Developmental Expression of Kv Potassium Channels at the Axon Initial Segment of Cultured Hippocampal Neurons

Axonal outgrowth and the formation of the axon initial segment (AIS) are early events in the acquisition of neuronal polarity. The AIS is characterized by a high concentration of voltage-dependent sodium and potassium channels. However, the specific ion channel subunits present and their precise localization in this axonal subdomain vary both during development and among the types of neurons, probably determining their firing characteristics in response to stimulation. Here, we characterize the developmental expression of different subfamilies of voltage-gated potassium channels in the AISs of cultured mouse hippocampal neurons, including subunits Kv1.2, Kv2.2 and Kv7.2. In contrast to the early appearance of voltage-gated sodium channels and the Kv7.2 subunit at the AIS, Kv1.2 and Kv2.2 subunits were tethered at the AIS only after 10 days in vitro. Interestingly, we observed different patterns of Kv1.2 and Kv2.2 subunit expression, with each confined to distinct neuronal populations. The accumulation of Kv1.2 and Kv2.2 subunits at the AIS was dependent on ankyrin G tethering, it was not affected by disruption of the actin cytoskeleton and it was resistant to detergent extraction, as described previously for other AIS proteins. This distribution of potassium channels in the AIS further emphasizes the heterogeneity of this structure in different neuronal populations, as proposed previously, and suggests corresponding differences in action potential regulation.

Colocalization of α-actinin and Synaptopodin in the Pyramidal Cell Axon Initial Segment

The cisternal organelle that resides in the axon initial segment (AIS) of neocortical and hippocampal pyramidal cells is thought to be involved in regulating the Ca(2+) available to maintain AIS scaffolding proteins, thereby preserving normal AIS structure and function. Through immunocytochemistry and correlative light and electron microscopy, we show here that the actin-binding protein ?-actinin is present in the typical cistenal organelle of rodent pyramidal neurons as well as in a large structure in the AIS of a subpopulation of layer V pyramidal cells that we have called the "giant saccular organelle." Indeed, this localization of ?-actinin in the AIS is dependent on the integrity of the actin cytoskeleton. Moreover, in the cisternal organelle of cultured hippocampal neurons, ?-actinin colocalizes extensively with synaptopodin, a protein that interacts with both actin and ?-actinin, and they appear concomitantly during the development of these neurons. Together, these results indicate that ?-actinin and the actin cytoskeleton are important components of the cisternal organelle that are probably required to stabilize the AIS.

Modelización de ensayos de corte directo de escollera en cajón de 1x1 m2

En el presente proyecto se redacta un estudio sobre la resistencia al corte de escolleras en cajón de 1x1m2.Con este estudio se intentará valores aproximados al ángulo de rozamiento. Estos parámetros se distinguirán en función si las escolleras son directamente vertidas o si han sufrido una previa compactación. Para ello se han realizado una serie de ensayos, con materias traído de varias zonas de España al Laboratorio de Geotecnia de CEDEX. Los datos obtenidos se han interpretado de varias formas. Primero se ha analizado las curvas resultantes de la tensión tangencial en función del desplazamiento. Después se ha aplicado un análisis de la relación entre el ángulo de rozamiento y la tensión normal. Por otra parte se han utilizado dos modelos, uno de hiperbólico y otro modelo rotura. Dentro del modelo de rotura se han tomado dos criterios. Un primer criterio según el criterio de rotura de Morh-Coulomb y un segundo criterio no lineal sino parabólico. Con los resultados obtenidos de las interpretaciones, se ha realizado una comparativa. Por una parte se han comparada con los datos que dieron otros autores y por otra parte con los resultados obtenidos en los ensayos de corte directo en caja de 30x30 cm2. Las conclusiones obtenidas, un valor del ángulo de rozamiento sería de 42° para escolleras que no han sido compactadas y de 47° para escolleras que hayan sido compactadas.

Implantación de un sistema de depósito, devolución y retorno en el mercado retail español

“Quien contamina, paga“, con esta premisa surgió la idea de este Trabajo Fin de Máster, en adelante TFM, cuyo objetivo era identificar medidas alternativas reales para una optimización del proceso actual de gestión de residuos sólidos urbanos ante una sociedad cada vez más superpoblada y con mayores ratios de consumo. Cada español genera anualmente un volumen de 485 Kg de residuos, de los cuales únicamente el 33 % son reciclados y pueden volver a un flujo normal de uso, especialmente preocupante durante los últimos años es el auge de los productos envasados, tanto de bebidas como de alimentos , cuya utilización se ha duplicado en la última década. La motivación de este trabajo Fin de Máster ha sido la de poner de manifiesto que la sostenibilidad con el medioambiente puede ir de la mano de la rentabilidad y del progreso. Durante este TFM se ha estudiado y analizado la viabilidad económica de implantación de un nuevo modelo de depósito, devolución y retorno en el mercado retail español y como con la adopción de este nuevo sistema se pueden lograr beneficios tanto para el propio minorista, como para el medio ambiente con ratios de reciclado superiores al 98%. La preocupación por el medio ambiente empieza a ser una constante entre los consumidores españoles y dicha preocupación comienza a ser influenciadora en las decisiones de compra (productos eco, sostenibilidad…). Nuestra propuesta consiste en dotar a los principales distribuidores del sector retail español de un sistema de depósito, devolución y retorno para envases de bebidas capaz de generar diferenciación, innovación y rentabilidad frente a la competencia. Dicho sistema consiste en pagar un depósito por cada envase de bebida que se adquiera y su correspondiente devolución en la siguiente compra, una vez que se devuelva vacío al establecimiento. Para ello se ha analizado el sector de la distribución en España, especialmente la distribución de bebidas. Se trata de un sector muy competitivo, que presenta varios formatos en función del tamaño del establecimiento (Hipermercados, Supermercados, tiendas tradicionales). Las principales empresas distribuidoras (Carrefour, Mercadona, Alcampo, Eroski, DIA) se encuentran en procesos de cambios estratégicos para lograr atraer a más consumidores hacia sus tiendas, por lo que nuestra propuesta podrá añadir valor a la hora de influenciar en la decisión del lugar de compra. En nuestro caso, nos dirigiremos principalmente a las grandes empresas distribuidoras, Hipermercados de más de 2.500 m2 ,que cuentan con más de 500 puntos de venta y distribución donde existe la posibilidad real de implantar un SDDR. Además se ha realizado un estudio de mercado sobre la influencia de dicho sistema en el consumidor final, donde se ha detectado dos segmentos principales cuya decisión de compra se vería muy influenciada por la implantación de un SDDR, un segmento Sénior, entre 45-54 años, preocupados por el medio ambiente y con poder adquisitivo suficiente como para que el pago del depósito no sea bloqueante, y un segmento Junior, entre 18-24 años, también muy concienciado el medio ambiente, de capacidad económica menor pero qué influye en la decisión de compra de sus progenitores. Para llevar a cabo este plan de negocio será necesario una inversión inicial de 57.000 €, con unas expectativas de recuperación de dicha inversión en el primer año y una TIR del 56%, presentando un VAN de 127.961 € para los 7 años de vida del proyecto. Para dar a conocer a los clientes del Hipermercado los beneficios de utilizar un sistema SDDR, se realizarán campañas de marketing a través de diferentes canales, promociones de apertura, acciones de marketing exteriores y planes de fidelización. La organización e implantación en el Hipermercado será muy sencilla con roles claramente diferenciados, únicamente involucraría a unos 9 recursos definidos y en aproximadamente 3 meses desde el inicio del proyecto ya se podría ofertar dicho servicio a los clientes del Hipermercado. Además se han analizado los principales riesgos a los que se enfrentaría el negocio, ponderándose en una matriz impacto-probabilidad. Se han establecido medidas correctoras en el caso que dicho riesgo aflore. Habrá que tener especialmente precaución con la pérdida de ventas durante el arranque del negocio en el caso que esto ocurra, por lo que se deberá controlar el gasto, fomentar la captación de clientes y mantener un fondo de maniobra lo suficientemente elevado como para absorber dicho riesgo.---ABSTRACT---“Polluters pay”, with this premise this TFM aimed at identifying real alternative measures for optimization of the current process of solid waste management in a crowded society and with greater consumption ratios. Spaniards generates an annual volume of 485 kg of waste; only 33 % are recycled and can return to a normal flow. Specially concern is the increased of packaged product in recent years, mainly drink and food, their use has been duplicated in the last decade. The motivation for this Thesis was to highlight that sustainability, profitability and progress can go together. During this TFM has been studied and analyzed the economic feasibility of implementing a new model of deposit , refund and return in the Spanish retail market and as with the adoption of this new system can achieve benefits for the retailer itself therefore to the environment with ratios above 98% recycled. Concern for the environment is becoming a constant among Spanish consumers , and this concern is becoming influencer in purchasing decisions ( eco, sustainability ... ) . Our proposal is to provide the main distributors of the Spanish retail sector a system of deposit, refund and return for beverage containers capable of generating differentiation, innovation and profitability over the competition. This system is to pay a deposit for each beverage container they purchase and their corresponding return in the next purchase, once they return empty to the establishment. For this we have analyzed the distribution sector in Spain, especially the distribution of beverages. This is a highly competitive industry, which features various formats depending on the size of establishments (hypermarkets, supermarkets, traditional shops). The main distribution companies (Carrefour, Mercadona, Alcampo, Eroski, DIA) are in the process of strategic changes in order to attract more consumers to their stores, so that our approach can add value in influencing the decision of place shopping. In our case, we will go mainly to large distributors, Hypermarkets of over 2,500 m2, which have more than 500 outlets and distribution where there is a real possibility of implementing a SDDR. It has also conducted a market study on the influence of that system on the final consumer, which has detected two main segments whose purchasing decisions would be greatly influenced by the introduction of a SDDR, a Senior segment, 45-54 years concerned about the environment and purchasing power enough that the deposit is not blocking, and a Junior Segment, aged 18-24, also concern with environment, lower economic capacity but what influences the decision purchase of their parents). To carry out this business plan will require an initial investment of 57,000 €, with expectations of recovery of such investment in the first year and an IRR of 56%, with an NPV of € 127,961 for the 7 years of the project . To publicize hypermarket customers the benefits of using a SDDR system, marketing campaigns conducted through different channels, opening promotions, marketing activities and external loyalty schemes. The organization and implementation in the Hypermarket is easy with distinct roles, involve only about 9 resourced and in about 3 months from the start of the project and could offer this service to customers in the hypermarket. We have also analyzed the main risks and established corrective measures to surface that risk . We should take caution with lost sales during startup of the business, such as control spending, customer retention and maintaining enough working capital.

Sequence of a 189-kb segment of the chromosome of Rhodobacter capsulatus SB1003

Cosmids from the 1A3–1A10 region of the complete miniset were individually subcloned by using the vector M13 mp18. Sequences of each cosmid were assembled from about 400 DNA fragments generated from the ends of these phage subclones and merged into one 189-kb contig. About 160 ORFs identified by the CodonUse program were subjected to similarity searches. The biological functions of 80 ORFs could be assigned reliably by using the WIT and Magpie genome investigation tools. Eighty percent of these recognizable ORFs were organized in functional clusters, which simplified assignment decisions and increased the strength of the predictions. A set of 26 genes for cobalamin biosynthesis, genes for polyhydroxyalkanoic acid metabolism, DNA replication and recombination, and DNA gyrase were among those identified. Most of the ORFs lacking significant similarity with reference databases also were grouped. There are two large clusters of these ORFs, one located between 45 and 67 kb of the map, and the other between 150 and 183 kb. Nine of the loosely identified ORFs (of 15) of the first of these clusters match ORFs from phages or transposons. The other cluster also has four ORFs of possible phage origin.

The active oligomeric state of the minimalistic influenza virus M2 ion channel is a tetramer

The influenza A virus M2 integral membrane protein is an ion channel that permits protons to enter virus particles during uncoating of virions in endosomes and also modulates the pH of the trans-Golgi network in virus-infected cells. The M2 protein is a homo-oligomer of 97 residues, and analysis by chemical cross-linking and SDS/PAGE indicates M2 forms a tetramer. However, a higher order molecular form is sometimes observed and, thus, it is necessary to determine the active form of the molecule. This was done by studying the currents of oocytes that expressed mixtures of the wild-type M2 protein (epitope tagged) and the mutant protein M2-V27S, which is resistant to the inhibitor amantadine. The composition of mixed oligomers of the two proteins expressed at the plasma membrane of individual oocytes was quantified after antibody capture of the cell surface expressed molecules and it was found that the subunits mixed freely. When the ratio of wild-type to mutant protein subunits was 0.85:0.15, the amantadine sensitivity was reduced to 50% and for a ratio of 0.71:0.29 to 20%. These results are consistent with the amantadine-resistant mutant being dominant and the oligomeric state being a tetramer.

Interaction of batrachotoxin with the local anesthetic receptor site in transmembrane segment IVS6 of the voltage-gated sodium channel

The voltage-gated sodium channel is the site of action of more than six classes of neurotoxins and drugs that alter its function by interaction with distinct, allosterically coupled receptor sites. Batrachotoxin (BTX) is a steroidal alkaloid that binds to neurotoxin receptor site 2 and causes persistent activation. BTX binding is inhibited allosterically by local anesthetics. We have investigated the interaction of BTX with amino acid residues I1760, F1764, and Y1771, which form part of local anesthetic receptor site in transmembrane segment IVS6 of type IIA sodium channels. Alanine substitution for F1764 (mutant F1764A) reduces tritiated BTX-A-20-α-benzoate binding affinity, causing a 60-fold increase in Kd. Alanine substitution for I1760, which is adjacent to F1764 in the predicted IVS6 transmembrane alpha helix, causes only a 4-fold increase in Kd. In contrast, mutant Y1771A shows no change in BTX binding affinity. For wild-type and mutant Y1771A, BTX shifted the voltage for half-maximal activation ≈40 mV in the hyperpolarizing direction and increased the percentage of noninactivating sodium current to ≈60%. In contrast, these BTX effects were eliminated completely for the F1764A mutant and were reduced substantially for mutant I1760A. Our data suggest that the BTX receptor site shares overlapping but nonidentical molecular determinants with the local anesthetic receptor site in transmembrane segment IVS6 as well as having unique molecular determinants in transmembrane segment IS6, as demonstrated in previous work. Evidently, BTX conforms to a domain–interface allosteric model of ligand binding and action, as previously proposed for calcium agonist and antagonist drugs acting on l-type calcium channels.

Selective Perturbation of Apical Membrane Traffic by Expression of Influenza M2, an Acid-activated Ion Channel, in Polarized Madin–Darby Canine Kidney Cells

The function of acidification along the endocytic pathway is not well understood, in part because the perturbants used to modify compartmental pH have global effects and in some cases alter cytoplasmic pH. We have used a new approach to study the effect of pH perturbation on postendocytic traffic in polarized Madin–Darby canine kidney (MDCK) cells. Influenza M2 is a small membrane protein that functions as an acid-activated ion channel and can elevate the pH of the trans-Golgi network and endosomes. We used recombinant adenoviruses to express the M2 protein of influenza virus in polarized MDCK cells stably transfected with the polymeric immunoglobulin (Ig) receptor. Using indirect immunofluorescence and immunoelectron microscopy, M2 was found to be concentrated at the apical plasma membrane and in subapical vesicles; intracellular M2 colocalized partly with internalized IgA in apical recycling endosomes as well as with the trans-Golgi network marker TGN-38. Expression of M2 slowed the rate of IgA transcytosis across polarized MDCK monolayers. The delay in transport occurred after IgA reached the apical recycling endosome, consistent with the localization of intracellular M2. Apical recycling of IgA was also slowed in the presence of M2, whereas basolateral recycling of transferrin and degradation of IgA were unaffected. By contrast, ammonium chloride affected both apical IgA and basolateral transferrin release. Together, our data suggest that M2 expression selectively perturbs acidification in compartments involved in apical delivery without disrupting other postendocytic transport steps.

Asparagine-proline sequence within membrane-spanning segment of SREBP triggers intramembrane cleavage by Site-2 protease

The NH2-terminal domains of membrane-bound sterol regulatory element-binding proteins (SREBPs) are released into the cytosol by regulated intramembrane proteolysis, after which they enter the nucleus to activate genes encoding lipid biosynthetic enzymes. Intramembrane proteolysis is catalyzed by Site-2 protease (S2P), a hydrophobic zinc metalloprotease that cleaves SREBPs at a membrane-embedded leucine-cysteine bond. In the current study, we use domain-swapping methods to localize the residues within the SREBP-2 membrane-spanning segment that are required for cleavage by S2P. The studies reveal a requirement for an asparagine-proline sequence in the middle third of the transmembrane segment. We propose a model in which the asparagine-proline sequence serves as an NH2-terminal cap for a portion of the transmembrane α-helix of SREBP, allowing the remainder of the α-helix to unwind partially to expose the peptide bond for cleavage by S2P.

Isolation and characterization of the chicken m2 acetylcholine receptor promoter region: Induction of gene transcription by leukemia inhibitory factor and ciliary neurotrophic factor

We have isolated the promoter region and determined the start sites of transcription for the gene encoding the chicken m2 (cm2) muscarinic acetylcholine receptor. Transfection experiments, using cm2-luciferase reporter gene constructs, demonstrated that a 789-bp genomic fragment was sufficient to drive high level expression in chicken heart primary cultures, while an additional 1.2-kb region was required for maximal expression in mouse septal/neuroblastoma (SN56) cells. Treatment of SN56 cells with the cytokines ciliary neurotrophic factor and leukemia inhibitory factor increases expression of endogenous muscarinic acetylcholine receptors and results in a 4- to 6-fold induction of cm2 promoter driven luciferase expression. We have mapped a region of the cm2 promoter that is necessary for induction by cytokines.

A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity

This study addresses the properties of a newly identified internal ribosome entry site (IRES) contained within the mRNA of the homeodomain protein Gtx. Sequential deletions of the 5′ untranslated region (UTR) from either end did not define distinct IRES boundaries; when five nonoverlapping UTR fragments were tested, four had IRES activity. These observations are consistent with other cellular IRES analyses suggesting that some cellular IRESes are composed of segments (IRES modules) that independently and combinatorially contribute to overall IRES activity. We characterize a 9-nt IRES module from the Gtx 5′ UTR that is 100% complementary to the 18S rRNA at nucleotides 1132–1124. In previous work, we demonstrated that this mRNA segment could be crosslinked to its complement within intact 40S subunits. Here we show that increasing the number of copies of this IRES module in the intercistronic region of a dicistronic mRNA strongly enhances IRES activity in various cell lines. Ten linked copies increased IRES activity up to 570-fold in Neuro 2a cells. This level of IRES activity is up to 63-fold greater than that obtained by using the well characterized encephalomyocarditis virus IRES when tested in the same assay system. When the number of nucleotides between two of the 9-nt Gtx IRES modules was increased, the synergy between them decreased. In light of these findings, we discuss possible mechanisms of ribosome recruitment by cellular mRNAs, address the proposed role of higher order RNA structures on cellular IRES activity, and suggest parallels between IRES modules and transcriptional enhancer elements.

A double-stranded RNA element from a hypovirulent strain of Rhizoctonia solani occurs in DNA form and is genetically related to the pentafunctional AROM protein of the shikimate pathway

M2 is a double-stranded RNA (dsRNA) element occurring in the hypovirulent isolate Rhs 1A1 of the plant pathogenic basidiomycete Rhizoctonia solani. Rhs 1A1 originated as a sector of the virulent field isolate Rhs 1AP, which contains no detectable amount of the M2 dsRNA. The complete sequence (3,570 bp) of the M2 dsRNA has been determined. A 6.9-kbp segment of total DNA from either Rhs 1A1 or Rhs 1AP hybridizes with an M2-specific cDNA probe. The sequences of M2 dsRNA and of PCR products generated from Rhs 1A1 total DNA were found to be identical. Thus this report describes a fungal host containing full-length DNA copies of a dsRNA element. A major portion of the M2 dsRNA is located in the cytoplasm, whereas a smaller amount is found in mitochondria. Based on either the universal or the mitochondrial genetic code of filamentous fungi, one strand of M2 encodes a putative protein of 754 amino acids. The resulting polypeptide has all four motifs of a dsRNA viral RNA-dependent RNA polymerase (RDRP) and is phylogenetically related to the RDRP of a mitochondrial dsRNA associated with hypovirulence in strain NB631 of Cryphonectria parasitica, incitant of chestnut blight. This polypeptide also has significant sequence similarity with two domains of a pentafunctional polypeptide, which catalyzes the five central steps of the shikimate pathway in yeast and filamentous fungi.

Expression of homeobox genes shows chelicerate arthropods retain their deutocerebral segment

Expression patterns of six homeobox containing genes in a model chelicerate, the oribatid mite Archegozetes longisetosus, were examined to establish homology of chelicerate and insect head segments and to investigate claims that the chelicerate deutocerebral segment has been reduced or lost. engrailed (en) expression, which has been used to demonstrate the presence of segments in insects, fails to demonstrate a reduced deutocerebral segment. Expression patterns of the chelicerate homologs of the Drosophila genes Antennapedia (Antp), Sex combs reduced (Scr), Deformed (Dfd), proboscipedia (pb), and orthodenticle (otd) confirm direct correspondence of head segments. The chelicerate deutocerebral segment has not been reduced or lost. We make further inferences concerning the evolution of heads and Hox genes in arthropods.

Segment-specific pattern of sympathetic preganglionic projections in the chicken embryo spinal cord is altered by retinoids

Sympathetic preganglionic neurons exhibit segment-specific projections. Preganglionic neurons located in rostral spinal segments project rostrally within the sympathetic chain, those located in caudal spinal segments project caudally, and those in midthoracic segments project either rostrally or caudally in segmentally graded proportions. Moreover, rostrally and caudally projecting preganglionic neurons are skewed toward the rostral and caudal regions, respectively, of each midthoracic segment. The mechanisms that establish these segment-specific projections are unknown. Here we show that experimental manipulation of retinoid signaling in the chicken embryo alters the segment-specific pattern of sympathetic preganglionic projections and that this effect is mediated by the somitic mesoderm. Application of exogenous retinoic acid to a single rostral thoracic somite decreases the number of rostrally projecting preganglionic neurons at that level. Conversely, disrupting endogenous synthesis of retinoic acid in a single caudal thoracic somite increases the number of rostrally projecting preganglionic neurons at that level. The number of caudally projecting neurons does not change in either case, indicating that the effect is specific for rostrally projecting preganglionic neurons. These results indicate that the sizes of the rostrally and caudally projecting populations may be independently regulated by different factors. Opposing gradients of such factors along the longitudinal axis of the thoracic region of the embryo could be sufficient, in combination, to determine the segment-specific identity of preganglionic projections.

A short segment within the cytoplasmic domain of the neural cell adhesion molecule (N-CAM) is essential for N-CAM-induced NF-κB activity in astrocytes

The neural cell adhesion molecule (N-CAM) is expressed on the surface of astrocytes, where its homophilic binding leads to the activation of the transcription factor NF-κB. Transfection of astrocytes with a construct encompassing the transmembrane region and the cytoplasmic domain of N-CAM (designated Tm-Cyto, amino acids 685–839 in the full-length molecule) inhibited this activation up to 40%, and inhibited N-CAM-induced translocation of NF-κB to the nucleus. N-CAM also activated NF-κB in astrocytes from N-CAM knockout mice, presumably through binding to a heterophile. This activation, however, was not blocked by Tm-Cyto expression, indicating that the inhibitory effect of the Tm-Cyto construct is specific for cell surface N-CAM. Deletions and point mutations of the cytoplasmic portion of the Tm-Cyto construct indicated that the region between amino acids 780 and 800 were essential for inhibitory activity. This region contains four threonines (788, 793, 794, and 797). Mutation to alanine of T788, T794, or T797, but not T793, abolished inhibitory activity, as did mutation of T788 or T797 to aspartic acid. A Tm-Cyto construct with T794 mutated to aspartic acid retained inhibitory activity but did not itself induce a constitutive NF-κB response. This result suggests that phosphorylation of T794 may be necessary but is not the triggering event. Overall, these findings define a short segment of the N-CAM cytoplasmic domain that is critical for N-CAM-induced activation of NF-κB and may be important in other N-CAM-mediated signaling.