31 research outputs found

    “Mecanismo de acción de lípidos antitumorales sintéticos en Saccharomyces cerevisiae

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    [ES] Los lípidos antitumorales (ATLs) son una familia de compuestos sintéticos, de relevancia médica por su citotoxicidad específica frente a determinados protozoos y células cancerosas. Se han propuesto diversos modos de acción, pues los ATLs interactúan con los lípidos biológicos, modulando la estructura de las membranas celulares y la homeostasis lipídica y provocando efectos pleiotrópicos. Se han estudiado además varios ATLs en distintos tipos celulares. Es por tanto necesario dilucidar qué mecanismos biológicos median la citotoxicidad, cómo interactúan y qué relevancia presenta cada mecanismo según el compuesto concreto en cada tipo celular. Mientras que los estudios previos sobre el modo de acción de los ATLs han sido mayoritariamente realizados en células de mamífero, en esta tesis doctoral se utilizó la levadura de gemación Saccharomyces cerevisiae. Este organismo ha sido empleado como modelo para el estudio de fármacos, pues combina la similitud con las células de mamífero ¿misma arquitectura celular eucariota, homólogos en alrededor de un 30% de sus genes¿ con una mayor facilidad y rapidez de cultivo, manejo y manipulación genética. El trabajo aquí planteado se basa en screenings de colecciones -bien de knock-outs, bien de células con una dosis génica aumentada- que permitan aislar cepas resistentes o sensibles a los fármacos estudiados. Posteriormente se identifican procesos, mecanismos y estructuras celulares implicados en la toxicidad a los fármacos. Experimentos de biología celular adicionales permiten ampliar y completar dicha información. Específicamente se analizaron la incorporación y translocación de ATLs, el efecto de los mismos sobre la translocación de la ATPasa Pma1 y el papel de la función mitocondrial en la toxicidad por ATLs. Los resultados esperados de este trabajo, además de novedosos, tienen un amplio enfoque dada la metodología empleada. Esto permite plantear un modelo del mecanismo de acción de los ATLs que explique los fenotipos observados. Además de la información relevante al mismo organismo modelo empleado, el estudio paralelo de dichos fenómenos en otros modelos biológicos de interés sanitario abre potenciales nuevas vías de estudio del mecanismo de los ATLs.[EN] The work here raised bases in screenings of collections - good of knockouts, good of cells with a dose génica increased - that allow to isolate vine-stocks resistant or sensitive to the studied medicaments. Later processes, mechanisms and cellular structures are identified implied in the toxicity to the medicaments. Additional experiments of cellular biology allow to extend and to complete the above mentioned information. Specifically the incorporation was analyzed and translocación of ATLs, the effect of the same ones on the translocación of the ATPasa Pma1 and the paper of the function mitocondrial in the toxicity for ATLs. The results expected from this work, besides new, have a wide approach given the used methodology. This there allows to raise a model of the mechanism of action of the ATLs who explains the observed phenotypes. Besides the relevant information to the same organism used model, the parallel study of the above mentioned phenomena in other biological models of sanitary interest opens potential new routes of study of the mechanism of the ATLs

    RasGAP Shields Akt from Deactivating Phosphatases in Fibroblast Growth Factor Signaling but Loses This Ability Once Cleaved by Caspase-3.

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    Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G2/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G2/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G2/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling

    The role personal responsibility norms play in sustainable development for university students: The impact of Service-Learning Projects

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    Nº artigo: 7330[Abstract] Sustainable development is a desirable, global challenge that is as complex as it is necessary.While individual actions are positive, effective progress must start from a collective commitment. Inline with this, recent research points to an urgent need for an increased effort to make sustainabilityeducation a key element in the basic literacy of all people. This study takes on this challenge of envi-ronmental education and changing attitudes in order to reach sustainable community developmentwhile relying on the principles of the value-belief-norm model (VBN). A quasi-experimental researchdesign was used to analyze the impact of learning service projects on the activation of personalnorms of university students. The results show that although no statistically significant differencesappear between pre-test and post-test measurements, there is a clear trend towards improvement ofpersonal norms about sustainable development, which encourages further research in this area

    Mecanismos de acción de lípidos antitumorales sintéticos en Saccharomyces cerevisiae

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    Memoria presentada por Álvaro Cuesta Marbán para optar al grado de Doctor por la Universidad de Salamanca y realizada en Instituto de Biología Molecular y Celular del Cancer de Salamanca del CSIC.Presentaciones del trabajo en congresos: 1. 8th International Meeting on Yeast Apoptosis (2-6 mayo 2011, University of Kent, Canterbury, UK) con presentación de la ponencia Genome‐wide screenings and subcellular localization analyses uncover major events in the mechanism of action antitumor lipids in Saccharomyces cerevisiae. 2. Reunión del grupo de Control del Crecimiento, la Proliferación y la Supervivencia Celular perteneciente a la Red Temática de investigación Cooperativa en Cáncer (11-12 abril 2011), con presentación de la ponencia Mecanismos de acción de lípidos antitumorales en Saccharomyces cerevisiae por medio de screenings químico-genómicos. 3. XXXIII Congreso de la Sociedad española de bioquímica y biología molecular (14-17 septiembre 2010, Córdoba, España), con presentación del poster Mecanismo de acción de éter-lípidos antitumorales en Saccharomyces cerevisiae por medio de screenings químico-genómicos 4. Encuentro Syracuse Biomaterials Institute: 2nd Annual Offsite Meeting and Poster Session, Universidad de Syracuse, New York, con presentación del poster Identification of Potential Pharmacological Targets of Anti-tumor Lipids through Genomic Screening Congreso 2009 Northeast Regional Yeast Meeting, Universidad de Cornell, Ithaca (New York)Los lípidos antitumorales (ATLs) son una familia de compuestos sintéticos, de relevancia médica por su citotoxicidad específica frente a determinados protozoos y células cancerosas. Se han propuesto diversos modos de acción, pues los ATLs interactúan con los lípidos biológicos, modulando la estructura de las membranas celulares y la homeostasis lipídica y provocando efectos pleiotrópicos. Se han estudiado además varios ATLs en distintos tipos celulares. Es por tanto necesario dilucidar qué mecanismos biológicos median la citotoxicidad, cómo interactúan y qué relevancia presenta cada mecanismo según el compuesto concreto en cada tipo celular. Mientras que los estudios previos sobre el modo de acción de los ATLs han sido mayoritariamente realizados en células de mamífero, en esta tesis doctoral se utilizó la levadura de gemación Saccharomyces cerevisiae. Este organismo ha sido empleado como modelo para el estudio de fármacos, pues combina la similitud con las células de mamífero (misma arquitectura celular eucariota, homólogos en alrededor de un 30% de sus genes) con una mayor facilidad y rapidez de cultivo, manejo y manipulación genética. El trabajo aquí planteado se basa en screenings de colecciones -bien de knock-outs, bien de células con una dosis génica aumentada- que permitan aislar cepas resistentes o sensibles a los fármacos estudiados. Posteriormente se identifican procesos, mecanismos y estructuras celulares implicados en la toxicidad a los fármacos. Experimentos de biología celular adicionales permiten ampliar y completar dicha información. Específicamente se analizaron la incorporación y translocación de ATLs, el efecto de los mismos sobre la translocación de la ATPasa Pma1 y el papel de la función mitocondrial en la toxicidad por ATLs. Los resultados esperados de este trabajo, además de novedosos, tienen un amplio enfoque dada la metodología empleada. Esto permite plantear un modelo del mecanismo de acción de los ATLs que explique los fenotipos observados. Además de la información relevante al mismo organismo modelo empleado, el estudio paralelo de dichos fenómenos en otros modelos biológicos de interés sanitario abre potenciales nuevas vías de estudio del mecanismo de los ATLs.Peer reviewe

    Alteration of plasma membrane organization by an anticancer lysophosphatidylcholine analogue induces intracellular acidification and internalization of plasma membrane transporters in yeast

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    The lysophosphatidylcholine analogue edelfosine is a potent antitumor lipid that targets cellular membranes. The underlying mechanisms leading to cell death remain controversial, although two cellular membranes have emerged as primary targets of edelfosine, the plasma membrane (PM) and the endoplasmic reticulum. In an effort to identify conditions that enhance or prevent the cytotoxic effect of edelfosine, we have conducted genome-wide surveys of edelfosine sensitivity and resistance in Saccharomyces cerevisiae presented in this work and the accompanying paper (Cuesta-Marbán, Á., Botet, J., Czyz, O., Cacharro, L. M., Gajate, C., Hornillos, V., Delgado, J., Zhang, H., Amat-Guerri, F., Acuña, A. U., McMaster, C. R., Revuelta, J. L., Zaremberg, V., and Mollinedo, F. (January 23, 2013) J. Biol. Chem. 288,), respectively. Our results point to maintenance of pH homeostasis as a major player in modulating susceptibility to edelfosine with the PM proton pump Pma1p playing a main role. We demonstrate that edelfosine alters PM organization and induces intracellular acidification. Significantly, we show that edelfosine selectively reduces lateral segregation of PM proteins like Pma1p and nutrient H+-symporters inducing their ubiquitination and internalization. The biology associated to the mode of action of edelfosine we have unveiled includes selective modification of lipid raft integrity altering pH homeostasis, which in turn regulates cell growth. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.This work was supported in part by a Natural Sciences and Engineering Research Council of Canada discovery grant, a seed grant from the University of Calgary, a Natural Sciences and Engineering Research Council of Canada University Faculty award (to V. Z.), Canadian Institutes of Health Research Grant 14124 (to C. R. M.), Spanish Ministerio de Economia y Competitividad Grants SAF2008-02251 and SAF2011-30518, Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, co-funded by the Fondo Europeo de Desarrollo Regional of the European Union Grants RD06/0020/1037 and RD12/0036/0065, European Community's Seventh Framework Programme FP7-2007-2013 Grant HEALTH-F2-2011-256986, (PANACREAS), and Junta de Castilla y León Grants CSI052A11-2 and CSI221A12-2 (to F. M.).Peer Reviewe

    Human initiator caspases trigger apoptotic and autophagic phenotypes in Saccharomyces cerevisiae

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    Caspases are a family of proteases that participate in the progression and execution of the apoptotic program. However, regulation of the caspase activation and their substrates has not yet been fully elucidated. Here we explore the effect of the ectopic expression of the human initiator caspases-8 and -10 in Saccharomyces cerevisiae. Our results showed that the expression of human CASP10 and CASP8 triggers certain apoptotic markers such as a massive production of reactive oxygen species (ROS), chromatin condensation and phosphatidylserine externalization, finally leading to cell death. In response to hydroxyurea (HU), yeast cells expressing caspase-10 did not reduce the replication of DNA and escaped to the intra-S checkpoint of the cell cycle. In addition, caspase-10 expression induced yeast vacuolization and a vacuole-associated phenotype resembling autophagy. Other intracellular alterations such as disorganization of the actin cytoskeleton, cell wall damage, and aberrations within the endoplasmic reticulum lumen were also associated with caspase-10 expression. Furthermore, caspase-induced cell death was completely dependent on the proteolytic activation of the enzyme but, in contrast, was not dependent on either of the endogenous yeast apoptotic proteins Aif1 and Mca1 or the mitochondria. © 2008 Elsevier B.V.This work was supported by grants from Ministerio de Ciencia e Innovación of Spain (AGL2005-07245-C03-03 to J. L. R., and SAF2008-02251 and RD06/0020/1037 - Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III to F. M.), Junta de Castilla y León (SA008B08) to A. J. Fundación “la Caixa” (BM05-30-0) and NIH grant GM621284 to A. M. N.. P. L. S. and A. C. M. are recipients of FPU predoctoral fellowships from the Spanish Ministerio de Educación y Ciencia.Peer Reviewe

    Mitochondria and lipid raft-located FOF1-ATP synthase as major therapeutic targets in the antileishmanial and anticancer activities of ether lipid edelfosine

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    [Background]:Leishmaniasis is the world's second deadliest parasitic disease after malaria, and current treatment of the different forms of this disease is far from satisfactory. Alkylphospholipid analogs (APLs) are a family of anticancer drugs that show antileishmanial activity, including the first oral drug (miltefosine) for leishmaniasis and drugs in preclinical/clinical oncology trials, but their precise mechanism of action remains to be elucidated.[Methodology/Principal Findings]:Here we show that the tumor cell apoptosis-inducer edelfosine was the most effective APL, as compared to miltefosine, perifosine and erucylphosphocholine, in killing Leishmania spp. promastigotes and amastigotes as well as tumor cells, as assessed by DNA breakdown determined by flow cytometry. In studies using animal models, we found that orally-administered edelfosine showed a potent in vivo antileishmanial activity and diminished macrophage pro-inflammatory responses. Edelfosine was also able to kill Leishmania axenic amastigotes. Edelfosine was taken up by host macrophages and killed intracellular Leishmania amastigotes in infected macrophages. Edelfosine accumulated in tumor cell mitochondria and Leishmania kinetoplast-mitochondrion, and led to mitochondrial transmembrane potential disruption, and to the successive breakdown of parasite mitochondrial and nuclear DNA. Ectopic expression of Bcl-XL inhibited edelfosine-induced cell death in both Leishmania parasites and tumor cells. We found that the cytotoxic activity of edelfosine against Leishmania parasites and tumor cells was associated with a dramatic recruitment of FOF1-ATP synthase into lipid rafts following edelfosine treatment in both parasites and cancer cells. Raft disruption and specific FOF1-ATP synthase inhibition hindered edelfosine-induced cell death in both Leishmania parasites and tumor cells. Genetic deletion of FOF1-ATP synthase led to edelfosine drug resistance in Saccharomyces cerevisiae yeast.[Conclusions/Significance]:The present study shows that the antileishmanial and anticancer actions of edelfosine share some common signaling processes, with mitochondria and raft-located FOF1-ATP synthase being critical in the killing process, thus identifying novel druggable targets for the treatment of leishmaniasis.This work was supported by grants from the Spanish Ministerio de Economia y Competitividad (SAF2014-59716-R and BIO2014-56930-P), Instituto de Salud Carlos III (RD12/0036/0065 from Red TemaÂtica de Investigación Cooperativa en Cáncer, cofunded by the EU's European Regional Development Fund ± FEDER),European Community's Seventh Framework Programme FP7-2007-2013 (grant HEALTH-F2-2011-256986, PANACREAS), and Spain-UK International Joint Project grant from The Royal Society-CSIC (2004GB0032). CG was supported by the RamoÂn y Cajal Program from the Spanish Ministerio de Ciencia e Innovación.AÂCM was recipient of Formación de Profesorado Universitario predoctoral fellowship from the Spanish Ministerio de Ciencia e Innovación.Peer reviewe
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