La Península Ibérica en el transporte masivo de mercancías entre Europa y África : futuras autopistas del mar

La competitividad del transporte de mercancías depende del estado y funcionamiento de las redes existentes y de sus infraestructuras, no del modo de transporte. En concreto, la rentabilidad o la reducción de los costes de producción del transporte marítimo se vería incrementado con el uso de buques de mayor capacidad y con el desarrollo de plataformas portuarias de distribución o puertos secos, ya que el 90% del comercio entre la Unión Europea y terceros países se realiza a través de sus puertos a un promedio de 3,2 billones de toneladas de mercancías manipuladas cada año y el 40% del tráfico intraeuropeo utiliza el transporte marítimo de corta distancia. A pesar de que los puertos europeos acogen anualmente a más de 400 millones de pasajeros, los grandes desarrollos se han producido en los puertos del norte de Europa (Róterdam, Amberes, Ámsterdam). Los países del Sur de Europa deben buscar nuevas fórmulas para ser más competitivos, ya sea mediante creación de nuevas infraestructuras o mediante refuerzo de las existentes, ofreciendo los costes de los puertos del Norte. El fomento del transporte marítimo y fluvial como alternativa al transporte por carretera, especialmente el transporte marítimo de corta distancia, ha sido impulsado por la Comisión Europea (CE) desde 2003 a través de programas de apoyo comunitario de aplicación directa a las Autopistas del Mar, a modo de ejemplo, cabría citar los programas Marco Polo I y II, los cuales contaron con una dotación presupuestaria total de 855 millones de euros para el período 2003 – 2013; en ese período de tiempo se establecieron objetivos de reducción de congestión vial y mejora del comportamiento medio ambiental del sistema de transporte de mercancías dentro de la comunidad y la potenciación de la intermodalidad. El concepto de Autopista del Mar surge en el Libro Blanco de Transportes de la Comisión Europea “La política europea de transportes de cara al 2010: La hora de la verdad” del 12 de diciembre de 2001, en el marco de una política europea para fomento y desarrollo de sistemas de transportes sostenibles. Las Autopistas del Mar consisten en rutas marítimas de corta distancia entre dos puntos, de menor distancia que por vía terrestre, en las que a través del transporte intermodal mejoran significativamente los tiempos y costes de la cadena logística, contribuyen a la reducción de accidentes, ruidos y emisiones de CO2 a la atmósfera, permite que los conductores pierdan horas de trabajo al volante y evita el deterioro de las infraestructuras terrestres, con el consiguiente ahorro en mantenimiento. La viabilidad de una Autopista del Mar depende tanto de factores de ubicación geográficos, como de características propias del puerto, pasando por los diferentes requerimientos del mercado en cada momento (energéticos, medio ambientales y tecnológicos). Existe un elemento nuevo creado por la Comisión Europea: la red transeuropea de transportes (RTE-T). En el caso de España, con sus dos accesos por los Pirineos (La Junquera e Irún) como únicos pasos terrestres de comunicación con el continente y con importantes limitaciones ferroviarias debido a los tres anchos de vía distintos, le resta competitividad frente al conjunto europeo; por el contrario, España es el país europeo con más kilómetros de costa (con más de 8.000 km) y con un emplazamiento geográfico estratégico, lo que le convierte en una plataforma logística para todo el sur de Europa, por lo que las Autopistas del Mar tendrán un papel importante y casi obligado para el desarrollo de los grandes corredores marítimos que promueve Europa. De hecho, Gijón y Vigo lo han hecho muy bien con sus respectivas líneas definidas como Autopistas del Mar y que conectan con el puerto francés de Nantes-Saint Nazaire, ya que desde ahí los camiones pueden coger rutas hacia el Norte. Paralelamente, la Unión Europea ha iniciado los pasos para el impulso de la primera Autopista del Mar que conectará España con el mercado de Reino Unido, concretamente los Puertos de Bilbao y Tilbury. Además, España e Italia sellaron un acuerdo internacional para desarrollar Autopistas del Mar entre ambos países, comprometiéndose a impulsar una docena de rutas entre puertos del litoral mediterráneo español y el italiano. Actualmente, están en funcionando los trayectos como Barcelona-Génova, Valencia-Civitavecchia y Alicante- Nápoles, notablemente más cortos por mar que por carretera. Bruselas identificó cuatro grandes corredores marítimos que podrían concentrar una alta densidad de tráfico de buques, y en dos de ellos España ya tenía desde un principio un papel crucial. La Comisión diseñó el 14 de abril de 2004, a través del proyecto West-Mos, una red de tráfico marítimo que tiene como vías fundamentales la denominada Autopista del Báltico (que enlaza Europa central y occidental con los países bálticos), la Autopista de Europa suroriental (que une el Adriático con el Jónico y el Mediterráneo más oriental) y también la Autopista de Europa occidental y la Autopista de Europa suroccidental (que enlazan España con Reino Unido y la Francia atlántica y con la Francia mediterránea e Italia, respectivamente). Para poder establecer Autopistas del Mar entre la Península Ibérica y el Norte de Europa primará especialmente la retirada de camiones en la frontera pirenaica, donde el tráfico pesado tiene actualmente una intensidad media diaria de 8.000 unidades, actuando sobre los puntos de mayor congestión, como por ejemplo los Alpes, los Pirineos, el Canal de la Mancha, las carreteras fronterizas de Francia y Euskadi, y proponiendo el traslado de las mercancías en barcos o en trenes. Por su parte, para contar con los subsidios y apoyos europeos las rutas seleccionadas como Autopistas del Mar deben mantener una serie de criterios de calidad relacionados con la frecuencia, coste “plataforma logística a plataforma logística”, simplicidad en procedimientos administrativos y participación de varios países, entre otros. Los estudios consideran inicialmente viables los tramos marítimos superiores a 450 millas, con un volumen de unas 15.000 plataformas al año y que dispongan de eficientes comunicaciones desde el puerto a las redes transeuropeas de autopistas y ferrocarril. Otro objetivo de las Autopistas del Mar es desarrollar las capacidades portuarias de forma que se puedan conectar mejor las regiones periféricas a escala del continente europeo. En lo que a Puertos se refiere, las terminales en los muelles deben contar con una línea de atraque de 250 m., un calado superior a 8 m., una rampa “ro-ro” de doble calzada, grúas portainer, y garantizar operatividad para un mínimo de dos frecuencias de carga semanales. El 28 de marzo de 2011 se publicó el segundo Libro Blanco sobre el futuro del transporte en Europa “Hoja de ruta hacia un espacio único europeo de transporte: por una política de transportes competitiva y sostenible”, donde se definió el marco general de las acciones a emprender en los próximos diez años en el ámbito de las infraestructuras de transporte, la legislación del mercado interior, la reducción de la dependencia del carbono, la tecnología para la gestión del tráfico y los vehículos limpios, así como la estandarización de los distintos mercados. Entre los principales desafíos se encuentran la eliminación de los cuellos de botella y obstáculos diversos de nuestra red europea de transporte, minimizar la dependencia del petróleo, reducir las emisiones de GEI en un 60% para 2050 con respecto a los niveles de 1990 y la inversión en nuevas tecnologías e infraestructuras que reduzcan estas emisiones de transporte en la UE. La conexión entre la UE y el norte de África provoca elevados niveles de congestión en los puntos más críticos del trayecto: frontera hispano-francesa, corredor del Mediterráneo y el paso del estrecho. A esto se le añade el hecho de que el sector del transporte por carretera está sujeto a una creciente competencia de mercado motivada por la eliminación de las barreras europeas, mayores exigencias de los cargadores, mayores restricciones a los conductores y aumento del precio del gasóleo. Por otro lado, el mercado potencial de pasajeros tiene una clara diferenciación en tipos de flujos: los flujos en el período extraordinario de la Operación Paso del Estrecho (OPE), enfocado principalmente a marroquíes que vuelven a su país de vacaciones; y los flujos en el período ordinario, enfocado a la movilidad global de la población. Por tanto, lo que se pretende conseguir con este estudio es analizar la situación actual del tráfico de mercancías y pasajeros con origen o destino la península ibérica y sus causas, así como la investigación de las ventajas de la creación de una conexión marítima (Autopista del Mar) con el Norte de África, basándose en los condicionantes técnicos, administrativos, económicos, políticos, sociales y medio ambientales. The competitiveness of freight transport depends on the condition and operation of existing networks and infrastructure, not the mode of transport. In particular, profitability could be increased or production costs of maritime transport could be reduced by using vessels with greater capacity and developing port distribution platforms or dry ports, seeing as 90% of trade between the European Union and third countries happens through its ports. On average 3,2 billion tonnes of freight are handled annualy and 40% of intra-European traffic uses Short Sea Shipping. In spite of European ports annually hosting more than 400 million passengers, there have been major developments in the northern European ports (Rotterdam, Antwerp, Amsterdam). Southern European countries need to find new ways to be more competitive, either by building new infrastructure or by strengthening existing infrastructure, offering costs northern ports. The use of maritime and river transport as an alternative to road transport, especially Short Sea Shipping, has been driven by the European Commission (EC) from 2003 through community support programs for the Motorways of the Sea. These programs include, for example, the Marco Polo I and II programs, which had a total budget of 855 million euros for the period 2003-2013. During this time objectives were set for reducing road congestion, improving the environmental performance of the freight transport system within the community and enhancing intermodal transport. The “Motorway of the Sea” concept arises in the European Commission’s Transport White Paper "European transport policy for 2010: time to decide" on 12 December 2001, as part of a European policy for the development and promotion of sustainable transport systems. A Motorway of the Sea is defined as a short sea route between two points, covering less distance than by road, which provides a significant improvement in intermodal transport times and to the cost supply chain. It contributes to reducing accidents, noise and CO2 emissions, allows drivers to shorten their driving time and prevents the deterioration of land infrastructure thereby saving on maintenance costs. The viability of a Motorway of the Sea depends as much on geographical location factors as on characteristics of the port, taking into account the different market requirements at all times (energy, environmental and technological). There is a new element created by the European Commission: the trans-European transport network (TEN-T). In the case of Spain, with its two access points in the Pyrenees (La Junquera and Irun) as the only land crossings connected to the mainland and major railway limitations due to the three different gauges, it appears less competitive compared to Europe as a whole. However, Spain is the European country with the most kilometers of coastline (over 8,000 km) and a strategic geographical location, which makes it a logistics platform for the all of Southern Europe. This is why the Motorways of the Sea will have an important role, and an almost necessary one to develop major maritime corridors that Europe supports. In fact, Gijon and Vigo have done very well with their respective sea lanes defined as Motorways of the Sea and which connect with the French port of Nantes-Saint Nazaire, as from there trucks can use nort-heading routes. In parallel, the European Union has taken the first steps to boost the first Motorway of the Sea linking Spain to the UK market, specifically the ports of Bilbao and Tilbury. Furthermore, Spain and Italy sealed an international agreement to develop Motorways of the Sea between both countries, pledging to develop a dozen routes between ports on the Spanish and Italian Mediterranean coasts. Currently, there are sea lanes already in use such as Barcelona-Genova, Valencia-Civitavecchia and Alicante-Naples, these are significantly shorter routes by sea than by road. Brussels identified four major maritime corridors that could hold heavy concentrate shipping traffic, and Spain had a crucial role in two of these from the beginning. On 14 April 2004 the Commission planned through the West-Mos project, a network of maritime traffic which includes the essential sea passages the so-called Baltic Motorway (linking Central and Western Europe with the Baltic countries), the southeast Europe Motorway (linking the Adriatic to the Ionian and eastern Mediterranean Sea), the Western Europe Motorway and southwestern Europe Motorway (that links Spain with Britain and the Atlantic coast of France and with the French Mediterranean coast and Italy, respectively). In order to establish Motorways of the Sea between the Iberian Peninsula and Northern Europe especially, it is necessary to remove trucks from the Pyrenean border, where sees heavy traffic (on average 8000 trucks per day) and addressing the points of greatest congestion, such as the Alps, the Pyrenees, the English Channel, the border roads of France and Euskadi, and proposing the transfer of freight on ships or trains. For its part, in order to receive subsidies and support from the European Commission, the routes selected as Motorways of the Sea should maintain a series of quality criteria related to frequency, costs "from logistics platform to logistics platform," simplicity in administrative procedures and participation of several countries, among others. To begin with, studies consider viable a maritime stretch of at least 450 miles with a volume of about 15,000 platforms per year and that have efficient connections from port to trans-European motorways and rail networks. Another objective of the Motorways of the Sea is to develop port capacity so that they can better connect peripheral regions across the European continent. Referring ports, the terminals at the docks must have a berthing line of 250 m., a draft greater than 8 m, a dual carriageway "ro-ro" ramp, portainer cranes, and ensure operability for a minimum of two loads per week. On 28 March 2011 the second White Paper about the future of transport in Europe "Roadmap to a Single European Transport Area – Towards a competitive and resource efficient transport system" was published. In this Paper the general framework of actions to be undertaken in the next ten years in the field of transport infrastructure was defined, including internal market legislation, reduction of carbon dependency, traffic management technology and clean vehicles, as well as the standardization of different markets. The main challenges are how to eliminate bottlenecks and various obstacles in our European transport network, minimize dependence on oil, reduce GHG emissions by 60% by 2050 compared to 1990 levels and encourage investment in new technologies and infrastructure that reduce EU transport emissions. The connection between the EU and North Africa causes high levels of congestion on the most critical points of the journey: the Spanish-French border, the Mediterranean corridor and Gibraltar Strait. In addition to this, the road transport sector is subject to increased market competition motivated by the elimination of European barriers, greater demands of shippers, greater restrictions on drivers and an increase in the price of diesel. On the other hand, the potential passenger market has a clear differentiation in type of flows: flows in the special period of the Crossing the Straits Operation (CSO), mainly focused on Moroccans who return home on vacation; and flows in the regular session, focused on the global mobile population. Therefore, what I want to achieve with this study is present an analysis of the current situation of freight and passengers to or from the Iberian Peninsula and their causes, as well as present research on the advantages of creating a maritime connection (Motorways of the Sea) with North Africa, based on the technical, administrative, economic, political, social and environmental conditions.

La transformación de las grandes estaciones europeas con la llegada de la Alta Velocidad. El caso de Atocha

La aparición del tren de alta velocidad en Europa en las últimas décadas del siglo XX supuso el resurgir de un medio de transporte en progresivo declive desde la popularización del automóvil y del avión. La decadencia del ferrocarril había supuesto en muchos casos el abandono, o incluso la demolición, de estaciones históricas y el deterioro de su entorno urbano. Como reacción a esa desatención surgió, también en el último cuarto de siglo, una mayor conciencia social preocupada por la conservación del patrimonio construido del ferrocarril. La necesidad de adaptación de las grandes estaciones de ferrocarril para dar servicio al nuevo sistema de transporte, junto con el interés por poner en valor sus construcciones históricas y su céntrico entorno, ha dado como resultado la realización de importantes transformaciones. El objeto de la presente investigación es el estudio de las transformaciones que han sufrido las grandes estaciones europeas del siglo XIX con la llegada del tren de alta velocidad, profundizando de manera especial en el caso más significativo que tenemos en nuestro país: la estación de Atocha. En el ámbito europeo es donde se localizan los ejemplos más relevantes de estaciones que tuvieron gran trascendencia en el siglo XIX y que ahora, con la llegada de la Alta Velocidad, vuelven a recuperar su grandeza. En España, el crecimiento de la Alta Velocidad en los últimos años ha sido extraordinario, hasta situarse como el segundo país del mundo con más kilómetros de líneas de alta velocidad en operación y, en consecuencia, se ha construido un gran número de estaciones adaptadas a este servicio. El caso más notable es el de la estación de Atocha, que desde la llegada del AVE en 1992 hasta el día de hoy, se ha convertido en uno de los complejos ferroviarios más importantes del mundo. El trabajo parte del estudio de otros referentes europeos, como las Gares de París, la estación de St Pancras en Londres y de otras cinco estaciones del centro de Europa –Amsterdam Centraal, Antwerpen Centraal, Köln Hauptbahnhof, Frankfurt (Main) Hauptbahnhof y la Gare de Strasbourg–, para establecer el marco analítico sobre el que se profundiza con la estación de Atocha. El proceso de transformación de la estación de Atocha se ha gestado a través de una serie de proyectos que han ido configurando la estación hasta el momento actual y planteando la previsión de futuro: el proyecto del Plan General de Madrid, el concurso de ideas para el diseño de la estación, la estación de Cercanías, la estación de Alta Velocidad y Largo Recorrido, la ampliación de esta para separar los flujos por niveles, los Estudios Informativos del Nuevo Complejo Ferroviario de la Estación de Atocha y su primera fase de construcción. Estos siete proyectos son objeto de un análisis en tres niveles: análisis cronológico, análisis funcional y análisis formal. La estación de Atocha fue la primera estación histórica europea en sufrir una gran transformación vinculada a la llegada de la Alta Velocidad. Aporta el entendimiento de la estación como un todo y la intermodalidad como sus principales valores, además de la gran mejora urbana que supuso la «operación Atocha», y adolece de ciertas carencias en su desarrollo comercial, vinculadas en parte a la presencia del jardín tropical, y de un pobre espacio en las salas de embarque para los pasajeros de salidas. La estación de Atocha completa su transformación a partir de su renovación funcional, manteniendo la carga simbólica de su historia. De la confrontación del caso de Atocha con otras importantes estaciones europeas resulta la definición de las principales consecuencias de la llegada de la Alta Velocidad a las grandes terminales europeas y la identificación de los elementos clave en su transformación. Las consecuencias principales son: la potenciación de la intermodalidad con otros medios de transporte, el desarrollo comercial no necesariamente destinado a los usuarios de los servicios ferroviarios, y la puesta en valor de la antigua estación y de su entorno urbano. Por su parte, los elementos clave en la transformación de las grandes estaciones tienen que ver directamente con la separación de flujos, el entendimiento de la estación por niveles, la dotación de nuevos accesos laterales y la construcción de una nueva gran cubierta para los nuevos andenes. La preeminencia de unos elementos sobre otros depende del carácter propio de cada estación y de cada país, de la magnitud de la intervención y, también, de la estructura y composición de los equipos encargados del diseño de la nueva estación. En la actualidad, nos encontramos en un momento interesante respecto a las estaciones de Alta Velocidad. Tras el reciente atentado frustrado en el Thalys que viajaba de Ámsterdam a París, se ha acordado establecer controles de identidad y equipajes en todas las estaciones de la red europea de alta velocidad, lo que implicará modificaciones importantes en las grandes estaciones que, probablemente, tomarán el modelo de la estación de Atocha como referencia. ABSTRACT The emergence of the high speed train in Europe in the last few decades of the 20th century represented the resurgence of a means of transport in progressive decline since the popularization of the car and the airplane. The railway decay brought in many cases the abandonment, or even the demolition, of historical stations and the deterioration of its urban environment. In response to that neglect, a greater social awareness towards the preservation of the railway built heritage raised up, also in the last quarter-century. The need for adaptation of the great railway stations to serve the new transport system, along with the interest in enhancing the historical buildings and its central locations, had resulted in important transformations. The subject of current investigation is the study of the transformations that the great 19th century European stations have experienced with the arrival of the high speed rail, deepening in particular in the most significant case we have in Spain: Atocha railway station. At European level is where the most relevant examples of stations which have had a great significance in the 19th century and now, with the arrival of the high speed train, have regain their greatness, are located. In Spain, the growth of the high speed rail over the past few years has been outstanding. Today is the second country in the world with the longest high speed rail network in operation and, therefore, with a great number of new stations adapted to this service. The most remarkable case is Atocha station. Since the arrival of the AVE in 1992, the station has become one of the world's most important railway hub. The research starts with the study of other European reference points, as the Gares of Paris, St Pancras station in London and five other stations of Central Europe –Amsterdam Centraal, Antwerpen Centraal, Köln Hauptbahnhof, Frankfurt (Main) Hauptbahnhof y la Gare de Strasbourg–, to establish the analytical framework that will be deepen with Atocha station. The transformation process of Atocha station has been created through a number of projects that have forged the station to date and have raised the sights in the future: the project of the General Urban Development Plan, the ideas competition for the station design, the Suburban train station, the High Speed and Long Distance station, its enlargement in order to separate passenger flows in different levels, the 'Masterplans' for the new Atocha transport hub and its first phase of construction. These seven projects are under scrutiny at three levels: chronological analysis, functional analysis and formal analysis. Atocha station was the first European historical station to undergo a great transformation tied to the arrival of the high speed rail. It brings the understanding of the station as a whole and the intermodality as its greatest values, besides the great urban improvement of the 'Atocha operation', and suffers from certain shortcomings in its commercial development, partly linked to the presence of the tropical garden, and from a poor space in the departure lounges. Atocha station completes its transformation on the basis of its functional renewal, keeping the symbolic charge of its history. The confrontation of Atocha case with the great European stations results in the definition of the principal consequences of the high speed rail arrival to the great European terminals and the identification of the key elements in its transformation. The principal consequences are: the empowering of the intermodality with other means of transport, of the commercial development, not necessarily intended for railway services users, and the enhancement of the old station and its urban environment. On the other hand, the key elements in the transformation of the great stations are directly related with the separation of passenger flows, the understanding of the station in different levels, the placement of new lateral accesses and the construction of a new deck over the new platforms. The pre-eminence of some elements over the others depends on the particular nature of each station and each country, on the scale of the intervention and also in the structure and composition of the teams in charge of the new station design. Nowadays, this is an interesting time concerning the high speed rail stations. After the recent foiled terrorist attempt in the Thalys train travelling from Amsterdam to Paris, it was agreed to establish passenger and luggage controls in every European high speed rail station. This will mean important changes in these great stations, which probably will take Atocha station's model as a reference.

The Aer protein and the serine chemoreceptor Tsr independently sense intracellular energy levels and transduce oxygen, redox, and energy signals for Escherichia coli behavior

We identified a protein, Aer, as a signal transducer that senses intracellular energy levels rather than the external environment and that transduces signals for aerotaxis (taxis to oxygen) and other energy-dependent behavioral responses in Escherichia coli. Domains in Aer are similar to the signaling domain in chemotaxis receptors and the putative oxygen-sensing domain of some transcriptional activators. A putative FAD-binding site in the N-terminal domain of Aer shares a consensus sequence with the NifL, Bat, and Wc-1 signal-transducing proteins that regulate gene expression in response to redox changes, oxygen, and blue light, respectively. A double mutant deficient in aer and tsr, which codes for the serine chemoreceptor, was negative for aerotaxis, redox taxis, and glycerol taxis, each of which requires the proton motive force and/or electron transport system for signaling. We propose that Aer and Tsr sense the proton motive force or cellular redox state and thereby integrate diverse signals that guide E. coli to environments where maximal energy is available for growth.

hCTR1: A human gene for copper uptake identified by complementation in yeast

The molecular mechanisms responsible for the cellular uptake of copper in mammalian cells are unknown. We describe isolation of a human gene involved in this process by complementation of the yeast high-affinity copper uptake mutant, ctr1. Besides complementing ctr1 growth defect on nonfermentable media, the human gene also rescues iron transport and SOD1 defects in ctr1 yeast. Overexpression of the gene in yeast leads to vulnerability to the toxicity of copper overload. In addition, its expression in ctr1 yeast significantly increases the level of cellular copper, as demonstrated by atomic absorption. We propose this gene as a candidate for high-affinity copper uptake in humans and by analogy have named it hCTR1. The hCTR1 and yeast CTR1 predicted transmembrane proteins are 29% identical, but the human protein is substantially smaller in both the extracellular metal-binding and intracellular domains. An additional human gene similar to hCTR1, here named hCTR2, was identified in a database search. Both hCTR1 and hCTR2 are expressed in all human tissues examined, and both genes are located in 9q31/32. These studies, together with the previously recognized functional and sequence similarity between the Menkes/Wilson copper export proteins and CCC2 in yeast, demonstrate that similar copper homeostatic mechanisms are used in these evolutionarily divergent organisms.

Nuclear Import of Sterol Regulatory Element–binding Protein-2, a Basic Helix-Loop-Helix–Leucine Zipper (bHLH-Zip)–containing Transcription Factor, Occurs through the Direct Interaction of Importin β with HLH-Zip

The sterol regulatory element–binding protein-2 (SREBP-2) is produced as a large precursor molecule attached to the endoplasmic reticulum membrane. In response to the sterol depletion, the N-terminal segment of the precursor, which contains a basic helix-loop-helix–leucine zipper domain, is released by two sequential cleavages and is translocated to the nucleus, where it activates the transcription of target genes. The data herein show that released SREBP-2 uses a distinct nuclear transport pathway, which is mediated by importin β. The mature form of SREBP-2 is actively transported into the nucleus when injected into the cell cytoplasm. SREBP-2 binds directly to importin β in the absence of importin α. Ran-GTP but not Ran-GDP causes the dissociation of the SREBP-2–importin β complex. G19VRan-GTP inhibits the nuclear import of SREBP-2 in living cells. In the permeabilized cell in vitro transport system, nuclear import of SREBP-2 is reconstituted only by importin β in conjunction with Ran and its interacting protein p10/NTF2. We further demonstrate that the helix-loop-helix–leucine zipper motif of SREBP-2 contains a novel type of nuclear localization signal, which binds directly to importin β.

Coilin Shuttles between the Nucleus and Cytoplasm In Xenopus Oocytes

Coiled bodies are discrete nuclear organelles often identified by the marker protein p80-coilin. Because coilin is not detected in the cytoplasm by immunofluorescence and Western blotting, it has been considered an exclusively nuclear protein. In the Xenopus germinal vesicle (GV), most coilin actually resides in the nucleoplasm, although it is highly concentrated in 50–100 coiled bodies. When affinity-purified anti-coilin antibodies were injected into the cytoplasm of oocytes, they could be detected in coiled bodies within 2–3 h. Coiled bodies were intensely labeled after 18 h, whereas other nuclear organelles remained negative. Because the nuclear envelope does not allow passive diffusion of immunoglobulins, this observation suggests that anti-coilin antibodies are imported into the nucleus as an antigen–antibody complex with coilin. Newly synthesized coilin is not required, because cycloheximide had no effect on nuclear import and subsequent targeting of the antibodies. Additional experiments with myc-tagged coilin and myc-tagged pyruvate kinase confirmed that coilin is a shuttling protein. The shuttling of Nopp140, NO38/B23, and nucleolin was easily demonstrated by the targeting of their respective antibodies to the nucleoli, whereas anti-SC35 did not enter the germinal vesicle. We suggest that coilin, perhaps in association with Nopp140, may function as part of a transport system between the cytoplasm and the coiled bodies.

Molecular characterization of the phenylacetic acid catabolic pathway in Pseudomonas putida U: The phenylacetyl-CoA catabolon

Fourteen different genes included in a DNA fragment of 18 kb are involved in the aerobic degradation of phenylacetic acid by Pseudomonas putida U. This catabolic pathway appears to be organized in three contiguous operons that contain the following functional units: (i) a transport system, (ii) a phenylacetic acid activating enzyme, (iii) a ring-hydroxylation complex, (iv) a ring-opening protein, (v) a β-oxidation-like system, and (vi) two regulatory genes. This pathway constitutes the common part (core) of a complex functional unit (catabolon) integrated by several routes that catalyze the transformation of structurally related molecules into a common intermediate (phenylacetyl-CoA).

VanX, a bacterial d-alanyl-d-alanine dipeptidase: Resistance, immunity, or survival function?

The zinc-containing d-alanyl-d-alanine (d-Ala-d-Ala) dipeptidase VanX has been detected in both Gram-positive and Gram-negative bacteria, where it appears to have adapted to at least three distinct physiological roles. In pathogenic vancomycin-resistant enterococci, vanX is part of a five-gene cluster that is switched on to reprogram cell-wall biosynthesis to produce peptidoglycan chain precursors terminating in d-alanyl-d-lactate (d-Ala-d-lactate) rather than d-Ala-d-Ala. The modified peptidoglycan exhibits a 1,000-fold decrease in affinity for vancomycin, accounting for the observed phenotypic resistance. In the glycopeptide antibiotic producers Streptomyces toyocaensis and Amylocatopsis orientalis, a vanHAX operon may have coevolved with antibiotic biosynthesis genes to provide immunity by reprogramming cell-wall termini to d-Ala-d-lactate as antibiotic biosynthesis is initiated. In the Gram-negative bacterium Escherichia coli, which is never challenged by the glycopeptide antibiotics because they cannot penetrate the outer membrane permeability barrier, the vanX homologue (ddpX) is cotranscribed with a putative dipeptide transport system (ddpABCDF) in stationary phase by the transcription factor RpoS (σs). The combined action of DdpX and the permease would permit hydrolysis of d-Ala-d-Ala transported back into the cytoplasm from the periplasm as cell-wall crosslinks are refashioned. The d-Ala product could then be oxidized as an energy source for cell survival under starvation conditions.

All kinesin superfamily protein, KIF, genes in mouse and human

Intracellular transport is essential for morphogenesis and functioning of the cell. The kinesin superfamily proteins (KIFs) have been shown to transport membranous organelles and protein complexes in a microtubule- and ATP-dependent manner. More than 30 KIFs have been reported in mice. However, the nomenclature of KIFs has not been clearly established, resulting in various designations and redundant names for a single KIF. Here, we report the identification and classification of all KIFs in mouse and human genome transcripts. Previously unidentified murine KIFs were found by a PCR-based search. The identification of all KIFs was confirmed by a database search of the total human genome. As a result, there are a total of 45 KIFs. The nomenclature of all KIFs is presented. To understand the function of KIFs in intracellular transport in a single tissue, we focused on the brain. The expression of 38 KIFs was detected in brain tissue by Northern blotting or PCR using cDNA. The brain, mainly composed of highly differentiated and polarized cells such as neurons and glia, requires a highly complex intracellular transport system as indicated by the increased number of KIFs for their sophisticated functions. It is becoming increasingly clear that the cell uses a number of KIFs and tightly controls the direction, destination, and velocity of transportation of various important functional molecules, including mRNA. This report will set the foundation of KIF and intracellular transport research.

Behavioral responses of Escherichia coli to changes in redox potential.

Escherichia coli bacteria sensed the redox state in their surroundings and they swam to a niche that had a preferred reduction potential. In a spatial redox gradient of benzoquinone/benzoquinol, E. coli cells migrated to form a sharply defined band. Bacteria swimming out of either face of the band tumbled and returned to the preferred conditions at the site of the band. This behavioral response was named redox taxis. Redox molecules, such as substituted quinones, that elicited redox taxis, interact with the bacterial electron transport system, thereby altering electron transport and the proton motive force. The magnitude of the behavioral response was dependent on the reduction potential of the chemoeffector. The Tsr, Tar, Trg, Tap, and CheR proteins, which have a role in chemotaxis, were not essential for redox taxis. A cheB mutant had inverted responses in redox taxis, as previously demonstrated in aerotaxis. A model is proposed in which a redox effector molecule perturbs the electron transport system, and an unknown sensor in the membrane detects changes in the proton motive force or the redox status of the electron transport system, and transduces this information into a signal that regulates phosphorylation of the CheA protein. A similar mechanism has been proposed for aerotaxis. Redox taxis may play an important role in the distribution of bacterial species in natural environments.

Defective forebrain development in mice lacking gp330/megalin.

gp330/megalin, a member of the low density lipoprotein (LDL) receptor gene family, is expressed on the apical surfaces of epithelial tissues, including the neuroepithelium, where it mediates the endocytic uptake of diverse macromolecules, such as cholesterol-carrying lipoproteins, proteases, and antiproteinases. Megalin knockout mice manifest abnormalities in epithelial tissues including lung and kidney that normally express the protein and they die perinatally from respiratory insufficiency. In brain, impaired proliferation of neuroepithelium produces a holoprosencephalic syndrome, characterized by lack of olfactory bulbs, forebrain fusion, and a common ventricular system. Similar syndromes in humans and animals are caused by insufficient supply of cholesterol during development. Because megalin can bind lipoproteins, we propose that the receptor is part of the maternal-fetal lipoprotein transport system and mediates the endocytic uptake of essential nutrients in the postgastrulation stage.

Expression cloning of the Golgi CMP-sialic acid transporter.

Translocation of nucleotide sugars across the membrane of the Golgi apparatus is a prerequisite for the synthesis of complex carbohydrate structures. While specific transport systems for different nucleotide sugars have been identified biochemically in isolated microsomes and Golgi vesicles, none of these transport proteins has been characterized at the molecular level. Chinese hamster ovary (CHO) mutants of the complementation group Lec2 exhibit a strong reduction in sialylation of glycoproteins and glycolipids due to a defect in the CMP-sialic acid transport system. By complementation cloning in the mutant 6B2, belonging to the Lec2 complementation group, we were able to isolate a cDNA encoding the putative murine Golgi CMP-sialic acid transporter. The cloned cDNA encodes a highly hydrophobic, multiple membrane spanning protein of 36.4 kDa, with structural similarity to the recently cloned ammonium transporters. Transfection of a hemagglutinin-tagged fusion protein into the mutant 6B2 led to Golgi localization of the hemagglutinin epitope. Our results, together with the observation that the cloned gene shares structural similarities to other recently cloned transporter proteins, strongly suggest that the isolated cDNA encodes the CMP-sialic acid transporter.

Cell-cell communication regulates the effects of protein aspartate phosphatases on the phosphorelay controlling development in Bacillus subtilis.

Rap phosphatases are a recently discovered family of protein aspartate phosphatases that dephosphorylate the Spo0F--P intermediate of the phosphorelay, thus preventing sporulation of Bacillus subtilis. They are regulators induced by physiological processes that are antithetical to sporulation. The RapA phosphatase is induced by the ComP-ComA two-component signal transduction system responsible for initiating competence. RapA phosphatase activity was found to be controlled by a small protein, PhrA, encoded on the same transcript as RapA. PhrA resembles secreted proteins and the evidence suggests that it is cleaved by signal peptidase I and a 19-residue C-terminal domain is secreted from the cell. The sporulation deficiency caused by the uncontrolled RapA activity of a phrA mutant can be complemented by synthetic peptides comprising the last six or more of the C-terminal residues of PhrA. Whether the peptide controls RapA activity directly or by regulating its synthesis remains to be determined. Complementation of the phrA mutant can also be obtained in mixed cultures with a wild-type strain, suggesting the peptide may serve as a means of communication between cells. Importation of the secreted peptide required the oligopeptide transport system. The sporulation deficiency of oligopeptide transport mutants can be suppressed by mutating the rapA and rapB genes or by introduction of a spo0F mutation Y13S that renders the protein insensitive to Rap phosphatases. The data indicate that the sporulation deficiency of oligopeptide transport mutants is due to their inability to import the peptides controlling Rap phosphatases.

Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter.

The presence of a proton-coupled electrogenic high-affinity peptide transporter in the apical membrane of tubular cells has been demonstrated by microperfusion studies and by use of brush border membrane vesicles. The transporter mediates tubular uptake of filtered di- and tripeptides and aminocephalosporin antibiotics. We have used expression cloning in Xenopus laevis oocytes for identification and characterization of the renal high-affinity peptide transporter. Injection of poly(A)+ RNA isolated from rabbit kidney cortex into oocytes resulted in expression of a pH-dependent transport activity for the aminocephalosporin antibiotic cefadroxil. After size fractionation of poly(A)+ RNA the transport activity was identified in the 3.0- to 5.0-kb fractions, which were used for construction of a cDNA library. The library was screened for expression of cefadroxil transport after injection of complementary RNA synthesized in vitro from different pools of clones. A single clone (rPepT2) was isolated that stimulated cefadroxil uptake into oocytes approximately 70-fold at a pH of 6.0. Kinetic analysis of cefadroxil uptake expressed by the transporter's complementary RNA showed a single saturable high-affinity transport system shared by dipeptides, tripeptides, and selected amino-beta-lactam antibiotics. Electrophysiological studies established that the transport activity is electrogenic and affected by membrane potential. Sequencing of the cDNA predicts a protein of 729 amino acids with 12 membrane-spanning domains. Although there is a significant amino acid sequence identity (47%) to the recently cloned peptide transporters from rabbit and human small intestine, the renal transporter shows distinct structural and functional differences.

A mammalian arginine/lysine transporter uses multiple promoters.

The mCAT-2 gene encodes a Na(+)-independent cationic amino acid (AA) transporter that is inducibly expressed in a tissue-specific manner in various physiological conditions. When mCAT-2 protein is expressed in Xenopus oocytes, the elicited AA transport properties are similar to the biochemically defined transport system y+. The mCAT-2 protein sequence is closely related to another cationic AA transporter (mCAT-1); these related proteins elicit virtually identical cationic AA transport in Xenopus oocytes. The two genes differ in their tissue expression and induction patterns. Here we report the presence of diverse 5' untranslated region (UTR) sequences in mCAT-2 transcripts. Sequence analysis of 22 independent mCAT-2 cDNA clones reveals that the cDNA sequences converge precisely 16 bp 5' of the initiator AUG codon. Moreover, analysis of genomic clones shows that the mCAT-2 gene 5'UTR exons are dispersed over 18 kb. Classical promoter and enhancer elements are present in appropriate positions 5' of the exons and their utilization results in regulated mCAT-2 mRNA accumulation in skeletal muscle and liver following partial hepatectomy. The isoform adjacent to the most distal promoter is found in all tissues and cell types previously shown to express mCAT-2, while the other 5' UTR isoforms are more tissue specific in their expression. Utilization of some or all of five putative promoters was documented in lymphoma cell clones, liver, and skeletal muscle. TATA-containing and (G+C)-rich TATA-less promoters appear to control mCAT-2 gene expression. The data indicate that the several distinct 5' mCAT-2 mRNA isoforms result from transcriptional initiation at distinct promoters and permit flexible transcriptional regulation of this cationic AA transporter gene.