Muñoz, José V. Sorlí, José Alfredo Martínez, and Miguel Angel Martínez-González, for the PREDIMED Study Investigators

* Departament de Nutrició i Bromatologia.

Abstract

Background: Observational cohort studies and a secondary prevention trial have shown an inverse association between adherence to the Mediterranean diet and cardiovascular risk. We conducted a randomized trial of this diet pattern for the primary prevention of cardiovascular events.

Methods: In a multicenter trial in Spain, we randomly assigned participants who were at high cardiovascular risk, but with no cardiovascular disease at enrollment, to one of three diets: a Mediterranean diet supplemented with extra-virgin olive oil, a Mediterranean diet supplemented with mixed nuts, or a control diet (advice to reduce dietary fat). Participants received quarterly individual and group educational sessions and, depending on group assignment, free provision of extra-virgin olive oil, mixed nuts, or small nonfood gifts. The primary end point was the rate of major cardiovascular events (myocardial infarction, stroke, or death from cardiovascular causes). On the basis of the results of an interim analysis, the trial was stopped after a median follow-up of 4.8 years.

Results: A total of 7447 persons were enrolled (age range, 55 to 80 years); 57% were women. The two Mediterranean-diet groups had good adherence to the intervention, according to self-reported intake and biomarker analyses. A primary end-point event occurred in 288 participants. The multivariable-adjusted hazard ratios were 0.70 (95% confidence interval [CI], 0.54 to 0.92) and 0.72 (95% CI, 0.54 to 0.96) for the group assigned to a Mediterranean diet with extra-virgin olive oil (96 events) and the group assigned to a Mediterranean diet with nuts (83 events), respectively, versus the control group (109 events). No diet-related adverse effects were reported.

Conclusions: Among persons at high cardiovascular risk, a Mediterranean diet supplemented with extra-virgin olive oil or nuts reduced the incidence of major cardiovascular events. (Funded by the Spanish government's Instituto de Salud Carlos III and others; Controlled-Trials.com number, ISRCTN35739639.).

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Fsp27/CIDEC is a CREB target gene induced during early fasting in liver and regulated by FA oxidation rate 

Anna Vilà-Brau, Ana Luísa De Sousa-Coelho, Joana F. Gonçalves, Diego Haro, and Pedro F. Marrero*

* Departament de Bioquímica i Biologia Molecular.

Abstract

FSP27 [cell death-inducing DFFA-like effector c (CIDEC) in humans] is a protein associated with lipid droplets that downregulates the fatty acid oxidation (FAO) rate when it is overexpressed. However, little is known about its physiological role in liver. Here, we show that fasting regulates liver expression of Fsp27 in a time-dependent manner. Thus, during the initial stages of fasting, a maximal induction of 800-fold was achieved, whereas during the later phase of fasting, Fsp27 expression decreased. The early response to fasting can be explained by a canonical PKA-CREB-CRTC2 signaling pathway because: i ) CIDEC expression was induced by forskolin, ii ) Fsp27 promoter activity was increased by CREB, and iii ) Fsp27 expression was upregulated in the liver of Sirt1 knockout animals. Interestingly, pharmacological (etomoxir) or genetic ( Hmgcs2 interference) inhibition of the FAO rate increases the in vivo expression of Fsp27 during fasting. Similarly, CIDEC expression was upregulated in HepG2 cells by either etomoxir or HMGCS2 interference. Our data indicate that there is a kinetic mechanism of autoregulation between short- and long-term fasting, by which free FAs delivered to the liver during early fasting are accumulated/exported by FSP27/CIDEC, whereas over longer periods of fasting, they are degraded in the mitochondria through the carnitine palmitoyl transferase system.

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Dietary resveratrol prevents Alzheimer's markers and increases life span in SAMP8  

David Porquet, Gemma Casadesús, Sergi Bayod, Alberto Vicente, Anna M. Canudas, Jordi Vilaplana, Carme Pelegrí, Coral Sanfeliu, Antoni Camins, Mercè Pallàs,* Jaume del Valle

* Departament de Farmacologia i Química Terapèutica, Unitat de Farmacologia i Farmacognòsia.

Abstract

Resveratrol is a polyphenol that is mainly found in grapes and red wine and has been reported to be a caloric restriction (CR) mimetic driven by Sirtuin 1 (SIRT1) activation. Resveratrol increases metabolic rate, insulin sensitivity, mitochondrial biogenesis and physical endurance, and reduces fat accumulation in mice. In addition, resveratrol may be a powerful agent to prevent age-associated neurodegeneration and to improve cognitive deficits in Alzheimer ' s disease (AD). Moreover, different findings support the view that longevity in mice could be promoted by CR. In this study, we examined the role of dietary resveratrol in SAMP8 mice, a model of age-related AD. We found that resveratrol supplements increased mean life expectancy and maximal life span in SAMP8 and in their control, the related strain SAMR1. In addition, we examined the resveratrol-mediated neuroprotective effects on several specific hallmarks of AD. We found that longterm dietary resveratrol activates AMPK pathways and pro-survival routes such as SIRT1 in vivo. It also reduces cognitive impairment and has a neuroprotective role, decreasing the amyloid burden and reducing tau hyperphosphorylation.

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The ‘paradigm of extremes': extremely low genetic diversity in an extremely narrow endemic species, Coristospermum huteri (Umbelliferae)

Jordi López-Pujol,* M. Carmen Martinell, Sergi Massó, Cèsar Blanché, Llorenç Sáez

* Departament de Productes Naturals, Biologia Vegetal i Edafologia, Unitat de Botànica.

Abstract

Low levels of genetic diversity in endemic species are generally attributable to the small size of their populations. This lack of genetic variability will, predictably, be more evident in those species that occur in only one or a very few localities with a total population consisting of a few dozen individuals, or sometimes fewer (i.e. ‘extremely narrow endemics', ENEs). We used allozyme electrophoresis to survey the genetic variability of Coristospermum huteri , an endemic species from the island of Majorca (Balearic Islands, W. Mediterranean Basin) with a single natural population of about 100 individuals. As expected, allozyme variability was virtually nil for this species ( P = 8.3 %, A = 1.08, He = 0.022), which seems to be a general rule for ENEs (mean He = 0.057). A founder effect associated with a dispersal event from the continent is probably behind the lack of genetic diversity in this highly threatened species. Preservation of the mountain summit where the plant is found (Puig Major) is essential for the survival of C. huteri , and would also guarantee the conservation of other ENEs and rare and threatened species.

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Evidence for a new binding mode to GSK-3: Allosteric regulation by the marine compound palinurin

Axel Bidon-Chanal,* Ana Fuertes, Diana Alonso, Daniel I. Pérez, Ana Martínez, F. Javier Luque, Miguel Medina

* Departament de Fisicoquímica

Abstract

Glycogen synthase kinase 3beta (GSK-3beta) is widely recognised as a relevant player in the pathogenesis of several highly prevalent disorders such as Alzheimer's disease, mood disorders, diabetes and cancer. Therefore, this enzyme constitutes a highly attractive therapeutic target for the development of selective inhibitors as new promising drugs for the treatment of these pathologies. We describe here the isolation and biochemical characterization of the marine natural sesquiterpene palinurin as a GSK-3beta inhibitor. Experimental studies performed for characterizing the inhibitory mechanism indicate that GSK-3beta inhibition by palinurin cannot be competed out by ATP nor peptide substrate. Molecular modelling techniques have enabled us to propose an unconventional binding mode to GSK-3beta. Moreover, molecular dynamics simulations have identi?ed an allosteric mechanism by which binding of palinurin leads to GSK-3beta inhibition. The inhibitory activities determined for a series of structurally related analogues support the proposed binding mode of palinurin, which is the ?rst compound described to target this allosteric site. The results offer new opportunities for designing and developing selective inhibitors with novel mechanisms of action.

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Atomic force microscopy: A versatile tool to probe the physical and chemical properties of supported membranes at the nanoscale  

Laura Picas, Pierre-Emmanuel Milhiet, Jordi Hernández-Borrell *

* Departament de Fisicoquímica.

Abstract

Atomic force microscopy (AFM) was developed in the 1980s following the invention of its precursor, scanning tunneling microscopy (STM), earlier in the decade. Several modes of operation have evolved, demonstrating the extreme versatility of this method for measuring the physicochemical properties of samples at the nanoscopic scale. AFM has proved an invaluable technique for visualizing the topographic characteristics of phospholipid monolayers and bilayers, such as roughness, height or laterally segregated domains. Implemented modes such as phase imaging have also provided criteria for discriminating the viscoelastic properties of different supported lipid bilayer (SLB) regions. In this review, we focus on the AFM force spectroscopy (FS) mode, which enables determination of the nanomechanical properties of membrane models. The interpretation of force curves is presented, together with newly emerging techniques that provide complementary information on physicochemical properties that may contribute to our understanding of the structure and function of biomembranes. Since AFM is an imaging technique, some basic indications on how real-time AFM imaging is evolving are also presented at the end of this paper.

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Establishment of an in vitro photoallergy test using NCTC2544 cells and IL-18 production  

Valentina Galbiati, Verónica Martínez, Sara Bianchi, Montserrat Mitjans,* Emanuela Corsini

* Departament de Fisiologia

Abstract

Differentiation between photoallergenic and phototoxic reactions induced by low molecular weight compounds represents a current problem. The use of keratinocytes as a potential tool for the detection of photoallergens as opposed to photoirritants is considered an interesting strategy for developing in vitro methods. We have previously demonstrated the possibility to use the human keratinocyte cell line NCTC2455 and the production of interleukin-18 (IL-18) to screen low molecular weight sensitizers. The purpose of this work was to explore the possibility to use the NCTC2544 assay to identify photoallergens and discriminate from phototoxic chemicals.

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Synthesis and anti-influenza A virus activity of 2,2-dialkylamantadines and related compounds

Eva Torres, Roser Fernández, Stéphanie Miquet, Merce Font-Bardia, Evelien Vanderlinden, Lieve Naesens, and Santiago Vázquez*

* Departament de Farmacologia i Química Terapèutica, Unitat de Química Farmacèutica.

Abstract

The M2 channel protein on the virus membrane of influenza A is the main target of adamantane-based drugs, the most successful of which are M2 ion channel blockers amantadine and rimantadine. However, most of the circulating strains are resistant or rapidly acquire resistance by mutating the M2 protein. Accordingly, novel anti-influenza virus drugs are urgently needed.

Here, Torres et al. (DOI: 10.1021/ml300279b) report the synthesis of new amantadine analogues using a novel synthetic approach. Several of the new compounds display low micromolar activity against the influenza A/H1N1 virus subtype while not being cytotoxic. The results suggest that these compounds are not targeting the M2 protein of the virus, as amantadine does, but acting through a different mechanism of action. This new approach to 2,2-dialkyl-1-aminoadamantanes could find a broad application in synthetic and medicinal chemistry.

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New cationic nanovesicular systems containing lysine-based surfactants for topical administration: Toxicity assessment using representative skin cell lines

Daniele Rubert Nogueira, M. Carmen Morán, Montserrat Mitjans, Verónica Martínez, Lourdes Pérez, M. Pilar Vinardell *

* Departament de Fisiologia

Abstract

Cationic nanovesicles have attracted considerable interest as effective carriers to improve the delivery of biologically active molecules into and through the skin. In this study, lipid-based nanovesicles containing three different cationic lysine-based surfactants were designed for topical administration. We used representative skin cell lines and in vitro assays to assess whether the cationic compounds modulate the toxic responses of these nanocarriers. The nanovesicles were characterized in both water and cell culture medium. In general, significant agglomeration occurred after 24 h incubation under cell culture conditions. We found different cytotoxic responses among the formulations, which depended on the surfactant, cell line (3T3, HaCaT, and THP-1) and endpoint assayed (MTT, NRU, and LDH). Moreover, no potential phototoxicity was detected in fibroblast or keratinocyte cells, whereas only a slight inflammatory response was induced, as detected by IL-1alpha and IL-8 production in HaCaT and THP-1 cell lines, respectively. A key finding of our research was that the cationic charge position and the alkyl chain length of the surfactants determine the nanovesicles resulting toxicity. The charge on the alpha-amino group of lysine increased the depletion of cell metabolic activity, as determined by the MTT assay, while a higher hydrophobicity tends to enhance the toxic responses of the nanovesicles. The insights provided here using different cell lines and assays offer a comprehensive toxicological evaluation of this group of new nanomaterials.

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Determination of maslinic acid, a pentacyclic triterpene from olives, in rat plasma by high-performance liquid chromatography

  Glòria Lozano-Mena, M. Emília Juan, Andrés García-Granados, and Joana M. Planas*

* Departament de Fisiologia

Abstract

Maslinic acid, a pentacyclic triterpene from olives, has been reported to exert beneficial effects on health, including anticarcinogenic activity. Despite its importance, little is known about its bioavailability in both humans and animals. A fundamental step for this evaluation consisted of measuring this compound in blood. Therefore, a simple high-performance liquid chromatography (HPLC) method with diode array detection has been developed. Maslinic acid contained in plasma was extracted twice using ethyl acetate. After centrifugation, the organic fraction was evaporated to dryness and the residue was reconstituted with methanol/water (75:25, v/v) and analyzed by HPLC. The method was validated by obtaining a linear correlation (r2 = 0.999) and an average recovery of 99%. Precision expressed as the coefficient of variation ranged from 1.23 to 9.06%. The oral administration of maslinic acid (50 mg/kg) to rats and its subsequent detection in plasma showed that the method is suitable for absorption, distribution, and metabolism studies.

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Combined drug action of 2-phenylimidazo[2,1-b]benzothiazole derivatives on cancer cells according to their oncogenic molecular signatures  

Alessandro Furlan, Benjamin Roux, Fabienne Lamballe, Filippo Conti, Nathalie Issaly, Fabrice Daian, Jean-François Guillemot, Sylvie Richelme, Magali Contensin, Joan Bosch,* Daniele Passarella, Oreste Piccolo, Rosanna Dono, Flavio Maina

* Departament de Farmacologia i Química Terapèutica, Unitat de Química Orgànica

Abstract

The development of targeted molecular therapies has provided remarkable advances into the treatment of human cancers. However, in most tumors the selective pressure triggered by anticancer agents encourages cancer cells to acquire resistance mechanisms. The generation of new rationally designed targeting agents acting on the oncogenic path(s) at multiple levels is a promising approach for molecular therapies. 2-phenylimidazo[2,1-b]benzothiazole derivatives have been highlighted for their properties of targeting oncogenic Met receptor tyrosine kinase (RTK) signaling. In this study, we evaluated the mechanism of action of one of the most active imidazo[2,1-b]benzothiazol-2-ylphenyl moiety-based agents, Triflorcas, on a panel of cancer cells with distinct features. We show that Triflorcas impairs in vitro and in vivo tumorigenesis of cancer cells carrying Met mutations. Moreover, Triflorcas hampers survival and anchorage-independent growth of cancer cells characterized by ‘‘RTK swapping'' by interfering with PDGFRbeta phosphorylation. A restrained effect of Triflorcas on metabolic genes correlates with the absence of major side effects in vivo. Mechanistically, in addition to targeting Met, Triflorcas alters phosphorylation levels of the PI3K-Akt pathway, mediating oncogenic dependency to Met, in addition to Retinoblastoma and nucleophosmin/B23, resulting in altered cell cycle progression and mitotic failure. Our findings show how the unusual binding plasticity of the Met active site towards structurally different inhibitors can be exploited to generate drugs able to target Met oncogenic dependency at distinct levels. Moreover, the disease-oriented NCI Anticancer Drug Screen revealed that Triflorcas elicits a unique profile of growth inhibitory-responses on cancer cell lines, indicating a novel mechanism of drug action. The anti-tumor activity elicited by 2-phenylimidazo[2,1-b]benzothiazole derivatives through combined inhibition of distinct effectors in cancer cells reveal them to be promising anticancer agents for further investigation.

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Population genetics of the “ Aeromonas hydrophila species complex”  

Mª Carmen Fusté, Maribel Farfán, David Miñana-Galbis, Vicenta Albarral, Ariadna Sanglas and José Gaspar Lorén

* Departament de Microbiologia i Parasitologia Sanitàries, Unitat de Microbiologia

Abstract

Population genetics studies the genetic variability of individuals in a population based on the allele frequencies at several genes or loci and tries to explain this variability in terms of mutation, selection or genetic recombination. The statistical analysis of these frequencies allows models of evolution to be established, which will help us to understand and predict the past and present gene flow in the population. Bacteria should be easier to analyze, because they are haploid, with no complications arising from dominance or over-dominance and we can usually deduce the genotype directly from the phenotype. Unfortunately, this hope of simplicity is not borne out. In order to establish the population structure and divergence of the species included the “Aeromonas hydrophila species complex” (AHC) we studied a set of strains representative of the AHC, in which we analyzed the nucleotide sequences (total or partial) of 6 housekeeping genes: cpn60 (555 bp), dnaJ (891 bp), gyrB (1089 bp), mdh (936 bp), recA (1065 bp), rpoD (843 bp), giving a total fragment length of 5379 bp. The results obtained from the linkage disequilibrium analysis and sequence divergence show that the AHC is composed of four robust groups that basically correspond with the phenotypically described species A. hydrophila, A. bestiarum, A. popofii and A. salmonicida. The genetic structure of the AHC appears to confirm that the entities phenotypically described as species form cohesive groups in which genetic recombination plays a limited role in reducing genetic variation and can be defined as biological species .

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Thioflavin-S staining coupled to flow cytometry. A screening tool to detect in vivo protein aggregation  

Alba Espargaró, Raimon Sabaté,* and Salvador Ventura

* Departament de Fisicoquímica

Abstract

Amyloid deposits are associated with an increasing number of human disorders, including Alzheimer's and Parkinson's diseases. Recent studies provide compelling evidence for the existence of amyloid-like conformations in the insoluble bacterial inclusion bodies (IBs) produced during the recombinant expression of amyloidogenic proteins. This makes prokaryotic cells a physiologically relevant system to study the mechanisms of in vivo amyloid deposition. We show here that the application of flow cytometry to detect Thioflavin-S (Th-S) fluorescence provides a fast, robust, quantitative, non-invasive method to screen for the presence of in vivo intracellular amyloid-like aggregates in bacteria, with potential application in the analysis of the impact of genetic mutations or chemical compounds on the aggregation of disease-associated polypeptides.

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Assessment of developmental delay in the zebrafish embryo teratogenicity assay  

E. Teixidó, E. Piqué,* J. Gómez-Catalán, J.M. Llobet

* Unitat de Toxicologia

Abstract

In this study we analyzed some aspects of the assessment of developmental delay in the zebrafish embryotoxicity/teratogenicity test and explored the suitability of acetylcholinesterase (AChE) activity as a biochemical marker and as a higher throughput alternative to morphological endpoints such as head–trunk angle, tail length and morphological score. Embryos were exposed from 4 to 52 h post-fertilization (hpf) to a selection of known embryotoxic/teratogen compounds (valproic acid, retinoic acid, caffeine, sodium salicylate, glucose, hydroxyurea, methoxyacetic acid, boric acid and paraoxon-methyl) over a concentration range. They were evaluated for AChE activity, head–trunk angle, tail length and several qualitative parameters integrated in a morphological score. In general, the different patterns of the concentration–response curves allowed distinguishing between chemicals that produced growth retardation (valproic and methoxyacetic acid) and chemicals that produced non-growth-delay related malformations. An acceptable correlation between the morphological score, AChE activity and head–trunk angle as markers of developmental delay was observed, being AChE activity particularly sensitive to detect delay in the absence of malformations

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Exploration of forbidden Povarov processes as a source of unexpected reactivity: a multicomponent Mannich–Ritter transformation  

Sara Preciado, Esther Vicente-García, Salomé Llabrés, F. Javier Luque, Rodolfo Lavilla*

* Departament de Farmacologia i Química Terapèutica, Unitat de Química Orgànica

Abstract

When a door closes, a window opens! The use of geometrically or electronically restricted imines for Povarov-type processes does not afford the anti-Bredt tetrahydroquinolines, leading instead to highly functionalized structures through novel reaction pathways. The exploration of forbidden routes constitutes a valuable approach in the search for new multicomponent reactions.

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Protein aggregation: Mechanisms and functional consequences

Gaetano Invernizzi , Elena Papaleo, Raimon Sabaté,* Salvador Ventura

* Departament de Fisicoquímica

Abstract

Understanding the mechanisms underlying protein misfolding and aggregation has become a central issue in biology and medicine. Compelling evidence show that the formation of amyloid aggregates has a negative impact in cell function and is behind the most prevalent human degenerative disorders, including Alzheimer's, Parkinson's, and Huntington's diseases or type 2 diabetes. Surprisingly, the same type of macromolecular assembly is used for specialized functions by different organisms, from bacteria to human. Here we address the conformational properties of these aggregates, their formation pathways, their role in human diseases, their functional properties and how bioinformatics tools might be of help to study these protein assemblies.

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Effects of MDMA (ecstasy) and two of its metabolites on rat embryos in vitro  

M. Barenys, * B. Flick, N. Boix, B. Almeida, J. Joglar, S. Klug, J.M. Llobet

* Unitat de Toxicologia

Abstract

MDMA consumers are young people of childbearing age. Consequently, developmental exposure to this drug is a potential public health concern. Several studies have addressed MDMA neurotoxicity in adults; however, knowledge of the effects of MDMA on developing embryos is limited. After administration, MDMA is metabolized species specifically via two main pathways. One leads to the formation of MDA and the other to the formation of HHMA. Here we evaluated the embryotoxic effects of MDMA, and also those of MDA, a main metabolite of MDMA in rats, and HHMA, a main metabolite in humans. For this purpose, we used the whole embryo culture (WEC). Our results show a concentration-dependent embryotoxic effect of MDMA, MDA and HHMA at a concentration range of 25–50 microgram/ml. The embryotoxic potential of the parent compound and the two metabolites was comparable in vitro.

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Predicting orally disintegrating tablets formulations of ibuprophen tablets: An application of the new SeDeM-ODT expert system

Johnny Edward Aguilar-Díaz, Encarna García-Montoya, * José María Suñe-Negre, Pilar Pérez-Lozano, Montserrat Miñarro, José Ramón Ticó

* Departament de Farmàcia i Tecnologia Farmacèutica

Abstract

This article provides a new innovative tool for pharmaceutical preformulation to predict whether a disintegrant excipient or mixture of powder containing API + excipients is suitable to obtain a bucodispersible tablet by direct compression or not.

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A multilevel strategy for the exploration of the conformational flexibility of small molecules

  Flavio Forti, Claudio N. Cavasotto, Modesto Orozco, Xavier Barril,* and F. Javier Luque*

* Departament de Fisicoquímica

Abstract

Predicting the conformational preferences of flexible compounds is still a challenging problem with important implications in areas such as molecular recognition and drug design. In this work, we describe a multilevel strategy to explore the conformational preferences of molecules. The method relies on the predominant-state approximation, which partitions the conformational space into distinct conformational wells. Moreover, it combines low-level (LL) methods for sampling the conformational minima and high-level (HL) techniques for calibrating their relative stability. In the implementation used in this study, the LL sampling is performed with the semiempirical RM1 Hamiltonian, and solvent effects are included using the RM1 version of the MST continuum solvation model. The HL refinement of the conformational wells is performed by combining geometry optimizations of the minima at the B3LYP (gas phase) or MST-B3LYP (solution) level, followed by single point MP2 computations using Dunning's augmented basis sets. Then, the effective free energy of a conformational well is estimated by combining the MP2 energy, supplemented with the MST-B3LYP solvation free energy for a conformational search in solution, with the local curvature of the well sampled at the semiempirical level. Applications of this strategy involve the exploration of the conformational preferences of 1,2-dichloroethane and neutral histamine in both the gas phase and water solution. Finally, the multilevel strategy is used to estimate the reorganization cost required for selecting the bioactive conformation of HIV reverse transcriptase inhibitors, which is estimated to be at most 1.3 kcal/mol.

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Ultrastructural changes of sparkling wine lees during long-term aging in real enological conditions

Rebeca Tudela, Joan J. Gallardo-Chacón, Núria Rius,* Elvira López-Tamames, and Susana Buxaderas

* Departament de Microbiologia i Parasitologia Sanitàries, Unitat de Microbiologia

Abstract

Ultrastructural changes of lees of three series of sparkling wines produced using the traditional method during long-term aging (4 years) were assessed by high-pressure freezing in combination with transmission electron microscopy. The stratified structure of the cell wall disappeared throughout aging. After 18 months, the microfibrous material of the cell wall appeared more diffuse and the amorphous midzone of the inner wall layer was progressively degraded. From 30 months onward, the cell wall consisted of a tangled structure of fibers. In spite of these changes, the cell wall of yeasts remained unbroken at 48 months of wine aging. Cell membrane breakage was observed for the first time in lees of Saccharomyces cerevisiae. An increase in the thickness of the periplasmic space owing to plasmolysis and of the number of cells with less cytoplasmic content was observed during aging. Morphological evidence of microautophagy was detected for the first time in S. cerevisiae in enological conditions.

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Counter-ion effect on surfactant-DNA gel particles as controlled DNA delivery systems

M. Carmen Morán,* Tania Alonso, Filipe S. Lima, M. Pilar Vinardell, M. Graça Miguel, and Björn Lindman

* Departament de Fisiologia

Abstract

The ability to entrap drugs within vehicles and subsequently release them has led to new treatments for a number of diseases. Based on an associative phase separation and interfacial diffusion approach, we developed a way to prepare DNA gel particles without adding any kind of cross-linker or organic solvent. Among the various agents studied, cationic surfactants offered particularly efficient control for encapsulation and DNA release from these DNA gel particles. The driving force for this strong association is the electrostatic interaction between the two components, as induced by the entropic increase due to the release of the respective counter-ions. However, little is known about the influence of the respective counter-ions on this surfactant-DNA interaction. Here we examined the effect of different counter-ions on the formation and properties of the DNA gel particles by mixing DNA (either single- (ssDNA) or double-stranded (dsDNA)) with the single chain surfactant dodecyltrimethylammonium (DTA). In particular, we used as counter-ions of this surfactant the hydrogen sulfate and trifluoromethane sulfonate anions and the two halides, chloride and bromide. Effects on the morphology of the particles obtained, the encapsulation of DNA and its release, as well as the haemocompatibility of these particles are presented, using counter-ion structure and DNA conformation as controlling parameters. Analysis of the data indicates that the degree of counter-ion dissociation from the surfactant micelles and the polar/hydrophobic character of the counter-ion are important parameters in the final properties of the particles. The stronger interaction with amphiphiles for ssDNA than for dsDNA suggests the important role of hydrophobic interactions in DNA.

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Interaction of mephedrone with dopamine and serotonin targets in rats

José Martínez-Clemente, Elena Escubedo, David Pubill, Jorge Camarasa*

* Unitat de Farmacologia i Farmacognòsia, Departament de Farmacologia i Química Terapèutica

Abstract

We described a first approach to the pharmacological targets of mephedrone (4-methyl-methcathinone) in rats to establish the basis of the mechanism of action of this drug of abuse. Experimental procedures: We performed in vitro experiments in isolated synaptosomes or tissue membrane preparations from rat cortex or striatum, studying the effect of mephedrone on monoamine uptake and the displacement of several specific radioligands by this drug. Results: In isolated synaptosomes from rat cortex or striatum, mephedrone inhibited the uptake of serotonin (5-HT) with an IC50 value lower than that of dopamine (DA) uptake (IC50=0.31±0.08 and 0.97±0.05 uM, respectively). Moreover, mephedrone displaced competitively both [3H] paroxetine and [3H]WIN35428 binding in a concentration-dependent manner (Ki values of 17.55±0.78 uM and 1.53±0.47 uM, respectively), indicating a greater affinity for DA than for 5-HT membrane transporters. The affinity profile of mephedrone for the 5-HT2 and D2 receptors was assessed by studying [3H]ketanserin and [3H] raclopride binding in rat membranes. Mephedrone showed a greater affinity for the 5-HT2 than for the D2 receptors. Discussion: These results provide evidence that mephedrone, interacting with 5-HT and DA transporters and receptors must display a similar pattern of other psychoactive drugs such as amphetamine-like compounds.

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Huprine - tacrine heterodimers as anti-amyloidogenic compounds of potential interest against Alzheimer's and prion diseases  

Carles Galdeano, Elisabet Viayna, Irene Sola, Xavier Formosa, Pelayo Camps, Albert Badia, M. Victòria Clos, Júlia Relat, Míriam Ratia, Manuela Bartolini, Francesca Mancini, Vincenza Andrisano, Mario Salmona, Cristina Minguillón, Gema C. González-Muñoz, M. Isabel Rodríguez-Franco, Axel Bidon-Chanal, F. Javier Luque, and Diego Muñoz-Torrero*

* Unitat de Química Farmacèutica, Departament de Farmacologia i Química Terapèutica

Abstract

A family of huprine-tacrine heterodimers has been developed to simultaneously block the active and peripheral sites of acetylcholinesterase (AChE). Their dual site binding for AChE, supported by kinetic and molecular modeling studies, results in a highly potent inhibition of the catalytic activity of human AChE and, more importantly, in the in vitro neutralization of the pathological chaperoning effect of AChE toward the aggregation of both the beta-amyloid peptide (Abeta) and a prion peptide with a key role in the aggregation of the prion protein. Huprine - tacrine heterodimers take on added value in that they display a potent in vitro inhibitory activity toward human butyrylcholinesterase, self-induced Abeta aggregation, and beta-secretase. Finally, they are able to cross the blood-brain barrier, as predicted in an artificial membrane model assay and demonstrated in ex vivo experiments with OF1 mice, reaching their multiple biological targets in the central nervous system. Overall, these compounds are promising lead compounds for the treatment of Alzheimer 's and prion diseases.

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Identification of new aminoacid amides containing the imidazo[2,1- b ]benzothiazol-2-ylphenyl moiety as inhibitors of tumorigenesis by oncogenic Met signaling

Alessandro Furlan, Francesco Colombo, Andrea Kover, Nathalie Issaly, Cristina Tintori, Lucilla Angeli, Vincent Leroux, Sébastien Letard, Mercedes Amat, Yasmine Asses, Bernard Maigret, Patrice Dubreuil, Maurizio Botta, Rosanna Dono, Joan Bosch,* Oreste Piccolo, Daniele Passarella, Flavio Maina

* Unitat de Química Orgànica, Departament de Farmacologia i Química Terapèutica

Abstract

The Met receptor tyrosine kinase is a promising target in anticancer therapies for its role during tumor evolution and resistance to treatment. It is characterized by an unusual structural plasticity as its active site accepts different inhibitor binding modes. Such feature can be exploited to identify distinct agents targeting tumor dependence and/or resistance by oncogenic Met. Here we report the identification of bioactive agents, featuring a new 4-(imidazo[2,1- b ]benzothiazol-2-yl)phenyl moiety, targeting cancer cells dependent on oncogenic Met. One of these compounds (7c; Triflorcas) impairs survival, anchorage-independent growth, and in vivo tumorigenesis, without showing side effects. Our medicinal chemistry strategy was based on an in-house Met-focused library of aminoacid-amide derivatives enriched through structure-based computer modeling, taking into account the Met multiple-binding-mode feature. Altogether, our findings show how a rational structure-based drug design approach coupled to cell-based drug evaluation strategies can be applied in medicinal chemistry to identify new agents targeting a given oncogenic-dependency setting

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Long-term treadmill exercise induces neuroprotective molecular changes in rat brain  

S. Bayod, J. del Valle, A. M. Canudas, J. F. Lalanza, S. Sanchez-Roige, A. Camins, R. M. Escorihuela, and M. Pallàs *

* Departament de Farmacologia i Química Terapèutica, Unitat de Farmacologia i Farmacognòsia

Abstract

Exercise enhances general health. However, its effects on neurodegeneration are controversial, and the molecular pathways in the brain involved in this enhancement are poorly understood. Here, we examined the effect of long-term moderate treadmill training on adult male rat cortex and hippocampus to identify the cellular mechanisms behind the effects of exercise. We compared three animal groups: exercised (30 min/day, 12 m/min, 5 days/wk, 36 wk), handled but nonexercised (treadmill handling procedure, 0 m/min), and sedentary (nonhandled and nonexercised). Moderate long-term exercise induced an increase in IGF-1 levels and also in energy parameters, such as PGC-1alpha and the OXPHOS system. Moreover, the sirtuin 1 pathway was activated in both the exercised and nonexercised groups but not in sedentary rats. This induction could be a consequence of exercise as well as the handling procedure. To determine whether the long-term moderate treadmill training had neuroprotective effects, we studied tau hyperphosphorylation and GSK3beta activation. Our results showed reduced levels of phospho-tau and GSK3beta activation mainly in the hippocampus of the exercised animals. In conclusion, in our rodent model, exercise improved several major brain parameters, especially in the hippocampus. These improvements induced the upregulation of sirtuin 1, a protein that extends life, the stimulation of mitochondrial biogenesis, the activation of AMPK, and the prevention of signs of neurodegeneration.These findings are consistent with other reports showing that physical exercise has positive effects on hormesis.

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A metabolomic approach differentiates between conventional and organic ketchups

Anna Vallverdú-Queralt, Alexander Medina-Remón, Isidre Casals-Ribes, Mercedes Amat, and Rosa Maria Lamuela-Raventós*

* Departament de Nutrició i Bromatologia

Abstract

The agronomic environments in which tomatoes are cultivated potentially affect the levels of antioxidants and other metabolites in commercial products. In this study, biochemical and metabolomic techniques were used to assess the differences between ketchups produced by organic and conventional systems. An untargeted metabolomic approach using QToF-MS was used to identify those nutrients that have the greatest impact on the overall metabolomic profile of organic ketchups as compared to conventional ones. Individual polyphenols were quantified using LC-ESI-QqQ. This multifaceted approach revealed that the agronomic environment in which tomatoes are grown induces alterations in the content of antioxidant capacity, phenolics, and other metabolites in ketchups. Organic cultivation was found to provide tomatoes and tomato-derived products with a significantly higher content of antioxidant microconstituents, whereas glutamylphenylalanine and N-malonyltryptophan were detected only in conventional ketchups.

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Total synthesis of Aeruginazole A  

Paolo Bruno, Stella Peña, Xavier Just-Baringo, Fernando Albericio, and Mercedes Álvarez*

* Unitat de Química Orgànica, Departament de Farmacologia i Química Terapèutica

Abstract

The first total synthesis of Aeruginazole A, prepared via a convergent strategy that involved both solid-phase peptide synthesis and solution phase chemistry and that enabled conservation of the stereochemistry of the intermediates, is reported.

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Highly stereoselective double ( R )-phenylglycinol-induced cyclocondensation reactions of symmetric aryl bis(oxoacids)  

Mercedes Amat,* Carlos Arróniz, Elies Molins, Carmen Escolano* and Joan Bosch

* Unitat de Química Orgànica, Departament de Farmacologia i Química Terapèutica

Abstract

The double cyclocondensation of symmetric pyridyl bis(oxoacids) 2b and 3b with ( R )-phenylglycinol stereoselectively gave access to bis-phenylglycinol-derived oxazolopyrrolidine 9 and oxazolopiperidone 10 , respectively. Application of the stereocontrolled cyclocondensation reaction to phenyl bis- gamma -oxoacid 4b provided 11 , which was converted to the corresponding enantiopure di(pyrrolidinyl)benzene 22 . The absolute configuration of the new stereogenic centers generated in the key cyclocondenstion step was unambiguously established by X-ray crystallographic analysis .

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Progress on lamellarins  

Daniel Pla, Fernando Albericio, and Mercedes Álvarez*

* Unitat de Química Orgànica, Departament de Farmacologia i Química Terapèutica

Abstract

This review covers recent literature on the lamellarins, a family of marine natural products, and related analogs, encompassing synthetic strategies for total synthesis, structure–activity relationships (SAR), and studies on mechanisms of biological action, namely in the context of anti-tumor activity. It reviews work published from January 2008 to December 2010.

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Effect of E1(64–81) hepatitis G peptide on the in vitro interaction of HIV-1 fusion peptide with membrane models. 

Maria Jesús Sánchez-Martín,* M. Antònia Busquets, Victoria Girona, Isabel Haro, M. Asunción Alsina, Montserrat Pujol

* Departament de Fisicoquímica

Abstract

One way to gain information about the fusogenic potential of virus-derived synthetic peptides is to examine their interfacial properties and subsequently to study them in monolayers and bilayers. Here, we characterize the physicochemical surface properties of the peptide E1(64–81), whose sequence is AQLVGELGSLYGPLSVSA. This peptide is derived from the E1 structural protein of GBV-C/HGV which was previously shown to inhibit leakage of vesicular contents caused by the HIV-1 fusion peptide (HIV-1 FP). Mixed isotherms of E1(64–81) and HIV-1 FP were obtained and their Brewster angle microscopy (BAM) and atomic force microscopy (AFM) images showed that the peptide mixture forms a different structure that is not present in the pure peptide images. Studies with lipid monolayers (1,2-dimyristoyl- sn -glycero-3-[phospho-rac-(1-glycerol)] (DMPG) and 1,2-dipalmitoyl- sn -glycero-3-phospho- rac -(1-glycerol) (DPPG)) show that both peptides interact with all the lipids assayed but the effect that HIV-1 FP has on the monolayers is reduced in the presence of E1(64–81). Moreover, differential scanning calorimetry (DSC) experiments show the capacity of HIV-1 FP to modify the properties of the bilayer structure and the capacity of E1(64–81) to inhibit these modifications. Our results indicate that E1(64–81) interacts with HIV-1 FP to form a new structure, and that this may be the cause of the previously observed inhibition of the activity of HIV-1 FP by E1(64–81).

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