Estrategias de Apropiación, Transformación y Usos en la Fábrica de Tabacos de Madrid

Tabacalera se presenta como un importante caso de estudio en cuanto a la posibilidad de gestión de dos actores completamente opuestos, el Centro Social Autogestionado La Tabacalera y Tabacalera Promoción del Arte, en los que se hace hincapié en la distinción de las distintas maneras de resolución que se han generado en un mismo edificio. Se pretende, mediante el análisis del caso específico, Tabacalera de Lavapiés, con una serie de cartografías, entrevistas y el contacto directo con sus participantes, rescatar estrategias y mecanismos de actuación que promuevan la gestión participativa, usuario/comunidad y sector público. Tomando la experiencia de este caso específico, se pretende desvelar métodos de actuación para aplicarse en contextos diferentes, concretamente en realidades latinoamericanas, donde el desarrollo desordenado y acelerado de las ciudades ha generado una serie de inmuebles de valor histórico en situación de ocupación informal con potencialidad de reactivación. Entender el fenómeno Tabacalera es una oportunidad en la que un referente promueve además de la reactivación de un inmueble en desuso, la participación de la comunidad de manera directa, proponiendo mediante una serie de técnicas de observación e implicación directa dentro de su estructura de funcionamiento, el estudio del real impacto inmediato en el edificio y la forma en la cual colectivos sociales y usuarios, se apropian de su espacio inmediato. Tabacalera is presented as an important case study as to the possibility of managing two completely opposing players, the Centro Social Autogestionado La Tabacalera and Tabacalera Promoción del Arte, where he emphasizes different ways of resolution have been generated by same building. It is intended by analyzing the specific case, Tabacalera de Lavapies, with a series of maps, interviews and direct contact with your participants, rescue strategies and mechanisms of action that promote participatory management, user / community and public sector. Taking the experience of this specific case, trying to reveal methods of action that could be applied in different contexts, specifically Latin American realities, where the disorderly and accelerated urban development has generated a number of properties of historical value in situations of informal employment with potential of reactivation. You understand the Tabacalera phenomenon is exhibited as an opportunity in which a reference also promotes the reactivation of a building into disuse the participation of the community directly, proposing through a series of techniques of observation and direct involvement within their structure working, studying real immediate impact on the building and the way in which social groups and users, appropriate their immediate space.

Los paisajes del desecho: reactivación de los lugares del deterioro

Actualmente en nuestro planeta producimos 1.300 millones de toneladas de residuos urbanos al año. Si los extendemos sobre la superficie de un cuadrado de lado 100 m (una hectárea) alcanzarían una altura de 146 km. ¿Cuál es el origen de nuestros residuos? ¿A dónde va esta basura? ¿Cómo nos afecta? ¿Tiene alguna utilidad? Se trata de un problema antiguo que, en los últimos tiempos, ha adquirido una nueva dimensión por el tipo y la cantidad de residuos generados. Las primeras preocupaciones de la ciudad por ordenar estos problemas dieron lugar al establecimiento de espacios o lugares específicos para la acumulación de los residuos urbanos: los vertederos. Los desechos hoy se generan más rápidamente que los medios disponibles para reciclarlos o tratarlos. Los vertederos de residuos urbanos son y seguirán siendo, a corto y medio plazo, soluciones válidas por ser un método de gestión relativamente barato, sobre todo en los países en vías de desarrollo. Como consecuencia y necesidad de lo anterior, se plantea demostrar que la recuperación y la transformación de estos vertederos de residuos urbanos (lugares del deterioro), una vez abandonados, es posible y que además pueden dar lugar a nuevos espacios públicos estratégicos de la ciudad contemporánea. Son espacios de oportunidad, vacíos monumentales producto de una reactivación arquitectónica y paisajística realizada a partir de complejos procesos de ingeniería medioambiental. Pero las soluciones aplicadas a los vertederos de residuos urbanos desde mediados del siglo XX se han realizado exclusivamente desde la ingeniería para tratar de resolver cuestiones técnicas, un modelo agotado que ya no puede gestionar la magnitud que este problema ha alcanzado, haciéndose necesaria e inevitable la participación de la arquitectura para abrir nuevas líneas de investigación y de acción. En estos primeros compases del siglo XXI existe una “nueva” preocupación, un “nuevo” interés en los paradigmas de lo ecológico y de la sostenibilidad, también un interés filosófico (que igualmente otorga un nuevo valor al residuo como recurso), que dirigen su mirada hacia un concepto de paisaje abierto y diferente a modelos anteriores más estáticos, recuperando como punto de partida el ideal pintoresco. El landscape urbanism se consolida como una disciplina capaz de dar respuesta a lo natural y artificial simultáneamente, que sustituye a las herramientas tradicionales de la arquitectura para solucionar los problemas de la ciudad contemporánea, incorporando las infraestructuras de gran escala, como un vertedero de residuos urbanos, y los paisajes públicos que generan como el verdadero mecanismo de organización del urbanismo de hoy. No se trata solo de un modelo formal sino, lo que es más importante: de un modelo de procesos. Esta nueva preocupación permite abordar la cuestión del paisaje de manera amplia, sin restricciones, con un alto grado de flexibilidad en las nuevas propuestas que surgen como consecuencia de estos conceptos, si bien los esfuerzos, hasta la fecha, parecen haberse dirigido más hacia el fenómeno de lo estético, quedando todavía por explorar las consecuencias políticas, sociales, económicas y energéticas derivadas de los residuos. También las arquitectónicas. El proyecto del landscape urbanism se ocupa de la superficie horizontal, del plano del suelo. Desde siempre, la preparación de este plano para desarrollar cualquier actividad humana ha sido un gesto fundacional, un gesto propio necesario de toda arquitectura, que además ahora debe considerarlo como un medio o soporte biológicamente activo. En términos contemporáneos, el interés disciplinar radica en la continuidad y en la accesibilidad del suelo, diluyendo los límites; en que funcione a largo plazo, que se anticipe al cambio, a través de la flexibilidad y de la capacidad de negociación, y que sea público. La recuperación de un vertedero de residuos urbanos ofrece todas estas condiciones. Un breve recorrido por la historia revela los primeros ejemplos aislados de recuperación de estos lugares del deterioro, que han pasado por distintas fases en función de la cantidad y el tipo de los desechos producidos, evolucionando gracias a la tecnología y a una nueva mirada sobre el paisaje, hasta desarrollar una verdadera conciencia de lo ecológico (nacimiento de una ideología). El Monte Testaccio en Roma (siglos I-III d.C.) constituye un caso paradigmático y ejemplar de vertedero planificado a priori no solo como lugar en el que depositar los residuos, sino como lugar que será recuperado posteriormente y devuelto a la ciudad en forma de espacio público. Una topografía de desechos generada por acumulación, organizada y planificada durante tres siglos, que nos hace reflexionar sobre los temas de producción, consumo y proyecto arquitectónico. El Monte Testaccio revela una fuente de inspiración, un arquetipo de gestión sostenible de los recursos y del territorio. A través de la experiencia en la recuperación y transformación en espacios públicos de casos contemporáneos, como el antiguo vertedero de Valdemingómez en Madrid o el de El Garraf en Barcelona, se han analizado las técnicas y las soluciones empleadas para establecer nuevas herramientas de proyecto planteadas en clave de futuro, que revelan la importancia de los procesos frente a la forma, en los cuales intervienen muchos factores (tanto naturales como artificiales), entre ellos la vida y el tiempo de la materia viva acumulada. Son lugares para nuevas oportunidades y ejemplos de una nueva relación con la naturaleza. La reactivación de los vertederos de residuos, a través del proyecto, nos propone una nueva topografía construida en el tiempo, el suelo como soporte, como punto de encuentro de la naturaleza y los sistemas tecnológicos de la ciudad que posibilitan nuevos modos de vida y nuevas actividades. Los vertederos de residuos son inmensas topografías naturales surgidas de procesos artificiales, atalayas desde las que divisar un nuevo horizonte, un nuevo mundo, un nuevo futuro donde sea posible lograr la reversibilidad de nuestros actos del deterioro. Pero la voluntad de estas recuperaciones y transformaciones no consiste exclusivamente en su reintegración al paisaje, sino que han servido como muestra de las nuevas actitudes que la sociedad ha de emprender en relación a los temas medio ambientales. ABSTRACT Here on our planet we currently produce 1.3 billion tonnes of urban waste per year. If we were to spread this over a surface of 100m2 (one hectare), it would reach a height of 146km. What is the origin of this waste? Where does our refuse go? How does it affect us? Does it have any uses? We are dealing with an old problem which, in recent times, has taken on a new dimension due to the type of waste and the amount generated. Cities’ first concerns in resolving these problems gave rise to the establishment of areas or specific places for the accumulation of urban waste: landfills. These days, waste is generated more quickly than the available resources can recycle or process it. Urban waste landfills are and will continue to be, in the short and mid-term, valid solutions, given that they constitute a relatively cheap method for waste management, especially in developing countries. Consequently and necessarily, we plan to demonstrate that it is possible to recover and transform these urban waste landfills (areas of deterioration) once they have been abandoned and that they can give rise to new strategic public areas in contemporary cities. They are areas of opportunity, monumental vacancies produced by an architectural reactivation of the landscape, which is achieved using complex processes of environmental engineering. But the solutions applied to urban waste landfills throughout the 20th century have used engineering exclusively in the attempt to resolve the technical aspects. This is a worn-out model which can no longer handle the magnitude which the problem has attained and therefore, there is an inevitable need for the participation of architecture, which can open new lines of research and action. In these first steps into the 21st century, there is a “new” concern, a “new” interest in the paradigms of environmentalism and sustainability. There is also a philosophical interest (which assigns the new value of ‘resource’ to waste) and all is aimed towards the concept of an open landscape, unlike the previous, more static models, and the intention is to recover picturesque ideals as the starting point. Landscape urbanism has been established as a discipline capable of simultaneously responding to the natural and the artificial, replacing the traditional tools of architecture in order to resolve contemporary cities’ problems. It incorporates large scale infrastructures, such as urban waste landfills, and public landscapes which are generated as the true organisational mechanism of modern day urbanism. It is not merely a formal model, it is more important than that: it is a model of processes. This new concern allows us to address the matter of landscape in a broad way, without restrictions, and with a great degree of flexibility in the new proposals which come about as a consequence of these concepts. However, efforts to date seem to have been more directed at aesthetic aspects and we have yet to explore the political, social, economic and energetic consequences derived from waste – nor have we delved into the architectural consequences. The landscape urbanism project is involved with the horizontal surface, the ground plane. Traditionally, the preparation of this plane for the development of any human activity has been a foundational act, a necessary act of all architecture, but now this plane must be considered as a biologically active medium or support. In contemporary terms, the discipline’s interest lies in the continuity and accessibility of the land, diffusing the limits; in long term functionality; in the anticipation of change, via flexibility and the ability to negotiate; and in it being a public space. The recovery of an urban waste landfill offers all of these conditions. A brief look through history reveals the first isolated examples of recovery of these spaces of deterioration. They have gone through various phases based on the quantity and type of waste produced, they have evolved thanks to technology and a new outlook on the landscape, and a real environmental awareness has been developed (the birth of an ideology). Monte Testaccio in Rome (1st to 3rd Century AD) constitutes a paradigmatic and exemplary case of a landfill that was planned a priori not only as a place to deposit waste but also as a place that would be subsequently recovered and given back to the city in the form of a public space. This spoil mound, generated by organised and planned accumulation over three centuries, makes us reflect on the themes of production, consumption and architectural planning. Monte Testaccio reveals a source of inspiration, an archetype of the sustainable management of resources and land. Using our experience of contemporary cases of land recovery and its transformation into public spaces, such as the former Valdemingómez landfill in Madrid or the Garraf in Barcelona, we analysed the techniques and solutions used in order to establish new project tools. These are proposed with an eye on the future, seeing as they reveal the importance of the processes over the form and involve many factors (both natural and artificial), including the life and age of the accumulated living matter. They are places for new opportunities and examples of our new relationship with nature. The reactivation of landfills, via this project, is a proposal for a new topography built within time, using the ground as the support, as the meeting point between nature and the technological systems of the city which make it possible for new ways of life and new activities to come about. Landfills are immense natural topographical areas produced by artificial processes, watchtowers from which to discern a new horizon, a new world, a new future in which it will be possible to reverse our acts of deterioration. But the intention behind these recoveries and transformations does not only hope for landscape reintegration but it also hopes that they will also serve as a sign of the new attitudes that must be adopted by society with regard to environmental matters.

Sistemas urbanos emergentes: procesos informales de gestión y producción de espacio público

Esta investigación analiza la capacidad del urbanismo de albergar sistemas emergentes de gestión urbana. El objetivo de esta tesis es describir estos procedimientos informales, realizando un exhaustivo recorrido a través de diversas experiencias de alteración del modo de gestión urbana al uso, para dar lugar a una sistematización de estos procesos que siente las bases para plantear nuevas formas de gestión urbana. Es decir, el fin no es tanto hacer una clasificación de las intervenciones más relevantes en materia urbana, sino descubrir su estructura, un orden para el desorden, una taxonomía en esas actuaciones que permita reformular las formas actuales de gestión de lo público. Se ha tratado de dibujar un mapa de oportunidades posibles en torno a la gestión del espacio urbano. A través de los procesos estudiados, esta investigación analiza los éxitos y fracasos obtenidos en los últimos cinco años de gestión alternativa de espacios vacantes, buscando extraer formulas susceptibles de incorporarse al modo actual de hacer ciudad. Este estudio pretende abordar un aspecto parcial, aunque fundamental del urbanismo, dando prioridad a lo público, sin la intención de negar el resto de formas del espacio urbano. Se concentra en los parámetros relativos a la interpretación de lo público: desatiende al conjunto urbano para descifrar ese fragmento. Así, el interés principal de esta tesis se concentra en la producción del espacio público. Al entender este ámbito como no exclusivo del urbanismo, otras actividades como el arte, la filosofía, la antropología o la sociología han aportado aspectos fundamentales dentro de esta investigación. De esta forma se ofrece un acercamiento poliédrico desde puntos de vista interdisciplinares, tratando de descubrir con rigor las posibilidades contrastadas de mejora del espacio urbano producido por los procesos emergentes de gestión informal de lo público. Mediante un metodología creada ex profeso (una estructura definida por tres líneas estratégicas: Gobernanza, Complejidad y Cohesión Social, que se desglosan en seis conceptos clave cada una) esta investigación analiza los aspectos relativos a la producción informal del espacio público de los sistemas emergentes. En relación con la Gobernanza, una parte de esta tesis se dedica al estudio de la informalidad, la transversalidad, la apropiación, la participación, la transparencia y las redes. La Complejidad se ha estudiado en su relación con la diversidad urbana, el uso sostenible del espacio público, la reactivación, la producción de espacio público, la ecología urbana y los procesos creativos. Para Cohesión Social se ha investigado la innovación en equipamientos, los procesos bottom-up, el rizoma, la identidad, los sistemas líquidos y el empoderamiento. Esta máquina para analizar se prueba en tres soportes urbanos: la escena urbana, los edificios-contenedores y los vacíos urbanos. Se han estudiado cinco situaciones urbanas en la ciudad de Madrid, seleccionados por ser las prácticas urbanas más relevantes de los últimos cinco años. Si bien este análisis nace de estos cincos casos, el modelo podría ser útil para otras investigaciones interesadas en los aspectos informales de la gestión urbana. Esta investigación se propone reclamar otras dimensiones más innovadoras que, si bien se escapan a los modos habituales de trabajar del urbanista, se revelan intensamente en los modos actuales de reclamar y producir espacio urbano. Se ha pretendido descubrir el espacio urbano como contenedor situacional del sentido público, potencial multiplicador de efectos funcionales como la gestión, la temporalidad, la participación, la rehabilitación, la multiplicidad de usos, la corrección del compacidad; efectos perceptuales y estéticos en la escena urbana; efectos políticos, de formación, de adquisición de poder; efectos sociales como la generación de nuevas redes, la creación de identidades. Se pretende que esta investigación arroje conclusiones que puedan tener aplicabilidad en la gestión y planificación del espacio público de ciudades y entornos urbanos inmersos a procesos semejantes. ABSTRACT This research examines the ability of urban planning to accommodate emerging urban management systems. The purpose of this thesis is to describe such informal procedures, by carrying out an exhaustive review across the different approaches to urban management, in order to systematize them as the basis for new ways of urban management. The aim is not, that is, to classify relevant urban interventions, but to recognize the intrinsic structure, an order for disorder, a taxonomy in those proceedings that could help reformulating existing forms of public management. The attempt has been to draw a map of possible opportunities around the management of urban space. Through the processes that have been the object of study, this research analyzes the successes and failures over the last five years of alternative management of vacant spaces, seeking to extract formulas that are likely to join the current way of making city. The focus of this urban study is the interpretation of public concepts, considered as key dimensions, not obliterating the other forms of urban space, but trying to make an in-depth analysis of this fragment within the global urban concept. Other activities intrinsically associated to public space generation, such as art, philosophy, anthropology or even sociology, are also contributing to this research. This interdisciplinary approach tries to enrich the research by recognizing the informal elements participating in emergent public management processes. The ex profeso devised methodology is structured by three main strategies: governance, complexity and Social Cohesiveness. Governance considers elements associated to informality, transversal dynamics, appropriation, participation, transparency and networking. Complexity encompasses elements of urban diversity, sustainable usage of public space, reactivation, urban space production, urban ecology and creative processes. Social Cohesiveness includes elements for equipment innovation, bottom-up processes, rhizome, identity, liquid systems and empowerment. This analyzing machine is tested on three urban scenarios: the urban scene, container buildings and empty spaces. Five relevant urban situations in Madrid have been selected, all of them developed in last five years, thus establishing the basis for other experiences of informal urban management. The result of the research, confirms the relevance of additional dimensions to traditional approaches for urban development. The urban space is recognized as a situational container of public sense and a potential multiplier of functional effects such as public management, temporality, participation, rehabilitation, multiplicity of uses, correction of compactness; perceptual and esthetic effects on the urban scene; politic effects, training, empowerment; social effects as generating new networks and identity creation. To sum up, the purpose of this thesis is to share the conclusions obtained by evaluating reference scenarios that could be applied in similar processes for cities and urban environment management and planning.

Essential role of germinal vesicle material in the meiotic cell cycle of Xenopus oocytes

In almost all animal species, immature oocytes are arrested naturally in the first meiotic prophase, with a large nucleus called the germinal vesicle. A number of previous studies showed that both activation of maturation/M phase-promoting factor (MPF) (assayed by semiquantitative cytological methods) and some other maturational events occur essentially normally in enucleated oocytes from many amphibian species and mice. Hence, for nearly three decades, it has generally been believed that nuclear material is dispensable for MPF activation and the meiotic cell cycle in vertebrate oocytes. Here, we have challenged this view by examining the histone H1 kinase activities and the molecular forms of MPF in experimentally manipulated Xenopus oocytes. We show that oocytes injected with nuclear material undergo much more rapid MPF activation and maturation than uninjected control oocytes. Conversely, enucleated oocytes, unlike nucleated counterparts, undergo only weak MPF activation in meiosis I and no detectable MPF reactivation in meiosis II, the latter accompanying inhibitory tyrosine phosphorylation of cdc2 kinase, the catalytic subunit of MPF. These results argue strongly that nuclear material is indispensable for the meiotic cell cycle, particularly MPF reactivation (or cdc2 tyrosine dephosphorylation) on entry into meiosis II, in Xenopus oocytes. The classical and general view may thus need reconsideration.

Severe genital herpes infections in HIV-infected individuals with impaired herpes simplex virus-specific CD8+ cytotoxic T lymphocyte responses

The specific mechanisms underlying the varied susceptibility of HIV-infected (HIV+) individuals to opportunistic infections (OI) are still incompletely understood. One hypothesis is that quantitative differences in specific T cell responses to a colonizing organism determine the development of an AIDS-defining OI. We evaluated this hypothesis for herpes simplex virus (HSV) infection, a common OI in HIV+ patients. Using limiting dilution analyses, the frequency of HSV-specific CD8+ cytotoxic T lymphocyte precursors (pCTL) and proliferative precursors were quantitated in peripheral blood mononuclear cells from 20 patients coinfected with HIV and HSV-2. The frequency of HSV-specific CD8+ pCTL in HSV+HIV+ individuals was significantly lower than in HSV+HIV− individuals (1 in 77,000 vs. 1 in 6,000, P = .0005) and was not different than in HSV-HIV− individuals (1 in 100,000, P = .24). HIV+ patients who suffered more severe genital herpes recurrences had significantly lower HSV-specific CD8+ pCTL frequencies than those patients with mild recurrences (1 in 170,000 vs. 1 in 26,000, P = .03). In contrast, no significant difference was seen in proliferative precursor frequencies between those patients with mild vs. severe genital herpes (1 in 3,800 vs. 1 in 6,600, P > .5). Quantitative differences in pCTL frequency to HSV appear to be the most important host factor influencing the frequency and severity of HSV reactivation in HIV+ patients. Studies to reconstitute such immunity, especially in people with acyclovir-resistant HSV, appear warranted.

Protein phosphatase 2C dephosphorylates and inactivates cystic fibrosis transmembrane conductance regulator

cAMP-dependent phosphorylation activates the cystic fibrosis transmembrane conductance regulator (CFTR) in epithelia. However, the protein phosphatase (PP) that dephosphorylates and inactivates CFTR in airway and intestinal epithelia, two major sites of disease, is not certain. We found that in airway and colonic epithelia, neither okadaic acid nor FK506 prevented inactivation of CFTR when cAMP was removed. These results suggested that a phosphatase distinct from PP1, PP2A, and PP2B was responsible. Because PP2C is insensitive to these inhibitors, we tested the hypothesis that it regulates CFTR. We found that PP2Cα is expressed in airway and T84 intestinal epithelia. To test its activity on CFTR, we generated recombinant human PP2Cα and found that it dephosphorylated CFTR and an R domain peptide in vitro. Moreover, in cell-free patches of membrane, addition of PP2Cα inactivated CFTR Cl− channels; reactivation required readdition of kinase. Finally, coexpression of PP2Cα with CFTR in epithelia reduced the Cl− current and increased the rate of channel inactivation. These results suggest that PP2C may be the okadaic acid-insensitive phosphatase that regulates CFTR in human airway and T84 colonic epithelia. It has been suggested that phosphatase inhibitors could be of therapeutic value in cystic fibrosis; our data suggest that PP2C may be an important phosphatase to target.

Mössbauer and electron paramagnetic resonance studies of chloroperoxidase following mechanism-based inactivation with allylbenzene

We have used Mössbauer and electron paramagnetic resonance (EPR) spectroscopy to study a heme-N-alkylated derivative of chloroperoxidase (CPO) prepared by mechanism-based inactivation with allylbenzene and hydrogen peroxide. The freshly prepared inactivated enzyme (“green CPO”) displayed a nearly pure low-spin ferric EPR signal with g = 1.94, 2.15, 2.31. The Mössbauer spectrum of the same species recorded at 4.2 K showed magnetic hyperfine splittings, which could be simulated in terms of a spin Hamiltonian with a complete set of hyperfine parameters in the slow spin fluctuation limit. The EPR spectrum of green CPO was simulated using a three-term crystal field model including g-strain. The best-fit parameters implied a very strong octahedral field in which the three 2T2 levels of the (3d)5 configuration in green CPO were lowest in energy, followed by a quartet. In native CPO, the 6A1 states follow the 2T2 ground state doublet. The alkene-mediated inactivation of CPO is spontaneously reversible. Warming of a sample of green CPO to 22°C for increasing times before freezing revealed slow conversion of the novel EPR species to two further spin S = ½ ferric species. One of these species displayed g = 1.82, 2.25, 2.60 indistinguishable from native CPO. By subtracting spectral components due to native and green CPO, a third species with g = 1.86, 2.24, 2.50 could be generated. The EPR spectrum of this “quasi-native CPO,” which appears at intermediate times during the reactivation, was simulated using best-fit parameters similar to those used for native CPO.

Suppression of gene silencing: A general strategy used by diverse DNA and RNA viruses of plants

In transgenic and nontransgenic plants, viruses are both initiators and targets of a defense mechanism that is similar to posttranscriptional gene silencing (PTGS). Recently, it was found that potyviruses and cucumoviruses encode pathogenicity determinants that suppress this defense mechanism. Here, we test diverse virus types for the ability to suppress PTGS. Nicotiana benthamiana exhibiting PTGS of a green fluorescent protein transgene were infected with a range of unrelated viruses and various potato virus X vectors producing viral pathogenicity factors. Upon infection, suppression of PTGS was assessed in planta through reactivation of green fluorescence and confirmed by molecular analysis. These experiments led to the identification of three suppressors of PTGS and showed that suppression of PTGS is widely used as a counter-defense strategy by DNA and RNA viruses. However, the spatial pattern and degree of suppression varied extensively between viruses. At one extreme, there are viruses that suppress in all tissues of all infected leaves, whereas others are able to suppress only in the veins of new emerging leaves. This variation existed even between closely related members of the potexvirus group. Collectively, these results suggest that virus-encoded suppressors of gene silencing have distinct modes of action, are targeted against distinct components of the host gene-silencing machinery, and that there is dynamic evolution of the host and viral components associated with the gene-silencing mechanism.

Inhibition of human telomerase in immortal human cells leads to progressive telomere shortening and cell death

The correlation between telomerase activity and human tumors has led to the hypothesis that tumor growth requires reactivation of telomerase and that telomerase inhibitors represent a class of chemotherapeutic agents. Herein, we examine the effects of inhibition of telomerase inside human cells. Peptide nucleic acid and 2′-O-MeRNA oligomers inhibit telomerase, leading to progressive telomere shortening and causing immortal human breast epithelial cells to undergo apoptosis with increasing frequency until no cells remain. Telomere shortening is reversible: if inhibitor addition is terminated, telomeres regain their initial lengths. Our results validate telomerase as a target for the discovery of anticancer drugs and supply general insights into the properties that successful agents will require regardless of chemical type. Chemically similar oligonucleotides are in clinical trials and have well characterized pharmacokinetics, making the inhibitors we describe practical lead compounds for testing for an antitelomerase chemotherapeutic strategy.

Bicarbonate is an essential constituent of the water-oxidizing complex of photosystem II

It is shown that restoration of photoinduced electron flow and O2 evolution with Mn2+ in Mn-depleted photosystem II (PSII) membrane fragments isolated from spinach chloroplasts is considerably increased with bicarbonate in the region pH 5.0–8.0 in bicarbonate-depleted medium. In buffered solutions equilibrated with the atmosphere (nondepleted of bicarbonate), the bicarbonate effect is observed only at pH lower than the pK of H2CO3 dissociation (6.4), which indicates that HCO3− is the essential species for the restoration effect. The addition of just 2 Mn2+ atoms per one PSII reaction center is enough for the maximal reactivation when bicarbonate is present in the medium. Analysis of bicarbonate concentration dependence of the restoration effect reveals two binding sites for bicarbonate with apparent dissociation constant (Kd) of ≈2.5 μM and 20–34 μM when 2,6-dichloro-p-benzoquinone is used as electron acceptor, while in the presence of silicomolybdate only the latter one remains. Similar bicarbonate concentration dependence of O2 evolution was obtained in untreated Mn-containing PSII membrane fragments. It is suggested that the Kd of 20–34 μM is associated with the donor side of PSII while the location of the lower Kd binding site is not quite clear. The conclusion is made that bicarbonate is an essential constituent of the water-oxidizing complex of PSII, important for its assembly and maintenance in the functionally active state.

HIV-1 Tat transactivator recruits p300 and CREB-binding protein histone acetyltransferases to the viral promoter

In cells infected with HIV type 1 (HIV-1), the integrated viral promoter is present in a chromatin-bound conformation and is transcriptionally silent in the absence of stimulation. The HIV-1 Tat protein binds to a stem-loop structure at the 5′ end of viral mRNA and relieves this inhibition by inducing a remodeling of the nucleosome arrangement downstream of the transcription-initiation site. Here we show that Tat performs this activity by recruiting to the viral long terminal repeat (LTR) the transcriptional coactivator p300 and the closely related CREB-binding protein (CBP), having histone acetyltransferase (HAT) activity. Tat associates with HAT activity in human nuclear extracts and binds to p300 and CBP both in vitro and in vivo. Integrity of the basic domain of Tat is essential for this interaction. By a quantitative chromatin immunoprecipitation assay we show that the delivery of recombinant Tat induces the association of p300 and CBP with the chromosomally integrated LTR promoter. Expression of human p300 in both human and rodent cells increases the levels of Tat transactivation of the integrated LTR. These results reinforce the evidence that p300 and CBP have a pivotal function at both cellular and viral promoters and demonstrate that they also can be recruited by an RNA-targeted activator. Additionally, these findings have important implications for the understanding of the mechanisms of HIV-1 latency and reactivation.

The Endogenous and Cell Cycle-dependent Phosphorylation of tau Protein in Living Cells: Implications for Alzheimer’s Disease

In Alzheimer’s disease the neuronal microtubule-associated protein tau becomes highly phosphorylated, loses its binding properties, and aggregates into paired helical filaments. There is increasing evidence that the events leading to this hyperphosphorylation are related to mitotic mechanisms. Hence, we have analyzed the physiological phosphorylation of endogenous tau protein in metabolically labeled human neuroblastoma cells and in Chinese hamster ovary cells stably transfected with tau. In nonsynchronized cultures the phosphorylation pattern was remarkably similar in both cell lines, suggesting a similar balance of kinases and phosphatases with respect to tau. Using phosphopeptide mapping and sequencing we identified 17 phosphorylation sites comprising 80–90% of the total phosphate incorporated. Most of these are in SP or TP motifs, except S214 and S262. Since phosphorylation of microtubule-associated proteins increases during mitosis, concomitant with increased microtubule dynamics, we analyzed cells mitotically arrested with nocodazole. This revealed that S214 is a prominent phosphorylation site in metaphase, but not in interphase. Phosphorylation of this residue strongly decreases the tau–microtubule interaction in vitro, suppresses microtubule assembly, and may be a key factor in the observed detachment of tau from microtubules during mitosis. Since S214 is also phosphorylated in Alzheimer’s disease tau, our results support the view that reactivation of the cell cycle machinery is involved in tau hyperphosphorylation.

Distinct Actions and Cooperative Roles of ROCK and mDia in Rho Small G Protein-induced Reorganization of the Actin Cytoskeleton in Madin–Darby Canine Kidney Cells

Rho, a member of the Rho small G protein family, regulates the formation of stress fibers and focal adhesions in various types of cultured cells. We investigated here the actions of ROCK and mDia, both of which have been identified to be putative downstream target molecules of Rho, in Madin–Darby canine kidney cells. The dominant active mutant of RhoA induced the formation of parallel stress fibers and focal adhesions, whereas the dominant active mutant of ROCK induced the formation of stellate stress fibers and focal adhesions, and the dominant active mutant of mDia induced the weak formation of parallel stress fibers without affecting the formation of focal adhesions. In the presence of C3 ADP-ribosyltransferase for Rho, the dominant active mutant of ROCK induced the formation of stellate stress fibers and focal adhesions, whereas the dominant active mutant of mDia induced only the diffuse localization of actin filaments. These results indicate that ROCK and mDia show distinct actions in reorganization of the actin cytoskeleton. The dominant negative mutant of either ROCK or mDia inhibited the formation of stress fibers and focal adhesions, indicating that both ROCK and mDia are necessary for the formation of stress fibers and focal adhesions. Moreover, inactivation and reactivation of both ROCK and mDia were necessary for the 12-O-tetradecanoylphorbol-13-acetate–induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. The morphologies of stress fibers and focal adhesions in the cells expressing both the dominant active mutants of ROCK and mDia were not identical to those induced by the dominant active mutant of Rho. These results indicate that at least ROCK and mDia cooperatively act as downstream target molecules of Rho in the Rho-induced reorganization of the actin cytoskeleton.

Gradual Phenotypic Conversion Associated with Immortalization of Cultured Human Mammary Epithelial Cells

Examination of the process of immortal transformation in early passages of two human mammary epithelial cell (HMEC) lines suggests the involvement of an epigenetic step. These lines, 184A1 and 184B5, arose after in vitro exposure of finite lifespan 184 HMEC to a chemical carcinogen, and both are clonally derived. Although early-passage mass cultures of 184A1 and 184B5 maintained continuous slow growth, most individual cells lost proliferative ability. Uniform good growth did not occur until 20–30 passages after the lines first appeared. Early-passage cultures expressed little or no telomerase activity and telomeres continued to shorten with increasing passage. Telomerase activity was first detected when the telomeres became critically short, and activity levels gradually increased thereafter. Early-passage cultures had little or no ability to maintain growth in transforming growth factor-β (TGFβ); however, both mass cultures and clonal isolates showed a very gradual increase in the number of cells displaying progressively increased ability to maintain growth in TGFβ. A strong correlation between capacity to maintain growth in the presence of TGFβ and expression of telomerase activity was observed. We have used the term “conversion” to describe this process of gradual acquisition of increased growth capacity in the absence or presence of TGFβ and reactivation of telomerase. We speculate that the development of extremely short telomeres may result in gradual, epigenetic-based changes in gene expression. Understanding the underlying mechanisms of HMEC conversion in vitro may provide new insight into the process of carcinogenic progression in vivo and offer novel modes for therapeutic intervention.

Molecular mechanism for silencing virally transduced genes involves histone deacetylation and chromatin condensation

Virally transduced genes are often silenced after integration into the host genome. Chromatin immunoprecipitation and nuclease sensitivity experiments now demonstrate that silencing of the transgene is characterized by deacetylation of histone H4 lysines and chromatin condensation. Trichostatin A treatment results in dramatic reactivation of gene expression that is preceded by histone acetylation and chromatin decondensation. Analysis of individual histone H4 lysines demonstrate that chromatin domain opening is coincident with rapid acetylation of histone H4 K5, K12, and K16 and that maintenance of the open domain is correlated with acetylation of histone H4 K8. Removal of trichostatin A results in rapid deacetylation of histone H4 K8, chromatin condensation, and transcription silencing. The results suggest that deacetylation of histone H4 lysines and coincident chromatin condensation are critically involved in the silencing of virally transduced genes.