Enhanced anticancer activity and endocytic mechanisms by polymeric nanocarriers of n-butylidenephthalide in leukemia cells

Purpose The purpose of this study was to investigate the antitumor mechanisms of n-butylidenephthalide (BP) and to further examine the delivery efficacy of polycationic liposome containing PEI and polyethylene glycol complex (LPPC)-encapsulated BP in leukemia cells. Method MTS, flow cytometric and TUNEL assays were performed to assess cell viability and apoptosis. BP and BP/LPPC complex delivery efficiency was analyzed by full-wavelength fluorescent scanner and fluorescence microscope. The expressions of cell cycle- and apoptosis-related proteins were conducted by Western blotting. Results The results showed that BP inhibited leukemia cell growth by inducing cell cycle arrest and cell apoptosis. LPPC-encapsulated BP rapidly induced endocytic pathway activation, resulting in the internalization of BP into leukemia cells, causing cell apoptosis within 1 h. Conclusions LPPC encapsulation enhanced the cytotoxic activity of BP and did not influence the effects of BP induction that suggested LPPC-encapsulated BP might be developed as anti-leukemia drugs in future (AU)

Fate of Bacillus cereus within phagocytic cells

In this study we assessed the interaction of different strains of Bacillus cereus with murine peritoneal macrophages and cultured phagocytic cells (Raw 264.7 cells). Association, internalization, intracellular survival, routing of bacteria to different compartments and expression of MHCII were assessed in cells infected with different strains of B. cereus in vegetative form. Association values (adhering + internalized bacteria) and phagocytosis were higher for strain B10502 than those for strains 2 and M2. However, after 90 min interaction, intracellular survival was higher for strain 2 than for strains M2 and B10502. Acquisition of lysosomal markers by B. cereus containing vacuoles (BcCV), assessed by LAMP1 and Lysotracker labelling occurred shortly after internalization. The highest ratio of LAMP1(+)-BcCV was found for strain M2. This strain was able to survive longer than strain B10502 which routes to LAMP1 containing vacuoles to a lesser extent. In addition, strain M2 stimulated expression of MHCII by infected cells. Confocal analyses 60 or 90 min post-infection showed different percentages of co-localization of bacteria with Lysotracker. Results suggest strain-dependent interaction and intracellular killing of B. cereus by phagocytic cells. These findings could be relevant for the pathogenic potential of Bacillus cereus strains

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Expression of soluble sCD163 in serum of psoriatic patients is modulated by Goeckerman therapy

Background: CD163 is the monocyte/macrophage receptor for haptoglobin-haemoglobin complexes. The aim of this study was to assess the kinetics in the expression of CD163 on monocytes and the concentration of soluble sCD163 in serum of psoriatic patients in order to examine the effect of Goeckerman therapy. Methods: sCD163 was measured in 71 patients before and after therapy, and in 57 healthy donors. A subgroup of 40 patients and 25 controls was used to assess the expression of membrane CD163. sCD163 was evaluated by ELISA. Flow cytometry method was used to determine the expression of membrane CD163 on monocytes, expressed as mean fluorescence index (MFI). Results: Before therapy, the serum level of sCD163 was significantly higher in our patients than in controls (P = 0.0154). However, we observed a profound decrease in sCD163 in our patients after therapy (P = 0.0037). Similar to sCD163, pre-treatment expression of CD163 on monocytes was significantly more enhanced in patients than that in controls (P = 0.0078). There was a trend towards down-regulation of the expression after therapy, nonetheless, the change was not statistically significant compared to the values before therapy (P = 0.8666). This was also confirmed by comparison with controls which displayed lower expression of CD163 than patients after therapy (P = 0.0019). The disease activity, expressed as PASI score, was significantly decreased in our patients by GT (P = 0.0001). Conclusions: While sCD163 level in psoriatic patients was diminished after GT therapy, CD163expression on monocytes was altered only to a minor extent (AU)

La megalina es el receptor para la endocitosis mediada por caveolas de la albúmina y se requiere para la síntesis del factor neurotrófico ácido oleico / Megalin is the receptor for caveola-mediated endocytosis of albumin and it’s required for the synthesis of the neurotrophic factor oleic acid

Objetivo: Estudiar el mecanismo de endocitosis de la albúmina en astrocitos de rata en cultivo primario. Material y Métodos: Se utilizaron neonatos (1 día de vida) de ratas Wistar para obtener astrocitos en cultivo primario. Los astrocitos se incubaron con albúmina al 2% o albúmina-FITC al 0,5%, durante 20 minutos, a 4ºC para los estudios de unión y durante 20 minutos a 4ºC, seguido de 10 minutos a 37ºC, para los estudios de internalización. Para los experimentos de pérdida de función, los astrocitos se transfectaron con 50nM de siRNA específico para el silenciamiento de la proteína diana durante 72 h. El ácido oleico presente en el medio de cultivo de astrocitos incubados con albúmina al 2% durante una hora se cuantifico por HPLC. Resultados: la megalina y la caveolina-1, pero no la clatrina, colocalizan con la albúmina en la membrana plasmática. El silenciamiento de la megalina y de la caveolina-1 reduce la capacidad de la albúmina para unirse a la membrana e internalizarse y reduce la capacidad de los astrocitos para sintetizar el factor neurotrófico ácido oleico. El silenciamiento de la clatrina no modifica la internalización de la albúmina ni la síntesis del ácido oleico. Conclusiones: la albúmina se internaliza mediante endocitosis dependiente de caveolas vía megalina en los astrocitos y que este proceso se requiere para la síntesis del factor neurotrófico ácido oleico (AU)

Aim: Study of the endocytic pathway of albumin in rat astrocytes from primary culture. Material y Methods: One day postnatal Wistar rats were used to obtain astrocytes form primary culture. Astrocytes were incubated with 2% albumin or 0.5% FITC-albumin during 20 minutes at 4ºC for binding experiments or during 20 minutes at 4ºC followed by 10 minutes at 37ºC for internalization experiments. For lossof- function experiments, astrocytes were transfected during 72h with 50 nM of siRNA specific for target protein, followed by the binding and internalization experiments. Oleic acid present in the culture medium of astrocytes incubated with 2% albumin during one hour was quantified by HPLC. Results: We observed that megalin and caveolin-1, but not clathrin, co-localize with albumin in the membrane. Megalin and caveolin-1 silencing by siRNA reduces albumin binding and internalization, as well as oleic acid synthesis in astrocytes. Nonetheless, clathrin silencing do not modify albumin internalization or oleic acid synthesis in astrocytes Conclusions: albumin is internalized in a caveola-dependent mechanism via megalin and that this event is required for the synthesis of the neurotrophic factor oleic acid (AU)

Glycoprotein clearance is rapid and suppressed by mannan in chicken embryos

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Carbohydrates are thought to function as tags that mark circulatory glycoproteins for rapid clearance. Scavenger endothelial cells (SECs) play the primary role in clearing glycoproteins via receptor-mediated endocytosis in adult animals. We found that horseradish peroxidase (HRP), a glycoprotein, was removed quickly, mostly by receptor mediation from the chicken embryo circulation, but bovine serum albumin was not. The half-life of HRP in the circulation varied with the embryo stage and fell rapidly from 0.73 h at embryonic day 4 (E4) to 0.23 h at E5, with no great difference among stages after E5. HRP clearance was far slower at E3.5 than at E5, but was obviously suppressed by mannan. These results imply that the function of clearing glycoprotein or waste macromolecules from the circulation via receptor-mediated endocytosis appears early in the embryo (AU)

Absorción de fármacos por vía tópica. Papel de la conjuntiva / Ocular drug absorption by topical route. Role of conjunctiva

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Roles of Protein Phosphatase Type 1 in Schizosaccharomyces pombe

Las proteína fosfatasas están consideradas reguladores globales en muchosprocesos biológicos. Se clasifican en distintas familias según su especificidad desustrato, sus mecanismos de catálisis y sus relaciones evolutivas. Los holoenzimasde proteína fosfatasas de tipo 1 (PP1) están compuestos por un pequeño númerode subunidades catalíticas y un amplio número de subunidades reguladoras. Elgenoma de S. pombe codifica dos subunidades catalíticas muy relacionadas, Dis2y Sds21. Hemos fusionado la proteína verde fluorescente «mejorada» (EGFP) alextremo amino-terminal de los genes endógenos de dis2+ y sds21+. Hemos descritoque Dis2 y Sds21 se localizan en distintos compartimentos y estructuras celulares,como los centromeros-kinetocoros, núcleo, un anillo en el ecuador de células endivisión, vesículas endocíticas y extremos celulares. Cada una de estaslocalizaciones sugiere diferentes funciones para las subunidades catalíticas de PP1que interaccionan con distintas proteínas reguladoras. Esto convierte a PP1 en unenzima multifuncional que actúa como un regulador global en muchos procesoscelulares

Protein phosphatases are considered to be global regulators in many biologicalprocesses. They are divided in families on the basis of substrate specificity,mechanisms of catalysis and evolutionary relations. Protein Phosphatase Type 1(PP1) holoenzymes are composed of a small number of catalytic subunits and anarray of regulatory, targeting, subunits. The S. pombe genome encodes two highlyrelated catalytic subunits, Dis2 and Sds21. We fused enhanced green fluorescence protein (EGFP) coding sequences to the aminus-termini of endogenous dis2+ andsds21+ genes. We have described that Dis2 and Sds21 localize in differentcell compartments and structures as centromeres- kinetochores, nucleoli, a ringat the cell equator in dividing cells, endocytic vesicles and the cell tips. Each ofthese locations suggests different functions of a single catalytic PP1 subunitmediated by its interaction with different targeting proteins. This converts PP1into a multifunctional enzyme that acts as a global regulator in many cellularprocesses

Synthetic particulate antigen delivery systems for vaccination

La encapsulación de un antígeno (proteína, péptido o DNA)en partículas sintéticas constituye un punto de partida razonablepara el diseño de vacunas subcelulares efectivas. Al dotarlo delas dimensiones de una partícula, se favorece su visualización porcélulas presentadoras de antígeno (APC), como las células dendríticas(DC). Utilizando distintos materiales y procedimientosde fabricación, la tecnología farmacéutica puede construir partículascon un amplio rango de tamaños y variadas propiedadesde superficie. Es nuestro objetivo con esta revisión analizar cómoestas propiedades físicas afectan a la intensidad y al tipo de respuestainmunitaria que producen. Así, partículas de distinto tamañopodrían poner en juego distintas APC y subtipos de DC. Susuperficie puede ser recubierta con ligandos que promueven laendocitosis receptor-específica y algún inmunoestimulante intrínsecoco-encapsulado junto con el antígeno dentro de la mismapartícula. El mecanismo de captura antigénica y el tipo de señalde activación pueden ser decisivos para potenciar y modular larespuesta inmunitaria al tipo deseado, Th2/humoral o Th1/citotoxica.Adicionalmente, las partículas que son de naturaleza poliméricapueden liberar lentamente el antígeno encapsulado, deforma continua o pulsátil, evitando la necesidad de administracionesrepetidas normalmente requerida para las vacunas tradicionales.Las partículas sintéticas para la encapsulación de antígenosconstituyen por tanto, una buena herramienta que puede hacerposible el diseño de vacunas seguras y efectivas con una sola administración,algunos de los atributos de una vacuna ideal. Existenciertos problemas tecnológicos, pero no parecen insalvables

Synthetic particles as carriers for antigens display a greatpotential and versatility for the design of effective vaccines. Supportedby multiple raw materials and preparation procedures,they can be engineered over a wide range of sizes, variable surfacenature and patterns of antigen release. It is our goal in thisreview to evaluate how these physical characteristics decide thetype of immune response the particles will develop, aiming at amore rational design of effective and safer synthetic subunit vaccines.So, as a function of their size, they will be uptaken by differentantigen presenting cell (APC) or dendritic cell (DC) subsets;certain ligands can be attached to the particle surfaces to promotereceptor-specific endocitosis and any immunopotentiatorco-encapsulated with the antigen in the same particle. The mechanismof antigen uptake and the signal for activating DC can bedecisive to increase and skew a Th1/cytotoxic or Th2/humoralresponse. Also, the antigen release rate has influence on the generationof immunological memory and an adequate profile of antigenrelease from these particles can mimic boosters of the traditionalvaccines.The current tendencies are oriented towards the developmentof new vaccines containing perfectly characterized antigens, establishedand safe, and with composition and preparation methodsrigorously controlled. The use of synthetic particles as vectorsmay be considered as adjuvants that accomplish with these requisites,being able to induce a response even at the mucosal level.There are technological handicaps faced by developers of syntheticparticles based vaccines but do not appear to be insurmountable

Modulación negativa del TCR por un mecanismo de todo-o-nada / All-or-none downregulation of the T cell antigen receptor

El contacto del TCR con MHC/antígeno resulta en su modulación y desaparición de la superficie celular. Recientemente hemos descrito que este proceso ocurre por al menos dos mecanismos: uno es dependiente de transmisión de señales, predomina a bajas concentraciones de antígeno y resulta en la modulación en trans de moléculas de TCR no contactadas. El otro requiere contacto directo y es independiente de transmisión de señales. En este artículo describimos que el TCR es modulado en una forma discontinua, es decir las células estimuladas oscilan de un estado no-modulado a otro completamente modulado sin transición aparente por estados intermedios, cuando las células T son estimuladas con altas dosis de antígeno o de anticuerpos anti-TCR. El fenómeno se reproduce cuando un receptor quimérico, que contiene la parte extracelular y transmembránica de CD8 y el tallo citoplásmico de CD3 , es entre cruzado con anticuerpos inmovilizados a un substrato. Este proceso de modulación de "todo-o-nada" no requiere de transmisión de señales o de polimerización del citoesqueleto de actina. El análisis por microscopía confocal muestra que el anticuerpo estimulante es tomado del substrato y concentrado junto con la quimera en un polo de la célula donde se constituye un sitio de nucleación para la formación de vesículas endocíticas. El efecto de "todo-o-nada" puede explicarse por la concentración lenta del TCR o de la quimera, seguido de la internalización rápida de los receptores agregados (AU)