Safety, feasibility, and long-term results of percutaneous closure of atrial septal defects using the Amplatzer septal occluder without periprocedural echocardiography.

OBJECTIVES We sought to assess the safety and efficacy of percutaneous closure of atrial septal defects (ASDs) under fluoroscopic guidance only, without periprocedural echocardiographic guidance. BACKGROUND Percutaneous closure of ASDs is usually performed using simultaneous fluoroscopic and transthoracic, transesophageal (TEE), or intracardiac echocardiographic (ICE) guidance. However, TEE requires deep sedation or general anesthesia, which considerably lengthens the procedure. TEE and ICE increase costs. METHODS Between 1997 and 2008, a total of 217 consecutive patients (age, 38 ± 22 years; 155 females and 62 males), of whom 44 were children ≤16 years, underwent percutaneous ASD closure with an Amplatzer ASD occluder (AASDO). TEE guidance and general anesthesia were restricted to the children, while devices were implanted under fluoroscopic guidance only in the adults. For comparison of technical safety and feasibility of the procedure without echocardiographic guidance, the children served as a control group. RESULTS The implantation procedure was successful in all but 3 patients (1 child and 2 adults; 1.4%). Mean device size was 23 ± 8 mm (range, 4-40 mm). There was 1 postprocedural complication (0.5%; transient perimyocarditis in an adult patient). At last echocardiographic follow-up, 13 ± 23 months after the procedure, 90% of patients had no residual shunt, whereas a minimal, moderate, or large shunt persisted in 7%, 1%, and 2%, respectively. Four adult patients (2%) underwent implantation of a second device for a residual shunt. During a mean follow-up period of 3 ± 2 years, 2 deaths and 1 ischemic stroke occurred. CONCLUSION According to these results, percutaneous ASD closure using the AASDO without periprocedural echocardiographic guidance seems safe and feasible.

On the Closure of the Tame Automorphism Group of Affine Three-Space

We provide explicit families of tame automorphisms of the complex affine three-space which degenerate to wild automorphisms. This shows that the tame subgroup of the group of polynomial automorphisms of C3 is not closed, when the latter is seen as an infinite-dimensional algebraic group.

Flexibility in Airline Business Models with Core Competence as an Indicator.

The airline industry is often unstable and unpredictable forcing airlines to restructure and create flexible strategies that can respond to external operating environmental changes. In turbulent and competitive environments, firms with higher flexibility perform better and the value of these flexibilities depends on factors of uncertainty in the competitive environment. A model is sought for and arrived at, that shows how an airline business model will function in an uncertain environment with the least reduction in business performance over time. An analysis of the business model flexibility of 17 Airlines from Asia, Europe and Oceania, that is done with core competence as the indicator reveals a picture of inconsistencies in the core competence strategy of certain airlines and the corresponding reduction in business performance. The performance variations are explained from a service oriented core competence strategy employed by airlines that ultimately enables them in having a flexible business model that not only increases business performance but also helps in reducing the uncertainties in the internal and external operating environments.

The flexibility matrix o timber composite beams with a discrete connection system

This work presents a method for the analysis of timber composite beams which considers the slip in the connection system, based on assembling the flexibility matrix of the whole structure. This method is based on one proposed by Tommola and Jutila (2001). This paper extends the method to the case of a gap between two pieces with an arbitrary location at the first connector, which notably broadens its practical application. The addition of the gap makes it possible to model a cracked zone in concrete topping, as well as the case in which forming produces the gap. The consideration of induced stresses due to changes in temperature and moisture content is also described, while the concept of equivalent eccentricity is generalized. This method has important advantages in connection with the current European Standard EN 1995-1-1: 2004, as it is able to deal with any type of load, variable section, discrete and non-regular connection systems, a gap between the two pieces, and variations in temperature and moisture content. Although it could be applied to any structural system, it is specially suited for the case of simple supported and continuous beams. Working examples are presented at the end, showing that the arrangement of the connection notably modifies shear force distribution. A first interpretation of the results is made on the basis of the strut and tie theory. The examples prove that the use of EC-5 is unsafe when, as a rule of thumb, the strut or compression field between the support and the first connector is at an angle with the axis of the beam of less than 60º.

Using the Own Flexibility of a Climbing Robot as a Double Force Sensor

Force sensors are used when interaction tasks are carried out by robots in general, and by climbing robots in particular. If the mechanics and electronics systems are contained inside the own robot, the robot becomes portable without external control. Commercial force sensors cannot be used due to limited space and weight. By selecting the links material with appropriate stiffness and placing strain gauges on the structure, the own robot flexibility can be used such as force sensor. Thus, forces applied on the robot tip can be measured without additional external devices. Only gauges and small internal electronic converters are necessary. This paper illustrates the proposed algorithm to achieve these measurements. Additionally, experimental results are presented.

Concienciación para la conservación del patrimonio urbano : los instrumentos de gestión para la recuperación del barrio madrileño de Embajadores

La tesis trata la evolución de la conciencia de la conservación de los centros históricos y de los instrumentos de gestión utilizados en las políticas de rehabilitación del casco antiguo de Madrid, desde las primeras experiencias de los años ‘80 hasta el perfeccionamiento del complejo mecanismo contemporáneo. El proceso de rehabilitación ha demostrado ser extremadamente coherente en cuanto a los objetivos y en cuanto al tema de los instrumentos ha seguido la tendencia de diversificar y flexibilizar. En Madrid se ha utilizado el concepto de rehabilitación en sentido integral, abarca una amplia gama de acciones que implican por una parte, intervenciones directas sobre los edificios y por otra, la valorización del espacio público, de los edificios representativos, la introducción de dotaciones y también un componente social. El tema se analizará desde la perspectiva del barrio de Embajadores, un área muy especial del casco antiguo de la ciudad. Desde 1998 hasta la actualidad allí funciona el Área de Rehabilitación Integral con el nombre de Lavapiés. El barrio de Embajadores representa un caso de extrema complejidad dada su tradición, que mantiene hasta el momento, de barrio popular que ha tenido como consecuencia negativa un grave estado de deterioro de los edificios y de sus estructuras urbanas. Además, presenta una composición socio-cultural muy heterogénea ya que siempre ha alojado oleadas de inmigrantes que llegaban a la capital tanto de las provincias como del extranjero. Los ‘80 marcan en Madrid el inicio de la preocupación por la rehabilitación urbana y de los primeros intentos de definir unos mecanismos. Durante este período el barrio de Embajadores es escenario de los primeros proyectos piloto de rehabilitación. Se sientan las bases del sistema de subvenciones, que empujará hacia un modelo de rehabilitación generalizada. A principios de los años ‘90, la insatisfacción dejada por de los resultados obtenidos, la Administración decide reorganizar la estrategia y se procede a la rehabilitación integral por áreas, concentrando los esfuerzos geográfica y temporalmente. La idea ya se había utilizado diez años antes, pero sin encontrar nunca la voluntad política de aplicarla. En comparación con el casco antiguo, el barrio de Embajadores se dejó para el final del proceso para verificar los métodos en zonas más restringidas en cuanto a superficie y con menos problemas. Desde 1998 hasta hoy, período en que funciona el Área de Rehabilitación Integral de Lavapiés, los distintos tipos de actuaciones, de producción arquitectónica, urbanística y sociocultural, han mostrado su eficacia, de forma que los cambios que aportaron al barrio son evidentes para todos. Mientras que las Áreas de Rehabilitación del centro una vez alcanzados los objetivos, se preparan para su clausura, a nivel administrativo se están definiendo nuevas direcciones y nuevos objetivos para el casco antiguo: subastas para incentivar intervenciones de mejoramiento energético y del nivel de sostenibilidad de los edificios madrileños. La primera parte de la tesis establece las premisas presentando algunos puntos relevantes relativos al desarrollo histórico de los documentos y normativas más importantes que tendrán eco en el proceso de concienciación de la cultura de la conservación del patrimonio urbano de los centros históricos. Después, se habla de la experiencia de rehabilitación urbana en Europa, en España en general y en Madrid en particular, con referencia a los modelos de enfoque, al marco legislativo y a las experiencias relacionadas con el casco antiguo, y en definitiva a la creación de la conciencia de la rehabilitación y conservación de los centros de las ciudades. La segunda parte se centra en el caso de estudio, el barrio de Embajadores. En primer lugar, se presenta en detalle el barrio: ubicación, datos característicos, edificios representativos y flujos que generan, trama urbana, datos sobre la formación histórica, tipologías y características de construcción de los edificios y, para finalizar, algún dato demográfico para introducir la compleja problemática social. Más adelante se exponen las primeras experiencias de rehabilitación: dos proyectos piloto de intervención sobre las viviendas realizados en los años ‘80, uno privado y el otro público. La parte central del trabajo trata sobre la fase intensiva de rehabilitación, proceso puesto en marcha en el año ’98 y que corresponde al período en el que Embajadores es Área de Rehabilitación Integral. Se presentan y analizan los objetivos de la rehabilitación y luego los instrumentos de orden administrativo, normativo y legal que regulan las actuaciones en el barrio. Sucesivamente se afronta la rehabilitación de la parte residencial, en régimen privado y público, que fue el modelo principal que la Administración llevó a cabo. Se presenta todo el proceso, desde la decisión sobre el tipo de rehabilitación, las subvenciones y las soluciones técnicas adoptadas así como la rehabilitación de casos especiales de edificios, que por sus características, el alto nivel de degradación o porque ocupados por inquilinos con dificultades económicas, se realizó con intervención pública. Las enormes dificultades derivadas de la complicada gestión y de las incongruencias de las normativas también se analizan en este trabajo. El autor presenta un dosier de una veintena de trabajos llevados a cobo en el Barrio de Embajadores a lo largo de más de 15 años de experiencia profesional propia. Se intenta, en primer lugar, evaluar la actuación pública en comparación con la privada en cuanto a los modelos propuestos, a las problemáticas que generan y a los resultados obtenidos. Una segunda línea de argumentación se refiere a la relación que se establece entre los instrumentos que se promueven y los que obligan a la conservación y a la rehabilitación de los edificios. Quizás el elemento más interesante, por su conocida innovación, de la experiencia de la obligación de conservación a cargo de los propietarios y la introducción de una revisión periódica del estado de los edificios (ITE), que conduce a su mantenimiento permanente a lo largo del tiempo. El ultimo instrumento aparecido y de por si el más novedoso se refiere a la política de rehabilitación energética iniciada en los últimos años por el Ayuntamiento de Madrid. ABSTRACT Starting from the early experiences of the ‘80s and tackling the complex and improved contemporary techniques of development, the present thesis focuses on raising awareness of the conservation of urban heritage and deals with the evolution of rehabilitation policies adopted in the historical center of Madrid. The rehabilitation process has proven to be extremely coherent in terms of its objectives and, consequently, the subject of the instruments has forged ahead a trend of diversification and flexibility. Madrid has used the concept of rehabilitation in a comprehensive manner, encompassing a wide range of actions, which involved on the one hand, direct interventions on buildings and on the other, the appreciation of the public space with its representative edifices and endowments. Confident that the social components have not been neglected during the rehabilitation proceedings, the topic will be analysed in relation to the neighbourhood of Embajadores, an authentic area of the old town. From 1998 onwards, this quarter serves as a Comprehensive Rehabilitation Area under the name of Lavapies. Considering the tradition and popularity of the district, Embajadores represents a challenging case for rehabilitation projects, aiming to improve the severe state of deterioration of buildings and urban structures. Having a heterogeneous sociocultural dynamic, the neighbourhood has always hosted waves of immigrants who come to the capital both from other provinces and abroad. The 1980s in Madrid marked the beginning of concerns with urban regeneration and the first attempts to define feasible restoration techniques. During this period, the district of Embajadores has benefited from various pilot projects. The financial investments have significant effects on the overall environment, so the model of general rehabilitation has been encouraged throughout the years. In the early 90s, left by the dissatisfaction of the results obtained, the authorities proposed to reorganise the strategy and proceed to the full rehabilitation of areas, concentrating efforts geographically and temporally. The idea had already been used ten years before, but never encountered the political force to be implemented. Compared to the old town, the Embajadores neighbourhood has been left for the end of the process, which focused on verifying the methods in more restricted areas that caused fewer disruptions. For the Comprehensive Rehabilitation Area of Lavapies, the various types of administrative implementations from architectural to urban and socio-cultural productions have proved effective. The improvements experienced by the neighbourhood from 1998 are obvious to all. However, while the areas of rehabilitation of the city centre have achieved their objectives and prepare for closure, the administration is still generating new directions and new targets for the ancient surroundings: auctions to encourage the improvement of energy interventions and the sustainability level for the buildings of Madrid. Therefore, the first part of the thesis establishes the premises and introduces some relevant points concerning the entire city. It describes the historical development of urban projects, indicating the events that will echo in the rehabilitation process. This paper then turns to discuss the experience of urban regeneration in Spain, emphasising the case of Madrid with reference to models of approach, legislative frameworks and appreciation of the old structures. The second part reveals an in depth case study of the district of Embajadores. At first, the thesis provides insights into the neighbourhood: location, general data on representative buildings and propagated trends, urban, historical data on training, building typologies and characteristics. The section introduces some demographic data to complete the portrait of the elaborate social problems encountered in this district, also outlining the first experiences of rehabilitation: two pilot projects on housing intervention made in the 80s, one private and one public. The central part of the thesis deals with the intensive phase of rehabilitation developments launched in the year '98 when Embajadores joined the Comprehensive Rehabilitation Area. It analyses the goals of rehabilitation and the instruments of administrative, regulatory and governing legal proceedings of the neighbourhood. The following chapter addresses the privately and publicly founded plan of residential rehabilitation that constituted the main model conducted by the authorities. It describes the entire process: deciding the type of restoration, subsidies and technical solutions as well as the degree of rehabilitation for special buildings, which either have a high level of degradation or require public intervention when tenants face financial difficulties. The administrative difficulties caused by the enormous bureaucratic machinery and the inconsistencies in regulations are also discussed in this paper. Thus, a just evaluation of the public and private performances regarding the proposed models of rehabilitation, along with the problems they generate and the results obtained is desired throughout the thesis. A second line of argument concerns the relationship established between the instruments that are promoted and requiring conservation and the building restoration. Perhaps the most interesting and innovative element of the new rehabilitation policies is the owners' obligation to preserve their properties and the introduction of a periodic review for the state of the buildings (ITE). These thorough and meticulous regulations lead to an ongoing maintenance of constructions, preventing them from severe or sudden deteriorations.

Flexibility, innovation and sustainability in business models as determinants of firm performance heterogeneity.

La comprensión actual de la heterogeneidad de las empresas académico rendimiento en el entorno entra industria necesita un mayor desarrollo. Gestión estratégica y el discurso sobre la literatura empresarial necesita mayor explicación de por qué los modelos de negocio aparentemente similares en el mismo sector actúan de forma diferente. También qué factores del entorno sectorial y operativo determina el surgimiento y funcionamiento de los modelos de negocio sostenibles e innovadoras permanecen sin explorar. Un marco se conceptualiza acompañado de estudios de caso sobre la compañía aérea y las industrias de energía renovable. El estudio lleva a una visión basada en los recursos de los modelos de negocio que las empresas alcancen posiciones heterogéneas de recursos. Una explicación para la heterogeneidad firme desempeño que se busca por intermediación conocimiento que genera valor a partir de la utilización eficaz de los recursos de conocimientos adquiridos a entornos entra y inter-empresa. Un marco para la aparición de nuevos modelos de negocios verdes se conceptualiza y deducciones se obtienen mediante un estudio de caso sobre la base de la industria de biocombustibles de energías renovables para explicar la dinámica de los mercados verdes y cómo se puede crear valor sostenible y capturó e innovadora de los modelos de negocios verdes. El marco desarrollado proporciona una visión cíclica de la flexibilidad del modelo de negocio en la que se propaga la acumulación de recursos basada en el conocimiento del modelo de negocio a través de los ambientes dentro y inter-empresa. Estrategias de conocimiento de corretaje del resultado ambientes inter e dentro-firma en un mejor desempeño del modelo de negocio. La flexibilidad del modelo de negocio que adquiere está determinada por la eficiencia con la acumulación de recursos está alineado con su ambiente externo. Las características de la que el modelo de negocio alcanza ventajas competitivas, como la innovación y la flexibilidad se atribuyen a la heterogeneidad de los recursos. La investigación se extiende a la literatura orientación de servicio al conceptualizar y medir la orientación a servicios como un requisito clave para la innovación del modelo de negocio, mientras que aboga por la necesidad de identificar correctamente las competencias básicas de la empresa, especialmente relevante en el contexto de la empresa orientada a los servicios, donde la creación de valor requiere recursos y la prestación eficiente de los servicios. La investigación trata de llegar a una descripción de los modelos de negocio sostenibles verdes y argumenta que la innovación, la flexibilidad y la sostenibilidad son los tres habilitadores básicos de los cuales el concepto de modelo de negocio verde puede evolucionar la explotación de nuevos mecanismos de mercado y los mercados para crear y capturar valor en el mantenimiento de su innovadora ambiente externo. La investigación integra efectivamente los conceptos de corretaje de conocimientos y modelos de negocio a partir de un recurso basado en la acumulación de vista y al mismo tiempo llega a la heterogeneidad rendimiento de los modelos de negocio aparentemente similares dentro de la misma industria. La investigación indica cómo se producen perturbaciones del mercado en una industria como resultado de modelos de negocio innovador y flexible, y cómo los nuevos modelos de negocio evolucionan en base a estos trastornos. Avanza la comprensión de cómo la estrategia de núcleo competencia y la innovación del modelo de negocio construcciones se comportan en el esfuerzo de la empresa de servicios para obtener una ventaja competitiva sostenible. Los resultados tienen implicaciones en el rendimiento de las empresas que empiezan sin recursos distintos de los suyos, o que utilizan un modelo de negocio imitado, para lograr un mejor rendimiento a través de la evolución del modelo de negocio alineado con las exitosas estrategias de conocimiento de corretaje. Dicho marco puede permitir a las empresas a evaluar y elegir un modelo de negocio basado en la innovación, la flexibilidad, la sostenibilidad y las opciones para el cambio. La investigación se suma a la literatura acumulación de recursos, explicando cómo los recursos pueden ser efectivamente adquirida para crear valor. La investigación también se suma a la literatura empresarial verde, explicando cómo las empresas crear y capturar valor en los nuevos mecanismos dinámicos de mercado.

Origin of the gases released from the Acqua Passante and Ermeta wells (Mt. Amiata, central Italy) and possible environmental implications for their closure

The Mt. Amiata volcano (Tuscany, central Italy) hosts the second largest geothermal field of Italy. Its SW and NE sectors are characterized by the presence of several CO2-rich (mayor que95% by vol.) gas discharges. An intense Hg mining activity had taken place from the 19th century up to the end of the ?70s, particularly close to Abbadia San Salvatore, during which two drillings (Acqua Passante and Ermeta) intercepted a CO2-rich gas fertile horizon. The related gases are emitted in the atmosphere since 1938 and 1959, respectively, causing severe concerns for the local air quality. In this work the results of a geochemical and isotopic survey carried out on these gas emissions from March 2009 to January 2014 are presented. CO2 fluxes from both the two wells and soil from an area of about 653,500 m2 located between them were measured. The two wells are emitting up to 15,000, 92 and 8 tons y-1 of CO2, CH4 and H2S, respectively, while the computed soil CO2 output was estimated at 4,311 ton y-1. The spatial distribution of the CO2 soil flux suggests the presence of preferential patterns, indicating sites of higher permeability. Since the local municipality is evaluating the possibility to plug the Ermeta vent, a temporarily closure should first be carried out to test the possible influence of this operation on the diffuse soil degassing of deep-originated CO2 in the surrounding area. This implies that diffuse soil gases should carefully be monitored before proceeding with its definitive closure.

Two binding modes reveal flexibility in kinase/response regulator interactions in the bacterial chemotaxis pathway

The crystal structure at 2.0-Å resolution of the complex of the Escherichia coli chemotaxis response regulator CheY and the phosphoacceptor-binding domain (P2) of the kinase CheA is presented. The binding interface involves the fourth and fifth helices and fifth β-strand of CheY and both helices of P2. Surprisingly, the two heterodimers in the asymmetric unit have two different binding modes involving the same interface, suggesting some flexibility in the binding regions. Significant conformational changes have occurred in CheY compared with previously determined unbound structures. The active site of CheY is exposed by the binding of the kinase domain, possibly to enhance phosphotransfer from CheA to CheY. The conformational changes upon complex formation as well as the observation that there are two different binding modes suggest that the plasticity of CheY is an essential feature of response regulator function.

Adjustment of conformational flexibility is a key event in the thermal adaptation of proteins

3-Isopropylmalate dehydrogenase (IPMDH, E.C. 1.1.1.85) from the thermophilic bacterium Thermus thermophilus HB8 is homologous to IPMDH from the mesophilic Escherichia coli, but has an approximately 17°C higher melting temperature. Its temperature optimum is 22–25°C higher than that of the E. coli enzyme; however, it is hardly active at room temperature. The increased conformational rigidity required to stabilize the thermophilic enzyme against heat denaturation might explain its different temperature-activity profile. Hydrogen/deuterium exchange studies were performed on this thermophilic-mesophilic enzyme pair to compare their conformational flexibilities. It was found that Th. thermophilus IPMDH is significantly more rigid at room temperature than E. coli IPMDH, whereas the enzymes have nearly identical flexibilities under their respective optimal working conditions, suggesting that evolutionary adaptation tends to maintain a “corresponding state” regarding conformational flexibility. These observations confirm that conformational fluctuations necessary for catalytic function are restricted at room temperature in the thermophilic enzyme, suggesting a close relationship between conformational flexibility and enzyme function.

Structural flexibility in transcription complex formation revealed by protein–DNA photocrosslinking

The Oct-1 POU domain binds diverse DNA-sequence elements and forms a higher-order regulatory complex with the herpes simplex virus coregulator VP16. The POU domain contains two separate DNA-binding domains joined by a flexible linker. By protein–DNA photocrosslinking we show that the relative positioning of the two POU DNA-binding domains on DNA varies depending on the nature of the DNA target. On a single VP16-responsive element, the POU domain adopts multiple conformations. To determine the structure of the Oct-1 POU domain in a multiprotein complex with VP16, we allowed VP16 to interact with previously crosslinked POU-domain–DNA complexes and found that VP16 can associate with multiple POU-domain conformations. These results reveal the dynamic potential of a DNA-binding domain in directing transcriptional regulatory complex formation.

Reducing the flexibility of retinal restores a wild-type-like photocycle in bacteriorhodopsin mutants defective in protein–retinal coupling

The thermal re-isomerization of retinal from the 13-cis to the all-trans state is a key step in the final stages of the photocycle of the light-driven proton pump, bacteriorhodopsin. This step is greatly slowed upon replacement of Leu-93, a residue in van der Waals contact with retinal. The most likely role of this key interaction is that it restricts the flexibility of retinal. To test this hypothesis, we have exchanged native retinal in Leu-93 mutants with bridged retinal analogs that render retinal less flexible by restricting free rotation around either the C10—C11 (9,11-bridged retinal) or C12—C13 (11,13-bridged retinal) single bonds. The effect of the analogs on the photocycle was then determined spectroscopically by taking advantage of the previous finding that the decay of the O intermediate in the Leu-93 mutants provides a convenient marker for retinal re-isomerization. Time-resolved spectroscopic studies showed that both retinal analogs resulted in a dramatic acceleration of the photocycling time by increasing the rate of decay of the O intermediate. In particular, exchange of native retinal in the Leu-93 → Ala mutant with the 9,11-bridged retinal resulted in an acceleration of the decay of the O intermediate to a rate similar to that seen in wild-type bacteriorhodopsin. We conclude that the protein-induced restriction of conformational flexibility in retinal is a key structural requirement for efficient protein–retinal coupling in the bacteriorhodopsin photocycle.

Tuning of activation thresholds explains flexibility in the selection and development of T cells in the thymus

Immature CD4+CD8+ thymocytes expressing T-cell antigen receptors (TCR) are selected by TCR-mediated recognition of peptides associated with major histocompatibility complex molecules on thymic stromal cells. Selection ensures reactivity of the mature cells to foreign antigens and tolerance to self. Although much has been learned about the factors that determine whether a thymocyte with a given specificity will be positively or negatively selected, selection as an aspect of the developmental process as a whole is less well-understood. Here we invoke a model in which thymocytes tune their response characteristics individually and dynamically in the course of development. Cellular development and selection are driven by receptor-mediated metabolic perturbations. Perturbation is a measure of the net intracellular change induced by external stimulation. It results from the integration of several signals and countersignals over time and therefore depends on the environment and the maturation stage of the cell. Individual cell adaptation limits the range of perturbations. Such adaptation renders thymocytes less sensitive to the level of stimulation per se, but responsive to environmental changes in that level. This formulation begins to explain the mechanisms that link developmental and selection events to each other.

Abscisic acid-induced stomatal closure mediated by cyclic ADP-ribose

Abscisic acid (ABA) is a plant hormone involved in the response of plants to reduced water availability. Reduction of guard cell turgor by ABA diminishes the aperture of the stomatal pore and thereby contributes to the ability of the plant to conserve water during periods of drought. Previous work has demonstrated that cytosolic Ca2+ is involved in the signal transduction pathway that mediates the reduction in guard cell turgor elicited by ABA. Here we report that ABA uses a Ca2+-mobilization pathway that involves cyclic adenosine 5′-diphosphoribose (cADPR). Microinjection of cADPR into guard cells caused reductions in turgor that were preceded by increases in the concentration of free Ca2+ in the cytosol. Patch clamp measurements of isolated guard cell vacuoles revealed the presence of a cADPR-elicited Ca2+-selective current that was inhibited at cytosolic Ca2+ ≥ 600 nM. Furthermore, microinjection of the cADPR antagonist 8-NH2-cADPR caused a reduction in the rate of turgor loss in response to ABA in 54% of cells tested, and nicotinamide, an antagonist of cADPR production, elicited a dose-dependent block of ABA-induced stomatal closure. Our data provide definitive evidence for a physiological role for cADPR and illustrate one mechanism of stimulus-specific Ca2+ mobilization in higher plants. Taken together with other recent data [Wu, Y., Kuzma, J., Marechal, E., Graeff, R., Lee, H. C., Foster, R. & Chua, N.-H. (1997) Science 278, 2126–2130], these results establish cADPR as a key player in ABA signal transduction pathways in plants.

Cell locomotion and focal adhesions are regulated by substrate flexibility

Responses of cells to mechanical properties of the adhesion substrate were examined by culturing normal rat kidney epithelial and 3T3 fibroblastic cells on a collagen-coated polyacrylamide substrate that allows the flexibility to be varied while maintaining a constant chemical environment. Compared with cells on rigid substrates, those on flexible substrates showed reduced spreading and increased rates of motility or lamellipodial activity. Microinjection of fluorescent vinculin indicated that focal adhesions on flexible substrates were irregularly shaped and highly dynamic whereas those on firm substrates had a normal morphology and were much more stable. Cells on flexible substrates also contained a reduced amount of phosphotyrosine at adhesion sites. Treatment of these cells with phenylarsine oxide, a tyrosine phosphatase inhibitor, induced the formation of normal, stable focal adhesions similar to those on firm substrates. Conversely, treatment of cells on firm substrates with myosin inhibitors 2,3-butanedione monoxime or KT5926 caused the reduction of both vinculin and phosphotyrosine at adhesion sites. These results demonstrate the ability of cells to survey the mechanical properties of their surrounding environment and suggest the possible involvement of both protein tyrosine phosphorylation and myosin-generated cortical forces in this process. Such response to physical parameters likely represents an important mechanism of cellular interaction with the surrounding environment within a complex organism.