Urban transport interchanges: Importance-Performance analysis for evaluating perceived quality

As the world becomes more urbanised, public transport in cities must seek to provide viable alternatives to individual car transport. At an urban level, interchanges in public transport networks provide easy transfers between and within different transport modes and facilitate seamless travel. This study proposes a methodological framework with which to identify the factors that travellers view as key elements of an urban transport interchange. An attitudinal survey was undertaken in order to collect information about users? needs and perceptions in the Moncloa interchange in Madrid, Spain. The results obtained from an Importance-Performance Analysis (IPA) show that aspects related to the signposting of different facilities and transport services, the internal design of the interchange and the surrounding area, and safety and security are the greatest strengths of the interchange.

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.

El recorrido: evaluación de las cualidades sensoriales y formales del medio urbano como parte de los criterios de caminabilidad. La ciudad de Taipei como caso de estudio

La presente investigación aborda el análisis de las cualidades del entorno urbano que inciden en la experiencia perceptiva de su recorrido. Los intereses que la motivan tienen que ver con la recuperación de la ciudad para el viandante y se sitúa dentro de un marco de intereses más amplio como es el de la ciudad sostenible. En primer lugar, se contextualiza el tema de la experiencia del recorrido a través de disciplinas diferentes como son el cine, la literatura, la arquitectura, el urbanismo, la arquitectura del paisaje, la psicología y la estética ambiental. De este modo, se refuerza el argumento en torno al papel que desempeñan la forma y otros estímulos sensoriales en la experiencia del espacio, ya sea éste urbano, arquitectónico o natural. La experiencia que las personas viven al desplazarse por un entorno viene definida por infinitos factores que van desde la configuración física del entorno recorrido, hasta el aprendizaje cultural de la persona que camina; desde los estímulos sensoriales que se reciben, hasta las condiciones físicas que posibilitan la funcionalidad del recorrido; desde las sutilezas de índole secuencial que se desvelan a medida que avanzamos, hasta las condiciones higrotérmicas en el momento en que se lleva a cabo el desplazamiento. Sin embargo, factores como las motivaciones y atenciones personales del que anda, así como sus recuerdos de experiencias anteriores, deseos, imaginación y estado de ánimo, también definen nuestra experiencia al caminar que, indudablemente, se encuentra ligada a nuestra personalidad y, por lo tanto, no puede ser exacta a la de otra persona. Conscientes de que todos estos factores se funden en un fenómeno global, y que no se dan en la vida por separado, nos hemos esforzado por independizar las cualidades sensoriales y formales, únicamente a efectos de investigación. La importancia del tema a investigar reside en las conexiones que existen entre las cualidades del medio construido y la experiencia cotidiana de su recorrido, en la medida en que dicha vinculación establece relaciones con la calidad de vida, la salud de los ciudadanos y sus sentimientos de identidad. En este sentido, el problema clave encontrado es que si la definición de la experiencia del recorrido urbano es demasiado amplia, se vuelve nebulosa e inoperativa, pero si se precisa demasiado, se pueden excluir variables importantes. Hemos concentrado nuestros esfuerzos en buscar un medio que permita hacer operativo el estudio de aspectos que, por su propia naturaleza, son difíciles de controlar y aplicar en la práctica. En este sentido, el campo de estudio que aúna interés en el tema, producción científica y vocación práctica, es el de la caminabilidad de las ciudades. Por esta razón, hemos situado nuestro trabajo dentro del marco de criterios que este campo establece para valorar un entorno caminable. Sin embargo, hemos detectado que los trabajos sobre caminabilidad tienden a desarrollarse dentro de la disciplina del planeamiento de transporte y, con frecuencia, siguiendo las mismas pautas que la investigación sobre el transporte motorizado. Además, la tendencia a proporcionar datos numéricos producto de la medición controlada de variables, para respaldar iniciativas dentro de los ámbitos de toma de decisiones, lleva consigo un progresivo alejamiento de los aspectos más sutiles y próximos de la experiencia de las personas y la especificidad de los lugares. Por estas razones, hemos estimado necesario profundizar en la experiencia peatonal explorando otras líneas de trabajo como son la habitabilidad de las ciudades, políticas llevadas a cabo para mejorar la calidad del espacio público o las certificaciones de sostenibilidad en el ámbito del urbanismo. También se han estudiado las aproximaciones gráficas a la representación y simulación de recorridos urbanos por su importancia en la comunicación y promoción de caminar. Para detectar las problemáticas implicadas en la definición de una herramienta que permita valorar, de manera operativa, la calidad de la experiencia del recorrido urbano, la metodología de valoración propuesta se ha basado en la combinación de distintos métodos y en la conjugación de tres aproximaciones: valoración por parte del investigador en calidad de experto, valoración realizada por el investigador en el papel de usuario y valoración por el ciudadano. El desarrollo de esta investigación se ha visto condicionado por la intención de aplicar lo estudiado en un caso práctico. El marco de criterios que los trabajos sobre caminabilidad de las ciudades establecen para que se dé un entorno caminable, se puede resumir en que concurran los siguientes factores: mezcla de usos, densidad de población y edificación relativamente altas, destinos públicos accesibles a pie, un alto grado de seguridad con respecto al tráfico y actos delictivos, alta funcionalidad (dimensiones, pendientes, etc.) y atractivo. Una vez definido el modelo de ciudad en que es pertinente realizar una investigación sobre las cualidades del entorno urbano responsables de que éste se perciba como atractivo, se escogió la ciudad de Taipei, entre otras razones, por cumplir con los requisitos restantes. Con respecto al caso práctico, el objetivo es detectar fortalezas y debilidades del área central de la ciudad de Taipei en cuanto a la experiencia perceptiva que proporciona a los viandantes. ABSTRACT This research addresses the analysis of the qualities of the urban environment that affect the perceptual experience of walking. The interests lying behind it are related to the recovery of the city for pedestrians, and is framed within the sustainable city framework of interests. Firstly, the issue of the experience of walking is contextualized through different disciplines such as film, literature, architecture, urban planning, landscape architecture, environmental psychology and aesthetics. This way, the argument about the role that form and other sensory stimuli play in the experience of space, whether it is urban, architectural or natural, is strengthened. The walking experience of people is defined by factors ranging from the physical configuration of the environment to the cultural background of the person who walks, from the sensory stimuli that are perceived to the physical conditions that enable the functionality of the walk, from the subtleties of sequential nature that are revealed as we move around to hygrothermal conditions at the specific time of walking. Nevertheless, factors such as personal motivations and attentions of the walker, as well as memories of past experiences, desires, imagination and mood, also define our walking experience that is undoubtedly linked to our personality and, therefore, it cannot be exactly the same as other people's experience. Being aware that all these factors come together in a total phenomenon and that, in real life, they do not exist separately, we focused on separating sensory and formal qualities only for research purposes. The importance of the research topic lies in the connections between the qualities of the built environment and the quotidian experience of walking through it, to the extent that such link establishes relationships with the quality of life, health and feelings of identity of citizens. In this sense, the key problem encountered is that, if the definition of urban walking experience is too broad, it becomes nebulous and non‐operational, but if it is too precise, important variables can be excluded. We concentrated our efforts on finding a way to operationalize the study of questions that, by their very nature, are difficult to control and apply in practice. In this regard, the field of study that combines interest in the topic, scientific production and practical purpose, is the walkability of cities. For this reason, we placed our work within the framework of the set of criteria that this field establishes for assessing an environment as walkable. However, we found that works on walkability tend to be developed within the discipline of transportation planning and often following the same guidelines that research on motorized transport. Furthermore, the tendency to provide numerical data as a result of the controlled measurement of variables in order to support initiatives in decision making areas, involves a progressive distancing from the proximity and the most subtle aspects of the experience of people, and from the specificity of places. For these reasons, we considered it was necessary to deepen into the study of pedestrians experience, by exploring other lines of work such as the livability of cities, implemented policies to improve the quality of the public space or sustainability certifications in urbanism. Graphic representation and simulation of urban walks are also studied due to their important role in communication and promotion of walking. In order to find the issues involved in defining a tool to assess, in an operational way, the quality of urban walking experience, the proposed assessment methodology is based on the combination of different methods and the synthesis of three approaches: assessment by the researcher as expert, assessment by the researcher playing the role of user and assessment by the citizens. The development of this research has been conditioned by our intention of applying it in a case study. The framework of criteria that works on walkability of cities set to define a walkable environment, can be summarized in the following factors: mix of uses, population and building density rather high, public destinations accessible on foot, high levels of safety in terms of traffic and crime, high functionality (dimensions, slopes, etc.) and attractiveness. After defining the model of city in which it is relevant to conduct an investigation on the qualities that are responsible of the attractiveness of the environment; Taipei City was selected because it meets the remaining requirements, among other reasons. Regarding the case study, the goal is to identify strengths and weaknesses in the central area of Taipei City in terms of the perceptual experience of pedestrians.

Functionality of major histocompatibility complex class II molecules in mice doubly deficient for invariant chain and H-2M complexes

By combining two previously generated null mutations, Ii° and M°, we produced mice lacking the invariant chain and H-2M complexes, both required for normal cell-surface expression of major histocompatibility complex class II molecules loaded with the usual diverse array of peptides. As expected, the maturation and transport of class II molecules, their expression at the cell surface, and their capacity to present antigens were quite similar for cells from Ii°M° double-mutant mice and from animals carrying just the Ii° mutation. More surprising were certain features of the CD4+ T cell repertoire selected in Ii°M° mice: many fewer cells were selected than in Ii+M° animals, and these had been purged of self-reactive specificities, unlike their counterparts in Ii+M° animals. These findings suggest (i) that the peptides carried by class II molecules on stromal cells lacking H-2M complexes may almost all derive from invariant chain and (ii) that H-2M complexes edit the peptide array displayed on thymic stromal cells in the absence of invariant chain, showing that it can edit, in vivo, peptides other than CLIP.

Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin–ankyrin G119 skeleton in Madin Darby canine kidney cells

Spectrin (βIΣ∗) and ankyrin (AnkG119) associate with Golgi membranes and the dynactin complex, but their role in vesicle trafficking remains uncertain. We find that the actin-binding domain and membrane-association domain 1 (MAD1) of βI spectrin together form a constitutive Golgi targeting signal in transfected MDCK cells. Expression of this signal in transfected cells disrupts the endogenous Golgi spectrin skeleton and blocks transport of α- and β-Na,K-ATPase and vesicular stomatitis virus-G protein from the endoplasmic reticulum (ER) but does not disrupt the formation of Golgi stacks, the distribution of β-COP, or the transport and surface display of E-cadherin. The Golgi spectrin skeleton is thus required for the transport of a subset of membrane proteins from the ER to the Golgi. We postulate that together with polyfunctional adapter proteins such as AnkG119, Golgi spectrin forms a docking complex that acts prior to the cis-Golgi, presumably with vesicular–tubular clusters (VTCs or ERGIC), to sequester specific membrane proteins into vesicles transiting between the ER and Golgi, and subsequently (probably involving other isoforms of spectrin and ankyrin) to mediate cargo transport within the Golgi and to other membrane compartments. We hypothesize that this vesicular spectrin–ankyrin adapter-protein trafficking (or tethering) system (SAATS) mediates the capture and transport of many membrane proteins and acts in conjunction with vesicle-targeting molecules to effect the efficient transport of cargo proteins.

Altered phosphorylation and intracellular distribution of a (CUG)n triplet repeat RNA-binding protein in patients with myotonic dystrophy and in myotonin protein kinase knockout mice

Myotonic dystrophy (DM) is associated with expansion of CTG repeats in the 3′-untranslated region of the myotonin protein kinase (DMPK) gene. The molecular mechanism whereby expansion of the (CUG)n repeats in the 3′-untranslated region of DMPK gene induces DM is unknown. We previously isolated a protein with specific binding to CUG repeat sequences (CUG-BP/hNab50) that possibly plays a role in mRNA processing and/or transport. Here we present evidence that the phosphorylation status and intracellular distribution of the RNA CUG-binding protein, identical to hNab50 protein (CUG-BP/hNab50), are altered in homozygous DM patient and that CUG-BP/hNab50 is a substrate for DMPK both in vivo and in vitro. Data from two biological systems with reduced levels of DMPK, homozygous DM patient and DMPK knockout mice, show that DMPK regulates both phosphorylation and intracellular localization of the CUG-BP/hNab50 protein. Decreased levels of DMPK observed in DM patients and DMPK knockout mice led to the elevation of the hypophosphorylated form of CUG-BP/hNab50. Nuclear concentration of the hypophosphorylated CUG-BP/hNab50 isoform is increased in DMPK knockout mice and in homozygous DM patient. DMPK also interacts with and phosphorylates CUG-BP/hNab50 protein in vitro. DMPK-mediated phosphorylation of CUG-BP/hNab50 results in dramatic reduction of the CUG-BP2, hypophosphorylated isoform, accumulation of which was observed in the nuclei of DMPK knockout mice. These data suggest a feedback mechanism whereby decreased levels of DMPK could alter phosphorylation status of CUG-BP/hNab50, thus facilitating nuclear localization of CUG-BP/hNab50. Our results suggest that DM pathophysiology could be, in part, a result of sequestration of CUG-BP/hNab50 and, in part, of lowered DMPK levels, which, in turn, affect processing and transport of specific subclass of mRNAs.

Expression, stability, and membrane integration of truncation mutants of bovine rhodopsin

Premature termination of protein synthesis by nonsense mutations is at the molecular origin of a number of inherited disorders in the family of G protein-coupled seven-helix receptor proteins. To understand how such truncated polypeptides are processed by the cell, we have carried out COS-1 cell expression studies of mutants of bovine rhodopsin truncated at the first 1, 1.5, 2, 3, or 5 transmembrane segments (TMS) of the seven present in wild-type opsin. Our experiments show that successful completion of different stages in the cellular processing of the protein [membrane insertion, N-linked glycosylation, stability to proteolytic degradation, and transport from the endoplasmic reticulum (ER) membrane] requires progressively longer lengths of the polypeptide chain. Thus, none of the truncations affected the ability of the polypeptides to be integral membrane proteins. C-terminal truncations that generated polypeptides with fewer than two TMS resulted in misorientation and prevented glycosylation at the N terminus, whereas truncations that generated polypeptides with fewer than five TMS greatly destabilized the protein. However, all of the truncations prevented exit of the polypeptide from the ER. We conclude that during the biogenesis of rhodopsin, proper integration into the ER membrane occurs only after the synthesis of at least two TMS is completed. Synthesis of the next three TMS confers a gradual increase in stability, whereas the presence of more than five TMS is necessary for exit from the ER.

Benthic–pelagic links and rocky intertidal communities: Bottom-up effects on top-down control?

Insight into the dependence of benthic communities on biological and physical processes in nearshore pelagic environments, long considered a “black box,” has eluded ecologists. In rocky intertidal communities at Oregon coastal sites 80 km apart, differences in abundance of sessile invertebrates, herbivores, carnivores, and macrophytes in the low zone were not readily explained by local scale differences in hydrodynamic or physical conditions (wave forces, surge flow, or air temperature during low tide). Field experiments employing predator and herbivore manipulations and prey transplants suggested top-down (predation, grazing) processes varied positively with bottom-up processes (growth of filter-feeders, prey recruitment), but the basis for these differences was unknown. Shore-based sampling revealed that between-site differences were associated with nearshore oceanographic conditions, including phytoplankton concentration and productivity, particulates, and water temperature during upwelling. Further, samples taken at 19 sites along 380 km of coastline suggested that the differences documented between two sites reflect broader scale gradients of phytoplankton concentration. Among several alternative explanations, a coastal hydrodynamics hypothesis, reflecting mesoscale (tens to hundreds of kilometers) variation in the interaction between offshore currents and winds and continental shelf bathymetry, was inferred to be the primary underlying cause. Satellite imagery and offshore chlorophyll-a samples are consistent with the postulated mechanism. Our results suggest that benthic community dynamics can be coupled to pelagic ecosystems by both trophic and transport linkages.

Effect of Carbohydrate Position on Lysosomal Transport of Procathepsin L

To study the role of carbohydrate in lysosomal protein transport, we engineered two novel glycosylation signals (Asn-X-Ser/Thr) into the cDNA of human procathepsin L, a lysosomal acid protease. We constructed six mutant cDNAs encoding glycosylation signals at mutant sites Asn-138, Asn-175, or both sites together, in the presence or absence of the wild-type Asn-204 site. We stably transfected wild-type and mutant cDNAs into NIH3T3 mouse fibroblasts and then used species-specific antibodies to determine the glycosylation status, phosphorylation, localization, and transport kinetics of recombinant human procathepsin L containing one, two, or three glycosylation sites. Both novel glycosylation sites were capable of being glycosylated, although Asn-175 was utilized only 30–50% of the time. Like the wild-type glycosylation at Asn-204, carbohydrates at Asn-138 and Asn-175 were completely sensitive to endoglycosidase H, and they were phosphorylated. Mutant proteins containing two carbohydrates were capable of being delivered to lysosomes, but there was not a consistent relationship between the efficiency of lysosomal delivery and carbohydrate content of the protein. Pulse-chase labeling revealed a unique biosynthetic pattern for proteins carrying the Asn-175 glycosylation sequence. Whereas wild-type procathepsin L and mutants bearing carbohydrate at Asn-138 appeared in lysosomes by about 60 min, proteins with carbohydrate at Asn-175 were processed to a lysosome-like polypeptide within 15 min. Temperature shift, brefeldin A, and NH4Cl experiments suggested that the rapid processing did not occur in the endoplasmic reticulum and that Asn-175 mutants could interact with the mannose 6-phosphate receptor. Taken together, our results are consistent with the interpretation that Asn-175 carbohydrate confers rapid transport to lysosomes. We may have identified a recognition domain in procathepsin L that is important for its interactions with the cellular transport machinery.

Proteasome inhibition interferes with Gag polyprotein processing, release, and maturation of HIV-1 and HIV-2

Retrovirus assembly and maturation involve folding and transport of viral proteins to the virus assembly site followed by subsequent proteolytic cleavage of the Gag polyprotein within the nascent virion. We report that inhibiting proteasomes severely decreases the budding, maturation, and infectivity of HIV. Although processing of the Env glycoproteins is not changed, proteasome inhibitors inhibit processing of Gag polyprotein by the viral protease without affecting the activity of the HIV-1 viral protease itself, as demonstrated by in vitro processing of HIV-1 Gag polyprotein Pr55. Furthermore, this effect occurs independently of the virus release function of the HIV-1 accessory protein Vpu and is not limited to HIV-1, as proteasome inhibitors also reduce virus release and Gag processing of HIV-2. Electron microscopy analysis revealed ultrastructural changes in budding virions similar to mutants in the late assembly domain of p6gag, a C-terminal domain of Pr55 required for efficient virus maturation and release. Proteasome inhibition reduced the level of free ubiquitin in HIV-1-infected cells and prevented monoubiquitination of p6gag. Consistent with this, viruses with mutations in PR or p6gag were resistant to detrimental effects mediated by proteasome inhibitors. These results indicate the requirement for an active proteasome/ubiquitin system in release and maturation of infectious HIV particles and provide a potential pharmaceutical strategy for interfering with retrovirus replication.

Characterization of Chlamydomonas reinhardtii Zygote-Specific cDNAs That Encode Novel Proteins Containing Ankyrin Repeats and WW Domains1

Genes that are expressed only in the young zygote are considered to be of great importance in the development of an isogamous green alga, Chlamydomonas reinhardtii. Clones representing the Zys3 gene were isolated from a cDNA library prepared using zygotes at 10 min after fertilization. Sequencing of Zys3 cDNA clones resulted in the isolation of two related molecular species. One of them encoded a protein that contained two kinds of protein-to-protein interaction motifs known as ankyrin repeats and WW domains. The other clone lacked the ankyrin repeats but was otherwise identical. These mRNA species began to accumulate simultaneously in cells beginning 10 min after fertilization, and reached maximum levels at about 4 h, after which time levels decreased markedly. Genomic DNA gel-blot analysis indicated that Zys3 was a single-copy gene. The Zys3 proteins exhibited parallel expression to the Zys3 mRNAs at first, appearing 2 h after mating, and reached maximum levels at more than 6 h, but persisted to at least 1 d. Immunocytochemical analysis revealed their localization in the endoplasmic reticulum, which suggests a role in the morphological changes of the endoplasmic reticulum or in the synthesis and transport of proteins to the Golgi apparatus or related vesicles.

Changes in global gene expression patterns during development and maturation of the rat kidney

We set out to define patterns of gene expression during kidney organogenesis by using high-density DNA array technology. Expression analysis of 8,740 rat genes revealed five discrete patterns or groups of gene expression during nephrogenesis. Group 1 consisted of genes with very high expression in the early embryonic kidney, many with roles in protein translation and DNA replication. Group 2 consisted of genes that peaked in midembryogenesis and contained many transcripts specifying proteins of the extracellular matrix. Many additional transcripts allied with groups 1 and 2 had known or proposed roles in kidney development and included LIM1, POD1, GFRA1, WT1, BCL2, Homeobox protein A11, timeless, pleiotrophin, HGF, HNF3, BMP4, TGF-α, TGF-β2, IGF-II, met, FGF7, BMP4, and ganglioside-GD3. Group 3 consisted of transcripts that peaked in the neonatal period and contained a number of retrotransposon RNAs. Group 4 contained genes that steadily increased in relative expression levels throughout development, including many genes involved in energy metabolism and transport. Group 5 consisted of genes with relatively low levels of expression throughout embryogenesis but with markedly higher levels in the adult kidney; this group included a heterogeneous mix of transporters, detoxification enzymes, and oxidative stress genes. The data suggest that the embryonic kidney is committed to cellular proliferation and morphogenesis early on, followed sequentially by extracellular matrix deposition and acquisition of markers of terminal differentiation. The neonatal burst of retrotransposon mRNA was unexpected and may play a role in a stress response associated with birth. Custom analytical tools were developed including “The Equalizer” and “eBlot,” which contain improved methods for data normalization, significance testing, and data mining.

Molecular kinesis in cellular function and plasticity

Intracellular transport and localization of cellular components are essential for the functional organization and plasticity of eukaryotic cells. Although the elucidation of protein transport mechanisms has made impressive progress in recent years, intracellular transport of RNA remains less well understood. The National Academy of Sciences Colloquium on Molecular Kinesis in Cellular Function and Plasticity therefore was devised as an interdisciplinary platform for participants to discuss intracellular molecular transport from a variety of different perspectives. Topics covered at the meeting included RNA metabolism and transport, mechanisms of protein synthesis and localization, the formation of complex interactive protein ensembles, and the relevance of such mechanisms for activity-dependent regulation and synaptic plasticity in neurons. It was the overall objective of the colloquium to generate momentum and cohesion for the emerging research field of molecular kinesis.

Short-Lived and Phosphorylated Proteins Contribute to Carrier-Mediated Efflux, but Not to Influx, of Auxin in Suspension-Cultured Tobacco Cells1

Auxin is transported across the plasma membrane of plant cells by diffusion and by two carriers operating in opposite directions, the influx and efflux carriers. Both carriers most likely play an important role in controlling auxin concentration and distribution in plants but little is known regarding their regulation. We describe the influence of modifications of the transmembrane pH gradient and the effect of agents interfering with protein synthesis, protein traffic, and protein phosphorylation on the activity of the auxin carriers in suspension-cultured tobacco (Nicotiana tabacum L.) cells. Carrier-mediated influx and efflux were monitored independently by measuring the accumulation of [14C]2,4-dichlorophenoxyacetic acid and [3H]naphthylacetic acid, respectively. The activity of the influx carrier decreased on increasing external pH and on decreasing internal pH, whereas that of the efflux carrier was only impaired on internal acidification. The efflux carrier activity was inhibited by cycloheximide, brefeldin A, and the protein kinase inhibitors staurosporine and K252a, as shown by the increased capability of treated cells to accumulate [3H]naphthylacetic acid. Kinetics and reversibility of the effect of brefeldin A were consistent with one or several components of the efflux system being turned over at the plasma membrane with a half-time of less than 10 min. Inhibition of efflux by protein kinase inhibitors suggested that protein phosphorylation was essential to sustain the activity of the efflux carrier. On the contrary, the pharmacological agents used in this study failed to inhibit [14C]2,4-dichlorophenoxyacetic acid accumulation, suggesting that rapidly turned-over proteins or proteins activated by phosphorylation are not essential to carrier-mediated auxin influx. Our data support the idea that the efflux carrier in plants constitutes a complex system regulated at multiple levels, in marked contrast with the influx carrier. Physiological implications of the kinetic features of this regulation are discussed.

Thapsigargin-induced transport of cholera toxin to the endoplasmic reticulum.

Cholera toxin is normally observed only in the Golgi apparatus and not in the endoplasmic reticulum (ER) although the enzymatically active A subunit of cholera toxin has a KDEL sequence. Here we demonstrate transport of horseradish peroxidase-labeled cholera toxin to the ER by electron microscopy in thapsigargin-treated A431 cells. Thapsigargin treatment strongly increased cholera toxin-induced cAMP production, and the formation of the catalytically active A1 fragment was somewhat increased. Binding of cholera toxin to the cell surface and transport of toxin to the Golgi apparatus were not changed in thapsigargin-treated cells, suggesting increased retrograde transport of cholera toxin from the Golgi apparatus to the ER. The data demonstrate that retrograde transport of cholera toxin can take place and that the transport is under regulation. The results are consistent with the idea that retrograde transport can be important for the action of cholera toxin.