In vitro reconstitution of light-harvesting POR-protochlorophyllide complex with protochlorophyllides a and b

NADPH:protochlorophyllide oxidoreductase (POR; EC1.1.33.1) is a key enzyme for the light-induced greening of angiosperms. In barley, two POR proteins exist, termed PORA and PORB. These have previously been proposed to form higher molecular weight light-harvesting complexes in the prolamellar body of etioplasts (Reinbothe, C., Lebedev, N., and Reinbothe, S. (1999)Nature 397, 80–84). Here we report the in vitro reconstitution of such complexes from chemically synthesized protochlorophyllides (Pchlides) a andb and galacto- and sulfolipids. Low temperature (77 K) fluorescence measurements revealed that the reconstituted, lipid-containing complex displayed the same characteristics of photoactive Pchlide 650/657 as the presumed native complex in the prolamellar body. Moreover, Pchlide F650/657 was converted to chlorophyllide (Chlide) 684/690 upon illumination of the reconstituted complex with a 1-ms flash of white light. Identification and quantification of acetone-extractable pigments revealed that only the PORB-bound Pchlide a had been photoactive and was converted to Chlide a, whereas Pchlide b bound to the PORA remained photoinactive. Nondenaturing PAGE of the reconstituted Pchlide a/b-containing complex further demonstrated a size similar to that of the presumed native complexin vivo, suggesting that both complexes may be identical.

In situ conversion of protochlorophyllide b to protochlorophyllide a in barley. Evidence for a novel role of 7-formyl reductase in the prolamellar body of etioplasts

We recently put forth a model of a protochlorophyllide (Pchlide) light-harvesting complex operative during angiosperm seedling de-etiolation (Reinbothe, C., Lebedev, N., and Reinbothe, S. (1999) Nature 397, 80–84). This model, which was based on in vitro reconstitution experiments with zinc analogs of Pchlide a and Pchlide b and the two NADPH:protochlorophyllide oxidoreductases (PORs), PORA and PORB, of barley, predicted a 5-fold excess of Pchlide b, relative to Pchlide a, in the prolamellar body of etioplasts. Recent work (Scheumann, V., Klement, H., Helfrich, M., Oster, U., Schoch, S., and Rüdiger, W. (1999) FEBS Lett. 445, 445–448), however, contradicted this model and reported that Pchlide b would not be present in etiolated plants. Here we demonstrate that Pchlide b is an abundant pigment in barley etioplasts but is rather metabolically unstable. It is rapidly converted to Pchlide a by virtue of 7-formyl reductase activity, an enzyme that had previously been implicated in the chlorophyll (Chl) b to Chl a reaction cycle. Our findings suggest that etiolated plants make use of 7-formyl reductase to fine tune the levels of Pchlide b and Pchlidea and thereby may regulate the steady-state level of light-harvesting POR-Pchlide comple

Azotobacter vinelandii nitrogenase: “Kinetics of nif gene expression and insights into the roles of FdxN and NifQ in FeMo-co biosynthesis”

The Molybdenum-nitrogenase is responsible for most biological nitrogen fixation activity (BNF) in the biosphere. Due to its great agronomical importance, it has been the subject of profound genetic and biochemical studies. The Mo nitrogenase carries at its active site a unique iron-molybdenum cofactor (FeMoco) that consists of an inorganic 7 Fe, 1 Mo, 1 C, 9 S core coordinated to the organic acid homocitrate. Biosynthesis of FeMo-co occurs outside nitrogenase through a complex and highly regulated pathway involving proteins acting as molecular scaffolds, metallocluster carriers or enzymes that provide substrates in appropriate chemical forms. Specific expression regulatory factors tightly control the accumulation levels of all these other components. Insertion of FeMo-co into a P-cluster containing apo-NifDK polypeptide results in nitrogenase reconstitution. Investigation of FeMo-co biosynthesis has uncovered new radical chemistry reactions and new roles for Fe-S clusters in biology.

Al Este del Retiro

La zona de Madrid al Este del Retiro ha estado indefectiblemente condicionada en su tardío desarrollo urbano por su posición a espaldas del Real Sitio. La construcción hacia 1640 de las tapias que rodeaban los reales jardines transformó la red de caminos que partían hacia oriente, aisló los terrenos ubicados más al Este de la ciudad, con la que ya sólo se podrían comunicar por las carreteras de Aragón y Valencia, y condenó las expectativas de desarrollo urbano reduciendo los precios de las propiedades, lo cual determinó durante décadas los usos y la arquitectura de la zona. El Anteproyecto de Ensanche de Carlos María de Castro constituye el germen a partir del cual, durante un lento proceso de casi cien años, fue configurándose la ciudad que hoy conocemos. La identificación en el Archivo de Villa del primer plano general del Ensanche trazado por Castro, del cual anteriores trabajos advirtieron de su existencia aunque se desconocía su localización, es la principal aportación de esta investigación. Por un lado, este primer plano general del Ensanche manuscrito es, por sí mismo, un documento de indudable importancia en la historia del urbanismo madrileño. En segundo lugar, el análisis de su contenido arroja nueva luz sobre la propuesta original de Castro, parcialmente censurada por la Dirección General de Obras Públicas antes de la aprobación del plan en 1860. Especialmente en lo referente a la zona de Madrid al Este del Retiro, proyectada como barrio obrero del Ensanche, este documento ha aportado un enfoque desconocido hasta ahora sobre el paisaje urbano concebido por Castro para la más ambiciosa propuesta planteada en mucho tiempo al problema de la vivienda obrera. Finalmente, el análisis de la factura del plano revela la superposición de varias capas de dibujo, evidenciando que durante un tiempo fue un documento vivo, utilizado como plano de trabajo por el equipo de Castro durante aproximadamente diez años, hasta la destitución del ingeniero en 1868. Posteriores análisis del plano sobre otros ámbitos de la ciudad arrojarán sin duda nuevos datos sobre el proceso proyectual del conjunto del Ensanche. Pero la dinámica de lo real, sintetizable en múltiples factores de índole social, económica y legislativa, transformó durante las primeras décadas de andadura del Ensanche la ciudad proyectada por Castro al Este del Retiro. El dibujo de la ciudad, entendido como herramienta de análisis y empleado con éxito en trabajos de investigación realizados por otros autores en la misma línea, ha permitido deducir la reconstitución gráfica del estado de la ciudad en diferentes momentos singulares del desarrollo urbanístico de la zona, así como de la propuesta original de barrio obrero de Castro. No hay que olvidar que, a pesar del escaso interés que suscitaba entre los inversores inmobiliarios el ámbito geográfico de estudio de esta tesis, fue objeto, durante casi un siglo, de numerosas propuestas de ordenación y urbanización que, aunque no llegaron a materializarse, fueron configurando una suerte de desarrollo virtual de la ciudad paralelo al devenir de la realidad. De esta forma, el dibujo se constituye en esta tesis como fuente de información, herramienta de pensamiento y resultado de la investigación en sí mismo, ilustrando y contribuyendo al mejor conocimiento de la forma urbana. ABSTRACT The area of Madrid to the East of the Retiro has been inevitably conditioned in its late urban development by its position behind the Royal Site. The construction of the walls surrounding the royal gardens around 1640 transformed the network of roads departing eastward, isolated land located to the East of the city, with which already only could communicate by roads of Aragon and Valencia, condemned the expectations of urban development by reducing the prices of the properties, and determined for decades uses and architecture of the area. The Carlos María de Castro preliminary design of City Expansion is the germ from which, during a slow process of almost one hundred years, the city which we know today was setting up. The discovery in the City Archive of the City Expansion first drawing traced by Castro, which previous investigations warned of its existence although its location was unknown, is the main contribution of this research. Firstly, this hand drawn general plan of the city expansion is by itself a document of undoubted importance in the history of Madrid urbanism. Secondly, the analysis of its content sheds new light on Castro´s original proposal, partially censored by the Dirección General de Obras Públicas before the approval of the plan in 1860. Especially concerning the area of Madrid to the East of the Retiro, projected as a workingclass district of the City Expansion, this document has provided an unknown up to now approach on the urban landscape designed by Castro for the more ambitious proposal put forward in a long time to the problem of worker housing. Finally, analysis of hand drawn plan reveals the superposition of several layers of drawing, demonstrating that for a time it was a living document, used as a work plan by the Castro team for approximately ten years, until the dismissal of the engineer in 1868. Subsequent analysis of the drawing on other areas of the city will have no doubt new data on the design process of the whole City Expansion. But the dynamics of reality, synthesizable on multiple factors in social, economic and legislative, transformed during the first decades of existence of the City Expansion designed by Castro to the East of the Retiro. Drawing of the city, understood as a tool of analysis and used successfully in research works done by other authors on the same line, has allowed to deduct graphic reconstitution of the city status in different and singular moments in the urban development of the area, as well as the original Castro´s proposal of working-class district. It should not be forgotten that, despite the lack of interest which raised among investors the geographic scope of this thesis study, it was the object, for nearly a century, of numerous proposals for urbanization which, although they didn´t materialize, were setting up a sort of virtual development of the city parallel to the becoming of the reality. In this way, drawing is used in the thesis as a source of information, tool of thought and outcome of the research itself, illustrating and contributing to a better understanding of urban form.

Configuración formal de los rosetones románicos de la ciudad de Zamora

La presente tesis estudia los rosetones románicos de la ciudad de Zamora. La elección del tema tiene como objetivo profundizar en el conocimiento de estos elementos ya que la información existente sobre ellos es muy escasa. El análisis de estos rosetones se ha realizado desde una perspectiva globalizadora que abarca aspectos tales como los geográficos, morfológicos, funcionales, compositivos, constructivos, geométricos, ornamentales, otros. Así mismo, para el desarrollo de esta investigación se ha considerado necesario el estudio de temas históricos, estilísticos, simbólicos, religiosos, culturales, etc., que aportan el marco contextual que permiten su mejor entendimiento. El estudio de cada rosetón ha permitido implementar y desarrollar un método de trabajo analítico basado en el estudio particular de una serie de aspectos como los anteriormente mencionados, así como plantear una estrategia que permite la reconstitución gráfica de los rosetones, basándose en un sistema de módulos que facilitan trabajar de acuerdo a las proporciones de los elementos; hecho que permite acercarnos con gran exactitud a la representación del objeto real cuando se carece de medidas. El desarrollo de esta investigación ha llevado a establecer entre otras cosas que la definición de “ventana circular” que se le atribuye a los rosetones románicos no es acertada, puesto que la función que cumplen en el edificio religioso es más bien de carácter simbólico. ABSTRACT This thesis studies the Romanesque rose windows of the Zamora city. The choice of topic is intended to deepen the knowledge of these elements as the existing information about them is very scarce. The analysis of these rose windows was made from a global perspective covering aspects such as geographic, morphological, functional, compositional, construction, geometric, ornamental, other. Also, for the development of this research it was considered necessary to study historical, stylistic, symbolic, religious, cultural issues, etc., that provide the contextual framework that allow for better understanding. The study of each rose windows has allowed implement and develop a method of analytical work based on the particular study a number of issues such as those mentioned above, as well as devise a strategy that allows the graphic reconstitution of the rose windows, based on a system of modules facilitate work according to the proportions of the elements; made with great precision approach allows the representation of the real thing when it lacks measures. The development of this research has led to establish among other things that the definition of "circular window" that is attributed to the Romanesque rose windows is not successful because the role in the religious building is rather symbolic.