Induction of apoptosis and enhancement of chemosensitivity in human prostate cancer LNCaP cells using bispecific antisense oligonucleotide targeting Bcl-2 and Bcl-xL genes

OBJECTIVE: To determine whether a specifically designed bispecific (Bcl-2/Bcl-xL) antisense oligonucleotide (ASO) induces apoptosis and enhances chemosensitivity in human prostate cancer LNCaP cells, as Bcl-2 and Bcl-xL are both anti-apoptotic genes associated with treatment resistance and tumour progression in many malignancies, including prostate cancer. MATERIALS AND METHODS: Inhibition of Bcl-2 and Bcl-xL expression by the bispecific ASO was evaluated using real-time reverse transcription-polymerase chain reaction and Western blotting, while growth inhibition and induction of apoptosis were analysed by a crystal violet assay, flow cytometry and Western blotting of apoptosis-relevant proteins. The effect of combined treatment with bispecific ASO and chemotherapy or small-interference RNA (siRNA) targeting the clusterin gene was also investigated. RESULTS: Bispecific ASO reduced Bcl-2 and Bcl-xL expression in LNCaP cells in a dose-dependent manner. There was cell growth inhibition, increases in the sub-G0-G1 fraction, and cleavage of caspase-3 and poly(ADP-Ribose) polymerase proteins in LNCaP cells after bispecific ASO treatment. Interestingly, Bcl-2/Bcl-xL bispecific ASO treatment also resulted in the down-regulation of Mcl-1 and up-regulation of Bax. The sensitivity of LNCaP cells to mitoxantrone, docetaxel or paclitaxel was significantly increased, reducing the 50% inhibitory concentration by 45%, 80% or 90%, respectively. Furthermore, the apoptotic induction by Bcl-2/Bcl-xL bispecific ASO was synergistically enhanced by siRNA-mediated inhibition of clusterin, a cytoprotective chaperone that interacts with and inhibits activated Bax. CONCLUSIONS: These findings support the concept of the targeted suppression of Bcl-2 anti-apoptotic family members using multitarget inhibition strategies for prostate cancer, through the effective induction of apoptosis.

Modulation of bcl-xL in tumor cells regulates angiogenesis through CXCL8 expression

In this paper, we investigated whether bcl-xL can be involved in the modulation of the angiogenic phenotype of human tumor cells. Using the ADF human glioblastoma and the M14 melanoma lines, and their derivative bcl-xL-overexpressing clones, we showed that the conditioned medium of bcl-xL transfectants increased in vitro endothelial cell functions, such as proliferation and morphogenesis, and in vivo vessel formation in Matrigel plugs, compared with the conditioned medium of control cells. Moreover, the overexpression of bcl-xL induced an increased expression of the proangiogenic interleukin-8 (CXCL8), both at the protein and mRNA levels, and an enhanced CXCL8 promoter activity. The role of CXCL8 on bcl-xL-induced angiogenesis was validated using CXCL8-neutralizing antibodies, whereas down-regulation of bcl-xL through antisense oligonucleotide or RNA interference strategies confirmed the involvement of bcl-xL on CXCL8 expression. Transient overexpression of bcl-xL led to extend this observation to other tumor cell lines with different origin, such as colon and prostate carcinoma. In conclusion, our results showed that CXCL8 modulation by bcl-xL regulates tumor angiogenesis, and they point to elucidate an additional function of bcl-xL protein.

Human midbrain precursors activate the expected developmental genetic program and differentiate long-term to functional A9 dopamine neurons in vitro. Enhancement by Bcl-XL

Understanding the molecular programs of the generation of human dopaminergic neurons (DAn) from their ventral mesencephalic (VM) precursors is of key importance for basic studies, progress in cell therapy, drug screening and pharmacology in the context of Parkinson's disease. The nature of human DAn precursors in vitro is poorly understood, their properties unstable, and their availability highly limited. Here we present positive evidence that human VM precursors retaining their genuine properties and long-term capacity to generate A9 type Substantia nigra human DAn (hVM1 model cell line) can be propagated in culture. During a one month differentiation, these cells activate all key genes needed to progress from pro-neural and prodopaminergic precursors to mature and functional DAn. For the first time, we demonstrate that gene cascades are correctly activated during differentiation, resulting in the generation of mature DAn. These DAn have morphological and functional properties undistinguishable from those generated by VM primary neuronal cultures. In addition, we have found that the forced expression of Bcl-XL induces an increase in the expression of key developmental genes (MSX1, NGN2), maintenance of PITX3 expression temporal profile, and also enhances genes involved in DAn long-term function, maintenance and survival (EN1, LMX1B, NURR1 and PITX3). As a result, Bcl-XL anticipates and enhances DAn generation.

Gestión intelectual de las prácticas comunicativas en arquitectura : S, M, L, XL, el gran evento = The intellectual management of communication practices in architecture : S, M, L, XL, the big event

La tesis doctoral se centra en la posibilidad de entender que la práctica de arquitectura puede encontrar en las prácticas comunicativas un apoyo instrumental, que sobrepasa cualquier simplificación clásica del uso de los medios como una mera aplicación superficial, post-producida o sencillamente promocional. A partir de esta premisa se exponen casos del último cuarto del siglo XX y se detecta que amenazas como el riesgo de la banalización, la posible saturación de la imagen pública o la previsible asociación incorrecta con otros individuos en presentaciones grupales o por temáticas, han podido influir en un crecimiento notable de la adquisición de control, por parte de los arquitectos, en sus oportunidades mediáticas. Esto es, como si la arquitectura hubiera empezado a superar y optimizar algo inevitable, que las fórmulas expositivas y las publicaciones, o más bien del exponer(se) y publicar(se), son herramientas disponibles para activar algún tipo de gestión intelectual de la comunicación e información circulante sobre si misma. Esta práctica de “autoedición” se analiza en un periodo concreto de la trayectoria de OMA -Office for Metropolitan Architecture-, estudio considerado pionero en el uso eficiente, oportunista y personalizado de los medios. Así, la segunda parte de la tesis se ocupa del análisis de su conocida monografía S,M,L,XL (1995), un volumen que contó con gran participación por parte de sus protagonistas durante la edición, y de cuyo proceso de producción apenas se había investigado. Esta publicación señaló un punto de inflexión en su género alterando todo formato y restricciones anteriores, y se ha convertido en un volumen emblemático para la disciplina que ninguna réplica posterior ha podido superar. Aquí se presenta a su vez como el desencadenante de la construcción de un “gran evento” que concluye en la transformación de la identidad de OMA en 10 años, paradójicamente entre el nacimiento de la Fundación Groszstadt y el arranque de la actividad de AMO, dos entidades paralelas clave anexas a OMA. Este planteamiento deviene de cómo la investigación desvela que S,M,L,XL es una pieza más, central pero no independiente, dentro de una suma de acciones e individuos, así como otras publicaciones, exposiciones, eventos y también artículos ensayados y proyectos, en particular Bigness, Generic City, Euralille y los concursos de 1989. Son significativos aspectos como la apertura a una autoría múltiple, encabezada por Rem Koolhaas y el diseñador gráfico Bruce Mau, acompañados en los agradecimientos de la editora Jennifer Sigler y cerca de una centena de nombres, cuyas aportaciones no necesariamente se basan en la construcción de fragmentos del libro. La supresión de ciertos límites permite superar también las tareas inicialmente relevantes en la edición de una publicación. Un objetivo general de la tesis es también la reflexión sobre relaciones anteriormente cuestionadas, como la establecida entre la arquitectura y los mercados o la economía. Tomando como punto de partida la idea de “design intelligence” sugerida por Michael Speaks (2001), se extrae de sus argumentos que lo esencial es el hallazgo de la singularidad o inteligencia propia de cada estudio de arquitectura o diseño. Asimismo se explora si en la construcción de ese tipo de fórmulas magistrales se alojaban también combinaciones de interés y productivas entre asuntos como la eficiencia y la creatividad, o la organización y las ideas. En esta dinámica de relaciones bidireccionales, y en ese presente de exceso de información, se fundamenta la propuesta de una equivalencia más evidenciada entre la “socialización” del trabajo del arquitecto, al compartirlo públicamente e introducir nuevas conversaciones, y la relación inversa a partir del trabajo sobre la “socialización” misma. Como si la consciencia sobre el uso de los medios pudiera ser efectivamente instrumental, y contribuir al desarrollo de la práctica de arquitectura, desde una perspectiva idealmente comprometida e intelectual. ABSTRACT The dissertation argues the possibility to understand that the practice of architecture can find an instrumental support in the practices of communication, overcoming any classical simplification of the use of media, generally reduced to superficial treatments or promotional efforts. Thus some cases of the last decades of the 20th century are presented. Some threats detected, such as the risk of triviality, the saturation of the public image or the foreseeable wrong association among individuals when they are introduced as part of thematic groups, might have encouraged a noticeable increase of command taken by architects when there is chance to intervene in a media environment. In other words, it can be argued that architecture has started to overcome and optimize the inevitable, the fact that exhibition formulas and publications, or simply the practice of (self)exhibition or (self)publication, are tools at our disposal for the activation of any kind of intellectual management of communication and circulating information about itself. This practice of “self-edition” is analyzed in a specific timeframe of OMA’s trajectory, an office that is considered as a ground-breaking actor in the efficient and opportunistic use of media. Then the second part of the thesis dissects their monograph S,M,L,XL (1995), a volume in which its main characters were deeply involved in terms of edition and design, a process barely analyzed up to now. This publication marked a turning point in its own genre, disrupting old formats and traditional restrictions. It became such an emblematic volume for the discipline that none of the following attempts of replica has ever been able to improve this precedent. Here, the book is also presented as the element that triggers the construction of a “big event” that concludes in the transformation of OMA identity in 10 years. Paradoxically, between the birth of the Groszstadt Foundation and the early steps of AMO, both two entities parallel and connected to OMA. This positions emerge from how the research unveils that S,M,L,XL is one more piece, a key one but not an unrelated element, within a sum of actions and individuals, as well as other publications, exhibitions, articles and projects, in particular Bigness, Generic City, Euralille and the competitions of 1989. Among the remarkable innovations of the monograph, there is an outstanding openness to a regime of multiple authorship, headed by Rem Koolhaas and the graphic designer Bruce Mau, who share the acknowledgements page with the editor, Jennifer Sigler, and almost 100 people, not necessarily responsible for specific fragments of the book. In this respect, the dissolution of certain limits made possible that the expected tasks in the edition of a publication could be trespassed. A general goal of the thesis is also to open a debate on typically questioned relations, particularly between architecture and markets or economy. Using the idea of “design intelligence”, outlined by Michael Speaks in 2001, the thesis pulls out its essence, basically the interest in detecting the singularity, or particular intelligence of every office of architecture and design. Then it explores if in the construction of this kind of ingenious formulas one could find interesting and useful combinations among issues like efficiency and creativity, or organization and ideas. This dynamic of bidirectional relations, rescued urgently at this present moment of excess of information, is based on the proposal for a more evident equivalence between the “socialization” of the work in architecture, anytime it is shared in public, and the opposite concept, the work on the proper act of “socialization” itself. As if a new awareness of the capacities of the use of media could turn it into an instrumental force, capable of contributing to the development of the practice of architecture, from an ideally committed and intelectual perspective.

Adaptación de GNU Radio a procesadores KeyStone II 66AK2xxx

En el presente proyecto se ha procedido a implantar la herramienta de procesado software GNU Radio en la tarjeta EVMK2H, que es un módulo de evaluación fabricado por Texas Instruments que incorpora un System on Chip (SoC) 66AK2H14 de la familia Keystone II, el cual dispone de 4 núcleos ARM y 8 núcleos DSP. Previamente a la instalación de GNU Radio, hubo que configurar la tarjeta, así como instalar el software necesario. De igual manera, se realizó una primera aproximación para comprender el funcionamiento de los sistemas de comunicación entre núcleos de que hace uso la tarjeta, y de los que se hizo uso posteriormente en el proyecto. Tras el portado de GNU Radio se ha comprobado el correcto funcionamiento del mecanismo de comunicación entre núcleos ARM y DSP con un par de aplicaciones de prueba. ABSTRACT. In the present project it was performed the implementation of the software processing toolkit GNU Radio into the EVMK2H board, which is an evaluation module from Texas Instruments that includes a 66AK2H14 System on Chip (SoC) from the Keystone II family, that provides 4 ARM cores and 8 DSP cores. Before installing GNU Radio, it was necessary to configure the board, and as well installing other needed software. Also, a first approach was performed to understand the way the communication system between cores included in the board works, which was used later in the project. After porting GNU Radio, some test applications have been written to test the correct operation of the communication mechanism between ARM and DSP cores.

Bcl-XL interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation

Recent studies indicate that Caenorhabditis elegans CED-4 interacts with and promotes the activation of the death protease CED-3, and that this activation is inhibited by CED-9. Here we show that a mammalian homolog of CED-4, Apaf-1, can associate with several death proteases, including caspase-4, caspase-8, caspase-9, and nematode CED-3 in mammalian cells. The interaction with caspase-9 was mediated by the N-terminal CED-4-like domain of Apaf-1. Expression of Apaf-1 enhanced the killing activity of caspase-9 that required the CED-4-like domain of Apaf-1. Furthermore, Apaf-1 promoted the processing and activation of caspase-9 in vivo. Bcl-XL, an antiapoptotic member of the Bcl-2 family, was shown to physically interact with Apaf-1 and caspase-9 in mammalian cells. The association of Apaf-1 with Bcl-XL was mediated through both its CED-4-like domain and the C-terminal domain containing WD-40 repeats. Expression of Bcl-XL inhibited the association of Apaf-1 with caspase-9 in mammalian cells. Significantly, recombinant Bcl-XL purified from Escherichia coli or insect cells inhibited Apaf-1-dependent processing of caspase-9. Furthermore, Bcl-XL failed to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting that Bcl-XL regulates caspase-9 through Apaf-1. These experiments demonstrate that Bcl-XL associates with caspase-9 and Apaf-1, and show that Bcl-XL inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans.

Regulation of Bcl-xL expression in human keratinocytes by cell–substratum adhesion and the epidermal growth factor receptor

Cell–substratum adhesion is an essential requirement for survival of human neonatal keratinocytes in vitro. Similarly, activation of the epidermal growth factor receptor (EGF-R) has recently been implicated not only in cell cycle progression but also in survival of normal keratinocytes. The mechanisms by which either cell–substratum adhesion or EGF-R activation protect keratinocytes from programmed cell death are poorly understood. Here we describe that blockade of the EGF-R and inhibition of substratum adhesion share a common downstream event, the down-regulation of the cell death protector Bcl-xL. Expression of Bcl-xL protein was down-regulated during forced suspension culture of keratinocytes, concurrent with large-scale apoptosis. Similarly, EGF-R blockade was accompanied by down-regulation of Bcl-xL steady-state mRNA and protein levels to an extent comparable to that observed in forced suspension culture. However, down-regulation of Bcl-xL expression by EGF-R blockade was not accompanied by apoptosis; in this case, a second signal, generated by passaging, was required to induce rapid and large-scale apoptosis. These findings are consistent with the conclusions that (i) Bcl-xL represents a shared molecular target for signaling through cell-substrate adhesion receptors and the EGF-R, and (ii) reduced levels of Bcl-xL expression through EGF-R blockade lower the tolerance of keratinocytes for cell death signals generated by cellular stress.

Activation of Fas by FasL induces apoptosis by a mechanism that cannot be blocked by Bcl-2 or Bcl-xL

Fas activation triggers apoptosis in many cell types. Studies with anti-Fas antibodies have produced conflicting results on Fas signaling, particularly the role of the Bcl-2 family in this process. Comparison between physiological ligand and anti-Fas antibodies revealed that only extensive Fas aggregation, by membrane bound FasL or aggregated soluble FasL consistently triggered apoptosis, whereas antibodies could act as death agonists or antagonists. Studies on Fas signaling in cell lines and primary cells from transgenic mice revealed that FADD/MORT1 and caspase-8 were required for apoptosis. In contrast, Bcl-2 or Bcl-xL did not block FasL-induced apoptosis in lymphocytes or hepatocytes, demonstrating that signaling for cell death induced by Fas and the pathways to apoptosis regulated by the Bcl-2 family are distinct.

Epistatic and independent functions of Caspase-3 and Bcl-XL in developmental programmed cell death

The number of neurons in the mammalian brain is determined by a balance between cell proliferation and programmed cell death. Recent studies indicated that Bcl-XL prevents, whereas Caspase-3 mediates, cell death in the developing nervous system, but whether Bcl-XL directly blocks the apoptotic function of Caspase-3 in vivo is not known. To examine this question, we generated bcl-x/caspase-3 double mutants and found that caspase-3 deficiency abrogated the increased apoptosis of postmitotic neurons but not the increased hematopoietic cell death and embryonic lethality caused by the bcl-x mutation. In contrast, caspase-3, but not bcl-x, deficiency changed the normal incidence of neuronal progenitor cell apoptosis, consistent with the lack of expression of Bcl-XL in the proliferative population of the embryonic cortex. Thus, although Caspase-3 is epistatically downstream to Bcl-XL in postmitotic neurons, it independently regulates apoptosis of neuronal founder cells. Taken together, these results establish a role of programmed cell death in regulating the size of progenitor population in the central nervous system, a function that is distinct from the classic role of cell death in matching postmitotic neuronal population with postsynaptic targets.

Xenopus Bcl-XL selectively protects Rohon-Beard neurons from metamorphic degeneration

Amphibian metamorphosis involves extensive, but selective, neuronal death and turnover, thus sharing many features with mammalian postnatal development. The antiapoptotic protein Bcl-XL plays an important role in postnatal mammalian neuronal survival. It is therefore of interest that accumulation of the mRNA encoding the Xenopus Bcl-XL homologue, termed xR11, increases abruptly in the nervous system, but not in other tissues, during metamorphosis in Xenopus tadpoles. This observation raises the intriguing possibility that xR11 selectively regulates neuronal survival during postembryonic development. To investigate this hypothesis, we overexpressed xR11 in vivo as a green fluorescent protein (GFP)-xR11 fusion protein by using somatic and germinal transgenesis. Somatic gene transfer showed that the fusion protein was effective in counteracting, in a dose-dependent manner, the proapoptotic effects of coexpressed Bax. When GFP-xR11 was expressed from the neuronal β-tubulin promoter by germinal transgenesis we observed neuronal specific expression that was maintained throughout metamorphosis and beyond, into juvenile and adult stages. Confocal microscopy showed GFP-xR11 to be exclusively localized in the mitochondria. Our findings show that GFP-xR11 significantly prolonged Rohon-Beard neuron survival up to the climax of metamorphosis, even in the regressing tadpole tail, whereas in controls these neurons disappeared in early metamorphosis. However, GFP-xR11 expression did not modify the fate of spinal cord motoneurons. The selective protection of Rohon-Beard neurons reveals cell-specific apoptotic pathways and offers approaches to further analyze programmed neuronal turnover during postembryonic development.

Christ and the nations : one of a series of Keystone graded elective courses for young people and adults, for use in Sunday school classes, brotherhoods, young people's societies, and other study groups /

Mode of access: Internet.

De la necessite de l'Appel des Eglises de France au futur Concile general de la Constitution Unigenitus : pour la défense de l'ancienne doctrine, de la morale, de la discipline & de la police de l'Eglise, & de la liberté des Ecoles catholiques, attaquées par cette Constitution, & par l'Instruction Pastorale de l' Assemblee des XL. Evêques.

Mode of access: Internet.

Los XL libros d'el compendio historial de las chronicas y vniuersal historia de todos los reynos de España /

Contiene: Los XL libros d'el compendio historial de las chronicas ..., con port. común a toda la obra ; Compendio historial de las chronicas y vniuersal historia de todos los Reynos d'España, donde se escriuen las vidas de los Reyes de Castilla y Leon : prosiguese tambien la sucession de los emperadores occidentales y orientales ..., con port. propia ; Compendio historial de las chronicas y vniuersal historia de todos los reynos d'España donde se escriuen las vidas de los Reyes de Nauarra : escrivese tambien la sucession de todos los reyes de Francia y obispos de la Santa Yglesia de Pamplona ..., con port. propia (iniciando una nueva secuencia de pag.) ; Compendio historial de las chronicas y vniuersal historia de todos los Reynos d'España donde se ponen en summa los condes, señores de Aragon, con los Reyes d'el mesmo reyno y condes de Barcelona, y Reyes de Napoles y Sicilia : en la fin d'estos Principes se escriue vn breue tratado de las insignias y deuisas de los escudos de armas ..., con port. (sin pie de imp.) y colofón conjunto al final de la edición.

S. Aurelii Augustini Hipponensis Episcopi Sermones Noui : numero XL : ex diuersis antiquis exemplaribus collecti /

Mode of access: Internet.