45 research outputs found

    Biolog铆a molecular y celular de la alteraci贸n de la homeostasis del pirofosfato inorg谩nico en eucariotas

    Get PDF
    Texto completo descargado desde TeseoEl PPi es un componente celular que se genera en muchas reacciones anab贸licas de bios铆ntesis de pol铆meros y metabolitos (Geigenberger, Hajirezaei et al. 1998; Stitt 1998; Rojas-Beltr谩n, Dubois et al. 1999; Farre, Bachmann et al. 2001; Sonnewald 2001). Su hidr贸lisis es esencial para que esas reacciones discurran en la direcci贸n correcta y se regenere el Pi necesario para las reacciones de fosforilaci贸n. La hidr贸lisis del PPi es una reacci贸n bioqu铆mica universal catalizada por pirofosfatasas inorg谩nicas (PPasas, EC 3.6.1.1). Existen algunos estudios que muestran la necesidad de PPasas para el correcto desarrollo del anabolismo celular (Heinonen et al., 2001), as铆 como el efecto negativo de la deficiencia de estas enzimas tanto en procariotas como en eucariotas. Sin embargo, estos estudios no van m谩s all谩 de postular que la carencia sPPasa genera parada del crecimiento en procariotas (Escherichia coli) (Chen et al., 1990) y es letal en eucariotas (Saccharomyces cerevisiae) (Giaever et al., 2002). En este trabajo de Tesis doctoral, se ha realizado un estudio detallado del mecanismo molecular de la toxicidad del exceso de PPi producido por la deficiencia en la sPPasa en eucariotas, as铆 como sobre el papel fisiol贸gico y bioqu铆mico de dichas enzimas en los diferentes subcompartimentos celulares en los que se localizan. Objetivos: 1. Determinar el mecanismo fisiol贸gico de la toxicidad por exceso de PPi en el modelo eucariota S. cerevisiae. 2. Analizar la relevancia de la localizaci贸n n煤cleo-citos贸lica de la sPPasa Ipp1p en S. cerevisiae. 3. Expresar heterologamente diferentes sPPasas en S. cerevisiae y l铆neas celulares de mam铆fero. 4. Reproducir en la levadura el escenario metab贸lico del PPi citos贸lico de c茅lulas vegetales. Conclusiones: 1. La ausencia de la sPPasa Ipp1p en la levaduras S. cerevisiae con metabolismo fermentativo genera muerte celular por autofagia, siendo uno de los mecanismos implicados el desbalance de la raz贸n NAD+/NADH debido a la inhibici贸n de la s铆ntesis de NAD+ por el exceso de PPi. 2. La ausencia de Ipp1p genera parada del ciclo celular en fase S en S. cerevisiae con metabolismo respiratorio. Esta parada es reversible si se retira el exceso de PPi y se reactiva la sPPasa. 3. Ipp1p es una enzima n煤cleo-citos贸lica, cuyos niveles tanto de prote铆na como de actividad catal铆tica son menores en el compartimento nuclear que en el citosol. Esta diferencia se debe a una estricta regulaci贸n de los niveles prote铆cos de la Ipp1p nuclear, mediante degradaci贸n proteas贸mica. Esta regulaci贸n es dependiente de la actividad catal铆tica de hidr贸lisis de PPi y no de los niveles de polip茅ptido en s铆. 4. La alta homolog铆a tanto en la conformaci贸n espacial del sitio activo, como del mecanismo catal铆tico, permite intercambiar sPPasas de diversas procedencias y diferentes familias en sistemas modelo eucari贸ticos como levadura o c茅lulas de mam铆fero. 5. La compartimentalizaci贸n nuclear de Ipp1p permite la eficiente complementaci贸n en levadura de la deficiencia en V-ATPasa mediante la expresi贸n de una H+-PPasa localizada en la membrana vacuolar, pudiendo ser este un escenario metab贸lico del PPi similar al que tiene lugar en c茅lulas vegetales

    Intracellular proton pumps as targets in chemotherapy: V-ATPases and cancer

    Get PDF
    Cancer cells show a metabolic shift that makes them overproduce protons; this has the potential to disturb the cellular acid-base homeostasis. However, these cells show cytoplasmic alkalinisation, increased acid extrusion and endosome-dependent drug resistance. Vacuolar type ATPases (V-ATPases), toghether with other transporters, are responsible to a great extent for these symptoms. These multisubunit proton pumps are involved in the control of cytosolic pH and the generation of proton gradients (positive inside) across endocellular membrane systems like Golgi, endosomes or lysosomes. In addition, in tumours, they have been determined to play an important role in the acidification of the intercellular medium. This importance makes them an attractive target for control of tumour cells. In the present review we portray the major characteristics of this kind of proton pumps, we provide some recent insights on their in vivo regulation, an overview of the consequences that V-ATPase inhibition carries for the tumour cell, such as cell cycle arrest or cell death, and a brief summary of the studies related to cancer made recently with commercially available inhibitors for this kind of proton pump. Some new approaches to affect V-ATPase function are also suggested in the light of recent knowledge on the regulation of this proton pump.Junta de Andaluc铆a PAIDI BIO-261 P07-CVI-3082Ministerio de Ciencia e Innovaci贸n BFU2007-61887 BFU2010-1562

    Intraorganellar acidification by V-ATPases: a target in cellproliferation and cancer therapy

    Get PDF
    V-ATPases are multicomponent proton pumps involved in the acidification of singlemembrane intracellular compartments such as endosomes and lysosomes. They couple thehydrolysis of ATP to the translocation of one to two protons across the membrane. Acidification ofthe lumen of single membrane organelles is a necessary factor for the correct traffic of membranesand cargo to and from the different internal compartments of a cell. Also, V-ATPases are involved inregulation of pH at the cytosol and, possibly, extracellular milieu. The inhibition of V-ATPases hasbeen shown to induce apoptosis and cell cycle arrest in tumour cells and, therefore, chemicals thatbehave as inhibitors of this kind of proton pumps have been proposed as putative treatment agentsagainst cancer. The present review will summarize the major types of V-ATPase inhibitors and theirmechanisms of action and put them in relation to the patents registered so far for the treatment of cancer.Junta de Andaluc铆a PAIDI BIO-261 P07-CVI-3082Ministerio de Ciencia e Innovaci贸n BMC2007-6188

    Nutrici贸n, inflamaci贸n y riesgo metab贸lico en ni帽os y adolescentes europeos

    Get PDF
    El estudio de los factores de riesgo cardio-metab贸licos es importante para intentar prevenir enfermedades futuras. Estos factores de riesgo aparecen cada vez a edades m谩s tempranas como en la adolescencia, o incluso en la infancia, y parecen estar asociados con algunos estilos de vida como la alimentaci贸n. Se ha observado que la la inflamaci贸n cr贸nica de bajo grado se relaciona con factores de riesgo cardio-metab贸licos. Por lo tanto, el estudio del estado inflamatorio en ni帽os y adolescentes es necesario para evaluar esta relaci贸n desde sus or铆genes y, de esta manera, poder entender sus mecanismos de aparici贸n. Es por ello que el objetivo general de esta Tesis Doctoral es evaluar la relaci贸n entre la inflamaci贸n, valorada mediante una serie de marcadores inflamatorios, la ingesta y las alteraciones cardio-metab贸licas asociadas con la obesidad en ni帽os y adolescentes europeos. Esta memoria se ha realizado por compendio de publicaciones, incluyendo seis art铆culos.La presente Tesis Doctoral se ha llevado a cabo teniendo en cuenta los resultados de dos grandes estudios europeos: el estudio IDEFICS (Identification and Prevention of Dietary- and Lifestyle- induced Health Effects in Children and Infants) y el estudio HELENA (Healthy Lifestyle in Europe by Nutrition in Adolescence). En el estudio IDEFICS se obtuvo informaci贸n de m谩s de 16.000 ni帽os, con edades comprendidas entre 2 y 9 a帽os, procedentes de ocho pa铆ses europeos (Italia, Estonia, Chipre, B茅lgica, Suecia, Alemania, Hungr铆a y Espa帽a). La medida incial se realiz贸 durante el curso 2007-2008. Estos ni帽os fueron re-evaluados dos a帽os despu茅s del incio del estudio. Se seleccionaron sujetos de este estudio para valorar la asociaci贸n entre la dieta y la prote铆na C-reactiva de alta sensibilidad (PCR-hs), como marcador de inflamaci贸n. En el primer art铆culo se valor贸 la asociaci贸n entre los 谩cidos grasos, medidos en sangre total, y la inflamaci贸n. Los 谩cidos grasos son componentes de la dieta que se relacionan con el estado inflamatorio, especialmente en el caso de los 谩cidos grasos de cadena larga. Se observ贸 que los acidos grasos omega-6 (suma de omega 6 y 谩cido linoleico) se asociaron con valores bajos de PCR-hs, en chicos, y con valores altos de PCR-hs en chicas (谩cido araquid贸nico, suma de omega 6 altamente insaturados y relaci贸n acido araquid贸nico/linoleico). En el segundo art铆culo se observ贸 una asociaci贸n clara entre la frecuencia de consumo de algunos alimentos y la PCR-hs. Espec铆ficamente, la elevada frecuencia de consumo de vegetales se relacionaba inversamente con la inflamaci贸n mientras que otros tipos de alimentos, como las bebidas azucaradas o la mayonesa, se relacionaban directamente con la inflamaci贸n.En el tercer art铆culo, tres tipos de patrones diet茅ticos fueron identificados y mantenidos a lo largo del seguimiento: el patr贸n 鈥漵aludable鈥, el patr贸n de 鈥減rote铆na animal y carbohidratos refinados鈥 y el patr贸n 鈥渄ulces y alimentos procesados鈥. En el an谩lisis transversal, realizado al final del seguimiento, se observ贸 que aquellos ni帽os incluidos en el patr贸n 鈥漝ulces y alimentos procesados鈥 mostraban una mayor probabilidad de tener la PCR-hs elevada, en comparaci贸n con aquellos asignados a un patr贸n 鈥漵aludable鈥. De igual manera, se observ贸 que aquellos incluidos en un patr贸n de 鈥渄ulces y alimentos procesados鈥 mantenido en el tiempo, es decir, desde la valoraci贸n inicial hasta la medida de seguimiento, mostraban mayor probabilidad de tener valores m谩s elevados de la PCR-hs, en comparaci贸n con los incluidos en un patr贸n saludable en las dos valoraciones. En el estudio HELENA, realizado entre 2006 y 2007, se valoraron m谩s de 3.000 adolescentes de 10 ciudades europeas: Atenas, Heraklion, Dortmund, Gante, Lille, Pecs, Roma, Estocolmo, Viena y Zaragoza. Las edades de los adolescentes participantes estaban entre 12,5 y 17,5 a帽os. Con datos de este estudio, se valoraron las asociaciones entre el riesgo cardio-metab贸lico y la inflamaci贸n en la adolescencia. La American Heart Asociation (AHA) ha propuesto un 铆ndice de salud cardiovascular ideal (ISCI) que incluye cuatro comportamientos y tres factores saludables. Los criterios relacionados con los comportamientos son: no haber fumado, ser f铆sicamente activo, tener un IMC normal y tener una alimentaci贸n saludable, mientras que los factores saludables incluidos son valores normales de: tensi贸n arterial, colesterol total y glucosa. Mediante el uso de este 铆ndice se valor贸 la relaci贸n entre la salud cardiovascular y la inflamaci贸n, la cual fue medida mediante un 铆ndice inflamatorio y, a su vez, mediante los biomarcadores que compon铆an el citado 铆ndice individualmente: PCR, el factor C3 y C4 del complemento, leptina y el recuento de gl贸bulos blancos. En este cuarto art铆culo, se observ贸 que puntuaciones superiores del 铆ndice de salud cardiovascular se relacionaban inversamente con los valores del 铆ndice inflamatorio y, adem谩s, con algunos de sus componentes individualmente. En el quinto art铆culo, se observ贸 que la composici贸n corporal juega un papel importante en la relaci贸n entre la resistencia a la insulina y la inflamaci贸n, medida con varios marcadores inflamatorios. La asociaci贸n entre la resistencia a la insulina y el factor C3 del complemento fue especialmente relevante para aquellos adolescentes con mayores niveles de adiposidad. Finalmente, en el 煤ltimo art铆culo, se valor贸 la asociaci贸n entre la salud metab贸lica y varios marcadores inflamatorios seleccionados, teniendo en cuenta la presencia o no de sobrepeso/obesidad. La existencia de sobrepeso/obesidad y un estatus metab贸lico alterado se asocia con marcadores inflamatorios, siendo la PCR, C3 y C4 los marcadores m谩s relacionados con esta condici贸n. C3 y C4 se asociaron consistentemente con la salud cardio-metab贸lica.En resumen, los resultados de esta Tesis Doctoral confirman la existencia de una asociaci贸n entre alimentaci贸n y PCR-hs desde la infancia y, a su vez, que los marcadores de riesgo cardio-metab贸lico est谩n presentes desde la adolescencia y se asocian con distintos marcadores inflamatorios, en esta etapa de la vida. Estos resultados ponen de manifiesto la importancia del desarrollo de estrategias de prevenci贸n precoz, teniendo en cuenta la promoci贸n de estilos de vida saludables, para evitar el desarrollo de los factores de riesgo cardio-metab贸lico y la aparici贸n de un estado inflamatorio cr贸nico de bajo grado, asociado a los mismos.<br /

    Conserved gene modules regulate light signals in algae and plants

    Get PDF
    In the Plant Development Unit (PDU) we aim to discover mechanisms that allowed photosynthetic organisms to reach the level of developmental complexity shown today. Plants are particularly good models as they have been evolving as light autotrophs for millions of years, ever since the first bacteria developed oxygenic photosynthesis and killed 99% of existing species in the process. But light is not only the main source of energy for plants, it is also one of the main regulators of their development, as endo-symbiotic cyanobacteria (chloroplasts) perfected their physiological synchronization with the emerging eukaryotes (1). Another important aspect of plant evolution was the transit to the aerial world and the acquisition of characteristics that allowed them to successfully colonise this new habitat (2). In the PDU we have followed the evolution of the day length response (photoperiod) that coordinates the daily physiological activities of plants and can be also used to regulate seasonal behaviours such as winter recesses or flowering time (3). When gene expression networks from photoperiod experiments from microalgae, bryophytes and higher plants are compared, a common nodular structure is discovered (4). Following these discoveries, we have isolated ancestor algal genes that show the same function as higher plants in the response to photoperiod such as the CONSTANS-DOF module (5). We are currently investigating common regulatory mechanisms in photoperiod sensing such as the effect on the circadian clock, senescence, retrograde signalling (6) and protein stability (7

    CONSTANS鈥揊KBP12 interaction contributes to modulation of photoperiodic flowering in Arabidopsis

    Get PDF
    Flowering time is a key process in plant development. Photoperiodic signals play a crucial role in the floral transition in Arabidopsis thaliana, and the protein CONSTANS (CO) has a central regulatory function that is tightly regulated at the transcriptional and post-translational levels. The stability of CO protein depends on a light-driven proteasome process that optimizes its accumulation in the evening to promote the production of the florigen FLOWERING LOCUS T (FT) and induce seasonal flowering. To further investigate the post-translational regulation of CO protein we have dissected its interactome network employing in vivo and in vitro assays and molecular genetics approaches. The immunophilin FKBP12 has been identified in Arabidopsis as a CO interactor that regulates its accumulation and activity. FKBP12 and CO interact through the CCT domain, affecting the stability and function of CO. fkbp12 insertion mutants show a delay in flowering time, while FKBP12 overexpression accelerates flowering, and these phenotypes can be directly related to a change in accumulation of FT protein. The interaction is conserved between the Chlamydomonas algal orthologs CrCO鈥揅rFKBP12, revealing an ancient regulatory step in photoperiod regulation of plant development.Ministerio de Ciencia BIO2014-52425-P, BIO2017-83629-RJunta de Andaluc铆a P08-AGR-03582, BIO-281European Union GA83831

    The evolution of the ventilatory ratio is a prognostic factor in mechanically ventilated COVID-19 ARDS patients

    Get PDF
    Background: Mortality due to COVID-19 is high, especially in patients requiring mechanical ventilation. The purpose of the study is to investigate associations between mortality and variables measured during the first three days of mechanical ventilation in patients with COVID-19 intubated at ICU admission. Methods: Multicenter, observational, cohort study includes consecutive patients with COVID-19 admitted to 44 Spanish ICUs between February 25 and July 31, 2020, who required intubation at ICU admission and mechanical ventilation for more than three days. We collected demographic and clinical data prior to admission; information about clinical evolution at days 1 and 3 of mechanical ventilation; and outcomes. Results: Of the 2,095 patients with COVID-19 admitted to the ICU, 1,118 (53.3%) were intubated at day 1 and remained under mechanical ventilation at day three. From days 1 to 3, PaO2/FiO2 increased from 115.6 [80.0-171.2] to 180.0 [135.4-227.9] mmHg and the ventilatory ratio from 1.73 [1.33-2.25] to 1.96 [1.61-2.40]. In-hospital mortality was 38.7%. A higher increase between ICU admission and day 3 in the ventilatory ratio (OR 1.04 [CI 1.01-1.07], p = 0.030) and creatinine levels (OR 1.05 [CI 1.01-1.09], p = 0.005) and a lower increase in platelet counts (OR 0.96 [CI 0.93-1.00], p = 0.037) were independently associated with a higher risk of death. No association between mortality and the PaO2/FiO2 variation was observed (OR 0.99 [CI 0.95 to 1.02], p = 0.47). Conclusions: Higher ventilatory ratio and its increase at day 3 is associated with mortality in patients with COVID-19 receiving mechanical ventilation at ICU admission. No association was found in the PaO2/FiO2 variation

    CIBERER : Spanish national network for research on rare diseases: A highly productive collaborative initiative

    Get PDF
    Altres ajuts: Instituto de Salud Carlos III (ISCIII); Ministerio de Ciencia e Innovaci贸n.CIBER (Center for Biomedical Network Research; Centro de Investigaci贸n Biom茅dica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research

    Diagnostic assessment of deep learning algorithms for detection of lymph node metastases in women with breast cancer

    Get PDF
    Importance Application of deep learning algorithms to whole-slide pathology images can potentially improve diagnostic accuracy and efficiency. Objective Assess the performance of automated deep learning algorithms at detecting metastases in hematoxylin and eosin鈥搒tained tissue sections of lymph nodes of women with breast cancer and compare it with pathologists鈥 diagnoses in a diagnostic setting. Design, Setting, and Participants Researcher challenge competition (CAMELYON16) to develop automated solutions for detecting lymph node metastases (November 2015-November 2016). A training data set of whole-slide images from 2 centers in the Netherlands with (n鈥=鈥110) and without (n鈥=鈥160) nodal metastases verified by immunohistochemical staining were provided to challenge participants to build algorithms. Algorithm performance was evaluated in an independent test set of 129 whole-slide images (49 with and 80 without metastases). The same test set of corresponding glass slides was also evaluated by a panel of 11 pathologists with time constraint (WTC) from the Netherlands to ascertain likelihood of nodal metastases for each slide in a flexible 2-hour session, simulating routine pathology workflow, and by 1 pathologist without time constraint (WOTC). Exposures Deep learning algorithms submitted as part of a challenge competition or pathologist interpretation. Main Outcomes and Measures The presence of specific metastatic foci and the absence vs presence of lymph node metastasis in a slide or image using receiver operating characteristic curve analysis. The 11 pathologists participating in the simulation exercise rated their diagnostic confidence as definitely normal, probably normal, equivocal, probably tumor, or definitely tumor. Results The area under the receiver operating characteristic curve (AUC) for the algorithms ranged from 0.556 to 0.994. The top-performing algorithm achieved a lesion-level, true-positive fraction comparable with that of the pathologist WOTC (72.4% [95% CI, 64.3%-80.4%]) at a mean of 0.0125 false-positives per normal whole-slide image. For the whole-slide image classification task, the best algorithm (AUC, 0.994 [95% CI, 0.983-0.999]) performed significantly better than the pathologists WTC in a diagnostic simulation (mean AUC, 0.810 [range, 0.738-0.884]; P鈥<鈥.001). The top 5 algorithms had a mean AUC that was comparable with the pathologist interpreting the slides in the absence of time constraints (mean AUC, 0.960 [range, 0.923-0.994] for the top 5 algorithms vs 0.966 [95% CI, 0.927-0.998] for the pathologist WOTC). Conclusions and Relevance In the setting of a challenge competition, some deep learning algorithms achieved better diagnostic performance than a panel of 11 pathologists participating in a simulation exercise designed to mimic routine pathology workflow; algorithm performance was comparable with an expert pathologist interpreting whole-slide images without time constraints. Whether this approach has clinical utility will require evaluation in a clinical setting

    8-Dehydrosterols induce membrane traffic and autophagy defects through V-ATPase dysfunction in Saccharomyces cerevisae

    Get PDF
    8-Dehydrosterols are present in a wide range of biologically relevant situations, from human rare diseases to amine fungicide-treated fungi and crops. However, the molecular bases of their toxicity are still obscure. We show here that 8-dehydrosterols, but not other sterols, affect yeast vacuole acidification through V-ATPases. Moreover, erg2螖 cells display reductions in proton pumping rates consistent with ion-transport uncoupling in vitro. Concomitantly, subunit Vph1p shows conformational changes in the presence of 8-dehydrosterols. Expression of a plant vacuolar H+-pumping pyrophosphatase as an alternative H+-pump relieves Vma鈭-like phenotypes in erg2螖-derived mutant cells. As a consequence of these acidification defects, endo- and exo-cytic traffic deficiencies that can be alleviated with a H+-pumping pyrophosphatase are also observed. Despite their effect on membrane traffic, 8-dehydrosterols do not induce endoplasmic reticulum stress or assembly defects on the V-ATPase. Autophagy is a V-ATPase dependent process and erg2螖 mutants accumulate autophagic bodies under nitrogen starvation similar to Vma鈭 mutants. In contrast to classical Atg鈭 mutants, this defect is not accompanied by impairment of traffic through the CVT pathway, processing of Pho8螖60p, GFP-Atg8p localisation or difficulties to survive under nitrogen starvation conditions, but it is concomitant to reduced vacuolar protease activity. All in all, erg2螖 cells are autophagy mutants albeit some of their phenotypic features differ from classical Atg鈭 defective cells. These results may pave the way to understand the aetiology of sterol-related diseases, the cytotoxic effect of amine fungicides, and may explain the tolerance to these compounds observed in plants.Peer reviewe
    corecore