Transport of prion protein across the blood-brain barrier.

The cellular form of the prion protein (PrP(c)) is necessary for the development of prion diseases and is a highly conserved protein that may play a role in neuroprotection. PrP(c) is found in both blood and cerebrospinal fluid and is likely produced by both peripheral tissues and the central nervous system (CNS). Exchange of PrP(c) between the brain and peripheral tissues could have important pathophysiologic and therapeutic implications, but it is unknown whether PrP(c) can cross the blood-brain barrier (BBB). Here, we found that radioactively labeled PrP(c) crossed the BBB in both the brain-to-blood and blood-to-brain directions. PrP(c) was enzymatically stable in blood and in brain, was cleared by liver and kidney, and was sequestered by spleen and the cervical lymph nodes. Circulating PrP(c) entered all regions of the CNS, but uptake by the lumbar and cervical spinal cord, hypothalamus, thalamus, and striatum was particularly high. These results show that PrP(c) has bidirectional, saturable transport across the BBB and selectively targets some CNS regions. Such transport may play a role in PrP(c) function and prion replication.

Endocannabinoid transport revisited.

Endocannabinoids are arachidonic acid-derived endogenous lipids that activate the endocannabinoid system which plays a major role in health and disease. The primary endocannabinoids are anandamide (AEA, N-arachidonoylethanolamine) and 2-arachidonoyl glycerol. While their biosynthesis and metabolism have been studied in detail, it remains unclear how endocannabinoids are transported across the cell membrane. In this review, we critically discuss the different models of endocannabinoid trafficking, focusing on AEA cellular uptake which is best studied. The evolution of the current knowledge obtained with different AEA transport inhibitors is reviewed and the confusions caused by the lack of their specificity discussed. A comparative summary of the most important AEA uptake inhibitors and the studies involving their use is provided. Based on a comprehensive literature analysis, we propose a model of facilitated AEA membrane transport followed by intracellular shuttling and sequestration. We conclude that novel and more specific probes will be essential to identify the missing targets involved in endocannabinoid membrane transport.

Selective Transport of Zinc, Manganese, Nickel, Cobalt and Cadmium in the Root System and Transfer to the Leaves in Young Wheat Plants

• Background and Aims The uptake, translocation and redistribution of the heavy metals zinc, manganese, nickel, cobalt and cadmium are relevant for plant nutrition as well as for the quality of harvested plant products. The long-distance transport of these heavy metals within the root system and the release to the shoot in young wheat (Triticum aestivum ‘Arina’) plants were investigated. • Methods After the application of 65Zn, 54Mn, 63Ni, 57Co and 109Cd for 24 h to one seminal root (the other seminal roots being excised) of 54-h-old wheat seedlings, the labelled plants were incubated for several days in hydroponic culture on a medium without radionuclides. • Key Results The content of 65Zn decreased quickly in the labelled part of the root. After the transfer of 65Zn from the roots to the shoot, a further redistribution in the phloem from older to younger leaves was observed. In contrast to 65Zn, 109Cd was released more slowly from the roots to the leaves and was subsequently redistributed in the phloem to the youngest leaves only at trace levels. The content of 63Ni decreased quickly in the labelled part of the root, moving to the newly formed parts of the root system and also accumulating transiently in the expanding leaves. The 54Mn content decreased quickly in the labelled part of the root and increased simultaneously in leaf 1. A strong retention in the labelled part of the root was observed after supplying 57Co. • Conclusions The dynamics of redistribution of 65Zn, 54Mn, 63Ni, 57Co and 109Cd differed considerably. The rapid redistribution of 63Ni from older to younger leaves throughout the experiment indicated a high mobility in the phloem, while 54Mn was mobile only in the xylem and 57Co was retained in the labelled root without being loaded into the xylem.

Lindane induced cellular dysfunction in MDCK cells: The role of the peripheral benzodiazepine receptor

The central nervous system GABAA/Benzodiazepine (GABAA/BZD) receptors are targets for many pharmaceutical agents and several classes of pesticides. Lindane is an organochlorine pesticide, although banned from production in the U.S. since 1977, still imported for use as an insecticide and pharmaceutically to control ectoparasites (ATSDR, 1994). Lindane functions as a GABA/BZD receptor antagonist within the central nervous system (CNS). Outside of the CNS, peripheral BZD receptors have been localized to the distal tubule of the kidney. Previous research in our laboratory has shown that incubation of renal cortical slices with lindane can produce an increase in kallikrein leakage, suggesting a distal tubular effect. In this study, Madin Darby Canine Kidney (MDCK) cells were used as an in vitro system to assess the toxicity of lindane. This purpose of this study was to determine if interactions between a renal distal tubular BZD-like receptor and lindane could lead to perturbations in renal distal cellular chloride (Cl−) transport and mitochondrial dysfunction and ultimately, cellular death. ^ Pertubations in renal chloride transport were measured indirectly by determining if lindane altered cell function responsiveness following osmotic stress. MDCK cells pre-treated with lindane and then subjected to osmotic stress remained swollen for up to 12 hours post-stress. Lindane-induced dysfunction was assessed through stress protein induction measured by Western Blot analysis. Lindane pretreatment delayed Heat Shock Protein 72 (HSP72) induction by 36 hours in osmotically stressed cells. Pretreatment with 1 × 10 −5 M LIN followed by osmotic stress elevated p38 and Stress Activated Protein Kinase (SAPK/JNK) at 15 minutes which declined at 30 minutes. Lindane appeared to have no effect on Endoplasmic Reticulum Related Kinase (ERK) induction. Lindane did not effect osmotically stressed LLC-PKI cells, a control cell line. ^ Lindane-treated MDCK cells did not exhibit necrosis. Instead, apoptosis was observed in lindane-treated MDCK cells in both time- and dose-dependent manners. LLC-PKI cells were not affected by LIN treatment. ^ To better understand the mechanism of lindane-induced apoptosis, mitochondrial function was measured. No changes in cytochrome c release or mitochondrial membrane potential were observed suggesting the mitochondrial pathway was not involved in lindane-induced apoptosis. ^ Further research will need to be conducted to determine the mechanism of lindane-induced adverse cellular effects. ^

Making Transport Safer: V2V-Based Automated Emergency Braking System

An important goal in the field of intelligent transportation systems (ITS) is to provide driving aids aimed at preventing accidents and reducing the number of traffic victims. The commonest traffic accidents in urban areas are due to sudden braking that demands a very fast response on the part of drivers. Attempts to solve this problem have motivated many ITS advances including the detection of the intention of surrounding cars using lasers, radars or cameras. However, this might not be enough to increase safety when there is a danger of collision. Vehicle to vehicle communications are needed to ensure that the other intentions of cars are also available. The article describes the development of a controller to perform an emergency stop via an electro-hydraulic braking system employed on dry asphalt. An original V2V communication scheme based on WiFi cards has been used for broadcasting positioning information to other vehicles. The reliability of the scheme has been theoretically analyzed to estimate its performance when the number of vehicles involved is much higher. This controller has been incorporated into the AUTOPIA program control for automatic cars. The system has been implemented in Citroën C3 Pluriel, and various tests were performed to evaluate its operation.

System supporting public transport based on geolocation

El propósito de este proyecto de fin de Grado es el estudio y desarrollo de una aplicación basada en Android que proporcionará soporte y atención a los servicios de transporte público existentes en Cracovia, Polonia. La principal funcionalidad del sistema será consultar la posición de un determinado autobús o tranvía y mostrar su ubicación con exactitud. Para lograr esto, necesitaremos tres fases de desarrollo. En primer lugar, deberemos implementar un sistema que obtenga las coordenadas geográficas de los vehículos de transporte público en cada instante. A continuación, tendremos que registrar todos estos datos y almacenarlos en una base de datos en un servidor web. Por último, desarrollaremos un sistema cliente que realice consultas a tiempo real sobre estos datos almacenados, obteniendo la posición para una línea determinada y mostrando su ubicación con un marcador en el mapa. Para hacer el seguimiento de los vehículos, sería necesario tener acceso a una API pública que nos proporcionase la posición registrada por los GPS que integran cada uno de ellos. Como esta API no existe actualmente para los servicios de autobús, y para los tranvías es de uso meramente privado, desarrollaremos una segunda aplicación en Android que hará las funciones del lado servidor. En ella podremos elegir mediante una simple interfaz el número de línea y un código específico que identificará a cada vehículo en particular (e.g. podemos tener 6 tranvías recorriendo la red al mismo tiempo para la línea 24). Esta aplicación obtendrá las coordenadas geográficas del teléfono móvil, lo cual incluye latitud, longitud y orientación a través del proveedor GPS. De este modo, podremos realizar una simulación de como el sistema funcionará a tiempo real utilizando la aplicación servidora desde dentro de un tranvía o autobús y, al mismo tiempo, utilizando la aplicación cliente haciendo peticiones para mostrar la información de dicho tranvía. El cliente, además, podrá consultar la ruta de cualquier línea sin necesidad de tener acceso a Internet. Almacenaremos las rutas y paradas de cada línea en la memoria del teléfono móvil utilizando ficheros XML debido al poco espacio que ocupan y a lo útil que resulta poder consultar un trayecto en cualquier momento, independientemente del acceso a la red. El usuario también podrá consultar las tablas de horarios oficiales para cada línea. Aunque en este caso si será necesaria una conexión a Internet debido a que se realizará a través de la web oficial de MPK. Para almacenar todas las coordenadas de cada vehículo en cada instante necesitaremos crear una base de datos en un servidor. Esto se resolverá mediante el uso de MYSQL y PHP. Se enviarán peticiones de tipo GET y POST a los servicios PHP que se encargarán de traducir y realizar la consulta correspondiente a la base de datos MYSQL. Por último, gracias a todos los datos recogidos relativos a la posición de los vehículos de transporte público, podremos realizar algunas tareas de análisis. Comparando la hora exacta a la que los vehículos pasaron por cada parada y la hora a la que deberían haber pasado según los horarios oficiales, podremos descubrir fallos en estos. Seremos capaces de determinar si es un error puntual debido a factores externos (atascos, averías,…) o si por el contrario, es algo que ocurre muy a menudo y se debería corregir el horario oficial. ABSTRACT The aim of this final Project (for University) is to develop an Android application thatwill provide support and feedback to the public transport services in Krakow. The main functionality of the system will be to track the position of a desired bus or tram line, and display its position on the map. To achieve this, we will need 3 stages: the first one will be to implement a system that sends the geographical position of the public transport vehicles, the second one will be to collect this data in a web server, and the last one will be to get the last location registered for the desired line and display it on the map. For tracking the vehicles, we would need to have access to a public API that should be connected with each bus/tram GPS. As this doesn’t exist in Krakow or at least is not available for public use, we will develop a second android application that will do the server side job. We will be able to choose in a simple interface the line number and a code letter to identify each vehicle (e.g. we can have 6 trams that belong to the line number 24 working at the same time). It will take the current mobile geolocation; this includes getting latitude, longitude and bearing from the GPS provider. Thus, we will be able to make a simulation of how the system works in real time by using the server app inside a tram and at the same time, using the client app and making requests to display the information of that tram. The client will also be able to check the path of the desired line without internet access. We will store the path and stops for each line locally in the phone memory using xml files due to the few requirements of available space it needs and the usefulness of checking a path when needed. This app will also offer the functionality of checking the timetable for the line, but in this case, it will link to the official Mpk website, so Internet access will be required. For storing all the coordinates for each vehicle at every moment we will need to create a database on a server. We have decided that the easiest way is to use Mysql and PHP for the deployment of the service. We will send GET and POST requests to the php files and those files will make the according queries to our database. Finally, based on all the collected data, we will be able to get some information about errors in the system of public transport timetables. We will check at what time a line was in each specific stop and compare it with the official timetable to find mistakes of time. We will determine if it is something that happens occasionally and related to external factors (e.g. traffic jams, breakdowns…) or if on the other hand, it is something that happens very often and the public transport timetables should be looked over and corrected.

Functional characterization of Rhizobium leguminosarum bv. viciae DmeRF, a cation diffusion facilitator system involved in nickel and cobalt resistance

In prokaryotes, nickel is an essential element participating in the structure of enzymes involved in multiple cellular processes. Nickel transport is a challenge for microorganisms since, although essential, high levels of this metal inside the cell are toxic. For this reason, bacteria have developed high-affinity nickel transporters as well as nickel-specific detoxification systems. Ultramafic soils, and soils contaminated with heavy metals are excellent sources of nickel resistant bacteria. Molecular analysis of strains isolated in the habitats has revealed novel genetic systems involved in adaptation to such hostile conditions.

Ontology-driven legal support-system in air transport passenger domain

This paper aims to present a preliminary version of asupport-system in the air transport passenger domain. This system relies upon an underlying on-tological structure representing a normative framework to facilitatethe provision of contextualized relevant legal information.This information includes the pas-senger's rights and itenhances self-litigation and the decision-making process of passengers.Our contribution is based in the attempt of rendering a user-centric-legal informationgroundedon case-scenarios of the most pronounced incidents related to the consumer complaints in the EU.A number ofadvantages with re-spect to the current state-of-the-art services are discussed and a case study illu-strates a possible technological application.

The maturity-onset diabetes of the young (MODY1) transcription factor HNF4α regulates expression of genes required for glucose transport and metabolism

Hepatocyte nuclear factor 4α (HNF4α) plays a critical role in regulating the expression of many genes essential for normal functioning of liver, gut, kidney, and pancreatic islets. A nonsense mutation (Q268X) in exon 7 of the HNF4α gene is responsible for an autosomal dominant, early-onset form of non-insulin-dependent diabetes mellitus (maturity-onset diabetes of the young; gene named MODY1). Although this mutation is predicted to delete 187 C-terminal amino acids of the HNF4α protein the molecular mechanism by which it causes diabetes is unknown. To address this, we first studied the functional properties of the MODY1 mutant protein. We show that it has lost its transcriptional transactivation activity, fails to dimerize and bind DNA, implying that the MODY1 phenotype is because of a loss of HNF4α function. The effect of loss of function on HNF4α target gene expression was investigated further in embryonic stem cells, which are amenable to genetic manipulation and can be induced to form visceral endoderm. Because the visceral endoderm shares many properties with the liver and pancreatic β-cells, including expression of genes for glucose transport and metabolism, it offers an ideal system to investigate HNF4-dependent gene regulation in glucose homeostasis. By exploiting this system we have identified several genes encoding components of the glucose-dependent insulin secretion pathway whose expression is dependent upon HNF4α. These include glucose transporter 2, and the glycolytic enzymes aldolase B and glyceraldehyde-3-phosphate dehydrogenase, and liver pyruvate kinase. In addition we have found that expression of the fatty acid binding proteins and cellular retinol binding protein also are down-regulated in the absence of HNF4α. These data provide direct evidence that HNF4α is critical for regulating glucose transport and glycolysis and in doing so is crucial for maintaining glucose homeostasis.

Alternative splicing of the rat sodium/bile acid transporter changes its cellular localization and transport properties

Bile secretion involves the structural and functional interplay of hepatocytes and cholangiocytes, the cells lining the intrahepatic bile ducts. Hepatocytes actively secrete bile acids into the canalicular space and cholangiocytes then transport bile acids in a vectorial manner across their apical and basolateral plasma membranes. The initial step in the transepithelial transport of bile acids across rat cholangiocytes is apical uptake by a Na+-dependent bile acid transporter (ASBT). To date, the molecular basis of the obligate efflux mechanism for extrusion of bile acids across the cholangiocyte basolateral membrane remains unknown. We have identified an exon-2 skipped, alternatively spliced form of ASBT, designated t-ASBT, expressed in rat cholangiocytes, ileum, and kidney. Alternative splicing causes a frameshift that produces a 154-aa protein. Antipeptide antibodies detected the ≈19 kDa t-ASBT polypeptide in rat cholangiocytes, ileum, and kidney. The t-ASBT was specifically localized to the basolateral domain of cholangiocytes. Transport studies in Xenopus oocytes revealed that t-ASBT can function as a bile acid efflux protein. Thus, alternative splicing changes the cellular targeting of ASBT, alters its functional properties, and provides a mechanism for rat cholangiocytes and other bile acid-transporting epithelia to extrude bile acids. Our work represents an example in which a single gene appears to encode via alternative splicing both uptake and obligate efflux carriers in a bile acid-transporting epithelial cell.

Expression of scavenger receptor class B type 1 (SR-BI) promotes microvillar channel formation and selective cholesteryl ester transport in a heterologous reconstituted system

In the “selective” cholesteryl ester (CE) uptake process, surface-associated lipoproteins [high density lipoprotein (HDL) and low density lipoprotein] are trapped in the space formed between closely apposed surface microvilli (microvillar channels) in hormone-stimulated steroidogenic cells. This is the same location where an HDL receptor (SR-BI) is found. In the current study, we sought to understand the relationship between SR-BI and selective CE uptake in a heterologous insect cell system. Sf9 (Spodoptera frugiperda) cells overexpressing recombinant SR-BI were examined for (i) SR-BI protein by Western blot analysis and light or electron immunomicroscopy, and (ii) selective lipoprotein CE uptake by the use of radiolabeled or fluorescent (BODIPY-CE)-labeled HDL. Noninfected or infected control Sf9 cells do not express SR-BI, show microvillar channels, or internalize CEs. An unexpected finding was the induction of a complex channel system in Sf9 cells expressing SR-BI. SR-BI-expressing cells showed many cell surface double-membraned channels, immunogold SR-BI, apolipoprotein (HDL) labeling of the channels, and high levels of selective HDL-CE uptake. Thus, double-membraned channels can be induced by expression of recombinant SR-BI in a heterologous system, and these specialized structures facilitate both the binding of HDL and selective HDL-CE uptake.

Dynamics of Immature Secretory Granules: Role of Cytoskeletal Elements during Transport, Cortical Restriction, and F-Actin-dependent Tethering

Secretory granules store neuropeptides and hormones and exhibit regulated exocytosis upon appropriate cellular stimulation. They are generated in the trans-Golgi network as immature secretory granules, short-lived vesicular intermediates, which undergo a complex and poorly understood maturation process. Due to their short half-life and low abundance, real-time studies of immature secretory granules have not been previously possible. We describe here a pulse/chase-like system based on the expression of a human chromogranin B-GFP fusion protein in neuroendocrine PC12 cells, which permits direct visualization of the budding of immature secretory granules and their dynamics during maturation. Live cell imaging revealed that newly formed immature secretory granules are transported in a direct and microtubule-dependent manner within a few seconds to the cell periphery. Our data suggest that the cooperative action of microtubules and actin filaments restricts immature secretory granules to the F-actin-rich cell cortex, where they move randomly and mature completely within a few hours. During this maturation period, secretory granules segregate into pools of different motility. In a late phase of maturation, 60% of secretory granules were found to be immobile and about half of these underwent F-actin-dependent tethering.

Retrograde axonal transport of glial cell line-derived neurotrophic factor in the adult nigrostriatal system suggests a trophic role in the adult.

The recently cloned, distant member of the transforming growth factor beta (TGF-beta) family, glial cell line-derived neurotrophic factor (GDNF), has potent trophic actions on fetal mesencephalic dopamine neurons. GDNF also has protective and restorative activity on adult mesencephalic dopaminergic neurons and potently protects motoneurons from axotomy-induced cell death. However, evidence for a role for endogenous GDNF as a target-derived trophic factor in adult midbrain dopaminergic circuits requires documentation of specific transport from the sites of synthesis in the target areas to the nerve cell bodies themselves. Here, we demonstrate that GDNF is retrogradely transported by mesencephalic dopamine neurons of the nigrostriatal pathway. The pattern of retrograde transport following intrastriatal injections indicates that there may be subpopulations of neurons that are GDNF responsive. Retrograde axonal transport of biologically active 125I-labeled GDNF was inhibited by an excess of unlabeled GDNF but not by an excess of cytochrome c. Specificity was further documented by demonstrating that another TGF-beta family member, TGF-beta 1, did not appear to affect retrograde transport. Retrograde transport was also demonstrated by immunohistochemistry by using intrastriatal injections of unlabeled GDNF. GDNF immunoreactivity was found specifically in dopamine nerve cell bodies of the substantia nigra pars compacta distributed in granules in the soma and proximal dendrites. Our data implicate a specific receptor-mediated uptake mechanism operating in the adult. Taken together, the present findings suggest that GDNF acts endogenously as a target-derived physiological survival/maintenance factor for dopaminergic neurons.