Molecular tools to improve chestnut management: El Bierzo as a case study

The European chestnut (Castanea sativa Mill.) is a multipurpose species that has been widely cultivated around the Mediterranean basin since ancient times. New varieties were brought to the Iberian Peninsula during the Roman Empire, which coexist since then with native populations that survived the last glaciation. The relevance of chestnut cultivation has being steadily growing since the Middle Ages, until the rural decline of the past century put a stop to this trend. Forest fires and diseases were also major factors. Chestnut cultivation is gaining momentum again due to its economic (wood, fruits) and ecologic relevance, and represents currently an important asset in many rural areas of Europe. In this Thesis we apply different molecular tools to help improve current management strategies. For this study we have chosen El Bierzo (Castile and Leon, NW Spain), which has a centenary tradition of chestnut cultivation and management, and also presents several unique features from a genetic perspective (next paragraph). Moreover, its nuts are widely appreciated in Spain and abroad for their organoleptic properties. We have focused our experimental work on two major problems faced by breeders and the industry: the lack of a fine-grained genetic characterization and the need for new strategies to control blight disease. To characterize with sufficient detail the genetic diversity and structure of El Bierzo orchards, we analyzed DNA from 169 trees grafted for nut production covering the entire region. We also analyzed 62 nuts from all traditional varieties. El Bierzo constitutes an outstanding scenario to study chestnut genetics and the influence of human management because: (i) it is located at one extreme of the distribution area; (ii) it is a major glacial refuge for the native species; (iii) it has a long tradition of human management (since Roman times, at least); and (iv) its geographical setting ensures an unusual degree of genetic isolation. Thirteen microsatellite markers provided enough informativeness and discrimination power to genotype at the individual level. Together with an unexpected level of genetic variability, we found evidence of genetic structure, with three major gene pools giving rise to the current population. High levels of genetic differentiation between groups supported this organization. Interestingly, genetic structure does not match with spatial boundaries, suggesting that the exchange of material and cultivation practices have strongly influenced natural gene flow. The microsatellite markers selected for this study were also used to classify a set of 62 samples belonging to all traditional varieties. We identified several cases of synonymies and homonymies, evidencing the need to substitute traditional classification systems with new tools for genetic profiling. Management and conservation strategies should also benefit from these tools. The avenue of high-throughput sequencing technologies, combined with the development of bioinformatics tools, have paved the way to study transcriptomes without the need for a reference genome. We took advantage of RNA sequencing and de novo assembly tools to determine the transcriptional landscape of chestnut in response to blight disease. In addition, we have selected a set of candidate genes with high potential for developing resistant varieties via genetic engineering. Our results evidenced a deep transcriptional reprogramming upon fungal infection. The plant hormones ET and JA appear to orchestrate the defensive response. Interestingly, our results also suggest a role for auxins in modulating such response. Many transcription factors were identified in this work that interact with promoters of genes involved in disease resistance. Among these genes, we have conducted a functional characterization of a two major thaumatin-like proteins (TLP) that belongs to the PR5 family. Two genes encoding chestnut cotyledon TLPs have been previously characterized, termed CsTL1 and CsTL2. We substantiate here their protective role against blight disease for the first time, including in silico, in vitro and in vivo evidence. The synergy between TLPs and other antifungal proteins, particularly endo-p-1,3-glucanases, bolsters their interest for future control strategies based on biotechnological approaches.

Genes encoding beta-mannanases in Brachypodium distachyon seeds have a role not only in cell wall softening upon germination sensu stricto but also during post-germinative reserve mobilization

En la coleorriza en semillas de Brachypodium son abundantes los mananos y estos van desapareciendo conforme progresa la germinación (entre 12-27 h), al mismo tiempo se observa un pico de actividad endo-beta-mananasa. Se ha establecido que de los 6 miembros de la familia MAN en B. distachyon 3 se expresan en el embrión en germinación y BdMAN3 también es abundante en la aleurona.

Optimization of the Integrated Guidance and Control for a Dual Aerodynamic Control Missile

Las futuras misiones para misiles aire-aire operando dentro de la atmósfera requieren la interceptación de blancos a mayores velocidades y más maniobrables, incluyendo los esperados vehículos aéreos de combate no tripulados. La intercepción tiene que lograrse desde cualquier ángulo de lanzamiento. Una de las principales discusiones en la tecnología de misiles en la actualidad es cómo satisfacer estos nuevos requisitos incrementando la capacidad de maniobra del misil y en paralelo, a través de mejoras en los métodos de guiado y control modernos. Esta Tesis aborda estos dos objetivos simultáneamente, al proponer un diseño integrando el guiado y el control de vuelo (autopiloto) y aplicarlo a misiles con control aerodinámico simultáneo en canard y cola. Un primer avance de los resultados obtenidos ha sido publicado recientemente en el Journal of Aerospace Engineering, en Abril de 2015, [Ibarrondo y Sanz-Aranguez, 2015]. El valor del diseño integrado obtenido es que permite al misil cumplir con los requisitos operacionales mencionados empleando únicamente control aerodinámico. El diseño propuesto se compara favorablemente con esquemas más tradicionales, consiguiendo menores distancias de paso al blanco y necesitando de menores esfuerzos de control incluso en presencia de ruidos. En esta Tesis se demostrará cómo la introducción del doble mando, donde tanto el canard como las aletas de cola son móviles, puede mejorar las actuaciones de un misil existente. Comparado con un misil con control en cola, el doble control requiere sólo introducir dos servos adicionales para accionar los canards también en guiñada y cabeceo. La sección de cola será responsable de controlar el misil en balanceo mediante deflexiones diferenciales de los controles. En el caso del doble mando, la complicación añadida es que los vórtices desprendidos de los canards se propagan corriente abajo y pueden incidir sobre las superficies de cola, alterando sus características de control. Como un primer aporte, se ha desarrollado un modelo analítico completo para la aerodinámica no lineal de un misil con doble control, incluyendo la caracterización de este efecto de acoplamiento aerodinámico. Hay dos modos de funcionamiento en picado y guiñada para un misil de doble mando: ”desviación” y ”opuesto”. En modo ”desviación”, los controles actúan en la misma dirección, generando un cambio inmediato en la sustentación y produciendo un movimiento de translación en el misil. La respuesta es rápida, pero en el modo ”desviación” los misiles con doble control pueden tener dificultades para alcanzar grandes ángulos de ataque y altas aceleraciones laterales. Cuando los controles actúan en direcciones opuestas, el misil rota y el ángulo de ataque del fuselaje se incrementa para generar mayores aceleraciones en estado estacionario, aunque el tiempo de respuesta es mayor. Con el modelo aerodinámico completo, es posible obtener una parametrización dependiente de los estados de la dinámica de corto periodo del misil. Debido al efecto de acoplamiento entre los controles, la respuesta en bucle abierto no depende linealmente de los controles. El autopiloto se optimiza para obtener la maniobra requerida por la ley de guiado sin exceder ninguno de los límites aerodinámicos o mecánicos del misil. Una segunda contribución de la tesis es el desarrollo de un autopiloto con múltiples entradas de control y que integra la aerodinámica no lineal, controlando los tres canales de picado, guiñada y cabeceo de forma simultánea. Las ganancias del autopiloto dependen de los estados del misil y se calculan a cada paso de integración mediante la resolución de una ecuación de Riccati de orden 21x21. Las ganancias obtenidas son sub-óptimas, debido a que una solución completa de la ecuación de Hamilton-Jacobi-Bellman no puede obtenerse de manera práctica, y se asumen ciertas simplificaciones. Se incorpora asimismo un mecanismo que permite acelerar la respuesta en caso necesario. Como parte del autopiloto, se define una estrategia para repartir el esfuerzo de control entre el canard y la cola. Esto se consigue mediante un controlador aumentado situado antes del bucle de optimización, que minimiza el esfuerzo total de control para maniobrar. Esta ley de alimentación directa mantiene al misil cerca de sus condiciones de equilibrio, garantizando una respuesta transitoria adecuada. El controlador no lineal elimina la respuesta de fase no-mínima característica de la cola. En esta Tesis se consideran dos diseños para el guiado y control, el control en Doble-Lazo y el control Integrado. En la aproximación de Doble-Lazo, el autopiloto se sitúa dentro de un bucle interior y se diseña independientemente del guiado, que conforma el bucle más exterior del control. Esta estructura asume que existe separación espectral entre los dos, esto es, que los tiempos de respuesta del autopiloto son mucho mayores que los tiempos característicos del guiado. En el estudio se combina el autopiloto desarrollado con una ley de guiado óptimo. Los resultados obtenidos demuestran que se consiguen aumentos muy importantes en las actuaciones frente a misiles con control canard o control en cola, y que la interceptación, cuando se lanza cerca del curso de colisión, se consigue desde cualquier ángulo alrededor del blanco. Para el misil de doble mando, la estrategia óptima resulta en utilizar el modo de control opuesto en la aproximación al blanco y utilizar el modo de desviación justo antes del impacto. Sin embargo la lógica de doble bucle no consigue el impacto cuando hay desviaciones importantes con respecto al curso de colisión. Una de las razones es que parte de la demanda de guiado se pierde, ya que el misil solo es capaz de modificar su aceleración lateral, y no tiene control sobre su aceleración axial, a no ser que incorpore un motor de empuje regulable. La hipótesis de separación mencionada, y que constituye la base del Doble-Bucle, puede no ser aplicable cuando la dinámica del misil es muy alta en las proximidades del blanco. Si se combinan el guiado y el autopiloto en un único bucle, la información de los estados del misil está disponible para el cálculo de la ley de guiado, y puede calcularse la estrategia optima de guiado considerando las capacidades y la actitud del misil. Una tercera contribución de la Tesis es la resolución de este segundo diseño, la integración no lineal del guiado y del autopiloto (IGA) para el misil de doble control. Aproximaciones anteriores en la literatura han planteado este sistema en ejes cuerpo, resultando en un sistema muy inestable debido al bajo amortiguamiento del misil en cabeceo y guiñada. Las simplificaciones que se tomaron también causan que el misil se deslice alrededor del blanco y no consiga la intercepción. En nuestra aproximación el problema se plantea en ejes inerciales y se recurre a la dinámica de los cuaterniones, eliminado estos inconvenientes. No se limita a la dinámica de corto periodo del misil, porque se construye incluyendo de modo explícito la velocidad dentro del bucle de optimización. La formulación resultante en el IGA es independiente de la maniobra del blanco, que sin embargo se ha de incluir en el cálculo del modelo en Doble-bucle. Un típico inconveniente de los sistemas integrados con controlador proporcional, es el problema de las escalas. Los errores de guiado dominan sobre los errores de posición del misil y saturan el controlador, provocando la pérdida del misil. Este problema se ha tratado aquí con un controlador aumentado previo al bucle de optimización, que define un estado de equilibrio local para el sistema integrado, que pasa a actuar como un regulador. Los criterios de actuaciones para el IGA son los mismos que para el sistema de Doble-Bucle. Sin embargo el problema matemático resultante es muy complejo. El problema óptimo para tiempo finito resulta en una ecuación diferencial de Riccati con condiciones terminales, que no puede resolverse. Mediante un cambio de variable y la introducción de una matriz de transición, este problema se transforma en una ecuación diferencial de Lyapunov que puede resolverse mediante métodos numéricos. La solución resultante solo es aplicable en un entorno cercano del blanco. Cuando la distancia entre misil y blanco es mayor, se desarrolla una solución aproximada basada en la solución de una ecuación algebraica de Riccati para cada paso de integración. Los resultados que se han obtenido demuestran, a través de análisis numéricos en distintos escenarios, que la solución integrada es mejor que el sistema de Doble-Bucle. Las trayectorias resultantes son muy distintas. El IGA preserva el guiado del misil y consigue maximizar el uso de la propulsión, consiguiendo la interceptación del blanco en menores tiempos de vuelo. El sistema es capaz de lograr el impacto donde el Doble-Bucle falla, y además requiere un orden menos de magnitud en la cantidad de cálculos necesarios. El efecto de los ruidos radar, datos discretos y errores del radomo se investigan. El IGA es más robusto, resultando menos afectado por perturbaciones que el Doble- Bucle, especialmente porque el núcleo de optimización en el IGA es independiente de la maniobra del blanco. La estimación de la maniobra del blanco es siempre imprecisa y contaminada por ruido, y degrada la precisión de la solución de Doble-Bucle. Finalmente, como una cuarta contribución, se demuestra que el misil con guiado IGA es capaz de realizar una maniobra de defensa contra un blanco que ataque por su cola, sólo con control aerodinámico. Las trayectorias estudiadas consideran una fase pre-programada de alta velocidad de giro, manteniendo siempre el misil dentro de su envuelta de vuelo. Este procedimiento no necesita recurrir a soluciones técnicamente más complejas como el control vectorial del empuje o control por chorro para ejecutar esta maniobra. En todas las demostraciones matemáticas se utiliza el producto de Kronecker como una herramienta practica para manejar las parametrizaciones dependientes de variables, que resultan en matrices de grandes dimensiones. ABSTRACT Future missions for air to air endo-atmospheric missiles require the interception of targets with higher speeds and more maneuverable, including forthcoming unmanned supersonic combat vehicles. The interception will need to be achieved from any angle and off-boresight launch conditions. One of the most significant discussions in missile technology today is how to satisfy these new operational requirements by increasing missile maneuvering capabilities and in parallel, through the development of more advanced guidance and control methods. This Thesis addresses these two objectives by proposing a novel optimal integrated guidance and autopilot design scheme, applicable to more maneuverable missiles with forward and rearward aerodynamic controls. A first insight of these results have been recently published in the Journal of Aerospace Engineering in April 2015, [Ibarrondo and Sanz-Aránguez, 2015]. The value of this integrated solution is that it allows the missile to comply with the aforementioned requirements only by applying aerodynamic control. The proposed design is compared against more traditional guidance and control approaches with positive results, achieving reduced control efforts and lower miss distances with the integrated logic even in the presence of noises. In this Thesis it will be demonstrated how the dual control missile, where canard and tail fins are both movable, can enhance the capabilities of an existing missile airframe. Compared to a tail missile, dual control only requires two additional servos to actuate the canards in pitch and yaw. The tail section will be responsible to maintain the missile stabilized in roll, like in a classic tail missile. The additional complexity is that the vortices shed from the canard propagate downstream where they interact with the tail surfaces, altering the tail expected control characteristics. These aerodynamic phenomena must be properly described, as a preliminary step, with high enough precision for advanced guidance and control studies. As a first contribution we have developed a full analytical model of the nonlinear aerodynamics of a missile with dual control, including the characterization of this cross-control coupling effect. This development has been produced from a theoretical model validated with reliable practical data obtained from wind tunnel experiments available in the scientific literature, complement with computer fluid dynamics and semi-experimental methods. There are two modes of operating a missile with forward and rear controls, ”divert” and ”opposite” modes. In divert mode, controls are deflected in the same direction, generating an increment in direct lift and missile translation. Response is fast, but in this mode, dual control missiles may have difficulties in achieving large angles of attack and high level of lateral accelerations. When controls are deflected in opposite directions (opposite mode) the missile airframe rotates and the body angle of attack is increased to generate greater accelerations in steady-state, although the response time is larger. With the aero-model, a state dependent parametrization of the dual control missile short term dynamics can be obtained. Due to the cross-coupling effect, the open loop dynamics for the dual control missile is not linearly dependent of the fin positions. The short term missile dynamics are blended with the servo system to obtain an extended autopilot model, where the response is linear with the control fins turning rates, that will be the control variables. The flight control loop is optimized to achieve the maneuver required by the guidance law without exceeding any of the missile aerodynamic or mechanical limitations. The specific aero-limitations and relevant performance indicators for the dual control are set as part of the analysis. A second contribution of this Thesis is the development of a step-tracking multi-input autopilot that integrates non-linear aerodynamics. The designed dual control missile autopilot is a full three dimensional autopilot, where roll, pitch and yaw are integrated, calculating command inputs simultaneously. The autopilot control gains are state dependent, and calculated at each integration step solving a matrix Riccati equation of order 21x21. The resulting gains are sub-optimal as a full solution for the Hamilton-Jacobi-Bellman equation cannot be resolved in practical terms and some simplifications are taken. Acceleration mechanisms with an λ-shift is incorporated in the design. As part of the autopilot, a strategy is defined for proper allocation of control effort between canard and tail channels. This is achieved with an augmented feed forward controller that minimizes the total control effort of the missile to maneuver. The feedforward law also maintains the missile near trim conditions, obtaining a well manner response of the missile. The nonlinear controller proves to eliminate the non-minimum phase effect of the tail. Two guidance and control designs have been considered in this Thesis: the Two- Loop and the Integrated approaches. In the Two-Loop approach, the autopilot is placed in an inner loop and designed separately from an outer guidance loop. This structure assumes that spectral separation holds, meaning that the autopilot response times are much higher than the guidance command updates. The developed nonlinear autopilot is linked in the study to an optimal guidance law. Simulations are carried on launching close to collision course against supersonic and highly maneuver targets. Results demonstrate a large boost in performance provided by the dual control versus more traditional canard and tail missiles, where interception with the dual control close to collision course is achieved form 365deg all around the target. It is shown that for the dual control missile the optimal flight strategy results in using opposite control in its approach to target and quick corrections with divert just before impact. However the Two-Loop logic fails to achieve target interception when there are large deviations initially from collision course. One of the reasons is that part of the guidance command is not followed, because the missile is not able to control its axial acceleration without a throttleable engine. Also the separation hypothesis may not be applicable for a high dynamic vehicle like a dual control missile approaching a maneuvering target. If the guidance and autopilot are combined into a single loop, the guidance law will have information of the missile states and could calculate the most optimal approach to the target considering the actual capabilities and attitude of the missile. A third contribution of this Thesis is the resolution of the mentioned second design, the non-linear integrated guidance and autopilot (IGA) problem for the dual control missile. Previous approaches in the literature have posed the problem in body axes, resulting in high unstable behavior due to the low damping of the missile, and have also caused the missile to slide around the target and not actually hitting it. The IGA system is posed here in inertial axes and quaternion dynamics, eliminating these inconveniences. It is not restricted to the missile short term dynamic, and we have explicitly included the missile speed as a state variable. The IGA formulation is also independent of the target maneuver model that is explicitly included in the Two-loop optimal guidance law model. A typical problem of the integrated systems with a proportional control law is the problem of scales. The guidance errors are larger than missile state errors during most of the flight and result in high gains, control saturation and loss of control. It has been addressed here with an integrated feedforward controller that defines a local equilibrium state at each flight point and the controller acts as a regulator to minimize the IGA states excursions versus the defined feedforward state. The performance criteria for the IGA are the same as in the Two-Loop case. However the resulting optimization problem is mathematically very complex. The optimal problem in a finite-time horizon results in an irresoluble state dependent differential Riccati equation with terminal conditions. With a change of variable and the introduction of a transition matrix, the equation is transformed into a time differential Lyapunov equation that can be solved with known numerical methods in real time. This solution results range limited, and applicable when the missile is in a close neighborhood of the target. For larger ranges, an approximate solution is used, obtained from solution of an algebraic matrix Riccati equation at each integration step. The results obtained show, by mean of several comparative numerical tests in diverse homing scenarios, than the integrated approach is a better solution that the Two- Loop scheme. Trajectories obtained are very different in the two cases. The IGA fully preserves the guidance command and it is able to maximize the utilization of the missile propulsion system, achieving interception with lower miss distances and in lower flight times. The IGA can achieve interception against off-boresight targets where the Two- Loop was not able to success. As an additional advantage, the IGA also requires one order of magnitude less calculations than the Two-Loop solution. The effects of radar noises, discrete radar data and radome errors are investigated. IGA solution is robust, and less affected by radar than the Two-Loop, especially because the target maneuvers are not part of the IGA core optimization loop. Estimation of target acceleration is always imprecise and noisy and degrade the performance of the two-Loop solution. The IGA trajectories are such that minimize the impact of radome errors in the guidance loop. Finally, as a fourth contribution, it is demonstrated that the missile with IGA guidance is capable of performing a defense against attacks from its rear hemisphere, as a tail attack, only with aerodynamic control. The studied trajectories have a preprogrammed high rate turn maneuver, maintaining the missile within its controllable envelope. This solution does not recur to more complex features in service today, like vector control of the missile thrust or side thrusters. In all the mathematical treatments and demonstrations, the Kronecker product has been introduced as a practical tool to handle the state dependent parametrizations that have resulted in very high order matrix equations.

A PRÁXIS DO MERCADO EDITORIAL EVANGÉLICO. SIMILARIDADES E DIFERENÇAS ENTRE A PRODUÇÃO E DISTRIBUIÇÃO DO LIVRO EVANGÉLICO E DO LIVRO SECULAR NO BRASIL.

A presente pesquisa tem como objetivo estudar o segmento editorial evangélico no Brasil, buscando identificar a práxis da sua produção, comercialização e marketing, comparando-a ao segmento editorial secular. Comparar o perfil do leitor evangélico com o perfil do leitor brasileiro em geral e, ainda, a produção e venda do segmento editorial evangélico com a produção e venda do segmento editorial secular também fazem parte dos objetivos da pesquisa. A metodologia incluiu pesquisa bibliográfica e documental e um estudo de casos múltiplos holísticos, envolvendo as editoras Mundo Cristão e Ediouro-Agir. Verificou-se que a práxis da produção do livro evangélico é semelhante ao do livro secular e que o mercado editorial evangélico cresce mais que o mercado secular. Outro resultado relevante desta pesquisa de dissertação foi a verificação de que o leitor evangélico lê mais que o brasileiro em geral, dados estes, obtidos utilizando-se da mesma metodologia e base de dados dos índices oficiais.(AU)

A PRÁXIS DO MERCADO EDITORIAL EVANGÉLICO. SIMILARIDADES E DIFERENÇAS ENTRE A PRODUÇÃO E DISTRIBUIÇÃO DO LIVRO EVANGÉLICO E DO LIVRO SECULAR NO BRASIL.

A presente pesquisa tem como objetivo estudar o segmento editorial evangélico no Brasil, buscando identificar a práxis da sua produção, comercialização e marketing, comparando-a ao segmento editorial secular. Comparar o perfil do leitor evangélico com o perfil do leitor brasileiro em geral e, ainda, a produção e venda do segmento editorial evangélico com a produção e venda do segmento editorial secular também fazem parte dos objetivos da pesquisa. A metodologia incluiu pesquisa bibliográfica e documental e um estudo de casos múltiplos holísticos, envolvendo as editoras Mundo Cristão e Ediouro-Agir. Verificou-se que a práxis da produção do livro evangélico é semelhante ao do livro secular e que o mercado editorial evangélico cresce mais que o mercado secular. Outro resultado relevante desta pesquisa de dissertação foi a verificação de que o leitor evangélico lê mais que o brasileiro em geral, dados estes, obtidos utilizando-se da mesma metodologia e base de dados dos índices oficiais.(AU)

INFORMAR E ENTRETER: O PROCESSO DE PRODUÇÃO DA NOTÍCIA NO HOJE EM DIA

Sabe-se que o entretenimento está incorporado no cotidiano da sociedade moderna de maneira que ultrapassa apenas os momentos de lazer e ócio, fazendo-se presente nos ambientes profissionais, na educação e na informação. Em meio a essa nova concepção, a comunicação também percebe alterações, seja pela interatividade promovida pela midiatização, pelo novo aspecto da audiência, mais exigente e conectada, ou ainda pela hibridização de gêneros e formatos. Neste contexto, surgem programas de entretenimento, especialmente televisivos, que incorporam aspectos técnicos e práticos do jornalismo, incluindo a narrativa e os processos produtivos. Tais programas veiculam um conteúdo denominado de infotenimento, que mescla informação e entretenimento e, assim, a representação jornalística da vida social fica vinculada à distração e ao divertimento. A dissertação faz uma análise do processo de produção do conteúdo noticioso dentro de um programa de entretenimento e variedades que apresenta grande quantidade de conteúdo jornalístico em sua composição: o Hoje Em Dia, da Rede Record. Trata-se de um estudo qualitativo, do tipo estudo de caso, com uso de técnicas de observação direta, entrevistas semiabertas, pesquisa documental e análise de conteúdo. A questão da reprodutibilidade técnica de padrões e a inserção de elementos cotidianos aliados à indústria cultural e à comunicação em massa também é retratada nesta dissertação. A principal intenção é entender como é a dinâmica da produção jornalística em programas que visam entreter e, ao mesmo tempo, informar. De que maneira esses produtos híbridos da modernidade são pensados e como a teoria do infotenimento pode ser aliada às mediações comunicacionais da cultura.

Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane

Translocation of mitochondrial precursor proteins across the mitochondrial outer membrane is facilitated by the translocase of the outer membrane (TOM) complex. By using site-specific photocrosslinking, we have mapped interactions between TOM proteins and a mitochondrial precursor protein arrested at two distinct stages, stage A (accumulated at 0°C) and stage B (accumulated at 30°C), in the translocation across the outer membrane at high resolution not achieved previously. Although the stage A and stage B intermediates were assigned previously to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crosslinking indicate that the presequence of the intermediates at both stage A and stage B is already on the trans side of the outer membrane. The mature domain is unfolded and bound to Tom40 at stage B whereas it remains folded at stage A. After dissociation from the TOM complex, translocation of the stage B intermediate, but not of the stage A intermediate, across the inner membrane was promoted by the intermembrane-space domain of Tom22. We propose a new model for protein translocation across the outer membrane, where translocation of the presequence and unfolding of the mature domain are not necessarily coupled.

Structure of the HIV-1 integrase catalytic domain complexed with an inhibitor: A platform for antiviral drug design

HIV integrase, the enzyme that inserts the viral DNA into the host chromosome, has no mammalian counterpart, making it an attractive target for antiviral drug design. As one of the three enzymes produced by HIV, it can be expected that inhibitors of this enzyme will complement the therapeutic use of HIV protease and reverse transcriptase inhibitors. We have determined the structure of a complex of the HIV-1 integrase core domain with a novel inhibitor, 5ClTEP, 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-propenone, to 2.1-Å resolution. The inhibitor binds centrally in the active site of the integrase and makes a number of close contacts with the protein. Only minor changes in the protein accompany inhibitor binding. This inhibitor complex will provide a platform for structure-based design of an additional class of inhibitors for antiviral therapy.

Glycosylation differences between the normal and pathogenic prion protein isoforms

Prion protein consists of an ensemble of glycosylated variants or glycoforms. The enzymes that direct oligosaccharide processing, and hence control the glycan profile for any given glycoprotein, are often exquisitely sensitive to other events taking place within the cell in which the glycoprotein is expressed. Alterations in the populations of sugars attached to proteins can reflect changes caused, for example, by developmental processes or by disease. Here we report that normal (PrPC) and pathogenic (PrPSc) prion proteins (PrP) from Syrian hamsters contain the same set of at least 52 bi-, tri-, and tetraantennary N-linked oligosaccharides, although the relative proportions of individual glycans differ. This conservation of structure suggests that the conversion of PrPC into PrPSc is not confined to a subset of PrPs that contain specific sugars. Compared with PrPC, PrPSc contains decreased levels of glycans with bisecting GlcNAc residues and increased levels of tri- and tetraantennary sugars. This change is consistent with a decrease in the activity of N-acetylglucosaminyltransferase III (GnTIII) toward PrPC in cells where PrPSc is formed and argues that, in at least some cells forming PrPSc, the glycosylation machinery has been perturbed. The reduction in GnTIII activity is intriguing both with respect to the pathogenesis of the prion disease and the replication pathway for prions.

Emergence of a highly pathogenic simian/human immunodeficiency virus in a rhesus macaque treated with anti-CD8 mAb during a primary infection with a nonpathogenic virus

Although simian/human immunodeficiency virus (SHIV) strain DH12 replicates to high titers and causes immunodeficiency in pig-tailed macaques, virus loads measured in SHIVDH12-infected rhesus monkeys are consistently 100-fold lower and none of 22 inoculated animals have developed disease. We previously reported that the administration of anti-human CD8 mAb to rhesus macaques at the time of primary SHIVDH12 infection resulted in marked elevations of virus loads. One of the treated animals experienced rapid and profound depletions of circulating CD4+ T lymphocytes. Although the CD4+ T cell number partially recovered, this monkey subsequently suffered significant weight loss and was euthanized. A tissue culture virus stock derived from this animal, designated SHIVDH12R, induced marked and rapid CD4+ cell loss after i.v. inoculation of rhesus monkeys. Retrospective analyses of clinical specimens, collected during the emergence of SHIVDH12R indicated: (i) the input cloned SHIV remained the predominant virus during the first 5–7 months of infection; (ii) variants bearing only a few of the SHIVDH12R consensus changes first appeared 7 months after the administration of anti-CD8 mAb; (iii) high titers of neutralizing antibody directed against the input SHIV were detected by week 10 and persisted throughout the infection; and (iv) no neutralizing antibody against SHIVDH12R ever developed.

Identification of semaphorin E as a non-MDR drug resistance gene of human cancers

To improve cancer chemotherapy, a better understanding of the molecular mechanisms of drug resistance is essential. To identify the molecules responsible for drug resistance that is unrelated to MDR1 or MRP gene products, a eukaryotic expression cDNA library of cis-diamminedichloroplatinum(II) (CDDP)-resistant ovarian cancer TYKnuR cells was introduced into Cos-7 cells. After repeated CDDP selection, cDNA homologous to murine semaphorin E was isolated from surviving cells. Human semaphorin E (H-sema E) was overexpressed in CDDP-resistant cell lines and was readily induced not only by diverse chemotherapeutic drugs but also by x-ray and UV irradiation. Transfection of H-sema E conferred a drug-resistant phenotype to CDDP-sensitive cells. In addition, the aberrant expression of H-sema E protein was detected immunohistochemically in 14 of 42 (33.3%) recurrent squamous cell carcinomas removed at autopsy after extensive radiochemotherapy. Recently, another member of the semaphorin family, CD100, was shown to significantly improve the viability of B lymphocytes. These results suggest the involvement of semaphorins in diverse cell survival mechanisms.

Disruption of a Dynamin Homologue Affects Endocytosis, Organelle Morphology, and Cytokinesis in Dictyostelium discoideum

The identification and functional characterization of Dictyostelium discoideum dynamin A, a protein composed of 853 amino acids that shares up to 44% sequence identity with other dynamin-related proteins, is described. Dynamin A is present during all stages of D. discoideum development and is found predominantly in the cytosolic fraction and in association with endosomal and postlysosomal vacuoles. Overexpression of the protein has no adverse effect on the cells, whereas depletion of dynamin A by gene-targeting techniques leads to multiple and complex phenotypic changes. Cells lacking a functional copy of dymA show alterations of mitochondrial, nuclear, and endosomal morphology and a defect in fluid-phase uptake. They also become multinucleated due to a failure to complete normal cytokinesis. These pleiotropic effects of dynamin A depletion can be rescued by complementation with the cloned gene. Morphological studies using cells producing green fluorescent protein-dynamin A revealed that dynamin A associates with punctate cytoplasmic vesicles. Double labeling with vacuolin, a marker of a postlysosomal compartment in D. discoideum, showed an almost complete colocalization of vacuolin and dynamin A. Our results suggest that that dynamin A is likely to function in membrane trafficking processes along the endo-lysosomal pathway of D. discoideum but not at the plasma membrane.

Chicken Erythroid AE1 Anion Exchangers Associate with the Cytoskeleton During Recycling to the Golgi

Chicken erythroid AE1 anion exchangers receive endoglycosidase F (endo F)-sensitive sugar modifications in their initial transit through the secretory pathway. After delivery to the plasma membrane, anion exchangers are internalized and recycled to the Golgi where they acquire additional N-linked modifications that are resistant to endo F. During recycling, some of the anion exchangers become detergent insoluble. The acquisition of detergent insolubility correlates with the association of the anion exchanger with cytoskeletal ankyrin. Reagents that inhibit different steps in the endocytic pathway, including 0.4 M sucrose, ammonium chloride, and brefeldin A, block the acquisition of endo F-resistant sugars and the acquisition of detergent insolubility by newly synthesized anion exchangers. The inhibitory effects of ammonium chloride on anion exchanger processing are rapidly reversible. Furthermore, AE1 anion exchangers become detergent insoluble more rapidly than they acquire endo F-resistant modifications in cells recovering from an ammonium chloride block. This suggests that the cytoskeletal association of the recycling anion exchangers occurs after release from the compartment where they accumulate due to ammonium chloride treatment, and prior to their transit through the Golgi. The recycling pool of newly synthesized anion exchangers is reflected in the steady-state distribution of the polypeptide. In addition to plasma membrane staining, anion exchanger antibodies stain a perinuclear compartment in erythroid cells. This perinuclear AE1-containing compartment is also stained by ankyrin antibodies and partially overlaps the membrane compartment stained by NBD C6-ceramide, a Golgi marker. Detergent extraction of erythroid cells in situ has suggested that a substantial fraction of the perinuclear pool of AE1 is cytoskeletal associated. The demonstration that erythroid anion exchangers interact with elements of the cytoskeleton during recycling to the Golgi suggests the cytoskeleton may be involved in the post-Golgi trafficking of this membrane transporter.

Trypanosoma cruzi Calreticulin Is a Lectin That Binds Monoglucosylated Oligosaccharides but Not Protein Moieties of Glycoproteins

Trypanosoma cruzi is a protozoan parasite that belongs to an early branch in evolution. Although it lacks several features of the pathway of protein N-glycosylation and oligosaccharide processing present in the endoplasmic reticulum of higher eukaryotes, it displays UDP-Glc:glycoprotein glucosyltransferase and glucosidase II activities. It is herewith reported that this protozoan also expresses a calreticulin-like molecule, the third component of the quality control of glycoprotein folding. No calnexin-encoding gene was detected. Recombinant T. cruzi calreticulin specifically recognized free monoglucosylated high-mannose-type oligosaccharides. Addition of anti-calreticulin serum to extracts obtained from cells pulse–chased with [35S]Met plus [35S]Cys immunoprecipitated two proteins that were identified as calreticulin and the lysosomal proteinase cruzipain (a major soluble glycoprotein). The latter but not the former protein disappeared from immunoprecipitates upon chasing cells. Contrary to what happens in mammalian cells, addition of the glucosidase II inhibitor 1-deoxynojirimycin promoted calreticulin–cruzipain interaction. This result is consistent with the known pathway of protein N-glycosylation and oligosaccharide processing occurring in T. cruzi. A treatment of the calreticulin-cruzipain complexes with endo-β-N-acetylglucosaminidase H either before or after addition of anti-calreticulin serum completely disrupted calreticulin–cruzipain interaction. In addition, mature monoglucosylated but not unglucosylated cruzipain isolated from lysosomes was found to interact with recombinant calreticulin. It was concluded that the quality control of glycoprotein folding appeared early in evolution, and that T. cruzi calreticulin binds monoglucosylated oligosaccharides but not the protein moiety of cruzipain. Furthermore, evidence is presented indicating that glucosyltransferase glucosylated cruzipain at its last folding stages.

Molecular identification of human G-substrate, a possible downstream component of the cGMP-dependent protein kinase cascade in cerebellar Purkinje cells

G-substrate, an endogenous substrate for cGMP-dependent protein kinase, exists almost exclusively in cerebellar Purkinje cells, where it is possibly involved in the induction of long-term depression. A G-substrate cDNA was identified by screening expressed sequence tag databases from a human brain library. The deduced amino acid sequence of human G-substrate contained two putative phosphorylation sites (Thr-68 and Thr-119) with amino acid sequences [KPRRKDT(p)PALH] that were identical to those reported for rabbit G-substrate. G-substrate mRNA was expressed almost exclusively in the cerebellum as a single transcript. The human G-substrate gene was mapped to human chromosome 7p15 by radiation hybrid panel analysis. In vitro translation products of the cDNA showed an apparent molecular mass of 24 kDa on SDS/PAGE which was close to that of purified rabbit G-substrate (23 kDa). Bacterially expressed human G-substrate is a heat-stable and acid-soluble protein that cross-reacts with antibodies raised against rabbit G-substrate. Recombinant human G-substrate was phosphorylated efficiently by cGMP-dependent protein kinase exclusively at Thr residues, and it was recognized by antibodies specific for rabbit phospho-G-substrate. The amino acid sequences surrounding the sites of phosphorylation in G-substrate are related to those around Thr-34 and Thr-35 of the dopamine- and cAMP-regulated phosphoprotein DARPP-32 and inhibitor-1, respectively, two potent inhibitors of protein phosphatase 1. However, purified G-substrate phosphorylated by cGMP-dependent protein kinase inhibited protein phosphatase 2A more effectively than protein phosphatase 1, suggesting a distinct role as a protein phosphatase inhibitor.