Desarrollo de aplicaciones android usando la tecnología wifi-direct

Este proyecto de fin de grado pretende demostrar la importancia y la utilidad de la creación de redes de dispositivos móviles conectados entre sí. Para ello se explicarán varios tipos de redes inalámbricas que permiten estas conexiones directas entre dispositivos sin la necesidad de un servidor. En estas redes inalámbricas se destacan las redes P2P y las redes Ad-hoc, las cuales se explicarán posteriormente. El despliegue de estas redes se puede encontrar en un amplio rango de campos como puede ser la agricultura, la medicina e incluso en el ámbito militar. Es objetivo de este proyecto, además, el estudio de la tecnología Wi-Fi Direct creada por la Wi-Fi Alliance. Como se explicará a lo largo del proyecto, Wi-Fi Direct está basado en las redes P2P. Esta tecnología permite a los dispositivos cercanos crear redes P2P a través de la red Wi-Fi sin la necesidad de un punto de acceso a Internet. Por otro lado, una gran cantidad de los dispositivos móviles que existen actualmente poseen el sistema operativo Android. Android ha incorporado en sus dispositivos más recientes la tecnología Wi-Fi Direct. Debido a ello han ido surgiendo aplicaciones que usando esta tecnología consiguen desde enviar ficheros hasta indicar la localización de un usuario. Esta tecnología combinada con este tipo de dispositivos puede ser muy útil para utilizar en casos de emergencia donde las infraestructuras de comunicaciones no estén disponibles ya que al no necesitar un punto de acceso a internet es posible la comunicación entre un usuario en peligro y otro que se encuentre dentro de un radio cercano. Por estos motivos otro de los principales objetivos de este proyecto es la implementación de una aplicación para dispositivos Android que use la tecnología Wi-Fi Direct para realizar varias funcionalidades diferentes, como es el intercambio de ficheros entre dispositivos y la creación de un chat para la comunicación simultanea entre dos dispositivos. Con esto se pretende conocer mejor el funcionamiento de la tecnología Wi-Fi Direct y demostrar su utilidad en los dispositivos móviles como son los dispositivos Android. ABSTRACT. This final degree Project tries to demonstrate the importance and utility of networking mobile devices. For this purpose several types of wireless networks will be explained. These networks allow direct connections between devices. The most prominent Wireless networks are P2P and Ad-hoc which will be explained later. The use of these networks can be found in a wide range of fields such as agriculture medicine, and even in the military sector. Besides, other aim of this project is the study of Wi-Fi Direct Technology which is created by Wi-Fi Alliance. As it explained along the project, Wi-Fi Direct is based on P2P networks. This technology lets nearby devices create P2P networks through Wi-Fi network without an Internet access point. On the other hand, a large number of mobile devices have the Android OS. Android has integrated Wi-Fi Direct technology in its latest devices. Because of this applications have emerged that using this technology they get from sending files to send the user’s location. This technology combined with these devices can be very useful to use in emergencies where communications infrastructures are not available. Since not need an Internet access point, communication between a user in danger and another within close radius is possible. For these reasons another of the main aims of this project is the implementation of an application for Android devices which use Wi-Fi Direct technology to perform several different functionalities, such as file exchange or chat for simultaneous communication between devices. This is intended to better understand the operation of Wi-Fi Direct technology and prove its utility on mobile devices such as Android devices.

Rem : a los dos lados del espejo = (Rem at both sides of the mirror)

Cuando se trata de Rem Koolhaas, su espejo no refleja una sola imagen sino múltiples, es un prisma poliédrico. Su espejo nos devuelve el Rem mediático, el intelectual, el conceptualizador, el constructor, el analista, el periodista, el actor... En el caso de esta investigación, fijamos el punto de mira en el Rem COMUNICADOR. “Rem a los dos lados del espejo” se enmarca en una investigación sobre los medios de comunicación de arquitectura, su reflejo en la producción arquitectónica y viceversa. Se trata de llegar a discernir si comunicación y producción arquitectónica colisionan y confluyen en el caso de grandes comunicadores como Rem Koolhaas, si el mensaje y el medio transmisor adquieren las mismas cualidades. Centrándose en la figura de Rem Koolhaas, la tesis aborda la evolución de su faceta comunicativa y las transformaciones sucesivas en el campo de la comunicación arquitectónica, en paralelo a su evolución conceptual a lo largo de su trayectoria. La investigación, por tanto, no se centra tanto en su componente teórica o en la práctica arquitectónica de OMA, sino en la exposición de su producción al mundo, especialmente a través de sus ensayos y libros. “Delirious New York” y “SMLXL” son un reflejo del momento conceptual en que se inscriben, y contienen mucha información sobre los referentes gráficos que irremediablemente han influido en su composición. Especialmente, la aparición de “SMLXL” supuso un revulsivo para el mundo de la comunicación arquitectónica, porque puso el foco sobre la importancia de dejar atrás un discurso narrativo linea y unifocal, para afrontar la comunicación barajando múltiples variables, y aproximaciones, en un proceso similar al desarrollo de un proyecto de arquitectura. Presenta un diseño muy novedoso y una edición extremadamente cuidada, que atiende a parámetros mucho más ambiciosos que los meramente narrativos. Profundiza en la necesidad de una temática global, planteando cuál es la aproximación más apropiada para cada uno de los proyectos que describe, transmitiendo al lector una percepción más allá de lo estrictamente visual, más próximo a lo sensorial. Además, su enorme repercusión a nivel internacional y el gran interés que despertó (no solamente entre los arquitectos, sino también entre diseñadores gráficos, publicistas, personas provenientes de todo tipo de tendencias artísticas y público en general), provocó la globalización del fenómeno de las publicaciones arquitectónicas y puso de manifiesto la importancia de la comunicación como una disciplina en sí misma, dentro de la producción arquitectónica en la era actual. A pesar de la importancia de “SMLXL” a todos los niveles, la presente tesis plantea que, donde realmente se culmina esa experiencia comunicativa, es en “Content”, al incluir nuevos parámetros relacionados con la fusión conceptual de continente y contenido. Es en esta publicación donde el objeto de la comunicación y la expresión de la misma se convierten en un único elemento, que se rige por leyes similares. En este caso, la ley fundamental es la aplicación hasta sus máximas consecuencias de la “cultura de la congestión”, tanto en el mensaje como en el medio, generando lo que hemos convenido en denominar “comunicación congestiva”. Esta concepción deviene en que necesariamente se materialice como un producto efímero, desechable, casi virtual, porque responde a las condiciones de un momento muy concreto y específico y fuera de ese contexto pierde su significación.. La “cultura de la congestión” empieza a surgir en los planteamientos de Koolhaas en la Architectural Association School of Architecture de Londres, bajo la tutela de Elia Zenghelis. Posteriormente se desarrolla en su manifiesto retroactivo sobre Manhattan, “Delirious New York”, donde declara la guerra abierta al urbanismo del movimiento moderno y afirma que la ciudad realmente contemporánea es aquella que es fruto de un desarrollo no planificado, hiperdensa y posible gracias a los avances tecnológicos de su era. Finalmente comienza a materializarse en la Diploma Unit 9 de la AA, donde entra como profesor en 1975, dejando una huella indeleble en las generaciones posteriores de arquitectos que pasaron dicha unidad. Rem Koolhaas es ante todo un intelectual y por ello, todo el constructo teórico sobre la metrópolis comienza a reflejarse en su obra a través de OMA desde el comienzo de su producción. Podemos decir a grandes rasgos que su carrera está marcada por dos hitos históricos fundamentales que determinan tres etapas diferenciadas en su producción. En sus primeros años de profesión, Koolhaas sigue fascinado por la metrópolis urbana y la aplicación del método paranoico crítico a su producción arquitectónica. Es un arquitecto profundamente surrealista. Entiende este método como una estrategia de conocimiento y aproximación al mundo que le rodea: “dejar salir el inconsciente pero sostenerlo con las muletas de la racionalidad”. Pero lo que en realidad le interesa es su aplicación a la gran escala, el “Bigness”, y por ello, participa en proyectos muy ambiciosos de los que surgen conceptos que, más allá de resultar premiados o no, han dejado una huella ideológica en el devenir de la arquitectura. Entre estos proyectos, cabe destacar su propuesta para el Parque de la Villette o la Très Grande Bibliotèque de París. Sus proyectos de esta época destilan una gran carga conceptual, que devienen en unos interiores sorprendentes pero una apariencia exterior sobria o incluso podríamos decir "povera", por el uso de materiales efímeros, poco habituales en la macro-arquitectura hasta ese momento. Súbitamente, en 1997, explotó el denominado “Efecto Bilbao”, de la mano de Frank Gehry (1). El Museo Guggenheim de Bilbao, con su espectacularidad, sus formas pregnantes e imposibles, impacta al mundo. Nace la era de la “Arquitectura del Espectáculo”; la transformación de la ciudad a través de ICONOS que actúen como nodos de atracción y concentración en torno a los cuales supuestamente se revitaliza la actividad económica, cultural y sociopolítica de la ciudad, como si a través de un único gesto se pudieran regenerar todos los tejidos internos de la urbe. Rem Koolhaas comprende rápidamente que la aproximación a la ciudad ha cambiado y, sobre todo, el mercado. En el mundo de la globalización, la única manera de llegar a materializar el “Bigness”, es encerrando sus ejercicios intelectuales en formas pregnantes, bellas, icónicas, espectaculares. Koolhaas encuentra su marca personal en la estética “Stealth”, proveniente de los aviones de combate facetados para evitar los radares, elaborados en los años 80. De esta época surgen proyectos como la Casa da Música de Oporto o la Biblioteca de Seattle; ambos edificios son iconos facetados, de belleza pregnante, que dejan una huella indeleble en la ciudad y provocan, al igual que el Guggenheim, un cierto efecto de recuperación y revitalización en el entorno en que se asientan, al menos de manera temporal. En cualquier caso, Koolhaas nunca abandona los ejercicios meramente teóricos, pero segrega su actividad en dos: OMA produce aquello que tiene vocación de ser construido y se rige por los parámetros del mercado global y AMO, la otra cara del espejo de Rem, aplica el pensamiento arquitectónico a campos no explorados, sin la dependencia de agentes externos, pudiendo permitirse ser un laboratorio puramente experimental. En este escenario, llega el 11 de septiembre de 2001 y el ataque a las Torres Gemelas de Nueva York tiene efectos devastadores a todos los niveles, significando, en un período de tiempo sorprendentemente corto, un cambio en el orden mundial. Rem Koolhaas da entonces un giro de 180 grados, dirige su mirada hacia China, donde entiende que sus aportaciones tienen un beneficio social más directo que en occidente. (2) Para presentar al mundo su nuevo cambio de rumbo y la creación del “Think Tank” AMO, plantea una gran exposición en la NeueGallerie de Berlín bajo el título de “Content”, experiencia paralela a la edición del libro con el mismo título, que inicialmente nace como “catálogo de la exposición, pero que internamente siempre se concibió como el documento más trascendente en el estudio desde “SMLXL”. Sin embargo, en muchos aspectos se trata de su opuesto: una publicación con formato revista, de tapa blanda, con paginado muy fino, formato de "folleto de supermercado" y contenido hiperdenso. Es un experimento efímero, fugaz, ligero, barato, de “usar y tirar”. De hecho, está fuera de stock, ya no se edita. Probablemente Rem Koolhaas desaprobaría que se hiciera una investigación que pusiera el foco sobre el mismo, porque diez años después de su publicación seguramente opine que su vigencia ha caducado. Sin embargo, muestra con una claridad meridiana el estado conceptual y vital de OMA en el momento de su publicación y representa, además un verdadero hito en la comunicación arquitectónica, un punto de no retorno, el máximo exponente de lo que hemos denominado “comunicación congestiva”. La presente tesis plantea que “Content” contiene la esencia de la mayor aportación de Rem Koolhaas al mundo de la arquitectura: la transformación profunda y definitiva de la comunicación arquitectónica mediante la convergencia del estado conceptual y la transmisión del mismo. Su legado arquitectónico y conceptual ha marcado a todas las generaciones posteriores de manera indeleble. Sus ensayos, sus teorías, sus proyectos y sus edificaciones ya pertenecen a la historia de la arquitectura, sin ninguna duda. Pero es su revisión del concepto de la comunicación en arquitectura lo que ha tenido y tendrá un reflejo inmediato en las generaciones futuras, no solamente en la comunicación sino en su arquitectura, a través de un intercambio biyectivo. El planteamiento a futuro sería determinar qué sucede tras “Content”, tras la hiperdensidad máxima, tras la cultura de la congestión visual; qué es lo que propone Koolhaas y qué se va a plantear también en el mundo de la comunicación arquitectónica. Para ello, estudiaremos en profundidad sus últimos proyectos relacionados con la comunicación, como su propuesta para la Biennale de Arquitectura de Venecia de 2014, su intensa investigación sobre el “Metabolismo” en “Project Japan: Metabolism Talks...”, o la dirección de sus últimos planteamientos territoriales. En los últimos tiempos Rem Koolhaas habla de “Preservación”, de “Sobriedad”, de “Esencialismo”, de “Performance”... El autor intelectual de la cultura de la congestión habla ahora de la “low density”...como no podía ser de otra manera en la otra cara del espejo. En definitiva, el color blanco como suma de todos los colores, todas las longitudes de onda del espectro visible recibidas al tiempo. ABSTRACT When talking about Rem Koolhaas, the mirror does not only reflect one but numerous images: it is nothing but a polyhedral prism. His mirror gives us the image of Rem the media celebrity, the intellectual, the conceptualizer, the builder, the analyst, the journalist, the actor... This research sets the spotlight on Rem the COMMUNICATOR. "Rem on both sides of the mirror" belongs to a research on architectural media, its influence on the architectural production and vice versa. It is aimed at getting to discern whether communication and architectural production collide and converge in the case of great communicators such as Rem Koolhaas, and whether the message and transmission media acquire the same features. Focusing on the figure of Rem Koolhaas, this thesis addresses the evolution of his communicative facet and the successive transformations in the field of architectural communication, parallel to the conceptual evolution he underwent throughout his career. Therefore, this research is not so much focused on his theoretical component or on the OMA’s architectural practice, but on the exhibition of his production to the world, especially through his essays and books. "Delirious New York" and "SMLXL" hold up a mirror to the conceptual moment they are part of, and contain a great deal of information about the graphic references that have inevitably influenced his work. Specially, the launch of "SMLXL" was a salutary shock for the architectural communication world, since it set the spotlight on the importance of leaving a linear and unifocal narrative behind in order to face communication considering multiple variables and approaches, based on a process similar to the development of an architectural project. It offers a very innovative design and an extremely careful editing, which deals with parameters much more ambitious than those merely narrative. It explores the need for a global subject and suggests the most appropriate approach for each of the projects described, giving the reader a closer insight to the sensory that goes beyond what’s strictly visual. In addition, its huge international impact and the great interest shown, not only by architects but also by graphic designers, publishers, people from all kinds of artistic trends and the general public, led to the globalisation of the architectural publications phenomenon and brought the importance of communication as a discipline in itself, within the architectural production in the age at hand, to light. Despite the importance of "SMLXL" at all levels, this thesis suggests that the communication experience really culminates in "Content", for it includes new conceptual parameters associated with the container-content conceptual fusion. It is in this book where the purpose of communication and the expression of such become a single element, ruled by similar laws. In this particular case, the fundamental law is to implement the "culture of congestion" to its extreme consequences in both the message and the media, leading to what we have agreed to refer to as "congestive communication”. This concept leads to its inevitable materialisation into an ephemeral, disposable, almost virtual product, because it meets the conditions of a very concrete and specific time, and outside that context it loses its significance. The "culture of congestion" emerged in Koolhaas’ approaches under the guidance of Elia Zenghelis, in the Architectural Association School of Architecture of London. Subsequently, his retroactive manifesto on Manhattan, "Delirious New York" developed it, waging an all-out war against the modern movement urbanism and maintaining that the really contemporary cities are those hyperdense ones that rise as a result of an unplanned development and thanks to the typical technological advances of their time. Finally it began to materialise in the Diploma Unit 9 of the AA, in which he started lecturing in 1975, leaving an indelible mark on subsequent generations of architects who passed that unit. First and foremost, Rem Koolhaas is an intellectual and, therefore, all the theoretical construct in the metropolis began to be reflected in his work through OMA since the beginnings of his production. Broadly speaking, we can say that his career is influenced by two essential historic events, which determine three different stages in his production. In the early years of his career, Koolhaas was still fascinated by the urban metropolis and the implementation of the paranoiac-critical method to his architectural production. He was then a deeply surreal architect. He understood this method as a knowledge strategy and an approach to the world around him: "let the subconscious out but hold it with the crutches of reasonableness”. However, he was actually interested in its implementation on a broad scale, the "Bigness", and therefore, he took part in ambitious projects that led to the accrual of concepts that, beyond being rewarded, left an ideological impression on the evolution of architecture. These projects included his proposal for the Parc de la Villette or the Très Grande Bibliotèque in Paris. The projects he carried out during this period showed a great conceptual background, which evolved into surprising interiors but a sober, or even "povera", exterior appearance, thanks to the use of ephemeral materials that were atypical in the macro-architecture field until that moment. Suddenly, in 1997, the so-called "Bilbao effect" boomed thanks to Frank Gehry (1). The Guggenheim Museum of Bilbao amazed the world with its spectacular nature and its pregnant and impossible shapes. It was the beginning of the era of “The architecture of spectacle”: the transformation of the city through ICONS that would act as nodes of attraction and gathering, around which the economic, cultural and socio-political activity of the city was supposed to be revitalized, as if through a single gesture all internal tissues of the city could be rebuilt. Rem Koolhaas quickly realized that the approach to the city, and especially to the global market, had changed. In the world of globalisation, the only way to get to materialise such "Bigness" was by keeping his intellectual exercises in pregnant, beautiful, iconic and spectacular shapes. Koolhaas found his personal brand in the Stealth aesthetic, resulting from the eighties American combat aircrafts whose shape was faceted in order to avoid radars. Projects such as the Casa da Música in Porto or the Seattle Library date from this period; both buildings are faceted icons of pregnant beauty that left an indelible mark on the city and caused, like the Guggenheim, some degree of recovery and revitalization on the environment in which they were based, at least temporarily. In any case, Koolhaas never gave the merely theoretical exercises up, but he segregated his work in two: OMA produced what was destined to be built and ruled by the parameters of the global market and AMO, Rem’s other side of the mirror, applied the architectural thought in unexplored fields, notwithstanding external agents and being able to work as a purely experimental laboratory. In light of this backdrop, September 11th 2001 came and the attacks on the Twin Towers in New York had devastating effects at all levels, leading to a change in the world order, in a surprisingly short period of time. Rem Koolhaas made a 180° turn directing his vision towards China, where he believed his contributions would have a more direct social benefit than in the Western world. (2) In order to introduce his new course of direction and the creation of the AMO "Think Tank", he planned a major exhibition in the Neue Nationalgalerie of Berlin under the title "Content", in parallel with edition of the book with the same title, which was at first the "exhibition catalog” but, deep down, was always conceived as the most important document of the Office since "SMLXL". However, in many ways it was just the opposite: a publication characterised by its magazine format, soft cover, very fine paging, "supermarket brochure" form and hyperdense content. It was an ephemeral, brief, light, cheap and "disposable" experiment. In fact, it is currently out of stock and out of print. Rem Koolhaas would probably disapprove of a research that sets the spotlight on him, for he would probably say that his validity has expired given that it has been ten years since its publication. However, it shows OMA’s conceptual and vital status at the time of its publication with crystalline clarity and it is also a true milestone in architectural communication. A point of no return. The epitome of the so-called "congestive communication ". This thesis suggests that "Content" contains the essence of Rem Koolhaas’ greatest contribution to the world of architecture: the deep and definitive transformation of architectural communication through the convergence of the conceptual state and the transmission thereof. His architectural and conceptual legacy has left an indelible mark on all subsequent generations. There is no doubt his essays, theories, projects and buildings already belong to the history of architecture. But it is his review on the concept of communication in architecture that has had and shall have an immediate influence on future generations, not only in their communication but also in their architecture, through a bijective exchange. Future approaches should try to determine what happens after "Content", after the maximum hyperdensity, after the visual culture of congestion; what shall Koolhaas suggest as well as what shall happen in the world of architectural communication. To this end, we shall study his latest communication-related projects, such as the design of the Venetian Architecture Biennale in 2014, his intensive research on the "Metabolism" in "Project Japan: Metabolism Talks ...", or the course of his latest territorial approaches in depth. Most recently, Rem Koolhaas has talked about "Preservation", "Sobriety" of "Essentialism", "Performance", etc. The mastermind of the culture of congestion now speaks of the "low density"... as it could not be otherwise, on the other side of the mirror. Summarizing, the white color as the sum of all colors; all wavelengths of the visible spectrum received at the same time.

Neurotrophic factors [activity-dependent neurotrophic factor (ADNF) and basic fibroblast growth factor (bFGF)] interrupt excitotoxic neurodegenerative cascades promoted by a PS1 mutation

Although an excitotoxic mechanism of neuronal injury has been proposed to play a role in chronic neurodegenerative disorders such as Alzheimer’s disease, and neurotrophic factors have been put forward as potential therapeutic agents, direct evidence is lacking. Taking advantage of the fact that mutations in the presenilin-1 (PS1) gene are causally linked to many cases of early-onset inherited Alzheimer’s disease, we generated PS1 mutant knock-in mice and directly tested the excitotoxic and neurotrophic hypotheses of Alzheimer’s disease. Primary hippocampal neurons from PS1 mutant knock-in mice exhibited increased production of amyloid β-peptide 42/43 and increased vulnerability to excitotoxicity, which occurred in a gene dosage-dependent manner. Neurons expressing mutant PS1 exhibited enhanced calcium responses to glutamate and increased oxyradical production and mitochondrial dysfunction. Pretreatment with either basic fibroblast growth factor or activity-dependent neurotrophic factor protected neurons expressing mutant PS1 against excitotoxicity. Both basic fibroblast growth factor and activity-dependent neurotrophic factor stabilized intracellular calcium levels and abrogated the increased oxyradical production and mitochondrial dysfunction otherwise caused by the PS1 mutation. Our data indicate that neurotrophic factors can interrupt excitotoxic neurodegenerative cascades promoted by PS1 mutations.

Direct adenovirus-mediated gene transfer of interleukin 1 and tumor necrosis factor α soluble receptors to rabbit knees with experimental arthritis has local and distal anti-arthritic effects

Adenoviral vectors were used to deliver genes encoding a soluble interleukin 1 (IL-1)-type I receptor-IgG fusion protein and/or a soluble type I tumor necrosis factor α (TNFα) receptor-IgG fusion protein directly to the knees of rabbits with antigen-induced arthritis. When tested individually, knees receiving the soluble IL-1 receptor had significantly reduced cartilage matrix degradation and white blood cell infiltration into the joint space. Delivery of the soluble TNFα receptor was less effective, having only a moderate effect on white blood cell infiltration and no effect on cartilage breakdown. When both soluble receptors were used together, there was a greater inhibition of white blood cell infiltration and cartilage breakdown with a considerable reduction of synovitis. Interestingly, anti-arthritic effects were also seen in contralateral control knees receiving only a marker gene, suggesting that sustained local inhibition of disease activity in one joint may confer an anti-arthritic effect on other joints. These results suggest that local intra-articular gene transfer could be used to treat systemic polyarticular arthritides.

A role for TFIIH in controlling the activity of early RNA polymerase II elongation complexes

TFIIH is a multifunctional RNA polymerase II transcription factor that possesses DNA-dependent ATPase, DNA helicase, and protein kinase activities. Previous studies have established that TFIIH enters the preinitiation complex and fulfills a critical role in initiation by catalyzing ATP-dependent formation of the open complex prior to synthesis of the first phosphodiester bond of nascent transcripts. In this report, we present direct evidence that TFIIH also controls RNA polymerase II activity at a postinitiation stage of transcription, by preventing premature arrest by very early elongation complexes just prior to their transition to stably elongating complexes. Unexpectedly, we observe that TFIIH is capable of entering the transcription cycle not only during assembly of the preinitiation complex but also after initiation and synthesis of as many as four to six phosphodiester bonds. These findings shed new light on the role of TFIIH in initiation and promoter escape and reveal an unanticipated flexibility in the ability of TFIIH to interact with RNA polymerase II transcription intermediates prior to, during, and immediately after initiation.

On the reaction mechanism of l-lactate oxidase: Quantitative structure-activity analysis of the reaction with para-substituted l-mandelates

The rate constants for reduction of the flavoenzyme, l-lactate oxidase, and a mutant (in which alanine 95 is replaced by glycine), by a series of para-substituted mandelates, in both the 2-1H- and 2-2H- forms, have been measured by rapid reaction spectrophotometry. In all cases, significant isotope effects (1H/2H = 3–7) on the rate constants of flavin reduction were found, indicating that flavin reduction is a direct measure of α-C-H bond breakage. The rate constants show only a small influence of the electronic characteristics of the substituents, but show a good correlation when combined with some substituent volume parameters. A surprisingly good correlation is found with the molecular mass of the substrate. The results are compatible with any mechanism in which there is little development of charge in the transition state. This could be a transfer of hydride to the flavin N(5) position or a synchronous mechanism in which the α-C-H is formally abstracted as a H+ while the resulting charge is simultaneously neutralized by another event.

CP12 provides a new mode of light regulation of Calvin cycle activity in higher plants

CP12 is a small nuclear encoded chloroplast protein of higher plants, which was recently shown to interact with NAD(P)H–glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.13), one of the key enzymes of the reductive pentosephosphate cycle (Calvin cycle). Screening of a pea cDNA library in the yeast two-hybrid system for proteins that interact with CP12, led to the identification of a second member of the Calvin cycle, phosphoribulokinase (PRK; EC 2.7.1.19), as a further specific binding partner for CP12. The exchange of cysteines for serines in CP12 demonstrate that interaction with PRK occurs at the N-terminal peptide loop of CP12. Size exclusion chromatography and immunoprecipitation assays reveal the existence of a stable 600-kDa PRK/CP12/GAPDH complex in the stroma of higher plant chloroplasts. Its stoichiometry is proposed to be of two N-terminally dimerized CP12 molecules, each carrying one PRK dimer on its N terminus and one A2B2 complex of GAPDH subunits on the C-terminal peptide loop. Incubation of the complex with NADP or NADPH, in contrast to NAD or NADH, causes its dissociation. Assays with the stromal 600-kDa fractions in the presence of the four different nicotinamide-adenine dinucleotides indicate that PRK activity depends on complex dissociation and might be further regulated by the accessible ratio of NADP/NADPH. From these results, we conclude that light regulation of the Calvin cycle in higher plants is not only via reductive activation of different proteins by the well-established ferredoxin/thioredoxin system, but in addition, by reversible dissociation of the PRK/CP12/GAPDH complex, mediated by NADP(H).

Protein S is inducible by interleukin 4 in T cells and inhibits lymphoid cell procoagulant activity

Extravascular procoagulant activity often accompanies cell-mediated immune responses and systemic administration of pharmacologic anticoagulants prevents cell-mediated delayed-type hypersensitivity reactions. These observations suggest a direct association between coagulation and cell-mediated immunity. The cytokine interleukin (IL)-4 potently suppresses cell-mediated immune responses, but its mechanism of action remains to be determined. Herein we demonstrate that the physiologic anticoagulant protein S is IL-4-inducible in primary T cells. Although protein S was known to inhibit the classic factor Va-dependent prothrombinase assembled by endothelial cells and platelets, we found that protein S also inhibits the factor Va-independent prothrombinase assembled by lymphoid cells. Thus, protein S-mediated down-regulation of lymphoid cell procoagulant activity may be one mechanism by which IL-4 antagonizes cell-mediated immunity.

Guanine nucleotide exchange factor GEF115 specifically mediates activation of Rho and serum response factor by the G protein α subunit Gα13

Signal transduction pathways that mediate activation of serum response factor (SRF) by heterotrimeric G protein α subunits were characterized in transfection systems. Gαq, Gα12, and Gα13, but not Gαi, activate SRF through RhoA. When Gαq, α12, or α13 were coexpressed with a Rho-specific guanine nucleotide exchange factor GEF115, Gα13, but not Gαq or Gα12, showed synergistic activation of SRF with GEF115. The synergy between Gα13 and GEF115 depends on the N-terminal part of GEF115, and there was no synergistic effect between Gα13 and another Rho-specific exchange factor Lbc. In addition, the Dbl-homology (DH)-domain-deletion mutant of GEF115 inhibited Gα13- and Gα12-induced, but not GEF115 itself- or Gαq-induced, SRF activation. The DH-domain-deletion mutant also suppressed thrombin- and lysophosphatidic acid-induced SRF activation in NIH 3T3 cells, probably by inhibition of Gα12/13. The N-terminal part of GEF115 contains a sequence motif that is homologous to the regulator of G protein signaling (RGS) domain of RGS12. RGS12 can inhibit both Gα12 and Gα13. Thus, the inhibition of Gα12/13 by the DH-deletion mutant may be due to the RGS activity of the mutant. The synergism between Gα13 and GEF115 indicates that GEF115 mediates Gα13-induced activation of Rho and SRF.

An inhibitor of HIV-1 protease modulates proteasome activity, antigen presentation, and T cell responses

Inhibitors of the protease of HIV-1 have been used successfully for the treatment of HIV-1-infected patients and AIDS disease. We tested whether these protease inhibitory drugs exerted effects in addition to their antiviral activity. Here, we show in mice infected with lymphocytic choriomeningitis virus and treated with the HIV-1 protease inhibitor ritonavir a marked inhibition of antiviral cytotoxic T lymphocyte (CTL) activity and impaired major histocompatibility complex class I-restricted epitope presentation in the absence of direct effects on lymphocytic choriomeningitis virus replication. A potential molecular target was found: ritonavir selectively inhibited the chymotrypsin-like activity of the 20S proteasome. In view of the possible role of T cell-mediated immunopathology in AIDS pathogenesis, the two mechanisms of action (i.e., reduction of HIV replication and impairment of CTL responses) may complement each other beneficially. Thus, the surprising ability of ritonavir to block the presentation of antigen to CTLs may possibly contribute to therapy of HIV infections but potentially also to the therapy of virally induced immunopathology, autoimmune diseases, and transplantation reactions.

Isolation of a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP ribosylation factor (ARF) 1 and ARF3 that contains a Sec7-like domain

Brefeldin A (BFA) inhibited the exchange of ADP ribosylation factor (ARF)-bound GDP for GTP by a Golgi-associated guanine nucleotide-exchange protein (GEP) [Helms, J. B. & Rothman, J. E. (1992) Nature (London) 360, 352–354; Donaldson, J. G., Finazzi, D. & Klausner, R. D. (1992) Nature (London) 360, 350–352]. Cytosolic ARF GEP was also inhibited by BFA, but after purification from bovine brain and rat spleen, it was no longer BFA-sensitive [Tsai, S.-C., Adamik, R., Moss, J. & Vaughan, M. (1996) Proc. Natl. Acad. Sci. USA 93, 305–309]. We describe here purification from bovine brain cytosol of a BFA-inhibited GEP. After chromatography on DEAE–Sephacel, hydroxylapatite, and Mono Q and precipitation at pH 5.8, GEP was eluted from Superose 6 as a large molecular weight complex at the position of thyroglobulin (≈670 kDa). After SDS/PAGE of samples from column fractions, silver-stained protein bands of ≈190 and 200 kDa correlated with activity. BFA-inhibited GEP activity of the 200-kDa protein was demonstrated following electroelution from the gel and renaturation by dialysis. Four tryptic peptides from the 200-kDa protein had amino acid sequences that were 47% identical to sequences in Sec7 from Saccharomyces cerevisiae (total of 51 amino acids), consistent with the view that the BFA-sensitive 200-kDa protein may be a mammalian counterpart of Sec7 that plays a similar role in cellular vesicular transport and Sec7 may be a GEP for one or more yeast ARFs.

Cloning and expression of a cDNA encoding a bovine brain brefeldin A-sensitive guanine nucleotide-exchange protein for ADP-ribosylation factor

A 200-kDa guanine nucleotide-exchange protein (p200 or GEP) for ADP-ribosylation factors 1 and 3 (ARF1 and ARF3) that was inhibited by brefeldin A (BFA) was purified earlier from cytosol of bovine brain cortex. Amino acid sequences of four tryptic peptides were 47% identical to that of Sec7 from Saccharomyces cerevisiae, which is involved in vesicular trafficking in the Golgi. By using a PCR-based procedure with two degenerate primers representing sequences of these peptides, a product similar in size to Sec7 that contained the peptide sequences was generated. Two oligonucleotides based on this product were used to screen a bovine brain library, which yielded one clone that was a partial cDNA for p200. The remainder of the cDNA was obtained by 5′ and 3′ rapid amplification of cDNA ends (RACE). The ORF of the cDNA encodes a protein of 1,849 amino acids (≈208 kDa) that is 33% identical to yeast Sec7 and 50% identical in the Sec7 domain region. On Northern blot analysis of bovine tissues, a ≈7.4-kb mRNA was identified that hybridized with a p200 probe; it was abundant in kidney, somewhat less abundant in lung, spleen, and brain, and still less abundant in heart. A six-His-tagged fusion protein synthesized in baculovirus-infected Sf9 cells demonstrated BFA-inhibited GEP activity, confirming that BFA sensitivity is an intrinsic property of this ARF GEP and not conferred by another protein component of the complex from which p200 was originally purified.

Direct repeat sequences in the Streptomyces chitinase-63 promoter direct both glucose repression and chitin induction

The chi63 promoter directs glucose-sensitive, chitin-dependent transcription of a gene involved in the utilization of chitin as carbon source. Analysis of 5′ and 3′ deletions of the promoter region revealed that a 350-bp segment is sufficient for wild-type levels of expression and regulation. The analysis of single base changes throughout the promoter region, introduced by random and site-directed mutagenesis, identified several sequences to be important for activity and regulation. Single base changes at −10, −12, −32, −33, −35, and −37 upstream of the transcription start site resulted in loss of activity from the promoter, suggesting that bases in these positions are important for RNA polymerase interaction. The sequences centered around −10 (TATTCT) and −35 (TTGACC) in this promoter are, in fact, prototypical of eubacterial promoters. Overlapping the RNA polymerase binding site is a perfect 12-bp direct repeat sequence. Some base changes within this direct repeat resulted in constitutive expression, suggesting that this sequence is an operator for negative regulation. Other base changes resulted in loss of glucose repression while retaining the requirement for chitin induction, suggesting that this sequence is also involved in glucose repression. The fact that cis-acting mutations resulted in glucose resistance but not inducer independence rules out the possibility that glucose repression acts exclusively by inducer exclusion. The fact that mutations that affect glucose repression and chitin induction fall within the same direct repeat sequence module suggests that the direct repeat sequence facilitates both chitin induction and glucose repression.

ATPases and phosphate exchange activities in magnesium chelatase subunits of Rhodobacter sphaeroides

Three separate proteins, BchD, BchH, and BchI, together with ATP, insert magnesium into protoporphyrin IX. An analysis of ATP utilization by the subunits revealed the following: BchH catalyzed ATP hydrolysis at the rate of 0.9 nmol per min per mg of protein. BchI and BchD, tested individually, had no ATPase activity but, when combined, hydrolyzed ATP at the rate of 117.9 nmol/min per mg of protein. Magnesium ions were required for the ATPase activities of both BchH and BchI+D, and these activities were inhibited 50% by 2 mM o-phenanthroline. BchI additionally catalyzed a phosphate exchange reaction from ATP and ADP. We conclude that ATP hydrolysis by BchI+D is required for an activation step in the magnesium chelatase reaction, whereas ATPase activity of BchH and the phosphate exchange activity of BchI participate in subsequent reactions leading to the insertion of Mg2+ into protoporphyrin IX.

Upstream A-tracts increase bacterial promoter activity through interactions with the RNA polymerase α subunit

Upstream A-tracts stimulate transcription from a variety of bacterial promoters, and this has been widely attributed to direct effects of the intrinsic curvature of A-tract-containing DNA. In this work we report experiments that suggest a different mechanism for the effects of upstream A-tracts on transcription. The similarity of A-tract-containing sequences to the adenine- and thymine-rich upstream recognition elements (UP elements) found in some bacterial promoters suggested that A-tracts might increase promoter activity by interacting with the α subunit of RNA polymerase (RNAP). We found that an A-tract-containing sequence placed upstream of the Escherichia coli lac or rrnB P1 promoters stimulated transcription both in vivo and in vitro, and that this stimulation required the C-terminal (DNA-binding) domain of the RNAP α subunit. The A-tract sequence was protected by wild-type RNAP but not by α-mutant RNAPs in footprints. The effect of the A-tracts on transcription was not as great as that of the most active UP elements, consistent with the degree of similarity of the A-tract sequence to the UP element consensus. A-tracts functioned best when positioned close to the −35 hexamer rather than one helical turn farther upstream, similar to the positioning optimal for UP element function. We conclude that A-tracts function as UP elements, stimulating transcription by providing binding site(s) for the RNAP αCTD, and we suggest that these interactions could contribute to the previously described wrapping of promoter DNA around RNAP.