Fibronectin modulates thymocyte-thymic epithelial cell interactions following Trypanosoma cruzi infection

Developing thymocytes interact with thymic epithelial cells (TECs) through cell-cell interactions, TEC-derived secretory moieties and extracellular matrix (ECM)-mediated interactions. These physiological interactions are crucial for normal thymocyte differentiation, but can be disrupted in pathological situations. Indeed, there is severe thymic atrophy in animals acutely infected with Trypanosoma cruzi due to CD4+CD8+ thymocyte depletion secondary to caspase-mediated apoptosis, together with changes in ECM deposition and thymocyte migration. We studied an in vitro model of TEC infection by T. cruzi and found that infected TEC cultures show a reduced number of cells, which was likely associated with decreased proliferative capacity, but not with increased cell death, as demonstrated by bromodeoxyuridine and annexin-V labelling. The infected TEC cultures exhibited increased expression of fibronectin (FN), laminin (LM) and type IV collagen. Importantly, treatment with FN increased the relative number of infected cells, whereas treatment with anti-FN or anti-LM antibodies resulted in lower infection rates. Consistent with these data, we observed increased thymocyte adhesion to infected TEC cultures. Overall, these results suggest that ECM molecules, particularly FN, facilitate infection of the thymic epithelium and that the consequent enhancement of ECM expression might be associated with changes in TEC-thymocyte interactions.

Neurofilament triplet proteins are restricted to a subset of neurons in the rat neocortex.

The cellular localisation of neurofilament triplet subunits was investigated in the rat neocortex. A subset of mainly pyramidal neurons showed colocalisation of subunit immunolabelling throughout the neocortex, including labelling with the antibody SMI32, which has been used extensively in other studies of the primate cortex as a selective cellular marker. Neurofilament-labelled neurons were principally localised to two or three cell layers in most cortical regions, but dramatically reduced labelling was present in areas such as the perirhinal cortex, anterior cingulate and a strip of cortex extending from caudal motor regions through the medial parietal region to secondary visual areas. However, quantitative analysis demonstrated a similar proportion (10-20%) of cells with neurofilament triplet labelling in regions of high or low labelling. Combining retrograde tracing with immunolabelling showed that cellular content of the neurofilament proteins was not correlated with the length of projection. Double labelling immunohistochemistry demonstrated that neurofilament content in axons was closely associated with myelination. Analysis of SMI32 labelling in development indicated that content of this epitope within cell bodies was associated with relatively late maturation, between postnatal days 14 and 21. This study is further evidence of a cell type-specific regulation of neurofilament proteins within neocortical neurons. Neurofilament triplet content may be more closely related to the degree of myelination, rather than the absolute length, of the projecting axon.

Distintiu de garantia de qualitat ambiental per a equipaments culturals: aplicació pilot en biblioteques

El DGQA per a Equipaments Culturals és un distintiu per a la certificació ambiental de biblioteques i museus. L’objectiu del projecte és avaluar el distintiu mitjançant una aplicació pilot en 26 biblioteques de la província de Barcelona, i proposar millores tant per al distintiu com per a les biblioteques. També es pretén fer recerca sobre l’ecoetiquetatge de serveis, ja que és un àmbit poc desenvolupat, i sobre el sistema d’estudi, les biblioteques. Una anàlisi de les ecoetiquetes a nivell mundial, considerant 4 macroregions, ha permès caracteritzar les ecoetiquetes i determinar la situació actual dels serveis en l’ecoetiquetatge. Hi han variacions en el nombre de categories entre ecoetiquetes, i el percentatge de categories de serveis és, en general, reduït (8% de mitjana). Els subsectors serveis dominants són els d’Hosteleria, serveis de neteja i comerç. No hi ha cap experiència de certificació ambiental de serveis culturals, per tant, l’ecoetiquetatge de serveis culturals és un àmbit nou. El sistema d’estudi són 26 biblioteques de la província de Barcelona. L’aplicació del distintiu a aquestes s’ha dut a terme realitzant treball de camp a cadascuna per tal de determinar el seu estat ambiental. Posteriorment, s’han analitzat les dades per establir el grau de compliment de cadascuna. La majoria de les biblioteques (85%) compleixen més de la meitat dels criteris bàsics, i un 60% superen la puntuació mínima de compliment dels criteris opcionals. Els resultats obtinguts han permès avaluar la viabilitat de la implantació del distintiu, a través de l’anàlisi de cadascun dels seus criteris. Per tal de millorar les possibilitats d’èxit en la implantació del distintiu, s’han aportat un seguit de propostes a nivell individual i col·lectiu. S’ha realitzat una fitxa per a cadascuna de les 26 biblioteques, per tal d’orientar els gestors de les biblioteques en l’adopció de mesures per a l’acompliment dels criteris. També s’han inclòs propostes a l’avaluació dels criteris, dirigides als gestors del distintiu.

High level expression of human neuropeptide Y receptors in mammalian cells infected with a recombinant vaccinia virus.

Neuropeptide Y (NPY) is a 36 amino acid peptide present in the central and peripheral nervous system. Numerous studies point to a role of NPY in cardiovascular regulation. NPY effects are mediated through stimulation of specific cell surface G protein-coupled receptors. To allow biochemical studies of the receptor and of its interaction with the ligand, we have developed a potent expression system for NPY receptors using a recombinant vaccinia virus. A human NPY receptor cDNA was fused to a strong vaccinia virus promoter and inserted into the viral genome by homologous recombination. Recombinant viruses were isolated and tested for their ability to induce NPY binding site expression following infection of mammalian cell lines. Using saturation and competition binding experiments we measured a Bmax of 5-10 x 10(6) NPY binding sites per cell. The Kd for the binding of NPY is about 20 nM. Labelling of infected cells with a fluorochrome-labelled NPY indicated that the recombinant protein integrates into the cell membrane.

Influence of the Paracoccidioides brasiliensis14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells

The fungal strain Paracoccidioides brasiliensisremains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensismolecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensisuses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis.

Molecular and physiological characterisation of lonotropic receptors in the sacculus in drosophila

Chemosensation is the detection of chemical signals in the environment that enable an animal to make informed decisions about food choice, mate preference or predator detection. Dissecting the molecular and neural mechanisms by which animals detect chemical cues is an important goal towards understanding how they interact with the environment. An attractive system to dissect the mechanisms of chemosensation is the olfactory system. One of the most-investigated olfactory systems is that of Drosophila melanogaster, a model organism that is amenable to a powerful combination of genetic and physiological analyses. Embedded within the antennal olfactory organ of Drosophila is an unusual sensory structure called the sacculus. The sacculus is comprised of three distinct chambers, each lined with several sensilla housing two to three neurons. Previous morphological, anatomical and surgical studies of sacculus neurons have implicated sacculus neurons in chemosensation, hygrosensation and/or thermosensation. While a subset of sacculus neurons have been physiologically characterised as temperature sensors, the role of this organ has remained largely mysterious, due to its inaccessibility to peripheral electrophysiological analysis. Recently a new family of olfactory receptors, the lonotropic Receptors (IRs), was identified. Five IRs are expressed in sacculus neurons providing the first selective molecular markers for these cells. In this thesis I describe the molecular, physiological and anatomical characterisation of these neurons. Genetic labelling of specific populations of sacculus neurons with anatomical (CD8:GFP) reporters has identified neurons in sacculus chambers I and II express IR40a+IR93a together with their co- receptor IR25a, while neurons in chamber III express IR64a with its co-receptor IR8a. Both these sets of neurons project to two distinct glomeruli in the antennal lobe; IR40a neurons project to the column and arm, IR64a neurons project to DC4 and DP1m. Through a live optical imaging screen I showed that these neurons are indeed olfactory and IR64a neurons recognise acidic ligands, while IR40a neurons recognise amine ligands. IR40a and IR64a neurons are in fact composed of anatomically and physiologically distinct subpopulations, strongly implying the existence of other factors that define their functional properties. My thesis identifies the sacculus as a specialised olfactory organ capable of detecting acids and bases, which are of widespread importance to insects. The data from my thesis along with data from other labs show the sacculus is composed of different populations of olfactory sensory neurons and thermosensory neurons. Comparative genomic analysis of sacculus IRs across insects reveals them to be among the most conserved of this receptor repertoire, suggesting that the sacculus represents an evolutionarily ancient insect olfactory acid-base sensor. - La détection des produits chimiques se trouvant dans l'environnement (perception chimiosensorielle) permet à un animal de choisir sa nourriture, son partenaire ou encore d'identifier ses prédateurs. Décortiquer les mécanismes moléculaires et neuronaux grâce auxquels les animaux détectent ces signaux chimiques permet de comprendre comment ces animaux interagissent avec leur environnement. Un système intéressant pour décortiquer ces mécanismes de perception chimiosensorielle est le système olfactif, de la drosophile (Drosophila melanogaster), aussi appelée mouche du vinaigre. C'est un animal modèle très utile grâce à la combinaison d'outils génétiques puissants et d'analyses physiologiques facilement réalisables. Dans l'antenne de la drosophile, qui est l'organe olfactif principal de cet animal, se trouve une structure appelée sacculus. Celui-ci est composé de trois chambres distinctes, chacune comprenant plusieurs sensilles à l'intérieur desquelles se trouvent deux à trois neurones. De précédentes études morphologiques et anatomiques des ces neurones ont déterminé qu'ils sont impliqués dans la perception des odeurs, de l'humidité et de la température. Malgré ceci, la fonction principale de cet organe reste largement inconnue, principalement car il est inaccessible aux analyses électrophysiologiques. Récemment, une nouvelle famille de soixante-six récepteurs olfactifs, nommés Récepteurs lonotropiques (IRs), a été découverte chez la drosophile. Cinq IRs sont exprimés dans les neurones du sacculus. Pour la première fois, une sélection de marqueurs moléculaires est disponible pour l'étude de ces cellules. Dans cette thèse, les caractéristiques moléculaires, physiologiques et anatomiques des neurones du sacculus sont décrites. Ces populations de neurones situés dans le sacculus ont été marquées avec des gènes rapporteurs (CD8:GFP). Ceci a montré que les récepteurs IR40a et IR93a sont exprimés ensemble avec le co-récepteur IR25a dans les chambres I et II, tandis que les neurones de la chambre III expriment IR64a avec son co-récepteur IR8a. Ces deux groupes de neurones projettent vers deux glomérules distincts du lobe antennaire : les neurones IR40a projettent vers la column et le arm, alors que les neurones IR64a projettent vers DC4 et DP1m. Un screen d'imagerie optique a démontré que ces neurones sont en effet des neurones olfactifs, et que les neurones IR64a reconnaissent des ligands acides, tandis que les neurones IR40a reconnaissent des ligands aminés. De plus, les neurones IR40a et IR64a sont séparés en sous-populations distinctes anatomiquement et physiologiquement, et d'autres facteurs permettant de définir leurs propriétés fonctionnelles sont probablement impliqués. Cette thèse identifie ainsi le sacculus comme un organe olfactif spécialisé capable de détecter des acides et amines, lesquels sont très importants pour les insectes. Toutes les données collectées durant cette thèse, combinées aux données d'autres laboratoires, montrent que le sacculus est composé de différentes populations de neurones olfactifs et thermosenseurs. Ces IRs sont très conservés parmi les insectes, suggérant que le sacculus représente révolution d'un ancien détecteur olfactif d'acides et de bases chez l'insecte. - Tous les animaux sont capables de percevoir les signaux chimiques dans leur environnement, comme les odeurs ou le goût, via différents organes. L'odorat est le sens qui permet de percevoir les odeurs, et il est implique des neurones olfactifs qui se trouvent dans le nez des mammifères ou les antennes des insectes. La capacité d'un neurone olfactif à détecter une molécule odorante dépend des types de récepteurs olfactifs qu'il exprime. Il existe deux grandes familles de récepteurs qui perçoivent les odeurs : les Récepteurs Olfactifs, ORs, et Récepteurs lonotropiques IRs, qui détectent différents types d'odeurs avec différents mécanismes. Lorsqu'un récepteur reconnaît une molécule odorante, il convertit ce signal en un signal électrique qui est ensuite transmis au centre olfactif dans le cerveau. La drosophile (Drosophila melanogaster), aussi appelée mouche du vinaigre, est utilisée comme animal modèle pour étudier l'odorat, parce que son génome entier a été séquencé et que ses gènes sont facilement manipulables. De plus, l'anatomie du système olfactif de la mouche est similaire à celui des mammifères, malgré qu'il possède moins de neurones, ce qui le rend moins complexe. Ma thèse a pour objectif d'étudier les Récepteurs lonotropiques dans un organe spécifique, appelé le sacculus, situé dans les antennes. Les neurones du sacculus exprimant des IRs envoient leurs projections au centre olfactif du cerveau, suggérant que ces neurones perçoivent les odeurs. Une technique d'imagerie optique a été utilisée sur le cerveau de mouches vivantes afin de mesurer la réponse des neurones du le sacculus à différentes odeurs. J'ai démontré que ces récepteurs détectent des acides et des amines, qui sont très importants pour les insectes. Par exemple, les acides se retrouvent dans les fruits mûrs sur lesquels les mouches vont se nourrir, s'accoupler et poser leurs oeufs, et les amines sont souvent produites par des bactéries pouvant être nuisible pour la mouche. La principale découverte de ma thèse est donc l'identification du sacculus comme un organe capable de détecter deux des principales odeurs importantes pour la mouche. Ces récepteurs sont aussi présents dans d'autres insectes où ils jouent peut-être des rôles différents. Les acides et les amines se retrouvent aussi dans les excrétions (comme la sueur ou l'urine) de beaucoup de mammifères, qui pourraient potentiellement être dangereux pour la mouche, mais qui attirent les moustiques se nourrissant de leur sang.

Harmonic Nanocrystals for Biolabeling: A Survey of Optical Properties and Biocompatibility.

Nonlinear optical nanocrystals have been recently introduced as a promising alternative to fluorescent probes for multiphoton microscopy. We present for the first time a complete survey of the properties of five nanomaterials (KNbO(3), LiNbO(3), BaTiO(3), KTP, and ZnO), describing their preparation and stabilization and providing quantitative estimations of their nonlinear optical response. In the light of their prospective use as biological and clinical markers, we assess their biocompatibility on human healthy and cancerous cell lines. Finally, we demonstrate the great potential for cell imaging of these inherently nonlinear probes in terms of optical contrast, wavelength flexibility, and signal photostability.

Distinct neuronal circuits underlying the bahavioral and physiological components of the fear response

Abstract : Expression of fear involves changes in a number of behavioral and physiological parameters that are triggered by the central amygdala (CeA). The fear circuit also includes a series of brain stem nuclei that are the final effectors of the changes induced by the fear reaction. The CeA expresses many different neuropeptide receptors that can modulate fear responses. Today, the precise organization and the modulation of projections from the amygdala to the brain stem are still poorly understood. The aim of this project was to better understand the organization and the modulation of the fear circuit. To investigate this we first determined whether the CeA is composed of separate neuronal populations, where each one projects to specific brain stem nuclei, or whether single CeA neurons project to several nuclei. For this purpose, we first selected two brain stem nuclei implicated in the modulation of different components of the fear reactions, the periaqueductal gray (implicated in freezing) and the nucleus of solitary tract (implicated in heart rate modulation). We then performed double injections of two different retrograde tracers in these two nuclei and we quantified the subsequent presence of co-labelling in the CeA. We found that neurons projecting to the PAG and to the NTS are organized in separate populations. Subsequent electrophysiological recordings of the two populations revealed that PAG and NTS projecting neurons also have different electrophysiological characteristics. We then verified in vitro whether the neurons projecting to different brain stem nuclei express specific combinations of neuropeptide receptors, and whether a neuropeptide acting pre-synaptically (oxytocin) specifically modulates one of these two projections. We did not find differences at the level of expression of neurópeptide receptors, but we observed that oxytocin, a neuropeptide with anxiolytic properties, modulates PAG projecting neurons without affecting NTS projecting neurons. As oxytocin appeared to specifically modulate projections to the PAG, involved in the modulation of the freezing reaction, but did not affect the projections to the NTS, implicated in the modulation of cardiovascular parameters, we verified how this modulation translates in living animals. We investigated the effects of infra-amygdala injection of oxytocin on cardiovascular and behavioral changes induced by contextual fear conditioning. We found that oxytocin decreased the freezing response without affecting the cardiovascular system. Finally, as neuropeptides are considered potential future anxiolytics, we investigated whether diazepam and oxytocin, acting on the same circuit, had additive effects. This question was addressed exclusively with an in vitro electrophysiological approach. We obtained that oxytocin and diazepam, when co-applied, had an additive effect on both synaptic transmission and neuronal activity. These results open new perspectives for the possible clinical applications of oxytocin. Résumé : L'expression de la peur est accompagnée par de nombreux changements physiologiques et comportementaux qui sont déclenchés par l'amygdale centrale (CeA). Le circuit inclue aussi une série de noyaux du tronc cérébrale qui sont les effecteurs des différentes composantes de la réaction de peur. On sait que CeA envoie des projections aux noyaux du tronc cérébral et que ces neurones expriment une grande variété de récepteurs aux neuropeptides. Par contre, l'organisation des projections, ainsi que la modulation de ces projections par les neuropeptides reste encore peu connue. Avec ce projet, on premièrement voulu déterminer si CeA est composée de populations neuronales séparées qui projettent vers un noyau spécifique, ou bien si chaque neurones envoie des projections vers plusieurs noyaux. A ce propos, on a effectué des doubles injections de deux traceurs rétrogrades différentes dans deux noyaux du tronc cérébral impliqués dans des différentes composantes des réactions de peur. On a injecté la substance grise périaqueducale (PAG), qui est impliquée dans la réponse d'immobilisation, ainsi que le noyau du tractus solitaire (NTS) qui est responsable des changements cardiovasculaires. On a ensuite quantifié la présence de neurones contenant les deux traceurs dans CeA. On a trouvé que la plupart des neurones de l'amygdale centrale projettent vers un noyau spécifique, et on peut donc dire que l'amygdale semble être composée de populations neuronales séparées. On a ensuite mesuré les caractéristiques électrophysiologiques de ces deux projections et on a trouvé des différences substantielles concernant la résistance membranaire, la capacitance, le potentiel membranaire de repos ainsi que la fréquence des potentiels d'action spontanés. Puis, comme beaucoup de neuropéptides dans l'amygdale exercent un effet modulatoire sûr les réactions de peur et sur l'anxiété, on a étudié les effets directs et indirects d'une série de neuropeptides sur les différentes projections pour évaluer s'il y a des neuropeptides qui agissent spécifiquement sur une. On n'a pas trouvé de différences entre neurones qui projettent vers le PAG et neurones qui projettent vers le NTS concernant les effets de neuropeptides qui agissent directement sur ces cellules. Par contre, on a trouvé que l'ocytocine, un neuropeptide qui se lie à des récepteurs dans la partie latérale de l'amygdale centrale et inhibe de façon indirecte les neurones de l'amygdala centrale médiale, module les projections vers le PAG sans affecter celles qui vont vers le NTS. Comme le PAG est impliqué dans la réponse d'immobilisation, alors que le NTS est impliqué dans la modulation cardiovasculaire, on a ensuite étudié les effets de l'ocytocine injectée dans l'amygdale de rat vivants sur les réactions de peur conditionnées. On a trouvé que l'ocytocine diminue la réponse d'immobilisation sans par contre affecter la réponse cardiovasculaire. Pour terminer, on a vérifié si l'ocytocine potentialise les effets d'un médicament anxiolytique, le diazeparn. Avec une étude in vitro on a trouvé qu'une co-application d'ocytocine et diazeparn résulte en un effet additionnel à la fois sur la transmission synaptique ainsi que sur l'activité neuronale des neurones de l'amygdale centrale médiale. Ces résultats ouvrent des nouvelles perspectives pour une potentielle utilisation clinique de l'ocytocine.

Several neuronal and axonal types form long intrinsic connections in the cat primary auditory cortical field (AI).

Intrinsic connections in the cat primary auditory field (AI) as revealed by injections of Phaseolus vulgaris leucoagglutinin (PHA-L) or biocytin, had an anisotropic and patchy distribution. Neurons, labelled retrogradely with PHA-L were concentrated along a dorsoventral stripe through the injection site and rostral to it; the spread of rostrally located neurons was greater after injections into regions of low rather than high characteristic frequencies. The intensity of retrograde labelling varied from weak and granular to very strong and Golgi-like. Out of 313 Golgi like retrogradely labelled neurons 79.6% were pyramidal, 17.2% multipolar, 2.6% bipolar, and 0.6% bitufted; 13.4% were putatively inhibitory, i.e. aspiny or sparsely spiny multipolar, or bitufted. Individual anterogradely labelled intrinsic axons were reconstructed for distances of 2 to 7 mm. Five main types were distinguished on the basis of the branching pattern and the location of synaptic specialisations. Type 1 axons travelled horizontally within layers II to VI and sent collaterals at regular intervals; boutons were only present in the terminal arborizations of these collaterals. Type 2 axons also travelled horizontally within layers II to VI and had rather short and thin collateral branches; boutons or spine-like protrusions occurred in most parts of the axon. Type 3 axons travelled obliquely through the cortex and formed a single terminal arborization, the only site where boutons were found. Type 4 axons travelled for some distance in layer I; they formed a heterogeneous group as to their collaterals and synaptic specializations. Type 5 axons travelled at the interface between layer VI and the white matter; boutons en passant, spine-like protrusions, and thin short branches with boutons en passant were frequent all along their trajectory. Thus, only some axonal types sustain the patchy pattern of intrinsic connectivity, whereas others are involved in a more diffuse connectivity.

Mophological analysis of the human vasculature from medical imaging. Application to cerebral and coranary arteries

In this project, we have investigated new ways of modelling and analysis of human vasculature from Medical images. The research was divided in two main areas: cerebral vasculature analysis and coronary arteries modeling. Regarding cerebral vasculature analysis, we have studed cerebral aneurysms, internal carotid and the Circle of Willis (CoW). Aneurysms are abnormal vessel enlargements that can rupture causing important cerebral damages or death. The understanding of this pathology, together with its virtual treatment, and image diagnosis and prognosis, includes identification and detailed measurement of the aneurysms. In this context, we have proposed two automatic aneurysm isolation method, to separate the abnormal part of the vessel from the healthy part, to homogenize and speed-up the processing pipeline usually employed to study this pathology, [Cardenes2011TMI, arrabide2011MedPhys]. The results obtained from both methods have been also compared and validatied in [Cardenes2012MBEC]. A second important task here the analysis of the internal carotid [Bogunovic2011Media] and the automatic labelling of the CoW, Bogunovic2011MICCAI, Bogunovic2012TMI]. The second area of research covers the study of coronary arteries, specially coronary bifurcations because there is where the formation of atherosclerotic plaque is more common, and where the intervention is more challenging. Therefore, we proposed a novel modelling method from Computed Tomography Angiography (CTA) images, combined with Conventional Coronary Angiography (CCA), to obtain realistic vascular models of coronary bifurcations, presented in [Cardenes2011MICCAI], and fully validated including phantom experiments in [Cardene2013MedPhys]. The realistic models obtained from this method are being used to simulate stenting procedures, and to investigate the hemodynamic variables in coronary bifurcations in the works submitted in [Morlachi2012, Chiastra2012]. Additionally, another preliminary work has been done to reconstruct the coronary tree from rotational angiography, and published in [Cardenes2012ISBI].

Biochemical, morphological and content characterization of synaptobrevin 2-expressing vesicles in astrocytes

Neuron-astrocyte reciprocal communication at synapses has emerged as a novel signalling pathway in brain function. Astrocytes sense the level of synaptic activity and, in turn, influence its efficacy through the regulated release of ''glio- transmitters'' such as glutamate, ATP or D-serine. A calcium- dependent exocytosis is proposed to drive the release of gliotransmitters but its existence is still debated. To shed light onto the mechanisms controlling the storage and the release of gliotransmitters and namely D-serine, we have developed a new method for the immunoisolation of synaptobrevin 2-positive vesicles from rat cortical astrocytes in culture. The purified organelles are clear round shape vesicles of excellent purity as judged by electron microscopy. Immunoblotting analysis revealed that isolated vesicles contain most of the major proteins already described for neuron-derived vesicles. In addition, we have analyzed the content for various amino acids of these vesicles by means of chiral capillary electro- phoresis coupled to laser-induced fluorescence detection and liquid chromatography coupled to mass spectrometry. Post- embedding immunogold labelling of the rat neocortex and hippocampus further revealed the expression of D-serine and glutamate in astrocyte processes contacting excitatory sy- napses. Our results provide significant support for the existence of secretory glial vesicles storing chemical substances like D- serine and glutamate and thus point to the co-release of amino acids by exocytosis in astrocytes.

ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells.

Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and "pump" functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34), the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy.

On the Segregation of Genetically Modified, Conventional, and Organic Products in European Agriculture: A Multi-market Equilibrium Analysis, October 2005

Evaluating the possible benefits of the introduction of genetically modified (GM) crops must address the issue of consumer resistance as well as the complex regulation that has ensued. In the European Union (EU) this regulation envisions the “co-existence” of GM food with conventional and quality-enhanced products, mandates the labelling and traceability of GM products, and allows only a stringent adventitious presence of GM content in other products. All these elements are brought together within a partial equilibrium model of the EU agricultural food sector. The model comprises conventional, GM and organic food. Demand is modelled in a novel fashion, whereby organic and conventional products are treated as horizontally differentiated but GM products are vertically differentiated (weakly inferior) relative to conventional ones. Supply accounts explicitly for the land constraint at the sector level and for the need for additional resources to produce organic food. Model calibration and simulation allow insights into the qualitative and quantitative effects of the large-scale introduction of GM products in the EU market. We find that the introduction of GM food reduces overall EU welfare, mostly because of the associated need for costly segregation of non-GM products, but the producers of quality-enhanced products actually benefit.

Specific binding of antigenic peptides to cell-associated MHC class I molecules.

T lymphocytes recognize antigen in the form of peptides that associate with specific alleles of class I or class II major histocompatibility (MHC) molecules. By contrast with the clear MHC allele-specific binding of peptides to purified class II molecules purified solubilized class I molecules either bind relatively poorly or show degenerate specificity. Using photo-affinity labelling, we demonstrate here the specific interaction of peptides with cell-associated MHC class I molecules and show that this involves metabolically active processes.

Patterns of plant subcellular responses to successful oomycete infections reveal differences in host cell reprogramming and endocytic trafficking.

Adapted filamentous pathogens such as the oomycetes Hyaloperonospora arabidopsidis (Hpa) and Phytophthora infestans (Pi) project specialized hyphae, the haustoria, inside living host cells for the suppression of host defence and acquisition of nutrients. Accommodation of haustoria requires reorganization of the host cell and the biogenesis of a novel host cell membrane, the extrahaustorial membrane (EHM), which envelops the haustorium separating the host cell from the pathogen. Here, we applied live-cell imaging of fluorescent-tagged proteins labelling a variety of membrane compartments and investigated the subcellular changes associated with accommodating oomycete haustoria in Arabidopsis and N. benthamiana. Plasma membrane-resident proteins differentially localized to the EHM. Likewise, secretory vesicles and endosomal compartments surrounded Hpa and Pi haustoria revealing differences between these two oomycetes, and suggesting a role for vesicle trafficking pathways for the pathogen-controlled biogenesis of the EHM. The latter is supported by enhanced susceptibility of mutants in endosome-mediated trafficking regulators. These observations point at host subcellular defences and specialization of the EHM in a pathogen-specific manner. Defence-associated haustorial encasements, a double-layered membrane that grows around mature haustoria, were frequently observed in Hpa interactions. Intriguingly, all tested plant proteins accumulated at Hpa haustorial encasements suggesting the general recruitment of default vesicle trafficking pathways to defend pathogen access. Altogether, our results show common requirements of subcellular changes associated with oomycete biotrophy, and highlight differences between two oomycete pathogens in reprogramming host cell vesicle trafficking for haustoria accommodation. This provides a framework for further dissection of the pathogen-triggered reprogramming of host subcellular changes.