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.

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.

Tracking molecular evolution of photosynthesis by characterization of a major photosynthesis gene cluster from Heliobacillus mobilis

A DNA sequence has been obtained for a 35.6-kb genomic segment from Heliobacillus mobilis that contains a major cluster of photosynthesis genes. A total of 30 ORFs were identified, 20 of which encode enzymes for bacteriochlorophyll and carotenoid biosynthesis, reaction-center (RC) apoprotein, and cytochromes for cyclic electron transport. Donor side electron-transfer components to the RC include a putative RC-associated cytochrome c553 and a unique four-large-subunit cytochrome bc complex consisting of Rieske Fe-S protein (encoded by petC), cytochrome b6 (petB), subunit IV (petD), and a diheme cytochrome c (petX). Phylogenetic analysis of various photosynthesis gene products indicates a consistent grouping of oxygenic lineages that are distinct and descendent from anoxygenic lineages. In addition, H. mobilis was placed as the closest relative to cyanobacteria, which form a monophyletic origin to chloroplast-based photosynthetic lineages. The consensus of the photosynthesis gene trees also indicates that purple bacteria are the earliest emerging photosynthetic lineage. Our analysis also indicates that an ancient gene-duplication event giving rise to the paralogous bchI and bchD genes predates the divergence of all photosynthetic groups. In addition, our analysis of gene duplication of the photosystem I and photosystem II core polypeptides supports a “heterologous fusion model” for the origin and evolution of oxygenic photosynthesis.

Peptide nucleic acid–DNA duplexes: Long range hole migration from an internally linked anthraquinone

The discovery that peptide nucleic acids (PNA) mimic DNA and RNA by forming complementary duplex structures following Watson–Crick base pairing rules opens fields in biochemistry, diagnostics, and medicine for exploration. Progress requires the development of modified PNA duplexes having unique and well defined properties. We find that anthraquinone groups bound to internal positions of a PNA oligomer intercalate in the PNA–DNA hybrid. Their irradiation with near-UV light leads to electron transfer and oxidative damage at remote GG doublets on the complementary DNA strand. This behavior mimics that observed in related DNA duplexes and provides the first evidence for long range electron (hole) transport in PNA–DNA hybrid. Analysis of the mechanism for electron transport supports hole hopping.

Preferential Zn2+ influx through Ca2+-permeable AMPA/kainate channels triggers prolonged mitochondrial superoxide production

Synaptically released Zn2+ can enter and cause injury to postsynaptic neurons. Microfluorimetric studies using the Zn2+-sensitive probe, Newport green, examined levels of [Zn2+]i attained in cultured cortical neurons on exposure to N-methyl-d-asparte, kainate, or high K+ (to activate voltage-sensitive Ca2+ channels) in the presence of 300 μM Zn2+. Indicating particularly high permeability through Ca2+-permeable α-amino3-hydroxy-5-methyl-4-isoxazolepropionic-acid/kainate (Ca-A/K) channels, micromolar [Zn2+]i rises were observed only after kainate exposures and only in neurons expressing these channels [Ca-A/K(+) neurons]. Further studies using the oxidation-sensitive dye, hydroethidine, revealed Zn2+-dependent reactive oxygen species (ROS) generation that paralleled the [Zn2+]i rises, with rapid oxidation observed only in the case of Zn2+ entry through Ca-A/K channels. Indicating a mitochondrial source of this ROS generation, hydroethidine oxidation was inhibited by the mitochondrial electron transport blocker, rotenone. Additional evidence for a direct interaction between Zn2+ and mitochondria was provided by the observation that the Zn2+ entry through Ca-A/K channels triggered rapid mitochondrial depolarization, as assessed by using the potential-sensitive dye tetramethylrhodamine ethylester. Whereas Ca2+ influx through Ca-A/K channels also triggers ROS production, the [Zn2+]i rises and subsequent ROS production are of more prolonged duration.

GTP bound to chloroplast thylakoid membranes is required for light-induced, multienzyme degradation of the photosystem II D1 protein

Even though light is the driving force in photosynthesis, it also can be harmful to plants. The water-splitting photosystem II is the main target for this light stress, leading to inactivation of photosynthetic electron transport and photooxidative damage to its reaction center. The plant survives through an intricate repair mechanism involving proteolytic degradation and replacement of the photodamaged reaction center D1 protein. Based on experiments with isolated chloroplast thylakoid membranes and photosystem II core complexes, we report several aspects concerning the rapid turnover of the D1 protein. (i) The primary cleavage step is a GTP-dependent process, leading to accumulation of a 23-kDa N-terminal fragment. (ii) Proteolysis of the D1 protein is inhibited below basal levels by nonhydrolyzable GTP analogues and apyrase treatment, indicating the existence of endogenous GTP tightly bound to the thylakoid membrane. This possibility was corroborated by binding studies. (iii) The proteolysis of the 23-kDa primary degradation fragment (but not of the D1 protein) is an ATP- and zinc-dependent process. (iv) D1 protein degradation is a multienzyme event involving a strategic (primary) protease and a cleaning-up (secondary) protease. (v) The chloroplast FtsH protease is likely to be involved in the secondary degradation steps. Apart from its significance for understanding the repair of photoinhibition, the discovery of tightly bound GTP should have general implications for other regulatory reactions and signal transduction pathways associated with the photosynthetic membrane.

Roles of a conserved arginine residue of DsbB in linking protein disulfide-bond-formation pathway to the respiratory chain of Escherichia coli

The active-site cysteines of DsbA, the periplasmic disulfide-bond-forming enzyme of Escherichia coli, are kept oxidized by the cytoplasmic membrane protein DsbB. DsbB, in turn, is oxidized by two kinds of quinones (ubiquinone for aerobic and menaquinone for anaerobic growth) in the electron-transport chain. We describe the isolation of dsbB missense mutations that change a highly conserved arginine residue at position 48 to histidine or cysteine. In these mutants, DsbB functions reasonably well aerobically but poorly anaerobically. Consistent with this conditional phenotype, purified R48H exhibits very low activity with menaquinone and an apparent Michaelis constant (Km) for ubiquinone seven times greater than that of the wild-type DsbB, while keeping an apparent Km for DsbA similar to that of wild-type enzyme. From these results, we propose that this highly conserved arginine residue of DsbB plays an important role in the catalysis of disulfide bond formation through its role in the interaction of DsbB with quinones.

Plastid redox state and sugars: Interactive regulators of nuclear-encoded photosynthetic gene expression

Feedback regulation of photosynthesis by carbon metabolites has long been recognized, but the underlying cellular mechanisms that control this process remain unclear. By using an Arabidopsis cell culture, we show that a block in photosynthetic electron flux prevents the increase in transcript levels of chlorophyll a/b-binding protein and the small subunit of Rubisco that typically occurs when intracellular sugar levels are depleted. In contrast, the expression of the nitrate reductase gene, which is induced by sugars, is not affected. These findings were confirmed in planta by using Arabidopsis carrying the firefly luciferase reporter gene fused to the plastocyanin and chlorophyll a/b-binding protein 2 gene promoters. Transcription from both promoters increases on carbohydrate depletion. Blocking photosynthetic electron transport with 3-(3′, 4′-dichlorophenyl)-1,1′-dimethylurea prevents this increase in transcription. We conclude that plastid-derived redox signaling can override the sugar-regulated expression of nuclear-encoded photosynthetic genes. In the sugar-response mutant, sucrose uncoupled 6 (sun6), plastocyanin-firefly luciferase transcription actually increases in response to exogenous sucrose rather than decreasing as in the wild type. Interestingly, plastid-derived redox signals do not influence this defective pattern of sugar-regulated gene expression in the sun6 mutant. A model, which invokes a positive inducer originating from the photosynthetic electron transport chain, is proposed to explain the nature of the plastid-derived signal.

The Relationship between Photosynthesis and a Mastoparan-Induced Hypersensitive Response in Isolated Mesophyll Cells1

The G-protein activator mastoparan (MP) was found to elicit the hypersensitive response (HR) in isolated Asparagus sprengeri mesophyll cells at micromolar concentrations. The HR was characterized by cell death, extracellular alkalinization, and an oxidative burst, indicated by the reduction of molecular O2 to O2⋅−. To our knowledge, this study was the first to monitor photosynthesis during the HR. MP had rapid and dramatic effects on photosynthetic electron transport and excitation energy transfer as determined by variable chlorophyll a fluorescence measurements. A large increase in nonphotochemical quenching of chlorophyll a fluorescence accompanied the initial stages of the oxidative burst. The minimal level of fluorescence was also quenched, which suggests the origin of this nonphotochemical quenching to be a decrease in the antenna size of photosystem II. In contrast, photochemical quenching of fluorescence decreased dramatically during the latter stages of the oxidative burst, indicating a somewhat slower inhibition of photosystem II electron transport. The net consumption of O2 and the initial rate of O2 uptake, elicited by MP, were higher in the light than in the dark. These data indicate that light enhances the oxidative burst and suggest a complex relationship between photosynthesis and the HR.

The Involvement of Hydrogen Peroxide in the Differentiation of Secondary Walls in Cotton Fibers1

H2O2 is a widespread molecule in many biological systems. It is created enzymatically in living cells during various oxidation reactions and by leakage of electrons from the electron transport chains. Depending on the concentration H2O2 can induce cell protective responses, programmed cell death, or necrosis. Here we provide evidence that H2O2 may function as a developmental signal in the differentiation of secondary walls in cotton (Gossypium hirsutum) fibers. Three lines of evidence support this conclusion: (a) the period of H2O2 generation coincided with the onset of secondary wall deposition, (b) inhibition of H2O2 production or scavenging the available H2O2 from the system prevented the wall differentiation process, and (c) exogenous addition of H2O2 prematurely promoted secondary wall formation in young fibers. Furthermore, we provide support for the concept that H2O2 generation could be mediated by the expression of the small GTPase Rac, the accumulation of which was shown previously to be strongly induced during the onset of secondary wall differentiation. In support of Rac's role in the activation of NADPH oxidase and the generation of reactive oxygen species, we transformed soybean (Glycine max) and Arabidopsis cells with mutated Rac genes. Transformation with a dominantly activated cotton Rac13 gene resulted in constitutively higher levels of H2O2, whereas transformation with the antisense and especially with dominant-negative Rac constructs decreased the levels of H2O2.

Influences of aging and caloric restriction on the transcriptional profile of skeletal muscle from rhesus monkeys

In laboratory rodents, caloric restriction (CR) retards several age-dependent physiological and biochemical changes in skeletal muscle, including increased steady-state levels of oxidative damage to lipids, DNA, and proteins. We have previously used high-density oligonucleotide arrays to show that CR can prevent or delay most of the major age-related transcriptional alterations in the gastrocnemius muscle of C57BL/6 mice. Here we report the effects of aging and adult-onset CR on the gene expression profile of 7,070 genes in the vastus lateralis muscle from rhesus monkeys. Gene expression analysis of aged rhesus monkeys (mean age of 26 years) was compared with that of young animals (mean age of 8 years). Aging resulted in a selective up-regulation of transcripts involved in inflammation and oxidative stress, and a down-regulation of genes involved in mitochondrial electron transport and oxidative phosphorylation. Middle-aged monkeys (mean age of 20 years) subjected to CR since early adulthood (mean age of 11 years) were studied to determine the gene expression profile induced by CR. CR resulted in an up-regulation of cytoskeletal protein-encoding genes, and also a decrease in the expression of genes involved in mitochondrial bioenergetics. Surprisingly, we did not observe any evidence for an inhibitory effect of adult-onset CR on age-related changes in gene expression. These results indicate that the induction of an oxidative stress-induced transcriptional response may be a common feature of aging in skeletal muscle of rodents and primates, but the extent to which CR modifies these responses may be species-specific.

Nitric oxide partitioning into mitochondrial membranes and the control of respiration at cytochrome c oxidase

An emerging and important site of action for nitric oxide (NO) within cells is the mitochondrial inner membrane, where NO binds to and inhibits members of the electron transport chain, complex III and cytochrome c oxidase. Although it is known that inhibition of cytochrome c oxidase by NO is competitive with O2, the mechanisms that underlie this phenomenon remain unclear, and the impact of both NO and O2 partitioning into biological membranes has not been considered. These properties are particularly interesting because physiological O2 tensions can vary widely, with NO having a greater inhibitory effect at low O2 tensions (<20 μM). In this study, we present evidence for a consumption of NO in mitochondrial membranes in the absence of substrate, in a nonsaturable process that is O2 dependent. This consumption modulates inhibition of cytochrome c oxidase by NO and is enhanced by the addition of exogenous membranes. From these data, it is evident that the partition of NO into mitochondrial membranes has a major impact on the ability of NO to control mitochondrial respiration. The implications of this conclusion are discussed in the context of mitochondrial lipid:protein ratios and the importance of NO as a regulator of respiration in pathophysiology.

Differential Control of Xanthophylls and Light-Induced Stress Proteins, as Opposed to Light-Harvesting Chlorophyll a/b Proteins, during Photosynthetic Acclimation of Barley Leaves to Light Irradiance

Barley (Hordeum vulgare L.) plants were grown at different photon flux densities ranging from 100 to 1800 μmol m−2 s−1 in air and/or in atmospheres with reduced levels of O2 and CO2. Low O2 and CO2 partial pressures allowed plants to grow under high photosystem II (PSII) excitation pressure, estimated in vivo by chlorophyll fluorescence measurements, at moderate photon flux densities. The xanthophyll-cycle pigments, the early light-inducible proteins, and their mRNA accumulated with increasing PSII excitation pressure irrespective of the way high excitation pressure was obtained (high-light irradiance or decreased CO2 and O2 availability). These findings indicate that the reduction state of electron transport chain components could be involved in light sensing for the regulation of nuclear-encoded chloroplast gene expression. In contrast, no correlation was found between the reduction state of PSII and various indicators of the PSII light-harvesting system, such as the chlorophyll a-to-b ratio, the abundance of the major pigment-protein complex of PSII (LHCII), the mRNA level of LHCII, the light-saturation curve of O2 evolution, and the induced chlorophyll-fluorescence rise. We conclude that the chlorophyll antenna size of PSII is not governed by the redox state of PSII in higher plants and, consequently, regulation of early light-inducible protein synthesis is different from that of LHCII.

Analysis of Respiratory Chain Regulation in Roots of Soybean Seedlings1

Changes in the respiratory rate and the contribution of the cytochrome (Cyt) c oxidase and alternative oxidase (COX and AOX, respectively) were investigated in soybean (Glycine max L. cv Stevens) root seedlings using the 18O-discrimination method. In 4-d-old roots respiration proceeded almost entirely via COX, but by d 17 more than 50% of the flux occurred via AOX. During this period the capacity of COX, the theoretical yield of ATP synthesis, and the root relative growth rate all decreased substantially. In extracts from whole roots of different ages, the ubiquinone pool was maintained at 50% to 60% reduction, whereas pyruvate content fluctuated without a consistent trend. In whole-root immunoblots, AOX protein was largely in the reduced, active form at 7 and 17 d but was partially oxidized at 4 d. In isolated mitochondria, Cyt pathway and succinate dehydrogenase capacities and COX I protein abundance decreased with root age, whereas both AOX capacity and protein abundance remained unchanged. The amount of mitochondrial protein on a dry-mass basis did not vary significantly with root age. It is concluded that decreases in whole-root respiration during growth of soybean seedlings can be largely explained by decreases in maximal rates of electron transport via COX. Flux via AOX is increased so that the ubiquinone pool is maintained in a moderately reduced state.

The NAD(P)H Dehydrogenase in Barley Thylakoids Is Photoactivatable and Uses NADPH as well as NADH1

An improved light-dependent assay was used to characterize the NAD(P)H dehydrogenase (NDH) in thylakoids of barley (Hordeum vulgare L.). The enzyme was sensitive to rotenone, confirming the involvement of a complex I-type enzyme. NADPH and NADH were equally good substrates for the dehydrogenase. Maximum rates of activity were 10 to 19 μmol electrons mg−1 chlorophyll h−1, corresponding to about 3% of linear electron-transport rates, or to about 40% of ferredoxin-dependent cyclic electron-transport rates. The NDH was activated by light treatment. After photoactivation, a subsequent light-independent period of about 1 h was required for maximum activation. The NDH could also be activated by incubation of the thylakoids in low-ionic-strength buffer. The kinetics, substrate specificity, and inhibitor profiles were essentially the same for both induction strategies. The possible involvement of ferredoxin:NADP+ oxidoreductase (FNR) in the NDH activity could be excluded based on the lack of preference for NADPH over NADH. Furthermore, thenoyltrifluoroacetone inhibited the diaphorase activity of FNR but not the NDH activity. These results also lead to the conclusion that direct reduction of plastoquinone by FNR is negligible.