ABSTRACT
RESUMEN La quinasa dependiente de ciclina 5 (CDK5) regula diversas funciones en neuronas, células endoteliales y epiteliales, entre ellas la dinámica del citoesqueleto. Así mismo, se ha reportado que componentes del citoesqueleto, tales como, filamentos de actina y microtúbulos juegan un rol importante durante la infección por el virus dengue (DENV). El objetivo del presente trabajo fue evaluar por dos métodos, inhibición química y silenciamiento génico, la participación de CDK5 durante la infección por DENV-2. La actividad antiviral de roscovitina fue evaluada usando ensayos de Unidades Formadoras de Placa (PFU). La eficiencia de transfección y el silenciamiento de CDK5, empleando miARNs artificiales, se determinó por citometría de flujo. El efecto sobre la proteína de envoltura viral y elementos del citoesqueleto se evidenció mediante microscopia avanzada de fluorescencia y análisis de imágenes. Roscovitina mostró actividad antiviral en etapas pre y post-infectivas en una forma dependiente de la dosis. El tratamiento con roscovitina y miRCDK5 mostró ser efectivo reduciendo la cantidad de CDK5 en células no infectadas. En células infectadas y transfectadas con miRCDK5, así como tratadas con el inhibidor, se observó una reducción significativa de la proteína de envoltura viral; sin embargo, no se encontró reducción significativa de CDK5. Además, el tratamiento con roscovitina indujo cambios celulares morfológicos evidentes en células infectadas. Los resultados indican la potencial participación de CDK5 durante la infección por DENV-2, posiblemente mediando la traducción proteica o la replicación del genoma viral a través de la regulación de la dinámica del citoesqueleto. Se requieren datos adicionales para esclarecer la mecanística del fenómeno usando métodos alternativos.
ABSTRACT Cyclin-Dependent Kinase 5 (CDK5) regulates several functions in neurons, endothelial, and epithelial cells, including the cytoskeleton dynamics. Likewise, it has been reported that some cytoskeleton elements, such as actin filaments and microtubules, play an essential role during Dengue virus (DENV) infection. This work aimed to evaluate the role of CDK5 during DENV-2 infection by two methods, chemical inhibition, and gene silencing. The antiviral activity of roscovitine was evaluated using Plaque Forming Units (PFU) assay. The transfection efficiency and knockdown of CDK5, using artificial miRNAs, was carried out by flow cytometry. The effect on the viral envelope protein and cytoskeleton elements was evidenced by advanced fluorescence microscopy and image analysis. Roscovitine showed antiviral activity in pre and post-infection stages in a dose-dependent manner. Treatment with roscovitine and miRCDK5 decrease the amount of CDK5 in uninfected cells. In cells infected and transfected with miRCDK5, as well as treated with the inhibitor, a significant reduction of the viral envelope protein was observed; however, no significant reduction of CDK5 was found. Also, evident morphological cellular changes were observed during the treatment with roscovitine in infected cells. The results indicate the potential participation of CDK5 during DENV-2 infection, possibly mediating protein translation or replication of the viral genome through the cytoskeletal dynamics regulation. Additional data are required to clarify the mechanistic of these phenomena using alternative methods.
ABSTRACT
Introdução: A qualidade da conservação obtida em um órgão e a duração do armazenamento seguro são dependentes do tipo de solução e tempo de conservação, estando sujeitas a intercorrências imprevisíveis. Em virtude disso, o parênquima renal e, particularmente, o túbulo contornado proximal (TCP), local onde ocorre necrose tubular aguda (NTA), são susceptíveis às mudanças na homeostase funcional do órgão, durante a conservação e durante a reperfusão pós-implante. O TCP reabsorve cerca de 70% de Na+ e H2O filtrados; sua membrana apical tem células especializadas com microvilosidades para aumentar a área de transporte unidirecional de solutos do lúmen para o sangue. O principal componente estrutural da bordadura em escova, filamentos de F-actina, interage com uma variedade de proteínas transmembranas, inclusive moléculas transportadoras de íons. Esta função é regulada por parâmetros fisiológicos e hormonais, sendo a Angiotensina II (AII) uma das principais envolvidas com o citoesqueleto de actina e proteínas associadas, mediada principalmente por seus receptores AT1. Métodos: Neste trabalho, túbulos isolados de fragmentos frescos e de fragmentos conservados por períodos de 1 e 24h nas soluções de Euro - Collins (ECO) e de Belzer (UW) foram tratados com AII, losartan e AII+losartan, para medida do volume de absorção de fluido (Jv=nl. min_1. mm-1 (microperfusão in vitro), e medida da intensidade (média) de pixel da fluorescência dos receptores AT1 e do citoesqueleto de actina (imunoistoquímica). Resultados: a) Demonstrou-se que AII (10-12M) estimula absorção fluida (Jv), que é diminuída com losartan (10-6M) em TCP frescos e também em TCP conservados em soluções...
Introduction: Depending on the preservation quality and duration, considering the solution composition and the preservation method, unexpected intercurrences may occur. In consequence, the renal parenchyma and particularly the proximal convoluted tubule (PCT), site where the acute tubular necrosis (NTA) occurs, are susceptible to changes in the organ functional homeostasis during preservation in the post implantation reperfusion period. The PCT reabsorbs approximately 70% of filtered Na+ and H2O, and its apical membrane has specialized cells with microvilli to increase the area of unidirectional solute transport from the lumen to the blood. The major structural component of brush border microvilli is the filamentous F-actin, which has been shown to interact with a variety of transmembrane proteins, including ion transport molecules. This function is regulated by physiological and hormonal parameters, such as angiotensin II (AII) interacting with actin cytoskeleton and associated proteins, mediated mainly through type I receptors (AT1R). After via different signaling pathways having been triggered, the varied functions are started, including fluid absorption (Jv), which is directly related to the actin cytoskeleton. Methods: Fresh (no preserved) and tubules preserved for 1h and 24hrs in Euro-Collins (EC) and Belzer (UW) solutions, treated with AII, losartan and AII+losartan, evaluated by "in vitro" microperfusion technique (fluid absorption (Jv=nl. min-1. mm-1), and by immunohistochemical technique (AT1 receptor measurement and actin cytoskeleton). Results: a) Our results showed that AII (10-12M) (physiological concentration) stimulates...